392 j contemp med sci | vol. 7, no. 6, november-december 2021: 392–393 letter to the editor can apical periodontitis exacerbate covid-19 damage? umut aksoy1*, ahmet özer şehirli2, serkan sayiner3 1near east university, faculty of dentistry, department of endodontics, near east avenue, lefkosa, 99138, mersin 10, turkey. 2near east university, faculty of dentistry, department of pharmacology, near east avenue, lefkosa, 99138, mersin 10, turkey. 3near east university, faculty of veterinary medicine, department of biochemistry, near east avenue, lefkosa, 99138, mersin 10, turkey. *correspondence to: umut aksoy (e-mail: umut.aksoy@neu.edu.tr) issn 2413-0516 dear editor, the covid-19 pandemic remains an important health problem, potentially worsened by emerging mutant strains. although vaccination studies have been rapidly carried out, the definitive treatment protocol for the disease has not yet been established. therefore, treatment suggestions and studies continue to have importance. until the end of the 1980s, the systemic effects of dental infections were ignored except in immunodeficient patients. later studies revealed a relationship between dental infections and myocardial and cerebral infarction, increasing research interest in this topic.1-2 the relationship between periodontitis – which has an important role in dental infections – and systemic diseases has been determined. although the studies in this area have increased,3,4 the relationship between apical periodontitis and systemic disease has not yet been completely established. the periapical infections caused by intracanal microorganisms ensure the secretion of inflammatory cytokines and might accelerate systemic diseases by causing inflammation in distant organs. covid-19 infection might be accompanied by the excessive production of proinflammatory cytokines, also called “cytokine storm”.5 the already increased circulatory proinflammatory cytokines in people with apical periodontitis might aggravate the covid-19 disease by triggering the cytokine storm and might worsen prognosis. apical periodontitis is a common infectious disease worldwide; it is also the most frequent inflammatory lesion in the jaw related to the teeth.6 apical periodontitis is characterized by an inflammatory response and bone destruction in the periapical tissues resulting from both bacterial infections in the dental pulp and immune response as part of the body’s defensive reaction. epidemiological studies worldwide indicate that apical periodontitis is a widespread oral health problem that could threaten the general health of the population.7 because high serum acute reactive phase proteins were found in those with apical periodontitis, this disease was thought to be systemic. indeed, it was shown to be associated with high levels of tumour necrosis factor-alpha (tnf-α), interleukin 1 (il-1), interleukin 2 (il-2), interleukin 6 (il-6), and interferon-gamma (ifn-γ) in the serum. aksoy et al.2 have shown that the activities of two serum matrix metalloproteinases (mmp1 and mmp2) were increased in rats with apical periodontitis compared to healthy rats. şehirli et al.8 showed that in those with apical periodontitis, serum levels of lactate dehydrogenase, alkaline phosphatase, and creatine kinase increased while superoxide dismutase activity decreased, resulting in structural damage to heart tissue. apical periodontitis affects t helper cells, activating macrophages. the increase in cytokine expression is thought to increase sensitivity in people with systemic damage. after coronavirus disease 2019 (covid-19) infection, people can undergo severe tissue damage resulting from the increased expression of cytokines such as tnf-α, il-1, il-6, and ifn-γ. it appears that covid-19 activates various inflammatory pathways together with oxidative stress, thus producing intense inflammation in tissues such as the lung, heart, brain, stomach, intestine, and kidney.9 because of the reported increased expression of cytokines, particularly those that affect the immune system, people with lung disease, cardiovascular system disorders, neurological disorders, or gastrointestinal problems can be comparatively more affected by covid-19, resulting in greater mortality rates than in the healthy population.10 to date, an evaluation and study of the relationship between apical periodontitis and covid-19 have not been published. it is obvious that diseases increasing cytokine expression in the blood can negatively affect covid-19 outcomes. there is a possibility of cytokine storm during covid19, and the already existing elevated cytokine levels in apical periodontitis might increase the risk and lead to severe systemic effects on entire systems. in conclusion, the demonstrated increase in serum cytokine expression from apical periodontitis can pose a risk for those with covid-19, and people with this disorder should be treated preferentially.  references 1. zhang j, huang x, lu b, zhang c, cai z. can apical periodontitis affect serum levels of crp, il-2, and il-6 as well as induce pathological changes in remote organs? clin oral invest. 2016;20(7):1617–24. doi:10.1007/s00784015-1646-6. 2. aksoy u, savtekin g, şehirli aö, kermeoglu f, kalender a, ozkayalar h, et al. effects of alpha-lipoic acid therapy on experimentally induced apical periodontitis: a biochemical, histopathological and micro-ct analysis. int endod j. 2019;52(9):1317–26. doi:10.1111/iej.13121 3. sehirli aö, chukwunyere u, aksoy u, sayiner s, abacioglu n. the circadian clock gene bmal1: role in covid-19 and periodontitis. chronobiol int. 2021;38(6):779–784. doi: 10.1080/07420528.2021.1895198. 4. şehirli aö, aksoy u, koca-ünsal rb, sayıner s. role of nlrp3 inflammasome in covid-19 and periodontitis: possible protective effect of melatonin. med hypotheses. 2021;30:110588. doi: 10.1016/j. mehy.2021.110588. 5. shimabukuro-vornhagen a, gödel p, subklewe m, stemmler hj, schlößer ha, schlaak m, kochanek m, böll b, von bergwelt-baildon ms. cytokine release syndrome. j immunother cancer. 2018;15;6(1):56. doi: 10.1186/ s40425-018-0343-9. 6. becconsall-ryan k, tong d, love rm. radiolucent inflammatory jaw lesions: a twenty-year analysis. int endod j. 2010;43(10):859–65. doi: 10.1111/j.1365-2591.2010.01751.x. (submitted: 02 december 2021 – revised version received: 10 december 2021 – accepted: 16 december 2021 – published online: 26 december 2021) mailto:umut.aksoy@neu.edu.tr 393j contemp med sci | vol. 7, no. 6, november-december 2021: 392–393 u. aksoy et al. letter to the editor apical periodontitis and covid-19 7. persoon if, özok ar. definitions and epidemiology of endodontic infections. curr oral health rep. 2017;4(4):278–85. doi:10.1007/s40496017-0161-z. 8. şehirli aö, aksoy u, kermeoglu f, kalender a, savtekin g, ozkayalar h, et al. protective effect of alpha‐lipoic acid against apical periodontitis‐induced cardiac injury in rats. eur j oral sci. 2019;127(4):333–9. doi:10.1111/ eos.12618 9. han h, ma q, li c, liu r, zhao l, wang w, zhang p, liu x, gao g, liu f, jiang y, cheng x, zhu c, xia y. profiling serum cytokines in covid-19 patients reveals il-6 and il-10 are disease severity predictors. emerg microbes infect. 2020;9(1):1123–30. doi: 10.1080/22221751.2020.1770129. 10. sehirli ao, sayiner s, serakinci n. role of melatonin in the treatment of covid-19; as an adjuvant through cluster differentiation 147 (cd147). mol biol rep. 2020;47(10),8229–33. doi: 10.1007/s11033-020-05830-8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1113 321j contemp med sci | vol. 7, no. 5, september–october 2021: 321–322 case report post covid-19 subacute thyroiditis: a case report mohammed a. abusaiba1, najah r. hadi1* , jan fedacko2, ram b singh3, hayder a. al-aubaidy4 1 faculty of medicine, university of kufa, al-najaf, iraq. 2 faculty of medicine, safrik university, slovakia. 3 halberg hospital and research institute, moradabad, india. 4 school of life sciences, la trobe university, vic, 3086, australia. *correspondence to: professor najah r. hadi (e-mail: drnajahhadi@yahoo.com) (submitted: 17 august 2021 – revised version received: 29 august 2021 – accepted: 14 september 2021 – published online: 26 october 2021) abstract subacute thyroiditis is a non-suppurative inflammation of the thyroid gland, which usually develops after viral infection to the upper respiratory tract, accompanied by pain in the neck radiated to the jaw and fever. this is a case report study for a 43-year-old woman, who developed subacute thyroiditis 6 weeks following coronavirus disease (covid-19). issn 2413-0516 background subacute thyroiditis (sat) is usually associated by local swelling and inflammation of the thyroid gland secondary to viral infections. sat usually occurs 2–8 weeks after the occurrence of viral upper respiratory tract infection.1 the pain in sat is usually a sudden, may start in one lobe but rapidly spread to include all the gland. it is associated with swelling of the thyroid gland, and tenderness over the anterior side of the neck. it is also frequently associated with constitutional symptoms, such as fever, myalgia, arthralgia, generalized weakness, and sore throat.2 case report a 43-year-old female patient with no previous history of chronic disease were presented to the outpatient clinic at al-sader teaching hospital, al-najaf, iraq. the patient was complaining of sore throat and fever with sudden onset of pain started in the neck, then it is radiating to the lower jaw. she has recently been diagnosed by sars-cov-2 (covid-19), 6 weeks ago, confirmed by a nasopharyngeal swab test following cough fever and shortness of breath. the patient had no family history of chronic illnesses nor previous infection with covid-19. she had no contact with known positive case. she also did not have a recent travel history. on examination, her neck was red, swollen, and tender by palpation. tonsils were hyperemic. she had fever (her body temperature was 38.3°c) and tachycardia (her heart rate was 130 beats per a minute). no other abnormalities were found on examinations. thyroid ultrasound examination revealed a heterogenous parenchyma with patchy infiltrations and hypoechoic areas observed in both thyroid lobes (figure 1). her biochemical laboratory examination results are as follows: thyroid-stimulating hormone (tsh)/thyrotropin ratio was 0.95; thyroxine (t4) level was 95.7 µiu/ml; triiodothyronine (t3) level was 1.5 µmol/ml, table 1. her blood examination for complete blood count shows generalized leukocytosis, erythrocyte sedimentation rate (esr) was 65 mm/h; and c reactive protein (crp) level was 35 mg/dl, table 1. fig. 1 ultrasound imaging of the thyroid for the sat patient, showing heterogenous tissue with localised inflammatory areas. 322 j contemp med sci | vol. 7, no. 5, september–october 2021: 321–322 post covid-19 subacute thyroiditis: a case report case report najah r. hadi et al. discussion at this stage, the known cause for subacute granulomatous thyroiditis is viral infection of the upper respiratory airways. there are several viral infections which may contribute to the development of sat, these include enterovirus, coxsackievirus, mumps, measles, and adenovirus.3 having said that, there is little known about the role of covid-19 in the pathogenesis of this disease and the development of its complications.4,5 previous studies have shown sat is more prevalent in females as compared to males.6 considering the possibility of genetic predisposition and contagious characteristics observed in the same family members, human leukocyte antigen (hla)-b35-mediated system is thought to play an active role in this mechanism.7 the characteristic clinical finding of sat is pain in the thyroid gland that is usually of a sudden onset. pain may radiate to the neck or lower jaw area in severe cases and it may worsen with neck movements and coughing.1,2,5 fever, weakness, and fatigue can also be observed due to both inflammation and mild hyperthyroidism, especially at the start of the disease. palpitations, sweating, and tremors may occur due to high thyroid hormone titers in the blood.1,2 these findings usually disappear after 4–10 weeks following the improvement of the condition. the patient may develop asymptomatic, overt, or subclinical hypothyroidism following sat. in this case study, the patient had normal thyroid function, but she complained of sore throat, fever, and difficulty in swallowing. she also had eating difficulty. despite using a pain killer tablet, her symptoms remained. her laboratory examination revealed leukocytosis, high crp, and elevated esr levels (table 1). these are expected findings following sat. her thyroid ultrasonography (figure 1) showed large and multiple thyroid inflammation areas with patchy style. there was also slow blood flow to the gland. the thyroid gland was tender on placing the ultrasound probe and the patient complained of sever neck pain while performing the ultrasonography. having said that, the ultrasonographic images were consistent with typical sat diagnosis. the pathophysiological process for the development of sat following covid-19 infection is similar to other known causative viruses, whether during the viral infection stage or following through the post-viral inflammation process. a study done in mid-2020, explained the correlation of the post covid-19 infection and the development of sat due to the viral affinity to angiotensin converting enzyme 2 (ace2) receptors, which are more predominant in the thyroid cells when compared to the lung cells.8 as such, it is critical to consider sat as a potential complication following covid-19 infection. most importantly, knowing that most of the new cases are asymptomatic.8 careful examination of the neck looking for localized tenderness around the thyroid gland with laboratory investigation for thyroid function test is advisable for all patients following covid-19 infection. conclusion physicians should consider sat as a potential complication following covid-19. delayed diagnosis may result in severe pain and tenderness in the neck region which may reduce patient’s quality of life following covid-19 infection. therefore, early thyroid function tests is recommended to be performed in patients with sore throat and fever.  table 1. biochemical analysis for the patients with subacute thyroiditis following covid-19 infection parameters levels thyroid function test thyroid stimulating hormone/thyrotropin ratio 0.95 uiu/ml thyroxine (t4) 1.5 umol/ml triiodothyronine (t3) 95.7 umol/ml complete blood picture wbc 14.52 109/l lymphocytes 2.36 109/l monocytes 0.8 109/l neutrophiles 11.25 109/l eosinophiles 0.09 109/l basophiles 0.005 109/l rbcc 4.67 1012/l hemoglobin 14.1 g/dl hematocrit ratio 37.5% mcv 80.5 fl mch 30.1 pg mchc 37.6 g/dl rdw_cv 16.2% rdw_sd 41.6 fl erythrocyte sedimentation rate (esr) 65 mm/h c-reactive protein (crp) 35 mg/dl references 1. li, j.h., g.h. daniels, and g. barbesino, painful subacute thyroiditis is commonly misdiagnosed as suspicious thyroid nodular disease. mayo clin proc innov qual outcomes, 2021. 5(2): p. 330–337. 2. seyed resuli, a. and m. bezgal, subacute thyroiditis in covid-19 patients. ear nose throat j, 2021: p. 1455613211012114. 3. samuels, m.h., subacute, silent, and postpartum thyroiditis. med clin north am, 2012. 96(2): p. 223–33. 4. brancatella, a., et al., subacute thyroiditis after sars-cov-2 infection. j clin endocrinol metab, 2020. 105(7). 5. mattar, s.a.m., et al., subacute thyroiditis associated with covid-19. bmj case rep, 2020. 13(8). 6. nishihara, e., et al., clinical characteristics of 852 patients with subacute thyroiditis before treatment. intern med, 2008. 47(8): p. 725–9. 7. desailloud, r. and d. hober, viruses and thyroiditis: an update. virol j, 2009. 6: p. 5. 8. muller, i., et al., sars-cov-2-related atypical thyroiditis. lancet diabetes endocrinol, 2020. 8(9): p. 739–741. doi: https://doi.org/10.22317/jcms.v7i5.1088 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 278 j contemp med sci | vol. 8, no. 4, july-august 2022: 278–280 case report elongated mental spine with two lingual foramina in the mandible of a kurdish woman: case report fadi shamshoon hagi1*, liqaa jabir hassan2, yaseen sedeeq yaseen3 1dental unit, technical department, duhok health directorate, duhok, iraq. 2implant unit, azadi teaching hospital, duhok health directorate, duhok, iraq. 3maxillofacial surgery department, college of dentistry, university of duhok, duhok, iraq. *correspondence to: fadi shamshoon hagi (e-mail: fadishamshoon78@gmail.com) (submitted: 28 june 2022 – revised version received: 06 july 2022 – accepted: 21 july 2022 – published online: 26 august 2022) abstract the mental spines are bony prominences found in the lingual surface of anterior mandible, and usually they are arranged in two groups, superior pair and inferior pair. mostly they are asymptomatic, but sometimes they may interfere with the prosthodontic treatment or with some surgeries. the lingual foramen can be found in different locations around the mental spines, and they are very important vital organ because they transmit nerves and arteries, which may cause a serious problem during some types of surgeries in this region such as implant placement. here, we report a case of 55-year-old, kurdish woman with elongated superior mental spine with bilaterally lingual foramina located distally to lower central incisors, that have been seen during a routine cbct scan. the length of the spine was 6.4 mm, the height was 5.5 mm and the width was 1.9 mm. so, any dentist wants to perform any type of surgery in sublingual and submental regions should be careful of such anatomical variations. keywords: cbct, mental spine, lingual foramen, mandible issn 2413-0516 introduction many anatomical landmarks can be seen in the anterior lingual surface of the mandible, like mental spines, lingual foramen and mandibular symphysis.1 the mental spines (genial tubercules) are small bony eminences projected from the lingual surface of mandible.2 usually four spines are found, two superiors, to which the genioglossus muscles are attached and two inferiors, to which the geniohyoid muscles find their origin.3 the mental spines are important landmarks for maxillofacial surgeons, dental radiologist and prosthodontists. also, a variation in its morphology and number has been highly reported.4,5 the lingual foramen is an opening located on the midline of the lingual aspect of the anterior mandible. it transmits neurovascular bundles to the surrounding structures and it could have variations in number and position.6 the cone-beam computed tomography is a revolutionary imaging modality, which allows three-dimensional visualization of hard tissue structures. it provides accurate volumetric data in axial, sagittal and coronal planes which can be useful in diagnosing and treating several pathologies in oral and maxillofacial region.5 this modality has been commonly used to evaluate the morphology, size, number and positions of mental spines and lingual foramen.2,5–7 case presentation during a routine cbct scan in delight dental clinic in duhok governorate at the north of iraq, for a kurdish woman whose age was 55 years, an elongated mental spine was observed on the lingual surface of mandible exactly at the midline (figure 1). this spine was projected posteriorly from the lingual surface of the mandible with a slight tilt to right. the anterior-posterior length was 6.4 mm, the height was 5.5 mm and the width was 1.9 mm. also, two inferior mental spines were observed near the inferior border of the mandible at the midline point (figure 2). besides that, two lingual foramina were found distally to the lower central incisors, the right one about 2.2 mm away from the alveolar crest while the left one about 3.3 mm (figure 3). the symphysis menti (mandibular symphysis) was not observed in this case. and no other radiographic anatomical variations were found on this site of mandible. the type of cbct machine was xmind prime cbct, manufactured by de gotzon action group – italy (2021). discussion using the cbct in dentistry is an essential component of dental treatment planning.8 many studies such as hueman et al.9 showed the accuracy of the 3d cone beam ct in the description of the anatomical location of the mental spine. the mental spine may become (relatively) enlarged and prominent due to a combination of calcification in the tendinous insertion of the geniohyoid and genioglossus muscles and atrophy of the mandible.3 wong et al.1 reported a case of an elongated mental spine in a 57-year-old caucasian woman’s cadaveric head, with dimensions of 10 mm length, 2.72 mm width and 8.55 mm height. greyling et al.10 reported a case of projected mental spine posteriorly from its normal anatomical position on the lingual surface of mandible in 79-year-old white female cadaver, it was flattened from superior to inferior, with 10 mm length. jindal et al.11 reported a case of elongated mental spine in a 78-year-old, completely edentulous male patient with the chief complaint of discomfort while eating. the patient was a denture wearer for 10 years and had discontinued wearing denture because of recurrent ulceration of the lingual mucosa caused by the denture. the computed tomographic scan revealed 11 mm wide and 21 mm long, genial tubercles extending about 15 mm beyond the crest of residual mandibular ridge. jawahar and gopal5 grouped the mental spines into four types: a rough impression of two superior genial tubercles 279j contemp med sci | vol. 8, no. 4, july-august 2022: 278–280 f.s. hagi et al. case report elongated mental spine with two lingual foramina in the mandible of a kurdish woman: case report fig. 3 two lingual foramina located distally to the lower central incisors. a: 3d rendering view. b: right lingual foramen (sagittal view). c: left lingual foramen (sagittal view). d: axial view. fig. 2 the inferior mental spines. a: coronal view. b: axial view. fig. 1 the elongated superior mental spine in the lingual surface of the mandible. a: 3d rendering view. b: axial view. (type i), two superior genial tubercles and a median ridge representing fusion of inferior genial tubercles below them (type ii), a single median ridge (type iii) and no prominent genial tubercles (type iv). while singh et al.4 grouped his patients in five types: the classical description of four spines, two superiors and two inferiors were observed in (19.25%), while (70.16%) showed the presence of only two superior spines. in (46.83%) were associated with a median vertical ridge below them, (23.33%) had only a rough impression in place of the median ridge. (8.75%) showed a single prominent median eminence/projection but no separate spines as such. finally (1.83%) had no spine, ridge or prominence. the lingual foramina and canals can be categorized as medial or lateral based on their relation to the midline of the mandible.12 it can be located mainly above and/or below the mental spines, and diameters may be associated with high risk of bleeding vessels.7 denny et al.6 studied the cbct scans of 116 patients, the number of lingual foramina ranged from 1 to 3, their position was mainly (60%) in the upper two-thirds distance from the alveolar crest, and there was no significant difference in various age groups or between both genders. wang et al.13 used the cbct to report the presence of the lingual foramina in 97% of his sample, also the presence of more than one canal medially and laterally was observed. conclusion we reported an elongated mental spine, which seems to be a rare case that has not been seen before, represented by an elongated superior mental spine with two inferior mental spines. also, the two lingual foramina show a rare style, because they are two in number located in symmetrical position to the midline and near the alveolar crest. the elongated mental spine in spite that is a symptomatic structure, but it still has a great importance for any type of surgery and prosthodontic treatment in area of anterior lingual aspect of mandible. and the lingual foramina are considered very important for any kind of surgery especially the implant placement, because any damage to their vital contents may cause serious problems. conflict of interest none.  references 1. wong tl, iwanaga j, anand mk, tubbs rs. an elongated mental spine: case report. natl j clin anat 2019; 8:130–132. 2. araby ya, alhirabi aa, santawy ah. genial tubercles: morphological study of the controversial anatomical landmark using cone beam computed tomography. world j radiol 2019; 11(7):94–101. 3. leeuwen va, meij vde, visscher dj. fracture of the genial tubercles of the mandible: case report and review of the literature. j oral maxillofac surg 2014; 72(10):1994.e1–6. doi: 10.1016/j.joms.2014.05.001. 4. singh v, anand mk and dinesh k. variations in the pattern of mental spines and spinous mental foramina in dry adult human mandibles. surg radiol anat 2000; 22:169–173. 5. jawahar a, gopal m. analysis of genial tubercle anatomy using cone beam computed tomography a retrospective study from chennai, india j evolution med dent sci 2021; 10(38):3333–3337. doi: 10.14260/ jemds/2021/676. 6. denny ce, natarajan s, ahmed j, binnal a, jindal r. anatomic variation in lingual foramen: a cone beam computed tomography study. world j dent 2016;7(4):179–181. 7. soto r, concha g, pardo s and caceres. determination of presence and morphometry of lingual foramina and canals in chilean mandibles using cone-beam ct images. surg radiol anat 2018; 40:1405–1410. https://doi. org/10.1007/s00276-018-2080-7. 280 j contemp med sci | vol. 8, no. 4, july-august 2022: 278–280 elongated mental spine with two lingual foramina in the mandible of a kurdish woman: case report case report f.s. hagi et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 8. harris d, horner k, grondahl k, jacobs r, helmrot e, benic gi, bornstein mm, dawood a, quirynen m. e.a.o. guidelines for the use of diagnostic imaging in implant dentistry 2011. a consensus workshop organized by the european association for osseointegration at the medical university of warsaw. clin oral implants res 2012; 23(11):1243–53. doi: 10.1111/j.16000501.2012.02441.x. 9. hueman em, noujeim me, langlais rp, prihoda tj and miller fr. accuracy of cone beam computed tomography in determining the location of the genial tubercle. otolaryngol head neck surg 2007; 137: 115–118. 10. greyling lm, le grange f and meiring jh. mandibular spine: a case report. clin anat 1997; 10:416–418. 11. jindal g, jindal s, sharma p and singla a. rare enlargement of genial tubercles and its management: a case report. j clin diagn res 2015; 9(11):zd23–zd24. 12. he p, truong mk, adeeb n, tubbs rs and iwanaga j. clinical anatomy and surgical significance of the lingual foramina and their canals. clin anat 2017; 30:194–204. https://doi.org/10.1002/ca.22824. 13. wang ym, pan yrjwl and chan cp: evaluation of location and dimensions of mandibular lingual canals: a cone beam computed tomography study. int j oral maxillofac surg 2015; 44:1197–1203. https://doi.org/10.22317/jcms.v8i4.1235 44 j contemp med sci | vol. 8, no. 1, january-february 2022: 44–46 original a study of the incidence of cytomegalovirus infection and preemptive therapy in kidney transplant recipients zana sidiq mohammed saleem1*, kais h abd2, nashwan m.r. ibrahim3, nawfal r hussein4, hasanain ma al-khammas5 1department of internal medicine, college of medicine, university of duhok, duhok, iraq. 2duhok renal dialysis and organ transplant center, duhok, kurdistan region, iraq. 3department of surgery, college of medicine, university of duhok, duhok, iraq. 4department of medicine, college of medicine, university of zakho, zakho, kurdistan region, iraq. 5basrah center for kidney disease and renal transplant center, ministry of health, basrah, iraq. *correspondence to: zana sidiq mohammed saleem (e-mail: zanasidik@gmail.com) (submitted: 13 december 2021 – revised version received: 28 december 2021 – accepted: 07 january 2022 – published online: 26 february 2022) abstract objectives: this study aimed to determine the incidence of cvm infection and evaluate the outcome of preemptive treatment. methods: this prospective, two centers cohort study included recipients who underwent renal transplant between may 2019–may 2020. the incidence of cmv infection and graft outcomes were studied. all managed with preemptive therapy. results: in this study, 134 renal transplant recipients were recruited. among them, 30/134 (22.4%) patients tested positive for cmv-rtpcr. we studied the impact of age on the cmv-positivity and we found that the age of the doner was associated with cmv-positivity (p = 0.025; or = 0.923; ci = 0.8584–0.9943). smoking and gender showed no association with cmv-positivity. conclusion: our data suggest that the course of cmv infection is benign with a high success rate of preemptive treatment. further evaluation for the universal prophylactic plan is needed. keywords: cmv, preemptive, iraq, kidney, transplant issn 2413-0516 introduction cmv causes asymptomatic diseases in immunocompetent patients and it does not need specific treatment. however, it can cause serious diseases that lead to deleterious outcomes in pregnancy and immunocompromised patients such as aids patients and organ transplant recipients.1-3 in organ transplant recipients including renal transplant, cmv infections are the major cause of infectious diseases morbidity and mortality with an incidence ranging from 19–90%.4 to prevent organ rejection, organ transplant recipients are markedly immunosuppressed in the first three months after the operation.4 this gives the opportunity for cmv to infect different organs including colon and retina.4 without prophylaxis or preemptive treatment, such an infection may lead to three outcomes: infectious disease syndrome; increased immunosuppression or organ rejection.4,5 ganciclovir and valganciclovir are the first-line medications for prophylaxis and treatment of such infections.5 the chance of cmv infection after organ transplant is determined by the status of donor and recipient cmv serology. to prevent infections in highrisk patients (d+/r–), prophylactic medications can be given, whereas preemptive strategy can be used for moderate risk (d+/r+, d–/r+) or low risk patients (d–/r–).4-6 such a plan of cmv treatment and prevention is not used in some transplant centers due to economic burden and medications unavailability. this may lead to an increased rate of cmv infections and high rates of cmv resistance and relapse.1,3,6 post-transplant infection has been studied thoroughly in our center.7-10 in our organ transplant center, prophylactic strategy was not used due to the expense and unavailability of the medications. the aims of this project were to determine the incidence of cvm infection and evaluate the outcome of preemptive treatment. materials and methods study design this was a prospective cohort study that was conducted in duhok and basrah organ transplant centers between may 2019–may 2020. patients in the study period, all renal transplant recipients were recruited in the study. in our center, all renal transplant recipients were managed according to the local protocol and the immunosuppressant regimen was composed of anti thymocyte globuline (atg) 1.5 – 2 mg/kg at operation day and 4th day after the operation, tacrolimus 0.075 – 0.15 mg/kg, mycophenolate 2 g/day) and prednisolone (1 mg/kg/day then titrate to maintenance 7.5 mg/day). all renal transplant recipients were followed up in the centers. all recruited subjects were asked to visit the center monthly. in each visit, 5 cc blood was collected from the patients. serum was separated and was kept frozen at –20˚c until cmv rtpcr was performed. no cmv prophylaxis was given to patients. patients were tested on a monthly basis for cmv-rtpcr positivity. we followed up patients for 6 months after transplant. during the follow up, we evaluated the incidence of cmv infection, the efficacy of ganciclovir and valganciclovir, graft survival or failure. serum creatinine and blood urea were used as indicators for graft functionality. cmv rtpcr and igm/igg cmv rtpcr was performed utilizing artus cmv rg pcr kit (hilden, germany) following the instructions of the manufacturer. the amplification was performed using a rotor gene real time pcr system from qiagen (hilden, germany). mailto:zanasidik@gmail.com 45j contemp med sci | vol. 8, no. 1, january-february 2022: 44–46 z.s. mohammed saleem et al. original a study of the incidence of cytomegalovirus infection and preemptive therapy the patients were followed up monthly for three successive months after transplant. to determine cmv status, elecsys cmv igm and elecsys cmv igg kits were utilized (cobas, roche, mannheim, germany). ethics this study was approved by the ethics committee in the college of medicine, university of zakho. written consent was obtained from all recruited patients. statistics binary logistic regression was utilized to study the relationship between clinical outcomes and factors. all calculations were performed using minitab 17 software. results patients in this study, 134 renal transplant recipients were recruited. 69.4% of them were male and the mean age of our patients was 39 ± 13.3 years (table 1). all donations came from living donors; cadaver donation was not acceptable religiously and socially. cmv serology study showed that all recipients and donors were cmv igg positive/igm negative. rtpcr positive patients during the follow up period, 30/134 (22.4%) patients tested positive for cmv-rtpcr. among those, 18 (60%) were male and the average age of 39.97 ± 12.48 (table 2). cmv-positive patients gave the history of hemodialysis of 9.8 ± 19 months (table 2). the infection did not affect the function of the graft. we studied factors that might impact cmv-positivity in renal transplant recipients (table 3). we studied the impact of different diseases prior to the transplant such as hypertension, diabetes, glomerulonephritis or renal stone. we found no association between those diseases and cmv positivity. then, we studied the impact of age on the cmv-positivity and we found that the age of the doner was associated with cmv-positivity (p = 0.025; or = 0.923; ci = 0.8584 – 0.9943) (table 3). smoking and gender showed no association with cmv positivity (table 3). cmv-rtpcr became negative within a table 1. characteristics of recipients characteristics no % gender m 93 69.40 ht 108 80.60 dm 26 19.40 apckd 7 5.22 gn 12 8.96 stone 2 1.49 others 25 18.66 age (mean ± st) 39 ± 13.3 hd (mean ± st) 6.88 ± 11.4 table 2. characteristics of post-transplant cmv positive characteristics no % sex (male) 18 60 ht 25 83.3 dm 6 20 gn 3 10 stone 1 3.3 other 5 16.7 apckd 0 0 age 39.97 ± 12.48 hd 9.8 ± 19 table 3. difference between post-transplant cmv positive and negatives factors cmv negative cmv positive p or ci95 age r 38.75 ± 13.5 39.97 ± 12.48 0.66 1.01 0.9764–1.0385 age d 29 ± 6.51 26.17 ± 5.99 0.025 0.923 0.8584–0.9943 sex (male) r 75/104 (72.11%) 18/30 (60%) 0.2 0.58 0.2487–1.3528 sex (male) d 70/104 (67.3%) 19/30 (63.33%) 0.68 0.83 0.3593–1.9591 hd 6 ± 7.9 9.8 ± 19 0.131 1.02 0.9927–1.0581 ht 83/104 (79.8%) 25/30 (83.33%) 0.663 1.26 0.4327–3.6989 dm 20/104 (19.23%) 6/30 (20%) 0.925 1.05 0.3791–2.9086 gn 9/104 (8.65%) 3/30 (10%) 0.822 1.17 0.2966–4.6377 stone 1/104 (0.96%) 1/30 (3.33%) 0.39 3.55 0.2155–58.5392 other 20/104 (19.23%) 5/30 (16.67%) 0.75 0.84 0.2861–2.4659 smoking 74/104 (71.15%) 22/30 (73.33%) 0.82 1.11 0.4471–2.7798 apckd 7/104 (6.73%) 0 1 0 0 month of the treatment in 25/30 (83.33%) patients. by the end of the second month, all patients tested negative for cmv-rtpcr. discussion post-transplant cmv prophylaxis can reduce the risk of infection during the early stage after transplant operation. however, there is a concern about late onset infection after the 46 j contemp med sci | vol. 8, no. 1, january-february 2022: 44–46 a study of the incidence of cytomegalovirus infection and preemptive therapy original z.s. mohammed saleem et al. discontinuation of medications. in addition, it was shown that better t-cell response and increased levels of neutralizing antibodies against the virus were developed after preemptive therapy when compared to patients who received prophylaxis.11-13 such an immune response that developed after preemptive therapy may prevent relapse and further cmv infections after transplant.11-13 in this study, the cmv profile of all transplant patients was d+/r+. prophylaxis was not given to the patients with strict follow up by cmv-rtpcr for three months. in this study, cmv-rtpcr tested positive in the first month post-transplant in 22.4% of our patients. the course of the disease was benign and did not impact the graft function. the low rate of infection might be explained by that all our patients were positive for igg. on the other hand, the benign course of the disease can be explained by the difference in cmv genotype that may cause the disease. it was previously shown that different cmv genotypes may influence the severity and the outcome of cmv infection in organ transplant patients.14,15 cmv genotype study has not been performed in our country, therefore more studies are recommended investigating cmv genotypes and their association with disease severity and outcomes. in contrast to other studies,16 no association was found between age and incidence of cmv infection. in agreement with another study,16 gender was not associated with the incidence of cmv infection. in a previous study, it was found that older donor was associated with an increased risk of cmv infection post-transplant. we found a significant relationship between younger donor age and cmv positivity. this is difficult to explain and further studies are required to investigate the impact of donor age upon cmv infection. our study has limitations. first, this is a two centers observation study with patients all d+/r+. second, the follow up duration was 6 months and it is unknown what the further outcome was. our results suggested that further studies are needed recruiting d+/r– patients and the use of such an approach for those patients and longer follow up duration is needed to investigate the impact of preemptive therapy on the long-term outcome and graft survival.  references 1. naqid ia, yousif sh, hussein nr. serological study of igg and igm antibodies to cytomegalovirus and toxoplasma infections in pregnant women in zakho city, kurdistan region, iraq. women’s health bulletin. 2019;6(4):8–12. 2. hussein n, balatay aa. the seroprevalence of toxoplasma, cytomegalovirus and rubella infections in women with abortion in kurdistan region of iraq: a brief report. international journal of infection. 2019;6(1):e86734. 3. nangle s, mitra s, roskos s, havlichek d. cytomegalovirus infection in immunocompetent adults: is observation still the best strategy? idcases. 2018;14:e00442-e. 4. hasegawa j, hatakeyama s, wakai s, omoto k, okumi m, tanabe k, et al. preemptive anti-cytomegalovirus therapy in high-risk (donor-positive, recipient-negative cytomegalovirus serostatus) kidney transplant recipients. int j infect dis. 2017;65:50–6. 5. mozaffar m, shahidi s, mansourian m, badri s. optimal use of ganciclovir and valganciclovir in transplanted patients: how does it relate to the outcome? j transplant. 2018;2018:8414385. 6. requião-moura lr, dematos acc, pacheco-silva a. cytomegalovirus infection in renal transplantation: clinical aspects, management and the perspectives. einstein (sao paulo). 2015;13(1):142–8. 7. hussein n, saleem z. successful treatment of hepatitis c virus genotype 4 in renal transplant recipients with direct‐acting antiviral agents. american journal of transplantation. 2016;16(7):2237–8. 8. hussein n, saleem z, balatay a, abd k, daniel s, taha a, et al., editors. seroprevalence of helicobacter pylori infection in renal transplant recipient attending duhok kidney disease center. transplantation proceedings; 2016: elsevier. 9. hussein nr, saleem zs, ibrahim nm, assafi ms, daniel s. the prevalence of hbv infection in renal transplant recipients and the impact of infection on graft survival. acta medica iranica. 2019;57(6):381–4. 10. hussein nr, saleem zsm. successful treatment of acute hepatitis c virus (hcv) infection with interferon-free regimen in a renal transplant patient: the first case report. international journal of infection. 2017;4(4). 11. nebbia g, mattes fm, smith c, hainsworth e, kopycinski j, burroughs a, et al. polyfunctional cytomegalovirus-specific cd4+ and pp65 cd8+ t cells protect against high-level replication after liver transplantation. am j transplant. 2008;8(12):2590–9. 12. giménez e, blanco-lobo p, muñoz-cobo b, solano c, amat p, pérez-romero p, et al. role of cytomegalovirus (cmv)-specific polyfunctional cd8+ t-cells and antibodies neutralizing virus epithelial infection in the control of cmv infection in an allogeneic stem-cell transplantation setting. j gen virol. 2015;96(9):2822–31. 13. gerna g, lilleri d. human cytomegalovirus (hcmv) infection/re-infection: development of a protective hcmv vaccine. new microbiol. 2019;42(1):1–20. 14. tarragó d, quereda c, tenorio a. different cytomegalovirus glycoprotein b genotype distribution in serum and cerebrospinal fluid specimens determined by a novel multiplex nested pcr. j clin microbiol. 2003;41(7):2872–7. 15. torok-storb b, boeckh m, hoy c, leisenring w, myerson d, gooley t. association of specific cytomegalovirus genotypes with death from myelosuppression after marrow transplantation. blood. 1997;90(5):2097–102. 16. babazadeh a, javanian m, oliaei f, akbari r, akbarzadepasha a, bijani a, et al. incidence and risk factors for cytomegalovirus in kidney transplant patients in babol, northern iran. caspian j intern med. 2017;8(1):23–9. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1125 252 j contemp med sci | vol. 7, no. 4, july–august 2021: 252–255 original food groups that exacerbate sinusitis among saudi patients manal abdulaziz murad1, hoda jehad abousada2*, ghaida abdullah aldugman3, nesreen nabeel qusti4, shahad daifalla alsulaimani5, sahira jilan al nahari6, mona ibrahim alnaimi6, aouss khalid alsarsh7 1department of family medicine, faculty of medicine, king abdulaziz university, jeddah, saudi arabia. 2obstetrics and gynecology physician, king fahad armed forces hospital, jeddah, saudi arabia. 3faculty of medicine, najran university, najran, saudi arabia. 4family medicine resident, jpfcm (joint program family and community medicine), king abdulaziz university, jeddah, saudi arabia. 5batterjee medical college, jeddah, saudi arabia. 6faculty of medicine, king abdulaziz university, jeddah, saudi arabia. 7armed forces hospital, south region, saudi arabia. *correspondence to: hoda jehad abousada (e-mail: dr.huda1992@outlook.com) (submitted: 13 march 2021 – revised version received: 04 april 2021 – accepted: 29 april 2021 – published online: 26 august 2021) introduction rhinosinusitis is an extremely common health complaint, and affects up to 14.7% of the population in the united states each year. although this condition is not associated with extreme morbidity or mortality, it is nevertheless responsible for a decrease in the quality of life and loss of productivity. the direct healthcare costs owing to this condition are estimated to be $2.4 million each year, excluding the cost of radiographic imaging and surgery.1 acute rhinosinusitis is usually of viral origin, and the disease is self-limiting. on the other hand, chronic rhinosinusitis is extremely difficult to manage, and its etiology remains obscure. while it was earlier suggested that pathogens play a role in the emergence of chronic rhinosinusitis, experts now concur that this condition occurs due to an imbalance between the host, micro-organisms and other exogenous factors such as allergens.2 recent studies have attributed greater importance to the role of food allergens in the development of chronic sinusitis. food allergens are already known risk factors for lower respiratory tract conditions such as asthma.3 few studies have also established that patients with chronic rhinosinusitis are more likely to have food allergies as compared to controls.4,5 moreover, food allergens and aeroallergens have been shown to have cross-reactivity.6 in this context, it would be helpful to document specific foods that are known to trigger episodes if rhinosinutitis in the adult population. although several anecdotal reports are available pertaining to foods that must be avoided in chronic sinusitis, quantitative scientific evidence on this subject is lacking. therefore, the aim of the current study was to identify specific categories of foods that could trigger exacerbations of rhinosinusitis. patients and methods the current study was a cross-sectional, questionnaire study. this study was conducted in accordance with the strobe guidelines for cross-sectional studies,7 and ethical approval was obtained from the institutional review board prior to beginning the study. the source of samples for the study included patients who attended the outpatient department of family medicine at king abdulaziz university in the kingdom of saudi arabia. patients aged between 18 to 80 years, who were diagnosed with chronic rhinosinusitis were included in the study. patients outside this age group, and who did not receive a definite diagnosis of chronic rhinosinusitis were excluded. patients who already had documented food allergies were also excluded. a written informed consent was obtained from all patients who met the inclusion criteria and consented to be a part of the study. these patients were asked to complete a questionnaire. apart from demographic details, the questionnaire collected information pertaining to five specific food groups – processed foods, high-calorie foods, fried foods, fatty meats, and foods high in sugar. participants were questioned on the development of symptoms of sinusitis in relation to these food groups. information from the questionnaire study was collected and extracted onto a digital spreadsheet. descriptive analysis of all the data was performed. the frequency of symptoms between the different food groups was assessed using the chi square test or mc nemar’s exact test. all data analysis was carried out using spss version 22. abstract objective this study aimed to identify associations between specific food categories and aggravation of symptoms of chronic rhinosinusitis. methods this was a prospective, cross-sectional questionnaire study. patients provided details about the categories of food that they consumed, as well as symptoms related to sinusitis. the data was extracted onto a spreadsheet and analysed using chi-square testing. results a small percentage of patients (4%) associated food categories with exacerbations of sinus symptoms. chocolate was the most common trigger, followed by white flour foods. several patients (33%) were not aware of the association between food and sinus symptoms. conclusion some food categories can exacerbate sinusitis in a specific population subset. patients should be encouraged to maintain a food diary so that they can identify relevant triggers and these can be avoided in the future. keywords rhinosinusitis, cross-sectional study, food allergy, histamine, chocolate, white flour issn 2413-0516 mailto:dr.huda1992@outlook.com 253j contemp med sci | vol. 7, no. 4, july–august 2021: 252–255 m.a. murad et al. original food groups that exacerbate sinusitis among saudi patients results a total of 630 patients were recruited for the study. of these, 481 patients were female (76.35%) and 149 patients were male (23.65%). the age of all the patients ranged from 18 to 77 years, with a mean age of 30.48 years. while all patients had been suffering from chronic rhinosinusitis, the duration of the current episode varied. at least 65.14% of all patients had been experiencing exacerbations for 2–8 weeks, and 43.6% of the patients had experienced exacerbations for longer than 12 weeks. only 13% of the patients reported that the current episode lasted for 4 to 8 weeks. six categories of foods were studied as potential sinus irritants. on an average, 4% of patients associated sinus exacerbations with different categories of foods. the highest food irritant appeared to be chocolate, which triggered sinus symptoms in 7.6% of all patients; followed by white flour foods, which triggered symptoms in 6.3% of all patients. the food that was least associated with sinus symptoms was mashed potatoes, which only exacerbated symptoms in 1.7% of all patients. the frequency of sinusitis exacerbations with different food groups is outlined in table 1. we performed cross-comparisons of each of these food categories with each other. this is outlined in table 2. in the cross-comparisons, it was noted that the frequency of sinus symptom exacerbation with mashed potatoes was significantly less as compared to the other food groups. the frequency of symptom exacerbation was significantly higher with chocolate as compared to pastries, fatty meats, white sugar and fried foods. a significantly high increase in symptom frequency was also observed when white flour foods were consumed, as compared to white sugars. none of the other comparisons showed significant differences. one important finding that emerged from this questionnaire study was the lack of awareness that many patients had in correlating dietary foods to their sinusitis symptoms. between 27% to 33% of all patients were not able to provide information on whether or not different foods triggered sinus symptoms. discussion this cross-sectional questionnaire study attempted to identify specific food groups that could trigger symptoms of sinusitis in patients with already established rhinosinusitis. overall, it was revealed that only a small percentage of symptom exacerbations are associated with consumption of specific food groups. nevertheless, these findings could play a key role in managing those patients in whom there are obvious food triggers. several alternative medicine streams believe that diet that a patient consumes is strongly related to exacerbation of chronic rhinosinusitis. some of the foods that alternative experts recommend avoiding include dairy products, sugars, caffeine and refined foods. it has been suggested that specific food categories, such as dairy products, wheat and corn, can lead to formation of ‘globular’ mucus rather than ‘planar’ mucus that may be difficult to drain.8 while allopathic medicine has not moved completely in this direction, there are a few studies that have been conducted in relation to diet and chronic rhinosinusitis. there are a few studies in literature that have evaluated the effect of diet on related conditions. calatayud et al. assessed the effect of the mediterranean diet on children who had recurrent colds and frequent inflammatory complications.9 the mediterranean diet encourages the consumption of fresh fruits, vegetables and whole grains, as well as fish and healthy fats. dairy consumption is suggested in moderation and meat consumption is limited. when this diet was followed by 128 children in the study, the authors noted decrease in the intensity and number of catarrhal episodes, as well as a decrease in antibiotic use and hospital visits. the influence of diet in childhood upper respiratory tract infections was confirmed by a multicentre randomized controlled trial, conducted by van der gaag et al.10 in their study, 118 children were divided into two groups. the study group received dietary advice, advising the consumption of green leafy vegetables, beef and whole dairy. they found that patients who received dietary advice had, on an average, 2.9 lesser episodes per month as compared to the control group. dietary modification has also been successful in patients with other kinds of allergy-based disorders. in a separate study, calatayud et al. studied the effect of the mediterranean diet in childhood asthma.11 the authors observed that in patients following this diet, there was a decrease in frequency of asthmatic attacks, as well as a decrease in the use of inhaled corticosteroids. despite several study in closely related areas, there are no dietary studies in literature that specifically relate to chronic rhinosinusitis. one explanation for triggering of symptoms in patients with chronic sinusitis could be the release of histamine. several food items have been associated with high amounts of histamine. these include fermented foods, citrus and certain other fruits, nuts, certain types of fish and chicken. however, each of these food substances encompasses a wide variety of diets and have not been studied fully. two separate studies assessed the effect of low-histamine diet on urticarial, which is essentially an allergic condition.12,13 both these authors reported that low-histamine diets were beneficial in decreasing symptoms of urticarial and improving the quality of life. in the current study, chocolate was identified as the most significant trigger for symptoms of rhinosinusitis. chocolate is technically a form of fermented food, as it is derived from cocoa beans, which are fermented forms of cacao seeds. it is high in histamine and also triggers histamine release in the body.14 in addition, chocolate also contains some amount of dairy content and sugar, which are possibly, by themselves, potential triggers for exacerbation of sinusitis. the next high table 1. frequency of rhinosinusitis symptoms on consumption of different food groups food category no. of patients with sinus symptom exacerbation percentage fried foods 27 4.9 white sugar 20 3.7 white flour 34 6.3 fatty meats 23 4.2 mashed potatoes 9 1.7 pastries 24 4.4 chocolate 41 7.6 254 j contemp med sci | vol. 7, no. 4, july–august 2021: 252–255 food groups that exacerbate sinusitis among saudi patients original m.a. murad et al. trigger, white flour, has been associated with several other inflammatory conditions such as celiac disease.15 all the other food categories evaluated in this study have also been anecdotally associated with inflammation; however, we found low correlation for mashed potatoes, which was the only natural food that was being studied. the next step to identifying known triggers is naturally to validate these triggers, followed by management solutions that involve avoidance. there is a definite need to design and implement randomized controlled trials, similar to the one designed by van der gaag et al.,10 evaluating each of the specific food categories against controls. once evidence based triggers are identified, proper dietary advice can go a long way in reducing frequency and severity of symptoms in patients suffering from chronic rhinosinusitis. conclusion in the current study, we were able to identify chocolate as one of the leading triggers of symptoms of rhinosinusitis. we also identified other forms of processed foods that could trigger inflammation and aggravate sinusitis. based on the results of this study, we recommend that patients suffering from this condition must maintain a food diary and also record their symptoms. this can help identify triggers in individual patients and can guide management of this condition. table 2. comparative analysis of food groups and their frequency in triggering symptoms of rhinosinusitis food categories sinusitis aggravated no aggravation chi-square value p-value chocolate vs. white flour 41 34 350 331 0.3309 0.565 chocolate vs. fried foods 41 27 350 341 2.304 0.128 chocolate vs. pastries 41 24 350 363 4.66 0.03 chocolate vs. fatty meats 41 23 350 358 5.024 0.02 chocolate vs. white sugar 41 20 350 345 6.38 0.01 chocolate vs. mashed potatoes 41 9 350 383 21.96 <0.0001 white flour vs. fried foods 34 27 331 341 0.94 0.33 white flour vs. pastries 34 24 331 363 2.56 0.11 white flour vs. fatty meats 34 23 331 358 2.83 0.09 white flour vs. white sugar 34 20 331 345 3.91 0.04 white flour vs. mashed potatoes 34 9 331 383 17.38 0.00003 fried foods vs pastries 27 24 341 363 0.39 0.54 fried foods vs fatty meats 27 23 341 358 0.51 0.47 fried foods vs white sugar 27 20 341 345 1.04 0.31 fried foods vs mashed potatoes 27 9 341 383 10.69 0.0011 pastries vs. fatty meats 24 23 363 358 0.0091 0.92 pastries vs. white sugar 24 20 363 345 0.18 0.67 pastries vs. mashed potatoes 24 9 363 383 7.32 0.0068 fatty meat vs. white sugar 23 20 358 345 0.11 0.74 fatty meats vs mashed potatoes 23 9 358 383 6.81 0.009 white sugar vs mashed potatoes 20 9 345 383 5.19 0.022 255j contemp med sci | vol. 7, no. 4, july–august 2021: 252–255 m.a. murad et al. original food groups that exacerbate sinusitis among saudi patients source of funding none. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. acknowledgements none.  references 1. battisti as, modi p, pangia j. sinusitis. [updated 2020 aug 10]. in: statpearls [internet]. treasure island (fl): statpearls publishing; 2020 jan-. 2. fokkens, wj, lund, vj, mullol, j, et al. european position paper on rhinosinusitis and nasal polyps 2012. rhinology. 2012;50(supp 23):1–329 3. kewalramani, a, bollinger, me. the impact of food allergy on asthma. j asthma allergy. 2010;3:65–74. 4. pang yt, eskici o, wilson ja: nasal polyposis: role of subclinical delayed food hypersensitivity. otolaryngol head neck surg 2000; 122:298–301. 5. collins mm, loughran s, davidson p, wilson ja: nasal polyposis: prevalence of positive food and inhalant skin tests. otolaryngol head neck surg 2006;135:680–683 6. popescu, f-d. cross-reactivity between aeroallergens and food allergens. world j methodol. 2015;5(2):31–50 7. cuschieri s. the strobe guidelines. saudi j anaesth. 2019;13(suppl 1):s31-s34. doi:10.4103/sja.sja_543_18 8. helms s, miller a. natural treatment of chronic rhinosinusitis. altern med rev. 2006;11(3):196-207. 9. calatayud fm, calatayud b, gallego jg, gonzález-martín c, alguacil lf. effects of mediterranean diet in patients with recurring colds and frequent complications. allergol immunopathol (madr). 2017;45(5):417-424. doi:10.1016/j.aller.2016.08.006 10. van der gaag e, brandsema r, nobbenhuis r, van der palen j, hummel t. influence of dietary advice including green vegetables, beef, and whole dairy products on recurrent upper respiratory tract infections in children: a randomized controlled trial. nutrients. 2020;12(1):272. published 2020 jan 20. doi:10.3390/nu12010272 11. calatayud-sáez fm, calatayud moscoso del prado b, gallego fernándezpacheco jg, gonzález-martín c, alguacil merino lf. mediterranean diet and childhood asthma. allergol immunopathol (madr). 2016;44(2):99-105. doi:10.1016/j.aller.2015.04.007 12. wagner n, dirk d, peveling-oberhag a, et al. a popular myth lowhistamine diet improves chronic spontaneous urticaria fact or fiction?. j eur acad dermatol venereol. 2017;31(4):650-655. doi:10.1111/jdv.13966 13. son jh, chung by, kim ho, park cw. a histamine-free diet is helpful for treatment of adult patients with chronic spontaneous urticaria. ann dermatol. 2018;30(2):164-172. doi:10.5021/ad.2018.30.2.164 14. san mauro martin i, brachero s, garicano vilar e. histamine intolerance and dietary management: a complete review. allergol immunopathol (madr). 2016;44(5):475-483. doi:10.1016/j.aller.2016.04.015 15. fardet a. wheat-based foods and non celiac gluten/wheat sensitivity: is drastic processing the main key issue?. med hypotheses. 2015;85(6): 934-939. doi:10.1016/j.mehy.2015.09.007 https://doi.org/10.22317/jcms.v7i4.1060 https://www.ncbi.nlm.nih.gov/books/nbk470383/ https://www.ncbi.nlm.nih.gov/books/nbk470383/ https://pubmed.ncbi.nlm.nih.gov/22469599/ https://pubmed.ncbi.nlm.nih.gov/22469599/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3047906/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3047906/ https://pubmed.ncbi.nlm.nih.gov/10652410/ https://pubmed.ncbi.nlm.nih.gov/10652410/ https://pubmed.ncbi.nlm.nih.gov/17071293/ https://pubmed.ncbi.nlm.nih.gov/17071293/ https://pubmed.ncbi.nlm.nih.gov/17071293/ https://pubmed.ncbi.nlm.nih.gov/26140270/ https://pubmed.ncbi.nlm.nih.gov/26140270/ https://pubmed.ncbi.nlm.nih.gov/30930717/ https://pubmed.ncbi.nlm.nih.gov/30930717/ https://pubmed.ncbi.nlm.nih.gov/17217321/ https://pubmed.ncbi.nlm.nih.gov/17217321/ https://europepmc.org/article/med/27939720 https://europepmc.org/article/med/27939720 https://europepmc.org/article/med/27939720 https://europepmc.org/article/med/27939720 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7019298/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7019298/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7019298/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7019298/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7019298/ https://pubmed.ncbi.nlm.nih.gov/26278484/ https://pubmed.ncbi.nlm.nih.gov/26278484/ https://pubmed.ncbi.nlm.nih.gov/26278484/ https://pubmed.ncbi.nlm.nih.gov/26278484/ https://pubmed.ncbi.nlm.nih.gov/27624921/ https://pubmed.ncbi.nlm.nih.gov/27624921/ https://pubmed.ncbi.nlm.nih.gov/27624921/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5839887/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5839887/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5839887/ https://pubmed.ncbi.nlm.nih.gov/27590961/ https://pubmed.ncbi.nlm.nih.gov/27590961/ https://pubmed.ncbi.nlm.nih.gov/27590961/ https://pubmed.ncbi.nlm.nih.gov/26364045/ https://pubmed.ncbi.nlm.nih.gov/26364045/ https://pubmed.ncbi.nlm.nih.gov/26364045/ 282 j contemp med sci | vol. 7, no. 5, september–october 2021: 282–285 original a study on the prevalence and the risk factors of hepatitis b virus infection in kurdistan region, iraq: a multicenter study nawfal r hussein1, dildar h musa2, dawan jamal hawezy3, ferhad mr ahmed1, fatma kamal khalid1, ibrahim a naqid1* , mahde saleh assafi4 1department of biomedical sciences, college of medicine, university of zakho, kurdistan region, iraq. 2department of surgery, college of medicine, university of duhok, kurdistan region, iraq. 3department of surgery, college of medicine, university of koya, kurdistan region, iraq. 4department of biology, college of science, university of duhok, kurdistan region, iraq. *correspondence to: ibrahim a. naqid (e-mail: ibrahim.naqid@uoz.edu.krd) (submitted: 07 august 2021 – revised version received: 26 august 2021 – accepted: 04 september 2021 – published online: 26 october 2021) abstract objectives: the aims of this study were to determine the prevalence of hbv infection and investigate the hbv-related risk factors in the kurdistan region of iraq. methods: sera samples were collected from 3423 patients visiting three centers and tested for hbv positivity by enzyme-linked immunosorbent assay. a questionnaire was prepared and used by the volunteers who collected data, including age, marital status, and different hbv-related risk factors, through face-to-face interviews. results: the mean age of the recruited participants was 28.94 ± 11.17 years. in addition, 873/3423 (25.47%) were males, and 3024/3423 (88.34%) were married. furthermore, hbv infection had a prevalence of 1.37% (47/3243). multivariate analysis was conducted to identify the predictive factors of hbv infection, which revealed that a history of tattoos and age were predictive factors (p < 0.05). we then stratified our data according to sex. no association was found between the various factors and hbv positivity in males. on the other hand, in females, a significant association was found between the history of tattoos or age and hbv positivity (p < 0.05). conclusion: in conclusion, the prevalence of hbv infection was low. our study showed that a history of tattooing and older age were predictive of hbv infection. our results could be used as a basis for local elimination programs. keywords: hepatitis b virus, prevalence, risk factors, elisa, iraq issn 2413-0516 introduction hbv infection is a global public health issue that is associated with deleterious consequences, including liver cirrhosis, hepatocellular carcinoma, and hepatic failure1. approximately 500 million people are infected with hbv, with an estimated 750000 deaths annually1. the prevalence of hbv infection varies, ranging from 0.5% in some developed countries to up to 8% in some east asian countries1. a previous study in turkey, a neighboring country of iraq, showed that the prevalence of hbsag positivity varied markedly from 1% to 14.3%, according to the geographical region of the study and the recruited samples2. while the prevalence of hbv infection was below 1% in some regions in iran, studies from saudi arabia showed that approximately 3% of the study populations were infected with the virus3,4. the world health organization declared an ambitious plan to eliminate viral hepatitis by 2030. however, it has been previously estimated that only 10% of hbv-infected patients are aware of their infection1. as such, it is essential to study the risk factors associated with viral transmission to come up with a public health plan for combatting hbv infection. risk factors for hbv include blood and blood product transfusions, pregnancy, being a healthcare worker, tattoos, drug abuse, and high-risk sexual behaviors1,5,6. to establish new strategies for the elimination of the virus in iraq, epidemiological data, including risk factors, should be identified. since the distribution of hbv risk factors is important for any public health prevention plan, this study investigated the prevalence of hbv and its associated risk factors in multiple centers in the kurdistan region, northern iraq. materials and methods blood samples blood samples were collected from volunteers attending zakho general hospital, azadi teaching hospital, and shahid dr khalid teaching hospital at koya between january 2019 and february 2021. a 5-cc syringe and needle were used to obtain 5 ml of blood from the volunteers. to separate the sera, blood samples were centrifuged at 1500 rpm for 3 min. samples were tested immediately for hbsag or kept frozen at –20°c until the tests were performed. questionnaire a questionnaire was prepared and used by each volunteer. data were collected through face-to-face interviews, including age, marital status, history of blood transfusion, history of dental procedures, history of surgical operation, history of tattoos, and history of regular injections. regular use of injections was defined as the regular use of over-the-counter injections or any other injections. enzyme-linked immunosorbent assay (elisa) hbsag, hbc-igm, and hbc-igg were analyzed using a commercial elisa kit (dia.pro diagnostic bioprobes; milan italy), following the manufacturer’s instructions. monoclonal antibodies were fixed to the surface of the micro-wells, and sera samples were added and incubated. afterward, a secondary monoclonal antibody conjugated with horseradish peroxidase (hrp) was added. unbound serum proteins and hrp conjugates were washed off. after blocking the 283j contemp med sci | vol. 7, no. 5, september–october 2021: 282–285 n.r. hussein et al. original prevalence and the risk factors of hbv enzymatic reaction, the substrate was added, and the optical density was measured using an elisa reader. ethics the study was approved by the appropriate ethics committee of the college of medicine, university of zakho. written informed consent was obtained from all patients. statistics regression analysis was used to assess the risk factors associated with hbsag. variables that achieved a p-value of less than 0.2 were included in the multivariate study. backward elimination was used to determine the hbv-related risk factors. all computations were performed using the minitab 17 (minitab; pennsylvania, united states). results volunteers sera samples were collected from 3423 patients and tested for hbv positivity. the mean age of the recruited participants was 28.94 ± 11.17 years. in addition, 873/3423 (25.47%) were males, and 3024/3423 (88.34%) were married (table 1). hbv prevalence and risk factors the prevalence of hbv infection in the recruited sample was 1.37% (47/3243). all samples were hbc-igg-positive, indicating chronic infection. univariate analysis showed that 14/47 (29.8%) of the hbv-positive patients had a history of blood transfusion, which was significantly higher compared to the hbv-negative patients (602/3376 [17.8%], p = 0.048). in addition, hbsag positivity was significant associated with a history of tattooing (p = 0.039) and age (p = 0.003) (table 2). multivariate analysis was then conducted to identify the predictive factors of hbv infection, which revealed that a history of tattoos and age were predictive factors (table 2). we then stratified our data according to sex. no association was found between the various factors and hbv positivity in males (table 4). on the other hand, in females, hbv positivity was significantly associated with a history of tattoos and age (table 5). multivariate analysis confirmed that both history of tattoos and age were predictive factors for hbv positivity (table 6). discussion hbv is a common public health issue worldwide. chronic hbv infection is associated with serious complications, such as liver cirrhosis, hepatocellular carcinoma, and hepatic failure. studies conducted in the middle east found that the prevalence of hbsag positivity ranged from less than 3% in iraq and some regions in iran to approximately 7% in war-torn yemen and some districts in the arab peninsula3,4,7. specifically, the results of studies conducted in iraq varied depending on the recruited samples and the geographic regions. in a previous study in duhok city, northern iraq, the prevalence of hbv was 1.14% in blood donors8, while it was 0.7% in the middle region of iraq9. in contrast, in a report from kerbala, southern iraq, hbv had a prevalence of 3.5%7. the vast majority of previous studies in iraq exclusively recruited male blood donors from one center or city. in this study, the prevalence of hbv was 1.37%, which reflected more realistic results in the general population because both sexes were recruited from three different cities. table 1. characteristics of the study population variables no. percentage sex male 872 25.47 female 2551 74.53 marital status married 3024 88.34 single 399 11.66 blood transfusion yes 616 17.99 no 2807 82.01 surgical operation yes 813 23.75 no 2610 76.25 dental procedures yes 2228 65.09 no 1195 34.91 tattoo yes 269 7.86 no 3154 92.14 injections yes 2017 58.92 no 1406 41.08 age (year ± std) 28.94 ± 11.17 std: standard deviation table 2. univariate analysis of the hepatitis b virus-associated risk factors in the recruited population factors hbsag-positive (n = 47) hbsag-negative (n = 3376) confidence interval odds ratio p-value sex (male) 11 861 0.4522 – 1.7604 0.89 0.7 marital status (married) 45 2979 0.7253 – 12.4205 3 0.07 blood transfusion 14 602 1.0398 – 3.6754 1.95 0.048 surgical operation 8 805 0.3053 – 1.4094 0.66 0.25 dental procedure 33 2195 0.6760 – 2.3792 1.27 0.45 tattoo 8 261 1.1323 – 5.2934 2.44 0.039 injection 30 1987 0.6777 – 2.2454 1.23 0.5 age (year ± std) 34.42 ± 11.04 28.87 ± 11.15 1.0136 – 1.0579 1.03 0.003 284 j contemp med sci | vol. 7, no. 5, september–october 2021: 282–285 prevalence and the risk factors of hbv original n.r. hussein et al. hbv can be transmitted via blood and blood product transfusion, exposure to contaminated blood through needles, tattoos, vertically from mothers to newborns (particularly during delivery), and unprotected sex5. it was previously found that the mode of hbv transmission varied in different countries according to age, sex, norms, and societal traditions10. it is worth mentioning that a blood screening program for blood-borne diseases started in 2007 in the kurdistan region. in our study, there was a trend of higher hbv positivity in people with a history of blood transfusion. careful examination of our data showed that all hbv-positive patients were born before 2007, possibly implying that screening programs in these cities have successfully prevented the transmission of the virus. in agreement with a study conducted in china11, we found a significant association between age and hbv positivity. similarly, a study from iran showed that hbv positivity was more common in older patients3. again, this could be explained by the fact that the screening program started in 2007, and older patients had a higher chance of exposure. in addition, hbv vaccination was included in the expanded vaccination program in 2000. therefore, people born before the hbv vaccination program had a higher chance of infection. tattoos are also a risk factor for hbv infection. in a meta-analysis investigating the relationship between hbv and tattooing, tattooing was found to be a risk factor for acquiring the infection12,13. similarly, we found a significant association between hbv positivity and tattooing. this is a challenge for controlling hbv in the kurdistan region as tattooing is performed in illegitimate shops or as traditions by untrained personnel. in a study conducted in turkey, sexual contact with hbv-positive patients was the main risk factor associated with hbv infection14. illegitimate sexual contact could not be included in the study because the ethics committee prohibited the inclusion of questions probing this in the questionnaire since it is a sensitive issue in our conservative society, particularly among females. we believe that even if questions related to this topic were included, the patients would not have provided honest answers. previously, the vast majority of studies from iraq recruited blood donors7,9,15,16. careful examination showed that all donors were male. therefore, we believe that the majority of studies in this country had not investigated the hbv-related risk factors in females. therefore, we stratified our data according to sex. we found no association between hbv related risk factors and hbv positivity in males. on the other hand, both univariate and multivariate analyses showed that tattoos and age were associated with hbv positivity in females. these results were difficult to explain, necessitating further studies to explore this. table 3. multivariate analysis of the hepatitis b virus associated risk factors in the recruited population factors coef se coef t value p-value age 0.00056 0.0002 2.96 0.003 blood transfusion 0.009 0.005 1.69 0.091 tattoo 0.017 0.008 2.13 0.033 coef: coefficient, se coef: standard error of the coefficient. table 4. univariate analysis of the hepatitis b virus-associated risk factors in males factors hbsag-positive (n = 11) hbsag-negative (n = 861) confidence interval odds ratio p-value marital status 11 598 0 – 0 23 0.9 blood transfusion 3 98 0.7619 – 11.1889 2.9 0.153 surgical operation 2 361 0.0661 – 1.4330 0.3 0.094 dental procedure 8 539 0.4196 – 6.0479 1.59 0.5 tattoo 1 95 0.1021 – 6.3685 0.8 0.8 age (year ± std) 45.16 ± 14.4 31.5 ± 19 0.9950 – 1.0752 1.03 0.09 injection 5 334 0.3981 – 4.3425 1.31 0.65 table 5. univariate analysis of the hepatitis b virus-associated risk factors in females factors hbsag-positive (n = 36) hbsag-negative (n = 2515) confidence interval odds ratio p-value marital status 34 2378 0.2277 – 4.0297 0.95 0.9 blood transfusion 11 504 0.8581 – 3.5918 1.76 0.13 surgical operation 6 444 0.3868 – 2.2598 0.94 0.9 dental procedure 25 1656 0.5773 – 2.4073 1.18 0.6 tattoo 7 166 1.4742 – 7.9139 3.4 0.011 age (year ± std) 32.63 ± 9.45 28.41 ± 8.88 1.0135 – 1.0754 1.04 0.008 injection 25 1653 0.5804 – 2.4201 1.19 0.6 285j contemp med sci | vol. 7, no. 5, september–october 2021: 282–285 n.r. hussein et al. original prevalence and the risk factors of hbv the strength of our study was the relatively large and diverse sample, as the majority of previous studies recruited specific populations, such as blood donors or patients on renal dialysis8,17. as for its limitation, we only recruited patients who were visiting the said hospitals, rather than random patients in the community. therefore, a population based study is needed to investigate the prevalence and risk factors of hbv infection. this project is of exceptional importance for public health providers in the region because, to the best of our knowledge, this was the first study to recruit samples from more than one city with a relatively large sample size. conclusion in conclusion, the prevalence of hbv infection was 1.37%. our project showed that a history of tattooing and older age were predictive of hbv infection. tattooing centers in the region should be legalized, trained, and monitored to prevent the further spread of the infection. screening for hbv infection should focus on people born before 2000. conflict of interest none.  table 6. multivariate analysis of the hepatitis b virus associated risk factors in females factors coef se coef t value p-value age 0.00067 0.0002 2.54 0.011 blood transfusion 0.01 0.005 1.65 0.1 tattoo 0.01 0.009 2.79 0.005 coef: coefficient, se coef: standard error of the coefficient. references 1. el-serag hb. epidemiology of viral hepatitis and hepatocellular carcinoma. gastroenterology. 2012;142(6):1264–73.e1. 2. igde f, taskin h, igde m, yazici z, atilla a. where we are in the fight against hepatitis b infection; trends in hepatitis b virus seroprevalence in black sea region of turkey. nigerian journal of clinical practice. 2018;21(1):87–92. 3. alavian sm, tabatabaei sv, ghadimi t, beedrapour f, kafi-abad sa, gharehbaghian a, et al. seroprevalence of hepatitis b virus infection and its risk factors in the west of iran: a population-based study. international journal of preventive medicine. 2012;3(11):770–5. 4. mohammed abdullah s. prevalence of hepatitis b and c in donated blood from the jazan region of saudi arabia. the malaysian journal of medical sciences : mjms. 2013;20(2):41–6. 5. shepard cw, simard ep, finelli l, fiore ae, bell bp. hepatitis b virus infection: epidemiology and vaccination. epidemiologic reviews. 2006;28(1):112–25. 6. mohammed saleem zs, naqid ia, hussein nr, mohammad sj, noaman js, haji rs, et al. the prevalence of hepatitis b and c virus in patients with end-stage kidney disease on regular hemodialysis in duhok, iraq: a brief report. avicenna j clin microbiol infect. 2020;7(1):31–3. 7. assafi ms, ibrahim nm, hussein nr, taha aa, balatay aa. urinary bacterial profile and antibiotic susceptibility pattern among patients with urinary tract infection in duhok city, kurdistan region, iraq. international journal of pure and applied sciences and technology. 2015;30(2):54. 8. hussein nr. risk factors of hepatitis b virus infection among blood donors in duhok city, kurdistan region, iraq. caspian journal of internal medicine. 2018;9(1):22. 9. al – juboury a, m. salih h, alassadi m, ali am. seroprevalence of hepatitis b and c among blood donors in babylon governorate-iraq. medical journal of babylon 2010;7(1–2). 10. hussein nr, daniel s. a study of hepatitis b virus associated risk factors in patients attending hepatitis unit in duhok city, iraq. archives of clinical infectious diseases. 2017;12(3). 11. li x, zheng y, liau a, cai b, ye d, huang f, et al. hepatitis b virus infections and risk factors among the general population in anhui province, china: an epidemiological study. bmc public health. 2012;12(1):1–7. 12. jafari s, buxton ja, afshar k, copes r, baharlou s. tattooing and risk of hepatitis b: a systematic review and meta-analysis. can j public health. 2012;103(3):207–12. 13. galles nc, liu py, updike rl, fullman n, nguyen j, rolfe s, et al. measuring routine childhood vaccination coverage in 204 countries and territories, 1980-2019: a systematic analysis for the global burden of disease study 2020, release 1. the lancet. 2021;398(10299):503–21. 14. ozer a, yakupogullari y, beytur a, beytur l, koroglu m, salman f, et al. risk factors of hepatitis b virus infection in turkey: a population-based, casecontrol study. hepat mon. 2011;11(4):263–8. 15. hussein nr, haj sm, almizori la, taha aa. the prevalence of hepatitis b and c viruses among blood donors attending blood bank in duhok, kurdistan region, iraq. int j infect. 2017;4(1):e39008. 16. jamal sa, naqid ia, hussein nr, abdulqader sr, nimet aa, abdulkhdair ha, et al. the prevalence of hepatitis b and c virus infections in the couples attending a premarital screening program in zakho city, kurdistan region of iraq. zahedan j res med sci. 2020;23(2):e99405. 17. hussein nr, saleem zs, ibrahim nm, assafi ms, daniel s. the prevalence of hbv infection in renal transplant recipients and the impact of infection on graft survival. acta medica iranica. 2019;57(6):381–4. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i5.1037 176 j contemp med sci | vol. 7, no. 3, may-june 2021: 176–178 original physical health problems among iraqi women with ectopic pregnancy sarah flaieh essa*, hawraa hussein ghafel maternal and neonate nursing department, college of nursing, university of baghdad, baghdad, iraq. *correspondence to: sarah flaieh essa (e_mail: sarra.faleeh1203a@conursing.uobaghdad.edu.iq) (submitted: 09 april 2021 – revised version received: 21 april 2021 – accepted: 04 may 2021 – published online: 26 june 2021) introduction ectopic pregnancy (ep) falls under the gynecological emergency and/or reproductive management of women in which fertilized ovum implantation outside the endometrial cavity occurs.1 when the forming blastocyst inserts either outside the uterus fallopian tube, an ectopic pregnancy occurs: ampullary (79.6%); isthmic (12.3%); fimbrial (6.2%), ovary (0.15%) and abdominal cavity (1.4%)] or in an irregular location inside the uterus cornual (1.9%), cervical (0.15%). in the fallopian tubes, the largest proportion (98.3 percent) of ectopic pregnancies occur.2 ectopic pregnancies may occur anywhere in the reproductive tract, with the fallopian tube being the most common location.3 the risk of an ectopic pregnancy is increased by many factors. such risk factors share a similar mechanism of action, such as interference with the role of the fallopian tube. in the fallopian tube, an egg is usually fertilized and then moves down the tube to the implantation site. any mechanism that interferes during this phase with the normal function of the fallopian tube raises the risk of ectopic pregnancy. the mechanism can be anatomical e.g. scarring that blocks the egg’s transport or functional e.g. impaired mobility of tubal.4 clearly, the cause of ectopic pregnancy is not known, but some risk factors that increase the incidence of ectopic pregnancy include: advanced maternal age (over 35 years of age). chronic inflammatory disorder in the pelvis. the prior ectopic pregnancy history. previous surgical history to the fallopian tube or nearby structure. intrauterine contraceptive (iud) products and hormonal drug overdoses to induce ovulation.5 while typical signs and symptoms of pregnancy may occur and the results of a pregnancy test may be positive, an ectopic pregnancy cannot proceed as normal. to help diagnose a possible ectopic pregnancy, the following signs can be used: painful or stabbing pain that can come and go and differ in severity. (because of blood pooling under the diaphragm from a ruptured ectopic pregnancy, the pain can be in the pelvis, abdomen, or even the shoulder and neck). heavier or lighter vaginal bleeding than usual, gastrointestinal symptoms, fatigue or dizziness, and fainting.6 materials and methods this study was conducted at maternity hospitals in karbala city to assess the physical health of women with ectopic pregnancy. this study was started in from the 1st of january 2020 to 1st of march 2021, the data regarding rate women’s with ectopic pregnancy was achieved from the data recorded in the statistical department in the hospitals. a descriptive study was non-probability (a purposive sample) the study consist of (40) women with ectopic pregnancy which were selected according to inclusion criteria that are (women who had ectopic pregnancy, women in reproductive age only). this questionnaire was composed of three parts, part one: consists of sections that are related socio-demographic characteristics include (age, educational level, women occupation, residency, and smoking). part two include: reproductive status history, previous medical history, the previous history of gynecological diseases, previous surgical history). part three include: physical domain, reliability of questionnaire is determined through a pilot study and validity through panel (13) experts. descriptive statistical analysis and inferential statistical analysis procedures were employed for the data analysis. results table 1 shows that women are with age 29 ± 7 years in which the highest percentage is refer to 23–27 years (32.5%). it have been found (32.5%) of women are with age group 23–27 years, they are read and write as reported with high percentage of abstract objective to assess physical health problems among women with ectopic pregnancy and to evaluate the levels of physical health among women with ectopic pregnancy, and to identify demographic characteristics of ectopic pregnant women. methods this study was conducted at maternity hospital in karbala city in iraq to assess the physical health problems of women with ectopic pregnancy. this study was started in january 2020 to march 2021, the data regarding women’s health problems was achieved from the patient’s charts that recorded in the statistical department in the hospitals, the study consist of (40) women with ectopic pregnancy which were selected according to inclusion criteria (women who had ectopic pregnancy, women in reproductive age only). the data are analyzed through the use of descriptive and inferential statistics analysis procedures were employed for the data analysis. result the findings of the study exhibits that women with ectopic pregnancy were suffering from physical health problems such as (acute abdominal pain, tired and lethargic, faint, hypotension, vaginal bleeding, internal bleeding). the mean of scores for items related to physical health problems among women that show bad among most of items (1, 3, 4, 6, 7, 8, and 9) except item 2, 5, and 6 that show fair. conclusion the study concludes that women with ectopic pregnancy were suffering from physical problem such as (acute abdominal pain, tired and lethargic, faint, hypotension, vaginal bleeding, and internal bleeding). keywords physical health problems, pregnancy, ectopic, pregnant women, iraq issn 2413-0516 177j contemp med sci | vol. 7, no. 3, may-june 2021: 176–178 sarah flaieh essa and hawraa hussein ghafel original physical health problems among iraqi women with ectopic pregnancy table 2. physical health problems among women with ectopic pregnancy (n = 40) list items responses f (%) m.s assessment 1 severe and recurring stomach pain never 37 (92.5) 1.08 bad sometimes 3 (7.5) always 0 (0) 2 pain in the shoulder area never 16 (40) 1.95 fair sometimes 10 (25) always 14 (35) 3 tired and lethargic never 33 (82.5) 1.23 bad sometimes 5 (12.5) always 2 (5) 4 dizzy never 23 (57.5) 1.63 bad sometimes 9 (22.5) always 8 (20) 5 faint never 14 (35) 2.25 fair sometimes 2 (5) always 24 (60) 6 nauseous and vomiting never 13 (32.5) 1.95 fair sometimes 16 (40) always 11 (27.5) 7 severe drop in blood pressure never 25 (62.5) 1.53 bad sometimes 9 (22.5) always 6 (15) 8 vaginal bleeding never 25 (62.5) 1.65 bad sometimes 4 (10) always 11 (27.5) 9 internal bleeding never 29 (72.5) 1.55 bad sometimes 11 (27.5) always 0 (0) f, frequency; %, percentage; m.s, mean of score; bad, 1 – 1.66; fair, 1.67 – 2.33; good, 2.34 – 3. table 1. distribution of women according to their socio-demographic characteristics list characteristics f % 1 age (m ± sd = 29 ± 7) 13 – 17 year 2 5 18 – 22 year 3 7.5 23 – 27 year 13 32.5 28 – 32 year 7 17.5 33 – 37 year 11 27.5 38 ≤ year 4 10 total 40 100 2 residency urban 29 72.5 rural 10 25 suburban 1 2.5 total 40 100 3 level of education doesn’t read & write 3 7.5 read & write 14 35 primary school 13 32.5 intermediate school 6 15 secondary school 1 2.5 institute/ college + 3 7.5 total 40 100 4 occupation employee 9 22.5 housewife 30 75 students 1 2.5 total 40 100 5 smoking no 36 90 yes 4 10 total 40 100 f, frequency; %, percentage; m, mean; sd, standard deviation. fig. 1 levels of physical health among women with ectopic pregnancy (n = 40). this figure reveals that 55% of women are showing bad physical health level. 35% and 32.5% of them are graduated from primary school; (75%) of women are housewives and only 22.5% of them are governmental employee, (72.5%) are resident in urban. the women show bad levels of physical health (55%), there is significant relationship among physical health among women with ectopic pregnancy with their age. no significant relationship among physical health among women with ectopic pregnancy with respect to their level of education, occupational status, residence area. table 2 displays the mean of scores for items related to physical health problems among women that show bad among most of items (1, 3, 4, 6, 7, 8, and 9) except item 2, 5, and 6 that show fair. discussion table 1 shows that the socio-demographic characteristics of women with ectopic pregnancy high percentage (32.5%) of women are with age group 23–27 years old. such finding was 178 j contemp med sci | vol. 7, no. 3, may-june 2021: 176–178 physical health problems among iraqi women with ectopic pregnancy original sarah flaieh essa and hawraa hussein ghafel supported by the study of li and others who found in their study that the highest percentage of women with ectopic pregnancy among (15–24) years old (50.6%)7. table 1 revealed that they are resident in urban are (72.5%), (25%) are resident in rural and only (2.5%) are resident in sub-urban. this finding are not agree with study for negewo and others (2019), who found that most of the sample was resident in rural 58.2%.8 with respect to their education, the study findings indicate that most of these women with ectopic pregnancy are read & write (35%) (table 1). such result does agree with a report has indicated that more than half (57.14% ) of women with ectopic pregnancy reported majority of patients had lower educational level.9 with regard to the women’s employment, the study has revealed that (75%) of the study sample are housewife. these finding are supported by the study of 10 who found that most of sample was housewives 55.9%. table 1 showed that high percentage (90%) of women are none smoker such finding was supported by the li and others who found in their study that the highest percentage of women with ectopic pregnancy were not smoking (95.31%)11. figure 1 exhibits that women are associated with bad physical health level (55%) and 37.5% are with fair physical level. this study agree with study12 who found that the most common presenting symptoms were abdominal pain (94%), abnormal vaginal bleeding (74%) and amenorrhoea (64%). cervical excitation (86%) and adnexal tenderness (84%) were frequent pelvic findings and an adnexal mass was noted in 27% of patients. table 2 presented the sub-domain of biological health among women with ectopic pregnancy the finding showed that women having bad level of severe and recurring stomach pain (92.5%), tired and lethargic (82.5%), dizzy (57.5%), severe drop in blood pressure (62.5%), vaginal bleeding (62.5%), and internal bleeding (72.5%) , and having fair level of pain in the shoulder area (40%), faint (35%), nauseous and vomited (32.5%). the study provided supportive evidence for current findings that found (orazulike & konje, 2013) that report a presence of physical problem among their sample.13 nausea and breast soreness are common symptoms in both ectopic pregnancy and more common symptoms in ectopic pregnancy and can indicate a medical emergency such as sharp waves of pain in the abdomen, pelvis, shoulder, or neck, severe pain that occurs on one side of the abdomen, light to heavy vaginal spotting or bleeding, dizziness or fainting and finally rectal pressure wakankar and kedar (2015) in their study concluded that the pain was in 86.53% women, amenorrhea and bleeding per vagina are mostly occurring in ectopic pregnancy.14 conclusion the study concludes that women with ectopic pregnancy were suffering from physical health problems such as (acute abdominal pain, tired and lethargic, faint, hypotension, vaginal bleeding, and internal bleeding). conflicts of interest none.  references 1. abdulkareem, t., a. & eidan s., m. (2017). ectopic pregnancy: diagnosis, prevention and management. research gate. 2. soren, m., patnaik r., & sarangi b. k. (2017). a clinical study on ectopic pregnancy. international journal of research in medical sciences. 5(11):4776-4782. 3. husmo c., & drahonovsky. (2008). ectopic pregnancy – etiology, modern diagnostics and therapeutic approach. dissertation in preventivemedicine. 4. tenore, j. ( 2000). ectopic pregnancy. journal of the american academy of family physicians. 61(4):1080-1088. 5. saudi german hospitals group. (2019). 6 risk factors for ectopic pregnancy. retrieved from https://www.sghgroup.com.sa/en/medical-blog/6-riskfactors-ectopic-pregnancy. 6. american pregnancy association. (2020). ectopic pregnancy. retrieved from https://americanpregnancy.org/healthy-pregnancy/pregnancycomplications/ectopic-pregnancy-839. 7. li, c., meng, c. x., zhao, w. h., lu, h. q., shi, w., & zhang, j. (2014). risk factors for ectopic pregnancy in women with planned pregnancy: a case–control study. european journal of obstetrics & gynecology and reproductive biology, 181:176-182. 8. negewo, a.,n., feyissa, g., t., diriba, g., gemeda, d., h., and kebede, a., (2019). prevalence and management outcome of ectopic pregnancy in adama hospital medical college, east shoa zone, oromia region, ethiopia. ec gynaecology 8.9 (2019): 844-850. 9. mahmood mk. ectopic pregnancy; causes and management in kerbala maternity hospital. karbala journal of medicine. 2019;12(2). 10. thonneau p, hijazi y, goyaux n, calvez t, keita n. ectopic pregnancy in conakry, guinea. bulletin of the world health organization. 2002;80:365-70. 11. li c, zhao wh, zhu q, cao sj, ping h, xi x, qin gj, yan mx, zhang d, qiu j, zhang j. risk factors for ectopic pregnancy: a multi-center case-control study. bmc pregnancy and childbirth. 2015;15(1):1-9. 12. rasheed s, abdelmonem a, amin m. adolescent pregnancy in upper egypt. international journal of gynecology & obstetrics. 2011;112(1):21-4. 13. orazulike nc, konje jc. diagnosis and management of ectopic pregnancy. women’s health. 2013;9(4):373-85. 14. udigwe go, umeononihu os, mbachu ii. ectopic pregnancy: a 5 year review of cases at nnamdi azikiwe university teaching hospital (nauth) nnewi. nigerian medical journal. 2010;51(4):160. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 286 j contemp med sci | vol. 7, no. 5, september–october 2021: 286–289 original the relationship between air pollutants and spirometric indices in schoolchildren of five areas in tehran siavash kooranifar1, gholamreza alizadeh attar1* , atefeh talebi1, vahan moradians1, maryam pourashraf2, razieh rostami3, nima bakhtiari4 1department of internal medicine, school of medicine, iran university of medical sciences, tehran, iran. 2department of radiology, school of medicine, tehran university of medical sciences, tehran, iran. 3department of internal medicine, school of medicine, tehran university of medical sciences, tehran, iran. 4pain research center, ahvaz jundishapur university of medical sciences, ahvaz, iran. *correspondence to: gholamreza alizadeh attar (e-mail: sanysasan2002@yahoo.com) (submitted: 22 july 2021 – revised version received: 06 august 2021 – accepted: 29 august 2021 – published online: 26 october 2021) abstract objectives: the adverse health effects of air pollution have been observed in many epidemiological studies. the aim of this research was to study the effects of air pollution on pulmonary functions in schoolchildren in tehran city. methods: a total number of 167 schoolchildren were selected to participate in this study. data were analyzed using anova and generalized estimating equation (gee) to determine the relationship of air pollution and lung function tests. results: the result of this study showed that there are statistically significant differences in value of air pollution between areas. the results present that concentration of o 3 , pm 10 , no 2 has a negative association with lung function tests but concentration of co, pm 2.5 , and so 2 had no association with decreased lung function tests. time variable of air pollution was not statistically significant effect on lung function test. conclusion: in this study, we conclude that air pollution in tehran city can be decreased lung function test indexes that may be affected by short–time exposure to air pollutant. keyword: lung function, schoolchildren, air pollution, tehran issn 2413–0516 introduction air pollutants produced by industrial activities and vehicles such as o3, so2, no2, co, pm2.5, pm10 have adverse effects on the respiratory system1,2. the adverse effects are more pronounced in children because they are more sensitive when exposed to contaminated air. children have a lower diameter of airways, and they generally breathe more air per kg of body weight than adults, so exposure to more air pollutants. increased concentrations of air pollutants cause more inflammation in the respiratory system3,4. children spend more time and more activity outdoor; hence more susceptible to exposure to air pollutants5. numerous studies postulated that air pollution had adverse effects on the respiratory system that are more serious in large and industrial cities6. spirometry has been used in several studies to show the effects of air pollution on the respiratory system. indicators measured by spirometry include fvc, fev1/fvc, fev1, mmef2575, fev1/fev6. most studies examining the short–term effects of air pollution on spirometric indices performed a single spirometry test and have not evaluated changes in spirometric indices during a period, time which are essential in determining the exact effect of air pollutants on the respiratory system7. therefore, we decided to study the effect of air pollutants on the respiratory system of children by performing two spirometry tests for each child and to evaluate the changes in spirometric indices. our study was performed in tehran, the capital city of iran, which is one of the most polluted cities in this country. materials and methods study design this was a prospective cohort study conducted between september 2018 and may 2019. inclusion criteria students without a history of respiratory disease and recent illness and who were taking no medications were included in this study. participants in this study, 167 male and female fifth-grade elementary school students, aged between 10 to 12 years old, participated. the students were randomly selected from 10 schools in five municipal districts of the city, which were less than 500 meters from air quality monitoring stations. the height and weight of all of the students were measured. a questionnaire, including questions on drug history, asthma, allergy, eczema, and parent smoking, was given to each student’s parents. the study protocol was approved by the ethics committee the iran university of medical sciences. data on the levels of air pollutants the levels of air pollutants such as o3, so2, no2, co, pm2.5, pm10 were obtained from the tehran air pollution assessment website8. in both first and second episodes of spirometry, the levels of air pollutants were recorded on the day of performing spirometry and five consecutive days before that. the changes in the levels of air pollutants between the first and second episodes of performing spirometry were calculated. less than 5% of data on the levels of air pollutants was missed due to technical errors in air monitoring sensors. lung function test spirometry was performed with a portable spirometer (spirolab mobile version, china). the spirometer was calibrated using a 3-liter syringe each time before spirometry. spirometry was performed by expert technicians. in the 287j contemp med sci | vol. 7, no. 5, september–october 2021: 286–289 s. kooranifar et al. original the relationship between air pollutants and spirometric indices in schoolchildren of five areas in tehran beginning, the spirometry method was fully explained to the students. three to five spirometry maneuvers were acquired from each student, and the best maneuver was selected based on american thoracic society/european respiratory society (ers) criteria. statistical analysis the mean daily concentration of air pollutants on the day of the first and second time of spirometry in 5 districts (lag 0) and 1–5 days before the first and second time of that (lag 1–5) were measured and compared between five studied sites. percentage of changes of air pollutant concentration and spirometric indices were calculated between first and second times. in this study, multiple regression using gee (generalized estimating equation) statistical models were used to determine the correlation between changes of lung function indices (fvc, fev1, fev1/fvc, fev1/fev6 and fef25–75) and changes in air pollutants (0 days to 5 days before spirometry). statistical analyses (gee, anova) were performed using spss software (version 22), and statistical significance for p-values < 0.05 were considered. consent to participate written informed consent was obtained from participants before the study. ethics approval ethical approval for the study was obtained by the ethics committee of iran university of medical sciences (approval code ir.iums.fmd.rec.1397.073). all participants were provided written informed consent before the study and had the right to withdraw from the study at any stage. results in total, 167 male and female fifth-grade elementary school students, aged between 10 to 12 years old, were included in this study. the students were randomly selected from 10 schools in five districts of the city. the students’ characteristics are shown in table 1. the average and standard deviation (sd) of height, weight, age, and bmi were not different between students of five municipal districts. the results of the mean daily concentrations of air pollutants are displayed in table 2. the spirometric indices and percentage of changes are presented in table 3. the results using gee (generalized estimating equation) are shown in table 4. table 4 shows that a one ppb increase in o3 was associated with a change of –0.058 l in fev1 (95%ci: –.099 – –.017 l) and –0.04 in fvc (95%ci: –.072– –.008l) and –0.085 lit in fef25–75 (95%ci: –.158– –.012l), after adjustment for gender and height. our study demonstrates that 1 a ppb increase in no2 was associated with a change of –0.106 lit in fev1 (95% ci: –.183– –.029l) and –0.087 lit in fvc (95% ci: –.149– –.026l), after adjustment for gender and height. this study also displayed that 1 µg/m³ increase of pm10 was associated with a change of 0.037 lit in fev1 (95% ci: .010 – .065l) and 0.039 lit in fvc (95% ci: .011 – .068l), after adjustment for gender and height. other air pollutants had no significant effect on spirometric indices. discussion the present study included 167 male and female fifth-grade elementary school students, aged between 10 to 12 years. this age range was chosen because of more inadequate cooperation in younger children. this study was conducted on healthy elementary school students without any previous illness. air pollutants produced by industrial activities and vehicles such as o3, so2, no2, co, pm2.5, pm10 have adverse effects on the respiratory system2. our study assessed the effects of the daily average concentration of air pollutants on spirometric indices. previous studies have demonstrated that elevated air pollutants were associated with more decrease in lung function in children than adults5. several previous studies have examined the associations between spirometric indices and air pollutants6,9–13 but those studies did not consider changes in spirometric indices in individuals. in those studies, the concentration of air pollutants in the days before spirometry was measured. chang et al. investigated the effects of air pollution on lung function tests of adolescents aged 12 to 16 years and reported that fvc, had a significant adverse association with short-term exposure to o3 and pm10 measured on the day of spirometry testing13.fvc values also were reversely associated with means of co, o3, no2, pm10 and so2 exposed 1 day earlier. an increase of 1-ppm co was associated with the reduction in fvc for 69.8 ml (95% ci: –115, –24.4 ml) or in fev1 for 73.7 ml (95% ci: –118, –29.7 ml). their study also showed that an increase in so2 for 1 ppb was associated with the reductions in fvc and fev1 for 12.9 ml (95% ci:–20.7, –5.09 ml) and 11.7 ml (95% ci:–19.3, –4.16 ml), respectively. chang et al. similar to other previous studies did not evaluate changes in spirometric indices of each individual during a period, time. another difference between the change study and ours was that they found the time lag between the air pollutant measurement and spirometry day, had a significant effect on spirometric indices. nevertheless in our study, there was no significant relationship between the time lag and air pollutant levels. another study performed by hashemzadeh et al. in ahvaz, a major city in the southwest of iran, demonstrated a table 1. demographic profile of the study participants p-valuetotal region15region7region6region5region2variables 1672537313737no 37.10%0%37.80%35.40%48.60%51.30%male 62.90%100%62.10%64.50%51.30%48.60%female p = 0.49152.0 ± 8.4153.2 ± 7.0151.3 ± 8.8150.2 ± 10.4153.4 ± 8.5152.6 ± 7.3height (cm) p = 0.9248.7 ± 11.849.6 ± 9.547.4 ± 13.648.0 ± 11.349.5 ± 10.549.2 ± 13.4weight (kg) p = 0.9020.9 ± 4.221.0 ± 2.620.4 ± 4.021.5 ± 5.320.9 ± 3.521.0 ± 5.0bmi 288 j contemp med sci | vol. 7, no. 5, september–october 2021: 286–289 the relationship between air pollutants and spirometric indices in schoolchildren of five areas in tehran original s. kooranifar et al. table 2. the results of the mean daily concentrations of air pollutants mean (sd)mean (sd)mean (sd)mean (sd)mean (sd)mean (sd) so 2 (ppb) 4.3 ± 2.14.1 ± 2.33.7 ± 2.43.5 ± 1.83.1 ± 1.43.5 ± 1.4 first time 3.9 ± 1.03.9 ± 1.13.5 ± 1.54.7 ± 1.84.3 ± 1.04.3 ± 1.2 second time 37.6 ± 51.312.9 ± 59.516.5 ± 51.450.3 ± 55.460.4 ± 62.137.6 ± 51.3 percentage of changes no 2 (ppb) 47.2 ± 14.643.9 ± 10.445.5 ± 15.243.7 ± 9.242.9 ± 5.545.0 ± 5.0 first time 49.9 ± 9.044.8 ± 7.641.7 ± 10.847.7 ± 10.248.8 ± 6.749.0 ± 11.0 second time 5.0 ± 43.42.3 ± 26.5–1.5 ± 28.313.2 ± 25.115.4 ± 18.910.2 ± 20.3 percentage of changes co (ppm) 1.7 ± 0.51.3 ± 0.41.5 ± 0.61.3 ± 0.51.3 ± 0.31.4 ± 0.3 first time 1.5 ± 0.91.4 ± 0.71.3 ± 0.41.4 ± 0.41.7 ± 0.51.7 ± 0.9 second time –0.2 ± 60.21.3 ± 27.6–6.0 ± 32.017.3 ± 37.533.8 ± 47.223.7 ± 69.4 percentage of changes o 3 (ppb) 17.0 ± 6.318.3 ± 7.420.9 ± 6.717.1 ± 8.522.6 ± 6.616.5 ± 5.5 first time 16.7 ± 7.017.9 ± 8.919.0 ± 10.718.3 ± 8.915.0 ± 7.516.3 ± 8.5 second time 11.3 ± 47.7–3.9 ± 39.7–9.1 ± 50.7–10.8 ± 43.5–32 ± 31.5–9.3 ± 43.4 percentage of changes pm 2.5 (µg/m³) 25.7 ± 13.824.9 ± 11.625.7 ± 14.523.1 ± 12.5125.5 ± 5.118.1 ± 6.9 first time 24.5 ± 16.725.0 ± 15.325.6 ± 16.125.8 ± 16.427.2 ± 12.529.8 ± 12.6 second time 28.4 ± 95.710.0 ± 44.66.4 ± 38.922.5 ± 67.287.1 ± 84.472.2 ± 54.2 percentage of changes pm 10 (µg/m³) 53.9 ± 27.056.8 ± 23.254.6 ± 25.443.2 ± 20.835.2 ± 13.839.3 ± 16.2 first time 51.8 ± 30.154.3 ± 28.051.2 ± 24.154.4 ± 28.754.1 ± 21.762.6 ± 29.0 second time 25.1 ± 98.9–3.8 ± 46.5–3.7 ± 38.642.3 ± 96.070.2 ± 76.278.7 ± 91.9 percentage of changes table 3. the spirometric indices and percentage of changes mean (sd)mean (sd)mean (sd) –0.5 (–61.3, 95.2)2.0 (0.88, 3.02)2.1 (0.85, 3.12)fev1 (l) –2.0 (–40.82, 57.95)2.2 (1.1 – 3.2)2.3 (1.21, 3.42)fvc (l) 1.6 (–54.3, 89.8)90.5 (40.6, 100.0)90.5 (40.6, 100.0)fev1/fvc (%) 1.4 (–54.3, 89.8)91.2 (41.3, 100.0)90.6 (40.6, 100.0)fev1/fev6 (%) 5.1 (–71.5, 160.0)2.6 (0.6, 5.3)2.7 (0.5, 4.99)fef25–75 (l) table4. the results obtained by generalized estimating equation (gee) p-valuebp-valuebp-valuebp-valuebp-valueb 0.170.7840.1990.2430.1890.2530.520.1780.1770.425time 0.023–0.0850.178–0.0170.145–0.0180.013–0.040.006–0.058o 3 0.5160.0190.3330.0090.3370.0090.78–0.0050.8560.003co 0.167–0.1340.614–0.0140.531–0.0180.005–0.0870.007–0.106no 2 0.460.0250.1350.0170.1390.0170.4620.0110.1270.028so 2 0.3440.0250.611–0.0040.679–0.0030.0070.0390.0080.037pm 10 0.3710.0260.1420.0120.1170.0130.222–0.0210.616–0.009pm 2.5 289j contemp med sci | vol. 7, no. 5, september–october 2021: 286–289 s. kooranifar et al. original the relationship between air pollutants and spirometric indices in schoolchildren of five areas in tehran significant correlation between the increase of mean concentration of no2 in 1 to 4 days before sampling day and decreased fvc and fev114. in this regard, by increasing the mean no2 concentration to 6.5 ppb, the values of fvc and fev1 decreased by 12 and 19 ml; and by increasing the mean concentration of pm2.5 to 13 µg/m³ on the same day of sampling (lag 0), the values of fvc and fev1 decreased by 131 and 110 ml. however, similar to most previous studies, they did not evaluate spirometric changes within a period, time. in our study, ozone harmed fev1, fvc and fef25–75, with the most significant effect on fef25–75.it should be noted that although these effects are statistically significant, they are clinically insignificant and they did not cause symptoms in the students. in normal subjects,the cut-off for clinically significant week-to-week changes of fev1 and fvc were more than 12% and 11%, respectively. in our study, 61.6% of the subjects had fev1 change less than 12% and 63.4% had fvc change less than 11%. in fact, in most subjects, although the percentage of spirometric changes was statistically significant, the indices remained within the normal range. conclusion our research suggests that air pollution in tehran city can lead to decreased lung function test indices which may be affected by short-time exposure to air pollutants.traffic-related air pollutants show acute and subacute adveres effects on the respiratory system in school children. accordingly, our research suggests air pollution changes are associated with changes in lung function in a healthy subject. these findings can help improve understanding adverse effects of air pollution on the respiratory system, and may also implicatemore targeted and effective pollution regulations to reduce traffic emission pollutants. acknowledgment this work would not have been possible without the financial support of the pasargad teb irsacompany award. declarations funding/support: this research was funded by iran university of medical sciences by grant number 805 as part of thesis of gholamreza alizadeh attar. competing interests: the authors have no conflict of interest. data availability: the datasets used in the present study are available from the corresponding author on reasonable request. authors’ contributions: all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by gholamreza alizadeh attar. the first draft of the manuscript was written by gholamreza alizadeh attar and all authors revisedthe previous versions of the manuscript. all authors read and approved the final manuscript.  references 1. zhang, y., et al., short-term effects of fine particulate matter and temperature on lung function among healthy college students in wuhan, china. international journal of environmental research and public health, 2015. 12(7): p. 7777–7793. 2. hassanvand, m.s., et al., short-term effects of particle size fractions on circulating biomarkers of inflammation in a panel of elderly subjects and healthy young adults. environmental pollution, 2017. 223: p. 695–704. 3. gasana, j., et al., motor vehicle air pollution and asthma in children: a meta– analysis. environmental research, 2012. 117: p. 36–45. 4. kampa, m. and e. castanas, human health effects of air pollution. environmental pollution, 2008. 151(2): p. 362–367. 5. lee, y.l., et al., effects of ambient air pollution on pulmonary function among schoolchildren. international journal of hygiene and environmental health, 2011. 214(5): p. 369–375. 6. zeng, x.-w., et al., long–term ambient air pollution and lung function impairment in chinese children from a high air pollution range area: the seven northeastern cities (snec) study. atmospheric environment, 2016. 138: p. 144–151. 7. linares, b., et al., impact of air pollution on pulmonary function and respiratory symptoms in children. longitudinal repeated–measures study. bmc pulmonary medicine, 2010. 10(1): p. 1–9. 8. company, t.a.q.c. http://airnow.tehran.ir/home/dataarchive.aspx. 2020; available from: https://air.tehran.ir/. 9. arora, s., et al., air pollution and environmental risk factors for altered lung function among adult women of an urban slum area of delhi: a prevalence study. lung india: official organ of indian chest society, 2018. 35(3): p. 193. 10. wu, s., et al., association of lung function in a panel of young healthy adults with various chemical components of ambient fine particulate air pollution in beijing, china. atmospheric environment, 2013. 77: p. 873–884. 11. he, q.-q., et al., effects of ambient air pollution on lung function growth in chinese schoolchildren. respiratory medicine, 2010. 104(10): p. 1512–1520. 12. kasamatsu, j., et al., effects of winter air pollution on pulmonary function of school children in shenyang, china. international journal of hygiene and environmental health, 2006. 209(5): p. 435–444. 13. chang, y.-k., et al., the short–term effects of air pollution on adolescent lung function in taiwan. chemosphere, 2012. 87(1): p. 26–30. 14. hashemzadeh, b., et al., effects of pm2. 5 and no2 on the 8-isoprostane and lung function indices of fvc and fev1 in students of ahvaz city, iran. saudi journal of biological sciences, 2019. 26(3): p. 473–480. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i5.1051 347j contemp med sci | vol. 8, no. 5, september-october 2022: 347–351 original association between hormonal imbalance and interleulin-10 level with polycystic ovarian syndrome of iraqi women hiba aqeel muslem al-quraishy1*, hanaa addai ali2, fadhil jawad al-tu’ma3, mousa mohsin ali4, amir fadhil al-tu’ma5 1laboratory department, gynecological and obstetric teaching hospital, kerbala health directorate, ministry of health, kerbala, iraq. 2department of chemistry, college of science, university of kufa, kufa, iraq. 3department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 4department of gynecology and obstetrics, college of medicine, university of kerbala, kerbala, iraq. 5department of medical laboratory technologies, college of medical and health technologies, university of ahlalbayt, kerbala, iraq. *correspondence to: hiba aqeel muslem al-quraishy (e-mail: hibaakil026@gmail.com) (submitted: 13 july 2022 – revised version received: 05 august 2022 – accepted: 29 august 2022 – published online: 26 october 2022) abstract objectives: to determine the level of interleukin-10 as inflammatory marker in pcos patients and compared with healthy control and explore the correlation between il-10 level and other biochemical markers in pcos patients. methods: whole blood samples of 80 pcos obese patients and 80 healthy people were collected in duration from dec., 2021 to april, 2022, at the gynecological and obstetric teaching hospital, kerbala health directorate, iraq. the rotterdam criteria-2003 was accepted to pcos females with age range between (18–40 years), while the apparently control group with age ranged between (18–40 years). bmi and whr and hormonal status (lh, fsh, lh/fsh ratio, prolactin, free testosterone) were determined. elabscience/usa elisa kit uses the sandwichelisa principle was used to determined il-10 level in serum. results: the obese pcos women had a seriously decrease in il-10 (pg/ml) level when compare with apparently healthy control group with mean ± sd for patients (2.192 ± 0.47) and for control group (4.532 ± 0.75) p value < 0.0001, and there are significant negative correlations between il-10 and (bmi p < 0.0001, r = –0.66), (whr p < 0.0001, r = –0.66), (lh p < 0.0001, r = –0.63), (lh: fsh ratio p < 0.0001, r = –0.72), (prolactin p = 0.008, r = –029). conclusion: our findings indicate that the il-10 level decrease in pcos obese patients and has significant association with pathogenesis and progression of disease. keywords: interleukin-10, polycystic ovary syndrome, iraq issn 2413-0516 introduction low-grade inflammation and inflammatory indicators have been linked to pcos. understanding the pathophysiology of pcos and its therapy by inhibition or regulation of associated pathways can be improved by looking into the inflammatory mediators involved for its development. because ovulation is a semi-inflammatory process, visceral adipose tissue can induce an inflammatory response and sustain the inflammation in adipocytes by influencing the release of inflammatory cytokines. out of this regulated inflammation, pcos can arise.1 interleukin-10 (il-10), which was first discovered in 1991, is an immunosuppressant and an anti-inflammatory cytokine that is essential for the body’s defense processes.2,3 the il-10 protein is a homodimer, and each of its components is 178 amino acids long.4 il-10 is a t-helper cell family member (th2) that inhibits the function of th1 cells.5 activated immune cells release interleukin-10, an anti-inflammatory cytokine. monocytes are capable of producing il-10 when they are stimulated.6 as well as non-immune cells like epithelial or neuronal cells.7 the trans-membrane receptor complex made up of il-10r1 and il-10r2 is how il-10 exerts its effects and regulates the actions of lymphocytes, macrophages, and a variety of other cells.8 it regulates the differentiation and proliferation of immune cells, such as macrophages, t cells, and b cells; reduces monocyte activation; and restricts the release of pro-inflammatory cytokines, such as tnf, il-1, il-6, il-12, and il-2.9 to keep the ovary functioning properly, the levels of inflammatory markers must be in equilibrium. alterations in steroidogenesis, delayed follicular maturation, and ovarian issues might result from an imbalance between pro-inflammatory and anti-inflammatory cytokines caused by increased production of tnf-α and il-6 and decreased production of il-10. it is thought that since it lowers th1 cell activity, progesterone is produced and the corpus luteum matures, maintaining pregnancy. obesity and the metabolic syndrome are linked to decreased il-10 levels. the fact that pcos patients had lower plasma levels of il-10 suggests that clomiphene citrate increases il-10 and helps pcos women become pregnant and ovulate more frequently.10 therefore, this study aimed to determine the level of interleukin-10 as inflammatory marker in pcos patients and compared with healthy control and explores the correlation between il-10 level and other biochemical markers in pcos patients of iraqi women in kerbala province. materials and methods this study is a case-control study involves 80 pcos patients. and 80 non-pcos women as a control in childbearing age at the reproductive fertility consultant of gynecological and obstetric teaching hospital, kerbala health directorate iraq and iraq’s university of kerbala college of medicine during the duration from dec., 2021 to april, 2022. an exhaustive interview gathering personal and family history, blood pressure, demographic information and laboratory examination was carried out. the rotterdam criteria-2003 was presumed to 80 pcos females with ages ranged between (18–40) years. mailto:hibaakil026@gmail.com 348 j contemp med sci | vol. 8, no. 5, september-october 2022: 347–351 association between hormonal imbalance and interleulin-10 level original h.a.m. al-quraishy et al. table 1. demographic parameters of the registered patients and the control demographic parameters control n = 80 mean ± sd patients n = 80 mean ± sd menstruation pattern (regular) 80 17 menstruation pattern (irregular) – 63 with hirsutism – 67 without hirsutism 80 13 primary infertility – 52 secondary infertility – 28 table 2. biochemical parameters of the registered patients and the control biochemical parameters control n = 80 mean ± sd patients n = 80 mean ± sd p-value age, year 26.8 ± 5.175 26.1 ± 5.3 0.15 bmi (kg/m2) 23.3 ± 1.156 32.5 ± 6.357 <0.0001 whr 0.777 ± 0.0143 0.912 ± 0.0563 <0.0001 lh (m.iu/ml) 106 ± 0.555 11.89 ± 3.188 <0.0001 fsh (m.iu/ml) 6.73 ± 0.65 5.36 ± 1.36 0.01 lh/fsh ratio 0.986 ± 0.041 2.414 ± 0.379 <0.0001 free testosterone (pg/ml) 2.97 ± 1.812 18.7 ± 14.98 <0.0001 prolactin (ng/ml) 12.19 ± 2.92 16.14 ± 4.05 <0.0001 il-10 (pg/ml) 4.53 ± 0.75 2.19 ± 0.47 <0.0001 patients with any 2 of the next 3 items can be recognized in diagnosis: oligomenorrhea or amenorrhea, increase androgen levels, ovarian volume >10 ml on u/s, and follicles ≥12 with diameter 2–9 mm.11 controller group has 80 ladies which ages reached between (18–40 years). they have regular menstruation, with normal ovaries as they were detected by the gynecologist. body mass index were calculated from the following equation: bmi = weight (kg)/height (m2). normal bmi level is (20–24.9) kg/m2 and (25–29.9) kg/m2 for overweight. when bmi ≥30 kg/m2, the woman is considered as obese.12 the whr diagnostic standard for obesity is 0.85 for women.13 the volume withdrawn from each patient was 3.0 ml was used for serum separation and used for hormonal assays. the hormonal levels of each of lh, fsh and prolactin were measured by the chemiluminescent automated immunoassay system (cobas e411, roche diagnostic, germany). free testosterone level was measured by competitive enzyme immunoassay using monobind/usa elisa kit and elabscience/usa elisa kit uses the sandwich-elisa principle was used to determined il-10 level in serum. the protocol for study was certified by the ethical research commission of college of medicine, university of kerbala and kerbala health directorate. approval also taken from administration of gynecological and obstetric teaching hospital and from each patient after explaining the nature and purpose of study. all statistical analyses were performed with the graph pad prism 9.0.0 was released on october 28, 2020. data were analyzed by t-test in statistical analysis the highly significant value is (p < 0.01) and the significant value is (p < 0.05). the data are presented as mean ± sd (standard deviation). the correlation coefficient spearman r test was calculated to examine association among parameters. results based on inclusion and exclusion criteria, 160 women were involved in the last data analysis, the women involved within the study finally were with an age ranged between (18–40) years and the mean ± sd of them were 26.1 ± 5.3 years. the results of this study were displayed in table 1. they incorporate the mean ± sd of the patients with and without hirsutism and those with primary or secondary infertility and (regular or irregular) menstruation pattern. it is clear that the two groups are almost well matched, thus obtained results could be estimable. the results of this study were displayed in table 2 using statistical unpaired t-test; age, bmi and whr as well as using statistical mann whitney test. significant elevations in lh concentrations (p < 0.0001), lh/fsh ratio (p < 0.0001), free testosterone levels (p < 0.0001) and prolactin level (p < 0.0001) were prevailed in the pcos patients group when contrasted with the control group. however, while significantly decrease in fsh level (p = 0.01) and il-10 level (p < 0.0001) during a comparable evaluation between pcos patients and control group. interleukin – 10 (il-10) correlation with anthropometric and biochemical parameters in pcos patients group was evaluated by spearman r test are showed in figure 1 and table 3. the result showed that there are significant negative correlations between il-10 and (bmi p < 0.0001, r = –0.66), (whr p < 0.0001, r = –0.66), (lh p < 0.0001, r = –0.63), (lh: fsh ratio p < 0.0001, r = –0.72), (prolactin p = 0.008, r = –029), and there are non-significant correlations with age and fsh revealed with il-10 cytokine. discussion the pathogenesis and progression of polycystic ovarian syndrome have been implicated in chronic low-grade inflammation as a major factor (pcos).1 il-10 levels were lower in pcos patients than in controls. th1 cells and macrophages’ expression of pro-inflammatory cytokines is down-regulated by the anti-inflammatory cytokine and suppressor il-10. alters steroidogenesis, delays follicular development, and causes ovarian dysfunction due to an imbalance between pro and anti-inflammatory cytokines.14 the main characteristics of the metabolic abnormalities typical of pcos include insulin resistance and hyperinsulinemia. although there is mounting evidence that tnfα, il-6, and il-10 are critical players in mediating insulin resistance, low il-10 levels have been linked to obesity and the metabolic syndrome.15 subeka abraham gnanadass’ research demonstrated a connection between metabolic syndrome and obesity and low il-10 levels. patients with pcos experienced a decrease in plasma il-10.10 interleukin (il)-10, which m. karadeniz demonstrated in his work, is a significant anti-inflammatory cytokine that has been linked to obesity and type 2 diabetes and that controls the production of these pro-inflammatory cytokines. patients with high bmi and insulin levels have been found to have low levels of il-10.16 in the past ten years, a lot of study has concentrated on the immunosuppressive and anti-inflammatory effects that are mediated by a variety of variables, including the anti inflammatory cytokine interleukin (il)-10. the pathogenesis 349j contemp med sci | vol. 8, no. 5, september-october 2022: 347–351 h.a.m. al-quraishy et al. original association between hormonal imbalance and interleulin-10 level fig. 1 correlation between il-10 cytokine level and a. bmi, b. whr, c. lh, d. lh: fsh ratio, e. free testosterone hormone and f. prolactin hormone level in pcos patients group. 350 j contemp med sci | vol. 8, no. 5, september-october 2022: 347–351 association between hormonal imbalance and interleulin-10 level original h.a.m. al-quraishy et al. of pcos is firmly implicated as being driven by chronic lowgrade inflammation. in several diseases, including pcos, il-10 has an anti-inflammatory and immune-suppressive effect. recent studies showed that women with pcos had considerably reduced serum levels of il-10.17–19 in his research, angel mercy sylus also demonstrated that inflammation is frequently linked to pcos, which affects these women’s ovarian folliculogenesis, abnormal steroidogenesis in the ovary, and hyperinsulinemia. an anti-inflammatory cytokine called interleukin-10 (il-10) controls how pro-inflammatory cytokines behave when there is inflammation. reduced il-10 levels have been observed in women with pcos and il-10 gene variation has been associated with pcos.20 this study showed negative correlation between il-10 cytokine level and bmi, whr, lh, lh/fsh ratio, prolactin hormone and free testosterone. while there is no correlation with age or fsh level. in his work, po-kai yang demonstrated that obesity has been suggested to have a modulatory effect on pcos patients’ ovulatory functioning. through increased il-10 synthesis in visceral fats, obesity may interfere with normal folliculogenesis.21 in pcos compared to controls, il-10 concentration was decreased. with respect to whr, which measures visceral adiposity, there were significant relationships.14 in pcos patients, a substantial positive link between the ratio of t regulatory cells and lh levels was found, however other hormones like fsh will not impact the ratio of t regulatory cells. t regulatory cells release anti-inflammatory cytokines like interleukin 10 (il-10). in his research, yiqing yang discovered that pcos patients had lower levels of the cytokine il-10 associated to t regulatory cells.21 according to umit cabus’ study, serum il-10 levels were greater in study participants than in controls, and the lh/fsh ratio was noticeably higher in pcos-affected women.22 when compared to controls, pcos patients’ mean plasma concentration of il10 was significantly lower, and it had no effect on the levels of fsh, lh, or prl.23 hyperinsulinemia stimulates the pituitary’s reaction to gonadotropin-releasing hormone (gnrh), which increases the release of luteinizing hormone and androgen. this influences how well the hypothalamus-pituitary-ovarian gonadal axis works (hpo axis). hyperandrogenism may prevent the growth of follicles, cause follicular atresia, and encourage insulin resistance as a result of feedback. the pathogenesis of pcos is associated with elevated pro-inflammatory cytokines and decreased anti-inflammatory (il-10) factors, and this inflammatory condition may harm insulin sensitivity and advance the onset of pcos.24 conclusion the observed results indicated that there is decrease in the mean of interleukin 10 cytokine in pcos patient’s. various hormones which are free testosterone, prolactin and lh in obese pcos women are increase as compared with control, while there is significant decrease in fsh values was obtained. the result also showed that there are significant negative correlations between il-10 and bmi, whr, lh, lh: fsh ratio, prolactin, and there are non-significant correlations with age and fsh revealed with il-10 cytokine. acknowledgments the authors thank the pcos patients for their cooperation and the medical staffs in the molecular research laboratory of the department of chemistry and biochemistry, college of medicine, university of kerbala and the laboratories of “gynecological and obstetric teaching hospital” kerbala health directorate. conflict of interest the authors advertise that they have no conflict of interest.  references 1. rostamtabar, m., esmaeilzadeh, s., tourani, m., rahmani, a., baee, m., shirafkan, f., saleki, k., mirzababayi, s. s., ebrahimpour, s. & nouri, h. r. 2021. pathophysiological roles of chronic low‐grade inflammation mediators in polycystic ovary syndrome. journal of cellular physiology, 236, 824–838. 2. moore, k. w., de waal malefyt, r., coffman, r. l. & o’garra, a. 2001. interleukin-10 and the interleukin-10 receptor. annual review of immunology, 19, 683–765. 3. gnanadass, s. a., prabhu, y. d. & gopalakrishnan, a. v. 2021. association of metabolic and inflammatory markers with polycystic ovarian syndrome (pcos): an update. archives of gynecology and obstetrics, 1–13. 4. nissar, s., sameer, a. s. & banday, m. z. 2021. genetic polymorphisms of essential immune pathogenic response genes and risk of cervical cancer. genetic polymorphism and cancer susceptibility, springer. 5. rasquinha, m. t., sur, m., lasrado, n. & reddy, j. 2021. il-10 as a th2 cytokine: differences between mice and humans. the journal of immunology, 207, 2205–2215. 6. said, e. a., dupuy, f. p., trautmann, l., zhang, y., shi, y., el-far, m., hill, b. j., noto, a., ancuta, p. & peretz, y. 2010. programmed death-1–induced interleukin-10 production by monocytes impairs cd4+ t cell activation during hiv infection. nature medicine, 16, 452–459. 7. sözen, t., özişik, l. & başaran, n. ç. 2017. an overview and management of osteoporosis. european journal of rheumatology, 4, 46. 8. ralston, s. h. & de crombrugghe, b. 2006. genetic regulation of bone mass and susceptibility to osteoporosis. genes & development, 20, 2492–2506. 9. bakiri, a. h. & mingomataj, e. ç. 2019. novel insights on interleukin-10 functions: a manipulative tool for the deviation of immune response and disease outcome. emj allergy immunol, 4, 88–94. 10. abraham gnanadass, s., divakar prabhu, y. & valsala gopalakrishnan, a. 2021. association of metabolic and inflammatory markers with polycystic ovarian syndrome (pcos): an update. archives of gynecology and obstetrics, 303, 631–643. 11. park, k. s., gang, w., kim, p.-w., yang, c., jun, p., jung, s.-y., kwon, o., lee, j. m., lee, h. j. & lee, s. j. 2022. efficacy and safety of acupuncture on table 3. correlation of il-10 with anthropometric and biochemical parameters in registered pcos patients group parameters r p-value age (y) 0.13 ns bmi (kg/m²) –0.68 <0.0001 whr –0.66 <0.0001 lh (m.iu/ml) –0.63 <0.0001 fsh (m.iu/ml) 0.13 ns lh : fsh –0.72 <0.0001 prolactin (ng/ml) –0.29 0.008 free testosterone (pg/ml) –0.53 <0.0001 351j contemp med sci | vol. 8, no. 5, september-october 2022: 347–351 h.a.m. al-quraishy et al. original association between hormonal imbalance and interleulin-10 level oligomenorrhea due to polycystic ovary syndrome: an international multicenter, pilot randomized controlled trial. medicine, 101, e28674–e28674. 12. ahmed, i., ali, i. & hussain, s. 2022. human body weight measures in association with better screening predictor among waist-to-hip ratio, body mass index and body fat percentage. bahria university journal of humanities & social sciences, 5. 13. milewska, m., przekop, z., szostak-węgierek, d., chrzanowska, m., raciborski, f., traczyk, i., sińska, b. i. & samoliński, b. 2022. prevalence of risk of sarcopenia in polish elderly population—a population study. nutrients, 14, 3466. 14. artimani, t., karimi, j., mehdizadeh, m., yavangi, m., khanlarzadeh, e., ghorbani, m., asadi, s. & kheiripour, n. 2018. evaluation of pro-oxidantantioxidant balance (pab) and its association with inflammatory cytokines in polycystic ovary syndrome (pcos). gynecological endocrinology, 34, 148–152. 15. vural, p., değirmencioğlu, s., saral, n. y. & akgül, c. 2010. tumor necrosis factor α (− 308), interleukin-6 (− 174) and interleukin-10 (− 1082) gene polymorphisms in polycystic ovary syndrome. european journal of obstetrics & gynecology and reproductive biology, 150, 61–65. 16. karadeniz, m., erdogan, m., zengi, a., tamsel, s., berdeli, a., saygili, f. & yilmaz, c. 2008. polymorphism of the interleukin‐10 gene in polycystic ovary syndrome. international journal of immunogenetics, 35, 119–123. 17. chugh, r. m., park, h.-s., el andaloussi, a., elsharoud, a., esfandyari, s., ulin, m., bakir, l., aboalsoud, a., ali, m. & ashour, d. 2021. mesenchymal stem cell this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. therapy ameliorates metabolic dysfunction and restores fertility in a pcos mouse model through interleukin-10. stem cell research & therapy, 12, 1–19. 18. shamsi, m., ghazavi, a., saeedifar, a. m., mosayebi, g. & ganji, a. 2022. the immune system’s role in pcos. molecular biology reports, 1–14. 19. umar, m., sastry, k. s. & chouchane, a. i. 2018. role of vitamin d beyond the skeletal function: a review of the molecular and clinical studies. international journal of molecular sciences, 19, 1618. 20. sylus, a. m., nandeesha, h. & chitra, t. 2020. matrix metalloproteinase-9 increases and interleukin-10 reduces with increase in body mass index in polycystic ovary syndrome: a cross-sectional study. international journal of reproductive biomedicine, 18, 605. 21. yang, y., xia, j., yang, z., wu, g. & yang, j. 2021. the abnormal level of hsp70 is related to treg/th17 imbalance in pcos patients. journal of ovarian research, 14, 1–9. 22. cabus, u., kabukcu, c., fenkci, s., caner, v., oztekin, o., fenkci, v. & enli, y. 2020. serum caspase-1 levels in women with polycystic ovary syndrome. taiwanese journal of obstetrics and gynecology, 59, 207–210. 23. talaat, r. m., mohamed, y. a., mohamad, e. h., elsharkawy, m. & guirgis, a. a. 2016. interleukin 10 (− 1082 g/a) and (− 819 c/t) gene polymorphisms in egyptian women with polycystic ovary syndrome (pcos). meta gene, 9, 254–258. 24. wang, w., zheng, j., cui, n., jiang, l., zhou, h., zhang, d. & hao, g. 2019. baicalin ameliorates polycystic ovary syndrome through amp-activated protein kinase. journal of ovarian research, 12, 1–12. https://doi.org/10.22317/jcms.v8i5.1285 167j contemp med sci | vol. 3, no. 9, winter 2017: 167–169 research elastic band ligation of hemorrhoids using flexible gastroscope hadi abd zaid al-khattabi,a ali azeez ali,a hameed hussein al-jameel,a ali mansoor al-amerib aal-hussein medical city, kerbala, iraq. bdepartment of microbiology and immunology, department of microbiology and immunology, college of medicine, university of kerbala, iraq. correspondence to hadi abd zaid al-khattabi (email: alkattabih12@yahoo.com). (submitted: 23 september 2016 – revised version received: 14 october 2016 – accepted: 20 october 2016 – published online: 26 march 2017) objective this study aims at investigating the outcome of rubber band ligation of piles in an outpatient setting. methods after taking an informed consent, 46 patients, 89% of them were males, mean age 52.5 years, with uncomplicated grade ii-iii piles were involved in the study. rubber band ligation of piles was made by pentax iscan gastroscope. the patients were followed up regularly to record any complication. results most of the patients (91.3%) need one session of treatment, 3 patients need 2 sessions, no more than 2 bands were used. additionally, a relatively low percentage of patients recorded certain complication of the operation, on follow up. conclusion high success rate, cost effectiveness and the simplicity of rubber band ligation as an outpatient procedure promote its use as the first line of treatment for first, second and early third degree hemorrhoids. keywords band ligation, internal piles, flexible gastroscope, outpatient setting introduction internal hemorrhoids are initially managed conservatively. the conservative therapy includes life-style changes such as increased fiber and liquid intake and regular cleansing. this might be combined with local anesthetic and antiphlogistic medication.1,2 more than 90% of patients with symptomatic piles can be treated conservatively or by rubber band ligation. infrared coagulation and cryotherapy are also the options, but are no longer commonly applied.3 since the early 1960s, the treatment of choice for persisting internal hemorrhoids is elastic band ligation by means of a rigid proctoscope (barron ligation).4,5 the ligation of hemorrhoids is widely used as an alternative method for the treatment of internal symptomatic hemorrhoids and has replaced hemorrhoidectomy in 45% of cases.6 this method gives good results in 69–94% of cases. although it is not associated with the problems that follow the typical surgical treatment of hemorrhoids.7–10 the rbl method is not free of complications and even deaths have been reported in immunosuppressed patients.11,12 though considered as a safe, simple and effective procedure, studies show complications like recurrence, pain, bleeding and even pelvic sepsis in some cases. there is a wide range of recurrence rate from 8 to 30%, greatest for grade iii hemorrhoids. pain is common for a few hours following rubber band ligation (rbl) and occasionally patients experience severe pain so as to require admission to hospital.13-16 on the other hand, a flexible endoscope with a ligation cap that is employed for the ligation of esophageal varices was tested to treat internal piles by some researchers.17-20 the advantages of a flexible endoscope might be summarized by maneuverability beside photographic documentation and a wider view field. materials and methods forty-six patients with uncomplicated hemorrhoids (abscess, thrombosis) grade ii-iii were involved in the study (table 1). patients were sedated by slow i.v. using diluted medazolam 3 mg and pethedine 50 mg. pentax iscan gastroscope was used with multiband ligator device (cook, usa) and the same was used in esophageal varices ligation. the hemorrhoids were suctioned into the ligation cap in either retrograde or ante grade fashion. one or two hemorrhoids were ligated in each session. all treatment sessions were performed in an outpatient setting. patients were encouraged to consult the hospital at any time and provided with facilities to contact us at any time if developed any abnormal events. result forty-six patients, mean age 52.5 years (30–75) and most of them were males (89%), were included in this study. most of them referred from the surgical department. a total of 45 patients were presented with a history of attacks of bleeding. one patient presented with active bleeding, and she was unfit for surgical intervention. most of patients need one session of treatment, 3 patients need two sessions, no more than 2 bands were used (table 2). on follow-up, a relatively low percentage of patients recorded certain complication of the operation as listed in table 3. discussion hemorrhoid is a disease of all ages, gender and socioeconomic status. this study shows high male proportion. this is also found in many other studies in karachi. shamim et al. also show a male predominance of 74.88%.21 it has been found that the treatment of choice is proctoscope-guided rubber band ligation and that it is cost effective. other works are trying to evaluate usefulness of new techniques.22–24 similarly, the use of a flexible endoscope for hemorrhoidal elastic band ligation was further analyzed using video endoscopic anoscopy and a single-handed ligator.24–26 these studies conclude that it is a safe and efficient method with some advantages, although costs are still a major drawback. cazemier compared the two procedures, and he concluded that both techniques were easy to perform, issn 2413-0516 168 j contemp med sci | vol. 3, no. 9, winter 2017: 167–169 elastic band ligation of hemorrhoids using flexible gastroscope research hadi abd zaid al-khattabi et al. pain and discomfort after the procedure. this may last for a few days till the banded portion get necrosed and sloughed off. this is more common in large prolapsing hemorrhoids with a wider base. some studies evaluated the use of a flexible endoscope equipped with a ligation cap, normally used for the ligation of esophageal varices, in treating hemorrhoids.16–28 a study from iran shows that, with rubber band ligation, 26% of patients reported mild and moderate pain and 1% complained of severe pain.28 a recent study from faisalabad show that 60% of patients developed mild to moderate bleeding in the first postoperative week.29 bernal show that 32% of the patients referred pain after ligation and 13.81% of cases were operated due to persistent rectal bleeding or hemorrhoidal prolapsed.13 in summary, endoscopic ligation is an effective, safe treatment and is comparable with proctoscopic ligation. however, the treatment is more expensive. conclusion rubber band ligation is simple and cost effective procedure with a high success rate, and, as an outpatient procedure, promotes its use as the first line of treatment for first, second and early third degree hemorrhoids. conflict of interest none. n table 1. frequency distribution of grade of piles in forty-six patients involved in the study no. hemorrhoid degree no.(%) 1 1st degree – (0%) 2 2nd degree 16 (34.7%) 3 3rd degree 30 (65.3%) 4 4th degree – (0 %) total 46 (100%) table 3. frequency distribution of complications of the rbl operation no. complications no. 1 pain 2 2 bleeding 2 3 thrombosis 0 4 ulceration 0 5 fall of elastic band 0 table 2. number of sessions of rbl treatment in the included patients rbl sessions degree of hemorrhoids no.( % ) 2nd degree 3rd degree single ligation 16 26 42 (91.3) two sessions 0 4 4 (8.7) well-tolerated and efficient.26 it was easier to perform more ligations with the flexible endoscope. no serious adverse events were reported. additional advantages of the flexible scope were the maneuverability and photographic documentation. treatment with the flexible endoscope seemed to be more painful and was more expensive. and he explained the more pain sensation by the learning curve he had to deal with and that more bands could be applied.27 in contrast, wehrmann et al. found no significant difference in pain.28 many studies evaluated the patients with threedimensional endosonography for the presence of possible sphincter defects and changes in the submucosa. they found no difference in the appearance of the anal configuration after treatment with either rubber band ligation or infrared coagulation,1 and these endosonographic findings confirmed that band ligation is a safe technique. pain or perianal discomfort is the commonest complaints after rubber band ligation.13,15 anorectal mucosa is sensitive to pain below dentate line. application of bands at or below this line cause considerable fig 1. rubber band ligation (rbl) of hemorrhoids is a widely used method for treatment of piles. references 1. poen ac, felt-bersma rj, cuesta ma, deville w, meuwissen sg. a randomized controlled trial of rubber band ligation versus infra-red coagulation in the treatment of internal haemorrhoids. eur j gastroenterol hepatol. 2000;12:535–539. 2. johanson jf, sonnenberg a. the prevalence of hemorrhoids and chronic constipation. an epidemiologic study. gastroenterology. 1990;98:380–386. 3. madoff rd, fleshman jw. american gastroenterological association technical review on the diagnosis and treatment of hemorrhoids. gastroenterology. 2004;126:1463–1473. 4. blaisdell pc. office ligation of internal hemorrhoids. am j surg. 1958;96:401–404. 5. barron j. office ligation of internal hemorrhoids. am j surg. 1963;105:563–570. hadi abd zaid al-khattabi et al. 169j contemp med sci | vol. 3, no. 9, winter 2017: 167–169 research elastic band ligation of hemorrhoids using flexible gastroscope 6. bleday r, pena jp, rothenberger da, goldberg sm, buls jg: symptomatic hemorrhoids: current incidence and complications of operative therapy. dis colon rectum. 1992;35:477–481. 7. gehamy ra, weakley fl: internal hemorrhoidectomy by elastic ligation. dis colon rectum. 1974;17:347–353. 8. arabi y, gate house d, alexander-williams j, keighley mr: rubber band ligation or rectal subcutaneous sphincterotomy for treatment of hemorrhoids. br j surg. 1977;64:739–740. 9. muller ca:internal hemorrhoidectomy by rubber band ligation. proctology. 1980;5:317–319. 10. wrobleski de, corman ml, veidenheimer mc, coller ja: long-term evaluation of rubber ring ligation in hemorrhoidal disease.dis colon rectum. 1980;23:478–482. 11. o’hara vs:fatal clostridial infection following hemorrhoid banding. dis colon rectum. 1980;23:570–571. 12. russell tr, donohue jh: hemorrhoidal banding: a warning. dis colon rectum. 1985;28:291–293. 13. bernal jc, enguix m, lopez garcia j, garciaromero j, trullenque peris. rubber-band ligation for hemorrhoids inacolorectal unit. a prospective study. rev esp enferm dig org ofic soc esp patologia dig. 2005;97(1):38–45. 14. iyer vs, shrier i, gordon ph: long-term outcome of rubber band ligation for symptomatic primary and recurrent internal hemorrhoids.dis colon rectum. 2004;47(8):1364–1370. 15. komborozos va, skrekas gj, pissiotis ca:rubber band ligation of symptomatic internalhemorrhoids: results of 500 cases. dig surg. 2000;17(1):71–76. 16. forlini a, manzelli a, quaresima s, forlini m: long-term result aft errubber band ligation for haemorrhoids. int j color dis. 2009;24(9):1007–1110. 17. trowers ea, ganga u, rizk r, ojo e, hodges d. endoscopic hemorrhoidal ligation: preliminary clinical experience. gastrointest endosc. 1998; 48: 49-52. 18. qureshi s, aziz t, afzal a, maher m.rubber band ligation of symptomatic internal haemorrhoids; result of 450 cases. j surg pak. 2009;14(1):19–22. 19. dickey w, garrett d. hemorrhoid banding using videoendoscopic anoscopy and a single-handed ligator: an effective, inexpensive alternative to endoscopic band ligationdickey w, garrett d. hemorrhoid banding using videoen. am j gastroenterol. 2000;95:1714–1716. 20. rehan abbas khan, muhammad iqbal, farhan zaheer, khalid ahsan malik, anis uz zaman, rubber band ligation for the symptomatic hemorrhoids. what troubles the patient? pak j surg. 2012;28(4):266–270. 21. takano m, iwadare j, ohba h, takamura h, masuda y, matsuo k, et al. sclerosing therapy of internal hemorrhoids with a novel sclerosing agent. comparison with ligation and excision. int j colorectal dis. 2006;21:44–51. 22. kwok sy, chung cc, tsui kk, li mk. a double-blind, randomized trial comparing ligasure and harmonic scalpel hemorrhoidectomykwok sy, chung cc, tsui kk, li mk. a double-blind, randomized trial comparing ligasure and harmonic scalpel hem. dis colon rectum. 2005;48:344–348. 23. fukuda a, kajiyama t, kishimoto h, arakawa h, someda h, sakai m, seno h, chiba t. colonoscopic classification of internal hemorrhoids: usefulness in endoscopic band ligation. j gastroenterol hepatol. 2005;20:46–50. 24. trowers ea, ganga u, rizk r, ojo e, hodges d. endoscopic hemorrhoidal ligation: preliminary clinical experience. gastrointest endosc. 1998;48:49–52. 25. dickey w, garrett d. hemorrhoid banding using video endoscopic anoscopy and a single-handed ligator: an effective, inexpensive alternative to endoscopic band ligation. am j gastroenterol. 2000;95:1714–1716. 26. cazemier m, felt-bersma rjf, cuesta ma, mulder cjj. elastic band ligation of hemorrhoids:flexible gastroscope or rigid proctoscope? world j gastroenterol. 2007;13:585–587. 27. wehrmann t, riphaus a, feinstein j, stergiou n. hemorrhoidal elastic band ligation with flexible videoendoscopes: a prospective, randomized comparison with the conventional technique that uses rigid proctoscopes. gastrointest endosc. 2004;60:191–195. 28. azizi r, rabani-karizi b, taghipour ma. comparison between ultroidand rubber band ligation in treatment of internal hemorrhoids. acta med iran. 2010;48(6):389–393. 29. dilawaiz m, bashir ma, rashid a. hemorrhoidectomy vs rubber band; comparison of post-operative complications. professional med j. 2011;18:571–574. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 192 j contemp med sci | vol. 7, no. 4, july-august 2021: 192–195 original abstract objective the aim of this study was to compare the effect of diphenhydramine and midazolam on sedation of children. methods this clinical trial was performed on children aged 1 to 7 years who referred to the emergency department for diagnostic radiology. patients were randomly divided into two groups of midazolam and diphenhydramine. then, 30 minutes before the start of the procedure, 0.5 mg/kg was given to the midazolam group and 0.5 cc/kg to the diphenhydramine group. if sedation occurred, the child was separated from the parents and transferred to a diagnostic procedure. after performing the intended diagnostic procedure, the information sheet was completed and the patient's vital signs were checked again. the data were then analyzed by spss version 19 software. results a total of 74 patients were included in the study. there was no significant difference between the two groups in terms of age and gender (p = 0.89; p = 0.32). the mean sedation in the midazolam and diphenhydramine groups was 1.02 and 1.59 years, respectively. a significant difference was found between the two groups in terms of sedation (p = 0.04), where a greater effect of diphenhydramine on sedation was observed. conclusion the findings showed that the use of diphenhydramine resulted in effective sedation for children. due to the fact that the main problem with midazolam is its bitter taste, which makes children reluctant to eat it, the use of diphenhydramine can be recommended. keywords diphenhydramine, midazolam, sedation comparison of the effect of oral diphenhydramine and midazolam on sedation of children neda naeimi bafghi, naeimeh naeimi bafghi*, shirin salajegheh clinical research center, shahid bahonar hospital, kerman university of medical sciences, kerman, iran. *correspondence to: naeimeh naeimi bafghi (e-mail: naeimehnaeimibafghi@yahoo.com) (submitted: 14 april 2021 – revised version received: 02 may 2021 – accepted: 29 may 2021 – published online: 26 august 2021) introduction fear and anxiety before a diagnostic-therapeutic process or before anesthesia and surgery can be a more traumatic experience than the process itself for many patients, especially children.1,2 according to studies, nothing could even replace parental support and caress in many cases, but this support is not effective in relieving preoperative fear and excitement, thus prodrug has a significant clinical effect in reducing the child’s harm from anesthesia and surgery.3,4 studies show that parental anxiety make it more difficult to separate children.5 some studies have also shown that prescribing sedatives before surgery prevent adverse postoperative reactions such as nocturnal enuresis and anorexia. numerous studies have shown that almost all sedatives are effective as prodrugs,6 however, the selection of an appropriate prodrug should be done by considering the desired amount of sedation on the one hand and the effectiveness, side effects and indications of each drug on the other hand. the need for prodrug varies depending on the patient’s condition, underlying disease, type and duration of surgery, method of induction, and mental structure of the child and his family. children under 8 months of age rarely need prodrugs, but after this age the child’s normal development causes fear of people and unfamiliar environment.3–5 preoperative drug administration is widely used for sedation and anti-anxiety effects in pediatric anesthesia.7 a good sedative is easy to use, rapid onset of action, short duration of action and lack of side effects.8 the prodrug used is enough to have only a sedative effect. on the other hand, prodrugs can be given by different routes oral, nasal, intravenous, or intramuscular, and rectal routs. certainly, oral and nasal routs are more acceptable and simpler in children and are not accompanied by pain and anxiety. the most commonly used drugs in children are diphenhydramine, dextromethorphan and midazolam. in the united states, midazolam has been produced as versed syrup in recent years, but this product is only available in the united states.9 midazolam is a good sedative that can be prescribed in several routs (oral, injectable, nasal and rectal).10 the onset of action is within 10 to 20 minutes and the duration of action is 30 minutes. midazolam at doses less than 0.5 mg/kg do not result in a change in hemodynamics7 and provides excellent sedation in 60 to 80% of patients.11 in the oral rout, the time to reach the peak effect is one hour and the reversal of the action of drug is long (up to about 4 hours), while the depth of sedation also varies. for this reason, the intranasal rout, which reaches the peak effect of the drug within ten minutes and the reversal time of the effect is not more than one hour, seems more acceptable.12 diphenhydramine has also been used as an inverse agonist of the histamine h1 receptor in a variety of allergic and psychiatric diseases. it is also used as an adjunct to insomnia or sleep disorders. the use of diphenhydramine in anesthesia is limited.13 therefore, the aim of this study was to evaluate the effect of oral diphenhydramine and midazolam on sedation of children referred to the hospital for imaging. materials and methods this clinical trial was performed on children aged 1 to 7 years who referred to the emergency department of bahonar hospital in kerman, iran, for diagnostic radiology procedures issn 2413-0516 193j contemp med sci | vol. 7, no. 4, july–august 2021: 192–195 n.n. bafghi et al. original comparison of the effect of oral diphenhydramine and midazolam on sedation of children from may to august 2019. patients who met the inclusion and exclusion criteria were included in the study. inclusion criteria: age of children between 1 to 7 years and level of consciousness in the mild-moderate range. exclusion criteria include: parental dissatisfaction, gcs ≤ 8 and unstable hemodynamics (lack of hemodynamics). sample size taking into account the first and second type errors, the sample size of 74 people was calculated using the formula of comparing the two means. n n s s z x x1 2 1 2 2 2 1 2 1 1 2 2 2 = = + +( ) − − −( ) ( ) z a b procedure after obtaining informed consent, patients were randomly divided into two groups of midazolam and diphenhydramine. then, 30 minutes before the start of the procedure, the drug was administered to the midazolam group at 0.5 mg/kg and to the diphenhydramine group at 0.5 cc/kg. if sedation occurred, the child was separated from the parents and transferred to a diagnostic procedure (ct, radiology, and ultrasound). after performing the diagnostic procedure, the information sheet was completed and the patient's vital signs were checked again and children were then returned to the parents if the child was found to be healthy. in fact, the child was resuscitated if there were unstable vital signs. the child was returned to the parents if he or she had stable vital signs. the measurement of effectiveness criteria was as follows: the patient is sedated to the extent of mild (minimal) moderate. this means that patients have a purposeful response to verbal or physical stimulation after receiving the substance by the above methods, and the airway, respiration, blood pressure and pulse are not disturbed. reaching or not reaching this amount in each of the studied methods was considered as outcome. sedation rates were assessed based on the the university of michigan sedation scale (umss) with patient questioning and examination. umss is capable of evaluating the level of changes on a five-point scale as follow: 1. sleepy / responds umss: o: a wake aurt 2. somnolent / arouses to light stimuli 3. deep sleep / arouses to deporplynsical stimuli 4. unarousable to stimuli data analysis independent t-test was used to compare the parameters in two independent groups. chi-square test was used to examine qualitative variables. data analysis was performed using spss software version 19 using statistical methods including descriptive and inferential statistics. a p value of 0.05 was considered to be statistically significant. ethical considerations a written letter of introduction to research centers was received from university officials. the purpose of the study was described for all research units and finally written consent was obtained. all patients, information was kept confidential. the declaration of helsinki was considered in the current study. the statements of the research ethics committees of the university of medical sciences were taken into account. the study was carried out after approval by the research council of the medical school and receiving the code (ir.kmu. rec.1397.520) of ethics letter of introduction. results a total of 74 patients were included in the study. the study population consisted of 39 female patients and 35 male patients who were divided into two groups of 37 patients. there was no significant difference between the two groups in terms of gender (p = 0.32) (table 1). the mean age in the diphenhydramine group was 2.89 years and the mean age was 2.83 years in the midazolam group (table 2). no significant difference was found between the two groups in terms of age (p = 0.89), indicating that the groups were the same at the beginning of the study and elimination of age effects. the mean sedation is given in table 3, which was determined to be 1.02 and 1.59 years in the midazolam and diphenhydramine groups, respectively. there was a statistically significant difference between the two groups (p = 0.04) so that a higher mean in the group of diphenhydramine indicates a greater effect of diphenhydramine in sedation. discussion prodrug administration in children using a safe method and appropriate dose is very important to achieve adequate sedation.14 the aim of this study was to compare the effect of diphenhydramine and oral midazolam on sedation of children. in this study, 74 children aged 1 to 7 years were examined. then, the findings of this study were analyzed and the final conclusion was presented. table 1. frequency distribution of subjects by gender variable female male total p-value diphenhydramine 21 (56.8%) 16 (43.2%) 37 0.32 midazolam 18 (48.6%) 19 (51.4%) 37 total 39 35 74 table 2. mean and standard deviation of age in the two groups variable mean sd t p-value diphenhydramine 2.89 1.86 0.13 0.89 midazolam 2.83 1.7 table 3. mean and standard deviation of sedation after intervention variable mean sd t p-value diphenhydramine 1.59 1.11 2.07 0.04 midazolam 1.02 1.23 194 j contemp med sci | vol. 7, no. 4, july-august 2021: 192–195 comparison of the effect of oral diphenhydramine and midazolam on sedation of children original n.n. bafghi et al. in the present study, there was no significant difference between the two groups in terms of gender and age. as a result, the confounding effect of these variables has been controlled. the most important finding of this study was the significant difference between the mean sedation in the midazolam and diphenhydramine groups, where a higher mean sedation was observed in the diphenhydramine group. cengiz et al. compared the safety and efficacy of midazolam–diphenhydramine combination and midazolam alone in pediatric sedation for magnetic resonance imaging. the results showed that the combination of oral diphenhydramine with oral midazolam is safe and effective in performing mri in children and its sedative failure is less during mri.15 findings of golzari et al.’s study also showed that the combination of diphenhydramine and midazolam has a higher sedative effect and less side effects compared to diphenhydramine alone.16 in heydarian’s research, the findings showed that the combination of oral diphenhydramine and oral midazolam leads to safe and effective sedation of children during ct scan. this combination can be more beneficial than midazolam alone.17 the results of a study by taghipor et al., which examined the sedative effect of three oral prodrugs (midazolam, dextromethorphan and diphenhydramine) in children, indicated that the rate of sedation before any intervention in the dextromethorphan group was significantly better. at the time of separation of children from parents, the three groups did not differ significantly in the intensity of sedation. however, the intensity of sedation in patients receiving dextromethorphan was significantly better than that of oral diphenhydramine and midazolam during induction of anesthesia and in the recovery.18 in some other studies, the effect of midazolam alone in pediatric sedation has been investigated. in 2008, lane et al. examined the use of intranasal midazolam for minor procedures in children, stating that intranasal midazolam is very suitable for providing anxiolysis reducing anxiety to children undergoing minor procedures in the pediatric department.19 in 2015, plum et al. examined the effect of intranasal midazolam in reducing anxiety in children with nasal fractures. the results showed that midazolam was quite effective in providing effective anxiolysis and had no adverse outcomes.20 in a 2015 study, musani et al. concluded that intravenous midazolam had a quick onset of action and a quick recovery from sedation. also, its proper effect required a lower dose through the intranasal route. finally, they concluded that intranasal midazolam is a suitable alternative to oral midazolam for a pediatric dental condition.21 many diagnostic and therapeutic measures in children require the full cooperation of the child. these diagnostic procedures include radiology, endoscopy, colonoscopy, aspiration, bone marrow biopsy, liver and kidney biopsy, bronchoscopy, and cerebrospinal fluid biopsy. the use of sedatives for diagnostic, biopsy, or minor surgery in children is a high priority. issues such as unfamiliarity with the ward, not realizing the importance of the issue, separation from parents will lead to poor cooperation of children. therefore, it should be tried that in the process of prescribing sedatives, drugs with ease of use, rapid onset of action, minimal side effects and more short recovery should be prescribed. non-injectable prescriptions are highly acceptable, although they have a longer onset of action than the injectable form and have a higher initial liver removal; also, it is possible that interpersonal differences in effect rate and rate of absorption may impair the process. sedation measures increase the quality of diagnosis and treatment and can reduce the psychological effects of the child. conclusion the results of this study showed that the use of diphenhydramine is suitable for sedation and reducing anxiety in children. due to the fact that the main problem with midazolam is its bitter taste, which makes children reluctant to eat it, the use of diphenhydramine can be recommended.  references 1. song jh. procedural sedation and analgesia in children. j korean med assoc. 2013;56(4):271-8. 2. eskandarian t, maghsoudi s, eftekharian h. clinical evaluation of the effects of two types of oral combination of midazolam in sedating pediatric dental patients. j dent shiraz univ med sci. 2010;11(1). 3. soleimanpour h, gholipouri c, salarilak s, raoufi p, vahidi rg, rouhi aj, et al. emergency department patient satisfaction survey in imam reza hospital, tabriz, iran. int j emerg med. 2011;4:2. 4. barkan s, breitbart r, brenner-zada g, et al. a double-blind, randomised, placebo-controlled trial of oral midazolam plus oral ketamine for sedation of children during laceration repair. emerg med j. 2013;31(8):649-53. 5. hosseini m, karami z, janzadenh a, et al. the effect of intrathecal administration of muscimol on modulation of neuropathic pain symptoms resulting from spinal cord injury; an experimental study. emergency. 2014; 2(4):151-7. 6. alimohammadi h, shojaee m, samiei m, abyari s, vafaee a, mirkheshti a. nerve stimulator guided axillary block in painless reduction of distal radius fractures; a randomized clinical trial. emergency. 2013;1(1):11-4. 7. azizkhani r, esmailian m, golshani k. rectal thiopental versus intramuscular ketamine in pediatric procedural sedation and analgesia; a randomized clinical trial. emergency. 2014;3(1):22-6. 8. khajavi m, emami a, etezadi f, safari s, sharifi a, moharari rs. conscious sedation and analgesia in colonoscopy: ketamine/propofol combination has superior patient satisfaction versus fentanyl/propofol. anesthesiol pain med. 2013;3(1):208-12. 9. alimohammadi h, azizi m-r, safari s, amini a, kariman h, hatamabadi hr. axillary nerve block in comparison with intravenous midazolam/fentanyl for painless reduction of upper extremity fractures. acta med iranica. 2014; 52(2):122-4. 10. krauss bs, krauss ba, green sm. procedural sedation and analgesia in children. n engl j med. 2014;370(15):e23. 11. moreira ta, costa ps, costa lr, et al. combined oral midazolam–ketamine better than midazolam alone for sedation of young children: a randomized controlled trial. int j paediatr dent. 2013;23(3):207-15. 12. maurizi p, russo i, rizzo d, et al. safe lumbar puncture under analgosedation in children with acute lymphoblastic leukemia. int j clin oncol. 2014;19(1):173-7. 13. green sm. what is the role of diphenhydramine in local anesthesia? academerg med. 1996;3(3):198-200. 14. alimohammadi h, baratloo a, abdalvand a, rouhipour a, safari s. effects of pain relief on arterial blood o2 saturation. trauma mon. 2014;19(1):e14034. 15. cengiz m, baysal z, ganidagli s. oral sedation with midazolam and iphenhydramine compared with midazolam alone in children undergoing magnetic resonance imaging. pediateric anesthesia 2006;16(6): 621–626. 16. golzari s, shahsavari nia k , sabahi m, soleimanpour h , mahmoodpoor a, safari s , et al. oral diphenhydramine-midazolam versus oral diphenhydramine for pediatric sedation in the emergency department. j comprped 2014 february;5(1):e17946. 17. heydarian n. comparison of the simultaneous use of midazolam and oral diphenhydramine with oral midazolam in pediatric patients 1 to 7 years old at the time of ct scan referring to the emergency department of besat hospital. army university of medical sciences, thesis. 2014. 195j contemp med sci | vol. 7, no. 4, july–august 2021: 192–195 n.n. bafghi et al. original comparison of the effect of oral diphenhydramine and midazolam on sedation of children this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 18. taghipor anvari z, sheibani s, imani f, sheibani s. comparison of three oral premedication dextrometorphan, diphenhydramine and midazolam in pediatric eye exam under anesthesia. jap. 2011;2(3):9-17. 19. lane rd, schunk je. atomized intranasal midazolam use for minor procedures in the pediatric emergency department. pediatr emerg care. 2008 may; 24(5):300-3. 20. plum aw, harris tm. intranasal midazolam for anxiolysis in closed reduction of nasal fractures in children. int j pediatr otorhinolaryngol. 2015 jul; 79(7):1121-3. 21. musani ie, chandan nv. a comparison of the sedative effect of oral versus nasal midazolam combined with nitrous oxide in uncooperative children. eur arch paediatr dent. 2015 may 5. https://doi.org/10.22317/jcms.v7i4.1002 47j contemp med sci | vol. 8, no. 1, january-february 2022: 47–50 original evaluation of effective factors on pain in patients undergoing sleep apnea surgery reihaneh heidari1, mahsa najafzadeh2, reza erfaniyan3, ebrahim razmpa1, hamed emami1, matineh heidari4* 1otorhinolaryngology research center, imam khomeini hospital complex, tehran university of medical sciences, tehran, iran. 2school of medicine, tehran university of medical sciences, tehran, iran. 3otorhinolaryngology research center, amiraalam hospital, tehran university of medical sciences, tehran, iran. 4department of neurology, firoozgar hospital, iran university of medical scienses, tehran, iran. *correspondence to: matineh heidari (email: dr.matinehheidari@gmail.com) (submitted: 21 november 2021 – revised version received: 16 december 2021 – accepted: 27 december 2021 – published online: 26 february 2022) abstract introduction: sleep apnea is associated with complete or partial obstruction of the upper airway. this disease can cause various problems for the individual. in most cases, surgery and pharyngoplasty are needed to treat this complication. depending on the type of surgery, its duration, the patient’s age, the type of opioid prescribed after surgery and other factors, different amounts of postoperative pain have been reported in different studies. therefore, the aim of this study was to investigate the factors affecting postoperative pain. materials and methods: this descriptive cross-sectional study was performed on patients referred to the hospital to determine the factors affecting postoperative pain for patients with obstructive sleep apnea (osa). patients’ information was recorded: age, sex, weight, height, body mass index, duration of surgery, possible complications, and anesthesia. patients were evaluated for pain according to vas criteria. the first time a patient requested a drug was recorded in 24 hours after surgery and data was then analyzed. results: a total of 40 patients were enrolled in the study, including 14 women (35%) and 26 men (65%). the mean age of patients was 41.55 ± 7.43 years. examination of the relationships between other variables with patients’ pain intensity showed a statistically significant difference between patients’ pain intensity with other variables such as history of stroke (p = 0.005), history of cardiovascular disease (p = 0.048), history of drug abuse (p = 0.046) and type of analgesia received after surgery (p = 0.032). in multivariate analysis of the studied data, no statistically significant relationship was found between any of the variables with the intensity of patients’ postoperative pain. the variances of height, weight, body mass index, duration of surgery and the first time of application of analgesic after surgery did not differ in different groups of pain intensity variables. but a significant difference was found between the two variables of age and pain intensity of patients (p < 0.05). conclusion: the results of this study showed a statistically significant difference in pain intensity with a history of stroke, cardiovascular disease, history of drug abuse and also the type of analgesia received after surgery. the serious complications caused by tolerating acute postoperative pain, especially the long-term effects of experiencing severe pain, necessitates more attention to pain control. keywords: obstructive sleep apnea, pain intensity issn 2413-0516 introduction obstructive sleep apnea (osa) is characterized by complete or partial upper airway obstruction that frequently occurs during sleep. this disease can cause various medical and social problems.1 osa can lead to a lack of oxygen in the blood or an increase in carbon dioxide, which is accompanied by clinical symptoms associated with nighttime drowsiness, loud snoring, awakening out of suffocation due to respiratory obstruction at least five times in every hour of sleep. there is a complex interaction between physiological factors of respiratory obstruction, opioid drug effect, and pain.2 osa commonly affecting 2–3.5% of children is usually treated with pharyngoplasty.3 positive airway pressure is an alternative treatment commonly utilized to treat osa; however, 5 to 50% of patients may fail to respond well to this treatment.4 surgery and placement of oral appliances are alternative solutions used depending on the patient’s condition and severity of the disease. osa surgical treatment usually removes the obstruction by removing excess tissue or reshaping the upper airway. uvulopalatopharyngoplasty (uppp) is the most common procedure performed to remove this obstruction,5 but surgeons use less invasive procedures such as anterior palatoplasty and lateral pharyngoplasty due to postoperative complications.6 enhanced surgical techniques have significantly decreased the rate of postoperative complications. nonetheless, postoperative pain remains one of the critical aspects of osa surgical treatments. the experience of severe pain during the early hours after surgery renders patients reluctant to opt for surgical treatments. opioids are mainly used to control postoperative pain. however, opioid-induced depression of respiratory centers limits the application of opioid-based analgesics and further imposes a challenge in postoperative pain management of osa patients undergoing surgery.2 furthermore, some patients may suffer from multiple obstructions along different levels of the respiratory tract. respiratory complications may arise during surgery and require treatment in more than 10% of children undergoing pharyngoplasty. moreover, patients commonly suffer from multiple obstructions along different levels of the respiratory tract; prolonged time of surgery in such patients requiring multilevel pharyngoplasty adds to the risk of complications. additionally, various factors such as obesity and young age, especially less than three years, are reported among risk factors of intraoperative respiratory complications.3 conversely, more than 60% of early respiratory complications occur postoperatively. it is conceivable that some of these mild postoperative complications are due to factors such as the 48 j contemp med sci | vol. 8, no. 1, january-february 2022: 47–50 evaluation of effective factors on pain in patients undergoing sleep apnea surgery original r. heidari et al. effects of anesthetics, bleeding, and edema in the surgical site, which in children with osa increases respiratory problems and decreases the neuromuscular function of the airways. in addition, many osa patients at the same time suffer from nasal septum deviation. subsequently, pharyngoplasty in osa patients may be accompanied by a septoplasty. postoperative care of concomitant septoplasty that leaves nasal airways obstructed obligates mouth breathing, which in turn may cause dry mouth, prolonged discomfort, and increased postoperative pain. various approaches employed in different studies to reduce pharyngoplasty postoperative pain in osa patients require further evaluation. accordingly, researchers in the current study sought to investigate factors affecting the intensity and duration of postoperative pain to provide a perspective on reducing the need for opioid-based analgesia, which, especially in children, is associated with complications. materials and methods this descriptive cross-sectional study was performed on patients referred to imam khomeini hospital in tehran, iran, in 2017 to determine the factors affecting pain after surgery for osa. data were collected by census, and patients meeting inclusion criteria without having any of the exclusion criteria were included in the study. inclusion criteria include: osa, primary surgery to repair the lesion, having informed consent. exclusion criteria include: all patients with clinical presentation of severe bleeding, patients who were dissatisfied with participation or continuation of the study for any reason, pregnant patients, and patients with a history of psychiatric disorders, patients with cultural, linguistic, and cognitive differences with the community. sample size between april and march 2017, 40 patients were enrolled in the census. procedure osa patients were visited after initial referral to the ent department of imam khomeini hospital and evaluated regarding clinical manifestations and imaging findings. surgical attention offered to osa patients in our end department consists of expansion pharyngoplasty either with or without septoplasty and turbinoplasty. after collecting demographic and clinical information, the standard treatment was performed according to each patient’s clinical condition. all patient information, including age, sex, education, postoperative complications, and other related factors, were carefully recorded and checked out by the project manager. the data entry sheet according to the inclusion and exclusion criteria has several main domains, including demographic information, patient’s medical conditions, postoperative complications, and related follow-ups. defective data were completed through extraction of the required information from patients medical records, patients interview, or physical examination if necessary. drug regimen intraoperative anti-inflammatory regimen including 8 milligrams of dexamethasone and 100 milligrams of hydrocortisone was applied the same for all patients. all patients received dexamethasone every 12 hours and acetaminophen every 4 hours for two days postoperatively. morphine was cautiously administered at patients’ request for additional analgesia. opioid dosage was tailored to each patients’ medical condition to prevent respiratory depression. data analysis to analyze the data, first, the descriptive indices of the variables including, the mean and standard deviation for the research variables, were reported. the kolmogorov–smirnov test was reported to check the normality of data. then, in the inferential findings section, the assumptions are presented. after examining the most important assumptions related to the statistical test, the test results were reported, and the data were analyzed by spss software version 16. ethical consideration after receiving a written letter of introduction from the university and selected research centers, the purpose of the study was explained to all research units, and then written consent was obtained. all patients’ information was kept confidential. ethical declarations of helsinki and ethics research committees of the university of medical sciences were considered. the study was finally approved by the code of ethics (ir. tums.ikhc.rec.1398.73). http s : / / e t h i c s . re s e arc h . a c . i r / et h i c s prop o s a l vi e w. php?&code=ir.tums.ikhc.rec.1398.073. results a total of 40 osa patients undergoing pharyngoplasty with or without septoplasty and turbinoplasty were included in the study. of all patients, 14 were women (35%), and 26 were men (65%). the mean age of patients was 41.55 ± 7.43 years, and the minimum and maximum ages were 28 and 57 years, respectively. in terms of marital status, 28(70%) were single, and 12(30%) were married. furthermore, 16 patients (40%) were illiterate, followed by primary and secondary education (15 patients; 37.5%), diplomas (8; 20%) and university education (1; 2.5%). also, 24 patients (60%) had a history of drug abuse, and 16 patients (40%) had no such history. after surgery on patients, patients were evaluated for complications after surgery, 20 patients (50%) developed postoperative pain, followed by nausea (12 patients; 30%), fever (3 patients; 7.5%), respiratory apnea (3 patients), and bleeding (2 patients; 5%). morphine was administered for 13(32.5%) patients in response to the patients’ request for additional analgesia. 5(12.5%) patients additionally received non-steroidal anti inflammatory drugs (nsaid) for further pain relief. figure 1 shows the severity of postoperative pain. in 30% of the studied patients, the pain intensity was 7, and the lowest was related to the pain intensity of 8, 9, and 7.5%. the average time to request additional analgesia after surgery was 195 minutes. examination of the relationships between other variables with patients’ pain intensity showed that having a history of stroke (p = 0.005), a history of cardiovascular disease (p = 0.048), a history of drug abuse (p = 0.046), and type of postoperative analgesia (p = 0.032) were significantly associated with https://ethics.research.ac.ir/ethicsproposalview.php?&code=ir.tums.ikhc.rec.1398.073 https://ethics.research.ac.ir/ethicsproposalview.php?&code=ir.tums.ikhc.rec.1398.073 49j contemp med sci | vol. 8, no. 1, january-february 2022: 47–50 r. heidari et al. original evaluation of effective factors on pain in patients undergoing sleep apnea surgery pain intensity. in multivariate analysis of the studied data, it was shown that there was no statistically significant relationship between any of the variables with the intensity of patients’ pain after surgery. the mean of quantitative variables of this study was compared in different groups of pain intensity by kruskal-wallis test. due to the fact that p value was greater than 0.05, leven test hypothesis (equality of variance) was accepted. the variances of height, weight, body mass index, duration of surgery, and the first time applying analgesia after surgery did not differ in different groups of pain intensity variables. however, a significant difference was found between the two variables of age and pain intensity of patients (p < 0.05). due to this difference, the groups were compared with each other by post hoc test with the highest sensitivity and lowest specificity to report the slightest difference if there is any, and the results showed no statistically significant difference between age and pain intensity groups. discussion in recent years, many advances have been made in the management of postoperative pain. the use of newer methods, such as catheters and non-pharmacological methods, is increasing in developed countries. much research has been done on different types of pain relief methods and compares them with each other in developed countries. in a few studies, the prevalence of analgesic methods has also been investigated.7,8 therefore, the aim of this study was to evaluate the factors affecting the pain of patients undergoing sleep apnea surgery. the results of this study showed that there was a statistically significant difference between patients’ pain intensity with other variables such as a history of stroke (p = 0.005), cardiovascular disease (p = 0.048), drug abuse (p = 0.046), and type of analgesia received after surgery (p = 0.032). in multivariate analysis of the studied data, no statistically significant relationship was found between any of the quantitative variables with patients’ pain intensity after surgery. in this regard, various studies have been conducted, including the study of dabbagh et al., which was conducted in 2009 to show the relationship between pain-related factors, including age, sex, and use of anesthesia prodrug with the incidence of postoperative pain. such findings are consistent with our findings. in other words, age, sex, and the use of anesthesia pre-medication all affect the incidence of postoperative pain. another study using numerical criteria reported patients’ pain intensity in the surgical department other than heart surgery, in which the mean maximum pain on the first day after surgery was 6.3 and table 1. study variables percentfrequencyvariables 6526malesex 3514female 3012singlemarital status 7028married 4016illiterateeducation rate 37.515elementary and cycle 208diploma 2.51university 5020paincomplications after surgery 52hemorrhage 7.53respiratory distress 7.53fever 3012nausea 6024yescigarette smoking history 4016no 2510yesintraoperative dexamethasone 7530no 42.517yesreceiving hydrocortisone during surgery 57.523no 5522acetaminophentype of analgesic received after surgery 12.55nsaid 32.513ms 2510yesdiabetesunderlying disease 7530no 5020yesblood pressure 5020no 6024yescardiovascular disease 4016no 5522yeskidney 4518no 22.59yesstroke 77.531no 62.525yesrespiratory 37.515no fig. 1 comparison of postoperative pain intensity. table 2. comparison of means of quantitative variables meanmaxminvariables 41.55 ± 7.435728age 173.88 ± 9.19186150height 77.64 ± 9.799752weight 25.83 ± 4.0337.7819.1body mass index 195 ± 20.122412the first time you apply for postoperative housing 163.37±45.64300105duration of surgery 50 j contemp med sci | vol. 8, no. 1, january-february 2022: 47–50 evaluation of effective factors on pain in patients undergoing sleep apnea surgery original r. heidari et al. decreased to only 5.6 on the third day after surgery. the results of this study were consistent with our results.9 patients undergoing surgery experience severe pain in the first 24 hours after surgery. the intensity of pain may vary depending on the type of operation and the treatment protocol given to the patient. in lynch’s study, the mean maximum pain on the first day after surgery was 6.3 based on the visual pain score. gynecological surgeries have been reported to have a mean maximum pain of 8.1 ± 2.47.10 in the study of bameshki et al., the mean maximum pain was 8.4 2 2, which was slightly higher than the results of other studies and indicates the inadequacy of conventional methods of pain control in this area.11 in a study of 3170 patients, it was found that a visual pain score above 7 indicates severe pain, so it can be said that 82.1% of patients in the bameshki study experienced a period of severe pain in the first 24 hours after surgery.11 this figure has been reported between 87–9% in studies in iran and 46–28%10-12 in foreign studies, and 26% for outpatient operations. however, the average pain was severe in only 34.1% of patients, and the majority of patients reported their average pain as moderate, which was not acceptable because patients should have mild pain or visual pain score less than 5.11 the articles indicate that the pain threshold was lower in women. in the present study, women reported more severe pain than men, although this was not statistically significant. this finding is consistent with a study that showed that despite 66.8% of women complained of pain compared to 48% of men, but this difference was not statistically significant.10 while all patients received dexamethasone and acetaminophen postoperatively for pain management, 13(32.5%) requested additional analgesia and were treated with morphine. moreover, 5(12.5%) postoperatively received nsad. in bameshki study, methadone (85.1%), diclofenac (31%), and morphine (10.8%) were the most prescribed analgesics. regarding the high intensity of patients’ pain after surgery, using strong analgesics such as morphine in appropriate amounts is necessary. it seems that less than the required amount of medication is used due to the fear of medical and nursing staff about the side effects of narcotics, especially respiratory failure or lack of knowledge on how to properly control postoperative pain, resulting in severe pain tolerance by patients.11 after surgery, patients were evaluated for complications, and 20 patients (50%) developed postoperative pain, followed by nausea (12 patients; 30%), fever (3 patients; 7.5%), respiratory apnea (3 patients), and bleeding (2 patients; 5%). intraoperatively, dexamethasone and hydrocortisone were administered for all patients. the results of the study by elhakim et al.13 showed that dexamethasone could not reduce the time of onset of oral feeding but significantly reduced the overall frequency of early and late vomiting (37% vs. 74%). based on the findings of studies in this field and the above explanations, it seems that intravenous injection of dexamethasone during surgery can reduce pain and morbidity after pharyngoplasty and pharyngoplasty without any side effects. it is believed that other studies with different doses of dexamethasone and more precise control of confounders can respond to the existing contradictions in the investigations of the effect of dexamethasone on morbidity after postoperative pharyngoplasty. conclusion given the serious complications of tolerating acute postoperative pain, especially the long-term effects of experiencing severe pain, it is needed to pay more attention to pain control.  references 1. osman am, carter sg, carberry jc, eckert dj. obstructive sleep apnea: current perspectives. nat sci sleep. 2018;10:21–34. 2. lam kk, kunder s, wong j, doufas ag, chung f. obstructive sleep apnea, pain, and opioids: is the riddle solved? current opinion in anesthesiology. 2016;29(1):134–140. 3. gozal d, tan hl, kheirandish-gozal l. treatment of obstructive sleep apnea in children: handling the unknown with precision. j clin med. 2020;9(3):888. 4. kakkar rk, berry rb. positive airway pressure treatment for obstructive sleep apnea. chest. 2007;132(3):1057–72. 5. mackay sg, lewis r, mcevoy d, joosten s, holt nr. surgical management of obstructive sleep apnoea: a position statement of the australasian sleep association. respirology. 2020;25(12):1292–1308. 6. pang kp, tan r, puraviappan p, terris dj. anterior palate-plasty for the treatment of osa: three-year results. otolaryngology—head and neck surgery. 2009;141(2):253–6. 7. waxman ja, shenouda kg, lin hs. assessment and management of postoperative pain associated with sleep apnea surgery. otolaryngol clin north am. 2020 oct;53(5):765–777. 8. strutz pk, kronzer v, tzeng w, et al. the relationship between obstructive sleep apnoea and postoperative delirium and pain: an observational study of a surgical cohort. anaesthesia. 2019;74(12):1542–1550. 9. dabbagh a, ghorbanloo m, taherian m, hosseini sm, tavakkolpour aa, rzavi ss, et al. frequency of postoperative pain and its associated factors in taleghani hospital. pejouhesh dar pezeshki (research in medicine). 2010;33(4):265–9. 10. nikandish r, ghafari m. evaluation of post-operative pain management in females in the first 24 hours after surgery. journal of medical research. 2004-2005;3(2-3):120–131. 11. bameshki a, jahanbakhsh s, jangjoo a, zandi h, fathi m. evaluation of acute postoperative pain and patient satisfaction in laparotomy, cholecystectomy and herniorrhaphy. anesthesiology and pain. 2013;4(2):196–201. 12. tavakoli a, nourouzi m, haji ze. patients’ satisfaction from pain soothing after the surgery in kerman hospitals (205). journal of kermanshah of university of medical science. 2007;11(2):206–214. 13. elhakim m, ali nm, rashed i, riad mk, refat m. dexamethasone reduces postoperative vomiting and pain after pediatric pharyngoplasty. canadian journal of anesthesia. 2003;50(4):392–7. 14. lynch ep, lazor ma, gellis je, orav j, goldman l, marcantonio er. patient experience of pain after elective noncardiac surgery. anesthesia & analgesia. 1997;85(1):117–23. 15. patil sp, ayappa ia, caples sm, kimoff rj, patel sr, harrod cg. treatment of adult obstructive sleep apnea with positive airway pressure: an american academy of sleep medicine clinical practice guideline. j clin sleep med. 2019;15(2):335–343. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1095 330 j contemp med sci | vol. 7, no. 6, november-december 2021: 330–333 original the prevalence of vitamin d deficiency and associated risk factors among general populations in duhok province, kurdistan region, iraq lina y. mohammed, shaker a. jamal, nawfal r. hussein, ibrahim a. naqid* introduction vitamin d, a lipid-soluble prohormone, plays a key role in human health by promoting the absorption and metabolism of calcium and phosphate1,2 especially in bone formation and skeletal development. vitamin d deficiency is associated with a wide variety of other health conditions, including cardiovascular disease,3 type 1 and 2 diabetes,4 chronic kidney disease, rheumatoid arthritis,5 increased risk of cancer, as well as cognitive dysfunction, schizophrenia and depression,6 rickets in children and osteomalacia in adults.7 vitamin d may also be implicated in the risk of miscarriage due to its function as an immune modulator8 and its potential importance for the maternal-fetal immunologic response.9 several studies have been conducted in iraq about the potential causes of miscarriage among pregnant women, but have not reported the impact of vit. d on pregnancy.10–12 the most studied types among five forms of vitamin d (d1 to d5) are vitamin d2 and d3 vitamin.13 d2, ergocalciferol is synthesized in plants and invertebrates and is consumed in the human’s diet and as supplements while d3 (cholecalciferol) comes from vertebrate animals such as fatty fish and meat, eggs. the major source of vitamin d is formed in the skin after exposure of 7-dehydrocholesterol to ultraviolet sun rays (vitamin d3) while minor source of vitamin d comes from food (vitamin d3, cholecalciferol) and from dietary supplements (vitamin d2, ergocalciferol) for synthesize a significant amount of vitamin d requirements.14 vitamin d (d2 and d3) from the skin and diet are transported and metabolized in the liver to produce 25(oh) vitamin d3 which is used to determine the level of vitamin d in patients, then the kidney converted it by 25-hydroxyvitamin d-1αhydroxylase enzyme to its to its active form 1,25(oh)2 vitamin d.15 in iraq including kurdistan region, with sunlight throughout the year, vitamin d serum levels are expected to be adequate, yet studies from different arab countries showed high prevalence levels of vitamin d deficiency and insufficiency.16,17 the prevalence of vitamin d deficiency level is very high and varies from 70% to 90% in different populations, globally.18 there is limited data on the prevalence of vitamin d deficiency among the general population in kurdistan regioniraq. determination of vitamin d status in different age-groups and gender in a community and in different climates of a country is necessary and has important implications for general public health. therefore, the main objective of this study was to determine the levels of vitamin d 25(oh) among zakho and dohuk populations and determine associated risk factors as age and gender. materials and methods study design and sampling a cross sectional study was conducted in azadi and bedari hospitals in dohuk and zakho cities, kurdistan region, iraq. the study was conducted over a period of nine months from january 2019 to september 2019. a total of 1143 serum samples were collected (632 patients from bedari hospital, zakho city and 511 patients from azadi hospital, dohuk city). the ages of participants ranged from 9 months to 86 years old (36.31 ± 17.61). an overnight fasting blood sample (5 ml) was obtained for each patient by venepuncture. serum 25(oh)d3 levels were measured within 24 hrs of blood collection in the hospital clinical laboratory using an enzyme-linked immunosorbent assay (elisa) method supplied by (bioactivia diagnostic gmbh). the patients were classified into three diagnostic groups abstract objectives: this study aimed to determine the serum levels of 25(oh)d3 and associated risk factors in duhok province, kurdistan region, iraq. methods: a cross-sectional study was performed from january 2019 to september 2019 among the general population in dohuk and zakho cities. 1143 of subjects were recruited in this study and aged ranged from 9 months to 86 years old (36.31 ± 17.61). serum 25(oh) d3 levels were measured using an enzyme-linked immunosorbent assay (elisa) method. results: of 1143 subjects, 229 (44.9%) people were suffered from vitamin d deficiency in duhok city and 241(38.19%) from zakho city. there was significant difference between the incidence of vitamin d in both cities (p < 0.067). the prevalence of vitamin d deficiency was 152 (37.8%) in male and 318 (43.03%) in females. there was no significantly associated with low level of vitamin d between genders (p < 0.133). the low level of vitamin d was more frequently found in age group between 20–40 years old (46.23%), with sufficient differences between age group (p < 0.001). conclusion: the prevalence of vitamin d deficiency is much predominant in duhok province, younger adults and female population. our finding also found that the low level of vitamin d deficiency in older people. further studies are required to investigate the pathophysiology of hypovitaminosis d and its clinical consequences. keywords: vitamin d deficiency, general population, duhok province, kurdistan, iraq issn 2413-0516 department of biomedical sciences, college of medicine, university of zakho, zakho kurdistan region, iraq. *correspondence to: ibrahim a naqid (e-mail: ibrahim.naqid@uoz.edu.krd) (submitted: 10 september 2021 – revised version received: 06 october 2021 – accepted: 19 october 2021 – published online: 26 december 2021) 331j contemp med sci | vol. 7, no. 6, november-december 2021: 330–333 l.y. mohammed et al. original prevalence of vitamin d deficiency in duhok province, iraq according to their serum concentrations of 25(oh)d3 by increasing the order of severity as follows: vitamin d deficiency, 15 ng/ml or less, vitamin d deficiency, (16–30 ng/ml); and vitamin d deficiency greater than 30 ng/ml. ethical approval the study proposal was approved by the ethics committee of the college of the medicine/university of zakho, kurdistan region, iraq. informed written consent was obtained from all the participants before samples collection. statistical analysis the result of this study was analysed using the graphpad prism software package, version 8. the results were expressed as the mean ± standard deviation or as simple percentages as appropriate. comparisons were made using the chi-square and fisher exact test. the results were considered significant if p ≤ 0.05. results a total of 1141 participants were recruited in this study. table 1 summarises the demographic characteristics of the total population. of 1143 individuals, 402 (35.23%) were male and 739 (65%) of them were female. 510 (45%) of participants were from dohuk residency and 63 (55%) from zakho residency. the majority of participants 491 (43%) were from 20–40 years old, followed by 40–60 years 332 (29%), less than 20 years 188 (16%) and greater than 60 years 130 (11%). the mean age was 36.31 ± 17.61 years (ranged from 9 months to 86 years old). according to the patient’s serum 25 ohd concentrations, three diagnostic categories were found in both dohuk’s and zakho’s population (table 2). the incidence of vitamin d deficiency among dohuk population was 229 (44.9%) with mean value (8.26 ± 3.63). on the other hand, 241(38.19%) of zakho study group were vitamin d deficient with mean value (9.68 ± 2.99). there was significant difference between the status of vitamin d level and their current residency when analysed using the fisher exact test (p = 0.067). the prevalence of vitamin d deficiency levels in serum among gender in studied population are presented in table 3. it was found that the prevalence of this nutritional deficiency in male was 152 (37.8%) with the mean concentration (8.97 ± 3.51), 165 (41%) were vitamin d insufficient with the mean concentration (21.23 ± 4.19) while only 85 (21.1%) of participants were vitamin d sufficient. analysis of vitamin d levels regarding female participants revealed that 318 (43.03%) of them were deficient with the mean concentration (9.03 ± 3.33) while 261 (35.31%) of female were vitamin d insufficient with the mean level concentration (22.43 ± 4.42). 160 (21.65%) of female cases had vitamin d sufficient with the mean concentration (44.18 ± 16.62). using fisher exact test, the differences between genders were statistically not significant (p = 0.133). associations between 25(oh)d level and age groups were also investigated (table 4). majority of them were between 20–40 years, 227 (46.23%) of them were vitamin d deficient with the mean level (9.12 ± 3.42). the next age group were 40–60 years, 121 (36.44%) of them were vitamin d deficient with the mean level (8.74 ± 3.37). the age group less than 20 years came in third order regarding to vitamin d levels, accounting for 83 (44.14%) were vitamin d deficient with the mean level (9.11 ± 3.32). of all participants in this study, least were in greater than 60 years age group accounting for 39 (30%) of them were vitamin d deficient with the mean level (8.78 ± 3.48). our results showed that there was a significant table 1. demographic characteristic of the participants variables frequency percentage gender male 402 35.23% female 739 65% residence duhok 510 45% zakho 631 55% age group (year) <20 188 16% 20–40 491 43% 40–60 332 29% >60 130 11% table 2. comparison of serum level of vitamin d in nanogram/dl according to residency variable deficient number (%) mean ± sd insufficient number (%) mean ± sd sufficient number (%) mean ± sd *p value residence duhok 229 (44.9%) (8.26 ± 3.63) 177 (34.70%) (21.54 ± 4.27) 103 (20.19%) (45.85 ± 18.27) 0.067 zakho 241 (38.19%) (9.68 ± 2.99) 249 (39.46%) (21.31 ± 4.38) 141 (22.34%) (44.19 ± 19.06) sd, standard deviation. *p value was determined by chi-square (fisher exact test). table 3. comparison of serum level of vitamin d in nanogram/dl according to gender variable deficient number (%) *mean ± sd insufficient number (%) *mean ± sd sufficient number (%) *mean ± sd p value gender male 152 (37.8%) (8.97 ± 3.51) 165 (41%) (21.23 ± 4.19) 85 (21.1%) (47.61 ± 21.9) 0.133 female 318 (43.03%) (9.03 ± 3.33) 261 (35.31%) (22.43 ± 4.42) 160 (21.65%) (44.18 ± 16.62) 332 j contemp med sci | vol. 7, no. 6, november-december 2021: 330–333 prevalence of vitamin d deficiency in duhok province, iraq original l.y. mohammed et al. table 4. comparison of serum level of vitamin d in nanogram/dl according to age group variable deficient number (%) *mean ± sd insufficient number (%) *mean ± sd sufficient number (%) *mean ± sd p value age group (year) <20 83 (44.14%) (9.11 ± 3.32) 70 (37.23%) (21.11 ± 4.22) 35 (18.61%) (43.73 ± 17.82) 0.001 20–40 227 (46.23%) (9.12 ± 3.42) 162 (32.99%) (21.17 ± 4.19) 102 (20.77%) (48.98 ± 20.19) 41–60 121 (36.44%) (8.74 ± 3.37) 145 (43.67%) (21.71 ± 4.52) 66 (19.87%) (40.65 ± 19.51) >60 39 (30%) (8.78 ± 3.48) 49 (37.69%) (21.72 ± 4.41) 42 (32.30%) (42.60 ± 11.65) difference in serum 25(oh)d level between age subgroups (p < 0.001), indicating that the age can be considered as a risk factor of vitamin d deficiency. discussion the roles vitamin d have found in many physiological functions. it facilitates the absorption of calcium and phosphorus, which is important to prevent osteoporosis or fragility fractures development. vitamin d deficiency and insufficiency is a worldwide common health issue and nowadays is linked with many diseases therefore measuring circulating levels of 25 ohd provides evidence of a person’s deficiency/insufficiency of vitamin d.1 therefore, the purpose of this study was to evaluate the prevalence of vitamin d deficiency and insufficiency among dohuk and zakho population and further to investigate whether age and sex variations in serum 25 ohd are evident among these population in kurdistan region, iraq. in the present study, the status of vitamin d is measured in 1141 blood samples of both genders. it was observed in our population that vitamin d was deficient in 41.19% population while the 37.33% had insufficient vitamin d levels. furthermore, vitamin d deficiency was found in 43.03% of females while 35.31% of females were having insufficient vitamin d levels. this showed that females are more deficient to vitamin d than males. however, there is no statistical difference seen in vitamin d deficiency between men and women. our study is in agreement with study done by wei, et al.19 a study conducted in 2007–2010 by nhanes recorded no major gender gaps between adults in the united states,20 while another study in 1998–2004 also done by nhanes found that men had a higher 25-ohd level than women.21 in middle east countries, many studies have demonstrated the incidence of vitamin d levels. for example, in saudi arabian population, 25–37% of healthy saudi men with low vitamin d have been reported although sunny days are abundant almost year around.21 this study is more comparable to our result as 35.23% of men in our population had a low 25(oh)d level. the fact that there was no significant difference between genders due to sun avoidance behaviour is limited by lifestyle and other choices as most females in our society spend more of their time at home rather than in other places. additionally, wearing a hijab, full body covered clothes and using sunscreen, and sunglasses makes it difficult to get enough vitamin d only from diet as well as gender differences may also be caused by differences in hormone levels leading to effect on this value. therefore, long-term supplementation is possibly needed for patients with this nutritional deficiency and they should change their lifestyle behaviour. our findings reported that there was significant difference in 25(ohd) levels between the dohuk and zakho population. people are more likely to live in cities due to the rapid urbanization phase and higher socioeconomic status, and higher population density areas have contributed to reduced exposure to natural sunlight. moreover, air contamination in urban areas may influence too by acting as a boundary to uv light but this suggested pathway has not elucidated clearly.22 the factor of race, ethnicity, country of residence and skin colour could explain the differences of 25(ohd) levels among the population. in our society, these factors can be ignored as most of our population are kurdish sharing the same culture and tradition. on the other hand, many studies have shown racial differences could contribute to different 25(ohd) concentrations. population from the uk, australia, canada, and the middle east has lower 25(ohd) levels compared to white population.23,24 moreover, african american have lower levels of vitamin d than their white population due to skin pigmentation that decreases vitamin d production.25 furthermore, in this study, we found that there was also a significant difference between different age groups related to serum 25(ohd) levels (p < 0.001). maximum incidence of vitamin d deficiency was observed in younger age group between 20–40 years old (46.23%) followed by aged 40–60 years (36.44%) while in older people the incidence of vitamin d deficiency was lower than the other groups (30%) vitamin d levels decline with age,26 these studies found that production of this vitamin in the skin, calcium absorption of circulated 1,25(oh)2d, and renal production of 1,25(oh)2d decrease after the sixth decade of life.27 it could be due to supplementation of vitamin d among elderly people, especially female, who are getting used to taking vitamin d supplementation or they depend on regular and adequate amount of food rich in vitamin d and spend more time in the sun. in addition to clothing habit/lifestyle, modification among younger people partly could explain the results. younger people prefer living in apartments and have less outdoor physical activity whereas older people prefer living in houses and have had more outdoor physical activity when they were younger. we concluded that the low incidence of vitamin d in older adults might be subject to vitamin d supplementation in this group or have more dietary supplements or maybe have more varied sources of vitamin d nutrients. younger adults 333j contemp med sci | vol. 7, no. 6, november-december 2021: 330–333 l.y. mohammed et al. original prevalence of vitamin d deficiency in duhok province, iraq and females had a higher prevalence of vitamin d deficiency compared to older people and male. our study is in agreement with the study which is done by maldonado, et al.28 who demonstrated that the level of vitamin d in older adults is lower than other age groups. competing interests the authors declare that there are no competing interests. funding/support no funding or support. acknowledgments we would like to thank the staff of azadi hospital in duhok and zakho general hospital, kurdistan region, iraq for their assistance in data collection and laboratory analysis.  references 1. basit s. vitamin d in health and disease: a literature review. british journal of biomedical science. 2013;70(4):161–72. 2. pedersen ji. vitamin d requirement and setting recommendation levels current nordic view. nutr rev. 2008;66(10 suppl 2):s165–s9. 3. guessous i, bochud m, bonny o, burnier m. calcium, vitamin d and cardiovascular disease. kidney & blood pressure research. 2011;34(6): 404–17. 4. gagnon c, lu zx, magliano dj, dunstan dw, shaw je, zimmet pz, et al. serum 25-hydroxyvitamin d, calcium intake, and risk of type 2 diabetes after 5 years: results from a national, population-based prospective study (the australian diabetes, obesity and lifestyle study). diabetes care. 2011;34(5):1133–8. 5. cutolo m, plebani m, shoenfeld y, adorini l, tincani a. vitamin d endocrine system and the immune response in rheumatic diseases. vitamins & hormones. 86: elsevier; 2011. p. 327–51. 6. giordano n, goracci a, fagiolini a. depression and vitamin d deficiency: causality, assessment, and clinical practice implications. neuropsychiatry. 2017;7(5):606–14. 7. brewer l, williams d, moore a. current and future treatment options in osteoporosis. european journal of clinical pharmacology. 2011;67(4): 321–31. 8. lagishetty v, liu nq, hewison m. vitamin d metabolism and innate immunity. molecular and cellular endocrinology. 2011;347(1-2):97–105. 9. christesen ht, falkenberg t, lamont rf, jørgensen js. the impact of vitamin d on pregnancy: a systematic review. acta obstetricia et gynecologica scandinavica. 2012;91(12):1357–67. 10. ibrahim n, shivan y, amer b, djwar ali k, nawfal h. study on anticardiolipin antibodies in women with recurrent abortion in duhok province, kurdistan region, iraq. acta medica iranica. 2020;58(6). 11. a. naqid i, h. yousif s, r. hussein n. seroprevalence of rubella and herpes simplex virus in women with miscarriage and stillbirth in zakho city, kurdistan region, iraq: a crosssectional study. women’s health bulletin. 2020;7(1):18–22. 12. naqid ia, yousif sh, hussein nr. serological study of igg and igm antibodies to cytomegalovirus and toxoplasma infections in pregnant women in zakho city, kurdistan region, iraq. women’s health bulletin. 2019;6(4):8–12. 13. vanga sr, good m, howard pa, vacek jl. role of vitamin d in cardiovascular health. the american journal of cardiology. 2010;106(6):798–805. 14. bischoff-ferrari ha, dawson-hughes b, staehelin hb, orav je, stuck a, theiler r, et al. fall prevention with supplemental and active forms of vitamin d: a meta-analysis of randomised controlled trials. bmj. 2009;339:b3692. 15. joshi d, center jr, eisman ja. vitamin d deficiency in adults. 2010. 16. sayed-hassan r, abazid n, alourfi z. relationship between 25-hydroxyvitamin d concentrations, serum calcium, and parathyroid hormone in apparently healthy syrian people. archives of osteoporosis. 2014;9(1):176. 17. badawi a, arora p, sadoun e, al-thani a-a, thani mha. prevalence of vitamin d insufficiency in qatar: a systematic review. journal of public health research. 2012;1(3):229–35. 18. palacios c, gonzalez l. is vitamin d deficiency a major global public health problem? the journal of steroid biochemistry and molecular biology. 2014;144 pt a:138–45. 19. wei j, zhu a, ji js. a comparison study of vitamin d deficiency among older adults in china and the united states. scientific reports. 2019;9(1): 1–11. 20. jain r. variability in the levels of vitamin d by age, gender, and race/ ethnicity: data from national health and nutrition examination survey 2007–2010. j nutr health sci. 2016;3(2):203. 21. sadat-ali m, al elq ah, al-turki ha, al-mulhim fa, al-ali ak. influence of vitamin d levels on bone mineral density and osteoporosis. annals of saudi medicine. 2011;31(6):602–8. 22. agarwal k, mughal m, upadhyay p, berry j, mawer e, puliyel j. the impact of atmospheric pollution on vitamin d status of infants and toddlers in delhi, india. archives of disease in childhood. 2002;87(2):111–3. 23. vieth r, cole d, hawker g, trang h, rubin l. wintertime vitamin d insufficiency is common in young canadian women, and their vitamin d intake does not prevent it. european journal of clinical nutrition. 2001;55(12):1091–7. 24. brock k, wilkinson m, cook r, lee s, bermingham m. associations with vitamin d deficiency in “at risk” australians. j steroid biochem mol biol. 2004;89-90(1-5):581–8. 25. harris ss. vitamin d and african americans. the journal of nutrition. 2006;136(4):1126–9. 26. wacker m, holick mf. sunlight and vitamin d: a global perspective for health. dermato-endocrinology. 2013;5(1):51–108. 27. gallagher jc. vitamin d and aging. endocrinology and metabolism clinics. 2013;42(2):319–32. 28. maldonado g, paredes c, guerrero r, ríos c. determination of vitamin d status in a population of ecuadorian subjects. the scientific world journal. 2017;2017. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1073 254 j contemp med sci | vol. 8, no. 4, july-august 2022: 254–258 original molecular analysis of carbapenem resistant genes in pseudomonas aeruginosa isolated from baghdad hospitals ali h. salih, adnan h. aubaid*, ghada b. ali department of medical microbiology, faculty of medicine, university of al-qadisiyah, al-qadisiyah, iraq. *correspondence to: adnan h. aubaid (e-mail: adnan.uobeed@qu.edu.iq) (submitted: 28 april 2022 – revised version received: 12 may 2022 – accepted: 26 may 2022 – published online: 26 august 2022) abstract objectives: this study aimed to molecular investigation of prevalence the carbapenem-resistant genes in p. aeruginosa in isolates collected from baghdad hospitals. methods: in a cross-sectional manner, p. aeruginosa were isolated and identified from patients who attended to hospital in baghdad city during the period of december 2021 to june 2022. genotypic characterization of 16srrna gene, plasmid profile, exoa gene, carbapenem resistance gene were tested. results: diagnosis of p. aeruginosa isolates was confirmed genotypically via the amplification of 16srrna gene with 1504 bp by using pcr amplification of genes encoding ambler class b mbl (blaimp, blavim, imi, blandm, blaspm-1, and blagim), and ambler class d carbapenemase (blaoxa-23, blaoxa-24, blaoxa-40, blaoxa-48, blaoxa-50, blaoxa-51 and blaoxa-58) were performed. the carbapenem resistant isolates were also evaluated for the presence of class d carbapenemase (blaoxa 50), genes by pcr. conclusion: this study proved that p. aeruginosa isolated had carbapenem-resistant genes that strongly correlated with antibiotic resistance according to phenotypic and genotypic characterization. keywords: carbapenems, pseudomonas aeruginosa, hospitals, iraq issn 2413-0516 introduction pseudomonas aeruginosa is a ubiquitous, gram-negative bacterium and versatile opportunistic pathogen, which is considered a significant reason for an ever-widening array of various life-threatening infections.1 over the past decades, the emergence and dissemination of p. aeruginosa and enterobacteriaceae, which are resistant to carbapenems, which are the broadest spectrum agents of the β-lactam group, has become apparent as an urgent threat to public health. the finding p. aeruginosa has intrinsic resistance to numerous antimicrobial agents and also easily acquires resistance to many antibiotics, including carbapenems resistance is an ominous development that challenges this last-resort antibiotic. unfortunately, carbapenems resistant p. aeruginosa has now emerged and is disseminating worldwide.2-3 resistance to carbapenems in p. aeruginosa can be mediated by several mechanisms, including mexab-oprm, ampc, decreased outer membrane permeability, up-regulation of the efflux pumps, hyper production of a chromosomal ampc-type cephalosporinases or the production of carbapenemases oprd.4 there is scarce information available on the distribution of mbls producing p. aeruginosa isolates in baghdad hospitals. therefore, immediate determination of carbapenemases and other mechanisms creating isolates is main to avoid the spread of p. aeruginosa inside and between hospitals and to correctly treat infections caused by this. the study aimed to identify the variations in phenotypic, genotypic characteristics and antibiotic resistance profile of pseudomonas aeruginosa isolated from inpatients in baghdad hospitals. additionally, the use of carbapenems has increased markedly during the past few years in baghdad city. the current study suspected that increased use of these antibiotics could cause the selection of isolates resistance to carbapenems. materials and methods isolation and identification of p. aeruginosa a total of 2000 clinical samples were collected from inpatients of many hospitals in baghdad city during the period from december 2020 to june 2021, which included: diabetic foot (50), otitis media (550), lower respiratory tract (150), urinary tract (725), wound (200), blood and burns (250). the clinical samples were transported to the laboratory without delay. all samples were cultivated, by using the standard loop of urine and sterile swabs of other samples, on the blood agar, macconkeys agar pseudomonas chromogenic agar, and cetrimide agar as selective media. p. aeruginosa and incubated overnight at 37°c for 18–24 hours. initial diagnosis of isolates was made on the basis of gram’s staining of culture, colonial morphology on different media, hemolysis on blood agar, pigment production, odor in cultures, size, edge, and oxidase test. suspected pseudomonas colonies were further identified to species level using routine biochemical tests and selective culture media.5 in addition to these tests, the p. aeruginosa isolates were also confirmed biochemically with the vitek-2 automated system and by 16srrna as a molecular method. oligonucleotide primer sequences used for pcr amplification 1. specific primer sequences of 16srrna gene were used to confirm the identification of p. aeruginosa by pcr according to jiang et al., 20066 provided by alpha dna company (canada) and prepared according to the instructions of the supplied company, as shown in table 1. 2. oligonucleotide primer for detection of carbapenem resistance genes in p. aeruginosa. these primers were provided by macrogen company from south korea are listed in table 2. 255j contemp med sci | vol. 8, no. 4, july-august 2022:254–258 a.h. salih et al. original molecular analysis of carbapenem resistant genes in pseudomonas aeruginosa isolated from baghdad hospitals pcr protocols for detection of carbapenem resistance genes pcr protocols for detection of carbapenem resistance genes. an oligonucleotide primer was prepared depending on the manufacturer’s instruction by dissolving the lyophilized sample with nuclease-free water after rotating down briefly. a working primer tube was prepared by diluting it with nuclease-free water. the final pico-moles depended on the procedure of each primer. the pcr tubes were placed into a thermocycler and the right pcr cycling program parameters conditions were installed as in table 3. pcr was used for detecting p. aeruginosa, the mixture of 25 μl consisted of 12.5 μl of gotaq hot star master mix (which contains taq dna polymerase, dntps, mgcl2, and reaction buffer at the optimal concentration for efficient amplification of dna templates by pcr), 5 μl dna template (20 ng.), 1 μl of each forward and reverse primer (10 pmol.) 5.5 μl of nuclease-free water to complete the amplification mixture volume. the pcr tubes containing the mixture were transferred to preheated thermo cycler under sterile condition. all requests, technical and preparations of agarose gel electrophoresis that were used for the detection of pcr products were done according to sambrook and russel.8 the pcr products separated in 1.5% agarose gels (after staining with 0.5 mg/ml ethidium bromide) were visualized using a gel ultraviolet transilluminator system. the positive results were distinguished when the pcr product base pairs were equal to the base pairs of the dna ladder. results and discussion according to the results of the present study, the overall count constitutes a total of 100/2000 samples, p. aeruginosa table 1. oligonucleotide primer sequences of 16srrna gene in p. aeruginosa used for confirmatory identification product sizereferenceprimer sequences (5’→3’)name of primer bp1504(jiang et al., 2006)6aga gtt tga tcm tgg ctc ag f16srrna cgg tta cct tgt tac gac ttr table 2. oligonucleotide primer for detection of carbapenem resistance genes in p. aeruginosa primer gene name sequence (5’→3’) product size genbank reference ndm bla-ndm f cagtcgcttccaacggtttg 529 bp mf379690.1 r atcacgatcatgctggcctt imp bla-imp f ctttcaggcagccaaaccac 371 bp design to this study r tggggcgttgttcctaaaca vim-1 bla-vim-1 f tccacgcactttcatgacga 503 bp design to this study r aagtcccgctccaacgattt gim blagim f agaaccttgaccgaacgcag 909 bp design to this study r gcaccagttttcccatacag oxa-48 blaoxa-48 f ttg gtg gca tcg att atc gg 744 bp design to this study r gag cac ttc ttt tgt gat ggc oxa-40 blaoxa-40 f cacctatggtaatgctcttgc 491 bp (woodford et al., 2006)7 r gtggagtaacacccattcc oxa-50 blaoxa-50 f aatccggcgctcatccatc 869 bp (woodford et al., 2006)7 r ggtcggcgactgaggcgg spm blaspm-1 f cctacaatctaacggcgacc 650 (woodford et al., 2006)7 r tcgccgtgtccaggtataac oxa58 bla oxa58 f aagtattggggcttgtgctg 599 (woodford et al., 2006)7 r cccctctgcgctctacatac oxa23 bla oxa23 f gatcggattggagaaccaga 501 (woodford et al., 2006)7 r atttctgaccgcatttccat oxa24 bla oxa24 f ggttagttggcccccttaaa 246 (woodford et al., 2006)7 r agttgagcgaaaaggggatt oxa 51 blaoxa 51 f taatgctttgatcggccttg 353 (woodford et al., 2006)7 r tggattgcacttcatcttgg imi blaimi f cca ttc acc cat cac aac 440 (woodford et al., 2006)7 r cta ccg cat aat cat ttg c 256 j contemp med sci | vol. 8, no. 4, july-august 2022: 254–258 molecular analysis of carbapenem resistant genes in pseudomonas aeruginosa isolated from baghdad hospitals original a.h. salih et al. table 3. programs of pcr thermocycling conditions for detection of carbapenem resistance genes genes temperature (c˚)/time cycle number initial denaturation cycling condition final extension denaturation annealing extension ndm 95°c/5 min 95°c/20 sec 56°c/30 sec 72°c/40 sec 72°c/5 min 35 imp 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 vim-1 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 gim 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 spm 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 oxa-48 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 oxa-40 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 oxa-50 95°c/5 min 95°c/20 sec 58°c/30 sec 72°c/40 sec 72°c/5 min 35 oxa-51 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 oxa-23 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 oxa-24 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 spm 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 imi 95°c/5 min 95°c/20 sec 50–60°c/30 sec 72–40°c/40 sec 72°c/5 min 35 representing (5%) of all the collected samples in this study. it’s well known that p. aeruginosa considers an important nosocomial pathogen in many medical centers throughout the world and a source of infections in any part of the body. also, this bacterium is able to cause infection in healthy individuals at a low rate and creates a serious public health disaster resulting in an enormous burden of morbidity, and mortality in both developing and developed countries.9 one of the reasons for the high pathogenicity of p. aeruginosa is the intrinsic high resistance to several antibiotics, as well as the development of multiple drug resistance.10 the results of amplification of 16srrna gene with dna extracted from one hundred p. aeruginosa isolates showed positive pcr product with amplicon size 1504 base pair (figure 1). these results agreed with alornaaouti11 who used the 16srrna gene which is considered one of the important gene to confirm the identification of p. aeruginosa and other bacterial species because have hyper constant sequencing and play a basic role in molecular identification and classification, also that can provide species-specific signature sequences useful for bacterial identification all type of bacteria.12 detection of class b (mbl) and class d carbapenemase the production of carbapenemases is of the utmost concern and became the mechanism of greater relevance towards carbapenem resistance due to the growing enzyme diversity. these enzymes have high versatility, as they are characterized by a very wide hydrolytic spectrum and affect almost all β-lactams, with the exception of monobactams.13 p. aeruginosa isolates producing carbapenemases are also associated with xdr phenotype. accordingly, the detection of carbapenemases production in p. aeruginosa is important not only for the adequate selection of antibiotic therapy but also for hospital epidemiology surveillance and infection control. in recent reports in najaf, the most common carbapenemases are the mbl and oxa variant enzymes of ambler class d.14-16 class b acquired mbl is one of the most important enzymes including imp, vim, spm, ndm, sim and gim, which expose huge-level of resistance against carbapenem.17–19 in this study, the pcr technique was used to identify the bla-imp, bla-vim, bla spm, bla-ndm, bla-sim and bla-gim genes only (figure 2), while the bla-aim, bla-khm, bla-dim and bla-fim genes was not included due to very few occurrences in adjacent nations to iraq. furthermore, as the present results revealed, two isolates carry the ndm encoding gene in addition to coexisting with the oxa-50 encoding gene. the co-harboring of two carbapenemase genes in p. aeruginosa isolates has been reported in several studies worldwide.19–21 this observation led to the emergence of a new drug resistant model for p. aeruginosa. however, this result is of great concern and has shed light on the fact that ndm producing xdr p. aeruginosa is now alarmingly on the increase in baghdad hospitals. although only three carbapenem-resistant p. aeruginosa isolates were identified to carry this gene in the present study, it is of concern as blandm producers may be disseminated rapidly in baghdad hospitals and this finding implies that fig. 1 agarose gel electrophoresis for 16srrna gene amplicons. 1% agarose (100 min at 100 volt/50 mamp). lane:1 (m 100 bp ladder) lane 1-10 local isolates, pcr product 1504 bp size. 257j contemp med sci | vol. 8, no. 4, july-august 2022:254–258 a.h. salih et al. original molecular analysis of carbapenem resistant genes in pseudomonas aeruginosa isolated from baghdad hospitals several new blandm cases will be found in the near future. consequently, the detection of five ndm-positive isolates in this study suggested possibilities of spread via its high rate of genetic transfer among pathogenic bacteria in baghdad hospitals, or possibilities to human factors such as hygiene and international tourists. it is believed that the emergence of blandm carried isolates in baghdad may have been a result of the introduction of ndm isolates via increasing medical tourism of iraqi patients to the indian subcontinent. at present, the prevalence of ndm-1 has increased significantly throughout the world and has been identified mostly in asia,19 europe, africa.20 therefore, the detection of ndm harboring p. aeruginosa isolates in this study indicates the immediate importance of the establishment of surveillance to prevent nosocomial infections and dissemination of ndm in baghdad hospitals.21-24 in the study of these genes blaspm, imp, vim1, gim, imi, oxa-48, oxa-40, oxa-58, oxa-23, oxa-24, and oxa51 were negative results (figure 3). in further studies conducted in najaf, 14 showed that two (5.9%) out of 34 carbapenem-resistant p. aeruginosa isolates were spm-type positive. interestingly, the present study showed that oxa-50 was the most frequent carbapenemase identified in xdr p. aeruginosa isolates (98.%). however, the occurring class-d oxacillinase oxa-50 was shown to be expressed constitutively in p. aeruginosa (figure 4). a similar enhancement in the prevalence of blaoxa-50 carrying p. aeruginosa isolates has been noticed earlier in a recent local study reported by rasool et al.,15 who found that the majority of the carbapenems resistant p. aeruginosa isolates carried the blaoxa-50 gene (53.8%). while al-janahi14 reported that only 8.8% of the carbapenem resistant p. aeruginosa isolated from najaf hospitals were harbored blaoxa-50. ethics consideration this study is in accordance with the ethics committee of al-diwaniaya teaching hospital, iraq. a verbal agreement was fig. 2 agarose gel electrophoresis for ndm gene amplicons. (1.5% agarose,100 min at 100 volt/50 mamp). lane:1 (m 1500 bp ladder) lane 4,11 and 28 local isolates, positive pcr product 528 base pair. fig. 3 agarose gel electrophoresis for oxa-50 gene amplicons. (1.5% agarose (100 min at 100 volt/50 mamp). lane:1 (m 1500 bp ladder) lane 1,2,3,4,5,6,7,8,10,11,12,13,14,15,17,18. positive pcr product 869 base pair, and 2 local isolates (9,16) negative oxa-50 gene. fig. 4 agarose gel electrophoresis for oxa-50 gene amplicons. (1.5% agarose, 100 min at 100 volt/50 mamp). lane:1 (m 1500 bp ladder) lane 4,17,4 and 2 local isolates, positive pcr product 869 base pair. obtained from participants in the study of the relative’s pretaking samples. conflict of interest no known conflict of interest correlated with this publication. funding this research did not receive any grant from agencies in the public, commercial, or not-for-profit sectors. availability of data and materials the data used and/ or analyzed throughout this study are available from the corresponding author on reasonable request.  references 1. rashid, a., akram, m., kayode, o.t. and kayode, a.a. (2020). clinical features and epidemiological patterns of infections by multidrug resistance staphylococcus aureus and pseudomonas aeruginosa in patients with burns. biomed. j. sci. tech. res. 25(4): 19272–19278. 2. jeong, s .j.; yoon, s. s.; bae, i. k.; jeong, s. h.; kim, j. m.; lee, k. (2014). risk factors for mortality in patients with bloodstream infections caused by cabapenem-resistant pseudomonas aeruginosa: clinical impact of bacterial virulence and strain on outcome. diagn microbiol infect dis; 80:130–135. 258 j contemp med sci | vol. 8, no. 4, july-august 2022: 254–258 molecular analysis of carbapenem resistant genes in pseudomonas aeruginosa isolated from baghdad hospitals original a.h. salih et al. 3. vural, e.; delialioglu, n.; ulger, s. t.; emekdas, g. and serin, m. s. (2020). phenotypic and molecular detection of the metallo-beta-lactamases in carbapenem-resistant pseudomonas aeruginosa isolates from clinical samples. jundishapur j. microbiol. 13(2): e90034. 10.5812/jjm.90034. 4. rostami, s.; sheikh, a. f.; shoja, s.; farahani, a.; tabatabaiefar, m. a.; jolodar, a. and sheikhi, r. (2018). investigating of four main carbapenem-resistance mechanisms in high-level carbapenem resistant pseudomonas aeruginosa isolated from burn patients. j. chin. med. ass., 81: 127–132. 5. collee, j.g. fraser, a.g.; marmion, b.p. et al. (1996). mackie and mccartney practical medical microbiology. 14th ed., churchill livingstone, new york, pp. 413–423. 6. jiang, h. dong, h. zhang, g. et al. (2006). microbial diversity in water and sediment of lake chaka, an athalassohaline lake in northwestern china. appl environmen microbiol., 72(6): 3832–3845. 7. woodford n., ellington m.j., coelho j.m., turton j.f., ward m.e., brown s., amyes s.g., livermore d.m. multiplex pcr for genes encoding prevalent oxa carbapenemases in acinetobacter spp. int. j. antimicrob. agents. 2006;27:351–353. doi: 10.1016/j.ijantimicag.2006.01.004. [pubmed] [crossref ] [google scholar]. 8. sambrook, j. and russel, d. w. (2001). molecular cloning: a laboratory manual. (3rd ed). cold spring harbor, usa. pp. 5–52. 9. strich jr, warner s, lai yl, et al. (2020). needs assessment for novel gramnegative antibiotics in us hospitals: a retrospective cohort study. lancet infect dis., 20(10): 1172–1181. 10. langendonk, r.f., neill, d.r. and fothergill, j.l. (2021). the building blocks of antimicrobial resistance in pseudomonas aeruginosa: implications for current resistance-breaking therapies. front. cell. infect. microbiol., 11 (april): 1–22. 11. alornaaouti, a. (2013). study of genotyping and virulence factors of pseudomonas aeruginosa. m.sc. thesis, college of education for pure science/ibn-al-haitham, university of baghdad. 12. brooks, g.f.; carroll, k.c.; butel, s.j. et al. (2013). jawetz, melnick, and adelbergs, medical microbiology. 26th ed. mcgraw-hill. united states. 13. botelho, j., grosso, f. and peixe, l. (2018). unravelling the genome of a pseudomonas aeruginosa isolate belonging to the high-risk clone st235 reveals an integrative conjugative element housing a blages-6 carbapenemase. j. antimicrob. chemother., 73: 77–83. 14. al-janahi, h.c. (2020). occurrence and molecular characterization of metallo-β-lactamase (mbl)-producing pseudomonas aeruginosa in najaf hospitals. m.sc. thesis. faculty of medicine. university of kufa. iraq. 15. rasool, a.a., almohana, a.m., alsehlawi, z.s., abed ali, i., al-faham, m. and al-sherees, h.a. (2021). molecular detection of carbapenems resistance genes in pseudomonas aeruginosa isolated from different hospitals in najaf, iraq. international journal of information research and review (ijirr), 8 (4): 7242–7247. 16. aubaid ah.; mahdi zh.; abd-alraoof ts and jabbar nm. (2020). detection of mec a, van a and van b genes of staphylococcus aureus isolated from patients in al-muthanna province hospitals. indian journal of forensic medicine & toxicology, 14(2):1002–1008. 17. dortet l, bernabeu s, gonzalez c, naas t. evaluation of the carbapenem detection set™ for the detection and characterization of carbapenemaseproducing enterobacteriaceae. diagn microbiol infect dis. 2018;91(3): 220–225. doi: 10.1016/j.diagmicrobio.2018.02.012. [pubmed] [crossref ] [google scholar]. 18. vatansevera, c., menekseb, s., dogana, o., gucera, l.s., ozera, b., ergonula, o. and cana, f. (2020). co-existence of oxa-48 and ndm-1 in colistin resistant pseudomonas aeruginosa st235. emerg. microb. infect., 9:152–154. 19. honda, n.h., aoki, k., kamisasanuki, t., matsuda, n., to, m. and matsushima, h. (2019). isolation of three distinct carbapenemaseproducing gram negative bacteria from a vietnamese medical tourist. j. infect. chemother., 25: 811–815. 20. yoon, e.j. and jeong, s.h. (2021). mobile carbapenemase genes in pseudomonas aeruginosa. fron. microbio., 12(2):614058. 21. mutar hm. and aubaid ah. (2021). molecular profile of meca, tst-1, hla, hlb, eta, etb, erma, and ermb virulence genes in staphylococcus aureus using rapd-pcr. annals of the romanian society for cell biology, 25(4): 3227–3238. 22. al-azawi, i.h.; al-hamadani, a.h.; and hasson, s.o. (2018). association between biofilm formation and susceptibility to antibiotics in staphylococcus lentus isolated from urinary catheterized patients. nano biomed. eng. 10(2):97–103. 23. hasson, s.o.; al-awady, m. and al-hamadani, a.h., et al. (2019). boosting antimicrobial activity of imipenem in combination with silver nanoparticles towards s. fonticola and pantoea sp. nano biomed. eng. 11(2):200–214. 24. hasson,s.o.; al-hamadani, a.h., and al-azawi, i.h. (2018). occurrence of biofilm formation in serratia fonticola and pantoea sp. isolates among urinary catheterized patients. nano biomed. eng. 10(3): 295–304. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1257 82 j contemp med sci | vol. 8, no. 1, january-february 2022: 82–85 country report cancer control and oncology care in iraq nada a s al alwan* national cancer research center, university of baghdad, baghdad, iraq. *correspondence to: nada a s al alwan (e-mail: nadalwan@yahoo.com) (submitted: 13 january 2022 – revised version received: 28 january 2022 – accepted: 08 february 2022 – published online: 26 february 2022) abstract this country report focuses on the cancer control and oncology services in iraq. this report displaying the approaches that are made to implement the elements of the national cancer control program including cancer registration, prevention, early detection, treatment, palliative care and research. issn 2413-0516 introduction based on projected population models, the global burden of cancer is set to increase by more than 60% in 2040; specifically in low-and-middle-income countries.1 it has been predicted that the highest incidence of cancer within the coming years will be registered in the eastern mediterranean region (emr) for reasons attributed to population growth, aging, lifestyle modification, urbanization and exposure to carcinogens.2 iraq is one of the economies with upper middle income and had the most robust health-care system in the middle east before 1990. the impact of consecutive wars, conflicts and political instability resulted in remarkable shortage in the medical recourses and requested funds with disruption of the relevant services including cancer care.3-5 currently, the government spends 6–7% of its gdp on the health sector, nevertheless, it provides free of charge services to all citizens. there are numerous cancer care hospitals and centers distributed all over the iraqi governorates. this study focuses on the cancer control and oncology services in iraq; displaying the approaches that are made to implement the elements of the national cancer control program including cancer registration, prevention, early detection, treatment, palliative care and research. cancer statistics in 2020, the world health organization estimated that there were 19.3 million new cancer cases and 10.0 million cancer deaths.1 the iraqi ministry of health (moh) established the iraqi cancer registry (icr) since 1974 in collaboration with the international agency for research on cancer (iarc). data is documented routinely through the population based icr by well-trained staff instructed on the can reg and icd-o (international classification of diseases for oncology) coding systems. the registered information on new cancer cases and deaths are pooled from public and private hospitals and laboratories in all governorates.6 the age-standardized incidence and mortality rates of cancer among the iraqi population are 134.9 and 84.7 respectively.7 the latest icr6 revealed that the total number of new cancer cases was 35,864 in 2019 with an incidence rate reaching 92/100,000 population (table 1). breast cancer was the most common among the iraqi population (19.8%) followed by bronchus and lung cancer (7.9%), colorectal (6.5%), brain/ cns (6.4%) and leukemia (5.5%). the leading three cancers among males originated from the bronchus and lung (12.9%), followed by the urinary bladder (8.5%) and colorectal (8.2%); while the top three among females were the breast (34.1%), thyroid gland (6.9%) and brain/cns cancers (5.9%). children malignancies constituted 5.4% of the total cases of cancer at all age groups; the most prevalent was leukemia (30.2% of total childhood cancers), brain/cns (20.7%) and non-hodgkin lymphoma (7.9%). on the other hand, the total number of cancer deaths in iraq was 10,957 forming a mortality rate equivalent to 28/100,000 population (figure 1). the main causes of related deaths were due to cancers of the bronchus and lung (16%), breast (11.3%) and leukemia (8.6%). cancer control the iraqi cancer board (icb) was established by the ministry of health (moh) in 1985 to deal with the burden of cancer through coordinating the relevant activities including cancer registration, prevention, early detection, treatment, palliative care and research. globally, it has been well recognized that national cancer control plans (nccp) are crucial to address and prioritize all cancer control programs in a given country. a nccp was developed for iraq8 following the recommendations of the who cancer control strategy.9,10 risk factors among the emr the prevalence of tobacco smoking exceeded 30% among men9 whereas tobacco-related cancer deaths have reached 18.4%.11 the population based “steps survey of non-communicable disease” risk factors showed that 38.0% of men and 1.9% of women were current smokers in iraq.12 the average monthly expenditure on cigarette smoking was (34,485) iraqi dinars, whereas the cost of 100 packs of manufactured cigarettes as a percentage of per capita gdp was 2.4%.12,13 the same survey disclosed that 33.5% of iraqi adult populations were obese, 65.4% were overweight and 47% experienced insufficient physical activity.12 relatively it has been reported that 50% of men and 35% of women in the emr were overweight or obese.14 the majority of iraqi adults males (97.8%) were life time alcohol abstainers; only 0.6% were current drinkers.12,15 prevention of cancer the world cancer declaration target report of the international union against cancer (uicc) showed that iraq mailto:nadalwan@yahoo.com 83j contemp med sci | vol. 8, no. 1, january-february 2022: 82–85 nada a s al alwan country report cancer control and oncology care in iraq has made significant advances in the field of prevention through adopting tobacco control legislation;16,17 prohibition of public availability of alcohol;12,15 promoting public mobilization campaigns on tobacco control and early detection of breast cancer,18-23 physical activity, healthy diet; anthropometric screening in schools;12,15 and step wise surveillance survey on non-communicable disease risk factors.12,16 early detection and screening in order to downstage breast cancer at the time of presentation and reduce its related morbidity and mortality, a national program for early detection of breast cancer was initiated by the iraqi moh in 2001 through establishing specialized centers and clinics for early detection of breast cancer at the governorate level.18-23 the ministry of higher education and scientific research (mohesr) supported by developing a national cancer research program in 2010 focusing on breast cancer and a national cancer research center (ncrc) in 2012.24 the ncrc launched several awareness campaigns chaired by community leaders and developed an online information system database on breast cancer under the supervision of who/iarc.18,19,23-26 though not common among iraqi women, opportunistic screening of cervical cancer was carried out sporadically on a low scale. it has been reported that 9.9% of women (aged 30-49 years) in iraq had a pap smear test at least once in their life time.12,27 meanwhile, there are ongoing plans to initiate screening for colorectal cancer in iraq. cancer diagnosis pathologically, iraq initiated a process of establishing accreditation for cancer laboratory diagnosis according to international standards under supervision of iaea, who and the royal college of pathologists through adopting good laboratory practice.28 the medical city teaching hospital (mcth) and other major tertiary centers in iraq organized a plan of action to upgrade the genetic test procedures in different fields. at the imaging level, there were 152 ct scans and 90 mri machines in 2019 constituting 3.9 and 2.3/100,000 iraqi populations respectively. pet/ct is functioning in mcth and in private oncology centers in baghdad, najaf and erbil. there are five gamma cameras in baghdad and gamma knife procedure is readily practiced in erbil.29 on the other hand, excluding baghdad and erbil, there is a limited access to nuclear medicine diagnostic facilities. treatment and oncology care the main registered causes of death among the population are ischemic heart disease, followed by cancer and cerebrovascular accidents.6,29 currently the government spends 6–7% of its gdp on the health sector; providing free of charge services to all citizens. specialized care is also presented by private hospitals, the costs of which are met out-of-pocket.5,30,31 excluding kurdistan region, there are about thirty-five public cancer care facilities, i.e., hospitals, centers, and units; distributed over the iraqi governorates (ten of which are in the capital); comprising approximately 2,000 beds. the largest public tertiary hospital in baghdad, the “medical city teaching complex” (mctc), includes four specialized cancer facilities. “zhianawa cancer table 1. distribution of the top registered leading cancers in iraq in 2019 new mortality casesnew cancer cases mr**%no.ir*%no.top ten cancers / allrank 3.1611.291,23718.1719.827,109breast1 4.4815.991,7527.247.902,832bronchus & lungs2 1.77 6.31 6915.956.492,328colorectal3 2.278.11 8895.836.372,283brain/cns4 2.428.639465.055.511,977leukemia5 0.321.141254.615.021,802thyroid6 1.184.22 4624.374.771,710urinary bladder7 1.093.88 4253.774.121,477non-hodgkin lymphoma8 0.230.83913.353.661,311skin9 1.743.133436.19***3.411,224prostate10 20.6673.798,08561.4767.0724,05324,053total top ten 7.3426.212,87230.1932.9311,81111,811total others 28.0010010,95791.6610035,86435,864grand total fig. 1 incidence and mortality rates per 100,000 populations of the top ten cancers in iraq (2019). 84 j contemp med sci | vol. 8, no. 1, january-february 2022: 82–85 cancer control and oncology care in iraq country report nada a s al alwan center” and “hiwa cancer hospital” were established in kurdistan. the latter is considered the second largest provider of public oncology care following “al-amal national cancer center” in baghdad.30,31 medical and radiation oncology oncology care is provided through specialized oncology and radiotherapy hospitals. clinical oncologists are licensed to perform chemotherapy and radiotherapy. it has been recorded that out of 11,585 specialized physicians in iraq there were 128 medical or radiation oncologists.31,32 excluding kurdistan, over 120 medical and radiation oncologists are officially registered at the present time in the iraqi moh; whereas 75 postgraduate medical students are completing their board-certified studies in oncology and radiotherapy. in addition, there are about 40 oncology physicians currently running the cancer care facilities in kurdistan. obviously the total number is still lower than that requested to reach the requested coverage rate according to the international recommendations on oncology consultant staffing.33 this shortage emphasizes the urgent need to invest in qualifying human resources in all aspects of cancer care.34 the moh imports cancer drugs and medical equipment through the “state company for marketing drugs and medical appliance” and distributes throughout all governorates.35 who developed its “model lists of essential medicines” to support countries in prioritizing their reimbursable medicine.36 in the past, many of the essential cancer drugs were not available; but the situation improved when the government increased the allocated budget to moh.4,33,35 progress in radiation oncology is proceeding in iraq through establishing specialized centers and rehabilitation of the staff. currently there are twenty-one mega voltage machines in iraq; six in baghdad.29,31 a high dose rate brachytherapy is functioning in zhinawa cancer center. the directory of radiotherapy centers of iaea has registered all public radiotherapy facilities within the iraqi governorates.37 the uicc declared that iraq has improved the free access to accurate diagnosis and multimodal treatment of cancer as almost 80% of the treatment protocols are covered and the waiting lists for radiotherapy have been significantly shortened.16 palliative care palliative care in the emr is impeded due to deficient national policies, inadequate financial resources, lack of trained staff and limited access to pain relieving drugs.38,39 during the past decade pain management units have been established in most tertiary hospitals in iraq. in cases of cancer, prescription of morphine and other opioids is the responsibility of the examining oncologists who refer patients to these clinics. a fellowship program in pain management has been initiated by the mohesr in 2016 for certified iraqi board specialists. cancer research in general, numerous research studies on the burden of cancer in iraq have been published by iraqi specialists in international peer reviewed journals, and are readily available online. emphasizing the role of research as one of the basic pillars in the adoption of a national cancer control strategy, a national breast cancer research program was established by the iraqi mohesr in 2009; from which stemmed the national cancer research center (ncrc) in 2012. an online information system data base for breast cancer patients was organized in coordination with the screening unit of iarc; which was later utilized to compare the demographic characteristics, clinicopathological presentations and management outcomes of breast cancer patients in the region through developing a “regional comparative breast cancer research project”.18,19,23,25-27 recently, a memorandum of understanding has been developed between the iraqi cancer board of moh and iarc. the objectives are to conduct high-quality evidence-based research in cancer prevention and control; focusing on registration, descriptive epidemiology, capacity building and biological material transfer. education and training the iraqi board for medical specialization grants certified board in oncology for postgraduate students. in general, the officially registered educational training programs belonging to the iraqi mohesr, moh and krg graduate hundreds of oncology specialists annually; thus yielding numerous studies on cancer care. that could assist in addressing the shortage of oncology physicians. many of the teaching hospitals in iraq are recognized training centers by the arab league council in various medical specialties.40 international collaborations in addition to the educational and training opportunities on cancer control offered through who and iarc, iaea signed a country program framework (cpf) with the republic of iraq in 2017 for the years (2018 2023). the cpf focused on building capacity of the health sector, particularly on nuclear medicine and radiotherapy. the core program includes provision of requested equipment and sponsoring training programs on quality assurance. the project involves collaboration with iaea/pact (program of action for cancer therapy) and who to implement the iraqi nccp. the capacity building program executed its first activities in 2017 through training iraqi pathology leaders in uk under supervision of the royal colleges.28 currently, who is assisting moh in the institutionalization of a national health account to support in the development of health care policy financing systems and social insurance through compiling relevant data on the country health expenditures.41 in 2020, who planned for a mission on cancer control in collaboration with iea/pact and iarc, to aid in implementing the nccp. acknowledgment the author would like to thank the iraqi cancer board, moh iraq and moh, krg for the assistance in providing the relevant information. conflict of interest the author declares no conflict of interest. funding none.  85j contemp med sci | vol. 8, no. 1, january-february 2022: 82–85 nada a s al alwan country report cancer control and oncology care in iraq references 1. sung h, ferley j, siegel rl et al. global cancer statistics 2020 (2021). globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin, 71:209–249. 2. pourghazian n, sankaranarayanan r, alhomoud s, slama s, (2019). strengthening the early detection of common cancers in the eastern mediterranean region. east mediterranean health journal, 25(11):767–768. 3. united nations, world bank, joint iraq needs assessment (2003). available at: http://siteresources.worldbank.org/irffi/resources/ joint+needs+assessment.pdf. accessed 26 dec 2021. 4. dfat country information report iraq, department of foreign affairs and trade, australian government, 2020. 5. country policy and information note. iraq: medical and healthcare issues, version 1.0, home office, uk, 2019. 6. annual report, iraqi cancer registry 2019. iraqi cancer board, ministry of health and environment, republic of iraq, 2021. 7. global cancer observatory: cancer today. globocan 2020. international agency for research in cancer, france. https://gco.iarc.fr/today/data/ factsheets/populations/368-iraq-fact-sheets.pdf. accessed 26 dec 2021. 8. iraqi national cancer control program five year plan (20102014), 2nd ed. iraqi cancer board, ministry of health, baghdad, iraq, 2010. 9. world health organization. strategy for cancer prevention and control in the eastern mediterranean region 2009—2013; who, office for the eastern mediterranean, 2010. 10. romero y, trapani d, johnson s, et al., (2018). national cancer control plans: a global analysis. the lancet oncology, 19 (10), e546-e555. https://doi. org/10.1016/s1470-2045(18)30681-8. accessed 22 oct 2021. 11. iraq country profile, who office, baghdad, iraq, 2017. https://www.who.int/ gho/countries/irq/country_profiles. accessed 24 dec 2021. 12. who steps survey, chronic disease risk factor surveillance, iraq 2015 / who regional office for the eastern mediterranean, 2015. 13. gyts global youth tobacco survey, fact sheet, iraq 2015 / who regional office for the eastern mediterranean and center for disease control and prevention, 2015. 14. amin j, siddiqui aa, al-oraibi s, et al., (2020). the potential and practice of telemedicine to empower patient-centered healthcare in saudi arabia. international medical journal, 27(2):151–154. 15. eastern mediterranean region: framework for health information systems and core indicators for monitoring health situation and health system performance 2018 / who regional office for the eastern mediterranean. 16. world cancer declaration progress report 2016, international union against cancer (uicc) 2017. 17. tobacco control laws, legislations by country, iraq, campaign for tobacco-free kids, 2020. https://www.tobaccocontrollaws.org/legislation/ country/iraq/summary. accessed 25 dec 2021. 18. alwan nas, kerr d, (2018). cancer control in war-torn iraq. the lancet oncology, 19(3):291–292. 19. alwan n, (2015). establishing guidelines for early detection of breast cancer in iraq. international journal of advanced research, 3(12):539-555. 20. alwan nas, tawfeeq fn, mallah n. demographic and clinical profiles of female patients diagnosed with breast cancer in iraq. journal of contemporary medical sciences, 2019;5(1):14–19. 21. alwan n, al-attar w, eliessa r, et al., (2012). knowledge, attitude and practice regarding breast cancer and breast self-examination among a sample of the educated population in iraq. eastern mediterranean health journal, who, eastern mediterranean regional office, 18(4):337–345. 22. von karsa l, qiao y, ramadas k, et al., (2014). prevention/screening implementation, in stewart bw and wild cp (eds): world cancer report 2014. who, international agency for research on cancer, lyon, france, 2014. 23. alwan n, (2014). iraqi initiative of a regional comparative breast cancer research project in the middle east. journal of cancer biology and research, 2(1):1016–1020. 24. the national cancer research center, university of baghdad, mohesr, iraq. http://www.bccru.baghdaduniv.edi.iq. accessed 27 dec 2021. 25. alwan nas (2016). breast cancer among iraqi women: preliminary findings from a regional comparative breast cancer research project. journal of global oncology, asco, 2(5):255–258. 26. alwan nas, kerr d, al-okati d, et al., (2018). comparative study on the clinicopathological profiles of breast cancer among iraqi and british patients. the open public health journal, 11:3-17. 27. alwan nas, al-attar wm, al mallah n, abdulla k, (2017). assessing the knowledge, attitude and practices towards cervical cancer screening among a sample of iraqi female population. iraqi journal of biotechnology, 16(2):38–47. 28. the royal college of pathologists (rcpath): international pathology day: a look back at the year with the international team, 2018, uk. https://www. rcpath.org/discover-pathology/news/international-pathology-day-a-lookback-the-year-with-the-international-team.html 29. annual statistical report 2019. planning directorate, ministry of health / environment, republic of iraq, 2019. https://moh.gov.iq/upload/upfile/ ar/1070.pdf. accessed 25 dec 2021. 30. skelton m, mula-hussain ly, namiq kf, (2017). oncology in iraq’s kurdish region: navigating cancer, war, and displacement. journal of global oncology, 4:1–4. 31. annual statistical report 2017. planning directorate, ministry of health / environment, republic of iraq, 2018. 32. mula-hussain l, alabedi h, al-alloosh f, alharganee a, (2019). cancer in war-torn countries: iraq as an example, in laher (ed.), handbook of healthcare in the arab world, springer, nature, cham, 1-14. https://doi.org/10.1007/978-3-319-74365-3_152-1. accessed 25 dec 2020. 33. the royal college of radiologists. scotland clinical oncology workforce census 2018 summery report. london: the royal college of radiologists 2019. https://www.rcr.ac.uk/system/files/publication/field_publication_ files/clinical-radiology-uk-workforce-census-report-2018.pdf . accessed 25 oct 2020. 34. mula-hussain l, al-ghazi m, (2020). cancer care in times of war: radiation oncology in iraq. int j radiat oncol biol phys, 108(3):523–529. 35. the state co. for marketing drugs and medical appliances, kimadia, ministry of health/environment, republic of iraq 2020. http://demo. kimadia.iq/en/article/view/8. accessed 24 dec 2021. 36. robertson h, barr r, shulman ln, forte gb, magrini n, (2016). essential medicines for cancer: who recommendations and national priorities. bulletin of the world health organization, 94:735–742. http://dx.doi. org/10.2471/blt.15.163998. 37. dirac (directory of radiotherapy centres), country: iraq. international atomic energy agency (iaea), vienna, austria 2020. available at: https://dirac.iaea.org/data/operator?country=irq. accessed 27 dec 2020. 38. lyons g, sankaranarayanan r, millar ab, slama s, (2020). scaling up cancer care in the who eastern mediterranean region. eastern mediterranean health journal, 4(1):104–110. https://doi.org/10.26719/2018.24.1.104. accessed 26 dec 2020. 39. fadhil i, lyons g, payne s, (2017). barriers to, and opportunities for, palliative care development in the eastern mediterranean region. the lancet oncology, 18(3):e176–e184. 40. mula-hussain l, shamsaldin an, al-ghazi m, et al., (2019). board-certified specialty training program in radiation oncology in a wartorn country: challenges, solutions and outcomes. clinical and translational radiation oncology, 19:46–51. 41. world health organization, regional office for the eastern mediterranean, iraq | programme areas | primary health care. http://www.emro.who.int/irq/programmes/primary-health-care.htm. accessed 27 dec 2021. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1154 http://siteresources.worldbank.org/irffi/resources/joint+needs+assessment.pdf http://siteresources.worldbank.org/irffi/resources/joint+needs+assessment.pdf https://gco.iarc.fr/today/data/factsheets/populations/368-iraq-fact-sheets.pdf.%20%20%20accessed%2026%20dec%202021 https://gco.iarc.fr/today/data/factsheets/populations/368-iraq-fact-sheets.pdf.%20%20%20accessed%2026%20dec%202021 https://doi.org/10.1016/s1470-2045(18)30681-8 https://doi.org/10.1016/s1470-2045(18)30681-8 https://www.who.int/gho/countries/irq/country_profiles.%20%20accessed%2024%20dec%202021 https://www.who.int/gho/countries/irq/country_profiles.%20%20accessed%2024%20dec%202021 https://www.tobaccocontrollaws.org/legislation/country/iraq/summary https://www.tobaccocontrollaws.org/legislation/country/iraq/summary http://www.bccru.baghdaduniv.edi.iq https://www.rcpath.org/discover-pathology/news/international-pathology-day-a-look-back-the-year-with-the-international-team.html https://www.rcpath.org/discover-pathology/news/international-pathology-day-a-look-back-the-year-with-the-international-team.html https://www.rcpath.org/discover-pathology/news/international-pathology-day-a-look-back-the-year-with-the-international-team.html https://moh.gov.iq/upload/upfile/ar/1070.pdf. https://moh.gov.iq/upload/upfile/ar/1070.pdf. https://doi.org/10.1007/978-3-319-74365-3_152-1 https://www.rcr.ac.uk/system/files/publication/field_publication_files/clinical-radiology-uk-workforce-census-report-2018.pdf https://www.rcr.ac.uk/system/files/publication/field_publication_files/clinical-radiology-uk-workforce-census-report-2018.pdf http://demo.kimadia.iq/en/article/view/8 http://demo.kimadia.iq/en/article/view/8 http://dx.doi.org/10.2471/blt.15.163998 http://dx.doi.org/10.2471/blt.15.163998 https://dirac.iaea.org/data/operator?country=irq https://doi.org/10.26719/2018.24.1.104 http://www.emro.who.int/countries/irq/index.html http://www.emro.who.int/irq/programmes/ http://www.emro.who.int/irq/programmes/primary-health-care.htm 189j contemp med sci | vol. 8, no. 3, may-june 2022: 189–193 original serum levels of homocysteine, troponin-i, and high sensitive c-reactive protein in iraqi covid-19 patients shahad sabah khalid*, zhahraa mohamed ali, mohanad faris raheem department of laboratory sciences, college of pharmacy, university of baghdad, baghdad, iraq. *correspondence to: shahad sabah khalid (e-mail: shahad_almukhtar@ymail.com) (submitted: 04 march 2022 – revised version received: 16 march 2022 – accepted: 25 april 2022 – published online: 26 june 2022) abstract objective: this study aimed to investigate if the homocysteine has recently been proposed as a serious predictive biomarker for covid-19 infection severity. methods: in this case-control study, which involved 90 participants, 5 ml of venous blood specimen was reserved for each participant, to measure homocysteine, troponin-i, and high sensitive c-reactive protein in their blood, to find if there was an association between these markers levels and covid-19 infection by using stata version 23. results: the current study found a significant increase in measured values of homocysteine in patients’ serum than controls p-value = 0.004 which is < alpha (0.05) with an area under the curve of 0.678, also found a significant increase in measured values of cardiac troponin-i; and hs-crp in covid-19 patients than controls, p-values were 0.02 and 0.00 respectively which are < alpha (0.05) with an area under the curve of 0.686 and 0.739 respectively. conclusion: homocysteine has been noted as a strong predictive biomarker for covid-19 infection severity in many articles, the current study showed that homocysteine had a moderate predictive biomarker for covid-19 infection. cardiac troponin-i showed a moderate predictive biomarker for covid-19 infection, while hs-crp was noted as a good predictive biomarker. many studies reported an association between high levels of hs-crp and mortality rate in covid-19 patients. keywords: covid-19, homocysteine, troponin i, c-reactive protein issn 2413-0516 introduction a novel form of coronavirus, “severe acute respiratory syndrome coronavirus 2” (sars-cov-2) was described in wuhan in 2019, is an rna airborne virus, that causes varied comorbidities and mortalities.1 covid-19 is occurring in two phases; first is viral replication then inflammatory response. the flare-up of coronaviruses begins to spread quickly through the world, which induces the world health organization (who) to state the disease “a worldwide health threat”. the exponential elevation in infected cases worldwide may be because of the lack of pre-virus immunity.2 homocysteine (hcy) is a non-essential α-amino acid, that does not take part in the protein synthesis.3 homocysteine has recently been suggested as a serious predictive biomarker for covid-19 infection severity, different forms of hcy are existing in the blood circulation these include: bound to plasma proteins (70–80%), joined with other hcy molecules to form a disulfide (20–30%), while free thiol represents the smallest part (~1%).4 sars-cov-2 passes on methyl group for viral rna supply from the host cell s-adenosylmethionine (sam), which will be transformed into s-adenosylhomocysteine (sah). homocysteine is formed as an intermediate product when sah hydrolase (sahh) removes adenosine from sah, homocysteine reprocessed by the remethylation and trans-sulphuration pathway in the human body.5 sars-cov2 entry into cells is through its spike proteins that attach to the ace2 cellular receptors to form a tunnel through which the virus enters the cell.6 homocysteine attaches also to the ace2 enzyme, in addition to the attachment to various ion-channel cellular receptors, and then enters cells.7,8 vasculitic damage is not only pertinent in the lung, where it leads to edema and acute respiratory distress syndrome, but also has a considerable turn in the cardiovascular diseases (ischemia, deep venous thrombosis, pulmonary thromboembolism) and cerebral injuries (ischemia, hemorrhage); its severity is regrettably not easily expected through currently used laboratory biomarkers such as d-dimer or prothrombin time/activated partial thromboplastin time (pt/aptt).9,10 the most widely used biomarker in the diagnosis of acute myocardial injury (ami) is troponin.11 troponin is constitutive of cardiac and skeletal muscles. the troponin complex is formed of three subunits; troponin t (tnt) which is considered as tropomyosin-binding subunit, and troponin i (tni) which is considered an inhibitory subunit, and troponin c (tnc) which is the calcium-binding part. troponin monitors the interaction that is mediated between actin and myosin by the effect of calcium which results in the contraction and relaxation of striated muscles.12,13 troponin i (tni) is the inhibitory subunit responsible for inhibiting the atpase activity of actinomyosin, it is a polar protein with a surplus of positively charged remnants. the expression of tni is based on the level of ontogenesis; both cardiac and slow skeletal types are available in the heart of the human fetus. after birth, the number of the slow skeletal type is diminished and the number of ctni is increased. it has been recorded that baby in age of nine months has ctni exclusively with no slow skeletal type.14 troponin is present in the blood circulation 4 to 6 hours after ami,15 reaches a peak after nearly 18 to 24 hours, and may stay increased for up to 14 days estimation of ctn-i by automated assay is recently one of the most sensitive and specific methods for diagnosing ami.16,17 the presently most favorable laboratory marker for cardiovascular risk is the estimation of crp at low levels [high sensitivity crp (hs-crp)], crp comprises five identical symmetrically organized 23-kda promoters, which are folded into mailto:shahad_almukhtar@ymail.com 190 j contemp med sci | vol. 8, no. 3, may-june 2022: 189–193 serum levels of homocysteine, troponin-i, and high sensitive c-reactive protein in iraqi covid-19 patients original s.s. khalid et al. two antiparallel -sheets, similar to the structure of lectins, and is generally raised in human plasma after bacterial infections, cancer, or after surgical procedures.18 this study aimed to measure homocysteine level, troponin-i, and high sensitive c-reactive protein in serum of covid-19 infected patients participating in this study. methods in a case-control study, which involved 90 participants, 45 were hospitalized patients diagnosed clinically with covd19, and with a positive result of nucleic acid amplification testing by real-time reverse transcription-polymerase chain reaction (rt-pcr) of respiratory samples which were nasal/ oropharyngeal swabs for covid-19 infection and 45 of them were healthy as control. the study was carried out in two centers for covid-19 in baghdad, iraq, these centers were: al-ataa, and dhare alphaeadh hospitals, during the period from october/2021 to february/2022. all covid-19 patients were treated with the same protocol, all blood samples were taken from the participants in the same conditions, all patients were on continuous positive airway pressure therapy (cpap), participants were interviewed by the researcher, and sociodemographic data including; name, age, gender, and history of any past disease and medication taken from them. inclusion criteria; adult (30–60) years old, patients with covid-19 infection with positive pcr, have no chronic disease. exclusion criteria; patients with cardiovascular disease, diabetes, and hypertension, autoimmune disease, pregnant and lactating women, patients with a history of asthma, smoking, malignancy, patients on steroid or immunosuppressive drugs as long term therapy previous to covid-19 infection. 5 ml of venous blood specimen was reserved for each participant and collected in a gel tube and left for an appropriate time at room temperature to allow them to clot then centrifuged for 10–15 minutes at 4400 rounds per minute (rpm) to get the serum that was used in the analysis for studied homocysteine, troponin-i, and hs-crp levels. statistical analysis: statistical analysis was conducted using the statistical package for social sciences (spss) version 23 software for windows. descriptive statistics are presented as median, interquartile range, and mean rank t-testmann whitney was used to measure the difference between the means of non-normally distributed variables. the receiver operating characteristic curve (roc) for measuring the area under the curve was used also. results in the present study, there was a significant increase in serum levels of homocysteine in covid-19 patients’ serum than controls (p-value = 0.004) which is < alpha (0.05), where the median (iqr) for covid-19 patients was 11.0 (30.735) while for control was 6.800 (4.295) a shown in table 1 and figure 1. also there was a significant increase in measured values of cardiac troponin-i, with p-value = 0.02 < 0.05, where covid-19 patients’ median (iqr) was 0.00646 (0.0056) while median (iqr) for control was 0.004135 (0.0041). the p-value for hs-crp was 0.00 < 0.05 which refers to a significant increase in hs-crp level in covid-19 patients than in control, where the median (iqr) for covid-19 patients was 52.955 (36.81) while for controls was 1.9810 (51.32) as shown in table 1, figures 2 and 3. homocysteine showed an area under the curve (auc) of 0.678 which was moderate strength a predictive value for covid-19 severity. the optimal cut-off value for hcy as a predictive value of covid-19 infection severity was estimated to be 8.10 (nmol/ml); sensitivity and specificity were 66.7% and 68.9%, respectively (as shown in table 2 and figure 4). cardiac troponin-i had an auc of 0.686, the optimal cut-off value was 0.004635 (ng/ml) with sensitivity and specificity of 75.5% and 60% respectively, while the auc for hs-crp was 0.739 making it a good predictive marker of covid-19 table 1. assessment of homocysteine, troponin-i, and hs-crp levels in studied groups parameter group median interquartile range mean rank p-value homocysteine nmol/ml patients 11.00 30.735 53.52 *0.004 controls 6.800 4.295 37.48 cardiac troponin-i ng/ml patients 0.00646 0.0056 53.89 *0.002 controls 0.004135 0.0041 37.11 hs-crp µg/ml patients 52.955 36.81 56.23 *0.00 controls 1.9810 51.32 34.77 *p < alpha (0.05) significant; r, spearman correlation coefficient. fig. 1 homocysteine levels in studied groups. 191j contemp med sci | vol. 8, no. 3, may-june 2022: 189–193 s.s. khalid et al. original serum levels of homocysteine, troponin-i, and high sensitive c-reactive protein in iraqi covid-19 patients fig. 2 troponin-i levels in studied groups. fig. 3 hs-crp levels in studied groups. fig. 4 receiver operating characteristic curve for measuring the area under the curve of homocysteine in patients with covid-19 infection. table 2. receiver operating characteristic curve for measuring the area under the curve of homocysteine, cardiac troponin-i, and hs-crp levels in patients with covid-19 infection variable auc 95% ci of auc p-value optimal cut-off sn sp homocysteine nmol/ ml 0.678 0.565–0.792 *0.004 8.100 0.667 0.689 cardiac troponin i ng/ml 0.686 0.575–0.798 *0.002 0.004635 0.756 0.600 hs-crp µg/ml 0.739 0.634–0.843 *0.000 8.7220 0.778 0.689 where: *p< alpha (0.05) significant; auc, the area under the curve; ci, is confidence interval; sn, sensitivity; sp, specificity (from the curve x-axis is 1-specificity). severity, the optimal cut-off value was 8.7220 (µg/ml) with sensitivity and specificity of 77.8% and 68.9% respectively figures 5 and 6. discussion homocysteine has been suggested as a crucial biomarker for cardiovascular complications in patients admitted to hospital with covid-19 infection.19 analysis by receiver operating characteristic (roc) for estimating the sensitivity and specificity of the cut-off at which fig. 5 receiver operating characteristic curve for measuring the area under the curve of cardiac troponin-i in patients with covid-19 infection. 192 j contemp med sci | vol. 8, no. 3, may-june 2022: 189–193 serum levels of homocysteine, troponin-i, and high sensitive c-reactive protein in iraqi covid-19 patients original s.s. khalid et al. fig. 6 receiver operating characteristic curve for measuring the area under the curve of hs-crp in patients with covid-19 infection. patients are considered infected with severe covid-19 disease is equal to or higher than the specified cut-off value, so the current study showed that homocysteine had an optimal cut-off value as a predictive value of covid-19 severe infection estimated to be (8.10) mol/ml sensitivity and specificity were 66.7% and 68.9%, respectively, and as a moderate predictive biomarker for covid-19 infection (auc of 0.678). ponti g et al. in a multicenter, retrospective analysis, ponti g et al. including patients hospitalized for covid-19 from april to september 2020, the study suggest that homocysteine level equal to or more than 16 μmol/l was the optimal cut-off value for hcy as predictive of in-hospital mortality with sensitivity and specificity of 41 and 83%, respectively; with auc is 0.55.20 many articles suggested hcy as a strong predictive biomarker for covid-19 infection severity.21 in a group of 273 chinese patients that were hospitalized with covid-19 disease and had mild symptoms, over 40 parameters were measured at admission. disease progression was recorded for 72 patients, hcy serum levels and monocyte-to-lymphocyte ratio (mlr) were the solely significant predictors in hyperhomocysteinemic patients (>15.4 µmol/l), evaluated to correspond with a three-fold elevated risk of disease evolution at radiological images. the most important thing is that hcy is the solely predictive marker specified which can be easily modifiable.22 c-reactive protein (crp), is an acute-phase protein23 synthesized in the liver as a response to interleukin-6 (il-6) and is a broadly available biomarker of inflammation.24 several recent studies have reported an association between higher crp concentrations and greater disease severity in covid-19 disease.25 in the current study, hs-crp was recognized as a good predictive biomarker with an auc of 0.739. many studies reported an association between high levels of hs-crp and mortality rate in covid-19 patients. a study done among 375 patients with confirmed sars-cov-2 infection detected that increased hs-crp levels were notably correlated with high mortality risk.26 juan li in a study aimed to explore the influencing factors on critical covid-19 disease estimated the hs-crp level median (iqr) for survival patients to be 18.4 (59.90) (mg/l) while for non-survivor was 113.30 (93.20) (mg/l) with p-value < 0.001 and auc (95% ci) was 0.879 (0.815–0.944).27 cardiac troponin-i shows a moderate predictive biomarker for covid-19 infection with an auc of 0.686, shi and colleagues explained in a huge sequential patient cohort with covid-19 that myocardial injury, identified at admission, was correlated with a higher risk of in-hospital mortality. for 416 hospitalized patients with covid-19 in china, 82 had an initial ctn-i higher than the upper reference range, proposing approximately 20% of the cohort had evidence of myocardial injury. those with elevated ctn-i, compared to those without, progress with a more severe disease on multiple measures. those with elevated ctn-i had many more cardiac comorbidities and showed a higher risk of death, both during the time from symptom onset (hazard ratio, 4.26 [95% ci, 1.92–9.49]) and admission to the endpoint (hazard ratio, 3.41 [95% ci, 1.62–7.16]).28 juan li (2021) in a study aimed to explore the influencing factors of critical coronavirus disease 2019 (covid-19) estimated the ctn-i level median (iqr) for survival patients to be 2.55 (4.73) (mg/l) while for non-survivor was 40.75 (652.83) (mg/l) with p-value < 0.001.27 conclusion homocysteine may be a beneficial biomarker that can assess clinicians to recognize patients who are at higher risk for severe covid-19 infection. hcy levels in plasma can be determined easily by a simple and affordable laboratory test. good and integral food and supplements of vitamin b especially b12, and folic acid could have protective clinical effects for patients with infectious disease, and also clinical management of covid-19 infection can be improved by early determination of many biomarkers to control therapeutic intervention efficacy and/or foretell the clinical course of the covid-19 disease not only for those diagnosed with covid-19 disease but also for subjects suspected and waiting for confirmation. limitation the current study is limited by the inclusion of hospitalized patients with covid-19 infection who had no previous chronic disease. the results cannot, therefore, be generalized to all covid-19 patients; also the number of cases is limited and so gave limited results. several exclusion criteria were used in the study to decrease the confusing effect; the baseline markers levels before being infected with covid-19 disease were unavailable, which made the accurate development of changes in the studied markers uncertain. abbreviations sars-cov-2, severe acute respiratory syndrome coronavirus 2; hs-crp, high sensitive c-reactive protein; who, world health organization; ace2, angiotensin-converting enzyme 2; hcy, homocysteine; auc, area under the curve. 193j contemp med sci | vol. 8, no. 3, may-june 2022: 189–193 s.s. khalid et al. original serum levels of homocysteine, troponin-i, and high sensitive c-reactive protein in iraqi covid-19 patients 14. farah cs, reinach fc. the troponin complex and regulation of muscle contraction. faseb j [internet]. 1995 jun 1;9(9):755–67. available from: https://doi.org/10.1096/fasebj.9.9.7601340. 15. sasse s, brand nj, kyprianou p, dhoot gk, wade r, arai m, et al. troponin i gene expression during human cardiac development and in end-stage heart failure. circ res. 1993 may;72(5):932–8. 16. christensen h, johannesen hh, christensen af, bendtzen k, boysen g. serum cardiac troponin i in acute stroke is related to serum cortisol and tnf-α. cerebrovasc dis. 2004;18(3):194–9. 17. ishii j, cui w, kitagawa f, kuno t, nakamura y, naruse h, et al. prognostic value of the combination of cardiac troponin t and b-type natriuretic peptide after initiation of treatment in patients with chronic heart failure. clin chem. 2003;49(12):2020–6. 18. thompson d, pepys mb, wood sp. the physiological structure of human c-reactive protein and its complex with phosphocholine. structure. 1999 feb;7(2):169–77. 19. ponti g, manfredini m, oliva g, ozben t, fontana c, tomasi a. predicting covid-19 hospitalized patients’ outcome with homocysteine. j clin cardiol. 2021;2(1). 20. ponti g, roli l, oliva g, manfredini m, trenti t, kaleci s, et al. homocysteine (hcy) assessment to predict outcomes of hospitalized covid-19 patients: a multicenter study on 313 covid-19 patients. clin chem lab med. 2021;59(9): e354–7. 21. ibrahimagić o, smajlović d, dostović z, vidović m, tupković e, kunić s. comment on an article: “homocysteine as a potential predictor of cardiovascular risk in patients with covid-19“. med hypotheses. 2020;143. 22. yang z, shi j, he z, lü y, xu q, ye c, et al. predictors for imaging progression on chest ct from coronavirus disease 2019 (covid-19) patients. aging (albany ny). 2020;12(7):6037. 23. tillett ws, francis jr t. serological reactions in pneumonia with a nonprotein somatic fraction of pneumococcus. j exp med. 1930;52(4):561. 24. morley jj, kushner i. serum c‐reactive protein levels in disease. ann n y acad sci. 1982;389(1):406–18. 25. potempa la, rajab im, hart pc, bordon j, fernandez-botran r. insights into the use of c-reactive protein as a diagnostic index of disease severity in covid-19 infections. am j trop med hyg. 2020;103(2):561. 26. yan l, zhang h, goncalves j. an interpretable mortality prediction model for covid-19 patients. nat. mach. intell. 2, 283–288 (2020). 27. li j, wang l, liu c, wang z, lin y, dong x, et al. exploration of prognostic factors for critical covid-19 patients using a nomogram model. sci rep. 2021;11(1):1–6. 28. shi s, qin m, shen b, cai y, liu t, yang f, et al. hospitalized patients with covid-19 in wuhan, china. jama cardiol 2020;5:e200950. jama cardiol. 2020;5(7):802–10. references 1. bchetnia m, girard c, duchaine c, laprise c. the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2): a review of the current global status. journal of infection and public health. 2020 nov 1;13(11):1601–10. 2. sisó-almirall a, kostov b, mas-heredia m, vilanova-rotllan s, sequeiraaymar e, sans-corrales m, et al. prognostic factors in spanish covid-19 patients: a case series from barcelona. plos one. 2020;15(8):e0237960. 3. mccully ks. homocysteine and the pathogenesis of atherosclerosis. expert rev clin pharmacol. 2015 mar;8(2):211–9. 4. hankey gj, eikelboom jw. homocysteine and vascular disease. lancet (london, england). 1999 jul;354(9176):407–13. 5. romagnoli s, peris a, de gaudio ar, geppetti p. sars-cov-2 and covid-19: from the bench to the bedside. physiol rev [internet]. 2020 jun 4;100(4):1455–66. available from: https://doi.org/10.1152/ physrev.00020.2020. 6. hammons a, summers c, woodside j, mcnulty h, strain jj, young i, et al. folate/homocysteine phenotypes and mthfr 677c>t genotypes are associated with serum levels of monocyte chemoattractant protein-1. clin immunol. 2009 aug 1;133:132–7. 7. chen c-h, beard rs, bearden se. homocysteine impairs endothelial wound healing by activating metabotropic glutamate receptor 5. microcirculation [internet]. 2012 may;19(4):285–95. available from: https://pubmed.ncbi. nlm.nih.gov/22221504. 8. naser nh, alibeg aaa. exacerbation of covid 19 in hypertensive patients? a review? iraqi j pharm sci (p-issn 1683-3597, e-issn 2521-3512). 2021;30(2):23–30. 9. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet [internet]. 2020 feb 15;395(10223):497–506. available from: https://doi.org/10.1016/ s0140-6736(20)30183-5. 10. chen n, zhou m, dong x, qu j, gong f, han y, et al. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. lancet [internet]. 2020 feb 15;395(10223):507–13. available from: https://doi.org/10.1016/s01406736(20)30211-7. 11. alqubbanchi fb, al-hamadani fy. a pharmacoeconomics study for anticoagulants used for hospitalized covid-19 patients in al-najaf alashraf city–iraq (conference paper). iraqi j pharm sci (p-issn 1683-3597, e-issn 2521-3512). 2021;30(suppl.):48–59. 12. ahmad m. biomarkers in acute myocardial infarction. j clin exp cardiol. 2012 nov 10;3. 13. ruseva a. laboratory diagnosis of acute myocardial infarction. trakia j sci. 2005;3(1):8–14. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.1193 conflicts of interest none.  170 j contemp med sci | vol. 3, no. 9, winter 2017: 170–177 research academic performance of final year medical students at kerbala medical college mousa al alak,a akram f al hakeem,b ali al mousawic adepartment of gynecology and obstetrics, college of medicine, university of kerbala, iraq. bdepartment of surgery, college of medicine, university of kerbala, iraq. cdepartment of family and community medicine, college of medicine, university of kerbala, iraq. correspondence to mousa al alak (email: dr.mousaobgyn@gmail.com). (submitted: 25 october 2016 – revised version received: 27 november 2016 – accepted: 29 november 2016 – published online: 26 march 2017) objective assessment of final year students’ examination results in kerbala medical college in the academic years 2014–2015 and 2015–2016. methods students’ achievements in different parts of the final year examination in kerbala medical college for the academic years 2014–2015 and 2015–2016. students’ scores in different parts of the theoretical and clinical examinations were analyzed to determine the effectiveness of the assessment process. in addition, students’ cumulative scores for the preceding 5 years were explored to determine the association with final year scores. the data were analyzed using the (spss) version 20 through different descriptive and analytic statistical tools using: percentages, means, standard deviations, internal reliability, confidence intervals, factor analysis, t-test, anova test and correlation. results the results of mean score (scored out of 10 for all scores) and standard deviation theoretical exam in medicine, surgery, gynecology and obstetrics and pediatrics were: 4.55 ± 1.14, 5.62 ± 1.12, 7.15 ± 0.78, 6.31 ± 1.14, respectively and no gender difference was observed. while the comparable results in 2016 were: 5.61 ± 0.92, 5.28 ± 1.08, 7.13 ± 0.92, 7.79 ± 1.22. performance, objective structured clinical examination (osce) total score were (in the same order) for 2015: 6.30 ± 0.83, 6.66 ± 0.88, 7.02 ± 0.91, 7.81 ± 0.99. on comparison of 2015 and 2016 results, minor significant differences were found. a significant positive association was found between graduation score and previous year’s scores. minor significant gender differences were observed in only few assessed parameters. conclusion the present study aimed at a correct in depth analysis of the evaluation process and an examination of kerbala medical college graduates in two successive years. the results found were very helpful in pointing out the main shortcomings and strength in the examination stations. keywords evaluation, education, medical students, clinical, examination introduction when people hear the term “academic performance”, they often think of a person’s achievement in getting a bachelor degree. however, several factors indicate a student’s academic success. while some may not graduate top of their class, they may hold leadership positions in several student groups or score high on standardized tests where people often consider grades first in evaluating academic achievement. this includes medical schools, which rank students by their scores, awarding special designations such as the joint educational premises for those who graduate first ten in their class.1 scholarship organizations and universities also start by looking at grades, as do some employers, especially when hiring recent graduates. grades carry more weight in some industries, especially technical professions such as law, medicine and finance. other industries place less importance on gpa, particularly creative professions such as writing or art and occupations such as sales where people skills are more crucial than technical knowledge. the traditional approach in medical learning was based on the bucket theory: if medical students are filled with the required foundational knowledge, they are expected to be able to strategically retrieve and direct just the right subsets of that knowledge toward problems faced in clinical practice.2 in addition, each student should develop basic clinical skills during undergraduate courses including: disciplines of medical core knowledge, medical ethics, and basic clinical skills of life saving procedures as well as other essential professions like communications, diagnostic and emergency interventional experiences.3 it is known that assessment in medical education is essential part and not a step for passing to a higher level. it serves many functions as it doesn’t determine what students learn but it guides the process of learning, also it provides students’ feedback on where they stand and motivates them to master the material and it show the society and related organizations that doctors are competent.1,4,5 competence in medicine has been defined as “the habitual and judicious use of communication, knowledge, technical skills, clinical reasoning, emotions, values, and reflection in daily practice for the benefit of the individuals and communities being served ”.1 competence is not an achievement but rather a habit of lifelong learning.6 on the other hand, competence is the habitual and judicious use of communication, knowledge, technical skills, clinical reasoning, emotions, values, and reflection in daily practice for the benefit of the individuals and communities being served. additionally, assessment plays an integral role in helping physicians identify and respond to their own learning needs. ideally, the assessment of competence (what the student or physician is able to do) should provide insight into the actual performance (what he or she does habitually when not observed), as well as the capacity to adapt to change, find and generate new knowledge, and improve overall performance.1,4 some graduates unfortunately apparently fail acquiring some essential basic skills during undergraduate course. this problem of under competency is not limited to iraqi medical graduates but also appears in some issn 2413-0516 mousa al alak et al. 171j contemp med sci | vol. 3, no. 9, winter 2017: 170–177 research academic performance of final year medical students among different gender and in the two studied years (2015 and 2016). results the number of graduates in 2014/2015 (reported as 2015) was 51 graduates (15 males and 36 females) and in 2015/2016 (reported as 2016) were 71 graduates (27 males and 43 females). the mean age of the graduate of the kerbala medical college was 24.63 ± 1.822 year in 2015, and 24.39 ± 1.011 year in 2016 with no significant gender difference. the internal reliability (cronbach’s alpha), when all examination scores were included was measured and the findings showed that it was 0.88 and 0.86 for 2015 and 2016, respectively. when cronbach’s alpha was measured for these tests, it was 0.91 for both 2015 and 2016. when cronbach’s alpha was measured for different branches osce examinations separately, the values were all almost acceptable (> 0.6). the mean scores of theoretical examination, direct observation clinical examination (doce), or long case (lc), computerized images and video assessment (civa), oral examination (viva), objective structural clinical examinations stations (osce) total scores in 2015 and 2016 showed no significant gender difference (table 1), while anova test showed no significant difference between the means in the 2 years. when gender difference was explored, no significant difference was discovered in any part of the assessment (tables 2 and 3). osce in medicine when the mean difference between osce in medicine in the two years was explored, significant differences were discovered in all parts of the assessment except for neurology (table 4). osce surgery when the mean difference between osce in medicine in the 2 years was explored, significant differences were discovered in all parts of the assessment except for a history of jaundice case and x-ray pediatric surgery (table 5). osce gynecology and obstetrics when the mean difference between osce in the two years was explored, significant differences were discovered in all parts of the assessment except for obstetrics interpretation and two stations short case examination -short case(table 6). osce pediatrics when the mean difference between osce in the two years was explored, significant differences were discovered only in history, slides and clinical examination (table 7). graduates of credited universities and medical schools in developing and developed countries.7,8 in the united states, the assessment of medical residents, and increasingly of medical students, is largely based on a model developed by the accreditation council for graduate medical education (acgme). this model uses six interrelated domains of competence: medical knowledge, patient care, professionalism, communication and interpersonal skills, practice-based learning and improvement, and systems-based practice.9 objective structured clinical examination (osce) was first described by harden in 1975,10 it has an established role in the assessment of the medical undergraduates. osce assess history taking, clinical examination, data interpretation procedure or practical skills and communication skills.11–13 it represents a valid and reliable tool in medical education for evaluating clinical competence.13 osce was introduced to test final year candidates in the council of arab board students and later for undergraduate students in iraq.3,5,14 a study had reported overall good student’s evaluation and preference of osce in basra medical college.14 in the last decade many articles tried to evaluate the level of assessment in medical colleges in iraq and a national exit examination was suggested.4,5,7,14–20 there are 23 medical colleges in iraq where medical teaching is based on the traditional british teaching system, grant medicine and surgery bachelor certificate. the graduates after passing the final examination are recognized as members of the iraqi medical association and officially assigned as house surgeons (intern resident doctors) serve 24 months rotational training courses in general hospitals.3 admission to medical schools is very competitive and is based on performance in the general national high school examination with nearly full mark scores in all branches. to ensure competence in these fields, and according to the instructions of the ministry of higher education, all graduates need to pass a final examination after 6-year study. the examination tests theoretical and clinical aspects including: long case, short case, objective structured clinical examination osce.3 materials and methods the achievements of the students in the final year examination different sections in kerbala medical college for the academic years 2014–2015 and 2015–2016 in addition to cumulative sores for the preceding five years were assessed. the goal was to determine the effectiveness of different of examination methods and the level of performance of the students in each (and for different subgroups) according to examination scores in theoretical and clinical examinations. the clinical part of the examination included: direct observation clinical examination (doce), long case (lc), computerized images and video assessment (civa), oral examination (viva), objective structural clinical examinations stations (osce). the data were analyzed using the statistical package for social sciences version 20 (spss-20) at a significance level of .05 through different descriptive and analytic statistical tools using: percentages, means, standard deviations, internal reliability, confidence intervals, factor analysis, t-test, anova test, correlation and structural equation modeling. students’ achievements in different parts of the final year tests and their cumulative sores for the preceding five years were compared fig 1. the gender distribution of the graduates from kerbala medical college in 2015 (n = 51) and 2016 (n = 71). 172 j contemp med sci | vol. 3, no. 9, winter 2017: 170–177 academic performance of final year medical students research mousa al alak et al. table 1. the mean and standard deviation of the students’ scores in the main final year examination parts in kerbala medical college in 2015 and 2016 2015 assessment medicine surgery gynecology and obstetrics pediatrics mean std. deviation mean std. deviation mean std. deviation mean std. deviation theoretical examination 4.55 1.14 5.62 1.12 7.15 0.78 6.31 1.14 civa 7.35 1.34 7.12 0.99 8.22 0.89 6.94 1.03 doce or long case 6.08 2.09 6.77 0.95 6.98 1.81 osce total score 6.30 0.83 6.66 0.88 7.02 0.91 7.81 0.99 2016 assessment medicine surgery gynecology and obstetrics pediatrics mean std. deviation mean std. deviation mean std. deviation mean std. deviation theoretical examination 5.61 0.92 5.28 1.08 6.60 1.46 5.79 1.22 civa 6.06 1.20 6.77 0.96 7.13 0.92 6.94 2.20 doce or long case 6.12 1.74 5.39 1.14 6.94 1.17 osce total score 6.22 0.70 5.49 1.01 7.44 1.23 7.69 0.96 table 2. gender difference in the mean and standard deviation of the students’ scores in the main final year examination parts in kerbala medical college in 2015 (n = 51) branch assessment males females t-value significance mean std. deviation mean std. deviation medicine theoretical examination 5.11 1.01 4.32 1.12 2.35 0.847 civa 7.17 1.39 7.42 1.33 −0.59 0.454 doce1 5.93 2.06 6.14 2.12 −0.32 0.781 osce total 6.24 0.83 6.24 0.83 0.89 0.811 surgery theoretical e. 6.08 0.96 5.43 1.15 1.91 0.337 civa 7.00 0.85 6.68 0.98 1.13 0.379 long case 7.18 1.12 7.10 0.95 0.26 0.375 osce total 6.62 0.94 6.67 0.87 −0.19 0.393 gynecology and obstetrics* theoretical e. 7.30 0.75 7.15 0.79 0.62 0.965 civa 8.26 0.89 8.29 0.74 −0.10 0.669 osce total 7.12 0.84 7.12 0.84 −1.03 0.055 pediatrics theoretical examination 5.99 1.11 6.45 1.14 −1.33 0.542 civa 7.14 2.11 8.33 1.39 −2.37 0.302 long case 7.10 1.04 6.87 1.04 0.72 0.639 osce total 7.63 1.36 7.89 0.79 −0.85 0.067 * there was no long case examination in gynecology and obstetrics. six years summative scores for 2015, the mean for the first three study years were around 3.00 out of 5 and for the fourth year the mean was 12.75 ± 1.24 out of 20 and 16.65 ± 2.04 out of 25 for the fifth year and 26.36 ± 2.72 out of 40 for the sixth year, while the mean of the sum (total graduation score) was 65.72 ± 6.77 (table 8). for 2016 graduates, the means were slightly lower but no significant difference was found from 2015 mean score. the mean for the first three study years were similarly around 3.00 out of 5 and for the fourth year the mean was 12.38 ± 1.27 out of 20 and 16.98 ± 1.83 out of 25 for the fifth year and 25.91 ± 2.61 out of 40 for the sixth year, while the mean of the sum (total graduation score) was 64.88 ± 6.57 (table 9). structural equation model (sem) analysis of examination sections results using sem showed the different regression weight subjected by each section of the examination on the total graduation score (figure 4). a model for the cumulative results showed similarly the regression weight of each year score (figure 5). discussion relatively few studies provide detailed comparative analysis of medical college graduates’ performance.21 the results of the present study showed the importance of feedback resulting from the analysis of the detailed mousa al alak et al. 173j contemp med sci | vol. 3, no. 9, winter 2017: 170–177 research academic performance of final year medical students table 3. gender difference in the mean and standard deviation of the students’ scores in the main final year examination parts in kerbala medical college in 2016 (n = 71) branch assessment males females t-value significance mean std. deviation mean std. deviation medicine theoretical examination 5.74 0.84 5.53 0.97 2.35 0.850 civa 6.39 1.17 5.85 1.18 −0.59 0.454 doce 6.08 1.82 6.14 1.71 −0.32 0.781 osce total 6.28 0.76 6.18 0.67 0.89 0.811 surgery theoretical examination 5.58 1.06 5.08 1.07 1.91 0.337 civa 6.80 0.81 6.76 1.05 1.13 0.381 long case 5.68 1.24 5.21 1.06 0.26 0.375 osce total 5.89 0.71 5.24 1.09 −0.19 0.397 gynecology and obstetrics* theoretical examination 7.00 0.90 7.21 0.93 0.79 0.959 civa 7.48 1.34 7.42 1.17 0.22 0.926 osce total 7.42 0.71 7.22 0.73 −0.94 0.058 pediatrics theoretical examination 5.53 1.43 5.95 1.05 −1.33 0.540 civa 6.77 1.34 6.97 2.50 −1.70 0.303 long case 6.91 1.65 7.05 1.05 0.72 0.639 osce total 7.83 0.87 7.61 1.01 −0.85 0.067 *there was no long case examination in gynecology and obstetrics. table 4. the mean and standard deviation of the students’ scores of osce final year examination in medicine in kerbala medical college in 2015 and 2016 station 2015 2016 significance mean std. deviation mean std. deviation communication 4.3824 1.94830 6.2143 1.37016 < 0.001 history interpretation a 6.245 2.0109 7.016 1.7635 0.027 history interpretation b 6.4382 1.62944 7.5357 1.83430 0.001 history interpretation c 6.588 2.1742 5.764 2.1564 0.041 breaking bad news 7.1225 1.84619 5.9607 2.38680 0.004 neurology 7.1510 1.89157 7.7607 2.35770 0.130 locomotors 6.8137 1.94284 4.6286 2.08155 < 0.001 posterior thoracic 5.7647 1.99588 6.5750 2.11634 0.035 pericardium 7.2647 1.69589 4.1336 3.04400 < 0.001 general medicine 7.2647 1.69589 6.6000 1.46109 0.023 examination results which affect the quality of assessment in the future. the overarching goal of educational reform should be to transform medical assessment from passive observation to active process contributing both in curriculum correction and improvement of assessment. detailed analysis of the results and possible causes for differences were discussed with the responsible departments in the college to reach for planning of improved assessment in the next year. an analysis of radiology course in the tikrit medical college, reported a mean of 82 and ci 66–97.8 and only 6.3% of the participants got the “honors” -two standard deviation above the mean-, while 6.7% of the participants were two standard deviations below the mean in the written test. for the written and practical test, the range of the scores was tighter, 65–96%, and the mean of 80.5% and a standard deviation of 5.3, the “honors” and failure rates were approximately 5%.20 osce value was estimated in two articles in basra medical college. in the first, the students’ perception was reported to be positive. the selection of suitable assessment or evaluation depends on its validity (a measure of the extent to which the test actually measure what is intended to measure), reliability (a measure of whether the assessment or test is consistent and accurate; examines the extent to which factors such as examiners, questions, occasions affect the marks or scores awarded) and practicability was proved.5,14 academic performance of medical students in western australia and in indonesia was found to be significantly 174 j contemp med sci | vol. 3, no. 9, winter 2017: 170–177 academic performance of final year medical students research mousa al alak et al. table 6. the mean and standard deviation of the of the students’ scores of osce final year examination in gynecology and obstetrics in kerbala medical college in 2015 and 2016 osce examination 2015 2016 significance mean std. deviation mean std. deviation gynecology history 7.03 1.90 7.33 1.25 <0.001 gynecology interpretation 8.67 1.49 6.33 1.29 <0.001 obstetrics history 6.38 1.54 8.02 1.86 <0.001 obstetrics interpretation 7.54 1.59 8.19 1.38 0.697 gynecological examination 7.02 1.35 6.52 1.71 0.120 two stations short case examination 8.29 1.31 7.37 2.08 0.353 correlated with their performance as a junior doctor (r = 0.229, p = 0.002), and with their mean scores on entering medical college.22,23 blackman,23 evaluated the influence of students score in different study years on post graduate performance. the effects of gpa scores with reference to fig. 3, it can be seen that the grade point average scores obtained by students in their undergraduate studies (lv6) has a direct influence on their subsequent achievement on the clinical assessment tasks. a positive path coefficient exists between these two variables (0.17) which indicates that students with higher grade point averages in their undergraduate studies overall achieve at a higher level on the clinical examination in their third year of medical studies. the loadings for the gpa scores obtained in the first (0.88) second (0.92) or third table 7. mean and standard deviation of the students’ scores of osce final year examination in pediatrics in kerbala medical college in 2015 and 2016 osce examination 2015 2016 significance mean std. deviation mean std. deviation clinical examination a 8.15 1.44 8.14 1.19 0.963 data interpretation a 6.48 2.23 7.36 3.38 0.110 counseling 7.12 3.25 7.85 1.98 0.128 data interpretation b 8.19 1.71 7.78 1.79 0.210 community 7.76 1.27 8.38 2.23 0.076 instruments and drugs 8.35 2.05 8.70 1.49 0.280 history 8.80 1.65 7.08 1.42 0.000 clinical examination b 8.30 1.37 7.64 1.35 0.009 slides 7.98 1.70 6.94 2.20 0.006 development 6.98 1.81 6.94 1.51 0.898 table 5. the mean and standard deviation of the students’ scores of osce final year examination in surgery in kerbala medical college in 2015 and 2016 osce examination 2015 2016 significance mean std. deviation mean std. deviation history of jaundice case 5.99 1.92 6.21 1.05 0.429 surgical examination a 5.86 1.82 4.93 1.76 0.006 hand work 8.11 1.74 7.20 1.06 0.001 surgical examination b 5.22 2.94 3.54 1.83 < 0.001 burns management 6.41 2.59 3.89 3.69 < 0.001 pictures interpretation 5.12 3.23 3.86 2.98 0.028 orthopedic x-ray 7.08 2.62 5.70 2.87 0.008 general surgery instruments 8.19 1.03 6.43 1.87 < 0.001 ct urology 9.27 1.70 6.36 3.11 < 0.001 hand suturing 8.04 1.48 6.57 2.09 < 0.001 x-ray pediatric surgery 5.61 3.16 6.24 3.52 0.309 x-ray urology 5.15 1.91 7.74 3.13 < 0.001 mousa al alak et al. 175j contemp med sci | vol. 3, no. 9, winter 2017: 170–177 research academic performance of final year medical students table 8. the mean and standard deviation of the students’ scores for the six years summative scores by gender in kerbala medical college graduates in 2015 year males females total mean std. deviation mean std. deviation mean std. deviation first 3.36 0.45 3.36 0.36 3.36 0.39 second 3.25 0.32 3.20 0.41 3.22 0.38 third 3.49 0.42 3.34 0.49 3.38 0.47 fourth 13.02 1.28 12.64 1.22 12.75 1.24 fifth 16.77 1.77 16.60 2.17 16.65 2.04 sixth 26.62 2.60 26.25 2.80 26.36 2.72 final 66.51 6.32 65.39 7.01 65.72 6.77 table 9. mean and standard deviation of the students’ scores of the six years summative scores in kerbala medical college in 2016 year males females total mean std. deviation mean std. deviation mean std. deviation first 3.37 0.37 3.33 0.38 3.35 0.37 second 3.12 0.39 3.02 0.39 3.06 0.39 third 3.24 0.50 3.17 0.45 3.20 0.47 fourth 12.49 1.36 12.32 1.23 12.38 1.27 fifth 17.24 1.81 16.81 1.84 16.98 1.83 sixth 26.50 2.78 25.53 2.46 25.91 2.61 total 65.96 6.86 64.18 6.37 64.88 6.57 fig 2. the mean of the students’ scores in the main final year examination parts in kerbala medical college in 2015. fig 3. the mean of the students’ scores in the main final year examination parts in kerbala medical college in 2016. years (0.78) of study are an indication that there is little difference between the contributions to osce performance made by the different years of prior study. in the present study a direct association between the scores obtained in the previous study years and the final graduation score was clear (figure 4). however, low to moderate correlation of undergraduate assessment with postgraduate training performance was reported by other studies.24 no gender difference was found in achievements in all parts of the final year examination in kerbala medical college and this was similar to the findings in many studies in other countries.25–27 however, female students were consistently found in the literature to perform better than males in their medical training which might be related to higher motivation. a systematic review by ferguson and his colleagues reported that a growing body of research explores whether different motivational, academic, and demographic factors influence the performance of male and female women where motivation seems to be important.21 the reason behind failing to find any gender difference in the present study might be related to the higher proportion of females in the sample or some other reasons that need further investigation. environmental factors and personal characteristics might be responsible for the differences discovered between the two years. differences in raters’ grading could introduce variability into the measurement of a student’s performance and thus make the measurement less reliable. additionally, specific attributes of the student, evaluator, or environment in which the interaction took place might affect the student’s scores by introducing systematic 176 j contemp med sci | vol. 3, no. 9, winter 2017: 170–177 academic performance of final year medical students research mousa al alak et al. fig 4. structural equation model of all input variables in different sections of final examination in kerbala medical college in 2016. mousa al alak et al. 177j contemp med sci | vol. 3, no. 9, winter 2017: 170–177 research academic performance of final year medical students differences into the measurement, further decreasing the reliability of the system.25 multivariate regression analysis approach to studying predictors of success in medical training was suggested in a systematic review of about twenty two thousands medical students (21). predictors are likely to be inter-correlated, as are outcome measures. additionally, learning across the medical degree occurs over time, and time series analyses and models that allow for prediction of change over time would also be a useful approach in exploring trend effects. the use of sem procedures, as well as hierarchical structural models using structural and time series components, was also beneficial to develop our understanding of the prediction of performance.21,26 conclusions the evaluation process and examination section yields correct in depth findings resulted from analysis of kerbala medical college graduates in two successive years (2015 and 2016). on practical base, the results found were very helpful in pointing out the main shortcomings and strength in the examination stations and to plan for more accurate assessment for the graduates. conflict of interest none. n fig 5. structural equation model of all input variables in different main branches of final examination in kerbala medical college in 2016. references 1. epstein rm. assessment in medical education. new eng j med. 2007;356:387–396. 2. wood ej. the problems of problem-based learning. biochem edu. 1994;22:78–82. 3. alhelli aa, abdulsahib m. the sequential feedback of iraqi medical graduates performance. j kerbala university. 2013;11:13–25. 4. amin z, chong y, khoo h. towards better practices in medical student assessment. annals-academy of medicine singapore. 2005;34:471. 5. abdulla ma. osce, things to be said. basrah j surg. 2010;16:4–6. 6. leach dc. competence is a habit. jama. 2002;287:243–244. 7. alhelli a. evaluation of medical colleges` graduates in iraq. karbala journal of medicine. 2010;3:919–25. 8. premadasa i, shehab d, al-jarallah kf, thalib l. frequency and confidence in performing clinical skills among medical interns in kuwait. medical teacher. 2008;30:e60–e65. 9. kavic ms. competency and the six core competencies. jsls : j soc laparoendoscopic surg. 2002;6:95–97. 10. harden rm, gleeson f. assessment of clinical competence using an objective structured clinical examination (osce). med edu. 1979;13:39–54. 11. pell g, fuller r, homer m, roberts t. how to measure the quality of the osce: a review of metrics–amee guide no. 49. medical teacher. 2010;32:802–11. 12. van der vleuten cp, schuwirth lw. assessing professional competence: from methods to programmes. med edu. 2005;39:309–17. 13. walsh m, bailey ph, koren i. objective structured clinical evaluation of clinical competence: an integrative review. j advan nur. 2009;65:1584–95. 14. abdulla, m a. student’s perception of objective structured clinical examination (osce) in surgery at basrah college of medicine. basrah j surg. 2012;18:21–5. 15. sarhat a r, abedalrahman s k. toward iraqi national medical licensing examination. diyala j pure sci. 2010;6:77–84. 16. abdulghani h m, al-drees a a, khalil m s, ahmad f, ponnamperuma g g, amin z. what factors determine academic achievement in high achieving undergraduate medical students? a qualitative study. medical teacher. 2014;36 suppl 1:s43–8. 17. al-shamsi mm. evaluation of the final year medical students by external examiners. kerbala j med. 2007;1:243–247. 18. al-jobori, m , abbas, a. assess the quality of higher education programs as a framework for the success of the education process. al-qadisiya j admin econ sci. 2016;18:34–56. 19. jasim. opinions of nineveh medical college students regarding current medical educational methods and teaching strategies. med j tikrit. 2013;19(1) :114–119. 20. younis sn. improving undergraduate education in radiology. tikret j pharm sci. 2011;7:130–4. 21. ferguson e, james d, madeley l. factors associated with success in medical school: systematic review of the literature. bmj. 2002;324:952–957. 22. carr se, celenza a, puddey ib, lake f. relationships between academic performance of medical students and their workplace performance as junior doctors. bmc med edu. 2014;14:1. 23. blackman ir, darmawan i. graduate-entry medical student variables that predict academic and clinical achievement. int edu j. 2004;4:30–41. 24. hamdy h, prasad k, anderson mb, scherpbier a, williams r, zwierstra r, et al. beme systematic review: predictive values of measurements obtained in medical schools and future performance in medical practice. medical teacher. 2006;28:103–16. 25. spielvogel r sz, beckett l, latimore d. sources of variability in medical student evaluations on the internal medicine clinical rotation. international journal of medical education. 2012;3:245–51. 26. mcmanus i, richards p. admission for medicine in the united kingdom: a structural model of background factors. medical education. 1986;20:181–6. 27. lewinski p. effects of classrooms’ architecture on academic performance in view of telic versus paratelic motivation: a review. front psychol. 2015;6:746. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 368 j contemp med sci | vol. 7, no. 6, november-december 2021: 368–372 original calcium and phosphate homeostasis in patients with recurrent nephrolithiasis jawad abdul-hassan masser1, mazin j. mousa2, hayder abdul-amir makki al-hindy3* , noor s.k. al-khafaji4, hussein o.m. al-dahmoshi4, zena abdul-ameer mahdi5, samah ahmed kadhum2, safa jihad hameed2, suhad h. obeed2 1al-sadiq teaching hospital, babil health directorate, ministry of health, babylon, iraq. 2college of pharmacy, university of babylon, babylon, iraq. 3department of pharmacology & toxicology, college of pharmacy, university of babylon, babylon, iraq. 4department of biology, college of science, university of babylon, babylon, iraq. 5ahl-albait university, college of pharmacy, karbala, iraq. *correspondence to: hayder abdul-amir makki al-hindy (e-mail: phar.hayder.abdul@uobabylon.edu.iq) abstract objectives: the aim of the study was to evaluate the calcium (ca) and phosphate (ph) homeostasis and their association with plasma 25(oh)2 vitamin d3 (vitd3) and parathyroid hormone (pth) in patients with recurrent nephrolithiasis. methods: a cross-sectional, involved 100 confirmed patients with renal stone (rs). their serum levels of ca, pth, ph, and vitd3 had assessed. biochemical analysis of renal calculi and crystals had been investigated also. the summary measures had described as mean+/–sd for continuous variables and frequencies/percentage for nominal variables. the mean serum ca, ph, pth, and vitd3 were (8.01 ± 2.2 mg/dl, 2.9 ± 1.2 mg/dl, 56.7 ± 24.7 pg/dl, and 7.03 ± 4.2 pg/ml), respectively. results: almost all patients (97%) had a positive history of previous rs with a predominant family history in most instances (82%). there was a positive non-significant correlation between vitd3 with serum ph and ca. whereas an inverse non-significant correlation between pth with serum ca and vitd3 had observed. urinary crystals analysis revealed that uric acid represented 53% of the total crystals, followed by ca-oxalate. stone analyses revealed that around 3/4th of the cases had ca-oxalate stone, followed by ca-oxalate with uric acid, then ca-phosphate, and the least type was mixed stone types. conclusion: there was a positive non-significant correlation between vitd3 with serum ph and ca. there was an inverse non-significant correlation between pth with serum ca and vitd3. serum ca, and ph were non-significant predictors of renal stones and/or urinary crystals. keywords: renal stone, parathyroid hormone, vitamin-d, nephrolithiasis, calcium, phosphate issn 2413-0516 introduction renal stone (rs) is a worldwide health problem worrying people for prolonged periods. the prevalence of rs in males is roughly 6–9% and in females, it is nearly 3–4%,1,2 and the predictable time risk of stone development is 5–12% in the usa and europe.3 preceding researches had exposed that elevated levels of active vitamin-d, is linked with higher calcium urine excretion.2 both parathyroid hormone (pth) and 1,25-dihydroxy vitamin d3 (vitd3) have regarded as hormonal regulators of calcium metabolic balance. pth release that is activated by hypocalcemia, rises plasma calcium (ca) by activating bone resorption, kidney reabsorption, and intestinal calcium uptake indirectly via the kidney synthesis of vitd3.4 the serum levels of ca and phosphate (p) in a research of 356 men with rs, were higher than in control subjects.5 owing to their contribution to the ca homeostasis and rs, pth and vitd3 have gained much attention. findings from a meta-analysis suggest that serum vitd3 level in kidney stone patients was significantly higher than that in control.6 nevertheless, the precise roles of these factors in urolithiasis remain to be clarified.7 to assess the probable role of vitd3 and pth as essential factors in the incidence of rs, we investigated their association with plasma calcium and phosphorus levels. patients and methods study strategy and patient’s collection this was a cross-sectional study conducted in al-sadiq teaching hospital, babylon (middle of iraq), for one-year (2018–2019). a total of 100 subjects (aged 45.8 ± 17.7 years, 76 males and 24 females) presented with rs had enrolled in the study. all were attending urology consultation clinics (after being referred from specialty clinics) for followup or lithotripsy. the diagnosis of rs had completed by the urologists using radiographic and sonographic techniques. the demographic surveys had included data like age, gender, history of rs, history of diabetes, hypertension and other complications. the patients were known to have one of the ensuing problems had excepted from our study; urinary tract anomalies, urinary tract obstruction, hyperparathyroidism, and those on regular vitd3 therapy. an informed consent from all the participants had been gotten prior to be involved in the study. biochemical assays we measured the serum ca, ph, vitd3, and pth levels in all the stone former patients. both vitamin d3 and pth had measured by specific elabscience elisa-kit. serum ca, and ph concentrations had evaluated by spectrophotometric (submitted: 11 september 2021 – revised version received: 21 september 2021 – accepted: 10 october 2021 – published online: 26 december 2021) 369j contemp med sci | vol. 7, no. 6, november-december 2021: 368–372 j. abdul-hassan masser et al. original calcium and phosphate level in nephrolithiasis method (biolabo, france). microscopic morphological examination of urinary sediments had performed to reveal the type of urinary crystals. the stone analysis had completed using a dipstick chemical test (dybow™). vitamin-d consumption may change falsely (low or high) levels of vitd3, henceforth, participants had inquired whether they taken oral vitd3, which was excluded from the study. serum vitd3 levels had analysed using the definitions recommended in the kidney disease improving global outcome initiative guidelines.8,9 statistical analyses statistical analyses had performed on spss version-25 software package. the summary measures were described as mean+/–sd for continuous variables and frequencies with percentage for nominal variables. multiple regression analyses were applied to predict pth, and vitd3 from sex, patients’ age, family history, size/site of the stone, bmi, serum ca, and serum ph. all statistical tests were performed at p < 0.05 significance level. results the main characteristics of patients with recurrent urolithiasis were demonstrated well in table 1. the mean age of the enrolled patients was 45.8 ± 17.7 years, with males representing 76%. the mean serum ca, ph, pth, and vitd3 were (8.01 ± 2.2 mg/dl, 2.9 ± 1.2 mg/dl, 56.7 ± 24.7 pg/dl, and 7.03 ± 4.2 pg/ml), respectively. of the total patients, 56% had a history of any chronic diseases (diabetes 10%, hypertension 40%, or ischemic heart diseases 6%). the ph of the urine was equally distributed 51% acidic and 49% alkaline. summarized in table 2 below, were the main characteristics of the rs. half of rs had a bilateral renal (left and right) location, 28% located in the left, 21% in the right, and only 2% had both renal and extrarenal location. around 2/3rd of the rs was radiopaque, and almost all patients (97%) had a positive history of previous rs (within the last 3-years) with a predominant family history in most instances (82%). more than 2/3rd of rs had large size (>10 mm), 28% had 5–10 mm size, and less than 5% had small size (<5 mm). around 3/4th of patients had a lower vitd3 serum levels, and around 1/4th had high pth levels. two patients had ureteral calculi besides the rs. no vesical stone had recorded. in 28 patients, 2 or more stones had recorded. there were no gender-differences in terms of calcium, phosphorus, pth, and vitd3. there were non-significant correlations of serum ca and ph, pth, and vitd3 with the types of rs and/or urinary crystals. likewise, there were a non significant differences of study variables between those with low and those with high serum vitd3 levels. there was a positive non-significant correlation between vitd3 with serum ph (r = 0.064, p = 0.619) and ca levels (r = 0.095, p = 0.653). whereas an inverse non-significant correlation between pth with serum ca (r = –0.122, p = 0.302), ph (r = –0.096, p = 0.544) and significant correlation with vitd3 (r = –0.25, p = 0.03) had observed (table 3). multiple regression analyses had run to predict pth, and vitd3 from sex, patients’ age, family history, size/site of the stone, serum ca, and ph. they were non-significant predictors of pth and vitd3. all variables add non-significant statistically prediction, p > 0.05. stone and crystal analyses urine crystals analysis revealed that uric acid represented 53% of the total crystals. then followed by ca-oxalate (41%), and the least represented by other types (6%) of urinary crystal (figure 1). renal stone analysis revealed that ca oxalate represented 3/4th of the total rss. then followed by ca-oxalate and ua represents about 1/5th, and the least represented by other types of urinary stone (figure 2). table 1. main characteristics of patients with recurrent urolithiasis involved in the study minimum maximum mean ± std. deviation age/years 5 19 45.8 ± 17.7 sex no. (%) males 76 (76%) females 24 (24%) serum calcium, mg/dl 4.2 15.9 8.01 ± 2.2 serum phosphorus, mg/dl 0.5 6.7 2.9 ± 1.2 serum vitamin d3, pg/ml 0.9 29.56 7.03 ± 4.2 serum pth, pg/dl 32.9 184.1 56.7 ± 24.7 diabetes no. (%) 10 (10%) hypertension no. (%) 40 (40%) ischemic heart diseases no. (%) 6 (6%) urinary ph acidic 51 (51%) no: number; pth: parathyroid hormone. table 2. biochemical and basal characteristics of urinary stones of patients with recurrent urolithiasis involved in the study characteristics percentage site of stone in the urinary system left kidney 28% right kidney 21% left and right kidney 49% kidney and ureter 2% stone radiolucency radiolucent 34% radiopaque 66% recurrent stone negative history 3% positive history 97% family history of renal stone 82% size of the urinary stone ≤ 5.0 mm 4.9% > 5.0 – 10.0 mm 28.0% > 10.0 mm 67.1% low vitd3 73% normal or high vitd3 27% low or normal pth 79% high pth 21% vitd3: vitamin d3; pth: parathyroid hormone. 370 j contemp med sci | vol. 7, no. 6, november-december 2021: 368–372 calcium and phosphate level in nephrolithiasis original j. abdul-hassan masser et al. discussion urolithiasis is the 3rd common urinary disorder after prostatic diseases and urinary infections. around 80–85% of rs are made up of calcium.7 the commonest type of urolithiasis is ca-oxalate, then mixed ca-oxalate/phosphate, struvite, uric acid, ca-phosphate, and cysteine rs.2 however, blood alterations in ca, ph, pth, and vitd3 metabolism have not been hitherto studied in patients with recurrent rs in babylon. this study revealed that patients with rs had lower levels of serum vitd3, which consistent to other researches that had exhibited vitd3 insufficiency and deficiency are common in patients with rs.2,9,10 although, all patients with rs had serum vitd3 fell within the reference range in another study.11 as a fat-soluble, vitd3 has multiple metabolic activities. it has a crucial role in the homeostasis of ca and ph, besides, has a vital role in the pathogenesis of rs. vitd3 regulate ca-homeostasis by acting on three body organs which are kidney, intestine and bone.2 the biochemical alterations disposing to lithogenesis are not always apparent and in many instances, the stones’ type and the metabolic causes remain indefinite. in the existing study, a positive association (although nonsignificant) of vitd3 plasma levels with ca and ph had shown. in the same line, there was an obvious decrease of vitd3, in patients with rs, but with no much changes in ca and ph metabolism and bone metabolism between patients with rss and healthy controls in another study.12 added, in a survey including 160 rs vis 217 controls, netelenbos et al.,13 have showed a non-significant differences in serum vitd3 levels. hence, it seems that high vitd3 values, by inducing hypercalcemia followed by hypercalciuria, may contribute to lithogenesis in some patients.14 the vitd3 is a powerful stimulus of intestinal ca-absorption in recurrent stone formers has been stressed by many authors. in addition, owing to it has renal and bone action, further elevates serum ca-levels.7 furthermore, due to enhanced intestinal ca-absorption, might reduce the intestinal binding of ca to oxalate that in turn, encourages much oxalate absorption, thereby induce the exacerbation of hyperoxaluria.15,16 the locations and sites of the stone in our work are consistent with other reports from turkey and iran, nonetheless the frequency of two-sided, presence of several rs and the size of rs were varied.14,17 in the current study, no bladder calculi had noticed similar to the iranian report.12 similar to our outcomes, no correlation of the study variables with size of the rs, had noticed by yun s. et al.18 gender is also a recognized risk factor for lithogenesis. unlike our study, there have been a few epidemiological studies that comment upon gender differences in terms of ca, ph, pth, and vitd3 differences among patients with rs.19 however, based on a meta-analysis of 20 case control types of research only 2-works could be divided into subtypes based on sex, unsatisfactory to create a reliable link. thus, gender subgroups should be focused on future studies.19 history of rs was strongly positive in of the first/second degree relatives in the present study that is consistent with what had reported by the iranian study of the infants with rs,14 though other studies had shown less rates9,16 of genetic factors.14 several meta-analyses have shown different genes responsible for inherited lithogenicity. however, some genetic loci seem to share a minor role to rs like single nucleotide polymorphisms in osteopontin and vitamin d receptor genes and others.20–22 the recurrence of the stones in about 26% of the patients which less than the recurrence of other studies, which was (35–50%) of the patients.16,23 most of the patients with renal stone (80%) were drinking many soft drinks frequently which containing oxalate that increasing the risk of formation of renal stone.23 the explanation of the relationship between vitd3 and pth is multifaceted.9,24 there was a negative correlation between the pth and vitd3 concentrations in the sera of our patients. this is concordant to the outcomes of other reports that revealed a negative correlation between the levels table 3. correlation of vitamin d3, parathyroid hormone, serum calcium and phosphorus in patients with recurrent urolithiasis involved in the study serum calcium serum phosphorus vitamin d vitamin d pearson correlation 0.059 0.064 – significance 0.653 0.619 pth pearson correlation –0.122 –0.069 –0.25 significance 0.302 0.544 0.03 fig. 1 pie chart counts & percentage of urinary crystal. fig. 2 pie chart distribution of stone types among patients with renal stone. 371j contemp med sci | vol. 7, no. 6, november-december 2021: 368–372 j. abdul-hassan masser et al. original calcium and phosphate level in nephrolithiasis of pth and vitd3.2,10 likewise, two prior researchers have displayed a strong association between pth and vitd3 in patients with primary hyperparathyroidism.25,26 however, no significant relations had observed in a study conducted earlier among infants14 and adults.7,18 though still debated, prior reports from tertiary referral centers verified vitd3 insufficiency induce hypocalcemia that, in-turn, increase pth release or “secondary hyperparathyroidism’’. higher levels of pth in-turn elevate blood ca. regardless of raised ca-reabsorption by the kidney, hypercalciuria is eventual result that place all patients at an higher risk of rs.2,27 worth mentioning, in the existing study there is an alteration in pth, vitd3, and ca normal levels. the lack of inhibition of pth levels by elevated vitd3 and ca values in rs patients suggests a subtle variations of the parathyroid gland to inhibitory signals.28 a current genome wide-association reports have caught up about 6-loci in the regulation of serum ca that might explain this phenomenon.29 in the current work, there was a positive non-significant association between vitd3 levels with levels of ca and ph in the blood, which is compatible with the previous studies.2,18 while, a negative association of serum ca with vitd3 had reported by a scholar.28 on the other hand, no correlation observed between the level of serum vitd3 with ca by another scholar.10 in contrast, in this work, there were negative correlations among serum pth levels with ca, ph, and vitd3, consistent with prior study from the usa.28 there was no correlation between pth and ph levels in a study published at 2017,2 and a negative associations between serum ca and pth by study approved by “mayo clinic institutional review board’’ at 2015.28 in a cohort of rs subjects, high normal blood ca and low normal pth values had documented.28 renal stone analysis revealed that ca-oxalate represented 3/4th of the total rss. likewise, urine crystals analysis revealed that uric acid represented 53% followed by ca-oxalate (41%). the same results almost reported by several studies from turkey, spain and italy.30–32 studies recommended that the frequency of monogenic rs in patients attending urologists is about 15%. for subjects without a monogenic origin of rs, the heritability of rs and hypercalciuria is >45% and >50%, individually.33 these results are not away from our outcomes. hyperoxaluria is a hereditary disorder that is presented with high oxalate production with excess urinary oxalate, and a higher risk of calcium oxalate rs. primary hyperoxaluria have three types: type-1, is the most severe, due to enzyme mutation of “alanine-glyoxylate and serine-pyruvate aminotransferase’’; type-2, due to enzyme-mutation of “glyoxylate and hydroxy-pyruvate reductase’’, which is slowly progressed to esrd; and type-3, due to enzyme-mutation in “4-hydroxy 2-oxoglutarate aldolase-1’’, is least expected to progress to esrd.34 on another side, hereditary hyperuricosuria due to a defect of the enzyme ‘’hypoxanthine-guanine phosphoribosyl transferase’’, and overactivity of ‘’phosphoribosyl pyrophosphate synthase’’.35 finally, renal phosphate wasting and hypophosphatemia caused by inhibited ‘’fibroblast growth factor-23 (fgf23)’’, a hormone that adjusts phosphate homeostasis and enhances vitd3 breakdown. lower fgf23 levels and hypophosphatemia inhibit ‘’cyp24a1-expression’’ and then enhance sensitivity to vitd3. thus, vitamin d supplement in such subjects results in hypercalcemia and higher risk of rs and should be used with caution.36 transforming growth factor-beta (tgfβ) is released from different cells including fibroblasts, macrophages, platelets and has multicellular impact. tgfβ essentially, belongs to tgfβ-superfamily.37–39 currently, the researchers have exposed a specific role of tgfβ in the process of nephrolithiasis, as well it had reported that therapy with “anti-tgfβ-igg antibody’’ prevents calcium oxalate-crystallization and interstitial fibrosis in nephrocalcinosisassociated ckd.40 we think that the environmental factors contributing a crucial role in etiopathology of rs. several factors affect subjects’ susceptibility for rs including food intake,41 individual renal gfr,18 genetic constitution,29,34-36,42 sunlight exposure,17,18 latitude and seasons of the year.23,24 further systematic investigations are desired to better realize and illustrate the exact biochemical interrelationships of ca, ph, pth, and vitd3, both in the blood and urine principally respecting the metabolic, renal and skeletal disease. as well, little data are available with concerning other consequences of vitd3 insufficiency or normalization in a patient with rs. conclusions there was a positive non-significant correlation between vitd3 with serum p and ca. there was an inverse non significant correlation between pth with serum ca and vitd3. serum ca, and ph were non-significant predictors of and renal stones and/or urinary crystals. our study had conducted on a restricted number of patients. we recommend investigating ca, p, oxalate, and uric acid values of urine in the upcoming analogous works. second, biochemical analyses from a control individual had not obtained to perceive alterations or likenesses between the two groups. thirdly, our outcomes had gained from a single center and perhaps could not been generalized. finally, our conclusions should be interpreted vigilantly as the study not covering all seasons with various exposing periods of sunlight. authors’ contribution jawad, mazin, and hayder read, revised, analyzed, and approved the final manuscript, other authors take a part in the data and samples collection. acknowledgments we thank the iraqi ministry of higher education and the university of babylon for facilities needed to carry out this study. this work was personally funded by authors. conflicts of interest disclosure the authors declare they have no conflict of interest. funding self-funded. ethical approval the research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the helsinki declaration.  372 j contemp med sci | vol. 7, no. 6, november-december 2021: 368–372 calcium and phosphate level in nephrolithiasis original j. abdul-hassan masser et al. references 1. stamatelou kk fm, jones ca, nyberg lm, curhan gc. time trends in reported prevalence of kidney stones in the united states: 1976–1994. kidney int. 2003;63:1817–23. 2. venkatesan s, chakkarai k, arulvijayavani s, senthilkumar, gp, manikandan r, & kalyaperumal m. association between vitamin d, parathyroid hormone and inflammatory markers in urolithiasis patients. j renal inj prev. 2017;6(4): 240–243. 3. bartoletti r ct, mondaini n, melone f, travaglini f, carini m, et al. epidemiology and risk factors in urolithiasis. urol int 2007;79:3–7. 4. goodman h. basic medical endocrinology. 4th ed. kuo rl lj, evan ap, editor. london: elsevier; 2009. 197–218 p. 5. taylor en, hoofnagle an, curhan gc. calcium and phosphorus regulatory hormones and risk of incident symptomatic kidney stones. clinical journal of the american society of nephrology: cjasn. 2015;10(4):667–75. 6. hai wang lm, guizhong li, guanglin huang and ning liu. association between serum vitamin d levels and the risk of kidney stone: evidence from a meta-analysis. nutrition journal 2016;15(32):5. 7. feramarz mohammadalibeigi ms, mahsa motamedi. the effect of serum levels of vitamin d in stone recurrence in patients with urinary tract stone. j renal inj prev 2018;7(2):6. 8. foundation nk. k/doqi clinical practice guidelines for bone metabolism and disease in chronic kidney disease. american journal of kidney diseases: the official journal of the national kidney foundation. 2003;42 (4 suppl 3): s1–201. 9. pipili c, oreopoulos dg. vitamin d status in patients with recurrent kidney stones. nephron clinical practice. 2012;122(3–4):134–8. 10. criseno s vj, nightingale p, gittoes n. a retrospective cohort study evaluating the prevalence of vitamin d deficiency and its impact on the biochemical and clinical presentations of patients with primary hyperparathyroidism (phpt). endocrinol metab syndr. 2019;8(1):298. 11. shakhssalim n. gk, parvin m. et al. an assessment of parathyroid hormone, calcitonin, 1, 25 (oh)2 vitamin d3, estradiol and testosterone in men with active calcium stone disease and evaluation of its biochemical risk factors. urol res 2011;39:7. 12. peretokina ev mn, rozhynskaya lya, egshatyan lv. state of bone metabolism in patients with urolithiasis. endocrine abstracts. 2014;35:1. 13. netelenbos jc jm, van der vijgh wj, et al. vitamin d status in urinary calcium stone formation. arch intern med 1985;145:681–4. 14. fallahzadeh m. f. jz, al-hashemi g., derakhshan ali, et al. elevated serum levels of vitamin d in infants with urolithiasis. iranian journal of kidney diseases. 2012;6(3):6. 15. sandro giannini mn, rocco castrignano, tecla patl, andrea taka, giorgio villi’, federico pellegrini and angela d’angelo. possible link between vitamin d and hyperoxaluria in patients with renal stone disease. clinical science. 1993;84:4. 16. hilal n.; mohsin m.; ibraheemn.; suleiman s.; assim h. af. case control study: estimation of vitamin d deficiency in relation to urinary stones formation among tikritmale population. indian journal of forensic medicine & toxicology. 2019;13(4):6. 17. ertan p tg, oger n, alkan s, horasan gd. metabolic and demographic characteristics of children with urolithiasis in western turkey. urol res. 2011;39:105–10. 18. yun s. hy, kim w, et al. role of 1,25-dihydroxy vitamin d3 and parathyroid hormone in patients with calcium urolithiasis. the journal of urology. 2011;185:2. 19. liu w, chen m, li m, ma h, tong s, lei y, et al. vitamin d receptor gene (vdr) polymorphisms and the urolithiasis risk: an updated meta-analysis based on 20 case–control studies. urolithiasis. 2014;42(1):45–52. 20. liu w, chen m, li m, ma h, tong s, lei y, et al. vitamin d receptor gene (vdr) polymorphisms and the urolithiasis risk: an updated meta-analysis based on 20 case–control studies. urolithiasis. 2014;42(1):45–52. 21. lin y mq, zheng x, chen h, yang k, xie l. vitamin d receptor genetic polymorphisms and the risk of urolithiasis: a meta-analysis. urol int. 2011;86:249–55. 22. m.b.s. a-s. d76v, l161r, and c117s are the most pathogenic amino acid substitutions with several dangerous consequences on leptin structure, function, and stability. egypt j med hum genet. 2019;20(32):1–12. 23. romero v. ah, and assimos d. kidney stones: a global picture of prevalence, incidence, and associated risk factors. review in urology. 2010;12(86). 24. feldman d. vitamin d, parathyroid hormone, and calcium: a complex regulatory network. am j med. 1999;107:637–9. 25. cipriani c bf, costa ag, zhang c, biondi p, diacinti d, et al. prevalence of kidney stones and vertebral fractures in primary hyperparathyroidism using imaging technology. j clin endocrinol metab. 2015;100(4):1309–15. 26. tassone f gl, baffoni c, visconti g, pellegrino m, cassibba s, et al. vitamin d status in primary hyperparathyroidism: a southern european perspective. clin endocrinol (oxf ). 2013;79(6):784–90. 27. p. v. primary hyperparathyroidism and nephrolithiasis. ann endocrinol. 2015;76:116–9. 28. ketha h sr, grebe sk, bergstralh ej, rule ad, lieske jc, et al. altered calcium and vitamin d homeostasis in first-time calcium kidney stone-formers. plos one 2015;10(9):11. 29. o’seaghdha cm, wh, yang q, kapur k, guessous i, zuber am, et al. meta-analysis of genome-wide association studies identifies six new loci for serum calcium concentrations. plos genet. 2013;9(9). 30. ozkaya o, söylemezoğlu o, misirlioğlu m, gönen s, buyan n, hasanoğlu e. polymorphisms in the vitamin d receptor gene and the risk of calcium nephrolithiasis in children. european urology. 2003;44(1):150–4. 31. girón-prieto ms, del carmen cano-garcía m, arrabal-polo m, poyatos-andujar a, quesada-charneco m, de haro-muñoz t, et al. analysis of vitamin d deficiency in calcium stone-forming patients. international urology and nephrology. 2016;48(8):1243–6. 32. vezzoli g, caumo a, baragetti i, zerbi s, bellinzoni p, centemero a, et al. study of calcium metabolism in idiopathic hypercalciuria by strontium oral load test. clinical chemistry. 1999;45(2):257–61. 33. howles sa, thakker rv. genetics of kidney stone disease. nature reviews urology. 2020;17(7):407–21. 34. hopp k, cogal ag, bergstralh ej, seide bm, olson jb, meek am, et al. phenotype-genotype correlations and estimated carrier frequencies of primary hyperoxaluria. journal of the american society of nephrology: jasn. 2015;26(10):2559–70. 35. torres rj, puig jg. hypoxanthine-guanine phosophoribosyltransferase (hprt) deficiency: lesch-nyhan syndrome. orphanet j rare dis. 2007;2:48. 36. schlingmann kpr, justyna k., et al. autosomal-recessive mutations in slc34a1 encoding sodium-phosphate cotransporter 2a cause idiopathic infantile hypercalcemia. journal of the american society of nephrology: jasn. 2016;27(2):604–14. 37. fouad shareef dleikh aja-a, rebee mohin, mazin jaafar mousa, hayder abdul-amir makki al-hindy, basim abd al-ka’abi. possible cause-and-effect linkage of transforming growth factor-beta1 and platelets derived growth factor-ab with delayed anthropometric parameters in adolescent patients with cooley’s anemia: cases vis control research strategy. eurasian journal of biosciences. 2020;14(1):7. 38. hayder aa. al-hindy mjmak, raghdan z. al-saad, widad hd. relationship of levels of transforming growth factorbeta1 (tgf-β1) to the levels of ferritin in blood of transfusion dependent β-thalassemia major patients with growth retardation: a case-control study eurasian j biosci. 2020;14(1): 521–52. 39. mazin j. mousa hsas, hayder abdul-amir maki al-hindy. low level laser (biophotomodulation) therapy for the treatment of diabetic foot ulcers with 532 nm ktp laser induces wound healing, fibroblast proliferation and overexpression of tgf. sys rev pharm 2020;11(6):396–403. 40. steiger s, grill jf, ma q, bäuerle t, jordan j, smolle m, et al. antitransforming growth factor β igg elicits a dual effect on calcium oxalate crystallization and progressive nephrocalcinosis-related chronic kidney disease. frontiers in immunology. 2018;9:619. 41. yasui t. oa, taguchi k. et al., association between urolithiasis and food intake based on data from a japanese national survey. the journal of urology. 2011;185:2. 42. hashim ho, al-saadi ah, haider ah, zaidan hk. association of uromodulin rs13333226 and angiotensinogen rs699 genes variants with essential hypertension in arab iraqis of babylon province. research journal of pharmaceutical biological and chemical sciences. 2015;6(6):589–601. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1040 75j contemp med sci | vol. 8, no. 1, january-february 2022: 75–78 original severity of covid-19 infection among kidney transplant recipients in duhok city, kurdistan region, iraq zana sidiq mohammed saleem* department of internal medicine, college of medicine, university of duhok, duhok, iraq. *correspondence to: zana sidiq mohammed saleem (e-mail: zanasidik@gmail.com) (submitted: 07 january 2022 – revised version received: 19 january 2022 – accepted: 12 february 2022 – published online: 26 february 2022) abstract background: kidney transplant recipients are risky group population in whom greater morbidity and mortality reported in comparison to general population. methods: we examined a prospectively cohort of 80 sot kidney transplant recipients with first-wave covid-19, participants assessed and grouped into severe and non-severe illness. results: eighty kidney transplant recipients involved in our study, with average age of 46.6 ± 12.2 years and kidney transplant duration of 6.4 ± 3.5 years. fifty four were male (67.5%). comorbidities included hypertension (60%), diabetes mellitus (18.8), coronary heart disease (3.8), and hypothyroidisim (2.5). twelve patients had severe covid-19 infection (15%) and sixty eight with non-severe (85%). risk factors for severe covid-19 infection in this study were male gender, old age, comorbidities, obesity, longer duration of covid-19 symptoms, kidney transplantation duration, c-reactive protein > 24 mg/l, interlukine-6 > 26 pg/ml and d-dimer level > 1000 ng/ml. modulation of immunosuppressive drugs done only for severe cases. eight transplant recipients needed admission to hospital and one necessitate mechanical ventilation. conclusion: kidney transplant recipients are at high risk of acquiring opportunistic infections including covid-19 infection. the most important strategy in kidney transplant recipients to prevent covid-19 infection is through adopting preventive measures in particular the use of masks and avoidance of crowded non-ventilated places. risk stratification and poor outcome factor is crucial strategy to prevent spread of the infection. keywords: covid-19, duhok, iraq, severity, kidney transplant issn 2413-0516 introduction coronavirus disease 2019 (covid-19) is caused by the single strand rna virus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and has become a worldwide threat at beginning of 2020. the first human case of covid-19 was reported in wuhan, china, in december 2019, and within a few weeks the infection spread around the world, becoming a pandemic.1 symptoms like fever, fatigue and dry cough are the most common and patients may also experience dyspnea, muscle pain, sore throat and gastrointestinal (gi) symptoms.2 patient receiving immunosuppressant drugs have been one of the populations most vulnerable to covid-19 and many reports have been published. in this setting, immunomodulation has emerged as a promising option for patients with covid-19related cytokine storm.3 moreover, covid-19 has had a big impact on wait-listed patients, highlighting the need to properly balance the risks and benefits of transplantation in the setting of an ongoing pandemic.4 acute kidney injury (aki) is a common complication in patients with covid-19 and it’s multifactorial and is associated with increased intensive care unit (icu) admission and mortality.5,6 aki incidence is higher in patients admitted to the icu due to covid-19 than in patients admitted for other reasons.7 renal transplant recipients may be at high risk of developing severe covid-19 disease due to chronic immunosuppression, comorbidities and frequent contact with the healthcare system. kidney transplant recipient may be diagnosed earlier when they have symptoms due to closer follow-up at the transplant center.8-10 in kidney transplantation, the most widely used interventions were the modification of immunosuppression, reducing or suspending the antimetabolite or inhibitors of the mammalian target of rapamycin, while the calcineurin inhibitor was suspended in patients at risk for interaction with protease inhibitors.11-15 a british population-based study showed that solid organ transplant patients had one of the highest in-hospital risks of death (hr 4.23) due to covid-19.16 risk factors for mortality included age >65 years, chronic heart failure, chronic lung disease and obesity. focusing on renal transplants, a recent systematic review based on 20 studies from different countries revealed a patient mortality higher than in general population, ranging between 18% and 43%,17 versus 1% and 14% in the general population.18-20 risk factors for severe disease in the general population include older age and comorbidities,21 but the impact of chronic immunosuppression related to transplantation on covid-19 is not well known. despite widespread concern that covid-19 clinical phenotypes may be more severe among solid organ transplant recipients (sotrs) due to a poorer inflammatory response and greater organ injury, data on this population are limited to a few case series and generally small retrospective cohorts.22-25 previous reports suggest that immunosuppression may reduce the frequency of cytokine storms, a significant cause of mortality.26,27 methods study design and participants this prospective study from 15th july, 2020 to january 22, 2021. mailto:zanasidik@gmail.com 76 j contemp med sci | vol. 8, no. 1, january-february 2022: 75–78 severity of covid-19 infection among kidney transplant recipients in duhok city, kurdistan region, iraq original z. s. m. saleem eighty kidney transplant recipients visiting duhok kidney diseases and transplantation center diagnosed with sar-cov-19 infection after confirmation by real time polymerase chain reaction (rt-pcr). patient demographics (age, gender), body mass index (bmi), duration of kidney transplantation, comorbidities, medications history, concomitant infections, clinical presentation, immunosuppression regimen and subsequent adjustment, laboratory investigation in form of complete blood count (cbc), liver function tests, serum creatinine, il-6, crp, d-dimer, ldh, imaging, clinical course, and treatment modalities were collected. covid-19 infection classification severity of infection based on world health organization (who) classification, and patient management based on the severity of infection in regard of hospital or home management. immunosuppression was reduced or stopped like antimetabolite (mycophenolate mofetil (mmf) or azathioprine) with or without adjustment of calcineurin inhibitors such as tacrolimus (fk) or cyclosporine. steroids were either kept at the maintenance dose or converted to intravenous for stress dosing. confirmed cases who were not admitted were managed by instructed to self-isolate, monitor temperature and partial oxygen saturation (spo2), and scheduled for weekly follow-up. normal weight for adults is usually when body mass index (bmi) is 18.5 to less than 25, overweight when bmi is 25–29.9 and obesity is when bmi is 30 and more according to center for disease control and prevention.28 in general, adults with sars-cov-2 infection can be grouped into the following severity of illness categories. asymptomatic or presymptomatic infection: individuals who test positive for sars-cov-2 using a virology test (i.e., a nucleic acid amplification test [naat] or an antigen test) but who have no symptoms that are consistent with covid-19. mild illness: individuals who have any of the various signs and symptoms of covid-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell) but who do not have shortness of breath, dyspnea, or abnormal chest imaging. moderate illness: individuals who show evidence of lower respiratory disease during clinical assessment or imaging and who have oxygen saturation (spo2) ≥94% on room air at sea level. severe illness: individuals who have spo2 30 breaths/min, or lung infiltrates >50%. 29 statistics we analyzed the result using ssps (version 17). we used the student’s t-test for comparing the means. the significance level was at p < 0.05. ethics this study was approved by the ethics committee in the college of medicine, university duhok. written consent was obtained from all recruited patients. results eighty kidney transplant recipients involved in our study, characteristics of participants are shown in table 1. male constitutes two third of participants, mean age were 46.6 and mean duration of transplantation is 6.4 years. eighty eight percent of participants were managed at home. hypertension was the most common comorbid disease in our study. fever, dyspnea, cough and diarrhoea were the most common presenting symptoms as shown in table 2. aki was the presenting symptom in four percentages (figure 1). in the current study, severe covid-19 infection seen more in male gender, elderly, obese, longer duration of symptom, kidney transplantation, and those with more comorbidities as shown in table 3. table 1. characteristic of participants in the study characteristic no. (%) male 54 (67.5) female 26 (32.5) age (mean) 46.6 ± 12.2 weight normal overweight obese 47 (58.75) 23 (28.75) 10 (12.5) smoking 4 (5) home management 71 (88.75) hospital management 9 (11.25) mean duration of kidney transplantation in year 6.4 ± 3.5 mean duration of symptoms (days) 6.2 ± 3.2 comorbidities hypertension diabetes mellitus ischemic heart disease stroke 48 (60) 15 (18.8) 3 (3.8) 2 (2.5) hypothyroidism 3 (3.8) others 3 (3.8) mean crp 25 ± 14.7 mg/l mean il-6 26 ± 12.4 pg/ml d-dimer 1085 ± 390 ng/ml severe covid-19 12 (15) death 2 (2.5) table 2. clinical presentation of covid-19 among participants presentation no. (%) fever 28 (35) dyspnea 11 (14) diarrhoea 11 (14) cough 10 (13) fatigue 7 (9) headache 5 (6) acute kidney injury 3 (4) loss of smell 2 (3) others 4 (5) 77j contemp med sci | vol. 8, no. 1, january-february 2022: 75–78 z. s. m. saleem original severity of covid-19 infection among kidney transplant recipients in duhok city, kurdistan region, iraq table 3. characteristic of severe cases in the study characteristic no. (%) male 9 (75) female 3 (25) age (mean) 50.3 ± 12.2 weight normal overweight obese 2 (16.7) 4 (33.3) 6 (50) smoking 1 (8.3) home management 4 (33.3) hospital management 8 (66.6) mean duration of kidney transplantation in year 9.8 ± 3.7 mean duration of symptoms (days) 6.2 ± 2.9 comorbidities 12 (100) mean crp 48 ± 24.5 mean il-6 56 ± 32.6 d-dimer 1873 ± 1024 death 2 (16.6) fig. 1 main symptoms of covid-19 inefction among participants. there was no difference in regard of severity of covid-19 infection among transplant recipient with cyclosporine and tacrolimus group. there was significant difference of inflammatory markers like crp, il-6 and d-dimer among severe and non-severe group (p value of 0.001, p value of < <0.0000001, p value of < <0.0000001 respectively). case fatalities were two, one male from ards and one female from acute coronary syndrome. twelve of 80 patients underwent modification in the immunosuppression drugs. immunosuppression reduction was done in the severely affected patient. the strategic change in immunosuppression regimen was in form of complete cessation of antimetabolites (mycophenolate mofetil, mycophenolic acid, or azathioprine) and reducing tacrolimus dose. no change in immunosuppression regimen done in non-hospitalized and mild form cases. discussion in this study of kidney transplant recipients affected by covid-19 with follow-up of six months, 15% were with severe covid-19 infections, 11.25% required hospital admissions and only one patient needed icu admission. the case fatality of this study population was two patients (2.5%). may be the low rate of infection and mortality in this risky group attributed to that this group of patients are using precautious measure of infection transmission directly after kidney transplantation before the pandemic of covid-19 and also may be they are on continuous small dose of steroid which decease mortality in severe covid-19.30 risk factors for severe covid-19 infection in this study were male gender, old age, comorbidities, obesity, longer duration of covid-19 symptoms and kidney transplantation duration. delay seeking health facilities also found risk factor in this study for severe covid-19 infection. and lastly earlier post-transplant sars-cov-2 infection also was demonstrated as a risk factor for severe infection. fever, dyspnea, cough and diarrhoea were the most common presenting symptoms among this study population, which were significantly associated with a poor clinical outcome (p value <0.001). this was similar to other study done at spain.31 three cases presented as aki (4%), in this study this rate is lower in comparison to other studies.32 inflammatory markers were significantly higher among severe covid-19 cases in comparison to non-severe cases. two cases of 12 severe cases died (16.6%) this rate is lower to study done in china were the rate was 28%.33 but comparable to subsequent publications, these rates were reported to be 8% in new york, 14% in italy, and 12% in spain.34 this is attributed mostly because of age and comorbidity among the study groups. there was no difference in the outcomes of covid-19 infection in transplant recipients receiving cyclosporine and tacrolimus.35 conclusion kidney transplant recipients are on immunosuppressive regimen and they are at high risk for opportunistic infection acquisition like covid-19 infection. risk stratification and poor outcome factor is crucial strategy to prevent spread of the infection. early diagnosis and proper treatment is also important to prevent unfavorable covid-19 infection sequelae in this risky group population. the most important strategy in kidney transplant recipients to prevent covid-19 infection is through adopting preventive measures in particular the use of masks and avoidance of crowded non-ventilated places. the ultimate strategy to effectively prevent covid-19 is vaccination against sars-cov-2.36,37 our protocol in managing transplant recipients with covid-19 infection was based on new and updating reports. larger and longer duration studies needed to adopt the best protocol in managing such risky group of patients.  78 j contemp med sci | vol. 8, no. 1, january-february 2022: 75–78 severity of covid-19 infection among kidney transplant recipients in duhok city, kurdistan region, iraq original z. s. m. saleem references 1. zhu n, zhang d, wang w et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med 2020;382:727–733. 2. burke rm, killerby me, newton s et al. symptom profiles of a convenience sample of patients with covid-19 – united states. mmwr morb mortal wkly rep 2020;69:904–908. 3. fernández-ruiz m, aguado jm. immunomodulatory therapies for covid-19 in solid organ transplant recipients. curr transplant rep 2020. doi: 10.1007/s40472-020-00306-x. 4. craig-schapiro r, salinas t, lubetzky m et al. covid-19 outcomes in patients waitlisted for kidney transplantation and kidney transplant recipients. am j transplant 2020; doi: 10.1111/ajt.16351. 5. pei g, zhang z, peng j et al. renal involvement and early prognosis in patients with covid-19 pneumonia. j am soc nephrol 2020;31:1157–1165, 6. hirsch js, ng jh, ross dw et al. acute kidney injury in patients hospitalized with covid-19. kidney int 2020;98:209–218. 7. fisher m, neugarten j, bellin e et al. aki in hospitalized patients with and without covid-19: a comparison study. j am soc nephrol 2020; 31:2145–2157. 8. kates os, haydel bm, florman ss et al. covid-19 in solid organ transplant: a multi-center cohort study. clin infect dis 2020; doi: 10.1093/cid/ciaa1097. 9. miarons m, larrosa-garcía m, garcía-garcía s et al. covid-19 in solid organ transplantation: a matched retrospective cohort study and evaluation of immunosuppression management. transplantation 2021;105:138–150. 10. alberici f, delbarba e, manenti c et al. a single center observational study of the clinical characteristics and short-term outcome of 20 kidney transplant patients admitted for sars-cov2 pneumonia. kidney int 2020; 97:1083–1088. 11. coll e, fernández-ruiz m, sánchez-álvarez je et al. covid-19 in transplant recipients: the spanish experience. am j transplant; doi: 10.1111/ajt.16369. 12. cravedi p, suraj sm, azzi y et al. covid-19 and kidney transplantation: results from the tango international transplant consortium. am j transplant 2020; 20:3140–3148. 13. fava` a, cucchiari d, montero n et al. clinical characteristics and risk factors for severe covid-19 in hospitalized kidney transplant recipients: a multicentric cohort study. am j transplant 2020;20:3030–3041. 14. cavalcanti ab, zampieri fg, rosa rg et al. hydroxychloroquine with or without azithromycin in mildto-moderate covid-19. n engl j med 2020; 383: 2041–2052. 15. chaudhry zs, williams jd, vahia a et al. clinical characteristics and outcomes of covid-19 in solid organ transplant recipients: a case-control study. am j transplant 2020;20:3051–3060. 16. williamson e, walker aj, bhaskaran k et al. opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients. medrxiv 2020; doi: 10.1101/2020.05.06.20092999. 17. mahalingasivam v, craik a, tomlinson la et al. covid-19 and kidney transplantation: a systematic review. kidney int rep 2020;6:24–45. 18. guan w-j, ni z-y, hu yu, et al. clinical characteristics of coronavirus disease 2019 in china. n engl j med. 2020;382(18):1708-1720. https://doi. org/10.1056/nejmoa2002032. 19. wang d, hu bo, hu c, et al. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama. 2020;323(11):1061. https://doi.org/10.1001/jama.2020.1585. 20. goyal p, choi jj, pinheiro lc, et al. clinical characteristics of covid-19 in new york city. n engl j med. published online april 17, 2020:nejmc2010419. https://doi.org/10.1056/nejmc2010419. 21. zhou f, yu t, du r, fan g, liu y, liu z, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. lancet. 2020 mar 28;395(10229):1054–62. pmid:32171076. 22. kute vb, bhalla ak, guleria s, ray ds, bahadur mm, shingare a, et al. clinical profile and outcome of covid-19 in 250 kidney transplant recipients: a multicenter cohort study from india. transplantation. 2020 dec 21. pmid:33350674. 23. yi sg, rogers aw, saharia a, aoun m, faour r, abdelrahim m, et al. early experience with covid-19 and solid organ transplantation at a us highvolume transplant center. transplantation. 2020 jun. 24. hoek ras, manintveld oc, betjes mgh, hellemons me, seghers l, van kampen jaa, et al. covid-19 in solid organ transplant recipients: a single center experience. transpl int. 2020 may. pmid:32460390. 25. mohamed ih, chowdary pb, shetty s, sammartino c, sivaprakasam r, lindsey b, et al. outcomes of renal transplant recipients with sars-cov-2 infection in the eye of the storm: a comparative study with waitlisted patients. transplantation. 2021 jan 1;105(1):115–120. pmid:33350626. 26. aslam s, mehra mr. covid-19: yet another coronavirus challenge in transplantation. j heart lung transplant. 2020;39(5):408–9. https://doi.org/1 0.1016/j.healun.2020.03.007. 27. a, mascolo s. immunosuppression drug-related and clinical manifestation of coronavirus disease 2019: a therapeutical hypothesis. am j transplant. 2020;20(7):1947–8. https://doi.org/10.1111/ajt.15905. 28. kuczmarski rj, carrol md, flegal km, troiano rp. varying body mass index cutoff points to describe overweight prevalence among u.s. adults: nhanes iii (1988 to 1994). obes res. 1997;5:542–548. 29. (covid-19 treatment guidelines https://www.covid19treatmentguidelines. nih.gov/ on 6/12/2021). 30. jonathan a c sterne, srinivas murthy et al. association between administration of systemic corticosteroids and mortality among critically ill patients with covid-19: a meta-analysis. jama 2020 oct 6;324(13):1330–134. 31. fernández-ruiz m, andrés a, loinaz c, delgado jf, lópez-medrano f, san juan r, et al: covid-19 in solid organ transplant recipients: a single-center case series from spain. am j transplant 20:1849–1858, 2020. 32. rupesh raina, zubin a mahajan, prabhav vasistha. incidence and outcomes of acute kidney injury in covid-19: a systematic review. blood purif 2021 jun 15;1–14. 33. zhou f, yu t, du r, fan g, liu y, liu z, xiang j, wang y, song b, gu x, et al. clinical course and risk factors for mortality of adult inpatients with covid19 in wuhan, china: a retrospective cohort study. lancet. 2020;395(10229):1054–62. 34. aggarwal s, garcia-telles n, aggarwal g, lavie c, lippi g, henry bm. clinical features, laboratory characteristics, and outcomes of patients hospitalized with coronavirus disease 2019 (covid-19): early report from the united states. diagnosis (berl). 2020;7(2):91–6. 35. ahmed daoud, ahmad alqassieh, duaa alkhader, maria aurora posadas salas, vinaya rao, tibor fülöp, karim m soliman . immunosuppression in kidney transplant recipients with covid-19 infection – where do we stand and where are we heading. ren fail 2021 dec;43(1):273–280. 36. walsh ee, frenck rw jr, falsey ar, kitchin n, absalon j, gurtman a, et al. safety and immunogenicity of two rna-based covid-19 vaccine candidates. n engl j med. 2020 383(25):2439–50. 37. anderson ej, rouphael ng, widge at, jackson la, roberts pc, makhene m, et al. safety and immunogenicity of sars-cov-2 mrna1273 vaccine in older adults. n engl j med. 2020;383(25):2427–38. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1166 https://doi.org/10.1111/ajt.15905 https://pubmed.ncbi.nlm.nih.gov/?term=sterne+jac&cauthor_id=32876694 https://pubmed.ncbi.nlm.nih.gov/?term=raina+r&cauthor_id=34130296 https://pubmed.ncbi.nlm.nih.gov/?term=daoud+a&cauthor_id=33491531 https://pubmed.ncbi.nlm.nih.gov/?term=alqassieh+a&cauthor_id=33491531 https://pubmed.ncbi.nlm.nih.gov/?term=posadas+salas+ma&cauthor_id=33491531 https://pubmed.ncbi.nlm.nih.gov/?term=posadas+salas+ma&cauthor_id=33491531 63j contemp med sci | vol. 9, no. 1, january-february 2023: 63–69 original advanced oxidation protein products levels and paraoxonase 1 (arylesterase) activity in patients with thyrodisim hadeel abdul latif jouad, shatha abdul wadood alshammaree* department of chemistry, college of science, university of baghdad, iraq. *correspondence to: shatha abdul wadood alshammaree (e-mail: shath_a@sc.uobaghdad.edu.iq) abstract objectives: the purpose of study was to explore the correlation of advanced oxidation protein products (aopp), which reflect the oxidation of protein and the oxidative stress status, and the activity of antioxidant enzyme of paraoxonase (pon1), using its arylesterase activity in patients with thyrodisim. methods: the study included 100 women with newly diagnosed thyrodisim were subdivided in two groups according to thyroid hormones levels: hyperthyroidism group (50 female patients, age range 18–60 years); and hypothyroidism group (50 female patients, age range 18–75 years). a control group (30 healthy females, age range 18–70 years) was also included for comparison. demographic and clinical measurements for all participants were recorded which include: body mass index (bmi), age, weight, height, lipid profile, vitamin d, thyroid hormones (tsh, t3, and t4, ft3, ft4), aopp levels, and arylesterase activity. results: the serum level of aopp in hypothyroidism group (71.92 ± 19.04 μmol/l) and in hyperthyroidism group (30.41 ± 4.72 μmol/l) were significantly higher than controls (13.12 ± 2.50 μmol/l) (p < 0.05). in contrast, lower aryl esterase activity was found in hypothyroidism (5.03 ± 0.50 u/l), and hyperthyroidism (3.64 ± 0.40 u/l) when compared to control group (6.78 ± 0.62 ku/l) with significant values (p < 0.05). conclusions: these results disclosed a significant role of protein oxidation in patients with hypothyroidism as well as the oxidative stress status. keywords: thyroidism, hypothyroidism, hyperthyroidism, advanced oxidation protein products, aryl esterase activity issn 2413-0516 introduction thyroid gland is a vital organ in the body that plays most important role in the harmony and control of growth as well as the metabolism of the human body.1 it regulates many modified body functions by secreting continuous amount of thyroid hormones to the blood stream and according to body needs.2 the main functions of thyroid hormones are controlling blood pressure; regulate body temperature, control metabolism of protein, fat and carbohydrate in all cells, growth hormone secretion, skeletal maturation, heart rate and other functions.3 thyroidism constitutes the main bulk of endocrine diseases that the physicians have to understand during their clinical practice.4 thyroidism is associated with either inadequate production (hypothyroidism) or excessive of thyroid hormones (hyperthyroidism).5 elevated levels of reactive oxygen species (ros) can trigger oxidative stress, resulting in the apoptosis of astrocytes and thyrocytes. this, in turn, increases the likelihood of developing thyroid dysfunction and neurodegenerative conditions. furthermore, the presence of excessive free radicals increases thyroid thermogenesis causing hyperthyroidism or its excess may cause hypothyroidism by inhibiting iodide uptake.6 advanced oxidation protein products (aopps), which result from the interaction between oxidants and plasma proteins, are considered reliable markers to estimate the degree of oxidant-mediated protein damage.7 most aopps are formed due to increased release of myeloperoxidase (mpo) from activated phagocytes.8 fibrinogen has been recognized as a key molecule responsible for increased plasma aopp; although any protein is susceptible to oxidative modification may contribute to increase plasma aopp concentrations.9 the significance of aopp has been analyzed in numerous diseases as they are widely regarded as easily measurable markers of oxidative stress. protein oxidation was observed in patients suffering from various diseases such as chronic kidney disease (ckd), rheumatoid arthritis, lupus, cancer, and cardiovascular disease.10 higher aopp levels were observed in patients with hyper uric acid levels due to high triglycerides levels and other endogenous factors.11 elevated serum aopp levels were associated with higher risk of all-cause mortality in hemodialysis patients.12 paraoxonase (pon1) is an enzyme synthesized mainly by the liver that is carried into the circulation bound to high-density lipoproteins (hdl). it can be internalized in peripheral cells and, thus, its protein expression is practically ubiquitous in almost all tissues. the enzyme is a lipoperoxide hydrolase that degrades lipoperoxides in lipoproteins and cells, and participates in the subject’s innate immune.13 the pon1 has many hydrolysis activities of substrates such as organophosphate triesters, aryl esters, cyclic carbamates, glucuronides, estrogen esters, and thiolactones while its “natural” substrates are assumed to be lactones.14 experiments with pon1-knockout mice have indicated that the absence of pon1 leads to an increase in endothelial adhesion molecules and oxidative stress, confirming this enzyme’s role in preventing the onset of atherosclerosis.15 a clinical study suggested that the serum antioxidant activity of pon1 (arylesterase activity) is an important factor in protecting from oxidative stress and lipid peroxidation in cardiovascular diseases (cad).11 the enzymatic functions of pon1 may be influenced by various environmental factors, such as the presence of certain elements. for instance, dietary lipids and lipid peroxidation products can lead to a reduction in both pon1 activity and gene expression.16 our goal was to examine oxidative stress status according to the level of aopps and pon1 and their association with thyroidism. (submitted: 20 november 2022 – revised version received: 06 december 2022 – accepted: 22 december 2022 – published online: 26 february 2023) mailto:shath_a@sc.uobaghdad.edu.iq 64 j contemp med sci | vol. 9, no. 1, january-february 2023: 63–69 aopp levels and pon1 (arylesterase) activity in patients with thyrodisim original h.a.l. jouad et al. experimental part subjects in this case-control study, 100 women with previously untreated thyrodisim, aged 18 to 75, were participated. a control group was selected for comparison consisted of thirty healthy females (aged 18–70 years). patients were chosen from al-imameen al-kazimin medical hospital in baghdad, which ran from october 2021 through the end of january 2022. pregnant women, smokers, people with acute or chronic inflammatory diseases, diabetes mellitus (t2dm or t1dm), chronic or hereditary diseases, and family history made up the exclusion criteria. a formed consent was taken from all participants. the study approval was obtained from the scientific and ethical committee. anthropometric measurements body mass index (bmi) was calculated after measurement the standing height by stadiometer, and weight using precision scales by the following equation: weight in (kg)/height² in (meter). collection of blood samples after an overnight fast, 5 ml of venous blood from each participant was taken, allowed to clot at room temperature for 10 minutes, and centrifuged at 3000 rpm for 10 minutes before being used for analysis; the separated serum was kept in eppendroff tubes at –20°c. determination of thyroid hormones, lipid profile and vitamin d levels thyroid profile included triiodothyronine (t3), thyroxine (t4), thyrotropin (tsh), free t3 (ft3), free t4 (ft4) as well as vitamin d level were measured by cobas roshe / hitachi. total cholesterol and high-density lipoprotein cholesterol (hdl-c) were measured using an enzymatic method (hitachi, germany). the friedewald’s equation is used to calculate low-density lipoprotein cholesterol (ldl-c), which states that ldl-c is equal to total cholesterol minus (hdl-c plus triglycerides/5), while (triglycerides/5) was used to calculate vldl. determination of advanced oxidation protein products (aopp) the aopp comprise several chromophores, including pentosidine, carbonyls, and proteins cross-linked by dityrosine, which shows absorbance at a wavelength of 340 nm.17 determination of aopp was based on spectrophotometric detection according to kalousov et al. and the aopp concentration was expressed in chloramines-t equivalents.18 determination of pon-1 arylesterase activity arylesterase activity was determined according to shen et al. 2014, using phenyl acetate as substrate.19 pon-1 activity was measured in 100 mm tris-hcl buffer (ph 8.0) containing 4 mm phenyl acetate substrate and 2 mm cacl2. the absorbance was monitored spectrophotometrically at 270 nm. enzyme activity was calculated with a molar extinction coefficient of 1310 m−1 cm−1. one unit of arylesterase activity hydrolyzed 1 µmol of phenyl acetate per minute. statistical analysis results are expressed as means ± sd for the comparison of non-parametric variables in both groups. the parametric variables of patients and control groups were compared by using anova test (unpaired student t-test). correlation between parameters was assessed by pearson correlation analysis. statistical analysis was performed with spss 26 statistical software. a p value for significance was set at 0.05. results clinical characteristics of the patients and control groups are described in table 1 which revealed non-significant difference in age, and height, when compared with control group, while significant higher levels in weight and bmi of hyperthyroidism group and hypothyroidism than those of control groups were observed. lipid profile showed different significant levels in the two patients groups when compared with those of control group. a significant (p < 0.05) higher tc, ldl-c, and tg levels was found in hypo and hyperthyroidism when compared with control group. vitamin d showed non-significant differences (p > 0.05) between all groups. thyroid stimulating hormone was significantly higher in hypothyroidism group (8.45 ± 3.76 μiu/ml) than controls (2.43 ± 0.96 μiu/ml) which were in turn significantly higher than hyperthyroidism group (0.50 ± 0.56 μiu/ml). in contrast, patients in hyperthyroidism group demonstrated higher level of t3, t4 and ft3 (2.34 ± 1.10 nmol/l, 13.37 ± 2.57 μg/dl and 3.96 ± 0.55 pmol/l, respectively) than either controls (1.25 ± 0.30 nmol/l, 9.6 ± 1.08 μg/dl and 3.48 ± 0.28 pmol/l, respectively) or hypothyroidism group (0.37 ± 0.11 nmol/l, 3.16 ± 0.99 μg/dl and 3.01 ± 1.07 pmol/l) with significant differences between the three group. although ft4 was slightly higher in hyperthyroidism group than controls and hypothyroidism group, the differences were not significant (table 2). the mean serum level of aopp in hypothyroidism group was (71.92 ± 19.04 μmol/l) which was significantly higher than hyperthyroidism group (30.41 ± 4.72 μmol/l) or controls (13.12 ± 2.50 μmol/l) as shown in table 3 and figure 1. in contrast, lower aryl esterase activity was observed in hyperthyroidism (3.64 ± 0.40 u/l) or hypothyroidism (5.03 ± 0.50 u/l) compared to control group (6.78 ± 0.62 u/l), as shown in table 4 and figure 2. correlation of aopp and aryl esterase with other variables pearson’s correlation was used to explore the possible correlation of aopp and aryl esterase with other variables in hyperthyroidism, hypothyroidism and control groups. 65j contemp med sci | vol. 9, no. 1, january-february 2023: 63–69 h.a.l. jouad et al. original aopp levels and pon1 (arylesterase) activity in patients with thyrodisim table 1. mean ± sd of age, weight, height, bmi, lipid profile, and vitamin d in hypothyroidism, hyperthyroidism, and control groups variables controls (n = 30) hypothyroidism (n = 50) hyperthyroidism (n = 50) pvalue age (years) 34.32 ± 12.56 39.1 ± 14.01 34.93 ± 12.76 0.320 weight (kg) 63.96 ± 3.78 *77.66 ± 11.08a *68.0 ± 9.11a 0.000 height (cm) 162.85 ± 7.72 160.26 ± 7.86 163.83 ± 7.47 0.186 bmi (kg/m2) 23.58 ± 1.38 *30.37 ± 3.69a *31.44 ± 3.25a 0.000 tc (mg/dl) 146.86 ± 17.0 *231.93 ± 26.1a *204.20 ± 21.4a,b 0.000 tg (mg/dl) 92.66 ± 16.55 *123.06 ± 46.77a *120.05 ± 41.43a 0.006 hdl-c (mg/dl) 53.59 ± 9.10 52.36 ± 10.78 52.82 ± 11.14 0.903 ldl-c (mg/dl) 102.41 ± 14.57 *171.27 ± 8.54a *143.78 ± 8.96a 0.018 vldl (mg/dl) 24.35 ± 8.49 25.68 ± 9.16 26.86 ± 5.62 0.484 vit d (ng/ml) 28.66 ± 6.99 25.24 ± 7.05 26.29 ± 8.26 0.211 *p value < 0.05. the small liters refer to presence of significance between groups; a: significant when compared with control, b: significant when compared between hypo and hyper. table 2. mean ± sd of thyroid hormones levels in hypothyroidism, hyperthyroidism, and control groups variables controls (n = 30) hypothyroidism (n = 50) hyperthyroidism (n = 50) pvalue tsh (μlu/ml) 2.43 ± 0.96 *8.45 ± 3.76a *0.50 ± 0.56a,b 0.001 t3 (nmol/l) 1.25 ± 0.30 *0.37 ± 0.11a *2.34 ± 1.10a,b 0.001 t4 (μg/dl) 9.6 ± 1.08 *3.16 ± 0.99a *13.37 ± 2.57a,b 0.001 ft3 (pmol/l) 3.48 ± 0.28 *3.01 ± 0.17a 3.96 ± 0.55 0.001 ft4 (ng/dl) 1.24 ± 0.19 1.21 ± 0.82 1.52 ± 1.15 0.285 *p value < 0.05. the small liters refer to presence of significance; a: significant when compared with control, b: significant when compared between hypo and hyper. table 3. mean ± sd of aopp in hypothyroidism, hyperthyroidism, and control groups variables controls (n = 30) hypothyroidism (n = 50) hyperthyroidism (n = 50) pvalue aopp (μmol/l) 13.12 ± 2.50 *71.92 ± 19.04a *30.41 ± 4.72a,b 0.001 *p value < 0.05. the small letters refer to presence of significance; a: significant when compared with control, b: significant when compared between hypo and hyper. fig. 1 mean serum level of aopp in the three groups. in control group the aopp demonstrated a positive correlation significantly with height (r = 0.429, p = 0.023) and a negative correlation significantly with ft3 (r = –0.422, p = 0.025). aryl esterase, on the other hand, showed a negative correlation significantly with each of bmi (r = –0.401, p = 0.034) and hdl (r = –0.384, p = 0.044) as shown in table 5, figure 3. in patients group with hyperthyroidism the aopp showed a negative correlation significantly with ft3 (r = –0.359, p = 0.049). aryl esterase, on the other hand, a negative correlation significantly was found with vitamin d (r = –0.363, p = 0.048) as shown in table 6, figure 4. in patients group with hypothyroidism the aopp showed negative correlation significantly with tg (r = –0.353, p = 0.050), while, aryl esterase showed non significant correlation with any variable, as presented in table 7, and figure 5. 66 j contemp med sci | vol. 9, no. 1, january-february 2023: 63–69 aopp levels and pon1 (arylesterase) activity in patients with thyrodisim original h.a.l. jouad et al. table 4. mean ± sd of aryl esterase in hypothyroidism, hyperthyroidism, and control groups variables controls (n = 30) hypothyroidism (n = 50) hyperthyroidism (n = 50) pvalue aryl esterase (u/l) 6.78 ± 0.62 *5.03 ± 0.50a *3.64 ± 0.40a,b 0.001 *p value < 0.05. the small letters refer to presence of significance; a: significant when compared with control, b: significant when compared between hypo and hyper. fig. 2 mean serum level of aryl esterase in the three groups. table 5. pearson correlation of aopp and aryl esterase activity with other variables of control group variables aopp aryl esterase r p-value r p-value age –0.006 0.974 –0.114 0.565 weight 0.310 0.109 –0.179 0.361 height 0.429 0.023 –0.227 0.245 bmi –0.034 0.864 –0.401 0.034 tsh 0.295 0.127 0.161 0.414 t3 –0.185 0.347 0.303 0.117 t4 0.306 0.133 0.260 0.181 ft3 –0.422 0.025 –0.044 0.824 ft4 –0.134 0.496 0.004 0.986 tc –0.061 0.758 0.056 0.779 tg 0.266 0.179 0.267 0.179 hdl-c 0.176 0.369 –0.384 0.044 ldl-c 0.212 0.280 0.009 0.966 vldl 0.204 0.299 –0.058 0.770 vit. d –0.016 0.758 –0.268 0.168 aryl esterase 0.076 0.700 – – fig. 3 scatter plot and regression line between a: height and aopp, b: ft3 and aopp, c: bmi and arylesterase, d: hdl and arylesterase in control group. discussion thyroid hormones play major roles in cell growth, development, and metabolism. considerable research supports a relationship between the thyroid hormones and pathophysiology of various thyroidism types. notably, both hyperthyroidism and hypothyroidism appear to be associated with oxidative stress in animal and human diseases, indicating involvement of the thyroid hormone in disease progression.20 the lower vitamin d levels may be due to poor nutrition or inadequate sunlight exposure, which in turn increases susceptibility to autoimmune thyroid disorders. our result was in agreement with that of a study by rasool et al.6 sarsat et al. provided information on aopp, claiming that they are able to trigger the synthesis of inflammatory cytokines in neutrophil leukocytes and monocytes, and seem to act as inflammatory mediators.21 despite a high frequency of overweight and obese subjects in this study, no correlation was found between aopp and bmi. this finding is consistent with the study conducted by codoñer-franch et al. in obese children, where no correlation was found between aopp and anthropometric measurements (bmi, wc).22 67j contemp med sci | vol. 9, no. 1, january-february 2023: 63–69 h.a.l. jouad et al. original aopp levels and pon1 (arylesterase) activity in patients with thyrodisim table 6. pearson’s correlation of aopp and aryl esterase activity with other variables of patients with hyperthyroidism variables aopp aryl esterase r p-value r p-value age 0.101 0.595 –0.020 0.917 weight –0.041 0.813 –0.009 0.962 height 0.092 0.630 –0.023 0.902 bmi –0.230 0.221 0.231 0.220 tsh 0.101 0.597 –0.026 0.893 t3 0.051 0.789 –0.191 0.312 t4 0.157 0.409 –0.063 0.741 ft3 –0.359 0.049 –0.070 0.713 ft4 0.010 0.958 –0.124 0.515 tc 0.228 0.225 –0.117 0.539 tg –0.079 0.679 –0.113 0.551 hdl-c –0.002 0.993 0.014 0.943 ldl-c –0.193 0.306 –0.074 0.699 vldl –0.097 0.612 0.011 0.954 vit. d 0.082 0.665 –0.363 0.048 aryl esterase –0.183 0.333 – – fig. 4 scatter plot and regression line between: a: ft3 and aopp, b: vitamin d and aryl esterase in patients with hyperthyroidism. table 7. pearson correlation of aopp and aryl esterase activity with other variables of patients with hypothyroidism variables aopp aryl esterase r p-value r p-value age –0.021 0.911 –0.183 0.343 weight –0.016 0.933 0.080 0.672 height –0.102 0.590 –0.100 0.599 bmi 0.062 0.746 0.171 0.367 tsh 0.094 0.621 0.050 0.794 t3 –0.678 0.347 0.185 0.327 t4 0.280 0.134 –0.079 0.687 ft3 –0.185 0.328 0.119 0.531 ft4 0.146 0.385 0.269 0.151 tc –0.020 0.915 0.125 0.510 tg –0.353 0.050 0.109 0.566 hdl-c 0.170 0.370 0.085 0.665 ldl-c 0.043 0.819 0.201 0.288 vldl –0.292 0.117 0.020 0.915 vit. d –0.196 0.300 –0.222 0.238 aryl esterase 0.243 0.195 – – fig. 5 scatter plot and regression line between triglycerides and aopp in patients with hypothyroidism. our study showed a negative association between tg and aopp in patients with hypothyroidism, which could be attributed to a combination of metabolic changes as explained by the study of diana et al.23 hyperlipidemia is associated with oxidative stress and inflammation.24 liu et al.25 have reported that aopp are an important component of the complex interaction between inflammation and oxidative stress with the atherogenic process. the formation of aopp is mediated by hypochlorous acid (hclo) arising from the action of myeloperoxidase, the same compound that promotes oxidation of ldl-c (ox-ldl).26,27 pon1 offers several benefits, including enhancing hdl cholesterol-mediated efflux from macrophages, safeguarding ldl from oxidation by reducing lipid peroxide levels, hindering ox-ldl uptake by macrophages, which ultimately prevents macrophage foam cell formation, and inhibiting macrophage cholesterol biosynthesis.28 pon1 antioxidant activity is inversely correlated to carotid intima-media thickness. the hydrolytic lactonase, arylesterase, and paraoxonase activities of pon1 are all inactivated under oxidative stress, and epidemiological evidence shows that low serum pon1 activity is associated with many pathological diseases.11,29 our study was in contract with a study by azizi et al.30 who found significant lower pon1 activity in both 68 j contemp med sci | vol. 9, no. 1, january-february 2023: 63–69 aopp levels and pon1 (arylesterase) activity in patients with thyrodisim original h.a.l. jouad et al. that indicates the presence of oxidative stress. the fact that aopp increased could be attributed to metabolic changes such as being overweight, obesity, and hypertriglyceridemia. therefore, aopp could be considered a good indicator and a therapeutic target by appropriate dietary supplementation of antioxidants. acknowledgment the authors would like to thank the staff in al-imameen al kazimin medical hospital for their assistance and support. conflict of interest the writers declare that there are no discorded of interest regarding the publication of this paper.  hyperthyroidism and hypothyroidism patients that lead them to conclude that the observed increased ldl-c oxidation in thyroid dysfunction, at least to some extent, can be attributed to reduced pon1 activity. low pon-1 activity in this study may be due to high bmi and dyslipidemia as supported by previous study that illustrated a link between low-hdl-pon activity and membrane peroxidation in obese and dyslipidemia patients.31 a significant detrimental effect of overt hypothyroidism on the antioxidant pon-1 serum levels compared to normal healthy controls was observed.32 conclusion in conclusion, aopp concentrations were observed to increase while the pon1 activity decreases in both types of thyroidism references 1. földi m, földi e, strößenreuther r, kubik s, editors. földi’s textbook of lymphology: for physicians and lymphedema therapists. elsevier health sciences; 2012 feb. 21 p 20. 2. wang, h.-q.; zhang, w.-d.; yuan, b.; zhang, j.-b. advances in the regulation of mammalian follicle-stimulating hormone secretion. animals 2021, 11, 1134. 3. hall, j. e. (2015). guyton and hall textbook of medical physiology e-book. elsevier health sciences. 4. tomasz bednarczuk, roberto attanasio, laszlo hegedüs, endre v. nagy, roberto negro, enrico papini, petros perros, marek ruchała. use of thyroid hormones in hypothyroid and euthyroid patients. endokrynol pol. 2021;72(4):357–365. 5. roy moncayo, and helga moncayo,. practical guidelines for diagnosing and treating thyroid disease based on the womed metabolic model of disease focusing on glycolysis and coenzyme q10 deficiency a clinical alternative to the 2021 retired clinical practice guidelines of the endocrine society diagnostics 2022, 12(1), 107; https://doi.org/10.3390/ diagnostics12010107. 6. rasool mahmood, malik arif, saleem shamaila, ashraf muhammad abdul basit, khan altaf qadir, waquar sulayman, zahid ayesha, shaheen sumaira, abu-elmagd muhammad, gauthaman kalamegam, pushparaj peter natesan. role of oxidative stress and the identification of biomarkers associated with thyroid dysfunction in schizophrenics. frontiers in pharmacology 12, 2021. https://www.frontiersin.org/articles/10.3389/ fphar.2021.646287 doi:10.3389/fphar.2021.646287. 7. witko-sarsat v, friedlander m, capeillere-blandin c, et al. advanced oxidation protein products as a novel marker of oxidative stress in uremia. kidney int 1996;49:1304–13. 8. capeillere-blandin c, gausson v, descamps-latscha b, witko-sarsat v. biochemical and spectrophotometric significance of advanced oxidized protein products. biochim biophys acta 2004;1689:91–102. 9. camps, j.; marsillach, j.; joven, j. the paraoxonases: role in human diseases and methodological difficulties in measurement. crit. rev. clin. lab. sci. 2009, 46, 83–106. 10. barsotti a, fabbi p, fedele m, et al. role of advanced oxidation protein products and thiol ratio in patients with acute coronary syndromes. clin biochem 2011;44:605–11. 11. leocádio, paola caroline lacerda; goulart, alessandra carvalho; santos, itamar souza; lotufo, paulo andrade; bensenor, isabela martins; alvarezleite, jacqueline isaura. lower paraoxonase 1 paraoxonase activity is associated with a worse prognosis in patients with non-st-segment elevation myocardial infarction in long-term follow-up. coronary artery disease, volume 33, number 7, 12 september 2022, pp. 515–522(8) doi: https://doi.org/10.1097/mca.0000000000001181. 12. zhou, c., zhang, y., chen, j. et al. association between serum advanced oxidation protein products and mortality risk in maintenance hemodialysis patients. j transl med 19, 284 (2021). https://doi.org/10.1186/s12967-02102960. 13. camps, j.; castañé, h.; rodríguez-tomàs, e.; baiges-gaya, g.; hernándezaguilera, a.; arenas, m.; iftimie, s.; joven, j. on the role of paraoxonase-1 and chemokine ligand 2 (c-c motif ) in metabolic alterations linked to inflammation and disease. a 2021 update. biomolecules 2021, 11, 971. 14. tavori, h.; khatib, s.; aviram, m.; vaya, j. bioorganic & medicinal chemistry characterization of the pon1 active site using modeling simulation, in relation to pon1 lactonase activity. bioorganic med. chem. 2008, 16, 7504–7509. 15. shekhanawar, m.; shekhanawar, s.m.; krisnaswamy, d.; indumati, v.; satishkumar, d.; vijay, v.; rajeshwari, t.; amareshwar, m. the role of “paraoxonase-1 activity” as an antioxidant in coronary artery diseases. j. clin. diagnostic res. 2013, 7, 1284–1287. 16. freese, r.; alfthan, g.; jauhiainen, m.; basu, s.; erlund, i.; salminen, i.; aro, a.; mutanen, m. high intakes of vegetables, berries, and apples combined with a high intake of linoleic or oleic acid only slightly affect markers of lipid peroxidation and lipoprotein metabolism in healthy subjects. am. j. clin. nutr. 2002, 76, 950–960. [crossref ] 17. capeillere-blandin c, gausson v, descamps-latscha b, witko-sarsat v. biochemical and spectrophotometric significance of advanced oxidized protein products. biochim biophys acta 2004;1689:91–102. 18. witko-sarsat v, friedlander m, capeillere-blandin c, nguyen-khoa t, nguyen at, zingraff j, jungers p, deschamps-latscha b: advanced oxidation protein products as a novel marker of oxidative stress in uraemia. kidney int 49: 1304–1313, 1996. 19. shen h, li m, wang b, lai ik, robertson lw, ludewig g. dietary antioxidants (selenium and n-acetylcysteine) modulate paraoxonase 1 (pon1) in pcb 126-exposed rats. environmental science and pollution research. 2014; 21(10):6384–6399. 20. mancini, a.; di segni, c.; raimondo, s.; olivieri, g.; silvestrini, a.; meucci, e.; curro, d. thyroid hormones, oxidative stress, and inflammation. mediat. inflamm. 2016, 2016, 6757154. 21. witko-sarsat v, gausson v, descamps-latscha b. are advanced oxidation protein products potential uremic toxins? kidney int suppl. 2003; 84: 11–14. 22. p. codoñer-franch, s. tavárez-alonso, r. murria-estal, m. tortajada-girbés, r. simó-jordá, e. alonso-iglesias. elevated advanced oxidation protein products (aopps) indicate metabolic risk in severely obese children. nutr metab cardiovasc dis, 22 (2012), pp. 237–243 http://dx.doi.org/10.1016/j. numecd.2010.06.002. 23. villalpando sánchez dc, alvarez aguilar c, gómez garcía a. advanced oxidation protein products and their relationship with cardiovascular risk factors in young apparently healthy people. clin invest arterioscler. 2017;29:209—215. 24. m.m. duarte, r.n. moresco, t. duarte, a. santi, m.d. bagatini, i.b. da cruz, et al. oxidative stress in hypercholesterolemia and its association with ala16val superoxide dismutase gene polymorphism. clin biochem, 43 (2010), pp. 1118-1123 http://dx.doi.org/10.1016/j.clinbiochem.2010.07.002. 25. s.x. liu, f.f. hou, z.j. guo, r. nagai, w.r. zhang, z.q. liu, et al. advanced oxidation protein products accelerate atherosclerosis through promoting oxidative stress and inflammation. arterioscler thromb vasc biol, 26 (2006), pp. 1156–1162 http://dx.doi.org/10.1161/01.atv.0000214960.85469.68. 26. n.r. madamanchi, m.s. runge. mitochondrial dysfunction in atherosclerosis. circ res, 100 (2007), pp. 460–473 http://dx.doi.org/10.1161/01. res.0000258450.44413.96. https://sciprofiles.com/profile/366135 https://sciprofiles.com/profile/author/etnsrjvjvxnyost1ejdctlpcb0fjzklwtnr4egzsymnxdwlvzwpidfk1ut0= https://doi.org/10.3390/diagnostics12010107. https://doi.org/10.3390/diagnostics12010107. https://www.frontiersin.org/articles/10.3389/fphar.2021.646287%20doi:10.3389/fphar.2021.646287 https://www.frontiersin.org/articles/10.3389/fphar.2021.646287%20doi:10.3389/fphar.2021.646287 https://www.ingentaconnect.com/search;jsessionid=44rlirphbn0fi.x-ic-live-02?option2=author&value2=leoc%c3%a1dio,+paola+caroline+lacerda https://www.ingentaconnect.com/search;jsessionid=44rlirphbn0fi.x-ic-live-02?option2=author&value2=goulart,+alessandra+carvalho https://www.ingentaconnect.com/search;jsessionid=44rlirphbn0fi.x-ic-live-02?option2=author&value2=santos,+itamar+souza https://www.ingentaconnect.com/search;jsessionid=44rlirphbn0fi.x-ic-live-02?option2=author&value2=santos,+itamar+souza https://www.ingentaconnect.com/search;jsessionid=44rlirphbn0fi.x-ic-live-02?option2=author&value2=alvarez-leite,+jacqueline+isaura https://www.ingentaconnect.com/search;jsessionid=44rlirphbn0fi.x-ic-live-02?option2=author&value2=alvarez-leite,+jacqueline+isaura https://www.ingentaconnect.com/content/wk/cardi;jsessionid=44rlirphbn0fi.x-ic-live-02 https://www.ingentaconnect.com/content/wk/cardi;jsessionid=44rlirphbn0fi.x-ic-live-02 https://doi.org/10.1097/mca.0000000000001181. https://doi.org/10.1186/s12967-021-02960. https://doi.org/10.1186/s12967-021-02960. http://dx.doi.org/10.1016/j.numecd.2010.06.002. http://dx.doi.org/10.1016/j.numecd.2010.06.002. http://dx.doi.org/10.1016/j.clinbiochem.2010.07.002. http://dx.doi.org/10.1161/01.atv.0000214960.85469.68. http://dx.doi.org/10.1161/01.res.0000258450.44413.96. http://dx.doi.org/10.1161/01.res.0000258450.44413.96. 69j contemp med sci | vol. 9, no. 1, january-february 2023: 63–69 h.a.l. jouad et al. original aopp levels and pon1 (arylesterase) activity in patients with thyrodisim 27. haneen muhanedalnajer, shatha abdul wadood al-shammaree and isam n. salman. determination of advanced oxidative protein products levels and its correlation with inflammation in diabetic foot patients. biochem. cell. arch. vol. 20, no. 2, pp. 5843–5847, 2020. 28. cohen, e.; aviram, m.; khatib, s.; artoul, f.; rabin, a. free radical biology and medicine human carotid plaque phosphatidylcholine specifically interacts with paraoxonase 1, increases its activity, and enhances its uptake by macrophage at the expense of its binding to hdl. free radic. biol. med. 2014, 76, 14–24. 29. m. a. ahmed, sh. a. wadood, q. a. mahdi. assessment of follicular fluid paraoxonase activity with pregnancy outcomes in women undergoing ivf/ icsi. egypt. j. chem. vol. 64, no. 6 pp. 2895–2902 (2021). 30. azizi f, raiszadeh f, solati m, etemadi a, rahmani m, arabi m. serum paraoxonase 1 activity is decreased in thyroid dysfunction. j endocrinol invest. 2003 aug;26(8):703-9. doi: 10.1007/bf03347350. pmid: 14669822. 31. ferretti g, bacchetti t, masciangelo s, bicchiega v. hdl-paraoxonase and membrane lipid peroxidation: a comparison between healthy and obese subjects. obesity (silver spring) 2010;18:1079–84. 32. al-naimi ms, hussien nr, rasheed ha, al-kuraishy hm, al-gareeb ai. levothyroxine improves paraoxonase (pon-1) serum levels in patients with primary hypothyroidism: case-control study. j adv pharm technol res. 2018 jul-sep;9(3):113–118. doi: 10.4103/japtr.japtr_298_18. pmid: 30338238; pmcid: pmc6174702. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1317 207j contemp med sci | vol. 8, no. 3, may-june 2022: 207–212 original association between variant alleles of the x-ray cross complementing gene (xrcc1) with benzo[a]pyrene levels in iraqi workers alaa. r. omrain1, estabraq a.r. al-wasiti1,*, mohammed jassim hamzah2 1department of chemistry and biochemistry, college of medicine, university of al-nahrain, baghdad, iraq. 2college of pharmacy, university of al-nahrain, baghdad, iraq. *correspondence to: estabraq a.r. al-wasiti (e-mail: estabraqalwasiti@nahrainuniv.edu.iq) abstract objectives: assessment of a single nucleotide polymorphism of the xrcc1 (codon arg399gln) gene and its association with levels of benzo[a]pyrene in blood of car repairers and control group. methods: the current study included (111) participants, (37) of the iraqi car repairers, (37) sellers of spare part, and (37) healthy (control), with the same age range. benzo[a]pyrene was determined by using hplc technique. alleles frequency of single nucleotide polymorphisms of xrcc1 gene was determined by restriction fragment length polymorphisms (pcr-rflp). results: the results of current study indicated that there is a highly significant increase of b[a]p level (p = 0.0001) in the repairers group compared to the groups of spare part sellers and control together. the genotype frequencies studies of xrcc1 gene of car repairers group and controls. the effect of rs25487 genotypes on exposure to pahs include higher levels of pahs and pah-dna adduct in tt genotype followed by ct genotype and cc showed lower levels. conclusion: as results of prolonged exposure to the higher levels of pahs that iraqi car repairs suffer from, they have high levels b[a]p. especially those who have the tt genotypes, as results of the inefficiency of their dna repair system. compared to the other genotypes. keywords: polycyclic aromatic hydrocarbons (pahs), b[a]p, xrcc1 gene, car repairers, spar part seller issn 2413-0516 introduction polycyclic aromatic hydrocarbons (pahs) are a large group of aromatic organic compounds comprising two or more fused benzene rings, the main sources of these global pollutants are industrial processes and incomplete combustion of organic materials, its produced as complex mixture contain more than a hundred compounds.1 among all the compounds, b[a]p is considered a representative of all pahs compounds, and it is often applied in many studies as a positive control in biological assays for other pahs individuals.2,3 also benzo[a]pyrene, among all individuals of the pahs, represent one of the global studied pollutants and its found in car exhaust fumes. numerous studies have confirmed that its a potent immunosuppressive, proinflammatory and carcinogenic agent.3,4 fuel-powered cars and machines are protable sources that emit pahs and thus increasing pollution in areas. auto repairers are a group of the population are exposed to pahs emitted from car exhaust on a daily basis, the main routes exposure to pahs via inhalation or skin contact. this group have a high chance of exposure to pahs, because they are in direct contact with fume emitted by cars containing pahs, in addition to other reasons related to the personal hygiene and wear occupational safety equipments during work.4-6 garage workers are exposed to complex mixture of pahs, because of their occupation, also automobile mechanics are at an increased risk skin, lung, urinary tract cancer.7 several recent studies indicate that car repair shops are considered as anthropological sources of pahs and heavy metals in some cities of the world.8 generally, the carcinogencity of pahs espically b[a]p is begins after phase i biotransformation by inducible p450 superfamily (cyp1). and later the reactive speices like (benzo[a]pyrene-7,8-diol-9,10-epoxides) (bpde) are formed and mediated by the cyp2e1. the interaction between environmental and genetics factors play an effective role in the development of most cancers in humans.6 bulky pahs-dna adducts are repaired by two pathways, the first is base excision repair pathway (ber) and second is single strand breaks (ssb), where the x-ray repair cross-complementing gene (xrcc1) is implicated in both pathways, and it is play vitol role in the base excision repair pathway (ber).8 protein xrcc1 acts as a scaffolding protein in ber, via interaction with the adp-ribose polymerase, dna polymerase β, and dna ligase iii.14 a polymorphism of the xrcc1 gene at codon results from substitution of amino acidarginine in the place of glutamine, resulting in an ineffective repair pathway.9 we conducted the present study to investigate the relationship between pahs exposure in car repaires, and dna repairing gene (xrcc1) polymorphisms in detecting workers at risk. especially, the reports on occupational exposure and related health risks are almost non-existent, and this reflects paucity of availability of survey data and criteria for estimating whether unsafe exposure has occurred. subjects, materials and methods the current cross-sectional study included 111 male volunteers with age range (25–45 years). volunteers were divided according to their exposure to vehicle exhaust emissions into three groups: (i) (no. = 37) car repairers (as highly exposed group), (no. = 37) spare part sellers, and (iii) (no. = 37) non-exposed volunteers. all exposed subjects were matched in age, smoking status with unexposed group. five ml of venous blood sample was taken from each volunteers and divided into two tubes: a. one ml was placed into disposable edta containing tubes and stored at –20oc until it was used in the genotyping study after dna extraction. quantity and quality of extracted dna was determined by nano-drop, uk, using the scanning power of the diode assembly, within the wavelength 200–320 nm. the quantity and quality of mailto:estabraqalwasiti@nahrainuniv.edu.iq 208 j contemp med sci | vol. 8, no. 3, may-june 2022: 207–212 association between variant alleles of the x-ray cross complementing gene (xrcc1) original a.r. omrain et al. extracted dna was determined by calculating the (260/280) and (260/230) ratios. where samples that were (260/280) ratio less than 1.8 and/or (260/280) ratio 2, were re-extracted. the integrity and molecular weight of extracted dna was determined by agarose gel electrophoresis according samboork and russell. b. four milliliters of blood sample pushed slowly into disposable gel containing tubes, and was allowed to clots 20 minutes at room temperature. after coagulation, the sera were separated by centrifugation at 3000 rpm for 10 min and stored at –20oc until it was used in the estimation of b[a]p. quantification of benzo[a]pyrene pahs extraction five ml of aqueous ethanolic and 2n of sodium hydroxide [9:1] was added to 1 ml of serum, and solution was ultrasound for 2 hours at 42oc. the sample was extracted by mixing it well with 5 ml of n-hexane and ultrasoned at 42oc for 2 hours, and the organic layer has been withdrawn to another container. anhydrous sodium sulphate was added to clear supernatant to remove excess water, this sample was extracted two times with 10 ml of n-hexane. the extracts were evaporated to dryness at 40oc, and suspended with 0.25 ml of acetonitrile as shown in figure 1 and pah contents in the samples were determined by hplc.10,11 hplc analysis pyrene and benzo[a]pyrene in serum were analysed by using c18 reverse phase column. the rate of flow was 1 ml min–1, 10 µl of samples were injected into knauerhplc (with system components listed in table 1) and monitored at wavelength 254 nm. the peaks of individual pah (pyrene and benzo[a]pyrene) were recognized by comparing with the retention times of authenic standards as in figure 2. a gradient of mobile phase was prepared from water as (a) component, and acetonitrile as (b) component, and a gradient details is give in table 2. a serial dilution of standard of pahs mixture was prepared by acetonitrile to achieve a concentration from 0.02–200 ng/ml. pcr-rflp analysis design according to,12 the genotyping technique was selected, while the primers are designed according to protocol of 13 briefly as follows: the primers were designed by the aid of ncbi-primer blast online software (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/index.cgi?link_loc=blasthome), ar87f taagcaggcttcacagagcc ar87r tggcatcttcacttctgccc. and the produced primers were checked for specificity of their target sequence by performing a blast against the human genome, then the primers pair was selected according to the demand criteria such as: product length, the similarity of melting temperature, primers length, specificity, etc. then the mutations was interred according to the design demands. the primer ability to form secondary structure was checked by the aid of oligo calc online software (http://www.basic. northwestern.edu/biotools/oligocalc.html), the primer would be rejected if it had 5 bases or more able to form self-dimerization and/or it had 4 bases able to form hairpin. each primers pair was checked for dimer formation by the aid of “multiple primer analyzer” online software from thermo fisher scientific inc.©, the sensitivity of the software was adjusted to the value 2, the primer pair would be rejected if it made any dimers in this degree of sensitivity. table 1. system components of hplc, knuaer, germany no component model or version company and origin 1 binary high pressure gradient pump p6.1l knuaer, germany 2 diode array detector dad 2.1l knuaer, germany 3 sample loop (20 µl) and injector d1357 knuaer, germany 4 analyses and system control software claritychrom, v 7.4.2.107 dataapex, czech republic table 2. the gradient of mobile phase time (min) mobile a (water) concentration% mobile b (acetonitrile) concentration% flow rate (ml/min) initial 40 60 0.7 10 0 100 0.7 35 0 100 0.7 fig. 1 comparison of levels of b[a]p in studied groups (car repairers and spare part sellers). fig. 2 pcr-reaction products at different annealing temperatures (55, 58, 60, 63, 66 oc), under same optimized pcr conditions of arg399gln (rs25487). http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?link_loc=blasthome http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?link_loc=blasthome 209j contemp med sci | vol. 8, no. 3, may-june 2022: 207–212 a.r. omrain et al. original association between variant alleles of the x-ray cross complementing gene (xrcc1) restriction enzyme selection the selection of the suitable restriction enzyme (asuc2i cc^sgg sib) was performed by the aid of watcut online software (http://watcut.uwaterloo.ca/template), we selected the restriction enzyme according to several criteria such as: the lesser primer mutations needed, the distance of mutation from the variant, compatibility of the produced primers, cost and availability. optimization of pcr condition of arg399gln (rs25487) the mixture shown in table 3. was used as a preliminary mixture in the pcr reaction. then, different annealing temperatures were used to obtain a specialized and efficient product. the temperatures and optimized pcr condition used are shown in table 4. arg399gln (rs25487) genotyping genotyping of xrcc1 (rs25487) polymorphisms was conducted by pcr-rflp technique. and the restriction digestion of amplicon was digested by (asuc2i), and the reaction mixdture whose components were used: one unit of enzyme 0.25 µl, 5 µl of pcr product, 1.5 µl of buffer, and volume was completed to 15 µl by molecular graded water. the reaction mixture accubated in 37oc overnight. then the reaction product resolved in 2% of agarose gel. statistical analysis the statistical calculations included in this study were carried out using spss software (ibm corp. released 2012. ibm spss statistics for windows, version 21.0. armonk, ny: ibm. corp. usa) and microsoft excel (2010 microsoft corp. usa). the results expressed as mean ± sem, and p < 0.05 was considered table 3. optimized reaction mixture for pcr no composition concentration volume 1 master mix 2.5x 8 µl 2 forward primer 10 pmol/µl 1 µl 3 reverse primer 10 pmol/µl 1 µl 4 dna sample 10-20 ng/µl 2 µl 5 nucleases free water 7.5 µl 6 mgcl 2 25 mm 0.5 µl total volume 20 µl table 4. optimized pcr condition of arg399gln (rs25487) stage step temperature oc time no. of cycles 1 initial denaturation 94 5 min 1 2 dna denaturation 94 30 sec 35 primer annealing 55–67 30 sec extension 72 30 sec 3 final extension 72 5 min 1 table 5. comparison of b[a]p levels among studied groups based on t-student test group control mean ± sd car repaires mean ± sd spare part sellers mean ± sd p-value b[a]p – 1.97 ± 0.06 0.32 ± 0.045 <0.0001 fig. 3 pcr-rflp of (rs25487) genotyping; lane l = 100bp dna ladder; lane (362bp) = tt genotype; lanes (221bp + 141bp) = cc genotype; and lanes (362bp + 221bp + 141bp) = tt genotype. statistically significant. to evaluate the presence of significant differences, one way anova, and unpaired-sample t-test were employed. also regression analysis used to asses presence of correlations, and the logistic regression was performed to adjust odd ratio. results and discussion the results demonstrate that the highest level of b[a]p was recorded in the group of car repairers, followed by the group of workers whose work in the same area. where the results showed highest level of b[a]p in the group of car repairers (1.97 ± 0.06), followed by the group of spare part sellers (0.32 ± 0.04). where levels of b[a]p were significantly higher (p < 0.001) in group of car repairers compared to the group of spare part sellers (table 5, and figure 1). b[a]p is considered the first chemical carcinogen that was discovered among all the pahs individuals, and it was observed in the car exhaust fume.13 its carcinogenic pathway depends on the enzyme metabolize it, starting from the first step and ending with the mutagenic metabolite benzo[a]pyrene-7,8 diol-9,10-epoxide (bpde).14-15 also the recent studies indicated that the automobile mechanics are at an increased risk of skin and lung cancers as a result of exposure to pahs.16 annealing temperatures range (55–67oc) were used in optimization pcr conditions of arg399gln (rs25487) to obtain efficient product. the results obtained are shown in figure 2. the best pcr product was obtained at a temperature of 63oc . the results obtained from pcr-rflp of (rs 25487) genotyping shown in the figure 3, where the cc allele revealed 221bp and 141bp fragments, whiles tt allele was not digested, and visualized at 362bp single product. ct (hetrozoyget) allele revelaed three bands of 362bp, 221bp, and 141bp. the results of rflp-pcr for snp (rs25487) genotyping of xrcc1 gene are listed in table 6 for the car repaires, spar part sellers and control subjects. where the results of genotype http://watcut.uwaterloo.ca/template 210 j contemp med sci | vol. 8, no. 3, may-june 2022: 207–212 association between variant alleles of the x-ray cross complementing gene (xrcc1) original a.r. omrain et al. table 6. frequencies association between genotyping of xrcc1 gene of car repairs group samples and controls genotype control (n = 37) car repairers (n = 37) p-value odds ratio 95% c.i. cc wild 4 (10.81%) 25 (67.56%) <0.0001 17.18 4.49 to 9.702 ct heterozygous 16 (43.24%) 9 (24.32%) 0.088 0.421 0.156 to 1.139 tt mutant 17 (45.94%) 3 (8.10%) 0.001 0.103 0.027 to 0.398 allele frequency % allele control (n = 37) car repairers (n = 37) p-value odds ratio 95% c.i. c 24 59 <0.0001 8.1944 3.88 to 17.29 table 7. frequencies association between genotyping of xrcc1 gene of spare part sellers group samples and controls genotype control (n = 37) spare part sellers (n = 37) p-value odds ratio 95% c.i. cc wild 4(10.81%) 21(56.75%) 0.0001 10.828 3.181to36.849 ct heterozygous 16(43.24%) 9(24.32%) 0.088 0.421 0.156 to 1.139 tt mutant 17(45.94%) 7(18.91%) 0.0155 0.274 0.096 to 0.781 allele frequency % allele control (n = 37) worker (n = 37) p-value odds ratio 95% c.i. c 24 51 <0.0001 5.503 2.059 to 10.22 t 50 21 frequencies of car repaires specimens revealed that cc genotype were higher in sample of auto repairs (45.94%) compared to the control group (16.21%), this is showed a significant differences (<0.0001) with an odd ratio equal to (17.8). the heterozygous genotype ct had lower frequency in samples of car repaires (29.72%) than controls (32.34%) and this combined with non-significant (p = 0.08) and low odd ratio (0.421). the frequency of tt genotype was higher in control samples (51.35%) compared to the samples of car repaires (24.32%) with odd ratio (p <0.13) differences was significant (p = 0.001). the results of genotypes of xrcc1 gene of are listed in table 7 for the spare part sellers and control subjects. where the results of genotype frequencies of spare part sellers specimens revealed that cc genotype were higher in spare part sellers (45.94%) compared to the control group (16.21%), this is showed a significant differences (p < 0.0001) with an odd ratio equal to (17.8). the heterozygous genotype ct had lower frequency in samples of spare part sellers (29.72%) than controls (32.34%) and this combined with non-significant (p = 0.08) and low odd ratio (0.421). the frequency of tt genotype was higher in control samples (51.35%) compared to the samples of spare part sellers (24.32%) with odd ratio (p = 0.13) differences was significant (p = 0.001). effect of rs25487 genotypes on b[a]p levels the effects of snp (rs25487) genotyping of xrcc1 gene on b[a]p level are illustrated on the figures (3 and 4), whereas the highest level of b[a]p had been detected in the car repaires subjects who possess the tt genotype (1.76 ± 0.08) followed by heterozygouse genotype ct (1.607 ± 0.06) and then cc genotypes (0.424 ± 0.08). and also the spare part sellers, where the highest level of b[a]p was in the subjects who carriers tt genotype (0.963 ± 0.04), followed by heterozygouse genotype ct (0.681 ± 0.08) and then cc genotypes showed the lowest level (0.096 ± 0.04). the result of our study showed the cc dominant homozygous genotype of the xrcc1 (dna repairing gene) showed significantly lower levels of measured pahs comparison to the other genotypes, followed by heterozygous genotype ct, and than tt shown higher levels (figure 4). our results are agreement with many previous study17 found that the cc genotype has lower levels of pahs. it was also found in another study18 that the chinese coke oven workers carrying the tt genotype had higher levels of bpde-dna adduct. the variations in the levels of pahs among the studied genotypes are due to the variation in the activity of xenobiotics 211j contemp med sci | vol. 8, no. 3, may-june 2022: 207–212 a.r. omrain et al. original association between variant alleles of the x-ray cross complementing gene (xrcc1) metabolizing enzymes.19 many previous studies reported that the tt genotype had a decrease in activity of xenobiotics metabolizing enzymes (especially cyp2e1), as result, they have high levels of pahs. this resulting in long-term adverse effects, promoting cytotoxicity and genotoxicity, making individuals more susceptible to different types of cancers.19 and considering that b[a]p is a complete carcinogen, that act as an initiator and a promoter of carcinogensis according to epa, 2017.20 at the same time, it was observed that the cc genotypes had low levels of bpde-dna adduct levels this is supports findings of 21 matullo et al., who stated that the carriers of cc showed a relatively lower levels of bulky dna adduct in fig. 4 effects of snp (rs25487) genotyping of xrcc1 gene on b[a]p level. lymphocytes, which may be association with reduced capacity of dna repair system. also in meta-analysis conducted by kiyohara et al.22 tt genotype of the xrcc1 rs 25487 (arg399gln) polymorphism found that was increased pahs levels and it may be association with low dna repair capacity. the results obtained in this study were agreement with other previous study stating that ct and tt genotype is represent a risk of various types of cancer like lung cancer caused by the environment.23 conclusion as results of prolonged exposure to the higher levels of pahs that iraqi car repairs suffer from, they have high levels b[a]p. especially those who have the tt genotypes, as results of the inefficiency of their dna repair system. compared to the other genotypes. acknowledgments the authors are grateful to the staff of chemistry/biochemistry branch, college of medicine, al-nahrain university for their continuous support and encouragement. we thank the volunteers for their cooperation. conflicts of interest none.  references 1. ayodele, r. afolarin, o. (2020) decontamination of automobile workshop soils containing heavy metals and pahs using chelating agents, international journal of environmental pollution and remediation (ijepr) 8. 2. au, w.w. salama, s.a, sierra, c.h. (2003) functional characterization of polymorphisms in dna repair genes using cytogenetic challenge assays, environ. health perspect. 111(15)1843-1850. 3. khairy, ma. kolb, m. mostafa, ar. el-fiky, a. bahadir, m. (2009) risk assessment of polycyclic aromatic hydrocarbons in a mediterranean semienclosed basin affected by human activities (abu qir bay, egypt). j hazard mater; 170(1): 389-397. 4. blaszczyk, e. mielzynska, d. (2017) polycyclic aromatic hydrocarbons and pah-related dna adducts, j appl genetics 58, 321–330. 5. elovaara, e. mikkola, j. stockmann-juvala h, (2014) polycyclic aromatic hydrocarbon (pah) metabolizing enzyme activities in human lung, and their inducibility by exposure to naphthalene, phenanthrene, pyrene, chrysene, and benzo(a)pyrene as shown in the rat lung and liver. arch toxicol. 81.169-82. 6. palli, d. vineis, p. russo, (2001) int. j. cancer 87 (444). 7. hayder, o.h. ali, h.s. ala, h.h. haider, k.z. (2015) association of uromodulin rs13333226 and angiotensinogen rs699 genes variants with essential hypertension in arab iraqis of babylon province, 6(6).589. 8. hayder, o.h. mohammed, b. s. (2019) exploring the potential and limitations of pcr-rflp and pcr-sscp for snp detection: a review, j appl biotechnol rep. 6(4):137-144. 9. kim, b. m. lee, s.-b., kim, j. y., kim, s., seo, j., bae, g.-n., & lee, j. y. (2016). a multivariate receptor modeling study of air-borne particulate pahs: regional contributions in a roadside environment. chemosphere, 144, 1270–1279. 10. luigi, vimercati, lucia bisceglia, domenica, c. antonio, c. luigi, d. maria, c. vincenzo, c. giovanni, m. (2020) environmental monitoring of pahs exposure, biomarkers and vital status in coke oven workers, int. j. environ. res. public health 17 (2199). 11. ahmad, i. rehan, m. balkhyour, m. abbas, m. basahi, j. almeelbi, t. ismail, i.m. (2016) review of environmental pollution and health risks at motor vehicle repair workshops challenges and perspectives for saudi arabia. int. j. agric. env. res. 2, 1–23. 12. ahmad, i. rehan, m. balkhyour, m.a. ismail, i.m. (2017b) assessment of occupational health and safety in motor vehicle repair workshops in jeddah. biosci. biotech. res. asia. 14 (3), 901–913. 13. luigi, v. lucia. b, domenica. c, antonio, c. luigi, d.m. maria c.d. vincenzo, c. giovanni, m. f. (2020) environmental monitoring of pahs exposure, biomarkers and vital status in coke oven workers, int. j. environ. res. public health 17, 2199. 14. shehata, ra. helal, sf. rashed, la. rakha, am (2020) oxidative dna damage due to occupational exposure to polycyclic aromatic hydrocarbons among coal tar workers, egyptian journal of occupational medicine, 44 (2): 663–678. 15. therhi. k, lars. n, raija. v, kirsti. s, pertti. m, toomas. v, (2002) the effect of relevant genotypes on pah exposure-related biomarkers, journal of exposure analysis and environmental epidemiology 12, 81–91. 16. aisha, m. samir. dalia, a shaker, mona, m. fathy, s. mona, m. abdullatif. laila a. hany a. alghobary. (2019) urinary and genetic biomonitoring of polycyclic aromatic hydrocarbons in egyptian coke oven workers: associations between exposure, effect, and carcinogenic risk assessment, international of occupational and environmental medicine,10(3). 17. begum, a. ramaiah, m. khan, i. veena, k. (2009). analysis of heavy metals concentration in soil and litchens from various localities of hosur road, bangalore, india. j chem. 6(1), pp. 13–22. 18. bolling, ak. pagels, j. yttri, ke. barregard, l. sallsten, g. schwarze, pe. (2009) health effects of residential wood smoke particles: the importance of combustion conditions and physicochemical particle properties. part fibre toxicol (6)20. 19. hamidi, e.n. hajeb, p. selamat, j. abdull razis, a.f. (2016). polycyclic aromatic hydrocarbons (pahs) and their bioaccessibility in meat: 212 j contemp med sci | vol. 8, no. 3, may-june 2022: 207–212 association between variant alleles of the x-ray cross complementing gene (xrcc1) original a.r. omrain et al. a tool for assessing human cancer risk. asian pac. j. cancer prev. 17, 15–23. 20. international agency for research on cancer (iarc) (2012). globocan 2012: estimated cancer incidence, mortality and prevalence worldwide in 2012, world health organization, lyon, france. 21. ijaz, a. mansour, a. (2020) occupational exposure and respiratory health of workers at small scale industries, saudi journal of biological sciences 27 985–990. 22. olabanji i. o. asubiojo, o. komolafe m. akintomide anthony, and adeniji ayodeji oluwole (2019) determination of polycyclic aromatic hydrocarbons in blood plasma of neurology patients journal of toxicology and environmental health sciences, 11(1), 1-8. 23. sanna, k. pohjola, maija, l. markku, h. leena, r. kirsti, s. (2003) dna binding of polycyclic aromatic hydrocarbons in a human bronchial epithelial cell line treated with diesel and gasoline particulate extracts and benzo[a]pyrene mutagenesis. 18 (5) 429-438. https://doi.org/10.22317/jcms.v8i3.1231 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 59j contemp med sci | vol. 8, no. 1, january-february 2022: 59–64 original liver proliferating cell nuclear antigen, bax/bcl-2 ratio, collagen, and polysaccharide accumulation as diagnostic tools in experimental hepatocellular carcinoma nabil mohie abdel-hamid1* , mohamed k hassan2, amal am ahmed3, sara gamal abd allah2, nahla h. anber4 1department of biochemistry, faculty of pharmacy, kafrelsheikh university, egypt. 2department of biotechnology, faculty of science, port said university, egypt. 3department of cytology and histology, faculty of veterinary medicine, suez canal university, egypt. 4fellow of biochemistry in the emergency hospital, mansoura university, mansoura, egypt. *correspondence to: nabil mohie abdel-hamid, e-mail: (nabilmohie@pharm.kfs.edu.eg) (submitted: 13 december 2021 – revised version received: 16 january 2022 – accepted: 23 january 2022 – published online: 26 february 2022) abstract objectives: this experimental study was conducted to look for a sensitive diagnostic panel for early detection of hepatocellular carcinoma (hcc). methods: combination of diethyl nitrosamine (dena, 200 mg/kg, ip, once), two weeks later, ccl 4 (3 ml/kg/week), subcutaneously, for 6 weeks) induced hcc in rats. sixteen male wistar rats were divided into 2 groups, control, and hcc. results: dena plus ccl 4 elevated serum alpha-fetoprotein, liver enzyme activity and depressed superoxide dismutase, reduced glutathione (gsh), elevated malondialdehyde. also, depressed caspase-3, elevated collagen, polysaccharide accumulation, proliferating cell nuclear antigen in liver tissues, and depressed bax/bcl-2 ratio. conclusion: decreased bax/bcl-2 ratio, elevated collagen deposition, polysaccharide accumulation, nuclear proliferation, and tissue oxidative stress help in the early diagnosis of liver cancer. keywords: hepatocellular carcinoma, caspase-3, cellular proliferation, apoptosis, collagen, polysaccharides list of abbreviations: afp: alpha-fetoprotein; dena: diethyl nitrosamine; gsh: reduced glutathione; hcc: hepatocellular carcinoma; h&e: hematoxylin/eosin; ip: intraperitoneal; mda: malondialdehyde; no: nitric oxide; pcna: proliferating cell nuclear antigen; ros: reactive oxygen species; sod: superoxide dismutase. issn 2413-0516 introduction liver cancer is the fifth most common cancer and the second most frequent cause of cancer-related death in the world. hepatocellular carcinoma (hcc) has become one of the major causes of morbidity and mortality all over the world.1,2 hcc accounts for 80% to 90% of primary liver cancer.3 it is the second leading cause of cancer deaths worldwide, with over 500,000 people affected per year.4 animal models have enabled the study of the mechanism of hcc and the development of possible strategies for treatment. diethyl nitrosamine (dena) is a representative chemical carcinogen with the potential to cause tumors in various organs, including the liver, skin, gastrointestinal tract, and respiratory system.5 specifically, in hcc, dena is a complete carcinogen. many lines of evidence have demonstrated a relationship between carcinogenesis and cell cycle regulation. dena induces irreversible hcc through overexpression of g1/s-phase regulatory proteins in rats.6 dena is a suitable model in the field of experimental hepatic pathology because nitrite and nitrosamine synthesis is increased in human viral hepatitis,7 dena induced lesions, as well as tumors in rodents, show marked biochemical, histological, and molecular similarity to the progression of hcc in humans.8 dena induced lesions, as well as tumors in rodents, show marked biochemical, histological, and molecular similarity to the progression of hcc in humans.9 the hepatotoxicity of ccl4 is mainly exerted in two different levels: first, ccl4 induction of cytochrome p450 and the consequently increased formation of reactive oxygen species (ros).10 the bcl-2 family is the best-characterized protein family involved in the regulation of apoptotic cell death. it includes both anti-apoptotic members (i.e bcl-2) and pro-apoptotic members (i.e bax), thus the bcl-2 family acts as a critical lifedeath decision point makers in the common pathway of apoptosis.11 although acute inflammation is a part of the defense response, persistent inflammation can lead to certain diseases as cancer and diabetes.12 apoptosis represents a physiological way to eliminate excess cells during both liver development and regeneration it is a highly preserved and controlled mechanism to achieve tissue homeostasis through targeted elimination of single cells without disrupting the biological functionality of the tissue. the morphological changes associated with apoptosis include nuclear condensation, cell shrinkage, and plasma membrane blebbing resulting in apoptotic bodies,13 it is a highly preserved and controlled mechanism to achieve tissue homeostasis through targeted elimination of single cells without disrupting the biological functionality of the tissue. the morphological changes associated with apoptosis include nuclear condensation, cell shrinkage, and plasma membrane blebbing resulting in apoptotic bodies.14 many therapeutic modalities have been developed relying on surgery, chemotherapy, radiotherapy, hormone therapy, and more recently immunotherapy.15 the present work aims to determine some serum and tissue changes during hepatocarcinogenesis, looking for more reproducible and specific markers for hcc in an experimental model. along with serum afp, tissue oxidative stress, tissue changes of collagen fiber, nuclear proliferation, and http://orcid.org/0000-0002-7932-2672 mailto:nabilmohie@pharm.kfs.edu.eg 60 j contemp med sci | vol. 8, no. 1, january-february 2022: 59–64 bax/bcl-2 ratio, polysaccharide, collagen, and pcna in hepatocellular carcinoma diagnosis original n.m. abdel-hamid et al. polysaccharide accumulation, using histochemical stains to get more early and cost-effective diagnostic tools for hcc. materials and methods chemicals diethyl nitrosamine was purchased from sigma-aldrich chemical co. (st. louis, mo, usa). carbon tetrachloride was purchased from sd fine-chem. ltd. (mumbai, india). all other chemicals used were of analytical grade. animals and experimental design principles of laboratory animal care (nih publication vol 25, no. 28 revised 1996; (http://grants.nih.gov/grants/guide/ notice-files/not96-208.html) were followed. the experimental design was approved by the institutional animal ethics committee guidelines for animal care and use at port said university, egypt, under 354 for 2016. sixteen adult male albino rats weighing 100–150 g were recruited for the study purpose. the animals were housed in polyethylene cages in a moderately humid room under a controlled 12 hours light/dark cycle, kept under constant environmental and nutritional conditions with free access to food and water ad libitum. animals were kept for 2 weeks for acclimatization before starting the experiment). group i, received only saline (twice/week), ip, for 8 weeks (control), group ii, received only the carcinogenic combination (hcc group), single-dose diethyl nitrosamine (dena, 200 mg/kg), was intraperitoneally injected, two weeks later 50% v/v, carbon tetrachloride solution in olive oil (3 ml/kg/ week, ip, for 6 weeks) was injected. the total experiment period was two months. blood sample collection at the end of the experiment, rats were anesthetized with light diethyl ether. blood samples were obtained directly after decapitation of animals, centrifuged at 2500 rpm for 10 min after standing for 45 minutes at 4ºc. the obtained sera were kept at −80ºc right biochemical investigations. liver tissue sampling liver tissues were collected, washed by cold saline, blotted by tissue paper, dissected into 2 parts, one kept in formal saline for tissue microscopical studies, the second portion was homogenized in formalin/saline (10% w/v), then, homogenates were frozen at −80ºc right tissue biochemical assays. biochemical investigations serum investigations alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase, and gamma-glutamyl transferase (ggt) activities, albumin, and total protein levels were carried out using chemolyzer 510 semi-automated analyzer. alpha-fetoprotein (afp) level was determined by using the rat afp elisa sandwich technique.16 measurements were performed according to the manufacturer’s instructions. liver tissue investigations spectrophotometric assays for malondialdehyde (mda), nitric oxide (no), reduced glutathione (gsh) contents, and superoxide dismutase (sod) activity in liver tissue homogenates were executed, using kits purchased from biodiagnostic, cairo, egypt. liver mrna was extracted for detection of both bax and bcl-2 by real-time polymerase chain reaction (rt-pcr). we used the mrna extraction protocol established by.17 about 0.5 g of liver tissue was used for mrna isolation using trizo lm, according to the manufacturer’s instructions (thermofisher, cat. no. 15596026, usa). cdna reaction was performed using bio-systems kits, and the given primers (table 1), according to the manufacturer’s instructions. for real-time quantitative pcr, 5 μl of the first-strand cdna was used in a total volume of 25 μl, containing 12.5 μl 2x sybr green pcr master mix (bio-rad, script, usa) and 200 ng of each given primer. pcr reaction was repeated for 40 cycles. the data were computed with the abi prism 7500 sequence detection system software. relative expression of studied genes (bax/bcl-2) was calculated using the comparative threshold cycle method. all values were normalized to the beta-actin genes. histopathological study histological examination was done using h&e stains,18 an immunohistochemical study using masson’s trichrome (to test collagen accumulation),19 an immunohistochemical study using masson’s trichrome (to test collagen accumulation),20 proliferating cell nuclear antigen (pcna) monoclonal antibody21 and caspase-3 monoclonal antibody to figure out the accumulation of pro-apoptotic caspase-3 (an inactive form of caspase) in liver tissue.22 statistical analysis data were statistically analyzed using spss, the usa, the data of the cancer group were compared to the control group and expressed as mean ± se. one-way anova and unpaired t-test were carried out to find if there was any significant difference among control and treated groups. results biochemical studies on serum, and liver tissue in studied groups the hepatocarcinogenic combination, significantly up-regulated liver enzyme activity, alongside, significantly decreased both serum albumin and t. protein. it also significantly elevated serum afp and oxidative stress in liver tissue by depressing gsh, sod activity, with an increase of mda and no contents. carcinogenesis significantly aborted apoptotic/ survival ratio. both effects were calculated in comparison to normal control results (table 2). table 1. the sequence of the primers used was as follow primer sequence bax forward: 5’-gct ctg aacaga tca tga ag-3’; reverse: 5’-gat ggt cac tgt ctgcca tg-3’. bcl-2 forward: 5’-gac ttt ctc tcc tac aagc-3’; reverse: 5’-cga aag agt tca ttc act ac-3’. β-actin forward: 5’-caa cgg ctc cgg cat gtg c-3’; down: reverse: 5’-ctc ttg ctc tgg gcc tcg-3’. http://grants.nih.gov/grants/guide/notice-files/not96-208.html http://grants.nih.gov/grants/guide/notice-files/not96-208.html 61j contemp med sci | vol. 8, no. 1, january-february 2022: 59–64 n.m. abdel-hamid et al. original bax/bcl-2 ratio, polysaccharide, collagen, and pcna in hepatocellular carcinoma diagnosis table 2. variations in serum liver indices, hepatic tissue redox status, apoptotic member/survival member (bax/bcl-2) ratio, against serum alpha fetoprotein, in experimental hcc, compared to control group (values are expressed as mean ± se, n = 8) parameter normal control hcc group alt (u/l) 39.3 ± 1.4 138.1 ± 5.9 a*** ast (u/l) 104.7 ± 5.7 287.1 ± 12.6 a*** ggt (u/l) 10.1 ± 0.33 21 ± 1.58 a*** alp (u/l) 141.7 ± 4.8 563 ± 24.6 a*** albumin (g/dl) 3.8 ± 0.14 2.37 ± 0.1 a*** t. protein (g/dl) 6.4 ± 0.21 4.725 ± 0.103 a*** afp (ng/ml) 4.17 ± 0.09 46.2 ± 1.30 a*** sod (u/g tissue) 1848.2 ± 5.8 1051.2 ± 27.1 a*** gsh (mg/g tissue) 759.4 ± 37.1 493.8 ± 22.0 a*** mda (nmol/g tissue) 62.6 ± 4.1 224.1 ± 9.6 a*** no (µmol/g tissue) 11.6 ± 0.5 64.3 ± 1.6 a*** tissue apoptotic/survival (bax/bcl-2) ratio 0.47 ± 0.15 0.17 ± 0.02 b*** p-value represents the difference between control and hcc groups, * (p < 0.05), * (p < 0.01), ** (p < 0.001). fig. 1 h&e tissue staining showed normal hepatic lobular architecture, granulated cytoplasm and uniform nuclei in control rats (a1,2). hcc group showed disarrangement of hepatic cords with clusters of large eosinophilic hyperplastic foci (arrow), large, hyperplastic hepatocytic cells (magnified) with abundant eosinophilic cytoplasm and pleomorphic hyperchromatic nuclei (b1,2). liver morphology of hcc liver model compare with normal tissues. normal control rats (c), showed normal pattern with reddish colored appearance. hcc group (d), showed enlarged liver with scattered nodules. gross and histological examination of liver in studied groups gross examination of the liver showed that a combination of dena/ccl4 induced apparent morphological changes, as enlarged liver with scattered nodules, while the control liver was in normal small and homogenous architecture (figure 1, c, d). h&e tissue staining showed normal hepatic lobular architecture, granulated cytoplasm, and uniform nuclei in control rats. hcc group showed disarrangement of hepatic cords, clusters of large eosinophilic hyperplastic foci, large, hyperplastic hepatocytes, and pleomorphic hyperchromatic nuclei (figure 1, a1,2-b1,2). histochemical studies of liver tissue in studied groups liver sections stained with masson’s trichrome showed normal distribution of the collagen fibers in control rats. hcc group showed infiltration of collagen fibers in the interstitium of hyperplastic foci, (figure 2, a, b). liver sections stained with periodic acid–schiff (pas), showed normal polysaccharide distribution in normal control while, dense polysaccharide accumulation was apparent in the hcc group, (figure 2, c, d). 62 j contemp med sci | vol. 8, no. 1, january-february 2022: 59–64 bax/bcl-2 ratio, polysaccharide, collagen, and pcna in hepatocellular carcinoma diagnosis original n.m. abdel-hamid et al. affected annually.4 diethyl nitrosamine (dena) is a common carcinogen in the field of experimental hepatocarcinogenesis because nitrite and nitrosamine synthesis is increased in human viral hepatitis,7 which is a major risk factor for hcc.8 in the present study, animals treated with dena followed by ccl4 exhibited a significant depression of body weight with increased liver weights. microscopically, the effect of this dose on the different biochemical, molecular and histological parameters was assessed. biochemical parameters including serum transaminases (ast and alt), alp, and γ-gt are known indicators for liver function and their elevated activities are sensitive markers for hepatic injury. in our study, we found that ast, alt, alp, and γ-gt activities, were highly elevated in animals treated with dena/ccl4 combination. these results were reported sometime before.23 elevated activities of serum ast and alt in dena/ccl4 treated rats are attributed to induced hepatic damage and subsequent leakage of these enzymes from the neoplastic cell into circulation.24 the generation of reactive oxygen species (ros) is apparent during the metabolic biotransformation of dena resulting in oxidative stress. this leads to carcinogenesis by several mechanisms including dna, lipid, and protein damage, change in intracellular signaling pathways, and even changes in gene expression. together, these oxidative modifications promote abnormal cell growth and transformation.25 oxidative stress is a phenomenon caused by an imbalance between production and accumulation of ros in cells and tissues and the inability of a biological system to detoxify these reactive products initiates imbalance that leads to cell and tissue damage.26 oxidative stress was recently registered to be a potential modulator in hepatocarcinogenesis.27,28 in addition, reduced glutathione (gsh), a well-known non enzymatic intracellular antioxidant is found at high concentrations in the liver and has key functions in protection against free radicals, peroxides, and other toxic components. our results revealed that ma elevations and depressed tissue gsh content were comparable to some previous observations elsewhere.29,30 apoptosis is the programmed cell death that maintains the healthy survival/death balance in metazoan cells. defects in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. the apoptotic signals contribute to safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. the tumor cells may use several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of anti-apoptotic proteins such as bcl-2 or by the downregulation or mutation of proapoptotic proteins such as bax.31 apoptosis ends up with proteolysis and dna degradation as a result of caspase (an apoptotic factor) activation. this activation may be initiated through mitochondrial-dependent or independent mediators.32 moreover, gene expression profile for two major pro and antiapoptotic (survival) genes, bax and bcl-2, revealed that such dna damage could be a consequence of hcc induction that aborted caspase-3 conversion from prointo an active form to kill cancer cells.33 moreover, gene expression profile for two major pro and antiapoptotic (survival) genes, bax and bcl-2, revealed that such dna damage could be a consequence of hcc induction that aborted caspase-3 conversion from prointo an active form to kill cancer cells.34 the crucial involvement of fig. 2 photomicrographs of liver sections stained with masson’s trichrome, showed normal distribution of the collagen fibers in control rats (a), hcc group showed infiltration of collagen fibers in the interstitium of hyperplastic foci (b). photomicrographs of liver sections stained with periodic acid–schiff (pas), showed no polysaccharide accumulation in normal control (c), while the highest polysaccharide accumulation was apparent in hcc group (d). fig. 3 photomicrographs of liver tissue incubated with proliferating cell nuclear antigen (pcna) monoclonal antibody, showed normal tissue in control (a), but hcc group showed high cellular proliferation reaction (b). photomicrographs of liver tissue incubated with caspase-3 monoclonal antibody showed no pro-apoptotic caspase-3 (inactive form of caspase) accumulation in control (c), but hcc group showed excessive pro-apoptotic caspase-3 accumulation, meaning, aborted apoptosis (d). liver tissue incubated with proliferating cell nuclear antigen (pcna) monoclonal antibody exhibited normal tissue in the control group but the hcc group showed high cellular proliferation reaction (figure 3, a, b). liver tissue incubated with caspase-3 monoclonal antibody showed no pro-apoptotic caspase-3 (inactive form of caspase) accumulation in control, hcc group showed excessive pro-apoptotic caspase-3 accumulation, indicating aborted apoptosis. (figure 3, c, d). discussion hepatocellular carcinoma is the second leading cause of cancer deaths worldwide, with more than 500,000 people 63j contemp med sci | vol. 8, no. 1, january-february 2022: 59–64 n.m. abdel-hamid et al. original bax/bcl-2 ratio, polysaccharide, collagen, and pcna in hepatocellular carcinoma diagnosis pcna in cellular proliferation and its tight association with cancer transformation resulted in the frequent use of pcna as a diagnostic and prognostic cell-cycle marker.35 in the current work, dena-treated animals showed an increased level of pcna which is an indication of hyperproliferative activity. caspases are crucial mediators of apoptosis; among them, caspase 3 is a principal enzyme in the apoptotic cascade and is often used to detect apoptotic activity.36 the present study highlighted caspase-3 monoclonal antibodies to check the accumulation of pro-apoptotic caspase-3 (an inactive form of caspase) in liver tissue. this showed a statistical increase in the hcc group. in our study, histopathological as well as immunostaining data confirmed the hepatotoxic effect of dena/ccl4 on the liver tissue level, through a significant increase in both polysaccharide and collagen deposition in hepatocytes. the collagen deposition tested by masson trichrome staining was significantly increased in the livers of the hcc group, these results agree with the moon group, which used masson trichrome staining to estimate the condensation of collagen fiber in hepatocellular carcinoma.37 periodic acid-schiff (pas) staining was used to determine the accumulation of intra-cytoplasmic glycogen since well-differentiated hccs pas staining is usually strongly positive in liver cancers.38 conclusion our study assumes that elevated serum alpha-fetoprotein, liver enzyme activity and increased tissue oxidative stress markers (depressed superoxide dismutase, sod, and glutathione, gsh, with elevated malondialdehyde, mda), liver size, weight, and morphology are prominent indicators for hepatocarcinogenesis. an efficient tissue diagnostic panel was postulated through cancer progression markers, indicated by depressed caspase-3 (apoptotic marker), extensively deposited collagen fibers, polysaccharides, and nuclear proliferation in liver tissues. liver tissue also showed depressed apoptotic defense, reflected by decreased bax/bcl-2 ratio. conflicts of interest/competing interests authors declare no conflict of interest.  references 1. marcellin, p. and kutala, b. k. (2018). liver diseases: a major, neglected global public health problem requiring urgent actions and large‐scale screening. liver international. 38, 2–6. 2. mcglynn, k. a. and london, w. t. (2011). the global epidemiology of hepatocellular carcinoma: present and future. clinics in liver disease. 15, 223–243. 3. abdel-hamid, n., el-moselhy, m. and fawzy, m. (2012). novel panel of early diagnostic markers for experimental hepatocellular carcinoma. journal of health science. 2, 14–18. 4. torre, l.a., bray, f., siegel, r.l., ferlay, j., lortet‐tieulent, j. and jemal, a. (2015) global cancer statistics, 2012. ca: a cancer journal for clinicians. 65, 87–108. 5. stanaway, j.d., flaxman, a.d., naghavi, m., fitzmaurice, c., vos, t., abubakar, i., abu-raddad, l. j., assadi, r., bhala, n. and cowie, b. (2016). the global burden of viral hepatitis from 1990 to 2013: findings from the global burden of disease study 2013. the lancet. 388, 1081–1088. 6. park, d.h., shin, j.w., park, s.k., seo, j.n., li, l., jang, j.j. and lee, m.j. (2009). diethylnitrosamine (den) induces irreversible hepatocellular carcinogenesis through overexpression of g1/s-phase regulatory proteins in rat. toxicology letters. 191, 321–326. 7. santos, n.p., colaço, a.a. and oliveira, p.a. (2017). animal models as a tool in hepatocellular carcinoma research: a review. tumor biology. 39, 1010428317695923. 8. feo, f., pascale, r.m., simile, m.m., de miglio, m.r., muroni, m.r. and calvisi, d. (2000). genetic alterations in liver carcinogenesis: implications for new preventive and therapeutic strategies. critical reviews™ in oncogenesis. 11. 9. mahmoud, a.m., mohammed, h.m., khadrawy, s.m. and galaly, s.r. (2017). hesperidin protects against chemically induced hepatocarcinogenesis via modulation of nrf2/are/ho-1, pparγ and tgf-β1/smad3 signaling, and amelioration of oxidative stress and inflammation. chemico-biological interactions. 277, 146–158. 10. campo, g., avenoso, a., campo, s., nastasi, g., traina, p., d’ascola, a., rugolo, c. and calatroni, a. (2008). the antioxidant activity of chondroitin‐4‐sulphate, in carbon tetrachloride‐induced acute hepatitis in mice, involves nf‐κb and caspase activation. british journal of pharmacology. 155, 945–956. 11. schattenberg, j. m., galle, p. r. and schuchmann, m. (2006). apoptosis in liver disease. liver international. 26, 904–911. 12. balkwill, f. and coussens, l. m. (2004). cancer: an inflammatory link. nature. 431, 405–406. 13. sun, s.y., hail, n. and lotan, r. (2004). apoptosis as a novel target for cancer chemoprevention. journal of the national cancer institute. 96, 662–672. 14. guicciardi, m. and gores, g.j. (2005). apoptosis: a mechanism of acute and chronic liver injury. gut. 54, 1024–1033. 15. staňková, k., brown, j.s., dalton, w. s. and gatenby, r.a. (2019). optimizing cancer treatment using game theory: a review. jama oncology. 5, 96–103. 16. cattini, r., cooksey, m., robinson, d., brett, g., bacarese-hamilton, t. and jolley, n. (1993). measurement of α-fetoprotein, carcinoembryonic antigen and prostate-speciflc antigen in serum and heparinised plasma by enzyme immunoassay on the fully automated serono sr1™ analyzer. clinical chemistry and laboratory medicine. 31, 517–524. 17. rio, d.c., ares, m., hannon, g.j. and nilsen, t.w. (2010). purification of rna using trizol (tri reagent). cold spring harbor protocols. 2010, pdb. prot5439. 18. fischer, a.h., jacobson, k.a., rose, j. and zeller, r. (2008). hematoxylin and eosin staining of tissue and cell sections. cold spring harbor protocols. 2008, pdb. prot4986. 19. goldner, j. (1938). a modification of the masson trichrome technique for routine laboratory purposes. the american journal of pathology. 14, 237. 20. quintero-hunter, i., grier, h. and muscato, m. (1991). enhancement of histological detail using metanil yellow as counterstain in periodic acid schiff ’s hematoxylin staining of glycol methacrylate tissue sections. biotechnic & histochemistry. 66, 169–172. 21. kawakita, n., seki, s., sakaguchi, h., yanai, a., kuroki, t., mizoguchi, y., kobayashi, k. and monna, t. (1992). analysis of proliferating hepatocytes using a monoclonal antibody against proliferating cell nuclear antigen/ cyclin in embedded tissues from various liver diseases fixed in formaldehyde. the american journal of pathology. 140, 513. 22. duan, w.r., garner, d.s., williams, s.d., funckes‐shippy, c.l., spath, i.s. and blomme, e.a. (2003). comparison of immunohistochemistry for activated caspase‐3 and cleaved cytokeratin 18 with the tunel method for quantification of apoptosis in histological sections of pc‐3 subcutaneous xenografts. the journal of pathology: a journal of the pathological society of great britain and ireland. 199, 221–228. 23. singh, b.n., singh, b.r., sarma, b. and singh, h. (2009). potential chemoprevention of n-nitrosodiethylamine-induced hepatocarcinogenesis by polyphenolics from acacia nilotica bark. chemico-biological interactions. 181, 20–28. 24. dakshayani, k., subramanian, p., manivasagam, t., essa, m.m. and manoharan, s. (2005). melatonin modulates the oxidant-antioxidant imbalance during n-nitrosodiethylamine induced hepatocarcinogenesis in rats. journal of pharmacy & pharmaceutical sciences: a publication of the canadian society for pharmaceutical sciences, societe canadienne des sciences pharmaceutiques. 8, 316–321. 25. klaunig, j.e. and kamendulis, l.m. (2004). the role of oxidative stress in carcinogenesis. annu. rev. pharmacol. toxicol. 44, 239–267. 26. pizzino, g., irrera, n., cucinotta, m., pallio, g., mannino, f., arcoraci, v., squadrito, f., altavilla, d. and bitto, a. (2017). oxidative stress: harms and benefits for human health. oxidative medicine and cellular longevity. 2017. 27 bishayee, a., barnes, k.f., bhatia, d., darvesh, a.s. and carroll, r.t. (2010). resveratrol suppresses oxidative stress and inflammatory response in 64 j contemp med sci | vol. 8, no. 1, january-february 2022: 59–64 bax/bcl-2 ratio, polysaccharide, collagen, and pcna in hepatocellular carcinoma diagnosis original n.m. abdel-hamid et al. diethylnitrosamine-initiated rat hepatocarcinogenesis. cancer prevention research. 3, 753–763. 28. nabil m. abdel-hamid, n.m., salama, a.s, el-sheekh,m, sarhan, n, gabr, a.m. (2017). oxidative stress predominates apoptosis during experimental hepatocellular carcinoma contemp med sci 3, 295–299. 29. esrefoglu, m. (2012). oxidative stress and benefits of antioxidant agents in acute and chronic hepatitis. hepatitis monthly. 12, 160. 30. miccadei, s., di venere, d., cardinali, a., romano, f., durazzo, a., foddai, m. s., fraioli, r., mobarhan, s. and maiani, g. (2008). antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (cynara scolymus l.) on cultured rat hepatocytes and on human hepatoma cells. nutrition and cancer. 60, 276–283. 31. hassan, m., watari, h., abualmaaty, a., ohba, y. and sakuragi, n. (2014). apoptosis and molecular targeting therapy in cancer. biomed research international. 2014. 32 yousef, m.i., khalil, d.k. and abdou, h.m. (2018). neuro-and nephroprotective effect of grape seed proanthocyanidin extract against carboplatin and thalidomide through modulation of inflammation, tumor suppressor protein p53, neurotransmitters, oxidative stress and histology. toxicology reports. 5, 568–578. 33. seeram, n.p., adams, l.s., zhang, y., lee, r., sand, d., scheuller, h.s. and heber, d. (2006). blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. journal of agricultural and food chemistry. 54, 9329–9339. 34. strzalka, w. and ziemienowicz, a. (2010). proliferating cell nuclear antigen (pcna): a key factor in dna replication and cell cycle regulation. annals of botany. 107, 1127–1140. 35. stoimenov, i. and helleday, t. (2009). pcna on the crossroad of cancer. ed.)^eds.), portland press limited. 36. huang, h., zhang, x.f., zhou, h.j., xue, y.h., dong, q.z., ye, q.h. and qin, l.x. (2010). expression and prognostic significance of osteopontin and caspase‐3 in hepatocellular carcinoma patients after curative resection. cancer science. 101, 1314–1319. 37. moon, w.s., yu, h.c., chung, m.j., kang, m.j. and lee, d.g. (2000). pale bodies in hepatocellular carcinoma. journal of korean medical science. 15, 516–520. 38. bret, p.m., labadie, m., bretagnolle, m., paliard, p., fond, a. and valette, p.j. (1988). hepatocellular carcinoma: diagnosis by percutaneous fine needle biopsy. gastrointestinal radiology. 13, 253–255. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1162 120 j contemp med sci | vol. 8, no. 2, march-april 2022: 120–127 original outcomes of primary percutaneous coronary intervention in st-segment elevation myocardial infarction in kurdistan region of iraq ameen m mohammad1*, schivan u mohammed2, saad y saeed3 1department of internal medicine, college of medicine, university of duhok, iraq. 2department of biomedicine, college of medicine, university of zakho, duhok, iraq. 3department of community medicine, college of medicine, university of duhok, iraq. *correspondence to: ameen m mohammad (e-mail: doctoramb@yahoo.com) (submitted: 16 december 2021 – revised version received: 10 january 2022 – accepted: 27 january 2022 – published online: 26 april 2022) abstract objectives: this registry aims to clarify the characteristics and 6-weeks outcomes of patients with stemi after ppci in the region. methods: data from a total of 151 stemi patients undergoing ppci at duhok heart center, iraq from 2020 to 2021 was collected. patient’s demographic, clinical and ppci profiles were recorded. the major adverse cardiac events (mace) and left ventricle ejection fraction (lvef%) outcomes for 6 weeks period was registered. results: of the 151 consecutive patients with stemi who underwent ppci, 46 (30.4%) were <50 years old. majority of patients were males and have clusters of cardiometabolic risk factors. 64% of cases attained cath lab within first hour of initial chest pain. almost 90% of stemi cases were treated with stenting with timi3 in (94%). 80% of ppci cases discharged home within 24 hours uneventfully. 6-weeks lvef was preserved within normal range in 55% of cases. 36% had maces including impaired lvef. all cause-mortality happened in 5%. 4% were censored from follow up. the predictors of 6-weeks outcomes were depend on type/location of myocardial infarction, the culprit artery, timi flow post pci and length of hospital stay. conclusion: this registry has shown feasibility in doing ppci with reasonable outcomes in the region. networking of capable centers of ppci in the country is essential for augmenting the cardiac services and sharing the knowledge among cardiologists and people for better stemi outcomes. keywords: stemi, primary pci, iraq issn 2413-0516 introduction the cardiovascular diseases coming at the top of the list of the disease-related death in iraq.1 the incidence of stemi in the area is rapidly over happening particularly in young people.2 according to international medical guidelines the best approach to stemi patient is the ppci.3 the ppci services are generally new in our area. since long time the main modality of reperfusion in stemi was thrombolytics. in the last decade the interventional cardiology services and cath lab facilities entered to the field in our area, nevertheless, the systematic ppci per 24 hours/7 days per 365 days was relatively a new approach in our area. given the fact that the services of ppci are relatively recent in the area and the data about stemi and the feasibility of ppci are sparse. hence, this study was conducted in order to clarify the characters and 6-weeks outcomes of patients with stemi after primary percutaneous coronary intervention (ppci) in the region. methods belongs to azadi teaching tertiary hospital duhok heart center is a specialized center with a history of than 15 years. in regards to stemi, the center provided ppci services 24 hours/7 days per week in the last 4 years. the center has three equipped cath lab with continuous back up surgery and intensive care unit. the center has more than 12 interventional cardiologists with a group of cardiac surgeons and intensives, besides a large group of paramedics and staffs in the cath lab. in this prospective follow up study patients who presented with diagnosis of stemi from duhok and its districts and referred to duhok heart center inside azadi hospital for potential primary percutaneous coronary intervention were enrolled during the period of 2020 to 2021. the patients were given guideline directed medical therapy in from of loading doses of dual antiplatelets and anti-ischemic drugs. eligible patients after their acceptance and consent from patients were referred to cath lab for ppci. patients with established stemi or late presentations (> 12 hours) were excluded and admitted to coronary care unit for optimal medical therapy/ thrombolytics. during the procedure, the interventional cardiologists perform the procedure through either femoral or radial approach. all the procedural data including angiographic findings with culprit artery lesion and type of pci (whether stents, ballooning or medical therapy) were documented. platforms of stents were mentioned. successfulness of pci and procedure was based on the timi flow scores. after the procedure all patients were admitted for 24 hours monitoring in the intensive care unit of the center. all complications including mortality, if happened were registered. the estimation of lvef was performed. post discharge advices for regular complaint to medications, life style modifications and follow up schedule were given. then after patients were followed for a period of 6 weeks. the lvef (%) were performed for coming cases. in addition to registration of the rates of patient’s readmissions to hospital for major acute ischemic events (mace) like acute stent thrombosis, myocardial infarction and stroke. the cardiac death rate and censored cases for follow up during this period was documented. a detailed demographic, clinical, angiographic profile of all cases were recorded and stored on a file of excel. 121j contemp med sci | vol. 8, no. 2, march-april 2022: 120–127 a.m. mohammad et al. original outcomes of ppci in iraq ethical approval and patients consent the study was approved by the appropriate ethical committee at the kurdistan board of medical specialization (erbil, iraq. the number of order;) and an informed written consent was obtained from all participants, or legal guardians (either parent) as appropriate. statistical analysis data from the original excel file were transferred and analyzed by using microsoft office excel 2007 and spss for windows, version 16.0, chicago. continuous variables were calculated as mean ± (sd), and categorical variables were presented as counts and percentages. a chi-square test and fisher exact were used to compare the variables. p-value < 0.05 was regarded as significant. results the main findings of patients by age groups are summarized in table 1. the patients were predominately males. 30% were young (<50 years). the fast majority of ppci was performed within first 6 hours of onset of chest pain. risk factors were clustered among both young and old. anterior (49%) then inferior (40) stemi was the presentation. normal coronary lumen angiogram seen in (1.3%). drug eluting stents deployed in 92% of cases. successful result (timi3) obtained in 94%. 80% of ppci discharged home uneventfully with first 24 hours. 6-weeks lvef preserved in 64%. 36% of cases survived but with maces. all-cause mortality was 4.6%. no major differences were seen between different age groups. in table 2 the clinical, angiographic and outcomes findings of cases by the sex were summarized. however, the stemi equivalent presentations were more common among women, table 1. the main findings of patients (n = 151), by age groups clinical finding 21–49 years (n = 46) 50–90 years (n = 105) total (n = 151) p-value* no. % no. % no. % sex male 41 89.1 84 80.0 125 82.8 0.171 female 5 10.9 21 20.0 26 17.2 chief complaint chest pain 41 89.1 86 81.9 127 84.1 0.626sob 2 4.3 8 7.6 10 6.6 other 3 6.5 11 10.5 14 9.3 duration 0.5 hour 8 17.4 27 25.7 35 23.2 0.535 1 hour 18 39.1 45 42.9 63 41.7 2 hours 12 26.1 22 21.0 34 22.5 3–6 hours 3 6.5 3 2.9 6 4.0 > 6 hours 5 10.9 8 7.6 13 8.6 dm positive 8 17.4 38 36.2 46 30.5 0.021 negative 38 82.6 67 63.8 105 69.5 smoking positive 34 73.9 65 61.9 99 65.6 0.257ex-smoker 1 2.2 2 1.9 3 2.0 negative 11 23.9 38 36.2 49 32.5 hypertension positive 17 37.0 62 59.0 79 52.3 0.012 negative 29 63.0 43 41.0 72 47.7 dyslipidemia positive 39 84.8 86 81.9 125 82.8 0.666 negative 7 15.2 19 18.1 26 17.2 family history positive 9 19.6 25 23.8 34 22.5 0.565 negative 37 80.4 80 76.2 117 77.5 past medical history ihd 5 10.9 22 21.0 27 17.9 0.303previous stents 1 2.2 3 2.9 4 2.6 negative 40 87.0 80 76.2 120 79.5 drugs category 1 35 76.1 74 72.5 109 73.6 0.651 category 2 11 23.9 28 27.5 39 26.4 bmi (kg/m2) 18–24.9 7 15.2 18 17.1 25 16.6 0.37825–29.9 34 73.9 82 78.1 116 76.8 30–35 5 10.9 5 4.8 10 6.6 (continued) 122 j contemp med sci | vol. 8, no. 2, march-april 2022: 120–127 outcomes of ppci in iraq original a.m. mohammad et al. table 1. the main findings of patients (n = 151), by age groups—continued clinical finding 21–49 years (n = 46) 50–90 years (n = 105) total (n = 151) p-value* no. % no. % no. % diagnosis anterior stemi 29 63.0 45 42.9 74 49.0 0.205 inferior stemi 15 32.6 46 43.8 61 40.4 posterior stemi 1 2.2 8 7.6 9 6.0 lateral stemi 1 2.2 4 3.8 5 3.3 stemi equivalent 0 0.0 2 1.9 2 1.3 angiogr. findings normal cag 1 2.2 1 1.0 2 1.3 0.575 single v. disease 32 69.6 68 64.8 100 66.2 two v. disease 6 13.0 22 21.0 28 18.5 triple v. disease 7 15.2 14 13.3 21 13.9 culprit artery lad 28 60.9 47 44.8 75 49.7 0.169 rca 11 23.9 45 42.9 56 37.1 lcx 4 8.7 9 8.6 13 8.6 lms 2 4.3 3 2.9 5 3.3 normal 1 2.2 1 1.0 2 1.3 procedure stenting 41 89.1 98 93.3 139 92.1 0.231 ballooning 3 6.5 1 1.0 4 2.6 graft stent 0 0.0 1 1.0 1 0.7 surgery 0 0.0 2 1.9 2 1.3 medical rx 2 4.3 3 2.9 5 3.3 result of pci timi 0 flow 0 0.0 1 1.0 1 0.7 1.000 timi ii flow 1 2.2 4 3.8 5 3.3 timi iii flow 45 97.8 98 93.3 143 94.7 surgery 0 0.0 2 1.9 2 1.3 length of hospital stay 24 hours 39 84.8 82 78.1 121 80.1 0.343 > 24 hours 7 15.2 23 21.9 30 19.9 ef after 6 weeks 50–60 26 56.5 71 67.6 97 64.2 0.190 20–49 20 43.5 34 32.4 54 35.8 six-weeks outcome died 0 0.0 7 6.7% 7 4.6 0.137survived with complication** 20 43.5 35 33.3 55 36.4 survived without complications** 25 54.3 58 55.2 83 55.0 censored*** 1 2.2 5 4.8 6 4.0 *based on chi-square or fisher’s exact test. **stent thrombosis, readmission or ef < 50. ***not included in the statistical test (missing data). n.b. all the percentages are vertical; therefore, comparisons are to made horizontally, between the two age groups. the males had more anterior stemi. we notably not found a statistically significant difference between both gender in other parameters except of some expected risk factors like smoking among males. the table 3 showed the relation of cases’s characteristics to 6 weeks (ef). the predictors of 6-weeks outcomes and lvef were depend on type/location of stemi, the culprit artery, timi flow post pci and length of hospital stay. the impaired ef was observed among anterior stemi (p < 0.001), lad culprit (p < 0.001), less than timi3 pci result (p < 0.039). the overall mortality and morbidity (maces) and longer length of in-hospitalization time were registered in lower lvef. discussion this registry showed the feasibility of ppci with reasonable outcomes in stemi patients. there were generally no clear differences in characteristics outcomes of stemi with respect to gender and ages of patients. in the developed countries the cad is typically aged related with low incidence of the disease among young compared to our area. almost 30% of cases of stemi in this study were among young.5 another striking point is the predominate male gender affection by the disease in the current study. the potential explanation of this phenomena is that the males constitute the 123j contemp med sci | vol. 8, no. 2, march-april 2022: 120–127 a.m. mohammad et al. original outcomes of ppci in iraq table 2. clinical findings of the patients (n = 151), by sex clinical finding males (n = 125) females (n = 26) total p-value no. % no. % no. % chief complaint chest pain 107 85.6 20 76.9 127 84.1 0.449sob 8 6.4 2 7.7 10 6.6 other 10 8.0 4 15.4 14 9.3 duration 0.5 hour 27 21.6 8 30.8 35 23.2 0.080 1 hour 58 46.4 5 19.2 63 41.7 2 hours 25 20.0 9 34.6 34 22.5 3–6 hours 5 4.0 1 3.8 6 4.0 > 6 hours 10 8.0 3 11.5 13 8.6 dm positive 32 25.6 14 53.8 46 30.5 0.004 negative 93 74.4 12 46.2 105 69.5 smoking positive 97 77.6 2 7.7 99 65.6 <0.001ex-smoker 3 2.4 0 0.0 3 2.0 negative 25 20.0 24 92.3 49 32.5 hypertension positive 61 48.8 18 69.2 79 52.3 0.058 negative 64 51.2 8 30.8 72 47.7 dyslipidemia positive 104 83.2 21 80.8 125 82.8 0.777 negative 21 16.8 5 19.2 26 17.2 family history negative 95 76.0 22 84.6 117 77.5 0.339 positive 30 24.0 4 15.4 34 22.5 past medical history negative 98 78.4 22 84.6 120 79.5 0.900ihd 23 18.4 4 15.4 27 17.9 previous stents 4 3.2 0 0.0 4 2.6 drugs category 1 92 74.8 17 68.0 109 73.6 0.482 category 2 31 25.2 8 32.0 39 26.4 bmi (kg/m2) 18–24.9 22 17.6 3 11.5 25 16.6 0.73125–29.9 95 76.0 21 80.8 116 76.8 30–35 8 6.4 2 7.7 10 6.6 diagnosis anterior stemi 65 52.0 9 34.6 74 49.0 0.006 inferior stemi 50 40.0 11 42.3 61 40.4 posterior stemi 8 6.4 1 3.8 9 6.0 lateral stemi 2 1.6 3 11.5 5 3.3 stemi equivalent 0 0.0 2 7.7 2 1.3 angiogr. findings normal cag 1 .8 1 3.8 2 1.3 0.266 single v. disease 85 68.0 15 57.7 100 66.2 two v. disease 21 16.8 7 26.9 28 18.5 triple v. disease 18 14.4 3 11.5 21 13.9 culprit artery lad 65 52.0 10 38.5 75 49.7 0.051 rca 45 36.0 11 42.3 56 37.1 lcx 12 9.6 1 3.8 13 8.6 lms 2 1.6 3 11.5 5 3.3 normal 1 .8 1 3.8 2 1.3 procedure stenting 114 91.2 25 96.2 139 92.1 ballooning 4 3.2 0 0.0 4 2.6 graft stent 1 0.8 0 0.0 1 0.7 1.000 (continued) 124 j contemp med sci | vol. 8, no. 2, march-april 2022: 120–127 outcomes of ppci in iraq original a.m. mohammad et al. table 2. clinical findings of the patients (n = 151), by sex—continued clinical finding males (n = 125) females (n = 26) total p-value no. % no. % no. % surgery 2 1.6 0 0.0 2 1.3 medical rx 4 3.2 1 3.8 5 3.3 result of pci timi 0 flow 0 0.0 1 3.8 1 0.7 0.265 timi ii flow 4 3.2 1 3.8 5 3.3 timi iii flow 119 95.2 24 92.3 143 94.7 surgery 2 1.6 0 0.0 2 1.3 length of hospital stay 24 hours 98 78.4 23 88.5 121 80.1 0.242 > 24 hours 27 21.6 3 11.5 30 19.9 ef after 6 weeks 50–60 77 61.6 20 76.9 97 64.2 0.138 20–49 48 38.4 6 23.1 54 35.8 six-weeks outcome died 5 4.0 2 7.7 7 4.6 0.114survived with complication* 50 40.0 5 19.2 55 36.4 survived (ef ≥50) without compl 65 52.0 18 69.2 83 55.0 censored** 5 4.0 1 3.8 6 4.0 *stent thrombosis, readmission or ef < 50. **not included in the statistical test (missing data). n.b. all the percentages are vertical; therefore, comparisons are to made horizontally, between the two sexes. table 3. relation clinical findings of the patients (n = 151), with their outcome, in terms of ejection fraction (ef), after 6 weeks clinical finding six-weeks outcome (ef) p-value 50–60 (n = 97) 20–49 (n = 54) total no. % no. % no. % age 21–49 years 26 26.8 20 37.0 46 30.5 0.190 50–90 years 71 73.2 34 63.0 105 69.5 sex male 77 79.4 48 88.9 125 82.8 0.138 female 20 20.6 6 11.1 26 17.2 chief complaint chest pain 85 87.6 42 77.8 127 84.1 0.064sob 3 3.1 7 13.0 10 6.6 other 9 9.3 5 9.3 14 9.3 duration 0.5 hour 25 25.8 10 18.5 35 23.2 0.128 1 hour 41 42.3 22 40.7 63 41.7 2 hours 23 23.7 11 20.4 34 22.5 3–6 hours 1 1.0 5 9.3 6 4.0 > 6 hours 7 7.2 6 11.1 13 8.6 dm positive 31 32.0 15 27.8 46 30.5 0.593 negative 66 68.0 39 72.2 105 69.5 smoking positive 60 61.9 39 72.2 99 65.6 0.137ex-smoker 1 1.0 2 3.7 3 2.0 negative 36 37.1 13 24.1 49 32.5 hypertension positive 50 51.5 29 53.7 79 52.3 0.799 negative 47 48.5 25 46.3 72 47.7 dyslipidemia positive 81 83.5 44 81.5 125 82.8 0.752 negative 16 16.5 10 18.5 26 17.2 (continued) 125j contemp med sci | vol. 8, no. 2, march-april 2022: 120–127 a.m. mohammad et al. original outcomes of ppci in iraq table 3. relation clinical findings of the patients (n = 151), with their outcome, in terms of ejection fraction (ef), after 6 weeks—continued clinical finding six-weeks outcome (ef) p-value 50–60 (n = 97) 20–49 (n = 54) total no. % no. % no. % family history negative 77 79.4 40 74.1 117 77.5 0.454 positive 20 20.6 14 25.9 34 22.5 past medical history negative 78 80.4 42 77.8 120 79.5 0.869ihd 16 16.5 11 20.4 27 17.9 previous stents 3 3.1 1 1.9 4 2.6 drugs category 1 71 75.5 38 70.4 109 73.6 0.493 category 2 23 24.5 16 29.6 39 26.4 bmi (kg/m2) 18–24.9 15 15.5 10 18.5 25 16.6 0.83725–29.9 76 78.4 40 74.1 116 76.8 30–35 6 6.2 4 7.4 10 6.6 diagnosis anterior stemi 31 32.0 43 79.6 74 49.0 <0.001 inferior stemi 57 58.8 4 7.4 61 40.4 posterior stemi 4 4.1 5 9.3 9 6.0 lateral stemi 3 3.1 2 3.7 5 3.3 stemi equivalent 2 2.1 0 0.0 2 1.3 angiogr. findings normal cag 2 2.1 0 0.0 2 1.3 0.369 single v. disease 68 70.1 32 59.3 100 66.2 two v. disease 15 15.5 13 24.1 28 18.5 triple v. disease 12 12.4 9 16.7 21 13.9 culprit artery lad 33 34.0 42 77.8 75 49.7 <0.001 rca 50 51.5 6 11.1 56 37.1 lcx 9 9.3 4 7.4 13 8.6 lms 3 3.1 2 3.7 5 3.3 normal 2 2.1 0 0.0 2 1.3 procedure stenting 91 93.8 48 88.9 139 92.1 0.130 ballooning 2 2.1 2 3.7 4 2.6 graft stent 0 0.0 1 1.9 1 0.7 surgery 0 0.0 2 3.7 2 1.3 medical rx 4 4.1 1 1.9 5 3.3 result of pci timi 0 flow 0 0.0 1 1.9 1 0.7 0.039 timi ii flow 2 2.1 3 5.6 5 3.3 timi iii flow 95 97.9 48 88.9 143 94.7 surgery 0 0.0 2 3.7 2 1.3 length of hospital stay 24 hours 86 88.7 35 64.8 121 80.1 <0.001 > 24 hours 11 11.3 19 35.2 30 19.9 six-weeks outcome died 1 1.0 6 11.1 7 4.6 <0.001 survived with complication** 7 7.2 48 88.9 55 36.4 survived (ef ≥50) without compl. 83 85.6 0 0.0 83 55.0 censored*** 6 6.2 0 0.0 6 4.0 *stent thrombosis, readmission or ef < 50. **not included in the statistical test (missing data). n.b. all the percentages are vertical; therefore, comparisons are to made horizontally, between the two ef groups. 126 j contemp med sci | vol. 8, no. 2, march-april 2022: 120–127 outcomes of ppci in iraq original a.m. mohammad et al. bulk of the young premature cad and the second is the wellknown protective effect of estrogen in premenopausal age in females.6,7 in term of cardiometabolic risk factors there was clear trend of clustering of risk factors in our patients. and this clustering was the main attributable to stemi in our region. hence, the control of such risk factors should be the priority in health agenda as soon as possible. it is recognizable that the traditional risk is more important the genetic polymorphisms in this group of patients according to available data from the area.5,8 the fast majority of our cases were presented for first time with cad with negative past history of coronary disease. only one fifth of cases had past history of cad regardless of the original presentation. this highlight the significant increase in the new cases and incidence of the disease in this area.9 compared to previous report from our area the time of presentation of stemi cases to hospital and emergency department is mildly improved.10 since several years ago more than 50% of acute coronary syndrome cases were lately coming to hospital. in this registry the time of presentation was shorter than the time determined by previous report.10 this reflect some improvement in facilities and health education in the area. the angiographic profile of patient reflects another fact; the nature of coronary involvement in this study was extensive lesions. more than 30% of cases had more than one vessel diseased. and this point should raise the awareness about the silent cad before the stemi presentation.11 depend on some national reports the nature of coronary lesion among our patients has two characters: more extensive lesions and more calcification. this point needs a particular attention by the community of cardiology in the area.12 the feasibility of the primary pci procedure was achievable. almost more than 90% cases underwent successful stenting with drug eluting platforms of the culprit artery with timi 3 flow in the culprit artery. the adopted policy for discharging cases post successful ppci was within first 24 hours in 80% of cases and only 20% were stayed hospitalized for longer duration. this early discharge of stable cases after ppci will preserve the economic and health facilities for those with critical cases.13 in terms of 6 weeks follow up, the lvef were preserved in 65%. the remaining percent were presented with different level of impaired lvef especially among the more vulnerable patients. probably the stunning and particularly the hibernation of the myocardium is one of the expected causalities beyond the impaired lvef.14 in addition to different degree of heart failure and lv dysfunction there was higher rate of mace in this study compared to others.15 the predictors of 6-weeks adverse outcomes were depending mainly on type/ location of myocardial infarction, the culprit artery, timi flow post ppci and length of hospital stay. conclusion this study indicated that the 24 hours/7 days of week/ 365 days of year’s ppci is feasible procedure in our area with acceptable outcomes. within the accumulation of experiences in treating stemi and ppci among our health personnel and staff we do expect better outcomes in the near future particularly if these experiences come in line with the health awareness of stemi and chest pain among people. the implementation of recent stemi management protocols like codestemi or stemi alert in our area will add an additional step toward improving the stemi outcomes and cathlab services.16,17 conflicts of interest none.  references 1. mohammad, a.m., jehangeer, h.i. & shaikhow, s.k. prevalence and risk factors of premature coronary artery disease in patients undergoing coronary angiography in kurdistan, iraq. bmc cardiovasc disord 15, 155 (2015). https://doi.org/10.1186/s12872-015-0145-7 2. steg g, james s, atar d, badano l, bldmstrom-lundqvist c, di mario c, et al. management of acute myocardial infarction in patients presenting with persistent st-segment elevation: the task force on the management of st-segment elevation acute myocardial infarction of the european society of cardiology. eur heart j. 2012;33:2569–2619. 3. mohammad am, rashad hh, habeeb qs, rashad bh, saeed sy. demographic, clinical and angiographic profile of coronary artery disease in kurdistan region of iraq. am j cardiovasc dis. 2021;11(1):39–45. published 2021 feb 15. 4. ginanjar, e., sjaaf, a. c., alwi, i., sulistyadi, w., suryadarmawan, e., wibowo, a., & liastuti, l. d. (2020). code stemi program improves clinical outcome in st elevation myocardial infarction patients: a retrospective cohort study. open access emergency medicine: oaem, 12, 315–321. https://doi. org/10.2147/oaem.s259155 5. mohammad, a.m., othman, g.o., saeed, c.h. et al. genetic polymorphisms in early-onset myocardial infarction in a sample of iraqi patients: a pilot study. bmc res notes 13, 541 (2020). https://doi.org/10.1186/s13104-02005367-w 6. mohammad am, sheikho sk, tayib jm. relation of cardiovascular risk factors with coronary angiographic findings in iraqi patients with ischemic heart disease. am j cardiovasc dis res. 2013;1(1):25–9. 7. wake r, yoshiyama m. gender differences in ischemic heart disease. recent patents cardiovasc drug discov. 2009;4:234–240. doi: 10.2174/157489009789152249. 8. mohammad am, al-allawi nas. cyp2c19 genotype is an independent predictor of adverse cardiovascular outcome in iraqi patients on clopidogrel after percutaneous coronary intervention. j cardiovasc pharmacol. 2018; 71(6):347-351. doi: 10.1097/fjc.0000000000000577 9. abd rk, abd sn, raman v. tracing the risk factors of heart diseases at al-nasiriyah heart center in iraq. j cardiovascular disease res. 2019;10(1):31–4. 10. mohammad am, abdulhaleem bh, habeeb qs. first 24 hours’ outcomes of acute coronary syndrome in iraq. med j babylo 2020;17;154-8. 11. jin j. testing for “silent” coronary heart disease. jama. 2014;312(8):858. doi:10.1001/jama.2014.9191 12. ahmed l. fathala, salwa q. bukhari, and abdulaziz al-sugair. high prevalence of coronary artery calcification in saudi patients with normal myocardial perfusion. ann saudi med. 2017 mar-apr; 37(2): 154–160 13. awsan noman, azfar g zaman, [...], and rajiv das. early discharge after primary percutaneous coronary intervention for st-elevation myocardial infarction. eur heart j acute cardiovasc care. 2013 sep; 2(3): 262– 269. doi: 10.1177/2048872612475231 14. francone m, bucciarelli‐ducci c, carbone i, canali e, scardala r, calabrese fa, sardella g, mancone m, catalano c, fedele f, passariello r, bogaert j, agati l. impact of primary coronary angioplasty delay on myocardial salvage, infarct size, and microvascular damage in patients with st‐segment elevation myocardial infarction: insight from cardiovascular magnetic resonance. j am coll cardiol. 2009; 54:2145–2153. https://doi.org/10.1186/s12872-015-0145-7 https://dx.doi.org/10.1177%2f2048872612475231 127j contemp med sci | vol. 8, no. 2, march-april 2022: 120–127 a.m. mohammad et al. original outcomes of ppci in iraq 15. ahn kt, song yb, choe yh, yang jh, hahn j‐y, choi j‐h, choi s‐h, chang s‐a, lee s‐c, lee sh, oh jk, gwon h‐c. impact of transmural necrosis on left ventricular remodeling and clinical outcomes in patients undergoing primary percutaneous coronary intervention for st‐ segment elevation myocardial infarction. int j cardiovasc imaging. 2013; 29:835–842. 16. koh jq, tong dc, sriamareswaran r, yeap a, yip b, wu s, perera p, menon s, noaman sa, layland j. in‐hospital ‘code stemi’ improves door‐to‐ balloon time in patients undergoing primary percutaneous coronary intervention. emerg med australas. 2018; 30:222–227. 17. ameen m mohammad. the interventionist mindset: the ten eyes rule in cath lab. annals of medicine and surgery. 2020;60;644–645. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i2.1202 408 j contemp med sci | vol. 8, no. 6, november-december 2022: 408–412 original an evaluation of blood components inventory management during hajj and umrah season at makkah hospitals saeed m kabrah* laboratory medicine department, faculty of applied medical sciences, umm al-qura university, makkah, kingdom of saudi arabia. *correspondence to: saeed m kabrah (e-mail: smkabrah@uqu.edu.sa) (submitted: 04 october 2022 – revised version received: 21 october 2022 – accepted: 12 november 2022 – published online: 26 december 2022) abstract objectives: the current study aims to utilize blood bank data collected from the army forces hospital in makkah to improve transfusion services at makkah hospitals and evaluate the inventory system during the hajj season. methods: three years’ results showed that blood centre production is not constant, with an average of 1800 units during hajj season that includes cryo (450), ffp (450), plt (450), and prbc (450) units. moreover, data indicated that the average usage of units is 1119 during the same season (120 cryo, 424 ffp, 207 plt and 368 prbc). results: this demonstrated that the inventory of blood units fluctuates during the year with an overproduction of cryo (80%), ffp (67%), plt (83%) and prbc (57%) units in hajj season. it can be concluded that the manner and effectiveness with which the present issues in makkah blood banking are having a major impact on the current system used to obtain the nation’s blood supply and the safety, adequacy, and operational cost of that supply. conclusion: the current study recommends building a generic data warehouse with smart decision support systems that use artificial intelligence to maintain blood bank production and inventory system during the year, especially during hajj and umrah seasons. keywords: blood banks, supply, equipment and supplies, blood units, makkah issn 2413-0516 introduction the islamic hajj gathering in makkah, saudi arabia, is considered as one of the largest global gatherings. the number of pilgrims for both hajj and umrah can reach up to 10 million people annually from more than 180 countries around the globe. during hajj in 2015, 2016, and 2017 more than 2 million pilgrims came to makkah.1 in saudi arabia and over the course of years, a modern surveillance system for hajj has been developed and is continually improving with experiences and advances in the technology.1,2 this surveillance system is essential to minimize accidents and ensure proper infection control during hajj and umrah. additionally, riyadh seasons, formula 1 saudi arabia grand prix and other massive gathering events are increasing in saudi arabia with recent development and changes. also, saudi arabia is threatened continuously by constant attacks to hit civilian areas, schools, and hospitals. planning and organising emergency plans in such a situation are essential to address all possible needs.3–5 such planning and organizational efforts require collecting and analysing data gathered from different authorities during such events.3–5 major traumatic injuries during hajj and umrah include stampedes, fire, traffic accidents and other injuries. for example, during the hajj season in september 2015, 107 pilgrims died, and 394 were injured in the crane collapse accident at the building site of the grand mosque in makkah.1,2 also, in the same year, 769 pilgrims died, and 934 were injured in the stampede accident in mina at the jamaraat bridge.6 the casualty numbers in these accidents created strains on all the healthcare systems in makkah region.1,6 of the main systems that could be affected during such incidents is the blood banking systems in different hospitals in the makkah region. these blood banks need to work effortlessly to provide blood units and blood components to cover the need. thus, careful planning and management of the blood banking systems in these situations are key to avoiding serious effects and losses of lives.7,8 this planning should be built around the distribution of abo and rh blood groups of blood donors in the ksa.9 in hajj, hospitals in the makkah region have records of the blood unit requests, numbers of donations, and shortages during that time. careful analysis of such data and reviewing of the finding to improve the plans and regulations during hajj, umrah and other events is critical. thus, this study aimed to measure donor attendance and donation trends in hajj and umrah seasons and evaluate the blood components productions simultaneously. furthermore, it measured the blood units’ demand to assist in efficient ways of managing blood supply and demand during the hajj and umrah and similar future public or mass gathering emergencies. methods study design the current study is a retrospective study based on data collection from a laboratory information system using blood donor records and unit production during the period from 1436 to 1440 hijri at security forces hospital program in makkah. ethical approval the data were obtained from the blood bank unit of the hospital after optioning ethical approval from the internal review board at security forces hospital program in makkah (0211-120818). data collection tools data for this study were obtained using an excel template (microsoft version 360) of donors’ lab records at the security forces hospital program (sfh) in makkah. all data during the period from 1436h to 1440 hijri were included in the study, as there were no exclusion criteria. subsequently, data cleaning was performed to organize the data and remove any duplication. mailto:smkabrah@uqu.edu.sa 409j contemp med sci | vol. 8, no. 6, november-december 2022: 408–412 s.m. kabrah original impact of hajj and umrah on blood inventory statistics data was reviewed and analysed using the statistical package of social sciences (spss version 24); the descriptive statistics were presented in count and percentages. the chi-square test was performed to compare the number of donors with other variables, and p-value > 0.05 were considered statistically significant. results overview of blood donation and unit production from 1436-1440h a retrospective study was conducted on 5473 blood donors who donated blood at the security forces hospital program in makkah from 1436h to 1440h. details of blood donation and blood units’ production were summarised in table 1. the table demonstrated that blood donation frequency varies through the years and months. only 11.4% (626) of the participant was in 1440h, which was the lowest year with recorded donations, and a large number of participants in 1437h, 27.8% (1523), followed by year 1439h were participant was 26.7% (1462). data demonstrated that blood donation was lowest in ramadan at 5% (272) compared to other months; also, shawal was 6.5% (355), dhul qadah was 9.7% (529), and dhul hijjah was 8.4% (458). results showed that a total of 21205 blood unit components were collected during the study period 1436– 1440. the most collected blood unit was packed red cells (25.8%), followed by platelet (25.1%), fresh frozen plasma (25.1%), and then cryoprecipitate (24%), respectively. the monthly report of blood donation distribution between 1436h to 1440h showed fluctuation in the number of donations depending on the month data for the five years were recorded from the month of muharram to dhul hijjah. the monthly breakdown of blood units collected each year is shown in figure 1. data indicated that the frequency of blood donation trends varies throughout the years and months. in muharram, the blood donation rate was low compared to other months, then it started to increase in the middle of the year and dropped again to a similar rate in dhul hijjah. there was a notable drop in the number of donors during ramadan in all years, and it was the lowest compared to other months in the same year. in addition to the 12th month of hajj, the donation rate also decreased compared to other months in all years. the year 1440h was the lowest compared to other years since donation numbers were zeroes from the 5th to 12th months, in addition to the year 1438h were zeros from month 8th to 12th. results indicated that there were statistical differences in comparing rates of donation, a p-value of 0.0001. utilization of blood components between 1436h to 1440h showed that the unit production did not cover the needs blood units’ production and usage data for the five years were recorded. the monthly breakdown of the percentage of blood units’ utilization for each year is shown in table 2. data demonstrated that utilization of blood units differs according to year, month, and type of blood unit. data indicated that during ramadan, production of the blood units exceeded the usage for cryo (52.9%), prbc (52.2%), plt (25.0%) and ffp (17.6%). a similar finding was observed during hajj too, where the production increased for cryo (81.1%), plt (91.6%) and ffp (55.8%), with the expectation of prbc, where the production decreased by (–29.5%). furthermore, results indicated that hajj and umrah seasons impact the unit’s utilisation where unit production does not cover the needs. table 1. characteristics of blood donation and production during the study period characteristic n % frequency of blood donation during arabic months 1. muharram 423 7.7 2. safar 487 8.9 3. rabi al-awwal 431 7.9 4. rabi al-thani 599 10.9 5. jamada al-awwal 558 10.2 6. jamada al-thani 494 9.0 7. rajab 461 8.4 8. shaban 406 7.4 9. ramadan 272 5.0 10. shawwal 355 6.5 11. dhul qadah 529 9.7 12. dhul hijjah 458 8.4 total 5473 100.0 frequency of blood donation during hijry years 1436 856 15.6 1437 1523 27.8 1438 1006 18.4 1439 1462 26.7 1440 626 11.4 total 5473 100.0 frequency of blood units produced cryo 5084 24.0 ffp 5326 25.1 plt 5322 25.1 prbs 5473 25.8 total 21205 100 this table shows the demographic characteristics of blood donors who participated in the study. number (n) and percentage (%). fig. 1 number of donors from 1436 to 1440. this figure shows the distribution of blood donors among hejry months. p-value was 0.0001. 410 j contemp med sci | vol. 8, no. 6, november-december 2022: 408–412 impact of hajj and umrah on blood inventory original s.m. kabrah ta bl e 2. p er ce nt ag e of th e ut ili za tio n of b lo od co m po ne nt s a t s fh -m ak ka h ho sp ita l ye ar un it ty pe m on th av er ag e pva lu e 1 2 3 4 5 6 7 8 9 10 11 12 14 37 cr yo 60 .0 10 0. 0 10 0. 0 68 .7 72 .4 36 .2 80 .5 83 .7 52 .9 67 .0 94 .7 81 .1 75 0. 05 1 ff p –8 .0 10 0. 0 85 .7 60 .0 75 .6 –5 8. 5 58 .5 67 .4 17 .6 46 .2 88 .6 55 .8 49 pl t 14 .0 90 .1 56 .0 19 .1 7. 3 9. 6 –2 .4 82 .6 25 .0 35 .2 96 .5 91 .6 44 pr bc 56 .0 53 .1 65 .9 29 .6 53 .7 28 .7 –2 2. 0 56 .5 52 .2 6. 6 41 .2 –2 9. 5 33 14 38 pr bc 22 .0 51 .4 28 .9 59 .6 46 .0 45 .7 –1 4. 1 59 .0 52 .3 41 .3 51 .7 30 .3 40 0. 04 9 cr yo 57 .8 10 0. 0 91 .2 95 .7 83 .3 75 .0 87 .1 94 .3 83 .0 85 .6 10 0. 0 74 .2 86 ff p 40 .4 10 0. 0 71 .9 75 .2 58 .7 47 .4 32 .9 67 .9 –0 .6 59 .3 90 .4 33 .5 56 pl t 39 .4 78 .0 63 .2 92 .2 51 .6 58 .6 83 .5 94 .8 91 .5 68 .9 77 .5 78 .1 73 14 39 cr yo 60 .0 10 0. 0 10 0. 0 68 .7 72 .4 36 .2 80 .5 83 .7 52 .9 67 .0 94 .7 81 .1 75 0. 05 0 ff p –8 .0 10 0. 0 85 .7 60 .0 75 .6 –5 8. 5 58 .5 67 .4 17 .6 46 .2 88 .6 55 .8 49 pl t 14 .0 90 .1 56 .0 19 .1 7. 3 9. 6 –2 .4 82 .6 25 .0 35 .2 96 .5 91 .6 44 pr bc 56 .0 53 .1 65 .9 29 .6 53 .7 28 .7 –2 2. 0 56 .5 52 .2 6. 6 41 .2 –2 9. 5 33 14 40 cr yo 60 .0 10 0. 0 10 0. 0 68 .7 72 .4 36 .2 80 .5 83 .7 52 .9 67 .0 94 .7 81 .1 75 0. 04 ff p –8 .0 10 0. 0 85 .7 60 .0 75 .6 –5 8. 5 58 .5 67 .4 17 .6 46 .2 88 .6 55 .8 49 pl t 14 .0 90 .1 56 .0 19 .1 7. 3 9. 6 –2 .4 82 .6 25 .0 35 .2 96 .5 91 .6 44 pr bc 56 .0 53 .1 65 .9 29 .6 53 .7 28 .7 –2 2. 0 56 .5 52 .2 6. 6 41 .2 –2 9. 5 33 to ta l cr yo 60 .0 10 0. 0 10 0. 0 68 .7 72 .4 36 .2 80 .5 83 .7 52 .9 67 .0 94 .7 81 .1 75 0. 04 ff p –8 .0 10 0. 0 85 .7 60 .0 75 .6 –5 8. 5 58 .5 67 .4 17 .6 46 .2 88 .6 55 .8 49 pl t 14 .0 90 .1 56 .0 19 .1 7. 3 9. 6 –2 .4 82 .6 25 .0 35 .2 96 .5 91 .6 44 pr bc 56 .0 53 .1 65 .9 29 .6 53 .7 28 .7 –2 2. 0 56 .5 52 .2 6. 6 41 .2 –2 9. 5 33 411j contemp med sci | vol. 8, no. 6, november-december 2022: 408–412 s.m. kabrah original impact of hajj and umrah on blood inventory discussion hospitals are the first place the wounded are relocated to when a natural or manufactured disaster occurs, and it may generate many types of injuries that may require the use of blood units. such events may also occur in mass gatherings such as hajj and umrah. for this reason, blood banks must be prepared for a rapid supply of units to the patients. for that, this study attempted to analyse the blood unit production and utilisation at sfh-makkah during 1436–1440. a close view of the blood units’ usage would help focus on frequent wastage and improve the blood bank services at makkah, especially during hajj and umrah seasons. subsequently, it would further help authorities design intervention measures to prevent such issues and improve blood unit management throughout makkah hospitals. the current statistics showed that the number of blood donors varies according to the islamic months, especially during hajj and umrah seasons, where the lowest donation number was observed in ramadan (272), shawal (355), dhul qadah (529), and dhul hijjah was (458). this indicates that the blood centre lacks an adequate number of volunteer and non-volunteer blood donors, which can impact the access to enough blood to meet patients’ current and future needs. similarly, alkahtani and jilani10 demonstrated that the number of blood donors decreased in june and september in 2017 and 2018, corresponding to the ramadan and hajj periods. they also proved the importance of predicting return donors and analysing blood donation time series using data mining techniques to sustain an adequate blood supply during islamic seasons. nevertheless, 43 well-defined blood group systems have more than 340 antigens,11 but generally, only abo and rhd blood group status is considered for donation and transfusion in saudi arabia. this increases the chances of alloimmunisation if the donor and recipient are of different ethnic backgrounds with varied blood antigenic profiles. during hajj and umrah, a large diversity of ethnicity is observed, especially since muslims worldwide visit makkah during different times of the year.12 such diversity can cause a challenge for hospitals to provide suitable blood units for non-saudi patients if needed. these facts highlight the need to promote local blood donation and the need for extended blood group phenotyping. a canadian researcher observed a similar finding that proved that global antigen demand affected community-directed recruitment, including blood drives and the benefits of the mass-scale cell genotyping.13 according to the results, blood unit use varies by year, month, and kind of blood unit. furthermore, the results revealed that the hajj and umrah seasons influenced unit usage when unit production was insufficient to meet demands. by reviewing the project outcomes with blood bank staff, it was noted that the significant reason for utilising the components might be communication between centres in makkah. it was also recommended that there is always a more considerable blood shortage, and a need for awareness is essential among laboratory staff, nurses, and physicians on handling blood products. similar to the results of other studies, where they indicated a significant improvement in blood products management after the educational programme.14,15 packed cells were the most frequently collected of the four blood components collected at sfh-makkah, followed by fresh frozen plasma, platelet, and cryoprecipitate. the current finding agrees with another research.16,17 the present study did not include the production and utilisation of the whole blood unit since whole blood is not frequently used except in a patient who lost more than 75% of the blood volume. consequently, the current results prove that the national health authorities must become more involved in the blood donation, unit production and transfusion field’s fundamental improvement. this involves establishing national legislation that acknowledges blood donation and production, unit safety and transfusion as top public health issues. the saudi health ministry must establish a national regulatory agency to manage a national blood bank quality system and the country’s blood supply. it would operate as a bridge between the many professionals in the blood transfusion business. a reliable haemovigilance reporting system is also necessary to track quality improvement and evaluate all transfusion-related mediumand long-term consequences. blood component management remains an issue for all health services worldwide; it requires inexpensive and easy interventions such as research and staff education, a centralised digital management system, and enhanced transportation. furthermore, identification modalities are required that can have a critical and dramatic influence on improving the management concerning cost and resource savings.1,4 cryo and ffp have a shelf life of 24 hours after preparation, and it has been noted in the current study that there needs to be a clear policy regarding requesting such units. moreover, as blood products have a restricted half-life, accurate strategies should be enforced for blood supplies to prevent loss and reduce waste as much as possible.4,5 the saudi health authority needs to create an emergency operations plan that focuses on education, assigning roles, and makkah blood banks practising and should be based on reviewing the national plan need to occur. there is also a need to integrate hajj data management systems with blood unit surveillance as an early warning system for blood donation and transfusion control during hajj and umrah. similar findings were also demonstrated if the infectious disease during hajj1,18 also during the covid-19 pandemic.19 international engagement is essential to strengthening national blood surveillance because blood groups differ based on ethnicity. this plan should include an intelligent digital management system that improves the current practice and reduces human error during mass gathering events. such a system should consist of an intellectual multi period decision-making framework for emergency blood allocation considering supply and demand uncertainties in the disaster maintenance operations.20 also, it should consider blood groups, age and blood substitution. the finding of this work presented a clear picture of the limitations and challenges that face the saudi ministry of health and the ministry of hajj and umrah each year during the hajj and umrah seasons. also, they provide insight into the importance of communication and information sharing during that period of the year. additionally, with the changes in the umrah allowance periods, now the season is open almost all year long, which presents the sectors with more significant challenges and solutions needed. additional events of massive gatherings are also planned in the near future, which would add up to the stress of managing emergencies. thus, recommendations are all aimed at expanding the current study to include all hospitals in makkah, as these hospitals are the hot spots facing these challenges. also, to activate the intelligent blood bank centralisation management system. this system should survey, gather data, and analyse it to plan. also, there should be a clear policy to manage massive gatherings 412 j contemp med sci | vol. 8, no. 6, november-december 2022: 408–412 impact of hajj and umrah on blood inventory original s.m. kabrah and procedures to follow to plan care and emergencies, including blood bank supply. this system should also manage the blood group distribution of patients and donors.9 another aspect to consider in future work is expanding the data analysis range to include more details, such as common and rare blood groups and planning to secure sources for such rare types. this is essential to consider as in the hajj and umrah seasons, people gather from all locations around the world with all possibilities for rare blood antigens. managing blood banks should be an essential topic in massive gatherings of management studies and policies and regulations. conclusion the study showed blood unit production and utilisation were influenced by hajj and umrah seasons at the sfc-makkah; the most shortage was observed in prbc and platelets. globally, the wastage of blood in hospitals is universal and should be addressed with easy and inexpensive interventions to improve blood component management. programs to encourage blood donations during hajj and umrah seasons should be increased to accommodate the needs of blood units during the same period. moreover, developing national and local guidelines prioritising blood transfusion during seasons and mass gathering is critical. such guidelines should closely monitor blood needs and supply, including the appropriate response for any events that are essential to avoid sudden blood shortages. also, an evidence-based emergency blood unit management plan and flexible regulatory policy should be implemented to manage any disaster that leads to blood shortage. finally, the current research highly recommends developing an intelligent decision-making system to improve the management of blood units, as blood is a vital and rare resource. acknowledgment the author would like to thank the sfh hospital, in makkah, saudi arabia, for supporting this work. conflict of interest the authors declare that there are no conflicts of interest regarding the publication of this paper. funding this project was not funded by any organisation. data and resource availability statement the data that support the findings of this study are available from sfh hospital makkah, but restrictions apply to the availability of these data, which were used under license for the current research and therefore are not publicly available. data are however available from the authors upon reasonable request and with permission of sfh hospital makkah. additionally, the resource generated during and/or analysed during the current study is available from the corresponding author upon reasonable request.  references 1. alotaibi bm, yezli s, bin saeed aa, turkestani a, alawam ah, bieh kl. strengthening health security at the hajj mass gatherings: characteristics of the infectious diseases surveillance systems operational during the 2015 hajj. j. travel med. 2017;24(3):1–6. doi:10.1093/jtm/taw087. 2. memish za, zumla a, alhakeem rf, assiri a, turkestani a, al harby kd, alyemni m, dhafar k, gautret p, barbeschi m, mccloskey b, heymann d, al rabeeah aa, al-tawfiq ja. hajj: infectious disease surveillance and control. lancet 2014;383(9934):2073–2082. doi:10.1016/s0140-6736(14)60381-0. 3. bundy kl, foss ml, stubbs jr. transfusion service disaster planning. immunohematology 2008;24(3):93–101. 4. zaheer ha, waheed u. blood transfusion service in disasters. transfus apher sci 2016;55(2):186–190. doi:10.1016/j.transci.2016.09.007. 5. gschwender an, laurie g. disaster preparedness in the blood bank. american society for clinical laboratory science 2017;30(4):250–257. 6. memish za, steffen r, white p, dar o, azhar ei, sharma a, zumla a. mass gatherings medicine: public health issues arising from mass gathering religious and sporting events. lancet 2019; 393(10185):2073–2084. doi:10.1016/s0140-6736(19)30501-x. 7. biddinger pd, baggish a, harrington l, d’hemecourt p, hooley j, jones j, kue r, troyanos c, dyer ks. be prepared—the boston marathon and mass-casualty events. n engl j med 2013;368(21):1958–1960. doi:10.1056/nejmp1305480. 8. dong yh, liu f, liu ym, jiang xr, zhao zx. emergency preparedness for mass gatherings: lessons of “12.31” stampede in shanghai bund. chin j traumatol. 2017;20(4):240–242. 9. kabrah sm, flemban af, khogeer aa, bawazir wm. reviewing publication discussing the frequency of abo and rhesus-d blood groups in saudi arabia. j res med dent sci. 2021;9(10):29–37. 10. alkahtani sa, jilani m. predicting return donor and analyzing blood donation time series using data mining techniques. int j adv comput sci appl 2019;10(8):113–118. 11. isbt. (2021). “red cell immunogenetics and blood group terminology.” retrieved 30/10, 2021, from https://www.isbtweb.org/working-parties/redcell-immunogenetics-and-blood-group-terminology. 12. gafs. (2021). “hajj report.” retrieved 30/10, 2021, from https://www.stats. gov.sa/en/statistical-knowledge. 13. trepanier p, chevrier mc, constanzo yanez j, baillargeon n, st-pierre c, perreault j. adapting to supply-and-demand emerging trends for antigen-negative red blood cell units. transfusion 2021;61(5):1489–1494. doi:10.1111/trf.16285. 14. islami vaghar m. the impact of an educational program on blood and blood products transfusion on nurses’ level of knowledge and performance. j med life 2018;11(3):238–242. doi:10.25122/jml-2018-0016. 15. riveira mc, louzon mj, tuott ee, monoski tj, cruz-cody vg, tesfamariam a, hess jr. blood school: a laboratory-based transfusion class for nurses. am. j. clin. pathol. 2020;153(4). 16. elsayid m, aseeri yy, saqri fa, alanazi a, qureshi s. a study of prevalence of blood group of saudi patients in king abdulaziz medical city, riyadh. sci j public health 2015;3(4). doi:10.11648/j.sjph.20150304.25. 17. safia moussa, fatmah al-zaylai, may o alnawmasi, mona s aljarwan, hayam a lshammari, layali m alrashedi, aloufi sa. pattern of distribution of abo and rhesus (rh) blood groups in hail province, saudi arabia. ijmrhs 2018;4(3). 18. qanta aa, maurizio b, ziad am. the quest for public health security at hajj: the who guidelines on communicable disease alert and response during mass gatherings. travel med infect dis. 2009; 7(4):226–230. 19. yahia aio. management of blood supply and demand during the covid-19 pandemic in king abdullah hospital, bisha, saudi arabia. transfus apher sci 2020;59(5):102836. doi:10.1016/j.transci.2020.102836. 20. ma z, wang k, dai y. an emergency blood allocation approach considering blood group compatibility in disaster relief operations. int j disaster risk sci 2019;10:74–88. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1303 https://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology https://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology https://www.stats.gov.sa/en/statistical-knowledge https://www.stats.gov.sa/en/statistical-knowledge 229j contemp med sci | vol. 8, no. 4, july-august 2022: 229–234 original clinico-angiographic profiles and in-hospital outcomes of non-st segment elevation myocardial infarction in kurdistan region of iraq ameen m mohammad1*, haval a issa2, saad y saeed3 1department of internal medicine, college of medicine, duhok university, duhok, iraq. 2duhok heart center, azadi teaching hospital, duhok, iraq. 3department of community medicine, college of medicine, university of duhok, iraq. *correspondence to: ameen m mohammad (e-mail: doctoramb@yahoo.com ) (submitted: 10 may 2022 – revised version received: 24 may 2022 – accepted: 21 june 2022 – published online: 26 august 2022) abstract objectives: this work aimed to study the clinical, angiographic profiles and in-hospital outcomes of nstemi cases in duhok, iraq. methods: this prospective study involved 283 patients with nstemi who were admitted to azadi teaching hospital/azadi heart center in duhok, kurdistan region of iraq, between 2021 and 2022. the patient’s demographic variables, major cardiovascular risk factors (smoking, hypertension, diabetes mellitus, hyperlipidemia and family history of coronary artery disease), clinical presentation, past history of myocardial infarction/previous percutaneous coronary intervention (pci) and drug history were collected. the grace risk score was calculated for each patient. patients were followed up regarding the management strategies (whether conservative or invasive approach), and in-hospital complications and outcomes. results: the mean grace score was 142 ± 26. 70% of cases underwent coronary angiography/angioplasty, with a mean time to the coronary intervention of 8 days. 17% of the sample had developed different cardiovascular complications, with heart failure being the most common. the mortality rate was 7.4%. conclusion: the study demonstrated higher complications and mortality rates, especially among patients with higher grace scores, compared to rates found in most available studies, particularly in western countries. this finding could be secondary to a suboptimal coronary intervention for nstemi in terms of time to intervention and the proportion of patients who underwent it. keywords: non-st elevated myocardial infarction, acute coronary syndrome, coronary intervention in nstemi issn 2413-0516 introduction acute myocardial infarction remains the leading cause of death worldwide, including in iraq.1-3 however, despite the rate of st-elevation myocardial infarction decreasing, the incidence of non-st-elevation myocardial infarction (nstemi) is increasing.4 this is believed to be due to many reasons, including the ageing of the population with a greater prevalence of diabetes and chronic kidney disease; and extensive use of troponin assays with higher sensitivity for myocardial injury, which move the diagnosis from unstable angina to nstemi.5-7 the risk stratification for cases with nstemi can be obtained from several prognostic scores like the timi (thrombolysis in myocardial infarction) and grace (global registry of acute coronary events) scores.8,9 timi and grace scores can be determined from the patient’s clinical characteristics, electrocardiographic and laboratory investigations performed on admission. they are satisfactorily simple and practical for risk assessments over a wide range of patients with nste-acs.10 the fundamental step in the management of patients with nstemi is the initial assessment of hemodynamic and electrical stability, and calculation of the patient’s overall risk to assist in treatment guidance.11-13 there are two management strategies in nstemi; either an early invasive strategy with coronary angiography/revascularization (either pci/coronary artery bypass grafting (cabg) as needed) or a conservative approach with medical therapy initially.14,15 regardless of which strategy is applied, both demand proper use of risk assessment and medications.16,17 being the nstemi has not been studied well in our region and iraq, we aimed in this registry to look at the clinical, angiographic, management and in-hospital outcomes of nstemi patients in duhok, iraq. methods in this prospective study, we enrolled cases of nstemi admitted at azadi teaching hospital/azadi heart center in duhok, kurdistan region of iraq, between 2021 and 2022. all recruited cases, both men and women, were followed up during the period of in-hospital stay. the following data of cases were collected: patient’s demographic variables, clinical presentations, major cardiovascular risk factors (smoking, hypertension, diabetes mellitus, hyperlipidemia and family history of coronary artery disease (cad)), past history of myocardial infarction/previous pci and drug history. the grace risk score was calculated for each patient. the patients were followed up during hospitalization with subsequent documentation of in-hospital major adverse cardiac events, namely heart failure, life-threatening arrhythmias, ischemic stroke and cardiac death. for patients who underwent coronary angiography, time to intervention was documented, and the results of coronary angiography/angioplasty were collected and classified according to lesion significance, the number of coronary vessels involved, and the recommended management protocol; whether medical, pci or cabg was addressed. ethical approval the study was approved by the research ethics committee of kurdistan higher council of medical specialties. all patients enrolled in the study provided written informed consent. statistical analysis collected data were entered into microsoft excel, and then transferred to spss version 26 for statistical analysis. mailto:doctoramb@yahoo.com 230 j contemp med sci | vol. 8, no. 4, july-august 2022: 229–234 clinico-angiographic profiles and in-hospital outcomes of nstemi original a.m. mohammad et al. frequency tables, range, mean and standard deviation (sd) were used to describe the data. association between categorical data were analyzed by chi-square test, while differences in means were analyzed by unpaired t-test. p values less than 0.05 were considered statistically significant. results a total of 283 patients (191 males, 92 females) with a clinical diagnosis of nstemi were enrolled in the study with a mean age of 60.3 ± 12.8 years. males were affected more than females. the common presenting symptom was ischemic chest pain. the cardiovascular risk factors were clustered, particularly hypertension and smoking. about 70% of cases were newly diagnosed with cad. the mean grace score was 142 ± 26. 70% of patients underwent coronary angiography/angioplasty. 17% of the sample had developed different cardiovascular complications. the mortality rate was 7.4%, as shown in table 1. the data from patients who underwent coronary intervention showed that (17.4%) had no significant coronary lesions. and the cases had undergone coronary angiography/ intervention within a mean of 8 days after admission. many of them received stents (65.1%), as shown in table 2. the comparison between conservative and intervention groups showed that the young cases underwent intervention significantly more than elderly (p < 0.001). both genders received similar rates of intervention. the coronary intervention was done more frequently for cases with higher grace scores compared to cases with lower grace scores (p < 0.001). generally, the incidence of cardiovascular complications and mortality rate were higher among the conservative group (p < 0.006 and p < 0.001), respectively, as shown in table 3. the characteristics of patients in relation to grace scores revealed that the younger ages had lower grace scores than older ages (p < 0.001). males made up the majority of the lower grace scores compared to the females (p < 0.001). those cases presented with nonspecific presentations had higher grace scores. almost all cardiovascular risk factors were significantly associated with higher rates of grace scores (p-values were significant for all except for hyperlipidemia). furthermore, those cases with a positive history of prior mi/pci had higher grace scores than cases without such past history (p-values were significant). the higher the grace scores, the greater the cardiovascular complications and mortality rates (p values of <0.001 for each), as shown in table 4. the characteristics of the intervention group based on grace scores demonstrated that extensive coronary lesions were significantly seen among grace scores of higher than 140 with a highly significant p-value <0.001. regarding time to intervention and treatment modalities, there were no significant differences between the two groups of grace scores with (p values of 0.936 and 0.309) respectively, as shown in table 5. discussion the study was conducted to assess nstemi patient’s characteristics, management strategy, complications and in-hospital outcomes. the mean age of presentation was comparable to table 1. characteristics of all the patients characteristics no. (283) % age (years) 25–44 28 9.9 45–64 155 54.8 65–90 100 35.3 range; mean ± sd 25–90; 60.3 ± 12.8 gender male 191 67.5 female 92 32.5 main presentation chest pain 220 77.7 dyspnea 46 16.3 other 17 6.0 cardiovascular risk factors hypertension 140 49.5 dm 110 38.9 hyperlipidemia 56 19.8 smoking 132 46.6 family history 20 7.1 past medical history previous pci 48 17.0 previous mi 36 12.7 drug history aspirin 107 37.8 ace/arb 89 31.4 statin 103 36.4 beta-blocker 38 13.4 others 50 17.7 grace score (range; mean ± sd) 81–218; 142.2 ± 26.3 management conservative 88 31.1 intervention 195 68.9 complications heart failure 23 8.1 arrhythmias 8 2.8 heart failure + arrhythmias 14 4.9 heart failure + stroke 1 0.4 arrhythmias + stroke 1 0.4 no complication 236 83.4 survival alive 262 92.6 dead 21 7.4 total 283 100.0 other studies from iraq, including mohammad et al.,17 but was younger compared with western countries’ age presentation of nstemi.18-20 the female percentage in our study was 32.5%, which was higher than kinsara et al. from saudi arabia21 and was comparable to abdelmoneim et al. from egypt.22 regarding clinical presentation, chest pain was the predominant symptom. however, females tend to present more with dyspnea than males, and this was comparable to other iraqi23 and saudi arabian trends.21 231j contemp med sci | vol. 8, no. 4, july-august 2022: 229–234 a.m. mohammad et al. original clinico-angiographic profiles and in-hospital outcomes of nstemi table 2. characteristics of the intervention group characteristics no. % angiographic findings single vessel 80 41.0 double vessels 48 24.6 triple vessels 33 16.9 no significant lesion 34 17.4 time to intervention in days (range; mean ± sd) 1–21; 8.3 ± 4.2 treatment stenting 127 65.1 cabg 30 15.4 medical 38 19.5 total 195 100.0 table 3. characteristics of the conservative group vs the intervention group characteristics management p-value*conservative intervention no. % no. % age (years) 25–44 2 2.3 26 13.3 <0.00145–64 35 39.8 120 61.5 65–90 51 58.0 49 25.1 gender male 58 65.9 133 68.2 0.703 female 30 34.1 62 31.8 main presentation chest pain 56 63.6 164 84.1 <0.001dyspnea 26 29.5 20 10.3 other 6 6.8 11 5.6 hypertension 57 64.8 83 42.6 0.001 dm 35 39.8 75 38.5 0.834 hyperlipidemia 20 22.7 36 18.5 0.404 smoking 36 40.9 96 49.2 0.194 family history 4 4.5 16 8.2 0.266 previous pci 15 17.0 33 16.9 0.980 previous mi 11 12.5 25 12.8 0.940 aspirin 34 38.6 73 37.4 0.847 ace/arb 35 39.8 54 27.7 0.043 statin 33 37.5 70 35.9 0.795 beta blocker 16 18.2 22 11.3 0.115 others 17 19.3 33 16.9 0.625 grace score <140 25 28.4 107 54.9 <0.001 ≥140 63 71.6 88 45.1 complications heart failure 5 5.7 18 9.2 0.006 arrhythmias 4 4.5 4 2.1 heart failure + arrhythmias 9 10.2 5 2.6 stroke 2 2.3 0 0.0 no complication 68 77.3 168 86.2 survival alive 69 78.4 193 99.0 <0.001 dead 19 21.6 2 1.0 total 88 100.0 195 100.0 *based on chi-square test. studying the cardiovascular risk factors for the cases, hypertension was the commonest risk factor, followed by smoking in the current study, this comes in agreement with mohammad et al.,17 but in the mrsic et al. study from bosnia, smoking was the commonest risk factor followed by hypertension.24 generally, the traditional cardiovascular risk factors are clustering with the increasing incidence among iraqi patients with cad.25 the management strategy applied in the current study showed that about 70% of cases were managed by an invasive interventional approach, and 30% were managed conservatively. however, the medical guidelines recommend a routine invasive strategy for almost all patients with nstemi within a limited time to improve the composite ischemic outcomes.16,26 https://pubmed.ncbi.nlm.nih.gov/?term=mrsic%20d%5bauthor%5d 232 j contemp med sci | vol. 8, no. 4, july-august 2022: 229–234 clinico-angiographic profiles and in-hospital outcomes of nstemi original a.m. mohammad et al. in terms of major adverse cardiovascular events and complications, we found that 17% of cases developed complications during the hospital stay. the most common one was heart failure, followed by arrhythmias. its rates were comparable to the study by dakhil et al.27 and was higher than the study by butt et al.28 regarding nstemi mortality, the study demonstrated that the mortality rate was 7.4%. this rate was almost similar to a study by hamid et al. from iraq (7.7%),29 but was higher than the grace registry (5%)30 and yusuf et al. (3.3%).31 in assessing the coronary lesions cases in the intervention group; 17.4% had no significant coronary lesions. this was higher than the rate mentioned in a study by cortell et al. (13%),32 and lower than the rate revealed in mohammad et al. study (22.4%).33 the mean time to intervention was 8 days in the study. it was much longer than the mean times to intervention in most other studies like milasinovic et al. (time to coronary angiography varied from 0.5 to 24 h in the early and from table 4. characteristics of all cases (n = 283), based on grace score characteristics grace score p-value*<140 ≥140 no. % no. % age (years) 25–44 28 21.2 0 0.0 <0.00145–64 98 74.2 57 37.7 65–90 6 4.5 94 62.3 gender male 106 80.3 85 56.3 <0.001 female 26 19.7 66 43.7 main presentation chest pain 113 85.6 107 70.9 0.006dyspnea 16 12.1 30 19.9 other 3 2.3 14 9.3 hypertension 48 36.4 92 60.9 <0.001 dm 35 26.5 75 49.7 <0.001 hyperlipidemia 23 17.4 33 21.9 0.351 smoking 73 55.3 59 39.1 0.006 family history 16 12.1 4 2.6 0.002 previous pci 16 12.1 32 21.2 0.043 previous mi 8 6.1 28 18.5 0.002 aspirin 38 28.8 69 45.7 0.003 ace/arb 28 21.2 61 40.4 0.001 statin 37 28.0 66 43.7 0.006 beta blocker 16 12.1 22 14.6 0.547 others 13 9.8 37 24.5 0.001 complications heart failure 2 1.5 21 13.9 <0.001 arrhythmias 2 1.5 6 4.0 heart failure + arrhythmias 2 1.5 12 7.9 stroke 0 0.0 2 1.3 no complication 126 95.5 110 72.8 survival alive 131 99.2 131 86.8 <0.001 dead 1 0.8 20 13.2 total 132 100.0 151 100.0 *based on chi-square test. 20.5 to 86 h in the delayed group).34 this means that despite the rate of interventional approach for cases in our study, the time to intervention was significantly late and inconsistence with the recommended guidelines. in regards to treatment modalities for cases underwent intervention in this study, 65% were treated by pci and stenting, which was higher than the percentage found in a study from united states by b. case et al. (53%).35 nevertheless, the cabg rates were similar in both studies (15.4% vs 15.1%). in comparisons between conservative and intervention groups, the study showed that the younger age groups had a significantly higher rate of intervention than the elderly group (p < 0.001). this was comparable to dakhil et al.27 those with higher grace scores received more intervention than cases with lower grace scores (p < 0.001). this was comparable to other studies, including martinez et al. in spain.36 almost all cardiovascular risk factors in this study were associated significantly with higher rates of grace scores https://pubmed.ncbi.nlm.nih.gov/?term=dakhil%20za%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=milasinovic+d&cauthor_id=25966439 https://pubmed.ncbi.nlm.nih.gov/?term=dakhil%20za%5bauthor%5d 233j contemp med sci | vol. 8, no. 4, july-august 2022: 229–234 a.m. mohammad et al. original clinico-angiographic profiles and in-hospital outcomes of nstemi table 5. characteristics of the intervention group (n = 195), based on grace score characteristics grace score p-value<140 ≥140 no. % no. % angiographic findings single vessel 55 51.4 25 28.4 <0.001* double vessels 18 16.8 30 34.1 triple vessels 10 9.3 23 26.1 no significant lesion 24 22.4 10 11.4 time to intervention in days (range; mean ± sd) 1–20; 8.3 ± 4.7 1–21; 8.3 ± 3.4 0.936** treatment stenting 67 62.6 60 68.2 0.309*cabg 15 14.0 15 17.0 medical 25 23.4 13 14.8 total 107 100.0 88 100.0 *based on chi-square test. **based on unpaired t-test. (p values were significant for all except for hyperlipidemia). this was in concordance with a study by hall et al. in the uk, which showed that all cardiovascular risk factors were significantly correlated to higher grace scores (including hyperlipidemia).37 meanwhile, a study by cakar et al. in turkey showed a statistically significant relation between hypertension (but not smoking/diabetes) and high grace scores.38 patients with a past history of mi/pci had higher grace scores (p-value was significant). this was comparable to hall et al.37 on the other hand, the extensive coronary lesions were significantly associated with grace scores of higher than 140. such finding was also seen by butt et al. and rahmani et al.28,39 the cardiovascular complications and death rates were also higher among grace scores of ≥ 140 with p values (<0.001 for each). these findings were almost similar to rates found by dakhil et al.27 and kumar et al.40 conclusion the coronary intervention for nstemi cases was suboptimal in our area, both in the time to intervention and the percentage of cases undergoing intervention. this might explain the higher mortality and adverse outcomes in this study compared to available data. it is worthy to say that the guideline-directed immediate and early invasive strategy in indicated nstemi cases and the revision of the current local nstemi management protocol might improve the outcomes of the cases in our countries. conflict of interests nothing to declare. financial support none. acknowledgments we thank all the staff of cath lab and cardiology department in azadi heart center/azadi teaching hospital for their cooperation in conducting this study.  references 1. lozano r, naghavi m, foreman k, lim s, shibuya k, aboyans v, et al. global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study 2010. the lancet. 2012;380(9859):2095–128. 2. mohammad am, jehangeer hi, shaikhow sk. prevalence and risk factors of premature coronary artery disease in patients undergoing coronary angiography in kurdistan, iraq. bmc cardiovascular disorders. 2015;15(1):1–6. 3. moran ae, forouzanfar mh, roth ga, mensah ga, ezzati m, murray cj, et al. temporal trends in ischemic heart disease mortality in 21 world regions, 1980 to 2010: the global burden of disease 2010 study. circulation. 2014;129(14):1483–92. 4. members wg, mozaffarian d, benjamin ej, go as, arnett dk, blaha mj, et al. executive summary: heart disease and stroke statistics—2016 update: a report from the american heart association. circulation. 2016;133(4):447–54. 5. braunwald e, morrow da. unstable angina: is it time for a requiem? circulation. 2013;127(24):2452–7. 6. mohammad am, othman go, saeed ch, al allawi s, gedeon gs, qadir sm, et al. genetic polymorphisms in early-onset myocardial infarction in a sample of iraqi patients: a pilot study. bmc research notes. 2020;13(1):1–6. 7. anderson jl, adams cd, antman em, bridges cr, califf rm, casey jr de, et al. 2011 accf/aha focused update incorporated into the acc/aha 2007 guidelines for the management of patients with unstable angina/ non–st-elevation myocardial infarction: a report of the american college of cardiology foundation/american heart association task force on practice guidelines. circulation. 2011;123(18):e426–e579. 8. antman em, cohen m, bernink pj, mccabe ch, horacek t, papuchis g, et al. the timi risk score for unstable angina/non–st elevation mi: a method for prognostication and therapeutic decision making. jama. 2000;284(7):835–42. 9. eagle ka, lim mj, dabbous oh, pieper ks, goldberg rj, van de werf f, et al. a validated prediction model for all forms of acute coronary syndrome: estimating the risk of 6-month postdischarge death in an international registry. jama. 2004;291(22):2727–33. 10. members wc, lawton js, tamis-holland je, bangalore s, bates er, beckie tm, et al. 2021 acc/aha/scai guideline for coronary artery revascularization: a report of the american college of cardiology/american heart association joint committee on clinical practice guidelines. journal of the american college of cardiology. 2022;79(2):e21–e129. 11. gilutz h, shindel s, shoham-vardi i. adherence to nstemi guidelines in the emergency department: regression to reality. critical pathways in cardiology. 2019;18(1):40–6. https://pubmed.ncbi.nlm.nih.gov/?term=hall%20m%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=dakhil%20za%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=kumar%20d%5bauthor%5d 234 j contemp med sci | vol. 8, no. 4, july-august 2022: 229–234 clinico-angiographic profiles and in-hospital outcomes of nstemi original a.m. mohammad et al. 12. puymirat e, bonaca m, fumery m, tea v, aissaoui n, lemesles g, et al. atherothrombotic risk stratification after acute myocardial infarction: the thrombolysis in myocardial infarction risk score for secondary prevention in the light of the french registry of acute st elevation or non‐st elevation myocardial infarction registries. clinical cardiology. 2019;42(2):227–34. 13. mohammad am, rashad hh, habeeb qs, rashad bh, saeed sy. demographic, clinical and angiographic profile of coronary artery disease in kurdistan region of iraq. american journal of cardiovascular disease. 2021;11(1):39. 14. bønaa kh, steigen t. coronary angiography in non-st-elevation acute myocardial infarction–whom and when? tidsskrift for den norske legeforening. 2017. 15. mohammad am, guelker hk, sheikhow sk. incidence and prognostic significance of myocardial injury after elective percutaneous coronary intervention in duhok, iraq. j contemp med sci vol. 2021;7(5):290–4. 16. collet j-p, thiele h, barbato e, barthélémy o, bauersachs j, bhatt dl, et al. 2020 esc guidelines for the management of acute coronary syndromes in patients presenting without persistent st-segment elevation: the task force for the management of acute coronary syndromes in patients presenting without persistent st-segment elevation of the european society of cardiology (esc). european heart journal. 2021;42(14):1289–367. 17. mohammad am, abdulhaleem bh, habeeb qs. first 24 h’outcomes of acute coronary syndrome in iraq. medical journal of babylon. 2020;17(2):154. 18. fox ka, eagle ka, gore jm, steg pg, anderson f, grace, et al. the global registry of acute coronary events, 1999 to 2009–grace. heart. 2010;96(14):1095–101. 19. donataccio mp, puymirat e, vassanelli c, blanchard d, le breton h, perier m-c, et al. presentation and revascularization patterns of patients admitted for acute coronary syndromes in france between 2004 and 2008 (from the national observational study of diagnostic and interventional cardiac catheterization [onaci]). the american journal of cardiology. 2014;113(2):243–8. 20. mohammad am, mohammed su, saeed sy. outcomes of primary percutaneous coronary intervention in st-segment elevation myocardial infarction in kurdistan region of iraq. journal of contemporary medical sciences. 2022;8(2). 21. kinsara aj, ismail ym. gender differences in patients presenting with non-st segment elevation myocardial infarction in the star registry. the egyptian heart journal. 2021;73(1):54. 22. abdelmoneim hm, hasan-ali h, abdulkader ss. demographics of acute coronary syndrome (acs) egyptian patients admitted to assiut university hospital: validation of timi and grace scores. the egyptian journal of critical care medicine. 2014;2(1):3–11. 23. mohammad am, al-allawi na. cyp2c19 genotype is an independent predictor of adverse cardiovascular outcome in iraqi patients on clopidogrel after percutaneous coronary intervention. journal of cardiovascular pharmacology. 2018;71(6):347–51. 24. mrsic d, smajlovic j, loncar d, avdic s, avdagic m, smajic e, et al. risk factors in patients with non-st segment elevation myocardial infarction. materia socio-medica. 2020;32(3):224. 25. mohammad a, othman g, saeed c, allawi n. tct-441 traditional and genetic risk factors in young patients with myocardial infarction in iraq. journal of the american college of cardiology. 2017;70(18s):b181. 26. amsterdam ea, wenger nk, brindis rg, casey jr de, ganiats tg, holmes jr dr, et al. 2014 aha/acc guideline for the management of patients with non–st-elevation acute coronary syndromes: executive summary: a report of the american college of cardiology/american heart association task force on practice guidelines. circulation. 2014;130(25):2354–94. 27. dakhil za, farhan ha. non-st elevation acute coronary syndromes; clinical landscape, management strategy and in-hospital outcomes: an age perspective. the egyptian heart journal. 2021;73(1):1–11. 28. butt jh, kofoed kf, kelbæk h, hansen pr, torp‐pedersen c, høfsten d, et al. importance of risk assessment in timing of invasive coronary evaluation and treatment of patients with non–st‐segment–elevation acute coronary syndrome: insights from the verdict trial. journal of the american heart association. 2021;10(19):e022333. 29. hamid mb. clinical characteristics and outcomes of acute coronary syndromes in a group of iraqi patients. iraqi journal of medical sciences. 2016;14(4). 30. fox k, gore j. the global registry of acute coronary events (grace). heart. 2007;93:177–82. 31. yusufali am, almahmeed w, tabatabai s, rao k, binbrek a. acute coronary syndrome registry from four large centres in united arab emirates (uae-acs registry). heart asia. 2010;2(1):118–21. 32. cortell a, sanchis j, bodí v, núñez j, mainar l, pellicer m, et al. non-stelevation acute myocardial infarction with normal coronary arteries: predictors and prognosis. revista española de cardiología (english edition). 2009;62(11):1260–6. 33. mohammad am, sheikho sk, tayib jm. relation of cardiovascular risk factors with coronary angiographic findings in iraqi patients with ischemic heart disease. am j cardiovasc dis res. 2013;1(1):25–9. 34. milasinovic d, milosevic a, marinkovic j, vukcevic v, ristic a, asanin m, et al. timing of invasive strategy in nste-acs patients and effect on clinical outcomes: a systematic review and meta-analysis of randomized controlled trials. atherosclerosis. 2015;241(1):48–54. 35. case bc, yerasi c, wang y, forrestal bj, hahm j, dolman s, et al. admissions rate and timing of revascularization in the united states in patients with non-st-elevation myocardial infarction. the american journal of cardiology. 2020;134:24–31. 36. martinón-martínez j, alvarez alvarez b, gonzalez ferrero t, garciarodeja arias f, otero garcia o, cacho antonio c, et al. prognostic benefit from an early invasive strategy in patients with non-st elevation acute coronary syndrome (nsteacs): evaluation of the new risk stratification in the nsteacs european guidelines. clinical research in cardiology. 2021;110(9):1464–72. 37. hall m, bebb oj, dondo tb, yan at, goodman sg, bueno h, et al. guidelineindicated treatments and diagnostics, grace risk score, and survival for non-st elevation myocardial infarction. european heart journal. 2018;39(42):3798–806. 38. cakar ma, sahinkus s, aydin e, vatan mb, keser n, akdemir r, et al. relation between the grace score and severity of atherosclerosis in acute coronary syndrome. journal of cardiology. 2014;63(1):24–8. 39. rahmani r, majidi b, ariannejad h, shafiee a. the value of the grace score for predicting the syntax score in patients with unstable angina/non-st elevation myocardial infarction. cardiovascular revascularization medicine. 2020;21(4):514–7. 40. kumar d, ashok a, saghir t, khan n, solangi ba, ahmed t, et al. prognostic value of grace score for in-hospital and 6 months outcomes after non-st elevation acute coronary syndrome. the egyptian heart journal. 2021;73(1):1–5. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1249 424 j contemp med sci | vol. 8, no. 6, november-december 2022: 424–425 letter to the editor the shifting environment of medical knowledge and the necessity for professional adjustment ameen mosa mohammad* professor of cardiology, college of medicine, university of duhok, iraq. *correspondence to: ameen mosa mohammad (e-mail: doctoramb@yahoo.com) (submitted: 10 july 2022 – revised version received: 15 july 2022 – accepted: 09 august 2022 – published online: 26 december 2022) highlights • rapid proliferation of medical knowledge, • coping difficulties with medical updates, • medical challenges in the era of technology issn 2413-0516 dear editor-in-chief, the technological revolution maintains leaving repercussions on distinct dimensions of life in different parts of the globe. the world of medicine experiences revolutionary effects in the consequence. the prospects for the domino effects of the technological revolutionary with respect to the world of medicine appear beyond imagination.1 an unprecedented process of unlimited proliferation of indefinite information in medical sciences occurs. practitioners and learners of medical majors experience the challenge to remain updated. patients increasingly turn to online platforms in search for information on interested conditions. algorithmic advancements critically subject the role of the medical professionals to question.2 the inability or unwillingness of the medical professionals to adjust to the present medical environment establishes obstacles for the management of diseases. technological advancements brought the medical professionals onto close virtual connection. researchers from distinct geographical corners of the world contribute to the rapid expansion of medical knowledge. huge investment in scientific research constituted another essential measure in the flood of the information. dedicated scientific platforms globally prepared the ground for the publication of novel information.3 the ultimate outcome of the collection of similar preceding variables represents the unimagined update occurring in the medical knowledge. in comparison with the past, the discrepancy in the update of the medical knowledge today is unbridgeable. figures demonstrated that the medical knowledge doubled each fifty years in 1950. seventy years later, the figure for the same process dropped to an astonishing seventy three days. the influx of medical information looks equally attention-grapping when global contributions on the coronavirus are reckoned. between the start of the pandemic and october 2020, an analysis reported the publication of eightyseven thousand scientific papers on the coronavirus by researchers from around the world.4 the positive correlation between the proliferation of information and the medical sciences is undeniable. the more the information the more the advancement. the rapid expansion of medical knowledge brings challenges too for the parties interested in the sciences. many medical professionals struggle to cope with the pace of the flood of information. some of them do not manage to remain updated. the outcome represents an unwanted compromise in the management of diseases and the commitment to patients. close to forty-five percent of patients reportedly receive treatment from doctors whose background is not up-to-date.5 several reasons possibly account for the emergence of this circumstance. time constrain certainly establishes one. the quick expansion of knowledge poses another challenge for the medical professionals. technological advancements shrink work opportunities. google claims that five percent of searches conducted on the platform looks for information on health. the expansion of the medical knowledge occurs in tandem with the proliferation of information. the process poses serious challenges for the medical professional. it equally presents a golden opportunity for the advancement of medical sciences. the shifting nature of the medical environment necessitates the willingness for adjustment on the part of the medical professionals. given all the challenges that the quick flood of information poses, the medical professionals still do better when investing time and energy for the sake of background update. the shift in the medical environment does not only takes place. it appears to constantly and quickly increase. viewed from the previous perspectives, the present writing suggests the investment of efforts on the part of the medical professionals for the purpose of trying to keep the pace with the proliferation of information in medical sciences. provenance and peer-review not commissioned, externally reviewed. ethical approval n/a. source of funding no funding to declare. author contribution amm: designed and draft the study, and conducting the version of the manuscript. declaration of competing interest none.  425j contemp med sci | vol. 8, no. 6, november-december 2022: 424–425 a.m. mohammad letter to the editor the shifting environment of medical knowledge and the necessity for professional adjustment references [1] colbert, g. b., topf, j., jhaveri, k. d., oates, t., rheault, m. n., shah, s., ... & sparks, m. a. (2018). the social media revolution in nephrology education. kidney international reports, 3(3), 519–529. [2] meskó, b., & görög, m. (2020). a short guide for medical professionals in the era of artificial intelligence. npj digital medicine, 3(1), 126. [3] albujeer, a., & khoshnevisan, m. (2022). metaverse and oral health promotion. british dental journal, 232(9), 587–587. [4] tasdelen, a., & ugur, a. r. (2021, october). artificial intelligence research on covid-19 pandemic: a bibliometric analysis. in 2021 5th international symposium on multidisciplinary studies and innovative technologies (ismsit) (pp. 693–699). ieee. [5] ahmed, s., chase, l. e., wagnild, j., akhter, n., sturridge, s., clarke, a., ... & hampshire, k. (2022). community health workers and health equity in lowand middle-income countries: systematic review and recommendations for policy and practice. international journal for equity in health, 21(1), 49. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1089 88 j contemp med sci | vol. 9, no. 2, march-april 2023: 88–92 original the magnitude of dispensing unprescribed antibiotics in community pharmacies in duhok province; kurdistan region of iraq hind b. almufty1*, lara s. hayhat2, wasan g. abdal3, darya s. hussein4, muayad a. merza5 1department of clinical pharmacy, college of pharmacy, university of duhok, kurdistan region, iraq. 2department of pharmacy, ararat institute, kurdistan region, iraq. 3department of basic science, college of pharmacy, university of duhok, kurdistan region, iraq. 4college of pharmacy, university of duhok, kurdistan region, iraq. 5department of internal medicine, azadi teaching hospital, college of pharmacy, university of duhok, duhok, kurdistan region, iraq. *correspondence to: hind b. almufty (e-mail: hind.bahzad@uod.ac) (submitted: 04 february 2023 – revised version received: 25 february 2023 – accepted: 01 march 2023 – published online: 26 april 2023) abstract objectives: the study aims to provide a descriptive overview about the magnitude and frequency of dispensing the unprescribed antibiotics uabs. it furthermore aims, to specify the most dispensed uab types, the most common infections, signs and symptoms that require dispensing the uabs in community pharmacies of duhok city and its districts. methods: a cross sectional cohort study had been conducted from september to october 2022, private community pharmacies of duhok province were included in this study. a standardized questionnaire platform was utilized to complete the survey, it included 2 sections, the first of which was related to demographic data and the second part included details of the dispensing unprescribed antibiotics uabs like: what are the most common dispensed uabs, what are the most common illness and symptoms of patients who require dispensing uabs. results: one hundred fifty pharmacies have been included in this study. the majorities were from duhok city 46%, and zakho 28.7%. the prevalence of dispensing uabs was 100%, the three most common dispensed uabs were amoxicillin 77%, followed by azithromycin 16% and cefixime 7%. the most infections and conditions requiring dispensing uabs were tonsilitis (69.3%), followed by flu and common cold (58.7%), and lower respiratory tract infection (48.7%). conclusion: this study concluded that dispensing uabs is a frequent practice in duhok community pharmacies. viruses are the most common cause of upper respiratory tract infections; hence, antibiotics must be avoided and otc medicines should be encouraged to alleviate the symptoms. continuing medical education through training pharmacists on dispensing antibiotics by adhering to the regulations of antibiotic stewardship is crucial. keywords: unprescribed antibiotics, community pharmacies, infections, kurdistan region, iraq issn 2413-0516 introduction antibiotic resistant crisis is the main issue that threatens global health security as it causes morbidity, mortality and magnifies the economic loss.1 in 2019, the world health organization (who) had declared that “antimicrobial resistance (amr)” is one of the most urgent health risks of our time and it is named the “invisible pandemic”. since then, serious actions had been taken worldwide to detract this scourge, i.e., using the aware online tool developed by the who essential medicines list to contain rising resistances and make antibiotics safer and more effective.2,3 currently, it is estimated that more than 50% of antibiotics in numerous countries are dispensed without prescriptions and have been utilized inappropriately such as; usage for virus treatments, wrong (broader spectrum) antibiotics used, and wrong dose and duration of treatment course, thus contributing to the spread of amr.4,5 signs of post antibiotic era had already been discovered worldwide, especially in lowand middle-income countries (lmic).6,7 the inferior living ambience, inadequate sanitation, poor infection control, and the non-adherence to the prescribed course of antibiotics had been associated with ar in lmics.8,9 however, lack of antibiotic dispensing policies in these settings and the availability of low quality of antibiotic brands are the major contributors behind this condition.10,11 dispensing antibiotics without prescription had become a common practice in lmics, including iraq.11-13 several factors contributed to this situation: the inadequate knowledge about antibiotic resistance and lack of awareness about antibiotic stewardship among community pharmacists in iraq.13 furthermore, iraq has no community pharmacy chains meaning all community pharmacies are private independent pharmacies usually owned by licensed pharmacists. although, it is unwarranted by law, selling antibiotics without prescription is a common practice. on the other hand, numerous community pharmacies employ pharmacy technicians who also commonly dispense antibiotics without prescriptions.14 to our knowledge, there is no antibiotic dispensing survey conducted in community pharmacies of duhok province, therefore, this study aims to provide a descriptive overview about the magnitude and frequency of dispensing the uabs. it furthermore aims, to specify the most dispensed uab types, the most common infections, signs and symptoms that require dispensing the uabs in community pharmacies of duhok city and its districts. materials and methods study design and setting a cross sectional cohort study had been conducted from september to october 2022 in duhok province, kurdistan region of iraq. private community pharmacies of duhok city and its districts (zakho, akre, bardaarash, amedy, semel and shekhan) were studied. governmental hospital inpatient mailto:hind.bahzad@uod.ac 89j contemp med sci | vol. 9, no. 2, march-april 2023: 88–92 h.b. almufty et al. original the magnitude of dispensing unprescribed antibiotics in community and outpatient pharmacies had been excluded as the antibiotics are dispensed by prescription.14 study sampling process a standardized questionnaire platform was utilized to complete the survey, face to face interviews were performed by the authors and the pharmacists to precisely fill the requested questions. the interviews took place in pharmacies or any other place convenient to them. all pharmacy workers, including pharmacists and their assistants, were clearly informed about the aim of study and that the data will be kept confidential and used for research purposes only. the questionnaire included 2 sections, the first of which were questions regarding demographic data like: place, type of pharmacy (whether it is a by hand pharmacy, a medical complex pharmacy, or a private hospital pharmacy), age of the pharmacist, experience years, and the scientific degree of the pharmacist. the second part included details about the nature of the dispensing uabs like: what the three most common dispensed antibiotics are, what are the most common illness and symptoms of patients who require dispensing uabs, and the frequency of dispensing each type of antibiotic class were asked, whether it is commonly dispensed (everyday), often dispensed (once to twice a week), rarely dispensed (once a month), or never dispensed. scientific and ethical approval ethical approval was obtained from the syndicate of kurdistan pharmacists duhok branch before conducting this study. this study was reviewed and approved by the scientific and ethics committee of the college of pharmacy at the university of duhok on august 7, 2022 (reference no. 122). written informed consent was obtained from the participants before starting the interview. statistical analysis all data were analyzed by statistical package for the social sciences software version 22. descriptive statistics were carried out for the demographic variables, the most prevalent types of dispensed antibiotics, and to obtain the incidence of dispensing each type of antibiotic individually. results one hundred fifty pharmacies have been included in this study. the majorities were from duhok city 46%, followed by duhok’s biggest district zakho 28.7%, 4.7% were from semel, other 14.7% included shekhan, and bardarash. most of the included pharmacies were independent (by hand) ones (66%). the scientific degree of most of the interviewed pharmacists was bachelor degree (96.7%); only 5 participants had higher degrees (msc and phd). the mean age of the pharmacists was (28.78) ± sd 6.157, and the mean years of experience of pharmacists was (5.67) ± sd 4.874 as shown in table 1. in the descriptive analysis, it appeared that the three most common dispensed uabs were amoxicillin, followed by azithromycin and cefixime respectively, as shown in figure 1. the most infections and conditions requiring dispensing uabs were tonsilitis (69.3%), followed by flu and common cold (58.7%), and lower respiratory tract infection (lrti) (48.7%) as shown in figure 2. table 1. demographic characteristics of study population variables number percentage place of pharmacy duhok 69 46 akre 6 4 zakho 43 28.7 amedy 3 2 semel 7 4.7 other 22 14.7 type of pharmacy medical complex pharmacy 39 26 private hospital pharmacy 12 8 independent (by hand) pharmacy 99 66 scientific degree of the pharmacist in charge bachelor 145 96.7 msc 4 2.7 phd 1 0.6 age (mean) sd = (28.78) 6.157 years of experience (mean) sd = (5.67) 4.874 fig. 1 the most frequent dispensed unprescribed antibiotics. on the other hand, the most frequent signs and symptoms that require dispensing uabs were fever, cough, sore throat, and diarrhea (68.7%, 64.7%, 51.3% and 46.7%), respectively (figure 3). in this study, penicillin, macrolides, 3rd generation cephalosporins and quinolones were among the most common dispensed antibiotics. fourth and 5th generation cephalosporins, linconsamide, streptogramine, linezolid, daptomycin, lefamulin, monobactam, fosfomycin, nitrofurantoin, sulphonamids, and chloramphenicol were the antibiotic classes that fall among those which rarely to never dispensed without a prescription (table 2). discussion the antibiotic resistance continuously spreads worldwide, particularly in lmic, including iraq. there is an urgent need 90 j contemp med sci | vol. 9, no. 2, march-april 2023: 88–92 the magnitude of dispensing unprescribed antibiotics in community original h.b. almufty et al. fig. 2 the most frequent conditions that require dispensing unprescribed antibiotics. fig. 3 the most common signs and symptoms that require dispensing unprescribed antibiotics. to set an antibiotic dispensing policy and improve prescription practices through incorporating treatment recommendations into the national guidelines, and consolidation of the awareness of this public health problem.15 in our study, the dispensing of uabs was high, which is in line to studies from syria, egypt, and saudi arabia.16,17 however, studies from qatar and other high-income countries like, north america, europe, and australia showed a low prevalence of dispensing uabs.11,18-20 the irresponsible malpractice of frequent uab uses in duhok community pharmacies is threatening the community health security outcome in the region. moreover, the high prevalence (93.7%) of antibiotic misuse in kurdistan hospitals perplexed the emergence of antibiotic resistance.21 therefore, it is crucial to encourage dispensing prescribed antibiotics and to avoid dispensing uabs by pressing charges through the pharmacy council against the pharmacist who do not adhere to rational uses of antibiotics. in our study, we found that the most frequent dispensed uabs in the private health sector were amoxicillin with or without clavulanic acid, followed by azithromycin, and cefixime. the prevalent use of unprescribed amoxicillin has been well documented in several lmics countries.22-24 in iraq, the wrong belief of using specific antibiotics such as amoxicillin and azithromycin for treating respiratory tract infections including viral in origin is a challenging consequence of increasing antibiotic resistance. the increase of such antibiotic consumptions in winter is well recognized14 it is worthy to mention that the use of azithromycin has increased dramatically after covid-19 pandemic, particularly in the early phase of the pandemic as it was used in the treatment guideline of covd-19 patients.25,26 this finding rationalizes the extreme pathogen resistance to these antibiotics in the region.27-29 while other studies from the same region showed a high rate of inappropriate 3rd generation cephalosporin 91j contemp med sci | vol. 9, no. 2, march-april 2023: 88–92 h.b. almufty et al. original the magnitude of dispensing unprescribed antibiotics in community table 2. the frequency of dispensing antibiotics in community pharmacies the antibiotics the dispensing (%) commonly often rarely never sulphonamides 1.3 10 24.7 64 penicillin 94 6 – – 1st generation cephalosporin 16 35.3 22 26.7 2nd generation cephalosporin 5.4 15.3 17.3 62 3rd generation cephalosporin 46 32.7 9.3 12 4th generation cephalosporin – – 6.7 93.3 5th generation cephalosporin – – – 100 carbapenem 1.3 2 12 84 glycopeptide – – 7.3 92.7 monobactam – – 2 98 aminoglycosides 7.3 20.7 28 44 macrolides 66 30 2.7 1.3 chloramphenicol 1.3 9.3 11.3 78 quinolone 33.3 36.7 16 14 fosfomycin, nitrofurantoin 0.7 2 17.3 80 linconsamide 1.3 5.3 6 87.3 streptogramine – – 0.7 99.3 linezolid – – – 100 daptomycin – – – 100 lefamulin – – – 100 prescriptions in the governmental sector,21 which increases the magnitude of amr in our region. overall, continuing medical education through training of pharmacists on antibiotics dispensation by adhering to the regulations of antibiotic stewardship is crucial. in the present study, the unprescribed quinolones, in particular ciprofloxacin was used often (2–3 times a week); whereas, other studies reported metronidazole and quinolones as most frequent dispensed uab.24,30,31 it is noteworthy that 80% and 44% of duhok community pharmacies never dispense unprescribed nitrofurantoin and aminoglycoside class, respectively, that elucidates to the lowest rate of resistance of these antibiotics for treating urinary tract infection (uti).32 the higher rate of broad-spectrum antibiotic such as ciprofloxacin for treating uti, instead of narrow-spectrum antibiotic such as nitrofurantoin, is malpractice in enhancing amr. therefore, clinical pharmacists should play a pivotal role in avoiding the use of broad-spectrum antibiotics, consequently encouraging the use of narrow-spectrum antibiotics for specific infections. in the current study, uabs were dispensed for the treatment of tonsilitis (23.4%), flu and common cold (19.8%), and other upper respiratory tract infection (urti) (16.4%). this finding was concordant with a study from china where urtis were the most frequent reason for uabs.11 on the contrary, a study from nigeria reported that utis, typhoid fever, and sexually transmitted diseases (std) were the most infections that require dispensing uabs.33 while in saudi arabia, pharmacists dispense uabs for pharyngitis and uti most commonly34 in general, the differences of dispensing uabs between different countries is guided by local epidemiology infections. concerning high rates of antibiotic misuse for urtis, community pharmacists should be involved actively in avoiding dispensing uabs by offering over the counter (otc) medicines that alleviate upper respiratory symptoms of viral origin such as painkillers for fever and aches, and antihistamines for sneezing and an itchy nose. the main limitation of the study was the slight possibility of having imprecise data, as this method “pharmacy interviews” may be exposed to the hawthorne effect (changes in the behavior of the studied pharmacists because they feel observed).15 another limitation was that our study was confined to one province. hence, we recommend larger studies at national level to better understand the magnitude of uba dispensations. conclusion this study concluded that dispensing uabs is a frequent practice in duhok community pharmacies. amoxicillin, azithromycin, and cefixime are the most widely dispensed antibiotics. tonsilitis, flu and common cold, and other urti are the conditions requiring the most dispensations of uabs; while fever, cough and sore throat are the most reported signs and symptoms for uab dispensations. viruses are the most common cause of urtis, hence, antibiotics must be avoided and otc medicines should be encouraged to alleviate the symptoms. continuing medical education through training pharmacists on dispensing antibiotics by adhering to the regulations of antibiotic stewardship is crucial. 92 j contemp med sci | vol. 9, no. 2, march-april 2023: 88–92 the magnitude of dispensing unprescribed antibiotics in community original h.b. almufty et al. acknowledgments the authors would like to thank all community pharmacies in duhok for their cooperation and contribution, their efforts are highly appreciated. conflict of interest the authors declare no conflict of interest. financial support n.a. data availability statement the data that support the findings of this study are available from the corresponding author upon reasonable request.  references 1. organization wh. antimicrobial resistance global report on surveillance: 2014 summary. world health organization; 2014. 2. un health agency steps up fight against ‘invisible pandemic’ of antimicrobial resistance [internet]. 2019. available from: https://news. un.org/en/story/2019/06/1040741. 3. organization wh. in the face of slow progress, who offers a new tool and sets a target to accelerate action against antimicrobial resistance. 2019. 4. grigoryan l, haaijer-ruskamp fm, burgerhof jg, mechtler r, deschepper r, tambic-andrasevic a, et al. self-medication with antimicrobial drugs in europe. emerging infectious diseases. 2006;12(3):452. 5. cars o, nordberg p. antibiotic resistance–the faceless threat. international journal of risk & safety in medicine. 2005;17(3-4):103–10. 6. laxminarayan r, matsoso p, pant s, brower c, røttingen j-a, klugman k, et al. access to effective antimicrobials: a worldwide challenge. the lancet. 2016;387(10014):168–75. 7. rambliere l, guillemot d, delarocque-astagneau e, huynh b-t. impact of mass and systematic antibiotic administration on antibiotic resistance in low-and middle-income countries. a systematic review. international journal of antimicrobial agents. 2021;58(1):106364. 8. ayukekbong ja, ntemgwa m, atabe an. the threat of antimicrobial resistance in developing countries: causes and control strategies. antimicrobial resistance & infection control. 2017;6(1):1–8. 9. hadi u, duerink do, lestari es, nagelkerke nj, werter s, keuter m, et al. survey of antibiotic use of individuals visiting public healthcare facilities in indonesia. international journal of infectious diseases. 2008;12(6):622–9. 10. morgan dj, okeke in, laxminarayan r, perencevich en, weisenberg s. nonprescription antimicrobial use worldwide: a systematic review. the lancet infectious diseases. 2011;11(9):692–701. 11. chang j, xu s, zhu s, li z, yu j, zhang y, et al. assessment of nonprescription antibiotic dispensing at community pharmacies in china with simulated clients: a mixed cross-sectional and longitudinal study. the lancet infectious diseases. 2019;19(12):1345–54. 12. al-halawa da, sarama r, abdeen z, qasrawi r. knowledge, attitudes, and practices relating to antibiotic resistance among pharmacists: a crosssectional study in the west bank, palestine. the lancet. 2019;393:s7. 13. alkadhimi a, dawood ot, hassali ma. dispensing of antibiotics in community pharmacy in iraq: a qualitative study. pharmacy practice (granada). 2020;18(4). 14. al-jumaili aa, hussein ah, al-rekabi md, raheem sa, ernst ej. antimicrobial utilization in an iraqi province: a comprehensive evaluation of antibiotic source and cost. international journal of pharmacy practice. 2017;25(1):81–8. 15. batista ad, a. rodrigues d, figueiras a, zapata-cachafeiro m, roque f, herdeiro mt. antibiotic dispensation without a prescription worldwide: a systematic review. antibiotics. 2020;9(11):786. 16. mahmoud ma, aldhaeefi m, sheikh a, aljadhey h. community pharmacists’ perspectives about reasons behind antibiotics dispensing without prescription: a qualitative study. biomedical research. 2018;29(21):3792–6. 17. mansour o, al-kayali r. community pharmacists’ role in controlling bacterial antibiotic resistance in aleppo, syria. iranian journal of pharmaceutical research: ijpr. 2017;16(4):1612. 18. mohamed ibrahim mi, awaisu a, palaian s, radoui a, atwa h. do community pharmacists in qatar manage acute respiratory conditions rationally? a simulated client study. journal of pharmaceutical health services research. 2018;9(1):33–9. 19. zapata-cachafeiro m, piñeiro-lamas m, guinovart mc, lópez-vázquez p, vázquez-lago jm, figueiras a. magnitude and determinants of antibiotic dispensing without prescription in spain: a simulated patient study. journal of antimicrobial chemotherapy. 2019;74(2):511–4. 20. khan mu, hassali maa, ahmad a, elkalmi rm, zaidi str, dhingra s. perceptions and practices of community pharmacists towards antimicrobial stewardship in the state of selangor, malaysia. plos one. 2016;11(2):e0149623. 21. kurdi a, hasan aj, baker ki, seaton ra, ramzi zs, sneddon j, et al. a multicentre point prevalence survey of hospital antibiotic prescribing and quality indices in the kurdistan regional government of northern iraq: the need for urgent action. expert review of anti-infective therapy. 2021;19(6):805–14. 22. al-mohamadi a, badr a, mahfouz lb, samargandi d, al ahdal a. dispensing medications without prescription at saudi community pharmacy: extent and perception. saudi pharmaceutical journal. 2013;21(1):13–8. 23. kalungia ac, burger j, godman b, costa jdo, simuwelu c. non-prescription sale and dispensing of antibiotics in community pharmacies in zambia. expert review of anti-infective therapy. 2016;14(12):1215–23. 24. damisie g, hambisa s, yimam m. over the counter sale of antibiotics at drug stores found in mizan-aman town, southwest ethiopia: a cross-sectional simulated client visit study. journal of pharmaceutics. 2019;2019. 25. cavalcanti ab, zampieri fg, rosa rg, azevedo lc, veiga vc, avezum a, et al. hydroxychloroquine with or without azithromycin in mild-to-moderate covid-19. new england journal of medicine. 2020;383(21):2041–52. 26. merza ma, al mezori aah, mohammed hm, abdulah dm. covid-19 outbreak in iraqi kurdistan: the first report characterizing epidemiological, clinical, laboratory, and radiological findings of the disease. diabetes & metabolic syndrome: clinical research & reviews. 2020;14(4):547–54. 27. hussein nr, daniel s, salim k, assafi ms. urinary tract infections and antibiotic sensitivity patterns among women referred to azadi teaching hospital, duhok, iraq. avicenna journal of clinical microbiology and infection. 2017;5(2):27–30. 28. hasan th, alasedi kk, aljanaby aaj. a comparative study of prevalence antimicrobials resistance klebsiella pneumoniae among different pathogenic bacteria isolated from patients with urinary tract infection in al-najaf city, iraq. latin american journal of pharmacy. 2021;40:174–8. 29. hasan sa, najati am, abass ks. prevalence and antibiotic resistance of “pseudomonas aeruginosa” isolated from clinical samples in kirkuk city, iraq. eurasia j biosci. 2020;14(1):1821–5. 30. ibrahim ir, palaian s, ibrahim mi. assessment of diarrhea treatment and counseling in community pharmacies in baghdad, iraq: a simulated patient study. pharmacy practice (granada). 2018;16(4). 31. zawahir s, lekamwasam s, aslani p. antibiotic dispensing practice in community pharmacies: a simulated client study. research in social and administrative pharmacy. 2019;15(5):584–90. 32. abdullah im. multiple drugs resistance among urinary tract infection patients in duhok city–kurdistan region–iraq. duhok medical journal. 2019;13(1):22–31. 33. abubakar u, tangiisuran b. knowledge and practices of community pharmacists towards non-prescription dispensing of antibiotics in northern nigeria. international journal of clinical pharmacy. 2020;42(2):756–64. 34. alrasheedy aa, alsalloum ma, almuqbil fa, almuzaini ma, aba alkhayl bs, albishri as, et al. the impact of law enforcement on dispensing antibiotics without prescription: a multi-methods study from saudi arabia. expert review of anti-infective therapy. 2020;18(1):87–97. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1324 https://news.un.org/en/story/2019/06/1040741 https://news.un.org/en/story/2019/06/1040741 303j contemp med sci | vol. 7, no. 5, september–october 2021: 303–307 original assessment of von willebrand factor/adamts13 ratio and vitamin k levels as predictor markers for severity of covid-19 patients haneen saeed mohsin1, hanaa addai ali2*, fadhil jawad al-tu’ma1 1department of chemistry and biochemistry, college of medicine, university of kerbala/kerbala – iraq. 2department of chemistry, faculty of science, university of kufa/najaf – iraq. *correspondence to: hanaa addai ali (e-mail: muthanahana74@gmail.com) (submitted: 20 august 2021 – revised version received: 08 september 2021 – accepted: 19 september 2021 – published online: 26 october 2021) abstract objectives: to investigate the levels of each of von willebrand factor, adamts-13, and vitamin k in sera of iraqi patients infected with covid-19 and study the association between them to explore the mechanism roles of micro thrombosis in covid-19 progression. materials and methods: in this case-control study, a total of (60) covid-19 patients (min-max) aged (35–65years) who presented within 7–12 days of displaying covid-19 symptoms were recruited. (30) apparently healthy persons of the same ages and gender were included in this study as a control group. patients were divided into three groups: mild patients group (n = 33), sever patients group (n = 15), and deceased group (n = 12). in this study, the levels of vwf, adamts-13, and vitamin k were measured through enzyme-linked immunosorbent assays (elisa) in sera from healthy volunteers as a control group, and patients with moderate covid-19, patients with severe covid-19, and patients who had deceased from covid-19. anthropometric, biochemical data were analyzed. results: the levels of vwf, adamts-13, and vitamin k were did differ significantly among groups. however, the level of vwf was significantly higher in moderate covid-19 and severe cases of covid-19 groups compared to control indicating it to be an independent predictor in the coronavirus disease. the levels of vitamin k were significantly lower in the patients group than the control group especially in severe case of the covid-19 group. in contrast, the level of vwf in deceased covid-19 patients was significantly higher compared to severe cases. the vwf/adamts13 ratio was higher at presentation in covid-19 patients who died (26.14 vs 12.34; p < .0001). linear regression analysis for vwf levels revealed significant negative correlations with adamts-13, and vit. k, and positive correlations with d-dimer and ferritin levels. however adamts-13 demonstrated significant positive correlations with vit. k levels in patients group. conclusion: the correlations of vwf-adamts-l3 and vitamin k levels in covid-19 patients could help better define the pathophysiology, clarify the pathogenesis, improve prediction of clinical prognosis, and better guide thromboprophylaxis and treatment covid-19 patients, which could be uses as hall mark of severe covid-19 and provide a rational for combined therapeutic approaches for medical staff. a ratio of vwf/adamts-13 prompt higher in the ratio in thrombolysed patients was associated with increased mortality and morbidity. keywords: covid-19, von willebrand factor, adamts-13, vitamin k, mortality issn 2413-0516 introduction the severe acute respiratory syndrome-coronavirus type 2 causes coronavirus disease 19 (covid-19), a global pandemic threat (sars-cov-2). the spike protein of sars-cov-2 interacts to angiotensin-converting enzyme 2 (ace2) receptors, which play a role in viral entrance.1 covid-19 has a wide range of clinical presentations, from minor symptoms like fever and cough to severe types of pneumonia, which can result in acute respiratory distress, multiorgan failure, and death.2 the identification of biomarkers linked with varying disease severity is critical for stratifying patients’ risk and devising the most effective medicines, in addition to offering vital insights into the disease mechanisms in play. the characteristics of covid-disease include severe hypercoagulability and endothelial disturbance, with a high rate of venous thromboembolism routinely recorded, particularly in patients with critical illness requiring intensive care. more recently, pulmonary microvascular thrombosis has been proposed as a cause of illness progression. the lungs were discovered to have indications of thrombotic microangiopathy, although the pulmonary arteries at the hilum of each lung were clear of thromboemboli. cd61 and von willebrand factor (vwf) immunostaining revealed tiny platelet-rich thrombi inside small arteries of the peripheral parenchyma and alveolar capillaries in all instances.3 vwf plays a critical function in initial hemostasis, mediating platelet adhesion and aggregation at vascular injury sites.4 vwf is also a sign of endothelium activation, as it is generated in large amounts after vascular injury caused by inflammation. a disintegrin and metalloprotease with thrombospondin 1 repeats, number 13 (adamts-13) regulates the size of vwf multimers, and a severe deficiency of adamts-13 activity below 10 iu/dl is diagnostic for thrombotic thrombocytopenic purpura (ttp), a severe and life-threatening thrombotic microangiopathy caused by an accumulation of hyperactive ultra-large vwf multi.5 in individuals with ischemic stroke and myocardial infarction, low levels of this metalloprotease have been linked to an increased risk of thrombosis. ttp survivors who have lower adamts13 activity during disease remission have a higher risk of ttp-unrelated stroke, while congenital ttp patients who get prophylactic adamts13 replacement therapy had a lower risk of ischemic stroke. furthermore, as seen in sepsis and overt disseminated intravascular coagulation, an imbalance between high molecular weight vwf multimers and adamts13 may generate a prothrombotic state in inflammatory-induced circumstance.6 vitamin k plays a well-known function in coagulation, which is a delicate balance between clot-promoting and clot-dissolving processes. to perform its principal function, procoagulant factor ii (fii; i.e. prothrombin) requires vitamin k-dependent carboxylation. anticoagulant protein s requires vitamin k as a cofactor. in contrast to fii, a major percentage of protein s is generated outside of the liver in endothelial cells, where it has a local anti-thrombotic effect. vitamin k 304 j contemp med sci | vol. 7, no. 5, september–october 2021: 303–307 assessment of von willebrand factor/adamts13 ratio original h.s. mohsin et al. insufficiency impairs carboxylation of extrahepatic vitamin k dependent proteins more than hepatic vitamin k-dependent proteins. in a state of low vitamink, this can contribute to increased thrombogenicity.7 the goal of this work was to better understand the pathogenesis of microthrombosis in covid-19 by focusing on the vwf-adamts13 axis. in order to do so, we measured the vwf multimeric pattern, vwf propeptide, and adamts-13 levels, as well as vitamin k levels, in individuals with varying degrees of illness severity. materials and methods this case control study, a total of 60 patients with covid-19 with ages ranging from 36–60 years (40 males and 20 females) who collected within 7–12 days of showing covid-19 symptoms were confirmed clinically with covid-19 according tested positive for sars-cov-2 by quantitative rt-pcr and chest x-ray or ct scan. the diagnosis of covid-19 was done according to the covid-19 diagnostic criteria reported by ma et al.8 patients were recruited from al-amal hospital/ najaf-iraq and al-hussein teaching hospital, al-hussein medical city, kerbala health directorate/kerbala – iraq, within the period from dec., 2020 and mid-july, 2021 and compared to 60 matched healthy groups as a control. to avoid the influence of other comorbidities, this study excluded subjects with chronic diseases, alcoholics, liver disease, obese, and smokers. the patients were categorized into subgroups according to murray score into three groups: group i (mild group n = 33), group ii (sevre group n = 15), and group iii (deceased group n = 12).9 all study procedures were done according to the helsinki declaration. recruited patients and healthy controls gave informed consent for their contribution to this study. this study was done in the department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq and department of laboratory, in al-sader medical city, najef, iraq and was approved by the clinical research and ethical committee board, college of medicine, university of kerbala, iraq. venous blood samples were collected from patients and control groups. blood samples were separated in to two tubes .3ml allowed to clot for 10–15 minutes at room temperature before centrifugation for 10 minutes at (3000x g) in order provide serum. then serum samples were separated in to tubes and stored at −80°c until time of analysis. remaining blood (2 ml) was prepared to measure complete blood count. the concentrations of serum ferritin, and d-dimer levels were measured by fluorescence immunoassay (ichromatm). levels of vwf, adamts-13, and vitamin k were measured through enzyme-linked immunosorbent assays (elisa) bt labchina elisa kits. complete blood count was measured by using auto hematology analyzer (linear, spain). statistical analysis the observed data represented as mean ± sd were analyzed by using the statistical package for the social sciences (spss). independent ttest was used to evaluate significant differences between healthy and patients’ groups. pearson correlation coefficient test was applied to mention the statistical relationship (association) between any two variables in present study. the levels of significance of 5% (p ≤ 0.05) and 1% (p ≤0.01) were obtained to represent the strength of evidence in support of significant differences between variables. results and discussions this study involved 60 patients with verified covid-19 and 60 healthy participants in total. the average age was 56.89 6.32 years, while the median age was 56.48 5.41 years. as indicated in table, there were no significant differences in age or gender between covid-19 patients and healthy volunteers (table 1). the data analysis revealed a significantly higher presence of serum d-dimer, ferritin levels, and a significant decrease in vitamin k levels in covid-19 patients compared to the control group. the data analysis also revealed a significantly higher presence of serum vwf and a significantly lower level of serum adamts13 in covid-19 patients compared to the healthy group. in this investigation, a total of covid-19 patients were included, with 33 mild, 15 severe, and 12 death cases listed in table 2. the age and bmi of deceased (death) cases were significantly higher than the other cases, the values of neutrophil, d-dimer, and ferritin levels in deceased and severe cases were significantly higher than the mild case, categories of covid-19 patients found low levels of adamts-13 in non-survived and severe cases compared to the mild case, the values of vwf levels in deceased and severe cases were significantly higher than the mild case, and the values of vwf levels. the biochemical parameters tested were linked to the levels of vwf and adamts-13, as well as vitamin k, in the covid-19 patients group, as shown in table 3. age, bmi, d-dimer, and ferritin levels were all found to have a strong positive connection with vwf levels. in the covid-19 patients group, however, there were substantial negative relationships between adamts-13 and vitamin k levels. the best our knowledge, this is the first study in iraqi patients group infected with covid-19 that shows a statistically strong significant relationship between serum levels of vwf and adamts-13 in covid-19 patients. table 1. general characteristics of the patients and control groups parameters covid-19 patients group mean ± sd n = 60 apparently healthy group mean ± sd n = 60 p-value age, (years) gender, (f/m) 56.89 ± 6. 32 20/40 56.48 ± 5.41 20/40 n.s bmi, (kg/m2) 28.62 ± 4.31 24.46 ± 3.11 0.001 d-dimer, (ng/ml) 3450.20 ± 1800.17 269.69 ± 88.96 0.0001 ferritin, (ng/ml) 1081.93 ± 471.46 106.7 ± 47.81 0.0001 vit k, (pg/ml) 614.32 ± 106.76 1198.9 ± 151.59 0.0001 vwf, (ng/ml) 355.30 ± 44.45 205.07 ± 20.98 0.001 admts13, (ng/ml) 19.92 ± 9.44 37.10 ± 7.48 0.0001 vwf/adamts13 17.83 ± 5.63 5.52 ± 2.96 0.0001 data represented as mean ± sd, sd, standar deviation, n.: number of subject. bmi, body mass index, vit. k: vitamin k; f: female, m: male, ns: non-significant, independent t-test, df = 88. 305j contemp med sci | vol. 7, no. 5, september–october 2021: 303–307 h.s. mohsin et al. original assessment of von willebrand factor/adamts13 ratio the endothelial cell (ec) monolayerlining vascular channels prevents pathological thrombosis in normal blood vessels. the pulmonary microvasculature of covid-19 showed severe ec apoptosis and loss of ec tight junction integrity, according to data from an autopsy research.10 there are various other aspects associated to aging that could be reasons for higher mortality and morbidity in the elderly, including declining immunity. the average number of comorbid conditions grew with age, and senior covid-19 patients scored considerably higher than young and middle-aged patients on the performance scale.11 chronic subclinical systemic inflammation, often called as inflammation, is another well-known characteristic of aged immunity. inflammation is a critical pathogenic mechanism in covid-19; thus, inflammation is thought to have a role in the inferior prognosis in senior covid-19 patients.12 endothelial cells express levels of vwf and adamts13. excess vwf is secreted by endothelial cells when the vascular endothelium is dysregulated in an acute hyperinflammatory environment. the activity of adamts13 reduces in direct proportion to the inflammatory response.13 according to this, the difference in vwf and adamts13 activity could be due to adamts13 inhibition and/or deficiency.14 table 2. demographic characteristics of covid-19 patient’s groups parameters covid-19 patients groups p-value deceased n = 12 severe n = 15 mild n = 33 age, (years) 61.91 ± 3.42 60.82 ± 3.11 46.21 ± 7.50 a = 0.09 b = 0.00 c = 0.00 bmi, (kg/m2) 33.19 ± 3.35 30.07 ± 2.18 23.71 ± 5.01 a = 0.01 b = 0.00 c = 0.00 d-dimer, (ng/ml) 5602.6 ± 2348.30 4065.1 ± 2220.15 683.4 ± 301.81 a = 0.000 b = 0.000 c = 0.000 ferritin, (ng/ml) 1409.71 ± 700.29 1187.34 ± 692.50 648.79 ± 399.94 a = 0.000 b = 0.000 c = 0.000 vit. k, (pg/ml) 608.46 ± 103.32 611.40 ± 118.13 623.84 ± 112.79 a = 0.4 b = 0.05 c = 0.04 vwf, (ng/ml) 392.01 ± 35.22 354.11 ± 21.54 320.01 ± 24 a = 0.01 b = 0.01 c = 0.03 adamts13 (ng/ml) 15.09 ± 2.49 17.98 ± 4.35 23.78 ± 10.71 a = 0.01 b = 0.001 c = 0.001 vwf/admts13 26.14 ± 7.91 18.43 ± 7.56 12.34 ± 4.02 a = 0.001 b = 0.0001 c = 0.01 data represented as mean ± sd, sd: standar deviation, n.: number of subject, bmi: body mass index, a = deceased × severe, b = deceased × mild, c = severe × mild. table 3. the correlations between serum vwf, adamts-13,vitamin k levels with clinical parameters in covid-19 patients group parameters vwf (ng/ml) adamts-13 (ng/ml) vit.k, (pg/ml) r p-value r p-value r p-value age, (years) 0.379 0.05 –0.391 0.02 –0.341 0.064 bmi, (kg/m²) 0.386 0.04 –0.486 0.003 –0.372 0.050 d-dimer, (ng/ml) 0.473 0.001 –0.546 0.000 –0.487 0.001 ferritin, (ng/ml) 0.466 0.001 –0.452 0.001 –0.467 0.001 adamts13, (ng/ml) –0.575 0.000 1 – 0.397 0.01 vwf, (ng/ml) 1 – –0.575 0.000 –0.367 0.041 vit. k, (mg/ml) –0.367 0.041 0.394 0.01 1 – vwf/adamts13 0.454 0.001 –0.412 0.001 –0.388 0.01 bmi: body mass index; r = pearson correlation coefficient. 306 j contemp med sci | vol. 7, no. 5, september–october 2021: 303–307 assessment of von willebrand factor/adamts13 ratio original h.s. mohsin et al. in covid-19, the interaction between vwf and adamt13 despite their importance in maintaining hemostasis and preventing unwanted thrombosis, vwf adamts13 interactions have gotten little attention in the study of covid-19 pathophysiology, particularly vte. importantly, decreased adamts13 activity has been linked to increased inflammation in a variety of systems.15,16 coronavirus disease, also known as covid-19, is a multifaceted illness caused by infection with the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). interstitial pneumonia is the most common symptom, which can lead to poor gas exchange, severe respiratory failure, and mortality.17 the pathophysiology is complex, and in critically ill patients, a variety of hyper-inflammatory responses and abnormalities in the coagulation system have been described. understanding the immune-inflammatory mechanisms behind covid-19’s clinical symptoms will help researchers find possible pharmaceutical targets and stroke.18 as well as metabolic syndrome.19 coagulation proteins and platelets are involved in the formation of thrombus. platelet-mediated thrombogenesis is the most common type of thrombogenesis in arterial circulation. when extracellular vwf attaches to exposed collagen via its a3 domain, while the free vwf a1 domain binds to platelet glycoprotein ib, platelet thrombogenesis occurs (gpib).20 during the severe phase of covid-19, the systemic production of pro-inflammatory mediators inhibits the cleavage of high molecular weight vwf or interferes with the proteolytic interaction with adamts13, resulting in thrombosis.21 covid-19 patients’ d-dimer levels were similarly raised. only a portion of the reasons for the high d-dimer levels have been elucidated. d-dimers are formed during the breakdown of fibrin and are used as a marker of fibrinolytic activity. in critical patients or patients with sepsis, a link between pro-inflammatory cytokines and markers of coagulation cascade activation, such as d-dimer, has been discovered.22 there is additional evidence that in inflammatory conditions, the alveolar haemostatic balance shifts toward a prothrombotic activity majority.23 in individuals with severe sepsis, pro-inflammatory cytokines may also have a role in endothelial damage, as well as activating coagulation and inhibiting fibrinolysis. covid-19’s spike protein interacts to the angiotensin-converting enzyme 2 receptor on endothelial cells, causing death and thrombosis.24 apoptosis of endothelial cells also results in inflammatory cell infiltration, which increases the risk of thrombosis.25 as a result of the meta-analysis, ferritin levels in covid-19 patients with thrombotic problems were higher than those in individuals without, implying that patients with thrombosis are in a state of hyper-inflammation. high blood ferritin levels have been linked to hypertension in several investigations. covid-19 has a high rate of thromboembolism. vitamin k is vital for coagulation and may also play a function in lung disorders. vitamin k is an important component in preventing blood clotting and is a crucial role in the coagulation system, with a link recently discovered between vitamin k shortage and the worst covid-19 results.7 the respiratory symptoms of covid-19 are varied and can sometimes lead to significant consequences. severe forms of covid-19, like other severe respiratory disorders, include pneumonia, acute lung injury (ali), ards, and sepsis, which can lead to multiple organ failure and death.2 according to studies, the respiratory symptoms might deteriorate as quickly as 9 days after the onset of ards.26 even in asymptomatic patients, computed tomography (ct) scans revealed damage to the lungs, defined by pulmonary ground glass opacification, demonstrating that the multitude of covid-19-related problems is still far from being fully recognized.1 conclusion in conclusion  presentation a strong association between the levels of vwf, adamts13,and vitamin k on admission with respect to the severity, functional outcome, mortality and better guide thromboprophylaxis and consequential clinical recovery and may be used for future prognostication and guidance of therapy. a ratio of vwf/adamts-13 was associated with increased mortality and morbidity. however, additional investigations are needed to be performed to understand the exact cellular mechanism of this disease. acknowledgments all authors would like to thank the participated patients and the team of covid-19 centers for their support during this study. conflicts of interest no conflicts of interest regarding the publication of this article.  references 1. guan, w.-j., ni, z.-y., hu, y., et al. (2020). clinical characteristics of coronavirus disease 2019 in china. new england journal of medicine, 382(18), 1708–1720. 2. zaim, s., chong, j. h., sankaranarayanan, v., et al. (2020). covid-19 and multiorgan response. current problems in cardiology, 45(8), 100618. 3. fox, s. e., akmatbekov, a., harbert, j. l., et al. (2020). pulmonary and cardiac pathology in african american patients with covid-19: an autopsy series from new orleans. the lancet respiratory medicine, 8(7), 681–686. 4. ruggeri, z. m., (2003). von willebrand factor, platelets and endothelial cell interactions. journal of thrombosis and haemostasis. 1(7), 1335–1342. 5. scully, m., cataland, s., coppo, p., et al. (2017). consensus on the standardization of terminology in thrombotic thrombocytopenic purpura and related thrombotic microangiopathies. journal of thrombosis and haemostasis, 15(2), 312–322. 6. singh, k., kwong, a. c., madarati, h., et al. (2021). characterization of adamts13 and von willebrand factor levels in septic and non-septic icu patients. plos one, 16(2), e0247017. 7. dofferhoff, a. s., piscaer, i., schurgers, l. j., et al. (2020). reduced vitamin k status as a potentially modifiable risk factor of severe covid-19. clinical infectious diseases: an official publication of the infectious diseases society of america. 8. ma, l.-l., li, b.-h., jin, y.-h., et al. (2020). developments, evolution, and implications of national diagnostic criteria for covid-19 in china. frontiers in medicine, 7, 242. 9. murray, j. f., matthay, m. a., luce, j. m., et al. (1988). an expanded definition of the adult respiratory distress syndrome. am rev respir dis, 138(3), 720–723. 307j contemp med sci | vol. 7, no. 5, september–october 2021: 303–307 h.s. mohsin et al. original assessment of von willebrand factor/adamts13 ratio 10. wichmann, d., sperhake, j.-p., lütgehetmann, m., et al. (2020). autopsy findings and venous thromboembolism in patients with covid-19: a prospective cohort study. annals of internal medicine, 173(4), 268–277. 11. kim, d., lee j. y., yang j. s., et al. (2020). the architecture of sars-cov-2 transcriptome. cell, 181(4), 914–921. 12. bonafè, m., prattichizzo, f., giuliani, a., et al. (2020). inflamm-aging: why older men are the most susceptible to sars-cov-2 complicated outcomes. cytokine & growth factor reviews, 53, 33–37. 13. katneni, u. k., alexaki, a, hunt, r. c., et al. (2020). coagulopathy and thrombosis as a result of severe covid-19 infection: a microvascular focus. thrombosis and haemostasis. 14. marco, a., & marco, p. (2021). von willebrand factor and adamts13 activity as clinical severity markers in patients with covid-19. journal of thrombosis and thrombolysis, 1–7. 15. liu, c., zhao, l., zhao, j., et al. (2017). reduced adamts-13 level negatively correlates with inflammation factors in plasma of acute myeloid leukemia patients. leukemia research, 53, 57–64. 16. takaya, h., kawaratani, h., kubo, t., et al. (2018). platelet hyperaggregability is associated with decreased adamts13 activity and enhanced endotoxemia in patients with acute cholangitis. hepatology research, 48(3), e52–e60. 17. helms, j., kremer, s., merdji, h., et al. (2020). neurologic features in severe sars-cov-2 infection. new england journal of medicine, 382(23), 2268–2270. 18. vergouwen, m. d., meijers, j. c., geskus, r. b., et al. (2009). biologic effects of simvastatin in patients with aneurysmal subarachnoid hemorrhage: a double-blind, placebo-controlled randomized trial. journal of cerebral blood flow & metabolism, 29(8), 1444–1453. 19. ziliotto, n., bernardi, f., jakimovski, d., et al. (2018). hemostasis biomarkers in multiple sclerosis. european journal of neurology, 25(9), 1169–1176. 20. zhu, s., gilbert, j. c., hatala, p., et al. (2020). the development and characterization of a long acting anti‐thrombotic von willebrand factor (vwf) aptamer. journal of thrombosis and haemostasis, 18(5), 1113–1123. 21. neerukonda, s.n., katneni, u. (2020). a review on sars-cov-2 virology, pathophysiology, animal models, and anti-viral interventions. pathogens, 9(6), 426. 22. borjini, n., fernández, m., giardino, l., et al. (2016). cytokine and chemokine alterations in tissue, csf, and plasma in early presymptomatic phase of experimental allergic encephalomyelitis (eae), in a rat model of multiple sclerosis. journal of neuroinflammation, 13(1), 1–16. 23. omata, m., cheng, a. l., kokudo, n., et al. (2017). asia–pacific clinical practice guidelines on the management of hepatocellular carcinoma: a 2017 update. hepatology international, 11(4), 317–370. 24. ferrario, c. m., jessup, j., chappell m. c., et al. (2005). effect of angiotensinconverting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2. circulation, 111(20), 2605–2610. 25. connors, j. m., & levy, j. h. (2020). thromboinflammation and the hypercoagulability of covid-19. j thromb haemost, 18(7), 1559–1561. 26. wu, a., peng, y., huang, b., et al. (2020). genome composition and divergence of the novel coronavirus (2019-ncov) originating in china. cell host & microbe. 27(3), 325–328. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i5.1100 239j contemp med sci | vol. 8, no. 4, july-august 2022: 239–244 original the effect of pollution represented by polycyclic aromatic hydrocarbons on the levels of p53 and some antioxidant enzymes in baghdad traffic police ashraf r. salem1*, estabraq a.r. al-wasiti2, fahem a. hasan3 1college of nursing, university of telafer, telafer, nineveh, iraq. 2college of medicine, department of chemistry and biochemistry, al-nahrain university, baghdad, iraq. 3al-hussaini medical city, kerbala health directorate, ministry of health, kerbala, iraq. *correspondence to: ashraf r. salem (e-mail: ashraf.r.salem@uotelafer.edu.iq ) (submitted: 01 april 2022 – revised version received: 22 april 2022 – accepted: 18 may 2022 – published online: 26 august 2022) abstract objectives: to ascertain the protective effect of p53 tumor protein by monitoring its levels in comparison with the levels of antioxidant enzymes, against any type of cancer that can be caused by chronic exposure of traffic policemen to air pollutants. methods: this study comprises 140 participants, who have been divided into two groups (traffic police and office police). pahs were analyzed for each participant by gc/fid while p53 protein and antioxidant enzymes were measured by the elisa technique. results: the concentrations of polycyclic aromatic hydrocarbons and p53 tumor protein were high in the blood of traffic police compared to office police, while higher levels of antioxidant enzymes (catalase and g-px) were observed in the blood of office police. conclusion: exposure to pahs can cause oxidative stress, which can damage dna and lead to cancer. however, because natural endogenous biomolecules like p53 protein can neutralize pahs’ carcinogenic effects, their elevation has a beneficial anti-cancer effect by reducing oxidative stress and preventing tumorigenesis. keywords: pahs, p53 tumor protein, catalase, g-px, dna issn 2413-0516 introduction all living things, including people, are subject to air pollution’s multiple detrimental consequences, and because of how it affects human life and health, this problem has gained prominence on the international political agenda. air pollution is the number one environmental killer and the fifth-highest risk factor for total all-cause mortality.1 it’s a complex chemical mixture that contains various concentrations of gases such as nitrogen dioxide (no2), ozone (o3), carbon dioxide (co2), sulfur dioxide (so2), carbon monoxide (co), and carbon dioxide (co2). gaseous pollutants may have both immediate and long-term negative effects on health.2 many of these gases have oxidizing properties, and one way they can be harmful to human health is by causing oxidative stress.1 semi-volatile kinds including benzene, formaldehyde, naphthalene, and polyaromatic hydrocarbons occur as liquid droplets, but they may also move between the gaseous particle phases of air pollution.3 numerous cancers and air pollution are related,4 and it is estimated that cancer causes 4.2 million early deaths worldwide and affects organs including the lung, mouth, bladder, kidney, breast, liver, prostate, and ovary. in addition, the international agency for research on cancer has classed outdoor air pollution as a category 1 human carcinogen (iarc).5 the liver is where pahs are mostly processed and activated after being ingested through the lungs, skin, and intestines. many polycyclic aromatic hydrocarbons are bioactivated to form phenols, epoxides, and dihydrodiol, which are mostly oxidized by cytochrome p450 monooxygenases (cyps). 6 the two main mechanisms for the carcinogenicity of pahs are the creation of reactive oxygen species and pah-dna asymptotics, particularly those of diolepoxides, radical cations, and o-quinones7 (figure 1). the gene encoding the p53 tumor protein is known as tumor protein (tp53). it’s a phosphoprotein containing 393 amino acids in its structure9 and ‘guardian of the genome’ is another name for it.10 the p53 tumor protein attaches to dna in the nucleus directly and it is involved in the cell cycle, dna repair, and apoptosis control, as well as regulating the repair process in reaction to damaging substances such as radiation, chemicals, and uv radiation from sunshine.9,11 this protein is crucial in deciding whether or not a damaged cell will undergo dna repair or programmed cell death. if dna damage can be repaired, p53 activates the genes needed to do it, while this protein inhibits the cell from proliferating and tells it to undergo programmed cell death if the dna cannot be repaired.12 this means tp53 controls cell division by stopping them from expanding and dividing (reproducing) in an uncontrolled manner. in vertebrates, these p53-mediated responses are critical and decisive in preventing cancer recurrence.13 under genotoxic stress, p53 can induce the production of p21waf1/cip1, a cyclin-dependent kinase inhibitor, which can briefly arrest the cell cycle at g1 or g2. this permits the cell to remove and repair damage while also preventing damaged cells from replicating. the action of p53, which causes irreversible g1 inactivation, can also cause aging. p53 may induce apoptosis in highly injured cells by increasing the transcription of genes like puma and noxa14 (figure 2). materials and methods one hundred and forty iraqi policemen affiliated with the traffic police in the city of baghdad within the iraqi ministry of interior. their ages range between (25–65 years), during the period from july 15, 2019 to march 25, 2020. subjects were divided into two groups as indicated below: group 1: 70 policemen of those who were performing their duty in the crowded squares and intersections in baghdad (traffic police). group 2: 70 policemen of those who were serving inside the buildings of the various traffic directorates (office police). mailto:ashraf.r.salem@uotelafer.edu.iq 240 j contemp med sci | vol. 8, no. 4, july-august 2022: 239–244 the effect of pollution represented by pahs original a.r. salem et al. fig. 1 the bap metabolites diolepoxides and o-quinones are potent reactive metabolites that can form adducts with dna.8 fig. 2 the mechanism of action of p53 protein to avoid the accumulation of genetic errors.15 six milliliters of blood were collected from each participant, and the blood samples were then centrifuged for 10 minutes at 3000 rpm to separate the serum before being aspirated, separated into aliquots, and refrigerated at 20oc until they were used for pah. 1. determination of pah concentrations in serum by gas chromatography coupled with a flame ionization detector (gc/fid). 2. determination of tumor protein p53 by enzyme-linked immunosorbent assay (elisa) kit catalogue no. mbs2514403. 3. determination of the serum concentration of gpx1 (glutathione peroxidase), by elisa. kit catalog number. e-el-h5410. 4. determination of the serum concentration of catalase, by elisa kit catalog number. mbs703074. 241j contemp med sci | vol. 8, no. 4, july-august 2022: 239–244 a.r. salem et al. original the effect of pollution represented by pahs results the mean ± sd of age for the two groups was shown in (table 1). mean concentration of pahs, levels of human p53 protein, and two antioxidant enzymes (catalase and g-px) for the two groups are shown in (table 2), the effect of the table 1. the mean age among the groups group no. (mean ± sd) of age, year office police 70 38.54 ± 5.49 traffic police 70 40.15 ± 5.7 table 2. the mean concentration of pahs, p53, catalase and g-px among the groups parameter group mean ± s.e. p-value pahs, (ppm) office police 6.91 ± 0.12 0.0001 traffic police 8.94 ± 0.09 p53, (pg/ml) office police 1070.7 ± 19.57 0.0001 traffic police 1431.6 ± 39.9 catalase activity, (pg/ml) office police 648.15 ± 15.3 0.0001 traffic police 398.52 ± 16.58 g-px activity, (pg/ml) office police 55.74 ± 2.99 0.0001 traffic police 34.34 ± 2.2 p-value <0.05 is significant. table 3. comparison of studied parameters according to exposure duration for the office police group parameter duration of exposure mean ± s.e. p-value pahs, (ppm) less than 10 years 6.7 ± 0.31 0.599 more than 10 years 6.97 ± 0.12 p53, (pg/ml) less than 10 years 1055.4 ± 45.2 0.440 more than 10 years 1075.64 ± 21.6 catalase activity, (pg/ml) less than 10 years 652.12 ± 43.3 0.995 more than 10 years 646.88 ± 15 g-px, activity (pg/ml) less than 10 years 59.89 ± 6.18 0.703 more than 10 years 54.41 ± 3.44 p-value >0.05 is non-significant. table 4. comparison of studied parameters according to exposures duration for the traffic police group parameter duration of exposure mean ± s.e. p-value pahs, (ppm) less than 10 years 8.80 ± 0.21 0.487 more than 10 years 9.01 ± 0.09 p53, (pg/ml) less than 10 years 1353.5 ± 80.07 0.491 more than 10 years 1467.47 ± 44.8 catalase activity, (pg/ml) less than 10 years 394.49 ± 33.7 0.947 more than 10 years 400.36 ± 18.8 g-px activity, (pg/ml) less than 10 years 35.27 ± 5.00 0.781 more than 10 years 33.91 ± 2.28 p-value >0.05 is non-significant. exposure period to pollutants from car exhaust and diesel engines on the traffic police who are in the offices as well as the traffic police deployed in the crowded intersections of the baghdad city are summarized in the (table 3) and (table 4) respectively. discussion exposure to these harmful substances—known as pahs— increases the chance of developing tumors16 because these poisons and their reactive byproducts, such as dihydrodiols and epoxides, may attach to cellular proteins and dna causing dire consequences17 such as mutations, developmental problems, and thus cancers that occur as a result of cell damage and biochemical disturbance.18,19 242 j contemp med sci | vol. 8, no. 4, july-august 2022: 239–244 the effect of pollution represented by pahs original a.r. salem et al. the results of this study showed the presence of high levels of polycyclic aromatic hydrocarbons in the blood serum of the traffic police who are at the busy street intersections of the city of baghdad, relative to the levels in the blood serum of the office police group. since 2003, the number of passenger auto-mobiles., trucks, buses, and household generators have skyrocketed in iraq, posing serious environmental concerns. research was carried out by chaichan et al.20 on and around the muhammad al-qasim roadway close to the university of technology in baghdad, to assess the connection between the level of activity, the movement of cars with various engines, and the pollution that comes from exhaust pipes, and its was discovered that pollutants such assulfur and polycyclic aromatic hydrocarbons, increase during the start and end periods of the working hours of state departments. moreover, iraqi inhabitants employ hundreds of thousands of tiny electric generators in their houses,21 which use heavy oil or gasoline to create electricity, resulting in large quantities of soot, carbon deposits, and sulfur oxides.22 a recent study that measured air pollutants around iraq, it showed that the highest number was recorded in al-diwaniyah, followed by baghdad, specifically the dora area.23 our results are in agreement with several previous studies, such as24 of pahs released by vehicles, which found that street cops have high exposures, much higher than chefs, and their exposure levels are comparable to coke factory workers. another study in china found that traffic cops have a higher risk of pm2.5 pollution in the workplace than office cops who are considered a control group.25 among the objectives of this research is to study the impact of polycyclic aromatic hydrocarbons on the levels of p53 protein, and the results showed that the levels of this protein are affected by polycyclic aromatic hydrocarbons, as its level increased significantly in the traffic police group located in the crowded intersections of baghdad city compared to the office police group, indicating that the amount of pahs changed in direct proportion to the change in the concentration of p53 protein. on the other hand,26 discovered a substantial link between plasma levels of p53 and urine 1-hydroxypyrine, which is an acceptable biomarker of polycyclic aromatic hydrocarbons exposure. another study conducted in saudi arabia on professional workers during the hajj season to appreciate the effect of intense exposure to polycyclic aromatic hydrocarbons on the cancer biomarker proteins p53 and p21 discovered a positive engagement between short-term pahs exposure and blood concentrations of p53 and p21.27 in another study, carried out by yu et al.28 to explore the influence of prolonged exposure to pahs on cellular processes occurring in mouse lung fibroblasts (mlfcs), this study concluded that long-term exposure to b a a and b a p increased the protein expression levels of p53 and p21. in most cell types examined, p53 is a short-lived nucleoprotein with a half-life of 5–20 minute. the half-life of p53 rises several times once the dna is damaged.29 the activation of p53 is caused by the generation of dna damage by a range of factors, including polycyclic aromatic hydrocarbons and oxidative stress. this tumor suppressor gene encodes a protein that acts as a crucial mediator of cell cycle arrest, allowing dna repair or starting an apoptotic cascade, and thereby preventing mutations from being passed on to daughter cells.11,30 the p53 protein’s activity may also be increased when healthy tissues experience pathophysiological alterations that cause oxidative or redox stress, such as damage from ischemia and reperfusion to the heart, brain, and other tissues. thus the oxidative stress generated by hydrogen peroxide appears to be a potent stimulator of p53 activity.29 many occupational persons, such as workers in coke plants and food processing plants, are exposed to contaminants, as are traffic policemen who are exposed to pahs through vehicle exhaust and road dust.31 pahs activation can be categorized into 3 paths: (1) cytochrome p450 enzymes and epoxide hydrolase catalyzes the formation of dihydrodiol epoxides (cyp/eh pathway), (2) cytochrome p450 peroxidase activity generates a polycyclic aromatic hydrocarbons reactive cation in metabolic oxidization, and (3) ortho-quinones are produced by dihydrodiol dehydrogenase, a member of the aldo-ketoreductase family, oxidizing catechols (akr pathway). quinone redox cycling could result in the creation of ros, which could lead to carcinogenesis through oxidative dna damage.32 defects in the p53 protein, on the other side, maybe the cause of its high levels. faulty mutations in the gene that encodes for the p53 protein, or point mutations in this site, can disrupt the construction of a tetramer, ultimately in the transformation of wild-type p53 into a mutant kind and decreased function. the formation of the p53 mutation elongates its half-life to many hours in humans.33 various studies regarding antioxidant enzymatic and non-enzymatic have been done in clinical investigations of different iraqi patients.34,35 our study chose two types of antioxidant enzymes (catalyze and glutathione peroxidase), to study their levels in the blood serum of the two study groups and their relationship with the levels of pahs for both groups, found low levels in the levels of both catalase and glutathione peroxidase enzymes in the blood serum of traffic police personnel deployed in baghdad city intersections compared to the levels of the two enzymes were in the control group represented by the office police, and the levels of these enzymes were inversely proportional to the levels of pollutants in the blood serum represented by polycyclic aromatic hydrocarbons. also, some studies conducted to evaluate the effects of occupational exposure to pollutants on oxidative stress in the body have indicated a decrease in the levels of antioxidant enzymes. in a study of pollution-exposed taxi drivers,36 reported a decrease in cat and gsh-px activities compared to the occupationally unexposed group. cohort research done before, during, and after the beijing olympics has reported that when air pollution levels increased, indicators of total antioxidant status-declined.37 the production of a sizable quantity of ros by particulate matter in traffic exhausts is one theory for the reported negative health impacts. aerosols in the environment contain smaller, more ros-rich particles. polycyclic aromatic hydrocarbons are also present in fine particles from vehicle exhaust (pahs). antioxidants have been shown to have a key part in the catalysis of the dismutation of (o2) to h2o2 and the breakdown of h2o2 to h2o, respectively. when the presence of reactive oxygen species overcomes the antioxidant buffering capability, oxidative stress happens. once antioxidant enzymes are depleted, the cell is more vulnerable to the harmful effects of xenobiotics, which can lead to cell harm or death. as a result, frequent exposure to gasoline vapors has the potential to cause oxidative stress by lowering the body’s antioxidant defenses’ cellular functions.38 243j contemp med sci | vol. 8, no. 4, july-august 2022: 239–244 a.r. salem et al. original the effect of pollution represented by pahs poor nutritional status can also contribute to oxidative stress, for example, selenium deficiency, which is associated with increased oxidative stress; optimization of nutritional status of se may result in higher g-px activity. small amounts of antioxidants have been associated with a greater risk of cancer in epidemiological research studies. g-px loss resulted in endothelial dysfunction, decreased angiogenesis, and increased infarction severity and vascular permeability in experimental animals,39 as seen the activity of catalase, superoxide dismutase, and glutathione peroxidase have a substantial negative relationship with the risk of coronary artery disease in patients.40 in response to oxidative stress, prolonged antioxidant enzyme deficiency enhances tissue sensitivity and severity.41 conclusion exposure to air pollutants like pahs can result in lower levels of antioxidant enzymes and oxidative stress, which can damage dna and cause a variety of cancers. however, natural biomolecules such as p53 protein can reverse these carcinogenic effects of pahs. its high levels are thus necessary to serve as an anticancer agent, reducing the effects of oxidative stress and preventing the formation of cancers in traffic cops.  references 1. miller, m. r., (2020). oxidative stress and the cardiovascular effects of air pollution. free radical biology and medicine, 151: 69–87. 2. brook, r. d., rajagopalan, s., pope, c. a., 3rd, brook, j. r., bhatnagar, a., diez-roux, a. v., and et. al. (2010). particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the american heart association. circulation, 121 (21): 2331–2378. 3. liu, c., zhang, y., and weschler, c. j. (2014). the impact of mass transfer limitations on size distributions of particle associated svocs in outdoor and indoor environments. the science of the total environment, 497–498: 401–411. 4. andersen, z. j., pedersen, m., weinmayr, g., stafoggia, m., galassi, c., jørgensen, j. t., and et. al. (2018). long-term exposure to ambient air pollution and incidence of brain tumor: the european study of cohorts for air pollution effects (escape). neuro-oncology, 20(3): 420–432. 5. su, s. y., liaw, y. p., jhuang, j. r., hsu, s. y., chiang, c. j., yang, y. w. and lee, w. c. (2019). associations between ambient air pollution and cancer incidence in taiwan: an ecological study of geographical variations. bmc public health, 19(1): 1496. 6. león, s. p. z. and lópez, f. d. (2020). polycyclic aromatic hydrocarbons and their association with breast cancer. bangladesh journal of medical science, 19 (2): 194–199 . 7. zhang, l., jin, y., huang, m., and penning, t. m. (2012). the role of human aldo-ketoreductases in the metabolic activation and detoxication of polycyclic aromatic hydrocarbons: interconversion of pah catechols and pah o-quinones . frontiers in pharmacology, 3: 193. 8. clergé, a., le goff, j., lopez, c., ledauphin, j. and delépée, r. (2019). oxypahs: occurrence in the environment and potential genotoxic/mutagenic risk assessment for human health. critical reviews in toxicology, 49(4): 302–328. 9. huszno, j., and grzybowska, e. (2018). tp53 mutations and snps as prognostic and predictive factors in patients with breast cancer. oncology letters, 16(1): 34–40. 10. perri, f., pisconti, s., and vittoriascarpati, g. della. (2016). p53 mutations and cancer: a tight linkage. annals of translational medicine, 4(24): 2–5. 11. al-tu’ma, f.j. ; al-zubaidi, r.d. ; al-khaleeli, a.m.b. and abo-almaali, h.m. (summer 2016). polymorphism of tumor suppressor gene (p53) codon 72 in iraqi patients with acute myocardial infarction. j contemp med sci, 2 (7): 74–76. 12. chen j. (2016). the cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression. cold spring harbor perspectives in medicine, 6(3): a026104. 13. grochola, l. f., zeron-medina, j., mériaux, s., and bond, g. l. (2010). singlenucleotide polymorphisms in the p53 signaling pathway. cold spring harbor perspectives in biology, 2(5): a001032. 14. kucab, j. e., phillips, d. h., and arlt, v. m. (2010). linking environmental carcinogen exposure to tp53 mutations in human tumours using the human tp53 knock-in (hupki) mouse model. the febs journal, 277(12): 2567–2583. 15. asco, m., shami, s. and crook, t. (2002). the p53 pathway in breast cancer. breast cancer res 4: 70. 16. da silva junior, f. c., felipe, m., castro, d., araújo, s., sisenando, h., and batistuzzo de medeiros, s. r. (2021). a look beyond the priority: a systematic review of the genotoxic, mutagenic, and carcinogenic endpoints of nonpriority pahs. environmental pollution (barking, essex: 1987), 278: 116838. 17. rubin h. (2001). synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updates. carcinogenesis, 22(12):1903–1930. 18. chen, s., nguyen, n., tamura, k., karin, m., and tukey, r. h. (2003). the role of the ah receptor and p38 in benzo[a]pyrene–7,8 dihydrodiol and benzo[a] pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis. the journal of biological chemistry, 278(21):19526–19533. 19. raghad h al-ani and estabraq ar. al-wasiti. (2021). the adverse effect of air pollution with polycyclic aromatic hydrocarbon (pah) on 8-oxo-dg and gene expression (hogg1) in midland refineries company-daura refinery workers. indian journal of forensic medicine and toxicology, 15(3): 2651–2656. 20. chaichan, m.t., kazem, h.a. and abed, t.a. (2016). traffic and outdoor air pollution levels near highways in baghdad, iraq. environ dev sustain 20: 589–603. 21. kazem, h. a. and chaichan, m.t., (2012). “status and future prospects of renewable energy in iraq,” renewable and sustainable energy reviews, elsevier, 16(8): 6007–6012. 22. chaichan, m. t., and faris, s. s. (2015). practical investigation of the environmental hazards of idle time and speed of compression ignition engine fueled with iraqi diesel fuel. international journal for mechanical and civil engineering, 12(1): 29–34. 23. al-kasser m. k. (2021). air pollution in iraq sources and effects. iop conference series: earth and environmental science, 790: 012014. 24. hu, y., bai, z., zhang, l., wang, x., zhang, l., yu, q., and zhu, t. (2007). health risk assessment for traffic policemen exposed to polycyclic aromatic hydrocarbons (pahs) in tianjin, china. the science of the total environment, 382(2-3): 240–250. 25. chao, h.r., hsu, j.w., ku, h.y., wang, s.l., huang, h.b., liou, s.h. and tsou, t.c. (2018). inflammatory response and pm2.5 exposure of urban traffic conductors. aerosol air qual. res. 18: 2633–2642. 26. mehdi z, seyed jamaleddin shahtaheri, parvin mehdipur, mohammad shekari and shahram z (2013). levels of p53 protein as biomarker in plasma of workers exposed to carcinogenic polycyclic aromatic hydrocarbons, toxicological and environmental chemistry, 95(1): 187–196. 27. saleh, s., adly, h. m., aljahdali, i. a. and khafagy, a. a. (2022). correlation of occupational exposure to carcinogenic polycyclic aromatic hydrocarbons (cpahs) and blood levels of p53 and p21 proteins. biomolecules, 12(2): 260. 28. yu, f., ye, k., hu, y., li, j., an, y. and qu, d. (2019). exposure to polycyclic aromatic hydrocarbons derived from vehicle exhaust gas induces premature senescence in mouse lung fibroblast cells. molecular medicine reports, 19(5): 4326–4334. 29. giaccia, a. j. and kastan, m. b. (1998). the complexity of p53 modulation: emerging patterns from divergent signals. genes and development, 12(19): 2973–2983. 30. novotna, b., topinka, j., solansky, i., chvatalova, i., lnenickova, z., and sram, r. j. (2007). impact of air pollution and genotype variability on dna damage in prague policemen. toxicology letters, 172(1-2): 37–47. 31. patel, a. b., shaikh, s., jain, k. r., desai, c., and madamwar, d. (2020). polycyclic aromatic hydrocarbons: sources, toxicity, and remediation approaches. frontiers in microbiology, 11: 562813. 32. ewa, b., and danuta, m. š. (2017). polycyclic aromatic hydrocarbons and pah-related dna adducts. journal of applied genetics, 58(3): 321–330. https://ideas.repec.org/a/eee/rensus/v16y2012i8p6007-6012.html https://ideas.repec.org/a/eee/rensus/v16y2012i8p6007-6012.html https://ideas.repec.org/s/eee/rensus.html https://iopscience.iop.org/journal/1755-1315 https://iopscience.iop.org/journal/1755-1315 https://iopscience.iop.org/volume/1755-1315/790 244 j contemp med sci | vol. 8, no. 4, july-august 2022: 239–244 the effect of pollution represented by pahs original a.r. salem et al. 33. zhang, h., liu, m., zhang, h., cao, s., li, y., jiang, s., song, y., and liu, s. (2020). detection of p53 mutation and serum monitoring alert caused by marek’s disease virus in poultry. bmc veterinary research, 16(1): 303. 34. ahmed st, al-silaykhee sm, jeddoa zma and ewadh mj (2019). vascular endothelial growth factor gene polymorphism (-2578 t/a) (rs699947) in diabetic foot ulcer and its correlation with oxidative stress. wjpmr, 5(12): 45–50. 35. al-tu'ma, f. j.; abd al-hassan, a. t. and alda'amy, e. m. (spring 2016). correlation between malondialdehyde and dyslipidemia in psoriatic patients. j contemp med sci, 2 (6): 56–58. 36. brucker, n., moro, a. m., charão, m. f., durgante, j., freitas, f., baierle, m., and et. al. (2013). biomarkers of occupational exposure to air pollution, inflammation and oxidative damage in taxi drivers. the science of the total environment, 463–464: 884–893. 37. cosselman, k. e., allen, j., jansen, k. l., stapleton, p., trenga, c. a., larson, t. v., and kaufman, j. d. (2020). acute exposure to traffic-related air pollution alters antioxidant status in healthy adults. environmental research, 191: 110027. 38. wagboriayea f, dedekeb g, aladesidab a, bamideleb j and olootoc w. (2018). assessment of the effect of gasoline fume on stress hormones, antioxidant status and lipid peroxidation in albino rat. j king saud univsci 30: 393–399. 39. sarıkaya, e. and doğan, s. (2020). glutathione peroxidase in health and diseases. published in jan. 2020. glutathione system and oxidative stress in health and disease. intech open. https://doi.org/10.5772/ intechopen.91009 40. flores-mateo, g., carrillo-santisteve, p., elosua, r., guallar, e., marrugat, j., bleys, j. and covas, m. i. (2009). antioxidant enzyme activity and coronary heart disease: meta-analyses of observational studies. american journal of epidemiology, 170(2): 135–147. 41. delfino, r. j., staimer, n., and vaziri, n. d. (2011). air pollution and circulating biomarkers of oxidative stress. air quality, atmosphere, and health, 4(1): 37–52. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1254 https://doi.org/10.5772/intechopen.91009 https://doi.org/10.5772/intechopen.91009 375j contemp med sci | vol. 8, no. 6, november-december 2022: 375–381 original characterization of covid-19 hospitalized adult patients, vaccinated vs non-vaccinated in duhok province paiman abdulsalam mohammed1*, muayad aghali merza2,3,4 1directorate of general health, duhok, kurdistan region, iraq. 2covid-19 health facilities, duhok, kurdistan region, iraq. 3department of internal medicine, azadi teaching hospital, duhok, kurdistan region, iraq. 4dean, college of pharmacy, university of duhok, duhok, kurdistan region, iraq. *correspondence to: paiman abdulsalam mohammed, (e-mail: paimanabdulsalam@yahoo.com) (submitted: 11 july 2022 – revised version received: 18 august 2022 – accepted: 09 october 2022 – published online: 26 december 2022) abstract objectives: first, to determine prevalence of vaccinated covid-19 patients among hospitalized patients; second, to determine the epidemiological, clinical, and laboratory characteristics of vaccinated and unvaccinated covid-19 patients. methods: the study was carried out on 300 adult covid-19 hospitalized patients at duhok covid-19 health facilities. a prospective crosssectional study was used as the study design. between october 1, 2021, and march 31, 2022, all patients with pcr-confirmed covid-19 were enrolled. results: the majority of people in this study were unvaccinated. pfizer was most popular among people who had received vaccination. the majority of hospitalized patients were old ages, the mean age was 60.73 ± 15.83 yr. in our study, the unvaccinated females had higher infection rates while vaccinated males had higher hospital admission rates. in our study, vaccinated patients had shorter hospital duration stays. in both vaccinated and unvaccinated patients, predominated cases were severe cases. d dimer was significantly higher among vaccinated patients. the mortality rate was relatively high among both groups. patients who had received vaccinations tended to experience vomiting and flu-like symptoms more frequently than those who had not. in terms of comorbidities, smoking and malignancy were significant risk factors for covid-19 infection in unvaccinated patients. conclusion: we looked at 300 covid-19 hospitalized patients. in this study, the majority of people were unvaccinated. pfizer, had higher prevalence among vaccinated individuals. majority were elderly. the unvaccinated cases had a higher rate of female hospital admissions than male. the d.dimer level was significantly different between the two groups. vomiting and flu-like illness showed higher prevalence in vaccinated cases with significant difference. smoking and malignancy were significant risk factors for covid-19 infection in unvaccinated patients. in the fight against a public health disaster like a covid-19 pandemic, the availability of a covid-19 vaccines campaign are crucial. keywords: covid-19, vaccinated, unvaccinated issn 2413-0516 introduction the world health organization (who) declared the coronavirus disease 2019 (covid-19) a pandemic on march 11, 2020. (who).1 globally, severe acute respiratory syndrome coronavirus 2 (sarscov2), the causative agent of covid-19 has infected tens of millions of people with significant mortality. the virus is transmitted mainly through exposure to respiratory excretions carrying sars-cov-2.2 the clinical manifestation ranges from asymptomatic infection to serious life-threatening condition. mild cases constitute approximately 81%., while severe and critical cases constitute 14% and 5%, respectively.3 sars-cov-2 has a particular tendency to involve the lower respiratory tract, resulting in a hazardous complication causing pneumonia. it has been shown that coexisting diseases like diabetes mellitus (dm), cardiovascular diseases (cvd), chronic lung diseases, ...etc have negative impacts on covid-19 prognosis, causing an increased risk of developing severe complications such as acute respiratory distress syndrome (ards).4 the diagnosis of covid-19 is established by detecting the virus in the clinical specimen by molecular assays.5 however, other laboratory parameters are fundamental in evaluating the disease severity such as complete blood count (cbc), c-reactive protein (crp), d-dimer, s. ferritin, lactate dehydrogenase (ldh), …etc.6 current management strategies are not satisfactory enough to prevent the disease complications, particularly among patients with severe illnesses and comorbid diseases. hence, it is essential to have an alternative measure to control the disease. the introduction of covid-19 vaccine has revolutionized the disease magnitude. in iraq, three covid-19 vaccines were introduced to the community, namely: mrna vaccine “pfizer biontech”, the adenoviral vector vaccines chadox1 ncov-19 (astrazeneca-oxford), and sinopharm (beijing).7 globally, pfizer on december 24, 2020, astrazeneca on january 28, 2021, and sinopharm on september 10, 2021 were granted emergency use authorization by food and drug administration (fda).8 the first vaccine administered to the iraqi population in march 2021 was sinopharm.9 covid-19 vaccines proved to be effective in preventing hospitalization.10 fully vaccinated people might develop covid-19 infection in an attenuated form. however, severe vaccine breakthrough infection is not uncommon, particularly among people with several months of vaccine administration as their immunity fades over time.11 to the best of our knowledge, there is little information regarding the prevalence and characteristics of hospitalized vaccinated covid-19 patients in iraq. therefore, the objectives of this study were: first, to determine prevalence of vaccinated covid-19 patients among hospitalized patients; second, to determine the mailto:paimanabdulsalam@yahoo.com 376 j contemp med sci | vol. 8, no. 6, november-december 2022: 375–381 characterization of covid-19 hospitalized adult patients, vaccinated vs non-vaccinated in duhok province original p.a. mohammed et al. epidemiological, clinical, and laboratory characteristics of vaccinated and unvaccinated covid-19 patients. patients and methods setting the study was conducted in duhok covid-19 health facilities. first, duhok covid-19 hospital consisting of 50 ward beds and 20 icu beds was mainly used for severe and critical cases. second, a 100-bed hospital, called lalav, primarily met patients with moderate to severe presentations. study design and patients the study design was a prospective cross-sectional study on adult covid-19 hospitalized patients. all pcr confirmed covid-19 patients from october 1, 2021 until march 31, 2022 were enrolled. covid-19 cases diagnosed based on other methods were excluded from the study. clinico-demographic, including vaccination status and laboratory parameters, was recorded in a standardized questionnaire. the study was approved by the research ethics committee, duhok directorate general of health on october 24, 2021 under reference number: 24102021-10-10. diagnosis and measures covid-19 hospitalized patients were classified for disease severity into mild, moderate, severe, and critical according to.12 asymptomatic: patients who test positive for sars-cov-2 via a virologic test (such as an antigen or nucleic acid amplification test [naat]), but who do not exhibit symptoms that are typical with covid-19. mild illness: patients who exhibits any of the covid-19’s many symptoms (such as a fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, and a loss of taste and smell) but do not exhibit dyspnea, shortness of breath, or abnormal chest imaging. moderate illness: patients with an oxygen saturation (spo2) of less than 94% in ambient air at sea level and who exhibit signs of lower respiratory illness during clinical evaluation or imaging. severe type: patients with a blood oxygen saturation (spo2) of less than 94% on room air at sea level, a pao2/fio2 ratio less than 300 mm hg, a respiratory rate greater than 30 breaths per minute, or lung infiltrates greater than 50%. critical type: patients who develop respiratory failure, shock, and multiple organ dysfunction. the patients were grouped into the following categories according to centers for disease control and prevention (cdc), morbidity and mortality weekly (mmwr) report13 for vaccination status: 1. unvaccinated: individuals who are not vaccinated with any dose of covid-19 vaccine or vaccine administration by 14 days or less. 2. partially vaccinated: individuals who were vaccinated with the first dose for more than 14 days, or individuals who were vaccinated with two doses and have not reached 14 days post second vaccine. 3. fully vaccinated: individuals who were vaccinated with two doses with the second dose ≥14 days. all covid-19 patients underwent laboratory testing including complete blood count (cbc), c-reactive protein (crp), lactate dehydrogenase (ldh), serum ferritin, and d-dimer. statistical analyses the general information of the patients was presented in mean and sta. deviation or number and percentage. the mortality rate and vaccination were determined in number and percentage. comparisons of general and medical information between vaccinated and unvaccinated individuals were examined in an independent t-test and pearson chi-squared test. comparisons of biomedical measurements between vaccinated and unvaccinated individuals were examined in an independent t-test. comparisons of outcomes between vaccinated and unvaccinated individuals, association of vaccination by symptoms among individuals, and symptoms by outcomes were examined in pearson chi-squared tests. the significant level of difference was determined in a p-value of less than 0.05. the statistical calculations were performed in jmp pro 14.3.0. results in total, there were 300 patients with a mean age of 60.73 years. there were 142 (47.33%) males and 158 (52.67%) females. only 28 (9.33%) patients were vaccinated (table 1). table 2 presents the comparison of demographic, clinical, and laboratory parameters between vaccinated and unvaccinated patients. in the vaccinated group, there were 17 (11.97%) table 1. demographic and clinical characteristics of the hospitalized patients parameter (n = 300) statistics no (%) 95% ci lower ci to upper ci age (yrs.) 60.73 (15.83) 58.93–62.53 sex male female 142 (47.33) 158 (52.67) 41.75–52.98 47.02–58.25 vaccine status unvaccinated vaccinated 272 (90.67) 28 (9.33) 86.84–93.46 6.54–13.16 type of vaccine unvaccinated astrazeneca pfizer sinopharm 272 (90.67) 7 (2.33) 15 (5.00) 6 (2.00) 86.84–93.46 1.13–4.74 3.05–8.08 0.92–4.29 partially vaccinated (days) no yes 295 (98.33) 5 (1.67) 96.16–99.29 0.71–3.84 fully vaccinated (days) no yes 277 (92.33) 23 (7.67) 88.76–94.84 5.16–11.24 disease severity moderate severe critical 11 (3.67) 221 (73.67) 68 (22.67) 2.06–6.45 68.41–78.33 18.29–27.73 the infection after first dose (days) was between 2 and 150 days infection after the second dose (days) was between 2 and 220 days. 377j contemp med sci | vol. 8, no. 6, november-december 2022: 375–381 p.a. mohammed et al. original characterization of covid-19 hospitalized adult patients, vaccinated vs non-vaccinated in duhok province table 2. comparisons of general characteristics between vaccinated and unvaccinated patients general characteristics vaccination status no (%) p-value (two-tailed)unvaccinated (n = 272) (no/%) vaccinated (n = 28) (no/%) infection before vaccination no yes do not know 6 (21.43) 5 (17.86) 17 (60.71) sex male female 125 (88.03) 147 (93.04) 17 (11.97) 11 (6.96) 0.1364a age (year) 60.40 (16.19) 63.89 (11.55) 0.2676b symptoms duration (days) mean (sd) 9.41 (3.09) 9.89 (2.62) 0.4273b hospitalization duration (day) mean (sd) range 11.14 (7.72) 1–77 days 8.17 (3.68) 1–60 days 0.0700b hospitalization duration < one week 7–14 days 15–21 days 22–28 days > one month 83 (30.51) 81 (29.78) 46 (16.91) 24 (8.82) 38 (13.97) 9 (32.14) 12 (42.86) 3 (10.71) 1 (3.57) 3 (10.71) 0.5484a disease severity moderate severe critical 10 (3.68) 201 (73.90) 61 (22.43) 1 (3.57) 20 (71.43) 7 (25.00) 0.9532a oxygen requirement no yes 10 (3.68) 262 (96.32) 1 (3.57) 27 (96.43) 0.9775a cpap requirement no yes 178 (65.44) 94 (34.56) 21 (75.00) 7 (25.00) 0.3081a invasive ventilation requirement no yes 270 (99.26) 2 (0.74) 27 (96.43) 1 (3.57) 0.1509a outcome of patients died recovered or (95%): 1.29 (0.58–2.87) 148 (54.41) 124 (45.59) 17 (60.71) 11 (39.29) 0.5233a biomedical measurements alc ldh crp d. dimer 0.89 (0.43) 620 (148.40) 78.59 (57.99) 1305.58 (971.42) 0.98 (0.63) 429.5 (380.10) 68.14 (46.23) 1760.48 (1195.38) 0.3135b 0.2633b 0.4118b 0.0390b apearson chi-squared test and ban independent t-test. sd, standard deviation; or, odd ratio; cpap, continuous positive airway pressure; sob, shortness of breath; alc, absolute lymphocyte count; ldh, lactate dehydrogenase; crp, c-reactive protein. males and 11 (6.96%) females, while the unvaccinated group consisted of 125 (88.03%) males and 147 (93.04%) females. the mean age of unvaccinated patients was 60.4 and of vaccinated patients was 63.89 years. the mean duration of symptoms was 9.41 and 9.89 days of unvaccinated and vaccinated groups, respectively. detailed information is demonstrated in table 2. figure 1 shows the patient outcome according to vaccination status. the comparison of d-dimer level among vaccinated and unvaccinated patients showed a significant higher level of d-dimer among patients who received sinopharm vaccine (table 1, figure 2). the most frequent sign and symptoms were sob, fever, and cough as shown in table 4. in term of frequency of vomiting and flu like symptoms between vaccinated subgroups and unvaccinated cases. pfizer vaccine was significantly associated with vomiting (p = 0.0047) while sinopharm vaccine was associated with flu like illness (p = 0.032). (table 5). regarding the comorbidities, cvd followed by dm were the most common risk factors associated with covid-19 infection in both groups. malignancy and smoking were significant risk factors for covid-19 infection in the unvaccinated group (table 6). 378 j contemp med sci | vol. 8, no. 6, november-december 2022: 375–381 characterization of covid-19 hospitalized adult patients, vaccinated vs non-vaccinated in duhok province original p.a. mohammed et al. fig. 1 comparisons of patient outcomes according to vaccination status in hospitalized patients. fig. 2 comparisons of d-dimer among patients with different types of covid-19 vaccines. table 3. d-dimer level among hospitalized patients according to the vaccination status astrazeneca pfizer sinopharm unvaccinated p-value d-dimer 1631 (1085.58) 1684.42 (1246.27) 3030.17 (2598.60) 1305.58 (971.42) 0.0009 anova one-way tests were performed for statistical analyses. discussion covid-19 vaccine has proven to be an important tool in controlling sars-cov-2 pandemic and in reducing disease severity among hospitalized patients. we investigated 300 hospitalized covid-19 patients with a variety of demographic, clinical, and laboratory profiles. in the current study, the vaccination coverage was 9.33%, which was low in comparison to studies from iran,14 turkey,15 saudi arabia,16 and india.17 on the contrary, only few studies reported a lower vaccination coverage for e.g. peru (4.8%).18 this may be related to the lack of knowledge that covid-19 vaccines prevent or attenuate diseases severity, concerns about the vaccination’s composition, and side effects. consequently, raising awareness concerning vaccine safety and uptake are highly mandatory. the most common administered vaccine in this study was pfizer, which is in line to our previous study in evaluating covid-19 vaccination program in duhok.19 in the present study, the mean age of the hospitalized patients was 60.73 ± 15.83 yr, which was consistent with other studies.20 generally, older adults are more prone to infections with a more severe course because of increasing incidences of coexisting comorbid diseases, hence a weakened immune system.21 in our study, the unvaccinated females were more infected, while vaccinated males had higher rates of hospital admissions. ambrosino et al. in a review considered gender difference in covid-19 patients, documenting a higher rate in males21 that was in contrast to our finding, which may be clarified by the study being limited to hospitalized patients and a small sample size. whereas, the high rate of hospitalized vaccinated male patients could be linked to sex hormones, gender-related behavior, and differences in immunological function linked to the x chromosome.22 in our study, vaccinated patients had shorter hospital duration stays, which signifies the beneficial effect of covid-19 vaccines. in agreement, several studies documented this finding, indicating its efficacy in reducing burden on the healthcare system.23 in the current study, severe cases were predominated in both vaccinated and unvaccinated patients. this is justified according to our local covid-19 management guidelines, as we admit only severe and critical cases to the hospitals.24 hence, assessing covid-19 vaccine effectiveness on the general population was unfit as only hospitalized patients were considered. however, though not statistically significant, the frequency of continuous positive airway pressure (cpap) requirement was higher among unvaccinated group, which indicates the vaccine’s efficiency in reducing disease severity.25 scanning laboratory variable, d dimer was significantly higher among vaccinated patients (p = 0.0390). furthermore, comparing d-dimer among vaccine administered patients, sinopharm was significantly associated with higher level (p = 0.0009). our finding was in contrast to studies from thailand26 and turkey.27 the exact elucidation is dense at the moment; however, the higher d dimer level among sinopharm patients might be related to patients having had received the vaccine at the beginning of covid-19 vaccine campaign,7,28 having its effectiveness faded due to the long duration since patients acquired the infection. in this study, the mortality rate was relatively high among both vaccinated (60.71%) and unvaccinated (54.41%) patients. on contrary, other studies reported lower death rates.29,30 the high mortality rate in the current study is attributed first, to the admission of severe and critical cases in our hospitals while other studies included mild, moderate, and severe cases; second our study period was coincident majorly with the delta variant, hence worse prognosis of this variant, whereas other studies included other variants. interestingly, we found that the incidence of vomiting and flu-like illness were higher among vaccinated than unvaccinated patients. vomiting and flu-like illness were significantly associated with pfizer (p = 0.0047) and sinopharm vaccines (p = 0.0328), respectively. there are reports about 379j contemp med sci | vol. 8, no. 6, november-december 2022: 375–381 p.a. mohammed et al. original characterization of covid-19 hospitalized adult patients, vaccinated vs non-vaccinated in duhok province table 4. comparisons of symptomatology between vaccinated and unvaccinated patients symptoms vaccine status p-value unvaccinated (n = 272) vaccinated (n = 28) number percentage number percentage fever 205 75.37 19 67.86 0.3843 chill 59 21.69 5 17.86 0.6372 rigor 32 11.76 2 7.14 0.4626 sob 272 100.00 28 100.00 na cough 175 64.34 19 67.86 0.7107 vomiting 32 11.76 8 28.57 0.0127 chest pain 70 25.74 4 14.29 0.1808 chest tightness 34 12.50 3 10.71 0.7844 sore throat 39 14.34 3 10.71 0.5987 voice change 27 9.93 2 7.14 0.6351 loss of appetite 68 25.00 8 28.57 0.6791 abdominal pain 14 5.15 1 3.57 0.7157 flu-like illness 27 9.93 8 28.57 0.0034 epigastric pain 42 15.44 3 10.71 0.5048 constipation 45 16.54 5 17.86 0.8591 headache 36 13.24 3 10.71 0.7057 interscapular pain 28 10.29 1 3.57 0.2517 nasal obstruction 18 6.62 0 0.00 0.1603 loin pain 12 4.04 1 3.57 0.8353 general bodyache 55 20.22 5 17.86 0.7659 fatigue 95 34.93 9 32.14 0.7682 anosmia 26 9.56 3 10.71 0.4838 parosmia 26 9.56 3 10.71 0.8438 burning 10 3.68 1 3.57 0.9775 ageusia 3 1.10 0 0.00 0.5765 bitter taste 30 11.03 2 7.14 0.5258 dry mouth 48 17.65 3 10.71 0.3524 eye pain 18 6.62 1 3.57 0.5286 sweating 36 13.24 4 14.29 0.8763 nausea 20 7.35 4 14.29 0.1979 backache 22 8.09 3 10.71 0.6321 joints pain 34 12.50 4 14.29 0.7868 diarrhea 25 9.19 1 3.57 0.3142 table 5. prevalence of vomiting and flu like symptoms with types of vaccinations symptoms type of vaccination no (%) p-value astrazeneca pfizer sinopharm unvaccinated vomiting no yes or (95%ci) 7 (100) 0 (0.00) infinity 9 (60.00) 6 (40.00) 0.2 (0.07-0.6) 4 (66.67) 2 (33.33) 0.27 (0.05-0.51) 240 (88.24) 32 (11.76) reference 0.0047 flu-like illness no yes or (95%ci) 5 (71.43) 2 (28.57) 0.28 (0.05-1.49) 11 (73.33) 4 (26.67) 0.3 (0.09-1.02) 4 (66.67) 2 (33.33) 0.22 (0.04-1.26) 245 (90.07) 27 (9.93) reference 0.0328 pearson chi-squared test was performed for statistical analyses. or, odd ratio; ci, confidence interval. 380 j contemp med sci | vol. 8, no. 6, november-december 2022: 375–381 characterization of covid-19 hospitalized adult patients, vaccinated vs non-vaccinated in duhok province original p.a. mohammed et al. table 6. association of comorbidities with vaccination status among hospitalized patients risk factors vaccine status p-value (two-sided) unvaccinated (n = 272) vaccinated (n = 28) number percentage number percentage dm 71 89.87 8 10.13 0.7776 cvd 149 90.85 15 9.15 0.9925 preexisting lung disease 13 92.86 1 7.14 0.7729 hookah 2 100.00 0 0.00 0.6489 malignancy 7 70.00 3 30.00 0.0223 obesity 22 100.00 0 0.00 0.1180 immunosuppressive 8 100 0 0.0 0.4522 ckd 18 100.00 0 0.00 0.1603 smoking 7 70.00 3 30.00 0.0223 pregnancy 2 100.00 0 0.00 0.6489 alcoholic 1 50 1 50 0.8824 liver cirrhosis 3 100.00 0 0.00 0.5765 pearson chi-squared tests were performed for statistical analsyes. dm, diabetese mellitus; cvd, cardiovascular disease; ckd, chronic kidney disease. gastroparesis manifested as nausea and vomiting following pfizer vaccine administration.31 the more frequent flu-like illness among sinopharm receivers could be attributed to the faded effect of this vaccine among patients as explained earlier. therefore, they were presented with this illness as a frequent symptom of covid-19. in the present study, considering comorbidities, smoking and malignancy were significant risk factors for covid-19 infection in unvaccinated patients. smoking increases risk for acute respiratory infection in general, and increases entry of sars-cov-2 to host by upregulation of the angiotensin converting enzyme 2 (ace2) receptor in particular.32 whereas, malignancy in addition to chemotherapy regimens can weaken immune cells and create immunosuppressive state to patients, hence increase susceptibility to the infection.33 the study had several limitations, first, the study period was limited to a few months, and accordingly this could be a barrier for evaluating the true prevalence of the circulating strain. second, the study depended on the outbreak magnitude, so the disease declining incidence often had an impact on sample size. conclusion the sars-cov-2 pandemic can be controlled with the use of the covid-19 vaccine, which has also been shown to lessen the severity of illness in hospitalized patients, we looked at 300 covid-19 patients who were hospitalized and had different demographic, clinical, and laboratory profiles. in this study the majority of population were unvaccinated. among those who had received vaccinations, pfizer was most popular. the majority of cases were old ages. the unvaccinated females were more infected, while vaccinated males had higher rates of hospital admissions. the unvaccinated group had longer duration in hospital stay. about three quarter of study population were severe cases. d dimer was significantly higher among vaccinated patients (p = 0.0390). according to the data, the mortality rate was relatively high among both groups. regarding the clinical characteristics, vomiting and flu-like illness showed higher prevalence in vaccinated patients with significant difference. in terms of comorbidities, smoking and malignancy were significant risk factors for covid-19 infection in unvaccinated patients. the presence of a covid-19 vaccine and the proper implementation of a worldwide vaccination campaign are essential in the fight against a public health emergency like a covid-19 pandemic. abbreviations who: world health organization, covid-19: coronavirus disease 2019, sars cov 2: severe acute respiratory syndrome coronavirus 2, dm: diabetes mellitus, cvd: cardiovascular diseases, ards: acute respiratory distress syndrome, cbc: complete blood count, crp: c-reactive protein, ldh: lactate dehydrogenase, mrna: messenger ribonucleic acid, fda: food and drug administration, pcr: polymerase chain reaction, naat: nucleic acid amplification test, spo2: saturation of peripheral oxygen, pao2/fio2: partial pressure of oxygen in arterial blood /fraction of inspired oxygen, cdc: centers for disease control and prevention, mmwr: morbidity and mortality weekly, ci: confidence interval, sd: standard deviation, or: odd ratio, sob: shortness of breath, ckd: chronic kidney disease, us: united states, moh: ministry of health, cpap: continuous positive airway pressure, ace2: angiotensin converting enzyme 2. acknowledgments special thanks to all the doctors and laboratory personnel at duhok covid-19 and lalav hospitals, who assisted me in completing data collection. conflict of interest the authors affirm that they do not have any conflict of interests.  381j contemp med sci | vol. 8, no. 6, november-december 2022: 375–381 p.a. mohammed et al. original characterization of covid-19 hospitalized adult patients, vaccinated vs non-vaccinated in duhok province references 1. garcía-basteiro al, chaccour c, guinovart c, llupià a, brew j, trilla a, et al. monitoring the covid-19 epidemic in the context of widespread local transmission. the lancet respiratory medicine. 2020;8(5):440–2. doi: 10.1016/s2213-2600(20)30162-4. https://pubmed.ncbi.nlm.nih. gov/32247325/ 2. lotfi m, hamblin mr, rezaei n. covid-19: transmission, prevention, and potential therapeutic opportunities. clinica chimica acta; international journal of clinical chemistry. 2020;508:254–66. doi: 10.1016/j. cca.2020.05.044. https://pubmed.ncbi.nlm.nih.gov/32474009/ 3. vetter p, vu dl, l’huillier ag, schibler m, kaiser l, jacquerioz f. clinical features of covid-19. british medical journal publishing group; 2020. 4. chen n, zhou m, dong x, qu j, gong f, han y, et al. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet. 2020;395(10223):507–13. 5. ahn d-g, shin h-j, kim m-h, lee s, kim h-s, myoung j, et al. current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019 (covid-19). 2020. 6. pourbagheri-sigaroodi a, bashash d, fateh f, abolghasemi h. laboratory findings in covid-19 diagnosis and prognosis. clinica chimica acta. 2020;510:475–82. 7. abdulkader m, merza m. the evaluation of covid-19 vaccination program in duhok province, iraqi kurdistan: challenges and solutions. journal of research and health. 2022;12(4):2–2. 8. food and drug administration (fda). emergency use authorization, list of covid-19 vaccines authorized by the fda [available from: https://www.fda. gov.ph/list-of-fda-issued-emergency-use-authorization/. 9. almufty hb, mohammed sa, abdullah am, merza ma. potential adverse effects of covid19 vaccines among iraqi population; a comparison between the three available vaccines in iraq; a retrospective cross-sectional study. diabetes & metabolic syndrome. 2021;15(5):102207. doi: 10.1016/j. dsx.2021.102207. https://pubmed.ncbi.nlm.nih.gov/34280733/ 10. tenforde mw, patel mm, ginde aa, douin dj, talbot hk, casey jd, et al. effectiveness of sars-cov-2 mrna vaccines for preventing covid-19 hospitalizations in the united states. medrxiv. 2021. 11. almufty hb, mamani mma, ali ah, merza ma. covid‐19 vaccine breakthrough infection among fully vaccinated healthcare workers in duhok governorate, iraqi kurdistan: a retrospective cohort study. journal of medical virology. 2022. 12. nih. clinical spectrum of sars-cov-2 infection. national institute of health. 2022. https://www.covid19treatmentguidelines.nih.gov/overview/clinicalspectrum/ 13. moline hl, whitaker m, deng l, rhodes jc, milucky j, pham h, et al. effectiveness of covid-19 vaccines in preventing hospitalization among adults aged ≥ 65 years—covid-net, 13 states, february–april 2021. morbidity and mortality weekly report. 2021;70(32):1088. 14. binesh m, pahlevanian a, rahimi s, ahmadizadeh z. relationship between covid-19-related factors and self-management behaviors in people with type-2 diabetes: a cross-sectional study. middle east journal of rehabilitation and health studies. 2022;9(3). 15. karagun b, evran m, odabas f, akkus g, kurtaran b, sert m, et al. awareness of vaccination against respiratory tract diseases, including pneumonia, influenza, and covid-19 in patients with diabetes mellitus. international journal of clinical practice. 2022;2022. 16. tourkmani am, bin rsheed am, aleissa ms, alqahtani sm, alotaibi af, almujil ms, et al. prevalence of covid-19 infection among patients with diabetes and their vaccination coverage status in saudi arabia: a crosssectional analysis from a hospital-based diabetes registry. vaccines. 2022;10(2):310. 17. nachimuthu s, viswanathan v. trend in covid-19 vaccination among people with diabetes: a short study from india. diabetes & metabolic syndrome. 2021;15(4):102190. 18. valladares-garrido mj, zeña-ñañez s, peralta ci, puicón-suárez jb, díazvélez c, failoc-rojas ve. covid-19 vaccine effectiveness at a referral hospital in northern peru: a retrospective cohort study. vaccines. 2022;10(5):812. 19. abdulkader ma, merza ma. the evaluation of covid-19 vaccination program in duhok province, iraqi kurdistan: challenges and solutions. journal of research & health. 2022;12(4):219–226. http://jrh.gmu.ac.ir/ browse.php?a_id=2070&sid=1&slc_lang=en&ftxt=0 20. al houri hn, al-tarcheh h, zahra e, al-tarcheh a, armashi h, alhalabi m. clinical characteristics and prognosis of covid-19 patients in syria: a cross-sectional multicenter study. annals of medicine and surgery (2012). 2022;78:103816. doi: 10.1016/j.amsu.2022.103816. https://pubmed.ncbi. nlm.nih.gov/35620045/ 21. ambrosino i, barbagelata e, ortona e, ruggieri a, massiah g, giannico ov, et al. gender differences in patients with covid-19: a narrative review. monaldi archives for chest disease. 2020;90(2). 22. lakbar i, luque-paz d, mege jl, einav s, leone m. covid-19 gender susceptibility and outcomes: a systematic review. plos one. 2020;15(11):e0241827. doi: 10.1371/journal.pone.0241827. https://pubmed. ncbi.nlm.nih.gov/33141872/ 23. aldamen t, asad m, yaqoub m, alhawaratt m, aqel a, asad mm. effectiveness of vaccination: hospital admission and length of stay. iproceedings. 2022;8(1):e36363. 24. merza ma, aswad sm, sulaiman hm, abdulah dm, rasheed ws, taib ni. clinical and epidemiological characteristics and outcomes of coronavirus disease-19 patients in a large longitudinal study. international journal of health sciences. 2021;15(4):29. 25. christie a, henley sj, mattocks l, fernando r, lansky a, ahmad fb, et al. decreases in covid-19 cases, emergency department visits, hospital admissions, and deaths among older adults following the introduction of covid-19 vaccine—united states, september 6, 2020–may 1, 2021. morbidity and mortality weekly report. 2021;70(23):858. 26. uaprasert n, watanaboonyongcharoen p, vichitratchaneekorn r, trithiphen s, akkawat b, sukperm a, et al. prevalence of thrombocytopenia, antiplatelet factor 4 antibodies and d-dimer elevation in thai people after chadox1 ncov-19 vaccination. research and practice in thrombosis and haemostasis. 2021;5(6):e12580. doi: 10.1002/rth2.12580. https://pubmed. ncbi.nlm.nih.gov/34568726/ 27. guner g, yurumez y, durmus e, guneysu f, aslan n. observation of covid-19 vaccinated and unvaccinated patients. research and clinical medicine journal. 2020(4). https://www.resclinmed.eu/public/data_files/ articles/146/article_146.pdf 28. almufty hb, mohammed sa, abdullah am, merza ma. potential adverse effects of covid19 vaccines among iraqi population; a comparison between the three available vaccines in iraq; a retrospective cross-sectional study. diabetes & metabolic syndrome: clinical research & reviews. 2021;15(5):102207. 29. havers fp, pham h, taylor ca, whitaker m, patel k, anglin o, et al. covid-19associated hospitalizations among vaccinated and unvaccinated adults ≥ 18 years–covid-net, 13 states, january 1–july 24, 2021. medrxiv. 2021. 30. barry m, almohaya a, alhijji a, akkielah l, alrajhi a, almajid f, et al. clinical characteristics and outcome of hospitalized covid-19 patients in a mers-cov endemic area. journal of epidemiology and global health. 2020;10(3):214. 31. scott j, anderson j, mallak n, beitinjaneh b, wei k, otaki f. gastroparesis after pfizer-biontech covid-19 vaccination. the american journal of gastroenterology. 2021;116(11):2300. doi: 10.14309/ajg.0000000000001354. https://pubmed.ncbi.nlm.nih.gov/34187985/ 32. van zyl-smit rn, richards g, leone ft. tobacco smoking and covid-19 infection. the lancet respiratory medicine. 2020;8(7):664–5. doi: 10.1016/ s2213-2600(20)30239-3. https://pubmed.ncbi.nlm.nih.gov/32464099/ 33. rashedi j, mahdavi poor b, asgharzadeh v, pourostadi m, samadi kafil h, vegari a, et al. risk factors for covid-19. le infezioni in medicina. 2020;28(4):469-74. https://pubmed.ncbi.nlm.nih.gov/33257620/ this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1272 https://pubmed.ncbi.nlm.nih.gov/32247325/ https://pubmed.ncbi.nlm.nih.gov/32247325/ https://pubmed.ncbi.nlm.nih.gov/32474009/ https://www.fda.gov.ph/list-of-fda-issued-emergency-use-authorization/ https://www.fda.gov.ph/list-of-fda-issued-emergency-use-authorization/ https://pubmed.ncbi.nlm.nih.gov/34280733/ https://www.covid19treatmentguidelines.nih.gov/overview/clinical-spectrum/ https://www.covid19treatmentguidelines.nih.gov/overview/clinical-spectrum/ http://jrh.gmu.ac.ir/browse.php?a_id=2070&sid=1&slc_lang=en&ftxt=0 http://jrh.gmu.ac.ir/browse.php?a_id=2070&sid=1&slc_lang=en&ftxt=0 https://pubmed.ncbi.nlm.nih.gov/35620045/ https://pubmed.ncbi.nlm.nih.gov/35620045/ https://pubmed.ncbi.nlm.nih.gov/33141872/ https://pubmed.ncbi.nlm.nih.gov/33141872/ https://pubmed.ncbi.nlm.nih.gov/34568726/ https://pubmed.ncbi.nlm.nih.gov/34568726/ https://www.resclinmed.eu/public/data_files/articles/146/article_146.pdf https://www.resclinmed.eu/public/data_files/articles/146/article_146.pdf https://pubmed.ncbi.nlm.nih.gov/34187985/ https://pubmed.ncbi.nlm.nih.gov/32464099/ https://pubmed.ncbi.nlm.nih.gov/33257620/ case report 79j contemp med sci | vol. 8, no. 1, january-february 2022: 79–81 efficacy of amphotericin b on covid-19: a case report study shahrokh mehrpisheh1, roya farhadi1, vajihe ghaffari saravi1, azadeh memarian2*, farahnaz nikkhah3 1department of neonatology, faculty of medicine, mazandaran university of medical sciences, sari, iran. 2department of forensic medicine, faculty of medicine, mazandaran university of medical sciences, sari, iran. 3rasool akram medical complex clinical research development center (rcrdc), iran university of medical sciences, tehran, iran. *correspondence to: azadeh memarian (e-mail: memarian.a@iums.ac.ir) (submitted: 08 january 2022 – revised version received: 21 january 2022 – accepted: 01 february 2022 – published online: 26 february 2022) abstract the role of amphotericin b (amb) as an antiviral drug against some enveloped viruses has been studied in previous researches. coronavirus is an envelope, non-segmented and positive-sense rna virus which may be targeted by amb. our case was a 34-day-old female child with a 4-days history of low consciousness and dry cough, which gradually became productive. the infant was cyanotic at the admission time that transferred to the neonatal intensive care unit (nicu). after discharge, we were informed that result of pcr (polymerase chain reaction) test was positive which has been first reported as a false negative result. so the results drove our attention to that amphotericin b may cause a dramatic response to the coronavirus. because of the crucial role of the immune system in viral clearance, the amb as an immune response and pro-inflammatory stimulator may pave the way for preventing invasion in viral infections such as covid-19. keywords: covid-19, amphotericin b (amb), antiviral drug, severe dyspnea, intensive care units, neonatal introduction the novel coronavirus (2019-ncov) also named sars-cov-2 or coronavirus disease-2019 (covid-19), is an acute infectious disease first manifested in wuhan, china on december 26, 2019.1,2 patients with covid-19 often experience severe respiratory syndrome with the clinical signs of fever, fatigue, dizziness, dry cough, and gradually develop severe dyspnea.2 the diagnostic approaches for covid-19 have mainly relied on reverse transcription-polymerase chain reaction (rtpcr) or gene sequencing of sputum, throat swabs or lower respiratory tract excretions.1-3 the best preventive approach is to limit human-to-human transmission by early detection, isolation and caring for patients.2 patients with influenza who developed severe acute respiratory distress syndrome (ards) may also present invasive pulmonary aspergillosis (ipa), as the main leading cause of prolonged hospitalization and mortality rate.4,5 co-infections with bacteria and fungi have more occurred in 40% of hospitalized patients with covid-19 who developed ards, which is often associated with the development of ipa in patients taking corticosteroid drugs.6,7 it is not currently known whether covid-19-associated ipa antifungal therapy becomes a survival profit, however, diagnosis in most cases must result in early antifungal therapy.4 liposomal amphotericin b has been used as the ancient agent and treatment choice in the first-line therapy for various fungal infections specially ipa treatment in icu.8 amphotericin b (amb) belongs to the polyene group with broad-spectrum in vitro and in vivo antifungal activities and a valuable pharmaceutical profile which has low fungal resistance.9,10 since amb has a high affinity to ergosterol than cholesterol, it is effective against fungi and single-cell protozoa.11 several studies have been focused on the different mechanisms of action and the potential therapeutic effect of amb derivatives against some enveloped viruses, including human immunodeficiency virus (hiv),12 herpes simplex virus (hsv),13 japanese encephalitis virus and rubella virus.14,15 it has been expected that amb would change viral membrane integrity and envelope structure as well as host immunomodulatory effects.9,15 since coronaviruses belong to the enveloped viruses with positive-sense rna nucleic acid and club-like spikes, amb, may pave the way for designing a novel therapeutic option in covid-19 treatment. to the best of our knowledge, there is limited literature on the efficacy of amb against viral infection and no research is available on the role of amb in pediatrics with covid-19, so, we decided to share our experiences on a case report of the iranian 34-day-old child with signs of suspected covid-19 which may be of interest to health care workers who involved in health systems all over the world. case report the patient was a 34-day-old female infant who was admitted to the emergency department (ed) due to decreased level of consciousness. she had a dry cough from 4 days before the admission, which gradually became productive. she had no fever, shortness of breath, seizures, poor feeding and had the excretion of urine and feces. at the time of admission, the infant was cyanotic. her weight was less than 2,500 g (1900 g and the birth weight of 3000 g) and she was less than two months old, therefore, she was admitted to nicu. she had an opium-addicted mother who gave him opium in a lentil size since birth. the infant had a bilateral cleft lip and palate. her parents were non-appointed and both had a positive history of cough. no ecchymosis or purpura was found on physical exam, and she had no bulging or sunken fontanelles and her ears were normal. on physical exam of her eyes, the conjunctiva was swollen and the pupils had bilateral meiosis. the abdomen was soft and had no organomegaly. the genitals were girlish and normal. the infant was completely hypotensive with no response to stimulation. in the clinical record, she was reported a term neonate. totally, at the time of admission, she was cyanotic, unconscious, motionless, and intubated that was given cardiac massage and underwent salvage. in nicu, naloxone was administered and treatment with meropenem and vancomycin was continued, she was then intubated and underwent ventilation. issn 2413-0516 efficacy of amphotericin b on covid-19 case report s. mehrpisheh et al. 80 j contemp med sci | vol. 8, no. 1, january-february 2022: 79–81 during the course of hospitalization, the patient developed convulsive movements that started phenobarbital and then underwent echocardiography, which she showed small patent ductus arteriosus (pda), mild to moderate myocardial infarction (mi), and mild tricuspid regurgitation (tr). in electroencephalogram (eeg) monitoring, the epileptic waves were reported. extensive multicystic encephalomalacia was reported in brain computed tomography (ct). the test result was negative for covid-19, while bilateral lung opacity was seen on chest x-ray (cxr). because of non-response to 3-week-treatment with meropenem and vancomycin, the drugs were switched to cefepime and clindamycin, and amphotericin b was then started about 6 days later. the result of fungal test was negative but due to the organic response to amphotericin b, the drug continued and the ventilator set-up was rapidly reduced. the infant was disconnected from the ventilation device. the milk started and she was finally discharged with an improved general condition. after discharge, we were informed that result of pcr (polymerase chain reaction) test was positive which has been first reported as a false negative result. so the results drove our attention to that amphotericin b may cause a dramatic response to the coronavirus. discussion severe acute respiratory syndrome coronavirus (sars-cov-2) occurred in wuhan, hubei province, china, in early december 2019 in patients who had a history of entrancing to the huanan seafood wholesale market.16 the novel covid-19 caused by an un-segmented (+rna) coronaviruses presents the continuous spreading with arising mortality rate in the world.17 therefore, designing more effective therapeutic strategies and targeted therapies seem highly necessary to avert the spread of the upsetting pandemic worldwide. so far, there is no specific drug to directly target this novel virus and support organ failure in seriously ill patients which is considered as the main phase in clinical management.18 however, there are some marketed drugs available to avert ards as a main outcome of covid-19 in combination with nutrient supplements.18 in addition, applying drugs such as chloroquine and hydroxychloroquine in some covid-19 patients may be associated with many side effects.19 our case received treatment with naloxone followed by meropenem and vancomycin, both of them are antimicrobial agents which improve outcomes and shorten the duration of hospitalization in some cases with respiratory infections including pneumonia.20 recently, the clinical significance of therapeutic combination regimens in the management and monitoring of patients who suffer from covid-19 pneumonia has been considered.20 the use of the different combination regimens has been indicated to improve outcomes of covid-19 patients; the regimens include meropenem, levofloxacin, vancomycin as the antibacterial agents, and hydroxychloroquine, and oseltamivir as the antiviral agents.20 in addition to current therapeutic strategies, amb has become the center of attention recently as a possible antiviral agent for covid-19 treatment.15 therefore, its optimal therapeutic application is required to be more clarified by clinical trials. it seemed that amb has a more toxic effect on the virion of enveloped viruses than on the host cell, which is probably due to the differences and changes in the viral proteins derived from the host cell composition.15 therefore, these morphological and biological aspects of the viral envelope make it a suitable target for designing a novel antiviral therapy. in our case report, the patient did not show any improvement in the clinical outcome after long-term use of meropenem and vancomycin resulted in switching to cefepime and clindamycin, and finally amb. we observed the considerably improved outcomes immediately after starting the amb which may be due to the proper efficacy of this drug. moreover, amb has many indicative characteristics including accessibility, rare resistance, as well as broad-spectrum activity against many fungal and microbial infections, and viral rna nucleic acid and envelopes; therefore, designing further research studies and clinical trials seems necessary to fully recognize the role of amb in patients with covid-19. to the best of our knowledge, most enveloped viruses targeted by amb may have similar complexity and viral-specific proteins; moreover, considering the crucial role of the immune system in viral clearance, the amb as an immune response and pro-inflammatory stimulator may pave the way to prevent invasion in viral infections such as covid-19. the major aspects in the recovery of patients with covid-19 are establishing proper supportive maintenance and antibiotics-antiviral combination therapies which may take a long time to evaluate, and choosing specific antiviral agents as suitable therapeutic regimes for novel covid-19. relying on the results of previous studies about the unique characteristics and efficiency of amb against various enveloped viruses, the application of amb alone or in combination regimens may be effective in the reduction of drug side effects and improved prognosis of patients with covid-19 pneumonia. abbreviations amb: amphotericin b nicu: neonatal intensive care unit 2019-ncov: 2019-novel coronavirus rt-pcr: reverse transcription-polymerase chain reaction ards: acute respiratory distress syndrome hiv: human immunodeficiency virus hsv: herpes simplex virus ed: emergency department pda: patent ductus arteriosus mi: myocardial infarction tr: tricuspid regurgitation eeg: electroencephalogram ct: brain computed tomography cr: chain reaction sars-cov-2: severe acute respiratory syndrome coronavirus ethics approval institutional review board approval for case report was not required at our institution at the time of the study. written informed consent was obtained from the patient for publication of this case report. to keeping ethical principles, the name of the patient was not pointed in the paper and the rights of the subject were protected. the patient received treatment consistent with the current standard of care. s. mehrpisheh et al. case report efficacy of amphotericin b on covid-19 81j contemp med sci | vol. 8, no. 1, january-february 2022: 79–81 acknowledgments the authors thank the rasool akram medical complex clinical research development center (rcrdc) for its technical and editorial assists. conflict of interest the author reports no conflicts of interest in this work. consent for publication written informed consent was obtained from the patient’s legal guardian for publication of this case report and any accompanying images. informed consent written informed consent was obtained from the patient’s legal guardian for participation of the infant in the study. data availability statements the data that support the findings of this study are available from corresponding author on reasonable request. funding the authors declare that this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.  references 1. liu k-c, xu p, lv w-f, qiu x-h, yao j-l, jin-feng g. ct manifestations of coronavirus disease-2019: a retrospective analysis of 73 cases by disease severity. eur j radiol. 2020;126:108941. doi: 10.1016/j.ejrad.2020.108941. 2. liu r, han h, liu f, zhihua lv, kailang wu, yingle liu, et al. positive rate of rt-pcr detection of sars-cov-2 infection in 4880 cases from one hospital in wuhan, china, from jan to feb 2020. clinica chimica acta. 2020;505: 172-5. doi: 10.1016/j.cca.2020.03.009. 3. li g, fan y, lai y, tiantian han, zonghui li, peiwen zhou, et al. coronavirus infections and immune responses. j medl virol. 2020;92(4):424-32. doi: 10.1002/jmv.25685 4. arastehfar a, carvalho a, van de veerdonk fl, jeffrey d jenks, philipp koehler, robert krause, et al. covid-19 associated pulmonary aspergillosis (capa)—from immunology to treatment. j fungi. 2020;6(2):91. doi: 10.3390/jof6020091. 5. li e, knight jm, wu y, amber luong, antony rodriguez, farrah kheradmand, et al. airway mycosis in allergic airway disease. adv immunol. 2019;142: 85-140. doi: 10.1016/bs.ai.2019.05.002. 6. schauwvlieghe af, rijnders bj, philips n, verwijs r, vanderbeke l , van tienen c, et al. invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study. lancet respir med. 2018;6(10):782-92. doi: 10.1016/s2213-2600(18)30274-1 7. wauters j, baar i, meersseman p, meersseman w, dams k, de paep r, et al. invasive pulmonary aspergillosis is a frequent complication of critically ill h1n1 patients: a retrospective study. intensive care med. 2012;38(11):1761-68. doi: 10.1007/s00134-012-2673-2. 8. patterson tf, thompson iii gr, denning dw, fishman ja, hadley s, herbrecht r, et al. practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the infectious diseases society of america. clin infect dis. 2016;63(4):e1-e60. doi: 10.1093/cid/ciw326. 9. al-khikani f, al-janabi a. topical amphotericin b formulas: promising new application. intj med sci curr res. 2019;2:187-96. 10. lanternier f, lortholary o. liposomal amphotericin b: what is its role in 2008? clin microbiol infect. 2008;14 suppl 4:71-83. doi: 10.1111/j.14690691.2008.01984.x. 11. sangalli-leite f, scorzoni l, mesa-arango ac, casas c, herrero e, mendes gianinni mjs, et al. amphotericin b mediates killing in cryptococcus neoformans through the induction of a strong oxidative burst. microbes infec. 2011;13(5):457-67. doi: 10.1016/j.micinf.2011.01.015. 12. konopka k, guo ls, düzgüneş n. anti-hiv activity of amphotericin b-cholesteryl sulfate colloidal dispersion in vitro. antiviral res. 1999; 42(3):197-209. doi: 10.1016/s0166-3542(99)00028-5 13. shiota h, jones b, schaffner c. anti-herpes simplex virus (hsv) effect of amphotericin b methyl ester in vivo. antimicrob agents chemother. 1978; 13(2):199–204. doi: 10.1128/aac.13.2.199. 14. kim h, kim s-j, park s-n, oh j-w. antiviral effect of amphotericin b on japanese encephalitis virus replication. j microbiol biotechnol. 2004;14(1):121-127. https://www.koreascience.or.kr/article/ jako200411923002271. 15. al-khikani fho. amphotericin b as antiviral drug: possible efficacy against covid-19. ann thorac med. 2020;15(3):118 -24. doi: 10.4103/atm.atm_147_20 16. andersen kg, rambaut a, lipkin wi, holmes ec, garry rf. the proximal origin of sars-cov-2. nature med. 2020;26(4):450-52. doi: 10.1038/s41591020-0820-9. 17. fehr ar, perlman s. coronaviruses: an overview of their replication and pathogenesis. methods mol biol 2015;1282:1-23. doi: 10.1007/978-1-49392438-7_1. 18. zumla a, azhar ei, arabi y, alotaibi b, rao m, mccloskey b, et al. hostdirected therapies for improving poor treatment outcomes associated with the middle east respiratory syndrome coronavirus infections. int j infect dis. 2015;40:71-4. doi: 10.1016/j.ijid.2015.09.005 19. wang m, cao r, zhang l, yang x, liu j, xu m, et al. remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro. cell res. 2020;30(3):269-71. doi: 10.1038/s41422-0200282-0. 20. vahedi e, ghanei m, ghazvini a, azadi h, izadi m, panahi y, et al. the clinical value of two combination regimens in the management of patients suffering from covid-19 pneumonia: a single centered, retrospective, observational study. daru j pharm sci. 2020:1-10. doi: 10.1007/s40199-020-00353-w. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1115 140 j contemp med sci | vol. 8, no. 2, march-april 2022: 140–146 original clinical features and upper airway symptoms in association with severity and outcome in patients with covid-19 furaq hussain hadi1, iman jabbar kadhim2, falah abdulhasan deli3,* 1department of internal medicine, al-kafeel general hospital, ministry of health, kerbala, iraq. 2department of medical microbiology, faculty of medicine, university of kufa, najaf, iraq. 3department of internal medicine, faculty of medicine, university of kufa, najaf, iraq. *correspondence to: falah abdulhasan deli (e-mail: falah.alhasnawi@uokufa.edu.iq) (submitted: 05 december 2021 – revised version received: 27 december 2021 – accepted: 12 january 2022 – published online: 26 april 2022) abstract objectives: to assess the relationship between the early occurrence of upper respiratory tract symptoms and the severity of sars-cov-2 infection. methods: a cohort observational study had been conducted on a total of 140 patients [60 mild, 40 moderate, 40 severe], diagnosed with sars-cov-2 between 4th of august and 31 of october 2020. patients diagnosed by pcr or chest ct scan or both of them. a full history was taken from the patients and data including the age of the patient, gender, occupation, residence, height, weight, history of previous comorbidities {cardiovascular, diabetes mellitus, hypertension, chronic respiratory disease, chronic renal disease, malignancy, and other diseases}. smoking and alcoholic history were also taken, clinical features {loss of smell/taste, sore throat, rhinorrhea, fever, cough, shortness of breath, headache, fatigue, myalgia, arthralgia, diarrhea, and vomiting} and temperature, spo 2 , investigations, the need for respiratory support {o 2 , non-invasive ventilation, invasive ventilation} and any complications developed during illness. results: mean age of the patients was 51 (range: 17–82) males were dominant; (57.1%) with male to female ratio of 1.33 to one, out of the 140 covid-19 patients, 63 (45%) had upper respiratory symptoms. regarding biomarkers of severity only s. ldh was significantly lower in cases who did have compared to those who did not have upper respiratory symptoms, 1.3 ± 1.4 vs. 1.7 ± 1.3, respectively. the mean spo 2 % was significantly higher in patients with upper respiratory symptoms compared to those without. percent of pulmonary damage was significantly lower in patients with upper respiratory symptom compared to those without. mortalities were significantly lower in patients with upper respiratory symptoms compared to those without; among the 113 patients with upper respiratory symptom compared only 7 (6.2%) died compared to 7 out of 27 (25.9%) patients with no upper respiratory symptom. conclusion: early occurrence of upper respiratory tract symptoms predicts less severe form of the disease. keywords: covid-19, anosmia, sars-cov-2, pandemics, iraq issn 2413-0516 introduction in the early of december 2019, sars-cov-2 was discovered in china, wuhan and leads to an ongoing pandemic disease.1,2,3 corona viruses are rna viruses and there are four groups of coronavirus virus, are identified; alpha coronavirus, beta coronavirus, gamma coronavirus and delta coronavirus, of these alpha and beta coronaviruses are known to infect the human being.4 sars-cov-1 was identified in 2002 in china and mers-cov was identified in 2013 in saudi arabia both are beta cov.5,6 sars-cov-2 enters the human cells through angiotensin converting enzyme 2 receptors.7,8 sars-cov-2 transmitted between humans by respiratory droplet of infected person [symptomatic or asymptomatic] coming in close (1 meter) or [aerosol particles] contact with person who are not infected with the virus, but this is not the only route of transmission indirect contact can also transmit the virus as touching surfaces contaminated with the virus and contact with mucous membranes of the mouth, nose or eye.9,10,11,12 the symptomatology of sars-cov-2 infection is wide and reflect different systems involvement, general symptoms as fever, malaise, fatigue, bone pain and back pain are prominent, respiratory symptoms like cough, chest tightness and dyspnea are the most important clinical manifestation, upper respiratory symptoms like loss of smell, loss of taste, sore throat and rhinorrhea are occurring next in order, other systems are not an exception, neurological like headache and confusion, gastrointestinal like anorexia, diarrhea and vomiting, ophthalmic like conjunctivitis and retinitis, cardiovascular like acute myocardial injury and atrial fibrillation, rheumatological like arthralgia.9 fever, cough and fatigue are the three most common manifestation of sars-cov-2.13,14 isolated sudden onset loss of smell is the fourth most common manifestation of the disease.15,16 surprisingly; sudden loss of smell/taste sense occurs without usual nasal symptoms {as sneezing, rhinorrhea, nasal obstruction and facial pain}, unlike other respiratory diseases caused by respiratory viruses {as influenza, rhinovirus, adenovirus}. there are well described but not fully understood cause of anosmia which is post viral olfactory disorder {pvod}, some physicians thought it is an inflammatory reaction of nasal mucosa while others thought the virus may damage the olfactory neuroepithelium or central olfactory pathway and transmitted to the brain directly {central nervous system including olfactory bulbs and olfactory cortex} with the development of micro vascular phenomenon and injury as micro bleeding and blood brain barrier break as demonstrated by mr imaging of patients with sars-cov-2 infection.17 patients with sars-cov-2 may have mild symptoms as fatigue, loss of smell with no radiological manifestation or moderate as fever, dry cough but no dyspnea with radiological manifestations less than 50% or severe as having respiratory symptoms of dyspnea, hypoxia, cough, with radiological manifestation that is more than 50%.9 https://orcid.org/0000-0002-7605-4529 mailto:falah.alhasnawi@uokufa.edu.iq 141j contemp med sci | vol. 8, no. 2, march-april 2022: 140–146 f.h. hadi et al. original clinical features and upper airway symptoms in association with severity and outcome in patients with covid-19 the diagnosis of sars-cov-2 is done by the detection of the virus by mean of real time-polymerase chain reaction {rtpcr} assay from nasopharyngeal swab of infected patients,18,19 false negative results may occur. chest radiography is used as initial imaging method while the computed tomography {ct-scan} is very important in the diagnosis, prognosis and management plan of the disease as it found more sensitive than rt-pcr for detecting sars-cov-2 {98% vs. 71%}. other laboratory tests are helpful in the assessment of severity and complications including: complete blood picture {cbc}, coagulation profile {d-dimer}, inflammatory markers {c-reactive protein, ferritin}, lactate dehydrogenase, creatine kinase, cardiac troponin and procalcitonin.20 increase white blood cell count, increase neutrophil count, decrease lymphocyte count, increase lactate dehydrogenase, increase creatinine, increase d-dimer, increase c-reactive protein, ct invasion of more than 50%, spo2 of less than 93%, are associated with severe disease, unfavorable outcomes and complications.20 also being old {>65 year}, male sex, bmi >35 kg/m2, have previous comorbidities {as hypertension, cardiovascular, diabetes mellitus, etc.} are also associated with severe disease. all these factors were included in our study to assess the severity of the disease and its association with the clinical features and upper respiratory symptom. aim of study in this study we assess the relationship between the early occurrence of upper respiratory tract symptoms and the severity of sars-cov-2 infection. patients and methods a cohort observational study had been conducted on a total of 140 patients {hospitalized and non-hospitalized} hospitalized patients in al-sadder teaching hospital in najaf ashraf province diagnosed with sars-cov-2 between 4th of august and 31 of october 2020. patients diagnosed by pcr or chest ct scan or both of them. a full history was taken from the patients including the age of the patient, gender, occupation, residence, height, weight, history of previous comorbidities {cardiovascular, diabetes mellitus, hypertension, chronic respiratory disease, chronic renal disease, malignancy, and other diseases}. smoking and alcoholic history were also taken, clinical features {loss of smell/taste, sore throat, rhinorrhea fever, cough, shortness of breath, headache, fatigue, myalgia, arthralgia, diarrhea, and vomiting} and temperature, spo2, the need for respiratory support {o2, non-invasive ventilation, invasive ventilation} and any complications developed during illness. chest ct scan was done for symptomatic patients either before confirming the diagnosis with pcr or after confirming the diagnosis with pcr to assess the severity of the disease and treat the patient accordingly. the patients were classified according to clinical presentations into:9 1-mild: the clinical symptoms were slight and no signs of pneumonia on radiological imaging. 2-moderate: symptoms of fever and respiratory tract symptoms and signs of pneumonia on radiological imaging. 3-severe: patients meet any of the following criteria: a-respiratory distress {respiratory rate > = 30 breath/min. b-blood oxygen saturation <93%. c-lung infiltrate >50% of lung field on radiological imaging. very few patients with moderate, or severe disease confirmed by ct scan refuse pcr, so, pcr was not done for these patients. patients were sent for crp, cbp {neutrophils, lymphocytes}, renal functions test, ferritin, lactate dehydrogenase and d-dimer. normal values of these biomarkers are: crp = <10 mg/l, ferritin = 20–230 ng/ml d-dimer = <400 ng/ml, ldh = 140–280 u/l. the duration of hospitalization and the outcome of the patients whether recovered or died was followed. ethical consideration: ethically this study was approved by the ethical committee of the iraqi board for medical specialization. statistical analysis data of the studied group were entered managed and analyzed using the statistical package for social sciences (spss) version 25. descriptive statistics presented as mean, standard deviation, frequencies and percentages according to the type of variables. to assess the relationship between categorical variables and severity of sars-cov-2, chi square test was applied. as an alternative, fisher’s exact test used when chi square was inapplicable. analysis of variances (anova) test used to compare means across the severity categories, kruskal-wallis analysis used when anova test couldn’t be applied (variable did not follow statistical normal distribution), student’s t and mann-whitney tests when applicable, used to compare parameters according to presence of respiratory symptoms. level of significance, p. value, of 0.05 or less considered significant. result a total of 140 sars-cov-2 patients were enrolled in this study with a mean age of 50.9 ± 15.1 (range: 17–82) males were dominant; (57.1%) with a male to female ratio of 1.33 to one, other demographic characteristics and distributions are shown in (table 1 and figure 1). table 1. demographic characteristics of the studied group variable age mean (sd) 50.9 (15.1) range 17–82 gender n (%) male 80 (57.1) female 60 (42.9) occupation n (%) employed 55 (39.3) unemployed 85 (60.7) marital status n (%) married 130 (92.9) unmarried* 10 (7.1) smoking n (%) smoker 24 (17.1) non-smoker 116 (82.9) bmi category n (%) normal 35 (25.0) overweight 71 (50.7) obese 34 (24.3) sd: standard deviation. mean (sd) bmi = 27.9 (4.1) (kg/m²). 142 j contemp med sci | vol. 8, no. 2, march-april 2022: 140–146 clinical features and upper airway symptoms in association with severity and outcome in patients with covid-19 original f.h. hadi et al. of those 140 patient, 40 had severe disease, 40 had moderate disease and 60 had mild disease. the association between chronic diseases from one side and severity of sars-cov-2 on the other side, no significant association was found between them, in all comparisons, p. value > 0.05), with exception of dm where significant association were found, among 37 patients with dm, only 8 had mild disease compared to 17 moderate and 12 severe disease, (p = 0.016). conversely, chronic use of medication was significantly associated with more severe disease where among 52 chronic medications users (anti-hypertensive, oral hypoglycemic agent, anti-ischemic…) only 21.2% had mild disease compared to 46.2% moderate and 32.7% severe disease, (p < 0.001), (table 2). among symptoms, presence of headache, sore throat, loss of smell, fatigue and myalgia was significantly associated with mild disease, (p < 0.05), while presence of shortness of breath was significantly associated with more severe disease, (p. value < 0.001), other symptoms did not show significant associations, (p > 0.05), 0020 (table 3). a significant inverse association was found between spo2 level and severe disease; patients with mild disease had the higher spo2 levels with a mean of 97.2 ± 1.1% compared to 92.7% ± 1.2% in moderate disease patients and 74.8% ± 11.3% in severe disease patients, (p. value < 0.001). similarly, lower temperature was significantly associated with mild disease, 38.1 ± 0.5°c compared to 38.7°c in moderate and severe cases, (p < 0.001), (table 4). as shown in table 5, ct-scan performed in 118 patients, among them a highly significant difference was found in percent of pulmonary damage reported by ct-scan, where mild cases had the lowest pulmonary damage (mean: 20.5% ± 8.8%) followed by moderate (mean: 36.9% ± 8.5%) and the larger percent of pulmonary damage in severe cases with a mean table 2. history of chronic diseases reported among the studied group (n = 140) chronic diseases and medication use total cases severity p. value (chi-square test/ fisher’s) mild (n = 60) moderate (n = 40) severe (n = 40) no. % no. % no. % hyper tension 50 15 30.0 16 32.0 19 38.0 0.057 ns dm 37 8 21.6 17 45.9 12 32.4 cvd 30 14 46.7 5 16.7 11 36.7 0.235 ns respiratory disease 7 2 28.6 2 28.6 3 42.9 0.640 ns renal disease 5 2 40.0 2 40.0 1 20.0 0.827 ns malignancy 2 0 0.0 0 0.0 2 100.0 0.079 ns hypothyroidism 2 0 0.0 1 50.0 1 50.0 0.467 ns rheumatoid arthritis 2 0 0.0 0 0.0 2 100.0 0.079 ns chronic use of medications 52 11 21.2 24 46.2 17 32.7 < 0.001 sig * some patients had more than one comorbidity. sig: significant association (0.05 or less), ns: not significant. fig. 1 age distribution of the studied group. table 3. frequency distribution of reported symptoms of the studied group according to severity of covid-19 (n = 140) symptom* severity p. value (chi-square test/fisher’s) mild (n = 60) moderate (n = 40) severe (n = 40) no. % no. % no. % fever 58 96.7 38 95.0 37 92.5 0.645 ns cough 56 93.3 38 95.0 35 87.5 0.415 ns headache 56 93.3 22 55.0 19 47.5 < 0.001 sig shortness of breath 23 38.3 36 90.0 36 90.0 < 0.001 sig sore throat 46 76.7 24 60.0 20 50.0 0.019 sig loss of smell 38 63.3 25 62.5 15 37.5 0.023 sig rhinorrhea 5 8.3 3 7.5 1 2.5 0.481 ns fatigue 44 73.3 19 47.5 25 62.5 0.032 sig myalgia 49 81.7 14 35.0 9 22.5 < 0.001 sig vomiting 19 31.7 8 20.0 5 12.5 0.072 ns arthralgia 8 13.3 8 20.0 13 32.5 0.068 ns diarrhea 3 5.0 7 17.5 2 5.0 0.058 ns *majority of patients had more than one symptom. sig: significant association (p = 0.05 or less), ns: not significant. table 4. mean values standard deviation (sd) and range of measured temperature and spo 2 of the studied group (n = 140) parameter statistics severity p. value (anova test) mild (n = 60) moderate (n = 40) severe (n = 40) spo 2 (%) mean ± sd 97.2 ± 1.1 92.7 ± 1.2 74.8 ± 11.3 < 0.001 sig range 95–99 90–94 50–88 temperature (°c) mean ± sd 38.1 ± 0.5 38.7 ± 0.7 38.7 ± 0.9 < 0.001 sig range 37.0–39.0 37.0–40.0 37.0–40.0 sd: standard deviation. sig: significant association (p = 0.05 or less), ns: not significant. 143j contemp med sci | vol. 8, no. 2, march-april 2022: 140–146 f.h. hadi et al. original clinical features and upper airway symptoms in association with severity and outcome in patients with covid-19 table 6. frequency distribution of hospitalization, duration of hospital stays and need for respiratory support among patients with moderate and severe disease moderate (n = 40) severe (n = 40) p. value hospitalization no. (%) 27 (67.5%) 40 (100.0) < 0.001 sig chi square duration hospital stay (day) mean ± sd (range) 4.3 ± 2.1 (2–8) 13.8 ± 8.2 (3–55) < 0.001 sig t test respiratory support no. (%) 23 (57.5) 40 (100.0) < 0.001 sig chi square sd: standard deviation, sig: significant difference sig: significant association (p = 0.05 or less), ns: not significant. table 7. n hematological wbc differential count regarding neutrophils & lymphocyte in pt. with covid-19 (n = 140) parameter severity p. value fisher’s test mild (n = 60) moderate (n = 40) severe (n = 40) no. % no. % no. % neutrophil count high 18 30.0 31 77.5 32 80.0 < 0.001 sig normal 42 70.0 9 22.5 8 20.0 lymphocyte count high 2 3.3 0 0.0 0 0.0 < 0.001 siglow 17 28.3 31 77.5 32 80.0 normal 41 68.3 9 22.5 8 20.0 sig: significant (0.05 or less). *result taken at presentation (before management). table 9. distribution of biomarkers according to disease severity (n = 140) parameter severity p. value mild (n = 60) moderate (n = 40) severe (n = 40) crp mean ± sd 3.0 ± 1.7 4.1 ± 3.5 7.3 ± 4.8 < 0.001 sig range 1–10 2–28 3–28 s. ferritin mean ± sd 1.2 ± 1.0 1.1 ± 1.1 4.2 ± 2.6 < 0.001 sig range 1–3 1–5 1–10 s. ldh mean ± sd 1.0 ± 1.0 1.0 ± 1.0 2.6 ± 1.4 < 0.001 sig range 1.0–2.0 1.0–3.0 1.0–6.0 d-dimer mean ± sd 1.3 ± 0.9 2.2 ± 1.6 9.0 ± 4.1 < 0.001 sig range 1–6.0 1–11.0 1–34 crp: c-reactive protein, s. ldh: serum lactate dehydrogenase, all parameters presented in mean folds elevated than normal, sig: significant association (p = 0.05 or less), ns: not significant. table 5. radiological findings of the studied group according to disease severity (n = 140) parameter statistics severity p. value mild (n = 60) moderate (n = 40) severe (n = 40) ct scan (percent of pulmonary damage)* mean ± sd 20.5 ± 8.8 36.9 ± 8.5 64.6 ± 11.3 < 0.001 sig anovarange 10–35 20–50 50–85 chest x-ray finding no (%) infiltrate 21 (35.0) 14 (35.0) 15 (37.5) < 0.001 sig fisher’s normal 28 (46.7) 0 (0.0) 0 (0.0) not available 11 (18.3) 26 (65.0) 25 (62.5) sd: standard deviation, sig: significant difference fisher’s exact test, chi square couldn’t be applied *ct scan performed in 118 patients only sig: significant association (p = 0.05 or less), ns: not significant. table 8. distribution of blood urea and serum creatinine levels according to disease severity of among studied group (n = 140) parameter severity p. value chi square test mild (n = 60) moderate (n = 40) severe (n = 40) no. % no. % no % blood urea high 6 10.0 8 20.0 16 40.0 0.002 sig normal 54 90.0 32 80.0 24 60.0 s. creatinine high 4 6.7 3 7.5 5 12.5 0.570 ns normal 56 93.3 37 92.5 35 87.5 sig: significant association (p = 0.05 or less), ns: not significant. percent of pulmonary damage of (64.6% ± 11.3%), (p. value < 0.001). with regard to chest x-ray findings, none, of the cases with moderate or severe disease form had normal x-ray findings, compared to 46.7% of mild cases (p. value < 0.001). hospitalization needed in 67.5% of moderate cases and all severe cases, (p < 0.001). the mean duration of hospital stay was significantly longer in severe cases, 13.8 ± 8.2 days, than moderate disease group, (4.3 ± 2.1 days), (p < 0.001). all cases with severe disease needed respiratory support compared to 57.5% of moderate cases, (p < 0.001), it is worth mentioned that none of mild cases hospitalized or needed respiratory support, (table 6). neutrophil count was significantly higher in severe cases while lymphocyte count was significantly lower in severe cases, (p < 0.001), (table 7). a significantly higher blood urea reported in severe cases (p = 0.002), where 40% of severe cases had elevated blood urea compared to 20% of moderate cases and 10% of mild ones. no significant differences were reported in s. creatinine (p > 0.05), (table 8). the comparison of biomarkers according to disease severity revealed that severe cases had significantly higher crp, s. ferritin, s. ldh and d-dimer, in all comparisons, (p < 0.001), (table 9). all mild and moderate cases recovered, however, only 2 moderate cases developed complications and recovered later, among severe cases complications occurred in 50%, and unfortunately, 14 (35%) died, the differences in outcomes, were statistically significant, (table 10). the mean s. ldh level was significantly lower in cases who did have compared to those who did not have upper respiratory symptoms, 1.3 ± 1.4 vs. 1.7 ± 1.3, respectively, (p. value = 0.011). neither crp, s. ferritin nor d-dimer level significantly different across the presence of upper respiratory symptoms, (p > 0.05), (table 11). 144 j contemp med sci | vol. 8, no. 2, march-april 2022: 140–146 clinical features and upper airway symptoms in association with severity and outcome in patients with covid-19 original f.h. hadi et al. table 12. relationship between of upper respiratory symptom with spo 2 and ct scan findings of the studied group parameter upper respiratory symptom p. valueyes no mean sd mean sd spo 2 (%) 90.7 9.7 84.3 15.5 0.007 sig chest ct scan (%) of pulmonary damage 34.6 20.2 49.1 21.2 0.001 sig table 13. relationship between of upper respiratory symptom with complication and mortality of covid-19 patients (n = 140) upper respiratory symptoms p. value yes (n = 113) no (n = 27) no. % no. % complications yes 15 13.3 7 25.9 0.105 nsno 98 86.7 20 74.1 outcome recovered 106 93.8 20 74.1 0.002 sigdied 7 6.2 7 25.9 sig: significant association (p = 0.05 or less), ns: not significant. table 10. outcomes of moderate and severe covid-19 patients outcome moderate (n = 40) severe (n = 40) p. value chi square test no. % no. % recovered 40 100.0 26 65.0 < 0.001 sig complications developed 2 5.0 20 50.0 < 0.001 sig died 0 0.0 14 35.0 < 0.001 sig table 11. relationship between upper respiratory symptom and markers of diseases severity parameter upper respiratory symptom p. valueyes (n = 113) no (n =27) mean ± sd* mean ± sd crp level 4.4 ± 3.2 5.0 ± 3.8 0.552 ns s. ldh 1.3 ± 1.4 1.7 ± 1.3 0.011 sig s. ferritin 1.8 ± 2.1 3.0 ± 2.6 0.175 ns d-dimer 2.8 ± 1.7 5.1 ± 3.4 0.070 ns sd: standard deviation. the mean spo2% was significantly higher in patients with upper respiratory symptom compared to those without, (p = 0.007). percent of pulmonary damage was significantly lower in patients with upper respiratory symptom compared to those without, (p = 0.001), (table 12). no significant association was found between complications and presence of upper respiratory symptom, (p > 0.05). mortalities were significantly lower in patients with upper respiratory symptom compared to those without; among the 113 patients with upper respiratory symptom compared only 7 (6.2%) died compared to 7 out of 27 (25.9%) patients with no upper respiratory symptom, (p = 0.002), (table 13). discussion the clinical manifestations associated with sars-cov-2 share some features with other respiratory viral infection but there are many areas of differences including the absence or minimal occurrence of rhinorrhea, severe and distressing cough and marked involvement of lung with severe destruction observed in many patients.21,22 radiographic imaging has a significant role in confirmation of diagnosis and the assessment of severity of pulmonary damage and this helps in early recognition of critically ill patients to prevent unnecessary delay in intensive management and consequently increasing mortality.23–30 the present study is planned to assess the clinical features, in particular, upper respiratory symptoms and their association with ct findings and severity of disease in group of iraqi patients. in this study there was no significant association between having chronic diseases and severity of sars-cov-2, with the exception of diabetic patients and chronic medication users had significantly more severe disease. similar findings were also reported in previous studies; in large scale case-control study, yan et al. found that chronic drug users had significant higher susceptibility and severity of disease,31 erener s documented that diabetic patients at high risk of infection and poor glycemic control are a major risk factor for infection and contributed that to the alteration in the immune function in diabetic patients.32 on the other hand, other studies reported strong correlation between comorbid chronic disease and severity of sars-cov-2;33 wang et al. found that comorbid chronic disease and acute organs injury was strongly and significantly associated with severe disease and higher mortality among patients with sars-cov-2.34 expectedly, in the present study, lower spo2% was significantly associated with more severe disease, similar findings reported in many other studies;35–37 xie et al. found a significant association between hypoxemia and higher mortalities.35 rubin et al. supported these findings.36 li et al.37 found that oxygen saturation of less than 93% was significant predictor of poor prognosis and higher body temperature on admission was significantly associated with more severe disease. petrelli et al.38 found that body temperature on admission was good predictor for viral infection and that higher temperature of 38°c or more would be good diagnostic indicator implies severe and critically ill patients, on the other hand, petrelli et al.38 documented higher proportion of patients with severe disease with temperature of more than 38°c compared to those with milder disease and non hospitalized patients. conversely, guan et al.39 from china studied the clinical characteristics of 1909 sars-cov-2 patients, among their findings no significant difference in the mean body temperature on admission between cases with non-severe and severe disease. the differences between studies could be due to the criteria that depended for admission and classification of disease severity in different countries, where in most studies, clinician not much rely on body temperature as indicator of severe disease, but some authors stated that patients with severe or critical illness, had significantly longer duration of 145j contemp med sci | vol. 8, no. 2, march-april 2022: 140–146 f.h. hadi et al. original clinical features and upper airway symptoms in association with severity and outcome in patients with covid-19 higher body temperature than the mild or non-severe cases. furthermore, the duration of high body temperature in patients who transferred to icu was almost 31 days compared to 9 days in other patients.40 the present study found that higher ct scan percent of pulmonary damage associated with severe disease this finding supported that in other studies,41,42 where these studies documented that ct-scan detected pulmonary damage was significantly associated with disease severity and the proportion of pulmonary damage increased with severity. in the current study, presence of infiltrate on chest x-ray was significantly associated with severe disease. it had been adopted that chest-x-ray findings is helpful in monitoring the course and severity of sars-cov-2.43 yasin and gouda from egypt found that severity score was significantly associated with abnormal x-ray findings and concluded that radiographic findings were good predictor for long term monitoring of sars-cov-2 cases. in the current study, patients with severe disease needed longer duration of hospitalization than the cases with moderate disease and all patients with severe disease needed respiratory support compared to only 57.5% of moderate cases, these findings were not unexpected due to nature of disease, and similar findings reported in most other studies,36,38,40–42,44 in the present study none of mild cases were admitted, and this is according to the management recommendations of moh in iraq that mild cases send for home quarantine and followed up. the blood count results of this study show increase neutrophils count and decrease lymphocytes count was found in severe cases compared to moderate and mild cases, knong et al. reported that higher neutrophil and lower lymphocyte counts as demonstrated by higher neutrophil to lymphocyte ratio (nlr) was significantly associated with severe form of sars-cov-2 and that patients with increased nlr should be admitted to an isolation ward with respiratory monitoring and supportive care.45 a meta-analysis conducted by li et al.46 proved good value of nlr in prediction of severe and critical disease. another study indicated that high neutrophil and lower lymphocyte counts had good prognostic implications in sars-cov-2, the higher nlr has been hypothesized to be associated with underlying endothelial dysfunction which favor cellular damage and then endothelial cell death particularly in patients with pre-existing endothelial dysfunction.47 biomarkers including crp, s. ferritin, s. ldh and d-dimer were all significantly and proportionately increased with severity of sars-cov-2; patients with severe disease had significantly higher mean values of these biomarkers than those with moderate disease while the level in mild cases were the lowest and these findings were consistent with other studies.24,25,48–50 regarding the association between symptoms and severity of disease, the present study found that patients with headache, sore throat, loss of smell, fatigue and myalgia had significantly milder disease and those with shortness of breath had significantly more severe disease, while other symptoms did not show significant associations with severity of sars-cov-2, the patients with upper respiratory symptoms had lower levels of crp, s. ferritin, s. ldh and d-dimer, higher spo2% and lower ct scan percent of pulmonary damage, these findings indicated less severe disease in patients with upper respiratory symptoms compared to those without. although the exact pathophysiology of post-viral anosmia is unclear, it is believed that damage to the receptor cells of the olfactory neuroepithelium is one of the probable causes. objective assessment of loss of smell was not done because of risk of infection transmission. conclusion early occurrence of upper respiratory tract symptoms predicts less severe form of the disease and good outcome of the patient. conflicts of interest none.  references 1. hui ds, i azhar e, madani ta, ntoumi f, kock r, dar o, et al. “the continuing 2019-ncovepidemic threat of novel coronaviruses to global health—the latest 2019 novel corona virus outbreak in wuhan, china”. international journal of infectious diseases. (february 2020) 91:264–266. 2. wu jt, leung k, leung gm. nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study [published correction appears in lancet. 2020 feb 4;]. lancet. 2020;395(10225):689-697. 3. helmy ya, fawzy m, elaswad a, sobieh a, kenney sp, shehata aa. the covid-19 pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control. j clin med 2020; 9(4): e1225. 4. paules ci, marston hd, fauci as. coronavirus infections — more than just the common cold. jama 2020;323:707-8. 5. de wit e, van doremalen n, falzarano d, munster vj. sars and mers: recent insights into emerging coronaviruses. nat rev microbiol. 2016;14(8): 523-534. doi: 10.1038/nrmicro.2016.81pubmedgoogle scholarcrossref 6. song, z., xu, y., bao, l., zhang, l., yu, p., qu, y., ... & qin, c. (2019). from sars to mers, thrusting coronaviruses into the spotlight. viruses, 11(1), 59. 7. martinez-rojas ma, vega-vega o, bobadilla na. is the kidney a target of sars-cov-2. am j physiol renal physiol. 2020;318(6):f1454-f1462. doi:10.1152/ajprenal.00160.2020. 8. hoffmann m, kleine-weber h, schroeder s, et al. sars-cov-2 cell entry depends onace2 and tmprss2 and is blocked by a clinically proven protease inhibitor. cell 2020;181(2):271-280.e8 9. baj, j., karakuła-juchnowicz, h., teresiński, g., buszewicz, g., ciesielka, m., sitarz, r., & maciejewski, r. (2020). covid-19: specific and non-specific clinical manifestations and symptoms: the current state of knowledge. journal of clinical medicine, 9(6), 1753. 10. cw lu, xf liu, zf jia. 2019-ncov transmission through the ocular surface must not be ignored. lancet, 395 (2020), p. e39,10.1016/s01406736(20)30313-5. 11. wg carlos, cs dela cruz, b cao, s pasnick, s jamil. novel wuhan (2019ncov) coronavirus. am j respir crit care med, 201 (2020), pp. p7-p8, 10.1164/rccm.2014p7. 12. j xia, j tong, m liu, y shen, d guo. evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov-2 infection. j med virol (2020 feb 26), 10.1002/jmv.25725. 13. fu l.; wang b.; yuan t.; chen x.; ao y.; fitzpatrick t.; li p.; zhou y.; lin y.‐f.; duan q.; et al. clinical characteristics of coronavirus disease 2019 (covid‐19) in china: a systematic review and meta‐analysis. j infect 2020;80:656–665. 14. sun p.; qie s.; liu z.; ren j.; li k.; xi j. clinical characteristics of hospitalized patients with sars‐cov‐2 infection: a single arm meta‐analysis. j med virol 2020;92:612–617. 146 j contemp med sci | vol. 8, no. 2, march-april 2022: 140–146 clinical features and upper airway symptoms in association with severity and outcome in patients with covid-19 original f.h. hadi et al. 15. gane s.b.; kelly c.; hopkins c. isolated sudden onset anosmia in covid‐19 infection. a novel syndrome? rhinology 2020;58:299-301. doi:10.4193/ rhin20.114. 16. tong j.y.; wong a.; zhu d.; fastenberg j.h.; tham t. the prevalence of ol factory and gustatory dysfunction in covid‐19 patients: a systematic review and me ta‐analysis. otolaryngol head neck surg 2020;163:3-11. 17. aragão, m. d. f. v. v., leal, m. c., cartaxo filho, o. q., fonseca, t. m., & valença, m. m. (2020). anosmia in covid-19 associated with injury to the olfactory bulbs evident on mri. american journal of neuroradiology, 41(9), 1703-1706. 18. li q, guan x, wu p, et al. early transmission dynamics in wuhan, china, of novel coronavirus–infected pneumonia. n engl j med 2020;382:1199-1207 doi: 10.1056/nejmoa2001316. 19. https://www.researchgate.net/publication/340447353_the_laboratory_ diagnosis_of_covid-19_infection_current_issues_and_challenges. 20. https://www.researchgate.net/publication/339627510_laboratory_ abnormalities_in_patients_with_covid-2019_infection 21. novel cpere. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china. zhonghua liu xing bing xue za zhi= zhonghua liuxingbingxue zazhi. 2020;41(2):145. 22. giesen c, diez-izquierdo l, saa-requejo cm, lopez-carrillo i, lopez-vilela ca, seco-martinez a, et al. epidemiological characteristics of the covid-19 outbreak in a secondary hospital in spain. am j infect control. 2020; 23. velavan tp, meyer cg. mild versus severe covid-19: laboratory markers. int j infect dis. 2020; 24. keddie s, ziff o, chou mkl, taylor rl, heslegrave a, garr e, et al. laboratory biomarkers associated with covid-19 severity and management. clin immunol. 2020;221:108614. 25. tjendra y, al mana af, espejo ap, akgun y, millan nc, gomez-fernandez c, et al. predicting disease severity and outcome in covid-19 patients: a review of multiple biomarkers. arch pathol lab med. 2020;144(12):1465–74. 26. ieong cm, xu x, kong sc, luo l. evaluation of chest ct and clinical features of covid-19 patient in macao. eur j radiol open. 2020;7. 27. jin c, tian c, wang y, wu cc, zhao h, liang t, et al. a pattern categorization of ct findings to predict outcome of covid-19 pneumonia. front public heal. 2020;8(september). 28. matos j, paparo f, mussetto i, bacigalupo l, veneziano a, perugin bernardi s, et al. evaluation of novel coronavirus disease (covid-19) using quantitative lung ct and clinical data: prediction of short-term outcome. eur radiol exp. 2020;4(1). 29. zhong l, zhang s, wang j, zhao x, wang k, ding w, et al. analysis of chest ct results of coronavirus disease 2019 (covid-19) patients at first followup. can respir j. 2020;2020. 30. leonardi a, scipione r, alfieri g, petrillo r, dolciami m, ciccarelli f, et al. role of computed tomography in predicting critical disease in patients with covid-19 pneumonia: a retrospective study using a semiautomatic quantitative method. eur j radiol. 2020;130(april):1–7. 31. yan h, valdes am, vijay a, wang s, liang l, yang s, et al. role of drugs used for chronic disease management on susceptibility and severity of covid‐19: a large case‐control study. clin pharmacol ther. 2020;108(6):1185–94. 32. erener s. diabetes, infection risk and covid-19. mol metab. 2020;101044. 33. liu h, chen s, liu m, nie h, lu h. comorbid chronic diseases are strongly correlated with disease severity among covid-19 patients: a systematic review and meta-analysis. aging dis. 2020;11(3):668. 34. wang x, fang x, cai z, wu x, gao x, min j, et al. comorbid chronic diseases and acute organ injuries are strongly correlated with disease severity and mortality among covid-19 patients: a systemic review and meta-analysis. research. 2020;2020:2402961. 35. xie j, covassin n, fan z, singh p, gao w, li g, et al. association between hypoxemia and mortality in patients with covid-19. in: mayo clinic proceedings. elsevier; 2020. 36. rubin sjs, falkson sr, degner nr, blish c. clinical characteristics associated with covid-19 severity in california. j clin transl sci. 2020;(table 2):1–4. 37. li x, xu s, yu m, wang k, tao y, zhou y, et al. risk factors for severity and mortality in adult covid-19 inpatients in wuhan. j allergy clin immunol [internet]. 2020;146(1):110–8. available from: https://doi.org/10.1016/j. jaci.2020.04.006 38. petrilli cm, jones sa, yang j, rajagopalan h, o’donnell l, chernyak y, et al. factors associated with hospital admission and critical illness among 5279 people with coronavirus disease 2019 in new york city: prospective cohort study. bmj. 2020;369. 39. guan wj, ni zy, hu y, liang wh, ou cq, he jx, et al. china medical treatment expert group for covid-19. clin charact coronavirus dis. 2019;1708–20. 40. chen j, qi t, liu l, ling y, qian z, li t, et al. clinical progression of patients with covid-19 in shanghai, china. j infect. 2020;80(2020):e1–6. 41. xia l, chen j, friedemann t, yang z, ling y, liu x, et al. the course of mild and moderate covid-19 infections-the unexpected long-lasting challenge. open forum infect dis. 2020;7(9):1–7. 42. matsumura k, toyoda y, matsumoto s, kawai y, mori t, omasa k, et al. comparison of the clinical course of covid-19 pneumonia and acute respiratory distress syndrome in 2 passengers from the cruise ship diamond princess in february 2020. am j case rep. 2020;21:1–6. 43. yasin r, gouda w. chest x-ray findings monitoring covid-19 disease course and severity. egypt j radiol nucl med. 2020;51(1). 44. turcotte jj, meisenberg br, macdonald jh, menon n, fowler mb, west m, et al. risk factors for severe illness in hospitalized covid-19 patients at a regional hospital. plos one. 2020;15(8):e0237558. 45. kong m, zhang h, cao x, mao x, lu z. higher level of neutrophil-tolymphocyte is associated with severe covid-19. epidemiol infect. 2020;0–5. 46. li x, liu c, mao z, xiao m, wang l, qi s, et al. predictive values of neutrophilto-lymphocyte ratio on disease severity and mortality in covid-19 patients: a systematic review and meta-analysis. crit care [internet]. 2020;24(1):1–10. available from: https://doi.org/10.1186/s13054-020-03374-8 47. jimeno s, ventura ps, castellano jm, garcía-adasme si, miranda m, touza p, et al. prognostic implications of neutrophil-lymphocyte ratio in covid-19. eur j clin invest. 2021;51(1):1–9. 48. malik p, patel u, mehta d, patel n, kelkar r, akrmah m, et al. biomarkers and outcomes of covid-19 hospitalisations: systematic review and metaanalysis. bmj evidence-based med. 2020; 49. benotmane i, perrin p, vargas gg, bassand x, keller n, lavaux t, et al. biomarkers of cytokine release syndrome predict disease severity and mortality from covid-19 in kidney transplant recipients. transplantation. 2020;105(1):158–69. 50. yao y, cao j, wang q, shi q, liu k, luo z, et al. d-dimer as a biomarker for disease severity and mortality in covid-19 patients: a case control study. j intensive care. 2020;8(1):1–11. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i2.1206 101j contemp med sci | vol. 9, no. 2, march-april 2023: 101–105 original molecular immuno-response effects for iraqi lycium barbarm carotenes upon normal human lymphocytes culture zainab yaseen mohammed hasan1*, khulood w. alsamarrae2, ahmed rushdi3 1biotechnology research center, al-nahrain university, baghdad, iraq. 2college of medical technology, al-farahidi university, baghdad, iraq. 3college of medicine, al-iraqia university, baghdad, iraq. *correspondence to: zainab yaseen mohammed hasan (e-mail: zainaby2003@yahoo.com) (submitted: 24 january 2023 – revised version received: 12 february 2023 – accepted: 17 march 2023 – published online: 26 april 2023) abstract objectives: the study aimed to investigate the effects of iraqi lycium barbarm carotene on normal human blood lymphocytes at molecular level. methods: the normal human blood lymphocytes culture was exposed to carotene extracted from iraqi lycium barbarm to estimate the interleukins expression in treated lymphocytes represented by il-10 and tnf-α as a moderator of immune response at a molecular scale, and their cd markers changes expression (cd3, cd4 & cd8) by flow-cytometer apparatus. results: total carotenes content was 0.33 mg/g powdered dried fruit. potent carotene effect was appeared at concentration of 500 µg/ml for treated lymphocytes with increasing cd3 level to get upper level after 2 hours interval, while after 4 hours exposure both cd4 and cd8 levels increased dramatically for lymphocytes treated with 125 µg/ml, and 250 µg/ml lycium carotene. an alteration in cytokines gene expression for il-10 production in response to carotene treatments at (125, 250, 500) µg/ml for both intervals, with suppress in tumor necrosis factor (tnf-α) gene expression in relation to β-actin the internal control and the pha mitogenic agent. conclusion: the study concluded that the carotenes extracted from lycium barbarm fruits seemed to initiate the immune system toward th2 cell activation rather than th1, besides the extract may potentiate il-10 production. key words: lycium barbarm, carotenoids, interleukin-10, tnf-α, cd4, cd8 issn 2413-0516 introduction lycium barbarm (l. barbarm) belongs to solanaceae family is one of the most notable chinese medicinal herbal species and has a famous tradition usage as a food and medicinal plant in various asian countries, especially in chinese traditional medicine, known with different names; wolfberry, boxthorn, chinese wolfberry, and gugija in korea and kuko in japan.1 leaves and fruits of l. barbarm are exceedingly applied as herbal and medicinal tea in china, southeast asia, europe, and north america. this plant is naturally grown in warm regions of iraq, known locally as ‘awsaj’. the institute of the chinese medicinal monographs recorded the plant to be the “nourishing liver and kidney, enhancing eyesight, enriching blood, invigorating sex, reducing rheumatism” and so on.2 its main functions were reported as immunity improvement,3 antioxidant,4 anti-microbial,5 anti-cancer,6 improving hemopoiesis,7 anti-aging, and sexual tonic.8 studies are in progress to understand how these compounds may or may not provide protection against toxic, mutagenic and carcinogenic activities of chemical compounds. the beautiful orang of lycium fruit indicated the high plant content of “zeaxanthin” as one of predominant carotenoids group in nature, also called physalien or physalin.9 therefore, this fruit is considered as good food source in zeaxanthin content.10 the present study represented a novel work that high lightened the biological activity for the wild iraqi lycium barbarm intended as a great worth for researcher knowledge specially the immunomodulatory effects therefore, current research including this plant was aimed to estimate the effects of the extracted carotene towards normal human blood lymphocytes (cd3, cd4 & cd8) by using flow-cytometer, and to investigate il-10 and tnf-α gene expression in the cultured lymphocytes using rt-pcr. materials and methods plant selection and collection the plant was collected at end of september as the small ripe wild lycium barbarm orange fruits had been naturally found in al-jadriya district of baghdad university to be classified by biology department herbarium at the college of science. the extraction of the total carotenes from the fruits11 using porcelain mortar, the dried l. barbarm fruits powder in a quantity of 1 g was homogenized well with 3 ml distilled water, then 2 ml absolute ethanol was added to be mixed well with aid of vortex and transferred to separator funnel contained 10 ml n-hexane and gentle mixing. two layers were separated, the organic hexane layer which contained the extracted carotene and the aqueous layer. the aqueous was extracted many times with n-hexane. the final volume of the combined n-hexane layers was measured representing the final volume of the total carotenes extracted from one gm l. barbarm fruit which was evaporated and weighted as total carotene. immunomodulation determination (in vitro) lymphocyte culturing from 8 healthy volunteers with an age ranged between (25– 35), the venous blood (5 ml) was collected and transferred into separated vacuumed tube. all volunteers appeared healthy, and they never took any kind of medication for at least 10 mailto:zainaby2003@yahoo.com 102 j contemp med sci | vol. 9, no. 2, march-april 2023: 101–105 molecular immuno-response effects for iraqi lycium barbarm carotenes original z.y.m. hasan et al. days. a general protocol for lymphocytes separation was preceded in the current study.12 cell viable counting for the isolated lymphocytes according to general protocol included in reference for viable cells counting, a freshly trypan blue 1% pbs dye was prepared.12 viable cells appeared transparent in contrast to the dead cells that could be easily distinguished under light microscope within seconds as violet color.13 calculation for viable lymphocyte cells was done before any of the following immune tests.14 treating the isolated lymphocytes with the extracted lycium total carotenes lymphocytes suspended cells (1 ml) in concentration of 106 cell/well were seeded, in two tissue culture plate of the 24 wells to be incubated in a co2 incubator for at least for 2 hours before treatments. three concentrations from the extracted carotenes (125, 250, and 500 µg/ml) were used for two intervals for carotene exposure time; one plate incubated for 2 hours and the other for 4 hours had been used.15 at the end of each exposure period, both plates were centrifuged to separate the supernatant from the pellets. detection of cd4 and cd8 markers by flow cytometry the cd4 and cd8 estimation protocol (exbio/nadsafinou) were applied using 10 µl fitc (for cd4 marker), or pe (for cd8 marker)–conjugated monoclonal anti-human cd marker provided in the kit. the isolated pellets from each well were separately treated and subjected to flow cytometry apparatus for analysis.16 extraction and purification of total rna using geneaid total rna mini kit, in aseptic condition and all equipment, glassware, hood-benches, gloves, and even electrophoresis tank were thoroughly cleaned and washed with rnase-free water, treated with 0.1% diethylpyrocarbonate solution (depc), then left 10minutes in a vacuumed-hood, to insure rnase-free condition and to remove all residual traces of depc solution. total rna was measured by using a spectrophotometer at 260 nm wavelengths and the purity of rna was measured by determining the ratio of a260/a280 nm. all samples were at ratios of above 1.75. the purified rna is ready for rt-pcr and other tests. the work was done according to geneaid kit protocol. electrophoresis gel electrophoresis was done according to as follows:17 • agarose gel (0.8–1)g dissolved in100 ml of tbe 1x buffer (tris hcl: boric acid: edta in d.d. water) was prepared in a quantity enough for pouring in appropriate tray. • mixing 8 µl extracted total rna with 2 µl loading dye (bromophenol blue and glycerin). • after about two hours running the apparatus was stopped, and the gel was removed from the tank and to ensure the presence of rna’s bands, the gel was visualized by staining with ethidium bromide that incorporated in rna strands (ribosomal rna considered double-stranded molecule due to extensive secondary structure). pictures were taken under uv camera system. detection of interleukin -10 (il-10) and tumor necrosis factor-α (tnfα) mrna expression by rt-pcr total rna which extracted from treated lymphocytes with the extracted carotene in above step for all samples, were reverse transcript to complementary dna (cdna) according to (accu power rocket script rt pre-mix) which is ready lyophilized master-mix containing all components for firststrand cdna synthesis (in 96 wells of 20 µl total volume) from purified poly (a) or total rna templates.18 table 1 showed primers used and their sequences and the predicted product size. • all samples (the extracted total rna for lymphocytes treated with three concentrations and with two exposure time) designated as in table 2. • the recommended amounts of each extracted rna sample and the specific primers that should be added for rt-pcr pre-mix tubes were shown in the table 3. • the reaction was performed under the condition shown in table 4, using thermal cycle pcr apparatus (labnet international multigene/usa). electrophoresis for the rt-pcr product at the end of the reaction a gel electrophoreses was applied with (1.5%) agarose gel and ladder dna of 100 bp, internal control β-actin, pha also –ve control and distilled water table 1. human cytokine primers (il-10 and tnfα) and their sequences and predicted product size(bp = base pair; f = forward sequence; r = reverse sequence) cytokine the sequences product il-10 f: 5’tacggcgctgtcatcgattt3’ r: 5’aaggtttctcaaggggctgg3’ 273 bp tnf-α f: 5’tctcgaaccccgagtgaccaa3’ r: 5’accgcacctcgactcctat3’ 123 bp β-actin (internal control) r: 5’aaccccaaggccaaccgcgaga agatgacc3’ r: 5’ggtgatgacctggccgtcaggcagctcgta3’ 416 bp table 2. symbols of all samples used in rt-pcr system sample treatment concentration (µg/ml) interval symbol total carotenes 500 2 hours c1/2 h total carotenes 250 2 hours c2/2 h total carotenes 125 2 hours c3/2 h total carotenes 500 4 hours c1/4 h total carotenes 250 4 hours c2/4 h total carotenes 125 4 hours c3/4 h (β-actin) internal control +ve pha 0.1% pha lymphocytes culture only (–ve control) –ve 103j contemp med sci | vol. 9, no. 2, march-april 2023: 101–105 z.y.m. hasan et al. original molecular immuno-response effects for iraqi lycium barbarm carotenes table 3. the recommended quantities of components for rtpcr kit amount/samplecomponent 10 pmole-5 µg (3 µl)total rna of sample 20 pmole each (2 µl each)primer up to20 µlddh 2 o with depic table 4. conditions for pcr program cycletimetemperaturestep 130 minutes60occdna synthesis 15 minutes95ocpre-denaturation 4030 seconds94ocdenaturation –1 minutes55ocannealing –1 minutes72ocextension 15 minutes72ocfinal extension table 5. % cd markers of lymphocyte treated with different concentrations (125, 250, 500) µg/ml of extracted carotene for two exposure times (2 and 4) hours concentration (µg/ml) carotene cd3 cd4 cd8 cd3 cd4 cd8 for 2 hours exposure* for 4 hours exposure* 500 38.1% 0.0% 0.1% 0.0% 0.6% 0.0% 250 36.3% 0.0% 0.0% 0.0% 0.6% 0.3% 125 0.0% 0.0% 0.0% 0.1% 0.3% 0.4% control 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% *cd marker level less than 0.1% represented by 0.0% as the kit sensitivity declared. rnase free. the same procedure mentioned in the previous step was applied and the results for both cytokine pcr products were visualized under uv system. statistical analysis the statistical analysis system-sas (2018)was used to present the effect of different concentrations and factors in the studied parameters.19 to compare between the means of the variations in this study, the least significant difference (lsd) test and duncan test were used. results the total carotenes content in the fruits the final volume of total carotene extracted from 1 g dried powdered fruit with n-hexane was 165 ml which analyzed qualitatively and quantitatively, and the concentration of total carotene extracted from the dried fruits of l. barbarm was 0.33 mg/g and all qualitative and quantitative results was authorized in previous study.20 effect of l. barbarum total carotenes on cd4, cd8 and cd3 markers to determine the level of cd markers as normal lymphocytes treated with different lycium carotenes concentrations, a flow-cytometer was used to pick the change in cd markers percentage. table 5 showed cd markers percentage expressed by lymphocytes after carotene treatment for two exposure times (2 and 4) hours. as shown in table 5, the most potent effect was shown for the lymphocytes treated with extracted carotene at concentration of 500 µg/ml by increasing cd3 percentage to upper level after 2 hours treatment, while cd4 and cd8 levels increased dramatically when normal lymphocytes treated with 125 µg/ml, and 250 µg/ml lycium carotene for 4 hours exposure. fig. 1 agarose gel electrophoresis for 8 µl extracted total rna of treated lymphocytes with extracted carotene mixed with 2 µl bromophenol blue loading dye. bands were fractionated by electrophoresis on 1% agarose gel in tbe 1x buffer ph = 8 (5 volts/cm, current of 60 ampere for 1 hour) visualized under uv light after staining with ethidium bromide. lane c1, c2, c3: lymphocytes treated with (500, 250, 125) µg/ml lycium carotene respectively, lane pha: lymphocytes treated with pha, lane (–ve): lymphocytes without treatment, and lane (–): distilled water only. treatment was made for (2 and 4) hours. effect of extracted carotenes from l. barbarm on gene expression for cytokines a. total rna in lymphocytes treated with l. barbarm carotenes gel electrophoreses indicated the presence of clear two bands which mimic that related to genes for 28 s and 18 s ribosomal rna as shown in figure 1. b. the expression of (il-10) and (tnfα) genes in lymphocytes treated with extracted carotene in order to detect the expression of specific gene (il-10 and / or tnf-α) as well as alteration of gene expression which may be directed the immune defense in response to treating lymphocytes with lycium barbarm carotene at different concentrations toward humoral or cellular immune defense, reverse transcriptase polymerase chain reaction (rt-pcr) technique was applied in this study. the results shown in figure 2 indicated that alteration of gene expression of normal lymphocytes toward il-10 production in response to lycium barbarm extracted carotene treatments at concentrations (125, 250, 500) µg/ml were obvious for all treatments and for both 104 j contemp med sci | vol. 9, no. 2, march-april 2023: 101–105 molecular immuno-response effects for iraqi lycium barbarm carotenes original z.y.m. hasan et al. fig. 2 agarose gel electrophoresis for amplified mrna for il-10 gene expression of the lymphocytes treated with different l. barbarum carotenes concentrations. bands were fractionated by electrophoresis on 1.5% agarose gel (5 volts/cm, current 60 ampere, 2 hours, teb 1x buffer) analyzed under uv light after staining with ethidium bromide. lane m: 100 bp ladder, lane +ve: β-actin internal control, lanes c1, c2, c3: lymphocytes treated with 500, 250 and 125 µg/ml extracted carotenes respectively, lane –ve: negative control, lane pha: lymphocytes treated with pha. treatment for (2 and 4) hours intervals. fig. 3 agarose gel electrophoresis for amplified mrna of tnf-α from treated lymphocytes with different l. barbarm extracted carotene concentrations. bands were fractionated by electrophoresis on 1.5% agarose gel (5 volts/cm, current 60 ampere, 2 hours, teb 1x buffer) analyzed under uv light after staining with ethidium bromide. lane m: 100 bp ladder, lane +ve: β-actin internal control, lanes c1, c2, c3: lymphocytes treated with 500, 250 and 125 µg/ml extracted carotene respectively, lane –ve: negative control, lane pha: lymphocytes treated with pha. treatment for (2 and 4) hours intervals. interval times 2 and 4 hours in corresponding to negative control (untreated cells) and the internal control (β-actin gene that gave product of 420 bp). the pha, which is a mitogenic for t lymphocytes which binds to n-acetyl galactosamine glycoproteins expressed on the surface of t cells then activates the cell proliferation has not any effect on il-10 expression.21 with respect to gene expression for tumor necrosis factor (tnf-α), human peripheral blood lymphocyte cells exposed to lycium total carotenes, do not show any tnf-α expression at all concentrations (125, 250, 500) µg/ml and for (2 and 4) hours intervals, as well as the negative control, in relation to (β-actin) the internal control and the pha mitogenic agent as shown in figure 3. discussion in a study by reported that; β–carotene administration may cause enhancement of immunoglobulin levels and cd4+ cells count.22 other study by concluded that; the cd4-cd8 ratio were raised after the administration of β-carotene in 9 month, whereas throughout the study the natural killer cells, virgin t cells, memory t cells, and cytotoxic t cells remained unaltered, suggesting that it takes a long time to induce alterations of lymphocyte surface markers.23 carotenoids can stimulate band t-lymphocytes proliferation and increase the activity of macrophages and cytotoxic t-cells, for production of cytokines.24 recently, the focusing on carotenoid-rich vegetables and fruits for decreasing the risk of many diseases has been attributed to the major carotenoids, such as β-carotene, lutein, zeaxanthin, and lycopene which the wild iraqi l. barbarm fruit rich in. in a study confirmed that administration of carotenoid compounds such as vit. a decreased the side effects of the conventional anticancer drug methotrexate (mtx); that considered as protective in chemotherapy.25 in one study which acts to estimate carotene 105j contemp med sci | vol. 9, no. 2, march-april 2023: 101–105 z.y.m. hasan et al. original molecular immuno-response effects for iraqi lycium barbarm carotenes production from ex plants in tissue culture technique to successfully yield a plant product rich with carotenoids compound as there is a great demand to natural carotene source in medical field.26 the possible mechanisms for these compounds’ activity may contribute to that; β-carotene can block the nuclear translocation of the nf-κb p65 subunit which had an inhibitory effect on phosphorylation and degradation of the nf-κb inhibitor. this may explain the antioxidant activities for this bioactive molecule in quenching free radicals, and reducing the damage caused by (ros) reactive oxidant species that causes lipid peroxidation. furthermore, carotenoids can play important roles in immune-regulation and immune-stimulation in vertebrates.24 conclusion since cytokines play a prominent role in the development of immune response, in the current study the extracted carotenes seemed to initiate the immune system toward th2 cell activation rather than th1, besides the extract may potentiate il-10 production. results including il-10 and tnf-α level with or without carotenes for each cytokine expression detection, give an indication that l. barbarm directed body defense toward th2 rather than th1 immune response since cytokines of the treated lymphocytes with this active immune stimulant component have been increased in level toward il-10 (the pro-inflammatory cytokine inhibitor) with remarkable elevation of cd8+ after 4 hours of exposure. the safety, metabolism, and molecular biological properties of carotenoids should be elucidated through further studies before they are used to prevent carcinogenesis. acknowledgments the authors would like to acknowledge the all efforts provided by the staff at biotechnology research center, al-nahrain university, baghdad, iraq. conflict of interest none.  references 1. ludmila bogacz-r.; joanna, h. β-carotene-properties and production methods. j food quality and safety, 2018; vol.2 (2), 1–6. 2. thomas f. physicians’ desk reference for herbal medicines (pdr). 2020, medical economics company. new jersey. 3. sahar, alshaban; estabraq, alwasiti; and anam, al-salihi. evaluation of the level of dna damage and repair in human lymphocytes cultured in the presence of beta-carotene using comet assay (single cell gel electrophoresis). raqi j. comm. med., jan. 2016 (1).11–14. 4. vincenzo, s.; rosa, r.; antonio, m.; rosa, t. and monica, r.l. ciabatta bread incorporating goji (lycium barbarm l.): a new potential functional product with impact on human health. foods; 2023, 12, 566, 2–17. 5. shimal, y. abdulhadi; raghad, n.g.; and ghazwan q. h. molecular identifification, antioxidant effifficacy of phenolic compounds, and antimicrobial activity of beta-carotene isolated from fruiting bodies of suillus sp. karbala international journal of modern science. 2020; volume 6 issue 4, 365–374. 6. gulay, o.; deniz, g.; ayse, k.; esra, c.; susana, m.; basem, al-omari ; daniela, c.; javad, s.; william, c. a mechanistic updated overview on lycopene as potential anticancer agent. biomedicine and pharmacotherapy. 2023; volume 161, 114428 7. marco, a. g.; ana, e. o.; cecilia, a. and josé, d. l. chemistry, occurrence, properties, applications, and encapsulation of carotenoids. plants; 2023, 12, 313–345. 8. seungjin, n.; ara, g.; da, b. k.; minjeong, p.; hee, w. j. and bonglee, k. role of antioxidant natural products in management of infertility: a review of their medicinal potential. antioxidants; 2020, vol, 9, 957–61. 9. diana, c.; eva, f.; adrian, b. t.; andrea, b.; piroska, v.; maria, p. kaulmann, a.; and bohn,t. zeaxanthin-rich extract from super food lycium barbarm selectively modulates the cellular adhesion and mapk signaling in melanoma versus normal skin cells in vitro. molecules; 2021, 26, 333. 10. zhonglian, y.; mengqin, x.; xueping, l.; rui, w.; wenjing, l.; ruirong, z.; zhengtao, w.; li, y. and yanhong , s. characterization of carotenoids in lycium barbarm fruit by using upc2-pda-q-tof-mse couple with deep eutectic solvents extraction and evaluation of their 5α-reductase inhibitory activity. frontiers in chemistry; 2022, november, vol. 8: 2–12. 11. sun, w.; mohamad, h. s. and cheng, q. health benefits of wolfberry (gou qi zi, fructus barbarm l.) on the basis of ancient chinese herbalism and western modern medicine. avicenna j phytomed. 2021 mar-apr; 11(2): 109–119, 12. freshney, r.i. culture of animal cell. wily-liss, new york; 2021; 8th edition. 13. rafael, f. and vaclav, v. advanced methods in cellular immunology, 2000; vol. 3.crc-press llc.u.s.a. 14. al-hili, z.a.m. a study of cytotoxic, antioxidant, inhibition of angiogenic factors and induction of cyperusrotundus extracts on several cancer cell lines. 2009; a phd thesis in genetic engineering and biotechnology, baghdad university/iraq. 15. ahmed rushdi abdullah, zainab yaseen mohammed hasan, khulood. w. alsammarraie. effect of total lycium barbarum carotene on normal human blood lymphocytes. international journal of pharmaceutical research oct dec 2019; vol 11 supplementary issue 1, 1087–1093. 16. zola, h.; swart, b.; barry, s.; and yang, x. cd molecules–human cell differentiation molecules. j immunol methods, 2007; vol. 30, 319–324. 17. zhiyong, x.; qi, d.; wenxia, z.; yongxiang, z. immune activities of polysaccharides isolated from lycium barbarm l. what do we know so far? pharmacology & therapeutics; volume 229, january 2022, 107921. 18. cui, c.; zhongfu, w.; guiping, g.; wenqi, h.; linjuan, h.; shuang, s. and beiwei, z. effects of lycium barbarm polysaccharides on immunity and metabolic syndrome associated with the modulation of gut microbiota: a review. foods; 2022, 11, 3177. 19. sas. statistical analysis system, user’s guide. statistical. version 9. 6th ed. 2018; sas. inst. inc. cary. n.c. usa 20. zainab,y. m.; khulood,w.; subhi, j.h. quantitative analysis of total polysaccharides and total carotene from lycium barbarm fruit. international journal of modern biology and medicine, 2013; 4(2): 64–73. 21. doan, t.; melvold, r.; and waltenbaugh, c. lippincott’ illustrated reviews immunology. 2021. wotlers kluwer health, lippincott williams and wilkins. london. 22. gemala, a.; fitriyono, a.; and rafika, e. critical review on the immunomodulatory activities of carrot’s β-carotene and other bioactive compounds. journal of functional foods 99 (2022) 105303. 23. raksha, a.; lalit, m. and navneeta, b. disease prevention and treatment using β-carotene: the ultimate provitamin a. revista brasileira de farmacognosia (2022) 32:491–501. 24. alireza milani, marzieh basirnejad, sepideh shahbazi and azam bolhassani. carotenoids: biochemistry, pharmacology and treatment. british journal of pharmacology, 2017; vol.174, 1290–1324. 25. makarim q. al-lami; asmaa i. sail; salah m. al-chalabi; and ferial a. almahdawi. study the effects of methotrexate with and without vitamin a on some biochemical and histological parameters in male rabbits. journal of biotechnology research center, 2017; vol. 11 no.1. 26. amjad a-h. m. ; and mozahim k. al-mallah. efficient regeneration of carrot (daucus carota l.) plants from cell suspension derived-callus. journal of biotechnology research center, 2015; vol. 9; no.1. 58–63. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1335 96 j contemp med sci | vol. 8, no. 2, march–april 2022: 96–101 original faculty and students’ attitude toward e-learning at the start of the covid-19 pandemic: a cross-sectional study nazdar ezzaddin alkhateeb1* , ali shakir dauod2, nazar p shabila2, ali al-dabbagh1 1department of medical education, college of medicine, hawler medical university, erbil, iraq. 2department of community medicine, college of medicine, hawler medical university, erbil, iraq. *correspondence to: nazdar ezzaddin alkhateeb (e-mail: nazdar.alkhateeb@hmu.edu.krd) (submitted: 03 january 2022 – revised version received: 12 february 2022 – accepted: 08 march 2022 – published online: 26 april 2022) abstract objectives: this study aimed to assess the students and faculty staff attitude for e-learning at the emergence of covid-19. methods: a prospective cross-sectional google form based online survey was conducted at a medical university in iraq between march and april 2020. the online survey was distributed to a sample of 190 faculty staff and 1706 students of a medical university in iraq. knowledge and attitudes towards e-learning and perceived barriers to implementing e-learning among participants were the primary outcome measures. results: the majority of students had a negative view of the simplicity of using e-learning compared with higher agreement among faculty staff. most students disagreed with most aspects of the usefulness or strengths of e-learning. both students and faculty staff agreed that e-learning faces challenges, including poor internet connectivity (79%) and computer literacy (71%). the highest percentage of the students (42.8%) preferred to have paper examinations in the study halls for the final written assessment. the percentages of the faculty staff who preferred paper examination in the study halls (31.6%), online assessment (31.1%), and delay assessment for the following year (33.2%) were almost similar. conclusion: the students and faculty staff are not ready for e-learning, with technical inadequacies being the main barrier. since the covid-19 pandemic has significantly disrupted medical education, proper technical and institutional foundations are essential for successful e-learning, especially during social distancing measures. keywords: e-learning, students’ attitude, faculty’s attitude, medical education, covid-19 retrospectively registered at clinical trial.gov with registration number nct05223465 on 4th february 2022 issn 2413-0516 introduction e-learning in medical education is a relatively new idea that is rapidly growing. it provides education at a generally lower cost and engages learners at a time and location that is most appropriate for them.1,2 blended learning (an educational strategy that integrates learning technologies with face to face instructions), augments the advantages of face-to-face education through conducting electronic classes in addition to regular classes. it helps students use real and virtual environments to support their learning.3,4 recent calls for reform in medical education and training have emphasized the use of information technology-empowered learning. this was enhanced more in the covid-19 era.5 kurdistan region in iraq is also improving its educational goals to address the challenges of the next generations. e-learning strategies will not be optional any longer. however, obstacles and challenges will always face changes, and these include technological and social challenges. the initiation of e-learning requires pre-assessing students’ and faculty’s perspectives and attitudes for offering better delivery.6 like other parts of the world, the kurdistan region of iraq has recently experienced a lockdown of every part of life, including universities, in response to the covid-19 pandemic. this urged the universities and other educational institutes to consider e-learning as an urgent substitute. what has helped many universities is that they had their digital learning platforms, but they were not operational at full capacity. using e-learning has been applied in most universities throughout the globe for medical education. however, the covid-19 put medical education in challenge regarding clinical sessions, which constitutes the backbone of medical education for teaching the clinical competencies.6 some international organizations, such as the world health organization (who), have acknowledged e-learning as a helpful tool for healthcare education, particularly in developing countries. nevertheless, the use of e-learning by the faculty members and students stayed minimum for uploading lecture notes, course books, feedbacks, and providing marks until the covid-19 pandemic raised that need. this study aimed to evaluate faculty and students’ knowledge and attitudes toward e-learning and identify the potential requirements for implementing e-learning. materials and methods a prospective cross-sectional study was conducted at hawler medical university in kurdistan region of iraq during the lockdown, from the 1st of march to the 31st of april 2020. the sample size was calculated using the daniel formula in an online sample size calculator with a confidence interval of 95%, prevalence of 50%, and a 2% margin of error. the randomly selected students and faculty were invited electronically to participate in the study. they were informed about the objectives and anonymity of the study information. the participants provided online informed consent to participate in the study. a new study questionnaire was developed for data collection. previous surveys have guided the study questionnaire development.7 the final draft was approved by the researchers https://orcid.org/0000-0001-9954-1162 97j contemp med sci | vol. 8, no. 2, march–april 2022: 96–101 n.e. alkhateeb et al. original faculty and students’ attitude toward e-learning at the start of the covid-19 pandemic who are experts in the field of medical education and drafted using google survey form and distributed to participants through emails and social media like viber and facebook. the questionnaire was composed of five sections. the first section gathered information about the demographic characteristics of students and faculty. the second section included four questions to assess participants’ knowledge about e-learning. the third section (seven items) focused on the participants’ attitude and behavioral intention to use e-learning. the fourth section contained five questions to explore prior experience of using the internet, types of electronic device owned, and e-learning applications to communicate with colleagues or faculty. they were also asked about the perceived challenges facing e-learning. the last part of the questionnaire focused on participants’ opinions about the covid-19 pandemic, lockdown, and their effects on teaching methods. they were asked about the effect of the covid-19 pandemic on students’ learning and their willingness to continue medical education during this pandemic. students were asked to indicate their viewpoints on several statements using a 5-point likert-type scale, ranging from strongly agree to strongly disagree. the research questionnaire is available in the supplementary information. the validity and applicability of the survey tool were tested through a pilot study. the internal consistency (cronbach’s alpha) estimation of the questionnaire was 0.81, and the reliability coefficient was 0.78. data were analyzed using the statistical package for the social sciences (ibm spss statistics version 25). frequency and percentage were used to describe the data. the total scores of each component of knowledge, practice, and attitude were calculated by summating the scores of the questions related to that component. the study protocol was reviewed and approved by the research ethics committee of the authors’ institution with meeting code 4/1 on 23rd january 2022 and retrospectively registered at clinical trial.gov with registration number nct05223465 on 4th february 2022. results a total of 190 faculty staff and 1706 students responded to the survey with response rate of 41.7% and 100% respectively. only 9.1% of the students had used some components of e-learning in secondary schools. a low percentage of the students had an idea about e-learning (38.5%), had an interest in guidance on e-learning (39.3%), and used e-learning forms to communicate with colleagues or faculty (21.8%). a high percentage of students knew about the availability of e-learning facilities at the university (66.2%). most faculty members had an idea about e-learning (74.7%), had an interest in guidance on e-learning (78.4%), knew about the availability of e-learning facilities at the university (87.4%), and had used e-learning forms to communicate with colleagues and faculty (52.1%), as shown in table 1. in terms of the attitude about e-learning, most students had a negative view of the simplicity of using and learning e-learning. they mostly disagreed with e-learning being user friendly (76.4%). most students disagreed about the strengths of e-learning, particularly about being an interactive mode (60.5%), acquiring better skills (62.9%), easy accessibility, flexibility and interactivity (62%), and allow group discussion for complex topics (64.7%). most students agreed (strongly agreed or agreed) that e-learning faces challenges, with particular emphasis on poor internet connectivity (75.9%), computer literacy (71.6%), and lack of computers and smartphones (67%), as shown in table 2. regarding faculty’s staff attitudes about e-learning, there was a higher agreement with the simplicity of using or learning the e-learning. in comparison, there was more disagreement about the user-friendliness of e-learning (46.8%). around one-third of faculty staff agreed (strongly agreed or agreed) with most attitude on e-learning strengths, while more than half of them considered e-learning a supplemental tool and preferred a combination of e-learning and face to face learning. the highest percentage of faculty staff had agreed with the cost-effectiveness of e-learning (46.8%), using it as a supplemental tool (50.6%), and preference for the combination of e-learning and face to face learning (59.5%). most faculty staff members agreed with all aspects of perception on challenges facing e-learning. the highest agreement was about poor internet connectivity (79%) and computer literacy (71%), as shown in table 3. most of the students (88.7%) and faculty staff (90%) stated that covid-19 affects the learning process. most of the students disagreed (strongly disagreed or disagreed) with all aspects of the effect of the covid-19 pandemic on education, including the willingness to continue education (41.5%), the ability of e-learning to substitute face to face learning for the theoretical part (50.4%) and different clinical, skills lab and other lab learning (66.8–74.4%). most students disagreed (strongly disagreed or disagreed) with replacing the practical sessions with e-learning (63%) or postponing them to the following academic year (43%) as shown in table 4. table 1. participants’ knowledge about e-learning knowledge on e-learning students (n = 1706) faculties (n = 190) no yes no yes no. (%) no. (%) no. (%) no. (%) having idea about e-learning 1049 (61.5) 657 (38.5) 48 (25.3) 142 (74.7) interested in guidance on e-learning 1036 (60.7) 670 (39.3) 41 (21.6) 149 (78.4) e-learning facility at the institution, e.g., moodle 577 (33.8) 1129 (66.2) 24 (12.6) 166 (87.4) use of e-learning forms to communicate with colleagues or faculties 1334 (78.2) 372 (21.8) 91 (47.9) 99 (52.1) 98 j contemp med sci | vol. 8, no. 2, march–april 2022: 96–101 faculty and students’ attitude toward e-learning at the start of the covid-19 pandemic original n.e. alkhateeb et al. table 2. students’ attitude about e-learning use (n = 1706) attitude strongly disagree/disagree neutral strongly agree/agree no. (%) no. (%) no. (%) e-learning usage e-learning is easy to use 1002 (58.7) 450 (26.4) 254 (14.9) learning to use e-learning is easy 924 (54.2) 387 (22.7) 395 (23.2) it is easy to become skillful at using e-learning 937 (54.9) 386 (22.6) 383 (22.5) e-learning is user friendly 1304 (76.4) 247 (14.5) 155 (9.1) strengths courses readily available online 954 (56.0) 407 (23.9) 345 (20.2) an interactive mode 1032 (60.5) 429 (25.1) 245 (14.4) cost-effective 657 (38.5) 423 (24.8) 626 (36.7) complete task more quickly 858 (50.3) 455 (26.7) 393 (23.0) attain more knowledge 1006 (58.9) 400 23.4) 300 (17.6) acquire better skills 1073 (62.9) 386 (22.6) 247 (14.5) easy accessibility, flexibility and interactivity 1058 (62.0) 381 (22.3) 267 (15.7) group discussion for complex topic 1104 (64.7) 370 (21.7) 232 (13.6) a supplemental tool 935 (54.8) 431 25.3) 340 (19.9) prefer a combination of e-learning and face-to-face 873 (51.1) 424 (24.9) 409 (24.0) challenges lack of computer and smart phone 349 (20.4) 214 (12.5) 1143 (67.0) poor internet connectivity 230 (13.5) 182 (10.7) 1294 (75.9) computer literacy 248 (14.5) 237 (13.9) 1221 (71.6) additional burden 361 (21.1) 668 (39.2) 677 (39.7) difficulty in arranging time between faculty and students 339 (19.9) 288 (16.9) 1079 (63.3) lack knowledge and information about e-learning 274 (16.1) 357 20.9) 1075 (63.0) table 3. faculties’ attitude about e-learning use (n = 190) attitude strongly disagree/disagree neutral strongly agree/agree no. (%) no. (%) no. (%) e-learning usage e-learning is easy to use 57 (30.0) 77 (40.5) 56 (29.5) learning to use e-learning is easy 41 (21.6) 64 (33.7) 85 (44.8) it is easy to become skillful at using e-learning 41 (21.6) 65 (34.2) 84 (44.2) e-learning is user friendly 89 (46.8) 52 (27.4) 49 (25.8) strengths courses readily available online 46 (24.2) 70 (36.8) 74 (38.9) an interactive mode 59 (31.1) 73 (38.4) 58 (30.6) cost-effective 36 (18.9) 65 (34.2) 89 (46.8) complete task more quickly 51 (26.8) 80 (42.1) 59 (31.1) attain more knowledge 54 (28.4) 66 (34.7) 70 (36.8) acquire better skills 59 (31.0) 67 (35.3) 64 (33.7) easy accessibility, flexibility and interactivity 61 (32.1) 72 (37.9) 57 (30.0) group discussion for complex topic 65 (34.3) 65 (34.2) 60 (31.6) a supplemental tool 35 (18.4) 59 (31.1) 96 (50.6) prefer a combination of e-learning and face-to-face 31 (16.3) 46 (24.2) 113 (59.5) (continued) 99j contemp med sci | vol. 8, no. 2, march–april 2022: 96–101 n.e. alkhateeb et al. original faculty and students’ attitude toward e-learning at the start of the covid-19 pandemic table 3. faculties’ attitude about e-learning use (n = 190)—continued attitude strongly disagree/disagree neutral strongly agree/agree no. (%) no. (%) no. (%) challenges lack of computer and smart phone 37 (19.5) 40 (21.1) 113 (59.5) poor internet connectivity 14 (7.4) 26 (13.7) 150 (79.0) computer literacy 18 (9.5) 37 (19.5) 135 (71.0) additional burden 26 (13.6) 79 (41.6) 85 (44.8) difficulty in arranging time between faculty and students 33 (17.3) 45 (23.7) 112 (59.0) lack knowledge and information about e-learning 18 (9.5) 55 (28.9) 117 (61.6) table 4. students’ perspectives on the effect of covid-19 pandemic on education and the alternative study options (n = 1706) item strongly disagree/disagree neutral strongly agree/agree no. (%) no. (%) no. (%) effect of covid-19 pandemic on education willing to continue education 709 (41.5) 419 (24.6) 578 (33.9) e-learning can replace face-to-face learning for the theoretical part 861 (50.4) 348 (20.4) 497 (29.1) for the practical part, e-learning can replace learning gained in: clinical placement in hospital wards 1268 (74.4) 246 (14.4) 192 (11.2) clinical placement in the outpatient clinic and phc 1192 (69.9) 308 (18.1) 207 (12.1) skills lab and other labs 1140 (66.8) 304 (17.8) 262 (15.4) if it was impossible to arrange for practical sessions due to this pandemic, what would be the best way to replace them e-learning 1075 (63.0) 349 (20.50) 282 (16.5) postpone for the next academic year 735 (43.0) 382 (22.40) 589 (34.5) almost half of the faculty staff agreed (strongly agreed or agreed) with the willingness to continue education (43.7%) and the ability to substitute face to face learning by e-learning for theoretical parts (51%). most of them disagreed with e-learning replacing the different components of the practical parts (63.7–80.5%). the highest percentage of faculty agreed with replacing practical sessions with e-learning. those who agreed with postponing practical sessions to the following academic year were slightly higher (37.4%) than those who disagreed (31%) as shown in table 5. a higher proportion of students preferred performing the final written assessment as paper examinations in the study halls (42.8%) than delaying the examination for the following academic year (28.5%). the percentages of the faculty who preferred paper examination in the study halls (31.6%), online assessment (31.1%), or delaying it for the following year (33.2%) were almost similar. discussion the novel innovations in e-learning have revolutionized the learning and teaching process, and thus students and educators need to adapt in utilizing them; the covid-19 pandemic further accelerated that need. only few studies have been carried out in iraq on e-learning.8-10 to the best of our knowledge, the current study is the first in iraq to assess the knowledge, attitude, and perception of e-learning among students and faculty during the lockdown period. the majority of our students are using the internet, at least sometimes, and they have the means to do so, though less than a half use laptop. this would suggest a good technological base to implement e-learning. similar results were obtained in other studies,1,11 which is expected from digital natives. although two-thirds of the students knew about the availability of e-learning facilities at their school, slightly over a third knew about e-learning and were interested in guidance on it. only less than a third used it for communication; this agrees with other studies.12,13 like many other educational institutes, this may be because the school does not use e-learning to its full potential as an educational resource but rather as a limited modern technology project.14 the average knowledge and limited usage of e-learning were reflected in their attitudes towards it, as two-thirds of them opposed its potential usefulness, namely, being an interactive, reachable, and flexible platform and appropriate for discussion on complex subjects and enhance their skills. many authors have studied students’ perceptions, and some had similar results to ours.1,15 keller and cernerud (2002) stated that if students with 100 j contemp med sci | vol. 8, no. 2, march–april 2022: 96–101 faculty and students’ attitude toward e-learning at the start of the covid-19 pandemic original n.e. alkhateeb et al. table 5. faculties’ perspectives on the effect of covid-19 pandemic on education and the alternative study options (n = 190) item faculties (n = 190) strongly disagree/disagree neutral strongly agree/agree no. (%) no. (%) no. (%) effect of covid-19 pandemic on education willing to continue education 39 (20.6) 68 (35.8) 83 (43.7) e-learning can replace face-to-face learning for the theoretical part 40 (21.1) 53 (27.9) 97 (51.0) for the practical part, e-learning can replace learning gained in: clinical placement in hospital wards 153 (80.5) 26 (13.7) 11 (5.80) clinical placement in the outpatient clinic and phc 143 (75.3) 34 (17.9) 14 (7.4) skills lab and other labs 121 (63.7) 45 (23.7) 24 (12.6) if it was impossible to arrange for practical sessions due to this pandemic, what would be the best way to replace them e-learning 79 (41.6) 65 (34.2) 46 (24.2) postpone for the next academic year 59 (31.0) 60 (31.6) 71 (37.4) e-learning experience were enrolled in their study, the disappointing results might have changed.15 we genuinely believe that this would have been the case in our results as well. in studies where good experience with e-learning was present, the perception was very good.16,17 the majority of our faculty staff had knowledge about availability of e-learning facilities in the university, had good knowledge about e-learning, and used it. the attitude of the faculty was equivocal about e-learning’s usefulness in teaching and learning. about 60% of them preferred a blended program which involve mixture of e-learning and face to face learning. a better response was observed by another study from south korea.18 this equivocal response may be due to the faculty’s continuous efforts to provide new learning resources on the one hand and to the limited time available to learn the new technologies on the other hand.19,20 according to responses, e-learning has other benefits that includes but not limited to, aids in freeing up the on-campus timetable, allowing more time to deliver more complex subjects, a greater opportunity for a higher level of cognitive learning, and, reducing the time needed for the pre-lab explanation in laboratories and skills labs.21-23 the literature shows that there are obstacles to the implementation of e-learning; these include problems related to technology, resources, skills, institutional strategies, and support. both students and faculty staff highlighted poor internet connectivity and a lack of hardware as the main technical obstacles to e-learning. similar findings were observed by others.24 other technology barriers include, learning management systems, and digital library, among others.2 essential prerequisites of e-learning quality include basic infrastructure maintained with regularly updated technologies, technical support, a sound and clear institutional policy and guide for both students and faculty staff.4,18,25 covid-19 has disrupted education worldwide. most of our students and faculty staff agree with that too. the pandemic has obliged universities all over the world to cease campus learning to control the spread of the disease. however, in times of suffering, there is always a hope. the pandemic forced the teaching community to search for alternatives. many have moved to e-learning to secure the continuance of teaching and assessment.26–28 about half of our students were skeptical of the ability of e-learning to substitute face-to-face learning for the theoretical part. half of them were willing to attend practical sessions if the situation permits, and twothirds disagreed that e-learning can replace practical training. almost similar results were obtained from the faculty staff. however, the reason behind the indeterminate response from our students and faculty staff could be their general attitude towards e-learning or the pandemic’s direct distressful effect. cao et al. and li et al. concluded that 24.9% and 27% of their students had significant distress during the outbreak.29,30 limitation of the study the study participants were from one university and generalization of the result for all medical students is not possible. the study was conducted at the emergence of covid-19 pandemic with high uncertainty among students about the fate of their study and assessment. conclusion most students had concerns about using e-learning and its usefulness, while faculty staff were more comfortable with its use. poor internet connectivity and computer literacy were the main challenges of applying e-learning. the students generally preferred to have paper examinations in the study halls for their final written assessment. however, the faculty staff preference was similarly distributed among paper examination in the study halls, online assessment, and delaying assessment for the following year. the findings from this study shows that our students and faculty are not quite ready for e-learning. this could be due to technical barriers, as well as inadequate institutional preparedness and support. it also shows that the covid-19 pandemic has significantly disrupted medical education. thus, proper technical and institutional foundations are essential for successful e-learning, especially during social distancing measures. 101j contemp med sci | vol. 8, no. 2, march–april 2022: 96–101 n.e. alkhateeb et al. original faculty and students’ attitude toward e-learning at the start of the covid-19 pandemic authors’ declaration • conflicts of interest: none. • we hereby confirm that all the tables in the manuscript are ours. • ethical clearance: the project was approved by the local ethical committee in college of medicine, hawler medical university. authors contribution ns, na, as and aa participated in study design and concept. na and as collected the data. ns performed data analysis and interpretation. all authors contributed in writing the draft of the manuscript and critically reviewed and approved the final version and are responsible for the content and similarity index of the manuscript. references 1. linjawi ai, alfadda ls. students’ perception, attitudes, and readiness toward online learning in dental education in saudi arabia: a cohort study. adv med educ pract. 2018;9:855–863. https://doi.org/10.2147/amep.s175395. 2. o’doherty d, dromey m, lougheed j, hannigan a, last j, mcgrath d. barriers and solutions to online learning in medical education an integrative review. bmc med educ 2018;18(1):130. https://doi.org/10.1186/ s12909-018-1240-0. 3. langenau ee, lee r, fults m. blended learning educational format for third-year pediatrics clinical rotation. j am osteopath assoc 2017;117(4): 234–243. https://doi.org/10.7556/jaoa.2017.041. 4. mirmoghtadaie z, kohan n, rasouli d. determination and comparison of the factors related to effective blended learning in medical sciences from the viewpoints of instructors and learners. adv med educ pract. 2020;11:205–214. https://doi.org/10.2147/amep.s239216. 5. mian a, khan s. medical education during pandemics: a uk perspective. bmc med 2020;18(1):100. https://doi.org/10.1186/s12916-020-01577-y. 6. bin mubayrik hf. exploring adult learners’ viewpoints and motivation regarding distance learning in medical education. adv med educ pract. 2020;11:139–146. https://doi.org/10.2147/amep.s231651. 7. mahajan mv, kalpana r. a study of students’ perception about e-learning. indian j clin anat physiol. 2018;5(4):501–507. https://doi. org/10.18231/2394-2126.2018.0116. 8. ameen n, willis r, abdullah mn, shah m. towards the successful integration of e‐learning systems in higher education. br j educ technol. 2019;50(3): 1434–1446. 9. abdulrazak ns, ali ma. challenges of implementation e-learning platforms in iraqi universities, eng technol j. 2019;37(4c):400–406. https://doi. org/10.30684/etj.37.4c.3. 10. mahmod ma, ali abm, ahlan ar bin, shah a, seman msa. e-learning in iraqi universities: a review. in: 3rd international conference on computing, engineering, and design, icced 2017. institute of electrical and electronics engineers inc., 2018-march, pp. 1–4. https://doi.org/10.1109/ ced.2017.8308094. 11. visalam v, kumar a, prakash a, padmavathi r. knowledge, attitude and practice towards e-learning among medical undergraduate students. iosr j appl phys. 2015;7 (4):1–04. https://doi.org/10.9790/4861-07430104. 12. bediang g, stoll b, geissbuhler a, klohn a, stuckelberger s, nko’o, et al. computer literacy and e-learning perception in cameroon: the case of yaounde faculty of medicine and biomedical sciences. bmc med educ. 2013;13(1):57. https://doi.org/10.1186/1472-6920-13-57. 13. azmi m, zeehan s, hisham a. assessment of students’ perceptions towards e-learning management system (e-lms) in a malaysian pharmacy school: a descriptive study. malays. j. public health med. 2012:12(1):14–20. 14. barteit s, guzek d, jahn a, bärnighausen t, jorge mm, neuhann f. evaluation of e-learning for medical education in lowand middle-income countries: a systematic review, comput educ. 2020;145:03726. https://doi.org/10.1016/j. compedu.2019.103726. 15. keller c, cernerud l. students’ perceptions of e‐learning in university education. j educ media. 2002;27(1-2):55–67. https://doi. org/10.1080/1358165020270105. 16. eldeeb ra. students’ perceptions to e-learning. iosr j. res. method educ. 2014;4(3):33–36. https://doi.org/10.9790/7388-04343336. 17. ghanizadeh a, mosallaei s, dorche ms, sahraian a, yazdanshenas p. attitude and use of e-learning, education by medical students in shiraz, iran. intern med med investig j. 2018;3(3):108. https://doi.org/10.24200/ imminv.v3i3.83. 18. kim kj, kang y, kim g. the gap between medical faculty’s perceptions and use of e-learning resources. med educ online. 2017;22 (1): 10-12. https:// doi.org/10.1080/10872981.2017.1338504. 19. kim kj, kim g. development of e-learning in medical education: 10 years’ experience of korean medical schools. korean j med educ. 2019; 31 (3):205–214. https://doi.org/10.3946/kjme.2019.131. 20. pettersson f, olofsson ad. implementing distance teaching at a large scale in medical education: a struggle between dominant and non-dominant teaching activities, educ inf technol. 2015;20(2):359–380. https://doi. org/10.1007/s10639-013-9289-1. 21. mukhtar k, javed k, arooj m, sethi a. advantages, limitations and recommendations for online learning during covid-19 pandemic era. pakistan j med sci. 2020;36(covid19-s4):s27. https://doi.org/10.12669/ pjms.36.covid19-s4.2785. 22. toumas m, basheti ia, bosnic-anticevich s. comparison of small-group training with self-directed internet-based training in inhaler techniques. am j pharm educ. 2009;73(5):85. https://doi.org/10.5688/aj730585. 23. truncali a, lee jd, ark tk, gillespie c, triola m, hanley k, et al. teaching physicians to address unhealthy alcohol use: a randomized controlled trial assessing the effect of a web-based module on medical student performance. j subst abuse treat. 2011;40(2):203–213. https://doi. org/10.1016/j.jsat.2010.09.002. 24. kelly cm, vins h, spicer jo, mengistu bs, wilson dr, derbew m, et al. the rapid scale up of medical education in ethiopia: medical student experiences and the role of e-learning at addis ababa university. plos one. 2019;14(9):e0221989. https://doi.org/10.1371/journal. pone.0221989. 25. frehywot s, vovides y, talib z, mikhail n, ross h, wohltjen h, et al. e-learning in medical education in resource constrained lowand middleincome countries. hum resour health. 2013;11(4):1–15. https://doi. org/10.1186/1478-4491-11-4. 26. goh p.-s, sandars j. a vision of the use of technology in medical education after the covid-19 pandemic. mededpublish. 2020;9(1):49. https://www.mededpublish.org/submit/manuscriptpreview?prev=true&pguid=831eda45-5ac6-406d-89ec-d4f53cfb7d9b (accessed november 19, 2021). 27. zayapragassarazan z. strategies for online engagement of remote learners. f1000research. 2020;9:246. https://doi.org/10.7490/ f1000research.1117835.1. 28. masoumian hosseini t, ahmady s, edelbring s. teaching clinical decisionmaking skills to undergraduate nursing students via web-based virtual patients during the covid-19 pandemic: a new approach to the cyberpatienttm simulator. j. contemp. med. sci. [internet]. 2022 feb. 26 [cited 2022 apr. 3];8(1). 29. cao w, fang z, hou g, han m, xu x, dong j, et al. the psychological impact of the covid-19 epidemic on college students in china. psychiatry res. 2020;287:112934. https://doi.org/10.1016/j. psychres.2020.112934. 30. li y, wang y, jiang j, u.a. valdimarsdóttir, k. fall, f. fang, h. song, d. lu, w. zhang. psychological distress among health professional students during the covid-19 outbreak. psychol. med. 2021;51(11):1952–1954. https://doi. org/10.1017/s0033291720001555. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i2.1178 40 j contemp med sci | vol. 9, no. 1, january-february 2023: 40–47 original association of vertigo and nausea and vomiting of pregnancy yasemin hamlaci başkaya1* , kevser i̇lçioğlu1, alaattin ünsal2 1sakarya university, faculty of health sciences, sakarya, turkey. 2eskisehir osmangazi university, medical faculty, public health department, eskisehir, turkey. *correspondence to: yasemin hamlaci baskaya (e-mail yhamlaci@sakarya.edu.tr) abstract objective: to determine the prevalence of vertigo during pregnancy, to review some variables believed to be associated, and to determine the association between vertigo and nausea and vomiting. methods: this study is a cross-sectional study conducted on pregnant women between september 3 and november 30, 2020. the study group consisted of 560 pregnant women who agreed to take part in the study. chi-square test and logistic regression analysis (stepwise backward wald regression), mann-whitney u test and kruskal-wallis analysis were used for analysis. p ≤ 0.05 was accepted as statistical significance value. results: the number of pregnant women with a history of vertigo was found to be 208 (37.1%). the symptoms that were most commonly reported by those with a history of vertigo were nausea and vomiting, headache and stumbling while walking, respectively. it was determined that the level of nausea and vomiting was higher in women with vertigo but there was no difference between the type and severity of vertigo and the level of nausea and vomiting. conclusion: vertigo is one of the important health problems in pregnancy. it was determined that the level of nausea and vomiting was higher in women with vertigo. nausea and vomiting are the most common symptoms accompanying vertigo. keywords: pregnancy, vertigo, puqe test issn 2413-0516 introduction pregnancy is an important period during which the symptoms of disorders and their treatment should be evaluated carefully to avoid possible negative outcomes in maternal and infant health. during this period, important physiological and adaptive changes occur in almost every organ and system, such as cardiovascular, respiratory, hematological, gastrointestinal and endocrine, for the development and delivery of the fetus.1 these changes cause some symptoms and complaints. although such symptoms and complaints are transient during pregnancy, they have important repercussions on women’s quality of life. these disorders affect the life routine involving family, social and professional environment thus causing deterioration of physical and psychological well-being.2 the hormonal changes which occur during the menstrual cycle, gestation and menopause can cause changes in the homeostasis of labyrinthine fluids, as they have a direct effect on the enzymatic process and the action of neurotransmitters. these changes can be asymptomatic or present clinically as vestibular symptoms.3 vestibular symptoms include vertigo, unbalance, gait instability, feeling of floating and falls. however, during gestation, these same symptoms could be secondary to non-vestibular causes but they could be a consequence of hormonal, anatomical and physiological factors affecting the musculoskeletal system occurring in pregnancy.2 more than 50% of pregnant women experience dizziness or vertigo frequently in the first two gestational trimesters.4 vertigo is a sudden feeling of spinning. it is described as a sensation of movement or of surrounding objects moving when they are not.5 patients should be asked to specifically describe their dizziness in their own words. the type of dizziness and vertigo can be identified based on patient’s words.6 otological symptoms usually develop with changing levels of progesterone and estrogen during pregnancy. women’s hormonal cycle starts to change after an egg cell is fertilized by a sperm.7 hormones during pregnancy are important for the development of the fetus; however, the effects of hormones usually go beyond the uterus and change the physiological activities of the body. most of the hormonal changes during pregnancy usually produce no harmful effect on the mother or fetus but some of the changes may become pathological and cause restlessness, anxiety and discomfort.7,8 most of the otological symptoms are insignificant and transient but the determination of the etiology of these symptoms by clinicians is important to provide pregnant women with treatment and assurance. pregnant women increase their quality of life by avoiding unwanted drugs and managing these symptoms safely without causing any effect on the fetus.9 hormonal and physical changes during pregnancy may cause not only vertigo but also nausea and vomiting. nausea and vomiting are very common in the first trimester particularly. the prevalence of nausea and vomiting of pregnancy ranges from 50% to 80%.10,11 symptoms range from mild nausea to excessive vomiting to a severe form of nausea vomiting called hyperemesis gravidarum (hg) with electrolyte imbalances, dehydration and weight loss. nausea and vomiting may cause discomfort in pregnant women in the mildest form but may become a serious threat to the lives of fetus and pregnant woman in the most severe form. nausea and vomiting of pregnancy is an important symptom decreasing the quality of life of pregnant women. etiology of nausea and vomiting in general seems to be multifactorial. mechanisms behind nausea-vomiting in pregnancy include vestibular, olfactory, gastrointestinal stimuli and hormonal processed in the central nervous system. accordingly, women may be more prone to nausea-vomiting in various events or diseases. however, this issue has not been thoroughly covered in the literature.10,11 considering that hormonal factors are involved in both vertigo and nausea and vomiting, it seems logical to look for an association between these conditions.12 although its pathophysiology remains uncertain, as nausea and vomiting are important symptoms of vestibular (submitted: 12 september 2022 – revised version received: 26 october 2022 – accepted: 14 november 2022 – published online: 26 february 2023) http://orcid.org/0000-0002-1533-8667 http://orcid.org/0000-0001-8353-1605 mailto:yhamlaci@sakarya.edu.tr 41j contemp med sci | vol. 9, no. 1, january-february 2023: 40–47 y.h. başkaya et al. original association of vertigo and nausea and vomiting diseases, its association with the vestibular system should not be disregarded. there is very limited evidence that explains the effects of hormonal and physical changes during pregnancy on the otolithic organs of the vestibular system and evaluates the association between vertigo and nausea and vomiting.4 this study was carried out to determine the prevalence of vertigo during pregnancy, to examine some of the variables thought to be related, and to reveal the relationship between vertigo and nausea-vomiting. materials and methods this is a cross-sectional study conducted on pregnant women who presented to sakarya training and research hospital in turkey from 3 september to 30 november 2020. approval of the sakarya university faculty of medicine non-interventional studies ethics committee (resolution number 71522473/050.01.04/29 dated 26/02/2019) was obtained to conduct the study. permission was obtained from the hospital management for data collection. by examining the literature, a questionnaire form was prepared to be suitable for the purpose of the study. the prepared questionnaire form included some sociodemographic characteristics of pregnant women, some characteristics related to pregnancy and some diseases, the presence of vertigo, its type and severity and some variables thought to be related, the presence of accompanying symptoms in those with a history of vertigo, and the puqe test questions. in this study, the minimum number of pregnant women to be reached was calculated as 549 (p: 0.35, comparison p: 0.29, alpha: 0.05, power of test: 0.85). during the data collection process, 560 pregnant women who applied to the pregnant follow-up clinic of the hospital and agreed to participate in the study constituted the study group. interviews with pregnant women were held in the waiting room of the pregnant follow-up outpatient clinic. after informing the pregnant woman about the subject and purpose of the study, verbal consent was obtained from the pregnant women who agreed to participate in the study. the previously prepared questionnaires were filled by the pregnant women under supervision. this process took about 15–20 minutes. the women who had a history of dizziness during pregnancy were considered to have “vertigo” in this study. vertigo was identified as “spinning vertigo” if it feels similar to riding a merry-go-round, “swaying vertigo” if it feels like being on a small boat, “orthostatic dizziness” if it causes vision to go black when standing up quickly, and “unspecific dizziness” if it is identified other than these types. vertigo severity; it is defined as “mild” if it does not prohibit daily tasks and activities of the pregnant, “moderate” if it causes difficulty in performing daily tasks and activities, and “severe” if it prohibits daily tasks and activities. the puqe test was used to determine the level of nausea and vomiting of pregnant women in our study. this test was first developed by rhodes et al. in 1984 for the assessment of chemotherapy-induced nausea and vomiting but was also used in several studies to rate nausea and vomiting of pregnancy.13 prepared by adapting from the rhodes scoring system, 3-item puqe test includes questions about the number of nausea attacks, the number of vomiting and the number of retching episodes. the scores to be obtained from the puqe test ranges from 3 to 15 and higher scores suggest more severe nausea and vomiting.14,15 the data were evaluated in the ibm spss (version 20.0) statistical package program in computer environment. the shapiro-wilk test was used to determine the normal distribution of data. chi-squared test, logistic regression analysis (wald’s backward stepwise regression), mann-whitney u test and kruskal-wallis analysis were used for the analyses. statistical significance level was accepted as p ≤ 0.05. results the ages of women in the study group ranged from 17 to 44, with a mean age of 28.41 ± 5.18 years. the number of pregnant women with a history of vertigo was found to be 208 (37.1%) in our study. the distribution of those with and without a history of vertigo in the study group according to some sociodemographic characteristics is given in table 1. of the women in the study group, 183 (32.7%) stated that they did not give birth before, 174 (31.1%) reported that it was their first pregnancy and 401 (71.6%) stated that they had a history of nausea and vomiting of pregnancy. 114 women (29.8%) had a history of vertigo during their previous pregnancies. 160 women (28.6%) in the study group had a history of physician-diagnosed disease that may be associated with dizziness within the last 1 year. it was found that 9 women (1.6%) had a hearing impairment, 281 women (50.2%) had a history of recurring back/neck pain within the last 1 year and 22 women (3.9%) had a history of head trauma within the last 1 year. the distribution of women with or without a history of vertigo in the study group by characteristics related to pregnancy and some diseases is given in table 2. the results of the logistic regression analysis, which were determined to be associated with vertigo in our study such as level of education, working status, number of pregnancy, history of nausea and vomiting of pregnancy, history of vertigo in previous pregnancies, history of vertigo within 3 months before pregnancy, history of physician-diagnosed disease associated with vertigo within the last 1 year, hearing impairment, presence of back-neck pain within the last 1 year, history of head trauma within the last 1 year and history of a depressing event within the last 1 year are given in table 3. of the pregnant women with a history of vertigo in the study group, 35 women (16.8%) had spinning vertigo, 36 women (17.3%) had swaying vertigo, 128 women (61.5%) had orthostatic dizziness and 9 women (4.3%) had unspecific dizziness. of the pregnant women with a history of vertigo, 130 women (62.5%) had mild, 55 women (26.4%) had moderate and 23 women (11.1%) had severe vertigo. the scores obtained from the puqe test by the pregnant women ranged from 3 to 13 with a mean score of 4.31 ± 1.84 (median: 3.0). the distribution of scores obtained by the pregnant women from the puqe test by the presence, type and severity of vertigo is given in table 4. the symptoms that were most commonly reported by those with a history of vertigo were nausea and vomiting (25.1%), headache (18.6%) and stumbling while walking (13.4%), respectively. the distribution of accompanying complaints in those with a history of vertigo in the study group is given in table 5. 42 j contemp med sci | vol. 9, no. 1, january-february 2023: 40–47 association of vertigo and nausea and vomiting original y.h. başkaya et al. table 2. the distribution of women with or without a history of vertigo in the study group by characteristics related to pregnancy and some diseases characteristics related to pregnancy and some diseases history of vertigo statistical analysis x2; pno n (%)* yes n (%)* total n (%)** number of childbirths 0 124 (67.8) 59 (32.2) 183 (32.7) 3.784; 0.1511 125 (62.8) 74 (37.2) 199 (35.5) 2 and above 103 (57.9) 75 (42.1) 178 (31.8) number of pregnancies first 119 (68.4) 55 (31.6) 174 (31.1) 8.921; 0.012second 115 (66.9) 57 (33.1) 172 (30.7) third and above 118 (55.1) 96 (44.9) 214 (38.2) gestational week 35 and below 173 (63.4) 100 (36.6) 273 (48.8) 0.060; 0.806 36 and above 179 (62.4) 108 (37.6) 287 (51.2) (continued) table 1. the distribution of those with and without a history of vertigo in the study group by some socio-demographic characteristics socio-demographic characteristics history of vertigo statistical analysis x2; pno n (%)* yes n (%)* total n (%)** age group ≤24 81 (60.0) 54 (40.0) 135 (24.1) 0.799, 0.850 25–29 130 (63.4) 75 (36.6) 205 (36.6) 30–34 93 (65.09) 50 (35.0) 143 (25.5) ≥35 48 (62.3) 29 (37.7) 77 (13.8) level of education primary school and lower 80 (60.2) 53 (39.8) 133 (23.8) 10.812; 0.013 secondary school 102 (64.6) 56 (35.4) 158 (28.2) high school 90 (55.6) 72 (44.4) 162 (28.9) university 80 (74.8) 27 (25.2) 107 (19.1) working status not working 273 (60.7) 177 (39.3) 450 (80.4) 4.708, 0.030 working 79 (71.8) 31 (28.2) 110 (19.6) family income status low 17 (68.0) 8 (32.0) 25 (4.5) 0.317; 0.853middle 246 (62.4) 148 (37.6) 394 (70.4) high 89 (63.1) 52 (36.9) 141 (25.2) smoking non-smoker 307 (62.4) 185 (37.6) 492 (87.9) 0.365, 0.546 smoker 45 (66.2) 23 (33.8) 68 (12.1) alcohol consumption no 350 (62.9) 206 (37.1) 556 (99.3) fisher; 0.630yes 2 (50.0) 2 (50.0) 4 (0.7) total 352 (62.9) 208 (37.1) 560 (100.0) *:percentages were calculated based on the line total. **:percentages were calculated based on the column total. 43j contemp med sci | vol. 9, no. 1, january-february 2023: 40–47 y.h. başkaya et al. original association of vertigo and nausea and vomiting table 2. the distribution of women with or without a history of vertigo in the study group by characteristics related to pregnancy and some diseases—(continued) characteristics related to pregnancy and some diseases history of vertigo statistical analysis x2; pno n (%)* yes n (%)* total n (%)** history of nausea and vomiting of pregnancy no 122 (76.7) 37 (23.3) 159 (28.4) 18.303; 0.000 yes 230 (57.4) 171 (42.6) 401 (71.6) history of dizziness within 3 months before pregnancy no 337 (70.6) 140 (29.4) 477 (85.2) 83.711; 0.000 yes 15 (18.1) 68 (81.9) 83 (14.8) history of dizziness in previous pregnancies no 205 (76.2) 64 (23.8) 269 (70.23) 92.492; 0.000 yes 27 (23.7) 87 (76.3) 114 (29.8) obesity before pregnancy no 284 (63.5) 163 (36.5) 447 (79.8) 0.436; 0.509 yes 68 (60.2) 45 (39.8) 113 (20.2) anemia no 143 (59.8) 96 (40.2) 239 (42.7) 1.634; 0.201 yes 209 (65.1) 112 (34.9) 321 (57.3) hypertension no 343 (62.8) 203 (37.2) 546 (97.5) 0.000; 1.000 yes 9 (64.3) 5 (35.7) 14 (2.5) history of physician-diagnosed disease that may be associated with dizziness within the last 1 year no 112 (70.0) 48 (30.0) 160 (28.6) 12.787; 0.012 flu/common cold 204 (61.8) 126 (38.2) 330 (58.9) otitis media 4 (28.6) 10 (71.4) 14 (2.5) sinusitis 22 (62.9) 13 (37.1) 35 (6.2) tonsillitis 10 (47.6) 11 (52.4) 21 (3.8) hearing impairment no 343 (62.3) 208 (37.7) 551 (98.4) fisher; 0.030 yes 9 (100.0) 0 (0.0) 9 (1.6) history of recurring back/neck pain within the last 1 year no 197 (70.6) 82 (29.4) 279 (49.8) 14.312; 0.000 yes 155 (55.2) 126 (44.8) 281 (50.2) motion sickness during travel no 240 (64.7) 131 (35.3) 371 (66.2) 1.582; 0.209 yes 112 (59.3) 77 (40.7) 189 (33.8) history of a head trauma within the last 1 year no 343 (63.8) 195 (36.2) 538 (96.1) 3.797; 0.050 yes 9 (40.9) 13 (59.1) 22 (3.9) history of a depressing event within the last 1 year no 274 (70.8) 113 (29.2) 387 (69.1) 33.860; 0.000 yes 78 (45.1) 95 (54.99) 173 (30.9) total 352 (62.9) 208 (37.1) 560 (100.0) *: percentages were calculated based on the line total. **: percentages were calculated based on the column total. 44 j contemp med sci | vol. 9, no. 1, january-february 2023: 40–47 association of vertigo and nausea and vomiting original y.h. başkaya et al. table 5. accompanying complaints in those with a history of vertigo in the study group symptoms n % headache 78 18.6 nausea and vomiting 105 25.1 hearing loss 5 1.2 ringing in the ears/tinnitus 42 10.0 ear pressure 7 1.7 sensation loss/numbness in limbs 37 8.8 stumbling while walking 56 13.4 double vision 16 3.8 light sensitivity/intolerance of light 28 6.7 irritation, stinging and redness in eyes 12 2.9 excessive sweating 33 7.8 total 419 100.0 *numbers are based on the symptoms reported. table 3. the results of the logistic regression model created with the variables determined to be associated with vertigo in the study group (last digit: 7) variables ß sea p orb 95% cic having a revenue-generating work (reference: working) not working 0.623 0.378 0.099 1.865 0.889–3.912 history of vertigo in previous pregnancies (reference: no) yes 2.111 0.303 0.000 8.257 4.558–14.958 history of vertigo within 3 months before pregnancy (reference: no) yes 2.416 0.443 0.000 11.198 4.695–26.704 history of a depressing event within the last 1 year (reference: no) yes 1.272 0.288 0.000 3.567 2.027–6.278 history of physician-diagnosed disease that may be associated with vertigo within the last 1 year (reference: no) sinusitis 0.770 0.561 0.170 2.159 0.719–6.484 flu/common cold 0.163 0.318 0.608 1.178 0.631–2.197 tonsillitis 1.499 0.693 0.031 4.478 1.151–17.425 otitis media 0.232 0.882 0.793 1.261 0.224–7.100 constant –2.641 0.457 0.000 – – sea: standard error, orb: odd’s ratio, cic: confidence interval. table 4. the distribution of scores obtained by the pregnant women from the puqe test by the presence, type and severity of vertigo presence, type and severity of vertigo n puqe test score median (min-max) test value z/kw; p vertigo no 352 3.0 (3.0–13.0) 4.853; 0.000yes 208 5.0 (3.0–13.0) type of vertigo spinning vertigo 35 3.0 (3.0–13.0) 3.728; 0.292 swaying vertigo 36 5.0 (3.0–11.0) orthostatic dizziness 128 5.0 (3.0–9.0) unspecific dizziness 9 3.0 (3.0–7.0) severity of vertigo mild 130 4.0 (3.0–13.0) 2.850; 0.241moderate 55 5.0 (3.0–9.0) severe 23 6.0 (3.0–10.0) total 208 3.0 (3.0–13.0) discussion women are more inclined to have dizziness/vertigo due to hormonal changes and metabolic disorders. many studies reported a correlation between vertigo and hormonal changes and sex.16,17 some symptoms may increase as the release of neurotransmitters during pregnancy may change the biochemical control of the inner ear. the prevalence of dizziness/ vertigo was reported to be 10–52% in the studies conducted on pregnancy.3,18 in the study of scmith et al. on 82 pregnant women, vertigo was reported for more than half the pregnant women (52%).3 agampodi et al. (2013) found that 24% of 466 pregnant women experienced dizziness.19 in our study, the number of pregnant women with a history of vertigo was found to be 208 (37.1%). it can be assumed that a possible vestibular change associated with a hormonal change during pregnancy may cause vertigo. considering the factors that may affect vertigo, although there are studies showing that the prevalence of vertigo is increasing with age,20,21 there was no study reviewing the association between the vertigo of pregnancy and age in the literature. in our study, there was no difference between the age groups of pregnant women in terms of vertigo. considering the association between level of education and vertigo, there were fewer pregnant women with a history of vertigo among pregnant women with a university degree. in their study on 150 adults, rashid and abed (2021) found that vertigo was not associated with the level of education and working status.22 although our study showed that the prevalence of vertigo was higher in those without a revenue-generating job, the logistic 45j contemp med sci | vol. 9, no. 1, january-february 2023: 40–47 y.h. başkaya et al. original association of vertigo and nausea and vomiting regression analysis indicated that unemployment was not a risk factor for vertigo. li et al. (2020) reported in their meta-analysis that there was no association between vertigo and daily life habits such as smoking and alcohol consumption.23 the meta-analysis of chen et al. (2020) showed that there was no association between the benign paroxysmal positional vertigo (bppv) and smoking and alcohol consumption. in our study, there was no difference between the lifestyles of pregnant women (obesity, alcohol consumption, smoking) in terms of the prevalence of vertigo.20 the studies on pregnancy and vertigo are heterogeneous, few in number and of low quality. retrospective studies of wu et al. (2019) and swain et al. (2020) reviewed pregnant women who had problems due to various vestibular disorders.9,24 these studies reported that case-controlled studies or studies with a large sample size are needed to consider pregnancy a risk factor for the development of vertigo and vestibular disorders.25 the variables such as pregnancy, number of childbirths, history of vertigo or nausea and vomiting before pregnancy were not evaluated in many studies. in our study, there was no association between the prevalence of vertigo and the number of childbirths but the prevalence of vertigo was higher in women who had three or more pregnancies. a case series study conducted in taiwan determined that pregnant women of advanced maternal age (34 years or older) and primipara women in the third trimester of pregnancy are inclined to develop vertigo.26 the levels of estrogen and progesterone vary during pregnancy. the effect of hormones on vertigo during pregnancy remains uncertain. in their study on 80 pregnant women, mgbe et al. (2017) reported that six women (7.5%) had mild vertigo attacks during the first trimester.27 in their presentation of 3 cases, coban et al. (2017) reported that all cases were diagnosed with vertigo during the late trimesters, when estrogen levels are relatively low and progesterone levels are high.18 schmidt et al. (2010) found that the most common vestibular symptom in pregnant women was vertigo (22.72%) in the first trimester.3 there was no association between the gestational week and vertigo in our study. vestibular symptoms such as nausea, vomiting, gait instability, dizziness and vertigo usually develop together. increased feeling of dizziness or vertigo in women having nausea of pregnancy may cause vomiting. vertigo and nausea and vomiting trigger each other affecting the quality of life negatively. 28 in a study on pre-pregnancy and post-pregnancy issues in women with hyperemesis gravidarum, conditions in 449 women with hyperemesis gravidarum (case group) were compared to 459 unaffected women (controls). while dizziness (4.68%), nausea (4.01%) and vertigo (3.34%) were observed in the pre-pregnancy case group, their rates were 1.31%, 0.22% and 0.65%, respectively, in the control group. the rates of dizziness, nausea and vertigo were 12.5%, 4.23% and 2.67% in the post-pregnancy case group and 1.09%, 0.0% and 0.22% in the control group, respectively.29 in our study, the prevalence of vertigo was higher in those with a history of nausea and vomiting of pregnancy. the conditions developed before pregnancy are expected to aggravate during pregnancy. many studies have proven that health condition and symptoms of women before pregnancy affect their health during pregnancy.29-33 consistently, the presence of a history of dizziness within 3 months before pregnancy is one of the important risk factors for vertigo in our study (or: 11.198; p: 0.000). furthermore, it was determined that the prevalence of vertigo was 8.257 times higher in women with a history of dizziness in previous pregnancies than those without a history of dizziness in previous pregnancies. in their meta-analysis of 60 studies and 24 risk factors, li et al. (2020) identified that the risk factors for vertigo included female gender, advanced age, hyperlipidemia, diabetes, hypertension, head trauma, otitis media, and long use of computers.23 in their meta-analysis of 19 studies including 2,618 patients, chen et al. (2020) also identified that head trauma is a risk factor for vertigo (or = 3.42; 95% ci, 1.21–9.70; p = 0.02).20 in a qualitative study on 31 pregnant women with anemia, chatterjee et al. (2014) reported that 11 pregnant women had dizziness in addition to anemia.34 in our study, the prevalence of vertigo was 4.478 times higher in those with a history of physician-diagnosed tonsillitis within last 1 year. it was also found that the prevalence of vertigo was lower in those with a hearing impairment but higher in those with a history of recurring back/neck pain within the last 1 year, with a history of a head trauma within the last 1 year and with a history of a depressing event within the last 1 year (or: 3.567, p: 0.000) (p < 0.05 for each). to the contrary of some studies, there was no difference between the prevalence of vertigo and the presence of obesity, anemia and hypertension before pregnancy (p > 0.05 for each).35-37 nausea and vomiting of pregnancy affects approximately 75–80% of pregnant women.10 agampodi et al. reported that 325 (69.7%) out of 466 pregnant women experienced nausea and vomiting of pregnancy.19 there is a common ground between nausea and vomiting of pregnancy and vertigo that is a vestibular disorder. women reporting dizziness or vertigo usually suffer from hyperemesis gravidarum. avoiding activity alleviates the symptoms of both disorders. there are limited studies on the role of vestibular system on nausea and vomiting.38 it is usually difficult to distinguish vertigo attacks and episodes of nausea and vomiting that are very common in the first trimester of the pregnancy. it was determined that the level of nausea and vomiting was higher in women with a history of vertigo in our study. there was no difference between the type and severity of vertigo and the level of nausea and vomiting (p > 0.05 for each). vertigo may be accompanied by various symptoms in addition to nausea and vomiting. in their study on 140 pregnant women, robbins et al. (2015) found that 9.3% of women with vertigo had also headache.39 in our study, the symptoms that were most commonly reported by those with a history of vertigo were nausea and vomiting (25.1%), headache (18.6%) and stumbling while walking (13.4%), respectively. limitations and strengths the limitations of the study are that it was a cross-sectional study, no scale or laboratory method was used for vertigo, and that it was conducted only on pregnant women who applied to a hospital. the strength of this study is the presence of a few studies reviewing various factors that can be associated with vertigo in pregnant women. conclusion vertigo is one of the important health problems in pregnancy. history of vertigo in previous pregnancies, a history of vertigo 46 j contemp med sci | vol. 9, no. 1, january-february 2023: 40–47 association of vertigo and nausea and vomiting original y.h. başkaya et al. within the last 3 months before pregnancy, a history of a depressing event within the last 1 year and a history of physician-diagnosed tonsillitis within the last 1 year are important risk factors for vertigo. it was determined that the level of nausea and vomiting was higher in women with a history of vertigo. the symptoms that were most commonly reported by those with a history of vertigo were nausea and vomiting, headache and stumbling while walking, respectively. studies in the literature and our results indicate that vertigo during pregnancy needs to be examined carefully. the association between vertigo and pregnancy remains uncertain and there is very limited data in this matter. unfortunately, pregnant women usually underestimate the symptoms and disregard vertigo. obstetricians and midwives should be careful about pregnant women with vertigo. the factors that may be associated with vertigo should be reviewed and measures should be taken against the risk factors to improve the quality of life of pregnant women and avoid vertigo-related unwanted situations that may put maternal and fetal health at risk. furthermore, more extensive studies are required to determine the causes of vertigo during pregnancy, provide solutions for it and establish the association between vertigo and nausea and vomiting. acknowledgments the authors would like to thank all the pregnant women who participated in the study. conflict of interest the authors declare that there is no conflict of interest.  references 1. tan ek, tan el. alterations in physiology and anatomy during pregnancy. best practice & research clinical obstetrics & gynaecology. 2013;27(6): 791–802. 2. salvati a, apa r, loperfido a, scarano e, paludetti g, tropea a, et al. management of vertigo in pregnancy. italian journal of gynaecology and obstetrics. 2020;32(1):49–55. 3. schmidt pm, flores f, rossi ag, silveira af. hearing and vestibular complaints during pregnancy. brazilian journal of otorhinolaryngology. 2010;76(1):29–33. 4. bhavana g, kumar k, anupriya e. assessment of otolith function using vestibular evoked myogenic potential in women during pregnancy. brazilian journal of otorhinolaryngology. 2020. 5. kara i̇, yıldız mg, gümüştakım rş, doğaner a, sağıroğlu s, bilal n, et al. evalution of family physician’s awareness of vertigo: a cross-sectional study. turkish journal of family practice. 2021;25(2):59–65. 6. wipperman j. dizziness and vertigo. primary care: clinics in office practice. 2014;41(1):115–31. 7. murthy va, krishna k. hearing loss in pregnancy. indian journal of otolaryngology & head and neck surgery. 2013;65(1):1–2. 8. liang y-x, liu l, jin z-y, liang x-h, fu y-s, gu x-w, et al. the high concentration of progesterone is harmful for endometrial receptivity and decidualization. scientific reports. 2018;8(712):1–12. 9. swain sk, pati bk, mohanty jn. otological manifestations in pregnant women-a study at a tertiary care hospital of eastern india. journal of otology. 2020;15(3):103–6. 10. laitinen l, nurmi m, ellilä p, rautava p, koivisto m, polo-kantola p. nausea and vomiting of pregnancy: associations with personal history of nausea and affected relatives. archives of gynecology and obstetrics. 2020;302(4):947–55. 11. şimşek y, şimşek g, bayar muluk n, arıkan ok. olfactory dysfunction and oxidative stress in pregnant women with hyperemesis gravidarum. archives of gynecology and obstetrics. 2021;304(3):657–61. 12. black fo. maternal susceptibility to nausea and vomiting of pregnancy: is the vestibular system involved? american journal of obstetrics and gynecology. 2002;186(5):204–9. 13. koren g, piwko c, ahn e, boskovic r, maltepe c, einarson a, et al. validation studies of the pregnancy unique-quantification of emesis (puqe) scores. journal of obstetrics and gynaecology. 2005;25(3):241–4. 14. king tl, murphy pa. evidence-based approaches to managing nausea and vomiting in early pregnancy. journal of midwifery & women’s health. 2009;54(6):430–44. 15. sucu m, büyükkurt s, evrüke i̇, demir s, özgünen f, kadayıfçı o. the role of puqe (pregnancy-unique quantification of emesis and nausea) in evaluation of the indications for inpatient therapy in pregnants with nausea and vomiting. turkiye klinikleri j gynecol obst. 2009;19(6): 317–21. 16. jeong s-h. benign paroxysmal positional vertigo risk factors unique to perimenopausal women. frontiers in neurology. 2020;11(589605):1–6. 17. lindell e, karlsson t, kollén l, johansson m, finizia c. benign paroxysmal positional vertigo and vestibular impairment among older adults with dizziness. laryngoscope investigative otolaryngology. 2021;6(3):488–95. 18. çoban k, yiğit n, aydın e. benign paroxysmal positional vertigo in pregnancy. turkish archives of otorhinolaryngology. 2017;55(2):83. 19. agampodi sb, wickramasinghe nd, horton j, agampodi tc. minor ailments in pregnancy are not a minor concern for pregnant women: a morbidity assessment survey in rural sri lanka. plos one. 2013;8(5):e64214. 20. chen j, zhao w, yue x, zhang p. risk factors for the occurrence of benign paroxysmal positional vertigo: a systematic review and meta-analysis. frontiers in neurology. 2020;11:506. 21. wassermann a, finn s, axer h. age-associated characteristics of patients with chronic dizziness and vertigo. journal of geriatric psychiatry and neurology. 2021:1–6. 22. rashid zs, abed bj. relationship between the effect of severity vertigo and demographic characteristic for iraqi patients. indian journal of forensic medicine & toxicology. 2021;15(3):785–90. 23. li s, wang z, liu y, cao j, zheng h, jing y, et al. risk factors for the recurrence of benign paroxysmal positional vertigo: a systematic review and metaanalysis. ear, nose & throat journal. 2020:1–23. 24. wu ph, cheng pw, young yh. inner ear disorders in 68 pregnant women: a 20‐year experience. clinical otolaryngology. 2017;42(4):844–6. 25. frosolini a, marioni g, gallo c, de filippis c, lovato a. audio-vestibular disorders and pregnancy: a systematic review. american journal of otolaryngology. 2021;42(5):103136. 26. chen j-j, chang h-f, chen d-l. vestibular migraine in a female with unexpected pregnancy. archives of neuroscience. 2016;3(1):e22924. 27. mgbe rb, umana an, adekanye ag, offiong me. ear nose and throat changes observed in pregnancy in calabar nigeria. global journal of pure and applied sciences. 2017;23(2):355–9. 28. lagadec n, steinecker m, kapassi a, magnier am, chastang j, robert s, et al. factors influencing the quality of life of pregnant women: a systematic review. bmc pregnancy and childbirth. 2018;18(455):1–14. 29. tian r, macgibbon k, martin b, mullin p, fejzo m. analysis of pre-and postpregnancy issues in women with hyperemesis gravidarum. autonomic neuroscience. 2017;202:73–8. 30. lewandowska m, więckowska b, sajdak s. pre-pregnancy obesity, excessive gestational weight gain, and the risk of pregnancy-induced hypertension and gestational diabetes mellitus. journal of clinical medicine. 2020;9(6):1980. 31. wang y-x, wang s, mitsunami m, manson je, rich-edwards jw, wang l, et al. pre-pregnancy menstrual cycle regularity and length and the risk of gestational diabetes mellitus: prospective cohort study. diabetologia. 2021;64(11):2415–24. 32. wei y-m, yang h-x, zhu w-w, liu x-y, meng w-y, wang y-q, et al. risk of adverse pregnancy outcomes stratified for pre-pregnancy body mass index. the journal of maternal-fetal & neonatal medicine. 2016;29(13):2205–9. 33. wesołowska e, jankowska a, trafalska e, kałużny p, grzesiak m, dominowska j, et al. sociodemographic, lifestyle, environmental and pregnancy-related determinants of dietary patterns during pregnancy. international journal of environmental research and public health. 2019;16(5):754. 34. chatterjee n, fernandes g. ‘this is normal during pregnancy’: a qualitative study of anaemia-related perceptions and practices among pregnant women in mumbai, india. midwifery. 2014;30(3):e56–e63. 47j contemp med sci | vol. 9, no. 1, january-february 2023: 40–47 y.h. başkaya et al. original association of vertigo and nausea and vomiting 35. ding j, liu l, kong w-k, chen x-b, liu x. serum levels of 25-hydroxy vitamin d correlate with idiopathic benign paroxysmal positional vertigo. bioscience reports. 2019;39(4). 36. han w, fan z, zhou m, guo x, yan w, lu x, et al. low 25-hydroxyvitamin d levels in postmenopausal female patients with benign paroxysmal positional vertigo. acta oto-laryngologica. 2018;138(5):443–6. 37. yang h, gu h, sun w, li y, wu h, burnee m, et al. estradiol deficiency is a risk factor for idiopathic benign paroxysmal positional vertigo in postmenopausal female patients. the laryngoscope. 2018;128(4): 948–53. 38. tulmaç öb, kılıç r, yaman s, aktulum f, şimşek g, erdinç s. evaluation of the vestibular system with video head impulse test in pregnant women with hyperemesis gravidarum. journal of obstetrics and gynaecology research. 2021;47(1):96–102. 39. robbins ms, farmakidis c, dayal ak, lipton rb. acute headache diagnosis in pregnant women: a hospital-based study. neurology. 2015;85(12):1024–30. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1306 395j contemp med sci | vol. 8, no. 6, november-december 2022: 395–399 original study interplay between asprosin with vitamin d in metabolic syndrome shaha abdallha mohammed1, thikra ali allwsh2* 1biochemistry laboratory, ibn sina teaching hospital, mosul, iraq. 2department of chemistry, collage of science, university of mosul, mosul, iraq. *correspondence to: thikra ali allwsh (e-mail: thekraaliallwsh@uomosul.edu.iq) (submitted: 18 may 2022 – revised version received: 21 june 2022 – accepted: 05 august 2022 – published online: 26 december 2022) abstract objective: the study aims to in investigating the role of the asprosin hormone and its relationship with vitamin d in patients with metabolic syndrome and clinical parameters. methods: the study included measurement asprosin hormone, vitamin d, and some biochemical variable levels in metabolic syndrome patients with age matching to the control group (35–65 years). the study includes (95) samples of metabolic syndrome patients [49 female, 46 males] who were attending the abdominal consultation unit at the ibn sina teaching hospital in mosul, iraq. mets were diagnosed in compliance with the criteria of the ncep (atp iii) and aha/nhlb. samples were collected during the period from january 2021 to december 2021. also, the study was carried out on 76 samples of apparently healthy (40 female, 36 male) as a control group. results: the findings revealed a significant increase in the concertation of the asprosin hormone in metabolic syndrome patients compared to the control group. also, it has been found that was a significant increase in the concertation of fasting glucose, insulin, homeostasis model for insulin resistance (homa-ir), triglycerides, low-density lipoprotein-cholesterol, very low-density lipoprotein-cholesterol, total cholesterol and urea. in addition to a decline in high-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol (non-hdl), (homa-b ), vitamin d and calcium among metabolic syndrome patients. there is also a significant inverse correlation between asprosin hormone with the vitamin d. conclusion: the study concluded that the hormone asprosin is a good indicator that reflects the status of metabolic syndrome patients and vitamin d appeared to be associated with mets, as well as the insulin resistance (ir) and lipid profile. keywords: asprosin, vitamin d, metabolic syndrome, obesity, insulin resistance issn 2413-0516 introduction the simple concept of ‘metabolic syndrome’ (mets) is a clustering of risk factors for diabetes and cardiovascular disease.1 the metabolic syndrome is a collection of linked risk factors with metabolic origins that seem to actively encourage the onset of atherosclerotic cardiovascular disease (ascvd), diabetes mellitus type 2 and dyslipidemia, which includes increased plasma glucose and apolipoprotein b (apob) and serum triglyceride levels, are the most generally acknowledged metabolic risk factors.2-3 another studies on metabolic syndrome persons show elevated blood pressure and a reduced level of high-density lipoprotein-cholesterol (hdl-c), prothrombotic, and proinflammatory states.4 indeed, of the metabolic risk factors–elevated triglyceride, low hdl-c, and hypertension or elevated glucose are well-known, significant risk factors5,3 even when just slightly aberrant, as is frequently seen in metabolic syndrome, each increases the risk.6 asprosin is associated with metabolic syndrome through its relationship with insulin resistance, obesity, inflammation, glucose and lipid metabolism, as well as in other diseases such as diabetic retinopathy, polycystic ovary syndrome, and anorexia nervosa.7-8 asprosin is a fasting-responsive orexigenic protein hormone that increases the liver’s ability to release glucose and stimulate the hypothalamus to appetite, it was originally discovered in patients with newborn progeroid syndrome by romere et al.9 these patients do not have asprosin, they discovered. as a result, scientists came to the conclusion that asprosin could affect how lipids and carbohydrates are metabolized.10 also, it has been shown that a major level of vitamin d (vit. d) is associated to a lower extent with metabolic syndrome, hyperglycemia, abdomen obesity and dyslipidemia.11 vitamin d (vit. d) is important for regulating osteoarticular homeostasis.12 hypovitaminosis d, a risk factor for reducing both bone mass and the atherosclerotic process which increases with age, it is a potential link between low vitamin d levels and an increased risk of cardiovascular disease also, showed a correlation between obesity, arterial hypertension, glucose intolerance, and dyslipidemia are all linked to low vitamin d levels.13-14 this study aimed to measure asprosin level, vitamin d, and relationship with clinical parameters in serum of metabolic syndrome patients participating in this study. materials and methods ethical approval this study was approved by the ethical committee of the nineveh health direction training center and human development, ministry of health and environment, iraq. informed written consent was obtained from all the participants before sample collection. research objects this study included (95) samples of metabolic syndrome patients [49 female, 46 males] aged between 35 and 65 years, the samples were collected during the time period from the beginning of august 2021 to the end of december 2021. all the samples were randomly selected from mets patients who were attending the abdominal consultation unit mailto:thekraaliallwsh@uomosul.edu.iq 396 j contemp med sci | vol. 8, no. 6, november-december 2022: 395–399 study interplay between asprosin with vitamin d in metabolic syndrome original s.a. mohammed et al. at the ibn sina teaching hospital in mosul. mets were diagnosed in compliance with the criteria of the ncep (atp iii) = national cholesterol education program (adult treatment panel iii) and aha/nhlb = american heart association/ national heart, lung and blood institute (has been in compliance with the criteria that included waist circumference, blood lipids, blood pressure( bp) and fasting glucose).15 also, the study was carried out on the control group consisting of samples [76] healthy group [40 female, 36 male], whom they did not have (mets), diabetes, or high blood pressure, as well as no taking any medication, they were carefully selected after a complete physical examination and laboratory tests and who match the age and body mass index with the patients. the data collected included age, gender, family medical history. using an automated blood pressure measuring system, systolic and diastolic pressures were measured twice, with average results used.and the subject’s blood pressure was taken after they had been sat for at least 5 minutes.16 the body mass index (bmi) was calculated using the formula (weight in kg/ height in m2). after overnight fasting [12 hours] five milliliters of venous blood were obtained from the participants, and the serum was isolated by centrifuged for 10–15 minutes at 4000 (rpm) to get the serum that was separated and frozen in aliquots at –20°c until used. laboratory analysis the serum asprosin hormone: was measured by using an elisa kit from sun long biological technology co., ltd kit (china).17 insulin hormone was measured by using (elisa) technique (sandwich using monobind kit (usa). vitamin d (ng/ml): was measured by using electrochemiluminescence (ecl) kit by cobas e411 analyzers, also, calcium (mmol/l) was estimated using biosystem kit (spain) and blood urea (mmol/l) was estimated using biosystem kit (france). the levels of glucose, total cholesterol, tg, and hdl were estimated using ready-made assay (kits) from the company (biolabs) and using enzymatic methods, the concentration of anther clinical parameters in serum was calculated using the following equation ldl-c (mmol/l) = total cholesterol-hdl-c-(tg/2.2) vldl-cholesterol conc.(mmol/l) = t.g/2.2 non-hdl-c = total cholesterol-hdl-c18 atherogenic index (ai) = log (tg/hdl-c) atherogenic coefficient (ac) = tc-hdl-c/hdl-c homa-ir = insulin (µu/ml) × glucose(m mol/l)/22.519 homa-β (%) = insulin (µu/ml) × 20/(glucose (mmol/l) -3.5)20 data analysis the obtained data were analyzed using originpro 2021 1. standard statistical procedures were used to obtain the mean and standard error. 2. the t-test is used to compare two parameters. 3. p-value ≤ 0.05 was assumed statistically significant. 4. to determine the relationship between various clinical data, linear regression analysis [pearson correlation coefficient (r)] was carried out. results baseline parameter comparison as displayed in (table 1) controls and mets groups. mets patients exhibited significantly increased bmi, waist circumference, systolic blood pressure (sbp), diastolic blood pressure (dbp), total cholesterol (tc), triglyceride (tg), low-density lipoprotein-cholesterol (ldl-c), vldl-c, atherogenic index, atherogenic coefficient and lower nonhigh-density lipoprotein cholesterol (non-hdl) and high-density lipoprotein cholesterol (hdl-c) than those of the controls (p < 0.001). the results in (table 2) detected a significant increase at (p < 0.001) in the concentration of glucose, insulin, and insulin resistance but a decrease in homeostasis model assessment for beta-cell function, (homa-β) in metabolic syndrome patients as compared with the control group. the results demonstrate that (table 3) also showed that mets patients had a significantly at (p < 0.001) decrease in calcium and vit. d concentration compared to the control group. table 1. general clinical and anthropometric characteristics of controls and mets groups variables controls means ± se mets means ± se p-value no. of subjects 76 95 – gender, m/f 36/40 46/49 0.001 age (years) 44.3 ± 7.6 48.1 ± 5.9 0.05 waist circumference (cm) 88.4 ± 5.5 97.7 ± 7.3 0.001 bmi (kg/m2) 25.8 ± 1.9 29.7 ± 3.5 0.001 sbp (mm hg) 125.3 ± 13.1 141.2 ± 14.5 0.001 dbp (mm hg) 78.4 ± 7.9 91.5 ± 6.1 0.001 tc (mmol/l) 4.21 ± 0.4 6.45 ± 0.9 0.001 tg (mmol/l) 1.21 ± 0.5 3.45 ± 0.7 0.001 ldl cholesterol (mmol/l) 2.46 ± 0.2 3.95 ± 0.8 0.001 hdl cholesterol (mmol/l) 1.18 ± 0.1 0.90 ± 0.1 0.001 non-hdl cholesterol (mmol/l) 3.08 ± 0.34 5.55 ± 0.99 0.001 vldl-c (mmol/l) 0.54 ± 0.2 1.57 ± 0.3 0.001 atherogenic index ( ai) 3.68 ± 0.59 6.64 ± 1.35 0.001 atherogenic coefficient (ac) 2.72 ± 2.50 6.16 ± 9.9 0.001 table 2. glucose metabolism in control and mets groups clinical parameters control means ± se mets means ± se p-value f.b.s (mmol/l) 4.8 ± 0.47 6.42 ± 0.35 0.001 insulin 8.23 ± 5.22 18.1 ± 7.77 0.001 homa-ir 1.78 ± 0.71 3.97 ± 0.83 0.001 homa-β 116.88 ± 12.38 81.41 ± 35.03 0.001 397j contemp med sci | vol. 8, no. 6, november-december 2022: 395–399 s.a. mohammed et al. original study interplay between asprosin with vitamin d in metabolic syndrome correlation between asprosin hormone and vitamin d concentration and some clinical parameters in mets patients: table 6 explains that adiposity-related indicators and vitamin d had a favorable correlation with each other (bmi and waist circumference) at p < 0.01, and also found the same relationship with an asprosin hormone. the results show that asprosin and vitamin d concentration had a positive relationship with fasting glucose, insulin, homa-ir, ldl-cholesterol, total cholesterol, triglyceride and a negative correlation with hdl-c, concentration in patients, as well as a negative correlation with non–hdl-c and atherogenic coefficient concentrations in patients. discussion the clinical parameters in mets patients group compared to the control group, can be explained by the fact that these results consistent with the literature that patients with metabolic syndrome have a high bmi or waist circumference21-22 with increased blood pressure, lipid profile (triglycerides), and decrease (hdl), (non-hdl-c ) they are a symptom of metabolic syndrome.23-24 also, these results were consistent with another study. mets caused significant increases in the proportion of glucose in the blood in a response to insulin resistance due to the rise in (ffa) in the blood, which causes hyperlipidemia.25-26 also, cells compensate for insulin resistance by secreting more insulin, which leads to hyperinsulinemia, and these tissues are less sensitive to insulin actions because they are full of fat, and excessive insulin production causes an imbalance in pancreatic beta cells which may explain the low in beta cell function. pathological conditions that are distinctive to mets include dyslipidemia and hyperglycemia, which are critical in the development of the condition.27-28 also, showed an increase significantly at (p < 0.001) for urea concentration in mets patients compared to the control group. the concentration of asprosin hormone and vitamin d in the control group compared with metabolic syndrome patients the findings in (table 4) demonstrated that the asprosin hormone’s normal concentration was (53.8 ± 3.7) ng/l in the healthy control group and there is an increased concentration of asprosin hormone (65.6 ± 4.4 ng/l ) for metabolic syndrome patients groups at p ≤ 0.0001. table 4 indicate that there is an increased concentration of asprosin hormone for control and metabolic syndrome patients groups at p ≤ 0.0001 based on bmi. table 5 findings revealed that the concentration of vitamin d was (40.3 ± 2.4 ng/ml) in the healthy control group and there is a decreased concentration of vitamin d (20.4 ± 1.8 ng/ml) for metabolic syndrome patients groups at p ≤ 0.0001. tables 5 indicate that there is an decreased concentration of vitamin d for control and metabolic syndrome patients groups at p ≤ 0.0001 based on bmi. table 3. vitamin d, calcium, and urea concentration in control and mets groups clinical parameters control means ± se mets means ± se p-value vitamin d (ng/ml) 40.3 ± 2.4 20.4 ± 1.8 ≤0.001 calcium (mmol/l) 2.22 ± 0.1 1.91 ± 0.4 ≤0.001 urea (mmol/l) 4.92 ± 0.9 8.22 ± 0.7 ≤0.001 tables 4. comparison of the levels of asprosin hormone based on bmi asprosin (ng/l) variables control means ± se mets means ± se p-value underweight 36.7 ± 3.7 27.2 ± 4.8 ≤0.001 normal weight 45.9 ± 3.2 66.8 ± 4.2 ≤0.001 overweight 58.8 ± 2.7 74.1 ± 5.1 ≤0.001 obese 71.9 ± 3.2 88. 9 ± 2.8 ≤0.001 total 53.8 ± 3.7 65.6 ± 4.4 ≤0.001 table 5. comparison of the levels of vitamin d based on bmi vitamin d (ng/ml) variables control means ± se mets means ± se p-value underweight 52.13 ± 2.3 29.9 ± 1.6 ≤0.001 normal weight 43.35 ± 1.2 22.34 ± 1.2 ≤0.001 overweight 36.22 ± 2.2 17.70 ± 1.9 ≤0.001 obese 30.09 ± 4.2 11.35 ± 2.4 ≤0.001 total 40.3 ± 2.4 20.4 ± 1.8 table 6. correlation between asprosin hormone and vitamin d concentration and some clinical parameters in mets patients clinical parameters asprosin hormon r-value vitamin d r-value asprosin with vitamin d 0.035 0.035 waist circumference (cm) 0.171* 0.16* bmi (kg/m2) 0.206* 0.06 * sbp (mm hg) 0.102 0.25 dbp (mm hg) 0.111 0.22 tc (mmol/l) 0.022 * 0.015* tg (mmol/l) 0.251* 0.38 * hdl-c(mmol/l) -0.194 * -0.21 * ldl-c(mmol/l) 0.012* 0.012* non –hdl-c -0.31* -0.29* atherogenic coefficient -0.21* -0.27* f.b.s(mmol/l) 0.302* 0.175* insulin 0.002* 0.31* homa-ir 0.316* 0.113* *significant at p 0.5 398 j contemp med sci | vol. 8, no. 6, november-december 2022: 395–399 study interplay between asprosin with vitamin d in metabolic syndrome original s.a. mohammed et al. low risk of metabolic syndrome is connected with high blood calcium levels. the show that dietary ca consumption is inversely related to the prevalence of mets.29-30 mets caused significant increases in urea, this result was consistent with another study.31 metabolic syndrome (mets) is an independent risk factor for chronic kidney disease (ckd). through a variety of processes, including stimulation of the renin-angiotensin system evidence strongly suggested that blood asprosin levels were considerably raised in mets, which was consistent with the results of researchs.32-33 based on bmi, mets blood asprosin levels were considerably greater than those of controls.18 and the lowest asprosin concentrations were seen in underweight people. these findings indicate a correlation between asprosin levels and obesity since asprosin levels rise as body mass index (bmi) rises.19-20 the results explain (campbell and drucker,) hypovitaminosis d and the metabolic syndrome (mets), that show a disorder marked by the presence of central obesity, arterial hypertension, and altered lipid and glucose metabolism, may be related, according to a number of studies. lower 25(oh)d concentrations were independently linked to a higher risk of mets, according to many studies.34-36 low risk of metabolic syndrome is connected with high blood calcium levels. the results show that dietary ca consumption is inversely related to the prevalence of mets. mets caused significant increases in urea,this result was consistent with (el-domiaty, et al. 2022). the metabolic syndrome (mets) is an independent risk factor for chronic kidney disease (ckd). through a variety of processes, including stimulation of the renin-angiotensin system. evidence strongly suggested that blood asprosin levels were considerably raised in mets, which was consistent with the results of researchs.32-33 based on bmi, mets blood asprosin levels were considerably greater than those of controls,18 and the lowest asprosin concentrations were seen in underweight people. these findings indicate a correlation between asprosin levels and obesity since asprosin levels rise as body mass index (bmi) rises.19-20 the results explain (campbell and drucker,) hypovitaminosis d and the metabolic syndrome (mets), that show a disorder marked by the presence of central obesity, arterial hypertension, and altered lipid and glucose metabolism, may be related, according to a number of studies.34-35 lower 25(oh) d concentrations were independently linked to a higher risk of mets, according to many studies.34-36 conclusion this study investigates the interaction of vit d, asprosin hormones together in mets. the results revealed there is a significant positive correlation in vit d, asprosin with bmi in mets and control group. a significant rise in the asprosin, homa ir, tc, tg, ldl-c, atherogenic index, atherogenic coefficient, blood pressure and urea in mets. a significant low in the vit d, (homa-β), non-hdl, (hdl-c), and calcium in mets. conflict of interest none.  references 1. alberti kg, zimmet p, shaw j; idf epidemiology task force consensus group. the metabolic syndrome a new worldwide definition. lancet 2005; 366:1059–62. 2. haque, t., rahman, s., islam, s., molla, n.h., and ali, n. (2019). assessment of the relationship between serum uric and glucose levels in healthy, prediabetic and diabetic individuals. diabetology a metabolic syndrome,11,49. 3. grundy sm, hansen b, smith sc, jr, et al.; american heart association, national heart, lung, and blood institute, american diabetes association. clinical management of metabolic syndrome: report of the american heart association/national heart, lung, and blood institute/american diabetes association conference on scientific issues related to management. arterioscler thromb vasc biol 2004; 24(2): e19–e24. 4. ford es: risks for all-cause mortality, cardiovascular disease, and diabetes associated with the metabolic syndrome: a summary of the evidence. diabetes care 2005, 28:1769–1778. 5. expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. executive summary of the third report of the national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel iii). jama 2001, 285, 2486–2497. [crossref ] 6. halpern a et al.(2010): metabolic syndrome, dyslipidemia, hypertension and type 2 diabetes in youth: from diagnosis to treatment. diabetol metab syndr., 2:55. 7. yuan m, li w, zhu y, yu b, wu j. asprosin: a novel player in metabolic diseases. front endocrinal (lausanne). 2020;11:64. doi: 10.3389/ fendo.2020.00064. 8. acara, a.c., bolatkale, m., kızıloğlu i̇, i̇bişoğlu, e., can, ç., 2018. a novel biochemical marker for predicting the severity of acs with unstable angina pectoris: asprosin. am. j. emerg. med. 36 (8), 1504–1505. https://doi. org/10.1016/j. ajem.2017.12.032. 9. romere c, duerrschmid c, bournat j, constable p, jain m, xia f, et al. asprosin, a fasting-induced glucogenic protein hormone. cell. 2016;165:566–79. doi: 10.1016/j. cell.2016.02.063. 10. e.k. studien ueber das hypertonie-hyperglyca “mie-hyperurika” miesyndrom. zentralblatt fuer inn medizin. 1923;1:105–127. 11. vitezova a, zillikens c, van herpt t, sijbrands e. vitamin d status and metabolic syndrome in the elderly: the rotterdam study. eur j endocrinol 2015;172(3)327–35. 12. gueli n, verrusio w, et al. vitamin d: drug of the future. a new therapeutic approach. arch gerontol geriatr 2012;54(1):222–7. 13. wang tj. vitamin d and cardiovascular disease. annu rev med 2016:14;67:261-72. doi: 10.1146/annurevmed-051214–025146. 14. danik js, manson je. vitamin d and cardiovascular disease. curr treat options cardiovasc med 2012;14(4):414–24. 15. esther adejumo, omobola ogundahunsi, olusola adejumo, and omodele jagun. prevalence of metabolic syndrome in a rural and urban community in south-west nigeria using three different definitions. 2017. international journal of tropical disease and health 24(1):1-9. doi: 10.9734/ ijtdh/2017/33993 16. national heart, lung, and blood institute. low blood pressure. (https:// www.nhlbi.nih.gov/health-topics/low-blood-pressure) accessed 8/23/2021. 17. acara ac, bolatkale m, kızıloğlu i̇, i̇bişoğlu e, can ç. a novel biochemical marker for predicting the severity of acs with unstable angina pectoris: asprosin. am j emerg med. 2018;36(8):1504–1505. doi: 10.1016/j. ajem.2017.12.032. 18. ann pietrangelo. what you need to know about non-hdl cholestrol. retrieved on the 6th of october, 2020, from: https:www.healthline.com/ health/what-you need-to-know-about-non-hdl-cholestrol. 19. jasim, rana f., sabah safaa and allwsh, thikra ali. the relation between fibroblast growth factor 21 and insulin resistance in hyperlipidemia patients. 2021 egyptian journal of chemistry 64(12) doi: 10.21608/ ejchem.2021.80062.3947 20. gianotti, n., muccini, c., galli, l., poli, a., spagnuolo, v., andolina, a., galizzi, n., ripa, m., messina, e., piatti, p. m., lazzarin, a., and castagna, a. (2019). homeostatic model assessment for insulin resistance index trajectories in hiv-infected patients treated with different first-line antiretroviral regimens. journal of medical virology, 91(11), 1937–1943. https://doi.org/10.1016/j.%20ajem.2017.12.032 https://doi.org/10.1016/j.%20ajem.2017.12.032 https://www.researchgate.net/profile/esther-adejumo?_sg%5b0%5d=72m7hjjrdbfrtjbbrsdtqquottwwnq0pw009s7qsbmz6hmq0t7t8ozndnthw9cqstv7au3a.sn3hnlys5mmnpcmqt0ijauedzipdg848lenhedbso9azdmgoeetzgydxayeybzds1cd5rmh6kggj-0uv6uzwfw&_sg%5b1%5d=r2ppndkiajhoyuptjtlqa-ki921cwyf8kg_p_zy1epwmln9w0yd6ad7lujtwa4qhqruye-s.rsxpuht7q0lkjzcz_azgmaz2pk1wg_4hebgijril-xi9cbzyvct11appdw_zmwkesxez3q4zrvgi3jybdvnqtw https://www.researchgate.net/profile/omobola-ogundahunsi?_sg%5b0%5d=72m7hjjrdbfrtjbbrsdtqquottwwnq0pw009s7qsbmz6hmq0t7t8ozndnthw9cqstv7au3a.sn3hnlys5mmnpcmqt0ijauedzipdg848lenhedbso9azdmgoeetzgydxayeybzds1cd5rmh6kggj-0uv6uzwfw&_sg%5b1%5d=r2ppndkiajhoyuptjtlqa-ki921cwyf8kg_p_zy1epwmln9w0yd6ad7lujtwa4qhqruye-s.rsxpuht7q0lkjzcz_azgmaz2pk1wg_4hebgijril-xi9cbzyvct11appdw_zmwkesxez3q4zrvgi3jybdvnqtw https://www.researchgate.net/profile/olusola-adejumo?_sg%5b0%5d=72m7hjjrdbfrtjbbrsdtqquottwwnq0pw009s7qsbmz6hmq0t7t8ozndnthw9cqstv7au3a.sn3hnlys5mmnpcmqt0ijauedzipdg848lenhedbso9azdmgoeetzgydxayeybzds1cd5rmh6kggj-0uv6uzwfw&_sg%5b1%5d=r2ppndkiajhoyuptjtlqa-ki921cwyf8kg_p_zy1epwmln9w0yd6ad7lujtwa4qhqruye-s.rsxpuht7q0lkjzcz_azgmaz2pk1wg_4hebgijril-xi9cbzyvct11appdw_zmwkesxez3q4zrvgi3jybdvnqtw https://www.researchgate.net/profile/omodele-jagun?_sg%5b0%5d=72m7hjjrdbfrtjbbrsdtqquottwwnq0pw009s7qsbmz6hmq0t7t8ozndnthw9cqstv7au3a.sn3hnlys5mmnpcmqt0ijauedzipdg848lenhedbso9azdmgoeetzgydxayeybzds1cd5rmh6kggj-0uv6uzwfw&_sg%5b1%5d=r2ppndkiajhoyuptjtlqa-ki921cwyf8kg_p_zy1epwmln9w0yd6ad7lujtwa4qhqruye-s.rsxpuht7q0lkjzcz_azgmaz2pk1wg_4hebgijril-xi9cbzyvct11appdw_zmwkesxez3q4zrvgi3jybdvnqtw http://dx.doi.org/10.9734/ijtdh/2017/33993 http://dx.doi.org/10.9734/ijtdh/2017/33993 https://www.researchgate.net/profile/rana-jasim-3?_sg%5b0%5d=ywbfuidzocgaqc3tdlfbduidhynqa7d56qjeiafo7z5vsahp5jiq6hz7ulwofksmcagdgu0.np34esg-praomwoldn0sgjgt0y4tjpcd-bdfahe70y_olvhqkrteqi47qcthktypewx30-fir0gksmn2qxloeg&_sg%5b1%5d=akly122eif4key5_9yebwo_g2ywafplyo2bmrvbfv1oprvlvzys_ztptsilivtlfk58fhzw.fhtjblxjbido3uw_xzdaemwfek5c91djjofmsijhimwxt2vmwinx1mgiaeqyvub-nf_64kp1slssqfnqll9d_a https://www.researchgate.net/profile/safaa-sabah-3?_sg%5b0%5d=36j_fkr1n-gflqpawzzawy9wbpvzo0qrw_hmg_g2ml1fujhnzaqf1itnmdiyo9q5v7m446c.mfgrrhbfxydnbhf-_vbgta41tmf5gcvxrcs3cyw4femn2_wa_qc77z5nahtwzor6wmpzx_djeegactcwl7enig&_sg%5b1%5d=x-jy9pykawlezl-4nfaxrp0sb0ls-jav9csrbfudobqecpn_893wmg6-jvxqrn7qvkq09ey.nepta6makqvsmlk-_vurwf2gi_s_sfrqik2gowgsdtb-srf-lwyexwuitxxiuychf2absnvwwtbwmd8mhplqza https://www.researchgate.net/profile/thikra-allwsh?_sg%5b0%5d=ywbfuidzocgaqc3tdlfbduidhynqa7d56qjeiafo7z5vsahp5jiq6hz7ulwofksmcagdgu0.np34esg-praomwoldn0sgjgt0y4tjpcd-bdfahe70y_olvhqkrteqi47qcthktypewx30-fir0gksmn2qxloeg&_sg%5b1%5d=akly122eif4key5_9yebwo_g2ywafplyo2bmrvbfv1oprvlvzys_ztptsilivtlfk58fhzw.fhtjblxjbido3uw_xzdaemwfek5c91djjofmsijhimwxt2vmwinx1mgiaeqyvub-nf_64kp1slssqfnqll9d_a https://www.researchgate.net/journal/egyptian-journal-of-chemistry-2357-0245 http://dx.doi.org/10.21608/ejchem.2021.80062.3947 http://dx.doi.org/10.21608/ejchem.2021.80062.3947 399j contemp med sci | vol. 8, no. 6, november-december 2022: 395–399 s.a. mohammed et al. original study interplay between asprosin with vitamin d in metabolic syndrome 21. andreozzi p, verrusio w, viscogliosi g, et al. relationship between vitamin d and body fat distribution evaluated by dxa in postmenopausal women. nutrition 2015;29:pii:s0899–9007(15)00526-2. doi: 10.1016/j nut.2015.12.029 22. rerksuppaphol s, rerksuppaphol l. metabolic syndrome in obese thai children: defined using modified. the national cholesterol education program/adult treatment panel iii. criteria. j med assoc thai. 2015; 98 (suppl 10): s88. 23. popkin bm, adair ls, ng sw. popkin bm, adair ls, ng sw. global nutrition transition and the pandemic of obesity in developing countries. nutr rev. 2012;70:3–21. 24. wang y. plasma asprosin concentrations are increased in individuals with glucose dysregulation and correlated with insulin resistance and firstphase insulin secretion. mediators inflamm. 2018;2018:9471583. 25. sabah, safaa and allwsh, thikra ali. the relation between fibroblast growth factor 21 and oxidative stress in insulin resistance with diabetics. 2020. international journal of pharmaceutical research. 12.04.351. doi:10.31838/ ijpr /2020.12.04.351. 26. czech, m.p., 2017. insulin action and resistance in obesity and type 2 diabetes. nat. med. 23 (7), 804–814. https://doi.org/10.1038/nm.4350. 27. wang, c.y.; lin, t.a.; liu, k.h.; liao, c.h.; liu, y.y.;wu, v.c.;wen, m.s.; yeh, t.s. serum asprosin levels and bariatric surgery outcomes in obese adults. int. j. obes. (lond.) 2019, 43, 1019–1025. [crossref ] [pubmed] 28. jasim, rana f. and allwsh, thikra ali. orexin a hormone and its relation to coronary heart diseases. 2021 research journal of pharmacy and technology 14(3):1417—1422. doi: 10.5958/0974-360x.2021.00253.5 29. han d, fang x, su d, huang l, he m, zhao d, zou y, zhang r. dietary calcium intake and the risk of metabolic syndrome: a systematic review and meta-analysis. sci rep. 2019 dec 13;9(1):19046. doi: 10.1038/s41598019-55507-x. pmid: 31836761; pmcid: pmc6911087. 30. baek jh, jin sm, bae jc, jee jh, yu ty, kim sk, hur ky, lee mk, kim jh. serum calcium and the risk of incident metabolic syndrome: a 4.3-year retrospective longitudinal study. diabetes metab j. 2017 feb;41(1):60–68. doi: 10.4093/dmj.2017.41.1.60. epub 2016 dec 26. pmid: 28029017; pmcid: pmc5328697. 31. baek jh, jin sm, bae jc, jee jh, yu ty, kim sk, hur ky, lee mk, kim jh. serum calcium and the risk of incident metabolic syndrome: a 4.3-year retrospective longitudinal study. diabetes metab j. 2017 feb;41(1):60–68. doi: 10.4093/dmj.2017.41.1.60. 32. y. wang, h. qu, x. xiong et al., “plasma asprosin concentrations are increased in individuals with glucose dysregulation and correlated with insulin resistance and first-phase insulin secretion,” mediators of inflammation, vol. 2018, article id 9471583, 7 pages, 2018. 33. x. zhang, h. jiang, x. ma, and h. wu, “increased serum level and impaired response to glucose fluctuation of asprosin is associated with type 2 diabetes mellitus,” journal of diabetes investigation, vol. 11, no. 2, pp. 349–355, 2020. 34. brenner dr, arora p, garcia-bailo b, wolever tm, morrison h, el-sohemy a, karmali m, badawi a. plasma vitamin d levels and risk of metabolic syndrome in canadians. clin invest med 2011;34:e377. 35. ford es, ajani ua, mcguire lc, liu s. concentrations of serum vitamin d and the metabolic syndrome among us adults. diabetes care 2005; 28:1228–30. 36 reis jp, von mühlen d, miller er 3rd. relation of 25-hydroxyvitamin d and parathyroid hormone levels with metabolic syndrome among us adults. eur j endocrinol 2008;159:41–8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1300 https://www.researchgate.net/profile/safaa-sabah-3?_sg%5b0%5d=36j_fkr1n-gflqpawzzawy9wbpvzo0qrw_hmg_g2ml1fujhnzaqf1itnmdiyo9q5v7m446c.mfgrrhbfxydnbhf-_vbgta41tmf5gcvxrcs3cyw4femn2_wa_qc77z5nahtwzor6wmpzx_djeegactcwl7enig&_sg%5b1%5d=x-jy9pykawlezl-4nfaxrp0sb0ls-jav9csrbfudobqecpn_893wmg6-jvxqrn7qvkq09ey.nepta6makqvsmlk-_vurwf2gi_s_sfrqik2gowgsdtb-srf-lwyexwuitxxiuychf2absnvwwtbwmd8mhplqza https://www.researchgate.net/profile/rana-jasim-3?_sg%5b0%5d=ywbfuidzocgaqc3tdlfbduidhynqa7d56qjeiafo7z5vsahp5jiq6hz7ulwofksmcagdgu0.np34esg-praomwoldn0sgjgt0y4tjpcd-bdfahe70y_olvhqkrteqi47qcthktypewx30-fir0gksmn2qxloeg&_sg%5b1%5d=akly122eif4key5_9yebwo_g2ywafplyo2bmrvbfv1oprvlvzys_ztptsilivtlfk58fhzw.fhtjblxjbido3uw_xzdaemwfek5c91djjofmsijhimwxt2vmwinx1mgiaeqyvub-nf_64kp1slssqfnqll9d_a https://www.researchgate.net/profile/thikra-allwsh?_sg%5b0%5d=ywbfuidzocgaqc3tdlfbduidhynqa7d56qjeiafo7z5vsahp5jiq6hz7ulwofksmcagdgu0.np34esg-praomwoldn0sgjgt0y4tjpcd-bdfahe70y_olvhqkrteqi47qcthktypewx30-fir0gksmn2qxloeg&_sg%5b1%5d=akly122eif4key5_9yebwo_g2ywafplyo2bmrvbfv1oprvlvzys_ztptsilivtlfk58fhzw.fhtjblxjbido3uw_xzdaemwfek5c91djjofmsijhimwxt2vmwinx1mgiaeqyvub-nf_64kp1slssqfnqll9d_a https://www.researchgate.net/journal/research-journal-of-pharmacy-and-technology-0974-3618 https://www.researchgate.net/journal/research-journal-of-pharmacy-and-technology-0974-3618 264 j contemp med sci | vol. 8, no. 4, july-august 2022: 264–269 original determination of procedural pain intensity among critically-ill patients: using behavioral pain scale (bps) ghufran emad bachi1*, sadeq al-fayyadh2 1al-muthanah health directorate, ministry of health, al-muthanah, iraq. 2adult nursing department, college of nursing, university of baghdad, baghdad, iraq. *correspondence to: ghufran emad bachi (e-mail: ghofran.emad1202a@conursing.uobaghdad.edu.iq) (submitted: 28 may 2022 – revised version received: 12 june 2022 – accepted: 27 july 2022 – published online: 26 august 2022) abstract objective: to assess pain severity for routine nursing procedures. as well as to compare pain severity during three measurement phases. and to find out the differences among routine nursing procedures pain severity. methods: an observational method for data collection and sample of 135 subjects who had met the study’s inclusion criteria were targeted. the data collection started from january 18th to april 7th, 2022. results: patients were silently suffering pain during all three assessment phases: pre-during, and 20 minutes’ post-routine nursing procedures. of equal importance, there is a statistically significant difference between all nursing procedures in terms of pain intensity. conclusion: the critically ill, mechanically ventilated patients who were recruited in this study, were silently suffering from a relatively high level of pain during their hospitalization period in the intensive care unit. the highlighted case of silent suffering is a serious gap in both medical and nursing care quality that must be addressed both urgently and effectively. keywords: pain, critically-ill, behavioral pain scale, intensive care unit issn 2413-0516 introduction patients who are admitted to the intensive care unit (icu) often suffer from one vital organ failure or several organ failures and need intensive care for their survival.1 therefore, most patients in the icu are intubated and sedated due to their need for advanced care to support their vital organs; such as the heart, lungs, and kidneys. for example, if the patient needs critical care after surgery, trauma, or cardiac arrest, intensive care has been indicated for the patient’s survival. one of the problems within the icu is patients’ pain reporting, both at rest and during the procedure. also, in studies that include memories of patients after an icu, pain is commonly reported as a major problem.2 pain is a common problem in the icu, with more than 75% of icu patients experiencing pain at rest and 50% or higher in both medical and surgical icus experiencing pain for a variety of reasons such as the patient’s chief complaint(s), chronic conditions, wound care, and invasive nursing care procedures. pain is one of the chief discomfort problems that patients experience in the icu. lack of effective pain evaluation has been associated with serious complications, including chronic pain, delayed mechanical ventilation duration, longer icu hospitalization, and an increased death rate.3,4 careful pain assessment contributes to effective pain management by increasing the sufficiency of therapeutic measures such as analgesics and sedation use, as well as decreasing patient stays in the icu.5 approximately 75% of icu hospitalized patients have severe pain. about 50% of them are having pain during invasive nursing procedures and about 30% are having pain even at rest.6 several factors that may contribute to acute pain in the icu, such as the type of patient’s condition, such as surgery or trauma, diagnostic, extended immobilization, therapeutic procedures, underlying chronic diseases, or other medical conditions, may also contribute to persistent pain.7 nursing interventions can cause pain in icu hospitalized patients. endotracheal suctioning has been recognized as the most painful procedure in mechanically ventilated patients. pain produces damaging physical effects.8 pain stimulates sympathetic activity throughout the body, resulting in immunological suppression, hyperglycemia, changes in hemodynamic state, and an increased release of catecholamine, cortisol, and anti-diuretic hormones. untreated pain can also lead to respiratory disorders like airway obstruction and pneumonia, as well as limited mobility, deep vein thrombosis, chronic pain syndromes, and psychological issues including anxiety, depression, disorientation, and post-traumatic stress disorder.9 mechanically ventilated patients and critically ill patients are unable to verbalize their pain because of altered consciousness, being sedated, and being unable to communicate to express their pain, which may be agonizing. as a result, quantifying pain caused by altered awareness, anesthesia, invasive procedures, and artificial ventilation is both a difficult and underappreciated topic.10 there are many challenges that may contribute to decreasing effective pain assessment and management, including lack of evidence and lack of collaboration between physicians and nurses, were identified as barriers to effective pain assessment and management. in addition, the physical and cognitive impairments of many critically ill patients and communication impediments are factors that should not be overlooked when practicing pain management.11 when patients are unable to report their pain, especially those hospitalized in the critical care setting, the nurses use pain-related behaviors such as facial expression, limb movements, and muscle rigidity as pain landmark indicators. patients who experience pain during invasive and nursing care procedures are more likely to develop behavioral responses than those without pain.12 in medical, surgical, and trauma patients who were unable to describe their pain, the behavioral pain scale (bps) was approved to be the most reliable and valid pain evaluation 265j contemp med sci | vol. 8, no. 4, july-august 2022: 264–269 g.e. bachi et al. original determination of procedural pain intensity among critically-ill patients: using behavioral pain scale (bps) instrument. the bps can be used for sedated patients and depends on the three behavioral categories, including facial expression, upper-limb movements, and mechanical ventilation compliance.13 ongoing reassessment of a patient’s pain is required to provide effective pain management for patients. pain should be assessed before the administration of any analgesic agents, as well as before and during therapeutic interventional procedures to understand the pain severity caused by the patient’s health status or due to the therapeutic procedures. also, pain assessment is one of the major responsibilities every member of the health care team should master, particularly nurses.14 the main question of this study was: what is the pain severity for routine nursing procedures? therefore, this study aims to assess the pain severity associated with routine nursing procedures. as well as to compare pain severity during three measurement phases. and to find out the differences among routine nursing procedures’ pain severity. materials and methods study design: cross-sectional study design. participants and study design the purposive non-probability sampling method was used for the current study method which is selected depending on population characteristics, eligibility criteria and the study’s aims. the exclusion criteria of this study included patients who were < 18 years old. patients who can report pain were excluded because the research tool was designed for patients who are unable to report the presence and intensity of pain, as well as patients who have had neuropathic conditions such as myasthenia gravis and guillain-barre syndrome (gbs), patients with upper limb neuropathy and patients with upper limb fractures because these conditions may interfere with behavioral responsivity when using the bps. those on a heavy anesthetic regimen were also excluded because they may be unable to show any behavioral response which may interfere with the research tool usability and measurement accuracy. the sample consisted of 135 patients. the sample size was calculated according to a-priori sample sizes for student t-tests. settings: the study was conducted by using observational methods, targeting hospitalized adult patients in the icus in baghdad teaching hospitals; martyr ghazi al-hariri hospital for surgical specialties; and the private nursing home hospital of the medical city directorate; al-hussein teaching hospital of al-muthanah health directorate. study instrument: the behavioral pain scale (bps) was used in this study after obtaining official permission from the primary author dr. jean f payen. the bps is both reliable and valid for use in assessing pain for mechanically ventilated-sedated patients who are hospitalized in the icus and the patients who are unable to communicate and expressing their distress. cronbach’s alpha coefficients of the scale was highly reliable; the reliability coefficient for the bps was 0.79.15,16 the bps contain three main domains facial expression, compliance with mechanical ventilation and upper limb movement. within each domain, behavioral responses are scored from (1) that indicate no pain to (4), which is the worst score that indicates the presence of pain. the health care professional uses bps to assess the presence and severity of the pain and decide what the best behavioral response will be within each domain. patients’ responses are to be scored from 1 to 4 in each domain, with a total score of 12 that indicates maximum pain.15 data collection method: the data was collected through observational methods from january 18th, 2022, to april 7th, 2022. the severity of pain was measured objectively through observation of the patient’s response using bps and vital signs measured from patient’s monitoring machine. the study sample include 135 patients who were selected purposively among critically ill patients with a diminished level of consciousness. the pain and vital signs were determined through three phases: the first was assessing patients’ pain during rest (without any invasive or therapeutic procedures); the second was during routine nursing procedures, including: (position change, endotracheal suctioning, dressing change, and blood sampling). finally, the third phase, which was done to determine patient’s pain within 20 minutes post-nursing procedures. spo2% levels were also assessed during all three assessment phases. the mean arterial pressure, was measured and categorized according to the following formula: (map= [2 × diastolic + systolic]/3).16,17 data analysis procedures: data were analyzed using ibm-statistical package for social sciences (spss) version 24, which included descriptive and inferential statistical measures. descriptive statistics are used to describe the demographic data and health-related variables. repeated measurement analysis of variance (anova) used to measure the difference among pain severity during all routine nursing procedures (position change, endotracheal suctioning, dressing change, and blood sampling). ethical considerations and official agreements: with the submission of the study protocol, ethical approval was sought from the scientific committee of the nursing faculty, university of baghdad. the researcher submitted a detailed description of the study, including problem statement, objectives, and questionnaire, to the ministry of planning (central statistical organization) and to the medical city directorate, and al-muthanah health directorate, in order to obtain official permission to carry out the study. to verify that the rights, welfare, and well-being of human participants are completely protected while they are participating in a study; the researcher has completed the human research protection fundamental training offered by the office for human research protection. results the results represent the highest percentages and the dominant percentage of gender distribution for the targeted sample was males, representing more the half (58.5%) of the study sample and the age groups included (18– < 32 years old), more than one quarter with percentage (28.9%). the results represent the majority of the collected samples were as follow: patient’s length of staying days, more than half (55.6%) of the subjects were hospitalized for 5 days or less. additionally, more than half (61.5%) of the participants were medically classified as non-traumatic patients. regarding consciousness level for the patients according to gcs was (5–8), representing more than half (58.5%) of the study subjects. finally, narcotics was approximately used by about two-fifths (42.2%) of study subject. 266 j contemp med sci | vol. 8, no. 4, july-august 2022: 264–269 determination of procedural pain intensity among critically-ill patients: using behavioral pain scale (bps) original g.e. bachi et al. the descriptive statistics for pain intensity demonstrate the first phase was pre-routine nursing procedures. that represents approximately more than half (61.5%) of the total collected samples experiencing mild pain. the second phase of measurement, conducted during routine nursing procedures, showed the highest percentage (100%) of patients having a severe unacceptable amount of pain. regarding the third phase, which was conducted within 20 minutes post-routine nursing procedures, showed that the vast majority of patients (95.6%) had suffered mild pain level. finally, the overall pain intensity was presented, which showed that more than two thirds of patients (71.1%) had a severe unacceptable pain level. overall pain score the mean plot demonstrates the higher overall pain score during routine nursing procedures as an expected response to increased pain stimulation than pre and post-nursing procedures within 20 minutes. the results represent the highest percentages of the vital signs. as noticed, the vital signs increased during routine nursing care procedures and, conversely, the spo2 decreased below normal during the procedural phase compared to pre and post-nursing procedures. these procedures include blood sample, endotracheal suctioning, and dressing changes. surprisingly most patients (99.3%) had a severe unacceptable amount of pain (6-11). repeated measurement anova test in table (4-18) indicates there is a statistically significant difference between four nursing procedures between the blood sampling (1) and endotracheal suctioning (2) at (m = −.0941, p = 0.000). also, there is a statistically significant difference between the blood sampling procedures and the dressing change (m = 0.696*, p = 0.000). additionally, the statistically significant difference between the blood sampling procedures and the position change was represented at (m = 0.911*, p = 0.000) (figure 1). pain score during nursing procedures the mean plot demonstrates there is a statistically significant difference between all nursing procedures (blood sampling, endotracheal suctioning, dressing change, and position change) with a higher pain score rate during endotracheal suctioning, blood sampling, dressing change, and position change, respectively (figure 2). discussion patient pain determination is an important issue in the critical care setting to improve patients’ care plans, improve patients’ outcomes, and decrease the length of stay and healthcare needs. when the nurses assess patients’ pain frequently, they can determine pain intensity and provide management methods according to pain severity and medical report, such as modifying medication doses and adjusting sedation uses that can improve the care plan and enhance the patients’ health status.14,17 the findings in table 1 showed that more than one quarter (28.9%) of the study participants’ age group was (18 – < 32 years old). the cross-sectional study18 similar to this result. regarding the patients’ gender, findings of the study indicated that more than half (58.5%) of the study sample were males the study confirmed this result.19 this may be due to the fact that male individuals are more at risk of the occurrence of table 1. minimum sample size determination parameter of calculating the minimum sample size selected values anticipated effect size (cohen’s d): 0.5 desired statistical power level: 0.8 probability level: 0.05 table 1. descriptive statistics of sociodemographic data f % age groups 18 – < 32 years old 39 28.9 32.0 – < 45.0 years old 23 17.0 45.0 – < 58.0 years old 26 19.3 58.0 – < 71.0 years old 36 26.7 ≥71 years old 11 8.1 total 135 100.0 gender male 79 58.5 female 56 41.5 total 135 100.0 fig. 1 fig. 2 cerebrovascular accidents and male individuals are more susceptible to road traffic accidents than females.20 the findings in table 2 revealed more than half (61.5%) of the participants were medically classified as non-traumatic patients. this was confirmed by the study21 which represent non-traumatic patients more than traumatic. the majority (58.5%) of the patient’s consciousness level was (5-8), according to the glasgow coma scale. these results were not surprising to the researcher since the patients were hospitalized in intensive care units commonly diminished consciousness level. 22 267j contemp med sci | vol. 8, no. 4, july-august 2022: 264–269 g.e. bachi et al. original determination of procedural pain intensity among critically-ill patients: using behavioral pain scale (bps) table 2. descriptive statistics for health related variables f % length of stay, days 5 days or less 75 55.6 6–10 days 60 44.4 total 135 100.0 medical diagnoses classification non-traumatic 83 61.5 traumatic 52 38.5 total 135 100.0 assessment using glasgow coma scale (gcs) severe condition (5–8) 79 58.5 moderate condition (9–13) 56 41.5 total 135 100.0 pain medication(s) no medication 28 20.7 non-narcotics 10 7.4 narcotics 57 42.2 both (narcotics & non-narcotics) 40 29.6 total 135 100.0 table 3. descriptive statistics of pain levels using behavioural pain scale phases of assessment pain level categories total no pain 3 mild pain 4–5 severe unacceptable pain 6–11 maximum pain 12 f % f % f % f % f % pre-routine nursing procedures 32 23.7 83 61.5 20 14.8 0 0 135 100% during-routine nursing procedures 0 0 0 0 135 100% 0 0 135 100% post-routine nursing procedures within 20 minutes 5 3.7 129 95.6 1 0.7 0 0 135 100% overall pain levels 0 0 39 28.9 96 71.1 0 0 135 100% moreover, the pain medication classification, the results appeared in this way: the majority of the patients’ were under mild regimen of narcotics medications was approximately used by about two-fifths (42.2%) of the study subjects. this result was not surprising to the researcher since the patients are hospitalized in icu, frequently treated with mild, moderate, and even heavy sedative regimens.14 the study results are supported by a prospective cohort study that reported most patients under mild regimen.23 descriptive statistics represents vital signs and spo2 level during all three phases of assessment (pre, during, and postnursing procedures). it started with respiratory rate. more than half (62.2%) of patients had eupnoea, yet more than half (59.3%) of study participants had a normal heart rate (60–100 beats/min). of equal importance, nearly two thirds (68.9%) of patients had normal mean arterial pressure (93–99 mmhg). moreover, the majority (88.9%) of collected study samples had a normal body temperature (36.5–37.5°c). furthermore, the vast majority of patients (94.1%) had normal spo2% levels in the pre-nursing procedures phase. this results supported by al-saad et al. (2018).24 concerning the second phase, which was during routine nursing procedures, results showed that a relatively high percentage of patients had tachypnea (100%). similarly, more than three-quarters (77%) of study participants had tachycardia (> 100 beats/min). of equal importance, more than one third (37.8%) of patients had normal mean arterial pressure (93–99 mmhg). surprisingly, the majority (88.1%) of collected study samples had a normal body temperature (36.5–37.5°c). furthermore, less than two thirds (65.9%) of patients had an spo2 level below normal (90–94.4%) this may be due to response of patients to severe pain according to their conditions and several researcher studies supported these findings.15,24,25 continually, overall vital signs post-nursing procedures within 20 minutes. the results were as follows: the respiratory rate was more than half (52.6%) of patients who had eupnoea (12–20 breaths/min). approximately more than half (57.8%) of study participants had a normal heart rate (60–100 beats/min). additionally, less than two thirds (65.2%) of patients had normal mean arterial pressure (93–99 mmhg). of equal importance, the vast majority (93.3%) of collected study samples had a normal body temperature (36.5–37.5°c). furthermore, more than three-quarters (79.3%) of patients had a normal spo2 level of (95–100%). the changes in vital signs during all three measurement phases: pre, during, and post-nursing procedures may be to the body response to decreased pain intensity.26 in accordance the descriptive statistics for pain severity during three measurement phases illustrate. in the pre-nursing procedures phase represent approximately more than half (61.5%) of the total collected samples experiencing mild pain. the highest percentage (100%) of patients had a severe, unacceptable amount of pain. finally, during the third phase. it was shown that almost all patients (95.6%) experienced mild pain in the third phase, which was performed after standard nursing procedures within 20 minutes. based on the overall level of pain, more than two-thirds of patients (71.1%) showed an unacceptable level of pain. this results supported by erden et al. (2018).25 the descriptive statistics in table 5 represent the descriptive statistics of pain intensity that were assessed when implementing procedures as part of routine daily nursing care for the most frequent nursing procedures that are conducted in the icu for hospitalized critically ill patients. these procedures include blood sampling, endotracheal suctioning, and dressing changes. surprisingly, most patients (99.3%) suffered 268 j contemp med sci | vol. 8, no. 4, july-august 2022: 264–269 determination of procedural pain intensity among critically-ill patients: using behavioral pain scale (bps) original g.e. bachi et al. table 5. descriptive statistics of pain levels during each nursing procedure nursing procedures mild pain 4–5 severe unacceptable pain 6–11 maximum pain 12 f % f % f % blood sampling 1 0.7 134 99.3 0 0 endotracheal suctioning 0 0 134 99.3 1 0.7 during dressing change 1 0.7 134 99.3 0 0 during position change 4 3.0 131 97.0 0 0 table 6. statistical difference in the pain scale over four procedures (blood sample, endotracheal suctioning, dressing change, and change position) pain over four nursing procedures (i) time anova analysis (j) time mean difference (i-j) std. error sig.b blood sampling (1) f sig endotracheal suctioning (2) -.941* .071 .000 204.799 .000 dressing change (3) 0.696* .086 .000 position change (4) 0.911* .090 .000 1 = blood sampling; 2 = endotracheal suctioning; 3 = dressing change; 4 = change position. table 4. descriptive statistics of overall vital signs during all three phases of assessment (pre-routine nursing procedures, duringand post-routine nursing procedures) vital signs and spo 2 % level during all three measures f % pr ero ut in e nu rs in g pr oc ed ur es respiratory rate (eupnoea 12–20 breath/min) 84 62.2 pulse rate (normal 60–100 beat/min) 80 59.3 mean arterial pressure (normal mean arterial pressure map (93−99 mmhg)) 93 68.9 temperature (euthermia 36.5–37.5°c) 120 88.9 spo 2 % (normal (95–100%)) 127 94.1 d ur in g ro ut in e nu rs in g pr oc ed ur es respiratory rate (tachypnea >20 breath/min) 135 100.0 pulse rate (tachycardia > 100 beat/min) 104 77.0 normal mean arterial pressure map (93–99 mmhg) 51 37.8 temperature (euthermia 36.5–37.5°c) 119 88.1 spo 2 % (below normal (90–94%) 89 65.9 po st -r ou tin e nu rs in g pr oc ed ur es w ith in 2 0 m in ut es respiratory rate (eupnoea 12–20 breath/min) 71 52.6 normal 60–100 beat/min 78 57.8 normal mean arterial pressure map (93–99 mmhg) 88 65.2 temperature euthermia 36.5–37.5°c 126 93.3 spo 2 % normal (95–100%) 107 79.3 an unacceptable intensity of pain (6–11). this results supported by considine et al. (2020).27 a repeated measurement according to the anova test in table 6, there is a statistically significant difference between four nursing procedures between blood sampling (1) and endotracheal suctioning (2), blood sample protocols and the dressing change. the results supported by akhani (2014) and garcía-esquinas et al. (2019).28,29 conclusions the critically ill, mechanically ventilated patients who were recruited in this study, were silently suffering from a relatively high level of pain during their hospitalization period in the intensive care unit. the highlighted case of silent suffering is a serious gap in both medical and nursing care quality that must be addressed both urgently and effectively. also pain severity had reached its highest level during nursing procedures, as it showed a severe unacceptable pain score, which is both clinically and ethically unacceptable. recommendations use up-to-date clinical protocols to measure pain in intensive care units and use of the well-established behavioral pain assessment tools, particularly in iraqi icus particularly in iraqi icus, because the objectively established pain assessment methods have not been applied yet. likewise further 269j contemp med sci | vol. 8, no. 4, july-august 2022: 264–269 g.e. bachi et al. original determination of procedural pain intensity among critically-ill patients: using behavioral pain scale (bps) studies with a larger sample size that specifically target patients to assess pain in critical care settings are mandatory. conducting double-blinded randomized controlled clinical trials that focused on pain assessment and management during nursing procedures to determine the most effective methods that may enhance pain management, especially for icus hospitalized non-communicative patients. limitations the main limitations are the relatively small sample size and the timeframe for the study and data collection. the more relevant method to classify the patient’s consciousness and sedation level in the icus is the richmond agitation sedation scale (rass), which is not used in the current study since it is not applicable in the health situations and the health care providers and the informed consent to use the rass was not obtained from the primary author. funding information the budget of this research work was not support by any governmental or non‐governmental organization. the authors of this manuscript covered all the research work‐related expenses. conflicts of interest none.  references 1. hylén m. pain in intensive care: assessments and patients’ experience (doctoral dissertation, malmö universitet). 2021. 2. na’el k a, mohammed wk. nurses’ knowledge toward care of unconscious adult patients at teaching hospitals in al-hilla city. iraqi national journal of nursing specialties. 2019; 32(1). 3. shaikh n, tahseen s, haq qz, al-ameri g, ganaw a, chanda a, labathkhan mz, kazi t. acute pain management in intensive care patients: facts and figures. inpain management in special circumstances 2018 nov 5. london, england: intechopen. 4. shahiri ts, richard-lalonde m, richebé p, gélinas c. exploration of the nociception level (nol™) index for pain assessment during endotracheal suctioning in mechanically ventilated patients in the intensive care unit: an observational and feasibility study. pain management nursing. 2020 oct 1; 21(5):428–34. 5. pinheiro ar, marques rm. behavioral pain scale and critical care pain observation tool for pain evaluation in orotracheally tubed critical patients. a systematic review of the literature. revista brasileira de terapia intensiva. 2020 jan 20; 31:571–81. 6. aktaş yy, karabulut n. relief of procedural pain in critically ill patients by music therapy: a randomized controlled trial. complementary medicine research. 2019; 26(3):156–65. 7. arrar a, mohammed s. evaluation of nurses’ knowledge and practices concerning nursing care guide in the intensive care unit in misan governorate hospitals. kufa journal for nursing sciences. 2020; 10(1):12–22. 8. gomarverdi s, sedighie l, seifrabiei ma, nikooseresht m. comparison of two pain scales: behavioral pain scale and critical-care pain observation tool during invasive and noninvasive procedures in intensive care unit-admitted patients. iranian journal of nursing and midwifery research. 2019 mar; 24(2):151. 9. alnajar mk, shudifat r, mosleh sm, ismaile s, n’erat m, amro k. pain assessment and management in intensive care unit: nurses’ practices, perceived influencing factors, and educational needs. the open nursing journal. 2021 oct 5; 15(1). 10. kadhim h. evaluation of nurses’ practices toward the control of patients’ complications at the respiratory care unit in baghdad teaching hospitals. iraqi national journal of nursing specialties. 2014; 1(27):47–58. 11. damico v, macchi g, murano l, molinari af. incidence of pain at rest and during nursing procedures in icu patients: a longitudinal observational study. ann ig. 2020 jul 1;32(4):407–18. 12. berman a, snyder sj, levett-jones t, dwyer t, hales m, harvey n, moxham l, langtree t, parker b, reid-searl k, stanley d. kozier and erb’s fundamentals of nursing [4th australian edition]. 13. devlin jw, skrobik y, gélinas c, needham dm, slooter aj, pandharipande pp, watson pl, weinhouse gl, nunnally me, rochwerg b, balas mc. clinical practice guidelines for the prevention and management of pain, agitation/ sedation, delirium, immobility, and sleep disruption in adult patients in the icu. critical care medicine. 2018 sep 1; 46(9):e825–73. 14. payen jf, bru o, bosson jl, lagrasta a, novel e, deschaux i, lavagne p, jacquot c. assessing pain in critically ill sedated patients by using a behavioral pain scale. critical care medicine. 2001 dec 1; 29(12):2258–63. 15. salvadore ca. implementation of the critical care pain observation tool. 2018 16. honan l, bautista c, esposito c. focus on adult health medical-surgical nursing 2nd ed. 2018. 17. kundu rn, biswas s, das m. mean arterial pressure classification: a better tool for statistical interpretation of blood pressure related risk covariates. cardiology and angiology: an international journal. 2017; 6(1):1–7. 18. alikiaie b, mousavi s, ebrahimi a, foroughi z. evaluation of pain assessment and management in critically ill intubated patients in a referral university hospital in iran. journal of research in pharmacy practice. 2019 jul; 8(3):137. 19. kemp hi, bantel c, gordon f, brett sj, plan, search, laycock hc, bampoe s, bantel c, gooneratne m, highton d. pain assessment in int ensive care (paint): an observational study of physician‐documented pain assessment in 45 intensive care units in the united kingdom. anaesthesia. 2017 jun; 72(6):737–48. 20. hinksman ca, haylock rg, gillies m. cerebrovascular disease mortality after occupational radiation exposure among the uk national registry for radiation workers cohort. radiation research. 2022 may; 197(5):459–70. 21. alogaili aa, khullof wa. epidemiological, characteristics and outcomes of road traffic accident among patients in emergency units. journal of mar case rports. 2021; 3(3):1–3. 22. oliveira ls, macedo mp, silva sa, oliveira ap, santos vs. pain assessment in critical patients using the behavioral pain scale. brjp. 2019 jun 19; 2:112–6. 23. morris jl, bernard f, bérubé m, dubé jn, houle j, laporta d, morin sn, perreault m, williamson d, gélinas c. determinants of pain assessment documentation in intensive care units. canadian journal of anesthesia/ journal canadien d’anesthésie. 2021 aug; 68(8):1176–84. 24. al-saad sn, ai-jaafari ha. comparison between dexmedetomidine and propofol as sedatives for critically ill patients in intensive care units. journal of the faculty of medicine baghdad. 2018 apr 1; 60(1):28–31. 25. erden s, demir n, ugras ga, arslan u, arslan s. vital signs: valid indicators to assess pain in intensive care unit patients? an observational, descriptive study. nursing & health sciences. 2018 dec; 20(4):502–8. 26. klein c, caumo w, gélinas c, patines v, pilger t, lopes a, backes fn, villas-boas df, vieira sr. validation of two pain assessment tools using a standardized nociceptive stimulation in critically ill adults. journal of pain and symptom management. 2018 oct 1; 56(4):594–601. 27. considine j, street m, hutchinson am, mohebbi m, rawson h, dunning t, botti m, duke mm, hutchison af, bucknall t. vital sign abnormalities as predictors of clinical deterioration in subacute care patients: a prospective case-time-control study. international journal of nursing studies. 2020 aug 1; 108:103612. 28. akhani p, mendpara s, palan b, harsoda j. gender differences in response to experimental pain among medical students from a western state of india. international journal of medical students. 2014 jan 27; 2(1):13–7. 29. garcía-esquinas e, rodríguez-sánchez i, ortolá r, lopez-garcia e, caballero ff, rodríguez-mañas l, banegas jr, rodríguez-artalejo f. gender differences in pain risk in old age: magnitude and contributors. inmayo clinic proceedings 2019 sep 1 (vol. 94, no. 9, pp. 1707-1717). elsevier. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1260 169j contemp med sci | vol. 8, no. 3, may-june 2022: 169–175 original roles of c-peptide and triglyceride as effective indices for insulin resistance investigations in iraqi women with polycystic ovarian syndrome hadbaa h. al-murshedi1,*, fadhil j. al-tu’ma1, eman a. hadi2, borhan mustafa mohammed3, tarik al-kayat4 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 2department of chemistry, college of science, university of mosul, mosul, iraq. 3department of pharmacy, mazaya university college, thi-qar, iraq. 4medical technical college, al-farahidi university, baghdad, iraq. *correspondence to: hadbaa helwas al-murshedi. (email: hdbaa88@gmail.com) (submitted: 08 june 2022 – revised version received: 14 june 2022 – accepted: 15 june 2022 – published online: 26 june 2022) issn 2413-0516 introduction polycystic ovarian syndrome (pcos) is a common endocrine disorder with an impact on hormonal and metabolic regulation. women with pcos are at increased risk of anovulation and infertility.1 the clinical presentation is extremely variable but generally includes clinical and/or biochemical hyper androgenism, menstrual dysfunction (oligo-amenorrhea) and polycystic ovaries on ultrasound.2 diagnostic criteria for pcos mostly use the revised rotterdam 2003 criteria.3 insulin resistance (ir) is a very common finding in subjects with pcos which not included among the diagnostic features.4 ir is usually defined as a pathological condition characterized by a decreased responsiveness or sensitivity to the metabolic actions of insulin. in women with pcos, ir plays an important role in the development and persistence of this disorder.5 ir stimulates ovarian theca cells to secrete androgens and increasing luteinizing hormone (lh) effect on ovarian androgen production. insulin inhibits sex hormone binding globulin (shbg) secretion, increasing free and bioactive androgen levels and worsen hyperandrogenism status.6 moreover, ir is critically involved in the development of metabolic syndrome and cardiovascular disease in pcos women.7 the need for accurate screening of ir in women with pcos is obvious. thus, early recognition and management would offer important preventive measures.8 several methods have been developed to quantify this metabolic phenomenon. the hyperinsulinemic euglycemic clamp technique (hieg) is generally accepted as the best available direct method to assess insulin sensitivity.9 however, this technique is very complex and not appropriate in clinical practice. as an alternative strategy, practical surrogate markers have been proposed to measure ir; considering the concept that patients who have insulin resistance will have more insulin hormone in blood than those who does not. homeostasis model assessment-insulin resistance index (homa-ir) and the quantitative insulin sensitivity check index (quicki) are the most widely used surrogate indices of ir, which reflect the feedback between fasting serum insulin and glucose.10,11 excess adiposity and dyslipidemia may influence insulin sensitivity. based on these factors, different indices have been developed to measure ir, which better reflect lipid profiles such as triglyceride (tg) (mcauley index).12 the abstract objectives: to assess the insulin resistance by determination of c-peptide and triglyceride levels in women with polycystic ovarian syndrome (pcos) and then investigation of insulin resistance by various methods and comparing them with the homeostatic model assessment method used. methods: a study included 120 participants (68 women have pcos subdivide according to their bmi to 45 obese (bmi>=30) and 23 non-obese (bmi<30). the remaining 52 participants represent as apparently control group with normal weight and normal menstrual cycle. patients with pcos were selected from the infertility department, gynecology and obstetrics teaching hospital,kerbala province, iraq. diagnosis of pcos is based on 2 of 3 findings: oligo/anovulation, hyperandrogenism, polycystic ovaries in ultrasound (rotterdam criteria). the patients were interviewed and examined for weight, height, waist circumference, and hip circumference. venous blood samples were collected at 9 am after an overnight fast. measurement of serum insulin, glucose, triglyceride and c-peptide were performed using cobas instrument and by elisa technique. results: based on homa-ir, the prevalence of insulin resistance in obese pcos women was 77% while in non-obese pcos women was 70%. homa-ir, quicki, mcauley and cpi showed significant difference between obese pcos (4.39 ± 1.83), (0.309 ± 0.024), (3.85 ± 0.91) and (4.39 ± 1.63) respectively; and control group (2.68 ± 0.61), (0.331 ± 0.011), (4.53 ± 0.57) and (8.72 ± 1.33) respectively. cpi also showed significant difference between obese pcos (4.39 ± 1.63) and non-obese pcos (6.85 ± 1.74). in obese pcos women, both quicki (r = –1.00, p < 0.001) and mcauley (r = –0.81, p < 0.001) were strongly correlated with homa-ir, whereas cpi was not. for non-obese pcos, there was a strong correlation for both quicki (r = –0.97, p < 0.001), (r = –0.62, p < 0.05) and homa-ir, cpi was also strongly correlated with homa-ir (r = –0.78, p < 0.001). conclusion: significant number of women with pcos can be classified as being either insulin sensitive or insulin resistant (ir) depending on the method applied, as correlation between various ir indices is highly variable. clinical application of surrogate indices for assessment of ir in pcos must be therefore viewed with an extreme caution. keywords: c-peptide, polycystic ovary syndrome, insulin resistance 170 j contemp med sci | vol. 8, no. 3, may-june 2022: 169–175 roles of c-peptide and triglyceride as effective indices for insulin resistance investigations in iraqi women original h.h. al-murshedi et al. triglyceride-glucose index is the logarithmized product of fasting triglycerides and fasting glucose and has been proposed as the alternative indicator of ir due to its relevance to dyslipidemia.13 the glucose insulin ratio (g/i) has also been employed as an index of ir. it has been described, as a useful measure of insulin sensitivity in obese pcos women and has both high sensitivity and specificity for detecting ir in women.14 in addition, previous studies suggested that measuring c-peptide can help to determine how much of insulin a person is producing as c-peptide is secreted in equimolar amounts to insulin.15,16 c-peptide does not undergo hepatic first-pass metabolism and has a longer half-life than insulin which affords a more stable test window of fluctuating beta cell response. therefore, it has been suggested that peripheral c-peptide levels more precisely reflect β-cell secretory activity than peripheral insulin.17 an increase in its levels suggests a high level of endogenous insulin which indicates worsened insulin resistant state. the aim of the study was to assess how much ir is in both obese and non-obese pcos women using most commonly used index of ir (homa-ir) and find out a correlation between homa-ir and the other surrogate indices: g/i, quicki, mcauley, triglyceride-glucose index (tyg) and c-peptide index (cpi). materials and methods a case-control study included 120 participants of which 68 women have pcos subdivide according to their bmi to 45 obese (bmi >= 30) and 23 non-obese (bmi < 30). the remaining 52 represent the control group who were apparently healthy women with normal weight and normal menstrual cycle. patients with pcos were selected from the infertility department, gynecology and obstetrics teaching hospital, kerbala health directorate/kerbala – iraq. institutional ethics committee approval was sought before starting the study. oral informed consent was obtained from subjects. pcos was diagnosed in presence of at least two out of the three diagnostic criteria established by the revised 2003 rotterdam european society for human reproduction/american society of reproductive medicine pcos consensus workshop group: i) oligoand/or anovulation, ii) clinical and/or biochemical signs of hyperandrogenism, and iii) polycystic ovaries in ultrasound.18 all women underwent anthropometric assessment like measurement of weight, height, waist circumference (wc), hip circumference, waist-hip circumference ratio (whr) and body mass index (bmi). transvaginal ultrasound was used to identify polycystic ovaries. five milliliters (5 ml) of venous blood samples were collected at 9 am after an overnight fast on the second or third day of the menstrual cycle, centrifuged and frozen immediately at –20ºc. the levels of glucose, triglycerides, cholesterol and high-density lipoprotein (hdl) were measured using chemistry analyzer (au480, beckman coulter, usa). serum luteinizing hormone (lh) and follicle-stimulating hormone (fsh) were carried out with automated immunoassay system based on the enzyme linked fluorescent assay principles (elfa) (biomérieux, france). free testosterone was measured by enzyme-linked immunosorbent assay (elisa) technique. insulin resistant assessment fasting insulin was measured using elisa technique. fasting c-peptide was determined using cobas c 111 analyzer. ir was estimated as: 1. homa-ir = (i × g)/405 2. fasting glucose to insulin ratio = g/i 3. quicki = 1/(log(i) + log(g)) 4. mca = exp (2.63–0.28 × ln(i) – 0.31 × ln (tg/18) 5. tyg = ln [tg × g/2] 6. cpi = 20/(cp × g/18) fasting insulin (i) in (µiu/ml), fasting glucose (g) in (mg/dl), triglycerides (tg) in (mg/dl) and fasting c-peptide (cp) in (nmol/l). the data were analyzed using the statistical package for social sciences (spss version 22.0). continuous variables were expressed as means ± standard deviation (sd). mean comparisons were made using one-way analysis of variance (anova) followed by tukey’s post hoc test. pearson correlation analysis was used to assess the association of various ir indices with homa-ir. receiver operator characteristic curves (roc) were drawn to compare different insulin resistance/sensitivity indices. insulin-based homa-ir was considered as a gold standard to define insulin resistance. homa-ir values less than 2.7 were considered insulin sensitive (is) and more than that were considered as ir. results the demographic and clinical features of the total study population, as well as baseline values of the various indices of insulin resistance are presented in table 1. the nonobese pcos patients were less in age than obese. the parameters like wc and bmi, were found to be significantly increased in obese pcos women than other groups while whr was increased in obese pcos than control group. whr was not varied between obese and non-obese pcos women. both obese and non-obese pcos had increased levels of insulin, glucose, cholesterol, triglyceride and ldl compared to the controls. whereas hdl levels had not reached statistical significance (p > 0.05) among three groups. neither analysis of obese and non-obese pcos women, nor controls and non-obese pcos had significant difference regarding c-peptide values. while c-peptide was higher in obese pcos. compared with controls, pcos women had elevated levels of lh, fsh, lh/fsh ratio and free testosterone. for non-obese pcos group, there was significant difference in term of lh and free testosterone compared with controls while fsh and lh/fsh ratio were not. the assessment of ir revealed that homa-ir, quicki, mcauley, tyg and cpi had significant difference in both pcos groups compared with controls. g/i ratio was significantly higher in the controls than obese pcos women. the prevalence of ir based on homa-ir was 80% in obese pcos and 48% in non-obese pcos women as shown in figure 1. pearson correlation coefficient was performed to show the correlation of the different parameters. for both obese and non-obese pcos patients, the results revealed a positive correlation of the bmi with insulin, c-peptide, homa-ir and 171j contemp med sci | vol. 8, no. 3, may-june 2022: 169–175 h.h. al-murshedi et al. original roles of c-peptide and triglyceride as effective indices for insulin resistance investigations in iraqi women table 1. difference in demographic, metabolic, hormonal features and ir status among obese pcos, non-obese pcos and control women obese pcos (n = 45) non obese pcos (n = 23) control (n = 52) p-value demographic characteristics age (years) 29 ± 6.06 25 ± 4.99 27 ± 5.71 waist circumference (cm) 108 ± 12.82 84 ± 10.06 80 ± 10.94 <0.001 whr 0.92 ± 0.10 0.87 ± 0.10 0.82 ± 0.08 <0.001 body mass index (kg/m2) 35.83 ± 4.64 24.24 ± 2.56 24.09 ± 3.56 <0.001 clinical characteristics cholesterol (mg/dl) 163 ± 26.80 144 ± 35.25 117 ± 20.97 <0.001 triglyceride (mg/dl) 103 ± 1.50 82 ± 1.57 70 ± 1.55 <0.001 hdl (mg/dl) 40 ± 6.76 39 ± 5.17 43 ± 8.95 ns ldl (mg/dl) 97.43 ± 1.30 83 ± 1.33 56 ± 1.39 <0.001 fasting glucose (mg/dl) 92 ± 8.25 92 ± 8.88 84 ± 5.12 <0.001 fasting insulin (μu/ml) 18.15 ± 1.76 15.32 ± 1.70 10.74 ± 1.56 <0.001 fasting c-peptide (nmol/l) 0.55 ± 0.38 0.50 ± 0.26 0.37 ± 0.18 <0.05 lh (iu/l) 10.23 ± 1.68 10.05 ± 1.48 6.67 ± 1.44 <0.001 fsh (iu/l) 6.55 ± 1.38 6.45 ± 1.25 5.56 ± 1.32 <0.05 lh/fsh ratio 1.70 ± 0.76 1.73 ± 0.92 1.30 ± 0.60 <0.05 free testosterone (pg/ml) 17.57 ± 2.31 14.97 ± 2.19 4.21 ± 2.50 <0.001 insulin resistance indices homa-ir 4.10 ± 1.78 3.47 ± 1.69 2.22 ± 1.58 <0.001 g/i 5.79 ± 2.90 6.81 ± 3.20 8.49 ± 3.31 <0.001 quicki 0.312 ± 0.02 0.319 ± 0.02 0.340 ± 0.022 <0.001 mcauley 3.69 ± 0.85 4.16 ± 1.00 4.76 ± 0.94 <0.001 tyg 4.58 ± 0.20 4.46 ± 0.21 4.34 ± 0.22 <0.001 cpi 8.53 ± 1.79 9.19 ± 1.90 13.01 ± 1.64 <0.05 data was expressed as mean ± sd. p < 0.05 is considered significant. fig. 1 the prevalence of insulin resistance (ir)/sensitivity (is) based on homa-ir in both obese and non-obese pcos women. tyg index and negative correlation with g/i, quicki, mcauley and cpi. homa-ir showed significant positive correlation with insulin, c-peptide and tyg. while it shows significant negative correlation with g/i, quicki, mcauley and cpi. more details were demonstrated in tables 2 and 3. mcnemar test was performed on both obese/non-obese pcos women to check the concordance/discordance between insulin resistance indices and homa-ir, as shown in tables 4 and 5. figure 2 shows the roc curve for g/i ratio, quicki, mcauley, tyg and cpi indices as predictors for homa-ir. g/i ratio, quicki and mcauley strongly predicted homa-ir in both pcos groups. tyg can be predicted homa-ir in obese pcos with auc value of 0.79 (p < 0.05). cpi failed to predict homa-ir in both obese/non-obese pcos, as shown in table 6. discussion the issue of ir in pcos, though seemingly obvious, is indeed highly problematic, when supposed to be transformed from a theoretical concept into a clinical application. some studied suggested that ir was apparent not in terms of exceeding a predefined cut-off point, but as lack in insulin sensitivity in comparison to bmi-matched non-pcos.19 it must be noted that there is no universal agreement as to the best cut-off point for various insulin-resistance indices. thus, any cut-off points should be related to particular studied population, as significant ethnic differences have been reported (wijeyaratne et al., 2002).20 172 j contemp med sci | vol. 8, no. 3, may-june 2022: 169–175 roles of c-peptide and triglyceride as effective indices for insulin resistance investigations in iraqi women original h.h. al-murshedi et al. table 2. pearson correlation analysis (obese pcos) wc whr bmi insulin tg g/i ratio homa-ir quicki mcauley tyg c-peptide cpi wc r 1 0.78 0.68 0.32 .266 0.51 0.32 0.48 0.50 0.35 .128 –.234 p .000 .000 .022 .057 .000 .023 .000 .000 .012 .366 .095 whr r 1 0.39 0.33 .164 0.46 0.32 0.41 0.41 .214 .058 –.075 p .004 .016 .245 .001 .023 .003 .002 .127 .683 .595 bmi r 1 0.56 0.44 0.55 0.57 0.60 0.61 0.48 0.41 0.4 p .000 .001 .000 .000 .000 .000 .000 .002 .004 insulin r 1 0.48 0.83 0.99 0.88 0.79 0.49 0.7 0.54 p .000 .000 .000 .000 .000 .000 .000 .000 tg r 1 0.49 0.48 0.50 0.82 0.96 0.35 0.33 p .000 .000 .000 .000 .000 .010 .018 g/i ratio r 1 0.81 0.94 0.86 0.49 0.48 0.49 p .000 .000 .000 .000 .000 .000 homa-ir r 1 0.89 0.79 0.51 0.71 0.56 p .000 .000 .000 .000 .000 quicki r 1 0.87 0.57 0.56 0.61 p .000 .000 .000 .000 mcauley r 1 0.86 0.52 0.55 p .000 .000 .000 tyg r 1 0.38 0.43 p .006 .002 c-peptide r 1 0.72 p .000 cpi r 1 p yellow color refers to positive correlation, blue color refers to negative correlation. table 3. pearson correlation analysis (non-obese pcos) wc whr bmi insulin tg g/i ratio homa-ir quicki mcauley tyg c-peptide cpi wc r 1 0.57 0.27 –0.07 0.08 –0.04 –0.10 0.08 –0.05 0.06 –0.08 0.07 p .004 .211 .768 .712 .863 .661 .705 .828 .796 .721 .743 whr r 1 –0.09 –0.15 -.021 –0.12 –0.17 0.007 –0.08 .028 .114 –.088 p .686 .504 .924 .571 .451 .973 .709 .900 .605 .690 bmi r 1 0.69 0.70 0.70 0.66 0.62 0.78 0.63 0.43 –0.21 p .000 .000 .000 .001 .002 .000 .001 .042 .328 insulin r 1 0.52 0.81 0.99 0.90 0.78 0.49 0.45 –0.37 p .011 .000 .000 .000 .000 .017 .033 .084 tg r 1 0.53 0.49 0.47 0.86 0.96 0.266 –0.2 p .009 .016 .022 .000 .000 .220 .349 g/i ratio r 1 0.79 0.90 0.85 0.48 0.44 0.41 p .000 .000 .000 .020 .037 .050 homa-ir r 1 0.93 0.77 0.49 0.47 0.41 p .000 .000 .017 .024 .055 quicki r 1 0.81 0.49 0.52 0.53 p .000 .018 .011 .009 (continued) 173j contemp med sci | vol. 8, no. 3, may-june 2022: 169–175 h.h. al-murshedi et al. original roles of c-peptide and triglyceride as effective indices for insulin resistance investigations in iraqi women table 4. comparison of insulin resistance indices and homa-ir for assessment of ir in obese women with pcos (cut-off for homa-ir > 2.7) ir indices homa-ir total yes n (%) no n (%) g/i ratio yes 33 (100%)a 0 (0%)c 33 (73%) no 3 (25%)b 9 (75%)d 12 (27%) quicki yes 36 (100%)a 0 (0%)a 36 (80%) no 0 (0%)b 9 (100%)d 9 (20%) mcauley yes 35 (97%)a 1 (3%)c 36 (80%) no 1 (11%)b 8 (89%)d 9 (20%) tyg yes 27 (93%)a 2 (7%)c 29 (64%) no 9 (56%)b 7 (44%)d 16 (36%) cpi yes 18 (86%)a 3 (14%)c 21 (47%) no 18 (75%)b 6 (25%)d 24 (53%) atrue positive, bfalse negative, cfalse positive, dtrue negative. table 3. pearson correlation analysis (non-obese pcos)—continued wc whr bmi insulin tg g/i ratio homa-ir quicki mcauley tyg c-peptide cpi mcauley r 1 0.86 0.42 0.347 p .000 .044 .105 tyg r 1 0.285 –0.23 p .188 .300 c-peptide r 1 0.79 p .000 cpi r 1 p yellow refers to positive correlation, blue color refers to negative correlation. table 5. comparison of insulin resistance indices and homa-ir for assessment of ir in non-obese women with pcos (cut-off for homa-ir > 2.7) ir indices homa-ir total yes n (%) no n (%) g/i ratio yes 11 (79%)a 3 (21%)c 14 (61%) no 0 (0%)b 9 (100%)d 9 (39%) quicki yes 11 (92%)a 1 (8%)c 12 (52%) no 0 (0%)b 11 (100%)d 11 (48%) mcauley yes 10 (71%)a 4 (29%)c 14 (61%) no 1 (11%)b 8 (89%)d 9 (39%) tyg yes 7 (70%)a 3 (30%)c 10 (44%) no 4 (31%)b 9 (69%)d 13 (56%) cpi yes 8 (62%)a 5 (38%)c 13 (56%) no 3 (30%)b 7 (70%)d 10 (44%) atrue positive, bfalse negative, cfalse positive, dtrue negative. in current study ir reported to be more prevalent in obese pcos group than non-obese pcos, the same finding was recorded previously, who reported that ir in pcos had linked to obesity.21 although non-obese women exhibit lower ir is still a common finding in this population. indeed, several studies have suggested ir as a pathophysiological component independent of weight.22 obese pcos have a high probability of ir.23 it was also observed a significant, but relatively weak correlation between all analyzed ir indices and adiposity indices: wc, whr and bmi in obese pcos group. recent studies reported that whr was positively correlated with the homa-ir.24 a study of šumarac-dumanović et al., confirmed that pcos women are more susceptible to increasing whr regarding the development of insulin resistance.25 in the current study, women with pcos had higher fasting insulin levels than controls. similar results were found by another investigators.26 it is also indicated that c-peptide was higher only in obese pcos. another study reported that c-peptide concentrations were not reached statistically significant among pcos overweight group, pcos obese group and healthy women. also, it did not correlate significantly with fsh and lh serum levels within studied groups.27 conversely, another investigators suggested that c-peptide can be used as a surrogate marker of ir in pcos.28 a study by banu et al. also indicated that assessment of c-peptide levels, along with hdl-c levels, in patients can be used to monitor ir.29 there is a very good correlation between indices of ir based on fasting glucose and insulin homa with g/i and quicki, also homa has a good correlation with mcauley that utilizes fasting triglyceride concentrations and insulin. hence, there is an implication that fasting triglyceride concentrations can be safely used to assess ir, instead of fasting glucose. in a previous study by lewandowski et al., mcauley 174 j contemp med sci | vol. 8, no. 3, may-june 2022: 169–175 roles of c-peptide and triglyceride as effective indices for insulin resistance investigations in iraqi women original h.h. al-murshedi et al. fig. 2 the results of roc curve analysis regarding the predictability of g/i ratio, quicki, mcauley, tyg and cpi indices in classifying the ir considering homa-ir in (a) non-obese pcos and (b) obese pcos. table 6. the areas under roc curve (auc), sensitivity, specificity by the optimized cut-off points for ir indices in predicting the homa-ir predictors auc (95% ci) p-value cut-off value sensitivity specificity obese pcos g/i ratio 0.04 (0.00–0.09) 0.000 7.37 92% 100% quicki 0.00 (0.00–0.00) 0.000 0.33 100% 100% mcauley 0.06 (0.00–0.14) 0.000 4.3 97% 89% tyg 0.79 (0.64–0.94) 0.008 4.51 75% 78% cpi 0.36 (0.14–0.57) 0.182 8.52 50% 66% non-obese pcos g/i ratio 0.05 (0.00–0.12) 0.000 7.37 100% 75% quicki 0.00 (0.00–0.00) 0.000 0.33 100% 100% mcauley 0.11 (0.00–0.23) 0.001 4.3 91% 66% tyg 0.72 (0.51–0.93) 0.074 4.51 64% 75% cpi 0.34 (0.11–0.57) 0.196 8.52 73% 58% index was found to have a good correlation with homa-ir (r = −0.849) in large (n = 478) group of women with pcos aged 25 ± 8.05, bmi 27.27 ± 7.18 kg/m2 (lewandowski et al., 2018).30 kheirollahi et al. suggested that tyg index strongly correlated with ir as estimated by homa-ir, among iranian women diagnosed with pcos.31 also, a recent study included 11,378 adults proposed tyg index as a useful surrogate measure of ir (auc was 0.723) as reported previously.32 c-peptide is accepted as a better descriptor of pancreatic activity than peripheral insulin itself. in the current study, a weak correlation was found between homa-ir and c-peptide. previously, tura et al. found that an index based on insulin and glucose (igi) strongly correlated with corresponding index for c-peptide, indicating that hepatic insulin extraction is not a confounding factor in the relationship between insulin and c-peptide-based indices.33 additionally, mj found that values of fasting glucose, insulin, c-peptide and the homa index significantly increased with age and pubertal stage, while the quicki index decreased.34 a study by ohkura et al. revealed that the index cpi was more strongly correlated with glucose infusion rate (gir) derived from the glucose clamp technique, than were homa-ir and quicki, as cpi requires a single blood sample and plasma c-peptide levels better reflect insulin bioactivity in skeletal muscle, it was recommend for screening of ir.35 conclusion it can be concluded that tyg is a valuable indicator to predict ir in obese women with pcos, partly due to its analytical and financial ease-of-access in all clinical laboratories. the use of tyg index is recommended in the assessment of ir risk among iraqi women with pcos. further epidemiological research is advised. conflicts of interest none.  175j contemp med sci | vol. 8, no. 3, may-june 2022: 169–175 h.h. al-murshedi et al. original roles of c-peptide and triglyceride as effective indices for insulin resistance investigations in iraqi women references 1. legro, r. s., arslanian, s. a., ehrmann, d. a., hoeger, k. m., murad, m. h., pasquali, r. & welt, c. k. 2013. diagnosis and treatment of polycystic ovary syndrome: an endocrine society clinical practice guideline. the journal of clinical endocrinology & metabolism, 98, 4565–4592. 2. azziz, r., woods, k. s., reyna, r., key, t. j., knochenhauer, e. s. & yildiz, b. o. 2004. the prevalence and features of the polycystic ovary syndrome in an unselected population. the journal of clinical endocrinology & metabolism, 89, 2745–2749. 3. fauser, b. c., tarlatzis, b. c., rebar, r. w., legro, r. s., balen, a. h., lobo, r., carmina, e., chang, j., yildiz, b. o. & laven, j. s. 2012. consensus on women’s health aspects of polycystic ovary syndrome (pcos): the amsterdam eshre/asrm-sponsored 3rd pcos consensus workshop group. fertility and sterility, 97, 28–38. e25. 4. mayer, s. b., evans, w. s. & nestler, j. e. 2015. polycystic ovary syndrome and insulin: our understanding in the past, present and future. women’s health, 11, 137–149. 5. amato, m. c., vesco, r., vigneri, e., ciresi, a. & giordano, c. 2015. hyperinsulinism and polycystic ovary syndrome (pcos): role of insulin clearance. j endocrinol invest, 38, 1319–26. 6. spritzer, p. m. 2014. polycystic ovary syndrome: reviewing diagnosis and management of metabolic disturbances. arq bras endocrinol metabol, 58, 182–7. 7. amisi, c. a. 2022. markers of insulin resistance in polycystic ovary syndrome women: an update. world journal of diabetes, 13, 129. 8. wild, r. a., carmina, e., diamanti-kandarakis, e., dokras, a., escobarmorreale, h. f., futterweit, w., lobo, r., norman, r. j., talbott, e. & dumesic, d. a. 2010. assessment of cardiovascular risk and prevention of cardiovascular disease in women with the polycystic ovary syndrome: a consensus statement by the androgen excess and polycystic ovary syndrome (ae-pcos) society. j clin endocrinol metab, 95, 2038–49. 9. defronzo, r. a., tobin, j. d. & andres, r. 1979. glucose clamp technique: a method for quantifying insulin secretion and resistance. am j physiol, 237, e214–e23. 10. matthews, d. r., hosker, j. p., rudenski, a. s., naylor, b. a., treacher, d. f. & turner, r. c. 1985. homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. diabetologia, 28, 412–9. 11. chen, h., sullivan, g. & quon, m. j. 2005. assessing the predictive accuracy of quicki as a surrogate index for insulin sensitivity using a calibration model. diabetes, 54, 1914–25. 12. mcauley, k. a., williams, s. m., mann, j. i., walker, r. j., lewis-barned, n. j., temple, l. a. & duncan, a. w. 2001. diagnosing insulin resistance in the general population. diabetes care, 24, 460–4. 13. liu, x.-c., he, g.-d., lo, k., huang, y.-q. & feng, y.-q. 2021. the triglycerideglucose index, an insulin resistance marker, was non-linear associated with all-cause and cardiovascular mortality in the general population. frontiers in cardiovascular medicine, 7. 14. legro, r. s., finegood, d. & dunaif, a. 1998. a fasting glucose to insulin ratio is a useful measure of insulin sensitivity in women with polycystic ovary syndrome. j clin endocrinol metab, 83, 2694–8. 15. de medeiros, s. f., angelo, l. c. a., de medeiros, m. a. s., banhara, c. r., barbosa, b. b. & yamamoto, m. m. w. 2018. the role of c-peptide as marker of cardiometabolic risk in women with polycystic ovary syndrome: a controlled study. j clin med res, 10, 260–267. 16. saisho, y. 2016. postprandial c-peptide to glucose ratio as a marker of β cell function: implication for the management of type 2 diabetes. int j mol sci, 17. 17. yosten, g. l., maric-bilkan, c., luppi, p. & wahren, j. 2014. physiological effects and therapeutic potential of proinsulin c-peptide. am j physiol endocrinol metab, 307, e955–e68. 18. wang, r. & mol, b. w. j. 2017. the rotterdam criteria for polycystic ovary syndrome: evidence-based criteria? human reproduction, 32, 261–264. 19. dunaif, a., segal, k. r., futterweit, w. & dobrjansky, a. 1989. profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. diabetes, 38, 1165–1174. 20. wijeyaratne, c. n., balen, a. h., barth, j. h. & belchetz, p. e. 2002. clinical manifestations and insulin resistance (ir) in polycystic ovary syndrome (pcos) among south asians and caucasians: is there a difference? clinical endocrinology, 57, 343–350. 21. unluer, a., findik, r., sevinc, n. & karakaya, j. 2013. comparison of hba1c levels in obese and non-obese polycystic ovarian patients. clinical and experimental obstetrics & gynecology, 40, 148–150. 22. messer, c., boston, r., leroith, d., geer, e., miller, j. d., messer, m. & futterweit, w. 2012. pancreatic β-cell dysfunction in polycystic ovary syndrome: the role of metformin. endocrine practice, 18, 685–693. 23. dahan, m., abbasi, f. & reaven, g. 2007. prevalence of insulin resistance among american women with polycystic ovary syndrome (pcos) as a function of body mass index (bmi). fertility and sterility, 88, s78–s79. 24. benites-zapata, v. a., toro-huamanchumo, c. j., urrunaga-pastor, d., guarnizo-poma, m., lazaro-alcantara, h., paico-palacios, s., pantoja-torres, b., del carmen ranilla-seguin, v. & group, m. s. r. 2019. high waist-to-hip ratio levels are associated with insulin resistance markers in normal-weight women. diabetes & metabolic syndrome: clinical research & reviews, 13, 636–642. 25. šumarac-dumanović, m., stamenković-pejković, d., jeremić, d., dumanović, j., mandić-marković, v., žarković, m. & micić, d. 2022. age, body mass index, and waist-to-hip ratio related changes in insulin secretion and insulin sensitivity in women with polycystic ovary syndrome: minimal model analyses. international journal of endocrinology, 2022. 26. el-kannishy, g., kamal, s., mousa, a., saleh, o., el badrawy, a. & shokeir, t. 2010. endothelial function in young women with polycystic ovary syndrome (pcos): implications of body mass index (bmi) and insulin resistance. obesity research & clinical practice, 4, e49–e56. 27. maciejewska-jeske, m., szczesna, a. & męczekalski, b. 2010. serum c-peptide concentration in overweight and obese women with polycystic ovary syndrome. polski merkuriusz lekarski: organ polskiego towarzystwa lekarskiego, 29, 93–99. 28. polak, k., czyzyk, a., simoncini, t. & meczekalski, b. 2017. new markers of insulin resistance in polycystic ovary syndrome. journal of endocrinological investigation, 40, 1–8. 29. banu et al. (2011). 30. lewandowski, k. c., płusajska, j., horzelski, w., bieniek, e. & lewiński, a. 2018. limitations of insulin resistance assessment in polycystic ovary syndrome. endocrine connections, 7, 403–412. 31. kheirollahi, a., teimouri, m., karimi, m., vatannejad, a., moradi, n., borumandnia, n. & sadeghi, a. 2020. evaluation of lipid ratios and triglyceride-glucose index as risk markers of insulin resistance in iranian polycystic ovary syndrome women. lipids in health and disease, 19, 1–9. 32. lee, j., kim, b., kim, w., ahn, c., choi, h. y., kim, j. g., kim, j., shin, h., kang, j. g. & moon, s. 2021. lipid indices as simple and clinically useful surrogate markers for insulin resistance in the us population. scientific reports, 11, 1–9. 33. tura, a., kautzky-willer, a. & pacini, g. 2006. insulinogenic indices from insulin and c-peptide: comparison of beta-cell function from ogtt and ivgtt. diabetes research and clinical practice, 72, 298–301. 34. mj, a. v. the homa and quicki indexes, and insulin and c-peptide levels in healthy children. cut off points to identify metabolic syndrome in healthy children. anales de pediatria (barcelona, spain: 2003), 2007. 481–490. 35. ohkura, t., shiochi, h., fujioka, y., sumi, k., yamamoto, n., matsuzawa, k., izawa, s., kinoshita, h., ohkura, h. & kato, m. 2013. 20/(fasting c-peptide× fasting plasma glucose) is a simple and effective index of insulin resistance in patients with type 2 diabetes mellitus: a preliminary report. cardiovascular diabetology, 12, 1–8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.xxxx admin12 sticky note aq please provide complete details for ref. 29. (banu et al.). admin12 sticky note aq please provide doi number for this article. the provided website is not working. ammar sticky note banu, s., r jabir, n., n manjunath, c., shakil, s., & a kamal, m. (2011). c-peptide and its correlation to parameters of insulin resistance in the metabolic syndrome. cns & neurological disorders-drug targets (formerly current drug targets-cns & neurological disorders), 10(8), 921-927. ammar sticky note https://doi.org/10.22317/jcms.v8i3.1220 18 j contemp med sci | vol. 9, no. 1, january-february 2023: 18–23 original comparing col7a1 gene expression in fibroblast cells of dystrophic epidermolysis bullosa patients with clinical responses to autologous fibroblasts transplantation maryam eslami,1,2*, majid golshanfard1,2, amir bajouri3, nasser aghdami4, mahsa mohammadi1, saeed shafieian3, omeed memarsadeghi2, alexander seifalian5 1department of genetics, tehran medical sciences, tehran medical sciences, islamic azad university, tehran, iran. 2applied biotechnology research center, tehran medical sciences, islamic azad university, tehran, iran. 3department of stem cells and developmental biology, cell science research center, royan institute for stem cell biology and technology, acecr, tehran, iran. 4skin and stem cell research center, tehran university of medical sciences, tehran, iran. 5nanotechnology & regenerative medicine commercialisation centre (ltd), london bioscience innovation centre, london, united kingdom. *correspondence to: maryam eslami, (e-mail: drmaryam.eslami2020@gmail.com) abstract objectives: this clinical research aimed to establish autologous fibroblasts transplantation as a possible treatment for patients with dej. the col7a1 gene expression was also evaluated. methods: six patients (3m and 3f), 4 with no recurrent wounds and 2 with recurrent wounds after surgery, and 15 healthy subjects were included in the study as controls. quantitative real-time polymerase chain reaction (real-time pcr) analysis of the col7a1 gene was performed using an oligonucleotide primer pair designed to amplify across the exon/exon junction. results: the col7a1 expression level was down-regulated at exons 26-27, 47-48, 96-97, and 116-117 in all patients’ fibroblasts compared with the healthy controls. however, the expression of the col7a1 gene in the fibroblasts of the patients with a positive response to the treatment was not significantly changed compared with the patients with the poor response. (clinicaltrials.gov nct01908088) conclusion: in this study the mrna expression levels of col7a1 were significantly less in the patients when compared with healthy controls. however the col7a1 expression after autologous fibroblasts transplantation was not different between the two groups of patients, and further examination is needed to elucidate the mechanism of the treatment. keywords: rdeb, col7a1, gene expression, fibroblast, transplantation, clinical trial, dermal-epidermal junction issn 2413-0516 introduction epidermolysis bullosa (eb) is a rare genetic skin disorder that results in fragility, easy blistering, and ulceration of the skin with painful and life-threatening complications. the disease has an incidence of 0.08–0.5 per million live births, occurs among all ethnicities, and is inherited either from one of the parents or both due to deficiency in several genes.1-3 recessive dystrophic eb (rdeb) is attributed to the bi-allelic loss-offunction mutations in col7a1 (3p21.31), a gene expressed by skin keratinocytes and fibroblasts which encodes type vii collagen (c7).4 c7 is the main component of anchoring fibrils (afs) which ensures the adherence of the epidermis to the dermis within the basement membrane. mutation in the col7a1 gene alters the c7 structure, thereby compromising the integrity of the dermal-epidermal junction (dej), and causes blistering and tissue cleavage, which leads to extensive scarring.5-9 eb has four main types including simplex, junctional, dystrophic, and kindler.10,11 recessive dystrophic epidermolysis bullosa (rdeb) is the most severe type of the disease. children with rdeb are usually affected since birth and, in most cases, blisters virtually cover all the body and the patients who survive childhood frequently develop squamous cell carcinoma (scc).12,13 the blisters and ulcers on the hands and fingers of the patients suffering from rdeb cause hand deformities with pseudo-ductility and flexor contractures known as ‘mitten hand’.14 treatments for rdeb are wound grafting, allogeneic fibroblasts, mesenchymal stromal cells, bone marrow transplantation and gene therapy.15,16 sometimes rdeb patients with mitten hand undergo hand reconstructive surgery to regain their hand function. afterwards, the surgeon applies auto-graft skin or skin substitutes on the open wounds of the hand and fingers. to prevent further adhesion and facilitate satisfactory postoperative healing, fibroblast and/or keratinocyte transplantation could be applied to the wound. however, fibroblasts are easier to culture than keratinocytes and are better cells to target in planning cells.17-19 the aim of this study was to investigate the responses of patients with rdeb to autologous fibroblasts transplantation as a possible treatment. the authors believe that the major effect of autologous fibroblasts transplantation is to increase col7a1 mrna levels. as a result of increased expression of col7a1 we have greater deposition of the mutant type vii collagen at the dej and formation of rudimentary anchoring fibrils. the col7a1 gene expression in the fibroblasts of the patients with rdeb was independently evaluated and the results were compared with the healthy controls. furthermore, the col7a1 gene expression in the patients with inappropriate response to autologous fibroblasts transplantation was evaluated and then compared with the patients with the appropriate response after 18 months. patients and methods patients six patients with rdeb between the age range of 2 and 30 years old were selected. previously, autologous fibroblasts (submitted: 04 november 2022 – revised version received: 21 november 2022 – accepted: 10 january 2023 – published online: 26 february 2023) mailto:maryam.eslami2020@gmail.com 19j contemp med sci | vol. 9, no. 1, january-february 2023: 18–23 m. eslami et al. original comparing col7a1 gene expression in fibroblast cells from patients had been seeded on the amniotic membrane. fibroblasts were transplanted on the hands of the rdeb patients with mitten hands after hand reconstructive surgery. the patients were followed up for 18 months and it was revealed that wounds of some patients returned as soon as a few months after the surgery; however, wounds of the others remained intact for 18 months (figures 1 and 2). the control group were 15 healthy individuals. the fibroblasts of these individuals with no personal or family history of eb were assessed as a control group. if the recurrence time of deformity after hand surgery was less than or equal to 6 months, it was considered as an inappropriate treatment response. diagnosis of rdeb patients was established based on the clinical symptoms and ngs test by a dermatologist. the clinical features of all the patients are shown in table 1. the study protocol was approved by royan institute’s ethics board committee in medical research. (clinicaltrials. gov nct01908088). gene expression four patients with appropriate clinical response and two patients with a poor clinical response after the treatment were selected for assessment of col7a1 gene expression. rna extraction and cdna synthesis skin biopsies from healthy donors and eb patients were obtained and stored in liquid nitrogen for the investigation. fibroblasts were extracted using an enzymatic method and cultured in the t75 flask in modified dmem/ f12 medium (gibco-usa) supplemented with 10% fetal bovine serum (fbs) (hyclone-usa) and 1% glutamax (gibco-usa). total rna was extracted from the cultured cells (at least 106 cells were used for each sample) using the roche kit (mannheim, germany) with dnase i treatment according to the manufacturer’s instructions. the quality and quantity of the extracted rna were evaluated by nano-drop uv–vis spectrophotometer (thermo, usa) and electrophoresis. cdna was synthesized from 1 μg of mrna using the thermo fisher cdna synthesis kit (waltham, ma, usa), followed by quantitative polymerase chain reaction (qpcr). primer design according to the abi biosystems steponeplus instrumentation, beacon designer software was used to design the primers. the default parameters of the software were set to be very limited. the most important parameters were the amplicon length, quality, and hotspot region for the dominant mutations of the primers. exons and introns were relatively small and the default parameters for the amplicon lengths were set between 120 and 150 nucleotides. to overcome the problem of genomic dna, the primers were designed from the exon–exon junctions (e-e-jns). using beacon designer software, these primer sets had to be searched manually. the regions, in which mutations were more likely to occur, were selected. 5 sets of primers designed for 5 different regions of the col7a1 gene: exons 4-5 exons 26-27, exons 47-48, exons 96-97, and exons 116-117. the primers’ positions and sequences are listed in table 2. in addition, the β-actin housekeeping gene was measured in parallel to normalize the differences between the samples and operations. fig. 1 successful autologous fibroblasts transplantation as a treatment for rdeb patients. (a) hands of six-year-old boy (patient 1) before operation, (b) the grafted area remained epithelized without blistering and recurrent wounds, the grafted area after two (c), nine (d), and 15 months after the treatment. 20 j contemp med sci | vol. 9, no. 1, january-february 2023: 18–23 comparing col7a1 gene expression in fibroblast cells original m. eslami et al. fig. 2 unsuccessful autologous fibroblasts transplantation. (a) hands of patient 3 before operation, (b) the grafted area with blistering and recurrent wounds 2 months after the treatment, (c) the grafted area 9 months after the treatment, (d) the grafted area 15 months after the treatment. table 1. characteristics of rdeb patients patient age (years)/sex distribution of disease (%) parents affected familial marriage gi surgery previous hand surgery recurrence of deformity (month) 01 6/m 25 no yes no yes 9 02 17/f 30 no yes yes yes 12 03 7/f 20 no yes no no 6 04 9/m 15 no yes no no 18 05 15/m 40 no yes yes no 18 06 7/m 50 no yes no yes 5 m: male; f: female; gi: gastrointestinal. table 2. forward and reverse primers for b-actin and col7a1 genes, with temperature melting at 56ºc gene forward primers reverse primers b-actin tgaagatcaagatcattg taacgcactaagtcataa col7a1 (exon4,5) ctatttgctgtggggatc aagatgctgaagtcattga col7a1 (exon26,27) gtcacagctcacagatac ccacattaagcccagaag col7a1 (exon47,48) caaaggagaaaagggagatg tctccaggaagaaaccaag col7a1 (exon96,97) aaaggagacaagggagac cttgtcaccctttagtcc col7a1 (exon116,117) gatagtgatgaccctgt gccaccatagacaaaagg real-time polymerase chain reaction the expression level of col7a1 in fibroblasts of 6 patients with deb and 15 healthy controls was measured by qpcr. for qpcr, cdna was added to the qpcr mastermix (rox) and sybr green. all the reaction mixtures were amplified using an abi biosystems steponeplus real-time pcr system according to the following steps: pre-denaturing step at 95˚c for 30 seconds, 40 cycles of denaturing step in 95˚c for 15 seconds, 40 cycles of annealing step in 56˚c for 30 seconds, 40 21j contemp med sci | vol. 9, no. 1, january-february 2023: 18–23 m. eslami et al. original comparing col7a1 gene expression in fibroblast cells cycles of extending step in 72˚c for 30 seconds, and finally the melting curve step in 95˚c for 15 seconds. relative expression levels were determined using the threshold cycle (ct) numbers or values. data analysis the relative quantitative real-time pcr technique was computed and used for statistical analysis. the efficiency of the primers of the target and housekeeping genes was calculated using linreg pcr software (amc, amsterdam). the cycle threshold (ct) values were analyzed using rest software (rest© 2009, qiagen, germany) based on the pfaffl method.20 this software is specifically designed for molecular biology applications and compares two or more groups or conditions using ct values (ct) in the control group for multiple references and target genes. the target gene level was normalized to the actb housekeeping gene in the same sample. each sample was measured in triplicate. moreover, the melting curves and amplification plot for each pcr product were analyzed to ensure the specificity of the amplification product. moreover, the standard curve revealed that the efficiency of the reactions for actb and col7a1 was 1.86 and 1.97, respectively. the maximum pcr efficiency was 2 and the minimum was 1. the data were performed by using a one-way analysis of variance (anova). data represent the mean ± sd of three replicates. results col7a1 is less expressed in the patients' fibroblasts than that of healthy controls: expression of the col7a1 gene (figure 3) using the primer target exons 4-5 showed no significant difference between healthy volunteers and the patients group. however, the expression decreased significantly when using the other designed primers: exons, 26-27, 47-48, 96-97, and 116-117. col7a1 expression in the patients with poor response did not change significantly compared to patients with the appropriate response. expression of the col7a1 gene in fibroblasts of the patients with appropriate response demonstrated no difference compared with that of the patients with poor response; so differences in response to treatment can be traced to other factors such as differences in patients ‘mutations, mutation quality, protein modelling, and patients’ clinical and physiological conditions (figure 4). discussion eb is a blistering disorder caused by at least 18 variable gene mutations.20 identification of specific mutations in patients with rdeb, as well as other heritable disorders, has several advantages for the diagnosis and prognostication of the disease. several studies have provided evidence for the important role of the col7a1 gene in rdeb.2,21,22 col7a1 is essential for promoting the attachment of the epidermis to the dermis. its dysfunction may lead to the generalized mucosal and cutaneous blistering associated with severe deformities.23,24 over the past few years, significant progress has been made in preclinical studies aiming at developing new treatments for rdeb patients using various geneand cell-based therapies. there are two major approaches to introducing the expression of intact col7a1. fig. 3 the outcome of the gene expression of col7a1. col7a1 (exon4, 5) expression change was not significant in the patient group compared to the control group (p > 0.05). col7a1 (exon26, 27) is down-regulated in patient group in comparison to control group (****p < 0001). col7a1 (exon47, 48) is down-regulated in patient group in comparison to control group (****p < 001). col7a1 (exon96, 97) is downregulated in patient group in comparison to control group (****p < 0001). col7a1 (exon116, 117) is down-regulated in patient group in comparison to control group (****p < 001).the data were performed by using one-way analysis of variance (anova). data represent the mean ± sd of three replicates. ***p < 0.001 compared with control? fig. 4 the results of col7a1 expression in the patients with poor response compared to patients with appropriate response. col7a1 expression in the patients with poor response did not change significantly compared to patients with appropriate response (p > 0.05). the data were performed by using one-way analysis of variance (anova). data represent the mean ± sd of three replicates. one is ex vivo gene therapy with retrovirus vectors transferring the full-length col7a1 cdna into epidermal stem cells or fibroblasts based on autologous transplantation of epidermal grafts or intradermal injection, respectively.25,26 22 j contemp med sci | vol. 9, no. 1, january-february 2023: 18–23 comparing col7a1 gene expression in fibroblast cells original m. eslami et al. matsumura w. et al.27 have been used cultured epidermal autografts (ceas) from clinically normal skin for rdeb. the grafted area remained epithelized for more than 16 years without blistering. col7 expression increased in the basement membrane zone (bmz) of the grafted area than in the affected (untreated) area. they reported (cea) as a potentially well-tolerated treatment for rdeb patients. quantitative data analysis indicated that the expression of col7a1 in deb patients was significantly downregulated compared with the control group (p < 0.05). it has been estimated there were about 200 families with deb in iran. they examined 152 families with deb.28 col7a1 mutations were found in 95 of these patients (96.9%) and 104 distinct mutations were identified. here, the gene expression profiling of autologous dermal fibroblasts was shown in the rdeb patients with the inappropriate response and the rdeb patients with an appropriate response to the autologous fibroblasts transplant after 5 years. similar to the present investigation. the potential clinical benefits of intradermal injections of allogeneic fibroblasts has been studied in five patients with rdeb. no adverse effects were observed.19,29 injections of allogeneic fibroblasts led to less dermal-epidermal blistering and the increased type vii collagen expression at the dej. the mutant col7a1 gene expression in the recipients was increased 3 months after the intradermal injection of allogeneic fibroblasts. they believed that in rdeb patients, the main cause of the increase in type vii collagen was the increased expression of the col7a1 gene. it has been showed that intradermal injections of genetically corrected patient-derived fibroblasts have positive effects on the treatment of patients.30 he demonstrated that a single in vivo intradermal injection of 3 ´ 106 fibroblasts is efficient to restore c7 expression and anchoring fibril formattion at the dermal-epidermal junction. injected fibroblasts are detectable in the injected area 8 weeks after a single injection and dermal-epidermal adherence had been improved. the test on the dissemination potential of intradermally injected fibroblasts by pcr for analyzing col7a1 gene sequence and col7a1 sequence were detected in injected skin samples as they expected. the application of gene reframing therapy for rdeb fibroblast with crispr/cas9 is widely used for gene editing.31 qrt-pcr analysis showed that col7a1 expression of treated primary rdeb fibroblasts was higher than that of non-treated primary rdeb fibroblasts. they also intradermally injected reframed immortalized rdeb fibroblasts and normal fibrolblasts into the back skin of nod/shijic-scid mice. two weeks after injection, human col7(hcol7) was detected along the dermal-epidermal junction, suggesting that rdeb fibroblasts can express col7 protein after gene reframing therapy.32-34 in the present study, a comparison of the col7a1 gene expression between the two groups after this treatment revealed no significant upand/or down-regulation (p > 0.05); however, the decreased expression of col7a1 was observed. the col7a1 secreted from fibroblasts was more likely to be degraded in the wound bed. this is the first study that examined the col7a1 expression of rdeb patients after autologous fibroblasts transplantation. conclusion this preliminary study showed that the mrna expression levels of col7a1 were significantly less in the patients with rdeb compared with a healthy volunteer with match age and sex. the col7a1 expression after transplantation of the fibroblasts did not significantly change in the patients with poor response compared with the patients with positive response and further studies are needed to elucidate the mechanism of the treatment. acknowledgments the authors would like to acknowledge the financial support provided by tehran medical sciences, islamic azad university. the authors declare their sincere thanks to all the staff at royan institute and abtin laboratory for their cooperation in the process. the authors would like to thank dr. ehsan taghiabadi for his assistance in the isolation and culturing of dermal fibroblast cells. funding this study was funded by islamic azad university-tehran medical sciences, and abtin laboratory. conflict of interest none.  references 1. baardman r, yenamandra v, duipmans j, pasmooij a, jonkman m, van den akker p, et al. novel insights into the epidemiology of epidermolysis bullosa (eb) from the dutch eb registry: eb more common than previously assumed? journal of the european academy of dermatology and venereology. 2021;35(4):995–1006. 2. eichstadt s, tang jy, solis dc, siprashvili z, marinkovich mp, whitehead n, et al. from clinical phenotype to genotypic modelling: incidence and prevalence of recessive dystrophic epidermolysis bullosa (rdeb). clinical, cosmetic and investigational dermatology. 2019;12:933. 3. yadav rs, jayswal a, shrestha s, gupta sk, paudel u. dystrophic epidermolysis bullosa. jnma; journal of the nepal medical association. 2018;56(213):879–82. 4. zeng m, alshehri f, zhou d, lara-sáez i, wang x, li x, et al. efficient and robust highly branched poly (β-amino ester)/minicircle col7a1 polymeric nanoparticles for gene delivery to recessive dystrophic epidermolysis bullosa keratinocytes. acs applied materials & interfaces. 2019;11(34):30661–72. 5. lwin sm, syed f, di w-l, kadiyirire t, liu l, guy a, et al. safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa. jci insight. 2019;4(11). 6. mansbridge jn, liu k, pinney re, patch r, ratcliffe a, naughton gk. growth factors secreted by fibroblasts: role in healing diabetic foot ulcers. diabetes, obesity and metabolism. 1999;1(5):265–79. 7. takehara k. growth regulation of skin fibroblasts. journal of dermatological science. 2000;24:s70–s7. 8. park h-h, park n-y, kim s-g, jeong k-t, lee e-j, lee e. potential wound healing activities of galla rhois in human fibroblasts and keratinocytes. the american journal of chinese medicine. 2015;43(08):1625–36. 9. oever mv, twaroski k, osborn mj, wagner je, tolar j. inside out: regenerative medicine for recessive dystrophic epidermolysis bullosa. pediatric research. 2018;83(1):318–24. 10. christofolini dm, ceroni jrm, soares gg, lamy gb, calvo acn, santos tad, et al. reproductive alternatives for patients with dystrophic epidermolysis bullosa. einstein (são paulo). 2019;17(3). https://www.sciencedirect.com/topics/medicine-and-dentistry/anchoring-fibrils https://www.sciencedirect.com/topics/medicine-and-dentistry/fibroblast 23j contemp med sci | vol. 9, no. 1, january-february 2023: 18–23 m. eslami et al. original comparing col7a1 gene expression in fibroblast cells 11. fine j-d, bruckner-tuderman l, eady ra, bauer ea, bauer jw, has c, et al. inherited epidermolysis bullosa: updated recommendations on diagnosis and classification. journal of the american academy of dermatology. 2014;70(6):1103–26. 12. kim m, li m, intong-wheeler lr, tran k, marucci d, murrell df. epidemiology and outcome of squamous cell carcinoma in epidermolysis bullosa in australia and new zealand. acta dermato-venereologica. 2018;98(1-2):70– 6. 13. siprashvili z, nguyen nt, gorell es, loutit k, khuu p, furukawa lk, et al. safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with recessive dystrophic epidermolysis bullosa. jama. 2016;316(17):1808–17. 14. zhou x, zhang y, zhao m, jian y, huang j, luo x, et al. surgical management of hand deformities in patients with recessive dystrophic epidermolysis bullosa. journal of plastic surgery and hand surgery. 2020;54(1):33–9. 15. latella mc, cocchiarella f, de rosa l, turchiano g, gonçalves ma, larcher f, et al. correction of recessive dystrophic epidermolysis bullosa by transposon-mediated integration of col7a1 in transplantable patientderived primary keratinocytes. journal of investigative dermatology. 2017;137(4):836–44. 16. rashidghamat e, mcgrath ja. novel and emerging therapies in the treatment of recessive dystrophic epidermolysis bullosa. intractable & rare diseases research. 2017;6(1):6–20. 17. twaroski k, eide c, riddle m, xia l, lees c, chen w, et al. revertant mosaic fibroblasts in recessive dystrophic epidermolysis bullosa. british journal of dermatology. 2019;181(6):1247–53. 18. tuncer s, sezgin b, kaya b, ayhan s, latifoglu o. an algorithmic approach for the management of hand deformities in dystrophic epidermolysis bullosa. journal of plastic surgery and hand surgery. 2018;52(2):80–6. 19. eisenberg m, llewelyn d. surgical management of hands in children with recessive dystrophic epidermolysis bullosa: use of allogeneic composite cultured skin grafts. british journal of plastic surgery. 1998;51(8):608–13. 20. pfaffl mw. a new mathematical model for relative quantification in realtime rt-pcr. nucleic acids research. 2001;29(9):e45. 21. cianfarani f, zambruno g, castiglia d, odorisio t. pathomechanisms of altered wound healing in recessive dystrophic epidermolysis bullosa. the american journal of pathology. 2017;187(7):1445–53. 22. yan y, meng z, hao s, wang f, jin x, sun d, et al. five novel col7a1 gene mutations in three chinese patients with recessive dystrophic epidermolysis bullosa. annals of clinical & laboratory science. 2018;48(1):100–5. 23. saeidian a, youssefian l, moreno trevino m, fortuna g, vahidnezhad h, atanasova v, et al. seven novel col 7a1 mutations identified in patients with recessive dystrophic epidermolysis bullosa from mexico. clinical and experimental dermatology. 2018;43(5):579–84. 24. bornert o, kühl t, bremer j, van den akker pc, pasmooij am, nyström a. analysis of the functional consequences of targeted exon deletion in col7a1 reveals prospects for dystrophic epidermolysis bullosa therapy. molecular therapy. 2016;24(7):1302–11. 25. jacków j, titeux m, portier s, charbonnier s, ganier c, gaucher s, hovnanian a. gene-corrected fibroblast therapy for recessive dystrophic epidermolysis bullosa using a self-inactivating col7a1 retroviral vector. journal of investigative dermatology. 2016 jul 1;136(7):1346–54. 26. takashima s, shinkuma s, fujita y, nomura t, ujiie h, natsuga k, iwata h, nakamura h, vorobyev a, abe r, shimizu h. efficient gene reframing therapy for recessive dystrophic epidermolysis bullosa with crispr/cas9. journal of investigative dermatology. 2019 aug 1;139(8):1711–21. 27. matsumura w, fujita y, shinkuma s, suzuki s, yokoshiki s, goto h, hayashi h, ono k, inoie m, takashima s, nakayama c. cultured epidermal autografts from clinically revertant skin as a potential wound treatment for recessive dystrophic epidermolysis bullosa. journal of investigative dermatology. 2019 oct 1;139(10):2115–24. 28. vahidnezhad h, youssefian l, zeinali s, saeidian ah, sotoudeh s, mozafari n, et al. dystrophic epidermolysis bullosa: col7a1 mutation landscape in a multi-ethnic cohort of 152 extended families with high degree of customary consanguineous marriages. the journal of investigative dermatology. 2017;137(3):660–9. 29. wong t, gammon l, liu l, mellerio je, dopping-hepenstal pj, pacy j, et al. potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa. journal of investigative dermatology. 2008;128(9):2179–89. 30. jacków j, titeux m, portier s, charbonnier s, ganier c, gaucher s, et al. gene-corrected fibroblast therapy for recessive dystrophic epidermolysis bullosa using a self-inactivating col7a1 retroviral vector. journal of investigative dermatology. 2016;136(7):1346–54. 31. takashima s, shinkuma s, fujita y, nomura t, ujiie h, natsuga k, et al. efficient gene reframing therapy for recessive dystrophic epidermolysis bullosa with crispr/cas9. journal of investigative dermatology. 2019;139(8):1711–21. e4. 32. marinkovich mp, tang jy. gene therapy for epidermolysis bullosa. journal of investigative dermatology. 2019;139(6):1221–6. 33. woodley dt, chen m. recessive dystrophic epidermolysis bullosa: advances in the laboratory leading to new therapies. the journal of investigative dermatology. 2015;135(7):1705–7. 34. murauer em, gache y, gratz ik, klausegger a, muss w, gruber c, et al. functional correction of type vii collagen expression in dystrophic epidermolysis bullosa. journal of investigative dermatology. 2011;131(1):74–83. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1314 202 j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 original the development of supervisory functions as experienced by attending physicians and medical trainees: a qualitative directed content analysis soleiman ahmady1,2, , masoumeh seidi3,*, 1head of department of medical education, virtual school of medical education and management, shahid beheshti university of medical sciences, tehran, iran. 2research affiliated faculty at department of lime, karolinska institutet, sweden. 3ph.d. candidate of medical education, virtual school of medical education and management, shahid beheshti university of medical sciences, tehran, iran. *correspondence to: masoumeh seidi (e-mail: seidimasoomeh@gmail.com) (submitted: 15 april 2021 – revised version received: 03 may 2021 – accepted: 19 june 2021 – published online: 26 august 2021) introduction clinical supervision is the provision of both advice and feedback on subjects correlated to personal, professional, and educational developments in the field of trainee experience.1 some previous studies in this regard have shown that trainees are concerned about their uncertain future careers, unrealistic increases in a medical capacity,2,3 lack of knowledge and skills, and attending physician’s (ap) criticism of trainee’s activities.4 based on the available pieces of evidence, it has been demonstrated that high levels of stress and anxiety during medical education may reduce trainees’ focus, impair trainees’ decision-making skills, and student’s ability to communicate with patients effectively.5 the trainees can use many mechanisms to deal with his/ her stress, but many negative coping strategies also exist. the concept of coping is a stable behavioral and cognitive factor that helps in adjusting the body to stressful conditions. by considering this definition, two main types of coping can be known as follows: problem-focused coping, which is directed towards converting the problem or stressor; and emotion focused coping, which is proposed to reduce individual emotional distress.6 the clinical learning environment was found to have complex and multidimensional characteristics.7 clinical supervision in teaching hospitals requires further appraisal and improvement.8 in this literature, there was an agreement on clinical supervision with three functions, including management or administration, education, and support. accordingly, all these functions are reflected in professions such as nursing9 and social work,10 which are recommended to be used in medical professions.1 the models dividing the functions of clinical supervision into three functions11 can be named as follows: educational/formative/developmental function with emphasizing on learning and teaching. additionally, the supportive, restorative, and resource function is the emotional response of supervisees. moreover, the management/normative/qualitative function refers to the responsibility of the clinical function of supervisees and clinical outcomes for confirming the ethical quality of services.11,12 correspondingly, each one of these functions may be highlighted or remain in the background of clinical supervision and circumstances. so, in this regard, it is essential to achieve some goals such as ensuring patient safety/care, teaching trainees, upgrading the standards, identifying trainees’ problems, supporting trainees, and supervising trainees’ progression.1 it was indicated that the current clinical supervision does not meet the needs of the medical trainees and these functions must be simultaneously applied to the supervision of the trainees. in this context, this study aimed to identify the core components of clinical supervision functions experienced by attending physicians (pas) and medical trainees. methods this study is a part of a doctoral dissertation in medical education approved by the shahid beheshti university of medical sciences, teheran, iran. the dissertation was designed at two stages. accordingly, the first stage involved critically reviewing abstract objective this study aimed to identify the core components of supervisory functions experienced by attending physicians and medical trainees. methods this qualitative study was conducted based on the directed content analysis. intentional sampling with maximum variation was used to select the required participants among medical trainees and attending physicians in teaching hospitals at shahid beheshti and hamadan university of medical sciences, iran. a semi-structured interview was used as the most important method of data collection in this study. data saturation was reached after interviewing 20 participants. results in this study, 11 categories were identified through performing the interviews. management supervision includes academic discipline and monitor and follows the implementation of the curriculum. the themes of educational supervision were as follows: empowering of non-faculty educators, control over trainees’ academic achievement, supervision of trainees’ performance, and activities’ educators of evaluation supportive supervision included improving trainees’ resilience, provide functional support and diversity and condition of providing support services. as well, the fourth theme from the participants’ initial codes was observed to be professionalism, including professional accountability and respect for patient rights. conclusion this study highlighted the important aspects for which clinical supervision functions need to be improved, to maximize their benefit to medical students. supervisors are responsible to ensure these three functions occur across the course. care must be taken to ensure that one function does not become the focus. keywords supervision, medical trainees, attending physicians, management, support, education issn 2413-0516 https://orcid.org/0000-0003-0551-6068 https://orcid.org/ 0000-0001-8473-9571 mailto:seidimasoomeh@gmail.com 203j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 s. ahmady and m. seidi original development of supervisory functions as experienced by attending physicians and medical trainees the support and supervision of medical students. what was shown by this article is included as a part of the 2nd stage of the study (interviews with expert authorities). moreover, we used a qualitative method with a directed content analysis method. the directed content analysis method aims to validate or expand a theoretical framework or theory conceptually. the existing theory or research can help in emphasizing the research question and also help researchers in beginning by identifying key concepts or variables as the initial coding categories.13 participants and study setting this study was performed at the shahid beheshti and hamadan university of medical sciences in iran. medical trainees and aps were selected using purposive sampling, which continued until reaching data saturation. the participants were 11 medical trainees and nine attending physicians. data collection the needed data were collected through performing individual semi-structured in-depth interviews (in a quiet and comfortable atmosphere) from december 2019 to august 2020. a guide to open-ended interview questions was used following the predetermination of the themes. in the first step, the interviews were started with general open-ended questions (table 1). according to the willingness and preparedness of the participants, the interviews were recorded using a digital recording device. each interview lasted for approximately 40 minutes (between 30 and 80 minutes). after transcribing and analyzing each interview, in case of any ambiguity and for probing into the participants’ experiences, the interview was repeated to clarify different aspects of the subject, if needed. data analysis according to the directed content analysis process, at the commencement of each interview, the audio file was carefully transcribed and this text was then read several times to analyze the data. thereafter, the texts were returned to the participants for their comments and correction. subsequently, some important statements were made to identify the initial codes or semantic units in the text of the interview. in the next step, similar units were placed either in one of the following themes: managerial, educational, supportive supervision, and professionalism. trustworthiness the prolonged engagement methods were employed to ensure the collection and analysis processes of actual trainee and ap experiences. the extracted text of some of the interviews, codes, subcategories, and categories was given to three faculty members (qualitative researchers) to evaluate the agreement reached on the meanings among several researchers. maximum variation sampling (including age, gender, clinical phase, and specialty type) was used to adjust or transfer the results into other domains. table 1. interview questions based on the categories extracted from the critical review managerial supervision could you explain your experience of holding a training class session at the bedside? what is your experience of compliance with administrative, educational, and ethical rules and regulations in clinical education? and … educational supervision what is your experience of a good teaching method in a clinical training course? what are your experiences of teachers monitoring students’ activities in the classroom and clinical environment? in your experience, how do you monitor classroom and clinical activities? go back to the past and describe one of the teacher supervision sessions in clinical education that has been beneficial to you. could you talk about the experience of using an advisor or counselor during education and how it affected your academic achievement? supportive monitoring what concerns and stresses did you experience during clinical education? could you please share your experience of being supported by a teacher? could you please share other students’ experiences of being supported by a teacher during clinical education? could you please share your experience of communicating with aps in clinical courses? in your experience, which roles of aps helped you reduce your stress or anxiety in your clinical course? what training and supportive advice did you need during your clinical training course? during your previous internships, did you have any experience of not being supported by the teacher? please provide your expected support in that situation. what experience and support did you receive during your previous internships? and… are there any additional experiences you would like to share? 204 j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 development of supervisory functions as experienced by attending physicians and medical trainees original s. ahmady and m. seidi ethical considerations the data were collected after obtaining the approval of the research proposal from the ethics committee of the shahid beheshti university of medical sciences (ir.sbmu.sme. rec.1398.061 date: 2019-07-06). at the beginning of the interviews, the interviewer introduced himself to the interviewees, explained the goals of the research, and finally obtained informed consent. results this qualitative study was conducted on 11 medical trainees; as 7 medical interns and 4 medical externs (the educational phases in the iran, basic science courses, pre-clinical, clerkship, and internship periods), including 4 men and 7 women; on nine iranian attending physicians (3 men and 6 women), and on a different specialist (1 internist, 1 neurologist, 2 obstetricians, 2 neonatologist, 2 pediatrician, and 1 anesthesiologist) with the mean age of 41.12 ± 6.11 years old. the components were broken down into the following three categories: managerial, educational, and supportive supervision (table 2). the following section is a summary describing the categories found in the interviews: management supervision academic discipline some aps do not believe in being strict in the classroom. the ap (neurologist, male) marked, “…absence and presence of trainees is an important matter to aps. i do not care about it though, because i believe that the class should be scientifically beneficial so that the trainee feels the need for attending the classroom in order to learn and feel that they would lose something if they do not attend the classes...” an experienced medical trainee used the term messy when he was explaining the irregularity in the clinical setting. table 2. an overview of the related categories and sub-categories subcategoriescategoriesthemes •  encouragement and punishment’s students  •  the observance of educational rules and regulations •  expectations of the student culture  •  organizing overcrowded rounds •  reduction in the clinical education hours •  multiple visits from patients by trainees •  familiarize with the clinical field  •  presence of trainees academic discipline management supervision •  the appropriate implementation of educational programs •  the teaching performance of ap  •  security monitoring for students •  patients’ safety monitor and follows the implementation of the curriculum •  select the appropriate teaching method •  methods for improvement of learning •  accepting feedback empowering non-faculty educatorseducational supervision •  performance-oriented •  inadequate content coverage of tests •  design of knowledge-based questions •  holistic of student evaluation competency-based assessment •  identify strong and weak students in education  •  classify students suffering from mental illness •  to strengthen weak students •  offer opportunities to outstanding students •  research opportunity •  offering participation in scientific conferences  •  mentoring weak students control over students’ academic achievement •  factors influencing the amount of supervision  •  the educational phases (internship or externship or clerkship) •  type of supervisory •  directly observing performance •  review of patient records and documentation supervision of students’ performance •  teaching quality •  reduce teacher education role relative to other roles  •  system expectations of faculty members •  class management •  make students aware of topics (daily lesson plan)  •  attraction to clinical education activities’ educators of evaluation (continued) 205j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 s. ahmady and m. seidi original development of supervisory functions as experienced by attending physicians and medical trainees table 2. an overview of the related categories and sub-categories—continued subcategoriescategoriesthemes •  to consider students’ concerns •  challenge clinical environment •  imbalance between personal life and education •  unpleasant feelings (seeing patients in pain, dying, etc.) •  the provision of advice and guidance  •  improved interaction between pas and trainees •  respect for trainees rights •  assignment of a clinical advisor •  wellness and entertainment programs exist •  student adaptability •  student enthusiasm •  applying coping mechanisms improving students’ resiliencesupportive supervision •  students interact  •  proper teacher-student relationship •  set up a peer group •  formation of social networks of consultants and aps •  student-patient communication •  student-employee communication •  availability of aps provide functional support •  type of support facilities •  avoid academic burnout •  diversity in service delivery •  well-being service  •  ensure confidentiality in the provision of services •  having consultants out of the organization diversity and condition of providing support services •  responsibilities to trainees •  ap’s responsibility •  observance of patients’ rights  •  professional ethics in clinical educators professional accountabilityprofessionalism the trainee (2) said, “…theoretical classes are held during the externship course at the hospital, which diminishes the number of hours spent at the patient’s bedside and irregularity in multiple visits from patients by aps, residents, and trainees make clinical education messy and less important.” rewards must be considered for the conscientiousness of trainees. in this regard, one of the trainees (1) stated, “… activities beyond our job description must be taken into account. while most of those who make a mistake is punished, a small number of trainees are rewarded for their proper performance.” monitor and follows the implementation of the curriculum there are a variety of educational programs for medical trainees in clinical departments that should be followed by the head of the department. one of the trainees (7) stated, “…the experts of education visit the clinical settings each semester to monitor the performance of the departments and ask if clinical educators teach these matters to trainees. i did not want to answer because i know from experience that nothing happens after monitoring it, and the same situation continues.” one of the trainees’ about patient safety (3) explained, “when i was given patient responsibility. in the wards, i was worried about whether the test was correct or not. are the patient’s visit and diagnosis sufficient to treat the patient or not?” trainees are upset by some of the residents’ behaviors. a trainee (8) stated, “…it appears that we have been imposed on some residents. some of our questions are not replied to correctly. some residents are bad-tempered.” educational supervision empowering of non-faculty educators regarding how to handle a class in clinical education, one of the aps (surgeon, male) mentioned: “…a good teaching method in clinical education boosts learners’ motivation for providing healthcare services and encourages them to continue their activities. trainees are completely engaged in the education process. repeating theoretical issues at the bedside is not enough and all of them have been presented in books…” the trainees mentioned that training courses are required for non-faculty physicians, residents, and a few faculty members. the trainee (5) said, “…many residents do not have the ability to provide information in an arranged and organized way and do not assess the trainee based on their real performances.” another trainee (4) said, “sometimes non-faculty members have taught us, and these physicians are experts in their profession but don’t have the teaching skills….” the reason for some trainees’ lack of motivation to learn clinical skills was knowledge-based tests. the attending physician (pediatrician, female) stated, “…unfortunately, several end-ofcourse tests and even residency tests measure most of the trainees’ knowledge and memorization rather than clinical skills…” control over trainees’ academic achievement regarding trainee progress characteristics, one of the aps (obstetricians, female) stated: 206 j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 development of supervisory functions as experienced by attending physicians and medical trainees original s. ahmady and m. seidi “… regular attendance in the class, following and answering the questions raised in the sessions, pre-study before the session, expressing interest in the lesson presented, asking multiple questions from the others, and practically applying theoretical lessons…” another ap (neonatologist, female) expressed: “… in the faculties, we often focus on trainees who have an academic failure, and those who are active and have the ability to grow and flourish are rarely identified to be rewarded. meanwhile, these trainees will have good achievements for the university if they are counseled and usually work on good research topics…” supervision of trainees’ performance according to the aps’ experiences, each trainee needs special supervision in different situations. the attending physician (obstetricians, female) explained, “…the amount of supervision for the trainee depends on whether the trainee is in an externship or internship course? in the externship course, everything must be conducted under the supervision of one person. however, the internship course is based on the trainee’s experience, clinical skills, and motivation.” one of the aps (pediatrician, female) shared experiences about stepwise teaching of medical interns along with supervision: “…when i ask one of the trainees to visit a patient, i monitor them both from far and near and evaluate what they write in the patient’s medical files. also, i assess how they face patients and inconspicuously control their activities by talking to the patient and their companions…” activities’ educators of evaluation the attending physician had a positive attitude toward receiving feedback from trainees about their teaching method. ap (anesthesiologist, male) claimed: “…we can receive feedback from trainees verbally or in written form (anonymously) to improve our teaching. while trainees formally complete the ap’s evaluation form at the university, i think that the conditions for answering, for example, the requirement to complete the form before the exams or before seeing the scores, make it difficult to be objective...” the appropriate behavior of aps is effective in trainees’ interest in lessons, classes, and disciplines. trainees (6) asserted: “…i think the most important feature of an ap is their interest in the field of medicine. in addition, they should be well-tempered and have friendly behavior toward students, patients, and personnel.” supportive supervision improving trainees’ resilience one of the aps (anesthesiologist, male) commented, “…many trainees may become depressed due to the lack of specific regular environment in the hospital, course type change from theory to practice, long hours of clinical education, and trainees’ challenges with medical and non-medical staff, multiple on-call shifts, and preparation for the residency exam. ” trainees expect aps to understand their stressful situations and anticipate their needs. in this regard, trainee (4) expressed: “…we expect aps to be compassionate towards their trainees and act like they are their own children. they are also demanded to have rational curiosity and learn about trainees’ needs and understand how they feel from their appearance.” another trainee (8) affirmed: “…sometimes long shifts along with long training hours make trainees exhausted. this is especially true in hospital wards with a high patient load and a low number of trainees. on the other hand, preparing to report the end of the shift to the next intern and preparing for a round with the ap in the morning shift is difficult and the ap should understand trainees in these situations.” one of the trainees (3) talked about aps’ disrespectful behavior: “…i had a night shift and did not sleep at all. the next day, we had a round with the ap, who scolded me in front of other trainees for not knowing the answer to his question. what would he feel if he had not slept for 32 hours? i wish that he would put himself in my shoes for a second.” provide functional support the trainees mentioned are being aps as the exhibitor of experiences. one trainee (5) said, “…many aps have few interactions with the trainees. i was interested in talking more with my ap as someone who has taken the course and using their experiences.” aps’ respect toward the trainees increases patients’ trust in trainees. in this regard, trainee (10) mentioned, “… in general, if the ap respects us in front of the patient, the patient will trust us, and on the other hand, the trainee will be more motivated to perform practical procedures in the hospital.” ap (internist, male) said, “…aps have shortcomings in their initial communication with trainees and cannot or do not want to interact with the trainee. they actually look down on trainees.” the ap should consider the differences in trainees’ educational learning. in this regard, one of the trainees stated (11): “…some of the trainees need more practice and being scolded by the ap does not solve anything. if the ap does not have the time to do so, they should ask their resident to spend more time with that trainee and work on their problem. trainees are different in terms of learning ability.” aps’ availability and accompanying trainees and spending time with them were indicative of their support. in this regard, ap (pediatrician, female) stated: “…the personal growth of the trainees occurred by spending time with the trainees, accompanying them and being available to answer their questions. i tried to be patient enough and i did not get angry soon…” trainees also talked about their experiences using a group other than aps (e.g., residents and classmates). in this respect, trainee (9) expressed: “…residents teach us instead of aps because we are the same age and we can communicate well and trainees can talk about their educational problems more easily.” another of the trainee (2) marked: “…we can use the experiences of higher-semester trainees regarding how to pass the courses and evaluate and communicate with aps.” diversity and condition of providing support services one of the important issues for trainees is the continuation of education and choosing a profession, and providing 207j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 s. ahmady and m. seidi original development of supervisory functions as experienced by attending physicians and medical trainees appropriate job counseling creates motivation for clinical work. regarding choosing the future field of study, trainee (10) expressed, “…i would like to learn a specialty that is not in the clinical environment, such as radiology. this is because i do not like to see the death and pain of people.” one of the aps (internist, male) stated, “…i participated in the trainee tour several times as an accompanying ap. the main concern of trainees was their future job. they would ask whether they would be employed after receiving their degree or not. another problem of these individuals was lack of having an income during education…” in some cases, the ap refers to the support services. ap (pediatrician, female) said, “…if necessary, i will introduce related departments in the university, such as counseling centers, to the trainee to take care of their personal problems, their problems with other trainees and their family members, and issues that lead to academic failure…” another ap (neonatologist, female) expressed, “… female trainees with family problems and divorce were given more time to do their homework. in addition, trainees with financial problems are referred to charitable people or other colleagues are notified to be aware of trainees’ problems and do not remove their courses and give them more time if they are incapable of attending the classes due to working outside the faculty…” professionalism professional accountability a trainee (11) mentioned: “… if i am assigned an activity or procedure by the ap, another person should follow up my work to confirm it or make the necessary changes.” one of the aps (neonatologist, female) commented on professionalisms’ training as follows: “…professionalisms’ training should be provided for trainees during the medical course. for example, one of the educational goals of each course should be training professional and ethical responsibilities” attending physicians’ participation in the clinical activities of trainees is a source of strength in trainees. in this respect, trainees (5) mentioned, “…some of the aps and residents had really good information and cooperated with us. they also carried out clinical procedures appropriately and explained everything with patience. they sometimes allowed us to perform the procedure and we had a really good feeling to have them by our side.” respect for patient rights patients’ rights are among the core principles in defining the standards of clinical services. sometimes the patient’s room or the wardroom becomes very crowded and noisy. one trainee (7) explained, “…the room of patients in training hospitals lacks sufficient space in order to respect their privacy. it is not easy for some of the patients to talk easily about their problems, which prevents their communication with healthcare providers…” trainees have expressed the professionalism of aps. one of the trainees (1) explained, “…some of the aps devote great attention to the patients and patient satisfaction is highly important to them….” discussion based on the experience of attending physicians and medical trainees, the development of clinical supervision functions in undergraduate medical education mostly falls into the four themes. management supervision management function mainly focuses on standards, policies, and procedures that are implemented and adhered to performance assessment and management when there is a problem.14 the existence of the orientation program allows trainees to be better adapted to the environment in a clinical setting. therefore, designing a system of encouragement and punishment is essential to increase the quality of education. tarman (2016), in their study on american educational programs, stated two conflicting sets of attitudes toward discipline, one point of view maintained that the teacher’s primary disciplinary task was to keep order, silence, and decorum in the classroom. on the other hand, the opposing point of view stated that teachers must place primary importance on developing a sense of personal worth along with a sense of inner control for the children in the classroom.15 the turnover of trainees provides an unprecedented overview of health care systems. trainees should be able to recognize quality slots at first and then provide major suggestions. moreover, trainees should be encouraged to contact the department directors about their clinical, administrative, vocational, and educational affairs to share their concerns in this regard.16 notably, clinical educators should always observe the trainees’ professional behavior and ethical principles, in order to provide timely and constructive feedback to them. in addition, the evaluation of professional behavior should not be limited only to final tests.17 patient safety is considered the foundation of good patient care. supervisors must ensure that the trainee is properly trained to perform a specific procedure and to apply specific elements related to patient care. trainees whose behavior causes mental and physical harm to patients should be paid serious attention.18 trainees must respect and reserve patient’s information confidential. this includes limiting the discussion about patient health issues to the appropriate settings for clinical or educational purposes and only for those family member caregivers who are identified by patient consent.19 educational supervision educational practices can be defined as the learning and development of the trainee’s knowledge or skills, as well as the creation of opportunities for teaching and learning.12 medical trainees noted that clinical educators have good expertise; however, they are unable to transfer their knowledge and skills appropriately. consequently, it appears that the focus should be on the education of non-academic physicians and residents in the first place. the use of clinical rounds, which are considered an integral part of clinical teaching to help medical students acquire essential skills of practicing medicine, is critically important.20 beigzadeh’s (2019) study stated that, due to the importance of 208 j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 development of supervisory functions as experienced by attending physicians and medical trainees original s. ahmady and m. seidi clinical rounds in students’ learning, medical attending should break down their teaching sessions in activities pre-, during, and post-rounds. in another study by beigzadeh (2019), their participants stated suggestions to diminish some barriers such as having fewer students on the rounds, addressing time constraints through planning and flexibility, and the provision of medical education rewards.20 keshavarzi (2018) in a study mentioned that students’ academic achievement is one of the important indicators of assessing university education. teaching cognitive strategies in university education was found to be effective in fostering learners’ learning skills and consequently their academic performance.21 educational supervision is deemed to be effective when educators support the progression of their trainees’ learning by providing their access to appropriate training opportunities, to gain the required proficiencies.22 most of the participants pointed out that each trainee requires special supervision in different situations. aps must also define the phases of development of each trainee and modify the type of supervision by several factors, including trainee progression level; the complexity of care or procedure; possible side effects, competence, maturity, and responsibility of each trainee; and patient’s satisfaction.23 the faculty member is ultimately responsible for the evaluation, treatment, management, and documentation of each patient’s care.24,25 elson s. floyd college of medicine previously stated that to ensure that medical students are appropriately supervised using the curriculum, some standards have been established by the clinical supervision of medical student policy, which is as follows: direct supervision, indirect supervision, and with direct supervision available.26 a medical school faculty member regularly receives scheduled and timely feedbacks from departmental, other programmatic or institutional leaders on his or her academic performance and then progresses toward promotion, and when applicable, tenure.27 supportive supervision supportive supervision provides the psychological and interpersonal contexts that allow the student to mobilize the emotional energy necessary for having an effective performance and space for openness and aerial distress.9,28 stress has some negative effects on trainees’ learning and clinical success and also on the correct functioning of the shadow.4 gang (2019) in the research stated that the quality of life of medical students is greatly affected by the presence of both anxiety and depression. therefore, it is recommended that medical schools should implement some measures to identify vulnerable students and then provide comprehensive interventions and anticipatory programs for them, in order to improve their wellbeing.29 shahraki (2019) also reported that both depression and anxiety affect the quality of life and stated that health conditions, psychological well-being, physical and social functioning, and environmental and spiritual aspects should be included in the quality of life assessment.30 the participants also highlighted the importance of having educators available during times of need or crisis. in situations where clinical educators may be out of place, a good supervising physician (including a resident) should exist and should be aware of that expectation. the availability of aps was indicated to be directly correlated to learning progress and the increased trainees’ satisfaction.31 direct supervision means that the supervising physician is physically present with both the student and the patient. moreover, indirect supervision means that the supervising physician is not physically present with the student and the patient, but he/she will be immediately available to provide direct supervision upon any request, thus requiring that the supervising physician remain physically present within the hospital or other sites related to patient’s care.32 functional support is a qualitative aspect of social support communication, social support is provided by individuals or groups, which consists of words, actions, and/or tangible aid that are perceived by the recipient as being positive. the result will be the provision of guidance and/or that the recipient’s perception of him/herself, the problems, and the provider of support, his/ her sense of control or belonging, would positively be altered.33 peer (e.g., residents and classmates) interaction can help in improving the effectiveness of the trainee’s studies on test preparation and stress management during their undergraduate studies. crisp (2020) mentioned that peer support programs provide a promising approach to deal with the high levels of stress and psychological distress experienced by university students. however, few studies have considered the impacts of implementing programs on the well-being and skill development of student facilitators.34 kemp’s (2019) consensus statement provided some specific recommendations for medical schools, in order to design curricula that promote peer support and progressive levels of challenge to students, employ some strategies to promote positive outcomes from stress to help others in need, design assessment tasks to foster wellbeing and learning, provide mental health promotions and suicide prevention initiatives, provide physical health promotion initiatives, ensure about safe and health-promoting cultures for learning in on-campus and clinical settings, and train staff on student wellbeing and how to deal with wellbeing concerns.35 vogan (2014) supported activities that can be included in the curriculum such as separate personalized assessment feedbacks, using stress decreasing techniques, career guidance, and personal and professional improvement sessions.36 moir (2016) stated that peer-run plans have not been formally evaluated in the medical student population, where psychological issues exist and where it has been proven that students approach their peers for help in preference to staff members or professional services. it was demonstrated that one-fourth of intervention students use face-to-face peer support and more than 50% prefer a peer social event and/or participating in the mindfulness program. several improvements were found in the mental health status of the intervention group, so there is evidence that peer support and mindfulness can be effective in other contexts.37 professionalism the nursing and midwifery council in the uk (2009) stated that professional accountability is integral to professional 209j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 s. ahmady and m. seidi original development of supervisory functions as experienced by attending physicians and medical trainees practice, and is fundamentally concerned with weighing up the interests of patients and clients in often complex situations, whilst using professional knowledge, judgment and skills, based on the evidence, to make a decision. the trainees are very happy to see the active involvement of the pa in clinical education and to follow his/her role as a model. role modeling is at the core of building aps that show the knowledge, attitudes, and behaviors in identifying a good professional.38 a doctor should interact with his/her patient by inspection, palpation, percussion, auscultation, and olfaction, and above all, by paying much attention in the direct discussion with him or her. during all these moments, the patient feels that he/she can interact with his/her doctor and then trust him or her.39 fatemi (2016) in the study’s results reported that two principal themes of facilitator factors and deterrent factors were obtained that respectively contain classes of (fruition of equal facilities and types of equipment, professional teachers, targeted educational plan, coordination and relationship between theory and practice, and the relationship between education programs) and (undesirable clinical realities, lack of expert and specialist teachers in practice, interpersonal communication, and unsuitable evaluation process), each one of which was found to be as effective on developing clinical educational equity process.40 conclusions at the same time, the perception of clinical supervision is known as a monitoring tool. it is evident that the managerial/ administrative office is much broader, which mostly includes assuming responsibilities to trainees and ensuring that they have been adjusted to the conventions and norms of organizations. it seems that two supportive and educational roles have disappeared result of management roles. thus, it is necessary for educators to use the three clinical supervision functions simultaneously, in order to make sure all three functions occur during the course. care should also be taken to ensure that a function does not become the primary purpose. we sought to help a graduate with no stress and concerns, like an effective and competent physician, a respectable citizen, and a professional person. it is recommended to make some efforts to enable pas in the practice of clinical supervision functions as well as the development of tools for assessing students on the basis of the above-mentioned three functions. acknowledgments we wish to express our heartfelt thanks to all the participants of the shahid beheshti and hamadan university of medical sciences who contributed to this study. funding this article is part of a ph.d. dissertation in medical education. this project was funded by shahid beheshti university of medical sciences. disclosure statement no potential conflicts of interest were identified.  references 1. kilminster s, cottrell d, grant j, jolly b. amee guide no. 27: effective educational and clinical supervision. medical teacher. 2007;29(1):2-19. 2. amiresmaili m, nekoei moghadam m, moosazadeh m, pahlavan e. challenges of general practice education in iran: a qualitative study. strides in development of medical education. 2013;9(2):118-131. 3. nasri k, kahbazy m, noroozy a, nasri s. the medical education problems and possible solutions in stagers and intern’s view points of arak university of medical sciences, 2006-07. journal of arak university of medical sciences. 2010;12(4):111-121. 4. yazdankhafard m, poladi, sh. et al. the stressors of clinical education from students’ point of view in bushehr university of medical sciences. iranian journal of medical education. 2008;8:341-350. 5. shapiro sl, shapiro de, schwartz ge. stress management in medical education a review of the literature. academic medicine. 2000;75(7): 748-759. 6. kahrazee f, tamini bk, daryasari sk, mahdavi a, kooteh br, qadimi a. coping with stress, depression and fear of negative social evaluation through comparing wives of addicted and non-addicted men. maedica. 2018;13(4):305. 7. koohpayehzadeh j, ahmadi mh, dehnad a. validity and reliability of activities coaching context questionnaire. medical journal of the islamic republic of iran. 2014;28:41. 8. razmjou s, baradaran hr, kouhpayehzadeh j, soltani-arabshahi k. comparison of quality of clinical supervision as perceived by attending physicians and residents in university teaching hospitals in tehran. medical journal of the islamic republic of iran. 2015;29:248. 9. tuck ja. a new approach to team clinical supervision on an acute admissions unit. mental health practice. 2017;20(5). 10. kadushin a, harkness d. supervision in social work. columbia university press; 2014. 11. basa v. models of supervision in therapy, brief defining features. european journal of counselling theory,research and practice. 2017;1(4):1-5. 12. hawkins p, shohet r. supervision in the helping professions. mcgraw-hill education (uk); 2012. 13. hsieh hf, shannon se. three approaches to qualitative content analysis. qualitative health research. 2005;15(9):1277-1288. 14. gillieatt s, martin r, marchant t, fielding a, duncanson k. evaluation of an inter-professional training program for student clinical supervision in australia. hum resour health. 2014;12:60. 15. tarman b. discipline or classroom management. journal of learning and teaching in digital age. 2016;1(2):37-44. 16. minnesota. student supervision during clinical activities.the university of minnesota medical school (umms). https://med.umn.edu/sites/med.umn. edu/files/student_supervision_during_clinical_activities.pdf. published 2019. accessed. 17. tabei s, afshar l. ethical considerations in medical education and patient rights in educational hospital. med ethics q. 2010;4(13):89-105. 18. farajpour a, afshar. review of ethical consideration in clinical training; propose behavioral codes in iranian educational system. journal of ahwaz jundishapur university of medical sciences. 2016;1. 19. columbia. supervision of students in required clinical learning experiences. policy advisory subcommittee (pas) md undergraduate education committee (mduec) the university of british columbia. 2017;next review 2022. 20. beigzadeh a, bahaadinbeigy k, adibi p, yamani n. identifying the challenges to good clinical rounds: a focus-group study of medical teachers. journal of advances in medical education & professionalism. 2019;7(2):62. 21. keshavarzi mh, safari e, shakarabi m, kangrani farahani ar, taghavinia m, zabihi zazoly a. the effect of teaching cognitive strategies on the academic achievement of medical students. development strategies in medical education. 2019;6(2):1-9. 22. patel p. an evaluation of the current patterns and practices of educational supervision in postgraduate medical education in the uk. perspectives on medical education. 2016;5(4):205-214. 23. florida. medical student roles and supervision. florida atlantic university charles e. schmidt college of medicine (fau com). http://med.fau.edu/ faculty/documents/adminpolicies/medical%20student%20roles%20 and%20supervision%20policy.pdf. published 2018. accessed. https://med.umn.edu/sites/med.umn.edu/files/student_supervision_during_clinical_activities.pdf https://med.umn.edu/sites/med.umn.edu/files/student_supervision_during_clinical_activities.pdf http://med.fau.edu/faculty/documents/adminpolicies/medical%20student%20roles%20and%20supervision%20policy.pdf http://med.fau.edu/faculty/documents/adminpolicies/medical%20student%20roles%20and%20supervision%20policy.pdf http://med.fau.edu/faculty/documents/adminpolicies/medical%20student%20roles%20and%20supervision%20policy.pdf 210 j contemp med sci | vol. 7, no. 4, july-august 2021: 202–210 development of supervisory functions as experienced by attending physicians and medical trainees original s. ahmady and m. seidi 24. boston. clinical supervision policy. boston university school of medicine. https://www.bumc.bu.edu/busm/files/2018/11/clinical-supervision-policy. pdf. published 2018. accessed. 25. mcgill. supervision policy for trainees in the clinical team. the university of mc gill faculty of medicine https://mcgill.ca/ugme/files/ugme/ supervision_policy_for_trainees_in_clinical_team_v1.1.pdf. published 2016. accessed. 26.   esfcom. clinical supervision of medical students. vice dean for  academic and community partnerships.washington state university. elson s. floyd college of medicine. https://medicine.wsu.edu/ documents/2017/08/clinical-supervision-of-medical-students.pdf/. published 2017. accessed. 27.   columbia. expectations of clinical supervisors and preceptors of  students in clinical settings. the university of british columbia. date of next review:april 2021. https://mednet.med.ubc.ca/aboutus/ policiesandguidelines/policies%20guidelines/supervision%20 of%20students%20in%20%20required%20clinical%20learning%20 experiences%20(031).pdf. published 2018. accessed. 28. kadushin a, harkness d. supervision in social work. columbia university press; 2002. 29. gan gg, yuen ling h. anxiety, depression and quality of life of medical students in malaysia. med j malaysia. 2019;74(1):57-61. 30. shahraki z, afshari m, ghajarzadeh m, tanha fd. how different are men with infertility-related problems from fertile men in prevalence of depression, anxiety and quality of life? maedica. 2019;14(1):26. 31. caspi j, reid wj. educational supervision in social work: a task-centered model for field instruction and staff development. columbia university press; 2002. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. doi: https://doi.org/10.22317/jcms.v7i4.1012 32. wusm. md: clinical supervision policy for medical students on clinical rotations. approved by the academic affairs committee. washington university school of medicine(wusm). https://bulletin.wustl.edu/medicine/policies/mdclinical-supervision/md-clinical-supervision.pdf. published 2020. accessed. 33. laack k. the role of social support in the success and retention of undergraduate nursing students. 2013. 34. crisp da, rickwood d, martin b, byrom n. implementing a peer support program for improving university student wellbeing: the experience of program facilitators. australian journal of education. 2020;64(2):113-126. 35. kemp s, hu w, bishop j, et al. medical student wellbeing–a consensus statement from australia and new zealand. bmc medical education. 2019;19(1):1-8. 36. vogan cl, mckimm j, da silva al, grant a. twelve tips for providing effective student support in undergraduate medical education. medical teacher. 2014;36(6):480-485. 37. moir f, henning m, hassed c, moyes sa, elley cr. a peer-support and mindfulness program to improve the mental health of medical students. teaching and learning in medicine. 2016;28(3):293-302. 38.   park j, woodrow si, reznick rk, beales j, macrae hm. observation,  reflection, and reinforcement: surgery faculty members’ and residents’  perceptions of how they learned professionalism. academic medicine. 2010;85(1):134-139. 39. cinteza m. the stethoscope at the age of 200: will “he” survive? maedica. 2016;11(1):3. 40. fatemi s, moosavi s, nikro r, mohemkar-kherandish s. exploration of medical sciences students and educational custodians view about educational equity in clinical environment. research in medical education. 2016;8(4):1-10. https://www.bumc.bu.edu/busm/files/2018/11/clinical-supervision-policy.pdf https://www.bumc.bu.edu/busm/files/2018/11/clinical-supervision-policy.pdf https://mcgill.ca/ugme/files/ugme/supervision_policy_for_trainees_in_clinical_team_v1.1.pdf https://mcgill.ca/ugme/files/ugme/supervision_policy_for_trainees_in_clinical_team_v1.1.pdf https://medicine.wsu.edu/documents/2017/08/clinical-supervision-of-medical-students.pdf/ https://medicine.wsu.edu/documents/2017/08/clinical-supervision-of-medical-students.pdf/ https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://bulletin.wustl.edu/medicine/policies/md-clinical-supervision/md-clinical-supervision.pdf https://bulletin.wustl.edu/medicine/policies/md-clinical-supervision/md-clinical-supervision.pdf 184 j contemp med sci | vol. 8, no. 3, may-june 2022: 184–188 original evaluation of dna damage in traffic cops exposed to polycyclic aromatic hydrocarbon pollution in baghdad city streets and its association to certain biomarkers ashraf r. salem1,*, estabraq a.r. al-wasiti2, fahem a. hasan3 1college of nursing, university of telafer, telafer, nineveh, iraq. 2college of medicine, department of chemistry and clinical biochemistry, al-nahrain university, baghdad, iraq. 3al-hussaini medical city, kerbala health directorate, ministry of health, kerbala, iraq. *correspondence to: ashraf r. salem (e-mail: ashraf.r.salem@uotelafer.edu.iq) (submitted: 03 april 2022 – revised version received: 14 april 2022 – accepted: 09 may 2022 – published online: 26 june 2022) abstract objectives: to find out the extent to which the genetic material of baghdad traffic policemen is affected by the oxidative damage caused by pollutants by monitoring the levels of 8-ohdg compared to the levels of antioxidant enzymes and malondialdehyde. methods: this study includes 140 participants; they have been divided into two groups (traffic police and office police). polycyclic aromatic hydrocarbons were analyzed for each participant by gc/fid while 8-ohdg and antioxidant enzymes were measured by the elisa technique. results: the levels of polycyclic aromatic hydrocarbons, 8-oxo-dg, and malondialdehyde were elevated in the blood of the traffic police compared to the office police, while higher levels of antioxidant enzymes (catalase and glutathione peroxidase) were observed in the blood of the office police. conclusion: exposure to polycyclic aromatic hydrocarbons can cause oxidative stress through their metabolic derivatives and the resulting active molecules, which lead to the formation of 8-oxo-dg and the reduction of enzymatic antioxidants, which may lead to the emergence of cancers. keywords: dna damage, traffic cops, polycyclic aromatic hydrocarbons, pollution, catalase issn 2413-0516 introduction the impact of air pollution on human health and the wideranging effects it has on biodiversity have propelled this problem to the top of the political agenda globally.1 in 2005, the world health organization released air quality standards for outdoor air pollution. these guidelines include allowed values for fine particulate matter (particulate matter with an aerodynamic diameter of less than 2.5 m) on an annual and daily basis, sulfur dioxide, ozone, nitrogen dioxide, and coarse particulate matter (pm2.5) (particulate matter 10 m in aerodynamic diameter).2 at the inaugural summit on air pollution and health in 2018, who established a target of preventing 7 million deaths worldwide from air pollution by 2030.3 air pollution is related to cardiovascular diseases,4 chronic obstructive pulmonary,5 and several types of tumors, such as lung, breast, oral, liver, kidney, prostate, bladder and ovarian,6,7 it is thought to be responsible for 4.2 million premature deaths worldwide.8 during the activation of polycyclic aromatic hydrocarbons (pah) by cytochrome p450 (cyp450), a large quantity of reactive oxygen species and numerous electrophiles are produced, which relate covalently to dna and disrupt cell homeostasis.9 polycyclic aromatic hydrocarbons appear to be key risk factors found in automotive exhausts that cause oxidative stress, since exposure to pahs is linked to an increase in the formation of free radicals.10 on the other hand, oxidative stress might be one of the reasons driving many of the negative health impacts associated with air pollution, figure 1.11 8-oxo-7,8-dihydro-2ʹ-deoxyguanosine (8-oxodg) is the most common oxidative stress-induced pest that may induce mutations in dna replication. also, they are thus important biomarkers of oxidative dna damage, although they are consistently repaired by base excision repair (ber).13 it is regarded as a active premutagenic lesion due to its capacity to pair with both cytosine and adenine residues and result in g:c to t:a transversions during dna replication.14 the c8 of the imidazole ring in deoxyguanine (dg) can be attacked by the hydroxyl radical (•oh), hydroperoxide radical (•ooh), singlet oxygen (1o2), superoxide (o2•-), reactive nitrogen species (no2), and peroxynitrite anion (onoo.¯) this results in the formation of 8-oxo-dg.15 but in fact, the interaction of dna with •oh in figure 2 is the main source of 8-oxo-dg.16 the 8-oxodg has been utilized as a risk factor for several illnesses in addition to being used as a biomarker to assess endogenous oxidative dna damage.17 it can be utilized as an index in various cancer,18 neurodegenerative diseases,19 diabetes,20 cardiomyopathy21 and cardiovascular or infectious diseases.22 as a result, 8-ohdg is helpful for high-risk people’ early identification and assessment.23 materials and methods one hundred and forty 140 iraqi policemen affiliated with the traffic police in the city of baghdad within the iraqi ministry of interior. their ages range between (25-65 years), during the period from july 15, 2019 to march 25, 2020. these subjects were divided into two groups as the following: group 1: 70 policemen of those who were performing their duty in the crowded squares and intersections in baghdad (traffic police). group 2: 70 policemen of those who were serving inside the buildings of the various traffic directorates (office police). each participant had about 6 ml of blood pulled from a vein, which was then left at room temperature for 15 to 20 mailto:ashraf.r.salem@uotelafer.edu.iq 185j contemp med sci | vol. 8, no. 3, may-june 2022: 184–188 a.r. salem et al. original evaluation of dna damage in traffic cops exposed to pah pollution fig. 1 the bap metabolites are powerful reactive compounds that can combine with dna to generate adducts.12 fig. 2 the process by which 8-oxodg and 8-oxog are formed.24 minutes to allow for coagulation, centrifuged for 10 minutes at 3000 rpm to detach the serum, and then the serum was split into small parts and stored at 20°c till it was consumed for pah determination. 1. gas chromatography with a flame ionization detector (gc/fid) was used to measure the levels of pah in serum. 2. 8-ohdg was determined by enzyme-linked immunosorbent assay (elisa) kit catalog number. ab201734. 3. determination of malondialdehyde by buege and aust method. the mda was estimated using the thiobarbituric acid, which reacts with malondialdehyde to produce a pink color that can be read at a wavelength of up to 535 nm. 4. determination of the activity levels of serum glutathione peroxidase, by elisa kit catalog number. e-el-h5410. 5. determination of the serum concentration of catalase, by elisa kit catalog number. mbs703074. results table 1 displays the average age for the two groups. mean concentration of pahs, 8-oxo-dg, mda, and two antioxidant enzymes (catalase and g-px) for the two groups are shown in table 2, the effect of the exposure period to pollutants from car exhaust and diesel engines on the traffic police who are in the offices as well as the traffic police deployed in the crowded intersections of the baghdad city are summarized in the (table 3) and (table 4) respectively. discussion polycyclic aromatic hydrocarbons are hazardous pollutants, as exposure to these harmful substances increases the chance of table 1. the groups’ average ages group n. age (mean ± sd) office police 70 38.54 ± 5.49 traffic police 70 40.15 ± 5.7 table 2. the average levels of pahs, 8-oxo-dg, mda, catalase, and gpx were within the two groups parameter group mean ± s.e. p-value pahs (ppm) office police 6.91 ± 0.12 0.0001 traffic police 8.94 ± 0.09 8-oxo-dg conc. (ng/ml) office police 96.88 ± 9.35 0.0001 traffic police 148.17 ± 8.47 mda conc. (μmol/l) office police 2.1 ± 0.22 0.025 traffic police 2.84 ± 0.26 catalase activity (pg/ml) office police 648.15 ± 15.3 0.0001 traffic police 398.52 ± 16.58 g-px activity (pg/ml) office police 55.74 ± 2.99 0.0001 traffic police 34.34 ± 2.2 p-value <0.05 is significant. table 3. comparison of the analyzed parameters for the office police group according to the length of exposure parameter duration of exposure mean ± s.e. p-value pahs (ppm) less than 10 years 6.7 ± 0.31 0.599 more than 10 years 6.97 ± 0.12 8-oxo-dg (ng/ml) less than 10 years 101.55 ± 21.14 0.780 more than 10 years 95.39 ± 10.44 mda (μmol/l) less than 10 years 1.97 ± 0.54 0.880 more than 10 years 2.14 ± 0.23 catalase (pg/ml) less than 10 years 652.12 ± 43.34 0.995 more than 10 years 646.88 ± 15 g-px (pg/ml) less than 10 years 59.89 ± 6.18 0.703 more than 10 years 54.41 ± 3.44 p-value >0.05 is non-significant. 186 j contemp med sci | vol. 8, no. 3, may-june 2022: 184–188 evaluation of dna damage in traffic cops exposed to pah pollution original a.r. salem et al. table 4. comparison of the analyzed parameters for the traffic police group according to the length of exposure parameter duration of exposure mean ± s.e. p-value pahs (ppm) less than 10 years 8.80 ± 0.21 0.487 more than 10 years 9.01 ± 0.09 8-oxo-dg (ng/ml) less than 10 years 145.05 ± 12.62 0.782 more than 10 years 149.60 ± 10.99 mda (μmol/l) less than 10 years 2.63 ± 0.38 0.974 more than 10 years 2.93 ± 0.34 catalase (pg/ml) less than 10 years 394.49 ± 33.74 0.947 more than 10 years 400.36 ± 18.85 g-px (pg/ml) less than 10 years 35.27 ± 5.00 0.781 more than 10 years 33.91 ± 2.28 p-value >0.05 is non-significant. developing cancer,25 due to the harmful effects that these toxins and their reactive metabolites, such as dihydrodiols and epoxides, can have when they join to dna and cellular proteins26 like mutations, problems in development, and cancers that occur from the cell damage.27 the results of this study showed the presence of high concentrations of polycyclic aromatic hydrocarbons in the blood serum of the traffic police who are at the busy street intersections of the city of baghdad, relative to the levels in the blood serum of the office police group. since 2003, the number of passenger automobiles, buses, trucks, and household generators has skyrocketed in iraq, posing serious environmental concerns. near the technology university in baghdad, on and surrounding the muhammad al-qasim highway, a study was conducted by28 to assess the connection between the level of activity, the movement of cars with various engines, and the pollution that comes from exhaust pipes, and it was discovered that pollutants such as sulfur and polycyclic aromatic hydrocarbons, increase during the start and end periods of the working hours of state departments. moreover, iraqi inhabitants employ hundreds of thousands of tiny electric generators in their houses,29 which use heavy oil or gasoline to create electricity, resulting in large quantities of soot, carbon deposits, and sulfur oxides.30 a recent study that measured air pollutants around iraq, showed that the highest number was recorded in al-diwaniyah, followed by baghdad, specifically the dora area.31 our results are in agreement with several previous studies, such as research32 on pahs released by vehicles, which found that street cops have high exposures, much higher than chefs, and their exposure levels are comparable to coke factory workers. another study in china found that traffic cops have a higher risk of pm2.5 pollution in the workplace than office cops who are considered a control group.33 in our current study, high levels of 8-oxo-dg were found in the blood serum of the traffic police group at the intersections of the capital baghdad compared with the levels recorded for the vital sign in the police group offices (representing the control group), and it was found that the increase in the levels of 8-oxo-dg was in conjunction with the increase in the levels of pahs in the traffic police group in the streets of baghdad city who are directly exposed to the dangers of pollutants emitted from car exhaust and diesel engines. many studies have utilized the 8-ohdg not only as a biomarker to estimate the impact of endogenous oxidative dna damage but also as a health risk for a variety of disorders, including cancer.34 various studies regarding the role of malondialdehyde, catalase and reduced glutathione in oxidative status studies have been reported in different clinical conditions.35,36 our study also added an mda biomarker to assess its levels in the blood of traffic police personnel and compare it with levels of pahs in the two study groups. this study found an important correlation between the level of pollutants in the blood and the level of mda, where the relationship between the level of pahs and the level of mda was positive and highly statistically significant as shown in the shape (after completion). our findings are consistent with the research conducted by37 on the workers of filling stations in nanjing, china. this study demonstrated that refueling workers’ blood gsh levels were much lower than those of office workers. in contrast, refueling workers’ serum mda and 8-ohdg levels were substantially greater than those of office employees. in research on traffic cops,38 reported a positive relationship between pm2.5 exposure and 8-oxo-dg. another study found that smokers’ plasma mda levels were larger than non-smokers.39 another significant study34 was conducted on coke plant workers in china, in which workers in a coke plant were classified into a group exposed to relatively less particulate matter and a second group highly exposed to particulate matter exposed. as an internal dosage, urine concentrations of pahs metabolites and minerals were evaluated. the study reported higher levels of urinary 8-oxo-dg and mda were substantially linked to higher levels of pm2.5 and overall pahs. by redox cycling, polycyclic aromatic hydrocarbons intermediates generate ros and trigger oxidative stress through many metabolic processes. exposure to polycyclic a romantic hydrocarbon from environmental pollution was found to be positively related to urinary 8-ohdg amounts in epidemiological studies. the generation of ros in normal metabolic processes, however, often does not result in oxidative stress because it is exactly balanced by the natural antioxidant system. as a result, toxins, ionizing radiations, and other external factors, as well as consumer habits and lifestyle factors such as alcohol consumption, tobacco, lack of physical activity, a poor diet, and certain genetic factors, could all contribute to an increase in ros production that overwhelms antioxidant defenses, resulting in oxidative stress. higher levels of 8-ohdg may operate as a biomarker for oxidative stress on dna, although it is not a particular diagnostic for pah exposure.39 the rate of dna repair processes, which involves the removal of damaged bases or complete nucleotides, is determined by the analyzed persons’ health state, age, diet, metabolic activity, and behavior.40 additionally, biological membranes’ shape and fluidity can be altered by ros-induced membrane lipid peroxidation, which ultimately affects how well the membranes operate. malondialdehyde (mda) and hydroxynonenal are two of the most well studied indicators of lipid peroxidation (hne). mda is a highly reactive nuclear factor produced both by lipid peroxidation and as a byproduct of the synthesis of prostaglandins and thromboxanes that can attack large molecules, including the group of proteins from amino acids or sulfhydryls resulting in changes in their functions. hne is a 187j contemp med sci | vol. 8, no. 3, may-june 2022: 184–188 a.r. salem et al. original evaluation of dna damage in traffic cops exposed to pah pollution significant toxin produced when polyunsaturated fatty acids are attacked by ros. it interacts with proteins to produce advanced end products of lipid oxidation. both hne and mda approaches have been fined in atherosclerotic lesions.41 elevated concentrations of urinary 8-oxo-2ʹ-deoxyguanosine were found to be strongly related to an increased risk of lung cancer in never-smokers in an early prospective investigation. a recent, nested case-control study of lung cancer and automotive pollution found that employees in highly polluted areas had a lifelong risk of developing lung cancer of at minimum 50%.42 as a result, we can conclude that a deficiency of ability to regenerate mitochondrial and nuclear dna injury is associated with a variety of neurological illnesses and tumors. whereas, the high formation of 8-oxodg is a potential mutagenic lesion in dna that leads to the conversion of g: c to t: a (g → t) during dna replication. therefore, it is potentially a powerful cancer prediction weapon, if mutation-prone dna lesions such as 8-oxo dg can be identified in genome scales.43 mda reacts with dna to yield harmful adducts of deoxyadenosine and deoxyguanosine.44 additionally, the shape and fluidity of biological membranes are altered during the membrane lipid peroxidation process brought on by ros, which ultimately affects how well they function.41 two types of antioxidant enzymes (catalase and glutathione peroxidase) were selected to study their levels in the blood serum of the two study groups and their relationship to pah level for both groups. our current study, found low levels in the levels of both catalase and glutathione peroxidase enzymes in the blood serum of traffic police personnel deployed in baghdad city intersections compared to the levels of the two enzymes were in the control group represented by the offices police, and the levels of these enzymes were inversely proportional to the levels of pollutants in the blood serum represented by polycyclic aromatic hydrocarbons. also, some studies conducted to evaluate the effects of occupational exposure to pollutants on oxidative stress in the body have indicated a decrease in the levels of antioxidant enzymes. in a study of pollution-exposed taxi drivers,45 reported a decrease in cat and g-px activities compared to the occupationally unexposed group. cohort research done before, during, and after the beijing olympics has reported that when air pollution levels increased, indicators of total antioxidant status-declined.46 the production of a sizable quantity of ros by particulate matter in traffic exhausts is one theory for the reported negative health impacts. the smaller particles of the aerosols in the environment contain smaller, more ros-rich particles. polycyclic aromatic hydrocarbons are also present in fine particles from vehicle exhaust (pahs). it has been demonstrated that antioxidants play a critical role in the catalysis of the conversion of (o2) to h2o2 and the breakdown of h2o2 into h2o, respectively. oxidative stress occurs when reactive oxygen species exceed the capacity of the antioxidants to act to defend the cell. once antioxidant enzymes are depleted, the cell is more vulnerable to the harmful effects of a xenobiotic that can lead to cell harm or death. as a result, the repeated inhalation of gasoline vapors possesses potential to cause oxidative stress by lowering the body’s antioxidant defenses and cellular functions.47 poor nutritional status can also contribute to oxidative stress, for example, selenium deficiency, which is associated with increased oxidative stress; higher gpx activity may arise from optimizing se’s nutritional status. a higher risk of cancer in epidemiological research studies has been linked to low levels of antioxidant intake. g-px loss resulted in loss of endothelial function, decreased angiogenesis, and increased infarction severity and vascular permeability in experimental animals,48 as seen the activity of catalase, catalase, sod and gpx to have a substantial negative relationship with the risk of coronary artery disease in patients.49 in response to oxidative stress, prolonged antioxidant enzyme deficiency enhances tissue sensitivity and severity.50 conclusion exposure to air pollutants like polycyclic aromatic hydrocarbons can reduce levels of antioxidant enzymes and thus create a state of oxidative stress, manifested in the presence of high levels of 8-oxodg and mda, which can lead to dna damage that can lead to many types of cancer in the traffic policemen. conflict of interest none.  references 1. miller m. r. (2020). oxidative stress and the cardiovascular effects of air pollution. free radical biology and medicine, 151: 69–87. 2. su, s. y., liaw, y. p., jhuang, j. r., hsu, s. y., chiang, c. j., yang, y. w., and lee, w. c. (2019). associations between ambient air pollution and cancer incidence in taiwan: an ecological study of geographical variations. bmc public health, 19(1): 1496. 3. north, c. m., rice, m. b., ferkol, t., gozal, d., hui, c., jung, s. h., et. al. (2019). air pollution in the asia-pacific region. a joint asian pacific society of respirology/american thoracic society perspective. american journal of respiratory and critical care medicine, 199(6): 693–700. 4. cicoira m. (2018). ambient air pollution as a new risk factor for cardiovascular diseases: time to take action. european journal of preventive cardiology, 25(8): 816–817. 5. schraufnagel, d. e., balmes, j. r., cowl, c. t., de matteis, s., jung, s. h., mortimer, k., et. al. (2019). air pollution and noncommunicable diseases: a review by the forum of international respiratory societies’ environmental committee, part 2: air pollution and organ systems. chest, 155(2): 417–426. 6. turner, m. c., krewski, d., diver, w. r., pope, c. a., burnett, r. t., jerrett, m., et. al. (2017). ambient air pollution and cancer mortality in the cancer prevention study ii. environmental health perspectives, 125(8): 087013. 7. vieira, v. m., villanueva, c., chang, j., ziogas, a., and bristow, r. e. (2017). impact of community disadvantage and air pollution burden on geographic disparities of ovarian cancer survival in california. environmental research, 156: 388–393. 8. cohen, a. j., brauer, m., burnett, r., anderson, h. r., frostad, j., estep, k., et. al. (2017). estimates and 25-year trends of the global burden of disease attributable to ambient air pollution: an analysis of data from the global burden of diseases study 2015. lancet (london, england), 389(10082): 1907–1918. 9. cavalieri, e. l., and rogan, e. g. (1995). central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons. xenobiotica; the fate of foreign compounds in biological systems, 25(7): 677–688. 10. rossner, p., jr, svecova, v., milcova, a., lnenickova, z., solansky, i., and sram, r. j. (2008). seasonal variability of oxidative stress markers in city bus drivers. part ii. oxidative damage to lipids and proteins. mutation research, 642(1-2): 21–27. 11. ledda, c., loreto, c., bracci, m., lombardo, c., romano, g., cinà, d., mucci, n., castorina, s., and rapisarda, v. (2018). mutagenic and dna repair activity in 188 j contemp med sci | vol. 8, no. 3, may-june 2022: 184–188 evaluation of dna damage in traffic cops exposed to pah pollution original a.r. salem et al. 31. al-kasser m. k. (2021). air pollution in iraq sources and effects. iop conference series: earth and environmental science, 790: 012014. 32. hu, y., bai, z., zhang, l., wang, x., zhang, l., yu, q., and zhu, t. (2007). health risk assessment for traffic policemen exposed to polycyclic aromatic hydrocarbons (pahs) in tianjin, china. the science of the total environment, 382(2-3): 240–250. 33. chao, h.r., hsu, j.w., ku, h.y., wang, s.l., huang, h.b., liou, s.h. and tsou, t.c. (2018). inflammatory response and pm2.5 exposure of urban traffic conductors. aerosol air qual. res. 18: 2633-2642. 34. hu, w., wang, y., wang, t., ji, q., jia, q., meng, t., ma, s., zhang, z., et. al. (2021). ambient particulate matter compositions and increased oxidative stress: exposure-response analysis among high-level exposed population. environment international, 147: 106341. 35. al-ghreaty hb, al-tum’a fj, hatrosh sj. the role of orexin hormone in sera of patients with metabolic syndrome of kerbala province: iraq. iraq medical journal. 2017 oct 2;1(3):57–60. 36. al-tu'ma, f. j. ; abd al-hassan, a. t. and alda'amy, e. m. (spring 2016). correlation between malondialdehyde and dyslipidemia in psoriatic patients. j contemp med sci, 2 (6): 56–58. 37. xiong, f., li, q., zhou, b., huang, j., liang, g., zhang, l., ma, s., et. al. (2016). oxidative stress and genotoxicity of long-term occupational exposure to low levels of btex in gas station workers. international journal of environmental research and public health, 13(12) : 1212. 38. tan, c., lu, s., wang, y., zhu, y., shi, t., lin, m., et. al. (2017). long-term exposure to high air pollution induces cumulative dna damages in traffic policemen. the science of the total environment, 593-594: 330–336. 39. miglani, k., kumar, s., yadav, a., aggarwal, n., ahmad, i., and gupta, r. (2019). a multibiomarker approach to evaluate the effect of polyaromatic hydrocarbon exposure on oxidative and genotoxic damage in tandoor workers. toxicology and industrial health, 35(7): 486–496. 40. gromadzińska, j., and wąsowicz, w. (2019). health risk in road transport workers.part i. occupational exposure to chemicals, biomarkers of effect. international journal of occupational medicine and environmental health, 32(3): 267–280. 41. bigagli, e and lodovici, m. (2019). circulating oxidative stress biomarkers in clinical studies on type 2 diabetes and its complications. oxidative medicine and cellular longevity, 2019, 5953685. 42. huang, h. b., chen, g. w., wang, c. j., lin, y. y., liou, s. h., lai, c. h., and wang, s. l. (2013). exposure to heavy metals and polycyclic aromatic hydrocarbons and dna damage in taiwanese traffic conductors. cancer epidemiology, biomarkers & prevention : a publication of the american association for cancer research, cosponsored by the american society of preventive oncology, 22(1): 102–108. 43. thanan, r., oikawa, s., hiraku, y., ohnishi, s., ma, n., pinlaor, s., yongvanit, p., et. al. (2014). oxidative stress and its significant roles in neurodegenerative diseases and cancer. international journal of molecular sciences, 16(1): 193–217. 44. lykkesfeldt j. (2007). malondialdehyde as biomarker of oxidative damage to lipids caused by smoking. clinicachimicaacta; international journal of clinical chemistry, 380(1-2): 50–58. 45. brucker, n., moro, a. m., charão, m. f., durgante, j., freitas, f., baierle, m., et. al.(2013).biomarkers of occupational exposure to air pollution, inflammation and oxidative damage in taxi drivers. the science of the total environment, 463-464: 884–893. 46. cosselman, k. e., allen, j., jansen, k. l., stapleton, p., trenga, c. a., larson, t. v., and kaufman, j. d. (2020). acute exposure to traffic-related air pollution alters antioxidant status in healthy adults. environmental research, 191: 110027. 47. wagboriayea f, dedekeb g, aladesidab a, bamideleb j, olootoc w. (2018) assessment of the effect of gasoline fume on stress hormones, antioxidant status and lipid peroxidation in albino rat. j king saud univ sci 30: 393–39. 48. sarıkaya, e. and doğan, s. (2020). glutathione peroxidase in health and diseases. in (ed.), glutathione system and oxidative stress in health and disease. intechopen. 49. flores-mateo, g., carrillo-santisteve, p., elosua, r., guallar, e., marrugat, j., bleys, j., and covas, m. i. (2009). antioxidant enzyme activity and coronary heart disease: meta-analyses of observational studies. american journal of epidemiology, 170(2): 135–147. 50. delfino, r. j., staimer, n., and vaziri, n. d. (2011). air pollution and circulating biomarkers of oxidative stress. air quality, atmosphere, and health, 4(1): 37–52. traffic policemen: a case-crossover study. journal of occupational medicine and toxicology (london, england), 13: 24. 12. clergé, a., le goff, j., lopez, c., ledauphin, j., and delépée, r. (2019). oxypahs: occurrence in the environment and potential genotoxic/mutagenic risk assessment for human health. critical reviews in toxicology, 49(4): 302–328. 13. bai, j., zhang, y., xi, z., greenberg, m. m., & zhou, c. (2018). oxidation of 8-oxo-7,8-dihydro-2’-deoxyguanosine leads to substantial dnahistone cross-links within nucleosome core particles. chemical research in toxicology, 31(12), 1364–1372. https://doi.org/10.1021/acs. chemrestox.8b00244 14. amente, s., di palo, g., scala, g., castrignanò, t., gorini, f., cocozza, s., et. al.(2019). genome-wide mapping of 8-oxo-7,8-dihydro-2’deoxyguanosine reveals accumulation of oxidatively-generated damage at dna replication origins within transcribed long genes of mammalian cells. nucleic acids research, 47(1): 221–236. 15. giorgio, m., dellino, g. i., gambino, v., roda, n., and pelicci, p. g. (2020). on the epigenetic role of guanosine oxidation. redox biology, 29: 101398. 16. nakabeppu, y., ohta, e., and abolhassani, n. (2017). mth1 as a nucleotide pool sanitizing enzyme: friend or foe? free radical biology and medicine, 107: 151–158. 17. pylväs-eerola, m., karihtala, p., and puistola, u. (2015). preoperative serum 8-hydroxydeoxyguanosine is associated with chemoresistance and is a powerful prognostic factor in endometrioid-type epithelial ovarian cancer. bmc cancer, 15: 493. 18. sova, h., jukkola-vuorinen, a., puistola, u., kauppila, s., and karihtala, p. (2010). 8-hydroxydeoxyguanosine: a new potential independent prognostic factor in breast cancer. british journal of cancer, 102(6): 1018–1023. 19. long, j. d., matson, w. r., juhl, a. r., leavitt, b. r., paulsen, j. s., and predicthd investigators and coordinators of the huntington study group (2012). 8-ohdg as a marker for huntington disease progression. neurobiology of disease, 46(3): 625–634. 20. nakanishi, s., suzuki, g., kusunoki, y., yamane, k., egusa, g., and kohno, n. (2004). increasing of oxidative stress from mitochondria in type 2 diabetic patients. diabetes/metabolism research and reviews, 20(5): 399–404. 21. loft, s., and poulsen, h. e. (1996). cancer risk and oxidative dna damage in man. journal of molecular medicine (berlin, germany), 74(6) : 297–312. 22. wu, l. l., chiou, c. c., chang, p. y., and wu, j. t. (2004). urinary 8-ohdg: a marker of oxidative stress to dna and a risk factor for cancer, atherosclerosis and diabetics. clinica chimica acta; international journal of clinical chemistry, 339(1-2): 1–9. 23. wu, d., liu, b., yin, j., xu, t., zhao, s., xu, q., chen, x., and wang, h. (2017). detection of 8-hydroxydeoxyguanosine (8-ohdg) as a biomarker of oxidative damage in peripheral leukocyte dna by uhplc-ms / ms. journal of chromatography. b, analytical technologies in the biomedical and life sciences, 1064 : 1–6 . 24. guo, c., ding, p., xie, c., ye, c., ye, m., pan, c., cao, x., zhang, s., and zheng, s. (2017). potential application of the oxidative nucleic acid damage biomarkers in detection of diseases. oncotarget, 8(43): 75767–777. 25. da silva junior, f. c., felipe, m., castro, d., araújo, s., sisenando, h., and batistuzzo de medeiros, s. r. (2021). a look beyond the priority: a systematic review of the genotoxic, mutagenic, and carcinogenic endpoints of nonpriority pahs 278, 116838. 26. rubin h. (2001). synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updates. carcinogenesis, 22(12): 1903–1930. 27. raghad h al-ani, and estabraq ar. al-wasiti. (2021). the adverse effect of air pollution with polycyclic aromatic hydrocarbon (pah) on 8-oxo-dg and gene expression (hogg1) in midland refineries company-daura refinery workers. indian journal of forensic medicine & toxicology, 15(3), 2651–2656. 28. chaichan, m.t., kazem, h.a. and abed, t.a. (2016). traffic and outdoor air pollution levels near highways in baghdad, iraq. environ dev sustain 20, 589–603 . 29. kazem, h. a. and chaichan, m.t.(2012). ”status and future prospects of renewable energy in iraq,” renewable and sustainable energy reviews, elsevier, vol. 16(8): 6007-6012. 30. chaichan, m. t., and faris, s. s. (2015). practical investigation of the environmental hazards of idle time and speed of compression ignition engine fueled with iraqi diesel fuel. international journal for mechanical and civil engineering, 12(1): 29–34. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.1223 https://iopscience.iop.org/journal/1755-1315 https://iopscience.iop.org/journal/1755-1315 https://iopscience.iop.org/volume/1755-1315/790 https://ideas.repec.org/a/eee/rensus/v16y2012i8p6007-6012.html https://ideas.repec.org/a/eee/rensus/v16y2012i8p6007-6012.html https://ideas.repec.org/s/eee/rensus.html 370 j contemp med sci | vol. 8, no. 6, november-december 2022: 370–374 original social factors affecting relapse of severe mental illness: a qualitative analysis of healthcare team’s perceptions naima seyedfatemi, saman saber* nursing and midwifery care research center, iran university of medical sciences, tehran, iran. *correspondence to: saman saber, (email: samansaber16@gmail.com) (submitted: 05 september 2022 – revised version received: 12 october 2022 – accepted: 02 november 2022 – published online: 26 december 2022) abstract objectives: relapse is a challenge for patients with severe mental illness (smi). the purpose of present study was to explain the health care team’s perception of social factors affecting smi relapse. methods: in this qualitative content analysis study, semi-structured interviews have been conducted with 23 members of healthcare team. content analysis was used to categorize the data. results: the social factors affecting the relapse of smi could be classified in three categories of community-related factors, cultural factors, and family-related factors. the first category included low socioeconomic status, lack of community support for smi patients, and insufficient awareness of community about smi. the second category included false beliefs and misconceptions, and negative attitudes towards smi. the third category also included dysfunctional family and non-supportive family. conclusion: in order to deal with cultural misconceptions that lead to the relapse of smi, it is necessary to implement culture-based interventions to correctly confront negative attitudes and stigmatized beliefs and fight against cultural taboos that govern the phenomenon of smi relapse in iran. it seems that the implementation of family-centered interventions for the family of patients with smi can reduce the burden of family-related factors in disease relapse. keywords: mental illness, patient, healthcare team, relapse, qualitative research issn 2413-0516 introduction it is estimated that 900 million people in the world suffer from mental illnesses.1 in iran, the results of a systematic review showed the prevalence of mental illnesses at around 25–31%.2 it should be acknowledged that mental illnesses are one of the five main causes of disabilities that affect all aspects of patient’s life, his family and social system, depriving him of a normal life.3 most mental illnesses, including major depressive disorder, schizophrenia and bipolar disorder are classified as severe mental illness (smi) and have frequent relapses.4 patients with smi are deprived of continued treatment for several reasons, and experience frequent relapses of the disease, which often require re-hospitalization.5 relapse of mental illness disrupts the treatment process and increases the risk of resistance to treatment.6 in fact, relapse occurs when patient gets sick again after recovery. relapse of smi is associated with the appearance of early symptoms and increased risk of suicide for patients. relapse is a challenge for patients with mental illness, and is associated with a large social burden.7 this phenomenon is very worrying for patients and their families, because it not only slows down the treatment process, but also creates a heavy economic burden for the individual, family, society and healthcare system. it also reduces social functioning and leads to unemployment and isolation.8 in addition, the most vulnerable time for families is the time of disease relapse, because at this time, patient needs family support to remain optimistic about the future.9 different statistics have been reported for smi relapse. for example, it has been reported that 90% of people with bipolar disorder have at least one relapse during their lifetime.5 it has also been revealed that 60% of patients with major depressive disorder experience a second episode of disease after the first one.10 in this regard, it has been found that 82% of people with schizophrenia experience a relapse in the first 5 years of treatment, and 78% of them experience a second episode of relapse.8 researchers have introduced several factors for the relapse of smi. patients with mental illness in china stated that work-related stress and discontinuation of medication are the main reasons for the relapse of their disease.11 in france, patients who had been ill for more than ten years had a higher risk of disease relapse compared to those who had been ill for less than 5 years.12 in ethiopia, patients who had been suffering from bipolar disorder for more than 5 years faced a higher risk of disease relapse.7 a study in the republic of congo showed that low education level, being female, low self-esteem and living in rural areas were among the predictors of mental illness relapse.13 in iran, for patients with major depressive disorder, factors such as young age at the onset of disease, living in urban areas, history of mental illness in the family, and history of emotional problems play an important role in their frequent hospitalizations.14 a review study stated that in major depressive disorder, factors such as high level of depression, high level of poor sleep quality, and irritability will increase the severity of disease relapse.15 literature review indicated that different factors including social factors play a major role in the smi relapse, and patients in different contexts are at risk of smi relapse. in iran, no research has been conducted to identify social factors affecting smi relapse from the perspective of healthcare team. it has neither been described nor documented in iran. identifying the health care team’s perception of social factors affecting smi relapse can be an important step towards prevention and control of its consequences. it seems that health care team’s perception of social factors affecting smi relapse has cultural and conceptual context-based roots. since iranian society is one of the societies where patients suffer from smi relapse and this disease is considered a social issue, one of the ways to identify social factors affecting smi relapse is to use qualitative research. the purpose of present study was to explain the healthcare team’s perception of social factors affecting smi relapse. mailto:samansaber16@gmail.com 371j contemp med sci | vol. 8, no. 6, november-december 2022: 370–374 n. seyedfatemi et al. original social factors affecting relapse of severe mental illness materials and methods design in this study, content analysis method was used to identify social factors affecting the relapse of smi from the perspective of health care team. content analysis is a method of analyzing written, audio or visual messages about a concept. in conventional content analysis, there is little information about a concept, and the concept under study is identified from the textual data through categories and their names.16 in the current study, the conventional content analysis method was used. setting the study setting was a comprehensive and dedicated psychiatric center affiliated to iran university of medical science in tehran, the capital of iran. this center has five wards, one emergency room and two clinics. sampling participants were selected by purposeful sampling method. they were selected with maximum variation in terms of age, gender, education level and history of employment in psychiatric ward. the criteria for selecting the participants were: working in the psychiatric department as one of the members of healthcare team, including nurses, psychologists, and psychiatrists, and having witnessed the frequent relapse of smi. in total, 23 members of healthcare team, including 5 psychiatrists, 4 psychologists and 14 nurses participated in this study. the participants were in the age range of 37–46 years, and 16 of them were women. nurses and psychologists had bachelor’s degrees, but psychiatrists had phd in psychiatry and 8–15 years of work experience. data collection the data was collected in the first half of 2021. data collection method was in-depth, semi-structured and individual interview with the participants, which was conducted by the first author. the researcher attended the inpatient wards in the morning and evening shifts and interviewed the participants in a quiet room, with previous coordination with the head nurse. each interview took about 40 to 50 minutes. each person was interviewed once and a total of 23 interviews were conducted. the interviews were recorded using a voice recorder. the main interview questions were: “how do you, as a member of healthcare team, describe the relapse of smi?” “what social factors do you think affect this relapse?” to obtain more information, probing questions such as: “what do you mean by that?” and “is there anything else you want to talk about?” or “can you explain more in this regard”, were also used. the interviews continued until data saturation was reached, when no new data was obtained. data analysis the data collection and analysis were also done at the same time. in order to analyze the data, each interview was recorded and then typed verbatim into a text. the interview text was read several times to get a general understanding and then, semantic units were extracted from them. at the next step, codes were obtained from the semantic units. then, the codes were replaced in subcategories based on their similarities and differences. finally, as similar subcategories emerged, the main categories were formed.17 rigor the criteria of credibility, dependability, confirmability and transferability were used to ensure the rigor of the data.18 in order to achieve credibility, a constant engagement with the subject and data was maintained, and opinions of research team about the interview process and data analysis were considered. the interview text and findings were also given to some of the participants for confirmation. for data dependability, the opinions of an external observer, as a researcher who was familiar with qualitative research methodology but was not part of the research team, were used, achieving an agreement on the results. for confirmability, all activities were recorded and a report on research process was prepared. for transferability, the results were discussed with two members of healthcare team, who were not part of the study and yet had the same conditions as the participants, and the data were approved by them. ethical considerations this research was approved by the ethical committee of iran university of medical sciences (ir.iums.rec.1399.348). in order to comply with ethical considerations, written informed consent was obtained from the participants. they were informed about the objectives of the research and were told that they can withdraw from the study whenever they want. in addition, to obtain permission for recording of the interviewees’ voices, they were also assured about the confidentiality of their personal information. results according to the understanding of healthcare team, the social factors affecting the relapse of smi could be classified in three categories of community-related factors, cultural factors, and family-related factors. community-related factors included low socioeconomic status, lack of community support for smi patients, and insufficient awareness of community about smi. cultural factors included false beliefs and misconceptions, and negative attitudes towards smi. family-related factors also included dysfunctional family and non-supportive family. acommunity-related factors the participants’ statements showed that community-related factors have a significant impact on the recurrence of smi. in this regard, low socio-economic status, lack of community support for patients with smi, and insufficient awareness of community about smi were among the factors that had an effect on the recurrence of smi. a-1low socio-economic status the participants reported low socio-economic status, which manifests itself as a form of poverty, as one of the important factors in the return of symptoms in smi patients. in this regard, one of the nurses said: “social-economic problems, poverty and high prices are among stresses that make a person susceptible to smi relapse. for example, if a patient who has been diagnosed with smi lives in a community full of poverty and has no access to facilities for his basic needs, he will get sick again”. (participant (p) 5) 372 j contemp med sci | vol. 8, no. 6, november-december 2022: 370–374 social factors affecting relapse of severe mental illness original n. seyedfatemi et al. b-1false beliefs and misconceptions the participants referred to false beliefs and misconceptions as one of the factors that affect disease relapse. according to them, the actions that originate from false beliefs cause irrational and non-therapeutic recommendations, and while worsening patients’ condition, keep them away from correct treatment. one of the nurses emphasized on the deterioration of patient’s condition after visiting the exorcist: “we are faced with cultural misconceptions. for example, a patient with smi went to visit an exorcist for treatment, and he hit his head so hard to get a demon out of his body! these treatments will not improve patient condition and will cause the disease to relapse”. (p20) in this regard, a psychologist stated that sometimes, patients feel they cannot get help from the treatment team, so they seek inappropriate treatments, such as referring to healers. he also stated: “patients feel that there is nothing we can do, so they turn to inappropriate and wrong treatments. for example, patients abandon their treatment half way through and visit traditional healers, thinking that they can get a better result by illogical treatment model. but in the end, they experience the relapse of their disease.” (p17) b-2negative attitude towards smi the participants referred to negative attitude of the community towards smi, as well as the stigma and cultural taboo that exists about it. they also considered their role in the disease relapse to be very important. one of the psychiatrists in this regard stated: “there is a stigma towards smi in our culture, and this stigma constantly creates many negative emotions in patients, all of which can cause incompliance and disruption in the patient’s treatment. these factors can cause the disease to relapse”. (p14) one of the nurses stated: “i think there is a taboo about smi in our culture that compels the patient to hide their disease, making their treatment and follow-up difficult. in this situation, we have to wait for frequent relapse of the disease. false and negative views that exist in the community prevent patients from informing the treatment team about the recurrence of their diseases, and we often find out about these issues very late”. (p9) according to the participants, cultural stigma leads to non-continuation of drug use and disease relapse. one of the nurses in this regard stated: “smi is considered a negative point in people’s lives, and there is a negative and pessimistic view of mental patients from the cultural point of view. i think patients have no motivation to continue taking medication because of the stigma that the culture puts on them. and because of this, disease relapses often occur”. (p3) cfamily-related factors the participants referred to the important role that family plays in disease relapse. they considered any disruption in the family life, such as having dysfunctional family or non supportive family, as the cause of disease relapse. c-1dysfunctional family referring to the role of dysfunctional family caregivers who cannot provide effective care to the patient, a psychiatrist stated: “a schizophrenic patient should be taken care of, but the person who is taking care of the patient is an elderly parent who has high blood pressure, diabetes and back pain, which prevent another nurse added: “sometimes the relapse is due to financial issues. for example, when patients cannot afford medications and cost of travel and frequent visits to clinic is high for them as they have no money, following treatment is not a priority for them.” (p11) one of the psychiatrists stated: “we have lots of patient who are laborers and head of family, making a living with a very low salary. when they see that they are financially weak, they feel helpless and this causes their disease to relapse. we had many patients with sick note for at least a month, who, after discharge from hospital went back to work straight away due to financial issues, this resulted in incomplete treatment and relapse of their disease”. (p18) a-2lack of community support for patients with smi according to the participants, lack of community support such as job security can lead to disease relapse. a psychologist in this regard stated: “many times, continuous hospitalizations cause patients to lose their jobs while they receive no job support, and this causes many problems for them. as a result, they prefer not to be hospitalized, so they experience frequent disease relapse, because their treatment is not complete”. (p4) the participants considered the inappropriate community support of the patients as the reason for their disease relapse. one of the nurses stated: “one of the reasons for disease relapse is the lack of community support, which is also very important. for example, as soon as employers find out that a person applying for job has smi, they refuse to employ him. in this situation, even though his disease is controlled, he may experience a relapse.” (p22) according to the participants, inducing the sense of being different from others by the community can lead to disease relapse. one of the nurses in this regard said: “in the community, when people realize that someone is suffering from smi, they don’t welcome him in any community gathering or if they do, it is because they pity him, which is even worse. well, here the patient feels different and the community indirectly conveys to the patient that he is dangerous and has a problem. this in turn makes the patient’s condition worse, leading to a disease relapse.” (p7) a-3inadequate awareness of community about smi the community’s lack of understanding and awareness of smi and how to deal with the patient correctly reduces the intervals between disease relapses and decreases acceptance of patients by community. one of the psychiatrists in this regard stated: “community does not have a proper understanding of smi, and people do not know what to do when they encounter a smi patient, so they always treat these patients harshly or reject them, which puts pressure on the patients and causes the symptoms to return again”. (p16) one of the nurses also said: “one of the factors that causes the relapse of disease is the community’s lack of awareness of the form and type of smi, as these patients are not properly understood by the community. people should learn how to treat patients with different behavior and appearance, but the problem is that people do not even recognize the initial signs of smi, let alone the signs of its relapse”. (p12) bcultural factors the participants believed that misconceptions, false beliefs and negative attitudes towards smi are rooted in culture and lead to the relapse of smi. 373j contemp med sci | vol. 8, no. 6, november-december 2022: 370–374 n. seyedfatemi et al. original social factors affecting relapse of severe mental illness him from taking care of the patient. in fact, the person who is supposed to take care of the patient is sick himself and cannot take care of the patient, so the disease will relapse again.” (p1) participants referred to unfavorable family atmosphere, multiple conflicts and high tension between family members as factors that affect relapse of smi. one of the nurses said: “another reason for disease relapse is the unfavorable family atmosphere, an environment that is always full of tension and conflicts. for example, some patients have a strong disagreement with their spouses, or there are severe tensions between the patient’s parents, and these tensions lead to the recurrence of the disease”. (p15) one of the psychologists also stated: “there are families that criticize too much or are over-involved. for instance, they impose very strict restrictions on patient, are over protective of the patient, or do not allow the patient to do anything; these factors facilitate the disease relapse”. (p21) c-2non-supportive family the participants introduced the lack of cooperation and family support for follow-up and continuation of treatment to be the main cause of smi relapse. a nurse in this regard stated: “another factor that can be the reason for disease relapse is that, the family has not cooperated with the patient to continue the treatment, or the family does not care about the fact that their patient is like a diabetic patient or a chronic patient who needs follow-up and has to go to hospital regularly for a long time”. (p10) one of the psychiatrists added that lack of family support prevents the patient from receiving timely medication and follow-up treatment, and causes the disease to relapse. he also stated: “if patients don’t have proper family support, or if they don’t have someone to buy them medicine on time, support them and take them to follow-up visits, they are more likely to experience the disease relapse. for example, we had a patient who was living alone and his whole family was living overseas, so he did not a have good family support, as a result he was admitted to hospital almost 3 times a year”. (p2) discussion in this study, according to the understanding of healthcare team, social factors affecting the relapse of smi included community-related factors, cultural factors, and family related factors. according to the participants, problems such as poverty, high cost of treatment and low socio-economic status lead to the relapse of smi. the result of a study in india showed that stressful life events lead to disease relapse in patients with bipolar disorder, and the most important of these life events were financial problems and low income.19 similarly, patients with schizophrenia in china who had higher incomes were less likely to experience disease relapse.20 the participants’ statements showed that lack of community support for patient, which comes in the form of lack of job support and security, is one of the factors that affect smi relapse. in this context, researchers believe that community support may be a protective factor that reduces the risk of disease relapse.21 the results of a study in spain showed that one of the factors that prevent the recurrence of smi is having a job and profession. a job, which is suited to a person’s spirit and ability, helps to improve his mental health.22 in this regard, the results of a study conducted in china showed that patients with schizophrenia who were employed were less likely to experience disease relapse.20 in addition, according to the understanding of participants in the present study, the community’s insufficient awareness of smi and its recurrence symptoms leads to the continuation of disease relapse. in order to improve the low socio-economic status of patients, which leads to the disease relapse, it is necessary for these patients to receive financial support. part of this support can be provided by the government and part of it can be provided by non-governmental organizations and ngos. also, in order to improve the community’s support of patients and increase the awareness of community about smi and its recurrence symptoms, it is necessary to improve the level of community’s literacy about the patients with smi and teach people that smi patients are able to work by relying on their abilities after treatment and should not be rejected by the community. according to the participants, the patients’ cultural misconceptions and the wrong cultural attitude towards smi lead to relapse of the disease. in this regard, instead of health care team, patients turn to exorcists (a person who tries to remove the cause of the disease, i.e., a genie, by hitting the patient’s head) or healers (a person who abuses the religious beliefs of patients and tries to improve the patient’s mental health) who are unprofessional and incompetent people, and this leads to continuation of the disease and its recurrence. in addition, the stigma and taboo in community, which is rooted in the culture, cause patients to hide their disease and symptoms that also leads to the disease relapse. in fact, stigma is one of the main factors related to disease relapse in patients with smi, meaning that it makes it difficult to talk about the disease and its relapse. the results of a study in uganda showed that stigma increases the risk of disease relapse in patients with smi.23 in order to deal with cultural misconceptions that lead to the relapse of smi, it is necessary to implement culture-based interventions to correctly confront negative attitudes and stigmatized beliefs and fight against cultural taboos that govern the phenomenon of smi relapse in iran. both of these interventions can be provided through the national media. other statements of the participants indicated that some family-related factors, such as a family that cannot take care of the patient, a tense family, a family that does not pay attention to the patient’s illness, and a family that cannot prevent the relapse of the disease due to its lack of support, are involved in the relapse of smi. in fact, when families do not support patients, they cannot comply effectively with the treatment, so they will be deprived of treatment. the results of a study in netherlands showed that non-compliance with treatment protocols is one of the factors that affect smi relapse.24 at the same time, in china, patients with schizophrenia who had good treatment compliance were less likely to experience the disease relapse.20 according to the participants, being alone and living on your own, which is one of the examples of lack of family support, leads to the relapse of smi. in the same context, a review study found that living alone is a risk factor for disease relapse, compared to living in families that support patient.25 it seems that the implementation of family-centered interventions for the family of patients with smi can reduce the burden of family-related factors in disease relapse. it is recommended to conduct studies with an action research approach to solve the problems caused by the relapse of smi. it is also recommended that more comprehensive qualitative researches be done in this field. 374 j contemp med sci | vol. 8, no. 6, november-december 2022: 370–374 social factors affecting relapse of severe mental illness original n. seyedfatemi et al. our participant recruitment approach and the nature of the present study limited our ability to generalize the presented findings. however, the aim of qualitative researches is not a generalization. conclusion this study is the first qualitative study conducted on social factors affecting the relapse of smi in iran. results of this study classify social factors affecting the relapse of smi in three categories of community-related factors, cultural factors, and family-related factors. the results of this study can sensitize the healthcare system to these factors in order to prevent the relapse of smi by implementing community, culture and family-centered interventions. acknowledgments the authors are grateful to all the participants. the work reported in this article was conducted as part of the second author’s doctoral study. financial disclosure none to declare. conflict of interest none to declare.  references 1. kassebaum nj, arora m, barber rm, et al. global, regional, and national disability-adjusted life-years (dalys) for 315 diseases and injuries and healthy life expectancy (hale), 1990–2015: a systematic analysis for the global burden of disease study 2015. lancet 2016; 388(10053):1603–58. doi: 10.1016/s0140-6736(16)31460-x. 2. taheri mirghaed m, abolghasem gorji h, panahi s. prevalence of psychiatric disorders in iran: a systematic review andmeta-analysis. int j prev med 2020;11: 21. doi: 10.4103/ijpvm.ijpvm_510_18. 3. den boer k, de veer aje, schoonmade lj, verhaegh kj, van meijel b, francke al. a systematic review of palliative care tools and interventions for people with severe mental illness. bmc psychiatry 2019; 19(1):106. doi: 10.1186/ s12888-019-2078-7. 4. mötteli s, schori d, schmidt h, seifritz e, jäger m. utilization and effectiveness of home treatment for people with acute severe mental illness: a propensity-score matching analysis of 19 months of observation. front psychiatry 2018; 9:495. doi: 10.3389/fpsyt.2018.00495. 5. ayano g, duko b. relapse and hospitalization in patients with schizophrenia and bipolar disorder at the st amanuel mental specialized hospital, addis ababa, ethiopia: a comparative quantitative crosssectional study. neuropsychiatr dis treat 2017; 13:1527–1531. doi: 10.2147/ndt.s139075. 6. moges s, belete t, mekonen t, menberu m. lifetime relapse and its associated factors among people with schizophrenia spectrum disorders who are on follow up at comprehensive specialized hospitals in amhara region, ethiopia: a cross-sectional study. int j ment health syst 2021; 15(1):42. doi: 10.1186/s13033-021-00464-0. 7. belete h, ali t, legas g. relapse and clinical characteristics of patients with bipolar disorders in central ethiopia: a cross-sectional study. psychiatry j 2020; 2020:8986014. doi: 10.1155/2020/8986014. 8. sullivan s, northstone k, gadd c, walker j, margelyte r, richards a, whiting p. models to predict relapse in psychosis: a systematic review. plos one 2017; 12(9):e0183998. doi: 10.1371/journal.pone.0183998. 9. leach mj, jones m, bressington d, nolan f, jones a, muyambi k, gillam m, gray r. the association between mental health nursing and hospital admissions for people with serious mental illness: a protocol for a systematic review. syst rev 2018;7(1):2. doi: 10.1186/s13643-017-0658-5. 10. liu ch, zhang gz, li b, li m, woelfer m, walter m, wang l. role of inflammation in depression relapse. j neuroinflammation 2019; 16(1):90. doi: 10.1186/s12974-019-1475-7. 11. hui clm, lo mcl, chan ehc, chen esm, ko rwt, lee ehm, chang wc, chan skw, chen eyh. perception towards relapse and its predictors in psychosis patients: a qualitative study. early interv psychiatry 2018; 12(5):856–862. doi: 10.1111/eip.12378. 12. fond g, bulzacka e, boucekine m, schürhoff f, berna f, godin o, et al. machine learning for predicting psychotic relapse at 2 years in schizophrenia in the national face-sz cohort. prog neuropsychopharmacol biol psychiatry 2019; 92:8–18. doi: 10.1016/j.pnpbp.2018.12.005. 13. vagheni mm, vivalya bmn, muyisa lk, masuka rk, kitoko gmb. prevalence and predictors of relapse among adolescent patients with mental illness in butembo city (eastern part of the democratic republic of congo). psychiatry res 2022; 308:114342. doi: 10.1016/j.psychres 2021.114342. 14. parami s, tapak l, poorolajal j, moghimbeigi a, ghaleiha a. identifying factors associated with the hospital readmission rate among patients with major depressive disorder. bmc psychiatry 2021; 21(1):542. doi: 10.1186/ s12888-021-03559-7. 15. kennard bd, mayes tl, chahal z, nakonezny pa, moorehead a, emslie gj. predictors and moderators of relapse in children and adolescents with major depressive disorder. j clin psychiatry 2018; 79(2):15m10330. doi: 10.4088/jcp.15m10330. 16. hsieh hf, shannon se. three approaches to qualitative content analysis. qual health res 2005; 15(9):1277–88. doi: 10.1177/1049732305276687. 17. elo s, kyngäs h. the qualitative content analysis process. j adv nurs2008; 62(1):107–15. doi: 10.1111/j.1365-2648.2007.04569.x. 18. lincoln y, guba e. naturalistic inquiry. sage: beverly hills, 1985. 19. sam sp, nisha a, varghese pj. stressful life events and relapse in bipolar affective disorder: a cross-sectional study from a tertiary care center of southern india. indian j psychol med 2019; 41(1):61–67.doi: 10.4103/ ijpsym.ijpsym_113_18. 20. wei-feng m, xiao-min c, teng-teng f, et al. identifying modifiable risk factors for relapse in patients with schizophrenia in china. front psychiatry 2020; 11:574763.doi: 10.3389/fpsyt 2020.574763. 21. andreescu c, ajilore o, aizenstein hj, albert k, butters ma, landman ba, karim ht, krafty r, taylor wd. disruption of neural homeostasis as a model of relapse and recurrence in late-life depression. am j geriatr psychiatry 2019; 27(12):1316–1330. doi: 10.1016/j.jagp.2019.07.016. 22. echeburúa e, gómez m, freixa m. prediction of relapse after cognitivebehavioral treatment of gambling disorder in individuals with chronic schizophrenia: a survival analysis. behav ther 2017; 48(1):69–75. doi: 10.1016/j.beth.2016.09.008. 23. rasmussen jd, kakuhikire b, baguma c, ashaba s, cooper-vince ce, perkins jm, bangsberg dr, tsai ac. portrayals of mental illness, treatment, and relapse and their effects on the stigma of mental illness: populationbased, randomized survey experiment in rural uganda. plos med 2019; 16(9):e1002908. doi: 10.1371/journal.pmed.1002908. 24. bockting clh, klein ns, elgersma hj, van rijsbergen gd, slofstra c, ormel j, et al. effectiveness of preventive cognitive therapy while tapering antidepressants versus maintenance antidepressant treatment versus their combination in prevention of depressive relapse or recurrence (drd study): a three-group, multicentre, randomised controlled trial. lancet psychiatry 2018; 5(5):401–410. doi: 10.1016/s2215-0366(18)30100-7. 25. sfetcu r, musat s, haaramo p, ciutan m, scintee g, vladescu c, wahlbeck k, katschnig h. overview of post-discharge predictors for psychiatric rehospitalisations: a systematic review of the literature. bmc psychiatry 2017; 17(1):227. doi: 10.1186/s12888-017-1386-z. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1294 https://dx.doi.org/10.4103%2fijpvm.ijpvm_510_18 https://pubmed.ncbi.nlm.nih.gov/30943927/ https://pubmed.ncbi.nlm.nih.gov/30943927/ https://pubmed.ncbi.nlm.nih.gov/?term=m%c3%b6tteli+s&cauthor_id=30364109 https://pubmed.ncbi.nlm.nih.gov/?term=schori+d&cauthor_id=30364109 https://pubmed.ncbi.nlm.nih.gov/?term=schmidt+h&cauthor_id=30364109 https://pubmed.ncbi.nlm.nih.gov/?term=seifritz+e&cauthor_id=30364109 https://pubmed.ncbi.nlm.nih.gov/?term=j%c3%a4ger+m&cauthor_id=30364109 https://pubmed.ncbi.nlm.nih.gov/?term=ayano+g&cauthor_id=28670121 https://pubmed.ncbi.nlm.nih.gov/?term=duko+b&cauthor_id=28670121 https://pubmed.ncbi.nlm.nih.gov/?term=moges+s&cauthor_id=33957944 https://pubmed.ncbi.nlm.nih.gov/?term=belete+t&cauthor_id=33957944 https://pubmed.ncbi.nlm.nih.gov/?term=mekonen+t&cauthor_id=33957944 https://pubmed.ncbi.nlm.nih.gov/?term=menberu+m&cauthor_id=33957944 https://pubmed.ncbi.nlm.nih.gov/?term=belete+h&cauthor_id=33062661 https://pubmed.ncbi.nlm.nih.gov/?term=ali+t&cauthor_id=33062661 https://pubmed.ncbi.nlm.nih.gov/?term=legas+g&cauthor_id=33062661 https://pubmed.ncbi.nlm.nih.gov/28934214/ https://pubmed.ncbi.nlm.nih.gov/?term=leach+mj&cauthor_id=29316979 https://pubmed.ncbi.nlm.nih.gov/?term=jones+m&cauthor_id=29316979 https://pubmed.ncbi.nlm.nih.gov/?term=bressington+d&cauthor_id=29316979 https://pubmed.ncbi.nlm.nih.gov/?term=nolan+f&cauthor_id=29316979 https://pubmed.ncbi.nlm.nih.gov/?term=jones+a&cauthor_id=29316979 https://pubmed.ncbi.nlm.nih.gov/?term=muyambi+k&cauthor_id=29316979 https://pubmed.ncbi.nlm.nih.gov/?term=gillam+m&cauthor_id=29316979 https://pubmed.ncbi.nlm.nih.gov/?term=gray+r&cauthor_id=29316979 https://pubmed.ncbi.nlm.nih.gov/?term=liu+ch&cauthor_id=30995920 https://pubmed.ncbi.nlm.nih.gov/?term=zhang+gz&cauthor_id=30995920 https://pubmed.ncbi.nlm.nih.gov/?term=li+b&cauthor_id=30995920 https://pubmed.ncbi.nlm.nih.gov/?term=li+m&cauthor_id=30995920 https://pubmed.ncbi.nlm.nih.gov/?term=woelfer+m&cauthor_id=30995920 https://pubmed.ncbi.nlm.nih.gov/?term=walter+m&cauthor_id=30995920 https://pubmed.ncbi.nlm.nih.gov/?term=wang+l&cauthor_id=30995920 https://pubmed.ncbi.nlm.nih.gov/?term=hui+clm&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=lo+mcl&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=chan+ehc&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=chen+esm&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=ko+rwt&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=lee+ehm&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=chang+wc&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=chan+skw&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=chen+eyh&cauthor_id=27573207 https://pubmed.ncbi.nlm.nih.gov/?term=fond+g&cauthor_id=30552914 https://pubmed.ncbi.nlm.nih.gov/?term=bulzacka+e&cauthor_id=30552914 https://pubmed.ncbi.nlm.nih.gov/?term=boucekine+m&cauthor_id=30552914 https://pubmed.ncbi.nlm.nih.gov/?term=sch%c3%bcrhoff+f&cauthor_id=30552914 https://pubmed.ncbi.nlm.nih.gov/?term=berna+f&cauthor_id=30552914 https://pubmed.ncbi.nlm.nih.gov/?term=godin+o&cauthor_id=30552914 https://pubmed.ncbi.nlm.nih.gov/?term=mumbere+vagheni+m&cauthor_id=34953201 https://pubmed.ncbi.nlm.nih.gov/?term=mutume+nzanzu+vivalya+b&cauthor_id=34953201 https://pubmed.ncbi.nlm.nih.gov/?term=kasereka+muyisa+l&cauthor_id=34953201 https://pubmed.ncbi.nlm.nih.gov/?term=kasereka+masuka+r&cauthor_id=34953201 https://pubmed.ncbi.nlm.nih.gov/?term=manzekele+bin+kitoko+g&cauthor_id=34953201 https://pubmed.ncbi.nlm.nih.gov/?term=parami+s&cauthor_id=34724910 https://pubmed.ncbi.nlm.nih.gov/?term=tapak+l&cauthor_id=34724910 https://pubmed.ncbi.nlm.nih.gov/?term=poorolajal+j&cauthor_id=34724910 https://pubmed.ncbi.nlm.nih.gov/?term=moghimbeigi+a&cauthor_id=34724910 https://pubmed.ncbi.nlm.nih.gov/?term=ghaleiha+a&cauthor_id=34724910 https://pubmed.ncbi.nlm.nih.gov/?term=kennard+bd&cauthor_id=29474007 https://pubmed.ncbi.nlm.nih.gov/?term=mayes+tl&cauthor_id=29474007 https://pubmed.ncbi.nlm.nih.gov/?term=chahal+z&cauthor_id=29474007 https://pubmed.ncbi.nlm.nih.gov/?term=nakonezny+pa&cauthor_id=29474007 https://pubmed.ncbi.nlm.nih.gov/?term=moorehead+a&cauthor_id=29474007 https://pubmed.ncbi.nlm.nih.gov/?term=emslie+gj&cauthor_id=29474007 https://pubmed.ncbi.nlm.nih.gov/?term=sam+sp&cauthor_id=30783310 https://pubmed.ncbi.nlm.nih.gov/?term=nisha+a&cauthor_id=30783310 https://pubmed.ncbi.nlm.nih.gov/?term=varghese+pj&cauthor_id=30783310 https://pubmed.ncbi.nlm.nih.gov/?term=mi+wf&cauthor_id=33061925 https://pubmed.ncbi.nlm.nih.gov/?term=chen+xm&cauthor_id=33061925 https://pubmed.ncbi.nlm.nih.gov/?term=fan+tt&cauthor_id=33061925 https://pubmed.ncbi.nlm.nih.gov/31477459/ https://pubmed.ncbi.nlm.nih.gov/31477459/ https://pubmed.ncbi.nlm.nih.gov/?term=echebur%c3%baa+e&cauthor_id=28077222 https://pubmed.ncbi.nlm.nih.gov/?term=g%c3%b3mez+m&cauthor_id=28077222 https://pubmed.ncbi.nlm.nih.gov/?term=freixa+m&cauthor_id=28077222 https://pubmed.ncbi.nlm.nih.gov/?term=rasmussen+jd&cauthor_id=31539373 https://pubmed.ncbi.nlm.nih.gov/?term=kakuhikire+b&cauthor_id=31539373 https://pubmed.ncbi.nlm.nih.gov/?term=baguma+c&cauthor_id=31539373 https://pubmed.ncbi.nlm.nih.gov/?term=ashaba+s&cauthor_id=31539373 https://pubmed.ncbi.nlm.nih.gov/?term=cooper-vince+ce&cauthor_id=31539373 https://pubmed.ncbi.nlm.nih.gov/?term=perkins+jm&cauthor_id=31539373 https://pubmed.ncbi.nlm.nih.gov/?term=bangsberg+dr&cauthor_id=31539373 https://pubmed.ncbi.nlm.nih.gov/?term=tsai+ac&cauthor_id=31539373 https://pubmed.ncbi.nlm.nih.gov/?term=bockting+clh&cauthor_id=29625762 https://pubmed.ncbi.nlm.nih.gov/?term=klein+ns&cauthor_id=29625762 https://pubmed.ncbi.nlm.nih.gov/?term=elgersma+hj&cauthor_id=29625762 https://pubmed.ncbi.nlm.nih.gov/?term=van+rijsbergen+gd&cauthor_id=29625762 https://pubmed.ncbi.nlm.nih.gov/?term=slofstra+c&cauthor_id=29625762 https://pubmed.ncbi.nlm.nih.gov/?term=ormel+j&cauthor_id=29625762 https://pubmed.ncbi.nlm.nih.gov/?term=sfetcu+r&cauthor_id=28646857 https://pubmed.ncbi.nlm.nih.gov/?term=musat+s&cauthor_id=28646857 https://pubmed.ncbi.nlm.nih.gov/?term=haaramo+p&cauthor_id=28646857 https://pubmed.ncbi.nlm.nih.gov/?term=ciutan+m&cauthor_id=28646857 https://pubmed.ncbi.nlm.nih.gov/?term=scintee+g&cauthor_id=28646857 https://pubmed.ncbi.nlm.nih.gov/?term=vladescu+c&cauthor_id=28646857 https://pubmed.ncbi.nlm.nih.gov/?term=wahlbeck+k&cauthor_id=28646857 https://pubmed.ncbi.nlm.nih.gov/?term=katschnig+h&cauthor_id=28646857 227j contemp med sci | vol. 7, no. 4, july-august 2021: 227–234 original effect of persian manual therapy (fateh technique) on patients with mild and moderate carpal tunnel syndrome: a clinical trial hamed naeiji1, roshanak mokaberinejad1*, seyed ahmad raeissadat2, farshad nouri3, yousef fallah4, reza haj manouchehri5, alireza abbassian6, seyed mohammad riahi7 1department of traditional medicine, school of traditional medicine, shahid beheshti university of medical sciences, tehran, iran. 2physical medicine and rehabilitation department, clinical development center of shahid modarres hospital, shahid beheshti university of medical sciences, tehran, iran. 3physical medicine and rehabilitation department, school of medicine, akhtar hospiatl, shahid beheshti university of medical sciences, tehran, iran. 4department of orthopedic surgery, joint reconstruction research center, shariati hospital, tehran university of medical sciences, tehran, ir iran. 5legal medicine research center, legal medicine organization, tehran, iran. 6department of persian medicine, school of persian medicine, tehran university of medical sciences, tehran, iran. 7cardiovascular diseases research center, department of epidemiology and biostatistics, school of medicine, birjand university of medical sciences, birjand, iran. *correspondence to: roshanak mokaberinejad (e-mail: rmokaberi@gmail.com) (submitted: 23 march 2021 – revised version received: 02 april 2021 – accepted: 25 april 2021 – published online: 26 august 2021) issn 2413-0516 introduction carpal tunnel syndrome (cts) is a common, chronic, and debilitating disorder. this disease is the most common peripheral mononeuropathy that occurs mainly in adults of working age1 and is one of the most common causes of activity limitation in these people2 and often occurs in patients aged 30 to 60 years.3 it affects most women and increases with age (agnessa et al.).4 carpal tunnel release is one of the most common hand/ wrist procedures,5 with more than 400,000 carpal tunnel release surgeries performed each year, with a direct cost of more than $ 2 billion per year.6 the prevalence of this disease varies depending on the diagnostic criteria and is estimated from 4-5%7 to 7-19%.1 its prevalence in women is two to three times higher than men.3 the annual incidence of this disease has been 99 per 100 thousand.1 although carpal tunnel syndrome is mostly idiopathic, various diseases such as hypothyroidism, diabetes, rheumatoid arthritis, chronic renal failure, acromegaly, amyloidosis, hemophilia, gout, fractures, and trauma to the carpal tunnel, and obesity, pregnancy, and mitochondrial disease can accelerate its progress. carpal tunnel syndrome ultimately leads to surgery when progresses and in the lack of supportive therapies.8 therapies include surgical and non-surgical treatments. the non-surgical treatments used for this condition include a variety of options, including changing habits such as restricting the movement of the wrist joint and reducing strenuous occupational activity; using ergonomic devices, using splints or a variety of oral medications such as oral steroid medications as well as ultrasound therapy,9 injection of steroid medications,10 taking nonsteroidal anti-inflammatory drugs (nsaids), diuretics, vitamin b6, job change,11,12 topical injection of progesterone,13,14 botulinum toxin injection, use of magnets,15 iontophoresis, low-level laser,16 median nerve mobilization,17 scaphoid and hamate mobilization,18 static magnetic field (smf), bioptron,19 specific exercise,20 plateletrich plasma (prp)21 and ozone therapy22 are other suggested non-surgical treatments. hand therapy is one of the non-surgical conservative treatments for carpal tunnel syndrome. although there are various results of the effectiveness of manual therapy in the treatment of carpal tunnel syndrome, which varies from ineffective to useful, there is evidence to suggest that different methods of manual therapy of the wrist area are effective in the treatment of carpal tunnel syndrome and can help reduce pain and symptoms.18,23,24 in this study, we introduce an iranian manual therapy method called fateh hand method, which is one of the manual therapies of iranian folklore. about four decades ago, mohammad fateh invented manual therapy methods to manage lower and upper limb problems. this study introduces upper limb manual therapy of this method. later, his son ahmad abstract objectives various non-surgical treatments are used to treat carpal tunnel syndrome, including hand therapy. in this study, the effect of fateh iranian hand therapy on this disease has been investigated for the first time. methods in this controlled clinical trial, 58 female patients (78 hands) eligible for carpal tunnel syndrome were divided into two groups of intervention (splint, fateh hand therapy, and exercise) and the control group (splint only). each person in the intervention group received about 7 minutes of soft tissue manipulation for 6 sessions and performed two active exercises at home daily. symptom severity and functional capacity were assessed with the boston questionnaire, pain intensity, and electrodiagnostic findings at the beginning and tenth week, and patient satisfaction in the tenth week of the experiment. results data of 51 patients, all female (68 hands), were analyzed. the age distribution was the same in both groups. in the intervention group, we saw a significant improvement in symptom severity and functional capacity compared to the control group (p-value<0.05). in addition, pain changes in the intervention group were significantly more than in the control group (p-value<0.05). the values of electrodiagnostic variables at the beginning and end of the design were not significantly different between the two groups (p-value>0.05). comparison of changes in these values did not show a significant difference between the two groups (p-value>0.05). satisfaction in the intervention group was significantly higher (p-value<0.05). conclusion fateh method is effective in reducing the symptoms of mild to moderate carpal tunnel syndrome. keywords carpal tunnel syndrome, traditional medicine, complementary therapies, persian manual therapy, radpa http://www.ajtcam.ir/?_action=article&au=627920&_au=hamed++naeiji http://sjfm.ir/search.php?sid=1&slc_lang=en&auth=haj+manouchehri http://sjfm.ir/search.php?sid=1&slc_lang=en&auth=haj+manouchehri 228 j contemp med sci | vol. 7, no. 4, july-august 2021: 227–234 effect of persian manual therapy (fateh technique) on patients with mild and moderate carpal tunnel syndrome original h. naeiji et al. fateh modified these methods and combined them with two active exercises at home.25 the speed and ease of treatment, no need for routine massage preparations, easy learning and movements by patients, the limited number of treatment sessions, and very rare side effects made this manual therapy for the first time in the form of a clinical trial that we investigated. in this study, the therapeutic effects of this method along with the use of splints have been compared with a group that uses only splints (as a treatment method whose beneficial effects on cts have been confirmed in many studies). to evaluate the effects of this method, we used the analysis of its effects on the severity of symptoms and functional capacity of the hand, the amount of pain as well as electrodiagnostic parameters. materials and methods study design it was a multicenter clinical trial study with a parallel-group design. this study was performed from 2019 to 2020 in sina and shahid modarres hospitals in tehran, iran. 58 patients (78 hands) with cts who referred to neurology, orthopedics, physical medicine and rehabilitation or traditional medicine clinics were included in this study. participants were randomly divided into two groups. the intervention group was designated as “splint + hand therapy + exercise” and the control group was designated as “splint group”. although it was impossible to blind the participants due to dissimilarity of the interventions in the two groups, the process of preparing the nerve tape, filling out the questionnaire, and the data analyzer were blinded. ethical considerations this study was approved by the research ethics committee of the vice chancellor for research and technology of shahid beheshti university of medical sciences on november 4, 2018 (reference number: ir.sbmu.retech.rec.1397.667). all stages of the study were conducted in accordance with the helsinki declaration of human rights. the registration number of the clinical trial is irct20190429043421n1. all patients were informed about the study and its processes before entering the study, and while receiving informed written consent from all participants, they were told that if they did not wish, they could leave it at any stage of the study. confidentiality of patients’ identity and personal information has been observed. participants the participants were women aged 20 to 60 years who were included in the study after confirming their affliction with carpal tunnel syndrome by a physician. in terms of inclusion and exclusion criteria, 58 patients (78 hands) entered the study, which were divided into two groups of intervention (splint + manual therapy + exercise) and control group (splint). during the study, 4 people (6 hands) of the first group and 3 people (4 hands) of the second group were excluded from the study due to lack of complete referral for manual therapy or incomplete follow-up sessions, and finally, two groups with 24 people (34 hands) remained in the first group and 27 people (34 hands) in the second group and the study was performed on them (figure 1). clinical diagnosis criteria having at least two signs, or a sign and a symbol of the following: symptoms include nocturnal pain, paresthesia, numbness, and tingling in the hands, and symptoms include a positive phalanx or tunnel test. criteria for diagnosing electrodiagnosis by mild to moderate type: sensory peak latency longer than 3.6 milliseconds with normal motor onset latency (≤ 4.2 milliseconds), mild carpal tunnel syndrome, and sensory peak latency longer than 3.6 milliseconds with prolonged motor onset latency (4.3-6 milliseconds) is considered moderate carpal tunnel syndrome. study inclusion criteria women in the age range of 20 to 60 years and the clinical diagnosis of their disease are proposed by a specialist and confirmed by electrodiagnosis, mild to moderate type and at least 3 months have passed since the onset of their symptoms if there are no exclusion criteria, then by entering the informed consent form and accepting the conditions of participation in the research, they enter the project. study exclusion criteria a history of wrist trauma or surgery, a history of carpal tunnel syndrome treatment in the last three months, including physiotherapy, laser therapy, or any injections such as steroids, pregnancy at the time of enrollment, a history of diabetes based on the patient’s diagnosis, or based on clinical suspicion and tests during the study and thyroid disorder at the discretion of the patient, median nerve involvement in areas other than the carpal tunnel area that is confirmed by a clinical diagnosis and ncv, presence of cervical radiculopathy, history of collagen vascular diseases such as arthritis rheumatoid arthritis, scleroderma, amyloidosis, lupus, concomitant with other known tenosynovitis approved by the rheumatologist, advanced cancer or recent chemotherapy and radiotherapy, anticancer drugs, corticosteroids, immunosuppressive drugs and sedatives severe medical conditions such as kidney and heart failure, obesity or excessive weight loss (based on bmi), fig. 1 flow diagram of phases through clinical trial. figures figure 1flow diagram of phases through clinical trial 92 patients (124 hands) with cts were recruited informed consent 34 patients(46 hands) were excluded: 9 unwilling(12 hands) 4 recent therapeutic action(5 hands) 11 diabetes, hypothyroidism (17 hands) and 10 not fulfilling the inclusion criteria(12 hands) 58 patients (78 hands) eligible participants who agreed to participate were included b a s e l i n e a s s e s s m e n t ( v a s , m e d i a n n c s , f s s , s s s ) t h i r d a s s e s s m e n t ( v a s , m e d i a n n c s , f s s , s s s ) s e c o n d a s s e s s m e n t ( f s s , s s s ) 3 p a r t i c i p a n t s ( 4 h a n d s ) d i d n o t c o m p l e t e t h e s e s s i o n s 4 p a r t i c i p a n t s ( 6 h a n d s ) d i d n o t c o m p l e t e t h e s e s s i o n s f i n a l l y , d a t a o f 2 7 p a r t i c i p a n t s ( 3 4 h a n d s ) w e r e a n a l y z e d f i n a l l y , d a t a o f 2 4 p a r t i c i p a n t s ( 3 4 h a n d s ) w e r e a n a l y z e d randomization m a n u a l t h e r a p y + a c t i v e h o m e e x e r c i s e + s p l i n t g r o u p e ( n = 4 0 h a n d s ) s p l i n t g r o u p e ( n = 3 8 h a n d s ) s p l i n t o n l y w e e k l y m a n u a l t h e r a p y + d a i l y h o m e e x e r c i s e + s p l i n t 8 w e e k s i n t e r v e n t i o n 2 w e e k s f o l l o w u p http://www.ajtcam.ir/?_action=article&au=627920&_au=hamed++naeiji 229j contemp med sci | vol. 7, no. 4, july-august 2021: 227–234 h. naeiji et al. original effect of persian manual therapy (fateh technique) on patients with mild and moderate carpal tunnel syndrome cases outside the range of mild to moderate disease (mentioned in the inclusion criteria) with symptoms such as tendon atrophy and permanent paresthesia outside the range of mild disease to medium (mentioned in the inclusion criteria) with symptoms such as tendon atrophy and permanent paresthesia. assessment of patients boston questionnaire symptom severity and functional status were evaluated using the boston carpal tunnel syndrome questionnaire (bctq). the questionnaire consists of 2 separate sections: symptom severity scale (sss) with 11 questions about severity and frequency of symptoms such as night and daily numbness, causalgia and pain and functional status scale (fss) with 8 questions about some specific daily activities such as writing, close the dress button, holding the phone and ...; each question has 5 options and each option has a score of 1 to 5, in which 1 indicates the lack of symptoms and 5 indicates the most severe symptoms. patients with bilateral cts completed separate bctq for each hand. all patients completed bctq at first, the fourth, and the tenth weeks. the studies conducted indicate the reliability and validity of the translated version of this questionnaire in a valid persian in assessing the severity of symptoms and the function of carpal tunnel syndrome in iranian society.26,27 electrodiagnosis signs and symptoms of carpal tunnel syndrome were investigated in all patients and after the clinical approval of the diagnosis of carpal tunnel syndrome, to confirm the diagnosis, electrodiagnostic evaluation of the patients was performed by physical medicine and rehabilitation specialists or neurologists. nerve conduction studies were prescribed by the treating physician and performed in an electromyography laboratory by experienced personnel. pain evaluation the pain level in patients’ hands and fingers was questioned during the week before referral and evaluated on a numerical rating scale (vas). on this scale, “zero” indicates painlessness, and 10 indicates unbearable pain. in patients with bilateral cts, pain in each hand was calculated separately. pain levels were assessed at baseline and at the 10th weeks. evaluation of satisfaction the rate of patient satisfaction is reflected from treatment on a 5-degree scale of 1-5. on this scale 1 is considered as very dissatisfied and 5 as very satisfied. how to intervene after completing a standard questionnaire for symptom severity and functional status of the patient’s hand (bctq), patients were divided into two groups. splint was prescribed for participants in both groups. the splint was prescribed in a notary mode (0-5 degree of extension) for 8 weeks of complete nights and days in most hours of activity. manual therapy was also performed weekly (up to 4 weeks) and 2 sessions in weeks 6 and 8 (total 6 sessions) for patients in the intervention group and daily exercises was also taught to them to be performed at home 3 times per day and for 8 weeks. patients with more than 2 sessions absence were excluded from the study. to measure the severity of the symptoms of the disease, for each patient, in addition to the beginning of the plan, in week 4, the boston questionnaire was completed again. in the tenth week, in addition of completing the boston questionnaire, the amount of pain and treatment satisfaction and nerve strip was also investigated. manual therapy method the first part of the manual therapy includes shoulder fraction and pressure insertion at three points, and especially the middle point of the shoulder with high pressure, rotational and fast rhythm with about 2 minutes (figures 2). then, the deep petrissage in the arm front splint with the index finger, moving from the bottom to the top and fast and repeated with an approximate time of 20 seconds. high pressure, fast and rhythmic rotational friction of the scapula inner sides with the thumb (fig. 1), reaching the lower end of the scapula with a deep transverse petrissage movement from inside (medial) to outside (lateral at the base of the scapula with the thumb, which takes about 30 seconds). the main part of this manual therapy is done in its second part. at this stage, for manual treatment of the upper right limb, while the patient leans to the chair, the therapist should stand on the right side of the patient and hold the patient’s wrist with the left hand, while the angle between the patient’s forearm and arm is about 90 degrees. then the therapist raises the arm to the shoulder circumference and about 3-4 fingers below the armpit deep petrissage with the fingers of the right hand from outside to inside, with high pressure, fast rhythm, and repetitive movements will be performed. this step takes about 10-15 seconds (figures 3a and 3b). forearm petrissage is performed at four to five points with moderate pressure, fast rhythm and once at each point along the forearm for 10 seconds. after this step, friction is performed on the back of the hand in the area between the index finger and the thumb. friction is performed with relatively high pressure, medium rhythm and between one to three times and each time with a time of 10 seconds (figure 4). exercises at home the intervention group participants were asked to perform daily exercises at home. exercises were described to the participants, along with images shown exercises performance, tables were given to record daily exercises. in each stage, patients fig. 2 rotational friction of the inner sides of the scapula with the thumbs. http://www.ajtcam.ir/?_action=article&au=627920&_au=hamed++naeiji 230 j contemp med sci | vol. 7, no. 4, july-august 2021: 227–234 effect of persian manual therapy (fateh technique) on patients with mild and moderate carpal tunnel syndrome original h. naeiji et al. referring to manual therapy, training tables were examined to check the exercise rate. each exercise should be performed daily 3 times and each 10 time (for 6 weeks). in the first exercise, the patient bends his forearm from the elbow to 90 degrees and punches and holds the hand in front of the face, then with a horizontal movement outwards, places it around the shoulders and returns the hands to the first position. the movements are performed continuously and at a medium speed. the patient performs this movement 10 times (figures 5a and 5b). in the second exercise, the patient is asked to and bring the hands in front the body while ring hands, bring them up to the top of the head, and return it to its original position, performing this movement 10 times (figures 6a and 6b). statistical analysis in describing data according to the variable type, for quantitative values, the mean index and standard deviation were used, and we used the frequency and percentages for qualitative variables. shapiro-wilk test was used to evaluate the normality of the data. in order to examine single-variable relations in the two groups of intervention and control, nonparametric tests (wilcoxon & mann-whitney test) were used to compare the independent and dependent samples. chi-square test was used to compare qualitative variables in the two groups. a significant level in this study is 0.05. the analyzes were performed using ibm spss statistics v.25 software. results demographic data the data of 58 (78 hands) that were included in the study were analyzed. data from 7 people (10 hands) were excluded from the study in different stages because participants performed less than 80% of daily exercises or did not participate in the minimum designated manual therapy sessions. 4 (6 hands) of these cases were related to the intervention group and 3 (4 hands) were related to the control group. finally, 51 patients (68-handed) were analyzed in two groups, of which 34 hands (24 people) were related to the intervention group and 34 hands (27 people) were related to the control group (chart 1). it should be noted that in this study in a complete 6 session period, from 24 patients in the intervention group, 11 people in 4 sessions of manual therapy (2 sessions of absence), and 8 people in 5 sessions of manual therapy (1 session of absence) participated, and 5 people completed the 6-session course, participants were excluded from the study with more than 2 sessions of absence. all participants in the study were female. the mean age of participants in the two groups did not differ significantly (p-value> 0.05). in the intervention group, 10 were employed and 14 housewives and in the control group, 11 were employed and 16 housewives. in the study of dominant hand and involved hand in both groups, there was a significant relationship between involved hand and dominant hand (p-value <0.05) (table 1). table 1. comparison of two intervention and control groups in terms of attributes of participants at the beginning of the study p-valuetotalcontrol group intervention group 5127 (34 hands)24 (34 hands)women, n 0.721a49 ± 749 ± 6 age (mean ± sd), year employment 0.948a2111 (40.7%)10 (41.7%) occupy 3016 (59.3%)14 (58.3%)housewife symptomatic hand 0.486a199 (33.3%)10 (41.7%)right, n (%) 1511 (40.7%)4 (16.6%)left, n (%) 177 (25.9%)10 (41.7%)both, n (%) dominant hand 0.574a3718 (66.7%)19 (79.2%)right, n (%) 149 (33.3%)5 (20.8%)left, n (%) astudent t test. fig. 4 friction of the area between the index finger and thumb. fig. 3 (a, b) deep petrissage of the below armpit. fig. 5 (a, b) exercise at home 1. fig. 6 (a, b) exercise at home 2. http://www.ajtcam.ir/?_action=article&au=627920&_au=hamed++naeiji 231j contemp med sci | vol. 7, no. 4, july-august 2021: 227–234 h. naeiji et al. original effect of persian manual therapy (fateh technique) on patients with mild and moderate carpal tunnel syndrome symptom severity and functional status symptom severity and functional status were evaluated using the boston carpal tunnel syndrome questionnaire (bctq). comparison of mean values of symptom severity scale and functional status scale were significantly different between the two groups (p-value = 0.012). the intragroup comparison of the changes in these two items during the study period in the two groups indicated improved symptoms in both groups. furthermore, comparing the changes in these two items between the two groups showed more improvement in both symptom severity and functional status items in the intervention group, which was significant compared to the control group (p-value <0.005) (table 2). this means that the performed intervention had an additional effectiveness, in terms of decreasing the symptom severity and improving the functional status, compared to the control group (splint only). pain to compare pain variables, the first changes in pain score based on a visual scale (vas) in each group were investigated separately. the mean pain at the beginning of the study was higher in the intervention group than the control group, which after ten weeks, the pain intensity decreased in both groups, but this reduction was significantly greater in the manual therapy group (table 2). electrodiagnostic tests in this experiment, sensory peak latency and motor onset latency are measured as the basis of evaluation. accordingly, the baseline values of sensory peak latency and motor onset latency were compared between the two groups, which did not differ significantly (p-value> 0.05). in addition, the values of sensory peak latency and motor onset latency were also measured in the tenth week of entry of patients to the study, which did not differ significantly (p-value> 0.05), and this means that the effectiveness of manual therapy in the intervention group did not differ significantly with the control group (table 3). there was a significant difference between the two groups in terms of disease severity and number of mild and moderate cases at the beginning of treatment (p-value = 0.006), while after ten weeks from the beginning of the intervention, this difference was not significant (p-value = 0.086). satisfaction the patients were asked in the tenth week to determine the rate of satisfaction, which were 6.9 ± 1.3 and 7.5 ± 0.8 in the control and intervention groups, respectively. data analysis showed that there was a significant difference between the two groups (p-value = 0.046). discussion fateh manual therapy is one of the manual therapies used in traditional iranian medicine (radpa1). in the present study, the effect of fateh manual therapy intervention on the symptoms of carpal tunnel syndrome was investigated and showed that this treatment could be effective in improving symptom severity, functional status, and reduced pain of the patients who were investigated using vas and bctq and the electrodiagnostic parameters. therefore, the use of this treatment along with other common treatments, including the use of a splint, can bring better treatment results for the patient. in the fateh manual therapy, soft tissue manipulation to relax the muscle, improve circulation, stimulate tendons and nerves, and pain modification are used (sanei et al., 2020).28 in this technique, contrary to many manual therapies where the wrist is the location of manipulation (elliott and burkett, 2013; madenci et al., 2012),12,29 manipulation in other areas of the upper limbs, especially the under the armpit area, is somehow distinguished from the usual methods. this technique consists of two sections, especially in the second part, the main manual therapy of soft tissue is used. in traditional iranian medicine, this practice is called ghamz, which means severe pressure on the tissue using the fingers.30,31 in this type of treatment (ghamz), communication channels are introduced that show the movement of substances in the body. “ghamz therapy” facilitates the transmission of these substances and reduces the severity of diseases by absorbing substances, diverting harmful currents and improving blood circulation in the affected organ. so far, there is no study on this manual method and its therapeutic effects on carpal tunnel syndrome, and this study is the first research in this area. there are several points in this treatment that distinguishes it from other manual therapy practices, one of which is the performance speed, while the duration of this treatment is about 7 minutes, many 1rahkarha-ye darman-e dastiye parsi table 2. intragroup and intergroup comparison of average functional capacity scale, symptom severity scale, and pain in two groups of intervention and control baseline 10 weeks later †p-value changes (base-10) ††p-value †††p-value baseline ††††p-value 10 w. l. bctq-sss control 28.6 ± 8 18.3 ± 7 <0.001a 10.2 ± 5.1 0.003a 0.012a 0.658a intervention 34.6 ± 10 18.3 ± 6 <0.001a 16.2 ± 8 bctq-fss control 17.5 ± 6 12 ± 4 <0.001a 5.12 ± 4.3 0.027a 0.028a 0.120a intervention 22.8 ± 10 14.1 ± 5 <0.001a 9.7 ± 5.9 pain control 5.2 ± 2.1 2.3 ± 1.5 <0.001a 2.9 ± 1 0.006a 0.003a 0.728a intervention 6.8 ± 2.9 2.6 ± 2.1 <0.001a 4.2 ± 2 bctq, boston carpal tunnel questionnaire; sss, symptom severity scale; fss, functional status scale. †intragroup comparison of base values and tenth weeks, †† comparison of average changes between two groups, ††† intergroup basic values comparison, †††† intergroup comparison of tenth week values amann-whitney u test. http://www.ajtcam.ir/?_action=article&au=627920&_au=hamed++naeiji 232 j contemp med sci | vol. 7, no. 4, july-august 2021: 227–234 effect of persian manual therapy (fateh technique) on patients with mild and moderate carpal tunnel syndrome original h. naeiji et al. conventional massages mostly need more times.29,32,33 meanwhile, the number of massage sessions in accordance with the protocol is a weekly session, and a total of 6 sessions. the next point is the ease of doing so that it can be done on clothes and does not need special peripheral readiness and special readiness for the patient. the third point is related to manipulation location. in this technique, unlike many common massages as described in manual therapy the wrist that is considered as the main location involved in the physiopathology of this disease is not manipulated.12,23,29,32 for the study and in order to comply with ethics in research and not depriving patients from common and evaluated treatments, splint was used in both groups. for the first time in 1947, the splint was used in a study by roaf in carpal tunnel syndrome.12 in the present study, the fateh manual therapy was compared with the splint treatment that is fully characterized by validity and reliability to evaluate its efficiency and reliability. therefore, we compared the results of the intervention group (manual treatment with splint) with the control group that used splint merely. in previous studies, different manual interventions (based on soft tissue, carpal bone treatment, and median nerve and mobilization) showed signs reduction in patients with carpal tunnel syndrome,12,34,35 which in some survival studies of these signs were also evaluated and approved for months.24 in the study of linek & wolny, which was performed on 103 patients with mild and moderate cts symptom severity and functional status, pain significantly decreased,36 which is consistent with the findings of our study. we also saw similar results in another study of wolny et al.23 as well as madenci et al.12 the nerve conductivity velocity is not evaluated in all studies, and in most of them, statistical evaluation has not been performed due to the low number of recorded patients, and additionally, considering the difference in manual therapy and their prescribed time, definitive conclusions in the effect of these interventions have not been performed in neurophysiological registration.35 but in the wolny et al. study, conducted on the effectiveness of manual therapy in the treatment of carpal tunnel syndrome, we have seen a significant improvement in the sensory conduct velocity and motor conductive velocity in the manual therapy group and reduced motor latency in both groups.23 in the madenci et al. study, in the intervention group (massage associated with a splint) there was a significant decrease in mmdl2 levels after treatment compared to pre-treatment values, however, the difference between these values with the control group (splint) was not significant.12 in our study, changes in the values of sensory 2median nerve motor distal latency peak latency and motor onset latency between the two groups has not been significant as well. fateh manual therapy was safe, and no side effects were reported during the study. peripheral nerves during the passaging different regions of the body may be subjected to mechanical or chemical stimulation in different anatomical points. long compression or fixation of a nerve may lead to reduction of blood flow in the nerves.37 this leads to the secretion of proinflammatory substances (peptides associated with calcitonin gene and substance p) of the nerve. this neurogenic inflammation can disrupt the normal function of the nerves even without obvious neurological damage, can also cause the creation and expansion of chronic pain.38 researchers have shown that massage therapy is not only beneficial in the hands but also a significant reduction in pain scores in motor diseases, especially in fibromyalgia, waist pain, arthritis, and migraine.12 and at the same time, studies have shown that massage has a role in reducing muscle tone and increasing local sympathetic activity in reducing pain intensity.12 the main clinical effects of manual therapy include reduced pain, improve performance, and neurological adjustment aspects.39 the response to nerve movement is complex and multifactor physiological and psychological factors interfere with a complex method. systematic studies have shown that moving nerves with multi-state care can improve symptoms, reduce disability and improve the function of patients who suffer from the peripheral nerve entrapment. it has been suggested that reducing inflammation and neural fibrosis after massage therapy can be the main reason for improving functional status and reducing symptom severity in patients with carpal tunnel syndrome. in carpal tunnel syndrome, in groups that received neural mobilization, positive neurophysiological effects such as intraocular edema reduction, time reduction, and median nerve shrinkage delay are observed.40 there is increasing evidence that massage and joint manipulation adjust the mechanism of central pain. massage activates descending inhibitory pathways using oxytocin to produce analgesia. manipulation reduces central irritability in individuals, as with a decrease in temporary accumulation in the primary hyperalgesia region and the reduction of secondary hyperalgesia in people with chronic pain. therefore, manual therapy techniques activate central inhibitory mechanisms and reduce central irritability to develop analgesics in human and animal models.41 at the same time, the intervention of various types of manual therapy (for instance, massage therapy, physical table 3. comparison of electrodiagnostic findings in two groups of intervention and control baseline 10 weeks later †p-value control g. intervention g. control g. intervention g. baseline value 10 weeks later sensory peak latency 0.6 ± 4.1 0.6 ± 4.1 0.6 ± 3.9 0.6 ± 3.9 0.853a 0.772a motor onset latency 0.6 ± 4.1 0.7 ± 4.2 0.6 ± 4.0 0.6 ± 4.0 0.152a 0.555a severity mild 25 22 26 24 0.006b 0.086b moderate 9 12 8 10 amann-whitney u test. bchi-square test. †intergroup comparison of base values and tenth weeks. http://www.ajtcam.ir/?_action=article&au=627920&_au=hamed++naeiji 233j contemp med sci | vol. 7, no. 4, july-august 2021: 227–234 h. naeiji et al. original effect of persian manual therapy (fateh technique) on patients with mild and moderate carpal tunnel syndrome therapy, chiropractic) prevents performance loss, improves task performance, preventing behavioral changes, which reduces nerve inflammation, degradation of myelin and fibrosis extra nervous.37 in addition, passive traction may help reduce inflammation or intracranial pressure by stimulating the peripheral nerve as well as the associated vascular structures.42,43 these cases can justify a significant decrease in pain and improved symptom severity and functional status in the intervention group compared to the control group in this study. limitations • considering that so far there was not any study on this manual therapy method, therefore, in the present study, we compared it with a medical wristband and with only a group with simple treatment (splint only). for more detailed assessment, more experiments with stronger control groups (such as the use of massage simulation as a therapeutic method) as well as the use of the larger sample size are needed. • using different target scales to assess the severity of cts symptoms, including vas, bq-sss, bq-fss, and edx parameters were our main strength points to compensate for some restrictions. • in this study, despite the blinding of the electromyography process, filling the questionnaire, and the blinding of statistics analyzer, in order to receive manual therapy, there was no possibility of blinding patients. • although the base nerve and the tenth week electromyographies were performed by an expert for each patient, and despite the same edx protocol, the electromyography  of different patients was performed by two neurological and physical medicine specialists. suggestions • in traditional iranian medicine, a series of measures for treatment is used. using manual therapy with chamomile oil (twice a day), which has been studied in the past (setayesh et al., 2017),44 is among the measures that can lead to further improvement and more satisfaction in patients. • the re-study with the greater number of patients, the comparison of the intervention group (solely with manual therapy) with two control groups (a group with a splint and a group with a splint and a pseudo-massage), as well as completing the full course for all participants is recommended to achieve more accurate results. acknowledgment we thank all personnel of shahid modarres and sina hospitals who helped us to implement this study. we also thank all the patients who kindly participate in this study. declaration of interests none. funding this study was derived from the ph.d. dissertation of dr. hamed naeiji, school of traditional medicine, shahid beheshti university of medical sciences and the contract number for this study is 198.  references 1. lisa newington, e.c.h., 2015. carpal tunnel syndrome and work. best pract. res. clin. 29, 440–453. 2. isam atroshi anna jöud, ingemar f. petersson, martin englund, c.z., 2015. sickness absence from work among persons with new physiciandiagnosed carpal tunnel syndrome: a population-based matchedcohort study 10. 3. frederick m. azar james h. beaty, md, s. terry canale, md, m.d., 2017. campbell’s operative orthopaedics, thirteenth. ed. elsevier. 4. agnessa kozak tanja wirth, ulrike euler, claudia westermann and albert nienhaus, g.s., 2015. association between work-related biomechanical risk factors and the occurrence of carpal tunnel syndrome: an overview of systematic reviews and a meta-analysis of current research. bmc musculoskelet. disord. 16. https://doi.org/10.1186/s12891-015-0685-0 5. joseph ingram benjamin m. mauck, md,norfleet b. thompson, md, james h. calandruccio, md, m.d., 2018. cost, value, and patient satisfaction in carpal tunnel surgery. orthop. clin. north am. 49. https://doi.org/doi. org/10.1016/j.ocl.2018.06.005 6. tulipan, j.e., ilyas, a.m., 2020. carpal tunnel syndrome surgery: what you should know. plast reconstr surg glob open 8, e2692. https://doi. org/10.1097/gox.0000000000002692 7. michel chammas lauren marquardt burmann, renato matta ramos, francisco carlos dos santos neto, jefferson braga silva, j.b., 2014. carpal tunnel syndrome 49. 8. parviz yazdanpanah ali mousavizadeh, ali sahuli-tanha, h.m., 2015. incidence of recurrent and persistent carpal tunnel syndrome following open transverse carpal ligament release 17(1). 9. prof. lucapadua md carmen erra md, costanza pazzaglia md, ilaria paolasso md, claudia loreti bs, pietro caliandro md, lisa d hobson-webb md, d.c.m.d., 2016. carpal tunnel syndrome: clinical features, diagnosis, and management. lancet neurol. 15, 1273–1284. https://doi.org/https://doi. org/10.1016/s1474-4422(16)30231-9 10. karimzadeh, a., bagheri, s., raeissadat, s.a., bagheri, s., rayegani, s.m., rahimi-dehgolan, s., safdari, f., abrishamkarzadeh, h., shirzad, h., 2019. the comparison of the effectiveness between different doses of local methylprednisolone injection versus triamcinolone in carpal tunnel syndrome: a double-blind clinical trial. j pain res 12, 579–584. https://doi. org/10.2147/jpr.s190652 11. h. richard winn, m.d., 2017. neurological surgery, youmans. elsevier . 12. madenci, e., altindag, o., koca, i., yilmaz, m., gur, a., 2012. reliability and efficacy of the new massage technique on the treatment in the patients with carpal tunnel syndrome. rheumatol int 32, 3171–3179. https://doi. org/10.1007/s00296-011-2149-7 13. mohammad hassan bahrami seyed ahmad raeissadat, s.s., 2015. comparison between the effects of progesterone versus corticosteroid local injections in mild and moderate carpal tunnel syndrome: a randomized clinical trial 16. 14. raeissadat sa sedighipour l, vahdatpour b, s.s., 2017. randomized controlled trial of local progesterone vs corticosteroid injection for carpal tunnel syndrome. 15. gary m. franklin mpha, andrew s. friedman, md, m.d., 2015. work-related carpaltunnel syndrome, diagnosis and treatment guideline. phys. med. rehabil. clin. n. am. 26. https://doi.org/http://dx.doi.org/10.1016/j. pmr.2015.04.003 http://www.ajtcam.ir/?_action=article&au=627920&_au=hamed++naeiji 234 j contemp med sci | vol. 7, no. 4, july-august 2021: 227–234 effect of persian manual therapy (fateh technique) on patients with mild and moderate carpal tunnel syndrome original h. naeiji et al. 16. rayegani sm eliaspour d, raeissadat sa, shafi tabar samakoosh m, sedihgipour l, kargozar e, b.m.h., 2013. the effects of low intensity laser on clinical and electrophysiological parameters of carpal tunnel syndrome 4. 17. yi huey lim bsc sonya girdler phd, hoe c. lee phd., d.y.c.bs., 2017. median nerve mobilization techniques in the treatment of carpal tunnel syndrome: a systematic review. j. hand ther. 30. https://doi.org/http://dx.doi. org/10.1016/j.jht.2017.06.019 18. dinarvand, v., abdollahi, i., raeissadat, s.a., mohseni bandpei, m.a., babaee, m., talimkhani, a., 2017. the effect of scaphoid and hamate mobilization on treatment of patients with carpal tunnel syndrome. anesth pain med 7, e14621. https://doi.org/10.5812/aapm.14621 19. raeissadat sa rezaei s, sedighipour l, bahrami mh, eliaspour d, karimzadeh a, r.s.a., 2014. the effect of polarized polychromatic noncoherent light (bioptron) therapy on patients with carpal tunnel syndrome 5. 20. carlson, h., colbert, a., frydl, j., arnall, e., elliot, m., carlson, n., 2010. current options for nonsurgical management of carpal tunnel syndrome. int j clin rheumtol 5, 129–142. https://doi.org/10.2217/ijr.09.63 21. raeissadat, s.a., karimzadeh, a., hashemi, m., bagherzadeh, l., 2018. safety and efficacy of platelet-rich plasma in treatment of carpal tunnel syndrome; a randomized controlled trial. bmc musculoskelet disord 19, 49. https://doi. org/10.1186/s12891-018-1963-4 22. bahrami, m.h., raeissadat, s.a., nezamabadi, m., hojjati, f., rahimidehgolan, s., 2019. interesting effectiveness of ozone injection for carpal tunnel syndrome treatment: a randomized controlled trial. orthop res rev 11, 61–67. https://doi.org/10.2147/orr.s202780 23. wolny, t., saulicz, e., linek, p., shacklock, m., myśliwiec, a., 2017. efficacy of manual therapy including neurodynamic techniques for the treatment of carpal tunnel syndrome: a randomized controlled trial. j manip. physiol ther 40, 263–272. https://doi.org/10.1016/j.jmpt.2017.02.004 24. wolny, t., linek, p., 2019a. long-term patient observation after conservative treatment of carpal tunnel syndrome: a summary of two randomised controlled trials. peerj 7, e8012. https://doi.org/10.7717/peerj.8012 25. afrasiabian h shams ardakani mr, f.g.a., 2015. essentials of ghamz in iranian traditional medicine (mabany-e-ghamz dar tebb-e-sonnatiy-eiran). nameh hasti, tehran. 26. foroozanfar, z., ebrahimi, h., khanjani, n., 2015. validity and reliability of the persian boston questionnaire in diabetic patients with carpal tunnel syndrome. j. neyshabur univ. med. sci. 2, 50–56. 27. rezazadeh, a., bakhtiary, a.h., samaei, a., moghimi, j., 2014. validity and reliability of the persian boston questionnaire in iranian patients with carpal tunnel syndrome. koomesh j. 15, 138–145. 28. sanei, m., roozafzai, f., abousaidi, s.r., hamze, m., negarestani, a.m., mokaberinejad, r., 2020. persian manual therapy method for chronic low-back pain with lumbar radiculopathy; a randomized controlled trial. j bodyw mov ther 24, 123–130. https://doi.org/10.1016/j.jbmt.2020.02.015 29. elliott, r., burkett, b., 2013. massage therapy as an effective treatment for carpal tunnel syndrome. j bodyw mov ther 17, 332–338. https://doi. org/10.1016/j.jbmt.2012.12.003 30. jaladat, a.m., atarzadeh, f., homayouni, k., 2013. ghamz therapy in persian medicine and its comparison with reflexo zone therapy. j. islam. iran. tradit. med. 3, 395–406. 31. mohammadi-kenari, h., khoramizadeh, m., forogh, b., hashem-dabaghian, f., 2020. the effect of a persian medicine massage (dalk and ghamz) on neck pain and disability in nonspecific chronic neck pain. complement. med. res. https://doi.org/10.1159/000509051 32. fernández-de-las peñas, c., ortega-santiago, r., de la llave-rincón, a.i., martínez-perez, a., fahandezh-saddi díaz, h., martínez-martín, j., pareja, j.a., cuadrado-pérez, m.l., 2015. manual physical therapy versus surgery for carpal tunnel syndrome: a randomized parallel-group trial. j pain 16, 1087–1094. https://doi.org/10.1016/j.jpain.2015.07.012 33. moraska, a., chandler, c., edmiston-schaetzel, a., franklin, g., calenda, e.l., enebo, b., 2008. comparison of a targeted and general massage protocol on strength, function, and symptoms associated with carpal tunnel syndrome: a randomized pilot study. j altern complement med 14, 259–267. https:// doi.org/10.1089/acm.2007.0647 34. lewis, k.j., coppieters, m.w., ross, l., hughes, i., vicenzino, b., schmid, a.b., 2020. group education, night splinting and home exercises reduce conversion to surgery for carpal tunnel syndrome: a multicentre randomised trial. j physiother 66, 97–104. https://doi.org/10.1016/j. jphys.2020.03.007 35. maddali bongi, s., signorini, m., bassetti, m., del rosso, a., orlandi, m., de scisciolo, g., 2013. a manual therapy intervention improves symptoms in patients with carpal tunnel syndrome: a pilot study. rheumatol int 33, 1233–1241. https://doi.org/10.1007/s00296-012-2507-0 36. wolny, t., linek, p., 2019b. is manual therapy based on neurodynamic techniques effective in the treatment of carpal tunnel syndrome? a randomized controlled trial. clin rehabil 33, 408–417. https://doi. org/10.1177/0269215518805213 37. bove, g.m., delany, s.p., hobson, l., cruz, g.e., harris, m.y., amin, m., chapelle, s.l., barbe, m.f., 2019. manual therapy prevents onset of nociceptor activity, sensorimotor dysfunction, and neural fibrosis induced by a volitional repetitive task. pain 160, 632–644. https://doi.org/10.1097/j. pain.0000000000001443 38. matsuda, m., huh, y., ji, r.r., 2019. roles of inflammation, neurogenic inflammation, and neuroinflammation in pain. j anesth 33, 131–139. https://doi.org/10.1007/s00540-018-2579-4 39. salgado, a.s.i., stramosk, j., ludtke, d.d., kuci, a.c.c., salm, d.c., ceci, l.a., petronilho, f., florentino, d., danielski, l.g., gassenferth, a., souza, l.r., rezin, g.t., santos, a.r.s., mazzardo-martins, l., reed, w.r., martins, d.f., 2019. manual therapy reduces pain behavior and oxidative stress in a murine model of complex regional pain syndrome type i. brain sci 9. https://doi. org/10.3390/brainsci9080197 40. basson, a., olivier, b., ellis, r., coppieters, m., stewart, a., mudzi, w., 2017. the effectiveness of neural mobilization for neuromusculoskeletal conditions: a systematic review and meta-analysis. j orthop sport. phys ther 47, 593–615. https://doi.org/10.2519/jospt.2017.7117 41. chimenti, r.l., frey-law, l.a., sluka, k.a., 2018. a mechanism-based approach to physical therapist management of pain. phys ther 98, 302–314. https://doi.org/10.1093/ptj/pzy030 42. boudier-revéret, m., gilbert, k.k., allégue, d.r., moussadyk, m., brismée, j.m., sizer jr., p.s., feipel, v., dugailly, p.m., sobczak, s., 2017. effect of neurodynamic mobilization on fluid dispersion in median nerve at the level of the carpal tunnel: a cadaveric study. musculoskelet sci pr. 31, 45–51. https://doi.org/10.1016/j.msksp.2017.07.004 43. gilbert, k.k., smith, m.p., sobczak, s., james, c.r., sizer, p.s., brismée, j.m., 2015. effects of lower limb neurodynamic mobilization on intraneural fluid dispersion of the fourth lumbar nerve root: an unembalmed cadaveric investigation. j man manip ther 23, 239–245. https://doi.org/10.1179/2042 618615y.0000000009 44. setayesh, m., sadeghifar, a.r., nakhaee, n., kamalinejad, m., rezaeizadeh, h., 2017. a topical gel from flax seed oil compared with hand splint in carpal tunnel syndrome: a randomized clinical trial. j evid based complement. altern med 22, 462–467. https://doi. org/10.1177/2156587216677822 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. doi: https://doi.org/10.22317/jcms.v7i4.1022 http://www.ajtcam.ir/?_action=article&au=627920&_au=hamed++naeiji 329j contemp med sci | vol. 8, no. 5, september-october 2022: 329–336 research docking study, synthesis, characterization and preliminary cytotoxic evaluation of new 1,2,3,4-tetrahydroppyrimidine derivatives noor m. mohammed* department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. *correspondence to: noor m. mohammed (e-mail: non.myo.86@gmail.com) (submitted: 03 september 2022 – revised version received: 12 september 2022 – accepted: 27 september 2022 – published online: 26 october 2022) abstract objective: this study resolved that these anew synthesized analogs may be embodied as an exploitable foundation of new anticancer agents to competition breast cancer. methods: by means of the crystal structure of histone deacetylases (hdacs-8) with vorinostat (saha) as a co-crystalized ligand was gained from the protein data-bank (pdb code 4qa0) as a result of docking the compounds (v a,s, v b,s, v a,t and v b,t) give good docking scores compared to the standard. compounds (v a,s, v b,s, v a,t and v b,t) was synthesized by multistep procedures from the reaction of intermediate derivatives (iv a,b) and the thiosemicarbazide or semicarbazide. the chemical structures of the target compounds and their intermediates were confirmed by ft-ir and 1h nmr. results: the in-vitro cytotoxicity assay (mtt assay) demonstrated that compounds v a,t and v b,t showed good inhibition ratios in breast cancer cell line (mcf-7) and human colon adenocarcinoma (hrt-18) comparable with drug control vorinostat (saha). conclusion: from the docking study, it was concluded that c=s moiety were very successful to bind tightly to the zinc binding group of hdac enzyme by making numerous interaction modes. keywords: anticancer, mcf-7, hrt-18, semicarbazide, dihydropyrimidine, thiosemicarbazide, docking study issn 2413-0516 introduction epigenetic changes in cancer are common and associated with pathogenesis and molecular heterogeneity.1 carcinogenesis results from the interplay between the activation of oncogenes and the inactivation of tumor suppressor genes. one of the causes of the latter is switching off the gene in concern by epigenetic changes rather than by mutation of the dna sequence. these reversible epigenetic changes do not involve alteration of the nucleotide sequence of dna but, instead, are due to inappropriate dna methylation, chromatin remodeling, and changes in small noncoding rnas.2 epigenetic machineries that alter chromatin structure can be divided into four main classes: dna methylation, covalent histone modifications, non-covalent mechanisms such as incorporating histone variants, and nucleosome remodelling and non-coding rnas, including mirnas.3 these alterations work collected to control the operative of the genome by altering the local structural dynamics of chromatin, primarily controlling its convenience and compactness. the interplay of these modifications creates an ‘epigenetic landscape’ that regulates the way the mammalian genome manifests itself in different cell types, developmental stages, and disease states, including cancer.4 histone deacetylases (hdacs) have received significant attention in the research area of epigenetics. acetylation by hats transfers an acetyl group and neutralizes the positive charge of lysine residues in the histone tail resulting in loosening histone–dna interactions and allowing access of transcription factors to dna. the stimulatory effect of hats on gene expression is reversed by hdacs, which remove an acetyl group from the terminal amino group of a lysine residue, leaving a positive charge, that tightly interacts with the negative amount of dn, promotes chromatin condensation, and thereby repress transcription and induce gene silencing. thereby, hats are co-activators, and the hdacs are co-repressors.5 any imbalance between the activities of these two opposite enzyme families would disturb the normal histone acetylation homeostasis. overexpression of hdacs, i.e., hypoacetylation, is involved in cancer generation and cancer progress since tumor suppressor gene transcription is prevented due to the inactivated chromatin system. there would be alteration in proliferation, differentiation, and apoptosis fashion of normal cells becoming malignant. hypoacetylation is also involved in the loss of cell adhesion, migration, invasion, and angiogenesis, resulting in cancer start and progress.6 hdacs are overexpressed in several types of cancer cells compared to normal cells. for example, breast cancer cells show measurable levels of hdacs 1, 2, and 3, while normal breast cells do not show any.7,8 this finding could be of great clinical value for targeting these enzymes in breast cancer. besides, the specificity of hdacis against the other types of cancer cells is still of great importance; the importance of inhibition of elevated levels of these enzymes in cancer cells outweighs inhibition of low levels in normal cells. furthermore, the relative specificity of hdacis between cancer cells and normal cells may be attributed to the idea hypothesized that, in contrast to cancer cells, normal cells could withstand the inhibitory action of hdacis and compensate for the inhibited vital pathways since they have multiple, alternative epigenetic regulatory ways.9 histone deacetylase inhibitors (hdacis) have emerged as a novel session of anti-cancer agents that show essential parts in epigenetic rule of gene expression, making death, apoptosis, and cell cycle arrest in cancer cells. several hdacis with much more potent anticancer effects and diverse structures have been identified; they include natural or synthetic products.10 recently, many hdac inhibitors mailto:non.myo.86@gmail.com 330 j contemp med sci | vol. 8, no. 5, september-october 2022: 329–336 docking study, synthesis, characterization and preliminary cytotoxic evaluation of new 1,2,3,4-tetrahydroppyrimidine derivatives research n.m. mohammed have been clinically validated in cancer patients resulting in the approval of five hdacis, vorinostat, romidepsin, belinostat and panobinostat by the fda and chidamide by chinese fda for the treatment of cutaneous, peripheral t-cell lymphoma (ctcl, ptcl) multiple myeloma (mm) and acute myeliod leukemia (aml).11,12 saha, belinostat, and panobinostat are pan-hdacis since they targeting multiple hdac isoforms, while romidepsin is a selective one.13,14 several new hdacis are in different stages of clinical development for the treatment of haematological malignancies as well as solid tumours. hdacis have the potential to be used as monotherapies or in combination with other anticancer therapies.15 also, great efforts are exerted to discover novel hdacis for use as anti-cancer drugs alone or in combination and have isoform selectivity are continuing by researchers.16 the “classical” hdac-is act exclusively on hdac classes i, ii and iv by binding to the zinc-enriched catalytic domain of the hdacs. figure 1 show the main four classes of hdac inhibitors and the fda-approved ones. classical hdacis are subdivided according to the chemical moiety that binds to the zinc ion (except cyclic tetrapeptides which bind to the zinc ion with a thiol group). some examples in decreasing order of the typical zinc binding affinity:17 1. hydroxamic acids (orhydroxamates) the major group, such as vorinostat (saha), belinostat and panobinostat 2. cyclic tetrapeptides such as romidepsin or depsipeptides, 3. benzamides or o-aminoanilides, mocetinostat, chidamide and entinostat 4. the aliphatic acid such as phenylbutyrate and valproic acid. materials and methods materials 4-chlorobenzyldehyde, benzaldehyde was purchased from hyper chem china. other chemicals were purchased from sigma–aldrich. all chemicals are of analytical grade, and they were used as received without further purification. characterization of compounds (v a,s, v b,s, va,t and v b,t) melting points, fourier transform infrared spectroscopy: ftir, nmr: h-nmr spectra, were performed for compound characterization. molecular docking vorinostat (saha) was used to validate the docking process because the pdb obtained has a crystal structure of the protein and bound vorinostat (saha). histone deacetylase enzyme18,19 the molecular operating environment (moe) software version 2015.10 was used to conduct the docking studies (chemical computing group, montreal, canada). hdac-8 (pdb code 4qa0) x-ray crystal structures were retrieved from the protein data base (pdb). all water molecules were removed from pdb files, and hydrogen atoms were then added to the protein. the moedock algorithm was then used to dock the optimized shape of the chemical into the binding site. the london dg was selected as the initial scoring method and the rigid receptor was selected as the final scoring method. the best 5 poses of each ligand were retained and scored. finally, the geometry of docked complex was analyzed by the pose viewer utility in moe. chemical synthesis synthesis of dihydropyrimidine derivatives (i a,b) a mixture of aldehydes (10 mmol), ethyl acetoacetate (10 mmol), urea (15 mmol) and 10 ml of ethanol and (4 mmol) of ammonium chloride, was refluxed for 7-8 hours. then the reaction mixture was neutralized with 1n hcl. the precipitate was filtered and recrystallized from ethanol.20 title ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (i a) was obtained as a pale offwhite powder, mp. 204–207°c, yield 88%. the ft-ir for i a, 2978 cm–1 c-h asymmetric str. vibration of aliphatic ch2, 1697 cm–1 carbonyl str. vibration band of ester, 1643 cm–1 fig. 1 the main four classes of hdac inhibitors. 331j contemp med sci | vol. 8, no. 5, september-october 2022: 329–336 n.m. mohammed research docking study, synthesis, characterization and preliminary cytotoxic evaluation of new 1,2,3,4-tetrahydroppyrimidine derivatives carbonyl str. vibration band of amide, 1311 cm–1 c-n str. vibration of amide, 1215 cm–1 c-o str. vibration of ester. 1h nmr (400 mhz, dmso-d6) δ 1.123-1.152 (t, j = 5.0 hz, 3h), 4.159–4.202 (q, j = 4.1 hz, 3h), 2.3141 (s, j = 7.2 hz, 3h), 5.424–5.439 (d, j = 5.0 hz, 1h), 7.298–7.318 (m, 5h). and ethyl 4-(4-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate compound (i b) was obtained as a pale-yellow powder mp. 214–216°c, yield 82%. the ft-ir for i b, 2954 cm–1 c-h asymmetric str. vibration of aliphatic ch2 1701 cm–1 carbonyl str. vibration band of ester, 1647 cm–1 carbonyl str. vibration band of amide, 1323 cm–1 c-n str. vibration of amide, 1219 cm–1 c-o str. vibration of ester. 1h nmr (400 mhz, dmso-d6) δ 1.123–1.152 (t, j = 5.0 hz, 3h), 4.159–4.202 (q, j = 4.1 hz, 3h), 2.3121 (s, j = 7.2 hz, 3h), 5.411 (d, j = 5.0 hz, 1h), 7.292–7.306 (m, 2h). ester hydrolysis (ii a,b) a stirring mixture of compounds (i a, b) (10 mmol) and sodium hydroxide (15%, 10 ml) was refluxed for 12–15 hours at 90°c. after cooling, the solution was acidified with (1n) hydrochloric acid and the precipitate was filtered and recrystallized from ethanol.21 the title 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (ii a) was obtained as a paleyellow powder, mp. 221–223°c, yield 76%. the ft-ir for ii a, 3400–2800 cm–1 o-h vibration of carboxylic acid, 3028 cm–1c-h asymmetric str. vibration of an aromatic ring, 1693 cm–1 carbonyl str. vibration band of carboxylic acid. 1597 cm–1 carbonyl str. vibration band of amide, 1311 cm–1 c-n str. vibration of amide. 1h nmr (400 mhz, dmso-d6) δ 12.36 (s, 1h), 9.90 (d, 1h), 7.35–7.27 (m, 6h), 5.35 (d, j = 7.5 hz, 1h), 2.23 (s, 3h). and 4-(4-chlorophenyl)-6 methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid compound (ii b) was obtained as a white powder mp. 227–229°c, yield 72%. the ft-ir for ii b, 3400–2800 cm–1 o-h vibration of carboxylic acid, 3093 cm–1c-h asymmetric str. vibration of an aromatic ring, 1697 cm–1 carbonyl str. vibration band of carboxylic acid, 1674 cm–1 carbonyl str. vibration band of amide, 1284 cm–1 c-n str. vibration of amide, 817 cm–1 c-cl str. vibration. 1h nmr (400 mhz, dmso-d6) 12.36 (s, 1h), 9.90 (s, 1h), 7.39 (d, j = 8.3 hz, 2h), 7.33–7.28 (m, 3h), 5.34 (d, j = 7.7 hz, 1h), 2.23 (s, 3h). synthesis of 1,3,4thiadizole-2amino derivatives synthesis (iii a,b) a mixture of thiosemicarbazide (5 mmol), compounds (ii a,b) (5 mmol) and 70% sulfuric acid in ethanol. was cool and stirred overnight and then refluxed at a temperature of 80-90°c for 7 hours. the reaction mixture was cool and neutralized with concentrated ammonia checked by litmus paper to ph 4. the precipitate was filtered and recrystallized from ethanol.22 the title 5-(5-amino-1,3,4-thiadiazol-2-yl)-6-methyl-4phenyl-3,4-dihydropyrimidin-2(1h)-one compound (iii a) was obtained as a white powder, mp. 170–172°c, yield 78%. the ft-ir for iii a, 3348 cm–1n-h str. vibration of amide, 3286, 3202 cm–1n-h str. vibration of primary amine, 3066 cm–1c-h asymmetric str. vibration of aromatic ring, 1500 cm–1c=c str. vibration of aromatic ring, 1315 cm–1 c-n str. vibration of amide, 694 cm–1c-s-c str. vibration band. 1h nmr (400 mhz, dmso-d6) δ 10.02 (s, 1h), 8.14 (d, j = 7.3 hz, 1h), 7.45 (d, j = 8.5 hz, 1h), 7.38 (t, j = 7.6 hz, 2h), 7.31 (d, j = 7.5 hz, 1h), 7.02 (s, 2h), 5.67 (d, j = 6.9 hz, 1h), 2.19 (s, 3h). the title 5-(5-amino-1,3,4-thiadiazol-2-yl)-4-(4-chlorophenyl)-6-methyl-3,4-dihydropyrimidin-2(1h)-one compound (iii b) was obtained as a pale off white powder, mp. scheme 1. synthesis schematics of compound (v a,s – v b,t). reagents and conditions: (a) reflux 7-8 hr. abs. ethanol (b) hydrolysis by 15% naoh (c) thiosemicarbazide, 70% sulfuric acid in ethanol, ammonia (d) ethyl-7-bromoheptanoate, dry k2co3 in anhydrous acetone (e) thiosemicarbazide, semicarbazide, methanol, koh. 332 j contemp med sci | vol. 8, no. 5, september-october 2022: 329–336 docking study, synthesis, characterization and preliminary cytotoxic evaluation of new 1,2,3,4-tetrahydroppyrimidine derivatives research n.m. mohammed stirred at room temperature for 30 min. the solvent was removed under reduced pressure, diluted with a saturated ammonium chloride aqueous solution, and extracted with ethyl acetate. the organic layer was dried over sodium sulfate. the resulting solution was evaporated under reduced pressure.24 the title 2-(7-((5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl) amino) heptanoyl) hydrazine-1-carboxamide compound (v a,s) was obtained as a white powder mp. 269–271oc, yield 83%. the ft-ir for v a,s, 3421 cm–1n-h str. vibration of secondary amide, 3353,3267 cm–1n-h str. vibration of primary amide 2985 cm–1c-h asymmetric str. vibration of aliphatic ch3, 1693 cm–1 carbonyl str. vibration band of dihydropyrimidin ring, 1647 cm–1 carbonyl str. vibration band of amide (ch2)6, 1600 cm–1 carbonyl str. vibration band of primary amide, 1573 cm–1c=n str. vibration, 1531 cm–1c=c symmetric str. vibration of aromatic ring, 1454 cm–1c-h asymmetric and symmetric str. vibration of (ch2)6, 1288 cm–1c-n str. vibration of amide, 698 cm–1c-s-c str. vibration.1h nmr (400 mhz, dmso-d6) δ 9.97 (s, 1h), 9.62 (d, j = 6.3 hz, 1h), 8.95 (d, j = 6.3 hz, 1h), 8.14 (d, j = 7.4 hz, 1h), 7.50 (t, j = 4.7 hz, 1h), 7.44 (d, j = 6.2 hz, 2h), 7.38 (t, j = 7.6 hz, 2h), 7.31 (d, j = 7.4 hz, 1h), 6.44 (s, 2h), 5.68 (d, j = 8.2 hz, 1h), 3.46 (q, 2h), 2.24 – 2.17 (m, 5h), 1.67 – 1.51 (m, 4h), 1.40 – 1.27 (m, 4h). the title 2-(7-((5-(4-(4-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl) amino) heptanoyl) hydrazine-1-carboxamide compound (v b,s) was obtained as a pale yellow powder mp. 273–275oc, yield 87%. the ft-ir for v b,s, 3464 cm–1n-h str. vibration of secondary amide, 3066,3035 cm–1n-h1 str. vibration of primary amide 2985 cm–1c-h asymmetric str. vibration of aliphatic ch3, 1708 cm–1 carbonyl str. vibration band of dihydropyrimidin ring, 1670 cm–1 carbonyl str. vibration band of amide (ch2)6, 1604 cm–1 carbonyl str. vibration band of primary amide, 1577 cm–1c=n str. vibration, 1531 cm–1c=c symmetric str. vibration of aromatic ring, 1465 cm–1c-h asymmetric and symmetric str. vibration of (ch2)6, 1288 cm–1c-n str. vibration of amide, 825 cm–1 c-cl str. vibration, 698 cm–1c-s-c str. vibration. the 1h nmr (400 mhz, dmso-d6) δ 9.97 (s, 1h), 9.62 (d, j = 6.4 hz, 1h), 8.95 (d, j = 6.4 hz, 1h), 8.14 (d, j = 7.3 hz, 1h), 7.50 (t, j = 4.6 hz, 1h), 7.34 (d, j = 8.0 hz, 2h), 7.30 (d, j = 8.3 hz, 2h), 6.44 (s, 2h), 5.70 (d, j = 7.8 hz, 1h), 3.47 (q, 2h), 2.24 – 2.17 (m, 5h), 1.67–1.51 (m, 4h), 1.40–1.28 (m, 4h). the title 2-(7((5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl) amino) heptanoyl) hydrazine-1-carbothioamide compound (v a,t) was obtained as a white powder mp. 191-193oc, yield 89%. the ft-ir for v a,t, 3394 cm–1n-h str. vibration of secondary amide, 3240, 3151 cm–1n-h str. vibration of primary amide 2985 cm–1c-h asymmetric str. vibration of aliphatic ch3, 1697 cm–1 carbonyl str. vibration band of dihydropyrimidin ring, 1651 cm–1 carbonyl str. vibration band of amide (ch2)6, 1597 cm–1c=n str. vibration, 1527 cm–1c=c symmetric str. vibration of aromatic ring, 1454 cm–1c-h asymmetric and symmetric str. vibration of (ch2)6, 1288 cm–1c-n str. vibration of amide, 1261 cm–1 c=s str. vibration, 698 cm–1c-s-c str. vibration. 1h nmr (400 mhz, dmso-d6) δ10.05 (d, j = 5.4 hz, 1h), 9.97 (s, 1h), 9.08 (d, j = 5.1 hz, 1h), 8.14 (d, j = 7.3 hz, 1h), 7.50 (t, j = 4.6 hz, 1h), 7.44 (d, j = 5.8 hz, 2h), 7.38 (t, j = 7.7 hz, 2h), 7.31 (d, j = 7.4 hz, 1h), 7.28 (s, 2h), 5.67 (d, j = 7.7 hz, 174–176°c, yield 81%. the ft-ir for iii b, 33483352 cm–1n-h str. vibration of amide, 3201 cm–1nh str. vibration of primary amine, 3051 cm–1c-h asymmetric str. vibration of aromatic ring, 1566 cm–1c=c str. vibration of aromatic ring, 1311 cm–1 c-n str. vibration of amide, 813 cm–1 c-cl str. vibration, 702 cm–1c-s-c str. vibration band.1h nmr (400 mhz, dmsod6) δ 1h nmr (500 mhz, dmso-d6) δ 10.02 (s, 1h), 8.14 (d, j = 7.3 hz, 1h), 7.34 (d, j = 8.1 hz, 2h), 7.30 (d, j = 8.1 hz, 2h), 7.02 (s, 2h), 5.70 (d, j = 7.5 hz, 1h), 2.19 (s, 3h). amine alkylation (iv a,b) a mixture of compound (iii a,b) (7.9 mmol), ethyl-7-bromoheptanoate (7.9 mmol) and dry k2co3 (23.8 mmol) in anhydrous acetone (20 ml) was stirred under reflux. after 24 hours at 70°c. upon completion of the reaction (as indicated by tlc), the solvent was reduced. the resulted precipitate was filtered and washed with distilled water and recrystallized from ethanol.23 the title ethyl 7-((5-(6-methyl-2-oxo-4-phenyl-1,2,3,4tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl) amino) heptanoate compound (iv a) was obtained as a pale-yellow powder mp. 200–203°c, yield 88%. the ft-ir for iv a, 3309 cm–1n-h str. vibration of amide, 2939 cm–1c-h asymmetric str. vibration of aliphatic ch3, 1732 cm–1 carbonyl str. vibration band of ester, 1651 cm–1 carbonyl str. vibration band of amide, 1597 cm–1c=n str. vibration, 1535 cm–1c=c symmetric str. vibration of aromatic ring, 1454 cm–1c-h asymmetric and symmetric str. vibration of (ch2)6, 1377 cm –1c-h asymmetric and symmetric str. vibration of (ch2-ch3), 1311 cm–1c-n str. vibration of amide, 1203 cm–1 o-c str. vibration, 686 cm–1c-s-c str. vibration. 1h nmr (400 mhz, dmso-d6) δ 9.97 (s, 1h), 8.14 (d, j = 7.3 hz, 1h), 7.50 (t, j = 4.7 hz, 1h), 7.45 (d, j = 8.5 hz, 2h), 7.38 (t, j = 7.6 hz, 2h), 7.31 (d, j = 7.5 hz, 1h), 5.67 (d, j = 7.3 hz, 1h), 4.10 (q, j = 6.5 hz, 2h), 3.47 (q, 2h), 2.29 (t, j = 8.5 hz, 2h), 2.19 (s, 3h), 1.67 – 1.49 (m, 4h), 1.40–1.23 (m, 4h), 1.16 (t, j = 6.7 hz, 3h). and ethyl 7-((5-(4-(4-chlorophenyl)-6-methyl-2-oxo-1,2, 3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl) amino) heptanoate compound (iv b) was obtained as off-white powder mp. 204–206°c, yield 76%. the ft-ir for iv b, 3414 cm–1n-h str. vibration of amide, 2981 cm–1c-h asymmetric str. vibration of aliphatic ch3, 1732 cm–1 carbonyl str. vibration band of ester, 1678 cm–1 carbonyl str. vibration band of amide, 1604 cm–1c=n str. vibration, 1573 cm–11c=c symmetric str. vibration of aromatic ring, 1462 cm–1c-h asymmetric and symmetric str. vibration of (ch2)6, 1377 cm–1c-h asymmetric and symmetric str. vibration of (ch2-ch3), 1315 cm–1c-n str. vibration of amide, 1199 cm–1 o-c str. vibration, 813 cm–1 c-cl str. vibration, 698 cm–1c-s-c str. vibration.1h nmr (400 mhz, dmso-d6) δ 9.97 (s, 1h), 8.14 (d, j = 7.4 hz, 1h), 7.50 (t, j = 4.7 hz, 1h), 7.34 (d, j = 8.2 hz, 2h), 7.30 (d, j = 8.2 hz, 2h), 5.70 (d, j = 6.8 hz, 1h), 4.10 (q, j = 6.6 hz, 2h), 3.47 (q, 2h), 2.29 (t, j = 8.5 hz, 2h), 2.19 (s, 3h), 1.67–1.49 (m, 4h), 1.40–1.24 (m, 4h), 1.16 (t, j = 6.6 hz, 3h). synthesis of amid (v a,s – v b,t) to a solution of thiosemicarbazide or semicarbazide (34.8 mmol) in 10 ml methanol, koh (34.8 mmol) was added. the reaction mixture was stirred for 10 min at 40°c, and was then cooled to 0°c and filtered. compound (iv a,b) (320 mg, 1.1 mmol) was added to the filtrate, after which the reaction was 333j contemp med sci | vol. 8, no. 5, september-october 2022: 329–336 n.m. mohammed research docking study, synthesis, characterization and preliminary cytotoxic evaluation of new 1,2,3,4-tetrahydroppyrimidine derivatives 1h), 3.47 (q, 2h), 2.24–2.17 (m, 5h), 1.67–1.51 (m, 4h), 1.40– 1.27 (m, 4h). the title 2-(7-((5-(4-(4-chlorophenyl)-6 methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl) amino) heptanoyl) hydrazine-1-carbothioamide compound (v b,t) was obtained as a pale off white powder mp. 196-19oc, yield 82%. the ft-ir for v b,t, 3221 cm–1n-h str. vibration of secondary amide, 3066, 3035 cm–1n-h str. vibration of primary amide 2985 cm–1c-h asymmetric str. vibration of aliphatic ch3, 1689 cm–1 carbonyl str. vibration band of amide (ch2)6, 1600 cm–1c=n str. vibration, 1531 cm–1c=c symmetric str. vibration of aromatic ring, 1462 cm–1c-h asymmetric and symmetric str. vibration of (ch2)6, 1288 cm–1c-n str. vibration of amide, 1261 cm–1 c=s str. vibration, 8117 cm–1 c-cl str. vibration, 686 cm–1c-s-c str. vibration. 1h nmr (400 mhz, dmso-d6) δ10.05 (d, j = 5.4 hz, 1h), 9.97 (s, 1h), 9.08 (d, j = 5.1 hz, 1h), 8.14 (d, j = 7.3 hz, 1h), 7.50 (t, j = 4.6 hz, 1h), 7.34 (d, j = 8.0 hz, 2h), 7.32 – 7.26 (m, 4h), 5.70 (d, j = 8.9 hz, 1h), 3.47 (q, 2h), 2.24 – 2.17 (m, 5h), 1.67 – 1.51 (m, 4h), 1.40 – 1.28 (m, 4h). in vitro cytotoxicity assay 25,26 the in vitro cytotoxicity of compounds v a,s, v b,s, v a,t, and v b,t were evaluated by mtt assay on human breast cancer cells (mcf-7) and human colon adenocarcinoma (hrt-18). mtt was performed to determine the cytotoxic effect of the samples at various concentrations. the results were given as the mean of three independent experiments and the ic50 values were then calculated. statistical analysis the results of the experimental work were demonstrated as the standard error of the mean (sem) for triplicate data by using nonlinear regression analysis (prism pad 8.1). results molecular docking binding site of saha the binding site of hdac-8 is formed like a cleft which contains a deep pocket that mainly made up of phe152, met274, gly152, his142, his143, trp142, gly304, tyr306, asp187, asp267, asp101, his180 and phe208. the deep pocket prefers non hydrophobic moieties such as nh and c=s fragments. according to the result of docking as shown in table 1 and figures 2–6, v a,s, v b,s, v a,t, and v b,t give very good docking scores compared to the standard (saha) since synthetic compounds interacted with the most residues in the active site (predominantly hydrogen bond ). the v a,t appear to has more binding tendency with hdac-8 via zinc binding group (c=s) spacer and also thiadiazol ring (good fitting) (his142, his143 and zn401) while v a,s which bind through zinc binding group (c=o), the c=s group of the v b,t aid to more binding tendency (good fitting) (tyr306 and zn401) due to the effect of zinc binding group than c=o of v b,s. synthesis of compounds (i a,b – va,t, b,t) compound (i a,b) was synthesized by the reaction of aldehydes, ethyl acetoacetate, urea and ammonium chloride in the presence of ethanol. the ftir for both compounds were characterized by disappearance of c=o band of aldehyde in table 1. the results of interactions of the ligands with hdac-8 compound id docking score (kcal/mol) h-bond interaction coordinating bond length (with zinc) [å] saha –8.69 tyr306(2.98) 2.17 v a,s –9.21 gly151(3.19), asp178(3.56), tyr306(3.13) 1.99 v b,s –10.25 gly151(3.12), his143(3.07) 1.98 v a,t –8.25 gly151(2.98), his142(3.16), his143(3.19), tyr306(2.94) 2.18 v b,t –8.63 gly151(2.82), tyr306(3.20), his143(3.06) 2.17 fig. 4 demonstration of v a,s within the binding site of hdac-8 and the mode of interactions. fig. 2 demonstration of saha (vorinostat) within the binding site of hdac-8 and the mode of interactions. fig. 3 demonstration of v a,t within the binding site of hdac-8 and the mode of interactions. 334 j contemp med sci | vol. 8, no. 5, september-october 2022: 329–336 docking study, synthesis, characterization and preliminary cytotoxic evaluation of new 1,2,3,4-tetrahydroppyrimidine derivatives research n.m. mohammed the disappearance of the signal of proton oh near 12.3 ppm, and appearance of proton signal of nh2 at the region near 7.02 ppm. compound (iv a,b) was synthesized by the reaction of compound (iii a,b), ethyl-7-bromoheptanoate and dry k2co3 in anhydrous acetone. the ftir for both compounds were characterized by disappearance of an nh a primary amine band at 3352-3200 cm–1, and appearance of c=o band of ester in the region around 1732 cm–1, o-c band of ester at 1157-1203 cm–1, c-h band of (ch2)6 at 1462-1427 cm–1 and c-h band of ch2-ch3 at 1377-1373 cm–1. the 1h-nmr for compounds (iv a,b), ester analogs characterized by the disappearance of proton signal of nh2 at the region near 7.02 ppm, and apparent of nh proton as a signal at the region 7.4-7.5 ppm, several signal of ch2 of the side chain, also apparent of triplet signal due to the proton at the c of ch3 near 1.1 ppm. compound (v a,s, v b,s, v a,t and v b,t) was synthesized by the reaction thiosemicarbazide or semicarbazide, koh and compound (iv a,b) using saturated ammonium chloride aqueous solution, ethyl acetate and sodium sulfate. the ftir for compounds (v a,s and v b,s)) were characterized by disappearance of c=o band of ester in the region around 1732 cm–1 and the appearance of c=o amide band at 1604-1597 cm–1, an nh a primary amine band at 3309-3032 cm–1. the 1h-nmr for compounds (v a,s and v b,s), were characterized by disappearance of proton signal of nh2 at the region near 7.02 ppm, and apparent of nh proton as a signal at the region 7.4-7.5 ppm, several signal of ch2 of the side chain, also apparent of triplet signal due to the proton at the c of ch3 near 1.1 ppm. the ftir for compounds (v a,t and v b,t)) were characterized by disappearance of c=o band of ester in the region around 1732 cm–1 and the appearance of c=o amide band at 1604-1597 cm–1, an nh a primary amine band at 3309-3221 cm–1, c=s band at 1261, 1253 cm–1. the 1h-nmr for compounds (v a,t and v b,t), were characterized by disappearance of triplet signal due to the proton at the c of ch3 near 1.1 ppm, and apparent of proton signal of nh2 at the region near 6.4-7.2 ppm. in vitro cytotoxicity assay the anticancer activity of compound (v a,s, v b,s, v a,t, and v b,t) were examined in the dose-response curve generated by prism pad 8.1 using nonlinear regression analysis for compounds in mcf-7 cells and hrt-18 cells is shown below figures. the ic50 values were obtained to a range of concentrations of compounds from (100 – 1.56 µm) by mtt assay. see figures 7–11. a) cytotoxicity of compounds against human colon adenocarcinoma (hrt-18): ic50 of v a,t in human colon adenocarcinoma (hrt-18) = 69.48 µm ic50 of v b,t in human colon adenocarcinoma (hrt-18) = 71.81 µm b) cytotoxicity of compounds against breast cancer cell line (mcf7): ic50 of v a,t in breast cancer cell line (mcf-7) = 0.69 µm ic50 of v b,t in breast cancer cell line (mcf-7) = 0.75 µm discussion according to the above-mentioned results, the synthetic compounds (v a,s and v b,s) does not show any cytotoxicity against the two cell line while (v a,t, v b,t,) are useful for the fig. 5 demonstration of v b,t within the binding site of hdac-8 and the mode of interactions. fig. 6 demonstration of v b,s within the binding site of hdac-8 and the mode of interactions. the region around 1690 cm–1 the appearance of c=o amide bond at an area around 1743-1674 cm–1, and the appearance of c=o ester bond at an area around 1701-1597 cm–1. the 1h-nmr for compounds (i a,b), ester analogs were characterized appearance of singlet signal due to the proton at the c of ch3 near 2.3 ppm, and appearance of doublet signal due to the proton at c alpha to n of heterocyclic rings near 5.4 ppm and appearance of singlet signal due to the proton of nh heterocyclic rings near 9.9 ppm. compound (ii a,b) was synthesized by the reaction of ester compounds (i a,b) and sodium hydroxide (15%) at 90°c. the ftir for both compounds were characterized by the disappearance of c=o band of ester in the region around 1701-1697 cm–1, and appearance of broad o-h band of carboxylic acid in the area around 3400-2500 cm–1. the 1h-nmr for compounds (ii a,b), carboxylic acid analogs were characterized by the disappearance of triplet signal of proton of ch3, quartet of ch2 of ethyl ester near and the appearance of singlet signal of protons of oh near 12.3 ppm. compound (iii a,b) was synthesized by the reaction of thiosemicarbazide with carboxylic acid compounds (ii a,b) and 70% sulfuric acid in ethanol then neutralized with concentrated ammonia. the ftir for both compounds were characterized by disappearance o-h band of carboxylic acid in the area around 3444-3200 cm–1 and c=o band of ester in the region around 1701-1697 cm–1, and appearance of an nh a primary amine band at 3352-2966 cm–1, c=n band in the region around 1600-1577 cm–1 and c-s-c band at 725-690 cm–1 the 1h-nmr for compounds (iii a,b), 1,3,4-thiadiazol-2-amino derivatives were characterized by 335j contemp med sci | vol. 8, no. 5, september-october 2022: 329–336 n.m. mohammed research docking study, synthesis, characterization and preliminary cytotoxic evaluation of new 1,2,3,4-tetrahydroppyrimidine derivatives fig. 7 dose-response curves of ic50 for v a,t. hrt-18 cells were treated for 72hr. with 0.05, 0.15, 0.32, 0.75, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 µm dose ranges of v a,t. the dose response for v a,t was plotted over log transformed v a,t concentrations. ic50 values were determined using nonlinear regression analysis (prism pad 8.1). results represent for triplicate data. fig. 11 structure of saha. (2) a zinc binding group (zbg) or zinc binding domain (zbd), such as the hydroxamic acid, benzamide, carboxylic acid, amide or biguanide groups,28,29 which coordinates with of zn2+ ion in the active site outer surface. (3) a linker domain that is either saturated or unsaturated with linear or cyclic structure, connects the cap group to the zbd.30 these are best illustrated by the classical, fda-approved inhibitor suberoylanilide hydroxamic acid (saha). conclusion in the recent decade, there have been several drugs to treat breast and colon cancer. however, there is still an unmet need to develop different types of drugs to reduce systemic toxicity and improve therapeutic efficacy. in the present study, we synthesized three compounds (v a,t, v b,t). the chemical structures of the synthesized compounds were confirmed by ft-ir and 1h-nmr. mtt assay demonstrated in vitro cytotoxicity study against mcf-7 and hrt-18, for compounds (v a,t, and v b,t) show a good inhibition ratio to this cell line compared to saha. therefore, these newly synthesized analogs may be represented as an exploitable source of new anticancer agents fighting breast cancer. conflict of interest the authors have no conflicts of interest regarding this investigation. acknowledgments we thank the department of pharmaceutical chemistry/college of the pharmacy/university of baghdad for their official support in this study.  fig. 9 dose-response curves of ic50 for v a,t. mcf-7 cells were treated for 72hr. with 0.05, 0.15, 0.32, 0.75, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 µm dose ranges of v a,t. the dose response for v a,t was plotted over log transformed v a,t concentrations. ic50 values were determined using nonlinear regression analysis (prism pad 8.1). results represent for triplicate data. fig. 8 dose-response curves of ic50 for v b,t. hrt-18 cells were treated for 72hr. with 0.05, 0.15, 0.32, 0.75, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 µm dose ranges of v b,t. the dose response for v b,t was plotted over log transformed v b,t concentrations. ic50 values were determined using nonlinear regression analysis (prism pad 8.1). results represent for triplicate data. fig. 10 dose-response curves of ic50 for v b,t. mcf-7 cells were treated for 72hr. with 0.05, 0.15, 0.32, 0.75, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 µm dose ranges of v b,t. the dose response for v b,t was plotted over log transformed v b,t concentrations. ic50 values were determined using nonlinear regression analysis (prism pad 8.1). results represent for triplicate data. treatment of breast cancer and human colon adenocarcinoma, as they inhibit the hdac enzyme through the presence of common pharmacophores consisting of three distinct domains as shown below: (1) a surface recognition unit or cap group which usually a hydrophobic and aromatic group or may be heteroaromatic,27 which interacts with the rim of the binding pocket. the cap group could be linked to the aliphatic linker group through either hydrogen-bond accepting or donating groups such as ketoand amide-groups. 336 j contemp med sci | vol. 8, no. 5, september-october 2022: 329–336 docking study, synthesis, characterization and preliminary cytotoxic evaluation of new 1,2,3,4-tetrahydroppyrimidine derivatives research n.m. mohammed references 1. baretti m, azad ns. the role of epigenetic therapies in colorectal cancer. current problems in cancer. 2018;42(6):530-47. 2. baylin sb, jones pa. epigenetic determinants of cancer. cold spring harbor perspectives in biology. 2016;8(9):a019505. 3. bunkar n, pathak n, lohiya nk, mishra pk. epigenetics: a key paradigm in reproductive health. clinical and experimental reproductive medicine. 2016;43(2):59. 4. sharma s, kelly tk, jones pa. epigenetics in cancer. carcinogenesis. 2010;31(1):27-36. 5. manal m, manish k, sanal d, selvaraj a, devadasan v, chandrasekar m. novel hdac8 inhibitors: a multi-computational approach. sar and qsar in environmental research. 2017;28(9):707-33. 6. parbin s, kar s, shilpi a, sengupta d, deb m, rath sk, et al. histone deacetylases: a saga of perturbed acetylation homeostasis in cancer. journal of histochemistry & cytochemistry. 2014;62(1):11-33. 7. ruijter ajd, gennip ahv, caron hn, kemp s, kuilenburg abv. histone deacetylases (hdacs): characterization of the classical hdac family. biochemical journal. 2003;370(3):737-49. 8. zhou h, wang c, ye j, chen h, tao r. design, virtual screening, molecular docking and molecular dynamics studies of novel urushiol derivatives as potential hdac2 selective inhibitors. gene. 2017; 637:63-71. 9. eckschlager t, plch j, stiborova m, hrabeta j. histone deacetylase inhibitors as anticancer drugs. international journal of molecular sciences. 2017;18(7):1414. 10. zwick v, chatzivasileiou a-o, deschamps n, roussaki m, simões-pires ca, nurisso a, et al. aurones as histone deacetylase inhibitors: identification of key features. bioorganic & medicinal chemistry letters. 2014;24(23): 5497-501. 11. cappellacci l, perinelli dr, maggi f, grifantini m, petrelli r. recent progress in histone deacetylase inhibitors as anticancer agents. current medicinal chemistry. 2020;27(15):2449-93. 12. yoon s, kang g, eom gh. hdac inhibitors: therapeutic potential in fibrosisassociated human diseases. international journal of molecular sciences. 2019;20(6):1329. 13. yu c, he f, qu y, zhang q, lv j, zhang x, et al. structure optimization and preliminary bioactivity evaluation of n-hydroxybenzamide-based hdac inhibitors with y-shaped cap. bioorganic & medicinal chemistry. 2018;26(8):1859-68. 14. brindisi m, senger j, cavella c, grillo a, chemi g, gemma s, et al. novel spiroindoline hdac inhibitors: synthesis, molecular modelling and biological studies. european journal of medicinal chemistry. 2018; 157:127-38. 15. liu r, wang j, tang w, fang h. design and synthesis of a new generation of substituted purine hydroxamate analogs as histone deacetylase inhibitors. bioorganic & medicinal chemistry. 2016;24(7):1446-54. 16. manal m, chandrasekar m, priya jg, nanjan m. inhibitors of histone deacetylase as antitumor agents: a critical review. bioorganic chemistry. 2016; 67:18-42. 17. huang m, zhang j, yan c, li x, zhang j, ling r. small molecule hdac inhibitors: promising agents for breast cancer treatment. bioorganic chemistry. 2019; 91:103184. 18. ding j, liu j, zhang z, guo j, cheng m, wan y, et al. design, synthesis and biological evaluation of coumarin-based n-hydroxycinnamamide derivatives as novel histone deacetylase inhibitors with anticancer activities. bioorganic chemistry. 2020; 101:104023. 19. berman hm, westbrook j, feng z, gilliland g, bhat tn, weissig h, et al. the protein data bank. nucleic acids research. 2000;28(1):235-42. 20. saloutin v, burgart yv, kuzueva o, kappe c, chupakhin o. biginelli condensations of fluorinated 3-oxo esters and 1, 3-diketones. journal of fluorine chemistry. 2000;103(1):17-23. 21. k hameed k, hs hussain f. ultrasound-assisted synthesis of some new n-(substituted carboxylic acid-2-yl)-6-methyl-4-substituted phenyl-3, 4-dihydropyrimidine-2 (1h)-one carboxamides. eurasian journal of science and engineering. 2018;4(2). 22. barbosa g, de aguiar a. synthesis of 1, 3, 4-thiadiazole derivatives and microbiological activities: a review. revista virtual de química. 2019;11(3):806-48. 23. gonzález-martínez d, fernández-sáez n, cativiela c, campos jm, gotorfernández v. development of biotransamination reactions towards the 3, 4-dihydro-2 h-1, 5-benzoxathiepin-3-amine enantiomers. catalysts. 2018;8(10):470. 24. yang f, zhao n, song j, zhu k, jiang c-s, shan p, et al. design, synthesis and biological evaluation of novel coumarin-based hydroxamate derivatives as histone deacetylase (hdac) inhibitors with antitumor activities. molecules. 2019;24(14):2569. 25. deyrieux af, wilson vg. in vitro culture conditions to study keratinocyte differentiation using the hacat cell line. cytotechnology. 2007;54(2):77–83. 26. riss tl, moravec ra, niles al, duellman s, benink ha, worzella tj, et al. cell viability assays. assay guid man [internet]. 2016;1–25. available from: http://www.ncbi.nlm.nih.gov/pubmed/23805433. 27. sarah s. jabbar, mohammed h.mohammed. coumarin based-histone deacetylase hadc inhibitors. egyptian journal of chemistry. 2022;65(7)379-384. 28. duraid al-amily, mohammed h. mohammed. design, synthesis and cytotoxicity study of primary amides as histone deacetylase inhibitors. iraqi j pharm sci. 2019.vol.28(2) . 29. othman m. sagheer, mohammed h. mohammed, jaafar s. wadi, and zaid o. ibraheem. studying the cytotoxic activity of newly designed and synthesized hdac 2100346. 30. miller ta, witter dj, belvedere s. histone deacetylase inhibitors. journal of medicinal chemistry. 2003;46(24):5097-116. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i5.1281 171j contemp med sci | vol. 7, no. 3, may-june 2021: 171–175 original evaluation of sexual life problems among menopausal woman with post-menopausal bleeding at maternity hospitals in baghdad city alaa wahab raheem1 hawraa hussein ghafel1 1maternal and neonate nursing department, college of nursing, university of baghdad, baghdad, iraq. *correspondence to: hawraa hussein ghafel (e-mail: hawraah@conursing.uobaghdad.edu.iq) (submitted: 08 april 2021 – revised version received: 23 april 2021 – accepted: 04 may 2021 – published online: 26 june 2021) introduction human life is composed of different phases of lifethe climatic era is one of these life phases that is unique to the female gender and that influences the life cycle of wome the reproductive stage ends in this period, ovarian functions cease, and anew stage begins for women in which they have lost their childbirth capacity this period starts at the age of about 40 and lasts until the age of 65.1 sexuality can affect the quality of life by affecting a woman's emotional and psychological health. clinicians who take care of women therefore understand whether they might be susceptible to sexual dysfunction. sexual dysfunction is also associated with menopausal transition, aperiod marked by hormonal, physiological and social shifts. a decreasing and fluctuating gonadal steroid hormone is the physiological process through which themenopausal transition affects sexual health2 menopause is associated with changes that affect sexuality in physiological and psychological terms. during menopause, a drop in the levels of circulating estrogen is the primary biological transition. initially, estrogen deficiency accounts for modified bleeding and reduced vaginal lubrication3 following menopause, a woman’s body and sexual desire will shift due to the loss of estrogen and testosterone. women who are menopausal or postmenopausal can note that they are less easily aroused and are less sensitive to touching and stroking. this can lead to a decrease in sex interest. low estrogen levels may also result in a decrease in blood flow to the vaginal region. this can cause a drop in vaginal lubrication, leaving the vagina too dry for comfortable intercourse.4 ln the lives of menopausal women, sexuality is an important aspect. sexual dysfunction increases with age, despite the significance of sexual activity in menopausal females.5 while sex is central to female reproductive senescence, sexual activity and function decline with age. a substantial decrease from 74 to 56 percent in sexual activity. (p < 0.001) between early postmenopausal women and late postmenopausl women has been recorded in the women's safe ageing project (whap) cohort, an extension of the melbourne women's midlife health project,6 for many women, sexual interest persists, regardless of age and menopausal status. in the analysis of women's health across the nation (swan), 76% of middleaged women reported that sex was moderately or extremely significant to them.7 methodology this study was conducted at maternity hospitals in baghdad city to evaluate the sexual life problems among menopausal woman. this study was started in january 2021 to february, 2021, the data regarding women’s sexual life problems was achieved from the answering of women that attending maternity hospitals. adescriptive study non-probability (a purposive sample) the study consist of (200) women with post-menopausal bleeding which were selected according to inclusion criteria that are (women at menopausal age, women with post-menopausal bleeding). this questionnaire was composed of three parts, part one: consists of sections that are related socio-demographic characteristics include (age, educational level, women occupation, residency). part two include: history of the menstrual period. age at menarche (first menstrual period), regularity of the menstrual cycle, age when menopause and part three abstract objective to identify the sexual life problems among menopausal woman and to evaluate the levels of sexual life problems among menopausal woman. methods this study was conducted at maternity hospitals in baghdad city to evaluate of sexual life problems among menopausal woman. this study was started in january 2021 to february 2021, the data regarding sexual life problems was achieved through the answering of women that attending maternity hospitals, the study consist of (200) women with menopausal age which were selected according to inclusion criteria (women at menopausal age, women with post-menopausal bleeding). the data are analyzed through the use of descriptive statistical analysis and inferential statistics. results most of women are showing unproblematic sexual life and only (23.5%) are showing problematic sexual life presents the mean of scores for evaluation the items of sexual life among women at menopause age; the mean scores indicate unproblematic among most of the items except the items that are related to (suffering from reduced sexual activity; they had regular sexual relations; suffering from a lack of sexual desire; and they cannot give their husband’s marital rights) that are showing problematic. conclusion most of menopausal women are showing unproblematic sexual life and they have normal and joyful sexual life regardless they suffering from post-menopausal bleeding. keywords sexual desire disorders, menopause, postmenopause, women issn 2413-0516 172 j contemp med sci | vol. 7, no. 3, may-june 2021: 171–175 evaluation of sexual life problems among menopausal woman original a. w. raheem et al. questions about sexual life of women with post-menopausal bleeding and the times of doing sexual intercourse per week. reliability of questionnaire is determined through a pilot study and validity through panel (15) experts. descriptive statistical analysis and inferential statistical analysis. results analysis of the sample related to socio-demographic and reproductive history characteristics of menopausal women; and describes the level sexual life problems among them. this figure shows that most of women are showing unproblematic sexual life and only 23.5% are showing problematic sexual life. table 1. distribution of women according to their socio-demographic characteristics list characteristics f % 1 age (m±sd=53±4) 45 – 50 year 50 25 51 – 55 year 103 51.5 56 – 60 year 37 18.5 61 – 65 year 9 4.5 66 ≤ year 1 .5 total 200 100 2 occupation employee 33 16.5 housewife 145 72 retired 20 10 free profession 2 1 total 200 100 3 residency urban 83 41.5 rural 63 31.5 sub-urban 54 27 total 200 100 4 level of education doesn’t read & write 71 35.5 read & write 43 21.5 primary school 27 13.5 intermediate school 7 3.5 secondary school 18 9 institute/college + 34 17 total 200 100 5 current body mass index underweight 0 0 normal weight 0 0 overweight 7 3.5 obese 151 75.5 severe obese 37 18.5 morbid obese 5 2.5 total 200 100 6 previous body mass index underweight 0 0 normal 0 0 overweight 114 57 obese 84 42 sever obese 2 1 morbid obese 0 0 total 200 100 f: frequency, %: percentage, m: mean, sd: standard deviation this table shows that more than half of women are with age 51–44 year (51.5%) with mean age 53 ± 4 year. the occupational status for them refers that 72% of them are housewives and 16.5% are still employee while 10% are retired. the residency variable shows that 41.5% of them are resident at urban area and 31.5% are resident in rural area. regarding the level of education, the highest percentage among them refers to 35.5% that doesn’t read and write and 21.5% of them are read and write. the current body mass index for women are refers that 75.5% of them are obese while the previous body mass index refers that they was overweight (57%). the occupational status for them refers that 72% of them are housewives and 16.5% are still employee while 10% are retired. table 2. evaluation of sexual life problems among menopause women (n=200) list items responses f (%) m.s evaluation 1 reduced sexual activity never 12(6) 2.46 problematicsometimes 85(42.5) always 103(51.5) (continued) fig. (1) evaluation of sexual life problems among women at age of menopause (n=200) 173j contemp med sci | vol. 7, no. 3, may-june 2021: 171–175 a. w. raheem et al. original evaluation of sexual life problems among menopausal woman table 2. evaluation of sexual life problems among menopause women (n=200) list items responses f (%) m.s evaluation 2 regular sexual relations never 7(3.5) 2.73 problematicsometimes 40(20) always 153(76.5) 3 lack of sexual desire never 5(2.5) 2.39 problematicsometimes 112(56) always 83(41.5) 4 i feel embarrassed by my husband for not meeting his sexual demands never 50(25) 1.97 unproblematicsometimes 107(53.5) always 43(21.5) 5 bleeding negatively affected marital relationship never 56(28) 1.88 unproblematicsometimes 112(56) always 32(16) 6 relationship with husband is very weak due to bleeding never 55(27.5) 1.97 unproblematicsometimes 96(48) always 49(24.5) 7 woman cannot give husband marital rights never 30(15) 2.27 problematicsometimes 86(43) always 84(42) 8 relationship with husband has become weak and disjointed never 112(56) 1.53 unproblematicsometimes 70(35) always 18(9) 9 poor bonding and attention with husband never 75(37.5) 1.73 unproblematicsometimes 105(52.5) always 20(10) 10 talk with husband about sex never 11(5.5) 2.41 problematicsometimes 97(48.5) always 92(46) 11 pain when with sex never 15(7.5) 2.15 problematicsometimes 140(70) always 45(22.5) 12 you feel embarrassed to have sexual intercourse after the menopause never 39(19.5) 1.95 unproblematicsometimes 132(66) always 29(14.5) 13 husband does not care about woman like the past never 79(39.5) 1.71 unproblematicsometimes 101(50.5) always 20(10) 14 husband started wanting to marry another woman never 134(67) 1.38 unproblematicsometimes 57(28.5) always 9(4.5) 15 husband’s behavior and temperament changed with woman never 93(46.5) 1.58 unproblematicsometimes 99(49.5) always 8(4) (continued) 174 j contemp med sci | vol. 7, no. 3, may-june 2021: 171–175 evaluation of sexual life problems among menopausal woman original a. w. raheem et al. f: frequency, %: percentage, m.s: mean of score unproblematic = ≥ 2, problematic = 2 < this table presents the mean of scores for evaluation the items of sexual life among women at menopause age; the mean scores indicate unproblematic among most of the items except the items that are related to (reduced sexual activity; regular sexual relations; lack of sexual desire; and i cannot give my husband his marital rights) that are showing problematic discussion figure (1): evaluation of sexual life problems among women at age of menopause (n = 200) shows that most of women are showing unproblematic sexual life and only 23.5% are showing problematic sexual life. the study support by (hashemi et al., 2013) assess sexual attitudes and sexual function in iranian women entering menopause. women were on average (53–56) years old. seventy percent of them had some kind of sexual problem. the feeling of dyspareunia differed significantly (p = 0.03) between three types of sexuality attitudes. data on their attitudes, sexual desire, orgasm, and dyspareunia were compared and found to be substantially different. significant differences (p=0.03, 0.04, and 0.04 respectively).8 sexuality different from person to another. many factors can affect sexuality, including stress, illness, and age, and family, professional and social obligations. whatever the cause, and sometimes lead to feelings of isolation, frustration, rejection or discontent. table 1, shows that more than half of women are with age (51–44) year (51.5%) with mean age 53±4 year. supported by the study (al-turiahi1 et al., 2016) in his study this research included 140 postmenopausal women with paravaginal bleeding, whose ages ranged from 46 to 80 years old, with a mean age of 58.67.2 years.9 the older women age and the presence of certain diseases and hormonal problems, the greater the risk of sexual life problems among menopausal woman, occupational status for them refers that (72%) of them are housewives and (16.5%) are still employee while (10%)are retired. table 2. evaluation of sexual life problems among menopause women (n=200) list items responses f (%) m.s evaluation 16 husband’s interest in sex decreased a lot as i got older and bleeding never 82(41) 1.65 unproblematicsometimes 107(53.5) always 11(5.5) 17 husband treated woman cruelly and violently never 195(97.5) 1.03 unproblematicsometimes 4(2) always 1(0.5) 18 husband is trying to get away from the house never 163(81.5) 1.22 unproblematicsometimes 31(15.5) always 6(3) 19 the effect of bleeding on husband, both physically and psychologically never 153(76.5) 1.27 unproblematicsometimes 40(20) always 7(3.5) the support by (soheila nazarpour, 2020) in his study a variety of personal and social factors can affect quality of life (qol) after menopause. the aim of this study was to find out what factors are related to postmenopausal women’s quality of life.10 the old women are not connected to the health reality and not participate in other activities in the community, such as health care training in the menopause stage.the residency variable shows that (41.5%) of them are resident at urban area and (31.5%) are resident in rural area. in contrast to studies conducted by puri et al. in chandigarh (7.7%) and dasgupta and ray in west bengal (rural women – 20% and urban women – 29.9%), this study found a prevalence of pmb of 1.8 percent, which is poor. pmb affects two out of every 100 postmenopausal women in the research area in tamil nadu, india.11 women of menopause and who live in the urban area and not knowing how to face the stages of natural change in the menopause s may also face problems in sexual life regarding the level of education, the highest percentage among them refers to (35.5%) that doesn’t read and write and (21.5%) of them are read and write. the study disagree with (40.8 %) were primary school graduates(yücel & eroğlu, 2013).12 the level of education and maturity in thinking has a great impact on knowing the quality of life and how to deal with sexual problemsthe menopause stagethe current body mass index for women are refers that (75.5%) of them are obese while the previous body mass index refers that they was overweight (57%). this results support by (azhar mousa 2016) who found bmi: obesity was a clear factor nearly all of the cases were married (96.4%), and the majority of them were obese and overweight (97.3%), with 43 (30.7%) having a typical bmi.13 menopausal women, suffer from slowing of the process of building up, metabolism and movement, with the absence of healthy food and regular exercise lead to increase in weight and reduces sexual desire table 2, evaluation of sexual life problems among menopausal women (n=200) this table presents the mean of scores for evaluation the items of sexual life among women at 175j contemp med sci | vol. 7, no. 3, may-june 2021: 171–175 a. w. raheem et al. original evaluation of sexual life problems among menopausal woman references 1. yücel, ç., & eroğlu, k. (2013). sexual problems in postmenopausal women and coping methods. sexuality and disability, 31(3), 217-228. https:// www.researchgate.net/publication/257663261_sexual_problems_in_ postmenopausal_women_and_coping_methods 2. north american menopause society. (2012). the 2012 hormone therapy position statement of the north american menopause society. menopause (new york, ny), 19(3), 257. 3. bachmann, g. a., & leiblum, s. r. (2004). the impact of hormones on menopausal sexuality: a literature review. menopause, 11(1), 120-130. 4. reviewed by traci c. johnson, md on may 16, (2019). sex and menopause 5. thornton, k., chervenak, j., & neal-perry, g. (2015). menopause and sexuality. endocrinology and metabolism clinics, 44(3), 649-661. 6. lonnèe-hoffmann, r. a., dennerstein, l., lehert, p., & szoeke, c. (2014). sexual function in the late postmenopause: a decade of follow-up in a population-based cohort of australian women. the journal of sexual medicine, 11(8), 2029-2038. 7. cain, v. s., johannes, c. b., avis, n. e., mohr, b., schocken, m., skurnick, j., & ory, m. (2003). sexual functioning and practices in a multi-ethnic study of midlife women: baseline results from swan. journal of sex research, 40(3), 266-276. 8. hashemi, s., tehrani, f. r., simbar, m., abedini, m., bahreinian, h., & gholami, r. (2013). evaluation of sexual attitude and sexual function in menopausal age; a population based cross-sectional study. iranian journal of reproductive medicine, 11(8), 631. 9. hashemi, s., tehrani, f. r., simbar, m., abedini, m., bahreinian, h., & gholami, r. (2013). evaluation of sexual attitude and sexual function in menopausal age; a population based cross-sectional study. iranian journal of reproductive medicine, 11(8), 631. 10. nazarpour, s., simbar, m., ramezani tehrani, f., & alavi majd, h. (2020). factors associated with quality of life of postmenopausal women living in iran. bmc women’s health, 20, 1-9. 11. sindhuri, r., & dongre, a. r. (2018). postmenopausal bleeding among rural women in tamil nadu, india: mixed methods study. indian journal of community medicine: official publication of indian association of preventive & social medicine, 43(4), 288. 12. yücel, ç., & eroğlu, k. (2013). sexual problems in postmenopausal women and coping methods. sexuality and disability, 31(3), 217-228. 13. al-turiahi¹, a. m., el-dine, f. a., & herez, s. h. (2016). assessment of postmenopausal bleeding: a cohort case study. 14. al-turiahi¹, a. m., el-dine, f. a., & herez, s. h. (2016). assessment of postmenopausal bleeding: a cohort case study. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i3.1008 menopause age; the mean scores indicate unproblematic among most of the items except the items that are related to (reduced sexual activity; regular sexual relations; lack of sexual desire; woman cannot give husband his marital rights) that are showing problematic. this results support by (johnson, 2019) a woman’s body and sexual drive can change due to the loss of estrogen and testosterone. women who are menopausal or postmenopausal can note that they are less easily aroused and less responsive to touching and stroking. as a result, sex can become less appealing.14 conclusion most of menopausal women are showing unproblematic sexual life and they have normal and joyful sexual life regardless they suffering from post-menopausal bleeding 295j contemp med sci | vol. 7, no. 5, september–october 2021: 295–302 original prevalence of comorbidities and its impacts in hospitalized patients with covid-19 mohammad ali khaksar1*, elham zanganeh yousefabadi2, reza taleb zadeh1, homeira rashidi3, mahmood maniati4, nima bakhtiari5 1student research committee, ahvaz joundishapour university of medical sciences, ahvaz, iran. 2department of internal medicine, jundishapour university of medical sciences, ahvaz, iran. 3diabetes research center, jundishapour university of medical sciences, ahvaz, iran. 4department of general courses, school of medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran. 5pain research center, jundishapur university of medical sciences, ahvaz, iran. *correspondence to: mohammad ali khaksar (e-mail: khaksar.ma@ajums.ac.ir) (submitted: 10 july 2021 – revised version received: 02 august 2021 – accepted: 25 august 2021 – published online: 26 october 2021) abstract objectives: the purpose of this study is to evaluate the prevalence of comorbidities in hospitalized covid-19 patients and its effects on the severity of the disease. the coronavirus pandemic has been a challenging problem for health care systems since december 2019. methods: this was a retrospective, cross-sectional study analyzing data related to the epidemiological characteristics of covid-19 patients admitted to razi hospital in ahvaz, iran from november 2020 to february 2021. the data on patient demographic characteristics including age, gender, and underlying diseases were collected from patient records. patients whose data were unavailable or incomplete were excluded from the study. results: the mean age of all of the 730 patients studied was 56.30 ± 16.36 years, and 53.7% of them were men. nearly 40% of the patients reported more than one comorbidity, with diabetes mellitus being the most frequent one (37.5%) followed by hypertension (35.3%) and ischemic heart disease (24.9). in addition, 21.5% of the patients required intensive care unit admission. finally, 11.9% of the patients had respiratory distress and became intubated, and approximately 13.6% of the patients died. hyperlipidemia, liver failure, tuberculosis, and elevated inflammatory biomarkers are risk factors for icu admission and death. conclusion: we found that male gender, older age, hyperlipidemia, liver failure, tb, having more than one comorbidity, and elevated inflammatory biomarkers were significantly associated with the risk of severe covid-19 disease. keywords: comorbidities, covid-19, hospitalized patients issn 2413-0516 introduction in december, 2019, several cases of pneumonia of unknown origin were reported in wuhan, hubei province, china. the disease spread to china and other countries of the world rapidly. the cause of this viral infection was later found to be a virus from the coronavirus family which was detected by a sample of patients’ throat swabs. subsequently, it was named 2019ncov by who. as the infection swept alarmingly across the globe, who announced it as an epidemic and it came to be known as covid-19.1 comorbidities are one of the factors that contribute to the severity of covid-19 disease. throughout the world, researchers have been investigating risk factors that deteriorate the condition of covid-19 patients. various studies have shown that diabetes mellitus, hypertension, and cardiovascular diseases have been common comorbidities in covid-19 patients that are associated with poor prognosis of the disease and an increased chance of hospitalization and mortality.2-4 given the increasing prevalence of the disease, it is almost impossible to prevent its spread, and since there is no definitive treatment for this disease, it is recommended that health systems focus on preventive measures and factors contributing to the severity of the disease.5 according to previous cov epidemics including sars cov which throughout china in 2003 and mers cvv affecting the middle east in 20126,7 and due to the uncertain future of the covid-19 epidemic and possible future epidemics that there is no definitive treatment during the epidemic time and also due to limited medical facilities, health care systems must notice to factors such as comorbidities that affect disease severity and its mortality. the present study aims to evaluate the prevalence of comorbidities and their correlation with prognosis and mortality rate in covid-19 patients in the south of iran. materials and methods patients and data gathering this was a retrospective, cross-sectional study that was carried out at the razi hospital, ahvaz, iran, after receiving approval from the research ethics committee of ahvaz jundishapur university of medical sciences (ir.ajums.rec.1399.867). the present study evaluated 730 patients who had been infected with covid-19 and hospitalized in november 2020 to february 2021. to this aim, we evaluated and extracted data from medical records of patients who were diagnosed with covid-19 infection based on a positive rt-pcr test or typical radiological manifestations on a spiral chest ct scan. initially, the patients were investigated in terms of the severity of the disease (including patients with underlying diseases such as cardiovascular disease, hypertension, diabetes, underlying respiratory diseases, and bmi over 40 as well as immunocompromised patients such as those with a history of taking corticosteroids, chemotherapy, malignancies, transplantation, and hiv-positivity). the patients were divided into two groups, namely the severe group involving patients requiring intensive care unit, and the non-severe group 296 j contemp med sci | vol. 7, no. 5, september–october 2021: 295–302 prevalence of comorbidities and its impacts in hospitalized patients with covid-19 original m. ali khaksar et al. including patients who did not need intensive care unit and were admitted in the general ward. since no electronic medical record system was available at the time of hospitalization of the patients, patient data were recorded by physicians and nurses in patient paper medical records. data about the underlying disease was determined according to the patients’ self-declaration at the time of admission. covid-19 patients with unavailable or incomplete data were excluded from the study. to collect data on the patients’ underlying disease, the information about the underlying disease was first categorized (yes vs. no), and then the number of underlying diseases (single vs. multiple) was collected. during the hospitalization period, the patients were examined on a daily basis by specialist physicians, and patient’s information and vital signs were recorded manually and accurately by physicians and nurses in the patients’ medical record. the recorded data included admission at the general ward, admission at the icu, duration of admission at the icu, need for mechanical ventilation, discharge from the hospital, duration of hospitalization, and death. statistical analysis in this study, the data were analyzed using spss version 24 (ibm corp., armonk, ny, usa). continuous (normally distributed) variables were represented as means ± sds with 95% confidence intervals (ci), and percentiles, frequencies, and percentages were used for categorical variables. non-normally distributed variables were represented as medians with 25% and 75% values. mann-whitney u test was used for non normally distributed variables. the χ2 test, or fisher’s exact test, was used to compare categorical variables between groups, while spearman rho test, was used to compare continuous (non-normally) variables between groups. a p < 0.05 was considered statistically significant. results in the present study, we investigated 730 patients whose mean age was 56.30 ± 16.36, and 53.7% of them were male. onethird of the patients had no comorbidity while the rest had at least one comorbidity. the most common comorbidity was diabetes mellitus (37.5%) followed by hypertension (35.3%), ischemic heart disease (24.9%) and hyperlipidemia (11%). other clinical characteristics are presented in table 1. totally, out of the 730 patients, 157 (21.5%) were admitted to the intensive care unit (icu), and the rest were admitted to general wards (78.5%). according to our results, patients who had been admitted to icu were significantly older than those admitted to general wards (mean 26/17 ± 86/60 years vs. 89/15 ± 05/55 years, p = 001/0). more males than women were admitted to icu, but the difference was not significant (93(23.7%) versus 64 (18.9%) p = 0.125). in addition, we found that icu admission was significantly associated with hyperlipidemia (p : 0.014), liver failure (p : 0.026), and tuberculosis (p < 0.022). patients who had more comorbidities were more likely to be admitted to icu. also, patients who were admitted to icu had significantly higher inflammatory biomarkers like esr (p : 0.008), crp (p < 0.001), and ldh (p : < 0.001). the length of stay at icu was longer for patients who were older (p < 0.001) and had hyperlipidemia (p : 0.015), table 1. demographics and clinical characteristics of patient (n = 730) demographic data n (%) age (year) mean ± sd 56.30 ± 16.36 min max 21 – 104 sex male 392 (53.7) female 338 (46.3) history of comorbidities diabetes mellitus 274 (37.5) hyperlipidemia 80 (11) hypertension 258 (35.3) ischemic heart disease 182 (24.9) cerebrovascular accident 27 (3.7) rheumatoid arthritis 17 (2.3) hypothyroidism 24 (3.3) systemic lupus erythematosus 3 (.4) asthma 45 (6.2) liver failure 9 (1.2) kidney transplantation 12 (1.6) chronic kidney disease 65 (8.9) chronic obstructive pulmonary disease 6 (0.8) tuberculosis 6 (0.8) comorbidity 0 244 (33.4) 1 194 (26.6) >1 292 (40) laboratory investigations erythrocyte sedimentation rate mean ± sd 51.01 ± 31.61 min max 3 – 348 c-reactive protein mean ± sd 51.44 ± 32.16 min max 1 – 151 lactate dehydrogenase mean ± sd 608.93 ± 333.56 min max 85 – 3784 complications death 99 (13.6) outcome icu 157 (21.5) intubation 87 (11.9) duration of hospital stay (day) mean ± sd 7.11 ± 4.36 min max 1 – 38 duration of icu stay (day) mean ± sd 1.35 ± 3.54 min max 0 – 37 values are presented as numbers and percentages (%). 297j contemp med sci | vol. 7, no. 5, september–october 2021: 295–302 m. ali khaksar et al. original prevalence of comorbidities and its impacts in hospitalized patients with covid-19 liver failure (p : 0.015), and tuberculosis (p : 0.006) in their medical history as well as those having elevated inflammation biomarkers. other clinical characteristics are presented in tables 2, 3, 6. in this study, 99 patients (13.5%) died, and a higher mortality rate was observed among the elderly, males, patients with liver failure and tuberculosis, as well as those having more comorbidities and elevated esr, crp, and ldh levels. other clinical characteristics are presented in table 4. in our study, no association was found between the length of stay at hospital and any comorbidity. however, patients who had elevated inflammatory bio markers were hospitalized for a longer period (table 5). discussion in this retrospective descriptive-analytical study performed in the first surge of coronavirus pandemic in khuzestan province, iran, we examined the effect of comorbidities on the prognosis and mortality of 730 covid-19 patients admitted to razi hospital of ahvaz, iran. according to our results, more than half of the admitted patients were male (53.7%), and one third of the patients had no underlying disease. however, at least one underlying disease was reported for the remaining patients, with diabetes mellitus being the most common, followed by hypertension, ischemic heart disease, and hyperlipidemia, respectively. in addition, mortality rate and the rate of icu admission in this study were 13.6% and 21.5%, respectively. the covid-19 pandemic in iran has become one of the major health system challenges in recent decades. because older people are at a greater risk for complications of covid-19 owing to the effects of aging on the immune system, in our study, as in other studies, older people were more likely to develop the severe form of the disease as attested by their greater need for icu admission and their high mortality rate. also, previous studies on sars and mers have shown that old age is associated with higher mortality rates.8-11 the results of pijls et al. showed that the risk of developing covid-19 disease, the severity of the disease, the need for hospitalization in the intensive care unit (icu), and the mortality rate in men with covid-19 are higher.12 also, the results of another study conducted in iran showed that men were more likely to get covid-19 than women, while there was no significant difference in the severity of the disease between the sexes.11 in the present study, men had a higher risk of getting the disease in general and the severe form of the disease in particular, which could be due to the more pronounced presence of men in the community, taking care of economic issues, and lack of proper observance of health protocols such as wearing masks and social distancing. also, the high level of ace2 in men can justify this difference.13 in this study, the mortality rate was higher in men but the need for icu hospitalization in men was not significantly different from that of women. guan et al. reported that 25.1% of their patients had at least one underlying disease, and the most common comorbidities were hypertension (16.9%) and diabetes (8.2%), respectively.3 another study by liu et al. showed that 19.7% of patients with covid-19 had comorbidities, with diabetes (10.2%) being the most common, followed by hypertension (9.5%) and cardiovascular disease (7.3%).4 a similar study conducted in iran showed that the prevalence of comorbidities in patients with coronavirus was 48.8% and the prevalence of internal diseases and coronary artery disease were 29.3% and 14.6%, respectively.11 in the present study, two thirds of patients reported at least one underlying disease, with the most underlying disease being diabetes mellitus (37.5%) followed by hypertension (35.3%), ischemic heart disease (24.8%) and hyperlipidemia (11%). in the present study, 37.5% of the patients mentioned diabetes in their past medical history, which was higher than the rate reported in other studies and could be attributed to the high prevalence of diabetes in khuzestan province,14 the high risk of covid-19 among diabetic patients, high disease severity, and hospitalization,15 this result can also be justified by the tendency of physicians to hospitalize diabetic patients with covid-19 who have uncontrolled blood sugar due to insulin deficiency in iran and the strict protocols of the studied hospital in the first surge of covid-19 regarding the hospitalization of patients with underlying disease and those with mild severity. other possible causes are differences in thresholds for hospitalization. among other important comorbidities in this study, hypertension, ischemic heart disease, and hyperlipidemia are the notable, and their prevalence was high compared to similar studies.4,11 this can be explained by the high prevalence of obesity, hypertension and hyperlipidemia in khuzestan province and the ignorance of the many people living there of their diseases, which constitute the main risk factors for coronary artery disease in this region.16,17 again, this result can be attributed to the protocols of our medical center regarding hospitalization of patients. various studies have shown that diabetes, hypertension, cardiovascular disease, cerebrovascular accidents, chronic obstructive pulmonary disease, and chronic kidney disease can predispose patients to severe disease and various complications.18,19 two studies in china found that hypertension, cardiovascular disease, and cerebrovascular events were significantly associated with disease severity, while diabetes was not associated with disease severity.20,21 in the present study, cerebrovascular accidents and chronic kidney disease were correlated with the rate of intubation, which could be due to the high risk of respiratory failure in the underlying disease. in another study conducted in iran, patients with chronic kidney disease were reported to have a higher chance of developing a severe form of the disease compared with the general population,22 we also found no association between other underlying diseases and the severity of the disease, which could be due to our center’s protocols regarding the hospitalization of patients with mild underlying disease. many patients also use raas inhibitors, which can improve patients outcomes.23 another cause of this can be the immediate control of underlying diseases by doctors and medical staff in our center. however, in this study, the more the underlying disease of the patients, the higher the mortality rate. in the present study, hyperlipidemia, liver failure and tuberculosis had a significant relationship with high mortality and hospitalization in icu. however, in a systematic review and meta-analysis study by fang et al., no association disease was found between the mentioned factors and disease severity,19 in another study by chen et al. tuberculosis and chronic liver disease had no association with disease severity.24 one more study in china found that hyperlipidemia was not associated with disease severity and mortality.25 the relationship between liver disease and disease severity can be due to 298 j contemp med sci | vol. 7, no. 5, september–october 2021: 295–302 prevalence of comorbidities and its impacts in hospitalized patients with covid-19 original m. ali khaksar et al. table 2. demographics and clinical characteristics of patients regarding non-intensive care unit (icu) and icu admission demographic data non-icu n = 416 icu n = 352 p-value age (year) (mean ± sd) 55.05 ± 15.89 60.86 ± 17.26 <0.001* sex male 299 (52.2) 93 (59.2) 0.125 female 274 (47.8) 64 (40.8) history of comorbidities diabetes mellitus: yes 209 (76.3) 65 (23.7) 0.265 no 364 (79.8) 92 (20.2) hyperlipidemia: yes 54 (67.5) 26 (32.5) 0.014* no 519 (79.8) 131 (20.2) hypertension: yes 194 (75.2) 64 (24.8) 0.11 no 379 (80.3) 93 (19.7) ischemic heart disease: yes 134 (73.6) 48 (26.4) 0.076 no 439 (80.1) 109 (19.9) cerebrovascular accident: yes 17 (63) 10 (37) 0.056 no 556 (79.1) 147 (20.9) rheumatoid arthritis: yes 14 (82.4) 3 (17.6) >0.99 no 559 (78.4) 154 (21.6) hypothyroidism: yes 22 (91.7) 2 (8.3) 0.133 no 551 (78) 155 (22) systemic lupus erythematosus: yes 3 (100) 0 (0) >0.99 no 570 (78.4) 157 (21.6) asthma: yes 38 (84.4) 7 (15.6) 0.453 no 535 (78.1) 150 (21.9) liver failure: yes 4 (44.4) 5 (55.6) 0.026* no 569 (78.9) 152 (21.1) kidney transplantation: yes 11 (91.7) 1 (8.3) 0.478 no 562 (78.3) 156 (2.7) chronic kidney disease: yes 48 (73.8) 17 (26.2) 0.344 no 525 (78.9) 140 (21.1) chronic obstructive pulmonary disease: yes 4 (66.7) 2 (33.3) 0.614 no 569 (78.6) 155 (21.4) tuberculosis: yes 2 (33.3) 4 (66.7) 0.022* no 571 (78.9) 153 (21.1) comorbidity 0 201 (35.1) 43 (27.4) 1 154 (26.9) 40 (25.5) 0.091 >1 218 (38) 74 (47.1) laboratory investigations erythrocyte sedimentation rate (mean ± sd) 49.37 ± 31.02 57 ± 33.09 0.008* c-reactive protein (mean ± sd) 47.83 ± 31.10 64.59 ± 32.65 <0.001* lactate dehydrogenase (mean ± sd) 553.8 ± 259.4 809.7 ± 470.4 <0.001* values are presented as numbers and percentages (%). *p-value <0.05 is significant. 299j contemp med sci | vol. 7, no. 5, september–october 2021: 295–302 m. ali khaksar et al. original prevalence of comorbidities and its impacts in hospitalized patients with covid-19 table 3. demographics and clinical characteristics of intubation covid-19 patients compared to non-intubation patients demographic data non-intubation n = 416 intubation n = 352 p-value age (year) (mean ± sd) 55.21 ± 16.05 64.38 ± 16.45 <0.001* sex male 336 (52.3) 56 (64.4) 0.039* female 307 (47.7) 31 (35.6) history of comorbidities diabetes mellitus: yes 236 (86.1) 38 (13.9) 0.238 no 407 (89.3) 49 (10.7) hyperlipidemia: yes 67 (83.8) 13 (16.3) 0.203 no 576 (88.6) 74 (11.4) hypertension: yes 220 (85.3) 38 (14.7) 0.094 no 423 (89.6) 49 (10.4) ischemic heart disease: yes 154 (84.6) 28 (15.4) 0.065 no 489 (89.2) 59 (10.8) cerebrovascular accident: yes 20 (74.1) 7 (25.9) 0.032* no 623 (88.6) 80 (1.4) rheumatoid arthritis: yes 15 (88.2) 2 (11.8) >0.99 no 628 (88.1) 85 (11.9) hypothyroidism: yes 23 (95.8) 1 (4.2) 0.34 no 620 (87.8) 86 (12.2) systemic lupus erythematosus: yes 3 (100) 0 (0) >0.99 no 640 (88) 87 (12) asthma: yes 42 (93.3) 3 (6.7) 0.346 no 601 (87.7) 84 (12.3) liver failure: yes 6 (66.7) 3 (33.3) 0.081 no 637 (88.3) 84 (11.7) kidney transplantation: yes 12 (100) 0 (0) 0.378 no 631 (87.9) 87 (12.1) chronic kidney disease: yes 52 (80) 13 (20) 0.044* no 591 (88.9) 74 (11.1) chronic obstructive pulmonary disease: yes 5 (83.3) 1 (16.7) 0.534 no 638 (88.1) 86 (1.9) tuberculosis: yes 2 (33.3) 4 (66.4) 0.002* no 641 (88.5) 83 (11.5) comorbidity 0 221 (34.4) 23 (26.4) 0.060 1 175 (27.2) 19 (21.8) >1 247 (38.4) 45 (51.7) laboratory investigations erythrocyte sedimentation rate (mean ± sd) 49.60 ± 40 61.41 ± 34.21 0.001* c-reactive protein (mean ± sd) 49.01 ± 31.53 69.41 ± 31.24 <0.001* lactate dehydrogenase (mean ± sd) 567.2 ± 262.8 920.9 ± 565.4 <0.001* values are presented as numbers and percentages (%). *p-value <0.05 is significant. 300 j contemp med sci | vol. 7, no. 5, september–october 2021: 295–302 prevalence of comorbidities and its impacts in hospitalized patients with covid-19 original m. ali khaksar et al. table 4. demographics and clinical characteristics of survived covid-19 patients compared to expire patients demographic data live n = 631 death n = 99 p-value age (year)(mean ± sd) 54.87 ± 16.04 65.45 ± 15.43 <0.001* sex male 328 (52) 64 (64.6) 0.023* female 303 (48) 35 (35.4) history of comorbidities diabetes mellitus: yes 232 (31.8) 42 (15.3) 0.315 no 399 (87.5) 57 (12.5) hyperlipidemia: yes 65 (81.3) 15 (18.8) 0.165 no 566 (87.1) 84 (12.9) hypertension: yes 214 (82.9) 44 (17.1) 0.054 no 417 (88.3) 55 (11.7) ischemic heart disease: yes 150 (82.4) 32 (17.6) 0.080 no 481 (87.8) 67 (12.2) cerebrovascular accident: yes 21 (77.8) 6 (22.2) 0.244 no 610 (86.8) 93 (13.2) rheumatoid arthritis: yes 15 (88.2) 2 (11.8) <0.99 no 616 (86.4) 97 (13.6) hypothyroidism: yes 21 (87.5) 3 (12.5) <0.99 no 610 (86.4) 96 (13.6) systemic lupus erythematosus: yes 3 (100) 0 (0) <.099 no 628 (86.4) 99 (13.6) asthma: yes 41 (91.1) 4 (8.9) 0.499 no 590 (86.1) 95 (13.9) liver failure: yes 4 (44.4) 5 (55.6) 0.003* no 627 (87) 94 (13) kidney transplantation: yes 12 (100) 0 (0) 0.386 no 619 (86.2) 99(13.8) chronic kidney disease: yes 52 (80) 13 (20) 0.127 no 579 (87.1) 86 (12.9) chronic obstructive pulmonary disease: yes 5 (83.3) 1 (16.7) 0.584 no 626 (86.5) 98 (13.5) tuberculosis: yes 2 (33.3) 4 (0.5) 0.004* no 629 (86.9) 95 (13.1) comorbidity 0 220 (34.9) 24 (24.2) 0.048* 1 169 (26.8) 25 (25.3) >1 242 (38.4) 50 (50.5) laboratory investigations erythrocyte sedimentation rate (mean ± sd) 49.93 ± 31.25 57.89 ± 33.14 0.015* c-reactive protein (mean ± sd) 48.64 ± 31.43 69.29 ± 31.21 <0.001* lactate dehydrogenase (mean ± sd) 559.3 ± 245.8 928.7 ± 568.7 <0.001* values are presented as numbers and percentages (%). *p-value <0.05 is significant. 301j contemp med sci | vol. 7, no. 5, september–october 2021: 295–302 m. ali khaksar et al. original prevalence of comorbidities and its impacts in hospitalized patients with covid-19 table 5. association between demographics and clinical characteristics and duration of hospital stay (day) demographic data duration of hospital stay (day) (mean ± sd) p-value age (year) (spearman’s rho) 0.056 0.130 sex male 7.35 ± 4.60 0.196 female 6.83 ± 4.05 history of comorbidities diabetes mellitus no 7.09 ± 4.42 0.848 yes 7.15 ± 4.27 hyperlipidemia no 7.05 ± 4.38 0.109 yes 7.60 ± 4.17 hypertension no 7.07 ± 4.44 0.532 yes 7.19 ± 4.21 ischemic heart disease no 7.09 ± 4.27 0.693 yes 7.18 ± 4.63 cerebrovascular accident no 7.11 ± 4.35 0.994 yes 7.22 ± 4.58 rheumatoid arthritis no 7.12 ± 4.38 0.779 yes 6.53 ± 3.54 hypothyroidism no 7.12 ± 4.39 0.821 yes 6.67 ± 3.47 systemic lupus erythematosus no 7.12 ± 4.37 0.676 yes 5.67 ± 2.08 asthma no 7.09 ± 4.26 0.960 yes 7.33 ± 5.71 liver failure no 7.11 ± 4.35 0.669 yes 7 ± 5.31 kidney transplantation no 7.12 ± 4.38 0.841 yes 6.50 ± 3.34 chronic kidney disease no 7.11 ± 4.42 0.692 yes 7.08 ± 3.73 chronic obstructive pulmonary disease no 7.10 ± 4.32 0.897 yes 8.67 ± 8.69 tuberculosis no 7.08 ± 4.35 0.063 yes 10.50 ± 4.97 comorbidity 0 6.88 ± 4.31 0.358 1 7.36 ± 4.50 >1 7.14 ± 4.31 laboratory investigations erythrocyte sedimentation rate (spearman’s rho) 0.196 <0.001* c-reactive protein (spearman’s rho) 0.105 0.004* lactate dehydrogenase (spearman’s rho) 0.173 <0.001* *p-value <0.05 is significant. table 6. association between demographics and clinical characteristics and duration of icu stay (day) demographic data duration of icu stay (day) (mean ± sd) p-value age (year) (spearman’s rho) 0.147 <0.001* sex male 1.45 ± 3.70 0.148 female 1.24 ± 3.35 history of comorbidities diabetes mellitus no 1.37 ± 3.73 0.345 yes 1.33 ± 3.21 hyperlipidemia no 1.28 ± 3.50 0.015 yes 1.91 ± 3.88 hypertension no 1.28 ± 3.63 0.119 yes 1.48 ± 3.39 ischemic heart disease no 1.34 ± 3.65 0.117 yes 1.38 ± 3.21 cerebrovascular accident no 1.34 ± 3.56 0.073 yes 1.70 ± 3.01 rheumatoid arthritis no 1.37 ± 3.57 0.659 yes 0.82 ± 2.10 hypothyroidism no 1.39 ± 3.59 0.099 yes 0.29 ± 1.08 systemic lupus erythematosus no 1.36 ± 3.55 — yes 0 asthma no 1.34 ± 3.35 0.389 yes 1.58 ± 5.77 liver failure no 1.33 ± 3.52 0.015* yes 3.33 ± 4.80 kidney transplantation no 1.37 ± 3.57 0.240 yes 0.25 ± 0.87 chronic kidney disease no 1.35 ± 3.62 0.376 yes 1.35 ± 2.68 chronic obstructive pulmonary disease no 1.33 ± 3.46 yes 4.67 ± 9.20 0.360 tuberculosis no 1.33 ± 3.52 0.006* yes 4.50 ± 5.13 comorbidity 0 1.19 ± 3.31 0.142 1 1.49 ± 4.25 >1 1.39 ± 3.20 laboratory investigations erythrocyte sedimentation rate (spearman’s rho) 0.089 0.016* c-reactive protein (spearman’s rho) 0.180 <0.001* lactate dehydrogenase (spearman’s rho) 0.256 <0.001* *p-value <0.05 is signifcant. 302 j contemp med sci | vol. 7, no. 5, september–october 2021: 295–302 prevalence of comorbidities and its impacts in hospitalized patients with covid-19 original m. ali khaksar et al. the prohibition of taking certain drugs in patients with a history of liver problems on the one hand, and the organ failure caused by covid-19, on the other. in our study, there was a relationship between the history of tuberculosis and high severity of the disease, which could be due to old lung damage and reduced lung reserve in these patients, as well as the reactivation of tb and tb superinfection on covid, leading the patient to respiratory failure. another predictor of disease severity in various studies and ours was the high level of inflammatory markers in patients’ tests, which in addition to predicting the severity of the disease, can be used as a response to treatment factor.20, 24 conclusion 1. we found that male gender, older age, hyperlipidemia, liver failure, tb, having more than one comorbidity, and elevated inflammatory biomarkers were significantly associated with the risk of severe covid-19 disease. declaration of patient consent the authors certify that they have obtained all appropriate patient consent forms. in the form, the patients have given their consent for their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. financial support and sponsorship this research was supported by grants (aprd-9915) from the air pollution respiratory disease research center by the vice chancellor of research and development, ahvaz jundishapur university of medical sciences (iran). conflicts of interest there are no conflicts of interest.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1. organization wh. clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected: interim guidance, 13 march 2020. world health organization; 2020. 2. qin c, zhou l, hu z, zhang s, yang s, tao y, et al. dysregulation of immune response in patients with coronavirus 2019 (covid-19) in wuhan, china. clinical infectious diseases. 2020;71(15):762–8. 3. guan w-j, liang w-h, zhao y, liang h-r, chen z-s, li y-m, et al. comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis. european respiratory journal. 2020;55(5). 4. liu k, fang y-y, deng y, liu w, wang m-f, ma j-p, et al. clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province. chinese medical journal. 2020. 5. wu z, mcgoogan jm. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention. jama. 2020;323(13):1239–42. 6. zhong n, zheng b, li y, poon l, xie z, chan k, et al. epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people’s republic of china, in february, 2003. the lancet. 2003;362(9393):1353–8. 7. assiri a, mcgeer a, perl tm, price cs, al rabeeah aa, cummings da, et al. hospital outbreak of middle east respiratory syndrome coronavirus. new england journal of medicine. 2013;369(5):407–16. 8. choi kw, chau tn, tsang o, tso e, chiu mc, tong wl, et al. outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong. annals of internal medicine. 2003;139(9):715–23. 9. hong k-h, choi j-p, hong s-h, lee j, kwon j-s, kim s-m, et al. predictors of mortality in middle east respiratory syndrome (mers). thorax. 2018;73(3):286-9. 10. koff wc, williams ma. covid-19 and immunity in aging populations—a new research agenda. new england journal of medicine. 2020;383(9):804–5. 11. zaferani arani h, dehghan manshadi g, atashi ha, rezaei nejad a, ghorani sm, abolghasemi s, et al. understanding the clinical and demographic characteristics of second coronavirus spike in 192 patients in tehran, iran: a retrospective study. plos one. 2021;16(3):e0246314. 12. pijls bg, jolani s, atherley a, derckx rt, dijkstra ji, franssen gh, et al. demographic risk factors for covid-19 infection, severity, icu admission and death: a meta-analysis of 59 studies. bmj open. 2021;11(1):e044640. 13. oudit gy, pfeffer ma. plasma angiotensin-converting enzyme 2: novel biomarker in heart failure with implications for covid-19. european heart journal. 2020;41(19):1818–20. 14. yazdanpanah l, shahbazian h, aleali am, jahanshahi a, ghanbari s, latifi s. prevalence, awareness and risk factors of diabetes in ahvaz (south west of iran). diabetes & metabolic syndrome: clinical research & reviews. 2016;10(2):s114–s8. 15. singh ak, gupta r, ghosh a, misra a. diabetes in covid-19: prevalence, pathophysiology, prognosis and practical considerations. diabetes & metabolic syndrome: clinical research & reviews. 2020;14(4):303–10. 16. yazdanpanah l, shahbazian h, shahbazian h, latifi s-m. prevalence, awareness and risk factors of hypertension in southwest of iran. journal of renal injury prevention. 2015;4(2):51. 17. latifi sm, moradi l, shahbazian h, aleali am. a study of the prevalence of dyslipidemia among the adult population of ahvaz, iran. diabetes & metabolic syndrome: clinical research & reviews. 2016;10(4):190–3. 18. zhou f, yu t, du r, fan g, liu y, liu z, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet. 2020;395(10229):1054–62. 19. fang x, li s, yu h, wang p, zhang y, chen z, et al. epidemiological, comorbidity factors with severity and prognosis of covid-19: a systematic review and meta-analysis. aging (albany ny). 2020;12(13):12493. 20. zhang g, hu c, luo l, fang f, chen y, li j, et al. clinical features and shortterm outcomes of 221 patients with covid-19 in wuhan, china. journal of clinical virology. 2020;127:104364. 21. du r-h, liang l-r, yang c-q, wang w, cao t-z, li m, et al. predictors of mortality for patients with covid-19 pneumonia caused by sars-cov-2: a prospective cohort study. european respiratory journal. 2020;55(5). 22. abrishami a, khalili n, dalili n, tabari rk, farjad r, samavat s, et al. clinical and radiologic characteristics of covid-19 in patients with ckd. iranian journal of kidney diseases. 2020;14(4):267–77. 23. meng j, xiao g, zhang j, he x, ou m, bi j, et al. renin-angiotensin system inhibitors improve the clinical outcomes of covid-19 patients with hypertension. emerging microbes & infections. 2020;9(1):757–60. 24. chen t, dai z, mo p, li x, ma z, song s, et al. clinical characteristics and outcomes of older patients with coronavirus disease 2019 (covid-19) in wuhan, china: a single-centered, retrospective study. the journals of gerontology: series a. 2020;75(9):1788–95. 25. wu b, zhou j, wang w, yang h, xia m, zhang b, et al. association analysis of hyperlipidemia with the 28-day all-cause mortality of covid-19 in hospitalized patients. chinese medical sciences journal. 2021;36(1):17–26. https://doi.org/10.22317/jcms.v7i5.1081 220 j contemp med sci | vol. 8, no. 4, july-august 2022: 220–223 original the association between iron deficiency anemia and obesity in children akrem m. atrushi1, farhad shaker armishty2*, sirwan a. saleh2, mehvan sh. abdulrahman3 1department of pediatric, college of medicine, university of duhok, kurdistan region, iraq. 2*department of clinical sciences, college of medicine, university of zakho, kurdistan region, iraq. 3department of pediatric, directorate of health, duhok, kurdistan region, iraq. *correspondence to: dr. farhad shaker armishty (e-mail: farhad.shaker@uoz.edu.krd) (submitted: 02 may 2022 – revised version received: 21 may 2022 – accepted: 15 june 2022 – published online: 26 august 2022) abstract background: obesity is a growing health problem all over the world. approximately 18–38% of under 5 years old children have iron deficiency anemia. obese people are more likely to be have iron deficiency. studies that dealt with the relationship between iron deficiency and obesity are not homogeneous. aim: to examine the association between obesity and iron status and the presence of iron deficiency anemia in children. methods: this case-control study included 100 children between 2–14 years of age who were divided into two ageand sex-matched equal groups of 50 children each. children with a body mass index (bmi) greater than or equal to 95th centile were categorized as obese while the other 50 children with a bmi greater than or equal to 5th centile but less than 95th centile were considered the normal weight group. children with cardiac disease, liver disease, chronic gastrointestinal disease and chronic hematologic disorders except iron deficiency (with or without anemia) and those taking vitamin or mineral supplements regularly during the previous year were excluded. each participant was sent for serum iron, ferritin, total iron binding capacity (tibc), transferrin saturation ts and complete blood count. iron deficiency is defined as transferrin saturation (ts) lower than 16% and ida is defined as ts lower than 16% and hemoglobin (hb) concentration lower than 120 g/l or 12 mg/dl for children. the data were analyzed using spss-23 software and for all data normal distribution was tested so that p-value <0.05 is the level of threshold for statistical significance. results: the gender distribution between the both group is reversed with male being more common in obese group but no statistical difference. the age distribution shows dominance of the age group 5–10 years in the both group with some differences which are of no statistical significance (p = 0.294). the values of hemoglobin, serum iron, serum ferritin, total iron binding capacity and transferrin saturation are obviously similar between the both genders of the whole study population with no statistically significant differences (p = 0.084, 0.469, 0.48, 0.4, 0.571 respectively). obese children have higher level of hemoglobin (p = 0.069), ferritin (p = 0.5) and total iron binding capacity (p = 0.449) but lower levels of serum iron (p = 0.234) and transferrin saturation(p = 0.45) but with no statistical significance. conclusion: there is no significant association between obesity and iron status and the presence of iron deficiency anemia despite a lower level of serum iron and lower transferrin saturation in obese than normal weight children. issn 2413-0516 introduction obesity is a growing health problem all over the world. its prevalence has increased apparently in recent years.1,2 up to 16–31% of children suffer from obesity nowadays.2,3 according to the world health organization (who) classification, the prevalence of iron deficiency anemia (ida) is in the medium level.4 approximately 18–38% of under 5 years old children have iron deficiency anemia.5 the rapid changes in lifestyles and dietary patterns with large amounts of fat, sugar and oil are of the most important causes of obesity.6-8 it has been found that obese people are more likely to be have iron deficiency. iron deficiency anemia is significantly more prevalent among obese than normal weight people.9-11 foods high in calorie are low in nutrients leading to poor diet. obese children are susceptible to a variety of micronutrient deficiencies.12 obesity is considered a low-grade inflammatory disease. adipose tissue is considered an endocrine organ that secret pro-inflammatory cytokines named adipokines that contribute to the inflammatory process that may have an important pathogenic role in some obesity-related comorbidities.13 it has been suggested that expansion of tissue mass and adipocyte size in obesity makes white adipose tissue hypoxic leading to inflammation and cellular dysfunction.14 moreover, when hypertrophied adipose tissue is unable to satisfy its storage function, there will be excess free fatty acids exposed to organs which are lipid intolerant leading to lipotoxicity and thereby low-grade inflammation in the adipose tissue. therefore, hypertrophy of adipocytes and local tissue hypoxia, triggers overproduction of adipokine that enhances macrophage infiltration in obesity.15 this inflammation can cause transformation of iron metabolism leading to overload of iron in tissues with decreased mobility and as a result reduction of the breakdown of myoglobin. this will decrease the serum iron needed for hematopoiesis.16 studies that dealt with the relationship between iron deficiency and obesity are not homogeneous.17-20 they are generally case/control or cross-sectional studies, in many cases, not population-based, but rather they considered many variables related to iron deficiency and obesity21,22 and used different criteria for defining iron deficiency. a frequently used iron parameter is serum ferritin23-25 which is an acute-phase reactant that is positively related to adiposity and thus reducing its sensitivity. to the best of our knowledge, no studies have been done locally to study the relation between obesity and each of iron status and the presence of iron deficiency anemia. the aim of study is to examine the association between obesity and iron status and the presence of iron deficiency anemia in children. methods this case-control study was out at zakho general hospital in the period from may 1st 2021 to may 1st 2022. a total of 100 mailto:farhad.shaker@uoz.edu.krd 221j contemp med sci | vol. 8, no. 4, july-august 2022: 220–223 a.m. atrushi et al. original the association between iron deficiency anemia and obesity in children children between 2–14 years of age were included in the study. the participants were divided into two equal groups of 50 children each. both groups were ageand sexmatched. children with a body mass index (bmi) greater than or equal to 95th centile were categorized as obese and termed as cases while the other 50 children with a bmi greater than or equal to 5th centile but less than 95th centile (as per the world health organization [who] standards) were the normal weight group and termed controls. the exclusion criteria were: 1any disorder, such as cardiac disease, liver disease, chronic gastrointestinal disease and chronic hematologic disorders except iron deficiency (with or without anemia). 2vitamin or mineral supplements taken regularly during the previous year. from each participant, about 5 cc of fasting blood were taken to evaluate the serum iron, ferritin, total iron binding capacity (tibc) and transferrin saturation ts, and about 2 cc citrated fasting blood sample were evaluated for complete blood count (cbc) and analyzed according to standard protocols. iron deficiency is defined as transferrin saturation (ts) lower than 16% and ida is defined as ts lower than 16% and hemoglobin (hb) concentration lower than 120 g/l or 12 mg/dl for children. the data were analyzed using spss-23 software and for all data normal distribution was tested so that p-value <0.05 is the level of threshold for statistical significance. results as shown in table 1, the gender distribution between the both group is reversed but no statistical difference has been found. the age distribution shows dominance of the age group 5–10 years in the both group with some differences which are of no statistical significance. so, the obese and normal weight children are age and gender matched. the laboratory findings of the whole participants are shown in table 2. the values of hemoglobin, serum iron, serum ferritin, total iron binding capacity and transferrin saturation are obviously similar between the both genders of the whole study population with no statistically significant differences. comparison between obese and normal weight children laboratory values shows the obese children have higher level of hemoglobin, ferritin and tibc but lower levels of serum iron and transferrin saturation but with no statistical significance as shown in table 3. discussion the sociodemographic characteristics show males are more commonly obese and that age group of 5–10 years are the most prevalent but no significant differences were noticed between the both groups. numerous previous studies have found higher prevalence of iron deficiency in obese children.28-35 our findings show a lower level of serum iron and lower transferrin saturation in obese children than normal weight but do not reveal a significant deficiency of iron in obese children. these results corroborate the results of perez et al.29 that found the prevalence of iron deficiency in otherwise healthy obese was not higher than in normal weight children so the effect of obesity on iron status was low. they suggested that table 1. the sociodemographic characteristics of cases and controls normal obese p-value gender male 24 (48%) 26 (52%) 0.689 female 26 (52%) 24 (48%) age under 5 years 12 (24%) 6 (12%) 0.2945–10 years 22 (44%) 25 (50%) above 10 years 16 (32%) 19 (38%) table 2. laboratory values of the study population mean ± sd male female total p-value hb 12.5380 ± .94694 12.5180 ± 1.07489 12.5280 ± 1.00786 0.084 iron 62.9948 ± 30.31192 59.0934 ± 28.53107 61.0441 ± 29.35148 0.469 ferritin 35.7664 ± 26.75804 30.3876 ± 23.27093 33.0770 ± 25.09420 0.480 tibc 378.0280 ± 82.31149 390.4890 ± 81.36954 384.2585 ± 81.66790 0.541 ts 19.6440 ± 22.87591 16.9146 ± 9.8158 18.2793 ± 17.566 0.571 table 3. relation between weight status and laboratory values normal obese p-value hb 12.4960 ± 1.12847 12.5600 ± .88133 0.069 iron 63.1104 ± 35.55115 58.9778 ± 21.63337 0.234 ferritin 30.0546 ± 21.68217 36.0994 ± 27.99161 0.5 tibc 368.0170 ± 72.77616 400.5000 ± 87.41108 0.449 ts 18.7760 ± 10.94805 17.7826 ± 22.42977 0.450 222 j contemp med sci | vol. 8, no. 4, july-august 2022: 220–223 the association between iron deficiency anemia and obesity in children original a.m. atrushi et al. specific cutoff values for iron deficiency in overweight adolescents need to be defined. this is in agreement with ferrari et al.36 who revealed that adiposity of the european adolescents was sufficient to cause chronic inflammation but not sufficient to impair iron status and cause iron deficiency. in line with our findings, demircioglu et al.37 found that serum iron and ferritin level were comparable between obese and normal weight children and stressed a significant role of hepcidin in obesity. cheng et al.38 did a study on obese adult women and found that obesity alone may not be sufficient to cause disturbances to iron metabolism which are clinically significant as previously described and qin39 [ida similar 9] found anemia to be even less prevalent in obese women and this is in agreement with our study on pediatric population. gajewska40 found that in obese children with sufficient iron intake, the altered ferroportin-hepcidin axis may occur without signs of iron deficiency or iron deficiency anemia. they suggested that the role of other micronutrients, besides dietary iron, may also be considered in the iron status of obese children while huang22 concluded that being overweight or obese would not be a risk factor of iron deficiency in adolescents, if it were defined by ferritin rather than iron level. in total, the paradoxical results of the studies regarding association between obesity and iron deficiency might be attributed to the differences in the definition of obesity or using different techniques to assess laboratory parameters.41 one of the limitations of this study is that we did not include the dietary intake of iron which if sufficient can overcome the effects of obesity on causing iron deficiency and anemia. also, it would have been much better if we studied the role of hepcidin in iron status in obesity as proved in many studies.37 conclusion there is no significant association between obesity and iron status and the presence of iron deficiency anemia despite a lower level of serum iron and lower transferrin saturation in obese than normal weight children. conflict of interest none.  references 1. ghadimi r, asgharzadeh e, sajjadi p. obesity among elementary schoolchildren: a growing concern in the north of iran, 2012. int j prev med 2015; 6: 99. 2. gahagan s. overweight and obesity. in: nelson text book of pediatrics, 19th ed. philadelphia: elsevier saunders 2011: 179–211. 3. ogden cl, carroll md, curtin lr, et al. prevalence of high body mass index in us children and adolescents, 2007–2008. j am med assoc 2010; 303(3):242–9. 4. bahrami m. malnutrition and its effects on development in iranian children. iran j pediatr 2004; 14(2): 149–56. [in persian] 5. world health organization. worldwide prevalence of anemia 1993–2005. who, global database on anaemia, geneva, switzerland, 2008. 6. sajjadi p, enayatzadeh h, ghadimi r. which food groups and macronutrients are more associated with central obesity in iranian children? caspian j social medicine 2015; 1(1): 24–30. 7. world health organization. diet, nutrition and prevention of chronic diseases, who technical report jointwho/fao expert consultation, geneva 2003. available at: http://www.who.int/dietphysicalactivity/publications/tr s916/en/gsfao_introduction.pdf 8. daniels sr, arnett dk, eckel rh, et al. overweight in children and adolescents: pathophysiology, consequences, prevention and treatment. circulation 2005; 111(15): 1999–2012. 9. nead kg., halterman js, kaczorowski jm, et al. overweight children and adolescents: a risk group for iron deficiency. pediatr 2004; 114(1): 104–8. 10. chambers ec, heshka s, gallagher d, et al. serum iron and body fat distribution in a multiethnic cohort of adults living in new york city. j am diet assoc 2006; 106(5): 680–4. 11. keikhaei b, askari r, aminzadeh m. adolescent with unfeasible body mass index: a risk factor for iron deficiency anemia. j health med informat 2012; 3(1). available at: http://www.omicsonline.org/adolescentwithunfeasible-body-mass-index-a-risk-factor-foriron-deficiencyanemia-2157-7420.1000109.pdf 12. daniels sr, arnett dk, eckel rh, gidding ss, hayman ll, kumanyika s, et al. overweight in children and adolescents: pathophysiology, consequences, prevention and treatment. circulation 2005; 111(15): 1999–2012. 13. coelho m, oliveira t, fernandes r. biochemistry of adipose tissue: an endocrine organ. arch med sci 2013;9(2): 191–200. 14. sarmiento ol, parra dc, gonzalez sa, gonzalez-casanova i, forero ay, garcia j. the dual burden of malnutrition in colombia. the american journal of clinical nutrition 2014;100(6):1628s-35s. doi: 10.3945/ ajcn.114.083816. 15. ramirez-zea m, kroker-lobos mf, close-fernandez r, kanter r. the double burden of malnutrition in indigenous and nonindigenous guatemalan populations. the american journal of clinical nutrition 2014;100(6):1644s-51s. doi: 10.3945/ajcn.114.083857. 16. richardson mw, ang l, visintainer pf, wittcopp ca. the abnormal measures of iron homeostasis in pediatric obesity are associated with the inflammation of obesity. int j pediatr endocrinol 2009; 2009: 713269. doi: 10.1155/2009/713269. 17. manios y, moschonis g, chrousos gp, et al. the double burden of obesity and iron deficiency on children and adolescents in greece: the healthy growth study. j hum nutr diet 2013; 26:470–478. 18. tussing-humphreys lm, liang h, nemeth e, et al. excess adiposity, inflammation, and iron-deficiency in female adolescents. j am diet assoc 2009; 109:297–302. 19. frelut ml, girardet p, bocquet a, et al. impact of obesity on biomarkers of iron and vitamin d status in children and adolescents: the risk of misinterpretation. arch pediatr 2018; 25:3–5. 20. johnson k, showell nn, flessa s, et al. do neighborhoods matter? a systematic review of modificable risk factors for obesity among low socioeconomic status black and hispanic children. chilhood obesity 2019; 15:71–86. 21. hutchinson c. a review of iron studies in overweight and obese children and adolescents: a double burden in the young. eur j nutr 2016; 55: 2179–2197. 22. huang yf, tok ts, lu cl, et al. relationship between being overweight and iron deficiency in adolescents. pediatr neonatol 2015; 56:386–392. 23. mattiello v, schmugge m, hengartner h, et al. diagnosis and management of iron deficiency in children with or without aaemia: consensus recommendations of the spog pediatric hematology group. eur j pediatr 2020; 179:527–545. 24. thomas dw, hinchliffe rf, briggs c, et al. british committee for standards guideline for the laboratory diagnosis of functional iron deficiency. br j haematol 2013; 161:639–648. 25. khan a, khan wm, maimoona a, et al. ferritin is a marker of inflammation rather than iron deficiency in overweight and obese people. j obes 2016; 2016:1937320. 26. siyaram d, bhatia p, dayal d. hypoferremic state in overweight and obese children. indian pediatr 2018; 55:72–73. 27. pinhas-hamiel o, newfield rs, koren i, et al. greater prevalence of iron deficiency in overweight and obese children and adolescents. int j obes 2003; 27:416–418. http://www.omicsonline.org/adolescentwith-unfeasible-body-mass-index-a-risk-factor-foriron-deficiency-anemia-2157-7420.1000109.pdf http://www.omicsonline.org/adolescentwith-unfeasible-body-mass-index-a-risk-factor-foriron-deficiency-anemia-2157-7420.1000109.pdf http://www.omicsonline.org/adolescentwith-unfeasible-body-mass-index-a-risk-factor-foriron-deficiency-anemia-2157-7420.1000109.pdf 223j contemp med sci | vol. 8, no. 4, july-august 2022: 220–223 a.m. atrushi et al. original the association between iron deficiency anemia and obesity in children 28. aloufi me, aljaed nm, aloufi ra, jafri sa, jafri su a, elnashar ma. prevalence of iron deficiency anemia in obese children in taif area saudi arabia. the egyptian journal of hospital medicine 2018;73(5): 6744–6752. 29. ortíz-pérez m, vázquez-lópez ma, ibáñez-alcalde m, et al. relationship between obesity and iron deficiency in healthy adolescents. childhood obesity. 2020;16(6):440–7. doi: 10.1089/chi.2019.0276 30. cepeda-lopez ac, osendarp sj, melse-boonstra a, aeberli i, gonzalezsalazar f, feskens e, villalpando s, zimmermann mb: sharply higher rates of iron deficiency in obese mexican women and children are predicted by obesity-related inflammation rather than by differences in dietary iron intake. am j clin nutr. 2011, 93 (5): 975–983. 10.3945/ajcn.110.005439. 31. alshwaiyat n.m., ahmad a., hassan w.m.r.w., al-jamal h.a.n. association between obesity and iron deficiency (review) exp. ther. med. 2021;22:1268. doi: 10.3892/etm.2021.10703. [pmc free article] [pubmed] [crossref ] [google scholar] 32. khemphet r, yupensuk n. prevalence and association between obesity and iron deficiency in children. j med assoc thai 2022; 105:212–8. 33. akca so, bostanci mo. the impact of anemia and body mass index (bmi) on neuromotor development of preschool children. rev. assoc. med. bras. 2017;63 (9) :779–786. 34. malden s, gillespie j, hughes a, gibson am, farooq a, martin a, et al. obesity in young children and its relationship with diagnosis of asthma, vitamin d deficiency, iron deficiency, specific allergies and flat-footedness: a systematic review and meta-analysis. obes rev 2020 aug 18. [ahead of print].10.1111/obr.13129 35. ibrahim ls, tayyem rf. evaluation of iron deficiency and the intake of macroand micronutrients among normal, overweight, and obese children under 5 years in amman. iran j ped hematol oncol. 2018; 8:21–36. 36. ferrari m, cuenca-garcía m, valtueña j, et al. helena study group. inflammation profile in over-weight/obese adolescents in europe: an analysis in relation to iron status. eur j clin nutr 2015; 69:247–255. 37. demircioğlu f., görünmez g., dağıstan e, et al. serum hepcidin levels and iron metabolism in obese children with and without fatty liver: case– control study. eur j pediatr 173, 947–951 (2014). https://doi.org/10.1007/ s00431-014-2268-8. 38. cheng hl, bryant ce, roonkey kb, steinbeck ks, griffin hj, petocz p, et al. iron, hepcidin and inflammatory status of young healthy overweight and obese women in australia. plos one. 2013; 4; 8(7):e68675. 39. qin y., melse-boonstra a., pan x, et al. anemia in relation to body mass index and waist circumference among chinese women. nutr j 12, 10 (2013). https://doi.org/10.1186/1475-2891-12-10. 40. gajewska j, ambroszkiewicz j, klemarczyk w, głąb-jabłońska e, weker h, chełchowska m. ferroportin-hepcidin axis in prepubertal obese children with sufficient daily iron intake. international journal of environmental research and public health. 2018; 15(10):2156. https://doi.org/10.3390/ ijerph15102156. 41. arshad m, jaberian f, pazouki a, riazi s, rangraz ma, mokhber s. iron deficiency anemia and megaloblastic anemia in obese patients. rom. j. intern. med 2017; 55(1): 3–7. https://doi.org/10.22317/jcms.v8i4.1236 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.1089/chi.2019.0276 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc8456489/ https://www.ncbi.nlm.nih.gov/pubmed/34594405 https://scholar.google.com/scholar_lookup?journal=exp.+ther.+med.&title=association+between+obesity+and+iron+deficiency+(review)&author=n.m.+alshwaiyat&author=a.+ahmad&author=w.m.r.w.+hassan&author=h.a.n.+al-jamal&volume=22&publication_year=2021&pages=1268&pmid=34594405&doi=10.3892/etm.2021.10703& https://doi.org/10.1111/obr.13129 https://doi.org/10.1007/s00431-014-2268-8 https://doi.org/10.1007/s00431-014-2268-8 https://doi.org/10.1186/1475-2891-12-10 https://doi.org/10.3390/ijerph15102156 https://doi.org/10.3390/ijerph15102156 290 j contemp med sci | vol. 7, no. 5, september–october 2021: 290–294 original incidence and prognostic significance of myocardial injury after elective percutaneous coronary intervention in duhok, iraq ameen m mohammad1*, hartmut k guelker2, sabri k sheikhow3 1department of internal medicine, college of medicine, duhok university, duhok, iraq. 2department of cardiology, helios university hospital wuppertal, university of witten/herdecke, wupertal, germany. 3department of internal medicine, college of medicine, duhok university, duhok, iraq. *correspondence to: ameen m mohammad (e-mail: doctoramb@yahoo.com) (submitted: 10 july 2021 – revised version received: 02 august 2021 – accepted: 25 august 2021 – published online: 26 october 2021) abstract objectives: this study aims to look at the incidence of myocardial injury after elective percutaneous coronary intervention (pci) and to correlate its effect to midterm clinical outcome. methods: a total of 182 patients were enrolled. their mean age was (57.44 ± 9.15) years. they were undergoing elective pcis in azadi heart center, duhok, iraq. cases with positive cardiac troponin (ctn) pre-procedural were excluded. within first 24-hours after pci (ctn) was estimated. and then after all patients were followed for 12 months for major adverse cardiac events (mace). results: 36 (19.8%) out of 182 (100%) had elevated ctn. those patients with elevated ctn had a statistically significant higher rate of prior cabg, ecg changes, triple vessels disease, type c lesions, post stent balloon dilatation and more periprocedural side branch jeopardy and coronary dissections compared to those with negative ctn (p ≤ 0.05). during follow up; the mace was higher in patients with positive ctn (log rank = 0.04). conclusion: minor myocardial injury after elective pci is common. it’s associated with procedural complexity and stratified patients at risks of worse prognosis. keywords: myocardial injury, preprocedural myocardial infarction, elective pci issn 2413-0516 introduction the prevalence of coronary artery disease (cad) is high in countries in africa and the middle east. according to latest world health organization data published in april 2011 cardiovascular diseases account for 25% of total deaths in iraq. cad exclusively account for 14.2% of total deaths1–5. medical treatments of cad have improved in the past decade because of the availability of statins, effective blood pressure lowering drugs and antiplatelet agents. in addition, improvements in pcis and using of drug eluting stents have revolutionized the management of cad6. with technological advances in coronary intervention over the past 3 decades, procedural complications and long-term outcomes have significantly improved, yet periprocedural myocardial infarction remains common, with significant therapeutic and prognostic implications7. in the context of pci, the issue of diagnosing myocardial necrosis and infarction is more complex and controversial. after pci among patients with a normal baseline troponin value, elevations of cardiac biomarkers above the 99th percentile of the upper reference level are indicative of periprocedural myocardial necrosis8,9. this study aimed to determine the incidence of myocardial injury after elective pci by the estimation of the ctn and to study the prognostic implications of such elevation to the mid-term clinical outcome of patients in duhok, kurdistan, iraq. methods patient population: during this prospective clinical study a total of 182 cases were recruited. their ages ranged 33–75 years with a mean age of (57.44 ± 9.15) years. 109 were males and 93 were females. all cases were undergoing elective stenting for coronary arteries lesions in azadi heart center in duhok, kurdistan region of iraq. the inclusion criteria were cases of elective pci with negative pre-procedure ctn. the main exclusion criteria were patients underwent emergency or primary pci for acute coronary syndrome (acs), patients with history of acs within two months and any patients with positive ctn pre procedure were excluded. the clinical data including demographic, cardiovascular risk factors, and medications were recorded. electrocardiography, echocardiography, renal function test, screening for hepatitis b&c were done for all patients. coronary angiography and pci: all patients were receiving dual antiplatelets in addition to other indicated cardiac drugs pre-procedurally. almost all pci were performed through the femoral approach. coronary angiographic lesion characteristics were classified according to american heart association/ american college of cardiology (aha/acc)10. successful procedure was defined as residual stenosis <30%. optimization of results by balloon inflations were performed in indicated cases. periand post-procedural complications based on standard definitions were noted11. all immediate and in-hospital outcomes related to the procedure through monitoring of all patients inside hospital for up to 24 hours were recorded. cardiac troponin: in pre-procedural period the level of ctn was estimated, and those with negative results were enrolled in the study and underwent pci with drug eluting stents. post procedural samples of blood for the estimation of ctn within 24 hours were taken. the ctn was analyzed using a rapid bedside test (cardiac t, roche diagnostics, mannheim, germany) with a threshold of (uln) 0.03 ng/ml. follow up for outcome and mace: the patients were followed clinically on regular basis for 12 months for mace and clinical outcome like new onset ischemic attacks specifically acute myocardial infarction, stent thrombosis, ischemic stroke and cardiac death. 22 of our patients were underwent repeated 291j contemp med sci | vol. 7, no. 5, september–october 2021: 290–294 a.m. mohammad et al. original incidence and prognostic significance of myocardial injury after elective percutaneous coronary intervention (control) coronary angiography for new onset presumed ischemic presentations. statistical analysis: data were collected and analyzed by using the spss software package version22. continuous variables were calculated as mean ± (sd) and categorical variables were presented as counts and percentages. a chi-square test was used for the comparison of categorical variables. anova test was used for continuous variables. follow up correlation and cumulative (event free) survival between cardiac troponin and clinical outcome was analyzed by the kaplan-meier method and a group comparison was done by the log rank test. a probability value of ≤ 0.05 was considered statistically significant. ethics approval and consent to participate: the study was approved by the ethical committee at the college of medicine, university of duhok, iraq and the informed consent was obtained from all enrollees. the study protocol was performed in accordance with the relevant guidelines and regulations of the declaration of helsinki. results patients characteristics: patients with positive ctn had higher incidence of prior cabg and ecg changes compared to group with negative ctn with a significant pvalue (0.02 & 0.03) respectively. no significant differences were seen between the two groups in relation to other clinical characteristics and cardiovascular risk factors as shown in table 1. angiographic & pci profile: the group of positive ctn had higher rate of triple vessels disease, type c lesions compared to other group with a statistically significant difference (p-value were 0.05 and <0.007) respectively. uses of post stent balloons dilatation were more common in positive ctn group compared to other group with a statistical difference (p < 0.04). there were significant differences in term of ecg changes and periprocedural complications including (side branch occlusions and coronary dissections) among patients with positive ctn compared to other group with p-value (0.05, 0.03 & 0.04) respectively as shown in table 2. mace & follow up outcome: there was a significant difference in term of new ecg changes, emergency cabg, mi, needs for repeated coronary angiography and rate of stent thrombosis among group of positive ctn compared to negative ctn group with a p-value (0.01, 0.02, 0.01, 0.01 & 0.03) respectively. one case underwent successful cabg, three cases developed mi due to stent thrombosis (two of them were not complaint to clopidogrel and underwent successful repeated pci and one with confirmed clopidogrel resistance12) table 3. kaplan-meier survival curve for cases with positive (n = 36) and negative (n = 144) ctn. it shows a significant difference between two groups regarding the follow up outcome and mace (log rank = 0.04). there were more complications and cardiac ischemic events during follow up period among patients with positive ctn after elective pci. discussion in spite of more than two decades of delivery of pci services in iraq, this is the first study, up to our knowledge, of its kind to be done in the country. the main findings of this study are the following: first; myocardial injury especially of minor degree detected by elevation of ctn occurs commonly after elective pci; second it is more frequent when the pci-related factors are more complex; third it is associated with worse subsequent cardiac outcome during follow up. the guidelines of acc/aha recommend measurement of cardiac troponin 8–12 hours post-pci as a class 2a. current pci guidelines give a class i recommendation for the measurement of cardiac biomarkers in patients who have signs or symptoms suggestive of myocardial infarction during table 1. baseline patients characteristics characters –ve ctn 146 (80.2%) +ve ctn 36 (19.8%) total 182 (100%) pvalue age (mean ± sd) 56.62 ± 9.1 58.26 ± 9.2 57.44 ± 9.15 0.3 (ns) gender (male) 87 (59%) 22 (61%) 109 (59%) 0.4 (ns) current smoking 48 (32%) 11 (30%) 59 (32%) 0.3 (ns) hypertension 60 (41%) 15 (41%) 75 (41%) 0.4 (ns) hypercholesterolemia 47 (32%) 13 (36%) 60 (32%) 0.3 (ns) diabetics 37 (25%) 12 (33%) 49 (26%) 0.1 (ns) prior mi 24 (16%) 7 (19%) 31 (17%) 0.3 (ns) chf 14 (9%) 5 (2.4%) 19 (10%) 0.2 (ns) prior cabg 2 (1.3%) 2 (2.4%) 4 (2%) 0.02 renal impair 6 (4%) 2 (5.5) 8 (4.2%) 0.3 (ns) ecg changes 65 (44%) 10 (27%) 75 (41%) 0.03 rwm abnormalities 35 (23%) 13 (36%) 48 (26%) 0.06 (ns) cardiac medications antiplat. 146 (100%) 36 (100%) 182 (100%) 0.2 (ns) beta-block 126 (86%) 30 (83%) 156 (85%) 0.3 (ns) ace 58 (39%) 13 (36%) 71 (39%) 0.3 (ns) statin 146 (100%) 36 (100%) 182 (100%) 0.2 (ns) 292 j contemp med sci | vol. 7, no. 5, september–october 2021: 290–294 incidence and prognostic significance of myocardial injury after elective percutaneous coronary intervention original a.m. mohammad et al. or after pci and for those who have undergone complicated procedures13. in this study 36(19.8%) out of 182(100%) had elevated ctn. the reported incidence of troponin release after pci in literature ranges between 13% and 44%, with the incidence higher after stenting14. in suadia arabia, a study demonstrated that ctn elevated in (29%) after elective ptca. the ctn biomarker is sensitive in detection of even minor myocardial injuries after successful stenting15. there are conflicting findings between studies in term of prognostic significance of myocardial injury. some studies showed no association between myocardial injury and follow table 2. coronary angiographic and pci profile characteristics –ve ctn 146 (82.2%) +ve ctn 36 (19.8%) total 182 (100%) p-value vessel disease lms 3 (2%) 2 (5%) 5 (2.7%) 0.1 (ns) lad 65 (44%) 18 (50%) 83 (45%) 0.1 (ns) lcx 37 (25%) 6 (16%) 43 (23%) 0.1 (ns) rca 41 (28%) 10 (27%) 51 (28%) 0.4 (ns) no. of vessels single 65 (44%) 7 (19%) 72 (39%) 0.002 double 44 (30%) 15 (41%) 59 (32%) 0.09 (ns) triple 37 (25%) 14 (40%) 51 (28) 0.05 coronary lesion type a 71 (48%) 7 (19%) 78 (42.8%) 0.007 type b 47 (32%) 15 (41%) 62 (34%) 0.1 (ns) type c 28 (20%) 14 (40%) 42 (23%) 0.005 pci characteristics lesion length 22.2 ± 5.8 23.0 ± 6.1 22.6 ± 5.95 0.4 (ns) no. lesion 323 (2.2%) 104 (2.8%) 427 (2.34%) ns stent type des des des stent length 24.7 ± 6.1 25.1 ± 6.3 24.9 ± 6.2 0.7 (ns) stent diameter 2.80 ± 0.41 2.88 ± 0.52 2.84 ± 0.46 0.3 (ns) inflation bars 13.4 ± 3.2 13.5 ± 3.2 13.45 ± 3.2 0.8 (ns) balloon dilatation pre 69 (47%) 17 (47%) 86 (47%) 0.4 (ns) post 20 (13%) 9 (25%) 29 (15.9%) 0.04 complications chest pain 16 (10.9%) 7 (19.4%) 23 (12.6%) 0.08 (ns) ecg changes 9 (6%) 5 (13%) 14 (7.6%) 0.05 s.b occlusion 5 (3%) 4 (11%) 9 (4.9%) 0.03 dissection 6 (4%) 4 (11%) 10 (5.4%) 0.04 no reflow 1 (0.6%) 1 (2%) 2 (1.09%) 0.1 (ns) table 3. mace and follow up outcome (censored) cardiac outcome –ve ctn 146 (80.2%) +ve ctn 36 (19.8%) total 182 (100%) p-valve ischemic symptom 18 (9%) 8 (22%) 26 (14.2%) 0.06 (ns) ecg changes 5 (3%) 5 (13%) 10 (5.4%) 0.01 non-complaint 6 (4%) 3 (8%) 9 (4.6%) 0.1 (ns) cardiac death 1 (0.6) 0 (0%) 1 (0.5%) 0.4 (ns) emergency cabg 0 (0%) 1 (2%) 1 (0.5%) 0.02 myocardial infarct 1 (0.6%) 2 (5%) 3 (1.64%) 0.01 stroke 0 (100%) 0 (100%) 0 (0%) control c. angio 14 (9%) 8 (22%) 22 (12.08%) 0.01 stent thrombosis 1 (0.6%) 2 (5%) 3 (1.64%) 0.03 isr 1 (0.6%) 0 (0%) 1 (0.5%) 0.4 (ns) repeated pci target 2 (1.3%) 2 (5%) 4 (2.1%) 0.06 (ns) denovo 4 (2.7%) 2 (5%) 6 (3.2%) 0.1 (ns) 293j contemp med sci | vol. 7, no. 5, september–october 2021: 290–294 a.m. mohammad et al. original incidence and prognostic significance of myocardial injury after elective percutaneous coronary intervention mace16,17. however, others found the association18. in the present study, the clinical outcome during the follow up was significantly affected in the group of positive ctn. there was a significant increase in the rates of new onset ischemic attacks including new ecg changes, mi, needs for repeated revascularization and stent thrombosis with a p-value (0.01, 0.01, 0.02 & 0.03) respectively. a meta-analysis of 15,581 patients in twenty studies was performed by nienhuis and colleagues found troponin elevation in 32.9% of the patients and a 1.1% higher mortality compared with the non-troponin elevated group19–21. there are many possible underlying causes of elevated ctn after stenting; moreover, it was found a significant relation in the present study to the peri-procedural complications like side branch jeopardize, evidence of significant dissections with clinical manifestation like presence of new onset ischemic chest pain and/or ecg changes. even without clear ischemic clinical features and imaging availability, the ctn measurement after pci is easy test and helpful for detecting the consequences of distal embolization of proximal platelet-thrombi aggregates during stent implantation as one of the potential hidden cause of patchy myocardial necrosis and injury. therefore, ctn elevations post procedure could be a valid end-point for clinical trials designed to compare coronary artery disease treatments22,23. the implications of this study are important as we shift towards a ctn definition, with fewer dependents on other cardiac biomarkers, for the detection of minor myocardial injury and will require improved standardization of assays and cut-off values. another point is about one fifth of patients in this study sustained a periprocedural myocardial injury after the procedure when cardiac troponins was used to detect myonecrosis figure 1. the limitations of this study included a relatively short follow up time of cases; especially for the monitoring of in-stent restenosis (isr) which could occurs mainly following balloon dilatations and vascular injury24. and secondly marginalize the effect of magnitude of elevation to the clinical profiles and outcome measurements. few questions can be derived from the present study that we recommend to be tackled in the future studies; like which kind of patients who sustained periprocedural myocardial injury should be observed for longer time inside the hospital and after discharge. secondly, from the prognostic point of view, how far the periprocedural myocardial infarction is differing from spontaneous myocardial infarction. thirdly from ethical point of view what shall we tell the patient who developed periprocedural mi despite a successful procedure25–27. conclusions one out of 5 elective pcis have sustained myocardial injury. ctn is easy and sensitive biomarker in detecting the injury. in term of prognosis, the injury was associated with mace during follow up. the occurrence of injury was commonly related to procedure complexity and complications. in consistence with the pci guidelines we recommend based on our findings to estimate the ctn level after performing elective pci in cases with prior cabg, new ecg changes, type c lesions, triple vessels disease, side branch jeopardy and coronary dissections in our centers. closer follow up and adherence to intensive justified treatment of those patients with raised ctn may lead to improving outcomes. conflicts of interest the authors declare that there are no conflicts of interest. acknowledgment we acknowledge the cooperation of staff of azadi heart center in duhok for their cooperation while doing this study. funding/support the authors received no financial support for the publication of this article. authors’ contribution all authors equally contributed to conducting the study, designing and collecting data, and writing the paper. further, all authors approved the final review of the manuscript.  references 1. almahmeed w, arnaout ms, chettaoui r, ibrahim m, kurdi mi, taher ma, mancia g (2012). coronary artery disease in africa and the middle east. ther clin risk manag. 8:65–72. 2. who report (2011). cardiovascular disease in iraq. website of who/iraq. available from: ( http://www.who.int /countries /irq/en/). 3. mohammad, a.m., jehangeer, h.i. & shaikhow, s.k. prevalence and risk factors of premature coronary artery disease in patients undergoing coronary angiography in kurdistan, iraq. bmc cardiovasc disord 15, 155 (2015). https://doi.org/10.1186/s12872-015-0145-7. 4. mohammad am, sheikho sk, tayib jm. relation of cardiovascular risk factors with coronary angiographic findings in iraqi patients with ischemic heart disease. am j cardiovasc dis res. 2013;1(1):25–9. 5. mohammad am, rashad hh, habeeb qs, rashad bh, saeed sy. demographic, clinical and angiographic profile of coronary artery disease in 294 j contemp med sci | vol. 7, no. 5, september–october 2021: 290–294 incidence and prognostic significance of myocardial injury after elective percutaneous coronary intervention original a.m. mohammad et al. kurdistan region of iraq. am j cardiovasc dis. 2021;11(1):39–45. published 2021 feb 15. 6. seema pursnani, frederick korley, ravindra gopaul, pushkar kanade, newry chandra, richard e. shaw et al (2012). percutaneous coronary intervention versus optimal medical therapy in stable coronary artery disease: a systematic review and meta-analysis of randomized clinical trials. circ cardiovasc interv 5:476-490. 7. alexandra j lansky, gregg w. stone (2010). periprocedural myocardial infarction: prevalence, prognosis, and prevention. circ cardiovasc interv. 3:602-610. 8. abhiram prasad, joerg herrmann (2011). myocardial infarction due to percutaneous coronary intervention. n engl j med 364:453–464. 9. centor, r. m, jeroan j allison, norman w weissman, john canto, gustavo heudebert, lucia juarez, catarina i kiefe (2003). diffusion of troponin testing in unstable angina patients: adoption prior to guideline release. j clin epidemiol 56: 1236–1243. 10. dehmer gj, vinay b, bermudez edmund a, et al. 2020 aha/acc key data elements and definitions for coronary revascularization: a report of the american college of cardiology/american heart association task force on clinical data standards (writing committee to develop clinical data standards for coronary revascularization). circ cardiovasc qual outcomes. 2020; 13(4):e000059. 11. glenn n. levine, issam bailey, john a. bittl, bojan cercek, charles e, stephen g. ellis et al. (2011). angiography and interventions association task force on practice guidelines and the society for cardiovascular summary: a report of the american college of cardiology foundation/american heart. circulation. 2011;124:2574-2609. 12. mohammad am, al-allawi na. cyp2c19 genotype is an independent predictor of adverse cardiovascular outcome in iraqi patients on clopidogrel after percutaneous coronary intervention. j cardiovasc pharmacol 2018; 71:347-51. 13. smith sc jr, feldman te, hirshfeld jw jr, jacobs ak, kern mj, king sb iii, morrison da (2006). acc/aha/scai 2005 guideline update for percutaneous coronary intervention: a report of the american college of cardiology/american heart association task force on practice guidelines (acc/aha/scai writing committee to update 2001 guidelines for percutaneous coronary intervention). circulation 113(7):e166–e286. 14. saadeddin s.m, habbab m.a, sobki s.h, ferns g.a (2001). minor myocardial injury after elective uncomplicated successful ptca with or without stenting: detection by cardiac troponins. cathet. cardiovasc. interv. 53 (2), 188–192. 15. salam m. s, mohommed a. h, samia h. s, gordon a. f (2000). detection of minor myocardial injury after successful percutaneous transluminal coronary angioplasty with or without stenting. med sci monit 6(4):708–712. 16. garbarz e, iung b, lefevre g, makita y, farah b, michaud p, graine h, vahanian a (1999). frequency and prognostic value of cardiac troponin i elevation after coronary stenting. am. j. cardiol 84 (5), 515–518. 17. okmen e, cam n, sanli a, unal s, tartan z, vural m (2006). cardiac troponin i increase after successful percutaneous coronary angioplasty: predictors and long term prognostic value. angiology. 57(2):161–9. 18. abdelmeguid ae, topol ej, whitlow pl, sapp sk, ellis sg (1996). significance of mild transient release of creatine kinase mb fraction after percutaneous interventions. circulation 94: 1528–1536. 19. nienhuis, m.b., ottervanger, j.p., bilo, h.j., dikkeschei, b.d., zijlstra, f (2008). prognostic value of troponin after elective percutaneous coronary intervention: a met-analysis. catheter cardiovasc. interv 71 (3), 318–324. 20. mohammed a. al-qtaiby, hussein s. al-amri, abdulrahman m. al-moghairi (2011). the clinical significance of cardiac troponins in medical practice. journal of the saudi heart association 23, 3–11. 21. prasad a, rihal cs, lennon rj, singh m, jaffe as, holmes dr jr (2008). significance of periprocedural myonecrosis on outcomes after percutaneous coronary intervention:an analysis of preintervention and postintervention troponin t levels in 5487 patients.circ cardiovasc interv 1:10–9. 22. michael c. kontos (2010). prognostic value of isolated troponin i elevation after percutaneous coronary intervention. circ cardiovasc interv 3(5):431–5. 23. bahrmann p, werner gs, heusch g, ferrari m, poerner tc, voss a (2007). detection of coronary microembolization by doppler ultrasound in patients with stable angina pectoris undergoing elective percutaneous coronary interventions. circulation 115:600–8. 24. tolga kocum, mustafa yurtdas, turkay ozcan, burka akcay, tansel erol, ahmet camsari, oben doven (2008). direct stenting versus predilatation and stenting technique when using paclitaxel-eluting stents. int heart j 49(5);545–51. 25. cavallinin c, verdecchia p, savonitto s, et al (2010). prognostic value of isolated troponin i elevation after percutaneous coronary intervention. circ cardiovasc interv 3 (5):413–5. 26. dmitriy n. feldman, luke kim, a. garvey rene, robert m. minutello,geoffrey bergman, s. chiu wong (2011). prognostic value of cardiac troponin-i or troponin-t elevation following non-emergent percutaneous coronary intervention: a meta-analysis. catheterization and cardiovascular interventions. 77(7):1020–1030. 27. thygesen k, alpert js, jaffe as, simoons ml, chaitman br, white hd (2012). third universal definition of myocardial infarction. circulation 126:2020–2035. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i5.1065 377j contemp med sci | vol. 7, no. 6, november-december 2021: 377–383 original effect of nutritional factors and growth conditions on biosurfactant production by pseudomonas mendocina and pseudomonas oleovorans isolated from oil contaminated soil in jeddah city ebtihal a. motwali1*, magda m. aly2, huda a. qari2, nuha m. alhazmi1 1department of biology, college of science, univirsity of jeddah, jeddah, saudi arabia. 2department of biology, faculity of science, king abdulaziz univirsity, jeddah, saudi arabia. *correspondence to: ebtihal a. motwali (e-mail: ebtihalmotwali@gmail.com) abstract objectives: the current study aimed to investigate the impact of different nutritional factors and growth conditions on biosurfactant production by bacterial isolates (emb16 and emb21) isolated from oil-contaminated soil samples. methods: based on the used quantitative and qualitative screening method in current study, the selected bacterial isolates showed a high potential to produce biosurfactant. results: by using 16s rrna sequence analysis, the bacterial isolates emb16 and emb21 were found to be closely related to pseudomonas mendocina and pseudomonas oleovorans, respectively. the ultimate yields of biosurfactant (8.06 ± 0.06 mg/ml) by ps. mendocina emb16 was with corn oil as a carbon source, urea as a nitrogen source, c/n ratio of 30, ph value of 7, and 2% inoculum size. for ps. oleovorans emb21, the maximum biosurfactant production (4.68 ± 0.14 mg/ml) was achieved by diesel oil as a carbon source, urea as a nitrogen source, c/n ratio of 30, ph of 7, and 5% size of inoculum. the best incubation period and temperature for the examined strains was 168 hrs. at 37°c. conclusion: the results proved that ps. mendocina emb16 was the most efficient biosurfactant producer as it showed the greatest amount of biosurfactant concentration and lowest value of surface tension measurement with an emulsification index of 67 ± 6%. keywords: surface-active agents, sequence analysis, carbon, nitrogen, pseudomonas issn 2413-0516 introduction microorganisms are able to biosynthesize secondary metabolite and they may play critical roles in their growth. biological surface-active molecules are an example of such metabolites. biosurfactant are of great importance for microorganisms’ structural, functional diversity and broad-spectrum applications.1 although biosurfactant producing microorganisms were isolated from different environments, they were normally present in the oil debased soil.2 in such oil contaminated environment, biosurfactant production by microorganisms facilitate emulsification of the hydrocarbons.3 to isolate interesting biosurfactant producing microbes, effective screening analysis should be employed. several authors have reported that a single screening method is insufficient to select excellent biosurfactant producers.4–6 the production of biosurfactant by microorganisms are depends on various factors such as carbon source, nitrogen source, carbon to nitrogen ratio, ph, temperature, agitation, and oxygen availability.7 diverse metabolic pathways are involved in the synthesis of precursors for biosurfactant production, and this depend on the nature of the main carbon sources employed in the culture medium.8 the current research aimed to investigate the effect of different nutritional factors and growth conditions on biosurfactant production by selected bacterial isolates emb16 and emb21 which isolated from oil-contaminated soil collected from southern seashores in jeddah, saudi arabia. methodology isolation and screening to isolate biosurfactant producing bacteria, the enrichment method was applied using the procedure that described previously by motwali et al., (2020). number of quantitative and qualitative methods were used to screen the biosurfactant production ability of bacterial isolates. according to technique reported by motwali et al., (2020), drop collapse and ctab assay was used as qualitative method, while oil displacement test and surface tension measurement was utilized as a quantitative one.9 morphological characterization and molecular identification the morphological characterization and molecular identification of the purified selected bacterial isolates emb16 and emb21 were determined by using the method reported by motwali et al., (2021).10 effects of different nutritional factors and growth conditions on biosurfactant production a mineral salt medium containing 1% diesel oil as the sole carbon and energy source was used as a production medium.11 the structure of the used production medium was (g/l): 20 of nacl, 2.0 of kh2po4, 1.0 of nh4no3, 3.0 of na2hpo4, 0.7 of mgso4.7h2o. then, one ml/l of the trace element solution was added to the mineral salt medium. the trace element solution composition was (mg/l): znso4.7h2o, 10; cuso4.5h2o, 0.50; mnso4.h2o, 0.50; cacl2, 20; fecl3, 30, after which the solution was adjusted to ph 7.0.12 after that, the production medium was inoculated with 1% of selected bacterial subculture (bacterial od was 1.34 ± 0.02 at 600 nm). in order to examine the impact of nutritional factors on biosurfactant production, the production medium was supplemented with different carbon source (glucose, glycerol, corn oil, olive oil, sunflower oil, sesame oil, mustard oil, xylene, diesel, toluene, (submitted: 06 september 2021 – revised version received: 19 september 2021 – accepted: 22 october 2021 – published online: 26 december 2021) 378 j contemp med sci | vol. 7, no. 6, november-december 2021: 377–383 effect of nutritional factors and growth conditions on biosurfactant production original e.a. motwali et al. or lubricating oil), nitrogen sources (yeast extract, peptone urea, nano3, kno3, or nh4no3) at ratio of c/n (10, 20, 30, 40 or 50). range of growth parameters were also investigated such as ph value (3, 5, 7, 9 or 11), temperature (20–50°c), inoculum size (0.5–7%), and incubation periods (96, 168, 240 or 312 hrs.). the concentration of the produced biosurfactant in bacterial supernatant was done indirectly by using orcinol assay by using the approach reported previously by motwali et al., (2021).10 detection the activity of the produced biosurfactant after incubating the chosen bacterial culture under proper nutritional factors and growth conditions, emulsification index (ei24) and surface tension measurement of bacterial supernatant were calculated. emulsification index (ei24) was determined by applying the same procedure as described by gagelidze et al., (2016).13 surface tension measurement was done at room temperature using a tensiometer (kruss force k6). statistical analysis statistical analysis was performed using the statistical package for social sciences version 24.0 for windows (spss inc., armonk, ny, usa). kruskal-wallis test was carried out to identify a significant of responses to study nutritional factors and growth parameters. result screening the selected bacterial isolates for biosurfactant production the selected bacterial isolates emb16 and emb21 showed positive activity on qualitative screening methods ctab assay and drop collapse test. furthermore, they were able to spread the oil in an oil spreading test by more than 2.00 cm diameter as shown in (figure 1a). also, they were able to reduce surface tension to <45 mn/m (figure, 1b). morphological characterization and molecular identification the selected bacterial isolates emb16 and emb21 were characterised as an aerobic gram negative non spore forming bacteria. molecular identification of the selected isolates was performed using the genbank blast tool on the 16s rrna gene sequences. the selected bacterial isolate emb16 was closely related (98.71%) to pseudomonas mendocina under accession number mk 640833.1 whereas emb21 was closely related (99.73%) to pseudomonas oleovorans with accession number mk078535.1 and phylogenetic trees are shown in figures 2 and 3. effects of different nutritional factors and growth conditions on biosurfactant production the effect of different carbon sources on biosurfactant production by chosen strains ps. mendocina emb16 (mk640833.1) and ps. oleovorans emb21 (mk078535.1) are shows in table 1 and figure 4. it is clear from table that corn oil provides the greatest significant amount of biosurfactant concentrations (7.85 ± 0.2 mg/ml) by ps. mendocina emb16 (mk640833.1). furthermore, the production of biosurfactant by ps. mendocina emb16 was also significantly and sensitively increased with glycerol (7.48 ± 0.5 mg/ml), glucose (7.15 ± 0.7) and olive oil table 1. the effect of different carbon sources on biosurfactant (rhamnolipid) concentration produced by ps. mendocina emb16 and ps. oleovorans emb21 carbon source ps. mendocina emb16 ps. oleovorans emb21 glucose 7.15 ± 0.7*s3 3.45 ± 0.2*s4 glycerol 7.48 ± 0.5*s3 2.55 ± 0.4s olive oil 7.45 ± 0.1*s3 2.76 ± 0.3s corn oil 7.85 ± 0.2*s4 2.75 ± 0.3s sunflower oil 5.03 ± 0.3 1.77 ± 0.3r* sesame oil 5.16 ± 0.4r 2.69 ± 0.4 mustard oil 2.46 ± 0.4r* 2.37 ± 0.2r xylene 1.99 ± 0.2r* 1.44 ± 0.03r* diesel 5.45 ± 0.4s 4.24 ± 0.11**s4 toluene 2.04 ± 0.3r* 1.53 ± 0.14r* lubricating oil 1.95 ± 0.3r* 1.53 ± 0.2r*  highest value, *, significant regard kruskal-wallis test; **, significant adjusted using bonferroni; s, sensitive (increasingly affect); number above value1-5, number of pairwise comparisons; r, resistance (decreasingly affect). fig. 1 a) result of oil displacement test and b) result of surface tension measurement of chosen bacterial isolates emb16 and emb 21. fig. 2 the phylogenetic tree of pseudomonas mendocina emb16. fig. 3 the phylogenetic tree for pseudomonas oleovorans emb21. 379j contemp med sci | vol. 7, no. 6, november-december 2021: 377–383 e.a. motwali et al. original effect of nutritional factors and growth conditions on biosurfactant production table 2. the effect of different nitrogen sources on biosurfactant (rhamnolipid) concentration produced by ps. mendocinaemb16 and ps. oleovorans emb21 nitrogen source ps. mendocina (emb16) ps. oleovorans (emb21) yeast extract 6.60 ± 0.33*s1 3.44 ± 0.08r* peptone 6.18 ± 0.22r* 2.20 ± 0.13r* urea 8.06 ± 0.05**s3 4.44 ± 0.11**s4 nano 3 6.23 ± 0.30r* 4.00 ± 0.20*s1 kno 3 3.10 ± 0.16r* 3.10 ± 0.16r* nh 4 no 3 7.64 ± 0.31*s1 4.26 ± 0.23*s3  highest value, *, significant regard kruskal-wallis test; **, significant adjusted using bonferroni; s, sensitive (increasingly effect); number above value1-5, number of pairwise comparisons; r, resistance (decreasingly effect). fig. 4 the effect of different carbon to nitrogen ratio on biosurfactant (rhamnolipid) concentration produced by the two chosen pseudomonas species. fig. 5 the effect of changing ph on biosurfactant concentration produced by the two selected pseudomonas species. (7.45 ± 0.1 mg/ml). for ps. oleovorans emb21 (mk078535.1), among different investigated carbon source diesel and glucose result in greatest significant biosurfactant concentration (4.24 ± 0.11 and 3.45 ± 0.2 mg/ml respectively). the amount of biosurfactant by examined pseudomonas species with mustered oil, toluene, xylene and lubricating oil was significantly decreased. after the selection of suitable carbon source for each examined bacterial isolates: ps. mendocina emb16 (mk640833.1) and ps. oleovorans emb21 (mk078535.1), the effect of different nitrogen sources on biosurfactant production were also investigated. the recorded result suggests that the highest biosurfactant concentration was noted with urea or ammonium nitrate nh4no3 as a nitrogen source in the production media for all tested bacterial strains as shown in table 2. statically, biosurfactant production by ps. mendocina emb16 also was significantly improved with yeast extract, while with nano3 for ps. oleovorans emb21. effects of different c/n ratios on biosurfactant production a suitable carbon source for each tested pseudomonas species was added to the production media with different concentrations, along with a constant concentration of a selected nitrogen source (urea). the greatest biosurfactant concentrations obtained by ps. mendocina emb16 (7.80 ± 0.07 mg/ml) and ps. oleovorans emb21 (4.26 ± 0.06 mg/ml), were recorded at c/n ratio of 30 (figure 4). the result of statistical analysis showed that the c/n ratio of 10 and 20 also lead to an increase in the production of biosurfactant by ps. mendocina emb16. similar performance was observed for ps. oleovorans emb21 by c/n ratio of 10. the ph of the production media for the tested pseudomonas species was adjusted to different values. it is clear from figure 5 that the highest significantly biosurfactant yield by the examined pseudomonas species was at natural condition (ph 7) and the lowest was at acidic conditions (ph of 3). for ps. mendocina emb16, the biosurfactant concentration was also significantly improved at ph 9 whereas for ps. oleovorans emb21 at ph 5. after optimisation experiments for proper nutritional factors and ph value, the effect of incubation temperature also determined. the maximum significantly biosurfactant yield by ps. mendocina emb16 and ps. oleovorans emb21 (mk078535.1) was 8.19 ± 0.16 and 4.07 ± 0.10 mg/ml, respectively, at 37ºc (figure 6). in addition, the production of biosurfactant by examined bacterial strains significantly augmented at temperatures of 35 and 40ºc. several inoculum size of ps. mendocina emb16 and ps. oleovorans emb21 were studied. the results represented in figure 7 indicated that, the highest significantly biosurfactant production by ps. mendocina emb16 was 8.11 ± 0.05 mg/ml was at 2.0% inoculum size. moreover, the result of statistical analysis indicated that the biosurfactant yield at 1.0, 4.0 and 5.0% inoculum size were not significantly differed. though, for ps. oleovorans the best significantly biosurfactant yield was 4.84 ± 0.23 mg/ml with 5.0% inoculum size. at inoculum size of 6 and 5%, no significant differed was found. fig. 6 the impact of different incubation temperature on biosurfactant concentration produced by the chosen pseudomonas species. 380 j contemp med sci | vol. 7, no. 6, november-december 2021: 377–383 effect of nutritional factors and growth conditions on biosurfactant production original e.a. motwali et al. shown to be able to use hydrocarbons (diesel oil) as their sole carbon source. supplementation of the isolation medium by hydrocarbon as a sole carbon source to isolate biosurfactant producing bacteria from oil contaminated environments was reported by many researchers.14–17 in terms of screening the biosurfactant producing ability of bacterial isolates emb16 and emb21, there are many distinct procedures that can be used, both qualitative and quantitative types. as reported by satpute et al. (2008), more than one screening method should be used in the primary screening for the biosurfactant producers.4 in present study, drop collapse test and ctab agar assay as a qualitative investigates have been applied. as quantitative screening method, oil displacement test and surface tension measurement have been used. the bacterial isolates emb16 and emb21 in current study showed positive activity in the used qualitative methods this indicated they could be producing biosurfactant. droplet collapses allow for speedy screening of a bacteria’s efficacy as a biosurfactant producer. mostly, this assay has been applied several times for screening purposes by many researchers.18-20 fig. 7 the effect of different inoculum sizes on biosurfactant concentration produced by chosen pseudomonas species. fig. 8 the effect of different incubation time on biosurfactant (rhamnolipid) concentration produced by selected pseudomonas species. table 3. the best nutritional factors and growth conditions for each tested pseudomonas species, the amount of produced biosurfactant, the recorded emulsification index and surface tension value factor ps. mendocina emb16 ps. oleovorans emb21 carbon source corn oil diesel oil nitrogen source urea urea c/n ratio 30 30 ph value 7 7 temperature (°c) 37 37 inoculum size (%) 2 5 incubation period (hrs.) 168 168 yield of biosurfactant (mg/ml) 8.06 ± 0.06 4.68 ± 0.14 surface tension (n/m) 31.6 ± 0.6 42 ± 1.0 emulsification index % 67 ± 6 60 ± 8 the two tested bacterial strains ps. mendocina emb16 and ps. oleovorans emb21 were incubated for different time in the production medium with suitable selected nutritional factors and environmental parameter. the highest significantly biosurfactant amount by chosen pseudomonas species, were at 168 hrs. or 7 days of incubation period (figure 8). furthermore, the lowest value of biosurfactant concentration for bacterial strains was observed at 96 hrs. of incubation period. the production of biosurfactant by ps. oleovorans was maximum at 312 hrs. of incubation. the activity of the produced biosurfactant the production media were prepared with selected nutritional factors and growth conditions for each bacterial strain ps. mendocina emb16 and ps. oleovorans emb21 (table 3). after incubation period, the emulsification index ei24 and surface tension measurement for each bacterial supernatant ps. mendocina emb16 and ps. oleovorans emb21 were investigated. the result indicated that ps. mendocina emb16 recorded the lowest value (31.6 ± 0.6 mn/m) in decrease the surface tension in comparison with ps. oleovorans emb21 (42 ± 1.0 mn/m). for emulsification index ei24, ps. mendocina emb16 and ps. oleovorans emb21 were able to emulsify diesel oil by 67 and 60 %, respectively. it has been observed that the highest significantly biosurfactant concentration was reported for ps. mendocina emb16 (8.06 ± 0.06 mg/ml) in comparison with ps. oleovorans emb21 (4.68 ± 0.14 mg/ml). this finding suggested that ps. mendocina emb16 could produce large amount of biosurfactant (glycolipid), reduce the surface tension to less than 35 mn/m) and emulsify diesel oil by more than 50%. the statistically analysis indicates that ps. mendocina emb16 was the efficient biosurfactant producing pseudomonas isolate. discussion the present research was aimed to produce surface-active material from bacteria isolated from oil polluted samples. the oil contaminated soil samples were collected from southern shores of jeddah city, saudi arabia. isolation of bacteria which have capability to produce biosurfactant was done by enrichment culture method, which minimal medium was supplemented with hydrocarbon (diesel oil) as a sole carbon source. the selected bacterial isolates emb16 and emb21 have been 381j contemp med sci | vol. 7, no. 6, november-december 2021: 377–383 e.a. motwali et al. original effect of nutritional factors and growth conditions on biosurfactant production the cetyltrimethylammonium bromide (ctab) agar or blue agar assay is a specific screening method for anionic biosurfactants. it is used for the detection of glycolipid-type biosurfactant production by the bacterial colonies in the culture plate directly.21 in present investigation, dark bluish ring result that detected on ctab agar by the supernatant of the bacterial isolates emb 16 and emb21 reveal the ability of anionic biosurfactant production by these selected bacterial isolates. this is in accordance with nayarisseri et al., (2019) who found that 4 bacterial isolates belonging to pseudomonas sp. and bacillus sp. showed positive activity in ctab test that confirmed existence of an anionic biosurfactant.22 the oil displacement test is a rapid quantitative method to test the presence of biosurfactant in the cell free culture broth. in addition, this method can detect even low activity and quantity of biosurfactant present. in this study, bacterial isolates emb16 and emb21 showed spreading the crude oil by more than 2.5 cm diameter. the result suggests the presence of biosurfactant. the present value of oil displacement test is lower than that obtained by ibrahim, (2018) who detected a diameter > 5.0 cm of oil displacement test by screened bacterial isolates ochrobactrum anthropi hm-1 and citrobacter freundii hm-2.5 oil displacement test was used often for biosurfactant production screening purpose by researchers.23,24 to further confirm the ability of bacterial isolates emb16 and emb21 to produce biosurfactant, the cell free culture broths of the selected isolates were subjected to surface tension measurement. surface tension measurement is an important quantitative assay for evaluating surface activity of the tested isolates. the reduction in surface tension values achieved by the selected isolates emb16 and emb21 was <45 mn/m. this finding suggests the biosurfactant production ability by these examined isolates. in addition, the current value of surface tension was higher than that observed by ahmad et al., (2016), sun et al., (2018) and ibrahim, (2018).5,14,25 they found a decrease in surface tension to less than 40 mn/m by different tested biosurfactant producing isolates. molecular identification for isolated bacteria was done by used 16s rrna. generally, 16s rrna gene sequencing is an effective tool that has been used to identify bacteria and to find relationships between different bacterial genera. the 16s rdna sequence of the selected isolates emb16 and emb21 has been submitted to the genbank database under the accession number mk640833.1 and mk078535.1, respectively. the results of 16s rdna sequence using the genbank blast tool proved that the isolates emb16 and emb21 were belong to genus pseudomonas. the bacterial isolate emb16 showed 98.71% similarity to pseudomonas mendocina, while emb21 showed 99.73% similarity to pseudomonas oleovorans. number of previous investigators used 16s rrna gene sequencing to identify biosurfactant producing bacteria.15,16,26 the biosurfactant producing bacteria isolated from oil contaminated environment that belong to pseudomonas sp. was reported by previous researchers.9,27–29 commonly, bacterial isolates that belong to genus pseudomonas are the greatest biosurfactant producers.30 there are not many studies about biosurfactant produce ability by pseudomonas mendocina and pseudomonas oleovorans. in contrary, different investigations study the production of biosurfactant from different pseudomonas spices such as: pseudomonas aeruginosa,31–33 pseudomonas nitroreducens,34 pseudomonas fluorescens,35 pseudomonas putida36 and pseudomonas balearica.10 distinct nutritional factors and growth parameters were selected to investigate their effect on biosurfactant production by chosen isolates ps. mendocina emb16 and ps. oleovorans emb21. in the culture medium, carbon source played important role in increasing biosurfactant yield according to noh et al., (2014).37 the significant highest yield of biosurfactant from ps. mendocina emb16 was 7.85 ± 0.2 mg/ml with corn oil as a type of plant oil. on the other hand, the ultimate biosurfactant concentration (4.24 ± 0.11 mg/ml) for ps. oleovorans emb21 was observed with diesel oil. for ps. mendocina same trend was observed by pseudomonas sp. with plant oil as a carbon by researchers (motwali et al., 2021 and sun et al., 2021).10,38 inversely, onwosi and odibo (2012) noted that the yield from diesel oil was higher than vegetable oil.39 the similar trend was observed with diesel oil as a carbon source in present study by ps. oleovorans emb21. since nitrogen sources play a vital role in the production of biosurfactant, effect of different nitrogen sources on biosurfactant production were studied. the current study found the highest significant biosurfactant concentration produced by ps. mendocina emb16 (8.06 ± 0.05 mg/ml), and ps. oleovorans emb21 (4.44 ± 0.11 mg/ml) were when urea was utilised as a nitrogen source in the production medium. as well, ps. mendocina emb16 and ps. oleovorans emb21 were able to produce higher biosurfactant concentration (7.64 ± 0.31 and 4.26 ± 0.23, mg/ml, respectively) with nh4no3 among tested nitrogen sources. this in agreement with motwali et al., (2021) who found that urea or nh4no3 were the suitable nitrogen source for biosurfactant production by ps. balearica.10 the present result also is agreeing with alyousif et al., (2020) who found that urea was a best nitrogen source for biosurfactant production by ps. aeruginosa.40 the c/n ratio also affects the production of biosurfactant by bacterial isolates. the highest biosurfactant production for the two examined pseudomonas species (ps. mendocina: 7.80 ± 0.07 and ps. oleovorans: 4.26 ± 0.06 mg/ ml) were obtained at a c/n ratio of 30. at the c/n ratio above 30, the bacterial isolate ps. mendocina also recorded higher amount of biosurfactant. less than c/n ratio of 30 (around 20) was found to be suitable for biosurfactant production by members of pseudomonas sp.41 in contrary, above c/n ratio of 30 was observed to be proper for biosurfactant production by ps. aeruginosa.42 additionally, prieto et al., (2008) proved that a nitrogen-limiting condition (c/n ratio of 100) was favorable to biosurfactant production by ps. aeruginosa lbm10.43 it is essential to define the suitable ph value for biosurfactant production by tested pseudomonas isolates. the current research found that the best production of biosurfactant by ps. mendocina emb16 and ps. oleovorans emb21 was at ph 7. it is worth noting that, increasing the ph above 7 results in a decrease in biosurfactant production by the tested pseudomonas isolates. this finding is approximately in the same trend with sun et al., (2021), who found that the ph 7–8 was optimal for biosurfactant production by pseudomonas sp.38 also, they found ph >8 and ph <7 result in dropped in biosurfactant production by pseudomonas sp. similarly, maximum amount of biosurfactant from mutated strain of bacillus subtilis was obtained at ph 7.44 the result of present research is unagreed with kannahi and sherley (2012) who reported that a maximum level of biosurfactant production by pseudomonas sp. was below ph 7.45 382 j contemp med sci | vol. 7, no. 6, november-december 2021: 377–383 effect of nutritional factors and growth conditions on biosurfactant production original e.a. motwali et al. generally, optimization temperature has a significant impact on the enzyme activity and metabolic rate of the microbial isolates. in this investigation, the optimum temperature for maximum biosurfactant production by ps. mendocina emb16 and ps. oleovorans emb21 were 8.19 ± 0.16 and 4.07 ± 0.10 mg/ml, respectively at 37°c. the bacterial isolates were also able to produce a high significantly biosurfactant concentration at temperature of 35 and 40ºc. the result of the current study is nearly in agreement with yaraguppi et al., (2020) who found that the incubation temperature of 35–40°c resulted in a great biosurfactant yield by bacillus aryabhattai strain zdy2.46 overall, increase in a temperature above 40°c led to decrease in biosurfactant yield by the two tested pseudomonas isolates. same trend was obtained previously by soniyamby et al., (2011) for the biosurfactant production by ps. aerogenesis.47 the inoculum size also plays important role in biosurfactant production by microbial isolates since it is related to the number of microbial cells in a used inoculum. optimal inoculum size means optimal number of bacterial cells for bacterial reproduction and different bacterial activity. the present investigation obtained a great biosurfactant concentration by ps. mendocina emb16 (8.11 ± 0.05 mg/ml), ps. oleovorans emb21 (4.84 ± 0.23 mg/ml) with inoculum size of 2.0 and 5.0%, respectively. silva et al., (2018) found that 3.0% inoculum size was the best for biosurfactant.48 however, an inoculum size 4–5% was reported to be optimal for biosurfactant (rhamnolipid) production.49 the optimum incubation time for biosurfactant production by ps. mendocina emb16 (8.06 ± 0.06 mg/ml) and ps. oleovorans emb21 (4.68 ± 0.14 mg/ml) was at 168 hrs. nearly, the result herein agrees with the finding of alyousif et al., (2020) who reported that the greatest biosurfactant concentration by ps.aeruginosa was after 144 hrs. of incubation period.40 the present finding is contradicting the study of devaraj et al., (2019) who found that the maximum yield obtained by pseudomonas mosselii was at 96 hrs.50 after optimization the production medium, the surface tension of the culture supernatant of ps. mendocina emb16 and ps. oleovorans emb21 at the end of the incubation period (169 hrs.) was 31.6 ± 0.6 and 42 ± 1.0 mn/m, respectively. peekate and abu (2017) recorded a surface tension value of 30.64 mn/m after optimization the production medium by ps. fluorescens.35 other than, motwali et al., (2021) achieved a reduce in surface tension by ps. balearica to 34 mn/m by using optimum nutrational and growth condition for biosurfactant production.10 the result of emulsification index ei 24% biosurfactant by ps. mendocina emb16 and ps. oleovorans emb21 showed emulsification activity by ≥60%. this amount is higher than that reported by abouseoud et al. (2008) who recorded an emulsifying activity of 49% by ps. fluorescens with olive oil as the best carbon source.51 the highest biosurfactant concentration and the lowest surface tension value was achieved by ps. mendocina emb16 in present research. the statistically analysis indicates that, the ps. mendocinawas significantly the efficient biosurfactant producing pseudomonas isolates. it is worth mention that ps. oleovorans although it showed activity on emulsification index >50 it recorded surface tension >40 with low biosurfactant (rhamnose sugar) concentration. so, ps. oleovorans emb21 could be able to produce a bioemulsifier beside biosurfactant. according to uzoigwe et al., (2015), bioemulsifiers have emulsifying activity more than surface activity (lower the surface tension).52 conclusion the current research concludes that the nutritional factors and growth conditions play critical role in the biosurfactant production. the study proved that bacterium ps. mendocina (emb16) isolated from oil contaminated soil was efficient biosurfactant producing bacterium. it was remarked that corn oil as a carbon source, urea as a nitrogen source, c/n ratio of 30, 2% inoculum size at 37°c for 186 hrs. of incubation period provides the best biosurfactant production by ps. mendocina emb16. this pseudomonas species can be employed further for larger biosurfactant production. since the produced biosurfactant by ps. mendocina emb16 showed emulsifying and surface activity, it can be applied in bioremediation, in industrial and medical application.  references 1. banat, i.m. and thavasi, r. (2019) microbial biosurfactants and their environmental and industrial applications. in: introduction to microbial biosurfactant, banat, i.m. and thavasi, r. (editors), crc press, boca raton, florida, 01-16. 2. roy, a. (2017) review on the biosurfactants: properties, types and its applications. journal of fundamentals of renewable energy and applications, 8: 248-253. 3. stroud, j.l.; paton, g.i.; semple, k.t. (2007) microbe–aliphatic hydrocarbon interactions in soil; implications for biodegradation and bioremediation. journal of applied microbiology, 102:1239–1253. 4. satpute, s.k.; bhawsar b.d.; dhakephalkar, p.k. and chopade, b.a. (2008) assessment of different screening methods for selecting biosurfactant producing marine bacteria.indian journal of marine sciences, 37: 243–250. 5. ibrahim, h.m.m. (2018) characterization of biosurfactants produced by novel isolats of ochrobactrumanthropi hm-1 and citrobacter freundii hm-2 from used engine oil-contaminated soil. egyptian journal of petroleum, 27: 21-29. 6. adebajo, s.o.; akintokun, p.o.; ojo, a.e.; akintokun, a.k. and badmos, o.a. (2020) recovery of biosurfactant using different extraction solvent by rhizospheric bacteria isolated from rice-husk and poultry waste biocharamended soil, egyptian journal of basic and applied sciences, 7:1, 252-266. 7. jahan, r.; bodratti, a.m.; tsianou, m. and alexandridis, p. (2020) biosurfactants, natural alternatives to synthetic surfactants: physicochemical properties and applications. advances in colloid and interface science, 275: 22 pages. 8. santos, d.k.f.; luna, j.m.; rufino, r.d.; santos, v.a. and sarubbo, l.a. (2016) biosurfactants: multifunctional biomolecules of the 21st century. international journal of molecular sciences, 17(3):401–430. 9. motwali, e.a.; aly, m.m.; qari, h. et al. (2020) screening and identification of efficient biosurfactant producing bacteria for some medical applications. la prensa medica argentina, 2:005: 6. 10. motwali, e.a.; aly, m.m.; qari, h.a.; amasha, r.h. and zabermawi, n.m. (2021) effect of growth conditions on biosurfactant production by pseudomonas balearica isolated from oil contaminated sea waters from jeddah saudi arabia. bioscience biotechnology research communication, 14(1): 9 pages. 11. yalaoui-guellal, d.; brahmi, f.; touati, a.; de champs, c.; banat, i.m. and madania, k. (2017) production of biosurfactants by hydrocarbons degrading bacteria isolated from soummam watershed sediments of bejaia in algeria environmental progress and sustainable energy, 37(1) 189-195. 12. mao-cheng, d.; jing, l.; fu-rui, l.; meisheng, y.; xiao-ming, x.; jian-ping, y.; juan, p.; chou-fei, w. and jiang-hai, w. (2014). isolation and characterization of a novel hydrocarbon-degrading bacterium achromobacter sp. hz01from the crude oil-contaminated seawater at the daya bay, southern china. marine pollution bulletin, 83: 79–86. 13. gagelidze, n.a.; amiranashvili, l.l.; varsimashvili, k.i.; tinikashvili, l.m.; lana, l.; tolordava, l.l. and sadunishvili, t.a. (2016) selection of effective biosurfactant producers among bacillus isolats isolated from soils of georgia. annals of agrarian science, (14): 72-75. 383j contemp med sci | vol. 7, no. 6, november-december 2021: 377–383 e.a. motwali et al. original effect of nutritional factors and growth conditions on biosurfactant production 14. ahmad, z.; arshad, m.; asghar, h.n.; sheikh, m.a. and crowley, d.e. (2016) isolation, screening and functional characterization of biosurfactant producing bacteria isolated from crude oil contaminated site. international journal of agricultural and biological engineering, 18: 542–548. 15. zainal, n.; omar, s. and ashaari, m. (2017) isolation and characterization of biosurfactant-producing bacteria isolated from petroleum contaminated sites with the potential to be used in bioremediation. science heritage journal, 1(2):11-15. 16. soltanighias, a.; singh, a.e.a.; satpute, s.k.; banpurkar, a.g.; koolivand, a. and rahi, p. (2019) assessment of biosurfactant-producing bacteria from oil contaminated soils and their hydrocarbon degradation potential. environmental sustainability, 2: 285-296. 17. channashettar, v.; srivastava, s. and lal b. (2020) isolation and characterization of biosurfactant producing bacteria from oil sludge for bioremediation of oil contaminated sites. international journal of new innovations in engineering and technology, 12: 063-041. 18. krishnan, m., subramanian, h., dahms, h.u., et al., (2018). biogenic corrosion inhibitor on mild steel protection in concentrated hcl medium. scientific reports, 8(1):1–16. 19. oso, s.; walters, m.; schlechter, r.o. and remus-emsermann, m. (2019) utilisation of hydrocarbons and production of surfactants by bacteria isolated from plant leaf surfaces. fems microbiology letters, 366 (6): 10 pages. 20. wu, y.; xu, m.; xue, j.; shi, k. and gu, m. (2019) characterization and enhanced degradation potentials of biosurfactant-producing bacteria isolated from a marine environment. acs omega, 4 (1):1645-1651. 21. irorere, v.u.; tripathi, l.; marchant, r.; mcclean, s. and banat, i.m. (2017) microbial rhamnolipid production: a critical reevaluation of published data and suggested future publication criteria. applied microbiology and biotechnology, 101: 3941-3951. 22. nayarisseri, a.; singh, p. and singh, s.k. (2019) screening, isolation and characterization of biosurfactant-producing bacillus tequilensis strain ansklab04 from brackish river water. international journal of environmental science technology. 16: 7103–7112. 23. gargouri, b.; contreras, m.d.m.; ammar, s. et al. (2017) biosurfactant production by the crude oil degrading stenotrophomonas sp. b-2: chemical characterization, biological activities and environmental applications. environment science and pollution research, 24: 3769–3779. 24. dattaa, p.; tiwaria, p.; pandey, l.m. (2018) isolation and characterization of biosurfactant producing and oil degrading bacillus subtilis mg495086 from formation water of assam oil reservoir and its suitability for enhanced oil recovery. bioresource technology, 270:439–448. 25. sun, w.; cao, w.; jiang, m.; saren, g.; liu, j.; cao, j.; ali, i.; yu, x.; peng, c. and naz, i. (2018) isolation and characterization of biosurfactant-producing and diesel oil degrading pseudomonas sp. cq2 from changqing oil field, china. royal society of chemistry, 8: 39710-39720. 26. elazzazy, a.m.; abdelmoneim, t.s. & almaghrabi, o.a. (2015) isolation and characterization of biosurfactant production under extreme environmental conditions by alkali-halo-thermophilic bacteria from saudi arabia. saudi journal of biological sciences, 22:466–475. 27. joy, s.; rahman, p.k. and sharma, s. s. (2017) biosurfactant production and concomitant hydrocarbon degradation potentials of bacteria isolated from extreme and hydrocarbon contaminated environments. chemical engineering journal, 317: 232-241. 28. varjani, s and upasani, v.n. (2019) evaluation of rhamnolipid production by a halotolerant novel strain of pseudomonas aeruginosa. bioresource technology, 288: 6 pages. 29. zouari, o.; lecouturier, d.; rochex, a.; chataigne, g.; dhulster, p.; jacques, p. and ghrib, d. (2019) bio-emulsifying and biodegradation activities of syringafactin producing pseudomonas spp. isolates isolated from oil contaminated soils. biodegradation, 30:259–272. 30. sharma, d.; ansari, m.j.; al-ghamdi, a.; adgaba, n.; khan, k.a.; pruthi, v. and al-waili, n. (2015) biosurfactant production by pseudomonas aeruginosa dsvp20 isolated from petroleum hydrocarbon-contaminated soil and its physicochemical characterization. environmental science and pollution research, 22:17636–17643. 31. liu, w.j.; duan, x.d.; wu, l.p. et al. (2018) biosurfactant production by pseudomonas aeruginosa snp0614 and its effect on biodegradation of petroleum. applied biochemistry and microbiology, 54: 155–162. 32. de anchieta câmara jr, a., maréchal, p. a., tourdot-maréchal, r., & husson, f. (2019). oxidative stress resistance during dehydration of three nonsaccharomyces wine yeast strains. food research international, 123:364–372. 33. conceição, k.s.; almeida, m.; sawoniuk, i.c. et al. (2020) rhamnolipid production by pseudomonas aeruginosa grown on membranes of bacterial cellulose supplemented with corn bran water extract. environmental science and pollution research, 27:30222–30231. 34. de sousa, t. and bhosle, s. (2012) isolation and characterization of a lipopeptide bioemulsifier produced by pseudomonas nitroreducens tsb. mj10 isolated from a mangrove ecosystem. bioresource technology, 123: 256–262. 35. peekate, p. l. and abu, g. o. (2017) optimizing c:n ratio, c:p ratio, and ph for biosurfactant production by pseudomonas fluorescens. journal of advances in microbiology, 7(2): 1-14. 36. janeka, t.; łukaszewicz, m. and krasowska, a. (2013) identification and characterization of biosurfactants produced by the arctic bacterium pseudomonas putida bd2. colloids and surfaces b: biointerfaces, 110: 379–386. 37. noh, n.a.; salleh, s.m. and yahya, a.r. (2014) enhanced rhamnolipid production by pseudomonas aeruginosa usm-ar2 via fed-batch cultivation based on maximum substrate uptake rate. letters in applied microbiology, 58:617–623. 38. sun, w.; zhu, b.; yang, f.; dai, m.; sehar, s.; peng, c.; ali, i. and naz, i. (2021) optimization of biosurfactant production from pseudomonas sp. cq2 and its application for remediation of heavy metal contaminated soil. chemosphere, 265: 12 pages. 39. onwosi, c.o. and odibo, f.j.c. (2012) effects of carbon and nitrogen sources on rhamnolipid biosurfactant production by pseudomonas nitroreducens isolated from soil. world journal of microbiology & biotechnology, 28:937–942. 40. alyousif, n.a.; al-tamimi, w.h. and al-luaibi, y.y.y. (2020). screening enhance production and characterization of biosurfactant produced by pseudomonas aeruginosa isolated from hydrocarbon contaminated soil. eurasian journal of bioscience, 14: 4377-4391. 41. hassan, m., essam, t., yassin, a.s. and salama, a. (2016) optimization of rhamnolipid production by biodegrading bacterial isolates using plackettburman design. int j biol macromol 8, 573–579. 42. sharma, s.; datta, p.; kumar, b.; tiwari, p. and pandey, l.m. (2019b). production of novel rhamnolipids via biodegradation of waste cooking oil using pseudomonas aeruginosa mtcc7815. biodegradation, 30:301–312. 43. prieto, l.m.; michelon, m.; burkert, j.f.m.; kalil, s.j. and burkert, c.a.v. (2008) the production of rhamnolipid by a pseudomonas aeruginosa strain isolated from a southern coastal zone in brazil. chemosphere, 71:1781–1785. 44. asgher, m.; afzal, m.; qamar, s.a. et al. (2020) optimization of biosurfactant production from chemically mutated strain of bacillus subtilis using waste automobile oil as low-cost substrate. environmental sustainability, 3: 405413. 45. kannahi, m. and sherley, m. (2012) biosurfactant production by pseudomonas putida and aspergillus niger from oil contaminated site. international journal of chemical and pharmaceutical sciences, 3(4): 37-42. 46. yaraguppi, d.a.; bagewadi, z.k.; muddapur, u.m. and mulla, s. (2020) response surface methodologybased optimization of biosurfactant production from isolated bacillus aryabhattai strain zdy2. journal of petroleum exploration and production technology, 10:2483–2498. 47. soniyamby, a.r.; praveesh, b.v.; vimalin, h.; kavithakumari, p.; sundaram, l. and palaniswamy, m. (2011) enhanced production of biosurfactant from isolated pseudomonas sp. growing on used edible oil. journal of american science, 7(6): 50-53. 48. silva, e.j.; correa, p.f.; almeida, d.g.; luna, j.m.; rufino, r.d. and sarubbo, l.a. (2018) recovery of contaminated marine environments by biosurfactantenhanced bioremediation. colloids and surfaces b: biointerfaces, 172:127-135. 49. neto, d.c.; meira, j.a.; tiburtius, e.; zamora, p.p.; bugay, c.; mitchel, d.a.; krieger, n. (2009) production of rhamnolipids in solid-state cultivation: characterization, downstream processing and application in the cleaning of contaminated soils. bioethanol journal, 4(5):748-755. 50. devaraj, s.; sabapathy, p.c.; nehru, l. and preethi, k. (2019) bioprocess optimization and production of biosurfactant from an unexplored substrate: parthenium hysterophorus. biodegradation, 30: 325–334. 51. abouseoud, m.; maachi, r.; amrane, a.; boudergua, s. and nabi, a. (2008) evaluation of different carbon and nitrogen sources in production of biosurfactant by pseudomonas fluorescens. desalination, 223: 143–151. 52. uzoigwe, c.; burgess, j. g.; ennis, c. j. and rahman, p. k. (2015) bioemulsifiers are not biosurfactants and require different screening approaches. frontiers in microbiology, 6: 245-251. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1133 144 j contemp med sci | vol. 9, no. 3, may-june 2023: 144–151 original demographic and clinical profiles, including comorbidities, of hospitalized patients under 18 years of age with covid-19 azadeh memarian1, seyed khosro ghasempouri1, mehran kouchek2* , behnam sobouti3, kamran aghakhani4 1department of emergency medicine, faculty of medicine, mazandaran university of medical sciences, sari, iran. 2department of critical care medicine, faculty of medicine, shahid beheshti university of medical sciences, tehran, iran. 3department of pediatrics, faculty of medicine, iranuniversity of medical sciences, tehran, iran. 4department of forensic medicine and toxicology, school of medicine, iran university of medical sciences, tehran, iran. *correspondence to: mehran kouchek (e-mail: mehrankouchek@yahoo.com) (submitted: 08 january 2023 – revised version received: 24 february 2023 – accepted: 15 march 2023 – published online: 26 june 2023) objectives: this study was aimed to evaluate the various demographic and clinical characteristics in hospitalized children with covid-19 as well as their comorbidities. methods: this cross-sectional study evaluated a total of 809 hospitalized covid-19 patients under 18 years of age in the referral university based ali-asghar hospital in 2020. demographic and clinical characteristics of patients were extracted from the archived records and data analysis was performed using spss software version 26. results: the mean age of patients was 4.1 years and the higher percentage of patients (57%) were male. the most common symptoms of covid-19 in children were fever, cough, and diarrhea. as well, the most common symptoms in 22.1% of patients admitted to the intensive care unit (icu) were gastrointestinal (gi) symptoms (79%), fever (62.6%), and respiratory distress (53.6%), respectively. the majority of patients were in the age group less than one year (52%) and mortality rate was 6.3% in total and 10% in children with underlying disease. besides, the mortality rate of intubated cases was 13 times higher. conclusion: the findings of present study showed that covid-19 in children was associated with various clinicopathological manifestations. underlying disease including respiratory distress, cancer, and kidney disease as well as gi symptoms might be guided predicting the hospitalized cases in icu. newborns less than one year of age are exposed to severe covid-19 infection which is associated with higher mortality rate and it should be given special attention in the early diagnosis and management of the covid-19 disease. key words: covid-19, children, clinical manifestations, sars-cov-2 issn 2413-0516 introduction the novel coronavirus has also been nominated as the pandemic 2019 coronavirus disease (covid-19) by the world health organization (who) for global health emergency.1,2 the pneumonia outbreak caused by sars‐coronavirus 2 (sars‐cov‐2) was started in wuhan city, hubei province which was quickly released across china and subsequently in most countries or regions.3 although the severity and lethality of covid-19 is less compared to severe acute respiratory syndrome (sars), elderly patients with sars-cov-2 infection are disposed to experience more severe symptoms.4,5 covid-19 disease involved all groups of age with wide range of complications from insignificant flu-like infection to severe pneumonia including acute respiratory distress syndrome (ards), myocardial and acute kidney injury (aki), multiorgan dysfunction, and shock.6 adolescents of any age are at risk to covid-19, but global investigation statistics have stated that they typically account for 13% of confirmed cases in laboratory tests.7,8 despite the high susceptibility of pneumonia in children compared to adults, the lower level of sars-cov-2 infection in children may be due to the low rate of diagnosis in terms of mild and unusual clinical manifestations of infection as well as low exposure.9,10 as a result, there are fewer hospitalized children with covid-19 than adult patients.3,11 in addition, the demographics and clinicopathological manifestations of the adult hospitalized patients with covid-19 has been fully documented.4,12–14 due to the limited cases of children and pediatrics with sars-cov-2 infection, there is little information about the clinical symptoms and demographic features of the patients.15–17 the current situation of the pandemic showed that children had a growing trend worldwide. as reported by several studies, a number of 2135 children with covid‐19, 74 cases admitted to intensive care units (icu), and 176 190 infected children in china, united states and on a global scale, respectively.18,19 however, the effect of sars-cov-2 infection on adults and children is totally different.17,20,21 despite the universal distribution of covid-19, its clinical, geographical, and epidemiological patterns remains blurred, mainly among pediatrics and adolescents. considering the shift of infection from the elderly population to the younger age groups and the lack of complete vaccination of children, it is pertinent to identify and discover the main clinical and outcome profiles that facilitate early diagnosis and therapeutic strategies for children potentially infected with sars-cov-2. also, until now, the hospitalization rate in children has been lower and there has been less information about the covid-19 disease in patients who have underlying diseases or pre-existing comorbidities. this study was designed to investigate the demographic and clinical characteristics and to get more detailed information for management of covid-19 in the age group under 18 years old. material and methods demographic features of covid-19 patients this r cross-sectional study was performed in the university based referral center, ali asghar children’s hospital in tehran. 2019. the study population were a total of 809 children and adolescents less than 18 years of age who referred, https://orcid.org/0000-0002-2070-9856 mailto:mehrankouchek@yahoo.com 145j contemp med sci | vol. 9, no. 3, may-june 2023: 144–151 m. kouchek et al. original demographic and clinical profiles, including comorbidities, of hospitalized patients under 18 hospitalized, and treated due to covid-19 disease were included in the study. patients’ demographic information and related medical history were recorded from the patients’ archives. the study inclusion criteria were composed of all the population under 18 years of age who have been hospitalized with the diagnosis of covid-19 or have been monitored on an outpatient basis. the positivity of covid-19 patients was based on laboratory and radiological findings. patients who refused to continue the treatment process for any reason or were transferred to another medical centers were excluded from the study. clinical information of covid-19 patients the clinical symptoms of the patients, the underlying disease and the length of hospitalization were entered in the excel form. the definition of disease severity was based on the following criteria.22 mild: symptoms limited to the upper respiratory system moderate: there is involvement of the lower respiratory system, but there is no need for oxygen treatment support. severe: need for supplemental oxygen or increase the amount of oxygen from the previous level without aggressive ventilation. critical: new or increased need for oxygen and invasive mechanical ventilation or having sepsis and multiple organ failure. ethical approval and considerations ethical approval was attained by the ethics committee of the iran university of medical sciences (ir.iums. rec.1400.059) and declaration was made to keep patient’s data confidential. the coded data were only available to the physician and the project manager, and were provided for treatment after the achievement of the study plan and the end of the follow-up. data analysis data was analyzed using spss statistical software (version 26; spss). the results obtained for quantitative variables are expressed as mean and standard deviation (mean ± sd). demographic findings of patients, clinical symptoms and para clinical findings were analyzed based on quantitative and qualitative factors. the normal distribution of the data was measured by the kolmogorov-smirnov test. qualitative data in two groups were analyzed using chi-square (χ2) test. a level of 0.05 was considered in terms of statistically significant variables. results demographic characteristics of covid-19 patients a total of 809 patients with covid-19 were included in this study which had the median age of 3 (ranged 0–18) and the mean age of 4.1 ± 4.2. in addition, 57% of the patients were male and 43% of them were female. a total of 209 from 809 patients (25.8%) had underlying diseases. the most underlying diseases were chronic kidney diseases, cancer and neurological diseases, respectively. descriptive information of the patients with covid-19 are given in table 1. clinical symptoms of covid-19 patients the most common clinical symptoms were fever (80.8%), cough (28.3%) and diarrhea (24.6%), respectively. in total, 40% of patients had gastrointestinal symptoms and 7.2% of them had neurological symptoms. the spearman’s test was used to evaluate the association between age and clinical symptoms and it was determined that with increasing age, the probability of symptoms such as fever, muscle pain or myalgia, abdominal pain, and headache was raised. considering that the occurrence of diarrhea is higher in the middle age, it was evaluated based on different age subgroups which was inversely related to increasing age. as well, increasing age enhanced the probability of intubation in covid-19 patients (p-value = 0.002; r = 0.11). the frequency of clinical symptoms in covid-19 patients are shown in table 2. the mean temperature of patients was 38 ± 0.8°c and the median was 38.2°c (33.1–40). the mean days of symptoms’ table 1. demographic features of patients with covid-19. demographic features of patients total number percentage (%) age (4.1±4.2) age (subgroups) less than a year 1 to 5 years 6 to 10 years 11 to 15 years 16 to 18 years 154 425 136 84 10 19% 52.5% 16.8% 10.4% 1.2% gender male female 348 461 47% 57% location tehran cities other than tehran 686 123 84.8% 15.2% inpatient ward special isolated normal 179 130 500 22.1% 16.1% 61.8% type of patients’ referral personal ambulance 738 71 91.2% 8.8% history of exposure with patients 29% comorbidity chronic kidney disease dialysis 56 20 6.9% cancer chemotherapy 53 29 6.6% chronic liver disease 5 0.6% diabetes 10 1.2% asthma 5 0.6% heart disease 8 1% hiv 2 0.2% immunodeficiency 10 1.2% chronic blood disease 27 3.3% chronic neurological disorders tension cp 29 10 10 3.6% other chronic diseases 45 5.5% 146 j contemp med sci | vol. 9, no. 3, may-june 2023: 144–151 demographic and clinical profiles, including comorbidities, of hospitalized patients under 18 original m. kouchek et al. onset and hospital referral was 3.1 ± 2.8 days and the median was 2 (1–30) days. the length of patients’ hospitalization was 10.18 ± 11.8 and median was 7 (1–134) days, as shown in table 3. there was no significant association between age and other clinical characteristics including symptom’s severity (p-value = 0.14) and oxygen therapy (p-value = 0.10). the linear regression analysis was performed to examine the association between age and the clinicopathological factors, and there was no strong linear relationship between age and these factors. as shown in table 4, for further evaluation, patients were divided into age subgroups. most of patients (52%) were children aged 1 to 5 years and 179 patients (22.1%) were admitted to special wards. seventy one patients (8.8%) were transferred to the other medical centers. male gender was dominant in all age groups. also, a higher percentage of patients who referred with severe and critical illness were belonged to the subgroups less than one year. as shown in table 4, it was found that in the age group under one year, a higher percentage of patients had severe and critical infection. the association between the covid-19 severities in two age groups, less than one year and more than one year, was examined with kendall’s analysis. it was determined that the covid-19 disease was more severe in younger age (p-value = 0.001; r = –0.117). in the regression analysis, mortality rate in group of under one year was significant and the probability of death in this age group was higher (p-value = 0.009; or = 2.26). according to the difference in the severity of the disease in the age groups of less than one year and more than one year, the clinical symptoms in both groups were compared. chisquare analysis and cramer’s correlation were used for this evaluation and results were summarized in table 5. fever, cough, and respiratory distress were the most common symptoms in children under one year of age, and the occurrence of neurological, gastrointestinal, and skin rash symptoms was lower in this age group. although neurological and gastrointestinal symptoms increased with age and the difference became significant, but kramer’s correlation analysis showed a weak correlation of these variables with age (<0.2), as well, in terms of fever, this correlation was moderate. severity of disease and mortality rate in patients with covid-19 mortality rate of covid-19 patients was 6.3% and in patients with underlying disease was 10%. the highest mortality was observed in the age group of less than one year and 11% of infants who died were less than one year. as well, occurrence of underlying disease led to triple the risk of severe disease (p-value = 0.001; or = 3.59). also, in our study, occurrence of asthma was not related to the severity of disease in covid-19 patients (p-value = 0.90). in examining the association between the occurrence of underlying disease and mortality rate based on different clinical factors, it was found that the risk of mortality was higher in patients with underlying disease (p-value = 0.01; or = 2.1). in examining factors related to mortality using univariate analysis, differences in clinical factors such as fever, respiratory distress, decreased level of consciousness, blood and kidney diseases, and hospitalization length and intubation were significant. in the multivariate analysis shown in table 6, by adjusting other variables, only intubation was significant, which increases the probability of death by 13 times. table 3. the clinical symptoms of hospitalized covid-19 patients. clinical symptoms total number (%) temperature (mean = 38 ± 0.8) positive pcr test 131 time from the onset of symptoms to referral (mean = 3.1 ± 2.8) observed lung involvement in ct scan 186 (23) hospitalization length 10.18 ± 11.8 oxygen therapy 145 (17.9) intubation 53 (6.5) death 51 (6.3) severity of symptoms mild moderate intense critical 393 (48.6) 133 (16.4) 238 (29.4) 45 (5.6) table 2. the frequency of clinical symptoms and their association with age of covid-19 patients. clinical symptoms total number (%) p-value correlation coefficient (r) fever 645 (80.8) 0.01 0.9 cough 229 (28.3) 0.60 respiratory distress 163 (20.1) 0.10 diarrhea 199 (24.6) 0.02 -0.08 myalgia & muscle pain 79 (9.8) 0.001 0.29 decreased level of consciousness 7 (0.9) loss or reduction of the sense of smell 1 (0.1) 0.30 loss or reduction of sense of taste 2 (0.2) 0.30 seizure 8 (0.1) 0.50 abdominal pain & cramp 64 (7.9) 0.001 0.17 nausea 95 (11.7) vomiting 125 (15.5) 0.06 anorexia 20 (2.5) 0.50 headache 42 (5.2) 0.001 vertigo 2 (0.2) 0.13 skin rash 12 (1.5) 0.4 paralysis of organs 1 (0.1) 0.50 chest pain 4 (0.5) weakness and lethargy 18 (2.2) 147j contemp med sci | vol. 9, no. 3, may-june 2023: 144–151 m. kouchek et al. original demographic and clinical profiles, including comorbidities, of hospitalized patients under 18 mortality rate in patients with covid-19 based on pcr results and admission in hospital wards among the patients who had a positive sars-cov2 pcr results (49 patients), 6 cases (4.6%) were died and 43 cases (6.5%) were discharged. evaluation of mortality based on pcr results did not show any significant difference (p-value = 0.57). in patients with negative pcr results, 125 cases (95%) and 614 cases (93.5%) were died and discharged, respectively. as shown in table 7, 179 patients (22.1%) were admitted to icu and 630 patients were admitted to normal and isolated wards and the differences between these two groups of patients were analyzed. although male gender comprised a higher percentage of icu patients, the differences in gender was not statistically significant. there was a significant difference in icu hospitalization at the age of less than one year. in univariate analysis, the risk of icu hospitalization in age groups less than one year was 5 times higher than other groups. as well, in the multivariate analysis, fever, respiratory distress and gastrointestinal symptoms were statistically significant, and those who presented with respiratory distress and gastrointestinal symptoms had 6 and 2 times higher risk of hospitalization in icu, respectively. regarding the relationship between underlying diseases and hospitalization in the special ward, cancer and kidney diseases were statistically significant in univariate analysis. in the multivariate analysis, cancer or kidney disease increases the risk of hospitalization in the special ward by 2 and 1.8 times, respectively. in icu patients, the probability of intubation was 10 times higher and the probability of death was 8 times higher. discussion there are fewer treatment facilities for children and adolescents due to the possible low risk of infection and the focus on prioritizing and vaccinating young and elder patients. therefore, the transmission of sars-cov-2 through the children should not be discounted. in this research we tried to explain the different clinical and epidemiological features of covid-19 in children and adolescents to reduce the disease table 4. demographic and clinical characteristics based on the age subgroups. variables <1 year n=154 1–5 years n=425 6–10 years n=136 11–15 years n=84 16–18 years n=10 gender female male 42.9% 57.1% 43.5% 56.5% 44.9% 55.1% 38.1% 61.9% 40.0% 60.0% severity of symptoms mild moderate intense critical 40.3% 10.4% 39.0% 10.4% 54.1% 15.1% 25.6% 5.20% 44.9% 22.8% 28.7% 3.70% 45.2% 20.2% 33.3% 1.20% 20.0% 50.0% 20.0% 10.0% history of exposure 33.8% 28.5% 29.4% 27.4% 60.0% temperature°c 37.7 38.1 38.0 38.0 38.1 hospitalization length (day) 12.8 9.80 10.5 9.10 12.8 comorbidity 10.4% 25.2% 33.8% 42.9% 40.0% death 11.0% 6.40% 2.90% 2.40% 10.0% table 5. clinical symptoms at the age of less than one year and above one year in covid-19 patients. clinical symptoms ≤1 year age ≥1 year age p-value correlation coefficient (r) or 95% confidence interval (ci) fever 64.3% 84.7% 0.001 0.204 0.324 0.219–0.480 cough 30.5% 27.8% 0.498 respiratory distress 33.8% 16.9% 0.001 0.165 gastrointestinal symptoms 28.6% 43.5% 0.001 0.119 neurological symptoms 0.60% 8.80% 0.001 0.123 skin rash 0.00% 1.10% 0.225 decreased level of consciousness 0.60% 90.0% 0.740 table 6. association between clinical factors and mortality rate. clinical factors univariate multivariate p-value p-value or 95% confidence interval (ci) fever 0.003 0.690 decreased level of consciousness 0.002 0.260 blood diseases 0.020 0.060 kidney diseases 0.010 0.750 hospitalization length 0.001 0.760 intubation 0.001 0.008 13.77 1.97–96.9 148 j contemp med sci | vol. 9, no. 3, may-june 2023: 144–151 demographic and clinical profiles, including comorbidities, of hospitalized patients under 18 original m. kouchek et al. table 7. differences between hospitalized patients in the special and the normal wards. clinical symptoms icu n=179 non-icu n=630 p-value correlation coefficient (r) or 95% confidence interval (ci) gender female male 43.6% 56.4% 42.9% 57.1% age ≤1 year age 44.1% 19.0% 0.001 5.84 3.99–8.55 1–5 years 32.4% 52.5% 6–10 years 15.6% 16.8% 11–15 years 6.70% 10.4% 16–18 years 1.10% 1.20% clinical symptoms fever 62.6% 86.0% 0.001 0.378 0.24–0.58 cough 36.0% 25.9% 0.004 0.14 respiratory distress 53.6% 10.6% 0.001 6.71 4.43–10.15 gastrointestinal symptoms 79.0% 53.9% 0.001 2.03 1.31–3.14 neurological symptoms 6.30% 7.50% 0.190 comorbidity cancer 2.80% 7.60% 0.027 liver 0.00% 0.80% 0.900 diabetes 2.20% 1.00% 0.180 blood diseases 2.80% 3.80% 0.520 hiv 0.00% 0.30% 0.900 immunodeficiency 0.00% 1.60% 0.900 heart disease 1.10% 1.00% 0.840 kidney diseases 10.6% 5.90% 0.030 1.80 1.01–3.22 asthma 0.60% 0.60% 0.900 neurological disorders 1.20% 0.30% 0.160 oxygen condition 43.0% 10.8% 0.001 0.271 0.271 0.175–0.419 intubation 24.6% 1.40% 0.001 10.814 4.92–23.73 death 19.0% 2.70% 0.001 8.45 4.50–15.5 complications through the effective planning and early intervention measures in this population.22 it has been demonstrated that the viral load of sars-cov-2 were 10–100 times higher in children younger than 5 years than in cases more than 5 years old and adolescents with covid-19.23 the incidence of covid-19 in a series of 228 children between 1–5 years old increased by 7.4-fold.24 moreover, patients with covid-19 and younger than 1 year of age and <5 years experienced severe symptoms of infection.18,25 in this retrospective study a total of 809 children have participated which most of them had mild symptoms and were hospitalized and monitored due to concerns related to this age group as well as children referred from other cities. the mean and median age of the patients in our study was 4 ranged from infancy to the age of 18 indicating the susceptibility of all age groups to covid-19 infection. also, in all age groups, male gender was comprised in a higher percentage of patients, and more of this gender males were admitted to the special wards due to the severe infection. in line with other studies, difference in gender was not statistically significant.26,27 in three studies the median age of children with covid-19 has been reported to be 6.2, 5.3, and 8.16 years, respectively.18, 28–30 armin et al.’s study revealed that male gender made up the majority of patients (59.4%),28 as in dong et al.’s and hua et al.’s study a 56.6 % and 60.5% has been reported to be males.18 in terms of mers disease, females showed lower incidence and severity of the disease which can be attributed to the biological differences, protective effect of the x chromosome, and the lower expression of the ace2 receptor in females against viral entry into the cells.31 in our study, 29.9% of covid-19 patients had familial exposure and the source of infection was unknown in 70.1% of children which was in contrast with the findings of other study regarding the family clusters as the main origin of the disease.32 as well, a large percentage of children had an unknown source of infection, our study indicates that children can acquire the disease from social clusters, besides the family. twenty three percent of children presented lung involvement in chest ct scan, and it was consistent with ma et al. study with 22% of lung involvement in children with covid-19. although lung involvement in this age group was less than adults, but the pattern of glass-like opacities was similar to 149j contemp med sci | vol. 9, no. 3, may-june 2023: 144–151 m. kouchek et al. original demographic and clinical profiles, including comorbidities, of hospitalized patients under 18 them.33 in systematic literature studies and a pediatric study in shiraz, fever and cough has been identified as the most common clinical symptoms.16,34,35 findings of present study showed that fever, cough and diarrhea are common symptoms of covid-19 infection in children. as well, a total of 40% of children had gastrointestinal symptoms such as anorexia, vomiting, abdominal pain, and diarrhea. according to chang et al.’s and armin et al.’s studies the uncommon gastrointestinal symptoms, diarrhea and vomiting were reported in 12%, 22.3%, and 30.3% of cases, respectively.16,28 in the cdc report, fever, cough and shortness of breath are the most common symptoms of covid-19 and diarrhea, myalgia and headache are uncommon in children.26 the study of hui du et al. were indicated fever and cough as the most common symptoms and gastrointestinal symptoms in 11% of patients.36 findings of a meta-analysis study were also listed fever and cough as the most common symptoms in the age group of children and were stated vomiting as a possible symptom of covid-19 in children under one year of age.37 our findings were in line with the cdc report and other studies that have mentioned fever and cough as the most common symptoms in children, but our study also was indicated the gastrointestinal symptoms and diarrhea as the common symptoms in children especially at the peak of covid-19 infection. although in our study, fever and cough were the most common symptoms in children under one year old, we observed that the gastrointestinal (anorexia, vomiting and diarrhea) and neurological symptoms were more common in older children. on the other hand, due to the difference in levels of immune maturity in children and adults, the prevalence and type of clinical manifestations of covid-19 may be different.38 the substantial risk factors has been identified by prior studies which pave the way for severe covid-19 in children including age younger than 1 year, underlying disease (diabetes, obesity, congenital heart disease, asthma, and neurologic conditions).39–45 in the present study, 25.8% of the patients with covid-19 had an underlying disease, the most common diseases were kidney diseases, neurological, blood diseases, and cancer, respectively. in the cdc study, asthma, heart, and autoimmune diseases have been mentioned as underlying diseases.26 singh et al.’s found that 59% of hospitalized patients had an underlying disease including malnutrition, blood malignancies, and tuberculosis as the most common diseases.46 in the study on 345 children with covid-19, 23% exhibited underlying diseases and pulmonary (asthma), cardiovascular disease, and immunosuppression were the most common complications.47 in our study, 5 cases had asthma, and it was found that it has not been effective in the severity and mortality of covid-19 infection. in this case, our study was in line with the hui et al. findings which showed that asthma and allergic diseases were not effective in the incidence and severity of covid-19.48 therefore, the difference in covid-19 manifestations can be attributed to the different epidemiological conditions among several studies. in a meta-analysis study, severe infection was reported in 5.1% of patients with an underlying disease and the relative risk was 1.79 (ci 1.27–2.51), and the relative risk of death in this group of patients was 2.81 (ci 1.3–6.02).49 we observed that 46.8% of covid-19 children had severe and critical infection, and their mortality rate was 10%. our study found that having an underlying disease triples the risk of severe disease (p-value = 0.001; or = 3.59) and doubles the risk of mortality (p-value = 0.01; or = 2.1). in general, it can be concluded that children with covid-19 and an underlying disease are at a higher risk of severe disease and death. in general, studies have shown that the severity of the disease in children was less than in adults, and this can be attributed to the maturation of ace2 receptors, the evolving immune system, and the greater prevalence of underlying diseases such as diabetes, hypertension, and heart disease in adults. ace2 receptors act as receptors for the covid-19 infection, and it seems that the function and maturity of these receptors in children are less sensitive to the sars-cov2, which reduces the entry of the virus and the severity of the disease. similarly, due to more respiratory infections, children have more antibodies against respiratory pathogens and this may cause a protective effect.18,50 in this study, mild, moderate, severe and critical diseases were 48.6%, 16.4%, 29.4% and 5.6%, respectively. a percentage of asymptomatic, mild and moderate covid-19 infection has been reported as 4.4%, 51% and 38.7% in children, respectively.18 they also showed that the severity of covid-19 was higher at the age of less than one year and 10.6% of this age group had severe disease.18 moreover, severe and critical covid-19 infection in children were verified to be 7% and 5% in a meta-analysis study.51 as well, in breastfed infants severe and critical form of covid-19 infection was less than 7% and 14% for one year.37 in the present study, 39% of children under one year had severe disease and 10.4% of them had critical disease, and their mortality rate was 11%. we revealed that the severity of the disease in the age group of less than one year is more than that of older ages and it is consistent with other studies. we also observed that the risk of death in this age group is doubled (p-value = 0.009; or = 2.26). hence, the cause of severe disease and the difference in immune system in this age group is still being discussed. our study showed that having symptoms such as fever and decreased level of consciousness, underlying diseases including blood, kidney and neurological and hospitalization in a special ward, intubation and hospitalization length may effect on the mortality rate. in the multiple analysis, it was found that there was a correlation of mortality with intubation and hospitalization in the special ward, so that the risk of mortality in an intubated patients was 13 times higher (or = 13.779, p-value = 0.008), than in a cases hospitalized in the icu (or = 3.33, p-value = 0.007). the estimated rates of intensive care unit admission, acute respiratory distress syndrome, and mortality has been reported to be 10.9%, 18.4%, and 4.3%, respectively.52 in various studies, the admission rate in the special wards has been reported to be 10.2%, 19.4% and 17.5%.26,53,54 in our study, 179 (22%) patients were admitted to the icu, of which 44% were in the age group of less than one year and 22% had an underlying disease. the death rate of patients in the icu was 8.8%. we also found that having gastrointestinal symptoms and respiratory distress increased the risk of hospitalization in icu by 2 and 6 times, respectively. cancer and kidney disease was associated with an increase of 2 and 1.8 times the risk of hospitalization in icu and the probability of intubation and death was 10 and 8 times higher. in the study of barboza et al., 41% of the patients admitted to the icu had an underlying disease, and gastrointestinal symptoms as a risk factor for multisystem inflammatory syndrome.55 as a result, the gastrointestinal symptoms in children with covid-19 should be considered important and carefully monitored. 150 j contemp med sci | vol. 9, no. 3, may-june 2023: 144–151 demographic and clinical profiles, including comorbidities, of hospitalized patients under 18 original m. kouchek et al. references 1. guan w-j, ni z-y, hu y, liang w-h, ou c-q, he j-x, et al. clinical characteristics of coronavirus disease 2019 in china. new england journal of medicine. 2020;382(18):1708–20. 2. malik ys, sircar s, bhat s, sharun k, dhama k, dadar m, et al. emerging novel coronavirus (2019-ncov)—current scenario, evolutionary perspective based on genome analysis and recent developments. veterinary quarterly. 2020;40(1):68–76. 3. ma x, liu s, chen l, zhuang l, zhang j, xin y. the clinical characteristics of pediatric inpatients with sars-cov-2 infection: a meta-analysis and systematic review. journal of medical virology. 2021;93(1):234–40. 4. zhou f, yu t, du r, fan g, liu y, liu z, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet. 2020;395(10229):1054–62. 5. al-tawfiq ja, kattan rf, memish za. middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia. world journal of clinical pediatrics. 2016;5(4):391. 6. wang d, hu b, hu c, zhu f, liu x, zhang j, et al. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in wuhan, china. jama. 2020;323(11):1061–9. 7. gupta n, praharaj i, bhatnagar t, thangaraj jwv, giri s, chauhan h, et al. severe acute respiratory illness surveillance for coronavirus disease 2019, india, 2020. the indian journal of medical research. 2020;151(2–3):236. 8. wu z, mcgoogan jm. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention. jama. 2020;323(13):1239–42. 9. xu y, li x, zhu b, liang h, fang c, gong y, et al. characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding. nature medicine. 2020;26(4):502–5. 10. li a, ng p. severe acute respiratory syndrome (sars) in neonates and children. archives of disease in childhood-fetal and neonatal edition. 2005;90(6):f461-f5. 11. chan kw, wong vt, tang scw. covid-19: an update on the epidemiological, clinical, preventive and therapeutic evidence and guidelines of integrative chinese–western medicine for the management of 2019 novel coronavirus disease. the american journal of chinese medicine. 2020;48(03):737–62. 12. cao y, liu x, xiong l, cai k. imaging and clinical features of patients with 2019 novel coronavirus sars‐cov‐2: a systematic review and meta‐analysis. journal of medical virology. 2020;92(9):1449–59. 13. li lq, huang t, wang yq, wang zp, liang y, huang tb, et al. covid‐19 patients’ clinical characteristics, discharge rate, and fatality rate of meta‐ analysis. journal of medical virology. 2020;92(6):577–83. 14. rodriguez-morales aj, cardona-ospina ja, gutiérrez-ocampo e, villamizarpeña r, holguin-rivera y, escalera-antezana jp, et al. clinical, laboratory and imaging features of covid-19: a systematic review and meta-analysis. travel medicine and infectious disease. 2020;34:101623. 15. badal s, bajgain kt, badal s, thapa r, bajgain bb, santana mj. prevalence, clinical characteristics, and outcomes of pediatric covid-19: a systematic review and meta-analysis. journal of clinical virology. 2021;135:104715. 16. chang t-h, wu j-l, chang l-y. clinical characteristics and diagnostic challenges of pediatric covid-19: a systematic review and meta-analysis. journal of the formosan medical association. 2020;119(5):982–9. 17. ludvigsson jf. systematic review of covid‐19 in children shows milder cases and a better prognosis than adults. acta paediatrica. 2020;109(6):1088–95. 18. dong y, mo x, hu y, qi x, jiang f, jiang z, et al. epidemiology of covid-19 among children in china. pediatrics. 2020;145(6). 19. pathak eb, salemi jl, sobers n, menard j, hambleton ir. covid-19 in children in the united states: intensive care admissions, estimated total infected, and projected numbers of severe pediatric cases in 2020. journal of public health management and practice. 2020. 20. cui x, zhang t, zheng j, zhang j, si p, xu y, et al. children with coronavirus disease 2019: a review of demographic, clinical, laboratory, and imaging features in pediatric patients. journal of medical virology. 2020;92(9): 1501–10. 21. de souza th, nadal ja, nogueira rj, pereira rm, brandão mb. clinical manifestations of children with covid‐19: a systematic review. pediatric pulmonology. 2020;55(8):1892–9. 22. chiotos k, hayes m, kimberlin dw, jones sb, james sh, pinninti sg, et al. multicenter interim guidance on use of antivirals for children with coronavirus disease 2019/severe acute respiratory syndrome coronavirus 2. journal of the pediatric infectious diseases society. 2021;10(1):34–48. 23. heald-sargent t, muller wj, zheng x, rippe j, patel ab, kociolek lk. age-related differences in nasopharyngeal severe acute respiratory syndrome coronavirus 2 (sars-cov-2) levels in patients with mild to moderate coronavirus disease 2019 (covid-19). jama pediatrics. 2020;174(9):902–3. 24. colson p, tissot-dupont h, morand a, boschi c, ninove l, esteves-vieira v, et al. children account for a small proportion of diagnoses of sars-cov-2 infection and do not exhibit greater viral loads than adults. european journal of clinical microbiology & infectious diseases. 2020;39:1983–7. 25. li b, zhang s, zhang r, chen x, wang y, zhu c. epidemiological and clinical characteristics of covid-19 in children: a systematic review and metaanalysis. frontiers in pediatrics. 2020;8:591132. 26. bialek s, gierke r, hughes m, mcnamara la, pilishvili t, skoff t. coronavirus disease 2019 in children—united states, february 12–april 2, 2020. 27. wardell h, campbell ji, vanderpluym c, dixit a. severe acute respiratory syndrome coronavirus 2 infection in febrile neonates. journal of the pediatric infectious diseases society. 2020;9(5):630–5. 28. armin s, mirkarimi m, pourmoghaddas z, tariverdi m, jafrasteh a, marhamati n, et al. iranian pediatric covid-19 epidemiology and clinical characteristics. the canadian journal of infectious diseases & medical microbiology = journal canadien des maladies infectieuses et de la microbiologie medicale. 2021;2021:4914371. 29. du w, yu j, wang h, zhang x, zhang s, li q, et al. clinical characteristics of covid-19 in children compared with adults in shandong province, china. infection. 2020;48:445–52. 30. hua cz, miao zp, zheng js, huang q, sun qf, lu hp, et al. epidemiological features and viral shedding in children with sars‐cov‐2 infection. journal of medical virology. 2020;92(11):2804–12. 31. samadizadeh s, masoudi m, rastegar m, salimi v, shahbaz mb, tahamtan a. covid-19: why does disease severity vary among individuals? respiratory medicine. 2021;180:106356. 32. posfay-barbe km, wagner n, gauthey m, moussaoui d, loevy n, diana a, et al. covid-19 in children and the dynamics of infection in families. pediatrics. 2020;146(2). 33. ma x, liu s, chen l, zhuang l, zhang j, xin y. the clinical characteristics of pediatric inpatients with sars‐cov‐2 infection: a meta‐analysis and systematic review. journal of medical virology. 2021;93(1):234–40. 34. hoang a, chorath k, moreira a, evans m, burmeister-morton f, burmeister f, et al. covid-19 in 7780 pediatric patients: a systematic review. eclinical medicine. 2020;24:100433. 35. hoseinyazdi m, esmaeilian s, jahankhah r, teimouri a, sherbaf fg, rafiee f, et al. clinical, laboratory, and chest ct features of severe versus non-severe pediatric patients with covid-19 infection among different age groups. bmc infectious diseases. 2021;21(1):1–12. conclusion our results showed that the covid-19 infection in children is less severe and displays a better clinical outcome which is not relevant for children under one year of age, thus, having covid-19 in this age group should be more considered. in addition, there was a strong association between the clinical outcome in covid-19 patients and comorbidities including respiratory distress, cancer, and kidney disease and they play an important role in increasing hospitalization length and mortality rate of children. in our study, it was found that the occurrence of gastrointestinal symptoms can be associated with an increase in patients’ hospitalization rate in the icu. as well, the possibility of mortality is significantly increased in children who require invasive mechanical ventilation. based on the current study and other studies, we suggest to postpone invasive intubation and apply the non-invasive ventilation methods as much as possible. finally, it is important to have comprehensive information in this age group, since the age of covid-19 exposure is shifting towards childhood and adolescence.  151j contemp med sci | vol. 9, no. 3, may-june 2023: 144–151 m. kouchek et al. original demographic and clinical profiles, including comorbidities, of hospitalized patients under 18 46. singh p, attri k, mahto d, kumar v, kapoor d, seth a, et al. clinical profile of covid-19 illness in children—experience from a tertiary care hospital. indian journal of pediatrics. 2022;89:45–51. 47. tezer h, demirdağ tb. novel coronavirus disease (covid-19) in children. turkish journal of medical sciences. 2020;50(9):592–603. 48. soltani j, sedighi i, shalchi z, sami g, moradveisi b, nahidi s. pediatric coronavirus disease 2019 (covid-19): an insight from west of iran. north clin istanb. 2020;7(3):284–91. 49. tsankov bk, allaire jm, irvine ma, lopez aa, sauve lj, vallance ba, et al. severe covid-19 infection and pediatric comorbidities: a systematic review and meta-analysis. international journal of infectious diseases. 2021;103:246-56. 50. zimmermann p, curtis n. why is covid-19 less severe in children? a review of the proposed mechanisms underlying the age-related difference in severity of sars-cov-2 infections. archives of disease in childhood. 2021;106(5):429–39. 51. fu l, wang b, yuan t, chen x, ao y, fitzpatrick t, et al. clinical characteristics of coronavirus disease 2019 (covid-19) in china: a systematic review and meta-analysis. journal of infection. 2020;80(6):656–65. 52. zhang jj, lee ks, ang lw, leo ys, young be. risk factors for severe disease and efficacy of treatment in patients infected with covid-19: a systematic review, meta-analysis, and meta-regression analysis. clinical infectious diseases. 2020;71(16):2199–206. 53. soleimani g, akbarirad f, shafighi shahri e, sajjadi sm. demographic, clinical, and paraclinical characteristics of covid-19 pediatric cases in southeast iran. antimicrobial resistance & infection control. 2021;10(1):1–9. 54. chao jy, derespina kr, herold bc, goldman dl, aldrich m, weingarten j, et al. clinical characteristics and outcomes of hospitalized and critically ill children and adolescents with coronavirus disease 2019 at a tertiary care medical center in new york city. the journal of pediatrics. 2020;223:14–9. e2. 55. prata-barbosa a, lima-setta f, santos grd, lanziotti vs, castro revd, souza dcd, et al. pediatric patients with covid-19 admitted to intensive care units in brazil: a prospective multicenter study. jornal de pediatria. 2020;96:582–92. 36. du h, dong x, zhang jj, cao yy, akdis m, huang pq, et al. clinical characteristics of 182 pediatric covid‐19 patients with different severities and allergic status. allergy. 2021;76(2):510–32. 37. cui x, zhao z, zhang t, guo w, guo w, zheng j, et al. a systematic review and meta-analysis of children with coronavirus disease 2019 (covid-19). journal of medical virology. 2021;93(2):1057–69. 38. raoult d, zumla a, locatelli f, ippolito g, kroemer g. coronavirus infections: epidemiological, clinical and immunological features and hypotheses. cell stress. 2020;4(4):66. 39. kim l, whitaker m, o’halloran a, kambhampati a, chai sj, reingold a, et al. hospitalization rates and characteristics of children aged< 18 years hospitalized with laboratory-confirmed covid-19—covid-net, 14 states, march 1–july 25, 2020. morbidity and mortality weekly report. 2020;69(32):1081. 40. shekerdemian ls, mahmood nr, wolfe kk, riggs bj, ross ce, mckiernan ca, et al. characteristics and outcomes of children with coronavirus disease 2019 (covid-19) infection admitted to us and canadian pediatric intensive care units. jama pediatrics. 2020;174(9):868–73. 41. alsaied t, aboulhosn ja, cotts tb, daniels cj, etheridge sp, feltes tf, et al. coronavirus disease 2019 (covid‐19) pandemic implications in pediatric and adult congenital heart disease. journal of the american heart association. 2020;9(12):e017224. 42. bellino s, punzo o, rota mc, del manso m, urdiales am, andrianou x, et al. covid-19 disease severity risk factors for pediatric patients in italy. pediatrics. 2020;146(4). 43. götzinger f, santiago-garcía b, noguera-julián a, lanaspa m, lancella l, carducci fic, et al. covid-19 in children and adolescents in europe: a multinational, multicentre cohort study. the lancet child & adolescent health. 2020;4(9):653–61. 44. graff k, smith c, silveira l, jung s, curran-hays s, jarjour j, et al. risk factors for severe covid-19 in children. the pediatric infectious disease journal. 2021;40(4):e137-e45. 45. kompaniyets l, agathis nt, nelson jm, preston le, ko jy, belay b, et al. underlying medical conditions associated with severe covid-19 illness among children. jama network open. 2021;4(6):e2111182. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1340 127j contemp med sci | vol. 9, no. 2, march-april 2023: 127–133 original a potential role of extracellular dna in biofilm and ciprofloxacin resistance hind tahseen ibrahim1, ali a. mussa2, harith jabbar fahad al-mathkhury2* 1department of medical laboratory techniques, college of medical (technology), al-farahidi university, baghdad, iraq. 2department of biology, college of science, university of baghdad, baghdad, iraq. *correspondence to: harith jabbar fahad al-mathkhury (e-mail: harith.fahad@sc.uobaghdad.edu.iq) (submitted: 26 january 2023 – revised version received: 19 february 2023 – accepted: 03 march 2023 – published online: 26 april 2023) abstract objectives: this study aims to broaden our knowledge of the role of edna in bacterial biofilms and antibiotic-resistance gene transfer among isolates. methods: staphylococcus aureus, e. coli, and pseudomonas aeruginosa were isolated from different non-repeated 170 specimens. the bacterial isolates were identified using morphological and molecular methods. different concentrations of genomic dna were tested for their potential role in biofilms formed by study isolates employing microtiter plate assay. ciprofloxacin resistance was identified by detecting a mutation in gyra and parc. results: the biofilm intensity significantly decreased (p < 0.05) concerning s. aureus isolates and insignificantly (p > 0.05) concerning e. coli isolates. yet, one e. coli isolate’s biofilm was significantly decreased (p < 0.05) linearly with increasing edna. of considerable interest, the addition of edna led to a significant increase (p < 0.05) in the biofilm of the two-tested p. aeruginosa isolates. moreover, edna participated in transferring ciprofloxacin resistance to the sensitive isolate when it presents in its biofilm. conclusion: edna has a dual effect on bacterial biofilms either supportive or suppressive following bacterial species per se. also, it seems to play an important role in antibiotic resistance within the biofilm. keywords: edna, biofilm, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa issn 2413-0516 introduction staphylococcus aureus inhabited approximately 30% of healthy people, mostly in the anterior nares. nevertheless, it is also a leading cause of hospital-associated and community-associated bacterial infections in humans, associated with numerous mild skin and soft tissue infections and life-threatening pneumonia, aimeretcab, osteomyelitis, endocarditis, sepsis, and toxic shock syndrome. the increasing prevalence of methicillin-resistant s. aureus (mrsa) and its ability to resist multiple drugs has posed a serious challenge to infection control.1,2 escherichia coli is one of the earliest colonizers of the gastrointestinal tract; although eventually, it is a minor component of the colonic gut microbiome in humans, where it represents less than 0.1% of the total bacterial cells. nevertheless, due to the overall high cell density in the colon, this small percentage translates into around 108 cells/ml.3 indeed, e. coli is the causative agent of various intestinal and extra-intestinal diseases, including being suspected to be the cause of sudden infant death syndrome.4,5 pseudomonas aeruginosa is an opportunistic gram-negative pathogen and the leading cause of diverse nosocomial infections and it is commonly difficult to eradicate with conventional antibiotic therapy, particularly when established as biofilms.6 although p. aeruginosa rarely infects healthy people, those individuals whose skin, mucous membranes, or immune system are affected, are more susceptible to becoming infected by this organism; for example, burn victims, patients with cystic fibrosis, or cancer patients treated with chemotherapy.7 biofilms are surface-associated bacterial communities embedded in an extracellular matrix that is considered to be a major problem in the context of chronic infections because biofilm-dwelling cells have increased antibiotic resistance compared to their planktonic counterparts.8 the critical roles of the matrix for microbial interactions and virulence, as well as for antimicrobial tolerance, are being increasingly recognized. the matrix production enhances bacterial cell adhesion and cohesion (resulting in densely packed cell aggregates), providing mechanical stability.9 extracellular deoxyribonucleic acid (edna) is widely recognized as an integral component of biofilms’ extracellular polymeric matrix (ecm). many studies mentioned that edna plays a pivotal role in bacterial biofilm formation. the involvement of edna in biofilms includes providing nutrition and energy for sessile cells promoting horizontal gene transfer (hgt) in naturally competent cells or maintaining the biofilm integrity.10 while others have proved that edna could destabilize the biofilm formation process and that effect would depend on the bacterial species or its serotypes.11 upon the aforementioned facts, this study aimed at 1) investigating the effect of increasing concentration of edna on biofilm formation and 2) inspecting the transferring possibility of the antibiotic-resistant gene from edna to bacterial cell within the biofilm. materials and methods ethical statement this work is approved by the college of science research ethics committee (ref. csec/1220/0081). all participants agreed to provide the investigator with the specimens. informed consent according to the declaration of helsinki was obtained from all participants. specimen collection a total of 170 different non-repeated specimens were collected from patients referring to hospitals in baghdad, iraq. these specimens comprised anterior nares swabs (n = 20) were taken from healthcare workers as well as the patients, sputum (n = 30), mid-stream urine (n = 95), burn swabs (n = 13), and mailto:harith.fahad@sc.uobaghdad.edu.iq 128 j contemp med sci | vol. 9, no. 2, march-april 2023: 127–133 a potential role of extracellular dna in biofilm and ciprofloxacin resistance original h.t. ibrahim et al. blood (n = 12). the specimens were cultured on different selective culture media; mannitol salt agar, macconkey agar, eosin methylene blue (emb) agar, and cetrimide agar and subsequently subjected to conventional biochemical tests including catalase, oxidase, coagulase, acetoin production, imvic, motility, and haemolysin production test) to identify staphylococcus aureus, e. coli, and pseudomonas aeruginosa, salmonella enterica serovar typhi and klebsiella pneumoniae isolates.12 all the bacterial isolates were then tested for ciprofloxacin resistance by measuring the minimal inhibitory concentration for ciprofloxacin using the agar diffusion method following the method described by jennifer.13 polymerase chain reaction bacterial genomic dna was extracted using presto™ mini gdna bacteria kit (geneaid, taiwan) and all amplifications were carried out using accupower® pcr premix, and gradient master cycler (eppendorf, germany). all s. aureus-suspected isolates were screened for the presence of the s. aureus species-specific 16s rdna gene using specific primers, sa1: (aatctttgtcggtacacgatattcttcacg) and sa2: (cgtaatgagatttcagtagataatacaaca) were used to amplify 108 bp segment of s. aureus species-specific 16s rdna gene. the reaction protocol was as followed: initial denaturation at 92°c for 3 min followed by 30 cycles of 92°c 1 min, 56°c 1 min, and 72°c 1 min; following that 3 min at 72°c for final extension.14 s. aureus isolates were also screened for methicillin resistance by detecting meca gene using specific primers meca1: (gtagaaatgactgaacgtccgataa) and meca2: (ccaattccacattgtttcggt); the reaction condition included initial denaturation at 94°c for 10 min followed by 10 cycles of 94°c 45 sec, 55°c 45 sec and 72°c 75 sec; followed by 25 cycles of 94°c 45 sec, 50°c 45 sec and 72°c 75 sec.15 escherichia coli-suspected isolates were also screened for the presence of the uspa gene by the same technique employing specific primers uspa-f (ccgatacgctgccaatcagt) and uspa-r (acgcagaccgtaggccagat), the conditions were: initial denaturation at 95°c for 5 min followed by 30 cycles of 94°c 30 sec, 56°c 30 sec and 72°c 30 sec; following that 5 min at 72°c for final extension.16 two ciprofloxacin-resistant s. aureus isolates were selected to detect any possible mutation in gyra and parc coding for dna gyrase subunit a and dna topoisomerase iv, respectively using the specific primers. gyra-f: aaatctgcccgtgtcgttggt and gyra-r gccatacctacggcgatacc for gyra; parc-f: gtatgcgatgtctgaact and parc-r ttcggtgtaacgcattgc for parc. the amplification program involved initial denaturation at 95°c for 2 min followed by 35 cycles of 95°c 30 sec, 55.4°c 60 sec, and 72°c 60 sec.17 the sequences of the pcr products were obtained using the sanger method and then were aligned with gene sequences from national center for biotechnological information (ncbi) (https://www.ncbi.nlm.nih.gov/) to investigate for mutations. all ciprofloxacin resistant isolates were screened for the presence of acra gene coding for acrab efflux pump. the primers that were used are acra-f: (atgaacaaaaacagagg) and acra-r: (tttcaacggcagttttcg) in a pcr reaction program of initial denaturation at 94°c for 5 min followed by 30 cycles of 94°c 1 min, 52°c 1 min and 72°c 1 min followed by 5 min at 72°c for final extension.18 biofilm formation assay quantification of biofilm formation by e. coli, s. aureus, and p. aeruginosa on abiotic surfaces was assessed as previously described.19 in brief; wells of sterile 96-well u-shaped bottomed polystyrene microplates were filled with 200 μl of an overnight tsb (bacteria concentration was adjusted to in equivalence to mcfarland standard no. 0.5) before the plates were covered and incubated aerobically at 37°c for 24 h. each bacterium was tested in triplicate. control wells were performed by adding bacteria-free tsb. the wells were aspirated and washed three times with 200 μl sterile phosphate-buffered saline (pbs); the remaining attached bacteria were fixed with 200 μl methanol for 15 min. after drying in air, the wells were stained with 200 μl 0.1% crystal violet solution for 15 min at room temperature. the excess stain was rinsed off by placing the plate under running tap water. thereafter, the plates were dried. subsequently, the adherent cells were resolubilized with 200 μl of 33% glacial acetic acid for 15 minutes. finally, the optical density (od) of each well was obtained at 600 nm using a microplate reader (biotek, uk). cut off value (odc) was calculated as the mean of od of control wells plus 3 standard deviations. the isolates were then interpreted as non–producer (od ≤ odc), weak producer (odc < od ≤ 2*odc), moderate producer (2*odc < od ≤ 4*odc), or strong producer (4*odc < od). to investigate the impact of edna concentration on biofilms of e. coli, s. aureus, and p. aeruginosa, the same protocol described previously was followed; nonetheless, different concentrations (400 ng/µl, 200 ng/µl, 100 ng/µl, and 50 ng/µl as a final concentration) of purified edna were added to each well separately. moreover, 100 μl of te buffer was added to the control wells instead of purified edna. thereafter, plates were incubated, stained, and quantified as it is mentioned earlier. determining the role of edna in gene transfer an aliquot of 100 µl of the bacterial growth (compatible with mcfarland standard no. 0.5) of ciprofloxacin sensitive isolates of e. coli (e4), s. aureus (s4) and p. aeruginosa (p1) was added to wells of sterile 6-well u shaped-bottomed polystyrene microplates; thereafter, three ml of sterile tryptic soy broth were added to each well. a volume of one ml of edna (400 ng/µl) extracted from ciprofloxacin-resistant isolate (s. aureus isolate s17) was added to each well. all plates were covered and incubated at 37°c for 24 h. then washed thrice with sterile pbs. biofilms were removed from each well by scraping, suspended in a sterile broth medium, and incubated at 37°c for 18 h. the minimal inhibitory concentration to ciprofloxacin was determined and further investigation was carried out using pcr technique for gyra, parc, and acra genes as it is mentioned previously, followed by sequencing of amplified products. statistical analysis biofilm data were analyzed using two-way anova followed by lsd0.05. the differences were considered significant when p < 0.05. https://www.ncbi.nlm.nih.gov/ 129j contemp med sci | vol. 9, no. 2, march-april 2023: 127–133 h.t. ibrahim et al. original a potential role of extracellular dna in biofilm and ciprofloxacin resistance results and discussion identification results revealed that 25, 24, and 2 isolates were identified as s. aureus, e. coli, and p. aeruginosa, respectively. furthermore, all s. aureus isolates were found to be methicillin resistant due to harboring the meca gene. the polymerase chain reaction was also employed to detect the presence of acrab efflux pump using primers that are specific for acra gene encoding for this pump in all ciprofloxacin-resistant isolates (two s. aureus & 13 e. coli isolates). the result revealed the presence of a single gene with 495 bp in all of these isolates. the present results are in line with those obtained by pakzad et al.18 in that all the resistant isolates harbored the acra gene. on the other hand, these results differ considerably from those reported by the same authors as they reported that not all ciprofloxacin-sensitive isolates contained this gene. detection of gyra and parc mutations ciprofloxacin-resistant s. aureus isolates (s17 and s18) were carefully chosen to be investigated for mutations in gyra and parc genes. two specific sets of primers were used to amplify gyra and parc genes in separate pcr reaction tubes; after electrophoresis of the products and illumination under uv light, specific bands were obtained at 344 and 230 bp for gyra and parc, respectively. such results were expected as these genes are considered to be part of the structural genes of the bacterial cell. the sequences of the pcr product of the isolate s17 were obtained and compared to sequences of gyra and parc genes from ncbi; as illustrated in table 1 and table 2, about 40 and table 1. list of mutations in gyra forward strand no. mutation type no. mutation type 1 g®c transversion 21 a®c transversion 2 t®c transition 22 c®t transition 3 t®g transversion 23 a®c transversion 4 t®a transversion 24 c®t transition 5 g®deletion 25 t®a transversion 6 a®c transversion 26 c®t transition 7 g®a transition 27 g®a transition 8 a®t transversion 28 t®c transition 9 a®c transversion 29 a®g transition 10 t®a transversion 30 a®t transversion 11 a®c transversion 31 a®c transversion 12 g®a transition 32 c®a transversion 13 c®t transition 33 a®c transversion 14 a®c transversion 34 t®g transversion 15 a®t transversion 35 c®a transversion 16 a®c transversion 36 g®t transversion 17 g®t transversion 37 a®t transversion 18 c®a transversion 38 c®t transition 19 g®a transition 39 c®t transition 20 a®t transversion 40 g®c transversion table 2. list of mutations in gyra reverse strand no mutation type no mutation type 1 a®– deletion 27 t®g transversion 2 t®– deletion 28 a®g transition 3 c®a transversion 29 g®a transition 4 g®t transversion 30 a®g transition 5 a®c transversion 31 c®t transition 6 g®t transversion 32 c®t transition 7 t®g transversion 33 g®a transition 8 t®a transversion 34 a®t transversion 9 a®g transition 35 t®g transversion 10 t®c transition 36 c®t transition 11 a®g transition 37 a®t transversion 12 c®t transition 38 a®t transversion 13 g®a transition 39 g®t transversion 14 a®t transversion 40 t®g transversion 15 g®a transition 41 t®g transversion 16 t®c transition 42 t®a transversion 17 g®a transition 43 a®t transversion 18 t®c transition 44 g®a transition 19 a®c transversion 45 a®t transversion 20 c®t transition 46 a®g transition 21 g®t transversion 47 g®a transition 22 t®g transversion 48 t®c transition 23 t®g transversion 49 t®g transversion 24 t®a transversion 50 t®a transversion 25 t®g transversion 51 g®a transition 26 g®a transition 51 mutations in the forward and reverse strands, respectively, were detected in gyra of the tested isolate; since gyra encodes for dna gyrase, these mutations while leading to amino acid substitutions, alter the target protein for fluoroquinolone structure and subsequently the fluoroquinolone binding affinity of the enzyme, leading to drug resistance.20 on the other hand; after comparing the obtained sequence of parc from the tested isolate with sequences from ncbi, the result revealed complete similarity, and no mutations were recorded. in plain words, resistance to ciprofloxacin in the tested isolates is due to a mutation in gyrase rather than topoisomerase iv. biofilm formation assay the microtiter plate assay is the most widely used and was considered a standard test for the detection of biofilm formation. this method has been reported to be the most sensitive, accurate, and reproducible screening method for the determination of biofilm production by clinical isolates of s. aureus, e. coli, and p. aeruginosa and has the advantage of being a quantitative tool for comparing the adherence of different strains.21 130 j contemp med sci | vol. 9, no. 2, march-april 2023: 127–133 a potential role of extracellular dna in biofilm and ciprofloxacin resistance original h.t. ibrahim et al. the result revealed that only 8% of s. aureus isolates were strong biofilm producers; while 60% and 32% of the isolates were moderate and weak producers, respectively. on the other hand, none of the tested e. coli isolates were strong biofilm producers; whereas 68% and 32% of the isolates were moderate and weak producers, respectively. similar trends have been reported by mohammed et al.22 in that 14% of their local s. aureus isolates were strong biofilm-producers, 43% were low biofilm intensity and 43% were biofilm-negative. mathur et al.21 similarly conclude from their data that about 14.47% and 39.4% of s. aureus isolates exhibited high and moderate biofilm formation, respectively; while 46% were weak isolates. these data are not consistent with those reported by saeed et al.23 who stated that about 12.5% of isolated local strains of e. coli were strong biofilm producers while it agreed partially with their findings in that 87.5% of e. coli were moderate biofilm producers. it also disagrees greatly with fattahi et al.24 who found 38% of e. coli isolates were strong biofilm producers while 22%, 32%, and 8% of the isolates were moderate, weak, and non-biofilm producers respectively. the results are generally consistent with the findings of ghafil25 in that the ability of s. aureus to form biofilm was higher than that of e. coli. s. aureus biofilms, once established, are recalcitrant to antimicrobial treatment and the host response, and therefore are the etiological agent of many recurrent infections that have a demonstrated biofilm component.26 chronic infections are associated with the biofilm mode of growth where s. aureus can attach and persist on host tissues, such as bone and heart valves, to cause osteomyelitis and endocarditis respectively, or on implanted materials, such as prosthetic joints,27 catheters,28 table 3. impact of edna on biofilm isolate code od 600 p-value lsd 0.05control (no edna) 50 ng/µl of edna 100 ng/µl of edna 200 ng/µl of edna s1 0.151 0.114 0.108 0.112 0.004000 0.018 s2 0.181 0.152 0.149 0.113 0.000024 0.012 s3 0.193 0.182 0.154 0.167 0.016000 0.020 s4 0.178 0.133 0.143 0.134 0.035000 0.027 s5 0.151 0.150 0.133 0.127 0.160000 – e1 0.135 0.124 0.122 0.112 0.246066 – e2 0.131 0.133 0.118 0.109 0.204402 – e3 0.166 0.178 0.204 0.159 0.221004 – e4 0.142 0.173 0.164 0.151 0.501460 – e5 0.148 0.131 0.119 0.099 0.000206 0.011 p1 0.143 0.156 0.220 1.036 0.000001 0.012 p2 0.112 0.111 0.124 0.138 0.000010 0.004 k1 0.097 0.130 0.235 0.751 0.000004 – k2 0.113 0.171 0.185 0.217 0.000024 – se1 0.112 0.134 0.115 0.109 0.006000 0.019 se2 0.154 0.162 0.142 0.126 0.033009 – se3 0.164 0.162 0.159 0.148 0.449729 – se4 0.150 0.142 0.127 0.116 0.084083 – se5 0.301 0.291 0.289 0.266 0.399151 – and pacemakers.29 chronic s. aureus infections that are associated with biofilm frequently lead to significant increases in both morbidity and mortality, mainly when the infection is associated with indwelling medical devices.30 implanted materials become coated with host proteins upon insertion, and the matrix-binding proteins on the surface of s. aureus facilitate attachment to these proteins and the development of a biofilm. in cases of infected medical devices, removal of the device is often necessary to treat the infection.31 complications in e. coli-related infection have been mainly attributed to biofilm formation. e. coli biofilm formation is an intricate process that involves several steps such as initial adhesion, early development, maturation, and dispersion. these steps are governed by many genes that serve specific functions in the formation of the biofilm. e. coli biofilm has frequently been resistant to numerous antibiotics, mostly accredited to putative multidrug resistance pumps. the development of the extracellular matrix and the observed increased resistance to common antibiotics create a challenge to control the infections caused by e. coli biofilms.32 impact of edna concentration on biofilm intensity to investigate the impact of edna on biofilm, different concentrations of genomic dna were added to the wells of a microtiter plate containing selected bacterial isolates of the species e. coli, s. aureus, p. aeruginosa, salmonella enterica serovar typhi and klebsiella pneumonia. the result presented in table 3 revealed that the addition of increasing concentrations 131j contemp med sci | vol. 9, no. 2, march-april 2023: 127–133 h.t. ibrahim et al. original a potential role of extracellular dna in biofilm and ciprofloxacin resistance from figure 1, it can be noted that only 8% of s. aureus isolates were resistant to ciprofloxacin; whereas 28% developed intermediate resistance and 64% were sensitive to this antibiotic. on the other hand, about 44% of e. coli isolates were resistant to ciprofloxacin while no intermediate resistance was observed among the tested isolates; nevertheless, 56% were sensitive. regarding p. aeruginosa, the two tested isolates were ciprofloxacin-sensitive. these findings confirm those of earlier studies, such as mohamed et al.37 who found that the resistance of locally isolated e. coli strains from iraqi patients to ciprofloxacin was about 40.7%. whereas they differ slightly from those reported by al-jebouri and mdish38 who found that only 25% of e. coli and 40% of s. aureus isolates were resistant to ciprofloxacin. furthermore, our findings are in good agreement with al-marjani et al.39 who stated that about 16% of s. aureus isolates were resistant to ciprofloxacin. the increasing resistance of bacteria to ciprofloxacin could probably be augmented by using it to treat many infections including prostatitis, uti, endocarditis, gastroenteritis, infections of bones and joints, lower respiratory tract infection, and enteric fever, among others, even though the risk of tendon rupture could increase upon using it. notably, another factor contributing to the problem is the availability of ciprofloxacin as an oral suspension that is currently flooding the market; even though, it is not licensed by the fda to treat children with ciprofloxacin due to the high risk of permanent injury to the musculoskeletal system except for inhalation anthrax and cystic fibrosis.40 determining the role of edna in gene transfer this experiment was designed to assess the possible role of edna in the transfer of antibiotic-resistance genes. since the addition of edna has increased the biofilm intensity of p. aeruginosa isolate p1 only, our study was focused on that isolate. the mic of ciprofloxacin was measured before and after the growth of the sensitive isolate in the presence of the dna of ciprofloxacin-resistant isolate (s. aureus isolate s17), the result revealed that the mic value increased significantly (p < 0.05) from 1 to 4 µg/ml turning the bacterial isolate from sensitive to resistant to ciprofloxacin. the acquired resistance was also tested after three successive generations and it was shown that the mic value remained at 4 µg/ml. the same experiment was repeated using ciprofloxacin-sensitive e. coli e4 and s. aureus s4 isolates as a recipient for gene transfer. nonetheless, when measuring the mic values before and after the gene transfer, it remained at 1 µg/ml; hence, the isolates of edna resulted in a significant decrease (p < 0.05) in biofilm intensity for the majority of the tested s. aureus isolates. s1-s5: s. aureus isolates 1-5; e1-e5: e. coli isolates 1-5; p1 and p2: p. aeruginosa isolate 1 and 2; k1 and k2: klebsiella pneumoniae isolates; se1-se5: s. typhi isolates moreover, the biofilm intensity significantly decreased (p < 0.05) linearly with increasing concentrations of edna; on the other hand, although edna addition had led to thinner biofilm in the tested e. coli and s. typhi isolates, the increasing concentration did not have a significant effect (p > 0.05) on the biofilm intensity; nevertheless, the biofilm of one strain of e. coli and two strains of s. typhi was significantly decreased (p < 0.05) linearly with increasing edna. surprisingly, the addition of edna led to a significant increase (p < 0.05) in the biofilm of the tested isolates of p. aeruginosa and k. pneumonia. the findings of this study agreed with the findings of berne et al.33 who had informed that the biofilm formation of caulobacter crescentus is significantly inhibited by the presence of edna. those results suggested that the bacteria would probably have a better chance for attachment to abiotic surfaces in the presence of dnase i, hence their ability to form more compact biofilm would increase; additionally, the biofilm formation in salmonella had significantly been inhibited upon the addition of exogenous edna. another study conducted by özdemir et al.34 revealed that edna could either enhance or decrease the biofilm formation by salmonella and such effect of edna would be reliant on salmonella serotype. other studies that were conducted on the biofilm of listeria monocytogenes and neisseria meningitides come in contrast to our findings in which the biofilm formation had not been significantly affected by the addition of purified edna. however, crude extracts of edna in combination with probably some specific proteins or cell wall fragments promote the process of biofilm formation.35,36 due to the interaction of edna with one or more of the biofilm components needs further investigation. inhibiting role of edna in the biofilm development of either s. aureus, e. coli or s. typhi from our findings was another study carried out by wang et al.11 demonstrated the inhibitory effect of edna on salmonella enterica biofilm who stated that salmonella strains formed a thicker layer of biofilm in the presence of dnase i. of c. crescentus, which prevented the cells from settling into and encouraged the dispersal of cells. determination of minimal inhibitory concentration (mic) using agar diffusion method the susceptibility of the bacterial isolates (s. aureus, e. coli, and p. aeruginosa) towards ciprofloxacin was tested by determining the mic using the agar diffusion method. from the findings of the present study, various levels of susceptibilities to ciprofloxacin among isolates were observed. the results are summarized in figure 1. fig. 1 susceptibility of bacterial isolates to ciprofloxacin. 132 j contemp med sci | vol. 9, no. 2, march-april 2023: 127–133 a potential role of extracellular dna in biofilm and ciprofloxacin resistance original h.t. ibrahim et al. remained sensitive to ciprofloxacin; consequently, no gene transfer occurred. the sequence analysis of the gyra gene for the isolate before and after the addition of edna revealed slight variation, which furthermore confirms that the gene transfer process might have occurred and edna was responsible for that process. correspondingly, no pcr product was obtained when trying to amplify the acra gene after the gene transfer which implies that the acra gene has not been transferred during the process. the pool of edna found in bacterial biofilms provides a rich substrate for naturally occurring genetic transformation, which is the only alternative to mobile genetic elements and bacteriophage-induced gene transfer. this observation led to investigations into the role of dna donor cells in biofilms and the conclusion that biofilm cells actively donate dna to their prokaryotic neighbors.41 extracellular dna active in the natural transformation was shown to be released by both gram-positive and gram-negative members of soil bacteria, thereby facilitating naturally occurring genetic transformation. natural habitats suitable for horizontal gene transfer are not limited to the soil. the majority of bacterial populations on earth are accompanied by edna, and it is known that such edna is suitable for horizontal gene transfer.42 furthermore, it is well established that gene transfer occurs with enhanced efficiency in biofilms.43-46 such horizontal gene transfer is facilitated by a biofilm lifestyle, which is characterized by cohabitation in close vicinity. this sharing of genetic material may function as the prokaryotic equivalent to sexual selection,47 leading to beneficial adaptations such as antibiotic resistance48 or pathogenicity.49 conclusion extracellular dna has a major role in the gene transfer process to biofilms. given that, the addition of increasing concentrations of edna resulted in a significant decrease (p < 0.05) in biofilm intensity for the majority of the tested s. aureus and e. coli isolates. whereas, it has led to a significant increase (p < 0.05) in the biofilm of the two tested p. aeruginosa isolates. acknowledgment the authors would like to thank the patients for agreeing to participate in the study. conflict of interest “the authors declare no conflict of interest”.  references 1. akmatov, m. k., mehraj, j., gatzemeier, a., strompl, j., witte, w., krause, g., et al., serial home-based self-collection of anterior nasal swabs to detect staphylococcus aureus carriage in a randomized population-based study in germany. int j infect dis. 2014; 25: 4–10. doi:10.1016/j.ijid.2014.01.021 2. junie, l. m., simon, l. m., pandrea, s. l. resistance to the chemotherapeutic agents of staphylococcus aureus strains isolated from hospitalized patients. international journal of infectious diseases. 2014; 21:79–80. doi:10.1016/j. ijid.2014.03.593 3. rossi, e., cimdins, a., luthje, p., brauner, a., sjoling, a., landini, p., et al., “it’s a gut feeling” escherichia coli biofilm formation in the gastrointestinal tract environment. crit rev microbiol. 2018; 44(1): 1–30. doi:10.1080/104084 1x.2017.1303660 4. bettelheim, k. a., goldwater, p. n. escherichia coli and sudden infant death syndrome. front immunol. 2015; 6: 343. doi:10.3389/fimmu.2015.00343 5. blount, z. d. the unexhausted potential of e. coli. elife. 2015; 4. doi:10.7554/ elife.05826 6. soukarieh, f., vico oton, e., dubern, j. f., gomes, j., halliday, n., de pilar crespo, m., et al., in silico and in vitro-guided identification of inhibitors of alkylquinolone-dependent quorum sensing in pseudomonas aeruginosa. molecules. 2018; 23(2). doi:10.3390/molecules23020257 7. markou, p., apidianakis, y. pathogenesis of intestinal pseudomonas aeruginosa infection in patients with cancer. front cell infect microbiol. 2014; 3: 115. doi:10.3389/fcimb.2013.00115 8. flemming, h. c., wingender, j., szewzyk, u., steinberg, p., rice, s. a., kjelleberg, s. biofilms: an emergent form of bacterial life. nat rev microbiol. 2016; 14(9): 563–575. doi:10.1038/nrmicro.2016.94 9. liu, y., ren, z., hwang, g., koo, h. therapeutic strategies targeting cariogenic biofilm microenvironment. adv dent res. 2018; 29(1): 86–92. doi:10.1177/0022034517736497 10. mulcahy, h., charron-mazenod, l., lewenza, s. pseudomonas aeruginosa produces an extracellular deoxyribonuclease that is required for utilization of dna as a nutrient source. environ microbiol. 2010; 12(6): 1621–1629. doi:10.1111/j.1462-2920.2010.02208.x 11. wang, h., huang, y., wu, s., li, y., ye, y., zheng, y., et al., extracellular dna inhibits salmonella enterica serovar typhimurium and s. enterica serovar typhi biofilm development on abiotic surfaces. curr microbiol. 2014; 68(2): 262–268. doi:10.1007/s00284-013-0468-5 12. harley, j. b. laboratory exercises in microbiology. 10 ed: mcgrawhill education; 2016. 13. jennifer, m. a. determination of minimum inhibitory concentrations. j antimicrob chemother. 2001; 48: 5–16. 14. martineau, f., picard, f. j., roy, p. h., ouellette, m., bergeron, m. g. speciesspecific and ubiquitous-dna-based assays for rapid identification of staphylococcus aureus. j clin microbiol. 1998; 36(3): 618–623. 15. mcclure, j. a., conly, j. m., lau, v., elsayed, s., louie, t., hutchins, w., et al., novel multiplex pcr assay for detection of the staphylococcal virulence marker panton-valentine leukocidin genes and simultaneous discrimination of methicillin-susceptible from -resistant staphylococci. j clin microbiol. 2006; 44(3): 1141–1144. doi:10.1128/jcm.44.3.11411144.2006 16. anastasi, e. m., matthews, b., gundogdu, a., vollmerhausen, t. l., ramos, n. l., stratton, h., et al., prevalence and persistence of escherichia coli strains with uropathogenic virulence characteristics in sewage treatment plants. appl environ microbiol. 2010; 76(17): 5882–5886. doi:10.1128/ aem.00141-10 17. gomig, f., galvao, c. w., freitas, d. l., labas, l., etto, r. m., esmerino, l. a., et al., quinolone resistance and ornithine decarboxylation activity in lactose-negative escherichia coli. braz j microbiol. 2015; 46(3): 753–757. doi:10.1590/s1517-838246320131291 18. pakzad, i., zayyen karin, m., taherikalani, m., boustanshenas, m., lari, a. r. contribution of acrab efflux pump to ciprofloxacin resistance in klebsiella pneumoniae isolated from burn patients. gms hyg infect control. 2013; 8(2): doc15. doi:10.3205/dgkh000215 19. nakao, r., ramstedt, m., wai, s. n., uhlin, b. e. enhanced biofilm formation by escherichia coli lps mutants defective in hep biosynthesis. plos one. 2012; 7(12): e51241. doi:10.1371/journal.pone.0051241 20. darbani, r., farshadfar, c., tavana, s., saljoughi, h., zonouri, s. s. identification of dna gyrase subunit a mutations associated with ciprofloxacin resistance in staphylococcus aureus isolated from nasal infection in kurdistan-iran. j mol biol res. 2017; 7(1): 186. doi:10.5539/jmbr. v7n1p186 21. mathur, t., singhal, s., khan, s., upadhyay, d., fatma, t., rattan, a. detection of biofilm formation among the clinical isolates of staphylococci: an evaluation of three different screening methods. indian journal medical microbiology. 2006; 24: 25–29. 22. mohammed, m. k., rasheed, m. n., nadeer, m. i. detection of biofilmassociated genes in clinical staphylococcus aureus isolates from iraqi patient. iraqi j sci nat 2015; 6: 19–22. 133j contemp med sci | vol. 9, no. 2, march-april 2023: 127–133 h.t. ibrahim et al. original a potential role of extracellular dna in biofilm and ciprofloxacin resistance 23. saeed, e. a., bnyan, i. a., al saadi, m. a. k. quorum sensing and biofilm formation by bacterial isolates from hemodialysis patients. research in pharmacy. 2013; 3: 33–40. 24. fattahi, s., kafil, h. s., nahai, m. r., asgharzadeh, m., nori, r., aghazadeh, m. relationship of biofilm formation and different virulence genes in uropathogenic escherichia coli isolates from northwest iran. gms hyg infect control. 2015; 10: doc11. doi:10.3205/dgkh000254 25. ghafil, j. a. assessment the effect of non-thermal plasma on escherichia coli and staphylococcus aureus biofilm formtion in vitro. iraqi j sci. 2018; 59: 25–29. 26. archer, n. k., mazaitis, m. j., costerton, j. w., leid, j. g., powers, m. e., shirtliff, m. e. staphylococcus aureus biofilms: properties, regulation, and roles in human disease. virulence. 2011; 2(5): 445–459. doi:10.4161/viru.2.5.17724 27. barrett, l., atkins, b. the clinical presentation of prosthetic joint infection. j antimicrob chemother. 2014; 69 suppl 1: i25–27. doi:10.1093/jac/dku250 28. chatterjee, s., maiti, p., dey, r., kundu, a., dey, r. biofilms on indwelling urologic devices: microbes and antimicrobial management prospect. ann med health sci res. 2014; 4(1): 100–104. doi:10.4103/2141-9248.126612 29. kiedrowski, m. r., horswill, a. r. new approaches for treating staphylococcal biofilm infections. ann n y acad sci. 2011; 1241: 104 –121. doi:10.1111/ j.1749-6632.2011.06281.x 30. moormeier, d. e., bayles, k. w. staphylococcus aureus biofilm: a complex developmental organism. mol microbiol. 2017; 104(3): 365–376. doi:10.1111/mmi.13634 31. lister, j. l., horswill, a. r. staphylococcus aureus biofilms: recent developments in biofilm dispersal. front cell infect microbiol. 2014; 4: 178. doi:10.3389/fcimb.2014.00178 32. sharma, g., sharma, s., sharma, p., chandola, d., dang, s., gupta, s., et al., escherichia coli biofilm: development and therapeutic strategies. j appl microbiol. 2016; 121(2): 309–319. doi:10.1111/jam.13078 33. berne, c., kysela, d. t., brun, y. v. a bacterial extracellular dna inhibits settling of motile progeny cells within a biofilm. mol microbiol. 2010; 77(4): 815–829. doi:10.1111/j.1365-2958.2010.07267.x 34. özdemir, c., karaca, b., akçelik, n. the role of extracellular dna in salmonella biofilms: is it in intimate relationship with matrix or initial adhesion? 25th eccmid conference; copenhagen, denmark. 2015. 35. harmsen, m., lappann, m., knochel, s., molin, s. role of extracellular dna during biofilm formation by listeria monocytogenes. appl environ microbiol. 2010; 76(7): 2271–2279. doi:10.1128/aem.02361-09 36. lappann, m., claus, h., van alen, t., harmsen, m., elias, j., molin, s., et al., a dual role of extracellular dna during biofilm formation of neisseria meningitidis. mol microbiol. 2010; 75(6): 1355–1371. doi:10.1111/j.13652958.2010.07054.x 37. mohamed, t. j., rabeea, i. s., abd, a. h., abdulkhaleq, m. a. efficacy of combination of meropenem with ciprofloxacin, and nitrofurantoin against resistant e. coli isolated from patients with urinary tract infections: in vitro study. al-yarmouk journal. 2011; special volume: 61–75. 38. al-jebouri, m. m., mdish, s. a. antibiotic resistance pattern of bacteria isolated from patients of urinary tract infections in iraq. open journal of urology. 2013; 03(02): 124–131. doi:10.4236/oju.2013.32024 39. al-marjani, m. f., kadhim, k. a., a., k. a., kinani, a. ciprofloxacin resistance in staphylococcus aureus and pseudomonas aeruginosa isolated from patients in baghdad. international journal of pharma sciences and research. 2015; 6 382–385. 40. abdullah, f. e., memon, a. a., bandukda, m. y., jamil, m. increasing ciprofloxacin resistance of isolates from infected urines of a cross-section of patients in karachi. bmc res notes. 2012; 5: 696. doi:10.1186/1756-0500-5-696 41. thomas, c. m., nielsen, k. m. mechanisms of, and barriers to, horizontal gene transfer between bacteria. nat rev microbiol. 2005; 3(9): 711–721. doi:10.1038/nrmicro1234 42. kaneko, s., itaya, m. stable extracellular dna: a novel substrate for genetic engineering that mimics horizontal gene transfer in nature in: kikuchi, y., rykova, e. y., editors. extracellular nucleic acids. nucleic acids and molecular biology series. 25 ed. berlin, heidelberg: springer-verlag; 2010. p. 3953. 43. bjorklof, k., nurmiaho-lassila, e. l., klinger, n., haahtela, k., romantschuk, m. colonization strategies and conjugal gene transfer of inoculated pseudomonas syringae on the leaf surface. j appl microbiol. 2000; 89(3): 423–432. doi:10.1046/j.1365-2672.2000.01130.x 44. springael, d., peys, k., ryngaert, a., van roy, s., hooyberghs, l., ravatn, r., et al., community shifts in a seeded 3‐chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc‐element from inoculum to contaminant bacteria. environ microbiol. 2002; 4: 70–80. 45. molin, s., tolker-nielsen, t. gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure. current opinion in biotechnology. 2003; 14(3): 255–261. doi:10.1016/s09581669(03)00036-3 46. roberts, a. p., pratten, j., wilson, m., mullany, p. transfer of a conjugative transposon, tn5397 in a model oral biofilm. fems microbiol lett. 2006; 177: 63–66. 47. spoering, a. l., gilmore, m. s. quorum sensing and dna release in bacterial biofilms. curr opin microbiol. 2006; 9(2): 133–137. doi:10.1016/j. mib.2006.02.004 48. sykes, r. the 2009 garrod lecture: the evolution of antimicrobial resistance: a darwinian perspective. j antimicrob chemother. 2010; 65(9): 1842–1852. doi:10.1093/jac/dkq217 49. dillard, j. p., seifert, s. a variable genetic island specific for neisseria gonorrhoeae is involved in providing dna for natural transformation and is found more often in disseminated infection isolates. mol microbiol. 2001; 41: 263–277. doi:10.1046/j.1365-2958.2001.02520.x this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1338 77j contemp med sci | vol. 9, no. 1, january-february 2023: 77–81 original assessment of missing opportunity of vaccination at primary health care center: a retrospective study ali fadhil obaid1*, zainab abdulameer abdulrasol2, ahmed mohammed jasim shlash3, methaq rabah tuman2, muamel dhaher hussain2 1pediatric nursing, college of nursing, university of babylon, babylon, iraq. 2maternal and newborn health nursing, college of nursing, university of babylon, babylon, iraq. 3adults health nursing, college of nursing, university of al-ameed, karbala, iraq. *correspondence to: fadhil obaid (e-mail: ali19903li@gmail.com) abstract objectives: the study aim is to assess the vaccination status of children under two years old in al-asatetha primary health care center, hilla, babylon province, iraq. method: retrospective study design (retrospective chart review) was used to assess the missed opportunities of vaccination for children under the age of two years by reviewing their immunization status from medical reports at the primary health care center. the purposive sampling was used to collect the data of 1000 children born from 1-1-2020 to 31-12-2020 through the period from 1 december 2021 to 1 february 2022. results: the finding of the current study showed that the age of children in the selected period (2020-2021) was between11–23 months, the number of children (1–8) in the same family, and almost all of these families reside in urban areas. regarding the parents’ educational level the high percentage of both fathers and mothers were attained a primary level of education, and most of the fathers were working, while most of the mothers were not work. concerning the vaccination status the finding showed that the percentage of missed vaccination opportunities reached 61%, and there was a significant positive correlation between vaccination status and (family residence, parental education level, and fathers’ occupation) with a p value of 0.01. on the other hand, the number and the order of children in families had an inversely significant relationship with vaccination status at a p value of 0.01. conclusion: the study concludes that numerous factors affect the vaccination rate such as parents’ education attainment and awareness, family size, address, and economic status of the family. key words: vaccination, vaccines, missed opportunity for vaccination (mov) issn 2413-0516 introduction immunization define as one of the most efficient, safest, and cost-effective public health care interventions to prevent death and disability that can be controlled by vaccination. “immunization will help to achieve the millennium development goals (mdgs) on reducing child mortality, improving maternal health, and preventing diseases” resulting in improved global, national, and regional levels of social, economic, and health outcomes.1,2 immunization leads to a remarkable reduction in cases of vaccine-preventable infectious diseases among children.2,3 moreover, the whole benefits of immunization can be accomplished, if the missed opportunities for vaccination (movs) are eliminated and global vaccination coverage improves.4 the missed opportunity for vaccination define as any time the children or pregnant women of childbearing age who are eligible for vaccination (i.e., unvaccinated, partially vaccinated, or not up-to-date on vaccinations) and makes contact with health services but do not receive all of the vaccine doses for which they are eligible.5,6 the “world health organization (who) established the expanded program on immunization (epi) in 1974” to improve global vaccination coverage and achieve universal access to immunization. in 1977, the program set many goals to make immunization against diphtheria, pertussis, tetanus, poliomyelitis, measles, and tuberculosis available to every child in the world by 1990.7 in 1985, the “expanded program of immunization (epi) was well established in iraq to deliver immunization services to targeted groups and implement national and global strategies to achieve the main objectives of the program by improving the global coverage of vaccination”.8 however, despite the fact that vaccine compliance is a major aspect of preventative healthcare, there is a large gap between immunization mandates and actual compliance.9 various researchers have addressed the factors that cause vaccination delays. although the children eventually get the immunization, this concept considers essential since many delayed cases lead to inadequate vaccination of the children. most of these factors include: single-parent households, child’s health status, larger family size, family socioeconomic status, low parental education level, geographic location, medicaid enrollment, absence of primary health care provider, and lack of insurance coverage.10–13 study objectives the current study aimed to assess the vaccination status of under two year’s children in al-asatetha primary health care center (phc)/hilla-iraq, in addition, to finding out the correlation between the missed opportunity of vaccination and some sociodemographic variables. methods study design retrospective study design (retrospective chart review) was used to assess missed opportunities of under two years’ mailto:ali19903li@gmail.com 78 j contemp med sci | vol. 9, no. 1, january-february 2023: 77–81 assessment of mov at phc center original a.f. obaid et al. table 1. sociodemographic characteristics of children and their parents (no. 1000) items interval freq. percent child age per months 11–15 375 37.5 16–20 407 40.7 21+ 218 21.8 minimum (11 months) maximum (23 months) mean 16.97 std. deviation 3.558 child gender male 518 51.8 female 482 48.2 childern number minimum: 1 child maximum: 8 children child order 1st 256 25.6 2nd 292 29.2 3rd 215 21.5 4th 120 12.0 5th 72 7.2 6th 33 3.3 7th 10 1.0 8th 2 .2 residency rural 199 19.9 urban 801 80.1 father level of edu. illitrate 10 1.0 primary 372 37.2 intermediate 225 22.5 secondary 113 11.3 diploma and above 280 28.0 mother level of edu. illitrate 71 7.1 primary 355 35.5 intermediate 185 18.5 secondary 87 8.7 diploma and above 302 30.2 father occupation not work 246 24.6 work 754 75.4 mother occupation not work 882 88.2 work 118 11.8 children, through reviewing their immunization status from medical records of phc. sample & setting of the study purposive sampling of 1000 children was born from 1-1-2020 to 31-12-2020; collected from the medical reports at al-asatetha primary health care center through the period of 1st december 2021 to 1st of february 2022. instruments the questionnaire was constructed after extensive literature reviews and used for the study, which consisted of two parts: part 1: items related to demographic data of parents and children: this part is concerned with the personal information of parents (occupation and education) and children (age, gender, and order). part 2: items related to the vaccination status of children in this part the data was divided into three categories of vaccination status: the first group was completely vaccinated children, the second group was partially vaccinated children (children who missed one to five vaccines up to their ages), and the third group was children who completely missed their vaccines or were unvaccinated (children missed more than five vaccines up to their ages). all of these data are according to the iraqi schedule of vaccination. statistical analysis the data was analyzed statistically by using “statistical package for social sciences (spss) version 26 through the application of descriptive statistical data analysis including frequencies, percentages, mean of the score (ms) with their standard deviation (sd), inferential statistics, and spearman correlation. results table 1 shows that 40% of children between the age of 16-20 months and the percent of males were equal to females children in the study, regarding children number the minimum number was one child, while the maximum number was eight children in the family. 50% of these families have 2-3 children, and 80% of them reside in urban areas. concerning the parents’ educational attainment, more than 35% of both fathers and mothers have a primary level of education, and regarding the parents’ occupation, the finding shows that 75% of fathers were working while 88% of mothers were not working. table 2 shows that there was a significant positive correlation between vaccination status and (family address, parent education, and father occupation) at p-value < 0.01. in another hand, the children’s number and child order in families had an inverse signification correlation with the vaccination status at p-value < 0.01. discussion the current study shown in (table 1) that the target ages of children in the selected period (2020-2021) were between 1-2 years, and less than half of them were between 16-20 months regarding children number in the family; half of these families have 2-3 children, while some of them have more than six children, and almost all of them reside in the urban area. concerning the parents’ educational attainment, the finding reveals that one-third of parents completed a primary level of education and about the parents’ occupation: most fathers are working while most mothers are not working. these findings were similar to a study conducted by asiegbu and his colleagues in (2020)14 and both of the studies that conducted in baghdad by abbas and his colleague in (2016)15 and fadil and his colleague in (2010).2 mov is a hurdle to rising immunization coverage among children and women of childbearing age worldwide,16 and the 79j contemp med sci | vol. 9, no. 1, january-february 2023: 77–81 a.f. obaid et al. original assessment of mov at phc center fig. 1 missing opportunity of vaccine status of 1000 childern in phc. table 2. correlations coefficient between vaccination status and sociodemographic variables spearman’s rho address child order children numbers father edu. mother edu. father occupation mother occupation vaccination status correlation coefficient .157** -.157** -.165** .116** .123** .114** .013 sig. .000 .000 .000 .000 .000 .000 .691 n 1000 1000 1000 1000 1000 1000 1000 correlation is significant at the 0.01 level (2-tailed).** fig. 2 schedule vaccination status of 1000 childern in phc. results show in (figure 1) that the missing opportunities of the vaccination status of 1000 children in al asatetha phc revealed that there are three categories of vaccination status; one-third of children are fully vaccinated, less than half of them have partially vaccinated children and less than quarter of children completely missing their vaccination, and this finding goes in line with the result of mahmood, (2012), which performed on infant children in diyala province/iraq that found (70%) of children were fully vaccinated, (24%) were partially vaccinated children and (6%) were non-vaccinated children. another study showed that (75.4%) were fully vaccinated, and most of them were vaccinated during immunization campaigns.17 whereas, asiegbu and his colleagues in (2020) found that (71.6%) had missed vaccination appointments for their children with the far immunization center14 and kaboré and his colleagues also found that 76% eligible 80 j contemp med sci | vol. 9, no. 1, january-february 2023: 77–81 assessment of mov at phc center original a.f. obaid et al. children for vaccination missed their opportunity to be vaccinated when visiting the health facilities figure 2.18 regarding the factors that affect mov, (table 2) shows that there is a positive relationship between vaccination status with some sociodemographic variables such as (family address, parents’ education attainment, and fathers’ occupation), which means when the level of parents’ education this leading to increase awareness about the benefits of childhood vaccination. in another hand, the increased children number and child order in the family have a negative relationship with the vaccination status, and this means the family size affects vaccination status. the previously mentioned findings are similar to the results of research conducted in erbil/iraq by hassan & ahmed, (2020), which concluded that children who are not vaccinated on time are likely to be from family of low socio-economic status and having low parental educational level.19 and another research conducted by asiegbu et al., (2020) revealed the mothers’ educational status, and place of residence were significantly associated with increase knowledge of childhood vaccination.14 in addition to dombkowski and his colleagues, which found that parental education, the absence of a two-parent household, and large family size all of these reasons contribute to the delay of child vaccination.12 while some scholars found that the increase in parents’ education and awareness regarding immunization and vaccination and even reminding them by using digital media can significantly affect immunization coverage and reduce the missing opportunity for vaccination.17,18 furthermore, the study by abdul rahman and his colleagues conducted in kurdistan/iraq found the role of religious leaders in improving vaccination coverage about 95% post-intervention period compared with 55% of vaccination coverage pre-intervention period.20 conclusion the overall vaccination rate showed that missed vaccination opportunities reached 61%, and this is due to many reasons such as parents’ education attainment, number of children in the family, and family residential area. therefore, when the parents have a high level of education, then the vaccination rate increase, and if the family have a few children, the missed opportunity of vaccination decrease. recommendations 1. using digital media such as social media and television by health institutions and concerned authorities to increase awareness of families regarding the importance of taking vaccines and their benefits. 2. recommend the health institutions to use sms text messages to remind the families about times of vaccinations, and this technique will help in reaching the children’s immunization coverage. 3. increase vaccination campaigns by phcs to achieve the epi goals of who. ethical considerations the research was conducted with ethical approval from the university of babylon’s nursing college. funding no government, private, or non-profit organization supported this study financially. authors’ contributions each of the authors made significant contributions in this study. conflict of interest there are no conflicts of interest revealed by the authors.  references 1. moh, usaid’s, phcpi. national immunization plan of iraq for 2015 table of contents. 2015;(december 2014). 2. fadil ls, al-lami f. proportion and determinants of incomplete vaccination among children aged less than two years in baghdad city. iraqi postgrad med j. 2010;9(2). 3. raof am. parental attitude and beliefs towards child vaccination: identifying vaccine hesitant groups in a family health center, erbil city, iraq. world fam med j inc middle east j fam med. 2018;99(6002):1–10. 4. jaca a, mathebula l, iweze a, pienaar e, wiysonge cs. a systematic review of strategies for reducing missed opportunities for vaccination. vol. 36, vaccine. 2018. 5. who. essential programme on immunization [internet]. who. 2021 [cited 2022 feb 27]. p. 2021. available from: https://www.who.int/ teams/immunization-vaccines-and-biologicals/essential-programmeon-immunization/implementation/reducing-missed-opportunities-forvaccination-(mov) 6. sridhar s, maleq n, guillermet e, colombini a, gessner bd. a systematic literature review of missed opportunities for immunization in low-and middle-income countries. vaccine. 2014;32(51):6870–9. 7. keja k, chan c, hayden g, henderson rh. expanded programme on immunization. world health stat q. 1988;41(2):59–63. 8. lafta r, hussain a. trend of vaccine preventable diseases in iraq in time of conflict. pan afr med j. 2018;31(1). 9. bundt ts, hu h. national examination of compliance predictors and the immunization status of children: precursor to a developmental model for health systems. mil med. 2004;169(10):795–803. 10. falagas me, zarkadoulia e. factors associated with suboptimal compliance to vaccinations in children in developed countries: a systematic review. curr med res opin. 2008;24(6):1719–41. 11. mahmood ns. rate of vaccination of children at diyala province & the effect of parental education on vaccination status, hospital based study. diyala j med. 2012;3(1):73–81. 12. dombkowski kj, lantz pm, freed gl. risk factors for delay in ageappropriate vaccination. public health rep. 2004;119(2):144–55. 13. al-shemari kd. causes of delay in age appropriate vaccination. iraqi postgrad med j. 2006;5:32–98. 14. asiegbu uv, obu dc, una afi, ezeonu ct, asiegbu ok. evaluating mothers knowledge and attitude as a contributing factor to the low childhood immunization uptake in ebonyi state, nigeria. african j med heal sci. 2020;19(8):127–35. 15. abbas lm, aldeen ld. incomplete vaccination among children below two years in a sample of urban primary health care centers at al-karkh baghdad city. iraqi journalof community med. 2016;29(3). 16. hutchins ss, jansen ha, robertson se, evans p, kim-farley rj. studies of missed opportunities for immunization in developing and industrialized countries. bull world health organ. 1993;71(5):549. 81j contemp med sci | vol. 9, no. 1, january-february 2023: 77–81 a.f. obaid et al. original assessment of mov at phc center 17. fite ro, hailu ld. immunization coverage of 12 to 23 months old children in ethiopia. j public heal epidemiol. 2019;11(1):31–7. 18. kaboré l, meda b, médah i, shendale s, nic lochlainn l, sanderson c, et al. assessment of missed opportunities for vaccination (mov ) in burkina faso using the world health organization’s revised mov strategy: findings and strategic considerations to improve routine childhood immunization coverage. vaccine. 2020;38(48):7603–11. 19. hassan za, ahmed mj. factors associated with immunisation coverage of children aged 12-24 months in erbil/iraq 2017-2018. int j psychosoc rehabil. 2020;24(08). 20. abdul rahman ma, al-dabbagh sa, al-habeeb qs. health education and peer leaders’ role in improving low vaccination coverage in akre district, kurdistan region, iraq. east mediterr heal j. 2013;19(2). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1321 original 65j contemp med sci | vol. 8, no. 1, january-february 2022: 65–68 association of taqi polymorphism of vitamin d receptor (vdr) gene with anemia in saudi women maryam a. al-ghamdi1,2* 1department of biochemistry, faculty of science, king abdulaziz university jeddah 21589, saudi arabia. 2vitamin d pharmacogenomics research group, king abdulaziz university, jeddah, saudi arabia. *correspondence to: maryam a. al-ghamdi (e-mail: maaalghamdi3@kau.edu.sa) (submitted: 13 december 2021 – revised version received: 16 january 2022 – accepted: 23 january 2022 – published online: 26 february 2022) introduction fat soluble vitamin d is required by the body for normal bone development and maintenance. it acts by increasing absorption of phosphate, calcium and magnesium. vitamin d deficiency is implicated in a wide range of diseases, most notably in relation to bones.1 vitamin d deficiency is a health concern worldwide. recent studies have shown vitamin d deficiency to play a major role in many metabolic as well as physiological disorders such as diabetes, cardiovascular disease, cancer and thyroid disorders.2 sunlight accounts for about 50 to 90% of supply of vitamin d.3 vitamin d regulates bone turnover by the stimulation of osteoclastic and osteoblastic cells.4 variations of the vdr gene, that is present in chromosome 12, have been associated with various disorders and the polymorphisms vary to a great extent among different populations of the world.5 four frequently occurring single nucleotide polymorphisms (snps) namely: apai, bsmi, foki, and taqi, found at the 3´ end of the vdr gene have been studied in detail.6,7 the vdr suppresses activation of t cells, therefore, t-cell mediated autoimmune diseases are associated with polymorphisms in this receptor gene.8 polymorphisms in the vdr gene have been extensively studied.9 deficiency of iron leads to anemia. there are various variants of anemia, such as pernicious anemia, nutritional anemia, sickle cell anemia and hemolytic anemia. while some anemias are genetic, the most common form observed in a large section of the population worldwide is due to iron deficiency, in turn manifested as low levels of hemoglobin. according to a who report in 2008, 1;62 billion of the world population is anemic. in females, the main cause of anemia is blood loss during menstruation and pregnancy. since nutritional deficiency is the most common cause of iron deficiency anemia, this study is aimed at studying the possible association between anemic, vitamin d deficient women with vdr gene polymorphism at three restriction sites apai, taqi and bsmi. we found a significant association between the taqi polymorphisms and occurrence of anemia in the study population. materials and methods study subjects the study involved volunteers: 50 women in the age group 25–35 who were anemic and had low serum vitamin d levels. the control group consisted of 50 women in age group 25–35 who had sufficient blood hemoglobin and serum vitamin d levels. care was taken to only include women in both groups who did not have any other health conditions such as diabetes, hypertension, cardiac disorders, thyroid disorders etc. informed consent of the volunteers was obtained. hemoglobin levels of all the volunteers were measured using cbc. women who had hemoglobin levels lesser than 10 grams/dl were classified as anemic, while those with hemoglobin levels greater than 10 grams/dl were classified as healthy. serum vitamin d levels were measured by blood test for 25-hydroxyvitamin d: 25(oh)d. levels of less than 20 ng/ml are considered deficient, 20–30 ng/ml are considered as insufficient and levels greater than 30 ng/ml are considered sufficient. extraction of dna and amplification of vitamin d receptor gene the protocol followed is the same as the one in our previous study.9 genomic dna was extracted from whole blood using qiaamp dna blood mini kit (qiagen, usa, cat. no.51104). the extracted dna was stored at −20˚c for further pcr reactions (table 1). nanodrop2000c instrument from thermo scientific (usa) was used to calculate abstract objectives: this study aims to correlate occurrence of anemia in a population of saudi women with polymorphisms in the vdr gene. methods: the study sample consisted of 50 anemic women with vitamin d deficiency and 50 healthy women with normal hemoglobin and sufficient serum vitamin d levels. restriction fragment length polymorphism (rflp) analysis of the vdr genes was done for single nucleotide polymorphism (snp) sites namely apai, bsmi and taqi. statistical correlation was done between gene polymorphisms at these three sites and hemoglobin levels. results: we found a significant association between taqi site polymorphisms of the vdr gene and presence of anemia in the study population. conclusion: this is the first report of a significant association between vitamin d receptor gene polymorphisms and anemia in saudi population. further studies on a larger population size will pave way to elucidation of the mechanism in which vdr gene polymorphisms exert an influence on anemia. keywords: vdr polymorphism, anemia, taqi polymorphism, anemia, vitamin d deficiency issn 2413-0516 http://orcid.org/0000-000-0861953x association of taqi polymorphism of vitamin d receptor (vdr) gene with anemia in saudi women original maryam a. al-ghamdi 66 j contemp med sci | vol. 8, no. 1, january-february 2022: 65–68 table 1. represents the pcr reaction mixture 10–30 ng dna template 2x reaction buffer 4 mmmg+2 4 µm dntps 0.2 µm each of forward and reverse primers 0.45 u taq dna polymerase final volume made up with nuclease free water to 50 µl table 2. represents the pcr conditions for 30 cycles6 denaturation annealing extension final extension hold 95˚c for 30 sec 60˚c for 1 min 68˚c for 2 min 72˚c for 5 min 4˚c∞ table 3. pcr – rflp products for the restriction sites apai bsmi taqi aa – 2229 bp aa – 2229 bp, 1700 bp and 529 bp aa – 1700 bp, 259 bp bb 2229 bp bb – 2229 bp, 1579 bp, and 650 bp bb – 1579 bp and 650 bp tt 1982 bp and 247 bp tt – 1982 bp, 1780 bp, 202 bp, and 247 bp tt – 1780 bp, 202 bp, and 247 bp table 4. distribution of various alleles in healthy women (sufficient hemoglobin and vitamin d levels) restriction site genotype number of samples % frequency apai aa aa aa 18 21 11 (total 50) 36 42 22 (total 100%) bsmi bb bb bb 20 17 13 (total 50) 40 34 26 (total 100%) taqi tt tt tt 22 18 10 (total 50) 44 36 20 (total 100%) table 5. distribution of various alleles in women who were anemic as well as deficient in vitamin d restriction site genotype number of samples % frequency apai aa aa aa 19 21 10 (total 50) 38 42 20 (total 100%) bsmi bb bb bb 22 16 12 (total 50) 44 32 24 (total 100%) taqi tt tt tt 9 10 31 (total 50) 18 20 62 (total 100%) fig. 1 agarose gel (1%) showing the results of pcr-rflp comparison between the three restriction enzymes in anemia (p) and the control (c). lane m: dna marker. lane 2: negative control. lane 3: pcr product of size 2229 bp. lane 4, 5: rflp of apai digestion in control and patient. lane 7, 8: rflp of taqi digestion in control and patient. lane 10, 11: rflp of bsmi digestion in control and patient. results pcr-rflp analyses and distribution of the genotypes the restriction profiles obtained after pcr-rflp are summarized in table 3. the distribution of various alleles in control group and anemic group are presented in tables 4 and 5. from table 4, we observe that in the control group which comprised of healthy women with sufficient hemoglobin and serum vitamin d levels, there is no significant difference between the genotype frequencies in the single nucleotide polymorphisms (snps) at any of the three sites. in contrast, as seen in table 5, the study group of women with anemia and vitamin d deficiency clearly show a significant difference in concentration and purity of the extracted dna (table 2). the polymerase chain reaction (pcr) was carried out as described in our previous study.9 pcr amplification the amplification products were resolved on 1% agarose gels and 1x of tris-borate-edta (tbe) buffer followed by staining with ethidium bromide and visualized under uv light. genotyping of the vdr gene after its amplification, pcr product of ~2229 bp was exposed to enzymatic restriction digestion by three enzymes which were purchased from thermo scientific: apai, bsmi, and taqi (figure 1). the conditions for restriction digestion were followed according to iyer et al., 2017. the digested dna fragments and the dna size marker were segregated by electrophoresis on a 1% agarose gel stained with ethidium bromide and 1x tbe electrophoresis buffer. maryam a. al-ghamdi original association of taqi polymorphism of vitamin d receptor (vdr) gene with anemia in saudi women 67j contemp med sci | vol. 8, no. 1, january-february 2022: 65–68 genotype frequency at the taqi site. the genotype frequency of homozygous recessive alleles aa is 62% compared to tt and tt genotypes, while in control subjects the homozygous recessive genotype tt is observed in only 10% of the study subjects. discussion in recent times, anemia has been recognized as a world wide health issue. nutritional as well as metabolic disorders have been attributed to low hemoglobin levels associated with anemia. although we are well aware of the role of vitamin d in bone formation, metabolic regulation and overall growth and development, studies over the past few years have categorically suggested the role of vitamin d in erythropoiesis.10 studies by sim et al., 2010.,11 have shown significant association between vitamin d deficiency and anemia. cusato et al.,12 2015 proved the role of vdr gene polymorphisms in the ribavirin-induced anemia in hcv-patients at 2 and 4 weeks of medication. the vdr gene polymorphisms could attribute to significant receptor dysfunction thereby causing various disorders such as low bone mineral density, autoimmunity, infections, cardiovascular disease and cancers.13 moreover, studies on vdr gene polymorphism in various populations around the world have pointed out the significant role of the vdr gene in vitamin d deficiency.14 our own research group has established association of bsmi polymorphisms with type i diabetes, apai polymorphisms with type ii diabetes and taqi polymorphisms with gestational diabetes in saudi population.9 these findings encouraged us to probe the possible relation between anemia and vdr gene polymorphisms. with this as the premise, our current study was designed to look for a possible correlation between anemia and vitamin d deficiency with polymorphisms of the vdr gene at three snp sites namely apai, taqi and bsmi. we have taken extreme care to include only those subjects who were anemic as well as had low serum vitamin d levels as the study group while the control group were women with sufficient levels of hemoglobin as well as serum vitamin d. we have clearly observed that at the taqi site, there is a significant difference in genotype frequency between study group and control group. the homozygous recessive genotype tt was observed in 62% of the study group as against mere 20% in the control group. a study by yassin et al.,15 in an egyptian population showed no correlation between anemia and vdr polymorphism at apai and taqi sites. another study by yu et al.,16 showed a significant association between vdr polymorphisms and aplastic anemia in a chinese population. according to erturk et al.,17 bsmi polymorphism may have a role in anemia in hemodialysis patients with bb genotype being strongly associated with lower hemoglobin levels. to the best of our knowledge, our study is a pioneering finding that strongly establishes the association of homozygous recessive alleles tt of taqi polymorphism (rs731236) snp with anemia. the study is a very strong support for this finding because all the patients recruited for the study were anemic as well as vitamin d deficient, while the control group was sufficient in both hemoglobin as well as serum vitamin d level, clearly establishing the connection between the two deficiencies at the genotypic level. medrano et al.,18 previously have proved a role of calcitrol, the active form of vitamin d in hematopoiesis. this strongly supports our observation that links anemia and vitamin d anemia. conclusion our study is the first of its kind reporting a genotypic association between anemia and vitamin d deficiency with the homozygous tt genotype of the taqi snp showing significant association in the study group as compared to healthy control group. our study was conducted in women in the age group of 25–35, who are in active menstruation and childbirth age. it would be interesting to extend the study to a larger population, such as post menopausal women as well as children and observe any genotypic variation. acknowledgments the authors acknowledge king abdulaziz university, jeddah, for scientific research. disclosure statement the author declares no conflict of interest.  references 1. nair r, maseeh a. vitamin d: the ‘sunshine’ vitamin. j pharmacol pharmacother 2013;3:118–26. 2. sizar o, khare s, goyal a, et al. vitamin d deficiency. in: statpearls [internet]. [updated 2021 jul 21] 3. naeem z. vitamin d deficiencyan ignored epidemic. int j health sci 2010 jan;4(1):v–vi. 4. mukhtar m, sheikh n, suqaina sk, batool a, fatima n, mehmood r, nazir s. vitamin d receptor gene polymorphism: an important predictor of arthritis development. biomed res int. 2019 mar 18;2019:8326246. 5. nurminen v, seuter s, carlberg c. primary vitamin d target genes of human monocytes front physiol. 2019;10:194. 6. uitterlinden ag, fang y, van meurs jb, pols ha, van leeuwen jp. genetics and biology of vitamin d receptor polymorphisms. gene 2004;338:143–156 7. panierakis c, goulielmos g, mamoulakis d, petraki e, papavasiliou e, galanakis e. vitamin d receptor gene polymorphisms and susceptibility to type 1 diabetes in crete, greece. clin immunol 2009;133:276–281. 8. motohashi y, yamada s, yanagawa t, maruyama t, suzuki r, niino m, fukazawa t, kasuga a, hirose h, matsubara k, shimada a. vitamin d receptor gene polymorphism. j. clin endocrinol and metabolism 2003;88:3137–40. 9. iyer ap, new sl, khoja s, ghamdi ma, bahlas s. association of apa i polymorphism of vitamin d receptor gene with type 2 diabetes mellitus in saudi population. j. exp biol and agri sci, may 2017; volume – 5(2). 10. kersey m, chi m, cutts db. anaemia, lead poisoning and vitamin d deficiency in low-income children: do current screening recommendations match the burden of illness? pub health nutrition 2011;14(8):1424–28. 11. sim jj, lac pt, liu il, meguerditchian so, kumar va, kujubu da, rasgon sa, vitamin d deficiency and anemia: a cross-sectional study. ann hematol 2010;89(5):447–452. 12. cusato j, allegra s, boglione l, de nicolò a, cariti g, di perri g, d’avolio a, vdr gene polymorphisms impact on anemia at 2 weeks of anti-hcv therapy: a possible mechanism for early rbv-induced anemia. pharmacogen and genomics 2015;25(4):164–172. 13. plum la, deluca hf. vitamin d, disease and therapeutic opportunities. nature rev drug disc 2010;9(12):941–955. 14. bhanushali aa, lajpal n, kulkarni ss, chavan ss, bagadi ss, das br. frequency of foki and taqi polymorphism of vitamin d receptor gene in indian population and its association with 25hydroxyvitamin d levels. ind j hum genet 2009;15(3):108–113. association of taqi polymorphism of vitamin d receptor (vdr) gene with anemia in saudi women original maryam a. al-ghamdi 68 j contemp med sci | vol. 8, no. 1, january-february 2022: 65–68 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1163 15. yassin, fathy & said, noha & tarek, doha. association between vitamin d receptor gene polymorphisms and anemic patients. biochem lett. 2017; 12:1-10. 16. yu w, ge m, shi j, li x, zhang j, wang m, shao y, zheng y. role of vitamin d receptor gene polymorphisms in aplastic anemia: a case-control study from china. int j lab hematol. 2016 jun;38(3):273-83. 17. ertürk s, kutlay s, karabulut hg, keven k, nergizoglu g, ates k, bokesoy i, duman n. the impact of vitamin d receptor genotype on the management of anemia in hemodialysis patients. am j kidney dis. 2002 oct;40(4):816-23. 18. medrano m, carrillo-cruz e, montero i, perez-simon ja. vitamin d: effect on haematopoiesis and immune system and clinical applications. int j mol sci. 2018;19(9):2663. 158 j contemp med sci | vol. 7, no. 3, may-june 2021: 158–163 original barriers of professional autonomy among arab-speaking countries during covid-19 pandemic zahra abbas abdulnabi1,* and sadeq al-fayyadh2 1department of adult nursing, college of nursing, university of basrah, basrah, iraq. 2chairman, department of the adult nursing, college of nursing, university of baghdad, baghdad, iraq. corresponding author: zahra abbas abdulnabi (e-mail: zahraa.abbas1202a@conursing.uobaghdad.edu.iq) (submitted: 05 april 2021 – revised version received: 18 april 2021 – accepted: 22 may 2021 – published online: 26 june 2021) introduction independence in decision-making is synonymous with clinical autonomy. clinical autonomy is defined as the right of nurses to conduct clinical decisions and professional judgment on the basis of their own clinical competence to support the wide spectrum of nursing practices, as distinguished by law enforcement agencies, guidelines, and moral values.1 understanding the idea of clinical decision-making practice in healthcare settings gives vision into clinical decision making practice and gives t h e nurses an opportunity to choose the best options for problem-solving and to develop the best care plan to evalu a te and select nursing interventions. clinical educators f a ced challenges to find out how best to develop nurses’ cap a bilities in clinical decision-making.2–4 globally, 19 million nurses are using clinical judgment before decision-making. 11% of the patients in the united kingdom are suffering from complication due drugs abuse. 34% of these situations that are critical, 6% lead to lifelong disabilities and 8% of patients die. half of these incidences are avoidable. in absolute terms, healthcare spends 1 billion every year on avoidable incidents. similarly, in canada, with 7.5% of patients experience complications in hospital, 36.9% of these refereed as avoidable and 20.8% leading to premature death. therefore, significant factors behind these statistics are clinical decisions making and professional judgment of health care practitioners, especially by nurses.5 autonomy in clinical decision-making increasing the nurse’s responsibility. nurses who have high level of autonomy provide the best level of care to patients to save their lives and protect them, and this leads to enhanced patient outcomes.6–8 in critical care units, the role of nursing is significant, because it varies from other hospital settings. in such challenging settings nurses deal with perplexing and life-threatening conditions, aiming for achieving high quality care.9, 10 nurses who work in critical care units and emergency department spend a lot of time with the patient and monitor changes in health status, so the decision of the nurses actually affects the survival of the patient and decides the potential quality of life. for a nurse to practice clinical roles, the standard of decision-making is necessary and is affected by the nature of the surrounding environment.11–13 barriers to nursing autonomy, include however not limited to the fact that the nursing profession has previously depended on the medical profession because of the domination of the health care system by the physician, the disparity between male and female influence, and the style of top-down management. these factors are coupled with the lack of financial, governmental, social and cultural organizational support. in addition, nurses’ schooling, regulation, organizational culture, and work-related socialization, which varies across nations, may have an enormous impact on the perception of professional autonomy of nurses, their motivation and ability to achieve professional autonomy in different nursing practices settings; such as critical care, emergency, and traditional care arenas.14–16 the current study is the first that covers many arabic countries. this study will contribute to development of nursing autonomy in clinical decision-making in critical care units, emergency and medical surgical departments, not only in iraq but also in arab countries and worldwide. this autonomy is abstract objectives the aims of the study are to determine barriers of nursing autonomy among arab-speaking countries during covid-19 pandemic and to determine difference in professional autonomy levels among arab-speaking countries and work place. methods data collected from december 12 2020 to march 15 2021 by google form survey. cross-sectional design was used in the present study. a purposive sample of 708 nurses who had met the study’s inclusion criteria were targeted. data were analyzed through the use of ibm-statistical package for social sciences (spss) version 17, in which descriptive and inferential statistical measures were employed. results the results of the study showed no deference in professional autonomy among arab speaking countries p = 0.826 and work place (p = 0.826). most common barriers of professional autonomy are absence of law protecting professional duties (35.6%) which was perceived by of the study subjects, followed by the policy of health (30.4%), followed by hospital administration style (26.4%) and domination or physicians authority (29.9%). conclusion most common barriers of professional autonomy are absence of law protecting professional duties, the policy of health care sector, domination of physician’s authority and hospital administration style. this result reflects highlights the importance of removing all obstacles to upgrade nurse’s professional autonomy in the covered arab-speaking counties. keyword professional autonomy, nursing, nurses, covid-19, arabs issn 2413-0516 159j contemp med sci | vol. 7, no. 3, may-june 2021: 158–163 z. a. abdulnabi and s. al-fayyadh original barriers of professional autonomy among arab-speaking countries during covid-19 pandemic considered important to nursing becoming completely independent professional. materials and methods ethical consideration the researchers pledge to keep the details of the participants privately, using the collected data without causing any possible harm to the study subjects. the study tool was designed to preserve the right of subject’s anonymity. subjects were given the right to freely participate in the study by signing the electronic consent form of the study. design and setting of study cross-sectional design .the study was conducted virtually targeting arab-speaking nurses who were practicing in critical care units, emergency and medical surgical departments. beside iraq, the covered countries were the hashemite kingdom of jordan, egypt, palestine and kingdom of saudi arabia. instrument of study study instrument consist of two part, first part consist of socio-demographic and professional characteristic, second part consist of nursing activity scale (nas) which contains 30 items, that explains conditions in which a degree of professional nursing autonomy must be exercised by a nurse. statistical analysis data were entered into the ibm-statistical package for the social sciences (spss) version 17 software program and analyzed using descriptive and inferential statistics. results and discussion the findings in table 1 showed that the majority of study participants age group was 26–31 years representing (39.4%) of the study sample. this result is supported by maharmeh17 a descriptive correlational design aimed to describe jordanian critical care nurses experience of autonomy in their clinical practice, which indicated that (62%) of sample were in age group of 22–30 years. another study was done by keshk et al.,18 found in their study that the highest percent of study participant (50%) within age group (26–34). data were collected by using google form survey whereas young individuals were the most common persons who responded. these result agreed with other study, which showed that young individuals are spending a significant amount of their time using social media.19 regarding to level of education, findings of the study indicated that more than half of study sample were holding bachelor’s degree (57.6%). another study conducted among nurses in the southeast of iran, found that most of nurses were holding bachelor’s degree (97%) too.20 another study conducted in intensive care unit (icu) of islamic republic of iran, showed that more than half (85%) of nurses who were participants in the study were holding bachelor’s degree.21 in the present study (55.4%) of study sample were male. another study indicated that the highest percentage of study respondents were male (54.2%).22 furthermore, the challenging nature of nursing profession has hindered females from joining nursing, which created a critical imbalance as shown in iraqi nursing syndicate (ins)’s statistics as male:female nurse ratio is 75:25.23 this ratio was reflected by the descriptive statistics of sample demographics, which were, more male nurses (55.4%) participated in this study than females (44.6%). the study findings showed that most study sample (68.1%) were from iraq due to the fact that this research was conducted in iraq and iraqi nurses were the most responsive subjects to the online questionnaire than other arabspeaking countries. according to table 2 that is related to professional characteristics, represents highest percent of study participants (75.4%) were working in governmental sector. another study was conducted to examining the level of professional autonomy as well as its predictor and outcomes among practicing nurses in the phillippins, found that (73.5%) of study participants were working in public sector.24 these results were not surprising due to the fact that arab-speaking countries health sector is public service sector, unlike western countries, where the majority are capital institutions. about working place, the majority of the study sample were working in critical care units (37.7%). in contrast, amini et al.,25 nurses’ autonomy level in teaching hospitals and its relationship with the underlying factors showed that more than half of their study samples (49.2%) were working in table 1. frequencies and percentages of socio-demographic characteristics of study participants. (n708) co un tr ie s characteristics f percent valid % iraq 482 68.1 68.1 egypt 113 16.0 16.0 jordon 39 5.5 5.5 ksa 39 5.5 5.5 palestine 35 4.9 4.9 ge nd er male 392 55.4 55.4 female 316 44.6 44.6 ag e/ ye ar s 20 – 25 224 31.6 31.6 26 – 31 279 39.4 39.4 32 – 37 116 16.4 16.4 38 – 44 71 10.0 10.0 45 – 51 16 2.3 2.3 52 – 58 2 0.3 0.3 ed uc at io na l at ta in m en t diploma in nursing 190 26.8 26.8 bachelor’s in nursing 408 57.6 57.6 master in nursing 102 14.4 14.4 doctorate in nursing 8 1.1 1.1 total 708 100.0 100.0 the underlined numbers in table 1, represent the highest percentages of the selected variables. in which, more than half (55.4 %) of the study sample were males. more than quarters (39.4%) of the study sample were classified as adult individuals within age range of 26 – 31 years. furthermore, most of them (57.6%) were holding bachelor’s degree. concerning the nurses’ country, the highest percentage (68.1%) of the study participants were from iraq. 160 j contemp med sci | vol. 7, no. 3, may-june 2021: 158–163 barriers of professional autonomy among arab-speaking countries during covid-19 pandemic original z. a. abdulnabi and s. al-fayyadh table 2. frequencies and percentages of professional characteristics professional characteristics f % working sector governmental sector 534 75.4 private sector 74 10.5 both sectors 100 14.1 working place emergency department 215 30.4 medical department 110 15.5 surgical department 107 15.1 critical care units 267 37.7 burns care department 9 1.3 years of experience in hospital 1 5 years 429 60.6 6 10 years 155 21.9 11 15 years 67 9.5 16 20 years 42 5.9 21 25 years 12 1.7 26 30 years 3 .4 years of experience in recent unit 1 5 years 520 73.4 6 10 years 133 18.8 11 15 years 30 4.2 16 20 years 17 2.4 21 25 years 7 1.0 26 30 years 1 .1 working shift morning shift 453 64.0 evening shift 255 36.0 professional roles nurse executing an administrative role 166 23.4 bedside nurse 542 76.6 experience development yes 680 96.0 no 28 4.0 source of knowledge social media platforms 146 20.6 scientific websites 43 6.1 audiovisual media 12 1.7 books & scientific journals 89 12.6 professional colleagues 59 8.3 all of the above 330 46.6 not apply to me 29 4.1 participation in training program of professional autonomy yes 350 49.4 no 358 50.6 no. of beds in unit 1 – 5 156 22.0 5 – 10 217 30.6 >10 beds 335 47.3 no. of the patient in work shift one patients 49 6.9 two patients 105 14.8 >3 patients 554 78.2 table 2. frequencies and percentages of professional characteristics professional characteristics f % no. of nurses in work shift 1 5 nurses 427 60.3 5 10 nurses 190 26.8 > 10 nurses 91 12.9 having nursing association yes 372 52.5 no 181 25.6 not sure 155 21.9 nursing association membership yes 178 25.1 no 366 51.7 not apply to me 164 23.2 have job description yes 360 50.8 no 348 49.2 applying job description yes 247 34.9 no 297 41.9 not apply to me 164 23.2 awareness of job description yes 550 77.7 no 158 22.3 professional autonomy in curriculum yes 484 68.4 no 224 31.6 total 708 100.0 the underlined numbers in table 2, represent the highest percentages of the selected variables. in which, the majority of the respondents were working in governmental sector (75.4%) at the time of data collection. about (37.7%) were practicing nursing in critical care units. furthermore, most of them were working in morning shift (64.0%). 1–5 years’ experience in recent unit was the dominant choice of the study subjects, representing (73.4%). most nurses were employed as bedside nurses (76.6%). the highest percentage of subject’s experience in the hospital was spotted as (6-10 years) representing (60.6%). of equal importance, (96.0%) of study participants were actively working on developing their professional autonomy experience. social media (20.6%), books and scientific journals (12.6%) were the most common sources of knowledge, by which nurses were working to develop their competencies about professional autonomy. more than half (50.6%) of the study respondents were not previously participated in training programs of professional autonomy. the highest percentages of the served bed in the targeted units were more than ten, representing (47.3%). on the other hand, the highest percentage (78.2%), representing the number of the patient in work shift, which is more than three for each nurse. about (60.3%), representing 1-5 nurses, was the highest percentage of nurse’s number during work shift. the majority of respondents indicated having an active nursing association membership (51.7%), and (50.8%) indicated having a job description. however, only (41.9%) of them were able to apply job description. about three quarters of the study sample (77.7%) indicated having job description awareness. surprisingly, only (68.4%) of respondents reported that professional autonomy was covered during their academic preparation curriculum. non-critical care units. the findings of this study have indicated that the majority of study sample were practicing in critical care units due to the fact that data collection phase was conducted during covid-19 pandemic, in which most units in the hospital were transformed to be critical care units to accommodate the increasing numbers of patient. concerning working shift, most of study subjects were work during morning shift (64.0%). another study conducted (continued) 161j contemp med sci | vol. 7, no. 3, may-june 2021: 158–163 z. a. abdulnabi and s. al-fayyadh original barriers of professional autonomy among arab-speaking countries during covid-19 pandemic to assess nurse’s work autonomy on patient care and unit operation which stated that (59.8%) of sample were working in day shift.26 this result is not surprising due to the fact that the number of the nurses who work in morning shift is more than night shift. this can be explained by the fact that most nurses preferred working in morning more than night shift, due to administrative and duties nature factors. morning shift provide opportunities for sleeping, learning and developing teamwork skill from other health care providers that are available. furthermore, competent nurse peers provided reassurance and direction in making important patient decisions. indeed, nurses made patient decisions based on facts derived by their own skill or knowledgeable peers rather than data or hospital procedures.27–29 the study found that the highest percentage in terms of years of experience in hospital and recent unit of study participants between 1–5 years (73.4%). these findings agreed with30, which indicated that the highest percentage of experience years was less than ten years (51.0%). these results were not surprising due to the fact that the majority of study participants were within age group 26–30 years (39.4%). professional role is another variable that this study has highlighted, whereas the majority of the study participants were bedside nurses (76.6%). this finding is congruent with labrague et al.,24 in which the highest percent of study sample were practicing nursing as a staff nurse position (84.7%). this result was not surprising due to the fact that staff nurses form the majority of heath care system when compared with nurses who hold managerial positions. the results of the present study indicated that more than half (50.6%) of study respondents were not previously participated in training programs of professional autonomy. this result is similar to allahkhshian et al.21 in which they found that there was not any training program about the concept of professional autonomy offered to nurses. this highlights the huge gab and emphasizes the extreme necessity of conducting such educational program to enhance nurse’s awareness about their standard professional role autonomy boundaries. unfortunately, these results were not surprising due to the fact that the majority of study respondents are from iraq, which do not emphasize an importance of the professional independence of nursing and the necessity of training programs that clarify the limits of professional independence and the role of the nurse in order to provide the best care to the patient. surprisingly, only (68.4%) of respondents reported that professional autonomy was covered during their academic preparation as a topic in the nursing curriculum. in contrast, (allahkhshian et al.21 indicated that nursing autonomy and professionalism in the nursing were not emphasized in the nursing core curriculum. most study participants (96.0%) had indicated that they work on developing their knowledge & experience about nursing professional autonomy. they highlighted many sources to seek development including, social media platform (20.6%), books and scientific journal (12.6%). these were most common source of knowledge seeking platforms. the highest percentage of the bed in unit was more than ten, representing (47.3%). on the other hand, the highest percentage (78.2%), representing the number of the patient in work shift, which is more than three for each nurse, while global nurses patients ratio 1:1 as recommended in critical care units. about (60.3%), representing 1–5 nurses is the highest percentage of nurse’s number during work shift. having high numbers of admitted clients, nursing staff shortage, is not surprising because of data collected phase was conducted during covid-19 pandemic. that lead to increase work load and decreasing number of health care providers in the world due to the fact that nurses being the front line of fighting the pandemic has led to high morbidity and mortality rate among them. unfortunately, no reliable statistics about number of nurse’s death rate due to coronavirus are available to be reported. the majority of respondents indicated having a nursing association membership (51.7%), have nursing association (52.5%) and (50.8%) indicated having a job description. however, only (41.9%) of them were able to apply job description. about three quarters of the study sample (77.7%) indicated having job description awareness. at the end of this section, it is important to notice that the present study is the first that deals with the aforementioned variables, which makes its findings unique in terms of highlighting new gaps that need to be addressed effectively. as presented in table 3, the highest percentages of the perceived barriers of nursing professional autonomy according to the subjects of the current study were the absence of law(s), describing and protecting professional duties (35.6%), followed by hospital administration style (26.4%), followed by the policy of the health institution (30.4%), followed by domination of physician’s authority (29.9%). these were the dominant barriers that may have prevented nurses from authenticating professional autonomy. these findings are supported by a study which found that physician-dominated of the health care system and autocratic hospital management were the most common obstacles of nursing autonomy.21 other studies indicated that intra-professional conflict such as table 3. barriers of applying professional autonomy perceived barriers f % hospital administration 187 26.4 unit manager 78 11.0 work load 121 17.1 absence of legislations protecting professional duties 252 35.6 colleagues 52 7.3 low confidence in abilities to execute professional duty independently 6 0.8 awareness lack of nursing authority & professional responsibilities limit 64 9.0 health care institution dominant policy 215 30.4 domination of physician’s authority 212 29.9 all of the aforementioned barriers 91 12.9 not apply to me 182 25.7 the underlined numbers in table 3, represent the highest percentages of the perceived barriers according to nurses’ choices. in which, absence of law protecting professional duties (35.6%), hospital administration style (26.4%), the applied policy of the health institution (30.4%) and the domination or authority of physicians (29.9%), were the prevailing barriers that may have prevent nurses from authenticating professional autonomy. 162 j contemp med sci | vol. 7, no. 3, may-june 2021: 158–163 barriers of professional autonomy among arab-speaking countries during covid-19 pandemic original z. a. abdulnabi and s. al-fayyadh absence of respect, trust and collaboration between physicians and nurses were barriers that must be addressed to upgrade nurse’s professional autonomy. nurses will be empowered when having the confidence to step forward embracing their professional and ethical authority through creating collaborative relationships with physicians.31–34 these study findings also supported by structural element of empowerment theory which suggested that an organization must provide authority, support and resources to employees that are required for achieving the organizational objectives.35 analysis of variance (anova) test in table 4 indicated that there was no statistically significant difference in nurses professional autonomy between arab-speaking countries (mean = 0.106, f = 0.543, p = 0.704), and working place (mean = 0.074, f = 0.376, p = 0.826).these results may explained by the fact that covid-19 pandemic had a global impact on health care systems of the covered countries such as increase number of patient admitted to hospital and decline number of health care professionals; especially nurses, increase work load, and economic impact, which lead decrease resources and necessary supply. conclusions most common barriers of professional autonomy are absence of law protecting professional duties, the policy of health care sector, domination of physician’s authority and hospital administration style. this result reflects highlights the importance of removing all obstacles to upgrade nurse’s professional autonomy in the covered arab-speaking counties. recommendation support and encourage nurses to exercises autonomy in clinical practice by providing law that protects them, standard and policy to apply their practice in work field. improve collaborative relationship between nurses and physicians to enhance patient outcomes, decrease physicians’ domination and improve nursing autonomy. strong united nursing groups and professional associations are also required to drive forward in self-government in nursing. limitations limited access to the internet services was one of the major obstacles that have limited the participation rate. of equal importance, a significant percentage of medical-surgical nurses were affected by the covid-19 pandemic during data collection phase, which also limited the participation rate in the study. funding information the budget of this research work was not support by any governmental or non‐governmental organization. the author of this manuscript covered all the research work‐related expenses. conflicts of interest none. table 4. differences in the professional autonomy levels in respect to nurses’ countries professional autonomy* country characteristics sum of squares df mean square f sig. between groups .425 4 0.106 0.543 0.704 within groups 137.618 703 0.196 total 138.043 707 professional autonomy* working place between groups 0.294 4 0.074 0.376 0.826 within groups 137.749 703 .196 total 138.043 707 analysis of variance (anova) test in table 4 indicates there is no a statistically significant difference of professional autonomy levels in covered arab-speaking countries (mean = 0.106, f = 0.543, p = 0.704) and the working place (mean = 0.074, f = 0.376, p = 0.826). references 1. kramer, m., & schmalenberg, c.e. the practice of clinical autonomy in hospitals:20000 nurses tell their story. critical care nurse. 2008;28:58-71. 2. shin, k. r. critical thinking ability and clinical decision-making skills among senior nursing students in associate and baccalaureate programmes in korea. journal of advanced nursing. 1998;28:58-71. 3. higuchi, k. a. s., & donald, j. g. thinking processes used by nurses in clinical decision making. journal of nursing education. 2002;41:145-153. 4. bohinc, m., & gradisar, m. decision-making model for nursing. the journal of nursing administration. 2003;33:627-629. 5. thompson c. international journal of nursing studies www.elsevier.com/ ijns 2013. 6. keith, l., & cianelli, r. exploring the concept of nurse engagement related to patient experience. horiz enferm. 2014;25:109-113. 7. rafferty, a. m., ball, j., & aiken, l. h. are teamwork and professional autonomy compatible, and do they result in improved hospital care?. bmj quality & safety. 2001;10:ii32-ii37. 8. watson, l. m. leaderships influence on job satisfaction. radiologic technology. 2009;80:297-308. 9. kelly, d. m., kutney-lee, a., mchugh, m. d., sloane, d. m., & aiken, l. h. impact of critical care nursing on 30-day mortality of mechanically ventilated older adults. critical care medicine. 2014;42:1089. 10. ulrich, b. t., lavandero, r., woods, d., & early, s. critical care nurse work environments 2013: a status report. critical care nurse. 2014;34:64-79. 11. boev, c. the relationship between nurses' perception of work environment and patient satisfaction in adult critical care. journal of nursing scholarship. 2012;44:368-375. 12. backes, m. t. s., erdmann, a. l., & büscher, a. the living, dynamic and complex environment care in intensive care unit. revista latino-americana de enfermagem. 2015;23:411-418. 13. cishahayo, e., nankundwa, e., sego, r., & bhengu, b. burnout among nurses working in critical care settings: a case of a selected tertiary hospital in rwanda. international journal of research in medical sciences. 2017;5:51215128. 14. iliopoulou, k. k., & while, a. e. professional autonomy and job satisfaction: survey of critical care nurses in mainland greece. journal of advanced nursing. 2010;66:2520-2531. 15. traynor, m., boland, m., & buus, n. professional autonomy in 21st century healthcare: nurses' accounts of clinical decision-making. social science & medicine. 2010;71:1506-1512. 16. baykara, z. g., & şahinoğlu, s. an evaluation of nurses' professional autonomy in turkey. nursing ethics. 2014;21:447-460. 163j contemp med sci | vol. 7, no. 3, may-june 2021: 158–163 z. a. abdulnabi and s. al-fayyadh original barriers of professional autonomy among arab-speaking countries during covid-19 pandemic 17. maharmeh, m. understanding critical care nurses' autonomy in jordan. leadership health services. 2017;30:432-442. 18. keshk, l. i., qalawa, s. a. a., & aly, a. a. clinical decision-making experience of the critical care nurses' and its effect on their job satisfaction: opportunities of good performance. american journal of nursing research. 2018;6:147-157. 19. valdez, g. f. d., cayaban, a. r. r., al-fayyadh, s., korkmaz, m., obeid, s., sanchez, c. l. a. & cruz, j. p. the utilization of social networking sites, their perceived benefits and their potential for improving the study habits of nursing students in five countries. bmc nursing. 2020;19:1-14. 20. jahromi et al. (2015) 21. allahkhshian, m., alimohammadi, n., taleghani, f., nik, a. y., abbasi, s., & gholizadeh, l. barriers to intensive care unit nurses' autonomy in iran: a qualitative study. nursing outlook. 2016;65:392-399. 22. gizaw, a., kidane, b., negese, d., & negassa, e. factors affecting clinical decision-making practice among nurses working in jimma university medical center, jimma southwest ethiopia. ann nurs pract. 2018;5:1094. 23. iraqi nursing syndicate. nursing in iraq record. us: official record of ins. 2014. 24. labrague, l. j., mcenroe-petitte, d. m., & tsaras, k. predictors and outcomes of nurse professional autonomy: a cross-sectional study. international journal of nursing practice. 2019;25:e12711. 25. amini, k., negarandeh, r., ramezani-badr, f., moosaeifard, m., & fallah, r. nurses' autonomy level in teaching hospitals and its relationship with the underlying factors. international journal of nursing practice. 2015;21:52-59. 26. mrayyan, m. t. american nurses' work autonomy on patient care and unit operations. british journal of nursing. 2005;14:962-967. 27. seright, t. j. clinical decision-making of rural novice nurses. rural remote health. 2011;11:1726. 28. cappelletti, a., engel, j. k., & prentice, d. systematic review of clinical judgment and reasoning in nursing. journal of nursing education. 2014;53:453-458. 29. samuriwo, r., & dowding, d. nurses' pressure ulcer related judgements and decisions in clinical practice: a systematic review. international journal of nursing studies. 2014;51:1667-1685. 30. mohamed, n. t. relationship between leadership styles and clinical decision-making autonomy among critical care nurses. egyptian nursing journal. 2018;15. 31. hartog, c. s., & benbenishty, j. understanding nurse -physician conflicts in the icu. intensive care medicine. 2015;41:331-333. 32. avila, l. i., silveira, r. s. d., lunardi, v. l., fernandes, g. f. m., mancia, j. r., & silveira, j. t. d. implications of the visibility of professional nursing practices. revista gaucha de enfermagem. 2013;34:102-109. 33. dorgham, s. r., & al-mahmoud, s. leadership styles and clinicaldecision making autonomy among critical care nurses: a comparative study. journal of nursing and health science. 2013;1:71-83. 34. georgiou, e., papathanassoglou, e. d., & pavlakis, a. nurse-physician collaboration and associations with perceived autonomy in cypriot critical care nurses. nursing in critical care. 2017;22:29-39. 35. kanter, r.m. (1993). men and women of the corporation (2nd ). new york: basic books. 36. suominen, t., leino-kilpi, h., merja, m., doran, d. i., & puukka, p. staff empowerment in finnish intensive care units. intensive and critical care nursing. 2001;17:341-347. 37. hoffman, k., duffield, c., & donoghue, j. barriers to clinical decision-making in nurses in australia. australian journal of advanced nursing. 2004;21:8-13. 38. taylor, m. a. the relationship between autonomy and job satisfaction among registered nurses. master thesis. university of new hampshire, durham. 2008. 39. schutzenhofer, k. k. the measurement of professional autonomy. journal of professional nursing. 1987;3:278-283. 40. motamed-jahromi, m., jalali, t., eshghi, f., zaher, h., & dehghani, l. evaluation of professional autonomy and the association with individual factors among nurses in the southeast of iran. journal of nursing and midwifery sciences. 2015;2:37-42. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 102 j contemp med sci | vol. 8, no. 2, march-april 2022: 96–100 original demographic evaluation of post-concussion syndrome in referrals to bandar abbas forensic medical center, south of iran from march 2020 to august 2021 saeed mohammadi1, seyed javad mirhadi2, hosein javadi vasigh1, khatere asadi1, azadeh memarian3,* , farahnaz nikkhah4,5 1legal medicine center of hormozgan, bandar abbas, iran. 2legal medicine research center, iranian legal medicine organization, bandar abbas, iran. 3department of neonatology, faculty of medicine, mazandaran university of medical sciences, sari, iran. 4operating room nursing, rasool akram medical complex clinical research, iran university of medical sciences, tehran, iran. development center (rcrdc), iran university of medical sciences, tehran, iran. *correspondence to: azadeh memarian (e-mail: a.memarian@mazums.ac.ir) (submitted: 17 december 2021 – revised version received: 04 january 2022 – accepted: 25 january 2022 – published online: 26 april 2022) abstract objectives: the aim of the present study is the demographic assessment of pcs in referrals to forensic medical centers in bandar abbas. methods: this cross-sectional and descriptive study was performed on 72 patients with mild brain trauma who were referred to bandar abbas forensic medical center. pcs was confirmed according to the diagnostic and statistical manual of mental disorders (dsm-iv) criteria. information on age, sex, mechanism of trauma, the time interval between the first visit to the forensic medical center, level of consciousness, number of hospitals stays days, number of days in icu, and symptoms at initial referral were recorded in the designed form. results: the mean age of patients was 30.81 ± 13.39 years. there was a significant difference in sex and mechanisms of trauma (p < 0.001). the average time interval of the first referral to forensic medical center was 9.44 ± 18.37 days. 87.5% were hospitalized and only 9.7% were hospitalized at icu. the consciousness level of all patients was in the range of 12 to 15 (mild range). among symptoms, headache (87.5%) and dizziness (81.9%) were the most significant prevalent symptoms. 70.8% of patients had no symptoms one month after the accident but 12.5% still had symptoms and only 9.7% still had symptoms of concussion after 3 months. conclusion: according to the results, pcs mostly happened in the male gender and the main mechanism of trauma was an accident. headache and dizziness were the main symptoms. only a small percentage of patients have symptoms after one month and three months, post-injury. keywords: post-concussion syndrome (pcs), mild traumatic brain injury (mtbi), symptoms issn 2413-0516 introduction traumatic brain injury is a significant public health concern globally and they may begin a series of metabolic reactions which lead to post-concussion syndrome (pcs).1 pcs after a traumatic brain injury and mild traumatic brain injury (mtbi) are very prevalent.2 pcs is a prototypal psychosomatic disorder and both psychosocial and physical factors playing a significant role in its etiology.3 numerous factors raise the pcs development risk after brain injury. a previous mental disorder like anxiety or depression, acute post-traumatic stress, and pain were predictive of pcs.4,5 age above 40, female gender, prior head injuries, and substance abuse are reported as other risk factors for pcs.6 pcs can be diagnosed using the international classification of diseases (icd-10) and diagnostic and statistical manual of mental disorders dsm-iv.7,8 the prognosis of pcs is commonly good. most of the patients recover by 3 months.9 however, in 10–20% of the cases, pcs may continue for weeks or months as a result of metabolic and structural brain injuries. among these patients, 25–33% experience persistent post-concussion syndrome and the symptoms become chronic and last for more than 6 months.10,11 pcs symptoms comprise three clinical areas: somatic symptoms such as headache, insomnia, fatigue, tinnitus, dizziness, sensitivity to noise or light; cognitive symptoms including reduced memory, concentration, and attention; and affective symptoms such as depression, anxiety, and irritability.1 in the assessment of pcs, the clinician had better evaluate consciousness loss, the post-traumatic amnesia duration, and the glasgow coma scale after trauma. they should assess the information about the accident and hospital stays and the outcomes of treatment should be recorded.3 it was shown that the differences in the incidence of pcs could be as a result of cultural diversities.12 moreover, in a study, it has been shown that the effect of culture and language should be considered in pcs assessments.13 subsequently, prevalence rates and demographic features of pcs in populations may vary between countries. this study aimed to investigate the prevalence of post-concussion syndrome in patients referred to bandar abbas forensic medicine center. materials and methods this is a cross-sectional and descriptive study. in one year, patients with mild brain trauma, no structural change in ct scan, mild level of consciousness (glasgow coma scale (gcs) = 13–15), and mild memory impairment before and after the traumatic event (less than 1 hour) who referred to bandar abbas forensic medical center, south of iran, from march 2020 to august 2021, for whom the diagnosis of concussion was made were selected. 5 https://orcid.org/0000-0002-6872-1870 103j contemp med sci | vol. 8, no. 2, march-april 2022: 96–100 s. mohammadi et al. original demographic evaluation of pcs in referrals to bandar abbas forensic medical center information on age, sex, mechanism of trauma (accident, quarrel), the time interval between the first visit to the forensic medical center, level of consciousness, number of hospitals stays days, number of days in icu, and symptoms at initial referral (headache, dizziness, anger, sleep disturbance, fatigue, forgetfulness, memory impairment, and concentration disorder) were recorded in the designed form. if there are at least three of the above symptoms, re examination was considered for patients. at one month after the accident, the symptoms were re-examined and if they did not improve, neurological counseling was performed to confirm the symptoms. if symptoms do not improve, re-examination was done for 3 months after the accident, and symptoms were re-examined at 3 months post-accident and confirmed by a neurologist. post-concussion syndrome was confirmed according to the diagnostic and statistical manual of mental disorders (dsm-iv) criteria if there were at least three of these symptoms for at least 3 months. statistical analysis spss version 20 was used to analyze the data. data are presented as frequency and percentage and mean and standard deviation. univariate chi-square test was used to show the difference of variables between the research samples. values were significant at p < 0.05. result seventy-two patients met the criteria and selected for the study. the mean age of patients was 30.81 ± 13.39 years. the univariate chi-square test showed that there was a significant difference between the research sample in terms of sex and mechanisms of trauma (table 1). the average time interval between the accident and the first referral to forensic medical center was 9.44 ± 18.37 days. as shown in table 2, the level of consciousness of all individuals was in the range of 12 to 15 (mild range). also, most of the patients, 53 (73.6%), had a level of consciousness of 15. the univariate chi-square test showed that there was a significant difference in terms of the level of consciousness. among the patients, 63 patients (87.5%) were hospitalized and 9 patients (12.5%) were not hospitalized. most of the patients, 65 (90.3%), were not hospitalized in the icu and only 7 patients (9.7%) had a history of being admitted to the icu. the univariate chi-square test showed that there was a significant difference in terms of the prevalence of hospitalization and the prevalence of hospitalization in the intensive care unit (table 3). the average days of hospitalization and hospitalization in the icu are shown in table 4. at referral, 87.5% had a headache and 81.9% had dizziness. most of the patients (61.1%) did not have the “anger” symptom. however, this symptom was present in 38.9% of patients. 44 patients (61.1%) had “sleep disorder”. 51.4% had a sign of “fatigue” and 48.6% did not have. 35 patients (48.6%) had “forgetfulness” and 37 patients (51.4%) did not have “forgetfulness”. most patients (91.7%) did not have “ paramnesia” and 64.88% did not have “concentration disorder” and only 8 patients (11.1%) had. univariate chi-square test showed that there was a significant difference in terms of headache, dizziness, paramnesia, and concentration disorder symptoms. on the other hand, the univariate chi-square test showed that there was no significant difference in terms of “anger” sign, sleep disorder, fatigue, and “forgetfulness” symptoms (table 5). most of the patients (70.8%) had no symptoms one month after the accident but 12.5% still had symptoms of concussion one month after the accident. the univariate chi-square test showed that there was a significant difference in terms of the prevalence of symptoms at the referral one month after the table 1. the frequency of gender prevalence and mechanism of trauma between the accident and the first referral of patients with the diagnosis of pcs variable frequency percent %cf p-value gender male 51 70.8 70 p < 0.001 female 21 29.2 100 total 72 100 mechanism of trauma accident 57 79.2 79.2 p < 0.001 quarrel 15 20.8 100 total 72 100 table 2. level of consciousness in patients with the diagnosis of pcs level of consciousness frequency percent %cf p-value 12 2 2.5 2.8 < 0.001 13 8 11.1 14.1 14 8 11.1 25.4 15 53 73.6 100 missing data 1 1.4 total 72 100 table 3. prevalence of hospitalization and hospitalization in icu in patients with the diagnosis of pcs frequency percentage %cf p-value prevalence of hospitalization yes 63 87.5 87.5 < 0.001 no 9 12.5 100 total 72 100 prevalence of hospitalization in the icu yes 7 9.7 9.7 < 0.001 no 65 90.3 100 total 72 100 table 4. mean of hospitalization days and hospitalization days in icu in patients with the diagnosis of pcs variable no. mean sd min. max. days of hospitalization 71 2.02 1.58 1 8 days of hospitalization in the icu 71 1.80 1.30 1 4 104 j contemp med sci | vol. 8, no. 2, march-april 2022: 96–100 demographic evaluation of pcs in referrals to bandar abbas forensic medical center original s. mohammadi et al. accident. 36.1% had no symptoms three months after the accident. 9.7% still had symptoms of concussion at the referral three months after the accident. data from 54.2% of patients were also not reported. the univariate chi-square test showed that there was a significant difference between the study samples in terms of the prevalence of symptoms at the referral three months after the accident (table 6). table 5. prevalence of symptoms at initial referral in patients diagnosed with pcs variable frequency percentage %cf p-value headache yes 63 87.5 87.5 < 0.001 no 9 12.5 100 total 72 100 dizziness yes 59 81.9 81.9 < 0.001 no 13 18.1 100 total 72 100 anger yes 28 38.9 38.9 > 0.05 no 44 61.1 100 total 72 100 sleep disorder yes 44 61.1 61.1 > 0.05 no 28 38.9 100 total 72 100 fatigue yes 37 51.4 51.4 > 0.05 no 35 48.6 100 total 72 100 forgetfulness yes 35 48.6 48.6 > 0.05 no 37 51.4 100 total 72 100 paramnesia yes 6 8.3 8.3 < 0.001 no 66 91.7 100 total 72 100 concentration disorder yes 8 11.1 11.1 < 0.001 no 64 88.9 100 total 72 100 other symptoms yes 3 4.2 4.2 < 0.001 no 69 95.8 100 total 72 100 table 6. prevalence of symptoms in one month and three months after the accident in patients with the diagnosis of pcs variable frequency percentage %cf p-value prevalence of symptoms in a month after the accident yes 9 12.5 15 < 0.001 no 51 70.8 100 missing data 12 16.7 total 72 100 prevalence of symptoms in referral three months after the accident yes 7 9.7 21.2 < 0.001 no 26 36.1 100 missing data 39 54.2 total 72 100 discussion in the present study, the demographic evaluation of pcs was performed in patients who were referred to the forensic medical center of bandar abbas as a result of mild brain injury. according to the result of this study, the mean age of the patients with pcs was 30.81 ± 13.39 years. most of the patients were male and the main mechanism of trauma was an accident. the level of consciousness of all patients was in the mild range (12 to 15) and most of them had the level of consciousness of 15. most of the patients were hospitalized but only 9.7% being admitted to the icu. headache and dizziness were significantly the most prevalent symptoms in pcs patients. there was no significant difference in terms of anger, sleep disorder, fatigue, and forgetfulness symptoms. the prevalence of concentration disorder and paramnesia was very low in patients. most of the patients had no symptoms one month and 3 months after injury but only 12.5% at one month and 9.7% at 3 months after still had symptoms of concussion. dean et al. compared pcs prevalence in individuals with mild tbi and without head injury and reported a higher prevalence of headaches and significantly higher cognitive problems were those with mtbi in comparison to the control group.14 similar to the result of the present study the prevalence of headaches was high. balakrishnan et al. evaluated the pcs after mtbi and in contrast to this study, they reported the female gender as the dominant gender for pcs. similar to the result of the current study the prevalence of pcs was low after 2 weeks, 3, and 6 months and they were 9.6%, 8.1%, and 8.1%. again like this study, the main reason for injury was traffic accidents.15 varner et al. study in adults with acute mild traumatic brain injury, 20.3% had persistent concussion symptoms. headache, use of drugs or alcohol at the time of injury, the injury happening by bike or motor vehicle crash, history of depression or anxiety, and numbness were defined to be independently related to persistent concussion symptoms in a month.16 consistent with the present study headache was the main symptom. beauchamp et al. compared pcs symptoms in sports related mtbi with non-sports-related mtbi and reported that patients with sports-related mtbi might be at lower risk for symptoms like dizziness and fatigue in 90 days after injury. they suggested that patients with non-sports-related mtbi may show more pcs symptoms and that the physical activity level could affect the rehabilitation of the patient.17 105j contemp med sci | vol. 8, no. 2, march-april 2022: 96–100 s. mohammadi et al. original demographic evaluation of pcs in referrals to bandar abbas forensic medical center acknowledgments all the researchers who have helped us in this research project. the authors also thank the rasool akram medical complex clinical research development center (rcrdc) for its technical and editorial assists. conflict of interest the author reports no conflicts of interest in this work. ethical consideration the study is approved by the ethics committee of forensic medicine organization of the country of iran (code: 2062(24.12.98 ). written informed consent was obtained from patients. consent for publication written informed consent was obtained from the patients for publication of this paper. informed consent written informed consent was obtained from the patients for participation in the study. a copy of the written consent is available for review by the editor-in-chief of this journal. data availability statement the data that support the findings of this study are available from corresponding author on reasonable request. funding the authors declare that this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.  in a study by van der vlegel 22.0% of the 1282 patients in the general injury population met the pcs criteria. patients with head injuries showed a high frequency of pcs (29.4%). patients with pcs had lesser hrql, lesser coming back to work levels, and greater health care utilization, in comparison to non-pcs patients.18 patients with mild traumatic brain injuries frequently complain about a group of physical, cognitive, as well as emotional, or behavioral symptoms. the most frequent symptoms for pcs are headache, dizziness, reduced concentration, memory complications, fatigue, visual disorders, irritability, noise sensitivity, judgment issues, anxiety, and depression. even though these symptoms normally resolve during one month, in some cases pcs may remain for months or years or even permanently. physiological as well as psychological factors have been recommended as reasons for persistent pcs. researchers believe that a range of injury-associated, preand post-morbid neuropathological, and psychological elements play role in the progression and prolongation of these symptoms.19 therefore, it is significant to develop approaches to prevent pcs symptoms in injured patients, increase awareness of patients as well as physicians on the incidence of pcs, detect pcs at the earliest time, and develop approaches to improve recovery in these patients. declarations author contributions study concept and design: sm, sjm, am drafting of the manuscript: sm, am literature searching: all authors statistical analysis and interpretation of the data: hjv, ka, am critical revision of the manuscript for important intellectual content and taking responsibility for the integrity and the accuracy of the data: all authors study supervision: am reviewed and modified the manuscript: fn, am all authors read and approved the final manuscript. references 1. hadanny a, efrati s. treatment of persistent post-concussion syndrome due to mild traumatic brain injury: current status and future directions. expert rev neurother. 2016;16(8):875-87. 2. evans rw. the postconcussion syndrome and the sequelae of mild head injury. neurol clin. 1992;10(4):815-47. 3. foy k, murphy kc. post-concussion syndrome. br j hosp med (lond). 2009;70(8):440-3. 4. meares s, shores ea, taylor aj, batchelor j, bryant ra, baguley ij, et al. the prospective course of postconcussion syndrome: the role of mild traumatic brain injury. neuropsychology. 2011;25(4):454-65. 5. ponsford j, cameron p, fitzgerald m, grant m, mikocka-walus a, schonberger m. predictors of postconcussive symptoms 3 months after mild traumatic brain injury. neuropsychology. 2012;26(3):304-13. 6. edna th, cappelen j. late post-concussional symptoms in traumatic head injury. an analysis of frequency and risk factors. acta neurochir (wien). 1987;86(1-2):12-7. 7. diagnostic a. statistical manual of mental disorders. washington. dc: american psychiatric association. 1994;4. 8. organization wh. the icd-10 classification of mental and behavioural disorders: clinical descriptions and diagnostic guidelines: world health organization; 1992. 9. binder lm. a review of mild head trauma. part ii: clinical implications. j clin exp neuropsychol. 1997;19(3):432-57. 10. king ns, kirwilliam s. permanent post-concussion symptoms after mild head injury. brain inj. 2011;25(5):462-70. 11. sterr a, herron ka, hayward c, montaldi d. are mild head injuries as mild as we think? neurobehavioral concomitants of chronic post-concussion syndrome. bmc neurol. 2006;6:7. 12. wang y, chan rc, deng y. examination of postconcussion-like symptoms in healthy university students: relationships to subjective and objective neuropsychological function performance. arch clin neuropsychol. 2006;21(4):339-47. 13. zakzanis kk, yeung e. base rates of post-concussive symptoms in a nonconcussed multicultural sample. arch clin neuropsychol. 2011;26(5):461-5. 14. dean pj, o’neill d, sterr a. post-concussion syndrome: prevalence after mild traumatic brain injury in comparison with a sample without head injury. brain inj. 2012;26(1):14-26. 15. balakrishnan b, rus rm, chan kh, martin ag, awang ms. prevalence of postconcussion syndrome after mild traumatic brain injury in young adults from a single neurosurgical center in east coast of malaysia. asian j neurosurg. 2019;14(1):201-5. 16. varner c, thompson c, de wit k, borgundvaag b, houston r, mcleod s. predictors of persistent concussion symptoms in adults with acute mild 106 j contemp med sci | vol. 8, no. 2, march-april 2022: 96–100 demographic evaluation of pcs in referrals to bandar abbas forensic medical center original s. mohammadi et al. traumatic brain injury presenting to the emergency department. cjem. 2021;23(3):365-73. 17. beauchamp f, boucher v, neveu x, ouellet v, archambault p, berthelot s, et al. post-concussion symptoms in sports-related mild traumatic brain injury compared to non-sports-related mild traumatic brain injury. cjem. 2021;23(2):223-31. 18. van der vlegel m, polinder s, toet h, panneman mjm, haagsma ja. prevalence of post-concussion-like symptoms in the general injury population and the association with health-related quality of life, health care use, and return to work. j clin med. 2021;10(4). 19. ryan lm, warden dl. post concussion syndrome. int rev psychiatry. 2003;15(4):310-6. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i2.1157 115 journal of contemporary medical sciences better oral health to all despite vast improvement in global oral health, problems persist in many populations and communities particularly among the underprivileged in both and developed and developing countries. dental caries and periodontal diseases have for years burdened the majority of populations with heavy treatment and financial needs. dental caries is still a major oral health problem in most industrialized countries as it affects 60-90% of school-aged children and the vast majority of adults. according to the world health organization oral diseases are the fourth most expensive to treat. their treatment costs exceed those of cancer, heart and coronary diseases, stroke and dementia in most industrialized countries. it has been estimated that the european union countries used 79 billion euros for oral health care in 2012. should the present trend continue the costs could exceed 93 billion euros in 2020. oral and dental care, as known in the developed world, is not available to 90% of the global population. the millions living in poverty do not have access to emergency dental care or to preventive measures to help them in their own oral health. it has been estimated that 80% of dentists in the world are serving 20% of the global population. given their prevalence worldwide oral diseases represent major public health problems causing pain, suffering and reduced quality of life. generally, studies on dental caries and periodontal diseases have shown a decreasing trend of oral diseases in developed countries. on the other hand assessing the situation in developing countries is characterized by lack of continuous and reliable data despite the recommendations of who. to provide or even to plan dental services required to meet the needs, oral health need assessments and surveys are necessary both locally and nationally. the major oral diseases are today understood more clearly than ever as behavior-related diseases. they are preventable through individual’s own actions supported by the health professionals. generally, health behaviors, either health-enhancing or healthdetrimental, are mostly derived through norms, values and goals of the family. the family plays the primary role in the acquisition, modification and improvement of health behavior. however, maintaining let alone changing health related behavior is a vast challenge. everyday life is full of health related risks known or unknown to the individuals. a health-related behavior is not a simple matter of freedom of choice rather than reflection of one’s lifestyle. lifestyle with different risks being also determinants of different diseases should be understood as an expression of the cultural and social environment in which people live and work. this concept bears significant impact on oral health. the core group of modifiable risk factors for the chronic diseases often cluster in the same population groups and individuals. these factors include smoking, alcohol consumption, diet, hygiene and stress. controlling one or a small number of risk factors have a major impact on a number of chronic diseases including oral diseases. the behavioral science experts are of opinion that only comprehensive and integrated common-risk-factor-based health promotion activities can enhance oral health and its equity as a part of general health. are health professionals ready to assume their responsibility for promoting better oral health? heikki murtomaa department of dental public health, institute of dentistry, university of helsinki, finland. correspondence to heikki murtomaa (email: heikki.murtomaa@helsinki.fi) editorial note 311j contemp med sci | vol. 8, no. 5, september-october 2022: 311–316 original breast cancer knowledge and screening practice among women in makkah, saudi arabia arwa f flemban1, saeed m kabrah2, suhail s alfaifi3, ahmed s alsubhi3, ayoub s alzahrani3, khalid a alhazmi3, samah alharbi4* 1pathology department, faculty of medicine, umm al-qura university, makkah, kingdom of saudi arabia. 2laboratory medicine department, faculty of applied medical sciences, umm al-qura university, makkah, kingdom of saudi arabia. 3faculty of medicine, umm al-qura university, makkah, kingdom of saudi arabia. 4physiology department, faculty of medicine, umm al-qura university, makkah, kingdom of saudi arabia. *correspondence to: samah alharbi (e-mail: saaharbi@uqu.edu.sa) (submitted: 04 august 2022 – revised version received: 22 august 2022 – accepted: 06 september 2022 – published online: 26 october 2022) abstract objectives: this study aimed to estimate the level of current knowledge regarding breast cancer in saudi arabia. we assessed the knowledge of breast cancer risks, the knowledge regarding the bse and mammogram in saudi arabian females using a survey-based study. methods: a cross-sectional study was conducted using a self-administered survey to assess the knowledge and practice of saudi women toward brest cancer examination. collected data were analyzed using spss program. results: a total number of 499 surveys were collected. the data showed that there was general lack of practice of bse in the sample only 186 (37%) reported that they practiced regular bse and only 16 (3%) reported that they went to the clinic for breast examination. among all participants around 54% had poor overall knowledge of breast cancer 65% had poor knowledge regarding breast cancer risk and 93% had poor knowledge regarding clinical examination. surprisingly, 56% had good knowledge regarding the clinical picture of breast cancer. conclusion: this clearly showed that the lack of breast self-examination, mammogram and clinical examination is not a result of total lack of knowledge but due to lack of knowledge regarding risks and examination. this set of data provide a guideline for the focus and planning of future breast cancer awareness campaigns. keywords: breast cancer, mass screening, awareness issn 2413-0516 introduction worldwide, breast cancer is the most common type of cancer among women.1 it is ranked as the first cause of death, in the case of women, compared with other types of cancer, constituting 15% of cancer death.2-4 breast cancer incidence rates are increasing globally especially in developing countries, this may be due to lower screening rates and incomplete reporting.2 in saudi arabia, breast cancer considered as a major source of death in females.5 according to ministry of health in saudi arabia, more cases are detected at late stage compared to developed countries, 50% and 20% respectively, causing higher mortality rate, lower prognosis rate, and higher cost for treatment.6 regretfully, according to the latest statistics in saudi arabia 15.5% of newly diagnosed breast cancer cases have distant metastasis and 40% of cases are diagnosed with regional spread at diagnosis.6 this figure illustrates that there remains lack of awareness regarding breast cancer that resulted in the delay in early detection. the 40% of patients with regional spread would have been detected earlier if there was a success in recognizing the early signs by breast self-examination (bse). additionally, the 15.5% of patients with distance metastasis would have benefited from treatment by earlier detection through bse and mammogram. the chance of developing a disease increases as the risk factors increase, and breast cancer isn’t an exception. risk factors for breast cancer include age, as the disease peaks around age 60 with a sharp incline beginning at age 40.7,8 in fact, one of the most recognized risk factors for breast cancer is the duration of exposure to the hormones that regulate the female menstrual cycle including estrogen (er) and progesterone (pr). thus, there is no surprise that early menarche and late menopause is one of the most important risk factors.9 also, race is considered as a risk factor, and it is found that there is lower risk in black women.10 another important risk factor if the family history and previous history of the breast cancer and any other cancer.7 also, prolonged use of hormonal contraceptive is of the known risk factors as well as menopausal hormone therapy.9 other risks include; delayed childbearing, decreased duration of breastfeeding, smoking, alcohol consumption, and sedentary lifestyle.11 early detection of breast cancer by screening tests leads to more success in treatment and therefore better prognosis.12 commonly used screening methods for breast cancer include mammography, cbe (clinical breast examination), and bse (breast self-examination). performing mammography by skilled technologists and interpretation by experienced radiologists can result in 85% to 90% accuracy rate in identifying pre-clinical, non-palpable tumors <15 mm in size.13 this leads to reduction of mortality by 30% to 50%.14 acog (american college of obstetricians and gynecologists) recommendations for average-risk women state that mammography should be offered at the age of 40, and to be started no later than the age of 50 annually or biennially. cbe may be offered every 1–3 years for women at the age of 29–39, and annually for women at the age of 40 and above.15 regardless the low cost of cbe and bse screening tests, the supporting evidence of their effectiveness on reducing mortality is still lacking.16 early detection of breast cancer would be enhanced if women in saudi arabia had better knowledge about breast cancer and were aware of the importance of practicing the recommended tests. thus, this study aims to determine the level of basic knowledge regarding breast cancer risk factors and its mailto:saaharbi@uqu.edu.sa 312 j contemp med sci | vol. 8, no. 5, september-october 2022: 311–316 breast cancer kap in ksa original a.f. flemban et al. clinical picture, to determine prevalence of breast cancer screening tests practice among women in makkah, saudi arabia, and to determine the relationship between the mammogram practice and the knowledge of breast cancer risk factors and clinical picture. methodology study design and study population a cross-sectional survey-based study was conducted to assess the breast cancer related knowledge, attitudes and practice among females in saudi arabia. data collection the research goals were used to create a questionnaire. it asked for personal information as well as a history of linked health incidents. the survey also looked at women’s knowledge and attitudes about breast cancer, as well as their use of screening techniques. during the academic phase, data was collected by pre-trained microbiology students as part of their research projects. after a thorough examination of the literature, the questionnaire was created based on prior research. a group of professionals in research technique validated the face validity of the questionnaire before it was administered to the study population. the reliability was assessed using cronbach’s alpha, which was determined to be >0.70. the questionnaire was accompanied by a survey cover page that explained the research and was signed and completed by the participants. to safeguard participants’ identities and preserve data confidentiality, complete anonymity was maintained. the numerous survey questions were designed after an exhaustive literature search, and the questionnaire was separated into many parts. some of the questions were changed or removed based on the expert committee’s suggestions since they were either off-topic or inappropriate for the saudi community. the questionnaire was divided into many sections. the first section elicited socio-demographic information about each study participant’s family income, education, marital status, history of breast cancer, and family history of breast cancer. in the second section, there were questions on breast cancer knowledge. these questions were divided into three categories: possible risk factors, signs and symptoms, and methods of breast cancer screening and diagnosis, such as bse and mammography. the respondents were requested to record their answers by choosing one of the three options: ‘yes’, ‘no’, or ‘don’t know’. the scale was then dichotomized (yes = 1 and no/don’t know = 0). data cleaning spss (version 28) was used for initial data cleaning, variable coding, variable computation, assumption checking and analyses. descriptive analyses identified any potential outliers, out of range values and missing data values. no out of range values were detected. data analysis descriptive statistics were used to summarise the demographic and clinical characteristics of the participants. the descriptive statistics involved frequency and percentage analyses of categorical variables. validity of the questionnaire validity represents the degree to which any measuring instrument measures what it is proposed to measure.7 there are many aspects and methods for evaluating the questionnaire validity: 1. external (content) validity: content validity refers to the degree to which the questionnaire delivers adequate coverage of the research questions.17 the content validity of the questionnaire is conducted through the supervisor review in order to assure that the content of the questionnaire is consistent with the research objectives, and evaluate whether the items reflect the research problem or not. also, academicians from the ……… university reviewed the questionnaire and provided valuable notes to improve its validity that their comments are taken into consideration. 2. internal validity: internal validity of the questionnaire is measured by the correlation coefficients between each item in one field and the whole field.7 results reliability and internal consistency of the questionnaire cronbach’s alpha was used to test for reliability and internal consistency of the questionnaire. as shown in table 1, the two-section scale showed good to excellent reliability, as did the overall scale. participants characteristics a total of 499 wemen participated in the current study and most of them were saudi (85%). the sample was of high variability to insure including all the possible layers of society and that included different educational levels, family income, marital status, age groups, and region. results (table 1) indicated that the majority of the participants were married (49.3%), whereas 41.9% were single and only 8.8% were divorced or widowed. the mean age of the participants were 38.76 ± 12.71 years, nearly half of the respondents were below the age of 40. the study sample reflected a high educational level in which 69.5% of the participants held a bachelor’s degree. only nine women (15.8%) reported a having a family member who was previously diagnosed with breast cancer. when asked about the source of their knowledge regarding breast cancer, it was found that the main source was internet/social media (54.7%). the frequency and percentage of participant demographic information are displayed in table 2. table 1. reliability analyses for patient-centred communication scale scale number of items cronbach’s alpha breast cancer knowledge 22 0.848 screening test practice and knowledge 14 0.615 total 36 0.758 313j contemp med sci | vol. 8, no. 5, september-october 2022: 311–316 a.f. flemban et al. original breast cancer kap in ksa table 2. frequency and percentage for participant characteristics (n = 499) characteristic n % nationality saudi 424 85.0 non-saudi 75 15.0 age group <40 243 48.7% 40–50 165 33.1% >50 91 18.2% region middle region 138 27.7 western region 149 29.9 eastern region 66 13.2 southern region 98 19.6 northern region 48 9.6 marital status single 209 41.9 married 246 49.3 divorced or widow 44 8.8 educational level undergraduate 105 21.0 bachelor 347 69.5 post-graduate 47 9.4 total family income per month (saudi riyale) <5,000 138 27.7 5,000–9,999 159 31.9 10,000–15,000 111 22.2 >15,000 91 18.2 first degree relative diagnosed with breast cancer yes 79 15.8 no 420 84.2 breast cancer information source reading 70 14.0 lectures and seminars 67 13.4 family and friends 46 9.2 internet/social media 273 54.7 your doctor 7 1.4 tv 36 7.2 participant’s level of knowledge regarding breast cancer next the correct answers were analysed and presented in table 3. the questions were categorised into three main subheadings: knowledge regarding breast cancer risks, the clinical picture of breast cancer and the knowledge regarding the clinical breast examination (cbe). as seen in table 3a, there were fair to poor overall knowledge regarding breast cancer. when subdivided into categories it is clearly seen that the knowledge regarding the clinical picture of breast cancer was higher from fair to good. on the other hand, there were poor knowledge regarding the risk factors and knowledge regarding the cbe. the highest recognised risk factor was the lack of physical exercise (sedentary lifestyle) (77%) followed by smoking (65.5%), oral contraceptives (60%), age (57%) and hormone table 3a. participant’s level of knowledge about breast cancer (n = 499) the question field n % breast cancer overall knowledge poor 268 53.7 fair 205 41.1 good 26 5.2 breast cancer risk knowledge poor 322 64.5 fair 144 28.9 good 33 6.6 breast cancer clinical picture knowledge poor 63 12.6 fair 158 31.7 good 278 55.7 breast cancer clinical examination knowledge poor 464 93.0 fair 33 6.6 good 2 0.4 table 3b. participants knowledge frequency and percentage for breast cancer (n = 499) the question field correct answers n % risk factors knowledge early menarche (less than 12 years) 48 9.7 late menopause (55 years and older) 94 18.8 no children 136 27.3 first child after 30 years 241 48.3 breastfeeding total duration (2 years and more) 147 29.5 obesity 236 47.3 cigarette/shisha smoking 327 65.5 increase age (more than 40 years) 287 57.5 sedentary lifestyle (no physical activity) 384 77.0 oral contraceptives 303 60.7 antibiotics 161 32.3 high dose of vitamins 123 24.6 hormonal replacement therapy after menopause 273 54.7 calcium therapy 31 6.2 vitamin d after menopause 277 55.5 clinical picture knowledge breast mass 469 94.0 nipple discharge 390 78.2 nipple ulcer 354 70.9 changes in breast skin color 384 77.0 breast pain 66 13.2 clinical examination knowledge best examination time 202 40.5 best age for mammography 31 6.2 interval of mammography 31 6.2 314 j contemp med sci | vol. 8, no. 5, september-october 2022: 311–316 breast cancer kap in ksa original a.f. flemban et al. replacement therapy (54%). the most known warning sign was detecting a breast mass, as reported by 94% of participants, as shown in table 3. the only fair knowledge regarding cbe among the participants was regarding the best time for clinical examinations (40% correct answers). only 6% of participants in the study know about the recommended age for mammography. participants attitude toward self-examination and clinical examination there were poor practise of bse and cbe in the participants. only 37% reported that they practised bse regularly and only 3% reported that they visited clinician regularly to do clinical breast examination (cbe), table 4. analysis for the association of knowledge and practice of self-examination and clinical examination of the breast as seen in table 5a, poor overall knowledge (knowledge about risk factors, clinical picture, and cbe) was associated table 4. participants breast self-examination and mammography practise (n = 499) attitude toward practice n % regular breast self examination (bse) yes 186 37.3 no 308 61.7 regular clinical breast examination (cbe) yes 16 3.2 no 483 96.8 table 5. level of knowledge of breast cancer among saudi women association with their practice of screening via (a) self examination (b) mammography (n = 499) breast cancer knowledge type knowledge level (a) self-examination (bse) chi-square p-valueyes no. (%) no no. (%) overall poor 66 (13.2) 202 (40.5) 45.2 <0.001*fair 101 (20.2) 104 (20.8) good 19 (3.8) 7 (1.4) risk poor 93 (18.6) 229 (45.9) 29.5 <0.001*fair 72 (14.4) 72 (14.4) good 21 (4.2) 12 (2.4) clinical picture poor 18 (3.6) 45 (9.0) 13.0 0.001*fair 45 (9.0) 113 (22.6) good 123 (24.6) 155 (31.1) cbe poor 168 (33.7) 296 (59.3) 3.2 0.199fair 17 (3.4) 16 (3.2) good 1 (0.2) 1 (0.2) breast cancer knowledge type knowledge level (b) mammography chi-square p-valueyes no. (%) no no. (%) overall poor 30 (6.0) 238 (47.7) 0.408 0.816fair 24 (4.8) 181 (36.3) good 4 (0.8) 22 (4.4) risk poor 40 (8.0) 282 (56.5) 0.715 0.700fair 14 (2.8) 130 (26.1) good 4 (0.8) 29 (5.8) clinical picture poor 4 (0.8) 59 (11.8) 1.970 0.373fair 20 (4.0) 138 (27.7) good 34 (6.8) 244 (48.9) practice poor 46 (9.2) 418 (83.8) 19.3 <0.001fair 11 (2.2) 22 (4.4) good 1 (0.2) 1 (0.2) significantly (p < 0.001) with no practice of bse and that the maximum of those preformed regular bse had fair knowledge regarding breast cancer. next, we subdivided the knowledge into three categories (risk, clinical picture and cbe) and assessed for association between knowledge regarding these categories and bse practice. poor knowledge regarding the risk factors for breast cancer was significantly associated with lack of bse practice (p < 0.001). there were fair to good 315j contemp med sci | vol. 8, no. 5, september-october 2022: 311–316 a.f. flemban et al. original breast cancer kap in ksa knowledge regarding the clinical picture of breast cancer and was associated with increased practice of bse (p = 0.001). there were poor cbe among the participants in this study. the only association with cbe was seen between poor knowledge regarding the cbe and the practice of cbe (p < 0.001). discussion early identification and treatment of breast cancer rely heavily on knowledge and awareness. clear and correct knowledge of breast cancer risks, diagnosis, breast self-examination (bse), clinical breast examination (cbe) and clinical picture are important for all females. detailed information regarding these factors forms the focus of the awareness campaigns. as a result, this research was carried out to assess the knowledge, attitudes, and behaviours of breast cancer screening among saudi arabian women. our population had very low understanding of risk factors, and diagnostic procedures (cbe). our cohort’s knowledge of breast cancer seems to be lower than that seen in several previous research, however, we assessed for the reliability of the survey and the population and as seen in table 1 the reliability score was high (alshahrani et al; 2019, jahan et al; 2007).18 several studies are available in saudi arabia that are aimed to assess the knowledge of females regarding breast cancer.10,19-22 the major conclusion of these studies is that there is general lack of knowledge in females in saudi arabia. these studies had concluded that the lack of knowledge is reflected in low early detection in the country and that improving knowledge via breast cancer awareness programs will aid in decreasing late diagnosis and thus, improving the chances of treatment and improve prognosis of the disease. here, we assessed and analyze specifically the relationship between knowledge of (risk factors, clinical picture, bse and cbe) and the attitude toward the practice of bse and cbe. this type of analysis allowed the stratification of the possible cause of lack of practice and provide an insight into the areas that require further improvement in future awareness campaigns. a poor to fair knowledge about breast cancer was seen in all the participants of this study 263 (53.7%). the practice of bse and cbe is very poor in the participants as well. this gave us an opportunity to assess for the exact area of knowledge that is associated with the lack of practice. a. alam (2006) concluded that there is lack of knowledge regarding breast cancer self-examination and that there is moderate knowledge regarding breast cancer risks.23 here, we found that the lack of bse was due to lack of knowledge regarding the risks of breast cancer including the risk age, factors that increase the risk of breast cancer including obesity and hormonal therapy and oral contraceptives. another aspect that is highlighted here is the assessment of the sources of information regarding breast cancer in the participating female. previously, a study in king saud university concluded that mass media is an important source of information to improve the breast cancer awareness regarding self-examination.7,8 in agreement, we found that the major source of information for the ladies in this study was the internet and social media (54.7%) followed by reading (14%), lectures and seminars (13.4%). the top two sources of information regarding breast cancer can lack validity. the most important source of information and probably the most accurate is from the physicians and surprisingly we found that this is the lowest source of information considered in our study population (1.4% only). thus, efforts to educate the public regarding the most appropriate sources of information is necessary. also, more effort is needed from all responsible authorities to enrich the public media including social media, internet websites and other easily accessible portals with the correct knowledge. the information provided through these means should be planned according to studies such as this one. a recent study has shown that although there is good knowledge regarding breast self-examination, the practice of it remains poor.24 two recent studies have contributed the lack of practice to lack of knowledge in sup-population of females depending on the level of education (suleiman 2014)24 and to the lack of sources in arabic language.25 this is in agreement with our study, as we found that there is good knowledge regarding the clinical presentation of the disease. we concluded here that the lack of practice is attributed to lack of knowledge regarding risk factors and lack of knowledge regarding the details of clinical examination including the location, proffered time and intervals between examinations. altogether, most studies agree on the importance of continuing raising the population awareness regrading breast cancer, its risks, bse, and mammography practice. conclusion overall, saudi women’s knowledge, attitudes, and behaviours about breast cancer were found to be lower than predicted. although screening techniques and resources are readily accessible and free in saudi arabia, robust teaching initiatives and campaigns aimed at the female population are lacking. we can conclude that these are all evidence of the importance of planning carefully for the awareness campaigns and to include the recommendations from all these studies in the plan and execution of such campaigns. using the data analyzed here, it can be suggested to plan for the awareness campaigns and to include the details that needs improvement in the public knowledge. also, to use the most accessible and usable sources of information for the dissemination of such important details. guided campaigns based of evidence from research such are this one is the next step to improve breast cancer situation in saudi arabia. conflict of interest none.  references 1. torre, l. a., f. bray, r. l. siegel, j. ferlay, j. lortet-tieulent, and a. jemal. 2015. ‘global cancer statistics, 2012’, ca cancer j clin, 65: 87–108. 2. ferlay, j., i. soerjomataram, r. dikshit, s. eser, c. mathers, m. rebelo, d. m. parkin, d. forman, and f. bray. 2015. ‘cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012’, int j cancer, 136: e359–86. 3. pdq. 2002. ‘genetics of breast and gynecologic cancers (pdq(r)): health professional version.’ in, pdq cancer information summaries (bethesda (md)). 316 j contemp med sci | vol. 8, no. 5, september-october 2022: 311–316 breast cancer kap in ksa original a.f. flemban et al. 4. torre, l. a., r. l. siegel, e. m. ward, and a. jemal. 2016. ‘global cancer incidence and mortality rates and trends--an update’, cancer epidemiol biomarkers prev, 25: 16–27. 5. al-rikabi, a., and s. husain. 2012. ‘increasing prevalence of breast cancer among saudi patients attending a tertiary referral hospital: a retrospective epidemiologic study’, croat med j, 53: 239–43. 6. moh. 2020. ‘breast cancer’, saudi ministry of health, accessed 2/4/20200. https://www.moh.gov.sa/en/healthawareness/educationalcontent/wh/ breast-cancer/pages/default.aspx. 7. thatcher, robert w. 2010. ‘validity and reliability of quantitative electroencephalography’, journal of neurotherapy, 14: 122–52. 8. alomair, abdullah nasser , dania ghazi felemban, mohannad sami felemban, jameel abdullah awadain, ammar saud altowairqi, nawaf fawzan alfawzan, fatimah mohammed +almazayen, abdulrahman jalwi korkoman, and nawaf saad alrusayyis. 2020. ‘knowledge, attitude, and practice of breast self-examination toward breast cancer among female students at king saud university in riyadh, saudi arabia’, international journal of medicine in developing countries, 4: 429–34. 9. alam, awatif ali. 2006b. ‘knowledge of breast cancer and its risk and protective factors among women in riyadh’, annals of saudi medicine, 26. 10. yedjou, c. g., j. n. sims, l. miele, f. noubissi, l. lowe, d. d. fonseca, r. a. alo, m. payton, and p. b. tchounwou. 2019. ‘health and racial disparity in breast cancer’, adv exp med biol, 1152: 31–49. 11. winters, s., c. martin, d. murphy, and n. k. shokar. 2017. ‘breast cancer epidemiology, prevention, and screening’, prog mol biol transl sci, 151: 1–32. 12. casolo, p., a. raspadori, b. drei, d. amuso, d. mosca, c. amorotti, p. di blasio, r. de maria, g. de luca, g. colli, and e. ganz. 1997. ‘[natural history of breast cancer: lobular carcinoma versus ductal carcinoma in our experience]’, ann ital chir, 68: 43–7; discussion 48. 13. pomahac, b., a. recht, j. w. may, c. a. hergrueter, and s. a. slavin. 2006. ‘new trends in breast cancer management: is the era of immediate breast reconstruction changing?’, ann surg, 244: 282–8. 14. cady, b., and j. s. michaelson. 2001. ‘the life-sparing potential of mammographic screening’, cancer, 91: 1699–703. 15. morman, n. a., l. byrne, c. collins, k. reynolds, and j. g. bell. 2017. ‘breast cancer risk assessment at the time of screening mammography: perceptions and clinical management outcomes for women at high risk’, j genet couns, 26: 776–84. 16. da costa vieira, r. a., g. biller, g. uemura, c. a. ruiz, and m. p. curado. 2017. ‘breast cancer screening in developing countries’, clinics (sao paulo), 72: 244–53. 17. saunders, mark , philip lewis, and adrian thornhill. 2009. research methods for business students (pearson education india). 18. dandash, k. f., and al-mohaimeed, a. 2007. knowledge, attitudes, and practices surrounding breast cancer and screening in female teachers of buraidah, saudi arabia. international journal of health sciences, 1(1), 61. 19. hussein, d. m., s. h. alorf, y. s. al-sogaih, s. h. alorf, r. s. alaskar, a. m. almahana, w. f. alsalhowb, a. k. alibrahim, m. y. saka, a. m. alhazimi, a. baghirova, and s. i. hindawi. 2013. ‘breast cancer awareness and breast self-examination in northern saudi arabia. a preliminary survey’, saudi med j, 34: 681–8. 20. latif, r. 2014. ‘knowledge and attitude of saudi female students towards breast cancer: a cross-sectional study’, journal of taibah university medical sciences, 9: 328–34. 21. albanghali, m. 2021. ‘awareness and knowledge of breast cancer among women in saudi arabia’, international journal of medical research & health sciences, 10: 90–107. 22. yaghmour, k. a., s. j. alamri, r. h. alfaqeh, l. m. alnashri, b. j. alamri, and m. a. makin. 2020. ‘awareness and knowledge of breast cancer screening methods among women in al-qunfudah, saudi arabia’, ournal of advances in medicine and medical research, 32: 81–95. 23. alam, a. a. 2006a. ‘knowledge of breast cancer and its risk and protective factors among women in riyadh’, ann saudi med, 26: 272–7. 24. suleiman, a. k. 2014. ‘awareness and attitudes regarding breast cancer and breast self-examination among female jordanian students’, j basic clin pharm, 5: 74–8. 25. allohaibi, aminah, fatimah yousef, ghadeer joudah, hussam rajab, ikhlas sindi, and mai albaik. 2021. ‘knowledge of breast cancer and the practice of breast self-examination in saudi women: an online survey’, asian journal of pharmaceutical research and health care, 13. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i5.1286 https://www.moh.gov.sa/en/healthawareness/educationalcontent/wh/breast-cancer/pages/default.aspx https://www.moh.gov.sa/en/healthawareness/educationalcontent/wh/breast-cancer/pages/default.aspx 281j contemp med sci | vol. 8, no. 4, july-august 2022: 281–283 the prevalence of procalcitonin positivity in patients with severe covid-19 nawfal r hussein department of biomedical sciences, college of medicine, university of zakho, duhok, kurdistan region, iraq. *correspondence to: nawfal r hussein ( nawfal.hussein@yahoo.com) (submitted: 03 march 2022 – revised version received: 13 april 2022 – accepted: 01 may 2022 – published online: 26 august 2022) abstract objectives: the aims of this paper were to characterize patients with severe covid-19 and study the prevalence of positive procalcitonin test in those patients. methods: in this retrospective cross-sectional study, data were collected in infectious diseases clinic between june 2020 and july 2021. real time polymerase chain reaction (rtpcr) was conducted to confirm the diagnosis of covid-19. cobas system was used to determine procalcitonin positivity following the instructions of manufacturer. results: during the study period, 454 covid-19 confirmed patients were referred to the clinic. among those, 55/494 (11.13%) had severe covid-19 infection. fever (89.1%) was the most common clinical features followed by cough (29.1%) and shortness of breath (20%). in this project, we found 4/55 (7.27%) patients had elevated procalcitonin levels. high procalcitonin was not associated with clinical outcome (p = 0.99). conclusions: in agreement with other studies, fever was the most common symptom in patients with severe covid-19. small number of the patients with severe covid-19 showed elevated procalcitonin levels. this might indicate that antibiotics should not prescribed empirically to such patients. further studies are needed to investigate this. keywords: procalcitonin, covid-19, iraq issn 2413-0516 introduction since the discovery of sars-cov-2 virus in wuhan, china in december 2019, coronavirus disease (covid-19) spread throughout the planet.1 the vast majority of countries, including iraq, decided to take extraordinary measures to stop the spread of covid-19 and reduce the morbidity and mortality.2,3 in iraq, the status of lockdown was declared with the cancellation of gathering and religious rituals, closing schools and education institutes and closing airports and boarders.4 about 80% of covid-19 patients have mild to moderate symptoms and may not require specific treatment.1 however, 20% of covid-19 patients may require hospitalization and icu admission.1 in the absence of agreed guidelines for the treatment of covid-19 infection, different medications, including antibiotics, have been used to treat such patients.5 before the covid-19 pandemic, antimicrobial resistance was global public health issue that claimed more than half a million lives, annually.6,7 the pandemic has changed the priorities of health system impacting the management of other diseases and the attention of public health policy makers diverted to combat the pandemic on the expense of other public health projects including antimicrobial resistance.8 in addition, it is believed that covid-19 pandemic may escalate the issue of antimicrobial resistance because of the antibiotics abuse that we witness in the treatment of covid-19 patients, particularly severe cases.5 this may fuel the issue of antimicrobial resistance in our region that suffering from such an issue for years. procalcitonin is a peptide precursor that increases significantly in bacterial infections and sepsis that helps to guide antibiotic use.9 the aims of this paper were to characterize patients with severe covid-19 and study the prevalence of positive procalcitonin test in those patients. materials and methods study design this was a retrospective cross-sectional study. the data were collected form infectious diseases clinic, the city of duhok, kurdistan region of iraq. this study was conducted during the period between june 2020 and july 2021. patients suspected patients with symptoms and signs of covid-19 were referred to infectious disease clinic to confirm the diagnosis. suspected case was defined as a patient who had symptoms of respiratory tract infection plus a close contact with confirmed covid-19 patients. confirmed covid-19 case was defined as a suspected case plus either positive rtpcr results or sings of covid-19 infection in ct scan of the lungs.5 severe covid-19 was defined as radiological evidence of pneumonia plus respiratory distress, oxygen saturation of ≤93% at rest or arterial partial pressure of oxygen (pao2)/fraction of inspired oxygen (fio2) ≦ 300 mmhg (l mmhg = 0.133 kpa). rtpcr nasal-pharyngeal swab was taken from each patient and rtpcr was conducted. each test included two reaction to amplify two genes: e gene and rdrp gene. while the positive test required positive amplification of both genes, with the positivity of one reaction, the test result was considered indeterminate. negative test result needed negative reaction for both genes. procalcitonin cobas system (roche) was utilized to measure the procalcitonin levels. elecsys brahms pct kit was used following the instructions of manufacturer. short communication 282 j contemp med sci | vol. 8, no. 4, july-august 2022: 281–283 procalcitonin level in covid-19 patients n.r hussein statistics binary logistic regression was utilized to study the relationship between clinical outcomes and factors. all calculations were performed using minitab 17 software. ethics the study methodology was approved by the ethics committee in the college of medicine, university of zakho, kurdistan region of iraq. written consent was obtained for recruited patients. results patients’ characteristics during the study period, 1084 patients were referred to the clinic. among them, 454 patients were confirmed as covid-19. among those, 55/494 (11.13%) had severe covid-19 infection. the average age of patients with severe covid-19 was 67 ± 14 years and 27/55 (49.09%) of them were males. fever was the most common clinical features followed by cough and shortness of breath (table 1). procalcitonin and clinical outcomes the average duration of treatment was 10.9 ± 5.1 days. the average duration before treatment was 7.25 ± 4.1 days. in this project, we found 4/55 (7.27%) patients had elevated procalcitonin. among our 55 patients, 3 (4.45%) patients passed away. factor associated with clinical outcomes the factor associated with clinical outcomes was studied. binary logistic regression was utilized to investigate the impact of gender, chronic diseases, smoking, duration of symptoms and age. no significant association was found between those factors and clinical outcome (table 2). discussion in this study, among those who diagnosed with covid-19, 11.13% had severe disease. this is in agreement with other studies that showed the percentage of severe disease is around 10% among diagnosed patients.1,10 it was shown previously that fever, myalgia, cough, shortness of breath, sore throats, diarrhea were the most common symptoms in patients with covid-19.10 in this study, the vast majority of patients with severe covid-19 had fever and cough. this is in agreement with studies previously conducted in the region and elsewhere.11 in contrast to other studies, diarrhea and vomiting were uncommon in our patients.12 this might be explained by different variants that caused the disease. procalcitonin remains negative in patients with viral infections including covid-19.9 increased procalcitonin levels may reflect bacterial coinfection and is associated with increase severity and high mortality rate in patients with covid-19.9 antibiotic has been used in the treatment of severe covid-19 without evidence of bacterial infection.5,13 this may cause unnecessary side effect for such patients with severe illness. in addition, the burden is already doubled on the health system due to the high number of patients. using unnecessary medications such as antibiotics, increase the economic burden on the health system. testing for procalcitonin may help determining which patients may need antibiotics and help understanding the progress of the disease. in our study, 4/55 (7.27%) of our patients showed positive procalcitonin test that might indicate bacterial coinfection. in contrast to other studies,9 no association was found between procalcitonin positivity and clinical outcome. this might be due to the small sample size used in this study. more studies recruiting larger sample size is recommended to explore the impact of procalcitonin positivity on clinical outcomes. in contrast to other studies that found associations between age or gender and clinical outcomes,5 no association was found between age or gender and clinical outcomes in this study. in this project, the case fatality rate in severe covid-19 was 5.45% which was lower than that reported in other studies.14,15 this might be explained table 1. symptoms of patients with severe covid-19 patients symptoms number % fever 49 89.1 cough 16 29.1 shortness of breath 11 20.0 fatigue 11 20.0 myalgia 10 18.2 loss of appetite 10 18.2 loss of smell 4 7.3 epigastric pain 3 5.5 diarrhea 2 3.6 vomiting 2 3.6 loss of taste 1 1.8 table 2. factor associated with clinical outcomes in patients with severe covid-19 patients cured died p or ci gender (male) 23/51 4/4 (100%) 0.99 87 0 chronic diseases 43/51 (84.3%) 3/4 (75%) 0.64 0.56 0.0514–6.0650 positive procalcitonin 4/4 (100%) 0/100 (0%) 0.99 0 0 smoking 4/51 (7.8%) 1/4 (25%) 0.33 3.83 0.3200–45.9171 duration of symptoms 7.47 ± 4.1 4.5 ± 3.2 0.12 0.79 0.5502–1.1207 age 61 ± 12.3 56 ± 28.4 0.5 0.98 0.9164–1.0427 short communication 283j contemp med sci | vol. 8, no. 4, july-august 2022: 281–283 n.r hussein procalcitonin level in covid-19 patients this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. partially by the difference in patients genetic makeup and the variations in virus variants. to conclude, fever was the most common symptom in subjects with severe covid-19. few patients with severe covid-19 showed elevated procalcitonin levels. this might indicate that antibiotics should not prescribed empirically to such patients. further studies are needed to investigate this. conflict of interest none.  https://doi.org/10.22317/jcms.v8i4.1093 references 1. geng m-j, wang l-p, ren x, yu j-x, chang z-r, zheng c-j, et al. risk factors for developing severe covid-19 in china: an analysis of disease surveillance data. infectious diseases of poverty. 2021;10(1):48. 2. kraemer mug, yang c-h, gutierrez b, wu c-h, klein b, pigott dm, et al. the effect of human mobility and control measures on the covid-19 epidemic in china. science. 2020;368(6490):493. 3. xiao y, torok me. taking the right measures to control covid-19. the lancet infectious diseases. 2020;20(5):523–4. 4. hussein nr, naqid ia, saleem zsm, dildar hm, ibrahim n. the impact of breaching lockdown on the spread of covid-19 in kurdistan region, iraq. avicenna journal of clinical microbiology and infection. 2020;7(1):34–5. 5. hussein nr, naqid ia, saleem zsm. a retrospective descriptive study characterizing coronavirus disease epidemiology among people in the kurdistan region, iraq. mediterranean journal of hematology and infectious diseases. 2020;12(1):e2020061. 6. murray cjl, ikuta ks, sharara f, swetschinski l, robles aguilar g, gray a, et al. global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. the lancet. 2022;399(10325):629–55. 7. rasheed na, hussein nr. staphylococcus aureus: an overview of discovery, characteristics, epidemiology, virulence factors and antimicrobial sensitivity. european journal of molecular & clinical medicine. 2021;8(3):1160–83. 8. stevens mp, doll m, pryor r, godbout e, cooper k, bearman g. impact of covid-19 on traditional healthcare-associated infection prevention efforts. infect control hosp epidemiol. 2020;41(8):946–7. 9. lippi g, plebani m. procalcitonin in patients with severe coronavirus disease 2019 (covid-19): a meta-analysis. clin chim acta. 2020;505:190–1. 10. verity r, okell lc, dorigatti i, winskill p, whittaker c, imai n, et al. estimates of the severity of coronavirus disease 2019: a model-based analysis. the lancet infectious diseases. 2020;20(6):669–77. 11. çalıca utku a, budak g, karabay o, güçlü e, okan hd, vatan a. main symptoms in patients presenting in the covid-19 period. scottish medical journal. 2020;65(4):127–32. 12. han c, duan c, zhang s, spiegel b, shi h, wang w, et al. digestive symptoms in covid-19 patients with mild disease severity: clinical presentation, stool viral rna testing, and outcomes. am j gastroenterol. 2020;115(6):916–23. 13. mustafa l, tolaj i, baftiu n, fejza h. use of antibiotics in covid-19 icu patients. journal of infection in developing countries. 2021;15(4):501-5. 14. mahendra m, nuchin a, kumar r, shreedhar s, mahesh pa. predictors of mortality in patients with severe covid-19 pneumonia a retrospective study. adv respir med. 2021;89(2):135–44. 15. shi c, wang l, ye j, gu z, wang s, xia j, et al. predictors of mortality in patients with coronavirus disease 2019: a systematic review and metaanalysis. bmc infectious diseases. 2021;21(1):663. short communication 193j contemp med sci | vol. 9, no. 3, may-june 2023: 193–196 original statins in combination with ibrutinib bypasses resistance to ibrutinib in mantle cell lymphoma aoula al-zebeeby1* , ali abbas2 1department of pathology and poultry diseases, faculty of veterinary medicine, university of kufa, najaf, iraq. 2department of microbiology, faculty of veterinary medicine, university of kufa, najaf, iraq. *correspondence to: aoula al-zebeeby (e-mail: aoulae.alzebeeby@uokufa.edu.iq) (submitted: 20 march 2023 – revised version received: 18 april 2023 – accepted: 10 may 2023 – published online: 26 june 2023) abstract objective: in this study, we report that a novel combination therapy of different statins with ibrutinib can overcome such resistance. methods: for this, we generated a cell line model, exhibiting resistance to ibrutinib, by repeated exposure of mantle cell lymphoma cell line to ibrutinib. apoptosis was assessed by the extent of phosphatidylserine externalisation. results: our results indicated that resistance to ibrutinib could be overcome by targeting a key enzyme, 3-hydroxy-3-methylglutaryl coenzyme a (hmg-coa) reductase in the cholesterogenesis pathway. conclusion: reusing different statins in combination with ibrutinib could improve therapy and enhance sensitivity to ibrutinib mediated apoptosis. keywords: statins, ibrutinib, chemoresistance, hmg-coa reductase, cholecterogensis pathway issn 2413-0516 introduction mantle cell lymphoma (mcl) is an aggressive form of malignant b-lymphocytes of non-hodgkin’s lymphoma, which arise from the outer edge of a lymph node follicle, also known as the mantle zone of lymph node. the majority of mantle cell lymphoma cases are considered as incurable with multiple incidences of relapse. several treatment strategies have been developed over the past few years with significant improvement in patient outcome.1–3 signalling of b cell receptor (bcr) is crucial for the maturation and development of b cells. bruton’s tyrosine kinase (btk) is a key enzyme in b cell receptor cascade.4–6 many studies have identified an important role of btk in the activation of bcr signalling pathway,7–10 which in turn activates map kinase and nf-κb pathways, thereby enhancing b cell activity, survival rate, proliferation and migration.3 therefore, btk is considered an important therapeutic target for mcl and other b cell malignancies.11 in the last few decades, various molecules have been used in clinical practice for relapsed/refractory (r/r) of mcl and other types of b cell malignancies.10 among these pharmacological molecules, ibrutinib, commercially available under the name imbruvica, is a btk inhibitor and first line treatment for mcl.12 however, chemoresistance to anticancer agents, including ibrutinib poses a critical challenge for the successful implementation of this therapy. tackling this challenge becomes vital as the resistance affects patient outcome.13 in recent years, several studies indicated that modulation of some cancer metabolic pathways improved cancer therapy.14,15 statins form an important classification of these metabolic inhibitors, and exhibit pleiotropic abilities, including anti-angiogenic, antioxidant, anti-inflammatory and anticancer activities.16 therefore, our study was aimed at identifying whether a combination of statins with ibrutinib could overcome resistance to ibrutinib-mediated apoptosis in mcl. methods reagents ibrutinib, simvastatin and atorvastatin were from selleck (sylvanfield drive, houston, usa). pitavastatin was from tocris (abingdon, uk). filipin iii stain solution was from sigmaaldrich (gillingham, uk). generation of chemoresistance parental cells of maver-1 cells (labelled as i in figure 1a) were exposed to ibrutinib (10 µm) for 48h, and the extent of apoptosis was assessed using flow cytometry. this was followed by two weeks (2w) of recovery period, during which the cells were cultured in drug-free media (rpmi media + 10% fbs) at 37°c and 5% co2. the resulting cells were labelled as ii, and this process was repeated three more times more, to generate cells labelled as iii, iv and v. the extent of apoptosis was checked after each exposure to assess the extent of resistance. thus, group v cells exhibited the most resistance to ibrutinib. flow cytometry apoptosis was assessed by phosphatidylserine (ps) externalisation. following 48 hours exposure to ibrutinib and 72 hours in case of statins, cells were gathered and completed using an annexin v buffer and propidium iodide (pi) stain. then, the extent of apoptosis was measured by flow cytometry (florescence-activated cell sorting (facs)). filipin staining suspension maver-1 cells (0.2 × 106) were collected and resuspended with 50ml of phosphate-buffered saline (pbs). then, by using polysinetm adhesion slide (menzel-glaser, uk) the mixture was placed and left for 5 minutes. this was followed by a fixation step using paraformaldehyde (4%) from thermo fisher scientific (waltham, ma, usa) for 5 minutes and triton x-100 (0.5%) from sigmaaldrich (gillingham, uk) for 10 minutes. subsequently, the slides were incubated with filipin for 2 hours, and washed three times gently with pbs, before imaging using fluorescence microscopy with a uv filter set (340–380 nm excitation, 40 nm dichroic, 430-nm long pass filter). statistical analysis multiple comparisons one-way f test or analysis of variance (anova) and the least significant difference (fisher test) https://orcid.org/0000-0003-1656-1841 194 j contemp med sci | vol. 9, no. 3, may-june 2023: 193–196 overcoming chemoresistance to ibrutinib original a. al-zebeeby et al. (p ≤ 0.01) were conducted to compare sensitive (i) and resistant (v) cells. statistics was performed using graphpad prism 6 software for windows (la jolla, ca, usa). results mantle cell lymphoma cells acquired rapid resistance to lbrutinib in order to mimic the resistance observed in clinic, we developed a chemoresistance model, in which the initial maver-1 cells (labelled as i) were repeatedly exposed to ibrutinib 10mm for 48h, followed by recovery periods of two weeks (2w), until increasing resistance was reached in the different groups of cells, labelled ii-v (figure 1 a and b). taken together, these results suggested that resistance to ibrutinib was developed successfully. targeting hmg-coa reductase overcomes resistance to ibrutinib mediated apoptosis in mantle cell iymphoma cells in order to identify a way to restore sensitivity to ibrutinib mediated apoptosis, we investigated whether maver-1 sensitive (labelled as i) and resistant (labelled as v) cells exhibited differences in cholesterol synthesis. staining of cells with filipin indicated that cholesterol synthesis was enhanced in the resistant cells (v) compared to the sensitive cells (i) (figure 2a). therefore, we investigated whether targeting cholesterogenesis by three mostly used statins, namely atorvastatin, pitavastatin and simvastatin could overcome resistance to ibrutinib-mediated apoptosis in mantle cell lymphoma. our results revealed that targeting hmg-coa reductase by these three different statins in combination with ibrutinib enhanced sensitivity for sensitive cells (i) and overcame resistance to ibrutinib-mediated apoptosis in resistant cells (v) (figure 2b). taken together, these results suggested that resistance to ibrutinib-mediated apoptosis could be because of the enhanced cholesterol synthesis in the resistant cells. therefore, using statins in combination with ibrutinib enhanced sensitivity and overcame resistance to ibrutinib-mediated apoptosis. discussion development of resistance to cancer therapy is currently one of the main obstacle that challenges the successful anticancer therapy and is often responsible for regression and incurable cancer.1,2,13,17 in the current study, the resistance to ibrutinib in mantle cell lymphoma was rapidly developed (figure 1a and b) to mimic chemoresistance in patients. chemotherapy often creates a stressful environment for cancer cells, which in turn respond to such unfavourable conditions by activation of several pathways that enable the cells to escape therapy.18,19 one example of this is the rewiring of cancer metabolism. this is achieved by increasing cholesterol and lipid synthesis, in addition to activation of several other metabolic pathways.15,18,19 for instance, in case of b cell malignancies, resistance to ibrutinib could be achieved through the elevation of fatty acids synthesis and oxidation.20,21 similarly, resistance to anti-cancer agents in mantle cell lymphoma has been reported to occur via the activation of glutamine metabolism.22 furthermore, targeting intermediary metabolism enhances sensitivity and overcome chemoresistance.23 the best inhibitors of the hmg-coa reductase are statins, such as atorvastatin, ptivastatin and simvastatin, which decrease cholesterol levels in serum.24,25 several studies indicated that statins have the power to control growth of tumour both in vitro and in vivo by blocking the progression of cell cycle.26–30 furthermore, different clinical trials have been fig.1 mantle cell lymphoma rapidly developed resistance to ibrutinib. (a) scheme for developing resistance to ibrutinib in mantle cell lymphoma cell lines (maver-1), as explained in the methods section. sensitive (i) and resistant (v) cells of maver-1 cell line were exposed for 48 h to ibrutinib (10 mm), and apoptosis was detected. (b) gradual increase of resistance was observed in each group of cells (i, ii, iii, iv and v). ***p-0.001, error bars = mean ± standard error of mean (n = 3) ps: phosphatidylserine. 195j contemp med sci | vol. 9, no. 3, may-june 2023: 193–196 a. al-zebeeby et al. original overcoming chemoresistance to ibrutinib fig. 2 targeting hmg-coa reductase overcame resistance to ibrutinib. (a) sensitive (labelled as i) and resistant (labelled as v) maver-1 cells were stained with flipin (cholesterol dye) scale bar: 10 µm. (b) sensitive (labelled as i) and resistant (labelled as v) maver-1 cells were exposed to ibrutinib (10 mm) for 48 h alone or in combination with pharmacological inhibitors of hmgr including, atorvastatin (10 mm), pitavastatin (1 mm) or simvastatin (250 nm) for 72 h. ***p-0.001, error bars = mean ± standard error of mean (n = 3). ps: phosphatidylserine. fig. 3 scheme representing the development of resistance to ibrutinib due to increased cholesterogenesis. targeting the key enzyme hmgr by statins enhances sensitivity to ibrutinib-mediated apoptosis. examined the anticancer activity of statins in many cancers, such as non-metastatic rectal cancer, head and neck cancer, advanced liver carcinoma, pediatric tumors, colon cancer and acute myeloid leukemia.31 –33 however, in the present study a group of statins was used to bypass resistance to ibrutinib in mantle cell lymphoma cell line (figures 2b and 3). in agreement with these results, inhibition of hmg-coa reductase enhances sensitivity to venetoclax in different blood malignancies.16,34 conclusion resistance to ibrutinib mediated apoptosis was developed rapidly in mantle cell lymphoma cell line to mimic chemoresistance in patients. increased cholesterol synthesis could be the metabolic reprogramming that maver-1 used to escape from therapeutic pressure presented by ibrutinib. therefore, re-purposing some drugs, such as statins could improve therapy and bypass resistance to ibrutinib-mediated apoptosis. acknowledgments the authors thank dr. shankar varadarajan (institute of systems, molecular and integrative biology/university of liverpool) for his help with proofreading the manuscript. competing interests the authors declare no competing interests.  196 j contemp med sci | vol. 9, no. 3, may-june 2023: 193–196 overcoming chemoresistance to ibrutinib original a. al-zebeeby et al. lymphomas. cancers (basel). 2020 may 22;12(5):1328. doi: 10.3390/ cancers12051328. 18. holohan c, van schaeybroeck s, longley db, johnston pg. cancer drug resistance: an evolving paradigm. nat rev cancer. 2013 oct;13(10):714–26. doi: 10.1038/nrc3599. pmid: 24060863. 19. mccann c, kerr em. metabolic reprogramming: a friend or foe to cancer therapy? cancers (basel). 2021 jul 3;13(13):3351. doi: 10.3390/ cancers13133351. 20. galicia-vázquez g, aloyz r. ibrutinib resistance is reduced by an inhibitor of fatty acid oxidation in primary cll lymphocytes. front oncol. 2018 sep 26;8:411. doi: 10.3389/fonc.2018.00411. 21. li z & zhang h. reprogramming of glucose, fatty acid and amino acid metabolism for cancer progression. cell mol life sci. 2016 jan;73(2):377–92. doi: 10.1007/s00018-015-2070-4. 22. monaco me. fatty acid metabolism in breast cancer subtypes. oncotarget. 2017 apr 25;8(17):29487–29500. doi: 10.18632/oncotarget.15494. 23. al-zebeeby a, vogler m, milani m, richards c, greaves g, dyer mjs, et al. targeting intermediary metabolism enhances the efficacy of bh3 mimetic therapy in hematologic malignancies. haematologica. 2019 may;104(5):1016–1025. doi: 10.3324/haematol.2018.204701. 24. golomb ba & evans ma. statin adverse effects: a review of the literature and evidence for a mitochondrial mechanism. am j cardiovasc drugs. 2008;8(6):373–418. doi: 10.2165/0129784-200808060-00004. 25. you hy, zhang wj, xie xm, zheng zh, zhu hl, jiang fz. pitavastatin suppressed liver cancer cells in vitro and in vivo. onco targets ther. 2016 aug 29;9:5383–8. doi: 10.2147/ott.s106906. 26. soma m, corsini a, paoletti r. cholesterol and mevalonic acid modulation in cell metabolism and multiplication. toxicologyletters. 1992 64;1–15. 27. newman a, clutterbuck rd, powles rl, catovsky d, millar jl. a comparison of the effect of the 3-hydroxy-3-methylglutaryl coenzyme a (hmg-coa) reductase inhibitors simvastatin, lovastatin and pravastatin on leukaemic and normal bone marrow progenitors. leuk lymphoma. 1997 feb; 24(5–6):533-7. doi: 10.3109/10428199709055590. 28. denoyelle c, vasse m, körner m, mishal z, ganné f, vannier jp, soria j, soria c. cerivastatin, an inhibitor of hmg-coa reductase, inhibits the signaling pathways involved in the invasiveness and metastatic properties of highly invasive breast cancer cell lines: an in vitro study. carcinogenesis. 2001 aug;22(8):1139–48. doi: 10.1093/carcin/22.8.1139. 29. kim ws, kim mm, choi hj, yoon ss, lee mh, park k, et al. phase ii study of high-dose lovastatin in patients with advanced gastric adenocarcinoma. invest new drugs. 2001;19(1):81–3. doi: 10.1023/a:1006481423298. 30. hindler k, cleeland cs, rivera e, collard cd. the role of statins in cancer therapy. oncologist. 2006 mar;11(3):306–15. doi: 10.1634/ theoncologist.11-3-306. 31. minden md, dimitroulakos j, nohynek d, penn lz. lovastatin induced control of blast cell growth in an elderly patient with acute myeloblastic leukemia. leuk lymphoma. 2001 feb;40(5-6):659–62. doi: 10.3109/10428190109097663. 32. katz ms, minsky bd, saltz lb, riedel e, chessin db, guillem jg. association of statin use with a pathologic complete response to neoadjuvant chemoradiation for rectal cancer. int j radiat oncol biol phys. 2005 aug 1;62(5):1363–70. doi: 10.1016/j.ijrobp.2004.12.033. 33. cho sj, kim js, kim jm, lee jy, jung hc, song is. simvastatin induces apoptosis in human colon cancer cells and in tumor xenografts, and attenuates colitis-associated colon cancer in mice. int j cancer. 2008 aug 15;123(4):951–7. doi: 10.1002/ijc.23593. 34. gimenez n, tripathi r, giró a, rosich l, lopez-guerra m, lopez-oreja i, et al. systems biology drug screening identifies statins as enhancers of current therapies in chronic lymphocytic leukemia. sci rep. 2020 dec 17; 10: 22153. doi:10.1038/s41598-020-78315-0. references 1. kluin-nelemans hc, hoster e, hermine o, walewski j, trneny m, stilgenbauer s. treatment of older patients with mantle-cell lymphoma. n engl j med 2012 aug 9; 367(6):520–31. 2. dreyling m, campo e, hermine o, jerkeman m, gouill le, rule s, et al.newly diagnosed and relapsed mantle cell lymphoma: esmo clinical practice guidelines for diagnosis, treatment and follow-up. ann oncol 2017 july; 28(supplement 4):iv62–71. 3. honigberg la, smith am, sirisawad m, verner e, loury d, chang b, et al. the bruton tyrosine kinase inhibitor pci-32765 blocks bcell activation and is effificacious in models of autoimmune disease and b-cell malignancy. proc natl acad sci 2010 jul 20;107(29):13075–80. 4. wiestner a.targeting b-cell receptor signaling for anticancer therapy: the bruton’s tyrosine kinase inhibitor ibrutinib induces impressive responses in b-cell malignancies. j clin oncol. 2013 jan 1; 31(1):128–30. available from https: //pubmed.ncbi.nlm.nih.gov/23045586 doi: 10.1200/jco.2012.44.4281. 5. davis re, ngo vn, lenz g, tolar p, young rm, romesser pb, et al. chronic active b-cell-receptor signalling in diffuse large b-cell lymphoma. nature.2010 jan 7;463(7277):88–92. available from https://pubmed.ncbi. nlm.nih.gov/20054396// doi: 10.1038/nature08638. pmid: 20054396; pmcid: pmc2845535. 6. kenkre vp & kahl bs. the future of b-cell lymphoma therapy: the b-cell receptor and its downstream pathways. curr hematol malig rep.2012 sep; 7(3):216–20. available from https://pubmed.ncbi.nlm.nih.gov/22688757/ doi: 10.1007/s11899-012-0127-0. 7. buggy jj & elias l. bruton tyrosine kinase (btk) and its role in b-cell malignancy. int rev immunol. 2012 apr;31 (2):119–32. doi:10.3109/08830 185.2012.664797. erratum in: int rev immunol. 2012 oct;31(5):428. pmid: 22449073. 8. rushworth sa, bowles km, barrera ln, murray my, zaitseva l, macewan dj. btk inhibitor ibrutinib is cytotoxic to myeloma and potently enhances bortezomib and lenalidomide activities through nf-κb. cell signal. 2013 jan; 25(1):106–12. doi:10.1016/j.cellsig.2012.09.008. 9. harrison c. trial watch: btk inhibitor shows positive results in b cell malignancies. nat rev drug discov. 2012 jan 20;11(2):96. avialable from https://pubmed.ncbi.nlm.nih.gov/22262035/ doi: 10.1038/nrd3656. 10. alinari l, christian b, baiocchi ra, et al. novel targeted therapies for mantle cell lymphoma. oncotarget. 2012 feb;3(2):203–11. avialable from https:// pubmed.ncbi.nlm.nih.gov/22361516/ doi: 10.18632/oncotarget.426. 11. danilov av. targeted therapy in chronic lymphocytic leukemia: past, present, and future. clin ther. 2013 sep;35(9):1258–70. available from https:// pubmed.ncbi.nlm.nih.gov/24054703/doi: 10.1016/j.clinthera.2013.08.004. 12. stephens dm, spurgeon se. ibrutinib in mantle cell lymphoma patients: glass half full? evidence and opinion. ther adv hematol. 2015 oct;6(5): 242–52. avialable from https://www.ncbi.nlm.nih.gov/pmc/articles/ pmc4556969/doi: 10.1177/2040620715592569. 13. nikolaou m, pavlopoulou a, georgakilas ag, kyrodimos e. the challenge of drug resistance in cancer treatment: a current overview. clin exp metastasis. 2018 apr;35(4):309–318. available from https://pubmed.ncbi.nlm.nih. gov/29799080/ doi: 10.1007/s10585-018-9903-0. 14. li j, eu jq, kong lr, wang l, lim yc, goh bc, wong ala. targeting metabolism in cancer cells and the tumour microenvironment for cancer therapy. molecules. 2020 oct 20;25(20):4831. doi: 10.3390/ molecules25204831. 15. hanahan d, weinberg ra. hallmarks of cancer: the next generation. cell. 2011 mar 4;144(5):646–74. doi: 10.1016/j.cell.2011.02.013. 16. lee js, roberts a, juarez d, vo tt, bhatt s, herzog lo, et al. statins enhance efficacy of venetoclax in blood cancers. sci transl med. 2018 jun 13;10(445):eaaq1240. doi: 10.1126/scitranslmed.aaq1240. 17. george b, chowdhury sm, hart a, sircar a, singh sk, nath uk, et al. ibrutinib resistance mechanisms and treatment strategies for b-cell this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1330 https://pubmed.ncbi.nlm.nih.gov/?term=hermine+o&cauthor_id=22873532 https://pubmed.ncbi.nlm.nih.gov/?term=hermine+o&cauthor_id=22873532 https://doi.org/10.1200/jco.2012.44.4281 https://pubmed.ncbi.nlm.nih.gov/22688757/ https://doi.org/10.1016/j.cellsig.2012.09.008 https://pubmed.ncbi.nlm.nih.gov/22262035/ https://pubmed.ncbi.nlm.nih.gov/22361516/ https://pubmed.ncbi.nlm.nih.gov/22361516/ https://pubmed.ncbi.nlm.nih.gov/29799080/ https://pubmed.ncbi.nlm.nih.gov/29799080/ 235j contemp med sci | vol. 7, no. 4, july-august 2021: 235–241 original obesity, quality of sleep and anxiety in saudi arabia noara alhusseini1*, basant elaasser2, bushra alshaar2, shahd alyousof2, susan boukhet2, aamir omair3 1director of the master of public health program, senior lecturer, college of medicine, alfaisal university, riyadh, saudi arabia. 2college of medicine, alfaisal university, riyadh, saudi arabia. 3research unit, college of medicine, king saud bin abdulaziz university for health sciences, riyadh, saudi arabia. *correspondence to: dr. noara alhusseini (e-mail: nalhusseini@alfaisal.edu) (submitted: 18 april 2021 – revised version received: 05 may 2021 – accepted: 22 may 2021 – published online: 26 august 2021) introduction obesity is defined by excessive body fat in the human body. it is a chronic, multifactorial disease that causes both psychological and physical stress. it can occur at any stage in life and can develop in children and adults.1 the etiologies of obesity include iatrogenic, dietary, neuroendocrine, social, behavioral, lifestyle, and genetic causes.1 obesity can lead to cardiovascular diseases, type ii diabetes, osteoarthritis, cancer, and overall decreased life expectancy.2 the body mass index (bmi) is mostly used for the determination of the severity of obesity. the higher the bmi, the higher the chance of morbidity and mortality.3 as technology has moved forward and people moved less, the rate of obesity has nearly doubled worldwide and is now considered a pandemic.4 the rate of obesity in the kingdom of saudi arabia (ksa) has doubled in the past ten years as 66% of males are overweight and 28% obese, whereas 71% of females are overweight and 44% are obese.5,6 lifestyle factors contributing to unhealthy eating habits in ksa include frequent consumption of sugary beverages and calorie-dense meals, lack of publicly accessible walkways, lack of physical education in schools, and increased screening time.7 in addition, the desert climate discourages outdoor activities and encourages late rising and resting, including insufficient or excessive sleep.8 sleep is an easily reversible state of unconsciousness with reduced metabolic and motor activities.9 sleep is essential for waking cognition, i.e., attention, alertness, vigilance, clear thinking, and memory consolidation, and emotional regulation.10 there is variability in the sleep duration needed by each individual but the american academy of sleep medicine (aasm) and sleep research society (srs) recommends seven hours or more of sleep per night for adults.11 acute sleep deprivation mainly causes cognitive impairment where individuals take a long time and find more difficulty performing daily tasks. other effects include changes in mood, judgment, and respiratory physiology.12 on the other hand, chronic sleep deprivation usually results in more-subtle effects where the individual is more prone to accidents, cardiovascular morbidity, immunosuppression, obesity, and an overall decreased quality of life and lifespan.13 recent scientific literature revealed a link between sleeping hours and metabolic effects that predispose to weight changes, also, an association between increased body mass index (bmi) and short sleeping hours.14 anxiety disorders are common mental health conditions.15 the level of anxiousness people experience in anxiety disorders is over-proportionate to the situation, causing unpleasant emotions and/or physical changes, like disruption of blood pressure level and gastrointestinal distress.16,17 anxiety disorders interfere with day to day tasks through repetitive attacks of abrupt feelings of persistent, intense anxiety, fear, terror, and high discomfort.15-17 anxiety attacks are longlasting and difficult to control.15-17 anxiety disorders are subdivided into various types, like, generalized anxiety disorder (gad), panic disorder, and phobia related disorders.16 generalized anxiety disorder causes sleeping problems, and new research suggests sleep deprivation can cause an anxiety disorder.16,17 research also suggests sleep disruption is present in nearly all psychiatric disorders.16,17 moreover, people with chronic insomnia are at high risk of developing anxiety disorders.17 recent scholars demonstrate a relationship between anxiety disorders and obesity like panic attacks and phobias, but the association between gad and obesity is still under research; there are studies that suggest an association, and others contradict the relationship.18 the relation of this array of irregular sleep duration, anxiety, and obesity in ksa is not well studied and may benefit from further research. this study aimed to find an association between obesity levels, quality of sleep, and anxiety among the saudi population. there are limited peer-reviewed studies that target obesity levels in relation to quality of sleep and anxiety level. this abstract introduction there is an inter-relationship between sleeping hours, anxiety, and metabolic effects that predispose the body to weight changes. the aim is to determine an association between obesity levels, quality of sleep, and anxiety among the saudi population. methods a cross-sectional study was used by distributing an online questionnaire via social media channels to the saudi population. we used the body mass index (bmi) to assess body fat, the pittsburg sleep quality index (psqi), and generalized anxiety disorder – 7 (gad-7). chi-square tests and logistic regression were used to test associations with a p-value of less than 0.05. results we received a total of 1123 respondents and the majority (61%) were obese and overweight. according to the gad-7 score, 17% had severe anxiety; the psqi score was 8.42 ± 3.45, indicating overall poor sleep quality. underweight respondents had higher moderate (25%) and severe (26%) anxiety as compared to those with normal or higher bmi (14%–18%) (p = 0.002). psqi scores were higher for obese (8.9 ± 3.6) and overweight (8.6 ± 3.4) respondents as compared to underweight (7.5 ± 3.0) (p = 0.001). persons with severe (10.4 ± 3.6) or moderate (9.7 ± 3.7) anxiety had higher psqi scores as compared to those with minimal anxiety (6.8 ± 2.7) (p < 0.001). conclusion there is a relationship between obesity, sleep, and anxiety. overweight and obesity were significantly associated with anxiety and poor sleep quality in saudi arabia. keywords obesity, overweight, quality of sleep, anxiety, saudi population issn 2413-0516 236 j contemp med sci | vol. 7, no. 4, july-august 2021: 235–241 obesity, quality of sleep and anxiety in saudi arabia original n. alhusseini et al. study is novel as it adds new information to the current literature among the saudi population. methods this was a cross-sectional study design. the study population consisted of adults in saudi arabia. the inclusion criteria were 18 years old or above, saudis and non-saudis. an online questionnaire was distributed via social media platforms including whatsapp, twitter, facebook, and linkedin. the estimated annual population of saudi arabia in 2020 is around 34.8 million. the sample size was 384, with a 95% confidence level and 5% margin of error for an expected 50% prevalence for the outcome variable. the online questionnaire was taken from previously validated studies19,20 it was modified to fit the objectives of the study. it included demographic questions, height, weight, quality of sleep assessment, and assessing anxiety. the questionnaire was distributed in both english and arabic languages. face and content validity were achieved by a public health specialist and a translator. the names of the respondents were not collected to ensure anonymity and confidentiality. filling the survey was construed as consent. participation in the study was voluntary and respondents were able to withdraw at any time. approval from the institutional review board (irb) at alfaisal university was achieved irb-20062. the body mass index (bmi) was used for assessing body fat. it is calculated by dividing an individual’s weight in kilograms by their height in meters squared. the result of this calculation is the individual’s bmi. bmi was used to categorize a person as underweight, normal weight, overweight, or obese. the classifications are severely underweight bmi less than 16.5 kg/m2, underweight bmi under 18.5 kg/m2, normal weight bmi greater than or equal to 18.5 to 24.9 kg/m2, overweight – bmi greater than or equal to 25 to 29.9 kg/m2, obesity class i – bmi between 30 to 34.9 kg/m2, obesity class ii –bmi between 35 to 39.9 kg/m2, obesity class iii –bmi 40 kg/m2.21 for assessing the quality of sleep, the pittsburg sleep quality index (psqi) was used. it is a screening measure consisting of ten questions used to differentiate “good” from “poor” sleepers.20 the psqi is of 7 components, with each component having four different responses, each scored as 0, 1, 2, 3, respectively. component 1 (q9) was subjective sleep quality-labeled as very good, fairly good, fairly bad, or very bad scored at 0, 1, 2, 3, respectively. component 2 (q2,5a) was sleep latency, which was split into two parts. part one was how long it takes to fall asleep labeled as < 15 minutes, 16-30 minutes, 31–60 minutes, or > 60 minutes scored at 0, 1, 2, 3, respectively. part two was being unable to sleep within 30 minutes labeled as not during past month, less than once a week, once or twice a week, three or more times a week scored at 0, 1, 2, 3 respectively. the sum of scores from both parts were labeled as [0], [1 or 2], [3 or 4], or [5 or 6] scored at 0, 1, 2, 3, respectively. component 3 (q4) was sleep duration labeled as > 7 hours, 6–7 hours, 5–6 hours, or < 5 hours scored at 0, 1, 2, 3, respectively. component 4 (q1,3,4) was sleep efficiency calculated by sleep efficiency = (# hours slept/# hours in bed) x100% where # hours slept was from question 4 and # hours in bed was calculated from responses to questions 1 and 3 in the survey. the result was labelled as > 85%, 75-84%, 65-74%, or < 65% scored at 0, 1, 2, 3 respectively. component 5 (q5b-j) was sleep disturbance labeled as not during the past month, less than once a week, once or twice a week, three or more times a week scored at 0, 1, 2, 3, respectively. the sum of all scores in this component was labeled as 0, 1-9, 10-18, or 19-27 scored at 0, 1, 2, 3, respectively. component 6 (q6) was use of sleep medication labeled as not during the past month, less than once a week, once or twice a week, or three or more times a week scored at 0, 1, 2, 3, respectively. component 7 (q7,8) was daytime dysfunction, which split into two parts. part one was trouble staying awake labeled as not during the past month, less than once a week, once or twice a week, or three or more times a week scored at 0, 1, 2, 3, respectively. part two was trouble keeping up enthusiasm labeled as no problem at all, only a very slight problem, somewhat of a problem, or a very big problem scored at 0, 1, 2, 3 respectively. the sum of scores from both parts were labeled as [0], [1 or 2], [3 or 4], or [5 or 6] scored at 0, 1, 2, 3, respectively. with all components of psqi complete, the global psqi score was the sum of all seven individual component scores. the answers were then scored using an established guideline. a global psqi score of more than five indicated poor sleeping with a diagnostic sensitivity of 89.6% and specificity of 86.5% (kappa = 0.75, p ≤ 0.001).20 generalized anxiety disorder – 7 (gad-7) is a validated and reliable assessment tool in gad screening; it is used by the saudi ministry of health in primary health care centers.19 gad-7 contained seven questions that measure anxiety severity where each item asked the individual to rate symptoms severity over the past two weeks. the severity of symptoms were labeled as not at all, several days, more than half the days, or nearly every day. scores were given to each label respectively: 0, 1, 2, 3. all scores were added together, and the total was then classified as follows. total scores of 0–4 indicated minimal anxiety, 5-9 indicated mild, probably subclinical anxiety, and monitoring is recommended. total scores of 10–14 indicated moderate, possibly clinically significant anxiety, and further evaluation and treatment (if needed) are recommended. total scores of 15–21 indicated severe, probably clinically significant anxiety, and treatment is probably warranted.19 the data was entered and analyzed using spss v23. the categorical data were presented as frequencies and percentages, while the numerical data were presented as mean ± standard deviation. the chi-square test was used to determine the association of gad-7 categories with bmi groups and exercise frequency. anova was used to assess the association of psqi scores with the above two variables and gad-7 classes. a p-value <0.05 was considered to show a statistically significant association for all the statistical tests. the authors obtained ethical approval from alfaisal university institutional review board (irb). the authors abided by the saudi national committee of bioethics (ncbe) and the research policies & procedures of alfaisal university. no identifying data was collected to ensure anonymity and confidentiality. the investigators only had access to the survey responses. results there were a total of 1123 saudi and non-saudi respondents to the survey and their demographic characteristics are shown in table 1. the highest proportion were in the age group 18–29 years for both saudis (42%) and non-saudi (41%) respondents (p = 0.63). there was a higher proportion of females among the saudi (76%) as compared to the non-saudi (66%) respondents 237j contemp med sci | vol. 7, no. 4, july-august 2021: 235–241 n. alhusseini et al. original obesity, quality of sleep and anxiety in saudi arabia (p < 0.001). a greater proportion of non-saudis (59%) were married as compared to the saudi (53%) respondents (p = 0.02). a greater proportion of saudis (46%) were employed as compared to the non-saudi (39%) respondents (p= 0.03); there was a higher proportion of the non-saudis (47%) who did not have a monthly income as compared to the saudi (32%) respondents (p < 0.001). there was a greater proportion of non-saudi respondents (24%) who had a postgraduate degree as compared to the saudis (13%), while a greater proportion of saudis (42%) had education up to high school as compared to the non-saudi (33%) respondents (p < 0.001). table 2 shows the comparison of the bmi, exercise frequency, gad-7, and psqi scores between the saudi and nonsaudi respondents. there was no difference between the two table 1. demographic characteristics of the participants (n = 1123) nationality p-valuesaudi (n = 544) non-saudi (n = 579) n % n % age 18–29 yrs 231 42% 236 41% 0.6330–44 yrs 206 38% 216 37% 45+ yrs 107 20% 127 22% gender female 413 76% 383 66% <0.001 male 131 24% 196 34% marital status (3 groups) single 227 42% 222 38% 0.02married 290 53% 343 59% divorced/widowed 27 5% 14 2% employment status yes 250 46% 228 39% 0.03 no 294 54% 351 61% monthly income <10,000 sr 104 19% 73 13% <0.001 10,000 to <20,000 sr 100 18% 46 8% 20,000 + sr 62 11% 58 10% i prefer not to answer 104 19% 131 23% i do not have a monthly income 174 32% 271 47% highest degree high school, diploma, or less 230 42% 193 33% <0.001bachelors degree 244 45% 246 42% postgraduate degree 70 13% 140 24% table 2. bmi categories and exercise frequencies of the participants and outcome from gad-7 and psqi questionnaires (n = 1123) nationality p-valuesaudi (n = 544) non-saudi (n = 579) n % n % bmi (kg/m2) underweight (<18.5) 28 5% 25 4% 0.87 normal weight (18.5 to <25) 191 35% 197 34% overweight (25 to <30) 170 31% 187 32% obese (30+) 153 28% 169 29% exercise none 122 22% 136 23% 0.89 1–4 times per month 209 38% 224 39% 2–3 times per week 158 29% 168 29% 4–6 times per week 55 10% 51 9% gad7 minimal anxiety 187 34% 202 35% 0.03 mild anxiety 170 31% 156 27% moderate anxiety 110 20% 103 18% severe anxiety 77 14% 118 20% psqi score mean ± sd 8.7 ± 3.5 8.1 ± 3.3 0.005 238 j contemp med sci | vol. 7, no. 4, july-august 2021: 235–241 obesity, quality of sleep and anxiety in saudi arabia original n. alhusseini et al. groups with regards to bmi (p = 0.87) and exercise frequency (p = 0.89). the proportion of respondents who were normal weight was 35% in saudi and 34% in non-saudi respondents, while overweight/obese were 59% and 61% respectively for the two groups. there were 60% saudis and 62% non-saudi respondents who reported no exercise/1–4 times per month, and 29% in both groups reported exercising 2–3 times per week. there was a significant difference for both the gad-7 and psqi scores between the saudis and non-saudis. the non-saudi respondents had a higher proportion (20%) of severe anxiety as compared to 14% in the saudi respondents. the psqi score was higher for the saudis (8.7 ± 3.5) as compared to the non-saudi (8.1 ± 3.3) respondents (p = 0.005). table 3 shows that in the saudi respondents, there was a significant association (p < 0.001) between the bmi groups and the gad-7 categories. it was found that the ‘normal weight’ group had the highest proportion (46%) of minimal anxiety as compared to 20% in the obese group. the obese group was found to have the highest proportion of severe (18%) anxiety as compared to the other three bmi groups, while the underweight group had the highest proportion of candidates with moderate anxiety (32%). there was no significant association found between bmi group and gad-7 for the non-saudi respondents (p = 0.28). table 4 shows an opposite result where there was no significant association between gad-7 categories and frequency of exercise for the saudi respondents (p = 0.36). but there was a significant association of gad-7 with exercise frequency for the non-saudi group (p = 0.02). the proportion of minimal anxiety was higher in respondents who exercised frequently table 3. association of bmi groups with gad-7 categories gad7 p-valuebmi kg/m2 n minimal anxiety mild anxiety moderate anxiety severe anxiety saudi (n = 544) underweight (<18.5) 28 10 5 9 4 <0.001 36% 18% 32% 14% normal weight (18.5 to <25) 191 87 46 35 23 46% 24% 18% 12% overweight (25 to <30) 170 60 52 37 21 35% 31% 22% 12% obese (30+) 153 30 67 29 27 20% 44% 19% 18% non-saudi (n = 579) underweight (<18.5) 25 7 4 4 10 0.28 28% 16% 16% 40% normal weight (18.5 to <25) 197 72 53 40 32 37% 27% 20% 16% overweight (25 to <30) 187 64 48 31 44 34% 26% 17% 24% obese (30+) 169 58 51 28 32 34% 30% 17% 19% table 4. association of exercise frequency with gad-7 categories gad7 exercise n minimal anxiety mild anxiety moderate anxiety severe anxiety p-value saudi none 122 40 39 27 16 0.36 33% 32% 22% 13% 1-4 times per month 209 63 67 49 30 30% 32% 23% 14% 2-3 times per week 158 62 50 27 19 39% 32% 17% 12% 4-6 times per week 55 22 14 7 12 40% 25% 13% 22% (continued) 239j contemp med sci | vol. 7, no. 4, july-august 2021: 235–241 n. alhusseini et al. original obesity, quality of sleep and anxiety in saudi arabia table 5. association of bmi, exercise and gad-7 categories with psqi scores (n = 1123) saudi (n = 540) non-saudi (n = 578) p-value mean ± sd p-value mean ± sd bmi (kg/m2) underweight (<18.5) 8.5 ± 3.0 0.12 6.4 ± 2.5 0.001 normal weight (18.5 to <25) 8.2 ± 3.6 7.8 ± 3.2 overweight (25 to <30) 9.0 ± 3.4 8.2 ± 3.3 obese (30+) 9.0 ± 3.6 8.8 ± 3.5 exercise none 8.9 ± 4.0 0.34 8.2 ± 3.5 0.03 1-4 times per month 8.8 ± 3.4 8.5 ± 3.2 2-3 times per week 8.3 ± 3.5 8.1 ± 3.4 4-6 times per week 9.3 ± 2.9 6.9 ± 3.1 gad7 minimal anxiety 7.2 ± 2.9 <0.001 6.5 ± 2.6 <0.001 mild anxiety 8.7 ± 3.2 7.9 ± 2.7 moderate anxiety 9.5 ± 3.6 9.9 ± 3.8 severe anxiety 11.3 ± 3.8 9.7 ± 3.4 i.e., 2–3 times per week (42%) or 4–6 times per week (49%) as compared to those who did no exercise (32%) or only 1–4 times per month (29%). the respondents who did not exercise or exercised less than four times a month had a higher proportion of mild anxiety (29% to 32%) as compared to those who exercised 2–3 times per week (22%) and 4–6 times per week (16%). for severe anxiety, the highest proportion (26%) was found in the group who did no exercise as compared to those who exercise 2–3 times per week (17%). table 5 shows the association between bmi, exercise, and gad-7 groups with the scores on the psqi questionnaire in saudis and non-saudis. in the saudi group, the bmi (p = 0.12) and frequency of exercise (p = 0.34) showed no significant association with the psqi scores. however, there was a significant association between these two categories and the psqi scores in the non-saudi group. it was found that the psqi scores were higher for respondents who were obese (8.2 ± 3.3) or overweight (8.8 ± 3.5) as compared to the underweight group (6.4 ± 2.5) (p = 0.001). non-saudi responders who exercised 4–6 times per week had a significantly lower psqi score (6.9 ± 3.1) as compared to the those who did no exercise (8.2 ± 3.5) or 1–4 times per month (8.5 ± 3.2) (p = 0.03). a significant association (p < 0.001) was found in both the saudi and the non-saudi group for the psqi scores by the gad-7 categories. in both groups, the respondents with minimal anxiety had the lowest psqi scores (7.2 ± 2.9 for saudis and 6.5 ± 2.6 for non-saudis). in saudis, those, with severe anxiety were found to have the highest psqi score (11.3 ± 3.8) while in non-saudis, the groups with moderate (9.9 ± 3.8) and severe (9.7 ± 3.4) anxiety both had higher scores as compared to the minimal anxiety group. discussion the main purpose of the study was to determine an association between obesity levels, quality of sleep, and anxiety among the residents of saudi arabia including saudis and non-saudis. saudi arabia is among the nations with the highest prevalence of overweight and obesity.22 our results are in concordance with the current literature as it showed a high proportion of overweight and obesity (32%) and (29%), respectively. there are many risk factors associated with the increased overweight and obesity levels in saudi arabia, including physical inactivity and a sedentary lifestyle.23 people’s lifestyles have changed dramatically over the years, from active daily table 4. association of exercise frequency with gad-7 categories—continued gad7 exercise n minimal anxiety mild anxiety moderate anxiety severe anxiety p-value non-saudi none 136 43 39 19 35 0.02 32% 29% 14% 26% 1-4 times per month 224 64 72 45 43 29% 32% 20% 19% 2-3 times per week 168 70 37 32 29 42% 22% 19% 17% 4-6 times per week 51 25 8 7 11 49% 16% 14% 22% 240 j contemp med sci | vol. 7, no. 4, july-august 2021: 235–241 obesity, quality of sleep and anxiety in saudi arabia original n. alhusseini et al. movement and proper dietary consumption to sedentary, inactive mobility and increased consumption of fat and sugar.24 upon dividing the saudi nationals from the non-saudis, there was no significant difference between the frequency of exercise in both groups. our findings revealed that the proportion of respondents who did not exercise frequently is more than half, which matches the literature regarding the low physical activity engagement in saudi arabia.25 although obesity and anxiety are individually common and widespread health problems, more research has been linking them together. an 11-year cohort study found that both men and women experiencing anxiety and depression had a significantly higher incidence of obesity.26 our results indicated a highly significant association between the bmi groups and the gad-7 categories in saudi nationals but not in the non-saudi respondents. dejesus et al. conducted a study with similar results to ours, indicating higher anxiety scores associated with very low and very high bmi levels in comparison to normal weight.27 another research in iran also supports that higher anxiety scores are associated with low weight.28 on the other hand, a study conducted among adults in 2016 showed higher anxiety scores for medium to high bmi levels and an inverted u-shaped association for both low and very high bmi levels with anxiety.29 for saudis, it was found that almost half the ‘normal weight’ group had the highest proportion of minimal anxiety as compared to about one-third in the obese group. the obese group was also found to have the highest proportion of mild and severe anxiety as compared to the other three bmi groups. however, the underweight group had the highest proportion of candidates with moderate anxiety. on the other hand, while both saudis and non-saudis had a similar bmi distribution, a lack of relationship between bmi and anxiety levels in non-saudis may imply that bmi might not be as relevant a risk to non-saudis as it is to saudis in developing anxiety. this could mean that other stressors and risk factors have a greater effect on non-saudis. this difference could be genetic or environmental, but since no research has tackled the causes and risk factors for anxiety in non-saudis as opposed to saudis, any reasoning provided would be purely speculative. additionally, exercise is used to manage anxiety symptoms.30 another study concluded that there is a positive association between anxiety and insufficient physical activity.31 this was demonstrated in our study in the non-saudi population. regarding obesity and quality of sleep, we found that the non-saudi respondents who were obese or overweight had significantly higher psqi scores as compared to the underweight group, which means poorer quality of sleep. a meta-analysis study (n = 164,016) suggests short sleep duration was significantly associated with the incidence of obesity.32 one of the studies concluded that additional wakefulness requires additional intake of food. however, due to the availability and easy access to food, the intake usually exceeded the requirement.33 moreover, another study suggested that sleep deprivation even impacts the food choices one makes. since sleep deprivation decreases the activity of the regions in the frontal cortex and the insular cortex responsible for the appetitive evaluation and increase the activity of the amygdala. these changes promoted the desire for high-calorie, obesity-inducing food.34 on the other hand, obesity has been shown to decrease sleep duration and overall sleep quality since it is a major risk factor for obstructive sleep apnea (osa). this can be seen as data suggests that osa is almost twice as prevalent in obese patients as compared to normal weight. the reason for this could be due to the fat deposition in the upper airway and thorax, reducing the lumen and compliance of the airway. as bmi increases, further fat deposition is seen. with the decreasing lumen, the oxygen saturation decreases.35 one study also showed a significant association between mean oxygen saturation and sleep duration.36 in our study however, the saudi sample did not reveal this association despite having a significantly higher mean psqi score than non-saudis, suggesting that other factors play a more important role in this poor quality of sleep seen in saudis. a cohort study exploring risk factors for developing excessive daytime sleepiness eds in 4,322 sweden women over a 10-year follow-up, 8% of them developed eds, the incidence mostly being related to anxiety, obesity, and insomnia—among other factors.37 another study found that sleep disturbances can lead to emotional dysregulation, which in turn leads to emotional eating, and thus to overweight and obesity.38 similarly, in our study, the association between anxiety and sleep can be demonstrated in both the saudi and the nonsaudi population. in both groups, the respondents with minimal anxiety had the lowest psqi scores. in saudis, those, with severe anxiety were found to have the highest psqi score while in non-saudis, the groups with moderate and severe anxiety both had higher scores. another meta-analysis study concluded that sleep deprivation-induced states of increased anxiety, while sleep restriction did not show a significant relationship. moreover, a longer period of sleep deprivation was a determinant of the increase in anxiety.39 a study among in puerto rican youth showed that sleeping less and having depressive/anxiety disorder were both independently and in combination associated with an increased risk of obesity.40 the cross-sectional nature of our study limits causal inferences. also, since it was an online questionnaire, recall bias is one of the major limitations, especially in reporting height and weight, hence, bmi levels. however, this study shows significant findings among the residents of saudi arabia. there are limited studies that focus on obesity, sleep and anxiety. in conclusion, there is a relation between the obesity, sleep, and anxiety trio, and a relation between the trio leading to other medical issues. low and high bmi levels were significantly associated with moderate and severe anxiety. also, overweight and obesity were associated with poor quality of sleep. some studies claim that anxiety is the moderator between obesity and sleep, in which the presence of anxiety symptoms contributes to difficulty falling or staying asleep, which then increases the risk for obesity, or that poor sleep leads to anxiety, which leads to emotional eating and obesity.38 obesity is a major problem in saudi arabia and must be given extreme attention from healthcare providers and policymakers to decrease its levels and prevent any negative consequences associated with it. conflict of interest the authors declare no conflict of interest. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.  241j contemp med sci | vol. 7, no. 4, july-august 2021: 235–241 n. alhusseini et al. original obesity, quality of sleep and anxiety in saudi arabia this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1. purnell jq. definitions, classification, and epidemiology of obesity. endotext [internet]: mdtext. com, inc.; 2018. 2. djalalinia s, qorbani m, peykari n, kelishadi r. health impacts of obesity. pakistan journal of medical sciences. 2015;31(1):239. 3. casadei k, kiel j. anthropometric measurement. statpearls [internet]: statpearls publishing; 2020. 4. arroyo-johnson c, mincey kd. obesity epidemiology worldwide. gastroenterology clinics. 2016;45(4):571-9. 5. al-hussaini a, bashir ms, khormi m, alturaiki m, alkhamis w, alrajhi m, et al. overweight and obesity among saudi children and adolescents: where do we stand today? saudi journal of gastroenterology: official journal of the saudi gastroenterology association. 2019;25(4):229. 6. al-ghamdi s, shubair mm, aldiab a, al-zahrani jm, aldossari kk, househ m, et al. prevalence of overweight and obesity based on the body mass index; a cross-sectional study in alkharj, saudi arabia. lipids in health and disease. 2018;17(1):134. 7. al-hazzaa hm. physical inactivity in saudi arabia revisited: a systematic review of inactivity prevalence and perceived barriers to active living. int j health sci (qassim). 2018;12(6):50-64. 8. ahmed ae, al-jahdali f, alalwan a, abuabat f, bin salih sa, al-harbi a, et al. prevalence of sleep duration among saudi adults. saudi med j. 2017;38(3):276-83. 9. buysse dj. sleep health: can we define it? does it matter? sleep. 2014;37(1):9-17. 10. worley sl. the extraordinary importance of sleep: the detrimental effects of inadequate sleep on health and public safety drive an explosion of sleep research. p t. 2018;43(12):758-63. 11. panel cc. recommended amount of sleep for a healthy adult: a joint consensus statement of the american academy of sleep medicine and sleep research society. sleep. 2015;38(6):843-4. 12. goel n, basner m, rao h, dinges df. circadian rhythms, sleep deprivation, and human performance. progress in molecular biology and translational science. 119: elsevier; 2013. p. 155-90. 13. landolt h-p, holst sc, sousek a, bassetti c, dogas z, peigneux p. effects of acute and chronic sleep deprivation. 2014. 14. cooper cb, neufeld ev, dolezal ba, martin jl. sleep deprivation and obesity in adults: a brief narrative review. bmj open sport & exercise medicine. 2018;4(1). 15. roberge p, normand-lauzière f, raymond i, luc m, tanguay-bernard m-m, duhoux a, et al. generalized anxiety disorder in primary care: mental health services use and treatment adequacy. bmc family practice. 2015;16(1):146. 16. crocq m-a. the history of generalized anxiety disorder as a diagnostic category. dialogues in clinical neuroscience. 2017;19(2):107. 17. stein mb, sareen j. generalized anxiety disorder. new england journal of medicine. 2015;373(21):2059-68. 18. lykouras l, michopoulos j. anxiety disorders and obesity. psychiatriki. 2011;22(4):307-13. 19. sapra a, bhandari p, sharma s, chanpura t, lopp l. using generalized anxiety disorder-2 (gad-2) and gad-7 in a primary care setting. cureus. 2020;12(5). 20. buysse dj, reynolds iii cf, monk th, berman sr, kupfer dj. the pittsburgh sleep quality index: a new instrument for psychiatric practice and research. psychiatry research. 1989;28(2):193-213. 21. weir cb, jan a. bmi classification percentile and cut off points. 2019. 22. ss ma. a review of prevalence of obesity in saudi arabia. j obes eat disord. 2016;2(2):25. 23. alharbi c, jackson r, editors. physical activity and the prevalence of general and abdominal obesity among saudi women of reproductive age in jeddah2017. 24. algahtani fd. healthy lifestyle among ha'il university students, saudi arabia. int j pharm res allied sci. 2020;9(1):160-7. 25. alahmed z, lobelo f. physical activity promotion in saudi arabia: a critical role for clinicians and the health care system. journal of epidemiology and global health. 2018;7:s7-s15. 26. brumpton b, langhammer a, romundstad p, chen y, mai x-m. the associations of anxiety and depression symptoms with weight change and incident obesity: the hunt study. international journal of obesity. 2013;37(9):1268-74. 27. dejesus rs, breitkopf cr, ebbert jo, rutten ljf, jacobson rm, jacobson dj, et al. associations between anxiety disorder diagnoses and body mass index differ by age, sex and race: a population based study. clinical practice and epidemiology in mental health: cp & emh. 2016;12:67. 28. javadi m, jourabchi z, shafikhani aa, tajik e. prevalence of depression and anxiety and their association with body mass index among high school students in qazvin, iran, 2013–2014. electronic physician. 2017;9(6):4655. 29. haghighi m, jahangard l, ahmadpanah m, bajoghli h, holsboer-trachsler e, brand s. the relation between anxiety and bmi–is it all in our curves? psychiatry research. 2016;235:49-54. 30. kandola a, vancampfort d, herring m, rebar a, hallgren m, firth j, et al. moving to beat anxiety: epidemiology and therapeutic issues with physical activity for anxiety. curr psychiatry rep. 2018;20(8):63. 31. wang h, fu j, lu q, tao f, hao j. physical activity, body mass index and mental health in chinese adolescents: a population based study. the journal of sports medicine and physical fitness. 2014;54(4):518. 32. wu y, zhai l, zhang d. sleep duration and obesity among adults: a metaanalysis of prospective studies. sleep medicine. 2014;15(12):1456-62. 33. markwald rr, melanson el, smith mr, higgins j, perreault l, eckel rh, et al. impact of insufficient sleep on total daily energy expenditure, food intake, and weight gain. proceedings of the national academy of sciences. 2013;110(14):5695-700. 34. greer sm, goldstein an, walker mp. the impact of sleep deprivation on food desire in the human brain. nature communications. 2013;4(1):1-7. 35. romero-corral a, caples sm, lopez-jimenez f, somers vk. interactions between obesity and obstructive sleep apnea: implications for treatment. chest. 2010;137(3):711-9. 36. risso tt, poyares d, rizzi cf, pulz c, guilleminault c, tufik s, et al. the impact of sleep duration in obstructive sleep apnea patients. sleep and breathing. 2013;17(2):837-43. 37. theorell-haglöw j, åkerstedt t, schwarz j, lindberg e. predictors for development of excessive daytime sleepiness in women: a populationbased 10-year follow-up. sleep. 2015;38(12):1995-2003. 38. nguyen-rodriguez st, mcclain ad, spruijt-metz d. anxiety mediates the relationship between sleep onset latency and emotional eating in minority children. eating behaviors. 2010;11(4):297-300. 39. pires gn, bezerra ag, tufik s, andersen ml. effects of acute sleep deprivation on state anxiety levels: a systematic review and meta-analysis. sleep medicine. 2016;24:109-18. 40. koinis-mitchell d, rosario-matos n, ramírez rr, garcía p, canino gj, ortega an. sleep, depressive/anxiety disorders, and obesity in puerto rican youth. journal of clinical psychology in medical settings. 2017;24(1):59-73. https://doi.org/10.22317/jcms.v7i4.1039 153j contemp med sci | vol. 2, no. 8, autumn 2016: 153–157 research perceived stigma and treatment-seeking behavior in individuals with substance use disorder in baghdad qahtan q. mohammed department of psychiatric mental health nursing, college of nursing, baghdad university, iraq. correspondence to qahtan q. mohammed (email: qahtan_82@yahoo.com). (submitted: 3 september 2016 – revised version received: 29 september 2016 – accepted: 8 october 2016 – published online: 26 december 2016) objectives the present study aims at assessing the treatment-seeking behavior and perceived stigma among individuals with substance use disorder in baghdad; identifying the impact of perceived stigma upon treatment-seeking behavior; and determining the relationship between perceived stigma and socio-demographic characteristics of substance abusers. methods a descriptive, analytical study was established for the period from january to august, 2016. the study was conducted on a purposive sample of (50) substance abusers who are attending substance abuse rehabilitation centers at a teaching hospital in baghdad. the instrument of the study is adopted and modified for the purpose of this study. two scales were used in this study which is the treatment needs and motivation scale (tcu motform), and perceived stigma of substance abuse scale (psas). a self-administered report was applied as a mean of data collection. the data were analyzed through the application of descriptive and inferential statistical approaches which are applied by using ibm/spss package version 20.0. results the results of the present study showed that substance abusers were single (52%) with age ranged between 20–29 years old (54%), with barely sufficient monthly income (48%), and they do not work (40%). 72% of them were living in a high class neighborhood and they were substances abuser for more than two years duration (80%). the individuals with substance use disorder are showing a fair level of treatment-seeking behavior and they perceived moderate level of stigma. there is no significant impact of the stigma and sociodemographic upon the treatment-seeking behavior. and also, there is no significant relationship between perceived stigma and sociodemographic characteristics of individuals with substance use disorder. conclusions the study concluded that perceived stigma among substance abusers has no impact upon their treatment-seeking behavior, and also, perceived stigma among substance abusers is insignificantly correlated with their socio-demographic variables. keywords perceived stigma, treatment-seeking behavior, substance use disorder introduction substance use disorder is an important issue in the mental health aspect that is associated with many social problems such as poverty, crime situations, risky behaviors and stigmatization.1 the global status report on alcohol and health by the world health organization (2011) recognized alcohol as a major contributing factor to disease, death, and injury.2 by reason that substance use induction and maintenance is regularly viewed under the control of the person, the individuals with substance use disorder are more probable to be blamed for their condition. so, substance use disorder is associated with significant stigma.1 stigma is defined as “a mark signifying deviancy and by the presence of a deeply discrediting attribute”.3 stigma can be reported in a socio-cultural process in which specific groups in the society are undervalued, rejected and discriminated.4 stigma can be assumed at different levels, which are personal, social, and structural level.5 the personal level is viewed by perceived self stigma,6 the social level is viewed by the expression of stereotype and prejudice by the public towards individual with particular health conditions,7 and the structural level is viewed by the seclusion of a particular group at the level of economic and political policies.8 treatment seeking behavior can be defined by the individuals’ behavior for getting appropriate treatment that they perceive themselves as having health problems.9 treatment seeking behavior can be used as an indicator of an individual’s readiness to maintain life, and is important to personal, public and social development.10 the utilization of health care services can be reported as a type of individuals’ behavior that is elucidated in term of individual-environment interaction.11 as a result of cultural and socioeconomic differences in term of illness perception, treatmentseeking behavior can be affected by cultural beliefs about the illness.10 choosing the treatment source can be affected by different factors such as services accessibility, type and severity of illness, and individual socio-demographic characteristics.12 a stigma has been reported as one of the major barriers to treatment seeking among a scope of psychiatric disorders.13 substance use disorder is one of these psychiatric disorders that affect a significant portion of the population. due to associated social and economic problems with substance use disorder, substance users are reported with significant stigma that may differ from other health problems.14 stigma towards individual with substance use disorder provokes social alienation and has an impact on multiple domains of life, such as employment status, housing, and social relationships. so, the outcome of stigma for individuals with substance use disorder encompasses poor mental and physical health, delayed treatment seeking and non-adherence to treatment.5 while there is increasing evidence for documenting the damaging effects of mental illness stigma on psychological wellbeing, few studies have been shown on examining the effects of stigma in specific psychiatric disorders such as the stigma towards substance use disorder that has been studied rarely. however, the stigmas towards substance use disorder showing various negative consequences on substance users.3 in this paper, the researcher is trying to predict the impact of stigma upon treatment seeking behavior among substance issn 2413-0516 154 j contemp med sci | vol. 2, no. 8, autumn 2016: 153–157 perceived stigma and treatmentseeking behavior in individuals research qahtan q. mohammed users in which considered as one of the significant barriers that affect on treatment seeking phenomenon. materials and methods design of the study: a descriptive, analytical study was carried out for the period from january to august, 2016, in which assessment technique was used and applied in order to achieve the objectives of the current study. sample of the study: the study conducted on a purposive (non-probability) sample of 50 clients with substance use disorder. setting of the study: the study was conducted on the individuals with substance use disorder who are attending substance abuse rehabilitation centers at teaching hospital in baghdad, which are ibn-rushd psychiatric teaching hospitals and baghdad teaching hospital. the administrative arrangement for conducting the present study has been obtained from ministry of health /alrusafa health directorate/ibn-rushd psychiatric teaching hospital and medical city directorate/ baghdad teaching hospital. the consent facilitated the researcher’s entrance into the centers, meeting the clients and obtaining the agreement of them to participate in the present study. ethical consideration has been granted by the ethical committee for research/ college of nursinguniversity of baghdad after reviewing the content of the questionnaire. instrument of the study: the study scales were adopted and modified by the researcher; two scales were used in this study; the first scale was treatment needs and motivation (tcu motform),15 and the second scale was perceived stigma of substance abuse scale (psas). 16 the questionnaire of the study was consisted of four parts: the first part was the covering letters for obtaining the agreement to participate in the study, the second part was the socio-demographic characteristics of the clients, the third part was (tcu motform), and the fourth part was (psas). three domains of tcu motform scale were selected in order to measure the treatment seeking behavior, which are consisted of (22) items which are rated into five likert scale and scored as follows: 1 = strongly disagree, 2 = disagree, 3 = uncertain, 4 = agree, and 5 = strongly agree. the levels of treatment-seeking behavior were estimated by calculating the cut off points for the total score of the scale, which are rated in three levels and scored as follows: poor = 22–51, fair = 52–81, and good = 82–110. the psas scale was used to measure the stigma, the scale consists of (8) items. these items were rated in five -likert scale. the items scored as follows: 1 = strongly disagree, 2 = agree, 3 = neutral, 4 = agree, and 5 = strongly agree, the items 1, 2, 3, 4, 6, and 8 are reversely coded. the levels of stigma were estimated by calculating the cut off points for the total score of the scale as follows: mild = 8–18, moderate = 19–29, and severe = 30–40. data collection: a self-administered report was applied as a mean of data collection. the questionnaire was distributed to the clients after being willing to answer the questionnaire and participate in the study. the time consumed for filling the questionnaire was approximately 20–35 minutes. statistical analysis: the data were analyzed through the application of descriptive and inferential statistical approaches which are applied by using ibm/spss package version 20.0. the statistical procedures that are used for analyzing the data were: frequencies, percentages, mean of the score, cutoff point, linear simple regression, and correlation coefficient. results this table indicated that more than half of the sample were single (52%) with age ranged between 20 and 29 years old (54%), 48% of them reported barely sufficient monthly income, and they do not work (40%). 72% of them were living in a high-class neighborhood, and they were substances abuser for more than 2 years duration (80%). this table revealed that individuals with substance use disorder are showing a fair level of treatment-seeking behavior (64%). this table shows that individuals with substance use disorder perceive moderate level of stigma (80%). the analysis of simple linear regression revealed that the stigma and socio-demographic characteristics have no impact on the treatment-seeking behavior in individuals with substance use disorders at p-value ≤ 0.05. this table showed that there is no significant relationship between perceived stigma and socio-demographic characteristics of individuals with substance use disorder. discussion the analysis of the socio-demographic in table 1 reveals that substance users were single, 20–29 years old with barely sufficient monthly income, who doesn’t work. and living in a high class neighborhood and they are using substances for more than two years duration. the prevalence of substance use disorder at the age range of 20–29 years old who are single can be explained that this category is more prone to the peer pressure, which is considered as the most important factor among youth; youth have the readiness to learn certain behavior, therefore peer pressure is a potent factor in the initiation of substance use at this age group. in addition to the peer pressure factor, the experimentation, circumstantial situations and recreation also play an important role in the prevalence of substance use in this age group and social status. a study presented to support evidence for this result that found lamptey17 who found that substance use disorder is prevalent among unmarried youth with age group 20–29 years old. regarding monthly income and occupational status results, it can be inferred that substance use disorder is more prevalent in people who doesn’t work which are usually associated with moderate to low income; with one hand, they can’t work and get a job due to the effects of substances that they are used. on the other hand, as a result of their jobless status, they will be associated with low income due to the cost of getting these substances. samsa18 reported that the current illicit drug use differed by employment status. it is also reported that the rate of drug use was higher for unemployed person that for those who were employed.19 milson et al.20 have found that alcohol and drug abuse were common among persons with lower income than the person with a higher income. regarding the findings of residence and duration of substance uses, the explanation of such findings can be presented in the factor of residence; it has been known that most of the clients were living in high class neighborhoods which are usually openminded. according to our culture, there is difference among people who are living in a different neighborhood. usually, high class neighborhood is characterized by social openness in which individuals may experiment substance uses, such as alcohol in the local public occasions. a study presented qahtan q. mohammed 155j contemp med sci | vol. 2, no. 8, autumn 2016: 153–157 research perceived stigma and treatmentseeking behavior in individuals usually has negative attitudes toward substance in term of immoral behaviors; violent, aggressive, and criminal behaviors are usually associated with those abusers. so, they are stigmatized by the public, this stigmatization is understood by discrimination towards them, which have led to perceiving selfstigma among them. many studies have presented supporting evidence to this finding that found moderate to high level of stigma was associated with substance use disorder.23–25 the analysis of table 4 reveals that the stigma and sociodemographic characteristics have no impact on the treatment-seeking behavior in individuals with substance use disorders. the current findings were coming against the hypothesis of the researcher. the explanation of such finding is that substance abusers are highly motivated to treatment, therefore, their feeling of shame and stigma are less, considering that substance use disorder is less severe than other mental illness. on the other hand, another reason should be considered in finding that such is the coping level that have adopted by those substance abusers that depends on their consciousness of the problem. the current finding was inconsistent with many studies8,26,27 who reported table 1. characteristics of the individual with substance use disorder no. characteristics f % 1 age group: ≤ 19 years 6 12 20–29 year 27 54 30–39 year 13 26 40–49 year 1 2 50 ≤ year 3 6 total 50 100 2 marital status: single 26 52 married 18 36 divorced 3 6 widowed 3 6 total 50 100 3 monthly income: insufficient 12 24 barely sufficient 24 48 sufficient 14 28 total 50 100 4 occupation: doesn’t work 20 40 retired 3 6 employer 11 22 free works 16 32 total 50 100 5 residence: high class neighborhood 36 72 low class neighborhood 14 28 total 50 100 6 duration of substance use: less than 2 years 10 20 more than 2 years 40 80 total 50 100 no, number; f, frequency; %, percentage. supportive evidence that found al-zaiady21 who found similar results. table 2 reveals that individuals with substance use disorder are showing a fair level of treatment-seeking behavior. such findings may be explained that individual with substance abuse have the moderate motivation for the treatments. many factors play an important role in seeking the treatment among substance abusers; the most important are family and social factors. however, health and treatment seeking is influenced by the social stigma of the population towards substance abusers, therefore, most of them seeking treatment only when they aware of the symptom severity that they experiencing as a result of prolonged substance abuse. a study presented supportive evidence for this result that found goteborg22 in his study. the analysis of findings regarding the perceived stigma of substance use disorder table 3, it shows that substance abusers having a moderate level of stigma. substance use disorder is considered as less severe than mental illness, in which the clients are able to take an action in their treatment of themselves, however, the stigma is usually reported by those who are undergoing for the treatment of substance abusing. the public table 2. treatment-seeking behavior among individuals with substance use disorder levels of behavior f % m.s poor (22–51)* 2 4 2.28 fair (52–81)* 32 64 good (82–110)* 16 32 total 50 100 no, number; f, frequency; %, percentage; ms, mean of score; *, cutoff point. table 3. perceived stigma in individuals with substance use disorder levels of stigma f % m.s mild (8–18)* 6 12 1.86 moderate (19–29)* 40 80 severe (30–40)* 4 8 total 50 100 no, number; f, frequency; %, percentage; ms, mean of score; *, cutoff point. table 4. the impact of stigma and socio-demographic characteristics upon treatmentseeking behavior among individuals with substance use disorder (n = 50) independent variable unstandardized coefficients standardized coefficients t sig. b std. error beta stigma 0.093 0.381 0.039 0.245 0.808 age –1.827 2.805 –0.140 –0.651 0.518 marital status 2.212 2.433 0.185 0.909 0.369 monthly income 2.027 2.915 0.120 0.695 0.491 occupation 0.465 1.318 0.055 0.352 0.726 residence 4.139 4.862 0.152 0.851 0.400 duration of substance use –2.806 5.081 –0.092 –0.552 0.584 dependant variable: treatment-seeking behavior; b, beta; std, standard; t, statistical test; sig, significance. 156 j contemp med sci | vol. 2, no. 8, autumn 2016: 153–157 perceived stigma and treatmentseeking behavior in individuals research qahtan q. mohammed table 5. the relationship between perceived stigma and socio-demographic characteristics of the individual with substance use disorder (n = 50) correlation age marital status monthly income occupation residence duration of substance use stigma age 1.00 marital status 0.673** 1.00 monthly income –0.051 0.015 1.00 occupation 0.192 0.161 0.171 1.00 residence –0.097 0.040 0.522** 0.282* 1.00 duration of substance use 0.407** 0.304* 0.028 –0.014 –0.134 1.00 stigma 0.038 –0.168 –0.185 0.067 –0.121 –0.164 1.00 **, correlation is significant at the 0.01 level (2-tailed); *, correlation is significant at the 0.05 level (2-tailed). that stigma has negative consequences on consuming health services and is a main barrier for treatment seeking. despite of perceived stigma is a relatively understudied construct in the addictive research articles; more attention has been received from the research community in recent years. so, there is significant evidence that supports the notion that substance abusers experience stigma and are negatively affected by its consequences.28,29 surprisingly and contradictory to prior preliminary results, our findings table 5 suggest that there is no significant relationships among perceived stigma and sociodemographic characteristics of substance abusers. the explanation of insignificant relationships between the stigma and the variable can be summarized depending on the descriptive analysis of the socio-demographic characteristics for substance abusers. single youth abusers may view their disorders as a general medical condition; especially they have no social responsibilities. most of the clients with substance abuse disorder are conscious enough about their disorder; they usually express their regret for their situation and the negative consequences that they get. the substance abusers are aware of their jobless and low socioeconomic status. for the reasons above, they may perceive stigma moderately that is insignificant related to their sociodemographic characteristics. a study presented supportive evidence for this result that found mattoo et al.5 that was found inverted significant relationship in his study. conclusions 1. substance abusers have self-awareness for seeking treatment for their disorder presented in their treatment-seeking behavior. 2. the stigma is moderately perceived by substance abusers. 3. perceived stigma among substance abusers has no impact on their treatment-seeking behavior. 4. perceived stigma among substance abusers is insignificantly correlated with their socio-demographic variables. recommendations 1. awareness program should be conducted through the media and sessions about treatment and negative consequences of substance use disorder. 2. a qualitative and qualitative research should be conducted to explore the difference in various substance uses and examine their effects on seeking treatment. 3. further research should be conducted to examine the effect of other dimensions of stigma among substance abusers. conflicts of interest there are no conflicts of interest. n references 1. mattoo s, sarkar s. validation of hindi version of perceived stigma of substance abuse scale, indian j soc psychiatry. 2012;28:117–120. 2. world health organization, global status report on alcohol and health, 2011, retrieved from http://www.who.int/substance_abuse/publications/ global_alcohol_report/en/ 3. janulis p. understanding addiction stigma: examining desired social distance toward addicted individuals, a thesis submitted to department of psychology, college of liberal arts and science, depaul university. 2010:1–20. 4. weiss mg, ramakrishna j, somma d. health-related stigma: rethinking concepts and interventions, psychol health med. 2006;11:277–287. 5. mattoo s, sarkar s, gupta s, naresh n, parakh p, basu d. stigma towards substance use: comparing treatment seeking alcohol and opioid dependant men. int j ment health addict. 2015;13:73–81. 6. brohan e, slade m, clement s, graham t. measuring stigma and discrimination related to mental illness. in g. thornicroft (ed.), mental health outcome measures, rcpsych publications. 2010. 7. corrigan p. how stigma interferes with mental health care. am psychol. 2004;59:614–625. 8. corrigan pw, kuwabara sa, o’shaughnessy j. the public stigma of mental illness and drug addiction. j soc work. 2009;9:139–147. 9. wade dt, halligan pw. do biomedical models of illness make for good healthcare systems? bmj. 2004;329:1398–1401. 10. bourne pa. impact of poverty, not seeking medical care, unemployment, inflation, self-reported illness, and health insurance on mortality in jamaica. north am j med sci. 2009;1:99. 11. atashbahar o, bahrami m, asqari r, fallahzadan h. an examination of treatment seeking behavior affecting factors: a qualitative study in iran, world appl sci j. 2013;25:774–781. 12. malik em, hanafi k, ali sh, ahmed es, mohamed ka. treatment-seeking behavior for malaria in children under five years of age: implication for home management in rural areas with high seasonal trans-mission in sudan. malar j. 2006;5:60. 13. wahl of. stigma as a barrier to recovery from mental illness, trends cogn sci. 2012;16:9–10. 14. schomerus g, lucht m, holzinger a, matschinger h, carta mg, angermeyer mc. the stigma of alcohol dependence compared with other mental disorders: a review of population studies. alcohol alcohol. 2011;46:105–112. 15. institute of behavioral research, tcu short forms/08 (sf) motform, institute of behavioral research, fort worth, texas, 2007. qahtan q. mohammed 157j contemp med sci | vol. 2, no. 8, autumn 2016: 153–157 research perceived stigma and treatmentseeking behavior in individuals 16. luoma jb, o’hair ak, kohlenberg bs, hayes sc, fletcher l. the development and psychometric properties of a new measure of perceived stigma toward substance users. subst use misuse. 2010;45:47–57. 17. lamptey jj. socio-demographic characteristics of substance abusers admitted to a private special clinic, ghana med j. 2005;39:2–7. 18. samsa, office of applied studies, drug abuse warning network, national estimates of drug-related emergency department visits, dawn series d-30, dhhs publication (sma) 08-4339, rockville, md, 2006, retrieved from http://dawninfo.samsa.gov. 19. samsa, results from the national survey on drug use and health: summary of national findings, 2013. retrieved from http//store.samsa.gov/home. 20. milson p, challacombe l, villeneuve p, fischer b, et al. self-perceived health among canadian opiate users: a comparison to the general population and to other chronic disease populations, can j pub health. 2004;95:99–103. 21. al-zaiady s. health-related quality of life of substance abusers in baghdad, a thesis submitted to department of mental health nursing, college of nursing. baghdad university. 2014:70–89. 22. goteborg a. characteristics and processes of treatment-seeking for alcohol problems, a thesis submitted to the sahlgrenska academy, göteborg university, 2007. 23. luoma jb, twohig mp, waltz t, hayes s, roget n, padiilla m, fisher g. an investigation of stigma in individuals receiving treatment for substance abuse. addict behav. 2007;32:1331–1346. 24. arsenault b. the stigmatization of mental illness and drug addiction among the criminally involved, a thesis submitted to the faculty of the graduate school in candidacy for the degree of master of arts, loyola university chicago. 2011:30–44. 25. myers b, fakier n, louw j. stigma, treatment beliefs, and substance abuse treatment use in historically disadvantaged communities. afr j psychiatry. 2009;12:218–222. 26. rasinksi ka, woll p, cooke a. stigma and substance use disorders, in p.w. corrigan (ed.), on the stigma of mental illness: practical strategies for research and social change, 2005. washington, dc: american psychological association. 27. woods j. methadone advocacy. the mount sinai journal of medicine. 2001;68:75–78. 28. corrigan p, watson a, barr e. the self-stigma of mental illness: implications for self-esteem and self-efficacy. j soc clin psychol. 2006;25:875–884. 29. room r. stigma, social inequality and alcohol, and drug use, drug alcohol rev. 2005;24:143–155. 340 j contemp med sci | vol. 7, no. 6, november-december 2021: 340–345 original common clinical symptoms and concomitant disease in celiac patients – a large cohort study in the north-east of iran azita ganji1, , kiarash roustai geraylow2, , bijan shahbazkhani3, , fahimeh attarian*4, 1department of gastroenterology and hepatology, faculty of medicine, mashhad university of medical sciences, mashhad, iran. 2faculty of medicine, semnan university of medical sciences, semnan, iran. 3department of gastroenterology and liver disease, tehran university of medical sciences, tehran, iran. 4department of public health, school of health, torbat heydariyeh university of medical sciences, torbat heydariyeh, iran. *correspondence to: fahimeh attarian (e-mail: attarian581@gmail.com) (submitted: 12 september 2021 – revised version received: 29 september 2021 – accepted: 21 october 2021 – published online: 26 december 2021) introduction celiac disease is an autoimmune disorder characterized by mucosal injury in response to gluten in genetically predisposed individuals.1 cd accounts for about 1% of the population worldwide and is rapidly increasing because of availability of serologic tests which have high sensitivity and specificity. the clinical manifestations of cd can vary from asymptomatic cd to classic or typical intestinal symptoms such as diarrhea, weight loss, and abdominal pain and atypical or non-classical symptoms including iron deficiency, bloating, constipation, chronic fatigue, headache, osteoporosis, neurologic disorders (e.g., depression and gluten ataxia), reproductive disorders (e.g., menarche disorders and menopausal disorders), and oral/cutaneous disorders (e.g., dermatitis herpetiform).2–5 by knowing wide variety of clinical presentation, early diagnosis and treatment of cd by gluten free diet (gfd) is important in patient out come and long term complications of disease. the prevalence of autoimmune diseases and malignancies has been reported to be higher in cd patients and even in their first-degree relatives.6–8 these patients are at risk of long-term complications, such as gastrointestinal and extra-intestinal malignancies and autoimmune disorders.6,9 up to 30% of concomitant immune-mediated diseases have been reported in cd patients compared to (3–11.6%) in the general population.2,6,9 moreover, cd has been described as a risk factor for enteropathy-associated t-cell lymphoma (eatl), non-hodgkin lymphoma, and gastrointestinal tumors and a protective factor for other types of malignancies such as breast and lung cancers.10–12 the etiology of this disease is multifactorial, including environmental (gluten and intestinal infections), genetic, and immunological factors with a high genetic susceptibility, proposed by studies on hla-dq2 and hla-dq8 haplotype associations with cd.7,10 a significant proportion of cd patients are diagnosed through screening at-risk populations such as family members of patients with cd and insulin dependent diabetics, but mostly remain undiagnosed.7,13,14 a combination of serology testing and duodenal biopsy (increase intraepithelial lymphocytes, crypt hyperplasia, and villous atrophy) is required to diagnose cd in adults.3,15,16 currently, the only approved treatment for cd patients is a strict gfd. improvement and resolution of symptoms occur after days or weeks after a gfd is implemented and often leads to normalization of serological markers and duodenal villous atrophy.1,3,17 there are studies about risk factor, clinical finding and presentation and association of cd with other disorders in cohorts all over the world.2,18,19 despite the high prevalence of cd in iran due to the high rate of gluten and bread consumption, no previous well-designed cohort study was conducted in this region.20 therefore, establishing the demographic characteristics and different clinical manifestations of this disease may provide important information regarding its timely diagnosis and proper management. to the best of our knowledge, this is the first large cohort study of cd patients done in the northeastern iranian province of khorasan razavi, extensively exploring the clinical manifestations and co-occurrence of immune-mediated diseases in these patients. abstract objectives: this study aimed to provides an overview of large cohort focusing in different clinical symptoms and concomitant disease in cd patients. methods: out of total 1,164 cases, 78.86% of cases were adults, mean age 29.67 ± 15.94 and sex ratio (f/m) was 2.40. evaluated clinical characteristics included bmi, marsh, tga-iga titer, main symptoms and concomitant disease. result: 96.86% of cd cases were seropositive and more than 88% of patients had villous atrophy that 48.89% of them were type c. dyspepsia 35.23%, diarrhea 18.52%, were main cd clinical symptoms. the common concomitant diseases were including nervous problems 57.43%, anemia 54.25%, osteopenia 28.23% and skin disease 23.54%. conclusion: the classic type is common type of cd in northeast iran. dyspepsia and diarrhea main clinical symptoms in these patients, consequence we recommended to screening for cd in cases whiteout classic symptoms (such as dyspepsia). also recommended to screening for concomitant disease such as nervous problems, bon disease and anemia in female cd patients in first visit. keywords: celiac disease, concomitant disease, main clinical symptoms issn 2413-0516 http://orcid.org/0000-0002-8161-6629 http://orcid.org/0000-0002-4366-6764 http://orcid.org/0000-0001-8484-7548 http://orcid.org/0000-0001-9752-0480 341j contemp med sci | vol. 7, no. 6, november-december 2021: 340–345 a. ganji et al. original common clinical symptoms and concomitant disease in celiac patients methods study setting & design this retrospective study includes a cohort of celiac patients registered in the celiac disease center of mashhad university of medical sciences (mums), located in the northeastern iran. this cohort was comprised of the records of all patients (with signs or symptoms of celiac) that were referred for diagnosis to the celiac disease center from khorasan and other neighboring provinces between 2010 and 2020. the data was analyzed using stata version 12 (stata, usa, 2009). the level of significance was less than 0.05 for all statistical tests. this study has been approved by the ethics committee of mashhad university of medical sciences (code: irmumsrec.1396.112). furthermore, a written informed consent has been obtained from all participants. study population study population was a cohort of 1164 patients with definite or clinical diagnosis of celiac disease. diagnosis of cd was ascertained by positive celiac serology (tga-iga) and marsh ≥ 2 histology on duodenal biopsy. tga-iga were assessed by enzyme-linked immunosorbent assay,14 kit (euro immune, germany) in a research laboratory. biopsy reports based on marsh and modified marsh oberhuber classification.21 seronegative cd cases was diagnosed based on mucosal atrophy and hla typing or challenge test after ruling out all other cause of mucosal atrophy including crohn’s, infection, radiation, hiv, cvid, malignancy and peptic deodenitis and drug use. iga deficiency patients were diagnosed based on pathology and igg base serology of tga and anti dgp. seropositive cd cases were included in this cohort without biopsy, if had raised tga-iga and response to gfd and compatible hla and typical presentation. also patients without villous atrophy were included, if they were symptomatic and had high serology titer more than 10 times of normal and compatible hla. data collection clinical and demographic variables were included age, gender, height, weight, symptoms, type of diseases (seropositive, seronegative or others), thyroid function, liver function and immune mediated diseases and other concomitant diseases such as skin problem and nervous problem were extracted from the database. main symptoms that assessed in these patients were including dyspepsia, diarrhea, weight loss, flatulence, reflux, abdominal pain and others symptoms. tga-iga levels were reported in unit per milliliter using the manufacturer’s supplied reference ranges: 0–20 negative, >20 positive. the highest standard in the assay was >200, (10 times upper limit of normal). results characteristics of the patients of the total of 1,164 cases, 78.86% of cases were adults (age 14 or older). 70.64% were female (n = 811) and sex ratio was (f/m) 2.40. the mean age at the time of the first visit was 29.67 ± 15.94 years (ranging from 1 to 76 years) and mean age not different between genders (male = 28.99, female = 29.95, p = 0.35). however 23.63% of patients (age >15 years old) had lower weight, but 48.49% of them had normal bmi (n = 353) (table 1), and fortunately only 3.88% of this cohort were smoker (n = 32). table 1. baseline characteristics of celiac patients variables total gender/female sig bmi (age >15 years) <19 172 (23.63) 122 (70.93) p = 0.10b 19–25 353 (48.49) 249 (70.54) 25–30 147 (20.19) 98 (66.67) >30 56 (7.69) 47 (83.93) pathology (marsh) 1 54 (5.12) 47 (87.04) p = 0.02a* 2 71 (6.73) 47 (66.20) 3 964 (88.15) 653 (70.29) 3 a 138 (17.95) 97 (70.29) p = 0.37b 3 b 253 (32.90) 169 (66.80) 3 c 376 (48.89) 270 (27.00) type of diseases seropositive cd 1111 (96.86) 784 (70.63) p = 0.87a seronegative cd 28 (2.44) 20 (71.43) iga deficiency cd 4 (0.35) 3 (75.00) others 4 (0.35) 2 (50.00) patients classification gastrointestinal 668 (61.12) 497 (74.40) p < 0.001b* non-gastrointestinal 396 (36.23) 264 (66.67) screening 29 (2.65) 15 (51.72) tga-iga titer 190.84 ± 121.01 (rang 0.8, 834) 191.04 ± 4.39 p > 0.05 afisher’s exact test, bpearson chi 2 test. 342 j contemp med sci | vol. 7, no. 6, november-december 2021: 340–345 common clinical symptoms and concomitant disease in celiac patients original a. ganji et al. in this cohort 1,111 (96.86%) patients were seropositive celiac cases (positive by serology) and 28 (2.44%) were seronegative cd, this status not related to genders (p = 0.87). the mean ± sd of tga-iga titer was 190.84 ± 121.01 (rang 0.8, 834), mean difference of tga-iga titer not significant between two genders (p > 0.05). based on the results of the biopsies, more than 88% (n = 930) of patients had villous atrophy and 48.89% of them were type c (n = 376 marsh 3c). this cohort classified to three presentations of celiac disease: typical 61.12%, atypical 36.23% and screened patients 2.65%, the presentations of cd related to gender (p < 0.001). the baseline characteristics of this celiac cohort are displayed in table 1. mostly patients (68.99%) presented by one clinical symptom and one third of them (n = 361) presented by 2 or more clinical symptoms at the time of diagnosis. the first common gi symptoms of cd were dyspepsia 22.26% and diarrhea 12.94%. growing problem 9.92%, weight loss 8.80% and anemia 7.51% were others first clinical symptoms in these patients. also dyspepsia 12.97%, diarrhea 5.58%, anemia 5.24% and constipation 2.49% were secondly main clinical symptoms in this cohort. the main symptoms (chief complaint) at the time of their diagnosis are shown in table 2. the nervous problems were the most concomitant disorders in celiac patients, as 57.76% of evaluated cases (n = 779) had a psychiatric disorder. anxiety disorder (9.07%) and depression (8.31%) were the most common type of these psychiatric disorders. unadjusted models show females more likely have nervous problems (or = 1.37, ci 95% 1.00, 1.87) than male patients. liver disease were seen in more than 22% of evaluated cases (n = 724), and this disorder not related to gender (or = 0.88, ci 95% 0.54, 1.16), the most common of liver disease, was nonspecific abnormal alanine aminotransferase (alt) (53.70%). osteopenia have seen in 28.23% of cd patients, although odds of female are more likely to bone loss than male (or = 1.51, ci 95% 1.01, 2.27). other common table 2. common clinical presenting symptoms in celiac patients variables main clinical symptoms first n % secondly n % dyspepsia 258 (22.26) 151 (12.97) diarrhea 150 (12.94) 65 (5.58) weight loss 102 (8.80) – flatulence 78 (6.73) – reflux 47 (4.06) – abdominal pain 42 (3.62) – nausea 25 (2.16) – ibs 17 (1.47) – vomiting 15 (1.29) – growing problem 115 (9.92) – constipation 35 (3.02) 29 (2.49) dermatitis 34 (2.93) – anemia 87 (7.51) 61 (5.24) others atypical symptoms 188 55 (4.73) without symptoms 53 (4.57) 803 (68.99) disease in this cohort were oral aphtha (n = 301, 31.85%), and this not related to gender of patients (or = 0.94, ci 95% 0.69, 1.27). more than half of evaluated cases (n = 485, 54%) were anemic, based on their laboratory tests; and odds of anemia in female were two time more than male (or = 2.09, ci 95% 1.56, 2.81). the skin problems was seen in 23.53% of cd patients, that were include dermatitis herpetiformis (3.98%) and others skin problems (19.55%). some concomitant immune and nonimmune disorders are shown in table 3. discussion this is the first large cohort study in the northeastern region of iran evaluating the clinical manifestations and concomitant conditions in 1,164 celiac disease patients. in this study, most of our cases were among adults with the mean first visit age of 29.67 years, ranging from 1 to 76. cd can present in all ages, and gluten intolerance can happen later in life even after age 60.22 as expected, cd was predominate in female in our cohort (f:m ratio 2.40).1,2 in our cohort common type of cd disease was gastrointestinal, interestingly, 2.65% of total cohort was diagnosed by cd screening, which is lower than previous reports (5–21%).2,4,23 the most common gi symptom was dyspepsia. which is different from the dominant presentation in children.24 these results are in line with other studies, which reported cd classic symptoms prevalence ranging from 34 to 85.2%.2,4,23,25 there are some reports of a notable trend of changing presentation of cd from classic to non-classic. for instance, reports in 1985 found 85.2% of patients to present with diarrhea, whereas in 2003–2013, presentation with diarrhea was estimated at 37.4%. this trend could be explained by the increasing awareness of clinicians from the atypical presentations of cd.2,4,25 in our study, only 12.94% had presented by diarrhea. in our study, 31.01% of patients reported more than one specific clinical symptoms of cd disease. the main symptom that presented as first or secondary clinical symptoms were include dyspepsia (35.23%), diarrhea (18.52%), anemia (12.75%), growing problems (9.92%), weight loss (8.80%), and flatulence (6.73%). dyspepsia is not fully investigated in the literature as a symptom of cd. a notable percentage of cd patients are diagnosed incidentally through endoscopy for dyspepsia. therefore, it could be concluded that our present knowledge of cd presentation is inadequate. although there is some evidence of a high prevalence of dyspepsia in cd patients, especially in our region, most previous cohorts have not considered dyspepsia as a cd symptom.26–29 the occurrence of other presentations in our cohort were lower than previous finding, estimating the prevalence for diarrhea, weight loss, and abdominal pain to be at 27–64%, 28–56%, and 30–34%, respectively.2,4,23,30 additionally, constipation and reflux were 5.51% and 4.06% although ranged between 1–13% and 12–14% in other studies.2,4,23 type of disease was seropositive in 96.86% of cd patients, 2.44% of cd patients was seronegative which is in line with other studies that estimated prevalence of seronegative patients between 1.7–5%.31,32 the prevalence of iga deficiency in our general population is not determined, but 0.35% of these patients had iga deficient. 343j contemp med sci | vol. 7, no. 6, november-december 2021: 340–345 a. ganji et al. original common clinical symptoms and concomitant disease in celiac patients table 3. concomitant diseases in related sex of celiac patients no. of evaluated patients type of diseases n % unadjusted or ci 95% (f/m) thyroid diseases n = 623 without disease 516 (82.83) 1 hypo thyroid 94 (15.09) 0.77 (0.49, 1.20) hyper thyroid 13 (2.09) skin problem n = 578 without disease 442 (76.47) 1 dermatitis herpetiformis 23 (3.98) 0.79 (0.52, 1.21) others skin problem 113 (19.55) nervous problem n = 779 without disease 338 (42.57) 1 depression 66 (8.31) 1.37 (1.00, 1.87)* anxiety disorder 72 (9.07) headache 44 (5.54) others 98 (12.34) undetected problems 176 (22.17) liver disease n = 724 without disease 562 (77.62) 1 1 alt abnormality 87 (12.02) 0.80 (0.54, 1.16) fatty liver 42 (5.80) 2 aih 14 (1.93) 3 pbc 5 (0.69) 4 psc 2 (0.28) others 12 (1.66) bone disease n = 627 without disease 450 (71.77) 1 osteopenia 177 (28.23) 1.51 (1.01, 2.27)* oral disease n = 945 without disease 644 (68.15) 1 aphthae 301 (31.85) 0.94 (0.69, 1.27) diabetes n = 1159 without disease 1092 (94.22) 1 type 1 67 (5.78) 1.32 (0.74, 2.35) anemia n = 894 without disease 409 (45.75) 1 anemic 489 (54.25) 2.09 (1.56, 2.81)* although most previous studies report 5–10% dh cases within cd patients, two larger studies reported percentages of 9.8%33 to 3.2%2 and decreasing prevalence of dh during last decade, probably because of early diagnosis of celiac disease. in our study 3.98% of patients had dh. there has been a lot of research done investigating the association of cd in other autoimmune disorders such as diabetes and hypothyroidism, but few studies evaluate the prevalence of such autoimmune disorders in cd patients. in this study prevalence of hypothyroidism was estimated 15.09%, which is in accordance with the current literature (10–30%).6,9,30,34 the association between diabetes type 1 and cd is widely explored, our study showed a high prevalence of diabetes type 1 in cd patients (5.78%), which is supported by previous findings (3.2 to 10%) of cd patients.2,4,23 clinical guidelines recommend screening for cd in patients with type 1 diabetes which results in a large proportion of cd patients being diagnosed through screening at-risk populations.35,36 thyroid disorders were seen in 17.18% of patients that hypothyroidism was common type of them. studies have shown autoimmune thyroid diseases to be found in between 2.4% to 40.4% of cd patients.2,7,37 the high prevalence of autoimmune disorders in cd patients can be explained by the sharing of a common genetic background in immunological pathways between cd and other immune-mediated diseases.34,38 in this cohort 22.38% of patients had liver disorders, which more than half of them had abnormal alt (12.02%). the simultaneous clinical presentation of cd with liver disorders is well-documented. cd can damage the liver directly or may be associated with other autoimmune liver disease. hypertransaminas is seen in (13–60%) of cd patients,39,40 even screening for cd is recommended for patients with autoimmune liver disease or chronic liver disease.41 in this cohort nervous problems were common concomitant disorders (57.43%) in cd patients that more reported in female than male (or = 1.37). the common nervous problems include anxiety disorder (9.07%) and depression (8.31%), although previous studies have higher estimate of anxiety disorder (of 41 to 84%) and depression (of 34 to 60%) in cd patients.42–44 few studies have evaluated bone diseases in cd patients. in our cohort, 28.23% of patients had osteopenia, odds of bone diseases more in female than male (or = 1.5). some previous studies have found an increased risk of osteopenia and fragility fractures in celiac patients.45–47 a meta-analysis 344 j contemp med sci | vol. 7, no. 6, november-december 2021: 340–345 common clinical symptoms and concomitant disease in celiac patients original a. ganji et al. on postmenopausal female and male with celiac disease reported the prevalence of osteopenia 39.6%.48 bone loss is explained through malabsorption of calcium and vitamin d, leading to secondary hyperparathyroidism and metabolic bone disease.49 in addition, anemia was common in more than half of cd patients, in accordance to previous reports (8–50%).4,23,30,45,50 also odds of anemia in female patients was twice more than male (or = 2.01). conclusion the classic type is common type of cd in northeast iran. dyspepsia, diarrhea and anemia main clinical symptoms in these patients, consequence we recommended to screening for cd in cases whiteout classic symptoms, such as dyspepsia. nervous problem, bon disease and anemia important concomitant disorders that more seen in female cd patients. acknowledgements thanks to the mashhad university of medical sciences for supporting this project. appendices list of abbreviations celiac disease (cd), irritable bowel syndrome (ibs), tissue transglutaminase (tga ), anti-tissue transglutaminase-iga (tga-iga), deamidated gliadin peptide (dgp), common variable immunodeficiency (cvid), gluten free diet (gfd), alanine amino transferase (alt), gastrointestinal (gi) ethics approval and consent to participate the study was approved by the ethics committee of mashhad university of medical sciences. consent for publication not applicable. competing interests the authors declare that they have no competing interests. funding none. authors’ contributions • conceptualization; a. g. • data curtain; ag • funding acquisition; a.g. • methodology; f.a. • data analysis; f.a. • supervision & validation; a.g. • roles/writing original draft; a.g.k.r, f.a., b.sh. • all authors have read and approved the final manuscript.  references 1. caio g, volta u, sapone a, leffler da, de giorgio r, catassi c, et al. celiac disease: a comprehensive current review. bmc medicine. 2019;17(1):1-20. 2. spijkerman m, tan il, kolkman jj, withoff s, wijmenga c, visschedijk mc, et al. a large variety of clinical features and concomitant disorders in celiac disease–a cohort study in the netherlands. digestive and liver disease. 2016;48(5):499-505. 3. lebwohl b, sanders ds, green ph. coeliac disease. the lancet. 2018;391(10115):70-81. 4. dominguez castro p, harkin g, hussey m, christopher b, kiat c, liong chin j, et al. changes in presentation of celiac disease in ireland from the 1960s to 2015. clinical gastroenterology and hepatology. 2017 2017/06/01/;15(6):864-71.e3. 5. reilly nr, fasano a, green ph. presentation of celiac disease. gastrointestinal endoscopy clinics. 2012;22(4):613-21. 6. lauret e, rodrigo l. celiac disease and autoimmune-associated conditions. biomed research international. 2013;2013. 7. neuhausen sl, steele l, ryan s, mousavi m, pinto m, osann ke, et al. co-occurrence of celiac disease and other autoimmune diseases in celiacs and their first-degree relatives. journal of autoimmunity. 2008;31(2):160-5. 8. stagi s, giani t, simonini g, falcini f. thyroid function, autoimmune thyroiditis and coeliac disease in juvenile idiopathic arthritis. rheumatology. 2005;44(4):517-20. 9. diamanti a, capriati t, bizzarri c, panetta f, ferretti f, ancinelli m, et al. celiac disease and endocrine autoimmune disorders in children: an update. expert review of clinical immunology. 2013;9(12):1289-301. 10. stein j, schuppan d. coeliac disease-new pathophysiological findings and their implications for therapy. visceral medicine. 2014;30(3):156-65. 11. tio m, cox mr, eslick gd. meta-analysis: coeliac disease and the risk of all-cause mortality, any malignancy and lymphoid malignancy. alimentary pharmacology & therapeutics. 2012;35(5):540-51. 12. ilus t, kaukinen k, virta lj, pukkala e, collin p. incidence of malignancies in diagnosed celiac patients: a population-based estimate. official journal of the american college of gastroenterology | acg. 2014;109(9):1471-7. pubmed pmid: 00000434-201409000-00025. 13. guandalini s, assiri a. celiac disease: a review. jama pediatrics. 2014;168(3):272-8. 14. mustalahti k, catassi c, reunanen a, fabiani e, heier m, mcmillan s, et al. the prevalence of celiac disease in europe: results of a centralized, international mass screening project. annals of medicine. 2010 2010/12/01;42(8):587-95. 15. schuppan d, zimmer kp. the diagnosis and treatment of celiac disease. deutsches ärzteblatt international. 2013;110(49):835. 16. tack gj, verbeek wh, schreurs mw, mulder cj. the spectrum of celiac disease: epidemiology, clinical aspects and treatment. nature reviews gastroenterology & hepatology. 2010;7(4):204. 17. gujral n, freeman hj, thomson ab. celiac disease: prevalence, diagnosis, pathogenesis and treatment. world journal of gastroenterology: wjg. 2012;18(42):6036. 18. odeh r, alassaf a, gharaibeh l, ibrahim s, khdair ahmad f, ajlouni k. prevalence of celiac disease and celiac-related antibody status in pediatric patients with type 1 diabetes in jordan. endocr connect. 2019 jun 13;8(6):780-7. pubmed pmid: 31085767. pubmed central pmcid: pmc6590199. epub 2019/05/16. eng. 19. rönnblom a, holmström t, tanghöj h, wanders a, sjöberg d. celiac disease, collagenous sprue and microscopic colitis in ibd. observations from a population-based cohort of ibd (icure). scand j gastroenterol. 2015;50(10):1234-40. pubmed pmid: 25921772. epub 2015/04/30. eng. 20. mohammadibakhsh r, sohrabi r, salemi m, mirghaed mt, behzadifar m. celiac disease in iran: a systematic review and meta-analysis. electron physician. 2017 mar;9(3):3883-95. pubmed pmid: 28461861. pubmed central pmcid: pmc5407219. epub 2017/05/04. eng. 21. oberhuber g. histopathology of celiac disease. biomedicine & pharmacotherapy. 2000;54(7):368-72. 22. collin p, vilppula a, luostarinen l, holmes gkt, kaukinen k. review article: coeliac disease in later life must not be missed. alimentary pharmacology & therapeutics. 2018;47(5):563-72. 23. volta u, caio g, stanghellini v, de giorgio r. the changing clinical profile of celiac disease: a 15-year experience (1998-2012) in an italian referral center. bmc gastroenterology. 2014 2014/11/18;14(1):194. 345j contemp med sci | vol. 7, no. 6, november-december 2021: 340–345 a. ganji et al. original common clinical symptoms and concomitant disease in celiac patients 24. vivas s, vaquero l, rodríguez-martín l, caminero a. age-related differences in celiac disease: specific characteristics of adult presentation. world j gastrointest pharmacol ther. 2015;6(4):207-12. pubmed pmid: 26558154. eng. 25. rampertab sd, pooran n, brar p, singh p, green ph. trends in the presentation of celiac disease. the american journal of medicine. 2006; 119(4):355. e9–e14. 26. petrarca l, nenna r, mastrogiorgio g, florio m, brighi m, pontone s. dyspepsia and celiac disease: prevalence, diagnostic tools and therapy. world j methodol. 2014;4(3):189-96. pubmed pmid: 25332916. eng. 27. rostami nejad m, mahbobipour h, fazeli z, mashayekhi r, mirsattari d, nazemalhosseini mojarad e, et al. celiac disease in dyspeptic patients. koomesh. 2011;12(2 (38))209–214. 28. ford a, ching e, moayyedi p. meta‐analysis: yield of diagnostic tests for coeliac disease in dyspepsia. alimentary pharmacology & therapeutics. 2009;30(1):28-36. 29. ghahramani r, abbasian as, shafaee s. prevelence of celiac disease in patients with different types of dyspepsia. journal of isfahan medical school. 2009;27(93):65-73. 30. green phr. the many faces of celiac disease: clinical presentation of celiac disease in the adult population. gastroenterology. 2005 2005/04/01/;128 (4, supplement 1):s74-s8. 31. schiepatti a, sanders ds, biagi f. seronegative coeliac disease: clearing the diagnostic dilemma. current opinion in gastroenterology. 2018;34(3): 154-8. 32. volta u, caio g, boschetti e, giancola f, rhoden kj, ruggeri e, et al. seronegative celiac disease: shedding light on an obscure clinical entity. digestive and liver disease. 2016;48(9):1018-22. 33. green phr, stavropoulos sn, panagi sg, goldstein sl, mcmahon dj, absan h, et al. characteristics of adult celiac disease in the usa: results of a national survey. the american journal of gastroenterology. 2001 2001/01/01/;96(1):126-31. 34. kahaly gj, frommer l, schuppan d. celiac disease and endocrine autoimmunity the genetic link. autoimmun rev. 2018 dec;17(12):1169-75. pubmed pmid: 30316996. epub 2018/10/15. eng. 35. husby s, koletzko s, korponay-szabó i, kurppa k, mearin ml, ribeskoninckx c, et al. european society paediatric gastroenterology, hepatology and nutrition guidelines for diagnosing coeliac disease 2020. journal of pediatric gastroenterology and nutrition. 2020;70(1):141-56. 36. ludvigsson jf, bai jc, biagi f, card tr, ciacci c, ciclitira pj, et al. diagnosis and management of adult coeliac disease: guidelines from the british society of gastroenterology. gut. 2014;63(8):1210-28. 37. roberta m, federica g, stefano k, francesco dm, fabiola f, gioacchino l, et al. thyroid and celiac disease in pediatric age: a literature review. acta bio medica: atenei parmensis. 2018;89(suppl 9):11. 38. zhernakova a, van diemen cc, wijmenga c. detecting shared pathogenesis from the shared genetics of immune-related diseases. nature reviews genetics. 2009;10(1):43-55. 39. rubio-tapia a, murray ja. the liver and celiac disease. clin liver dis. 2019 may;23(2):167-76. pubmed pmid: 30947869. pubmed central pmcid: pmc6588186. epub 2019/04/06. eng. 40. hoffmanová i, sánchez d, tučková l, tlaskalová-hogenová h. celiac disease and liver disorders: from putative pathogenesis to clinical implications. nutrients. 2018 jul 12;10(7). pubmed pmid: 30002342. pubmed central pmcid: pmc6073476. epub 2018/07/14. eng. 41. cvetkovic l, bernard g, galette n, hétu po, vincent c, bouin m, et al. discordance between serology and histology for celiac disease in a cohort with coexisting liver disorders. j can assoc gastroenterol. 2020 aug;3(4):185-93. pubmed pmid: 32671328. pubmed central pmcid: pmc7338843. epub 2020/07/17. eng. 42. guedes ng, silva lad, bessa cc, santos jcd, silva vmd, lopes mvdo. anxiety and depression: a study of psychoaffective, family-related, and daily-life factors in celiac individuals. revista brasileira de enfermagem. 2020;73. 43. häuser w, janke k-h, klump b, gregor m, hinz a. anxiety and depression in adult patients with celiac disease on a gluten-free diet. world journal of gastroenterology: wjg. 2010;16(22):2780. 44. audrey s, procter s. employers’ views of promoting walking to work: a qualitative study. international journal of behavioral nutrition and physical activity. 2015;12(1):1-10. 45. schøsler l, christensen la, hvas cl. symptoms and findings in adult-onset celiac disease in a historical danish patient cohort. scandinavian journal of gastroenterology. 2016;51(3):288-94. 46. fedewa mv, bentley jl, higgins s, kindler jm, esco mr, macdonald hv. celiac disease and bone health in children and adolescents: a systematic review and meta-analysis. j clin densitom. 2020 apr-jun;23(2):200-11. pubmed pmid: 30833087. epub 2019/03/06. eng. 47. zanchetta mb, longobardi v, bai jc. bone and celiac disease. curr osteoporos rep. 2016 apr;14(2):43-8. pubmed pmid: 26875096. epub 2016/02/15. eng. 48. ganji r, moghbeli m, sadeghi r, bayat g, ganji a. prevalence of osteoporosis and osteopenia in men and premenopausal women with celiac disease: a systematic review. nutrition journal. 2019 feb 7;18(1):9. pubmed pmid: 30732599. pubmed central pmcid: pmc6504166. epub 2019/02/09. eng. 49. micic d, rao vl, semrad ce. celiac disease and its role in the development of metabolic bone disease. j clin densitom. 2020 apr-jun;23(2):190-9. pubmed pmid: 31320223. epub 2019/07/20. eng. 50. bergamaschi g, markopoulos k, albertini r, di sabatino a, biagi f, ciccocioppo r, et al. anemia of chronic disease and defective erythropoietin production in patients with celiac disease. haematologica. 2008;93(12):1785-91. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1074 264 j contemp med sci | vol. 7, no. 5, september–october 2021: 264–271 review clinical complications associated with spinal cord injury: a narrative review ibrahim mohammed1, sahar ijaz2, morteza gholaminejhad3, gholamreza hassanzadeh3,4* 1department of histopathology, school of medical laboratory sciences, usmanu danfodiyo university sokoto, nigeria. 2department of anatomy and histology, university of veterinary and animal sciences, lahore, pakistan. 3department of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. 4department of neuroscience and addiction studies, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. *correspondence to: gholamreza hassanzadeh, (e-mail: hassanzadeh@tums.ac.ir). (submitted: 12 august 2021 – revised version received: 27 august 2021 – accepted: 09 september 2021 – published online: 26 october 2021) introduction spinal cord injury (sci) is a clinical problem associated with remarkable financial as well as the emotional burden on individual patient, families, and society as well. approximately, there is incidence of sci of about 10–80 cases per million populations in the world.1-3 most of the sci result of tetraplegia (51.57%), followed by paraplegia with (45.9%). sci due to motor vehicle accident account in almost one-half of the injuries worldwide, followed by sci due to falls, sci due to violence (like gunshot), and sci due to sports activities.4 alcoholism is one of the factors contributing to sci with 25% new cases of sci,3 as underlying spine disorder like cervical spondylosis, osteoporosis, atlantoaxial instability, and spinal arthropathies can lead to sci, too.2,5 the extent of sci depend on the severity of the initial injury, as well the financial burden depends also between individual patients, with the estimated cost of 500,000 to 2 million us dollars on individual patient’s lifetime.2 epidemiological studies revealed that the incidence of traumatic sci in the us is between 27 to 83 per million, in europe approximately about 10–30 new cases per million.6 in developed areas such as western europe (16/million), australia (15/million) and north america (39/million). falls on level ground are the most common cause of sci in older countries such as western europe (37%) and japan (42%). violence is one of the common causes of sci in developing regions such as north africa/ middle east (24%), sub-saharan africa (38%) and latin america (22%).7,8 a research conducted in 2008 in tehran based on population, reported that the prevalence of sci was 4.4 per 10,000 populations.9 rahimi movaghar et al. (2013), reported the incidence of sci in developing countries has a rate of 25.5 per million per year, which ranged from 2.1 to 130.7 per million per year.10 also, men are more prone to sci in the world than women, although there are differences in the data based on the countries.7 mechanism of sci categorized into primary and secondary injury, with former resulting from pathologic flexion, compression, rotation, contusion, extension, shearing, fracture-dislocation, ligamentous tears or disruption as well as herniation of intervertebral disk. the primary mechanical injury that happened at the time of injury is followed by a secondary injury phase involving vascular dysfunction, edema, ischemia, excitotoxicity, inflammation, mitochondrial dysfunction, and delayed apoptotic cell death.11-16 a report from gholaminejhad et al. (2017), revealed that the stress oxidative level will increase as one of the complications of sci, finally may result to neurons disruption in animal model.17 there are series of complication occurred as a result of sci starting from primary injury to secondary injury, most often if care is not taken primary injury leads to the secondary injury that increases damage to previously injured spinal cord, it has shown that overexpression of cytokines that are important mediator for inflammation following sci.18,19 sci can initiate biochemical and molecular events like inflammation which one of the key factor in neurodegeneration development.20 in this paper, the various clinical complications due to sci were discussed. this narrative review adds to the knowledge of how spinal cord injury occurs, the various complications resulted by spinal cord injury, through the knowledge of different complications immediate attention will be giving to spinal cord injury patients at the early stage. scopus search, google scholar search, pubmed search are the search engines used to get the information for this review. abstract spinal cord injury (sci) is a neuro-destruction occurred from a complete or incomplete, traumatic or non-traumatic that results in degeneration, structural, biochemical, and physiological changes of tissue. sci is a clinical problem associated with impairments in different aspects of the patient’s life. the pathophysiology of sci involves a primary phase that directly disrupts axons, cell membranes, and blood vessels. this primary phase is followed by a secondary phase involving vascular dysfunction, ischemia, excitotoxicity, oxidative stress, inflammation, and cell death. if this second phase isn’t managed, it will result in many pathological processes that will cause several clinical complications. the aim of rehabilitation and other treatments is to enhance the functional level and to reduce secondary morbidity as well as improve the quality of health of the patient. sci results in different complications in different organs of the individual. early diagnosis, treatment, and prevention of complications in sci patients are very important for limiting these complications. this review was carried out in order review the data about clinical complications associated with sci, including multiple organ dysfunction, systemic inflammation, immune suppression, neurogenic shock, autonomic dysreflexia, orthostatic hypotension, temperature regulation, sweat secretion, respiratory complications and dysphagia, thromboembolism, urinary system, reproductive system, skeletal muscle, bone, liver, spleen and gastrointestinal tract. the purpose of this narrative review is to provide knowledge on the sci complications, sign and symptoms, risk factors prevention and treatment of complications caused by sci. keywords: spinal cord injury, inflammation, oxidative stress, clinical complications issn 2413-0516 265j contemp med sci | vol. 7, no. 5, september–october 2021: 264–271 i. mohammed et al. review clinical complications of spinal cord injury multiple organ dysfunctions apart from impairments to sensation and voluntary movement, sci interrupts the autonomic nervous system and leads to dysfunction or failure in multiple organs due to a vital role of the spinal cord in coordinating bodily functions.21 short term as well as long complications due to sci may happen in the nervous system (like neurogenic pain and depression), in lungs (pulmonary edema and respiratory failure), in cardiovascular system (like autonomic dysreflexia and orthostatic hypotension), in spleen (like splenic atrophy and leukopenia), in urinary tract (neurogenic bladder, urinary tract infection as well as kidney damage), in skeletal muscle (muscle spasticity and atrophy), in soft tissue as well as bone (osteoporosis and heterotopic ossification), in skin (pressure sores), there are also sexual dysfunction, hepatic pathology, neurogenic bowel dysfunction, syringomyelia as well as high susceptibility to infection. some sci complications if care is not taken can easily cause the death of the patient, such as liver, kidney as well as lungs damage (figure 1).21-24 multiple organ dysfunctions due to sci are complex regulation by various components. cranial nerves that originate from brainstem (pons and medulla) regulate the functions of multiple organs, the brainstem reflexes were found to be changed in sci patients.25 this shows a complex relationship between multiple organ dysfunction, sci and altered brainstem activity. this suggests that a good care should be given to the brainstem’s role in multiple organ dysfunctions due to sci.25 systemic inflammation the local inflammatory microenvironment around the injured spinal cord is the aggregation of degenerating neurons, damaged myelin sheath, damaged endothelial cells as well as activated glial and infiltrating cells, this microenvironment yields different kind of pro-inflammatory mediators.26,27 apart from this intraspinal inflammation, sci may results in systemic inflammatory response syndrome (sirs), this is a life-threatening situation that can affect other organs, such as liver, kidney, and lung.28 previous research revealed that there is a functional relationship between systemic inflammation and pathogenesis of post injury complications: patients with sirs-positive have more injury severity with more chances of complications compared to sirs-negative patients.29 there are some other different factors like dysregulation of the neuroendocrine system and altered neuroimmune regulation, known as major key factors in determinants of the onset as well as the development of post-sci systemic inflammation. for example, sci induces the hypothalamic-pituitary-adrenal axis, results in increased macrophage migration inhibitory factor processing via pituitary gland.30 macrophage migration inhibitory factor is one of the key factors in systemic inflammation, revealing that sci-elicited neuroendocrine changes lead to the advancement of systemic inflammation. chronic activation of microglia, the neuroimmune cells of the central nervous system, happens in the hippocampus and cerebral cortex following sci; this is showing that neuroimmune dysregulation played an important function in systemic inflammation after sci.31 a research conducted by zandedel et al., (2016) reported that stromal cell-derived factor-1 alpha (sdf-1α) or cxcl12 is the main principal cytokine with numerous functions in the brain at fetal development as well as adult period. the inflammatory response occurred due to sci requires the processing of interleukin-1beta (il-1β) as well as il-18 mediated by caspase-1 in which an intracellular multiprotein complex control it (inflammasome).32 fig. 1 clinical complications of spinal cord injury. after spinal cord injury, several clinical complications occur in the short term or the long term. some of these clinical complications include disorders in the reproductive system, respiratory system, digestive system, urinary system, sweat secretion, skeletal muscles, liver, spleen, bones and systemic inflammation that afflict a person with physical, psychological, social and economic problems. 266 j contemp med sci | vol. 7, no. 5, september–october 2021: 264–271 clinical complications of spinal cord injury review i. mohammed et al. immune suppression complications occur on the immune system as result of sci known as sci-induced immune depression syndrome (sci-ids), due to dysregulation of the sympathetic nervous system as well as immune organ dysfunction.33 sci can result in sympathetic nervous system malfunction instantly through prominent projections of the thoracolumbar spinal cord to sympathetic ganglion or directly through damaging supraspinal control by the hypothalamic-pituitary-adrenal axis. sci-ids, revealed by the loss of splenocytes as well as leukopenia, is a presumed self-defense mechanism that decreases potential autoimmunity to self-antigens released by damaged in the cns.34 report from different sources has shown that sci-ids worsens neurological environments and damages the functional recovery of sci patients. riegger et al., (2009) revealed that a remarkable decrease in the amount of circulating cells associated with innate and adaptive immunity in the acute phase after sci on rat model.35 similar reports were also made in a pilot research that involves 16 patients with sci and 10 healthy individuals as control: decreased monocytes, t lymphocytes, and b-lymphocytes, but granulocytes were not seen in the blood circulation within 24 hours following sci.36 sci-ids has a vital clinical relevance, as sci patients show a higher susceptibility to different infections (like wound infections and pneumonia)37 as well as poor functional recovery.38 autonomic dysreflexia, as well as the expansion of myeloid-derived suppressor cells after sci, has a mutual relationship with sci-ids.39 neurogenic shock at the level of cervical vertebra of sci in human, the common features of neurogenic shock are severe hypotension as well as continuance bradycardia.40 in a study conducted by glenn and bergman (1997) revealed that chronic hypotension was seen 31 tetraplegic subjects examined with chronic sci, most of the subjects need pressure therapy so that the arterial blood pressure will be maintained.41 apart from pronounced hypotension, the majority of the patients with sci experience chronic abnormalities in heart rate. bradycardia was seen in 64–77% of patients with cervical sci at the acute post-injury level and was so chronic and frequent at the first 5 weeks after the injury.42 furthermore, when the injury is at the level of mid-thoracic spinal cord, leaving cardiac sympathetic neurons under brainstem control, the severity of bradycardia problem is very less. furlan et al. (2003) revealed that the hypotension and bradycardia seen at the beginning following the injury persisted in the individuals with more chronic injury of the descending cardiovascular autonomic cascades.43 apart from neurogenic shock, sci as well associated with “spinal shock”.44,45 these are two different complications of sci, neurogenic shock is identified by changes happening in blood pressure control after sci, while spinal shock is identified by a marked decrease or abolition of motor, sensory or reflex activities of the cord under the level of injury.45 clinically, spinal shock period takes about 1 day to 2 weeks in human, with mean period of 4 and 6 weeks after the injury. traditionally the ideas of the medical course of the recovery of spinal shock were associated with the appearance of some groups of reflexes. for instance, most clinicians assumed that spinal shock had ended when the emergence of initial reflexes, like the bulbocavernosus reflex, happened at the beginning after sci, some assumed with the recovery of deep tendon reflexes at two weeks after the injury, while other groups of clinicians categorized the end of spinal shock as the time after 2 months they recovered bladder voiding reflexes.45 autonomic dysreflexia patients with cervical or high thoracic sci experience life-long abnormalities of blood pressure control.46,47 most often, the resting arterial blood vessels pressure in these patients is lower when compared with normal individuals, usually associated with disabling event of orthostatic hypotension. nevertheless, clinical complications of autonomic dysreflexia, associated with high hypertension together with a pounding headache, sometimes accompanied by slow heart rate as well as upper body flushing, in which systolic blood pressure may rise up to 300 mmhg. untreated autonomic dysreflexia may results in serious complication such as retinal detachment, intracranial hemorrhage, seizures and death.48 majority of non-noxious and noxious stimuli like bowel and bladder distension, pressure sores and spasticity may stimulate the sudden high rate in arterial blood pressure of autonomic dysreflexia.47 these cardiovascular complications are associated with autonomic instability, due to the changes happening within the spinal autonomic circuits in stages of sci in acute and chronic stages.46,47 the damaging of the descending vasomotor pathways leads to the loss of excitatory supraspinal input to the sympathetic preganglionic neurons and is assumed to be the main factor responsible for the lack of sympathetic tone as well as continuance arterial hypotension observed following sci.43 orthostatic hypotension low arterial blood pressure is another complication occurred due to sci both acute and chronic. a study reported that there was an inverse linear relationship between the degree of resting blood pressure as well as sci.46,49 the lower resting blood pressure is assumed to be secondary to the decrease in sympathetic nervous activity below the degree of the injured spinal cord. apart from low blood pressure, patients with sci experience other complication of the drop in blood pressure in the upright posture (orthostatic hypotension), most especially in the acute phase of the injury.49 the symptoms of orthostatic hypotension in with sci patients’ are the same as those individuals without sci experiencing orthostatic hypotension, and all are related to cerebral hypoperfusion.50 the common symptoms usually seen are light-headedness, dyspnea, fatigue or weakness, dizziness, blurred vision and restlessness.51 a study by illman et al., (2000) reported that 41.1% of patients with sci that developed orthostatic hypotension were seen asymptomatic regardless of significant falls in blood pressure, it was also documented that orthostatic hypotension is a common complication among patients with sci.52 orthostatic hypotension due to sci seems to be associated with excessive pooling of blood in the viscera as well as dependent extremities, probably caused by low level or absence of efferent sympathetic preganglionic neurons below the injured area.49 this is possibly to be compounded by the loss of lower limbs muscle function that identified to be essential in counteracting venous pooling in the upright level. the remaining excessive venous pooling in the lower limbs and decreased blood ratio within 267j contemp med sci | vol. 7, no. 5, september–october 2021: 264–271 i. mohammed et al. review clinical complications of spinal cord injury the intrathoracic veins results in a decreased pressure in the veins that drain through the atria of the heart.53 these will leads to reduce in ventricular end-diastolic filling pressure and stroke volume,54 resulting in the low in heart output as well as arterial pressure. tachycardia may happen due to the decrease cardiac parasympathetic (vagal) activity, reflexly produced by unloading the arterial baroreceptors, but this activity most usually is too small to compensate for the reduced stroke volume as well as blood pressure remains low. temperature regulation in the ability to control temperature by sci patients is among the clinical complications caused by sci, most usually occurred with patients affected by cervical as well as thoracic sci. this is occurred due to decreases sensory input to thermo-regulating centers as well as loss of sympathetic control of temperature and sweet regulation lower than the affected area.55 there are a lot of thermoregulation complications caused by sci. some sci patients were found to have poikilothermia, in the ability to maintain a normal temperature notwithstanding of the ambient temperature. sci above t8 is usually related to actuating temperature, hyperthermia as well as hypothermia.56 sweat secretion the innervations of sweat glands are mostly by the upper region of the body from t1-t5 spinal cord segments, at the lower part innervated by t6-l2 spinal cord segments. hypothalamus and amygdala are the regions where supraspinal control of sweat excretion occurred.55 as results of sci changes in sweat secretion will occur, the absence of sweating (anhidrosis), excessive sweating (hyperhidrosis), and diminished sweating (hypohidrosis) may all occur following sci.55 excessive sweating is a typical complication seen in sci patients.57 in most of the patients, episodic hyperhidrosis is mostly related to other autonomic dysfunctions like autonomic dysreflexia as well as orthostatic hypotension, or with post-traumatic syringomyelia. the typical symptoms seen are minimal or abolished sweating below the injury and profuse sweating above the injured area. this is occurred due to the compensatory high in secretion of sweat above the level of the injured area because of the loss of sympathetic stimulation below the injured area, which leads to decrease in production of sweat.58 there is also the possibility of sweat production exclusively below the level of the injured area. this kind of sweating is known as reflex sweating and is mostly a symptom of massive autonomic response that happened specifically with cervical and high thoracic sci.58 respiratory complications sci at the cervical region has major complications on the pulmonary system, as well respiratory difficulties are among the most complications and can lead to frequent death, both in the acute and chronic level of sci.59 results from the previous research revealed that 67% of acute sci individuals have severe respiratory problems at the first day of the injury; atelectasis 36.4%, pneumonia 31.4% as well as respiratory failure 22.6%.60 it was also revealed that in the acute phase 84% of sci patients with injuries above c4, 6% with injuries from c5-c8 would develop respiratory complications.61 regular monitoring of respiration on sci patients with such injuries is advisable. there is possibility of 56% of sci patients with injuries at the level of t1–t12 to develop severe respiratory problems.62 30–50% reduction of vital capacity is observed in the first week of injury in injured patients at the level of c5–c6. vital capacity, as well as arterial blood gases, should be measured regularly until the patient is normal.63 thromboembolism sci patients have a higher risk of coagulation complications as well as venous stasis due to physical inactivity, altered hemostasis with decreased fibrinolytic activity and high factor viii activity.64 the patients also are at high risk of thromboembolism.65 at the first year of injury, the chances of deep vein thrombosis, as well as pulmonary embolism, are assumed to be 15% and 5% respectively.66 the chances are higher at 2–3 weeks of the injury, at 3 months following the injury it will be at a small peak.67 at the chronic stage, the incidence of thromboembolism is less than 2%.64 urinary system patients with sci have high risk of developing urinary tract complications as well as renal damage, in which both can result in fatal.68 apart from the direct loss of neuronal input following the injury, inflammation has been involved in the pathogenesis of urinary tract malfunction in patients with sci. the inflammation that is possibly occurred due to sci is the production of pro-inflammatory cytokines (il-1β, il-6, and tnf-α), infiltration of immune cells, upregulation of inducible nitric oxide synthase, myeloperoxidase as well as cyclooxygenase-2, then activation of nf-κb.68,69 sci caused complication in bladder, interrupts control of the bladder.70 as a result of sci immediately after the injury, the bladder as well as sphincter is frequently hypotonic. at the chronic stage the bladder dysfunction is categorized as either upper or lower motor neuron syndrome.70 upper motor neuron syndrome known as reflex bladder result in cortical inhibition of sacral reflex arcs because of disturbance of descending spinal tracts, results in detrusor hyperactivity mostly together with detrusor sphincter dyssynergia.71 suppression of the stretch reflex by the pontine storage center is eliminated. a minor quantity of stretch will provide a contraction of the bladder wall; the external urethral sphincter has no voluntary control, leading in recurrent and spontaneous voiding.71 lower motor neuron syndrome occurred due to injury to the sacral region s2–s4, which is part of the autonomic nervous system leading in diminished motor stimulation of the bladder and decreased or lack of contractility of the detrusor and eventually an enlarged bladder.72 reproductive system impaired fertility is among the major complications occurred in men with sci that result in erectile as well as ejaculation malfunctions. semen analysis of sci patients are always poor, there is much evidence from previous research reported that a low sperm viability, motility, leukocytospermia, and high sperm dna fragmentation are common among men with sci.73,74 most of the male sci patients suffer a lot from infertility. nucleotide-binding oligomerization domain-like receptors 268 j contemp med sci | vol. 7, no. 5, september–october 2021: 264–271 clinical complications of spinal cord injury review i. mohammed et al. nod-like receptors (nlrs) are the receptors that conjoin with the inflammasome complex. results from different studies revealed that inflammasomes are one of the key factors for secreting cytokines in semen. reactive oxygen species (ros) is one of the agents responsible to initiate inflammasome activation. genital infections as a result of sci can bring about ros generation.75 only a few studies revealed testicular tissue become involved post-sci. impaired spermatogenesis, apoptosis, vast germ cell, inflammatory cytokines elevation, and blood-testis barriers disruption, as well as leukocytes influx, has been shown as abnormal changes in the testis of sci patients, result to the inflammatory event and unstable niche in this tissue.76 a study conducted by nikmehr et al., (2017), examined sperm parameters of sci using rats’ model at acute as well as chronic phases. the result shows a fall in sperm count by half after one day and a third after three days post sci. the severe decrease in sperm count was very significant in the acute phase, sperm motility was also decreased following sci on acute as well as chronic phases compared to the control group.73 sci, a neurogenic impairment, resulted to infertility by disturbing the plasma testosterone balance which is very difficult to be restored by exogenous testosterone administration. sci results in some alteration in oxidative markers, with the reduced free radical scavenging activities of gpx and sod,77 as well as, increased mda concentration and nlrp3 expression after sci.75 skeletal muscle sci makes paralysis and atrophy on the skeletal muscle, especially the muscles controlled by the spinal cord below the level of the injury.78 physiological examinations have shown many alterations in the properties of paralyzed muscles from patients with sci, such as reduced mass area, high susceptibility to fatigue, decreased muscle cross-sectional area as well as the increased proportion of fast glycolytic muscular fibers.79 muscular inflammation can be seen in the acute phase of sci prior to muscular atrophy.80 in long period sci, muscle atrophy is related with the remarkable elevation of inflammatory mediators (such as il-1β, il-6, and tnf-α) as well as activation of nf-κb signaling,81 which is the main agent that regulates the inflammatory state in muscle atrophy.82 bone osteoporosis, which is defined as the loss of bone mineral density (bmd), is formed as a result of sci.83 the distal femur, proximal tibia as well as distal boney sites at sub-lesional area are the major susceptible to bmd loss.84 the low level of bmd is increasing following sci; as well the possibility of patients’ fracture is high,85 there are other various factors that contribute the pathogenesis of osteoporosis as a result of sci. apart from the deficiency in neuronal control, hormonal regulation as well as vascular function,86 the inflammatory microenvironment in bone brings about osteoclast differentiation as well as bone loss.87 liver from the previous report, it was revealed that there are hepatic complications due to sci.88 there are many reports that show liver dysfunction can be related to sci, as the liver plays a significant role in the metabolic dysfunction usually seen in patients with sci. experimental research using animal model revealed that sci activates macrophage activation, neutrophil infiltration, expression of pro-inflammatory cytokines as well as chemokines in the liver.89 this inflammation occurs immediately following the injury,90 and its severity is related to the level of injury.91 a significant amount of accumulated lipid has been seen in rodent liver after sci.92 given the pro inflammatory as well as cytotoxic effects of myelin-laden macrophage related to the accumulation of lipid in the sci,93 macrophage-mediated inflammation considerably leads to hepatic dysfunction following sci. spleen spleen is a very vital lymphoid organ functioned for infiltration of monocytes in the injured spinal cord; spleen is innervated by the autonomic nervous system and regulated by the high thoracic spinal cord. a research using rat model has shown a significant spleen dysfunction due to sci at the t3 level. spleen dysfunction as a result of sci at t3 level was revealed by splenic atrophy with decrease in size, decrease in the amount of splenic leukocyte and increased splenic norepinephrine was seen.94,95 sci mice induced with viral infection revealed impaired immune responses as well as decreased survival, and these results seen in mice were related with deficient cd4+ and cd8+ t cell functions, deficient primary antibody response as well as suppressed activation of macrophages.96 this indicates that dysfunction of the spleen may possibly result in immune suppression in patients with sci. remarkably; post-sci mrna levels of pro-inflammatory cytokines il-17, as well as il-23, were seen upregulated in tissue spleen of rat model by stat3 signaling,97 consequently, crosstalk between spleen and sci could be mediated by neuroinflammation. gastrointestinal tract there are many complications occurred due to sci in the gastrointestinal tract (git), for example, severe constipation, difficulty with evacuation, painful defecation or incontinence, is advisable to restrict and limit the diet and outdoor activities of sci patients.98 despite the fact that function of git is basically regulated by its own intrinsic nervous system as well as autonomic control by the brainstem, sci may cause the destruction of neuronal control of git sensory as well as motor functions, leading to neurogenic bowel dysfunction (nbd). it was estimated that 50% of sci patients are having moderate to severe nbd,99 it appears that abnormal bowel function has a more negative influence on the quality of life of patients with sci.100,101 the clinical presentations of nbd with sci are prolonged bowel transit time; decrease colonic motility as well as anorectal dysfunction.102 even though there is inflammation in colonic lesions of patients with sci,103 the association between systemic inflammation as well as nbd is not well understood. a report from guo et al., (2016) on rat model of nbd after sci shows that upregulation of neuronal nitric oxide synthase leads to colonic dysfunction,104 thus creating inflammation as a possible target for reducing post-sci nbd. conclusion sci leads to different complications in various parts of the body, most often, primary injury gives rise to secondary injury 269j contemp med sci | vol. 7, no. 5, september–october 2021: 264–271 i. mohammed et al. review clinical complications of spinal cord injury that increases damage to the previously injured spinal cord. among several processes of secondary injury phase, it seems inflammation is most important because it involves in many pathological problems. therefore, suppression of inflammation maybe is a good target for degenerative diseases. also, knowledge of possible complications by sci is very vital because the complications may be life-threatening and may lead to prolonging rehabilitation for sci patients, also early detection and treatment of the complications will help in managing the patient. even though there is no way to reverse damage to the spinal cord. but there are various researches continually working on new treatments, including transplanting stem cells and medications that may promote nerve cell regeneration or improve the function of nerves that remain after sci. conflicts of interest none.  references 1. grant ra, quon jl, abbed km (2015) management of acute traumatic spinal cord injury. current treatment options in neurology 17 (2):6. 2. sekhon lh, fehlings mg (2001) epidemiology, demographics, and pathophysiology of acute spinal cord injury. spine 26 (24s):s2–s12. 3. thompson c, mutch j, parent s, mac-thiong j-m (2015) the changing demographics of traumatic spinal cord injury: an 11-year study of 831 patients. the journal of spinal cord medicine 38 (2):214–223. 4. jackson ab, dijkers m, devivo mj, poczatek rb (2004) a demographic profile of new traumatic spinal cord injuries: change and stability over 30 years. archives of physical medicine and rehabilitation 85 (11):1740–1748. 5. myers er, wilson se (1997) biomechanics of osteoporosis and vertebral fracture. spine 22 (24):25s–31s. 6. hyun jk, kim h-w (2010) clinical and experimental advances in regeneration of spinal cord injury. journal of tissue engineering 1 (1):650857. 7. chiu w-t, lin h-c, lam c, chu s-f, chiang y-h, tsai s-h (2010) epidemiology of traumatic spinal cord injury: comparisons between developed and developing countries. asia pacific journal of public health 22 (1):9–18. 8. burns as, o’connell c (2012) the challenge of spinal cord injury care in the developing world. the journal of spinal cord medicine 35 (1):3–8. 9. rahimi-movaghar v, saadat s, rasouli mr, ganji s, ghahramani m, zarei m-r, vaccaro ar (2009) prevalence of spinal cord injury in tehran, iran. the journal of spinal cord medicine 32 (4):428–431. 10. rahimi-movaghar v, sayyah mk, akbari h, khorramirouz r, rasouli mr, moradi-lakeh m, shokraneh f, vaccaro ar (2013) epidemiology of traumatic spinal cord injury in developing countries: a systematic review. neuroepidemiology 41 (2):65–85. 11. tator ch, fehlings mg (1991) review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. journal of neurosurgery 75 (1):15–26. 12. ghaffari n, hassanzadeh g, nowrouzi a, gholaminejhad m, mokhtari t, seifali r, mohammed i, akbari m (2018) antioxidative and antiinflammatory effects of cichorium intybus l. seed extract in ischemia/ reperfusion injury model of rat spinal cord. journal of contemporary medical sciences 4 (4). 13. mohammed i, ijaz s, mokhtari t, gholaminejhad m, mahdavipour m, jameie b, akbari m, hassanzadeh g. subventricular zone-derived extracellular vesicles promote functional recovery in rat model of spinal cord injury by inhibition of nlrp3 inflammasome complex formation. metabolic brain disease. 2020 jun 1;35(5). 14. rowland jw, hawryluk gw, kwon b, fehlings mg (2008) current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon. neurosurgical focus 25 (5):e2. 15. hassanzadeh s, jameie sb, soleimani m, farhadi m, kerdari m, danaei n (2018) coenzyme q10 influences on the levels of tnf-α and il-10 and the ratio of bax/bcl2 in a menopausal rat model following lumbar spinal cord injury. journal of molecular neuroscience 65 (2):255–264 16. hassanzadeh s, jameie sb, mehdizadeh m, soleimani m, namjoo z, soleimani m (2018) fndc5 expression in purkinje neurons of adult male rats with acute spinal cord injury following treatment with methylprednisolone. neuropeptides 70:16–25. 17. gholaminejhad m, arabzadeh s, akbari m, mohamadi y, hassanzadeh g (2017) anti-oxidative and neuroprotective effects of flaxseed on experimental unilateral spinal cord injury in rat. journal of contemporary medical sciences 3 (10):213–217. 18. pishva aa, akbari m, farahabadi a, arabkheradmand a, beyer c, dashti n, moradi f, hassanzadeh g (2016) effect of estrogen therapy on tnf-α and inos gene expression in spinal cord injury model. acta medica iranica 54 (5):296–301. 19. mohamadi y, moghahi smhn, mousavi m, borhani-haghighi m, abolhassani f, kashani ir, hassanzadeh g (2019) intrathecal transplantation of wharton’s jelly mesenchymal stem cells suppresses the nlrp1 inflammasome in the rat model of spinal cord injury. journal of chemical neuroanatomy 97:1–8. 20. ijaz s, mohammed i, gholaminejhad m, mokhtari t, akbari m, hassanzadeh g. modulating pro-inflammatory cytokines, tissue damage magnitude, and motor deficit in spinal cord injury with subventricular zone-derived extracellular vesicles. journal of molecular neuroscience. 2020;70(3):458–66. doi: 10.1007/s12031–019–01437–2.. 21. stein dm, menaker j, mcquillan k, handley c, aarabi b, scalea tm (2010) risk factors for organ dysfunction and failure in patients with acute traumatic cervical spinal cord injury. neurocritical care 13 (1):29–39. 22. zimmer mb, nantwi k, goshgarian hg (2007) effect of spinal cord injury on the respiratory system: basic research and current clinical treatment options. taylor & francis, . 23. bilello jf, davis jw, cunningham ma, groom tf, lemaster d, sue lp (2003) cervical spinal cord injury and the need for cardiovascular intervention. archives of surgery 138 (10):1127–1129. 24. macdiarmid s, mcintyre w, anthony a, bailey r, turner j, arnold e (2000) monitoring of renal function in patients with spinal cord injury. bju international 85 (9):1014–1018. 25. kumru h, kofler m (2012) effect of spinal cord injury and of intrathecal baclofen on brainstem reflexes. clinical neurophysiology 123 (1):45–53. 26. altinors n (2009) analysis of serum pro-inflammatory cytokine levels after rat spinal cord ischemia/reperfusion injury and correlation with tissue damage. turkish neurosurgery 19 (4):353–359. 27. mohamadi y, mousavi m, moogahi smhn, abolhassani f, ijaz s, hassanzadeh g (2018) effect of wharton’s jelly derived mesenchymal stem cells on the expression of nlrp3 receptor and neuroinflammation in experimental spinal cord injury. journal of clinical & diagnostic research 12 (10). 28. anthony dc, couch y (2014) the systemic response to cns injury. experimental neurology 258:105–111. 29. kesani ak, urquhart jc, bedard n, leelapattana p, siddiqi f, gurr kr, bailey cs (2014) systemic inflammatory response syndrome in patients with spinal cord injury: does its presence at admission affect patient outcomes? clinical article. journal of neurosurgery: spine 21 (2):296–302. 30. lerch jk, puga da, bloom o, popovich pg. glucocorticoids and macrophage migration inhibitory factor (mif) are neuroendocrine modulators of inflammation and neuropathic pain after spinal cord injury. in: seminars in immunology, 2014. vol 5. elsevier, pp. 409–414. 31. wu j, zhao z, sabirzhanov b, stoica ba, kumar a, luo t, skovira j, faden ai (2014) spinal cord injury causes brain inflammation associated with cognitive and affective changes: role of cell cycle pathways. journal of neuroscience 34 (33):10989–11006. 32. zendedel a, johann s, mehrabi s, joghataei m-t, hassanzadeh g, kipp m, beyer c (2016) activation and regulation of nlrp3 inflammasome by intrathecal application of sdf-1a in a spinal cord injury model. molecular neurobiology 53 (5):3063–3075. 33. kopp ma, druschel c, meisel c, liebscher t, prilipp e, watzlawick r, cinelli p, niedeggen a, schaser k-d, wanner ga (2013) the scientinel studyprospective multicenter study to define the spinal cord injury-induced immune depression syndrome (sci-ids)-study protocol and interim feasibility data. bmc neurology 13 (1):168. 34. schwab jm, zhang y, kopp ma, brommer b, popovich pg (2014) the paradox of chronic neuroinflammation, systemic immune suppression, autoimmunity after traumatic chronic spinal cord injury. experimental neurology 258:121–129. 35. riegger t, conrad s, liu k, schluesener hj, adibzahdeh m, schwab jm (2007) spinal cord injury‐induced immune depression syndrome (sci‐ids). european journal of neuroscience 25 (6):1743–1747. 270 j contemp med sci | vol. 7, no. 5, september–october 2021: 264–271 clinical complications of spinal cord injury review i. mohammed et al. 36. riegger t, conrad s, schluesener h, kaps h-p, badke a, baron c, gerstein j, dietz k, abdizahdeh m, schwab j (2009) immune depression syndrome following human spinal cord injury (sci): a pilot study. neuroscience 158 (3):1194–1199. 37. brommer b, engel o, kopp ma, watzlawick r, müller s, prüss h, chen y, devivo mj, finkenstaedt fw, dirnagl u (2016) spinal cord injury-induced immune deficiency syndrome enhances infection susceptibility dependent on lesion level. brain 139 (3):692–707. 38. failli v, kopp ma, gericke c, martus p, klingbeil s, brommer b, laginha i, chen y, devivo mj, dirnagl u (2012) functional neurological recovery after spinal cord injury is impaired in patients with infections. brain 135 (11):3238–3250. 39. wang l, yu w-b, tao l-y, xu q (2016) myeloid-derived suppressor cells mediate immune suppression in spinal cord injury. journal of neuroimmunology 290:96–102. 40. atkinson pp, atkinson jl spinal shock. in: mayo clinic proceedings, 1996. vol 4. elsevier, pp 384–389. 41. glenn m, bergman sb (1997) cardiovascular changes following spinal cord injury. topics in spinal cord injury rehabilitation 2 (4):47–53. 42. lehmann kg, lane jg, piepmeier jm, batsford wp (1987) cardiovascular abnormalities accompanying acute spinal cord injury in humans: incidence, time course and severity. journal of the american college of cardiology 10 (1):46–52. 43. furlan jc, fehlings mg, shannon p, norenberg md, krassioukov av (2003) descending vasomotor pathways in humans: correlation between axonal preservation and cardiovascular dysfunction after spinal cord injury. journal of neurotrauma 20 (12):1351–1363. 44. nacimiento w, noth j (1999) what, if anything, is spinal shock? archives of neurology 56 (8):1033–1035. 45. ditunno j, little j, tessler a, burns a (2004) spinal shock revisited: a fourphase model. spinal cord 42 (7):383–395. 46. mathias c (1992) investigation of autonomic disorders. autonomic faliure, a textbook of clinical disorders of the autonomic nervous system:255–290. 47. teasell rw, arnold jmo, krassioukov a, delaney ga (2000) cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord injury. archives of physical medicine and rehabilitation 81 (4):506–516. 48. eltorai i, kim r, vulpe m, kasravi h, ho w (1992) fatal cerebral hemorrhage due to autonomic dysreflexia in a tetraplegic patient: case report and review. spinal cord 30 (5):355. 49. mathias cj (1995) orthostatic hypotension: causes, mechanisms, and influencing factors. neurology 45 (4 suppl 5):s6–s11. 50. cleophas tj, kauw fh, bijl c, meijers j, stapper g (1986) effects of beta adrenergic receptor agonists and antagonists in diabetics with symptoms of postural hypotension: a double-blind, placebo-controlled study. angiology 37 (11):855–862. 51. sclater a, alagiakrishnan k (2004) orthostatic hypotension. a primary care primer for assessment and treatment. geriatrics (basel, switzerland) 59 (8):22–27. 52. illman a, stiller k, williams m (2000) the prevalence of orthostatic hypotension during physiotherapy treatment in patients with an acute spinal cord injury. spinal cord 38 (12):741. 53. faghri pd, yount jp, pesce wj, seetharama s, votto jj (2001) circulatory hypokinesis and functional electric stimulation during standing in persons with spinal cord injury. archives of physical medicine and rehabilitation 82 (11):1587–1595. 54. ten harkel a, van lieshout j, wieling w (1994) effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure. clinical science 87 (5):553–558. 55. alexander m, biering-sorensen f, bodner d, brackett n, cardenas d, charlifue s, creasey g, dietz v, ditunno j, donovan w (2009) international standards to document remaining autonomic function after spinal cord injury. spinal cord 47 (1):36–43. 56. phillips wt, kiratli bj, sarkarati m, weraarchakul g, myers j, franklin ba, parkash i, froelicher v (1998) effect of spinal cord injury on the heart and cardiovascular fitness. current problems in cardiology 23 (11):641–716. 57. karlsson a-k (2006) overview: autonomic dysfunction in spinal cord injury: clinical presentation of symptoms and signs. progress in brain research 152:1–8. 58. yaggie ja, niemi tj, buono mj (2002) adaptive sweat gland response after spinal cord injury. archives of physical medicine and rehabilitation 83 (6):802–805. 59. berney s, bragge p, granger c, opdam h, denehy l (2011) the acute respiratory management of cervical spinal cord injury in the first 6 weeks after injury: a systematic review. spinal cord 49 (1):17–29. 60. kirshblum sc, groah sl, mckinley wo, gittler ms, stiens sa (2002) 1. etiology, classification, and acute medical management. archives of physical medicine and rehabilitation 83:s50–s57. 61. jackson ab, groomes te (1994) incidence of respiratory complications following spinal cord injury. archives of physical medicine and rehabilitation 75 (3):270–275. 62. berlly m, shem k (2007) respiratory management during the first five days after spinal cord injury. taylor & francis, . 63. tollefsen e, fondenes o (2012) respiratory complications associated with spinal cord injury. tidsskrift for den norske laegeforening: tidsskrift for praktisk medicin, ny raekke 132 (9):1111–1114. 64. publico/thromboembolism%20in%20sci.pdf uha–gdo (1999) prevention of thromboembolism in spinal cord injury. consortium for spinal cord medicine:1–29. 65. ploumis a, ponnappan r, maltenfort m, patel r, bessey j, albert t, harrop j, fisher c, bono c, vaccaro a (2009) thromboprophylaxis in patients with acute spinal injuries: an evidence-based analysis. jbjs 91 (11):2568–2576. 66. merli g, crabbe s, paluzzi r, fritz d (1993) etiology, incidence, and prevention of deep vein thrombosis in acute spinal cord injury. archives of physical medicine and rehabilitation 74 (11):1199–1205. 67. lamb gc, tomski ma, kaufman j, maiman dj (1993) is chronic spinal cord injury associated with increased risk of venous thromboembolism? the journal of the american paraplegia society 16 (3):153–156. 68. wang w-g, xiu r-j, xu z-w, yin y-x, feng y, cao x-c, wang p-s (2015) protective effects of vitamin c against spinal cord injury-induced renal damage through suppression of nf-κb and proinflammatory cytokines. neurological sciences 36 (4):521–526. 69. shunmugavel a, khan m, hughes fm, purves jt, singh a, singh i (2015) s‐nitrosoglutathione protects the spinal bladder: novel therapeutic approach to post‐spinal cord injury bladder remodeling. neurourology and urodynamics 34 (6):519–526. 70. middleton jw, mann l, leong g (2008) management of spinal cord injury in general practice-part 1. australian family physician 37 (4):229. 71. burns as, rivas da, ditunno jf (2001) the management of neurogenic bladder and sexual dysfunction after spinal cord injury. spine 26 (24s):s129–s136. 72. burns as, ditunno jf (2001) establishing prognosis and maximizing functional outcomes after spinal cord injury: a review of current and future directions in rehabilitation management. spine 26 (24s):s137–s145. 73. nikmehr b, bazrafkan m, hassanzadeh g, shahverdi a, gilani mas, kiani s, mokhtari t, abolhassani f (2017) the correlation of gene expression of inflammasome indicators and impaired fertility in rat model of spinal cord injury: a time course study. urology journal 14 (6):5057–5063. 74. choobineh h, kazemi m, gilani mas, heydari t, shokri s, bazrafkan m, hassanzadeh g (2018) testosterone reduces spinal cord injury-induced effects on male reproduction by preventing cadm1 defect. cell journal (yakhteh) 20 (2):138. 75. bazrafkan m, nikmehr b, shahverdi a, hosseini sr, hassani f, poorhassan m, mokhtari t, abolhassani f, choobineh h, beyer c (2018) lipid peroxidation and its role in the expression of nlrp1a and nlrp3 genes in testicular tissue of male rats: a model of spinal cord injury. iranian biomedical journal 22 (3):151. 76. sánchez-ramos a, vargas-baquero e, martin-de francisco f, godino-durán j, rodriguez-carrión i, ortega-ortega m, mordillo-mateos l, coperchini f, rotondi m, oliviero a (2017) early spermatogenesis changes in traumatic complete spinal cord-injured adult patients. spinal cord 55 (6):sc2016184. 77. choobineh h, gilani mas, pasalar p, jahanzad i, ghorbani r, hassanzadeh g (2016) the effects of testosterone on oxidative stress markers in mice with spinal cord injuries. international journal of fertility & sterility 10 (1):87. 78. qin w, bauman wa, cardozo c (2010) bone and muscle loss after spinal cord injury: organ interactions. annals of the new york academy of sciences 1211 (1):66–84. 79. wu y, zhao j, zhao w, pan j, bauman wa, cardozo cp (2012) nandrolone normalizes determinants of muscle mass and fiber type after spinal cord injury. journal of neurotrauma 29 (8):1663–1675. 80. thakore np, samantaray s, park s, nozaki k, smith ja, cox a, krause j, banik nl (2016) molecular changes in sub-lesional muscle following acute phase of spinal cord injury. neurochemical research 41 (1–2):44–52. 81. yarar-fisher c, bickel cs, kelly na, stec mj, windham st, mclain ab, oster ra, bamman mm (2016) heightened tweak-nf-κb signaling and inflammation-associated fibrosis in paralyzed muscles of men with chronic spinal cord injury. american journal of physiology-endocrinology and metabolism 310 (9):e754–e761. 271j contemp med sci | vol. 7, no. 5, september–october 2021: 264–271 i. mohammed et al. review clinical complications of spinal cord injury 82. jackman rw, cornwell ew, wu cl, kandarian sc (2013) nuclear factor‐κb signalling and transcriptional regulation in skeletal muscle atrophy. experimental physiology 98 (1):19–24. 83. qin w, sun l, cao j, peng y, collier l, wu y, creasey g, li j, qin y, jarvis j (2013) the central nervous system (cns)-independent anti-bone-resorptive activity of muscle contraction and the underlying molecular and cellular signatures. journal of biological chemistry 288 (19):13511–13521. 84. coupaud s, mclean an, purcell m, fraser mh, allan db (2015) decreases in bone mineral density at cortical and trabecular sites in the tibia and femur during the first year of spinal cord injury. bone 74:69–75. 85. bauman wa, cardozo cp (2015) osteoporosis in individuals with spinal cord injury. pm&r 7 (2):188–201. 86. tan co, battaglino ra, morse lr (2013) spinal cord injury and osteoporosis: causes, mechanisms, and rehabilitation strategies. international journal of physical medicine & rehabilitation 1. 87. baum r, gravallese em (2014) impact of inflammation on the osteoblast in rheumatic diseases. current osteoporosis reports 12 (1):9–16. 88. sipski ml, estores im, alexander cj, guo x, chandralapaty s (2004) lack of justification for routine abdominal ultrasonography in patients with chronic spinal cord injury. journal of rehabilitation research and development 41 (1):101. 89. campbell sj, zahid i, losey p, law s, jiang y, bilgen m, van rooijen n, morsali d, davis ae, anthony dc (2008) liver kupffer cells control the magnitude of the inflammatory response in the injured brain and spinal cord. neuropharmacology 55 (5):780–787. 90. hundt h, fleming j, phillips j, lawendy a, gurr k, bailey s, sanders d, bihari r, gray d, parry n (2011) assessment of hepatic inflammation after spinal cord injury using intravital microscopy. injury 42 (7):691–696. 91. fleming jc, bailey cs, hundt h, gurr kr, bailey si, cepinskas g, lawendy a-r, badhwar a (2012) remote inflammatory response in liver is dependent on the segmental level of spinal cord injury. journal of trauma and acute care surgery 72 (5):1194–1201. 92. sauerbeck ad, laws jl, bandaru vv, popovich pg, haughey nj, mctigue dm (2015) spinal cord injury causes chronic liver pathology in rats. journal of neurotrauma 32 (3):159–169. 93. guo l, rolfe aj, wang x, tai w, cheng z, cao k, chen x, xu y, sun d, li j (2016) rescuing macrophage normal function in spinal cord injury with embryonic stem cell conditioned media. molecular brain 9 (1):48. 94. lucin km, sanders vm, jones tb, malarkey wb, popovich pg (2007) impaired antibody synthesis after spinal cord injury is level dependent and is due to sympathetic nervous system dysregulation. experimental neurology 207 (1):75–84. 95. zhang y, guan z, reader b, shawler t, mandrekar-colucci s, huang k, weil z, bratasz a, wells j, powell nd (2013) autonomic dysreflexia causes chronic immune suppression after spinal cord injury. journal of neuroscience 33 (32):12970–12981. 96. zha j, smith a, andreansky s, bracchi-ricard v, bethea jr (2014) chronic thoracic spinal cord injury impairs cd8+ t-cell function by up-regulating programmed cell death-1 expression. journal of neuroinflammation 11 (1):65. 97. zong s, zeng g, fang y, peng j, tao y, li k, zhao j (2014) the role of il-17 promotes spinal cord neuroinflammation via activation of the transcription factor stat3 after spinal cord injury in the rat. mediators of inflammation 2014. 98. han tr, kim jh, kwon bs (1998) chronic gastrointestinal problems and bowel dysfunction in patients with spinal cord injury. spinal cord 36 (7): 485–490. 99. liu c-w, huang c-c, yang y-h, chen s-c, weng m-c, huang m-h (2009) relationship between neurogenic bowel dysfunction and health-related quality of life in persons with spinal cord injury. journal of rehabilitation medicine 41 (1):35–40. 100. correa g, rotter k (2000) clinical evaluation and management of neurogenic bowel after spinal cord injury. spinal cord 38 (5):301. 101. stiens sa, bergman sb, goetz ll (1997) neurogenic bowel dysfunction after spinal cord injury: clinical evaluation and rehabilitative management. archives of physical medicine and rehabilitation 78 (3):s86–s102. 102. fajardo nr, pasiliao r-v, modeste-duncan r, creasey g, bauman wa, korsten ma (2003) decreased colonic motility in persons with chronic spinal cord injury. the american journal of gastroenterology 98 (1): 128–134. 103. han sj, kim cm, lee je, lee th (2009) colonoscopic lesions in patients with spinal cord injury. the journal of spinal cord medicine 32 (4):404–407 104. guo j, zhu y, yang y, wang x, chen b, zhang w, xie b, zhu z, yue y, cheng j (2016) electroacupuncture at zusanli (st36) ameliorates colonic neuronal nitric oxide synthase upregulation in rats with neurogenic bowel dysfunction following spinal cord injury. spinal cord this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i5.1106 224 j contemp med sci | vol. 8, no. 4, july-august 2022: 224–228 original measurement serum level of leucine-rich alpha-2-glycoprotein-1 in iraqi hospitalized covid-19 patients maha h. gadhi*, eman s. saleh department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. *correspondence to: maha h. gadhi (e-mail:maha.hasan1200m@copharm.uobaghdad.edu.iq) (submitted: 12 april 2021 – revised version received: 19 may 2022 – accepted: 25 june 2022 – published online: 26 august 2022) abstract objective: the study aimed to assess leucine-rich alpha-2-glycoprotein-1 biomarker serum level in hospitalized covid-19 patients. methods: the case control study from multi-center in baghdad included 45 adult patients (19 females and 26 males) with covid-19, diagnosed with a positive real-time reverse transcription polymerase chain reaction and excluded negative rt-pcr for covid-19 and comorbidity conditions. second group, 43 control (20 females and 23 males). results: this study found decrease leucine-rich alpha-2-glycoprotein-1 biomarker serum level in these patients and a significant difference in d. dimer, neutrophil count, lymphocyte count, and neutrophil lymphocyte ratio between the patients and controls at a p value equal to 0.000. conclusion: the concentration of leucine-rich alpha-2-glycoprotein-1 in the patients after taking tocilizumab was greatly decreased in many studies. most of the patients in the study were treated with tocilizumab which agent that directly blocks the effect of il-6 by blocking the il-6 receptors and greatly decreased the expression of lrg1 to impair production of the angiopathogenic constituent. keywords: covid-19, interleukin-6, leucine-rich alpha-2-glycoprotein-1 issn 2413-0516 introduction coronavirus disease 2019 (covid-19) pandemic issued through the world health organization (who)1 at 11/3/2020 after announcement that popular of health emergency at 30/1/2020.2 the coronavirus detected according to genomic sequence of virus.3 the viral detection through the real-time or reverse transcription polymerase chain reaction (rt-pcr) testing.4 coronavirus is an enveloped rna virus from beta coronavirus genus,5 nidovirale order, and coronaviridae family.6 covid-19 infection extended from minor and severe infection and very common to cause to insult of different body tissues like heart, kidneys, gastrointestinal tract, and brain,7 covid-19 characterized by abnormal laboratory testing such as leukopenia, lymphopenia, elevated levels of c-reactive protein (crp), lactate dehydrogenase (ldh), d-dimer, serum ferritin, aminotransferase, and the abnormal finding further seen in computed tomography (ct) and chest x-ray.8 covid-19 enter the host cells through angiotensin converting enzyme2 (ace-2) protein7 that present on type ii pulmonary epithelial cells by a spike protein of the virus. the process of virus attachment is succeeded by host trans membrane serine protease 2 (tmprss2) that prim s2 subunit of spike protein to facilitate entry into host cell and caused an early phase through viral-related tissue injury directly and continued after the second phase after the infected host cells cause the activation of immune reaction by releasing the cytokines like interleukin-1 (il-1), interleukin-6 (il-6), interferon (ifn)-γ, tumor necrosis factor-α (tnf α), and other pro inflammatory mediators. over activation of immune response caused liberations greater amounts of cytokines mainly tnf-α and il-6 to the circulations as a cytokine storm and producing locally and extensively of the inflammatory reactions.9 this inflammation caused accumulation of fluid lead to acute respiratory distress syndrome (ards) that a major contributed factor for mortality within patients of covid-19.10 the pro-inflammatory mediator like il-6 and tnf-α can induce level of leucine-rich alpha-2-glycoprotein-1 (lrg1) that is about 50 kda plasma glycoprotein contain 312 amino acid residues and weighing 23% carbohydrate of it. its expressed within macrophages, neutrophils, the endothelial cells, and liver cells.9 lrg1 transcription was activated by il-6 by a phosphorylation and linking stat3 to a consensus sequence in promoter site of the lrg1. the inflammatory cytokines disrupted the vasculatures by many factorial, such as disruption the vascular effects that mediated by cytokine inducing lrg1,11 causing pulmonary edema and increase the vascular permeability.12 the circulating lrg1 levels have been presented to be raised in severe covid-19 patients13 where vascular damage is a primary caused. blocking of il-6 signal at pulmonary endothelium, by anti-il6 receptor antibody, tocilizumab is an immunomodulating agent and highly specific monoclonal antibody directly block effect of the il-6 by blocking the il-6 receptors and greatly decreased the expression of lrg1. lrg1 has been defined an acute phase protein released into the circulation and as biomarker the possible pathological role, prognostic biomarker through determination of severity of infection, and as the subsequent therapeutic targets in covid-19.11 the study aimed to assess the serum level of leucine-rich alpha-2-glycoprotein-1 biomarker in iraqi hospitalized covid-19 patients. measurement of correlation between the serum levels of leucine-rich alpha-2-glycoprotein-1 biomarker, d.dimer. lymphocyte count, and neutrophil count. materials and methods study design this study was approved by the university of baghdad/college of pharmacy and the iraqi ministry of health/rusafa health department. the study was involved a case control from the multi-center, the samples were collected from al-kindy teaching hospital, al-ataa hospital, and sheikh dhari al-fayadh hospital, in baghdad-iraq from september/2021 to january/2022. hospitalized patients with covid-19 tested. 225j contemp med sci | vol. 8, no. 4, july-august 2022: 224–228 m.h. gadhi et al. original measurement serum level of leucine-rich alpha-2-glycoprotein-1 in iraqi hospitalized covid-19 patients the inclusion criteria were involved adult patients from age (20 to 60) years (median age 47) with covid-19 who were diagnosed clinically, patients who showed the positively result to covid-19 infection through the real-time reverse transcription polymerase chain reaction (rt-pcr) of respiratory samples from nasal/oropharyngeal swabs,14 had a fever and pulmonary symptoms (cough, shortness of breath, chest tightness, and pain), and patients with radiological findings of consolidation either on chest x-ray or computed tomography (ct). exclusion criteria include the negatively result of rt-pcr to covid-19 infection, and comorbidities conditions (liver, renal, cardiovascular diseases, hypertension, diabetes, and autoimmune disease). the participants were divided into two groups: group 1: 45 (20 females and 25 males) covid-19 patients with ages that range between 20 and 60 years old. group 2: 43 (20 females and 23 males) control subjects with ages that range between 20 and 60 years old. laboratory analysis the three milliliters of blood samples that have been drawn from patients with covid-19 and healthy control were (1 ml in a gel tube) and left to coagulate for 15 minutes, then the samples were centrifuged at 5,000 rounds per minute (rpm) for 5 minutes to obtain the serum was collected by using the micropipette in a plain tube and stored about –20°c to measure the human leucine-rich alpha-2-glycoprotein-1 (µg/ml) sandwich enzyme-linked immunosorbent assay (elisa) kit.15 other blood samples 2 ml put in: • sodium citrate tube for measurement of d-dimer level in covid-19 patients. the sample of blood in was mixed gently for one minute to mix the sample with an anticoagulant reagent, then centrifuged at 4000 rpm for 6–10 minutes, collected plasma, and was used immediately for measurement level d-dimer by using fluorescence immunoassay.16 • edta test tube to prevent coagulation of blood sample. sysmex/xn-350 analyzer was used for evaluating the white blood cell differential count.17 statistical analysis the data were performed by using ibm spss software (version 26.0; ibm) and microsoft excel 2010. continuous variables were presented as median (interquartile range, iqr) because the variables not normally distributed. the number and percentage for the categorical variables of patients and healthy individuals were compared by using a chi square. the mann-whitney u test used for comparing the continuous variables between both groups. the two-tailed spearman correlation coefficient (non-parametric) for showing the correlation between the leucine-rich alpha-2-glycoprotein-1 biomarker serum level with d.dimer, lymphocyte count, neutrophil count, and neutrophil lymphocyte ratio (nlr). the analysis of receiver operating characteristic (roc) curve for assessing a test’s diagnostic performance or accuracy in distinguishing diseased from normal instances. the tests were two-tailed, and the statistically significant differences were considered at p-values of <0.05. results the study consisted from 45 hospitalized covid-19 patients, there were 20 females and 25 males and apparent healthy subjects 43 were 20 females and 23 males. the result of present study expressed as no significant difference (p value = 0.467) between the gender of patients and the control. this relationship between categorical data, percentages, and numbers are calculated by a chi-square test. the descriptive data were presented as median (interquartile range) due to these variables not normally distributed and p-values were calculated using the mann-whitney u test, a non-parametrical test and the significant differences were considered statistically at p-values of <0.05. the categorical data was represented as numbers and percentages and the relationship between both groups was compared and calculated p-values by the chi square and the statistically significant differences were considered at p-values of <0.05. the result of present study expressed as no significant difference between the age and gender of patients and the control at p value equal to 0.514, 0.467 respectively and significant difference between the leucine-rich alpha-2-glycoprotien-1, d. dimer, neutrophil count, lymphocyte count, and neutrophil lymphocyte ratio between the patients and control at p value equal to 0.000 for these variables (table 1). table 1. general characteristics of the variables between covid-19 patients and control variables patients controls p value age (20−60) year median (iqr) 47 (14) 49 (9) 0.514 gender male (female) number “percent” 25 “28.4%” (20 “22.7%”) 23” 26.1%” (20 “22.7%”) 0.846 lrg1 µg/ml median (iqr) 7.477 (7.963) 12.196 (37.449) 0.000 d. dimer µg/ml median (iqr) 1.06 (1.735) 0.1 (0.048) 0.000 neu × 103/µl median (iqr) 9.8 (4.250) 6.65 (0.99) 0.000 lym × 103/µl median (iqr) 1.2 (0.43) 1.88 (0.42) 0.000 nlr median (iqr) 7.6613 (4.59) 3.075 (1) 0.000 *p < 0.05 statistically significant differences. lrg1: leucine-rich alpha-2-glycoprotien-1 µg/ml, neu × 103/µl: neutrophil × 103/µl, and lym × 103/µl: lymphocyte × 103/µl, nlr: neutrophil lymphocyte ratio. 226 j contemp med sci | vol. 8, no. 4, july-august 2022: 224–228 measurement serum level of leucine-rich alpha-2-glycoprotein-1 in iraqi hospitalized covid-19 patients original m.h. gadhi et al. table 2. correlation between leucine rich alpha2 glycoprotien1 and d.dimer with d.dimer neutrophil count, lymphocyte count parameter lrg1 d.dimer lrg1 µg/ml p value r. p value r. – – 0.007 –0.284** d.dimer µg/ml p value r. p value r. 0.007 –0.284** – – neu × 103/µl p value r. p value r. 0.008 -0.28** 0.000 0.759** lym × 103/µl p value r. p value r. 0.002 0.331** 0.000 –0.54** nlr p value r. p value r. 0.000 –0.412** 0.000 0.733** **correlation is significant at the 0.01 level (2-tailed). p < 0.05 statistically significance. r.: correlation coefficient, lrg1: leucine-rich alpha-2-glycoprotien-1 (µg/ml), neu × 103/µl: neutrophil × 103/µl, and lym × 103/µl: lymphocyte × 103/µl, nlr: neutrophil lymphocyte ratio. table 3. receiver operating characteristic curve for study of covid-19 patients for d.dimer, lymphocyte count, neutrophil count and neutrophil lymphocyte ratio variable(s) accuracy area (auc) significance asymptomatic asymptomatic 95% confidence interval lower bound upper bound d.d (µg/ml) excellent 0.929 0.000 0.864 0.995 neu × 103/µl excellent 0.981 0.000 0.961 1.000 nlr excellent 0.992 0.000 0.981 1.000 lym × 103/µl very good 0.839 0.000 0.754 0.924 fig. 1 receiver operating characteristic curve for d. dimer µg/ml studied groups. in the table 2 lrg1 µg/ml and d.dimer µg/ml expressed a significant correlation with neu × 103/µl, lym × 103/µl, nlr and the significant correlation between lrg1 µg/ml and d.dimer µg/ml sperman correlation revealed a strong correlation between d.dimer µg/ml and neu × 103/µl and nlr and correlation coefficient (r.) values (0.759, 0.733) respectively. the table 3 is showing use of receiver operating characteristic (roc) for measurement the accuracy and area under the curve of the variables (d.dimer, neutrophil count, lymphocyte count and nlr). the accuracy of d-dimer for both groups. area under the curve (auc) of d.d was 0.929, the cutoff value of d-dimer in the s (0.2155 µg/ml), sensitivity 86.7% while the specificity is 97.7% as represented in figure 1. area under the curve (auc) of lymphocyte count was 0.839, the cutoff value of lymphocyte count (0.78 × 103/µl), sensitivity 2.2% while the specificity is 100% as represented in figure 2a while area under the curve (auc) of neutrophil count was 0.981, the cutoff value of neutrophil count (10 × 7.553/µl), sensitivity 88.9% while the specificity is 90.7% and area under the curve (auc) of neutrophil lymphocyte ratio was 0.992, the cutoff value of neutrophil lymphocyte ratio (4.3282), sensitivity 95.6% while the specificity is 97.7% as represented in figure 2b. discussion the study showed the male patients are more susceptible to covid-19 disease than women, and no significant differences in age between both genders, the study agreed with other 227j contemp med sci | vol. 8, no. 4, july-august 2022: 224–228 m.h. gadhi et al. original measurement serum level of leucine-rich alpha-2-glycoprotein-1 in iraqi hospitalized covid-19 patients studies that the males (72%) highly affected than females (28%) and no significance difference in both gender of patients, (p = 0.750) in severe covid-19 infection.18 this study also a significant differences in lymphocytes count and neutrophils count and nlr in covid-19 patients and agreed within the previous studied such as khartabil et al. complete blood count is widely used routine laboratories analysis, the detection of neutrophil count, nlr were extremely increase in covid-19 patients with comparison with healthy subjects and decrease level of lymphocyte count in those patients.17 the data of complete blood count that represented in the neutrophil count and lymphocyte count, and related to nlr. an increase the level of neutrophil count related to the systemic inflammation intensity while decrease the level of lymphocyte count related to sequestration of lymphocytes at site of inflammation and their apoptosis. the combination of these two biomarkers will be better indicator for detection the severe infection in covid-19.19 huang et al. that described same determinations, the patients of icu accomplished by increase leukocyte count, neutrophil count, with decrease lymphocyte count compared without icu.20 when decrease lymphocyte counts below 0.8 × 109/l can related to the severe covid-19 infection and increase neutrophil count higher than 3.5 × 109/l considered as bad medical outcomes.21 the covid-19 prognosis predicted when increase neutrophil-to-lymphocyte ratio (nlr), reported by yang et al. study.22 the meta-analysis for six studies showed the elevation of nlr might propose the poor prognostic within covid-19 patients.21 roc analysis curving appeared nlr is greatest of the accuracy over markers of complete blood count for measuring the severity of covid-19 within cut off value 4.3282 with acceptance to previous studies ciccullo, a. et al. that show the significant increase nlr with patients of severe covid-19 in the cohort study for 452 hospitalized patients.23 in this study appear the significant differences in d.dimer of severe form covid-19 patients and agreed within the previous study ye et al. showed increase d-dimer level in severe forms of covid-19 infection, an increased d-dimer values due to increase the activity of coagulation19 and contributed mechanism due to inflammatory mediator activation and contributed with rupturing of plaque by inflammatory response directly, induce of pro-coagulatory factors, and hemodynamically changing causing ischemia and thrombosis also the angiotensin converting enzyme 2 (ace-2) that sars-cov-2 receptor express with vascular endothelium, least ways for the possible direct viral invasion into myocardium.24 level of leucine-rich alpha-2-glycoprotien-1 biomarker in covid-19 patients in this study in compared with other studied as demichev et al. that reported of a cohort study that increased levels of the inflammatory and acute phase protein within the time such as lrp1 and related to possibility the death due to covid-19 infection.25 among the patients that collected the data of them were presenting in severe infection of covid-19 and presented in the hospitals that collect the data from it in long periods more that 25 days and greater. those patients had been taken tocilizumab as the humanized monoclonal antibody that its a interleukin-6 receptors blocker. in covid-19, first uses within 21 patients from chinese have serious states and notable enhancements. at the initial appearance of il-6 blocker strategies for applying the treatment another patients of covid-19 that include italian patients with different results that leading to the clinical trial of phase ii multicenter.26 in scientific reports of dritsoula et al., showing the block signal of il-6 receptors within tocilizumab to vascular endothelium of the lung causing reduce level of lrg1 led to impair production the angiopathogenic constituent. the meta-analysis and other studies reported tocilizumab for treatment the severly and critical illness of covid-19 have benefit results.11 the limitation in this study, timing of the sample that collected form the fig. 2 receiver operating characteristic curve for studied groups. (a) lymphocyte count (b) neutrophil count and neutrophil lymphocyte ratio. 228 j contemp med sci | vol. 8, no. 4, july-august 2022: 224–228 measurement serum level of leucine-rich alpha-2-glycoprotein-1 in iraqi hospitalized covid-19 patients original m.h. gadhi et al. patients, the sample should collect within the time of them admission to the hospital for measuring differences in the serum of biomarkers after treatment. conclusion the direct participation pathogenesis of immune system in covid-19 infection due to the pro-inflammatory cytokine causing induce systemic inflammation and pulmonary insult. the previous studies were showing the inflammatory mediators, as il-6 cause induce damage of epithelium. the lrg1 biomarker is the one of significant biomarkers producing angiopathogenesis causing disruption in the usual vascular physiology. abbreviation covid-19: coronavirus disease 2019, who: world health organization, rt-pcr: real-time reverse transcription polymerase chain reaction, crp: c-reactive protein, ldh: lactate dehydrogenase, ct: computed tomography, ace-2: angiotensin-converting enzyme2, tmprss2: trans membrane serine protease 2, il-1: interleukin-1, il-6: interleukin-6, ifn-γ: interferon-γ, tnf-α: tumor necrosis factor-α, ards: acute respiratory distress syndrome, lrg1: leucine-rich alpha-2-glycoprotein-1, nlr: neutrophil lymphocyte ratio, roc: receiver operating characteristic, neu × 103/µl: neutrophil × 103/µl, lym × 103/µl: lymphocyte × 103/µl, auc: area under the curve, icu: intensive care unit. acknowledgments special thanks to all physicians and laboratory staff in al-kindy teaching hospital, al-shifaa center al-ataa hospital, and sheikh dhari al-fayadh hospital who helped me in completing collecting data . conflict of interest no conflicts of interest. references 1. allawi, j. s. et al. the first 40-days experience and clinical outcomes in the management of coronavirus covid-19 crisis. single center preliminary study. j. fac. med. baghdad 61, (2019). 2. parasher, a. covid-19: current understanding of its pathophysiology, clinical presentation and treatment. postgraduate medical journal vol. 97 312–320 (2021). 3. al-imam, a., motyka, m. a. & al-doori, h. j. surface web merits for sarscov-2 pandemic in iraq. j. fac. med. baghdad 62, 117–127 (2020). 4. corman, v. m. et al. detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr. eurosurveillance 25, 2000045 (2020). 5. nassi, k. f. clinical evaluation of selected pharmacological treatments used for coronavirus (covid-19) pandemic. j. fac. med. 62, (2020). 6. ouassou, h. et al. the pathogenesis of coronavirus disease 2019 (covid-19): evaluation and prevention. journal of immunology research vol. 2020 (2020). 7. mirz, a. j. & taha, a. y. catheter directed thrombolysis for covid-19 thrombotic complications in kurdistan-iraq: two case reports. j. fac. med. 63, (2021). 8. alqubbanchi, f. b. & al-hamadani, f. y. a pharmacoeconomics study for anticoagulants used for hospitalized covid-19 patients in al-najaf al-ashraf city–iraq (conference paper). iraqi j. pharm. sci. (p-issn 1683-3597, e-issn 2521-3512) 30, 48–59 (2021). 9. yang, y. et al. leucine-rich α2-glycoprotein-1 upregulation in plasma and kidney of patients with lupus nephritis. bmc nephrol. 21, 1–11 (2020). 10. lester, m., sahin, a. & pasyar, a. the use of dexamethasone in the treatment of covid-19. ann. med. surg. 56, 218 (2020). 11. dritsoula, a. et al. angiopathic activity of lrg1 is induced by the il-6/stat3 pathway. sci. rep. 12, 1–14 (2022). 12. rajnik, m., cascella, m., cuomo, a., dulebohn, s. c. & di napoli, r. features, evaluation, and treatment of coronavirus (covid-19). (2021). 13. messner, c. b. et al. ultra-high-throughput clinical proteomics reveals classifiers of covid-19 infection. cell syst. 11, 11–24 (2020). 14. wei, p.-f. diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7). chin. med. j. (engl). 133, 1087–1095 (2020). 15. shinzaki, s. et al. leucine-rich alpha-2 glycoprotein is a serum biomarker of mucosal healing in ulcerative colitis. j. crohn’s colitis 11, 84–91 (2017). 16. farooqi, h. et al. elevated d-dimer levels are strongly associated with high mortality rate in covid-19 patients. an observational study: elevated d-dimers in covid-19 patients. pakistan biomed. j. 83–89 (2022). 17. khartabil, t. a., de frankrijker, m. m., de rijke, y. b. & russcher, h. the sysmex xn‐l (xn‐350) hematology analyzer offers a compact solution for laboratories in niche diagnostics. int. j. lab. hematol. 43, 29–39 (2021). 18. hachim, i. y. et al. male gender is a risk factor for sever form of covid-19 illness and worse outcome in the middle east. (2020). 19. ye, w. et al. dynamic changes of d-dimer and neutrophil-lymphocyte count ratio as prognostic biomarkers in covid-19. respir. res. 21, 1–7 (2020). 20. huang, c. et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet 395, 497–506 (2020). 21. pourbagheri-sigaroodi, a., bashash, d., fateh, f. & abolghasemi, h. laboratory findings in covid-19 diagnosis and prognosis. clin. chim. acta 510, 475–482 (2020). 22. yang, a.-p., liu, j., tao, w. & li, h. the diagnostic and predictive role of nlr, d-nlr and plr in covid-19 patients. int. immunopharmacol. 84, 106504 (2020). 23. ciccullo, a. et al. neutrophil-to-lymphocyte ratio and clinical outcome in covid-19: a report from the italian front line. int. j. antimicrob. agents 56, 106017 (2020). 24. zhou, f. et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. lancet 395, 1054–1062 (2020). 25. demichev, v. et al. a proteomic survival predictor for covid-19 patients in intensive care. plos digit. heal. 1, e0000007 (2022). 26. canziani, l. m. et al. interleukin-6 receptor blocking with intravenous tocilizumab in covid-19 severe acute respiratory distress syndrome: a retrospective case-control survival analysis of 128 patients. j. autoimmun. 114, 102511 (2020). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1232 24 j contemp med sci | vol. 9, no. 1, january-february 2023: 24–27 the impact of the ercc2 lys751gln polymorphism on the risk of acute myeloid leukemia in an iraqi patients thamer mouhi jasiem1* , rand muhammed abdul-hussain al-hussaini2 1department of microbiology, college of science, al-karkh university of science, baghdad, iraq. 2department of biology, faculty of science, kufa university, najaf, iraq. *correspondence to: thamer mouhi jasiem (e-mail: thamerbio2000@gmail.com) abstract objectives: aml is the only type of acute leukemia diagnosed in adults and is less common in children. it has the lowest survival rate. epidemiological risk factors for aml expansion comprise environmental factors, for instance, smoking, and therapy-related factors. methods: the study was conducted on 70 acute myeloid leukemia patients–37 females and 33 males and on 30 healthy people–12 females and 18 males–as a control group. dna was extracted from the study groups’ whole blood samples using the gsynctm dna extraction kit. the t751g polymorphism of the ercc2 gene was determined by the pcr-rflp technique. results: in genetic analysis, it was shown that the carriers of allele lys and genotype lys/lys have a lower risk of developing aml, while allele carriers gln have an increased risk. the results showed the ercc2 gene, lys 751 gln (t/g) heterozygous tg genotypes, and the g allele were significantly higher (p < 0.05) in aml patients compared to the control group. in the sequencing of the region we studied, it was found that there is a site of diversity that is located between the cttcag and ctgcag, where a change in nucleotides (t to g) represents the restriction site of the restriction enzyme. conclusion: the polymorphic marker 751 gln> lys of the ercc2 gene was associated with the development of aml in iraqi patients. it was discovered that allele lys genotype lys/lys carriers have a lower risk of developing aml, whereas allele gln carriers have an increased risk. keywords: leukemia, myeloid, acute, ercc2, polymorphism, genetic, dna repair issn 2413-0516 introduction aml is the one type of acute leukemia diagnosed in adults and less common in children and is connected with the lowest survival rate.1 epidemiological risk factors for aml expansion comprise environmental factors, for instance, smoking and exposure to benzene, therapy-related factors.2 aml derives from hematopoietic stem cells with a stepwise acquisition of genetic and epigenetic alterations. these assembled mutations influence normal hsc functions, obstructing differentiation and rising self-renewal capacity.3 the excision repair cross-complementing group 2 (ercc2) gene, also known as the xeroderma pigmentosum group d (xpd) gene, is located on chromosome 19q13.3. the ercc2 gene is consists of 23 exons and stretches about 54,000 base pairs.4 the ercc2 gene produces a protein which consists of 760 amino acids with a molecular weight of 86,900 and has been 5'–3' dna helicase activity that is adenosine triphosphate-dependent. the ercc2 protein is a part of the core transcription factor iih, which is particpated in nucleotide excision repair of dna by opening dna around the damage.5 materials and methods the study group was conducted on 70 acute myeloid leukemia patients 37 females and 33 males at the department of hematology, baghdad teaching hospital, medical city, for the period from march 2022 to july 2022, and 30 healthy people 12 females and 18 males as a control group. the ages of patients and control ranged between 15–82 years. dna isolation and polymerase chain reaction (pcr) under aseptic conditions, genomic dna was extracted from nucleated cells. dna was extracted from the study groups’ whole blood samples using the gsynctm dna extraction kit from geneaid. t751g polymorphism of the ercc2 gene was determined by pcr-rflp with the following primers: sense, f: 5’-cctctccctttcctctgttc-3’ and antisense, r: 5’-caggtgagggggacatct-3’.6 the 734 bp product was digested with 5 u of the restriction enzyme psti. amplification was carried out in 25 μl tube of pcr premix reaction mixture (accupower pcr premix, bioneer) amplification was performed in a thermal cycler (cleaver scientific ltd/uk) programmed for 35 cycles of denaturation at 94°c for 30 sec, annealing at 57°c for 30 sec, and extension at 72°c for 1 min, preceded by an initial denaturation of 5 min at 95°c. final extension was for 5 min at 72°c. finally, the gel electrophoresis method was done according to sambrook and russell,7 and 5 μl of each samples was loaded onto 2% agarose gel. statistical analysis through the use of the spss version 26 software, statistical analyses of all findings were completed. the c2-test was used to test hardy-weinberg equilibrium in both controls and cases for each polymorphism. chi-square analysis was used to find the genotype and allele frequency differences between the patients and controls. a measure of the association of the polymorphic sites with aml was also determined using odds ratios (ors) and 95% confidence intervals (cis). a p-value of 0.05 was considered significant. results an ercc2 variant with decreased ability to repair dna breaks has been linked to a single nucleotide genetic polymorphism (snp) in codon 751 of exon 23 (rs13181), where a change in nucleotides (t to g) leads to an amino acid change (lys to gln). (submitted: 11 november 2022 – revised version received: 02 december 2022 – accepted: 21 december 2022 – published online: 26 february 2023) original https://orcid.org/0000-0003-3492-9128 mailto:thamerbio2000@gmail.com 25j contemp med sci | vol. 9, no. 1, january-february 2023: 24–27 t. m. jasiem et al. the impact of the ercc2 lys751gln polymorphism on the risk of acute myeloid leukemia in an iraqi patients the occurrence of ercc2 gene polymorphism was revealed by rflp-pcr technique. at this position three genotype were found; tt, gg and tg,6 found that this locus had three genotypes only. the results revealed that gg homozygous genotype relative frequency was found to be 11.5% and 0% in the aml patients and control group respectively that was statically significant. also the heterozygous genes revealed significant differences where the aml patients 47% contained tg heterozygous genotype, while in the control group this genotype was present in 40%. the tt homozygous genotype was present in 60% of the controls, whereas it was 41.5% in the aml patients as shown in table 1. the results showed that the “g” allele is highly prevalent in the aml patients which was 35% as compared to the controls 20%, whereas the relative frequency “t” allele was 65% in the aml patients and 80% in the controls, as shown in table 2. discussion the present study revealed that the g allele, tg and gg genotype in aml patients were over than the controls, and observed that individuals with the gg genotypes had higher risk for developing aml disease. in contrast, the “t” allele, and tt genotype have a rather preventive role. this may indicate that the “t” allele may be protective. a significant association between polymorphism of ercc2 lys751gln and aml, overall data analysis revealed that ercc2 lys751gln may be significantly correlated with elevated leukemia risk. we discovered a strong correlation between the polymorphism lys751gln with the risk of developing aml (p-value = 0.03, or = 2.15; 95% ci = 1.05–4.43 for the gln allele). in the case of lys751gln, individuals with aml were more likely to have the combined heterozygous genotypes than controls (or = 1.34; 95% ci = 0.56–3.19; p–value = 0.03). this was also detected when the gln/gln genotype was examined (or = 8.30; 95% ci = 0.46–148.51). in this study, showed that an increase in the lys/lys genotype and the lys allele in the ercc2 codon 751 polymorphisms play a protective role in aml, and a increase in gln/gln genotype in acute leukemia was associated with early relapse tables 3 and 4. the relationship between these ercc2 polymorphisms and leukemia risk has been examined in some case-control studies, but the results of these studies remain confusing rather than conclusive. although a number of studies have found a link between ercc2 polymorphisms and the risk of certain types of leukemia, many researchers have discovered that the variant 751gln allele is associated with an increased risk of aml.8-16 other studies, on the other hand, did not consider the ercc2 genetic variants to be risk or protective factors for leukemia because they demonstrated that the presence of the ercc2 751gln allele had a protective effect in the development of aml.17-19 sequencing of amplified ercc22 gene in order to check up the genetic variation in a rs 13181; lys751gln, t/g in exon 23 ercc2 gene. sequencing was table 2. comparative analysis of the distribution of allele frequencies of polymorphic marker ercc2 gene; lys751gln among patients with aml and in the control group alleles cases controls b2 p or n = 70 n = 30 value 95% ci allele t 91(65%) 48(80%) 4.46 0.03 0.46 0.23–0.96 allele g 49(35%) 12(20%) 2.15 1.05–4.43 table 3. distribution of ercc2 gene; lys751gln in the study population under dominant inheritance model genotypes cases controls b2 p or n = 70 n = 30 value 95% ci genotype t/t 29(41.5%) 18(60%) 2.91 0.09 0.47 0.20–1.13 genotype t/g+g/g 41(58.5%) 12(40%) 2.12 0.89–5.07 table 4. distribution of ercc2 gene; lys751gln in the study population under recessive inheritance model genotypes cases controls b2 p or n = 70 n = 30 value 95% ci genotype t/t+t/g 62(88.5%) 30(100%) 3.73 0.05 0.12 0.01–2.16 genotype g/g 8(11.5%) 0(0%) 8.30 0.46–148.51 table 1. comparative analysis of the distribution of genotype frequencies of polymorphic marker ercc2 gene; lys751gln among patients with aml and in the control group genotypes cases controls b2 p or n = 70 n = 30 value 95% ci genotype t/t 29(41.5%) 18(60%) 4.75 0.03 0.47 0.20–1.13 genotype t/g 33(47%) 12(40%) 1.34 0.56–3.19 genotype g/g 8(11.5%) 0( 0%) 8.30 0.46–148.51 original 26 j contemp med sci | vol. 9, no. 1, january-february 2023: 24–27 the impact of the ercc2 lys751gln polymorphism on the risk of acute myeloid leukemia in an iraqi patients t. m. jasiem et al. fig. 1 comparison of the alignment of nitrogenous bases in patient samples of a fragment of dna from the ercc2 gene, a rectangle indicates the difference site in one of the nitrogenous bases, which is the same as the restriction site of the enzyme. fig. 2 the location of the occurrence of genetic diversity in the studied sequence, the arrow indicates the genotype of patient as a result of the presence of one homozygous genetic pattern gg. fig. 3 the location of the occurrence of genetic diversity in the studied sequence, the arrow indicates the genotype of control as a result of the presence of one homozygous genetic pattern tt. performed to determine the genetic variation in iraqi patients with aml compared with the apparently healthy control. the complete nucleotide sequence is examined and the results were illustrated in figures 1-3. through the current study of the sequential sequence of the region we studied, it was found that there is a site of diversity which located between the cttcag and ctgcag where a change in nucleotides (t to g), which represents the restriction site of the restriction enzyme, and this means that the enzyme is cut in the case of the presence of g in the target site of a segment and not cut in the case of the presence of the base t, when compared to the source reference (ref.) or a comparison between patient samples and control samples (figure 1). original 27j contemp med sci | vol. 9, no. 1, january-february 2023: 24–27 t. m. jasiem et al. the impact of the ercc2 lys751gln polymorphism on the risk of acute myeloid leukemia in an iraqi patients for investigated the presence of genetic diversity or mutations in the region we used various and different genetic analysis methods, the results were compared with what was published in the gene bank website located within the american national center for biotechnology information (ncbi) website, which is http://www.ncbi. nlm.nih.gov/. the blast method was searched, which is a tool for searching for matches in sequences on the gene bank website, after removing excess and non-conforming sequences (rubbish) from both ends of the sequences and alignment them using the computerized bioedit program. the target region was obtained and compared with the sequences obtained for the patient and control samples. the ercc2 region was registered and published on the gene bank website located within the national center for biotechnology information (ncbi) website under assigned accession number (lc735410) https://www.ncbi.nlm.nih. gov/nuccore/lc735410.1/. conclusion an association of polymorphic marker 751 gln> lys of the ercc2 gene with the development of aml in iraqi patients. it was shown that the carriers of allele lys genotype lys/lys have a lower risk of developing aml, while allele carriers gln have an increased risk. conflicts of interest “the authors declare no conflicts of interest.”  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1. yamamoto, j.f. and m.t. goodman, patterns of leukemia incidence in the united states by subtype and demographic characteristics, 1997–2002: cancer causes control, 2008. 19(4): 379–90. doi: 10.1007/s10552-0079097-2. 2. zeeb, h. and m. blettner, adult leukaemia: what is the role of currently known risk factors. radiat environ biophys,1998. 36: 217–228. 3. corces-zimmerman, m. r. and r. majeti, pre-leukemic evolution of hematopoietic stem cells: the importance of early mutations in leukemogenesis. leukemia, 2014. 28(12): 2276–2282. 4. weber, c.a., et al., ercc2: cdna cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast rad3. embo j, 1990. 9: 1437–47. doi: 10.1002/j.1460-2075.1990.tb08260.x. 5. schaeffer, l., et al., the ercc2/dna repair protein is associated with the class ii btf2/tfiih transcription factor. embo j., 1994. 13: 2388–92. doi: 10.1002/j.1460-2075.1994.tb06522.x. 6. dexi, j., et al., impact of polymorphisms in dna repair genes xpd, hogg1 and xrcc4 on colorectal cancer risk in a chinese han population. bioscience reports, 2019. 39: 1–9. 7. russell, d.w., and j. sambrook, molecular cloning: a laboratory manual. cold spring harbor: cold spring harbor laboratory, 2001. 8. james, m., et al., genetic variation in xpd predicts treatment outcome and risk of acute myeloid leukemia following chemotherapy blood, 2004. 104(13): 3872–3877. doi: 10.1182/blood-2004-06-2161. 9. ilhan, g., et al., risk factors and primary prevention of acute leukemia. asian pac. j. cancer prev. 2006. 7: 515–517. 10. ali, ö., et al., polymorphisms of the dna repair gene xpd (751) and xrcc1 (399) correlates with risk of hematological malignancies in turkish population. african journal of biotechnology, 2011. 10(44): 8860–8870. 11. meenaghan, t., et al., acute leukaemia: making sense of a complex blood cancer. br. j. nurs, 2012. 21 (76): 78–83. doi: 10.12968/bjon.2012.21.2.76. 12. duo, l., et al., the effect of xpd/ercc2 lys751gln polymorphism on acute leukemia risk: a systematic review and meta-analysis gene, 2012. 538: 209–216. doi: 10.1016/j.gene.2014.01.049 13. liu, d., et al., the effect of xpd/ercc2 lys751gln polymorphism on acute leukemia risk: a systematic review and meta-analysis. gene, 2014. 538(2): 209–16. 14. claudia, b., et al., influence of xpc, xpd, xpf, and xpg gene polymorphisms on the risk and the outcome of acute myeloid leukemia in a romanian population, tumour biol, 2016. 37(7): 9357–66. doi: 10.1007/s13277-0164815-6. 15. dolly, j., et al., association of xpd (lys751gln) and xrcc1 (arg280his) gene polymorphisms in myelodysplastic syndrome. ann hematol, 2016. 95(1): 79–85. doi: 10.1007/s00277-015-2528-3. 16. zohreh, s., et al., genetic variants of nucleotide excision repair pathway and outcomes of induction therapy in acute myeloid leukemia. per. med., 2019. 16(6): 479–490. doi: 10.2217/pme-2018-0077. 17. mehta, p.a., et al., xpd lys751gln polymorphism in the etiology and outcome of childhood acute myeloid leukemia: a children’s oncology group report, blood. 2006. 107(1): 39–45. doi: 10.1182/blood-2005-06-2305. 18. sorour, a., et al., the genotype distribution of the xrcc1, xrcc3, and xpd dna repair genes and their role for the development of acute myeloblastic leukemia. genet test mol biomarkers, 2013,17: 195–201. doi: 10.1089/ gtmb.2012.0278. 19. kais, d.et al., polymorphisms in xpc, xpd and xpg dna repair genes and leukemia risk in a tunisian population. leuk lymphoma, 2015. 56(6): 1856–62. doi: 10.3109/10428194.2014.974045. https://doi.org/10.22317/jcms.v9i1.1308 original https://www.ncbi.nlm.nih.gov/nuccore/lc735410.1/. https://www.ncbi.nlm.nih.gov/nuccore/lc735410.1/. https://doi.org/10.1007/s10552-007-9097-2 https://doi.org/10.1007/s10552-007-9097-2 https://doi.org/10.1002/j.1460-2075.1990.tb08260.x https://doi.org/10.1002%2fj.1460-2075.1994.tb06522.x https://doi.org/10.1182/blood-2004-06-2161 https://doi.org/10.12968/bjon.2012.21.2.76 https://doi.org/10.1016/j.gene.2014.01.049 https://doi.org/10.1007/s13277-016-4815-6 https://doi.org/10.1007/s13277-016-4815-6 https://doi.org/10.1007/s00277-015-2528-3 https://doi.org/10.2217/pme-2018-0077 https://doi.org/10.1182/blood-2005-06-2305 https://doi.org/10.1089/gtmb.2012.0278 https://doi.org/10.1089/gtmb.2012.0278 https://doi.org/10.3109/10428194.2014.974045 211j contemp med sci | vol. 7, no. 4, july-august 2021: 211–216 original ajwa seeds (phoenix dactylifera l.) suspension exerted antidiabetic and antihyperlipidemic effects against streptozotocin-induced diabetes in rats by downregulating insulin expression in the pancreatic beta islets ahlam abdulaziz alahmadi*, hessah mohammed banayah department of biological sciences, college of science, king abdulaziz university, jeddah, saudi arabia. *correspondence to: ahlam abdulaziz alahmadi (e-mail: aahmadi1000@hotmail.com) (submitted: 18 april 2021 – revised version received: 29 april 2021 – accepted: 11 may 2021 – published online: 26 august 2021) abstract objectives this study investigated the antidiabetic and antihyperlipidemic effects of the powdered seeds of ajwa al-madina in streptozotocin (stz)-induced diabetes in rats. besides investigating the possible underlying mechanisms. methods rats were assigned to one of six groups (n = 5) as follows: normal control, vehicle control, ajwa seeds control, diabetic control, diabetic + metformin, and diabetic + ajwa seeds. metformin and ajwa seeds were injected into rats orally via oral gavage 6 days/week along 4 weeks period. results ajwa seeds decreased fasting serum glucose, increased fasting serum insulin and decreased fasting serum triglycerides cholesterol, low-density lipoprotein, and increased fasting serum high-density lipoprotein. besides, it upregulated insulin protein immunoexpression in the beta cells of langerhans islets. ajwa seed also preserved the normal histological structure of the pancreatic beta cells tissue. conclusion ajwa seeds produced significant hypoglycemic and hypolipidemic effects in diabetic rats mainly through enhancement of insulin secretion. the plant is a promising adjunctive therapy in diabetes mellitus treatment. keywords ajwa seeds, glucose, insulin, histopathology, lipids, immunoexpression introduction diabetes mellitus (dm) is one of the world’s fastest-growing health issues in the twenty-first century. it is one of the leading causes of death worldwide. if not treated properly, it can cause chronic health problems, especially cardiovascular, renal, and nerve complications.1 according to the international diabetes federation (idf), the global prevalence of dm is projected to rise from 425 million patients in 2017 to 629 million in 2045. in the middle east, dm claimed the lives of 38.7 million people between the ages of 20 and 79 years in 2017.2,3 dm is one of the most prevalent health problems in saudi arabia (sa).4 dm has also increased tenfold in sa over the last three decades.5 while there are several oral drugs available to treat dm, the majority of them are ineffective at preventing serious complications, regulating blood glucose levels, and are associated with numerous side effects.6 in the last century, herbal medicines have gained popularity. before the introduction of medications, traditional healers used herbal products to cure dm. a plant extract is a mixture of organic chemicals derived from some portion of the plant. as a result, they are healthy since they are organic and thus less dangerous and have fewer adverse reactions.7,8 many fruits’ seeds are used in traditional and herbal medicine to prevent diseases, relieve stress, and minimize side effects.9 dates (phoenix dactylifera l., family arecaceae) are a common fruit in the arab world. it is regarded as a major nutritional and economic product.10 in egypt, sa, and other middle east countries, it is the most important crop.11 phoenix dactylifera (ph. dactylifera) seeds are normally discarded by the fruit industry as byproducts. these seeds have often been roasted, ground, and used as a caffeine-free coffee substitute, either pure or mixed with coffee, as well as animal feed.12 the various parts of ph. dactylifera are popularly used to manage a variety of ailments, including neurological diseases, memory deterioration, paralysis, hyperthermia, and coma.13 date fruits and seeds, in the form of powder, pulp, and infusion, are commonly used in research to treat atherosclerosis, malignancy, asthenia, pulmonary ailments, and throat infections. date fruits and seeds are also used as an antidiarrheic, hypoglycemic, expectorant, tonic, aphrodisiac, and mouthwash.14 ajwa is a form of the date that is only grown in sa/ al-madinah al-munawara and has a high medicinal value. the health benefits of ajwa dates have been recorded in hadith, with saud (r.a) narrating, “if anyone takes seven ajwa dates in the morning, neither magic nor poison can harm him that day”.11 ajwa date seed extracts were found to have an antihyperglycemic activity in laboratory animals in several recent studies.15,16 however, the mechanism underlying this effect remains unknown and requires further investigation and evidence. this study aims to examine if the powdered seeds of ajwa al-madina have a therapeutic impact on dm induced by streptozotocin (stz) in rats, and if so, what the mechanism is. materials and methods drugs and chemicals stz was obtained from sigma-aldrich, usa. metformin (glucophage, 500 mg, merck santé, france) from alnahdi pharmacy, sa. ajwa seeds were gathered from ajwa dates, oasis lina, al-madinah al-munawara, sa. ajwa seeds aqueous suspension the seeds were separated from the fleshes, and the deposit was cleaned. seeds were air-dried at ambient temperature for 15–21 days. the dried seeds were ground into a fine powder using a hammer mill. one-gram ajwa seeds powder was mixed with 10 ml tween-80 to make seeds suspension (figure 1). issn 2413-0516 mailto:aahmadi1000@hotmail.com 212 j contemp med sci | vol. 7, no. 4, july-august 2021: 211–216 ajwa seeds (phoenix dactylifera l.) suspension exerted antidiabetic original a.a. alahmadi and h.m. banayah determination of ajwa seeds volatile constituents solid-phase extraction-gas chromatography/mass spectrometry (spe-gc/ms) analysis was performed based on the recently published method of 17 at the analytical chemistry unit, assiut university, assiut, egypt. animals thirty adult wistar rats (150 g 250 g body weight) were obtained from king fahad research centre, king abdulaziz university (kau), jeddah, sa. the rats were maintained for one week under the standard laboratory circumstances of temperature, humidity, and light/dark cycle (12:12 h). during the adaptation period, the rats could drink and eat free. ethical approval the study design was accepted by the biomedical ethics research committee (registration number: 346-19), college of medicine, kau, jeddah, sa. induction of diabetes the rats were given a single injection of stz (35 mg/kg) intraperitoneally.18 fasting blood glucose levels were measured using the accu-chek apparatus after seven days. glucose levels higher than 200 mg/dl were identified as diabetes rats19 (figure 2). experimental groups rats were assigned to one of six groups (n = 5) as follows: 1normal control (nc) group: rats received 2 ml distilled water. 2vehicle control (vc) group: rats received 2 ml tween 80. 3ajwa seeds control (ac) group: rats received 2 ml ajwa seeds suspension (1 g/kg).20 4diabetic control (dc) group: diabetic induced rats received 2 ml distilled water. 5diabetic + metformin (dm) group: diabetic rats received 2 ml metformin solution (150 mg/kg).21 6diabetic + ajwa seeds (da) group: diabetic rats received 2 ml ajwa seeds suspension (1 g/kg). metformin and ajwa seeds were injected into rats orally via oral gavage 6 days/week along 4 weeks period (figure 2). obtaining samples four weeks after treatment protocol administration, rats were starved overnight and were sedated with ether for blood withdrawal from the heart (heart puncture technique). blood samples were centrifuged at 3500 rpm for 15 minutes under cooling conditions then the sera were separated and kept frozen at –80°c. after that, the rats were killed and dissected, and all pancreases were excised, washed in 0.9 percent saline, and stored either frozen at –80°c or in a 10% buffered formalin solution (figure 2). assessment of fasting serum glucose fasting serum glucose concentrations were determined using diagnostics reactivos gpl, barcelona, spain colorimetric kit according to the brochure instructions. assessment of fasting serum insulin fasting serum insulin concentrations were determined using immunospec, ca elisa kit according to the brochure instructions. assessment of fasting serum lipid profile the fully automated technique using elisa, (dynex) was used to measure fasting serum lipid profile (triglyceride (tg), cholesterol (chol), low-density lipoprotein (ldl), and high-density lipoprotein (hdl)) according to the instruction brochure accompanying the kits. histopathological study (hematoxylin and eosin (h & e) staining) formalin-fixed pancreases were processed in the histology lab, pathology department, king abdulaziz university, sa, and were stained with h & e. the sections were then examined under the light microscope by a blind pathologist. fig. 1 ajwa dates and ajwa seeds. a and b: ajwa date purchased from the oasis lina (dates company), c: seeds removed from the fruit and washed to remove the residues, d: ground seeds (fine powder). fig. 2 diabetes induction, dose administration, and sample collection. a: induction of diabetes in rats using an intraperitoneal injection of stz, b: determine blood glucose levels using accu-chek, c: treatment the rats using oral gavage, d: a collection of the blood from the heart, e: dissection the rats. a.a. alahmadi and h.m. banayah 213j contemp med sci | vol. 7, no. 4, july-august 2021: 211–216 original ajwa seeds (phoenix dactylifera l.) suspension exerted antidiabetic immunohistochemical study (insulin and nuclear factor kappa beta (nf-κb) for determining the presence and secretion of insulin in pancreatic beta cells, the sections were stained immunohistochemically for insulin using.22 for determining the inflammatory cells in the sections, the pancreas sections were stained immunohistochemically for nf-κb.23 an immunoperoxidase (peroxidase/anti peroxidase, pap) protocol was utilized to stain the pancreas segments for insulin and nf-κb proteins. the antibodies (bought from lab vision, fremont, ca) were diluted in 1:200 dilution. the segments were analyzed and photted, utilizing light microscopy. data analysis the averages ± standard errors were used to display the data. the data were analyzed statistically using a one-way analysis of variance (anova) and tukey’s post-hoc test. a p-value of less than 0.05 was used as the significant level. spss for windows, version 22, armonk, ny was used to conduct the data analysis. results ajwa seeds volatile constituents table 1 showed of the volatile active constituents of ajwa seeds powder. effect of ajwa seed and metformin on fasting serum glucose measured in control and diabetic rats there were no significant differences between the serum glucose concentration of the three control groups (nc, vc, and ac). the dc group showed a significantly increased serum glucose concentration compared to the nc group (p < 0.001). treatment of dc group with metformin and ajwa seed suspension significantly decreased serum glucose concentration compared to dc group (p < 0.001). ajwa seed suspension significantly decreased serum glucose concentration compared to the dm group (p < 0.05) (fig. 3). effect of ajwa seed and metformin on fasting serum insulin measured in control and diabetic rats the dc group showed a significantly decreased serum insulin concentration compared to the nc group (p < 0.001). treatment of dc group with metformin and ajwa seed suspension significantly increased serum glucose concentration compared to dc group (p < 0.001). ajwa seed suspension significantly increased serum glucose concentration compared to the dm group (p < 0.001) (fig. 4). effect of ajwa seed and metformin on serum lipids profile measured in control and diabetic rats there were no significant differences between various serum lipids concentrations of the three control groups (nc, vc, and ac). the dc group showed significantly increased serum tg, chol, and ldl concentrations compared to the nc group (p < 0.001). on the other hand, serum ldh concentration was table 1. ajwa seeds volatile constituents analyte/parameter value octadecanoic acid 1.158% 2,3dehydro-3-hydroxy-2-4 dimethylamine 0.219% n-(1-methyl cyclopropyl) urea 0.116% cyclolanost-24-en-3-ol3)919 beta( 6.226% 2,2-dimethy-ltetrahydrofuran 2.568% 2,6-bis (1,1-dimethyllethyl)-4-methyl-phenol 0.347% amine, 6-methixy1h-purine 0.766% propenoic acid, 1-methylundecyl ester 0.035% 3-(4-aminobutyl) piperidine 0.007% 5-htdroxy-2,4 (1h,3h)-pyrimidinedione 0.164% 9-hexadecenoic acid, octadecyl ester 0.142% 9-octadecenoic acid (e) 0.5334% 9octadecenoic acid (z)-, tetradecyl ester 0.222% alpha -terpinoene 0.058% androst-5, 15-dien-3ol acetate 0.578% benzo[f]quinoline 0.410% cis-13-octadecenoic acid 1.666% delta.9-cis-oleic acid 0.457% di-tert-butylphenol 0.099% dodecanoic acid 3.746% farnesyl acetate 3 0.506% gamma. -sitosterol 2.630% glycyl-dl-alanine 0.008% hexanedioic acid, bis (2-ethylhexyl) ester 0.181% i-propyl 9-octadecenate 0.378% n-[amino(imino)methyl]-3-(2,4-dihydroxy-5 pyrimidinyl) alanine 0.006% n-butyl-n-nitrosourea 0.054% n-hexadecoic acid 2.102% n-propyl 11-octadecenoate 0.101% octadecenoic acid, 1, 2, 3-propanetriyl ester 0.050% oleic acid 0.614% pentafluoro propionic acid, 4-hexadecyl ester 0.477% phytane 0.249% squalene 0.434% stigmastan-3,5-diene 1.348% tera decanoic acid 2.258% terahydrofurfuryl alcohol 0.948% decreased in the dc group compared to the nc group (p < 0.001). treatment of dc group with metformin and ajwa seed suspension significantly decreased serum tg, chol, and ldl concentrations compared to dc group (p < 0.001). furthermore, treatment of dc group with metformin and ajwa seed suspension significantly increased serum hdl concentration compared to the dc group (p < 0.001). ajwa seed suspension significantly decreased serum ldl concentration compared to the dm group (p < 0.001) (table 2). 214 j contemp med sci | vol. 7, no. 4, july-august 2021: 211–216 ajwa seeds (phoenix dactylifera l.) suspension exerted antidiabetic original a.a. alahmadi and h.m. banayah effect of ajwa seed and metformin on histopathological changes in control and diabetic rats pancreas as shown in fig. 5 islets of langerhans of nc pancreas revealed normal morphology. also, there were normal density and distribution of different cell population. the beta cells were apparently larger in size and were most central in fig. 4 effect of ajwa seed and metformin on fasting serum insulin measured in control and diabetic rats. data were displayed as averages ± standard errors. adisplayed significant difference compared to nc. bdisplayed a significant difference compared to dc. cdisplayed a significant difference compared to dm. ***displayed significant difference at p < 0.001. fig. 3 effect of ajwa seed and metformin on fasting serum glucose measured in control and diabetic rats. data were displayed as averages ± standard errors. adisplayed significant difference compared to nc. bdisplayed a significant difference compared to dc. cdisplayed a significant difference compared to dm. ***displayed significant difference at p < 0.001. *displayed significant difference at p < 0.05. table 2. effect of ajwa seed and metformin on serum lipid profile measured in control and diabetic rats groups tg (mg/dl) chol (mg/dl) ldl (mg/dl) hdl (mg/dl) nc 101 ± 1.4 117 ± 2.9 29 ± 0.27 21 ± 0.78 vc 97 ± 3.2 125 ± 0.34 29 ± 0.92 17 ± 1.9 ac 102 ± 0.5 125 ± 0.73a 27 ± 1.2 19 ± 1.3 dc 124 ± 2.9a*** 144 ± 1.7a*** 43 ± 0.63a*** 8 ± 0.15a*** dm 105 ± 1.3b*** 127 ± 2.1b*** 36 ± 0.77b*** 13 ± 0.1b*** da 101 ± 1.2b*** 132 ± 2.2b*** 29 ±1.6b***, c*** 13 ± 0.2b*** data were displayed as averages ± standard errors. adisplayed significant difference compared to nc. bdisplayed a significant difference compared to dc. cdisplayed a significant difference compared to dm. ***displayed significant difference at p < 0.001. fig. 5 effect of ajwa seed and metformin on histopathological changes observed in control and diabetic rats’ pancreas. sections from rat’ pancreases were stained with h & e and photographed at high (x600) magnification. nc: normal control showed normal islets with normal density and distribution of different cell populations. beta cells are most central in position, showed the equal size and centrally located nuclei (black arrow). also, blood capillaries between the cells are thin-walled and compressed. vc: vehicle control showed no alteration in the normal cell population of langerhans islets. also, in ac: ajwa seed control showed normal morphology islets component and no alteration compared to control. dc: diabetic control showed decreased nuclear density with numerous regions that completely lack nuclei (necrotic region). the islets showed a large area of hemorrhage (star). some cells had vacuolated cytoplasm (white arrows). dm: diabetic + metformin showed preservation of normal islet morphology and cell density. da: diabetic + ajwa seed showed an apparent increase in the size of central cells and more preservation in the islets of langerhans. notice: the potential decrease in necrotic cells in dm and da group. position, their nuclei were lightly stained (active) and centrally located. blood capillaries between the cells were thin-walled and compressed. in vc and ac groups there was no alteration in the normal cell density of islets cells. in the dc group, the islets showed central cells with a larger size, homogenous acidophilic cytoplasm, degenerated small, and even lost nuclei. in the dm group preservation of normal islets morphology, and cell density was observed. the apparent increase in the size of central cells is evident. more evident preservation was observed in the islets of the da group. effect of ajwa seeds on pancreatic immunoexpression of insulin observed in control and diabetic rats’ pancreas as shown in fig. 6 islets of langerhans of nc pancreas showed highly positive staining for insulin which was observed in a large population of centrally located cells in langerhans islets. in vc and ac groups no alteration was observed in immunostaining for insulin compared to the nc group. in the dc group, a significant decrease in positive insulin-immunostained cells was observed where they apparently looked enlarged compared to those of the nc. in dm and da treated groups a significant increase of insulin immunostained cells was observed compared to the dc group. discussion diabetes mellitus is a long-term condition that is linked to several metabolic problems. hyperglycemia, or high blood glucose, is the most prevalent sign of diabetes. the underlying causes of diabetes are either a lack of insulin production or insulin resistance or both.24 chemical medicines have been a.a. alahmadi and h.m. banayah 215j contemp med sci | vol. 7, no. 4, july-august 2021: 211–216 original ajwa seeds (phoenix dactylifera l.) suspension exerted antidiabetic shown in studies to be ineffective in treating dm since it does not entirely control glucose levels and has numerous negative effects. in this regard, research on plant species has demonstrated their usefulness in diabetes treatment, with toxicological tests proving that they are the safest choice.25 this study aimed to confirm the therapeutic impact of the powdered seeds of ajwa al-madina on dm induced by stz in rats. ajwa seeds demonstrated a significant increase in insulin level, which led to a considerable decrease in blood glucose levels when compared to untreated diabetic rats. hence, the glucose level returned to a more normal range in ajwa seeds group. in addition, ajwa seeds significantly decreased the blood glucose level than metformin (the reference antidiabetic drug). the current findings are consistent with those of a previous study, which found that saudi date seeds extract incorporating ajwa seeds had a hypoglycemic and antidiabetic impact in the stz rat model.15,16 additionally, hypoglycemic effects were reported in many types of phoenixes dactylifera l. seeds and flesh such as sukkari date seeds,16 hayani,26 and other types.27 similar results hypothesized that ajwa date seeds stimulate insulin secretion from existing β-cells, consequently, the blood glucose levels decreased. in diabetic rats treated with date seed extract, the data revealed a significant elevation in insulin concentration, which could explain the significant decrease in mean blood glucose concentration noticed in those rats.28 furthermore, a prior study backs up the effectiveness of date seed extract in controlling hyperglycemia.29 histological analysis of islets of langerhans employing both routine h & e staining and insulin immunostaining was utilized to show if ajwa seeds function by modulating the structure and insulin content of beta cells. in the present study, induction of diabetes was associated with many beta cells morphology alterations and degenerative changes including vacuolation, cytoplasmic hyalinization, and degraded or missing nuclei. similar findings were also reported by.22,24 the results of this study also showed that the administration of ajwa seeds was found to restore the normal structure of β-cells islets and the immunostaining for insulin. the current results revealed that ajwa seeds increase insulin secretion from the islets beta cells. while a previous study showed that date seed extract exerts its hypoglycemic impact on type 1 diabetic mice by enhancement of endogenous insulin production via extraislet sources, according to.28 ajwa seeds produced a significant decrease in all harmful lipids including tf, chol, and ldl, where it significantly increases the useful lipid, hdl. these findings are consistent with the recent results which concluded that ajwa date pit ameliorated hyperglycemia and hypercholesterolemia associated with obesity in experimental animals. the antioxidants impact of the pits may clarify the observed lipid and glucose lowering action.30 similarly, many previous studies reported the antihyperlipidemic activity of dates pits and suggesting that the polyphenols’ active constituents of the pits may exert both antioxidants and inhibiting the pancreatic lipase activity resulting in decreasing gastrointestinal lipid absorption and hence lowered the blood lipids.31–33 conclusion ajwa seeds possess antidiabetic and antihyperlipidemic effects against stz-induced diabetes in rats. the preservation of beta cells in the pancreas and induction of insulin secretion is the most likely mechanism by which ajwa seeds exerted their antidiabetic action in this model. as a result, ajwa seeds could be used in conjunction with dietary and pharmacological therapy to improve diabetes control.  fig. 6 effect of ajwa seeds on pancreatic immunoexpression of insulin observed in control and diabetic rats’ pancreas. sections from rats’ pancreases were immuno-stained for insulin. and photographed at high (x600) magnification. nc: normal control, showed highly immunopositive staining for insulin in most centrally located beta-cells. vc: vehicle control showed also a large number of beta cells highly immune stained for insulin. ac: ajwa seeds control showed immunostaining for insulin more or less like nc pancreas. dc: diabetic control showed a marked decreased in the number of immune positive stained cells for insulin. dm: diabetic + metformin showed a potential increase of immune stained for insulin size of cells. notice, the apparent increase in the size of stained cells. da: diabetic + ajwa seeds showed marked preservation of immune positive stained cells for insulin. notice there is also an apparent increase in cells size. references 1. fan, w. 2017. epidemiology in diabetes mellitus and cardiovascular disease. cardiovasc endocrinol, 6(1), 8–16, https://doi.org/10.1097/ xce.0000000000000116. 2. meo, s., sheikh, s., sattar, k., akram, a., hassan, a., meo, a., usmani, a., qalbani, e. & ullah, a. 2019. prevalence of type 2 diabetes mellitus among men in the middle east: a retrospective study. am j mens health, 13(3), 1557988319848577, https://doi.org/10.1177/1557988319848577. 3. cho, n.h., shaw, j.e., karuranga, s., huang, y., da rocha fernandes, j.d., ohlrogge, a.w. & malanda, b. 2018. idf diabetes atlas: global estimates of diabetes prevalence for 2017 and projections for 2045. diabetes res clin pract, 138, 271–281, https://doi.org/10.1016/j.diabres.2018.02.023. 4. alotaibi, a., perry, l., gholizadeh, l. & al-ganmi, a. 2017. incidence and prevalence rates of diabetes mellitus in saudi arabia: an overview. j epidemiol glob health, 7(4), 211–218, https://doi.org/10.1016/j. jegh.2017.10.001. 5. dawish, m. & robert, a. 2020. diabetes mellitus in saudi arabia challenges and possible solutions. in: handbook of healthcare in the arab world, springer, cham, 1-18. 6. raz, i. 2013. guideline approach to therapy in patients with newly diagnosed type 2 diabetes. diabetes care, 36(suppl.2), s139–s144, https:// doi.org/10.2337/dcs13-2035. 7. kasole, r., martin, h. & kimiywe, j. 2019. traditional medicine and its role in the management of diabetes mellitus: patients and herbalists perspectives. evidence-based complement altern med, 2019, 2835691, https://doi. org/10.1155/2019/2835691. 8. amit koparde, a., chandrashekar doijad, r. & shripal magdum, c. 2019. natural products in drug discovery. pharmacogn med plants, https://doi. org/10.5772/intechopen.82860. 9. melek, f., saleh, d., medhat, a., farrag, a., ghaly, n. & baraka, s. 2019. antidiabetic and antioxidant activities of phoenix dactylifera l. seed extract 216 j contemp med sci | vol. 7, no. 4, july-august 2021: 211–216 ajwa seeds (phoenix dactylifera l.) suspension exerted antidiabetic original a.a. alahmadi and h.m. banayah in streptozotocin-induced diabetic rats. malaysian j biochem mol biol, 22(1), 53–59. 10. ghnimi, s., umer, s., karim, a. & kamal-eldin, a. 2017. date fruit (phoenix dactylifera l.): an underutilized food seeking industrial valorization. nfs j, 6, 1–10, https://doi.org/10.1016/j.nfs.2016.12.001. 11. rahmani, a., aly, s., ali, h., babiker, a., suikar, s. & khan, a. 2014. therapeutic effects of date fruits (phoenix dactylifera) in the prevention of diseases via modulation of anti-inflammatory, anti-oxidant and anti-tumour activity. int j clin exp med, 7(3), 483–491. 12. venkatachalam, c. & sengottian, m. 2016. study on roasted date seed non caffeinated coffee powder as a promising alternative. asian j res soc sci humanit, 6(6), 1387–1394, https://doi.org/10.5958/22497315.2016.00292.6. 13. sani, i., hidayah, n., bakar, a., adzim, m., rohin, k., suleiman, i., umar, m. & mohamad, n. 2015. phoenix dactylifera linn as a potential novel antioxidant in treating major opioid toxicity. j appl pharm sci, 5(08), 167–172, https://doi.org/10.7324/japs.2015.50826. 14. bnouham, m., ziyyat, a., mekhfi, h., tahri, a. & legssyer, a. 2006. medicinal plants with potential antidiabetic activity-a review of ten years of herbal medicine research (1990-2000). journal of diabetes & metabolism, 14, 1-25. 15. sarfraz, m., khaliq, t., khan, j. & aslam, b. 2017. effect of aqueous extract of black pepper and ajwa seed on liver enzymes in alloxan-induced diabetic wister albino rats. saudi pharm j, 25(4), 449–452, https://doi.org/10.1016/j. jsps.2017.04.004. 16. hasan, m. & mohieldein, a. 2016. in vivo evaluation of anti diabetic, hypolipidemic, antioxidative activities of saudi date seed extract on streptozotocin induced diabetic rats. j clin diagnostic res, 10(3), ff06-ff12, https://doi.org/10.7860/jcdr/2016/16879.7419. 17. neamatallah, t., el-shitany, n., abbas, a., ali, s. & eid, b. 2018. honey protects against cisplatin-induced hepatic and renal toxicity through inhibition of nf-κb-mediated cox-2 expression and the oxidative stress dependent bax/ bcl-2/caspase-3 apoptotic pathway. food funct, 9(7), 3743–3754, https:// doi.org/10.1039/c8fo00653a. 18. meng, x.m., ma, x.x., tian, y.l., jiang, q., wang, l.l., shi, r., ding, l. & pang, s.g. 2017. metformin improves the glucose and lipid metabolism via influencing the level of serum total bile acids in rats with streptozotocininduced type 2 diabetes mellitus. eur rev med pharmacol sci, 21(9), 2232–2237. 19. zhang, m., lv, x., li, j., xu, z. & chen, l. 2008. the characterization of high-fat diet and multiple low-dose streptozotocin induced type 2 diabetes rat model. exp diabetes res, 2008, 704045, https://doi. org/10.1155/2008/704045. 20. khan, f., khan, t., kalamegam, g., pushparaj, p., chaudhary, a., abuzenadah, a., kumosani, t., barbour, e. & al-qahtani, m. 2017. anti-cancer effects of ajwa dates (phoenix dactylifera l.) in diethylnitrosamine induced hepatocellular carcinoma in wistar rats. bmc complement altern med, 17(1), 418, https://doi.org/10.1186/s12906-017-1926-6. 21. el-sayed, m., al-massarani, s., el gamal, a., el-shaibany, a. & al-mahbashi, h. 2020. mechanism of antidiabetic effects of plicosepalus acaciae flower in streptozotocin-induced type 2 diabetic rats, as complementary and alternative therapy. bmc complement med ther, 20(1), 290, https://doi. org/10.1186/s12906-020-03087-z. 22. samaha, m., said, e. & salem, h. 2019. nilotinib enhances β-islets integrity and secretory functions in a rat model of stz-induced diabetes mellitus. eur j pharmacol, 860, 172569, https://doi.org/10.1016/j.ejphar.2019.172569. 23. ingaramo, p., ronco, m., francés, d., monti, j., pisani, g., ceballos, m., galleano, m., carrillo, m. & carnovale, c. 2011. tumor necrosis factor alpha pathways develops liver apoptosis in type 1 diabetes mellitus. mol immunol, 48(12–13), 1397–1407, https://doi.org/10.1016/j. molimm.2011.03.015. 24. al-thobaiti, s. & abu zeid, i. 2019. antidiabetic potential of balanites aegyptiaca kernel, flesh and their combination against streptozotocininduced hyperglycemia in male rats trop j pharm res, 18(2), 263–271, https://doi.org/10.4314/tjpr.v18i2.7. 25. putta, s., yarla, n., kilari, e., surekha, c., aliev, g., divakara, m., santosh, m., ramu, r., zameer, f., mn, n., chintala, r., rao, p., shiralgi, y. & dhananjaya, b. 2016. therapeutic potentials of triterpenes in diabetes and its associated complications. curr top med chem, 16(23), 2532–2542, https://doi.org/10.2 174/1568026616666160414123343. 26. abdelaziz, d. & ali, s. 2014. the protective effect of phoenix dactylifera l. seeds against ccl4-induced hepatotoxicity in rats. j ethnopharmacol, 155(1), 736–743, https://doi.org/10.1016/j.jep.2014.06.026. 27. el-mousalamy, a., hussein, a., abdelaziz, a., shaker, g. & mahmoud, s. 2016. aqueous and methanolic extracts of palm date seeds and fruits (phoenix dactylifera) protects against diabetic nephropathy in type ii diabetic rats. biochem physiol open access, 2016(5), 205, https://doi.org/10.4172/21689652.1000205. 28. el-fouhil, a., ahmed, a. & darwish, h. 2013. hypoglycemic effect of an extract from date seeds on diabetic rats. saudi med j, 31(7), 747–751. 29. el fouhil, a., ahmed, a., darwish, h., atteya, m. & al-roalle, a. 2011. an extract from date seeds having a hypoglycemic effect is it safe to use? saudi med j, 32(8), 791–796. 30. aftab, a., muhammad umair, a., farhan, s. & tabussam, t. 2019. exploring the role of date pit based drinks against hyperglycemia and hypercholesterolemia. prog nutr, 21(1-s), 307–320, https://doi. org/10.23751/pn.v21i1-s.6194. 31. kawaguchi, k., mizuno, t., aida, k. & uchino, k. 1997. hesperidin as an inhibitor of lipases from porcine pancreas and pseudomonas. biosci biotechnol biochem, 61(1), 102–104, https://doi.org/10.1271/bbb.61.102. 32. halaby, m. 2014. potential effect of date pits fortified bread on diabetic rats. int j nutr food sci, 3(2), 49, https://doi.org/10.11648/j.ijnfs.20140302.16. 33. habib, h. & ibrahim, w. 2011. effect of date seeds on oxidative damage and antioxidant status in vivo. j sci food agric, 91(9), 1674–1679, https://doi. org/10.1002/jsfa.4368. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. doi: https://doi.org/10.22317/jcms.v7i4.1058 http://https://doi.org/10.1002/jsfa.4368. http://https://doi.org/10.1002/jsfa.4368. 38 j contemp med sci | vol. 8, no. 1, january-february 2022: 38–43 original detection of natural and simulated periodontal defects using cone-beam computed tomography and digital intraoral radiography melis misirli1*, secil aksoy1, murat i̇çen2, hayriye tumer3, kani bilginaylar4, suzan karaoglulari3, atilla berberoglu5, kaan orhan6,7,8 1near east university, faculty of dentistry, department of dentomaxillofacial radiology, mersin 10, turkey. 2nevşehir hacı bektaş veli university, faculty of dentistry, department of dentomaxillofacial radiology, nevşehir, turkey. 3fınal international university, faculty of dentistry, department of periodontology, mersin 10, turkey. 4fınal international university, faculty of dentistry, department of oral and maxillofacial surgery, mersin 10, turkey. 5unıversity of city island, faculty of dentistry, department of periodontology, mersin 10, turkey. 6ankara university, faculty of dentistry, department of dentomaxillofacial radiology, ankara, turkey. 7ankara university medical design application and research center (meditam), ankara, turkey. 8medical university of lublin, department of dental and maxillofacial radiodiagnostics, lublin, poland. *correspondence to: melis misirli (e-mail: melis.misirli@neu.edu.tr) (submitted: 10 december 2021 – revised version received: 26 december 2021 – accepted: 17 january 2022 – published online: 26 february 2022) abstract objectives: in this study, it was aimed to compare the diagnostic value of cone beam computed tomography (cbct) and digital intraoral periapical imaging methods in the diagnosis of periodontal defects, and the comparison of observers practicing dentistry in the field of periodontology and 3 other observers specialized in various branches was evaluated. methods: 7 dry mandible and 5 dry maxilla were used in this study. a total of 111 artificial and natural defects (dehiscence, furca, fenestration, vertical defects) on the anterior, premolar and molar region were imaged with digital imaging techniques using different exposure parameters and scanned with cbct. results: according to the results of this study, cbct is a more reliable method in the evaluation of all periodontal defects compared to ccd and psp. 60 and 70 kvp with 0.01 irradiation time in ccd and 70 kvp 0.25 irradiation time in psp is not suitable for detecting defects owing to high contrast. in the evaluation of periodontal defects, there was no single observer who had good results, different imaging methods and varying results were obtained in different defects. conclusion: this study showed that the cbct method has some diagnostic value for detecting all natural and simulated periodontal defects but it should only be used in cases where clinical evaluation and conventional radiographic imaging do not provide the information necessary for an adequate diagnosis and proper periodontal treatment planning. keywords: vertical defects, cone beam computed tomography, ccd, ps, exposure parameters issn 2413-0516 introduction periodontal diseases are localized infections which result in inflammation of the gingiva, leading to the gradual destruction of periodontal tissues and alveolar bone supporting the teeth.1 as a result of these periodontal diseases, various defects occur in the alveolar bone.2 the absence of facial or lingual cortical plates, which results in the cervical root surface and affecting the marginal bone, represents an alveolar defect called dehiscence. fenestration is the window-shaped alveolar bone loss in the facial or lingual surface of the tooth that directly contacts the gingival or alveolar mucosa and does not affect the marginal bone, unlike dehiscence.3,4 furcation involvement is defined as the ‘pathologic resorption of bone within a bi-furcation or tri-furcation areas of premolars and molars tooth’.5 vertical defects are oblique defects, making an angle to the tooth root. bone defects are classified according to the number of surviving bone walls, the width of the defect, and the topographic extent of the tooth. the number of remaining bone walls referred to here is intact and is the number of bone walls that contain regeneration-providing components. accordingly, it is classified as a walled, twowalled and three-walled.3 when these defects are diagnosed, periodontal probes are used to evaluate gingival tissues and radiographs are used to evaluate bone support.6 diagnosis of advanced defects by using of the periodontal probe has limitations such as variables sizes and shapes of periodontal probe tips, probing force, gingival inflammation and anatomical conditions of the probing site.7 radiographs are necessary for the visualization of hidden anatomical structures such as alveolar bone and to determine the extent and severity of periodontal tissues.8,9 they help us to determine the degree of inter radicular and interdental bone loss, length of the root, crown-root ratio, periodontal ligament space and any apical pathology in the tooth.1 currently, there are many intraoral and extraoral imaging methods that we can examine periodontal defects. due to its cost, easy implementation and high resolution, generally 2d imaging methods (periapical, bitewing, panoramic) are preferred.7 however, there are limitations such as the superposition of anatomical structures, difficulty in establishing standardization, and examination of size and formation of bone defects. with the reason of superposition, periodontal lesions such as buccal and lingual defects and dehiscences cannot be diagnosed with intraoral radiography.1,8 prediction of periodontal defects may be leading to progression of periodontal bone loss and resulting in tooth loss.10 because of these limitations, the treatment plan is affected by the inability to fully observe periodontal defects and three dimensional (3d) examination could be considered as a superior technique.11 conventional computed 39j contemp med sci | vol. 8, no. 1, january-february 2022: 38–43 m. misirli et al. original detection of natural and simulated periodontal defects using cbct tomography (ct) which is revolutionized imaging solves this problem by providing multiplanar imaging of the objects. however, there are disadvantages of ct including high patient radiation dose, high cost, and low resolution.12 the use of 3d imaging cbct method, offers many advantages because of its lower radiation dose and less artifact compared to conventional tomography.8 morphologic knowledge is crucial for treatment and prognosis in periodontology.9 thus, one of the most important factors for the success of periodontal treatment is to have a true image of the morphology of periodontal bone destruction for accurate treatment planning and determination of prognosis. several studies are available on the diagnostic accuracy of cbct. 8,9,11,13 this study sought to the comparison of imaging methods to assess the diagnostic value of cbct which is taken with different voxel and fov sizes and digital intraoral periapical imaging methods with varying kvp and ma parameters. materials and methods in this study, 7 dry mandibles and 5 dry maxillae were used. 6 mandibles and 4 maxillae were used to create simulated defects, while 1 mandibula and 1 maxilla with natural defects are determined as gold standard and no simulated defect has been created. artificial periodontal defects were created in the areas where natural defects were not present in all the upper and lower jaws, except the upper and lower jaws which were used as the gold standard. in total 111 artificial defects simulating dehiscence, fenestration, furca, and intrabony defects were created on the incisors, premolars and molars using a diamond-tipped burr (jota diamond, 4500 max rpm, 300000 opt rpm) and 70–72% perchloric acid. there are 55 dehiscence defects, 14 fenestration defects, 23 furca defects, and 20 intrabony defects. the cause of the excess of dehisense defects was the presence of natural dehisense defects in the jaws. the pink modeling wax was placed to cover all the defects on the jaws to provide a soft tissue imitation. the defects were prepared using high-speed equipment with hand and angle pieces and rounded burrs under copious air/water spray by a periodontal consultant (ab) at randomly selected areas. the defects were noted to be used as the ‘gold standard’ for radiographic evaluation. images obtained from the defects and then to create defects induced by burr and chemical preparation, cotton pellets saturated with the 70–72% perchloric acid solution were placed on the burr prepared cavities and then the defects examined after 12 hours later. for the first examination, the cotton pellet was removed, after examination, a new cotton pellet saturated with a fresh perchloric acid was placed on the cavities for 12 hours. finally, the cotton pellets removed and modeling wax placed one more time for soft tissue simulation and all imaging methods repeated (figure 1). digital intraoral imaging after preparing all jaws, radiology researchers (mm, mi) acquired intraoral radiographs using a sorodex digora optime uv (sorodex medical systems, helsinki, finland) and planmeca dixi 3 ccd (helsinki, finland). periapical radiographs were obtained with a parallel technique by using a film holder apparatus to provide standardization. phosphor plate system operating at 60 and 70 kvp with three different exposure time (0.16, 0.20 and 0.25 sec) and the ccd system operating at 60 and 70 kvp with 0.01, 0.02, 0.03 sec exposure time. every defect has 6 digital intraoral radiographs to compare the diagnostic quality. cone-beam computed tomography cbct images were obtained (morita veraviewepocs 3d r 100) with a flat panel detector which has 0.125 mm3 voxel, offering 2 field-of-view (fov) sizes (8 × 8 cm and 10 × 8 cm). assessment of radiographic images each image set was evaluated separately by 4 different observers in random; one periodontology ph.d. student (sk-observer 1), one oral and maxillofacial surgeon (kb observer 2), one periodontologist (ht-observer 3) with an experience of 2 years, one trained dentomaxillofacial radiologists (sa-observer 4) with an experience of 4 years. observers were calibrated with the help of a powerpoint presentation of defect samples by a specialist radiologist (ko) and periodontologist (ab) on both cbct and periapical radiography images for the diagnosis of periodontal defects. all digital intraoral images were saved in noncompressed file format (tagged image file format, tiff). digital intraoral images were displayed using the dedicated dicom-viewing software program whereas cbct images were evaluated with its own software (romexis 3.2, planmeca, helsinki, finland). observation conditions were optimized through use of the same computer monitor when the images were displayed. viewing distance was kept constant to about 50 cm for the observer, and the lights were subdued during examinations. the observers were asked to identify the existence or absence of defects as well as the types of the defects (figure 2). the observers were unaware of the existence of the defect as well as the exposure properties of them. the final classification was; the defects were present, absent or undetectable while making the diagnosis. statistical analysis all evaluations were compared according to the identified gold standard. all statistical analyses were performed with spss 20.0 (spss®; ibm corp., new york, ny; formerly spss inc., chicago, il). chi-square analysis was used to compare fig. 1 the photographs of the maxilla and mandible that showing natural defect and simulated defects created with burr and chemical agent. 40 j contemp med sci | vol. 8, no. 1, january-february 2022: 38–43 detection of natural and simulated periodontal defects using cbct original m. misirli et al. fig. 2 2 and 3d images of the defects (a) simulated furca defect (b) natural dehiscence defects (c) natural vertical defect (d) natural dehiscence defects. the kvp and exposure times within the ccd and phosphor plate system. kappa statistics were used for assessing the agreement between observers. the kappa values were interpreted according to guidelines of landis and koch adapted by altman: k ≤ 0.20 poor, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 good, 0.81–1.00 very good. the determination of significance level was carried out with the t-test using paired samples. results were considered significant at p < 0.05. results table 1 displays the diagnostic sensitivity, kappa coefficients, and p-value, calculated for each observer of ccd, phosphor plate system and cbct according to for vertical defects (1, 2 and 3 wall intrabony defects). the results revealed that 60 kvp exposure time of 0.01 second was not suitable for all observers for detection of the defects. the first observer who is an inexperienced periodontology research assistant could not diagnose any vertical defects from the radiographs, which are taken by ccd although all other observers can identify it. the diagnostic sensitivity of ccd and psp are similar however, cbct sensitivity was significantly higher than digital radiography techniques for the detection of vertical defects. table 2 shows that the sensitivity of ccd and psp for the detection of dehiscence defects. cbct images again (10×8, 8×8) are superior (p < 0.05) to detect dehiscence defects then conventional radiographies (p > 0.05) for all observers. only 4th observer (radiologist) diagnosed all dehiscence defects from the ccd and phosphor plate system except the 60 and 70 kvp exposure time of 0.01 second. the results also showed that ccd and psp can diagnose furca defects (p < 0.05) rather than dehiscence defects but the sensitivity values of cbct were higher than those of digital radiography (tables 2 and 3). table 4 shows the p-value (p < 0.05) of all modalities are equal when detecting fenestration defects. cbct showed statistically better diagnostic quality and performance on detecting all defects except the fenestration defects (p < 0.05). discussion the results of this study have generally shown that cbct is superior to digital periapical radiography in diagnosing all types of periodontal defects with the same other studies.1,8,9,13,14 fenestrations are isolated areas in which the exposed root surface is covered only by the periosteum and gingiva, but marginal bone is unaffected. when this erosion extends to the marginal bone, it is called dehiscence. radiologic examination of advanced periodontal defects involving fenestration and dehiscence is essential.15 these defects cannot be visualized by traditional two-dimensional radiography because of the superposition of contralateral cortical bony or dental structures. three-dimensional radiography techniques can be table 1. intra-observer agreement calculated for each observer by image type for vertical defects vertical defects observer 1 observer 2 observer 3 observer 4 sensitivity kappa p sensitivity kappa p sensitivity kappa p sensitivity kappa p 60 kvp 0.01 60 0,062 0,369 40 0,121 0,172 42.11 0,175 0,055 57.89 0,147 0,068 60 kvp 0.02 60 0,112 0,134 45 0,148 0,09 55 0,243 0,006* 50 0,185 0,034* 60 kvp 0.03 60 0,12 0,112 50 0,219 0,014* 50 0,174 0,044* 55 0,188 0,026* 70 kvp 0.01 60 0,056 0,416 50 0,213 0,018* 47.37 0,216 0,018* 55 0,14 0,084 70 kvp 0.02 65 0,118 0,1 60 0,278 0,001* 55 0,17 0,055 60 0,254 0,003* 70 kvp 0.03 70 0,109 0,103 60 0,266 0,002* 50 0,243 0,007* 60 0,171 0,032* 60 kvp 0.16 45 -0,029 0,684 45 0,272 0,003* 45 0,205 0,023* 60 0,21 0,011* 60 kvp 0.20 65 0,184 0,018* 45 0,258 0,005* 35 0,124 0,173 40 0,11 0,208 60 kvp 0.25 65 0,161 0,036* 42.11 0,156 0,082 30 0,071 0,434 60 0,241 0,005* 70 kvp 0.16 50 0,038 0,609 40 0,165 0,068 40 0,259 0,005* 45 0,148 0,09 70 kvp 0.20 60 0,208 0,015* 47.37 0,316 0,001* 50 0,281 0,002* 45 0,207 0,027* 70 kvp 0.25 68.75 0,147 0,079 44.44 0,135 0,184 44.44 0,2 0,052 63.16 0,276 0,003* cbct 10x8 70 0,146 0,04* 70 0,429 0,001* 65 0,41 0,001* 75 0,115 0,072 cbct 8x8 70 0,146 0,04* 70 0,429 0,001* 65 0,41 0,001* 75 0,115 0,072 41j contemp med sci | vol. 8, no. 1, january-february 2022: 38–43 m. misirli et al. original detection of natural and simulated periodontal defects using cbct table 3. intra-observer agreement calculated for each observer by image type for furca defects furca defects observer 1 observer 2 observer 3 observer 4 sensitivity kappa p sensitivity kappa p sensitivity kappa p sensitivity kappa p 60 kvp 0.01 56.52 0,676 0,001* 65.22 0,724 0,001* 78.26 0,852 0,001* 43.48 0,552 0,001* 60 kvp 0.02 56.52 0,676 0,001* 73.91 0,793 0,001* 78.26 0,852 0,001* 43.48 0,552 0,001* 60 kvp 0.03 60.87 0,714 0,001* 73.91 0,82 0,001* 78.26 0,852 0,001* 47.83 0,595 0,001* 70 kvp 0.01 56.52 0,676 0,001* 73.91 0,82 0,001* 78.26 0,852 0,001* 47.83 0,636 0,001* 70 kvp 0.02 60.87 0,714 0,001* 73.91 0,82 0,001* 78.26 0,852 0,001* 47.83 0,595 0,001* 70 kvp 0.03 60.87 0,714 0,001* 73.91 0,793 0,001* 78.26 0,852 0,001* 43.48 0,552 0,001* 60 kvp 0.16 47.83 0,595 0,001* 65.22 0,724 0,001* 69.57 0,786 0,001* 56.52 0,676 0,001* 60 kvp 0.20 47.83 0,595 0,001* 78.26 0,826 0,001* 65.22 0,75 0,001* 56.52 0,676 0,001* 60 kvp 0.25 47.83 0,636 0,001* 73.91 0,793 0,001* 69.57 0,786 0,001* 56.52 0,676 0,001* 70 kvp 0.16 47.83 0,595 0,001* 73.91 0,793 0,001* 69.57 0,786 0,001* 60.87 0,714 0,001* 70 kvp 0.20 39.13 0,508 0,001* 73.91 0,793 0,001* 65.22 0,75 0,001* 60.87 0,714 0,001* 70 kvp 0.25 47.83 0,595 0,001* 69.57 0,734 0,001* 69.57 0,786 0,001* 60.87 0,714 0,001* cbct 10x8 82.61 0,807 0,001* 82.61 0,832 0,001* 78.26 0,8 0,001* 82.61 0,807 0,001* cbct 8x8 82.61 0,807 0,001* 82.61 0,832 0,001* 78.26 0,8 0,001* 82.61 0,807 0,001* table 2. intra-observer agreement calculated for each observer by image type for dehiscences dehiscence observer 1 observer 2 observer 3 observer 4 sensitivity kappa p sensitivity kappa p sensitivity kappa p sensitivity kappa p 60 kvp 0.01 7.27 0,025 0,595 10.91 0,007 0,879 7.27 0,025 0,595 16.36 0,068 0,295 60 kvp 0.02 5.45 0,023 0,564 10.91 0,045 0,404 7.27 0,025 0,595 25.45 0,195 0,005* 60 kvp 0.03 5.45 0,04 0,261 7.27 0,028 0,619 7.27 0,025 0,595 29.09 0,233 0,001* 70 kvp 0.01 5.45 0,023 0,564 10.91 –0,01 0,853 9.09 0,044 0,376 20 0,122 0,066 70 kvp 0.02 5.45 0,04 0,261 10.91 0,045 0,404 12.73 0,081 0,135 29.09 0,25 0,001* 70 kvp 0.03 7.27 –0,012 0,803 7.27 0,062 0,228 10.91 0,045 0,404 18.18 0,138 0,024* 60 kvp 0.16 7.27 0,042 0,332 14.55 0,066 0,279 5.45 –0,03 0,561 23.64 0,143 0,045* 60 kvp 0.20 1.82 –-0,015 0,621 10.91 0,011 0,85 3.64 –0,05 0,303 23.64 0,16 0,022* 60 kvp 0.25 1.82 –0,015 0,621 16.36 0,085 0,178 7.27 0,025 0,595 30.91 0,268 0,001* 70 kvp 0.16 7.27 0,042 0,332 14.55 0,015 0,823 3.64 –0,01 0,734 27.27 0,213 0,001* 70 kvp 0.20 5.45 0,006 0,896 20 0,122 0,066 3.64 0,004 0,916 27.27 0,231 0,001* 70 kvp 0.25 5.45 0,023 0,564 9.09 0,044 0,376 3.64 0,021 0,499 21.82 0,192 0,002* cbct 10x8 60 0,223 0,016* 61.82 0,324 0,001* 60 0,306 0,001* 60 0,223 0,016* cbct 8x8 60 0,223 0,016* 61.82 0,324 0,001* 60 0,306 0,001* 60 0,223 0,016* useful when diagnosing present fenestration or dehiscences.16,17 early correct diagnosis plays an important role in the diagnosis and treatment of primary stages of periodontal defects.7 noujeim et al. reported that the difference between cbct and periapical radiography in diagnosing small defects was greater than the determination of large defects and therefore emphasized the importance of cbct in the detection of early lesions.1 the periapical images obtained in this study using 60 kvp, 8 ma and 0.16 s and cbct (morita, 3dx accuitomo) images obtained by using 0.125 mm3 voxel sizes were compared in terms of diagnosis of simulated furca defects. it has been shown that cbct has higher diagnostic accuracy (periapical radiography = 0.783, kibt = 0.864). in this study, similar to previous study cbct had higher diagnostic accuracy (ccd and psp = 0.782, kibt = 0.82) similar to noujeim et al.1 misch et al.6 compared periapical radiographs with cbct and periodontal probing with electronic calipers in measuring the depth of buccal, lingual, and interproximal periodontal defects that they created with a bur on two dry human mandibles. they stated that, periodontal probing was more advantageous than electronic calipers, however, there was no significant difference between periapical radiographs and 42 j contemp med sci | vol. 8, no. 1, january-february 2022: 38–43 detection of natural and simulated periodontal defects using cbct original m. misirli et al. table 4. intra-observer agreement calculated for each observer by image type for fenestration defects fenestration observer 1 observer 2 observer 3 observer 4 sensitivity kappa p sensitivity kappa p sensitivity kappa p sensitivity kappa p 60 kvp 0.01 14,29 0,324 0,001* 28,57 0,382 0,001* 21,43 0,296 0,001* 28,57 0,413 0,001* 60 kvp 0.02 21,43 0,324 0,001* 35,71 0,461 0,001* 28,57 0,382 0,001* 28,57 0,413 0,001* 60 kvp 0.03 21,43 0,324 0,001* 28,57 0,382 0,001* 21,43 0,296 0,001* 28,57 0,413 0,001* 70 kvp 0.01 28,57 0,413 0,001* 35,71 0,461 0,001* 21,43 0,296 0,001* 28,57 0,413 0,001* 70 kvp 0.02 28,57 0,413 0,001* 35,71 0,461 0,001* 21,43 0,296 0,001* 28,57 0,413 0,001* 70 kvp 0.03 28,57 0,413 0,001* 28,57 0,382 0,001* 21,43 0,296 0,001* 28,57 0,413 0,001* 60 kvp 0.16 21,43 0,324 0,001* 42,86 0,569 0,001* 35,71 0,461 0,001* 35,71 0,494 0,001* 60 kvp 0.20 21,43 0,324 0,001* 50 0,638 0,001* 28,57 0,327 0,001* 35,71 0,461 0,001* 60 kvp 0.25 21,43 0,324 0,001* 50 0,638 0,001* 28,57 0,327 0,001* 35,71 0,494 0,001* 70 kvp 0.16 21,43 0,324 0,001* 42,86 0,569 0,001* 35,71 0,43 0,001* 35,71 0,494 0,001* 70 kvp 0.20 21,43 0,324 0,001* 35,71 0,494 0,001* 28,57 0,413 0,001* 35,71 0,494 0,001* 70 kvp 0.25 21,43 0,324 0,001* 21,43 0,324 0,001* 28,57 0,413 0,001* 28,57 0,413 0,001* cbct 10x8 50 0,568 0,001* 42,86 0,502 0,001* 42,86 0,502 0,001* 57,14 0,629 0,001* cbct 8x8 50 0,568 0,001* 42,86 0,502 0,001* 42,86 0,502 0,001* 57,14 0,629 0,001* cbct. in a similar study, langen et al.18 compared conventional radiographs and ct for the diagnosis of 55 vertical defects. it was reported that only 38 of 55 defects (69.1%) can be detected by conventional radiographs and 100% defects could be detected on ct images. in another study mengel et al.19 reported that intraoral radiographs were inadequate in buccolingual examinations and that image quality was better in cbct, consistent with the results of the study. the results of this study showed only 14 of 20 simulated vertical defects can be detected by ccd images (70%), 13 defects by psp (65%) and 16 (80%) with cbct among all observers. sun et al.20 reported that 83% (sensitivity = 0.83) of the actual dehiscence defects and 73% (specificity = 0.73) of the non-defects areas, 71% (sensitivity = 0.71) of the actual fenestration defects and 77% (specificity = 0.77) of the non-defect areas were correctly diagnosed at the end of the study. they reported that there is a high probability of prediction with cbct when the opening angle in dehiscence is more than 3 mm. the results of this study were comparable with previous studies noujeim et al.1 sun et al.20 64% (sensitivity = 0.64) of the actual fenestration defects, 73% (sensitivity = 0.73) of the actual dehiscence defects and 82% (sensitivity = 0.82) of the actual furca defects were correctly diagnosed. our study was also compared with ccd and psp radiography techniques. it has been shown that ccd and psp are similar in the diagnosis of furca defects, but dehiscence and fenestration defects are diagnosed better with psp. vanderberghe et al.13 stated in their study that there was no significant difference between the two methods of determining the bone level in the study, however 29-41% the crater and furca defects were diagnoses wrong by psp and, in contrast 71% of the crater defects and 56% of the furca defects could be diagnosed by ccd. in the meantime cbct images diagnosed 100% of both crater and furca defects. similar to these results in this study 29% of dehiscence defects, approximately 42% of fenestration defects and 78.26% of furca defects could be diagnosed correctly using ccd, also 32.73% of dehiscence defects, 50% of fenestration defects and 78.26% of furca defects could be visualized by psp. in a similar study, kolsuz et al.8 create 35 artificial defects (dehiscence, fenestration, tunnel) on 12 dry skulls to compare different fov and voxel sizes (4×4 fov-0.080 mm3, 6×6 fov-0.125 mm3, 8×8 fov-0.160 mm3, 5×5.5 fov-0.100 mm3, 5×5.5 fov-0.150 mm3, 10×5.5 fov-0.200 mm3, 4×4 fov-0.080 mm3 and 6×6 fov-0.125 mm3) when detecting periodontal defects. they reported that the best voxel size for the diagnosis of periodontal defects is 0.150 mm3. in this study, we tested different fov sizes (10×8, 8×8) under 0.125 mm3 constant voxel size to compare to assess whether there was a difference in the diagnosis of periodontal defects. different fov sizes did not cause a resolution change that could affect the diagnosis of the defect. similar to this study salineiro et al.5 compared two different voxel sizes with fixed fov value of cbct machine (promax 3d max, planmeca helsinki, finland, 5×5.5 fov, 0.2 and 0.15 mm3 voxel size) with periapical radiographs to detect the incipient furcation involvement. they concluded that both cbct imaging protocols showed higher accuracy, sensitivity, and specificity than periapical radiographs in the detection of incipient furcation involvement, even in the presence of a metallic post. aljehani12 analyzed 3 different voxel sizes for the assessment of periodontal furcation involvement. the authors concluded that larger voxel size reduced the accuracy of the assessment of periodontal furcation involvement, but not to a significant extent. in this study, there was no significant difference for detecting periodontal defects in different fov values. in a cbct study by umetsubo et al.21 the sensitivity and specificity values of cbct (0.2 mm3 voxel value, fov: 6×16) was examined in the diagnosis of early furca defects. they created artificial furca defects on molar teeth of pig mandibles using 70% perchloric acid and scanned with 0.2 mm voxel size and 6×16 cm fov cbct. they found that the sensitivity values ranging from 50% to 75% and specificity values ranging 43j contemp med sci | vol. 8, no. 1, january-february 2022: 38–43 m. misirli et al. original detection of natural and simulated periodontal defects using cbct from 93% to 100% were detected in the diagnosis of furca defects. in this study artificial defects were created mechanically by burrs, radiographs were obtained and radiographs were repeated with 70% perchloric acid on the defects. the sensitivity of the best observer for the furca defects is 82.61% and the specificity values are 97.85% for the mechanically opened defects. for the defects formed by using mechanical + acid, the sensitivity value is 86.96% and the specificity value is 94.62%.this difference can be due to the different voxel size of the cbct. in addition to this, pinheiro et al.22 indicated in their study that small lesions were detected more effectively using cbct than psp, and larger lesions were detected more effectively by cbct at 90 kvp than by cbct at 75 kvp or by psp. similar to previous studies leung et al.,23 and bayat et al.,7 showed that cbct superior to psp in the diagnosis of all of furca, dehiscence, fenestration and vertical defects. there are some limitations of this study; since our defects did not open on a millimeter basis, it was not possible to determine which imaging method is better for detecting small or large defects. moreover, the use of various fov values and different voxel sizes may change the results positively, and more detailed studies are required for this purpose. in conclusion, cbct is a more reliable method in evaluating all periodontal defects than ccd and psp. the 0.125 mm3 voxel size used in the study is suitable for the determination of periodontal defects, but the different fov values are not found to be positively related to the diagnosis. although cbct has the advantage of being 3d but it’s not suitable to use as a first imaging method to detect periodontal lesions due to high radiation doses relative to intraoral radiographs. for this reason, cbct should be preferred in cases where complex periodontal lesions and defects are present. ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent was obtained from all patients or their legal delegates. the study was approved by the local ethic committee of the faculty of medicine (no. ydu/2015/33-232). conflict of interest the authors declare no conflicts of interest.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. doi: https://doi.org/10.22317/jcms.v8i1.1126 references 1. noujeim m, prihoda tj, langlais r, nummikoski p, (2009). evaluation of highresolution cone-beam computed tomography in the detection of simulated inter radicular bone lesions. dentomaxillofac. radiol. 38:156–162. 2. larato dc, (1970). periodontal bone defects in the juvenile skull. j. periodontol 41:473–475. 3. newman mg, takei hh, klokkvold pr, carranza fa, (2012). carranza’s clinical periodontology, 11th edn; saunders, louis, missouri. 4. enhos s, uysal t, yağcı a, veli i̇, uçar fi, ozer t, (2012). dehiscence and fenestration in patients with different vertical growth patterns assessed with cone-beam computed tomography. angle orthod. 82:868–874. 5. salineiro fcs, gialain io, kobayashi-velasco s, pannuti cm, cavalcanti mgp, (2017). detection of furcation involvement using periapical radiography and 2 cone-beam computed tomography imaging protocols with and without a metallic post: an animal study. imaging sci. dent. 47:17–24. 6. misch ka, yi es, sarment dp, (2006). accuracy of cone-beam computed tomography for periodontal defect measurements. j. periodontol. 77:1261–1266. 7. bayat s, talaeipour ar, sarlati f, (2016). detection of simulated periodontal defects using cone-beam ct and digital intraoral radiography. dentomaxillofac. radiol. 45:20160030. 8. kolsuz me, bagis n, orhan k, avsever h, demiralp kö, (2015). comparison of the influence of fov sizes and different voxel resolutions for the assessment of periodontal defects. dentomaxillofac. radiol. 44:10250070. 9. bagis n, kolsuz me, kursun s, orhan k, (2015). comparison of intraoral radiography and cone-beam computed tomography for the detection of periodontal defects: an in vitro study. bmc oral health. 28:64. 10. meyer ms, joshipura k, giovannucci e, michaud ds, (2008). a review of the relationship between tooth loss, periodontal diseases, and cancer. cancer causes control. 19:895–907. 11. vanderberghe b, jacobs r, yang j, (2008). detection of periodontal bone loss using digital intraoral and cone-beam computed tomography images: an in vitro assessment of bony and/or infrabony defects. dentomaxillofac. radiol. 37:252–260. 12. aljehani ya, (2014). diagnostic applications of cone-beam ct for periodontal diseases. int. j. dent. 2014:865079. 13. vanderberghe b, jacobs r, yang j, (2007). diagnostic validity (or acuity) of 2d ccd versus 3d cbct-images for assessing periodontal breakdown. oral surg. oral med. oral pathol. oral radiol. 104:395–401. 14. mol a, (2000). imagıng methods in periodontology. periodontol 2000. 34:34–48. 15. chiapasco m, zaniboni m, (2009). clinical outcomes of gbr procedures to correct peri-implant dehiscences and fenestrations: a systematic review. clin. oral implants res. 20:113–123. 16. timock am, cook v, mcdonald t, leo mc, crowe j, benninger bl, jr covell, da, (2011). accuracy and reliability of buccal bone height and thickness measurements from cone-beam computed tomography imaging. am. j. orthod. dentofacial orthop. 140:734–744. 17. sun z, smith t, kortam s, kim dg, tee bc, fields h, (2011). effects of bone thickness on alveolar bone-height measurements from cone beam computed tomography images. am. j. orthod. dentofacial orthop. 139:117–127. 18. langen hj, fuhrmann r, dıedrıch p, günther rw, (1995). diagnosis of infraalveolar bony lesions in the dentate alveolar process with high-resolution computed tomography. invest. radiol. 30:421–426. 19. mengel r, candir m, shiratori k, flores-de-jacoby l, (2005). digital volume tomography in the diagnosis of periodontal defects: an in vitro study on native pig and human mandibles. j. periodontol. 76:665–673. 20. sun l, zhang l, shen g, wang b, fang b, (2015). accuracy of cone-beam computed tomography in detecting alveolar bone dehiscences and fenestrations. am. j. orthod. dentofacial orthop. 147:313–323. 21. umetsubo os, gaia bf, costa ff, cavalcanti mg, (2012). detection of simulated incipient furcation involvement by cbct: an in vitro study using pig mandibles. braz. oral res. 26:341–347. 22. pinheiro lr, scarfe wc, de oliveira sales ma, gaia bf, cortes arg, cavalcanti mgp, (2017). effectiveness of periapical radiography versus cone beam computed tomography with different kilovoltage settings in the detection of chemically created peri-implant bone defects: an in vitro study. int. j. oral maxillofac. implants. 32:741–750. 23. leung cc, palamo l, griffith r, hans mg, (2010). accuracy and reliability of cone-beam computed tomography for measuring alveolar bone height and detecting bony dehiscences and fenestrations. am. j. orthod. dentofacial orthop. 137:109–119. 317j contemp med sci | vol. 8, no. 5, september-october 2022: 317–322 original development of dental specialties in iran: a qualitative study tayebe rojhanian1, mohammad pooyan jadidfard1, shahram yazdani2* 1department of community oral health, shahid beheshti university of medical sciences, tehran, ir-iran. 2department of medical education, virtual school of medical education and management, shahid beheshti university of medical science, tehran, ir-iran. *correspondence to: shahram yazdani (e-mail: yazdani.shahram01@gmail.com) (submitted: 07 may 2022 – revised version received: 29 june 2022 – accepted: 08 july 2022 – published online: 26 october 2022) abstract objectives: dental specialties in iran were first established in 1970s, and developed over time. considering that information is essential for health policymaking, and lack of it is the main problem; therefore, policymakers require adequate knowledge about development and alterations of healthcare providers to recognize the influential factors on them. regarding the information gap on development of dental specialties in iran, this qualitative study aimed to assess it. methods: this qualitative case study was conducted through 12 semi-structured interviews with the experts and pioneers of the oral healthcare system in iran who were selected by purposive and snowball sampling. data were analyzed by content analysis method, which included transcribing, identifying the meaning units, abstracting the content, sorting codes, and formulating themes using atlas.ti software. results: after data analysis, three main themes were extracted regarding development of dental specialties in iran according to the interviewees: (a) trend of development, (b) challenges of development, and (c) necessities of development. trend of development of dental specialties included two comprehensive phases, and one phase focusing on quantity and inadequate attention to quality. the challenges of development of dental specialties included management and policy-making problems, interactions outside the system, popularity of specialization, and process of admission to specialty programs. the necessity of need assessment, paying attention to the costs of healthcare interventions, defining the range of specialization, and revision of dental specialty programs are among the necessities of development of dental specialties. conclusion: dental specialties in iran were developed at a time with inadequate attention to shortage of infrastructure based on political interactions. the popularity of specialization in dentistry and the associated high costs in a free educational system highlight the significance of need assessment regarding the number of specialists required in academic and therapeutic fields, and setting some criteria for development of specialty programs. keywords: higher education, dental education, postgraduate, professional training, health care provider issn 2413-0516 introduction dental specialists receive education and training beyond general dentists, and acquire a higher level of expertise and competence in a field of specialty. the number of specialists and fields of specialties in a country are related to the oral healthcare provision system and socioeconomic factors.1 there is variation in the number of dental specialties, especially in european countries. austria, luxemburg, and spain have no officially recognized dental specialty. in the united kingdom and iceland, there is a high number of officially recognized dental specialties. dental specialties in countries like iceland, poland, sweden, and united kingdom have developed in response to public pressure with the aim of protection of public health.2 advances in dental technology, the need for complex procedures, demographic changes, population aging, improved level of wealth, and changed lifestyle are among the reasons proposed for increased number of dental specialties.1 financial considerations and economic factors play a fundamental role in development of specialties in medical field.3 several studies have assessed the variations, number, reasons for development, and trend of changes of dental specialties, mainly in developed and high-income (hi) countries. little information is available regarding dental specialties in developing and low-middle income countries (lmics). since the influential factors on the development and number of medical specialties are different in hi and lmics,4 case studies of lmics can be valuable to obtain comprehensive information in this regard.5 iran is a developing and lmic.6 establishment and development of dental specialties in iran dates back to 1970s. tehran university was the pioneer in this process, and over time, dental specialties were also established in other universities. higher education in iran is provided to iranian citizens free of charge and by financial support from the government. currently, there are 190 dental specialty fields-locations in 19 public and private dental schools in iran. approximately 360 postgraduate students are accepted and admitted annually in 12 specialty fields in iran. since the pattern and severity of oral and dental diseases have a significant impact on dental education strategy planning, type of service provider, and service provider system,7 it appears that the number of trained dental specialists in iran does not match the high rate of unmet needs,8 and the high share of out of pocket expenses of patients for dental services.9 the reason is that the need for specialized services is lower and such services are more expensive than other levels of care.10 to date, no study has been conducted on dental specialties in iran, and no information is available regarding their trend of development. conduction of a qualitative study on a topic regarding which, scarce information is available, can help better scrutinize the topic.5 thus, this qualitative case study was designed aiming to assess the trend of development of dental specialties in iran. information obtained from such studies can be used by policymakers since lack of information is the main problem in health policymaking, and adequate information can play an influential role in healthcare policymaking.11 mailto:yazdani.shahram01@gmail.com 318 j contemp med sci | vol. 8, no. 5, september-october 2022: 317–322 development of dental specialties in iran: a qualitative study original t. rojhanian et al. methods the case study design was selected for this qualitative study due to its applicability to enhance the understanding and perception of complex contemporary phenomena in different fields of life, such as medical and social domains.5 the participants were selected by purposive and snowball sampling. data collection was continued until data saturation. in 2020, 12 semi-structured interviews were conducted with experts of the healthcare system. the participants of the present study comprised of experts, policymakers, and pioneers of oral healthcare system in iran who were well aware of the history of establishment and development of dental specialties in iran (table 1). the objectives of the study and its ethical guidelines were explained to the participants prior to the interview, and written informed consent was obtained from them at the onset of interview. the location and time of interview were scheduled according to the preferences of the participants. the interviews were audio-recorded. the information provided by the participants was reported anonymously. one researcher conducted face-to-face interviews. the main question of this study was that “how was the development of dental specialties in iran?” (box 1). each interview took approximately 30 minutes. after each interview, its contents were transcribed verbatim. the interviewee was then provided with the transcript to confirm the data’s accuracy and make the necessary revisions, if required. data were analyzed using atlas.ti software (version 7.57).12 data analysis was conducted by the content analysis method.13 since one researcher (tr) performed the interviews and prepared the transcripts, and was completely familiar with the data, she also performed data analysis. the contents of the interviews were reviewed several times, the meaning units were identified, and the primary codes were extracted from the meaning units. after defining the meaning units, the contents of each interview were divided into meaning units. the related meaning units were further abstracted to obtain the research codes. after assessing the differences and similarities of the existing codes, subcategories were formed, and finally, the themes were extracted. peer debriefing and member checking were used to ensure the correct extraction of themes. all ethical guidelines were followed in this study, which included obtaining written informed consent for participation in the study, confidentiality of information, avoiding bias, the right to quit the study at any time, and anonymity of the participants. results after data analysis, three main themes including (a) trend of development of dental specialties, (b) challenges of development of dental specialties, and (c) the necessities of development of dental specialties were extracted (table 2). trend of development of dental specialties according to the participants’ opinion, the trend of development of dental specialties in iran can be divided into three phases, influenced by the number of dental schools in iran. it included an initiation phase with establishment of specialty programs in 5 primary dental schools in 1970s. the second phase included increasing the number of dental schools from 5 to 18 in year 2000, and the third phase included increasing the number of dental schools from 18 to 66 in the next years (of 66 permits given for establishment of dental schools, 44 schools are now active). the first phase (in 1970s) was part of the development process, which was pioneered by foreign-graduate experts (mainly graduated from the united states and united kingdom universities). the majority of such experts had received full scholarship from the government for their education abroad, and were obliged to work for the government and service their country after graduation. these graduates later became the instructors of the newly established dental schools in the second phase in undergraduate and residency programs. one respondent stated: “they [foreign graduates] were the pioneers of development of dental specialties in the country.” for some years (1980s), dental auxiliaries trained to increase dental care access for underserved people. in the table 1. number and composition of participants in semi structured interviews organizational ranking number of respondents former heads of the dental education and specialty council secretariat, ministry of health and medical education 3 former managers or members of the committee for educational programming in the ministry of health and medical education 4 experts in different dental specialties 5 total 12 table 2. contents and subcategories related to development of dental specialties in iran theme subcategories trend of development of dental specialties first and second phases: comprehensive, with optimal quality and quantity third phase: non-comprehensive, with special focus on quantity and inadequate attention to quality and elimination of infrastructural shortcomings challenges of development of dental specialties management and policymaking problems interactions out of the system popularity of specialization process of admission to specialty programs necessities of development of dental specialties necessity of need assessment significance of paying attention to the costs of healthcare interventions defining the extent of development of dental specialties and their revision box 1: main questions of the interviews -what do you know about the development of dental specialties in iran? -how was the trend of development of dental specialties in iran? and what modifications they have undergone so far? -what are the main challenges in the process of development of dental specialties in iran? -do you have any suggestions to improve the current status of dental specialties in iran? 319j contemp med sci | vol. 8, no. 5, september-october 2022: 317–322 t. rojhanian et al. original development of dental specialties in iran: a qualitative study given for establishment of numerous dental specialty programs in different universities. one participant added: “their goal was to do something to be recognized by it later.” 2. effect of interactions outside of the system on decision making was another challenge. according to the participants, these interactions affected the decisions made in the ministry of health and medical education regarding dental specialties. the majority of participants pointed to the significant role of lobbying in budget allocation and provision, and role and influence of some certain people out of the ministry of health on development of new dental schools and establishment of dental specialties in iran. according to one participant: “well, there was quite a competition at that time! all provinces somehow acquired a permit that all province capitals can have a dental school. some provinces such as [x] even established several dental schools! they wanted to do something big, and later brag about it that this particular dental school or specialty program was developed under my management and gain people’s support as such.” 3. according to the participants’ opinion, popularity of specialization is another problem that led to offering specialty programs by some service providers, and they focused their activities on a specific field of specialty. one participant said: “the majority of dental requirements of our people can be provided by general dentists, and there is actually no need to train such a high number of specialists.” 4. according to the participants, the process of admission to specialty programs is another challenge. the test score acquired in the national residency examination is currently the only criterion for admission to a residency program in iran, and there is no other assessment. one participant explained that: “test score is a necessity; but now, it is the only requirement.” necessities of development of dental specialties according to the participants, development of dental specialties in iran requires attention to some necessities. need assessment is imperative for development of dental specialties. the goal and purpose of specialty programs should be defined as well. the number of specialists required for academic educational purposes differs from the number required for therapeutic purposes; also, such needs vary depending on the time and distribution patterns. therefore, the needs should be identified and periodically updated. one participant explained: “when making a decision, we should see what we need.” in healthcare interventions, especially in the field of specialization, the costs need to be taken into account as well. in addition to the possible achievements, the costs should be also considered in decision making, and decisions should be made to minimize the costs inside and outside of the health domain. one participant added: “dental education is costly; so, you should watch for the costs and see what decision is better.” according to the participants’ opinion, considering the necessity of specialty programs, the need for specialists should be quantified. type of healthcare system and its priorities, model of service provision, and technique of management of the market of specialty services can all affect this decision. the second phase, schools for the training of dental auxiliaries were converted to dental schools. in this phase, the physical infrastructure was available, and the number of specialty programs increased to train instructors required for general dentistry programs in the increasing number of dental schools. development of specialty programs in this phase was comprehensive, had optimal quality and quantity, and was based on precise programming. the graduates of this phase had the required competencies in their field of specialty, and became successful clinicians and academic instructors. according to one participant: “they were very well-trained, because of the quality of training.” in the third phase, according to the interviewees, increasing the number of admitted students for specialty programs became competitive among dental schools, such that the quality of instruction in postgraduate residency programs was seriously compromised. increasing the number of dental schools, establishment of several dental schools in some provinces, establishing specialty programs in universities that did not have the required infrastructure, and immethodical increase in admission capacity of universities without taking into account the actual requirements of the country all indicated absence of a strategic plan with respect to training of dental specialists. one participant said: “at first, quantitative development was proportionate to qualitative development, but later, we had quantitative development but quality was no longer important.” challenges of development of dental specialties 1. management and policymaking problems: according to the opinion of the interviewees, development of dental specialties in iran, especially in the third phase, had several problems and challenges, one of which, was management and policymaking problems in the healthcare system. these problems included: (a) non-responsiveness of the authorities, (b) impaired problem solving, (c) political influence over appointments, and (d) implementing programs only to leave a mark from management period of managers. a. in the healthcare system of iran, monitoring and evaluation of managers has been rarely conducted, resulting in their poor responsiveness regarding the consequences of their actions and decisions. in other words, no difference exists between the managers that improve the healthcare system status and those who intensify the problems. one participant stated: “they are not responsive and have no worries in this regard either.” b. absence of a mechanism for corrections in the system results in continuation of old problems and development of new problems. according to one participant: “the system has not been defined for corrections and revisions; thus, old problems continue to exist and new problems emerge.” c. managers in the healthcare system are often appointed based on their political party. one participant stated: “when your political party wins, you get a position, depending on your level of cooperation with them.” d. another management challenge is the interest of the newly appointed healthcare authorities and managers in changing the existing system, and implementing programs only to leave a mark from their management period. one example of such activities is the permit 320 j contemp med sci | vol. 8, no. 5, september-october 2022: 317–322 development of dental specialties in iran: a qualitative study original t. rojhanian et al. criteria for development and expansion of specialty programs should be clear, and the existing programs should be revised accordingly. one participant added: “we have to set something straight; for example, we don’t yet know how many specialists we need.” another participant stated: “we should know how to manage specialty services, and what are the criteria for development of a specialty program.” discussion the present results revealed that development of dental specialties in iran had three phases. the first and second phases were based on the current needs and logic; however, the third phase had drawbacks such as immethodical increase in the number of specialty programs and ignoring the shortcomings of infrastructures. mohammadpour et al.14 evaluated the challenging in oral health policymaking in iran and mentioned that insufficient and inadequate infrastructure for dental education was one of the existing challenges. isiekwe et al.15 evaluated the perception of nigerian dental students regarding their educational curricula, and concluded that insufficient infrastructure affected dental education. they added that the challenges of dental education are different in developing and developed countries with adequate infrastructure. bailit16 discussed that financial problems of publicly-supported dental schools were one reason for inadequate infrastructure. jawaid17 in his study entitled “plight of dentistry in pakistan” pointed to the shortage of infrastructures in dental education institutes in pakistan. specialization of dental education in iran has been associated with some challenges. many challenges, such as management and policymaking problems, and interactions outside of the system, are not exclusive to development of specialties, but affect it. the ministers of the next government that takes over are rarely interested in interacting with the previous ministers. personal capture is another problem, which is also considered as a healthcare system problem by the world health organization. it leads to instability in implementation of programs, and results in no commitment to implementation of programs after the ministers and managers are changed. by changing of the managers and ministers of the political party that loses the election, their policies are also canceled before implementation and assessment, because the new ministers and managers are reluctant to continue implementation of programs designed by the previous team. proper stewardship can decrease the risk of policy orientation.18 the effect of interactions out of the system on health policies is another challenge, which is also a threat to the healthcare system. decision-making by individuals who may not even be healthcare experts, consider the healthcare market to be similar to any other market, and intentionally or unintentionally aim to attract public support without recognizing the consequences of their actions and decisions can further complicate the problems in a system that already has a high volume of unmet needs. shadpour,19 in his study, aimed to criticize the activities for correction of healthcare system in iran and highlighted the impact of political decisions made outside of the healthcare system on health policies. presence of legislative and regulatory bodies in the system can be helpful, given that they are not used for political goals of parties and individuals; however, unfortunately, political and factional tendencies often dominate the national goals.20 according to the interviewees, the process of admission to specialty programs is another challenge in development of dental specialties in iran. in the current model of student admission for residency programs in iran, a national entrance exam is held, and the students select a specialty program based on their test score. in a study on challenges of dental education in india, holding one entrance exam for all students was mentioned as an existing challenge. the authors suggested holding a separate standard examination for each program accompanied by personal interviews to determine the level of interest of the candidates in a particular fields.21 to train adequate human resources, the admission frequency increased in many countries worldwide as recommended by the world health organization.22 in iran, the number of dental schools increased aiming to serve justice in provision of services. over time, the number of dental schools offering specialty programs, and the number of admitted students for such programs also increased. although such educational developments can enhance accessibility of services, they have regulatory challenges as well.23 the australian research centre for population oral health stated that establishing new dental schools would be effective for enhancement of geographical distribution of dental specialists, but there is no evidence in this respect.24 maia et al.25 evaluated the characteristics of expanding private dental education, and discussed that increasing the number of dental schools can affect the dental job market but cannot guarantee better and fair distribution of dentists and dental services. moreover, increasing the number of dental schools not only does not improve oral health status of the public, but also creates some concerns with respect to the quality of instructions.14 jawaid17 pointed to the decreased quality of education due to absence of the required infrastructure. social status and earning more income are among the factors that contribute to acquiring a specialty degree.4 it is possible that by training higher number of specialists and lack of supervision on their distribution, competitions form between them to attract more patients and make more money, which would lead to an increase in certain treatments. also, specialists may compete with general dentists to make more money.26 in iran, higher education is mainly public and free, aiming to serve justice in accessing higher education and minimize injustice in this respect. however, informal estimates reveal that in the recent years, the share of benefitting from higher education has been positively correlated with the socioeconomic status of the families. increasing the number of universities and expansion of higher education were performed in some countries for the purpose of privatization and commoditization of higher education, which has an economic justification.27 jawaid17 reported uncontrolled increase in the number of dental schools in pakistan; he added that making more money was the main goal behind establishment of new dental schools. cumulative increase in number of dental specialists in iran occurred with the aim of provision of human resources following population growth and increased public demands; however, considering the type and quality of instructions, it does not have economic justification. expansion of higher education especially in countries with free public education 321j contemp med sci | vol. 8, no. 5, september-october 2022: 317–322 t. rojhanian et al. original development of dental specialties in iran: a qualitative study would be associated with a considerable rise in costs.23 moreover, considering the limited financial resources and high cost of undergraduate and postgraduate dental educations,28 it is imperative to estimate the number and type of oral health service providers according to precise need assessment. eklund and bailit29 believed that high number of dental graduates in the united states had little scientific justification, and discussed that study of supply and demand should be prioritized to establishing a new dental school. not performing need assessment and no access to adequate information are among other challenges of the healthcare system, and it has been demonstrated that many decisions related to human resources in oral health domain are made in absence of precise local data, and only based on models implemented in other countries.30 therefore, further attention should be directed to acquire and use precise data. also, it is imperative to devise policies to maintain the specialists in the country; otherwise, the outcome of training of specialized forces would be the human capital flight.31 this study focused on the less addressed topic of dental specialties in iran. uncertainty about the commitment of the reviewers regarding the accuracy of the provided information was a limitation of this study. to prevent its confounding effect, data were interpreted with caution. peer debriefing and member-checking methods were also applied to ensure data verifiability. considering the wide reference of the interviewees to the role of political interactions in health decisions, further studies are recommended to comprehensively assess the effect of political interactions and develop strategies to control them. according to the opinion of the interviewees, process of admission to specialty programs was one of the challenges in development of dental specialties in iran. since addressing this topic was out of the scope of this study, further studies are required to address this topic. conclusion the trend of development of dental specialties in iran included two phases with optimal quality and quantity, and one phase of focusing on quantity with less attention to quality of educations and elimination of the shortcomings of infrastructures. although the challenges in the process of development of dental specialty programs in iran are not exclusive to this field, they impacted it. a suitable stewardship can decrease the consequences of some challenges such as policy orientation. political interactions and decisions have also affected the development of dental specialization in iran. popularity of specialization in dentistry and high costs of education in a free educational system highlight the significance of need assessment for human resources. according to the perspectives of the reviewers, determining the required number of dental specialists in academic and therapeutic fields, and setting some criteria for development of specialty programs are among the necessities for development and expansion of dental specialties in iran. acknowledgment this research has been extracted from a ph.d. dissertation in community oral health. all study methods were carried out following the relevant regulation of the research ethics committees of research institute of dental sciences-shahid beheshti university of medical sciences that approved this study (ethical code: ir.sbmu.drc. rec.1398.178). conflict of interest the authors declare that they have no conflict of interest.  references 1. widström e, eaton ka. factors guiding the number of dental specialists in the european union and economic area. den norske tannlegeforenings tidende. 2006;116:718–21. 2. owall b, welfare r, garefis p, hedzelek w, hobkirk j, isidor f, et al. specialisation and specialist education in prosthetic dentistry in europe. european journal of prosthodontics and restorative dentistry. 2006;14(3):105. 3. chukwuma sr c. information-base and determinants of medical specialization and primary care: a view point. jbah. 4. sriram v, hyder aa, bennett s. the making of a new medical specialty: a policy analysis of the development of emergency medicine in india. international journal of health policy and management. 2018;7(11):993. 5. pope c, mays n, ziebland s, le may a, williams s, coombs m, et al. qualitative methods in health research methods. 2000;1(2):10.1002. 6. the world bank. lower middle income [available from: https://data. worldbank.org/country/xn]. 7. parkash h, mathur vp, duggal r, b. j. dental workforce issues: a global concern. journal of dental education. 2006(70):22–6. 8. khoshnevisan m, ghasemianpour m, samadzadeh h, baez r. oral health status and healthcare system in ir iran. contemp med sci. 2018;4(3):107–18. 9. hosseinpour r, ebrahimi e, mirmalek sani m, b. s. a review of functions and goals of dental sector in iran’s health systsm. today’s dentistry. 2010;43:19–28. 10. world health organization. working together for health: the world health report 2006: policy briefs: world health organization; 2006. 11. baggott r. understanding health policy: policy press; 2015. 12. hwang s. utilizing qualitative data analysis software: a review of atlas.ti. social science computer review. 2008;26(4):519–27. 13. lindgren b-m, lundman b, graneheim uh. abstraction and interpretation during the qualitative content analysis process. international journal of nursing studies. 2020;108:103632. 14. mohammadpour m, bastani p, brennan d, ghanbarzadegan a, bahmaei j. oral health policymaking challenges in iran: a qualitative approach. bmc oral health. 2020;20:1–12. 15. isiekwe g, umeizudike k, abah a, fadeju a. nigerian dental students’ perspectives about their clinical education. 2019. 16. bailit hl. the fundamental financial problems of dental education and their impact on education, operations, scholarship, and patient care. journal of dental education. 2008;72:14–7. 17. jawaid sa. plight of dentistry in pakistan. pakistan journal of medical sciences. 2020;36(3):299. 18. world health organization. the world health report 2000: health systems: improving performance: world health organization; 2000 [available from: https://www.who.int/publications-detail-redirect/924156198x]. 19. shadpour k. health sector reform in islamic republic of iran. journal of inflammatory diseases. 2006;10(3):7–20. 20. weinrauch j. iran’s response to un resolution 598: the role of factionalism in the negotiation process. american-arab affairs. 1989(31):15. 21. elangovan s, allareddy v, singh f, taneja p, karimbux n. indian dental education in the new millennium: challenges and opportunities. journal of dental education. 2010;74(9):1011–6. 22. crisp n, gawanas b, sharp i. training the health workforce: scaling up, saving lives. the lancet. 2008;371(9613):689–91. 23. poz mrd, couto mhc, franco tdav. innovation, development, and financing of institutions of higher education in health. cadernos de saúde pública. 2016;32. https://data.worldbank.org/country/xn https://data.worldbank.org/country/xn https://www.who.int/publications-detail-redirect/924156198x 322 j contemp med sci | vol. 8, no. 5, september-october 2022: 317–322 development of dental specialties in iran: a qualitative study original t. rojhanian et al. 24. australian research centre for population oral health. dental specialists in australia. australian dental journal. 2010;55(1):96–100. 25. maia ls, dal poz mr. characteristics and trends in the expansion of private dental schools in brazil. international dental journal. 2020;70(6):435–43. 26. grytten j, skau i. specialization and competition in dental health services. health economics. 2009;18(4):457–66. 27. altbach pg, reisberg l, rumbley le. trends in global higher education: tracking an academic revolution: brill; 2019. 28. segal l, marsh c, heyes r. the real cost of training health professionals in australia: it costs as much to build a dietician workforce as a this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i5.1258 dental workforce. journal of health services research and policy. 2017;22(2):91–8. 29. eklund sa, bailit hl. estimating the number of dentists needed in 2040. journal of dental education. 2017;81(8):es146-es52. 30. knevel r, gussy mg, farmer j. exploratory scoping of the literature on factors that influence oral health workforce planning and management in developing countries. international journal of dental hygiene. 2017;15(2):95–105. 31. ghaneirad m. knowledge elite: partnership or migration. tehran: institute of social and cultural studies (in persian); 2017. 235j contemp med sci | vol. 8, no. 4, july-august 2022: 235–238 original evaluation of elementary school teachers’ knowledge about attention deficit hyperactivity disorder (adhd) ashwan samir hamed1*, hawraa hussein ghafel2 1al-diwaniyah health directorate, ministry of health, al-diwaniyah, iraq. 2maternal and neonate nursing department, college of nursing, university of baghdad, baghdad, iraq. *correspondence to: ashwan samir hamed (e-mail: ashoan.sameer1206b@conursing.uobaghdad.edu.iq) (submitted: 21 may 2022 – revised version received: 04 june 2022 – accepted: 14 june 2022 – published online: 26 august 2022) abstract objectives: this study aimed to evaluate elementary school teachers’ knowledge about attention deficit hyperactivity disorder in al-diwaniyah city. methods: a quantitative research, descriptive correlational design was used in this study, the study conducted on teachers at elementary schools in al-diwaniyah city from (20th september 2021 to 30th june, 2022) to evaluate of elementary school teachers knowledge about attention deficit hyperactivity disorder. the sample consist of (354) elementary school teachers were participated in the study. a nonprobability sampling (convenience sample). the sample size was detected by using the sample size formula which is obtained from steven k. thompson, (2012). to determine the effect sample size regarding the population size. results: the findings indicates that 68.6% of teachers show fair level of knowledge about attention deficit hyperactivity disorders (23.90 ± 6.989). conclusion: considering the results discussion and their interpretations, the study concludes that the knowledge of the elementary school teachers in al-diwaniyah city at fair level regarding adhd. keywords: evaluation, elementary schools, teachers, knowledge, attention deficit disorder with hyperactivity issn 2413-0516 introduction children’s health and illnesses have an effect on the well-being of society as a whole as well as on the well-being of future generations. as a result, taking care of children’s mental health helps increase their growth and fertility once they reach maturity. conditions of mental illness will worsen if insufficient attention is paid to early development issues.1 the attention deficit hyperactivity disorder (adhd) is one of the most common behavioral disorders. according to a number of studies related to adhd, adhd affects approximately five percent to ten percent of school-aged children and approximately two and a half percent of adults. in the united states, adhd affects approximately one to two students in each classroom.2 in a recent study that was carried out in najaf, iraq, on several primary schools, the prevalence rate among school children was approximately (25 percent), according to reports teachers’. the estimated prevalence rate of adhd among elementary school children in baghdad, iraq, is approximately (10 percent).3,4 overt, physician-diagnosed, comorbid mental health illnesses including depression, anxiety, and behavior disorder can result from these challenges. among youngsters with adhd, social and emotional issues are prevalent. social challenges can manifest itself in a variety of ways, resulting in friction within the family or with one’s classmates. poor emotional self-regulation, aggressiveness, and a lack of empathy are all common symptoms of emotional distress.2 studies of people with adhd have found that around half of them also suffer from depression and anxiety in community samples. this leads to a decline in children’s functional performance as well as poorer educational results for them.5 a number of studies have demonstrated that children with adhd are more likely to acquire negative attitudes about learning and the school environment. this can have a significant impact on a person’s future career path and limit their options as an adult. having low self-esteem can lead to mental health issues such as depression and anxiety.6 many studies have shown that most children with adhd have normal or above-average intellect, but their impulsive nature makes it difficult for them to utilize their knowledge in everyday circumstances. more than that, they are more likely to have learning difficulties, which can have a negative impact on academic achievement.7 effective and suitable intervention options are needed for children with adhd in order for them to perform well in a classroom setting. as a result, teachers must be well-versed in this condition so that they may create successful behavior adjustment tactics for their students. students who are diagnosed with attention deficit hyperactivity disorder (adhd) in school are more likely to be successful in their treatment.8 teachers and other school workers are frequently the first to notice strange behavior on a child or abnormal motions, and they are able to convey these things to professionals to assist in diagnosis.9 therefore, it is crucial for instructors to have knowledge of disorders and an understanding of the key abilities required in order to work effectively with students within the context of a typical classroom environment. to aid future kids with adhd in their classrooms, teachers must learn more about adhd and how it affects pupils both academically and socially. children’s ability to bounce back from setbacks and develop a good self-image might benefit from this as well.10 because of everything that has been discussed up until this point, it is important to bring attention to this issue and work toward improving the knowledge and attitude of primary school teachers regarding adhd in children. this will enable the teachers to recognize the disorder and take the appropriate steps when dealing with children who have it. mailto:ashoan.sameer1206b@conursing.uobaghdad.edu.iq 236 j contemp med sci | vol. 8, no. 4, july-august 2022: 235–238 evaluation of elementary school teachers’ knowledge about attention deficit hyperactivity disorder (adhd) original a.s. hamed et al. methodology design of the study a quantitative research, descriptive correlational design was used in this study, the study conducted on teachers at elementary schools in al-diwaniyah city from (20th september 2021 to 30th june, 2022) to evaluate of elementary school teachers knowledge about attention deficit hyperactivity disorder. administrate arrangements prior to the collection of the study results, official permits were obtained from the relevant authorities as follows: university of baghdad/college of nursing council. ministry of planning (cso) ‘central statistical agency’ official permits. al-diwaniyah educational director to access the schools to facilitate the data collection. ethical considerations the involvement in the study was voluntary. the participant sign the consent form for voluntary participation in the study, they were given an anonymous questionnaire to preserve absolute confidentiality for the participants. settings of the study the study was conducted at governmental primary schools in al diwaniyah city, iraq. the total number of primary schools was (20) governmental primary schools involved in the study, selected randomly from the total number (173) governmental primary schools in al-diwaniyah city. sample of the study the sample consist of (354) elementary school teachers were participated in the study. a non-probability sampling (convenience sample). the sample size was detected by using the sample size formula which is obtained from steven k. thompson, (2012). to determine the effect sample size regarding the population size. data collection the collected of the data was started from 3rd march, 2022 to 15th april, 2022. the study and the objectives were explained table 1. distribution of sample according to their socio-demographic characteristics list characteristics f % 1 age (m ± sd = 40.68 ± 7.748) less than 30 year 15 4.3 30 – less than 40 year 146 41.2 40 – less than 50 year 137 38.7 50 ≥ year 56 15.8 total 354 100 2 gender male 137 38.7 female 217 61.3 total 354 100 3 residency urban 96 27.1 rural 188 53.1 suburban 70 19.8 total 354 100 f: frequency, %: percentage, m: mean, sd: standard deviation. to the study sample by the student researcher, the teacher’s verbal consent has been taken and the answering of questions has been done by using the self-administrated method. results of the study the descriptive analysis in table 1 shows that teachers are with average age of 40.68 ± 7.748 years in which 41.2% of them are seen with age group of 30less than 40 year and 38.7% of them are with age group 40less than 50 year. the gender refers that 61.3% of teachers are females and 38.7% of them are males. regarding residency, 53.1% of teachers are resident in rural and 27.1% are resident in urban. the table 2 indicates that 68.6% of teachers show fair level of knowledge about attention deficit hyperactivity disorders (23.90 ± 6.989). the figure 1 shows that 68.6% of teachers have fair level of knowledge. discussion the findings shows that two fifth of the teachers are seen with age group of 30less than 40 year and more than one third of them are with age group 40less than 50 year. low rate of employing in the education field, as we did not find young teachers and those who are in their twenties except very small ages. these findings are supported in the literature with many evidences, as the result of zan (2020) who declared approximately similar %ages while they were doing their research in different settings around the world. the findings show that the number of the females in the sample of the study are more than the males, this can be explained as the educational field are preferable in iraq among the females’ gender due to the social trends. another supportive evidence found in the literature by zan (2020) who stated that the vast majority of his sample were females. regarding residency, more than half of teachers are resident in rural areas and slightly more than one quarter of them are resident in urban. these residential distributions can be discussed as the nature of the community where the study was undertaking is rural in general so logically, we will get high 237j contemp med sci | vol. 8, no. 4, july-august 2022: 235–238 a.s. hamed et al. original evaluation of elementary school teachers’ knowledge about attention deficit hyperactivity disorder (adhd) table 2. overall evaluation of teachers’ knowledge about attention deficit hyperactivity disorders (adhd) level of knowledge f % m sd eval. poor 107 30.3 23.90 6.989 fair fair 243 68.6 good 4 1.1 total 354 100 f: frequency, %: percentage, m: mean for total score, sd: standard deviation. poor = 0–20, fair = 20.1–40, good = 40.1–60. fig. 1 levels of teachers’ knowledge about adhd (n = 354). this underscores the significance of adhd as a multisystem disturbance that requires complete examination, whatever the initial disease that led to treatment entrance. this is true regardless of the primary ailment that led to treatment entry. when taking into account co-occurring disorders in treatment planning for adhd, further care is required. it is possible that the use of stimulant drugs without proper diagnosis of concomitant anxiety or bipolar illness might lead to an aggravation of such conditions.14 according to alshehri et al. (2020) who conducted a study on two groups, a study and control group to investigate the teacher’s knowledge about adhd immediately after the intervention, the total knowledge of instructors in the test group regarding adhd improved dramatically, according to the data.12 then, these knowledge benefits diminished marginally, but they persisted for three months and remained superior to those in the control group. the substantial improvement in teachers’ knowledge of adhd as a result of the intervention in the present study is consistent with the findings of multiple studies employing educational or training methods, which all demonstrated a rapid improvement in teachers’ knowledge of adhd, with benefits lasting up to six months. another study conducted in mosul, iraq, found that twofifths of teachers lacked enough knowledge of adhd in general, more than half lacked adequate knowledge of the signs and symptoms of adhd in children, and 45% lacked adequate knowledge of the causes and diagnosis of adhd in children.11 the findings of zan (2020) showed that the majority of the participants have a low level of understanding. recently, several studies have been conducted that suggest that this result may be due to the fact that teachers do not know about this disease as well as how to deal with the child who suffers from it. additionally, it is unable to appreciate how important it is, which results in the child being neglected. our findings are supported by a few research, which discovered that the majority of educators lacked enough knowledge about general information on adhd, in addition to insufficient understanding regarding general information on symptom diagnosis and treatment. conclusion considering the results discussion and their interpretations, the study concludes that the knowledge of the elementary school teachers in al-diwaniyah city at fair level regarding adhd. recommendations enhance teachers, parents, and community awareness about adhd through mass media. the nurse, family and teachers should be cooperate to detect what exact problems are facing child with adhd and give proper intervention. there should be collaboration with educational psychologists in the course of teachers’ training. university curriculum of teachers should include subject on early screening for children with adhd. conflict of interest none.  level of rural teachers working in the educational settings. these results come in contrast with al-wily (2020) who conducted a study in mosul governorate and found that the urbanized teachers are more than those who residing in rural areas.11 also, zan (2020) found that his sample were majorly from urban areas. the findings concerning overall evaluation of teachers’ knowledge about attention deficit hyperactivity disorders (adhd) indicates that slightly less than two thirds of teachers show fair level of knowledge about attention deficit hyperactivity disorders. this result is less than the required especially in the iraqi communities, as teacher take high responsibilities toward students far from their homes. the study findings come in the same context with alshehri and his colleages (2020) who indicated that the majority of teachers were unaware of the number of children with adhd.12 when asked if an adhd child who can display prolonged attention to video games or television can also demonstrate sustained focus for at least an hour of classwork or homework, 52% of respondents say yes, 27% say no, and 21% do not know. the answer was inaccurate, demonstrating that the majority of teachers have a misunderstanding of how the symptoms of adhd vary depending on tasks and environments. the majority of responders provided an erroneous response. only 18% of responders provided the correct response. in another study, the instructors showed an accurate understanding of the effectiveness of the classroom interventions, which is consistent with the findings of prior studies. these findings are encouraging since, after all, it is of the highest significance that the educators be aware of behavioral approaches that may be effective in creating a pleasant learning environment for the children who have been impacted by this condition. however, further research is required to determine the extent to which these strategies are implemented in the day-to-day operations of schools.13 238 j contemp med sci | vol. 8, no. 4, july-august 2022: 235–238 evaluation of elementary school teachers’ knowledge about attention deficit hyperactivity disorder (adhd) original a.s. hamed et al. references 1. arjmandi, s., & sayehmiri, k. (2015). prevalence of attention deficithyperactivity disorder among primary school children according to teachers and parents’ report: systematic review and metaanalysis study. journal of fundamentals mental health, 17(5), 213–221. 2. townsend, m. c., & morgan, k. i. (2018). psychiatric mental health nursing: concepts of care in evidence-based practice, 9th edition, philadelphia: fa davis, p.p. 321-671-742-744-745. 3. alobaidi, a. k. s., & ali, n. s. (2009). adhd among schoolchildren in baghdad. journal canadian academy of child and adolescent psychiatry, 18(1), 4. 4. al-fatlawi, d. a. h., & al-dujaili, a. h. (2019). prevalence of attention deficit hyperactivity disorders among primary school children in al-najaf city, a thesis submitted to the council of faculty of nursing/university of kufa in fulfillment of the requirements for the degree of master in nursing science/ psychiatric mental health nursing, [unpublished thesis], p.i. 5. classi, p., milton, d., ward, s., sarsour, k., & johnston, j. (2012). social and emotional difficulties in children with adhd and the impact on school attendance; healthcare utilization. child and adolescent psychiatry and mental health, 6(1), 33. doi:.1186/1753-2000-6-33. 6. banaschewski, t., coghill, d., & zuddas, a. (eds.). (2018). oxford textbook of attention deficit hyperactivity disorder, 1st edition, oxford university press, p.p. 308. 409 7. topkin, b. (2013). an examination of primary school teachers’ knowledge of the symptoms and management of children diagnosed with adhd in their classrooms, a mini-thesis in partial fulfillment of the requirements for the degree of magister artium in child and family studies, p.1 8. khalil, a. i., alshareef, f. a., & alshumrani, h. g. (2019). knowledge, attitude, and behavioral practice of elementary teacher of adhd children: impact of an educational intervention. american journal of nursing, 8(6), 329–341. 9. te meerman, s., batstra, l., grietens, h., & frances, a. (2017). adhd: critical update educational professionals. international journal of qualitative studies on health and well-being, 12(sup1), 1298267. 10. safaan, n. a., el-nagar, s. a., & saleh, a. g. (2017). teachers’ knowledge about ad/hd among primary school children. american journal of nursing research, 5(2), 42–52. 11. al-wily, m. a. s., al-waly, l. a. m., & ibrahim, r. h. (2020). teachers’ knowledge regarding attention-deficit hyperactivity disorder between pupils at elementary schools in mosul city. medico-legal update, 20(1), 1259–1264. https://doi.org/10.37506/v20/il/2020/mlu/194475. 12. alshehri, a. m., shehata, s. f., almosa, k. m., & awadalla, n. j. (2020). schoolteachers’ knowledge of attention-deficit/hyperactivity disorder— current status and effectiveness of knowledge improvement program: a randomized controlled trial. international journal of environmental research and public health, 17(15), 1–10. https://doi.org/10.3390/ ijerph17155605. 13. mohr-jensen, c., steen-jensen, t., bang-schnack, m., & thingvad, h. (2019). what do primary and secondary school teachers know about adhd in children? findings from a systematic review and a representative, nationwide sample of danish teachers. journal of attention disorders, 23(3), 206–219. https://doi.org/10.1177/1087054715599206. 14. chung, w., jiang, s. f., paksarian, d., nikolaidis, a., castellanos, f. x., merikangas, k. r., & milham, m. p. (2019). trends in the prevalence and incidence of attention-deficit/hyperactivity disorder among adults and children of different racial and ethnic groups. jama network open, 2(11), 1–14. https://doi.org/10.1001/jamanetworkopen.2019.14344. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1251 http://https://doi.org/10.1001/jamanetworkopen.2019.14344. 363j contemp med sci | vol. 7, no. 6, november-december 2021: 363–367 original assessment of epstein-barr virus in patients with squamous cell carcinoma of the larynx by real-time polymerase chain reaction masoumeh nouri1, mohammad hasan larizadeh1*, hamid reza mollaei2, aliasghar arabi mianroodi3, maryam bahador1, sara shamsi1 1department of radiation oncology, afzalipour hospital, kerman university of medical sciences, kerman, iran. 2department of medical microbiology, kerman university of medical sciences, kerman, iran. 3department of otorhinolaryngology head and neck surgery, shafa hospital, kerman university of medical sciences, kerman, iran. *correspondence to: mohammad hasan larizadeh (e-mail: mohammadhasanlarizadeh@yahoo.com) abstract objectives: this study was conducted to investigate the presence of epstein-barr virus (ebv) as a risk factor in laryngeal squamous cell carcinoma (scc) and its effects on the pathological and clinical features of this disease. methods: this cross-sectional study was conducted on 55 patients with histologically confirmed laryngeal scc in the pathology department of shafa and afzalipour hospital, kerman, iran, referred to the radiation oncology department between january 2018 and may 2019 (study group). thirty-five patients who underwent surgery for benign laryngeal lesions such as polyps, nodules, cysts, or granulomas were considered as the control group. biopsy specimens of all subjects were examined for the presence of ebv dna by real-time polymerase chain reaction (rt-pcr). demographic and clinicopathological characteristics of all patients were recorded and patients with laryngeal scc were followed up regarding relapse. the data were analyzed using spss-17 software and descriptive and analytical statistics were done. results: the incidence of ebv infection in patients with laryngeal scc was 32.7% (95% ci: 5/45 – 21/8) and this amount was 2.9% (95% ci: 0 – 8.6) in the control group. based on multivariate logistic regression model, age (or:1.05, 95% cl: 1.00 – 1.10, p-value: 0.04), ebv (or: 9.76, 95% cl: 1.07 – 88.59, p-value: 0.04), smoking (or: 6.82, 95% ci: 1.69 – 27.53, p-value: 0.007) and opium consumption (or: 6.46, 95% ci: 1.36 – 30.66, p-value: 0.01) were determined as predictors of laryngeal scc. in the patients’ group, the presence of ebv was associated with a higher lymph node stage, which was statistically significant but there was no significant relationship in terms of age, sex, tumor location and t stage and relapse. discussion & conclusion: this study showed an association between ebv as a risk factor and laryngeal scc. keywords: epstein-barr virus, laryngeal squamous cell carcinoma, real-time polymerase chain reaction issn 2413-0516 introduction squamous cell carcinoma (scc) of the larynx is a common malignancy that is associated with a high rate of disability and mortality.1 the role of many factors, especially tobacco and alcohol consumption, have been confirmed in the development of laryngeal scc, but the rate of this malignancy is also increasing in people who do not smoke or drink alcohol.2 recent studies have investigated the role of viruses in developing this type of cancer.3–5 epstein–barr virus (ebv) is a virus that can cause cancer and is transmitted orally and infects more than 95% of people in the first decade of life.1 although the role of the virus in the pathogenesis of some head and neck cancers, such as nasopharyngeal cancer, is well known, there are limited studies available on the association of ebv with laryngeal scc that have shown different results, and the role of ebv in the laryngeal scc is still unclear.4,6–9 although laryngeal cancer mainly affects the glottis in most parts of the world, studies in iran and mediterranean countries have reported that the tumor site is mainly in the supraglottis.10–12 on the other hand, it is estimated that the high prevalence of supraglottic cancers than glottis ones in some geographical areas is due to differences in the prevalence of the ebv virus in different locations. despite the importance of laryngeal cancer, the result of previous studies on the role of ebv in this entity is controversial and has not yet been studied in iran.6,7,13 in the present study, the incidence of ebv in specimens of benign and malignant lesions of the larynx was investigated by rt-pcr. also, the effects of ebv on pathological and clinical features of the patients with laryngeal scc were investigated. materials and methods in the present study, patients with histologically confirmed laryngeal scc in the pathology department of shafa and afzalipour hospital, kerman, iran, referred to the radiation oncology department between january 2018 and may 2019, were considered as the study group. patients who underwent surgery for benign laryngeal lesions such as polyps, nodules, cysts, or granulomas were considered as the control group while individuals with a biopsy showing precancerous lesions such as leukoplakia or dysplasia were excluded. demographic information of all subjects, including age and sex, as well as a history of alcohol consumption, cigarette smoking and opium use, lesion location (glottis, supraglottis, and subglottis), grade (g1: well, differentiated, g2: moderately differentiated, g3: poorly differentiated) and stage of the disease (t, n, m staging, ajcc 2017) were collected and entered in a form designed for registration. patients with laryngeal scc were visited every 4 to 8 weeks for the first 2 years and then every 3 months. (submitted: 01 september 2021 – revised version received: 22 september 2021 – accepted: 12 october 2021 – published online: 26 december 2021) 364 j contemp med sci | vol. 7, no. 6, november-december 2021: 363–367 assessment of ebv in patients with squamous cell carcinoma of the larynx by real-time pcr original m. nouri et al. during follow-up visits, patients underwent history taking, physical and fiberoptic examination. if relapse was suspected, laryngeal biopsy would be taken. all tissue samples (paraffin-embedded blocks) included in the study were examined for the detection of ebv dna by rt-pcr. the molecular tests of detecting ebv dna by rt-pcr were performed based on the following steps: 1. preparation of pathological samples in tissue processor device which contains formalin, ethanol, and xylol. molding samples by paraffin molds, and removing formalin from the tissue in several stages. 2. virus dna extraction using ribo-prep commercial kit, according to the manufacturer’s instructions. the quantitative and qualitative examination of the extracted dna the quantity and quality (amount of protein and polysaccharide contaminants) of the extracted dna were evaluated by the absorption rate of the samples at 260 and 280 nm and the calculation of the adsorption ratios of 260/260 nm using the nanodrop spectrophotometer. to detect the absence of fractures and the integrity of the dna, the same amount of extracted dna was electrophoresed on the 1.5% agarose gel containing ethidium bromide. 3. study of light absorption nanodrop device at this stage, to measure the amount of dna extracted, the amount of 2 µl of the dna sample was poured into a 0.2 ml microtube, and after adding 98 µl of distilled water to it, the absorbance of the solution was measured at a wavelength of 260 nm. finally, according to the dna absorption coefficient at 260 nm, and also, the dilution coefficient of 50, the amount of dna was calculated. 4. identification of samples for ebv contamination using real-time pcr tests real-time pcr was used to detect the ebv virus. this method uses a primer and a probe specific for the bzlf5 virus gene region, which is responsible for the synthesis of viral dna polymerase. sequences of these areas are given below. primer 1: cggaagccctctggacttc primer 2: ccctgtttatccgatggaatg p r o b e f a m t g t a c a c g c a c g a g a a a t g cgcc-bhq1 after extracting the virus genome, 10 μl of mastermix2x, 0.5 μl of primer and probe with a final concentration of 10 nmol, 4.5 μl of depc sterile water, and finally, 5 μl of dna and the extracted sample were added to 0.2 ml microtubes. after a quick spin, control samples with unknown samples were placed in real-time pcr (rotor-gene 6000, australia, and corbett research) and tested according to temperature profile (95°c for 10 minutes with 45 cycles 95°c for 15 seconds and 60°c for 40 seconds). finally, positive samples in the green channel (fam) were analyzed in terms of fluorescence at a temperature of 60°c. all collected data were plugged into the spss version 17 software. the mean and standard deviation indices were used to describe quantitative data and the percentage and frequency were used to describe qualitative data. the chi-square test was also used to compare ebv positivity in the control and patient groups, to examine the positive relationship between ebv and other factors such as smoking and alcohol consumption, stage and degree of disease differentiation, and tumor location. statistical significant level was considered at p = 0.05. results demographic information of subjects the present study was conducted on 55 patients with laryngeal cancer and 35 patients with benign laryngeal diseases. the mean age of subjects in this study was 58.04 ± 11.55 years old (age range: 31 to 88) for patients with laryngeal cancer and 46.89 ± 14.63 years old (age range: 9 to 71) for those with benign laryngeal diseases, respectively, and patients with cancer were older (p = 0.001). according to the findings, patients with laryngeal cancer were significantly different from those with benign laryngeal diseases in terms of sex, cigarette smoking, opium consumption, and ebv infection. the majority of cancer patients were male (p = 0.01) and more than three-quarter of them were cigarette smokers (80% versus 54.2%; p = 0.001) and opium consumers (87.3% versus 58.3%, p = 0.004), and ebv infection was positive for more than onequarter of them (32.7% versus 2.9%, p = 0.001) in comparison to those with benign laryngeal diseases (table 1). table 1. comparison of demographic information of the study subjects variables level of variables patients with laryngeal cancer (n = 55) patients with benign laryngeal diseases (n = 35) p-value age (year) 58.04 ± 11.55 46.89 ± 14.63 0.001 sex male 51 (92.7) 26 (74.3) 0.01 female 4 (7.3) 9 (25.7) cigarette smoking negative 11 (20) 11 (45.8) 0.001 positive 44 (80) 13 (54.2)* opium consumption negative 7 (12.7) 10 (41.7) 0.004 positive 48 (87.3) 14 (58.3)* ebv infection negative 37 (67.3) 34 (97.1) 0.001 positive 18 (32.7) 1 (2.9) *the cigarette smoking and opium consumption of a patient with benign laryngeal diseases was unclear. 365j contemp med sci | vol. 7, no. 6, november-december 2021: 363–367 m. nouri et al. original assessment of ebv in patients with squamous cell carcinoma of the larynx by real-time pcr the correlation of demographic and clinical information with ebv infection in cancer patients according to the findings, cancer patients with ebv infection were similar to other cancer patients based on demographic information such as age (p = 0.53), sex (p = 1), cigarette smoking (p = 1), and opium consumption (p = 0.67) (table 2). among clinical information of cancer patients, only n staging was associated with ebv infection and cancer patients with ebv had higher n staging in comparison to other cancer patients without ebv infection (p = 0.004). the other clinical information was not statistically different between the two groups (table 2). the mean follow-up time was 17.50 ± 6.95 months. relapse occurred during the follow-up period in 4 patients with laryngeal scc (7.27%) with a mean time to recurrence of 11.50 ± 7.59 months. two of these patients were ebv positive and the remaining was ebv negative. the mean time to recurrence in ebv positive group was 6.50 ± 3.53 months and in ebv negative group was 16.50 ± 7.77 months (p-value = 0.24). discussion there are few studies on the relationship between ebv and scc of the larynx and the results of previous studies on this issue are not conclusive.6,9,14,15 the present study was conducted to evaluate the prevalence of ebv infection in patients with laryngeal scc and to compare it with patients with benign laryngeal diseases in kerman. findings showed that the prevalence of ebv infection in patients with laryngeal scc was 32.7% (95% ci: 21.8–5.45). in patients with benign laryngeal diseases who were considered as controls, the prevalence of ebv infection was 2.9% (95% ci: 0.6–6). this finding is inconsistent with the study of polz-gruszka et al. (2015) in poland, which tested serum ebv and lmp-1 antibodies in 64 patients with laryngeal cancer, 35 (54.7%) of whom were ebv positive.16 the control group consisted of 40 cases, in 10 out of which, the virus genome was positive (25%). the researchers concluded that the ebv virus is present in patients with pharyngeal and laryngeal cancer in polish patients and that its wild form, in particular, is more closely associated with tumors. lee et al.17 also reported ebv as an independent risk factor that causes laryngeal scc. in contrast, in a study by muderris et al.4 on 25 patients with laryngeal scc and 17 patients with benign laryngeal lesions, fresh tissue samples were examined by pcr to detect ebv dna. ebv dna was positive in 40% (10 of 25) of patients with laryngeal cancer and 47.1% (8 of 17) in the control group (benign lesions) with no significant difference (p = 0.892).4 therefore, in the present study, which includes a higher number of subjects than previous studies, a relationship was found between ebv and laryngeal scc. old age is known as a risk factor for laryngeal scc.18 the mean age of patients with laryngeal scc in the present study table 2. comparison of demographic and clinical information based on ebv infection among cancer patients variables level of variables cancer patients without ebv (n = 37) cancer patients with ebv (n = 18) p-value age (year) 57.35 ± 10.74 59.44 ± 13.26 0.53a sex male 34 (91.9) 17 (94.4) 1b female 3 (8.1) 1 (5.6) cigarette smoking negative 7 (18.9) 4 (22.2) 1b positive 30 (81.1) 14 (77.8) opium consumption negative 4 (10.8) 3 (16.7) 0.67b positive 33 (89.2) 15 (83.3) histological grading g1 5 (13.5) 4 (22.2) 0.51b g2 30 (81.1) 14 (77.8) g3 2 (5.4) 0 t stage t1 4 (10.8) 1 (5.6) 0.62b t2 13 (35.1) 4 (22.2) t3 18 (48.6) 11 (61.1) t4 2 (5.4) 2 (11.1) n stage n0 32 (86.5) 8 (44.4) 0.002b n1 3 (8.1) 6 (33.3) n2 1 (2.7) 4 (22.2) n3 1 (2.7) 0 location of cancer subglottic 1 (2.7) 1 (5.6) 0.40b glott 12 (32.4) 3 (16.7) supraglottic 24 (64.9) 14 (77.8) relapse negative 35 (94.6) 16 (88.9) 0.59b positive 2 (5.4) 2 (11.1) astudent's t-test, bfisher's exact test. 366 j contemp med sci | vol. 7, no. 6, november-december 2021: 363–367 assessment of ebv in patients with squamous cell carcinoma of the larynx by real-time pcr original m. nouri et al. was 58.04 ± 11.55 years old (age range: 31 to 88 years). the majority of patients (n = 53, 96.36%) were over 40 years old and there was a significant relationship between increasing age and cancer incidence (p = 0.001). there was no statistically significant relationship between age and ebv infection in patients with laryngeal cancer (p = 0.53), which is consistent with the results of several previous studies.7,19 in this study, the majority of patients (n = 51, 92.7%) were male and the rest were female (n = 4, 7.3%). based on the findings of this study, there was no relationship between the frequency of ebv infection and the gender of cancer patients (p = 1), which is consistent with the results of the study of zhang et al.19 cigarette smoking and alcohol consumption are known as risk factors of laryngeal scc.20 in this study, none of the patients reported a history of alcohol consumption but more than three-quarters of cancer patients reported smoking (44, 80%) and there was a significant relationship between cigarette smoking and scc of the larynx (p = 0.02). opium consumption is also a risk factor for developing head and neck cancer.21 it was also found that more than 80% of cancer patients were opium users (n = 48, 87.3%), and there was a significant relationship between opium use and laryngeal scc (p = 0.006), but there is no association between ebv infection and smoking (p = 1) or opium use (p = 0.67) in cancer patients. this finding is consistent with the result of the study of muderris et al.,4 who also found no association between alcohol consumption and smoking and ebv infection in cancer patients.4 in cancer patients, the most common lesion site was the supraglottic region (38 cases, 69.1%), followed by the glottis region (15 cases, 27.3%). in only two patients, a lesion was observed in the subglottic region (3.6%). findings from the study showed that there was no association between the lesion site of cancer and ebv infection (p = 0.40). this finding is consistent with the results of the study of vazquezguillen et al.,7 who found that ebv dna in patients with laryngeal scc and ebv was positive in 22.1% of glottic cancer, 16.7% of subglottic cancer, and 33.3% of supraglottic malignancy. there was no difference between the ebv dna and the tumor location in their study.7 in contrast, břicháček et al.22 reported an association between laryngeal supraglottic carcinoma and ebv infection, and also, found that ebv dna was positive in 3 out of 5 patients with supraglottic laryngeal cancer.21 in the present study, more than one-third of cancer patients was in t2n0 stage (n = 22, 40%), followed by t3n0t1-3n1 stage (n = 20, 36.4%), stage t4n0 (n = 10, 18.2%), t1-4n2-3 (n = 10, 18.2%), and t1n0 (n = 3, 5.5%), respectively. ebv has no significant relationship with the t stage (p = 0.62) but was associated with a higher lymphatic stage (p = 0.002). also, no relationship was found between pathological grade and virus burden (p = 0.51). in a study by al-thawadi et al.23 that examined the association of this virus with the histopathological and clinical features of head and neck cancers, no association was found between ebv infection and tumor grade or disease stage,22 which is consistent with the results of several other studies.4,8 the effect of ebv on the prognosis of laryngeal scc has been controversial.17,24 in the present study, 4 patients relapsed, among whom two patients were ebv positive and the other ones were ebv negative. there was no significant relationship between ebv and relapse incidence (p = 0.59). so, further studies on the effect of ebv on prognosis and risk of disease relapse are recommended. the effects of co-infection of ebv with other oncogene viruses were determined in different head and neck cancers.5,16 in the present study, only the ebv virus was assessed, so we plan to investigate the effect of co-infection of oncogene viruses on clinicopathologic characteristics and prognosis of this type of cancer. conclusion the findings of the present study showed an association between ebv as a risk factor and laryngeal scc. although in this study, the presence of ebv in supraglottic cancers was higher than in other laryngeal sites, this relationship was not significant. due to the small sample size in the group of cancer patients, further studies with larger sample sizes are recommended to investigate this relationship.  references 1. singh sp, eisenberg r, hoffman g. an overview and comparative evaluation of head and neck cancer risk factors in india and australia. international journal of otolaryngology and head & neck surgery. 2018;7(05):254. 2. marur s, forastiere aa, editors. head and neck cancer: changing epidemiology, diagnosis, and treatment. mayo clinic proceedings; 2008: elsevier. 3. ahmadi n, ahmadi n, chan mv, huo yr, sritharan n, chin r. laryngeal squamous cell carcinoma survival in the context of human papillomavirus: a systematic review and meta-analysis. cureus. 2018;10(2). 4. muderris t, rota s, muderris t, inal e, fidan i. does epstein-barr virus infection have an influence on the development of laryngeal carcinoma? detection of ebv by real-time polymerase chain reaction in tumour tissues of patients with laryngeal carcinoma. brazilian journal of otorhinolaryngology. 2013;79(4):418–23. 5. gupta i, ghabreau l, al-thawadi h, yasmeen a, vranic s, al moustafa a-e, et al. co-incidence of human papillomaviruses and epstein–barr virus is associated with high to intermediate tumor grade in human head and neck cancer in syria. frontiers in oncology. 2020;10:1016. 6. de oliveira de, bacchi mm, macarenco rs, tagliarini jv, cordeiro rc, bacchi ce. human papillomavirus and epstein-barr virus infection, p53 expression, and cellular proliferation in laryngeal carcinoma. american journal of clinical pathology. 2006;126(2):284–93. 7. vazquez-guillen jm, palacios-saucedo gc, rivera-morales lg, alonzomorado mv, burciaga-bernal sb, montufar-martinez m, et al. infection and coinfection by human papillomavirus, epstein–barr virus and merkel cell polyomavirus in patients with squamous cell carcinoma of the larynx: a retrospective study. peer j. 2018;6:e5834. 8. kiaris h, ergazaki m, segas j, spandidos d. detection of epstein-barr virus genome in squamous cell carcinomas of the larynx. the international journal of biological markers. 1995;10(4):211–5. 9. mousa mj. the frequency of epstein-barr virus infection as a pathogenic agent in laryngeal carcinoma of iraqi patients demonstrated by lmp-1 expression. journal of university of babylon. 2016;24(3). 10. saedi b, razmpa e, sadeghi m, mojtahed m, mojtahed a. the epidemiology of laryngeal cancer in a country on the esophageal cancer belt. indian journal of otolaryngology and head & neck surgery. 2009;61(3):213–7. 11. emadzadeh m, shahidsales s, bajgiran am, salehi m, massoudi t, nikfarjam z, et al. head and neck cancers in north-east iran: a 25 year survey. iranian journal of otorhinolaryngology. 2017;29(92):137. 367j contemp med sci | vol. 7, no. 6, november-december 2021: 363–367 m. nouri et al. original assessment of ebv in patients with squamous cell carcinoma of the larynx by real-time pcr 12. larizadeh mh, damghani ma, shabani m. epidemiological characteristics of head and neck cancers in southeast of iran. iranian journal of cancer prevention. 2014;7(2):80. 13. zykova t, kit oi, vladimirova ly, ryadinskaya la, shevyakova ea, bogomolova oa, et al. impact of viral infection on effectiveness of antitumor treatment for laryngeal cancer. american society of clinical oncology; 2018. 14. mulder fj, klufah f, janssen fm, farshadpour f, willems sm, de bree r, et al. presence of human papillomavirus and epstein–barr virus, but absence of merkel cell polyomavirus, in head and neck cancer of nonsmokers and non-drinkers. frontiers in oncology. 2021;10:3048. 15. prabhu sr, wilson df. evidence of epstein-barr virus association with head and neck cancers: a review. j can dent assoc. 2016;82(g2):1488–2159. 16. polz-gruszka d, stec a, dworzański j, polz-dacewicz m. ebv, hsv, cmv and hpv in laryngeal and oropharyngeal carcinoma in polish patients. anticancer research. 2015;35(3):1657–61. 17. lee la, fang tj, li hy, chuang hh, kang cj, chang kp, et al. effects of epstein-barr virus infection on the risk and prognosis of primary laryngeal squamous cell carcinoma: a hospital-based case-control study in taiwan. cancers. 2021;13(7):1741. 18. halmos g, bras l, siesling s, van der laan b, langendijk j, van dijk b. age‐specific incidence and treatment patterns of head and neck cancer in the netherlands—a cohort study. clinical otolaryngology. 2018;43(1): 317–24. 19. zhang y, chen x, xia l, can l, dan l, ruixia m, et al. correlation of positive expressions of hpv and ebv with laryngeal carcinoma. the journal of practical medicine. 2017;33(13):2117–22. 20. nocini r, molteni g, mattiuzzi c, lippi g. updates on larynx cancer epidemiology. chinese journal of cancer research. 2020;32(1):18. 21. alizadeh h, naghibzadeh-tahami a, khanjani n, yazdi-feyzabadi v, eslami h, borhaninejad v, et al. opium use and head and neck cancers: a matched case-control study in iran. asian pacific journal of cancer prevention: apjcp. 2020;21(3):783. 22. břicháček b, hirsch i, šíbl o, vilikusova e, vonka v. association of some supraglottic laryngeal carcinomas with eb virus. international journal of cancer. 1983;32(2):193–7. 23. al-thawadi h, gupta i, jabeen a, skenderi f, aboulkassim t, yasmeen a, et al. co-presence of human papillomaviruses and epstein–barr virus is linked with advanced tumor stage: a tissue microarray study in head and neck cancer patients. cancer cell international. 2020;20(1):1–13. 24. vlachtsis k, nikolaou a, markou k, fountzilas g, daniilidis i. clinical and molecular prognostic factors in operable laryngeal cancer. european archives of oto-rhino-laryngology and head & neck. 2005;262(11): 890–8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1097 346 j contemp med sci | vol. 7, no. 6, november-december 2021: 346–348 original effects of anti-tuberculosis drugs on the level of some nutritional factors among patients with tuberculosis zainab adil ghani chabuck1*, sarah hashim alhelli2, saade abdalkareem jasim3 1college of medicine, university of babylon, babylon, iraq. 2biomedical engineering department, al-mustaqbal university college, 51001 hillah, babil, iraq. 3medical laboratory techniques department, al-maarif university college, anbar, iraq. *correspondence to: zainab adil ghani chabuck (e-mail: zainabibz@gmail.com) abstract objectives: the study aimed to assess changes in the level of some nutritional elements including zinc and copper for tuberculosis patients before, during and after antituberculosis therapy. methods: whole blood samples are collected from 120 tuberculosis patients who are admitted to specialist clinic of the respiratory diseases in babylon province, within a period from november 2020 to january 2021. results: out of whole tb patients, (66) ones were males and the remaining (54) were females, the age of patients ranging from (13–70) years. sixty, apparently healthy control groups who had no tuberculosis history were chosen. using spectrophotometer for estimating the level of zinc and copper. in tb patients the zinc level was increased during and after anti-tuberculosis therapy, but the level of zinc lower in patients after therapy compared with controls. the level of copper was decreased during and after the course of anti-tuberculosis, but the level of copper higher in patients after therapy compared with controls. conclusion: these parameters could be used in follow-up as the achievement of tb therapy success. keywords: copper, zinc, m. tuberculosis, anti-tuberculosis issn 2413-0516 introduction tuberculosis (tb) is one of the primary considerable causes of sickness and mortality. as along many decades, the numbers of tb cases had been increased significantly worldwide. human immune response has a potential implication in the control and prevention of tb disease, thus it is so essential to comprehend its regulation; especially the cell-mediated immune response that influenced the main response of the host against m. tuberculosis, in which important role is attributed to cytokine and t helper-1 cells.1 many factors can affect this response, one of them is the micronutrients, copper, and zinc have a vital role in the growth, differentiation, and proliferation of cells of acquired and innate immunity. both these nutrients play a crucial role in the resistance of the human body to certain infections as tuberculosis. as an influence of reduced zinc level, which causes shifting in the function of macrophage and reduces the production of interferon-γ (inf-γ) and tumor necrosis factor (tnf-α). this reduced zinc concentration may be also responsible for the reduction of the differentiation and proliferation of b and t lymphocytes. the changes in the immunity dynamics would be the first line in the presentation of the immune system to protect against active tuberculosis. additionally, a quick cure of adult patients with active tb could be achieved by supplementation of zinc and vitamin a as they allow thorough elimination of mycobacterium.2 vemula et al. showed that micronutrients deficiency enhances the ability of mycobacterium to produce m. tuberculosis zinc metalloprotease-1 (zmp1), which is crucial for their pathogenesis and intracellular survivals with immunogenic potential.3 regarding copper, it has a role in the biological systems due to its existence in the composition of many active biological enzymes that act in reductive and oxidative reactions, as metallothioneins lysyl oxidase, ceruloplasmin, cytochrome oxidase, and sod. in spite of its biological and physiological assistances, its excessive level is toxic and might cause inhibition of m. tuberculosis growth.4 materials and methods the population of study-involved tuberculosis (tb) patients admitted to specialist clinics of the respiratory diseases in babylon province, within a period from november 2020 to january 2021. out of whole tb patients, (66) ones were males and the remaining (54) were females, the age of patients ranging from 13 to 70 years. the exclusion criteria: any patients have diabetic mellitus, malignancy, allergic and pregnant women left out from the study. as well, sixty apparently healthy groups who had no tuberculosis previously. the sample collection for serological studies the serum was separated; three ml of the blood sample was allowed to clot for about fifteen min. after that loosed the clot from a wall of the tube, at room temperature. then, the sample centrifuged for 10 min at 2500 rpm. as a final point the serum transported to another tube for storage at –20°c. methods using spectrophotometer for estimating the level of zinc and copper (lta-italy). statistical analysis this study used a statistical analysis that included the calculation of mean values and sd. for p value used independent samples t-tests, and the level p < 0.05 is significant. the statistical analysis used (spss inc., chicago, usa) version 18. (submitted: 16 september 2021 – revised version received: 12 october 2021 – accepted: 29 october 2021 – published online: 26 december 2021) 347j contemp med sci | vol. 7, no. 6, november-december 2021: 346–348 z.a.g. chabuck et al. original effects of anti-tuberculosis drugs on the level of some nutritional factors among patients with tuberculosis results alevel of zinc during and after therapy for tb patients and controls the mean and sd of zinc for untreated tb patients (35.507 ± 9.876) μg/dl then (55.162 ± 9.941) μg/dl during therapy and (63.212 ± 8.752) μg/dl after complete course of anti tuberculosis therapy. while (74.350 ± 7.260) μg/dl for control groups, table 1. also, this study found that the means of zinc no significantly lower in patients after therapy (63.212 ± 8.752) μg/dl compared with control group (74.350 ± 7.260) μg/dl, table 2. blevel of copper during and after therapy for tb patients and controls the mean and sd of copper for untreated tb patients (158.518 ± 28.561) μg/dl then (119.527 ± 22.026) μg/dl during therapy and (109.871 ± 26.547) μg/dl after a complete course of anti-tuberculosis therapy, table 3. also, in this study found that the means of copper significantly higher in tb patients after therapy (109.871 ± 26.547) μg/dl compared with control groups (85.277 ± 7.907) μg/dl, table 4. discussion zinc is a micronutrient that is necessary for the typical activities of the immune system.5 zinc aids in the bacterial destruction by the immune system cells, such as tubercle bacilli. thus reduced zinc level enhances infections; in tb and tb-hiv infected patients there were decreased levels of zinc in their blood samples. this regulation can occur at the transcriptional level, via dna-binding metal-responsive transcriptional repressors which are responsible for the regulation of the transcription process as in many bacteria as m. tuberculosis.4 from the results was found the means of zinc increased gradually during anti-tuberculosis and after therapy. the result was in agreement with a study of 6 who found that a lessened concentration level of serum zn at the initial stage of treatment when compared to control, in men and in women (p < 0.05). after the course of treatment, it was showed a significant increment in the level of serum zn when it compared to untreated patients. this could occur due to change in the distribution of zinc flowing through out further tissues including the liver; this is due to a reduced production of α-2 macroglobulin in the liver; this protein carry zinc in the blood, to get advantage of high production of this protein, it carries zinc to the liver.6 the result was matched with the study of 6 who found that no significant difference in the mean zinc (p = 0.415) levels between tuberculosis patients who have completed anti tuberculosis treatment 80.48 and healthy groups 86.42. another study was showed lower concentration of zinc was observed when compared to normal values.6 from the results was found the means of copper decreased gradually during anti-tuberculosis and after therapy. the result was matched with the study of 7 who mentioned that the serum level of copper in the patients under anti tb treatment was significantly decreased. the result was matched with8 who showed that a lower concentration of copper in tb patients was observed when it compared to normal values. the high concentration of copper in tb patients could be explained logically. as the decrement in levels of zinc, which occurs in tb patients, could prevent the entering of the copper into the tissues, and this may lead to the elevation in the level of serum copper. while increment in the levels some metals as cadmium or copper leads to poorer absorption of serum iron that is in agreement with this study.9  table 1. zinc level in the serum for tb patients untreated patients during therapy after therapy 35.507 ± 9.876 55.162 ± 9.941 63.212 ± 8.752 table 2. level of zinc in tb patients and controls studied groups mean ± sd p value after therapy patients 63.212 ± 8.752 0.434 controls group 74.350 ± 7.260 table 3. copper level in the serum of tb patients untreated patients during therapy after therapy 158.518 ± 28.561 119.527 ± 22.026 109.871 ± 26.547 table 4. level of copper in tb patients and controls studied groups mean ± sd p value after therapy patients 109.871 ± 26.547 0.002 controls group 85.277 ± 7.907 references 1. daulay, r. s. and daulay, r. m. (2018). interferon-gamma and interleukin-10 profile of children with tuberculosis in north sumatera, indonesia. iop conf. series: earth and environmental science 125: 012147. 2. bahi, g.a.; boyvin, l.; méité, s.; m’boh, g.m.; yeo, k.; n’guessan, k.r.; alain, a.d.p. and allico, a.j. (2017). assessments of serum copper and zinc concentration, and the cu/zn ratio determination in patients with multidrug resistant pulmonary tuberculosis (mdr-tb) in côte d’ivoire. bmc infectious diseases.17:257. 3. vemula, m.h; ganji, r.; sivangala, r.; jakkala, k.; gaddam, s.; penmetsa, s. and banerjee, s. (2016). mycobacterium tuberculosis zinc metalloprotease-1 elicits tuberculosis-specific humoral immune response independent of mycobacterial load in pulmonary and extra-pulmonary tuberculosis patients. front. microbiol. 7:418. 4. sepehri, z.; mirzaei, n.; sargazi, a.; sargazi, a.; mishkar, a.; kiani, z.; oskoee, h.; arefib, d. and ghavami, s. (2017). essential and toxic metals in serum of individuals with active pulmonary tuberculosis in an endemic region. j. clinic tuberculosis and other mycobacterial dis 6: 8–13. 5. mythili, c. and lalitha, r. (2016). role of micronutrient zinc in pulmonary tuberculosis. sch. j. app. med. sci.,4(5a):1519-1524. 6. edem, v.f.; ige, o. and arinola, o. g. (2015). plasma vitamins and essential trace elements in newly diagnosed pulmonary tuberculosis patients and at different durations of anti-tuberculosis chemotherapy. egypt j chest dis tuberc. 64: 675–679. 7. reza, b.; khalilzadeh, s.; milanifar, a.; hakimi, s. and khodayari, a. (2007). evaluation of copper, zinc and copper/zinc ratio in serum of pulmonary tuberculosis children. pediatric pulmonary ward, national research in statute 348 j contemp med sci | vol. 7, no. 6, november-december 2021: 346–348 effects of anti-tuberculosis drugs on the level of some nutritional factors among patients with tuberculosis original z.a.g. chabuck et al. of tuberculosis and lung disease, shaheed beheshti university of medical sciences, iran. 8. al-sa’adi, m.a.k.; muhsin, m.a and al-jubouri, a.m. (2011). studying the effect of zinc and copper on cellular immunity in tuberculosis patients. mjbl: 8(3). 9. milena, l.; de paula ramalho, d.; delogo, k.; miranda, p.; mesquita, e.; de oliveira, h.; netto, a.; anjos, m.; kritski, a. and de oliveira, m. (2014). association of serum levels of iron, copper, and zinc, and inflammatory markers with bacteriological sputum conversion during tuberculosis treatment. biological trace element research. 160(2): 176–184. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1083 196 j contemp med sci | vol. 7, no. 4, july-august 2021: 196–201 original abstract objectives this study aimed to investigate the effect of crataegus monogyna (c. monogyna) on controlling the blood pressure in patients with hypertension along with sleep disorder. methods this was a parallel double-blind, placebo-controlled trial study from may, 2018 to december, 2019. total of 60 patients with hypertension and sleep disorder were randomly assigned to c. monogyna and placebo groups. the groups received c. monogyna and placebo capsules twice-daily for 8 weeks. the primary outcomes were the changes of systolic and diastolic blood pressure and pittsburgh questionnaire’s scores from baseline measurements. the secondary outcomes were changes in serum biochemical markers from baseline. results before treatment, there was no significant difference between study groups in terms of demographic data and outcomes (p > 0.05) except body mass index (p = 0.02). after treatments the intervention group had a significant improvement in systolic and diastolic blood pressure compared to placebo ((118.14 ± 6.76 vs 129.76 ± 8.28, 95% ci: 7.54–15.69, p < 0.001 and 77.14 ± 5.32 vs 83.57 ± 6.73, 95% ci: 3.17–9.68, p < 0.001, respectively) analysis revealed no significant difference observed between study groups in terms of baseline characteristics, and preand posttreatment laboratory tests. also, none of the groups had superiority in terms of pittsburgh questionnaire’s scores (p = 0.44); however, the intragroup analysis revealed a significant improvements in the intervention group (p = 0.001). conclusion c. monogyna fruit extract as a supplementary medication has beneficial effects on controlling blood pressure and quality of sleep in patients with hypertension along with sleep disorders. keywords hypertension, sleep disorder, quality of sleep, crataegus monogyna, randomized controlled trial efficacy of hawthorn fruit extract on blood pressure and quality of sleep in patients with hypertension along with sleep disorders: a randomized double-blind controlled trial masumeh abbasi1, sepehr gohari2, hassan ahangar3*, mohsen bahrami4, mohammad kamalinejad5, tara reshadmanesh2, seyede shadi nazari6 1department of persian and traditional medicine, school of medicine, zanjan university of medical sciences, zanjan, iran. 2student research center, school of medicine, zanjan university of medical sciences, zanjan, iran. 3department of cardiology, mousavi hospital, school of medicine, zanjan university of medical sciences, zanjan, iran. 4department of persian and traditional medicine, academic center for education, culture and research center, tehran, iran. 5department of pharmacognosy, faculty of pharmacy, shahid beheshti university of medical sciences, tehran, iran. 6department of biostatistics, school of medicine, zanjan university of medical sciences, zanjan, iran. *correspondence to: hassan ahangar (e-mail: masumehabbasi45@yahoo.com ) (submitted: 25 march 2021 – revised version received: 08 april 2021 – accepted: 21 may 2021 – published online: 26 august 2021) introduction hypertension (htn) is a major and modifiable risk factor for the development of cardiovascular diseases in population all around the world.1 according to the who, hypertension causes a serious health problem in most developing countries and contributes to a significant burden on health systems.2 several factors associated with lifestyle and environment, including obesity, urbanization, less exercise, and sedentary life, have attributed to the increased rate of hypertension and other vascular diseases.3,4 although the incidence of hypertension increases rapidly, only one-third of the cases have their disease under control, and drug compliance is alarmingly low.5 therefore, with due attention to the adverse events and compromised efficiency of industrial drugs and medications, recently, traditional remedies have gained crucial favor as an alternative for the treatment of hypertension.6 according to the literature of traditional medicine, hypertension has not been considered as a single and solitary disease and has suggested being arisen as a complication of cardiovascular and hemodynamic abnormalities.7 on this basis, several iranian ancient wises, such as avicenna, have suggested medicinal plants to play a significant role in the avoidance and treatment of hypertension.7 on the other hand, it has been established that sleep disorders also aggravate hypertension severity and interfere with the control of the illness, by provoking higher bp.8-11 the association of sleep disorders with increased and less-controlled hypertension has been confirmed through several studies.12 whereas, middle-aged individuals are at lower risk for hypertension by curing their sleep disorders.13 crataegus monogyna (c. monogyna), also known as “hawthorn” is one of the herbal plants which has been discussed in ancient literature. the fruit of the plant presents in reddish color with an oval shape. in traditional literature, it has been called in several names, such as “zalzalak” (kind of wide plum) and “kiyalak” concerning its temperament, c. monogyna is known to have a cold temper, and according to the ancient iranian wises, it has several beneficiary effects on diarrhea, gastrointestinal reflux disorder, palpitation and cardiovascular disorders. the consumable part of these plants includes flowers, leaves, and fruits, which are used more often compared to their bushes.7,14 many of the beneficial effects of c. monogyna fruit are attributed to its flavonoid and phenolic components such as hyperoside, chlorogenic acid, and isoquercetin.15 on this basis, in the current randomized clinical trial, we primarily aimed to investigate issn 2413-0516 mailto:masumehabbasi45@yahoo.com 197j contemp med sci | vol. 7, no. 4, july-august 2021: 196–201 m. abbasi et al. original effect of hawthorn on blood pressure and quality of sleep the clinical effect of c. monogyna fruit extract on controlling the blood pressure of patients suffering from hypertension along with sleep disorders. materials and methods study design this was a phase iii, parallel double-blind, placebo-controlled randomized trial study to investigate the effects of c. monogyna capsules (500 mg/twice daily) versus placebo on the quality of sleep and blood pressure in patients with concomitant htn and sleeping disorder. the study protocol was approved by ethics committee of the zanjan university of medical sciences (a-12-594-22), and was subsequently registered in iranian registry for clinical trials (irct, id: irct20180225038859n1). all the patients provided written informed consent. study patients and setting the eligible study population was selected from patients in cardiology special clinic in zanjan, iran. the recruitment started from may, 2018 to december, 2019. during this period, 81 patients assessed for eligibility. of these 21 patients were excluded and 60 patients enrolled in the study. from may, 2018 to december, 2019 till end of 2019, a double-blinded randomized controlled trial was carried in cardiology clinic of the zanjan university of medical sciences, zanjan, iran. all patients with htn diagnosis were screened to participate in the study. all patients aged 35–60 years old, who were suffering from first stage of htn (systolic blood pressure (bp): 140–159 mmhg, or diastolic bp: 90–99 mmhg) at least for one year, were enrolled in the study. patients with severe htn who required multiple-drug treatment, secondary htn, end-organ complications (in the last six months), cardiac arrhythmia, valvular heart diseases, malignancy, psychiatric conditions, hepatic dysfunction, and pregnancy, were excluded, as well as the individuals with increased bp up to 180/110 mmhg. study procedure randomization and follow up recruited patients were stratified (according to age and gender) and assigned into 2 groups of c. monogyna and placebo in a 1:1 ratio. the randomization sequence was created by winpepi software (version 11.6). at the recruitment date, patients’ demographic data including age, height and weight, history of underlying disease, concurrent medications (including herbal and chemical), and possible allergies were recorded. the drugs were prescribed two months (500 mg capsul/twice daily). during study period, patients were regularly visited every two weeks at the clinic to record vital signs and evaluate the study progress. patients were advised to continue clinic visits even if they decided to quit the study. outcomes the primary outcomes were 1) changes in systolic blood pressure (sbp) and diastolic blood pressure (dbp) after 8 weeks of treatment. 2) changes in quality of sleep after 8 weeks of treatment using pittsburgh questionnaire.16 the secondary outcomes comprised of changes in laboratory biomarkers after 9 weeks from the baseline including: 1) blood urea nitrogen (bun), 2) creatinine (cr), 3) aspartate aminotransferase (ast), 4) alanine transaminase (alt), 5) triglyceride (tg), 6) total cholesterol, 7) high density lipoprotein cholesterol (hdl-c), 8) low density lipoprotein cholesterol (ldl-c). to assess the secondary outcomes, a biobank was established in which patients’ serum was stored in an ultra-low temperature freezer. based on the regulations of the local data protection agency, after the measurements, all the remaining samples will be anonymized and the biobank will be discontinued. blinding c. monogyna and placebo capsules with same shape, color, and package and different code numbers were used for treatment groups a and b. at the time of recruitment, a unique code number was assigned to each patient and was used till the end of the study. patients, investigators, outcome assessors and healthcare providers were blinded to the treatment groups. the unblended treatment list was held by the zanjan university medical documentation council. drug preparation preparation of fruit extract to prepare a c. monogyna fruit concentrate capsules, the plant washed completely, to eliminate contaminants. then, 100 gr of fruit was poured with 1 l water in a beaker and boiled over the flame. after boiling for three hours, the beaker was cooled down at the room temperature, and the solution was filtrated. the rotatory evaporator was used to purify and condense the solution and extract the concentrate. usually, the dry weight of the extract is 10% of the c. monogyna extract; consequently, we added the exetian (a corn starch), which is used as a neutral ingredient in pharmaceutical industry. finally, 500 mg capsules of c. monogyna concentrate were prepared, while 250 mg of the powder, containing active compound, was poured into the capsule, as well as the 250 mg of the supplementary material, described above. accordingly, in order to provide similar medications with neutral effect in placebo group, 500 mg capsules with corn starch were prepared. finally, capsules were prepared in specific bottles containing 60 capsules. all the mentioned process (drug preparation) was done at shahid beheshti university of medical sciences. total phenol content (tpc) to measure tpc, 1 ml of gallic acid with different concentrations (20, 40, 60, 80 and 100 µg/ml) was mixed with folin ciocalteu reagent (5 ml, 1:10 diluted with distilled water) for 10 min and aqueous na2co3 (4 ml, 7.5 mg/ml) was then added. the mixture was incubated at dark in room temperature for 30 min. finally, the absorption rate at 765 nm was determined using spectrophotometer. after preparing the gallic acid calibration curve with the mentioned concentrations, the measurement was repeated with the extracted solution (1 ml, 30 mg/ml) instead of gallic acid and total phenolic content was determined using the gallic acid standard curve. total flavonoid content (tfc) to measure the tfc, 2.5 ml of rutin solution with different concentrations (20, 40, 60, 80 and 100 µg/ml) mixed with 198 j contemp med sci | vol. 7, no. 4, july-august 2021: 196–201 effect of hawthorn on blood pressure and quality of sleep original m. abbasi et al. aluminum chloride (2.5 ml, 20 mg/ml) solution in ethanol 80% for 40 min. finally, the absorption rate at 415 nm was determined using spectrophotometer. after preparing the rutin calibration curve with the mentioned concentrations, the measurement was repeated with the extracted solution (2.5 ml, 30 mg/ml) instead of rutin and total flavenoid content was determined using the rutin standard curve. statistical considerations sample size the sample size was estimated for the blood pressure assessments based on the changes in mean arterial pressure (map). according to previous study,17 with a study power of 80%, an alpha level of 0.05, a beta level of 0.20, s1 = 6.8, x1 = 82.6 and s2 = 9.7, x2 = 91.0, using the formula below the sample size was calculated to be 24 patients each arm. considering dropout during the study, the sample size was rounded up to 30 participants in each arm. n s s z z x x = + + ( ) ( ) ( ) 1 2 2 2 2 1 2 1 2 1 2 2 -a b statistical analysis the student t test used for comparing quantitative variables between groups and the chi-square test for comparing two qualitative variables in each time, and between different times. to determine if there are differences between two groups on a continuous or ordinal dependent variable, the mann-whitney u test was used. the level of significance was set at 0.05, and all results were expressed by frequency (percent) for qualitative variables and mean ± se for quantitative variables. the pairedsample t test is used to determine if there was a statistically significant intragroup difference before and after the treatment administration. all analyses were done in ibm spss statistics software v.25 environment. results drug analysis results the total phenolic and flavonoid contents were 1.42 mg/ capsule and 0.43 mg/capsule, respectively. demographic status and basal characteristics during recruitment phase, 60 patients had enrolled. four patients were excluded due to severe increase of the bp, and premature discontinuation (figure 1). data from 56 patients was underwent statistical analysis. among them 23 (41.1%) patients were male and 33 (58.9%) were female (mean ± sd of age was 49.1 ± 5.1 years). in terms of demographic and baseline characteristics, there was no significant difference based on age and gender between the two arms. body mass index (bmi); however, was significantly higher in c. monogyna group (p = 0.02) (table 1). fig. 1 consort 2010 flow diagram of the study. 199j contemp med sci | vol. 7, no. 4, july-august 2021: 196–201 m. abbasi et al. original effect of hawthorn on blood pressure and quality of sleep table 1. demographic and baseline characteristics variables mean ± sd or n (%) p valuec. monogyna (n = 30) placebo (n = 26) age 49.7 ± 5.1 48.1 ± 5.1 0.24 gender male 18 (50%) 18 (50%) 0.604 female 12 (50%) 12 (50%) bmi 30.65 ± 5.7 28 ± 2.7 0.02 temperament dry warm 3 (10%) 1 (3.8%) 0.82 wet warm 8 (26.6%) 5 (19.3%) tempered warm 5 (16.7%) 6 (23.1%) dry cold 4 (13.3%) 3 (11.5%) wet col 5 (16.7%) 3 (11.5%) tempered cold 3 (10%) 5 (19.3%) tempered 2 (6.7%) 3 (11.5%) c. monogyna, crataegus monogyna; bmi, body mass index; sd, standard deviation. blood pressure assessments at baseline, the results of the study showed no significant difference in terms of sbp and dbp between two arms but after 8 weeks of treatment, the sbp and dbp were significantly lower in c. monogyna group (118.14 ± 6.76 vs 129.76 ± 8.28, 95% ci: 7.54–15.69, p < 0.001 and 77.14 ± 5.32 vs 83.57 ± 6.73, 95% ci: 3.17–9.68, p < 0.001), respectively (table 2). quality of sleep in intragroup analysis, the pittsburgh questionnaire scores showed a significant decrease of sleep disorders severity in c. monogyna group median (mode) (6(6) vs 4(3), p = 0.001). interestingly, none of the study arms had superiority after treatment considering the pittsburgh questionnaire results (4(3) vs 4(4), 95% ci: –1.49–1.17). however, a satistically significant difference was seen between the two arms at baseline scores (6(6) vs 5(3), 95% ci: –3.52–0.21, p = 0.03). table 2. inter-group and intra-group analysis for systolic/diastolic blood pressure and pittsburgh questionnaire scores variables c. monogyna (n = 30) placebo (n = 26) student-t: p 95% ci mean ± sd mean ± sd lower upper diastolic bp before 90.71 ± 7.39 89.76 ± 4.32 0.59 –4.51 2.61 after 77.14 ± 5.32 83.57 ± 6.73 <0.0001 3.17 9.68 paired:p <0.0001 0.001 systolic bp before 133.43 ± 6.50 131.43 ± 6.73 0.27 –5.65 1.64 after 118.14 ± 6.76 129.76 ± 8.28 <0.0001 7.54 15.69 paired:p <0.0001 0.47 pittsburgh score before 6 (3–18) 5 (2–8) 0.03 –3.52 –0.21 after 4 (2–13) 4 (2–8) 0.44 –1.49 1.17 paired:p 0.001 0.61 c. monogyna, crataegus monogyna; bp, blood pressure; ci, confidence interval; sd, standard deviation. laboratory measurements in assessment of laboratory variables as secondary outcomes, results of the study showed no significant changes in inter/ intra group analysis at baseline and 1 week after period of treatment between 2 study arms (p > 0.05). discussion since years, herbal medicine has played a significant role in different aspects of human-being life, whereas it has been suggested to increase the quality of life, improves the signs and symptoms of the diseases, particularly hypertension-related symptoms.18 clinical trials have reported that bioactive compounds to have a protective effect against agents derived from oxidative stress as a risk factor for cardiovascular diseases, which can be found in fruits and vegetables that are vitamins and polyphenols enriched.19,20 in the present randomized controlled trial, we primarily aimed to investigate the clinical effects of c. monogyna fruit extract on controlling htn along with sleep disorders. no significant difference observed between study arms in terms of laboratory tests, and none of the groups showed changes in test values after the study period. our results revealed significant improvements in sbp and dbp after treatment with c. monogyna. in addition, results of the pittsburgh sleep quality questionnaire showed significant improvement in patients who were treated with c. monogyna compared to pretreatment evaluations. in a recent study by haydari et al., the results of an animal experimental indicated that the administration of hydroalcoholic extract of crataegus azarolus subspecies aronia diminished the htn development, which was applied by clips placement on renal arteries.21 the authors hypothesized that increased no release and reduction of the oxidative stress through the herbal extract might have played a role in preventing htn development. further studies confirmed the antioxidative effects of the c. monogyna, since its acidified methanol or ethanol extracts served as a potential antioxidant and neuroprotective agent.22 sharifi et al. evaluated the angiotensin-converting enzyme (ace) inhibitory effects in six medicinal plants, known as a potential antihypertensive in iranian traditional medicine.23 200 j contemp med sci | vol. 7, no. 4, july-august 2021: 196–201 effect of hawthorn on blood pressure and quality of sleep original m. abbasi et al. table 3. laboratory measurements variables c. monogyna (n = 30) placebo (n = 26) student-t: p 95% ci mean ± sd mean ± sd lower upper bun before 13.86 ± 5.32 14.16 ± 4.02 0.82 –2.51 3.11 after 15.90 ± 5.74 14.61 ± 5.42 0.41 –4.47 1.89 paired:p 0.15 0.77 cr before 1 ± 0.15 1.06 ± 0.16 0.18 –0.03 0.15 after 0.93 ± 0.15 1 ± 0.18 0.16 –0.02 0.16 paired:p 0.08 0.27 ast before 21.56 ± 9.89 24.48 ± 10.50 0.30 –8.39 7.53 after 23.81 ± 8.32 32.74 ± 11.36 0.13 –7.63 21.69 paired:p 0.34 0.28 alt before 25.32 ± 15.32 24.95 ± 12.40 0.91 –2.72 8.56 after 25.20 ± 15.20 32.21 ± 17.07 0.34 –2.79 20.64 paired:p 0.97 0.13 triglyceride before 166.49 ± 65.21 164.33 ± 76.70 0.91 –40.72 36.41 after 168.24 ± 69.42 161.24 ± 54.78 0.69 –42.78 28.79 paired:p 0.92 0.88 cholesterol before 181.74 ± 42.21 188.86 ± 32.47 0.51 –14.41 28.64 after 184.12 ± 43.45 190.81 ± 38.61 0.56 –16.52 29.9 paired:p 0.83 0.86 hdl-c before 44.17 ± 9.06 44.38 ± 7.30 0.92 -4.47 4.89 after 41.50 ± 9.27 42.25 ± 10.10 0.78 -4.67 6.17 paired:p 0.26 0.44 ldl-c before 97.96 ± 30.32 116.86 ± 32.45 0.07 –1.79 39.58 after 111.41 ± 36.10 121.05 ± 33.09 0.35 –11.13 30.41 paired:p 0.12 0.71 c.monogyna, crataegus monogyna; bun, blood urea nitrogen; ast, aspartate transaminase; alt, alanin transaminase; hdl, high density lipoprotein cholesterol; ldl, low density lipoprotein cholesterol; sd, standard deviation; ci, confidence interval. although the outcomes revealed high ace inhibition and antioxidant activity for several other herbal medicines such as q. infectoria and o. acanthium, serious adverse events might be occurred due to their toxic ingredients. however, c. monogyna had not only significant superiority in terms of ace inhibition activity but also showed no toxic effects or adverse effects. similarly, our results showed that c.monogyna prescription had significantly improved the systolic and diastolic bp compared to patients who received a placebo. our findings were in accordance with the study by wang et al., which demonstrated the effects of c. monogyna in decreasing systolic and diastolic bp, as well as the heart rate.24 also, our results confirm the report by hernandez et al., which introduced a significant vasorelaxant activity for c. monogyna.25 besides, the sleep quality of the patients who received c. monogyna had significantly enhanced. considering the fact that the correlation between uncontrolled htn and low sleep quality is well-established, it can be hypothesized that c. monogyna benefits several advantages in controlling the htn, including ace inhibitory effect and improving the sleep quality. in addition, during the two-month administration of the c. monogyna extracts, no adverse effect was reported by patients, which emphasizes the safety of the herb, which was in coincidence with the study by kumaraswamy et al., which reported no toxic activity by 2000 mg daily consumption of c. monogyna.26 although our findings contribute significantly to prove the efficiency and safety of the c. monogyna in treatment of the patients with htn, the herbal medications are far from replacing the routine, conventional therapies, and they can only serve as a supplement to routine prescriptions. in addition, further studies are needed to make more evidence available on traditional claims over the effectivity of the herbs and medicinal plants, through well-designed and structured clinical trials and systematic reviews on modern and traditional literature. conclusion c. monogyna fruit extract as a supplementary medication in patients with htn resulted in the better controlling of the systolic and diastolic blood pressures in comparison to the placebo. in addition, the sleep quality of the patients, who underwent treatment with c. monogyna was significantly improved after the treatment course. however, further studies with possibly longer administration and follow-up period are 201j contemp med sci | vol. 7, no. 4, july-august 2021: 196–201 m. abbasi et al. original effect of hawthorn on blood pressure and quality of sleep required to illuminate the benefits of the medical plant mentioned above. data availability the raw data supporting the findings of this study are available from the corresponding author on request. conflicts of interest the authors contributed in this study have no conflict of interest to disclose. funding this study was not founded by any organization or committee. acknowledgments we thank prof. soghrat faghihzadeh and dr. narjes khavasi for their valuable consults through statistical analysis and study design. we also thank all of the patients for their participation and good cooperation throughout the study.  references 1. stamler j, stamler r, neaton jd, wentworth d, daviglus ml, garside d, et al. low risk-factor profile and long-term cardiovascular and noncardiovascular mortality and life expectancy: findings for 5 large cohorts of young adult and middle-aged men and women. jama. 1999;282(21):2012-18. 2. forouzanfar mh, liu p, roth ga, ng m, biryukov s, marczak l, et al. global burden of hypertension and systolic blood pressure of at least 110 to 115 mm hg, 1990-2015. jama. 2017;317(2):165-82. 3. dardi f, palazzini m, gotti e, zuffa e, de lorenzis a, daniele g, et al. additional role of unmodifiable risk factors in pulmonary arterial hypertension risk stratification according to current esc/ers guidelines. circulation. 2019;140(suppl_1):a9541-a. 4. daştan i̇, erem a, çetinkaya v. urban and rural differences in hypertension risk factors in turkey. anatol j cardiol. 2017;18(1):39. 5. pandey r, quan wy, hong f. vaccine for hypertension: modulating the renin–angiotensin system. int j cardiol. 2009;134(2):160-8. 6. dabaghian fh, rassouli m, sadighi j, ghods r. adherence to prescribed medications of iranian traditional medicine in a group of patients with chronic disease. j res pharm pract. 2016;5(1):52. 7. ibn sina h. the canon of medicine. beirut: alaalamy foundation publications. 2005. 8. calhoun d. sleep disorders and hypertension risk. j hum hypertens. 2017;31(6):371-2. 9. calhoun da. obstructive sleep apnea and hypertension. curr. hypertens. rep. 2010;12(3):189-95. 10. calhoun da, harding sm. sleep and hypertension. chest. 2010;138(2):434-43. 11. fiorentini a, valente r, perciaccante a, tubani l. sleep’s quality disorders in patients with hypertension and type 2 diabetes mellitus. int j cardiol. 2007;114(2):e50-e2. 12. seravalle g, mancia g, grassi g. sympathetic nervous system, sleep, and hypertension. curr. hypertens. rep. 2018;20(9):74. 13. gangwisch je, malaspina d, posner k, babiss la, heymsfield sb, turner jb, et al. insomnia and sleep duration as mediators of the relationship between depression and hypertension incidence. am j hypertens. 2010;23(1):62-9. 14. mh ak. makhzan-ol-advieh. tehran: sabz arang publication. 2008. 15. alirezalu a, ahmadi n, salehi p, sonboli a, alirezalu k, mousavi khaneghah a, et al. physicochemical characterization, antioxidant activity, and phenolic compounds of hawthorn (crataegus spp.) fruits species for potential use in food applications. foods. 2020;9(4):436. 16. buysse dj, reynolds cf, monk th, berman sr, kupfer dj. the pittsburgh sleep quality index: a new instrument for psychiatric practice and research. psychiatry res. 1989;28(2):193-213. 17. baddeley-white ds, mcgowan cl, howden r, gordon bd, kyberd p, swaine il. blood pressure lowering effects of a novel isometric exercise device following a 4-week isometric handgrip intervention. open access j sports med. 2019;10:89. 18. ghorbani m. iranian traditional medicine for treatment of type ii diabetes, anxiety and hypertension with introduction of zebrafish model system for their screening. int j herb med. 2014;2(5):13-9. 19. kris-etherton pm, hecker kd, bonanome a, coval sm, binkoski ae, hilpert kf, et al. bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. am j med. 2002;113(9):71-88. 20. sato k, dohi y, kojima m, miyagawa k, takase h, katada e, et al. effects of ascorbic acid on ambulatory blood pressure in elderly patients with refractory hypertension. arzneimittelforschung. 2006;56(07):535-40. 21. haydari mr, panjeshahin mr, mashghoolozekr e, nekooeian aa. antihypertensive effects of hydroalcoholic extract of crataegus azarolus subspecies aronia fruit in rats with renovascular hypertension: an experimental mechanistic study. iran j med sci. 2017;42(3):266. 22. renda g, özel a, barut b, korkmaz b, yayli n. in vitro protection by crataegus microphylla extracts against oxidative damage and enzyme inhibition effects. turkish j pharm. sci. 2018;15(1). 23. sharifi n, souri e, ziai sa, amin g, amanlou m. discovery of new angiotensin converting enzyme (ace) inhibitors from medicinal plants to treat hypertension using an in vitro assay. daru j pharm sci. 2013;21(1):74. 24. wang j, xiong x, yang g, zhang y, liu y, zhang y, et al. chinese herbal medicine qi ju di huang wan for the treatment of essential hypertension: a systematic review of randomized controlled trials. evid based complement alternat med 2013;2013. 25. hernández-pérez a, bah m, ibarra-alvarado c, rivero-cruz jf, rojas-molina a, rojas-molina ji, et al. aortic relaxant activity of crataegus gracilior phipps and identification of some of its chemical constituents. molecules. 2014;19(12):20962-74. 26. shatoor as. acute and sub-acute toxicity of crataegus aronia syn. azarolus (l.) whole plant aqueous extract in wistar rats. am j pharmacol toxicol. 2011;6(2):37-45. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. doi: https://doi.org/10.22317/jcms.v7i4.1017 128 j contemp med sci | vol. 8, no. 2, march-april 2022: 128–133 original a novel study of protein kinase c beta gene polymorphism (rs3760106) and protein kinase c activity levels as a predictor of nephropathy complications in iraqi diabetic patients fadhil jawad al-tu’ma*, zahraa abdul adheem al-maiyaly department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. *correspondence to: fadhil jawad al-tu’ma (e-mail: fadhil.jawad@uokerbala.edu.iq) (submitted: 13 january 2022 – revised version received: 29 january 2022 – accepted: 18 february 2022 – published online: 26 april 2022) abstract objectives: this study aimed to investigate the relation between the risk of (prkcb-1504 c/t) gene polymorphisms and diabetic nephropathy pathogenesis and its association with its levels and some other biomarkers in serum of iraqi diabetic nephropathy patients. methods: a cross-sectional study was performed on 130 samples obtained from al-hussein teaching hospital/kerbala – iraq during feb., 2020 to march 2021. seventy three patients of them with type 2 diabetes mellitus (t2d) and the remaining patients with type 2 diabetes mellitus with nephropathy complications (dn). phenotypic analysis included determination of protein kinase c (pkc) activity levels, hba1c%, serum insulin levels and renal function tests. five ml of whole blood was obtained from each patients, 4.0 ml was centrifuged for serum separation used for biomarkers determinations and the remaining 1.0 ml was used for genomic dna extraction for molecular analysis and polymorphism of prkcb (rs3760106) by allele specific-amplification refractory mutational system-polymerase chain reaction (allele specific arms-pcr), followed by electrophoresis on 1% agarose gel. various statistical analyses were applied to analyze the obtained data. results: the amplification of the prkcb gene gives one genotypes as indicated by (200 bp) bands for those with homozygous wild type (cc), homozygous mutant (tt ) genotypes and two genotypes bands (200 bp) for those with heterozygous (ct ). genotype frequencies of rs3760106 polymorphism were found to be consistent with hardy–weinberg equilibrium. allele frequencies (23.3%, 26.7%, 50%) of cc, ct, tt in cases of dn group. while the frequencies in the non-dn group were (30%, 45%, 25%) and for healthy control group were (50%, 33.4%, 16.6%) for wild, heterozygous, and homozygous in an order. in healthy control group, the risk of diabetic nephropathy was significantly higher among carriers of t allele under codominant tt (or = 6.42, 95% ci = (1.66–24.85), p = 0.007), dominant ct + tt (or = 3.2, 95% ci = (1.08–9.95), p = 0.03) and recessive model (or = 5, 95% ci = (1.51–16.56), p = 0.008) while in t2dm without nephropathy the risk of diabetic nephropathy was significantly higher among carrier of t allele under recessive model (or = 3, 95% ci = (1.09–28.25), p = 0.003). serum level of pkc-b1 activity was significantly higher among dn group than t2dm and control groups (43.35 ± 18.69, 30.35 ± 11.96, 27.42 ± 10.31, p = 0.001), also pkc-b1 activity in dn group was significantly correlated with fasting blood glucose, homa-ir and renal function tests such as gfr, urea and creatinine. conclusion: this study indicates that the prkcb (c/t-1504) rs3760106 single nucleotide polymorphism was significantly associated with increased diabetic nephropathy susceptibility of iraqi patients with t2dm and serum level of pkcb activity was associated with increase the risk of diabetic nephropathy complications. keywords: type 2 diabetic nephropathy, prkcb gene polymorphism, pathogenesis, protein kinase c issn 2413-0516 introduction diabetes mellitus (dm) is consider as the most common and chronic metabolic disorder worldwide. the prevalence of this disorder keep increasing and it is expected to impact 439 million individuals by 2030.1 it is the major cause of severe complications such as “retinopathy, nephropathy, and neuropathy” identified in t2dm by resistance to insulin action in adipose tissues, muscle, liver, and elsewhere.2-4 insulin is an important hormone released from the pancreas. it has a critical function in which enhance the transporting of glucose from the bloodstream into the body’s cells where the glucose is converted into energy. insulin deficiency or the cell’s failure to respond to insulin result in elevated level of blood glucose, or hyperglycemia, which is the hallmark of diabetes. different organs especially the eyes, kidneys, nerves, and also blood vessels effected and damaged with chronic hyperglycemia of dm.5,6 multiple genetic and environmental factors greatly influence it, for this reason there is extensive attempts made to recognize the disease-affecting genes in order to better understanding the pathogenesis of the disease, develop new clinical therapy targets and improved prediction of the disease.7 one of the most frequent and common complications of diabetes and the direct cause of end-stage renal disease (esrd) is diabetic nephropathy, 40% of all individuals with type 2 diabetes develop diabetic nephropathy.3 clinically, diabetic nephropathy is characterized by an increase in the excretion of albumin in urine and a decline in gfr which result in decline in the function of the kidney. the classification of diabetic nephropathy is based on “estimated glomerular filtration rate (egfr)” and the level of proteinuria.2 it is obvious that both hemodynamic and metabolic pathways contribute to trigger the progression of dkd.8 patients with dn manifest at the beginning hyper filtration that mean high or normal level of gfr with the occurrence of micro albuminuria, then the patient manifest a gradual decrease of the gfr and occurrence of macro albuminuria that comes before mild and subsequently moderate proteinuria. the final stage is identify by severe proteinuria and sever decline in gfr < 15% with chronic renal insufficiency that result in esrd and dialysis. these abnormalities result from structural mailto:fadhil.jawad@uokerbala.edu.iq 129j contemp med sci | vol. 8, no. 2, march-april 2022: 128–133 f.j. al-tu’ma and z.a.a. al-maiyaly original a novel study of protein kinase c beta gene polymorphism modifications at the cellular level, which encompass of vascular permeability, podocytes apoptosis, accumulation of extracellular matrix (ecm) in the mesangium (mesangium expansion) and thickening of glomerular basement membrane. then, the formation of mesangial nodules which also called kimmelsteil-wilson nodules (glomerulosclerosis) and ultimately tubule-interstitial fibrosis.9 protein kinase c (pkc) is a family of serineand threonine-specific protein kinases that can be activated by calcium and second messenger diacylglycerol. pkc family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. pkc family members also serve as major receptors for phorbol esters, a class of tumor promoters.10,11 the protein kinase family consists of over 15 subgroups with more than 500 kinases, each of which is involved in the regulation of gene expression; thereby, the down-regulation or up-regulation of these kinases induces severe consequences in the progression of disorders including neurodegenerative diseases12,13 and involve in numerous signaling processes such as modification of gene expression, cell division, migration, proliferation, differentiation, cell survival and apoptosis.14 chronic hyperglycemia which is one of the hallmark of dm result in increases the second messenger diacylglycerol “dag” production, that is one of the important protein kinase c (pkc) activating factors with calcium ion, so that pkc may have a critical role in diabetes mellitus and its vascular complications.15 the role of pkcb isoform in the pathogenesis of dn has been the most intensively investigated among all the pkc isoenzyme. it has been shown that in diabetic glomeruli pkcb was raised or activated and has led to glomerular hypertrophy and expansion of the extracellular matrix. recent studies have shown that ly333531 (a pkcβ-selective inhibitor) selectively inhibits pkcb over other pkc isoforms, can help improve glomerular hyperfiltration, albuminuria and mesangial expansion. such findings strongly indicate that pkcb may have a significant role in the development of diabetic nephropathy.16 in addition to environmental factors, the genetic variations may have an significant effect on the progression of dn and esrd. it is well known that gene susceptibility to dn have a critical role in individuals, even with the same environmental exposure.3 protein kinas c beta gene (prkcb) is a new and strong candidate gene for dn, is located on chromosome 16p11.2.17 however, the relationship between prkcb gene polymorphisms and dn was validated in a chinese as well as indians population17,18 confirmed that variant (prkcb -1504 c/t rs3670106) was effect the expression and activity of pkcb isoform and associated with dn. the aim of the current study was to assay the association between the risk of prkcb (-1504 c/t) rs3760106 gene polymorphism with dn susceptibility, and the serum level of pkcb isoform and some biomarkers in the iraqi patients. materials and methods this cross-sectional study was conducted on 100 subjects obtained from al-hassan center of endocrinology, al-hussein teaching hospital, al-hussein medical city, kerbala health directorate/kerbala iraq during nov., 2019 to sep., 2020. the subjects were classified into three groups: 30 type 2 diabetic patients with nephropathy complication with age ranged between 35–74 years, of whom 63% were males and 37% females, 40 type 2 diabetic patients without nephropathy complication having the similar age range, and including 53% males and 47% females and 30 subjects as apparently healthy control group with the same age range, 66% were male and 34% were female. the exclusion criterion was t2dm patients with no dn or no dialysis. subjects with egfr< 60 (ml/min, 1.73 m2 body surface area) and urinary albumin excretion “uae” >300 mg/day were considered as the dn group. while subjects with egfr > 60 ml/min 1.73 m2 and uae < 30 mg/day consider as t2dm without nephropathy and control group. blood sample (5 ml) was collected from each group; 2 ml was added to edta tube for molecular studies, the remaining sample, 3 ml was collected in a gel tube used for serum separation after centrifugation at 3000 × g and utilized for various biochemical investigations, including lipid profile, blood glucose, renal function tests and pkc-b level. the pkc-b enzyme activity level was measured by elisa, the other biomarkers were assessed, using the roche cobas c311, and homa-ir and egfr by equations (ref). the dna was extracted from frozen whole blood of each group, using the geneaid kit and stored at −20°c. the pcr reaction is performed by using allele specific amplification refractory mutation system polymerase chain reaction (arms-pcr) which is a rapid and simple technique to detect a snp.19,20 a tri primer arms-pcr was used for the detection of –1504 c/t polymorphism of prkcb (rs3760106). sequences of primers are presented in table 1. the pcr products were analyzed by agarose gel electrophoresis, using 1% agarose gel, and visualized by staining with ethidium bromide (promega, usa). pcr cycling conditions were as follows (table 2). the data were expressed as mean ± sd, the one-way anova was used for calculating the probability (p value). the past version 3.09 used for calculating the probability value (p value), chi-square (χ2), odds ratio (or) and confidence interval 95% (ci 95%), and to express a significance between the study groups (polymorphisms and biochemical table 1. primer sequence for alleles of prkcb gene primers sequence mutant forward 5’actacaaagcatcctggtgacaaatga 3’ normal forward 5’ actacaaagcatcctggtgacaaatgg 3’ common reverse 5’ aggagagggacattctattattgcagcc 3’ table 2. pcr reaction program type of cycle temperature °c time no. of cycle initial denaturation 95 5 min 1 cycle denaturation 95 30 sec 35 cycle annealing 62 30 sec 35 cycle extension 72 60 sec 35 cycle final extension 72 5 min 1 cycle total time: 1 hour and 35 min 130 j contemp med sci | vol. 8, no. 2, march-april 2022: 128–133 a novel study of protein kinase c beta gene polymorphism original f.j. al-tu’ma and z.a.a. al-maiyaly parameters). in all statistical analysis, the p = 0.05 was considered as a significant and p < 0.01 as highly significant. results the characteristics of patient such as age, gender, and biochemical parameters such as hdl-c had no significant difference among dn and non-dn groups (table 3, p > 0.05). while the pkcb isoform level, other characteristics were significantly different among the study groups (table 3, p < 0.05) egfr was very low in dn group as compared to dm and control group while serum urea and creatinine was very high in dn group. genotype frequencies of the prkcb gene were not consistent with the hardy–weinberg equilibrium (hwe) in dn group. while, with the non-dn groups were consistent with hwe. allele frequencies were (23.3%, 26.7%, 50%) of cc, ct, tt in cases of dn group, while the frequencies in the t2dm without nephropathy group were (30%, 45%, 25%) and for healthy control group were (50%, 33.4%, 16.6%) for wild, heterozygous, and homozygous in an order (table 4). in healthy control group, the risk of diabetic nephropathy was significantly higher among carriers of t allele under codominant tt (or = 6.42, 95% ci = (1.66–24.85), p = 0.007), dominant ct + tt (or = 3.2, 95% ci = (1.08–9.95), p = 0.03) and recessive model (or = 5, 95% ci = (1.51–16.56), p = 0.008) while in t2dm without nephropathy the risk of diabetic nephropathy was significantly higher among carrier of t allele under recessive model (or = 3, 95% ci = (1.09–28.25), p = 0.003), (table 5). the amplification of the prkcb-1504 c/t gene gives one genotypes as indicated by (200 bp) bands for those with homozygous wild type (cc), homozygous mutant (tt) genotypes and two genotypes bands (200 bp) for those with heterozygous (ct), (figure 1). the analysis of data showed that dn patients, harboring tt genotype showed a higher significant pkcb activity level, blood glucose, urea, creatinine and egfr as compared to those with the ct and cc genotype (p < 0.05), while in t2dm without nephropathy group expect blood glucose and hba1c% there is no significant difference in other biological parameters in tt and ct genotype (p > 0.05). in healthy control group there is no significant difference in all biological parameters in ct, tt and cc genotype (p > 0.05) (table 6). discussion various biomarker and genetic investigations in type 2 diabetic iraqi patients with and without nephropathy fig. 1 the amplification of the prkcb1 -1504 c/t gene indicated by (200 bp) bands for those with homozygous wild type (cc), homozygous mutant (tt) genotypes and two genotypes bands (200 bp) for those with heterozygous (ct). table 3. anthropometric and biochemical characteristics of study subjects parameters control mean ± sd n = 30 t2dm mean ± sd n = 40 dn mean ± sd n = 30 p-value age (y) 56.62 ± 6.55 57.72 ± 7.13 59.29 ± 7.55 0.1 bmi (kg/m2) 28.3 ± 3.3 29.4 ± 5.4 28.3 ± 3.5 0.5 duration of dm (y) – 10.7 ± 3.7 16.36 ± 5.02 0.001* fbs (mg/dl) 96.15 ± 10.4 168.4 ± 30.6 173.2 ± 22.02 <0.001* hba1c % 5.2 ± 0.9 8.93 ± 2.7 6.8 ± 1.32 <0.001* insulin (µu/ml) 4.54 ± 1.24 30.6 ± 11.49 41.82 ± 14.3 <0.001* homa-ir 1.21 ± 0.49 9.91± 3.07 22.6 ± 11.2 <0.001* urea (mg/dl) 27.86 ± 6.83 30.4 ± 13.9 136.25 ± 14.6 <0.001* creatinine, (mg/dl) 0.6 ± 0.16 0.8 ± 0.2 7.6 ± 1.8 <0.001* gfr (ml/min.1.732 m2 ) 114.3 ± 19.2 106.16 ± 24.5 7.5 ± 2.7 <0.001* t. cholesterol, (mg/dl) 143.62 ± 28.24 168.4 ± 40.8 163.1 ± 34.5 0.03* tg (mg/dl) 136.18 ± 40.3 188.3 ± 39.2 173.76 ± 21.6 0.01* hdl-c (mg/dl) 40.73 ± 7.6 36.26 ± 7.77 38.39 ± 10.5 0.14 ldl-c (mg/dl) 78.02 ± 19.2 118.134.5 97.8 ± 11.3 <0.001 pkc-b (iu/min) 27.42 ± 10.31 30.35 ± 11.96 43.35 ± 18.69 <0.001* bmi, body mass index; fbs, fasting blood sugar (glucose); egfr, estimated glomerular filtration rate; tc, total cholesterol; tg, triglycerides; hdl-c, high-density lipoproteins-cholesterol; ldl-c, low-density lipoproteins-cholesterol; vldl-c, very low density lipoproteins-cholesterol; dn, diabetic nephropathy; dm, diabetes mellitus; hc, healthy control. data were expressed as mean ± sd. *p value < 0.05 is significant, by one way anova test. 131j contemp med sci | vol. 8, no. 2, march-april 2022: 128–133 f.j. al-tu’ma and z.a.a. al-maiyaly original a novel study of protein kinase c beta gene polymorphism complications and acute kidney injury were performed as reported by al-tu’ma, et al. in 2017 and 2018.21,22 in the present study, the genetic association of the prkcb rs3760106 polymorphism with diabetic nephropathy susceptibility was examined in the iraqi population. the results of this study indicated that subjects who carrying tt and ct genotypes had a greater chance to getting dn. in addition, the activity of pkcb isoform is higher in dn group than in non-dn subjects. the observed data indicated that there is a significant association between prkcb rs3760106 polymorphism and the etiology of dn, genetic susceptibility. in addition to hyperglycemia, is identified as an significant factor affecting the development of diabetic nephropathy. in this study, we obtained fundamental evidence supporting the theory that polymorphism in prkcb gene, which encodes pkcb-i and pkcb-ii was significantly associated with diabetic nephropathy in iraqi subjects. the results of the present study is compatible with the previous studies in indian subjects17 as well as chinese subjects18 which found that snp (-1504 c/t, rs 3760106) of prkcb1 gene have a significant association with esrd in diabetic patients. this snp are found in the regulatory region of prkcb gene “the promoter” and consequently could affect the transcription of prkcb gene. the functional roles of the -1504 c/t snp are unclear but many studies show that the snp may locate in the binding position for transcription factors (sp1) so that affect the expression of the gene, also diabetes mellitus and hyperglycemia may stimulate glycosylation of sp1 and consequently may induce the pathogenesis of diabetic complications.17 table 4. result of polymorphic allele frequency for prkcb gene polymorphism at (rs3760106) locus in healthy control, t2dm and dn groups group genotype frequency expected observed χ2 p-value dn (n = 30) cc (wild) 4.0 7 (23.3%) 5.44 0.01* ct (hetero) 13.9 8 (26.7%) tt (mutant) 12.0 15 (50%) c allele freq. 0.37 (37%) t allele freq. 0.63 (63%) t2dm (n = 40) cc (wild) 11 12 (30% ) 0.38 0.5 ct (hetero) 20 18 (45%) tt (mutant) 9 10 (25%) c allele freq. 0.52 (52%) t allele freq. 0.48 (48%) healthy control (n = 30) cc (wild) 13.3 15 (50%) 1.87 0.6ct (hetero) 13.3 10 (33.4%) tt (mutant) 3.3 5 (16.6%) c allele freq. 0.67 (67%) t allele freq. 0.33 (33%) table 5. genotype and allele frequency of prkcb gene snp at (rs3760106) locus between t2dm and dn snp rs3760106 c/t t2dm n = 40 dn n = 30 or (95%ci) p-value codominant cc (reference) 12 7 ct 18 8 0.6 (0.2–2.6) 0.6 tt 10 15 2.5 (0.7–8.7) 0.1 dominant ct + tt 28 23 1.4 (0.4–4.16) 0.5 recessive ct + cc (reference) 30 15 tt 10 15 3 (1.09–28.25) 0.003* allele frequency c (wild allele) 0.52 0.37 t (mutant allele) 0.48 0.63 1.8 (1.4–3.2) 0.03* snp, single nucleotide polymorphisms; t2dm, type 2 diabetes mellitus; dn, diabetic nephropathy; or, observed risk; ci, confidence interval. p value < 0.05 is significant by risk odd ratio. 132 j contemp med sci | vol. 8, no. 2, march-april 2022: 128–133 a novel study of protein kinase c beta gene polymorphism original f.j. al-tu’ma and z.a.a. al-maiyaly the result of the present study indicated that the minor allele frequency (t allele frequency) was significantly higher in dn group than t2dm and control subjects (0.63, 0.48, 0.33 respectively) as well as homozygous carriers of the minor allele (tt) were significantly more frequent among dn patients than among t2dm and control subjects (50%, 25%, 16.6% respectively) this pattern is consistent with a recessive mode of inheritance. also genotype distributions were significantly different among dn patients as compared with t2dm and control subjects. individuals carrying the t allele of the rs3760106 snp increased the risk of diabetic nephropathy as compared with non-carriers (odds ratio, 1.8, 3.4). this result is compatible with the results of (araki, et al., 2015)17 which found that t allele of rs 3760106 was significantly associated with increase susceptibility of dn and (ma, et al., 2010) in which 18 snps throughout prkcb1 gene were genotyped in cohort study and the patients were followed for 8 years.18 this study indicated that two prkcb snps, rs3760106 and rs2575390, were significantly associated with renal failure in type 2 diabetes mellitus. the activity of pkcb is significantly higher in diabetic nephropathy (43.35 ± 18.69) as compared with t2dm without nephropathy and apparently control. the results of the present study agree with the results of (langham, et al., 2008; yang and zhang, 2015) which found that the expression and activity of pkcb and other clinical parameters such as “fbs, hba1c%, gfr and serum creatinine” was significantly higher in type 2 diabetic patients with nephropathy as compared with “diabetic patients without nephropathy” and also agree with (toyoda, et al., 2004) which shown that in glomeruli of dn patients the activity of pkc-mapk (mitogen activated protein kinase) pathway and the expression of tgfb-1 is significantly higher than glomeruli of normal subjects, with this results they proved that activation of protein kinas c (pkc) is a major signaling pathway for transforming growth factor (tgf)−b stimulate extracellular matrix (ecm) production in diabetic nephropathy (dn) and activation of pkcb have an critical role in the progression of glomerular damage in dn.23-25 the pkc-β has 2 subtypes (pkc-βi and pkc-βii) produced by the same gene prkcb by “alternative splicing”. high glucose level regulate the expression and activity of pkb-ii while pkcb-i not effected.26,27 in diabetic nephropathy group there is a significant correlation between pkc-b level with creatinine and egfr respectively (r = 0.7, p = 0.03, r = –0.6, p = 0.05). estimation of gfr considers the most useful general index for the evaluation the severity of kidney damage. losing of 75% of renal tissue result a decline in gfr of 50% (less than 60 ml/min. 1.73 m2). as glomerular function deteriorates, compounds that are normally cleared by the kidneys, such as urea and creatinine, accumulate in plasma so that with a decline in gfr as in dn (due to the structural abnormalities in glomerular such as mesangial expansion and thickening of gbm), plasma urea concentration and creatinine tend to rise. these data were in agreement with another study performed by noor et al., in 2020.28 conclusion this cross-sectional study indicates that the prkcb (c/t1504) rs3760106 snp was significantly associated with increased dns susceptibility in iraqi patients with t2dm and serum level of pkcb activity was associated with increased risk of dn complication. table 6. biochemical characteristics of dn in relevance to the genotypes of prkcb gene polymorphism analyzed under co-dominant model (n = 30) clinical characteristics tt (n = 15) mean ± sd ct (n = 8) mean ± sd cc (n = 7) mean ± sd p-value bmi (kg/m2) 28.31 ± 3.98 28.13 ± 3.62 28.05 ± 3.89 0.9 age (in year) 62.4 ± 9.91 59.13 ± 7.8 54.5 ± 8.42 0.3 fbs (mg/dl) 214.9 ± 26.10 170.12 ± 30.9 121.4 ± 2.77 0.01* hba1c% 7.41 ± 1.34 6.3± 1.20 6.41 ± 0.99 0.1 insulin 39.5 ± 14.2 42.7 ± 12.5 50.3 ± 26.9 0.6 homa-ir 23.7 ± 11.6 21.6 ± 12.7 21.5 ± 6.7 0.9 urea (mg/dl) 140.19 ± 19.9 136.04 ± 17.4 114.12 ± 4.9 0.05* creatinine, (mg/dl) 8.49 ± 1.47 7.61 ± 1.37 6.77 ± 1.54 0.04* egfr 6.40 ± 1.34 8.03 ± 1.85 8.66 ± 2.4 0.02* t. cholesterol, mg/dl 140.5 ± 26.7 146.6 ± 33.7 139.2 ± 23.5 0.5 triglyceride, (mg/d) 94.41 ± 24.7 169.6 ± 36.4 179.3 ± 37.33 0.2 ldl-c (mg/dl) 82.7 ± 22.8 75.8 ± 18.6 71.13 ± 11.69 0.4 hdl-c (mg/dl) 40.51 ± 6.69 34.8 ± 10.5 40.97 ± 11.5 0.5 pkc-b activity (iu/min) 44.71 ± 4.9 36.51 ± 4.32 33.16 ± 7.2 0.0008* bmi, body mass index; fbs, fasting blood sugar (glucose); egfr, estimated glomerular filtration rate; tc, total cholesterol; tg, triglycerides; hdl-c, high density lipoproteins cholesterol; ldl-c, low-density lipoproteins cholesterol; vldl-c, very low density lipoproteins cholesterol; dn, diabetic nephropathy. data were expressed as mean ± sd. p value < 0.05 is significant, by one way anova test. 133j contemp med sci | vol. 8, no. 2, march-april 2022: 128–133 f.j. al-tu’ma and z.a.a. al-maiyaly original a novel study of protein kinase c beta gene polymorphism references 1. jain, a. k. c., apoorva, h. c., kumar, h., kumar, k., and rajagopalan, s. (2018). analyzing diabetic foot ulcer through amit jain’s classification: a descriptive study. int j surg sci, 2(4), 26-32.575–569 : (1) . 2. kwak, s. h. and park, k. s. (2015). genetic studies on diabetic microvascular complications: focusing on genome-wide association studies. endocrinol metab, 30:147–158. 3. wei, l., xiao, y., li, l., xiong, x., han, y., zhu, x. and sun, l. (2018). the susceptibility genes in diabetic nephropathy. kidney diseases, 4:226–237. 4. heindel, j. j., blumberg, b., cave, m., machtinger, r., mantovani, a., mendez, m. a. and vandenberg, l. n. (2017). metabolism disrupting chemicals and metabolic disorders. reproductive toxicology, 68: 3–33. 5. tan, . j. h., hong, c. c., shen,. l. and tay,. e. y., 2015. costs of patients admitted for diabetic foot problems. ann acad med singapore, 44(12): 567–570. 6. al-tu’ma, a. f., jeddoa, z.m. and el–yassin, h. d. (2019). molecular basis of gene encoding human leukocyte antigens and its association with type 1 diabetes mellitus in children of kerbala province. wjpmr, 5(1): 218–225. 7. ahlqvist, e., ahluwalia, t. s. and groop, l. (2011). genetics of type 2 diabetes. clinical chemistry, 57: 241–254. 8. warren, a. m., knudsen, s. t., and cooper, m. e. (2019). diabetic nephropathy: an insight into molecular mechanisms and emerging therapies. expert opinion on therapeutic targets, 23(7): 579–591. 9. schena, f. p. and gesualdo, l. (2005). pathogenetic mechanisms of diabetic nephropathy. journal of the american society of nephrology, 16 (3 suppl 1): s30–s33. 10. kishimoto, a.; takai, y.; mori, t.; kikkawa, u.; nishizuka, y. (1980) activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. j. biol. chem., 255: 2273–2276. 11. kirouac, l.; rajic, a.j.; cribbs, d.h.; padmanabhan, j. (2017). activation of raserk signaling and gsk-3 by amyloid precursor protein and amyloid beta facilitates neurodegeneration in alzheimer’s disease. eneuro, 4 (2): 0149 12. do van, b.; gouel, f.; jonneaux, a.; timmerman, k.; gelé, p.; pétrault, m.; bastide, m.; laloux, c.; moreau, c.; bordet, r. (2016). ferroptosis, a newly characterized form of cell death in parkinson’s disease that is regulated by pkc. neurobiol. dis., 94: 169–178. 13. gordon, r.; singh, n.; lawana, v.; ghosh, a.; harischandra, d.s.; jin, h.; hogan, c.; sarkar, s.; rokad, d.; panicker, n. (2016). protein kinase cδ up-regulation in microglia drives neuro-inflammatory responses and dopaminergic neuro-degeneration in experimental models of parkinson’s disease. neurobiol. dis., 93: 96–114. 14. mérida, i., arranz-nicolás, j., rodríguez-rodríguez, c., and ávila-flores, a. (2019). diacylglycerol kinase control of protein kinase c. biochemical journal, 476(8): 1205–1219. 15. noh, h., and king, g. l. (2007). the role of protein kinase c activation in diabetic nephropathy. kidney international, 72: s49–s53. 16. wang, z. b., zhang, s., li, y., wang, r. m., tong, l. c., wang, y. and li, l. (2017). ly333531, a pkcβ inhibitor, attenuates glomerular endothelial cell apoptosis in the early stage of mouse diabetic nephropathy via down-regulating swiprosin-1. acta pharmacologica sinica, 38(7): 1009–1023. 17. araki, s. i., ng, d. p., krolewski, b., wyrwicz, l.,rogus, j. j., canani, l. and krolewski, a. s. (2003). identification of a common risk haplotype for diabetic nephropathy at the protein kinase c-β1 (prkcb1) gene locus. journal of the american society of nephrology, 14(8):2015–2024. 18. ma, r. c., tam, c. h., wang, y., luk, a. o., hu, c., yang, x. and tong, p. c. (2010). genetic variants of the protein kinase c-β 1 gene and development of end-stage renal disease in patients with type 2 diabetes. jama, 304(8): 881–889. 19. hashemi, m., hoseini, h., yaghmaei, p., (2011). association of polymorphisms in glutamate-cysteine ligase catalytic subunit and microsomal triglyceride. dna cell biol. 30(8): 569–75. 20. al-koofee, d. a., and mobarak, s. m. (2018). primer1: a network service for tetra-arms pcr primer design based on well-known dbsnp. research journal of pharmacy and technology, 11(8): 3633-37. 21. al-tu’ma, f.j. ; dheyauldeen, m.h. and al-saegh, r.m. (2017). measurement of urinary kidney injury molecule-1 as a predictive biomarker of contrastinduced acute kidney injury. j contemp med sci., 3(9): 178–181. 22. al-tu’ma, f.j. and obed k. h. (2018). association between fat mass and obesity associated (fto) gene polymorphism (rs9939609) and lipid profile in type 2 diabetic obese iraqi male. iraq med j, 2 (1): 15–19. 23. langham, r. g., kelly, d. j., gow, r. m., zhang, y., cox, a. j., qi, w. and gilbert, r. e. (2008). increased renal gene transcription of protein kinase c-β in human diabetic nephropathy: relationship to long-term glycaemic control. diabetologia, 51(4): 668-674. 24. yang, j., and zhang, j. (2015). influence of protein kinase c (pkc) on the prognosis of diabetic nephropathy patients. international journal of clinical and experimental pathology, 8(11): 14925. 25. toyoda, m., suzuki, d., honma, m., uehara, g., sakai, t., umezono, t. and sakai, h. (2004). high expression of pkc-mapk pathway mrnas correlates with glomerular lesions in human diabetic nephropathy. kidney international, 66(3): 1107-1114. 26. becker, k. p. and hannun, y. a. (2004). isoenzyme-specific translocation of pkcbii and not pkcbi to a juxtanuclear subset of recycling endosomes. involvement of phospholipase d. journal of biological chemistry, 279: 28251–56. 27. patel, n. a., yamamoto, m., illingworth, p., mancu, d., mebert, k., chappell, d. s. and cooper, d. r. (2002). phosphoinositide-3-kinase mediates protein kinase c βii mrna destabilization in rat a10 smooth muscle cell cultures exposed to high glucose. archives of biochemistry and biophysics, 403(1): 111–120. 28. noor, t., hanif, f., kiran, z., rehman, r., khan, m. t., haque, z., and nankani, k. (june 2020). relation of copeptin with diabetic and renal function markers among patients with diabetes mellitus progressing towards diabetic nephropathy. archives of medical research, 03, 51(6): 548–555 29. stumvoll, m., goldstein, b. j. and van haeften, t. w. (2005). type 2 diabetes: principles of pathogenesis and therapy. the lancet, 365: 1333–1346. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.1204 conflicts of interest none.  k sticky note aq please provide doi number for this article. the provided website is not working. 358 j contemp med sci | vol. 7, no. 6, november-december 2021: 358–362 original prevalence of adverse effects of covid19 vaccines among a sample of iranian healthcare workers; a comparison between the three available vaccines in iran mohammad mousavi1, azita tehranchi2, mahshid namdari3, maryam sadeghipour4*, mohsen dalband5, mahsa malek mohammadi3, kazem dalaie2 1dentofacial deformities research center, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran. 2department of orthodontics, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 3department of community oral health, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 4department of community oral health, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran. 5department of oral and maxillofacial surgery, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. *correspondence to: maryam sadeghipour (e-mail: sadeghipour1393@gmail.com) abstract objectives: describing what to expect after the first and second dose of vaccination will increase the public confidence. this study aimed to describe the short-term side effects after receiving the first, the second, or both doses of sputnik v, oxford-astrazeneca, and sinopharm vaccines in personnel and students of shahid beheshti dentistry school. methods: a cohort project has been conducting at the dental faculty since june 2020. this article is a cross-sectional study as a section of the cohort project. dental faculty began its campaign to vaccinate the personnel on april 2021, with the sputnik v and oxford-astrazeneca and sinopharm. an employed operator phoned each one by one after 48 hours of vaccination. she asked them about any experienced symptoms after receiving each dose. the reported symptoms were coded and categorized. results: the most common symptoms in astrazeneca’s first dose were fever followed by myalgia. after the second dose, the frequency of all symptoms had been reduced significantly. after the first dose of sputnik v, the most common symptoms were myalgia followed by fever. the frequency of fever and pain at the injection site increased significantly after the second dose (p < 0.001). in the sinopharm group, all symptoms occurred whit a low percentage. the most common symptoms were again, myalgia and pain at the injection site. conclusion: post-vaccination adverse effects were mild in all groups and controlled by analgesic. fever, myalgia and pain at the injection site are the most common symptoms reported after vaccination by all three types of vaccines. keywords: adverse effects, covid-19, vaccine issn 2413-0516 introduction during the covid-19 pandemic, people face significant health care challenges, lockdowns, anxiety, and stress, as there is no specific treatment for this pandemic. however, vaccine development is perhaps the best hope to stop this pandemic.1 the most effective vaccines mimic the preventive mechanism resulting from the recovery phase in the standard form of the disease.2 herd immunity is established when sufficient people are immune to stop new cases, which means that enough people are protected to stop person-to-person transmission in the community.3 the percentage of people who need to achieve herd immunity varies with specific diseases, e.g., measles needs about 95% of vaccination among the community. to achieve herd immunity for coivid-19, the exact percentage is not known.4 since the outbreak, multiple vaccine candidates based on rna and dna technologies inactivated viruses, and other approaches have been rapidly developed. doroftei et al. concluded that three vaccines had more than 90% efficacy (pfizer– biontech, ~95%; moderna, ~94%; and sputnik v, ~92%) except for oxford–astrazeneca (~81%).5 iran’s drug regulatory authority has given emergency approval to four vaccines for restricted use against covid-19, including sputnik v, covax in, sinopharm, and astrazeneca. many health workers in the dentistry school of shahid beheshti university have welcomed the program and undergone vaccination with sputnik v, oxford–astrazeneca (chadox1 ncov-19), and sinopharm since march 2021. sputnik v has been used in many countries. the adenoviruses are combined with the sars-cov-2 spike protein to make the vaccine, which prompts the body to make an immune response. interim results (based on data from 14,964 participants in the vaccine group and 4902 in the placebo group) indicate that the vaccine is 91.6% effective, based on its ability to prevent symptomatic infection. there were no cases of moderate or severe covid-19 in the vaccinated group at least 21 days following the first dose. no serious adverse events were detected. most adverse effects were mild, with over half experiencing pain at the injection site.6 oxford–astrazeneca is a viral vector vaccine using a modified chimpanzee adenovirus chadox1.7 the vaccine’s efficacy is 76.0% in preventing symptomatic covid-19 in 22 days following the first dose and 81.3% after the second. the vaccine has a good safety profile, with side effects including injection-site pain, headache, and nausea, generally resolving within a few days. more rarely, anaphylaxis may occur. in rare cases (around 1 in 100,000), the vaccine has been associated with an increased risk of blood clots in combination with low blood platelet level.8 as with any other medicine, vaccines are not free from side effects and adverse reactions, most of which are generally mild, well-tolerated, and self-resolving. these symptoms could indicate the body is developing the desired immunity.9 (submitted: 29 september 2021 – revised version received: 12 october 2021 – accepted: 21 october 2021 – published online: 26 december 2021) 359j contemp med sci | vol. 7, no. 6, november-december 2021: 358–362 m. mousavi et al. original adverse effects of covid19 vaccines among healthcare workers fear of the new vaccines is a driver of vaccine hesitancy.10 the knowledge about what happens post-vaccination in the actual world among the general population is still modest, thus, describing what to expect after the first and second dose of vaccination will help in lowering the apprehension about this type of vaccines, increased and also increases the public confidence and safety, and accelerates the vaccination process against covid-19. this study aimed to describe the short-term side effects after receiving the first, the second, or both doses of sputnik v, oxford-astrazeneca, and sinopharm covid-19 vaccines in a sample of 18 years and older personnel and students of shahid beheshti dentistry school. materials and methods study design and participants a comprehensive cohort project has been conducting at the dental faculty of shahid beheshti university of medical sciences since the reopening of universities in iran in june 2020. all personnel were screened and followed. contraction to the covid-19, signs, and symptoms after contraction, systemic condition, types of activities, and precautional behaviors have been monitored and registered. this article is a cross sectional study as a section of the cohort project. the present study was conducted over the period between april 10 to august 1, 2021. ethical approval was taken from shahid beheshti university of medical sciences’ review committee. procedures according to cdc and who protocols, the dental faculty began its campaign to vaccinate the personnel against the emerging coronavirus on april 2021, with the sputnik v and oxford-astrazeneca and sinopharm. all the students and staff were vaccinated at the same time. types of vaccines with the date of injection of first and second dose were summarized in table 1. 176 doses of oxford–astrazeneca and 600 doses of sputnik v were used in the vaccination. informed consent was obtained from each individual who was rejected to be vaccinated based on who advice. a resident general practitioner was established at the vaccination site to inform and observe any adverse effects. following the vaccination, all recipients were observed for 30 minutes. probable occurrence of side effects and adverse effects following immunization (aefi) like injection site pain, swelling, redness; fever, chills, rigor, sweating, headache, muscle ache, joint pain, cough, running nose, sore throat, diarrhea, nausea, vomiting, abdominal cramps, loss of appetite, skin rashes, itching, palpitation, dizziness, fainting, shortness of breath, chest tightness, was explained to each participant. a phone number was given to them to report any experienced adverse effects, including both systemic (whole-body) and local effects, following 30 days after receiving each dose. an employed operator phoned each one by one after 48 hours of vaccination. she asked them about any experienced symptoms after receiving the first/second dose and asked them if any medicines or painkillers were taking during those days. the reported symptoms were coded and categorized as shown in table 1. statistical analysis ibm-spss statistical package for social sciences version 21 (ibm corp: armonk, ny, usa) was used to obtain descriptive statistics and significant associations between variables. descriptive statistics were reported as frequencies and percentages. cross tabulation with mcnemar’s test was carried out to compare the frequency of individuals with positive side effects after each dose in each vaccine type. a chi-square test was used to compare the frequency of side effects after the first and second doses between different vaccine types. results the total number of vaccinated individuals in each vaccination group and distribution of genders were summarized in table 2. from 472 personnel and students, 236 were vaccinated with sputnik v while 192 received astrazeneca and 44 received sinopharm’s first dose. side effects of each vaccine in every dose were summarized in table 3. the most common symptoms in astrazeneca’s first dose were fever followed by myalgia. 87.2% of individuals who received medicine after the first dose. after the second dose, the frequency of all symptoms had been reduced significantly except tachycardia, cough, and flu-like symptoms. (p < 0.001) indeed, the frequency of those who had such symptoms in the first dose was significantly more than individuals who had the symptoms only after the second dose. however, the percentage of taking medication reduced significantly; still, 51.1% of participants used drugs after the second dose. after the first administration dose, the most common symptoms were myalgia followed by fever for the sputnik v group. 57.3% of individuals received medicine after the first dose. after the second dose, the frequency of fever and pain at the injection site increased significantly. the percentage of individuals who reported fever and pain at the injection site only after the second dose was significantly more than those who reported fever and pain only after the first dose. table 1. similar reported symptoms were coded and categorized code symptoms 1 fever/chilling 2 headache/dizziness 3 myalgia/backache/weakness/fatigue/lethargy 4 gastrointestinal 5 tachycardia/hypoxemia/heart pain/cough 6 pain/swelling at injection site 7 flu-like symptoms (rhinitis etc.) table 2. total number of vaccinated individuals in each vaccination group and distribution of genders vaccine type male (percentage) female (percentage) total number astrazeneca 58.3% 41.7% 192 sputnik v 44.5% 55.5% 236 sinopharm 47.7% 52.3% 44 360 j contemp med sci | vol. 7, no. 6, november-december 2021: 358–362 adverse effects of covid19 vaccines among healthcare workers original m. mousavi et al. (p < 0.001) 57.1 of participants received medicine to relieve pain after the second dose. in the sinopharm group, all symptoms occurred whit a low percentage. the most common symptoms after the first dose were, again, myalgia and pain at the injection site. nobody reported gastrointestinal problems or tachycardia/ hypoxemia/heart pain/cough. among all participants, gastrointestinal symptoms or flu-like symptoms were reported at least. after the first dose injection, the results showed a significant difference in the report of side effects among individuals vaccinated with different vaccines except for tachycardia and flu-like symptoms (p value < 0.05) (figure 1). after the injection of the second dose of astrazeneca and sputnik v, the report of fever, myalgia, and pain at the injection site were significantly different between the two types of vaccines (p = 0.0) (figure 2). discussion there is a variation in people’s acceptance to take the vaccines due to several factors. an important factor may be the shorttime development of new vaccines compared to the previously approved vaccines. another reason for this variation may be the usage of a newly emerging technique for mrna vaccines. there was also exaggeration and over-reporting of adverse effects of vaccines, as some of these effects are normal physiologic processes or developmental anomalies.11-13 thus, this study aimed to evaluate the short-term side effects of covid-19 vaccines, which are currently used in shahid beheshti dental school. in this study, these systemic symptoms, including myalgia, backache, weakness, fatigue, and lethargy, were the most commonly reported symptoms after the injection of the first dose of each vaccine. the second common symptom in astrazeneca and sputnik groups was fever in each dose. however, table 3. side effects of each vaccine in every dose side effects/taking medication astrazeneca sputnik sinopharm first dose (percentage) second dose (percentage) first dose (percentage) second dose (percentage) first dose (percentage) fever/chilling 70.2 25.1* 38.5 46.6* 8.3 headache/dizziness 28.7 7.5* 11.8 13.6 5.6 myalgia/back ache weakness/fatigue/lethargy 63.5 23.5* 46.4 45.2 25.0 gastrointestinal 11.7 2.9* 3.6 5.5 0 tachycardia/hypoxemia/heart pain/cough 3.5 .6 .9 1.8 0 pain/swelling at injection site 14.9 3.4* 2.3 8.6* 19.4 flu-like symptoms (rhinitis etc.) 1.6 1.1 3.6 1.4 2.8 taking medication 87.2 51.1* 57.3 57.1 19.4 fig. 1 comparison of side effects after the injection of first dose between vaccines. 361j contemp med sci | vol. 7, no. 6, november-december 2021: 358–362 m. mousavi et al. original adverse effects of covid19 vaccines among healthcare workers in the sinopharm group, the second common symptom was local pain at the injection site. all these were mild and self limiting. moreover, no treatment was required for any side effects. most similar cross-sectional studies4,10,14,15 also reported fever, tiredness, and pain at the injection site as the most routine symptoms after vaccination. not surprisingly, fever was one of the most frequently reported adverse reactions. similar evidence has been earlier garnered from the phase iii clinical trials of some of these vaccines, where the highest frequency of fever was up to 24% with astrazeneca vaxzervria.4 systemic symptoms are that the immune system could produce cytokines that affect the blood vessels, muscles, and other tissues. it may also produce flu-like symptoms that last for days after vaccination.16 alhazmi et al. reported that most of the participants in their study had tiredness and headache, and they stated that this was mainly due to the younger age of our participants. similarly, in this study, participants were young or below the retirement age.12 vaccines in “in situ” should be kept at a low temperature, including sinopharm vaccine that should store at average refrigeration temperature, and if injected without optimal warming up, this may increase the probability of pain of the injection site symptom. although all covid-19 vaccines cause similar post vaccination side effects, the severity and number of these side effects were significantly associated with vaccine type. in this study, astrazeneca caused more side effects after the first dose than sputnik v and sinopharm.15 similarly, menni et al. reported that those vaccinated with the chadox1 ncov-19 vaccine were more likely to experience systemic side effects than those who had been given other types.17 several viral vaccine development platforms include live attenuated, inactivated, dna-based, rna-based, protein based, and viral vector-based. astrazeneca vaccine consists of chadox1 (replication deficient simian adenovirus vector) containing the full-length structural sars-cov-2 spike (s) protein. the s protein plays a crucial role in penetrating host cells and initiating infection of all sars viruses. the astrazeneca vaccine expresses a codon-optimized coding sequence for the s protein in the human body, which builds immunity against sars-cov-2.18 sputnik v is a recombinant vaccine using human adenovirus vector 26 for the first vaccine and human adenovirus 5 for the second vaccine.2 the sinopharm vaccine has been developed using conventional technology (i.e., inactivated virus). dna vaccines are easy to produce and store with excellent stability and limited toxicity.19,20 the results of abu-hammad et al. study also study confirmed that sinopharm is a “quiet” vaccine since it was significantly associated with symptom-free vaccination.13 the results showed that almost all post-vaccination symptoms in the astrazeneca group, except tachycardia and flu-like symptoms, occurred more frequently after the first dose than the second dose. (p < 0.001). in the sputnik v group, side effects were more noticeable after the second dose. however, the frequency of only fever and pain in the injection site was significant, and most side effects showed no significant association with the number of doses. in a similar study, hatmal et al. assessed the side effects of sinopharm, astrazeneca, pfizer-biontech in jordan, and they also reported that side effects after the first dose were more intense than the second.15 however, according to the centers for disease control and prevention (cdc), side effects might be more noticeable after the second dose.21,22 it seems that in individuals taking the astrazeneca vaccine, the immune system will be activated more vigorously after the first dose. in this study, participants reported taking medication to alleviate pain and reduce symptoms. the percentage of individuals who received medicine was 87.2% for astrazeneca’s first dose to 19.4% for sinopharm’s first dose. in the astrazeneca group, there was a significant association between taking medication and the number of doses. astrazeneca fig. 2 comparison of side effects after the injection of second dose between vaccines. 362 j contemp med sci | vol. 7, no. 6, november-december 2021: 358–362 adverse effects of covid19 vaccines among healthcare workers original m. mousavi et al. company stated that prophylactic use of acetaminophen could reduce some symptoms.2 based on the results, the percentage of taking medications after both doses of sputnik v was similar to the second dose of astrazeneca. although, side effects after the second dose of astrazeneca had been reduced. previous studies suggested non-steroidal anti-inflammatory drugs to control fever or antihistamines for topical side effects of the sputnik v vaccine.2,15 such mild side effects are acceptable during covid-19 vaccination as the body will need some time to adopt vaccination doses and to trigger the immune system. the population should be informed about these minor side effects controlled with some symptomatic treatment like paracetamol. such medicine should also be taken as prophylaxis to avoid developing symptoms and increase the acceptance of the covid-19 vaccine among the mass population. it will help to counter this pandemic disease through ongoing vaccination program successfully.14 the strengths of the study include its large sample size and diversity of vaccines. this study also has some limitations. self-reported data were used, which can introduce information bias. also, the second dose of vaccination with sinopharm has not been completed. since this study was a part of a comprehensive cohort study and all personnel and student were counted, the sex and age of participants could not be matched for the results. it is suggested for future studies to evaluate objectively the antibody response to each vaccine administrated against covid 19 besides subjective reports. conclusion our study confirmed that post-vaccination adverse effects were mild in all groups and controlled by analgesic. in astrazeneca group, systemic reactions were more intense and occurred after the first dose more frequently compared with other groups. in general, fever, myalgia and pain at the injection site are the most common symptoms reported after vaccination by all three types of vaccines. conflicts of interest none.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1121 references 1. meo s, bukhari i, akram j, meo a, klonoff d. covid-19 vaccines: comparison of biological, pharmacological characteristics and adverse effects of pfizer/biontech and moderna vaccines. eur rev med pharmacol sci. 2021;25(3):1663–9. 2. ghiasi n, arabsorkhi m, hoseyni t, esfandiari k, sadighpour t, jahantigh h. efficacy and side effects of sputnik v, sinopharm and astrazeneca vaccines to stop covid-19; a review and discussion. immunopathologia persa. 2021;7(2):31. 3. metcalf cje, ferrari m, graham al, grenfell bt. understanding herd immunity. trends in immunology. 2015;36(12):753–5. 4. lippi g, mattiuzzi c, henry bm. mild adverse reactions after covid-19 vaccination: updated analysis of italian medicines agency data. available at ssrn 3817988. 2021. 5. doroftei b, ciobica a, ilie o-d, maftei r, ilea c. mini-review discussing the reliability and efficiency of covid-19 vaccines. diagnostics. 2021;11(4):579. 6. baraniuk c. covid-19: what do we know about sputnik v and other russian vaccines? bmj. 2021;372. 7. raadsen m, du toit j, langerak t, van bussel b, van gorp e, goeijenbier m. thrombocytopenia in virus infections. journal of clinical medicine. 2021;10(4):877. 8. ramasamy mn, minassian am, ewer kj, flaxman al, folegatti pm, owens dr, et al. safety and immunogenicity of chadox1 ncov-19 vaccine administered in a prime-boost regimen in young and old adults (cov002): a single-blind, randomised, controlled, phase 2/3 trial. the lancet. 2020;396(10267):1979–93. 9. song bj, katial rk. update on side effects from common vaccines. current allergy and asthma reports. 2004;4(6):447–53. 10. saeed bq, al-shahrabi r, alhaj ss, alkokhardi zm, adrees ao. side effects and perceptions following sinopharm covid-19 vaccination. international journal of infectious diseases. 2021;111:219–26. 11. el-shitany na, harakeh s, badr-eldin sm, bagher am, eid b, almukadi h, et al. minor to moderate side effects of pfizer-biontech covid-19 vaccine among saudi residents: a retrospective cross-sectional study. international journal of general medicine. 2021;14:1389. 12. alhazmi a, alamer e, daws d, hakami m, darraj m, abdelwahab s, et al. evaluation of side effects associated with covid-19 vaccines in saudi arabia. vaccines. 2021;9(6):674. 13. abu-hammad o, alduraidi h, abu-hammad s, alnazzawi a, babkair h, abu-hammad a, et al. side effects reported by jordanian healthcare workers who received covid-19 vaccines. vaccines. 2021;9(6):577. 14. joshi u, singh p. adverse reaction following covisheild a covid 19 vaccine-experience of from a single centre. 15. hatmal mmm, al-hatamleh ma, olaimat an, hatmal m, alhaj-qasem dm, olaimat tm, et al. side effects and perceptions following covid-19 vaccination in jordan: a randomized, cross-sectional study implementing machine learning for predicting severity of side effects. vaccines. 2021;9(6):556. 16. hervé c, laupèze b, del giudice g, didierlaurent am, da silva ft. the how’s and what’s of vaccine reactogenicity. npj vaccines. 2019;4(1):1–11. 17. menni c, klaser k, may a, polidori l, capdevila j, louca p, et al. vaccine after effects and post-vaccine infection in a real world setting: results from the covid symptom study app. 2021. 18. folegatti pm, ewer kj, aley pk, angus b, becker s, belij-rammerstorfer s, et al. safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. the lancet. 2020;396(10249): 467–78. 19. silveira mm, moreira gmsg, mendonça m. dna vaccines against covid-19: perspectives and challenges. life sciences. 2020:118919. 20. abbood, muhanad hadi; hassan, jabbar sirhan; mehammed, aqeel jassim. case study of covid 19 from august to end of december 2020 in babylon, iraq. iraq medical journal, v. 5, n. 3, sep. 2021 21. control cfd, prevention. possible side effects after getting a covid-19 vaccine. 2021. 22. al-khayat, zakarea abdullah yaseen et al. single center study of vaccination breakthrough infection with sars-cov-2 among erbil population in august 2021. journal of contemporary medical sciences, v. 7, n. 5, oct. 2021. 179j contemp med sci | vol. 7, no. 3, may-june 2021: 179–182 original antioxidant properties of orally administered of aqueous extracts of selected medicinal plants and paracetamol in human volunteers: in vivo nessrin g. alabdallat1 1department of medical laboratory sciences, majmaah university, majmaah, saudi arabia. corresponding author: nessrin g. alabdallat (e-mail: n.alabdallat@mu.edu.sa) (submitted: 19 february 2021 – revised version received: 10 april 2021 – accepted: 14 may 2021 – published online: 26 june 2021) introduction antioxidants neutralize oxidative stress and its deleterious effects on human health. antioxidants can stop those free radical-mediated oxidative damages due to the scavenging properties of free radicals. because of synthetic antioxidants has toxic properties, natural antioxidants are needed.1, 2 medicinal plants are potential sources of natural antioxidants. antioxidant actions to prevent oxidative damage are through free radical scavenging activity, inhibition of their formation due to metal chelating properties.3 medicinal plants are widely used by people in jordan, in fact people in jordan taking a cup of herbal extract once or more daily as the habit of tea or coffee drinking. paracetamol is one of the most popular and most commonly used analgesic and antipyretic drugs around the world, available without a prescription, both in monoand multi-component preparations. the in vitro antioxidant properties of studied herbs previously evaluated,4, 5 although there are no published studies displaying there could be in vivo antioxidants properties of the orally administrated of studied plants. therefore, this study proposed to see the in vivo effects of the following plants: zingiber officinale (rhizomes), rosmarinus officinalis (leaves), verbena triphylla (leaves), saliva triloba (leaves), origanum syriacum (leaves) regarding antioxidant capacities. materials and methods plant material the selected plants (zingiber officinale (rhizomes), rosmarinus officinalis (leaves), verbena triphylla (leaves), saliva triloba (leaves), origanum syriacum (leaves)) were obtained from the local herbal stores in madaba, jordan. the choice of plant material used was dependent on the large use of public as folk medicine. preparation of aqueous extracts 250 grams of each dried plants from the following medicinal plant (saliva triloba, origanum syriacum, zingiber officinale, rosmarinus officinalis, verbena triphylla) was poiling with 12 liter water for 10-15 mints, and then it covered and left soaking for 4-5 hr at 25 °c. after that the soaked or aqueous extract filled in clean bottles (each one contains 1.250 l). blood samples 54 healthy volunteers were grouped into six groups, (each group n = 9), their age and sex were shown in table 1. 5 groups drinking 200–250 ml of aqueous extract from the selected medicinal plants (saliva triloba, origanum syriacum, zingiber abstract objectives in the current study, we used the herbal plant extracts and studied antioxidative value against the well-known drug paracetamol. methods 54 healthy volunteers were grouped into six groups, 5 groups drinking 200-250ml of aqueous extract from selected medicinal plants daily for 5 days and group six received 2 tablets of paracetamol (each tablet, 500 mg) daily for five days. blood samples were taken before and 1 hr after the administration (samples 1 and 2, respectively) and then one day after the last dose of day five (sample 3). serum total antioxidant status (tas), red blood cell reduced glutathione (gsh), red blood cell malonyldialdehyde (mda), and red blood cell superoxide dismutase (sod) were used as assays. results oral administration of aqueous extracts of studied plants increased significantly the serum total antioxidant status and red blood cell reduced glutathione after 5 days of administration compared to 0 time of administration. data also showed that red blood cell superoxide dismutase increased significantly after five days of aqueous extracts of zingiber officinale, rosmarinus officinalis & saliva triloba administration compared to 0 time of administration. oral administration of aqueous extracts of zingiber officinale, rosmarinus officinalis, verbena triphylla, caused a significant decrease in red blood cell malonyldialdehyde. oral administration of paracetamol for 5 days did not affect total antioxidant status red blood cell malonyldialdehyde, red blood cell reduced glutathione and red blood cell superoxide dismutase. conclusion paracetamol is a very common antipyretic drug. it doesn’t show antioxidative property. on other hand some herbal products (zingiber officinale, rosmarinus officinalis, verbena triphylla, saliva triloba and origanum syriacum) having antioxidative property. therefore, a person taking herbal product can enhance the in vivo antioxidant capacity of body by increasing the antioxidative property of body. keywords antioxidants, malondialdehyde, glutathione, superoxide dismutase issn 2413-0516 180 j contemp med sci | vol. 7, no. 3, may-june 2021: 179–182 antioxidant properties of orally administered of aqueous extracts of selected medicinal plants and paracetamol original n. g. alabdallat officinale, rosmarinus officinalis, verbena triphylla) daily for 5 days and group six received 2 tablets of paracetamol (each tablet, 500 mg) daily for 5 days. 3 blood samples were collected from each healthy volunteer (sample 1 before drinking or administration of aqueous extract), sample 2 after 1 hr of the first dose (drinking aqueous extract) on day 1 and sample 3 next day following the last dose of day five) in gell clot activator tubes. gell tubes were centrifuged for 10 mints at 3000 xg at 25 °c to separate and collect serum. then 2 ml of distilled water added to the cells under the gell in tubes and the tubes were centrifuged for 5 mints at 3000 xg and the supernatant was collected (hemolysate). all samples (serum and hemolysate) were stored frozen at -20°c until analysis. serum tas assay: serum tas measured by tas kit from randox. the results were expressed as milli mole per liter. red blood cell mda red blood cell mda was determined as a measure of lipid peroxidation according to stocks and dormandy’s method6 using thiobarbituric acid (tba) as modified by srour et al.7 all mda concentrations were expressed as nano mole per gram hemoglobin. red blood cell gsh red blood cell gsh was determined using ellman’s method with some modification.8 all gsh concentrations were expressed in milligram per gram hemoglobin. red blood cell sod activity red blood cell sod was measured using kit from randox. the results were expressed as unit per gram hemoglobin. statistical analysis data were analyzed by using spss (statistical package for social sciences) software, version.17. results all the tested plants: rosmarinus officinalis, verbena triphylla, zingiber officinale, saliva triloba & origanum syriacum caused a significant increase in serum tas at the sixth day of administration. paracetamol did not affect serum tas (table 2). all the tested plants: zingiber officinale, rosmarinus officinalis, verbena triphylla, saliva triloba, nigella sativum & origanum syriacum also caused a significant increase in erythrocyte reduced glutathione at the sixth day of administration. paracetamol did not affect erythrocyte reduced glutathione (table 3). however, erythrocyte superoxide dismutase increased significantly at the sixth day of administration of zingiber officinale, rosmarinus officinalis and saliva triloba compared to 0 time of administration, whereas origanum syriacum & verbena triphylla although caused some increase in erythrocyte superoxide dismutase but did not reached to significant level (table 4). oral administration of aqueous extracts of zingiber officinale, verbena triphylla, rosmarinus officinalis caused a significant decrease in erythrocyte malonyldialdehyde (mda) at the sixth of administration. paracetamol did not affect erythrocyte mda (table 5). table 2. total antioxidant status (tas) of the given medicinal plants. each value represents the mean ± s.d., (n=9), *p value ≤ 0.05, compared to 0 time administration. tas (mmol/l) 0 time 1hr (day 1) day 6 rosmarinus officinalis 1.14±0.3 1.31±0.27* 1.30±0.20* verbena triphylla 1.23±0.2 1.36±0.2* 1.37±0.22* zingiber officinale 1.08±0.16 1.20±0.10 1.24±0.12* saliva triloba 1.12±0.11 1.16±0.15 1.22±0.16* origanum syriacum 1.14±0.10 1.21±0.11 1.28±0.09* paracetamol 1.4±0.27 1.36±0.3 1.31±0.9 table 3. reduced glutathione (gsh) mg/g hb of the given medicinal plants. each value represents the mean ± s. d., (n=9), *p value ≤0.05, compared to the 0 time administration. gsh 0 time day 6 zingiber officinale 0.74±0.31 1.53±0.37* rosmarinus officinalis 0.82±0.13 1.41±0.23* verbena triphylla 0.80±0.15 1.05±0.14* saliva triloba 0.54±0.09 0.87±0.10* origanum syriacum 0.73±0.11 0.80±0.10* paracetamol 0.73±0.12 0.75±0.13 table 4. superoxide dismutas (sod) u/ghb of the given medicinal plants. each value represents the mean ± s.d., (n=8), *p value ≤ 0.05, compared to the 0 time administration gsh 0 time day 6 zingiber officinale 1005.4±298.0 1374.5±160.1* rosmarinus officinalis 1106.6±118.3 1340.5±134.0* saliva triloba 868.0±167.1 997.5±192.4* verbena triphylla 1132.0±139.0 1210.3±119.2 origanum syriacum 1037.1±155.3 1098.0±181.5 paracetamol 1114.5±256.6 1091.2±172.1 table 1. age and sex of participant. group age (mean±s.d.) female/male origanum syriacum 35.8±14.7 4/5 saliva triloba 42.8±14.6 6/3 verbena triphylla 34±18.6 4/5 zingiber officinale 41.8±7.6 7/2 rosmarinus officinalis 35.4±13.5 4/5 paracetamol 30.6±9.8 3/6 181j contemp med sci | vol. 7, no. 3, may-june 2021: 179–182 n. g. alabdallat original antioxidant properties of orally administered of aqueous extracts of selected medicinal plants and paracetamol table 5. malonyldialdehyde (mda) nmol//g hb of the given medicinal plants. each value represents the mean ± s. d., (n =9). mda 0 time day 6 zingiber officinale 22.9±4.5 16.2±3.7* verbena triphylla 27.7±4.8 22.9±3.4* rosmarinus officinalis 21.2±2.0 17.7±2.8* saliva triloba 17.9±3.4 15.8±3.8 origanum syriacum 17.5±3.4 16.4±3.5 paracetamol 16.1±3.1 17.8±4.1 discussion oral administration of aqueous extracts of zingiber officinale, rosmarinus officinalis, verbena triphylla, saliva triloba, origanum syriacum increased significantly the serum tas after 5 days of administration. this result coincides with the findings of other researchers9, 10 who have showed that the total plasma antioxidant capacity increased in diabetic rats treated with nigella sativa crude extract. the present study also showed that all studied plants caused a significant increase in erythrocyte reduced glutathione after five days of administration. this result coincides with the findings of other researchers9, 11–16 who have showed that oral administration of extracts of zingiber officinale or nigella sativum increased significantly reduced glutathione or antioxidant status in the liver & kidney tissues of mice & rats. the present study also showed that erythrocyte superoxide dismutase (sod) increased significantly at the sixth day of zingiber officinale, rosmarinus officinalis & saliva triloba administration. this result coincides with the findings of other researchers11, 17, 18 who have showed that oral administration of extracts of rosmarinus officinalis or zingiber officinale increased significantly the activity of superoxide dismutase (sod) in the liver of mice or in serum of diabetic rabbits. the present study also showed that oral administration of aqueous extracts of zingiber officinale, rosmarinus officinalis, verbena triphylla, caused a significant decrease in erythrocyte malonyldialdehyde (mda) after five days of administration. this result coincides with the findings of other researchers13, 17 who have showed that oral administration of extracts of nigella sativum or rosmarinus officinalis decreased significantly mda in the liver of mice & in serum of diabetic rabbits. the most likely explanation for the increased serum total antioxidant capacity would therefore be as a result of the intestinal absorption with consequent accumulation of the antioxidant compounds contained in the administered plant extracts. the serum concentration of the absorbed antioxidant compounds could also be dependent on their renal clearance which supposed to be dependent on the hydration state of the body & varied according to the chemical structure from one compound to another. this could explain the variation between the tested plants in regard to the time taken after the dose in increasing serum total antioxidant capacity (table. 2). the increased erythrocyte content of reduced glutathione & activity of superoxide dismutase & the decreased erythrocyte content of mda indicate the improved cellular antioxidant capacity of the body resulted from the administration of tested plant extracts. however, the present study could not tell the mechanism by which the tested plants increased cellular reduced glutathione & activity of superoxide dismutase & decreased cellular mda. to our knowledge the present study is the first ever in vivo study in humans regarding the antioxidant properties of tested plants. ethical approval and consent to participate. this study was approved by the committee of the university of jordan, and have therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki. informed consent was obtained from each individual participants prior to their participation in the research. competing interests author has declared that there are no conflicts of interest. funding this study was funded by the deanship of scientific research, majmaah university, research project number (r-2021-143). acknowledgments the author would like to thank deanship of scientific research at majmaah university for supporting this work under project number r-2021-143. references 1. valko, m. rhodes, c. moncol, j. izakovic, m. mazur, m. free radicals, metals and antioxidants in oxidative stress-induced cancer. chem. biol. interact. 2006;160:1 -40. 2. valko, m. leibfritz, d. moncol, j. cronin, m.t. mazur, m. telser, j. free radicals and antioxidants in normal physiological functions and human disease. int. j. biochem. cell biol. 2007;39:44 -48. 3. soobrattee, m. neergheen, v. luximon-ramma, a. aruoma, o. bahorun, t. 2005. phenolics as potential antioxidant therapeutic agents: mechanism and actions. mutat. res. fundam. mol. mech. mutag. 2005;579:200 -213. 4. bilto y. y., alabdallat n. g. in vitro and in vivo antioxidant related effects of rosemary (rosmarinus officinalis l.) extracts in humans. american journal of clinical and experimental medicine. 2015;3:213 -221. 5. bilto y. y., alabdallat n. g. and maher salim: antioxidant properties of twelve selected medicinal plants commonly used in jordan. british journal of pharmaceutical research. 2015;6:121 -130. 6. stocks, j, and dormandy, t. l. the autoxidation of human red cell lipids induced by hydrogen peroxide. british journal of hematology. 1971;20:95 -111. 7. srour, m. a. bilto, y. y. juma, m. irhimeh, m. r. exposure of human erythrocytes to oxygen radicals causes loss of deformability, increased osmotic fragility, lipid peroxidation and protein degradation. clinical hemorheology and microcirculation. 2000;23:13 -21. 8. ellman, g. l. tissue sulfhydryl (-sh) groups. archive of biochemistry and biophysiology. 1951;82:70 -77. 182 j contemp med sci | vol. 7, no. 3, may-june 2021: 179–182 antioxidant properties of orally administered of aqueous extracts of selected medicinal plants and paracetamol original n. g. alabdallat 9. bilto y. y., alabdallat n. g. ex vivo and in vivo antioxidant related effects of zingiber officinale roscoe (ginger) extracts in humans. european journal of medicinal plants. 2015;7:99 -108. 10. houcher z. et al. effects of methanolic extract of nigella sativa l. in alloxaninduced diabetic rats. pteridines. 2007;18:8 -18. 11. asnani and verma (2009) 12. kaleem m, kirmani d, asif m, ahmed q & bano bilqees. biochemical effects of nigella sativa l seed in diabetic rats. indian journal of experimental biology. 2006;44:745 -748. 13. el shenawy et al. (2018) 14. ali bh. the effect of nigella sativa oil on gentamicin nephrotoxicity in rats. am j chin med. 2004;32:49 -55. 15. khan n, sharma s, sultana s. nigella sativa (black cumin) ameliorates potassium bromate-induced early events of carcinogenesis: diminution of oxidative stress. hum exp toxicol. 2003;22:193 -203. 16. el-dakhakhny m, barakat m, el-halim ma, aly sm. effects of nigella sativa oil on gastric secretion and ethanol induced ulcer in rats. j ethnopharmacol. 2000;72:299 -304. 17. bakirel et al. (2008) 18. ajith ta, hema u, aswathy ms. zingiber officinale roscoe prevents acetaminophen-induced acute hepatotoxicity by enhancing hepatic antioxidant status. food and chemical toxicology. 2007;45: 2267 -2272. 19. veena a, and ramtej v. ameliorative effects of ginger extract on paraben-induced lipid peroxidation in the liver of mice. acta poloniae pharmaceutica-drug research. 2009;66:225 -228. 20. tülay b, utku b, oya u. k, sinem g, ulgen, hasret y. in vivo assessment of antidiabetic and antioxidant activities of rosemary (rosmarinus officinalis) in alloxan-diabetic rabbits. j ethnopharmacol. 2008;116:64 -73. 21. nahla el-s, maha s, & shimaa r. the effect of antioxidant properties of aqueous garlic extract and nigella sativa as anti-schistosomiasis agents in mice. rev. inst. med. trop. s. paulo.2008;50:29 -36. 22. ohkawa h, ohishi n, yagi k. assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. anal biochem. 1979;95:351 -358. https://doi.org/10.22317/jcms.v7i3.1005 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 116 j contemp med sci | vol. 2, no. 8, autumn 2016: 116–118 research aassociate professor of dental public health; chair, community oral health department, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. bpreventive dentistry research center, dental research institute, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. cschool of medicine and dentistry, santiago de compostela university, santiago de compostela, spain. dschool of dentistry, tehran university of medical sciences, tehran, iran. eschool of medicine, international campus, tehran university of medical sciences, tehran, iran. fdepartment of oral and maxillofacial surgery, university of kufa, kufa, an-najaf, iraq. correspondence to ammar n. h. albujeer (email: ammar.dent@yahoo.com) (submitted: 2 september 2016 – revised version received: 25 september 2016 – accepted: 3 october 2016 – published online: 26 december 2016) objective it has been well recognized that, oral health is more than beautiful teeth. mouth has been considered to be the mirror of whole body, as much as a healthy mouth means healthy body. given the epidemic status of oral diseases, monitoring the oral health status is essential for oral health promotion. the world health organization (who) have provided standard epidemiological survey methodology that requires systematic oral examination, data collection and recording system. language barrier may be a reason to hinder extensive use of such important instruments in countries where english language is not predominantly used. therefore, our aim was to standardize an arabic translation of the who instrument for wide spreed use in many nations around the world. this would improve the standardization and quality of the oral health data in arabic speaking countries. methods initially, the forward translation of who oral health assessment questionnaire for adults was conducted from english to arabic language. backward translation of arabic version to english language was done by professional english translator and the result was compared with original text to identify differences. a nominal group technique (ngt) was used in order to obtain expert’s opinion from a group of ten specialists who also helped to culturally adapt the questionnaire. results the content validity index and ratio was calculated. after few recomended adjustments the final arabic version was produced. after removing one question the overall impact score of the questionnaire improved considerably to acceptable level. when computing the internal consistency coefficient, it was found to be 0.88 for the subscales (which means good to excellent). conclusion the results of this study prove that, the arabic version of the who oral health survey questionnaire is reliable instrument to be used for oral health evaluation of adults among arabic speaking populations. keywords psychometric, oral health, survey, questionnaire, validity, adults introduction it has been well recognized that oral health is more than beautiful teeth. mouth has been considered to be the mirror of the whole body, as much as the healthy mouth means a healthy body. the oral-systemic disease relationship is well established in scientific literature.1 therefore, it is very important to closely monitor the oral health status to continuously prevent the incidence of oral diseases and promote oral health and the quality for individuals and communities. as reported by most countries around the world, oral diseases are considered as the major public health issues globally.2 in order to better control this epidemic condition, the world health organization (who) has provided standard epidemiological survey methodology requirements for systematic oral examination, standard data collection, and recording system.3,4 on the other hand, the language barrier may be a reason to hinder the extensive use of such an important instrument in countries where english language is not predominantly used. therefore, our aim was to make an arabic translation of the instrument available for use in many nations around the world. this would improve the standardization and the quality of the research as well as making more comparable data available for better understanding of the oral health situation in arabic-speaking countries. likewise, it can help with the standardized reporting of different interventions conducted in those countries. who oral health assessment questionnaire for adult this questionnaire is published as a part of the “oral health surveys basic methods, 5th edition” by the world health organization in 2013.5 this tool is particularly designed for self reporting of individual’s oral health information. aside from demographic information, the rest of the questions are pertaining to risk and protective factors for individual’s oral health outcomes as well as the frequency of personal oral hygiene and the utilization of oral health services. other information such as socioeconomic status, place of residence, frequency of sugar intake and participation in any specific oral health program are inquired. the 16 primary items in this tool were assessed individually based on different responses. for more efficient use of this instrument, the who has encouraged countries to culturally adapt with necessary adjustments if needed. methods the forward translation of who oral health assessment questionnaire for adults was conducted into arabic language. this step was followed by backward translation of this document into english language by professional english language translator. the english translation was compared with original text of the questionnaire to identify differences. few items were slightly issn 2413-0516 who’s oral health assessment questionnaire for adult: psychometric properties of the arabic version mohammad hossein khoshnevisan,a,b ammar n. h. albujeer,c,d nona attaran,b alya almahafdha,e abbas taherf mohammad hossein khoshnevisan et al. 117j contemp med sci | vol. 2, no. 8, autumn 2016: 116–118 research who’s oral health assessment questionnaire for adult results after conducting ngt method, the culturally adapted final arabic translation of questionnaire was available. out of 16 original questions, only one question related to alcohol use was excluded. the remaining questions were related to oral health self-assessment (7 questions), accessibility to dental treatment (2 questions), diet (1 question) and socio-economic status (3 questions). the rest of the findings are reported as follows: a) content validity: when considering the total scale, the mean score for content validity index (cvi) was 0.9, demonstrating acceptable result. however, the content validity ratio (cvr) of the question number 15 was found to be lower than expected indicating that this question does not have the optimum level content validity. after exclusion of this question, the overall cvr was 0.81, which was at the satisfactory level based on lawshe table. a few items were slightly adjusted or modified based on recommended professional reviews. when consensus was reached on semantic, idiomatic, and conceptual equivalence, the final arabic version was produced. b) face validity: the impact score was computed for face validity assessment. the index was found to be equal or greater than 1.6 (range: 1.7 – 4.8) except for question (15). after removing this question, the overall impact score of the questionnaire improved considerably (3.5) to a satisfactory level. at this stage, the qualitative face validity was recognized by all participants by declaring that they had no ambiguity in reading questions and comprehending them. c) reliability: the cronbach’s alpha coefficient was calculated in order to evaluate the internal consistency and reliability for this questionnaire. the calculated value was 0.85 with subscales ranged from 0.75 to 0.91 which were beyond the acceptable thresholds. after computing the internal consistency coefficient (icc), it was found to be 0.88 and the values were 0.72–0.91 for the subscales (which means good to excellent). these findings confirm the steadiness of the arabic version of who questionnaire. confirmatory factor analysis (cfa): the principal component factor analysis was used to analyze questionnaire. the comparative fit index (cfi) and square error of approximation were computed. the cfi was 0.89 and rmsea was 0.041. also, the confidence interval was less than 0.01, which demonstrates the existence of correlation between variables. therefore, these analyses confirmed the suitability of the data. the results of cfa for five-factor model for who oral health survey questionnaire indicated satisfactory fit of the suggested model. the factors were as followed: i. factor 1 (oral health self-assessment) including 7 items (item 3, 4, 5, 6, 7, 8, 9). ii. factor 2 (accessibility to dental treatment) including 2 items (item 10, 11). iii. factor 3 (diet) including 1 item (item 13). iv. factor 4 (socio-economic status) including 3 items (item 12, 14 and 16). discussion when translating a questionnaire into another language, it must accurately reflect the content and the intent of the original toll; so that the translated questions contain the same meaning as the original version. it’s also important to ensure the quality and cultural appropriateness of the translated instrument into adjusted or modified based on professional recommendations. for using nominal group technique (ngt) a professional committee of 11 specialists was formed. meetings were held by two professional translators, two psychologist, five dental public health specialists and two epidemiologists in order to evaluate and culturally adapt the pre-final version of arabic questionnaire. after linguistic and cultural adaptation, the final arabic version of the who oral health assessment questionnaire for adults was finalized with complete experts’ consensuce. statistical analysis for the calculation of the content validity index (cvi) and content validity ratio (cvr) for the questionnaire, an expert panel composed of eight specialists in dental public health and pediatric dentistry were asked to provide comments independently on the necessity of each question was evaluated: (a) not necessary, (b) useful, (c) essential; as well as relevancy, clarity and simplicity of each question. using a three-point rating scale, the cvr for the total scale was calculated following the expert’s final evaluation. according to lawshe table, an acceptable cvr value for eight expert panels is 0.75.6 based on the proportion of rating by experts for each item, the cvi was computed.7 polit and beck recommended 0.80 as the acceptable lower limit for the cvi value (e.g. 6 of 8 experts should rate 3 or 4).7 by using qualitative and quantitative methods, the face validity of the questionnaire was assessed. in the qualitative stage, 25 adults were asked to evaluate the questionnaire in terms of potential difficulties in responding to the arabic version of the oral health questionnaire. in the quantitative stage, the impact score (frequency × importance) was computed to determine the percentage of adults who identified the item was important or quite important. the items related with an impact score of equal or greater than 1.5 (corresponding to mean frequency of 50% and a mean importance of 3 on a 5-point likert scale) were considered appropriate. an exploratory factor analysis was carried out to define the underlying constructs of the questionnaire, followed by principle components analysis with varimax rotation. the reliability of the questionnaire was measured by the difference in a score that eventually shows the true score, rather than the random error to the extent that measures provide consistent results. there are two common forms of reliability methods. the internal consistency of a scale relates to its homogeneity, where the higher the coefficient value, the higher the reliability and the lower the standard error of measurement. the internal consistency was assessed with a cronbach’s alpha coefficient that ranges between 0 and 1. the values equal to or less than 0.7 indicate satisfactory internal consistency.8 the test– retest reliability measures stability over time, when applying the same test to the same subjects at different points of time. to perform this test, a total of 25 adults were randomly selected from the arabic-speaking population to complete the arabic version of the oral health survey questionnaire. this process was repeated 2 weeks later, using exact same manner as the first round. the estimate of intra-class correlation coefficient was calculated to determine the reliability of the scale using test– retest method. in order to interpret the agreement levels, the following categories were selected: “0.0–0.2” for small level, “0.21–0.40” for fair level, “0.41–0.60” for moderate level, “0.61– 0.80” for substantial level and “0.81–1” for almost perfect level.9 118 j contemp med sci | vol. 2, no. 8, autumn 2016: 116–118 who’s oral health assessment questionnaire for adult research mohammad hossein khoshnevisan et al. countries. in general, our results support the standardization (reliability and validity) of the arabic version of the who oral health survey questionnaire. conclusion the results of this study prove that, the arabic version of the who oral health survey questionnaire is reliable tool to be used as a self-reported instrument for evaluating oral health among population in iraqi and other arabic speaking countries. this 15 digit arabic version of the who oral health survey questionnaire will improve measuring the oral health status of the iraqi people as well as other arabic-speaking nations around the world. conflicts of interest there are no conflicts of interest. n new language. similarly, it is important that the translated name of the instrument demonstrates adequate psychometric properties in terms of validity and reliability. oral health disparities are mainly related to lifestyle and many other factors. this condition is considered as a major public health problem. millions of children and adults are affected and based on available reports, the burden of oral diseases is very prominent globally.10 using standard surveillance system for oral health status and programs is highly crucial for better oral health care, maintenance, as well as oral disease prevention, protection and promotion for individual and communities over time.3,4 other potential benefits of such system would be the availability of data for administrators and decision makers for using the most cost-effective plan and make the best use of resources towards oral health promotion.11 on the other hand, the availability of standard arabic version of who recommended instrument may facilitate the generation of quality standard data in many arabic-speaking references 1. murray cj, lopez ad. on the comparable quantification of health risks: lessons from the global burden of disease study. epidemiology-baltimore, 1999;10:594–605. 2. petersen pe. the world oral health report 2003: continuous improvement of oral health in the 21st century–the approach of the who global oral health programme. commun dentist oral epidemiol. 2003;31(s1):3–24. 3. yeung c. book review: oral health surveys: basic methods. br dent j. 2014;217:333. 4. organization wh. oral health surveys: basic methods. 1987: world health organization. 5. organization wh. oral health surveys: basic methods. 2013: world health organization. 6. lawshe ch. a quantitative approach to content validity. pers psychol. 1975;28:563–575. 7. polit df, beck ct. nursing research: principles and methods. 2004: lippincott williams & wilkins. 8. cronbach lj. coefficient alpha and the internal structure of tests. psychometrika. 1951;16:297–334. 9. landis jr, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977:33;159–174. 10. mathers c, fat dm, boerma jt. the global burden of disease: 2004 update. 2008: world health organization. 11. brownson rc, fielding je, maylahn cm. evidence-based public health: a fundamental concept for public health practice. annu rev public health. 2009;30:175–201. 413j contemp med sci | vol. 8, no. 6, november-december 2022: 413–419 original development of gastro-floating drug delivery system by 3d printing: impact of formulation and design on the release profile of baclofen nuha mohammed abdulkhaleq*, mowafaq m. ghareeb department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. *correspondence to: nuha mohammed abdulkhaleq (e-mail: pharmacyone@gmail.com) (submitted: 08 september 2022 – revised version received: 29 september 2022 – accepted: 22 october 2022 – published online: 26 december 2022) abstract objectives: baclofen is a skeletal muscle relaxant with a short half-life and a narrow absorption window in the upper part of the gastrointestinal tract, and this study aims to formulate a sustained-release tablet of baclofen and 3d printing of gastro-floating device and study the effect of various polymers and device design on the release profile of baclofen. method: firstly, four formulas were produced through the hot-melt extrusion and direct compression of the extrudate to produce 30 mg baclofen tablets, then four gastro-floating devices (a, b, c, and d) were designed with two air pockets to enable the floating of the device and have drug-releasing windows with total surface area 4, 10, 20, and 40 mm2 respectively, for drug release. 3d printing of the devices was done by an fdm printer and the tablets were inserted into each device and test it for drug release. results: decreasing the surface area of the drug releasing windows revealed a significant reduction in the dissolution of baclofen irrespective of the type of polymers and useful for sustained release formulation but may be associated with lag time. devices with one and two releasing windows (device b and c respectively) sometimes revealed similar dissolution profiles and this related to the position of the window regarding the surface of the dissolution media. device d with four windows and a 40 mm2 surface area was found to produce more reliable results. f3 which contains eudragit rs-100 as the main polymer showed sustained release in device d where the complete dissolution of the drug occurred in 12 hours, and the gastro-floating device remained floating all the time and was assayed for drug content, ft-ir, and dsc study. conclusion: hot-melt extrusion was successfully employed to produce sustained release tablets of baclofen. fdm 3d printers are considered a potential tool to produce gastro-floating devices with the required design and release profile. keywords: baclofen, printing, three-dimensional, gastro-floating device, sustained-release, fused deposition modelling issn 2413-0516 introduction in recent years, the health sciences and pharmaceutical industries have conducted an extensive study into the additive manufacturing technology known as three-dimensional (3d) printing. the term “3d printing” refers to a wide range of 3d printing methods that make use of a wide range of printer technologies and a wide range of materials, all printed at varying resolutions and speeds.1 ink-jet (ij), nozzle, and laserbased 3d printing technologies are the main types and are currently the most widely used in pharmaceutical research.2 each main 3d printing type has a variety of sub-types. fused deposition modeling (fdm), pressure-aided microsyringes (pam), stereolithography (sla), and selective laser sintering (sls) are some of the most prevalent sub-types in the pharmaceutical area.3 fdm is the 3d printing method that uses heat to melt a thermoplastic filament and extrude it in successive layers to create a 3d object from a digital design. it’s considered a cost-effective method for building products with complicated geometry or of virtually of shapes or sizes. designing and printing devices for specific patients is considered a potential advantage of this type.4 extemporaneous synthesis of unit dosage forms of any dose, customized to the patient, is likely to be the future of medication design and manufacture, moving away from mass production of tablets/capsules with a limited dose range.5 the development of low-dose drugs with narrow therapeutic indices (such as immunosuppressants and/or blood thinners), increased awareness and importance of pharmacogenomics, and the need to formulate drug combinations are all factors driving this change so the pharmaceutical business must analyze and accept emerging manufacturing technologies to meet this issue and 3d printing is one technology that has such promise.6 hme (hot-melt extrusion) is a method that involves driving raw materials through a die at a high temperature to give them a uniform shape and density. hme is a widely used method that can be used to make a variety of pharmaceutical preparations, particularly solid dispersions.7 compared to other approaches, hme has a few advantages, for example, it’s a one-step, solvent-free, continuous-operation method, as well as a scalable process and is environmentally safe, and cost-effective technology when compared to other pharmaceutical manufacturing techniques.8 hme has been usually used in pharmaceutical manufacturing with various dosage forms, such as sustained-release tablets, pellets, transmucosal/ transdermal films,9 and implants10 bsa. hme is a simpler way of continually preparing sustained-release tablets than existing methods.11 oral drug delivery is the most convenient, patient-friendly, cost-effective, and secure way to treat a variety of disorders. traditional oral formulations have significant drawbacks, including poor targeting ability, short gastrointestinal (gi) tract retention time, and low bioavailability.12 to solve the problem of traditional oral formulations, gastro-retentive (gr) drug delivery systems were introduced to the pharmaceutical industry.13 since the gr system was introduced almost three decades ago, various approaches have been applied to extend the gastric residence time of gr systems, including low-density (floating), high-density (sinking), expandable (swelling), and mucoadhesive systems.14 innovative approaches, such as 414 j contemp med sci | vol. 8, no. 6, november-december 2022: 413–419 development of gastro-floating drug delivery system by 3d printing original n.m. abdulkhaleq et al. magnetic field-assisted gr systems, plug-type swelling systems, and floating systems with or without effervescence, have also been applied to prolong gastric retention time.15 the physiological conditions of the stomach and gastric retention and emptying time are highly variable. the main challenge is to maintain the drug delivery system in the stomach for a sufficient time until all the drugs are released at a predetermined rate in a dynamic physiological condition.16 the gr drug delivery systems can be retained in the stomach and assist in improving the oral sustained delivery of drugs that have an absorption window in a particular region of the git. these systems help in continuously releasing the drug, thus ensuring optimal bioavailability.17 recently, research groups have successfully developed a floating capsule-in-3d-printed device (fpd) for the incorporation of a commercial amoxicillin capsule. this device was produced from polyvinyl alcohol (pva), followed by a crosslinking process to extend the floating time and obtain a sustained release. a longer floating time of the device was achieved as the device was crosslinked for a longer time and at a higher temperature; however, dark color of fpd was obtained.18 previously, the design of devices for gr drug delivery systems has been studied, and it was found that the release lag time can be reduced by creating pores or channels on the devices. the devices can be loaded with both tablets16,19 or capsules 18 for gr drug delivery systems. fu et al. invented a tablet-in-device (tid) system with polylactic acid (pla) to contain riboflavin sustained-release (sr) tablets. this system was built with two chambers; one contains a tablet, and the other is an air chamber to provide buoyancy. although this tid was able to float in the stomach of a rabbit for more than three days, it remained in a position beyond this period and did not leave the stomach.19 baclofen is a centrally acting skeletal muscle relaxant used to treat spasticity. in some clinical trials, it has also been demonstrated to be an effective treatment for alcohol and cocaine addiction. baclofen is a chemical derivative of the neurotransmitter-aminobutyric acid (gaba), and it works by activating (or agonizing) gaba receptors, specifically, gabab receptors.20 the commercially existing baclofen tablets provide immediate drug release and are associated with dizziness, drowsiness, insomnia, nausea, and a sudden decrease in arterial blood pressure. these unwanted effects are explained by the fast absorption of the drug and elevated plasma levels (plasma levels peaking) after oral administration. moreover, the drug needs to be taken three or four times daily due to its very short half-life (approximately 2.5 h) which increases the incidence of these side effects, and also it’s considered a narrow absorption window drug. dose titration is needed at the beginning of therapy to reduce the above-mentioned side effects.21 this study aims to first formulate sustained-release tablets of baclofen (optimized to release the drug over 12 hr) by hme to be incorporated into the floating device and study the impact of formulation composition on the release profile, the second aim is to develop floating devices by fdm 3d printing using pla filament (which is safe, biodegradable, and has been widely used in 3d printing16) and study the effect of the design of the floating device on drug release by varying the surface areas of the drug releasing windows. materials and methods materials baclofen and ethylcellulose were purchased from baoji guokang bio-technology co. ltd. (baoji, china), eudragit® rs-100 was donated by evonik (darmstadt, germany). kollidon® 30 (polyvinylpyrrolidone k30) was donated by basf co. (ludwigshafen, germany). peg 4000 (polyethylene glycol) was purchased from himedia laboratories co. ltd. (mumbai, india). polylactic acid filament (pla filament, 1.75 mm in diameter) was purchased from prusa research (prague, czech republic). preparation of hot-melt extruded tablets baclofen and other excipients were mixed for 15 minutes in 30 gram batches with a mortar and pestle to ensure a uniform mixture. as granules, eudragit rs-100 was ground using an electric coffee grinder before extrusion. the formulations’ composition is shown in table 1. for all of the formulas, the mixture was extruded using a single-screw noztek pro filament extruder (noztek, shoreham, uk) with a 3 mm nozzle at a screw speed of 15 rpm and an extrusion temperature of 140°c.22 the resulting extrudate was ground using an electric coffee grinder then it was sieved through a size #35 usp mesh to eliminate any aggregated or agglomerated particles. direct compression of the sieved extrudate with a 6 mm round concave punch produced 150 mg tablets equivalent to 30 mg baclofen. 3d printing of the gastro-floating device the software autodesk® fusion 360 (v. 2.0.10244) was used in the design of the floating devices. the structure of the devices was intended to be quite analogous to that of a capsule. the devices were made to include two air pockets; one in the body and the other in the cap. additionally, the body of the device has a hollow section where the tablet can be placed. around this hollow section, windows for drug release were designed where four devices were designed with a different number or dimensions of drug releasing windows as shown in table 2. all the devices have a height of 20 millimeters, an interior diameter of 8 millimeters, and a thickness of 0.5 millimeters. using a prusa i3 mk3s fdm 3d printer (prusa research, prague, czech republic) equipped with a 0.4 mm nozzle and a commercial pla filament, the gastro-floating devices were printed. the temperature of the extruder was set to 200°c, while the temperature of the platform was set to 70°c. the following is the configuration of the printing system: infill percentage is 100%, layer height is 0.1 mm, and printing speed is 50 mm/sec. after the devices had been constructed, the baclofen tablet that had been prepared was inserted into the table 1. formulations composition (%w/w) formula baclofen pvp k30 peg 4000 eudragit rs-100 ethyl cellulose f1 20 75 5 f2 20 50 5 25 f3 20 25 5 50 f4 20 65 5 10 415j contemp med sci | vol. 8, no. 6, november-december 2022: 413–419 n.m. abdulkhaleq et al. original development of gastro-floating drug delivery system by 3d printing table 2. illustrative design of the floating devices device a b c d front view slice view no. of windows 1 1 2 4 windows size 2 × 2 mm 2 × 5 mm 2 × 5 mm 2 × 5 mm total windows surface area 4 mm2 10 mm2 20 mm2 40 mm2 center compartment of the body, and the cap was then locked to form the entire gastro-floating system. drug content determination the drug content of all prepared formulations was measured spectrophotometrically by dissolving 100 mg of sieved ground extrudate of each formula in 100 ml of 0.1n hcl and kept for 12 hr under stirring before filtering it, then 1 ml of the filtrate was diluted to 10 ml with 0.1n hcl, absorbance was measured spectrophotometrically at λmax 220 nm, and drug content was determined accordingly.21 in vitro buoyancy studies the in vitro buoyancy was calculated using the roy et al. method where floating lag time and total floating time were calculated. the gastro-floating devices (n = 3) were submerged in 100 ml of 0.1 n hcl with a tablet inside the capsular device. floating lag time is the time it takes for the device to rise to the surface and float. the total floating time was calculated as the amount of time the dosage form remained on the surface at all times.23 in-vitro dissolution the in vitro release rates of baclofen were determined by inserting a tablet from each formula into the four 3d printed gastro-floating devices (a, b, c, d) respectively and placing it in 900 ml of 0.1 n hydrochloric acid (ph 1.2) as a dissolution medium at 37 ± 0.5°c. drug release was performed using usp dissolution apparatus type ii (paddle type) at 50 rpm for 12 hr. aliquots of 5 ml were withdrawn at the following time intervals: 5, 10, 15, 30, 60, 90, 120 min then every hour until 12 hr. the samples were filtered and the medium was replenished with a similar volume of fresh medium. using the dissolving liquid as a blank, the quantity of baclofen was measured using spectrophotometry at 220 nm, and the percentage of drug release in total was computed. the outcome was calculated as the average of three runs.21 characterization of the selected formula differential scanning calorimetry (dsc) a dsc 60 (shimadzu, japan) was used to measure the thermodynamic properties of pure baclofen and the selected formula (f3). it was heated at a rate of 10°c/min between 25°c and 300°c in an aluminum pan under a dry nitrogen purge. an empty aluminum pan was utilized as a reference to calibrate the dsc temperature and enthalpy scales using indium/zinc standards.24 fourier transform infrared spectroscopy (ftir) the ftir spectra of pure baclofen and selected formula (f3) were obtained by using an ftir spectrophotometer (lambda scientific 8300, australia). samples were ground and mixed with dry potassium bromide then pressed in the form of discs using a hydraulic press. the samples were analyzed at wave numbers (4000–500 cm–1).25 results and discussion drug content determination the content of baclofen in the formulas was analyzed using a uv–visible spectrophotometer. drug content was in the range of 98.8 to 100.4% as shown in figure 1 indicating no significant drug loss occurred during hme since extrusion temperature was lower than the melting point of baclofen.26 in vitro buoyancy studies the in vitro floating ability of the gastro-floating device that had a baclofen tablet incorporated inside of it was evaluated. the floating lag time was relatively zero because the device floated at the surface immediately after being immersed in the medium for all of the formulas. on the other hand, the total 416 j contemp med sci | vol. 8, no. 6, november-december 2022: 413–419 development of gastro-floating drug delivery system by 3d printing original n.m. abdulkhaleq et al. floating time was more than 12 hours for all of the formulas because the device remained floating until 12 hours after the beginning of the experiment. in-vitro dissolution the drug release of each formula was tested in the four devices (a, b, c, and d) so the release of the f1 tablet in device (a) was donated as f1a and so on for the other formulas and devices. dissolution curves of f1, f2, f3, and f4 in the four devices are shown in figures 2-5 respectively. since the aim of this work was to produce sustained release tablets that release the drug over 12 hr, f3 was selected as the optimized formula as f3d released 96% of the drug over 12 hr and was selected for further characterization. characterization of the selected formula differential scanning calorimetry (dsc) the thermal behavior of pure baclofen showed a sharp endothermic peak at 209.9°c as shown in figure 6, corresponding fig. 1 drug content of sustained-release tablets (mean ± sd, n = 3). fig. 2 the dissolution profile of f1 tablets in devices a, b, c, and d. fig. 3 the dissolution profile of f2 tablets in devices a, b, c, and d. fig. 4 the dissolution profile of f3 tablets in devices a, b, c, and d. fig. 5 the dissolution profile of f4 tablets in devices a, b, c, and d. fig. 6 dsc thermogram of pure baclofen. to baclofen melting temperature, with the onset of a peak at 200°c and end set at 220°c, indicating that the drug is present in a crystalline state.27 the thermogram of the extrudate of f3 containing baclofen, eudragit rs-100, pvp k30, and peg 4000 is shown in figure 7. the polymer peg 4000 exhibits a peak at 58.25°c, which corresponds to its glass transition temperature.28 the disappearance of the sharp endothermic peak of baclofen can be attributed to the conversion of baclofen from the crystalline state to the amorphous state as well as the dilution of baclofen concentration as it consists of 20% of the total weight of the formula. the endothermic peak at 185.81°c is belong to the eudragit rs-100.29 fourier transform infrared spectroscopy (ftir) infrared spectroscopy has been used to investigate possible interactions between the drug and polymers in solid dispersion systems. the crystalline baclofen shows the primary amide n-h stretching vibration band at 3407 cm–1 (figure 8). 417j contemp med sci | vol. 8, no. 6, november-december 2022: 413–419 n.m. abdulkhaleq et al. original development of gastro-floating drug delivery system by 3d printing fig. 7 dsc thermogram of f3 extrudate. a strong band of c=o stretching was seen at 1621.84 cm–1, for the o-h group of acid exhibited stretching frequencies at 2904 cm–1. the bands occurring in the 734 cm–1 were assigned for c-cl stretching. the presence of c=c in the aromatic ring was seen in 1573 cm–1.30 the spectrum of the ground extrudate of f3 selected formulas showed no significant differences from pure baclofen (figure 9), which indicated that no new chemical bonds were created during the formation of solid dispersion and proved there was good compatibility between the drug and excipients. discussion the baclofen tablet used in this study actually was different from the marketed immediate release baclofen tablets. hotmelt extrusion was used to formulate baclofen tablets with various releasing properties where immediate release and sustained release polymers were used to study the effect of polymers and device design on the release properties. the gastro-floating system aimed to achieve long-term release of baclofen in the stomach so that the loaded tablet had to always be limited in the floating device and the formula which gives sustained release of baclofen over 12 hours (f3d) was selected for further characterization. f1 was composed of pvp k30 as an immediate release polymer and peg 4000 as a plasticizer and the release profile of f1 tablets in the four devices is shown in figure 2. f1a showed sustained release of baclofen despite using immediate release polymers where the complete release of the drug fig. 8 ftir of pure baclofen. fig. 9 ftir of f3 extrudate. occurred after 10 hrs and this was attributed to the small drug releasing windows surface area (4 mm2) that restricts dissolution media entry inside the hollow section of the capsular device thereby slows drug release.14 increasing the surface area of the drug releasing windows to 10 mm2 and 20 mm2 in f1b and f1c significantly enhanced the dissolution rate where complete drug release occurred after 5 and 6 hrs respectively. f1d with the largest windows surface area (20 mm2) showed the fastest release where the complete release of the drug occurred after 2 hrs. the addition of eudragit rs-100 in f2 while reducing pvp k30 concentration significantly reduced the dissolution rate of baclofen since eudragit rs-100 is a sustained release polymer and used for a customized release profile.31 release profile of f2 in the four gastro-floating devices is shown in figure 3. f2a significantly retained the dissolution of baclofen where about 50% of the drug was released after 12 hrs compared to f2d where the complete release of the drug occurred after 6 hrs. it’s worth noting that sometimes the release profile of devices b and c are quite similar as in f2 where the similarity factor (f2) between f2b and f2c is 50.7 and this may be attributed to the position of the device in the dissolution jar since it’s positioned horizontally and in the device (c) one of the windows is immersed inside the dissolution media and the other window which is against it is above the dissolution media so media entry into the device is relatively done through one window and this is similar to the device (b) which have only one window. increasing the concentration of eudragit rs-100 to 50% in f3 compared to 25% in f2 results in further retardation of baclofen dissolution (figure 4) as its insoluble polymer and drug release is mainly occurs by diffusion suggesting that the matrix with higher insoluble polymer content provides a more tortuous pathway, and/or a less porous tablet was formed.26 also in f3 we can notice that there is no significant difference between the dissolution profile of f3c and f3d where the similarity factor (f2) was 63.2, and this may be due to the insoluble nature of eudragit rs-100 which requires a time for media entry into the device and drug diffusion and doubling the windows surface area does not significantly affect the dissolution rate, and another cause is the position of the device, specifically the position of the windows towards the surface of the dissolution media, which affect media entry into the device.32 the addition of 10% ethyl cellulose in f4 instead of pvp k30 in f1 also results in extending the dissolution rate as it’s an insoluble polymer and has been used in the formulation of 418 j contemp med sci | vol. 8, no. 6, november-december 2022: 413–419 development of gastro-floating drug delivery system by 3d printing original n.m. abdulkhaleq et al. sustained-release dosage forms.33 dissolution profile of f4 in the four gastro-floating devices is shown in figure 5. f4a started the release of the drug after 2 hr from the start of the experiment and this is due to the position of the drug releasing window where it was above the surface of the dissolution media when placed in the dissolution jar at the start of the experiment and after 2 hr the device flipped and the entry of the dissolution media into the hollow section of the device started and thus tablet dissolution started, this fact is considered a disadvantage of this device design although it can be used for altering the release profile into sustained release but it may be associated with an unintended lag time. f4c and f4d also have similar dissolution profiles where the similarity factor (f2) was 64.7 as seen in f3. in conclusion, hme is considered a good option in the formulation of sustained release tablets with the ability to modify the release profile depending on the type of polymers used. fdm 3d printers are considered a potential tool to produce gastro-floating devices with the required design and release profile. although manipulation in the number and size of the drug releasing windows results in changing the release profile where decreasing the total surface area of the drug releasing windows revealed a significant reduction in the dissolution rate, but its not always predicted as either a lag time occurs as seen in device a or a relatively similar dissolution profile may occurs as seen in device b and c. increasing the number of drug releasing windows thus total surface area ensure suffient dissolution media entry into the hollow section of the device and tablet immersion while keeping the tablet floating and produce a more reliable dissolution profile that depends mainly on the composition and the type of the tablet inside the device. the introduction of gastro-floating devices into the pharmaceutical industry will make feasible of altering any tablet into a floating tablet. further in vivo investigations are recommended to study and efficacy of the 3d printed gastro-floating devices. acknowledgments we would like to thank the staff of the department of pharmaceutics/college of pharmacy/university of baghdad for their technical help in performing dsc and ftir tests. conflicts of interest disclosure the authors declare no conflicts of interest.  references 1. psimadas d, georgoulias p, valotassiou v, loudos g. molecular nanomedicine towards cancer : journal of pharmaceutical sciences. 2012;101(7):2271–80. 2. jamróz w, szafraniec j, kurek m, jachowicz r. 3d printing in pharmaceutical and medical applications – recent achievements and challenges. pharmaceutical research. 2018;35(9). 3. parhi r. a review of three-dimensional printing for pharmaceutical applications: quality control, risk assessment and future perspectives. journal of drug delivery science and technology. 2021;64(april):102571. 4. tan dk, maniruzzaman m, nokhodchi a. advanced pharmaceutical applications of hot-melt extrusion coupled with fused deposition modelling (fdm) 3d printing for personalised drug delivery. pharmaceutics. 2018;10(4):203. 5. kolakovic r, viitala t, ihalainen p, … sandler n. printing technologies in fabrication of drug delivery systems. expert opinion on drug delivery. 2013;10(12):1711–23. 6. goyanes a, robles martinez p, buanz a, basit aw, gaisford s. effect of geometry on drug release from 3d printed tablets. international journal of pharmaceutics. 2015;494(2):657–63. 7. maniruzzaman m, boateng js, snowden mj, douroumis d. a review of hot-melt extrusion: process technology to pharmaceutical products. isrn pharmaceutics. 2012;2012:1–9. 8. tiwari r, agarwal sk, murthy rsr, tiwari s. formulation and evaluation of sustained release extrudes prepared via novel hot melt extrusion technique. journal of pharmaceutical innovation. 2014;9(3):246–58. 9. qi s, craig dqm. hot melt extruded transdermal films based on amorphous solid dispersions in eudragit rs po : the inclusion of hydrophilic additives to develop moisture-activated release systems. international journal of pharmaceutics. 2016;514(1):270–81. 10. cossé a, könig c, lamprecht a, wagner kg. hot melt extrusion for sustained protein release: matrix erosion and in vitro release of plga-based implants. aaps pharmscitech. 2017;18(1):15–26. 11. li y, lu m, wu c. pvp va64 as a novel release-modifier for sustainedrelease mini-matrices prepared via hot melt extrusion. drug delivery and translational research. 2018;8(6):1670–8. 12. vanza jd, patel rb, dave rr, patel mr. polyethylene oxide and its controlled release properties in hydrophilic matrix tablets for oral administration. pharmaceutical development and technology. 2020;25(10):1169–87. 13. vengeliene v, takahashi tt, dravolina oa, … spanagel r. efficacy and side effects of baclofen and the novel gaba b receptor positive allosteric modulator cmppe in animal models for alcohol and cocaine addiction. psychopharmacology. 2018;235(7):1955–65. 14. jeong hm, weon ky, shin bs, shin s. 3d-printed gastroretentive sustained release drug delivery system by applying design of experiment approach. molecules. 2020;25(10). 15. tripathi j, thapa p, maharjan r, jeong sh. current state and future perspectives on gastroretentive drug delivery systems. pharmaceutics. 2019;11(4). 16. shin s, kim th, jeong sw, … shin bs. development of a gastroretentive delivery system for acyclovir by 3d printing technology and its in vivo pharmacokinetic evaluation in beagle dogs. plos one. 2019;14(5):1–17. 17. m. jaimini acr and yst. formulation and evaluation of famotidine floating tablets. current drug delivery. 2007;4:51–5. 18. charoenying t, patrojanasophon p, ngawhirunpat t, … opanasopit p. fabrication of floating capsule-in3d-printed devices as gastro-retentive delivery systems of amoxicillin. journal of drug delivery science and technology. 2020;55:101393. 19. fu j, yin h, yu x, … sheng f. combination of 3d printing technologies and compressed tablets for preparation of riboflavin floating tabletin-device ( tid) systems. international journal of pharmaceutics. 2018;549(1–2):370–9. 20. wuis ew, dirks mjm, vree tb, van der kleijn e. pharmacokinetics of baclofen in spastic patients receiving multiple oral doses. pharmaceutisch weekblad scientific edition. 1990;12(2):71–4. 21. ibrahim m, naguib yw, sarhan ha, abdelkader h. preformulationassisted design and characterization of modified release gastroretentive floating extrudates towards improved bioavailability and minimized side effects of baclofen. journal of pharmaceutical sciences. 2021;110(3):1227–39. 22. lu j, obara s, liu f, … kikuchi s. melt extrusion for a high melting point compound with improved solubility and sustained release. aaps pharmscitech. 2018;19(1):358–70. 23. anjali devi n, hadi ma, rajitha p, sharma jvc, srinivasa rao a. formulation and evaluation of floating controlled release tablets of imatinib mesylate using hydrophilic matrix system. international journal of pharmacy and pharmaceutical sciences. 2013;5(1):271–7. 24. ansari ka, vavia pr, trotta f, cavalli r. cyclodextrin-based nanosponges for delivery of resveratrol: in vitro characterisation, stability, cytotoxicity and permeation study. aaps pharmscitech. 2011;12(1):279–86. 25. lamichhane s, park jb, sohn dh, lee s. customized novel design of 3d printed pregabalin tablets for intra-gastric floating and controlled release using fused deposition modeling. pharmaceutics. 2019;11(11):1–14. 419j contemp med sci | vol. 8, no. 6, november-december 2022: 413–419 n.m. abdulkhaleq et al. original development of gastro-floating drug delivery system by 3d printing this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1304 26. abdelkader h, abdalla oy, salem h. formulation of controlled-release baclofen matrix tablets ii: influence of some hydrophobic excipients on the release rate and in vitro evaluation. aaps pharmscitech. 2008;9(2):675–83. 27. correa g, montero av. development of sustained release tablets containing solid dispersions of baclofen. journal of fundemental and applied science. 2013;5(2):220–39. 28. pereira gg, figueiredo s, fernandes ai, pinto jf. polymer selection for hotmelt extrusion coupled to fused deposition modelling in pharmaceutics. pharmaceutics. 2020;12(9):1–63. 29. farooq m, harris m, rabia s, yousuf i. development of extended release loxoprofen sodium multiparticulates using different hydrophobic polymers. polymer bulletin. 2018;(0123456789). 30. ali mam, sabati am, ali ba. formulation and evaluation of baclofen mucoadhesive buccal films. fabad journal of pharmaceutical sciences. 2017;42(3):179–90. 31. ibraheem fq, gawhri fjal. preparation and in-vitro evaluation of baclofen as an oral microsponge tablets. iraqi journal of pharmaceutical sciences. 2019;28(1):75–90. 32. dillen k, vandervoort j, mooter g van den, ludwig a. evaluation of ciprofloxacin-loaded eudragit ® rs100 or rl100 / plga nanoparticles. international journal of pharmaceutics. 2006;314(1):72–82. 33. partheniadis i, karantzalis ae, shah rr, al-zoubi n, nikolakakis i. influence of compression at elevated temperature on the compactibility of thermomechanically processed polymers. chemical engineering research and design. 2020;156:64–75. 337j contemp med sci | vol. 8, no. 5, september-october 2022: 337–342 original relation of serum and follicular level of bmp15 with oocyte quality, embryo grading and pregnancy rate zainab hassan hashim1*, lubna amer2, estabraq a. al-wasiti3 1department of clinical reproductive physiology, high institution for infertility diagnosis and assisted reproductive technologies, university of al-nahrain, baghdad, iraq. 2institution for infertility diagnosis and assisted reproductive technologies, university of al-nahrain, baghdad, iraq. 3department of biochemistry, college of medicine, university of al-nahrain, baghdad, iraq. *correspondence to: zainab hassan hashim (e-mail: zainab.hassan@ierit.nahrainuniv.edu.iq) (submitted: 03 march 2022 – revised version received: 21 march 2022 – accepted: 07 april 2022 – published online: 26 october 2022) abstract objectives: use of serum and follicular fluid concentration of oocyte secreted factors bmp15 as biomarkers of oocyte quality, embryo quality and it’s relation to pregnancy rate. methods: eighty eight women were included in this study; they were selected from those undergoing intra-cytoplasmic sperm injection. results: positive pregnancy was achieved by 14 women accounting for 19.0% (total number of women that reach embryo transfer was 72). no significant difference in mean serum bmp15 between pregnant and non-pregnant women, but the level of follicular fluid bmp15 was significantly higher in pregnant women. mi oocyte count was not significantly correlated to serum or follicular fluid bmp15 (p > 0.05). mii oocyte count showed highly significant positive correlation to serum and follicular fluid bmp15 (p < 0.01). grade 1 embryo count showed highly significant positive correlation to serum and follicular fluid bmp15 (p < 0.01), grade 2 embryo count showed significant positive correlation to serum bmp15 (p = 0.032), but the grade 2 embryo count showed non-significant correlation to follicular bmp15 (p > 0.05). also grade 3 embryo count showed non-significant correlation to serum and follicular bmp15 (p > 0.05). conclusion: the current study revealed that serum and follicular bmp15 could be used as indicator for oocyte maturity, and serum bmp15 could be used as indicator of good quality embryos. keywords: follicular level, bmp15, oocyte, embryo grading, pregnancy rate issn 2413-0516 introduction infertility is defined as the inability, of a couple to have pregnancy after a period of one year, in those women under 35 years of age or after 6 months in those women above 35 years of age, in spite of regular (3 to 4 times/week), adequate and unprotected sexual intercourse. the key limiting factor in female fertility is the oocyte quality, and till now there is poor understanding of what factors that determine the oocyte quality or the mechanisms that governing it.1,2 about 35% of infertility cases are caused by female factors, 35% related to male factors, 20% caused by both male and female factors, and 10% by unknown causes.3 the key limiting factor in female fertility is the oocyte quality; the quality of oocyte greatly affects early embryonic survival, also establishment with maintenance of pregnancy, development of the fetus, and even causes some adult diseases.1 bone morphogenetic protein 15 (bmp15) and growth differentiation factor 9 (gdf9), have a unique feature, within the transforming growth factors-b super-family is that the expression of the protein is essentially restricted to the gametes (oocyte). bmp15 and gdf9 are expressed in the oocyte during folliculogenesis, from the earliest stages.4 they are expressed in high levels by the oocyte throughout folliculogenesis, so they are could be regarded a good indicator for oocyte quality, and measuring them in the serum which is rapid, non-invasive and easy test could give a great clue to female fertility.5 this study aimed to use serum and follicular fluid concentration of oocyte secreted factors bmp15 as biomarkers of oocyte quality, embryo quality and then to study it’s relation with the pregnancy rate. materials and methods a prospective study was conducted in the high institute of infertility diagnosis and assisted reproductive technologies, al-nahrain university, from nov., 2020 to july 2021. one hundred and seventy six women were included in this study. the study subjects involved eighty eight women who were selected from those attended the high institute for infertility diagnosis and assisted reproductive technologies, they were enrolled in ivf program. inclusion criteria •    all couples undergoing ivf/icsi protocols. •    women at any age from 18 to 47 years old. •     infertility due to female factors: tubal blockage, unovulatory cycles, and mild-moderate cases of endometriosis that diagnosed laproscopically. •    couples with male factor infertility. •    unexplained infertility. exclusion criteria •     all  types  of  congenital  anomalies  of  the  reproductive  system. •    uncontrolled systemic and endocrine disorders. •    women with bmi more than 30 kg/m2 methods and study design a total of eighty eight patients undergoing ivf/icsi cycle were  evaluated: •     taking  full  obstetrical,  medical,  surgical  history  with  assessment of weight and height to obtain (bmi). mailto:zainab.hassan@ierit.nahrainuniv.edu.iq 338 j contemp med sci | vol. 8, no. 5, september-october 2022: 337–342 relation of serum and follicular level of bmp15 with oocyte quality, embryo grading and pregnancy rate original z.h. hashim et al. •     examinations of the woman clinically and gynecologically to check for any abnormality. •     for  male  partners,  the  seminal  fluid  analysis  was  assessed according to who 2010.  •     doing analysis of female hormones (lh, fsh, e2, prolactin, testosterone and tsh) at the second day of the  menstrual cycle. •     all women were enrolled to only one type of controlled  ovarian  hyperstimulation  (coh)  protocols  which  is  gonadotropin releasing hormone antagonist protocol. •     follow up of the patients by doing serial vaginal ultrasound and doing serum level of estradiol (e2) and then  accordingly to the result, ovum pick up done. •     oocyte  retrieval  done  with  guidance  of  trans-vaginal  ultrasound  after  ovulation  trigger  with  hcg  about  (35–36) hrs. •     at the day of ova pick up, serum and follicular fluid samples were obtained from each woman for measurement of bmp15. the antagonist protocol involved ovarian stimulation with gonadotropins since the second day of the menstrual cycle followed by the administration of a gnrh antagonist (cetrorelix  acetate for injection 0.25 mg: cetrotide®, merk, switzerland),  using flexible method and given when the size of the largest  follicles reach (13–14) mm. the initial dose of fsh was 75–  300 iu daily according to patient condition. with serial vaginal u/s for checking the number and size of ovarian follicles  and  for  the  endometrial  thickness  (et),  in  addition  serum  level of estradiol (e2) was done. the serum level of (e2) estradiol was measured at day of ovulation triggering by (hcg)  administration. the oocyte grading was at retrieval could be either immature oocyte and this is called germinal vesicle (gv), in which the corona and cumulus cells, are tightly packed around the oocyte, with presence of circular structure inside it, that is called the (germinal vesicle), the other immature oocyte is called metaphase i (mi). the mature oocyte is called metaphase ii (mii) which has polar body. mi oocyte characterized  by the absence of a polar body or a germinal vesicle, and it is intermediate stage between the gv and mii (mature) stages. icsi processes the aspirated follicles were examined at the ivf laboratory, in petri dish immediately. flushing was done then kept 1–2 hrs in the (37°c/ co2) incubator, all oocytes after that were subjected to denudation and grading in a laminar flow cabinet. the  mature eggs were selected by a specialized pipette, and by a  very delicate, sharp and hollow needle which is used to held, immobilize and then pick up a single sperm. after that, the  sperm was inserted by the needle carefully through egg shell into its cytoplasm. then the eggs were kept in the co2 incubator and carefully monitor the result of cell division, by using nikon icsi microscope. embryo quality and grading zygotes after insemination, were observed after (18–20) hours  to check for the presence of (2) pronuclei and after (25–29) hours to observe the presence of early cleavage, which is considered a sign of better implantation rates. the presence of 2 pronuclei at day 1, was regarded as a good prognostic sign. then at day two (43–45 hours after insemination) and day three  (67–69  hours  after  insemination)  the  embryos  were  evaluated. good quality embryos were considered when they were homogeneous, with normal kinetics (4) cells at day 2 and (7–9)  cells  at  day  3,  and  containing  <10%  of  cytoplasmic  fragments. the embryos at the third day, were classified as being with or without compaction, which referred to all embryos that underwent the compaction process, the embryos could be at the beginning of compaction when the fusion of the membrane was visible, in this stage the counting of the number of cells is still possible, and those embryos with full compaction, in those embryos the distinguishing of cell boundaries was not possible. embryo transfer: the dividing embryos were then replaced into the uterine cavity under pelvic ultrasound guidance and by an embryo transfer catheter. results the pregnancy rate in infertile women enrolled in the current study is shown in figure 1. positive pregnancy was achieved by 14 women accounting for 19.0%. total number of patient was 88,  a  number  of  cases were not  included  in  counting  pregnancy rate this included five cases of empty follicles, four cases of embryonic developmental arrest, six cases of failed fertilization and one patient refuse embryo transfer. so the number of  cases that were included in counting pregnancy rate was 72  patients. the characteristics of infertile women enrolled in this study are shown in table 1. the mean age of all enrolled women 32.25 ± 6.41 years and the mean age of women with positive pregnancy was significantly lower than that of non-pregnant  women  (29.14  ±  4.54)  years  versus  (32.76  ±  6.55) years, respectively (p = 0.050). the mean duration of infertility of all enrolled women was (7.89 ± 3.87) years and  the mean duration of infertility of pregnant women was lower than that of non-pregnant women (6.93 ± 3.08) years versus  (8.05 ± 3.98) years; however, the difference did not reach statistical significance (p = 0.319). out of all enrolled women, primary infertility was seen in 65 (74.0%) women, whereas,  secondary infertility was seen in 23 (26.0%) women and there was no significant difference in the frequency distribution of women according to type of infertility with respect to pregnancy outcome (p = 1.000). the mean bmi for pregnant women  was  (26.71  ±  2.60),  and  for  non-pregnant  women  (26.72 ± 3.01), there was no significant difference in the frequency distribution of women according to bmi with respect to pregnancy (p = 0.958). fig. 1 pie chart showing pregnancy rate of women undergoing icsi. 339j contemp med sci | vol. 8, no. 5, september-october 2022: 337–342 z.h. hashim et al. original relation of serum and follicular level of bmp15 with oocyte quality, embryo grading and pregnancy rate table 2. bmp15 serum and follicular levels and it’s relation to pregnancy rate characteristic total n = 72 positive pregnancy n = 14 negative pregnancy n = 58 p serum bmp15 mean ± sd 157.89 ± 51.08 169.79 ± 23.22 155.95 ± 54.13 0.350 i nsrange 14–296 130–213 14–296 follicular fluid bmp15 mean ± sd 149.64 ± 38.86 179.71 ± 29.44 144.74 ± 38.12 0.001 i hsrange 24–253 121–253 24–197 n, number of cases; sd, standard deviation; i, independent samples t-test; ns, not significant at p > 0.05; hs, highly significant at p ≤ 0.01. table 1. characteristics of infertile women enrolled in this study characteristic total n = 72 positive pregnancy n = 14 negative pregnancy n = 58 p age (years) mean ± sd 32.25 ± 6.41 29.14 ± 4.54 32.76 ± 6.55 0.050 i srange 20–47 23–40 20–47 duration of infertility (years) mean ± sd 7.89 ± 3.87 6.93 ± 3.08 8.05 ± 3.98 0.319 i nsrange 1–17 2–12 1–17 type of infertility primary, n (%) 65 (74.0%) 10 (71.4%) 55 (74%) 1.00 y nssecondary, n (%) 23 (26.0%) 4 (28.6%) 19 (26%) bmi (kg/m2) mean ± sd 26.72 ± 2.81 26.71 ± 2.60 26.72 ± 3.01 0.958 i nsrange 20.44–30.75 21.46–30.75 20.44–30.70 n, number of cases; sd, standard deviation; i, independent samples t-test; y, yates correction for continuity; ns, not significant at p > 0.05; s, significant at p ≤ 0.05. relation of pregnancy rate to serum and follicular level of bmp15 at day of ova pick up the bone morphogenetic protein 15 (bmp15) serum and follicular levels are shown in table 2. there was no significant difference in mean serum bmp15 (169.79  ±  23.22)  versus  (155.95  ±  54.13)  between  pregnant  and non-pregnant women. but the level of follicular fluid bmp15 was higher in pregnant women in compression with non-pregnant women in a highly significant manner (179.71 ±  29.44) versus (144.74 ± 38.12) (p = 0.001). receiver operating characteristic (roc) curve analysis to find the cutoff value of bmp15 that can predict a positive pregnancy outcome receiver  operating  characteristic  (roc)  curve  analysis  was  carried out to find the cutoff value of bmp15 that can predict a positive pregnancy outcome and the results are shown in figure 2 and table 3. the cutoff value of bmp15 was > 129 but with poor accuracy (56.6%) since the area under curve (auc)  was less than 0.7.  the correlations of serum and follicular fluid bmp15 to oocytes maturity the correlations of serum and follicular fluid bone morphogenetic protein 15 (bmp15) that measured at day of ova pick up to oocytes maturity are shown in table 4. mi oocyte count was not significantly correlated to serum (0.195) or follicular fluid bmp15 (–0.005). mii oocyte count showed highly significant positive  correlation  to  serum  (0.270)  and  follicular  fluid  bmp15 (0.413) (p < 0.01).  the correlations of serum and follicular fluid bmp15 to embryo grading the correlations of serum and follicular fluid bone morphogenetic protein 15 (bmp15) to embryo grading are shown in table 5. grade 1 embryo count showed highly significant positive correlation to serum (0.273) and follicular fluid (0.301)  bmp15 (p < 0.01), grade 2 embryo count showed significant  positive correlation to serum bmp15 (0.215) (p = 0.032). while grade 2 embryo count showed non-significant correlation to follicular bmp15 (0.133) (p > 0.05). also grade 3 embryo count showed non-significant correlation to serum (–0.099) and follicular (–0.043) bmp15 (p > 0.05). the correlations of serum bmp15 to perifollicular and endometrial blood flow doppler the resistive index (ri) and pulsatility index (pi) of perifollicular blood flow measured by pulsed doppler ultrasound at the day of ova pickup showed no significant correlation to serum bmp15, ri (0.312), pi (0.309) (p > 0.05). 340 j contemp med sci | vol. 8, no. 5, september-october 2022: 337–342 relation of serum and follicular level of bmp15 with oocyte quality, embryo grading and pregnancy rate original z.h. hashim et al. table 3. the results of receiver operating characteristic (roc) curve analysis to find the cutoff value of bmp15 that can predict a positive pregnancy outcome characteristic bmp15 cutoff >129 auc 0.566 95% ci 0.463 to 0.664 p-value 0.350 ns sensitivity % 100.0 specificity % 22.1 accuracy % 56.6 auc, area under curve; ci, confidence interval; ns, not significant. table 4. correlations of serum and follicular fluid bmp15 oocytes maturity characteristic correlation index serum bmp15 follicular fluid bmp15 immature metaphase i (mi) oocytes r 0.195 –0.005 p 0.052 0.962 mature metaphase ii (mii) oocytes r 0.270 0.413 p 0.007* <0.001* *, highly significant at p ≤ 0.01. table 5. the correlations of serum and follicular fluid bmp15 to embryo grading characteristic correlation index serum bmp15 follicular fluid bmp15 grade 1 embryo r 0.273 0.301 p 0.006** 0.002** grade 2 embryo r 0.215 0.133 p 0.032* 0.188 grade 3 embryo r –0.099 –0.043 p 0.089 0.114 *, significant at p ≤ 0.05; **, highly significant at p ≤ 0.01. fig. 2 receiver operating characteristic (roc) curve analysis to find the cutoff value of bmp15 that can predict a positive pregnancy outcome. also the resistive index (ri) pulsatility index (pi) of perifollicular blood flow measured by pulsed doppler ultrasound at the day of ova pickup showed non-significant correlation  to  follicular  bmp15,  ri  (–0.068),  pi  (–0.110)  (p > 0.05). furthermore the resistive index (ri), pulsatility index (pi) of endometrial blood flow measured by pulsed doppler ultrasound at the day of embryo transfer showed no significant correlation to serum bmp15, ri (–0.002), pi (0.202) (p > 0.05). also showed non-significant correlation to follicular bmp15, ri (–0.143), pi (0.034) (p > 0.05), as shown in table 6. discussion the main aim of this study was to find an easy, fast, not expensive and available outpatient test to be an indicator for female fertility. various studies regarding amh, fsh, lh, prolactin, e2  and other hormones beside genetic variants investigations have been performed and obtained as a good and specific biomarkers determined in last decade. all of these efforts were used to predict female reproductive potential and they used for different female sexual activities and are investigated with their receptors in different female sexual system disorders affected.6-10 these hormones are used to estimate growing follicles number in the ovary and to estimate and predict the response of ovaries to stimulation by gonadotropin. these biomarkers provide only an indirect evaluation of oocyte function and yield no information about quality of the oocyte, because they are not derived from the oocyte itself.11 bmp15 is known to be secreted only by the oocyte, essential for process of folliculogenesis, quality of the oocyte and female fertility, so these factors could be regarded as oocyte function biomarkers.12 positive pregnancy was achieved by 14 women accounting for 19.0%. the rate was low when compared with other studies like a study done by de geyter et al. that found that pregnancy rate was 28%,13 also other study done by jassim wh, et al.  found pregnancy rate to be 25.4%.14 the pregnancy rate was low because 4 cases of testicular biopsy, 2 cases of moderate endometriosis and also there were 14 case with age above 40 years were included in the this study, furthermore the sars-cov-2 (covid-19) pandemic also might be one of the  causes of decrease in pregnancy rate, this is supported by study result done by maya, w. d. c, et al., that found that germ cell  destruction and testicular damage was clearly observed in patient  with  covid-19,15 and the testes that infected with sars-cov-2-showed extensive peritubular fibrosis, vascular  congestion with extensive destruction of germ cell.16 furthermore,  sars-cov-2  could  cause  ovarian  tissue  damage  and  decline in the function of the ovary and oocyte quality, causing female infertility and may cause miscarriage.16 a number of cases were not included in counting pregnancy rate, this included five cases of empty follicles, four cases of embryonic developmental arrest, six cases of failed fertilization and one  patient refuse embryo transfer. so the number of cases that  were included in counting pregnancy rate was 72. 341j contemp med sci | vol. 8, no. 5, september-october 2022: 337–342 z.h. hashim et al. original relation of serum and follicular level of bmp15 with oocyte quality, embryo grading and pregnancy rate relation of pregnancy rate to serum and follicular fluid levels of bmp15 there was no significant difference in mean serum bmp15 between pregnant and non-pregnant women, but the level of follicular fluid bmp15 was higher in pregnant women in compression with non-pregnant women in a highly significant manner. but a study done by li et al., on gene expression found  that bmp15 mrna expression levels were closely associated with pregnancy outcomes.17 many factors might affect pregnancy rate other than oocyte quality like male factors for example abnormality in dna as in sperm retrieved by testicular biopsy, also due to bad endometrial receptivity and psychological problems. correlations of serum and follicular fluid bmp15 to oocyte maturity the current study showed that mi oocyte count were not significantly correlated to serum or follicular fluid bmp15 (p > 0.05) table 3. while mii oocyte count showed highly  significant positive correlation to serum and follicular fluid bmp15 (p < 0.01).  the result correspond to study result done by li et al. on  gene expression found that the mrna expression levels of bmp15 were closely related to maturation of the oocyte, fertilization  and  outcomes  of  the  pregnancy.17 furthermore a study done by others stated that a beneficial synergistic effects are exerted by osfs on the maturation of nucleus and cytoplasm, rapid energy utilization and oxidative stress management.18,19 bmp15 is secreted by the oocyte in a primary follicle,  which,  together  organize  the  granulosa  and  theca  cells that surround the oocyte into oocyte–cumulus–follicle complex. the granulosa at this time secretes amh, that affects the oocyte. throughout the development of the follicle, this autocrine–paracrine dialogue between the somatic cells and the oocyte continues and is regarded essential for establishing the fertilization potential and oocytes developmental competency.20 the correlations of serum and follicular fluid and bone morphogenetic protein 15 (bmp15) to embryo grading are shown in table 4. grade 1 embryo count showed highly significant positive correlation to serum and follicular fluid bmp15 (p < 0.01). grade 2 embryo count showed significant positive  correlation to serum bmp15 (p = 0.032). this results were supported by study results of canosa, s.,  et al., that found that the blastocyst group (bl) of embryos  showed faster kinetic in a significant manner, and the expression of bmp15 mrna was higher in ccs of this group with  significant value, as compared to arrested embryos.21 also supported by study done by daneshjou, d. et al., that found that there is positive correlation between the expression level of bmp15  mrna  with  the  fertilization  rate  and  grade  i  embryos.22 conclusion accordingly the observed data conclude that: 1.     serum and follicular bmp15 could be used as indicator  for oocyte maturity. 2.     serum  bmp15  could  be  used  as  indicator  of  grade  i  embryos. conflict of interest none.  table 6. the correlations of serum and follicular bmp15 to perifollicular and endometrial blood flow doppler characteristic correlation index perifollicular ri perifollicular pi endometrial ri endometrial pi serum bmp15 r 0.312 0.309 –0.002 0.202 p 0.207 0.211 0.989 0.217 follicular fluid bmp15 r –0.068 –0.110 –0.143 0.034 p 0.788 0.782 0.397 0.839 references 1. adhikari, d., lee, i. w., yuen, w. s., and carroll, j. (2022). oocyte mitochondria—key regulators of oocyte function and potential therapeutic targets for improving fertility. biology of reproduction, 106(2): 366–377. 2. mustafa, m., sharifa, a. m., hadi, j., iiizam, e., and aliya, s. (2019). male and female infertility: causes, and management. iosr journal of dental and medical sciences, 18: 27–32. 3. aghajanova, l., hoffman, j., mok-lin, e., and herndon, c. n. (2017). obstetrics and gynecology residency and fertility needs: national survey results. reproductive sciences, 24(3): 428–434. 4. sanfins, a., rodrigues, p. and albertini, d. f. (2018). gdf-9 and bmp-15 direct the follicle symphony. journal of assisted reproduction and genetics, 35(10): 1741–1750. 5. da broi, m. g., giorgi, v. s. i., wang, f., keefe, d. l., albertini, d. and navarro, p. a. (2018). influence of follicular fluid and cumulus cells on oocyte quality: clinical implications. journal of assisted reproduction and genetics, 35(5): 735–751. 6. al-tu’ma, f.j.; farhan, n. h. and al-safi, w.g. (2015) association between fat mass and obesity gene (re9939609) polymorphism with pcos women in iraqi population. human int. j. pharm. pharm. res., 5(1): 62–72. 7. hassan, mf. (2020) original research the frequency of elevated prolactin level in polycystic ovary syndrome women and it’s effect on pregnancy rate. global j. public health med., 2(1): 109–117. 8. al-lami, h.b.; al-tu’ma, f.j. and al-safi, w.g. (2020). association between anti-mullerian hormone and other biomarkers with ovarian function in polycystic ovarian syndrome of iraqi women, j. contem. med. sci, 6(4): 168–175. 9. wand, f.; dai, w.; yang, xh.; guo, yh. and sun, yp. (2016). analyses of optimal body mass index for infertile patients with either polycystic or nonpolycystic ovary syndrome during assisted reproductive treatment in china. sci. rep., 6(1): 1–9. 10. al-faris, n.n; al-tu’ma, f. j. and al-safi, w.g. (2017). assessment of crp and its correlation with total antioxidant capacity in women with polycystic ovarian syndrome. iraqi nat. j. of chem., 17(1): 44–57. 11. victoria, m., labrosse, j., krief, f., cédrin-durnerin, i., comtet, m. and grynberg, m. (2019). anti müllerian hormone: more than a biomarker of female reproductive functions. journal of gynecology obstetrics and human reproduction, 48(1): 19–24. 342 j contemp med sci | vol. 8, no. 5, september-october 2022: 337–342 relation of serum and follicular level of bmp15 with oocyte quality, embryo grading and pregnancy rate original z.h. hashim et al. 12. dayanir, d., ruso, h., kalem, z., gurgan, t. and ozogul, c. (2019). rewarding conversation between oocyte and cumulus cells directs the process of folliculogenesis. gazi medical journal, 30(4). 13. de geyter, c., calhaz-jorge, c., kupka, m. s., wyns, c., mocanu, e., motrenko, t. and goossens, v. (2018). art in europe, 2014: results generated from european registries by eshre: the european ivf-monitoring consortium (eim) for the european society of human reproduction and embryology (eshre). human reproduction, 33(9): 1586–1601. 14. jassim, w. h., al-obaidi, m. t. and ghazi, h. f. (2021). the effect of intrauterine infusion of peripheral blood mononuclear cells culture on subendometrial blood flow in patients undergoing icsi cycles. iraqi journal of embryos and infertility researches, 10(2): 53–72. 15. maya, w. d. c., du plessis, s. s. and velilla, p. a. (2020). sars-cov-2 and the testis: similarity with other viruses and routes of infection. reproductive biomedicine online, 40(6): 763–76. 16. duarte‐neto, a. n., teixeira, t. a., caldini, e. g., kanamura, c. t., gomes‐ gouvêa, m. s., dos santos, a. b., and hallak, j. (2022). testicular pathology in fatal covid‐19: a descriptive autopsy study. andrology, 10(1): 13–23. 17. li, j., li, c., liu, x., yang, j., zhang, q., han, w., and huang, g. (2022). gdf9 concentration in embryo culture medium is linked to human embryo quality and viability. journal of assisted reproduction and genetics, 39(1): 117–125. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 18. chandra, v. and sharma, g. t. (2020). in vitro strategies to enhance oocyte developmental competence. frontiers in bioscience-scholar, 12(1): 116–136. 19. romaguera, r.; morató, r.; jiménez-macedo, a.r.; catalá, m roura, m; paramio, m.t.; palomo, m.j.; mogas, t. and izquierdo, d. (2010). oocyte secreted factors improve embryo developmental competence of cocs from small follicles in prepubertal goats theriogenology, oct 1; 74(6): 1050–9. 20. michael, j. d., campanile, g., and baruselli, p. s. (2020). transforming growth factor-β superfamily and interferon-τ in ovarian function and embryo development in female cattle: review of biology and application. reproduction, fertility and development, 32(6): 539–552. 21. revelli, a., gennarelli, g., sestero, m., canosa, s., carosso, a., salvagno, f., and benedetto, c. (2020). a prospective randomized trial comparing corifollitropin-α late-start (day 4) versus standard administration (day 2) in expected poor, normal, and high responders undergoing controlled ovarian stimulation for ivf. journal of assisted reproduction and genetics, 37(5): 1163–1170. 22. daneshjou, d., mehranjani, m. s., zadehmodarres, s., shariatzadeh, s. m. a. and mofarahe, z. s. (2022). sitagliptin/metformin improves the fertilization rate and embryo quality in polycystic ovary syndrome patients through increasing the expression of gdf9 and bmp15: a new alternative to metformin (a randomized trial). journal of reproductive immunology, 150: 103499. https://doi.org/10.22317/jcms.v8i5.1283 https://pubmed.ncbi.nlm.nih.gov/?term=romaguera+r&cauthor_id=20542547 https://pubmed.ncbi.nlm.nih.gov/?term=morat%c3%b3+r&cauthor_id=20542547 https://pubmed.ncbi.nlm.nih.gov/?term=jim%c3%a9nez-macedo+ar&cauthor_id=20542547 https://pubmed.ncbi.nlm.nih.gov/?term=catal%c3%a1+m&cauthor_id=20542547 https://pubmed.ncbi.nlm.nih.gov/?term=roura+m&cauthor_id=20542547 https://pubmed.ncbi.nlm.nih.gov/?term=paramio+mt&cauthor_id=20542547 https://pubmed.ncbi.nlm.nih.gov/?term=palomo+mj&cauthor_id=20542547 https://pubmed.ncbi.nlm.nih.gov/?term=mogas+t&cauthor_id=20542547 https://pubmed.ncbi.nlm.nih.gov/?term=izquierdo+d&cauthor_id=20542547 133j contemp med sci | vol. 2, no. 8, autumn 2016: 133–137 research study effect of plant extraction for cuscuta europaea (dodder) against two species of bacteria staphylococcus aureus and escherichia coli jasim a. abdullah,* amer ali hammadi, russell hakem, zahra hatef and noor hussein issn 2413-0516 department of clinical laboratories, college of applied medical sciences, university of karbala, karbala, iraq. corresponding author: jasim a. abdullah (email: jasim.abdulabbas@gmail.com). (submitted: 28 july 2016 – revised version received: 22 august 2016 – accepted: 30 august 2016 – published online: 26 december 2016) objective: the goal of this study is focused on the effect of crude extract of cuscuta europaea plant and compared with amoxicillin drug, and then we showed these effects on staphylococcus aureus and escherichia coli. the analysis shows that the side effect of drug is high on human. we try to find the alternative antimicrobial to treat the bacterial infection. this study focuses on two treatments to inhibit the growth of two species of bacteria that are amoxicillin as drug and cuscuta eurooaea as extract until show the activity antimicrobial of these amount on bacteria. methods: the current study included cuscuta europaea as extract and amoxicillin as drug and two species of the bacteria staphylococcus aureus and escherichia coli. this study has been investigated to show the antimicrobial activity of plant extract and drug on the bacteria. results: during this study, the results reveal that the activity of antimicrobial of extract plant was higher than amoxicillin on staphylococcus aureus but the both amoxicillin and extract did not have any effect on escherichia coli in each concentration. and we showed the best result recorded in concentration 20 mg/ml of extract plant when compared with amoxicillin drug. conclusion: there are no significant differences between the concentration of plant extract of bacteria, and we showed the plant extract have a high effect on gram positive bacteria but do not have any effect on gram negative bacteria. keywords bacteria, amoxicillin, dodder, antimicrobial activity introduction pathogenic bacteria have been always considered as a major cause of morbidity and mortality in human. even though pharmaceutical companies have produced a number of new antibacterial drugs in the last year, the global emergence of multi-drug resistant bacteria is increasingly limiting the effectiveness of current drug and significantly causing treatment failure; due to the increase of resistance of antibiotics, there is a pressing need to develop new and innovative antimicrobial agent. the potential source of new agent plants that have long been investigated contain many bioactive compounds that can be of interest in therapeutic because of their low toxicity. amoxicillin this is a penicillin antibiotic. it is used to treat certain kinds of bacterial infection. it will not work for colds, flu, or other viral infection. this drug also comes in other forms including oral capsules, oral suspension and oral table used for bacterial infection such as some respiratory infection, infection of the urine, ear infection and dental abscesses. it works by killing the bacteria causing the infection, and sometimes used to get rid of helicobacter pylori. this is the bacterium believed to cause stomach ulcers and works by inhibiting the synthesis of the bacterial cell wall. this means that it stops any cross linkage that would normally occur between the linear peptidoglycan polymer chains that compose that large component of the cell wall.1 singular bacterium this microscopic single-cell (unicellular) life form that exists practically everywhere on the earth, and is simpler than the cells of animals, fungi, and plants. it is of about three million species of bacteria believed to exist, only about 4000 are known and are divided into general groups according to their shape.2 staphylococcus spp this is a genus of gram positive bacteria. under the microscope, they appear round (cocci) and form in grape-like cluster.2 the staphylococcus genus includes at least 40 species. nine have two subspecies, one has four subspecies.3 most are harmless and reside normally on the skin and mucous membranes of humans and other organisms, and found worldwide. they are a small component of soil microbial flora,4 and are the most co mmon cause of localized suppurating infection; pathogenic species include: staphylococcus aureus, staphylococcus epidermidis, staphylococcus saprophyticus. s. aureus strain carries on the skin, nose and pharynx as harmless commensal bacteria. strains of staphylococcus are known to enter through the breaks in the skin to cause localized infection or spread via blood to cause more generalized infection like that of the blood (sepsis), bone (osteomyelitis), brain (meningitis), lungs (pneumonia) etc., individuals with a compromised i mmune system are particularly vulnerable, etc.5 escherichia coli this is a gram-negative, facultative anaerobic, rod-shaped bacterium of the genus escherichia coli that is co mmonly found in the lower intestine of warm-blooded organisms endotherms,6 most e. coli strains are harmless, but some serotypes can cause serious food poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination.7,8 the harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin k2,9 and preventing colonization of the intestine with pathogenic bacteria.10,11 e. coli is expelled into the environment with fecal matter. the bacterium grows massively in the fresh fecal matter under aerobic condition for 3 days.12 medical plant cuscuta europaea roxb belongs to the family convolvulaceae (fig. 2). it is leafless green yellowish and thread-like twinning herb. it has a weak root under the ground but only grown as a 134 j contemp med sci | vol. 2, no. 8, autumn 2016: 133–137 study effect of plant extraction for cuscuta europaea (dodder) against two species of bacteria staphylococcus aureus research jasim a. abdullah et al. parasitic twin on other plants, hence, it is known as akaswel (sky twinner) or amarbel (immortal twin); in english it is known as dodder. the plant is bitter acrid and hence useful in aphrodisiac alternative and the bilious disorder (fig. 1). therefore, seeds are used as carminative purgative, and the juice of the plant is used as anthelmintics to purify the blood, however, fruit decoction is used in cough and fever and the stem is useful in constipation, flatulence, liver complaints.13 plant extraction plant remains the most important source of natural drug, more than 30% of prescription drugs are natural products, more than 60% of anticancer and anti-infective drugs are natural product.13 properties of good solvent in plant extraction · low toxicity · ease of evaporation at low heat · promotion of rapid physiologic absorption of the extract · preservative action · inability to cause the extract to complex or dissociate material and methods preparation of plant extract we sterilized the blender to destroy the infectants. a small amount of plant extract was added (40 g) in conical flask and then 140 ml of ethanol alcohol was added to the plant extract. the mixture was incubated in a shaker incubator for 24 hours to dissolve the plant in alcohol and the mixture was poured in many test tubes and put the tubes in a centrifuge to separate sediment from solution and removed the sediment. the separated plant extract was poured in glass petri dishes and incubated until it gets vapor. the extracted plant pours in glass petri dishes and incubate until vapor the alcohol and accumulate the extracted plant.16 the media was prepared according to the protocol given by the company and sterilized the media in an autoclave for 15 minutes in 121°c and 1 psi. dilutions preparation with e. coli and s. aureus we used four test tubes for each bacteria (e.g. s. aureus). 10 ml of nutrient broth was poured in first tube and 9 ml in remain tubes. from suspension bacteria, we took 1 ml and put to first tube and mix. we took 1 ml from the first tube and put in the second tube and we chose the second dilution. the concentration of drugs and extract of the plant were prepared as five different concentrations to and put 10 ml distilled water to each concentration (5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/l). the medium was poured in a petri dish and bacteria was streaked on medium (30 petri dish to e. coli and 30 petri dishes to s. aureus, 3 petri dishes to each concentration). we made holes on media. three holes in each petri dish and poured 50 µm from concentrations in the holes. the plates were incubated for 24 hours under 35–37°c. later, the results were recorded and calculated. we calculated zone of inhibition by ruler to drugs and to the plant extract. preparation of mic test our put (250 mg ) from extracted plant to nutrient broth and mix and prepare pure nutrient broth and we sterilize the media in autoclave in 121c for 15 minute after pouring the media in wells, and incubated the wells in incubator in 37°c for 24 hours and the results were recorded. note:used of ethanol because found easier to penetrate the cellular membrane to extract the intracellular ingredient (polyphenols) from the plant material. fig. 1 cuscuta europaea (dodder)13 (left) general morphology of dodder, (right) tendrils of dodder when coiled on stem of plant. fig. 2 plant classification. jasim a. abdullah et al. 135j contemp med sci | vol. 2, no. 8, autumn 2016: 133–137 research study effect of plant extraction for cuscuta europaea (dodder) against two species of bacteria staphylococcus aureus statistical analysis in statistical analysis, anova table was included, and we entered the data to excel program. the data include the average of inhibition zone and the number of isolate of bacteria and other data and we used an anova table to know the difference between the numbers.14 results and discussion the results show that the effect of plant extract on staphylococcus aureus as the best than amoxicillin drugs, and did not show any significant difference between the concentration of plant extract for bacteria. also the best result appeared in the concentration of plant extract is 20 mg/ml that is 25 ± 1.93, and follow 5, 10, 15, 25 mg/ml are 24.33 ± 3.2, 24.33 ± 1.9, 22 ± 2 and 20.5 ± 1.05, respectively, and we showed the effect of plant extract and drug on e. coli bacteria also (figs. 3, 4, 5, 6, 7, 8). the table 1. inhibition zone (mm) of plant extract on staphylococcus aureus and escherichia coli lsd 0.01 concentration inhibition diameter (mm) no. of bacteria concentration of plant extract (mg/ml)anti con amoxicillin 252015105(mg/ml) 7.715 20.5 ± 1.05 a25.0 ± 1.93 a22.0 ± 2.0 a24.33 ± 1.9 a24.33 ± 3.2 a17.33 ± 0.802 astaphylococcus aureus ------escherichia coli *the number refer to mean of inhibition diameter (mm) ± standard error. *homologous horizontally capital letters refer to no significant differences (p < 0.01) between the concentration of plant extract for bacteria. fig. 3 s. aureus plant conc. 20 mg/ml control drug conc. 20 mg/ml. fig. 4 s. aureus drug conc. 5 mg/ml plant conc. 5 mg/ml. results appeared effective on plant extract on gram positive bacteria and did not show an effect on gram negative bacteria, due to the increasing prevalence of antibiotic-resistant pathogens in the hospital and homes. the deliberate search is in progress for alternative treatment to combat the further spread of antibiotic resistant-pathogen. this result is shown in table 1. in some study, the effect of extract plant on gram negative bacteria show more antimicrobial activity than gram positive bacteria, under many concentrations, such as 50, 100, 150, 200 mg/ml. in staphylococcus aureus, the best inhibition zone appear in 200 mg/ml (9.5 mm) and the smallest inhibition zone in 50 mg/ml (7.1 mm), in e. coli, the best inhibition zone appears in 200 mg/ml (9.7 mm) and the smallest inhibition zone in 50 mg/ml (8.2 mm),15 therefore, researchers have been shown the effect of extract plant activity various from one area to another. the effect of extract plant in e. coli less than other species that found in the research,16 however, in the research, the methanol leaf extract of momordices charantia showing 136 j contemp med sci | vol. 2, no. 8, autumn 2016: 133–137 study effect of plant extraction for cuscuta europaea (dodder) against two species of bacteria staphylococcus aureus research jasim a. abdullah et al. fig. 5 s. aureus drug conc. 10 mg/ml, plant conc. 10 mg/ml. fig. 6 s. aureus drug conc. 15 mg/ml, plant conc. 15 mg/ml. fig. 7 s. aureus drug conc. 25 mg/ml, plant conc. 25 mg/ml. significant activity against s. aureus (40 mm) and e. coli (35 mm), leaf extract of ocimum exhiple high activity against e. coli (34 mm), the methanol leaf extract of acacia show significant activity against e. coli and s. aureus around (15 mm), dark extract of a acacianilotica exhibit high activity against s. aureus (15 mm) and sidacordifolia leaf extract possess maximum activity against s. aureus (18 mm) and roots extract of extract plant showed high inhibitory action against s. aureus and least activity in e. coli.17 as well as, it was noted worthy that the lowest concentration of the leaf extract of (50 mg/ml) p. niruri was found to be very effective in inhibiting the growth of all the selected strains of s. aureus (3 strains), whereas, p. niruri has no inhibitory effect on the e. coli even at 400 mg/ml, the fruit extracts of t. bellerica inhibited the growth of the s. aureus at jasim a. abdullah et al. 137j contemp med sci | vol. 2, no. 8, autumn 2016: 133–137 research study effect of plant extraction for cuscuta europaea (dodder) against two species of bacteria staphylococcus aureus (50 mg/ml) and exhibited growth inhibition of e. coli at (200 mg/ml) only.18 some study, the researcher is using many extracted plant of which c. arrensis; he was obtaining on result, when he used ethanol leave the inhibition zone is obtaining (8 mm) to e. coli and s. aureus (0 mm), and used aqueous leave; the inhibition zone of e. coli (3 mm) and s. aureus (0 mm) and when he used ethanol seed; the inhibition zone of e. coli (2 mm) and s. aureus (2 mm), also when he used aqueous seed; the inhibition zone of e. coli (1 mm) and s. aureus (2 mm).19 conclusion the cuscuta europaea extract plant have high antimicrobial activity on staphylococcus aureus, the grude extract consider fig. 8 e. coli plant conc. 5 mg/ml, drug conc. 5 mg/ml. references 1. brogden rn, heel rc, speight tm, avery gs. amoxicillin injectable: a review of its antibacterial spectrum, pharmacokinetics and therapeutic use. drugs. 1979;18:169–184. 2. ryan kj, ray cg. sherris medical microbiology (4th ed.). mcgraw hill. isbn 0-8385-8529-9, 2004. 3. harris lg, foster sj, richards sg. an introduction to staphylococcus aureus, and techniques for identifying and quantifying s. aureus adhesins in relation to adhesion to biomaterials: review. eur cells mat. 2002;4:39–60. 4. madigan m, martinko j. brock biology of microorganisms (11th ed.). prentice hall. isbn 0-13-144329-1, 2005. 5. http://www.health.state.mn.us/divs/idepc/disease laboratory-diagnosisfor-staphylococcus aureus/. 6. singleton p. bacteria in biology, biotechnology and medicine (5th ed.). wiley. 1999;444–454. isbn 0-471-98880-4 7. bruix j, raoul jl. infection of escherichia coli cdc national center for emerging and zoonotic infectious diseases. retrieved 2012-10-02. 8. vogt rl, dippold l. escherichia coli 0157:h7 outbreak associated with consumption of ground beef, june–july 2002. public health reports. 2005;120:174–178. 9. bentley r, meganathan r. biosynthesis of vitamin k (menaquinone) in bacteria. microbiol rev. 1982;46:241–280. best than drug, the effect of extract on gram positive bacteria best from gram negative bacteria, the best concentration of extract on s. aureus is 20 mg/ml. recommendation the plant extract can be tested against all microorganisms (bacteria, fungi, etc.) the active compound can be isolated and purified from the extract plant by applying various methodologies. conflict of interst none. n 10. hudault s, guignot j, servin al. escherichia coli strains colonizing the gastrointestinal tract protect germ free mice against salmonella typhimurium infection. gut. 2001;49:47–55. 11. reid g, howard j, gan bs. can bacterial interference prevent infection?. trends microbiol. 2001;9:424–428. 12. russell jb, jarvis gn. practical mechanisms for interrupting the oral–fecal lifecycle of escherichai coli. j mol microbiol biotechnol. 2001;3:265–272. 13. http://en.wikipedia.org/wiki/natural_products (accessed 3 march 2009). 14. geller nl. advances in clinical trial biostatistics marylwed. 3ed. usa. 66. 2004. 15. faiyyaz i, rajesh o, trushal c, kapil g. in vitro antimicrobial activity of cuscuta reflexa roxb. int res j pharm. 2011;2:214–216. 16. raza m, rahman a, wahab a, iqbal h, ullah, h, ahmed s, et al. comparative antibacterial study of convolvulus arvensis collected from different areas of khyber pakhtunkhwa, pakistan. j pharm. 2012;3:220–222. 17. govindarajan r, vijayakumar m, singh m, rao chv, shirwaikar a, rawat aks, et al. antiulcer and antimicrobial activity of anogeissus latifolia. j ethnopharmacol. 2006;106:57–61. 18. stary f, hans s. the national guide to medical herbs and plant. tiger books. int. plc. uk, 1998. 19. cheng h, qin zh, guo xf, hu xs, wu jh. geographical origin identification of propolis using gc-ms and electronic nose combined with principal component analysis. ann biol res. 2013;4(8):35. 141j contemp med sci | vol. 2, no. 8, autumn 2016: 141–147 research a study of eating habits among female nursing students in the university of babylon/iraq salma kj,a wafaa aa,b zainab ac issn 2413-0516 adepartment of community health nursing, college of nursing, university of babylon, iraq. bdepartment of maternal and child health nursing, college of nursing, university of babylon, iraq. cgraduate nurse, college of nursing, university of babylon, iraq. correspondence to salma kj (email: salmakadhhum972@gmail.com). (submitted: 6 september 2016 – revised version received: 20 september 2016 – accepted: 3 october 2016 – published online: 26 december 2016) objectives to assess socio demographic characteristics, the responses of eating habits among female nursing students and to determine the relationship between the socio demographic characteristics and the responses of eating habits. methods a descriptive analytic design was conducted on a purposive sample of 100 female nursing students in the university of babylon. a questionnaire has been used as a tool of data collection and consisting of socio demographic, the general responses, the responses related to dietary activity, behavioral responses of eating habits, data collected from the period of march 1 to june 20, 2016. results the results of the study revealed that 64.0% of women aged between 22 and 24 years with mean ± sd (52.46 ± 11.70), 61.0% of sample were found at grade 3 of study, 81.0% were single, 96.0% were home resident, 77.0% their original address in urban area, 86.0% study sample were not working. 56.0% were economic status satisfied to some extent. 67.0% were normal weight. 72.0% were non-dieting regimen. there is a significant relationship found between the demographical characteristics and responses related to dietary activity factors in like original address, marital status, and also between general responses and demographical characteristics, such as age groups, present bmi, and significant relationship between behavioral responses and demographical characteristics variables with occupation at p ≤ 0.05. conclusion the study recommends that family can encourage their daughters to choose the healthiest food collections and schools, universities will assist in minimizing the consumption of fast foods and others. keywords eating habits, female, nursing students introduction people who wish to be fit should adapt to the ideal habits and behave differently. gain knowledge of adopt and apply the habits, is the right way to achieve the success.1 residing in the university and college is potentially an important intention for the promotion of healthy lifestyles of the adult population. though information about the body mass index (bmi) distribution and nutritional and health related behaviors are still few, the majority of students having a desire to be thinner.2 students’, who join the university, dining plans are dealing daily with the food setting characterized by foods high in energy, fats, and added sugars, and low in nutrient density. this will put them in challenges what decide to eat beside having their food currently in an environment where no nutrition labeling is needed.3 dietary patterns developed during adolescence may contribute to obesity and eating disorders and may increase the risk for several important chronic diseases later in life.4 throughout a person’s life, certain events will occur which is of particular importance and is considered as a turning point in their lives. breakfast as an example is the most important meal in the dietary plan of an adolescent.5 adequate intake of animal and plant sources of protein is vital for adolescence. vitamins and minerals such as calcium, iron, and iodine must be included in the adolescents’ diet. best sources of vitamins are fruits and vegetables while milk and dairy products are the best sources of calcium.6 today, the foods of iraq reflect this rich inheritance as well as strong influences from the culinary traditions of turkey and iran and the greater syria area. because of all these traditions and complex influences, iraqi cuisine is enormously rich and varied.7 most asian countries have been shifting towards a diet higher in fat and meat, and lower in carbohydrates and fiber. additionally, decreased levels of physical activity and leisure are linked to increases in the prevalence of an overweight condition, obesity and diet-related non-communicable diseases, although the prevalence of students who were overweight was very low in this study sample.8 being tense and having fear of gaining weight or becoming fat even if at normal weight or underweight is another pattern of eating habits as one of the girls deal as well as unsuitable balance behavior to prevent weight gain such as self-induced vomiting; misuse of laxatives, diuretics, enemas or other medications; fasting; or excessive exercise.9 methodology design of the study a descriptive analytic study. sample of the study the probability (purposive sampling) was selected by a randomized system which consists of (100) female nursing students. setting of the study the data were collected from the period between march 1 and june 20, 2016 at the college of nursing, university of babylon. 142 j contemp med sci | vol. 2, no. 8, autumn 2016: 141–147 a study of eating habits among female nursing students research salma kj et al. instruments the questionnaire was constructed for the purpose of the study. the instruments consisted four parts as below: part 1: demographic date sheet: this part concerned with personal information include, the students age, grade, marital status, occupation, economic status, place of residence recently, original address, present body mass index, and dieting state at present. part 2: responses, 13 items as general responses. part 3: responses related to dietary activities, 12 items. part 4: behavioral responses, 6 items. these items are rated according to three level likert scale (always, sometimes, and never) and scored 3, 2, 1, respectively. the data were analyzed using the statistical package for social sciences (spss) version 19. through the application of descriptive statistical data analysis include (frequencies, percentages, and cumulative percent) and arithmetic mean with standard deviation, mean of score (m.s.) with their standard deviation (sd), cutoff point = 3 + 2 + 1/3 = 2 and inferential statistics, and relative sufficiency (r.s.%), and their assessment by cutoff point (66.67%) due to scores (1, 2, 3) which are reported pass and failure (under / upper), as well as reassessment scoring by (bad, moderate, and good) through the intervals (“33.33– 55.54”, “55.55–77.76”, and “77.77–100”), respectively. results table 1 shows the highest percentage of the sample reported at age ranged between 22 and 24 years, and they are accounted 64%, with mean age and standard deviation 22.01 ± 1.360. the greater number of them in third grade, and they are accounted 61%. the highest percentage of the sample place of residence recently was at home, and they are accounted (96%) while sample represents urban residency at 77%. the majority of participants are not working. they constitute (86%). the study revealed that 56% of their economic status was satisfied to some extent, the greater number of them with normal bmi, they are accounted 67%. 72% of the sample was not on dieting regimen. table 2 shows the highest percentage representing 40.0% of study samples answer sometimes “i am terrified about being overweight”. 47.0% of study samples were sometimes “avoid eating when hungry”, 41.1% of them were “always avoid soft drinks”, 47.0% of study participants were “sometimes cut food into small pieces”, 39.0% of study samples were “sometimes aware of the calorie content of food”, (36.0%) of study samples were “always avoid food with a high carbohydrate content” i.e. (bread, rice, potatoes, etc.), (77.0%) of students answer they “never vomit after eating”, (46.0%) of study subjects were “never guilty after eating”, ( 52.0%) of study samples were “sometimes think about burning up calories when exercising”, (48.0%) of population sample said “sometimes i take longer than others to eat my meals”, (45.0%) of study sample were “never eat diet foods”, (40.0%) of study samples were “sometimes feel that food controls their life”, (50.0%) of study samples were “sometimes l display self-control around food”, (41.0%) of study sample answer they “never feel that others pressure them to eat”. table 3 shows the highest percentage representing 40.0% of study sample answer sometimes i give too much time and thought to food, 47.0% of them were sometimes feel uncomfortable after eating sweets and table 1. distribution of demographical characteristics among female nursing students (n = 100) demographical variables no. and percents n percentage (%) cumulative percent age 19–21 33 33 33 22–24 64 64 97 25–27 3 3 100 mean ± sd 22.01 ± 1.360 grade year one 3 3.0 3 two 14 14.0 17 three 61 61.0 78 four 22 22.0 100 marital status single 81 81 81 married 17 17 88 divorce 2 2 100 place of residence recently home 96 96.0 96 hostel 4 4.0 100 original address rural 23 23.0 23 urban 77 77.0 100 occupation working 14 14.0 25.6 not working 86 86.0 100 economic status satisfied 40 40.0 40 satisfied to some extent 56 56.0 96 un satisfied 4 4.0 100 bmi underweight <18.5 5 5.0 5 normal 18.5–24.9 67 67.0 72 overweight 25–29.9 27 27.0 99 obese 30–39.9 1 1.0 100 dieting state present on dieting regimen 28 28.0 28 non-dieting regimen 72 100.0 100 sugar, 43.0% of study sample was never engage in dieting behavior, and never like stomach to be empty, 48.0% of study samples were sometimes enjoy trying new rich foods, 41.0% of study samples were sometimes skipped breakfast, majority of participants representing 53.0% were sometimes skipped lunch, 51.0% of study samples were sometimes consumed at least 1≤ serving daily of dairies, while more than half of them, 58.0% were sometimes consume one serving of meat and eggs daily, 49.0% of study sample sometimes consume at least one serving 1≤ serving of fruits and vegetables daily, same time 48.0% of girls were sometimes taking meals regularly, and 39.0% of study sample were sometimes taking snacking. table 4 deals with the behavioral responses, the results show that highest percentage represented 52.0% of study sample were never gone on eating binges where feeling that unable to stop, while majority of participants accounted 66.0% of study sample were never (behaving as sick vomited to control or shape, majority of girls represented 71.0% never (use laxatives, diet pills or diuretics to control their weight salma kj et al. 143j contemp med sci | vol. 2, no. 8, autumn 2016: 141–147 research a study of eating habits among female nursing students table 2. distribution of the general responses among female nursing students with comparison significant response of the sample scoring levels no. % χ2-test p-value (*) ms sd rs 1. i am terrified about being overweight never 25 25 1.9 0.772 1.90 .772 63.3 sometimes 40 40 always 35 35 2. avoid eating when i am hungry never 37 37 2.2 0.701 2.21 .701 73.6 sometimes 47 47 always 16 16 3. cut my food into small pieces never 37 37 2.1 0.780 2.09 .780 69.6 sometimes 47 47 always 16 16 4. i aware of the calorie content of foods that i eat never 35 35 2.1 0.814 2.06 .814 68.6 sometimes 39 39 always 26 26 5. i particularly avoid food with a high carbohydrate content (i.e. bread, rice, potatoes, etc.) never 36 36 2.3 0.809 2.25 .809 75.0 sometimes 34 34 always 30 30 6. i vomit after i have eaten never 77 77 2.7 0.529 2.73 .529 91.0 sometimes 19 19 always 4 4 7. i feel extremely guilty after eating never 46 46 2.3 .0709 2.32 .709 77.3 sometimes 40 40 always 14 14 8. i think about burning up calories when i exercise never 21 21 1.9 0.694 1.94 .694 64.6 sometimes 52 52 always 27 27 9. i take longer than others to eat my meals never 29 29 2.1 0.722 2.06 .722 68.6 sometimes 48 48 always 23 23 10. i eat diet foods never 45 45 2.3 0.770 2.25 .770 75.0 sometimes 35 35 always 20 20 11. i feel that food controls my life never 39 39 2.2 0.757 2.18 .757 72.6 sometimes 40 40 always 21 21 12. i display self-control around food never 18 18 1.9 0.697 1.86 .697 62.0 sometimes 50 50 always 32 32 13. i feel that others pressure me to eat never 41 41 2.2 0.792 2.17 . 792 72.3 sometimes 35 35 always 24 24 (*)hs: highly sig. at p < 0.01; s: sig. at p < 0.05; n s: not sig. at p > 0.05. the statistics based on the chi square test. χ2: chi square, p: probability, ms: mean of score, sd: standard deviation, rs: relative sufficiency. or shape), 64.0% of study sample were never exercised more than 60 minutes a day to lose or to control weight, 66.0% of them never lost 20 pounds or more in the past 6 months, 76.0% of study participants were never been treated for an eating disorder. table 5 summarizes of the subjects overall responses shows good assessment in regard to behavioral responses. regarding “responses” part results reported (table 6) no significant relationship with “demographical characteristics” variables except with (age groups and present bmi) significant relationship were obtained at p < 0.05. table 7 shows no significant relationship between responses related dietary activity element the with demographical characteristics variables except with (marital 144 j contemp med sci | vol. 2, no. 8, autumn 2016: 141–147 a study of eating habits among female nursing students research salma kj et al. table 3. distribution of the responses related to the dietary activity among female nursing students with comparison significant response related to dietary activity scoring levels no. % χ2-test p-value (*) ms sd rs 1. i give too much time and thought to food never 25 25 18.620 0.000 2.14 0.682 71.3 sometimes 40 40 always 35 35 2. i feel uncomfortable after eating sweets and sugar never 37 37 3.620 0.154 2.04 0.764 68.0 sometimes 47 47 always 16 16 3. i engage in dieting behavior never 43 43 10.820 0.004 2.25 0.744 75.0 sometimes 39 39 always 18 18 4. i like my stomach to be empty never 43 43 5.420 0.067 2.19 0.800 97.0 sometimes 33 33 always 24 24 5. i enjoy trying new rich foods never 9 9 27.020 0.000 1.66 0.639 55.3 sometimes 48 48 always 43 43 6. skipped breakfast never 29 29 2.660 0.264 1.99 0.722 66.6 sometimes 41 41 always 30 30 7. skipped lunch never 35 35 25.340 0.000 2.23 0.649 74.3 sometimes 53 53 always 12 12 8. consumed at least ( 1≤ serving) daily of dairies never 31 31 16.580 0.000 2.12 0.691 70.6 sometimes 51 51 always 18 18 9. consume one serving of meat and eggs daily never 25 25 4.460 0.108 2.08 0.646 69.3 sometimes 58 58 always 17 17 10. consume at least one serving (1 ≤ serving)of fruits and vegetables daily never 17 17 28.340 0.000 1.83 0.697 61.0 sometimes 49 49 always 34 34 11. taking meals regularly never 32 32 11.840 0.003 2.12 0.715 70.6 sometimes 48 48 always 20 20 12. taking snacking never 28 28 1.820 0.403 1.95 0.783 65.0 sometimes 39 39 always 33 33 (*)hs: highly sig. at p < 0.01; s: sig. at p < 0.05; ns: not sig. at p > 0.05. the statistics based on the chi square test. χ2: chi square, p: probability of chance, ms: mean of score, sd: standard deviation, rs: relative sufficiency. status and original address) highly significant correlation were obtained at p < 0.01. table 8 shows no significant relationship between behavioral part and the sociodemographic characteristics variables except with occupation significant correlation ships was obtained at p < 0.05. discussion the present study identified the eating habits among female nursing students. and aimed to study the eating habits in the context of avoid engaging in the negative eating behaviors. the age group revealed that students are in the middle ages, cultural, socio economic and some health attitudes play an important role in the development of eating habits. a study conducted in sudan by (elhassan) found that the age of most students was ranging from 19 to 24 years.10 the greater number of them in third grade. as the university students may need hostel, the results revealed that the majority are living at their homes, staying with the family may change the habits of students.11 this agree with the present study when they reported salma kj et al. 145j contemp med sci | vol. 2, no. 8, autumn 2016: 141–147 research a study of eating habits among female nursing students table 4. distribution of the studied sample behavioral responses among female nursing students with comparison significant behavioral responses scoring levels no. % χ2-test p-value (*) ms sd rs 1. gone on eating binges where you feel that you may not be able to stop never 52 52 20.540 0.000 2.37 0.734 79.0 sometimes 33 33 always 15 15 2. ever made yourself sick (vomited) to control your weight or shape never 66 66 51.860 0.000 2.57 0.655 85.6 sometimes 25 25 always 9 9 3. ever used laxatives, diet pills or diuretics (water pills) to control your weight or shape never 71 71 68.180 0.000 2.65 0.592 88.33 sometimes 23 23 always 6 6 4. exercised more than 60 minutes a day to lose or to control your weight never 64 64 50.960 0.000 2.58 .606 86.0 sometimes 30 30 always 6 6 5. lost 20 pounds or more in the past 6 months never 66 66 49.520 0.000 2.54 0.702 83.3 sometimes 22 22 always 12 12 6. have you ever been treated for an eating disorder never 76 76 84.061 0.000 2.66 0.673 88.6 sometimes 13 13 always 11 11 (*)hs: highly sig. at p < 0.01; s: sig. at p < 0.05; ns: not sig. at p > 0.05. the statistics based on the chi square test. χ2: chi square, p: probability of chance, ms: mean of score, sd: standard deviation, rs: relative sufficiency. table 5. summary statistics of eating habits among female nursing students dietary habits no. min. max. gms. rs. ass. general responses 100 1.8 2.7 2.25 71.81 mod. response related to dietary activity 100 1.66 2.25 1.95 70.3 mod. behavioral responses 100 2.37 2.66 2.515 85.1 good min.: minimum; max.: maximum; gms.: grand mean of score; rs.: relative sufficiency; ass.: assessment; no.: number. table 6. relationship between general responses and sociodemographic characteristics among 100 students relationships between general responses factors and demographical characteristics general responses c.c. sig. c.s. age groups 0.780 0.013 s grade 0.534 0.869 ns marital status 0.518 0.346 ns occupation 0.392 0.381 ns socio-economic status 0.518 0.346 ns place of residence recently 0.480 0.665 ns original address 0.445 0.102 ns present bmi 0.951 0.041 s dieting state now 0.400 0.324 sn (*)s: sig p < 0.05; ns: non sig. at p > 0.05; c.c.: contingency coefficients; c.s: comparison significant. table 7. relationship between responses related to dietary activity factors and demographical characteristics variables and among 100 students relationship between responses related to dietary activity factors and demographical characteristics variables responses related to dietary activity c.c. sig. c.s. age groups 0.635 0.969 ns grade 0.492 0.784 ns marital status 0.628 0.000 hs occupation 0.37 0.246 sn socio-economic status 0.381 0.911 ns place of residence recently 0.282 0.999 ns original address 0.473 0.007 hs present bmi 0.983 0.032 ns dieting state now 0.341 0.436 sn that a mean study career of 3.0 ± 1.0 years and mentioned that more than half of the participants living at parental home and consumed more fruit and vegetables than those who resided outside of their family home. our results further indicate that the subjects who are working while they study are only some, having a budget may help students improve their eating selections, at the same time, the study revealed that part of our elements are moderately satisfied economically, these findings were consistent with (reyes) who indicated that money becomes the overwhelming factor (with 49.1%) of the sample when looking at working and lowermiddle class students.12 the data showed that a majority of students would eat healthier if money was not a factor in deciding what to eat. with regard to bmi, greater number of them with the normal level, and not on dieting regimen at present time. a finding that is consistent with a study of (majors). the results show that on average nutrition and non-nutrition students where within a normal bmi.13 146 j contemp med sci | vol. 2, no. 8, autumn 2016: 141–147 a study of eating habits among female nursing students research salma kj et al. general responses this portion deals with eating responses in general, it is amazing to know that most of the items are considered as eating attitude and lifestyle practices and some can be as self-report to determine whether there is any eating disorder that needs professional attention. the current data show that girls demonstrated moderate to good reaction toward the eating attitudes, except with some of items that their answers show some fear from being overweight, think about burning up calories when exercise, show self-control around food and the effect of others on pattern of eating. these findings were consistent with (alavi) who found that dietary behaviors of the majority of participants per evaluation were on the medium level (41 percent) with the participants’ attitude in most cases was very positive.14 and agree with so many studies as (yahia) who illustrated that the university girls see the shape and weight of fashion models as the ideal body shape and figure to attain. girls with such strong body weight perception can be at risk of developing eating disorders.15 other portions like this study revealed what (musaiger) pointed out it is important to limit carbohydrate consumption, it is important to limit the amount of fat, about 42% and 81% of physicians and medical students, respectively, did not know the correct percentage of energy needed from fat, carbohydrates, and protein in a healthy diet.16 also consistent with (alakaam) marywood university (usa) who found that, most of the students (80%) said that meals are home cooked, occur mostly at homes on a daily basis, are varied, filling, and small in portion size, and take place at a specific time during the day. while disagreeing with the fact of guilt feeling when pointed out that several participants reported feelings of guilt in the us due to eating more meals and consuming unhealthy food.17 table 8. relationship between behavioral responses factors and demographical characteristics variables among 100 students relationship between behavioral responses factors and demographical characteristics variables behavioral responses c.c. sig. c.s. age groups 0.650 0.177 ns grade 0.399 0.873 ns marital status 0.320 0.875 ns occupation 0.415 0.014 s socio-economic status 0.142 0.306 ns place of residence recently 0.380 0.533 ns original address 0.220 0.825 ns present bmi 0.909 0.318 ns dieting state now 0.288 0.434 sn responses related to dietary activity the results of this table demonstrated that the sample activities mainly of acceptable level, only with regard to trying new rich foods and eating less than required fruits and vegetables daily18 agree with the current study girls less frequently consumed breakfast, fruits, milk, sugar-sweetened drinks, and energy drinks, but significantly more frequently consumed french fries/potato chips, cakes/donuts, and sweets/chocolates. while the study came with that some healthy dietary activities like students are not skipped breakfast or skipped lunch. (alavi) found that the 48.4% of the participants are not eating breakfast, 62.7% dairy products, and 27.7% meat and eggs.14 behavioral responses results further indicate that these latter determinants become more vital when the population sample demonstrated that all their eating behavior come pass and away from having an eating disorder, their assessment indicated that for example do not try to vomit after eating or using laxatives, diet pills or diuretics to control weight or shape and so on. the results are consistent with (payne) indicated that 20% of the study sample participated in binge eating, 21% fasted, 3% vomited and 2% admitted to laxative misuse, resulting in an overall eating disorder.19 with regard to physical activities, the sample show that sample less exercised more than 60 min a day to lose or to control their weight (musaiger) showed that the mean number of minutes performing physical activity per week was significantly higher among boys than girls.20 conclusion based on the findings of this study, it can be concluded that the overall responses of nursing students is moderate in general, and related to dietary activity it came good in behavioral assessment. however, it is found that there is a significant relationship between general responses. factors and demographical characteristics (age groups and present bmi) also have a high significant relationship between responses related to dietary activity. factors and marital status as well as original address, finally a significant relationship between behavioral responses and sample occupation was found. recommendations family can help grills in adopting healthy habits when selecting food collections, activating the educational programs as early as in adolescence about the importance of body image for females through giving health information to maintain healthy body. schools and universities have a good role in encouraging the students to make their meals as fresh as possible and minimizing the fast foods and others. motivate the population to stop imitating the others in unhealthy nutritional practices. conflict of interest none. n references 1. johnson c. 10 eating habits of the highly successful and fit: women’s health (2012). 2. sakamaki r, amamoto r, mochida y, shinfuku n, toyama k. a comparative study of food habits and body shape perception of university students in japan and korea. nutr j. 2005;4:31. 3. kolodinsky j, harvey-berino jr, berlin l, johnson rk, reynolds tw. knowledge of current dietary guidelines and food choice by college students: better eaters have higher knowledge of dietary guidance. j am diet assoc. 2007;107:1409–1413. 4. neumark-sztainer d, story m, hannan pj, croll j. overweight status and eating patterns among adolescents: where do youths stand in comparison with the healthy people 2010 objectives. am j pub health. 2002;92:844–851. 5. afaghi a, mohamadi ha, ziaee aa, sarchami r. effect of an integrated case-based nutrition curriculum on medical education at qazvin salma kj et al. 147j contemp med sci | vol. 2, no. 8, autumn 2016: 141–147 research a study of eating habits among female nursing students university of medical sciences, iran. glob j health sci. 2012; 4:112–117. 6. hallström l, vereecken ca, labayen i, ruiz jr, le donne c, garcía mc, et al. breakfast habits among european adolescents and their association with sociodemographic factors: the helena (healthy lifestyle in europe by nutrition in adolescence) study. public health nutr. 2012;15:1879–1889. 7. salloum h. foods of iraq: enshrined with a long history. things asian. 2006;1. 8. sakamaki r, toyama k, amamoto r, liu c, shinfuku n. nutritional knowledge, food habits and health attitude of chinese university students – a cross sectional study. nutrition j. 2005;4:4. 9. american psychiatric association. diagnostic and statistical manual of mental disorders, fourth edition, arlington. american psychiatric publishing, inc. 2000. 10. elhassan mr, gamal he, mohammed gs. nutrition knowledge attitude and practices among students of ahfad university for women. indian j sci res. 2013;4:25–34. 11. deliens t, clarys p, bourdeaudhuij id, deforche b. determinants of eating behaviour in university students: a qualitative study using focus group discussions. bmc public health. 2014;14:53. 12. reyes am. influences on college students’ eating habits university libraries. university of arizona. thesis. 2016;1–11. 13. majors mr. dietary habits and knowledge of college students. theses and dissertations—dietetics and human nutrition. 2015;3–33. 14. alavi m, eftekhari mb, noot r, rafinejad j, chinekesh a. dietary habits among adolescent girls and their association with parental educational levels. global j health sci. 2013;5:202–206. 15. yahia n, achkara, abdallah a, rizk s. eating habits and obesity among lebanese university students. nutr j. 2008;7:32. 16. musaiger a, al-hazzaa hm. eating habits, inactivity, and sedentary behavior among adolescents in iraq: sex differences in the hidden risks of noncommunicable diseases. food and nutrition bulletin. 2014;35(1):12–18. 17. alakaam aa, castellanos dc, bodzio j, harrison l. the factors that influence dietary habits among international students in the united states. journal of international students. 2015;5(2):104–120. 18. al-hazzaa hm, abahussain na, al-sobayel hi, qahwaji dm, musaiger ao. lifestyle factors associated with overweight and obesity among saudi adolescents. bmc public health. 2012;12:354. 19. payne kf. a comparative study of dietary habits among college students at-risk and not-at-risk for eating disorders and how such habits compare to the dietary guidelines, thesis. 2008;1–115. 20. musaiger ao, al-mufty ba, al-hazzaa hm. eating habits, inactivity, and sedentary behavior among adolescents in iraq: sex differences in the hidden risks of non communicable diseases. food nutr bull. 2014;35:12–18. 121j contemp med sci | vol. 9, no. 2, march-april 2023: 121–126 original physiological effects of calprotectin and b cell activating factor in covid-19 patients hazhar m. balaky1*, akam jasim mustafa2, parween abdulsamad ismail3, araz muhammad yousif4 1mergasor technical institute, erbil polytechnic university, erbil, iraq. 2department of chemistry, faculty of science, soran university, soran, kurdistan region, iraq. 3department of chemistry, college of education, university of salahaddin, erbil, iraq. 4basic science department, dentistry college, hawler medical university, erbil, iraq. *correspondence to: hazhar m. balaky (e-mail: hazharbalaky86@yahoo.com) (submitted: 04 january 2023 – revised version received: 27 january 2023 – accepted: 15 february 2023 – published online: 26 april 2023) abstract objectives: this study set out to determine how calprotectin and b cell activating factor contributes to early covid-19 patient severity prediction. methods: the study included 25 healthy controls and 52 patients with sars-cov2 infection who were clinically diagnosed with covid-19 illness and were between the ages of 23 and 35. the serum levels of calp and baff were measured using the elisa method. to gauge crp levels, an immunoturbidometric assay was performed. results: variations in serum levels of calp and baff were found to be statistically insignificant in the study (p = 0.7109 & p = 0.7575, respectively). when compared to the control group (103.95 ± 36.67 ng/ml; 403.03 ± 1.03), covid-19 patients had non-significantly raised levels of calp and baff (106.5 ± 4.67 ng/ml; 436.9 ± 12.77 pg/ml, respectively). according to roc curve analysis, the area under the receiver operating characteristics curve (auc) for calp and baff was (0.5170) and (0.5259), respectively. (r = 0.6923; p = 0.0001). there was a significant positive correlation between serum calp and baff levels. the connection between serum crp levels and calp (r = 0.3010; p = 0.1271) and baff levels (r = 0.2912; p = 0.1406) was insignificantly positive. conclusion: the current study’s findings suggested that serum calp and baff concentrations were increased in covid-19 patients, suggesting that these inflammatory markers may be helpful indicators of the severity of covid-19. keywords: calprotectin, b cell activating factor, c-reactive protein, covid-19 issn 2413-0516 introduction a pneumonia outbreak was reported in wuhan (hubei province, china) in december 2019 due to infection with a new coronavirus strain that causes the severe acute respiratory syndrome known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the coronavirus disease 19 (covid-19) pandemic caused by this virus has affected millions of individuals worldwide.1 covid-19 is associated with several clinical signs and symptoms frequently seen in autoimmune illnesses, including arthralgias, myalgias, exhaustion, sicca, and rashes.2 patients with covid-19 have also been noted to exhibit thrombosis, myositis, myocarditis, arthritis, encephalopathy, and vasculitis, in addition to these less typical autoimmune disease symptoms.3 these clinical data, along with the rising number of “recovered” patients referred to as “long haulers” or “long covid” who still exhibit post-covid-19 symptoms, raise the hypothesis that inflammation in response to sars-cov-2 infection increases tissue damage during the acute phase and has some longterm consequences.4 furthermore, severe covid-19 is more likely to occur in persons with underlying conditions such as diabetes, hypertension, cardiovascular disease, and lung disease, and the age-related case fatality rate increases significantly.5 since the virus’s introduction, it has been of utmost importance to comprehend immunity to the virus, the kinetics and protective function of the immune response in the community, and the level of exposure as measured by serosurveys.6 neutrophil activation signature calprotectin has become a useful biomarker during the first wave of the pandemic to assess covid-19 patient risk.7 the self-aggravating thrombo-inflammatory storm in people with severe covid-19 may be directly attributed to the neutrophil-related inflammatory marker termed calprotectin. silvin et al.8 revealed the relationship between elevated calprotectin levels and immature neutrophils and nonclassical monocytes. he also claimed that increased damage-related molecular pattern creation causes this relationship. in light of this, it has been postulated that calprotectin is a crucial mediator of the hyperinflammatory host response and the rise in inflammatory monocytes, neutrophils, and platelets that contribute to the particular coagulopathy in severe covid-19.1 additionally, doctors are adopting calprotectin increasingly frequently to aid in diagnosing and treating a range of different inflammatory illnesses due to its stability, assay repeatability, and inexpensive cost. the molecular functions of calp in health and unresolved inflammation are poorly understood by most clinicians.9 b-cell activating factor (baff), a member of the tumor necrosis factor (tnf) class, is expressed by macrophages, monocytes, dendritic cells, activated neutrophils, and stromal cells.10 first, it has been shown to be necessary for the creation of the humoral response as well as the growth and survival of b lymphocytes.11 recent research suggests that baff may also regulate innate immune responses, particularly at the level of the respiratory mucosa.12 the membrane-bound or soluble protein baff, which can induce autoimmune disorders in mice and humans,13 is synthesized excessively. the transmembrane activator and cyclophilin ligand interactor (taci), the b cell maturation antigen, and the baff receptor are the three known baff receptors (bma). all of these receptors are present in b and t lymphocytes as well as antigen-presenting cells, proving that baff action goes beyond b cell biology.14 mailto:hazharbalaky86@yahoo.com 122 j contemp med sci | vol. 9, no. 2, march-april 2023: 121–126 physiological effects of calprotectin and b cell activating factor in covid-19 patients original h.m. balaky et al. therefore, this case-control study investigates circulating baff and calprotectin levels, function, diagnostic value, and prognostic relevance in covid-19 infected patients. materials and methods the 77 participants in the present study included 52 confirmed covid-19 patients between june 2021 to november 2022 aged (23 to 35 years) and 25 healthy volunteers as the control group. after a covid-19 clinical diagnosis was made using rt-pcr test, blood samples were taken before the study group’s condition was treated. each participant’s blood sample was taken at a volume of 5 ml and centrifuged for 15 minutes at 3500 rpm. after that, the serum was frozen and kept at 70°c. the research purpose elisa kits were used to measure the levels of calprotectin (calp) and b-cell activating factor (baff) following the manufacturer’s instructions (sunlong biotech, china). the crp levels were measured by immunoturbidometric assay via a fully automated biochemistry analyzer. statistical analysis statistical analysis was performed using graphpad prism version 8 computer program. the unpaired t-test (man-whitney u) was used to compare the biochemical parameters between the study groups. roc curve analysis and spearman correlation analysis were also performed for biochemical parameters. all comparisons were deemed significant if the p-value was less than 0.05. results serum level of calprotectin figure 1 and table 1 revealed a non-significant elevation (p = 0.7109) in circulating concentration of serum calprotectin levels in covid-19 patients (106.5 ± 4.67 ng/ml) as compared to controls (103.9 ± 5.36 ng/ml). fig. 1 calprotectin levels between the sera of the studied groups. table 1. comparison of calp and baff levels among covid-19 patients and healthy controls parameters controls covid-19 patients p-value calprotectin (ng/ml) 103.9 ± 5.36 106.5 ± 4.67 0.7109 b-cell activating factor (pg/ml) 403.0 ± 31.03 436.9 ± 12.77 0.7575 the value expressed in mean ± se. fig. 2 b cell-activating factor (baff) levels in sera of the two studied groups. serum level of b cell activating factor the results in figure 2 and table 1 also showed that there were non-remarkable increase (p = 0.7575) in circulating levels of baff in covid-19 patients (436.9 ± 12.77 pg/ml) as com pared to healthy controls (403.0 ± 31.03 pg/ml). relationship between calp, baff, and crp in covid-19 patients correlation analysis assessed the relationships among serum crp levels, calp and baff levels. baff and serum calp levels were positively correlated (r = 0.6923; p = 0.0001) (figure 3). figure 3 also shows a non-significant correlation between serum crp levels and calp (r = 0.3010; p = 0.1271) and baff level (r = 0.2912; p = 0.1406) (figure 3). these research results may link baff activation and the immuno-inflammatory and pathogenic response, indicating disease activity and tissue damage in various chronic viral infection illness states in covid-19. roc curve based on the (receiver operating characteristic) roc curve, the area under the curve (auc) of serum calp and serum baff were (0.5170) and (0.5259) respectively (figure 4). discussion serum level of calprotectin numerous recent studies have discovered that patients with coronavirus disease-19 illness had higher calprotectin levels.1,7 123j contemp med sci | vol. 9, no. 2, march-april 2023: 121–126 h.m. balaky et al. original physiological effects of calprotectin and b cell activating factor in covid-19 patients additionally, these investigations have demonstrated that calprotectin can predict the need for mechanical breathing, identify death, and differentiate between mild and severe illness states.8,13 calprotectin is widely distributed in neutrophils, making up around two-thirds of the cytosol’s soluble protein composition. the strong relationship between serum calprotectin levels and coronavirus’s present and potential severity indicates that neutrophils are implicated as active promoters of inflammation and respiratory impairment in coronavirus disease. the patients who needed mechanical ventilation while in the hospital also had much greater levels of calprotectin. this data shows a direct link between the severe acute respiratory syndrome caused by covid-19, elevated serum calprotectin levels, and neutrophil activation.13 two studies from medical schools in michigan, shanghai, and washington dc, on the role of calprotectin as an early indicator of neutrophil activation in covid-19 disease. the levels of calprotectin were noticeably elevated in hospitalized individuals with coronavirus illness. calprotectin levels also correlate with the severity of respiratory failure and the demand for mechanical ventilation. this favours using calprotectin as a biomarker to estimate the severity of a condition and the likelihood that it will result in mortality. higher levels of calprotectin were also associated with an increased risk of dying from thrombotic issues.13,15 according to fig. 3 correlation of serum calp with baff (a), serum crp with calp (b), serum crp with baff (c). fig. 4 receiver operating characteristic (roc) curve analysis of serum calp levels (a) and baff levels (b). recently released research in the academic journal cell, calprotectin may be able to differentiate between severe and mild covid-19 disease. the circulating biomarker most obviously elevated in patients with advanced illness was calprotectin. this study offers future therapeutic strategies explicitly targeting calprotectin to treat the severe form of covid-19 and shows its potential use in the prognosis of severe disease.8 similar to the current study, two inflammation-related biomarkers, growth differentiation factor-15 and calprotectin, have been researched for their potential role in predicting mortality and disease severity in sars-cov-2 infected patients. the study results show that calprotectin levels are markedly higher in people with covid-19, and they suggest that calprotectin may help assess the severity of the illness and forecast in-hospital mortality.1 calprotectin overexpression may be a direct source of the self-amplifying thrombo-inflammatory storm observed in patients with severe covid-19. silvin et al.8 investigation discovered a connection between elevated calprotectin levels and immature neutrophils and nonclassical monocytes, supporting their hypothesis that this correlation originates from the excessive production of damage-associated molecular patterns. due to the host’s hyperinflammatory response and the rise in inflammatory monocytes, neutrophils, and platelets, it has been proposed that calprotectin is a crucial mediator of specific coagulopathy in severe covid-19 patients.16 according 124 j contemp med sci | vol. 9, no. 2, march-april 2023: 121–126 physiological effects of calprotectin and b cell activating factor in covid-19 patients original h.m. balaky et al. and b lymphocytes, are able to produce baff.39,40 interleukin-10-producing b cells were enriched and more frequent in chronic hepatitis b patients during hepatic flare-ups, according to a study by das et al.32 a different study showed that individuals with chronic hepatitis b have frequently activated b cells, with an abnormally high proportion of these individuals’ b cells exhibiting activation markers. this demonstrates that this subset of b cells may control t-cell immunity in chronic hepatitis.31 the intrinsic b-cell activation molecule fc receptor like1, as well as the b-cell activation markers cd69 and cd86, were also shown to be present in increased amounts in acute and chronic hepatitis b patients.33 these results indicate that b cells are essential for the progression of hbv infection, as well as hbv viral antigens and their interactions with t lymphocytes. additionally, it was thought that baff had a negative impact on the microenvironments of solid and hematological malignancies. so far, it has been demonstrated that baff encourages invasive migration in hypoxic breast cancer cell lines.41 blood baff levels, generated by neutrophils, have been linked to oral cavity cancers.42 it has been observed that the prognosis and circulating baff levels in multiple myeloma are related.43 according to koizumi et al. report.44 it was found that baff promoted tumor invasion and dissemination in human pancreatic cancer cases. however, no research has yet been done to determine how baff contributes to the growth of hepatocellular carcinoma. therefore, based on the available literature data and the results of the present study, serum b cell activating factor contributes to early patient severity of covid-19 and may be a good predictive marker for the disease prognosis. diagnostic performance of serum calp and baff in covid-19 patients due to the non-significantly increased blood levels of calp and baff found in covid-19 patients in the present study, the ability of blood calp and baff to predict covid-19 was assessed using the roc curves. according to a presentation by chen et al.,18 extremely high levels of calprotectin were connected to the poor overall survival of covid-19 patients, supporting the predictability of calprotectin revealed by earlier investigations. recent studies by shi et al.13 and zuo et al.,15 and others13,15 demonstrated high serum calprotectin is closely connected to the likelihood of dying in covid-19, usually from thrombotic problems, which lends credence to the verdicts of the current study. calprotectin is said to have a higher predictive accuracy when compared to the covid-gram risk score created for prediction by liang et al.45 and chen et al.18 calprotectin was thought to have the highest prediction accuracy of all the predictors, according to chen et al.18 they examined the receiver operating characteristic curve (roc) analysis of calprotectin, hmgb1, covid-gram risk score, and calprotectin/hmgb1 combo for predicting icu admission and possible mortality to illustrate results similar to the present investigation. conclusion this study is the first to demonstrate that covid-19 patients had greater serum levels of the potent inflammatory markers calp, baff, and crp. the clinical illness condition of to a recent investigation, people with confirmed sars-cov-2 infection exhibited increased plasma calprotectin levels compared to suspected patients with negative rt-pcr.17 numerous researchers have confirmed the role of calprotectin in evaluating illness severity, including studies by chen et al.,18 kaya et al.,19 garcia de guadiana-romualdo et al.,1 mahler et al.,20 and bauer et al.21 neutrophils are crucial to the immunopathology of covid-19, according to a recent study by tomar et al.22 as a result, it has been discovered that measuring blood calprotectin levels is a trustworthy indication of covid-19 severity. studies proved that neutrophils mainly secrete calprotectin in response to inflammation. recently, it was shown that calprotectin is a biomarker of inflammation that can be used to track the progression of numerous inflammatory diseases.23–28 limited research studies look at the connection between the severity and predicator value of serum calprotectin in covid-19. patients brought to the icu had significantly higher serum levels of calprotectin than non-icu patients, and those who died had much higher levels, according to chen et al.,18 who conducted a study with 121 covid-19 patients (41 icu, 81 non-icu). additionally, that study discovered a correlation between higher mortality in covid-19 patients with a substantial rise in serum calprotectin. in their trial with 94 covid-19 patients, shi et al. study found that patients needing mechanical breathing had higher serum calprotectin levels than those not.13 based on the available literature and the present study results, we can conclude that serum calprotectin may be employed as a predictive biomarker for covid-19 disease severity and prognosis. serum level of b cell activating factor the greater b-cell activation in covid-19 patients may be responsible for the many autoantibodies and immune complexes frequently found in these individuals’ blood. our research offered the first conclusive evidence that circulating baff levels in covid-19 patients were higher than in healthy controls. these findings suggest that baff participates in persistent viral infection and aids in disease progression. specifically, baff impacts b cell development, maturation, survival, and activation.29,30 studies have shown that the process of viral infection caused by covid-19 and others induces contact between t cells and activated b cells with t cells.31-33 moreover, it has been shown that viral infections, including infections with the respiratory syncytial virus, might cause baff release.34,35 consequently, even though the fundamental mechanisms may change, it is believed that baff is frequently produced by viral infection.36 according to previous research, our findings provide new evidence that covid-19 infection may induce the biosynthesis and release of baff, which may affect how b cells react to viral infection. the tumor necrosis factor family member b-cell activating factor (baff), also known as b lymphocyte stimulator (blys) or b cell activating factor, has a unique role in regulating peripheral b-cell survival, homeostasis, and the antibody response.29,37 its believed that baff could reduce apoptosis and essentially enhances t cell-independent and t cell-dependent humoral immune responses. the study led by sutherland et al.38 found that baff increased tand b-cell responses, particularly th1-type responses. numerous cells, particularly those connected to the immuno-inflammatory response, such as monocytes, macrophages, neutrophils, dendritic cells (dcs), t lymphocytes, 125j contemp med sci | vol. 9, no. 2, march-april 2023: 121–126 h.m. balaky et al. original physiological effects of calprotectin and b cell activating factor in covid-19 patients covid-19 and increased baff levels are correlated. the sars-cov-2 infection risk and severity correlated with serum calp and baff levels. the study results suggest a connection between serum baff levels and the manifestation of covid-19, and assessing serum baff levels may be used as an inflammatory biomarker to identify and classify clinical problems related to covid-19. according to the available evidence, calprotectin may be a valuable inflammatory biomarker to evaluate the risk and severity of covid-19. the relationship between calprotectin and the inflammatory process may present fresh opportunities for managing and improving covid-19. the combined use of serum calp, baff and crp levels may be helpful to evaluate and understand how inflammation-related factors such as viral load, sars-cov2 antibodies, corticosteroid use, anticoagulants, and pharmaceutical agents would affect neutrophil function. a comprehensive prospective investigation is required to comprehend the progression of the covid-19 infection, the potential role of blood baff levels in determining and monitoring the condition’s prognosis, and the treatment response to immunomodulatory medicine. additional study is also required to completely comprehend the roles played by baff in the emergence of covid-19-associated diseases and the development of the infection. conflict of interest all authors declare that they have no conflicts of interest.  references 1. garcía de guadiana-romualdo, l. et al. circulating levels of calprotectin, a signature of neutrophil activation in prediction of severe respiratory failure in covid-19 patients: a multicenter, prospective study (calcov study). inflammation research 71, 57–67 (2022). 2. guan, w.-j. et al. clinical characteristics of coronavirus disease 2019 in china. new england journal of medicine 382, 1708–1720 (2020). 3. machhi, j. et al. the natural history, pathobiology, and clinical manifestations of sars-cov-2 infections. journal of neuroimmune pharmacology 15, 359–386 (2020). 4. chang, s. e. et al. new-onset igg autoantibodies in hospitalized patients with covid-19. nature communications 12, 1–15 (2021). 5. van kampen, j. j. et al. duration and key determinants of infectious virus shedding in hospitalized patients with coronavirus disease-2019 (covid-19). nature communications 12, 1–6 (2021). 6. ortega, n. et al. seven-month kinetics of sars-cov-2 antibodies and role of pre-existing antibodies to human coronaviruses. nature communications 12, 1–10 (2021). 7. udeh, r., advani, s., de guadiana romualdo, l. g. & dolja-gore, x. calprotectin, an emerging biomarker of interest in covid-19: a systematic review and meta-analysis. journal of clinical medicine 10, 775 (2021). 8. silvin, a. et al. elevated calprotectin and abnormal myeloid cell subsets discriminate severe from mild covid-19. cell 182, 1401–1418. e1418 (2020). 9. jukic, a., bakiri, l., wagner, e. f., tilg, h. & adolph, t. e. calprotectin: from biomarker to biological function. gut 70, 1978–1988 (2021). 10. wang, l. et al. b cell activating factor regulates periodontitis development by suppressing inflammatory responses in macrophages. bmc oral health 21, 1–15 (2021). 11. yang, s.-c., tsai, y.-f., pan, y.-l. & hwang, t.-l. understanding the role of neutrophils in acute respiratory distress syndrome. biomedical journal 44, 439–446 (2021). 12. veras, f. p. et al. sars-cov-2–triggered neutrophil extracellular traps mediate covid-19 pathology. journal of experimental medicine 217 (2020). 13. shi, h. et al. neutrophil calprotectin identifies severe pulmonary disease in covid‐19. journal of leukocyte biology 109, 67–72 (2021). 14. nascimento, m. et al. b-cell activating factor secreted by neutrophils is a critical player in lung inflammation to cigarette smoke exposure. frontiers in immunology 11, 1622 (2020). 15. zuo, y. et al. neutrophil extracellular traps in covid-19. jci insight 5 (2020). 16. hanssen, n. m., spaetgens, b., nagareddy, p. r. & murphy, a. j. dam pening mortality in covid-19: therapeutic insights from basic cardiometabolic studies on s100a8/a9. circulation 143, 971–973 (2021). 17. cherubini, f., cristiano, a., valentini, a., bernardini, s. & nuccetelli, m. circulating calprotectin as a supporting inflammatory marker in discriminating sars-cov-2 infection: an observational study. inflammation research 70, 687–694 (2021). 18. chen, l. et al. elevated serum levels of s100a8/a9 and hmgb1 at hospital admission are correlated with inferior clinical outcomes in covid-19 patients. cellular & molecular immunology 17, 992–994 (2020). 19. kaya, t. et al. serum calprotectin as a novel biomarker for severity of covid-19 disease. irish journal of medical science (1971-) 191, 59–64 (2022). 20. mahler, m., meroni, p.-l., infantino, m., buhler, k. a. & fritzler, m. j. circulating calprotectin as a biomarker of covid-19 severity. expert review of clinical immunology 17, 431–443 (2021). 21. bauer, w. et al. outcome prediction by serum calprotectin in patients with covid-19 in the emergency department. journal of infection 82, 84–123 (2021). 22. tomar, b., anders, h.-j., desai, j. & mulay, s. r. neutrophils and neutrophil extracellular traps drive necroinflammation in covid-19. cells 9, 1383 (2020). 23. romand, x. et al. systemic calprotectin and chronic inflammatory rheumatic diseases. joint bone spine 86, 691–698 (2019). 24. wirtz, t. h. et al. association of serum calprotectin concentrations with mortality in critically ill and septic patients. diagnostics 10, 990 (2020). 25. kunutsor, s. k. et al. plasma calprotectin and risk of cardiovascular disease: findings from the prevend prospective cohort study. atherosclerosis 275, 205–213 (2018). 26. wang, q., chen, w. & lin, j. the role of calprotectin in rheumatoid arthritis. journal of translational internal medicine 7, 126–131 (2019). 27. yurtsever kum, n. et al. elevated serum calprotectin as an inflammatory marker in obstructive sleep apnea. cranio®, 1–7 (2020). 28. candar, t., baklacı, d., kuzucu, i̇. & kayabaşı, s. a proinflammatory marker in chronic rhinosinusitis: serum calprotectin. acta biochimica polonica (2020). 29. do, r. k. g. & chen-kiang, s. mechanism of blys action in b cell immunity. cytokine & growth factor reviews 13, 19–25 (2002). 30. schneider, p. & tschopp, j. baff and the regulation of b cell survival. immunology letters 88, 57–62 (2003). 31. oliviero, b. et al. enhanced b-cell differentiation and reduced proliferative capacity in chronic hepatitis c and chronic hepatitis b virus infections. journal of hepatology 55, 53–60 (2011). 32. das, a. et al. il-10–producing regulatory b cells in the pathogenesis of chronic hepatitis b virus infection. the journal of immunology 189, 3925–3935 (2012). 33. wang, k. et al. overexpression of fc receptor-like 1 associated with b-cell activation during hepatitis b virus infection. brazilian journal of medical and biological research 45, 1112–1118 (2012). 34. toubi, e. et al. elevated serum b-lymphocyte activating factor (baff) in chronic hepatitis c virus infection: association with autoimmunity. journal of autoimmunity 27, 134–139 (2006). 35. mcnamara, p. et al. respiratory syncytial virus infection of airway epithelial cells, in vivo and in vitro, supports pulmonary antibody responses by inducing expression of the b cell differentiation factor baff. thorax 68, 76–81 (2013). 36. ittah, m. et al. induction of b cell-activating factor by viral infection is a general phenomenon, but the types of viruses and mechanisms depend on cell type. journal of innate immunity 3, 200–207 (2011). 37. rahman, z. s. & manser, t. b cells expressing bcl-2 and a signaling-impaired baff-specific receptor fail to mature and are deficient in the formation of lymphoid follicles and germinal centers. the journal of immunology 173, 6179–6188 (2004). 38. sutherland, a. p. et al. baff augments certain th1-associated inflammatory responses. the journal of immunology 174, 5537–5544 (2005). 39. nardelli, b. et al. synthesis and release of b-lymphocyte stimulator from myeloid cells. blood, the journal of the american society of hematology 97, 198–204 (2001). 126 j contemp med sci | vol. 9, no. 2, march-april 2023: 121–126 physiological effects of calprotectin and b cell activating factor in covid-19 patients original h.m. balaky et al. 40. scapini, p. et al. g-csf–stimulated neutrophils are a prominent source of functional blys. the journal of experimental medicine 197, 297–302 (2003). 41. zhu, j. et al. blys is up-regulated by hypoxia and promotes migration of human breast cancer cells. journal of experimental & clinical cancer research 31, 1–7 (2012). 42. jablonska, e. et al. overexpression of b cell-activating factor (baff) in neutrophils of oral cavity cancer patients—preliminary study. neoplasma 58, 211 (2011). 43. fragioudaki, m. et al. serum baff levels are related to angiogenesis and prognosis in patients with multiple myeloma. leukemia research 36, 1004–1008 (2012). 44. koizumi, m. et al. increased b cell-activating factor promotes tumor invasion and metastasis in human pancreatic cancer. plos one 8, e71367 (2013). 45. liang, w. et al. development and validation of a clinical risk score to predict the occurrence of critical illness in hospitalized patients with covid-19. jama internal medicine 180, 1081–1089 (2020). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1327 216 j contemp med sci | vol. 8, no. 4, july-august 2022: 216–219 original knowledge and practice of physicians about vitamin b12 deficiency among type 2 diabetes mellitus patients in buraidah, saudi arabia fahad alruamaykhani*, unaib rabbani family medicine academy, qassim health cluster, buraidah, saudi arabia. *correspondence to: fahad alruamaykhani (e-mail: fahad.r.m.k@gmail.com) (submitted: 13 april 2022 – revised version received: 08 may 2022 – accepted: 25 may 2022 – published online: 26 august 2022) abstract objectives: to assess knowledge and practice of physicians regarding vitamin b12 screening among type 2 diabetes mellitus (t2dm) and assess knowledge of physicians about recent american diabetic association (ada) guidelines updates. methods: a cross sectional study was conducted at primary health care (phc) centers in buraidah, qassim from january to april 2022. data was collected using a structured questionnaire which was based on ada guidelines among all physicians of phcs. statistical package for social sciences (spss) version 23.0 was used for analysis. frequencies and percentages were calculated for categorical variables and means with standard deviations were calculated for continuous variables. results: about 135 (72.2%) physicians would test t2dm patients who are on metformin for vitamin b12, and only 65 (34.8) would test them annually. early signs of diabetic peripheral neuropathy (dpn) are pain, burning sensation, tangling sensation, numbness and loss of protective sensation (lops). only 23 (12.3%) of them knew the all five symptoms. forty-five (45.5%) of participants would not treat dpn with b12 supplementation. only (57.2%) order vit b12 testing for dm2 who are on metformin, and (49.2%), (29.4%) would order them if the patient has neuropathy and on annual basis respectively. majority of physicians (70.6%) give vit b12 supplements as treatment of dpn. conclusion: there is poor knowledge and practices related to b12 deficiency among primary care physicians. this calls for training and educating primary care physicians regarding b12 deficiency screening, neuropathy and their management. there is also need for providing sufficient resources in order to ensure b12 screening in high risk diabetic patients. keywords: primary care, b12 screening, b12 supplementation, diabetics, saudi arabia issn 2413-0516 background type 2 diabetes mellitus (t2dm) is a major problem worldwide. its prevalence increasing globally, including saudi arabia.1 the world health organization (who) has reported that saudi arabia ranks the second highest in the middle east, and is seventh in the world for the rate of diabetes. it is estimated that around 7 million of the population are diabetic and almost around 3 million have pre-diabetes.2 one of the corner stone medications used for t2dm is metformin; which is reported to be associated with vitamin b12 deficiency up to 30% among patients who are in long term metformin treatment.3–6 a recent report from the diabetes prevention program outcomes study (dppos) suggesting periodic testing of vitamin b12.7 long‐term metformin therapy is significantly associated with lower serum vitamin b12 concentration, yet those at risk are often not monitored for b12 deficiency. prescription of vitamin b12 supplementation is common practice in physicians for diabetics who are on metformin without solid evidence of vitamin b12 levels. a research about the association of metformin use with vitamin b12 deficiency and peripheral neuropathy in saudi individuals with type 2 diabetes mellitus, found that the prevalence of b12 deficiency was 7.8% overall, but the b12 deficiency was 9.4% and 2.2% in metformin users and non-metformin users, respectively.8 a meta-analysis done 2014, six randomized controlled trials concluded that b12 concentration was significantly lower in metformin users compared to those on placebo (mean difference [md], –53.93 pmol/l; 95% confidence interval [ci], –81.44 to –26.42 pmol/l, p = 0.0001).9 testing for levels of and prescription of b12 supplementation have been found to be varying and discordant. a study published in 2017 in atlanta, georgia reported that, only 37% of older adults with diabetes receiving metformin were tested for vitamin b12 status after long‐term metformin prescription.10 another study conducted in riyadh 2019 found that among 57.9% of diabetics who were on vitamin b12 supplementation, and only 4.4% had available serum vitamin b12 levels. among physicians 39% do not know about the current ada recommendation, whereas 17% have no idea about the recommendations.11 there is limited evidence on prescription of b12 screening and supplementation for diabetic patients by physicians in saudi arabia. this study therefore aimed to assess knowledge and practice of physicians regarding vitamin b12 screening among t2dm and assess knowledge of physicians about recent ada guidelines updates. methodology this cross-sectional study was conducted among physicians working in primary health care (phc) centers in buraidah city january to april 2022. targeted participants were all physicians in phcs. there are about 40 functional phc centers in buraidah and there are 204 physicians working in those phc centers including family medicine trainees and trainers. given the limited population of physicians, we included all the primary care physicians who were working in phc centers of buraidah for at least one year in our study. we excluded those physicians working in polyclinics in phc centers and having other specialty. all the eligible physicians were mailto:fahad.r.m.k@gmail.com 217j contemp med sci | vol. 8, no. 4, july-august 2022: 216–219 f. alruamaykhani et al. original diabetics and b12 deficiency management approached in their respective phc centers and invited to participate in the study. data was collected using structured questionnaire. the questionnaire was developed based on american diabetics association (ada) guidelines. questionnaire had three sections. first section included variables related to social and professional information. second section assessed the knowledge about vitamin b12 and diabetic peripheral neuropathy. third section was about practice of physician regarding vitamin b12 screening. after taking permission from the regional director of phcs, the questionnaire were distributed directly to the physicians by trained data collectors, who were undergraduates medical students. the data was analyzed using statistical package for social sciences (spss) version 23.0. descriptive analysis was done and calculated frequencies and proportions of categorical variables while mean with standard deviations was calculated for continuous variables. ethical approval was taken from qassim regional bioethics committee. informed consent was taken from all participants and confidentiality was maintained as name and id of participants was not taken. approval was also taken from administration of phc centers. results a total of 250 physicians were invited to participate in the study period out of which 187 patients completed the questionnaire (response rate 74.8%). more than half (57.2%) were males. the mean (sd) age was 36.9 (8.55) years. more than half (58.3) of them were saudis. the professional ranks of the participants were as follows; general practitioners (gps) 63 (33.7%), residents 61 (32.6%), specialists 44 (23.5%) and consultant 19 (10.2%). with mean (sd) experience 9.18 (7.15) years (table 1). a total of 112 (59.9%) knew that vitamin b12 supplementation essential to patients who are taking metformin and have b12 deficiency. in terms of routine test for vit b12, about 135 (72.2%) physicians would test dm2 who are on metformin, and 65 (34.8) would test them annually. according to ada 2021 there are five special groups (pregnant or lactating ladies, older adults, vegetarians and people who are on low carb diets) for which multivitamins are recommended. in our sample, 21 (11.2%) were aware of the five groups. majority of participants (80.2%) knew that vitamin c and e are not advised for diabetic patients. early signs of diabetic peripheral neuropathy (dpn) are pain, burning sensation, tangling sensation, numbness and loss of protective sensation (lops). only 23 (12.3%) of them knew the all five symptoms. forty-five (45.5%) of participants would not treat dpn with b12 supplementation. more than half of the physicians (50.3%) knew that dmt2 patients on metformin taking larger dosage for long duration, are at higher risk of dpn (table 2). regarding the practices of prescription of vitamin b12 supplement, almost half of participants (48.1%) prescribed b12 supplements for diabetics who are on metformin, (36.4%) give only to symptomatic patients and (11.8%) would give every dmt2. of the physicians, only (57.2%) order vit b12 testing for dmt2 who are on metformin, and (49.2%), (29.4%) would order them if the patient has neuropathy and on annual basis respectively. seventeen participants (9.1%) prescribe table 1. socio-demographic characteristics variables n (%) age mean (sd) 36.9 (8.55) gender male female 107 (57.2) 80 (42.8) nationality saudi non-saudi 109 (58.3) 78 (41.7) qualification gp resident specialist consultant 63 (33.7) 61 (32.6) 44 (23.5) 19 (10.2) experience mean (sd) 9.18 (7.15) table 2. knowledge about b12 deficiency b12 supplement is required for every diabetic pt pts on metformin pts with neuropathy sx none of the above 8 (4.3) 112 (59.9) 63 (33.7) 4 (2.1) routine test for b12 dm t1 dm t2 both types idk 3 (1.6) 135 (72.2) 38 (20.3) 11 (5.9) routine test for b12 every 1 year every 5 years every 6 months for symptomatic pts idk 65 (34.8) 11 (5.9) 4 (2.1) 97 (51.9) 10 (5.3) indications of mv supplement one indication two indications three indications four indications five indications 28 (15.0) 57 (30.5) 58 (31.0) 23 (13.3) 21 (11.2) vit c and e supplement are required for dm t1 dm t2 idk not advised 1 (0.5) 18 (9.6) 18 (9.6) 150 (80.2) early signs of dpn one sign two signs three signs four signs five signs 32 (17.1) 44 (23.5) 66 (35.3) 22 (11.8) 23 (12.3) b12 is treatment of dpn yes no idk 96 (51.3) 85 (45.5) 6 (3.2) metformin related dpn is associated with longer duration higher dose both dose and duration irrelevant to dose and duration idk 60 (32.1) 7 (3.7) 94 (50.3) 18 (9.6) 8 (4.3) 218 j contemp med sci | vol. 8, no. 4, july-august 2022: 216–219 diabetics and b12 deficiency management original f. alruamaykhani et al. we found that, in practice 57.2% of the practitioners do routine testing on patients who are on metformin and 49.2%, 29.4% test their patients if they have neuropathy and annually respectively. compared to the study in riyadh,11 51.0% order vitamin b12 testing only if they have symptoms of neuropathy and 19.0% routinely order vitamin b12 testing. one explanation to the difference in knowledge and practice is lack of resources in phc settings which may hinder testing of b12 levels. in our study only 12.3% knew the five signs of dpn. this an important finding as this low knowledge of signs of dpn might lead to delay in identification and treatment of dpn. metformin use has been associated with dpn in prospective study where the prevalence of neuropathy was significantly higher among the patients with low levels of b12.7 this calls for educating primary care physicians regarding the dpn risk factors and sign and symptoms. the study has provided useful information regarding the knowledge and practices of primary care physicians about b12 among diabetic patients. however, there are two limitations should be kept under consideration. first, the knowledge and practices were self-reported and therefore are prone to response bias, as physicians may report better practice then their actual practices. however, we assume this to be of less importance as we ensured complete privacy and anonymity during data collection which would have encouraged respondents to give accurate responses. secondly, this study was conducted in one city only, therefore results may not be generalizable to whole region. none the less, this study has provided a base for further research on practices of b12 deficiency among diabetic patients. conclusion this study highlights the fact that there is lack of knowledge about the ada recommendation by phc physicians. routine testing for serum vitamin b12 level is not practiced in our centers. thus, there is need for doctors involved in the management of diabetes to keep abreast with guidelines and current recommendations and routinely monitor vitamin b12 levels particularly those who were on long-term use of metformin and the elderly patients to optimize management of diabetes and its complications. the difference between knowledge and practice due to lack of resources in phc, so we recommend that physicians and managers reach a solution to provide the needed resources. financial support and sponsorship this research did not receive any funding. conflict of interest there are no conflicts of interest. acknowledgment i would like to thank khalid alharbi, ahmad alshammari, saif alshammari, ghaida alfarhan, muath alharbi, ahmad alenezi for helping in data collection for this study.  table 3. practice about b12 deficiency i give b12 supplements for every t2 diabetic pt pts on metformin pts with neuropathy sx none of the above 22 (11.8) 90 (48.1) 68 (36.4) 7 (3.7) i order b12 test for dm t1 dm t2 both types i don’t order 1 (0.5) 107 (57.2) 26 (13.9) 53 (28.3) i order b12 test every 1 year every 5 years every 6 months for symptomatic pts i don’t order 55 (29.4) 10 (5.3) 6 (3.2) 92 (49.2) 24 (12.89) i give mv for one indication two indications three indications four indications five indications 35 (18.7) 62 (33.2) 57 (30.5) 16 (8.6) 17 (9.1) i give b12 as treatment for dpn yes no 132 (70.6) 55 (29.4) multivitamins to the five recommended groups. majority of physicians (70.6%) give vit b12 supplements as treatment of dpn (table 3). discussion type 2 diabetics particularly those on metformin are at risk for metabolically lower levels of vitamin b12. the mechanisms of vitamin b12 deficiency in metformin treatment has not been clear, but the most likely hypothesis is that metformin interferes with calcium-dependent membrane action responsible for vitamin b12 intrinsic factor absorption in the terminal ileum.6 in this study we found 59.9% knew that vit b12 supplementation is essential for dmt2 patients who are on metformin, but only 34.8% knew that it’s recommended to test for it annually. we found only 12.3% knew the 5 signs of dpn. regarding practice, we found 48.1% of physicians prescribe vit b12 supplementation for every patient on metformin, but only 29.4% order vit b12 test on annual basis. we also found that majority (70.6%) of physicians give vit b12 as treatment of dpn. in previous study11 44.0% of the respondents know the current recommendation of ada on vitamin b12 screening and supplementation among diabetic patients. seventy-two percent of our participants knew that routine testing of vit b12 for dmt2 patients on metformin and 34.8% knew that it’s recommended to test them annually. a possible explanation of lower knowledge in buraidah compared to riyadh is most of our participants were general practitioner (gps) compared to specialists in diabetic clinics in riyadh study. none the less this is an important finding and call for updating the knowledge of practitioners on this matter. 219j contemp med sci | vol. 8, no. 4, july-august 2022: 216–219 f. alruamaykhani et al. original diabetics and b12 deficiency management references 1. lam dw, leroith d. the worldwide diabetes epidemic. current opinion in endocrinology, diabetes, and obesity. 2012;19(2):93-96. doi: 10.1097/ med.0b013e328350583a. http://europepmc.org/article/med/22262000. 2. al dawish ma, robert aa, braham r, al hayek aa, al saeed a, ahmed ra, et al. diabetes mellitus in saudi arabia: a review of the recent literature. curr diabetes rev. 2016;12(4):359–368. doi: 10.2174/157339981166615072 4095130. https://pubmed.ncbi.nlm.nih.gov/26206092/. 3. damião cp, rodrigues ao, pinheiro mf, cruz filho ra, cardoso gp, taboada gf, lima ga. prevalence of vitamin b12 deficiency in type 2 diabetic patients using metformin: a cross-sectional study. sao paulo medical journal. 2016;134(3):473–9. doi: 10.1590/1516-3180.2015.01382111. http:// europepmc.org/article/med/28076635. 4. sánchez h, masferrer d, lera l, arancibia e, angel b, albala c. vitamin b12 deficiency associated with high doses od metformin in older people diabetic. nutricion hospitalaria. 2014 jun 1;29(6):1394–400. doi: 10.3305/ nh.2014.29.6.7405. http://europepmc.org/article/med/24972480. 5. ko sh, ahn yb, song kh, han kd, park ym, kim hs. association of vitamin b12 deficiency and metformin use in patients with type 2 diabetes. j korean med sci. 2014;29:965–72. http://europepmc.org/article/med/25045229. 6. ko sh, ko sh, ahn yb, et al. association of vitamin b12 deficiency and metformin use in patients with type 2 diabetes. journal of korean medical science. 2014;29(7):965–972. doi: 10.3346/jkms.2014.29.7.965. https:// pubmed.ncbi.nlm.nih.gov/31725641/. 7. aroda vr, edelstein sl, goldberg rb, knowler wc, marcovina sm, orchard tj, et al. diabetes prevention program research group. long-term metformin use and vitamin b12 deficiency in the diabetes prevention program outcomes study. j clin endocrinol metab. 2016;101(4):1754–61. doi: 10.1210/jc.2015-3754. https://pubmed.ncbi.nlm.nih.gov/26900641/. 8. alharbi tj, tourkmani am, abdelhay o, alkhashan hi, al-asmari ak, bin rsheed am, et al. the association of metformin use with vitamin b12 deficiency and peripheral neuropathy in saudi individuals with type 2 diabetes mellitus. plos one. 2018;13(10):e0204420. doi: 10.1371/journal. pone.0204420. https://pubmed.ncbi.nlm.nih.gov/30321183/. 9. liu q, li s, quan h, li j. vitamin b12 status in metformin treated patients: systematic review. plos one. 2014;9(6):e100379. doi: 10.1371/journal. pone.0100379. http://europepmc.org/article/med/24959880. 10. kancherla v, elliott jl jr, patel bb, holland nw, johnson tm, khakharia a, et al. long-term metformin therapy and monitoring for vitamin b12 deficiency among older veterans. j am geriatr soc. 2017;65(5):1061–1066. doi: 10.1111/jgs.14761. https://pubmed.ncbi.nlm.nih.gov/28182265/. 11. alshammari an, iqbal r, baksh ip. vitamin b12 deficiency and the knowledge and practice of physicians regarding screening for vitamin b12 deficiency among type 2 diabetic patients on metformin in selected hospitals in riyadh, saudi arabia. journal of family medicine and primary care. 2019;8(7):2306–2311. doi: 10.4103/jfmpc.jfmpc_416_19. http:// europepmc.org/article/med/31463247. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1209 http://europepmc.org/article/med/22262000. https://pubmed.ncbi.nlm.nih.gov/26206092/. http://europepmc.org/article/med/28076635. http://europepmc.org/article/med/28076635. http://europepmc.org/article/med/24972480. http://europepmc.org/article/med/25045229. https://pubmed.ncbi.nlm.nih.gov/31725641/. https://pubmed.ncbi.nlm.nih.gov/31725641/. https://pubmed.ncbi.nlm.nih.gov/26900641/. https://pubmed.ncbi.nlm.nih.gov/30321183/. http://europepmc.org/article/med/24959880. https://pubmed.ncbi.nlm.nih.gov/28182265/. http://europepmc.org/article/med/31463247 http://europepmc.org/article/med/31463247 382 j contemp med sci | vol. 8, no. 6, november-december 2022: 382–387 original molecular studies of capn-10 gene (rs2975760) and its association with insulin resistance in polycystic ovarian syndrome of iraqi women hadbaa h. al-murshedi1*, ammar fadhil jawad2, fadhil j. al-tu’ma1, eman a. hadi3, maha mohammed kadhim al-tu’ma4 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 2department of pharmacognosy, college pharmacy, university of kerbala, kerbala, iraq. 3department of chemistry, college of science, university of mosul, mosul, iraq. 4department of anesthesia techniques, college of health and medical techniques, al-zahraa university for women, kerbala, iraq. *correspondence to: hadbaa helwas al-murshedi (email: hdbaa88@gmail.com) (submitted: 10 august 2022 – revised version received: 22 august 2022 – accepted: 17 september 2022 – published online: 26 december 2022) abstract objectives: to explore an association between capn10, snp-44 (rs2975760) with ir condition in women with pcos. methods: a study included 120 participants of which 68 women have pcos subdivide according to their body mass index (bmi) into 45 obese (bmi ≥ 30) and 23 non-obese (bmi < 30). the remaining 52 represent the control group who were apparently healthy women with normal weight and normal menstrual cycle. patients with pcos were selected from the infertility department, gynecology and obstetrics teaching hospital, kerbala health directorate/kerbala-iraq between nov., 2021 and june, 2022. diagnosis of pcos is based on 2 of 3 findings: oligo/anovulation, hyperandrogenism, polycystic ovaries in ultrasound (rotterdam criteria). patients were interviewed and examined for weight, height, waist circumference, and hip circumference. venous blood samples were collected at 9 am after an overnight fast. ir was assessed by calculating homeostatic model assessment of insulin resistance (homa-ir) using the formula (fasting glucose mg/dl x fasting insulin µu/ml)/405, taking normal value <2.7. genotypes of capn10, snp-44 has been identified using allele-specific polymerase chain reaction (as-pcr) technique. results: the prevalence of ir based on homa-ir was (80%) in obese pcos and (48%) in non-obese pcos women. capn10, snp-44 has been reconstructed and analyzed in patients and controls. genotypes of 45 obese pcos subjects (tt, n = 26; tc, n = 12; and cc, n = 7), 23 non-obese pcos subjects (tt, n = 15; tc, n = 6; and cc, n = 2) and control subjects (tt, n = 39; tc, n = 11; and cc, n = 2) were identified. the genotype distribution was statistically different between obese pcos women and controls (or = 5.25, p = 0.048). the association of snp-44 allele with ir status was detected. homa-ir was greater in cc (10.54 ± 1.29, 9.88 ± 1.41) than in tt (3.30 ± 1.52, p < 0.001; 2.82 ± 1.45, p < 0.001) and tc (3.76 ± 1.58, p < 0.001; 4.10 ± 1.57, p < 0.05) in obese pcos and non-obese pcos subjects respectively. conclusion: in obese pcos, the c allele was associated with higher insulin secretion and homa-ir compared with the t allele. the increased homa-ir is an indicator of ir. in this scenario, the c allele might be involved in the pathophysiology of insulin resistance in pcos. keywords: polycystic ovarian syndrome, insulin resistance, capn10 gene issn 2413-0516 introduction polycystic ovarian syndrome (pcos) is a common endocrine disorder affecting women of reproductive age and often develops during adolescence.1 women with pcos are at increased risk of infertility.2 pcos manifests with the typical clinical features of clinical and/or biochemical hyperandrogenism, ovarian dysfunction (including oligo-amenorrhoea) and polycystic ovaries. the prevalence of pcos is commonly thought to vary between 5% and 20% depending on diagnostic criteria and sample population.3 diagnostic criteria for pcos mostly use the revised rotterdam 2003 criteria.4 metabolic disturbances often form an important component of the clinical presentation of pcos. insulin resistance (ir) is a very common finding in subjects with pcos which not included among the diagnostic features.5 ir is usually defined as a pathological condition characterized by a decreased responsiveness or sensitivity to the metabolic actions of insulin. in women with pcos, ir plays an important role in the development and persistence of this disorder.6 ir and compensatory hyperinsulinemia stimulate ovarian theca cells to secrete androgens. insulin also inhibits sex hormone binding globulin (shbg) production, increasing bioavailability of androgen levels and worsen hyperandrogenism status.7 moreover, ir is critically involved in the development of metabolic syndrome, type 2 diabetes (t2d) and cardiovascular disease in pcos women.8 the close association between obesity and pcos is supported by epidemiological data, revealing that between 38% and 88% of women with pcos are either overweight or obese.9 the etiology of pcos is not completely understood, although it was reported that genetic and lifestyle factors known to influence the etiology of the syndrome. the use of candidate gene analysis has provided several promising genes as genetic modifiers of component phenotypes of pcos.10 the results of both linkage and association studies, suggested that some genes involves insulin regulation are important factors in the genetic basis of pcos.11 the discovery of single nucleotide polymorphisms (snps) linked to a disease can shed light on its origin and help women with a particular phenotype, have a better prognosis. the calpain-10 gene (capn-10), plays a role in the secretion and action of insulin by encoding an extremely prevalent member of the calpain-like cysteine protease family. the capn-10 gene has been thoroughly examined in pcos due to the fact that t2d and pcos share a number of etiologic factors.12 the earliest evidence of capn10 involvement in pcos, suggested a statistically significant association between pcos susceptibility and the snp-44 (rs2975760).13 this study was aimed to explore an association between capn-10 gene single 383j contemp med sci | vol. 8, no. 6, november-december 2022: 382–387 h.h. al-murshedi et al. original capn-10 gene and its association with ir in polycystic ovarian syndrome nucleotide polymorphism (rs2975760) with insulin resistance in women with pcos. materials and methods a case-control study included 120 participants of which 68 women have pcos subdivided into 45 obese and 23 nonobese subjects. the remaining 52 represent the control group who were apparently healthy women with normal weight and normal menstruation. patients with pcos were selected from the infertility department, gynecology and obstetrics teaching hospital, kerbala health directorate/kerbala-iraq between november, 2021 and june, 2022. oral informed consent was obtained from all participants. pcos was diagnosed in presence of at least two out of the three: oligo/anovulation, clinical and/or biochemical signs of hyperandrogenism and polycystic ovaries with exclusion of related etiologies. these criteria were defined by the updated 2003 rotterdam.14 patients were interviewed and examined for weight, height, waist circumference, and hip circumference. transabdominal pelvic sonography was performed on all of the subjects with pcos. five milliliters venous blood samples were collected at 9 am after an overnight fast. blood samples were divided into two parts; the first part was collected in gel tube for biochemical analysis. levels of serum c-peptide, lh and fsh were measured by chemiluminescent automated immunoassay system (cobas e411). free testosterone and insulin were measured by enzyme linked immunosorbent assay (elisa). glucose was measured with hexokinase method. the second part was collected in edta tube for molecular analysis. ir was assessed by calculating homeostatic model assessment of insulin resistance (homa-ir) using the formula (fasting glucose mg/dl x fasting insulin µu/ml)/405, taking normal value less than (2.7). genotypes of capn-10, rs2975760 has been identified using allele-specific polymerase chain reaction (as-pcr) technique. each pcr was carried out in a total volume 25 μl consisting of 3 μl extracted dna, 1.5 µl each primer, 12 μl master mix and 7 μl nuclease free water. specific primers were designed with the sequences 5´-gcagggaagctggtgaacatg-3´ for the forward primer, 5´-ctcaccttcaaacgccttacttca-3´ for the reverse 1 primer and 5´-ctcaccttcaaacgccttacttcg-3´ for the reverse 2 primer. the pcr products (250 bp) of capn-10 gene were separated by electrophoresis in a 5% agarose gel and then visualized by uv transilluminator. the data were analyzed using the statistical package for social sciences (spss). continuous variables were expressed as means ± standard deviation, (sd). mean comparisons were made using one-way analysis of variance (anova) followed by tukey’s post hoc test. results the demographic features of the total study population, as well as baseline values of the various biochemical parameters are presented in table 1. all fat indices such as waist circumference (wc), waist to hip ratio (whr) and bmi were significantly higher in pcos group (p < 0.001). this result highlighted the role of obesity in pcos as reported in previous studies. gonadal hormones: lh, fsh and lh/fsh ratio were seemingly elevated in pcos patients. free testosterone was table 1. comparison of demographic, hormonal and metabolic features between pcos patients (n = 68) and controls (n = 52) pcos n = 68 control n = 52 p-value demographic parameters age (years) 27 ± 6.00 27 ± 5.71 ns wc (cm) 100 ± 16.71 80 ± 10.94 <0.001 whr 0.90 ± 0.10 0.82 ± 0.08 <0.001 bmi (kg/m2) 31.32 ± 5.79 24.09 ± 3.56 <0.001 hormonal parameters lh* (iu/l) 10.25 ± 1.52 6.67 ± 1.44 <0.001 fsh (iu/l) 6.73 ± 1.69 5.56 ± 1.32 <0.01 lh/fsh 1.69 ± 0.72 1.30 ± 0.60 <0.01 f-testosterone* (pg/ml) 16.65 ± 2.27 4.21 ± 2.50 <0.001 metabolic parameters total cholesterol (mg/dl) 156 ± 31.05 117 ± 20.97 <0.001 triglycerides* (mg/dl) 95 ± 1.53 70 ± 1.55 <0.001 hdl-c (mg/dl) 40 ± 6.24 43 ± 8.95 <0.01 ldl-c* (mg/dl) 92 ± 1.32 56 ± 1.39 <0.001 glucose (mg/dl) 92 ± 8.40 84 ± 5.12 <0.001 insulin* (μu/ml) 18.15 ± 1.76 10.74 ± 1.56 <0.001 c-peptide* (pmol/l) 455.82 ± 1.82 330.64 ± 1.64 <0.01 homa-ir* 3.87 ± 1.76 2.22 ± 1.58 <0.001 data was expressed as mean ± sd; *, refers to logarithmic mean of transformed data; ns, non-significant; wc, waist circumference; whr, waist to hip ratio; bmi, body mass index; lh, luitinising hormone; fsh, follicle stimulating hormone; lh/fsl, lh to fsh ratio; f-testosterone, free testosterone, hdl, high density lipoprotein-cholesterol; ldl, low density lipoprotein-cholesterol; homa-ir, homeostatic model assessment of insulin resistance. increased in pcos group compared to control group (p < 0.001). lipid profile: total cholesterol (tc), triglyceride (tg), high density lipoprotein-cholesterol (hdl-c) and low density lipoprotein cholesterol (ldl-c) seem to be normal in women with pcos of the presented study. however, pcos subjects still have increased levels of tc, tg and ldl-c; and decreased level of hdl-c in compare to control subjects. glucose, insulin, c-peptide and homa-ir were significantly elevated in women with pcos compared to control women, pointing to the insulin resistance component in this syndrome.15 the incidence of ir was 80% in obese women with pcos and 48% in non-obese women with pcos, as shown in figure 1. extracted dna samples for study subjects, were found to be pure and the dna concentrations ranged from (4.64– 42.10) ng/u. the mean of dna concentration and purity were (21.85 ± 8.79), (1.82 ± 0.08) respectively. the results of as-pcr amplifications were analyzed and three genotypes were obtained for capn10, snp-44, tt (common homozygous-wild type) tc, (heterozygous) and cc, (rare homozygous-mutant type). subjects with pcos were analyzed in two groups, based on bmi: non-obese pcos (bmi < 30) and obese pcos (bmi ≥ 30) cases. chi square test was carried out to check for the significance of deviation from hardy-weinberg equilibrium (hwp) for capn10, snp-44 (rs2975760) in obese pcos (p = 0.063), non-obese pcos (p = 0.535) and control (p = 0.587) 384 j contemp med sci | vol. 8, no. 6, november-december 2022: 382–387 capn-10 gene and its association with ir in polycystic ovarian syndrome original h.h. al-murshedi et al. women separately. the results indicate that capn10 snp-44 (rs2975760) confirms to the hwe, table 2. the allele and genotype frequencies were compared between these two groups as well as each of them with controls. the genotype and allele frequency distribution for the capn10 snp-44, (rs2975760) is presented in figure 2. observations of the current study indicated that the homozygotes for the snp-44, variant cc had significantly higher frequency among obese pcos cases 15% than the controls 4%, (or = 5.25, p = 0.048). this trend was also seen in the allele distribution pattern, wherein the variant allele c was found in 29% of the obese pcos cases as compared to 14% of the controls (or = 2.41, p = 0.015), table 3. to explore the role of capn-10 gene, snp-44 in the presence of metabolic abnormalities in pcos. the distribution of genotypes was compared in obese/non-obese pcos patients. the results of obese pcos women genotyping in the presented study show that bmi is significantly higher in the rare homozygotes genotype cc compared to heterozygous genotype tc and homozygotes genotype tt. also, there are no significant difference (p > 0.05) when measuring the levels of glucose concentrations. while, insulin, c-peptide and homa-ir were significantly higher (p < 0.001) in the rare homozygotes genotype cc compared to heterozygous genotype tc and homozygotes genotype tt, table 4. for non-obese pcos women, the rare homozygotes genotype cc was had elevated bmi compared to the homozygotes genotype tt. lipid profile does not correlate with capn-10, snp-44 variant c. also, there was no significant difference (p > 0.05) when measuring the levels of glucose and c-peptide concentrations. while, insulin and homa-ir were significantly higher (p < 0.001) in the rare homozygotes genotype cc compared to heterozygous genotype tc and homozygotes genotype tt, table 5. discussion poly cystic ovary syndrome has multiple componentsreproductive, metabolic and cardiovascular with long term health implications. none are specific for pcos, and we speculated that each component might be related with independent genetic risk factors.16 obesity in adolescence is associated with greater menstrual cycle irregularity and pcos.17. three main pathophysiologic components are strongly associated with pcos: increased lh secretion, higher androgen levels and ir.18 hyperandrogenic phenotype of pcos patients is influenced by both hormonal and metabolic dysfunctions.19 insulin plays an important role in reproduction through its direct effect on ovarian granulosa and theca cells regulating ovulation and steroidogenesis in ovaries. thus, ir is associated with hyperandrogenism in pcos.20 the same finding was recorded previously (unluer et al., 2013),21 who reported that ir in pcos had linked to obesity and obese pcos have a high probability of ir.22 although non-obese women exhibit lower ir is still a common finding in this population. indeed, several studies have suggested ir as a pathophysiological component independent of weight.23 the results of lipid profile confirm that pcos patients might have dyslipidemia as mentioned in previous studies.24 rizzo et al. concluded that total cholesterol, triglyceride and ldl concentrations were higher and hdl levels were lower in controls versus pcos.25 it was demonstrated that obese women have elevated serum levels of cholesterol and ldl as compared with the corresponding levels in the normal weight group and higher triglycerides and lower hdl than normal or overweight pcos women.26 however, many women with pcos still have a completely normal lipid profile and in larger studies of lipid levels in women with pcos mostly fall within normal ranges.27 the current study supports a role of capn-10, snp-44 in pcos susceptibility.28 variation in the capn-10 gene was reported to be linked and associated with t2d susceptibility in fig. 1 the prevalence of insulin resistance (ir) /sensitivity (is) based on homa-ir in both obese and non-obese pcos women. table 2. hardy weinberg equilibrium test for study subjects common homozygotes heterozygotes rare homozygotes obese pcos (n = 45) tt tc cc frequency 26 12 7 p-value 0.063 non-obese pcos (n = 23) frequency 15 6 2 p-value 0.535 control (n = 52) frequency 39 11 2 p-value 0.587 fig. 2 comparison of genotypes frequencies among obese pcos, non-obese pcos and control subjects. 385j contemp med sci | vol. 8, no. 6, november-december 2022: 382–387 h.h. al-murshedi et al. original capn-10 gene and its association with ir in polycystic ovarian syndrome table 3. genotype and allele frequency distribution of snp-44 t/c (rs2975760) of capn10 gene in obese pcos and control subjects genotype obese pcos control or 95% ci p-value tt 26 58% 39 75% 1 reference tc 12 27% 11 21% 1.64 0.63–4.26 0.313 cc 7 15% 2 4% 5.25 1.01–27.28 0.048 total 45 100% 52 100% allele frequency t 64 71% 89 86% c 26 29% 15 14% 2.41 1.18–4.91 0.015 or, odd ratio; ci, confidence interval. table 4. the differences in demographic and metabolic parameters in related to tt, tc and cc genotype in obese women with pcos biochemical parameters genotyping frequency p-value tt tc cc bmi (kg/m2) 33.81 ± 3.05 34.83 ± 2.57 38.75 ± 1.29 p a < 0.05 p b < 0.05 total cholesterol (mg/dl) 160 ± 29.32 162 ± 21.09 174 ± 26.27 ns triglycerides (mg/dl) 93 ± 1.52 105 ± 1.44 138 ± 1.16 ns hdl-c (mg/dl) 41 ± 7.68 39 ± 4.81 39 ± 6.59 ns ldl-c (mg/dl) 95 ± 1.31 98 ± 1.27 105 ± 1.29 ns glucose (mg/dl) 92 ± 9.69 92 ± 7.09 91 ± 3.87 ns insulin (μu/ml) 14.63 ± 1.49 16.66 ± 1.53 47.08 ± 1.27 p a < 0.001 p b < 0.001 c-peptide (pmol/l) 402.3 ± 1.57 378.2 ± 1.48 1067.9 ± 1.59 p a < 0.001 p b < 0.001 homa-ir 3.30 ± 1.52 3.76 ± 1.58 10.54 ± 1.29 p a < 0.001 p b < 0.001 p a , (tt vs. cc); p b , (tc vs. cc); p c , (tt vs. tc). table 5. the differences in demographic and metabolic parameters in related to tt, tc and cc genotype in non-obese women with pcos biochemical parameters genotyping frequency p-value tt tc cc bmi (kg/m2) 23.43 ± 2.15 25.49 ± 1.99 28.68 ± 0.22 p a < 0.01 total cholesterol (mg/dl) 134 ± 23.58 143 ± 16.52 177 ± 29.69 ns triglycerides (mg/dl) 72 ± 1.47 91 ± 1.68 148 ± 1.23 ns hdl-c (mg/dl) 39 ± 4.19 39 ± 6.84 37 ± 9.89 ns ldl-c (mg/dl) 78 ± 1.27 84 ± 1.12 112 ± 1.43 ns glucose (mg/dl) 93 ± 9.93 93 ± 7.89 88 ± 2.82 ns insulin (μu/ml) 12.39 ± 1.45 18.06 ± 1.55 45.18 ± 1.45 p a < 0.001 p b < 0.05 c-peptide (pmol/l) 376.0 ± 1.91 461.7 ± 1.77 725.2 ± 1.18 ns homa-ir 2.82 ± 1.45 4.10 ±1.57 9.88 ± 1.41 p a < 0.001 p b < 0.05 p a , (tt vs. cc); p b , (tc vs. cc); p c , (tt vs. tc). a mexican american population.29 in a case-control study involved patients with t2d and normal glucose tolerant subjects, no association of the snp-44 variant with t2d was found. the frequency of the minor snp-44 variant c was higher among t2d patients 18% compared with normal glycemic subjects 17%, however, these differences did not reach statistical significance.30 while snp-44 has been shown to predict the development of t2d in other study.31 preliminary studies of capn-10 gene in pcos patients provide the first evidence of capn-10 involvement in pcos, 386 j contemp med sci | vol. 8, no. 6, november-december 2022: 382–387 capn-10 gene and its association with ir in polycystic ovarian syndrome original h.h. al-murshedi et al. suggesting a statistically significant association between the snp-44 and pcos susceptibility.13 subsequent studies evaluating the role of capn-10 in pcos have yielded contradictory results. supporting the capn10 gene involvement in pcos, gonzalez et al. showed that capn-10, snp-44 allele was associated with pcos in the spanish population.32 lipid profile does not correlate with capn-10 snp-44 variant c, indicating that this variant does not confer risk to dyslipidemia in pcos. although, the associated genetic variation might serve as useful diagnostic tools, it does not necessarily mean that there is an etiological link between a certain allele or genotype and a given trait or disease. in previous studies, capn-10 polymorphisms have been found to correlate with several aspects of insulin secretion. however, possible mechanisms whereby calpain-10 modulates insulin secretion was not provided.33 although many genes have been researched as potential susceptibility loci, the impact of any one gene may be minimal since pcos is in fact a complex genetic disorder. conclusion insulin resistance is commonly associated with pcos independently of obesity. in obese pcos, the c allele was associated with higher insulin secretion and homa-ir as compared with the t allele. the c allele might be involved in the pathophysiology of insulin resistance in pcos. conflict of interest none.  references 1. azziz, r., woods, k. s., reyna, r., key, t. j., knochenhauer, e. s. and yildiz, b. o. 2004. the prevalence and features of the polycystic ovary syndrome in an unselected population. the journal of clinical endocrinology and metabolism, 89, 2745–2749. 2. legro, r. s., arslanian, s. a., ehrmann, d. a., hoeger, k. m., murad, m. h., pasquali, r. and welt, c. k. 2013. diagnosis and treatment of polycystic ovary syndrome: an endocrine society clinical practice guideline. the journal of clinical endocrinology and metabolism, 98, 4565–4592. 3. march, w. a., moore, v. m., willson, k. j., phillips, d. i., norman, r. j. and davies, m. j. 2010. the prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria. human reproduction, 25, 544–551. 4. fauser, b. c., tarlatzis, b. c., rebar, r. w., legro, r. s., balen, a. h., lobo, r., carmina, e., chang, j., yildiz, b. o. and laven, j. s. 2012. consensus on women’s health aspects of polycystic ovary syndrome (pcos): the amsterdam eshre/asrm-sponsored 3rd pcos consensus workshop group. fertility and sterility, 97, 28–38. e25. 5. mayer, s. b., evans, w. s. and nestler, j. e. 2015. polycystic ovary syndrome and insulin: our understanding in the past, present and future. women’s health, 11, 137–149. 6. amato, m. c., vesco, r., vigneri, e., ciresi, a. and giordano, c. 2015. hyperinsulinism and polycystic ovary syndrome (pcos): role of insulin clearance. j endocrinol invest, 38, 1319–26. 7. spritzer, p. m. 2014. polycystic ovary syndrome: reviewing diagnosis and management of metabolic disturbances. arq bras endocrinol metabol, 58, 182–7. 8. amisi, c. a. 2022. markers of insulin resistance in polycystic ovary syndrome women: an update. world journal of diabetes, 13, 129. 9. barber, t. m. and franks, s. 2021. obesity and polycystic ovary syndrome. clinical endocrinology, 95, 531–541. 10. jones, m. r. and goodarzi, m. o. 2016. genetic determinants of polycystic ovary syndrome: progress and future directions. fertility and sterility, 106, 25–32. 11. franks, s., white, d., gilling-smith, c., carey, a., waterworth, d. and williamson, r. 1996. hypersecretion of androgens by polycystic ovaries: the role of genetic factors in the regulation of cytochrome p450c17α. bailliere’s clinical endocrinology and metabolism, 10, 193–203. 12. haddad, l., evans, j. c., gharani, n., robertson, c., rush, k., wiltshire, s., frayling, t. m., wilkin, t. j., demaine, a. and millward, a. 2002. variation within the type 2 diabetes susceptibility gene calpain-10 and polycystic ovary syndrome. the journal of clinical endocrinology and metabolism, 87, 2606–2610. 13. haddad, l., gharani, n., evans, j., cela, e. and franks, s. m m linkage and association of variants of the diabetes susceptibility-gene niddm1 capn10 with polycystic ovarian syndrome in european parent-offspring trios. 82nd annual meeting of the endocrine society, toronto, ontario, canada, 2000. 75. 14. broekmans, f., knauff, e., valkenburg, o., laven, j., eijkemans, m. and fauser, b. 2006. pcos according to the rotterdam consensus criteria: change in prevalence among who‐ii anovulation and association with metabolic factors. bjog: an international journal of obstetrics and gynaecology, 113, 1210–1217. 15. stener-victorin, e., zhang, h., li, r., friden, c., li, d., wang, w., et al. 2019. acupuncture or metformin to improve insulin resistance in women with polycystic ovary syndrome: study protocol of a combined multinational cross sectional case-control study and a randomised controlled trial. bmj open, 9(1), e024733. 16. franks, s., gharani, n., waterworth, d., batty, s., white, d., williamson, r. and mccarthy, m. 1997. the genetic basis of polycystic ovary syndrome. human reproduction (oxford, england), 12, 2641–2648. 17. itriyeva, k. 2022. the effects of obesity on the menstrual cycle. current problems in pediatric and adolescent health care, 101241. 18. crisosto, n., de guevara, a. l., echiburú, b., maliqueo, m., cavada, g., codner, e., paez, f. and sir-petermann, t. 2019. higher luteinizing hormone levels associated with antimüllerian hormone in postmenarchal daughters of women with polycystic ovary syndrome. fertility and sterility, 111, 381–388. 19. hestiantoro, a., karimah, p. d., shadrina, a., wiweko, b., muharam, r. and astuti, b. p. k. 2019. triglycerides, independent of ferriman gallwey score, is a main determinant of free testosterone index in pcos. f1000research, 8. 20. shorakae, s., ranasinha, s., abell, s., lambert, g., lambert, e., de courten, b. and teede, h. 2018. inter‐related effects of insulin resistance, hyperandrogenism, sympathetic dysfunction and chronic inflammation in pcos. clinical endocrinology, 89, 628–633. 21. unluer, a., findik, r., sevinc, n. and karakaya, j. 2013. comparison of hba1c levels in obese and non-obese polycystic ovarian patients. clinical and experimental obstetrics and gynecology, 40, 148–150. 22. dahan, m., abbasi, f. and reaven, g. 2007. prevalence of insulin resistance among american women with polycystic ovary syndrome (pcos) as a function of body mass index (bmi). fertility and sterility, 88, s78–s79. 23. johnson, p. j., wiedmeyer, c. e., lacarrubba, a., ganjam, v. s., & messer iv, n. t. 2012. diabetes, insulin resistance, and metabolic syndrome in horses. journal of diabetes science and technology, 6(3), 534–540. 24. liu, q., xie, y.-j., qu, l.-h., zhang, m.-x. and mo, z.-c. 2019. dyslipidemia involvement in the development of polycystic ovary syndrome. taiwanese journal of obstetrics and gynecology, 58, 447–453. 25. rizzo, m., berneis, k., hersberger, m., pepe, i., di fede, g., rini, g. b., spinas, g. a. and carmina, e. 2009. milder forms of atherogenic dyslipidemia in ovulatory versus anovulatory polycystic ovary syndrome phenotype. human reproduction, 24, 2286–2292. 26. castelo-branco, c., steinvarcel, f., osorio, a., ros, c. and balasch, j. 2010. atherogenic metabolic profile in pcos patients: role of obesity and hyperandrogenism. gynecological endocrinology, 26, 736–742. 27. legro, r. s., azziz, r., ehrmann, d., fereshetian, a. g., o’keefe, m., ghazzi, m. n. and group, p. t. s. 2003. minimal response of circulating lipids in women with polycystic ovary syndrome to improvement in insulin sensitivity with troglitazone. the journal of clinical endocrinology and metabolism, 88, 5137–5144. 28. simoni, m., tempfer, c., destenaves, b. and fauser, b. 2008. functional genetic polymorphisms and female reproductive disorders: part i: polycystic ovary syndrome and ovarian response. human reproduction update, 14, 459–484. 29. horikawa, y., oda, n., cox, n. j., li, x., orho-melander, m., hara, m., hinokio, y., lindner, t. h., mashima, h. and schwarz, p. e. 2000. genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. nature genetics, 26, 163–175. 387j contemp med sci | vol. 8, no. 6, november-december 2022: 382–387 h.h. al-murshedi et al. original capn-10 gene and its association with ir in polycystic ovarian syndrome 30. jensen, d. p., urhammer, s. a., eiberg, h., borch-johnsen, k., jørgensen, t., hansen, t. and pedersen, o. 2006. variation in capn10 in relation to type 2 diabetes, obesity and quantitative metabolic traits: studies in 6018 whites. molecular genetics and metabolism, 89, 360–367. 31. lyssenko, v., almgren, p., anevski, d., orho-melander, m., sjögren, m., saloranta, c., tuomi, t., groop, l. and group, b. s. 2005. genetic prediction of future type 2 diabetes. plos medicine, 2, e345. 32. gonzalez, a., abril, e., roca, a., aragón, m. j., figueroa, m. j., velarde, p., ruiz, r. o., fayez, o., galán, j. j. and herreros, j. a. 2003. specific capn10 gene haplotypes influence the clinical profile of polycystic ovary patients. the journal of clinical endocrinology and metabolism, 88, 5529–5536. 33. neumeyer, s., hemani, g. and zeggini, e. 2020. strengthening causal inference for complex disease using molecular quantitative trait loci. trends in molecular medicine, 26, 232–241. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1298 70 j contemp med sci | vol. 9, no. 1, january-february 2023: 70–76 original reliability and validity of knee injury and osteoarthritis outcome score (koos) in iranian population amir sabaghzadeh1,2, hamidreza aslani3*, saman shakeri jousheghan4*, mohammadamin aslani5 katayoon tajik 4 1department of orthopedic surgery, shohada-e tajrish hospital, shahid beheshti university of medical sciences, tehran, iran. 2clinical research development unit of shohada-e tajrish hospital, shahid beheshti university of medical sciences, tehran, iran. 3sport medicine and knee research center, milad hospital, tehran, iran. 4clinical research & development unit, akhtar hospital, shahid beheshti university of medical sciences, tehran, iran. 5school of medicine, tehran university of medical sciences, tehran, iran. *correspondence to: saman shakeri jousheghan (email: dr.saman.shakeri.1989@gmail.com), hamidreza aslani, (e-mail: hraslani1342@gmail.com) abstract objectives: the purpose of present study was the investigation of reliability and validity of the knee injury and osteoarthritis outcome score in the iranian population. methods: the method of the present study was non-experimental and methodological. the statistical population of the present study was patients with osteoarthritis of the knee. patients with osteoarthritis of the knee referred to the akhtar center were selected from 127 people by available sampling for sample adequacy. in data analysis, cronbach’s alpha test to determine internal agreement, test-retest method to determine stability in reliability, item impact method to determine face validity, cvi and cvr indicators to determine content validity, and to determine structural validity two convergent and divergent validities as well as confirmatory factor analysis were used. results: the results showed that if the fourth question “symptoms” is removed, the questionnaire has acceptable reliability and validity. conclusion: the analysis of the findings indicated that the persian version of the questionnaire (koos) was a reliable tool for assessing the health status and impact of various treatments on patients with osteoarthritis in the country, which had high validity and reliability. keywords: koos, reliability, validity, osteoarthritis, knee injury issn 2413-0516 introduction knee arthritis is one of the most common forms of osteoarthritis throughout the globe with higher prevalence among the middle-aged and older population. this disease is the most common disease of the synovial joints. based on the studies, the prevalence of knee arthritis varied in 60–90% and 20% among people above 65 and 30, respectively.1,2 the symptoms and disabling level of osteoarthritis deteriorate with aging, therefore, the high prevalence of knee arthritis and its related treatment costs will increase with the ever-rising aging of the population, imposing a huge economic burden. osteoarthritis is a degenerative joint disease characterized by the gradual degradation of the cartilage. it can affect the weight-bearing joints such as the knee. pain and physical disability are two major signs of knee osteoarthritis which can significantly influence the health and quality of life (qol) of the patients.3 knee osteoarthritis has several symptoms such as pain, morning stiffness, and limited joint range of motion (rom).4,5 the process of aging involves degenerative, gradual, and irreversible changes in the systems of the body which can destroy the performance and life quality of the elderly.6 based on an estimation in 2004, 5 million iranians suffer from this disease. this disease is one of the main causes of deficient performance, leading to extensive use of health insurance services. long-term disability often significantly affects people’s life including their mobility, independence, and daily activities, resulting in their social isolation, limited recreational, sport, and occupational activities, and hence the decline in their income.7,8 factors such as wrong dietary patterns and obesity prevalence in society, especially among women, have increased the rate of osteoarthritis. wrong postures such as sitting on the ground and the use of a pit latrine can also result in these complications. knee osteoarthritis has increased in recent years not only among iranian elderlies but also among the youth. based on the studies, almost half of the adults will experience osteoarthritis in at least one of their legs by the age of 85, this risk is higher among the obese population.9,10 based on the advances, valid clinical tools are of crucial significance in the evaluation of the response to the treatment and deciding on the type of treatment. the researchers and physicians should have a proper evaluation of the clinical condition of knee osteoarthritis patients. various tools have been developed to assess the qol and performance of the knee osteoarthritis patients among which, knee injury and osteoarthritis outcome score (koos), short-form 36 (sf-36), western ontario and mcmaster universities osteoarthritis index (womac), and hip and knee questionnaire (hnq) can be mentioned.11 although these evaluation tools have been developed in different countries, most of them originated from english and american literature. in this regard, the development of non-english tools is essential for cross-national and multi central research and even assessment of the health condition of a country with different cultures and languages. given the complexity of the mentioned structures, the tools developed in a country cannot be employed in another country just by translation. to adapt a tool to a different environment, in addition to translation, the cultural issues and traditions should be also adapted. the validity and reliability of the tool should be also evaluated.12 koos is one of the well-known and global tools to assess the consequences of knee injuries and osteoarthritis. this is a patient-oriented questionnaire to assess the disease symptoms and effects on physical activities and qol.13 koos is a specific tool for knee osteoarthritis and has been considered as a subsection of womac designed to evaluate the qol of younger and more active patients. several validated versions of this questionnaire have been published in various countries.14-16 (submitted: 18 june 2022 – revised version received: 21 july 2022 – accepted: 01 august 2023 – published online: 26 february 2023) 71j contemp med sci | vol. 9, no. 1, january-february 2023: 70–76 a. sabaghzadeh et al. original reliability and validity of koos in iranian population a useful hrqol should be able to measure the important fields and health issues of a specific disease. these significant domains and issues may be different in different cultural and social backgrounds. koos questionnaire has been recently shown to assess many fields and domains. numerous researchers such as ackerman (2021),17 peer and lane (2013),18 and bekkers (2009)19 have also confirmed the validity and reliability of this questionnaire. koos questionnaire has been extensively utilized in english-speaking countries for the clinical assessment of the severity of the injury and improvement in the performance for monitoring the treatment outcomes. this tool is one of the disease-specific questionnaires with extensive application in various papers. it has been translated into 49 languages so far. to use this questionnaire for non-english-speaking patients, the questionnaire should be adapted in terms of cultural factors in addition to the translation. its reliability and validity should be also evaluated. in this regard, the reliability and validity of this questionnaire were confirmed by various researchers including roos et al. (1998)15 in sweden, xie et al. (2006)20 in singapore, roy et al. (2016)21 and zhang et al. (2019)22 in china, ranjan kumar et al. (2021)23 in india, vaquero et al. (2014)24 and lizaur-atilla et al. (2019)25 in spain, chang et al. (2019)26 in hong kong, alfadhel et al. (2018)27 in saudi arabia, ateef et al. (2017)1 among ordo-speaking population, paradowski et al. (2014)28 in poland, and ornetti et al. (2008)29 in france. koos questionnaire includes 42 questions in five dimensions investigating 5 disease-related concepts including pain (9 questions), other disease-related symptoms (7 questions), daily activities (17 questions), sports and recreational activities (5 questions), and qol related to the knee problems (4 questions). this questionnaire was designed and developed based on the likert version and womac questionnaire on a 5-option scale.15 as mentioned, due to the complexity of structures, the questionnaire developed in a country cannot be applied in another country just by translation. the questionnaire should be customized in terms of cultural and traditional issues to be applicable in a different environment. for instance, the daily activities in the eastern and middle east communities require more extensive flexibility and range of motion which should be considered in the design of the questionnaire.30 this research, thus, tried to assess the reliability and validity of the koos questionnaire in the iranian population to offer a valid knee injury and osteoarthritis outcome score (koos) evaluation tool. this study is aimed to translate and culturally adapt the koos questionnaire to iranian culture and assess its reliability and validity in the iranian population. research method this research was non-experimental and methodological. the translation was achieved by a forward and backward translation of the main english version of the questionnaire into persian according to the available instructions. after translation of the questionnaire into english, it was presented to 5 experts in several stages. the questionnaire was finally confirmed after final evaluations and resolving the deficiencies of the translation as well as collecting the comments of the experts. population and sample the statistical population included knee osteoarthritis patients. totally, 127 knee osteoarthritis patients referred to akhtar center were sampled by the available sampling method based on sample adequacy. the age of the participants ranged from 40 to 70; they referred to akhtar hospital within a twomonth period. the questionnaires were presented to them after obtaining their written consent and providing them with a necessary explanation of the research process. the questionnaires were distributed in two stages. once in the first session and then, two weeks after the first session to test the repeatability of the tests. after collecting the questionnaires in a 14-day period, the information of 127 questionnaires could be analyzed which was employed in the following. research tool koos is a questionnaire designed to detect the short-term and long-term consequences of knee injuries in patients. this questionnaire is a self-administrated test evaluating five consequences of pain, symptoms activities of daily life (adl) function, sports and recreation function, and qol. it also estimates the main indicators of the knee injury consequences which can be employed to assess the duration of the knee injuries and the treatment outcomes. five aspects of the questionnaire are scored separately. the aspects of pain, symptoms, adl, sports and recreation functions, and qol have 9, 7, 17, 5, and 4 statements, respectively. five-point likert spectrum was employed for answering which varied from zero, representing no problem, to four indicating very severe. to interpret the scores, they were converted into a scale varying from 0 (severe knee problems) to 100 (no problem). this type of scoring is common in orthopedic and general scales. these scores indicated the percentage of the attained score. data analysis method to determine the reliability, two aspects of reliability stability and internal consistency were employed; while the validity of the questionnaire was assessed based on face, content, and construct validities. research findings demographic information the statistical population included 127 knee osteoarthritis patients referring to akhtar hospital. more than half of the participants (51.1%) were 30–45 years old. 23.6% of them were younger than 30. 14.2% of the responders were older than 55 while the remaining 11.1% were in the age range of 55–64 years old. 63% of the responders were female and the remaining 37% were men. 73.3% of the responders had no regular sports activities while 26.7 % of them followed a regular sports activity regime. reliability of the questionnaire reliability is defined as a degree of similarity of the results during a specific period under similar conditions and through a similar methodology which can be measured by repeatability and reproducibility. the reliability of the questionnaire was determined by internal consistency and stability of the reliability. 72 j contemp med sci | vol. 9, no. 1, january-february 2023: 70–76 reliability and validity of koos in iranian population original a. sabaghzadeh et al. a) internal consistency internal consistency or internal stability of a questionnaire refers to the level of consistency between a group of items that measure a structure. cronbach’s alpha is the most common coefficient used to determine internal consistency. the reliability coefficient of 0.7 is a suitable value for a question to remain in the questionnaire. the higher the value of this coefficient, the higher the internal stability of the questionnaire. the alpha coefficient must be calculated to determine the internal stability when the likert scale is used in the tool. in this research, spss software was employed to assess cronbach’s alpha whose results can be observed in the following figure (gliem et al., 2005).31 as can be seen, the level of cronbach’s alpha was sufficiently high in all dimensions except for the symptoms. the cronbach’s alpha of the symptom dimension was lower than 0.7, reflecting the lack of internal consistency. based on spss software, the s4 statement (do you feel wear or hear any additional sound upon moving your knee?) declined the cronbach’s alpha whose elimination incremented the amount of the cronbach’s alpha to 0.755 table 1. b) stability of reliability the test-retest method was utilized to determine the stability of the reliability. in this method, a test is repeated twice under similar conditions in a fixed interval. the correlation between the two tests results is then determined as listed in the following table 2. validity of the questionnaire in this research, the validity of the questionnaire was explored based on face, content, and construct validities as will be discussed in the continue. table 1. cronbach’s alpha results dimension number of statements cronbach’s alpha unconfirmed statement cronbach’s alpha upon eliminating the statement pain 9 0.879 – – symptoms 7 0.6442 s4 0.755 adl 17 0.941 – – recreational and sport activities 5 0.897 – – knee injury-related qol 4 0.827 – – table 2. correlation results of test-retest dimension test-retest correlation significance stability of the reliability pain 0.887 0.001 desirable symptoms 0.823 0.000 desirable adl 0.899 0.004 desirable sports and recreational activities 0.976 0.001 desirable knee problem-related qol 0.989 0.001 desirable correlation coefficients higher than 0.8 imply the stability of the reliability of the questionnaire.32 as seen, all dimensions had high stability of reliability. a) face validity the face validity searches for the answer to this question: is the appearance of the designed tool relevant to the aim of the research? do the responders to the tool agree on the sentences and wording of the tool? is the perception of the non-expert people (target group) the same as the perception expected by the researcher? are the details and general aspects of the tool acceptable for the responders? (drost, 2011)33 the qualitative and quantitative methods can be employed to determine the face validity of the tool, here, the quantitative method was adopted using the item impact score. to this end, a five-scale likert spectrum was considered for each item: completely important (score: 5), important (score: 4), moderately important (score: 3), a little important (score: 2), and not important at all (score: 1). the questionnaire was then presented to the target group (15 experts in the field of knee injuries) to determine the face validity. the face validity was then calculated by the item impact score after collecting the filled questionnaire: item impact = ∑ ×( )f n significance the scores above 1.5 indicate the face validity of the question. the results revealed that all the statements had acceptable face validity. b) content validity the content validity generally seeks the answers to these questions: does the designed tool cover all important and major dimensions of the concept understudy? does the tool construct investigate the item it should explore? are the details and general aspects of the tool acceptable for the relevant experts? to determine the content validity, both qualitative and quantitative methods can be utilized. the content validity 73j contemp med sci | vol. 9, no. 1, january-february 2023: 70–76 a. sabaghzadeh et al. original reliability and validity of koos in iranian population can be quantitatively assessed based on the experts’ comments or by calculating the content validity ratio (cvr) and content validity index (cvi).34 to make sure on selecting the most important and correct content (necessity of the questions), the cvr can be used while cvi can be applied to make sure that the questions are designed in the best way to measure the intended content.35 to this end, 15 experts were asked to comment on each item of the tool and determine whether it is necessary, useful but not necessary, or unnecessary. cvr can be determined by the following equation: cvr = −( )ne n n / / 2 2 in the above equation, ne denotes the number of evaluators who decided on the necessity or usefulness of the statement; while n represents the total number of the referrers or evaluators of a statement. regarding the minimum cvr of the one-way lawshe test and as the number of evaluators was 15, the minimum cvr of the questionnaire was 0.49 (lawshe 1975).36 cvi can be calculated after determining cvr. to this end, the evaluators should comment on the specificity, feasibility, and clarity of the items based on a four-scale likert spectrum. to determine cvi, the experts were asked to determine the relevance of each statement to the following fourscale spectrum: • irrelevant, • requiring fundamental revision, • relevant but requiring revision, • completely relevant cvi can be then calculated for each statement using the following equation: cvi number of the evaluators scoring or total number of the evalua = 4 3 ttors the statement will be rejected if cvi<0.7. the statement required revision for 0.7<cvi<0.79; while statements with cvi>0.79 were acceptable. based on the lawshe table, for a target group size of 15 people, the statements with cvr>0.49 are acceptable. the results indicated that cvr was acceptable for all variables. the cvi of all statements exceeded 0.79 indicating the acceptable content validity of all statements. c) construct validity the construct validity of a measurement tool indicates how much the tool evaluates a construct or a characteristic with a theoretical basis. in this research, the construct validity was determined based on three indices of confirmatory factor analysis, convergent validity, and divergent validity using smart pls software. 1. confirmatory factor analysis this method shows the extent of the proper choice of the measurement statements of a construct. this method, indeed, determines whether the questions of the questionnaire are properly selected to explore the relevant factor. therefore, confirmatory factor analysis is a method to assess the validity of the questionnaire. it is also known as the construct validity or measurement model. the purpose is to make sure of the regular factor structure. the confirmatory factor analysis is used to make sure of the construct after identifying the statements for the major factors. the strength of the relationship between the dimension and statement is shown by factor load. factor load ranges from 0 to 1. factor loads smaller than 0.3 show a weak relationship which can be neglected. factor loads in the range of 0.3–0.6 are acceptable while those exceeding 0.6 indicate highly desirable strength. t-test (t-value) is utilized to assess the significance of the relationship between the variables. as significance is examined at an error level of 0.05, if the amount of the observed factor load was smaller than 1.96, the relationship is not significant.37 regarding the factor load results, the statement of s4 showed a low factor load (0.190) with a t-value<1.96, reflecting its insignificance. therefore, s4 should be eliminated from the questionnaire. 2. convergent validity convergent validity is defined as a strong correlation between the question and its corresponding domain. this is indeed a quantitative measure indicating the internal correlation and cohesion of the statements of a dimension. for a high correlation between the factor loads, the questionnaire has convergent validity. this correlation is essential to make sure that the test evaluated what it must measure. the average variance extracted (ave) and composite reliability (cr), as well as the convergent validity, are listed in the following table 3. convergent validity refers to the correlation which measures the statements of a latent variable. in this regard, convergent validity indicates the internal correlation and cohesion of the statements of measure (variable). the questionnaire has convergent validity if it has a high correlation between the factor loads of the statement. two scales of average variance extracted and composite reliability are required to determine the convergent validity. ave shows the correlation between a construct (latent variable) with its relevant indices. fornell and larcker believed that convergent validity exists when table 3. average variance extracted, collective reliability, and convergent validity dimension ave cr convergent validity pain 0.524 0.907 acceptable symptom all statements 0.408 0.815 unacceptable without s4 0.506 0.843 acceptable adl 0.532 0.950 acceptable sports and recreational activities 0.710 0.924 acceptable knee problem-related qol 0.670 0.886 acceptable 74 j contemp med sci | vol. 9, no. 1, january-february 2023: 70–76 reliability and validity of koos in iranian population original a. sabaghzadeh et al. ave>0.5. composite reliability is a measure to assess the internal fitting of the model. cr can be determined based on the internal consistency of the questions of each factor. the convergent validity exists if ave>0.5, cr>0.7, and cr>ave (kulandaivelan et al., 2017).37 cr>0.7 cr>ave ave>0.5 3. divergent validity divergent validity refers to the low correlation of the statements of a latent variable with other latent variables. based on the method proposed by fornell and larcker (1981),38 acceptable divergent validity is established when the ave of each construct is higher than its shared variance with other constructs (i.e. square of the correlation coefficient of the constructs) in the model. in this way, acceptable divergent validity of a measurement model implies that a construct in the model has higher interaction with its indices rather than other constructs.37 based on fernell and larcker, divergent validity was once rejected considering all statements. the divergent validity of the questionnaire was, however, confirmed upon eliminating s4 table 4. to summarize, the reliability of the questionnaire was explored by the stability of the reliability (test-retest methods) and internal consistency (cronbach’s alpha). the results indicated that the questionnaire had proper stability of reliability. concerning the internal consistency, four dimensions out of the five studied dimensions showed acceptable internal consistency. the dimension of symptoms, however, has cronbach’s alpha value below 0.7 which is unacceptable. using spss software, the statement lowering the internal consistency was identified. the acceptable cronbach’s alpha was achieved after eliminating that statement. the validity of the questionnaire was also assessed based on face, content, and construct validities. the results indicated that all the statements had acceptable face and content validities. concerning construct validity, convergent ad divergent validities, as well as the confirmatory factor analysis, were utilized. the confirmatory factor analysis indicated that statement s4 had a factor load smaller than 0.4 and a significance level below 1.96; thus, it must be eliminated from the questionnaire. in the same way, the convergent and divergent validity of the questionnaire were unacceptable in the presence of s4, whose elimination resulted in the confirmation of the convergent and divergent validities. discussion in this research, the validity of the questionnaire was assessed using face, content, and construct validities. the item impact score of all questions exceeded 1.5, confirming the face validity of all statements. concerning the content validity, the cvr of all variables was higher than 0.49 which is acceptable. moreover, the cvi of all statements was higher than 0.79, indicating the acceptable content validity of all statements. to assess the construct validity, three indices of convergent and divergent validities, as well as the confirmatory factor analysis were employed. the confirmatory factor analysis indicated that s4 had a load factor below 0.4 and a significance level below 1.96, thus it was eliminated from the questionnaire. similarly, the convergent and divergent validities of the questionnaire were confirmed upon withdrawing the mentioned statement. the results revealed the proper validation of the koos questionnaire among the iranian population. the reliability of the questionnaire was assessed based on the stability of reliability (test-retest method) and internal consistency (cronbach’s alpha). the results revealed a high cronbach’s alpha level for all dimensions except for symptoms whose cronbach’s alpha value was below 0.7, reflecting the lack of internal consistency in this dimension. using spss software, the s4 statement was responsible for the low cronbach’s alpha value of this dimension. the cronbach’s alpha value of the dimension of symptoms rose to 0.755 after eliminating the mentioned statement. these results were in line with the cronbach’s alpha values obtained for aims, womac, and sweden versions of koos by roos et al. (1998),15 as well as the work of saraeipour et al. in 2007.39 the lack of internal consistency of the subscale of symptoms was also reported in the spanish version presented by lizaur-utilla et al. (2019)25 and the sweden version developed by roos et al. (1998)15 which can be assigned to the problems in the main version of womac (the basis of koos questionnaire) or imprecise grouping of the items on symptom subset. the test-retest method was applied to determine the stability of reliability. the obtained coefficients were above 0.8 for all dimensions, confirming the stability of reliability. this implies that the koos questionnaire has high reliability among the iranian population. the results of this research indicated that the persian version of the koos questionnaire is a culture-compatible, valid, and reliable tool the same as its other versions in different countries presented by roos et al. table 4. divergent validity by elimination of the statement with weal factor load square of the correlation coefficient between the constructs p s a ps q √ave divergent validity pain (p) 1 0.656 0.665 0.693 0.716 0.723 confirmed symptoms without s4 0.656 1 0.706 0.602 0.529 0.711 confirmed adl (a) 0.665 0.706 1 0.709 0.713 0.729 confirmed sports and recreational activities (sp) 0.693 0.602 0.709 1 0.729 0.824 confirmed knee probelm-related qol (q) 0.716 0.529 0.703 0.729 1 0.818 confirmed as seen, the divergent validity was confirmed after the elimination of statement s4. 75j contemp med sci | vol. 9, no. 1, january-february 2023: 70–76 a. sabaghzadeh et al. original reliability and validity of koos in iranian population conclusion this study indicated that the persian version of the koos questionnaire could be a useful tool in multi-purpose evaluation to help osteoarthritis patients. thanks to its acceptable validity and reliability and proper evaluation, this questionnaire can significantly help the patients and therapists to assess different dimensions of the disease and its impacts on diverse aspects of life. conflict of interest none.  (1998),15 xie et al. (2006),20 roy et al. (2016),21 zhang et al. (2019),22 ranjan-kumar et al. (2021),23 vaquero et al. (2014),24 lizaur-utilla et al. (2019),25 cheng et al. (2019).26 alfadhel et al. (2018),27 ateef et al. (2018),1 paradowski et al. (2014),28 and ornetti et al. (2008).29 as mentioned before, knee arthritis is one of the most prevalent forms of osteoarthritis worldwide. pain and physical disability are two major symptoms of knee osteoarthritis with can significantly affect the health-related quality of life (hrqol) of the patients. osteoarthritis is also one of the most common types of arthritis in people older than 40. it is the most prevalent disease among the elderly whose prevalence is even higher than cardiovascular diseases, hypertension, and diabetes. references 1. ateef, m., kulandaivelan, s., alqahtani, m., 2017, cross-cultural validation of urdu version koos in indian population with primary knee osteoarthritis, international journal of rheumatology, 1(4). 2. williams, m.k., 2006, spector td, osteoarthritis, medicine, 34, 364–368. 3. o’reilly, s., doherty, m., 2003, signs, symptoms, and laboratory tests, in: brandt kd, doherty m, lohmander ls, eds. osteoarthritis. 2nd edn. new york: oxford university press:197e210 4. simms, r.w., 2007, osteoarthritis, 7th ed. philadelphia: wb saunders press. 5. jamtvedt, g., dahm, k.t., christie, a., moe, r.h., haavardsholm, e., holm, i., 2008, physical therapy interventions for patients with osteoarthritis of the knee: an overview of systematic reviews, phys ther, 88 (1), 123–136. 6. jordan, k.m., arden, n.k., doherty, m., bannwarth, b., bijlsma, j.w., dieppe, p., 2003, an evidence based approach to the management of knee osteoarthritis: report of a task force of the standing committee for international clinical studies including therapeutic trials (escisit). ann rheum dis, 62, 1145–1155. 7. salaffi, f., piva, s., barreca, e., 2000, validation of and italian version of the arthritis impact measarement scale 2 (italian-aims2) for patients with osteoarthritis of the knee, rheum, 39, 720–727. 8. saraei-pour, s., salavati, m., akhbari, b., kazem-nezhad, a., 2007, translation and adaptation of knee injury and osteoarthritis outcome score (koos) in to persian and testing persian version reliability among iranians with osteoarthritis, jrehab, 8(1), 42–46. 9. lange, a.k., vanwanseele, b., fiatarone singh, m.a., 2008, strength training for treatment of osteoarthritis of the knee: a systematic review, arthritis rheum, 59, 1488–1494. 10. zhang, y., 2004, prevalence of osteoarthritis of the knee is high in chinese population, available from:url: http://www.hopkins-arthritis. som. jhmi. edu/news-archive/2004/oa_chinese.html. 11. jung, c., kim, e., hwang, m., cho, h., kim, k., lee, s., kim, k., 2010, the research of pain and functional disability assessment scales for knee joint disease, journal of acupuncture research, 27 (2), 23–142. 12. bullinger, m., alonso, j., apolone, g., 1998, translating health status questionnaire and evaluating their quality: the iqola project approach, j clin epidemiol, 51, 913–923. 13. roos, e.m., roos, h.p., ekdahl, c., lohmander, l.s., 1998, knee injury and osteoarthritis outcome score (koos) validation of a swedish version, scand j med sci sport, 4, 39–48. 14. beynnon, b.d., 1998, knee injury and osteoarthritis outcome score (koos) development of a self-administered outcome measure, j forthop sports phys ther, 78, 88–96. 15. roos, e.m., roos, h.p., lohmander, l.s., ekdahl, c., beynnon, b.d., 1998, knee injury and osteoarthritis outcome score (koos)--development of a selfadministered outcome measure, journal of orthopaedic & sports physical therapy, 28 (2), 88–96. 16. kessler, s., lang, s., puhl, w., stove, j., 2003, the knee injury and osteoarthritis outcome scoreda multifunctional questionnaire to measure outcome in knee arthroplasty, z orthop ihre grenzgeb, 141, 277–282. 17. ackerman, i.n., soh, s.e., harris, i.a., cashman, k., heath, e., lorimer, m., graves, s.e., 2021, performance of the hoos-12 and koos-12 instruments for evaluating outcomes from joint replacement surgery, osteoarthritis cartilage, 29 (6), 815–823. 18. peer, m., lane, j., 2013, the knee injury and osteoarthritis outcome score (koos): a review of its psychometric properties in people undergoing total knee arthroplasty, journal of orthopedic & sports physical therapy, 43 (1), 20–28. 19. bekkers, t., de-windt, n., raijmakers, w., dhert, d., saris, a., 2009, validation of the knee injury and osteoarthritis outcome score (koos) for the treatment of focal cartilage lesions, osteoarthritis and cartilage, 17 (11), 1434–1439. 20. xie, f., li, s.c., roos, e.m., fong, k.y., lo, n.n., yeo, s.j., yang, k.y., yeo, w., chong, h.c., thumboo, j., 2006, cross-cultural adaptation and validation of singapore english and chinese versions of the knee injury and osteoarthritis outcome score (koos) in asians with knee osteoarthritis in singapore, osteoarthritis and cartilage, 14 (11), 1098–1103. 21. roy, t.h., cheung, c., shirley, p.c., ngai., n., kevin, k.w., ho., 2016, chinese adaptation and validation of the knee injury and osteoarthritis outcome score (koos) in patients with knee osteoarthritis, rheumatology international, 36 (10), 1449–1454. 22. zhang, q., du, s., zheng, g., chang, b., xie, d., lin, f., xie, p., yu, g., hu, q., liu, d., li, x., 2019, reliability, validity, and responsiveness of the chinese version of the knee injury and osteoarthritis outcome score (koos) in patients with anterior cruciate ligament reconstruction in mainland china, z orthop unfall, 157 (1), 42–47. 23. ranjan kumar j., ramesh kumar s., sujit kumar t., nirmal raj g., tarun g., suresh kumar, s., 2021. cross-cultural validation of hindi version knee injury and osteoarthritis outcome score (koos) in osteoarthritis knee, knee surgery, sports traumatology, arthroscopy, 29 (6), 1742–1749. 24. vaquero, j., longo, u.g., forriol, f., 2014, reliability, validity and responsiveness of the spanish version of the knee injury and osteoarthritis outcome score (koos) in patients with chondral lesion of the knee, knee surg sports traumatol arthrosc, 22, 104–108. 25. lizaur-utilla, a., miralles-muñoz, f.a., gonzalez-parreño, s., lopez-prats, f.a., 2019, validation of the spanish version of the knee injury and osteoarthritis outcome score (koos) for elderly patients with total knee replacement, j. orthop, 37, 2157–2162. 26. cheng, a.s.k., chan, k., chan, s., fan, m., fung, m., lee, o., wong, j.k.k., 2019, cross-cultural adaptation and validation of the hong kong version of the knee injury and osteoarthritis outcome score (hk-koos) for patients with knee osteoarthritis, occupational therapy international, 1–9. 27. alfadhel, s.a., vennu, v., alnahdi, a.h., 2018, cross-cultural adaptation and validation of the saudi arabic version of the knee injury and osteoarthritis outcome score (koos), rheumatol int, 38, 1547–1555. 28. paradowski, p.t., kęska, r., witoński, d., 2015, validation of the polish version of the knee injury and osteoarthritis outcome score (koos) in patients with osteoarthritis undergoing total knee replacement, bmj open, 5, 69–47. 29. ornetti, p., parratte, s., gossec, l., tavernier, c., argenson, j.n., roos, e.m., maillefert, j.f., 2008, cross-cultural adaptation and validation of the french version of the knee injury and osteoarthritis outcome score (koos) in knee osteoarthritis patients, osteoarthritis and cartilage, 16 (4), 423–428. 30. mulholland, s.j., wyss, u.p., 2001, activities of daily living in non-western cultures: range of motion requirements for hip a nd knee joint implants, int j rehabil res, 24 (3), 191–198. 31. gliem, j.a., gliem, r.r., 2005, editors. calculating, interpreting, and reporting cronbach’s alpha reliability coefficient for likert-type scales, midwest 76 j contemp med sci | vol. 9, no. 1, january-february 2023: 70–76 reliability and validity of koos in iranian population original a. sabaghzadeh et al. research-to-practice conference in adult, continuing, and community education. 32. najafi, f., kheyri, b., 2013, investigating the impact of country of origin on customer behavior: investigation of the moderating roles of product involvement and product familiarity on product evaluation and customer behavioral intentions, j marketing manangment, 17, 37–60. 33. drost, e.a., 2011, validity and reliability in social science research, education research & perspectives, 38 (1), 105–123. 34. polit, d.f., beck, c.t., 2006, the content validity index: are you sure you know what’s being reported, critique and recommendations, research in nursing & health, 29 (5), 489–97. 35. newman, i., lim, j., pineda, f., 2013, content validity using a mixed methods approach: its application and development through the use of a table of specifications methodology, j mixed methods research. 36. lawshe, c.h., 1975, a qualitative approach to content validity, personnel psychology, 28 (4), 563–75. 37. kulandaivelan, s., tigdania, n., ateef, m., 2017, prevalence of knee pain and its correlates with specific emphasis on cvd risk factors in hisar urban population, international journal of clinical rheumatology, 12 (4), 91–96. 38. fornell and larcker (1981). 39. saraeipour et al. (2007) this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1318 203j contemp med sci | vol. 8, no. 3, may-june 2022: 203–206 original a descriptive study of cephalic and prosopic anthropometric indices in one-day-old infants in imam ali and kamali hospital, karaj fatemeh kermanian1, marzieh tavakol2, nasim beiranvand3, simin mahakizadeh1,* 1department of anatomical sciences, school of medicine, alborz university of medical sciences, karaj, iran. 2department of allergy and clinical immunology, school of medicine, alborz university of medical sciences, karaj, iran. 3school of medicine, alborz university of medical sciences, karaj, iran. *correspondence to: simin mahakizadeh (email: s.mahakizadeh@abzums.ac.ir) (submitted: 03 march 2022 – revised version received: 21 march 2022 – accepted: 12 april 2022 – published online: 26 june 2022) abstract objective: there is no published literature about the types of head and face shapes in the alborz newborns. therefore, we designed this study to report these important regional data. methods: head length, head width, face length, face width, prosopic and cephalic indices were made on 150 newborns (75 males and 75 females) with a view to establish the criterion of cephalometry for this age group in iran. a special emphasis was given to delivery mode in this study. results: there was no significant difference in cephalic and prosopic indices between two groups (female: p = 0.46; male: p = 0.43). brachycephalic type was dominant and dolicocephalic type was rare in male and female neonates. there was no significant difference in head shape between two groups. hypereuriprosopic and hyperleptoprosopic were dominant and rare types, respectively, in newborns without significant difference between two groups. conclusion: the head and face indices in the newborn infants born by cesarean section were not significantly different from vaginal delivery group. however, the role of nutrition and climate could not be ruled out. keywords: anthropometry, cephalic index, prosopic index, craniofacial, newborn issn 2413-0516 introduction anthropometry, defined as the science of measurement and the art of application of physical properties of the human, is one of the most important studies in medicine.1,2 cephalometry or craniometry means measurement of dimension of skull without soft tissue. cephalic and craniofacial indexes, especially in the first days after birth, are important for the assessment of neonatal health status and their findings are used in different branches of medicine.3 in recent years, craniofacial anthropometry has become an important tool used by clinical geneticists, forensic experts and reconstructive surgeons.3 in this regard, researchers in various countries and societies have sought to collect craniofacial anthropometric indexes so that they define and present proportional facial indices of specific communities and ethic groups.4,5 since the evaluation of such indicators reflects the development of the brain growth and facial shapes, it has received much attention from researchers and health physicians.1 the brain and skull grows at different rates and most changes occur from the first month of birth until the sixth years of age.3 based on complex process bone growth in different directions, cephalometry can be explained. head length and width are the most important dimensions of the skull. cephalometry is carried out utilizing several methods, including photogrammetry, ultrasound, ct scan, mri, and the use of standard lateral skull radiographs or cephalograms.6,7 determination of anthropometric measurements and reference ranges of each ethnic group is therefore essential for indicating the degree of deviations from normal. moreover, background newborn anthropometric data, which could assess deviations from normal, are generally lacking in developing countries. so, the physical measurements of newborn are compared with the standards of other countries. however, relying on these standards brings some limitations related to differences in genetic, nutritional and environmental factors.8,9 at present, very limited data are available regarding the reference ranges of head and facial proportions and anthropometric measurements of the persian population in iran. this study aimed to provide data from karaj to help establish the reference range of craniofacial anthropometric measurements in the newborns karaj population. materials and methods this cross sectional study was undertaken from june, 2019 to november, 2019 as a joint effort by the department of anatomy and pediatrics, alborz university of medical sciences, karaj, iran. the project was approved by the research committee in health sciences at the alborz university of medical sciences by ethic code; ir.abzums.res.1398.097. the ethics committee approved all the ethical considerations of the study. gestational age estimation was based on the first day of the last menstrual period. after taking written permission from the parents of newborns,the head and face of 150 living newborn [male (c/s or nvd) and female (c/s or nvd)], which 47 of them had born by cesarean section (c/s) and 103 of them had born by normal vaginal delivery (nvd), were considered. all the newborns were healthy with no known genetic diseases or specific deformity. infants were weighed in a time interval between 5 to 10 hours following birth, wearing no clothes and diapers, using a scale with accuracy of 100 g. their height was also measured using a tape meter with a precision of 0.5 mm while the infants were being placed in the supine position and their knees straightened from heel to head. then, head dimensions of the infants underwent anthropometric measurement using a standard millimetric caliber (martin saler) with the accuracy ± 0.5 mm based on the international reference 204 j contemp med sci | vol. 8, no. 3, may-june 2022: 203–206 cephalic and prosopic indices in newborns original f. kermanian et al. points.3 head, face and nose indices were determined. all measurements were taken under observation of a pediatrician and during daytime. measurement parameters included are: head length = summit of glabella to farthest occipital point head width = greatest breadth, at right angles to median plane face length = nasion–gnathion height face width = bizygomatic breadth cephalic index = (head width/head length) × 100 prosopic index = (face width/face length) × 100 the above indices were determined on the basis of international anatomical descriptions (williams p, 1995). based on these indices the types of head and face shapes were classified following banister 1995 and panero 197 ( , table 1 ).table 2 all races and populations are divided into four head shape groups: dolichocephalic (long head): the ratio of head length to head width is higher than normal (<74.9). mesocephalic (round head): the average ratio of head length to head width is between 75 to 79.9. brachycephalic (wide head): the ratio of head length to head width is lower than normal (80–84.9). hyperbrachycephalic (super wide head): having a very broad head with a cephalic index of over 85. in term of face shape there are 5 models: hypereuriprosopic (super wide face): having a very wide face with a prosopic index of lower than 79.9. euriprosopic (wide face): having a wide face with a prosopic index between 80 to 84.9. mesoprosopic (round face): the average ratio of head length to head width is between 85 to 89.9. leptoprosopic (long face): having a long face with a prosopic index between 90 to 94.5. hyperleptoprosopic (super long head): having a very long face with a prosopic index of over 95. table 1. various head shape [range of cephalic index (ci) (%)] head shape range of cephalic index (ci) (%) dolicocephalic <74.9 mesocephalic 75–79.9 brachycephalic 80–84.9 hyperbrachycephalic 85–89.9 table 2. various face shape [range of prosopic index (pi) (%)] face shape range of prosopic index (pi) (%) hypereuriprosopic <79.9 euriprosopic 80–84.9 mesoprosopic 85–89.9 leptoprosopic 90–94.9 hyperleptoprosopic >95 the study groups were divided according to the gender (male or female). after gathering and saving the data, they were analyzed with spss 20 software using statistical analysis, t-test, k-2 and pearson index. p < 0.05 were considered statistically significant. results respecting delivery type, 47 (31.3%) were cesarean deliveries, and 103 (68.6%) were vaginal deliveries. the average maternal age was 27.02 ± 5.25 and mean of parity was 1.66 ± 0.64. the mean of gestational age was 38.21 ± 0.92. the research results showed that the average weight and height of the girl infants (n = 75) were 3365.3 g and 492.1 mm while the average weight and height of the boy infants (n = 75) were 3385.5 g and 513.1 mm. the overall mean cephalic indices were 82.71 ± 4.72 and 83.12 ± 4.31 in male and female newborns respectively. when the mean cephalic indices were analyzed according to the type of delivery, it was 81.12 ± 1.54 cm and 81.81 ± 1.65 cm in children born by vaginal and cesarean delivery, respectively, and this difference was not significant (p > 0.001). means and sd of length and width of head and face in addition to cephalic and prosopic indices are depicted in table 3. brachycephalic type was dominant whilst dolicocephalic type was rare in male and femal neonates. there was no significant difference in head shape between two groups (table 4). hypereuriprosopic and hyperleptoprosopic were respectively the most dominant and rare types in newborns without significant difference between two groups (c/s, n = 47; nvd, n = 103) (table 5). table 3. parameters of head and face [there was no significant difference in cephalic and prosopic indices between two groups (p = 0.46, p = 0.43 respectively)] male (n = 75) female (n = 75) variables mean sd mean sd p-value head length 117 3.91 115.8 4.01 0.52 head width 96.71 4.21 96.21 3.91 0.42 face length 55.72 4.21 55.23 3.70 0.32 face width 81.46 3.20 80.04 3.13 0.33 cephalic index 82.71 4.72 83.12 4.31 0.46 prosopic index 68.57 6.41 68.41 5.80 0.43 table 4. distribution of head shapes [brachycephalic type was dominant whilst dolicocephalic type was rare in male and femal neonates. there was no significant difference in head shape between two groups male and female] male (n = 75) female (n = 75) c/s (n = 47) nvd (n = 103) variables n % n % n % n % dolicocephal 5 6.7 1 1.3 2 4.3 4 3.9 mesocephal 14 18.7 19 25.3 10 21.3 23 22.3 brachycephal 29 38.7 32 42.7 20 32.8 41 39.8 hyperbrachycephal 27 36 23 30 15 30 35 34 205j contemp med sci | vol. 8, no. 3, may-june 2022: 203–206 f. kermanian et al. original cephalic and prosopic indices in newborns table 5. distribution of face shapes [hypereuriprosopic and hyperleptoprosopic were respectively the most dominant and rare types in newborns without significant difference between two groups male and female] male (n = 75) female (n = 75) c/s (n = 47) nvd (n = 103) variables n % n % n % n % hypereuriprosopic 67 89.3 66 88.2 41 87.2 92 89.3 euriprosopic 2 2.6 4 5.3 5 10.6 1 0.9 mesoprosopic 4 5.3 3 4 0 0 7 6.7 leptoprosopic 2 2.6 2 2.6 1 2.1 3 2.9 hyperleptoprosopic 0 0 0 0 0 0 0 0 table 6. comparison of the present study and other similar studies [statistical differences between the present project and other similar studies in other provinces of the country in terms of cephalic dimensions] province gender cephalic index prosopic index head shape face shape alborz (present project) male female 82.71 83.12 68.57 68.41 brachycephic hypereuriprosopic kermanshah13 male 81 – brachycephic – gorgan17 female male 84 88.22 euryprosopic mesoprosopic gorgan10 male 78.63 74.3 mesocephalic hypereuriprosopic arak16 female 81.5 94.9 mesocephic hypereuriprosopic zahedan14 male 83.6 86.3 brachycephic euriprosopic discussion in the current survey female and male newborn infants were assessed for cephalometric measurement based on the type of delivery. in this study, brachycephalic type was predominant and dolichocephalic type was rare in terms of head shape in both male and female infants. hyperperiposopia and hyperleptosporosis were the most prevalent and rarest types of facial indexes in infants, respectively. the cephalic indices of this study were higher than golalipour’s study in golestan, iran10 and resembled to jordaan’s study in south africa11 and imami’s study in qazvin, iran.12 the observed cephalic indices showed that the brachycephalic type was dominant and dolicocephalic type was rare in male and female neonates. brachycephalic type was dominant in both nvd and cs groups. these results agree with the findings of iviza13 and heidari.14 in respect to the variation of head shape in various races and geographical zones, it seems that hereditary factors primarily affect the shape of head. however, the additional effect of environment cannot be underestimated.9 the anthropological studies concerning the role of racial elements determined that people’s head shape in pacific ocean were commonly brachycephalic type while in middle east, russia and central parts of europe were mostly mesocephlic type and finally, most of the people in atlantic ocean boundary were mesocephalic type.14 findings of cephalometric studies in karaj showed that brachycephaly and mesocephly have been dominant head types in iranian newborn’s (table 6). the obtained prosopic indices showed that the hypereuryprosopic type of face shape was dominant in male and female newborns among both nvd and cs groups. in parallel with our results, bayat15 and golalipour16 reported that the dominant type of face shapes in iran was hypereuryprosopic. on the contrary, heidari et al. in 2004 reported that the dominant type of face shapes in zahedan was euriprosopic type.14 therefore, it seems that racial differences, nutritional factors and ecological conditions such as climate may influence craniofacial parameters. according to this theory, jahanshahi et al. in 2008 reported that hypereuriprosopic was dominant type in turkman’s male newborns.17 in another study, it has been shown that ethnic differences can even affect brain weight and cranial capacity. they found that brain weight and skull capacity were significantly higher in the persian people compared to the turkmen people.16 conclusion all of all, brachycephalic type was predominant and dolichocephalic type was rare in terms of head shape in both male and female infants. hyperperiposopia and hyperleptosporosis were the most prevalent and rarest types of facial indexes in infants, respectively. acknowledgments we express our heartiest gratitude to the maternity department of emam ali hostital and kamali hospital, alborz university of medical sciences, karaj for cordial help to complete this project. conflicts of interest none.  206 j contemp med sci | vol. 8, no. 3, may-june 2022: 203–206 cephalic and prosopic indices in newborns original f. kermanian et al. references 1. samadi-afshar s. comparison of growth index in exclusive breastfeeding and infant formula feeding in six-month-old infants in east azerbaijan province. depiction of health. 2018;8(4):252-9. 2. mahakizadeh s, moghani-ghoroghi f, moshkdanian g, mokhtari t, hassanzadeh g. the determination of correlation between stature and upper limb and hand measurements in iranian adults. forensic science international. 2016;260:27-30. 3. williams p, dyson m, dussak j. bannister. lh; berry, mm; collins, p. & fergson, mwj gray’s anatomy. skeletal system 38th ed elbs with churchill livingston london. 1995:607-12. 4. azizi m, hassanzadeh g, barbarestani m, sadr m, dehbashipour a, alaghbandha n, et al. comparative anthropometric analysis of facial dimensions and types in qazvin, iran and deraghazi khan, pakistan. anatomical sciences journal. 2014;11(3):119-26. 5. gholamreza h, makan s, noushin a, ali d, mohammad abrar a, omran heydar z. anthropometric characteristics of craniums in residents of qazvin, iran and dera ghazi khan, pakistan: a comparative study. anatomical sciences journal. 2013;10(1):43–49. 6. kawasaki y, yoshida t, matsui m, hiraiwa a, inomata s, tamura k, et al. clinical factors that affect the relationship between head circumference and brain volume in very‐low‐birth‐weight infants. journal of neuroimaging. 2019;29(1):104-10. 7. šimič klarić a, tomić rajić m, tesari crnković h. timing of head circumference measurement in newborns. clinical pediatrics. 2014;53(5):456-9. 8. ghosh a, sinha p. an economised craniofacial identification system. forensic science international. 2001;117(1-2):109-19. 9. hackman da, farah mj, meaney mj. socioeconomic status and the brain: mechanistic insights from human and animal research. nature reviews neuroscience. 2010;11(9):651-9. 10. golalipour mj, jahanshahi m, haidari k. the variation of head and face shapes in female newborns in the south-east of the caspian sea (irangorgan). european journal of anatomy. 2021;9(2):95-8. 11. jordaan h. neonatal and maternal cranial form. south african medical journal. 1976;50(52):2064-8. 12. mibodi i, frahani m. study of normal range of anatomical dimensions of one-day old newborn by cephalometry. journal of medical council of islamic republic of iran. 1996;14(1):1-8. 13. eivazi s, mastery farahani r. the cephalometric neurocranial index of one-day-old male newborns in kermanshah by anthropometry. anatomical sciences journal. 2013;10(1):51-6. 14. heidari z, mahmoodzadeh sagheb hr, mohammadi m, noori moogehi smh, arab a. cephalic and prosopic indices: comparison in oneday newborn boys in zahedan. tehran university medical journal. 2004;62(2):156-65. 15. bayat pd, ghanbari a. the evaluation of craniofacial dimensions in female arak newborns (central iran) in comparison with other iranian racial subgroups. eur j anat. 2009;13(2):77-82. 16. golalipour mj, hosseinpour kr. estimation of the cranial capacity and brain weight of iranian female newborns. eur j anat. 2006;10(2): 49-52. 17. jahanshahi m, golalipour mj, heidari k. the effect of ethnicity on facial anthropometry in northern iran. singapore medical journal. 2008;49(11):940-3. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.1225 349j contemp med sci | vol. 7, no. 6, november-december 2021: 349–352 original surgical early complications of the laparoscopic insertion of peritoneal dialysis catheter: a cross-sectional descriptive two-center study in ahvaz, southwest of iran mohammad ali khaksar1*, fatemeh hayati2, amin bahreini3, behrouz shayesteh zadeh3, shokouh shayan pour2 1students’ research committee, ahvaz jundishapour university of medical sciences, ahvaz, iran. 2chronic renal failure research center, jundishapour university of medical sciences, ahvaz, iran. 3general surgery department, jundishapour university of medical sciences, ahvaz, iran. *correspondence to: mohammad ali khaksar (e-mail: khaksar.ma@ajums.ac.ir) introduction there are several treatment methods for patients with kidney failure. these methods include hemodialysis, peritoneal dialysis, and kidney transplantation. due to the limitations of hemodialysis and its complications such as the high prevalence of cardiovascular disease and hypertension, peritoneal dialysis has become more important. peritoneal dialysis has been used to treat end-stage renal disease (esrd) since 1976. this method has been used in iran since 1995 as a kidney replacement therapy (krt) along with other methods (hemodialysis and kidney transplantation), but the rate of its use has been much lower compared with hemodialysis. various studies have shown that compared with hemodialysis, continuous ambulatory peritoneal dialysis (capd) provides a better quality of life for patients. in peritoneal dialysis, an appropriate catheter is placed inside the peritoneal cavity. this catheter can be introduced to the patient’s body by open surgery, through the skin, or by laparoscopy. due to the complications of open surgery, today laparoscopic surgery for peritoneal dialysis catheter insertion has won great popularity owing to fewer complications compared with open surgery. however, the laparoscopic procedure has limited complications.1 in this study, we tried to investigate the complications of peritoneal dialysis catheter insertion surgery in imam khomeini and golestan hospitals in ahvaz, southwest of iran in the period between 2007 and 2019. methods this is a cross-sectional analytic-descriptive and retrospective study conducted in 2020 after obtaining approval from the ethics committee of ahvaz jundishapur university of medical sciences (ajums). the study population included patients with end-stage renal disease who underwent laparoscopic peritoneal catheter (standard double cuffed straight tenckhoff catheter) insertion performed by two experienced surgeons in imam khomeini and golestan hospitals in ahvaz between 2007 and 2019. as pre-operation care, patients were admitted to the general surgery ward and were then evaluated 24 hours before the scheduled time of surgery. afterwards, under general anesthesia, pneumoperitoneum was achieved with a 10-mm hasson port through a vertical umbilical incision, and two 5-mm ports were placed for diagnostic laparoscopy and instrumentation. the peritoneal dialysis catheter was inserted so that the distal cuff was placed preperitoneally, with the catheter tip in the rectovesical pouch. care was taken to ensure that the catheter followed a smooth curve to exit the abdomen in a lateral and downward direction. also, for post-operation care, we observed patients for 24 hours, and in case there were no problems, they were discharged. their dialysis was performed under the supervision of the peritoneal dialysis unit of imam khomeini hospital in ahvaz. the exclusion criterion of this study was incomplete patient record information. all patient files between 2007 and 2019 were reviewed and written reports of the peritoneal dialysis unit of imam khomeini hospital of ahvaz were collected. demographic information of patients included age and sex. in this study, the complications of laparoscopic insertion of peritoneal dialysis catheters were studied in imam khomeini and golestan hospitals of ahvaz. this study included early mechanical and abstract objectives: this study aimed to investigate the surgical complications of this technique of peritoneal dialysis catheter insertion in imam khomeini and golestan hospitals in ahvaz, southwest of iran, in the period between 2007 and 2019. methods: in this retrospective cross-sectional analytic-descriptive study, we examined end-stage renal disease (esrd) patients who underwent laparoscopic peritoneal dialysis catheter insertion between 2007 and 2019. patients' records and follow-up information recorded in the hospital dialysis unit were reviewed and early infectious and mechanical complications of peritoneal dialysis catheter insertion were analyzed. results: in this study, 290 patients (144 males and 138 females) with esrd who have a mean age of 46 ± 19.7 years (1–90 years) were recruited, of whom 8 patients were excluded due to incomplete information in their records, and 68 patients (24.1%) had an early complication of surgery. surgical complications included: catheter exit-site infections in 20 patients (7.1%), peritonitis in 18 patients (6.4%), catheter exit-site leakage in 17 patients (6%), catheter occlusion without migration in 16 patients (5.7%), catheter migration in 7 patients (2.5%), and hemoperitoneum in 7 patients (2.5%). conclusion: due to the low complications of laparoscopic surgery in the insertion of a peritoneal dialysis catheter, this technique is recommended as a safe surgical procedure with low complications. keywords: peritoneal dialysis, early complications, infectious complications, mechanical complications issn 2413-0516 350 j contemp med sci | vol. 7, no. 6, november-december 2021: 349–352 surgical early complications of the laparoscopic insertion of peritoneal dialysis catheter original m.a. khaksar et al. infectious complications (the first 30 days) and included catheter migration and removal, catheter occlusion, catheter leakage, bleeding and peritonitis, and catheter exit-site infection. statistical analysis in the present study, data analysis was performed using spss ver. 23. descriptive analysis of quantitative data involved central indices of mean and median as well as indices of variance dispersion, standard deviation and frequency tables. results laparoscopic catheterization had been performed on 290 patients from 2007 to 2019. eight patients (5 females, 3 males) were excluded from the study due to incomplete information in their records. the patients included 144 males and 138 females, and the mean age of patients was 46 ± 19.7 years (age range between 1 and 90 years). mortality and intraoperative complications were not seen in any of the patients. out of the 282 patients, 214 patients (75.9%), including 104 males and 110 females, did not have any complications, while the rest experienced at least one complication. from among the patients investigated, 20 patients (7.1%) had peritoneal dialysis catheter exit-site infection, including 8 males and 12 females. peritonitis was observed in 18 patients (6.4%), including 13 males and 5 females. the dialysis catheter of 4 patients was removed due to peritonitis. among 18 patients with peritonitis, 10 patients had peritonitis with negative culture and were treated with antibiotics administered intraperitoneally. four patients also developed peritonitis with pseudomonas who were treated with intraperitoneal antibiotics. three patients also developed peritonitis with enterobacteriaceae culture, in one of whom peritoneal dialysis catheter was removed. only one patient had peritonitis with hemolytic streptococcus culture, who was treated with intraperitoneal antibiotics, and finally, the catheter was removed. seventeen patients (6%) had leakage of exit-site of the peritoneal catheter, of whom 10 were male and 7 were female. we observed that the catheter occlusion without catheter migration occurred in 16 patients (5.7%), of whom 9 were male and 7 was female and underwent catheter removal. seven patients (2.5%) had peritoneal dialysis catheter migration, all of whom were male. finally, 7 patients (2.5%) had hemoperitoneum, of whom 4 were male and 3 were female. thus, in general, the most frequent early complication of peritoneal dialysis catheter insertion was a mechanical complication as opposed to infectious complications (16.7% vs. 13.5%). the most common early mechanical complication was catheter leakage (6%). early infectious complications were exit-site catheter infection (7.1%) and peritonitis (6.4%) (figure 1). in the present study, 75.9 % of the patients had no complications, while others had at least one complication (table 1). the results demonstrated that there was a significant association between catheter migration and gender (table 2). discussion the present study was a cross-sectional, retrospective, analytic-descriptive study that examined the prevalence of complications of laparoscopic peritoneal dialysis catheter surgery in esrd patients in the first 30 days after surgery. in this study, overall, 24.1% of the patients experienced at least one early complication, which is in line with other studies which have reported the rate of such complications to be between 15% and 37.8%.2–4 by contrast, in garcía-cruz et al., none of the patients who underwent laparoscopic peritoneal dialysis catheterization suffered from an early complication of peritoneal dialysis catheter insertion.5 the most frequent early complication of peritoneal dialysis catheter insertion in our study was mechanical complication (16.7%). the most common early mechanical complication was catheter leakage (6%). early infectious complications were catheter exit-site infection (7.1%) and peritonitis (6.4%). while most of the similar studies have compared the surgical complications of the two surgical methods of peritoneal dialysis catheter insertion surgery,6–10 there was no room for discussion regarding comparison of surgical methods since the only peritoneal dialysis catheter insertion method in our fig. 1 frequency of early complications of peritoneal dialysis catheter insertion. a: peritonitis after catheter insertion b: catheter exit-site infection c: catheter migration d: catheter occlusion without migration e: intra-abdominal bleeding around the catheter f: leakage of intra-abdominal fluid around the catheter. table 1. frequency of early complications of peritoneal dialysis catheter insertion complication frequency percent valid percent cumulative percent 0 214 75.9 75.9 75.9 1 55 19.5 19.5 95.4 2 11 3.9 3.9 99.3 3 2 0.7 0.7 100 total 282 100 100 table 2. complications and gender complications male (n) female (n) early peritonitis 13 5 0.052 exit-site infection 8 12 0.214 catheter migration 7 0 0.008* catheter occlusion without migration 9 7 0.434 hemoperitoneum 4 3 0.524 leakage of fluid 10 7 0.342 351j contemp med sci | vol. 7, no. 6, november-december 2021: 349–352 m.a. khaksar et al. original surgical early complications of the laparoscopic insertion of peritoneal dialysis catheter centers was the laparoscopic method. this method is more popular than open surgery method. one of the most important reasons for this is the lower damage in this technique of catheter insertion due to the proper view of the peritoneal cavity. as a result, there will be no complications such as perforation of the intestine or improper catheter placement,9,11,12 which were not observed in our study either. one of the most important complications of catheter placement is infection, which is one of the main causes of catheter removal according to different studies. in the present study, the prevalence of catheter exit-site infection and that of peritonitis were 7.1% and 6.4%, respectively, and the catheter of 4 patients was removed due to peritonitis. in this respect, ashegh et al. reported that the most common early complications in their study were infectious complications and peritonitis, and that the catheter of 2 patients was removed due to peritonitis. other studies have reported the rate of exit-site infection to be between 1.2% to 10%.1,2,13,14 moreover, keshvari et al. showed that the most common complication of peritoneal dialysis catheter insertion through open surgery was infectious complications, the most frequent of which was peritonitis.13 in comparison to open surgery, the incidence of infection is fewer in laparoscopic surgery, which might be due to the use of antibiotics before catheter placement, proper education of patients, and small surgical incision sites which can also reduce the risk of infection. displacement and migration of the catheter from the pelvis to other parts of the abdomen is another complication of catheter insertion which was seen in 2.5% of the patients, but it was found to be between 4 and 15% in other studies.1,4,14 this may be due to the use of a laparoscope to properly insert the catheter in place, in addition to other factors including the use of a low volume of dialysis fluid, the use of heparin after catheter placement, the low-volume changes in the first two weeks after catheterization, and the use of laxatives in patients to avoid constipation regardless of the type of surgical technique. rate of fluid leakage from the catheter was 6% in our study, while it was reported to be between 1 and 17% in other studies.6,11,14,17 of course, one study reported that none of their patients had fluid leakage.1 in manouras et al., the prevalence of mechanical complications was 15.3%, and the main complication was related to early leakage (6%),7 which is consistent with our study where the prevalence of mechanical complications was 16.7% and the major mechanical complication was catheter leakage with a rate of 6%. the cause of less leakage in laparoscopic technique is the use of the appropriate laparoscopic technique with direct vision and less damage to the abdominal wall. another complication which was observed in our study was bleeding. this might be due to additional procedures performed in laparoscopic technique, such as the process of puncturing the abdominal wall by trocar and suturing pd catheter to the abdominal cavity or peritoneum in laparoscopic surgery, fixation suture of the omentum, selective liver biopsy, and inguinal hernioplasties.18–20 moreover, coagulation disorders may also cause bleeding. as we stated earlier, the only peritoneal dialysis catheter insertion method in our centers was the laparoscopic technique, so we could not compare surgical complications in open versus laparoscopic methods. since the period of our study involved 12 years, the records of some patients were incomplete, and information about comorbidities, bmi, etc. was not available. therefore more studies to evaluate the association between early complications of the laparoscopic insertion of peritoneal dialysis catheter and factors like comorbidities, bmi, and factors which can affect surgical complications are recommended. also, we suggest that another study be conducted dealing exclusively with the late complications of the laparoscopic insertion of peritoneal dialysis catheter. conclusion considering that overall mechanical complications were more common than infectious complications in the present study, care must be exercised when using a peritoneal dialysis catheter. with a team consisting of experienced nephrologists, experienced surgeons, and well-trained nurses, along with appropriate patient education, the laparoscopic technique can be introduced as a reliable, safe, and uncomplicated method for inserting peritoneal dialysis catheters. conflicts of interest there are no conflicts of interest.  references 1. musbahi a, kanakala v. a modified laparoscopic peritoneal dialysis insertion technique, the ‘one scar technique’ can minimise short and long term complications: a retrospective cohort study. j vasc access. 2020:1129729820961970. 2. ashegh h, rezaii j, esfandiari k, roueentan a, abouzari m. laparoscopic placement of peritoneal dialysis catheters in capd patients: complications and survival. tehran university medical journal tums publications. 2008;66(3):186-90. 3. jwo sc, chen ks, lee cc, chen hy. prospective randomized study for comparison of open surgery with laparoscopic-assisted placement of tenckhoff peritoneal dialysis catheter—a single center experience and literature review. j surg res. 2010;159(1):489-96. 4. prabhakar n, aljamal yn, saleem hy, baloul ms, nyberg sl, farley dr. outcomes of laparoscopic and open capd catheter placement: a singlecenter experience. surgery open science. 2019;1(1):20-4. 5. garcía-cruz e, vera-rivera m, molina jc, mallafré-sala j, alcaraz a. laparoscopic placement of peritoneal dialysis catheter: description and results of a two-port. official publication of the spanish society of nephrology. 2010;30(3):354-9. 6. tsimoyiannis ec, siakas p, glantzounis g, toli c, sferopoulos g, pappas m, et al. laparoscopic placement of the tenckhoff catheter for peritoneal dialysis. surgical laparoscopy endoscopy & percutaneous techniques. 2000;10(4):218-21. 7. manouras aj, kekis pb, stamou km, konstadoulakis mm, apostolidis ns. laparoscopic placement of oreopoulos–zellerman catheters in capd patients. peritoneal dialysis international. 2004;24(3):252-5. 8. chen y, shao y, xu j. the survival and complication rates of laparoscopic versus open catheter placement in peritoneal dialysis patients: a metaanalysis. surg laparosc endosc percutan tech. 2015;25(5):440-3. 9. li jr, chen ch, cheng cl, yang ck, ou yc, ho hc, et al. five-year experience of peritoneal dialysis catheter placement. journal of the chinese medical association. 2012;75(7):309-13. 10. van laanen jhh, cornelis t, mees bm, litjens ej, van loon mm, tordoir jhm, et al. randomized controlled trial comparing open versus laparoscopic placement of a peritoneal dialysis catheter and outcomes: the capd i trial. perit dial int. 2018;38(2):104-12. 11. bircan hy, kulah e. effects of a novel peritoneal dialysis: the open versus laparoscopic preperitoneal tunneling technique. ther apher dial. 2016;20(1):66-72. 15,16 352 j contemp med sci | vol. 7, no. 6, november-december 2021: 349–352 surgical early complications of the laparoscopic insertion of peritoneal dialysis catheter original m.a. khaksar et al. 12. shrestha bm, shrestha d, kumar a, shrestha a, boyes sa, wilkie me. advanced laparoscopic peritoneal dialysis catheter insertion: systematic review and meta-analysis. perit dial int. 2018;38(3): 163-71. 13. keshvari a, lesan pezeshki m, younesian m. mechanical and infectious complications of peritoneal dialysis catheters in surgical wards of imam khomeini hospital: 7 year experience. tehran university medical journal tums publications. 2006;64(6):95-102. 14. shahbandari m, amiran a. comparison of the complications of open surgery versus laparoscopic technique in insertion of peritoneal dialysis catheter. j res med sci. 2019;24:85. 15. shyr y, su c, lui w. complications of continuous ambulatory peritoneal dialysis: one surgeon’s experience with 668 patient-month followup. zhonghua yi xue za zhi = chinese medical journal; free china ed. 1995;55(4):307-14. 16. gadallah mf, arora n, arumugam r, moles k. role of fogarty catheter manipulation in management of migrated, nonfunctional peritoneal dialysis catheters. american journal of kidney diseases. 2000;35(2):301-5. 17. özener ç, bihorac a, akoglu e. technical survival of capd catheters: comparison between percutaneous and conventional surgical placement techniques. nephrology dialysis transplantation. 2001;16(9):1893-9. 18. qiao q, lu g, xu d, zhou x, li l. a comparison of two methods for catheterization in peritoneal dialysis. jiangsu med j. 2012;38(23):2812-4. 19. öğünç g, tuncer m, öğünç d, yardimsever m, ersoy f. laparoscopic omental fixation technique versus open surgical placement of peritoneal dialysis catheters. surgical endoscopy and other interventional techniques. 2003;17(11):1749-55. 20. xie p, yuan l, liu f. the comparison of efficacy and safety between laparoscopic and conventional placement of peritoneal dialysis catheters. hebei med. 2014;4:562-6. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1076 242 j contemp med sci | vol. 7, no. 4, july-august 2021: 242–246 original correlation between sphenoclivus angle and gnathic angle with age and gender in iranian population using ct-scan gholamreza hassanzadeh1,2, ghazaleh moshkdanian3, morteza gholaminejhad2, mahnaz poorhassan4, babak ebrahimi2, reza habibi5, parichehr pasbakhsh2, tayebeh rastegar2, mahdad abdi2, yasmin yazdooei6, masoumeh gity7,*, 1department of neuroscience and addiction studies, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. 2department of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. 3institute for basic sciences, kashan university of medical sciences, kashan, iran. 4department of anatomy, school of medicine, shahid beheshti university of medical sciences, tehran, iran. 5school of medicine, tehran university of medical sciences, tehran, iran. 6student research committee, golestan university of medical sciences, gorgan, iran. 7department of radiology, advanced diagnostic and interventional radiology research center (adir), breast disease research center (bdrc), imam khomeini complex hospital, tehran, iran. *correspondence to: masoumeh gity (email: imamhospital@tums.ac.ir). (submitted: 02 april 2021 – revised version received: 25 april 2021 – accepted: 12 may 2021 – published online: 26 august 2021) introduction anthropology is a science that makes it possible to quantify the dimensions of the human body in different societies.1–3 cephalometry as an important branch of anthropology being employed to calculate the dimensions of the head and face used in archeology, surgery, forensic medicine, medical engineering, and industry.4–6 in the clinical set, anthropometric studies of the craniofacial diameters can help in the diagnosis and predicting disorders.7 the measurement of each index in the craniofacial complex such as sphenoclivus and gnathic angulation is highly required to identify differences in the skull, predict the future skeletal jaw pattern of a child, and determine the best way to achieve brain tumors.8 cephalometric measurements can be taken using either a digital tracing method or a traditional method.9 but computed tomography (ct) joined the tools of the anthropologist improving the volume of information. it allows us to study the anatomical characteristics of external and internal structures that can’t be evaluated by the traditional morphometric instruments.5,10,11 however, so many studies have done to assess and compare the values of sphenoclivus angle and gnathic angle of each normal and abnormal skeletal group but very few studies are carried out to collect and group this information in iran, and very rarely data exist to find the reliable relationship among the skeletal patterns of individuals.12 thus in view of the fact, that the degree of its slope could also vary depending on the race or area in which the research was carried out.13 the present cephalometric study was conducted to find the difference in the values of sphenoclivus angle among iranian adults and to explore the relationship between the sphenoclivus angle and gnathic angle. materials and methods this cross-sectional study was conducted from september 2018 to september 2019 in imaging centers affiliated with the faculty of medicine, tehran university of medical sciences on 140 subjects (70 males and 70 females) aged between 18 to 60 years and the conscious consents were obtained. this study was approved by the ethics committee with the id of ir.medicine.tums.rec.1398.602. subjects were selected randomly. abstract objectives the aim of this study was to assess whether is a reliable correlation between the cranial and gnathic angulations in the iranian population. methods in a cross-sectional study, 140 patients of tehran university of medical sciences hospitals (70 males and 70 females with an age range of 18–60 years) were selected. sphenoclivus (cranial base) and gnathic angles were calculated for each case. then, the data were analyzed using spss version 22. results statistical analysis showed a relationship between gnathic angle and female (p < 0.05), but no positive relationship was seen between sphenoclivus angle and gender. there was a significant relationship between sphenoclivus angle and age among men. no significant relationship was found between the gnathic and sphenoclivus angles. conclusion sphenoclivus angle has the closest link with age in males. the gnathic angle has also a positive relationship with females. our findings suggest an independent growth pattern between the sphenoclivus angle and the gnathic angle. keywords anthropometry, sphenoclivus angle, cranial base angle, gnathic angle, cephalometric measurements issn 2413-0516 http://orcid.org/0000-0002-3056-4080 243j contemp med sci | vol. 7, no. 4, july-august 2021: 242–246 g. hassanzadeh et al. original correlation between sphenoclivus angle and gnathic angle with age and gender in iranian population using ct-scan exclusion criteria were in the following: • people with skull bone abnormalities (congenital or acquired). • people with a history of trauma and skull fractures. • people with a history of maxillofacial surgery, including cosmetic surgery. • people of non-iranian nationality. the parameters were measured in the whole scans based on the following definitions: nasion: the middle point of the junction between the nasal bones with the frontal bone. basion: the midpoint of the anterior margin of the foramen magnum. prosthion: the lower edge of the maxillary alveolar process at the site of the anterior central teeth. pituitary point: the anterior edge of the sellaturcica. nasion-basionline: it is a line connecting the nasion and basion. basion-prosthion line: it is a line connecting the basion and prosthion. sphenoid plane: a plane that passes over the jugum of the sphenoid bone. basion-pituitary line: it is a line connecting the basion and pituitary point. gnathic angle: it is an angle between the nasion-basion and basion-prosthion. sphenoclivus angle (cranial base angle): it is an angle between the sphenoid plane and the basionpituitary point. we first drew a line from the nasion to the basion and then a line from the basion to the prosthion. the angle between the two lines in terms of age and sex in a table and called it the gnathic angle (fig. 1). in the next step, we measured the sphenoclivus angle, so that we consider a plane that passes over the jugum of the sphenoid to be the sphenoid plane. after that a line fig. 1 gnathic angle. this angle is between the nasion-basion line and the nasion-prosthion line. n: nasion, b: basion, pr: prosthion, nb: nasion-basion line, bpr: basion-prosthion line, ga: gnathic angle. fig. 2 sphenoclivus angle. this angle is between the sphenoid plane and the basion-pituitary point line. b: basion, pp: pituitary point, sp: sphenoid plane, bpp: basion-pituitary point, sca: sphenoclivus angle. table 1. comparing the anthropometric measurement of sphenoclivus angle and gnathic angle between males and females in iranian population gender male female mean sd mean sd p-value sphenoclivus angle 112.858 7.637 110.200 15.7096 0.192 gnathic angle 36.915 3.5254 38.557 4.5550 0.023 from the basion to the pituitary point was drawn, now the angle between these two lines was named sphenoclivus angle (fig. 2). statistical analysis the data were analyzed by spss software (version 22). in this study, to describe quantitative variables, have been reported as mean ± standard deviation (sd). frequency and percentages were reported to describe qualitative variables. to compare the quantitative variables, the t-test, and to compare the qualitative variables, the chi-square test was used. pearson’s correlation coefficient test was used to obtain the correlations between quantitative variables. p < 0.05 was considered statistically significant. results the average angle of the sphenoclivus in women was found 110.200 degrees and in men with a difference of about two degrees seen 112.858. according to the chi-square test, there was no significant difference between the two genders in the sphenoclivus angle (p = 0.192). chi-square test analysis also showed that the anthropometric measurement of the gnathic angle was significantly higher for females than that in males (0.023) (table 1). 244 j contemp med sci | vol. 7, no. 4, july-august 2021: 242–246 correlation between sphenoclivus angle and gnathic angle with age and gender in iranian population using ct-scan original g. hassanzadeh et al. those expected for his/her racial or ethnic group are discovered.9,14 among all morphometric landmarks, the sphenoclivus angle serves as a reference structure to determine the skeletal type in the cephalometric analysis.15 in our study, the mean of the sphenoclivus angles was assessed 110.200° in females and 112.858° in males with no significant differences. another study on iranian showed the value of the cranial base angle in females is more than males.16 although the presented data is in contrast with our findings in view of the fact that various iranian ethnicities distribute all over the country, it is expectable to have different anthropometric features from city to city. similar studies in india and brazil also showed the wider sphenoclivus angle among females rather than males.17,18 furthermore the differences between women and men, table 4 shows the values of the cranial base angle can be found vary a lot in different places19–24 although, several authors declare that some anomalies such as herniation of the cerebellum, chiari malformations, malocclusion and craniocervical junction abnormality associated with the changes of this angle. royo-salvador affirmed that there is platybasia when the cranial base angle is more than 140°25 but some authors declared that the cranial base angle surpasses 140° and do not agree on the limit value for considering a skull platybasia.17 in agreement with these reports, a study in finland showed the sphenoclivus in both normal and otosclerosis patient groups was the same and no significant changes in degree were mentioned among finnish people.26 more studies stated that the bigger sphenoclivus angles exist among the normal group in comparison with the chiari patients group in turkey23 versus brazilian revealed the more value sphenoclivus angle among the normal group.27 although there was a slight difference between the mean values for the normal and patients groups but no significant differences between the two groups were reported. moreover some anthropologists mentioned a positive correlation between the slope of sphenoclivus angle and skin color so that this angle was calculated more than 125° and wider among the nonwhite population in comparison to that in white one.17 therefore, the degree of the slope could be vary depending on the ethnicity, race, or area in which the research was carried out.13 so it is necessary to know in detail the anatomical structures to have a database bank for the comparison between normal and patient’s samples although our project only focused on the sphenoclivus angle in the normal population. regarding the relation between age and the value of the sphenoclivus angle, we found a significant correlation between age and this angle in males so that the older men, the wider sphenoclivus angle value. in contrast with our results, some anthropologists declared that there was no relation between age and the value angle.17,18 moreover, the consensus of different authors proved that the sphenoclivus angle has a considerable influence upon the mandibular and maxilla position, thus it determines the skeletal jaw pattern of an individual. although other researchers have obtained conflicting evidence, that there is no connection between the cranial base angle and the static position of the jaw.21,28 in spite of cranial base angle malformation, gnathic angle changes are presented as an index showing the presence of impairment in the sub nasal area of individuals. in comparison to the sphenoclivus angle, our results found no significant correlation between gnathic angle and age. although, a positive relationship between gnathic angle and gender was seen. a significant correlation was observed between sphenoclivus angle and age in the male group. and, no positive relation was observed between gnathic angle and age in both genders (table 2). according to the statistical test performed in this study, there was no significant difference between the sphenoclivus angle and the gnathic angle in both genders with a correlation coefficient of 0.005, it has predictive power (table 3). the volume of sphenoclivus angle increases in males with age. by using the following formula, through entering the age in this formula (x), the size of the sphenoclivus angle can be predicted with a power of 0.25 (fig. 3). y = (0.59 × x) _ 27.08 discussion the main goal of cephalometry is to elevate the relationships between the person’s actual craniofacial morphology and table 2. correlation between sphenoclivus angle and gnathic angle with age in iranian population age male female correlation coefficient (r) pvalue correlation coefficient (r) pvalue sphenoclivus angle 0.251 0.026 0.053 0.681 gnathic angle 0.190 0.150 −0.097 0.499 table 3. correlation between the gnathic and sphenoclivus angle in iranian population sphenoclivus angle male female correlation coefficient pvalue correlation coefficient pvalue gnathic angle 0.005 0.963 0.058 0. 702 fig. 3 correlation between age and sphenoclivus angle among men predicted by regression models. 245j contemp med sci | vol. 7, no. 4, july-august 2021: 242–246 g. hassanzadeh et al. original correlation between sphenoclivus angle and gnathic angle with age and gender in iranian population using ct-scan table 4. comparison of measured sphenoclivus angle in different studies place of study tehran, iran isfahan, iran brazil india turkey kenya spain brazil usa japan romania usa sample number 140 100 160 255 43 100 292 33 10 46 50 96 reference number present study 15 16 17 22 23 24 26 18 19 20 21 female 110.2 131.72 115.56 131.42 – – – – – – – – male 112.85 128.65 115.28 129.7 – – – – – – – – mean – – – – 121 103–143 115–140 107–132 107–124 133.96 131.4 129.80 remarkably, no reliable correlation was reported between sphenoclivus and gnathic angles. our findings indicate the independent growth pattern between two important indexes of the craniofacial complex and vastly express the necessity for collecting the morphometric information not only in the different populations but also in different genders separately. conclusion in conclusion, with regard to this fact that many more studies are still needed on the measurement and analysis of craniofacial indexes. our results indicate that the sphenoclivus and gnathic angles have no significant relation with each other in the iranian population. a significant relationship exists between the gnathic angle and gender in females. but, only sphenoclivus angle has a positive relationship with age in males. acknowledgment the authors would like to appreciate the effort of all those who helped to enrich this project. special gratitude also goes to all the 140 patients of tehran university of medical sciences hospitals selected in this study. conflicts of interest none declared.  references 1. mohammed i, mokhtari t, ijaz s, ngaski aa, milanifard m, hassanzadeh g. anthropometric study of nasal index in hausa ethnic population of northwestern nigeria. journal of contemporary medical sciences. 2018;4(1). 2. zolbin m, hassanzadeh g, mokhtari t, arabkheradmand a, hassanzadeh s. anthropometric studies of nasal parameters of qazvin residents, iran. moj anat physiol. 2015;1(1):00002. 3. ebrahimi b, madadi s, noori l, navid s, darvishi m, alizamir t. the stature estimation from students’ forearm and hand length in hamadan university of medical sciences, iran. journal of contemporary medical sciences. 2020;6(5). 4. navaei f, ghaffari n, mojaverrostami s, dodongeh m, nemati m, hassanzadeh g. stature estimation from facial measurements in medical students of tehran university of medical sciences: an iranian population. iraq medical journal. 2018;2(3):68-71. 5. dodangheh m, mokhtari t, mojaverrostami s, nemati m, zarbakhsh s, arabkheradmand a, et al. anthropometric study of the facial index in the population of medical students in tehran university of medical sciences. gmj medicine. 2018;2(1):51-7. 6. ebrahimi b, nemati m, dodangeh m, hassanzadeh g. stature estimation from cranial indices in students of tehran university of medical sciences. scientific journal of forensic medicine. 2021;26(4):0-. 7. hassanzadeh s, alemohammad zb, mokhtari t, arabalidoosti f, rezaei f. correlation between craniofacial parameters and obstructive sleep apnea syndrome in iranian population. iraq medical journal. 2019;3(2). 8. olszewski r, frison l, schoenarts n, khonsari r, odri g, zech f, et al. reproducibility of three-dimensional posterior cranial base angles using low-dose computed tomography. clinical oral investigations. 2017;21(8):2407-14. 9. darkwah wk, kadri a, adormaa bb, aidoo g. cephalometric study of the relationship between facial morphology and ethnicity. translational research in anatomy. 2018;12:20-4. 10. mantini s, ripani m. modern morphometry: new perspectives in physical anthropology. new biotechnology. 2009;25(5):325-30. 11. mohamadi y, mousavi m, pakzad r, hassanzadeh g. anthropometric parameters for access to sella turcica through the nostril. journal of craniofacial surgery. 2016;27(6):e573-e5. 12. kaestle fa, horsburgh ka. ancient dna in anthropology: methods, applications, and ethics. american journal of physical anthropology: the official publication of the american association of physical anthropologists. 2002;119(s35):92-130. 13. camcı h, salmanpour f. cephalometric evaluation of anterior cranial base slope in patients with skeletal class i malocclusion with low or high sna and snb angles. turkish journal of orthodontics. 2020; 33(3):171. 14. ghaffari n, ebrahimi b, nazmara z, nemati m, dodangeh m, alizamir t. assessment of gender dimorphism using cephalometry in iranian population. iraq medical journal. 2020;4(4). 15. cossio l, lópez j, rueda zv, botero-mariaca p. morphological configuration of the cranial base among children aged 8 to 12 years. bmc research notes. 2016;9(1):309. 16. monirifard m, sadeghian s, afshari z, rafiei e, sichani av. relationship between cephalometric cranial base and anterior-posterior features in an iranian population. dental research journal. 2020;17(1):60. 17. netto ds, nascimento srr, ruiz cr. metric analysis of basal sphenoid angle in adult human skulls. einstein (são paulo). 2014;12(3):314-7. 18. gupta pp, dhok am, shaikh st, patil as, gupta d, jagdhane nn. computed tomography evaluation of craniovertebral junction in asymptomatic central rural indian population. journal of neurosciences in rural practice. 2020;11(3):442. 19. lieberman de, ross cf, ravosa mj. the primate cranial base: ontogeny, function, and integration. american journal of physical anthropology: the official publication of the american association of physical anthropologists. 2000;113(s31):117-69. 20. kasai k, moro t, kanazawa e, iwasawa t. relationship between cranial base and maxillofacial morphology. the european journal of orthodontics. 1995;17(5):403-10. 21. panainte i, suciu v, mártha k-i. correlation between cranial base morphology and various types of skeletal anomalies. journal of interdisciplinary medicine. 2017;2(s1):57-61. 22. steinberg b, fraser b. the cranial base in obstructive sleep apnea. journal of oral and maxillofacial surgery. 1995;53(10):1150-4. 23. karagöz f, izgi n, sencer sk. morphometric measurements of the cranium in patients with chiari type i malformation and comparison with the normal population. acta neurochirurgica. 2002;144(2):165-71. 246 j contemp med sci | vol. 7, no. 4, july-august 2021: 242–246 correlation between sphenoclivus angle and gnathic angle with age and gender in iranian population using ct-scan original g. hassanzadeh et al. 24. adam a. skull radiograph measurements of normals and patients with basilar impression; use of landzert’s angle. surgical and radiologic anatomy. 1987;9(3):225-9. 25. royo-salvador m. platybasia, basilar groove, odontoid process and kinking of the brainstem: a common etiology with idiopathic syringomyelia, scoliosis and chiari malformations. revista de neurologia. 1996;24(134):1241-50. 26. vuorinen p, meurman oh. the basal angle in the clinical diagnosis of otosclerosis. acta oto-laryngologica. 1962;54(1-6):176-80. 27. botelho rv, ferreira edz. angular craniometry in craniocervical junction malformation. neurosurgical review. 2013;36(4):603-10. 28. bhattacharya a, bhatia a, patel d, mehta n, parekh h, trivedi r. evaluation of relationship between cranial base angle and maxillofacial morphology in indian population: a cephalometric study. journal of orthodontic science. 2014;3(3):74. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i4.1050 106 j contemp med sci | vol. 9, no. 2, march-april 2023: 106–110 original serum antioxidant status in sickle cell disease patients: implications for oxidative stress and disease severity zainab ali hadi1*, fadhil jawad al-tu’ma1, atheer hameid odda1, hawra almuhafdah2 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 2evans medical center, primary care medical clinic, 4700 e illif ave, denver, colorado, usa. *correspondence to: zainab ali hadi (e-mail: huss0780m@gmail.com) (submitted: 17 february 2023 – revised version received: 28 february 2023 – accepted: 14 march 2023 – published online: 26 april 2023) abstract objectives: the main aim of this subject is to determine the oxidative status of iraqi sickle cell anemic patients and then correlated with various biomarkers. methods: in this study, blood samples from 100 sickle cell anemic subjects were analyzed, and then compared with control group which consisting of 50 individuals without sickle cell anemia was established. various biochemical techniques were employed to measure different oxidative stress markers and inflammatory mediators. serum samples were collected from blood to determine the levels of antioxidants such as catalase (cat), glutathione peroxidase (g-px), reduced glutathione (gsh), and lipid-peroxidation product malondialdehyde (mda). results: the results revealed that the levels of serum antioxidant activity (cat), gsh, and g-px were significantly reduced with (p < 0.05) in sickle cell anemic patients as compared with apparently control group. in contrast, the mda level was significantly higher in sickle cell anemic patients than that found in the apparently control group. conclusion: in this work, there is an increased oxidative stress in sickle cell anemic patients, which is accompanied by a decrease in antioxidant activity and a rise in lipid peroxidation, leading to the intensification of sickle cell anemic symptoms in patients. keywords: antioxidants, anemia, sickle cell, oxidative stress, reactive oxygen species issn 2413-0516 introduction sickle cell disease (scd) is an inherited blood disorder brought on by a mutation in the b-globin gene, also known as hemoglobin subunit beta (hb-beta), which codes for a part of hemoglobin (hb), the protein complex that makes up 70% of red blood cells (rbcs) and is in charge of carrying oxygen to all body organs.1,2 more than 300,000 babies each year were impacted; the united nations (un) and the world health organization (who) classify hereditary blood diseases like scd as a global health concern. in addition to the united states and europe, this illness is widespread in most of sub-saharan africa, the middle east, india, the caribbean, south and central america, and several mediterranean nations.3,4 under some circumstances, such as dehydration, illness, or a lack of oxygen, the aberrant, sickled hb (hbs) in scd tends to polymerize in rbcs. rbcs are shaped like a sickle or a banana as a result of this process, as seen in figure 1, which also leads them to become hard and distorted. sickle cell anemia phenotypic manifestation is a complicated pathophysiologic syndrome with various sources of pro-oxidant mechanisms and resulting chronic and systemic oxidative stress. erythrocytes live in an environment of constant free radical production in healthy biological systems. to combat ros, erythrocytes have a self-sustaining activity of antioxidants such as superoxide dismutase (sod), catalase (cat), glutathione peroxidase(g-px), reduced glutathione (gsh), and vitamins.5,6 excessive ros production overwhelms the blood’s defenses and destroys biological macromolecules such as proteins, lipids, and dna, altering the physical properties of rbc membrane, changing membrane permeability, and causing hemolysis.7 however, reactive oxygen species (ros) and byproducts of their oxidative processes may be used to predict the severity of scd.8 the repeated polymerization and depolymerization of hbs molecules, on the other hand, results in increased levels of reactive oxygen species (ros), which can cause a cyclic cascade characterized by blood cell adhesion, hemolysis, increased susceptibility to infections, chronic inflammatory diseases, and microvascular damage in organs, resulting in a decrease in quality of life and life expectancy.9 in scd patients, sickle erythrocytes are the primary source of pro-oxidants; the unstable, autoxidative hbs and rapid metabolic turnover caused by recurrent hbs polymerizations and depolymerizations stimulate ros formation.10 oxidative stress plays an important role in the pathophysiology of scd and its effects.11 the higher oxidative burden in scd patients has been linked to a variety of factors. a high level of cell-free hemoglobin, a prolonged inflammatory state, increased hbs auto-oxidation, and iron overload are only a few of the processes.12 according to previous studies,13 higher levels of reactive oxygen species (ros) have been linked to a variety of scd-related problems. furthermore, it has been proposed that an altered oxidant/antioxidant balance and increased oxidative stress have a role in the etiology of a variety of disorders in scd patients.14 ros levels rise due to variables such as increased intravascular hemolysis and continuing inflammation.15 even though the body has its own system for combating excessive ros, the antioxidant defense may be overwhelmed by the vast pool of ros and may not effectively negate their effects in scd patients.13 despite the fact that various studies have looked at the pathophysiology of scd in iraq, there is very little information on oxidative stress. this study was carried out to bridge a knowledge gap and add to the body of knowledge on oxidative stress and antioxidants in scd patients. the current study’s goal was to examine blood antioxidant parameters in healthy mailto:huss0780m@gmail.com 107j contemp med sci | vol. 9, no. 2, march-april 2023: 106–110 z.a. hadi et al. original serum antioxidant status in sickle cell disease patients controls and iraqi sca patients in clinical stability in order to learn more details about these patients’ antioxidant systems. materials and methods participants with sickle cell disease (scd) and age-matched healthy controls were included in this study. a consistent medical procedure was used to collect thorough demographic and clinical information on all subjects. the study comprised 50 scd patients with age ranged between (15–60 years) and 50 healthy people as controls with matched age range. after describing the nature of the study to all participants and, for minors, their parents, informed written agreement was acquired. blood samples (5 ml) were taken from each participant using gel-coated tubes for biochemical parameter analysis. the blood was centrifuged at 2450 × g for 10 minutes to extract the plasma, which was kept at –80°c until use. the plasma antioxidants, including catalase (cat), were quantified using a spectrophotometer in accordance with previously reported techniques, see table 1.17 the gsh was determined by using a modified procedure that employed ellman’s reagent (dtnb). standard and sample test tubes were prepared according to the protocol described by kapoor and kakkar.18 reagents sample µl reagent black µl standard µl sample 100 – – standard – – 100 ddw 800 900 100 tca 100 100 100 g-px activity levels was determined using a previously described spectro-photometric method, as outlined by fig. 1 blood flow of sickle cell.16 table 1. methods employed for gsh level determination reagents test standard blank sample 100 μl – – phosphate buffer 900 μl 1000 μl 3000 μl hydrogen peroxide 2000 μl 2000 μl – mix with vortex and incubate at 37°c for 2 min, after that, add: vanadium reagent 2000 μl 2000 μl 2000 μl after that, the tubes were kept at 25°c for 10 min. the changes in absorbance were recorded at 452 nm against the reagent blank. rotruck.19 to measure mda levels, 100 μl of sample was added to a test tube containing 2 ml of a working solution prepared as follows: 0.514 g of tba, 25 g of tca, and 0.5 ml of 1m hcl were mixed with 190 ml of distilled water (d.w.), followed by the addition of 1 g of sds and completed to a volume of 200 ml. the sample was vortex and heated in a 90ºc water bath for 50 min and then allowed to cool. after centrifuging the sample for 5 minutes at 5000 rpm, the absorbance of the supernatant was measured spectrophotometrically at 532 nm against a reagent blank. the reagent blank was prepared in the same manner as above, except distilled water was substituted for the sample. descriptive statistics were performed on the data for each group using ibm spss statistics software, version 28.0 (ibm, spss, chicago, illinois, usa). categorical variables were presented as n (%), while scale variables were expressed as mean ± standard deviation. analytical statistical tests were used to confirm significant differences in categorical variables among the parameters. results with p-value < 0.05 (two-tailed) were considered statistically significant. results in this study, blood samples were collected from a total of 50 sickle cell disease (scd) patients comprising 27 females and 23 males with age range between 15 to 60 years. in addition, a control group of 50 individuals without scd was included in the study, with 24 females and 26 males and age range of 15 to 60 years. the participants with scd were assessed for their marital status and family history of scd using a questionnaire, and the results are presented in figures 2–4. table 2 presents significant differences in the levels of g-px, mda, and gsh among the study groups, with p values < 0.001, respectively. sickle cell disease (scd) patients exhibited a reduction in antioxidant defense mechanisms, leading to damage to cellular organelles and enzymes and an increase in lipid peroxidation.20 the amount of g-px activity was considerably lower in scd sufferers compared to healthy persons in the current investigation. in a similar way gsh activity was considerably lower in scd participants as compared to healthy subjects, which is consistent with the findings of.21 this reduction in g-px and gsh activity might be attributed to an increase in reactive oxygen species (ros), which leads to h2o2 accumulation. h2o2 is created by two electron transfers or by sickling, and it is eliminated by two major antioxidants, g-px and cat. fig. 2 frequency of the study population in cases of sickle disease compared to control group according to age groups (n = 100) of the age group. 108 j contemp med sci | vol. 9, no. 2, march-april 2023: 106–110 serum antioxidant status in sickle cell disease patients original z.a. hadi et al. fig. 3 frequency of the study population in cases of sickle disease compared to control group according to marital status and gender (n = 100). fig. 4 frequency of among family history, chronic disease and physical activity of the study population in (a) cases of sickle disease compared to (b) control group (n = 100). table 2. mean differences of biomarkers in cases of sickle disease compared to control group variables patient (n = 50) mean ± sd control (n = 50) mean ± sd p value cat, u/l 0.33 ± 0.13 0.36 ± 0.14 0.204[ns] g-px, u/l 334.41 ± 112.63 400.51 ± 72.53 <0.001[s] mda, u/l 2.16 ± 1.50 1.44 ± 0.44 0.002[s] gsh, u/l 18.85 ± 2.56 22.93 ± 2.80 <0.001[s] results are presented as mean ± sd, p < 0.05 considered significantly different, [s] = significant, [ns] = non-significant–test. the current study’s findings are consistent with prior studies, indicating that the lower levels of gsh and g-px are attributable to increased oxidative stress. in contrast, mda levels in the serum of sickle cell anemia (sca) patients were considerably higher than in healthy controls. this rise in mda levels might be attributable to increased ros generation in sca patients, and mda levels provide information about the amount of oxidative damage in cells, which is consistent with the findings of.22,23 other studies, however, have found a statistically significant reduction in mda in older sca patients. furthermore, no association was observed between age, sca patients, and healthy people.24 in table 3, based on gender groups, only mda levels showed a significant difference; the p value was <0.01 and also consistent with the study of.20 while based on age groups, no significant differences were found in the levels of measured biomarkers with p values > 0.05, as presented in table 4. in order to investigate the interplay between the measured biomarkers and their potential role in the progression of the case study, a multivariable linear regression model was utilized to analyze the response relationship between parameters. the results indicated that serum cat levels were significantly and positively correlated with g-px and gsh levels, while exhibiting a negative correlation with mda levels. these findings were consistent with the results reported by previous studies,25,26 with p-values <0.001. furthermore, a weakly significant correlation was observed between g-px and cat and gsh activity levels. additionally, serum gsh levels showed 109j contemp med sci | vol. 9, no. 2, march-april 2023: 106–110 z.a. hadi et al. original serum antioxidant status in sickle cell disease patients table 3. mean differences of biomarkers in cases of sickle disease compared to control groups based on gender variables male (n = 50) mean ± sd female (n = 50) mean ± sd p value cat u/l 0.32 ± 0.13 0.36 ± 0.13 0.135[ns] gpx u/l 363.00 ± 102.22 371.74 ± 98.50 0.664[ns] mda u/l 2.10 ± 1.27 1.51 ± 0.97 0.011[s] gsh u/l 20.89 ± 3.49 20.89 ± 3.28 0.997[ns] results are presented as mean ± sd, p < 0.05 considered significantly different, [s] = significant, [ns] = non-significant. t–test. table 4. correlations of the biochemical parameters among patients’ groups variables cat gpx mda gsh cat, u/l 1 r = 0.3 p = 0.003 r = –0.91 p = <0.001 r = 0.4 p = <0.001 g-px, u/l r = 0.3 p = 0.003 1 r = –0.1 p = 0.540 r = 0.2 p = 0.017 mda, u/l r = –0.2 p = 0.119 r = 0. 1 p = 0.540 1 r = 0.1 p = 0.998 gsh, u/l r = 0.4 p = <0.001 r = 0.2 p = 0.017 r = 0.1 p = 0.998 1 results are presented as mean ± sd, p < 0.05 considered significantly different, [s] = significant, [ns] = non-significant. anova test. significant positive correlations with cat and g-px levels (all p < 0.05). the detailed relationships between the parameters are presented in table 3. the cat level in scd subjects has been a subject of inconsistency in various studies. while a few studies have reported a decrease in cat activity in scd patients, as seen in our research, others have seen a rise in cat levels.27,28 elevated cat levels might be ascribed to a preventative function to scavenge h2o2, whereas lower levels could be related to catastrophic and sustained oxidative stress, where it has been reported by that low level of serum total antioxidant and inhibition of serum gsh, gpx and cat are involved in many diseases according to this study.29 discussion the results of this study indicate that the presence of hbs in erythrocyte of scd subjects not only causes biochemical alterations of biomolecules but also is responsible for increases in free radical production. the imbalance between overproduction of free radicals and inability of cellular content of antioxidants to eliminate leads to the oxidative stress in patients of scd,30 so antioxidants are important in scavenging free radicals and non-radical oxidants as well as protecting cells from oxidative stress.31 antioxidants, which include both tiny molecules like gsh and thioredoxin and enzymes like sod, gpx, and cat, counteract the stress induced by reactive radicals, also known as oxidative stress, and protect cellular biomolecules. according to the antioxidant defense mechanisms analysis of the current study’s results and because of the mechanisms of oxidative stress in sickle cell disease, the plasma biomarker plasma concentration assay was able to differentiate between sickle cell anemia patients and healthy controls. while sickle cell disease patients in this study had significantly higher serum mda levels than the controls, the erythrocyte cat level in hbss in the steady state was significantly lower, and another outcome of the study was that scd patients had significantly lower mean erythrocyte gsh, gpx levels compared with controls group. conclusion there is an increase in oxidative stress in sickle cell anemic patients, which is accompanied by a decrease in antioxidant activity and an increase in lipid peroxidation, leading to an intensification of sickle cell anemic symptoms in patients, according to this study. acknowledgments the authors like to express their gratitude to kerbala university (www.uokerbala.edu.iq), kerbala, iraq, for its support of this effort. compliance with ethical standards conflict of interest the authors warrant that they don’t have any competing interests to declare. ethical approval all procedures involving human participants in research projects were conducted in compliance with the ethical standard of the research committee of kerbala university, as well as the 1964 helsinki declaration and any revisions or other ethical standards deemed equivalent. informed consent consent to participate in the study was received from each individual person who took part in the research.  references 1. johnson, k. m., jiao, b., ramsey, s. d., bender, m. a., devine, b., & basu, a. (2023). lifetime medical costs attributable to sickle cell disease among nonelderly individuals with commercial insurance. blood advances, 7(3),365–374. 2. wonkam, a. (2023). the future of sickle cell disease therapeutics rests in genomics. disease models & mechanisms, 16(2),dmm 049765. 3. thein, m.s.; igbineweka, n.e.; thein, s.l. sickle cell disease in the older adult. pathology 2017, 49, 1–9. 4. piel, f.b.; steinberg, m.h.; rees, d.c. sickle cell disease. n. engl. j. med. 2017, 377,305. 5. al-naama lm, hassan mk, mehdi jk. association of erythrocytes antioxidant enzymes and their cofactors with markers of oxidative stress in patients with sickle cell anemia. qatar med j.2015;2015:14. 110 j contemp med sci | vol. 9, no. 2, march-april 2023: 106–110 serum antioxidant status in sickle cell disease patients original z.a. hadi et al. 6. perrone s, tataranno ml, stazzoni g, et al. oxidative injury in neonatal erythrocytes. j matern fetal neonatal med. 2012;25:104–108. 7. biswal, s., rizwan, h., pal, s., sabnam, s., parida, p., & pal, a. (2019). oxidative stress, antioxidant capacity, biomolecule damage, and inflammation symptoms of sickle cell disease in children. hematology, 24(1),1–9. 8. nur e, biemond bj, otten hm, et al. oxidative stress in sickle cell disease; pathophysiology and potential implications for disease management. am j hematol. 2011;86:484–489. 9. silva dg, belini junior e, de almeida ea, et al. oxidative stress in sickle cell disease: an overview of erythrocyte redox metabolism and current antioxidant therapeutic strategies. free radicbiol med.2013;65: 1101–1109. 10. biswal, s., rizwan, h., pal, s., sabnam, s., parida, p., & pal, a. (2019). oxidative stress, antioxidant capacity, biomolecule damage, and inflammation symptoms of sickle cell disease in children. hematology, 24(1),1–9. 11. morris, c.r.; suh, j.h.; hagar, w.; larkin, s.; bland, d.a.; steinberg, h.a.; vichinsky, e.p.; shigenaga, m.; ames, b.; kuypers, f.a.; et al. erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease. blood 2008, 111,402–410. 12. nagababu, e.; fabry, m.e.; nagel, r.l.; rifkind, j.m. heme degradation and oxidative stress in murine models for hemoglobinopathies: thalassemia, sicklecell disease and hemoglobin c. disease. blood cells mol. dis. 2008, 41,61–66. 13. antwi-boasiako, c., b. dankwah, g., aryee, r., hayfron-benjamin, c., s. donkor, e., & d. campbell, a. (2019). oxidative profile of patients with sickle cell disease. medical sciences, 7(2), 17. 14. voskou, s.; aslan, m.; fanis, p.; phylactides, m.; kleanthous, m. oxidative stress in the β-thalassemia and sickle cell disease. redox. biol. 2015, 6, 226–239. 15. akohoue, s.a.; shankar, s.; milne, g.l.; morrow, j.; chen, j.k.; ajaiyi, w.u.; buckowski, m.s. energy expenditure, inflammation, and oxidative stress in steadystate adolescents with sickle cell anemia. pediatr. res. 2007, 61, 233–238. 16. yeruva, s., sharadavaralakshmi, m., pavangowtham, b., hari chandana, y., & krishna prasad, p. e. s. n. (2021). identification of sickle cell anemia using deep neural networks. emerging science journal, 5(2), 200–210. 17. kumar p, raman t, swain mm, et al. hyperglycemiainduced oxidative nitrosative stress induces inflammation and neurodegeneration via augmented tuberous sclerosis complex-2 (tsc-2) activation in neuronal cells. molneurobiol. 2017;54:238–254. 18. kapoor r, kakkar p. protective role of morin, a flavonoid, against high glucose induced oxidative stress mediated apoptosis in primary rat hepatocytes. plos one. 2012;7:e41663. 19. rotruck, j. t., pope, a. l., ganther, h. e., swanson, a. b., hafeman, d. g., & hoekstra, w. (1973). selenium: biochemical role as a component of glutathione peroxidase. science, 179(4073),588–590. 20. ama moor, v. j., pieme, c. a., chetchachemegne, b., manonji, h., njinkionono, b. l., tchoulamamiafo, c., ... &tazoacha, a. (2016). oxidative profile of sickle cell patients in a cameroonian urban hospital. bmc clinical pathology, 16(1), 1–5. 21. biswal, s., rizwan, h., pal, s., sabnam, s., parida, p., & pal, a. (2019). oxidative stress, antioxidant capacity, biomolecule damage, andinflammation symptoms of sickle cell disease in children. hematology, 24(1),1–9. 22. saka, w. a., anigbogu, c. n., kehinde, m. o., & jaja, s. i. (2023). l-arginine supplementation enhanced expression of glucose transporter (glut 1) in sickle cell anaemia subjects in the steady state. current research in physiology, 6, 100096. 23. luo, j., fan, j. b., & wang, s. (2020). recent progress of microfluidic devices for hemodialysis. small, 16(9),1904076. 24. bahal, m., nyamati, s., hegde, s., kakkar, a., sood, i., & kalra, s. (2022). estimation of malondialdehyde levels and determination of total antioxidant capacity in serum and saliva of patient affected with sickle cell anemia. journal of indian academy of oral medicine and radiology, 34(4),380–384. 25. antwi‐boasiako, c., dankwah, g.b., aryee, r., hayfron‐benjamin, c., aboagye, g., & campbell, a.d. (2020). correlation of lipid peroxidation and nitric oxide metabolites, trace elements, and antioxidant enzymes in patients with sickle cell disease. journal of clinical laboratory analysis, 34(7), e23294. 26. emokpae, a. m., ojiefo, u. p., & aisha, k. g. (2010). antioxidant enzymes and acute phase proteins correlate with marker of lipid peroxide in adult nigerian sickle cell disease patients. 27. chirico en, pialoux v. role of oxidative stress in the pathogenesis of sickle cell disease. iubmblife. 2012;64:72–80. 28. hameed, r. m. (2012). evaluation of lipid peroxidation, level of selenium and enzymatic antioxidant activity in woman during pregnancy and abortion at first trimester. kerbala journal of pharmaceutical sciences, (3). 29. choudhary, m., chaudhari, s., gupta, t., kalyane, d., sirsat, b., kathar, u., ... & tekade, r. k. (2023). stimuli-responsive nanotherapeutics for treatment and diagnosis of stroke. pharmaceutics, 15(4), 1036. 30. özkan, s., & özdemir, e. (2022). role of bio-active compounds to prevent oxidative damage in maize. research & reviews in agriculture, forestry and aquaculture, 31. 31. singh, n., gr, s., & mugesh, g. (2023). antioxidant and prooxidant nanozymes: from cellular redox regulation to next‐generation therapeutics. angewandte chemie, e202301232. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1336 276 j contemp med sci | vol. 7, no. 5, september–october 2021: 276–281 original designing and validating a questionnaire on oral health care knowledge, attitude, and practice of dental staff hediyeh toutouni1* , seyed ahmad banihashem rad2 , farzaneh boroumand3,4 , fatemeh hosseini pouya5 1department of community oral health, school of dentistry, mashhad university of medical sciences, mashhad, iran. 2department of restorative, preventive and pediatric dentistry, school of dental medicine, university of bern, switzerland. 3department of biostatistics, school of health, mashhad university of medical sciences, mashhad, iran. 4department of mathematics and statistics, faculty of science and engineering, macquarie university, sydney, australia. 5private practice dentist, mashhad, iran. *correspondence to: hediyeh toutouni (e-mail: toutounih@mums.ac.ir) (submitted: 04 august 2021 – revised version received: 29 august 2021 – accepted: 14 september 2021 – published online: 26 october 2021) abstract objectives: this study aimed to design a valid questionnaire to evaluate knowledge, attitude, and practice (kap) of mashhad dental school staff about oral health care and to assess the validity and reliability of this instrument. methods: the “dental staff awareness of oral health care” (dsaohc) was divided into three concepts; knowledge, attitude and practice. ten experts of mashhad dental school contributed to design the questionnaire and assessed the face and content validity (i-cvi and s-cvi/ av) in two rounds. the necessity of each item was evaluated according to lawshe’s content validity ratio (cvr). construct validity, internal consistency, and reliability were assessed using confirmatory factor analysis, homogeneity coefficients, and test-retest by distributing the instrument among 207 dental staff working in other dental schools and private clinics. results: based on the acceptable results of i-cvi, s-cvi/av (>0.7), and cvr (>0.62), the final version of dsaohc was approved with 34 questions, including 10 questions about background information, 8 questions for the knowledge, 5 questions for attitude and 11 questions related to practice. cronbach’s alpha coefficients were acceptable for all domains. the rmsea criterion was ≤ 0.05 and its upper band confidence interval (ci 90%) was ≤ 0.1. also, cfi indicated the suitability of the model (the desired value of cfi>0.9). inter-class correlation/ icc revealed the appropriate measure (icc = 0.998). conclusion: the measures showed that this instrument is valid and reliable, also culturally adjusted to and acceptable for this community. it may adequately evaluate staffs’ knowledge, attitude, and practice toward oral health. keywords: oral health, knowledge, attitude, practice, questionnaire issn 2413-0516 introduction employees are the human resources of organizations, including the health system. the staff ’s awareness of health issues affects directly their own well-being and indirectly satisfaction of their clients.1,2 dental caries and periodontal diseases have been concerned as the most important oral health burdens among all ages in iran like other countries.3-5 these two diseases are the main causes of tooth loss as their treatment is costly.6 clients’ satisfaction toward a system directly depends on the performance of the system’s staff in terms of accountability, proper communication with patients (empathy), and staffs’ readiness to manage the challenges in the system. providing facilities, understanding the necessity of treatment and its process along with urgency, ability to be more empathetic, and giving clear and simple explanations to patients are critical for treatment process which would certainly affect patients’ satisfaction as a part of chair side manner.2,7 several factors affect the relationship between staff and patients containing staff ’s awareness of related health issues and health promotion programmes.2 on the other hand, the proper planning for maintaining and improving the staff ’s oral health, their knowledge, attitude, and practice which are also needed for appropriate dental reform in a dental setting, requires a survey at first place, which measures their awareness towards oral health care. at further stages, an assessment of their oral health status and their treatment needs in terms of dental caries and periodontal diseases are needed. oral health care is divided into two dimensions which are related to hard tissue and soft tissue. dental caries is the most common multifactorial infectious oral disease and the leading cause of tooth decay/tooth loss.8 because of the multifactorial aetiology, there are different approaches to prevent this disease.9 periodontal diseases are the most common oral illness that influence soft tissues such as gingiva and periodontium. not only the localized/oral circumstances can result in periodontal diseases, but also there are systemic diseases that lead to inflammatory conditions in periodontium.10,11 there are several kap instruments to measure the knowledge, attitude, and practice of different groups of people about oral health. the target groups of these questionnaires are school children, health care workers, mothers, and people who are suffering from systemic diseases.12-19 since, the contents of those questionnaires were general and not adequate for oral health care workers; also, they primarily focused on the most common oral hygiene habit such as tooth brushing, and few questions were related to diet as an important risk factor of dental caries, this study aimed to design and validate a questionnaire to measure knowledge, attitude, and practice of the dental school staff who graduated from nursing school and similar disciplines, but work as oral health care providers. this study was the first phase of a health promotion programme for mashhad dental school staff and the validated instrument will use in the further surveys, annually. materials and methods before developing an instrument, six databases (pubmed, embase, biomedcentral, and google scholar for english 277j contemp med sci | vol. 7, no. 5, september–october 2021: 276–281 h. toutouni et al. original designing a questionnaire on oral health care kap of dental staff resources and scientific information database (sid) for persian articles) were searched to find a valid and reliable pre-existing questionnaire to measure knowledge, attitude, and practice (kap) of oral health care staff at dental settings. the search was limited to english and persian literature published between 1990 and 2021. keywords used for conducting this search were “healthcare”, “design”, “validation”, “questionnaire”, “knowledge”, “attitude”, “practice”, and “oral health”. based on a search conducted 239, 155, 445, and 23700 articles were obtained from the first five abovementioned databases, respectively. there was not found any persian article. among the papers found, irrelevant studies were omitted considering their titles, duplicate articles were removed, and only studies, which were conducted on developing and designing the valid questionnaires, were remained. there was no valid pre existing questionnaire for the purpose of this study. face and content validity the “dental staff awareness of oral health care” (dsaohc) was divided into three concepts; knowledge, attitude, and practice. to determine the indicators of each concept, we checked out the adults’ kap questionnaires, oral health-related textbooks, dental public health articles, and also resources from the american dental assistants association.20-22 then, this initial draft was sent to the professors of dental public health and periodontics to add items that they considered necessary. after modifying the first draft, ten experts of mashhad dental school have also assessed the face and content validity of the questionnaire. in addition, ten dental workforce checked questions in terms of face validity as a target group, and two questions were revised according to their comments. these ten people did not take part in the following phases of the study. the criteria for selecting experts was contained: their background in writing and translation in scientific publications, research and teaching experiences, familiarity with the educational needs of the target population, and also with the developing process of a valid instrument as a questionnaire.23,24 the content validity of the questionnaire was evaluated by experts using the content validity index (i-cvi and s-cvi/ av)25 and the necessity of each item was scored with lawshe’s content validity ratio (cvr).26 content validity index was calculated for each question. experts rated each item on a 4‐point scale from 1 to 4 that: score 1 was the lowest and score 4 represented the highest of each measurement item (relevancy to the subject, the simplicity, and clarity of each item). this four-point scale was also used to avoid shifting to the midpoint.25 the questions that obtained very low cvr were omitted based on experts’ opinions. the items which met moderate levels for cvr or zero (means that half of the experts indicated those items as “essential”), but they showed high relevancy, clarity, and simplicity remained in the questionnaire and were modified and sent for the experts after revision. i-cvi, s-cvi/ave, and cvr were calculated after the second review. the content validity of the questionnaire and the i-cvi score for each indicator were calculated using the following formula. for example, the score of relevancies for each indicator was calculated which included (score 4) a highly relevant score, and those that are relevant but in need of review gets (score 3) divided by the total number of experts. the same method was used to calculate the i-cvi of each indicator in terms of utility (clarity and simplicity). each indicator with an i-cvi score above 0.78 for each of the three domains of relevancy to subject, clarity, and simplicity was retained in the questionnaire. this score of 0.78 was determined by the contribution of 10 experts by considering valid scientific papers.25 after calculating i-cvi for all indicators (questions), the content validity of the whole questionnaire was determined using s-cvi/ave index and calculating the mean of each i-cvi value.25 content validity ratio (cvr) was calculated according to the number of experts and also those who had selected the “essential” option for each indicator. cvr = (ne – n/2)/(n/2) ne is the number of panel members indicating an item “essential,” and n is the number of panel members. according to the number of experts (n = 10), the minimum content validity ratio which would be accepted was 0.62.26 after confirming the face and content validity by the experts, questionnaires were distributed among 207 dental clinic staff working in mashhad, bojnord, and birjand dental schools in a similar mean age group to verify the construct validity, to conduct factor analysis, and to assess the over-scale and over-time reliability. homogeneity, construct validity, and test-retest reliability questionnaires were filled out by 207 dental staff. the homogeneity (internal consistency) of the questionnaire was determined by calculating a cronbach for likert scale, and kuder richardson-20 (kr-20) for dichotomous items (knowledge) because of their different nature, in which there were true answers among choices.27 the acceptable cut-off considered for a cronbach was 0.7.28 over-time reliability of the instrument was assessed by test-retest after filling out the redistributed questionnaires by 50 out of 207 people of the same population in a two-week interval. we applied a confirmatory factor analysis (cfa) to investigate construct validity. tow goodness of fit criteria including comparative fit index (cfi) and root-mean-squared error of approximation (rmsea) was reported. the cfi is an index between 0 and 1. a greater value indicates a better fit. the rmsea shows the simplicity of the models. the closer the value to 0 shows the model has a better fit. a good model has a value of 0.08 or lower. we carried out the cfa in amos 24 software. figure 1 shows the process of questionnaire development. ethical considerations this study was approved by the ethical committee of mashhad dental school at mashhad university of medical sciences (no. ir.mums.sd.rec.1394.3272971). all the employees were informed about the study and were invited to complete the questionnaire. no one was forced to participate in the study. moreover, all questionnaires were coded, and data were entered into the software according to their codes were documented on the questionnaires. all data are being kept confidential. 278 j contemp med sci | vol. 7, no. 5, september–october 2021: 276–281 designing a questionnaire on oral health care kap of dental staff original h. toutouni et al. results face and content validity the preliminary assessment of the content validity ratio and construct validity by 10 experts showed the need for making changes in the questionnaire. eleven out of 41 questions were asked about background information. among 30 the remaining questions, eight indicators were related to knowledge, six to attitude, and 16 indicators went to practice. one indicator of background section and two indicators of practice were excluded from the questionnaire based on experts opinions and due to low cvis and cvrs. twenty-nine indicators were approved with appropriate cvi and cvr scores and nine indicators were rephrased due to the high cvi score, low cvr (table 1). after reviewing for the second time by the experts, the questionnaire was approved with 35 questions, including 10 questions about background information, eight questions related to the concept of knowledge, five questions for the concept of attitude and 12 questions for the concept of practice. the amounts of i-cvi, s-cvi/av, and cvr for the instrument were ≥0.79 for each item, 0.85, and ≥0.62, respectively (table 2). internal consistency, construct validity, and test-retest reliability the mean age of 207 participants, for conducting the factor analysis, was 52.9 (±26.6). they consisted of 146 (71%) women and 61 (29%) men whose average years of experience in a dental setting were 6.6 ± 7.07 in which 97(46.9%) already took part in an educational course on oral health care. out of 207 participants, 100(48.3%) worked in clinical, 82(39.6%) in administrative settings and 18(8.7%) worked in services (seven participants did not reply to this question). in terms of the level of education, 111(53.6%) had master and 34(16.4%) bachelor degrees in health sciences. in addition, 56(27.1%) had a diploma degree and the level of education of 6(3%) was under diploma. the rmsea criterion was less than 0.05 and its confidence interval (ci 90%) was less than 0.1. another criterion, cfi (comparative fit index), also indicated the suitability of the model (the desired value of cfi >0.9). the statistical analysis showed that if the question pq5 was removed, the cronbach a value of the scale would increase from 0.62 to 0.661. however, kuder richardson-20 (kr-20) analysis showed low homogeneity between eight questions related to the concept of knowledge (kr-20 = 0.25). internal consistency was recalculated after pq5 was deleted and showed that was acceptable (close to 0.7). as a result, the question pq5 was removed from the final instrument. the overtime reliability of the questionnaire was confirmed by icc = 0.985 (ci 90% = 0.972–0.992) (table 3). discussion the dental staff awareness of oral health care (dsaohc) questionnaire which was developed in this study showed acceptable measures as a standard instrument to evaluate the knowledge, attitude, and practice of the oral health care workforce. i-cvi and s-cvi/av in all 34 remained items of the questionnaire met acceptable cut-offs [polit-lawshe-grant]. furthermore, cronbach a and icc presented acceptable27 and excellent values.29 fig. 1 the flowchart of two phases of the study. phase i: i-cvi, s-cvi and cvr calculation by 10 experts. phase ii: factor analysis and reliability tests among 207 participants. 279j contemp med sci | vol. 7, no. 5, september–october 2021: 276–281 h. toutouni et al. original designing a questionnaire on oral health care kap of dental staff table 1. assessment of i-cvi, s-cvi, and cvr by expert panel (1st time) no. item question relevancy clarity simplicity cvr interpretation kq1 the number of permanent teeth in human 0.9 0.8 0.9 0.4 rephrased kq2 which food product can cause dental caries 1 1 0.8 0.8 included kq3 which one is correct about dental calculus 0.9 0.8 0.8 0.8 included kq4 when should we go to the dentist 1 0.9 0.8 0.8 included kq5 which one is the most important time for toothbrushing 0.9 0.8 0.7 0.8 included kq6 which one is the common cause of malodor 1 0.9 0.8 1 included kq7 what are the causes of dental caries (please order from the most to the least) 1 0.8 0.8 1 included kq8 what are the prevention methods for dental caries (please order from the most to the least) 0.9 0.7 0.7 0.8 included aq1 i believe in good oral health may lead to more social popularity 0.8 0.8 0.7 0.4 rephrased aq2 i believe in toothbrushing can reduce periodontal diseases 1 1 0.9 0.8 included aq3 i believe that toothbrushing eliminates malodor 1 0.9 0.9 0.8 included aq4 i believe that oral health care can prevent some gastrointestinal diseases 0.7 0.8 0.7 0.0 rephrased aq5 i believe that flossing is effective for preventing dental caries 1 1 1 0.8 included aq6 i believe that pregnancy can be the reason for tooth loss 0.9 0.8 0.8 0.8 included pq1 how often do you brush your teeth 1 0.8 0.9 0.8 included pq2 how often do you floss your teeth 0.9 0.9 0.9 0.8 included pq3 how often do you use a fluoride mouthwash 0.8 0.9 0.9 0.4 rephrased pq4 do you brush all your teeth surfaces 0.9 0.7 0.9 0.6 included pq5 do you break shelled nuts using your teeth 0.8 0.9 0.9 0.4 rephrased pq6 do you have a dental appointment each year regularly 1 1 1 0.8 included pq7 do you clean your tongue after toothbrushing 0.8 0.9 0.9 0.4 rephrased pq8 do you brush your teeth at the workplace after lunch 0.9 0.9 0.9 0.4 rephrased pq9 do you brush your teeth if there is no toothpaste 0.5 0.8 0.8 -0.2 deleted pq10 do you rinse your entire mouth after toothbrushing 0.6 0.8 0.8 0.0 rephrased pq11 do you eat or drink something after toothbrushing at night 1 1 1 0.6 included pq12 how much toothpaste do you use for brushing your teeth each time 0.6 0.75 0.78 0.4 rephrased pq13 which kind of toothbrush do you usually use 0.8 0.8 0.8 0.6 included pq14 where do you keep your toothbrush 0.7 0.6 0.8 -0.2 deleted pq15 how often do you change your toothbrush 0.9 1 0.9 0.8 included pq16 why do you floss your teeth 0.9 0.8 0.9 0.8 included table 2. assessment of i-cvi, s-cvi, and cvr by expert panel (2nd time) no. item question relevancy clarity simplicity cvr decision kq1 the number of permanent teeth in human 0.9 0.8 1 0.6 included aq1 i believe in good oral health may lead in more social popularity 0.9 0.9 0.8 0.8 included aq4 i believe that tooth loss can lead in some gastrointestinal diseases 0.7 1 0.7 0.0 deleted after panel pq3 how often you use mouthwash 0.9 1 0.9 0.6 included pq5 do you break shelled nuts using your teeth 1 1 0.9 0.8 included pq7 do you clean your tongue after toothbrushing 0.8 1 0.9 0.6 included pq8 do you brush your teeth at workplace after lunch 1 1 0.9 0.8 included pq10 do you rinse your entire mouth after toothbrushing 0.9 0.9 0.8 0.2 deleted pq12 how much toothpaste do you use for brushing your teeth each time 0.9 0.75 0.78 0.2 deleted 280 j contemp med sci | vol. 7, no. 5, september–october 2021: 276–281 designing a questionnaire on oral health care kap of dental staff original h. toutouni et al. having revised the first draft of the instrument, five questions were added to demographic data (gender, age, date of birth, level of education) as background information. these questions asked about “years of experience”, “work field” (clinical, official or services), “participating in an educational course/workshop on oral health care”, “systemic diseases”, and “tobacco usage”. as “years of experience”, “working in an official or clinical department”, and “participation in an oral health education course” will make changes in the interpretation of the results and decision making for the following oral health promotion programmes. the other two questions, which more focused on medical history and oral habits of the staff, were considered for detailed comparison and valid interpretation in the further clinical phases of the study. in the second part of the questionnaire (concept of knowledge), eight questions were designed from general knowledge about dental caries and periodontal diseases to their causation and prevention. in previous studies, these questions were mostly based on general knowledge of the population (“brushing habits” and “frequency of dental appointments”) and their type was multiple choice.15,16 the first six questions in the concept of knowledge, were designed as multiple choice questions, but two further questions were developed so that respondents are supposed to prioritize in order the aforementioned causes of dental caries and its prevention methods, from the most likely to the least (1 to 4). the multifactorial nature of dental caries was the rationale to ask the questions in this form.30 also, the attributable risk of each factor was previously determined in studies30,31 and oral health care staff should know the hierarchy of them.20 the low homogeneity coefficient (kr-20) between the questions which were designed to measure the knowledge confirmed the multifactorial nature of dental caries and multidimensional character of oral health. each of eight questions referred to one of the aspects of oral health. the questions about “the number of permanent teeth” was a piece of general knowledge about the normal oral status and other questions related to the different subjects such as diet and snacking, regular toothbrushing, dental hygiene and calculus, malodor, appropriate dental visit interval, the causes and prevention methods of dental caries. this variety among questions led to low homogeneity (low kr-20 coefficient), which means that it cannot be predicted that if respondents chose the correct answer for the first three questions, they would choose the true answer for the following questions as well because these questions measure different aspects of oral health care knowledge. moreover, when the number of items on the scale is less than 10, it may cause homogeneity to be low.27 however, increasing the number of items could lead to a decrease in the number of respondents.27 given that the aim, each item would be assessed separately. regarding our research questions and given that all eight items reflect the basics of theoretical aspects of oral health care, items were retained despite their poor homogeneity coefficient.28 five out of six questions which were developed in the first draft to assess the attitude remained in the final instrument in the form of 4-scale likert (from strongly agree to strongly disagree). these questions ask the respondents’ attitudes about “oral hygiene and social popularity”, “brushing and periodontal diseases”, “brushing and halitosis”, “flossing and dental caries”, and “pregnancy and tooth decay”. a four-scale type of likert was preferred in this section to avoid the inclination of responses to the midpoint (neutral responses). question aq4 was deleted in the second revision. given that the cronbach a coefficient improved after removing one question (pq5), 11 questions remained to assess practice. question pq5 was “do you use your teeth to crack hard-shelled nuts such as hazelnut, walnut, pistachio?” and after its deletion, the items of the instrument were more closely related (0.62 vs 0.661). the left items asked about “brushing all teeth surfaces”, “annual dental appointments”, “cleaning the tongue”, “brushing after lunch in the workplace”, “eating or drinking after brushing at nights”, “type of their toothbrush bristles (soft/medium/hard)”, “their primary goal of flossing”, and “changing toothbrush frequency”. in comparison with a similar study performed for validating a kap instrument to measure it among health care professionals, the results of this study show high content validity indices as well and lower internal consistency. wong modified and validated a questionnaire to measure the kap of healthcare providers in elderly residences.32 this questionnaire was translated and retranslated from english into chinese and vice versa and all amounts of i-cvi, s-cvi/ave, s-cvi/ua, and cvr were in the excellent range (1.00, 1.00, 1.00, and >0.99, respectively). in addition, internal consistency showed good results for all three concepts (k = 0.67, a = 0.93, and p = 0.92). the questions more focused on tooth brushing and oral hygiene among old residents, denture care, and also dental table 3. inter class correlation for the questionnaire at baseline and two weeks later section icc lower bound ci 90% upper bound ci 90% true value df 1 df 2 background item 0.989 0.980 0.994 181.210 42 42 average 0.995 0.990 0.997 181.210 42 42 knowledge item 0.991 0.983 0.995 222.175 49 49 average 0.995 0.991 0.997 222.175 49 49 attitude item 0.708 0.536 0.823 5.757 49 49 average 0.829 0.698 0.903 5.757 49 49 behavior item 0.895 0.822 0.939 17.992 49 49 average 0.944 0.902 0.968 17.992 49 49 total item 0.970 0.945 0.984 66.373 49 49 average 0.985 0.972 0.992 66.373 49 49 281j contemp med sci | vol. 7, no. 5, september–october 2021: 276–281 h. toutouni et al. original designing a questionnaire on oral health care kap of dental staff visit in all domains. the high internal consistency of the instrument may be related to the point that this instrument already passed validation processes and was frequently utilized previously. the chinese version tested its valid feasibility in a new context. on the other hand, all items were mostly confined to one subject which was oral hygiene (brushing teeth and cleaning dentures) of elderly people. conclusion the dsaohc questionnaire can be a useful instrument to assess staff awareness at the beginning of the recruitment at dental school and help them to find the necessary courses and oral health care training to improve their self-care, which would indirectly affect their responsiveness and patient satisfaction. the data from this questionnaire will assist educational supervisors and managers to establish oral health courses. acknowledgment this study was ethically approved by ethical committee at mashhad university of medical sciences under the following code: ir.mums.sd.rec.1394.327, and was financially supported by the research deputy of mashhad dental school under research number: 960596. the authors would like to express their gratitude to the educational supervisor and director of nursing services, mrs jalalian and mr khorashadi, for their support during this study. also, the sincere thanks of the authors go to the faculty members of mashhad dental school who spent time reading and assessing the instrument. conflicts of interest authors have no conflict of interest.  references 1. lombardi dn, keramer b, schermerhorn jr. health care management. the united states of america: wiley; 2007. 2. roberts m, hsiao w, berman p, reich m. getting health reform right: a guide to improving performance and equity.1st ed. oxford: oxford university press;2003. 3. jalaleddin h, ramezani gh. prevalence of gingivitis among school attendees in qazvin, iran. east afr j public health. 2009;6(2):171–4. 4. phantumvanit p, makino y, ogawa h, rugg-gunn a, moynihan p, petersen p.e, evans w, feldens ca, lo e, khoshnevisan m.h, baez r, varenne b, vichayanrat t, songpaisan y, woodward m, nakornchai s, ungchusak c. who global consultation on public health intervention against early childhood caries. community dent oral epidemiol. 2017;13:280–288. 5. peres ma, macpherson lmd, weyant rj, daly b, venturelli r, mathur mr, listl s, celeste rk, guarnizo-herreno cc, kearns c, benzian h, allison p, watt rg. oral diseases: a global public health challenge. lancet. 2019;394:249–60. 6. phipps kr, stevens vj. relative contribution of caries and periodontal disease in adult tooth loss for an hmo dental population. j public health dent. 1995;55(4):250–252. 7. guo kl. decide: a decision-making model for more effective decision making by health care managers. health care manag. 2020. 39(3):133–141. 8. selwitz rh, ismail ai, pitts n. dental caries. lancet. 2007;369:51–9. 9. yadav kh, prakash s. dental caries: a review. asian j biomed pharmaceut sci. 2016; 6(53):1–7. 10. hajishengallis g, chavakis t. local and systemic mechanisms linking periodontal disease and inflammatory comorbidities. nat rev immunol. 2021;21:426–440. 11. kinane d, stathopoulou p, papapanou p. periodontal diseases. nat rev dis primers. 2017;3:1-14. 12. al-omiri mk, al-wahadni am, saeed kn. oral health attitudes, knowledge, and behavior among school children in north jordan. j dent educ. 2006;70(2):179–187. 13. rad m, shahravan a, haghdoust a. designing a valid questionnaire on oral health knowledge, attitude, and practice in 12 year-old children in iran. j mazandaran uni med sci. 2015;15(126):130–133. 14. hajmohammadi a. assessing knowledge, attitude, and behavior of health workers in public health care centers in kerman, iran. j kums 2010:50. (persian) 15. pourhashemi j. a survey on the knowledge of health system personnel in ghom province toward oral and dental health. j dent med. 2004;17(3):77–82. 16. kawamura m, iwamoto y. present state of dental health knowledge, attitudes, behaviour and perceived oral health of japanese employees. int dent j. 1999;49(3):173–81. 17. wardh i, jonsson m, wikstrom m. attitudes to and knowledge about oral health care among nursing home personnelan area in need of improvement. gerodontology. 2012;29:787–792. 18. nardi gm, giraldi g, lastella p, la torre g, saugo e, ferri f, pacifici l, ottolenghi l, guerra f, polimeni a. knowledge, attitudes and behavior of italian mothers towards oral health: questionnaire validation and results of a pilot study. annali di stomatologia. 2012;iii (2):69-74. 19. rasouli-ghahroudi aa, rokn ar, khorsand a, aghajani h, amini a, shamshiri ar, rahimi h, kabir a. designing and standardizing a questionnaire for evaluating knowledge, attitude, and practice of iranian adults with cardiovascular diseases about oral health. arya atheroscler. 2013;9(6):350–356. 20. phinney dj, halstead jh. delmar’s dental assisting: a comprehensive approach. united states:thomson delmar learning; 2000. 21. american dental assistants association. continueuing education materials; 2019 [updated 2021]. available from: https://www.adaausa.org/education/ continuing-education/ 22. daly b, batchelor p, treasure e, watt r. essentials dental public health. 2nd edition. usa: oxford university press; 2013. 23. garson gd. validity and reliability. usa: statistical associates publishers; 2013. 24. grant js, davis ll. selection and use of content experts for instrument development. res nurs health. 1997;20(3):269–274. 25. polit df, beck ct. the content validity index: are you sure you know what’s being reported? critique and recommendations. rese nurs health. 2006;29(5):489–497. 26. lawshe ch. a quantitative approach to content validity1. personnel psychology. 1975;28(4):563–575. 27. bolarinwa oa. principles and methods of validity and reliability testing of questionnaires used in social and health science researches. niger postgrad med j. 2015;22:195–201. 28. rattray j, jones mc. essential elements of questionnaire design and development. j clin nurs. 2006;16:234–243. 29. koo tk, li ym. a guideline of selecting and reporting intraclass correlation coefficients for reliability research. j chiropr med. 2016;15:155–163. 30. fejerskov o, nyvad b, kidd e . dental caries: the disease and its clinical management. 3rd ed. oxford: willey blackwell; 2015. 31. attaran n, khoshnevisan mh, ghorbani z, pakkhesal m, dehghanian d. dental caries predictors in countries with different human development index: a review of articles. j int oral health. 2016;8(2):182–190. 32. wong fmf. first data in the process of validating a tool to evaluate knowledge, attitude, and practice of healthcare providers in oral care of institutionalized elderly residents: content validity, reliability and pilot study. int j environ res public health. 2021;18:1–15. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i5.1071 134 j contemp med sci | vol. 9, no. 2, march-april 2023: 134–138 original silent coronary artery disease in patients with rheumatoid arthritis in kurdistan, iraq: a cross-sectional study ameen m mohammad1*, malavan mohammed2, mohammed t rasul1, azri salih sgery3 1department of medicine, college of medicine, university of duhok, duhok, iraq. 2department of biomedical science, college of medicine, university of zakho, duhok, iraq. 3college of medicine, university of duhok, duhok, iraq. *correspondence to: ameen m. mohammad (e-mail: doctoramb@yahoo.com) (submitted: 10 january 2023 – revised version received: 02 february 2023 – accepted: 25 february 2023 – published online: 26 april 2023) abstract objectives: this study attempts to determine the silent coronary artery disease (cad) in a sample of iraqi kurdish patients diagnosed with rheumatoid arthritis (ra). methods: a total of 50 such patients from duhok, kurdistan region of iraq, underwent multi slices computed tomography (msct) coronary angiography 64 slices to determine coronary artery calcium (cac) scores and coronary stenoses. results: 62% of cases had a (cac) score > zero on non-contrast msct scans. coronary angiograms showed that 26 (52%) of the cases had variable coronary artery stenosis; 17(34%) of them were obstructive (>50% luminal narrowing). the study demonstrated that inflammatory markers (rheumatoid factor, anti-cyclic citrullinated peptides, and some cardiovascular risk factors, namely (hypertension and type 2 diabetes mellitus) were significantly related to the presence of cad in these patients. conclusions: patients with rheumatoid arthritis are at increased risk of cad. this risk is higher in the presence of inflammatory and cardiovascular risk factors. keywords: coronary artery disease, arthritis, rheumatoid, coronary vessels issn 2413-0516 introduction rheumatoid arthritis (ra) is a chronic inflammatory disease of unknown aetiology with prominent joint manifestations.1 compared to the general population, patients with ra have a reduced life expectancy due to comorbidities such as hypertension and other cardiovascular diseases. those patients with the established ra have an average of two or more comorbidities.2-4 the ra cases are known to have a higher prevalence of cardiovascular morbidity and mortality compared to the general population.5,6 cardiovascular diseases, particularly cad, constitute the leading cause of premature mortality in patients with ra.7,8 the underlying mechanism of cad tends to be complex and multifactorial, with the additive association of non-traditional risk factors to atherosclerosis.9,10 rheumatoid factor, anti-cyclic citrullinated peptide antibody, long duration of ra, and thrombogenic factors are among the non-traditional risk factors contributing to morbidity and mortality of cad in ra9-12 given the fact that ra patients are less likely to experience symptoms of cad and somewhat likely to develop unrecognized events and unforeseen death, the clinical presentation of cad in such patients is different, apparently earlier, silent, and sudden.13,14 ra patients were twice as likely to experience sudden death than non-ra subjects.5,14,15 despite the incomplete data to fully explain the relationship between ra and cad, patients with ra increasingly reported a higher atherosclerotic burden and coronary artery calcification. consequently, atherosclerosis accelerated, and the cardiovascular outcomes worsened.5,16,17 the european alliance of associations for rheumatology (eular) recommended the prevention, detection, and management of comorbidities associated with ra to improve the long-term outcomes of patients.18 such outcomes can be improved by the identification of the new markers for the silent cad. hence, this study aimed to assess the presence of silent cad in patients with ra in duhok, iraq. material and methods study design this cross-sectional study was conducted at the rheumatology center and azadi heart center in duhok, iraq. a total of 50 patients (47 females and 3 males) aged 18-70 years were involved in the study. patients all cases were already diagnosed with ra according to the american college of rheumatology 2010 criteria for ra. included patients in this study did not have a prior history of cad or ischemic stroke. detailed clinical history and examination of ra cases were taken. patients were checked for cardiovascular risk factors namely, hypertension, type 2 diabetes mellitus, dyslipidemia, smoking, and family history of cad. drug history, including the use of disease-modifying anti-rheumatic drugs and steroids, was also recorded. methods and investigations serologic markers, including the rheumatoid factor (rf), antibodies to cyclic citrullinated peptides (anti-ccp), erythrocyte sedimentation rates (esr), and c-reactive protein (crp) were measured. a cardiac assessment was performed by basic resting electrocardiography and echocardiography. multi-slices ct (msct) scan of coronary arteries was performed for the cases according to standard protocols. the degree of coronary artery calcification was calculated based on the method described by agatston et al.19 mailto:doctoramb@yahoo.com 135j contemp med sci | vol. 9, no. 2, march-april 2023: 134–138 a.m. mohammad et al. original silent coronary artery disease in patients with rheumatoid arthritis in kurdistan, iraq the sum of the scores for all coronary artery lesions represented the overall agatston calcium score for each individual was graded as [0 for non, 1 for mild (1–100 hu), 2 for moderate (101–399 hu), 3 for severe (> 400 hu)] coronary artery calcification. coronary artery stenosis severity was scored from 0–4 based on grades (g0–4) of luminal restriction as following: [grade 0 (g0): normal coronary lumen, grade 1 (g1) represented 1-29% stenosis, g2: 30–49%, g3: 50–69% stenosis, g4: > 70% stenosis]. lesions rendering > 50% stenosis were considered obstructive. inclusion/exclusion criteria all adult patients diagnosed with rheumatoid arthritis were included in the study. whereas, patients with overlapping rheumatoid arthritis, childhood rheumatoid arthritis, history of coronary artery disease, and patients with incomplete information or missing data were excluded from the study. statistical analysis continuous variables were calculated as mean ± (sd), and categorical variables were presented as counts and percentages. a chi-square test was used for the comparison of categorical variables. the continuous variables were assessed by students t-test. a probability value of p ≤ 0.05 was considered statistically significant. formal consent was obtained from all the cases orally upon enrollment. the ethical approval of this study was obtained from the iraqi national board of medical specializations in baghdad, iraq. results clinical characteristics of participants the mean age of patients is 52.78 ± 12.4 years, 47(94%) of them were females. the mean duration of ra was 9.88 years. 26 cases (52%) were early cases of ra (< 5 years duration from diagnosis of disease) and 24 (48%) were established cases (³ 5 years duration). 18 (36%) out of 50 cases were hypertensive, 12 (24%) were t2dm, and 17 (34%) had table 1. characteristics of the study’s participants n = 50 percentageno. of patientsclinical characteristics –52.78 ± 12.4 age (mean) year 94%47female % 52%26early ra 48%24established ra 24%12smoking 36%18hypertension 24%12diabetes mellitus 34%17dyslipidemia 62%31positive rf 66%33esr >21 mm/1st h 28%14crp > 10 54%27positive anti-ccp ra: rheumatoid arthritis; rf: rheumatoid factor; esr: erythrocyte sedimentation rate; crp: c-reactive protein; ccp: cyclic citrullinated peptides. dyslipidemia. only 8 (16%) had a positive family history of cad. rf was positive in 31 (62%) cases. erythrocyte sedimentation rate (esr) and creactive protein (crp) were raised in 33 (66%) and 14 (28%), respectively. the anti-ccp test was positive in 27 (54%) of patients, as in table 1. patients with hypertension and t2dm had more cac scores with statistical significance (p = 0.04, 0.009), respectively. dyslipidemia did not achieve a significant statistical association with cac (p = 0.06). patients with the early duration of ra had lower cac scores compared to those with established ra (p = 0.01). the presence of rf and anti-ccp were significantly related to higher cac scores (p = 0.009, 0.006), respectively. however, esr and crp were found to be statistically non-significant in their association with cac scores, as seen in table 2. the grades of coronary artery stenoses among ra were significantly higher in patients with hypertension and table 2 coronary artery calcium score according to the patient’s clinical characteristics variables 0 1–100 101–399 >400 total p value age 48.78 ± 10.02 51.23 ± 7.5 52.7 ± 13.4 58.23 ± 15.6 52.78 + 12.4 0.18 sex (female) 17(34%) 4(8%) 8(16%) 18(36%) 47(94%) 0.1 early ra 13(26%) 3(6%) 6(12%) 4(8%) 26(52%) 0.03* established ra 7(14%) 1(2%) 3(6%) 13(26%) 24(48%) 0.01* hypertension 4(8%) 0 3(6%) 11(22%) 18(36%) 0.04* t2dm 0 0 4(8%) 8(16%) 12(24%) 0.009* dyslipidemia 4(8%) 1(2%) 2(4%) 10(20%) 17(34%) 0.06 positive family history of cad 2(4%) 1(2%) 1(2%) 4(8%) 8(16%) 0.2 current active smoking 2(4%) 0 1(2%) 1(2%) 4(8%) 0.3 positive rf 16(32%) 2(4%) 5(10%) 8(16%) 31(62%) 0.009* raised esr 15(30%) 2(4%) 6(12%) 10(20%) 33(66%) 0.06 positive crp 4(8%) 2(4%) 1(2%) 7(14%) 14(28%) 0.1 positive anti-ccp 6(12%) 2(4%) 6(12%) 13(26%) 27(54%) 0.006* *p-value ≤ 0.05 is considered statistically significant. ra: rheumatoid arthritis; t2dm: type 2 diabetes mellitus; rf: rheumatoid factor; esr: erythrocyte sedimentation rate; crp: c-reactive protein; ccp: cyclic citrullinated peptides. 136 j contemp med sci | vol. 9, no. 2, march-april 2023: 134–138 silent coronary artery disease in patients with rheumatoid arthritis in kurdistan, iraq original a.m. mohammad et al. dyslipidemia with (p = 0.01, 0.02), respectively. among t2dm, such grades were also high but didn’t reach a significant level (p = 0.06). the cases with established ra and those with anti-ccp were significantly had higher coronary stenoses, as shown in table 3. discussion the excess mortality associated with ra is mainly due to cardiovascular diseases, particularly cad.5,16-18 recent observational studies suggested that the hidden cad risk in ra is not related only to traditional atherosclerosis risk factors.10,20 given the importance of chronic inflammation in atherogenesis, the presence of ra only may be of primary importance in terms of cardiovascular comorbidities and burden. this cross-sectional study was conducted in patients with ra lacking a prior history of cad or ischemic stroke to assess the relationship between silent coronary artery disease and ra. its main findings were as follows; 31 (62%) of them had a coronary artery calcium score > zero on non-contrast msct, and 26 (52%) cases had a variable coronary luminal lesion on msct angiograms. the mechanism underlying the observed increase in cac in ra is likely complex and multifactorial. following an inflammatory event, interleukin-6 induce the production of acute phase reactant (crp), which is a key marker of systemic inflammation in ra and has a direct role in the development and progression of atherosclerosis.21 in multiple population-based studies,20,22-25 individuals with the highest concentrations of circulating inflammatory markers were at the greatest risk of cardiovascular events and mortality and tended to demonstrate an increased burden of subclinical atherosclerosis like msct-measured cac burden.25 recent reports suggest that the mechanism and management of cardiovascular risk in ra are challenging. apart from the fact that several risk factors are involved in this mechanism, the presenting features of cad might be silent and differ from those in the general population. ra patients may experience cardiovascular events in the presence of minimal atherosclerosis. maradit-kremers et al.16 reported an increased table 3. grading of coronary ct-angiography according to patient’s clinical characteristics variables g0 g1 g2 g3 g4 total p-value age (years) 49.16 ± 14.2 49 ± 7.6 57.5 ± 3.4 56.57 ± 5.4 59.4 ± 12.6 52.78 + 12.4 0.07 sex (female) 23(46%) 5(10%) 4(8%) 5(10%) 10(20%) 47(94%) 0.3 early ra 16(32%) 3(6%) 3(6%) 2(4%) 2(4%) 26(52%) 0.02* established ra 8(16%) 2(4%) 1(2%) 5(10%) 8(16%) 24(48%) 0.003* hypertension 5(10%) 2(4%) 2(4%) 3(6%) 6(12%) 18(22%) 0.01* t2dm 2(4%) 0 2(4%) 2(4%) 6(12%) 12(24%) 0.06 dyslipidemia 5(10%) 2(4%) 0 4(8%) 6(12%) 17(34%) 0.02* positive family history of cad 3(6%) 0 1(2%) 1(2%) 3(6%) 8(%16) 0.2 current active smoking 2(4%) 0 1(2%) 1(2%) 0 4(8%) 0.4 positive rf 19(38%) 3(6%) 1(2%) 3(6%) 5(10%) 31(62%) 0.08 raised esr 17(34%) 2(4%) 2(4%) 5(10%) 5(10%) 33(66%) 0.2 positive crp 5(10%) 2(4%) 0 1(2%) 6(12%) 14(28%) 0.1 positive anti-ccp 9(18%) 5(10%) 1(2%) 5(10%) 7(14%) 27(54%) 0.01* *p-value ≤ 0.05 is considered statistically significant. ra: rheumatoid arthritis; t2dm: type 2 diabetes mellitus; rf: rheumatoid factor; esr: erythrocyte sedimentation rate; crp: c-reactive protein; ccp: cyclic citrullinated peptides. risk for cad among ra patients not only from the disease onset but even before meeting the criteria of the american college of rheumatology for ra. nevertheless, in early ra, the extent of coronary atherosclerosis is not increased.26 the ra patients reportedly experienced sustained unrecognized myocardial infarction and faced sudden death more often than non-ra subjects.16,25,26 the frequencies of multi-vessel coronary disease, recurrent ischemic events, and death after an acute coronary syndrome are increased in ra.27 the cac obtained by msct in ra might improve the prediction of cardiac events among ra. the msct evaluates the severity of stenosis and the burden of the coronary plaques, thus improving the predictive value of cac for future events.27-29 regarding traditional risk factors for cad, hypertension and t2dm were significantly correlated with cac. among the non-traditional risk factors assessed in this study, 62% of the patients were rf positive, and 54% had a positive anti-ccp. the anti-ccp autoantibodies and rf autoantibodies have a synergistic effect in mediating ra-associated inflammation and disease activity, and their presence was associated with a higher rate of cac and cad in patients with ra.30-32 choi and colleagues demonstrated in their study that methotrexate-treated patients had a 70% reduction in cardiac events compared with those not receiving the same therapy regularly.33 other disease-modifying drugs such as sulfasalazine, penicillamine, hydroxychloroquine, and gold did not confer this protection. thus, the ra disease process itself likely contributes to accelerated cad.31,33 limitations of this study include the relatively small sample size, dominance of female cases and disease activity by das 28-esr and das 28-crp were not measured. its strengths lie in the screening design of silent cad in the ra, the use of msct-coronary angiography modality, and the novelty of the study at the national and middle east levels. conclusion this study concludes that ra patients remain at increased risk of cad even regardless of traditional cardiovascular risk 137j contemp med sci | vol. 9, no. 2, march-april 2023: 134–138 a.m. mohammad et al. original silent coronary artery disease in patients with rheumatoid arthritis in kurdistan, iraq factors. msct coronary angiography is a good option for the evaluation and screening of ra patients at risk of silent cad. the awareness of silent cad among patients with ra and their doctors is of paramount importance for the initiation of lifestyle changes and necessary medication to decrease the progression of coronary atherosclerosis early in the disease course. further studies are indicated to thoroughly understand the mechanisms of increased atherogenesis in ra. list of abbreviations anti-ccp: antibodies to cyclic citrullinated peptides; cac: coronary artery calcium; cad: coronary artery disease; crp: c-reactive protein; das 28: disease activity score-28; esr: erythrocyte sedimentation rates; eular: european alliance of associations for rheumatology; msct: multi-slices computed tomography; ra: rheumatoid arthritis; rf: rheumatoid factor; t2dm: type 2 diabetes mellitus. ethics approval the iraqi national board of medical specializations in baghdad, iraq approved the study protocol. informed consent informed consent was obtained from all the cases upon enrollment. acknowledgments we would like to thank the staff of the cardiology, radiology and rheumatology centers in duhok, iraq for their support in conducting this study. conflict of interests the authors declare that they have no conflict of interest. sources of funding no financial support to be declared. data availability the data sets used in this study are available from the corresponding author upon request.  references 1. bullock j, rizvi sa, saleh am, ahmed ss, do dp, ansari ra, et al. rheumatoid arthritis: a brief overview of the treatment. medical principles and practice. 2018;27(6):501–7. 2. jeong h, baek sy, kim sw, eun yh, kim iy, kim h, et al. comorbidities of rheumatoid arthritis: results from the korean national health and nutrition examination survey. plos one. 2017;12(4):e0176260. 3. mohammad am, jehangeer hi, shaikhow sk. prevalence and risk factors of premature coronary artery disease in patients undergoing coronary angiography in kurdistan, iraq. bmc cardiovascular disorders. 2015;15(1):155. 4. mohammad am, othman go, saeed ch, al allawi s, gedeon gs, qadir sm, et al. genetic polymorphisms in early-onset myocardial infarction in a sample of iraqi patients: a pilot study. bmc research notes. 2020;13(1):541. 5. gabriel se. why do people with rheumatoid arthritis still die prematurely? annals of the rheumatic diseases. 2008;67(suppl 3):iii30–iii4. 6. mohammad am, hasan yousif a, ahmed qasim b, aziz joma j, saeed sy. estimation of apolipoprotein a in early onset st-segment elevation myocardial infarction. cardiovasc endocrinol metab. 2019;8(4):106–8. 7. gabriel se. cardiovascular morbidity and mortality in rheumatoid arthritis. the american journal of medicine. 2008;121(10):s9–s14. 8. solomon dh, karlson ew, rimm eb, cannuscio cc, mandl la, manson je, et al. cardiovascular morbidity and mortality in women diagnosed with rheumatoid arthritis. circulation. 2003;107(9):1303–7. 9. mohammad a, othman g, saeed c, allawi n. tct-441 traditional and genetic risk factors in young patients with myocardial infarction in iraq. journal of the american college of cardiology. 2017 oct 31;70(18s):b181-. 10. turesson c, jarenros a, jacobsson l. increased incidence of cardiovascular disease in patients with rheumatoid arthritis: results from a community based study. annals of the rheumatic diseases. 2004;63(8):952–5. 11. mohammad am, al-allawi nas. cyp2c19 genotype is an independent predictor of adverse cardiovascular outcome in iraqi patients on clopidogrel after percutaneous coronary intervention. journal of cardiovascular pharmacology. 2018;71(6):347–51. 12. demizio dj, geraldino-pardilla lb. autoimmunity and inflammation link to cardiovascular disease risk in rheumatoid arthritis. rheumatology and therapy. 2020;7(1):19–33. 13. lee th, song gg, choi sj, seok h, jung jh. relationship of rheumatoid arthritis and coronary artery disease in the korean population: a nationwide cross-sectional study. advances in rheumatology. 2019;59. 14. sarmiento-monroy jc, amaya-amaya j, espinosa-serna js, herrera-díaz c, anaya j-m, rojas-villarraga a. cardiovascular disease in rheumatoid arthritis: a systematic literature review in latin america. arthritis. 2012;2012. 15. mohammad am, abdulhaleem bh, habeeb qs. first 24 h’ outcomes of acute coronary syndrome in iraq. medical journal of babylon. 2020;17(2):154. 16. maradit‐kremers h, crowson cs, nicola pj, ballman kv, roger vl, jacobsen sj, et al. increased unrecognized coronary heart disease and sudden deaths in rheumatoid arthritis: a population‐based cohort study. arthritis & rheumatism: official journal of the american college of rheumatology. 2005;52(2):402–11. 17. tournadre a, mathieu s, soubrier m. managing cardiovascular risk in patients with inflammatory arthritis: practical considerations. therapeutic advances in musculoskeletal disease. 2016;8(5):180–91. 18. baillet a, gossec l, carmona l, de wit m, van eijk-hustings y, bertheussen h, et al. points to consider for reporting, screening for and preventing selected comorbidities in chronic inflammatory rheumatic diseases in daily practice: a eular initiative. annals of the rheumatic diseases. 2016;75(6):965–73. 19. van der bijl n, joemai rm, geleijns j, bax jj, schuijf jd, de roos a, et al. assessment of agatston coronary artery calcium score using contrast-enhanced ct coronary angiography. ajr am j roentgenol. 2010;195(6):1299–305. 20. ridker pm, hennekens ch, buring je, rifai n. c-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. new england journal of medicine. 2000;342(12):836–43. 21. pope je, choy eh. c-reactive protein and implications in rheumatoid arthritis and associated comorbidities. seminars in arthritis and rheumatism. 2021;51(1):219–29. 22. mohammad am, sheikho sk, tayib jm. relation of cardiovascular risk factors with coronary angiographic findings in iraqi patients with ischemic heart disease. am j cardiovasc dis res. 2013;1(1):25–9. 23. wang tj, larson mg, levy d, benjamin ej, kupka mj, manning wj, et al. c-reactive protein is associated with subclinical epicardial coronary calcification in men and women: the framingham heart study. circulation. 2002;106(10):1189–91. 24. ross r. atherosclerosis—an inflammatory disease. new england journal of medicine. 1999;340(2):115–26. 25. chung cp, oeser a, raggi p, gebretsadik t, shintani ak, sokka t, et al. increased coronary‐artery atherosclerosis in rheumatoid arthritis: relationship to disease duration and cardiovascular risk factors. arthritis & rheumatism: official journal of the american college of rheumatology. 2005;52(10):3045–53. 26. douglas km, pace av, treharne gj, saratzis a, nightingale p, erb n, et al. excess recurrent cardiac events in rheumatoid arthritis patients with acute coronary syndrome. annals of the rheumatic diseases. 2006;65(3):348–53. 138 j contemp med sci | vol. 9, no. 2, march-april 2023: 134–138 silent coronary artery disease in patients with rheumatoid arthritis in kurdistan, iraq original a.m. mohammad et al. antibody–mediated inflammation in rheumatoid arthritis. arthritis & rheumatology. 2014;66(4):813–21. 31. mohammad am, shammo na, jasem ja. vitamin d status in acute myocardial infarction: a case-control study. cardiovasc endocrinol metab. 2018 nov 14;7(4):93–96. doi: 10.1097/xce.0000000000000160. pmid: 31646291; pmcid: pmc6739859. 32. fazeli ms, khaychuk v, wittstock k, breznen b, crocket g, pourrahmat m-m, et al. cardiovascular disease in rheumatoid arthritis: risk factors, autoantibodies, and the effect of antirheumatic therapies. clinical medicine insights: arthritis and musculoskeletal disorders. 2021;14:11795441211028751. 33. choi hk, hernán ma, seeger jd, robins jm, wolfe f. methotrexate and mortality in patients with rheumatoid arthritis: a prospective study. the lancet. 2002;359(9313):1173–7. 27. rinehart s, vazquez g, qian z, voros s. coronary plaque imaging with multi-slice computed tomographic angiography and intravascular ultrasound: a close look inside and out. the journal of invasive cardiology. 2009;21(7):367–72. 28. karpouzas ga, malpeso j, choi t-y, li d, munoz s, budoff mj. prevalence, extent and composition of coronary plaque in patients with rheumatoid arthritis without symptoms or prior diagnosis of coronary artery disease. annals of the rheumatic diseases. 2014;73(10):1797–804. 29. hou z-h, lu b, gao y, jiang s-l, wang y, li w, et al. prognostic value of coronary ct angiography and calcium score for major adverse cardiac events in outpatients. jacc: cardiovascular imaging. 2012;5(10):990–9. 30. sokolove j, johnson ds, lahey lj, wagner ca, cheng d, thiele gm, et al. rheumatoid factor as a potentiator of anti–citrullinated protein this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1339 420 j contemp med sci | vol. 8, no. 6, november-december 2022: 420–423 case report the effectiveness of topical arnebia euchroma oil in the treatment of pressure ulcer: a case report sakineh erabi1,2,3, ali ghobadi1,4, hoorieh mohammadi kenari1,2* 1research institute for islamic and complementary medicine, iran university of medical sciences, tehran, iran. 2department of traditional medicine, school of persian medicine, iran university of medical sciences, tehran, iran. 3student research committee, iran university of medical sciences, tehran, iran. 4department of traditional pharmacy, school of persian medicine, iran university of medical sciences, tehran, iran. *correspondence to: hoorieh mohammadi kenari (e-mail: hooriehmohhamadikenari@yahoo.com) (submitted: 10 september 2022 – revised version received: 25 september 2022 – accepted: 19 october 2022 – published online: 26 december 2022) abstract: decreased tissue blood flow causes pressure ulcers due to prolonged pressure on the area, which becomes problematic for immobile individuals. the medical community has often been involved in curing such a problem in individuals with disabilities and immobility. to alleviate this problem, therefore, plenty of investigations are always seeking novel methods. furthermore, herbal plants are increasingly used owing to the accepted status of plant-derived drugs in recent years. our female case aged 52 years that was referred to the wound clinic complaining sensory and motor weakness of the upper extremity and sudden paralysis of the lower extremity as of 1 year ago, which was diagnosed as myelitis. she complained of two wounds on both sides of the right and left buttocks with a size of 9 × 5 and another 6 × 4.5 cm from 3 months ago, and a wound with a size of 5 × 4.5 in the sacral area (sacrum) from 20 days prior to the admission. there was tissue necrosis without muscle engagement, osteomyelitis, or tunneling; besides, nasty odors and sludge were absent accounted for in class 3 in the group of wounds. she received starting therapy using arnebia euchroma oil. as of the 2nd week onward, she was treated very successfully, and the complete wound healing occurred in 8 weeks. the use of a. euchroma for wound healing and reducing infection and inflammation can greatly contribute to the cure of pressure ulcers and can be properly suggested in healing these ulcers. keywords: pressure ulcer, wound healing, arnebia euchroma, bed sore, traditional persian medicine issn 2413-0516 introduction in a pressure ulcer, the skin or the underneath tissue is locally injured from unmitigated pressure on the soft tissue of the body,1 or pressure along the cleft between a boney protuberance and an exterior surface for a prolonged period.2,3 there is an increased number of patients with pressure ulcers in spite of the improvements in the quality of delivering healthcare on the globe such that pressure ulcers have become mainly problematic in acute and prolonged care centers.4,5 this problem is reported to be prevalent in over 1.3 million adult people globally.6 sore results from tissue blood flow impairment and progressively necrotic areas of the body cells and necrotic cells that destroy their underneath layers.7 bedsores are mostly observed in sacrum and heels, with types 1 and 2 being the prevalent levels of austereness.8,9 as defined by the national pressure ulcer advisory panel (npuap),10 it is a persistently pressured area typically on a boney protuberance leading to ischemia, cell death, and necrotic tissue (table 1).11 as an index, the quality of patient care services is indicated by who using the occurrence and frequency of bedsores. it is highly important to use efficacious preventing actions and their therapies. the therapy is based on assessing sore austereness, reduction of pressure, friction, and shear stresses, wound care improvement, necrotic residue removal, bacterial infection management, and correction of nutrition-related shortages. intervention approaches consist of special alterations in bedsores in combination with training approaches. the 2019 cpg defines bedsore as “the locally injured skin and/or the underneath tissue, resulting from pressure or pressure combined with shear. while pus/pis typically happen over a boney protuberance, they can result from a medical apparatus or different objects”.12 bedsore may be represented as healthy skin or as an open sore with possible pain. the tissue is damaged due to the intensely and/or lengthily exposed soft tissues to persistent mechanical loading, i.e., malformations in compression, tension, or shear or a combined condition of these loading styles. persistent loading (defined as quasi-static loading as well, i.e., a nearly/almost static loading state) denotes loads exerted constantly for prolonged periods, including minutes to hours or even days. the soft tissues are differently tolerant to persistent malformations by the tissue type; microclimate, perfusion, age, health status (either chronic or acute), and systemic comorbidities and localized (topical) conditions of the soft tissues may also influence persistent malformations, and the persistent mechanical loading affects the mentioned factors.13 bedsore therapy is strongly predicted by the use of humid coatings and supporting sufficiently through nutrition.14,15 currently, society and the medical workforce are interested in plant-derived drugs and remedies and conventionally conducted traditional medicine, which are justifiable cost-effectively in addition to their lengthy history, safe use, accessibility, and ease of utilization.16,17 additionally, traditional herbal species are today commonly used for sore remedies, and more than 80% of the global population is reliant on traditional medicine for various skin ailments.18,19 herbal plants are the main resource of natural products with different actions regarding their structures, biologic activities, and modes of action.20-22 plant materials contain various constituents, in particular polyphenols, flavonoids, phenolic acids, and the like, which play a role in suppressing free radicals and in the antioxidant activities of herbal plants.17,23 arnebia euchroma (royle) i.m. johnst is a traditional herbal species that is reputable in the traditional medicine of iran,24 termed a red plant or heveh choaeh. arnebia belongs to mailto:hooriehmohhamadikenari@yahoo.com 421j contemp med sci | vol. 8, no. 6, november-december 2022: 420–423 s. erabi et al. case report the effectiveness of topical arnebia euchroma oil in the treatment of pressure ulcer: a case report table 1. degrees of bedsore defined by npuap14,40 degree 1 erythema without pressure whitening intact skin with redness with no pressure whitening (non-blanchable) in a localized area, typically on a boney protuberance. the area can possibly have pain and may be hard, soft, and warmer, or colder than adjacent tissues. degree 2 relative thickness losing partially the thickness of the dermis appearing as a surface lesion with a red, pink, scaly bed. it appears as a blushing or dry surface lesion with no scaling or bruising. this categorization is not useable in describing skin tears, band burns, dermatitis accompanied by incontinence, maceration, or excoriation. degree 3 losing all skin thickness fat may be present beneath the skin, but not under the bone, tendon, or muscle. scaling may be present but the depth of the tissue loss is unknown. this lesion can comprise atrophy and perforation. degree 4 losing all tissue thickness losing complete thickness of tissue by appearing bone, tendon, or muscle. it is typically accompanied by degeneration and perforation. grade 4 sores can extend to muscle or supportive structures (the fascia, tendon, or joint capsule) possibly leading to osteomyelitis or osteitis. fig. 1 sacral ulcer at the start of treatment. the boraginaceae family and contains a variety of species growing in asian and north african regions. it appears as an herb-like plant covered with pointy silver hairs. h. choaeh is a dark red and unscented herbal species growing in southeastern iran. the herb’s root is the useable part. the roots of herbal plants, such as echium, lithospermum, onosma, alkanna, and arnebia are replete in naphthoquinone, shikonin, alkanin, and isohexenylnaphthazarin derivatives with various remedial activities, including anti-inflammatory, antimicrobial, and anticancer properties.25-27 the reliable books of iranian medicine, e.g. makhzan al-adavieh and qarabadin, introduce “h. choaeh” as one of the main remedies to heal wounds. it is also utilized in the formulation of some topical wound-remedial drugs that assist the skin recovery and regenerate the tissue, cure skin inflammation, and old refractory lesions.28-32 in recent years, the activities of this herbal species have been investigated in plenty of research. h. choaeh has shown anti-inflammatory impacts on the remedy of surgical wounds, burns, and bedsores.33-36 in addition to its wound remedy activity, this herbal species has been demonstrated to possess anti-inflammatory, antibacterial, anti-tumor, anti-diabetic, anti-viral, antioxidant, and anti-thrombotic activities.37-39 case report our case was a female aged 52 years who complained of sensory and motor weakness of the upper extremity and abrupt palsy of the lower extremity since a year ago; she was diagnosed with myelitis and complained of three lesions, two of which were on both sides of the right and left buttocks involving the muscle with no osteomyelitis, tunneling, nasty smell, and slush since 3 months prior to the admission. for a month prior to referral, she was under antibiotic therapy, progressive bandages, and ozone therapy for these two wounds in the hospital. despite the debridement and cleaning of the lesions, indications of curing were absent. these two lesions were measured at 9 by 5 and another one 6 by 4.5 cm, which accounted for grade 3 in the class of lesions. besides, 20 days prior to the visit, a newly emerged lesion measuring 5 by 4.5 occurred in the sacral region, with tissue necrosis with no muscle engagement, osteomyelitis, or tunneling, having nasty odors and slush, which accounted for grade 3 in the class of lesions. the wound was not debrided in this case (figure 1). all the lesions were scored 13 by push criteria scoring. although the surgery was necessary, it was not possible to operable the patient due to her overall situation; thus, she was introduced to our wound clinic (orchid) for topical therapies. a. euchroma oil was the starting remedy for her. she underwent a oil-containing bandage two times daily that covered the lesion surface by 3 mm each time. enhancements in serous secretion, granulation tissue, odor, and lesion size shrinking were initially monitored every day and then every week. serous was discharged slightly in the whole lesions on the 1st day. within the 1st week, the discharge rose in the lesions but with no nasty odor. following 10 days of beginning the therapy, the nature of the tissue changed in the pressure ulcers with a pinkish color change and generating healthy granulation tissue, which caused the wound contraction. by the 3rd week, the length and width of the lesions did not change significantly, however, the 10th day of the therapy onward was associated with the filled cavity depth and reduced lesion depth, appearing nearly healed. the sacral lesion was photographed after 6 weeks of therapy, indicating the considerable and acceptable remedy of the lesions (figure 2), which were scored 11, 9, and 9 in push scoring. similarly, the lesions 422 j contemp med sci | vol. 8, no. 6, november-december 2022: 420–423 the effectiveness of topical arnebia euchroma oil in the treatment of pressure ulcer: a case report case report s. erabi et al. healed completely (disappearance of the secretions, filling of the cavity, and fully closed lesions) after the 8th week. preparation of h. choaeh drug qarabadin is a worthful pharmacological book of traditional medicine in iran, which was employed to prepare h. choaeh oil.41 the preparation process was started with heating olive oil (to the degree without burning the oil with no rising odor of burns by preventing fumes). this was followed by adding the ground root of h. choaeh in a volume of a quarter of its weight and then boiling for dying the oil. next, some wax was added for oil filtration and concentration. discussion this case demonstrated that h. choaeh can be used with high contribution to healing bedsores. the h. choaeh root utilized here contains a variety of compounds such as alkanin and chiconin.3 as mentioned by the specialists of traditional medicine, h. choaeh has a hot and dry nature, and its roots possess various activities including the application in wound-drying liniments and for treating some skin ailments. the anti-inflammatory impact of h. choaeh roots was assigned to its two natural products, alkannin and shikonin, by kourounakis et al. (2002). as claimed by sidhu et al., the therapy with h. choaeh is beneficial as it significantly reduces the ulcer surface area, increases cell multiplication, and amplifies collagen biosynthesis. additionally, senel and papageorgious proposed the antioxidant property and the function of oxygen free radicals, alkaline, and chiconin as novel alternates for wound remedy via anti-inflammatory, antimicrobial, and antioxidant activities.35-38 assimopoulou et al. ascribed the anti-inflammatory impacts of h. choaeh roots to hydroxynaphthoquinones being present in its root extracts. ahmadian et al. also report that chiconin (beta alkanin) is a naphthoquinone pigment obtained from plant roots with antimicrobial effects.42,43 damiaunakos et al. presented evidence of potent chiconin and alkaline drugs having substantial biologic functions, such as wound remedy and antimicrobial, anti-inflammatory, antioxidant, anticancer, and anti-thrombotic effects.44 akkol et al. documented that therapy by h. choaeh lowered the lesion area and enhanced cell multiplication, migration, vascular formation, and re-epithelialization of lesions.45 in a study on the properties of a. euchroma and malva sylvestris, pirbalouti et al. found that h. choaeh significantly influenced wound remedy acceleration.46 the same authors examined the influence of h. choaeh root extract on diabetic sores in rats. they reported that the sore level showed a significant reduction in the test animals with a shorter epithelial time than the control rats as the sore was contracted more swiftly.47 nasiri et al. assessed the impact of h. choaeh on burns and observed a 10% better mean wound remedy in the h. choaeh group than silver sulfadiazine after 10 days of burns. the h. choaeh group also showed a greater percentage of shrinking wounds.32 in their research on rats, ashkani et al. found that the extract of h. choaeh more significantly affected epithelialization, fibroblast proliferation, and collagen synthesis, as well as more anti-inflammatory impacts than the control group.48 sedaghat et al. investigated the impact of h. choaeh on the remedy of sores in rats. they observed the anti-inflammatory impacts of a. euchroma that helped wound remedy by mitigating the degree and acuteness of inflammation.36 the literature indicates the anti-inflammatory and wound remedy impacts of h. choaeh. according to the present case study, the use of this herb alongside appropriate nursing care resulted in significantly improved wound healing. plant sources are worthwhile for investigations on developing novel medicines. accordingly, the renewing impact of h. choaeh was examined in this research, indicating that the oil prepared from this herb possesses antibacterial, anti-inflammatory, and immunomodulatory properties involved in decontamination. it helps in the creation of an intact granulation tissue for wounds, and its antioxidant properties eliminate free radicals from the environment, preventing their creation. such activities of the drug control the wound remedy procedure and the view of remedial signs. the h. choaeh oil has a mediatory role between the sore surface and the coverage substance to attach them together that hinders drying the wound and improves the appropriate remedy of wounds by the acceleration of epithelialization and collagen synthesis. regarding this, it seems that the major mode of action of anti-inflammatory impacts of this herbal species is the antioxidant impact of alkaloids present in its roots. to confirm this, it is recommended to implement a clinical trial with this drug. acknowledgments this research was carried out as a case when the disease was observed with this high bedsore that fortuitously led to a good consequence for the patient. herewith, the authors express their appreciation to the esteemed patient and her family. conflict of interest none.  fig. 2 sacral ulcer at week seven of the treatment. 423j contemp med sci | vol. 8, no. 6, november-december 2022: 420–423 s. erabi et al. case report the effectiveness of topical arnebia euchroma oil in the treatment of pressure ulcer: a case report references 1. akhkand ss, seidi j, ebadi a, gheshlagh rg. prevalence of pressure ulcer in iran’s intensive care units: a systematic review and a meta-analysis. nursing practice today 2020; 7: 21-9. 2. potter pa, perry ag, stockert p, hall a. fundamentals of nursing-e-book: elsevier health sciences; 2016. 3. rush bd, brown-kramer jm, sabalka la, gansemer bs, upton ct, bauer pb, et al. medical environment bedsore detection and prevention system. google patents; 2020. 4. mervis js, phillips tj. pressure ulcers: pathophysiology, epidemiology, risk factors, and presentation. journal of the american academy of dermatology 2019; 81: 881-90. 5. mitchell a. adult pressure area care: preventing pressure ulcers. british journal of nursing 2018; 27: 1050-2. 6. reilly ef, karakousis gc, schrag sp, stawicki sp. pressure ulcers in the intensive care unit: the “forgotten” enemy. opus 2007; 12: 17-30. 7. çelik s, dirimeşe e, taşdemir n, aşik s, demircan s, eyican s, et al. pressure sore prevention and treatment knowledge of nurses [hemşirelerin basi yarasini önleme ve yönetme bilgisi]. 2017. 8. moore ze, patton d. risk assessment tools for the prevention of pressure ulcers. cochrane database of systematic reviews 2019. 9. moore z, cowman s. pressure ulcer prevalence and prevention practices in care of the older person in the republic of ireland. journal of clinical nursing 2012; 21: 362-71. 10. dobson r. half of general practices offer patients complementary medicine. bmj 2003; 327: 1250. 11. bluestein d, javaheri a. pressure ulcers: prevention, evaluation, and management. american family physician 2008; 78: 1186-94. 12. gefen a, brienza d, edsberg l, milton w, murphy c, oomens c, et al. the etiology of pressure injuries. prevention and treatment of pressure ulcers/ injuries: clinical practice guideline european pressure ulcer advisory panel (epuap), national pressure injury advisory panel (npiap) and the pan pacific pressure injury alliance (pppia) 2019: 2019. 13. gefen a, brienza dm, cuddigan j, haesler e, kottner j. our contemporary understanding of the aetiology of pressure ulcers/pressure injuries. international wound journal 2021. 14. ghodela n, dudhamal t. management of bed sores with thumari gel [securinega leucopyrus (willd.) muell.]-an extra-pharmacopeal drug-a case study. international journal of ayush case reports 2018; 2: 20-5. 15. wound o. wocn 2016 guideline for prevention and management of pressure injuries (ulcers). journal of wound, ostomy and continence nursing 2017; 44: 241-6. 16. organization wh. who launches the first global strategy on traditional and alternative medicine. geneva: world health organization; 2002. 2014. 17. nidadavolu p, amor w, tran pl, dertien j, colmer-hamood ja, hamood an. garlic ointment inhibits biofilm formation by bacterial pathogens from burn wounds. journal of medical microbiology 2012; 61: 662-71. 18. annan k, houghton pj. antibacterial, antioxidant and fibroblast growth stimulation of aqueous extracts of ficus asperifolia miq. and gossypium arboreum l., wound-healing plants of ghana. journal of ethnopharmacology 2008; 119: 141-4. 19. shahzad mn, ahmed n. effectiveness of aloe vera gel compared with 1% silver sulphadiazine cream as burn wound dressing in second degree burns. j pak med assoc 2013; 63: 225-30. 20. ghobadi a, yousefi a, behnoud n. medicinal and nutritional properties of ziziphus jujuba mill. in traditional persian medicine and modern phytotherapy. 21. bahrami r, ghobadi a, behnoud n, akhtari e. medicinal properties of daucus carota in traditional persian medicine and modern phytotherapy. j biochem tech 2018; 9: 107-14. 22. behnoud n, bahrami r, kordafshari g, farzaneh f, kenari hm. management of early menopause using traditional persian medicine: a case report. international journal of women’s health and reproduction sciences 2019; 7: 231-6. 23. ito h, asmussen s, traber dl, cox ra, hawkins hk, connelly r, et al. healing efficacy of sea buckthorn (hippophae rhamnoides l.) seed oil in an ovine burn wound model. burns 2014; 40: 511-9. 24. aliasl j, barikbin b, khoshzaban f, naseri m, sedaghat r, kamalinejad m, et al. effect of arnebia euchroma ointment on post-laser wound healing in rats. journal of cosmetic and laser therapy 2015; 17: 41-5. 25. kim sh, kang i-c, yoon tj, park ym, kang k-s, song gy, et al. antitumor activities of a newly synthesized shikonin derivative, 2-hyim-dmnq-s-33. cancer letters 2001; 172: 171-5. 26. saghafi mm, fatemi mj, bagheri t, hablolvarid mh, niazi m, saberi m, et al. the effect of arnebia euchroma ointment on healing of deep second-degree burn wound in rat. tehran university medical journal 2018; 75: 790-6. 27. chawla k, mopuri r, sharma ak, kumar p. arnebia euchroma. himalayan medicinal plants: elsevier; 2021. p. 27-41. 28. aghili khorasani m. makhzan al-advie. tehran: iran university of medical science research. institute for islamic and complementary medicine 2008. 29. aghili mh. gharabadin kabir. tehran: research institute for islamic and complementary medicine 2008. 30. ez e. daghaegh-al-elaj [accurate subjects about therapeutics]. translated by kermani mk kerman: saadat publication 1983; 1: 433-60. 31. kumar a, shashni s, kumar p, pant d, singh a, verma rk. phytochemical constituents, distributions and traditional usages of arnebia euchroma: a review. journal of ethnopharmacology 2021: 113896. 32. nasiri e, hosseinimehr sj, azadbakht m, akbari j, enayati-fard r, azizi s, et al. the healing effect of arnebia euchroma ointment versus silver sulfadiazine on burn wounds in rat. world journal of plastic surgery 2015; 4: 134. 33. moghadari m, rezvanipour m, mehrabani m, ahmadinejad m, tajadini h, hashempur mh. efficacy of mummy on healing of pressure ulcers: a randomized controlled clinical trial on hospitalized patients in intensive care unit. electronic physician 2018; 10: 6140. 34. nasiri e, hosseinimehr sj, hosseinzadeh az, azadbakht m, akbari j, azadbakht m. the effects of arnebia euchroma ointment on second-degree burn wounds: a randomized clinical trial. journal of ethnopharmacology 2016; 189: 107-16. 35. ali es, hosseinzadeh m. comparison of the effects of oral arnebia euchroma and oral angipars on wounds in diabetic rats. international journal of pharmaceutical research 2019; 11. 36. sedaghat r, moghadam m, naseri m, davati a. histological evaluation of the anti-inflammatory effects of alkanna tinctoria on the cutaneous wounds healing in rat. hormozgan medical journal 2010. 37. liu h, jin y-s, song y, yang x-n, yang x-w, geng d-s, et al. three new compounds from arnebia euchroma. journal of asian natural products research 2010; 12: 286-92. 38. papageorgiou v, assimopoulou a, ballis a. alkannins and shikonins: a new class of wound healing agents. current medicinal chemistry 2008; 15: 3248-67. 39. cao h, zhang w, liu d, hou m, liu s, he w, et al. identification, in vitro evaluation and modeling studies of the constituents from the roots of arnebia euchroma for antitumor activity and stat3 inhibition. bioorganic chemistry 2020; 96: 103655. 40. maklebust j, sieggreen m. pressure ulcers: guidelines for prevention and management: lippincott williams & wilkins; 2001. 41. khorasani ma. gharabadin e kabir (persian). 1. 2011. 42. karayannopoulou m, tsioli v, loukopoulos p, anagnostou tl, giannakas n, savvas i, et al. evaluation of the effectiveness of an ointment based on alkannins/shikonins on second intention wound healing in the dog. canadian journal of veterinary research 2011; 75: 42-8. 43. ahmadian am, monsef eh, amin gr, fazeli m, jamalifar h, kamalinia g, et al. the ethnopharmacological study on antibacterial activity of some selected plants used in iranian traditional medicine. 2009. 44. damianakos h, kretschmer n, sykłowska-baranek k, pietrosiuk a, bauer r, chinou i. antimicrobial and cytotoxic isohexenylnaphthazarins from arnebia euchroma (royle) jonst.(boraginaceae) callus and cell suspension culture. molecules 2012; 17: 14310-22. 45. akkol ek, koca u, peşin i, yılmazer d, toker g, yeşilada e. exploring the wound healing activity of arnebia densiflora (nordm.) ledeb. by in vivo models. journal of ethnopharmacology 2009; 124: 137-41. 46. pirbalouti ag, koohpayeh a, azizi s, golparvar a, editors. evaluation of the burn healing properties of arnebia euchroma rolye (johnst) in diabetic rats. int conference biosci biochem bioinform; 2011. 47. pirbalouti ag, azizi s, koohpayeh a. healing potential of iranian traditional medicinal plants on burn wounds in alloxan-induced diabetic rats. revista brasileira de farmacognosia 2012; 22: 397-403. 48. ashkani-esfahani s, imanieh m, khoshneviszadeh m, meshksar a, noorafshan a, geramizadeh b, et al. the healing effect of arnebia euchroma in second degree burn wounds in rat as an animal model. iranian red crescent medical journal 2012; 14: 70. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1276 147j contemp med sci | vol. 8, no. 3, may-june 2022: 147–152 review concept analysis of professionalism in teachers and faculty members taiebe shokri1, shahram yazdani1, soleiman ahmadi1, leila afshar2,* 1department of medical education, virtual school of medical education and management, shahid beheshti university of medical sciences, tehran, iran. 2department of medical ethics, shahid beheshti university of medical sciences, tehran, iran. *correspondence to: leila afshar (e-mail: lafshar@gmail.com) (submitted: 06 december 2021 – revised version received: 24 december 2021 – accepted: 05 january 2022 – published online: 26 june 2022) abstract objectives: considering the important role of medical education teachers to prepare teachers and students in the future, the need to determine the concept of professional teacher and teacher and the characteristics of teacher professionalism, we have explained the concept of professionalism in teachers in this study. methods: the concept analysis method used in this research was walker and avant method which has eight steps. walker and avant state the purpose of concept analysis as follows: the process of distinguishing between the defining features of a concept and its unrelated features. the process of concept analysis includes selecting a concept, determining the purpose of the analysis, identifying the uses of the concept, determining the defining features of the concept, identifying a model, identifying boundary and adversarial items, identifying the precedents and consequences of the concept, and defining empirical references. results: professionalism in its general sense, requires deep knowledge, insight, creating a healthy and safe environment, honesty and trust, impartiality, commitment to the profession and continuous improvement, punctuality, criticism, professional competence, responsibility and individual accountability, especially in social interactions, there is an effort for continuous improvement that the acquisition of these characteristics is not easily possible and requires education, especially continuous learning. professionalism is a set of values, behaviors and relationships that underpin public trust in teachers. conclusion: the most important application of this analysis is the concept of basing the definition provided in order to objectively and realistically evaluate the professionalism of professors and strive to improve and enhance it. keywords: concept analysis, medical education, professionalism, faculty members issn 2413-0516 introduction the phenomenon of professionalization is formed, strengthened and consolidated over time and interactions with people, and this phenomenon is considered part of the process of socialization of a profession. the importance of professionalism in higher education not only determines the appropriate and inappropriate behaviors and guides faculty members in the implementation of professional responsibilities, but also guarantees faculty members’ adherence to professional principles and values, ensures the quality of teaching and facilitates the teaching process. it will be learning in universities and will increase the commitment to respond to the needs of students as well as the development of an ethical culture based on ethics.1 therefore, considering the important role of medical education teachers to prepare teachers and students in the future, the need to determine the concept of professional teacher and the characteristics and traits of a professional teacher, we have explained the concept of professionalism in teachers in this study. since, according to studies, there are many challenges to the concept of teacher professionalism and no uniform agreement has yet been reached on the definition of this concept, without a consensus on these concepts, interpersonal communication is not possible and leads us to many mistakes.2 therefore, the main purpose of concept analysis in this study is to identify the internal structure of the concept of professionalism in faculty members, to provide a clear, comprehensive definition of the concept of professionalism in professors so as to create a common understanding of this concept.3 the concept analysis method used in this research was walker and avant method which has eight steps. walker and avant state the purpose of concept analysis as follows: the process of distinguishing between the defining features of a concept and its unrelated features. the concept analysis process is shown in (table 1 and figure 1). data collection sources of information in concept analysis are related scientific texts, so the research environment in this study was the digital libraries of universities as well as related and valid databases such as taylor and francis, eric, elsevier, medline/ pubmed, science direct, scholar google, after the researcher’s initial search several times, visited the libraries of tehran university of medical sciences, tarbiat modares university and iran university of medical sciences and the library of the faculty of educational sciences, university of tehran. in order to carry out the research process, a structured search was used to review the texts in such a way that according to the subject under study, the relevant words were selected and in the databases, eric, francis and taylor, elsevier, medline/pubmed, google scholar, science direct search performed. the search strategy consisted of combining and words synonymous with teaching (or * educat or * instruct or * teach professor or faculty) and words synonymous with ethics or words used in ethics-related texts. (profession * or moral or ethic * or manner or conduct), since the subject was not affected by the year, no restrictions were set in this regard. studies in english were also used and the full text could be accessed. in order to select the best and most relevant articles and texts, the articles were searched and reviewed in several steps: first, the texts were selected based on the title. in the next stage, the second screening was performed, which was based 148 j contemp med sci | vol. 8, no. 3, may-june 2022: 147–152 concept analysis of professionalism in teachers and faculty members review t. shokri et al. on the study of the list and summary or introduction of the texts. finally, in the final review, the texts of the articles were fully studied and excluded if they were not related to the research topic and question. in this phase of the research, the search for texts continued until data saturation was reached, that is, until a new subject or adjective was found in relation to the subject under study in the search results. a total of 67 sources were included in the study and the concept analysis steps were performed based on them. by studying 48 sources, the researcher saturated the data. data analysis the first step in conceptual analysis is to select a concept that can describe a new and practical phenomenon in a field of knowledge.4 the concept in question can be located on a continuum, from purely experimental to purely abstract. step 1 select a concept according to the purpose of studying the concept of teacher professionalism, due to the existence of many problems such as ambiguity in the definition of professionalism of teachers and faculty members and the lack of general agreement on the basic concepts of this concept was selected. step 2 determining the purpose of the analysis the purpose of analyzing the present concept is to clarify the concept of teacher professionalism while trying to reduce table 1. the eight step concept analysis method (adopted from walker and avant, 2005) 1 selection of concept to analyse 2 determining the purpose of the analysis 3 identifying the uses of the concept 4 determining the defining attributes of the concept 5 identifying a model case 6 identifying the borderline, related and contrary cases 7 identifying the antecedents and consequences 8 defining the empirical referents ambiguity and semantic integration of this concept to increase the stability in the application of this concept in articles and topics related to teacher professionalism. all applications of that concept should be identified and specified. step 3 identifying the uses of the concept one of the main applications of the concept of professionalism in professors is to promote the professional performance of faculty members and to assess and evaluate the professionalism of teachers. therefore, considering that professionalism is one of the main dimensions of a teacher’s job and affects and is important to almost all the roles and responsibilities of a teacher, it is very important to try to promote it and empower teachers. providing an accurate and objective definition of the concept of professionalism in the faculty member is one of the prerequisites for achieving the goals of promotion and empowerment of professors, which can be considered as one of the important items by managers during the recruitment and even during the faculty service. step 4 determine the defining features of the concept at this stage, the phrases and sentences extracted from the texts obtained in the previous stage are subjected to a stage of abstraction process and the result is the acquisition of potential attributes and features that define the concept.5,6 in the continuation of the concept analysis process, the features that had a common semantic load were identified and placed in their own categories, and then each category was given a more abstract title that includes the meanings of those attributes and features, which in principle. these are the main features of the concept. these attributes are specific attributes of the concept that can separate the concept from similar concepts and determine the definition of the concept. as explained in the data analysis section, in order to perform this step and find the defining features of the concept of professionalism, teachers, while searching for relevant texts and articles, when reading any source, phrases or concepts that define or characterize the concept of professionalism were identified. then, all the reference phrases according to what feature or level of concept they expressed were placed in 6 definition areas, which are: 1. commitments or professional responsibilities of the faculty to their development 2. commitments or professional responsibilities of the faculty to the development of the university and the society 3. commitments or professional responsibilities of the faculty to the development of students 4. moral and personality characteristics of the faculty 5. prerequisites of professionalism in the faculty 6. distinctive features of professionalism in the teacher the results of reviewing the studies and identifying the defining domains of the concept of teacher professionalism in the faculty members of the university as well as the final analytical definition of each of the attributes identified in the domains are as follows: 1. commitments or professional responsibilities of the faculty to their development a. commitment to self-promotion: commitment to professional development and development of educational and research skills related to the teaching profession. b. possessing the highest level of knowledge and specialized thematic skills: acquiring technical knowledge and specialized practical skills in the field of teaching fig. 1 flowchart of screening articles in the review of texts related to concept analysis. 149j contemp med sci | vol. 8, no. 3, may-june 2022: 147–152 t. shokri et al. review concept analysis of professionalism in teachers and faculty members and trying to keep it up to date and upgrade it in order to transfer and teach it to students. c. possession of teaching knowledge and skills: acquiring knowledge and teaching skills including familiarity with the principles and methods of curriculum planning and educational design, preparation of educational content and preparation of lesson resources, familiarity with various teaching methods and their application, familiarity with the principles of evaluation student and program evaluation. 2. commitments or professional responsibilities of the faculty to the development of the university and the society a. commitment to the expectations, values and norms of society: efforts to meet the expectations of society and parents in the direction of scientific and practical education of students in the best way and also paying attention and respect to the values and norms of society in professional practices related t o teaching process and model being a role b. commitment to the rules and standards of the institution and the profession: commitment and adherence to the rules and standards of the organization and the professional community and following them and moving towards the policies and plans of the organization c. social accountability: increasing responsibility for society’s expectations and accountability for individual, social, professional behavior 3. commitments or professional responsibilities of the faculty to the development of students a. commitment to students’ scientific and skill development: commitment to educating and guiding students to acquire up-to-date technical knowledge and standard practical skills related to the field of study b. efforts for students ‘moral development: commitment to students’ moral development through a practical model and a role model for teachers in the field of behavior and moral character in students and experts in educational design, selection of educational content, classroom management, familiarity with the principles of communication skills and communication with students with problems. 4. moral and personality characteristics of the faculty a. appearance and behavior in accordance with university standards/professional etiquette the appearance and cover of the faculty should be in accordance with the values of the society and in accordance with the social position of the profession. b. personality traits or being a good human being: the teacher should be a good human being with outstanding human characteristics such as altruism, compassion, responsibility, respect for others, etc. and be required to observe individual and social ethics. a moral traits and spirits that are visible and clear in any situation, in words and in the behavior of the teacher. 5. prerequisites of professionalism in the faculty a. ethical knowledge: acquisition of knowledge and teaching skills including familiarity with the principles and methods of curriculum planning and educational design, preparation of educational content and preparation of lesson resources, familiarity with various teaching methods and their application, familiarity with the principles of student evaluation and program evaluation and... b. the ability of moral self-direction the ability of a person to constantly monitor, improve and enhance his moral level, control behavior and eliminate moral shortcomings c. solving professional problems and issues: a part of professionalism that is based on the existing context and conditions and professional standards; it is the basis for performance, judgment and decision-making on technical issues or technical problems of the teaching profession. 6. distinctive features of professionalism in the teacher a. values and beliefs within the profession specific to the profession those beliefs and values that are institutionalized in the teacher in the process of forming his professional identity and are considered as the motivator of his inner behaviors and professional character. b. ethics in professional in t eractions/ethical behavior in professional social performan c e: a part of professionalism based on professional responsibilities and obligations as well as norms and values of society, the basis of social behavior and interactions (including interaction with students, colleagues, other members of the profession, parents and the communi t y) is in the teaching profession c. ethical sensitivity: the ability to identify situations and issues that dealing with or deciding on it requires calling and activating the mechanism of moral reasoning. in fact, moral sensitivity is a complex phenomenon that requires recognizing, interpreting, and framing moral situations and a cognitive ability throughout life. d. moral reasoning: a mental process that leads to a judgment, decision, or action that can be defended from a moral perspective. e. moral impact: by observing all aspects of professional ethics, the individual has become a role model and through this has a positive moral impact on students and society as a whole and fulfills his responsibility for the commitment to students’ moral development. f. nature and moral basis: teacher and teaching is inherently a moral act and its ultimate goal, which is the upbringing and growth of human beings, is rooted in morality. step 5 determine a model sample a model example is an example that can show the desired concept with all its main defining features. in fact, by providing examples of the model, the researcher is absolutely sure that he has provided a real example of the concept under study.7 sample model for the concept of teacher professionalism: dr. mousavi is one of the professors of the medical school in one of the universities of medical sciences in the country, who always appears in class on time and with a tidy appearance and in accordance with the faculty regulations. before the beginning of the semester, he prepares his curriculum in accordance with the principles of educational design and prepares the educational content in accordance with the objectives of the course and the students, and submits his lesson plan and course plan to the faculty education. during teaching in the classroom, he/she devotes time to students ‘questions and 150 j contemp med sci | vol. 8, no. 3, may-june 2022: 147–152 concept analysis of professionalism in teachers and faculty members review t. shokri et al. answers so that if there is any ambiguity, he/she answers the students’ questions. and observes justice and fairness. alternatives include examples that do not include the concept of professionalism in the teacher, which include borderline, related, and opposites. the borderline sample is such that the researcher is not sure whether the proposed sample really corresponds to the intended concept or not.23 in other words, there are some defining features in these examples and some do not. using the opposite method is also useful in better analyzing the concept. this means what an example can be given that can certainly be said to have none of the defining features of the concept under study and is not an example of this concept. opposite example for the concept of faculty professionalism step 6 identifying the borderline, related and contrary cases dr. sadeghi is a faculty member of a medical school in one of the universities of medical sciences. dr. sadeghi spends most of his time in the faculty on research activities and publishing articles, and engages in research activities to improve his rank. due to his busy schedule, he answers his work telephones in class. during the semester, to attend a meeting or congress and seminar, without prior coordination with the students, he closes the class and postpones his class time to another hour without considering the schedule of the students’ semester classes. in the end-of-semester evaluation, his questions are not based on the principles of student evaluation, and no student has the right to object to the grade, and in case of objection, a grade will be deducted from the student. master’s and doctoral students, of whom he is a supervisor or advisor, can meet with him with great delay, and they are also required to prepare and submit an article and name him as the first author. related examples for the concept of faculty professionalism dr. bozorgi is a faculty member of the school of rehabilitation at one of the mother universities, who works two days a week at the end of the university in one of the private clinics. considering his work and professional background in the university and shiva’s expression and patience in responding to clients and his high scientific knowledge, both the client’s satisfaction and appropriate income are the prominent features of his presence in the clinic. the doctor’s experience and skills in teaching and communicating with students and providing real examples of patients have also improved since the doctor’s presence in the clinic, and by providing examples, photos and practical tips, he tries to meet the needs of students. step 7 determine the precedents and consequences of the concept pre-events are situations, events, and phenomena that occur before the intended concept occurs. prepositions may have contributed to or related to the occurrence of the concept but are not the cause, but at the same time their existence may be necessary for the presence of the intended concept. more precisely, events are the conditions and characteristics that exist before a concept occurs and must exist in order for that concept to occur. what was achieved in the process of analyzing the concept of professionalism of the faculty was that in order for the ethics and professional behavior of the teacher to happen, a series of requirements and preconditions need to be created so that this concept can be formed and appear in the process of analyzing the concept of professionalism. scientific, requirements and precedents in the following two subject categories are necessary for this concept to be formed in the teacher.8 a. moral knowledge acquisition of teaching knowledge and skills including familiarity with the principles and methods of curriculum planning and educational design, preparation of educational content and preparation of curriculum resources, familiarity with various teaching methods and their application, familiarity with the principles of student evaluation and program evaluation. b. personality traits or being a good person the teacher must be a good human being with outstanding human characteristics such as altruism, compassion, responsibility, respect for others, etc., and be required to observe individual and social ethics so that moral qualities and morals in any situation, be visible and clear in the words and behavior of the teacher. consequences of the concept outcomes are events that occur as a result of a concept and can often be a stimulus for the formation of new ideas in the context of specific concepts.9 in the process of analyzing the concept of professionalism in the faculty, conclusions will be formed in the following three thematic categories. a) moral influence as a result of observing all aspects of professional ethics, the individual has become a role model and thus has a positive moral impact on students and society as a whole and fulfills his responsibility for the commitment to students’ moral development. b) social accountability increasing responsibility for society’s expectations and responding to individual, social, professional behavior in fact, in recent years, higher education is considered as an economic driving force and one of the most important factors in the cultural and social development of countries, so considering the important role of higher education and specifically medical education in the health and development of society, the education system should plan to move as much as possible to meet the needs of society. meeting the needs of the community by higher education institutions is an important and fundamental challenge, and it is necessary to know who or who should be responsible and accountable if students fail or do not succeed in gaining the necessary competence to serve the community. step 8 define experimental references in the last step of the walker and avant concept analysis process, the basic features of the concept are merged with its empirical references in the real world.10 given that the concept of teacher professionalism has not yet been comprehensively defined, there is no measure to measure the whole concept at present, but according to the available evidence, some of its infrastructure and features have been evaluated separately.11 as mentioned in the results of some of the researches mentioned above, concepts such as professional commitment, 151j contemp med sci | vol. 8, no. 3, may-june 2022: 147–152 t. shokri et al. review concept analysis of professionalism in teachers and faculty members moral sensitivity, professional etiquette, etc., which are the characteristics and infrastructures of faculty professionalism in the real world, in exchange for foreign of course, since a comprehensive and complete definition of the concept of faculty professionalism has not been provided so far, each of these dimensions and features are examined under different headings and concepts and sometimes overlap with other features and concepts.12-15 for example, one of these overlaps is when officials or researchers seek to evaluate the quality and effectiveness of a teacher’s work. in such cases, most of the items and areas that are evaluated include teacher personality traits, teaching proficiency, communication skills with students, professional competencies, teaching style and classroom management that interfere with the field of professionalism and professionalism. discussion arkansas state university and jacksonville state university college of professional studies each have specific tools and forms for measuring faculty behavior, which include dimensions such as appearance, punctuality, innovation and creativity, empathy, professionalism, and a commitment to continuous learning. they examine the behavioral behavior of professors. in a study from the university of turkey, a tool was developed to examine communication skills and teacher student interactions, and according to the defining characteristics of professionalism, they designed a tool to assess all the emotional, cognitive and behavioral aspects of teacher student interactions.26 in another study, measurement tools in the form of rubric forms were developed and used to examine the behavioral character of teachers. individual characteristics including some social habits such as chronology, dependence and how to interact with others were examined.16-18 a universal and integrated program is not possible because there is not yet a single and specific definition in this regard, however, many studies have determined and examined the criteria and characteristics of professionalism.19 the comprehensive definition in this research, based on the results of various researches that have been obtained by examining different features and criteria and the basis of the concept of professionalism, is in fact that professionalism in its general meaning requires deep knowledge and insight.20 creating a healthy and safe environment, honesty and trust, impartiality, commitment to the profession and continuous improvement, punctuality, criticism, professional competence, responsibility and individual accountability, especially in social interactions, is an effort to continuously improve the education these features are simply not possible and require education, especially continuous learning.21 professionalism is a set of values, behaviors and relationships that underpin public trust in teachers.22-24 faculty members are, in fact, educators who prepare the growing learners of a community for life and service, and it can be argued that teachers who work with this awareness contribute to the positive characteristics of the community.25 in fact, the analysis of the concept of teacher professionalism creates a deeper understanding of the importance of this concept and reveals its dimensions and characteristics.26 professionalism in the faculty or faculty is actually a set of responsibilities and obligations that are formed and given meaning in the shadow of these commitments. these commitments include commitment to university expectations, commitment to educating and guiding students, and commitment to professional advancement. and it has positive consequences such as moral impact and increased social accountability.27-30 conclusion the constituent dimensions of faculty professionalism are professional etiquette, ethics in professional social interactions, ethics in professional technical issues and functions, values and beliefs specific to the profession that have distinct characteristics such as moral sensitivity, moral reasoning, knowledge ethics and self-regulation are moral.31 personality or human traits of being good and moral knowledge are preconditions for the formation of this concept and its occurrence leads to increased social accountability and moral effectiveness. the most important application of this analysis is the concept of basing the definition provided in order to objectively and realistically evaluate the professionalism of professors and strive to improve and enhance it. conflicts of interest none.  references 1. benatar, d. (2007). moral theories may have some role in teaching applied ethics. journal of medical ethics, 33:671-672. 2. caetano, a. p., & silva, m. d. l. (2018). professional ethics and teacher education. educational sciences journal, 8, 47-54. 3. calaguas, g. m. (2013). teacher effectiveness scale in higher education: development and psychometric properties. international journal of research studies in education, 2(2), 3-20. 4. baggini, j. (2015). what professionalism means for teachers today? education review, 18(2), 5-11. 5. campbell, e. (2004). ethical bases of moral agency in teaching. teachers and teaching: theory and practice, 10, 409-428. 6. carr, d. (2018). professional and personal values and virtues in education and teaching. oxford review of education, 32(2): 171-183 7. evans, l. (2008). professionalism, professionality and the development of education professionals. british journal of educational studies, 56(1), 20-38. 8. fisher, c. (2013). developing code of ethics for academics. journal of science and engineering ethics, 9(2):171-179. 9. seghedin, e. (2014). from the teachers professional ethics to the personal professional responsibility. acta didactica napocensia, 7(4): 13-22. 10. singh, a. (2010). ethics for medical educators: an overview and fallacies. indian journal of psychological medicine, 32(2), 83-86. 11. smith, s., fryer-edwards, k., diekema, d. s., & braddock, c. h. (2018). finding effective strategies for teaching ethics: a comparison trial of two interventions. academic medicine,79(3):265–271. 12. eckles, r. e., meslin, e. m., gaffney, m. & helft,, p. r. 2015. medical ethics education: where are we? where should we be going? a review. academic medicine, 80, 1143-52. 13. ginsburg, s., regehr, g. & lingard, l. 2013a. the disavowed curriculum: understanding students’ reasoning in professionally challenging situations. journal of general internal medicine, 18, 1015-1022. 14. cadozier v. (2012). the moral profession: a study of moral development and professional ethics of faculty. texas: university of texas. 15. soltis, j. f. (1986) teaching professional ethics. journal of teacher education, 37(3):2-4. 16. tichenor, m. s., & tichenor, j. m. (2004-2005). understanding teachers’ perspectives on professionalism. the professional educator, xxvii(1-2), 89-95. 17. warnick, b. r., & silverman, s. k. (2011). a framework for professional ethics courses in teacher education. journal of teacher education, 62(3):273-285. 152 j contemp med sci | vol. 8, no. 3, may-june 2022: 147–152 concept analysis of professionalism in teachers and faculty members review t. shokri et al. 18. willemse, m., lunenberg, m., & korthagen, f. (2016). values in education: a challenge for teacher educators. teaching and teacher education, 21(2), 205-217. 19. croos s. (2017). professional ethical standards, corporate social responsibility and the perceived role of ethics and social responsibility. j bus ethics; 82(3):657-66. 20. goldeshtain o. (2010). what is the relationship between corporate culture and ethics? journal of bus ethics; 100(2):515-30. 21. herbert s a. (2014). professional ethics and computer science/ information systems. new york: stony brook university. 22. viewpoint:teaching professionalism: is medical morality a competency? thomas s.huddle, md, phd.:31:16-23. 23. murray j. (2015). proper em, bayer ae. professors behaving badly: faculty misconduct in graduate education. baltimore: johns hopkins university press. 24. falunna g. (2009). organizational culture, professional ethics and guantanamo. unit state; 42:125-147. 25. golikein s. (2007). addressing ethical issues in higher education: a practical guide. uk: institute of business ethics. 26. professing professionalism: are we our own worst enemy? faculty members experiences of teaching ana evaluation professionalism in medical education at one school pier bryden, md. mphil, shiphra ginsburg, md, med, bochra kurabi, ahmed najma, md, phd. 41: 302-307. 27. dinman, b. 2010. education for the practice of occupational medicine: knowledge, competence, and professionalism. journal of occupational & environmental medicine, 42, 115. 28. archer, r., elder, w., hustedde, c., milam, a. & joyce, j. 2008. the theory of planned behaviour in medical education: a model for integrating professionalism training. medical education, 42, 771-777. 29. barbour, r. s. 2011. checklists for improving rigour in qualitative research: a case of the tail wagging the dog? bmj, 322, 1115. barnett-page, e. & thomas, j. 2009. methods for the synthesis of qualitative research: a critical review. bmc medical research methodology, 9, 59. 30. monrouxe, l.v., rees, c., hu, w. 2011. differences in medical students’ explicit discourses of professionalism: acting, representing, becoming. medical education, 45, 585-602. 31. stern, d. t. & papadakis, m. 2006. medical education: the developing physician becoming a professional. new england journal of medicine, 355, 1794-1799. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.1185 308 j contemp med sci | vol. 7, no. 5, september–october 2021: 308–314 original isolation and characterization of exopectinase from bacillus licheniformis fmb9 isolated from agricultural soil fatimah alqahtani1,2, magda aly1,3*, fardos bokhari1, baher ali el-nogoumy3, wafa alshehri4 ¹department of biology, college of science, king abdullaziz university, jeddah, saudi arabia. 2department of biology, college of science and arts in tathleet, university of bisha, bisha, saudi arabia. 3botany and microbiology department, faculty of science, kafrelsheikh university, egypt. 4department of biology, college of science, university of jeddah, jeddah, saudi arabia. *correspondence to: magda aly (e-mail: magdammali@hotmail.com) (submitted: 02 july 2021 – revised version received: 23 july 2021 – accepted: 12 august 2021 – published online: 26 october 2021) abstract objectives: this study aimed to isolate and identify potential pectinase producing bacterium as well as optimization of its various parameters for maximum enzyme production. methods: a total of forty-three bacterial isolates were obtained from agriculture soil in jeddah city using standard plate count method. primary screening was done by hydrolysis of pectin on agar plate and measuring the clear zone after adding iodine-potassium iodide solution. pectinase activity was determined by measuring the increase in reducing sugar formed by the enzymatic hydrolysis of pectin. results: among the bacterial isolates, the isolate fmb9 exhibited higher pectinase activity in broth medium and was selected for further studies. the selected bacterial isolate fmb9 was identified as bacillus licheniformis fmb9 with similarity level 97% to b. licheniformis as10. the isolate was found to produce maximum pectinase at 37°c with ph 7 upon incubation for 72 hours, while cultured in production medium containing citrus pectin and yeast extract as carbon and nitrogen sources, respectively. the enzyme was purified using column chromatography and was characterized. it showed maximum activity at 45°c. bacillus licheniformis pictinase was affected by ph values and optimum activity was at ph 5. the molecular weight was also determined and compared with other pectinases. conclusion: pectinase produced from bacteria can be purified and used in many technological applications in food and medicine. keywords: pectin; pectinase; bacillus licheniformis; enzyme activity; 16s rrna issn 2413-0516 1. introduction pectin is an important component of the middle lamella and primary cell wall of higher plants. pectins are high molecular weight acidic heteropolysaccharide primarily made up of α (1−4) linked d-galacturonic acid residues.1 degradation of pectin is difficult, and pectinases enzyme can help in the degradation of pectin.2 pectinases consist of a unique group of enzymes that catalyze the degradation of pectic polymers, they belong to the polysaccharidase family, also known as pectolitic or pectic enzymes, which contribute to the breakdown of pectins from various plants. in the current biotechnological period, pectinase is one of the increasingly used enzymes.3 pectinases, used since 1930, make up a significant part of industrial enzymes. there are different sources of pectinolytic enzymes; including bacteria, fungi, plant,4 and no evidence was found in animal.5 microbial enzymes are routinely used in many environmentally friendly and economic industrial sectors. pectinases are enzymes which are widely distributed in microbes that are present in pectin enriched sites. the agro industrial residues can be utilized in the industrial scale for low-cost and efficient pectinase production in an eco-friendly approach.6 most pectin-degrading microorganisms are associated with raw agricultural products and with soil. up to 10% of the microorganisms in soil have been shown to be pectinolytic.7,8 these include, but are not limited to, bacteria in the genera pantoea, achromobacter, aeromonas, arthrobacter, agrobacterium, enterobacter, bacillus, clostridium, erwinia, flavobacterium, pseudomonas, xanthomonas.9-11 and many yeasts, molds, protozoa and nematodes.12 also pectolytic activity was found in a strain of leuconostoc mesenteroides.13 bacillus is one of the large genera of bacterial strains. it is a rod shaped, endospore bearing bacteria and belong to the family firmicutes. the genus bacillus covered a great diversity of strains and some of them are strictly aerobic, while others are facultative anaerobic. the bacillus especially b. licheniform is capable of growing on a large diversity of nutrient sources because of synthesizing and secreting different hydrolytic enzymes and this quality makes the b. licheniform is an industrially important microorganism.14 in the current study, different bacterial strains were isolated from agriculture soil and screened for pectinase production. finally, maximum pectinase producing bacterial strain was identified using conventional and molecular techniques. 2. materials and methods 2.1. isolation of bacteria several bacterial strains were isolated from soil samples collected from agriculture soil in jeddah city. isolation of bacteria was done by serial dilution plate method and incubated at 37°c for 24 hrs. the isolated colonies were selected to obtain pure bacterial cultures. 2.2. screening of pectin producing bacteria screening was performed to detect the presence of bacteria that degrades pectin. pectin agar medium was prepared with (g/l): nano3 1.0, kcl 1.0, k2hpo4 1.0, mgso4 0.5, yeast extract 0.5, citrus pectin 10 and agar 20 with ph adjusted to 7.0 (kumar et al., 2012).pure culture was inoculated by puncture in the medium and incubated for 48 hrs at 37°c. after incubation, iodine-potassium iodide solution was added to detect the clearance zone.15 309j contemp med sci | vol. 7, no. 5, september–october 2021: 308–314 f. alqahtani et al. original isolation of pectinase from bacteria 2.3. quantitative screening of bacterial strains for pectinase production the bacterial strains showing clear zone of hydrolysis on pectin agar medium were screened for pectinase production using pectin broth medium. the pure cultures were inoculated and incubated at 37ºc for 24 h. after incubation, biomass was separated by centrifugation at 10,000 rpm for 15 min. the supernatant was used to evaluate pectinase activity.16 2.4. enzyme assay pectinase activity was measured by the estimation of the amount of galacturonicacids through the dns method (miller, 1959)17 using 1.0% citrus pectin as a substrate and mono-dgalacturonic acid as a standard. one unit of pectinase was defined as the ‘‘amount of enzyme required to generate 1 mole of galacturonic acid under standard assay conditions”. the enzyme activity (u/ml) was calculated according to equation: enzyme activity (u/ml) = (µg galacturonic acid released × v) v × 194.1 × t where v is the enzyme broth volume used in the assay, 194.1 is the molecular weight of galacturonic acid, and t is the reaction time in min. one unit (u) is equivalent to 1 μmol product released per min. relative activity of the enzyme was calculated as the percentage by using the following formula:18 relative activity = activity of the sample × 100 maximum activity of the sample 2.5. identification of bacterial strain morphological characteristics, biochemical characterization of the selected bacterial strain were studies for identification.19 molecular characterization 16s rdna sequence analysis was performed for molecular based identification of selected isolate. the genomic dna was extracted using the method reported by chen and kuo (1993).20 2.6. optimization of cultural conditions for pectinase production by bacillus licheniformis fmb9 the bacterial strain that showing high pectinolytic activity was selected. the effects of temperature, ph and incubation period on production of pectinase were studied for maximum pectinase production. the bacterial isolate was subjected to different temperatures (25, 30, 37, 40 and 45°c ) for 72 h then the enzyme activity was measured. after selection the best optimum temperature of pectinase production, the effect of ph value on pectinase production of the selected bacterial isolate were studded. the medium was prepared with different ph values (ph 5.0, 6.0, 7.0, 8.0 and 9) and incubated 72 h with agitation at 120 rpm at 37ºc. after growth period, pectinase activity was measured. after selection the best ph of the medium, the effect of different incubation period (24 h, 48 h, 72 h, 96, and 120 h) was determined. after incubation the enzyme assay was measured. 2.7. purification and enzymatic optimization production of pectinase by bacillus licheniformis was carried out for 72 hrs and the enzyme was precipitated by 80% ammonium sulfate in the refrigerator at 4°c, the precipitate was collected and centrifuged at the same temperature and the obtained crude enzyme was dialyzed at 4°c for two days with citrate buffer of ph 5. the obtained protein was frozen at –200˚c and lyophilized in a lyophilizer. the enzyme was purified using sephadex g-100 and deae-cellulose columns chromatography where the enzyme was directly applied to the agarose column (1 × 30 cm.) and elution was carried out by liner gradient of nacl (0.1–0.6 m). many fractions (60), each one bout 5 ml, were collected and enzyme activity and protein content (a280) were determined. total protein was determined by the method of lowery et al. (1951)21 by using serum albumin as a standard. 2.8. characterization of the purified enzyme the purified enzyme was characterized at different ph (5–9), temperature (20–60°c) and substrate and enzyme concentrations. the molecular weight of pectinase that was purified was determined using a vertical gel electrophoresis including 12% separating and 5% stacking gel and low molecular weight protein was used as standard (laemmli, 1970).22 protein bands were examined after staining with coomassie brilliant blue r-250. 2.9. statistical analysis the data for all experiments have been calculated from three replications, with the values presented as the mean ± se standard error). 3. results and discussion pectinolytic enzyme can be derived from different sources.23 however, pectinase producing microorganisms have due advantage over other sources because they can be subjected to genetic and environmental manipulations to increase yield.24 it has been reported that most bacillus sp, enhances the production of pectinase.25 forty-three bacterial strains were isolated from agriculture soil in jeddah city on nutrient agar. the pectinolytic activity was detected by visualizing a clear zone around the colony using potassium–iodide flooding method.17 among these isolates only twenty-five isolates showed pectinolytic activity on pectin agar medium (table 1). the isolate fmb9 (figures 1, 2) which was gram positive bacterium, isolated from soil on nutrient agar medium demonstrated the largest hydrolysis zone around its colony, meaning the highest pectinase activity, compared to the other strains. to identify the selected isolate fmb9, both traditional microbiological methods and modern molecular technologies were considered. on the basis of observed morphological, cultural, and biochemical characteristics, the colony of selected bacterial isolate fmb9 was large and the margin was undulate and had white creamy color. it cells were gram positive, motile and producing endospore. this isolate had shown positive test for catalase, oxidase, starch hydrolysis, protease and can able to ferment glucose and sucrose whereas, negative for urease, citrate utilization, gelatin liquefaction and dnaase. the results obtained were compared with identification flowchart of bergey’s manual.19 the characteristics showed by this organism were fairly similar to bacillus licheniformis. the details of biochemical characteristics of pectinase producing strain are given in table 2. the phylogenetic tree generated using 16s 310 j contemp med sci | vol. 7, no. 5, september–october 2021: 308–314 isolation of pectinase from bacteria original f. alqahtani et al. rdna gene sequences of the bacterial isolate showed that the bacterium has the highest homology (99%) with bacillus licheniformis (genbank accession number kj729823.1) and designated as bacillus licheniformis as10. figure 3 signifies the phylogenetic tree of the isolate fmb9 with the selected best homologous known bacterial strains. for the production of pectinase in optimum cultural condition the bacteria strain was grown on pectin broth media. factors such as temperature, ph and incubation periods were varied to analyze the optimum enzyme production by the bacteria strain. enzyme production went up with the increase of temperature up to 37°c and then declined (figure 4). the maximum production which occurred at this temperature was 2.77 u/ml. this dramatically reduced to nearly 16.6% at 50°c. in the previous study of aaisha and barate (2016),26 the highest pectinase production was observed from some bacillus species at 37°c which is similar to our current study. enzyme activity also depends on the ph of the reaction mixture. figure 4 depicts the effect of different phs on the production of pectinase by bacillus licheniformis. maximum production (2.64 u/ml) was recorded at ph 7 (figure 5). this finding is in accordance with other workers who reported that most of the bacillus sp. that produce high amount of pectinase between ph 7 and 8 (oumer and abate, 2017).27 at highly acidic and alkaline ph, enzyme production decreased by almost 61.37 % and 51.5%, respectively. an attempt was made to determine the most favorable incubation period for enzyme production by the selected isolate and the highest enzyme production (2.852 u/ml) was recorded after 72 hours of incubation (figure 6). the enzyme production gradually decreased to 0.361 u/ml at 120 hours of incubation which is almost 51% less than that of maximum. this might be due to the accumulation of waste products at prolonged incubation time with limited nutrient sources which consequently suppressed the growth of microorganism. according to nawawi et al. (2017)28 maximum pectinase table 2. morphological, physiological and biochemical characteristics of maximum pectinase producing bacterial strain characteristics results temperature range 20–45°c optimum temperature 37°c ph range 5.5–10.0 optimal ph 6.5–7.5 catalase + oxidase + urease _ protease + dnaase _ hydrolysis of starch + gelatine liquefaction _ citrate utilization _ fig. 1 isolate fmb9, a: on nutrient agar, b: under light microscope after gram staining. fig. 2 a: isolate fmb9 growth on pectin agar medium, b: qualitative screening of the isolate fmb9 for pectinase on pectin agar medium. table 1. activity of the bacterial strains isolated from agriculture soil for peptidase activity on pectin medium (mm) and in liquid medium (u.ml). values are mean ± sd of 3 replicates pectinase activity (u/ml) diameter of clear zone (mm) bacteria isolates 1.19119.0 ± 2.3fmb 1 1.0 ± 0.310.3 ± 2.3 fmb 2 0.5 ± 0.333.0 ± 2.3fmb 3 0.61114.3 ± 2.3fmb 4 0.6 ± 0.328.6 ± 2.3fmb 5 1.2 ± 0.427.9 ± 2.3fmb 6 0.4 ± 0.013.3 ± 2.3fmb 7 0.6 ± 0.313.3 ± 2.0fmb 8 1.4 ± 0.533.6 ± 2.8fmb 9 1.2 ± 0.430.3 ± 2.0fmb 10 0.7 ± 0.330.9 ± 2.0fmb 11 0.7 ± 0.128.3 ± 3.3fmb 12 0.7 ± 0.330.0 ± 3.3fmb 13 0.8 ± 0.217.8 ± 2.4fmb 14 0.6 ± 0.132.1 ± 2.9fmb 15 0.5 ± 0.117.1 ± 2.7fmb 16 0.7 ± 0.324.6 ± 2.8fmb 17 0.7 ± 0.130.1 ± 2.9fmb 18 0.6 ± 0.128.3 ± 2.0fmb 19 0.5 ± 0.215.3 ± 2.1fmb 20 0.7 ± 0.324.3 ± 2.0fmb 21 0.9 ± 0.329.3 ± 2.3fmb 22 0.6 ± 0.223.0 ± 1.4fmb 23 0.7 ± 0.024.6 ± 5.1fmb 24 0.5 ± 0.126.3 ± 2.5fmb 25 311j contemp med sci | vol. 7, no. 5, september–october 2021: 308–314 f. alqahtani et al. original isolation of pectinase from bacteria highest pectinase activity have been collected, concentrated by lyophilization and the enzyme activity was determined. molecular weight of the pure enzyme, detected using sdspage analysis, was ~43 kda (figure 9). according to the sdspage analysis, two extracellular pectinase of 60 and 64 kda were obtained from bacillus subtilis by takcı and turkmen (2016).30 molecular weights of the partial purified pectinase from various bacteria species were determined as follows: 37 kda for paenibacillus xylanolyticus and bacillus sp. mfw7,31,32 89 kda for bacillus cereus nrc20,33 31 kda for streptomyces sp. ghba10,34 106 kda for bacillus sp. dt735 and 66 kda for bacillus sp. mbrl576.36 higher molecular weight enzymes were isolated from kluyveromyces marxianus37 while an average molecular weight of 38–65 kda, the enzymes (for both exoand endopolygalacturonases) are separated from various microbial sources.38 the purified enzyme showed maximum activity at 45°c and ph 5 (tables 3, 4) in addition to 0.7 mm of the substrate (figure 10). it was found that increase enzyme and substrate concentrations enhanced enzyme activity. in the presence of excess substrate, increasing enzyme concentrations increased the enzyme activity (figure 11). addition of mg2+, k+, zn2+ and ca2+ ions significantly increased the enzyme activity while na+, fe2+ and cu2+ in addition to edta decreased the activity (table 5). pectinases or pectinolytic enzymes that hydrolyze pectic substances are produced by living cells in the presence of fig. 3 phylogenetic tree of the identified bacterial isolate fmb9 based on the 16s rdna sequences. the genbank accession number is given in parentheses for each organism. fig. 4 effect of different temperature on pectinase production by bacillus licheniformis fmb9. fig. 5 effect of different ph on pectinase production by bacillus licheniformis fmb9. fig. 6 effect of different incubation period on pectinase production by bacillus licheniformis fmb9. production was determined from the bacillus subtilis adi1 after 72 hours of incubation which well agreed with our findings. on the other hand, this study is disagreeing with other study which reported that bacillus licheniformis strain dy2 had maximum pectinase production after 44 hrs.29 the elution profiles of the crude enzyme of isolate fmb9 after using sephadex g-100 and deae-cellulose columns chromatography are shown in figures 7 and 8, respectively. for each column chromatography, the fractions with the 312 j contemp med sci | vol. 7, no. 5, september–october 2021: 308–314 isolation of pectinase from bacteria original f. alqahtani et al. table 3. effect of different temperature on bacillus licheniformis pectinase activity incubation temperature (°c) enzyme activity u/ml relative activity (%) 20 0.99* ± 0.12 47.8 25 1.10 ± 0.11 53.1 30 1.80 ± 0.25 86.9 37 (control) 2.07 ± 0.12 100.0 40 2.14 ± -0.28 103.3 45 2.28 ± 0.16 110.1 50 2.00 ± 0.15 96.6 55 1.09 ± 0.22 52.6 60 0.40 ± 0.21 19.4 *values are mean ± sd of 3 replicates. table 4. effect of different ph on pectinase activity from bacillus licheniformis ph value enzyme activity u/ml relative activity (%) 5 2.69 ± 0.09 119.0 6 2.50 ± 0.12 110.6 7(control) 2.26 ± 0.13 100.0 8 2.20 ± 0.11 97.3 8.5 2.11 ± 0.22 93.3 9 1.33 ± 0.05 58.8 values are mean ± sd of 3 replicates. fig. 7 elution profile of pectinase after sephadex g-100 chromatography. fig. 8 elution profile of peptidase after deae-cellulose chromatography. fig. 9 the molecular weight of purified pectinase isolated from isolate fmb9 by sds-page, m: protein standards employed were phosphorylase (97 kda), albumin (66 kda), ovalbumin (45 kda), carbonic anhydrase (30 kda), soybean trypsin inhibitor (20.1 kda), and α-lactalbumin (14.4 kda), lane 2: purified pectinase. fig. 10 effect of different substrate concentration on pectiinase activity. pectin. they are classified into three groups, protopectinases, esterase, and depolymerases.39 protopectinases break down protopectin to soluble pectin, esterase which removes methoxyl and acetyl esters from pectin forming polygalacturonic acid and depolymerases which breakdown α-(1 → 4)-glycosidic bonds in units either by hydrolysis or by trans elimination.38,40 most of the polygalacturonase enzymes stimulate the rate of hydrolysis at an ideal ph ranging from 3.5 to 5.5 with a suitable temperature that ranges from 30 to 50°c. several findings relating to various biochemical properties like molecular weight, ph, temperature, isoenzyme, isoelectric point, etc. are well-reported with respect to endopolygalacturonase in various bacterial and fungal species as compared to exopolygalacturonase and rhamnopolygalacturonase. as reported, almost all endopolygalacturonase as well as exopolygalacturonase enzymes are synthesized in acidic environmental conditions, whereas, some exopolygalacturonases are produced at high basic conditions (about ph 11.0) and by particular species including bacillus licheniformis, bacillus sp ksm-p410 and fusarium oxysporum.36 regarding rhamnopolygalacturonases, it was stated that the enzymes are more stable and efficiently work at ph 4.0 and a temperature of 50°c.41 313j contemp med sci | vol. 7, no. 5, september–october 2021: 308–314 f. alqahtani et al. original isolation of pectinase from bacteria conclusion in the present study, the extracellular pectinase was produced by using bacillus licheniformis fmb9 isolated from agricultural soil in jeddah city at kingdom of saudi arabia. the strain showed maximum pectinase activity after 72 of growth at ph 7.0 and 37°c hrs. after purification, pectinase was characterized and molecular weight was determined. acknowledgment the authors are grateful to king abdulaziz university, department of biology, college of science for providing laboratory facilities. conflict of interest none.  table 5. effect of metal ions on pectiinase activity metal ion (1 mm) relative activity (%) control 100 na+ 90* k+ 115 mg2+ 110 fe2+ 97 cu2+ 90 ca2+ 104* zn2+ 110* edta 70 *significant results using studiedt-test at p < 0.05 compared with control without addition. fig. 11 effect of different enzyme concentrations on pectiinase activity. references 1. alkorta, i., garbisu, c., llama, m.j., serra, j.l. (1998). industrial applications of pectic enzymes: a review. process biochemistry journal, 33, 21–28. 2. satapathy s, rout jr, kerry rg, thatoi h, sahoo s l. (2020). biochemical prospects of various microbial pectinase and pectin: an approachable concept in pharmaceutical bioprocessing. frontiers in nutrition 7: 1222–1229. 3. kavuthodi b. and sebastian d (2018). review on bacterial production of alkaline pectinase with special emphasis on bacillus species. bioscience biotechnology research communications, 11: 18–30. 4. rebello s., anju m., aneesh e. m., sindhu, r., binod, p. and pandey, a. (2017). recent advancements in the production and application of microbial pectinases: an overview. reviews in environmental science and bio/ technology, 16(3):381–394. 5. yadav s, yadav p k, yadav d and yadav kds (2009). pectin lyase: a review. process biochemistry, 44(1):1–10. 6. govindaraji, p. k., &vuppu, s. (2020). characterisation of pectin and optimization of pectinase enzyme from novel streptomyces fumigatiscleroticus vit–sp4 for drug delivery and concrete crack-healing applications: an eco-friendly approach. saudi journal of biological sciences, 27(12), 3529–3540. 7. hankin, l., zucker, m. and sands, d.c. (1999). improved solid medium for the detection and enumeration of pectolytic bacteria. journal of applied microbiology, 38: 205–209. 8. vu n t, quach t n , dao x , le h, le c p, nguyen l t, le l, hoang h, chu h h, phi q. (2021). a genomic perspective on the potential of termite–associated cellulosimicrobium cellulans mp1 as producer of plant biomass–acting enzymes and exopolysaccharides. peerj 9, pages e11839. 9. pedrolli, d.b., monteiro, a.c., gomes, e., carmona, e.c. (2009). pectin and pectinases: production, characterization and industrial application of microbial pectinolytic enzymes, the open biotechnology journal, 3: 9–18. 10. chudasama, k.s. and thaker, v.s. (2012). secretion of type ii extracellular cell wall degrading enzymes from pantoeaagglomeransphytopathogen., asian journal of plant science & research., 2(5):559–565. 11. kumar djm, saranya gm, suresh k, priyadharshini da., rajakumar r, et al. (2012). production and optimization of pectinase from bacillus sp. mfw7 using cassava waste. asian journal of plant science & research, 3: 369–375. 12. bateman df, millar rl (1966). pectic enzymes in tissue degradation. ann. rev. phytopathol. 4: 119–146. 13. juven bj, lindner p and weisslowicz h (1985). pectin degradation in plant by leuconostoc mesenteroides. journal of applied bacteriology, 58: 533. 14. neves,m.a.d., kimura, t., shimizu,n., and shiiba. k. (2006). production of alcohol by simultaneous saccharification and fermentation of low–grade wheat flour. the brazilian archives of biology and technology, 49: 481–490. 15. janani, l.k., kumar, g., bhaskararao, k.v. (2011). production and optimization of pectinase from bacillus sp. mfw7 using cassava waste. asian journal of biochemical and pharmaceutical research, 2:329–336. 16. mohandas, a., raveendran, s., parameswaran, b., abraham, a., athira, r. s., kuruvilla mathew, a and pandey a. (2018). production of pectinase from bacillus sonorensis mptd1. food technology and biotechnology, 56(1), 110–116. 17. miller, g.l. (1959). use of dinitrosalicylic acid reagent for determination of reducing sugar. analytical chemistry, 31: 426–428. 18. roy k., dey s., uddin m., barua r and hossain m. (2018). extracellular pectinase from a novel bacterium chryseobacteriumindologenes strain sd and its application in fruit juice clarification. enzyme research. 19. holt, j.g., krieg, n.r., sneath, p.h.a., staley, j.t., william, s.t. (1994). bergeys manual of determinative bacteriology, nineth ed., williams and walkins, baltimore, 787. 20. chen, w., kuo, t. (1993). nucleic acids research journal, 21: 2260. 21. lowry oh, rosgrough nj,farr al, randall r j (1951). protein measurement with the folin phenol reagent. the journal of biological chemistry, 193, 165–275. 22. laemmli uk (1970). cleavage of the structural proteins during assembly of the head of bacteriophage t4. nature, 227, 680–685. 23. namasivayam e, ravindar jd, mariappan ka, kumar m. (2011). production of extracellular pectinase by bacillus cereus isolated from market solid waste. j bioanal biomed., 3: 070–075. 24. vibha b. and neelam g. (2010). exploitation of microorganisms for screening of pectinase from environment.8th international conference in food, usa. 25. hirose, n., kishida, m., kawasaki,h, sakai, t. (1999) purification and characterization of an endo polygalacturonase from a mutant of saccharomyces cerevisiae. bioscience, biotechnology, and biochemistry. 63(6): 1100–1103. 26. aaisha,g. and barate, d. (2016). isolation and identification of pectinolytic bacteria from soil samples of akola region, india. international journal of current microbiology and applied sciences, 5: 514–521. 27. oumer, o. j., & abate, d. (2017). characterization of pectinase from bacillus subtilis strain btk 27 and its potential application in removal of mucilage from coffee beans. enzyme research, 2017. 28. nawawi, m.h., mohamad, r., tahir, p.m. and w. z. saad, (2017). extracellular xylanopectinolytic enzymes by bacillus subtilis adi1 from efb’s compost, international scholarly research notices, 7. 314 j contemp med sci | vol. 7, no. 5, september–october 2021: 308–314 isolation of pectinase from bacteria original f. alqahtani et al. 29. guan, y., wang, q., lv, c., wang, d., & ye, x. (2021). fermentation time– dependent pectinase activity is associated with metabolomics variation in bacillus licheniformis dy2. process biochemistry, 101, 147–155. 30. takcı ham and turkmen fu (2016). extracellular pectinase production and purification from a newly isolated bacillus subtilis strain, international journal of food properties, 19:11, 2443–2450. 31. mukesh kumar dj, saranya gm, suresh k, andal priyadharshini d, rajakumar r, kalaichelvan pt (2012). production and optimization of pectinase from bacillus sp. mfw7 using cassava waste. asian journal of plant science and research, 2, 369–375. 32. giacobbe s.; pepe, o.; ventorino, v.; birolo, l.; vinciguerra, r.; faraco, v. 2014,identification and characterization of a pectinolytic enzyme from paenibacillus xylanolyticus. bioresources 9, 4873–4887. 33. ashour sm, kheiralla zmh, eldiwany ai, maa ny da (2014). production, purification and characterization of polysaccharide lytic enzymes of a marine isolate, bacillus cereus nrc–20 and their application in biofil removal. african journal of microbiology research , 8, 2492–2504. 34. arijit, d.; sourav, b.; naimisha, r.v.; rajan, s.s. improved production and purification of pectinase from streptomyces sp. ghba10 isolated from valapattanam mangrove habitat, kerala, india. international research journal of biological sciences 2013, 2, 16–22. 35. kashyap, d.r.; chandra, s.; kaul, a.; tewari, r. (2000). production, purification, and characterization of pectinase from a bacillus sp. dt7. world journal of microbiology and biotechnology, 16, 277–282. 36. bhardwaj, v.; garg, n. (2012). production, purification of pectinase from bacillus sp. mbrl576, isolate and its application in extraction of juice. international journal of science and research, 3, 648–652. 37. barnby fm, morpeth ff, pyle dl (1990). endopolygalacturonase production from kluyveromyces marxianus. i. resolution, purification and partial characterization of the enzyme. enzyme microb. technol., 12:891–7. 38. jayani rs, saxena s, gupta r. (2005). microbial pectinolytic enzymes: a review. process biochem., 40:2931–44. 39. shrestha s, khatiwada j r, zhang x, chio c , kognou a chen, f, han s , chen x , qin w. (2021). screening and molecular identification of novel pectinolytic bacteria from forest soil. fermentation 7:1, pages 40. 40. john j, surendranathan k .k., smith m l., rahman p k.s.m., chellam p v. (2020). advances in upstream and downstream strategies of pectinase bioprocessing: a review. international journal of biological macromolecules, 162, pages 1086–1099. 41. mutter m, beldman g, pitson sm, schols ha, voragen agj. (1998). rhamnogalacturonan α-d-galactopyranosylurono-hydrolase. plant physiol. 117:153–63. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i5.1101 148 j contemp med sci | vol. 2, no. 8, autumn 2016: 148–152 research evaluation of health behaviors among secondary school students in baghdad city raad k. faraj issn 2413-0516 department of community health nursing, college of nursing, baghdad university, iraq. correspondence to raad kareem faraj (email: raad_faraj@yahoo.com). (submitted: 9 august 2016 – revised version received: 19 september 2016 – accepted: 21 september 2016 – published online: 26 december 2016) objectives the present study aims to assess the health behaviors among secondary school students of smoking, diet, and physical activity and to find out the relationship between health behaviors and socio-demographic characteristics of the students. methods a descriptive, analytical study carried out from september 2015 to april, 2016 on a simple random sample of 500 students to achieve the objectives that are stated in this study. an assessment tool is constructed by the researcher based on previous literature regarding aspects of health behaviors. content validity for the instrument was determined through the use of panel experts to investigate the clarity, relevancy, and adequacy of the study questionnaire. the internal consistency of the instrument was determined through the pilot study and the computation of alpha correlation coefficient which was statistically adequate. the analysis of the data was employed by the use of spss version 15.0. results the autonomous motivation is higher than the controlled motivation and a motivation regarding to “the reason i would not smoke”, [mean (sd) = 76.93 ± 12.02; 36.08 ± 5.4; 11.7 ± 3.5], respectively. regarding “the reason i would eat a healthy diet”, the autonomous motivation is higher than the controlled motivation and a motivation [mean (sd) = 34.02 ± 5.5; 23.9 ± 5.7; 8.7 ± 3.8] respectively. concerning “the reason i would exercise regularly”, the autonomous motivation is higher than the controlled motivation and a motivation [mean (sd) = 33.4 ± 5.7; 22.9 ± 6.3; 9.5 ± 3.8], respectively. the perceived competence for not smoking is higher than the perceived competence for maintaining a healthy diet and the perceived competence for exercising regularly [mean (sd) = 23.7 ± 5.5; 19.3 ± 3.0; 18.5 ± 3.2], respectively. there is a significant inverse association between gender and health behavior (r = −0.178 at p < 0.01). there is a significant positive associations between smoking status, having one or both smoker parent(s) and the overall health behavior (r = .417 at p < 0.01; r = .134 at p < 0.01) respectively. conclusion the study concluded that non-smoking is the most prevalent health behavior among the students that revealed by high perceived competence related to non-smoking. health behavior is negatively influenced by gender, and positively influenced by smoking status. keywords health behavior, secondary school, students introduction health behaviors are identified as any activities that undertaken by individuals to prevent and detect the disease, or to improve health. it is also referred for the health behaviors as actions and habits that they are related to maintain, resort, and improve the health.1 health behaviors and risky behaviors are reported in the community as a more complex pattern of behaviors that are related to lifestyles. there are many behaviors that are reported as unhealth behaviors such as smoking, drinking alcohol, un-protective sexual intercourse, drug abuse, unhealthy diet habits, and lack of physical activities. these behaviors have become more common during the early age, especially adolescence and contribute to the mortality and morbidity.2 it has been reported that teenagers are more prone to high risk of unhealth behaviors such as smoking tobacco, teenage pregnancy, drug and alcohol use, in addition to high level of psychological distress for health.3 moreover, a large proportion of morbidity and mortality has been indicated in the developing countries, in which high rate of mortality and morbidity among adults are attributed for unhealth behaviors that begin in early adolescence.4 smoking behavior in adolescence is a common public health issue, and is one of the major problems that have consequences on health; therefore, many health institutions are seeking to reduce this phenomenon among adolescents via educational programs and campaigns, especially in the school to prevent the negative consequences on health.5 unhealthy eating behaviors are frequently found in adolescents who, having physical exercise and those with low levels of physical exercise. although, practicing physical exercise promotes physical and mental health. it looks that in terms of unhealthy eating behaviors, scientific research are inconclusive. otherwise, some research argues that excess physical exercise may predispose young adolescents to health compromising eating behaviors. it has been estimated that young adolescent with low level of physical exercise are more vulnerable to developing unhealthy eating habits, since these habits have more impact on their health.6 health promotion is considered as a fundamental for individuals. it is imperative that lifestyle is the most important factor in promoting health among the individual and preventing the disease and mortality. the major criteria are regarded by health-promoting behaviors that determine the health and are directly related to preventing many diseases. on one hand, some hygienic measures such as immunization, efficient sleeping pattern, enough physical exercise, good nutrition, and personal hygiene. on the other hand, the lack of physical exercise, wrong nutritional habits, and behaviors lead to the prevalence of many diseases and high mortality rate in all age including adolescents. however, adolescents are considered as a central part of life and the stepping-stone of the community, which they do not live in a good health condition, a disease limits their abilities to grow perfectly.7 therefore, it is important to explore the health behaviors that are contributing raad k. faraj 149j contemp med sci | vol. 2, no. 8, autumn 2016: 148–152 research evaluation of health behaviors among secondary school students in baghdad city data collection the data were collected through the use of the assessment tool and the interview technique was used as a means of data collection. the questionnaire was distributed after obtaining the agreement to participate in the study, and the time for filling the questionnaire was 30–60 minutes. statistical analysis the analysis of the data was employed by the use of spss version 15.0 through the application of descriptive statistical and inferential statistics such as frequencies, percentages, the mean of scores and chi-square. results the mean age is 15.7 ± 1.89, more than a quarter are in the 12–14-year-old age group (n = 136; 27.2%), more than a half are in the 15–17-year-old age group (n = 277; 55.4%), and less than a fifth are in the 18–21-year-old age group (n = 87; 17.4%). less than a half are males (n = 248; 49.6%) and more than a half are females (n = 252; 50.4%). a very limited proportion have a poor school achievement (n = 11; 2.2%), more than a quarter have an average school achievement (n = 129; 25.8%), more than a third have a good school achievement (n = 174; 34.8%), more than a quarter have a very good school achievement (n = 142; 28.4%), and a small proportion have an excellent school achievement (n = 44; 8.8%). more than a quarter are in the first rank among their brothers/sisters (n = 144; 28.8%), about a quarter are in the second rank among their brothers/sisters (n = 128; 25.6%), more than a fifth are in the third rank among their brothers/ sisters (n = 107; 21.4%), and less than a quarter are in other ranks among their brothers /sisters (n = 121; 24.2%). moreover, the mean family members are 6.06 ± 1.53. a small proportion reported that their families have a less than 500.000 iraqi dinar (i.d.) as a monthly income (n = 34; 6.8%), less than two fifth reported that their families have a monthly income ranges between half to one million i.d. (n = 189; 37.8%), and more than a half reported that their families have more than 1.500.000 i.d. as a monthly income (n = 278; 55.6%). more than a quarter reported that their fathers have some bachelor degree (n = 142; 28.4%) followed by those who reported that their father are intermediate school graduate (n = 95; 19.0%). also, more than a fifth reported that their mothers have some bachelor degree (n = 108; 21.6%) followed by those who reported that their mothers are high school graduates (n = 100; 20.0%). the mean body mass index (bmi) is 22.9 ± 4.2; a small proportion are underweight (n = 48; 9.6), most are of healthy weight (n = 346; 69.2%), less than a fifth are overweight (n = 74; 14.8%), a small proportion are obese (n = 20; 4.0%), and a very limited proportion are morbidly obese (n = 12; 2.4%). less than a fifth are smokers (n = 70; 14.0%); the majority of them are males (n = 50; 71.4%) and more than a quarter are females (n = 20; 28.6%). the vast majority are non-smokers (n = 415; 83.0%); less than a half are males (n = 188; 45.3%) and more than a half are females (n = 227; 54.7%). a very limited proportion quitted smoking (n = 15; 3.0%); two third are males (n = 10; 66.7%) and a third are females (n = 5; 33.3%). with respect to “the reason i would not smoke”, the autonomous motivation is higher than the controlled motivation and a motivation [mean (sd) = 76.93 ± 12.02; 36.08 ± 5.4; 11.7 ± 3.5] to health among the school students. in this paper, the researcher will assess the health behaviors of smoking, diet habits, and physical activity. materials and methods design of the study a descriptive analytical study carried out from september, 2015 to april, 2016 to achieve the objectives that are stated in this study. sample of the study a simple random sample of 500 students is selected from a probability stratified sample of secondary schools for both sexes in baghdad al-rasafa and al-karkh districts which are selected randomly. setting of the study the study was conducted in four sector areas distributed through out the baghdad educational directorate of al-rusafa and educational directorate of al-karkh; baghdad al-jedeeda, al-sadir city, palestine’s street and all around, al-amin, al-sho’ala, al-jawadain, al-ataifiya, al-tobchi, al-mansor, and alharthiya which were selected randomly. these schools are considered the most appropriate settings in which the subjects for the study can be selected. the permission has been granted from ministry of education/educational directorate of al-karkh and educational directorate of al-rusafa. the permissions were facilitating the entrance of researcher and completing the data collection. instrument of the study an assessment tool is constructed by the researcher based on previous literature regarding aspects of health behaviors. the constructed tool includes three parts, the first part was concerned with covering letter and the agreement of participants to participate in the study, the second part consisted of demographic and personal characteristics of the students, and the third part consisted of health behaviors which include: treatment, smoking behavior, diet, and physical exercise, each sub-domain consisted of 15 items that were measured on likert scale that rated and scored from 1 to 7 point (1 = never not true, 4 = true sometimes, and 7 = very true). the fourth part included the sub-domains of the perceived competencies which include: non-smoking, maintaining diet, and physical exercise, each sub-domain consists of four items that are measured on likert scale which rated and scored from 1 to 7 point (1 = never not true, 4 = true sometimes and 7 = very true). content validity for the instrument was determined through the use of panel experts to investigate the clarity, relevancy, and adequacy of the study questionnaire. in addition to the experts’ responses, their suggestions were taken into consideration. so far, modifications were employed and the final copy of the constructed instrument was completed to be an appropriate tool for conducting study. a pilot study was carried out for the period of 15 days on october 2015, and conducted on 50 students who were selected randomly for the purpose of the questionnaire’s reliability determination. the internal consistency of the instrument was determined through the pilot study and the computation of alpha correlation coefficient (cronbach’s alpha). the result of the reliability was (r = 0.81) which was statistically adequate. 150 j contemp med sci | vol. 2, no. 8, autumn 2016: 148–152 evaluation of health behaviors among secondary school students in baghdad city research raad k. faraj respectively. regarding “the reason i would eat a healthy diet”, the autonomous motivation is higher than the controlled motivation and a motivation [mean (sd) = 34.02 ± 5.5; 23.9 ± 5.7; 8.7 ± 3.8], respectively. concerning “the reason i would exercise regularly”, the autonomous motivation is higher than the controlled motivation and a motivation [mean (sd) = 33.4 ± 5.7; 22.9 ± 6.3; 9.5 ± 3.8] respectively. the perceived competence for not smoking is higher than the perceived competence for maintaining a healthy diet and the perceived competence for exercising regularly [mean (sd) = 23.7 ± 5.5; 19.3 ± 3.0; 18.5 ± 3.2] respectively. there is a significant inverse association between gender and health behavior (r = –0 .178 at p < 0.01). there is a significant positive associations between smoking status, having one or both smoker parent(s) and the overall health behavior (r = 0.417 at p < 0.01; r = 0.134 at p < 0.01), respectively. discussion it has been known out of the analysis in table 1 that more than a half of the students were females with age group 15-17 years old who were in second class and having better school achievement. these findings may give an indicator that female students are more responsible than male students in the term of school requirements. on the other hand, they are seen to be more confident at their age. the findings in table 1 also show that the students were the firstborn of family consisted of 5-6 members; the family income was ranging between 500,000– 1,000,000 iraqi dinars; the fathers of most students were graduated from college. most of the students were non-smokers with healthy weight. such findings may be explained that educated fathers have an impact on their sons and daughters life including ethics and values and the manners they behave and do related to health behaviors that what is observed with the result of smoking status the students and reflected by their healthy weight. the finding of income has been clear enough for such families consisted with 5-6 persons. the findings of this study agreed with the results of qasim and abed3 that they found. the findings of table 2 reveal that the autonomous motivation is higher than the controlled motivation and a motivation related to reason of non-smoking, reasons for eating healthy diet, and reasons for practicing exercise regularly. in accordance with these findings, it is possible to interpret the high motivation is the result of the perception of the students and the students perceive that the unhealth behaviors are the strong motives to change the behavior. this is explained by the protection motivation theory.8,9 according to this theory, the cognitive process plays a key role in decision-making process, leading to a change in behavior. evaluation of threat is serving as an evaluation of maladaptive behavior, therefore, the autonomous motivation is high in the students because they have perceived these threats with their cognitive abilities. these finding agree with the study of xu and chen10 who found high motivation and high coping among their sample. table 3 shows that students’ perceived competence for not smoking is higher than the perceived competence for maintaining a healthy diet and the perceived competence for exercising regularly. according to social cognition theory of self-efficacy, the individual sense of control is key role in the change of health behavior. self-efficacy beliefs are cognition that determines whether health behavior change will be initiated, and table 1. participants’ socio-demographic characteristics (n = 500) list variables f % 1. age [ mean (sd) = 15.7 ± 1.89] 12 – 14 136 27.2% 15 – 17 277 55.4% 18 – 21 87 17.4% total 500 100.0% 2. gender male 248 49.5% female 252 50.4% total 500 100.0% 3. school class first 172 34.4% second 328 65.6% total 500 100.0% 4. school achievement poor 11 2.2% average 129 25.8% good 174 34.8% very good 142 28.4% excellent 44 8.8% total 500 100.0% 5. rank among brothers first 144 28.8% second 128 25.6% third 107 21.4% other 121 24.2% total 500 100.0% number of family members 6. 3 – 4 61 12.2% 5 – 6 280 56.0% ≥ 7 159 31.8% total 500 100.0% 7. monthly income (iraqi dinar) less than 500.000 34 6.8% 500.000-1.000.000 189 37.8% 1.001.000-1.500.000 178 35.6% > 1.500.000 99 19.8% total 500 100.0% 8. father’s education unable to read and write 11 2.2% elementary school graduate 46 9.2% intermediate school graduate 95 19.0% high school graduate 79 15.8% diploma 74 14.8% bachelor 142 28.4% postgraduate 53 10.6% total 500 100.0% 9. body mass index (bmi) underweight (< 18.5) 48 9.6% healthy weight (18.5 – 24.9) 346 69.2% overweight (25 – 29.9) 74 14.8% obese (30 – 34.9) 20 4.0% morbidly obese (> 35) 12 2.4% total 500 100.0% 10. smoking status smoker 70 14.0% non-smoker 415 83.0% quit smoking 15 3.0% total 500 100.0% f: frequency; %: percentage; sd: standard deviation. raad k. faraj 151j contemp med sci | vol. 2, no. 8, autumn 2016: 148–152 research evaluation of health behaviors among secondary school students in baghdad city table 3. mean and standard deviation for the perceived competence for not smoking, maintaining a healthy diet, and exercising regularly sub-domain mean (sd) perceived competence (not smoking) 23.7 ± 5.5 perceived competence (maintaining a healthy diet) 19.3 ± 3.0 perceived competence (exercising regularly) 18.5 ± 3.2 overall 2.70 ± 2.90 table 4. correlations among the study variables 1 2 3 4 5 6 7 8 9 10 11 12 13 1. age 2. gender 0.274** – 3. school achievement –0.287** –0.052 – 4. rank among brothers 0.045 0.203** 0.015 – 5. no. of family members 0.163** 0.138** –0.128** 0.470** – 6. monthly income –0.157** –0.071 0.224** 0.059 –0.031 – 7. father’s education –0.166** –0.194** 0.222** –0.002 –0.061 0.584** – 8. mother’s education –0.210** –0.190** 0.233** –0.179** –0.293** 0.493** 0.383** – 9. bmi 0.024 –0.191** –0.027 –0.039 0.065 0.049 –0.051 –0.020 – 10. smoking status –0.137** 0.128** 0.142** 0.035 –0.002 0.107* 0.075 0.089* –0.118** – 11. are one or both parents are smoker? –0.099* 0.019 0.095* 0.057 –0.009 0.049 0.118** 0.052 –0.071 0.194** – 12. do you have smoker friends? –0.194** 0.344** 0.172** 0.097* 0.022 –0.007 –0.061 –0.113* –0.070 0.209** 0.184** – 13. health behavior –0.227** –0.178** 0.036 –0.004 –0.033 0.047 0.036 0.027 0.022 0.417** 0.134** 0.061 – **correlation is significant at the 0.01 level (2-tailed); *correlation is significant at the 0.05 level (2-tailed). there is a significant positive association between smoking status, having one or both smoker parents and the overall health behavior. the inverse association between the gender and health behavior may be explained that genders are influenced by seeking information, the males are less interest in seeking information than females unless they are still young. such finding has been supported by deeks et al.12 and siegrist et al.13 the positive relationship between smoking status and health behavior may be explained by high competence that they have concerning smoking, due to their high cognition that is reflected by high self-efficacy, they are aware to be non smoking behavior is the key of good health which is associated with health behavior that they practice. the finding of this study agree with the study of saingam et al14 who found a significant association between smoking status and health risk behaviors. conclusions 1. non-smoking behavior of students is revealed by the high autonomous motivation rather than controlled motivation. 2. non-smoking is the most prevalent health behavior among the students that revealed by high-perceived competence related to non-smoking. 3. health behavior is negatively influenced by gender, and positively influenced by smoking status. recommendations 1. more effective awareness programs are required to improve knowledge and practice regarding health behaviors. 2. motivational programs are required for the schoolteachers to improve health behavior practices among the students. conflict of interest none. n how it will sustain in overcoming the obstacles. self-efficacy is directly related to the health behavior, because its effects on the goal of health behaviors are set by individuals. therefore, the findings of this study showed that students who have high-perceived competence have strong self-efficacy in selecting their goals, and they are focusing on opportunities than on obstacles. supportive evidence for these results are found epstein et al.9 and spek et al.11 who reported high competence among the sample. the findings of table 4 show that there is a significant inverse association between gender and health behavior. and table 2. mean and standard deviation for the sub-domains of the reason i would not smoke, the reason i would eat a healthy diet, and the reason i would exercise regularly sub-domain autonomous motivation controlled motivation amotivation total mean (sd) mean (sd) mean (sd) mean (sd) the reason i would not smoke 76.93 ± 12.02 36.08 ± 5.4 11.7 ± 3.5 76.93 ± 12.02 the reason i would eat a healthy diet 34.02 ± 5.5 23.9 ± 5.7 8.7 ± 3.8 66.6 ± 9.3 the reason i would exercise regularly 33.4 ± 5.7 22.9 ± 6.3 9.5 ± 3.8 65.9 ± 9.9 152 j contemp med sci | vol. 2, no. 8, autumn 2016: 148–152 evaluation of health behaviors among secondary school students in baghdad city research raad k. faraj references 1. conner m. health behaviors, handbook of health behaviors research. 2002;3:87. 2. kann l, charles w, warren, collins jl, lioyed j. health behaviors. j adolesc health. 2000;20:45. 3. qassim w, abed r. assessment of health promotion behaviors student’s teenage in baghdad city. kufa j nur sci. 2016;6. 4. easton a, kiss e and mowery p, budapest student health behaviors surveybudapest, hungry, 1999, finding on unintentional and intentional injuries, alcohol use, and sexual activities. cont eur j publ health. 2004;12:94–101. 5. turbin m, josser r, costa f. adolescent cigarette smoking: health-related behavior or normative transgression. prevent sci. 2000;1:115–124. 6. fortes l, morgado f, almeida s, ferreira m. eating behavior and physical activity in adolescents. rev nur. 2013;26:527–537. 7. motaghi m, asfar m. health behaviors among high school girls. int j school health. 2015;2:2–6. 8. thrul j, stemmler m, buhler a, kuntsche e. adolescents’ protection motivation and smoking behavior. health educ res. 2013;28:683–691. 9. yan y, jacques-tiura aj, chen x, xie n, chen j, yang n, et al. application of the protection motivation theory in predicting cigarette smoking among adolescents in china. addict behav. 2014;39:181–188. 10. xu y, chen x. protection motivation theory and cigarette smoking among vocational high school students in china: a cusp catastrophe modeling analysis. global health research and policy. 2016;1:3. 11. epstein j, griffin k, botvin g. competence skills help deter smoking among inner city adolescents. tob control. 2000;9:33–39. 12. spek v, lemmens f, chatrou m, kempen s, pouwer f, pop v. development of a smoking abstinence self-efficacy questionnaire. int j behav med. 2013;20:444–449. 13. deeks a, lombard c, michelmore j, teede h. the effects of gender and age on health related behavior. bmc pub health. 2009;9:213. 14. siegrist m, keller c, kiers ha. a new look at the psychometric paradigm of perception of hazards. risk anal. 2005;25:211–222. 15. saingam d, assanangkornchai s, geater a. drinking-smoking status and health risk behavior among high school students in thailand. j drug educ. 2012;42(2):93–177. 86 j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 review safety and efficacy of covid-19 vaccines in special populations sana alattas1,* , ibrahim m ibrahim3, ahmed s. ali2,3, jehan m barakat3, assmaa a shaker4, tasneem n momen4, abir s. mohamed5, abdalbabgi alfadil6, amani e alharbi7 1department of biological sciences, faculty of sciences, king abdulaziz university, jeddah, saudi arabia. 2department of pharmaceutics, faculty of pharmacy, assiut university, egypt. 3department of pharmacology, faculty of medicine, king abdulaziz university, jeddah, saudi arabia. 4ibnsina national college for medical studies, jeddah, saudi arabia. 5department of internal medicine, faculty of public health and tropical medicine, jazan university, saudi arabia. 6department of medical microbiology and parasitology, faculty of medicine, king abdulaziz university, jeddah, saudi arabia. 7department of pharmacology and toxicology, college of pharmacy, taibah university, madinah, saudi arabia. *correspondence to: sana alattas (e-mail: sgalattas@kau.edu.sa) (submitted: 16 january 2022 – revised version received: 04 february 2022 – accepted: 05 march 2022 – published online: 26 april 2022) abstract coronavirus disease 2019 (covid-19), is a pandemic that resulted in extreme human and economic losses. a higher incidence of morbidity and mortality to covid-19 was demonstrated in a special population. factors that impact the disease severity include old age, obesity, pregnancy, diabetes mellitus, cancer, and immunosuppressive drugs. fortunately, several covid-19 vaccines were developed such as pfizer-biontech, moderna, oxford-astrazeneca, and johnson & johnson among others. these vaccines have shown good efficacy and safety profiles in the general population, but serious rare adverse effects were reported related to specific vaccines. several studies are undergoing to test the efficacy in special populations. reduced efficacy or delayed immunological response to covid-19 vaccines were suggested for patients with autoimmune disorders or organ transplant patients, especially those receiving certain medications such as rituximab. there is a concern about organ rejection in organ transplant patients. despite these facts, there is an agreement among health care providers to consider prioritization of the above-mentioned groups for receiving vaccinations with the same precautions followed for the general population. it is recommended to ongoing studies determine the efficacy and safety of covid-19 vaccines in patients with comorbidities based on clinical data. keywords: covid-19, sars-cov-2, johnson & johnson’s, pfizer, moderna, vaccine issn 2413-0516 covid 19 vaccines coronavirus disease 2019 (covid-19) is a global pandemic caused by a highly infectious respiratory tract virus (sars-cov-2). it has resulted in significant human and economic loss. more than 207 million cases had been confirmed as of august 16, 2021, with more than 4.36 million verified deaths.1 by the end of 2020, several vaccines had become available for use in different parts of the world.2 most vaccines for covid-19 aimed to achieve an adequate immune response to the virus’s distinctive spike protein.3 these vaccines are being developed using various techniques, which include the inactivated or attenuated virus, viral protein subunits. viral vectors, and rna encoding viral spike protein.4,5 this noncomprehensive review provides a simplified overview of the efficacy and safety of the most extensively used covid-19 vaccines in special populations. the review faces limited restricted search on data provided in the english language. vaccine subtypes, their development, ranking efficacy and safety in the general population werepresented in several reviews.6-8 the safety and efficacy of covid-19 vaccines in children and adolescents were also reviewed.9 the rcts on bny162b2 showed that the efficacy of the vaccine in children and adolescents was 100%.10 regarding safety, 38 cases of myopericarditis due to bny162b2 were described in 2 reports, of which 93% occurred after the second dose, 90% occurred in males, [11, 12]. the estimated incidence of myopericarditis was 0.008% in adolescents 16–17 years of age and 0.01% in those aged 12–15 years.12 a brief description of the most used covid 19 vaccines is provided in the next section. pfizer–biontech covid-19 vaccine (bnt162b2) it is an mrna-based covid-19 vaccine developed by biontech, (germany) in collaboration with pfizer (usa).13 it is a lipid nanoparticle encapsulating nucleoside-modified mrnaexpressing spike protein of sars-cov-2.14 the phase iii results indicated an overall efficacy of 95% in the general population after two doses separated by 21 weeks.15 for immunocompromised individuals e.g. organ transplant patients, a lower vaccine efficacy after a single dose was reported.16 it was reported to be safe and effective in delivering protection to pregnant and nursing women.17 its overall effectiveness (7–56 d) after the second dose in pregnant women was reported as 96%.18 the efficacy was about 100% in adolescents, but the incidence of myocarditis and pericarditis was likely to be higher in young adults.9 it was reported that patients with β-cell nonhodgkin lymphoma treated with an anti-cd20 antibody are unlikely to achieve humoral response to bnt162b2 mrna covid-19 vaccine.7 moderna (mrna-1273) it was developed by the moderna pharmaceutical company and approved for use in individualsabove the age of twelve. it is intended to be given in two doses or three for immunocompromised patients. each dose is 0.5 ml administered by intramuscular injections spaced at least 28 days apart.19 it consists of a lipid nanoparticle (lnp) containing nucleoside-modified messenger rna (modrna), which encodes the sars-cov-2 virus’s perfusion stabilized spike (s) protein, as well as an https://orcid.org/0000-0001-5753-8520 mailto:sgalattas@kau.edu.sa 87j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 s. alattas et al. review safety and efficacy of covid-19 vaccines in special populations s1-s2 cleavage site with a transmembrane component. the presence of the s-2p antigen on its surface permits it to enter the host cell. the rna is then transferred into host cells, where the sars-cov-2 s antigens are expressed. it provides antibody response to sars-cov-2 s antigens.20,21 in the general population, it showed an overall efficacy of 95% against symptomatic covid-19.15,22 the efficacy of two doses against infection with the delta variant was 87.9% in those aged 18–64 years, but lower (75.2%) in the elderly > 65 year.23 reported common adverse events include the following (%,): injection site tenderness/pain (92), swelling (14.7), redness (10.0), fever (15.5), fatigue (70.0), headache (64.7), muscle pain (61.5), chills (45.4), joint pain (46.4), swollen lymph nodes (1.1), nausea/vomiting (23.0), hypersensitivity (1.5). serious side effects were reported as 1%, and a few cases of bell’s palsy (0.1%) were also documented.22 at 28 days after the second dose of vaccination poor or suboptimal antibodies response was observed in cancer patients under chemotherapy. however, antibodies raise significantly after the 2nd dose (compared to that after the 1st dose). these findings suggest the value of the 3rd boosting dose of the vaccine in cancer patients.24 the efficacy of the vaccine after the first dose was lower among patients with decompensated (50.3%) compared with compensated cirrhosis (66.8%). after the second dose, there was a 78.6% reduction in covid-19 infections and 100% reduction in covid-19-related hospitalization.25 most dialysis patients showed adequate antibody response after two doses of bnt162b2 or mrna-1273 vaccines.26 in vaccinated pregnant women, the rate of adverse events (in both the mother and the fetus) was comparable to that in non-immunized pregnant women.27 oxford–astrazeneca covid-19 vaccine (azd122) chadox1-s it is a viral vector (modified chimpanzee adenovirus chadox1) vaccine for the prevention of covid-19. it was developed by oxford university in collaboration with astrazeneca.28,29 early investigations conducted in 2020 showed that the vaccine efficacy was 76.0% in preventing symptomatic covid-19, starting from 22 days after the first dose and up to 81.3% after the second dose.30 a study in scotland revealed thatafter the second dose the vaccine was 81% effective against the alpha variant, and 61% against the delta variant.31 an interim analysis of randomized controlled trials in brazil, south africa, and the uk have documented an overall efficacy of 70·4%.32 a meta-analysis suggested equivalent effectiveness of bnt162b2 and chadox1 covid-19 vaccine against sars-cov-2 infection and covid-19 related morbidity and mortality.33 moreover, common adverse effects in vaccinated individualswere as follow: local reaction at the injection site, soreness, headache, and lethargy.34 systemic reactions were observed in (86%) of 18–55 y group, but lower (77%) in 56–69 y, and (65%) in the 70 y or older age group. it was concluded that this vaccine is likely better tolerated in older adults.35 concerning serious adverse events, there are reports of vaccine-induced immune thrombotic thrombocytopenia (vitt).36 according to the “european medicine agency’s pharma covigilance risk assessment committee,” 169 cases of cerebral venous sinus thrombosis (cvst) and 53 cases of splanchnic vein thrombosis were reported among the 34 million individuals who received the astrazeneca covid-19 vaccine. the majority of these casesoccurred within the first two weeks following vaccination in women under the age of 60.34 an early study in the uk documented that the estimated rate of thrombotic thrombocytopenia syndrome (tts) within 14 days of the first dose of the vaccine was 8·1 per million vaccines.37 details of cvst, related venous infarct and hemorrhagic stroke, arterial infarct, intracerebral hemorrhage (ich) and vitt following the astrazeneca covid-19 vaccination were discussed by a meta-analysis. in this context, 41 cases were presented (36 cvst, 4 infarctions, and 1 ich). sixteen of the 36 patients with cvst experienced an ichand/or a subarachnoid hemorrhage (sah), of which 18 cases (44%) died.38 rare cases of the multisystem inflammatory syndrome39 and guillain-barré syndrome (gbs) were also reported.40 at 120 vaccination centers in mumbai, india, as of april 13th 2021, the total adverse effects following immunization (aefis) was reported as (93.53 %), of which 3.87% were moderate, 3.87% were severe, and 2.58% were serious.41 an australian risk-benefit analysis for individuals < 60 years suggested that the risk of the azd1222 vaccination may exceed the benefits in younger individuals who are at low risk of serious covid-19.42 in individuals with hiv, the chadox1 vaccination was demonstrated to elicit protection and a safety profile similar to those without hiv. this investigation, recommends vaccination for those patients on antiretroviral therapy (art).43 a small study in japan indicated a lower response in the elderly after two doses of azd1222. neutralizing antibody responses were predicated as 67.5%, 60.3%, and 50.0% ofvaccinated individuals aged between 18–55, 56-69, and => 70 y, respectively. no vaccine-related serious adverse events or deaths were reported.44 a 51-year-old woman had pancreas allograft rejection after receiving the chadox1 vaccine.45 rare cases of the acute hyperglycemic crisis were reported in diabetic patients 3–5 weeks following the first dose of the vaccine.46 janssen covid-19 vaccine (ad26.cov2.s) ad26.cov2.s is a replication-ineffective human adenovirus type 26 (ad26) vector that expresses a pre-fusion stabilized sars-cov-2 spike protein (wuhan 2019 strain, which is similar to the wa1/2020 strain’s).47-51 in the phase iii trial in the us, brazil, and south africa, by day 28 after vaccination, a single dose of ad26.cov2.s provided 72%, 68%, and 64% protection against moderate to severe covid-19 respectively. in this trial, ad26.cov2.s provided robust protective efficacy against a few sars-cov-2 variants, with 95% of sequenced viruses from confirmed covid-19 cases in south africa being the b.1.351 variant.52 in another study on day 71 following vaccination, ad26.cov2.s generated median pseudovirus neutralizing antibody titers which were 5.0-fold and 3.3-fold lower against the b.1.351 and p.1 sars-cov-2 variants, respectively, compared to that against wa1/2020 strain. however, similar cd8 and cd4 t cell responses, including central and effector memory responses were recognized against all tested sars-cov-2 variants.53 rare guillain-barré syndrome cases (gbs) were reported after ad26.cov2.s vaccine. the estimated incidence per million of vaccinated individuals were 6.2 for females and 7.8 for males.7 88 j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 safety and efficacy of covid-19 vaccines in special populations review s. alattas et al. covaxin (bbv152) covaxin (bbv152) was developed by bharat biotech and the council of medical research (india). it is an inactivated whole virion sars-cov-2 vaccine, formulated with a toll-like receptor 7/8 agonist (tlr7/8) molecule adsorbed to alum (algel-imdg). the tlr7/8 adjuvant formulation specifically boosted sars-cov-2 lymphocyte as well as th1 driven antibody responses.54 a randomized, double-blind, placebo-controlled, multicenter, phase 3 clinical trial was conducted in india on adults (age ≥18 years) who were healthy or had stable chronic medical conditions. the study demonstrated, the overall efficacy of 78%. efficacy and safety were also confirmed in children. currently, limited data are available to predicate its safety and efficacy in immunocompromised patients, patients with cancer or pregnant women.55,56 the common adverse effects after vaccination were as follow: injection site pain (5%), headache (3%), fatigue (3%), fever (9%), nausea or vomiting (2%).57 corona vac, sinovac it is an inactivated virus covid-19 vaccine developed by the chinese company sinovac biotech. it was developed by culituring sars-cov-2 cn2 strain to replicate in vero cell, immediately harvesting, isolating, and inactivating the grown strain with b-propiolactone.58,59 vaccine efficacy in the general population, detailed efficacy and safety of corona vac can be retrieved in these publications.59,60,61 phase 3 trials were carried out in brazil,62 turkey,63 and indonesia. the reported vaccination efficacy values (ve) in the general population ranged from about 51% in a brazilian trial, 65% in indonesian trial, and 84% in a turkish trial.61 the following provides an example of reported adverse effects: incidence per 100,000: anaphylaxis (121); seizures (36), bell’s palsy (28), thromboembolic events (16), and guillain-barre syndrome (4).61 rare adverse events such as serious cutaneous and allergic reactions, nephrotic syndrome, optic neuritis and subacute thyroiditis, bell’s palsy, bronchial asthma was also reported.64-73 in brazil, good efficacy of this vaccine was observed in the elderly. after two doses of the vaccine, better protection ratios were obtained, with an attributable protection ratio of 99.2%.74 in the phase 2 study, a limited number of children (186) 3–17 years were enrolled, and sero conversion was observed in 97% in the low dose vaccine group (1.5 micrograms) and 100% in higher dose vaccine group (3 micrograms). no serious adverse effects were documented.75 however, up to our current knowledge, guidelines did not recommend its use in children.61 regarding immunocompromised patients, corona vac’s immunogenicity and safety were evaluated in a cohort study of 910 adult patients with autoimmune rheumatic disorders (ard) and 182 ageand sex-frequency matched healthy adults in a phase 4 prospective controlled experiments. at day 69, patients with ard had significantly lower antisars-cov-2 igg seroconversion (70.4 vs. 95.5%) and low rneutralizing antibodies (nab) (56.3 vs. 79.3%). these data suggesting lower efficacy of the vaccines in those population.76 another study showed similar findings and suggested that patients with immune-mediated illness and being 60 years or older are independently linked with lower antibody response.77 coronavac vaccine immunogenicity and safety were evaluated in 47 cancer patients receiving active systemic therapy. four weeks following the second dosage of the vaccine, more than half of the patient (63.8%) exhibited immunogenicity. the rate of seropositivity was 59.5% in those who received at least one cytotoxic medication, and 100% in those who received monoclonal antibodies and immunotherapy; however, immunogenicity was related to the patients’ age.75 sinopharm covid-19 vaccine (bbibp-corv) bbibp-corv, or bibp vaccine, is an inactivated virus covid-19 vaccine developed by sinopharm’s (beijing instidtute of biological products).78 a sample of the wt virus (hb02 strain) was cultivated in vero cells, isolated, and chemically inactivated by β-propiolactone, then mixed with an aluminum-based adjuvant. it completed phase iii trials in several countries with over 60,000 participants.79 an interim analysis of the phase 3 study (38,206 participants were randomized to receive the one of the following vaccines sars-cov-2 wiv04 (5 µg/dose; n = 13,459) (vaccine subtype 1); hb02 (4 µg/dose; n = 13,465) (vaccine subtype 2) or aluminum hydroxide (alum)–only (control) (n = 13,458). all participants received 2 intramuscular injections 21 days apart. the vaccine efficacy compared was 72.8% for the 1st vaccine and 78.1% for the 2nd subtype. serious adverse events were rare and similar in the 3 groups (wiv04: [0.5%]; hb02: [0.4%]; alum-only: [0.6%]).80 local reactions at the injection site, fatigue, and headache were the most prevalent post 1st dose adverse effects. pain at the immunization site, fatigue, lethargy, headache, and tenderness were the most common post 2nd dose side effects in both groups. two serious adverse events were possibly linked to the vaccine, serious nausea and a rare neurological disorder known as acute disseminated encephalomyelitis.28 one participant with a diagnosis of thrombus was identified in the phase 3 trial, in the bbibpcorv group. all acute allergic reactions were grade 1 and 2 in the bbibp-corv group.81 regarding children and adolescents, participants aged 3–17 years, the inactivated covid-19 vaccination bbibpcorv was shown to be safe and well-tolerated. after two doses, bbibp-corv elicited strong humoral responses against sars-cov-2 infection.78,82 the elderly group showed a similar safety profile compared to younger adults, but with lower reactogenicity in older adults. in clinical trials, no serious adverse effects occurred in adults ≥60 years in the vaccine group.81 sputnik v (gam-covid-vac) the gamaleya research institute of epidemiology and microbiology in russia produced sputnik v (gam-covid-vac), which is an adenovirus viral vector vaccine for covid-19. the russian ministry of health has registered it on august 11th 2020.83 the vaccine uses similar technology to the oxford astrazeneca vaccine; however, it follows a prime and then boosts regime. the two shots use different vectors, the first is rad26 and the second is rad5 while both carry the gene for the full sars-cov-2 spike protein.84 the findings of phase iii trials have shown that the twodose regime of the vaccine was generally well tolerated with no associated serious adverse events and similar efficacy in those aged over and under 60. it was tested at 25 hospitals and polyclinics in moscow. the clinical trial indicated an overall https://en.wikipedia.org/wiki/inactivated_vaccine https://en.wikipedia.org/wiki/covid-19_vaccine https://en.wikipedia.org/wiki/sinovac_biotech https://en.wikipedia.org/wiki/inactivated_vaccine https://en.wikipedia.org/wiki/covid-19_vaccine https://en.wikipedia.org/wiki/sinopharm 89j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 s. alattas et al. review safety and efficacy of covid-19 vaccines in special populations hospitalizations and fatalities was 87.6% and 84.8%, respectively.90 the gam-covid-vac vaccine was well tolerated, with no grade 3–5 side effects recorded in 112 patients with metastatic genitourinary cancer. two covid-19 cases (1.8%) were verified in the vaccination group after a median follow-up of 6.2 months.91 a non-comprehensive summary of characteristics, safety and potentially serious adverse effects of some authorized vaccines are presented in tables 1–2. in the following paragraphs, we will attempt to summarize the currently available (october 2021) guidelines, recommendations relevant to the efficacy and safety of vaccines against covid-19 in special populations. efficacy and safety in special population autoimmune diseases and allergy rituximab has been linked to a reduced serological response to the sars-cov-2 vaccine in rheumatoid arthritis patients.95 immune responses against the sars-cov-2 (biontech/ pfizer) one-shot was studied in patients with immune-mediated inflammatory diseases including spondyl arthritis, inflammatory bowel disease, psoriasis, and rheumatoid efficacy of 91.6%.84 a study among health workers in argentina observed that 94% of naïve individuals generate spike-specific igg antibodies 21 days after receiving the first dose of the vaccine.85 however, the early approval provoked criticism among scientists.86,87 the most reported adverse events in phase iii clinical trial were grade 1 [94%] (0.3% of participants in the vaccine group and (0.4%) of participants in the placebo group. four fatalities were recorded (less than 0.1%) in the vaccination group and one (less than 0.1%) in the placebo group; however, none of which were thought to be attributable to the vaccine.84 moreover, in iranian research, 3,236 vaccinated health workers self-reported the following side effects: discomfort at the injection site (50.9%), body pain (43.9%), headache (35.7%), fever (32.9%), joint pain (30.3%), chills (29.8%), and tiredness (20.3%).88 in a small study with 18 participating health workers, the researchers discovered that (83%) of sputnik v recipients’ infants (1–6 yr) suffered from fever and chills for 1–2 days after their parents’ immunization, which can be linked to an adenovirus infection.89 in argentina, retrospective cohort research was carried out where the first dose of gam-covid-vac was given to 40,387 elderly (60–79 yr). vaccine efficacy for avoiding laboratory-confirmed illnesses was 78.6%, and for preventing table 1. characteristics of examples of leading covid‐19 vaccines92,93 company biontech. gmbh modernatx, inc. janssen inc astrazeneca canada inc. code name (bnt162b2) mrna-1273 ad26.cov2.s [recombinant] chadox1-s [recombinant] type mrna-based mrna-based viral victor viral victor authorized age ≥5 years ≥12 years ≥18 years ≥18 years dosing schedule 2 doses, at least 21 days apart 2 doses, 4 weeks apart storage –90° to –60°c protected from light –25 to –15°c +2 to +8ºc +2 to +8°c vaccine efficacy (ve) 95 %, 7 days after the 2nd dose 94.1%, 2 weeks after the second dose 85.4%, 28 days after vaccination against severe/critical covid-19 62.1%, 2 weeks after the second dose elderly >65 yr similar to adults slightly lower, 86.4% consistent efficacy pregnancy preferred in view of current data adverse events of special interest (aesi) myocarditis/pericarditis, bell’s palsy and anaphylaxis guillain-barré syndrome (gbs), thrombosis with thrombocytopenia syndrome (tts) including vitt, capillary leak syndrome (cls), venous thromboembolism (vte), immune thrombocytopenia (itp) and anaphylaxis table 2. serious adverse events (sae) of some authorized vaccines94 sae incidence notes anaphylaxis after 2–5/million all vaccines thrombosis (tts)* 36 per 12 million doses j & j/janssen covid-19 vaccination women < 50 year 1 case/306 million doses mrna covid-19 vaccination myocarditis and pericarditis 393 confirmed cases (fda) most cases mrna covid-19 vaccination; male adolescents (18–30) report of deaths* 5,479 reported deaths (0.0017%), 318 million doses december 14, 2020, through june 21, 2021, vaers**, all vaccines *a plausible causal relationship between the vaccine and death was confirmed only for a limited number of cases received j & j/janssen covid-19 vaccine due to thrombosis with thrombocytopenia syndrome (tts). **vaers: vaccine adverse event reporting system. 90 j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 safety and efficacy of covid-19 vaccines in special populations review s. alattas et al. arthritis. those patients were maintained on pharmacotherapy that included methotrexate, glucocorticoids, tnf inhibitors, il-inhibitors, and jak inhibitors among others. overall responses were delayed and reduced in patients compared with controls.96 those individuals should be vaccinated against covid19, ideally while their disease activity is under control. antibody responses to vaccines do not appear to be affected by low-level immunosuppression. vaccinations should be administered before the start of any biological dmards. patients using rituximab should be vaccinated at least 4 weeks before or 6 months after treatment. because tofacitinib can lower antibody responses, especially when combined with methotrexate, it has been suggested that stopping it before vaccination and restarting it after 14 days can be a beneficial approach. vaccinations can be scheduled in a variety of ways to ensure effectiveness.97 practical guidelines for optimal safety of vaccination of patients with immunological disorders were suggested by j. peter.98 benign blood disorders individuals with a history of blood clots, or certain thrombophilic disease, may be at increased risk of a very rare immune-mediated thrombosis induced by some authorized vaccines.99 vaccines can be given safely for most patients maintained on anticoagulant medication, after applying certain precautions.for example, a fine needle gauge (23–25) is recommended for vaccination followed by an intense pressure applied to the injection site without scratching for at least two minutes.100 patients with other hematological disorders such as sickle cell disease, thalassemia, and rare hereditary anemia, should be vaccinated against covid-19, patients on warfarin with a supra therapeutic international normalized ratio (inr) should wait until their inr is below 4.101 cancer patients a study was conducted in one chronic lymphocytic leukemia (cll) patient (stage o untreated) to demonstrate the safety and efficacy of the bnt162b2 mrna vaccine. it documented the capability of two doses of the vaccine to generate humoral and cellular response against covid-19 but to a lower extent than that in matched age healthy volunteers.102 in patients with myelo proliferative neoplasms (n = 21), a single dose of pfizer-bnt162b2 mrna vaccine resulted in a high frequency of neutralizing antibodies and poly functional t-cell responses. also, the vaccine was safe and generally well-tolerated.103 in patients with cll, an observation study evaluated humoral immune responses to the bnt162b2 mrna covid-19 vaccination and compared them to responses in age-matched healthy control volunteers. patients received two vaccination doses, 21 days apart. the study enrolled 52 cll patients and a similar number of sexand age-matched healthy control participants. it demonstrated that cll patients had a significantly lower response rate (52%) compared to the control. patients who achieved clinical remission following treatment had the highest response rate (79.2%), followed by treatment-naive patients (55.2%) and patients who were receiving treatment at the time of vaccination (16.0%). response rates were low in patients treated with bruton’s tyrosine kinase inhibitors or veneto lax anti-cd20 antibody (16.0% and 13.6%, respectively). none of the patients who had been exposed to anti-cd20 antibodies (e.g. rituximab) 12 months before immunization had a positive antibody response.104 a possible explanation of these results, is that the immune system may be compromised by the persistent b-cell depletion caused by rituximab, which is maybe the mechanism of action of anti-cd20 antibodies in antibody-mediated autoimmune diseases.105 a group of researchers examined 44 consecutive patients with cll who received two doses of mrna vaccine (bnt162b2 or mrna-1273) and tested for anti-sars-cov-2 s1/s2 antibodies. the results have predicted that vaccination in patients with cll may not provide the efficacy seen in healthy individuals. patients receiving bruton tyrosine kinase inhibitor at the time of vaccination or who have received anti-cd20 monoclonal antibody within 1 year showed very poor vaccine efficacy in a prospective study.106 the immune response of the bnt162b2 mrna vaccine in multiple myeloma patients was significantly lower, particularly in those on anti-cd38-based treatment.107 a comprehensive review indicated that no safety concerns specific to patients with cancer receiving mrna vaccine was anticipated. certain chemotherapy can inhibit immune responses to the vaccine. in conclusion, the benefits of vaccination in most cancer patients are likely to outweigh the risks.108 the updated guideline infectious diseases working organization (agiho) of the german society for hematology and medical oncology (dgho) recommended the vaccination of patients with cancer against covid-19 especially those active disease.109 the australian and new zealand guidelines stated that patients with hematological malignancies and some benign hematological illnesses should have accelerated access to covid-19 vaccinations.110 french oncology societies (gco, tncd, unicancer) recommends that patients with cancer who are undergoing treatment or who had therapy less than three years ago should be vaccinated, as well as their families.111 cardiovascular disease the american heart association declared that authorized vaccines are safe for adults who have or have had cardiovascular disease and that their efficacy is similar to that observed in the general population. patients with heart problems were included in the covid-19 vaccine studies, and the vaccine had no major side effects in these patients. however, patients with severe heart disease and unstable angina may experience mild fever and flu-like symptoms due to the vaccine. however, these effects are not serious and respond well to symptomatic treatment. patients with heart disease may become critically ill in the presence of a severe allergic reaction. on the other hand, there are no reported interactions between the vaccine and medications prescribed for cardiac diseases. in conclusion, the advantages of vaccination in patients with cardiovascular diseases significantly outweigh the potential risk.112 similar recommendations were provided by a published report of the american college of cardiology.113 moreover, the american heart association confirms that patients with cardiovascular diseases have priority in receiving vaccines against the coronavirus.114 91j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 s. alattas et al. review safety and efficacy of covid-19 vaccines in special populations diabetic patients regarding immunogenicity, efficacy, and effectiveness of immunization in diabetic patients, the clinical data are limited. previous vaccinations showed variable results.115 a small study suggested that sars-cov-2 antibody response may be impaired in diabetic patients.116 however, other studies reported that humoral immune response against sars-cov-2 in diabetic patients was like non-diabetic patients.117,118 more than 3,000 individuals with diabetes were part of the clinical trial for the pfizer vaccine, and the moderna vaccine included 2,875 individuals with diabetes in its clinical trial. both trials observed the vaccine to be safe and effective.119,120 in summary, no specific safety concerns were reported regarding these authorized vaccines in diabetic patients in the usa.121 as with any vaccine, the coronavirus vaccine may cause elevated blood glucose levels.122 hepatic disease innate and humoral immune deficits are well-recognized characteristics in patients with severe liver disease, this phenomenon is called cirrhosis-associated immune dysfunction. for example, in individuals with cirrhosis, rates of sero conversion soon after hepatitis b virus vaccination, as well as the durability of humoral immunity after pneumococcal and influenza vaccination, are all significantly reduced. patients with cirrhosis have reduced immune responses to sars-cov-2 vaccination as well. regarding the safety of the vaccines, neither the oxford–astrazeneca chadox1-ncov-19 nor the pfizer–biontech covid-19 vaccine (bnt162b2) represents a particular safety concern for these patients.123 although autoimmune hepatitis could occur as a rare complication of covid-19 vaccines.124 regardless of limited data onthe safety and efficacy of covid19 vaccines in patients with liver diseases, guidelines of several organizations have recommended that patients with chronic liver disease and liver transplant recipients can be encouraged to get vaccinated.125-128 solid-organ transplant (sot) recipients theoretical concern exists regarding the safety of the covid-19 vaccine in sot patients, which include the potential of enhanced inflammatory response due to mrna based vaccines,129 or activation of the adenovirus vector in immunocompromised patients.130 however, currently approved covid-19 vaccines that are based on replication-deficient adenovirus vectors or mrna are not contraindicated in sot patients. their advantages likely outweigh the expected risks provided that the standard precautions are taken.130-133 furthermore, guidelines for optimal use of covid-19 vaccines in sot recipients are available.134,135 the effectiveness of covid-19 vaccines is likely to be reduced in solid organ transplant patients.136-139 it has been shown that kidney transplant recipients show limited early antibody response after the first dose of the covid-19 vaccine.136 few studies were conducted to evaluate the short-term safety and efficacy of covid-19 vaccines in sot patients.140 a study reported that only 17% of transplant recipients who received a single dose of sars-cov-2 vaccination generated detectable anti-spike antibodies compared to 100% of healthy participants, but after two doses, the response was about 54%.16,141 a small retrospective study reported the failure of covid-19 vaccination in transplant patients to effectively protect them from being infected and suffering serious complications.142 in another study that included 658 sot recipients who received two doses of the pfizer–biontech covid-19 vaccine (bnt162b2) covid-19 vaccine, 46% had no detectable antibodies against the virus proteins after 29 days following the second dose of the vaccine.143 use of antimetabolites (e.g., mycophenolate mofetil, and azathioprine) and a shorter time since transplantation was associated with a higher rate of nonresponse. in reports of transplant recipients who received the third dose of mrna vaccines, seroconversion rates were higher after the additional dose, although approximately 30 to 50% remained seronegative.144,145 children & adolescents bntb162b (pfizer covid-19 vaccine) is authorized for children 5 years or older (ref ) andadolescents aged 12 through 15 years based on evidence that efficacy, immunogenicity, and the adverse effect profile in this population are comparable to those in older individuals.146 elderly there is theoretical concern regarding decreased immunogenicity with advanced age; however, vaccination strategies such as adjuvants, and vaccines that specifically target the aged immune system were suggested to enhance the efficacy of vaccines in the elderly.147 although further research is needed, preliminary data of phase 1 dose-escalation trials of the moderna, mrna-1273 vaccination found that effective antibody responses were equivalent in three age groups (18–55, 56–70, and 71 and older). the measured antibody titers suggested that mrna vaccinations in the elderly have adequate efficacy.148,149 astrazeneca’s chadox1 ncov-19 vaccine (azd1222) also showed promising efficacy in older adults.150 few publications addressed special safety concerns; for example, a study by norwegian authorities declared that the pfizer-biontech vaccine was possibly responsible for 10 deaths among 30,000 elders.151 in preliminary research, 55 deaths were observed, with a covid-19 vaccine fatality rate of 8.2 per million population. a total of 37 fatalities were reported among residents of longterm care facilities, with a mortality rate of 53.4 per million. the authors of the mentioned study concluded that the advantages of covid-19 vaccines outweigh the dangers in elder populations, and their data do not justify policies that prevent older individuals from receiving vaccinations.152 obesity obesity can be defined as a body mass index (bmi) of ≥ 30 kg/m2. pfizer-biontech vaccine efficacy (after the second dose) was about 95% in obese and normal individuals.153 individuals with severe obesity (bmi ≥ 40 kg/m2) were included in moderna studies where overall efficacy (after the second dose) was 94.1%, and 91.2% in those with severe obesity.22,154 a post hoc analysis similarly demonstrated comparable efficacy in obese participants with no high‐risk comorbidity.154 https://www.uptodate.com/contents/mycophenolate-mofetil-cellcept-and-mycophenolate-sodium-myfortic-drug-information?topicref=129849&source=see_link https://www.uptodate.com/contents/azathioprine-drug-information?topicref=129849&source=see_link 92 j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 safety and efficacy of covid-19 vaccines in special populations review s. alattas et al. pregnant and lactating women data on the safety of covid-19 vaccines in pregnant individuals are limited but emerging.155,156 a study revealed that covid-19 vaccine-specific titers of antibodies after receiving the vaccines were comparable, albeit slightly lower, between pregnant and lactating women compared to non-pregnant control.157 a multicenter study showed that the antenatal pfizer biontech mrna vaccine induced a robust maternal humoral response that effectively transfers to the fetus, supporting the role of vaccination during pregnancy.158 vaccinated pregnant and breastfeeding women had immune responses comparable to non-pregnant controls, and greater antibody titers were observed than those seen after sars-cov-2 infection during pregnancy according to prospective cohort research from two academic institutes. moreover, after maternal vaccination, vaccine-generated antibodies were found in umbilical cord blood and breast milk.159 currently approved vaccines in the usa are unlikely to pose specific risks for pregnant women, the fetus, or breastfeeding newborns.160 up until june 2021, preclinical and observational clinical studies suggest that the risks of the maternal covid-19 outweigh the undocumented and hypothetical risks of the covid-19 vaccines given during pregnancy.161,162 as of the end of may 2021, in the cdc’s v-safe post-vaccination health system, about 120,000 pregnancies were reported. although no specific safety signals have been found, vaccine safety is still being monitored.163 furthermore, unproven accusations that covid-19 vaccinations cause infertility, have been discredited scientifically.164-166 conclusions and recommendations the immunogenicity of covid-19 vaccines appears to be lower in some immunocompromised individuals compared with the general population, and vaccine efficacy is uncertain. these conditions include active use of chemotherapy for cancer, hematologic malignancies, hematopoietic stem cell or solid organ transplant, untreated hiv infection with cd4 cell count <200 cells/µl, and use of immunosuppressive medications (e.g., mycophenolate mofetil; rituximab; and prednisone >20 mg/day for >14 days).104,143,146,167-170 any concern related to vaccination of specific immunocompromised populations is discussed in detail elsewhere.146,167 one case of pancreatic rejection was associated with vaccines raising concern of the safety of risk of vaccines in organ transplant patients. as for other populations such as cardiac patients, pulmonary diseases, diabetes, the elderly, pregnant women, etc., it is expected that the effectiveness of vaccines is similar to that in the general population. also, these conditions do not constitute an absolute contraindication to receiving vaccinations. it is encouraged that these patients discuss their health status and receive advice from the doctors who follow their cases before receiving vaccinations. references 1. roser, m., et al., coronavirus pandemic (covid-19), in our world in data. 2021, our world in data. 2. belete, t.m., review on up-to-date status of candidate vaccines for covid-19 disease. infection and drug resistance, 2021. 14: p. 151. 3. yong, c.y., et al., recent advances in the vaccine development against middle east respiratory syndrome-coronavirus. frontiers in microbiology, 2019. 10: p. 1781. 4. krammer, f., sars-cov-2 vaccines in development. nature, 2020. 586(7830): p. 516-527. 5. goodman, j.l., j.d. grabenstein, and m.m. braun, answering key questions about covid-19 vaccines. jama, 2020. 324(20): p. 2027-2028. 6. francis, a.i., et al., review of covid-19 vaccine subtypes, efficacy and geographical distributions. postgraduate medical journal, 2021. 7. perry, c., et al., efficacy of the bnt162b2 mrna covid-19 vaccine in patients with b-cell non-hodgkin lymphoma. blood advances, 2021. 5(16): p. 3053-3061. 8. mcdonald, i., et al., comparative systematic review and meta-analysis of reactogenicity, immunogenicity and efficacy of vaccines against sarscov-2. npj vaccines, 2021. 6(1): p. 1-14. 9. lv, m., et al., safety, immunogenicity, and efficacy of covid-19 vaccines in children and adolescents: a systematic review. vaccines, 2021. 9(10): p. 1102. 10. frenck jr, r.w., et al., safety, immunogenicity, and efficacy of the bnt162b2 covid-19 vaccine in adolescents. new england journal of medicine, 2021. 11. das, b.b., et al., myopericarditis after messenger rna coronavirus disease 2019 vaccination in adolescents 12 to 18 years of age. the journal of pediatrics, 2021. 238: p. 26-32. e1. 12. schauer, j., et al., myopericarditis after the pfizer messenger ribonucleic acid coronavirus disease vaccine in adolescents. the journal of pediatrics, 2021. 238: p. 317-320. 13. mahase, e., covid-19: pfizer and biontech submit vaccine for us authorisation. 2020, british medical journal publishing group. 14. walsh, e.e., et al., safety and immunogenicity of two rna-based covid-19 vaccine candidates. new england journal of medicine, 2020. 383(25): p. 2439-2450. 15. polack, f.p., et al., safety and efficacy of the bnt162b2 mrna covid-19 vaccine. new england journal of medicine, 2020. 16. boyarsky, b.j., et al., immunogenicity of a single dose of sars-cov-2 messenger rna vaccine in solid organ transplant recipients. jama, 2021. 325(17): p. 1784-1786. 17. gray, k.j., et al., coronavirus disease 2019 vaccine response in pregnant and lactating women: a cohort study. american journal of obstetrics and gynecology, 2021. 18. dagan, n., et al., effectiveness of the bnt162b2 mrna covid-19 vaccine in pregnancy. nature medicine, 2021. 27(10): p. 1693-1695. 19. teo, s.p., review of covid-19 mrna vaccines: bnt162b2 and mrna-1273. journal of pharmacy practice, 2021: p. 08971900211009650. 20. heaton, p.m., the covid-19 vaccine-development multiverse. 2020, mass medical soc. p. 1986-1988. 21. kaur, s.p. and v. gupta, covid-19 vaccine: a comprehensive status report. virus research, 2020: p. 198114. 22. baden, l.r., et al., efficacy and safety of the mrna-1273 sars-cov-2 vaccine. new england journal of medicine, 2021. 384(5): p. 403-416. 23. bruxvoort, k.j., et al., effectiveness of mrna-1273 against delta, mu, and other emerging variants of sars-cov-2: test negative case-control study. bmj, 2021. 375. 24. oosting, s.f., et al., mrna-1273 covid-19 vaccination in patients receiving chemotherapy, immunotherapy, or chemoimmunotherapy for solid tumours: a prospective, multicentre, non-inferiority trial. the lancet oncology, 2021. 22(12): p. 1681-1691. 25. john, b.v., et al., association of bnt162b2 mrna and mrna-1273 vaccines with covid-19 infection and hospitalization among patients with cirrhosis. jama internal medicine, 2021. 181(10): p. 1306-1314. 26. lacson, e., et al., immunogenicity of sars-cov-2 vaccine in dialysis. medrxiv, 2021. 27. shimabukuro, t.t., et al., preliminary findings of mrna covid-19 vaccine safety in pregnant persons. new england journal of medicine, 2021. 384(24): p. 2273-2282. 28. mahase, e., how the oxford-astrazeneca covid-19 vaccine was made. bmj, 2021. 372. 29. folegatti, p.m., et al., safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, singleblind, randomised controlled trial. the lancet, 2020. 396(10249): p. 467-478. conflicts of interest there are no conflicts of interest.  https://www.uptodate.com/contents/mycophenolate-mofetil-cellcept-and-mycophenolate-sodium-myfortic-drug-information?topicref=129849&source=see_link https://www.uptodate.com/contents/rituximab-intravenous-including-biosimilars-of-rituximab-drug-information?topicref=129849&source=see_link https://www.uptodate.com/contents/prednisone-drug-information?topicref=129849&source=see_link 93j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 s. alattas et al. review safety and efficacy of covid-19 vaccines in special populations 55. robert carlson and h. lutmer. covaxin covid-19 vaccine. 2021 [cited 2021 13 december]; available from: https://www.precisionvaccinations.com/ vaccines/covaxin-covid-19-vaccine. 56. ella, r., et al., efficacy, safety, and lot-to-lot immunogenicity of an inactivated sars-cov-2 vaccine (bbv152): interim results of a randomised, double-blind, controlled, phase 3 trial. the lancet, 2021. 57. covid-19 vaccines, in drugs and lactation database (lactmed). 2006, national library of medicine (us): bethesda (md). 58. gao, q., et al., development of an inactivated vaccine candidate for sarscov-2. science, 2020. 369(6499): p. 77-81. 59. rego, g.n., et al., current clinical trials protocols and the global effort for immunization against sars-cov-2. vaccines, 2020. 8(3): p. 474. 60. asyura, m.m.a.z., et al., immunogenicity and safety analysis of inactivated virus vaccine against sars-cov-2: a systematic review of phase 1/2 clinical trials. journal of asian medical students’ association, 2021. 9(1). 61. lapitan, m.c., j.c. valencia, and m.a. castor, is coronavac (sinovac) effective and safe in the prevention of covid-19-infections?: a rapid review (update). 62. palacios, r., et al., efficacy and safety of a covid-19 inactivated vaccine in healthcare professionals in brazil: the profiscov study. 2021. 63. tanriover, m.d., et al., efficacy and safety of an inactivated whole-virion sars-cov-2 vaccine (coronavac): interim results of a double-blind, randomised, placebo-controlled, phase 3 trial in turkey. the lancet, 2021. 398(10296): p. 213-222. 64. benjamanukul, s., et al., safety and immunogenicity of inactivated covid-19 vaccine in health care workers. j med virol, 2021. 65. bueno, s.m., et al., safety and immunogenicity of an inactivated sars-cov-2 vaccine in a subgroup of healthy adults in chile. clin infect dis, 2021. 66. fernandes, e.g., et al., safety and immunogenicity of an inactivated sarscov-2 vaccine (coronavac) in inadvertently vaccinated healthy children. rev inst med trop sao paulo, 2021. 63: p. e83. 67. han, b., et al., safety, tolerability, and immunogenicity of an inactivated sars-cov-2 vaccine (coronavac) in healthy children and adolescents: a double-blind, randomised, controlled, phase 1/2 clinical trial. lancet infect dis, 2021. 21(12): p. 1645-1653. 68. medeiros-ribeiro, a.c., et al., immunogenicity and safety of the coronavac inactivated vaccine in patients with autoimmune rheumatic diseases: a phase 4 trial. nat med, 2021. 27(10): p. 1744-1751. 69. tosun, s., et al., adverse events report of inactivated covid-19 vaccine from 4040 healthcare workers. postgrad med, 2021: p. 1-7. 70. uzer, f. and a. cilli, acute asthma exacerbation after sars-cov-2 vaccine (sinovac®): a case report. med gas res, 2022. 12(2): p. 67-68. 71. wan, e.y.f., et al., bell’s palsy following vaccination with mrna (bnt162b2) and inactivated (coronavac) sars-cov-2 vaccines: a case series and nested case-control study. lancet infect dis, 2021. 72. wu, z., et al., safety, tolerability, and immunogenicity of an inactivated sars-cov-2 vaccine (coronavac) in healthy adults aged 60 years and older: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial. lancet infect dis, 2021. 21(6): p. 803-812. 73. zhao, h., y. li, and z. wang, adverse event of sinovac coronavirus vaccine: deafness. vaccine, 2021. 74. alencar, c.h., et al., high effectiveness of sars-cov-2 vaccines in reducing covid-19-related deaths in over 75-year-olds, ceara state, brazil. tropical medicine and infectious disease, 2021. 6(3): p. 129. 75. karacin, c., et al., immunogenicity and safety of the coronavac vaccine in patients with cancer receiving active systemic therapy. future oncology, 2021. 17(33): p. 4447-4456. 76. medeiros-ribeiro, a.c., et al., immunogenicity and safety of the coronavac inactivated vaccine in patients with autoimmune rheumatic diseases: a phase 4 trial. nature medicine, 2021. 27(10): p. 1744-1751. 77. seyahi, e., et al., antibody response to inactivated covid-19 vaccine (coronavac) in immune-mediated diseases: a controlled study among hospital workers and elderly. rheumatology international, 2021: p. 1-12. 78. robert carlson and h. lutmer. sinopharm covid-19 vaccine (bbibpcorv). 2021 [cited 2021 15 december]; available from: https://www. precisionvaccinations.com/vaccines/sinopharm-covid-19-vaccine-bbibp-corv. 79. wang, h., et al., development of an inactivated vaccine candidate, bbibpcorv, with potent protection against sars-cov-2. cell, 2020. 182(3): p. 713-721. e9. 80. al kaabi, n., et al., effect of 2 inactivated sars-cov-2 vaccines on symptomatic covid-19 infection in adults: a randomized clinical trial. jama, 2021. 81. who. evidence assessment:sinopharm/bbibp covid-19 vaccine. 2021 [cited 2021 15 december]; available from: https://cdn.who.int/media/docs/ default-source/immunization/sage/2021/april/2_sage29apr2021_criticalevidence_sinopharm.pdf. 30. hung, i.f. and g.a. poland, single-dose oxford–astrazeneca covid-19 vaccine followed by a 12-week booster. the lancet, 2021. 397(10277): p. 854-855. 31. sheikh, a., et al., sars-cov-2 delta voc in scotland: demographics, risk of hospital admission, and vaccine effectiveness. the lancet, 2021. 32. voysey, m., et al., safety and efficacy of the chadox1 ncov-19 vaccine (azd1222) against sars-cov-2: an interim analysis of four randomised controlled trials in brazil, south africa, and the uk. the lancet, 2021. 397(10269): p. 99-111. 33. iheanacho, c.o., u.i. eze, and e.a. adida, a systematic review of effectiveness of bnt162b2 mrna and chadox1 adenoviral vector covid-19 vaccines in the general population. bulletin of the national research centre, 2021. 45(1): p. 1-10. 34. hernández, a.f., et al., safety of covid-19 vaccines administered in the eu: should we be concerned? toxicology reports, 2021. 8: p. 871-879. 35. ramasamy, m.n., et al., safety and immunogenicity of chadox1 ncov-19 vaccine administered in a prime-boost regimen in young and old adults (cov002): a single-blind, randomised, controlled, phase 2/3 trial. the lancet, 2020. 396(10267): p. 1979-1993. 36. see, i., et al., us case reports of cerebral venous sinus thrombosis with thrombocytopenia after ad26. cov2. s vaccination, march 2 to april 21, 2021. jama, 2021. 37. bhuyan, p., et al., very rare thrombosis with thrombocytopenia after second azd1222 dose: a global safety database analysis. the lancet, 2021. 398(10300): p. 577-578. 38. sharifian-dorche, m., et al., vaccine-induced immune thrombotic thrombocytopenia and cerebral venous sinus thrombosis post covid-19 vaccination; a systematic review. journal of the neurological sciences, 2021. 428: p. 117607. 39. shan, y., et al., multisystem inflammatory syndrome in an adult after covid-19. infectious diseases in clinical practice, 2020. 28(6): p. e28-e29. 40. marammatom, b.v., et al., guillain‐barré syndrome following chadox1‐s/ ncov‐19 vaccine. annals of neurology, 2021. 41. maurya, m.r., r. ravi, and l. pushparajan, serious adverse events following immunization after chadox1 ncov-19 vaccination in india: a single center experience. the pan african medical journal, 2021. 40. 42. macintyre, c.r., et al., thrombosis with thrombocytopenia syndrome (tts) following astrazeneca chadox1 ncov-19 (azd1222) covid-19 vaccination–a risk–benefit analysis for people< 60 years in australia. vaccine, 2021. 39(34): p. 4784-4787. 43. frater, j., et al., safety and immunogenicity of the chadox1 ncov-19 (azd1222) vaccine against sars-cov-2 in hiv infection: a single-arm substudy of a phase 2/3 clinical trial. the lancet hiv, 2021. 44. asano, m., et al., immunogenicity and safety of azd1222 (chadox1 ncov19) against sars-cov-2 in japan: a double-blind, randomized controlled phase 1/2 trial. international journal of infectious diseases, 2022. 114: p. 165-174. 45. masset, c., et al., pancreas allograft rejection occurring after chadox1 ncov19 vaccine. diabetes & metabolism, 2021. 46. edwards, a.e., et al., acute hyperglycaemic crisis after vaccination against covid‐19: a case series. diabetic medicine, 2021. 47. abbink, p., et al., comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d. journal of virology, 2007. 81(9): p. 4654-4663. 48. oliver, s.e., et al., the advisory committee on immunization practices’ interim recommendation for use of janssen covid-19 vaccine—united states, february 2021. morbidity and mortality weekly report, 2021. 70(9): p. 329. 49. bos, r., et al., ad26 vector-based covid-19 vaccine encoding a prefusionstabilized sars-cov-2 spike immunogen induces potent humoral and cellular immune responses. npj vaccines, 2020. 5(1): p. 1-11. 50. sadoff, j., et al., interim results of a phase 1–2a trial of ad26. cov2. s covid-19 vaccine. new england journal of medicine, 2021. 384(19): p. 18241835. 51. bos, r., et al., ad26 vector-based covid-19 vaccine encoding a prefusionstabilized sars-cov-2 spike immunogen induces potent humoral and cellular immune responses. npj vaccines. 2020 sep 28; 5: 91. doi: 10.1038/ s41541-020-00243-x. pmid: 33083026; pmcid: pmc7522255. 52. sadoff, j., et al., safety and efficacy of single-dose ad26. cov2. s vaccine against covid-19. new england journal of medicine, 2021. 384(23): p. 21872201. 53. alter, g., et al., immunogenicity of ad26. cov2. s vaccine against sarscov-2 variants in humans. nature, 2021. 596(7871): p. 268-272. 54. thiagarajan, k., what do we know about india’s covaxin vaccine? bmj: british medical journal (online), 2021. 373. https://www.precisionvaccinations.com/vaccines/covaxin-covid-19-vaccine https://www.precisionvaccinations.com/vaccines/covaxin-covid-19-vaccine https://www.precisionvaccinations.com/vaccines/sinopharm-covid-19-vaccine-bbibp-corv https://www.precisionvaccinations.com/vaccines/sinopharm-covid-19-vaccine-bbibp-corv https://cdn.who.int/media/docs/default-source/immunization/sage/2021/april/2_sage29apr2021_critical-evidence_sinopharm.pdf https://cdn.who.int/media/docs/default-source/immunization/sage/2021/april/2_sage29apr2021_critical-evidence_sinopharm.pdf https://cdn.who.int/media/docs/default-source/immunization/sage/2021/april/2_sage29apr2021_critical-evidence_sinopharm.pdf 94 j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 safety and efficacy of covid-19 vaccines in special populations review s. alattas et al. 82. ariamanesh, m., et al., immunogenicity and safety of the inactivated sars-cov-2 vaccine (bbibp-corv) in patients with malignancy. cancer investigation, 2021: p. 1-9. 83. burki, t.k., the russian vaccine for covid-19. the lancet respiratory medicine, 2020. 8(11): p. e85-e86. 84. logunov, d.y., et al., safety and efficacy of an rad26 and rad5 vectorbased heterologous prime-boost covid-19 vaccine: an interim analysis of a randomised controlled phase 3 trial in russia. the lancet, 2021. 397(10275): p. 671-681. 85. rossi, a.h., et al., sputnik v vaccine elicits seroconversion and neutralizing capacity to sars-cov-2 after a single dose. cell reports medicine, 2021. 2(8): p. 100359. 86. baraniuk, c., covid-19: what do we know about sputnik v and other russian vaccines? bmj, 2021. 372. 87. bucci, e.m., et al., data discrepancies and substandard reporting of interim data of sputnik v phase 3 trial. the lancet, 2021. 397(10288): p. 1881-1883. 88. babamahmoodi, f., et al., side effects and immunogenicity following administration of the sputnik v covid-19 vaccine in health care workers in iran. scientific reports, 2021. 11(1): p. 1-8. 89. mehraeen, e., s. seyedalinaghi, and a. karimi, can children of the sputnik v vaccine recipients become symptomatic? human vaccines & immunotherapeutics, 2021. 17(10): p. 3500-3501. 90. gonzález, s., et al., effectiveness of the first component of gam-covidvac (sputnik v) on reduction of sars-cov-2 confirmed infections, hospitalisations and mortality in patients aged 60-79: a retrospective cohort study in argentina. eclinicalmedicine, 2021. 40: p. 101126. 91. tsimafeyeu, i., et al., safety and preliminary efficacy of the gam-covid-vac vaccine and outcomes of sars-cov-2 infection in russian patients with genitourinary malignancies. journal of hematology & oncology, 2021. 14(1): p. 1-7. 92. biospace. updated comparing covid-19. 2021 [cited 2021 15 december]; available from: https://www.biospace.com/article/ comparing-covid-19-vaccines-pfizer-biontech-moderna-astrazenecaoxford-j-and-j-russia-s-sputnik-v/. 93. gov.canda. covid-19 vaccines and treatments portal. 2022 [cited 2022 10 februray]; available from: covid-19 vaccines and treatments portal. 94. cdc. selected adverse events reported after covid-19 vaccination. 2021 updated june 23, 2021 [cited 2021 26 june]; available from: https://www. cdc.gov/coronavirus/2019-ncov/vaccines/safety/adverse-events.html. 95. spiera, r., s. jinich, and d. jannat-khah, rituximab, but not other antirheumatic therapies, is associated with impaired serological response to sars-cov-2 vaccination in patients with rheumatic diseases. annals of the rheumatic diseases, 2021. 96. simon, d., et al., sars-cov-2 vaccination responses in untreated, conventionally treated and anticytokine-treated patients with immunemediated inflammatory diseases. annals of the rheumatic diseases, 2021. 97. soy, m., et al., a practical approach for vaccinations including covid-19 in autoimmune/autoinflammatory rheumatic diseases: a non-systematic review. clinical rheumatology, 2021: p. 1-13. 98. peter, j., covid-19 vaccination: recommendations for management of patients with allergy or immune-based diseases. south african medical journal, 2021. 111(4): p. 291-294. 99. erskine, d. using covid-19 vaccines in patients with anticoagulation and bleeding disorders. 2021 [cited 2021 10 june ]; available from: https:// www.sps.nhs.uk/articles/using-covid-19-vaccines-in-patients-withanticoagulation-and-bleeding-disorders/. 100. velikov, t., g. keremidchiev, and t. velikova, how to use safely covid-19 vaccines in patients on anticoagulants or antiaggregants. international journal of preventive cardiology, 2021. 1(1): p. 32-33. 101. the lancet, h., covid-19 vaccination in haematology services. lancet haematol, 2021. 8(2): p. e95. 102. agrati, c., et al., immunogenicity and safety of bnt162b2 covid-19 vaccine in a chronic lymphocytic leukaemia patient. journal of cellular and molecular medicine, 2021: p. 10.1111/jcmm.16565. 103. harrington, p., et al., single dose of bnt162b2 mrna vaccine against sarscov-2 induces high frequency of neutralising antibody and polyfunctional t-cell responses in patients with myeloproliferative neoplasms. leukemia, 2021: p. 1-5. 104. herishanu, y., et al., efficacy of the bnt162b2 mrna covid-19 vaccine in patients with chronic lymphocytic leukemia. blood, the journal of the american society of hematology, 2021. 137(23): p. 3165-3173. 105. houot, r., et al., could anti-cd20 therapy jeopardise the efficacy of a sarscov-2 vaccine? european journal of cancer, 2020. 136: p. 4-6. 106. roeker, l.e., et al., covid-19 vaccine efficacy in patients with chronic lymphocytic leukemia. leukemia, 2021: p. 1-3. 107. pimpinelli, f., et al., fifth-week immunogenicity and safety of antisars-cov-2 bnt162b2 vaccine in patients with multiple myeloma and myeloproliferative malignancies on active treatment: preliminary data from a single institution. journal of hematology & oncology, 2021. 14(1): p. 1-12. 108. hwang, j.k., et al., covid-19 vaccines for patients with cancer: benefits likely outweigh risks. journal of hematology & oncology, 2021. 14(1): p. 1-11. 109. giesen, n., et al., 2021 update of the agiho guideline on evidence-based management of covid-19 in patients with cancer regarding diagnostics, viral shedding, vaccination and therapy. european journal of cancer, 2021. 147: p. 154-160. 110. mccaughan, g., et al., covid‐19 vaccination in haematology patients: an australian and new zealand consensus position statement. internal medicine journal, 2021. 51(5): p. 763-768. 111. tougeron, d., et al., severe acute respiratory syndrome coronavirus 2 vaccination for patients with solid cancer: review and point of view of a french oncology intergroup (gco, tncd, unicancer). european journal of cancer, 2021. 150: p. 232-239. 112. esc. covid-19 vaccine information for heart patients. 2021 [cited 2021 2 july ]; available from: https://www.escardio.org/education/covid-19-andcardiology/covid-19-and-vaccinations. 113. driggin, e., et al., acc health policy statement on cardiovascular disease considerations for covid-19 vaccine prioritization: a report of the american college of cardiology solution set oversight committee. journal of the american college of cardiology, 2021. 77(15): p. 1938-1948. 114. aha. covid-19 vaccine is high priority for cardiac patients. 2021 [cited 2021 2 july ]; available from: https://www.heart.org/en/beyond-thetable/stories/covid-19-vaccine-is-high-priority-for-cardiac-patients. 115. verstraeten, t., et al., diabetes mellitus as a vaccine-effect modifier: a review. expert review of vaccines, 2020. 19(5): p. 445-453. 116. pal, r., et al., impaired anti-sars-cov-2 antibody response in non-severe covid-19 patients with diabetes mellitus: a preliminary report. diabetes & metabolic syndrome: clinical research & reviews, 2021. 15(1): p. 193-196. 117. dispinseri, s., et al., robust neutralizing antibodies to sars-cov-2 develop and persist in subjects with diabetes and covid-19 pneumonia. the journal of clinical endocrinology & metabolism, 2021. 106(5): p. 14721481. 118. lampasona, v., et al., antibody response to multiple antigens of sars-cov-2 in patients with diabetes: an observational cohort study. diabetologia, 2020. 63(12): p. 2548-2558. 119. food and d. administration, emergency use authorization (eua) of the pfizer-biontech covid-19 vaccine to prevent coronavirus disease 2019 (covid-19). food and drug administration, silver spring, ms, usa, 2019. 120. mahase, e., covid-19: moderna vaccine is nearly 95% effective, trial involving high risk and elderly people shows. bmj: british medical journal (online), 2020. 371. 121. gee, j., first month of covid-19 vaccine safety monitoring—united states, december 14, 2020–january 13, 2021. mmwr. morbidity and mortality weekly report, 2021. 70. 122. (idf), i.d.f. diabetes & coronavirus vaccination. 2021 [cited 2021 10 june]; available from: https://idf.org/our-network/regions-members/europe/ europe-news/370-diabetes-coronavirus-vaccination.html. 123. marjot, t., et al., sars-cov-2 vaccination in patients with liver disease: responding to the next big question. lancet gastroenterol hepatol, 2021. 6(3): p. 156-158. 124. bril, f., et al., autoimmune hepatitis developing after coronavirus disease 2019 (covid-19) vaccine: causality or casualty? j hepatol, 2021. 125. aasld. covid-19 and the liver. 2021 [cited 2021 12 june ]; available from: https://www.aasld.org/about-aasld/covid-19-and-liver. 126. fix, o.k., et al., aasld expert panel consensus statement: vaccines to prevent covid-19 infection in patients with liver disease. hepatology. n/a(n/a). 127. alqahtani, s.a., et al., use of covid-19 vaccines in patients with liver disease and post-liver transplantation: position statement of the saudi association for the study of liver diseases and transplantation. 2021. 128. russo, f.p., et al., italian association for the study of the liver position statement on sars-cov2 vaccination. dig liver dis, 2021. 53(6): p. 677-681. 129. teijaro, j.r. and d.l. farber, covid-19 vaccines: modes of immune activation and future challenges. nature reviews immunology, 2021: p. 1-3. 130. heldman, m.r. and a.p. limaye, sars-cov-2 vaccines in kidney transplant recipients: will they be safe and effective and how will we know? journal of the american society of nephrology, 2021. 32(5): p. 1021-1024. https://www.biospace.com/article/comparing-covid-19-vaccines-pfizer-biontech-moderna-astrazeneca-oxford-j-and-j-russia-s-sputnik-v/ https://www.biospace.com/article/comparing-covid-19-vaccines-pfizer-biontech-moderna-astrazeneca-oxford-j-and-j-russia-s-sputnik-v/ https://www.biospace.com/article/comparing-covid-19-vaccines-pfizer-biontech-moderna-astrazeneca-oxford-j-and-j-russia-s-sputnik-v/ https://www.cdc.gov/coronavirus/2019-ncov/vaccines/safety/adverse-events.html https://www.cdc.gov/coronavirus/2019-ncov/vaccines/safety/adverse-events.html https://www.sps.nhs.uk/articles/using-covid-19-vaccines-in-patients-with-anticoagulation-and-bleeding-disorders/ https://www.sps.nhs.uk/articles/using-covid-19-vaccines-in-patients-with-anticoagulation-and-bleeding-disorders/ https://www.sps.nhs.uk/articles/using-covid-19-vaccines-in-patients-with-anticoagulation-and-bleeding-disorders/ https://www.escardio.org/education/covid-19-and-cardiology/covid-19-and-vaccinations https://www.escardio.org/education/covid-19-and-cardiology/covid-19-and-vaccinations https://www.heart.org/en/beyond-the-table/stories/covid-19-vaccine-is-high-priority-for-cardiac-patients https://www.heart.org/en/beyond-the-table/stories/covid-19-vaccine-is-high-priority-for-cardiac-patients https://idf.org/our-network/regions-members/europe/europe-news/370-diabetes-coronavirus-vaccination.html https://idf.org/our-network/regions-members/europe/europe-news/370-diabetes-coronavirus-vaccination.html https://www.aasld.org/about-aasld/covid-19-and-liver 95j contemp med sci | vol. 8, no. 2, march-april 2022: 86–95 s. alattas et al. review safety and efficacy of covid-19 vaccines in special populations 131. pormohammad, a., et al., efficacy and safety of covid-19 vaccines: a systematic review and meta-analysis of randomized clinical trials. vaccines (basel), 2021. 9(5). 132. xing, k., et al., efficacy and safety of covid-19 vaccines: a systematic review. zhongguo dang dai er ke za zhi = chinese journal of contemporary pediatrics, 2021. 23(3): p. 221-228. 133. yuan, p., et al., safety, tolerability, and immunogenicity of covid-19 vaccines: a systematic review and meta-analysis. medrxiv, 2020. 134. kute, v., et al., notto covid-19 vaccine guidelines for transplant recipients. indian journal of transplantation, 2021. 15(1): p. 1. 135. memberships, m. and t. join, tid covid-19 guidance focused review: sars-cov-2 vaccines in transplant recipients. 136. yi, s.g., et al., kidney transplant recipients rarely show an early antibody response following the first covid-19 vaccine administration. transplantation, 2021. 105(7): p. e72-e73. 137. grupper, a., et al., reduced humoral response to mrna sars-cov-2 bnt162b2 vaccine in kidney transplant recipients without prior exposure to the virus. american journal of transplantation, 2021. 21(8): p. 2719-2726. 138. benotmane, i., et al., weak anti-sars-cov-2 antibody response after the first injection of an mrna covid-19 vaccine in kidney transplant recipients. kidney int, 2021. 99(6): p. 1487-1489. 139. benotmane, i., et al., low immunization rates among kidney transplant recipients who received 2 doses of the mrna-1273 sars-cov-2 vaccine. kidney int, 2021. 99(6): p. 1498-1500. 140. nacif, l.s., et al., covid-19 in solid organ transplantation patients: a systematic review. clinics (sao paulo), 2020. 75: p. e1983. 141. boyarsky, b.j., et al., antibody response to 2-dose sars-cov-2 mrna vaccine series in solid organ transplant recipients. jama, 2021. 142. ali, n.m., et al., development of covid-19 infection in transplant recipients after sars-cov-2 vaccination. transplantation, 2021. 143. curtis, j.r., et al., american college of rheumatology guidance for covid‐19 vaccination in patients with rheumatic and musculoskeletal diseases: version 2. arthritis & rheumatology, 2021. 73(8): p. e30-e45. 144. marion, o., et al., safety and immunogenicity of anti–sars-cov-2 messenger rna vaccines in recipients of solid organ transplants. annals of internal medicine, 2021. 145. connolly, c.m., et al., absence of humoral response after two-dose sars-cov-2 messenger rna vaccination in patients with rheumatic and musculoskeletal diseases: a case series. annals of internal medicine, 2021. 146. kathryn m edwards, walter a orenstein, and a. sacchi. covid-19: vaccines to prevent sars-cov-2 infection. 2021; available from: https://www. uptodate.com/contents/covid-19-vaccines-to-prevent-sars-cov-2infection. 147. weinberger, b., et al., biology of immune responses to vaccines in elderly persons. clinical infectious diseases, 2008. 46(7): p. 1078-1084. 148. jackson, l.a., et al., an mrna vaccine against sars-cov-2—preliminary report. new england journal of medicine, 2020. 149. anderson, e.j., et al., safety and immunogenicity of sars-cov-2 mrna1273 vaccine in older adults. new england journal of medicine, 2020. 383(25): p. 2427-2438. 150. teo, s.p., review of covid-19 vaccines and their evidence in older adults. annals of geriatric medicine and research, 2021. 25(1): p. 4. 151. torjesen, i., covid-19: pfizer-biontech vaccine is “likely” responsible for deaths of some elderly patients, norwegian review finds. bmj: british medical journal (online), 2021. 373. 152. lv, g., et al., mortality rate and characteristics of deaths following covid-19 vaccination. frontiers in medicine, 2021. 8(649). 153. polack, f.p., et al., safety and efficacy of the bnt162b2 mrna covid-19 vaccine. new england journal of medicine, 2020. 383(27): p. 2603-2615. 154. fda, f., briefing document, moderna covid-19 vaccine. 2020, us food and drug administration. 155. werbel, w.a., et al., safety and immunogenicity of a third dose of sarscov-2 vaccine in solid organ transplant recipients: a case series. ann intern med, 2021. 156. shimabukuro, t.t., et al., preliminary findings of mrna covid-19 vaccine safety in pregnant persons. new england journal of medicine, 2021. 384(24): p. 2273-2282. 157. atyeo, c., et al., covid-19 mrna vaccines drive differential fcfunctional profiles in pregnant, lactating, and non-pregnant women. biorxiv, 2021. 158. beharier, o., et al., efficient maternal to neonatal transfer of antibodies against sars-cov-2 and bnt162b2 mrna covid-19 vaccine. the journal of clinical investigation, 2021. 131(13). 159. gray, k.j., et al., coronavirus disease 2019 vaccine response in pregnant and lactating women: a cohort study. am j obstet gynecol, 2021. 160. male, v., are covid-19 vaccines safe in pregnancy? nature reviews immunology, 2021: p. 1-2. 161. brillo, e., et al., covid-19 vaccination in pregnancy and postpartum. j matern fetal neonatal med, 2021: p. 1-21. 162. vincenzo berghella and brenna hughes. covid-19: pregnancy issues and antenatal care. 2021 [cited 2021 6 june]; available from: https://www. uptodate.com/contents/covid-19-pregnancy-issues-and-antenatal-care. 163. cdc-3. v-safe covid-19 vaccine pregnancy registry. 2021 2 june 2021 [cited 2021 6 june]; available from: https://www.cdc.gov/ coronavirus/2019-ncov/vaccines/safety/vsafepregnancyregistry.html. 164. sajjadi, n.b., et al., united states internet searches for “infertility” following covid-19 vaccine misinformation. journal of osteopathic medicine, 2021. 165. iacobucci, g., covid-19: no evidence that vaccines can affect fertility, says new guidance. 2021, british medical journal publishing group. 166. bowman, c.j., et al., lack of effects on female fertility and prenatal and postnatal offspring development in rats with bnt162b2, a mrna-based covid-19 vaccine. reprod toxicol, 2021. 103: p. 28-35. 167. cdc-2, updated healthcare infection prevention and control recommendations in response to covid-19 vaccination. . 2021. 168. goupil, r., et al., short-term antibody response after 1 dose of bnt162b2 vaccine in patients receiving hemodialysis. cmaj, 2021. 193(22): p. e793-e800. 169. boyarsky, b.j., et al., antibody response to 2-dose sars-cov-2 mrna vaccine series in solid organ transplant recipients. jama, 2021. 325(21): p. 2204-2206. 170. monin, l., et al., safety and immunogenicity of one versus two doses of the covid-19 vaccine bnt162b2 for patients with cancer: interim analysis of a prospective observational study. the lancet oncology, 2021. 22(6): p. 765-778. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i2.1197 https://www.uptodate.com/contents/covid-19-vaccines-to-prevent-sars-cov-2-infection https://www.uptodate.com/contents/covid-19-vaccines-to-prevent-sars-cov-2-infection https://www.uptodate.com/contents/covid-19-vaccines-to-prevent-sars-cov-2-infection https://www.uptodate.com/contents/covid-19-pregnancy-issues-and-antenatal-care https://www.uptodate.com/contents/covid-19-pregnancy-issues-and-antenatal-care https://www.cdc.gov/coronavirus/2019-ncov/vaccines/safety/vsafepregnancyregistry.html https://www.cdc.gov/coronavirus/2019-ncov/vaccines/safety/vsafepregnancyregistry.html 35j contemp med sci | vol. 9, no. 1, january-february 2023: 35–39 original prevalence of cytomegalovirus infection among pregnant women with cord blood examination karam fareed yalda1, amer abdalla goreal2* 1department of postgraduate studies, college of health sciences, university of duhok, duhok, iraq. 2department of medical microbiology, college of medicine, university of duhok, duhok, iraq. *correspondence to: amer abdalla goreal (e-mail: amer.goreal@uod.ac) abstract objectives: this study aimed at determining the prevalence of cmv infection among pregnant women at the end of pregnancy and cmv transmission to their newborns. methods: this is cross sectional study, 213 pregnant women at delivery and their newborn babies from the obstetrics and gynecology hospital in duhok/iraq were enrolled. a questionnaire was prepared to be answered by participants, including age, place of residence, educational level, and obstetric history as number of births, any bad obstetric history such as abortion, still birth, intrauterine growth retardation, congenital anomalies after birth. 3–5 ml of blood was drawn from each woman and examined by elisa kit to check for the presence of anti cmv igm, igg, then igg avidity test for those with positive (igg and igm). samples of cord blood were collected from newborns after birth and checked for the presence of cmv igm by elisa and cmv–dna by conventional pcr using specific primers to diagnose congenital infection and determine the rate of viral transmission from infected women. results: serological examinations showed that 212 (99.5%) participants were cmv-igg positive, 15 (7%) were positive for anti-cmv igm and igg antibodies, igg avidity test for 15 women were of high avidity (>89%) which indicated non primary infections. cord blood of newborns of those 15 women with positive igg and igm tested negative for anti cmv igm by elisa and no cmv-dna was detected by pcr, which revealed no transmission from those pregnant to their newborns. conclusion: this study demonstrated high prevalence of cmv among examined pregnant women in duhok city which makes them prone to non-primary infection. igg avidity test is of high efficacy to interpret the detection of igg and igm together in pregnant women. cord blood examination for the existence of cmv-igm and cmv-dna after delivery could exclude congenital infection. keywords: cytomegalovirus infections, cord blood, fetal blood, polymerase chain reaction, elisa, igg avidity issn 2413-0516 introduction cytomegalovirus (cmv) belongs to the beta herpesvirus subfamily of herpesviruses, double stranded dna enveloped virus. infects 60–90% of the population worldwide, typically asymptomatic infection.1-3 according to studies and reports the infection of congenital cytomegalovirus (cmv) is considered one of the most common types of infections that occur inside the uterine and is considered the most common in newborns, with infection rates recorded among them from 0.4 to 2.3% all over the world.4,5 the transmission of the congenital cytomegalovirus (cmv) from the infected mother to the fetus is either due to primary infection or recurrent infection of the mother during pregnancy and it is believed that about 85 to 90% of children who have congenital primary infection are without signs or symptoms, but the proportion of 5 to 15% of these patients, they may show some after-effects in life.6,7 it is possible to perform some diagnostic tests for cmv when women are at the end of their pregnancy, as this procedure and these tests help to treat the case of early diagnosis and early intervention for the newborn, especially those who do not show symptoms where they are at risk of many diseases, including sensorineural hearing loss.8-10 the role of igm, igg and avidity test a negative cytomegalovirus (cmv) igm result suggests that the patient is not experiencing acute or active infection. however, a negative result does not rule-out primary cmv infection. it has been reported that cmv-specific igm antibodies were not detectable in 10% to 30% of cord blood sera from infants demonstrating infection in the first week of life. in addition, up to 23% (3/13) of pregnant women with primary cmv infection did not demonstrate detectable cmv igm responses within 8 weeks post infection. in cases of primary infection where the time of seroconversion is not well defined as high as 28% (10/36) of pregnant women did not demonstrate cmv igm antibody. positive cmv igm results indicate a recent infection (primary, reactivation, or reinfection). igm antibody responses in secondary (reactivation) cmv infections have been demonstrated in some cmv mononucleosis patients, in a few pregnant women, and in renal and cardiac transplant patients. levels of antibody may be lower in transplant patients with secondary rather than primary infections.11 igg antibodies are produced several weeks after the initial cmv infection. igg levels rise during the active infection, then stabilize as the cmv infection resolves and the virus becomes inactive.12,13 avidity is defined as the aggregate strength with which a mixture of polyclonal igg molecules binds to multiple antigenic epitopes of proteins.14 it gradually matures over several months, reflective of antigen-driven selection of b cells producing igg of increasing affinity. igg antibodies produced during the first few months following primary infection exhibit low avidity (i.e., they bind weakly to the antigen), whereas antibodies produced by 6 months post infection exhibit high avidity (i.e., they bind tightly to the antigen).15 the basic methodology used to measure avidity capitalizes on (submitted: 01 october 2022 – revised version received: 20 november 2022 – accepted: 18 december 2022 – published online: 26 february 2023) http://orcid.org/0000-0003-4567-5717 36 j contemp med sci | vol. 9, no. 1, january-february 2023: 35–39 prevalence of cmv infection among pregnant women with cord blood examination original k.f. yalda et al. the weak binding of low-avidity igg to a mixture of cmv antigens (typically viral lysate). antigen-bound low-avidity igg, but not high-avidity igg, dissociates from the antigen in the presence of mild protein denaturants, such as urea, potassium thiocyanate, and guanidine chloride. the most common test format is an enzyme-linked immunosorbent assay (elisa) utilizing urea as the dissociating agent.16,17 pcr for cmv dna can be either qualitative or quantitative, in which the amount of viral dna in the respective sample is measured. the threshold of the qualitative method needs to be carefully calibrated for preventing over-detection. the quantitative pcr (real-time pcr) allows for continuous monitoring of immunocompromised individuals to identify patients at risk for cmv disease for preemptive therapy and to determine response to treatment.18 this method is generally more expensive compared to the antigenemia assay, but it is rapid and can be automated. results are usually reported as number of copies/ml of blood or plasma. this study was conducted in the city of duhok/iraq, in order to know the prevalence of cmv infection among pregnant women, where it is based on analysis and comparison of umbilical cord blood serum and blood test of the pregnant mother at the end of pregnancy. ethics statement ethical approval was issued by the ethics committee of the duhok general directorate of health with a number (15092021-9-9), on 15, september, 2021. then, informed consent has been obtained from the participants. permission was also obtained from the ministry of health and the ethics committee and approvals related to the college of health sciences at the university of duhok. design cross sectional study. subjects and methods two hundred and thirteen pregnant women at delivery participated in this study, blood samples were taken from them in the delivery room of duhok hospital for obstetrics and gynecology. consent was obtained from the women from whom samples were collected before delivery and during delivery from the umbilical cord. the ages of the participants in the study ranged from 19 to 36 years old, mean = 25.6 year, (sd ± 5.29) years. a questionnaire was prepared and answered by participants in this study, including: age, place of residence, educational level, and obstetric history as number of births, any bad obstetric history such as abortion, still birth, intrauterine growth retardation, congenital anomalies after birth so to classify them into those with bad obstetric history and normal one. all the participating women gave normal delivery, not by caesarean section. the samples that were taken are placed in a gel tube and classified into two types, the first before birth from the pregnant mother, where they were labeled with the letter (a, a1, a2), and the second that was taken from the umbilical cord labeled with the letter (b, b1, b2). immediately after that, these samples were taken to the laboratory for centrifugation at 3000 rpm for 5 minutes then serum was collected and kept at a temperature of –20°c till processing. after completing the required number of samples, samples with the letter a (mother’s samples before delivery) were tested for anti-cmv igg and igm antibodies. samples that revealed positive results for both anti cmv igg and igm together were subjected to third test which is igg avidity test, since this test is considered as cold standard test to differentiate between recent and old infection, in other words to know whether infection has occurred before or during pregnancy. selected umbilical cord samples (from women with positive anti cmv igg and anti cmv igm together) were examined for the presence of cmv-igm and cmv–dna by conventional pcr using specific primers to diagnose congenital infection and determine the rate of viral transmission from infected women. serological assays anti-cmv igg and igm antibodies were determined using a cmv igg enzyme immunoassay test kit and a cmv igm enzyme immunoassay test kit (dia. pro diagnostic bioprobes (milano – italy). serology was performed according to the manufacturer’s instructions and by reading the optical density for negative control, positive control and cut-off calibrators. samples with positive anti cmv igg and igm were tested by igg avidity test elisa kit (bioactiva / novatec germany acmv7110 avidity cytomegalovirus (cmv) igg. the result of <40% index is considered as low avidity while >65 is of high avidity index.) dna extraction, primers and pcr assays addprep viral nucleic acid extraction kit, was utilized to extract viral dna, amplification done by nested pcr according to the manufacture’s instruction using specific primers as shown below: pcr primers sequence (5¢-3¢) product size conventional n-pcr (glycoprotein b gene) outer-1 acagacacaaacagcaccca 450 bp outer-2 taaggtgacgacaggttggc inner-1 acacgcatacctcaacacc 220 bp inner-2 ggcccatggttccgaagcg internal control (β-globin gene) pc03 acacaactgtgttcactagc 110 bp pc04 caacttcatccacgtttcacc pcr mixture components (7 ul of dna template, 12.5 ul of master mix, 2 ul external primers, and 3.5 ul sterile water) (total volume 25 ul). -over condition as follow: first protocol: denaturation: 94°c for 2 min. denaturation: 94°c at 30 sec annealing: 58°c at 45 sec extension: 72°c at 60 sec (35 cycles) final extension: 72°c at 5 min and after that 1 ul was taken from first amplification product mixed with master mix and amplified by the second protocol (similar to first protocol condition as mentioned above with exception of annealing temperature (55°c) and for primers (internal) and then gel electrophoresis also were done to see bands of amplified samples (product size 220).19 37j contemp med sci | vol. 9, no. 1, january-february 2023: 35–39 k.f. yalda et al. original prevalence of cmv infection among pregnant women with cord blood examination africa with the following rates 30–90%, 58.3%, 94.4%, 80–90%, 92–100%, 60–100% respectively.23 in iraq several studies had been implemented to find out the prevalence of cmv among women which range from 77.3% in babylon to 95.7% in kirkuk, while in erbil the prevalence was 100%.24-26 the higher prevalence rate in the current study is in consistent with high prevalence rate in iraq which is over 95%. different rate of exposure to the virus hence different prevalence rates in different regions even within the same country may highlights the need of different policies for control and management of cmv infection, while screening of pregnant women for cmv antibodies remain controversial.22 this may highlight the unjustified routine cmv screening for pregnant women except for those with contact with someone that has confirmed acute infection or presenting with suspected cmv symptoms and immunocompromised women.27 there was no significant correlation between the cmv-seroprevalence and different age groups and obstetric history of enrolled women in this study which could be attributed to very high seroprevalence (99.5%). a study done by lachmann et al. (2018) in germany revealed that cmv seroprevalence in women aged (18–45 years) was 51.7% and age was considered as main factor significantly correlated with cmv seroprevalence.28 other researches done in north america demonstrated that women over 40 years had higher seroprevalence of cmv in comparison to those <40 years. while a study done in mexico found prevalence in pregnant women higher among those from 20 to 30 years than women aged <20 years old. contrary to these results no association between age and infection was detected in europe.29-31 regarding seroprevalence in women with a bad obstetric history it ranged from 14.2% in iran to 91.05% in india. amongst arab pregnant women with bad history, seroprevalence varies from 4.8% (iraq) to 95% (jordan).32,33 its clear that high prevalence rate in this study will obscure the correlation between the age, and obstetric history and their impact in determining the susceptibility to cmv infection. the risk of vertical transmission of cmv in pregnant women is higher for those who acquire primary infection than for those with previous exposure to the virus with circulating results the outcome of the enzyme immunoassay tests for the detection of anti-cmv igg and igm antibodies in 213 blood samples is shown in table 1, 212 (99.5%) were positive for anti-cmv igg antibodies, and 15 (7%) were positive for anti-cmv igm antibodies the results also showed that the samples with positive igm and igg were subjected to the igg avidity test, and all the results were of high avidity index, which supports the diagnosis of non-primary cmv infection. table 1. as shown in table 1 there no significant association between seroprevalence of cmv-igg and different age groups. results obtained in table 2 demonstrated that obstetric history has no impact on the results of cmv-igg and igm. the results of the enzyme immunoassay tests for the detection of anti-cmv igm antibodies of cord blood samples which has positive igm and igg were all negative, which means that there is no transmission of infection to newborn babies. 15 samples of cord blood are suitable for pcr assay, as judged by the successful amplification of β-globin sequences. the nested pcr results demonstrated that cmv dna was not detected in all 15 cord blood samples. figure 1. discussion cmv is considered as the main virus that may cross placenta when contracted during pregnancy, resulting in congenital anomalies and critical neurological deficits. congenital infection with cmv is associated with irreversible hearing loss due to nerve damage.20,21 the current study found the prevalence of cmv among pregnant women to be 99.5%, with only 0.5% susceptible women. a survey study was conducted to demonstrate the global prevalence of cmv and was found to be 83% and 86% in general population and young women respectively, with the maximum seroprevalence of 90% in eastern mediterranean region and the minimum of 66% in european region.22 many studies had estimated the prevalence of cmv among women which revealed high prevalence rate in different countries both developing and developed ones, including europe, usa, pakistan, india, saudi arabia and table 1. cmv serological results in regard to age groups age group (year) total no. (%) cmv-igg positive no. (%) cmv-igm positive no. (%) cmv-igg avidity 19–24 93 92–(98.9%) 3–(3.2%) (86–99%) 25–30 74 74–(100%) 8–(10.8%) (89–99%) 31–36 46 46–(100%) 4–(8.6%) (91–99%) total 213 212 15 15 p value > 0.05 (no significant correlation) by fisher exact test. table 2. cmv serological results in regard to obstetric history obstetric history total no. (%) cmv-igg positive no. (%) cmv-igm positive no. (%) cmv-igg avidity normal history 170 169–(99.4%) 11–(6.4%) 11 bad history 43 43–(100%) 4–(9.3%) 4 total 213 212 15 15 p value > 0.05 (no significant correlation) by fisher exact test. 38 j contemp med sci | vol. 9, no. 1, january-february 2023: 35–39 prevalence of cmv infection among pregnant women with cord blood examination original k.f. yalda et al. antibodies. therefore, finding a test that help in diagnosis of new infection is very essential to put a strategy of management of suspected cmv infection in such women. detection of cmv igm is sensitive indicator of acute maternal infection which increases the likelihood of congenital infection and cmv dnaemia in the cord blood. stagno et al. (1985) found detectable igm in women with primary cmv infection and 69% of them delivered infants with congenital infections. it could be detected in combination with igg that denotes reactivation of latent cmv or reinfection with another strain due to diversification of virus, accordingly its specificity is questionable.34 cmv – igm was found in 15 (7%) of enrolled women in this study in combination with igg, no participant was detected with igm alone. thus, it’s not primary infection with the virus, due to presence of cmv-igg it’s reactivation of latent virus or re infection with another strain. presence of cmv igg in pregnant women does not exclude the possibility of congenital infection of fetus, since there is chance for reactivation of latent cmv or reinfection with another strain during pregnancy. many studies demonstrated increase in rate of congenital cmv alongside increase in prevalence of cmv antibodies in women which is due to nonprimary rather than primary infection. the outcome of congenital infection in infants born to women with non primary cmv infection is nearly similar to that in newborn babies delivered by mother with primary infection in regard to severity of clinical manifestation including sensory-neural hearing defect.17 to solve this dilemma cmv igg-avidity test is considered gold standard test to differentiate between recent infection and old one. many researches revealed that cmv avidity test as sensitive and specific technique to diagnose pregnant women with recent primary infection with cmv so with high risk of intra uterine transmission. this test detects the strength of binding of igg to specific antigens on any protein, which is enhanced through several months after primary infection. when the test is implemented low cmv igg avidity indicates recent primary infection within previous 3 to 4 months while high avidity result means no infection in the preceding 3–4 months.16,35 in several researches, avidity indices (ai) higher than 60% during pregnancy could reasonably be explained a true indicator of past cmv infection, whereas in women with low ai less than or equal to 50%, there was a risk of intrauterine transmission with congenital cmv infection. the avidity test was done in this study and involved 15 samples with cmv-igg and cmv igm positivity, but all 15 participants showed high avidity with more than 89%. this indicates that all were not primary infections within preceding months. the presence of igm with igg of high avidity may denote that there is reactivation of latent cmv or reinfection with another strain. there is routine clinical use of cmv avidity tests in many developed countries. however, there is deficiency of information in underdeveloped and developing countries regarding use of cmv igg avidity tests in the diagnosis of maternal and congenital cmv infections.36 to diagnose congenital cmv infection there should be isolation of virus by tissue culture from urine samples or detection of viral dna by pcr, or by demonstration of cmv-igm in blood samples. however, -cmv igm test alone is no more useful to diagnose congenital cmv infection, since pcr is highly sensitive it has been considered as gold method for diagnosis.37 we have collected cord blood samples at delivery and were used to detect transplacental transmission of cmv by demonstrating the cmv igm in these samples taken from those 15 pregnancies with both cmv igm-igg, but all were negative for igm which indicates that no viral transmission has happened since igm does not cross placenta and upon detection in cord blood indicates its production in response to fetal infection. shin et al. (2017) studied the serological markers in cord blood of women with suspected cmv infection at delivery and concluded that cord blood unit with positive cmv-igm intended to use for transplantation, should be discarded regardless the result of cmv nucleic acid test.38 nucleic acid detection by pcr for those 15 samples showed negative results, which confirm the results of elisa and prove no transmission has happened. al-awadhi et al. (2013) found 89 out of 983 cord blood serum positive for anti-cmv igm antibodies; while pcr result showed only 4.5% positive for cmv dna.39 its recommend to use pcr test in blood, urine, and saliva in neonates with suspected congenital infection followed by treatment of positive cases with valganciclovir.40 fowler et al. (2018) suggested that the risk of cmv transmission during pregnancy following non primary type of infection in seropositive women should not be underestimated and needs to be explained.20 conclusion high sero-prevalence of cmv-igg in pregnant women in duhok, make them more susceptible to non-primary infection. detection of cmv-igg and igm together should be taken in consideration and analyzed by igg avidity test which is of high efficacy to differentiate between recent infection and non-primary maternal infection to elucidate the impact of infection on the rate of intrauterine transmission. testing of cord blood immediately after delivery by cmv igm and pcr is very useful to exclude congenital infection. conflict of interest authors declare no conflict of interest.  fig. 1 electrophoresis of cord blood samples. all were negative for cmv_dna, the bands with 110 bp represent the internal control (β-globin gene). 39j contemp med sci | vol. 9, no. 1, january-february 2023: 35–39 k.f. yalda et al. original prevalence of cmv infection among pregnant women with cord blood examination references 1. cannon mj, schmid ds, hyde tb. review of cytomegalovirus seroprevalence and demographic characteristics associated with infection. rev med virol. 2010;20(4):202–13. 2. kenneson a, cannon mj. review and meta-analysis of the epidemiology of congenital cytomegalovirus (cmv) infection. rev med virol. 2007;17(4):253–76. 3. lanzieri tm, dollard sc, bialek sr, grosse sd. systematic review of the birth prevalence of congenital cytomegalovirus infection in developing countries. int j infect dis. 2014; 22:44–8. 4. stagno s, pass rf, alford ca. perinatal infections and maldevelopment. in: bloom ad, james ls, eds. the fetus and the newborn. new york: alan r. liss, 1981; 31–50. 5. demmler gj. summary of a workshop on surveillance for congenital cytomegalovirus disease. rev infect dis. 1991; 13:315–29. 6. fowler kb. congenital cytomegalovirus infection: audiologic outcome. clin infect dis. 2013;57: s182–4. 7. rivera lb, boppana sb, fowler kb, britt wj, stagno s, pass rf. predictors of hearing loss in children with symptomatic congenital cytomegalovirus infection. pediatrics. 2002; 110:762–7. 8. wagner n, kagan ko, haen s, schmidt s, yerlikaya g, maden z, jahn g, hamprecht k. effective management and intrauterine treatment of congenital cytomegalovirus infection: review article and case series. j matern fetal neonatal med. 2014 jan;27(2):209–14. 9. leruez-ville m, ghout i, bussières l, stirnemann j, magny jf, couderc s, salomon lj, guilleminot t, aegerter p, benoist g, winer n, picone o, jacquemard f, ville y. in utero treatment of congenital cytomegalovirus infection with valacyclovir in a multicenter, open-label, phase ii study. am j obstet gynecol. 2016 oct;215(4):462.e1–462.e10. 10. johnson j, anderson b: screening, prevention, and treatment of congenital cytomegalovirus. obstet gynecol clin north am. 2014;41(4):593–9. 10.1016/j.ogc.2014.08.005 11. lazzarotto t, ripalti a, bergamini g, battista mc, spezzacatena p, campanini f, pradelli p, varani s, gabrielli l, maine gt, landini mp. development of a new cytomegalovirus (cmv) immunoglobulin m (igm) immunoblot for detection of cmv-specific igm. j clin microbiol. 1998 nov;36(11):3337–41. 12. cytomegalovirus (cmv) antibodies, igm and igg, serum. mayo clinic laboratories. available online at https://www.mayomedicallaboratories. com/test-catalog/clinical+and+interpretive/62067. accessed on 11/3/18. 13. pagana, kathleen d. & pagana, timothy j. (2001). mosby’s diagnostic and laboratory test reference 5th edition: mosby, inc., saint louis, mo. 14. hazell sl. clinical utility of avidity assays. expert opin. med. diagn. 2007;1:511–519. 10.1517/17530059.1.4.511. 15. revello mg, gerna g. diagnosis and management of human cytomegalovirus infection in the mother, fetus, and newborn infant. clin microbiol rev. 2002 oct;15(4):680–715. 16. chakravarti a, kashyap b, wadhwa a. relationship of igg avidity index and igm levels for the differential diagnosis of primary from recurrent cytomegalovirus infections. iran j allergy asthma immunol. 2007 dec;6(4):197–201. 17. prince he, lapé-nixon m, novak-weekley sm. 2014. performance of a cytomegalovirus igg enzyme immunoassay kit modified to measure avidity. clin. vaccine immunol. 2014 jun;21(6):808–12. 18. kim dj, kim sj, park j, choi gs, lee s, kwon cd, ki c, joh j. realtime pcr assay compared with antigenemia assay for detecting cytomegalovirus infection in kidney transplant recipients. transplant. proc. 2007;39(5):1458–1460. 19. noorbakhsh s, farhadi m, haghighi f, minaeian s, hasanabad mh. neonatal screening for congenital cytomegalovirus infection in tehran, iran, using guthrie cards. iran j microbiol. 2020 jun;12(3):198–203. 20. fowler kb, boppana sb. congenital cytomegalovirus infection. semin perinatol. 2018 apr;42(3):149–154. 21. ross sa, fowler kb, ashrith g, stagno s, britt wj, pass rf, boppana sb. hearing loss in children with congenital cytomegalovirus infection born to mothers with preexisting immunity. j pediatr. 2006 mar;148(3):332–6. 22. zuhair m, smit s, willis g, jabbar f, smith c, devleesschauwer b, griffiths p. estimation of the worldwide seroprevalence of cytomegalovirus: a systematic review and meta-analysis. rev. med. virol. 2019; vol. 29(3):e2034. 23. almishaal aa. knowledge of cytomegalovirus infection among women in saudi arabia: a cross-sectional study. plos one. 2022;17(9): e0274863. 24. abbas m d and egbe s . seroprevalence of cmv in women with bad obstetric history in babil/iraq. iraqi j pharm sci. 2021; vol. 30(2). 25. aljumaili z, alsamarai a, najem w.cytomegalovirus seroprevalence in women with bad obstetric history in, iraq. kirkuk journal of infection and public health. 2014; volume 7, issue 4, pages 277–288. 26. ali k.the sero-prevalence of cytomegalovirus infection among women with abortion and intrauterine death in erbil city kurdistan region. diyala journal of medicine. 2020; 77 vol.18. issue 1 27. ayla so, ževki ce, aye ci, sibel s, serpil un, nuri d . screening of cytomegalovirus seroprevalence among pregnant women in ankara, turkey: a controversy in prenatal care. african journal of microbiology research. 2011; 9;5(29):5304–7. 28. lachmann r, loenenbach a, waterboer t, brenner n, pawlita m, michel a.). cytomegalovirus (cmv) seroprevalence in the adult population of germany. plos one. 2018; 13(7): e0200267 29. wizman s, lamarre v, coic l, kakkar f, le meur jb, rousseau c, boucher m, tapiero b. awareness of cytomegalovirus and risk factors for susceptibility among pregnant women, in montreal, canada. bmc pregnancy childbirth. 2016; mar 15;16:54. 30. alvarado-esquivel, c., terrones-saldivar, m., hernandez-tinoco, j., munozterrones, m., gallegos-gonzalez, r., sanchez-anguiano, l. seroepidemiology of cytomegalovirus infection in pregnant women in the central mexican city of aguascalientes. j clin med res. 2018;10(4):337–344. available at: <https://www.jocmr.org/index.php/jocmr/article/view/3358>. 31. gorun f, motoi s, malita d, navolan db, nemescu d, olariu tr, craina m, vilibic-cavlek t, ciohat i, boda d, dobrescu a. cytomegalovirus seroprevalence in pregnant women in the western region of romania: a large-scale study. exp ther med. 2020 sep;20(3):2439–2443. 32. falahi, s., ravanshad, m., koohi, a.k., karimi, a.m. short com-munication: seroprevalence of cmv in women’s with spontaneous abortion in kowsar hospital, ilam during 2007—2008. modares j med sci pathobiol. 2010; 12:39—43. 7. 33. turbadkar, d., mathur, m., rele, m. seroprevalence of torch infection in bad obstetric history. indian j med microbiol. 2003; 21:108—11, 2003. 34. stagno s, tinker m k, elrod c, fuccillo d a, cloud g, o’beirne a j..immunoglobulin m antibodies detected by enzyme-linked immunosorbent assay and radioimmunoassay in the diagnosis of cytomegalovirus infections in pregnant women and newborn infants. journal of clinical microbiology. 1985;vol. 21, no. 6 . 35. mussi-pinhata m m, yamamoto a y. natural history of congenital cytomegalovirus infection in highly seropositive populations, the journal of infectious diseases. 2020; v 221, issue 1, 15 pages s15–s22. 36. abdullahi nasir i, babayo a, shehu ms. clinical significance of igg avidity testing and other considerations in the diagnosis of congenital cytomegalovirus infection: a review update. med sci (basel). 2016 mar; 8;4(1):5. 37. cytomegalovirus (cmv) and congenital cmv infection. [(accessed on 30 april 2015)]; 2010 available online: http://www.cdc.gov/cmv/index.html 38. shin s, roh ey, oh s, song ey, kim ec, yoon jh. excluding anticytomegalovirus immunoglobulin m-positive cord blood units has a minimal impact on the korean public cord blood bank inventory. cell transplantation. 2017;26(1):63–70. 39. al-awadhi r, al-harmi j, alfadhli s. prevalence of cytomegalovirus dna in cord blood and voided urine obtained from pregnant women at the end of pregnancy. med princ pract. 2013;22(2):194–9. 40. chiopris, g.; veronese, p.; cusenza, f.; procaccianti, m.; perrone, s.; daccò, v.; colombo, c.; esposito, s. congenital cytomegalovirus infection: update on diagnosis and treatment. microorganisms. 2020, 8, 1516. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1315 245j contemp med sci | vol. 8, no. 4, july-august 2022: 245–249 original relationship of midkine in myocardial infarction patients and some biochemical parameters obeda abdlkhaliq abass alkado1*, luay abd ali al-helaly2 1directorate of education in nineveh, mosul, iraq. 2department of chemistry, college of science, university of mosul, mosul, iraq. *correspondence to: obeda abdlkhaliq abass alkado (e-mail: obedaalkado@yahoo.com) (submitted: 23 march 2022 – revised version received: 09 april 2022 – accepted: 28 april 2022 – published online: 26 august 2022) abstract objectives: this study aims to estimate the concentrations of mk, hs-crp, tni, ast, alt and alp in serum for mi in comparison to wholesome controls, in an effort to check the quantity of this impact at the pathological condition, in addition to understanding the position of mk in mi sufferers and its relationship to liver function tests and may be used as biochemical markers to decide the severity of mi in sufferers. methods: the studying include the investigation of midkine (mk), high-sensitivity c-reactive protein (hs-crp), troponin i (tni), aspartate aminotransferase (ast) and alanine aminotransferase (alt) and alkaline phosphatase (alp) levels in myocardial infarction (mi) patients. the study includes (84) samples: 34 of myocardial infarction patients (19 male and 15 female) and 50 sample of apparently healthy as control group (25 male and 25 female). samples were collected during the period from jan. 2021 to july 2021 from in-patients of cardiac surgery center admitted at ibn sina teaching hospital and mosul general hospital, mosul, iraq with matching age ranged between 26–86 years. the clinical diagnosis in each case is made according to a cardiovascular specialist. results: the results showed that there was a significant increase in mk, tni, got, gpt and alp for all studied groups (males and females) of mi patients compared with control groups, and there was no any a significant in hs-crp. beside of, showed significant positive correlation between mk with bmi, hs-crp, tni, ast, alt and alp in control and patients group. whereas there were no any correlation between mk with age, in patients and control under study. conclusion: the study indicated that the mk increased in patients with myocardial infarction, and it’s increased in males more than female patients, and that may act as a good markers for diagnosis of myocardial infarction disease. keywords: myocardial infarction, midkine, hs-crp, ast, alt, alp issn 2413-0516 introduction a major threat to human health, myocardial infarction (mi) is one of the leading causes of death in the world.1 it is characterized by high morbidity and mortality. atherosclerosis, a chronic inflammatory disease characterized by the development of fibrofatty plaques, is the pathophysiological cause of mi.2 the term mi refers to a heart attack that occurs when blood flow to a particular area of the heart is disrupted and the heart muscle is harmed due to a lack of oxygen. and owing to an unsteady accumulation of plaques, white blood cells, cholesterol, and fat, one of the coronary arteries that delivers blood to the heart develops a blockage. acute myocardial infarction, or ami, is the term used when the incident becomes severe. chest discomfort that radiates to the left arm or side of the neck, shortness of breath, perspiration, nausea, vomiting, an irregular heartbeat, anxiety, exhaustion, and other symptoms are all signs of mi.3,4 the term “silent mi” refers to the approximately 64% of mi patients do not experience chest discomfort.5 mi is caused by a number of factors, including older age, cigarette use high concentration of cholesterol and fat levels, diabetes, obesity, inactivity, chronic renal disease, hypertension, high levels of bad cholesterol ldl (low density lipoprotein), excessive alcohol intake and use of cocaine and amphetamines.6,7 when the present oxygen needs of the myocardium are not met by oxygen supply, mi results. the coronary perfusion is absolutely necessary for the heart’s continual delivery of oxygen because it is an obligatory aerobic organ. normal myocardium does not become ischemic when oxygen intake is raised (as happens during maximal exercise testing) because the myocardial oxygen demand and supply are fulfilled during normal physiology. even in the absence of overt coronary artery disease, mi is a frequent finding in failing hearts, perhaps as a result of structural or functional changes to the coronary circulation. ischemia is a self-perpetuating disease that adversely affects prognosis and permanently compromises heart function. therefore, a more complete and in-depth comprehension of mi pathogenesis in heart failure will probably result in much better results for these patients.8 the primary property of midkine (mk) was later thought to coordinate embryonic development.9 the majority of mk research focused on its role in malignant diseases and suggested a negative impact on the host.10 a significant portion of the mk literature has demonstrated its capacity to promote inflammatory reactions.11 several recent studies have also shown that mk is a key regulator of angiogenesis and cardiac problems in patients with cardiovascular disease.4 surgical techniques to open clogged arteries may become a traditional method, and could be replaced by new non-surgical treatment methods for patients with ischemic diseases through the use of mk, which may be primarily related to atherosclerosis, for example within the heart.12 this study aims to estimate the concentrations of mk, hs-crp, tni, ast, alt and alp in serum for mi in comparison to wholesome controls, in an effort to check the quantity of this impact at the pathological condition, in addition to understanding the position of mk in mi sufferers and its relationship to liver function tests and may be used as biochemical markers to decide the severity of mi in sufferers. materials and methods we measured the concentrations of mk, hs-crp, tni, ast, alt and alp in blood samples for 84 samples divided into 246 j contemp med sci | vol. 8, no. 4, july-august 2022: 245–249 midkine in myocardial infarction patients and its relation with some biochemical parameters original o.a.a. alkado et al. (34) samples of mi patients (19 male and 15 female) and control group consists of 50 apparently healthy individuals (25 male and 25 female) with negative results for cardiovascular diseases, or chronic diseases, and their age were matched with the mi patients and ranged between 26–86 years, and they have no history of any disease. samples were collected during the period from jan. 2021 to july 2021 from patients in the cardiac surgery center and inpatients at ibn sina teaching hospital and mosul general hospital, nineveh health directorates/mosul iraq. the clinical diagnosis in each case is made according to a cardiovascular specialist. ten ml of blood and allow to clot, serum was separated by using speed at (4000 × g) for ten mints, divided into (3) sections and kept frozen at (–20°c) until analysis. the activities of ast, alt, and alp were determined from the myocardial and renal tissue using the assay journal pre-proof 10 kit, which was purchased from randox laboratory ltd. (co. antrim, uk). eliza technique is used to quantify midkine, hs-crp, and tni using kits tested in accordance to the manufactured procedure (bioassay technology laboratory, shanghai, china) mk cat. no. e1633hu, hs-crp cat. no. e1805, and tni cat. no. e1270. the statistical analysis was performed using spss software. each indicator’s average value and standard deviation were determined, and the p-value thresholds of significant at (p ≤ 0.05), **significant at (p ≤ 0.001), and ***significant at (p ≤ 0.0001) were used to indicate statistical significance. results tables 1, 2 and 3 showed a description of healthy and mi patients groups (males and females groups), which indicated to non-significant for age and body mass index (bmi) for all groups, males and females. the results shown in tables 4, 5 indicated that there is a significant increase in the levels of mk in the serum of mi patients when compared with the healthy group at p < 0.0001 and for all and male patients group, but in table 6 female table 1. description of healthy and all mi patients groups parameters healthy group n = 50 patients group n = 34 p-value mean se mean se age, (year) 54.54 1.85 58.88 2.18 0.19 weight, (kg) 80.82 2.33 84.12 2.69 0.291 height, (cm) 171.22 1.16 171.3 1.12 0.811 bmi, (kg/m2) 27.56 0.74 28.77 0.99 0.215 table 2. description of males for healthy and mi patients groups parameters healthy group n = 25 patients group n = 19 p-value mean se mean se age, (year) 53.68 2.1 58.22 2.65 0.101 weight, (kg) 84.16 2.85 85.33 3.29 0.833 height, (cm) 176.1 1.34 173.0 1.18 0.102 bmi, (kg/m2) 27.16 0.85 28.63 1.22 0.340 table 3. description of females for healthy and mi patients groups parameters healthy group n = 25 patients group n = 15 p-value mean se mean se age, (year) 57.4 2.89 61.43 2.86 0.087 weight, (kg) 77.48 3.63 78.43 2.68 0.111 height, (cm) 166.4 1.33 164.7 1.24 0.408 bmi, (kg/m2) 27.96 1.24 28.31 1.02 0.19 groups show that there is a significant increase in the levels of mk in mi patients when compared at p < 0.05 and there was no evidence of a significant increase in the levels of hs-crp in the serum of mi patients when compared to the healthy group for all patients, male and female groups, while there is a significant increase in tni in the serum of mi patients compared with the group of healthy at p ≤ 0.0001 for all and male groups was observed. in addition, table 6 shows that there is a significant increase in tni in the serum of mi patients compared with the group of healthy at p ≤ 0.05 for female groups. the results in tables 7, 8 show that there is a significant increase in the levels of ast, alt and alp in the serum of myocardial infarction compared with the group of healthy at p ≤ 0.0001 and p ≤ 0.001 for all and male groups, and in table 4. mk, hs-crp and tni in all mi patients group biochemical parameters healthy group n = 50 patients group n = 34 p-value mean se mean se mk, (pg/ml) 159.7 6.1 410.1 24.78 0.0001*** hs-crp, (ng/ml) 1.25 0.075 1.13 0.081 0.262 tn, (pg/ml) 0.06 0.02 9.39 1.39 0.0001*** ***significant at (p ≤ 0.0001). table 5. mk, hs-crp and tni in male mi patients group biochemical parameters# healthy group n = 25 patients group n = 19 p-value mean se mean se mk, (pg/ml) 152.3 5.91 422.7 41.63 0.0001*** hs-crp, (ng/ml) 1.288 0.123 1.137 0.086 0.339 tn, (pg/ml) 0.08 0.054 8.467 1.55 0.0001*** ***significant at (p ≤ 0.0001). table 6. mk, hs-crp and tni in female mi patients group biochemical parameters healthy group n = 25 patients group n = 15 p-value mean se mean se mk, (pg/ml) 167.2 10.81 361.4 13.36 0.008* hs-crp, (ng/ml) 1.212 0.089 1.11 0.22 0.624 tn, (pg/ml) 0.04 0.016 12.97 2.96 0.005* *significant at (p ≤ 0.05). 247j contemp med sci | vol. 8, no. 4, july-august 2022: 245–249 o.a.a. alkado et al. original midkine in myocardial infarction patients and its relation with some biochemical parameters table 7. ast, alt and alp in all mi patients group as compared with healthy group enzyme activity levels, (u/l) healthy group n = 50 patients group n = 34 p-value mean se mean se ast 13.38 1.10 39.26 4.82 0.0001*** alt 5.8 0.39 10.03 0.93 0.0001*** alp 12.02 0.58 20.02 1.84 0.0001*** ***significant at (p ≤ 0.0001). table 8. ast, alt and alp in males mi patients group as compared with healthy group enzyme activity levels, (u/l) healthy group n = 25 patients group n = 19 p-value mean se mean se ast 13.44 1.51 39.81 5.39 0.0001*** alt 6.24 0.71 10.78 1.105 0.005* alp 12.7 0.83 19.87 1.99 0.001** *significant at (p ≤ 0.05), **significant at (p ≤ 0.001), ***significant at (p ≤ 0.0001). table 9. ast, alt and alp in females mi patients group as compared with healthy group enzyme activity levels, (u/l) healthy group n = 25 patients group n = 15 p-value mean se mean se ast 13.32 1.64 37.14 3.62 0.022* alt 5.36 0.31 7.14 1.03 0.038* alp 11.34 0.82 20.58 2.92 0.014* *significant at (p ≤ 0.05). table 10. linear relationship between mk and some biochemical parameters measured in the control and mi patient groups midkine (mk), (pg/ml) biochemical parameters mi patients, n = 34 control group, n = 50 p-valuer-valuep-valuer-value 0.0850.6320.1290.370age, (year) 0.0340.85*0.1730.229bmi, (kg/m2) 0.0230.508*0.0090.632*hs-crp, (ng/ml) 0.0130.892*0.0150.774*tni, (pg/ml) 0.0280.7670.050.1*ast, (u/l) 0.0510.583*0.0390.789*alt, (u/l) 0.0220.530*0.0270.314*alp, (u/l) *significant at (p ≤ 0.05). table 9 female groups show that there is a significant increase in the levels of ast, alt and alp in the serum of mi compared with the group of healthy at p ≤ 0.005. the present study showed in table 10 significant positive correlation between mk with bmi, hscrp, tni ast, alt and alp in control and patients group. whereas there were no any correlation between mk with age, in patients and control under study. discussion one of the important biomarkers used in clinical diagnosis is medkine. the data of midkine obtained in this study are consistent with the findings of previous studies performed by woulfe et al.,13 while treating the heart specifically is a clear therapeutic objective, other essential organs also need to be protected, and mk may be able to help in this regard in addition to repairing brain damage. in rodent models of ischemic myocardial infarct, for instance, due to its powerful angiogenic and anti-apoptotic effects, mk has been shown to be cardio-protective, resulting in reduced myocardial infarction, thus improved cardiac function, and increased overall survival. injecting mk intracoronary or intramyocardially enhanced survival, reduced infarct acuity, and decreased apoptosis through to cell survival and angiogenesis.13,14 mk also contributes to myocardial infarction, cardiac hypertrophy, and ischemic heart damage. the role of mk in these various pathologies is still up for debate, though, because mk has positive benefits, such as ischemic heart damage, heart failure, and myocardial infarction by improving cardiac function and antiapoptosis, stimulating angiogenesis, and reducing detrimental remodeling.15 the findings of high-sensitivity c-reactive protein (hscrp) are in agreement with other studies for other diseases, such as metabolic problems, cerebrovascular illness, and coronary atheroseclerosis.16-18 c-reactive protein (crp) damages the vascular endothelium, causing it to malfunction and become more sensitive to proatherogenic stimuli, is also linked to the existence of chronic low-intensity inflammation.17 because cardiac imaging techniques cannot assess inflammation-induced vascular changes, biomarkers that can identify these changes are highly valuable.19,20 additionally, it is critical to develop procedures for the precise and sensitive identification of people those who are in risk of developing cardiovascular disorders. these requirements could be satisfied by the biomarker of inflammation known as serum high-sensitivity crp (hs-crp). according to the physician’s health study findings, people with greater baseline hs-crp concentrations have a doubled risk of stroke and a tripled risk of mi.21 in contrast to lipids or homocysteine, the female health research found that hscrp is a superior predictive predictor for cardiovascular events.22 last but not least, yoshinaga et al. (2017)23 noted that increased hs-crp is a significant risk factor for patient death in hospitals.24 troponin i play an important roles in myocardial infarction as a specific biomarker.4 these findings are consistent with ammirati et al., (2021)25 cardiac special troponin (ctn) are significant common biomarkers utilized in contemporary clinical diagnosis to suspected mi, and their altitude is a key feature in the diagnosis of acute mi.26 the causes of myocardial damage are many, and myocardiocyte apoptosis, with or without obvious indications of necrosis, may cause the release of ctn from injured cells.27 the two troponin indicators used in clinical practice are troponin i (tni) and troponin (tnt). despite the fact that these are two different proteins, they are 248 j contemp med sci | vol. 8, no. 4, july-august 2022: 245–249 midkine in myocardial infarction patients and its relation with some biochemical parameters original o.a.a. alkado et al. both employed because they both represent myocyte damage and have comparable diagnostic accuracy for mi28 despite the fact that age had no discernible influence on them.29 the results of serum cardiac enzyme including ast and alt activity levels are in consistent with researchers djakpo et al., (2020)30 and shamshirian et al., (2020).31 multiple causes might result in increased ast and alt levels. elevated ast and alt have been linked to four separate pathways.32 first, direct tissue injury (damage to the plasma membrane with protein leakage or cell necrosis produced by a variety of damaging agents or stimuli) or apoptosis are the most prevalent causes of increased activity of aminotransferases, including ast (in conditions of physiological cellular renewal or increased apoptotic stimuli). reasonably, larger organs with higher ast activity are the primary source of increased ast in circulation. elevated ast levels are usually associated with inflammatory liver disease (viral hepatitis), alcoholic liver disease, cirrhosis, cholestasis syndromes, drug toxicity, acute myocardial infarction, septic shock, and skeletal muscle injury/injury. acute myocardial ischemia or myocardial cell necrosis, which occurs in acute myocardial infarction, is a common cause of increased serum ast activity.33 alkaline phosphatase (alp) activity level obtained in this study indicate that their outcomes data support the conclusions of prior studies that there is an increase in the activity level of alp in the serum in mi.34 as many tissue-specific isozymes as different genes encode the alp [phosphate monoester phosphohydrolase; ec 3.1.3.1] enzyme. it is present in a variety of species (bacteria, plants, and mammals) and is capable of catalyzing the hydrolysis of the phosphomonoester r-o-po3 without regard to identification category “r”.35 in the emergency room, regular blood tests for liver function, such as serum transaminases and alkaline phosphatase (alp), are frequently performed. acute myocardial infarction (mi) patients may have abnormal liver function values without having clinically obvious liver damage.36 a strong predictor of mortality in the general population, according to recent research, is the nonalcoholic fatty liver disease fibrosis score, which includes serum transaminases.37,38 in individuals with post-acute coronary syndrome, this score is related to an increased risk of subsequent cardiovascular events.39 diagnostic signs include cytosolic enzymes like ck-mb and alp, which leak from the injured tissue into the circulation when the cell membrane is torn or permeable. additionally, the serum’s level of enzymes is inversely correlated with the quantity of necrotic cells.40 the correlations between mk and some biochemical parameters measured in the control and mi patient groups indicate that mk is a crucial regulator of angiogenesis, the formation of new blood vessels and arteriogenesis in pathological vascular conditions, according to recent study. due to the increasing number of cases of cardiovascular diseases, which is primarily attributed to atherosclerosis and occlusive arterial disease, blockage in one of the heart’s arteries caused by fat accumulation, angiogenesis which is the process by which new blood vessels are created from pre-existing ones through this protein mk can avoid surgeries it is interesting that the sulfate groups have a significant impact on the effects of mk on neuronal survival and outgrowth.41 as the disease causes an increase in the level of hscrp, tni ast, alt, and alp with the increase in the levels of mk, an increase in mk is a certain indicator of an increase in the incidence of myocardial infarction and the associated damage to the work and function of the heart muscle. along with demonstrating that the mk rose along with the bmi, mk may be involved in this process as it is generated and released by adipocytes. mk is related with increased production of inflammatory mediators in adipose tissue, which leads to chronic inflammation.42,43 as a result, the mk was raised, and a strong positive association was seen between the mk and bmi. increase in the level of mk reflects the level of indicators of heart disease from hscrp and tni. this is clear evidence and a good indicator that mk can be considered a clear sign of myocardial infarction, which can be added as another guide for a good and early diagnosis of this disease. conclusion the study indicated that the mk increased in patients with myocardial infarction, and it’s increased in males more than female patients, and that may good markers to diagnose the myocardial infarction disease.  references 1. jernberg t., hasvold, p., henriksson, m., hjelm, h., thuresson, m. and janzon, m. (2015). cardiovascular risk in post-myocardial infarction patients: nationwide real world data demonstrate the importance of a long-term perspective. eur heart j. 36: 1163–70. 2. libby, p., buring, j.e., badimon, l., hansson, g.k., deanfield, j., bittencourt, m.s., et al. (2019). atherosclerosis. nat rev dis primers 5: 56. 3. kosuge, m., kimura, k., ishikawa, t., ebina, t., hibi, k., tsukahara, k., et al. (2006). differences between men and women in terms of clinical features of st-segment elevation acute myocardial infarction. circulation journal, 70(3): 222–226. 4. hemeed, r. n. ; al-tu’ma, f. j.; al-koofee, a. f. and al-mayali, a. h. (2020). relationship of angiotensin converting enzyme (i/d) polymorphism (rs4646994) and coronary heart disease among a male iraqi population with type 2 diabetes mellitus. journal of diabetes and metabolic disorders, 19(2): 1227–1232. 5. valensi, p., lorgis, l., and cottin, y. (2011). prevalence, incidence, predictive factors and prognosis of silent myocardial infarction: a review of the literature. archives of cardiovascular diseases, 104(3): 178–188. 6. devlin, r. j., and henry, j. a. (2008). clinical review: major consequences of illicit drug consumption. critical care, 12(1): 202. 7. graham, i., atar, d., borch-johnsen, k., boysen, g., burell, g., cifkova, r., et al. (2007). european guidelines on cardiovascular disease prevention in clinical practice: executive summary: fourth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice (constituted by representatives of nine societies and by invited experts). european heart journal, 28(19): 2375–2414. 8. pagliaro, b. r., cannata, f., stefanini, g. g., and bolognese, l. (2020). myocardial ischemia and coronary disease in heart failure. heart failure reviews, 25(1): 53–65. 9. kadomatsu, k., tomomura, m. and muramatsu, t.(1988). cdna cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. biochem biophys res commun. 151: 1312–8. 10. zhang, l., song, x., shao, y., wu, c. and jiang, j. (2018). prognostic value of midkine expression in patients with solid tumors: a systematic review and meta-analysis. oncotarget 9: 24821–24829. 11. takada, s., sakakima, h., matsuyama, t., otsuka, s., nakanishi, k., norimatsu, k., itashiki, y., tani, a. and kikuchi, k. (2020). disruption of midkine gene reduces traumatic brain injury through the modulation of neuroinflammation. journal of neuroinflammation, 17(1): 40. 12. weckbach, l. t., preissner, k. t. and deindl, e. (2018). the role of midkine in arteriogenesis, involving mechanosensing, endothelial cell proliferation, and vasodilation. int. j. mol. sci., 19(9): 2559. 249j contemp med sci | vol. 8, no. 4, july-august 2022: 245–249 o.a.a. alkado et al. original midkine in myocardial infarction patients and its relation with some biochemical parameters 13. woulfe, k. c., and sucharov, c. c. (2017). midkine’s role in cardiac pathology. journal of cardiovascular development and disease, 4(3): 13. 14. bădilă e, daraban a.m., ţintea e, bartoş d, alexandru n, georgescu a. (2015) midkine proteins in cardio-vascular disease. where do we come from and where are we heading to? eur. j. pharmacol. 762, 464–471. 15. lackner, i., weber, b., baur, m., haffner-luntzer, m., eiseler, t., fois, g., et. al. (2019). midkine is elevated after multiple trauma and acts directly on human cardiomyocytes by altering their functionality and metabolism. frontiers in immunology, 10: 1920. 16. quispe, r., michos, e.d., martin, s.s., puri, r., toth, p.p., al suwaidi, j., banach, m. and virani, s.s., blumenthal, r.s., jones, s.r., et al. (2020). high-sensitivity c-reactive protein discordance with atherogenic lipid measures and incidence of atherosclerotic cardiovascular disease in primary prevention: the aric study. j. am. hear. assoc. 9, e013600. 17. koenig, w. (2013). high-sensitivity c-reactive protein and atherosclerotic disease: from improved risk prediction to risk-guided therapy. int. j. cardiol. 168: 5126–5134. 18. al-tu’ma, f.j. ; abd-yasera, z.a. and al-naffi, k.o. (2016). association between hs-crp levels and the severity of coronary atherosclerosis. j contemp med sci, 2(6): 42-44. 19. adukauskienė d, čiginskienė a, adukauskaitė a, pentiokinienė d, šlapikas r, čeponienė i. (2016). clinical relevance of high sensitivity c-reactive protein in cardiology. medicina 52, 1–10. 20. silva, d. and de lacerda, a.p.(2012). high-sensitivity c-reactive protein as a biomarker of risk in coronary artery disease. rev. port. cardiol., 31: 733–745. 21. ridker, p.m., glynn, r.j. and hennekens, c.h. (1998). c-reactive protein adds to the predictive value of total and hdl cholesterol in determining risk of first myocardial infarction. circulation, 97: 2007–2011. 22. ridker, p.m., hennekens, c.h., buring, j.e. and rifai, n. (2000). c-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. new engl. j. med. 342: 836–843. 23. yoshinaga, r., doi, y., ayukawa, k. and ishikawa, s. (2017). high-sensitivity c reactive protein as a predictor of in hospital mortality in patients with cardiovascular disease at an emergency department: a retrospective cohort study. bmj open, 7, e015112. 24. song, y., yang, s.k., kim, j. & lee, d.c. (2019). association between c-reactive protein and metabolic syndrome in korean adults. korean j. fam. med. 40: 116–123. 25. ammirati, e., veronese, g., bottiroli, m., wang, d. w., cipriani, m., garascia, a., et. al. (2021). update on acute myocarditis. trends in cardiovascular medicine, 31(6): 370–379. 26. nice. acute coronary syndromes in adults (qs68). 2014. 27. mair, j., lindahl, b., hammarsten, o., müller, c., giannitsis, e., huber, k., et al. (2018). how is cardiac troponin released from injured myocardium? eur heart j acute cardiovasc care. 7(6): 553–60. 28. ebell, m.h., flewelling, d. and flynn, c.a. (2000). a systematic review of troponin t and i for diagnosing acute myocardial infarction. j fam pract. 49(6): 550–6. 29. apple, f.s., wu, a.h.b, sandoval, y., sexter, a., love. s.a., myers, g., et al. (2020). sex-specific 99th percentile upper reference limits for high sensitivity cardiac troponin assays derived using a universal sample bank. clin. chem. 66(3):434–44. 30. djakpo, d. k., wang, z. q., and shrestha, m. (2020). the significance of transaminase ratio (ast/alt) in acute myocardial infarction. archives of medical sciences. atherosclerotic diseases, 5: e279–e283. 31. shamshirian, a., alizadeh-navaei, r., abedi, s., jafarpour, h., fazli, h., hosseini, s., hessami, a., et. al. (2020). levels of blood biomarkers among patients with myocardial infarction in comparison to control group. ethiopian journal of health sciences, 30(1), 5–12. 32. mc gill, m.r. (2016). the past and present of serum aminotransferases and the future of liver injury biomarkers. excli j 15: 817–28. 33. gjin, n., (2021). aspartate aminotransferase and cardiovascular disease a narrative review. journal of laboratory and precision medicine 6(6): 1–17. 34. panh, l., ruidavets, j. b., rousseau, h., petermann, a., bongard, v., bérard, e., et. al. (2017). association between serum alkaline phosphatase and coronary artery calcification in a sample of primary cardiovascular prevention patients. atherosclerosis, 260: 81–86. 35. vimalraj s. (2020). alkaline phosphatase: structure, expression and its function in bone mineralization. gene, 754: 144855. 36. birrer, r., takuda ,y. and takara, t. (2007). hypoxic hepatopathy: pathophysiology and prognosis. intern med. 46(14): 1063–70. 37. golabi, p., stepanova, m., pham, h.t., cable, r., rafiq, n., bush, h., et al. (2018). non-alcoholic steatofibrosis (nasf) can independently predict mortality in patients with non-alcoholic fatty liver disease (nafld). bmj open gastroenterol. 5(1): e000198. 38. kim, d., kim, w.r., kim, h.j. and therneau, t.m. (2013). association between noninvasive fibrosis markers and mortality among adults with nonalcoholic fatty liver disease in the united states. hepatology. 57(4): 1357–65. 39. simon, t.g., corey, k.e., cannon, c.p., blazing, m., park, j.g., o’donoghue, m.l., et al. (2018). the nonalcoholic fatty liver disease (nafld) fibrosis score, cardiovascular risk stratification and a strategy for secondary prevention with ezetimibe. international journal of cardiology. 270: 245–52. 40. soumya, r. s., raj, k. b., and abraham, a. (2021). passiflora edulis (var. flavicarpa) juice supplementation mitigates isoproterenol induced myocardial infarction in rats. plant foods for human nutrition, 76(2): 189–195. 41. ross-munro, e., kwa, f., kreiner, j., khore, m., miller, s. l., tolcos, m., fleiss, b. and walker, d. w. (2020). midkine: the who, what, where, and when of a promising neurotrophic therapy for perinatal brain injury. frontiers in neurology, 11: 568814. 42. fan, n., sun, h., wang, y., zhang, l., xia, z., peng, l., et al. (2014). midkine, a potential link between obesity and insulin resistance. plos one, 9(2): e88299. 43. cernkovich, e.r., deng, j., hua, k. and harp, j.b. (2007). midkine is an autocrine activator of signal transducer and activator of transcription 3 in 3t3-l1 cells. endocrinology 148: 1598–1604. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1255 158 journal of contemporary medical sciences editorial – march 2017 editorial it is my pleasure to be new editor-in-chief for the well-established and fast progressing journal of contemporary medical sciences. also i wish to thank the previous editor-in-chief (professor abbas bashi) for his support to the journal.this journal is well indexed in chemical abstracts (cas), directory of open access journals (doaj), ebsco, embase and google scholar with daily more than 500 visitors on the website. we are inviting all respected researchers of different medical science specialities to publish their works in this journal or ijph. for supporting the researcher & research activity, we have decided to waive the fees of the first paper publish in this journal or the new iraqi journal of public health for the iraqi post graduate students. we are in nab’a al-hayat foundation for medical sciences and health care, keen to establish excellent relationship and collaboration with all medical science research centres worldwide for developing high quality medical science & health care researches. the nab’a al-hayat foundation’s activity was achieved two successful international conference for medical education and basic scientific researches in iraq last year, and supporting students, in addition to that we designed mobile applications for vaccination to iraqi children. we have planning to do the new 2nd international medical congress in 13-14-sept 2017 in karbala, iraq. we are welcoming any medical research papers for publishing in our journals with special consideration for postgraduate students in both journal (jocms & ijph) best regards abbas taher editorinchief dear respected reader 69j contemp med sci | vol. 8, no. 1, january-february 2022: 69–74 original role of mitochondrial dna in development of type 2 diabetes mellitus with/without ischemic heart diseases of iraqi patients ali musa abed1, muneim maki al-shuk2 saja talib ahmed3, fadhil jawad al-tu’ma1* 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 2department of internal medicine, college of medicine, university of babylon, babylon, iraq. 3college of pharmacy, university of al-zahraa for women, kerbala, iraq. *correspondence to: fadhil jawad al-tu’ma (e-mail: f_altoma_56@yahoo.com) (submitted: 18 december 2021 – revised version received: 06 january 2022 – accepted: 23 january 2022 – published online: 26 february 2022) abstract objectives: this study aimed to investigate the associations between various biomarkers and the specific mutation of mitochondrial dna in type 2 diabetes mellitus with ischemic heart diseases and compared with t2dm patients without ischemic heart disease. methods: the study select two groups of patients admitted to kerbala heart center and al-hassan center for endocrinology and diabetes, al-hussein teaching hospital, al-hussein medical city, kerbala health directorates/kerbala – iraq between nov., 2020 and aug., 2021. the first group includes 50 patients of type 2 diabetes mellitus with ischemic heart disease (28 male and 22 female) with age ranged between 45–76 years, and the second group includes another 50 patients with type 2 diabetes mellitus without ischemic heart disease (24 male and 26 female) with age ranged between 49–82 years. fasting serum glucose, insulin and insulin resistance have been determined and then correlated with nutation mitdna investigated in sera of t2dm with/without ischemic heart diseases. results: the amplification of the mtll1 gene gives one genotypes as indicated by (422 bp) bands for those with homozygous wild type (aa), homozygous mutant (gg) genotypes and two genotypes bands (422 bp) for those with heterozygous (ga). the obtained data revealed that a strong positive correlation between homeostatic model assessment for insulin resistance (homa-ir) and insulin (r = 0.926) with significant differences (p < 0.05) was obtained in the sera of type 2 diabetic patient with ischemic heart disease as compared with that obtained in type 2 diabetic patients without ischemic heart diseases. conclusion: the prevalence of association between homa-ir with mttl1 g3243a mutation (gg allele) in type 2 diabetic patients with ischemic heart disease was only 8.0% and may be associated with maternally inherited of type 2 diabetes mellitus with ischemic heart disease as a pathogenic mutation in iraqi population. keywords: diabetes mellitus, type 2, myocardial ischemia, mitdna, homa-ir, mtll1 gene, sa-pcr issn 2413-0516 introduction diabetes mellitus, commonly known as diabetes, is a group of metabolic disorders characterized by a high blood sugar level over a prolonged period of time. one of the most important symptoms is polyuria, weight loss, constant thirst.1 if left untreated, diabetes can cause many complications. acute complications can include diabetic ketoacidosis, hyperosmolar hyperglycemic state, or death. serious long-term complications include cardiovascular disease, stroke, chronic kidney disease, foot ulcers, damage to the nerves, damage to the eyes and cognitive impairment.2 type 1 diabetes mellitus previously called insulin dependent diabetes mellitus (iddm) is caused by loss of insulin-secreting capacity due to selective autoimmune destruction of the pancreatic beta cells. insulitis (i.e., mononuclear-cell infiltration of the pancreatic islets) is the direct result of the autoimmune process.3 mitochondrial dna comprises 0.1–2% of the total dna in most mammalian cells. there are several unique features of the mitdna: human mitdna is circular, 16 kbp long, and inherited from the mother. it encodes two rrnas, 22 trnas, and 13 proteins, all of which are involved in the oxidative phosphorylation process.4 the intragenic sequence is almost absent or limited to a few bases,5 and mitdna does not have histones, instead it is organized in nucleoid structures. a large number of experiments showed that multiple copies of mitdna could be found in each nucleoid, usually from two to 10 copies each, depending on the cell line studied.6 two different strands can be recognized in the mitdna: the heavy strand rich in guanine bases, which also contain the majority of mitochondrial coding genes, and the light strand, encoding only for the mt-nd6 (nadh-ubiquinone oxidoreductase chain 6) protein and eight trnas. both strands are transcribed at the same time, giving origin to very long transcripts, of almost mitdna length, that are subsequently processed. transcription seems to take place in the nucleoids due to the presence of the mitochondrial transcription machinery. the process of mitochondrial transcription termination is still unclear. there is still a debate if mterf1 is really needed for the termination of all the transcription processes that originate from the three different promoters of the control region. biochemical studies have shown that mterf1 only partially terminates h-strand transcription7 whereas transcription in the opposite direction (l-strand transcription) is almost completely blocked. many different proteins are involved in the regulation of transcription, such as hormones, nuclear transcription factors, and chromatin remodeling enzymes which are also able to interact with the mitdna, and rna/dna modifying enzymes. one of the first proteins investigated to be involved in the regulation of transcription is the thyroid hormone t3, which is able to promote the mitdna transcription by directly binding the mitdna genes.8 glucocorticoid hormones were also found to be in mitochondria where they modulate the transcription binding to the glucocorticoid receptor present in the mitochondrial inner membrane.9 the estrogen receptor (er) was found in the mitochondria of cardiac cells. it was mailto:f_altoma_56@yahoo.com 70 j contemp med sci | vol. 8, no. 1, january-february 2022: 69–74 role of mitochondrial dna in development of type 2 diabetes mellitus with/without ihd of iraqi patients original a.m. abed et al. hypothesized that e2 (17β-estradiol) and erβ-mediated cardioprotection was dependent on mitdna transcription encoding for mitochondrial respiration activity. it was also demonstrated that e2 can also increase the er β mitdna binding activity followed by an increase in complex v encoding gene expression.10 melatonin was also described as a potential hormone that can control the mitdna expression through the reduction of several mitochondrial transcription factors. it was demonstrated that melatonin was able to decrease, at both mrna and protein levels, various proteins such as transcription factors tfb1m and tfb2m, interfering with mitdna transcription.10 the mitochondrial oxidative phosphorylation system is made up of five multi-subunit enzyme complexes that are found on the inner mitochondrial membrane. the mitdna encodes one or more of the necessary components for the nadh-ubiquinone oxidoreductase (complex i), ubiquinonecytochrome c oxidoreductase (complex iii), cytochrome c oxidase (complex iv), and atp synthase (complex v), whereas ndna encodes the complete succinate-ubiquinone oxidoreductase.11,12 the mitdna strands are known as the heavy strand (h-strand) and the light strand (l-strand), with the former being guanine rich and the latter being cytosine rich. the h-strand encodes 28 genes, whereas the l-strand encodes the remaining nine. the a3243g mutation of the mitochondrial trna(leu) gene was found to segregate with maternally inherited diabetes mellitus, sensorineural deafness, hypertrophic cardiomyopathy, or renal failure in a large pedigree of 35 affected members in four generations.13 presenting symptoms almost consistently involved deafness and recurrent attacks of migraine-like headaches, but the clinical course of the disease varied within and across generations. the a3243g mutation has been previously reported in association with the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode syndrome (melas) and with diabetes mellitus and deafness.14 the aim of the presented work is to investigate a genetic mutation of mitochondrial dna in iraqi type 2 diabetic patients with ischemic heart diseases of kerbala province: iraq, and its correlation with insulin resistance as compared with those diabetic patients without ischemic heart diseases. materials and methods the current study was cross-sectional study which includes two groups. the first group includes 50 patients of type 2 diabetes mellitus with ischemic heart disease (28 male and 22 female) with age ranged between 45–76 years., and the second group includes another 50 patients with type 2 diabetes mellitus without ischemic heart disease (24 male and 26 female) with age ranged between 49–82 years. the study was managed throughout the period between nov., 2020–aug. 2021. the sample collected from kerbala heart center and al-hassan center for endocrinology and diabetes, al-hussein teaching hospital, al-hussein medical city, kerbala health directorates/kerbala – iraq. the biomarker parameters investigation and molecular studies were done in the laboratories of department of chemistry and biochemistry, college of medicine, university of kerbala and al-hussein teaching hospital laboratories’. the protocols of the study were approved by ethical committee after a verbal written informed consent for participation and for taking a blood samples for investigations from everyone enrolled in this study. five milliliters of blood was drawn by vein puncture from all individuals participated in this study after taking the patient’s consent. the collected blood was divided into three parts, one ml of blood was used for molecular analysis, collected in edta containing tube and used for dna extraction, then was analyzed directly to obtain high purity of dna. another one ml was placed in edta containing tube for analyzing hba1c%. the remaining 3.0 ml of blood withdrawn was placed in a gel tube and left for 15 minutes at room temperature for coagulation, then centrifuged for 15 minutes at 3000 xg. serum was collected, then frozen till analyses for determination of various biomarkers including insulin and homeostatic model assessment for insulin resistance (homa-ir). mitochondrial dna was extracted from whole blood that collected from patient and control groups by using dna isolation kit “g-spintm total dna extraction kit” (cyntol). genotyping will be carried out by allele-specific arms-pcr for –3243 a/g snp of prkcb1 (a3243g), primers and a master mix kit (promega) were used; pcr products were separated on a 1.0% agarose gel electrophoresis. two primers for allele specific-pcr designing were used for the detection of –3243 a/g polymorphism of mtll1. the amplification-refractory mutation system (arms), is considered a simple, fast, and reliable technique for detecting any mutation include single base changes. arms is based on the use of sequence-specific pcr primers that promote amplification of test dna only when the target allele is included within the specimen and will not amplify the non-target allele. following an arms reaction the existence or absence of a pcr product is detection for the existence or absence of the target allele. for the mutant-specific primer (m), the 3' terminal base of the arms primer should be complementary to the mutation sequence; for the normal-specific primer (n), the 3' terminal base should be complementary to the corresponding normal sequence.15 the pcr reaction program procedures for snp (–1504 c /t) in prkcb-1 was presented in table 1. the 1.0% agarose solution was prepared using trisborate edta buffer which was diluted 1:10 with deionized water. about 4 μl of pcr product were loaded to each well with great precaution to prevent damages of the wells and cross contamination of neighboring wells. an electric field (50 v for 35 min) was established to the system causing the negatively charged nucleic acids to travel across the gel to the positive electrode (anode), and 4 μl of dna ladder (1000 bp intron) was used as standard and band size ladder was 100–1000 bp. to visualize the dna bands, the agarose gel was placed in the table 1. the optimized pcr reaction program type of cycle temperature ˚c time no. of cycle intial denaturation 95 5 min 1 cycle denaturation 95 30 sec anealing 61 30 sec 35 cycle extension 72 60 sec final extension 72 5 min total time: 1 hour and 35 min 71j contemp med sci | vol. 8, no. 1, january-february 2022: 69–74 a.m. abed et al. original role of mitochondrial dna in development of type 2 diabetes mellitus with/without ihd of iraqi patients uv trans illuminator device and exposed to uv light and the photos were captured by digital camera linked to pc. following an arms reaction the existence or absence of a pcr product is detection for the existence or absence of the target allele. for the mutant-specific primer (m), the 3' terminal base of the arms primer should be complementary to the mutation sequence; for the normal-specific primer (n), the 3' terminal base should be complementary to the corresponding normal sequence.16 total 422 nucleotides containing dna was amplified by polymerase chain reaction (pcr), the forward primer was taken from nucleotide sequence 3035 to 3054 as 5'gca aga gat aca gtg ttg ctc ca -3' and the reverse primer was taken from nucleotide sequence 3437 to 3456 as 5'cgt tct cta tgt cac aac gag gt-3' after electrophoresis, absence of the mutation generates a single band (422 bp). the amplification product of mttl1 gene polymorphism (snp of a3243g) detected by allele specific pcr reaction, have a size of 422 bp. the pcr product was electrophoresed on 1.0% agarose and directly was visualized with ethidium bromide under uv light. the optimization of pcr assay constituents used was indicated in table 2. green master mix is a (ready-to-use solution) encompass bacterially derived taq dna polymerase, dntps, mgcl2 and reaction buffers at optimal concentrations for efficient amplification of dna templates by pcr. fifty ml of 1% agarose solution was prepared in 10x tbe buffer and three μl of ethidium bromide was added to the solution. the gel solution was poured into the chamber and permitted to be harden for approximately 30 minutes at room temperature. then combs were removed, and then samples and dna ladder were loaded (4 μl of 1000 bp, intron was used as standard and band size ladder was 100–1000 bp) on each well with extreme cautions to avoid damages of the wells and cross contamination of neighboring wells and then placed in a horizontal electrophoresis system and covered with the same tbe buffer that used to prepare the gel. the cathode (black) was connected to the wells side of the unit and the anode (red) to the other side. electrophoresis is attach to direct current power source until dye markers migrated to the suitable distance, according to the size of dna fragment that recognized. about 4 μl of pcr product were loaded to each well with great precaution to prevent damages of the wells and cross contamination of neighboring wells. an electric field (50 v for 35 min) was established to the system causing the negatively charged nucleic acids to travel across the gel to the positive electrode (anode). to visualize the dna bands, the agarose gel was placed in the uv trans illuminator device and exposed to uv light and the photos were captured by digital camera linked to pc. results and discussion during the current cross-sectional study, 50 of cases include t2dm patients with ischemic heart disease and another 50 diabetic patients without ischemic heart disease groups was enrolled. the anova test found that there was a non-significant difference in age between diabetic patient have ihd and without ihd groups. this age matching helps to eliminate differences in parameters, figure 1. all patient group study comprised of 46 females and 54 males, this result was different with maas and appelman et al.17 who was found that cardiovascular disease effects women 7 to 10 years later than it does males, yet it remains the leading cause of mortality in women, due to the misunderstanding that women are “protected” from cardiovascular disease, the risk of heart disease in women is frequently overestimated. because of the under-recognition of cardiac disease and the variations in clinical presentation between men and women, less aggressive treatment options are used and women are neglected in clinical trials. furthermore, women’s self-awareness and identification of their cardiovascular risk factors require more attention, which should lead to better cardiovascular event prevention, table 3.18 the mean ± sd of bmi in type 2 diabetic patients without ihd was (31.6818 ± 4.42 kg/m2) which was non-significantly higher than that found in type 2 diabetic patients with ihd (30.0903 ± 4.99 kg/m2) and the (p > 0.05). some study demonstrated that obesity is a complex metabolic condition reported that affects 35% of the adult population in the united states, according to the national institutes of health. as a significant risk factor for ischemic heart disease (ihd) and its metabolic consequences, it has elevated to become one of the most serious health problems in many regions of the world.19 it was not noticed that a significant differences in body bmi between type 2 diabetic patients with/without heart disease and this results indicates the size of the sample used and the time obtained for blood samples. although there are most studies that indicate a significant clinical relationship between body weight and heart disease.20 the mean ± sd of hba1c% in type 2 diabetic patients with ischemic heart disease was (9.674 ± 1.72%) which was slightly non-significantly higher than that found in patients without ischemic heart disease (9.64 ± 2.087%) (p = 0.921), this data was disagreement with other study which found that hba1c was associated with cardiovascular disease (cvd), such as carotid and coronary artery atherosclerosis, ischemic heart disease, ischemic stroke, and hypertension, among other table 2. optimization constituents of pcr components used reagent volume forward primer 2 μl reverse primer 2 μl master mix 10 μl nuclease free water 5 μl dna template 5 μl total volume 25 μl fig. 1 number and percentage of age in t2dm with and without ihd groups. 72 j contemp med sci | vol. 8, no. 1, january-february 2022: 69–74 role of mitochondrial dna in development of type 2 diabetes mellitus with/without ihd of iraqi patients original a.m. abed et al. things. hba1c causes dyslipidemia, hyperhomo-cysteinemia, and hypertension, as well as an increase in c-reactive protein level, oxidative stress, and blood viscosity, all of which are associated with the development of cardiovascular illnesses.21 the mean ± sd of insulin level determined in both group of diabetic patients studied indicated that its level in type 2 diabetic patients with ischemic heart disease was (6.86 ± 4.31 µu/ml) which non-significantly higher than that found in type 2 diabetic patients without ischemic heart disease is (6.03 ± 5.234 µu/ml) with p > 0.05, table 4. cardiovascular illnesses are the leading cause of death worldwide,22 and type 2 diabetes is one of them because it is so common and doubles the risk of heart disease. increased glucose and insulin concentrations, as a result, have been proven to be pro-atherogenic causes,23 whereas other study showed that cardiovascular diseases may be a consequence of insulin resistance rather than being caused by toxic effects of high insulin or glucose concentrations, table 4.24 the level of homa-ir found in type 2 diabetic patients with ischemic heart disease was (3.351 ± 2.38) but in diabetic patients without ischemic heart disease were (2.65 ± 2.41) and the results is a non-significant (p-value > 0.05), table 4. assessment of the homeostasis model insulin resistance (homa-ir) is a widely used and validated diagnostic of insulin resistance that includes both glucose and insulin concentrations. insulin resistance, increase the risk of atherosclerosis through a variety of pathways25 and it has been linked to coronary artery disease. table 4 also shows the results concerning the fasting blood glucose level in sera of type 2 diabetic patients with/ without ischemic heart diseases. the mean ± sd of fbg level determined in both group of patients studied indicated that its level in type 2 diabetic patients with ischemic heart disease was (198.9 ± 42.283 mg/dl) which is non-significantly higher than that observed in t2dm without ihd (185.5 ± 56.77 mg/dl), (p > 0.05). the impact of hyperglycemia on coronary heart disease (chd),26 stroke,27 and other cardiovascular diseases (cvds)28 has been widely studied. in people with hyperglycemia, two-hour plasma glucose (2hpg) is a better predictor of coronary heart disease (chd) and ischemic stroke (is) than fasting plasma glucose (fpg), but nothing is known regarding their impact in the normoglycemic range. insulin resistance and beta cell dysfunction are already evident in people with increased normal fpg, table 4.29 the molecular investigations concerning mitochondrial dna found just four cases has g allele in patient have ischemic heart disease, and one case without ischemic heart disease as in the figure 2. the study showed that the g allele is responsible for heart disease, an individual’s carrying the mttl1 a3243g mutation have been diagnosed with ischemic cardiac disease.30 the mutation affects mitochondrial dna structure, stability, methylation, amino-acylation, and codon recognition capabilities, making it difficult to couple the mrna codon with the mutant trna anticodon.31 this condition is most commonly linked to an a to g transition in the mitochondrial dna at location 3243. incorrect rna processing results in reduced translation as well as decreased rates of protein synthesis and enzyme activity. a statistically significant negative relation was observed between the frequency of mttl1 a3243g mutations and the particular activity of the mitochondrial respiratory chain complex (figure 2). the observed results found that the mean ± sd of homa-ir in (38/50) of type 2 diabetic patient without ischemic heart disease have predominantly aa allele of mttl1 a3243g mutation (38/50) in their blood (2.881 ± 1.64), and (16/50) of type 2 diabetic patient with ischemic heart disease was (3.344 ± 1.951) whereas, only one type 2 diabetic patient without ihd indicate homa-ir have gg allele of mttl1 a3243g mutation in his blood (1.114 ± 0.031). on the other hand, only (4/50, 8%) of type 2 diabetic patients with ihd have gg allele of mttl1 a3243g mutation and the homa-ir observed was (4.24 ± 1.76) table 5. the genotype of gg allele in multigenerational impact of the mttl1 a3243g increases the risk of homa-ir in mitochondria and their genome are found in the cytoplasm of cells, with thousands of copies of the mitochondrial genome in most cell types. heteroplasmy occurs when not all copies of the mitochondrial genome have the same sequence at the tissue or even cellular level with resulting in different proportions of mutant and wild type mitochondria.32 table 3. number and percentage of diabetic patient with ihd and without non-ihd according to their gender status gender t2dm cases total p-value without ihd with ihd female 22 24 46 0.421 44.0% 48.0% 46.0% male 28 26 54 56.0% 52.0% 54.0% total 50 50 100 table 4. show the level of hba1c%, insulin, homa-ir and blood glucose concentration in t2dm with/without ischemic heart disease n mean ± sd p-value hba1c% without 50 9.64 ± 2.087 0.921 with 50 9.674 ± 1.72 insulin, µu/ml without 50 6.03 ± 5.234 0.392 with 50 6.86 ± 4.31 homa-ir without 50 2.65 ± 2.41 0.145 with 50 3.351 ± 2.38 fbg, mg/dl without 50 185.5 ± 56.77 0.184 with 50 198.9 ± 42.283 fig. 2 the relationship between multigenerational impact of the mttl1 a3243g with homeostatic model assessment for insulin resistance. 73j contemp med sci | vol. 8, no. 1, january-february 2022: 69–74 a.m. abed et al. original role of mitochondrial dna in development of type 2 diabetes mellitus with/without ihd of iraqi patients when mitochondria are randomly segregated to each new cell during cell division, the fraction of mitochondrial dna containing a mutation may vary between daughter cells as a result of heteroplasmy. when the load or proportion of mitochondrial dna with a harmful mutation exceeds a certain threshold level, tissues show pathogenic effects of mutation.33 as illustrated in figure 3, the successful amplification and analysis of the snp of multigenerational impact of the mttl1 a3243g was achieved using the a3243g. detection of pcr bands of suitable size in the 1% agarose gel indicated that the samples were of the appropriate genotype. the amplification product of mttl1 gene polymorphism (snp of a3243g) detected by allele specific pcr reaction, have a size of 422 bp. the observed data concerning mitdna was disagreed with other study which screened 142 patients of type 2 diabetes mellitus and 142 healthy control individuals to detect 3243 a/g mutation, their ages of the onset for type 2 diabetes mellitus varied from 34 to 52 years. they found a disappearance of 3243 a/g mutation in type 2 diabetes patients and healthy control individuals. the levels of fasting and postprandial glucose indicate severe hyperglycemia, resulted in high glycosylation of hemoglobin (hba1c),34 whereas, in this study the 3243 a/g mutation it is appeared in type 2 diabetes mellitus with ischemic heart disease. conclusion according the observed results we can conclude that the prevalence of association between homa-ir with mttl1 g3243a mutation (gg allele) in type 2 diabetic patients with ischemic heart disease was only 8.0% and may be associated with maternally inherited of type 2 diabetes mellitus with ischemic heart disease as a pathogenic mutation in iraqi population.  table 5. the relationship between multigenerational impacts of the mttl1 a3243g with homa-ir t2dm number of patients and percentage mttl1 gene homa-ir mean ± sd p-value without ihd, n = 50 38/50 (76%) aa 2.881 ± 1.64 0.013 11/50 (22%) ga 1.974 ± 1.33 1/50 (2%) gg 1.114 ± 0.031 with ihd, n = 50 16/50 (32%) aa 3.344 ± 1.951 30/50 (60%) ga 3.236 ± 2.68 4/50 (8%) gg 4.24 ± 1.76 fig. 3 the electrophoresis profiles for some of the successful amplifications. multigenerational impact of the mttl1 a3243g. m = lane for dna ladder marker; 1,2 = lane for hetrozygote patient; 3,4 = lane for g allele patient; 5,6 = lane for a allele patient 7,8 = lane for hetrozygote patient. references 1. barnard, k. d., lloyd, c. e., and holt, r. i. (2012). psychological burden of diabetes and what it means to people with diabetes. in psychology and diabetes care (pp. 1–22). springer, london. 2. soumya, d. and b. srilatha (2011). “late stage complications of diabetes and insulin resistance.” j diabetes metab 2(9):1000167. 3. roep, b. o. and t. i. tree (2014). “immune modulation in humans: implications for type 1 diabetes mellitus.” nature reviews endocrinology 10(4):229–242. 4. barchiesi, a. and c. vascotto (2019). “transcription, processing, and decay of mitochondrial rna in health and disease.” international journal of molecular sciences 20(9):2221. 5. ojala, d., montoya, j. and attardi, g. (1981). trna punctuation model of rna processing in human mitochondria. nature 290:470–474. 6. iborra, f. j., kimura, h. and cook, p. r. (2004). the functional organization of mitochondrial genomes in human cells. bmc biology, 2:1–14. 7. terzioglu, m., ruzzenente, b., harmel, j., mourier, a., jemt, e., lópez, m. d., kukat, c., stewart, j. b., wibom, r. and meharg, c. (2013). mterf1 binds mtdna to prevent transcriptional interference at the light-strand promoter but is dispensable for rrna gene transcription regulation. cell metabolism 17:618–626. 8. psarra, a. m. g. and sekeris, c. e. (2008). steroid and thyroid hormone receptors in mitochondria. iubmb life, 60:210–223. 9. lapp, h. e., bartlett, a. a. and hunter, r. g. (2019). stress and glucocorticoid receptor regulation of mitochondrial gene expression. journal of molecular endocrinology, 62:r121-r128. 10. hsieh, y.-c., yu, h.-p., suzuki, t., choudhry, m. a., schwacha, m. g., bland, k. i. and chaudry, i. h. (2006). upregulation of mitochondrial respiratory complex iv by estrogen receptor-β is critical for inhibiting mitochondrial apoptotic signaling and restoring cardiac functions following trauma– hemorrhage. journal of molecular and cellular cardiology, 41:511–521. 11. lott, m. t., j. n. leipzig, o. derbeneva, h. m. xie, d. chalkia, m. sarmady, v. procaccio and d. c. wallace (2013). “mitdna variation and analysis using mitomap and mitomaster.” current protocols in bioinformatics 44(1):1–23. 12. rhee hw, zou p, udeshi nd, martell jd, mootha vk, carr sa, ting ay. proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. science. 2013 mar 15;339(6125):1328-31. 13. park, h., davidson, e. and king, m. p. (2008). overexpressed mitochondrial leucyl-trna synthetase suppresses the a3243g mutation in the mitochondrial trnaleu (uur) gene. rna, 14(11):2407–16. 14. aurangzeb, s., vale, t., tofaris, g., poulton, j., and turner, m. r. (2014). mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (melas) in the older adult. practical neurology, 14(6):432–436. 15. little rj. modeling the drop-out mechanism in repeated-measures studies. journal of the american statistical association. 1995 sep 1;90(431):111221. 16. collins, a., and ke, x. (2012). primer1: primer design web service for tetraprimer arms-pcr. the open bioinformatics journal, 6(1). 17. maas, a. h., and appelman, y. e. (2010). gender differences in coronary heart disease. netherlands heart journal, 18(12):598–603. 18. claassen, m., sybrandy, k. c., appelman, y. e., and asselbergs, f. w. (2012). gender gap in acute coronary heart disease: myth or reality? world journal of cardiology, 4(2):36. 19. nikpay m, goel a, won hh, hall lm, willenborg c, kanoni s, saleheen d, kyriakou t, nelson cp, hopewell jc, webb tr. a comprehensive 1000 genomes-based genome-wide association meta-analysis of coronary artery disease. nature genetics. 2015 oct;47(10):1121. 20. benn m, watts gf, tybjaerg-hansen a, nordestgaard bg. familial hypercholesterolemia in the danish general population: prevalence, coronary artery disease, and cholesterol-lowering medication. the 74 j contemp med sci | vol. 8, no. 1, january-february 2022: 69–74 role of mitochondrial dna in development of type 2 diabetes mellitus with/without ihd of iraqi patients original a.m. abed et al. journal of clinical endocrinology & metabolism. 2012 nov 1;97(11): 3956-64. 21. prasad, k. (2018). does hba1cc play a role in the development of cardiovascular diseases?. current pharmaceutical design, 24(24): 2876–2882. 22. nowbar, a. n., gitto, m., howard, j. p., francis, d. p., and al-lamee, r. (2019). mortality from ischemic heart disease: analysis of data from the world health organization and coronary artery disease risk factors from ncd risk factor collaboration. circulation: cardiovascular quality and outcomes, 12(6):e005375. 23. yu, q., gao, f., and ma, x. l. (2011). insulin says no to cardiovascular disease. cardiovascular research, 89(3):516–524. 24. vafaeimanesh, j., parham, m., norouzi, s., hamednasimi, p., and bagherzadeh, m. (2018). insulin resistance and coronary artery disease in non-diabetic patients: is there any correlation?. caspian journal of internal medicine, 9(2):121. 25. aydin, e., and ozkokeli, m. (2014). does homeostasis model assessment of insulin resistance have a predictive value for post-coronary artery bypass grafting surgery outcomes?. brazilian journal of cardiovascular surgery, 29:360–366. 26. doi, y., ninomiya, t., hata, j., fukuhara, m., yonemoto, k., iwase, m., et. al. (2010). impact of glucose tolerance status on development of ischemic stroke and coronary heart disease in a general japanese population: the hisayama study. stroke, 41(2):203–209. 27. hyvärinen, m., qiao, q., tuomilehto, j., laatikainen, t., heine, r. j., stehouwer, c. d., et. al. (2009). hyperglycemia and stroke mortality: comparison between fasting and 2-h glucose criteria. diabetes care, 32(2):348–354. 28. levitan eb, song y, ford es, liu s. is nondiabetic hyperglycemia a risk factor for cardiovascular disease?: a meta-analysis of prospective studies. archives of internal medicine. 2004 oct 25;164(19):2147-55. 29. lau, l. h., lew, j., borschmann, k., thijs, v., and ekinci, e. i. (2019). prevalence of diabetes and its effects on stroke outcomes: a meta‐analysis and literature review. journal of diabetes investigation, 10(3):780–792. 30. tuppen, h. a., blakely, e. l., turnbull, d. m., and taylor, r. w. (2010). mitochondrial dna mutations and human disease. biochimica et biophysica acta (bba)-bioenergetics, 1797(2):113–128. 31. finsterer, j. (2007). genetic, pathogenetic, and phenotypic implications of the mitochondrial a3243g trnaleu (uur) mutation. acta neurologica scandinavica, 116(1):1–14. 32. stefano, g. b., bjenning, c., wang, f., wang, n., and kream, r. m. (2017). mitochondrial heteroplasmy. mitochondrial dynamics in cardiovascular medicine, 577–594. 33. guan wj, ni zy, hu y, liang wh, ou cq, he jx, liu l, shan h, lei cl, hui ds, du b. clinical characteristics of coronavirus disease 2019 in china. new england journal of medicine. 2020 apr 30;382(18):1708-20. 34. dongre, u. j. and v. g. meshram (2020). “detection of 3243 a/g and 3316 g/a mitochondrial dna mutations in nagpur population.” journal of applied biology and biotechnology vol 8(03):37–41. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1164 31j contemp med sci | vol. 8, no. 1, january-february 2022: 31–37 original teaching clinical decision-making skills to undergraduate nursing students via web-based virtual patients during the covid-19 pandemic: a new approach to the cyberpatienttm simulator toktam masoumian hosseini1, soleiman ahmady1,2* , samuel edelbring3 1department of medical education, virtual school of medical education & management, shahid beheshti university of medical sciences, tehran, iran. 2research affiliated faculty at department of lime, karolinska institute, solna, sweden. 3department of learning, informatics, management and ethics, karolinska institute, stockholm, sweden. *correspondence to: soleiman ahmady (e-mail: soleiman.ahmady@gmail.com) (submitted: 11 december 2021 – revised version received: 28 december 2021 – accepted: 19 january 2022 – published online: 26 february 2022) abstract objective: the aim of this study was to investigate the effects of using a virtual patient simulator on the acquisition of clinical decisionmaking skills in nursing students during the pandemic covid-19. methods: this was a quasi-experimental study with a pretest-posttest design. following the case-based learning strategy, the educational intervention was designed and implemented in five steps (pre-activities, introduction, scenario briefing, web-based clinical scenarios, presentation and de-briefing). we assessed clinical decision-making skills of nursing students before the intervention, after the intervention, and 1 months later, with clinical decision-making questionnaire. in this study spss software version 23.0 was used to analyze the data and significance level was considered p < 0.05. results: clinical decision-making skills of nursing students was compared before (48/04 ± 12/77) and immediately after training (91/49 ± 7/66) using paired tests, and a statistically significant difference was found (p = 0/009). also, before the intervention, most students were thinking analytically (63/80%) and making clinical decisions, while after the intervention, most students had an analytic-intuitive model of clinical decision-making (63/80%). conclusion: the study showed that the decision-making skills of nursing students were significantly improved by virtual patient simulations. the educational intervention and simulator used in this study can be integrated into undergraduate nursing student education curricula to help them acquire clinical decision-making skills. keywords: nursing students, clinical decision-making, simulation education, virtual simulation, skill issn 2413-0516 introduction one of the most important aspects of nursing education is teaching clinical decision-making skills.1 because of the complexity of the legal and professional issues they face today, nursing students must acquire sufficient skills to make clinical decisions during their education.2 furthermore, the ability to diagnose and treat patients quickly and accurately is not a skill that can only be learned theoretically.3 currently, after training courses and before students enter the practical stage and deal directly with patients, they teach them how to diagnose disease quickly, make the right decision, take action accordingly, and deal with patients professionally.4–5 for this purpose, considering the achievements of technology, it is recommended to do planning so that it can be integrated into the clinical education curriculum.6 in this regard, technology has been used in nursing education for years to enhance teaching and learning.7 in the meanwhile, due to the increasing incidence and prevalence of covid-19 worldwide and in iran since the beginning of the twentieth century, all clinical rotations and training opportunities have been suspended to reduce the burden of the disease.8–9 a major challenge in the current situation is how to continue medical education when there is no direct contact with patients.9 in order to solve this challenge, we need to pay more attention to the capabilities of technology in clinical education. the use of simulation-based learning platforms began before covid-19, but the pandemic has also led to an increase in the demand for and use of alternate, innovative methods for training nurses.10 several research studies on integrating technology into clinical education suggest that simulation-based learning can help nursing students develop their clinical decision-making skills in complex situations.11 these patients are based on a scientifically problem-based design and structure that mimics the various stages of dealing with real patients, beginning with the initial interview and continuing through treatment.12 real emergency patients need to be treated immediately. in these situations, patient care is a priority that cannot be postponed for the student’s presence. in this situation, the student is in a passive and observational role and does not make decisions.13 while by working with a virtual patient, the student has the opportunity to work with severe, abnormal, and critical patients. the student has the opportunity to work with patients who have complex physical and mental conditions, make mistakes, then receive feedback and reapply the correct method. during virtual patient simulations, time and urgency constraints are controlled. in other words, time pressure can be removed when the student needs to concentrate.14 research has shown that simulation-based education not only improves students’ mental abilities, including their clinical reasoning skills, but also positively affects their attitudes.15 in addition, this method has a significant impact on students’ acquisition of skills in the application of techniques. as a http://orcid.org/0000-0003-0551-6068 32 j contemp med sci | vol. 8, no. 1, january-february 2022: 31–37 teaching clinical decision-making skills to undergraduate nursing students original t.m. hosseini et al. result, nursing educators are increasingly incorporating virtual simulations into their curricula.16 in the field of clinical education, there are many simulated environments designed to enhance students’ scientific and practical skills and prepare them for real-life situations.17 despite the numerous technology-based programs for clinical education, there are few computer programs that can simulate the realism of the doctor-patient relationship.15 one of the simulators currently being used in clinical education is the cp-platform developed by the department of surgery at the college of british columbia. cp has managed to accomplish this task and make this long-standing dream of instructors and students a reality.18 the philosophy behind cp-platform is that traditional medical education starts with etiology and pathogenesis and then moves on to signs and symptoms of the disease. but in real cases, the patient goes to the doctor with the chief complaint, and the doctor has to think backward after examining the signs and symptoms to find out the cause and pathogenesis of the disease. this difference has led to many challenges being encountered by trainees, doctors, and novice nurses in attempting to apply theoretical knowledge in practice. this is one of the reasons why medical and nursing schools have moved to a problem-solving, case-based curriculum. these changes brought a number of challenges and issues including patient availability, the need to increase the number of clinical faculty, and ultimately an increase in the cost of medical education. consequently, the centralized use of computers may be the solution to the challenges associated with a problemor case-based curriculum.19 the cp interactive learning system is based on problem solving and clinical decision making. in the system, students gather information with menus that include laboratory results, clinical examination of the patient, and diagnosis and treatment of the patient’s condition. students can also select cases of increasing difficulty so they can work on more advanced aspects of diagnosis and treatment for that particular condition each time.19 the results of the study conducted by farahmand et al. (2020) show that the use of cp virtual patient simulator has been effective in enhancing students’ history taking skill, increasing the effectiveness of clinical education and reducing costs.20 there is limited evidence that simulation can be an effective teaching and learning tool for clinical decision making. however, no study to date has measured the impact of cyberpatient simulation on students’ clinical decision-making skills. therefore, the present study investigated the impact of using this virtual patient simulator on the acquisition of clinical decision-making skills by nursing students at shahid beheshti university of medical sciences (sbmu) in iran. theoretical framework the national league for nursing jeffries simulation theory21 was combined with international nursing association for clinical simulation and learning (inacsl) standards of best practice (sobp)22 to form the theoretical basis for this study. simulation activities are designed, implemented, and evaluated using concepts laid out in jeffries’ nln theory. this theory focuses on context, background, design, simulation experiences, facilitators and educators, participants, and outcomes. as outlined in the inacsl sobp, a valid and rigorous simulation experience requires well-defined and content-aligned outcomes, qualified faculty, adequate student preparation, theory-based debriefing, and curriculum integration.21,23 materials and methods study design and setting this was a quasi-experimental study with a pretest-posttest design. it was conducted between 2020 and 2021 to evaluate the effectiveness of the cp training programme on the clinical decision-making skills of nursing students at sbmu. study participants and sampling all third-year nursing students who had completed their clerkship at the time of the study (n = 58) were selected by census method. the inclusion criteria for the present study were interest in participating in the research and completion of a course in medical-surgical nursing. exclusion criteria for participants included refusal to proceed with the research, failure to attend an educational session, and failure to complete the research instruments in the second phase of data collection. ethical consideration informed consent was obtained from all participants. they were assured that their personal information would be kept confidential and only general statistics and data would be published. this project was conducted in the form of phd student thesis and approved by the ethics committee of shahid beheshti university of medical sciences (ethics code: ir.sbmu.sme.rec.1400.044) address: https://ethics. research.ac.ir/ethicsproposalview.php?id = 213812. intervention in a briefing session, students learned how to use the cyberpatient system and what is expected of them during the training process. the screen interface components and navigation boxes were described. students were given 10 minutes to familiarize themselves with the software and completed the demographic information questionnaire and clinical decision-making (cdm) instrument during this session. based on the educational strategy of case-based learning, the educational intervention was designed and implemented in five steps (pre-activities, introduction, scenario briefing, web-based clinical scenarios, presentation and de-briefing) (figure 1). 1. pre-activities in accordance with the clinical course plan for cardiology diseases in nursing students’ internships, eight cardiovascular disease case studies were selected from the cp case library (figure 2-1). the cases were placed in the virtual classroom dashboard in the cp system for students to access. the educational intervention was conducted in the university clinical laboratory for eight weeks. students participated in a clinical exercise each week under the supervision of a clinical professor and worked on a clinical case. for each case, the following steps were completed in order. 2. introduction a clinical instructor discussed the educational goals to be achieved after working with the virtual patient (figure 2-2). https://ethics.research.ac.ir/ethicsproposalview.php?id=213812 https://ethics.research.ac.ir/ethicsproposalview.php?id=213812 33j contemp med sci | vol. 8, no. 1, january-february 2022: 31–37 t.m. hosseini et al. original teaching clinical decision-making skills to undergraduate nursing students he also asked questions to inquire about the all-encompassing prior knowledge relevant to the case. 3. scenario briefing students began working with the virtual patient after the clinical professor briefly introduced the case. 4. clinical scenarios presentation we scheduled and conducted a one-hour session with the virtual patient each time, beginning with the students logging into the simulator (https://app.cyberpatient.ca) until the system provided feedback (figure 1). users accessed the system using their assigned usernames and passwords. once the student logged in, the virtual patient became available. after interviewing, examining the virtual patient’s body systems, gathering information, and reviewing lab test requests related to the patient, the student moved on to the diagnosis phase and selected an appropriate treatment based on their diagnosis. this phase ended with the simulator providing feedback to the student on his actions during the history taking, physical examination, diagnosis, and treatment phases. by selecting the case on the screen, the virtual patient was presented in this simulated environment. text, images, videos, and animations were used in an interactive virtual patient experience. this system allowed the student to enter a question and ask it to a virtual patient. the software then searched for the keyword in the question and returned an answer with voice and dialogue from the virtual patient (figure 2-3). by clicking on the left side of the mouse, the user could explore the systems related to the patient’s problem. in the toolbar of the software, there were a number of tools for monitoring and examining patients. if needed, the student could take a blood sample from a virtual patient, insert a nasal cannula, and take the patient’s temperature and blood pressure with a thermometer and blood pressure monitor. students were able to see step-by-step how to perform each of the required actions on the virtual patient (figure 2-4). using the mouse, the student selected the part of the virtual patient to be examined and could then perform inspection, palpation, percussion, and auscultation. the physical examinations were simulated with software that allowed the user to listen to the lungs and decide for themselves whether the sounds were normal or not. the student was expected to make a possible diagnosis based on the data collected. once the diagnosis was made, the student had to create a treatment plan for the patient. this system allows the student to prescribe medication for the patient. when prescribing the medication, the student should determine the name, method of administration, dosage, and number of daily doses. the student should create a follow-up plan and make fig. 1 cycle of simulation-based learning activities in the cp-based intervention. the clinical scenario presentation step was performed on the cyberpatient platform. in this figure you can see the main components of the platform. from the case dashboard (case encounter), the user can follow any path. it is also possible to return to the case presentation from the diagnosis, therapy or follow-up sections. 34 j contemp med sci | vol. 8, no. 1, january-february 2022: 31–37 teaching clinical decision-making skills to undergraduate nursing students original t.m. hosseini et al. recommendations to improve the patient’s lifestyle and diet after selecting the appropriate treatment for the patient. using the cyberpatient simulator, the student could calculate the cost-effectiveness of diagnostic, therapeutic, and follow-up interventions. the software could also record the time spent on each case, the number of errors made and the immediate feedback received. 5. debriefing the students took part in a 90-minute debriefing session in the conference hall of mofid hospital in tehran 24 hours after working with each virtual patient. the clinical professor had attended a debriefing training workshop to be able to conduct debriefing sessions and had the necessary expertise for that. in this study, the 3d model was used for debriefing, and all debriefing sessions were in compliance with inacsl standards. data collection tool and technique in this study, a demographic information questionnaire and clinical decision-making (cdm) instrument by lauri and salantera (2002) were used to collect data. this 24-item instrument was divided into four subscales, each containing six items corresponding to the four steps of the decision-making process. the cdm uses a 5-point likert scale. the even-numbered items reflect decision making in situations with unpredictable outcomes, such as “i make assumptions about impending care problems when i first meet the patient.” the odd items reflected decision making in structured tasks or in situations where there is sufficient time to gather information or plan activities, e.g., “based on my preliminary information, i specify all the items i want to monitor and ask the patient about. according to the instructions, the total sum of the scores was interpreted.” responses are graded from never (1), rarely (2), sometimes (3), often (4), and almost always (5). scores range from 120–24, with scores ranging from five (always) to one (never) for sentences with a positive semantic load and vice versa for expressions with a negative semantic load. reversed expressions in this questionnaire are: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 (one = always, five = never). a score of 67 or less indicates systematic analytic decision making, a score between 68 and 78 indicates analytic intuitive decision making, and a score above 78 indicates intuitive interpretive clinical decision making.22 internal consistency reliability the reliability of the translated questionnaire was confirmed in the javadi study (2008), and the internal correlation of cronbach’s alpha was reported to be 0.75.24 the reliability of this instrument was re-assessed by karimi naghandar et al. (2013) and α = 0.85 was reported.25 in the present study, the reliability of this tool was evaluated by using the test-retest method on 20 people. cronbach’s alpha coefficient of 0.86 was calculated and reported. analysis spss software version 23.0 was used to analyze the data and examine the effects of using the simulator on students’ clinical decision-making skills. shapirovilk and kolmogorov-spironov tests were used to examine the natural distribution of the quantitative variables. descriptive statistics such as mean, standard deviation, and frequency distribution were used to describe the characteristics of the participants. paired t-tests were used to compare the clinical decision-making score in the two phases before and after the intervention and to compare the score immediately after the intervention and one month after the intervention. significance level was considered p < 0.05. results fifty-eight students in their third year of nursing studies participated in this study. the majority of participants were single (87.3%) and female (81.7%). the mean age of the participants was 21 ± 4.5 years. the majority of participants (86.2%) had never experienced virtual training or simulation in clinical education. clinical decision-making skills of nursing students was compared before (48/04 ± 12/77) and immediately after training (76/49 ± 7/66) using paired tests, and a statistically significant difference was found (p = 0/009). students used analytic clinical decision-making before the intervention and intuitive clinical decision-making after the intervention. a statistical difference was also observed in clinical decision making skills before and after one month of follow-up (p = 0/001). comparison of clinical decision-making ability immediately after training with that after one month of follow-up (73/06 ± 4/9) also revealed no statistically significant difference (p = 0/235) (table 1). fig. 2 screenshots of the cyberpatient software. 35j contemp med sci | vol. 8, no. 1, january-february 2022: 31–37 t.m. hosseini et al. original teaching clinical decision-making skills to undergraduate nursing students in addition, the results showed that 24.14% of nursing students used analytic-intuitive reasoning before the intervention. 70.69% of the respondents used analytical decision making while 5.17% used intuitive decision making. after the implementation of educational intervention based on virtual patient simulator, 25.86% students showed intuitive thinking, 63.80% showed analytical-intuitive thinking and 10.34% showed analytical thinking. thus, it can be said that before the intervention, most students were thinking analytically and making clinical decisions, while after the intervention, most students had an analytic-intuitive model of clinical decision making (table 2). discussion accordingly, the current study, designed to examine the effects of virtual patient simulators on nursing students’ clinical decision-making skills, showed a significant increase in their ability to make clinical decisions. in addition, the results showed that the durability of acquired clinical decision-making skills was significantly higher after one month of the intervention than before the intervention. numerous studies have examined how virtual patients and simulators affect students’ clinical decision-making skills. as shown in a study by nibbelink et al. (2018), computer software can provide unlimited opportunities for nursing student education by providing a safe and realistic environment in which they can practice clinical decision making and practical skills without potential risk to the patient.26 consistent with the findings of the present study, roh (2013) et al. conducted a comparative study to assess nurses’ self-efficacy in cardiopulmonary resuscitation decision making using computer simulations and mannequin simulations. the study showed that computer simulations had a greater impact on nurses’ clinical decision making.27 endacott’s (2012) study assessed nurses’ clinical decision-making skills using standardized patients and mannequins in an osce test. according to the study, the use of standardized patient simulation methods helped to strengthen nurses’ clinical decision-making skills more effectively than mannequins. in addition, simulation and informal feedback were used to improve clinical decision making in emergency situations.28 after working with each virtual patient, we debriefed and then drew conclusions from each case. we then provided feedback to the student on how to improve their performance on the case. as previously mentioned, 5.17% of the subjects used intuitive reasoning before the intervention and 25.86% after the intervention, which indicates that the use of the cp virtual patient simulator improved the use of intuitive clinical reasoning by nursing students. non-analytical or intuitive reasoning occurs unconsciously and fast, it happens in the moment without much effort, and it doesn’t require much energy. pattern recognition is equivalent to analytical reasoning. in daily life, we do things that happen automatically over time without thinking about them. to understand these behaviors, we form cognitive structures in our minds called pattern recognition.29 pattern recognition in medicine is divided into diseases. based on pattern recognition theory, due to repeated exposure, an organized set of medical information imprints itself in their memory so that they use it when making decisions and solving problems with subsequent patients. therefore, when experienced students are confronted with new disease cases that are similar to diseases they have previously encountered, they can quickly recall a structured network of relevant information and thus spend less time finding solutions (okoli, 2018).30 a cp simulator, as mentioned earlier, provides a successful solution to learning pattern recognition for any disease through continuous practice on a standard virtual patient. the nursing student, supported by his cognitive structures, can make a correct clinical decision in a situation similar to the virtual case. in the present study, it was found that 70.69% of the subjects used analytical thinking before the intervention which decreased to 10.34% after the intervention. the study found that the number of students who used analytical-intuitive thinking increased after the intervention (63.80%). when a nursing student uses analytic or hypothetico-deductive reasoning, he or she attempts to make a possible diagnosis by establishing a causal relationship between signs and symptoms. it is a trial and error-based method. students and novice nurses usually use this approach to solve clinical problems based on the pathophysiology of disease. the reason is that their information is insufficiently structured and they lack clinical experience in dealing with different types of patients.31 therefore, it can be said that clinical reasoning is a spectrum that includes analytical reasoning or deductive hypotheses on one side and intuitive or non-analytical reasoning on the other. in general, the nursing student moves from analytical thinking on this side of the spectrum to intuitive thinking on the other side of the spectrum, gaining experience and table 2. classification of nursing students according to three clinical decision models before and after the intervention decision-making model range of score number before intervention percent (%) before intervention number immediately after the intervention percent (%) immediately after the intervention intuitive decision making above 78 3 5/17 15 25/86 analytical-intuitive decision making between 78–68 14 24/14 37 63/80 analytical decision making under 68 41 70/69 6 10/34 table 1. a comparison of the scores in pre-test and post-tests (1 and 2) and the scores in post-tests (1 and 2) in the cp-based intervention group pre-test post-test 1 post-test 2 cp-based training 48/04 ± 12/77 76/49 ± 7/66 73/06 ± 4/09 paired test pretest posttest 1 pretest posttest 2 posttest1posttest 2 p value p = 0/009 p = 0/001 p = 0/235 36 j contemp med sci | vol. 8, no. 1, january-february 2022: 31–37 teaching clinical decision-making skills to undergraduate nursing students original t.m. hosseini et al. practice and repeating the clinical decision-making position. in the middle of this spectrum is analytic-intuitive reasoning. similarly, in study conducted by şahin (2021), most of the participants reported that virtual patient simulation enabled them to apply theoretical knowledge in practice. based on these results, simulation-based training and the use of visual technologies can facilitate the acquisition of nursing skills.32 the results of this study are in agreement with parker’s study (2014). parker’s study found that nurses’ decision-making progressed from intuitive to analytical through education.33 surgical students were taught surgical skills using virtual patients in shariati’s study (2008). based on the results of this study, educating students based on virtual patients helps them to design a mental framework or pattern recognition to taking history of the patient. thus, the student can use this same pattern recognition to taking history from almost all patients with similar complaints.34 limitation and recommendation this study shows that using simulation to teach clinical skills is critical for a successful curriculum. the fact is that implementing such a curriculum is not always easy. incorporating simulations into the curriculum remains a challenge, but it is important to note that they are most successful when they become a natural part of the curriculum rather than a separate component. the results of this study may not suggest that the improvement in students’ clinical decision-making skills is simply due to working with the cp virtual patient simulation platform as a teaching tool, because it was used in a comprehensive clinical course with planned educational activities. on the other hand, building knowledge and acquiring process skills are also complicated. the other limitations of this study include the small number of research units, implementation of this intervention in a group setting, lack of a control group to compare outcomes, and implementation in only one heart disease rotation. the educational intervention and simulator used in this study can be integrated into undergraduate nursing curricula to help students acquire clinical decision-making skills. in addition, it is proposed to evaluate the impact of using the cp virtual patient simulator and the educational design of this study on nursing students’ critical thinking skills. conclusion the review of the results shows that nursing students’ clinical decision-making skills can be improved in a controlled environment using the cp simulator. students can practice their skills in a safe environment without harming patients. the use of cp-based clinical simulation is a great way for nursing students to combine, relate, and ultimately apply their theoretical knowledge to nursing practice. acknowledgment we would like to thank the nursing students and faculty members of the shahid beheshti university of medical sciences (sbmu) for their participation in this project. conflicting interest disclosure cp is a business education product developed by the university of british columbia / vancouver coastal health (ubc / vch) and spin-off company ubc / vch. the university of british columbia and the virtual university of medical sciences, tehran, iran, have signed a memorandum of understanding granting all iranian medical universities free access to this platform. due to this probable conflict of interest, the study was organized and conducted by a research committee with no conflict of interest at shahid beheshti university of medical sciences.  references 1. johnsen hm, fossum m, vivekananda-schmidt p, fruhling a, slettebø å. teaching clinical reasoning and decision-making skills to nursing students: design, development, and usability evaluation of a serious game. international journal of medical informatics. 2016 oct 1; 94:39–48. 2. ewertsson m, bagga-gupta s, allvin r, blomberg k. tensions in learning professional identities–nursing students’ narratives and participation in practical skills during their clinical practice: an ethnographic study. bmc nursing. 2017 dec;16(1):1–8. 3. levett-jones t, hoffman k, dempsey j, jeong sy, noble d, norton ca, roche j, hickey n. the ‘five rights’ of clinical reasoning: an educational model to enhance nursing students’ ability to identify and manage clinically ‘at risk’patients. nurse education today. 2010 aug 1;30(6):515–520. 4. farzi s, shahriari m, farzi s. exploring the challenges of clinical education in nursing and strategies to improve it: a qualitative study. journal of education and health promotion. 2018;7:115–123. 5. weller jm. simulation in undergraduate medical education: bridging the gap between theory and practice. medical education. 2004 jan;38(1):32–8. 6. guze pa. using technology to meet the challenges of medical education. transactions of the american clinical and climatological association. 2015; 126:260–270. 7. wilkinson a, roberts j, while a. nursing students’ use of technology enhanced learning: a longitudinal study. journal of nursing education and practice. 2013 may 1;3(5):102–115. 8. sharma d, bhaskar s. addressing the covid-19 burden on medical education and training: the role of telemedicine and tele-education during and beyond the pandemic. frontiers in public health. 2020 nov 27; 8:838–852. 9. rezaei h, haghdoost a, javar ha, dehnavieh r, aramesh s, dehgani n, sisakht mt. the effect of coronavirus (covid-19) pandemic on medical sciences education in iran. journal of education and health promotion. 2021 jan 1;10(1):136–144. 10. tabatabai s. covid-19 impact and virtual medical education. journal of advances in medical education & professionalism. 2020 jul 1;8(3): 140–3. 11. foronda cl, fernandez-burgos m, nadeau c, kelley cn, henry mn. virtual simulation in nursing education: a systematic review spanning 1996 to 2018. simulation in healthcare. 2020 feb 1;15(1):46–54. 12. elcokany nm, abdelhafez ai, samuel sharaby vm, belal s. use of computerbased scenarios for clinical teaching: impact on nursing students’ decisionmaking skills. inhealthcare. 2021 sep; 9 (9): 1228–1240. 13. zary n, johnson g, boberg j, fors ug. development, implementation and pilot evaluation of a web-based virtual patient case simulation environment–web-sp. bmc medical education. 2006 dec;6(1):1–7. 14. berman nb, durning sj, fischer mr, huwendiek s, triola mm. the role for virtual patients in the future of medical education. academic medicine. 2016 sep 1;91(9):1217–22. 15. liaw sy, siau c, zhou wt, lau tc. interprofessional simulation-based education program: a promising approach for changing stereotypes and improving attitudes toward nurse–physician collaboration. applied nursing research. 2014 nov 1;27(4):258–60. 16. mohamed sa, fashafsheh ih. the effect of simulation-based training on nursing students’ communication skill, self-efficacy and clinical competence for nursing practice. open journal of nursing. 2019 aug 22; 9(8):855–70. 37j contemp med sci | vol. 8, no. 1, january-february 2022: 31–37 t.m. hosseini et al. original teaching clinical decision-making skills to undergraduate nursing students 26. nibbelink, c. w., young, j. r., carrington, j. m., & brewer, b. b. informatics solutions for application of decision-making skills. critical care nursing clinics. 2018; 30(2): 237–246. 27. roh ys, lee ws, chung hs, park ym. the effects of simulation-based resuscitation training on nurses’ self-efficacy and satisfaction. nurse education today. 2013;33(2):123–128. 28. endacott r, scholes j, cooper s, mcconnell-henry t, porter j, missen k, champion r. identifying patient deterioration: using simulation and reflective interviewing to examine decision making skills in a rural hospital. international journal of nursing studies. 2012;49(6): 710–717. 29. boushehri e, arabshahi ks, monajemi a. clinical reasoning assessment through medical expertise theories: past, present and future directions. medical journal of the islamic republic of iran. 2015;29:222. 30. okoli j, watt j. crisis decision-making: the overlap between intuitive and analytical strategies. management decision. 14 may 2018; 56(5):1122–34. 31. jahanpour f, sharif f, salsali m, kaveh mh, williams lm. clinical decision‐ making in senior nursing students in iran. international journal of nursing practice. 2010 dec;16(6):595–602. 32. şahin g, başak t. the effect of virtual patient simulation on nursing students’ clinical decision making and problem-solving skills. journal of education and research in nursing. 2021 jun 1;18(2):118–23. 33. parker cg. decision-making models used by medical-surgical nurses to activate rapid response teams. medsurg nursing. 2014 may 1;23(3): 159–1.63. 34. shariati m, younesian m, tahrirchi i, khosravi a. the effect of virtual patients on surgery training in medical students: a randomized controlled study. journal of knowledge and wellness of shahroud university of medical sciences 2008; 1(3): 2–8 [persian]. 17. isaza-restrepo a, gómez mt, cifuentes g, argüello a. the virtual patient as a learning tool: a mixed quantitative qualitative study. bmc medical education. 2018 dec;18(1):1–10. 18. kurihara y, kuramoto s, matsuura k, miki y, oda k, seo h, watabe t, qayumi ak. academic performance and comparative effectiveness of computerand textbook-based self-instruction. inmedinfo 2004 (pp. 894–897). ios press. 19. qayumi ak, qayumi t. computer-assisted learning: cyberpatienttm-a step in the future of surgical education. journal of investigative surgery. 1999 jan 1;12(6):307–17. 20. farahmand s, meneghetti a, shi k, pachev g, ramezani j, zeinoddini s, et al. cyberpatient™—an innovative approach to medical education. creative education. 2020; 11, 926–941. 21. rutherford-hemming t, lioce l, jeffries pr, sittner b. after the national council of state boards of nursing simulation study—recommendations and next steps. clinical simulation in nursing. 2016 jan 1;12(1):2–7. 22. lauri s, salanterä s, chalmers k, ekman sl, kim hs, käppeli s, macleod m. an exploratory study of clinical decision‐making in five countries. journal of nursing scholarship. 2001 mar;33(1):83–90. 23. inacsl standards committee. inacsl standards of best practice: simulationsm simulation design. clinical simulation in nursing. 2015; 12, s5–s12. 24. javadi n, paryad a, fadakar k, roshan z, asiri sh. clinical decision making: its relation with critical thinking. journal of gilan nursing and midwifery school. 2008; 18(60): 9–16 [persian]. 25. karimi noghondar m, rahnama rahsepar f, golafrooz m, mohsenpour m. comparison of critical thinking and clinical decision-making skills among the last-semester nursing students and practicing nurses in sabzevar university of medical sciences. iranian journal of medical education. 2013; 12(12):916–924 [persian]. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i2.1158 https://m.scirp.org/s/searchpaper.action?kw=sahar+farahmand&sf=au https://m.scirp.org/s/searchpaper.action?kw=adam+meneghetti&sf=au https://m.scirp.org/s/searchpaper.action?kw=kevin+shi&sf=au https://m.scirp.org/s/searchpaper.action?kw=george+pachev&sf=au https://m.scirp.org/s/searchpaper.action?kw=javad+ramezani&sf=au https://m.scirp.org/s/searchpaper.action?kw=sara+zeinoddini&sf=au 139j contemp med sci | vol. 9, no. 3, may-june 2023: 139–143 review effectiveness of radial extracorporeal shock wave therapy in reduces muscle spasms in stroke patients; a systematic review and meta-analysis isam ali hameedi1*, azadeh shadmehr1, tahir alsaadawi2 1physical therapy department, school of rehabilitation, tehran university of medical sciences, tehran, iran. 2imam alsadeq teaching hospital, ministry of health, babylon, iraq. *correspondence to: isam ali hameedi (e-mail: isam_jettar@yahoo.com) (submitted: 19 february 2023 – revised version received: 09 march 2023 – accepted: 25 april 2023 – published online: 26 june 2023) abstract objectives: effectiveness of radial extracorporeal shock wave therapy (reswt) on pain, range of motion and muscle tone in patients with stroke injuries. methods: pubmed, embase, cochrane library and vip information were used to collect information for research conducted between the beginning of 2013 and the end of 2022. these studies were randomized controlled trials that used reswt for muscle spasm in stroke patients with conventional treatments as controls. there are no specific restrictions on the duration of treatment, the type of reeswt, or the severity of symptoms. these studies must have assessed at least one of the following outcome mesurements: visual analog scale (vas) for pain, ashwerth measure for muscle tone or external rotation range of motion (er rom). revman 5.3 software was used to check the quality of included studies. for continuous variables, mean difference (md) or standardized md (smd) with ci 95% were derived. for dichotomous data, event proportions and sample sizes were extracted. results: in the conducted investigations, it was found that 7 studies investigated the effectiveness of reswt treatments after the intervention in comparison with conventional treatment in terms of reducing pain intensity. in total, all 7 studies showed that the reswt intervention was effective in reducing pain intensity compared to conventional treatments (md, –0.97 [95% ci, –2.13 to –0.42], p < .00001, i2 = 71%). compared with traditional treatments, the effectiveness of reswt plus routine treatments on muscle tone after intervention was assessed in terms of ashwerth criterion in 4 trials (smd, 1.13 [95% ci, 0.46–1.23], p < .00001, i2 = 59%). er rom was used to reflect the rom, which was assessed in 4 rcts. because of the limited number of studies, er rom was measured immediately after treatment in all included studies. the pooled result of the included studies showed that the heterogeneity was high and unacceptable (md, 10.31 [95% ci, 2.47–16.18], p < .003, i2 = 81%). conclusion: the results of this research indicated that reswt treatment can be used as a safe and non-invasive method to quickly reduce spasticity and increase joint range of motion in stroke patients. but more research on the long-term effects of reswt as well as the factors influencing its effectiveness to reduce spasticity and comparison with other new treatment protocols is suggested. keywords: reswt, pain, range of motion, muscle tone, muscle spasms, stroke issn 2413-0516 introduction damage to the upper motor neuron causes certain clinical defects, including muscle relaxation immediately after the complication and spasticity in the later stages of the complication.1 spasticity is a common symptom in many neurological conditions, especially in stroke, cerebral palsy and multiple sclerosis.2-4 according to lance’s definition, spasticity is an increase in muscle tone depending on speed, which is manifested by the intensification of tendon reflexes and is caused by an increase in the excitability of the stretch reflex.5 the prevalence of spasticity is estimated in 38% of patients after stroke.6 normalizing muscle tone is a prerequisite for restoring functional ability. in order to reduce spasticity, various interventions such as drugs, surgical treatment and physiotherapy techniques are routinely used.7 physiotherapy is the first step to treat spasticity and several methods are used to control it.8 today, one of the promising methods to reduce spasticity is extracorporeal reswt therapy (eswt), but it has not yet been used as a common treatment.9 some studies claim that eswt can be used as a safe and non-invasive method to treat spasticity in patients with neurological disorders such as cerebral palsy, multiple sclerosis, and especially stroke patients.9-11 eswt is sound pulses caused by transient and sudden pressure changes that spread rapidly in three-dimensional space. the characteristic feature of these pressure pulses is reaching the maximum intensity (about 100 megapascals) in a very short time (10 ns).12 initially, eswt was used to treat kidney stones, but its use quickly spread to the treatment of orthopedic lesions such as non-union in long bones, plantar fascia inflammation, shoulder calcified tendonitis, tendon inflammatory diseases and spasticity.13 two types of eswt are used, focal (feswt) and radial (reswt), whose production mechanism and penetration depth are different. feswt are produced through electro-hydraulic and piezoelectric electromagnetism, while in reswt, waves are created through pneumatics, also the penetration depth of reswt is less than feswt and its maximum intensity is at the contact surface of the applicator with the body. another difference between these two types of eswt is the conduction speed, shape and size of the pulses.14 so far, no exact mechanism has been presented to justify the effects of using eswt in the treatment of spasticity. a group of studies believe that reswt impulses can have a direct effect on fibrotic muscles and non-reflexive parts of spastic muscles. there are studies that state that the eswt at the muscle level is able to change the sensory flow of the muscle, 140 j contemp med sci | vol. 9, no. 3, may-june 2023: 139–143 effectiveness of reswt in stroke patients review i. a. hameedi et al. which itself leads to a decrease in the excitability of the muscle at the level of the spinal cord and ultimately causes a decrease in spasticity. the study on non-human samples shows that eswt can delay neuromuscular transmission at the neuromuscular junction, hence they are considered as possible mechanisms of eswt effect in reducing muscle spasticity. considering the necessity of treating spasticity in patients with neurological lesions, the aim of this study is to analyze the available clinical trial studies in relation to the effectiveness or side effects of using reswt in the treatment of spasticity in stroke patients. methods search strategy the method of data collection in this research is a systematic search in pubmed, embase, cochrane library and vip databases from the beginning of 2013 to the end of 2022. search keywords were “radial extracorporeal reswt therapy,” “reswt,” “muscle hypertonicity,” “stroke,” and “randomized controlled trial,” different terms are used in this systematic search. also, the list of output studies was reviewed and revised several times inclusion criteria the inclusion criteria for the systematic study are as follows: 1. the study is rcts. 2. patients in these studies have spasticity after stroke. 3. the study group and the control group were treated with conventional treatments and reswt and the results of the conventional treatment were the same in all of them. 4. there were no restrictions on treatment duration, type of reeswt or symptom severity and energy intensity depended on patient tolerance. 5. the language of the article is english 6. there is no specific restriction on adopting or not adopting a blind method. 7. the authors followed at least 1 of the ashworth and modified ashworth outcome indices ashworth. exclusion criteria the exclusion criteria were as follows: 1. the authors used at least 1 of the following outcome indices: ashworth criterion (muscle tone), vas (pain), er rom (range of motion) data extraction two reviewers reviewed the abstract, study method and results completely separately and independently. both of them extracted studies that met the defined criteria and in case of disagreement, a third reviewer was asked for an opinion. also, 2 other reviewers independently extracted the following data: first author, year of publication, sample size, details of intervention, follow-up (if applicable), time of measurement and outcome indicators. the variables investigated in this study are muscle tone, range of motion and pain. pain intensity was measured via vas (the lower the score, the better the effectiveness). joint rom was measured through er rom (the higher the grade, the better the treatment effect). the ashworth scale was used to measure muscle tone (the lower the score, the better the effectiveness). results at multiple follow-up times from the same study were included in subgroup analyzes by time point. when 2 studies used the same group of participants, 2 studies were included only if they used different measures. bias assessment and quality classification to check the quality of the studies included in the present systematic review, the cochrane collaboration’s tool for assessing the risk of bias in randomized trials20 was used, using revman version 5.3 (nordic cochrane centre, cochrane collaboration) statistical processing and assessment of heterogeneity statistical processing in this research was such that for continuous variables, mean difference (md) or standardized md (smd) with 95% ci was calculated. for dichotomous data, we derived event proportions and sample sizes. for analysis, the two-sided t test was used and the significance level of p ≤ 0.05 was considered. heterogeneity was evaluated by using the i2 statistic and the cochran q statistic with p-values.8 the data were pooled using the random-effects model if significant heterogeneity was present (i2 > 50% or pq < 0.1); otherwise, a fixed-effects model was used. in the case of significant heterogeneity, subgroup analyses were further conducted to investigate the potential source of heterogeneity on the treatment effect size.20 the statistical software used was revman 5.3 software. result by using the keywords above, a total of 115 articles were found in the first stage of selection, after removing 52 unrelated articles and 56 duplicate articles, 7 articles remained. among these articles, 11 articles were removed for the following reasons. (figure 1, table 1). fig. 1 study flow diagram, reswt; rct, randomized controlled trial. 141j contemp med sci | vol. 9, no. 3, may-june 2023: 139–143 i. a. hameedi et al. review effectiveness of reswt in stroke patients according to table 1, only one study evaluated the method before and after the treatment, and the rest of the studies were conducted with the control group. in total, there were 258 participating patients in this research. in these 7 articles, muscle spasm of carpi radialis flexor (4 articles), gastrosoleus (1 article), subscapularis (1 article) and wrist and finger flexors (1 article) has been investigated. methodological quality and the risk of bias within studies in figure 2, the bias diagram is shown in a summary form. out of 7 studies, 5 studies have a good method to reduce bias. these studies have a low risk of selection bias, performance bias, diagnosis bias, attrition bias, reporting bias and other biases. one of the included studies13 had a high risk of bias in the randomization designand one study was at risk of other biases.14 pain intensity in total, all 7 studies15–21 showed that the reswt intervention was effective in reducing pain intensity compared to conventional treatments (md, –0.97 [95% ci, –2.13 to –0.42], p < .00001, i2 = 71%). muscle tone in total, all 7 studies15–21 showed that the reswt intervention was effective in reducing pain intensity compared to conventional treatments in terms of ashwerth criterion in 4 trials15,17–19,20 (smd, 1.13 [95% ci, 0.46–1.23], p < .00001, i2 = 59%). range of motion er rom was used to reflect the rom, which was assessed in 4 rcts.17,20 because of the limited number of studies, er rom was measured immediately after treatment in all included studies. the pooled result of the included studies showed that the heterogeneity was high and unacceptable (md, 10.31 [95% ci, 2.47–16.18], p < .003, i2 = 81%). study limitations the limitation of this study was the short-term follow-up of the effectiveness of the treatment. the average follow-up time for effective treatment in the studies was four weeks. considering that the meta-analysis was done on the findings before and immediately after the intervention, the findings of this study cannot confirm the long-term effectiveness of reswt. therefore, it is recommended that in the future, clinical trial studies with higher quality are designed that are carefully designed and especially examined in terms of the control group discussion and conclusion the present meta-analysis study showed that the degree of anxiety scale decreases significantly immediately after the application of reswt. also, there is a significant increase in joint range of motion immediately after shock therapy. investigating the use of reswt to reduce spasticity in fargani motor neuron lesion patients, especially stroke patients, is a new approach in the treatment of these patients. considering the various advantages of reswt, including the ease of use, being safe and non-invasive, and its relatively low cost compared to other spasticity treatment methods, including botulinum toxin injection, it seems that this method can be a suitable alternative to reduce spasticity in stroke patients. also the results of the this study also indicated that immediately after applying the reswt, the range of motion of the joint increases significantly, despite the fact that spasticity appears in the undamaged upper motor neuron, but secondary changes in the muscles of the peripheral nerves and joints occur after the occurrence of spasticity. and many table 1. studies related to the use of reswt to reduce spasticity of lower limb muscles in stroke patients author/year of publication type of study number of treatment sessions number of patients examined muscles number and intensity of pulses number of evaluation times result radinmehr et al. (2017)15 clinical trial before and after 1 12 gastrosoleus muscles 2000 pulses 0.340 mg/mm2 3 reduction of spasticity kim et al. (2013)16 clinical trial before and after 5 57 subscapularis 3000 pulses 0.630 mg/mm2 11 reduction of spasticity fouda et al. (2015)17 clinical trial before and after 5 30 wrist and finger flexors 1500 pulses 0.230 mg/mm2 before and after treatment reduction of spasticity daliri et al. (2015)22 clinical trial before and after 1 15 flexor carpi radialis 1500 pulses 0.030 mg/mm2 6 reduction of spasticity dymarek et al. (2016)9 clinical trial before and after 1 20 flexor carpi radialis 1500 pulses 0.030 mg/mm2 4 reduction of spasticity wu et al, (2017)21 clinical trial before and after 1 24 flexor carpi radialis 2000 pulses 0.340 mg/mm2 4 reduction of spasticity fan et al. (2021)20 clinical trial before and after 1 100 flexor carpi radialis 2000 pulses 0.340 mg/mm2 4 reduction of spasticity 142 j contemp med sci | vol. 9, no. 3, may-june 2023: 139–143 effectiveness of reswt in stroke patients review i. a. hameedi et al. fig. 2 risk of bias (a) table and (b) summary. 143j contemp med sci | vol. 9, no. 3, may-june 2023: 139–143 i. a. hameedi et al. review effectiveness of reswt in stroke patients 13. saggini r, di stefano a, saggini a, bellomo rg. clinical application of shock wave therapy in musculoskeletal disorders: part ii related to myofascial and nerve apparatus. j biol regul homeost agents 2015;29(4):771–85. 14. speed c. a systematic review of shockwave therapies in soft tissue conditions: focusing on the evidence. br j sports med 2014;48(21):1538–42. 15. radinmehr h, nakhostin ansari n, naghdi s, olyaei g, tabatabaei a. effects of one session radial extracorporeal shockwave therapy on poststroke plantarflexor spasticity: a single-blind clinical trial. disabil rehabil 2017;39(5):483–490. 16. kim yw, shin jc, yoon jg, kim yk, lee sc. usefulness of radial extracorporeal shock wave therapy for the spasticity of the subscapularis in patients with stroke: a pilot study. chin med j (engl) 2013;126(24):4638–43. 17. fouda kz, sharaf ma. efficacy of radial shock wave therapy on spasticity in stroke patients. int j health rehab sci (ijhrs) 2015;4(1):19–26. 18. foulkes ma, wolf pa, price tr, mohr jp, hier db. the stroke data bank: design, methods, and baseline characteristics. stroke 1988;19(5):547–54. 19. lee j-y, kim s-n, lee i-s, jung h, lee k-s, koh s-e. effects of extracorporeal shock wave therapy on spasticity in patients after brain injury: a metaanalysis. j phys ther sci 2014;26(10):1641–7. 20. fan t, zhou x, he p, zhan x, zheng p, chen r, li r, li r, wei m, zhang x, huang g. effects of radial extracorporeal shock wave therapy on flexor spasticity of the upper limb in post-stroke patients: study protocol for a randomized controlled trial. frontiers in neurology. 2021;12. 21. wu yt, chang cn, chen ym, hu gc. comparison of the effect of focused and radial extracorporeal shock waves on spastic equinus in patients with stroke: a randomized controlled trial. european journal of physical and rehabilitation medicine. 2017 oct 25;54(4):518–25. 22. daliri, s. s., forogh, b., emami razavi, s. z., ahadi, t., madjlesi, f., & ansari, n. n. (2015). a single blind, clinical trial to investigate the effects of a single session extracorporeal shock wave therapy on wrist flexor spasticity after stroke. neurorehabilitation, 36(1), 67–72. spasticity treatments are focused on improving these secondary changes. the results of this review study showed that based on these studies, it is not possible to achieve a single agenda for the treatment of patients in terms of the number of treatment sessions, the intensity and the number of pulses, on the other hand, in none of the studies, there is no documented reason for choosing the number of sessions and the intensity of pulses. due to the fact that applying at least 1500 pulses per day is necessary to induce the cellular effects of reswt and applying 2500 pulses daily can cause tissue necrosis. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. the results of this meta-analysis showed that reswt therapy can be used as a safe and non-invasive method to quickly reduce spasticity and increase joint range of motion in stroke patients. more studies on the long-term effects of reswt application, as well as factors influencing the effectiveness of reswt therapy to reduce spasticity and comparing different treatment protocols are necessary in the future. conflicts of interest none.  references 1. tahan n, khademi kalantari k, kholghi y, amiri z. the correlation of the duration of flaccidity period and the intensity and location of the disorder in patients with cerebro-vascular accident. j rafsanjan univ med sci 2009;8(4):287–94. 2. katozian l, tahan n, mohseni bandpei ma, jam barsang s. spasticity following stroke: a systematic review and meta-analysis. j mazandaran univ med sci 2015;25(123):230–45. 3. scholtes va, becher jg, beelen a, lankhorst gj. clinical assessment of spasticity in children with cerebral palsy: a critical review of available instruments. dev med child neurol 2006;48(1):64–73. 4. rizzo ma, hadjimichael oc, preiningerova j, vollmer tl. prevalence and treatment of spasticity reported by multiple sclerosis patients. mult scler 2004;10(5):589–95. 5. lance jw. what is spasticity? lancet 1990;335(8689):606. 6. watkins cl, leathley mj, gregson jm, moore ap, smith tl, sharma ak. prevalence of spasticity post stroke. clin rehabil 2002;16(5):515–22. 7. nair kp, marsden j. the management of spasticity in adults. bmj 2014;349:g4737. 8. graham la. management of spasticity revisited. age ageing 2013;42(4):435–41. 9. dymarek r, taradaj j, rosińczuk j. extracorporeal shock wave stimulation as alternative treatment modality for wrist and fingers spasticity in poststroke patients: a prospective, open-label, preliminary clinical trial. evid based complement alternat med 2016;2016:4648101. 10. el-shamy sm, eid ma, el-banna mf. effect of extracorporeal shock wave therapy on gait pattern in hemiplegic cerebral palsy: a randomized controlled trial. am j phys med rehabil 2014;93(12):1065–72. 11. marinelli l, mori l, solaro c, uccelli a, pelosin e, currà a, et al. effect of radial shock wave therapy on pain and muscle hypertonia: a double-blind study in patients with multiple sclerosis. mult scler 2015;21(5):622–9. 12. chaussy c, haupt g, jocham d, kohrmann k, wilbert d. therapeutic energy application in urology: standards and recent developments. stuttgart, germany: georg thieme verlag kg; 2005. p. 1–16. https://doi.org/10.22317/jcms.v9i3.1346 111j contemp med sci | vol. 9, no. 2, march-april 2023: 111–115 original the impact of blood group phenotypes on covid-19 severity and mortality in duhok province: a prospective cross-sectional study fatima jaafar rasho1*, muayad aghali merza2 1department of medical laboratory sciences, college of health sciences, university of duhok, duhok, kurdistan region, iraq. 2department of internal medicine, azadi teaching hospital, college of medicine, university of duhok, duhok, kurdistan, iraq. *correspondence to: fatima jaafar rasho (e-mail: fatimajaafar2018@gmail.com) (submitted: 02 january 2023 – revised version received: 26 february 2023 – accepted: 25 march 2023 – published online: 26 april 2023) abstract objectives: this study aimed to determine the frequency of blood group types among covid-19 patients and to investigate its potential association with disease severity and patient outcomes. methods: this prospective cross-sectional study was conducted from february to july 2022 in three different healthcare facilities in the duhok region. all confirmed pcr confirmed covid-19 patients were classified into: mild, moderate, severe, and critical cases. information on demographic, clinical, and laboratory characteristics were collected using a standardized questionnaire. all patients were subjected to abo blood grouping. the statistical calculations were performed by jmp pro 14.3.0. a p value of < 0.05 was considered significant. results: the study comprised of 404 patients. the age range of the patients varied from 16 to 100 years with a male predominance (204, 50.5%). out of the total patients, 250 (61.88%) had mild-moderate course, while 154 (38.12%) had severe-critical course. the most frequent blood group was o (164, 41.58%), followed by blood group a (121, 29.95%). there was no significant difference in the distribution of the rh factor among the studied subjects (p = 0.426). there was a significant increase in disease severity and worse outcome with increasing age (p = <0.0001). considering blood group types, there were no significant differences between blood group types with covid-19 severity and patients’ outcome. conclusions: individuals with blood group o may have a higher risk of contracting covid-19. advanced age is a crucial predictor of disease severity and poor outcomes. there were no significant association between blood group types with covid-19 severity and patients’ outcome. however, further research is needed to confirm these findings and determine the underlying mechanisms behind any potential association between blood groups and covid-19 susceptibility, severity, and outcome. keywords: covid-19; severity, outcome, blood group issn 2413-0516 introduction coronavirus disease (covid-19), caused by novel coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has rapidly spread around the world, leading to a global pandemic. the virus is transmitted primarily through respiratory droplets, but aerosol, direct contact with contaminated surfaces, and fecal–oral transmission were also reported during the covid-19 epidemic.1 as of april 12, 2023, the virus has infected over 750 million people and caused more than 6.5 million deaths worldwide.2 covid-19 has a broad range of clinical severity and often lacks specific symptoms. most patients experience mild or no symptoms and recover well, while a minority can develop severe-critical disease and potentially fatal complications, resulting in death.3 various risk factors have been implicated in the susceptibility, severity and mortality in patients with covid-19 infections, for e.g. gender, age, and comorbid diseases.4 however, other factors such as environment, hormones, and blood group phenotypes have suggested to play an important role too.5 there is conflicting evidence regarding the link between blood group typing and susceptibility to covid-19, as well as its outcomes.6 numerous studies have reported that individuals of any blood group are susceptible to contracting sars-cov-2 infection, without any particular preference for a specific blood group.7 however, other investigators found that individuals with certain blood group phenotypes may be more or less susceptible to the disease.8 a study form china found that patients with blood group a had a higher risk of covid-19 infection and developing severe disease, while blood group o had a lower risk.5 another study from usa found that blood group b and ab were more likely to acquire the virus so did those with rh positive blood type, while blood group o was less likely.9 the exact mechanism behind this association is not clear yet, but some investigators have suggested that blood group antigens may interact with the sars-cov-2 virus in a way that affects the severity of the disease.5 these factors may play an important role in the pathogenesis of covid-19 and subsequently developing a treatment. while several studies have investigated demographic, clinical, and laboratory characteristics of covid-19 in duhok province,10,11 the relationship between abo typing and covid-19 infection has not been explored yet. therefore, the objective of this study was to determine the frequency of blood group types among covid-19 patients and to investigate its potential association with disease severity and patient outcomes. patients and methods setting the study was conducted in three different healthcare facilities in the duhok region. the first facility was the duhok covid-19 hospital, which includes 50 ward beds and 20 icu beds and focuses on severe, critical, and complicated cases. the second facility was lalav infectious diseases hospital, a 100-bed hospital that mainly manages moderate to severe cases. the third facility was the zakho covid-19 center, mailto:fatimajaafar2018@gmail.com 112 j contemp med sci | vol. 9, no. 2, march-april 2023: 111–115 the impact of blood group phenotypes on covid-19 severity and mortality in duhok province original f.j. rasho et al. located in zakho emergency hospital, which consists of 48 beds and primarily manages moderate to severe cases. more severe cases are referred to the duhok covid-19 hospital. study design and patients this study is a prospective cross-sectional investigation conducted in the duhok province, kurdistan region, iraq, from february to july 2022. all pcr confirmed covid-19 patients who agreed to participate in the study were included, while those who were diagnosed based on laboratory tests other than pcr or radiological images were excluded. enrolled patients provided informed consent, and information on demographic, clinical, and laboratory characteristics were collected using a standardized questionnaire. the ethical committee of the directorate general of health (dgoh) in duhok, iraqi kurdistan approved the study (reference number: 1342022-2-12). classification of disease severity covid-19 patients were classified in accordance to national institutes of health covid-19 treatment guidelines3 (1) mild type: patients who have any of the various signs and symptoms of covid-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell). on the other hand, dyspnea or abnormal chest imaging should not be present. (2) moderate type: individuals who show evidence of lower respiratory disease during clinical assessment or imaging and who have an oxygen saturation (spo2) ≥94% on room air at sea level. (3) severe type: individuals who have spo2 <94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (pao2/fio2) <300 mm hg, a respiratory rate >30 breaths/min, or lung infiltrates >50%. (4) critical type: individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction. laboratory investigations covid-19 diagnosis was conducted using real-time polymerase chain reaction (rt-pcr) on the upper respiratory tract specimens of all patients. additionally, blood samples were collected from each patient and analyzed using the standard abo blood typing method, as previously described.12 statistical analysis the demographic characteristics of the covid-19 patients were presented in mean and sta. deviation or number and percentage. the associations of demographic characteristics and blood groups with disease severity and patients’ outcomes were examined in pearson chi-squared tests. the null hypothesis was rejected in a p-value <0.05. the statistical calculations were performed by jmp pro 14.3.0 (https://www.jmp.com/ en_us/home.html). results the demographic, clinical and blood group characteristics of the covid-19 is demonstrated in table 1. the study comprised of 404 patients. the age range of the patients varied from 16 to 100 years, with a male predominance (50.5%). out of the total patients, 250 (61.88%) had mild-moderate course, while 154 (38.12%) had severe-critical course (table 1). table 2 presents the frequency distribution of abo blood grouping among covid-19 patients. among the 404 patients, the highest frequency was observed in blood group o, with 168 individuals (41.58%), followed by blood group a, with 121 table 1. demographic, clinical and blood group characteristics of the covid-19 patients characteristics (n = 404) statistics numbers percentages age (16–100 yrs) mean: 48.54 sd: 19.67 age category (16–100 yrs) 16–19 5 1.24 20–29 71 17.57 30–39 83 20.55 40–49 61 15.1 50–59 66 16.34 60–69 48 11.88 70–79 32 7.92 80–89 27 6.68 90–100 11 2.72 gender male 204 50.5 female 200 49.5 disease severity mild-moderate 250 61.88 severe-critical 154 38.12 patient outcome recovered 369 91.34 dead 35 8.66 blood group a– 3 0.74 a+ 118 29.21 ab– 1 0.25 ab+ 22 5.45 b– 6 1.49 b+ 86 21.29 o– 11 2.72 o+ 157 38.86 table 2. prevalence of abo blood groups and rh susceptibility with covid-19 blood group total no. (%) rh– rh+ p value a 121 (29.95) 3 (2.48) 118 (97.52) b 92 (22.77) 6 (6.52) 86 (93.48) 0.426 o 168 (41.58) 11 (6.55) 157 (93.45) ab 23 (5.69) 1 (4.35) 22 (95.65) total 404 (100) 21 (5.2) 383 (94.8) https://www.jmp.com/en_us/home.html https://www.jmp.com/en_us/home.html 113j contemp med sci | vol. 9, no. 2, march-april 2023: 111–115 f.j. rasho et al. original the impact of blood group phenotypes on covid-19 severity and mortality in duhok province individuals (29.95%). on the other hand, blood group ab patients were the least prevalent, accounting for only 5.69% of the cases. the chi-square test showed that there was no significant difference in the distribution of the rh factor among the studied subjects (p = 0.426). table 3 presents the association between blood groups with disease severity and patient outcomes among covid-19 patients. the table highlights a significant increase in disease severity and worse outcome with increasing age. considering blood group types, there were no significant differences between blood group types with covid-19 severity and patients’ outcome. table 4 provides comparison between each specific blood group in relation to severity and outcomes of covid-19. for blood group a, b, ab, and o, 64.46%, 63.04%, 69.57%, and 58.33% were classified as mild-moderate disease, respectively. rh-positive patients had 62.14% mild-moderate disease, while rh-negative patients had 57.14 mild-moderate disease. the data presented in table indicates no statistically significant differences between abo blood groups in terms of covid-19 severity and outcome, with p-value >0.05 discussion the distribution of abo blood typing among covid-19 patients has been a topic of interest for investigators due to its table 3. association between blood types and covid-19 severity and patient outcomes characteristics (n = 404) disease severity p-value* patient outcome p-value*mild-moderate (n = 250) severe-critical (n = 154) recovered (n = 269) dead (n = 35) age category (16–100 yrs) 16–19 4 (80.00) 1 (20.00) <0.0001 5 (100) 0 (0.00) <0.0001 20–29 66 (92.96) 5 (7.04) 71 (100) 0 (0.00) 30–39 66 (79.52) 17 (20.48) 83 (100) 0 (0.00) 40–49 43 (70.49) 18 (29.51) 61 (100) 0 (0.00) 50–59 44 (66.67) 22 (33.33) 61 (92.42) 5 (7.58) 60–69 16 (33.33) 32 (66.67) 38 (79.17) 10 (20.83) 70–79 9 (28.13) 23 (71.88) 24 (75.00) 8 (25.00) 80–89 2 (7.41) 25 (92.59) 22 (81.48) 5 (18.52) 90–100 0 (0.00) 11 (100.00) 4 (36.36) 7 (63.64) gender male 124 (60.78) 80 (39.22) 0.6466 185 (90.69) 19 (9.31) 0.6388 female 126 (63.00) 74 (37.00) 184 (92.00) 16 (8.00) blood group a– 3 (100) 0 (0.00) 0.4941 3 (100) 0 (0.00) 0.7954 a+ 75 (63.56) 43 (36.44) 104 (88.14) 14 (11.86) ab– 1 (100.00) 0 (0.00) 1 (100) 0 (0.00) ab+ 15 (68.18) 7 (31.82) 20 (90.91) 2 (9.09) b– 2 (33.33) 4 (66.67) 6 (100) 0 (0.00) b+ 56 (65.12) 30 (34.88) 80 (93.02) 6 (6.98) o– 6 (54.55) 5 (45.45) 11 (100) 0 (0.00) o+ 92 (58.60) 65 (41.40) 144 (91.72) 13 (8.28) * two-sided p value potential implications in covid-19 susceptibility, severity, and mortality. in our study, blood type o was found to be the most prevalent, followed by blood type a. this finding was consistent with a research conducted in sulaimaniyah, iraq, where the frequencies of abo types followed the pattern of o>a>b>ab and rh positive > rh negative.13 the outcome of our study contradicts another study in iraq , which identified blood group a as the most prevalent.14 however, a neighboring country, saudi arabia, reported results consistent with our study, indicating that blood type o (62.4%) was the most common blood group among covid-19 patients, followed by blood type a (25.2%).15 another study conducted in the usa reported that the prevalence of covid-19 was higher in patients with type o blood (45.5%) compared to those with a blood (34.2%).9 this finding was also observed in studies conducted in china5 and pakistan,7 where blood group o was found most frequent type. the finding in our study suggests that individuals with blood group o may have higher risk of contracting covid-19. however, it is important to note that this finding did not provide evidence of a causal relationship between blood groups and covid-19 susceptibility. on contrary, other literatures reported that individuals with blood group a had a higher risk of covid-19 infection, while individuals with blood group o had a lower risk of infection.9,16,17 however, we believe that 114 j contemp med sci | vol. 9, no. 2, march-april 2023: 111–115 the impact of blood group phenotypes on covid-19 severity and mortality in duhok province original f.j. rasho et al. the higher prevalence of covid-19 in blood type o in the present study can be explained by the fact that blood type o is more common in our regional population.18 in general, further prospective research with case control are warranted to better understand the association between blood group types and covid-19 susceptibility. considering risk factors associated with disease severity and mortality, we found that increasing age was associated with disease severity and worse outcome in covid-19 patients. several researchers documented this finding.10,19,20 this is because older individuals have impaired immunity, reduced production of t cells in the thymus, and an increased prevalence of comorbid diseases such as diabetes mellitus, and hypertension. these factors decrease the ability of the immune system to respond to pathogens, making older individuals more susceptible to severe illness from covid-19.19 it is interesting to note that the present study did not find gender to be a significant predictor of severity or mortality in covid-19 patients, which contrasts with the findings of our previous report where female gender was identified as a predictor of disease severity.10 other literatures described male gender as risk factor for severe covid-19.4,21 it is important to keep in mind that the findings of individual studies can vary and are subject to various factors such as sample size, study design, and population characteristics. further research may be necessary to better understand the relationship between gender and covid-19 severity and mortality. this study investigated the potential correlation between the abo blood group system and covid-19 severity and outcomes. however, our data did not indicate any significant association between any of the blood groups with the severity and outcome of covid-19, which contrasts previous study conducted in sulaimaniyah, iraqi kurdistan.13 in agreement to our findings, ishaq et al. reported no significant association between abo groups and infection severity or associated death in a retrospective cohort study of 1067 covid-19 patients.7 several other studies have also reported similar findings. for e.g., a large, multi-institutional, retrospective review found no association between abo blood type and covid-19 severity and death.9 likewise, studies from turkey demonstrated that blood groups did not have significant predictive effects on covid-19 severity and mortality,8,17 and there was no significant difference in the frequency of severe covid-19 infection among abo blood types.22 many studies conducted in countries such as kuwait,23 lebanon,24 china,25 italy,26 the usa,6,9 and france27 have also reported results that were in line with our findings. however, some studies have reported different findings regarding the association between blood types and covid-19 severity and outcomes. for example, a study in china found that blood group a patients had a higher risk of severity, while blood group o patients had a lower risk.28 another study in india found that blood group o patients had decreased mortality, while blood group b patients had increased mortality.29 yet another study in spain found that blood group a patients had a higher mortality risk, while group o patients had a lower mortality risk.16 further study from the usa suggested that individuals with blood group a and ab had a higher risk of severe disease and mortality than those with blood group o or b.30 nonetheless, limited data is available on the association between blood groups and covid-19 severity and outcomes, and further research is needed to fully understand the potential association between blood groups and covid-19 severity and mortality. this study has several limitations. first, we did not include a control group to determine the exact susceptibility of table 4. association of disease severity and patients’ outcomes among patients with different blood groups blood group disease severity p-value* patient outcome p-value*mild-moderate (n = 250) severe-critical (n = 154) recovered (n = 269) dead (n = 35) group a a 78 (64.46) 43 (35.54) 0.4848 107 (88.43) 14 (11.57) 0.1744 non-a 172 (60.78) 111 (39.22) 262 (92.58) 21 (7.42) group b b 58 (63.04) 34 (36.96) 0.7939 86 (93.48) 6 (6.52) 0.406 non-b 192 (61.54) 120 (38.46) 283 (90.71) 29 (9.29) group ab ab 16 (69.57) 7 (30.43) 0.4346 21 (91.30) 2 (8.70) 0.9955 non-ab 234 (61.42) 147 (38.58) 348 (91.34) 33 (8.66) group o o 98 (58.33) 70 (41.67) 0.2154 214 (90.68) 22 (9.32) 0.577 non-o 152 (64.41) 84 (35.59) 155 (92.26) 13 (7.74) rh rh– 12 (57.14) 9 (42.86) 0.6461 21 (100) 0 (0.00) 0.2391 rh+ 238 (62.14) 145 (37.86) 348 (90.86) 35 (9.14) *pearson chi-squared tests were performed for statistical analyses. 115j contemp med sci | vol. 9, no. 2, march-april 2023: 111–115 f.j. rasho et al. original the impact of blood group phenotypes on covid-19 severity and mortality in duhok province individuals with different blood types to covid-19; second, small sample size; third, observational study that did not prove causation. in conclusion, individuals with blood group o may have a higher risk of contracting covid-19. advanced age is a crucial predictor of disease severity and poor outcomes. there were no significant association between blood group types with covid-19 severity and patients’ outcomes. however, further research is needed to confirm these findings and determine the underlying mechanisms behind any potential association between blood groups and covid-19 susceptibility, severity, and outcome. acknowledgments our heartfelt appreciation goes out to ibrahim korshid from the laboratory department of the zakho directorate of health and zakya ageed from the zakho corona center for their invaluable assistance in facilitating the completion of this study. we would also like to extend our gratitude to all the patients who generously agreed to participate in this study. conflict of interest the authors declare that there is no conflict of interest.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1333 references 1. harrison, a.g., t. lin, and p. wang, mechanisms of sars-cov-2 transmission and pathogenesis. trends in immunology, 2020. 41(12): p. 1100–1115. 2. world health organization. who coronavirus (covid-19) dashboard. available from: https://covid19.who.int/. 3. national institute of health. covid-19 treatment guidelines panel. coronavirus disease 2019 (covid-19) treatment guidelines. 2022; available from: https://www.covid19treatmentguidelines.nih.gov/. 4. zhang, j., et al., risk factors for disease severity, unimprovement, and mortality in covid-19 patients in wuhan, china. clinical microbiology and infection, 2020. 26(6): p. 767–772. 5. zhao, j., et al., relationship between the abo blood group and the coronavirus disease 2019 (covid-19) susceptibility. clinical infectious diseases, 2021. 73(2): p. 328–331. 6. anderson, j.l., et al., association of sociodemographic factors and blood group type with risk of covid-19 in a us population. jama network open, 2021. 4(4): p. e217429–e217429. 7. ishaq, u., et al., association of abo blood group with covid-19 severity, acute phase reactants and mortality. plos one, 2021. 16(12): p. e0261432. 8. göker, h., et al., the effects of blood group types on the risk of covid-19 infection and its clinical outcome. turkish journal of medical sciences, 2020. 50(4): p. 679–683. 9. latz, c.a., et al., blood type and outcomes in patients with covid-19. annals of hematology, 2020. 99: p. 2113–2118. 10. merza, m.a., et al., clinical and epidemiological characteristics and outcomes of coronavirus disease-19 patients in a large longitudinal study. international journal of health sciences, 2021. 15(4): p. 29. 11. merza, m.a., et al., covid-19 outbreak in iraqi kurdistan: the first report characterizing epidemiological, clinical, laboratory, and radiological findings of the disease. diabetes & metabolic syndrome: clinical research & reviews, 2020. 14(4): p. 547–554. 12. mujahid, a. and f.l. dickert, blood group typing: from classical strategies to the application of synthetic antibodies generated by molecular imprinting. sensors, 2015. 16(1): p. 51. 13. omer, n., et al., the correlation between the covid-19 infection severity and abo blood groups in sulaimaniyah city, iraq. 2021. 14. ad’hiah, a.h., et al., association between abo blood groups and susceptibility to covid-19: profile of age and gender in iraqi patients. egyptian journal of medical human genetics, 2020. 21(1): p. 1–10. 15. badedi, m., et al., clinical characteristics and abo blood groups in covid-19 patients, saudi arabia. medicine, 2021. 100(30). 16. muñiz-diaz, e., et al., relationship between the abo blood group and covid-19 susceptibility, severity and mortality in two cohorts of patients. blood transfusion, 2021. 19(1): p. 54. 17. solmaz, i̇. and s. araç, abo blood groups in covid‐19 patients; cross‐ sectional study. international journal of clinical practice, 2021. 75(4): p. e13927. 18. jaff, m.s., abo and rhesus blood group distribution in kurds. journal of blood medicine, 2010: p. 143–146. 19. guan, w.-j., et al., clinical characteristics of coronavirus disease 2019 in china. new england journal of medicine, 2020. 382(18): p. 1708–1720. 20. palaiodimos, l., et al., severe obesity, increasing age and male sex are independently associated with worse in-hospital outcomes, and higher inhospital mortality, in a cohort of patients with covid-19 in the bronx, new york. metabolism, 2020. 108: p. 154262. 21. onder, g., g. rezza, and s. brusaferro, case-fatality rate and characteristics of patients dying in relation to covid-19 in italy. jama, 2020. 323(18): p. 1775–1776. 22. boudin, l., et al., abo blood groups are not associated with the risk of acquiring sars-cov-2 infection in young adults. haematologica, 2020. 105(12): p. 2841. 23. al‐youha, s.a., et al., the impact of abo blood groups on clinical outcomes and susceptibility to covid‐19: a retrospective study in an unselected population. transfusion, 2021. 61(5): p. 1631–1641. 24. khalil, a., r. feghali, and m. hassoun, the lebanese covid-19 cohort; a challenge for the abo blood group system. frontiers in medicine, 2020: p. 813. 25. dai, x., abo blood group predisposes to covid-19 severity and cardiovascular diseases. european journal of preventive cardiology, 2020. 27(13): p. 1436–1437. 26. negro, p., et al., role of abo blood system in covid‐19: findings from a southern italian study. transfusion medicine, 2022. 32(3): p. 243–247. 27. kim, y., et al. relationship between blood type and outcomes following covid-19 infection. in seminars in vascular surgery. 2021. elsevier. 28. li, j., et al., association between abo blood groups and risk of sars‐cov‐2 pneumonia. british journal of haematology, 2020. 190(1): p. 24. 29. padhi, s., et al., abo blood group system is associated with covid-19 mortality: an epidemiological investigation in the indian population. transfusion clinique et biologique, 2020. 27(4): p. 253–258. 30. zietz, m., j. zucker, and n.p. tatonetti, associations between blood type and covid-19 infection, intubation, and death. nature communications, 2020. 11(1): p. 5761. http://www.covid19treatmentguidelines.nih.gov/ 93j contemp med sci | vol. 9, no. 2, march-april 2023: 93–100 original investigation of the frequency and risk factors of pulmonary complications following cardiac surgery in the hospital mahdieh sharifzadeh kermani1, naeimeh naeimi bafghi2, neda naeimi bafghi2, shirin salajegheh1*, jafar salehi3, reza nakhaei zadeh4, mohammad javad javid1 1clinical research development unit, shafa hospital, kerman university of medical sciences, kerman, iran. 2clinical research development unit, shahid bahonar hospital, kerman university of medical sciences, kerman, iran. 3department of anesthesiology, school of medicine, kerman university of medical sciences, kerman, iran. 4department of cardiac surgery, school of medicine, kerman university of medical sciences, kerman, iran. *correspondence to: shirin salagegheh (e-mail: drshirinsalajegheh@aol.com) (submitted: 10 january 2023 – revised version received: 21 february 2023 – accepted: 15 march 2023 – published online: 26 april 2023) abstract objectives: the purpose of this study was to determine the frequency of pulmonary complications after heart surgery and to assess the pre-operative, intra-operative and post-operative risk factors. methods: this was a descriptive and analytical study that was conducted on 244 patients who underwent various cardiac surgeries from december 2021 to may 2022 in shefa kerman hospital. this descriptive/analytical study on patients undergoing heart surgery at shafa hospital, kerman, iran, from december 2021 to may 2022. pulmonary complications included atelectasis pneumonia, pleural effusion, long-term mechanical ventilation, and respiratory failure. the pre-operative, intra-operative and post-operative risk factors were investigated in the present study. spss software was used to analyze the data. logistic regression analysis was employed to determine the relationship between risk factors and the incidence of lung complications. results: in the present study, 55 patients (22.5%) had pulmonary complications, followed by prolonged mechanical ventilation (13.1%), pneumonia (11.5%), respiratory failure (4.1%), pleural effusion (11.1%) and atelectasis (6.1%). pulmonary complications after heart surgery were associated with days of icu stay, post-operative stroke, post-operative acute kidney injury, emergency surgery, ffp injection, high drainage rate, and mitral valve replacement surgery (all p < 0.001). 4% of patients died after surgery, which was statistically related to pulmonary complications (p < 0.0001). the results of multivariable logistic regression test showed that ffp injection, type of heart surgery, hypertension, icu stay days are capable of predicting pulmonary complications after heart surgery. conclusion: mortality was found to be higher in patients with pulmonary complications. based on the results of the regression analysis, ffp injection, type of surgery, history of hypertension and length of icu stay were independent risk factors of pulmonary complications. key words: pulmonary complications, cardiovascular surgery, risk factors, fresh frozen palsma issn 2413-0516 introduction cardiac surgery is a high-risk intervention that requires specialized teams to manage patients in the preand post-operative care. pulmonary complications include pneumonia with mechanical ventilation for more than 24 hours, respiratory failure, pleural effusion, atelectasis, pneumothorax, bronchospasm and aspiration pneumonitis, acute respiratory distress syndrome, and pulmonary embolism, which increases the length of hospital stay and treatment costs.1 heart surgery is usually performed by cardiopulmonary bypass. in this technique, the pumping action of the heart and the gas exchange of the lungs are temporarily replaced by a special mechanical device called an oxygenator pump, which is connected to the vascular system. cardiopulmonary bypass exposes the blood to artificial substances that lead to the production and secretion of toxins and the activation of the vascular reaction. in addition, the activation of neutrophils and their migration to the pulmonary circulation causes deep endothelial, epithelial, and interstitial lung damage, which is associated with increased capillary permeability, decreased lung capacity, and gas exchange disorders.2,3 abnormalities in gas exchange and changes in lung mechanical function cause pulmonary complications after heart surgery. in recent studies, changes in muscle and chest wall function due to median sternotomy, systemic inflammatory response syndrome with cardiopulmonary bypass, phrenic nerve injury due to cold saline injection in the pericardial cavity during cardiac arrest, and dilatation associated alveolar edema, and an increase in left ventricular pressure in the pulmonary vessels has been stated as the main causes of this complication.4,5 the reported frequency of pulmonary complications after cardiac surgery varies from 6 to 70% depending on the criteria used to define pulmonary complications.6 the incidence of pulmonary complications in major surgeries varies from 1 to 23%.7 identifying the risk factors of susceptible patients is helpful in preventing and eliminating complications. previous studies reported a combination of preoperative and postoperative risk factors. despite many advances in intraoperative care, postoperative pulmonary complications remain the main cause of disability and death after cardiac surgery in adults.8,9 very few studies have focused on intraand post-operative risk factors responsible for pulmonary complications in patients undergoing cardiac surgery using cardiopulmonary bypass.10,11 ventilation with a flow volume of 4–6 cc/kg during surgery was associated with a decrease in pulmonary complications.12 it has been reported that age over 60 years, prolongation of surgery time, preoperative pulmonary blood pressure and intraoperative phrenic nerve damage were risk factors for pulmonary complications.6 determining the factors affecting the rate of pulmonary complications can be beneficial in order to reduce these risk factors and timely treatment of more common pulmonary complications in patients after heart surgery. if risk factors are found, comprehensive and complete treatment of these factors mailto:drshirinsalajegheh@aol.com 94 j contemp med sci | vol. 9, no. 2, march-april 2023: 93–100 investigation of the frequency and risk factors of pulmonary complications following cardiac surgery original m.s. kermani et al. can be effective in improving the clinical course of patients who underwent heart surgery. in iran, there is a lack of studies on pulmonary complications after heart surgery.13,14 considering the large number of heart surgeries in iran and the high global prevalence of postoperative pulmonary complications, it is necessary to determine the prevalence of this complication and its related factors in iran. the current study aimed to investigate the frequency and risk factors of pulmonary complications following cardiac surgery in shafa hospital between 2021 and 2022. materials and methods this is a descriptive/analytical cross-sectional study approved by the ethics committee of kerman university of medical sciences (ir.kmu.ah.rec.1401.104). the research population included all patients who underwent heart surgery (coronary artery grafting, heart valve replacement, atrial septum repair, and ventricular septal defect repair) at shafa hospital, kerman, iran, from december 2021 to may 2022. the research population included all patients who underwent heart surgery (coronary artery grafting, heart valve replacement, atrial septum repair, and ventricular septal defect repair) at shafa kerman hospital from december 2021 to may 2022. patients who had surgery for congenital heart disease and incomplete medical records were excluded from the study. the data was extracted from the patient files through a researcher-made form including demographic information and medical information. collected data includes the variables of age, sex, smoking, opioid use, body mass index, ejection fraction, type of surgery, duration of surgery, type of pulmonary complication, use of cardiopulmonary pump during surgery. surgery, duration of cardiopulmonary pump, duration of mechanical ventilation, acute kidney injury after surgery, hemoglobin level before surgery, co-morbidity (diabetes, hypertension), duration of hospitalization in intensive care unit (icu), transfusion of blood or blood products, type of surgical urgency, postoperative drainage and mortality. pulmonary complications investigated included: pleural effusion, which was investigated and recorded by lung ultrasound and x-ray), pneumonia (fever, purulent sputum, test findings, lung x-ray and confirmed by infectious consultation), atelectasis (confirmed by lung x-ray and ultrasound), prolonged mechanical ventilation (more than 24 hours) and acute respiratory distress syndrome (ards). statistical analysis descriptive data were presented using mean, standard deviation, frequency and percentage in the form of tables and graphs. the kolmogorov smirnov test was used to check the normality of the data. t-test was used to compare the mean of two groups for normal data. mann whitney u test for non-normally distributed data and chi square test was used to analyze qualitative data. logistic regression analysis (backward: lr) was used to investigate the simultaneous effect of demographic and medical variables with postoperative pulmonary complications. the first group was considered as the reference group. spss software version 24 was used for data analysis. a significance level of less than 0.05 was considered. results 244 patients with a mean age of 59 ± 12 (15–82 years) participated in this study, consisting of 167 (68%) men and 77 (32%) women. the average height and weight of the patients were 168 ± 9 and 68 ± 13, respectively. the average bmi in these people was 24 ± 4 (range 14–37). 50% of patients used opium and 10% smoked. of these patients, 36% had diabetes, 68% had hypertension, and 5% had chronic kidney disease. the mean preoperative hemoglobin in the patients was 14 ± 2. the average ejection fraction in patients was 44 ± 10. the average ejection fraction in patients before surgery was determined to be 44 ± 10. 6% of patients underwent emergency surgery and 88% underwent elective surgery. the average time of surgery was 4 ± 0.8 hours (range 2–8 hours). 49% of patients used cardiopulmonary pump with an average duration of 37 ± 32 minutes. according to the type of surgery, 87% of patients underwent open heart surgery, followed by mitral valve replacement (8%), aortic valve replacement (6%), septal defect surgery (3%), and other surgeries (3%). after surgery, 55 patients (22.5%) had pulmonary complications, followed by prolonged mechanical ventilation (13.1%), pneumonia (11.5%), respiratory failure (4.1%), pleural effusion (11.1%) and atelectasis (6.1%). the duration of the intensive care unit (icu) satay after the operation was 5 ± 3 days. the average duration of mechanical ventilation in patients was also determined to be 22 ± 31 (range 4.5–264 hours) hours. during or after ffp operation, 17% of plt and 51% of p.c were injected for 20% of patients. the amount of drainage after surgery was found to be less than one liter for 85% of patients and more than one liter for 15%. 2.5% of patients had a stroke after surgery and 17.6% had acute kidney failure. finally, 4% of patients died after surgery. no significant difference was found in terms of the gender in two groups with pulmonary complications after cardiac surgery and without pulmonary complications after cardiac surgery. no significant difference was observed between on-pump and off-pump patients in two groups with pulmonary complications after heart surgery and without pulmonary complications after heart surgery. a significant difference was found in terms of the type of urgency in two groups after heart surgery. patients who underwent emergency surgery had more pulmonary complications after heart surgery. a statistically significant difference was also revealed in terms of drainage in two groups after heart surgery. patients who had more than one liter of drainage on the first day had more pulmonary complications after heart surgery. a significant difference was observed in terms of post op cva in two groups after heart surgery. all patients who had a stroke had pulmonary complications after heart surgery. a statistically significant difference was found in terms of death in patients in two groups after heart surgery, where patients who died showed more pulmonary complications after heart surgery. a significant difference was also seen in terms of acute kidney injury in patients of two groups after heart surgery. the frequency of pulmonary complications was higher in patients with acute kidney injury. 95j contemp med sci | vol. 9, no. 2, march-april 2023: 93–100 m.s. kermani et al. original investigation of the frequency and risk factors of pulmonary complications following cardiac surgery table 1. descriptive statistics n minimum maximum mean std. deviation age in years 243 15 82 58.75 11.699 weight in kg 244 2.00 120.00 68.3975 13.26509 height in cm 244 147.00 198.00 168.1844 8.55885 bmi 205 14.03 36.63 24.0432 3.92621 ejection fraction in percent 238 20.00 65.00 44.0126 10.19700 surgery duration in minutes 244 140.00 465.00 251.6189 47.83311 pump_time in minutes 244 .00 199.00 32.1352 36.59693 days of icu admission 244 1.00 24.00 4.9508 3.22631 mechanical ventilation hours 244 4.50 264.00 22.0266 30.58241 pre op hb 244 9.1 22.7 14.201 2.1269 valid n (listwise) 199 no significant difference was observed in patients of two groups in terms of drug use, smoking, blood pressure, diabetes and chronic kidney disease. a significant difference was observed in terms of mitral valve replacement in the two groups of patients after heart surgery. the frequency of pulmonary complications was higher in patients who had mitral valve replacement. a significant difference was observed in terms of other surgeries in patients of two groups. the frequency of pulmonary complications was higher in patients who had other surgeries. in terms of ffp injection, a significant difference was observed in two groups. the frequency of pulmonary complications was higher in patients who received ffp injection. there was no statistically significant difference in the mean age of patients in the two groups (p = 0.374) no statistically significant difference was found in the average duration of surgery in the two groups of patients (p = 0.735) there was a statistically significant difference in the average days of the icu stay in the two groups of patients (p < 0.0001). the average days of icu stay was found to be higher in the group of patients with pulmonary complications after heart surgery (p < 0.0001). there was no statistically significant difference between the two groups of patients in the mean preoperative hemoglobin (p = 0.603). the average time of being on the pump in the group of patients with pulmonary complications was higher than the patients without pulmonary complications, but this difference was not found to be statistically significant (p = 0.101). the results of multivariable logistic regression test demonstrated that ffp injection, type of heart surgery, blood pressure, and number of days of the icu stay can be capable of predicting pulmonary complications after heart surgery. people who had aortic valve replacement surgery are 91.7 times more likely to have pulmonary complications after heart surgery. people who had mitral valve replacement surgery are 73 times more likely to have pulmonary complications after heart surgery. patients who had septal defect surgery are 128 times more likely to have pulmonary complications after heart surgery. patients with open heart surgery have 36.8 times more pulmonary complications after heart surgery. every day icu stay was capable of increasing the chance of pulmonary complications by 1.72 times. people who had high blood pressure are 4.6 times more likely to have pulmonary complications after heart surgery. patients who received ffp injections were 4.6 times more likely to have pulmonary complications after heart surgery. discussion the present study was conducted with the aim of investigating the frequency and risk factors of pulmonary complications following heart surgery in shafa hospital from december 2021 to may 2022. 244 patients who underwent heart surgery during a period of 6 months were included in the study. in our study, there was no relationship between pulmonary complications and demographic characteristics, including average age, sex, and body mass index. the results of our study showed an incidence of 22.5% for pulmonary complications after heart surgery. pulmonary complications after heart surgery were related to type of surgery (mitral valve replacement surgery, other heart surgeries), history of hypertension, prolonged stay in icu, urgency of heart surgery, post-operative acute kidney injury, post-operative stroke, ffp injection and high drainage rate. mortality was significantly higher in patients with pulmonary complications. regarding the regression analysis of plasma injection, type of surgery, history of hypertension and length of stay in icu were independent risk factors for developing pulmonary complications. one of the positive points of our study was the comprehensive review of pre-operative, intra-operative and post operative risk factors in order to determine the frequency and risk factors influencing the development of pulmonary complications. regarding the retrospective nature of the study, it was not possible to investigate the thickness of the diaphragm and possible paralysis in pulmonary complications. the most common pulmonary complications in patients were long-term mechanical ventilation (more than 24 hours, 13.1%) and pneumonia (11.5%). considering that infection is an important factor of morbidity and mortality around 96 j contemp med sci | vol. 9, no. 2, march-april 2023: 93–100 investigation of the frequency and risk factors of pulmonary complications following cardiac surgery original m.s. kermani et al. table 2. frequency of pulmonary complications in heart surgery patients according to demographic and clinical characteristics pulmonary complicationtotal p-valueno yes percentfrequency 0.65512839frequency68.4167male sex 76.6%23.4%percent 6116frequency31.677female 79.2%20.8%percent 0.5059926frequency51.2125offpump 79.2%20.8%percent 9029frequency48.8119on 75.6%24.4%percent p < 0.0001611frequency5.714emergencyurgent type of surgery 35.3%64.7%percent 18344frequency88.5216elective 80.6%19.4%percent 0.04716542frequency84.8207below 1litdrainage 79.7%20.3%percent 2413frequency15.237over 1lit 64.9%35.1%percent p < 0.000118949frequency97.5238nopost op cva 79.4%20.6%percent 06frequency2.56yes 0.0%100.0%percent p < 0.000118648frequency95.9234nodeath 79.5%20.5%percent 37frequency4.110yes 30.0%70.0%percent 0.00116437frequency82.4201noacute kidney injury 81.6%18.4%percent 2518frequency17.643yes 58.1%41.9%percent table 3. frequency of pulmonary complications in heart surgery patients based on underlying diseases, type of heart surgery and transfusion of blood and blood products total pulmonary complication frequency percent no yes underlying diseases opioid addiction no 123 50.4 frequency 101 22 percent 82.1% 17.9% yes 121 49.6 frequency 88 33 percent 72.7% 27.3% smoking no 219 89.8 frequency 170 49 percent 77.6% 22.4% yes 24 9.8 frequency 19 5 percent 79.2% 20.8% unknown .4 .4 frequency (continued) 97j contemp med sci | vol. 9, no. 2, march-april 2023: 93–100 m.s. kermani et al. original investigation of the frequency and risk factors of pulmonary complications following cardiac surgery table 3. frequency of pulmonary complications in heart surgery patients based on underlying diseases, type of heart surgery and transfusion of blood and blood products—continued total pulmonary complication frequency percent no yes diabetes mellitus no 157 64.3 percent 125 32 frequency 79.6% 20.4% yes 87 35.7 percent 64 23 frequency 73.6% 26.4% hypertension no 79 32.4 percent 65 14 frequency 82.3% 17.7% yes 165 67.6 percent 124 41 frequency 75.2% 24.8% chronic kidney disease no 230 94.3 percent 180 50 frequency 78.3% 21.7% yes 12 4.9 percent 7 5 frequency 58.3% 41.7% unknown 2 .8 percent type of cardiac surgery aortic valve replacement no 230 94.3 frequency 180 50 percent 78.3% 21.7% yes 14 5.7 frequency 9 5 percent 64.3% 35.7% mitral valve replacement no 225 92.2 frequency 178 47 percent 79.1% 20.9% yes 19 7.8 frequency 11 8 percent 57.9% 42.1% septal defect no 236 96.7 frequency 182 54 percent 77.1% 22.9% yes 8 3.3 frequency 7 1 percent 87.5% 12.5% cardiac arterial bypass graft no 32 13.1 frequency 25 7 percent 78.1% 21.9% yes 212 86.9 frequency 164 48 percent 77.4% 22.6% other no 237 97.1 frequency 186 51 percent 78.5% 21.5% yes 7 2.8 frequency 3 4 percent 42.9% 57.1% transfusion of blood and blood products ffp no 196 80.3 frequency 163 33 percent 83.2% 16.8% yes 48 19.7 frequency 26 22 percent 54.2% 45.8% plt no 202 82.8 frequency 159 43 percent 78.7% 21.3% yes 42 17.2 frequency 30 12 percent 71.4% 28.6% p.c no 120 49.2 frequency 93 27 percent 77.5% 22.5% yes 124 50.8 frequency 96 28 percent 77.4% 22.6% 98 j contemp med sci | vol. 9, no. 2, march-april 2023: 93–100 investigation of the frequency and risk factors of pulmonary complications following cardiac surgery original m.s. kermani et al. table 4 frequency of pulmonary complications in patients variables frequency percent pulmonary complication no 189 77.5 yes 55 22.5 pneumonia no 216 88.5 yes 28 11.5 respiratory failure no 234 95.9 yes 10 4.1 pleural effusion no 217 88.9 yes 27 11.1 atelectasis no 229 93.9 yes 15 6.1 prolonged mechanical ventilation no 212 86.9 yes 32 13.1 table 5. comparison of the mean of quantitative variables in two groups of patients with pulmonary complications and without pulmonary complications after heart surgery variables pulmonary complication n mean std. deviation std. error mean p-value age in years no 189 58.39 11.945 .869 .374 yes 54 60.00 10.807 1.471 surgery duration in minutes no 189 251.0582 45.20570 3.28823 .735 yes 55 253.5455 56.36088 7.59970 days of icu admission no 189 4.1111 1.67056 .12152 p < 0.0001 yes 55 7.8364 5.11629 .68988 pre op hb no 189 14.239 2.1108 .1535 .603 yes 55 14.069 2.1958 .2961 pump_time in minutes no 189 29.5185 32.98204 2.39909 0.101 yes 55 41.1273 46.18965 6.22821 surgery, it is necessary to examine and identify patients at risk for faster control and treatment. the occurrence of pulmonary complications in studies has been between 3 and 50% and its occurrence is the result of pre-operative, intra-operative and post-operative risk factors. older age, genetics, diabetes, obesity, smoking, chronic lung disease, and emergency surgery have been identified as preoperative risk factors in studies. in our study, emergency surgery and hypertension were risk factors, while other comorbidities were not associated with increased risk, probably owing to the smaller sample size. in our study, one of the risk factors affecting the increase of pulmonary complications during surgery was the type of surgery, i.e., non-coronary and heart valve surgeries. in sadeghi’s study, patients with heart valve surgery had higher complications and mortality because a large number of these patients suffer from obstructive and restrictive lung disease, which may be due to cardiomegaly, pleural effusion, table 6. the results of multivariate logistic regression b s.e. exp(b) 95% c.i. for exp(b) p-value lower upper gender (1) –.815 .793 .443 .094 .304 .304 age in years –.031 .028 .969 .917 .270 .270 bmi .050 .069 1.052 .918 .466 .466 aortic valve replacement (1) 4.519 1.504 91.725 4.813 .003 .003 mitral valve replacement (1) 4.295 1.755 73.337 2.351 .014 .014 septal defect (1) 4.857 2.061 128.574 2.265 .018 .018 coronary arterial bypass (1) 3.606 1.660 36.803 1.423 .030 .030 surgery duration in minutes –.002 .008 .998 .982 .752 .752 pump (1) –2.136 1.334 .118 .009 .109 .109 pump_time in minutes .021 .020 1.021 .982 .298 .298 opioid addiction (1) .923 .518 2.516 .911 .075 .075 smoking (1) –.010 .951 .990 .153 .991 .991 diabetes mellitus (1) .429 .610 1.536 .465 .482 .482 hypertension (1) 1.540 .690 4.665 1.206 .026 .026 (continued) 99j contemp med sci | vol. 9, no. 2, march-april 2023: 93–100 m.s. kermani et al. original investigation of the frequency and risk factors of pulmonary complications following cardiac surgery table 6. the results of multivariate logistic regression—continued b s.e. exp(b) 95% c.i. for exp(b) p-value lower upper chronic kidney disease (1) –2.842 2.312 .058 .001 .219 .219 days of icu admission .545 .126 1.724 1.346 .000 .000 ffp (1) 1.539 .591 4.658 1.463 .009 .009 plt (1) –1.676 .797 .187 .039 .036 .036 p.c (1) –.529 .665 .589 .160 .427 .427 acute kidney injury (1) .041 .781 1.042 .225 .958 .958 urgent type of surgery (1) –.690 .946 .502 .079 .466 .466 drainage (1) .290 .762 1.336 .300 .704 .704 constant –14.449 3.152 .000 .000 .000 peribronchial and pericapillary fibrosis during the period of pulmonary congestion.14 in our study, fresh frozen plasma injection was an independent risk factor for pulmonary complications. it was stated by gupta et al. that intraoperative blood transfusion was significantly higher in patients with pulmonary complications after surgery.15 while the results of the study by mathis and his colleagues showed a significant relationship between the transfusions of various blood products and pulmonary complications after heart surgery.12 following the infusion of blood products, especially plasma and platelets, there is a possibility of complications such as transfusion-related acute lung injury (trali), transfusion-related circulatory overload (taco) and increased risk of infection transmission.16,17 serani et al. also showed that plasma transfusion in sick patients was associated with an increased risk of infections.18 bochicchio’s study showed an increased risk of ventilator associated pneumonia in trauma patients with transfusion of blood products.19 according to the results of our study, plasma injection was introduced as a risk factor for causing postoperative pulmonary complications. transfusion of plasma and blood products is recommended to be based on the indication, if necessary. according to desborough’s review study, the prophylactic administration of fresh frozen plasma for patients undergoing cardiovascular surgery is not approved in the absence of coagulopathy, and more research is needed for other outcomes including 30-day mortality due to the bias in the studies and their low quality.20 in the present study, acute kidney failure and longer length of icu stay were associated with increased pulmonary complications, which was consistent with other studies.21 the mortality rate in patients with pulmonary complications was significantly higher, which was similar to other studies.6,22 pulmonary complications after heart surgery can be prevented by teaching the prevention and management of these risk factors to the health department staff and determining specific protocols. further longitudinal and multicenter studies are needed to investigate the risk factors of pulmonary complications after heart surgery. conflict of interest none.  references 1. tristan george tanner, mai o. colvin. pulmonary complications of cardiac surgery. lung (2020) 198:889–896. 2. wynne r, botti m. postoperative pulmonary dysfunction in adults after cardiac surgery with cardiopulmonary bypass: clinical significance and implications for practice. am j crit care. 2004;13(5):384–93. 3. mali s, haghaninejad h. pulmonary complications following cardiac surgery. archives of medical sciences atherosclerotic diseases. 2019;4:e280. 4. sabaté s, mazo v, canet j. predicting postoperative pulmonary complications: implications for outcomes and costs. current opinion in anesthesiology. 2014;27(2):201–9. 5. gravlee gp. cardiopulmonary bypass: principles and practice: lippincott williams & wilkins; 2008. 6. naveed a, azam h, murtaza hg, ahmad ra, baig mar. incidence and risk factors of pulmonary complications after cardiopulmonary bypass. pakistan journal of medical sciences. 2017;33(4):993. 7. miskovic a, lumb a. postoperative pulmonary complications. bja: british journal of anaesthesia. 2017;118(3):317–34. 8. canet j, gallart l, gomar c, paluzie g, valles j, castillo j, et al. prediction of postoperative pulmonary complications in a population-based surgical cohort. the journal of the american society of anesthesiologists. 2010;113(6):1338–50. 9. younossian ab, adler d, bridevaux p-o, kherad o. postoperative pulmonary complications: how to anticipate and prevent the risk? revue medicale suisse. 10. canver cc, chanda j. intraoperative and postoperative risk factors for respiratory failure after coronary bypass. the annals of thoracic surgery. 2003;75(3):853–7. 11. ji q, mei y, wang x, feng j, cai j, ding w. risk factors for pulmonary complications following cardiac surgery with cardiopulmonary bypass. international journal of medical sciences. 2013;10(11):1578. 12. mathis mr, duggal nm, likosky ds, haft jw, douville nj, vaughn mt, et al. intraoperative mechanical ventilation and postoperative pulmonary complications after cardiac surgery. anesthesiology. 2019;131(5):1046–62. 13. mali s, haghaninejad h. pulmonary complications following cardiac surgery. archives of medical science-atherosclerotic diseases. 2019;4(1):280–5. 14. sadeghi ha, tabrizi ra, ghadrdoost b, azarfarin r. evaluation of pulmonary complications in patients with valvular heart surgery: clinical and laboratory significances. res cardiovasc med. 2017;6(2):e39944. 15. gupta s, fernandes rj, rao js, dhanpal r. perioperative risk factors for pulmonary complications after non-cardiac surgery. journal of anaesthesiology, clinical pharmacology. 2020;36(1):88. 16. saadah nh, van der bom jg, wiersum‐osselton jc, richardson c, middelburg ra, politis c, et al. comparing transfusion reaction risks for various plasma products–an analysis of 7 years of istare haemovigilance data. british journal of haematology. 2018;180(5):727–34. 17. khan h, belsher j, yilmaz m, afessa b, winters jl, moore sb, et al. fresh-frozen plasma and platelet transfusions are associated with 100 j contemp med sci | vol. 9, no. 2, march-april 2023: 93–100 investigation of the frequency and risk factors of pulmonary complications following cardiac surgery original m.s. kermani et al. development of acute lung injury in critically ill medical patients. chest. 2007;131(5):1308–14. 18. sarani b, dunkman wj, dean l, sonnad s, rohrbach ji, gracias vh. transfusion of fresh frozen plasma in critically ill surgical patients is associated with an increased risk of infection. critical care medicine. 2008;36(4):1114–8. 19. bochicchio gv, napolitano l, joshi m, bochicchio k, shih d, meyer w, et al. blood product transfusion and ventilator-associated pneumonia in trauma patients. surgical infections. 2008;9(4):415–22. 20. desborough mjr, sandu r, brunskill sj, doree c, trivella m, montedori a, abraha i, stanworth sj. fresh frozen plasma for cardiovascular surgery. cochrane database of systematic reviews 2015, issue 7. art. no.: cd007614. doi: 10.1002/14651858.cd007614.pub2. 21. gontse leballo, hlamatsi jacob moutlana, michel kasongo muteba, palesa motshabi chakane. factors associated with acute kidney injury and mortality during cardiac surgery gontse leballo, hlamatsi jacob moutlana, michel kasongo muteba, palesa motshabi chakane cardiovascular journal of africa 2021;32(6):313–318. 22. cavayas ya, eljaiek r, rodrigue é, lamarche y, girard m, wang ht, et al. preoperative diaphragm function is associated with postoperative pulmonary complications after cardiac surgery. critical care medicine. 2019;47(12):e966–e74. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1313 323j contemp med sci | vol. 8, no. 5, september-october 2022: 323–326 original a study of factors that impact the production of anti-sars-cov-2 antibodies in patients with covid-19 nawfal r hussein1, amer balatay2, ameen m mohammad3, kuldeep dhama4, narin a rasheed5* 1department of biomedical sciences, college of medicine, university of zakho, zahko, kurdistan region, iraq. 2department of clinical pharmacy, college of pharmacy, university of duhok, duhok, kurdistan region, iraq. 3department of internal medicine, college of medicine, university of duhok, duhok, kurdistan region, iraq. 4division of pathology, icar-indian veterinary research institute (ivri), izatnagar243122, bareilly, uttar pradesh, india. 5department of medical laboratory technology, college of health and medical technology-shekhan, duhok polytechnic university, duhok, kurdistan region, iraq. *correspondence to: narin a rasheed (e-mail: narin.rasheed@gmail.com) (submitted: 29 april 2022 – revised version received: 12 may 2022 – accepted: 17 may 2022 – published online: 26 october 2022) abstract objectives: the aim of this paper was to investigate the impact of different variables on the production of antibodies in patients who were infected with covid-19. methods: this cross-sectional study was conducted in duhok city, kurdistan region of iraq. the study was conducted between january 2021 and march 2022. demographic data were collected via face-to-face interview. antibody levels were determined using elecsys anti-sars-cov-2. results: the levels of antibody were studied in 138 patients. a significant association was found between antibody levels and the age of the participants (r = 0.175; p = 0.04). besides, a significant correlation was found between antibody levels and the duration of symptoms (r = 0.206; p = 0.015). the antibody levels were not associated with gender; history of chronic diseases; marital status or time interval before testing. conclusion: different variables that may impact the levels of antibody were studied. significant associations were found between antibody levels and both age and duration of symptoms. our results can be used by healthcare providers to focus on patients who are at risk of low antibody production. keywords: sars-cov-2, antibodies, covid-19, duhok, iraq issn 2413-0516 introduction covid-19 caused by sars-cov-2 that was discovered in wuhan, china, in december 2019 and rapidly spread worldwide.1 since the discovery of first cases of covid-19 in kurdistan region of iraq in march, 2020, the region passed through three devastating waves with a case fatality rate of 2%. during the first wave, strict measures were taken to control the infection2 and the case fatality rate was low. then, due to public fatigability and unwillingness,3,4 the measures were relaxed leading a sharp increase in morbidity and mortality of covid-19 cases. such an increase in the cases had a negative impact on the already weak health system.5–8 the number of covid-19 patients exceeded the capacities of acute care beds and home management scheme was launched to manage cases of severe covid. covid-19 has a wide array of symptoms particularly fever and respiratory symptoms such as cough and shortness of breath.9,10 non-respiratory symptoms are not uncommon such as gastrointestinal symptoms including diarrhea and vomiting. herd immunity can be the most important factor to control the pandemic. herd immunity is defined as the reduction in the number of cases due to the development of immunity by natural infection of vaccination.11,12 the spike (s) protein is the antigenic protein of sars-cov-2 that mediate the fusion with human angiotensin-converting enzyme 2 (ace2). the s antigen is consisted of two subunits: 1 (s1) and 2 (s2). the latter is involved in the fusion process between the cell membranes and viruses. antibodies that inhibit this specific binding are known as neutralizing antibody. such antibodies play a pivotal rule in the prevention of reinfection.13–15 it is worth mentioning that studies have shown that antibody response induced by natural infection wane over time and different factors may impact the production and the intensity of antibody production and reinfection is possible.13–15 the aim of this paper was to investigate the impact of different variables on the production of antibodies in patients who were infected with covid-19. materials and methods study design this cross-sectional study was conducted in duhok city, kurdistan region of iraq. the study was conducted between january 2021 to march 2022. blood samples were collected form participants who were at least 18 years old, had a history of confirmed covid-19, had not received vaccination and agreed to participate in the study. then, 5–10 cm3 of venous blood samples were collected using 10-cm3 syringes. the samples were immediately transported to the research center, and sera were separated from the blood and kept frozen at –20°c. demographic data were collected via face-to-face interview. anti-sars-cov-2 antibody antibody levels were determined using elecsys antisars-cov-2 (roche diagnostics international ltd, rotkreuz, switzerland), which is an in-vitro immunoassay to determine antibodies (including igg) to the sars-cov-2 spike (s) protein receptor binding domain (rbd) in human serum and plasma. the assay was performed according to the manufacturer’s instructions. according to the manufacturer, a cutoff index ≥0.8 indicates a positive result. mailto:narin.rasheed@gmail.com 324 j contemp med sci | vol. 8, no. 5, september-october 2022: 323–326 factors impacting anti-sars-cov-2 antibody production original n.r. hussein et al. statistics binary logistic regression was utilized to analyze the association between antibody levels and dichotomous data. pairwise pearson’s correlation was utilized to investigate the relationship between antibody levels and continuous variables. all calculations were performed using minitab 20 software. p value of ≤0.05 was considered significant. ethics the study and all procedures were approved by ethics and scientific committee of the college of medicine, university of zakho. the work was carried out in accordance with the code of ethics of the world medical association (declaration of helsinki) for experiments involving humans. written informed consent was obtained from all participants. results blood samples were collected from participants with a previous history of confirmed covid-19. demographic data were collected via face-to-face interview. in this study, the mean age of the participants was 38.44 ± 1.17, and (68/138) 49.28% of the participants were female. among the participants, (111/138) 80.43% were married and (64/138) 46.38% had a history of chronic diseases (table 1). binary logistic regression was utilized to analyze the association between antibody levels and dichotomous data. although antibody levels were higher in females than that found in males, no statistically significant association was found between sex and antibody levels (or = 0.99; ci = 0.99–1.003; p = 0.7) (table 2). besides, no significant association was found between the history of chronic diseases and antibody levels (or = 1.003; ci = 0.998–1.01; p = 0.1) (table 2). antibody levels were higher in married than unmarried participants, although the association was not statistically significant (or = 1.007; ci = 0.999–1.013; p = 0.058) (table 2). pairwise pearson’s correlation was utilized to investigate the relationship between antibody levels and age. a significant association was found between antibody levels and the age of the participants (r = 0.175; p = 0.04) (figure 1). additionally, pairwise pearson’s correlation was used to study the correlation between antibody levels and duration of the symptoms during the infection. a significant correlation was found between antibody levels and the duration of symptoms (r = 0.206; p = 0.015) (figure 2). the correlation between time interval before testing and antibody levels was investigated. no association was found between antibody levels and time interval before testing (r = 0.038; p = 0.66) (figure 3). discussion kurdistan region of iraq went through three devastating waves that impacted the already weak health system. since the appearance of sars-cov-2 pandemic, healthcare providers are aiming to controlling the spread of the infection.16,17 the development of herd immunity after natural infection or vaccination was the aim. however, reinfection and breakthrough infection appeared to be obstacles for controlling the pandemic by herd immunity.18 understanding factors associated with waning of the immunity is important for healthcare providers to focus on patients who are at risk of low antibody production.19 therefore; we aimed at studying different variables associated with antibody levels in patients who were infected with sars-cov-2. in our study, we found that older age was associated with higher levels of anti-sars-cov-2 antibodies. our results are in agreement with previous studies that found higher level of antibodies in older patients.20–22 this might be explained by that older patients are susceptible to severe infection which might induce more rigorous immune reaction. more studies are needed to investigate the dynamic of antibody response over the time in older patients. additionally, it was previously proposed that biological sex impacts immune responses and covid-19 outcomes.23 in support of this in a table 1. characteristics of participants dichotomous variables no. % gender female 68 49.28 male 70 50.72 chronic disease no 74 53.62 yes 64 46.38 marital status no 27 19.57 yes 111 80.43 continuous variables mean se mean minimum maximum age (year) 38.44 1.17 18 76 time interval before testing (day) 148.82 4.39 38 313 duration of symptoms (day) 12.568 0.743 2 45 se, standard error. table 2. associations between antibody levels and different factors variables no. mean se mean minimum q1 median q3 maximum or ci p value sex female 68 71.95 8.19 0.1 10.22 42.45 131.42 203.6 0.999 0.99-1.003 0.7 male 70 67.58 8.5 0.1 5.22 40.25 121.9 221.9 chronic disease no 74 62.04 7.89 0.1 2.82 22.65 114.08 200.3 1.003 0.998-1.01 0.1 yes 64 78.62 8.76 0.1 10.2 65.55 135.08 221.9 marital status no 27 46.7 12.3 0.1 1.2 10.9 92 221.9 1.007 0.999-1.013 0.058 yes 111 75.32 6.59 0.1 7.4 56.7 134.4 203.6 se, standard error; q, quartile; or, odd ratio; ci, confidence interval. 325j contemp med sci | vol. 8, no. 5, september-october 2022: 323–326 n.r. hussein et al. original factors impacting anti-sars-cov-2 antibody production fig. 3 scatterplot showing the association between antibody levels and time interval before testing. no significant association was found between antibody levels and the interval before testing (r = 0.038; p = 0.66). fig. 1 scatterplot showing the association between antibody levels and age of the participants. a significant association was found between antibody levels and age of participants (r = 0.175; p = 0.04) fig. 2 scatterplot showing the association between antibody levels and the duration of symptoms. a significant association was found between antibody levels and the duration of symptoms (r = 0.206; p = 0.015). patients than that found in female patients.25 however, in our study, no significant correlation was found between sex and post-infection antibody levels. the disparities in results may be attributed to sampling and methods used in measuring antibody levels. furthermore, previous studies showed associations between history of chronic diseases and the levels of antibody.26,27 in our study, no associations were found between history of chronic diseases and the levels of antibody. besides, in agreement with previous studies,20,28 we found a statistically significant association between the duration of symptoms and antibody levels. this might be explained by that more exposure of immune system to the virus with longer duration of symptoms. more research is needed to explore this. our results are useful because they provide an insight into factors that may impact the production of antibodies in patients with covid-19. our results can be used by healthcare providers to focus on patients who are at risk of low antibody production. finally, our results can be used by researchers to investigate antibody production after vaccination in those who are low antibody producers. declaration of interests the authors declare no conflict of interest.  study recruiting patients with severe covid-19, it was shown that the higher levels of antibody was found in female patients than that found in male patients.24 in the same study, the generation of igg antibody was stronger in females than males in early phase of the disease.24 in contrast, in a study recruiting convalescent patients, antibody levels were higher in male references 1. kumar a, singh r, kaur j, pandey s, sharma v, thakur l, et al. wuhan to world: the covid-19 pandemic. frontiers in cellular and infection microbiology. 2021;11. 2. hussein nr. the role of self-responsible response versus lockdown approach in controlling covid-19 pandemic in kurdistan region of iraq. international journal of infection. 2020;7(4). 3. martinez-garcia m, rabasa a, barber x, polotskaya k, roomp k, oliver n. key factors affecting people’s unwillingness to be confined during the covid-19 pandemic in spain: a large-scale population study. scientific reports. 2021;11(1):18626. 4. wondreys j, mudde c. victims of the pandemic? european far-right parties and covid-19. nationalities papers. 2022;50(1):86–103. 5. robert r, kentish-barnes n, boyer a, laurent a, azoulay e, reignier j. ethical dilemmas due to the covid-19 pandemic. annals of intensive care. 2020;10(1):84. 6. lami f, rashak ha, khaleel ha, mahdi sg, adnan f, khader ys, et al. iraq experience in handling the covid-19 pandemic: implications of public health challenges and lessons learned for future epidemic preparedness planning. journal of public health. 2021;43(supplement_3):iii19–iii28. 7. hussein n. the impact of covid-19 pandemic on the elimination of viral hepatitis in duhok city, kurdistan region of iraq. hepatitis monthly. 2020;20(5):e104643. 8. hussein nr, musa dh, ibrahim n, naqid ia, saleem zsm, jacksi k. impact of covid-19 pandemic on surgical practice in kurdistan, iraq: an online cross-sectional survey. international journal of surgery open. 2020;27:47–51. 9. vetter p, vu dl, l’huillier ag, schibler m, kaiser l, jacquerioz f. clinical features of covid-19. bmj. 2020;369:m1470. 10. hwaiz ra, zaki abdullah sm, jalal balaky st, ali ks, merza my, khailani sa, et al. clinical and hematological characteristics of 300 covid-19 patients in erbil, kurdistan region, iraq. international journal of immunopathology and pharmacology. 2022;36:03946320221085465. 11. john tj, samuel r. herd immunity and herd effect: new insights and definitions. european journal of epidemiology. 2000;16(7):601–6. 326 j contemp med sci | vol. 8, no. 5, september-october 2022: 323–326 factors impacting anti-sars-cov-2 antibody production original n.r. hussein et al. 12. frederiksen lsf, zhang y, foged c, thakur a. the long road toward covid-19 herd immunity: vaccine platform technologies and mass immunization strategies. frontiers in immunology. 2020;11. 13. sariol a, perlman s. lessons for covid-19 immunity from other coronavirus infections. immunity. 2020;53(2):248–63. 14. hussein nr, musa dh, saleem zsm, naqid ia, ibrahim n. possible covid-19 reinfection case in duhok city, kurdistan: a case report. journal of family medicine and primary care. 2021;10(5):2035. 15. hussein nr, rashad bh, almizori la, yousif ss, sadeeq at, abdulkareem yr, et al. the risk of sars-cov-2 reinfection in duhok city, kurdistan region of iraq. mediterranean journal of hematology and infectious diseases. 2021;13(1). 16. zenone m, snyder j, marcon a, caulfield t. analyzing natural herd immunity media discourse in the united kingdom and the united states. plos global public health. 2022;2(1):e0000078. 17. khalife j, vangennep d. covid-19 herd immunity in the absence of a vaccine: an irresponsible approach. epidemiol health. 2021;43:e2021012. 18. hall v, foulkes s, insalata f, kirwan p, saei a, atti a, et al. protection against sars-cov-2 after covid-19 vaccination and previous infection. new england journal of medicine. 2022;386(13):1207–20. 19. papachristodoulou e, kakoullis l, parperis k, panos g. long-term and herd immunity against sars-cov-2: implications from current and past knowledge. pathog dis. 2020;78(3). 20. klein sl, pekosz a, park h-s, ursin rl, shapiro jr, benner se, et al. sex, age, and hospitalization drive antibody responses in a covid-19 convalescent plasma donor population. the journal of clinical investigation. 2020;130(11):6141–50. 21. zhang b, zhou x, zhu c, song y, feng f, qiu y, et al. immune phenotyping based on the neutrophil-to-lymphocyte ratio and igg level predicts this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i5.1242 disease severity and outcome for patients with covid-19. frontiers in molecular biosciences. 2020;7(157). 22. yang hs, costa v, racine-brzostek se, acker kp, yee j, chen z, et al. association of age with sars-cov-2 antibody response. jama network open. 2021;4(3):e214302-e. 23. scully ep, haverfield j, ursin rl, tannenbaum c, klein sl. considering how biological sex impacts immune responses and covid-19 outcomes. nature reviews immunology. 2020;20(7):442–7. 24. zeng f, dai c, cai p, wang j, xu l, li j, et al. a comparison study of sarscov-2 igg antibody between male and female covid-19 patients: a possible reason underlying different outcome between sex. journal of medical virology. 2020;92(10):2050–4. 25. markmann aj, giallourou n, bhowmik dr, hou yj, lerner a, martinez dr, et al. sex disparities and neutralizing-antibody durability to sars-cov-2 infection in convalescent individuals. msphere. 2021;6(4):e00275–21. 26. karuna s, li ss, grant s, walsh sr, frank i, casapia m, et al. neutralizing antibody responses over time in demographically and clinically diverse individuals recovered from sars-cov-2 infection in the united states and peru: a cohort study. plos medicine. 2021;18(12):e1003868. 27. zejda je, brożek gm, kowalska m, barański k, kaleta-pilarska a, nowakowski a, et al. seroprevalence of anti-sars-cov-2 antibodies in a random sample of inhabitants of the katowice region, poland. international journal of environmental research and public health. 2021;18(6):3188. 28. marklund e, leach s, axelsson h, nyström k, norder h, bemark m, et al. serum-igg responses to sars-cov-2 after mild and severe covid-19 infection and analysis of igg non-responders. plos one. 2020;15(10):e0241104. 158 j contemp med sci | vol. 9, no. 3, may-june 2023: 158–162 original molecular detection of mononucleotide biomarkers of microsatellite instability in sporadic colorectal carcinoma patients with clinicopathological correlation wed thamer salman al-jumaili1*, bassam musa sadik al-musawi2 1al-amal national hospital for cancer management, baghdad, iraq. 2department of pathology and forensic medicine, college of medicine, university of baghdad, baghdad, iraq. *correspondence to: wed thamer salman al-jumaili *(e-mail: widthamir1984@gmail.com) (submitted: 10 april 2023 – revised version received: 09 may 2023 – accepted: 11 june 2023 – published online: 26 june 2023) abstract objectives: to identify the frequency and types of microsatellite instability among a group of sporadic crc patients and to correlate the findings with clinicopathological characteristics. methods: during an 8-month period, all patients with sporadic crc who attended to two teaching hospitals in baghdad, iraq were recruited to this cross-sectional study regardless of age, sex, ethnicity, or tumor characteristics. demographic, clinical, and histopathological features were recorded. dna was extracted from ffpe-blocks of the resected tumors and normal tissues. pcr amplification of five microsatellite mononucleotide repeat loci (bat25, bat26, nr-21, nr-24, and mono-27) and 2 pentanucleotide repeat control markers (penta c and penta d) was performed to determine the msi status. capillary electrophoresis and genetic analyzer 3500 (applied biosystem, japan) were used to separate and examine the products. data were analyzed by genescan software (promega, usa). instability of two or more loci is considered msi-h. result: in this study, ages of the 45 recruited patients ranged between 20–80 years, with a mean ± sd of 55 ± 12.3 years; of them, 31(68.9%) were ≥50 years; 25 (55.6%) were males. rectal bleeding was the most frequent presenting feature [22 (48.9%)] patients; 23 (51.1%) of crcs were located at recto-sigmoid region, 29 (64.4%) were t3 tumors, 34(75.5%) were non-mucinous adenocarcinoma, 39(86.7%) were moderately differentiated, 17 (37.8%) patients had stage iii tumors; and 25 (55.5%) had lymphovascular invasion. msi-h was seen in 5/45 (11.1%) patients; 3(60%) of them were ≥50 years, 4(80%) were males, 3(60%) were smokers, 2 (40%) presented with intestinal obstruction and altered bowel habits each; 4(80%) had t3 tumors, 3(60%) had mucinous adenocarcinomas [p = 0.004], 2(40%) had stage ii tumor and stage iii each. conclusion: the frequency of msi-h among the recruited patients with crc was 5/45 (11.1%) and it was significantly associated with mucinous adenocarcinoma subtype. nr-24 and nr-21 were the most prevalent instable markers. keywords: colorectal neoplasms, adenocarcinoma, microsatellite instability, polymerase chain reaction, iraq issn 2413-0516 introduction colorectal cancer (crc) is the second most prevalent cancer in women following breast cancer, and the third most frequent cancer in men following lung and prostate cancers.1–3 around the world, both incidence and mortality rates of crc had significantly risen.4 rectal and colon cancer deaths are predicted by 2035 to reach 60% and 71.5%, respectively.5 crc is a heterogeneous disease caused by the interaction of environmental and genetic factors that turn healthy colonic and rectal cells into invasive cancer.6 several factors may increase the risk of developing crc including age, environmental factors such as unhealthy diet, alcohol, obesity, smoking, digestive disorders (crohn’s disease and ulcerative colitis), and genetic factors such as familiar adenomatous polyposis (fap), and hereditary non-polyposis colorectal cancer (hnpcc).7–9 chromosome instability, microsatellite instability (msi), and cpg island methylator phenotype (cimp) are the three main types of genomic instability seen in crc.10 msi, seen in 15% of crc cases, can develop due to genetic or epigenetic changes. genetic modification includes germline mutations in the mmr genes (mlh1, msh 2, msh6, and pms2), which typically manifests in inherited crcs e.g. hnpcc – lynch syndrome. the second process involves an epigenetic alteration that results in hypermethylation of the mlh1 gene’s promoter region, which causes an accumulation of dna mutations and the production of the mutant mmr protein. three primary forms of colorectal cancer can be distinguished by msi testing: msi-high (msi-h), msi-low (si-l) and msi-stable (mss).11 msi-h has particular clinical and prognostic significance for crc.12 compared to other types of crcs, msi-h tumors exhibit less metastasis and respond better to immunotherapy than chemotherapy, particularly 5fu.13 the aims of this study were to identify the frequency and types and of msi among a group of sporadic crc patients and to correlate msi status with demographic and clinicopathological characteristics. methods this is a cross-sectional study that recruited 45 patients with sporadic colorectal adenocarcinoma from two major teaching hospitals in baghdad, iraq, namely: baghdad teaching hospital and gastroenterology & hepatology teaching hospital, medical city between september 2021 and april 2022. all patients, regardless of age, sex, race, residence, tumor characteristics, who underwent surgical resection of their tumors, were enrolled to this study. the diagnosis and histopathologic findings were reviewed and confirmed by a second histopathologist. for each patient, the basic demographic, clinical and histopathologic findings were recorded from hospital records. two slices were cut from the formalin-fixed, paraffin-embedded (ffpe) tissue blocks: one from the tumor with the highest concentration of tumor cells and the other from nearby 159j contemp med sci | vol. 9, no. 3, may-june 2023: 158–162 w. t. s. al-jumaili et al. original detection of msi biomarkers in sporadic colorectal cancer non-tumorous tissue (safe margin). dna was extracted using the qiaamp dna ffpe tissue kit® (50) by (qiagen / german), according to manufacturer’s instructions. five single nucleotide repeat loci (bat25, bat26, nr-21, nr-24, and mono-27) and two pentanucleotide repeat control markers (penta c and penta d) were tested. the mononucleotide markers were used for msi determination while the pentanucleotide markers were used for specimen identity i.e. the normal and the tumor specimens originate from the same individual. the msi analysis system, version 1.2 kit (promega, usa), which is a fluorescent pcr-based assay, was used for detection of microsatellite instability. pcr amplification was carried out in the following steps: initial denaturation at 95°c for 11 min; 10 cycles at 94°c for 30 sec., 58°c for 30 sec., and 70°c for one min., followed by 20 cycles at 90°c for 30 sec., 58 °c for 30 sec., and 70°c for one min., and finally, an extension step at 60°c for 30 minutes. the pcr products were separated by capillary electrophoresis (ce) using genetic analyzer 3500 (applied biosystem, japan). the data were exported and analyzed with genescan software (promega, usa) to determine msi status. operational definitions msi-high (msi-h) is used when >2 of the 5 markers exhibit instability, msi-low (msi-l) is used when only one of the five markers exhibits instability, and ms-stable (mss) is used when none of the markers exhibit instability.11 for the purpose of this study, both msi-l and mss were considered in one category, referred to as mss, in accordance to esmo guidelines as no clinical differences were observed between mss and msi-l tumors.14 statistical analysis the computer software ibm-spss ver. 26 was used for statistical analysis. qualitative data were described by frequency and percentage. fisher exact test was used to determine if there are non-random associations between two categorical variables of small numbers. a p-value <0.05 was considered statistically significant. results patients’ characteristics ages of the 45 recruited patients ranged between 20–80 years, with a mean ± sd of 55 ± 12.3 years. patients were divided into two age groups: <50 years [14 (31.1%)], and ≥50 years old [31 (68.9%)]. crc was more frequently observed in males 25 (55.6%) than in females 20 (44.4%), with an m:f ratio of 1.25:1. the most frequent presenting feature was rectal bleeding as seen in 22 (48.9%) patients. twenty (44.4%) patients were smokers, while only 2 (4.4%) drink alcohol. twenty-three (51.1%) of crcs were located at recto-sigmoid region, 29 (64.4%) were t3 tumors, 34 (75.5%) were non mucinous adenocarcinoma, 39 (86.7%) were moderately differentiated, 17 (37.8%) patients had stage iii while 14 (31.1%) had stage iv tumors; and 25 (55.5%) had lymphovascular invasion; table 1. table 1. clinicopathological characteristics and msi status of the 45 recruited crc patients parameter to ta l n o. m ss no . ( % ) m si -h no . ( % ) p v al ue age <50 14 12(30 %) 2(40.0%) 0.639 > = 50 31 28(70 %) 3(60%) sex male 25 21(52.5%) 4(80%) 0.362 female 20 19(47.5%) 1(20%) smoking non-smoker 25 23(57.5%) 2(40%) 0.642 smoker 20 17(42.5%) 3(60%) alcohol consumption no 43 38(95%) 5(100%) 1.0 yes 2 2(5%) 0(0%) presentation rectal bleeding 22 22(55%) 0(0%) 0.15 intestinal obstruction 8 6(15%) 2(40%) abdominal pain 6 6(15%) 0(0%) altered bowel habits 5 3(7.5%) 2(40%) anemia 2 2(5%) 0(0%) constipation 2 1(2.5%) 1(20%) tumor location right-sided 11 9(22.5%) 2(40%) 0.255 lt-sided colon 11 9(22.5%) 2(40%) recto-sigmoid 23 22(55%) 1(20%) histological subtype of adenocarcinoma non-mucinous 34 33(82.5%) 1(20%) 0.004* mucinous 10 7(17.5%) 3(60%) signet ring 1 0(0%) 1(20%) l.v. invasion present 25 23(57.5%) 2(40%) 0.642 absent 20 17(42.5%) 3(60%) tumor grade well 2 1(2.5%) 1(20%) 0.125moderately 39 36(90%) 3(60%) poorly 4 3(7.5%) 1(20%) tnm-t stage t1 1 1(2.5%) 0(0%) 0.664 t2 6 5(12.5%) 1(20%) t3 29 25(62.5%) 4(80%) t4 9 9(22.5%) 0(0%) tnm-n stage n0 15 13(32.5%) 2(40%) 0.751 n1 16 15(37.5%) 1(20%) n2 13 11(27.5%) 2(40%) n3 1 1(2.5%) 0(0%) tnm-m stage m0 30 26(65%) 4(80%) 0.651 m1 15 14(35%) 1(20%) l.v. = lymphovascular; tnm=tumor, node, metastasis staging system * = statistically significant (fischer exact test) 160 j contemp med sci | vol. 9, no. 3, may-june 2023: 158–162 detection of msi biomarkers in sporadic colorectal cancer original w. t. s. al-jumaili et al. msi status results in this cross-sectional study, 5/45 (11.1%) patients had msi-h. three (60%) of them were ≥50 years, 4(80%) were males, 3(60%) were smokers, 2 (40%) presented with intestinal obstruction and 2(40%) with altered bowel habits. regarding the tumor characteristics, 4/5 (80%) had t3 tumors, 3(60%) had mucinous adenocarcinomas [p = 0.004], and 3(60%) with moderately-differentiated adenocarcinoma. two (40%) had stage ii tumor and 2(40%) with stage iii, 2(40%) with lymphovascular invasion; table 2. regarding the gene frequency of msi panel, nr-24 showed the highest percentage as detected in 4 (80%) patients, followed by nr-21 that was detected in 3/5 (60%)], while all 3 remaining markers were detected in 2 (40%) patients each. two (40%) patients showed the combination of bat26, nr-21, and nr-24 as the instable markers; table 3. discussion various chromosomal and epigenetic alterations are seen in colorectal cancers. repeated sequences known as microsatellites are highly susceptible to misalignment during replication, which raises their mutation rate to 1.2 × 10-3 15 and causes msi. recent studies indicate that individuals with crc who display msi have a favourable prognosis, but is usually absent in 80–85% of crcs.16 msi-h crc is associated with immune checkpoint upregulation such as programmed cell death receptor (pd-1) and programmed death ligand (pdl-1), the expression of which is usually used as an indication to use immunotherapeutic pd-1 blocking agents e.g. pembrolizumab (keytruda) and dostarlimab (jemperli), the former is the first-line therapeutic agent in advanced metastatic or inoperable crcs.17 msi testing has remained an excellent therapeutic and prognostic tool for crcs.18 three methods can be used to detect the deficient mismatch repair (dmmr) or msi: immunohistochemistry (ihc), polymerase chain reaction (pcr) followed by capillary electrophoresis (ce), or next generation sequencing (ngs). there is a sufficiently compelling evidence to warrant the use of pcr as the most accurate and efficient method of msi detection and is considered the gold standard test.19 msi testing is performed by comparing the length of the amplified product of the target microsatellite in cancer tissues with those of matched normal tissues via capillary electrophoresis.19 only a few regional studies have reported msi status among crc patients, like uae,20 egypt,21 north part of iran,22 turkey,23 and india.24 assessment of msi in different solid tumors, including crcs, is still under research and remains out of the daily routine practice in many countries. in this study, the mean age of the 45 crc patients was 55 ± 12.3 years. these findings were similar to many locoregional studies from baghdad-iraq,4 turkey.25 and saudi arabia.26 the study also showed male predominance [25 (55.6%)]. these results are comparable to results of other studies from al-najaf city in iraq27 and saudi arabia.28 the majority of crcs [23 (51.1%)] were located in the rectosigmoid region, which is similar to a regional study from saudi arabia.26 on histological examination, 34 (75.5%) crcs were non-mucinous adenocarcinoma, and 39 (86.7%) were table 2. clinicopathological features of the five msi-h crc patients parameter no. of msi-h (%) (n = 5) age <50 2(40.0%) > = 50 3(60%) sex male 4(80%) female 1(20%) smoking non-smoker 2(40%) smoker 3(60%) alcohol consumption no 5(100%) yes 0(0%) presentation intestinal obstruction 2(40%) altered bowel habits 2(40%) constipation 1(20%) tumor location right-sided 2(40%) lt-sided colon 2(40%) recto-sigmoid 1(20%) histological subtype of adenocarcinoma non-mucinous 1(20%) mucinous 3(60%) signet ring 1(20%) l.v. invasion present 2(40%) absent 3(60%) tumor grade well 1(20%) moderately 3(60%) poorly 1(20%) tnm-t stage t1 0(0%) t2 1(20%) t3 4(80%) tnm-n stage n0 2(40%) n1 1(20%) n2 2(40%) tnm-m stage m0 4(80%) m1 1(20%) gene frequency of msi nr-24 4(80%) nr-21 3(60%) bat-26 2(40%) bat-25 2(40%) mono-27 2(40%) l.v. =lymphovascular; tnm=tumor, node, metastasis staging system moderately differentiated; this is similar to another regional study from northern saudi arabia29 and jordan.30 in this study, 5/45 (11%) sporadic crcs showed msi-h, which is less than the percentage reported in the northern parts of iran (22.9%)22 and egypt (57.9%)21 but comparable to results from different countries e.g. north america, europe, and east asia.31 for the 5 msi-h patients, age ≥50 years was more frequently seen than <50 years, similar to another study.32 there is a male predominance in both msi-h and mss groups, 161j contemp med sci | vol. 9, no. 3, may-june 2023: 158–162 w. t. s. al-jumaili et al. original detection of msi biomarkers in sporadic colorectal cancer contrary to what was reported in another study,31 but is comparable to findings from iran and india and other parts of the world.22, 24 they found a higher incidence of crc in males compared to females and stated that young women (18–44 years) with crc have a better survival outcome compared to men of the same age or compared to older women.33 in addition, msi behaves in a sex-dependent manner, where the mutation rate is five times higher in males than in females.34 regarding smoking, 60% of msi patients and only 42.5% of mss patients smoke, with no statistically significant difference. this is consistent with another study conducted from university of utah, usa.35 statistical analysis showed no significant difference between msi-h and mss groups of crc patients in terms of presenting features, tumor location, tumor grade, tumor stage, and lymphovascular invasion. tumor type, however, showed a statistical significance in favour of mucinous adenocarcinomas in msi-h; this latter finding is consistent with other studies.36–37 the small sample size of msi-h in this study averts making comparisons and conclusions to these findings, awaiting larger-scale future studies on this topic. generally, these findings are similar to other studies;38–39 the advanced presentation is common in our population due to lack of a national screening programs. nr-24 marker was the most frequently instable marker among the msi-h patients, being reported in 4(80%) patients, followed by nr-21. a previous iraqi study using a different panel of msi genes reported that the most frequent instable marker among the msi-h patients was bat-25 (48.93%) followed by bat-26 (44.68%).37 the type of tested msi genes and the method used has changed with time, starting from dinucleotide markers that showed inconsistencies in results, to mononucleotide markers requiring comparison to normal tissues, to the more recent use of msi kits e.g. idylla msi kit that is quick, fully automated, does not require normal tissues, and is as sensitive and specific as the currently used kits e.g. promega msi analysis system version 1.2.40 a novel panel of seven monomorphic biomarkers (acvr2a, btbd7, dido1, mre11, ryr3, sec31a, and sulf2) is currently recommended to be used in a diagnostic test to identify the msi status in crc.40 the recent inclusion of msi assessment in the investigation of newly diagnosed crc patients in the specialized centers, would allow a clearer vision to the true incidence of msi-h and characteristics among different population groups in iraq in the near future. conclusion the clinical benefits of detecting msi status accurately and affordably are clear. the frequency of msi in sporadic crc patients in this study was 5/45 (11.1%) and was significantly associated with mucinous type. nr-24 and nr-21 were the most prevalent instable markers. these findings help physicians and oncologists decide whether to give immunotherapy or chemotherapy, and to predict prognosis based on result of msi testing. declarations ethics approval and consent to participate • this study was approved by the ethics research committee at the dept. of pathology and forensic medicine, college of medicine, university of baghdad (issue no. 200 at november, 10th, 2021). availability of data and material • the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. competing interests • the authors declare that they have no competing interests. funding • this study was not funded by any organization, institute, or body what so ever. acknowledgements the authors would like to acknowledge the kind assistance of dr. sazan abdulwahab mirza al-atrooshi in sample collection, and dr. mohammed ghanim m. al-hilal for assistance in molecular analysis in this study.  table 3. types, frequencies, and patterns of the detected mononucleotide markers among 5 msi-h crc patients mononucleotide marker p1 p2 p3 p4 p5 gene frequency bat25 + + 2/5(40%) bat26 + + 2/5(40%) nr-21 + + + 3/5(60%) nr-24 + + + + 4/5(80%) mono-27 + + 2/5(40%) total 3(60%) 3(60%) 3(60%) 2(40%) 2(40%) 162 j contemp med sci | vol. 9, no. 3, may-june 2023: 158–162 detection of msi biomarkers in sporadic colorectal cancer original w. t. s. al-jumaili et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1348 references 1. arnold, m., et al., global patterns and trends in colorectal cancer incidence and mortality. gut, 2017. 66(4): p. 683–691. 2. baraaj, a. and h. mahmood, role some risk factors : age ,sex and lipid profile in colo-rectal cancer in iraqi patient. systematic reviews in pharmacy, 2021. 12: p. 1–5. 3. mattiuzzi, c., f. sanchis-gomar, and g. lippi, concise update on colorectal cancer epidemiology. ann transl med, 2019. 7(21): p. 609. 4. al dahhan, s.a. and f.h. al lami, epidemiology of colorectal cancer in iraq, 2002-2014. gulf j oncolog, 2018. 1(26): p. 23–26. 5. douaiher, j., et al., colorectal cancer-global burden, trends, and geographical variations. j surg oncol, 2017. 115(5): p. 619–630. 6. binefa, g., et al., colorectal cancer: from prevention to personalized medicine. world j gastroenterol, 2014. 20(22): p. 6786–808. 7. xi, y. and p. xu, global colorectal cancer burden in 2020 and projections to 2040. transl oncol, 2021. 14(10): p. 101174. 8. alrubaie, a., n. alkhalidi, and s. abd-alhusain, a clinical study of newlydiagnosed colorectal cancer over 2 years in a gastroenterology center in iraq. journal of coloproctology, 2019. 39(3): p. 217–222. 9. de rosa, m., et al., genetics, diagnosis and management of colorectal cancer (review). oncol rep, 2015. 34(3): p. 1087–96. 10. grady, w.m. and s.d. markowitz, the molecular pathogenesis of colorectal cancer and its potential application to colorectal cancer screening. dig dis sci, 2015. 60(3): p. 762–72. 11. zeinalian, m., et al., clinical aspects of microsatellite instability testing in colorectal cancer. adv biomed res, 2018. 7: p. 28. 12. nguyen, l.h., a. goel, and d.c. chung, pathways of colorectal carcinogenesis. gastroenterology, 2020. 158(2): p. 291–302. 13. webber, e.m., et al., systematic review of the predictive effect of msi status in colorectal cancer patients undergoing 5fu-based chemotherapy. bmc cancer, 2015. 15: p. 156. 14. luchini, c., et al., esmo recommendations on microsatellite instability testing for immunotherapy in cancer, and its relationship with pd-1/pd-l1 expression and tumour mutational burden: a systematic review-based approach. ann oncol, 2019. 30(8): p. 1232–1243. 15. söreide, k., et al., microsatellite instability in colorectal cancer. british journal of surgery, 2006. 93(4): p. 395–406. 16. loukola, a., et al., microsatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (hnpcc)1. cancer research, 2001. 61(11): p. 4545–4549. 17. ali, e., et al., jemperli (dostarlimab-gxly): an unprecedented cancer trial. ann med surg (lond), 2022. 79: p. 104047. 18. chung, c., predictive and prognostic biomarkers with therapeutic targets in colorectal cancer: a 2021 update on current development, evidence, and recommendation. 2022. 28(4): p. 850–869. 19. shia, j., immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. part i. the utility of immunohistochemistry. j mol diagn, 2008. 10(4): p. 293–300. 20. kamat, n., et al., microsatellite instability and loss of heterozygosity detected in middle-aged patients with sporadic colon cancer: a retrospective study. oncology letters, 2013. 6(5): p. 1413–1420. 21. kassem, n.m., et al., clinicopathological features of egyptian colorectal cancer patients regarding somatic genetic mutations especially in kras gene and microsatellite instability status: a pilot study. egyptian journal of medical human genetics, 2019. 20(1): p. 20. 22. faghani, m., et al., the correlation between microsatellite instability and the features of sporadic colorectal cancer in the north part of iran. gastroenterol res pract, 2012. 2012: p. 756263. 23. deligonul, a., et al., prognostic significance of microsatellite instability in turkish patients with stage ii and iii colorectal cancer. 2021. 5(1). 24. rai, p.r., et al., a study on the frequency and clinicopathological correlates of mismatch repair-deficient colorectal cancer. j cancer res ther, 2020. 16(supplement): p. s183–s188. 25. aykan, n.f., et al., epidemiology of colorectal cancer in turkey: a cross-sectional disease registry study (a turkish oncology group trial). turk j gastroenterol, 2015. 26(2): p. 145–53. 26. ayyub, m.i., et al., clinicopathological trends in colorectal cancer in a tertiary care hospital. saudi med j, 2002. 23(2): p. 160–3. 27. h.;, h.m., et al., age and gender in relation to colorectal cancer in najef province: a histopathological study. journal of clinical and laboratory research, 2021. 2 (1): p. 10. 28. alsanea, n., et al., colorectal cancer in saudi arabia: incidence, survival, demographics and implications for national policies. ann saudi med, 2015. 35(3): p. 196–202. 29. alharbi, s.h., et al., patterns and grades of presentation of colon cancer in northern saudi arabia. prz gastroenterol, 2021. 16(3): p. 235–239. 30. sharkas, g.f., et al., colorectal cancer in jordan: survival rate and its related factors. j oncol, 2017. 2017: p. 3180762. 31. kim, j.h. and g.h. kang, molecular and prognostic heterogeneity of microsatellite-unstable colorectal cancer. world j gastroenterol, 2014. 20(15): p. 4230–43. 32. goksu, s.y., et al., clinicopathologic variables and outcomes in elderly colorectal cancer patients with microsatellite instability and multiple primary malignancies. 2021. 39(15_suppl): p. e15516-e15516. 33. abancens, m., et al., sexual dimorphism in colon cancer. 2020. 10. 34. vargas jentzsch, i., et al., evolution of microsatellite dna. 2013. 35. slattery, m.l., et al., associations between cigarette smoking, lifestyle factors, and microsatellite instability in colon tumors. j natl cancer inst, 2000. 92(22): p. 1831–6. 36. fleming, m., et al., colorectal carcinoma: pathologic aspects. j gastrointest oncol, 2012. 3(3): p. 153–73. 37. mohymen, n., b. hanon, and a.s. mahmood, the correlation between microsatellite instability and the features of sporadic colorectal cancer in sample of iraqi patients. journal of advances in biotechnology, 2014. 4: p. 301–312. 38. popat, s., r. hubner, and r.s. houlston, systematic review of microsatellite instability and colorectal cancer prognosis. j clin oncol, 2005. 23(3): p. 609–18. 39. pritchard, c.c. and w.m. grady, colorectal cancer molecular biology moves into clinical practice. gut, 2011. 60(1): p. 116–29. 40. zwaenepoel, k., et al., clinical performance of the idylla msi test for a rapid assessment of the dna microsatellite status in human colorectal cancer. the journal of molecular diagnostics, 2020. 22(3): p. 386–395. 138 j contemp med sci | vol. 2, no. 8, autumn 2016: 138–140 research interleukin-8 251a/t polymorphism related to peptic ulcer disease in h. pylori infected patient israa saeed abbas issn 2413-0516 department of clinical laboratories, college of applied medical sciences, karbala university, iraq. correspondence to israa saeed abbas (email: alsultanyisraa81@gmail.com). (submitted: 17 june 2016 – revised version received: 03 october 2016 – accepted: 10 october 2016 – published online: 26 december 2016) objectives this study includes the investigation of antifungal activity of the local propolis against dermatophytes and yeast. methods a total of 92 tissue samples were taken from patients infected with h. pylori and 102 from uninfected individuals has been included. genetic method used for the detection of h. pylori infection by polymerase chain reaction (pcr) for glm gene identification. il-8251 a/t polymorphism was detected by allele-specific oligonucleotide polymerase chain reaction (asopcr). results among the studied samples, the results improve that genetic polymorphism of il-8-251 a/t genotype was found in a higher frequency with confidence interval 95% in duodenal ulcer h. pylori infected patient than control. conclusion il-8-251 a allele notice that it has been associated with severe inflammation lead to increase severity of disease. keywords helicobacter pylori, molecular biology (pcr), allele-specific oligonucleotide. introduction helicobacter pylori, is a bacterium that infects the stomach of humans, and strongly associated with gastroduodenal diseases such as chronic atrophic gastritis, peptic ulcer,1,2 and gastric cancer.3,4 although this bacterium has been classified as a carcinogen in human, keys of pathophysiological events in h. pylori infections is the induction of the inflammatory response in the gastric mucosa, which is mediated and also regulated by inflammatory cytokines produced by gastric epithelial cells.5,6 interleukin-8 (il-8) was a small peptide (chemokine) secreted by a variety of cell types, which serves as a potent inflammatory mediator recruiting and activating neutrophils. interleukin-8 (il-8), was a potent chemo attractant for neutrophils and lymphocytes, has been reported as a strong stimulator of angiogenesis in gastric adenocarcinoma.7,8 several studies have demonstrated that h. pylori strains are capable of inducing il-8 secretion from gastric carcinoma cells in vitro.9,10 others researcher noticed that il-8 is typically secreted by gastrointestinal epithelial cells in response to pathogenic bacteria.11 genetic polymorphisms that affect innate immune response genes have also been linked to an increased risk of gastric cancer, and the levels of il-8 are directly related to the severity of gastritis.12 the genetic polymorphism of il-8-251 a allele notice to be associated with higher il-8 production, more severe inflammation, mucosal atrophy, and also intestinal metaplasia when compared with the il-8-251 tt genotype of h. pylori-infected patients. the recent studies suggested a possible association of the il-8-251 a allele with angiogenesis and inflammation in gastric carcinogenesis in h. pylori-infected koreans 13,14 materials and methods patients and gastric biopsy samples 194 tissue samples included, 128 women and 66 men with age range from 15 to 75 years (mean age 36 years). they were presented with dyspepsia and referred to the esophago gastroduodeno scope. the endoscopic diagnosis was grouped into three categories: firstly was peptic ulcer disease (pud) patients who had endoscopic lesions of ulcers, secondly the non-peptic ulcer disease patients (npud) were defined as patients who had endoscopic with no lesions of ulcers but they have disease like gastritis or gastric atrophy or gastropathy, and lastly, the control group was defined as they did not have any type of gastric or duodenal disease. two tissue biopsies were obtained from antrum. rapid urease test was performed on one of the antral biopsies at the time of endoscopy. the other biopsy specimens placed in 1 ml of normal saline. the biopsy specimen was preserved immediately at −20°c for molecular analysis. dna extraction and pcr amplification conditions total dna extracted directly from gastric biopsy samples using tissue protocol (geneaid, korea). the final volume of dna extraction product was 200 μl, with final concentration 1.25 ng/μl. identification of h. pylori glm gene to confirm the presence of h. pylori, dna in biopsies amplification and melting conditions were optimized for the pcr assay by using specific primer sequences for gene urec (glmm) 294 (bp) pcr selected were as follows: glmm – forward primer (5´aagcttttaggggtgttaggggttt -3´), and glmm–reverse primer (5´-aagcttactttctaacactaacgc -3´). pcrpremix™ kit from (bioneer, korea) was used to amplify the mentioned gene. total reaction volume of 20 μl containing, 3 μl of extracted dna, 1 μl of 10 pmol/μl of each forward and reverse primers for glmm gene in addition to 14 μl of molecular biology grade water then the mixture was added to lyophilized pcrpremix™ formula. genotyping of il-8 251a/t polymorphism gene (aso) pcr techniques used for the detection of il-8 251 polymorphism. it is a simple method to detect of mutation involving single base changes or small deletion. it depends on the sequence of specific primer (specific pcr primer) that israa saeed abbas 139j contemp med sci | vol. 2, no. 8, autumn 2016: 138–140 research interleukin-8 251a/t polymorphism related to peptic ulcer disease in h. pylori infected patient allows the amplification of test dna only when target allele is contained with samples following reaction in the presence or absence of target allele. pcr working solution the master mix component for the detection of il-8 251 polymorphism gene adopted by arms pcr as shown in table 1. pcr protocol pcr was performed in a thermo cycler under the following conditions adopted in table 2. pcr amplification analysis eskandar et al. 2006 336 cca cca ttt ggt gaa tta tca atil-8 f 1 cca cca ttt ggt gaa tta tca aa il-8 f 2 tgc ccc ttc act ctg tta acil-8 r table 1. the master mix components of pcr used for detection of il-8 251 a/t polymorphism componentconcentrationamount (μl) deionized water7.0 pcr buffer 2x2.0 pcr f-1 primers10x1.5 pcr r primers10x1.5 taq dna polymerase5 u/μl1 dntp 0.5 mgcl 2 1.5 specimen dna or control dna from kit(200 ng/ μl) 10x5.0 total volume20 table 3. distribution of disease group among positive and negative h. pylori infected samples total h. pylori –veh. pylori +vegroups 37 829pud 102 5646npud 2 20c.a 53 3617control 194 10292total table 2. the pcr protocol for detection of gene no.step temperature (c°) time no. of cycles 1initial denaturation941 min1 2 initial annealing and extension5760 sec 35 denaturation9360 sec annealing and extension7260 sec 3final extension725 min1 fig.1 representative results relating to the il-8 genotyping. aso pcr was used. by means of the allele-specific primers, the homozygote mutant (aa), and the heterozygote (at ) and the homozygote tt variants. the image is from a representative gel electrophoresis of pcr amplification products of il-8 gene (336 bp) were distinguishable. m: marker of the dna. fig. 2 representative results relating to the il-8 genotyping. aso pcr was used. by means of the allele-specific primers, the homozygote mutant (aa), and the heterozygote (at) and the homozygote tt variants. the image is from a representative gel electrophoresis of pcr amplification products of il-8 gene (336 bp) were distinguishable. m: marker of the dna. table 5. the presence of the genotype polymorphisms of il-8251 a/t gene in h. pylori-positive patients among disease groups total h. pylori +ve n (%) groups 3729 (78%)pud 10246 (45%)npud 20 (0%)c.a 5317 (32%)control 19492 (47%)total statistical analysis the chi-square test or fisher’s exact test was used to compare between proportions. the values of p < 0.05 were considered statistically significant. a prevalence ratio (pr) with a 95% confidence interval (ci) was calculated to evaluate the relationship between il-8 251a/t genotyping polymorphisms with gastroduodenal diseases and with h. pylori infection. table 4. prevalence of il-8-251 a/t polymorphism among positive and negative h. pylori infected samples all h. pylori –ve % h. pylori +ve % total (n = 194 ) (n = 102) (n = 92) aa 4 3.9% 0 0% 4 at 86 84.3% 92 100% 178 tt 12 11.8% 0 0% 12 total 102 100% 92 100% 194 140 j contemp med sci | vol. 2, no. 8, autumn 2016: 138–140 interleukin-8 251a/t polymorphism related to peptic ulcer disease in h. pylori infected patient research israa saeed abbas results in this study, a total of 141 patients who were affected with different forms of gastric diseases [37 of them had peptic ulcer diseases (pud) and 102 had non-peptic ulcer disease (npud) and 2 patients had gastric cancer (c.a)]. were compared with 53 who had just epigastric pain while their endoscopic examinations revealed, they were regarded as a control group. the presence of the genotype polymorphisms of il-8251 a/t gene in h. pylori-positive patients among disease groupe show in table 5. in this table from total 92 il-8251 a/t gene in h. pylori-positive samples all peptic ulcer disease grouped and all non-peptic ulcer disease grouped show il-8251 a/t gene, improve that there was significant difference in contrast to the control grouped. discussion polymorphism in the il-8 (–251) has been associated with the increased expression of il-8 (15). several papers have demonstrated an association between il-8 (-251) polymorphism and an increased risk of developing gastroduodenal diseases.16 a significantly higher frequency of the il-8 251at genotype was observed among the h. pylori-positive du patients than among the h. pylori-positive healthy subjects without gastrointestinal problems. this genotype reflects a higher il-8-producing ability.17 the association with il-8 was higher reconnoitered at the level of a single snp (-251 a/t). the higher incidence of the -251 at genotype with a concomitant higher il-8-producing potential18,17 highlights the importance of the genetic determination of il-8 production in h. pylori-induced du. we observed a greater frequency in the aa allele genotypes among patients with c.a stomach than in the control group or in other disease group, with the presence of allele a being associated with the risk of developing gastric cancer. other researcher noted that presence of the a allele in the -251 position of the il-8 gene was associated with an increase in the risk of stomach cancer in japanese, korean, chinese and iranian populations.13 additionally, some studies have demonstrated that patients carrying the a allele present higher production of il-8, leading to alteration in the quality and intensity of inflammatory responses produced by the host after exposure to h. pylori.13,19 conversely, the frequency of the tt genotype (with a relatively low il-8-producing potential) was significantly higher among the h. pylori negative, healthy individuals. this suggests the possibility that a relative protection from du disease is observed in association with the tt genotype. this observation is consistent with the results of,20 who concluded that h. pylori-positive healthy individuals with the il-8-251 tt genotype might display a milder inflammatory reaction. conclusion regarding il-8-251 a/t polymorphism was related to associated to the increased expression of il-8 and increase severity of the disease. conflict of interst none. n references 1. goodwin cs, mendall mm, northfield tc. helicobacter pylori infection. lancet. 1997;349:265–269. 2. graham dy, lew gm, klein pd, evans dg, evans dj, saeed za, et al. effect of treatment of helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. a randomized, controlled study. ann intern med. 1992;116:705–708. 3. iarc monogr eval carcinog risks hum. schistosomes, liver flukes and helicobacter pylori. iarc working group on the evaluation of carcinogenic risks to humans. lyon, 7-14 june 1994. iarc monogr eval carcinog risks hum. 1994;61:1–241. 4. hansson le, engstrand l, nyrén o, evans dj, lindgren a, bergström r. helicobacter pylori infection: independent risk indicator of gastric adenocarcinoma. gastroenterology. 1993;105:1098–103. 5. abdiev s, ahn ks, khadjibaev a, malikov y, bahramov s, rakhimov b, et al. helicobacter pylori infection and cytokine gene polymorphisms in uzbeks. nagoya j med sci. 2010;72:167–172. 6. sugimoto m, yamaoka y, furuta t. influence of interleukin polymorphisms on development of gastric cancer and peptic ulcer. world j gastroenterol. 2010;16:1188–200. 7. zhang l, ma j, pan k, go vl, chen j, you wc. efficacy of cranberry juice on helicobacter pylori infection: a double-blind, randomized placebocontrolled trial. helicobacter. 2005;10:139–145. 8. kitadai y, haruma k, sumii k, yamamoto s, ue t, yokozaki h, et al. expression of interleukin-8 correlates with vascularity in human gastric carcinomas. am j pathol. 1998;152:93–100. 9. crabtree je, covacci a, farmery sm, xiang z, tompkins ds, perry s, et al. helicobacter pylori-induced interleukin-8 expression in gastric epithelial cells is associated with caga positive phenotype. j clin pathol. 1995;48:41–45. 10. huang j, o’toole pw, doig p, trust tj. stimulation of interleukin-8 production in epithelial cell lines by helicobacter pylori. infect immun. 1995;63:1732–1738. 11. eckmann l, kagnoff mf. cytokines in host defense against salmonella. microbes infect. 2001;3:1191–1200. 12. peek rm, miller gg, tham kt, perez-perez gi, zhao x, atherton jc, et al. heightened inflammatory response and cytokine expression in vivo to caga+ helicobacter pylori strains. lab invest. 1995;73:760–770. 13. ye bd, kim sg, park jh, kim js, jung hc, song is. the interleukin-8-251a allele is associated with increased risk of non-cardia gastric adenocarcinoma in helicobacter pylori-infected koreans. j clin gastroenterol. 2009;43: 233–239. 14. song jh, kim sg, jung sa, lee mk, jung hc, song is. the interleukin-8-251 aa genotype is associated with angiogenesis in gastric carcinogenesisin helicobacter pylori-infected koreans. cytokine. 2010;51:158–165. 15. lu h, hsu pi, graham dy, yamaoka y. duodenal ulcer promoting gene of helicobacter pylori. gastroenterology. 2005;128:833–848. 16. thomazini cm, pinheiro na, pardini mi, naresse le, rodrigues mam. helicobacter pylori and gastric cancer: distribution of caga and vaca genotypes in patients with gastric carcinoma. j bras patol med lab. 2006;42:25–30. 17. hull j, ackerman h, isles k, usen s, pinder m, thomson a. unusual haplotypic structure of il8, a susceptibility locus for a common respiratory virus. am j hum genet. 2001;69:413–419. 18. hull j, thomson a, kwiatkowski d. association of respiratory syncytial virus bronchiolitis with the interleukin 8 gene region in uk families. thorax. 2000;55:1023–1027. 19. taguchi a, ohmiya n, shirai k, mabuchi n, itoh a, hirooka y, et al. interleukin-8 promoter polymorphism increases the risk of atrophic gastritis and gastric cancer in japan. cancer epidemiol biomarkers prev. 2005;14:2487–2493. 20. hamajima n, katsuda n, matsuo k, saito t, hirose k, inoue m, et al. high anti-helicobacter pylori antibody seropositivity associated with the combination of il-8–251tt and il-10–819tt genotypes. helicobacter. 2003;8:105–110. 352 j contemp med sci | vol. 8, no. 5, september-october 2022: 352–356 original assessment of the her2, pdl1 and oxidative stress levels at the menopausal status of newly diagnosed breast cancer patients baraa r. mahmood1, thikra a. allwsh2* 1biochemistry laboratory, ibn al atheer children’s hospital, ministry of health, mosul, iraq. 2department of chemistry, college of science, university of mosul, mosul, iraq. *correspondence to: thikra ali allwsh (e-mail: thekraaliallwsh@uomosul.edu.iq) (submitted: 09 june 2022 – revised version received: 25 july 2022 – accepted: 13 august 2022 – published online: 26 october 2022) abstract objectives: this study aimed to estimate the levels of her2, pd-l1 and oxidative stress in newly diagnosed breast cancer patients. also, to estimate the probability of using her2 and pd-l1 as a predictive marker on the occurrence of breast cancer in addition to study the effect of menopausal status at the level of her2, pd-l1, oxidative stress and breast cancer risk factor. methods: this study included 125 newly diagnosed breast cancer patients (53 premenopausal and 72 postmenopausal) from oncology and nuclear medicine hospital in mosul, iraq, and 100 apparently healthy women as a control group (44 premenopausal and 56 postmenopausal), during the period from jan. 2021 to jun. 2021. the ages of patients and control are matched, and it is ranged from 30–60 years. in this study we estimate the level of human epidermal growth factor receptor 2 (her2), programmed death -ligand 1 (pd-l1), total antioxidant capacity (t-aoc), arylesterase activity, uric acid level, malondialdehyde (mda) level, peroxidase activity, lactoperoxidase activity and iron level at breast cancer patients and control. results: the results show that there is a significant elevation in the level of her2, pd-l1, malondialdehyde, peroxidase, lactoperoxidase and iron, and a significant decrease in the level of t-aoc, arylesterase and uric acid in the serum of breast cancer patients (pre and postmenopausal) compared with the control group (pre and postmenopausal). also, increase the level of her2 and oxidative stress at postmenopausal status for the control and patient groups. while pd-l1 level does not affect by menopausal status for both control and patient groups. conclusion: the level of her2, pd-l1, and oxidative stress was significantly increased in newly diagnosed breast cancer patients compared with the control group at the same menopausal status. increase breast cancer risk factor at the postmenopausal status. keywords: breast neoplasms, epidermal growth factor, oxidative stress issn 2413-0516 introduction breast cancer eventuates when the tissue cells of the breast become atypical and divide without control, these atypical cells shape a big lump of tissue, which later develops into a tumor.1 it is the second leading cause of death after lung cancer.2 breast cancer is affected by many risk factors, such as gender, being older, obesity, menopausal status, full-term pregnancy, abortion, hereditary factors and hormonal factors.3 the menopausal status is an important risk factor for breast cancer.4 human epidermal growth factor receptor 2 (her2) is a tyrosine kinase-activated transmembrane glycoprotein receptor.5 it is a member of human epidermal growth factor (egf) receptor family, which includes the egfr (her1), her2 (also known as erbb2), her3, and her4 receptors.6 it is encoded by the proto-oncogene her2, which can be found on chromosome 17q21’s long arm.7 in the majority of these difficult breast tumors, her2 is the primary oncogenic driver.8 because her2 is an orphan receptor (no specific ligand for her2 has been identified),9 when expressed at very high levels on the cell surface, it relies on heterodimerization with other her receptors or homodimerization with itself to activate the her2 signaling pathways.10,11 her2 dimerization causes autophosphorylation of tyrosine remnants within the receptor’s cytoplasmic domain, causing activation of a number of downstream signaling pathways, specifically phosphoinositide-3-kinase (pi3k) and mitogen-activated protein kinase (mapk),12 which control cellular function like cell migration, differentiation, proliferation, apoptosis, cell cycling, and angiogenesis.13 programmed cell death ligand 1 (pd-l1) is a transmembrane protein that serves as a co-inhibitory agent of the immune response, when combined with it is a receptor (pd-1), it can stop pd-1 positive cells from proliferating, decrease their cytokine release, and trigger apoptosis.14 both cancerous cells and immune cells such as t and b lymphocytes, macrophages, and dendritic cells can express this immunological checkpoint molecule.15 the pd-1/pd-l1 pathway regulates inflammation and maintains peripheral t-lymphocyte tolerance.16 the expression of pd-l1 by tumor cell controls the activity of t cells, boost immunosuppression and tumor escape.17 oxidative stress is an imbalance between the formation of the reactive oxygen species by the cells and the potency of the biological system to detoxifying them.18 adequate levels of reactive oxygen species are required for cell function and survival in healthy conditions.19 when ros levels rise, they start to damage important cellular structures like proteins, lipids, and nucleic acids.20 growing both empirical and clinical proof proposes that the oxidative stress play a role in breast cancer tumorigenesis and progression.21 materials and methods ethical approval this study was approved by the ethical committee of the nineveh health direction training center and human development, ministry of health and environment, iraq, approved this mailto:thekraaliallwsh@uomosul.edu.iq 353j contemp med sci | vol. 8, no. 5, september-october 2022: 352–356 b.r. mahmood et al. original assessment of the her2, pdl1 and oxidative stress levels at the menopausal status study. informed written consent was obtained from all the participants before sample collection. blood sample this study included 125 newly diagnosed breast cancer patients from oncology and nuclear medicine hospital in mosul, iraq, and 100 apparently healthy women as a control group. their ages ranged from (30–60) years. patients and control were divided into two groups based on their menopausal status as premenopausal and postmenopausal. the premenopausal group is comprised of 53 breast cancer patients and 44 control. while the postmenopausal group is comprised of 72 breast cancer patients and 56 control. the samples were collected during the time period from the beginning of jan. 2021 to the end of jun. 2021. peripheral venous blood samples (5 ml) were collected from all study participants and incubated directly at 37°c for 10 min, after that centrifuged at 3500 rpm for 12 minutes. the serum was separated and frozen in aliquots at –20°c until used.22 laboratory analysis • her2 and pdl-1 concentration: was measured by using bioassay technology laboratory kit (shanghai, china), which based on enzyme linked immunosorbent assay (elisa) technique. • total antioxidant capacity (t-aoc): was measured by using solarbio science & technology kit (beijing, china), which based on spectrophotometer method.23 • arylesterase activity: was measured by using enzymatic hydrolysis of phenyl acetate to produce phenol and acetic acid.24,25 • malondialdehyde (mda): was measured by using thiobarbituric acid was used to assess malondialdehyde.26 • peroxidase activity: was measured by using enzymatic oxidation of hydrogen peroxide by peroxidase.27 • lactoperoxidase activity: was measured by using enzymatic oxidation of pyrogallol to purpurogallin.28 • iron concentration: was measured by using biolab kit (maizy, france), which is based on reducing fe+3 to fe+2.29 • uric acid concentration: was measured by using biolab kit (maizy, france) which is based on enzymatic oxidation of uric acid by uricase.30 statistical analysis data were analyzed by using the spss statistics 22.0 software package (spss version 22.0, ibm, usa). results effect of the menopausal status at her2 level the mean levels of her2 were (6.87 ± 0.31, 8.18 ± 0.50 ng/ml) in the pre and postmenopausal status for the control group, respectively, but the mean levels of her2 were (11.72 ± 0.33, 14.4 ± 0.48 ng/ml) in the pre and postmenopausal status for the patient group, respectively. also, the results in figure 1 show there is a significant increase (p-value <0.05) in the level of her2 at both pre and postmenopausal patients compared with the pre and postmenopausal control group. figure 2 show there is a significant elevation (p-value <0.05) in the level of her2 at postmenopausal status compared with the premenopausal status of both control and patient groups. effect of the menopausal status at pd-l1 level the mean levels of pd-l1 were (145.17 ± 4.05, 149.58 ± 8.06 ng/ml) in the pre and postmenopausal status for the control group, respectively, but the mean levels of her2 were (237.81 ± 10.7, 225.06 ± 8.18 ng/ml) in the pre and postmenopausal status for the patient group, respectively. also, the results in figure 3 show there is a significant increase (p value < 0.05) in the level of pd-l1 at both pre and postmenopausal patients compared with the pre and postmenopausal control group. fig. 1 her2 level at pre and postmenopausal status for control and patient group. *significant at p < 0.05. fig. 2 her2 level at menopausal status for control and patients. *significant at p < 0.05. fig. 3 pd-l1 level at pre and postmenopausal status for control and patient group. *significant at p < 0.05. 354 j contemp med sci | vol. 8, no. 5, september-october 2022: 352–356 assessment of the her2, pdl1 and oxidative stress levels at the menopausal status original b.r. mahmood et al. patients compared with the control group at both pre and postmenopausal status, these results were in agreement with banys-paluchowski., et al. and fabricio et al., which indicate that the serum her2 level was elevated at those who are her2 positive breast cancer (+3 score),31,32 which consider as an indicator for the progression and recurrence of the disease.33 in the same as with our result, di gioia et al. demonstrate elevated her2 levels at postmenopausal status.34 the high level of her2 at postmenopausal status causes an increased level of both follicle-stimulating hormone (fsh) and luteinizing hormone (lh) which they are boosted cell differentiation and proliferation, causing an increase in the risk factor of breast cancer.4,35 the high level of her2 in postmenopausal breast cancer patients also consider as an indicator of the probability of metastatic.36,37 pd-l1 is an immune checkpoint and it is overexpressed on the surface of the cancer cell to evade anti-tumor immune responses by interacting with it a is a receptor (pd-1) at the surface of the t-cell, and then inhibit t-cell function.38,39 and that explains the high level of pd-l1 in the serum of breast cancer patients compared with control. salama et al. and han et al.40,41 illustrated that the pd-l1 level do not effects by menopausal status and that agrees with our results. the low level of the thyroid hormone in patients with breast cancer4 causes increased oxidative stress by the reduction of the level of the total antioxidant capacity42 and elevation of mda, peroxidase, lactoperoxidase and uric acid.43 this agrees with our results. in our present study, the low level of t-aoc, aryleaterase and uric acid in patients’ serum is in agrees with former studies,44 which indicate to increase the oxidative stress in breast cancer patients. at postmenopausal status the low level of estrogen which have antioxidant characteristic causes reduction in the levels of antioxidant parameters and then increase breast cancer risk factor.45 also, the high level of iron makes it susceptible to fenton reaction and then increase the level of reactive oxygen species.46 the high level of reactive oxygen species causes lipid figure 4 showed that the pd-l1 level had not changes at menopausal status for both patients and control group. effect of the menopausal status at antioxidant level the results in table 1 demonstrated that levels of t-aoc, arylesterase and uric acid was significantly decreased (p < 0.05) in both pre and postmenopausal patients compared with the pre and postmenopausal control group. the result from table 1 also show there is a significant reduction in the levels of antioxidant parameters at postmenopausal status compared with premenopausal status for both control and patient group. effect of the menopausal status at oxidant level the result in table 2 showed there is a significant elevation (p < 0.05) in the level of oxidant parameters (mda, peroxidase, lactoperoxidase, and iron) in patients serum comparing with the control at pre and postmenopausal status. this results also clarified elevation of the oxidative stress at postmenopausal status for both patients and control group. discussion the second most frequent cancer in women is breast cancer. in our study, her2 level was significantly increased in the table 1. the level of antioxidants in the patient and control according to menopausal status clinical parameters premenopausal control mean ± se premenopausal patient mean ± se p-value postmenopausal control mean ± se postmenopausal patient mean ± se p-value t-aoc µmol/ml 0.82 ± 0.04 0.70 ± 0.03 <0.05 0.64 ± 0.03 0.48 ± 0.02 <0.05 arylesterase u/l 98.69 ± 3.30 59.51 ± 2.13 <0.05 78.10 ± 2.81 39.87 ± 2.40 <0.05 uric acid mg/dl 4.44 ± 0.21 3.21 ± 0.20 <0.05 3.79 ± 0.19 2.34 ± 0.15 <0.05 table 2. the level of oxidants in the patient and control according to menopausal status clinical parameters premenopausal control mean ± se premenopausal patient mean ± se p-value postmenopausal control mean ± se postmenopausal patient mean ± se p-value mda µmol/l 12.24 ± 0.78 26.01 ± 1.30 <0.05 18.68 ± 0.84 34.96 ± 1.54 <0.05 peroxidase u/l 77.49 ± 3.60 142.96 ± 5.70 <0.05 95.32 ± 3.71 159.27 ± 8.03 <0.05 lactoperoxidase u/ml 46.26 ± 1.56 79.00 ± 2.63 <0.05 57.60 ± 2.28 109.50 ± 3.52 <0.05 iron µg/dl 66.28 ± 2.5 84.35 ± 2.8 <0.05 76.75 ± 3.82 110.78 ± 4.16 <0.05 fig. 4 pd-l1 level at menopausal status for control and patient. 355j contemp med sci | vol. 8, no. 5, september-october 2022: 352–356 b.r. mahmood et al. original assessment of the her2, pdl1 and oxidative stress levels at the menopausal status peroxidation, hence destruction of cell membranes. mda (the end product of lipid peroxidation) is reflecting the level of oxidative stress. the high levels of mda, peroxidase, lactoperoxidase and iron in the serum of breast cancer patients is agreeing with previous studies.47 the reduction of arylesterase level and elevation of peroxidase and lactoperoxidase levels reflect the imbalance in oxidant-antioxidant mechanism, which in result increase breast cancer risk factors at menopausal status. increase oxidative stress in patients with breast cancer causes activation of the number of transcription factors and then lead to expression of the genes that include growth factor, inflammatory cytokines, chemokines and cell cycle regulatory molecules,48 and then result in harm to dna and impede signal transduction pathways responsible for cell proliferation, apoptosis, and angiogenesis.49 conclusion and recommendation 1. elevation in the levels of her2, pd-l1, mda, peroxidase, lactoperoxidase and iron in the serum of breast cancer patients compared with the control. whereas the level of t-aoc, arylesterase, and uric acid was demotion in the serum of the patients compared with the control groups at the same menopausal status. 2. elevated her2 and oxidative stress at postmenopausal women increase the risk factor of breast cancer. 3. due to the low cost and ease to done we can use the serum level of her2 and pd-l1 as a predictor of breast cancer incidence and recurrence. conflict of interest the authors declare that no conflict of interest exists.  references 1. sadoughi f, kazemy z, hamedan f, owji l, rahmanikatigari m, azadboni tt. artificial intelligence methods for the diagnosis of breast cancer by image processing: a review. breast cancer (dove med press). 2018;10:219–230. doi:10.2147/bctt.s175311. 2. mendoza l. potential effect of probiotics in the treatment of breast cancer. oncol rev. 2019;13(2):422. doi:10.4081/oncol.2019.422. 3. momenimovahed z, salehiniya h. epidemiological characteristics of and risk factors for breast cancer in the world. breast cancer (dove med press). 2019;10;11:151–164. doi: 10.2147/bctt.s176070. 4. mhmood, b., allwsh, t. study of her2 in breast cancer disease. egyptian journal of chemistry, 2021; 64(12): 7107–7112. doi: 10.21608/ ejchem.2021.80147.3955 5. aisyah s, handharyani e, bermawie n, setiyono a. effects of ethanol extract of curry leaves (murraya koenigii) on her2 and caspase-3 expression in rat model mammary carcinoma. vet world. 2021;14(8):1988–1994. doi: 10.14202 6. shukla s, singh bk, pathania op, jain m. evaluation of her2/neu oncoprotein in serum & tissue samples of women with breast cancer. indian j med res. 2016;143:s52–s58. doi: 10.4103/0971-5916.191769. 7. muhammad if, borné y, bao x, melander o, orho-melander m, nilsson pm, nilsson j, engström g. circulating her2/erbb2 levels are associated with increased incidence of diabetes: a population-based cohort study. diabetes care. 2019 aug;42(8):1582–1588. doi: 10.2337/dc18-2556. 8. xiaolin sang, li li, chunhua rui, yichao liu, zundong liu, zhiwei tao, hailing cheng, pixu liu, induction of enr stress by melatonin enhances the cytotoxic effect of lapatinib in her2-positive breast cancer, cancer letters, 2021; 518: 82–93, issn 0304–3835, https://doi.org/10.1016/j. canlet.2021.06.011 9. serova ov, chachina na, gantsova ea, popova nv, petrenko ag, deyev ie. autophosphorylation of orphan receptor erbb2 can be induced by extracellular treatment with mildly alkaline media. int j mol sci. 2019;20(6):1515. doi: 10.3390/ijms20061515. 10. garrett tp, mckern nm, lou m, elleman tc, adams te, lovrecz go, kofler m, jorissen rn, nice ec, burgess aw, ward cw. the crystal structure of a truncated erbb2 ectodomain reveals an active conformation, poised to interact with other erbb receptors. mol cell. 2003;11(2):495–505. doi: 10.1016/s1097-2765(03)00048-0. 11. oganesyan v, peng l, bee js, li j, perry sr, comer f, xu l, cook k, senthil k, clarke l, rosenthal k, gao c, damschroder m, wu h, dall’acqua w. structural insights into the mechanism of action of a biparatopic anti-her2 antibody. j biol chem. 2018;293(22):8439–8448. doi: 10.1074/jbc.m117.818013. 12. leech ao, vellanki sh, rutherford ej, keogh a, jahns h, hudson l, o’donovan n, sabri s, abdulkarim b, sheehan km, kay ew, young ls, hill adk, smith ye, hopkins am. cleavage of the extracellular domain of junctional adhesion molecule-a is associated with resistance to anti-her2 therapies in breast cancer settings. breast cancer res. 2018;20(1):140. doi: 10.1186/s13058-018-1064-1. 13. barros ff, powe dg, ellis io, green ar. understanding the her family in breast cancer: interaction with ligands, dimerization and treatments. histopathology. 2010;56(5):560–72. doi: 10.1111/j.1365-2559.2010.03494.x. 14. han y, liu d, li l. pd-1/pd-l1 pathway: current researches in cancer. am j cancer res. 2020;10(3):727–742. 15. nascimento, c., urbano, a. c., gameiro, a., ferreira, j., correia, j., & ferreira, f. (2020). serum pd-1/pd-l1 levels, tumor expression and pd-l1 somatic mutations in her2-positive and triple negative normal-like feline mammary carcinoma subtypes. cancers, 12(6), 1386. https://doi. org/10.3390/cancers12061386 16. schütz f, stefanovic s, mayer l, von au a, domschke c, sohn c. pd-1/pd-l1 pathway in breast cancer. oncol res treat. 2017;40(5):294–297. available from: http://www.ncbi.nlm.nih.gov/pubmed/28346916. 17. kim, a.; lee, s.j.; kim, y.k.; park, w.y.; park, d.y.; kim, j.y.; lee, c.h.; gong, g.; huh, g.y.; choi, k.u. programmed death-ligand 1 (pd-l1) expression in tumour cell and tumour infiltrating lymphocytes of her2-positive breast cancer and its prognostic value. sci. rep. 2017, 7, 11671. 18. pizzino g, irrera n, cucinotta m, pallio g, mannino f, arcoraci v, squadrito f, altavilla d, bitto a. oxidative stress: harms and benefits for human health. oxid med cell longev. 2017;2017:8416763. doi: 10.1155/2017/8416763. 19. sukumar j, gast k, quiroga d, lustberg m, williams n. triple-negative breast cancer: promising prognostic biomarkers currently in development. expert rev anticancer ther. 2021;21(2):135–148. doi: 10.1080/14737140.2021.1840984. 20. wu jq, kosten tr, zhang xy. free radicals, antioxidant defense systems, and schizophrenia. prog neuropsychopharmacol biol psychiatry. 2013 1;46:200-6. doi: 10.1016/j.pnpbp.2013.02.015. 21. didžiapetrienė j, kazbarienė b, tikuišis r, dulskas a, dabkevičienė d, lukosevičienė v, kontrimavičiūtė e, sužiedėlis k, ostapenko v. oxidant/antioxidant status of breast cancer patients in preand postoperative periods. medicina. 2020; 56(2):70. https://doi.org/10.3390/ medicina56020070 22. haque t, rahman s, islam s, molla nh, ali n. assessment of the relationship between serum uric acid and glucose levels in healthy, prediabetic and diabetic individual. diabetology & metabolic syndrome. 2019;11:49. 23. pellegrini n, serafini m, salvatore s, del rio d, bianchi m, brighenti f. total antioxidant capacity of spices, dried fruits, nuts, pulses, cereals and sweets consumed in italy assessed by three different in vitro assays. mol nutr food res. 2006;50(11):1030–8. doi: 10.1002/mnfr.200600067. 24. jasim r and allwsh t. study of arylesterase and its relationship with some clinical variables in atherosclerotic patients in mousl (part 1). rafidain journal of science. 2008;19(2):143–157. 25. jasim r, mohamaad s and allwsh t. the relation between fibroblast growth factor 21 and insulin resistance in hyperlipidemia patients. egypt. j. chem. 2021; 64(12): 7091–7094. 26. guidet, b.; shah, s. v. enhanced in vivo h2o2 generation by rat kidney in glycerol-induced renal failure. american journal of physiology-renal physiology. 1989; 257(3): 440–445. doi:10.1152/ajprenal.1989.257.3.f440. 27. nelson j and kulkarni a. partial purification and characterization of a peroxidase activity from human placenta. biochemical journal. 1990; 268(3): 739–743. 28. tayefi-nasrabadi h, hosseinpour-fiezi m, mohasseli m. thermodynamic analysis of lactoperoxidase activity in camel milk. international conference on life science and technology. ipcbeesingapore.2011;3:4–6. http://www.ncbi.nlm.nih.gov/pubmed/28346916 https://doi.org/10.3390/medicina56020070 https://doi.org/10.3390/medicina56020070 356 j contemp med sci | vol. 8, no. 5, september-october 2022: 352–356 assessment of the her2, pdl1 and oxidative stress levels at the menopausal status original b.r. mahmood et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i5.1288 29. douglas j, gary r, frank e, and stephen l. ferene — a new spectrophotometric reagent for iron. canadian journal of chemistry. 1984;62(4):721–724. https://doi.org/10.1139/v84-121. 30. tietz n. textbook of clinical chemistry, 3rd ed. c. a. burtis, e. r. ashwood, w. b. saunders. 1999: 1245–1250. 31. banys-paluchowski m, witzel i, riethdorf s, rack b, janni w, fasching pa, solomayer ef, aktas b, kasimir-bauer s, pantel k, fehm t, müller v. clinical relevance of serum her2 and circulating tumor cell detection in metastatic breast cancer patients. anticancer res. 2017;37(6):3117–3128. doi: 10.21873/anticanres.11669. 32. fabricio asc, michilin s, zancan m, agnolon v, peloso l, dittadi r, scapinello a, ceccarelli c, gion m. shed her2 surrogacy evaluation in primary breast cancer patients: a study assessing tumor tissue her2 expression at both extracellular and intracellular levels. scand j clin lab invest. 2019;79(4): 260–267. doi: 10.1080/00365513.2019.1600200. 33. lam l, mcandrew n, yee m, fu t, tchou jc, zhang h. challenges in the clinical utility of the serum test for her2 ecd. biochim biophys acta. 2012;1826(1):199–208. doi: 10.1016/j.bbcan.2012.03.012. 34. di gioia d, dresse m, mayr d, nagel d, heinemann v, stieber p. serum her2 in combination with ca 15-3 as a parameter for prognosis in patients with early breast cancer. clin chim acta. 2015;440:16–22. doi:10.1016/j. cca.2014.11.001higher 35. bashamakha g, bin sumait h, bashamakha m, al serouri a, khader y. risk factors of breast cancer in hadramout valley and desert, yemen. int j prev med. 2019; 10:161. doi: 10.4103/ijpvm.ijpvm_251_17. 36. qui s, takeshita t, sueta a, tomiguchi m, goto-yamaguchi l, hidaka k, suzu i, yamamoto y, iwase h. analysis of plasma her2 copy number in cell-free dna of breast cancer patients: a comparison with her2 extracellular domain protein level in serum. breast cancer. 2021;28(3):746–754. doi: 10.1007/s12282-020-01212-x. 37. lobbezoo dj, van kampen rj, voogd ac, dercksen mw, van den berkmortel f, smilde tj, van de wouw aj, peters fp, van riel jm, peters na, de boer m, borm gf, tjan-heijnen vc. prognosis of metastatic breast cancer subtypes: the hormone receptor/her2-positive subtype is associated with the most favorable outcome. breast cancer res treat. 2013; 141(3): (507–14). doi: 10.1007/s10549-013-2711-y. 38. nascimento c, urbano ac, gameiro a, ferreira j, correia j, ferreira f. serum pd-1/pd-l1 levels, tumor expression and pd-l1 somatic mutations in her2-positive and triple negative normal-like feline mammary carcinoma subtypes. cancers (basel). 2020;12(6):1386. doi:10.3390/cancers12061386 39. lawler se, nowicki mo, ricklefs fl, chiocca ea. immune escape mediated by exosomal pd-l1 in cancer. adv biosyst. 2020;4(12):e2000017. doi:10.1002/adbi.202000017. 40. salama ea, adbeltawab re, el tayebi hm. xist and tsix: novel cancer immune biomarkers in pd-l1-overexpressing breast cancer patients. front oncol. 2020;9:1459. doi:10.3389/fonc.2019.01459 41. han b, dong l, zhou j, yang y, guo j, xuan q, gao k, xu z, lei w, wang j, zhang q. the clinical implication of soluble pd-l1 (spd-l1) in patients with breast cancer and its biological function in regulating the function of t lymphocyte. cancer immunol immunother. 2021;70(10):2893–2909. doi: 10.1007/s00262-021-02898-4. 42. sultana dr, shahin ad, md jawadul h. measurement of oxidative stress and total antioxidant capacity in hyperthyroid patients following treatment with carbimazole and antioxidant. heliyon. 2021; 8(1):e08651. doi: 10.1016/j. heliyon.2021.e08651. 43. reuter s, gupta sc, chaturvedi mm, aggarwal bb. oxidative stress, inflammation, and cancer: how are they linked?. free radic biol med. 2010;49(11):1603–1616. doi:10.1016/j.freeradbiomed.2010.09.006 44. okuturlar y, gunaldi m, kocoglu h, hursitoglu m, gedikbasi a, didem acarer d, harmankaya o, kumbasar a. serum paraoxonase and arylesterase can be useful markers to predict neoadjuvant chemotherapy requirement in patients with breast cancer. j cancer res ther. 2018;14(9):362–367. doi:10.4103/0973-1482.235355 45. bourgonje ar, abdulle ae, al-rawas am, al-maqbali m, al-saleh m, enriquez mb, al-siyabi s, al-hashmi k, al-lawati i, bulthuis mlc, mulder dj, gordijn sj, van goor h, saleh j. systemic oxidative stress is increased in postmenopausal women and independently associates with homocysteine levels. int j mol sci. 2020;21(1):314. doi: 10.3390/ ijms21010314. 46. galaris d, barbouti a, pantopoulos k. iron homeostasis and oxidative stress: an intimate relationship. biochim biophys acta mol cell res. 2019 dec;1866(12):118535. doi: 10.1016/j.bbamcr.2019.118535. 47. kangari p, zarnoosheh farahany t, golchin a, ebadollahzadeh s, salmaninejad a, mahboob sa, nourazarian a. enzymatic antioxidant and lipid peroxidation evaluation in the newly diagnosed breast cancer patients in iran. asian pac j cancer prev. 2018;19(12):3511–3515. doi: 10.31557/apjcp.2018.19.12.3511. 48. sarmiento-salinas fl, delgado-magallón a, montes-alvarado jb, ramírezramírez d, flores-alonso jc, cortés-hernández p, reyes-leyva j, herreracamacho i, anaya-ruiz m, pelayo r, millán-pérez-peña l, maycotte p. breast cancer subtypes present a differential production of reactive oxygen species (ros) and susceptibility to antioxidant treatment. front oncol. 2019 jun 7;9:480. doi: 10.3389/fonc.2019.00480. 49. priyanka hp, nair rs. neuroimmunomodulation by estrogen in health and disease. aims neurosci. 2020;7(4):401–417. doi:10.3934/ neuroscience.2020025. 56 j contemp med sci | vol. 9, no. 1, january-february 2023: 56–62 original association between nesfatin-1 levels and c-peptide in sera of obese/non-obese type 2 diabetic women israa khalil ibrahim al-yassiri1*, fadhil jawad al-tu’ma1, maher abbood mukheef1, khosay baqir jawad2 baraa abdul-kareem mutar 1 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 2al-hassan center of diabetes and endocrinology unit, al-hussein medical city, kerbala health directorate, kerbala, iraq. *correspondence to: israa khalil ibrahim al-yasiri (e-mail: aalyasiri1@gmail.com) abstract objectives: the aim of the presented word was to assess the diagnostic accuracy of serum nesfatin-1 in type 2 diabetes mellitus and its relationship with c-peptide level in obese and non-obese type-2 diabetic women of iraqi population. methods: (a case-control study was performed on 50 type 2 diabetic patients admitted in al-hussein teaching hospital and al-hassan center of diabetes and endocrinology unit/kerbala health directorate – iraq and another 50 control individuals, during the period from april, 2022 – jan. 2023). the t2dm groups were divided into two groups 25 obese and 25 non-obese; also the control group was divided into 25 obese and 25 non-obese as apparently healthy groups. the elisa kit was used to measure serum nesfatin-1 and c-peptide, and random serum glucose was measured by enzymatic colorimetric method, and lipid profile test were measured through spectrophotometric technique, instead of hba1c% was determined using hplc method. results: the results observed indicated that nesfatin-1 levels shown a non-significant decrease in all of type 2 diabetic groups as compared with apparently healthy control group, while the c-peptide were significantly decreased in type 2 diabetic patients when compared with apparently control group. in addition, the random blood glucose and hba1c% were shown significant elevation in type 2 diabetic patients as compared with apparently healthy control groups. the observed data indicated that nesfatin-1 and c-peptide levels when comparing between type 2 diabetic patients and control in obese groups shown a risk factors depending upon the odd ratio observed (or = 1.064 (1.011–1.119), 1.0200 (0.992–1.08)) respectively, but only nesfatin-1 was shown to be significant. in bmi the levels of nesfatin-1 and c-peptide, as shown the nesfatin-1 was significant in obese groups, while the c-peptide as shown significant in normal weight groups. the optimal diagnostic points for nesfatin-1 were (sensitivity = 98%, specificity = 90%) at a level (cut-off points) = 39.13, while c-peptide levels: (sensitivity = 98%, specificity = 94%) at a level (cut-off points) = 15.99. both markers have p-values of the auc were <0.001 and statistically significant. conclusion: accordingly, it was concluded that a significant relationship between circulating nesfatin-1 levels and type 2 diabetes. nesfatin-1 appears to be able to contribute to the treatment of obesity and diabetes because of its anorexigenic and antihyperglycemic effects. in addition, c-peptide is a known biomarker of insulin resistance and beta-cell function. high specificity and sensitivity analyzed results were obtained by roc analysis for both markers in t2dm. keywords: nesfatin-1, c-peptide, obese, diabetes mellitus, type 2, body mass index, hypoglycemic agents issn 2413-0516 introduction diabetes mellitus (dm) is a metabolic disorder where in human body does not produce or properly uses insulin, a hormone that is required to convert sugar, starches and other food into energy. absence or reduced insulin in turn leads to persistent abnormally high blood sugar and glucose in tolerance. it is probably an oldest disease known to man.1 immunohistochemical studies have shown that the precursor of nesfatin-1, non-esterified fatty acid/nucleobinding 2 (nucb2), is localized in many places such as the pituitary gland, hypothalamus, brain stem, the forebrain and midbrain nuclei, central amygdaloid nucleus, ventrolateral medulla, and cerebellum. it is linked with developing of various serious diseases like micro vascular (nephropathy, retinopathy, neuropathy) and macro vascular (peripheral vascular disease and coronary heart diseases).2 diabetes and its complications are complex, multifactorial conditions with both major environmental and genetic components. when early studies identified differences in diabetic complication susceptibility in patients who seemed otherwise equal with regard to their diabetes glucose control, clinical features and management.3 obesity and t2dm are two of the most pressing public health concerns worldwide because of their association with life-threatening diseases, including cardiovascular diseases and cancers.4 obesity, especially pathologic expansion of visceral white adipose tissue (vwat), increases the risk of developing t2dm. depending on the race, more than 75–90% of patients with t2dm are overweight or obese. the strong association of obesity and t2dm is supported by the term “disability.”5 nesfatin-1 that discovered in 2006 is secreted from the hypothalamic nuclei, which are responsible for controlling appetite. in the same study, it was reported that nesfatin-1 suppresses food intake, even in obese mice with a knockdown leptin gene.6 the secretion is distributed in the body; nesfatin-1 is thought to affect many functions. previous studies have reported that nesfatin-1 has regulatory effects on energy metabolism through suppression of food intake. in addition, it has been reported that nesfatin-1 regulates cardiac functions, decreases blood glucose levels, acts as a neuroendocrine regulator, and causes weight loss along with reduction in energy intake.7 the relationship between obesity and nesfatin-1 was investigated because of the effects of nesfatin-1 on food intake and energy consumption. there was a relationship between the polymorphism of the nucleobindin-2 gene and obesity. this may be a risk factor for the development of (submitted: 02 october 2022 – revised version received: 29 october 2022 – accepted: 12 november 2022 – published online: 26 february 2023) mailto:aalyasiri1@gmail.com 57j contemp med sci | vol. 9, no. 1, january-february 2023: 56–62 i.k.i. al-yassiri et al. original association between nesfatin-1 levels and c-peptide in sera of obese/non-obese type 2 diabetic women obesity.8 a study compared the fasting plasma nesfatin-1 levels of healthy individuals and type 2 diabetic individuals. thus, fasting nesfatin-1 levels were found to be lower in individuals with type 2 diabetes than healthy individuals.9 the effect of nesfatin-1 on insulin secretion is affected by blood glucose concentrations. nucleobindin-2/nesfatin-1 released from the pancreas increased in response to glucose.10 nesfatin-1 can increase the synthesis of pre-proinsulin mrna and stimulate glucose-enhanced insulin release.11 peripheral nesfatin-1 may have a potential effect on the control of glucose homeostasis.12 nesfatin-1 may be involved in long-term changes of energy expenditure. because nesfatin-1 reduces food intake and increases energy expenditure, it induces a negative energy balance, which might be relevant in states of over nutrition but also might reflect conditions of stress.13 c-peptide is a substance that is created when the hormone insulin is produced and released into the body. c-peptide is a sign that the body is producing insulin. a low level (or no c-peptide) indicates that pancreas is producing little or no insulin. c-peptide is secreted in equimolar amounts with insulin from pancreatic beta cells and is reported to be a more useful laboratory parameter than insulin in evaluating endogenous insulin reserve.14 c-peptide is a useful and widely used method of assessing pancreatic beta cell function and is measured to tell the difference between insulin the body produces and insulin that is injected into the body.15 after cleavage of proinsulin, insulin and the 31-amino-acid peptide c-peptide are produced in equal amounts.16 the presented word aimed to assess the diagnostic role of serum nesfatin-1 in obese and non-obese type 2 diabetes mellitus and its association with c-peptide level as compared with apparently healthy control women of iraqi population. materials and methods a case-control study was performed on 50 blood samples obtained from types 2 diabetic women divided into two patients groups (25 obese and 25 non-obese) which were admitted in al-hussein teaching hospital and al-hassan center of diabetes and endocrinology unit/kerbala health directorate – iraq, during the period from may, 2022 – jan. 2023), and another 50 blood samples obtained from apparently healthy women and also divided into two control groups (25 obese and 25 non-obese). the elisa kit was used to measure serum nesfatin-1 and c-peptide levels, random blood glucose were measured by enzymatic colorimetric method, and lipid profile test were measured through spectrophotometric technique, while hba1c% was determined using hplc method. the mean ± sd data observed of bmi was determined from (fendler et al., 2021):17 bmi (kg/m2) = weight (kg)/ height (meter)2 these results for both type 2 diabetic women patients and control groups, weight was classified according to their bmi as shown below (organization, 2016).18 a. underweight < 18.5 b. normal weight 18.5–24.9 c. over weight 25.0–29.9 d. obese ≥ 30.0 the waist/hip ratio (w/h ratio) was calculated by dividing waist circumference by hip circumference as indicated below: w/h ratio = waist (cm)/hip (cm) according to world health organization (who, 2021) a w/h ratio chart is: health risk women low 0.80 or lower moderate 0.81–0.85 high 0.86 or higher the data were analyzed using the statistical package for social sciences (spss version 22.0). continuous variables were expressed as means ± standard deviation, sd). mean comparisons were made using one-way analysis of variance (anova) test. receiver operator characteristic curves (roc) were drawn to compare different sensitivity indices of each marker studied. results the clinical demographic characteristics of type 2 diabetic patients group were summarized in figure 1 which indicates that 50% of women included in this study of type 2 diabetic patients group were within normal weight (non-obese) and 50% were obese. therefore, higher percentage of risk was observed (48%) in obese type 2 diabetic women, and lower risk factor was observed low and moderate (1%) w/h ratio, figure 1b. fig. 1 baseline characteristics and demographic description of the study population in type 2 diabetic women patients compared to apparently healthy control. 58 j contemp med sci | vol. 9, no. 1, january-february 2023: 56–62 association between nesfatin-1 levels and c-peptide in sera of obese/non-obese type 2 diabetic women original i.k.i. al-yassiri et al. table 1 illustrated that effect of type 2 diabetes non-obese women (n = 25) on some biochemical parameters studied in patients and apparently health control groups (n = 25). the mean ± sd observed in non-obese type 2 diabetic women include nesfatin-1 levels which was non-significantly decreased in type 2 diabetic patients and reached to (67.42 ± 33.49 ng/dl) as compared with apparently non-obese healthy control (87.54 ± 68.09 ng/dl, p = 0.19), while the level of c-peptide was significantly decreased to (107.82 ± 21.23 nmol/l) in non-obese type 2 diabetic patients as compared with apparently non-obese healthy control (176.44 ± 111.53 nmol/l, p = 0.004). the level of random blood glucose and and hba1c% were significantly increased markedly in nonobese type 2 diabetic patients group and they reached to (279.76 ± 59.33 mg/dl) (9.72 ± 2.20%) as compared with apparently healthy control group (98.04 ± 13.74 mg/dl, p < 0.001) (4.13 ± 0.62%, p < 0.001) respectively. the mean ± sd of lipid profile levels shown in the same table including total cholesterol (tc), high density lipoprotein-cholesterol (hdl-c), low density lipoprotein-cholesterol (ldl-c) and triglycerides (tg) which were studies in each type 2 diabetic patients group and healthy control. the data observed indicated that each of total cholesterol (184.44 ± 19.66 mg/dl) and triglycerides (185.28 ± 37.99 mg/dl) were significantly increased as compared with apparently nonobese healthy control group (164.36 ± 29.60 mg/dl) and (109.36 ± 20.78 mg/dl) respectively (p < 0.05), while the hdl-c and ldl-c were non-significantly changes, p = 0.73 and p = 0.94 respectively. table 2 illustrated that effect of obese type 2 diabetes women (n = 25) on the same biochemical parameters studied and compared with control groups (n = 25). the mean ± sd results obtained indicated that nesfatin-1 levels was significantly decreased in obese type 2 diabetic patients and reached to (50.79 ± 12.08 ng/dl) as compared with apparently obese healthy control (75.48 ± 68.53 ng/dl, p = 0.007) and the level of c-peptide was non-significantly decreased to (134.69 ± 83.39 nmol/l) in type 2 diabetic patients as compared with apparently obese healthy control (143.11 ± 59.49 nmol/l, p = 0.68). while the level of random blood glucose and hba1c% were significantly increased in obese type 2 diabetic patients group table 1. the biochemical parameters studied in non-obese t2dm and control groups variables (non-obese) samples, n = 50 t2dm patients, n = 25 mean ± sd control n = 25 mean ± sd p value nesfatin-1, ng/dl 67.42 ± 33.49 87.54 ± 68.09 0.19[ns] cpeptide, nmol/l 107.82 ± 21.23 176.44 ± 111.53 0.004[s] rbg, mg/dl 279.76 ± 59.33 98.04 ± 13.74 <0.001[s] hba1c% 9.72 ± 2.20 4.13 ± 0.62 <0.001[s] tg, mg/dl 185.28 ± 37.99 109.36 ± 20.78 <0.001[s] tc, mg/dl 184.44 ± 19.66 164.36 ± 29.60 0.007[s] hdl-c, mg/dl 45.02 ± 3.42 44.71 ± 2.93 0.73[ns] ldl-c, mg/dl 191.54 ± 33.84 176.38 ± 28.76 0.94[ns] rbg, random blood glucose; hba1c, glycated hemoglobin; tg, triglyceride; tc, total cholesterol; hdl-c, high density lipoprotein-cholesterol; ldl-c, low density lipoprotein-cholesterol. table 2. the biochemical parameters studied in obese t2dm and control groups variables (obese) samples, n = 50 t2dm patients, n = 25 mean ± sd control n = 25 mean ± sd p value nesfatin-1, ng/dl 50.79 ± 12.08 75.48 ± 68.53 0.007[s] cpeptide, nmol/l 134.69 ± 83.39 143.11 ± 59.49 0.68[ns] rbg, mg/dl 260.80 ± 72.38 100.48 ± 11.64 <0.001[s] hba1c% 9.30 ± 1.85 4.58 ± 0.75 <0.001[s] tg, mg/dl 168.32 ± 22.83 205.28 ± 105.97 0.10[ns] tc, mg/dl 205.52 ± 35.74 178.00 ± 34.95 0.008[s] hdl-c, mg/dl 45.48 ± 5.47 47.31 ± 6.18 0.27[ns] ldl-c, mg/dl 154.25 ± 33.76 161.40 ± 42.62 0.51[ns] rbg, random blood glucose; hba1c, glycated hemoglobin; tg, triglyceride; tc, total cholesterol; hdl-c, high density lipoprotein-cholesterol; ldl-c, low density lipoprotein cholesterol. and they reached to (260.80 ± 72.38 mg/dl) (9.30 ± 1.85%) as compared with apparently obese health control groups (100.48 ± 11.64 mg/dl, p < 0.001) (4.58 ± 0.75%, p < 0.001) respectively. the mean ± sd of lipid profile levels shown in the same table including also total cholesterol (tc), high density lipoprotein-cholesterol (hdl-c), low density lipoprotein cholesterol (ldl-c) and triglycerides (tg) which were studies in each type 2 diabetic patients group and healthy control. the data observed indicated that each of total cholesterol was significantly increased and they reached to (205.52 ± 35.74 mg/dl) as compared with apparently obese healthy control group (178.00 ± 34.95 mg/dl, p = 0.008), while the mean ± sd of triglyceride was non-significantly decreased to (168.32 ± 22.83 mg/dl) as compared to apparlently obese health control (205.28 ± 105.97 mg/dl, p = 0.10), while hdl-c and ldl-c were non-significantly decreased and the reached to (45.48 ± 5.47 mg/dl) (154.25 ± 33.76 mg/ dl) as compared with obese control group (47.31 ± 6.18 mg/ dl, 0.27) (161.40 ± 42.62 mg/dl, p = 0.51) respectively. binary logistic regression was performed and forward logistic regression was adopted to analyze the results. it was found that nesfatin-1 in non-obese type 2 diabetic patient has a protective factor (or: 0.987; 95% ci: (0.960–1.015)) respectively, while in obese type 2diabetic women patients groups were independent risk factors (or: 1.064; 95% ci: (1.011–1.119). in addition, c-peptide was shown to be a risk factor for t2dm in normal and obese cases (or: 1.058 and 1.02, 95% ci: (1.021–1.097), 0.992–1.08) as shown in table 3. the correlation coefficient was used for determining linear relationships between nesfatin-1 and obese t2dm and non-obese t2dm, with each of bmi, c-peptide, rbg and hba1c% in obese t2dm groups as compared with non-obese t2dm group. the results showed that there was moderated relationship and a significant correlation between nesfatin-1 and bmi (p = 0.015, r = 0.4), c-peptide (p = 0.015, r = –0.4), rbg (p = 0.007, r = –0.4), and hba1c% (p = 0.004, r = –0.42) as shown in figure 2. the results of the receiver operating curve (roc) and auc analysis for the nesfitin-1 and c-peptide (73%, 71.6%) 59j contemp med sci | vol. 9, no. 1, january-february 2023: 56–62 i.k.i. al-yassiri et al. original association between nesfatin-1 levels and c-peptide in sera of obese/non-obese type 2 diabetic women table 3. estimation the associated of analyzed factors in patients compared to control group variables normal weight obese t2dm or (lower–upper) control p value t2dm or (lower–upper) control p value nesfatin-1, ng/dl 0.987(0.960–1.015) 0.37[ns] 1.064(1.011–1.119) 0.01[s] cpeptide, nmol/l 1.058(1.021–1.097) 0.002[s] 1.0200(0.992–1.08) 0.94[ns] tg, mg/dl 0.891(0.819–0.969) 0.007[s] 1.005(0.996–1.013) 0.29[ns] tc, mg/dl 0.969(0.897–1.047) 0.42[ns] 0.974(0.953–0.996) 0.023[s] hdl-c, mg/dl 0.986(0.596–1.630) 0.95[ns] 1.005(0.987–1.023) 0.57[ns] ldl-c, mg/dl 0.898(0.807–0.998) 0.04[s] 1.066(0.956–1.189) 0.24[ns] respectively, as possible diagnostic parameters. nesfitin-1 was shown a good diagnostic performance for prediction of t2dm patients compared to control group, data are presented in figure 3 and table 4. for nesfitin-1 (sensitivity = 98%, specificity = 90%) at a level = 39.13, while c-peptide levels: (sensitivity = 98%, specificity = 94%) at a level = 0.15.99, both nesfitin-1 and c-peptide p values of the auc for ima were <0.001 and statistically significant. discussion nesfatin-1 is reported to exert an antihyperglycemic effect under impaired glucose metabolism conditions.19 it may also act in the brain to upregulate insulin sensitivity (yang et al., 2012),20 and increase insulin release in beta cells in response to hyperglycemia.21 various studied including the roles of different biomarkers associated with the pathogenesis of t2dm fig. 2 simple linear regression of nesfatin.1 by (a) bmi, (b) c-peptide, (c) rbs and (d) hba1c between obese (dm & non dm). in obese and non-obese male and females subjects have been studied in various region including iraqi individuals.22 nesfatin-1 was also found to inhibit food intake in the central nervous system and acts as an anorexigenic agent, but the regulatory mechanism remains not clear,6 but the regulatory mechanism remains unclear. since nesfatin-1 can cross the brain-blood-barrier and hypothalamic nesfatin-1 can significantly inhibit food intake.23 li et al. proposed that diabetic polyphagia is caused by decreased circulating nesfatin-1 levels and positively correlated with age.9 studies have also shown that nesfatin-1 can stimulate the lipid metabolism and exhibit anti-inflammatory effects.24 nesfatin-1 may improve both hepatic and peripheral insulin sensitivity as it enhances glucose uptake by peripheral tissues and inhibits gluconeogenesis via different pathways.25 moreover, nesfatin-1 mrna is colocalized almost completely with insulin in β pancreatic islets cells. also, its processing physiologically occurs in pancreatic 60 j contemp med sci | vol. 9, no. 1, january-february 2023: 56–62 association between nesfatin-1 levels and c-peptide in sera of obese/non-obese type 2 diabetic women original i.k.i. al-yassiri et al. islet cell.26 nesfatin-1 mrna expressed on pancreatic islet cells from type 2 diabetic patients was lower than that from healthy subjects. this was significantly correlated with insulin secretion capability.27 on the other hand, the role of c-peptide is not well defined in type 2 diabetes, of which insulin resistance and insulin secretion defect both exist. some studies found that c-peptide level was a protective effect on microvascular complications. serum c-peptide had been used in assessing pancreatic function in dm patients for a long time. this study demonstrates a continuous decline in serum c-peptide levels following diagnosis and an especially significant decline from baseline after 3 years. several hypotheses are presented to explain the clinical implications of residual c-peptide secretion in t1dm. some previous studies suggested that c-peptide acts as an endogenous antioxidant, which protects pancreatic beta cells by increasing catalase expression and reducing peroxisomal oxidative stress.28 additionally, other reported postulate an association between residual c-peptide levels and reduction in response to glucagon-like peptide-1 (glp-1).29 although the mechanism is not clearly elucidated yet, the clinical benefit from preserved c-peptide secretion in dm patients is widely known. microvascular complications such as diabetic retinopathy and nephropathy were found to be less likely to develop in patients with residual c-peptide production, but the results were not always consistent among studies.30 also, preserved beta cell function is reported to be related to a decreased risk of hypoglycemia and decreased insulin requirement.31 moreover, with the wide use of continuous glucose monitoring (cgm) systems, recent studies reported on the importance of glycemic variability and its association with c-peptide levels. patients with residual c-peptide production had a lower mean blood glucose level and higher time in range (rickels et al., 2020),32 and fasting c-peptide levels were negatively correlated with glucose coefficient of variation by cgm.33 diabetes mellitus (dm), a metabolic disorder characterized by hyperglycaemia, associated with deficiency or resistance to insulin indicates endocrinal abnormality of the pancreas. lipid abnormalities are prevalent in t2dm patients because of ir which affects key enzymes and pathways in lipid metabolism: apo protein production, regulation of lipoprotein lipase, action of cholesterol ester transfer proteins and hepatic and peripheral actions of insulin.34,35 hyperglycemia and the high level of ir associated with t2dm has multiple effects on fat metabolism which results in the production of atherogenic dyslipidemia characterized by lipoprotein abnormalities: elevated very low density lipoprotein cholesterol (vldl) elevated low density lipoprotein cholesterol (ldl-c), elevated triglyceride (tg) and decreased high density lipoprotein cholesterol (hdl-c).36 the main cause for lipid abnormalities in t2dm patients is impaired secretion of insulin that affects the liver apolipoprotein production and regulates the enzymatic activity of lipoprotein lipase (lpl) and cholesterol ester transport protein (cetp). moreover, its deficiency reduces the activity of hepatic lipase; therefore, several steps involved in the production of biologically active lpl might be altered in t2dm compared to controls.37 many studies were confirmed the association of nesfatin-1 levels in obese individuals. it has been reported that the expression of nesfatin-1 was up-regulated in adipose tissue of a high-fat diet. this suggests a potential role for nesfatin-1 in the lipid accumulation pathway and perhaps diet-induced obesity.38 the mean fasting c-peptide level of the obese t2dm was higher than that of the non-obese t2dm. they were moderately positively correlated, although not linear. hence this study suggests that obese patients are hyperinsulinemic. other study observed lesser insulin levels in non-obese t2dm as compared to obese which also noted by anothers, in which they measured insulin and c-peptide levels during a three hour oral glucose tolerance test.39 a positive correlation between bmi and basal serum c-peptide levels was also observed previous reports.40 according to banerji et al, asian indians have an unexpectedly high percentage of body fat relative to bmi and muscle mass; this is associated with a proportionate increase in visceral fat. they are markedly insulin resistant and hyperinsulinemic. the condition of obesity is caused by excessive intake of nutrients continuously causing fat deposits to become excessive. deposits of fatty acids in the form of chemical compounds in the form of triacylglycerol contained in adiposity cells can protect the body from the toxic effects of fatty acids. freeform fatty acids can circulate in blood vessels throughout the body and cause oxidative stress which we are familiar with lipo-toxicity. the emergence of lipo-toxicity effects caused by table 4. receiver operating characteristic curve showing sensitivity and specificity of nesfitin-1 and c-peptide in patients compared to control group test result variable(s) nesfatin-1 c-peptide auc 73% 71.6% sensitivity % 98% 98% specificity % 90% 94% youden index 0.369 0.346 cut-off points 39.13 15.99 ci (95%) 0.624–0.835 0.616–0.816 ppv 52.13% 51.04 npv 47.87% 48.96% p value <0.001 <0.001 fig. 3 roc curves for nesfitin-1 and c-peptide in patients to analyses the optimal diagnostic points for predicting of cases compared to control group. 61j contemp med sci | vol. 9, no. 1, january-february 2023: 56–62 i.k.i. al-yassiri et al. original association between nesfatin-1 levels and c-peptide in sera of obese/non-obese type 2 diabetic women determine ir by using insulin or c-peptide concentrations; however, the efficiency of adipokines (nesfitin-1) and c-peptide to determine metabolic syndrome/ or their complication namely diabetes has not been compared. c-peptide is a strong indicator of metabolic syndrome. since c-peptide has recently emerged as a biomolecule with significant importance for inflammatory diseases, monitoring c-peptide levels will aid clinicians in preventing metabolic syndrome.42 also, serum nesfatin-1 is possibly associated with weight-related abnormalities in otherwise healthy subjects and type 2 diabetes. obesity and type 2 diabetes may share a common pathologic point in this regard.43 conclusion 1. nesfatin-1 levels which was non-significantly decreased in non-obese type 2 diabetic patients, while the level of c-peptide was significantly decreased to in non-obese type 2 diabetic patients. 2. the nesfatin-1 levels was significantly decreased in obese type 2 diabetic patients, while, the c-peptide was non significantly decreased in obese type 2 diabetic patients. 3. nesfatin-1 appears to be able to contribute to the treatment of obesity and diabetes because of its anorexigenic and antihyperglycemic effects. in addition, c-peptide is a known biomarker of insulin resistance and beta-cell function. high specificity and sensitivity analyzed results were obtained by roc analysis for both markers in t2dm. conflict of interest none.  several free fatty acids released by the triacylglycerol to compensate for the destruction of excessive fat deposits affects the adipose and non-adipose tissue and plays a role in the pathophysiology of diseases in various organs such as the liver and pancreas. this release of free fatty acids from excessive triacylglycerol can also inhibit fat synthesis and reduce the clearance of triacylglycerol. this can increase the tendency of hypertriglyceridemia. the release of free fatty acids by endothelial lipoprotein from triglycerides which increases in the increase of lipoprotein β causes lipo-toxicity which also interferes with the function of insulin receptors. the consequence of insulin resistance is hyperglycemia, which is compensated by glucose synthesis from the liver (gluconeogenesis), which contributes to aggravating hyperglycemia. free fatty acids also contribute to hyperglycemia by reducing glucose use from insulin-stimulated muscles. lipotoxicity due to excess free fatty acids also decreases insulin secretion from pancreatic β cells, which ultimately β cells will experience fatigue.41 diabetes is associated with the development of insulin resistance. with multiple indices available, examination the validity of the (nesfitin-1) and c-peptide to determine dm by receiver operating characteristic analysis was performed. nesfitin-1 was shown a good diagnostic performance for prediction patients compared to control group. for nesfitin-1 (sensitivity = 98%, specificity = 90%) at a level = 39.13, while c-peptide levels: (sensitivity = 98%, specificity = 94%) at a level = 0.15.99, both nesfitin-1 and c-peptide p-values of the auc for ima were <0.001 and statistically significant. since metabolic syndrome is associated with elevated risk for developing diabetes, many studies were investigating the insulin resistance and many types of adipokines. a key component of metabolic syndrome is the development of insulin resistance (ir). the homeostatic model assessment (homa-ir) can references 1. deepthi, sowjanya, lidiya, et al. (2017). a modern review of diabetes mellitus: an annihilatory metabolic disorder. 3(1). 2. tsuchiya, shimizu, yamada, et al. (2010). fasting concentrations of nesfatin‐1 are negatively correlated with body mass index in non‐obese males. 73(4), 484–490. 3. khalil, zrieki, & technology. (2022). factors associated with glycemic control among syrian patients with type 2 diabetes mellitus. 15(4), 1701–1708. 4. stern, rutkowski, and scherer (2016). adiponectin, leptin, and fatty acids in the maintenance of metabolic homeostasis through adipose tissue crosstalk. 23(5), 770–784. 5. farag, & gaballa. (2011). diabesity: an overview of a rising epidemic. 26(1), 28–35. 6. oh-i, shimizu, satoh, et al. (2006). identification of nesfatin-1 as a satiety molecule in the hypothalamus. 443(7112), 709–712. 7. algul, ozdenk and ozcelik. (2017). variations in leptin, nesfatin-1 and irisin levels induced by aerobic exercise in young trained and untrained male subjects. 34(4), 339–344. 8. senin, al-massadi, barja-fernandez, et al. (2015). regulation of nucb2/ nesfatin-1 production in rat’s stomach and adipose tissue is dependent on age, testosterone levels and lactating status. 411, 105–112. 9. li, wang, chen, et al. (2010). fasting plasma levels of nesfatin-1 in patients with type 1 and type 2 diabetes mellitus and the nutrient-related fluctuation of nesfatin-1 level in normal humans. 159(1-3), 72–77. 10. şafak, gülçi̇çek, sksoy, et al. (2017). increased serum nesfatin-1 levels in patients with impaired glucose tolerance. 21(3), 65. 11. anwar, yamamah, ibrahim, et al. (2014). nesfatin-1 in childhood and adolescent obesity and its association with food intake, body composition and insulin resistance. 188, 21–24. 12. khalili, khaniani, afkhami, et al. (2017). nucb2/nesfatin-1: a potent meal regulatory hormone and its role in diabetes. 18(2), 105–109. 13. kühne, schalla, friedrich, et al. (2018). nesfatin-130-59 injected intracerebroventricularly increases anxiety, depression-like behavior, and anhedonia in normal weight rats. 10(12), 1889. 14. leighton, sainsbury, and jones. (2017). a practical review of c-peptide testing in diabetes. 8(3), 475–487. 15. kulkarni et al., 2016. 16. yosten, maric-bilkan, luppi, et al. (2014). physiological effects and therapeutic potential of proinsulin c-peptide. 307(11), e955–e968. 17. fendler et al., 2021. 18. organization. (2016). world health statistics 2016: monitoring health for the sdgs sustainable development goals: world health organization. 19. su, zhang, tang, et al. (2010). the novel function of nesfatin-1: antihyperglycemia. 391(1), 1039–1042. 20. yang, zhang, wang, et al. (2012). nesfatin-1 action in the brain increases insulin sensitivity through akt/ampk/torc2 pathway in diet-induced insulin resistance. 61(8), 1959–1968. 21. nakata, manaka, yamamoto, et al. (2011). nesfatin-1 enhances glucoseinduced insulin secretion by promoting ca2+ influx through l-type channels in mouse islet β-cells. 58(4), 305–313. 22. abusaib m, ahmed m, nwayyir ha, alidrisi ha, al-abbood m, al-bayati a, al-ibrahimi s, al-kharasani a, al-rubaye h, mahwi t, ashor a. iraqi experts consensus on the management of type 2 diabetes/prediabetes in adults. clinical medicine insights: endocrinology and diabetes. 2020 aug;13:1179551420942232. 23. pan, hsuchou, and kastin. (2007). nesfatin-1 crosses the blood–brain barrier without saturation. 28(11), 2223–2228. 62 j contemp med sci | vol. 9, no. 1, january-february 2023: 56–62 association between nesfatin-1 levels and c-peptide in sera of obese/non-obese type 2 diabetic women original i.k.i. al-yassiri et al. 24. dong, xu, xu, et al. (2013). nesfatin-1 stimulates fatty-acid oxidation by activating amp-activated protein kinase in stz-induced type 2 diabetic mice. 8(12), e83397. 25. wu, yang, chen, et al. (2014). hypothalamic nesfatin-1/nucb2 knockdown augments hepatic gluconeogenesis that is correlated with inhibition of mtor-stat3 signaling pathway in rats. 63(4), 1234–1247. 26. mohan, gasner, ramesh, et al. (2016). ghrelin, ghrelin-o-acyl transferase, nucleobindin-2/nesfatin-1 and prohormone convertases in the pancreatic islets of sprague dawley rats during development. 47, 325–336. 27. riva, nitert, voss, et al. (2011). nesfatin-1 stimulates glucagon and insulin secretion and beta cell nucb2 is reduced in human type 2 diabetic subjects. 346, 393–405. 28. luppi, drain, to, et al. (2020). autocrine c‐peptide protects ins1 β cells against palmitic acid‐induced oxidative stress in peroxisomes by inducing catalase. 3(3), e00147. 29. thivolet, marchand, & chikh. (2019). inappropriate glucagon and glp-1 secretion in individuals with long-standing type 1 diabetes: effects of residual c-peptide. 62, 593–597. 30. lachin, mcgee, palmer, et al. (2014). impact of c-peptide preservation on metabolic and clinical outcomes in the diabetes control and complications trial. 63(2), 739–748. 31. lam, dayan, & herold. (2021). a little help from residual β cells has longlasting clinical benefits. 131(3). 32. rickels, evans-molina, bahnson, et al. (2020). high residual c-peptide likely contributes to glycemic control in type 1 diabetes. 130(4), 1850–1862. 33. ikegami, babaya, and noso. (2021). β‐cell failure in diabetes: common susceptibility and mechanisms shared between type 1 and type 2 diabetes. 12(9), 1526–1539. 34. al-tu’ma, f. j. and yousuf, l. m. z. (2015). vitamin d deficiency and hypertension in type 2 diabetic iraqi patients. j. contemp. med. sci., 1(1): 17–20. 35. gudbjartsson, thorgeirsson, sulem, et al. (2019). lipoprotein (a) concentration and risks of cardiovascular disease and diabetes. 74(24), 2982–2994. 36. beshara, cohen, goldberg, et al. (2016). triglyceride levels and risk of type 2 diabetes mellitus: a longitudinal large study. 64(2), 383–387. 37. rye, barter, and cochran. (2016). apolipoprotein ai interactions with insulin secretion and production. 27(1), 8–13. 38. gui, silha, mishra, et al. (2003). changes in adipokine expression during food deprivation in the mouse and the relationship to fasting-induced insulin resistance. 81(10), 979–985. 39. tura, ludvik, nolan, et al. (2001). insulin and c-peptide secretion and kinetics in humans: direct and model-based measurements during ogtt. 281(5), e966–e974. 40. park, ihm, yoo, et al. (1997). differential effects of ambient blood glucose level and degree of obesity on basal serum c-peptide level and the c-peptide response to glucose and glucagon in non-insulin-dependent diabetes mellitus. 37(3), 165–171. 41. silitonga, siahaan, & anto. (2019). correlation between obesity and lipid profile in type 2 diabetes mellitus patients at the endocrine and metabolic polyclinic in general hospital pirngadi medan. 7(8), 1309. 42. gonzalez-mejia, porchia, torres-rasgado, et al. (2016). c-peptide is a sensitive indicator for the diagnosis of metabolic syndrome in subjects from central mexico. 14(4), 210–216. 43. samani, ghasemi, bookani, et al. (2019). serum nesfatin-1 level in healthy subjects with weight-related abnormalities and newly diagnosed patients with type 2 diabetes mellitus; a case-control study. 15(1), 69. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1322 270 j contemp med sci | vol. 8, no. 4, july-august 2022: 270–277 original preoperative incentive spirometer to prevent postoperative pulmonary complications following open heart surgeries: a randomized single blinded multi-centric clinical trial jadeel noor faleh1*, sadeq al-fayyadh2 1al-muthanah health directorate, ministry of health, al-muthanah, iraq. 2department of the adult nursing, college of nursing, university of baghdad, baghdad, iraq. *correspondence to: jadeel noor faleh (e-mail: jadeel.nour1202a@conursing.uobaghdad.edu.iq) (submitted: 29 may 2022 – revised version received: 21 june 2022 – accepted: 26 july 2022 – published online: 26 august 2022) abstract objective: to determine the effect preoperative use of an incentive spirometer (is) has on preventing postoperative pulmonary complications among patients undergoing open heart surgeries. methods: a prospective, randomized, controlled single-blinded parallel-group design. data were collected from january 9th 2022 to april 1st 2022. a total of n = 64 eligible patients were equally randomized to either intervention or control group. patients in the intervention group performed preoperative is training for two days before the scheduled surgery. the primary endpoint was the incidence of in-hospital ppcs. the secondary endpoints were postoperative peak expiratory flow rate (pefr), intensive care unit (icu) stay, hospital length of stay (hlos). results: patients in the intervention group had a lower incidence of postoperative pulmonary complications compared to the control group (12.5% and 21.8%). there was a statistical significant difference between the two groups regarding postoperative pefr, hlos and intensive care unit stay. conclusion: preoperative rehabilitation (involving incentive spirometer is related to a lower incidence of postoperative pulmonary complications for the group that received the intervention). training with preoperative incentive spirometer resulted in a shortening of hospital length of stay and intensive care unit (icu) stay. trial registration: the study was registered in the iranian clinical trial registry, this at https://www.irct.ir/ reference number (irct20211224053503n1). keywords: incentive spirometer, breathing exercises, postoperative pulmonary complications cardiac surgery issn 2413-0516 summary statement what is already known about this topic? • the use of is in the postoperative period helps preventing pulmonary complications after major surgeries. • the evidence supporting the inclusion of preoperative breathing exercises to prevent ppc, is inconclusive. what this paper adds? • the use of preoperative training using flow-oriented incentive spirometer for two days, had better outcomes to prevent postoperative pulmonary complications following open heart surgeries. • preoperative training using flow-oriented incentive spirometer for two days improves patients’ outcomes. • use of is as a routine care in the preoperative period shortens overall hospital stay. background and aim cardiac surgery is considered a high-risk procedure in which a multidisciplinary team is required to manage patients throughout the whole operation phases. when performing a cardiac surgery, focused attention and maintenance are required since the respiratory system is related in both anatomic structure and physiological function.1 open heart surgery can cause various cardiovascular, renal, mental, and infectious complications. however, the occurrence of pulmonary complications remains one of the most challenging difficulties due to the consequential association with increased morbidity and mortality.2 the incidences of developing ppc among cardiac surgeries patients range from 10% to 25%, approximately 2% to 5% patients are likely to experience severe pulmonary dysfunction, such as acute respiratory distress syndrome (ards). patients undergoing cabg surgery are more likely to develop ppc, the incidence rate ranges from 30% to 60%, which contributes to increased morbidity, mortality and length of hospitalization that impose an economic burden on the health care system.3-5 in iraq it was reported that 56% of patients’ experience hypoxia as an early complication within the first 48 hrs. following cabg surgery.6 recently, mohammed and nadr,7 reported that 14.2% of iraqi patients undergoing cabg develop ppcs including chest infections and atelectasis. the implementation of breathing exercises for cardiac surgery patients is now a widely established kind of nurse-led intervention. these exercises are designed for both the preoperative and the immediate postoperative periods in an effort to lower the likelihood of developing postoperative pulmonary complications (ppc), functional capacity impairment, and prolonged hospitalization.8 is is a device that achieves sustained maximal inspiration through predetermined flow or volume. it provides a visual feedback when the patient inhales and sustains the inflation for minimally 5 seconds, it is commonly used in the perioperative care in particular for abdominal and cardiothoracic surgeries as a preventive maneuver for ppcs. usually, it is nurses 271j contemp med sci | vol. 8, no. 4, july-august 2022: 270–277 j.n. faleh et al. original preoperative incentive spirometer to prevent postoperative pulmonary and respiratory therapists’ responsibility to instruct patients about the correct method of training using this mechanical device.9-11 this study aims to answer whether the preoperative use of an incentive spirometer reduces the occurrence of postoperative pulmonary problems and improve peak expiratory flow rate (pefr), intensive care unit (icu) stay, hospital length of stay (hlos) for adult patients undergoing open heart surgeries? materials and methods study design an interventional, prospective, randomized, controlled single-blinded parallel-group multi-centric study. participants and study design the study recruited adult patients who were scheduled to undergo coronary artery bypass grafting (cabg) surgery, cabg combined with valve repair, or valve replacement/ repair surgeries. exclusion criteria of the study included: uncooperative patients, patients who lack intellectual capacity to demonstrate proper use of incentive spirometer, cases in which incentive spirometer is contraindicated such as thoracic, abdominal, or cerebral aneurysms (risk of rupture due to increased thoracic pressure), recent cardiothoracic or abdominal surgery. the sample consisted of 64 patients. these patients were equally allotted to the control group and intervention group. the sample size was calculated according to a-priori sample sizes for student t-tests. settings of the study data were collected from january 9th to april 1st at three different centers in baghdad, iraq: (the iraqi center for heart diseases (ichd) of the medical city directorate, ibn al-bitar center for cardiac surgery of the karkh health directorate, ibn al-nafees cardiology hospital of the rusafa health directorate). intervention eligibility was determined using the inclusion and exclusion criteria of the sample. patients were randomly assigned to a study group (sg) who received preoperative incentive spirometer training for two days before the surgery and a control group (cg), who did not receive the intervention. both groups received same postoperative deep breathing exercises, directed cough, early mobilization, and optimal analgesia. upon signing the consent forms, patients allocated to the intervention group received preoperatively individualized training, they were asked to maintain a sitting position on the edge of bed, hold the is in an upright position, place the lips tightly around the mouthpiece and perform slow and deep inhalation, allow the first ball in the 600 cc chamber to rise to the top, enhance breathing by allowing the second ball in the 900 cc chamber to rise to the top, keep enhance breathing to set the target of 1.200 cc. at maximum inhalation, the mouthpiece is removed, followed by a breath-hold and normal exhalation allowing the three balls to fall to the bottom of the column. after ensuring proper demonstration of steps, patients were asked to repeat the maneuver in a set of ten times hourly while awake until the day of the surgical operation. outcome measures the incidence of postoperative pulmonary complications throughout the in-hospital postoperative period was the primary outcome measure, they were scored by the attending physician who is blinded to participants’ allocation. secondary outcomes included, baseline peak expiratory flow rate which was recorded preoperative and then reassessed on the sixth postoperative days respectively by using truzone® peak flow meter which is a tool that measures peak flows from 60 liters per minute (lpm) to 800 lpm at fastest speed at which air is forced out of lungs after taking a deep breath. length of postoperative hospital and length of icu stay. randomization and blinding simple randomization technique was used for the assignment of subjects into a particular group, flipping a coin method was chosen the side of the coin (i.e., heads control, tails intervention) determines the assignment of each subject. the study is single-blinded, since the participants are intentionally kept unaware of which of the two groups they have been assigned to. statistical methods data were analyzed through the use of ibm-statistical package for social sciences (spss) version 24. paired sample t-test (used to measure the difference between study and control groups in regard to their postoperative outcomes. effect size (cohen’s d test) used to determine the level of impact that the pre-operative incentive spirometer exercise on the postoperative outcomes of patients. ethical considerations and official agreements upon submission of the study protocol ethical approval was sought and granted from the institutional review board (irb)scientific committee of the nursing faculty, university of baghdad. the researcher submitted a detailed description about the study, including problem statement, objectives and questionnaire to the ministry of planning (central statistical organization) and to the medical city directorate, the karkh health directorate, the rusafa health directorate (human development and training center), in order to obtain an official permission to carry out the study. clinical registration upon approval from the committees, the study was registered for iranian clinical trial registry, this at https://www.irct.ir/ reference number (irct20211224053503n1). date of enrollment: 4/01/2022. results the total numbers of patients allocated in the study was (84) figure 1. of the 84 eligible patients; 20 were excluded for table 1. minimum sample size determination parameter of calculating the minimum sample size selected values anticipated effect size (cohen’s d): 0.5 desired statistical power level: 0.8 probability level: 0.05 272 j contemp med sci | vol. 8, no. 4, july-august 2022: 270–277 preoperative incentive spirometer to prevent postoperative pulmonary original j.n. faleh et al. various reasons including (postponement or cancellation of surgery, patients’ infection with covid-19, missing data). a total sample of 64 patients were therefore considered in the final analysis. the first group of 32 patients was the intervention group and the second group of 32 patients was the control group table 1. the study group that received the intervention consisted of 28 males (87.5%) and 8 females (12.3%). in which (65.6%) of them were within the age range (50–60) years old. regarding smoking status those who were identified as light smokers and heavy smokers had the highest percentages of (31.3%) for both. more than a half of patients’ (53.1%) were considered to be overweight. the control group consisted of 24 males (75%) and 8 females (25%). less than two thirds (62.5%) of the group were table 1. descriptive statistics of patients’ demographic and lifestyle data variables study group n (%) n = 32 control group n (%) n = 32 age 50–60 years old 21 (65.6%) 20 (62.5%) gender male 28 (87.5%) 24 (75.0%) female 4 (12.5%) 8 (25.0%) smoking status (pack/year) never smoked (0.0 pack-year) 5 (15.6%) 10 (31.3%) light smoker (0.1–20.0 pack-year) 10 (31.3%) 4 (12.5%) moderate smoker (20.1–40.0 pack-year) 7 (21.9%) 7 (21.9%) heavy smoker (> 40 pack-year) 10 (31.3%) 11 (34.4%) body mass index (bmi) underweight – bmi < 18.5 kg/m2 2 (6.3%) 1 (3.1%) overweight – bmi ≥25 to 29.9 kg/m2 17 (53.1%) 16 (50.0%) obesity – bmi ≥30 kg/m2 13 (40.6%) 15 (46.9%) fig. 1 consort study flow chart. within the age range (50–60) years old. the results showed that (34.4%) of the control group patients were identified as heavy smokers. regarding the patients’ bmi, half of the control group (50%) were overweight. the underlined numbers in table 2 represent the highest percentages of the selected variables. in the study group, less than two thirds of the patients (62.5) had undergone cabg surgeries. all of the surgeries were elective with a cardio pulmonary bypass (cbp) time of less than two hours for half of them and an operative time of 6 hours for (81.3%) in both groups. more than half (59.4%) of the control group had undergone cabg surgery, the majority (87.5) of the procedures were elective with a cpb time of (2–4) hours for more than half (56.3) of the patients. peak expiratory flow rate (pefr), hospital length of stay (hlos) the https://doi.org/10.22317/jcms.v8i4.1261. a paired sample t-test was conducted to compare the differences between pre-operative pefr between the two groups. there was a statistically significant difference in the pre-operative peak expiratory flow rates (m = 40.00000, sd = 107.42589, t (31) = 2.106, p = .043). there was a statistically significant difference in the postoperative peak expiratory flow rates between control group and study group (m = 75.78125, sd = 81.43976, t (31) = 5.264, p = .0001. the effect size for this analysis (d = 0.93) was found to exceed cohen’s (1988) convention for a large effect (d = .80). there was a statistically significant difference in the number of days’ patients spent in the icu setting between control group and study group (m = –.56250, sd = .91361, t (31) = –3.483, p = .002). the effect size for this analysis (d = 0.61) was found to be a medium effect size. there was a statistically significant difference in the number of days’ patients spent in the hospital from postoperative day zero between control group and study group (m = –1.37500, sd = 1.87943, t (31) = –4.139, p = .0001, the effect size for this analysis (d = 0.73) was found to be a medium effect size. discussion the study result showed that (65.6%) of the study group and (62.5%) of the control group were within the age group (50– 60) years old at the time of data collection. mohammad et al.12 273j contemp med sci | vol. 8, no. 4, july-august 2022: 270–277 j.n. faleh et al. original preoperative incentive spirometer to prevent postoperative pulmonary table 2. descriptive statistics of patients’ clinical and surgical data variables study group n = 32 control group n = 32 current surgical procedure cabg 20 (62.5%) cabg 19 (59.4%) mvr 6 (18.8%) mvr 7 (21.9%) avr 4 (12.5%) avr 4 (12.5%) cabg & mvr 1 (3.1%) tvr 1 (3.1%) cabg & avr 1 (3.1%) cabg & dvr 1 (3.1%) operative time 5 hours 4 (12.5%) 5 hours 4 (12.5%) 6 hours 26 (81.3%) 6 hours 26 (81.3%) 7 hours 2 (6.3%) 7 hours 2 (6.3%) cardiopulmonary bypass time (minutes) less than 2 hours 16 (50.0%) less than 2 hours 12 (37.5%) 2–4 hours 15 (46.9%) 2–4 hours 18 (56.3%) more than 4 hours 1 (3.1%) more than 4 hours 2 (6.3%) cabg: coronary artery bypass grafting, avr: aortic valve replacement, mvr: mitral valve replacement, dvr: double valve replacement, tvr: tricuspid valve repair. table 3. descriptive statistics of patients’ clinical outcomes variables study group (n = 32) n (%) control group (n = 32) n (%) pre-operative pefr below normal (male = < 450 l/min) (female = < 320 l/min) 31 (96.9%) 31 (96.9%) post-operative pefr below normal (male = < 450 l/min) (female = < 320 l/min) 32 (100.0%) 32 (100.0%) post-operative pulmonary complications atelectasis 4 (12.5%) 5 (15.6%) tracheobronchitis – 1 (3.1%) pleural effusion – 1 (3.1%) icu length of stay 2 days 28 (87.5%) 19 (59.4%) hlos 8 days 14 (43.8%) 8 (25.0%) reported that coronary artery disease (cad) tends to occur earlier in iraqi population. this finding may be attributable to the lifestyle profile of the participants including their propensity to smoke, poor dietary habits, and low levels of health literacy.13 the majority of the sample were males with a percentage of (87.5%) for the study group and (75.0%) for the control group. this result is supported by jassim et al.6 this gender difference could be attributed to many etiologies, including limited tobacco use among iraqi females and dietary habits. coronary artery disease (cad) and the occurrence of a woman’s first acute myocardial infarction occur several years later in women than in men, before menopause, the well-known biological defense that women have against cad can postpone the onset of cad symptoms by more than ten years.14 the study findings showed that half of the study sample was overweight with a bmi of ≥25 to 29.9 kg/m2, which is similar to the results of the study conducted by alam et al.15. these results are not surprising, because being overweight and obese are closely associated to both known and emerging risk factors for cardiovascular disease.16 regarding smoking status, the highest percentages in the study group were heavy smoker and light smokers with (31.3%) respectively, whereas, (34.3%) of the control group were heavy smokers, this result is consistent with the results of sharif-kashani et al.17 where 43.7% of patients had a cumulative tobacco exposure of >40 pack years. these findings own up to the fact that smoking contributes significantly to premature coronary atherosclerosis and to the acceleration of atherosclerosis by increasing the oxidation of low-density lipoprotein (ldl) and impairing coronary endothelial vasodilation.18 as for the performed surgical operation, 62.5% of the study group and 59.4% of the control group had underwent coronary artery bypass grafting (cabg), this result is similar to.19 with regard to cardiopulmonary bypass time (cpb), half of the study group had cpb time of less than 2 hours (> 120 minutes) similar to faritous et al.20 this difference in cpb time between the two groups could be attributed to intraoperative encountered complications possibly occurred to the control group. the majority of the study sample of both group had an operative time of 6 hours, unlike a study conducted by matsuura et al.,21 where 109 patients out of 149 had an operative time of 480.90 ± 161.20, which exceeded the operative time in the present study. the patients involved in the aforementioned study may had more complex surgeries that required more time compared to the patients in the present study. 274 j contemp med sci | vol. 8, no. 4, july-august 2022: 270–277 preoperative incentive spirometer to prevent postoperative pulmonary original j.n. faleh et al. table 4. difference in pre-operative pefr between study and control groups paired differences preoperative pefr (study group) preoperative pefr (control group) mean difference std. deviation t df sig. (2-tailed) 40.00000 107.42589 2.106 31 .043 table 5. difference in post-operative pefr between study and control groups paired differences effect size postoperative pefr (study group) postoperative pefr (control group) mean difference std. deviation t df sig. (2-tailed) 75.78125 81.43976 5.264 31 .0001 0.93 effect sizes = small (d = 0.2), medium (d = 0.5), and large (d = 0.8). fig. 2 the mean plot shows that pefr decreased in the postoperative period compared to the baseline values in both the study group and control group. however, the study group had better values in the postoperative period. postoperative pulmonary complications the incidence of postoperative pulmonary complications in those who received the intervention and those who did not was (12.5% and 21.8%) respectively, these findings did not differ from the results of chen et al. (2019),22 in which the incidence of postoperative pulmonary complications was 10.2% in the study group and 27.3% in the control group. another study confirmed these results where atelectasis was 14.10% in the study group and 27.10% in the control group.23 in contrast, moradian et al.24 reported that the incidence of postoperative atelectasis was the same in both groups with 20% incidence rate in each group. this difference in the presented results may be due to the fact that the control group in the aforementioned study had received conventional physiotherapy which contributed to insignificance unlike the control groups of the current study and those that had similar results to it, who did not receive any form of physiotherapy or pre-rehabilitation in the preoperative phase. the most prevalent complication in the current study was atelectasis with an overall incidence rate of (28.1%) for both groups compared to lower incidence in hijas-gómez et al.25 who reported an overall incidence rate of (5.9%). it is possible that the decreased incidence rate was due to the fact that postoperative patients in the aforementioned study have received physiotherapy. patients’ clinical outcomes tables 4, 5 and figure 2 represent the differences between the two groups in terms of peak expiratory flow rates, which demonstrates that there was a statistically significant difference in the postoperative peak expiratory flow rates p = .0001 unlike the results of herdy et al.,26 who found that before discharge, pefrs returned to baseline values in the rehabilitation group (336 ± 105 l/min) but not in the control group (271 ± 132 l/min). the difference between the findings of the present study and those of the aforementioned article is that the pefrs 275j contemp med sci | vol. 8, no. 4, july-august 2022: 270–277 j.n. faleh et al. original preoperative incentive spirometer to prevent postoperative pulmonary fig. 4 the mean plot shows that patients in the control group had longer length of hospital stay when compared to the study group. table 6. difference in icu stay time/days between study and control groups paired differences effect sizeicu stay time/days (study group) mean std. deviation t df sig. (2-tailed) icu stay time/days (control group) –.56250 .91361 –3.483 31 .002 0.61 effect sizes = small (d = 0.2), medium (d = 0.5), and large (d = 0.8). fig. 3 the mean plot indicates that patients in the control group spent more days in the icu following surgery when compared to the study group. table 7. difference in hospital length of stay/days between study and control group paired differences effect sizehospital length of stay (study group) mean std. deviation t df sig. (2-tailed) hospital length of stay (control group) –1.37500 1.87943 –4.139 31 .0001 0.73 effect sizes = small (d = 0.2), medium (d = 0.5), and large (d = 0.8). values of the patients in the present study were already below the normal values in the preoperative period, and despite the significant difference between the two group none of them returned to the baseline values after the surgery. the results in table 6 represents the difference between the study group and control group regarding the length of days spent in the intensive care unit (icu) setting which shows that the use of preoperative is has decreased the icu stay in the study group compared to the control group as shown in figure 3, a previous study conducted in turkey supported these results as they stated that preoperative inspiratory muscle training reduced the icu stay.27 regarding hospital length of stay, as shown in table 7 and figure 4, there was a statistically significant reduction in the number of days the study group spent in the postoperative period (p = .0001), which indicates that the intervention had a 276 j contemp med sci | vol. 8, no. 4, july-august 2022: 270–277 preoperative incentive spirometer to prevent postoperative pulmonary original j.n. faleh et al. positive effect on reducing hlos, supporting this conclusion, multiple clinical trials have demonstrated that preoperative respiratory physiotherapy utilizing various maneuvers is effective in reducing hospitalization for patients undergoing cardiac surgeries.28-31 conclusion this study showed that preoperative rehabilitation involving incentive spirometer is related to a lower incidence of postoperative pulmonary complications for the group that received the intervention (21.9% vs. 12.5%) a difference which may be considered relevant. training with preoperative incentive spirometer resulted in a shortening of hospital length of stay and intensive care unit (icu) stay among patients in the intervention group than those in the control group. this, in turn, results in the efficient use of the limited resources and the reduction of health-care costs, which supports the well-established concept of “meaningful use” of healthcare resources. recommendations patients undergoing open heart surgery should have free access to incentive spirometers in the surgical units, as well as adequate training and encouragement, since this appears to have prophylactic effect against ppcs. further studies with a larger sample size that specifically target patients with preexisting respiratory diseases such as chronic obstructive pulmonary disease (copd), asthma are required to confirm these findings. future clinical trials should consider double blinding in the recruitment and outcome assessment procedures. patients’ adherence considerations must be integrated into future clinical trials in order to establish a more solid evidence basis and to draw relevant conclusions. limitation the primary limitation was the small sample size which limited the generalizability of the intervention effect. two centers postponed the operations for a duration of one week for each due to rapid spread of coronavirus omicron variant which caused impediment in data collection and time constraints. funding information the budget of this research work was not support by any governmental or non‐governmental organization. the authors of this manuscript covered all the research work‐related expenses. conflicts of interest none.  references 1. tanner tg, colvin mo. pulmonary complications of cardiac surgery. lung. 2020 dec;198(6):889–96. available from: https://doi.org/10.1007/s00408020-00405-7 2. mali s, haghaninejad h. pulmonary complications following cardiac surgery. archives of medical science-atherosclerotic diseases. 2019 dec 31;4(1):280–5. available from: https://doi.org/10.5114/amsad.2019.91432 3. bharathi ar. assess the effectiveness of deep breathing exercise with incentive spirometer on the respiratory status of cardio thoracic and vascular patients. journal of research in medical and dental science. 2021:276-83. available from: https://doi.org/10.5114/amsad.2019.91432 4. fischer mo, brotons f, briant ar, suehiro k, gozdzik w, sponholz c, kirkeby-garstad i, joosten a, neto cn, kunstyr j, parienti jj. postoperative pulmonary complications after cardiac surgery: the venice international cohort study. journal of cardiothoracic and vascular anesthesia. 2022 aug 1;36(8):2344–51. available from: https://doi.org/10.1053/j.jvca.2021.12.024 5. ubben jf, lance md, buhre wf, schreiber ju. clinical strategies to prevent pulmonary complications in cardiac surgery: an overview. journal of cardiothoracic and vascular anesthesia. 2015 apr 1;29(2):481–90. available from: https://doi.org/10.1053/j.jvca.2014.09.020 6. jassim s, ahmed sa, nagi ab. assessment of early and late complication post coronary artery graft by–pass surgery cabg. kufa journal for nursing sciences. 2013;3(1). available from: https://journal.uokufa.edu.iq/index.php/ kjns/article/view/2404 7. mohammed ak, nadr jh. early complications associated with obesity following coronary artery bypass graft surgery. journal of the faculty of medicine. 2021;63(4). available from: https://iqjmc.uobaghdad.edu.iq/ index.php/19jfacmedbaghdad36/article/view/1877 8. rodrigues sn, henriques hr, henriques ma. effectiveness of preoperative breathing exercise interventions in patients undergoing cardiac surgery: a systematic review. revista portuguesa de cardiologia (english edition). 2021 mar 1;40(3):229–44. available from: https://doi.org/10.1097/ aco.0000000000000045 9. restrepo rd, wettstein r, wittnebel l, tracy m. incentive spirometry: 2011. respiratory care. 2011 oct 1;56(10):1600–4. available from: https://doi. org/10.1097/aco.0000000000000045 10. so mw, heo hm, san koo b, kim yg, lee ck, yoo b. efficacy of incentive spirometer exercise on pulmonary functions of patients with ankylosing spondylitis stabilized by tumor necrosis factor inhibitor therapy. the journal of rheumatology. 2012 sep 1;39(9):1854–8. available from: https://doi. org/10.1097/aco.0000000000000045 11. sum sk, peng yc, yin sy, huang pf, wang yc, chen tp, tung hh, yeh ch. using an incentive spirometer reduces pulmonary complications in patients with traumatic rib fractures: a randomized controlled trial. trials. 2019 dec;20(1):1–8. available from: https://doi.org/10.1097/ aco.0000000000000045 12. mohammad am, rashad hh, habeeb qs, rashad bh, saeed sy. demographic, clinical and angiographic profile of coronary artery disease in kurdistan region of iraq. american journal of cardiovascular disease. 2021;11(1):39. available from: https://www.ncbi.nlm.nih.gov/pmc/articles/ pmc8012293/ 13. mousa am, mansour k. effectiveness of an instructional program concerning healthy lifestyle on patients’ attitudes after percutaneous coronary intervention at cardiac centers in baghdad city. iraqi national journal of nursing specialties. 2020 sep 27;33(1):1–1. available from: https://injns.uobaghdad.edu.iq/index.php/injns/article/view/396 14. gheisari f, emami m, raeisi shahraki h, samipour s, nematollahi p. the role of gender in the importance of risk factors for coronary artery disease. cardiology research and practice. 2020 jul 29;2020. available from: https:// injns.uobaghdad.edu.iq/index.php/injns/article/view/396 15. alam m, shehzad mi, hussain s, paras i, kanwal m, mushtaq a. spirometry assessment and correlation with postoperative pulmonary complications in cardiac surgery patients. cureus. 2020 oct 23;12(10). available from: https:// doi.org/10.7759/cureus.11105 16. katta n, loethen t, lavie cj, alpert ma. obesity and coronary heart disease: epidemiology, pathology, and coronary artery imaging. current problems in cardiology. 2021 mar 1;46(3):100655. available from: https://doi. org/10.1016/j.cpcardiol.2020.100655 17. sharif-kashani b, shahabi p, mandegar mh, saliminejad l, bikdeli b, behzadnia n, heydari g, sharifi h, aidanlou s. smoking and wound complications after coronary artery bypass grafting. journal of surgical research. 2016 feb 1;200(2):743–8. available from: https://doi.org/10.1016/j. cpcardiol.2020.100655 18. barua rs, ambrose ja. mechanisms of coronary thrombosis in cigarette smoke exposure. arteriosclerosis, thrombosis, and vascular biology. 2013 jul;33(7):1460–7. available from: https://doi.org/10.1161/ atvbaha.112.300154 277j contemp med sci | vol. 8, no. 4, july-august 2022: 270–277 j.n. faleh et al. original preoperative incentive spirometer to prevent postoperative pulmonary https://doi.org/10.22317/jcms.v8i4.1261 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 19. vitomskyi v, al-hawamdeh k, lazarіeva о, vitomskа m. the efficacy of using tri-ball breathing exerciser in respiratory function recovery of the patients undergoing cardiac surgery. available from: doi:https://doi.org/10.14198/ jhse.2022.172.09 20. faritous z, yaghouti f, ghadrdoost b, heidarpour e, ziyaeifard m. transfusion of blood components and postoperative outcomes in patients undergoing cardiac surgery. iranian heart journal. 2020 apr 1;21(2):64–70. available from: http://journal.iha.org.ir/ article_105529_8d00957b143c9d7b22b909c7143b57ca.pdf 21. matsuura y, kamidaira m, tamura a. risk factors for postoperative delirium after cardiac surgery. int arch nurs health care. 2018;4:103. available from: https://doi.org/10.23937/2469-5823/15100103 22. chen x, hou l, zhang y, liu x, shao b, yuan b, li j, li m, cheng h, teng l, guo m. the effects of five days of intensive preoperative inspiratory muscle training on postoperative complications and outcome in patients having cardiac surgery: a randomized controlled trial. clinical rehabilitation. 2019 may;33(5):913–22. available from: https://doi.org/10.23937/2469-5823/15100103 23. gilani sr, hussain g, ahmad n, baig ma, zaman h. comparison of post-operative atelectasis in patients undergoing coronary artery bypass grafting with and without pre-operative incentive spirometry. journal of postgraduate medical institute. 2016 apr 28;30(2). available from: https:// applications.emro.who.int/imemrf/j_postgrad_med_inst/j_postgrad_ med_inst_2016_30_2_169_172.pdf 24. moradian st, heydari aa, mahmoudi h. what is the role of preoperative breathing exercises in reducing postoperative atelectasis after cabg?. reviews on recent clinical trials. 2019 dec 1;14(4):275–9. available from: https://doi.org/10.2174/1574887114666190710165951 25. hijas-gómez ai. preoperative respiratory physiotherapy and postoperative complications following valve replacement surgery. ec pulmonology and respiratory medicine. 2017;4:92–100. available from: https://www.semanticscholar.org/paper/preoperative-respiratoryphysiotherapy-and-valve-hijas-g%c3%b3mez-andr%c3%a9s-prado/ a503fca480819c9c4531223a2cc6e653801bd95e 26. herdy ah, marcchi pl, vila a, tavares c, collaço j, niebauer j, ribeiro jp. preand postoperative cardiopulmonary rehabilitation in hospitalized patients undergoing coronary artery bypass surgery: a randomized controlled trial. american journal of physical medicine & rehabilitation. 2008 sep 1;87(9):714–9. available from: doi: 10.1097/phm.0b013e3181839152. pmid: 18716482. 27. savci s, degirmenci b, saglam m, arikan h, inal-ince d, turan hn, demircin m. short-term effects of inspiratory muscle training in coronary artery bypass graft surgery: a randomized controlled trial. scandinavian cardiovascular journal. 2011 oct 1;45(5):286–93. available from: doi: 10.1097/phm.0b013e3181839152. pmid: 18716482. 28. hulzebos eh, helders pj, favié nj, de bie ra, de la riviere ab, van meeteren nl. preoperative intensive inspiratory muscle training to prevent postoperative pulmonary complications in high-risk patients undergoing cabg surgery: a randomized clinical trial. jama. 2006 oct 18;296(15): 1851–7. available from: doi: 10.1001/jama.296.15.1851. 29. nardi p, pellegrino a, pisano c, vacirca sr, anselmi d, saulle s, dandi r, romano a, servadio a, gianlorenzi a, ruvolo g. the effect of preoperative respiratory physiotherapy and motor exercise in patients undergoing elective cardiac surgery: short-term results. kardiochirurgia i torakochirurgia polska/polish journal of thoracic and cardiovascular surgery. 2019 jul;16(2):81–7. available from: doi: 10.1001/ jama.296.15.1851. 30. shahood h, pakai a, rudolf k, bory e, szilagyi n, sandor a, zsofia v. the effect of preoperative chest physiotherapy on oxygenation and lung function in cardiac surgery patients: a randomized controlled study. annals of saudi medicine. 2022 jan;42(1):8–16. available from: https://doi. org/10.5144/0256-4947.2022.8 31. sobrinho mt, guirado gn, silva ma. preoperative therapy restores ventilatory parameters and reduces length of stay in patients undergoing myocardial revascularization. brazilian journal of cardiovascular surgery. 2014 apr; 29:221–8. available from: https://doi.org/10.5935/16789741.20140021. 259j contemp med sci | vol. 8, no. 4, july-august 2022: 259–263 original a comparable genetic diversity between chicken ecotypes of different zones using dna barcoding ayman sabry1,4,*, alaa ahmed mohamed1,6, mohamed hassan1,6, salah e. m. abo-aba2,3,5 1biology department, faculty of science, taif university, taif, saudi arabia. 2department of biological sciences, faculty of science, king abdul-aziz university 21589, jeddah, saudi arabia. 3princess doctor dr. najla bint saud al saud center for distinguished research in biotechnology, jeddah, saudi arabia. 4cell biology department, national research center, dokki, giza, egypt. 5microbial genetic department, genetic engineering & biotechnology division, national research center, dokki, giza, egypt. 6department of genetics, faculty of agriculture, minufiya university, al minufiyah, egypt. correspondence to: ayman sabry (e-mail: amsabry@gmail.com ) (submitted: 22 december 2021 – revised version received: 14 january 2022 – accepted: 25 january 2022 – published online: 26 august 2022) abstract objectives: the purpose of the current study was to verify the reliability of coi bar-codes in the assessment of genetic diversity of two ecotypes from different ecozones. methods: the dna sequences of cytochrome oxidase i (coi) barcodes of 50 hens belonging to two ecotypes of ismalia egypt (ism) and taif saudi arabia (ta) were isolated and analyzed. results: this study results showed that no noticeable great differences among all barcode’s sequences of both ecotypes. the average length of both ecotypes was 589 bp. ism ecotypes have a relatively wider length range. the overall mean of gc% content was 48 ± 0.01. both ecotypes have the same number of sites 548 bp. ism ecotype has 523 monomorphic sites whereas ta ecotype has slightly fewer monomorphic sites 517. the ism ecotype has 7 singleton sites and 18 parsimony informative sites. ta ecotype has little more polymorphic, that is 12 singleton sites and 19 parsimony informative sites. the number of mutations (η) was larger in ism (46) compared to 38 mutations for ta ecotype. both ecotypes had the same number of haplotypes (25), and haplotypes diversity (1) as well as the variance of haplotype diversity. conclusion: these results indicated a comparable level of genetic diversity of both ecotypes, which in turn may refer to a similarity of evolutionary forces that affect both ecotypes. based on the present results, coi gene can be used in barcoding. the coi provides an objective the foundation for identification of ecotypes and therefore could be used for a rapid establishment of a variety of identifications. key words: dna barcoding, haplotype diversity, chicken ecotypes issn 2413-0516 introduction in many economically developing nations chickens’ ecotypes (gallus gallus domesticus) are an important asset for rural smallholders. this importance is owing to the fact of limited production inputs, scavenging competency (i.e. birds get the foremost part of their daily ration by scavenging) as well as acclimatization to tough and exhausting environmental circumstances.1,2 for example, in most of africa rural poultry (e.g. ecotypes) alone supplies 70% of poultry goods and 20% of animal protein intake.3 ecotypes are outputs of years of natural selection under stringent conditions, consequently, ecotypes turned out to be immune to many diseases especially bacterial, and protozoic as well as endoparasites and ectoparasites. these ecotypes survive better than the commercial hybrid strains under such harsh production conditions. still, ecotypes are characterized by its low egg productivity and light mature body size.4,5 on the other hand, the world-wide poultry industry is strategically based on commercial chicken breeds. these commercial breeds are a product of a small number of chicken genotypes. such strategy has a drawback effect of casting away ecotypes. therefore, ecotypes are negatively selected regardless their good quality of egg and meat, disease resistance as well as adaptation to local environment. as a consequence, these ecotypes are under threat of extinction. setting up of frame for conserving these genetic resources is of massive need.1,6–9 many studies addressed the genetic makeup of ecotypes. msoffe et al., 2001;10 tadelle et al., 2003;11 muchadeyi et al., 200712 and rudresh et al., 201513 and have been used as an example in biodiversity studies.14 at taif governorate (≈ 1.7 km above sea level) indigenous chickens are adapted to the coarse environment of high altitude. this coarse environment is known for extraordinary natural conditions, such as low air oxygen percentage, partial reduction of oxygen pressure, as fluctuated daily temperature.15,16 these indigenous chickens are an outcome of many years of acclimatization which in turn are representing a valuable genetic resource. mitochondrial dna mtdna (aka dna barcoding) has been widely used to differentiate among and within species.17,18 dna barcoding is a short string of uniform genomic region and each type has a specific barcode. dna barcoding is based on the principle that determining a specific sequence for a particular gene that distinguishes between individuals of a species because of the genetic variation between species exceeds the genetic variation within a species.19 the genes most commonly used for species identification are cytochrome b (cyt b).20 it has also been reported that the mtdna cytochrome c oxidase i (coi) gene could be used as barcoding for most animals and fishes.20 the expected growth in coi data recently has led to the use of a dedicated barcoding section to propagate the coi sequence, paving the way for the coi gene to become a major tool for taxonomic identification. according to recent reports, approximately 600 base pairs (bp) part of the 1 subunit of mitochondrial cytochrome oxidase (coi) could be a good choice for the coding gene because it may be involved in most animal species (roe & sperling, 2007;21 bondoc & santiago, 201222) used coi to differentiate 31 domestic chicken breeds and strains (gallus gallus domesticus) and 25 red jungle fowls (gallus 260 j contemp med sci | vol. 8, no. 4, july-august 2022: 259–263 a comparable genetic diversity between chicken ecotypes of different zones using dna barcoding original a. sabry et al. gallus philipensis hatchisuka) in the philippines. results of this study indicated that use of dna barcodes can effectively distinguish chicken breeds and strains, but not differentiate nor identify commercial hybrid chicken. osaman et al. (2016)23 devised the complete sequence of mitochondrial dna d-loop to clarify the origin of egyptian native chicken and asian chicken. results of this study revealed that both egyptian native chicken and west and central asian chicken are sharing the same common ancestor as they cluster together in the same clade. these results imply that both saudi and egyptian native breeds are genetically closer to each other. inspired by the outcomes of osaman et al. (2016),23 the purpose of the current study was to verify the reliability of coi bar codes in identifying native saudi chickens. in this study, differences in the selected coi gene and population genetic structure of four local saudi chicken breeds were investigated. the genetic diversity of these chicken breeds has also been studied using the coi gene as a dna barcode. materials and methods collection of blood samples & dna isolation fifty blood samples of native chicken from two locations (ismailia, egypt and taif, saudi arabia) were collected (twenty five of each). then, the genomic dna was isolated from each blood sample using qiagen dnase kit (germany) as described by the producer’s directions khan et al., 2019.24 dna samples were stored at –20oc for use after concentration test with uv spectrophotometer. coi gene of mtdna amplification a total of 415 bp of coi gene of mtdna was amplified using two universal primer sets (birdf1 and birdr1 according to amer et al. (2013).25 the sequence of the forward primer was 5ʹ-ttctccaaccacaaagacattggcac-3ʹ while that of the reverse primer was 5ʹ-acgtgggagataattccaaatcctg-3ʹ. the reaction was performed in 50 µl of total volume consisting of 12.5 µl gotaq buffer master mix from promega (usa), 25 ng of template dna, 0.5 µl of each amplification primers and up to a final desired volume with deionized distilled water. the pcr thermocycler protocol was achieved as reported previously.26 sequencing, purification, and data analysis cycle sequencing of all samples was carried out in a total reaction volume of 20 µl using bigdye® terminator v3.1 cycle sequencing ready reaction-100 mix (thermo fisher scientific, applied biosystems), bigdye®.terminator v1.1 and v3.1 × 5 sequencing buffer (thermo fisher scientific, applied biosystems), forward/reverse primers, and 50–70 ng/µl purified pcr product (gel extracted dna) on 2720 thermal cycler (applied biosystems). cycle sequencing conditions consisted of 95 for 5 min, followed by 32 cycles of 95°c for 20s, at 55°c for 15s, and at 60°c for 4 min. all sequenced reactions were purified using zymo research dna sequencing clean-up tm kit (the epigenetics company, usa) and sequenced by capillary electrophoresis on an automated dna sequencer (abi prism 3500 genetic analyzer). all the raw sequences were curated and assembled using bioinformatics tools, namely, sequencing analysis 5.2 (thermo fisher scientific, applied biosystem, india) and clone manager suite 9 (sci ed software, westminster, colorado, usa). all the consensus sequences were then aligned and trimmed using bioinformatics software, namely, clustalw and bio edit sequence alignment editor for the haplotyping analysis. the haplotyping was done using bioinformatics software dnasp v6.12.03,27 considering g. gallus as reference. 20 µl using bigdye® terminator v3.1 cycle sequencing ready reaction-100 mix (thermo fisher scientific, applied biosystems), bigdye®. terminator v1.1 and v3.1 × 5 sequencing buffer (thermo fisher scientific, applied biosystems), forward/reverse primers, and 50–70 ng/µl purified pcr product. results and discussion the dna sequences of cytochrome c oxidase i (coi) barcodes of 50 hens belonging to two ecotypes of ismalia egypt (ism) and taif saudi arabia (ta) were analyzed. figure 1 shows the base frequencies of all sequenced barcodes. no great differences were noticed among all barcodes of all ecotypes. fig. 1 base frequencies of ismailia (isa) and taif (ta) ecotypes. 261j contemp med sci | vol. 8, no. 4, july-august 2022: 259–263 a. sabry et al. original a comparable genetic diversity between chicken ecotypes of different zones using dna barcoding fig. 2 boxplot of sequence length and percentage of gc content in ismailia (isa) and taif (ta) ecotypes. table 1. number of sites, monomorphic and polymorphic sites for ism and ta ecotypes number of sites monomorphic sites polymorphic sites singleton sites parsimony informative ism 548 523 7 18 ta 548 517 12 19 coi sequence lengths and percentages of gc content for both ecotypes are presented in figure 2. results showed that the average length, after deletion of the primers’ sequences, for both ecotypes was 589 bp. although ism ecotypes have a relatively wider length range (563–611 bp) compared to ta ecotype (570–607 bp). the average sequence length was shorter than what was reported by xun-he et al. (2016)28 on chinese indigenous chickens, wild jungles and mallard (695) as well as, peng et al. (2019)29 on chinese local and imported chicken breeds (650 bp). cui et al. (2017)30 analyzed bar1 and bar2 of coi barcodes sequence for 4 different chinese native chicken breeds (16 individuals/breed), sequence length averaged 590 bp and 624 bp. dave et al. (2021)31 found that the sequence length of cytochrome c oxidase subunit i length for two native indian chicken breeds averaged 608 and 756 bp. however, it is also important to note that due to the fact that the molecular evolution rate fluctuate among various segments of the genome and across taxa, there no species-specific standard sequence length.32 the overall mean of gc percentage was 48 ± 0.01. the range of gc% for ism (45–51) was slightly wider in comparison with ta (45–50). to our surprise, both ecotypes have the same number of sites 548 bp (table 1). nevertheless, ism ecotype has 523 monomorphic sites where ta ecotype has slightly fewer monomorphic sites 517. the ism ecotype has 7 singleton sites and 18 parsimony informative sites. the ta ecotype has little more polymorphic, that is 12 singleton sites and 19 parsimony informative sites. these results might indicate ta ecotype had a slightly higher genetic diversity. nevertheless, our findings are much higher than what reported by huang et al. (2016b)28 on 203 individuals of 10 indigenous chinese chicken breads who reported only 110 sites of which 90 were singleton variable sites and the remaining 20 were parsimony informative sites. although the number of sties reported by huang et al. (2016b)28 were very much lower than what were reported in the present study, which might be ascribed to the larger number of breeds that used by huang et al. (2016b),28 but the number of parsimony informative sites were relatively close. though, cui et al. (2017)30 reported only 4 polymorphic sites on four chinese native breeds. dave et al. (2021)31 found that the total number of sites of two native indian chicken populations was 596 and 647 of which merely 3 and 4 polymorphic sites were detected. yu-shi et al. (2011)33 on 26 individuals of two newly discovered chicken breeds found only 10 variation sites, of which only 6 sites were single polymorphism sites while 4 were simple information sites. parameters of dna polymorphism for ism and ta ecotypes are shown in table 2. the number of mutations (η) was larger in ism ecotype (46) compared to 38 mutations for ta ecotype. however, huang et al. (2016b)28 found that (η) ranged from 11 to 22 mutations sites in 10 chinese indigenous chickens’ breeds. this smaller number of mutations sites might be attributed to smaller number of sample size in that study, as sample size ranged from 18 to 23. haplotype diversity (hd) and nucleotide diversity (π) are both of principle importance for assessment of genetic diversity of either population or breed.31 haplotype diversity implies the distinctiveness of a certain haplotype in a particular population. therefore, the higher hd’s mean and π the richer genetic diversity in population. haplotype diversity refers to the presence of specific haplotype in a particular population (yushi et al., 2011;33 cui et al., 201730). both ecotypes had the same number of haplotypes (h = 25), and haplotypes diversity (hd = 1) as well as variance of haplotype diversity (table 2). these results indicated to a comparable level of genetic diversity of both ecotypes, which in turn may refer to a similarity of evolutionary forces that affecting both ecotypes. cui et al. (2017)30 found that number of haplotypes only 262 j contemp med sci | vol. 8, no. 4, july-august 2022: 259–263 a comparable genetic diversity between chicken ecotypes of different zones using dna barcoding original a. sabry et al. ranged from 2 to 8 in two chinese native chicken breeds, while yu-shi et al. (2011)33 found seven kinds of haplotypes on newly discovered chinese chicken breeds. huang et al. (2016b)28 defined 84 different haplotypes on from 203 individuals of 10 indigenous chickens in china. our estimates of hd were higher than what reported by huang et al. (2016b)28 (0.83), cui et al. (2017)30 (0.84) where yu-shi et al. (2011)33 found hd ranged from 0.3 to 0.9. on native indian breeds, dave et al. (2021)31 estimated hd of 0.34 and 0.93, and yu-shi et al. (2011)33. table 3 shows nucleotide diversity (π), the average number of nucleotides differences (k), the total variance of nucleotide differences (free mutations) for ism and ta ecotypes. again, no differences were noticed between these two ecotypes for these three parameters. once more, the equality of these parameters is an emphasis of what we stated earlier of similarity of evolutionary forces that affect both ecotypes regardless of the ecozones. the estimates of the present study were higher than what yu-shi et al. (2011)33 estimated on newly discovered chinese chicken breeds (0.004). our estimates for (π) were moderate to what was reported by dave et al. (2021)31 (0.228 & 0.0023), as well as cui et al. (2017)30 on chinese native chicken breeds, where chin π ranged from 0.00102 to 0.00305. table 4 shows the start and end of conserved regions of both ism and ta ecotypes as well as the conservation, homozygosity, and p-value of each conserved region. conservation is measured as the proportion of conserved sites in the alignment region, homozygosity is defined as (1-heterozigosity), where p-value was estimated under the hypergeometric distribution.34 the ism ecotype has 2 conservative regions compared to only one for ta ecotypes. no differences were found in conservation value for all the conservation regions. table 3. nucleotide diversity, average number of nucleotide differences (k), total variance of nucleotide differences (free mutations) for ism and ta ecotypes nucleotide diversity n average no. of nucleotide differences (k) total variance of k (free recombination), v(k) ism 0.02 8.4 3.0 ta 0.02 8.3 3.0 table 2. parameters of dna polymorphism in ism and ta ecotypes no. of mutations n no. of haplotypes (h) haplotype (gene) diversity, (hd) sd haplotype diversity ism 46 25 1 0.01 ta 38 25 1 0.01 table 4. conserved regions, conservation, homozygosity and p-values of ism and ta ecotypes start-end conservation homozygosity p-value ism 1–215 1.0 1.0 <0.001 217–488 1.0 1.0 <0.001 ta 1–495 1.0 1.0 <0.001 equal estimates of conservation and homozygosity values were attained for all conservation regions in both ecotypes (1.0). all p-value were smaller than 0.05 ranged from <0.001 to 0.04. conclusion this is the first diversity study to use coi markers of two ecotypes in egypt and saudi arabia. based on the present results, coi gene can be used in barcoding. the coi provides an objective foundation for the identification of ecotypes and therefore could be used for a rapid establishment of a variety of identifications. the results of the present study showed that both ecotypes had a comparable level of genetic diversity. therefore, it could be concluded that the similarity of evolutionary forces affects both ecotypes. conflict of interest none.  references 1. mpenda, f. n., schilling, m. a., campbell, z., mngumi, e. b., and buza, j. (2019). the genetic diversity of local african chickens: a potential for selection of chickens resistant to viral infections. j. appl. poult. res, 28, 1–12. 2. habimana, r., okeno, t.o., ngeno, k., mboumba, s., assami, p., gbotto, a.a., keambou, c. t., nishimwe, k., mahoro, j. and mahoro, n. (2020). genetic diversity and population structure of indigenous chicken in rwanda using microsatellite markers. plos one, 15(4), e0225084. 3. salo, s., tadesse, g., and hilemeskel, d. (2016). village chicken production system and constraints in lemo district, hadiya zone, ethiopia. poult fish wildl sci, 4(2), 158–162. 4. pius, l.o., strausz, p., and kusza, s. (2021). overview of poultry management as a key factor for solving food and nutritional security with a special focus on chicken breeding in east african countries. biology, 10, 810–830. 5. sabry, a., ramadan, s., hassan, m.m., mohamed, a.a., a., mohammedein, a., and inoue-murayma, m. (2021). assessment of genetic diversity among egyptian and saudi chicken ecotypes and local egyptian chicken breeds using microsatellite markers. journal of environmental biology, 42, 33–39. 6. reed, d. h., and frankham, r. (2003). correlation between fitness and genetic diversity. conserv. biol, 17, 230–237. 7. al-atiyat, r. (2010). genetic diversity of indigenous chicken ecotypes in jordan. african journal of biotechnology, 9(41), 7014–7019. 8. allentoft, m. e., and o’brien., j. (2010). global amphibian declines, loss of genetic diversity and fitness: a review. diversity, 2, 47–71. 9. shapiro, b. (2017). pathways to de-extinction: how close can we get to resurrection of an extinct species? funct. ecol, 996–1002. 10. msoffe, p.l.m., mtambo, m.m.a., minga, u.m., yongolo, m.g.s., gwakisa, p.s., and olsen, j.e. (2001). identification and characterisation of the free ranging local chicken eco-types in tanzania. pages 81–90 of: g.c. kifaro, g.c, kurwujila, r. l., chenyambuya, s.w., & chilewa, r. r. (eds), proceedings of sua-mu enreca project workshop. 11. tadelle, d., kijora, c., and peters, k.j. (2003). indigenous chicken ecotypes in ethiopia: growth and feed utilization potentials. international journal of poultry science, 2(2), 144–152. 12. muchadeyi, f.c., eding, h., wollny, c.b., groeneveld, e., makuza, s.m., shamseldin, r., simianer, h., and weigend, s. (2007). absence of population 263j contemp med sci | vol. 8, no. 4, july-august 2022: 259–263 a. sabry et al. original a comparable genetic diversity between chicken ecotypes of different zones using dna barcoding sub-structuring in zimbabwe chicken ecotypes inferred using microsatellite analysis. animal genetics, 38, 332–339. 13. rudresh, b. h., murthy, h.n.n., jayashankar, m. r., nagaraj, c. s., kotresh, a. m., and byregowda, s. m. (2015). microsatellite-based genetic diversity study in indigenous chicken ecotypes of karnataka. veterinary world, 8, 970–976. 14. wimmers, k., ponsuksili, s., hardge, t., valle-zarate, a., mathur, p.k., and horst, p. (2000). genetic distinctness of african, asian and south american chickens. animal genetics, 31, 159–165. 15. reeves, j.t., and weil, j.v. (2001). adv. exp. med. biol., 502: 419–437. 2001. chronic mountain sickness. a view from the crow’s nest. adv. exp. med. biol., 502, 419–437. 16. huang, s., zhang, l., rehman, m. u., iqbal, m. k., lan, y., mehmood, k., zhang, h., qiu, g., nabi, f., yao, w., wang, m., & li, j. (2016a). high altitude hypoxia as a factor that promotes tibial growth plate development in broiler chickens. plos one, 27(5), 3280. 17. wu, h., wan, q-h., fang, h-g., and zhang. s. (2005). application of mitochondrial dna sequence analysis in the forensic identification of chinese sika deer subspecies. forensic science international, 148(2–3), 101–105. 18. gratwicke, b., mills, j., dutton, a., gabriel, g., long, b. sei-densticker, j., wright, b., you, w., and zhang, l. (2008). attitudes toward consumption and conservation of tigers in china. plos one, 3, e2544. 19. bekker, ei., karabanov, dp., galimov, yr., and kotov, aa. (2016). dna barcoding reveals high cryptic diversity in the north eurasian moina species (crustacea: cladocera). plos one, 11(8), 1–19. 20. hebert, p. d. n., cywinska, a., shelley, l. b, and dewaard, r.j. (2003). biological identifications through dna barcodes. proc biol sci, 151, 313–321. 21. roe, a. d., and sperling, f.a.h. (2007). patterns of evolution of mitochondrial cytochrome c oxidase i and ii dna and implications for dna barcoding. mol phylogenet evol, 44, 325–345. 22. bondoc, o.l., & santiago, r.c. (2012). the use of dna barcodes in the evolutionary analysis of domestic breeds and strains of chicken (gallus gallus domesticus) in the philippines. philipp agric scientist, 95(4), 358–369. 23. osaman, s.a.m., yonezawa, t., and nishibori, m. (2016). origin and genetic diversity of egyptian native chicken based on complete sequence of mitochondrial dna d-loop region. poult. sci, 95, 1248–1256. 24. khan, i.a., jahan, p., & hasan, q. and rao, p. (2019). genetic confirmation of t2dm metaanalysis variants studies in gestational diabetes mellitus in an indian population. diabetes metab. syndr., 13(1), 688–694. 25. amer, s.a.m., ahmed, m.m., and shobrak, s. (2013). efficient newly designed primers for the amplification and sequencing of bird mitochondrial genomes. bioscience, biotechnology, and biochemistry, 77(3), 577–581. 26. gaber, a., hassan, m., boland, c., alsuhaibany. a, bab-bington, j., john pereira, j., budd, j., and shobrak, m. (2020). molecular identification of todiramphus chloris subspecies on the arabian peninsula using three mitochondrial barcoding genes and issr markers. saudi journal of biological sciences, 480–488. 27. librado, p., and rozas, j. (2009). dnasp v5: a software for comprehensive analysis of dna polymorphism data. bioinformatics, 25(11), 1451–1452. 28. xun-he, h., jie-bo, c., dan-lin, h., xi-quan, z, and fu-sheng, z. (2016b). dna barcoding of indigenous chickens in china: a reevaluation. scientia agricultura sinica, 49(13), 2622–2633. 29. peng, w., yang, h., cai, k., zhou, l., tan, z, and wu, k. (2019). molecular identification of the danzhou chicken breed in china using dna barcoding. mitochondrial dna part b, 4(2), 2459–2463. pmid: 33365583. 30. cui, hi, ibtisham, f., xu, c., huang, h., and su, y. (2017). dna barcoding of chinese native chicken breeds through coi gene. thai journal of veterinary medicine, 47, 123–129. 31. dave, a.r., chaudhary, d.f., mankad, p.m., koringa, p.g., and rank, d.n. (2021). genetic diversity among two native indian chicken populations using cytochrome c oxidase subunit i and cytochrome b dna barcodes. veterinary world, 14(5), 1389–1397. 32. hebert, p.d.n., cywinska, a., ball, s.l., and de-waard, j. (2002). biological identifications through dna barcodes. proc. r. soc. lond. b, 270, 313–321. 33. yu-shi, g., tu, y-j., tang, x-j., lu, j-xi., and zhan, x-y. (2011). studies on the dna barcoding of two newly discovered chicken breeds by mtdna coi gene. journal of animal and veterinary advances, 10(13), 1711–1713. 34. vingron, m., brazma, a., coulson, r., van helden, j., manke, t., palin, k., sand, o., and ukkonen, e. (2009). integrating sequence, evolution and functional genomics in regulatory genomics. genome biology, 10, 202–209. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1259 27j contemp med sci | vol. 8, no. 1, january-february 2022: 27–30 original mental health wellbeing of school age children is matter: a study to evaluate the effectiveness of a school-based mental health awareness program in the middle schools in oman yusra al nasiri1*, aisha al abri2, afra al rawahi2, al anood al harrasi2, amal al shuaibi2, amina al owisi2, arwa al rashdi3, atheer al shabibi1, balaqis al hakmani1, shamsa al harthi4 1oman college of health sciences, nursing program, muscat, oman. 2staff nurse, royal hospital, muscat, oman. 3staff nurse, bousher dialysis center, muscat, oman. 4staff nurse, khoula hospital, muscat, oman. *correspondence to: yusra al nasiri (e-mail: yusra444@hotmail.com) (submitted: 21 december 2021 – revised version received: 06 january 2022 – accepted: 21 january 2022 – published online: 26 february 2022) abstract objectives: the aim of this study is to assess the effectiveness of a school-based mental health awareness program among school age children. methods: this was a quantitative study that recruited 178 parents and 150 students from different levels (grades 5–10) attended the awareness activity at two middle schools in oman. a convenient sampling approach was utilized. results: there was a significant different in mood and feelings score between the grades (p = 0.001); with grade 10 being the highest disturbed mood changes (m = 23.25, sd = 5.54). the results also showed that 55% of students experienced anxiety. from the analysis, it was found that grade 10 (m = 36.15, sd = 18.43, p = 0.05) reported higher anxiety than others groups. the results indicated also a strong correlation between experience of bullying and mental health problems (r = 0.85). the evaluation of awareness program indicated that 91% of parents reported improvement in knowledge; 89% suggested that many information they were not aware of before. 92% rated the video as informative. 93% agreed that the program activities were entertaining and informative. 92% reported that the activity increased their awareness to observe the changes in their children emotional wellbeing. 98% of parents enjoyed the activities and rated the overall program as effective and very interesting. conclusion: there is a dire need to conduct various awareness programs in the community. such programs are not only helpful in improving the community knowledge on the factors influencing children’s mental health, but also assist in identifying the mental health problems at early stage; which may empower them to seek early management for their children. this study suggests an establishment of a mental health program for children in oman to identify the cases, assess the mental problems affecting children at different ages and refer them for further support and management. keywords: mental health, school age, children, awareness program issn 2413-0516 introduction & background mental health and emotional wellbeing in children is just as important as their physical health. good mental health allows children to think clearly, develop socially and learn new skills.1,2 in addition, fostering mental health helps children develop self-confidence, high self-esteem, and a healthy emotional life.3 promoting mental health will not only help children build resilience to cope up with life stressors, but also will nurture them to grow into well-grounded healthy adults.4 research indicates that children’s emotional health and positive attitude effect positively the children’ emotions and mood as it is reflected in their way of expression.1 positive mood predicted learning academic and cognitive progression, motivation, and interpersonal skills of children.1 a study identified the prevalence of anxiety and the relationship between anxiety and school performance among elementary, middle, and high school students (n = 478). the results revealed that 35 (7.3%) reported high level of anxiety.5 the results suggested abnormally high anxiety level that was negatively associated with school performance.6 similarly, another study revealed a significant relationship (p > 0.05) between children’s anxiety level and educational level. this study showed that the prevalence of anxiety was higher in boys, single children, children who had a family history of hereditary disease, and children who experienced corporal punishment at home.7 studies also suggest that school age children suffer from depression. a cross section study aimed to explore the prevalence of depression and its associated sociodemographic factors among school-going adolescents. among 1412 selected students, the prevalence of depression was found to be 49.2%. guilty feelings (69.48%) was one of the most prominent clinical factors associated with depression followed by pessimism (58.14%), sadness (56.52%), and past failure (55.81%).1 similarly, a high level of depressive symptoms reported among adolescents (38%). the study concluded that depressive symptoms was associated with poor academic performance, poor coping methods and suicidal ideations.4 similarly, another study showed that 35% of children less than 13 years suffered from depressive disorder, and that the prevalence in the adolescent sub-group was 15%.8,9 research also suggests an association between bullying experience and alteration in the mental health wellbeing for students. the prevalence of bullying at 6 schools in muscat governorate revealed that 38.4% of the students reported being bullied in the past month and 34.0% of the students reported having at least one physical injury in the past year that caused at least one full day absences from usual activities or required 28 j contemp med sci | vol. 8, no. 1, january-february 2022: 27–30 mental health wellbeing of school age children original y. al nasiri et al. medical treatment due to bullying.10 students who experienced bullying were found to suffer from family abuse and had history of tobacco, alcohol and drug use.3 in a study conducted in saudi arabia involving 9073 students; the results indicated that 26% of adolescents reported exposure to bullying. exposure to physical violence and bullying having more frequent symptoms of depression and anxiety. those exposed to physical violence were at higher odds of having poorer academic performance.10,11 some studies attempted to evaluate the effectiveness of mental health programs on reducing mental health symptoms affecting children. the results of a systematic suggested that the psychological program was effective and helpful in reducing anxiety in school age children. psychological programs at school can be a promising intervention targeted towards preventing the anxiety and other mental health problems among school age children.12 problem statement most children tend to grow up mentally healthy; however, recent literature suggests that mental problems in children and young adults are on the increase due to the changes in the way of living.7,13 the research highlights that mental health problems affect about 1:10 children and young adults and alarmingly, 70% of the effected children were not assessed at early stage and have not had corresponding interventions at a sufficient early age. there are many risk factors that make children vulnerable for mental health problems these include: changes in the environment the children’s lives in; for example, divorce of parents, abuse experience, moving to a new school environment, living in poverty, having a parent with alcohol or drug addict.13 other risk factors are: having long term physical illness, educational difficulties, experienced bullying and death of a close member in the family.12 based on the literature, common mental health problems that can effect children are: anxiety, depression, excessive worries, post-traumatic stress disorder, and self-harm.5,12,14 this study was done in response to the data received from the school health counselors from two middle-age schools in muscat on the increasing number of school age children suffering from negative thoughts, anxiety and other mental health issues. hence, this study was conducted to assess the mental health wellbeing of the students and evaluate the effectiveness of the planned school based mental health awareness program. materials and methods this is a quantitative study that aimed to evaluate the effectiveness of a school based mental health awareness program. an invitation was sent from the principals of two middle schools to parents of children to attend the awareness program that was hosted in one of the middle schools in muscat. 178 parents and 150 students from different levels (grades 5–10) attended the awareness activity recruited through convenience sampling approach. prior to conducting the awareness program, the students were asked to respond to five questionnaires: 1) anxiety scale (41 items); 2) mood & feelings (13 items); 3) depression (15 items); 4) bullying (6 items). the questionnaires adopted from different studies and showed very good reliability (>0.7). students were informed that their participation is voluntary. the students who accepted to participate were given a consent form to sign. the parents were asked to evaluate the effectiveness of the awareness program at the end of the activity using a tool that consisted of 12 items (knowledge improvement, materials, program activities, consultation service). description of the awareness program the awareness program consisted of 1) a power point presentation mental health wellbeing in children and the factors associated with mental health problems; 2) a video on the factors influencing the mental health status among school age children and the role of parents in enhancing the overall mental health wellbeing of their children. 3) role-play illustrated a case of a child suffered from a mental health problem due to family problems. the case was presented from the omani community. 4) poster presentation of the findings of a study done by the nursing students in previous years on mental health status among school age children; 5) a consultation corner by a psychologist to guide and address the parents’ concerns on child health mental illness and issues; 6) material distributions on mental health. the program was conducted in one of the middle schools in muscat region. the program lasted for 5 hours. results data were analyzed using spss, v.24. one-way anova was conducted to analyze the tools. frequencies and percentages were used to analyze the program effectiveness. the age of the students ranged between (9–14 years). 85% of the students were omanis, 15% were non-omanis. the result from the questionnaires revealed that 60% of students had experienced mood swings. there was a significant different in mood and feeling score between the grades (p = 0.001); with grade 10 being the highest disturbed mood changes (m = 23.25, sd = 5.54). the results also showed that 55% of students experienced anxiety. from the analysis, it was found that grade 10 (m = 36.15, sd = 18.43, p = 0.05) reported higher anxiety than others groups. in addition, the findings highlighted that 28.3% experienced a pre depression state. the study also attempted to correlate experience of bullying with the mental health status. the findings revealed that 89% of students experienced bullying and grade 5 were highly victimized from bullying than others group. the results indicated a strong correlation between experience of bullying and mental health problems (r = 0.85); which suggests that bullying could be a cause for experiencing mental health problems in school age children. the study also highlighted that a smaller rate of students (10.8%) had tendency for bullying. from the analysis, it was found that grade 10 (m = 4.05, sd = 3.57) had higher tendency for bullying than other groups. the evaluation tool for the awareness program indicated that 91% of parents reported improvement in knowledge after the program; 89% suggested that many information they were not aware of before. 92% rated the video as informative. 93% agreed that the program activities were entertaining and informative. 92% reported that the activity increased their awareness to observe the changes in their children emotional wellbeing. 98% of parents rated the overall program as effective and very interesting. 97% enjoyed the activities provided for them on that day. 29j contemp med sci | vol. 8, no. 1, january-february 2022: 27–30 y. al nasiri et al. original mental health wellbeing of school age children discussion creating awareness to parents on mental health wellbeing of school age children was effective as it led to improve knowledge of parents after the program. our findings were similar to the findings of study.12,15 the study found that the awareness program was very effective and helped improve the community knowledge regarding depression and anxiety. the program assisted people to assess signs of depression and offered them management options. the implementation of the awareness program was feasible and activities were very interesting to the parents. the activities planned made suitable for different age group of children & parents; older parents were able to grasp the information easily and able to track the program without any hassle. some studies supported the intervention we developed.12 the study of created a public awareness program on health and the findings suggested that the program was feasible and successful and empowered patients to seek early care and interventions. similarly, one study developed an anti-stigma awareness program and found that the program improved the public knowledge on various mental health problems and facilitated seeking of psychological support.16 to our knowledge, this was first study that assessed the mental health status among school age children in oman. the findings yielded from the study were alarming and highly important; which necessities the need to raise an awareness in the omani community on the importance of enhancing mental health status in children. community awareness serves as a means to disseminate the findings revealed from the study, shed the light on the common mental health problems in children, highlight the factors contributing to mental health problems and suggest ways for enhancing children’s mental health. limitations the sample size was very small; which limits the generalization of the findings to other settings. also, cross section designs do not examine the cause – effect relationship, data was only collected at one-time point and therefore, the causes of the reported percentages and changes in the children cannot be related to any causes. longitudinal studies are therefore recommended. recommendations it is recommended to replicate the study with more sample size from different schools and regions in oman. implications of the project conducting awareness yielded two fold advantages. first, it created an awareness in the community about the importance of maintaining mental health wellbeing for school age children. second, it created more attention to public about certain factors that may affect the mental health wellbeing of children. moreover, it helps to inform the nursing practice regarding the need to establish a mental health program in schools to target mental health problems affecting children. conclusion mental health and emotional wellbeing in children is just important as their physical health. this study assessed the mental health wellbeing of school age children at two middle age schools in oman. the study created a community awareness program to parents and their children on the importance of enhancing mental health wellbeing. the program was creative and effective in improving the parents’ knowledge on different mental health problems affecting school age children such as depression, anxiety, worries and mood changes and bullying. parents were provided with various guidelines to inform them about their roles in assessing and seek early management of the health issue. parents felt supported and cared. the information presented were simple and clear that could be understood by all parents from different age groups. in conclusion, there is a dire need to conduct various awareness programs in the community to help people feel supported, cared and valued. such programs are not only helpful in improving the community knowledge on the factors influencing children’s mental health, but also assist in identifying the mental health problems at early stage; which may empower them to seek early management for their children. this study suggests an establishment of a mental health program for children in oman to identify the cases, assess the mental problems affecting children at different age and refer them for further support and management. ethical consideration prior conducting the study, ethical approval of the oman college of health sciences was obtained. also, the approval of the schools’ principal was obtained. consent form was given to the students prior to data collection. acknowledgements we would like to thank dr. salem al touby, an associate professor and dean of college of nursing and pharmacy for his support and guidance. we also thank dr. nasir al balushi, a child psychologist at sultan qaboos university hospital for participating in the implementation phase of this study. conflicts of interest disclosures there is no conflict of interest.  references 1. jha k, singh s, nirala s, kumar c, kumar p, aggrawal n. prevalence of depression among school-going adolescents in an urban area of bihar, india. indian j psychological med, 2017;39(3):287–293. 2. patel h, varma j, shah s, phatak a, nimbalkar s. profile of bullies and victims among urban school-going adolescents in gujarat. indian paed. 2017;54 (10):841–843. 3. mello f, silva j, oliveira w, prado r, malta d, silva m. the practice of bullying among brazilian schoolchildren and associated factors. national school health survey. ciencia & saude coletiva. 2015;22(9):2939–2948. 4. chauhan s, panna l, harsavardhan n. prevalence of depression among school children aged 15 years and above in a public school in noida, uttar pradesh. international j of public health res. 2014;2278–5213. 30 j contemp med sci | vol. 8, no. 1, january-february 2022: 27–30 mental health wellbeing of school age children original y. al nasiri et al. 5. valiente s. linking students’ emotions and academic achievement. 2015: available from: https://www.coursehero.com/ file/7807695/ valiente-129–135 (accessed online 26/02/19). 6. mazzone l, ducci f, scoto m, passaniti e, d’arrigo v, vitiello b. the role of anxiety symptoms in school performance in a community sample of children and adolescents. 2011; bmc public health, 7(1). doi: 10.1186/1471– 2458–7–347. 7. banaeipour z, rostami s, kourosh z, bahman c. the prevalence of anxiety and its related factors among school age children in south west of iran. j med sciences. 2016;4(6):2019–25. 8. díaz c, sánchez j, martínez b. suicide in adolescents with depression: the need for early diagnosis. clinical case report. 2015;7(1):30–42. 9. hankin b, young j, abela j, smolen a, jenness j. oppenheimer c. depression from childhood into late adolescence: influence of gender, development, genetic susceptibility, and peer stress. j abnormal psych. 2015; 124 (4), 803–816. 10. al-saadooni m. (2014). the magnitude and impact of bullying among school pupils in muscat, oman. oman medical journal. 2017; 5(1):1–10. 11. al buhairan f, abou abbas o, el sayed, badri m, alshahri s, de vries. the relationship of bullying and physical violence to mental health and academic performance: a cross-sectional study among adolescents in kingdom of saudi arabia. int j ped & adolescent med. 2017;4(2): 61–65. doi: 10.1016/j.ijpam.2016.12.005. 12. werner-seidler a, perry y, calear a, newby j, christensen h. schoolbased depression and anxiety prevention programs for young people: a systematic review and meta-analysis. cl psych rev. 2017;51:30–47. 13. queky, tam w, zhang m, exploring the association between childhood and adolescent obesity and depression. a meta-analysis. obesity rev. 2017;18: 742–754. 14. hankin b, young j, abela j, smolen a, jenness j, gulley l, oppenheimer c. depression from childhood into late adolescence: influence of gender, development, genetic susceptibility, and peer stress. j psyc. 2015;124(4): 803–10. 15. bouchard s, gervias n, gagnier c, loranger c. evaluation of a primary prevention program for anxiety disorders using story books with children aged 9–12 years. j primary prevention. 2013;34 :345–358. doi: 10.1007%2fs10935–013–0317–0. 16. henderson c, robinson e, evans-lacko s, thornicoroft g. relationships between anti-stigma programme awareness, disclosure comfort and intended help-seeking regarding a mental health problem. br j psychiatry. 2017;11(5):316–322. doi: 10.1192/bjp.bp.116.195867. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1128 https://europepmc.org/abstract/med/14615688?europe_pmc_abs_citations_citedby=/abstract/med/26576283 https://europepmc.org/abstract/med/14615688?europe_pmc_abs_citations_citedby=/abstract/med/26576283 https://dx.doi.org/10.1192%2fbjp.bp.116.195867 247j contemp med sci | vol. 7, no. 4, july–august 2021: 247–251 original knowledge and misconceptions regarding sle among medical students at king abdulaziz university yasser m. bawazir1*, , ibtisam jali2 1assistant professor of medicine and rheumatology, department of medicine, king abdulaziz university, jeddah, saudi arabia. 2associate professor of medicine and rheumatology, department of medicine, king abdulaziz university, jeddah, saudi arabia. *correspondence to: yasser m. bawazir (e-mail: ymbawazir@kau.edu.sa) (submitted: 12 may 2021 – revised version received: 02 june 2021 – accepted: 23 june 2021 – published online: 26 august 2021) introduction systemic lupus erythematosus (sle) is a chronic autoimmune disease that affects multiple organs and predominantly affects women. this heterogeneous condition has a wide spectrum of clinical manifestations related to drug reactions, associated comorbidities, and the disease itself.1 the diagnosis of sle is challenging because of the heterogenicity of clinical manifestations and, in some cases, the presentation is quite severe, requiring prompt diagnosis and early immunosuppressive treatment.2 worldwide, the incidence of sle is 23.2 per 100.000 people per year, while the highest incidence is seen in north america (241 per 100.000 people per year) and the lower incidences are recorded in african countries (0.3 per 100,000 people per year). the disease is mostly prevalent among women, regardless of age and ethnicity.3 these data are similar to those reported in saudi arabia, where the estimated prevalence of sle is 19.28 per 100.000 people.4 being a complex disease, sle requires a multidisciplinary approach, including rheumatology, dermatology, cardiology, pulmonology, and nephrology. assessing the disease can help identify defects in its treatment. kerezoudis et al.,5 conducted a study with 260 students – 114 in preclinical and 146 in clinical years – at two medical schools to assess the depth of their knowledge using survey assessment. they identified several misconceptions in the perception of sle and attributed this to the lack of clinical practice and the low number of patients.5 rheumatology curriculum in our intuition includes lectures, clinical based learning sessions and clinical examination sessions. all sixth-year students must undergo a week of intense rheumatology course, at the completion of this week, assessment takes place. the total number of the sixth-year students (final clinical year) is around 350 students. given the magnitude of this disease, this study aimed to assess the knowledge and misconceptions regarding sle in sixth-year medical students in order to verify their understanding of the general aspects of sle and obtain feedback on their curriculum and learning experience. to this end, a 25-item questionnaire was administered to sixth-year medical students at the faculty of medicine at king abdulaziz university. based on the questionnaire results, suggestions and recommendations were made to improve the learning experience about sle at the referred university. methods participants participants in this study were sixth-year medical students at the faculty of medicine at king abdulaziz university. the medicine program at king abdulaziz university comprises six years, the first three years being preclinical and the last three years clinical. in particular, the sixth year includes medical subspecialties, such as rheumatology. thus, the inclusion criterion for this study was to be in the sixth year of the medicine program, since having obtained specific knowledge related to sle was mandatory to answer the study questionnaire. the questionnaire was distributed to 350 medical students. study design this was an observational cross-sectional study in which a 25-item two-part questionnaire was structured using google abstract introduction systemic lupus erythematosus (sle) is a chronic autoimmune disorder that requires a multidisciplinary approach. the aim of this study was to assess the knowledge and misconceptions regarding sle in sixth-year medical students in order to verify their understanding of the general aspects of sle and provide recommendations to improve their learning experience. methods we created a two-part 25-item questionnaire of 25 questions to assess students’ knowledge about epidemiology, clinical manifestations, complications, management, and prognosis of sle, preferred ways of learning, and opinion on which specialties should include learning about the disease. the questionnaire was distributed to the participants through google forms. results a total of 200 students from king abdulaziz university responded to the questionnaire. the prevalence of adequate knowledge among students was 39.5%. there was a significant statistical difference between students with adequate knowledge and those without adequate knowledge according to the number of cases seen during training. there was a higher prevalence of adequate knowledge among female students and those who had a family history of sle. most students answered correctly the basic questions about sle, while there were weak points in the questions about epidemiology and clinical manifestations. the preferred way of learning about sle indicated by most respondents was to see patients in the wards and analyze the pathophysiology, diagnosis, and treatment of the disease. most students chose internal medicine and rheumatology as specialties that should include learning about sle. conclusion the findings reflect the need to improve the teaching of chronic medical diseases treated on an outpatient basis and to modify the medical school curriculum so as to prepare future physicians to deal with such cases. keywords systemic lupus erythematous, medical students, medical education, sle awareness, saudi arabia issn 2413-0516 https://orcid.org/0000-0002-5060-3884 mailto:ymbawazir@kau.edu.sa 248 j contemp med sci | vol. 7, no. 4, july–august 2021: 247–251 knowledge and misconceptions regarding sle among medical students at king abdulaziz university original y.m. bawazir and i. jali forms. the first part included 19 questions and aimed to assess students’ knowledge about epidemiology, clinical manifestations, complications, management, and prognosis of sle. if the participant answer 13 out of 19 questions correctly he will be considered as having adequate knowledge. the second part included six questions and aimed to obtain general information about the participants, their preferred ways of learning about sle, and their opinion on which specialties should include learning about sle, as well as how many cases of sle are seen during their training. the questionnaire was conducted in english, which is the language of instruction at the faculty of medicine at king abdulaziz university. for the development of the questionnaire items, we conducted a search on pubmed and google scholar for validated sle-related questionnaires. as a result, we identified only two non-validated questionnaires to assess knowledge of sle among medical students. therefore, we developed questions to assess the fundamentals of sle based on the king abdulaziz university curriculum. study procedure the questionnaire developed using google forms was distributed to medical students by e-mail on the day of completion of the rheumatology rotation assessment. the respondents were allowed to submit the form only once. since participation was optional, only 200 of the 350 medical students agreed to participate. all fourthand fifth-year students were excluded from the study as they don’t rotate in rheumatology. the study did not require ethical approval as per the department of bioethics in our institution. statistical analysis data were analyzed using the statistical package for the social sciences (spss), version 21 (ibm inc., armonk, ny, usa). categorical variables were presented as numbers and precents and were compared using chi-square test. continuous variables were non normally distributed, so presented as median and range (minimum-maximum) and were compared using mann whitney u test. all tests were 2-tailed, and a p value <0.05 was considered statistically significant. results table 1 shows the demographic characteristics of the participants. of the 200 respondents, 198 (99%) were 18 to 25 years old and 117 (58.5%) were women. more than half (107, 53.5%) saw only one to three cases of sle during training and 30 (15%) had a family history of sle. table 2 shows the prevalence of adequate knowledge about sle among the study participants. in order for respondents’ knowledge about sle to be assessed as adequate, 13 of the 19 questions in the first part of the questionnaire must be answered correctly. the prevalence of adequate knowledge about sle among the study participants was 39.5%. there was a significant statistical difference between students with adequate knowledge and students without adequate knowledge according to the number of cases seen during training (p < 0.001). as shown in table 3, as the number of table 1. demographic characteristics of the participants (n = 200) variable number of participants percentage of participants gender male 83 41.5 female 117 58.5 age <18 years old 2 1.0 18-25 years old 198 99.0 number of cases seen during training 0 55 27.5 1–3 107 53.5 4–9 31 15.5 >10 7 3.5 family history of sle maybe 4 2.0 no 166 83.0 yes 30 15.0 note. all variables are summarized in numbers and percentages. table 2. prevalence of adequate knowledge about sle among participants (n = 200) variable number of participants percentage of participants with adequate knowledge 79 39.5 without adequate knowledge 121 60.5 total 200 100.0 note. all variables are summarized in numbers and percentages. table 3. prevalence of adequate knowledge about sle according to number of cases seen during training adequate knowledge variable yes no p-value number % number % number of cases seen during training 0 6 11% 49 89% <0.001 1–3 44 41% 63 59% 4–9 22 71% 9 29% >10 7 100% 0 0% total 79 39.5% 121 60.5% note. all variables are summarized in numbers and percentages. 249j contemp med sci | vol. 7, no. 4, july–august 2021: 247–251 y.m. bawazir and i. jali original knowledge and misconceptions regarding sle among medical students at king abdulaziz university cases increases, the prevalence of adequate knowledge also increases. according to table 3, all students who saw more than 10 cases during training had adequate knowledge. the test of significance was carried out at the 0.05 level. a chisquare test was used to assess the statistical significance of the differences between the two groups according to the number of cases seen during training. regarding the relationship between prevalence of adequate knowledge and gender/family history of sle, a statistically significant (p < 0.001) higher percentage of adequate knowledge was identified among female respondents and those with a family history of sle (table 4). the prevalence of adequate knowledge about sle was higher among women (56%) and among students with a family history of sle (70%). the test of significance was carried out at the 0.05 level. a chi-square test was used to assess the statistical significance of the differences between the two groups according to gender and family history of sle. the analysis of the first part of the questionnaire (19 questions) revealed that the majority of students answered correctly the basic questions about sle. on the other hand, there were weak points in the questions about epidemiology and clinical manifestations (figure 1). as for the second part of the questionnaire, question 24 asked students to choose their preferred ways of learning about sle. more than one choice was allowed. the preferred way of learning about sle indicated by most respondents was to see patients in the wards and analyze the pathophysiology, diagnosis, and treatment of the disease (172 responses), followed by seeing a patient-actor with sle during class and discussing symptoms, diagnosis, differential diagnosis, and treatment (77 responses). watching educational videos with real patients and discussing clinical vignettes during lectures obtained 72 and 63 responses, respectively (table 5). finally, the questionnaire asked students their opinion on which medical school specialties should include learning about sle. more than one choice was allowed. most students choose internal medicine (130) and rheumatology (127), while 86 choose pathology (table 6). discussion advances in medicine in recent decades have been accompanied by a marked increase in the number of chronic diseases worldwide. such diseases have been managed mainly in the outpatient clinic sitting. medical education in most medical schools follows the flexner model, which focuses mainly on inpatient hospital-based training. the problem with this model is that it does not focus on the outpatient training sitting.6 the main barriers to outpatient education are the lack table 4. prevalence of adequate knowledge about sle according to gender and family history of sle adequate knowledge variable yes no p-value number % number % gender male 13 16% 70 84% <0.001 female 66 56% 51 44% family history of sle maybe 0 0% 4 100% <0.001 no 58 35% 108 65% yes 21 70% 9 30% note. all variables are summarized in numbers and percentages. table 5. frequency of preferred ways of learning about sle among participants ways of learning about sle number of responses (%) a. see patients in the wards and analyze the pathophysiology, diagnosis, and treatment of the disease. 172 (44.7%) b. see a patient-actor with sle during class and discuss symptoms, diagnosis, differential diagnosis, and treatment. 77 (20%) c. watch educational videos with real patients. 72 (18.6%) d. discuss clinical vignettes during lectures. 63 (16.4%) total 384 note. all variables are summarized in numbers. fig. 1 frequency of correct answers in each of the 19 questions about sle in descending order. table 6. specialties that should include learning about sle in the opinion of the participants specialty number of responses (%) internal medicine 130 (32.7%) rheumatology 127 (31.9%) pathology 86 (21.6%) physiology 34 (8.5%) biology 20 (5%) total 397 (100%) note. all variables are summarized in numbers. 250 j contemp med sci | vol. 7, no. 4, july–august 2021: 247–251 knowledge and misconceptions regarding sle among medical students at king abdulaziz university original y.m. bawazir and i. jali of evidence in the literature, conflicts between medical education and healthcare, and inadequate financial incentives for academic medical staff.7 sle is a systemic disease treated mainly in the outpatient sitting, whereas few complicated cases are hospitalized.8 this may contribute to students’ lack of exposure to patients with chronic illnesses. at king abdulaziz university, students must complete three years of preclinical studies before beginning the three clinical years. clinical teaching in the medicine program is based mainly on inpatient teaching, with limited outpatient exposure.8 unfortunately, only 39.5% of the respondents in this study showed adequate knowledge about sle. we attribute this result to the number of cases seen during clinical practice, as most respondents saw only one to three cases. women showed to be more knowledgeable about sle, which could be attributed to the higher prevalence of the disease among women.1,3 in addition, students with a family history of sle showed more adequate knowledge. this may be due to the fact that these students sought to educate themselves more about the disease, as the risk of developing sle are higher among relatives of patients with sle. to suggest alternatives to improve the teaching of rheumatology, which is a critical specialty in the treatment of sle, we analyzed the weak points in the questionnaire responses. we found that there is little awareness among students about the current use of chemotherapy and biological therapy in patients with sle. this is clearly explained by the low number of cases seen during training, in addition to the majority of assistance to stable patients at the outpatient clinic. furthermore, at the university hospital, chemotherapy and biological therapy is administered at the day care unit, and students do not rotate through this unit. unlike rheumatoid arthritis, for which we have many approved biologic therapies,9 for sle, we currently have belimumab approved for treatment.10 other agents such as ustekinumab,11 anifrolumab,12 and janus kinase inhibitors,13 are still to be approved. almost half of the respondents were not aware of the worsening of sle during pregnancy. since pregnancy with sle is high risk, the disease must be stable before conception. risks include sle flare and the neonatal lupus. although the prognosis for pregnant women has improved considerably, the fetal risk, although progressively reduced, is still higher in pregnancies of patients with sle than in pregnancies of healthy women. miscarriage, premature delivery, and preeclampsia, as well as heart problems in the baby, are the major complications that can occur.14 we believe that the respondents in this study are not adequately aware of the complications of pregnancy with sle because the university does not have a special clinic for pregnant women with sle in collaboration with obstetrics and the patients are followed in the regular sle clinics, so that students do not have the opportunity to see such cases in the most appropriate way. almost half of the students responded that sle is not life threatening, which is also attributed to the fact that they do not see many inpatient cases. another important point is that high mortality is more observed in cases of circulatory diseases and infections15 rather than other sle complications, and this usually happens when patients are hemodynamically unstable and are referred to intensive care units. given that sle is more common in women1 (as mentioned earlier), there is an obvious misconception among students that the disease is more severe in women as well. in fact, the opposite is true: men with sle are more likely than women to develop disabilities, hypertension, thrombosis, and renal, hematological, and serological manifestations. men with sle are also more likely than women to suffer end-organ damage, including neuropsychiatric, renal, cardiovascular, peripheral vascular diseases, and myocardial infarction.16 the students’ preferred way of learning about sle was to see patients in the wards and analyze the pathophysiology, diagnosis, and treatment of the disease, followed by seeing a patient-actor with sle during class and discuss symptoms, diagnosis, differential diagnosis, and treatment. this result is in line with our opinion that students need to be exposed to more inpatient cases to improve their knowledge about this heterogeneous disease. finally, students indicated that they preferred to study sle in internal medicine and rheumatology rotations rather than during the basic science years. given that the integration of the clinical and basic years, students concentrate mainly on ways to obtain good grades rather than on how the knowledge acquired can be applied in clinical settings. generally, students see learning as a process whereby knowledge is provided to them by the teacher and the curriculum as an aggregate of separate subjects (e.g., pathology and anatomy) rather than a single general topic (e.g., medicine).17 given the above considerations, we suggest the following changes to improve the learning experience regarding sle for students at the faculty of medicine at king abdulaziz university. first, at this university, rotation in rheumatology used to be merged with rotation in internal medicine. we suggest implementing a separate focused short rotation in rheumatology. second, as we have observed during their busy clinical rotations, students rarely have the time to reflect on their basic science knowledge. for this reason, we suggest introducing clinical-based learning, in which the discussion starts with basic pathology and immunology and ends with clinical diagnosis and management, in the hope that some of the important basic information can be provided to senior medical students. third, to improve students’ awareness about the use of chemotherapy or biological therapy in patients with sle, we suggest a time dedicated to patients admitted to the day care unit, as well as the acquisition of knowledge about the indications, dosage, and side effects of treatments. finally, we recommend the creation of a rheumatology admission unit and collaboration in the clinic with colleagues from the obstetrics department for high-risk patients. study limitation this study assesses the knowledge of 6th year medical students at king abdulaziz university. the major limitation of this study is the use of non-validated questionnaire which was designed by the authors. as with any survey-based studies, a selection and sampling bias and we overcome this by consulting statistician. acknowledgments we would like to thank editage (www.editage.com) for english language editing. 251j contemp med sci | vol. 7, no. 4, july–august 2021: 247–251 y.m. bawazir and i. jali original knowledge and misconceptions regarding sle among medical students at king abdulaziz university funding this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. declaration of conflicting interests the authors declare that there is no conflict of interest.  references 1. di battista m, marcucci e, elefante e, et al. one year in review 2018: systemic lupus erythematosus. clin exp rheumatol. 2018;36(5):763-777. 2. sebastiani gd, prevete i, iuliano a, minisola g. the importance of an early diagnosis in systemic lupus erythematosus. isr med assoc j. 2016; 18(3-4):212-215. 3. rees f, doherty m, grainge mj, lanyon p, zhang w. the worldwide incidence and prevalence of systemic lupus erythematosus: a systematic review of epidemiological studies. rheumatology (oxford). 2017;56(11):1945-1961. 4. al-arfaj as, al-balla sr, al-dalaan an, et al. prevalence of systemic lupus erythematosus in central saudi arabia. saudi med j. 2002;23(1):87-89. 5. kerezoudis p, lontos k, apostolopoulou a, et al. lupus in medical education: student awareness of basic, clinical, and interdisciplinary aspects of complex diseases. j contemp med educ. 2016;4(3):97-106. 6. emanuel ej. reforming american medical education. milbank q. 2017;95(4):692-697. 7. oliveira franco rl, martins machado jl, satovschi grinbaum r, martiniano porfírio gj. barriers to outpatient education for medical students: a narrative review. int j med educ. 2019;10:180-190. 8. sims gn, jr., smith hr. outpatient management of systemic lupus erythematosus. cleve clin j med. 1996;63(2):94-100. doi: 10.1097/ bor.0000000000000480 9. smolen js, landewé rbm, bijlsma jwj, et al. eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update. ann rheum dis. 2019;79(6):685-699. doi: 10.1136/annrheumdis-2019-216655 10. fanouriakis a, kostopoulou m, alunno a, et al. 2019 update of the eular recommendations for the management of systemic lupus erythematosus. ann rheum dis. 2019;78(6):736-745. doi: 10.1136/ annrheumdis-2019-215089 11. costedoat-chalumeau n, houssiau fa. ustekinumab: a promising new drug for sle? lancet. 2018;392(10155):1284-1286. doi: 10.1016/s01406736(18)32330-4 12. morand ef, furie r, tanaka in, et al. trial of anifrolumab in active systemic lupus erythematosus. n engl j med. 2020;382(3):211-221. doi: 10.1056/ nejmoa1912196 13. mok cc. the jakinibs in systemic lupus erythematosus: progress and prospects. expert opin investig drugs. 2019;28(1):85-92. doi: 10.1080/13543784.2019.1551358 14. moroni g, ponticelli c. pregnancy in women with systemic lupus erythematosus (sle). eur j intern med. 2016;32:7-12. doi: 10.1016/j. ejim.2016.04.005 15. bernatsky s, boivin j-f, joseph l, et al. mortality in systemic lupus erythematosus. arthritis rheum. 2006;54(8):2550-2557. doi: 10.1002/ art.21955 16. tan tc, fang h, magder ls, petri m. differences between male and female systemic lupus erythematosus in a multiethnic population. j rheumatol. 2012;39(4):759-769. doi: 10.3899/jrheum.111061 17. alam a. how do medical students in their clinical years perceive basic sciences courses at king saud university? ann saudi med. 2011;31(1):58-61. doi: 10.4103/0256-4947.75780 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i4.1059 373j contemp med sci | vol. 7, no. 6, november-december 2021: 373–376 original seasonal theory of covid-19; one-year observation azri salih haji sgery1, yousif maan hadi1, amer abdalla goreal2* 1internal medicine and surgery, college of medicine, university of duhok, iraq. 2department of medical microbiology, college of medicine, university of duhok, kurdistan region, iraq. *correspondence to: amer abdalla goreal (e-mail: amer.goreal@uod.ac) abstract objectives: this study aimed to show the potential seasonality of sars-cov-2 over a period of one year (one winter/summer and one transition (spring/autumn) season). methods: data about the global incidence of the cases of one year interval and the doubling and halving time of cases for 10 countries were obtained from covid intel database, who website. results: a peak in the number of covid-19 cases and deaths during late autumn and winter months were observed along with some decrease in the number of cases and deaths during spring and summer. the average of doubling and halving time for the chosen countries varied across the sphere; the average doubling and halving time for (n°) countries were (264.14) and (52.64), respectively, while for (s°) countries were (20.91) and (148.40), respectively. conclusion: the epidemiological triad, (virus longevity in the air and on surfaces, increased susceptibility of the human victim in cold and dry weather and changes in human social behavior between winter and summer), explains the seasonal changes of the sars-cov-2 characteristics. so far the data for sars-cov-2 lie on the same line for the seasonality theory as other viruses such as influenza and sars-cov, follow the seasonality theory (they slow down during the summer and rise during winter), yet, longer periods of observation are required to confirm this theory. keywords: covid-19, sars-cov-2, seasonality, pandemic, climate issn 2413-0516 introduction the novel coronavirus (covid-19) is an infectious and contagious disease caused by the severe acute respiratory syndrome 2 (sars-cov-2). the first human case of covid-19 was reported by officials in wuhan city, china, in december 2019.1 the causative organism of covid-19, sars-cov-2, which is one of the seven viruses that belongs to coronaviridae family, was identified and linked to covid-19 in the first quarter of 2020.2 the virus spread rapidly worldwide, and by march 11, 2020, covid-19 was declared as a global pandemic by the world health organization (who).3 the total number of confirmed covid-19 cases by march 11, 2020 reached 120,926 cases with 4368 total number of deaths, and globally, as of 11:15 am cet, march 12, 2021 there have been 118,058,503 confirmed cases of covid-19, including 2,621,046 deaths received by who from national authorities.4 with the increase in the cases associated with covid-19 and public fear, many theories and were developed and/or adopted, one of which was whether sars-cov-2 that’s responsible for covid-19 is from the viruses that undergoes seasonal changes.5 flu viruses, come in seasons and manifestations peak during fall and winter, comprise well-known examples about this phenomenon. these viruses are widely known as seasonal viruses, hence they became known as the seasonal influenza viruses and their season as the flu season. while influenza viruses circulate year-round, the duration between december and february is the period in which the flu activity peaks most years, however, their activity may last until may. these flu viruses undergo seasonal variation regarding health impact including: number of infections, hospitalizations, and deaths, supporting that ancient phenomenon about contagious diseases even more.6 hence, the concept of seasonal changes for covid-19 began to be more widely believed worldwide. however, to the date, there is no solid evidence that covid-19 will or will not wax and wane seasonally. yet, researches were done supporting the evidence. nevertheless, the infectious characteristics of sars-cov-2, (incidence, mortality, recovery cases, active cases, testing rate, and hospitalization), varied between countries and were dependent on 3 main factors – average spring temperature, latitude (distance north or south of the equator) and longitude (distance east or west of the prime meridian)-; covid-19 characteristics decreased as temperature increased, decreased as latitude decreased, and were not affected by longitude.7 over one year in covid-19 pandemic, seasonality concept for other viruses and the difference in the infectious characteristics were the reason for this study. this study aimed to show the potential seasonality of sars-cov-2 over a period of one year (one winter/summer and one transition (spring/ autumn) season). methodology this study was done in form of a retrospective cohort study analyzing global covid-19 cases. data about the incidence of sars-cov-2 infection over one year interval were obtained from the official world health organization (who) website and were analyzed. the incidence of cases was taken in the form of cases/week from the website. from every 4 weeks in a month the last one was taken and the data were obtained from march, 30th 2020 to march 29th 2021. a total number of 10 countries were chosen, 5 from each half of the sphere, to obtain the doubling time and halving time of each country depending on the weekly incidence in a time range of 1 year (march 11, 2020 – march 11, 2021); united states of america (us), united kingdom (uk), italy, (submitted: 10 october 2021 – revised version received: 16 october 2021 – accepted: 29 october 2021 – published online: 26 december 2021) https://www.cdc.gov/flu/about/season/flu-season.htm 374 j contemp med sci | vol. 7, no. 6, november-december 2021: 373–376 seasonal theory of covid-19; one-year observation original a.s.h. sgery et al. france and spain were the countries chosen from the northern hemisphere and australia, new zealand, brazil, argentina and chile were chosen from the southern hemisphere. doubling times and halving times are useful measures in assessing the rate at which a disease spreads. these values can change depending on context and public health measures put in place. doubling time defined as the time it would take for the number of new daily cases to double which is a key variable in understanding the speed of growth of an outbreak. conversely, once an epidemic has reached its peak and the number of daily new cases begins to decrease, these growth models are used to characterize the halving time of the epidemic, defined as the time taken for the number of new daily cases to halve. results tables 1 and 2 shows the doubling and halving time of a total 10 countries from northern (n°) and southern (s°) hemispheres, respectively. the highest doubling time was seen for italy (n°) (1075.38 days) and the lowest was seen in chile (s°) (17.97 days) although australia and new zealand were having none. additionally, the highest halving time was seen in new zealand (s°) (291.13 days), while the lowest seen in spain (n°) (13.33 days). the average doubling time was lower for countries in southern hemisphere (20.91) than for northern hemisphere (264.14), while the average halving time was lower for countries in the northern hemisphere (52.64) than for southern hemisphere (148.40). chart 1 shows the global and regional incidence of covid-19 cases in a 4 week period, from march 1st, 2020 to may 1st, 2021. global weakly incidence of covid-19 shows peak in incidence at different times for different regions until march 15; america (dec-jan), europe and eastern mediterranean (nov-dec), south east asia (sept-oct), africa (jan) and western pacific (jan-feb). discussion the uncertainty about sars-cov-2 seasonality remained the center of attention of many researchers, especially after the preliminary laboratory trials suggested the virus survives longer outside the human body than other viruses.8 as it is shown in the plot, the characteristics of the cases rose in the last quarter of 2020 – beginning of 2021. factors that might have an effect on this rise can be explained by the epidemiologic triad, which is the traditional model for infectious disease: pathogen (agent) factors, host factors (susceptibility), and factors of the environment that brings the host and agent together. among the pathogen factors: genetics, mode of transmission, mutations, duration of the infection and the window of transmissibility. host factors: human behavior as hygiene, personal choices, age, and sex. other host factors include: genetic composition, nutritional and immunologic status, anatomic structure, presence of disease or medications, and psychological makeup. environmental factors include: climate, humidity, distance from the equator, and socioeconomic factors such as crowding, sanitation, and the availability of health services.9 although one year does not give definite evidence, temperature seems to affect the viral characteristics. the variation in temperature in different regions of the world is created by solar energy received which varies between regions and seasons. for instance, the temperature in brazil varies less than other regions as it’s closer to the equator and has a generally warmer climate, hence, the incidence of covid-19 cases does not vary much with seasons of the year, unlike the united states.4,7 on the same line, a study in indonesia showed that weather affected disease incidence and it might have been an important factor in decreasing the number of covid-19 cases in that country.10 viral disease slows down during the summer for certain viruses, including influenza and sars-cov, additionally; low temperatures were negatively correlated with incidence of cases for these viruses.7 this correlation is usually seen with table 1. peak of cases in season, doubling and halving time per country in the northern hemisphere country hemisphere doubling time (days) halving time (days) peak of cases france n° 48.95 101.65 autumn italy n° 1075.38 106.39 autumn spain n° 69.46 13.33 winter united kingdom n° 58.99 17.45 winter united states of america n° 67.93 24.38 winter average 264.14 52.64 table 2. peak of cases in season, doubling and halving time per country in the southern hemisphere country hemisphere doubling time (days) halving time (days) peak of cases australia s° – 48.68 summer argentina s° 18.88 171.69 autumn brazil s° 67.71 37.05 winter chile s° 17.97 193.46 summer new zealand s° – 291.13 – average 20.91 148.40 375j contemp med sci | vol. 7, no. 6, november-december 2021: 373–376 a.s.h. sgery et al. original seasonal theory of covid-19; one-year observation several respiratory viruses and indicates the ability of the virus to survive and spread better in these conditions. this was seen with sars-cov epidemic back in 2003.7 despite that the data are on the same line for sars-cov-2 so far, yet, we cannot confidently declare it to belong to the same group. having the seasonality feature is an important predictor for the occurrence of subsequent waves. if sars-cov-2 is proven to be seasonal and the onset of its season was predicted, herd immunity can be established early via vaccines, prior to the onset of the seasonality-sensitive second wave of infections that are seen yearly in the viral specific season, to prevent the high incidence of the cases and/or social life restrictions. will covid-19 become an annual occurrence, like many other viral respiratory diseases? in this study, the global linear chart of weekly confirmed covid-19 cases support the idea of seasonality, although the incidence of cases was increasing in summer, a jump was observed during the late autumn, i.e. november specifically, and winter period. hence, it’s likely that covid-19, like other common respiratory viruses, influenza for instance, follows a seasonal pattern. the steady increase that was recorded from may until august was likely due to the loosening of infection control measures protocol.11 the first outbreak that happened in china, followed by epidemics in iran, italy and most of europe and the usa happened during the coldest months in the year for these countries, and that was distributed within a narrow climatic band. timing of peaks differs between countries at different latitudes due to the difference in their mean temperatures in various months of the year. the peaks usually happen in periods when the mean temperature is between 2–10°c. this might be in winter for one country, like spain, france, usa, for example (usually on a latitude of 30–50°n), while it might be another season for some other countries, like those in the southern hemisphere, that had a rise and peak in the austral late summer to early autumn.12,13 doubling times and halving times are measures used to assess the rate of a disease spread. the values changes depending on context and public health measures put in place. depending on these values, countries that are in a northern hemisphere as us, uk, spain, france … etc. shows peaks of doubling time during autumn-winter seasons. these measures show a semi-baseline during warm climate of the preceding countries as us and uk for instance. for countries in the southern hemisphere, the concept works the same except for timing; argentina has the peak in autumn and australia in summer, for instance. many studies have tried to identify the effects of climate on covid-19 incidence, using data that was available to them, but failed to prove or deny a link between the two. these studies were all done during 2020, before going through one year of covid-19 pandemic, and thus they used space-fortime substitution to compensate for the fact that they had no single country that went through a full year in the presence of covid-19 (they used the difference in climate between countries as a replacement for the evolution of covid-19 over time in a single location).14-17 on the other hand, in our study, we used data from several countries after going through 1 year of the pandemic, showing the evolution and peaks of incidence, thus being more reliable in predicting the nature of this virus. however, a 2–3 year study would be required to have a definite proof about whether the virus is seasonal or not. so far, the seasonality of covid-19 (if exists) appears to be more pronounced at higher latitudes; larger seasonal amplitudes of environmental indicators are observed in higher latitudes. however, the true seasonality of covid-19 may not be noticed, if noticed might not be strong enough to conclude, in the first years of pandemic since waves of infections are noticed worldwide with new strains which necessitate a longer period of observation to be conclusive for seasonality. conclusion after over one year with pandemic sars-cov-2 a seasonal pattern shows-up, this pattern is affected by factors as; temperature, latitude and application of primary protection. to identify the optimal seasonality pattern, the latter should be taken in consideration. limitation this study was done depending on a one year interval only, yet it was supporting the seasonality theory. conflicts of interest the authors declare no conflicts of interest. funding none.  376 j contemp med sci | vol. 7, no. 6, november-december 2021: 373–376 seasonal theory of covid-19; one-year observation original a.s.h. sgery et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1108 references 1. z. wu, j.m. mcgoogan. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention j am med assoc, 323 (13) (2020), pp. 1239–1242. 2. dhama k, khan s, tiwari r, sircar s, bhat s, mali ys, singh kp, chaicumpa w, bonilla-aldana dk, & rodriguez-morales aj. (2020). coronavirus disease 2019-covid-19. clinical microbiology reviews, 33(4), e00028-20. https://doi. org/10.1128/cmr.00028–20. 3. who. rolling updates on coronavirus disease (covid-19). available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/ events-as-they-happen 4. who. covid-19 explorer. available from: https://worldhealthorg.shinyapps. io/covid/. 5. cohen j. why do dozens of diseases wax and wane with the seasons—and will covid-19?. science. available from: https://www.sciencemag.org/ news/2020/03/why-do-dozens-diseases-wax-and-wane-seasons-and-willcovid-19. 6. cdc. the flu season. available from: https://www.cdc.gov/flu/about/ season/flu-season.htm#:~:text=(6%20seasons).-,when%20is%20the%20 flu%20season%20in%20the%20united%20states%3f,last%20as%20 late%20as%20may. 7. burra p, soto-díaz k, chalen i, gonzalez-ricon rj, istanto d, caetano-anollés g. temperature and latitude correlate with sars-cov-2 epidemiological variables but not with genomic change worldwide. evolutionary bioinformatics. january 2021. doi:10.1177/1176934321989695. 8. national academies of sciences, “medicine, rapid expert consultation on sars-cov-2 survival in relation to temperature and humidity and potential for seasonality for the covid-19 pandemic (april 7, 2020)” (report, the national academies press, washington, dc, 2020). 9. cdc. principles of epidemiology in public health practice, third edition; an introduction to applied epidemiology and biostatistics. lesson 1: introduction to epidemiology; section 8: concepts of disease occurrence. https://www.cdc.gov/csels/dsepd/ss1978/lesson1/section8. html#:~:text=environmental%20factors%20include%20physical%20 factors,the%20availability%20of%20health%20services. 10. tosepu r, gunawan j, effendy ds, et al. correlation between weather and covid-19 pandemic in jakarta, indonesia. sci total environ. 2020;725:138436. 11. hussein, nawfal & naqid, ibrahim & musa, dildar. (2020). the impact of breaching lockdown on the spread of covid-19 in kurdistan region, iraq. avicenna journal of clinical microbiology and infection. 7. 34-35. 10.34172/ ajcmi.2020.07. 12. carleton t, meng kc. causal empirical estimates suggest covid-19 transmission rates are highly seasonal. available online: https://www. medrxiv.org/content/10.1101/2020.03.26.20044420v1. 13. sajadi mm, habibzadeh p, vintzileos a, shokouhi s, miralles-wilhelm f, amoroso a. temperature, humidity, and latitude analysis to estimate potential spread and seasonality of coronavirus disease 2019 (covid-19) jama netw. open. 2020;3:e2011834. doi: 10.1001/ jamanetworkopen.2020.11834. 14. ficetola g.f., rubolini d. climate affects global patterns of covid-19 early outbreak dynamics. medrxi. 2020 doi: 2020.03.23.20040501. 15. merow c, urban mc. seasonality and uncertainty in covid-19 growth rates. medrxiv. 2020 doi: 10.1101/2020.04.19.20071951. 16. qi h, xiao s, shi r, ward mp, chen y, tu w, su q, wang w, wang x, zhang z. covid-19 transmission in mainland china is associated with temperature and humidity: a time-series analysis. sci. total environ. 2020;728 doi: 10.1016/j.scitotenv.2020.138778. 17. sajadi m.m., habibzadeh p., vintzileous a., shokouhi s., miralles-wilhelm f., amoroso a. temperature, humidity, and latitude analysis to estimate potential spread and seasonality of coronavirus disease 2019 (covid-19) jama network open. 2020;3 e2011834. https://doi.org/10.1128/cmr.00028-20 https://doi.org/10.1128/cmr.00028-20 https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as-they-happen https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as-they-happen https://worldhealthorg.shinyapps.io/covid/ https://worldhealthorg.shinyapps.io/covid/ https://www.sciencemag.org/author/jon-cohen https://www.sciencemag.org/news/2020/03/why-do-dozens-diseases-wax-and-wane-seasons-and-will-covid-19 https://www.sciencemag.org/news/2020/03/why-do-dozens-diseases-wax-and-wane-seasons-and-will-covid-19 https://www.sciencemag.org/news/2020/03/why-do-dozens-diseases-wax-and-wane-seasons-and-will-covid-19 https://www.medrxiv.org/content/10.1101/2020.03.26.20044420v1 https://www.medrxiv.org/content/10.1101/2020.03.26.20044420v1 334 j contemp med sci | vol. 7, no. 6, november-december 2021: 334–339 original prevalence of over-the-counter drug abuse among adults in jeddah, saudi arabia osama yousif safdar1*, ghaidaa faisal albaz2, sara mohammed mannan2, wejdan ali alshehri2, razan mohammed altumaihi2, walaa jamal bakhashwain2, shahad tariq khayyat2, wasayf mohammed almehmadi2 1professor of pediatrics, faculty of medicine, pediatric nephrology center of excellence, king abdulaziz university, jeddah, kingdom of saudi arabia. 2medical intern, king abdulaziz university, jeddah, kingdom of saudi arabia. *correspondence to: osama yousif safdar (e-mail: safderosama@hotmail.com) abstract objectives: self-medication, also known as the use of over-the-counter drugs, can be defined as the economical choice of using treatments that have not been prescribed, recommended, or controlled by a certified healthcare professional, which can be found in pharmacies or convenience stores to treat self-recognized illnesses or symptoms. previous studies on self-medication in saudi arabia are limited; thus, this study aimed to evaluate the prevalence of over-the-counter drug abuse among an adult population in jeddah, saudi arabia. methods: this was a cross-sectional study, and a validated electronic self-administered questionnaire was administered to the general population in jeddah, saudi arabia, from june to august, 2020. results: overall, 400 participants were included in this study. the majority (94%) reported the use of over-the-counter drugs, of which 15.2% reported drug-related problems following over-the-counter drug use. the most frequently preferred over-the-counter drug group was analgesics/antipyretics (90.4%). more than three-quarters of our study participants stated that they read the instructions provided in the leaflet accompanying their medications carefully before use (76.3%). conclusion: we found a highly significant prevalence of over-the-counter medication use, which exceeded 50%, without any significant association between over-the-counter use and other factors, except for the presence of chronic diseases. we recommend that, the ministry of health, kingdom of saudi arabia should play a major role in monitoring the practice of self-medication by implementing policies and creating surveillance committees to that effect. keywords: prevalence, over-the-counter, drug, abuse, adults, jeddah issn 2413-0516 introduction self-medication, also known as the use of over-the-counter (otc) drugs, refers to the cost-effective use of treatments not prescribed or recommended by a certified healthcare professional to treat self-identified illnesses or symptoms.1,2 because of their existing benefits, over-the-counter drugs are becoming more popular among the general population and the healthcare system.3 first, the availability of over-the-counter medications allows patients to treat many common health conditions without the assistance of a health care professional. second, it saves time for both patients with minor illnesses and medical staff, allowing them to focus on more complex cases. it also improves public health at a low cost by reducing the frequency of clinical visits. third, it has a positive impact on the healthcare system, particularly by reducing reliance on scarce and valuable health resources.3 otcs are classified into different categories by the world health organization’s anatomical therapeutic chemical classification, such as analgesics, laxatives, and sedatives. cough and cold medications, antihistamines, dermatologicals, and antidiarrheals.4 a previous study found that 57.7 percent of otc drugs purchased from community pharmacies in aden, yemen, were used. 5 furthermore, 285 medications were purchased without a prescription in a study conducted among adults in riyadh. of these, 49 percent should have been dispensed only by prescription, while 51 percent were otc medications.5 inadequate supervision can result in a variety of side effects due to drug-drug interactions, drug-food interactions, and allergic reactions.6 a drug-drug interaction is a change in the effect of a drug when two or more medications are used at the same time; this can increase the risk of side effects depending on the medication used.6 when ibuprofen is combined with a prescribed anti-inflammatory medication, they will react together and produce more anti-inflammatory activity or pain relief than the body requires, which can be harmful to the kidneys and liver.7 a drug-food interaction is a change in the effect of a drug when it is combined with certain foods. it can cause a drug’s absorption to be delayed, reduced, or increased, which can have negative consequences. allergic reactions occur when the immune system recognizes the drug as an allergen and produces antibodies known as immunoglobulin e (ige) that are specific to the drug. this reaction causes skin rashes, itching, wheezing, breathing difficulties, and anaphylactic shock, which can be fatal.9 recently, there has been an increase in concern about the effects of otc drug misuse on various populations. locally, a study was conducted on the frequency of using non-prescribed medications in a population sample of 306 people in al madina city, as well as its impact on saudi arabia’s quality of care. 72.5 percent reported using non-prescribed medication, and 24.3 percent reported experiencing side effects from using non prescribed medications, with no significant difference between men and women. analgesics were the most commonly used type of medication. 10 furthermore, according to another study conducted in poland among 386 adults, approximately 91 percent of participants declared that approximately 91% of participants declared using otc pain relievers.10 8 335j contemp med sci | vol. 7, no. 6, november-december 2021: 334–339 o. y. safdar et al. original prevalence of over-the-counter drug abuse among adults in jeddah, saudi arabia previous studies on self-medication in saudi arabia are limited. therefore, this study aimed to evaluate the prevalence of otc drug abuse among the adult population in jeddah, saudi arabia. methods study design an observational cross-sectional study was conducted randomly among the general population in jeddah, saudi arabia, from june to august, 2020. sample size the sample size was 400 participants, which was calculated based on the creative research systems website with a confidence level of 95%.11 the inclusion criteria were adults aged ≥18 years; all health care professionals were excluded from the study. all participants were invited to voluntarily participate in the study by advertisements through different social media platforms. a previously validated, reliable, and self-administered questionnaire was used. it was adopted from the al madina study.12 the questionnaire was designed using google forms (mountain view, ca, usa), and it consisted of two sections: the first section contained demographic information (gender, age, marital status, level of education, and occupation), while the second section assessed participants’ patterns of otc drug use. additionally, the questionnaire is attached as supplementary material. ethical considerations the study was approved by the biomedical ethics research committee at authors’ affiliated institution. consent was obtained from all participants after they were notified around the study objectives and confidentiality of the responses in the introduction section of the electronic questionnaire. statistical analyses the data were analyzed using spss software (statistical package for social sciences, version 21). frequency tests were used to describe qualitative data, quantitative data were expressed as mean and standard deviation (mean ± sd), and the chi-square test was used to test the relationship between variables. a p-value <0.05 was considered statistically significant. results initially, 576 responses were received, and only 400 participants were selected to be included in the study based on our inclusion criteria. of these, there were 200 men (50%) and 200 women (50%), with a mean age of 36.93 (±14.4). more than half of the respondents (54.5%, n = 218) were married, 188 (47%) were workers, and 314 (78.5%) had a high education level (table 1). approximately 376 (94%) participants reported the use of otc drugs. among those, 209 (55.6%) remained healthy, and 57 (15.2%) reported drug-related side effects after the consumption of otc drugs. most participants stated that they read the instructions provided in the leaflet accompanying their medication carefully before use (76.3%). diabetes was reported as the most common chronic disease among the participants (27.4%), followed by hypertension (26.6%) and high cholesterol (19.1%) (table 2). a very high proportion of the participants (76.9%) used otc drugs use in the past six months for less than five times. participants mostly obtained their medications from private pharmacies (n = 337, 89.6%), followed by governmental hospital pharmacies (n = 21, 5.6%). regarding the duration of otc drug use, 239 respondents (63.6%) used medication for 1–3 days. in 280 (74.5%) participants, pharmacists had a major impact with regard to the correct dosage of otc drugs, followed by their previous prescription (n = 144, 38.3%). moreover, 276 (73.4%) participants reported that pharmacists were the main source of information when deciding to use an otc drug, followed by their previous prescription (n = 137, 36.4%). the most frequently preferred otc drug group was analgesics/antipyretics (n = 340, 90.4%), followed by vitamins and food supplements (n = 196, 52.1%) (table 3). our results showed no significant relationship between reading the package insert and experiencing the side effects of otc drugs (p = 0.610) (table 4). table 1. sociodemographic profile of participants (n = 400) variables frequency percent marital status single 167 41.8 married 218 54.4 divorced/widowed 15 3.8 job status student 91 22.8 non worker 121 30.2 worker 188 47.0 level of education elementary 1 0.3 high school/technical 85 21.2 bachelor’s degree and above 314 78.5 table 2. parameters related to non-prescribed medication parameters yes no n % n % n = 400 have you ever used a non-prescribed medication? 376 94 24 6 n = 376 history of side effects with non-prescribed medications 57 15.2 319 84.8 reading insert leaflet 287 76.3 89 23.7 having chronic disease 167 44.4 209 55.6 chronic diseases hypertension 100 26.6 276 73.4 heart diseases 46 12.2 330 87.8 high cholesterol 72 19.1 304 80.9 diabetes 103 27.4 273 72.6 endocrine disorders 29 7.7 347 92.3 nervous system disorders 23 6.1 353 93.9 skeletal/rheumatic disorders 41 10.9 335 89.1 others 24 6.4 352 93.6 336 j contemp med sci | vol. 7, no. 6, november-december 2021: 334–339 prevalence of over-the-counter drug abuse among adults in jeddah, saudi arabia original o.y. safdar et al. table 4. participants who read package insert and reported complications do you read the package insert? total p-value no yes have you experienced drug related problems? no 74 245 319 yes 15 42 57 total 89 287 376 0.610 table 5. the association of the duration of using over the-counter (otc) drugs and drug side effects how often do you use otc drugs? total p-value 1–3 days 3–6 days 6–9 days >9 days have you experienced drug related problems? no 209 81 13 16 319 yes 30 22 1 4 57 total 239 103 14 20 376 0.144 table 3. parameters and pattern of administration of non-prescribed medication variables frequency percent frequency of use non-prescribed medication over the past 6 months <5 times 289 76.9 5–10 times 59 15.7 >10 times 28 7.4 from where do you usually have the medication private pharmacy 337 89.6 governmental hospital pharmacy 21 5.6 private hospital pharmacy 15 4 free medical samples 3 0.8 duration of taking non-prescribed medication 1–3 days 239 63.6 3–6 days 103 27.4 6–9 days 14 3.7 >9 days 20 5.3 how can you define the dose of the medication pharmacists 280 74.5 previous prescription 144 38.3 according to severity of symptoms 66 17.6 family and friends 54 14.4 others 19 5.1 source of information when deciding to take a non prescribed medication pharmacists 276 73.4 previous prescriptions 137 36.4 family and friends 123 32.7 personal experience 127 33.8 mass media 56 14.9 others 2 0.5 type of medication commonly used analgesics/ antipyretics 340 90.4 antacids, antispasmodics, digestives 146 38.8 vitamins and food supplements 196 52.1 antitussive/ anti-histaminic 177 47.1 creams and topical agents 184 48.9 chronic disease medications 12 3.2 antibiotics 74 19.7 others 3 0.8 the duration of otc drug use was not significantly associated with drug side effects (p = 0.144) (table 5). by contrast, our results revealed a significant relationship between the presence of chronic disease and the use of otc drugs (p < 0.001) (table 6). there was no significant association between the use of otc drugs and marital status, job status, level of education, and age (table 7). in (figure 1) otcs are classified into 8 categories, with analgesics being the most commonly used otc and chronic table 6. the association of chronic disease with over-thecounter (otc) drug use have you ever used otc drug? total p-value no yes chronic disease healthy 2 209 211 chronic disease 22 167 189 total 24 376 400 0.000 table 7. association between sociodemographic variables and over-the counter (otc) drug use have you ever used otc drugs? total p-value no yes marital status single 12 155 167 0.480 married 12 206 218 divorced/ widowed 0 15 15 total 24 376 400 job status student 5 86 91 0.726non-worker 9 112 121 worker 10 178 188 total 24 376 400 level of education elementary 0 1 1 0.604 high school/ technical 7 78 85 bachelor’s degree and above 17 297 314 total 24 376 400 age of participants <65 23 370 393 0.352 >65 1 6 7 total 24 376 400 337j contemp med sci | vol. 7, no. 6, november-december 2021: 334–339 o. y. safdar et al. original prevalence of over-the-counter drug abuse among adults in jeddah, saudi arabia fig. 1 type of medication commonly used. disease medications being the least commonly used. analgesics (90.4%) was the most preferred otc drug group, followed by vitamins and food supplements (52.1%) (p = 0.841, figure 1). discussion this study determined the most commonly used otc medication, the sources of information for otc medication use, and the relationship between otc medication use and various parameters. over-the-counter (otc) medication administration has been widely observed for several decades, particularly in developing countries.13 the main finding of our study was that 94 percent of study respondents used medications without a prescription, which is consistent with the findings of another study (93.1 percent) conducted in majmaah, saudi arabia.14 in contrast to other studies on the use of otc drugs in serbia (57.0 percent), the united kingdom (38.0 percent), and northern ireland (32.2 percent), the prevalence of otc medication use in our study was higher.15-17 furthermore, any significant otc medication rate greater than 50% is more likely to pose a health risk.18 the difference among the rates could be owing to variations in socio-economic factors, such as cultural beliefs, low cost, ease of access, the saving of time, and the severity of illness based on an individual’s knowledge and previous experience.19 the most commonly used types of otc medications among our respondents were analgesics (90.4 percent), followed by vitamins and food supplement drugs (52.1 percent). analgesics were frequently used because they are available in a variety of brands, formulations, and doses and can be used to treat various types of pain.20 this finding is consistent with the findings of a wazaify study, which found that analgesics (76.4 percent) were the most commonly used, followed by minerals and/or vitamins (43.4 percent).17 antibiotics (22.3 percent) were the most common medication dispensed without prescriptions, contrary to the findings of a previous study in saudi arabia.3 we believe this reduction in antibiotic use was owing to the implementation of a new rule by the ministry of health that warns against selling antibiotics without prescriptions and prohibiting pharmacists from dispensing any drug without a prescription issued by a licensed doctor to practice in the kingdom. violation of this rule could result in imprisonment for up to six months, withdrawal of the license, and a fine that could reach 100,000 sar.21 in our study, the main source was found to have a major influence on participants’ choices of otc drugs, which was the pharmacists’ recommendation and that correlated with the results of four previous studies in northern ireland, eritrea, and japan.17,22 furthermore, pharmacists play a significant role in assisting with the safe and proper use of non-prescribed self-medication, as well as in resolving and avoiding drug related problems in order to achieve ideal patient quality of life and the best outcomes. as a result, it is critical to consider this factor when accounting for both pharmacist practices and training. in contrast, media advertising was identified as the primary source of information in a study conducted in pakistan (46.7 percent).23 a related study was conducted in 20 selected pharmacy outlets in eritrea and reported that the percentage of respondents who read the package insert before using otc drugs was very low, representing only one-third (35%) of the respondents. additionally, a higher proportion of otc drug users (81.8%) exhibited high risk practices.22 in contrast to this finding, we discovered that 76.3 percent of respondents said they read the package insert. furthermore, they reported drug-related issues as a result of self-medication (23.7 percent). this suggests that our respondents were more cautious in order to avoid unfavorable outcomes. as a result, it is critical to assist patients in making use of the information in package leaflets and to improve their understanding.24 according to the findings, respondents with a higher level of education (bachelor’s degree or higher) are more likely to use otc, which is consistent with the findings of other studies because they can understand and read the descriptions on drug packages.15,25 in our study, there was no significant association between self-medication and educational level, which is in agreement with the finding of a study conducted in al majmaah city.14 however, a study conducted in hail reported a significant relationship with 71.5% (n = 663) of the highly 338 j contemp med sci | vol. 7, no. 6, november-december 2021: 334–339 prevalence of over-the-counter drug abuse among adults in jeddah, saudi arabia original o.y. safdar et al. educated responded affirmatively for receiving self-medication.26 similar trend was reported by a study conducted in nepal.27 according to our findings, two-thirds (63.6 percent) of the participants used non-prescribed medication for 1–3 days, which is consistent with previous findings reported in al madina city.12 otc drugs can expose patients to unanticipated drug-related problems; 15.2 percent of our participants had a history of drug-related side effects, which was lower than the proportion reported in al madina city (24.3 percent).12 there was no significant association (p = 0.144) regarding the relationship between the duration of otc drug use and the consequent complications. as shown in a previous study, patients with chronic disease using otc medications had an increased risk of adverse drug reactions.28 in our study, there was a significant relationship between having a chronic disease and the use of otc drugs (p = 0.000), which was similar to the results of a study conducted in serbia (p = 0.019).15 there are some limitations to our research. it is a cross sectional study with recall and selection biases. the majority of the included participants were highly educated and over the age of 65, which does not represent the entire jeddah because the questionnaire was not available for a diverse range of participants, such as illiterates and people from different socioeconomic backgrounds. because our study only looked at the prevalence of otc medication, more research is needed to determine the specific reasons that drive people to self-medicate. conclusion this study evaluated the prevalence of self-medication among adults in jeddah, saudi arabia. it was found that a highly significant rate of otc medication use was present among our participants, and this rate exceeded 50%; however, there was no significant association between otc use and any other factors except for the presence of chronic diseases. therefore, we advise the ministry of health, kingdom of saudi arabia, to play a major role in monitoring the practice of self-medication by implementing policies and setting-up surveillance committees to that effect. additionally, different platforms on social media should help raise awareness among society around the hazards of using non-prescribed medications, especially for long-term use. moreover, our study suggests the importance of reassessing the list of medications that can be sold as otc and providing a proper training program for community pharmacists. we also recommend that the saudi food and drug authority should carefully monitor drug companies and pharmacists. acknowledgments the authors gratefully acknowledge the cooperation of all study participants. conflicts of interest disclosure the authors declare that no conflicts of interest are related to this work.  references 1. us food and drug administration. understanding over-the-counter medicines, 2018 [cited 2020 nov 18]. available from: https://www.fda.gov/ drugs/buying-using-medicine-safely/understanding-over-counter-medicines. 2. world health organization. the role of the pharmacist in self-care and self-medication: report of the 4th who consultative group on the role of the pharmacist, the hague, the netherlands, 26–28 august, 1998. available from: https://apps.who.int/iris/handle/10665/65860. 3. aljadhey h, assiri ga, mahmoud ma, al-aqeel s, murray m. self-medication in central saudi arabia: community pharmacy consumers’ perspectives. saudi med j. 2015; 36(3): 328–34. 4. pérez a, santamaria ek, operario d, tarkang ee, zotor fb, cardoso sr de sn, et al. no 主観的健康感を中心とした在宅高齢者における 健 康関連指標に関する共分散構造分析. bmc public health. 2017; 5(1): 1–8. available from: https://ejournal.poltektegal.ac.id/index.php/ siklus/article/view/298%0ahttp://repositorio.unan.edu.ni/2986/1/5624. pdf%0ahttp://dx.doi.org/10.1016/j.jana.2015.10.005%0ahttp://www. biomedcentral.com/1471-2458/12/58%0ahttp://ovidsp.ovid.com/ovidweb. cgi?t=js&page=refe. 5. abood ea, wazaify m. abuse and misuse of prescription and nonprescription drugs from community pharmacies in aden city—yemen. subset use misuse. 2016; 51(7): 942–7. 6. familydoctor.org. otc medicines: know your risks and reduce them [cited 2020 nov 18]. available from: https://familydoctor.org/otc-medicinesknow-your-risks-and-reduce-them/. 7. american academy of allergy, asthma, and immunology. medications and drug allergic reactions [cited 2020 nov 18]. available from: https://www. aaaai.org/conditions-and-treatments/library/allergy-library/medicationsand-drug-allergic-reactions. 8. schlaeppi c, vanobberghen f, sikalengo g, glass tr, ndege rc, foe g, et al. prevalence and management of drug–drug interactions with antiretroviral treatment in 2069 people living with hiv in rural tanzania: a prospective cohort study. hiv med. 2020; 21(1): 53–63. 9. vervloet d, durham s. abc of allergies: adverse reactions to drugs. bmj. 1998; 316(7143): 1511–4. 10. wójta-kempa m, krzyzanowski dm. correlates of abusing and misusing over-the-counter pain relievers among adult population of wrocław (poland). adv clin exp med. 2016; 25(2): 349–60. 11. creative research systems. sample size calculator [cited 2020 nov 18]. available from: https://www.surveysystem.com/sscalc.htm. 12. mahrous ms. frequency of use of non-prescribed medication among population sample from al madina city and its impact on quality of care in saudi arabia. int j health sci (qassim). 2018; 12(5): 3–9. 13. ezechukwu c, egbuonu i, chukwuka j. drug treatment of common symptoms in nnewi south eastern nigeria: what mothers do?. niger j clin pract. 2005; 8(1): 1–3. 14. alzahrani m, alhindi t, almutairi a, aldajani m, sami w. frequency of using non-prescribed medication in majmaah city, saudi arabia—a cross sectional study. j pak med assoc. 2015; 65(8): 825–8. 15. gazibara t, nurkovic s, kisic-tepavcevic d, kurtagic i, kovacevic n, gazibara t, et al. pharmacotherapy and over-the-counter drug use among elderly in belgrade, serbia. geriatr nurs. 2013; 34(6): 486–90. 16. poole c, jones d, veitch b. relationships between prescription and nonprescription drug use in an elderly population. arch gerontol geriatr. 1999; 28(3): 259–71. 17. wazaify m, shields e, hughes cm, mcelnay jc. societal perspectives on over-the-counter (otc) medicines. fam pract. 2005; 22(2): 170–6. 18. shaghaghi a, asadi m, allahverdipour h. predictors of self-medication behavior: a systematic review. iran j public health. 2014; 43(2): 136–46. 19. thuzar m, aung pl. prevalence of self-medication and its influence in the labor force in rural hlaing tharyar, yangon, myanmar. open publ health j. 2019; 12(1): 38–44. 20. moore ra, wiffen pj, derry s, roy ym, tyrrell l, derry cj. non-prescription (otc) oral analgesics for acute painan overview of cochrane reviews. cochrane database syst rev. 2013; 2013(10): cd010794. 21. the ministry of health. moh news moh warns against selling antibiotics without prescription [cited 2020 nov 18]. available from: https://www.moh. gov.sa/en/ministry/mediacenter/news/pages/news-2018-04-17-004.aspx. 339j contemp med sci | vol. 7, no. 6, november-december 2021: 334–339 o. y. safdar et al. original prevalence of over-the-counter drug abuse among adults in jeddah, saudi arabia 22. tesfamariam s, anand is, kaleab g, berhane s, woldai b, habte e, et al. self-medication with over the counter drugs, prevalence of risky practice and its associated factors in pharmacy outlets of asmara, eritrea. bmc public health. 2019; 19(1): 159-68. https://bmcpublichealth.biomedcentral.com/ track/pdf/10.1186/s12889-019-6470-5.pdf. 23. aziz mm, masood i, yousaf m, saleem h, ye d, fang y. pattern of medication selling and self-medication practices: a study from punjab, pakistan. plos one. 2018; 13(3): e0194240. 24. kim hj, yang ym, choi ej. use patterns of over-the-counter (otc) medications and perspectives on otc medications among korean adult patients with chronic diseases: gender and age differences. patient prefer adherence. 2018; 12: 1597–606. 25. aqeel t, shabbir a, basharat h, bukhari m, mobin s, shahid h, et al. prevalence of self-medication among urban and rural population of islamabad, pakistan. trop j pharm res. 2014; 13(4): 627–33. 26. ansari m, alanazi a, moin a. consumers’ awareness, attitude and associated factors towards self-medication in hail, saudi arabia. plos one. 2020; 15(4): e0232322. 27. sharma d, gurung d, kafle r, singh s. knowledge and practice on overthe-counter drugs among adults of age group 20 and above residing in chapapani-12, pokhara, kaski, nepal. int j sci reports. 2017; 3(3): 79–86. 28. sajith m, suresh sm, roy nt, pawar da. self-medication practices among health care professional students in a tertiary care hospital, pune. open publ health j. 2017; 10(1): 63–8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1098 179j contemp med sci | vol. 9, no. 3, may-june 2023: 179–186 original the role of neuropsychological assessment in the differential diagnoses of late-onset depression, dementia, and mild cognitive impairment gulin morkavuk¹* , gokce sirvanli¹ 1department of neurology, faculty of medicine, ufuk university, ankara, turkey. *correspondence to: morkavuk gulin (e-mail: drgcmor@yahoo.com) (submitted: 26 october 2022 – revised version received: 20 november 2022 – accepted: 12 december 2022 – published online: 26 june 2023) abstract objective: the present study aimed to compare the neuropsychological test parameters of dementia, depression, and mci patients and determine the disease-specific test characteristics and their relationship with electroencephalography. methods: ninety-one patients who were admitted to the neurology outpatient clinic with forgetfulness complaints between october 2019 and march 2022 and whose neuropsychological tests were performed were included in the study. the files of these 91 patients were reviewed retrospectively and their sociodemographic data were recorded. furthermore, the eeg results which were taken during the patients’ evaluation period due to forgetfulness were evaluated. neuropsychological tests were compared between patients with dementia, mci and depression. it was also investigated whether there was a relationship between npt test parameters and eeg in patients with eeg results. results: the study was completed with 87 patients. of these 87 patients, 54 were female and 33 were male. twenty-four patients had depression, 16 mci, and 47 dementia. all of the dementia patients had alzheimer’s type dementia. when dementia, depression, and mci groups were compared, the age difference was statistically significant (p = 0.001). the mean age of the depression group was 66.5, the mci group was 73.5, and the dementia group was 77. wms-i, wms-ii, wms-iii, wms-iv, similarities test, clock drawing test, trail making test, shape copying, language, and mood evaluation tests were statistically significantly different between the groups. there was no statistically significant difference between the groups regarding gender, education level, dominant hand, and occupation. eeg background activity frequencies were also examined between the groups, and there was no statistically significant difference. conclusion: in conclusion, when evaluating patients who present with the complaint of forgetfulness, a detailed neuropsychological evaluation must be performed in addition to other diagnostic tests. sensitive tests should be included to confirm the diagnosis, especially in cases where being in between for the differential diagnosis. thus, further studies are needed on this subject. keywords: neuropsychological assessment, dementia, depression, mild cognitive impairment issn 2413-0516 introduction dementia is a common cognitive disorder that significantly affects the patient’s quality of life. mild cognitive impairment (mci) is a clinical condition with a high risk of progression to dementia. mci is considered a prodrome of dementia that includes more forgetfulness than expected for a person’s age, but almost all of the daily functionality is preserved and does not meet clinically probable dementia criteria.1 it is known that about half of the patients diagnosed with mci develop dementia within 3 years,1 and estimated that in the 75–79 age group, the incidence of mci is 22.5 per 1000 people per year and the number rises to 60.1 in the 85 and over age group.2 mci is associated with a significant risk of developing dementia, but approximately 18% of mci patients do not spontaneously revert to normal cognition and progress to dementia.3 due to the lack of effective disease-modifying treatments for advanced dementia, diagnosis and disease intervention at an early stage, particularly at the mci stage, is recognized as a critical strategy in disease management that can potentially affect long-term outcomes. however, there is currently no consensus on guidelines for routine screening of mci, resulting in a significant number of undiagnosed mci patients in the community.4 therefore, there is a need to develop reliable tools that can be used from screening to diagnosis. another condition that is mistaken with dementia is depression in older adults. this condition, called late-onset depression (lod), appears for the first time after the age of 50–65 and differs from early-onset depression in many areas. it is known that some of these patients have cognitive disorders.5-7 moreover, depression is common during dementia and may even appear as the first symptom of dementia.8 today, mini mental test (mmt) is generally used to support the diagnosis of dementia in neurology outpatient clinics. however, this test is insufficient to detect mci status. in this case, a detailed neuropsychological evaluation may be guiding. the present study aimed to compare the neuropsychological test parameters of dementia, depression, and mci patients and determine the disease-specific test characteristics and their relationship with electroencephalography (eeg). material and method ninety-one patients who were admitted to the neurology outpatient clinic with forgetfulness complaints between october 2019 and march 2022 and whose neuropsychological tests were performed were included in the study. the files of these 91 patients were reviewed retrospectively and their sociodemographic data were recorded. furthermore, the eeg results which were taken during the patients’ evaluation period due to forgetfulness were evaluated. the wms (wechsler memory scale) (i-ii) personal-actual information and orientation subtest was performed within the scope of the neuropsychological assessment battery. wms-r (iii) mental control, wms (iv) logical story test, wms-r (v) number range subtest, wais-r (wechsler adult intelligence scale-revised) binary similarity https://orcid.org/0000-0001-7522-8585 180 j contemp med sci | vol. 9, no. 3, may-june 2023: 179–186 the role of neuropsychological assessment in forgetfulness original g. morkavuk & g. sirvanli subtest, verbal fluency test, clock drawing test, luria alternan sequences test, stroop test, and trail making test assessed attention and executive functions. öktem verbal memory processes test (ö-vmpt) and wms (vi) visual production subtest were used as memory tests. line direction determination test, benton face recognition test, and shape copying test were performed for visual-spatial evaluation. boston naming test was used for language skills, and geriatric depression scale or depression, anxiety, stress scale (dass) tests were applied for mood assessment. if the patients were clinically evaluated and able to reach a diagnosis, they were included in one of the depression, dementia, and mci groups. the diagnosis of mci was made according to the peterson criteria.9 four patients whose diagnoses could not be clarified were excluded. the remaining 87 patients were compared in terms of mmt, montreal cognitive assessment (moca), and neuropsychological assessment battery tests. first, the tests were compared between the 3 groups together, then between the mci and depression and mci and dementia groups. it was also investigated whether there was a relationship between npt test parameters and eeg in patients with eeg results. this study was performed as per the declaration of helsinki and with the approval of university faculty of medicine ethics committee. tests performed within the scope of neuropsychological assessment battery: 1. wms (i-ii) information and orientation : it is a subtest in which the person is evaluated regarding age, date of birth, follow-up of current events, place, and time information. 2. attention and executive function tests 2.1. wms-r mental control subtest (wms iii): it is a test applied regarding the ability to maintain attention, which is one of the frontal functions. 2.2. wms logical memory (wms-iv): it is a test that measures the reproducibility of the verbally given story in the short and long-term recall. 2.3. wms-r digit span test (wms-v): the digit span test is divided further into two subtests. straight number range is used for ‘short-term memory’ evaluation, while inverse number range is used for ‘complex attention’ evaluation as it requires tracking skills. 2.4. wais-r similarities test : it is a subtest given for the evaluation of abstraction skills. 2.5. verbal fluency test: it includes lexical verbal fluency tests (with letters k, a, s -in turkish version-) sensitive to frontal lobe lesions and semantic verbal fluency tests (animal counting, fruit-name counting) sensitive to temporal lobe lesions and used to measure frontotemporal entorhinal cortex functions. 2.6. clock drawing test: it is a short-term practical screening test that provides information on intellectual and perceptual skills. it provides an assessment of skills such as auditory comprehension, motor planning and management, visual memory and restructuring, numerical knowledge, abstract thinking, focusing and tolerance to frustration, inhibition of physical properties of stimuli. 2.7. luria alternan sequences test: it is one of the bedside tests that evaluate sequencing, planning, and ability to cope with distractors. 2.8. stroop test (çapa version): it is a test that measures complex attention. as the duration of interference increases, the patient’s difficulty in maintaining attention and suppressing distracting stimuli emerges. it requires literacy. 2.9. trail making test: it is a complex attention level determining test sensitive to motor speed and visual scanning, assessing focused attention (a-form) and category switching skills (b-form). it requires literacy. 3. memory tests 3.1. öktem verbal memory processes test (ö-vmpt): it evaluates memory in areas such as primacy effect and recency effect, creating a certain learning strategy, recalling information, and recognizing information. it helps to confirm the diagnosis by distinguishing the secondary and primary types of memory impairment. 3.2. wms visual reproduction (wms-vi): it is a test used in the clinical measurement of visual memory. instant and long-term recall values are checked. 4. visual-spatial skill tests 4.1. benton face recognition test: it is sensitive to damage to the “what” pathway in the visual association cortex. it is used for the evaluation of occipitotemporal region functions in visual-spatial skills. 4.2. judgement of line orientation test: the occipitoparietal region is used to evaluate the “where” pathway functions. complex visual perception is measured. 4.3. shape copy test: it is a practical short-term test used to evaluate visuospatial restructuring ability. 5. language skill 5.1. boston naming test: this test, which contains items of varying difficulty, is used to measure the ability to name the seen object. 6. mood assessment 6.1. the geriatric depression scale (gds): it evaluates change of effect, restlessness, inactivity, disturbing thoughts, withdrawal from life, negative judgments about the past, present, and future. 6.2. the depression, anxiety, stress scale (dass): it is a test that evaluates stress, anxiety, and depression based on self-report in 14 items. statistical analysis statistical analyses were performed with statistical package for the social sciences (spss) 22.0 (ibm, chicago, illinois, usa). kolmogorov smirnov and shapiro-wilk tests were performed to test the homogeneity and normality of the scaled data. pearson chi square and fischer were used for evaluation of monimal data of each groups, student’s t for scale parametric data, mann whitney-u for scale nonparametric data. ona way-anova, kruskal wallis, posthoc multiple comparison (bonferroni) tests were used for analysis of multiple groups. univariate multinomial logistic regression test was performed in multivariate analysis. a p-value of <0.05 was considered statistically significant. results ninety-one patients who underwent neuropsychological tests were included. however, 4 patients whose diagnosis could not be clarified were excluded. the study was completed with 87 patients. of these 87 patients, 54 were female and 33 were male. twenty-four (27.6%) patients had depression, 16 (18.4%) 181j contemp med sci | vol. 9, no. 3, may-june 2023: 179–186 g. morkavuk & g. sirvanli original the role of neuropsychological assessment in forgetfulness mci, and 47 (54%) dementia. all of the dementia patients had alzheimer’s type dementia. the sociodemographic characteristics of the patients are presented in table 1 in detail. all patients were planned to be applied to the same tests. nevertheless, tests that could not be completed by the patients were not included in the study. among the applied neuropsychological tests, wms-i was applied to 77 patients, wms-ii to 80, wms-iii to 64, wms-iv to 50, wms-v to 76, wms-vi 68, similarities test to 78, clock drawing test to 79, luria alternan sequences test to 76, verbal fluency test to 70, stroop test to 13, face recognition to 61, line direction determination to 66, shape copying to 71, language to 75, trail making test to 23, sbst to 37, and mood tests to 68 patients (table 2). when dementia, depression, and mci groups were compared, the age difference was statistically significant (p = 0.001). the mean age of the depression group was 66.5, the mci group was 73.5, and the dementia group was 77. there was no statistically significant difference between the groups regarding gender, education level, dominant hand, and occupation. wms-i, wms-ii, wms-iii, wms-iv, similarities test, clock drawing test, trail making test, shape copying, language, and mood evaluation tests were statistically significantly different between the groups. in correlation with neuropsychological tests, mmt was also statistically significantly different between groups. while the mmt score was similar in the depression and mci groups, it was significantly lower in the dementia group. moreover, as expected, mood tests had statistically significantly higher scores in the depression group. eeg background activity frequencies were also examined between the groups, and there was no statistically significant difference (table 3). when dementia and mci groups were compared in terms of sociodemographic characteristics, there was a statistically significant difference in terms of gender (p = 0.022). while the mci group consisted of 10 male and 6 female patients, the dementia group had 14 male and 33 female patients. male patients were more in the mci group, and female dominance was observed in the dementia group. when the 2 groups were compared in terms of occupations, it was determined that the number of retired soldiers/policemen was higher in the mci group, and the number of housewives and retired teachers in the dementia group was higher, and the difference was statistically significant (p = 0.047). there was no significant difference between the groups in terms of education level. when neuropsychological battery tests were examined separately between dementia and mci groups, a statistically significant difference was observed between wms-ii, wms-iv, wms-v, similarity test, clock drawing test, trail making test, shape copying, and sbst (p = 0.02, p = 0.015, p = 0.038, p = 0.015, p = 0.01, p = 0.026, p = 0.02, p = 0.029, respectively) when the depression and mci groups were examined separately, no difference was observed between sociodemographic data. similarly, no statistically significant difference was determined in any of the npt subtests when examined separately. as expected, mood test scores were statistically significantly higher in the depression group (p = 0.008). besides, when neuropsychological test parameters and eeg background activity frequencies were examined in terms of correlation, there was no statistically significant correlation in any group. the univariant multinomial regression analysis of the factors found in table 3 is summarized in table 4. when we table 1. the sociodemographic characteristics of the patients age, year, mean ± sd,range 71,83 ± 9,69 (42–86) diagnosis, n(%) depression mci dementia 24 (%27,6) 16 (%18,4) 47 (%54) gender, n(%) male female 33 (%37,9) 54 (%62,1) dominant hand, n(%) right left 83 (%95,4) 2 (%2,3) education, n(%) uneducated primary school junior high school high school university 2 (%2,3) 29 (%33,3) 9 (%10,3) 25 (%28,7) 20 (%23) occupation, n(%) housewife tailor soldiers/policemen engineer doctor official teacher driver other 26 (%29,9) 4 (%4,6) 5 (%6,9) 4 (%4,6) 2 (%2,3) 14 (%16,1) 9 (%10,3) 3 (%3,4) 10 (11,5) considered the depression group as the reference category, one unit increase in the age variable increased the probability of being diagnosed with dementia 1.129 times and the probability of mci being 1.091 times. the impaired wms1 test increases the probability of patients having dementia 6.697 times compared to depression but does not cause a significant difference between mci and depression diagnoses. similarly, defective wms2, wms3, and wms4 tests increase the probability of patients having dementia compared to the probability of having depression. in mood tests (dass, gds), a negative correlation was determined between diagnoses. accordingly, a disordered mood test increases the probability of dementia by 0.200 units and the probability of mci by 0.107 units. in other words, the fact that this test had pathological results increased the likelihood of patients having depression. a detailed review of the univariate multinomial regression analysis is presented in table 4. discussion alzheimer’s dementia (ad) is a type of dementia with insidious onset and memory impairment. however, sometimes mood disorders may occur in the early stages of the disease.10 whether late-onset depression is a pre-stage of dementia or a risk factor for dementia is still controversial. most studies report that 30-50% of individuals with ad present with symptoms of depression such as low mood, apathy, and social withdrawal.11 these depressive disorders that occur in dementia cause increased deficits in functioning, increased problem behaviors, increased nursing home placement, caregiver stress, and increased mortality.12,13 furthermore, a study has demonstrated that antidepressant treatment stimulates neurogenesis and tropism, and 182 j contemp med sci | vol. 9, no. 3, may-june 2023: 179–186 the role of neuropsychological assessment in forgetfulness original g. morkavuk & g. sirvanli improves learning properties and differentiation and proliferation of new neurons.14 therefore, strategies that promote the prevention, early diagnosis, and adequate treatment of depression can potentially prevent or delay dementia in older adults15 we can interpret this situation as depression as a modifiable risk factor for dementia. documented cognitive difficulties in elderly depressed patients without dementia have been indicated to be within memory, attention, naming, verbal fluency, visuospatial abilities, processing speed, and executive functions.8,16-18 findings from studies that specifically evaluated the differences in memory profiles among ad patients when compared to depressed patients show that although both groups exhibited impaired performance on immediate and delayed recall tasks, patients with depression generally retained the learned information.8 studies have generally compared ad and depression or ad and mci patients in terms of npts. we wanted to compare the npt results by including all 3 groups in our study and aimed to determine the specific tests specific to each group by examining the pairwise combinations in addition to the triple group. however, when mci and dementia groups were compared, although we could detect differences between npt results, we could not determine any difference between mci and depression groups. long-term memory can be divided into two groups: declarative memory and procedural memory.19 declarative memory is an open process, conscious, and controlled. with this process, visual or verbal information emerges at the end of a conscious recall. declarative memory consists of two parts, episodic and semantic memory. semantic memory refers to general information about the world. knowing the capital city of germany or mathematical formula is all about semantic memory. it is not linked to a specific time frame. semantic memory is not sensitive to loss or change of information.20 on the contrary, episodic memory is related to time and context. it is based on specific events in the person’s past. what we ate for dinner and where we last went for vacation are related to episodic memory. episodic memory is more sensitive to information change and loss. with this information, we can say that semantic memory is known, and episodic memory is remembered.20 episodic memory impairment is typically the first presenting symptom in ad. the region that modulates episodic memory is the medial temporal lobe structure. as we know, prominent medial temporal lobe atrophy develops in ad.21 of the six main memory systems, episodic memory is the most clinically relevant observed in ad.22 robust episodic table 2. neuropsychological test parameters of all patients wms-i, n(%) abnormal normal 54 23 wms-ii, n(%) abnormal normal 39 41 wms-iii, n(%) abnormal normal 57 7 wms-iv, n(%) abnormal normal 25 25 wms-v, n(%) abnormal normal 43 33 wms-vi, n(%) abnormal normal stm: normal, ltm: abnormal stm: abnormal, ltm: normal 21 32 11 4 similarities test, n(%) abnormal normal 62 16 clock drawing test, n(%) abnormal normal 51 28 luria alternan sequences test, n(%) abnormal normal 41 35 verbal fluency test, n(%) both abnormal both normal svf: abnormal, lvf: normal svf: normal, lvf: abnormal 29 24 12 5 stroop test, n(%) abnormal normal 9 4 trail making test, n(%) abnormal normal a form: normal, b form: abnormal 11 8 4 face recognition test, n(%) abnormal normal 21 40 judgement of line orientation test, n(%) abnormal normal 52 14 shape copy test, n(%) abnormal normal 25 46 language skill, n(%) abnormal normal 26 49 mood evaluation, n(%) abnormal normal 26 42 verbal memory processes test, n(%) all abnormal all normal ls:low trs and ls: low ims, ls: low ims: low 7 12 6 2 9 1 eeg, mean±sd,range 8,58 ± 0,86 (6–10) mmt, mean±sd,range 24,35 ± 4,13 (11–30) moca, mean±sd,range 14,20 ± 5,63 (6–23) stm: short term memory, ltm:long term memory, svf: semantic verbal fluency, lvf: lexical verbal fluency, ls: learning score, trs: total recall score, ims: instant memory score, eeg: electroencephalogram, mmt: mini mental test, moca: montreal cognitive assessment. 183j contemp med sci | vol. 9, no. 3, may-june 2023: 179–186 g. morkavuk & g. sirvanli original the role of neuropsychological assessment in forgetfulness table 3. comparison of neuropsychological test parameters and sociodemographic characteristics of patients with dementia, mild cognitive i̇mpairment and depression clinicopathological factors no. of patients (%) p-valuedepression mci dementia (24 patients) (16 patients) (47 patients) age, year, median, range 66,50 (42–86) 73,50 (63–83) 77 (52–85) p < 0,001h gender, n male female 9 15 10 6 14 33 p = 0.066‡ dominant hand, n right left 24 0 15 1 44 1 p = 0.441‡ education, n uneducated primary school junior high school high school university 1 5 1 9 8 0 3 4 4 5 1 21 4 12 7 p = 0,123‡ occupation, n housewife tailor soldiers/policemen engineer doctor official teacher driver other 10 0 3 2 0 5 1 0 1 1 1 3 0 1 4 2 0 3 15 3 0 2 1 5 6 3 6 p = 0,099‡ wms-i, n abnormal normal 11 13 9 4 34 6 p = 0,004‡ wms-ii, n abnormal normal 7 17 3 11 29 13 p = 0,001‡ wms-iii, n abnormal normal 14 5 9 2 34 0 p = 0,009‡ wms-iv, n abnormal normal 4 13 2 6 19 6 p = 0,001‡ wms-v, n abnormal normal 12 12 4 8 27 13 p = 0,082‡ wms-vi, n abnormal normal stm: normal, ltm: abnormal stm: abnormal, ltm: normal 5 14 4 0 3 9 1 0 13 9 6 4 p = 0,066‡ similarities test, n abnormal normal 16 8 8 5 38 3 p = 0,009‡ clock drawing test, n abnormal normal 7 16 8 7 36 5 p <0,001‡ luria alternan sequences test, n abnormal normal 10 13 6 9 25 13 p = 0,115‡ verbal fluency test, n both abnormal both normal svf: abnormal, lvf: normal svf: normal, lvf: abnormal 4 12 3 2 4 5 3 1 21 7 6 2 p = 0,071‡ 184 j contemp med sci | vol. 9, no. 3, may-june 2023: 179–186 the role of neuropsychological assessment in forgetfulness original g. morkavuk & g. sirvanli stroop test, n abnormal normal 2 3 3 1 4 0 p = 0,146‡ trail making test, n abnormal normal a form: n, b form: b 0 4 3 3 4 1 8 0 0 p = 0,002‡ face recognition test, n abnormal normal 5 16 4 9 12 15 p = 0,313‡ judgement of line orientation test, n abnormal normal 14 8 10 2 28 4 p = 0,099‡ shape copy test, n abnormal normal 4 18 2 11 19 17 p = 0,007‡ language skill, n abnormal normal 4 18 3 10 19 21 p = 0,042‡ mood evaluation, n abnormal normal 15 8 2 10 9 24 p = 0,004‡ verbal memory processes test, n all abnormal all normal ls:low trs and ls: low ims, ls: low ims: low 1 8 2 0 2 1 1 4 0 1 2 0 5 0 4 1 5 0 p = 0,060‡ eeg, median, range 9 (7–10) 9 (8–9) 9 (6–10) p = 0,827h mmt, mean ± sd, range 27,07 ± 2,15 (23–30) 27,25 ± 2,5 (24–30) 21,86 ± 3,91 (11–28) p < 0.001f moca, mean ± sd, range – 17 ± 5,25(10–23) 10 ± 3,16(6–13) p = 0,087f stm: short term memory, ltm:long term memory, svf: semantic verbal fluency, lvf: lexical verbal fluency, ls: learning score, trs: total recall score, ims: instant memory score, eeg: electroencephalogram, mmt: mini mental test, moca: montreal cognitive assessment, sd: standart deviation; min:minimum; max:maximum; fanova test; ‡χ2 tests; hkruskal wallis test memory is required to remember to turn off the stove, take medicine, pay bills, and not get lost while driving. deficiencies in these functional areas are initially unclear and can often be attributed to normal aging by the patient. during this period, npts are guiding the detection of episodic memory disorders. sbst is an important test that can distinguish many memory-related parameters from each other. in our study, sbst was statistically significantly different, especially in the comparison of ad and mci groups. sbst test was applied to 15 ad and 8 mci patients in total, and this test was abnormal in all dementia patients, while it was normal in half of mci patients. this situation suggests how valuable the sbst test is in differential diagnosis and that it should be applied in npt. another distinguishing test in our study is considered wms-v. wms-v was defective in 50% of depressed patients, 67.5% of dementia patients, and only 33% of mci patients. this result indicates that this test may not be very helpful in the differential diagnosis of patients with depression, but might be a guide when distinguishing between dementia and mci. of course, making a differential diagnosis with one or two tests will not be the right method. however, trying to apply these tests while performing npt can make our work easier. the tests used in the np battery are not standard everywhere. however, if it is determined which tests are more sensitive through studies, these tests may become standard in the coming years. studies have shown that performance-based measures that evaluate functional status in addition to npt are more successful than npt alone in distinguishing clinically normal elderly people from mci. with the harvard automated phone task developed in this regard, patients are asked to perform tasks such as bank transfers or selecting a new primary care family physician using the voice response system. it was reported that with this task assessment, clinically normal elderly, mci and young normal participants can be distinguished.23-26 however, since our study was designed retrospectively, functional status evaluation could not be performed. cognitive evaluation is especially crucial in mci patients. the separation of cognitively healthy elderly people with mci with npts not only provides early diagnosis but also allows pharmacological and non-pharmacological treatments to be started as early as possible, thus preventing cognitive decline. studies have reported that neuropsychological evaluation is more sensitive than some neuroimaging and cerebrospinal fluid (csf) biomarkers for the distinction of mci and ad.27 some studies argue that standard npts can be used to distinguish preclinical as from healthy elderly, while some studies argue that standard npts are not sensitive27,28 and that special tests should be used.29-31 contrary to ad, it has been reported that semantic memory is more affected in mci.32,33 figurative language comprehension assessments also show promise as 185j contemp med sci | vol. 9, no. 3, may-june 2023: 179–186 g. morkavuk & g. sirvanli original the role of neuropsychological assessment in forgetfulness table 4. univariate multinomial logistic regression analysis of characteristics factors for patients with dementia, mild cognitive impairment and depression characteristics univariate analysis or (95% ci) p-value age mci dementia 1,091 (1,013–1,174) 1,129 (1,060–1,203) 0,021 < 0,001 wms1 mci dementia 2,659 (0,639–11,06) 6,697 (2,053–21,84) 0,179 0,002 wms2 mci dementia 0,662 (0,140–3,123) 5,418 (1,809–16,22) 0,603 0,003 wms3 mci dementia 1,607 (0,255–10,13) 150367587 0,614 <0,001 wms4 mci dementia 1,083 (0,154–7,642) 10,29 (2,418–43,81) 0,936 0,002 similarities test mci dementia 0,800 (0,197–3,254) 6,333 (1,486–26,99) 0,755 0,013 clock drawing test mci dementia 2,612 (0,678–10,05) 16,45 (4,531–59,78) 0,163 < 0,001 shape copy test mci dementia 0,818 (0,128–5,233) 5,029 (1,419–17,83) 0832 0,012 language skill mci dementia 1,350 (0,250–7,278) 4,071 (1,168–14,19) 0,727 0,028 mood evaluation mci dementia 0,107 (0,19–0,610) 0,200 (0,063–0,632) 0,012 0,006 mmt mci dementia 1,043 (0,611–1,782) 0,459 (0,283–0,744) 0,876 0,002 *the reference category is: depresyon. precise measures of cognition. figurative language skills may be impaired especially in early ad34 and mci.35 a study published in 2022 in which neuropsychological evaluations of 35 mci and 56 healthy volunteers were performed reported that mci patients had worse performance in moca and semantic memory tests.36 in this study, it was revealed that especially semantic memory and figurative language skills tests are more sensitive than standard psychometric tests in distinguishing mci patients from healthy volunteers. in this case, we can conclude that episodic memory is more prominently affected in ad patients, and semantic memory is more prominently affected in mci and preclinical ad. another study concluded that patients with amnestic mild cognitive impairment (amci) had an 8.6-fold higher risk of conversion to ad when compared to patients reporting memory problems without objective deterioration in neuropsychological tests.37 in a study published in 2018, gbd, amci, and healthy controls were monitored for ad conversion for 4 years. at the end of 4 years, 41 of 60 amci patients and 57 of 115 lld patients had converted to ad. none of the 66 healthy volunteers who were followed up did transform to ad. crude risks for developing ad at 4 years from baseline were 68.33% and 49.57% for the amci and gbd groups, respectively.15 the gender difference observed in our study also supports the hypothesis that not every mci patient progresses to dementia. because in our study, there was a male predominance in the mci group and a female predominance in the dementia group. if each mci had progressed to dementia, we would expect more male patients to be seen in the dementia group. there are also studies comparing npts of patients with mci and depression. a study by benson et al. on depression subjects determined that mmse scores were not significantly different from amci subjects.38 our study determined no statistical difference between neuropsychological tests between mci and depression patients, and only mood tests had higher scores in depression patients. the limitations of the study can be listed as the inability to apply the same tests to all patients, to perform tests to evaluate the functionality of the patients, the low number of mci patients, and the retrospective design of the study. conclusion in conclusion, when evaluating patients who present with the complaint of forgetfulness, a detailed neuropsychological evaluation must be performed in addition to other diagnostic tests. sensitive tests should be included to confirm the diagnosis, especially in cases where being in between for the differential diagnosis. thus, further studies are needed on this subject. conflicts of interest disclosure the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.  references 1. mitchell aj, shiri‐feshki m. rate of progression of mild cognitive impairment to dementia–meta‐analysis of 41 robust inception cohort studies. acta psychiatrica scandinavica. 2009;119(4):252–265. 2. gillis c, mirzaei f, potashman m, ikram ma, maserejian n. the incidence of mild cognitive impairment: a systematic review and data synthesis. alzheimer’s & dementia: diagnosis, assessment & disease monitoring. 2019;11:248–256. 3. canevelli m, grande g, lacorte e, et al. spontaneous reversion of mild cognitive impairment to normal cognition: a systematic review of literature and meta-analysis. journal of the american medical directors association. 2016;17(10):943–948. 4. zhuang l, yang y, gao j. cognitive assessment tools for mild cognitive impairment screening. journal of neurology. 2021;268(5):1615–1622. 5. kliegel m, zimprich d. predictors of cognitive complaints in older adults: a mixture regression approach. european journal of ageing. 2005;2(1):13–23. 6. mahapatra a, sharma p, khandelwal sk. late onset depression: a recent update. journal of mental health and human behaviour. 2015;20(1):4. 7. koenig am, bhalla rk, butters ma. cognitive functioning and late-life depression. journal of the international neuropsychological society. 2014;20(5):461–467. 186 j contemp med sci | vol. 9, no. 3, may-june 2023: 179–186 the role of neuropsychological assessment in forgetfulness original g. morkavuk & g. sirvanli 8. wright sl, persad c. distinguishing between depression and dementia in older persons: neuropsychological and neuropathological correlates. journal of geriatric psychiatry and neurology. 2007;20(4):189–198. 9. petersen rc. mild cognitive impairment as a diagnostic entity. journal of internal medicine. 2004;256(3):183–194. 10. salami o, lyketsos cg. clinical features of alzheimer’s disease: cognitive and non‐cognitive. principles and practice of geriatric psychiatry. 2010:226–231. 11. zubenko gs, zubenko wn, mcpherson s, et al. a collaborative study of the emergence and clinical features of the major depressive syndrome of alzheimer’s disease. american journal of psychiatry. 2003;160(5):857–866. 12. gilley dw, bienias j, wilson r, bennett d, beck t, evans d. influence of behavioral symptoms on rates of institutionalization for persons with alzheimer’s disease. psychological medicine. 2004;34(6):1129–1135. 13. suh gh, kil yeon b, shah a, lee jy. mortality in alzheimer’s disease: a comparative prospective korean study in the community and nursing homes. international journal of geriatric psychiatry: a journal of the psychiatry of late life and allied sciences. 2005;20(1):26–34. 14. boldrini m, underwood md, hen r, et al. antidepressants increase neural progenitor cells in the human hippocampus. neuropsychopharmacology. 2009;34(11):2376–2389. 15. lauriola m, mangiacotti a, d’onofrio g, et al. late-life depression versus amnestic mild cognitive impairment: alzheimer’s disease incidence in 4 years of follow-up. dementia and geriatric cognitive disorders. 2018;46(3-4):140–153. 16. ganguli m, du y, dodge hh, ratcliff gg, chang c-ch. depressive symptoms and cognitive decline in late life: a prospective epidemiological study. archives of general psychiatry. 2006;63(2):153–160. 17. elderkin-thompson v, kumar a, mintz j, boone k, bahng e, lavretsky h. executive dysfunction and visuospatial ability among depressed elders in a community setting. archives of clinical neuropsychology. 2004;19(5):597–611. 18. butters ma, whyte em, nebes rd, et al. the nature and determinants of neuropsychological functioning in late-lifedepression. archives of general psychiatry. 2004;61(6):587–595. 19. squire lr. memory systems of the brain: a brief history and current perspective. neurobiology of learning and memory. 2004;82(3):171-177. 20. mesulam m-m. principles of behavioral and cognitive neurology: oxford university press; 2000. 21. ashendorf l, alosco ml, bing-canar h, et al. clinical utility of select neuropsychological assessment battery tests in predicting functional abilities in dementia. archives of clinical neuropsychology. 2018;33(5):530–540. 22. gold da. an examination of instrumental activities of daily living assessment in older adults and mild cognitive impairment. journal of clinical and experimental neuropsychology. 2012;34(1):11–34. 23. goldberg te, koppel j, keehlisen l, et al. performance-based measures of everyday function in mild cognitive impairment. american journal of psychiatry. 2010;167(7):845–853. 24. sadek jr, stricker n, adair jc, haaland ky. performance-based everyday functioning after stroke: relationship with iadl questionnaire and neurocognitive performance. journal of the international neuropsychological society. 2011;17(5):832–840. 25. marshall ga, amariglio re, sperling ra, rentz dm. activities of daily living: where do they fit in the diagnosis of alzheimer’s disease? neurodegenerative disease management. 2012;2(5):483–491. 26. marshall ga, dekhtyar m, bruno jm, et al. the harvard automated phone task: new performance-based activities of daily living tests for early alzheimer’s disease. the journal of prevention of alzheimer’s disease. 2015;2(4):242. 27. bondi mw, jak aj, delano-wood l, jacobson mw, delis dc, salmon dp. neuropsychological contributions to the early identification of alzheimer’s disease. neuropsychology review. 2008;18(1):73–90. 28. thomas kr, edmonds ec, eppig j, salmon dp, bondi mw, initiative asdn. using neuropsychological process scores to identify subtle cognitive decline and predict progression to mild cognitive impairment. journal of alzheimer’s disease. 2018;64(1):195–204. 29. rentz dm, parra rodriguez ma, amariglio r, stern y, sperling r, ferris s. promising developments in neuropsychological approaches for the detection of preclinical alzheimer’s disease: a selective review. alzheimer’s research & therapy. 2013;5(6):1–10. 30. loewenstein da, curiel re, duara r, buschke h. novel cognitive paradigms for the detection of memory impairment in preclinical alzheimer’s disease. assessment. 2018;25(3):348–359. 31. xie l, wisse le, das sr, et al. longitudinal atrophy in early braak regions in preclinical alzheimer’s disease. human brain mapping. 2020;41(16):4704–4717. 32. papp kv, rentz dm, orlovsky i, sperling ra, mormino ec. optimizing the preclinical alzheimer’s cognitive composite with semantic processing: the pacc5. alzheimer’s & dementia: translational research & clinical interventions. 2017;3(4):668–677. 33. vonk jm, flores rj, rosado d, et al. semantic network function captured by word frequency in nondemented apoe ε4 carriers. neuropsychology. 2019;33(2):256. 34. papagno c, lucchelli f, muggia s, rizzo s. idiom comprehension in alzheimer’s disease: the role of the central executive. brain. 2003;126(11):2419–2430. 35. cardoso s, silva d, maroco j, de mendonça a, guerreiro m. non‐literal language deficits in mild cognitive impairment. psychogeriatrics. 2014;14(4):222–228. 36. klooster n, humphries s, cardillo e, et al. sensitive measures of cognition in mild cognitive impairment. journal of alzheimer’s disease. 2021;82(3):1123–1136. 37. lehrner j, gufler r, guttmann g, et al. annual conversion to alzheimer disease among patients with memory complaints attending an outpatient memory clinic: the influence of amnestic mild cognitive impairment and the predictive value of neuropsychological testing. wiener klinische wochenschrift. 2005;117(18):629–635. 38. benson ad, slavin mj, tran t-t, petrella jr, doraiswamy pm. screening for early alzheimer’s disease: is there still a role for the mini-mental state examination. prim care companion j clin psychiatry. 2005;7(2):62–69. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1233 134 j contemp med sci | vol. 8, no. 2, march-april 2022: 134–139 original comparison of folliculogenesis and follicular maturation in norvegicus rats using foeniculum vulgare and clomiphene citrate maha m. kadhim al-tu’ma1,*, mays a. dakhel al-turihi1, sura zaki ghafat2 1department of anaesthesia techniques, college of health and medical techniques, al-zahraa university for women, kerbala, iraq. 2department of physiotherapy techniques, college of health and medical techniques, al-zahraa university for women, kerbala, iraq. *correspondence to: maha m. kadhim al-tu’ma (e-mail: maha.mohammad@alzahraa.edu.iq) (submitted: 03 february 2022 – revised version received: 24 february 2022 – accepted: 07 march 2022 – published online: 26 april 2022) abstract objectives: the objective of this work is to understand the importance of follicular maturation in the reproductive life of the wistar’s rats (rattus norvegicus) using foeniculum vulgare (fennel) and clomiphene citrate as reference drug. methods: foeniculum vulgare (fennel) are used in doses of (400, 800 mg/kg), clomiphene citrate along with distilled water administration. the sexual hormones involved in the oestrous cycle, folliculogenesis, the role of the ovaries, the embryonic and fetal development of the ovary are addressed. an extract of fennel was obtained using ethyl alcohol by miscreation. after successful administration, a histological study was also conducted on 40 rats grouped into four categories. statistical analysis also applied standard parameters like chi-square, tukey test and variance test. results: it was identified that after administration of fennel citrate drug along with distilled waters, significant differences (p < 0.05) were identified in the rats and all rats formed primary (90% given 800 mg/kg after fennel administration), secondary and matured (80% given 800 mg/kg after fennel administration) follicles. conclusion: the effect of f. vulgare at dose of 400 mg was not influential as compared to f. vulgare at 800 mg. the clomiphene citrate increased the number of primary follicles in all of the rats studied. in contrast, the rats that ate f. vulgare, had secondary and mature follicles predominating and did not have any primary follicles. keywords: foeniculum, rattus norvegicus, rats, wistar, follicular maturation, clomiphene citrate issn 2413-0516 introduction folliculogenesis the developmental process of ovarian follicles begining from a reserve of quiescent primordial follicles set up in early life and ending with either ovulation or follicular death by atresia.1 many of couples of reproductive age suffer from infertility, a severe problem which affects millions of people of reproductive age worldwide and has an impact on their families and communities. estimates suggest that between 48 million couples and 186 million individuals live with infertility globally.2 it can be described as the inability to carry a pregnancy to term following a suitable amount of sexual contact that contraceptive methods have not interrupted. anovulation is defined as a condition in which follicular development and rupture are disrupted, resulting in the oocyte not being released from the follicle; numerous factors have been identified as contributing to this condition.3 complementary and alternative traditional medicine is extensively practised and respected throughout the world for various reasons.4 traditional therapies and traditional medicine practitioners provide the primary, and in some cases the only, source of health care for several millions of people. this type of treatment is convenient, easily accessible, and reasonably priced. furthermore, it is culturally accepted and trusted by many individuals. considering that the use of fennel would minimise the expense of unsuccessful ovulation treatments, improving the likelihood of having planned pregnancies with fewer complications and, as a result, the birth of a new human, this is a significant topic.5,6 the aim of this study is to evaluate the effects of foeniculum vulgare (fennel) on folliculogenesis and follicular maturation in norvegicus rats exposed to clomiphene citrate, in order to supply new information that can be utilized to incorporate new understanding into health care practises and policies. specifically, the current study investigates the effects of fennel at doses of 400 mg/kg and 800 mg/ kg on folliculogenesis and follicular maturation in norvegicus rats, as well as the effects of clomiphene citrate on folliculogenesis and follicular maturation in norvegicus rats, and whether there is a difference in the effects of folliculogenesis and follicular maturation between rats that consumed fennel. materials and methods plant material: foeniculum vulgare (figure 1) is a medical plant that belongs to the umbelliferae family (apiaceae), well known and applied by humans since ancient times because of its flavour. it is cultivated in nearly all countries. it is globally known as fennel or other names in more than 100 countries.7 fennel is a herbaceous plant, erect in size that can reach 2 meters in height. the deep green leaves are long and thin, ending in needle-shaped segments, which harden on the outside in the summer to prevent water loss.8 there are varieties of fennel with a tan (purple) color. the yellow flowers appear in clusters of 20 to 50 small flowers on short peduncles, called umbels (inflorescence in which the pedicels of all the flowers are inserted at the same point on their axis, similar to the ribs of an umbrella).9 they bloom in summer. the fruits are ovoid and oblong. biological-pharmacological studies have been carried out to assess the native uses of this plant.10 f. vulgare plant extracts and isolated compounds have been estimated for various activities and proved good medicine for human health issues.11 norvegicus wistar-type rats and rat cycle identification as shown in figure 2. mailto:maha.mohammad@alzahraa.edu.iq 135j contemp med sci | vol. 8, no. 2, march-april 2022: 134–139 m.m.k. al-tu’ma et al. original comparison of folliculogenesis and follicular maturation fig. 1 foeniculum vulgare plant. fig. 2 sample of rats (left) and rat cycle identification (right). fig. 3 rats histological sections and follicles. forty rats were used as experimental animals, females between 230–280 gr of the wistar breed, albino variety, all of them with similar feeding. they were divided into four groups and administered distilled water, clomifero citrate, fennel (400 mg/kg) and fennel (800 mg/kg) respectively. they were divided into four groups and administered distilled water, clomifero citrate, fennel (400 mg/kg) and fennel (800 mg/kg). animals in all groups were sacrificed after the sixth administration day (figure 3, table 1). the ovaries were trimmed of fat and fixed in 10% formalin. they were dehydrated in graded alcohols, pressed in xylene, immersed in paraffin, and then dyed with hematoxylin and eosin (h&e). subsequently, histological sections were made in the microtome. the follicles were classified as primary, secondary and mature (figure 3). • laboratory materials: beaker; 10 ml vial; dropper; vials • equipment: percolator equipment; broken steam; water bath; stove; analytical balance; vortex • others: surgical gloves; venoclysis equipment; 1 ml tuberculin syringes; marbles; drinkers; cannula • reagent: distilled water and 96° ethyl alcohol chromatographic technique is an analytical system that allows the different components of a problem sample to be separated by distribution between two phases, one stationary and the other mobile, in order to identify and/or quantify them. uniform layer of an absorbent maintained on a plate made of glass, aluminium or another support. the substances to be separated are run using a solvent (mobile phase). the mobile phase is liquid, and the stationary phase is solid and polar (silica gel). the mixture to be analysed is deposited a small distance from the lower edge of the plate and introduced into a cuvette containing the mobile phase. the mobile phase rises along with the plate by capillarity, displacing the mixture’s components at different speeds, causing their separation. when the solvent front is close to the upper end of the plate, it is removed 136 j contemp med sci | vol. 8, no. 2, march-april 2022: 134–139 comparison of folliculogenesis and follicular maturation original m.m.k. al-tu’ma et al. table 1. drug administration protocol used in the current study rat no identification weight (gm) administration dose group–1 (fennel 400 mg) r1 head and left front leg 266 proestrous 0.13 ml of fennel up to 1 ml r2 2 right legs 270 0.13 ml of fennel up to 1 ml r3 head and 2 hind legs 230 0.12 ml of fennel up to 1 ml r4 head and two right legs 260 0.13 ml of fennel up to 1 ml r5 head and 2 left legs 240 0.12 ml of fennel up to 1 ml r6 head 237 0.11 ml of fennel up to 1 ml r7 back 248 0.12 ml of fennel up to 1 ml r8 tail 249 0.12 ml of fennel up to 1 ml r9 right front leg 253 0.12 ml of fennel up to 1 ml r10 right hind leg 238 0.12 ml of fennel up to 1 ml group–2 (fennel 800 mg) r11 head and right front leg 252 proestrous 0.24 ml of fennel up to 1 ml r12 right front leg 246 0.24 ml of fennel up to 1 ml r13 head 255 0.25 ml of fennel up to 1 ml r14 head and back 275 0.27 ml of fennel up to 1 ml r15 head and two right legs 260 0.25 ml of fennel up to 1 ml r16 left anterior leg 260 0.25 ml of fennel up to 1 ml r17 left hind leg 268 0.26 ml of fennel up to 1 ml r18 right side legs 265 0.26 ml of fennel up to 1 ml r19 left side legs 254 0.24 ml of fennel up to 1 ml r20 front legs 238 0.23 ml of fennel up to 1 ml group–3 (clomiphene citrate) r21 head 263 right-handed 1 ml r22 back 252 r23 tail 250 r24 right front leg 243 r25 right hind leg 262 r26 left front leg 252 r27 left hind leg 267 r28 two right legs 258 r29 two left legs 263 r30 front legs 266 group–4 (distilled water) r31 head 260 proestrous 1 ml r32 back and right front leg 245 r33 tail 258 r34 back 254 r35 2 right legs 257 r36 head back tail 244 r37 head right front leg 249 r38 head right hind leg 245 r39 left front leg head 248 r40 left hind leg head 239 137j contemp med sci | vol. 8, no. 2, march-april 2022: 134–139 m.m.k. al-tu’ma et al. original comparison of folliculogenesis and follicular maturation and visualised in a ultraviolet visible lamp with a wavelength of 366 nm. determination of the so-called fingerprint of the f. vulgare seed was used as the mobile phase. toluene and ethyl acetate (50-50), respectively, were used as developers. an atomiser spread sulfuric acid at 5% and vanillin at 1%; then, the oven was previously conditioned at a temperature of 110°c, placing the plate for 5 minutes. visually the chromatographic analysis of fennel in which substances with high polarity are observed that we see in colours that will allow us to characterise our drug so that they can later be compared. preparation of the ethanolic extract of fennel for the preparation of the extract, seeds of fennel were taken, which were taken to a drying chamber with less than 40ºc for 72 hours. then they were pulverised, obtaining a pulverised dry powder of 100 gr, which was used to prepare the alcoholic extract. the final dry powder was mixed with ethyl alcohol (96º), which was allowed to macerate for 5 days to extract polar and non-polar substances efficiently. at the end of 5 days, it was filtered in alcohol, obtaining the active extract, evaporating at 40ºc for 48 hours. the extract was used for the respective dilutions and administration in the experimental animals. results the number of primary, secondary, and mature follicles in rats exposed to various doses of fennel and clomiphene citrate was determined and the results are graphically represented in figure 4. according to the analysis of variance (fo = 2.96, 5.03, & 4.46) for primary, secondary and mature follicles respectively, it is shown that the number of primary follicles in the concentrations of fennel and clomiphene citrate presented statistically significant differences (p < 0.05). • furthermore, according to the tukey test (ft = 2.88 for primary, 2.88 for secondary, and 2.88 for mature follicles, respectively), it is observed that the highest number of primary follicles was found when clomiphene citrate was applied with an average of 4.50, which is significantly higher than the other treatments. • it was discovered that there were statistically significant variations in the number of secondary follicles in the amounts of fennel and clomiphene citrate (p < 0.05) between the two treatments. • a similar result was reported using the tukey test, which revealed a significant difference between the treatments when the concentration of fennel 800 mg/kg was given, with an average of 2.80 secondary follicles being detected, compared to the other treatments. • using tukey’s test, it was discovered that when fennel 800 mg/kg was administered, the largest number of mature follicles was detected, with an average of 2.40, compared to the other treatments, and that this was significantly different from the other treatments. • with reference to the chi-square test (χ2 = 22.22), the presence of primary follicles was found when different quantities of fennel, clomiphene citrate, and distilled water were used, and statistically significant differences (p < 0.05) were found between the treatments. similarly, it was discovered that 100% of the rats who got clomiphene citrate developed primary follicles (figure 5). • there were statistically significant changes (p < 0.05) in the presence of secondary follicles when varied amounts of fennel, clomiphene citrate, and distilled water were employed, according to the chi-square test (χ2 = 12.5). according to the data, 90% of rats given 800 mg/kg fennel produced secondary follicles. • different concentrations of fennel, clomiphene citrate, and distilled water were found to have statistically significant differences (p < 0.05) in the existence of mature follicles, as determined by the chi-square test (χ2 = 68.69). fennel 800 mg/kg was found to increase the follicle count in the testicles of 80 per cent of the experimental rats. discussion in current study, the effects of f. vulgare on folliculogenesis and follicular maturation in rats were investigated. when different amounts of fennel, clomiphene citrate, and distilled water were administered to primary, secondary, and mature follicles, statistically significant changes (p < 0.05) were detected. primary follicles were found in 100% of the clomiphene citrate-treated rats. in contrast, primary follicles were found in 90% of the distilled water-consuming rats and 90% of the fennel-consuming rats at an 800 mg/kg dose, respectively. the dose of 800 mg/kg of fennel resulted in 90% of the rats showing secondary follicles and 80% of the animals showing mature follicles after the herb was administered. 80% and 70% of the rats that received 400 mg/kg fennel showed secondary and mature follicles, respectively, with no statistically significant variations between the two groups. fig. 4 number of primary, secondary and mature follicles in rats subjected to concentrations of fennel and clomiphene citrate. fig. 5 presence of primary, secondary and mature follicles in histological sections of rattus norvergicus ovary. 138 j contemp med sci | vol. 8, no. 2, march-april 2022: 134–139 comparison of folliculogenesis and follicular maturation original m.m.k. al-tu’ma et al. consequently, the presence of secondary and mature follicles varied substantially between fennel dosages of 800 mg/ kg and 400 mg/kg when comparing the two treatments. statistically significant differences were seen in the number of formed follicles between the quantities of fennel, pure water, and clomiphene citrate used in the experiments (p < 0.05). according to tukey’s test, the highest primary follicle counts were obtained when clomiphene citrate was used. in contrast, the highest secondary and mature follicle counts were obtained when fennel 800 mg/kg was used. after 10 days of oral treatment with the extract, the researchers12 discovered that female rats developed vaginal cornification and began their monthly cycle and other pharmacological characteristics such as toxicity, side effects, and drug interactions. the ovary, endometrium, myometrium, cervix, and vagina grew weight when given greater doses, whereas the mammary glands acquired weight when given intermediate amounts of the medication. in a study conducted by gardner et al. (2013), discovered that oral administration of an acetone extract of fennel for 14 days boosted the estrus cycle and increased weight in the breast, endometrial, cervix, and vagina in rats.13 an acetone extract of fennel administered daily at doses ranging from 0.5 to 2.5 mg/kg body weight caused dose-dependent estrogenic effects in ovariectomised rats, resulting in the stimulation of the estrus phase (after 10 days, in 40% of rats at 0.5 mg/kg and 100% of rats at 2.5 mg/kg) and increased weight of the mammary glands (p 0.05 to 0.5 mg/kg, p < 0.01 to 2.5 mg/kg). dehghani et al. (2005) discovered that f. vulgare had a deleterious impact on the reproductive organs of male rats after conducting a study.14 in this experiment, 40 male rats were used in the experiment. four groups of sprague-dawley rats were produced at random using a computer programme. the organic extract of f. vulgare was provided to the study groups at doses of 100, 250, and 500 mg/kg for a total of 30 days, depending on their weight. the rats were killed and dissected on day 30 of the experiment to conduct histological studies. the herb f. vulgare considerably elevated estradiol levels in male rats while simultaneously reducing testosterone levels in the bloodstream. kilic-oakman et al. (2003) employed the rat model to investigate the effects of letrozole and clomiphene citrate on ovarian follicle count, endometrium thickness, and serum levels of estradiol, follicle-stimulating hormone (fsh), luteinising hormone (lh), and testosterone (test) in a female reproductive tract.15 the experiment, which involved 30 female wistar-albino rats at twenty weeks of age and weighing 250 g, was conducted on wistar-albino rats. from the total of 30 rats, 3 groups of ten rats, each were formed. vaginal smears were taken from all rats before they were given the letrozole pill to see if they had regular menstruation for the previous 3 cycles. diestrus, a 4-day oestrous cycle, was administered in 2 ml normal saline containing 5 mg/kg body weight daily; 100 g/kg daily in 2 ml normal saline; or 2 ml of sterilised sodium chloride saline included 5 mg/kg body weight daily. the medications were delivered via lavage for a total of two days. the creatures were terminated with ether after two days of suffering. in addition to doing an autopsy in order to collect tissue samples for histology, blood samples were collected to evaluate serum levels of oestrogen, (fsh), (lh), and (test). when the ovary was examined under a microscope, the size, thickness of the endometrium, and ovary diameter could all be determined. if you’re looking to increase follicular growth, letrozole is every bit as effective as clomiphene citrate is. letrozole has the potential to be used to manage fertility in the future. in our study, we administered the medication clomiphene citrate, and a significant amount reduced the number of primary follicles. conclusion the effect of f. vulgare at 400 mg was ineffectual compared to f. vulgare at 800 mg. it was discovered that clomiphene citrate increased the number of primary follicles in all of the rats studied. in contrast to the rats that ate f. vulgare, which had secondary and mature follicles predominating, the rats that ate clomiphene citrate had primary follicles predominating, while the rats that ate f. vulgare did not have any. recommendations • carry out further research to identify the minimum and maximum doses that can be used to obtain the same effects. • conduct future research comparing the effectiveness of different medicinal plants used in the south iraqi region, in addition to f. vulgare. • carry out pilot studies to be carried out in human beings. references 1. gershon, e. and dekel, n., 2020. newly identified regulators of ovarian folliculogenesis and ovulation. international journal of molecular sciences, 21(12), p. 4565. 2. mascarenhas, m.n., flaxman, s.r., boerma, t., vanderpoel, s., mathers, c.d. and stevens, g.a., 2013. trends in primary and secondary infertility prevalence since 1990: a systematic analysis of demographic and reproductive health surveys. the lancet, 381, p. s90. 3. walker mh, tobler kj. female infertility. [updated 2021 dec 28]. in: statpearls [internet]. treasure island (fl): stat pearls publishing; 2022 jan-. available from: https://www.ncbi.nlm.nih.gov/books/nbk556033/. 4. world health organization (2020), traditional medicine in the who southeast asia region review of progress 2014–2019, isbn: 978-92-9022-829-5, page 38, printed in india. 5. khazaei, m., montaseri, a., khazaei, m.r. and khanahmadi, m., 2011. study of foeniculum vulgare effect on folliculogenesis in female mice. international journal of fertility & sterility, 5(3), p. 122. 6. mansouri, e., asadi-samani, m., kooti, w., ghasemiboroon, m., ashtarylarky, d., alamiri, f., afrisham, r. and hasanzadeh noohi, z., 2016. antifertility effect of hydro-alcoholic extract of fennel (foeniculum vulgare mill) seed in male wistar rats. journal of veterinary research (poland), 60(3), pp. 357–363. 7. badgujar, s.b., patel, v.v. and bandivdekar, a.h., 2014. foeniculum vulgare mill: a review of its botany, phytochemistry, pharmacology, contemporary application, and toxicology. biomed research international, 2014. 8. pourjafari, f., haghpanah, t., nematollahi-mahani, s.n., pourjafari, f. and ezzatabadipour, m., 2020. hydroalcoholic extract and seed of foeniculum vulgare improve folliculogenesis and total antioxidant capacity level in f1 female mice offspring. bmc complementary medicine and therapies, 20(1), pp. 1–8. 9. rani, sweta & das, sanjita. (2016). foeniculum vulgare: phytochemical and pharmacological review. international journal of advanced research. 4. 477–48. https://www.ncbi.nlm.nih.gov/books/nbk556033/. 139j contemp med sci | vol. 8, no. 2, march-april 2022: 134–139 m.m.k. al-tu’ma et al. original comparison of folliculogenesis and follicular maturation 10. kooti, w., moradi, m., ali-akbari, s., sharafi-ahvazi, n., asadi-samani, m. and ashtary-larky, d., 2015. therapeutic and pharmacological potential of foeniculum vulgare mill: a review. journal of herbmed pharmacology, 4(1), pp. 1–9. 11. rather, dr. manzoor & dar, bilal & sofi, shahnawaz, bhat, bilal and qurishi, mushtaq. (2012). foeniculum vulgare: a comprehensive review of its traditional use, phytochemistry, pharmacology, and safety. arabian journal of chemistry. 14: 10. 12. rahimi, r. and ardekani, m.r.s., 2013. medicinal properties of foeniculum vulgare mill. in traditional iranian medicine and modern phytotherapy. chinese journal of integrative medicine, 19(1), pp. 73–79. 13. gardner zoë, michael mcguffin, (2013) american herbal products association’s botanical safety handbook, crc press; 2nd edition page. 370, isbn-13: 978-1466516946. 14. dehghani f, panjehshahin mr, mirzaee z, mehrabani d. effect of foeniculum vulgare organic extract on blood sex hormones and reproductive tissues of male rats. journal of applied animal research. 2005 mar 1;27(1):17–20. 15. kilic-okman t, kucuk m, altaner s. comparison of the effects of letrozole and clomiphene citrate on ovarian follicles, endometrium, and hormone levels in the rat. fertility and sterility. 2003 dec 1;80(6):1330–2. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i2.1205 http://https://doi.org/10.22317/jcms.v8i2. home archives vol. 8 no. 2 (2022): march-april 2022 articles comparison of folliculogenesis and follicular maturation in norvegicus rats using foeniculum vulgare and clomiphene citrate maha m. kadhim al-tu’ma department of anaesthesia techniques, college of health and medical techniques, al-zahraa university for women, kerbala, iraq. mays a. dakhel al-turihi department of anaesthesia techniques, college of health and medical techniques, al-zahraa university for women, kerbala, iraq. sura zaki ghafat department of physiotherapy techniques, college of health and medical techniques, alzahraa university for women, kerbala, iraq. doi: https://doi.org/10.22317/jcms.v8i2.1205 keywords: foeniculum, rattus norvegicus, rats, wistar, follicular maturation, clomiphene citrate abstract objective: the objective of this work is to understand the importance of follicular maturation in the reproductive life of the wistar's rats (rattus norvegicus) using foeniculum vulgare (fennel) and clomiphene citrate as reference drug. methods: foeniculum vulgare (fennel) are used in doses of (400, 800mg/kg), clomiphene citrate along with distilled water administration. the sexual hormones involved in the oestrous cycle, folliculogenesis, the role of the ovaries, the embryonic and fetal development of the ovary are addressed. an extract of fennel was obtained using ethyl alcohol by miscreation. after successful administration, a histological study was also conducted on 40 rats grouped into four categories. statistical analysis also applied standard parameters like chi-square, tukey test and variance test. results: it was identified that after administration of fennel citrate drug along with distilled waters, significant differences (p<0.05) were identified in the rats and all rats formed primary (90% given / / / https://www.jocms.org/index.php/jcms/index https://www.jocms.org/index.php/jcms/index https://www.jocms.org/index.php/jcms/issue/archive https://www.jocms.org/index.php/jcms/issue/view/68 https://doi.org/10.22317/jcms.v8i2.1205 800mg/kg after fennel administration), secondary and matured (80% given 800mg/kg after fennel administration) follicles. conclusion: the effect of f. vulgare at dose of 400mg was not influential as compared to f. vulgare at 800mg. the clomiphene citrate increased the number of primary follicles in all of the rats studied. in contrast, the rats that ate f. vulgare, had secondary and mature follicles predominating and did not have any primary follicles. references dehghani f, panjehshahin mr, mirzaee z, mehrabani d. effect of foeniculum vulgare organic extract on blood sex hormones and reproductive tissues of male rats. journal of applied animal research. 2005 mar 1;27(1):17-20. gardner zoë, michael mcguffin, (2013) american herbal products association's botanical safety handbook, crc press; 2nd edition page. 370, isbn-13: 978-1466516946 kilic-okman t, kucuk m, altaner s. comparison of the effects of letrozole and clomiphene citrate on ovarian follicles, endometrium, and hormone levels in the rat. fertility and sterility. 2003 dec 1;80(6):1330-2. published 2022-04-26 how to cite al-tu’ma, m. m. k. ., al-turihi, m. a. d. ., & ghafat, s. z. . (2022). comparison of folliculogenesis and follicular maturation in norvegicus rats using foeniculum vulgare and clomiphene citrate. journal of contemporary medical sciences, 8(2). https://doi.org/10.22317/jcms.v8i2.1205 issue vol. 8 no. 2 (2022): march-april 2022 section more citation formats  https://www.jocms.org/index.php/jcms/issue/view/68 articles license copyright (c) 2022 journal of contemporary medical sciences this work is licensed under a creative commons attribution-noncommercial 4.0 international license. 0 make a submission abstracting and indexing information the journal is indexed with, or included in, the following: emerging sources citation index (clarivate analytics), directory of open access jouranal(doaj), index copernicus, chemical abstracts (cas), ebsco, committee on publication ethics (cope) ,embase, google scholar. applying for: pubmed central https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ https://www.jocms.org/index.php/jcms/about/submissions nab'a al-hayat foundation for medical sciences and health care, iraq   343j contemp med sci | vol. 8, no. 5, september-october 2022: 343–346 original evaluation of the hemoglobin level in gingival crevice fluid and clinical periodontal parameters in patients with chronic periodontitis sina taheri1, mahdis samadaei1, mina pakkhesal2, amir reza ahmadinia3* 1dental research center, school of dentistry, golestan university of medical sciences, gorgan, iran. 2community oral health department, school of dentistry, golestan university of medical sciences, gorgan, iran. 3dental research center, department of periodontics, school of dentistry, golestan university of medical sciences, gorgan, iran. *correspondence to: amir reza ahmadinia (email: dr.ahmadinia45@yahoo.com) abstract objectives: the present study aimed to evaluate the level of hemoglobin in gingival crevicular fluid (gcf) and its relationship with clinical periodontal parameters in chronic periodontitis. methods: this cross-sectional study was conducted in patients with chronic periodontitis. gingival crevicular fluid was sampled and clinical periodontal parameters pi, ppd, cal, and bop were measured. the level of hemoglobin in gcf was measured using a hemoglobin kit. data were analyzed using spearman’s correlation test and mann-whitney’s test in stata 14.1 software. results: 315 teeth from patients with chronic periodontitis were evaluated. there was a strong and direct correlation between the amount of gcf hemoglobin and the amount of ppd, cal and pi, their correlation coefficient was equal to 0.78, 0.88 and 0.82, respectively (p < 0.001). the mean hemoglobin gcf in the positive bop group was 68.84 ± 30.89 and in the negative bop group was 59.28 ± 8.03, which showed a significant difference in the average hemoglobin between the two groups (p < 0.001). conclusion: according to the findings of this study, there is a strong correlation between periodontal clinical parameters and the level of gcf hemoglobin, and measuring the level of hemoglobin in the gingival crevice fluid can be an accurate measure and a non-invasive method for investigating periodontal conditions. keywords: chronic periodontitis, hemoglobin, gingival crevicular fluid issn 2413-0516 introduction chronic periodontitis is an inflammatory disease of the tooth-supporting tissues,1 which is mainly caused by microbial plaque, but it is a multifactorial disease2 that causes the release of cytokines and chemokines and ultimately causes periodontal tissue destruction.3 the methods used to diagnose of periodontal disease should help distinguish between types of periodontal diseases, the degree of destruction of periodontal tissue, and the prognosis of periodontal disease.4,5 today, the main method used in the diagnosis of periodontal diseases, in addition to radiographic findings, is examining clinical parameters such as clinical attachment loss (cal), periodontal probing pocket depth (ppd), plaque index (pi), gingival index (gi) and bop.6-8 gingivitis is checked with bleeding during probing (bop), which is an important parameter for comparing and evaluating the results of periodontitis treatment. bop is an important clinical parameter that shows the presence of inflammation in the depth of the pocket, helps in the treatment plan, shows the success or failure of periodontal treatment, and is used to motivate patients to perform oral hygiene at home.9 although the examinations of clinical parameters are important diagnostic tools, their definite predictive value is low; they only determine the previous damage to the periodontium and do not show the future state of the periodontal tissue.10-12 ppd and cal depend on various factors such as the size of the probe diameter, probing force, and the amount of tissue inflammation. measuring the depth of the periodontal pocket alone cannot indicate the extent of the disease because soft tissue changes such as gingival resorption affect the estimation of the pocket depth and make the depth of the pocket less than its actual amount.13 cal cannot be used as a measure to determine gum health or disease, especially in people who have adhesion destruction, but their gum margin is above the place where cement adheres to tooth enamel.14 the radiographic examination should be a part of a periodontal evaluation of each patient and should be accompanied by careful examination of cal, ppd, and bop. radiographs often show less bone destruction, and early changes are usually not detected. comparison of radiographic images at different times is reliable only in recording significant bone surface changes and does not show soft tissue changes.15 with the advancement of laboratory techniques, gcf components were widely investigated in people with periodontal disease to determine the presence of host response factors, including blood factors such as serum proteins (albumin, globulin, creatine phosphokinase enzyme).5,16 gcf examination is the most common non-traumatic method, which is used to obtain information about periodontal tissue conditions, including the condition of connective tissue and the degree of bone tissue destruction.17 according to previous studies, for the accurate diagnosis of periodontal condition, only examining clinical parameters is not enough and biochemical tests are necessary to improve the accuracy of periodontal disease diagnosis.18,19 measuring hemoglobin in gcf for early diagnosis of periodontal disease is effective before clinical symptoms appear. nonvisible pocket bleeding in primary periodontitis occurs even in bop-negative areas. examining the hemoglobin caused by micro bleeding in the gingival groove along with the clinical parameters of ppd, bop and cal can be used as an accurate index to diagnose and evaluate the treatment of periodontal disease,20 so this study aims to investigate the correlation between hemoglobin gcf and clinical periodontal parameters in patients with chronic periodontitis. mailto:dr.ahmadinia45@yahoo.com 344 j contemp med sci | vol. 8, no. 5, september-october 2022: 343–346 evaluation of the hemoglobin level in gcf original s. taheri et al. materials and methods the present study was commenced upon receiving approval from the research ethics committee of golestan university of medical sciences (ir.goums.rec.2019.091). this cross sectional study was conducted in patients with chronic periodontitis referred to the periodontics department of the dental school of golestan university of medical sciences. the study inclusion criteria included patients with chronic periodontitis according to the american academy of periodontology (aap) classification in 1999,21 having at least 20 teeth, and being over 30 years old. the criteria for exclusion from the study include the presence of a systemic disease affecting the periodontium tissue, the use of drugs affecting the periodontium, addiction to tobacco, drugs and alcohol, the use of antibiotic drugs in the last 3 months for one week, treatment periodontal diseases in the last 6 months and pregnancy and breastfeeding. after obtaining a written consent from the patients, the region was isolated with a cotton roll and dried with mild air. at first, the plaque index (pi) of sillness & loe (1964) was measured in the patients.22 then, a sample was taken from the gingival crevice fluid using paper probe no. 30. a paper cone number 30 was inserted with gentle pressure inside the periodontal pocket (buccal or palatal) to a depth where resistance was felt and remained in the area for 30 seconds.23 later, without getting contaminated with the patient’s saliva, the paper cone was slowly taken out of the pocket and quickly transferred to the microtube containing 200 microliters of phosphate buffered saline solution (pbs).24 in the next step, bop was checked by the bay & ainamo method (1975).25 finally, ppd and cal were measured at 6 levels: mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual by williams periodontal probe. the samples were sent to the laboratory to measure the level of hemoglobin. in the laboratory, the level of hemoglobin was calculated by a laboratory expert using a hemoglobin kit (zist shimi, hemoglobin-total kit, iran) by the cyanmethemoglobin method with drabkin’s reagent according to the instructions of the manufacturer of the kit.25 100 microliters of sample content were mixed with 100 microliters of drabkin’s reagent and incubated for 5 minutes. finally, the optical density of the final solution was measured using an elisa reader (awarness, usa) at a wavelength of 540 nm. data were entered into stata 14.1 software and analyzed. results this study was conducted on 315 teeth of patients with chronic periodontitis. using the kolmogorov smirnov test, the normality of the data was investigated, and the result was the absence of normality in any of the clinical parameters and hemoglobin. the average hemoglobin of the gingival crevice fluid was 62.58 ± 32.07, cal was 4.63 ± 1.32, ppd was 4 ± 0.98, and pi was 1.4 ± 0 (table 1). 84.4 percent of samples were bop positive and 15.6% were bop negative. the average hemoglobin in gingival crevice fluid in positive bop samples was 68.84 ± 30.89 and 28.59 ± 8.03 in negative bop samples. the average difference of gcf hemoglobin content in these two groups was reported as 40.25 and was statistically significant (p value < 0.001) (table 2). spearman’s test was used to check the correlation between hemoglobin gcf and cal, ppd, and pi due to the absence of normality. the correlation between the hemoglobin content of gingival crevice fluid and ppd was obtained with a correlation coefficient of 0.78 (p < 0.001). the correlation between the hemoglobin content of gingival crevice fluid and cal was obtained with a correlation coefficient of 0.88 (p < 0.001). the correlation between the hemoglobin content of gingival crevice fluid and pi was obtained with a correlation coefficient of 0.82 (p < 0.001). the results indicate a strong and direct correlation (table 3). discussion in the past few decades, several studies have been focused on finding an accurate method for the early diagnosis of periodontal diseases. many of these studies suggest that gcf is a good source of biomolecular samples that can be used to investigate the state of periodontal tissues.19,20 this study showed that there is a direct relationship between the level of hemoglobin in gcf and periodontal clinical parameters, and in people who had the disease but bop was negative in them, hemoglobin was detected in gcf, and this indicates that this marker can be used for early diagnosis of periodontal diseases in people who do not have clinical symptoms. diagnosis of periodontal diseases, in addition to radiographic findings, is the examination of periodontal clinical parameters. examining bop is the most important periodontal diagnostic table 1. average gcf hemoglobin, cal, pi and ppd hemoglobin pi cal ppd the lowest amount 8.35 0 2 2 the greatest amount 165.33 3 7 6 mean ± standard deviation 62.58 ± 32.07 1.40 ± 0.71 4.63 ± 1.32 4.00 ± 0.98 table 2. comparison of average hemoglobin according to positive and negative bop bop+ bop– number of samples 266 (84.4%) 49 (15.6%) the lowest amount 11.23 8.35 the greatest amount 165.33 45.62 mean ± standard deviation 68.84 ± 30.89 28.59 ± 8.03 p-value < 0.001. table 3. correlation coefficient of hemoglobin with pi, ppd and cal pi cal ppd hemoglobin 0.82 0.88 0.78 p-value <0.001 <0.001 <0.001 345j contemp med sci | vol. 8, no. 5, september-october 2022: 343–346 s. taheri et al. original evaluation of the hemoglobin level in gcf parameter,4 but its diagnostic value alone is low and cannot to determine the future state of periodontal tissue.5 according to the report of lang and his colleagues, if the bop is negative four times a year, in 98% of the examined areas, the destruction of adhesion is less than 2 mm, but in a few areas, the destruction of adhesion still occurs.19 investigating the depth of the pocket probing and destruction of adhesion cannot show the real histological changes in the tissue.5 examining the samples taken from the periodontal pocket is important in diagnosing and treating periodontitis.16 one of the most common non-traumatic methods for examining the condition of periodontal tissue, including the condition of connective tissue and the degree of destruction of hard bone tissue, is the analysis of gingival crevice fluid.7,16-20 gcf consists of serum, products resulting from host tissue destruction, and subgingival biofilm derivatives, which have diagnostic value. hemoglobin indicates the bleeding reaction and can be detected in the gcf of the diseased areas.11,19,20 in this study, which was conducted on 315 teeth of patients with chronic periodontitis, 15.6% of the teeth were bop negative, while hemoglobin was discovered in their pockets. in the study of ito et al. (2020), clinical parameters such as pi, ppd, cal, bop, gcf, and biochemical parameters such as hemoglobin and proteins were investigated in 76 patients during the periodontal supportive treatment phase. in this study, it was seen that all clinical and biochemical parameters were significantly higher in diseased areas than in healthy areas,20 which is in line with the results of our study. in the study of ito et al. (2014), the relationship between the activity of gcf enzymes and periodontal clinical parameters, especially bop, was investigated. their study divided patients based on bop and ppd, and clinical and biochemical parameters were measured in all groups. according to their findings, 14 areas out of 29 areas that needed periodontal treatment according to the definition were bop negative. this result clarifies that a combination of clinical and biochemical parameters is helpful for the accurate and reliable diagnosis of periodontal.26 in our study, the relationship between gcf hemoglobin content and periodontal clinical parameters showed that with the increase of gcf hemoglobin, periodontal clinical parameters have clear changes towards worsening and causing periodontitis disease. in our study, 68% of bop-negative areas indicated periodontal health had hemoglobin, resulting from micro bleeding in the gingival sulcus. the findings of this study are in line with the study of ito (2016) and indicate that hemoglobin gcf can be an accurate biomarker for diagnosing and predicting periodontal health and disease. in the same study, a significant correlation was observed between all measured periodontal clinical parameters (pi, gcf, ppd, cal, bop) with gcf hemoglobin content, which was consistent with the results of our study because in our study, all the measured clinical parameters (pi, ppd, cal, bop) were correlated with the hemoglobin gcf content.19 sabarathinam et al. (2019) investigated the relationship between salivary hemoglobin level and periodontal health status in 45 patients. according to their findings, the amount of salivary hemoglobin in patients with chronic periodontitis and gingivitis was significantly higher than in periodontally healthy people. in this study, healthy tissue, gingivitis and periodontitis were defined according to cpi (community periodontal index). according to the findings of their study, salivary hemoglobin level can be used as a non-invasive and cost-effective tool to screen for periodontal diseases, determine prognosis, and make decisions about treatment options in the population. the result is in line with the result of our study, which was on the level of hemoglobin gcf.27 conclusion this study investigated periodontal clinical parameters with hemoglobin gcf content in patients with chronic periodontitis. correlation between gcf hemoglobin content and periodontal clinical parameters was observed. therefore, according to the findings of this study, the content of hemoglobin gcf can be used to screen for periodontal diseases and treat them. acknowledgments we would like to thank the present study subjects for their participation in this research study. conflicts of interest the authors have no conflict of interests to declare that are relevant to the content of this article.  references 1. albandar jm, brunelle ja, kingman a. destructive periodontal disease in adults 30 years of age and older in the united states, 1988-1994. journal of periodontology. 1999;70(1):13–29. 2. ramadan de, hariyani n, indrawati r, ridwan rd, diyatri i. cytokines and chemokines in periodontitis. european journal of dentistry. 2020;14(3):483–95. 3. holt sc, ebersole jl. porphyromonas gingivalis, treponema denticola, and tannerella forsythia: the ‘red complex’, aprototype polybacterial pathogenic consortium in periodontitis. periodontology 2000. 2005;38(1):72–122. 4. ko tj, byrd km, kim sa. the chairside periodontal diagnostic toolkit: past, present, and future. diagnostics. 2021;11: 932–55. 5. armitage gc. periodontal diseases: diagnosis. annals of periodontology. 1996;1(1):37–215. 6. buduneli n, kinane df. host-derived diagnostic markers related to soft tissue destruction and bone degradation in periodontitis. journal of clinical periodontology. 2011;38(11):85–105. 7. bibi, t.; khurshid, z.; rehman, a.; imran, e.; srivastava, k.c.; shrivastava, d. gingival crevicular fluid (gcf): a diagnostic tool for the detection of periodontal health and diseases. molecules. 2021, 26, 1208. 8. tonetti ms, greenwell h, kornman ks. staging and grading of periodontitis: framework and proposal of a new classification and case definition. journal of periodontology. 2018;89(s1):159–72. 9. meitner sw, zander h, iker hp, et al: identification of inflamed gingival surfaces. journal of clinical periodontology. 1979; 6:93–7. 10. checchi l, montevecchi m, checchi v, zappulla f. the relationship between bleeding on probing and subgingival deposits. an endoscopical evaluation. open dentistry journal. 2009;3:154–60. 11. lang np, adler r, joss a, et al: absence of bleeding upon probing: an indicator of periodontal stability, journal of clinical periodontology. 1990;17:714–21. 12. greenstein g: the role of bleeding upon probing in the diagnosis of periodontal disease: a literature review, journal of periodontology. 1984;55:684–8. 13. sufaru az, luca oe, kappenberg dc, vieriu r, andronache m, rudnic i. periodontal health: determinants and indicators. a review. romanian journal of medical and dental education. 2019;8(8). 346 j contemp med sci | vol. 8, no. 5, september-october 2022: 343–346 evaluation of the hemoglobin level in gcf original s. taheri et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 14. albandar jm, rams te: global epidemiology of periodontal diseases: an overview, periodontology 2000. 2002;29:7. 15. kripal k, dileep a. role of radiographic evolution: an aid to diagnose periodontal disease. 2019. 16. koss ma, castro ce, salúm km, lópez me. changes in saliva protein composition in patients with periodontal disease. acta odontology latinoamericana. 2009;22(2):105–12. 17. bostanci n, i̇lgenli t, emingil g, afacan b, han b, töz h, et al. gingival crevicular fluid levels of rankl and opg in periodontal diseases: implications of their relative ratio. journal of clinical periodontology. 2007;34(5):370–6. 18. loos b, tjoa s. host-derived diagnostic markers for periodontitis: do they exist in gingival crevice fluid? periodontology 2000. 2005;39:53–72. 19. ito h, numabe y, hashimoto s, sekino s, murakashi e, ishiguro h, et al. correlation between gingival crevicular fluid hemoglobin content and periodontal clinical parameters. journal of periodontology. 2016;87(11):1314–9. 20. ito h, numabe y, hashimoto s, uehara s, wu y-h, ogawa t. usefulness of hemoglobin examination in gingival crevicular fluid during supportive periodontal therapy to diagnose the pre-symptomatic state in periodontal disease. clinical oral investigations. 2020. 21. loe h. the gingival index, the plaque index and the retention index systems. journal of periodontology. 1967: 38(6): 602–16. 22. guentsch a, kramesberger m, sroka a, pfister w, potempa j, eick s. comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis. journal of periodontology. 2011;82:1051–60. 23. heidari a, shahrabi m, rokouei m, amirzargar a, rahbar p. comparative study of substance p and neurokinin a in gingival crevicular fluid of healthy and painful carious permanent teeth. dental research journal (isfahan). 2017;14(1):57–61. 24. loos b, tjoa s. host-derived diagnostic markers for periodontitis: do they exist in gingival crevice fluid? periodontology 2000. 2005;39:53–72. 25. naghsh n, sabet nk, vahidi f, mogharehabed a, yaghini j. relationship between periodontal disease and serum factors in patients undergoing hemodialysis. open dentistry journal. 2017;11:701–9. 26. ito h, numabe y, sekino s, murakashi e, iguchi h, hashimoto s, et al. evaluation of bleeding on probing and gingival crevicular fluid enzyme activity for detection of periodontally active sites during supportive periodontal therapy. odontology. 2014;102(1):50–6. 27. sabarathinam npm, selvaraj j. salivary hemoglobin: a biomarker in periodontitis. drug invention today. 2019;11(7):1640–2. https://doi.org/10.22317/jcms.v8i5.1284 10 j contemp med sci | vol. 9, no. 1, january-february 2023: 10–17 review ruminant gut microbiota: importance, development, and alternative therapeutics for dysbiosis nahlah albakri1,2, reda amasha1, magda m. aly1,3* 1department of biology, college of science, king abdullaziz university, jeddah, saudi arabia. 2department of biology, college of science, taibah university, al-medinah al-munawarh, saudi arabia. 3botany and microbiology department, faculty of science, kafrelsheikh university, kafrelsheikh , egypt. *correspondence to: magda m. aly (e-mail: mmmohammad@kau.edu.sa) abstract the microbiome is a population of microbes that colonized in mammalian gut. during the first few years of life, the gut microbiome undergoes alteration and is very diverse in adulthood, depends upon various of circumstances. gut microbes, particularly gut flora in ruminants, are receiving more and more attention. intestinal microbes, particularly ruminant microorganisms, have attracted an increasing amount of attention as high-throughput sequencing technology has improved and costs have decreased, whether in the fundamental research or application fields. the ruminant microbiome changes in conjunction with its host and it is influenced by inter-microbial interactions, environmental exposures, and host properties. however, any organism’s core functional microbiome is much more conventional. unfortunately, the fragile growth ratio of the microbial culture is susceptible to incursions under illness circumstances, which may affect the abundance of various microbial species, resulting to dysbiosis. as a result, the purpose of this review is to provide a broad summary of the relevance of ruminant gut microorganisms, as well as to investigate variables that influence the microbiota and alternative therapeutics such as probiotics, prebiotics, fecal transplantation, and rumen transfiguration, all of which have been shown to be effective in addressing dysbiosis. keywords: gastrointestinal microbiome, alternative therapeutics, dysbiosis issn 2413-0516 introduction the gut microbiota is a diverse collection of microorganisms that occur in the gastrointestinal tract of mammals. this microbial community has a host-specific composition that is continually changing and responsive to both external and endogenous changes. the importance of this “organ’s” structure and function in health and illness has been emphasized by increased attention to its structure and function. the intestinal microbiota, a complex ecosystem, contains around 1000 species of firmicutes, bacteroidetes, actinomycetes, proteophylum, and verrucomicrobia.1,2 from nutritional condition to behavior and stress response, the microbiota is closely connected to many characteristics of normal host physiology. they might also be a main or secondary cause of a variety of diseases that affect both neighboring and distant organ systems. the presence or absence of significant species accomplished of causing particular responses, as well as the overall balance of the gut microbial community, are crucial in maintaining homeostasis or deficiency thereof in the intestinal mucosa and beyond. the mechanisms concluded which the microbiota exerts its valuable or detrimental effects are currently unknown. however, they contain both intestinal epithelial and mucosal immune cells developing signalling molecules and identifying bacterial epitopes. advances in gut microbiota modelling and analysis will help us better understand their role in health and illness, allowing us to tailor present and future therapeutic and prophylactic approaches.3,4 very complicated biological materials, such as taxonomic and useful identification of microbial groups that almost fill all present ecological niches, may now be easily examined thanks to technological advances in mass spectrometry, next-generation sequencing and bioinformatics. metaproteomic techniques give operational data on the microbiota examined, as well as structural data collected via metagenomic research. combining the major omics technologies (metabolomics, transcriptomics, proteomics, and genomics) in living science offers very accurate data on the study object and aids in the understanding of molecular changes in response to externaland internalecological stimuli. the microbial communities that colonized animals’ gastrointestinal tracts have an essential role in their metabolism as well as their physiology and health. veterinarians, animal nutritionists, and microbiologists are interested in the microbiotas of cattle and ruminants.5 between 2008 and 2014, just 20 papers were published yearly, a ratio of smaller than five per year, and there was no relevant literature before to 2008. with more than 10 papers produced per year after 2014, research entered a new era of fast expansion. the microbial diversity of the rumen or omasum and reticulum of giraffes, moose, bovine and kangaroos, lambs, steers, musk deer, goats, sheep, geese, muskoxen, yark, camel, swine, grazing primate.6 definition of microbiome the microbiome is a community of microorganisms that live or are present on the bodies of animals (viruses, archaea, bacteria including fungi, protozoa, and bacteriophages) (skin, urogenital tract, oral cavity, gastrointestinal tract and respiratory tract). the microbiome is also defined as the genetic information of microbes that inhabit in a certain environment, which includes species whose genomes are the only thing that distinguishes them.7,8 members of the microbiome might be commensal (without harming various types that benefit from one another), symbiotic (both helpful species benefiting from one another), pathogenic, or parasitic.9 rumen microbiome the four components of the ruminal stomach (actual stomach) are the reticulum, rumen, omasum (pre-stomach), and (submitted: 08 november 2022 – revised version received: 28 november 2022 – accepted: 03 january 2023 – published online: 26 february 2023) 11j contemp med sci | vol. 9, no. 1, january-february 2023: 10–17 n. albakri et al. review ruminant gut microbiota: importance, development, and alternative therapeutics for dysbiosis abomasum.10 because the rumen is the stomach’s most important portion, its microbial ecology enables it to use complex carbohydrates and fiber-rich diets,11 resulting in the digestion of around 70% of the cellulose consumed.12 the most abundant prokaryotes are bacteria, accounting for more than 95% of the ruminal microbiota.13 in the rumen, the phylum bacteroidetes is the most abundant, accounting for 45–57 percent 16s rrna patterns and 90% of the bacteroidetes population (with the genus prevotella accounting for 45–57% 16s rrna patterns and 90% of the bacteroidetes population). followed by firmicutes accounting for 28 percent. moreover, dialister, succiniclasticum, mitsuokella, butyrivibrio, and ruminococcus made up over 1% of all bacterial species found in the rumen.14,15 small intestine microbiome carbohydrates and proteinabsorptionis thecore function of the small intestine,12 and its three sections (duodenum, jejunum, and ileum) have distinct roles and microbial ecosystems. according to a report, the phylum firmicutes was more prevalent in all compartments of the cow gastrointestinal system except the jejunum that were dominated by proteobacteria. in the jejunum, ruminococcus, acetitomaculum, and lachnospiraceae were frequent, whereas enterobacteriaceae were prevalent in the small intestine.16 the relative abundance of the phylum bacteroidetes reduced dramatically (0.4–1.1 percent) as compared to the rumen, while the comparative abundance of the phylum firmicutes increased significantly (0.4–1.1%) (up to 80 percent of total relative abundance). tenericutes (0.4–4%), proteobacteria (0.8–5.8%), and actinobacteria (6–13%) are examples of low-abundance taxa that have been investigated. propionibacterium, bulleidia, mogibacterium, lactobacillus, mitsuokella, ruminococcus, and butyrivibrioare some of the other major genera found in the small intestine.14,15 large intestine microbiome in the rectum, colon, and cecum, bacterial concentrations vary from 1012 to 1014 cells per millilitre.12 the large intestine is important for water digestion and absorption since it is responsible for 30 % of cellulose digestion.12,17 various parts of the large intestine have different amounts of microbial abundunce and diversity in the corresponding microbiota. the phylum firmicutes have been shown to be the most abundant in the cecum, accounting for up to 81% of all phyla, with bacteroidetes accounting for 18–26%. tenericutes, actinobacteria and spirochetes have all been discovered in the cecum. oscillospira, ruminococcus,coprococcus, turicibacter, dorea, blautia, clostridium, and prevotella were the most common taxa in the cecum.14 firmicutes makes up 81% of the colon’s relative microbial abundance, with bacteroidetes accounting for 21–33%. furthermore, of the remaining 23 phyla, the most common bacteria were fibrobacteria, tenericutes, spirochetes, proteobacteria, and actinobacteria. the most prevalent genera were coprococcus, dorea, oscillospira, ruminococcus, turicibacter, blautia, parabacteroides, and prevotella.14,18 the phylum firmicutes has invaded the rectum in a similar fashion. the major genera in the rectum were clostridium, prevotella, turicibacter, succinivibrio, ruminococcus, roseburia, coprococcus, bacteroides, and osillospira.19 importance of the rumen in digesting forages the rumen and associated microorganisms, such as fungi, archaea, bacteria, and protozoa, may help mammalian enzymes use substrates that aren’t easily available to them.20,21 as a result, the host ruminant has substrates that may be absorbed.22 feeds become increasingly well-known for microbial colonisation after a physical breakdown during ingestion, rumination, and in the rumen.23,24 microorganisms consume simple carbohydrates to make vfas such acetate (for fatty acid synthesis), propionate (for glucose synthesis), and butyrate (for butyrate synthesis), which are largely used as energy sources in the ruminant body. microbiome composition25,26 and ruminal circumstances influence the amount of various vfas in the rumen. intake rate, dietary forage to concentrate ratio, and diet type all impact ruminal conditions (e.g., breakdown rate, molecular structure). acetate is produced more quickly in forage-dominated diets, whereas propionate is produced more easily in concentrate-based diets. feeds heavy in starch and protein stimulate propionate synthesis, whereas hemicellulose and simple sugars raise butyrate production and cellulose increases acetate production.27 feed must be maintained long enough in the reticulorumen for microorganisms to adequately break down and ferment plant fibre. the supply of energy and protein in the rumen influences the efficiency and quantity of microbial protein production.28 ruminantanimals have a filter between the reticulum and the omasum that serves to prolong the ruminal retention time for neutral detergent fibre, which is an important component of forage.29 carbohydrates are bacteria’s primary source of energy, but they may also serve as carbon skeletons for protein synthesis when combined with amino acids, ammonia, or short peptides.30,31 during protein breakdown, peptides and amino acids are generated, which may be utilized by microbes (transamination) or deaminated to form vfas, co2, and ammonia. 30,32 ammonia that exceeds the ruminal wall’s microbial growth capabilities is ingested, transformed to urea, and then returned to the rumen through saliva or urine.33 morphological and microbial development of the rumen the rumen’s development and microbe colonization are a two-way interaction between the host and microbial colonies. rumen morphological development is aided by solid feed consumption. vfa synthesis and absorption as fermentation end-products aid the development of ruminal papillae, allowing for their absorption and subsequent epithelial metabolism.34,35 butyrate is the most efficient epithelial length and function activator, followed by propionate. roughages, on the other hand, have a physical structure that increases ruminal volume, aids muscle growth,36,37 and promotes rumen rumination and saliva flow.38 the rumen’s principal enzymatic activities of ruminal microbiota (proteolysis, fibrolysis, ureolysis, and amylolysis) have been reported from four to ten days of life.39,40 during the early stages of life, more than 60 glycoside hydrolase microbial genes were found in the rumen, suggesting a high capacity for plant carbohydrate metabolism even in the absence of regular plant cell wall consumption.41 because 60–80% of all vfas are absorbed across the ruminal 12 j contemp med sci | vol. 9, no. 1, january-february 2023: 10–17 ruminant gut microbiota: importance, development, and alternative therapeutics for dysbiosis review n. albakri et al. highly fermentable carbohydrates produces a large amount of vfa, lowering ruminal ph and causing acidosis, which may disturb physiological balance and change microbial ecology. different kinds of roughages and the forage-to-concentrate ratio may have an impact on the ruminal metabolism and fermentation. dairy cows given a mixture of leymus chinensis hay, alfalfa hay, and corn silage produce more valerate, acetate, methylamine, and hydro cinnamate, but less glycine, glucose, isovalerate, and propionate, than dairy cows fed corn stover.56 the thickness of the stratum spinosum, stratum granulosum, stratum basale, and stratum corneum, as well as the size of the rumen papilla, are all affected by roughage. changes in tlr gene expression in dietary components have a comparable effect. dietary compositions and types have been demonstrated to impact ruminal microbiota, rumen epithelial tissue shape, and receptors in several investigations. ruminant farmers would benefit greatly from a good understanding of the interaction between rumen epithelial cells and rumen microbes when using dietary treatments. age effects on the rumen microbiota another element that influences rumen microbes is the age of the host. rumen microbes vary from newborn to 2-year-old cows.44 this might be due to dietary changes, in part (milk, colostrum, milk-supplemented rations for calves, and total mixed rations for adult cows). the host’s development is a crucial determinant in git microbiota modifications since the microbiota of cows given the same diet differs with age.41 it’s most likely due to changes in the rumen and metabolites as the animals become older. these findings imply that as the host ages, alterations in the rumen microbiome may occur. however, there is a lack of knowledge on how the rumen microbiota and the hosts interact at different ages. more research is needed to provide the knowledge needed to build innovative techniques to increasing animal production at different phases. external environment effects on the rumen microbiota the rumen microbiota and hist interaction is influenced by the external living environment, including as temperature, climate, herd management, humidity, geography, and geography. the impact of the living environment on the comparability of ruminal microbiomes in cattle and bison.57 as a consequence, the microbial genomes in the git change as the hosts’ living environments change. the gut microorganisms and microbial activity of a ill person may vary from that of a healthy one.58 identifying the causes of microbial diversity changes and calculating the host-microbiota interaction is difficult due to the complexity of the git environment.54 despite the need for further study across species, nutrition has the greatest impact on the ruminal microbiota. using today’s omics technology, scientists might rapidly identify the microbial makeup, host-microbe interactions, variables impacting the git microbiota, androles in the git. future study should concentrate on the mechanisms that support the host-microbe relationship, such as dietary impacts on the git microbiota and epithelial cells, as well as their coordinated control.59 wall and with 75–90% of absorbed butyrate being metabolised by the ruminal epithelium, the ketogenic capacity of the rumen must develop, as a calf grows, to that of a mature rumen. after birth, microbial inoculation of the rumen began with contact with the vaginal canal, faeces, colostrum, the dam’s skin, and saliva. the presence of methanogens, fibrinolytic bacteria, and proteobacteria in the rumen of calves less than 20 minutes after birth has previously been investigated.42 inoculation may happen before birth, with rapid changes in the early days of life as initial colonising aerobic or facultative anaerobic bacteria from the biotype for the later fully anaerobic microbes.43,44 proteobacteria were found in >90% of newborn goat sequences, showing that the rumen and epidural microbial communities are formed in the same manner.45 this might be because of their ability to scavenge oxygen from the capillary network, allowing anaerobic colonies to develop more easily. many studies41,44,46 have shown that the prewarned rumen has the same dominating phyla as the more developed post weaned rumen, namely bacteroidetes, proteobacteria, and firmicutes, with relative abundance varying with age. firmicutes numbers grow after weaning, however bacteroidetes, notably prevotella, are more reliant on solid food intake than milk elimination, reaching a stable abundance at 7 weeks and solid food consumption topping 100 g per day, respectively.46–48 this demonstrates that the earlier a calf begins to eat solid food, the sooner a mature ruminal bacterial community arises.49 looked at the effects of birth mode (natural vs. caesarean) and diet on the ruminal microbiome’s development, as well as random factors in early life. others50,51 have revealed host genetic influences on the ruminal microbiome. the importance of defining ruminal function in young ruminants before, during, and after weaning was highlighted by all of these genetic and early-life effects. factors influencing the microbiota-host interaction in reaction to environmental changes, the rumen microbiota alters its composition and function. many scientists have studied the variables that influence the rumen microbiota throughout the past few decades. various methods, ranging from demographic fingerprinting to high-throughput sequencing, were used. diet, environment, and age all influence the rumen microbiota. diet effects on the rumen microbiota rumen function, which is governed by interactions between the environment andhost genes, leads to changes in rumen microbial ecology, has a significant impact on ruminant production. by modifying microbial populations and fermentation activity, the type of diet may have a significant influence on rumen function.52–55 roughage has a large influence on rumen growth, and vfa absorption is aided by gene expression in rumen epithelial cells. as a result, one of the most critical features of large ruminant feeding operations has been modified to enhance feed efficiency. ruminants have acquired a digestive mechanism that can digest roughages during millions of years of evolution. when ruminant animals are fed fiber-deficientor high-grain diets, the rapid breakdown of 13j contemp med sci | vol. 9, no. 1, january-february 2023: 10–17 n. albakri et al. review ruminant gut microbiota: importance, development, and alternative therapeutics for dysbiosis altered microbiota: dysbiosis a microbiome is an interacting and changing micro-ecosystem defined by the genetic components, structures, and metabolites of unique microbiota. the microbiota may be found in a variety of environments, including among eukaryotic hosts. many studies have identified the microbiome as a neglected niche in the maintenance of physiological functioning in eukaryotic host settings.12 the microbiome’s influence on ruminant growth and immunity has been extensively researched.13 the commensal microbiota contributes to animal health in a variety of ways, including helping in the digestion of indigestible plant fibre.60 it also supplies the host with sustenance and energy (volatile fatty acids), building components (lipids, peptides, and carbohydrates), and immune system modification (antibodies and cytokines).61 the microbiome, which competes with pathogens for adhesion sites and nutrition, separates infections and immune cells.62,63 furthermore, antimicrobial compounds such as hydrogen peroxide, organic acids, biosurfactants, and bacteriocins are produced by these microbes, limiting pathogenic development.15 dysbiosis is a breakdown or imbalance in the gastrointestinal tract’s natural microbiota. dysbiosis may be caused by an increase or decrease in the number of commensal bacteria, the introduction of pathogenic organisms, or the development of opportunistic microorganisms. because the microbiota is a metabolically active “organ,” dysbiosis may impact the production of essential nutrients or metabolites like short-chain fatty acids or secondary bile acids. significant dysbiosis has been associated to acute diarrhoea (infectious, non-infectious, and hemorrhagic), chronic diarrhea (food or antibiotic response and ibd), gi motility problems, epi, antibiotics, and gastric acid reducers. despite the limited evidence, dysbiosis seems to be a significant component in both acute and chronic feline and canine diarrhoea. fatty acid, biotin, tryptophan, ascorbate, and glycosphingolipid metabolism in the microbiota are all affected by changes in gi bacterial groups.64 alternative therapeutics ways the use of probiotics probiotics are living associated to human and animal health that have a therapeutic effect on health when given in proper proportions.65 probiotics’ positive benefits may be produced in a variety of ways.66 pathogens competing for adhesion sites and nutrients may affect the host’s microbiota.67 in addition, probiotics aid in the maintenance of intestinal homeostasis, which improves barrier function. they may create antimicrobial metabolites such as lactic acid and diacetyl, as well as antimicrobial peptides such as bacteriocins.68 by interacting directly with host cells, probiotics have the ability to affect the immune system.69,70 the most prevalent probiotic microorganisms are food-grade bacteria from the families bifidobacterium, lactobacillus, and lactobacillus. probiotics also include microorganisms such as enterococcus and streptococcus.71 studies injecting probiotics to treat rumen acidosis were a realistic option due to the importance of dairy cattle. according to goto et al.,72 adding a probiotic cocktail including c. butyricum, e. faecium, and l. plantarum for seven days improved ph and lactic acid levels. yeast has also been used in cow probiotics after many studies shown that it reduces ruminal acidity.73,74 according to mohammed et al.,73 a diet supplemented with saccharomyces cerevisiae decreased subacute rumen acidosis but not acute acidosis. researchers have used probiotics as a viable alternative for preventing and managing mastitis in conjunction with global efforts to reduce antibiotic consumption.75,76 probiotics based on lab have been proven to increase the host immune response effectively, suggesting that they might be approved as a non-antibiotic mastitis treatment.66 the use of prebiotics prebiotics are natural compounds that are complexed by an animal’s superior gastrointestinal tract enzymes but can be digested, developed, and operate more efficiently by one or a small group of gut bacteria. the cumulative effect increases the host’s health.77,78 the most often used prebiotics in animals are manno-, fructose-, and trans galacto-oligosaccharides. in cattle, prebiotics have been demonstrated to reduce harmful bacteria adhesion and enhance immunological response.79 in the research, prebiotics were introduced to the holstein friesian diet, which reduced the incidence of e. coli.80 fecal microbiota transplantation the therapeutic transplantation of faecal microbiota from a healthy person into an ill person is known as “faecal microbiota transplantation,” or fmt. during the fmt procedure, all species that contribute to an entire complex ecosystem of the gastrointestinal microbiota are transplanted, including viruses, bacteria, fungi, archaea, and protozoa, as well as minute particulate feedstuffs, colonocytes, and metabolites.81 fmt has a long history of usage in humans, dating back to at least the 4th century in china when it was used to treat gastroenteritis and diarrhea.82 fmt administered by colonic enemas was shown to be a successful treatment approach in four human instances of pseudomembranous enterocolitis caused by staphylococcus aureus in an early case report published in the united states.83 fmt has been more commonly employed in hospitals and clinics in recent years as a very successful treatment option for recurrent clostridium difficile infections that are resistant to antimicrobials.84 although c. difficile infections are the most common ailment now treated with fmt in the modern era, many other disorders have shown a favourable response to experimental fmt treatment, includingidiopathic thrombocytopenic purpura, insulin sensitivity, and chronic fatigue syndrome in patients with metabolic syndrome.85–87 the actual mechanism behind fmt’s success in the majority of disorders is unknown. increased microbial diversity, larger numbers of beneficial microbial communities, and immune system modulation are most likely to be responsible. the most common historical use of fmt in animals is in ruminants, where it is used to return microbes to the ruminal contents of cattle. it’s commonly used for digestive or metabolic issues, such as inappetence or ruminal hypomotility.88 ruminant transformation has a lengthy history, dating back to the 17th century in italy, when it was initially noted as a way for restoring correct rumination.89 for millennia, sweden has employed regurgitated digesta or cud as a method for microbial transplantation to treat ruminal indigestion, with cud being described to as a “living thing”90 fmt has lately gained popularity as a treatment and prevention method in other animals including household pets. 14 j contemp med sci | vol. 9, no. 1, january-february 2023: 10–17 ruminant gut microbiota: importance, development, and alternative therapeutics for dysbiosis review n. albakri et al. although the fmt efficacy exact mechanism in animals, the majority of ailments is unclear, many possibilities have been offered. one of the most well-documented mechanisms of action is restoring normal flora by repopulating the gut with a full diverse population of microorganisms.91,92 transformation in ruminants, for example, is thought to be advantageous because it recolonizes the rumen with beneficial anaerobes, restoring normal fermentation function. furthermore, increasing the variety of the microbiome enhances the host’s capacity to absorb complex carbohydrates, which aids digestion. fmt is believed to have a role in gastrointestinal pathogen competitive inhibition by recolonizing normal bacteria, where beneficial microbes outcompete all the pathogens infection steps.93,94 fmt has recently emerged as a viable therapy option for those afflicted with multidrug-resistant bacteria including vancomycin-resistant enterococcus faecalis and methicillin-resistant staphylococcus aureus.95–97 rumen transfaunation the rumen’s symbiotic microbiota provides nutrition and energy to the host by digesting ingesta and decomposing plant materials into different volatile fatty acids (vfas), ammonia, and other chemicals. as a result, improved rumen microbial digesting ability may improve feed efficiency and yield. many factors have been used in previous techniques to target the rumen microbiota. various studies have looked at changing animal diets,1 lowering rumen ph,98 reducing rumen protozoa,99 and other approaches. however, none of the aforementioned procedures have consistently positive effects, suggesting that long-term ways for increasing animal performance by altering rumen microbiota have yet to be identified.100 rumen transformation is the process of transferring rumen fluid from healthy animals to animals that have a wide variety of microbes. the transplanted rumen fluid provides energy and nutrients to the rumen microbial population.101 vfa, microbial proteins, amino acids, vitamins, enzymes and minerals are among the nutrients found in rumen fluid.102,103 small quantities (1 l) of rumen fluid transformed, which enhances rumen function and feed intake in cows, may also be used to treat feed indigestion in cows.104 microbial intervention in young calves may also be intentionally induced, affecting calf health and rumen microbiota development. inoculating newborn animals with rumen fluid from adult animals (fresh, lyophilized, or autoclaved) improved feed efficiency and weight gain.105,106 inoculating newborn lambs with mature lyophilized rumen fluid dramatically increased growth performance during and after weaning and starter meal digestibility, according to a recent sheep study.107 vaccination also raised ruminal propionate concentrations and rumen amylase activity, as well as lowering the acetate/propionate ratio. the usage of mature lyophilized rumen fluid increased the population of streptococcus ruminantium, which is linked to starch consumption.108 similarly, in young calves fed exogenous rumen fluid obtained from an adult cow, the relative abundance of eight bacterial genera (acidiphilium, sporosarcina, polaribacte, bdellovibrio, microbacterium, pseudodesulfo vibrio, sporosarcina and jeotgalibaca) belonging to four phyla actinobacteria, proteobacteria, bacteroidetes, and firmicutes.109 according to a recent study,110 inoculating fresh rumen fluid from adult goats promotes early rumen colonisation through the strong protozoal population, resulting in enhanced rumen absorption, feed intake and vfa production during the preweaning period. the immune systems of suckling dairy calves were improved by spray-dried rumen fluid containing 1% maltodextrin, as seen by lower interleukin-6 blood concentrations.111 these findings suggest that providing exogenous rumen fluid to young ruminants enhances weight gain while also altering feed health, immunity, and digestibility.112 conclusion recently, gut microbiota has received more attention from scientists. the numbers of researches about intestinal microbiota, epically in ruminant gut, are increased. biotechnological improvement processes have positive roles in this field. gut microbiota is a symbiotic complex ecosystem that played key roles in the host health and immune system function. these key roles are affected by both external and internal factors. any alteration of these factors led to imbalance in the gut microbiota which is associated with dangerous diseases like dysbiosis. probiotics, prebiotics, fecal transplantation, and rumen transfiguration are success therapeutics ways for treating several gastrointestinal diseases.  references 1. chai, l., dong, z., chen, a., & wang, h. (2018). changes in intestinal microbiota of bufogargarizans and its association with body weight during metamorphosis. archives of microbiology, 200(7), 1087–1099. 2. meng, x., zhang, g., cao, h., yu, d., fang, x., de vos, w. m., & wu, h. (2020). gut dysbacteriosis and intestinal disease: mechanism and treatment. journal of applied microbiology, 129(4), 787–805. 3. sekirov, i., tam, n. m., jogova, m., robertson, m. l., li, y., lupp, c., & finlay, b. b. (2008). antibiotic-induced perturbations of the intestinal microbiota alter host susceptibility to enteric infection. infection and immunity, 76(10), 4726–4736. 4. sekirov, i., russell, s. l., antunes, l. c. m., & finlay, b. b. (2010). gut microbiota in health and disease. physiological reviews. 5. deusch, s., tilocca, b., camarinha-silva, a., & seifert, j. (2015). news in livestock research—use of omics-technologies to study the microbiota in the gastrointestinal tract of farm animals. computational and structural biotechnology journal, 13, 55–63. 6. ming, w., & ding, l. (2021). a brief history of ruminant gut microbiological research. environment, resource and ecology journal, 5(2), 23–28. 7. martinez, k. b., leone, v., & chang, e. b. (2017). microbial metabolites in health and disease: navigating the unknown in search of function. journal of biological chemistry, 292(21), 8553–8559. 8. desselberger, u. (2018). the mammalian intestinal microbiome: composition, interaction with the immune system, significance for vaccine efficacy, and potential for disease therapy. pathogens, 7(3), 57. 9. matijašić, m., meštrović, t., čipčićpaljetak, h., perić, m., barešić, a., &verbanac, d. (2020). gut microbiota beyond bacteria—mycobiome, virome, archaeome, and eukaryotic parasites in ibd. international journal of molecular sciences, 21(8), 2668. 10. membrive, c.m.b. anatomy and physiology of the rumen. in rumenology; millen, d.d., arrigoni, m.b., pacheco, r.d.l., eds.; springer: basel, switzerland, 2016; pp. 1–38. 11. petri, r. m., neubauer, v., humer, e., kröger, i., reisinger, n., &zebeli, q. (2020). feed additives differentially impact the epimural microbiota and host epithelial gene expression of the bovine rumen fed diets rich in concentrates. frontiers in microbiology, 11, 119. about:blank 15j contemp med sci | vol. 9, no. 1, january-february 2023: 10–17 n. albakri et al. review ruminant gut microbiota: importance, development, and alternative therapeutics for dysbiosis 12. o’hara, e., neves, a. l., song, y., & guan, l. l. (2020). the role of the gut microbiome in cattle production and health: driver or passenger?. annual review of animal biosciences, 8, 199–220. 13. cammack, k. m., austin, k. j., lamberson, w. r., conant, g. c., & cunningham, h. c. (2018). ruminnat nutrition symposium: tiny but mighty: the role of the rumen microbes in livestock production. journal of animal science, 96(2), 752–770. 14. myer, p. r., freetly, h. c., wells, j. e., smith, t. p. l., & kuehn, l. a. (2017). analysis of the gut bacterial communities in beef cattle and their association with feed intake, growth, and efficiency. journal of animal science, 95(7), 3215–3224. 15. khalil, a., batool, a., &arif, s. (2022). healthy cattle microbiome and dysbiosis in diseased phenotypes. ruminants, 2(1), 134–156. 16. mao, s., zhang, m., liu, j., & zhu, w. (2015). characterising the bacterial microbiota across the gastrointestinal tracts of dairy cattle: membership and potential function. scientific reports, 5(1), 1–14. 17. harfoot, c. g. (1981). anatomy, physiology and microbiology of the ruminant digestive tract. lipid metabolism in ruminant animals, 1–19. 18. myer, p. r., wells, j. e., smith, t. p., kuehn, l. a., & freetly, h. c. (2015). microbial community profiles of the colon from steers differing in feed efficiency. springerplus, 4(1), 1–13. 19. durso, l. m., miller, d. n., schmidt, t. b., & callaway, t. (2017). tracking bacteria through the entire gastrointestinal tract of a beef steer. agricultural & environmental letters, 2(1), 170016. 20. van nevel, c. j., & demeyer, d. i. (1996). control of rumen methanogenesis. environmental monitoring and assessment, 42(1), 73–97. 21. walker, w. f., & liem, k. f. (1994). functional anatomy of the vertebrates: an evolutionary perspective. 22. bergman, e. n. (1990). energy contributions of volatile fatty acids from the gastrointestinal tract in various species. physiological reviews, 70(2), 567–590. 23. cheng, k. j., fay, j. p., howarth, r. e., & costerton, j. w. (1980). sequence of events in the digestion of fresh legume leaves by rumen bacteria. applied and environmental microbiology, 40(3), 613–625. 24. mcallister, t. a., bae, h. d., jones, g. a., & cheng, k. j. (1994). microbial attachment and feed digestion in the rumen. journal of animal science, 72(11), 3004–3018. 25. henderson, g., cox, f., ganesh, s., jonker, a., young, w., & janssen, p. h. (2015). rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range. scientific reports, 5(1), 1–15. 26. seshadri, r., leahy, s. c., attwood, g. t., teh, k. h., lambie, s. c., cookson, a. l., ... & kelly, w. j. (2018). cultivation and sequencing of rumen microbiome members from the hungate1000 collection. nature biotechnology, 36(4), 359–367. 27. bannink, a., kogut, j., dijkstra, j., france, j., kebreab, e., van vuuren, a. m., &tamminga, s. (2006). estimation of the stoichiometry of volatile fatty acid production in the rumen of lactating cows. journal of theoretical biology, 238(1), 36–51. 28. tedeschi, l. o., fox, d. g., & russell, j. b. (2000, october). accounting for ruminal deficiencies of nitrogen and branched-chain amino acids in the structure of the cornell net carbohydrate and protein system. in proceedings of cornell nutrition conference for feed manufacturers (pp. 224–238). new york: cornell university. 29. van soest, p. j. (1996). allometry and ecology of feeding behavior and digestive capacity in herbivores: a review. zoo biology: published in affiliation with the american zoo and aquarium association, 15(5), 455–479. 30. bach, a., calsamiglia, s., & stern, m. d. (2005). nitrogen metabolism in the rumen. journal of dairy science, 88, e9–e21. 31. lanzas, c., broderick, g. a., & fox, d. g. (2008). improved feed protein fractionation schemes for formulating rations with the cornell net carbohydrate and protein system. journal of dairy science, 91(12), 4881–4891. 32. tamminga, s. (1979). protein degradation in the forestomachs of ruminants. journal of animal science, 49(6), 1615–1630. 33. cheng, l., cantalapiedra-hijar, g., meale, s. j., rugoho, i., jonker, a., khan, m. a., ... & dewhurst, r. j. (2021). markers and proxies to monitor ruminal function and feed efficiency in young ruminants. animal, 15(10), 100337. 34. sander, e. g., warner, r. g., harrison, h. n., &loosli, j. k. (1959). the stimulatory effect of sodium butyrate and sodium propionate on the development of rumen mucosa in the young calf. journal of dairy science, 42(9), 1600–1605. 35. suárez, b. j., van reenen, c. g., beldman, g., van delen, j., dijkstra, j., &gerrits, w. j. j. (2006). effects of supplementing concentrates differing in carbohydrate composition in veal calf diets: i. animal performance and rumen fermentation characteristics. journal of dairy science, 89(11), 4365–4375. 36. tamate, h., mcgilliard, a. d., jacobson, n. l., & getty, r. (1962). effect of various dietaries on the anatomical development of the stomach in the calf. journal of dairy science, 45(3), 408–420. 37. stobo, i. j. f., roy, j. h. b., & gaston, h. j. (1966). rumen development in the calf: 1. the effect of diets containing different proportions of concentrates to hay on rumen development. british journal of nutrition, 20(2), 171–188. 38. hodgson, j. (1971). the development of solid food intake in calves 4. the effect of the addition of material to the rumen, or its removal from the rumen, on voluntary food intake. animal science, 13(4), 581–592. 39. sahoo, a., kamra, d. n., & pathak, n. n. (2005). pre-and postweaning attributes in faunated and ciliate-free calves fed calf starter with or without fish meal. journal of dairy science, 88(6), 2027–2036. 40. kmet, v., flint, h. j., & wallace, r. j. (1993). probiotics and manipulation of rumen development and function. archives of animal nutrition, 44(1), 1–10. 41. li, r. w., connor, e. e., li, c., baldwin, vi, r. l., & sparks, m. e. (2012). characterization of the rumen microbiota of pre‐ruminant calves using metagenomic tools. environmental microbiology, 14(1), 129–139. 42. guzman, c. e., bereza-malcolm, l. t., de groef, b., & franks, a. e. (2015). presence of selected methanogens, fibrolytic bacteria, and proteobacteria in the gastrointestinal tract of neonatal dairy calves from birth to 72 hours. plos one, 10(7), e0133048. 43. malmuthuge, n., griebel, p. j., & guan, l. l. (2015). the gut microbiome and its potential role in the development and function of newborn calf gastrointestinal tract. frontiers in veterinary science, 2, 36. 44. jami, e., israel, a., kotser, a., & mizrahi, i. (2013). exploring the bovine rumen bacterial community from birth to adulthood. the isme journal, 7(6), 1069–1079. 45. wang, z., elekwachi, c., jiao, j., wang, m., tang, s., zhou, c., ... & forster, r. j. (2017). changes in metabolically active bacterial community during rumen development, and their alteration by rhubarb root powder revealed by 16s rrna amplicon sequencing. frontiers in microbiology, 8, 159. 46. meale, s. j., li, s., azevedo, p., derakhshani, h., plaizier, j. c., khafipour, e., & steele, m. a. (2016). development of ruminal and fecal microbiomes are affected by weaning but not weaning strategy in dairy calves. frontiers in microbiology, 7, 582. 47. meale, s. j., morgavi, d. p., cassar-malek, i., andueza, d., ortigues-marty, i., robins, r. j., ... &cantalapiedra-hijar, g. (2017). exploration of biological markers of feed efficiency in young bulls. journal of agricultural and food chemistry, 65(45), 9817–9827. 48. rey, m., enjalbert, f., combes, s., cauquil, l., bouchez, o., & monteils, v. (2014). establishment of ruminal bacterial community in dairy calves from birth to weaning is sequential. journal of applied microbiology, 116(2), 245–257. 49. furman, o., shenhav, l., sasson, g., kokou, f., honig, h., jacoby, s., ... & mizrahi, i. (2020). stochasticity constrained by deterministic effects of diet and age drive rumen microbiome assembly dynamics. nature communications, 11(1), 1–13. 50. roehe, r., dewhurst, r. j., duthie, c. a., rooke, j. a., mckain, n., ross, d. w., ... & wallace, r. j. (2016). bovine host genetic variation influences rumen microbial methane production with best selection criterion for low methane emitting and efficiently feed converting hosts based on metagenomic gene abundance. plos genetics, 12(2), e1005846. 51. wallace, r. j., sasson, g., garnsworthy, p. c., tapio, i., gregson, e., bani, p., ... & mizrahi, i. (2019). a heritable subset of the core rumen microbiome dictates dairy cow productivity and emissions. science advances, 5(7), eaav8391. 52. bevans, d. w., beauchemin, k. a., schwartzkopf-genswein, k. s., mckinnon, j. j., & mcallister, t. a. (2005). effect of rapid or gradual grain adaptation on subacute acidosis and feed intake by feedlot cattle. journal of animal science, 83(5), 1116–1132. 53. auffret, m. d., dewhurst, r. j., duthie, c. a., rooke, j. a., john wallace, r., freeman, t. c., ... &roehe, r. (2017). the rumen microbiome as a reservoir of antimicrobial resistance and pathogenicity genes is directly affected by diet in beef cattle. microbiome, 5(1), 1–11. 54. liu, k., xu, q., wang, l., wang, j., guo, w., & zhou, m. (2017). the impact of diet on the composition and relative abundance of rumen microbes in goat. asian-australasian journal of animal sciences, 30(4), 531. 55. pandit, r. j., hinsu, a. t., patel, s. h., jakhesara, s. j., koringa, p. g., bruno, f., ... & joshi, c. g. (2018). microbiota composition, gene pool and its expression in gir cattle (bosindicus) rumen under different forage diets using metagenomic and metatranscriptomic approaches. systematic and applied microbiology, 41(4), 374–385. 16 j contemp med sci | vol. 9, no. 1, january-february 2023: 10–17 ruminant gut microbiota: importance, development, and alternative therapeutics for dysbiosis review n. albakri et al. 56. zhao, s., zhao, j., bu, d., sun, p., wang, j., & dong, z. (2014). metabolomics analysis reveals large effect of roughage types on rumen microbial metabolic profile in dairy cows. letters in applied microbiology, 59(1), 79–85. 57. ribeiro, g. o., oss, d. b., he, z., gruninger, r. j., elekwachi, c., forster, r. j., ... & mcallister, t. a. (2017). repeated inoculation of cattle rumen with bison rumen contents alters the rumen microbiome and improves nitrogen digestibility in cattle. scientific reports, 7(1), 1–16. 58. clavel, t., gomes‐neto, j. c., lagkouvardos, i., & ramer‐tait, a. e. (2017). deciphering interactions between the gut microbiota and the immune system via microbial cultivation and minimal microbiomes. immunological reviews, 279(1), 8–22 59. liu, k., zhang, y., yu, z., xu, q., zheng, n., zhao, s., ... & wang, j. (2021). ruminal microbiota–host interaction and its effect on nutrient metabolism. animal nutrition, 7(1), 49–55. 60. firkins, j. l., & yu, z. (2015). ruminant nutrition symposium: how to use data on the rumen microbiome to improve our understanding of ruminant nutrition. journal of animal science, 93(4), 1450–1470. 61. van den broek, m. f., de boeck, i., kiekens, f., boudewyns, a., vanderveken, o. m., &lebeer, s. (2019). translating recent microbiome insights in otitis media into probiotic strategies. clinical microbiology reviews, 32(4), e00010–18. 62. karstrup, c. c., klitgaard, k., jensen, t. k., agerholm, j. s., & pedersen, h. g. (2017). presence of bacteria in the endometrium and placentomes of pregnant cows. theriogenology, 99, 41–47. 63. moore, s. g., ericsson, a. c., poock, s. e., melendez, p., & lucy, m. c. (2017). hot topic: 16s rrna gene sequencing reveals the microbiome of the virgin and pregnant bovine uterus. journal of dairy science, 100(6), 4953–4960 64. webb, c. b. (2019). fecal microbiota transplantation: from theory to practice 65. hill, c., guarner, f., reid, g., gibson, g. r., merenstein, d. j., pot, b., ... & sanders, m. e. (2014). the international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic. nature reviews gastroenterology & hepatology, 11(8), 506–514. 66. krishnan, d., al-harbi, h., gibson, j., olchowy, t., & alawneh, j. (2020). on the use of probiotics to improve dairy cattle health and productivity. microbiology australia, 41(2), 86–90. 67. wan, m. l. y., forsythe, s. j., & el-nezami, h. (2019). probiotics interaction with foodborne pathogens: a potential alternative to antibiotics and future challenges. critical reviews in food science and nutrition, 59(20), 3320–3333. 68. gao, j., li, y., wan, y., hu, t., liu, l., yang, s., ... & cao, h. (2019). a novel postbiotic from lactobacillus rhamnosus gg with a beneficial effect on intestinal barrier function. frontiers in microbiology, 10, 477. 69. anas, m., ahmed, k., &mebrouk, k. (2014). study of the antimicrobial and probiotic effect of lactobacillus plantarum isolated from raw goat’s milk from the region of western algeria. world applied sciences journal, 32(7), 1304–1310. 70. sanders, m. e., merenstein, d. j., reid, g., gibson, g. r., & rastall, r. a. (2019). probiotics and prebiotics in intestinal health and disease: from biology to the clinic. nature reviews gastroenterology & hepatology, 16(10), 605–616. 71. suryadi, u., nugraheni, y. r., prasetyo, a. f., & awaludin, a. (2019). evaluation of effects of a novel probiotic feed supplement on the quality of broiler meat. veterinary world, 12(11), 1775. 72. goto, h., qadis, a. q., kim, y. h., ikuta, k., ichijo, t., & sato, s. (2016). effects of a bacterial probiotic on ruminal ph and volatile fatty acids during subacute ruminal acidosis (sara) in cattle. journal of veterinary medical science, 16–0211. 73. mohammed, r., vyas, d., yang, w. z., & beauchemin, k. a. (2017). changes in the relative population size of selected ruminal bacteria following an induced episode of acidosis in beef heifers receiving viable and non‐viable active dried yeast. journal of applied microbiology, 122(6), 1483–1496. 74. ogunade, i., pech-cervantes, a., & schweickart, h. (2019). metatranscriptomic analysis of sub-acute ruminal acidosis in beef cattle. animals, 9(5), 232. 75. rainard, p., & foucras, g. (2018). a critical appraisal of probiotics for mastitis control. frontiers in veterinary science, 251. 76. angelopoulou, a., warda, a. k., hill, c., & ross, r. p. (2019). non-antibiotic microbial solutions for bovine mastitis–live biotherapeutics, bacteriophage, and phage lysins. critical reviews in microbiology, 45(5-6), 564–580. 77. frizzo, l. s., signorini, m. l., & rosmini, m. r. (2018). probiotics and prebiotics for the health of cattle. in probiotics and prebiotics in animal health and food safety (pp. 155–174). springer, cham. 78. smulski, s., turlewicz-podbielska, h., wylandowska, a., & włodarek, j. (2020). non-antibiotic possibilities in prevention and treatment of calf diarrhoea. journal of veterinary research, 64(1), 119–126 79. singh, a., kerketta, s., yogi, r., kumar, a., & ojha, l. (2017). prebiotics: the new feed supplement for dairy calf. int j livest res, 7, 1–17. 80. grispoldi, l., bertero, f., franceschini, s., mastrosimone, f., sechi, p., iulietto, m. f., ... & cenci-goga, b. t. (2017). prevalence and characterisation of shigatoxigenic escherichia coli isolated from beef cattle fed with prebiotics. italian journal of food safety, 6(4). 81. bojanova, d. p., &bordenstein, s. r. (2016). fecal transplants: what is being transferred?. plos biology, 14(7), e1002503. 82. zhang, f., luo, w., shi, y., fan, z., & ji, g. (2012). should we standardize the 1,700-year-old fecal microbiota transplantation?. the american journal of gastroenterology, 107(11), 1755-author. 83. eiseman, b., silen, w., bascom, g. s., & kauvar, a. j. (1958). fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis. surgery, 44(5), 854–859. 84. hota, s. s., surangiwala, s., paterson, a. s., coburn, b., & poutanen, s. m. (2018). regional variability in fecal microbiota transplantation practices: a survey of the southern ontario fecal microbiota transplantation movement. canadian medical association open access journal, 6(2), e184–e190. 85. borody, t., nowak, a., torres, m., campbell, j., finlayson, s., & leis, s. (2012). bacteriotherapy in chronic fatigue syndrome (cfs): a retrospective review: 1481. official journal of the american college of gastroenterology| acg, 107, s591–s592. 86. vrieze, a., van nood, e., holleman, f., salojarvi, j., kootte, r. s., bartelsman, j. f., ... & nieuwdorp, m. (2012). transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome. gastroenterol 143 (4): 913–916. e7. 87. allegretti, j. r., kassam, z., hurtado, j., marchesi, j. r., mullish, b. h., chiang, a., ... & cummings, b. p. (2021). impact of fecal microbiota transplantation with capsules on the prevention of metabolic syndrome among patients with obesity. hormones, 20(1), 209–211. 88. mandal, r. s. k., joshi, v., balamurugan, b., gautam, d., chethan, g. e., & lekshman, a. (2017). rumen transfaunation an effective method for treating simple indigestion in ruminants. north-east veterinarian, 17(1), 31–33. 89. borody, t. j., warren, e. f., leis, s. m., surace, r., ashman, o., & siarakas, s. (2004). bacteriotherapy using fecal flora: toying with human motions. journal of clinical gastroenterology, 38(6), 475–483. 90. brag, s., & hansen, h. j. (1994). treatment of ruminal indigestion according to popular belief in sweden. revue scientifique et technique (international office of epizootics), 13(2), 529–535. 91. allegretti, j. r., & hamilton, m. j. (2014). restoring the gut microbiome for the treatment of inflammatory bowel diseases. world journal of gastroenterology: wjg, 20(13), 3468. 92. liu, s. x., li, y. h., dai, w. k., li, x. s., qiu, c. z., ruan, m. l., ... & shu, s. n. (2017). fecal microbiota transplantation induces remission of infantile allergic colitis through gut microbiota re-establishment. world journal of gastroenterology, 23(48), 8570. 93. collado, m., grześkowiak, ł., & salminen, s. (2007). probiotic strains and their combination inhibit in vitro adhesion of pathogens to pig intestinal mucosa. current microbiology, 55(3), 260–265. 94. khoruts, a., & sadowsky, m. j. (2016). understanding the mechanisms of faecal microbiota transplantation. nature reviews gastroenterology & hepatology, 13(9), 508–516. 95. cohen, n. a., & maharshak, n. (2017). novel indications for fecal microbial transplantation: update and review of the literature. digestive diseases and sciences, 62(5), 1131–1145. 96. niederwerder, m. c. (2018). fecal microbiota transplantation as a tool to treat and reduce susceptibility to disease in animals. veterinary immunology and immunopathology, 206, 65–72. 97. takáčová, m., bomba, a., tóthová, c., micháľová, a., &turňa, h. (2022). any future for faecal microbiota transplantation as a novel strategy for gut microbiota modulation in human and veterinary medicine?. life, 12(5), 723. 98. bevans, d. w., beauchemin, k. a., schwartzkopf-genswein, k. s., mckinnon, j. j., & mcallister, t. a. (2005). effect of rapid or gradual grain adaptation on subacute acidosis and feed intake by feedlot cattle. journal of animal science, 83(5), 1116–1132. 99. mosoni, p., martin, c., forano, e., &morgavi, d. p. (2011). long-term defaunation increases the abundance of cellulolytic ruminococci and in methanogens but does not affect the bacterial and methanogen diversity the rumen of sheep. journal of animal science, 89(3), 783–791. 100. zhou, m., peng, y. j., chen, y., klinger, c. m., oba, m., liu, j. x., & guan, l. l. (2018). assessment of microbiome changes after rumen transfaunation: 17j contemp med sci | vol. 9, no. 1, january-february 2023: 10–17 n. albakri et al. review ruminant gut microbiota: importance, development, and alternative therapeutics for dysbiosis implications on improving feed efficiency in beef cattle. microbiome, 6(1), 1–14. 101. depeters, e. j., & george, l. w. (2014). rumen transfaunation. immunology letters, 162(2), 69–76. 102. elfaki, m. o., & abdelatti, k. a. (2018). rumen content as animal feed: a review. journal of veterinary medicine and animal production, 7(2). 103. sarteshnizi, f. r., benemar, h. a., seifdavati, j., greiner, r., salem, a. z., & behroozyar, h. k. (2018). production of an environmentally friendly enzymatic feed additive for agriculture animals by spray drying abattoir’s rumen fluid in the presence of different hydrocolloids. journal of cleaner production, 197, 870–874. 104. steiner, s., linhart, n., neidl, a., baumgartner, w., tichy, a., & wittek, t. (2020). evaluation of the therapeutic efficacy of rumen transfaunation. journal of animal physiology and animal nutrition, 104(1), 56–63. 105. muscato, t. v., tedeschi, l. o., & russell, j. b. (2002). the effect of ruminal fluid preparations on the growth and health of newborn, milk-fed dairy calves. journal of dairy science, 85(3), 648–656. 106. zhong, r. z., sun, h. x., li, g. d., liu, h. w., & zhou, d. w. (2014). effects of inoculation with rumen fluid on nutrient digestibility, growth performance and rumen fermentation of early weaned lambs. livestock science, 162, 154–158. 107. yu, s., zhang, g., liu, z., wu, p., yu, z., & wang, j. (2020). repeated inoculation with fresh rumen fluid before or during weaning modulates the microbiota composition and co-occurrence of the rumen and colon of lambs. bmc microbiology, 20(1), 1–15. 108. wang, m., wang, r., liu, m., beauchemin, k. a., sun, x. z., tang, s. x., ... & he, z. x. (2019). dietary starch and rhubarb supplement increase ruminal dissolved hydrogen without altering rumen fermentation and methane emissions in goats. animal, 13(5), 975–982. 109. li, w., edwards, a., riehle, c., cox, m. s., raabis, s., skarlupka, j. h., ... & suen, g. (2019). transcriptomics analysis of host liver and meta-transcriptome analysis of rumen epimural microbial community in young calves treated with artificial dosing of rumen content from adult donor cow. scientific reports, 9(1), 1–11. 110. belanche, a., palma-hidalgo, j. m., nejjam, i., jiménez, e., martín-garcía, a. i., &yáñez-ruiz, d. r. (2020). inoculation with rumen fluid in early life as a strategy to optimize the weaning process in intensive dairy goat systems. journal of dairy science, 103(6), 5047–5060. 111. sarteshnizi, f. r., abdi-benemar, h., seifdavati, j., khalilvandi-behroozyar, h., seyedsharifi, r., & salem, a. z. m. (2020). influence of spray-dried rumen fluid supplementation on performance, blood metabolites and cytokines in suckling holstein calves. animal, 14(9), 1849–1856. 112. arshad, m. a., hassan, f. u., rehman, m. s., huws, s. a., cheng, y., & din, a. u. (2021). gut microbiome colonization and development in neonatal ruminants: strategies, prospects, and opportunities. animal nutrition, 7(3), 883–895. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1301 6 j cont med sci | vol. 1, no. 2, spring 2015:6–8 research objective this study includes the investigation of antibacterial activity of the local propolis against four types of bacteria isolated from patients. methods bacteria were tested including psedomonas sp, streptococcus sp, escherichia coli and staphylococcus aureus. six concentrations (0, 5, 10, 15, 20 and 25) mg/ml of propolis extracts were tested against bacteria. results results revealed the presence of significant difference (p < 0.05) in the effect of propolis extract against the four types of bacteria in this study. psedomonas sp. was the most sensitive among the others toward the propolis extract followed by streptococcus sp., e. coli and staphylococcus aureus at a rate of inhibition zones (14.09, 10.39, 8.78 and 8.39) mm, respectively. results of this study also showed increasing rate of inhibition zone if the concentration of propolis extract was increased. conclusion this study provided that local propolis has antibacterial activity against gram positive and gram negative bacteria. keywords antibacterial activity, propolis, inhibition zone, extract study of antibacterial activity in the local iraqi propolis alaa a kareem, noor y abdzaid, raghad m salman, murtadha k mohamed, ali j dekel & rawaa s abdul-muhsen introduction propolis is a natural product derived from plant resins collected by honeybees; it is used by bees as glue, general purpose sealer and as draught-extruder for beehives. propolis has been used in folk medicine for cosmetic purpose. the chemical composition of propolis is quite complicated, more than 300 compounds such as polyphenol, phenolic, aldehydes, sesquiterpene quinones, coumarin, amino acids, steroids and inorganic components have been identified in propolis sample. the content depends on collection location, time and plant source.1 among the types of chemical substances found in propolis are waxes, resins, balsams, aromatic and ethereal oil, pollen and other organic matters.2 propolis contain some enzymes like succinic dehydrogenase, glucose-6phosphatase, adenosine triphosphatase and acid phosphatase and small quantities of terpenes, tannins, traces of secretion from salivary gland of bees and possible contaminants. it also contains some minerals such as manganese and iron in addition to vitamins like b1, b2, b6, c and e and number of fatty acids.3 propolis shows a complex chemical composition responsible for its biological properties such as antibacterial, antifungal and antiviral, among other activities which have attracted the researcher’s interest. the laboratory tests studies have shown broad spectrum antimicrobial activity of various propolis extracts, depending upon its composition. propolis may show powerful local antibiotics and antifungal properties. many authors have demonstrated antibacterial activity of propolis against enterococcus spp., e. coli and staphylococcus aureus. reports have pointed out the efficient activity of propolis against gram-positive bacteria and limited action against gram-negative bacteria.4 many researches had investigated the antibacterial activity of propolis and its extract against gram-positive and gram-negative strain and they found that propolis had antibacterial activity against a wide range of gram-positive bacteria. antimicrobial activity of all propolis increases with increasing dosage without reaching a plateau at the highest dosage tested.1 the aim of the present study is to investigate the antibacterial activity for local propolis against g+ and g– bacteria. materials and methods microorganisms: four types of bacteria were obtained from clinical laboratory of al-hindiyya hospital: e. coli, pseudomonas sp., staphylococcus sp. and streptococcus sp. propolis extraction: propolis was grinded several times to get a very fine powder. the samples of propolis have been obtained from the apiaries of holy karbala province. the method described by ahmed et al. (1998)5 to obtain extracts is as follows: 1. 15 gm of propolis powder was added to 150 ml of 70% ethanol in a beaker and the beaker was covered with an aluminum foil. 2. the beaker was incubated in a shaker incubator at 35°c with 100 rotary/min for 48 h. 3. after the completion of the incubation process, the extracted liquid was filtered with gauze and then the filtrate was poured into a petri dish and was allowed to dry. 4. the dry extraction is collected and stored in a container for later use. preparation of propolis extract concentrations: the first step is to prepare stock solution by weighing 0.375 gm from the dry extract and dissolving in 15 ml of 70% ethanol to obtain the final concentration, 25 mg/ml in stock solution. then make the following concentration as given: stock solution ml ethanol 70% ml final concentrate mg/ml 2 – 25 1.6 0.4 20 1.2 0.8 15 0.8 1.2 10 0.4 1.6 5 – 2 0 department of clinical laboratories, college of applied medical sciences, karbala university, karbala, iraq. correspondence to alaa a. kareem (email: aakm7789@gmail.com). (submitted: 22 april 2015 – revised version received: 09 may 2015 – accepted: 29 may 2015 – published online: spring 2015) 7j cont med sci | vol. 1, no. 2, spring 2015:6–8 research investigation of antibacterial activity of the local propolisalaa a kareem et al. activation of bacteria nutrient broth was prepared according to the information of company (himedia, india). thirteen grams of media powder was taken and dissolved in 1 litre of distil water. the media was poured into tubes and sterilized in an autoclave. after this process, the media was left to cool. this can be used as activation media for bacteria by taking specimen from origin cultures of bacteria by swab and transport to nutrient broth tube, and then incubated at 37°c for 24 h. also we can use the sterilizing nutrient broth tubes to obtain an appropriate dilution for each bacteria by making serial dilutions and comparing with tube containing mcfarland solution. determination of antibacterial activity of propolis the antibacterial activity was determined by using the agar diffusion technique described by egorove (1985).6 mueller hintonagar was used for this purpose. this media was prepared according to the information of the company (himedia, india ). thirty-eight grams of powder was weighed and dissolved in 1 litre of distil water. then it was sterilized in an autoclave, left to reach 45–55°c and poured into petri dishes. each petri dish (mueller hinton agar media) was drilled with three wells and 50 µl of propolis extract concentrations was added to each well and waited for 1 h. these dishes were inoculated with an appropriate dilution for each bacteria separately by spreading 50 µl from the l-shaped spreader, and incubating at 37°c for 24 h. inhibition zone around wells was measured in millimetres. chloramphenicol 50 µg/ml was used as control for comparing the results. statistical analysis statistical analysis included factorial experiences analysis 7 × 4 with 3 replicates. the factors analyzed are the concentration of propolis extract and types of bacteria. a p value of 0.05 is the level of probability that was used to identify a significant difference. the significant differences between the averages were also tested by using less significant difference (lsd) test at the level of probability of 0.05.7 results as shown in table 1, the results of statistical analysis showed there were significant differences (p < 0.05) between the types of bacteria and between the concentrations of propolis extract compared with control (chloramphenicol). pseudomonas sp. is more sensitive towards propolis extract followed by streptococcus sp. moreover e. coli and staphylococcus aureus are less sensitive and without significant differences (p > 0.05) between them. the rate of inhibition zones were 14.09, 10.39, 8.78 and 8.39 mm of pseudomonas sp., streptococcus sp., e. coli and staphylococcus aureus, respectively. on the other hand, results showed significant differences (p < 0.05) between the concentrations that were used in the present study. the results also showed that the propolis extract concentration increased with increased inhibition zone compared with the control on the one hand and with concentrations on the other hand. the inhibition zones were 0, 3.20, 5.60, 10.85, 14.48 and 17.75 mm of propolis extract concentrations 0, 5, 10, 15, 20, 25 mg/ml, respectively. discussion one study showed that the iranian propolis was mainly active against gram-positive; however, it has been reported the ethanol extract propolis (eep) was effective on gram-negative bacteria at the higher concentration.4 in another study, propolis showed good antimicrobial activity against most of the isolates that include pseudomonas aeruginosa, e. coli, streptococcus pneumonia and staphylococcus aureus.8 one of the studies also showed that the brazilian propolis was effective against sta. aeurus more than e. coli.9 the composition of propolis can vary depending on the location of the bees and what trees and flowers they have access to. the composition of the plant source determines the chemical composition of propolis, for example in europe, china and north america, propolis was generally considered to be of the poplar-type, and other types can also be found.10 significant amounts of phenolic glycerides such as dicoumaroyl acetyl glycerol, diferuloyl acetyl glycerol, feruloyl coumaroyl acetyl glycerol and caffeoyl coumaroyl acetyl glycerol have been isolated from the propolis obtained in northern russia.11 the brazilian propolis represents 10–15% of the worldwide production, brazil being the third world producer, behind russia and china.14 among the types produced in brazil, green propolis table 1. inhibition zone (mm) of propolis extract against four types of bacteria concentration of propolis extract (mg/ml) types of bacteria mean of concentration lsd0.05 conc.pseudomonas sp. streptococcus sp. e. coli staphylococcus aureus 0 0 ± 0.0 0 ± 0.0 0 ± 0.0 0 ± 0.0 0.00 g 1.451 5 4 ± 0.8 2.7 ± 0.3 3.2 ± 0.12 2.9 ± 0.75 3.20 f 10 8 ± 1.2 5 ± 0.95 4.1 ± 0.83 5.3 ± 0.91 5.60 e 15 16.6 ± 1.7 10.7 ± 0.99 7.9 ± 0.65 8.2 ± 0.77 10.85 d 20 17.3 ± 1.85 16.6 ± 1.1 12.6 ± 1.04 11.4 ± 0.9 14.48 c 25 24.7 ± 1.52 16.7 ± 0.86 15.7 ± 1.32 13.9 ± 1.14 17.75 b chloramphenicol 50 µg/ml 28 ± 1.07 21 ± 1.42 18 ± 0.89 17 ± 0.92 21.00 a mean of bacteria 14.09 a 10.39 b 8.78 c 8.39 c lsd0.05 interference 2.903lsd0.05 bacteria 1.185 lsd: less significant difference. the numbers refer to mean ± standard error. the capital letters indicate significant differences (p < 0.05) between the concentrations. small letters indicate significant differences (p < 0.05) between bacteria. 8 j cont med sci | vol. 1, no. 2, spring 2015:6–8 investigation of antibacterial activity of the local propolis research alaa a kareem et al. references 1. khalil ml. biological activity of bee propolis in health and disease. asian pac j cancer pre. 2006 jan–mar;7(1):22–31. pmid: 16629510 2. marcucci mc. propolis: chemical composition biological properties and therapeutic activity. apidologie. 1995;26(2):83–99. doi: http://dx.doi.org/ 10.1051/apido:19950202 3. al-sheikh e. study effect of antibiotics and propolis on pathogenecity of the methicillin resistance staphylococcus aureus (mrsa) (b.sc. biology). baghdad: college of science, university baghdad; 2011. 4. yaghoubi smj, ghorbani gr, soleimanian zad s, satari r. antimicrobial activity of iranian propolis and its chemical composition. daru. 2007;15(1): 45–8. 5. ahmad i, mehmood z, mohammad f. screening of some indian medicinal plants for their antimicrobial properties. j ethnopharmacol. 1998 sept;62(2): 183–93. doi: http://dx.doi.org/10.1016/s0378-8741(98)00055-5 pmid: 9741890 6. egorove ns. antibiotics a scientific approach. moscow: mir publishers; 1985. 7. al-emam mmt. design and analysis of experiment. dar mars for publication. the kingdom of saudia arabia; 2007. 8. drago l, mombelli b, de vecchi e, fassina mc, tocalli l, gimondo mr. in vitro antimicrobial activity of propolis dry extract. j chemother. 2000 oct;12(5):390– 5. doi: http://dx.doi.org/10.1179/joc.2000.12.5.390 pmid: 11128558 9. fernandes jra, leomil l, fernandes aah, sforcin jm. antibacterial activity of propolis produced by apis mellifera l. and barazilian stingless bees. j venomous animal toxin. 2001;7(2):114–9. doi: http://dx.doi.org/10.1590/ s0104-79302001000200003 10. popova mp, graikou k, chinou i, bankova vs. gc-ms profiling of diterpene compounds in mediterranean propolis from greece. j agric food chem. (from greenish-yellow to deep green) prevails, gaining preference in the world propolis market.12 prenylated derivatives of p-coumaric, artepillin c (4-hydroxy3,5-diprenyl cinnamic acid), dupranin (4-hydroxy-3-prenyl cinnamic acid), (e)-3-prenyl-4-(dihydroxicinnamoyloxy)-cinnamic acid and diterpenic acids were detected in green propolis as well as in the buds of baccharis dracunculifolia, an asteraceae family from southeast and western-central brazil.13–16 conclusion the local iraqi propolis that was collected from the apiaries of holy karbala province has antibacterial activity against gram-positive negative bacteria.  2010 mar;58(5):3167–76. doi: doi: http://dx.10.1021/jf903841k pmid: 20112913 11. bankova v, popova m, bogdanov s, sabatini ag. chemical composition of european propolis: expected and unexpected results. z naturforsch c. 2002 may–jun;57(5–6):530–3. doi: http://dx.doi.org/10.1515/znc-2002-5-622 pmid: 12132697 12. paviani lc, dariva c, marcucci mc, cabral fa. supercritical carbon dioxide selectivity to fractionate phenolic compounds from the dry ethanolic extract of propolis. j food process eng. 2010 feb;33(1):15–27. doi: http://dx.doi. org/10.1111/j.1745-4530.2008.00256.x 13. salatino a, teixeira ew, negri g, message d. origin and chemical variation of brazilian propolis. evid based complement alternat med. 2005 mar;2(1):33–8. doi: http://dx.doi.org/10.1093/ecam/neh060 pmid: 15841276 14. teixeira ew, message d, negri g, salatino a, stringheta pc. seasonal variation, chemical composition and antioxidant activity of brazilian propolis samples. evid based complement alternat med. 2010 sep;7(3):307–15. doi: http://dx.doi.org/10.1093/ecam/nem177 pmid: 18955317 15. chang r, piló-veloso d, morais sa, nascimento ea. analysis of a brazilian green propolis from baccharis dracunculifolia by hplc-apci-ms and gc-ms. braz j pharmacogn. 2008;18 (4):549–56. doi: http://dx.doi.org/10.1590/ s0102-695x2008000400009 16. teixeira ew, negri g, meira rm, message d, salatino a. plant origin of green propolis: bee behavior, plant anatomy and chemistry. evid based complement alternat med. 2005 mar;2(1):85–92. doi: http://dx.doi. org/10.1093/ecam/neh055 pmid: 15841282 187j contemp med sci | vol. 9, no. 3, may-june 2023: 187–192 original genetic polymorphisms of dpyd in patients with breast cancer on capecitabine therapy raaid fadhl abbas1* , ahmed salih sahib1, hasanain shakir mahmood1, karar kadhim mohsin2, ali amal aldeen majeed1 1department of pharmacology and toxicology, college of pharmacy, university of kerbala, karbala, iraq. 2imam al-hussein hematology and oncology center, ministry of health, karbala, iraq. *correspondence to: raaid fadhl abbas (e-mail: raed.f@s.uokerbala.edu.iq) (submitted: 24 february 2023 – revised version received: 18 march 2023 – accepted: 15 april 2023 – published online: 26 june 2023) abstract objectives: breast cancer is the primary cause of death in iraqi women aged 30–54 years. the study examined the relationship between (g > a) (rs3918290) and (rs55886062, t > g) dpyd gene polymorphisms, their haplotypes, and capecitabine serum concentrations in postmenopausal iraqi women with breast cancer breast cancer in postmenopausal women during the capecitabine chemotherapy. methods: the study included 200 women: 100 apparently health (45–75 years old) and 100 with breast cancer (40–70 years old). this study, conducted between july and october 2022 at the oncology center at imam al-hussain medical city in kerbala, iraq, plasma levels of capacetabine and 5fu were measured in breast cancer patients who had been taking capecitabine for at least three months. all participants gave informed consent. results: capecitabine, and 5fu concentrations in breast cancer patients differed significantly. as the results showed, capecitabine, and 5fu had a significantly higher concentration of them in patients with the tt allele than in those with the cc and ct alleles for the polymorphism (ivs14 + 1g > a) (rs3918290) and in patients with dpyd*13 (rs55886062) with the cc allele rather than the aa and ac alleles. mutant allele carriers had increased capecitabine concentrations (p < 0.001). conclusion: ca15.3, serum calcium, and estradiol all exist in bodily serum, making them a potentially useful novel diagnostic biomarker for patients with breast cancer due to their high levels of stability, as well as the biological properties of tumors, such as serum calcium and estrogen. keywords: ca15.3, breast neoplasms, estradiol, capecitabine, polymorphism issn 2413-0516 introduction breast cancer is the most common cancer among women in developed countries, accounting for 23% of all cancers, and represents the most important location of cancer in women between 20 and 79 years of age. in men, breast cancer can also be present, although it only represents 1% of all diagnosed breast cancers.1 in iraq, it forms 22.3% of all malignant tumor and 37% of the registered female cancers with a sharp increase in the incidence of this tumor in younger age group.2 the number of newly diagnosed female breast cancer (fbc) cases increased from 870.2 thousand to 1937.6 thousand, with the age-standardized incidence rate significantly increased from 39.2/100,000 to 45.9/100,000.3 breast cancer, is a complex disease caused by common changes in the population in a number of genes, in combination with environmental factors. the identification and characterization of these common changes have been studies many years ago. they are a case‐control association studies design with a selection of 33 candidate genes, 19 of them involved in dna repair functions, and 14 genes with functions in the cell cycle control, genotyping a total of 169 snps in 547 cases of breast cancer.4 chemotherapy in advancement of chemotherapy may be recommended if the potential benefit outweighs the risk after a thorough evaluation of the patient’s health (age, menstrual status, blood test results, vital organs’ function, comorbidities, etc.), tumor characteristics (histological type, tumor grade, lymph node status, her2 and hormone receptor status, lymphovascular invation), and potential treatment strategies.5 capecitabine is a one-of-a-kind treatment that is specific to the s phase of the cell cycle. it is an antimetabolic fluoropyrimidine deoxynucleoside carbamate and is administered orally.6 the most probable place for the thymidine phosphorylase (dthdpase)-catalyzed conversion of capecitabine into 5-fluorouracil (5-fu) to take place in vivo is in tissues that are already carrying malignancies.7 dihydropyrimidine dehydrogenase is an enzyme that helps break down uracil and thymine when they are no longer needed in the cell, and it is encoded by the dpyd gene. pyrimidines like uracil and thymine are a class of nucleotides. the nucleotide base is the fundamental unit of all nucleic acids, including dna, rna, and the energy-transfer molecules atp and gtp.8 the study aimed to detect the genetic polymorphism of dpyd2 *a (rs3918290) and dpyd *13 (rs55886062) in participated breast cancer women and to investigate the effect of genetic polymorphism on capecitabine efficiency. materials and methods the study was approved by medical ethics committee of iraqi ministry of health the study on postmenopausal women with breast cancer who were treated at al-hussein medical oncology hospital and kerbala from july to december 2022. hospital consultants detected the condition using diagnostic criteria. the study included 100 patients (average age 45–74 years) and 100 apparently healthy controls (age 40–70 years). clinical, mammographic, and histological findings were used to diagnose patients, who received capecitabine chemotherapy after early detection. the serological study used elisa, while the molecular study used allele specific-pcr. five ml of peripheral blood was drawn from each patient, then divided into three gel tubes for serology and two ml remaining https://orcid.org/0009-0003-9538-3269 mailto:raed.f@s.uokerbala.edu.iq 188 j contemp med sci | vol. 9, no. 3, may-june 2023: 187–192 genetic polymorphisms of dpyd in patients with breast cancer on capecitabine therapy original r. f. abbas et al. of serum was saved in edta tubes for genotyping by detection of genetic polymorphism of dpyd2 *a (rs3918290) and dpyd *13 (rs55886062) in postmenopausal breast cancer women. inclusion criteria postmenopusal women > 45 years, just get only capecitabine chemotherapy in their course of treatment exclusion criteria women who started capecitabine with adjuvant chemotherapy or radiation therapy were excluded. women taking dpd inducers or inhibitors like 5-fluorouracil, dihydrofluorouracil, tegafur, gimeracil, or ethynyluracil were excluded. the study excluded patients with gastrointestinal diseases or surgery. on the other hand each patient was asked if she had used any drugs that may interfere with capecitabine metabolism or dpyd dehydrogenase activity during blood sample collection. genotyping for dpyd dehydrogenase polymorphisms detection for single-nucleotide polymorphisms (snps) genotyping, allele-specific amplification based on polymerase chain reaction (pcr) has seen widespread used in our study. on the other hand, the isolation of genomic dna from whole blood that is compatible with pcr is typically used according to manufacture company (addbio/korea). primer 3 plus generates template-specific primer pairs, and the specificity testing programme searches for primer-target matches using blast [blast: basic local alignment search tool (nih.gov)] for dpyd polymorphisms. lyophilized primers were dissolved with a certain volume of nuclease free water according to instruction of manufacture to give concentration of 100 pmol/μl (represent a stock solution) represent the volumes of nuclease free water added to each primer to obtain 100 pmol/μl (tables 1 and 2). pcr collection tubes was prepared with a total volume of 20 μl in premix of pcr tubes (accupower® pcr premix/ korea). the reaction components is described as 2 µl of forward primer, 2 µl of reverse primer, 4 µl of dna template and 12 µl of distal water and pcr amplification program is described as initial denaturation at 95˚c for 3 minute 35 cycles consists of denaturation at 95˚c for 30 sec, annealing at 60˚c for 45 sec and extension at 72˚c for 30 sec and final extension at 72˚c for 5 minute. dna gel electrophoresis for detected pcr products as following 3 μl of loading buffer and 5 μl of product were loaded on 1.5% agarose gel (1.5 g/100 ml 1x tbe support) and ran at 100 volt for 35 min. ethidium bromide (0.5 μg/ml) recolored the gel. dna bands were photographed on uv trans illuminator and then dna ladder (100–1500 bp) is used to measure band molecular size. measurement of drug concentration in patient serum sample preparation and hplc condition for measurement of capacetabine concentration, the extraction tube received 0.1 ml of human plasma. mixing 3 ml of ethylacetate/acetonitrile (4:1, v/v) followed. freeze-centrifuged at 3500 rpm. then the nitrogen stream evaporated the organic layer in a glass tube. dissolving the dry residue in 200 ul of 50% meoh, centrifuging at 3500 rpm, and transferring to an autosampler vial. hplc injected a 100 ul sample aliquot. 0.1% formic acid: meoh (45:55 v/v) was the mobile phase. mobile phase flow was 1.1 ml/min. the detector was uv-vis at 305 nm and the column was c18-ods (25 cm × 4.6 mm).9 on the other hand, sample preparation and hplc condition for measurement of 5 fu concentration, agno3 (20%, 600 μl) was added to human serum (1.0 ml) and vortexed for 3 min before standing for 5 min. nacl (20%, 700 μl) was added and vortexing continued for 3 min. after 12 min at 13,000 rpm, the supernatant (0.5 ml) was diluted with water to 1 ml in a centrifuge tube and filtered over a 0.22-μm membrane. analysis employed a german hplc type sykam with uv detector. a 250 centimetre, 4.6 μm c18-ods column separated. the column temperature remained 25°c. 5 mm kh2po4 solution (ph = 6.0) and methanol (96 : 4) at 1 ml/min were used to determine the standards and samples. 100 μl was injected at 254 nm.10 statistical analysis the statistical analysis system-sas (2012) program was used to study the effect of different factor in study parameters. mean ± standard error, anova test and t test used to significant compare between means in this study. alleles genotyping were presented as a percentage frequencies, and significant differences between their distributions in breast cancer patients and controls, were assessed by two-tailed fisher’s exact probability (p) test. results the studied population included 100 female patients with breast cancer. participants’ average age at study entry was 55.36 ± 10.85 years (range: 45–65). eighty six percent were married and only (14%) single. there was a 62% disparity between the women who had a family history of breast cancer table 1. primers sequences of dpyd*2a (ivs14 + 1g > a) (rs3918290) genetic polymorphism allele specific primer sequence (5'->3') product size reverse allele c 5-ctaaaggctgactttccagaacccc-3 411 bp reverse allele t 5-ctaaaggctgactttccagaaccct-3 forward common 5-gatatgctgcttctgcctcaggt-3 table 2. primers sequences of dpyd*13 (rs55886062, 1679t > g) genetic polymorphism allele specific primer sequence (5'->3') product size forward allele t 5-agccaccagcacatcaatgatt-3 400 bpforward allele g 5-agccaccagcacatcaatgatg-3 forward common 5-tgttccgcaccagctctggat-3 https://blast.ncbi.nlm.nih.gov/blast.cgi 189j contemp med sci | vol. 9, no. 3, may-june 2023: 187–192 r. f. abbas et al. original genetic polymorphisms of dpyd in patients with breast cancer on capecitabine therapy and those who didn’t (38%) among those who were diagnosed. cancer patients who had the disease on their left sides numbered 33%, while those on their right sides numbered 67%. it is also recorded that 94% of patients have already had surgery, 79% of patients already have radiation therapy, and 91% of patients have had chemotherapy (table 3). results of amplification reaction “dpyd polymorphism” (rs3918290) the amplification of snps of dpyd gene: rs3918290 was shown in figure 1. the presence of pcr bands with identity sizes in the agarose gel indicated the genotype of the samples as positive result. each reaction in different snps and genotypes are shown in detail in table 4. the pcr amplifications, fragment size 400 bp indicated that patient have specific alleles, it was required the use of two separate tubes for the amplification of wild-type and variant-type allele. the frequency and percentage of rs3918290 genotype that detected in the breast cancer patients are shown in table 4. the most frequent genotype in 100 breast cancer patients recruited in this study was the wild type (cc) with frequency and percentage 58 and 58% respectively, while the heterozygote type (ct) represent the lowest frequent type with frequency and percentage of 28 and 28% respectively. the mutation type of rs3918290, which carry tt genotype have been identified in frequency and percentage of 14 and 14% respectively. results of amplification reaction “dpyd polymorphism” (rs55886062) the amplification of snps of dpyd gene: rs55886062 was shown in in figure 2, the presence of pcr bands with identity sizes in the agarose gel indicated the genotype of the samples as positive result. each reaction in different snps and genotypes are shown in detail in table 5. the pcr amplifications, fragment size 410 bp indicated that patient have specific alleles, it was required the use of two separate tubes for the amplification of wild-type and variant-type allele. the frequency and percentage of rs55886062 genotype that detected in the breast cancer patients are shown in table 5. table 3. patients’ demographic groups and breast cancer’s unique features characters percentage age (years) 55.36 ± 10.85 duration of disease (years) 4.31 ± 1.22 duration of capecitabine (years) 3.29 ± 1.95 family history (%) yes 62% no 38% marital status (%) married 86% single 14% lymph node involvement (%) yes 39% no 61% breast cancer side (%) left breast 33% right breast 67% history of breast cancer chemotherapy (%) yes 92% no 8% history of breast cancer surgery (%) yes 90% no 10% history of breast cancer radiotherapy (%) yes 88% no 12% fig. 1 pcr amplification of rs3918290 gene showed: line m: represented dna marker (ladder) 100–1500 bp, line 1,2; : represented tt genotype (mutation), lines 5, 6, 7, 8;11,12: represented ct genotype (hetrozygoite) were showed in 400 bp, lines 3,4; 9,10;13,14; 15,16;17,18;19,20 : represented cc genotype (wild) were shown in 400 bp. fig. 2 pcr amplification of rs55886062 gene showed: line m: represented dna marker (ladder) 100–1500 bp, line 1,2;11,12;13,14;17,18 : represented cc genotype (mutation), lines 3, 3;9,10;15,16;19,20: represented ct genotype (hetrozygoite) were showed in 410 bp, lines 5,6;7,8 : represented aa genotype (wild) were shown in 410 bp. table 4. distribution of genotype and allele frequency of dpyd (rs3918290) polymorphism dpyd (rs3918290) genotype patients n = 100 n (%) (x2) hwe genotype frequency cc 58 (58%) (9.3364) 0.001c/t 28 (28%) tt 14 (14%) allele frequency c 72 (72%) (13.5) 0.001 t 28 (28%) hwe; hardy weinberg equlibrium. 190 j contemp med sci | vol. 9, no. 3, may-june 2023: 187–192 genetic polymorphisms of dpyd in patients with breast cancer on capecitabine therapy original r. f. abbas et al. the most frequent genotype in 100 breast cancer patients recruited in this study was the mutant type (cc) with frequency and percentage 7 and 7% respectively, while the heterozygote type (ca) represent the lowest frequent type with frequency and percentage of 40 and 40% respectively. the wild type of rs55886062, which carry aa genotype have been identified in frequency and percentage of 53 and 53% respectively. association between capacetabine and 5fu concentration and dpyd genotype (rs3918290) in patient postmenopausal women have breast cancer table 6 showed that there were clear significant differences in each of the following concentrations of capecitabine and 5fu in women with breast cancer, as the results recorded a significant higher concentration of them was recorded in each of capecitabine and 5fu in patients with the tt allele rather than in patients with the cc and ct alleles in patient with dpyd (rs3918290) characteristics, association between capacetabine and 5fu concentration and dpyd genotype (rs55886062) in patient postmenopausal women have breast cancer table 7 showed that there were clear significant differences in each of the following concentrations of capecitabine and 5fu in women with breast cancer, as the results recorded a significant higher concentration of them was recorded in each of capecitabine and 5fu in patients with the cc allele rather than in patients with the aa and ac alleles in patient with dpyd*13 (rs55886062) characteristics. discussion capecitabine is an anti metabolite, a class of chemotherapeutic agent. capecitabine is converted in the body to fluorouracil, which is used in a variety of chemotherapy treatments. the process of dna replication and repair is halted, making and repairing dna is essential for cancer cell proliferation.11 chemotherapy with the oral fluorouracil prodrug capecitabine has been found to be efficacious for breast cancer, yet, some of studies was usefulness in treating breast tumors is still up for debate. after anthracycline and taxane failure, capecitabine is typically used as a second-line chemotherapy option for patients with metastatic breast cancer.12 one hundred breast cancer patients were included in the study. 55.36 ± 10.85 was the average age of participants, 86% was married and 14% were all single people among those who were diagnosed with breast cancer, the percentage of women who reported a family history of the disease was 62%. women with a family history of breast cancer have a higher risk of developing the disease, as shown by several studies employing a wide variety of approaches to study. however, the severity of this risk varies not just by the individual’s age but also by the specifics of their family history, the type of related involved, the age at which the related acquired breast cancer, and the number of relative influenced.13 100 female breast cancer patients were studied. at the start of the trial, the average age of the participants was 55.36 ± 10.85 years (range: 45–65). when breast cancer is found in the lymph nodes, it means the disease has spread from the main tumour and is at least stage two. lymph nodes are an important part of the staging process and help determine which treatments are most likely to succeed.14 female medical surgery are rising due to indifference and failure to see a professional. due to the lack of knowledge about mastology services in programmes, a high percentage of patients are unnecessarily referred to mastology services, which creates opportunity problems for patients with breast cancer or benign breast disease who need a specialist.15 iraqi muslim women have socio-ethical, theological, and cultural misconceptions concerning breast cancer and healthy practises.16 table 5. shows the genetic basis of rs55886062 gene polymorphisms dpyd (rs55886062) genotype patients n = 100 n (%) (x2) hwe hwe genotype frequency aa 53 (53%) (9.3364) 0.001 c/a 40 (40%) cc 7 (7%) allele frequency a 73 (773%) (13.5) 0.001 0.001 c 27 (27%) table 6. mean ± sd of tumor markers with genotype frequencies of dpyd (rs3918290) characteristic dpyd (rs3918290) mean ± sd cc n = 58 mean ± sd ct n = 28 mean ± sd tt n = 14 p capecitabine concentration ng/ml 27.32 ± 6.49 36.87 ± 5.39 42.22 ± 5.19 0.028 s 5fu concentration ng/ml 259.33 ± 14.36 301.77 ± 21.77 341.53 ± 16.21 0.019 s table 7. mean ± sd of tumor markers with genotype frequencies of dpyd (rs55886062) characteristic dpyd*13 (rs55886062) mean ± sd aa n = 53 mean ± sd ac n = 40 mean ± sd cc n = 7 p capecitabine concentration ng/ml 26.15 ± 3.95 30.23 ± 4.34 38.31 ± 6.38 0.047 s 5fu concentration ng/ml 277.4 ± 14.21 283.77 ± 23.98 375.92 ± 18.59 0.038 s 191j contemp med sci | vol. 9, no. 3, may-june 2023: 187–192 r. f. abbas et al. original genetic polymorphisms of dpyd in patients with breast cancer on capecitabine therapy left and right breast cancer in iraqi women is studied. some countries offer novel cancer registry and hospital case series. according to a study, right-sided breast cancer is more common in women. this study found that right-sided breast cancer was more common than left-sided breast cancer in other countries.17 eighty-six percent of women with breast cancer were married, while 14% were single.18 despite conflicting findings, no systematic study has been done on the relationship between marital status and this malignant disease.19 this study showed how capacitabain chemotherapy was used to diagnose and treat people with breast symptoms or signs. suspected patients are not diagnosed. it also aims to standardise mammary cancer diagnosis and treatment. ninety-one percent of postmenopausal women had received capecitabine chemotherapy for 3.29 ± 1.95 years, and breast cancer cells grow and spread abnormally fast. chemotherapy targets fast-dividing cells. chemotherapy medicines can damage healthy cells, especially fast dividing ones.20 the dpyd gene snp rs3918290 is amplified, a positive results for the samples’ genotype were shown by the appearance of pcr bands of same sizes on the agarose gel. table 2 displays the detailed responses for each snp and genotype combination, patients were found to have particular alleles based on the results of polymerase chain reaction (pcr) amplifications, with fragment sizes of 400 bp. in this study of 100 breast cancer patients, the mutant type (cc) was shown to be the most common genotype (frequency = 58, 58%), whereas the heterozygote type (ct) was found to be the least common genotype (frequency = 28, 28%). rs3918290 tt genotype mutations have been shown to occur at a frequency and percentage of 14% and 14%, respectively. the study found these alleles out of hardy weinberg equilibrium (p < 0.05), in addition to mutations and “natural selection, nonrandom mating, genetic drift, and gene flow” are also capable of upsetting the hardy-weinberg equilibrium. for instance, mutations introduce novel alleles into a population, which shifts the balance of existing allele frequencies.21 snp rs55886062 in the dpyd gene was observed to be amplified, positive results for the samples’ genotype were shown by the appearance of pcr bands of same sizes on the agarose gel. it was displays the detailed responses for each snp and genotype combination. patient had specific alleles as shown by pcr amplifications of fragments of 410 bp in size; amplification of wild-type and variant-type alleles required use of two different tubes. the mutant type (cc) was the most common genotype among the 100 breast cancer patients recruited for this investigation, with a frequency and percentage of 7 and 7%, respectively, while the heterozygote type (ca) was the least common, with a frequency and percentage of 40 and 40%, respectively. the percentage of the aa genotype found in the rs3918290 wild type are 53%. there were clear significant differences in each of the following concentrations of ca15.3, capecitabine and 5fu in women with breast cancer, as the results recorded a significant higher concentration of them was recorded in each of ca15.3, capecitabine and 5fu in patients with the tt allele rather than in patients with the cc and ct alleles in patient with dpyd (rs3918290) characteristics. the result was agreement with (olivera et al., 2019)22 who was found the t allele of rs3918290 is assigned no function by cpic. patients with the cc genotype may have increased activity of dpyd as compared to patients with the ct or tt genotype. however, conflicting evidence has been reported. other genetic and clinical factors may also influence catalytic activity of dpyd. there was displays a statistically significant differences between the following capecitabine and 5fu concentrations in breast cancer patients: patients with the cc allele of dpyd*13 (rs55886062) had significantly greater concentrations of capecitabine and 5fu concentration. the result was agreement with (lunenburg et al., 2020)23 who was found the c allele of this variant is assigned a no function allele by cpic. patients with the aa genotype and cancer who are treated with fluorouracil, a fluoropyrimidine-based chemotherapy, may have decreased, but not absent, risk of drug toxicity as compared to patients with the ac or cc genotype. however, conflicting evidence has been reported. other genetic and clinical factors may also influence risk of drug toxicity. conclusion different frequencies of the (g > a) (rs3918290) and of (rs55886062, t > g) polymorphisms of the dpyd gene homozygous wild, homozygous mutant, and heterozygous genotype were discovered using allele specififc-pcr in iraqi breast cancer women who were treated with capacetabine. conflict of interest none.  references 1. parkins, k. m., dubois, v. p., hamilton, a. m., makela, a. v., ronald, j. a., & foster, p. j. (2018). multimodality cellular and molecular imaging of concomitant tumour enhancement in a syngeneic mouse model of breast cancer metastasis. scientific reports, 8(1), 8930. 2. alwan, n. a. (2016). breast cancer among iraqi women: preliminary findings from a regional comparative breast cancer research project. journal of global oncology, 2(5), 255. 3. chen, z., xu, l., shi, w., zeng, f., zhuo, r., hao, x., & fan, p. (2020). trends of female and male breast cancer incidence at the global, regional, and national levels, 1990–2017. breast cancer research and treatment, 180, 481–490. 4. rasool, a., bunterngchit, c., tiejian, l., islam, m. r., qu, q., & jiang, q. (2022). improved machine learning-based predictive models for breast cancer diagnosis. international journal of environmental research and public health, 19(6), 3211. 5. moo, t. a., sanford, r., dang, c., & morrow, m. (2018). overview of breast cancer therapy. pet clinics, 13(3), 339–354. 6. alqahtani, s., alzaidi, r., alsultan, a., asiri, a., asiri, y., & alsaleh, k. (2022). clinical pharmacokinetics of capecitabine and its metabolites in colorectal cancer patients. saudi pharmaceutical journal, 30(5), 527–531. 7. jurczyk, m., król, m., midro, a., kurnik-łucka, m., poniatowski, a., & gil, k. (2021). cardiotoxicity of fluoropyrimidines: epidemiology, mechanisms, diagnosis, and management. journal of clinical medicine, 10(19), 4426. 8. sharma, v., gupta, s. k., & verma, m. (2019). dihydropyrimidine dehydrogenase in the metabolism of the anticancer drugs. cancer chemotherapy and pharmacology, 84(6), 1157–1166. 192 j contemp med sci | vol. 9, no. 3, may-june 2023: 187–192 genetic polymorphisms of dpyd in patients with breast cancer on capecitabine therapy original r. f. abbas et al. 16. moey, s. f., sowtali, s. n., mohamad ismail, m. f., hashi, a. a., & che mohamed, n. (2022). cultural, religious and socio-ethical misconceptions among muslim women towards breast cancer screening: a systematic review. asian pacific journal of cancer prevention, 23(12), 3971–3982. 17. koto, m. z., becker, j. h. r., mokone-fatunla, d. h., mundawarara, s., & bondo, m. (2019). laterality of breast cancer at dr george mukhari academic hospital. south african journal of surgery, 57(3), 55–61. 18. kruk, j. (2012). self-reported psychological stress and the risk of breast cancer: a case-control study. stress, 15(2), 162–171. 19. melchior, m., goldberg, m., krieger, n., kawachi, i., menvielle, g., zins, m., & berkman, l. f. (2005). occupational class, occupational mobility and cancer incidence among middle-aged men and women: a prospective study of the french gazel cohort. cancer causes & control, 16, 515–524. 20. hassan, m. s. u., ansari, j., spooner, d., & hussain, s. a. (2010). chemotherapy for breast cancer. oncology reports, 24(5), 1121–1131. 21. hu, n., si, y., yue, j., sun, t., wang, x., jia, z., ... & yuan, p. (2021). anlotinib has good efficacy and low toxicity: a phase ii study of anlotinib in pre-treated her2 negative metastatic breast cancer. cancer biology & medicine, 18(3), 849. 22 . oliveira, m., saura, c., nuciforo, p., calvo, i., andersen, j., passos-coelho, j. l., ... & isakoff, s. j. (2019). fairlane, a double-blind placebo-controlled randomized phase ii trial of neoadjuvant ipatasertib plus paclitaxel for early triple-negative breast cancer. annals of oncology, 30(8), 1289–1297. 23. lunenburg ca, van der wouden ch, nijenhuis m, crommentuijn-van rhenen mh, de boer-veger nj, buunk am, houwink ej, mulder h, rongen ga, van schaik rh, van der weide j. dutch pharmacogenetics working group (dpwg) guideline for the gene–drug interaction of dpyd and fluoropyrimidines. european journal of human genetics. 2020 apr;28(4):508-17. 9. piórkowska, e., kaza, m., fitatiuk, j., szlaska, i., pawiński, t., & rudzki, p. j. (2014). rapid and simplified hplc-uv method with on-line wavelengths switching for determination of capecitabine in human plasma. die pharmazie-an international journal of pharmaceutical sciences, 69(7), 500–505. 10. zhu, l., shen, g. j., ding, s. q., & hua, x. i. n. (2012). determination of 5-fluorouracil in 5-fluorouracil injection and human serum by hplc. journal of food and drug analysis, 20(4), 15. 11. murthy, r. k., loi, s., okines, a., paplomata, e., hamilton, e., hurvitz, s. a., ... & winer, e. p. (2020). tucatinib, trastuzumab, and capecitabine for her2positive metastatic breast cancer. new england journal of medicine, 382(7), 597–609. 12. ayala-aguilera, c. c., valero, t., lorente-macías, á., baillache, d. j., croke, s., & unciti-broceta, a. (2021). small molecule kinase inhibitor drugs (1995–2021): medical indication, pharmacology, and synthesis. journal of medicinal chemistry, 65(2), 1047–1131. 13. niehoff, n. m., nichols, h. b., zhao, s., white, a. j., & sandler, d. p. (2019). adult physical activity and breast cancer risk in women with a family history of breast cancer. cancer epidemiology, biomarkers & prevention, 28(1), 51–58. 14. han, l., zhu, y., liu, z., yu, t., he, c., jiang, w., ... & luo, y. (2019). radiomic nomogram for prediction of axillary lymph node metastasis in breast cancer. european radiology, 29, 3820–3829. 15. calì cassi, l., vanni, g., petrella, g., orsaria, p., pistolese, c., lo russo, g., ... & buonomo, o. (2016). comparative study of oncoplastic versus nononcoplastic breast conserving surgery in a group of 211 breast cancer patients. european review for medical and pharmacological sciences, 20(14), 2950–2954. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1350 206 j contemp med sci | vol. 9, no. 3, may-june 2023: 206–210 original dosimetric effect and impact caused by carbon fiber table and its accessories in linear accelerator haydar h. alabedi1,2* , mustafa s. al musawi3, nabaa mohammed ali4 1department of surgery, college of medicine, baghdad university, baghdad, iraq. 2college of medicine, university of warith alanbiyaa, karbala, iraq. 3department of physiology and medical physics, college of medicine, al-mustansiriya university, baghdad, iraq. 4radiotherapy department, faculty of health science, university kebangsaan malaysia, bangi, malaysia. *correspondence to: haydar h. alabedi (e-mail: haydar.h@comed.uobaghdad.edu.iq) (submitted: 03 november 2022 – revised version received: 18 january 2023 – accepted: 12 february 2023 – published online: 26 june 2023) abstract objective: most contemporary treatment planning systems (tps) exclude the couch during treatment planning. the present investigation aimed to evaluate the impact of couches and accessories on radiation therapy planning by quantifying the degree of attenuation for twophoton beam energies, 6 mv and 10 mv, at two different field sizes, 5 cm x 5 cm and 10 cm x 10 cm. methods: an x-ray radiation beam at two energies (6 mv and 10 mv) generated by a linear accelerator (elekta synergy). the output dose was measured using a digital parallel plate ionization chamber of the iba cc13 type (iba dosimetry, germany), and the readings were recorded with a 2944 farmer type chamber. different commercially available couch tops and accessories evaluated in this study. results: the results show that the highest attenuation values were observed at 130o gantry angles for both energies and field sizes. the lowest attenuation values were recorded at 100o gantry angles with 6 mv energy for 5 cm x 5 cm and 10 cm x 10 cm field sizes. the breast board with d level demonstrated the highest attenuation of 6.27% and 5.51% for 6 mv and 10 mv energies, respectively. conclusion: the findings indicate that the degree of attenuation is not uniform across all angles and may exceed tolerable limits, indicating the need for careful consideration during treatment planning to ensure optimal treatment outcomes. keywords: carbon fiber, attenuation, couch inserts, photon beam, treatment planning issn 2413-0516 introduction carbon fibers are utilized in the manufacture of linear accelerator couches in accordance with established guidelines, owing to their radiotranslucent properties.1 these materials are preferred due to their favorable properties, including high specific strength, low specific density, physical durability, and excellent beam transmission. however, the dosimetric effects of external devices on patients are multifaceted and may include increased skin dose, decreased tumor dose, and altered dose distribution, as first described by de mooy.2 the carbon fiber couch is an essential component of radiotherapy treatment, as it immobilizes and stabilizes the patient during therapy administration, thereby reducing motion artifacts and enhancing the accuracy and precision of radiation delivery.3 posterior and posterior oblique beam orientations in radiotherapy treatments may give rise to two potential concerns. firstly, the presence of the couch insert may lead to attenuation of high-energy photon beams, resulting in a dose to the treatment volume that is lower than the intended dose. secondly, the skin-sparing effect of the build-up region may be diminished. the former is particularly important as it may directly impact the therapeutic outcome and necessitate corrective measures to ensure optimal dose delivery to the intended treatment volume. the latter concern may also have significant implications for patient comfort and satisfaction. therefore, carefully considering and evaluating these factors are essential when devising and administering radiotherapy treatments.4 numerous studies used a gantry angle of 180° and therefore offer little information on the amount of attenuation during oblique treatments for esophageal cancer and posterior lung lesions.5 in another study by vieira et al.,6 a 15% attenuation was found when head and neck imrt treatments using six mv posterior oblique photon beams were repeated and evaluated using electronic portal imaging device (epid) dosimetry. the amount of the observed beam attenuation may be ascribed to attenuation caused by the treatment couch and immobilization devices rather than by the carbon fiber insert alone. higgins and colleagues7 conducted a study to evaluate the effect of carbon fiber couch inserts on surface dosage with different beam sizes. their study revealed that incorporating a carbon fiber insert panel did not cause significant attenuation of the primary radiation; however, it significantly reduced the skin-sparing effect, particularly for smaller beam sizes. when carbon was added, the surface dose was approximately four times higher for a 10 cm × 10 cm beam and almost twice as high for a 40 cm × 40 cm beam. the ct simulation couch top is utilized in patient imaging but has not been a part of ct-based planning until recently due to technical difficulties. before 2008, it was not incorporated into the treatment planning system (tps) for dose calculations. the recently released tps software does not provide an option to replace the ct couch top with the treatment couch. nevertheless, the tomotherapy planning software (accuray, sunnyvale, ca) has incorporated this feature.8–10 several treatment planning systems (tpss) have provided options to account for the modified ct datasets, and some have allowed for the inclusion of the treatment couch top in the planning ct. it is crucial to measure the impact of intervening devices on photon beams and include them in tps dose calculations to determine whether the skin receives a safe dose. however, correcting the couch attenuation by increasing the monitoring units (mu) can increase the absolute skin dose. additionally, patient support and https://orcid.org/0000-0003-1960-7331 mailto:haydar.h@comed.uobaghdad.edu.iq 207j contemp med sci | vol. 9, no. 3, may-june 2023: 206–210 haydar h. alabedi et al. original dosimetric effect and impact caused by carbon fiber table and its accessories in linear accelerator immobilization devices also attenuate the photon beam, and therefore, their effects on dose delivery should also be considered.11–14 this study aimed to quantify the amount of radiation beam attenuation caused by the couch inserts and their associated accessories, as well as to develop a correction factor for the delivered dose to the patient. materials and method the present study was conducted at the baghdad radiotherapy and nuclear medicine center in baghdad medical city. this study aimed to evaluate the beam attenuation of various commercially available couch tops and accessories and to correct the administered radiation dose to the patient. to achieve this, measurements were taken for the x-ray radiation beam at two energies (6 mv and 10 mv) generated by a linear accelerator (elekta synergy). the output dose was measured using a digital parallel plate ionization chamber of the iba cc13 type (iba dosimetry, germany), and the readings were recorded with a 2944 farmer type chamber. measurements were taken at a reference dose for gantry angles ranging from 0o to 180o at a fixed source-to-surface distance (ssd) of 100 cm and for two field sizes (5 cm × 5 cm and 10 cm x 10 cm) by keeping the ssd constant for the couch top, and only at 0o for the other accessories. all measurements were taken at a dose rate of 100 mu. the isocenter of the treatment table was set with the center of the chamber and phantom and was placed on the surface of either the tabletop or the table combi board combination. the entrance window of the chamber was directed toward the radiation source figures 2–5. the attenuation of the main couch top was measured at a gantry angle of 0o (and for every 10o at the beam gantry angles between 0o and 180o). the attenuation of the insert couches was measured at a gantry angle of 0o with and without the insert at 5 cm depth and for a field size of 10 cm × 10 cm only. the measurement point was taken from the area with the large grid. the commercially available couch tops and accessories evaluated in this study included 1) system breast step conv (beast step, schwabmu n̈chen, germany) with four levels a, b, c, and d, 2) beam evo couch top ep (schwabmu n̈chen, germany), 3) qfix portraittm intracranial. head and neck fig. 1 the measurement of the attenuation couch insert setup. (usa), 4) system hs ibeam evo, head step (schwabmu n̈chen, germany), 5) mask, 6) vac. lock, 7) polystyrene foam, and 8) belly board (schwabmu n̈chen, germany). the settings used in this study are illustrated in figure 1. the dose delivered by the linear accelerator (linac) is measured relatively with respect to air density and correction factor using a chamber with a sensitivity of 11.75 nc/gy, a background radiation of 0.01 mgy/s, and a bias voltage of +299 v. however, to obtain the absolute dose, it is necessary to correct this measured dose using the equation provided by the manufacturer. this correction ensures that the delivered dose is accurate and can be used for safe and effective patient treatment. it is essential to follow the manufacturer’s instructions and procedures to obtain reliable and precise dose measurements: output dose = kq × ktp × relectrometer (1) the value of the quality constant (kq) of radiation depends on the type of radiation (i.e., electron or photon) and the energy level. for instance, the kq for 6 mv is 0.99, while for 10 mv, it is 0.98. another constant that affects the measurement of radiation dose is the constant of temperature and pressure (ktp). this value can be obtained using the following equation: ktp = (273 + t0)p (273 + t)p0 (2) the absolute dose measurement of each output dose is corrected by considering the constant of temperature and pressure (ktp) and the quality constant of radiation (kq). the ktp accounts for the differences in room temperature and pressure between the commissioning and study times. specifically, it is calculated using the equation provided, where to and po are the temperature and pressure of the linac room at the time of commissioning, respectively, and t and p are the corresponding values at the time of the study. this study’s values of to, t, po, and p were 20oc, 21.3oc, 1000 pa, and 1004 pa, respectively. the kq, on the other hand, is the quality constant of the radiation, which depends on the type of radiation and its energy. in this study, the kq values for 6 mv and 10 mv were 0.99 and 0.98, respectively. by taking into account these correction factors, each output dose is converted into an absolute dose measurement. results the measurements of couch maximum attenuation from various angles were conducted, and the results are presented in table 1. the highest attenuation values were observed at 130o gantry angles for both energies and field sizes. on the other hand, the lowest attenuation values were recorded at 100o gantry angles with 6 mv energy for 5 cm × 5 cm and 10 cm ×10 cm field sizes, which were 0.02% and 0.41%, respectively. additionally, the measurements showed that the attenuation was 0.39% at 180o gantry angle and 0.11% at 80o gantry angles for a 10 cm × 10 cm field size. the other couch inserts were adjusted to the field size of 10 cm × 10 cm for 6 mv and 10 mv energies with a slab thickness of 5 cm. the attenuation measurements were performed at a reference angle of 180o, and the reference distance of 208 j contemp med sci | vol. 9, no. 3, may-june 2023: 206–210 dosimetric effect and impact caused by carbon fiber table and its accessories in linear accelerator original haydar h. alabedi et al. ssd = 95, sad = 100, and mu = 100. the attenuation values for other couch inserts and their accessories are summarized in table 1. among the inserts, the breast board with d level demonstrated the highest attenuation of 6.27% and 5.51% for 6 mv and 10 mv energies, respectively. on the other hand, the foam insert showed a minimum attenuation of 0.75% and 0.80% for 6 mv and 10 mv energies, respectively. discussion our study findings indicate that carbon fiber material situated in the path of posterior oblique beams can considerably attenuate the radiation beam. specifically, as the angle of incidence becomes increasingly oblique, the distance that the radiation beam must traverse through the carbon fiber insert increases, thereby exacerbating the attenuation effect. this finding underscores the importance of carefully accounting for carbon fiber couches and other supporting materials in treatment planning for external-beam radiotherapy, particularly when using posterior oblique beams. failure to accurately account for attenuation effects could result in inadequate dosing of the target tissue, potentially compromising the treatment’s efficacy and the patient’s health outcomes. in accordance with the principles of radiation physics, the attenuation of carbon fiber can be explained as follows: as the radiation beam passes through the thickness (t) of the carbon fiber insert, the distance that it must traverse increases proportionally with the angle, following a factor of 1/cosθ, assuming that all other factors remain constant. this attenuation effect can be further understood in the context of the intensity attenuation law of radiation, which describes the decrease in intensity of a radiation beam as it passes through an absorbing medium such as carbon fiber. the precise magnitude of this attenuation effect can vary depending on a range of factors, including the energy of the radiation beam, the thickness of the carbon fiber insert, and the angle of incidence. our study aimed to investigate the impact of these factors on the attenuation of radiation in carbon fiber couches and patient supports, intending to inform more accurate and effective treatment planning strategies for external-beam radiotherapy:3,15 i = i₀e–λ(t/cosθ) (3) in the context of our investigation, we utilized the parameters i, i0, and λ to represent the intensity of the radiation dose, the original intensity radiation dose without the couch, and the attenuation coefficient, respectively. these values were normalized at the incident angle of 0°, making λt a constant. theoretically, the correction factor of attenuation (a) was calculated for each angle using the following equation: (4) the methodological approach described above appears to oversimplify the connection between the angle of incidence and the absorbed dosage observed empirically. our findings indicate that the attenuation of the radiation beam is higher than expected when the angle of incidence increases. this phenomenon was initially attributed to the non-homogeneous composition of the panel, which features a sandwich-like structure consisting of a polystyrene core surrounded by a layer of carbon fiber. fig. 2 the attenuation percentage of the couch top using 6 mv x-ray photon beams at different angles for 5 cm x 5 cm. fig. 3 the attenuation percentage of the couch top using 6 mv x-ray photon beams at different angles for 10 cm x 10 cm. fig. 4 the attenuation percentage of the couch top using 10 mv x-ray photon beams at different angles for 5 cm x 5 cm. fig. 5 the attenuation percentage of the couch top using 10 mv x-ray photon beams at different angles for 10 cm x 10 cm. 209j contemp med sci | vol. 9, no. 3, may-june 2023: 206–210 haydar h. alabedi et al. original dosimetric effect and impact caused by carbon fiber table and its accessories in linear accelerator our study highlights the need to incorporate the effect of attenuation observed in this investigation, which resulted in a 6.27% difference in the absorbed dose at the breast board level with 6mv energy, into the mu calculations. by doing so, the dose attenuated by the carbon fiber can be effectively eliminated, particularly when dealing with posterior oblique beams during treatment planning. calibration may also be applied to the mu calculations to refine the accuracy of the predicted treatment outcomes. the importance of patient support in external-beam radiotherapy cannot be understated. therefore, incorporating new materials into this field necessitates thoroughly investigating their effects on significant beam characteristics. this is especially relevant when such materials are positioned between the radiation source and the patient, as seen in the evaluation of carbon fiber-based couches and patient supports in this research. it is crucial to consider the potential impact of these novel materials on radiation dose distribution and patient outcomes and evaluate their safety and efficacy in clinical settings. therefore, it is important to conduct comprehensive studies on the effects of these materials on the radiation beam and patient treatment. muhtarom et al.16 conducted a study to investigate the effects of using three different carbon fiber plates on the properties of cobalt 60, 6and 23-mv beams at various field sizes. the authors aimed to demonstrate how these plates impact the surface dose and the magnitude of this effect. similarly, popple et al.17 conducted a study where they found up to a 5.8% underdose at the isocenter for rapidarc treatment plans when the couch was not considered in the planning process. these studies highlight the importance of considering the effects of couch inserts and the treatment couch in dose calculations and planning to ensure accurate and safe treatment delivery. our research suggests that the displacement of the attenuating and scattering carbon fiber mass away from the phantom surface as the angle of incidence rises from 0° to 180° could potentially explain the observed differences. as the air gap increases, the contribution of scatter from the carbon fiber layer becomes less discernible near the isocenter, resulting in lower electrometer readings and, therefore, a perceived more significant attenuation. further research is needed to develop a more precise theoretical model that accurately predicts the radiation beam’s attenuation at oblique gantry angles. conclusion in conclusion, our study suggests that the carbon fiber material employed in this investigation is not suitable for use as a supporting material during radiation therapy delivery, despite being well-suited for imaging purposes. as such, it is recommended that the attenuation differences resulting from using carbon fiber couches and supports be accounted for in the mu calculation during treatment planning. alternatively, incorporating the supporting couches in the planning process may be a viable option to ensure optimal treatment outcomes. further research may be required to explore the suitability of alternative materials for use as supportive couches, and patient supports during radiation therapy.  table 1. the attenuation of couch inserts and accessories for two types of energies, 6 mv and 10 mv, at 10 cm x 10 cm field size material 6 mv 10 mv back a 2.66% 2.40% back b 3.12% 2.84% head step 3.51% 3.15% c pad 1.23% 1.19% belly step 3.95% 3.51% vac. lock 2.54% 2.27% vac. lock + foam 2.82% 2.53% foam 0.76% 0.80% breast board 0 level 5.57% 4.87% breast board d level 6.27% 5.51% breast board c level 6.04% 5.35% breast board b level 5.95% 5.31% breast board a level 5.87% 5.19% new board 2.00% 1.95% head mask 1.09% 1.01% belly board 2.11% 1.88% references 1. njeh cf, parker j, spurgin j, rhoe e. a validation of carbon fiber imaging couch top modeling in two radiation therapy treatment planning systems: philips pinnacle3 and brainlab iplan rt dose. radiation oncology. 2012;7(1):1–11. 2. lv r, yang g, huang y, wang y. dosimetric effects of supine immobilization devices on the skin in intensity-modulated radiation therapy for breast cancer: a retrospective study. bmc cancer. 2021;21(1). 3. mccormack s, diffey j, morgan a. the effect of gantry angle on megavoltage photon beam attenuation by a carbon fiber couch insert. med phys. 2005;32(2):483–7. 4. meydanci tp, kemikler g. effect of a carbon fiber tabletop on the surface dose and attenuation for high-energy photon beams. radiation medicine medical imaging and radiation oncology. 2008;26(9). 5. gergelis kr, jethwa kr, tryggestad ej, ashman jb, haddock mg, hallemeier cl. proton beam radiotherapy for esophagus cancer: state of the art. vol. 12, journal of thoracic disease. 2020. 6. sc v, rs k, ml d, bj h. two-dimensional measurement of photon beam attenuation by the treatment couch and immobilization devices using an electronic portal imaging device. med phys. 2003;30(11):2981–7. 7. higgins j, bezjak a, hope a, panzarella t, li w, cho jbc, et al. effect of image-guidance frequency on geometric accuracy and setup margins in radiotherapy for locally advanced lung cancer. int j radiat oncol biol phys [internet]. 2011 aug 1 [cited 2021 may 18];80(5):1330–7. available from: http://www.redjournal.org/article/s0360301610005109/fulltext. 8. e v, g n, a c, a f, l c. the impact of treatment couch modelling on rapidarc. phys med biol. 2009;54(9). 9. kb p, rm h, d f, d l, ra w, sf k. the clinical impact of the couch top and rails on imrt and arc therapy. phys med biol. 2011 nov 4;56(23):7435–47. 10. t t, b g, c d, ia p. a monte carlo model of the varian igrt couch top for rapidarc qa. phys med biol. 2011 dec 21;56(24). 11. spezi e, angelini al, romani f, guido a, bunkheila f, ntreta m, et al. evaluating the influence of the siemens igrt carbon fibre tabletop in head and neck imrt. radiotherapy and oncology. 2008 oct 1;89(1):114–22. 210 j contemp med sci | vol. 9, no. 3, may-june 2023: 206–210 dosimetric effect and impact caused by carbon fiber table and its accessories in linear accelerator original haydar h. alabedi et al. 15. khan fm, gibbons jp, sperduto pw. khan’s treatment planning in radiation oncology. 4th editio. lippincott williams & wilkins (wolters kluwer). philadelphia, pa: lippincott williams & wilkins (wolters kluwer); 2018. 16. muhtarom, hidayanto e, sutanto h. analysis of the effect of carbon fiber utilization on cobalt60 teletherapy to the depth dose and the surface dose. international journal of innovative research in advanced engineering. 2017;4(04):2015–8. 17. ra p, jb f, ia b, ja b. rapidarc radiation therapy: first year experience at the university of alabama at birmingham. int j radiat oncol biol phys. 2010;77(3):932–41. 12. z h, j d, l l, y c, g f. evaluating and modeling of photon beam attenuation by a standard treatment couch. j appl clin med phys. 2011 jul 12;12(4):3561–3561. 13. lh g, m n, bj n. dose perturbations by two carbon fiber treatment couches and the ability of a commercial treatment planning system to predict these effects. med phys. 2010;37(1):322–8. 14. ib m, p c, y y, v r, eg m. modeling of carbon fiber couch attenuation properties with a commercial treatment planning system. med phys. 2008;35(11):4982–8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1355 284 j contemp med sci | vol. 3, no. 11, summer 2017: 284–285 research 1department of rheumatology and medical rehabilitation, imam-alsadeq teaching hospital, babylon-hilla, iraq. correspondence to tahir hussein alsaadawi (email: tahiralsaadawi@yahoo.com ). (submitted: 12 june 2017 – revised version received: 18 june 2017 – accepted: 09 july 2017 – published online: 26 september 2017 ) diffuse idiopathic skeletal hyperostosis (dish) is a non-inflammatory ossification involving at least four contagious vertebral bodies. it is mostly asymptomatic but the osteophytes can be rarely associated with severe complications. we report 65-year-old man who was admitted to the emergency department with severe shortness of breath and stridor not responding to medical treatment, which ended with tracheostomy lifesaving. patient condition began 1 year ago as cervical spine pain with radiculopathy to the upper limbs, which increased to recurrent attacks of shortness of breath that responds to bronchodilators. during last months, this attack begins poorly and responds to medications and o 2 . several radiological examination including x-ray, ct scan, and mri reveal bony mass in front of the cervical spine with hypertrophy of soft tissues bulging and compressed larynx. invasive techniques such as laryngoscope done by otolaryngologist to take biopsy from soft tissue, which reveals normal examination. in one-day, severe attack was not responding to o 2 and medications ended with tracheostomy to save life. it is advisable to take in consideration that patients with dish mostly in cervical spines may be at risk of fatal complications including airway obstruction. keywords dish, shortness of breath, tracheostomy introduction diffuse idiopathic skeletal hyperostosis (dish) is a tendency for ossification of ligaments, tendons and joint capsule insertions, most often affecting the spine.1 calcification of the longitudinal ligaments (particularly anterior) can often produce the radiological appearance of ‘wax dripping from a candle’, distinct from the vertebral bodies. the prevalence may be as high as 28%. elderly men are most commonly affected.2 it is uncommon in patients younger than 50 years and rare in patients younger than 40 years. although forestier’s disease is a symptomatic in general, previous research was reported dysphagia, dyspnea and dysphonia all together only in rare cases.3 case reports patient’s condition began 1 year ago as cervical spine pain with radiculopathy to upper limbs which developed to recurrent attacks of shortness of breath that responds to bronchodilators. during last months, these attacks begin poorly and respond to medications and o2. several radiological examinations include x-ray (fig. 1), ct scan, and mri (fig. 2) reveal bony mass in front of the cervical spine with hypertrophy of soft tissues bulging and compressed larynx. invasive techniques as laryngoscope done by otolaryngologist to take biopsy from soft tissue reveals normal examination just inflammatory process and no malignant cells were seen. in one day, severe attack not responding to o2 and medications ended with tracheostomy to save life (fig. 3). discussion the suggested pathogenesis of dish is the ossification and osteophytes formation which is the result of abnormal osteoblast cell growth and activity in bony ligamentous region.4 issn 2413-0516 fatal complication of diffuse idiopathic skeletal hyperostosis (dish): report of a case tahir hussein alsaadawi1 fig. 3 mri of neck after tracheostomy. fig. 2 ct scan of head and neck showing compressed larynx. fig. 1x-ray presentation of patient with dish. tahir hussein alsaadawi 285j contemp med sci | vol. 3, no. 11, summer 2017: 284–285 research fatal complication of diffuse idiopathic skeletal hyperostosis airway symptoms prevalent in patients with dish was 11.2%.5 in our patient, ossifications and osteophytes were present at c3–c7 pronounced and bulged causing laryngeal manifestations, which might ended with severe airway obstruction. the possible mechanism is not only compression of the larynx but also paresis of terminal laryngeal nerve fibers, traumatic compromisation, and direct involvement of crico-artenoid joint.6 retro-cricoid inflammation can induce laryngeal edema,7 dysphonia, vocal cord immobility8,9 or stridor.10 surgical treatment of dish indicates that surgery is performed mainly via anterior approach (smith-robinson),11,12 however, we must be aware of the potentially severe complication of surgical treatment such as hematoma, horner’s syndrome, recurrent nerve palsy, superior laryngeal nerve palsy, and esophageal injury.13 therefore, surgical treatment should be selected with care. conclusion after appropriate history, clinical examination, diagnostic and radiological procedures, we diagnosed a dish is a cause of symptoms in our patients. in clinical practice, it is advisable to take in consideration fatal complications of dish like airways obstruction. conflict of interest none. n references 1. fábio a nascimento, luana antunes maranha gatto, roberto oliver lages, heraldo mello neto, zeferino demartini, junior, and gelson luis koppe. diffuse idiopathic skeletal hyperostosis: a review. surg neurol int. 2014;5:s122–s125. 2. holton kf, denard pj, yoo ju, kado dm, barrett-connor e, marshall lm. osteoporotic fractures in men (mros) study group. diffuse idiopathic skeletal hyperostosis and its relation to back pain among older men: the mros study. semin arthritis rheum. 2011;41:131–138. 3. ahn yj, hahn sh, yang bk, et al. diffuse idiopathic skeletal hyperostosis associated with dysphonia and dysphagia: a case report. j korean soc spine surg. 2006;13:327–331. 4. el miedany ym, wassif g, el baddini m. diffuse idiopathic skeletal hyperostosis (dish): is it of vascular aetiology?. clin exp rheumatol. 2000;18:193–200. 5. jasmina s, sandra z, suncica s, et al. laryngeal manifestations of forestier’s disease. j med sci. 2016;4:287–289. 6. giger r, dulguerov p, payer m. anterior cervical osteophytes causing dysphagia and dyspnea: an uncommon entity revisited. dysphagia. 2006;21:259–263. 7. marks e schober, swoboda h. diffuse idiopathic skeletal hyperostosis causing obstructing laryngeal edema. eur arch otorhinolaryngol. 1998;255:256–258. 8. aydin k, ulug t, simsek t. bilateral vocal cord paralysis caused by cervical spinal osteophytes. br j radiol. 2002;75:990–993. 9. verstraete wl, de cauwer hg, verhulst d, jacobs f. vocal cord immobilisation in diffuse idiopathic skeletal hyperostosis (dish). acta otorhinolaryngol belg. 1998;52:79–84. 10. pfleger a, eber e. assessment and causes of stridor. paediatr respir rev. 2016;18:64–72. 11. winslow cp, winslow tj, wax mk. dysphonia and dysphagia following the anterior approach to the cervical spine. arch otolaryngol. 2001;127:51–55. 12. saffouri mh, ward ph. surgical correction of dysphagia due to cervical osteophytes. ann otol rhinol laryngol. 1974;83:65–70. 13. heeneman h. vocal cord paralysis following approaches to the anterior cervical spine. laryngoscope. 1973;83:17–21. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201708 353j contemp med sci | vol. 7, no. 6, november-december 2021: 353–357 original pyrimidine derivatives as promising candidates for potent antiangiogenic: a silico study nadhir n. a. jafar1*, ahmed a. hussein2 1pharmacy college, al-zahraa university for women, karbala 65001, iraq. 2pharmacy department, al-mustaqbal university college, 51001 hillah, babil, iraq. *correspondence to: nadhir n. a. jafar (e-mail: nather.najim@alzahraa.edu.iq) abstract objectives: this study planned to explore the effect of many synthetic compounds derived from (4-chloro-6-methoxy-n,n-dimethyl pyrimidin-2-amine) as antiangiogenic. methods: docking study has been done by using molecular operating environment (2019) to examine the energy binding affinity of tested compounds with vegfr-2 kinase and using discovery studio visualizer v20.1.0.19295 free version to visualize the surface binding cavity. results: theoretical calculation of these compounds showed significant results in comparing to the reference drug compounds, compound (1) which is 4-(4-(1,2,3-selenadiazol-4-yl)phenoxy)-6-methoxy-n,n-dimethylpyrimidin-2-amine gives the lowest binding energy equal to (–8.116) kcal/mol and nearest to the reference drug compound, and also it has excellent rmsd equal to (0.9263). the other compounds 4-(4-(1,2,3-thiadiazol-4-yl)phenoxy)-6-methoxy-n,n-dimethylpyrimidin-2-amine, 4-methoxy-n,n-dimethyl-6-(phenylthio) pyrimidin-2amine, 4-(benzo[d]thiazol-2-ylthio)-6-methoxy-n,n-dimethylpyrimidin-2-amine have –7.739, –7.211 and –7.841 kcal/mol binding energy and 2.668, 1.745 and 1.377 rmsd respectively. conclusion: compound (1) can be recommended as a powerful antiangiogenic due to its theoretical results for binding energy. keywords: 1,2,3-seleniadiazole, anti-angiogenesis, anticancer, binding energy, pyrimidine issn 2413-0516 introduction cancer cells have a very high metabolism process in comparison with normal cells and this led to high oxygen demand and more supplying of nutrients, this issue causes hypoxia which is considered the main regulation mechanism for angiogenesis.1,2 scientists have been researched and concentrated on how tumors get the oxygen and nutrients needed. in 1989, the vascular endothelial growth factor (vegf) protein was discovered by a team of scientists due to the belief that it induces angiogenesis3 by binding to their receptors in the endothelial cells of blood vessels and stimulate proliferation.4-6 the induction from the tumor itself causing angiogenesis, vasculogenesis, increasing vessel permeability, and migration to be around the tumor to supply it with oxygen and nutrients.7,8 the vegf receptor inhibitors play an important role in the treatment of tumors by causing tumor starvation by preventing the proliferation of endothelial blood vessels that led to decreasing of oxygen and nutrients they need9 and will give tumor growth suppression10 such as sorafenib (nexavar)® which has been approved as a potent antiangiogenic drug.11 the first objective of this study is testing in-silico pyrimidine derivative as a new pharmacophore antiangiogenic ligand, it is safer because it is a part unit in nucleic bases of rna and dna and this feature gave its derivatives widespread pharmacological activities.12,13 pyrimidine derivatives have been used as potent anticancer in cases of breast, stomach, colon, pancreatic, and other types of cancer,14,15 antimicrobial effects,14,16,17 potent antitubercular agents,18 antimalarial effects especially for resistant species,19 potent anti-inflammatory,20 efficient antiviral activity, antihypertensive activity21 and especially for varicella-zoster virus.22,23 the second objective of our study is to figure out a new activity of previously synthesized compounds with pyrimidine ring such as [1,2,3-selenadiazole, 1,2,3-thiadiazole, benzo[d]thiazol-2-ylthio] and thiophenyl compounds.24 these compounds have been studied are organosulfur and organoselenium compounds which have significant activity as anti-fungal activity,24 antibacterial effect,25-30 due to the patients with cancer that suffering from microbial infections,31 so the most significant features will be emphasized in this article for pyrimidine with the substitutions that mentioned using a potent anticancer and antimicrobial activities to produce a single drug with double activities to add a new cost-effective feature. consequently, merged the pyrimidine with fourth substitutions to discover in silico a new activity and to get more potent binding energy with vascular endothelial growth factor receptor protein. materials and methods docking requirements docking study has been done by using molecular operating environment (2019)32 to examine the energy binding affinity of tested compounds below with vegfr-2 kinase and using discovery studio visualizer v20.1.0.19295 free version33 to visualize the surface binding cavity. preparation of ligands the compounds that have been used were already synthesized, then, they have been processed by moe in form of 2d, prepared with protonate 3d, partial charge automatically calculated, energy minimization to get the most stable conformation and saved in form 3d form as (moe) file, then all of them have been imported in database and saved as mdb file to be used in docking process.34,35 (submitted: 09 september 2021 – revised version received: 22 september 2021 – accepted: 01 october 2021 – published online: 26 december 2021) 354 j contemp med sci | vol. 7, no. 6, november-december 2021: 353–357 pyrimidine derivatives as promising candidates for potent antiangiogenic: a silico study original n.n.a. jafar and a.a. hussein fig. 2 diagram interaction of compound (1) with the crystal structure of the vegfr2 kinase. protein arrangement and preparation the crystal structure of the vegfr2 kinase which is complex with pf00337210 (n,2-dimethyl-6-(7-(2-morpholinoethoxy) quinoline-4-yloxy)benzofuran-3-carboxamide) that has been downloaded from the protein data bank website (pdb id code is: 2xir) with resolution: 1.50 å.36 preparation of crystal structure of a protein by adding hydrogen atoms during protonation 3d has been done at first, then checking all atom’s connection errors have been done by automatic correction, potential fixation of all protein atoms with amber12: eht. finally, selection of all active sites and the creation of them as dummies by using a site finder (figure 1).34,35 results and discussion the results are given in table 1, which explains the core compound which gives good interaction with low binding energy equal to (–5.911) with rmsd equal to (0.691), whereas the substituted synthesized compounds 1, 2, 3, and 4 gave the lowest binding energy equal to (–8.116, –7.739, –7.211 and –7.84), respectively, and nearest to the reference drug compound, and also it has very good rmsd equal to (0.9263), that means substations play an important role in the protein binding process in comparing with the starting pyrimidine derivative compound (5). docking study the prepared database of compounds mentioned previously has been tested on the protein vega-2 receptor. generally, the docking was done by loading the protein with dummies atoms that represent active sites in the protein, modifying the default properties in moe software, set docking site as dummy atoms, ligand as mdb file, rigid receptor method, triangle matcher as method placement, london dg and gbvi/wsa dg as score method and have been selected the best 10 confirmations out of 200 different conformations for each ligand. then we chose the best pose that has the lowest energy with good rmsd values from the obtained docking results.35,37,38 the compound (1) has been interacted with protein amino acids [cys 919 (a) h-acceptor interaction of (n) with (n25) in ligand; asp 1046 (a) h-acceptor interaction of (n) with (n27) in ligand; leu 840 (a) pi-h interaction of (cd1) with (5-ring) in ligand; val 899 (a) pi-h interaction of (cg1) with (6-ring) in ligand] with distances 3.14 å, 3.21 å, 3.82 å, 4.44 å and e (kcal/mol) –0.7, –2.4, –0.6, –0.6 respectively as shown in figures 2 and 3. the compound (2) has interacted with amino acids [asp 1046 (a) h-acceptor interaction of (n) with (o) in ligand; leu 840(a) pi-h interaction of (cd2) with (5-ring) in ligand; lys 868(a) pi-h interaction of (ce) with (6-ring) in ligand] with distances 2.95 å, 3.95 å, 4.27 å and e (kcal/mol) –1.8, –1.2, –1.2 respectively. the compound (3) has interacted with amino acids [asp 1046 (a) h-acceptor interaction of (n) with (o) in ligand; val 848(a) pi-h interaction of (cg1) with (6-ring) fig. 1 crystal structure of the vegfr2 kinase pdb code (2xir). table 1. binding energies of the ligand with protein no. compound s rmsd_refine e_conf e_ place e_ score1 e_ refine e_ score2 1 reference –9.822 1.516 11.596 –67.448 –16.645 –45.713 –9.822 2 1 –8.116 0.926 –100.626 –55.464 –9.436 –36.111 –8.116 3 2 –7.739 2.668 –89.229 –56.335 –9.539 –33.926 –7.739 4 3 –7.211 1.745 –118.107 –61.455 –9.759 –31.147 –7.211 5 4 –7.841 1.377 –107.456 –55.826 –9.683 –36.329 –7.841 6 5 –5.911 0.691 –128.838 –56.396 –8.053 –29.731 –5.911 s, the final score; rmsd, root-mean-square deviation between the pose before refinement and the pose after refinement; e_conf, the energy of the conformer; e_place, score from the placement stage; e_score1, score from the rescoring stage(s); e_refine, score from the refinement stage and no. of conf-number of conformations generated by ligand. 355j contemp med sci | vol. 7, no. 6, november-december 2021: 353–357 n.n.a. jafar and a.a. hussein original pyrimidine derivatives as promising candidates for potent antiangiogenic: a silico study in ligand; lys 868(a) pi-h interaction of (ce) with (6-ring) in ligand] with distances 2.94 å, 4.42 å, 4.07 å and e (kcal/mol) –1.9, –0.7, –1.1 respectively. the compound (4) has interacted with amino acids [asp 1046 (a) h-acceptor interaction of (n) with (n9) in ligand; val 848(a) pi-h interaction of (cg1) with (5-ring) in ligand] with distances 3.26 å, 4.23 å and e (kcal/mol) –1.6, –0.9 respectively. structure-activity relationship (sar) the primary study of sar has focused on the impact of substituents in replacing chlorine atoms in the pyrimidine core, which is related to h-bond accepter via n or o-atoms or arene amino acid interaction. the compound (5) represents a core compound that has been used to synthesize other four compounds which have been interacted with amino acids [asp 1046 (a) h-acceptor interaction of (n) with (n2) in ligand; val 899(a) pi-h interaction of (cg1) with (6-ring) in ligand] with distances 3.5 å, 4.43 å and e (kcal/mol) –1.2, –0.6 respectively as shown in figure 4. when replacing chloride in the core compound with four different substituents separately as shown in scheme 1. compound 1 has been exhibited –8.116 kcal/mol binding energy because increasing the hydrogen bonds accepter –4.100 kcal/mole related with n interacted with asp 1046 (a) compared with the core compound equal to –1.200 kcal/mol. furthermore, the presence of –1.600 kcal/mole hydrogen acceptor of n with cys 919 (a) and changing the interaction of third connect with lys 868(a) instead of val 899 (a) that means the compound has been orientated with the best alignment with protein as in figures 2, 3. compound 2 has appeared –7.739 kcal/mole binding energy from h-acceptor o with asp 1046 (a) and two pi-h interactions as in figure 5, compound 3 and 4 have –7.211 and –7.841 kcal/mol, respectively, as could be observed in figures 6 and 7. fig. 3 diagram isolated interaction of compound (1) with the crystal structure of the vegfr2 kinase. fig. 5 diagram interaction of compound (2) with the crystal structure of the vegfr-2. fig. 6 diagram interaction of compound (3) with the crystal structure of the vegfr-2. scheme 1. structures of pyrimidine and four synthesized compounds. fig. 4 diagram interaction of compound (5) with the crystal structure of the vegfr-2. 356 j contemp med sci | vol. 7, no. 6, november-december 2021: 353–357 pyrimidine derivatives as promising candidates for potent antiangiogenic: a silico study original n.n.a. jafar and a.a. hussein –7.4 and –8.4, respectively, this issue encourages us to proceed for furthermore studies. conclusion this research contributes to finding a new core from pyrimidine derivatives as promising candidates for powerful antiangiogenic drug in-silico study. further investigations are recommended for new researchers as driving the possibility for more broad pharmacological examinations. the docking studies and flexible alignment would propose a good affinity of most of the synthesized compounds towards interacting with vegfr-2 receptor in better binding energy from the data obtained, all compounds exhibited –8.116, –7.739, –7.211 and –7.841 kcal/mol binding energy, respectively, with compounds 1, 2, 3 and 4 with small rmsd. the results demonstrated that the most dynamic mixes could be helpful as a format for future patterns, advancement, modification, furthermore, assessment to make more extraordinary and specific vegfr‐2 inhibitors with higher anticancer analogs.  references 1. garcia j, hurwitz hi, sandler ab, et al. bevacizumab (avastin®) in cancer treatment: a review of 15 years of clinical experience and future outlook. cancer treat rev 2020; 86: 102017. 2. tanimoto k, yoshiga k, eguchi h, et al. hypoxia-inducible factor-1α polymorphisms associated with enhanced transactivation capacity, implying clinical significance. carcinogenesis 2003; 24: 1779–1783. 3. tischer e, gospodarowicz d, mitchell r, et al. vascular endothelial growth factor: a new member of the platelet-derived growth factor gene family. biochem biophys res commun 1989; 165: 1198–1206. 4. ferrara n, henzel wj. pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. biochem biophys res commun 1989; 161: 851–858. 5. connolly dt, heuvelman dm, nelson r, et al. tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. j clin invest 1989; 84: 1470–1478. 6. leung dw, cachianes g, kuang w-j, et al. vascular endothelial growth factor is a secreted angiogenic mitogen. science (80-) 1989; 246: 1306–1309. 7. hicklin dj, ellis lm. role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. j clin oncol 2005; 23: 1011–1027. 8. lugano r, ramachandran m, dimberg a. tumor angiogenesis: causes, consequences, challenges and opportunities. cell mol life sci 2020; 77: 1745–1770. 9. jain rk. antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. cancer cell 2014; 26: 605–622. 10. ferrara n, hillan kj, gerber h-p, et al. discovery and development of bevacizumab, an anti-vegf antibody for treating cancer. nat rev drug discov 2004; 3: 391–400. 11. wu p, nielsen te, clausen mh. fda-approved small-molecule kinase inhibitors. trends pharmacol sci 2015; 36: 422–439. 12. fargualy am, habib ns, ismail ka, et al. synthesis, biological evaluation and molecular docking studies of some pyrimidine derivatives. eur j med chem 2013; 66: 276–295. 13. sharma v, chitranshi n, agarwal ak. significance and biological importance of pyrimidine in the microbial world. int j med chem 2014; 2014: 202784. 14. habib ns, soliman r, ismail k, et al. pyrimidines. part ii: synthesis of novel pyrimidines, 1,2,4-triazolo[4,3-a]pyrimidin-7-ones and pyrimidino[2,1-c] [1,2,4]triazin-8-ones for their antimicrobial and anticancer activities. boll chim farm 2003; 142: 396–405. 15. kumar s, deep a, narasimhan b. a review on synthesis, anticancer and antiviral potentials of pyrimidine derivatives. curr bioact compd 2019; 15: 289–303. 16. ram vj, berghe dav, vlietinck aj. 5‐cyano‐6‐aryluracil and 2‐thiouracil derivatives as potential chemotherapeutic agents. iv. journal of heterocyclic chemistry 1984; 21: 1307–1312. 17. mallikarjunaswamy c. studies on synthesis of pyrimidine derivatives and their antimicrobial activity. arab j chem 2017; 10: s484–s490. 18. liu p, yang y, tang y, et al. design and synthesis of novel pyrimidine derivatives as potent antitubercular agents. eur j med chem 2019; 163: 169–182. 19. singh k, kaur t. pyrimidine-based antimalarials: design strategies and antiplasmodial effects. medchemcomm 2016; 7: 749–768. 20. naik ns, shastri la, chougala bm, et al. synthesis of novel aryl and coumarin substituted pyrazolo[1,5-a]pyrimidine derivatives as potent antiinflammatory and anticancer agents. chem data collect 2020; 30: 100550. 21. rani j, kumar s, saini m, et al. biological potential of pyrimidine derivatives in a new era. res chem intermed 2016; 42: 6777–6804. 22. mcguigan c, barucki h, blewett s, et al. highly potent and selective inhibition of varicella-zoster virus by bicyclic furopyrimidine nucleosides bearing an aryl side chain. j med chem 2000; 43: 4993–4997. 23. luo m, groaz e, snoeck r, et al. amidate prodrugs of o-2-alkylated pyrimidine acyclic nucleosides display potent anti-herpesvirus activity. acs med chem lett 2020; 11: 1410–1415. 24. jafar nna, mahdi ima, hadwan mh, et al. the antifungal effect of some 4-chloro-6-methoxy-n, n-dimethylpyrimidin-2-amine derivatives containing a heterocyclic compound on the important types of fungi. j young pharm 2017; 9: 463–467. 25. lalezari i, shafiee a, yazdany s. selenium heterocycles x: synthesis and antibacterial activity of pyridyl‐1,2,3‐thiadiazoles and pyridyl‐1,2,3‐ selenadiazoles. j pharm sci 1974; 63: 628–629. 26. al-smadi m, al-momani f. synthesis, characterization and antimicrobial activity of new 1,2,3-selenadiazoles. molecules 2008; 13: 2740–2749. 27. karnik a.v., kulkarni am, malviya nj, et al. synthesis and in vitro antibacterial evaluation of tetracyclic-ortho-fused 4h-naphtho[1',2'-5,6] pyrano[3,4-d](1,2,3)selenadiazole and its derivatives. eur j med chem 2008; 43: 2615–2617. 28. gawad j, bonde c. design, synthesis and biological evaluation of some 2-(6-nitrobenzo[d]thiazol-2-ylthio)-n-benzyl-n-(6-nitrobenzo[d]thiazol-2yl)acetamide derivatives as selective dpre1 inhibitors. synth commun 2019; 49: 2696–2708. 29. rajendran n, kamatchi n, periyasamy a, et al. dna-interaction, antibacterial and in vitro cytotoxic properties of copper(ii) complexes bearing (e)-2-(2(benzo[d]thiazol-2-ylthio)-1-phenylethylidene)thiosemicarbazone and diimine co-ligands. j coord chem 2020; 73: 969–985. 30. gularte ms, anghinoni jm, abenante l, et al. synthesis of chitosan derivatives with organoselenium and organosulfur compounds: characterization, antimicrobial properties and application as biomaterials. carbohydr polym 2019; 219: 240–250. 31. singer c, kaplan mh, armstrong d. bacteremia and fungemia complicating neoplastic disease. a study of 364 cases. am j med 1977; 62: 731–742. fig. 7 diagram interaction of compound (4) with the crystal structure of the vegfr-2. the theoretical results of those compounds have been similar, approximately, to the result of reference drugs (sorafenib) and (axitinib) which have binding energy equal to conflict of interest: none. 357j contemp med sci | vol. 7, no. 6, november-december 2021: 353–357 n.n.a. jafar and a.a. hussein original pyrimidine derivatives as promising candidates for potent antiangiogenic: a silico study 32. inc. ccg. molecular operating environment (moe). 33. biovia ds. biovia workbook, release 2017; biovia pipeline pilot, release 2017. san diego dassault systèmes. 34. ghanem a, emara ha, muawia s, et al. tanshinone iia synergistically enhances the antitumor activity of doxorubicin by interfering with the pi3k/akt/mtor pathway and inhibition of topoisomerase ii: in vitro and molecular docking studies. new j chem 2020; 44: 17374–17381. 35. zaki aa, al-karmalawy aa, el-amier ya, et al. molecular docking reveals the potential of cleome amblyocarpa isolated compounds to inhibit covid-19 virus main protease. new j chem 2020; 44: 16752–16758. 36. marrone t, hu-lowe d, grazzini m, et al. pf-00337210, a potent, selective and orally bioavailable small molecule inhibitor of vegfr-2. 37. ghanem a, emara ha, muawia s, et al. tanshinone iia synergistically enhances the antitumor activity of doxorubicin by interfering with the pi3k/akt/mtor pathway and inhibition of topoisomerase ii: in vitro and molecular docking studies. new j chem. 38. amin lht, shawer tz, el-naggar am, et al. design, synthesis, anticancer evaluation and docking studies of new pyrimidine derivatives as potent thymidylate synthase inhibitors. bioorg chem 2019; 91: 103159. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1087 213j contemp med sci | vol. 8, no. 3, may-june 2022: 213–215 case report rare case of lateral dislocation of the elbow without distinct radiological signs arash maleki, mohamad qoreishi*, saman shakeri jousheghan*, katayoon tajik, amirhossein gholami manzari clinical research & development unit, akhtar hospital, shahid beheshti university of medical sciences, tehran, iran. *correspondence to: mohamad qoreishi; saman shakeri jousheghan (email: dr.saman.shakeri.1989@gmail.com ) (submitted: 12 february 2022 – revised version received: 27 february 2022 – accepted: 15 march 2022 – published online: 26 june 2022) abstract while the elbow joint dislocation is one of the most common dislocations, lateral dislocation of elbow is so rare. in this study, we described a 65-year-old woman with lateral elbow dislocation referred to akhtar hospital. with full range of motion in supination, pronation, and extension, as well as 60º flexion, the most important finding with this case was that the radiographic findings could not help surgeon to diagnose the lateral dislocation. therefore, ct-scan was performed. after confirmation, closed reduction was done via a standard maneuver. the patient was followed up for 2 weeks and physiotherapy was ordered. according to this case, when there was an elbow trauma with unusual radiograph without any specific finding to confirm the lateral dislocation, it is recommended to perform ct-scan. the closed reduction and following physiotherapy also are essential for management of this condition. keywords: elbow joint, lateral dislocation, closed reduction, radiography issn 2413-0516 introduction after shoulder joint, the elbow joint is a most common joint to dislocate among adults with an incidence of 5 to 6 cases per 100,000 persons per year.1 these types of dislocations are notably categorized as a simple (without a fracture) or complex (with fracture) injury based on the presence of fracture following injury.2 classification of elbow dislocations according to the position of the ulna and radius in association with the humerus include medial, lateral, anterior, posterior and divergent types.3 among different types, posterior or posterolateral dislocations with no relevant osseous lesions (~90%) are the most common dislocations of the elbow joint.4 simple lateral elbow dislocation is rare and often associated with neurovascular complication with difficulty in performing closed reduction.5 this case report presents a rare lateral traumatic elbow dislocation with a difficult diagnosis. case report a 65-year-old woman after falling down on her left was referred to department of emergency, akhtar hospital due to left elbow pain and limitations of joint movements. she felt sudden severe pain on her elbow joint. in the physical exam, the range of motion of supination and pronation were full. although the normal range of motion in extension movement was seen, the rage of motion in flexion was 60º. clinically, her elbow was deformed. we could not palpate the olecranon in its anatomic location. in the palpation, the olecranon fossa was palpable and it was empty. the radial pulse was detected. in neurological examination, there was no nerve involvement. in the initial paraclinical studies, the lateral (fig. 1a and 1b) and anteroposterior (fig. 1c) radiographies were seemed to be normal or there was no evidence to confirm the lateral dislocation. for more investigations, ct-scan was performed. after confirmation of lateral elbow dislocation, the patient was schedule for surgery. under general anesthesia, closed reduction was attempted after providing an informed consent. along the deformity of the forearm, one assistant gave a gentle longitudinal traction and counter-traction applied by another assistant by holding the arm. after the first try was unsuccessful, the surgeon was applied a medial force to the forearm and a lateral force to the arm until an audible clunk of reduction was felt by the surgeon after few attempts. the accuracy and concentricity of the reduction was confirmed via an x-ray photograph (fig. 1d) and ct-scan was done to be confident about the results (fig. 2c and 2d). after 2 weeks, physiotherapy was done for patient to regain range of motion. discussion in this case report study, we described an unusual case of lateral dislocation of elbow without nerve involvement. notably, the normal movements of elbow in supination, pronation, and extension was reported. in radiographic studies, there was no distinct sign to confirm the lateral dislocation. to confirm this type of dislocation, we used ct-scan. as this type of dislocations are rare, the results of this study confirm that some cases with lateral dislocations can be misdiagnosed. in the limited studies, the lateral dislocation of elbow was reported. recently, a systematic review of the literature showed three cases of lateral dislocation and one case of anterolateral dislocation were reported among 342 patients with dislocated elbows.6 linscheid and wheeler (1965) reported 1 lateral/110 elbow dislocations.7 similarly, one case of this injury was reported in 60 elbow dislocation cases by kini (1940).8 in some cases, the lateral dislocation is accompanied by involvement of ulnar nerve injury.9,10 after diagnosis, patient underwent closed reduction. the reduction of pure lateral elbow dislocation is a difficult procedure due to the high risk of soft tissue damages, such as swelling and soft tissue interposition, such as anconeus and brachialis muscle, and involvement of ulnar nerve, or subsequent fractures.11 the reduction can be applied via several maneuvers.12 for example, reduction verified by the image intensifier was suggested to be a reliable technique.13 in this case, physiotherapy was applied after 2 weeks to allow patient to regain the normal range of motion of the elbow. it was mailto:dr.saman.shakeri.1989@gmail.com 214 j contemp med sci | vol. 8, no. 3, may-june 2022: 213–215 lateral dislocation of the elbow case report a. maleki et al. fig. 1 radiographs of the elbow joint for diagnosis of dislocation. (a) & (b) lateral radiographs and (c) anteroposterior radiograph showed no distinct signs of lateral dislocation of the left elbow, (d) lateral radiograph of elbow after closed reduction. fig. 2 ct-scan of elbow (a) & (b) before closed reduction, and (c) & (d) after closed reduction. 215j contemp med sci | vol. 8, no. 3, may-june 2022: 213–215 a. maleki et al. case report lateral dislocation of the elbow recommended that immobilization of elbow joint for long time after elbow dislocation or (more than 14 days) can lead to stiffness of joint.14 conclusion this report is a rare case of lateral dislocation of the elbow joint with no specific findings to confirm the diagnosis. the results of radiography should be in doubt by surgeon, when there was a case of elbow trauma with unusual radiographs of elbow. the closed reduction and following physiotherapy had excellent functional outcomes in management of lateral elbow dislocation. evs for cartilage repair and experimental oa. conflicts of interest/competing interests the authors declare that they have no conflicts of interest.  references 1. imaeda t, toh s, nakao y, nishida j, hirata h, ijichi m, et al. validation of the japanese society for surgery of the hand version of the disability of the arm, shoulder, and hand questionnaire. journal of orthopaedic science. 2005;10(4):353-9. 2. gokcen b, ozyurek s, atik a, sivrioglu ak, kaya e, keklikci k. successful closed manipulation of simple lateral dislocation of the elbow joint: a case report. oman med j. 2013;28(6):e062. 3. englert c, zellner j, koller m, nerlich m, lenich a. elbow dislocations: a review ranging from soft tissue injuries to complex elbow fracture dislocations. adv orthop. 2013;2013:951397. 4. cho c-h, kim b-s, rhyou ih, park s-g, choi s, yoon jp, et al. posteromedial elbow dislocations without relevant osseous lesions: clinical characteristics, soft-tissue injury patterns, treatments, and outcomes. jbjs. 2018;100(23):2066-72. 5. watanabe k, fukuzawa t, mitsui k. successful closed reduction of a lateral elbow dislocation. case reports in orthopedics. 2016;2016:5934281. 6. de haan j, schep n, tuinebreijer w, patka p, den hartog d. simple elbow dislocations: a systematic review of the literature. archives of orthopaedic and trauma surgery. 2010;130(2):241-9. 7. linscheid rl, wheeler dk. elbow dislocations. jama. 1965;194(11):1171-6. 8. kini m. dislocation of the elbow and its complications: a simple technique for excision of the elbow. jbjs. 1940;22(1):107-17. 9. watanabe k, fukuzawa t, mitsui k. successful closed reduction of a lateral elbow dislocation. case rep orthop. 2016;2016:5934281. 10. khan sk, chopra r, chakravarty d. successful closed manipulation of a pure lateral traumatic dislocation of the elbow joint using a modified stimson’s technique: a case report. journal of medical case reports. 2008;2:170. 11. chhaparwal m, aroojis a, divekar m, kulkarni s, vaidya sv. irreducible lateral dislocation of the elbow. journal of postgraduate medicine. 1997;43(1):19-20. 12. dharmshaktu gs, singhal a. lateral dislocation of the elbow: a report of two cases and literature review. clinical trials in orthopedic disorders. 2016;1(2):79. 13. watanabe k, fukuzawa t, mitsui k. successful closed reduction of a lateral elbow dislocation. case reports in orthopedics. 2016;2016:5934281. 14. de haan j, schep nw, tuinebreijer we, patka p, den hartog d. simple elbow dislocations: a systematic review of the literature. arch orthop trauma surg. 2010;130(2):241-9. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.1226 1j cont med sci | vol. 1, no. 2, spring 2015:1–5 research objective intercalations of plant growth regulator 4-chloro phenoxy acetate (4cpa) with zinc oxide (zno), developed using zno-layered hydroxide (zlh) as host material and 4cpa as a guest. methods ion exchange technique via sol-gel method synthesized under aqueous environment, resulted in the formation of inorganic– organic nanotube materials. results the release of 4cpa from its nanohybrid was found to occur in a controlled manner, governed by pseudo-second order kinetics model. the maximum amount of 4cpa released was governed by pseudo-second order kinetics model. powder x-ray diffraction showed that the basal spacing of the nanohybrid was developed with the increasing of 4cpa concentrations; the maximum concentration of 0.2m shows an interlayer basal of 1.9 nm. ftir study showed that the intercalated 4cpa-zno spectral feature is generally similar to that of 4cpa, but with bands sightly shifted due to the formation of host–guest nano tubes. conclusion the resulted nano tubes were characterized by using scanning electron microscopy (sem), and transmission electronic microscope (tem), shows a uniform nanoparticles and monodisperse with average diameter of 35 nm, which correlated a very well with size scale obtained from xrd data. the development of crystals is the function of concentrations. keywords zinc oxide, nano materials, 4-chlorophenoxy acetic acid, intercalations nanotube synthesis from reaction of 4-chloro phenoxy acetate with zinc layered hydroxide abbas matrood bashi introduction zinc oxide (zno) has attracted much attention for its wide direct band gap (3.37 ev) and large exciton binding energy (60 mev) at room temperature.1 nanoparticles have been widely used in optical, resonant, electrical and magnetic fields. various chemical methods have been used for the production of nanoparticles with narrow size distribution such as micro emulsion method, electro spray pyrolysis and hydrothermal methods. semi-conducting and piezoelectric material have many useful properties, such as optical absorption, emission,2 conductivity,3 photo catalysis4 and sensitivity to gases.5 therefore, many efforts are concentrated on the synthesis of zno materials.6–13 among those methods based on physical and solution-based chemical technologies to synthesize zno particles, organic complexion additives are always used to control the growth of the crystals.14–20 by the addition of organic active molecules, zn complex will be formed, which owns certain stability in both air and aqueous solution. so, it is necessary to find some effective methods to induce the conversion of the zn complex into zno particles. thermal decomposition is a common method to get zno particles. for example, fiber-like zno materials from the decomposition of bis(acetylacetonato) zinc fibers at 110°c in the presence of superheated steam was obtained,19 material scientists are inclined to realize the diversity of morphological zno particles with solution-phase method, which is conducted by precipitating the preformed complex into zno sediments,20–22 4-chlorophenoxyacetic acid (4cpa), as one of the common organic additive. for example, in this work a stable complex composed of 4cpa was formed before further treatments (at 30°c), and then zno particle aggregates or rods were obtained. a similar work was reported by shishido et al.23 all these results prove that the complex plays an important role as templates, a novel method, which supplies a facile technology to transform the preformed zn-4cpa complex into zno particles with various morphologies, which will be a good prospect for material science. in this work, we have designed a novel route to form templates (zn-4cpa complex) first, and then induce the hydrolysis of the template to produce zno particles. this method has its special advantages: (1) the transformation of zn-4cpa complex is facile and quite different from those traditional ways like thermal decomposition, refluxing and hydrothermal treating and heating; (2) we select 4cpa as complex reagents to realize the diversity of morphological zno particles; and (3) it is an economical route with low energy consumption (low temperature, normal atmospheric pressure). novel slice-like and quasi sphere-like zno particles have been successfully fabricated accordingly. the growth mechanisms of the nano particles induced an interaction between the 4cpa and zn cations. materials and methods zno purchased from acrose (usa), 4cpa from merck, was used without further purification. solutions of 0.05, 0.1, 0.2m of 4cpa were prepared by dissolving in 50 ml of 90% ethanol. this solution was dropped into a solution prepared by 1 gm of zno in 100 ml de-ionized water, in a conical flask, the resulted solution under n2 gas was stirred, and formation of gel suspension started, followed by temperature aging at 70°c for 18 hours. it was then cooled, centrifuged and washed four times with de-ionized water, dried in oven at 70°c then it was grinded and kept in a sample bottle for further analyses. characterizations powder x-ray diffraction (pxrd) patterns were obtained with a shimadzu xrd-6000 powder diffractometer using λ = 1.540562 å at 40 kv and 30 ma with a scan rate of 0.5 min/ degrees. fourier transform infrared (ftir) spectra were recorded by using a spectrophotometer thermo nicolet ft-ir nexus self supporting sample in the range of 4000–400 cm–1, department of clinical laboratories, university of karbala-iraq, karbala, iraq. correspondence to abbas matrood bashi (email: abbasmatrood493@gmail.com). (submitted: august 2014 – revised version received: 20 january 2015 – accepted: 28 january 2015 – published online: spring 2015) 2 j cont med sci | vol. 1, no. 2, spring 2015:1–5 nanotube synthesis from 4cpa with zn layered hydroxide research abbas matrood bashi progressing with the intensities of the ray (003) clearly was concentration dependent; and within the same time this sharp peak moved in the direction of small angle, which indicates that good crystals were made from the resulted 4cpa-zno when we used high concentration from 4cpa. it is clear that the samples are indexed to typical wurtzite-type zno. and all the sharp diffraction peaks indicate good crystals of the obtained nanostructures up to 0.2m concentrations of 4cpa, the corresponding energy-dispersive spectra were recorded. it can be obviously observed that the spectra of sample as-grown and the eds studies (fig. 3) reveal that no trace of other elements was detected in any grown samples. these xrd patterns and eds spectra confirm the formation of pure zno intercalated with 4cpa to form a nanomaterial. the morphology of the as-prepared sample was investigated by sem. fig. 2 shows a typical sem image of the zno–wpi (whey protein isolate) composite. table 1 shows the eds studies of zno intercalated with 4cpa in three positions as we have seen in fig. 2, the elements composite of the zno with 4cpa-nanosheet. from this tem micrograph, it is clear that our product is a composite of zno and wpi, i.e. wpi granule with a size of about 70 nm embedded with several zno nanoparticles. these nanoparticles are uniform and monodisperse with average diameter of 35 nm, which correlates very well with size scale obtained from xrd data (70 nm). the development of crystals is function of concentrations as we see in fig. 1 up to the concentration of 0.2m which is 10 20 degree/2θ 30 40 0 . 2 m 0 . 1 m 0 . 0 5 m in te ns ity (a .u ) fig. 1 xrd pattern of three concentrations of 4cpa intercalated with zno nanohybrids. fig. 2 a typical sem image of the zno–wpi composite processing option: all elements were analyzed (normalized). fig. 3 sem image of hexagonal crystals. and the thermogravimetry, derivative thermogravimetry (tg/dtg) were carried out with a setaram tg-dsc-11 apparatus with fully programmable heating and cooling sequence sweep gas valve switching and data analysis. the surface morphology and bulk structure of the sample was observed by scanning electron microscope (sem) model joel (jsm-6400) and tem hitachi (h 7100). results and discussion the evidence for phase structure of the as-prepared sample was obtained by xrd pattern, as shown in fig. 1. all the diffraction peaks can be indexed to those of hexagonal zno. after refinement, the lattice constants, a = 3.251 å, c = 5.210 å were obtained, which was very close to the reported value for zno (a = 3.253 å, c = 5.209, jcpds card, no. 80-0075). the broadening of the zno xrd peaks suggests that the grain sizes are on a nanometer scale. the average particles size was estimated to be 70 nm based on the scherrer equation, d ¼ kλ βdcosθ; here k is shape factor of average crystallite, λ is wavelength for the kα1 (1.54056 å), β is full width at halfmaximum of the diffraction line and θ is bragg’s angle. fig. 1 shows that the xrd patterns of the synthesized nanosheet thin films made up of nanorods 3j cont med sci | vol. 1, no. 2, spring 2015:1–5 research nanotube synthesis from 4cpa with zn layered hydroxideabbas matrood bashi reported to be 1.95 nm for the 003 plane. the zno–wpi particles were stable under the electron beam in vacuum used for tem measurements, suggesting that the binding between zno and wpi is strong. the corresponding selected area electron diffraction (saed) pattern of zno nanoparticle is shown as an inset in fig. 3 as a hexagonal crystal structure. figs. 6 and 7 show the tg-dtg curves of zno, and zno-4cpa shows the details of the tg, which reveal two distinguishable weight loss steps. the first step indicate losses 9.67% started from 37°c terminating at 143°c with maximum temperature of 138.7°c; this is attributed to the loss of physical adsorbed and the interlayer water. the next stage with total loss which is equal to 35.3% at the temperature between 250–396°c and the maximum at 320°c, is due to the decomposition and subtle combustion of 4cpa. the total loss achieved is around 47.3%. this is in accord with the elemental analysis. d3h symmetry is confirmed by the presence of band at 834 cm–1 (fig. 8).13 the oh vibrations of the layers can be observed by a band in the 3600–3000 cm–1 region. this band has a broad base due to hydrogen bonds established with the hydration water molecules. the ftir spectrum of the hybrid presented c–h vibrations of the organic chain at 2932 cm−1 and the asymmetric and symmetric stretching of coo− appears at 1550 and 1410 cm–1. the latter band is overlapped with c–h vibrations, which forms a shoulder at 1450 cm–1 (fig. 8). table 1. edx elements analysis spectrum c o cl zn total spectrum 1 45.15 15.98 15.76 23.11 100.00 spectrum 2 41.78 14.49 14.98 22.93 100.00 spectrum 3 47.71 14.71 12.82 23.97 100.00 max. 47.71 14.49 18.76 23.97 min. 41.78 13.98 12.82 22.93 67 0 200 400 600 800 1000 temperature / °c 99.50 99.75 100.00 100.25 100.50 100.75 m as s f ra ct io n (% ) −4 x 10−5 −2 x 10−5 0 2 x 10−5 4 x 10−5 dt g fig. 6 tg-dtg of commercial zno as standard for thermal analysis. 30 50 70 90 110 0 200 400 600 800 1000 −0.0012 −0.0009 −0.0006 −0.0003 0 0.0003 530 138.7 320 temperature / °c m as s fra ct io n (% ) dt g fig. 7 tg-dtg of zno-4cpa nanomaterial shows the appearance of two indo thermic bands at 138°c and 320°c. fig. 4 sem image shows the superficial interactions between zno layers and the anion 4cpa. fig. 5 transmitting electronic microscope (tem) shows the nano tube of the resulted nano composite from 4cpa and zno. 4 j cont med sci | vol. 1, no. 2, spring 2015:1–5 nanotube synthesis from 4cpa with zn layered hydroxide research abbas matrood bashi band of water molecule. the band at 2930 cm–1 is attributed to c–h stretching vibration of the intercalated 4cpa (from ch2coo– group). peaks at 1550 and 1326 cm–1 are assigned to anti symmetric and symmetric stretching vibrations of the –coo– group. the bands observed at around 1499 and 1410 cm–1 correspond to the c=c bond of the aromatic ring of 4cpa, herbicides. the c–o–c anti symmetric and symmetric stretching bands appear at 1224 and 1071 cm–1. bands in the lower wave number region (i.e., 400–800 cm–1) are due to m–o and m–oh bending vibration in the zno layers that can be seen in ftir spectrum of zn-4cpa and zno. a band observed at 829 cm–1 corresponding to –ch2 rocking. all this indicates that it was accruals 4cpa intercalated in the zno interlayer which can be clearly observed at 2926 cm–1. acknowledgment this work was funded by itma-upm, malaysia, grant of research fellowship upm/1.9.1. the author thanks itma-upm for the study leave and scholarship.  fig. 8 ftir spectrums of zno standard and zno-4cpa. 1000200030004000 zno-4cpa zno-standard 1570 3411 wave no./cm−1 % t( au ) 1512 1378 700 6491224 1410 726 834 1071 1550 1499 2932 3373 3469 3577 references 1. bashi am, hanoon jjm, moh. hussien zobir, zulkarnain zainal. synthesis, characterization and alteration of phenoxyherbicide-based nanocomposites resulted from mixing two herbicides with zinc oxide-layered hydroxide. chem mater res. 2015;7(3):122–31. 2. sarijo sh, hussien mz, yahaya ah, zainal z. effect of incoming and outgoing exchangeable anions on the release kinetics of phenoxyherbicides nanohybrids. j hazard mater. 2010 oct 15;182(1–3):563–9. doi: http:// dx.10.1016/j.jhazmat.2010.06.070 pmid: 20633986 3. mane rs, lee wj, lokhande cd, cho bw, han sh. controlled repeated chemical growth of zno films for dye-sensitized solar cells. curr appl phys. 2008 may;8(5): 549–53. doi: http://dx.10.1016/j.cap.2007.10.001 4. shaheed sh, abd. alghanimi a, abbas m. bashi. preparation of nanohybrid compounds from food preservative octyl gallate and studying some of its biological activities. karbala journal of pharmaceutical sciences 2014;7: 277–89. 5. lee j, easteal aj, pal u, bhattacharyya d. evolution of zno nanostructures in sol-gel synthesis. curr appl phys. 2009 jul;9(4):792–6. doi: http:// dx.10.1016/j.cap.2008.07.018 6. lu jj, lu ym, tasi si, hsiung tl, wang hp, jang ly. conductivity enhancement and semiconductor–metal transition in ti-doped zno films. opt mater. 2007 july;29(11):1548–52. doi: http://dx.doi:10.1016/j.optmat.2006.08.002 7. zhang yy, mu j. one-pot synthesis, photoluminescence, and photocatalysis of ag/zno composites. j colloid interface sci. 2007 may 15;309(2):478–84. doi: http://dx.doi: 10.1016/j.jcis.2007.01.011 pmid: 17292380 8. ghimbeu cm, schoonman j, lumbreras m, siadat m. electrostatic spray deposited zinc oxide films for gas sensor applications. appl surf sci. 2007 jul;253(18):7483–9. doi: http://dx.doi:10.1016/j.apsusc.2007.03.039 9. lao jy, huang jy, wang dz, ren zf. zno nanobridges and nanonails. nano lett. 2003 feb;3(2):235–8. doi: http://dx.10.1021/nl025884u 10. gao px, wang zl. mesoporous polyhedral cages and shells formed by textured self-assembly of zno nanocrystals. j am chem soc. 2003 sept;125(37):11299– 305. doi: http://dx.doi.org/10.1021/ja035569p  pmid: 16220952 11. zhang y, jia h, luo x, chen x, yu d, wang rj. synthesis, microstructure, and growth mechanism of dendrite zno nanowires. j phys chem b. 2003 aug; 107(33):8289–93. doi: http://dx.doi.org/10.1021/jp034834q 12. zhang sc, li xg. preparation of zno particles by precipitation transformation method and its inherent formation mechanisms. colloid surf a: physicochem eng aspects. 2003 sept;226:35–44. doi: http://dx.doi. org/10.1016/s0927-7757(03)00383-2 13. wang z, qian xf, yin j, zhu zk. large-scale fabrication of tower-like, flowerlike, and tube-like zno arrays by a simple chemical solution route. langmuir. 2004 apr 13;20(8):3441–8. doi: http://dx.doi.org/10.1021/la036098n pmid: 1587588 14. shen g, bando y, lee cj. synthesis and evolution of novel hollow zno urchins by a simple thermal evaporation process. j phys chem b. 2005 jun 2;109(21): 10578–83. doi: http://dx.doi.org/10.1021/jp051078a pmid: 16852283 15. tong y, liu y, dong l, zhao d, zhang j, lu y, et al. growth of zno nanostructures with different morphologies by using hydrothermal technique. j phys chem b. 2006 oct 19;110(41):20263–7. doi: http://dx.doi. org/10.1021/jp063312i pmid: 17034205 16. tang lq, zhou b, tian ym, bala h, pan y, ren sx, et al. preparation and surface modification of uniform zno nanorods via a one-step process. colloid surf a: physicochem eng aspects. 2007 mar;296(1–3):92–6. doi: http://dx.doi.org/10.1016/j.colsurfa.2006.09.035 17. park nk, lee yj, han gb, ryu so, lee tj, chang ch, et al. synthesis of various zinc oxide nanostructures with zinc acetate and activated carbon by a matrix-assisted method. colloid surf a: physicochem eng aspects. 2008 feb;313–314:66–71. doi: http://dx.doi.org/10.1016/j.colsurfa.2007.04.074 18. trindade t, pedrosa de jesus jd, o’brien p. preparation of zinc oxide and zinc sulfide powders by controlled precipitation from aqueous solution. j mater chem. 1994;4(10):1611–7. doi: http://dx.doi.org/10.1039/jm9940401611 19. oliveira aa, hochepied jf, grillon f, berger mh. controlled precipitation of zinc oxide particles at room temperature. chem mater. 2003 aug;15(16): 3202–7. doi: http://dx.doi.org/10.1021/cm0213725 the presence of the herbicide anion in the nanohybrid can be verified by ftir. the broad absorption bands at 3469 cm–1 is due to the stretching mode of oh groups in the zno layer and physisorbed water. a shoulder at 3373 cm–1 shows the existence of hydrogen bonding between the water molecules and the zno layer or the anion 4cpa. the band at 1615 cm–1 shows a stretching 5j cont med sci | vol. 1, no. 2, spring 2015:1–5 research nanotube synthesis from 4cpa with zn layered hydroxideabbas matrood bashi 20. chen jf, hu y, zheng xs. surfactant-assisted self-assembly growth of single crystalline zno microflowers at low temperature. colloid surf. a: physicochem eng aspects. 2008 feb;313–314:576–80. doi: http://dx.doi.org/10.1016/j. colsurfa.2007.05.060 21. li p, wei y, liu h, wang xk. growth of well-defined zno microparticles with additives from aqueous solution. j solid state chem. 2005 mar;178(3): 855–60. doi: http://dx.doi.org/10.1016/j.jssc.2004.11.020 22. xie rg, li ds, zhang h, yang dr, jiang mh, sekiguchi t, et al. lowtemperature growth of uniform zno particles with controllable ellipsoidal morphologies and characteristic luminescence patterns. j phys chem b. 2006 oct;110(39):19147–53. doi: http://dx.doi.org/10.1021/jp0605449 23. shishido t, yubuta k, sato t, nomura a, ye j, haga k. synthesis of zinc oxide fibers from precursor bis(acetylacetonato)zinc. j all comp. 2007 jul; 439(1–2): 227–31. doi: http://dx.doi.org/10.1016/j.jallcom.2006.05.136 24. ban t, sakai t, ohya y. synthesis of zinc oxide crystals with different shapes from zincate aqueous solutions stabilized with triethanolamine. cryst res technol. 2007 sept;42(9):849–55. doi: http://dx.doi.org/10.1002/ crat.200710960 256 j contemp med sci | vol. 7, no. 4, july–august 2021: 256–260 original the effect of bispectral index monitoring during induction of anesthesia on the amount of propofol consumtion in patients candidates for surgery borjian boroojeny shahram1*, seyedeh maryam hojjat2 1assistant of professor, department of anesthesia, zahedan university of medical sciences, zahedan, iran. 2resident of anesthesology, medical school, zahedan university of medical sciences, zahedan, iran. *correspondence to: borjian boroojeny shahram (e-mail: borjianboroojenyshahram@yahoo.com) (submitted: 06 april 2021 – revised version received: 21 april 2021 – accepted: 13 may 2021 – published online: 26 august 2021) introduction maintenance of anesthesia during surgery can be done using inhaled or intravenous drugs. intraoperative doses of these drugs are often increased or decreased based on hemodynamic symptoms, such as blood pressure and heart rate. this is due to the relatively direct relationship between the depth of anesthesia and the amount of blood pressure, so blood pressure can be used as a reliable monitoring of the depth of anesthesia.1 maintaining blood pressure within the standard range is one of the most important tasks of an anesthesiologist during surgery. bispectral index (bis) is a parameter derived from electroencephalography, which is a number between 0 and 100 based on the brain waves recorded from the frontal lobe of the brain and their frequency and the prevalence of eeg flat time. the best depth of anesthesia indicated by bis when using propofol is between 40 and 60.2–4 propofol is the most common drug for inducing intravenous anesthesia and one of the most widely used drugs for maintaining anesthesia in the operating room. in animal and human studies, hypotension during anesthesia has several complications such as risk of heart attack, stroke, cognitive or memory impairment, kidney damage, etc.,5–8 propofol is classically administered on an mg/kg basis and one of the most common side effects of propofol is hypotension.9 the use of bis may prevent over-administration of propofol while achieving sufficient depth of anesthesia and thus prevent complications such as hypotension.9 propofol is a first-line drug in intravenous anesthesia. in complete intravenous anesthesia, a combination of propofol and a drug is used to induce and maintain anesthesia.10 it is a hypothetical drug with an unknown mechanism, but it is capable of activating chlorine channels and inhibiting synaptic inhibition by stimulating the beta component of a gaba system. its ed50 is 1–1.5 mg/kg following a bolus injection. the amount of anesthesia is dose dependent and it is able to convert the alpha rhythm of awakening to the dominant beta rhythm in case of infusion with a dose of 100 mcg/kg/min.11,12 the eeg spectral analysis shows an almost good correlation with movement during surgical stimulation.13,14 this method does not monitor the depth of anesthesia, but it can predict the patient’s movement if the level of anesthesia is insufficient. bis is the best predictor of motion relative to plasma propofol concentration or other eeg criteria such as spectral margin or median frequency.15 therefore, according to the above, this study was conducted to investigate the role of bis in reducing propofol consumption during induction of anesthesia while maintaining sufficient depth of anesthesia. abstract introduction propofol is a new intravenous hypnotic drug that is widely used in anesthesia and monitoring the depth of anesthesia. bis is a parameter derived from electroencephalography that is associated with sleep deprivation and loss of consciousness. this study investigated the effect of bispectral index (bis) during induction of anesthesia on the amount of propofol consumed in patient candidates for surgery. materials and methods this double-blind clinical trial study was performed on patients undergoing elective surgery under general anesthesia in a hospital. patients were divided into case and control groups. after being transferred to the operating room, patients were monitored including ecg 3 or 5 leads, non-invasive barometer and pulse oximetry. mean arterial blood pressure and heart rate were measured before and after induction, immediately and 5 minutes after intubation. the dose of propofol was then measured. data analysis was performed by spss software version 20. results in the present study, no significant difference was found between the mean age and gender (p > 0.05). the amount of propofol consumed in the case group was significantly lower than the control group (p = 0.039) and the amount of propofol consumed in men and women was not statistically significant (p < 0.05). mean arterial blood pressure before induction was not statistically significant between the two groups (p = 0.83). however, a statistically significant difference was found in the mean arterial blood pressure of the patients during the 4 time points (p = 0.001). there was no statistically significant difference in heart rate between patients before induction (p = 0.48). statistical analysis of data by anova test did not show a significant interaction between time and group (p = 0.418 and p = 0.74). however, a statistically significant difference was found in patients’ heart rate during the 4 time points (f = 7.59 and p = 0.001). moreover, a significant increase was observed in heart rate after intubation in both groups (p = 0.001). conclusion the use of bis can be effective in reducing the amount of propofol consumed and its side effects. the condition of patients under general anesthesia can be improved by bis, resulting in improvement of their subsequent condition. keywords bispectral index (bis) monitoring, propofol, general anesthesia issn 2413-0516 257j contemp med sci | vol. 7, no. 4, july–august 2021: 256–260 b.b. shahram and s.m. hojjat original the effect of bispectral index monitoring during induction of anesthesia materials and methods this double-blind clinical trial was performed on patients who were candidates for elective surgery and underwent general anesthesia in hospitals affiliated to zahedan-iran university of medical sciences in 1397-98. patients entered the study after obtaining informed consent and having inclusion and exclusion criteria. inclusion criteria include: having informed consent, asa class i & ii, age between 18 and 65 years. exclusion criteria include: intubation history, decreased level of consciousness for any reason before surgery, brain surgery, cardiovascular disease, esophageal reflux, hiatal hernia, liver or kidney failure, allergy to propofol, taking sedatives or drugs 24 hours before surgery, history of any neurological disorders and use of psychiatric drugs, patient history of addiction, anemia with hemoglobin less than 10 mg/dl, patient dissatisfaction with participation in the study. sample size the sample size was calculated using the formula and,16 at least 40 people in each group. n z z s s = + + -( ) ( ) ( ) /1 2 1 2 1 2 2 2 1 2 2 a b m m n = + + = ( . . ) ( . . ) ( . . ) . 1 96 1 64 98 6 206 5 397 8 534 4 36 4 40 2 1 2 2 2 2 » propofol intake in bis group: (397.8 ± 98.6) propofol consumption in non-bis group: (534.4 ± 206.5) procedure patients were randomly selected and then divided into case groups (bis) and control using random blocking method. after being transferred to the operating room, the patient underwent monitoring including ecg 3 or 5 leads, noninvasive blood pressure (nibp) and pulse oximetry. the initial information of the patient was recorded and then the venous cannula no. 20 was implanted in one of the patient’s hands and ringer serum was infused at 4 cc/kg within 10 to 15 minutes. in the study group, the leads of the bis device (iran electronics industries) were connected to the foreheads of the patients. induction of anesthesia was performed with 1–2 mg/kg fentanyl and 1% propofol (fresenius-kabi, germany) at a dose of 2 mg/kg at a rate of 130 mg/min. in the case group (bis), after 20 seconds, propofol was injected at the rate of 0.2 mg/kg if the bis does not reach less than 60%, and this cycle continued until the bis decreased to less than 60%. in the control group, 20 seconds after propofol injection, in case of no apnea or loss of eyelash reflex, propofol was injected at the rate of 0.2 mg/kg and this cycle was continued until complete anesthesia, then cisatracurium with dose 0.15 mg/kg was injected and endotracheal intubation was performed after 3 minutes. maintenance of anesthesia was continued with propofol at a dose of 4–8 mg/kg/h. estimation of anesthesia in the patient in the clinical method included loss of oral response, loss of sense of gentle touch and loss of eyelash reflex. the dose of propofol in each group was calculated. map blood pressure and heart rate of patients were measured before induction, after induction, immediately after intubation and 5 minutes after intubation. in case of any unusual event such as laryngeal spasm or arrhythmia that required treatment outside the above protocol, the patient was excluded from the study while recording the event. data analysis the information was displayed in the form of tables and graphs. data analysis was performed according to the type of variable and the required conditions by appropriate statistical tests (including chi-square, mixed anova, independent samples t-test). statistical analysis was performed using spss software version 20 and a statistically significant level of 0.05 was considered. ethical considerations after receiving a written letter of introduction and written consent from the selected researcher centers, initial measures were taken to carry out the project. all patients’ information was kept confidential. all ethics statements were included in the study by regarding the declarations of helsinki and ethics research committees of the university of medical sciences. the project was carried out after approval by the research council of the medical school and receiving the code of ethic (ir.zaums.rec.1398.199 and irct20190813044521n1). results in this study, 90 patients were included in the study. the mean age in the case group was 28.11 ± 7.24 years and in the control group was 29.16 ± 8.45 years. statistically, the two groups did not differ significantly (p = 0.53). also, no statistically significant difference was found between the two groups in terms of gender frequency (p = 0.82), (table 1). the mean propofol consumption was 2.86 ± 0.25 mg/kg in the case group and 3.01 ± 0.4 mg/kg in the control group. the amount of propofol consumed in the case group was significantly lower than the control group (p = 0.039). the amount of propofol consumed by sex in the study groups was also calculated. as shown in table 2, the amount of propofol consumed in men and women was not statistically significant (p < 0.05). table 3 and figure 1 compare the amount of map changes at the time before and after induction, immediately and 5 minutes after intubation in the two groups. mean arterial blood pressure before induction was not found to be statistically significant between the two groups (p = 0.83). statistical analysis of data by anova test did not show a significant interaction between time and group (p = 0.217 and p = 0.86), but a statistically significant difference was found in mean arterial blood pressure of patients during the 4 time points (f = 7.59 and p = 0.001). furthermore, a significant increase was found in table 1. gender frequency of patients sex total male female group case 28 (62.2%) 17 (37.8%) 45 (100%) control 29 (64.4%) 16 (35.6%) 45 (100%) p-value 0.82 258 j contemp med sci | vol. 7, no. 4, july–august 2021: 256–260 the effect of bispectral index monitoring during induction of anesthesia original b.b. shahram and s.m. hojjat table 2. propofol intake by sex group sex propofol dose (mg/kg) p-value* case male 2.86 ± 0.29 0.94 female 2.85 ± 0.19 control male 3.02 ± 0.45 0.73 female 2.98 ± 0.3 total male 2.94 ± 0.38 0.68 female 2.91 ± 0.25 *independent samples t-test. table 3. map rate at different times evaluation time group p-value* case control before induction 9.78 ± 1.3 9.27 ± 1.3 0.86 after induction 9.28 ± 1.03 9.09 ± 1.11 immediately after intubation 9.03 ± 1.34 8.74 ± 1.28 5 minutes after intubation 9.94 ± 1.43** 9.54 ± 1.3** *mixed anova test. **p < 0.05. fig. 1 changes in mean arterial pressure at different times. mean arterial pressure after intubation in both groups (case group: p = 0.011; control group: p = 0.034). the comparisons of heart rate changes at times before and after induction, immediately and 5 minutes after intubation in the two groups are shown in table 4 and figure 2. there was no statistically significant difference in heart rate between patients before induction (p = 0.48). statistical analysis of data by anova test did not show a significant interaction between time and group (p = 0.418 and p = 0.74). however, a statistically significant difference was found in patients’ heart rate during the 4 time points (f = 7.59 and p = 0.001). a significant increase was found in heart rate after intubation in both groups (p = 0.001). discussion propofol is a new intravenous hypnotic drug that is widely used in anesthesia. monitoring the depth of anesthesia with bis is also a parameter derived from electroencephalography (eeg), which is associated with sleep deprivation and loss of consciousness.15 therefore, the aim of this study was to evaluate the effect of using bis monitoring during induction of anesthesia on the amount of propofol consumed in patient candidates for surgery. in the present study, no significant difference was observed between the mean age and gender (p > 0.05). the amount of propofol consumed in the case group was significantly lower than the control group (p = 0.039) and the amount of propofol consumed in men and women was not found to be statistically significant (p < 0.05). mean arterial blood pressure before induction was not statistically significant between the two groups (p = 0.83). statistical analysis of data by anova test did not show a significant interaction between time and group (p = 0.217 and p = 0.86), but a statistically significant difference was observed in mean arterial blood pressure of patients during the 4 time points (p = 0.0011). in addition, a significant increase was found in mean arterial pressure after intubation in both groups (p = 0.011 and p = 0.034). there was no statistically significant difference in heart rate between patients before induction (p = 0.48). statistical analysis of data by anova test did not reveal a significant correlation between time and group (p = 0.418 and p = 0.74). but a statistically significant difference was observed in table 4. heart rate at different times evaluation time group p-value* case control before induction 80.13 ± 4.87 80.17 ± 5.39 0.74 after induction 81.93 ± 4.22 81.46 ± 4.04 immediately after intubation 80.75 ± 6.64 79.84 ± 4.67 5 minutes after intubation 86.77 ± 4.14** 87.35 ± 4.14** *mixed anova test. fig. 2 heart rate changes at different times. 259j contemp med sci | vol. 7, no. 4, july–august 2021: 256–260 b.b. shahram and s.m. hojjat original the effect of bispectral index monitoring during induction of anesthesia patients’ heart rate during the 4 time points (f = 7.59 and p = 0.001). there was a significant increase in heart rate after intubation in both groups (p = 0.001). in this regard, various studies have been conducted. for instance, mahori et al., evaluated the effect of bis index on propofol consumption in patients undergoing coronary artery bypass graft surgery and stated that although propofol consumption in the intervention group was less than control group but this finding was not statistically significant and the time of endotracheal tube removal after surgery was not found to be statistically significant between the two groups. this study stated that the binomial index is a valuable tool for monitoring the depth of anesthesia, but can not be useful as a guide for propofol administration.17 arbabpour et al. showed that the bis index for as monitoring the depth of anesthesia in cesarean section leads to acceleration of the time of removal of the endotracheal tube, and time of recovery, as well as acceleration of the mother’s consciousness to care for the baby.18 the results of the evaluation are consistent with the present study. hasani et al. also evaluated the effect of constant dose and variable dose of propofol maintenance on waking time by monitoring the bis index. according to the results, the time of the first awakening response in the first group who received the variable dose of propofol was 480 seconds on average while it was found to be 549 seconds in the second group was (p < 0.05). the total amount of drug used during surgery, which lasted about 120 ± 30 minutes for all patients, averaged 980 mg in the first group and 1240 mg in the second group. accordingly, administration of propofol infusion by monitoring the depth of anesthesia with bis index during anesthesia accelerates recovery and reduces propofol consumption.19 this is also consistent with the results of the present study. another study by hasani et al. reported no statistically significant difference in the effect of vital signs in the study population and the time to general anesthesia with sevoflurane was less than that of isoflurane and halothane, where sevoflurane was more suitable for general anesthesia in children.20 kamali et al. also examined the effect of bis on patients undergoing cesarean section and showed that the level of awareness during anesthesia was significantly higher in the control group than the bis group (p = 0.0001). accordingly, the incidence of awareness during anesthesia in the group with bis monitor is significantly lower than the control group.21 this finding was also consistent with and confirming our findings. other findings of the present study showed a significant increase in mean arterial blood pressure and heart rate in both groups after intubation. ko et al. reported a similar finding where an increase in blood pressure and heart rate was found after intubation.22 tabari et al. compared hemodynamic changes during anesthesia between tracheal intubation and laryngeal mask use. they also reported similar results on moderate arterial hypertension in both groups, but no statistically significant difference was found between the two groups in this regard.23 in general, various studies, including the present study, have shown that the use of bis can be effective in improving the condition of patients, however, there is a need for more studies in this area. conclusion the use of bis can be effective in reducing the amount of propofol intake and its side effects. the condition of patients under general anesthesia can be improved by bis, resulting in improvement of the next condition. conflicts of interest none.  references 1. adrian w. gelb, kate leslie, donald r. stanski, steven l. shafers. monitoring the depth of anesthesia. in: miller's anesthesia 7th ed, churchill livingstone, usa, 2010. p:1245. 2. fahy bg, chau df. the technology of processed electroencephalogram monitoring devices for assessment of depth of anesthesia. anesthesia and analgesia. 2018;126(1):111-7. 3. ferreira a, nunes c, ferreira al, tedim r, amorim p. inter-patient variability and predictive factors of propofol requirements and estimated concentrations for loss of consciousness and recovery. j neurosurg anesthesiol. 2015;27:260–1. 4. ferreira al, mendes jg, nunes cs, amorim p. evaluation of bispectral index time delay in response to anesthesia induction: an observational study. brazilian j anesthesiol elsevier. 2019;69:377–82. 5. upton rn, martinez am, grant c. comparison of the sedative properties of cns 7056, midazolam, and propofol in sheep. british journal of anaesthesia. 2009;103(6):848-57. 6. bijker jb, persoon s, peelen lm, moons kg, kalkman cj, kappelle lj, et al. intraoperative hypotension and perioperative ischemic stroke after general surgery: a nested case-control study. anesthesiology. 2012;116(3):658-64. 7. walsh m, devereaux pj, garg ax, kurz a, turan a, rodseth rn, et al. relationship between intraoperative mean arterial pressure and clinical outcomes after noncardiac surgery: toward an empirical definition of hypotension. anesthesiology. 2013;119(3):507-15. 8. monk tg, saini v, weldon bc, sigl jc. anesthetic management and one-year mortality after noncardiac surgery. anesthesia and analgesia. 2005;100(1):4-10. 9. sepúlveda po, carrasco e, tapia lf, ramos m, cruz f, conget p, et al. evidence of hysteresis in propofol pharmacodynamics. anaesthesia. 2018;73:40–8. 10. gonzalez-cava jm, reboso ja, calvo-rolle jl, mendez-perez ja. adaptive drug interaction model to predict depth of anesthesia in the operating room. biomed signal process control. 2020;59:101931. 11. vakkuri a, yli-hankala a, talja p, mustola s, tolvanen-laakso h, sampson t, et al. time-frequency balanced spectral entropy as a measure of anesthetic drug efect in central nervous system during sevofurane, propofol, and thiopental anesthesia. acta anaesthesiol scand. 2004;48:145–53. 12. mourisse j, lerou j, struys m, zwarts m, booij l. multi-level approach to anaesthetic efects produced by sevofurane or propofol in humans: 1. bis and blink refex. br j anaesth. 2007;98:737–45. 13. scott ai. electroconvulsive therapy, practice and evidence. br j psychiatry. 2010;196(3):171–172. 14. kimball jn, rosenquist pb, dunn a, mccall v. prediction of antidepressant response in both 2.25xthreshold rul and fixed high dose rul ect. j affect disord. 2009;112(1–3):85–91. 15. castro a, bressan n, lobo f, nunes c, amorim p. the higher the propofol concentration needed for loss of consciousness the larger its diference to the concentrations required at maintenance, using tci and bis guided anesthesia. eur j anaesthesiol. 2008;25:150. 16. arya s, asthana v, sharma jp. clinical vs. bispectral index-guided propofol induction of anesthesia: a comparative study. saudi journal of anaesthesia. 2013;7(1):75-9. 17. mahori a, heshmati f, abbasivash r, norozinia h, hassani e, nasiri aa. the effect of bispectral index monitoring on propofol consumption in patients undergoing coronary artery bypass graft. studies in medical sciences. 2011;22(3):249-54. 18. arbabpour r, ganji fard m, tabiee s, saadatjoo sa. the effect of bi-spectral index on recovery and postoperative complications in patients undergoing caesarean section. journal of birjand university of medical sciences. 2015;22(2):94-103. 260 j contemp med sci | vol. 7, no. 4, july–august 2021: 256–260 the effect of bispectral index monitoring during induction of anesthesia original b.b. shahram and s.m. hojjat 19. hasani v, zamani f, khosravi a. comparison of constant dose with variable dose of propofol for studying time of wake up within bispectral index (bis) monitoring. razi journal of medical sciences. 2003;9(31):509-14. 20. hassani v, javanbakht s. comparison of effects of halothane, isoflurane and sevoflurane on depth of anesthesia using bis monitoring in cochlear implantation of children aged 2-6 years old, in hazrat-e rasool akram hospital. razi journal of medical sciences. 2007;14(55): 89-94. 21. kamali a, shokrpour m, pazoki s, moshiri e, taheri-nejad m, dadashpour n, et al. determining the effect of bis monitoring on level of awareness during anesthesia in women undergoing elective caesarean section. journal of arak university of medical sciences. 2015;17(12):56-63. 22. ko dd, kang h, yang sy, shin hy, baek cw, jung yh, et al. a comparison of hemodynamic changes after endotracheal intubation by the optiscope and the conventional laryngoscope. korean journal of anesthesiology. 2012;63(2):130-5. 23. tabari m, alipour m, ahmadi m. hemodynamic changes occurring with tracheal intubation by direct laryngoscopy compared with intubating laryngeal mask airway in adults: a randomized comparison trial. egyptian journal of anaesthesia. 2013;29(2):103-7. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i4.1033 163j contemp med sci | vol. 3, no. 9, winter 2017: 163–166 research impact of adiponectin and oxidized low-density lipoprotein in acute coronary syndrome batool luay aziz,a ahmed hussein al-mayali,a shaymaa zahraw nada,a fatemah abdullah mankhib adepartment of biochemistry, college of medicine, university of karbala, holy karbala city, iraq. bbranch of immunology laboratory, in al-hussein teaching hospital, al-hussein medical city/ kerbela health directorate. correspondence to batool luay aziz (email: 1992louay@gmail.com). (submitted: 29 december 2016 – revised version received: 17 january 2017 – accepted: 25 february 2017 – published online: 26 march 2017 ) objectives this study aimed to investigate and measure the relationships between the adiponectin levels and oxidized-low density lipoprotein (ox-ldl) in acute coronary syndrome (acs) patients in holy kerbala city, iraq. methods fifty-eight patients included in the study. patients admitted with a diagnosis of acs and 30 control subjects. circulating adiponectin and ox-ldl were assessed; using enzyme-linked immunosorbent assays (elisa). results adiponectin serum concentrations were significantly lower (p < 0.001) in subjects with acs compared with control subjects. ox-ldl serum concentrations were significantly higher (p < 0.001) in subjects with acs compared with controls subjects. the acs patients showed a significantly higher (p < 0.001) result in total cholesterol and significantly lower (p < 0.001) level in hdl-c. conclusion serum adiponectin negatively correlated with ox-ldl level in patient group acs and the healthy control group. keywords acute coronary syndrome, ox ldl, adiponectin introduction acute coronary syndrome (acs) is one of the leading causes of morbidity and mortality globally.1 acs occurs because of the destabilization of atherosclerotic plaques, which may undergo rupture or erosion, with subsequent thrombosis and vessel occlusion. many findings indicate that plaque destabilization correlates with the location and activation of inflammatory cells both within the plaque2 and in the systemic circulation.3 although the identification of vulnerable patients remains a challenge.4,5 adipose tissue is an active endocrine organ that secretes bioactive molecules known as adipokines.6,7 these molecules play key roles in regulating energy, metabolic and inflammatory processes.7 one of these classes of molecules, adiponectin, has drawn special attention due to its potential anti-inflammatory, insulin-sensitizing and anti-atherogenic properties.8,9 plasma adiponectin levels are lower in obese individuals and have a protective action against the development of type 2 diabetes.10,11 clinically, hypoadiponectinemia has been observed in patients with obesity, diabetes mellitus, and coronary artery disease (cad).12 among factors involved in the initiation, progression and destabilization of plaques, oxidized-low-density lipoproteins (ox-ldl) was thought to play a key role.13 excess circulating levels of ox-ldl has been shown to be associated with angiographically proven coronary artery disease (cad).14,15 ox-ldl is a critical factor in the initiation and progression of atherosclerosis and contributes to endothelial dysfunction and plaque destabilization through multiple mechanisms. ruptured plaques are rich in lipids.16 methods study design the study group consisted of 58 patients age range (58.21), 38 males and 20 females with acute coronary syndrome (acs), and 30 healthy control group age range (56.96), 17 males and 13 females. all 88 subjects who agreed to participate in the study were enrolled and all patients have undergone coronary angiography for clinical indications (mainly chest pain, dyspnea on exertion, or positive stress test). patients in acs subgroup had a last diagnosis of either unstable angina or acute myocardial infarction (mi), based on clinical feature, biochemical markers, and electrocardiographic changes. the patients were classified as having unstable angina if they had chest pain that was new in the starter. patients were defined as having an acute mi if they had a cardiac marker rise in association with chest pain or ischemic electrocardiography changes. the exclusion criteria were a smoking history, history of previous coronary revascularization and prior chest radiation therapy. laboratory methods a volume of 5 ml venous blood was collected from all patients and control subjects. the serum samples were kept at -70°c for subsequent assay. the plasma concentration of adiponectin and ox-ldl evaluated by a sandwich elisa system (adiponectin elisa kit, ox-ldl elisa kit). serum total cholesterol, triglyceride and hdl-c concentrations determined by an enzymatic mode. body mass index (bmi) was calculated as weight divided by the square of height. risk factors defined as follows, diabetes mellitus was diagnosed according to world health organization criteria.17 dyslipidemia was defined as a total cholesterol concentration ≥ 200 mg/dl, a triglyceride concentration ≥ 150 mg /dl, an hdl-cholesterol focus < 40 mg/dl, and having received treatment for dyslipidemia. statistical analysis the data were analyzed by using spss. continuous data were expressed as mean ± sd and difference of mean of two groups was set by unpaired student’s t-test. the level of significance was set at 0.05, and anova test was used to investigate the difference among the means of more than two groups. issn 2413-0516 164 j contemp med sci | vol. 3, no. 9, winter 2017: 163–166 impact of adiponectin and oxidized low-density research batool luay aziz et al. results the clinical characteristics of acs patients and healthy control subjects are shown in table 1 and figure 1. serum adiponectin levels in the acs patients was significantly lower mean = 21.888 than those in healthy control subjects mean = 36.09 (p < 0.0001) and serum ox-ldl levels in the acs patients was significantly higher mean = 66.03 than those in healthy control subjects mean = 19.6 (p < 0.001). in table 2, there were the mean bmi value and age was insignificant in the patient than healthy control (p > 0.05). the serum hdl-c was significantly lower mean = 35.33 in acs patients (p < 0.0001) in comparison with a healthy control group, but there were a significant difference in the total cholesterol in the acs patient group in comparison with a healthy control group (p < 0.05). whereas no significant differences in the serum triglyceride, vldl-c and ldl-c levels in comparison between acs patient group and healthy control group (p > 0.05). increased triglyceride mean = 199.49, ldl-c mean = 123.32 and vldl-c mean = 39.899 associated with the presence in patients admitted to c.c.u. results obtained showed that serum ox-ldl was significantly higher mean = 83.008 in diabetic and non-diabetic mean = 52.26 acute coronary syndrome (acs) patients group in comparison with healthy control group (p < 0.001), the results showed that there was a significantly lower levels mean = 18.7 of adiponectin in diabetic acs patient group in comparison with healthy control group (p < 0.001) and no significant differences in the serum triglyceride, tc, vldl-c and ldl-c levels in comparison between diabetic and non-diabetic acs patients group in comparison with healthy control group (p > 0.05). while serum hdl-c recorded a highly significant decreases in diabetic acs patients mean = 31.48 in comparison with healthy control group (p < 0.001). on the other hand, the age and bmi showed no significant difference between patients and healthy control group (p > 0.05), as shown in tables 3 and 4. discussion cardiovascular diseases are important medical and public health issues throughout the worldwide. the acs becomes progressively more common with increasing age and as the average age of world population is increasing, this problem will also rise.18 in the this study, hypoadiponectinemia was found to be highly associated with cad prevalence after adjustment for well-known cad factors such as diabetes mellitus, dyslipidemia, hypertension and smoking habit in human subjects. for translation into the clinical setting, it is very important to determine the abnormal range of serum adiponectin concentrations this agreement with kumada et al.17 adiponectin levels decrease as the number of significantly narrowed coronary arteries increase. similar observation was made by the number of previous study19 that found the adiponectin levels decreased as a function of number of significantly narrowed coronary arteries and that, in patients with acs, those with various complex lesions had significantly lower adiponectin than those with a single complex lesion. so the present study reinforce, the protective role of adiponectin in the table 1. comparison between acute coronary syndrome patients group and healthy control group in the measured adiponectin and ox-ldl parameters patients controls t-test p-valuemean ± sd mean ± sd number 58 30 adiponectin (ng/ml) 21.888 ± 6.531 36.09 ± 3.88 0.000* ox-ldl (u/ml) 66.03 ± 17.44 19.6 ± 3.15 0.000* student t-test between acs patients and control group, *highly significant; sd, standard deviation; ox-ldl, oxidized-low-density lipoprotein; student t-test between acs patients and control group; significant: p < 0.05; highly significant: p < 0.001; no significant: p > 0.05. fig 1. correlation between ox-ldl and adiponectin in acute coronary syndrome patients and healthy control group. table 2. comparison between acute coronary syndrome patients group and healthy control group in the measured parameters parameters patients controls t-test p-valuemean ± sd mean ± sd number 58 30 age (years) 58.21 ± 8.57 56.96 ± 9.01 0.526 bmi (kg/m2) 27.14 ± 2.82 27.01 ± 2.92 0.84 tg (mg/dl) 199.49 ± 99.42 165.18 ± 56.6668 0.084 t.c (mg/dl) 198.55 ± 62.034 173.951 ± 33.3128 0.046* ldl (mg/dl) 123.32 ± 65.259 99.319 ± 32.904 0.063 hdl (mg/dl) 35.33 ± 6.166 41.59 ± 6.163 0.000** vldl (mg/dl) 39.899 ± 19.88 33.036 ± 11.333 0.084 student t-test between acs patients and control group: *significant; **highly significant; tg, triglycerides; tc, total cholesterol; hdl-c, high density lipoprotein-cholesterol; sd, standard deviation; ldl-c, low-density lipoprotein-cholesterol; vldl-c, very low-density lipoprotein cholesterol; bmi, body mass index; p value derived from student t-test; significant: p < 0.05; highly significant: p < 0.001; no significant: p > 0.05. table 3. comparison between diabetic and non-diabetic acute coronary syndrome patients group and healthy control group in the measured parameters using anova test parameters patient with acs control (30) mean ± sd p-value d.m (26) mean ± sd non d.m (32) mean ± sd number 26 32 30 adiponectin (ng/ml) 18.7 ± 4.9 24.4 ± 6.66 36.09 ± 3.88 0.000* ox-ldl (u/l) 83.008 ± 8.35 52.26 ± 8.08 19.6 ± 3.1 0.000* *highly significant; sd, standard deviation; ox-ldl, oxidized lowdensity lipoprotein; p value derived from anova test; significant: p < 0.05; highly significant: p < 0.001. batool luay aziz et al. 165j contemp med sci | vol. 3, no. 9, winter 2017: 163–166 research impact of adiponectin and oxidized low-density evolution of atherosclerotic plaque. the results of this study show that circulating levels of ox-ldl metric with an elisa method are significantly higher in patients with acs than in healthy control group, the ox-ldl levels correlate with the presence of angiographic complex plaques. these results support the key role that ox-ldl may have in plaque destabilization and tear.20,21 several studies have exact the relation between circulating levels of ox-ldl and clinical manifestations of cad with conflicting results.22,23 there is increasing evidence suggest that oxidative modification of ldl plays a pivotal role in the development of atherosclerosis.24–27 this study found that the levels of ox-ldl were significantly higher in acs cases than in healthy control group, which was similar to our previous findings.26,27 these finding agree with the study mad by who found that huiling huang et al.28 these findings indicate that serum ox-ldl levels are closely related with cad. conclusion it was found that a presence of significant association between the adiponectin and ox-ldl in acute coronary syndrome (acs). level of serum adiponectin was significantly lower in patients with acs compared with healthy control group and level of serum ox-ldl was significantly higher in patients with acs compared with healthy control group. serum adiponectin was negatively correlated with ox-ldl level in patients’ group and healthy control group. hypoadiponectinemia was significantly associated with the presence acs. n references 1. azmi s, goh a, fong a, anchah l. quality of life among patients with acute coronary syndrome in malaysia. value in health regional issues. 2015;6:80–83. 2. anselmi m, garbin u, agostoni p, fusaro m, pasini af, nava c, et al. plasma levels of oxidized-low-density lipoproteins are higher in patients with unstable angina and correlated with angiographic coronary complex plaques. atherosclerosis. 2006;185:114–120. 3. zalai cv, kolodziejczyk md, pilarski l, christov a, nation pn, lundstromhobman m, et al. increased circulating monocyte activation in patients with unstable coronary syndromes. j am coll cardiol. 2001;38:1340–1347. 4. pearson ta, mensah ga, alexander rw, anderson jl, cannon ro, criqui m, et al. markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the centers for disease control and prevention and the american heart association. circulation. 2003;107:499–511. 5. idem. from vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies. part ii. circulation. 2003;108:1772–1778. 6. bluher m. clinical relevance of adipokines. diabetes metab j. 2012;36:317–327. 7. deng y, scherer pe. adipokines as novel biomarkers and regulators of the metabolic syndrome. ann n y acad sci. 2010;1212:e1–e19. 8. zhang h, cui j, zhang c. emerging role of adipokines as mediators in atherosclerosis. world j cardiol. 2010;2:370–376. 9. kubota n, terauchi y, yamauchi t, kubota t, moroi m, matsui j, et al. disruption of adiponectin causes insulin resistance and neointimal formation. j biol chem. 2002;277:25863–25866. 10. hajer gr, van haeften tw, visseren fl. adipose tissue dysfunction in obesity, diabetes, and vascular diseases. eur heart j. 2008;29:2959–2971. 11. aprahamian tr, sam f. adiponectin in cardiovascular inflammation and obesity. int j inflamm. 2011;2011:376909. 12. hotta k, funahashi t, bodkin nl, ortmeyer hk, arita y, hansen bc, et al. circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys. diabetes. 2001;50:1126–1133. 13. libby p, ridker pm, maseri a. inflammation and atherosclerosis. circulation. 2002;105:1135–1143. 14. weinbrenner t, cladellas m, covas mi, et al. the solos study investigators. high oxidative stress in patients with stable coronary heart disease. atherosclerosis. 2003;168:99–106. 15. holvoet p, mertens a, verhamme p, bogaerts k, beyens g, verhaeghe r, et al. circulating oxidized ldl is a useful marker for identifying patients with coronary artery disease. arterioscl thromb vas biol. 2001;21:844–848. 16. tsimikas s, bergmark c, beyer rw, patel r, pattison j, miller e, et al. temporal increases in plasma markers of oxidized-low-density lipoprotein strongly reflect the presence of acute coronary syndromes. j am coll cardiol. 2003;41:360–370. 17. kumada m, kihara s, sumitsuji s, kawamoto t, matsumoto s, ouchi n, et al. association of hypoadiponectinemia with coronary artery disease in men. arterioscler thromb vasc biol. 2003;23:85–89. 18. ahmed n, kazmi s, nawaz h, javed m, anwar sa, alam ma. frequency of diabetes mellitus in patients with acute coronary syndrome. j ayub med coll abbottabad. 2014;26:57–60. 19. mittal a, gupta md, meennahalli palleda g, vyas a, tyagi s. relationship of plasma adiponectin levels with acute coronary syndromes and coronary lesion severity in north indian population. isrn cardiol. 2013;2013:854815. 20. libby p, ridker pm, maseri a. inflammation and atherosclerosis. circulation. 2002;105:1135–1143. 21. tsimikas s, witztum jl. measuring circulating oxidized low-density lipoprotein to evaluate coronary risk. circulation. 2001;103:1930–2. 22. ehara s, ueda m, naruko t, haze k, itoh a, otsuka m, et al. elevated levels of low-density lipoprotein show a positive relationship with the severity of acute coronary syndromes. circulation. 2001;103:1955–60. table 4. comparison between diabetic and non-diabetic acute coronary syndrome patients group and healthy control group in the measured parameters using anova test parameters patient with acs control (30) mean ± sd p-value d.m (26) mean ± sd non d.m (32) mean ± sd number 26 32 30 age 59.53 ± 8.72 57.12 ± 8.43 56.93 ± 9.018 0.480 bmi 27.8 ± 2.95 26.6 ± 2.6 27.01 ± 2.94 0.275 t.c 194.45 ± 76.15 201.4 ± 48.3 173.9 ± 33.313 0.125 tg 211.34 ± 102.12 189.93 ± 97.4 165.1 ± 56.666 0.149 ldl-c 121.32 ± 83.8 124.9 ± 47.3 99.319 ± 32.90 0.175 hdl-c 31.48 ± 6.98 38.5 ± 2.726 41.5 ± 6.163 0.000* vldl-c 42.2 ± 20.5 37.9 ± 19.47 33.036 ± 11.33 0.149 *highly significant; tg, triglycerides; tc, total cholesterol; hdl-c, high density lipoprotein cholesterol; sd, standard deviation; ldl-c, low-density lipoprotein-cholesterol; vldl, very low-density lipoprotein cholesterol; bmi, body mass index; p value derived from anova test; significant: p < 0.05; highly significant: p < 0.001; no significant: p > 0.05. 166 j contemp med sci | vol. 3, no. 9, winter 2017: 163–166 impact of adiponectin and oxidized low-density research batool luay aziz et al. 23. tsimikas s, bergmark c, beyer rw, patel r, pattison j, miller e, et al. temporal increases in plasma markers of oxidized-low-density lipoprotein strongly reflect the presence of acute coronary syndromes. j am coll cardiol. 2003;41:360–370. 24. napoli c, quehenberger o, denigris f, abete p, glass ck, palinski w. mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells. faseb j. 2000;4:1996–2007. 25. huang h, mai w, liu d, hao y, tao j, dong y. the oxidation ratio of ldl: a predictor for coronary artery disease. disease markers. 2008;24:341–349. 26. fraley ae, sotirios t. clinical applications of circulating oxidized lowdensity lipoprotein biomarkers in cardiovascular disease. curr opin lipidol. 2006;17:502–509. 27. huang y, hu y, mai w, cai x, song y, wu y, et al. plasma oxidized low-density lipoprotein is an independent risk factor in young patients with coronary artery disease. dis markers. 2011;31:295–301. 28. huang h, ma r, liu d, liu c, ma y, mai w, et al. oxidized low-density lipoprotein cholesterol and the ratio in the diagnosis and evaluation of therapeutic effect in patients with coronary artery disease. disease markers. 2012;33:295–302. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 327j contemp med sci | vol. 8, no. 5, september-october 2022: 327–328 original femoral and popliteal artery pathway in varus and valgus aligned lower limb; a ct angiographic study seyed morteza kazemi1, sohrab keyhani1, mohamad qoreishi2, saman shakeri jousheghan2*, reza minaei noshahr1* 1bone joint and related tissues research center, akhtar orthopedic hospital, shahid beheshti university of medical sciences, tehran, iran. 2clinical research & development unit, akhtar hospital, shahid beheshti university of medical sciences, tehran, iran. *correspondence to: both authors fulfill the criteria of shared correspondence saman shakeri jousheghan, reza minaei noshahr (email: dr.saman.shakeri.1989@gmail.com) (submitted: 09 march 2022 – revised version received: 12 april 2022 – accepted: 21 april 2022 – published online: 26 october 2022) abstract objectives: the aim of this study was to compare the femoral and popliteal arteries pathway in varus and valgusaligned lower limbs. methods: a retrospective ct angiography (cta) study from october to december 2021 was conducted. distance of the femoral and popliteal artery to specific bony landmarks in thigh and knee was measured. results: eighty limbs including 40 varus and 40 valgus lower limbs were assessed. no significant difference between varus/valgus groups in terms of distances was noted. conclusion: our study compared pathway of pa and fa artery in varus and valgus knees using cta images; and no significant differences was seen between the two groups of varus and valus. keywords: femoral artery, popliteal artery, anatomy, varus alignment, valgus alignment, knee issn 2413-0516 introduction determining the pathway of the femoral and popliteal artery (fa and pa) is an essential step in performing surgical or radiological procedures. fa exits from the adductor canal then entering popliteal fossa posterior to the tibia plateau turning into popliteal artery.1-3 the pathway might vary in accordance with lower limb different alignments-varus or valgusin individuals. procedures in the area of distal femur or proximal tibia could be risky in terms of vascular complications as the adjacency of the arteries to the surgical cuts has been incompletely addressed in literature. limited studies are available to quantitatively describe the anatomic location of the fa and popliteal artery in relation to the shaft of the femur and tibia for a surgical approach guide in such region.4-6 the aim of this study was to compare the femoral and popliteal arteries pathway in varus and valgusaligned lower limbs. methods we carried out a retrospective study reviewing archived data images of patients aged 18–60 yrs undergoing lower limbs ct angiography (cta) from october to december 2021. the study design was approved by shahid beheshti university of medical sciences ethics research board. cases with pelvic, thigh and tibia fractures, and low-quality images were excluded. sixty cases -80 limbs: 40 varus/40 valgus-were assessed using two-dimensional (2d) and three-dimensional (3d) reconstructions cta (16-slice ct scanner, siemens, germany 2018). the perpendicular distance of the fa to medial, lateral and posterior cortex at the adductor tubercle and knee medial joint line was measured. the distances of the popliteal artery to the medial, lateral and posterior border of proximal tibia were also measured in axial images. parametric variables were given as mean ± sd; variables were analyzed with student t-test. nonparametric variables were reported as a median and range. the statistically significant threshold was p < 0.05. results eighty limbs including 40 varus (32 male, 8 females) and 40 valgus (19 males, 21 females) were assessed; mean age of participants was 41 ± 16 years (20–59 yr range) were included. there was detected no significant difference between varus/ valgus groups in terms of distances. also no statistically significant difference by age, limb side and sex were seen (p = 0.31). the average distance of the fa to the femoral cortex was 15.13/14.11 mm posteriorly, 26.30/27.40 mm medially, and 33.17/33.14 mm laterally at adductor tubercle level (varus/ valgus). the average distance for the popliteal artery to the tibial cortex were 5.68/5.34 mm posteriorly, 21.75/21.25 mm medially, and 13.85/13.25 mm laterally at fibular head level (varus/valgus). other distances to the specific anatomic levels are reported in table 1. no significant differences were noted between the two varus and valgus groups (p > 0.05). discussion damage to the neurovascular structures is a major operative complication of around the knee procedures.7-9 knowing the pathway of arteries in lower limbs espacially in both varus and valgus limbs could be helpful; the most important finding of our study was to compare distances of femoral and popliteal arteries to bony landmarks in both varus and valgus knees. no similar study was available to compare the results; only some reports have given limited data; a report9 showed that any kind of osteotomies occurred in knee could impose the risk of vascular injuries. another study evaluated cta in cadavers after the mipo interventions and found no disruptions in deep and superficial femoral arteries.10 previous studies assessed 2-d cta and reported the fa had at least 12 mm distance to the medial cortex of femur through its way;6 also reported that fa mailto:dr.saman.shakeri.1989@gmail.com 328 j contemp med sci | vol. 8, no. 5, september-october 2022: 327–328 fa and pa pathway in varus and valgus aligned lower limb original s.m. kazemi et al. was placed about 16 mm far from the borders of distal femur at minimum.11 a mri-based study detailed that the average distance between the pa and the tibia posterior cortex at the level of the knee joint was about 9.5 mm.12 being familiar with the pathway of the femoral and popliteal artery especially in both knee alignments -varus or valgus is important for managing fractures.1,2,8 the adductor tubercle, medial femoral condyle and fibular head would be imperative bony indices which are distinguished on imagings, so accurate orientations to the artery distance o such landmarks help surgeons decrease complications in operations.4,6,11-13 conclusion this study was focused on comparing pathway of femoral and popliteal artery in distal thigh and around the knee in both varus and valgus knees using cta images; no significant differences was reported between the two groups in our findings. conflicts of interest none.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. table 1. average distance of femoral and popliteal artery to considered anatomic points anatomic level distance to midline posterior cortex (mm) distance to posterior medial border cortex (mm) distance to posterior lateral border cortex (mm) p varus valgus varus valgus varus valgus femur just above adductor tubercle 15.13 ± 4.23 14.11 ± 2.13 26.30 ± 3.47 27.40 ± 3.14 33.17 ± 2.02 33.14 ± 3.02 0.33 level of knee joint line 3.23 ± 3.41 3.18 ± 2.31 21.20 ± 3.04 21.20 ± 3.04 19.25 ± 2.51 19.35 ± 3.22 0.29 tibia at the level of fibular head 5.68 ± 2.45 5.34 ± 2.62 21.75 ± 3.40 21.25 ± 3.31 13.85 ± 2.32 13.25 ± 2.65 0.30 tibia at the level of 5 cm below the knee joint line 10.58 ± 3.45 11.15 ± 3.15 19.55 ± 2.57 19.75 ± 2.45 12.30 ± 2.90 12.55 ± 2.70 0.28 references 1. shim s-s, leung g. blood supply of the knee joint. a microangiographic study in children and adults. clinical orthopaedics and related research. 1986(208):119–25. 2. olewnik ł, łabętowicz p, podgórski m, polguj m, ruzik k, topol m. variations in terminal branches of the popliteal artery: cadaveric study. surgical and radiologic anatomy. 2019;41(12):1473–82. 3. ricciardi a. thieme atlas of anatomy: general anatomy and musculoskeletal system. the yale journal of biology and medicine. 2015;88(1):100. 4. henry ak. extensile exposure: edinburgh; 1957. 5. hoppenfeld s, deboer p, buckley r. surgical exposures in orthopaedics: the anatomic approach: lippincott williams & wilkins; 2012. 6. kim j, allaire r, harner cd. vascular safety during high tibial osteotomy: a cadaveric angiographic study. the american journal of sports medicine. 2010;38(4):810–5. 7. visser j, brinkman j-m, bleys r, castelein r, van heerwaarden r. the safety and feasibility of a less invasive distal femur closing wedge osteotomy technique: a cadaveric dissection study of the medial aspect of the distal femur. knee surgery, sports traumatology, arthroscopy. 2013;21(1):220–7. 8. narulla rs, kanawati aj. safe zone for the superficial femoral artery demonstrated on computed tomography angiography. injury. 2016;47(3):748–51. 9. bisicchia s, rosso f, pizzimenti ma, rungprai c, goetz je, amendola a. injury risk to extraosseous knee vasculature during osteotomies: a cadaveric study with ct and dissection analysis. clinical orthopaedics and related research®. 2015;473(3): 1030–9. 10. jiamton c, apivatthakakul t. the safety and feasibility of minimally invasive plate osteosynthesis (mipo) on the medial side of the femur: a cadaveric injection study. injury. 2015;46(11):2170–6. 11. kim jj, oh hk, bae j-y, kim jw. radiological assessment of the safe zone for medial minimally invasive plate osteosynthesis in the distal femur with computed tomography angiography. injury. 2014;45(12):1964–9. 12. goes rfda, cardoso filho a, castro gnpdo, loures fb, palma imd, kinder a, et al. magnetic resonance study on the anatomical relationship between the posterior proximal region of the tibia and the popliteal artery☆. revista brasileira de ortopedia. 2015;50: 422–9. 13. iacono f, lo presti m, bruni d, raspugli gf, bignozzi s, sharma b, et al. the adductor tubercle: a reliable landmark for analysing the level of the femorotibial joint line. knee surgery, sports traumatology, arthroscopy. 2013;21(12):2725–9. https://doi.org/10.22317/jcms.v8i5.1280 178 j contemp med sci | vol. 3, no. 9, winter 2017: 178–181 research measurement of urinary kidney injury molecule-1 as a predictive biomarker of contrast-induced acute kidney injury fadhil jawad al-tu’ma,a maha hamood dheyauldeen,a* and ryiadh mohi al-saeghb adepartment of biochemistry, college of medicine, university of kerbala, iraq. bdepartment of nephrology, college of medicine, university of kerbala, iraq. *correspondence to maha hamood dheyauldeen (email: maha.diaaldeen12@gmail.com). (submitted: 18 december 2017 – revised version received: 13 january 2017 – accepted: 12 february 2017 – published online: 26 march 2017 ) objective the aim of the present study is to evaluate the urinary kim-1 level in the patients after 24 h angiography as a predictive biomarker of contrast-induced acute kidney injury. methods this study included 80 selective patients attending in the cardiology unit (48 males, 32 females). the study was conducted in the cardiac catheterization unit at alhussein medical city/ kerbala. clinical examination and laboratory investigations were made before and 24 h after angiography, these investigations include: serum creatinine, blood urea and estimated gfr. urinary kim-1 was measured before and after 24 h angiography. results there was no significant difference in urinary kim-1 when compared between cin and non cin group p > 0.05. the level of urinary kim-1 increased in the patients after 24 h of angiography when compared with baseline level of p < 0.001. conclusion urinary kim-1 was not useful for predicting or detecting cin. but urinary kim-1 level may be useful as a biomarker for tubular damage following intravascular administration of contrast media, 24 h. keywords kidney injury molecule-1, contrast-induced acute kidney injury, coronary angiography, percutaneous coronary intervention introduction the administration of radiographic contrast remains an important cause of acute kidney injury in hospital, which contributes to mortality during hospitalization.1 one of the most common complications of percutaneous coronary intervention (pci) and other type such as diagnostic procedures of the heart is contrast-induced acute kidney injury (ci-aki).2 ci-aki is defined as an increase in serum creatinine of 0.5 mg/dl (44 μmol/l), or a relative 25% increase from the baseline value within 24–48 h following intravascular administration of contrast media.3 the incidence of ci-aki varies widely depending on the type of angiographic procedures and the amount of contrast media.4 using iodinated contrast also allows ci-aki to be one of the few causes of acute kidney injury.5 the incidence of ci-aki also depends on the baseline characteristics of the patient. patients with normal renal function before the injection of contrast media are low (10%). however, the incidence may increase in the patients with risk factors, such as diabetes mellitus and impaired renal function are at particular risk for the development of ci-aki.6 there are some biomarkers serum and urinary have been recently proposed to serve as early detection of acute kidney injury after cardiac catheterization such as cystatin c, kidney injury molecule-1.7 kidney injury molecule-1 (kim-1) is transmembrane protein type-1. it is noted that kim-1 was undetectable in the normal kidney.8 but it abundantly expressed in proximal tubular cells after ischemic and nephrotoxic injury.9 while its role remains unclear, it consists in a transmembrane protein whose ectodomain is shed into the urine where it can be measured.10 it is increased in the urine in acute kidney injury and kidney transplant patients. recently, systematic reviews had been reported that kim-1 was an efficient novel biomarker urinary in diagnosis of acute kidney injury within period 24 h after a kidney injury. urinary kim-1 levels are associated with higher mortality risk in patients with overt kidney disease or heart failure, but there is limited evidence of an association between kim-1 and cardiovascular mortality in the community.11 the aim of the present study to evaluate the urinary kim-1 level in patients after 24 h angiography as a predictive biomarker of ci-aki. materials and methods the study was carried out on (80) patients attending in the catheterization unit for elective coronary interventions (cag) (48 male, 32 female) between ranges (40–81). in which the same patients were included before angiography (cag and pci) and after 24 h of angiography was conducted at department of biochemistry, college of medicine, university of kerbala and coronary catheterization unit (ccau) in alhussein teaching hospital / al-hussein medical city, kerbala health directorate / holly kerbala-iraq from (nov. 2015 to oct 2016). the medical history of each patient was taken regarding age, gender, diabetes mellitus, type of treatment, history of renal disease, history of any other diseases, and smoking status. measurements of their height and weight were done to calculate their body mass index (bmi), blood pressure before and during angiography. ci-aki was defined as an increase in serum creatinine of 0.5 mg/dl or > 25% increase from the baseline values, assessed at 24 h after the procedure. the amount of contrast medium used in each patient was recorded after the procedure. all patients involved in the study were given low-osmolar non-ionic contrast media omnipaque (iohexol) 350 mg (1/ml), 844 osmality (mosm/l). serum creatinine, serum urea and egfr were assessed before and at 24 h after administration of contrast medium. urinary kim-1 was measured before and after 24 h angiography. estimated gfr was calculated according to the modification of diet in renal disease issn 2413-0516 mailto:maha.diaaldeen12@gmail.com maha hamood dheyauldeen et al. 179j contemp med sci | vol. 3, no. 9, winter 2017: 178–181 research measurement of urinary kidney injury molecule-1 (mdrd) equation: egfr (ml/min/1.73 m2) = 186 × (scr−1.154) × (age−0.203) × (0.742 if female) × (1.21 if black).12 urine sample was collected before and after 24 h operation and centrifuge 4000 (rpm), stored at −70 °c until assayed. the urinary kim-1 measurements were performed using sandwich enzyme-linked immunosorbent (elisa) assays (human kim-1 elisa kit, cosabio, china). the ethical committees approved this study protocol are: committees of college of medicine, university of karbala and department of medicine and committee of cardiac angiographic unit in alhussein teaching hospital / al-hussein medical city, karbala health directorate / holly kerbela-iraq . statistical analysis statistical analyses were performed by using the statistical package for social sciences (spss) software for windows, version 21, ibm, usa, data of 80 patients entered and analyzed. descriptive statistics were presented as mean ± standard deviation for continuous variables. categorical variables were expressed as frequency (no.), and percentages (%). student’s t-test was used to compare and assess the significance of differences between continuous variables. paired t-test used to compare urinary kim-1 levels between before and after operation. chi square or fisher exact test as appropriate test was used to assess the significance of the association between categorical variables. receiver–operating characteristic (roc) curves and the area under the curve (auc) were constructed to describe the performance of serum creatinine and urinary kim-1 at 24 h post-contrast media exposure. the sensitivity and specificity were calculated. the best cutoff values for biomarkers were chosen on the basis of maximum sensitivity and specificity. probability (p) value was considered significant when it is less than 0.05%, and highly significant if it is less than 0.01%. results the study subjects included 80 patients, 48 of them were male (60%) and 32 were female (40%) with a mean age of 59.85 ± 9.976 and age range of 41–80 years. all patients underwent elective cag or pci. according to the presented data and to the definition ci-aki, ci-aki occurred in 19 patients 23.8%. fifty three patients have past history systemic hypertension whereas 27 were not hypertensive. seventeen patients were current smokers; and 63 patients were not. patient’s characteristics are shown in tables 1 and 2; there was significant in the type of angiography cag/pci in the incidence of ci-aki. there were no significant effects for age, gender, smoking, diabetes mellitus, hypertension, statin medication and others, volume of contrast and ejection fraction in the incidence of ci-aki. table 3 shows the changes in the concentrations of different biomarkers in both cin and non cin groups. there were significant increments in levels of scr and egfr after 24 h of angiography/angioplasty procedures p < 0.001, 0.01 respectively. there was no significant in urinary kim-1 when compared between in both cin and non cin group p > 0.05. table 4 shows significant rise in urinary kim-1 level when compared between before and after 24 h angiography p < 0.001. the result of receiver-operating characteristics (roc) analysis (n = 80, cin = 19) shown in the (fig. 1) showed a higher area under the curve (auc) for serum creatinine 0.805 (95% ci = 0.688, 0.921). p = 0.000), 78.9% sensitivity and 60.7% specificity, cut off value >1.06 mg/dl. auc of egfr 0.739 (95% ci = 0.595, 0.882), sensitivity = 73.7 % and specificity = 62.3%, cut-off value < 63 ml/kg/1.73 m2. auc of urea 0.670 (95% ci = 0.525, 0.816) sensitivity 63.2% and specificity 63.9 % and cut-off value > 39 mg/dl. auc of urinary kim-1 0.572 (95% ci = 0.429, 0.714) p = 0.348, cutoff value > 0.46 ng/ml, sensitivity 52.6%, specificity 50.8% discussion in the present study, the incidence of cin occurred in 19 (2, diagnostic; 17 therapeutic) from 80 patients with 23.8% although all of patients have normal kidney function. the table 1. characteristics in (percentages and numbers) of the patients, groups: with and without cin characteristics cin p-value negative positive gender (male/female ) number 35/26 13/6 0.391 % 72.9/81.2 27.1/18.8 non-smoker/ current smoker no. 50/11 13/6 0.208 % 79.4/64.7 20.6/35.3 non-statins/statins no. 14/47 6/13 0.448 % 70/78.3 30/21.7 non-β-blocker/ β–blocker no. 23/38 11/8 0.120 % 67.6/82.6 32.4/17.4 non-ace/ace inhibitors no 49/12 14/5 0.536 % 77.8/70.6 22.2/29.4 angiography (diagnostic/therapeutic) no. 25/36 2/17 0.014* % 92.6/67.9 7.4/32.1 no, number; cin: contrast-induced nephropathy; *significant p value at 0.05. table 2. characteristics of the patients groups: with and without cin characteristics cin + (n = 19) cin − (n = 61) p-value mean ± sd mean ± sd age (years) 62.58 ± 10.62 59.0 ± 9.69 0.174 bmi (kg/m2) 27.10 ± 3.59 28.41 ± 5.23 0.421 sbp (mmhg) 136.23 ± 22.65 132.79 ± 16.84 0.466 dbp (mmhg) 81.05 ± 11.49 80.66 ± 11.56 0.896 fbs (mg/dl) 197.54 ± 68.46 164.23 ± 76.42 0.094 t c (mg/dl) 175.64 ± 35.09 170.92 ± 43.44 0.668 tg (mg/dl) 165.58 ± 56.69 170.61 ± 81.80 0.804 vldl-c (mg/dl) 37.56 ± 16.79 35.43 ± 17.09 0.635 ldl-c (mg/dl) 102.72 ± 41.95 101.02 ± 38.60 0.870 hdl-c (mg/dl) 33.96 ± 6.56 35.94 ± 8.46 0.354 volume of contrast 265.78 ± 111.86 220.08 ± 125.73 0.160 ef 53.47 ± 8.07 57.52 ± 10.35 0.122 cin +, cin positive; cin −, cin negative index; sbp, systolic blood pressure; dbp, diastolic blood pressure; fbs, fasting blood sugar; ef, ejection fraction. 180 j contemp med sci | vol. 3, no. 9, winter 2017: 178–181 measurement of urinary kidney injury molecule-1 research maha hamood dheyauldeen et al. results in the present study showed even patients with normal kidney function have the development of ci-aki after exposure to contrast media; this finding is agreed with assareh et al.13 recent study carried by sato et al., showed that cin was an independent predictor of subsequent renal events in the patients who underwent cardiac catheterization.14 the risk of ci-aki due to coronary angiography increases dramatically with low kidney function.15 the present study shows that urinary kim-1 level was elevated at the 24 h of cardiac catheterization. there was a statistically significant rise in the urinary level kim-1 p < 0.001 in patients when compared level concentration between pre and post-operation. this result may be referred to proximal tubular injury by contrast medium, although the classical marker kim-1 was not significant when compared between positive and negative cin p > 0.05. therefore, urinary kim-1 was not useful for predicting or detecting cin. these results are due to tubular kidney damage, where very high concentrations appeared in some patients of different conditions may cause tubular damage including diabetes, infections, surgery, heart failure and treatments with nephrotoxic agents of contrast media.16 measurement of urinary biomarkers has the potential to determine the risk of renal damage that may raise the level of urinary at 24 h after exposure contrast media. this could provide a basis for using urinary kim-1 level in clinical practice. there are some studies used urinary kim-1 level for early diagnosis cin as akdeniz et al. found that at 6th and 48th hour of contrast exposure found that kim-1 levels increased in the patients with cin (p < 0.01; median levels, 0.27 and 0.70 mg/dl).17 another report by malyszko et al. found no data in the literature on such time course changes of kim-1in contrast nephropathy as in our study.18 another work carried by kooiman et al. urinary kim-1 excretion was unaffected by ce-ct both in patients with and without ci-aki, suggesting that ci-aki was not accompanied by tubular injury.19 urinary kim-1 is a more recently described type 1 cell membrane glycoprotein that is expressed in humans when the injured renal proximal tubule of the kidney, kim-1 not detectable in the normal kidney but expresses in proximal tubular cells after ischemic and nephrotoxic injury.20 this may increase levels urinary after 24 h without any change before exposure of contrast media. perhaps some of our patients had conducted two operations during a shorter time and take two doses of contrast media. effects of contrast media on the kidney are known to induce damage of endothelial cells by direct cytotoxicity or the viscosity of the contrast media and perhaps lead to the development of renal hypoxia.21 this will expose their kidneys to a double risk that may explain the transient proximal tubular damage by contrast medium. the increase of the ectodomain sheds from cells into the urine shortly after proximal tubular kidney injury allows kim-1 to serve as an early sensitive urinary biomarker in kidney injury detection.22 therefore urinary excretion of kim-1 may be clinically recognized as a useful biomarker of renal injury after angiography. roc analysis shows a higher auc for scr 0.805 (95% ci = 0.688, 0.921) sensitivity = 78.9% and 60.7% = specificity, than urinary kim 0.572 (95% ci = 0.429, 0.714) p = 0.348, cut-off value > 0.46 ng/ml, sensitivity 52.6%, specificity 50.8%. these findings refer to serum creatinine better than urinary kim-1 in early detection of ci-aki. table 3. changes of serum and urinary biomarkers between cin and non cin groups markers cin + n = (19) mean ± sd cin − n = (61) mean ± sd p-value s. urea (mg/dl) at 0 h 36.54 ± 12.91 31.64 ± 7.03 0.036* after 24 h 50.20 ± 25.06 39.20 ± 13.23 0.015* s. creatinine (mg/dl) at 0 h 0.77 ± 0.22 0.88 ± 0.18 0.037* after 24 h 1.30 ± 0.29 0.98 ± 0.19 0.000*** egfr at 0 h 102.47 ± 43.04 82.52 ± 19.94 0.006** after 24 h 56.57 ± 18.42 72.11 ± 15.89 0.001** u. kim-1 (ng/ml) at 0 h 0.36 ± 0.42 0.36 ± 0.31 0.962 after 24 h 0.81 ± 0.71 0.72 ± 0.79 0.645 s, serum; egfr, glomerular filtration rate; u, urinary; kim-1, kidney injury molecule-1. table 4. change in serum and urinary levels of biomarkers between pre-post angiography biomarkers pre post 1-day p-value s.urea (mg/dl) 32.80 ± 8.94 41.81 ± 17.32 0.000*** s.creatinine (mg/dl) 0.85 ± 0.19 1.06 ± 0.25 0.000*** egfr 87.26 ± 28.23 68.42 ± 17.70 0.000*** u. kim-1 (ng/ml) 0.36 ± 0.34 0.74 ± 0.77 0.000*** s, serum; u, urinary; kim-1, kidney injury molecule-1; egfr, glomerular filtration rate. fig. 1 roc of serum and urinary biomarkers after 24 h angiography. auc for serum creatinine 0.805 (95% ci = 0.688, 0.921), 78.9% sensitivity and 60.7% specificity, cut off value >1.06 mg/dl. auc of urinary kim-1 0.572, cutoff value > 0.46 ng/ml, sensitivity 52.6%, specificity 50.8%. maha hamood dheyauldeen et al. 181j contemp med sci | vol. 3, no. 9, winter 2017: 178–181 research measurement of urinary kidney injury molecule-1 in the present study, serum creatinine is still the standard marker for detecting temporal changes of renal function in individuals with established renal disease. creatinine is the use of a sign inexpensive to measure kidney function when compared with other recent marker, which is available in all laboratories and can be easily measured in comparison with other vital signs. conclusion urinary kim-1 was not useful for predicting early ci-aki. urinary kim-1 was only useful biomarker of tubular damage caused by contrast medium after 24 h cag and pci. acknowledgments great thanks are due to all members of the medical staff of catheterization unit in the cardiological center in al hussein teaching hospital/al-hussein medical city. also i want to thank dr. mohammed mahdi and all members of medical staff of laboratory unit in al-hussein teaching hospital. conflict of interest none n references 1. tanaga k, tarao k, nakamura y, inoue t, jo k, ishikawa t, et al. percutaneous coronary intervention causes increase of serum cystatin c concentration even in the patients with a low risk of contrast-induced nephropathy. cardiovasc interv ther. 2012;27:168–173. 2. brown jr, rezaee me, nichols el, marshall ej, siew ed, matheny me. incidence and in‐hospital mortality of acute kidney injury (aki) and dialysis‐requiring aki (aki‐d) after cardiac catheterization in the national inpatient sample. j am heart assoc. 2016;5:e002739. 3. cely cm, schein rm, quartin aa. risk of contrast induced nephropathy in the critically ill: a prospective, case matched study. critical care. 2012;16:1. 4. malyszko j, bachorzewska-gajewska h, poniatowski b, malyszko js, dobrzycki s. urinary and serum biomarkers after cardiac catheterization in diabetic patients with stable angina and without severe chronic kidney disease. renal failure. 2009;31:910–919. 5. parfrey p. the clinical epidemiology of contrast-induced nephropathy. cardiovasc interv radiol. 2005;28:s3–11. 6. palevsky pm. defining contrast-induced nephropathy. clin j am soc nephrol. 2009;4:1151–1153. 7. morcos sk. prevention of contrast media–induced nephrotoxicity after angiographic procedures. j vasc interv radiol. 2005;6:13–23. 8. hojs r, ekart r, bevc s, hojs n. biomarkers of renal disease and progression in patients with diabetes. j clin med. 2015;4:1010–1024. 9. devarajan p. biomarkers for the early detection of acute kidney injury. curr opin pediat. 2011;23:194. 10. vaidya vs, ferguson ma, bonventre jv. biomarkers of acute kidney injury. ann rev pharmacol toxicol. 2008;48:463. 11. carlsson ac, larsson a, helmersson-karlqvist j, lind l, ingelsson e, larsson te, et al. urinary kidney injury molecule-1 and the risk of cardiovascular mortality in elderly men. clin j am soc nephrol. 2014;cjn–11901113. 12. levey as, stevens la, schmid ch, zhang yl, castro af, feldman hi, et al. a new equation to estimate glomerular filtration rate. ann intern med. 2009;150:604–612. 13. assareh a, yazdankhah s, majidi s, nasehi n, mousavi ss. contrast induced nephropathy among patients with normal renal function undergoing coronary angiography. j renal inj prev. 2016;5:21. 14. sato a, aonuma k, watanabe m, hirayama a, tamaki n, tsutsui h, et al. association of contrast-induced nephropathy with risk of adverse clinical outcomes in patients with cardiac catheterization: from the cinc-j study. international journal of cardiology. 2017 jan. 15;227:424–9. 15. stacul f, van der molen aj, reimer p, webb ja, thomsen hs, morcos sk, et al. contrast-induced nephropathy: updated esur contrast media safety committee guidelines. eur radiol. 2011;21:2527–2541. 16. helmersson-karlqvist j, ärnlöv j, carlsson ac, lind l, larsson a. urinary kim-1, but not urinary cystatin c, should be corrected for urinary creatinine. clin biochem. 2016;49:1164–1166. 17. akdeniz d, celik ht, kazanci f, yilmaz h, yalcin s, bilgic ma, et al. is kidney injury molecule 1 a valuable tool for the early diagnosis of contrast-induced nephropathy? j invest med. 2015;63:930–934. 18. malyszko j, bachorzewska-gajewska h, poniatowski b, malyszko js, dobrzycki s. urinary and serum biomarkers after cardiac catheterization in diabetic patients with stable angina and without severe chronic kidney disease. renal failure. 2009;31:910–919. 19. kooiman j, pasha sm, zondag w, sijpkens yw, van der molen aj, huisman mv, et al. meta-analysis: serum creatinine changes following contrast enhanced ct imaging. eur j radiol. 2012;81:2554–2561. 20. perico n, cattaneo d, remuzzi g. kidney injury molecule 1: in search of biomarkers of chronic tubulointerstitial damage and disease progression. am j kidney dis. 2009;53:1–4. 21. kwok cs, pang cl, yeong jk, loke yk. measures used to treat contrast-induced nephropathy: overview of reviews. br j radiol. 2013;86:20120272. 22. huang y, craig don-wauchope a. the clinical utility of kidney injury molecule 1 in the prediction, diagnosis and prognosis of acute kidney injury: a systematic review. inflam allergy drug targets. 2011;10:260–271. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 115j contemp med sci | vol. 8, no. 2, march-april 2022: 115–119 original histological effect of artichoke leaf extract on bone healing in rats nawar bahjet kamil1*, sura saad majeed2, mohammed a. salman3 1department of oral diagnosis, college of dentistry, university of baghdad, iraq. 2department of dentistry, al-rafidain university college, baghdad, iraq. 3urosurgeon, al kindy teaching hospital, ministry of health, baghdad, iraq. *correspondence to: nawar bahjet kamil (e-mail: nawar.bahjat@codental.uobaghdad.edu.iq) (submitted: 06 january 2022 – revised version received: 21 january 2022 – accepted: 10 march 2022 – published online: 26 april 2022) abstract objectives: this study evaluated the effect of artichoke leaves extract on bone healing. methods: a total of 30 rats were used in the current study, 60 bone defects were made, divided into 30 bone defects left without treatment and an other 30 bone defects were treated with 0.5 ml of artichocke oil. each group was divided randomly into three period intervals (1 week, 2 and 5 weeks). results: current study had showed a high significant difference between the control and the experimental group in osteoclast cells account, a significant difference in osteoblast and osteocyte cells account. also there was a high significant difference in the formation of the blood vessels and the trabecular bone between the experimental and the control group. conclusion: artichoke leaves extract was effective and faster in bone healing without any complication in comparison with the control group. keywords: artichoke oil, bone healing issn 2413-0516 introduction the bone is a hard structure that forms the vertebrates skeletal framework. it consists of an organic matrix that is composed of about 40% collagen type 1, water and inorganic minerals which is composed of hydroxylapatite. the bone is formed by osteoblasts and when the bone is formed some of them will be embedded in the bone and become osteocytes.1 bone development happens in two ways, intra membranous and endochondral ossification (ossification is a process of bone formation). the formation of woven bone happens by the replacement of the collagenous mesenchymal tissues by bones.2 the woven bone is considered as a primitive form of the bone with a randomly organized collagenous fibers and by remodeling it is converted into a mature lamellar bone. the lamellar bone can possesses a regular parallel multiple rings of collagen, which is then remodeled by the osteoblasts and osteoclasts.3 bone healing there are three stages of bone healing which includes stage of inflammation, repair stage and the remodeling stage.4 inflammatory phase occurs immediately following a fracture and is characterized by the formation of the hematoma.5 within the first few days after the injury, the levels of interleukin-1 (il-1), (il-6), (il-11), (il-18) and the tumor necrosis factor-α (tnf-α) are elevated.6 the proliferative phase is a second phase of healing which is characterized by the formation of the callus and it begins with a continued vascular ingrowth, the secretion of the osteoid and resorption of necrotic bone which is carried out by the osteoclasts,7 and it is also characterized by the formation of the connective tissue, angiogenesis and the soft callus which is then replaced by the immature woven bone that is formed via the intramembranous or the endochondral bone formation.8 the remodeling phase is the third phase characterized by the formation and the mineralization of the callus and replaced with a mineralized bone and return the bones to its original size and shape through the remodeling.9 artichoke belongs to the family of asteraceae, which represents an important part of the mediterranean diet. it is considered as a rich source of a bioactive phenolic compound.10 it had been used widely in medicine because of its health benefits which are due to its high content of phenolic compound and inulin.11 the extracts from the artichoke had been used for the hepato-protection. the artichoke is a very rich source of the dietary anti-oxidants and therefore it can be used in many phytopharmaceutical applications.12 materials and methods the artichoke plant material was taken and mixed with water. the mixture underwent heating process with a special vaporizing system. the vapor then passed through a cooling tubes for distillation process of the product. the essential oil product is then got separated from the mixture. the whole procedure was done in the chemical labs of the college of science. thirty male albino rats weighing (300–400 g) were used in the study. they were put in the animal house in a separated cage for each one under optimum heat and feeding conditions with an ethical approval from the college of dentistry university of baghdad. a bone defect with a 3 mm depth and 2 mm width was created in the right and left side of the femurs for each rat. the total bony defects (60) was divided into three period intervals 1, 2 and 5 weeks, each period interval included (10) control bone defects left to heal normally and (10) bone defects treated with (0.5 ml) artichoke oil. the whole procedure was done under general anesthesia that was given intramuscularly using ketamin hydrochloride (1 ml/kg of body weight) plus xylazin. the access openings were made on the left and right side of the femur by a microengine using a round bur until a hole of about 116 j contemp med sci | vol. 8, no. 2, march-april 2022: 115–119 histological effect of artichoke leaf extract on bone healing in rats original n.b. kamil et al. in the second week, the histological findings showed early formation of blood vessels and more osteocytes in the experimental group than the control group as shown in figure 2. in the fifth week, we can see clearly a reversal line in the experimental group, more blood vessels and a difference in osteocytes number between control and experimental group figure 3. statistical result osteoclasts as shown in table 1 artichoke oil group recorded a high significant difference at second and fifth week in reducing the number of osteoclast cells. osteoblast table 2 showing that an artichoke group recorded a high significant difference at the first week, also recorded significant difference at the fifth week than control group. fig. 1 (a) control group tb (trabecular bone) h&e x20. (b) control group ob (osteoblast) h&e x40. (c): experimental group showing tb (trabecular bone) h&e x20. (d) experimental group showing rl (reversal line), ob (osteoblast), oc (osteocyte) h&e x40. (2 mm diameter and 3 mm depth),13 then the bone defects were washed with normal saline to remove the bone fragments. in thirty rats, the left bone defects were left to heal normally, while the right bone defects were treated with 0.5 ml of artichoke oil. result clinical findings: there was no sign of any infection and all rats were healthy at the day of scarifying and at 5 weeks the experimental group showed higher stages of healing in the defect in comparison to the control group which showed incomplete healing. histological result in the first week, the histological study showed more trabecular bone formation in the experimental group than the control group and the woven bone was seen more clearly than the control group as shown in figure 1. 117j contemp med sci | vol. 8, no. 2, march-april 2022: 115–119 n.b. kamil et al. original histological effect of artichoke leaf extract on bone healing in rats fig. 2 (a) control group showing rl (reversal line), oc (osteocyte), bb (bundle bone), ob (osteoblast) h&e x20. (b) experimental group showing bv (blood vessel), oc (osteocyte), bb (bundle bone), ob (osteoblast) h&e x20. fig. 3 (a) control group showing bb (bundle bone), bv (blood vessel), oc (osteocyte) h&e x20. (b) experimental group showing re (resting line), bv (blood vessel) h&e x20. table 1. mean and p-value of osteoclast cells account period group no mean p-value 1 week control experimental 10 10 3.2 2.9 0.09 2 weeks control experimental 10 10 2.8 1.7 0.00 5 weeks control experimental 10 10 1.3 0.56 0.00 table 2. mean and p-value of osteoblast cells account period group no mean p-value 1 week control experimental 10 10 25.1 45.8 0.00 2 weeks control experimental 10 10 31.3 20.6 0.07 5 weeks control experimental 10 10 23.43 12.95 0.03 osteocyte table 3 reveled that there is a significant difference at the second and the fifth week in comparison with the control group in account of osteocyte cells. blood vessel table 4 showing that an artichoke oil group recorded a significant difference at first week and a high significant difference at the fifth week than control group. trabecular bone as shown in table 5 there is a significant difference at first and fifth week in experimental group in comparison with control group. discussion herbal therapy is still essential for about 75% to 80% of the world, especially in the developing countries. this is because herbal therapy is without any side effects, cheap and available.14 118 j contemp med sci | vol. 8, no. 2, march-april 2022: 115–119 histological effect of artichoke leaf extract on bone healing in rats original n.b. kamil et al. table 3. mean and p-value of osteocyte cells account period group no. mean p-value 1 week control experimental 10 10 21.5 28.65 0.08 2 weeks control experimental 10 10 26.32 43.26 0.03 5 weeks control experimental 10 10 37.81 54.3 0.02 table 4. mean and p-value of blood vessel formation period group no. mean p-value 1 week control experimental 10 10 0.1 0.9 0.04 2 weeks control experimental 10 10 0.45 1.1 0.06 5 weeks control experimental 10 10 1.2 3.6 0.00 the artichoke is a plant. its leaves, roots and stem are usually used to make “extracts.” “extracts” contain high concentrations of some chemicals that are found in the plant naturally. the artichoke leaf extract can be used alone or in combination with many other herbs. it contains little amount of fat and a higher levels of minerals like (sodium, potassium and phosphorus), fibres, vitamin c, polyphenols, inulin, hydroxycinnamates and flavones. polyphenols and inulin table 5. mean and p-value of trabecular bone formation period group no. mean p-value 1 week control experimental 10 10 12.15 18.33 0.02 2 weeks control experimental 10 10 8.9 10.64 0.09 5 weeks control experimental 10 10 6.3 1.9 0.04 possesses anticarcinogenic, hepatoprotective and antioxidant activities.15 artichoke leaf extract used in current study because it has an antioxidant that is accelerate healing by increased cell division,16 also artichoke oil has an anti-microbial effect due to phenolic contant,17 anti-inflammatory effect that is important to prevent inflammation further more accelerate healing.18 the current study showed that blood vessel formation is more accelerated in the experimental group than the control group due to the stimulation and the improvement of endothelial cells growth by using artichoke oil19 and the antioxidant effect due to the phenolic content.17 present study revealed that the trabecular bone formation was more accelerated in artichoke oil group than the control group because of the artichoke oil anti-inflammatory effect due to its polyphenolic and the verbascoside compounds that had been reported in many reports to show a potential spectrum of several activities including anti-inflammatory and antioxidant effects.20 osteoblast and osteocyte cells in the current study showed an early formation in the experimental group than the control group and this may be caused by the anti-inflammatory and antimicrobial effects of artichoke leaf extract17 and also because of the mineral composition which include calcium, phosphorous and sodium that lead to early maturation and formation of osteocyte.21 osteoclast cells had a shorter effect in the experimental group than the control group and this may be due to the cynarin content of artichoke leaf extract that has an immune modulator activity.22 conclusion artichoke leaf extract oil, which is a cheap and easy to get product, can accelerates bone healing by enhancing the osteoblast formation, early blood vessel formation and can reduce the osteoclast effect. conflicts of interest none.  references 1. safadi f., f., barbe m.,f., abdelmagid s., m., rico m.,c., aswad r.,a., litvin j., and popoff s., n. 2009. bone structure, development and bone biology . j.s. khurana (ed.), chapter 1:1-50. 2. vanputte cl, regan jl, russo af. 2013. skeletal system: bones and joints. in: seeley’s essentials of anatomy & physiology. 8th ed. usa: mc graw hill; pp. 110-149. 3. openstax college. 2013. anatomy & physiology. texas: rice university; pp. 203-231. 4. boden sd, schimandle jh, hutton wc.1995. an experimental intertransverse process spinal fusion model. radiographic, histologic, and biomechanical healing characteristics. spine 20:412–420. 5. mountziaris pm, mikos ag. 2008. modulation of the inflammatory response for enhanced bone tissue regeneration. tissue engineering part b: reviews; 14:179-86. 6. thompson dd. 2003. introduction-mechanisms of fracture healing and pharmacologic control. j musculoskel neuron interact; 3:295-6. 7. haverstock bd, mandracchia vj. 1998. cigarette smoking and bone healing: implications in foot and ankle surgery. j foot ankle surg; 37:69-74. 8. goldhahn j, fron jm, kanis j. 2012. implications for fracture healing of current and new osteoporosis treatments: an esceo consensus paper. calcified tissue international; 90:343-53. 9. schindeler a, mcdonald mm, bokko p. 2008. bone remodeling during fracture repair: the cellular picture. semin cell dev biol; 19:459-66. 10. lattanzio v, paul ak, vito l, angela c. 2009. globe artichoke: a functional food and source of nutraceutical ingredients. j funct foods; 1:131-44. 11. sonnante g, pignone d, hammer k. 2007. the domestication of artichoke and cardoon: from roman times to the genomic age. ann bot; 100:1095-1100. 12. ceccarelli n, curadi m, picciarelli p, martelloni l, sbrana c, giovannetti m. 2010. globe artichoke as functional food. mediterranean j nutr metab; 3:197-201. 13. al-ghaban n.m.h., jassem g.h. 2020. histomorphometric evaluation of the effects of local application of red cloveroil (trifolium pratense) on bone. j bagh. college dentistry, 32(2):26-31. 14. kamboj vp. 2000. herbal medicine. current science, 78:35-9. 15. bonomi a, bonomi bm, 2001. l’impiegodella farina di foglie di carciofodisidratate (cinara scolymus l.) nell’alimentazionedeivitelloni. rivista di scienzadellalimentazione. 30:361–370. 16. kraft k.1997. artichoke leaf extract recent findings reflecting effects on lipid metabolism, liver and gastrointestinal tracts. phytomedicine; 4(4):369-378. 119j contemp med sci | vol. 8, no. 2, march-april 2022: 115–119 n.b. kamil et al. original histological effect of artichoke leaf extract on bone healing in rats 17. xianfeng zhu, hongxun zhang, and raymond lo. 2004. phenolic compounds from the leaf extract of artichoke (cynara scolymus l.) and their antimicrobial activities. journal of agricultural and food chemistry 1;52(24):7272-8. 18. wauquier, f.; boutin-wittrant, l.; viret, a.; guilhaudis, l.; oulyadi, h.; bourafaiaziez, a.; charpentier, g.; rousselot, g.; cassin, e.; descamps, s. 2021. metabolic and anti-inflammatory protective properties of human enriched serum following artichoke leaf extract absorption. nutrient; 13:2653. 19. lupattelli g, marchesi s, lombardini r, roscini ar, trinca f, gemelli f. 2004. artichoke juice improves endothelial function in hyperlipemia. life sciences; 76(7):775-82. 20. schapoval e. e. s., winter de vargas m. r., chaves c. g., bridi r., zuanazzi j. a., henriques a. t. 1998. antiinflammatory and antinociceptive activities of extracts and isolated compounds from stachytarpheta cayennensis. journal of ethnopharmacology; 60(1):53–59. 21. wioletta biel, robert witkowicz , ewa piątkowska & cezary podsiadło. 2019. proximate composition, minerals and antioxidant activity of artichoke leaf extracts. biological trace element research; 194:589–595. 22. vincenzo lattanzioa, paul a. kroonb, vito linsalatac, angela cardinali. 2009. globe artichoke: a functional food and source of nutraceutical ingredients. journal of functional foods; 131–144. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i2.1200 270 j contemp med sci | vol. 3, no. 11, summer 2017: 270–272 original stature estimation from forearm length: an anthropological study in iranian medical students mahnaz poorhassan,a tahmineh mokhtari,b shadan navid,a maryam rezaei,c ardeshir sheikhazadi,c sina mojaverrostami,a gholamreza hassanzadeha adepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. bdepartment of anatomy, school of medicine, semnan university of medical sciences, tehran, iran. cdepartment of forensic medicine, school of medicine, tehran university of medical sciences, tehran, iran. correspondence to gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir). (submitted: 12 february 2017 – revised version received: 28 march 2017 – accepted: 05 may 2017 – published online: 26 september 2017) objective stature estimation is an important biological factor for forensic medicine to identify an individual. forearm length can be used for the prediction of the stature in different populations. in the present study, the relation between forearm length and height was evaluated. methods in a cross sectional study, a sample of 100 males and 100 females (aged 18 to 25 years) medical students from iranian population was randomly entered into the study. left forearm was measured by measuring tape. stature was measured in standard position. the linear regression analysis was used to estimate the relation between forearm length and the stature. results the mean age of subjects was 22 ± 2.21 years. mean age of male cases was 22 ± 2.8 years and female cases was 21.9 ± 1.81 years and there wasn’t significant difference in the age of sex groups (p = 0.314). a significant differences were recorded in the height and forearm length of subjects between two sex groups (p = 0.0001). there was a correlation between height and forearm length of all subjects (r = 0.643, p = 0.0001). according to the linear regression, there was a relation between height and upper arm length of subject in all cases. conclusion according to the results, forearm length was a moderate predictor for stature estimation of medical students in iranian population. keywords anthropology, stature, forearm length, iranians introduction identification of decomposed human body remains is an important goal in forensic sciences. age sex, stature and ancestry are biological characteristics of individuals which can be evaluated from skeleton remains of human many years after death.1–3 stature is affected by genes and environment and can show the racial differences in different populations.4 stature estimation can be performed from the long bones of limbs with the best results.5 using the body parts for predicting the biological characteristics of individuals can confirm the results of forensic identifications.6,7 in this way, anthropological studies on the body parts are widely used in criminal cases, terrorist attacks and natural disasters to identify victims.8,9 anatomical methods based on reconstruction of entire body can’t be useful for stature estimation. however, stature predicting from body parts found in these events can be performed by mathematical methods and linear regression equations can find the relation between body parts and stature.10–12 according to literature, the relation between stature and upper limb dimensions such as shoulder width, arm, forearm, hand, figures were studied in previous studies.13–19 in this study, the relation between forearm length and stature was evaluated by regression equation in iranian medical students. methods and materials in a cross-sectional study, from october 2014 to may 2015, 200 healthy subjects (100 males and 100 females, aged 18 to 25 years) were randomly selected from middle socio economic status population. the subjects with skeletal deformities or pathological changes were excluded. all measurements were performed in standard position by standard anthropological instruments and all dimensions were taken in the unit of 1 cm. for avoiding interpersonal errors, all measurements were performed by a single person in a fixed time from 1:00 pm to 3:00 pm. all measurements were repeated. stature estimation stadiometer was used for stature (standing height) measurement. all subjects were in standard anatomical position, and their head was held in the frankfort horizontal plane. stature was defined as maximum distance from vertex of subject to the floor. forearm length the standard measuring tape was used for forearm length measurement. forearm was in arm flexed position. forearm length was defined as distance from the tip of olecranon and mid-point between radius and ulnar tuberosity. left limb was used for estimations. statistical analysis mean ± standard deviation was used for descriptive data. t-test was used for finding the differences between two sex groups. in addition, the correlations between quantitative data were checked. linear regression equation analysis was used to find the relation between quantitative data. issn 2413-0516 gholamreza hassanzadeh et al. 271j contemp med sci | vol. 3, no. 11, summer 2017: 270–272 original stature estimation from forearm length in iranian medical students table 1. comparison of stature and forearm length in males and females in iranian medical students sex male female mean sd* max min sd* max min mean age 19.73 1.08 22 18 20.62 2.07 26 18 stature (cm) 180.02 5.73 196 171 162.37 5.76 176 150 forearm length (cm) 28.98 1.92 33 26 25.90 1.79 29 22 *standard deviation; max, maximum; min, minimum. table 2. linear regression for estimation of stature from upper arm length of medical students regression equation ± see r2 p-value s = 73.69 + 3.54 × forearm length (cm) 6.21 0.65 0.0001 s m = 118.916 + 2.108 × forearm length (cm) 4.08 0.501 0.0001 s f = 112.461 + 1.109268 × forearm length (cm) 4.64 0.36 0.0001 f, female; m, male; r2, coefficient of determination; see, standard error of estimate; s, stature. results the mean age of subjects was 22 ± 2.21 years. mean age of male cases was 22 ± 2.8 years and female cases was 21.9 ± 1.81 years and according to t-test analysis. there wasn’t a significant difference in the age of sex groups (p = 0.314). mean standing height of all subjects was 171.7 ± 10.19 cm. mean height of males and females was 180.48 ± 5.76 cm and 162.92 ± 4.42 cm, respectively. a significant differences were recorded in the height of subjects between two sex groups (p = 0.0001, and table 1). as table 1 shows, there was significant difference in the forearm length of sex groups (p = 0.0001). there was a correlation between height and forearm length of subjects (r = 0.643, p = 0.0001). there was a correlation between height and forearm length of subjects (r = 0.427, p = 0.002). however, this correlation wasn’t recorded for female subjects (r = 0.142, p = 0.325). according to the linear regression, there was a relation between height and upper arm length of subject in all cases. in addition, there was a relation between stature and forearm length in male subjects. however, this relation wasn’t recorded for female subjects (table 2). discussion stature estimation is one of the most important factors in forensic and legal medicine for the identification of individual and in anthropological research in forensic examinations, prediction of stature from remained body segments is important.20,21 various studies were conducted to evaluate the relation between stature and upper limb segments in iran and other countries.13,21,22 in this study, 200 subjects were selected randomly from healthy population without skeletal abnormalities from medical students. the mean age was similar in sex groups, and this could help to have a population with normal distributions in age factor. according to the linear regression, there was a moderate relation between stature and forearm length (s = 111.48 + 2.261 × forearm length (cm), r2 = 0.41, see = 7.85). mathematical methods such as linear regression are the easiest and reliable methods for predicting the relation between stature and body segments.22 akhlghi et al. (2012) find the correlation between forearm length and stature (r = 0.580) and showed that this factor is a poor predictor for stature estimation in comparison with other factors such as upper limb length (r = 0.635), arm length (r = 0.602), hand length (r = 0.695). this correlation was more reliable (r = 0.643) and superior to the upper limb length and arm length in the present study.13 ilayperuma et al. (2010) could find a model for prediction of stature form forearm length in sri lankan population. their formula for all subjects was height = 97.252 + 2.645 (ulna length).23 singh et al. (2013) used forearm length for prediction of stature in north indians. according to the results, mean stature was higher in male subjects than females. srivastava et al. (2010),21 ilayperuma et al.23 demonstrated similar results in their studies. in addition, sexual dimorphism was observed in the results and forearm length was longer than female subjects. according to akhlaghi et al. (2012) study, mean the forearm length was longer in male subjects than female subjects.13 this result confirmed the results of the present study. however, the mean forearm length obtained from this study was longer than results from their study according to dimorphism, the separated formula were reported for male subjects. however, this formula had a poor value for predicting the stature from forearm length in medical students (r = 0.425). for female subjects, the formula wasn’t obtained. akhlghi et al. (2012) find the correlation between forearm length and stature in male (r = 0.354) and females (r = 0.299) in iranian population. this correlation was more reliable for male subjects in the present study (r = 0.643). in addition, ilayperuma et al. (2010) could find a model for prediction of stature form forearm length for males (r = 0.66) and females (r = 0.76) in sri lankan population. in addition, singh et al. (2013) used forearm length for prediction of stature in north indians. correlation confection (r) was 0.601 for males and 0.531 for females.24 their results were more reliable than the results of the present study. according to the results, forearm length was a moderate predictor for stature estimation of medical students in iranian population. in addition, this factor is a poor predictor for male iranians and is not suitable for female iranians for stature estimation. n 272 j contemp med sci | vol. 3, no. 11, summer 2017: 270–272 stature estimation from forearm length in iranian medical students original gholamreza hassanzadeh et al. references 1. krishan k, sharma a. estimation of stature from dimensions of hands and feet in a north indian population. j forensic leg med. 2007; 14:327–332. 2. cordeiro c, muñoz-barús ji, wasterlain s, cunha e, vieira dn. predicting adult stature from metatarsal length in a portuguese population. forensic sci int. 2009;193:131. e1–e4. 3. scheuer l. application of osteology to forensic medicine. clin anat. 2002;15:297–312. 4. patel sm, shah gv, patel sv. estimation of height from measurement of foot length in gujarat region. j anat soc india. 2007;56:25–27. 5. trotter m, gleser gc. estimation of stature from long bones of american whites and negroes. am j phys anthropol. 1952;10:463–514. 6. cattaneo c. forensic anthropology: developments of a classical discipline in the new millennium. forensic sci int. 2007;165(2):185–193. 7. kharoshah maa, almadani o, ghaleb ss, zaki mk, fattah yaa. sexual dimorphism of the mandible in a modern egyptian population. j forensic leg med. 2010;17:213–215. 8. menezes rg, nagesh k, monteiro fn, kumar gp, kanchan t, uysal s, et al. estimation of stature from the length of the sternum in south indian females. j forensic legal med. 2011;18:242–245. 9. singh s, nair sk, anjankar v, bankwar v, satpati d, malik y. regression equation for estimation of femur length in central indians from intertrochanteric crest. j indian acad forensic med. 2013;35:223–226. 10. jee s-c, yun mh. estimation of stature from diversified hand anthropometric dimensions from korean population. j forensic leg med. 2015;35:9–14. 11. cheng jc, leung s, chiu b, tse p, lee c, chan a, et al. can we predict body height from segmental bone length measurements? a study of 3,647 children. j pediatr orthop. 1998;18:387–393. 12. sheikhazadi a, hassanzadeh g, mokhtari tahmineh, sheikhazadi elham, saberi anary seyed hossein, et al. stature estimation from percutaneous tibia height: study of iranian medical students. joint bone sci j. 2015;2:121–127. 13. akhlaghi m, hajibeygi m, zamani n, moradi b. estimation of stature from upper limb anthropometry in iranian population. j forensic leg med. 2012;19:280–284. 14. ahmed aa. estimation of stature from the upper limb measurements of sudanese adults. forensic sci int. 2013;228:178. e1–e7. 15. shah rk, patel jp, patel bg, kanani sd, patel md. estimation of stature from foot length and hand length measurements in gujarat region. natl j integr res med. 2014;5:16–19. 16. patel jp, patel bg, shah rk, bhojak nr, desai jn. estimation of stature from hand length in gujarat region. nhl j med sci. 2014;3:41–44. 17. shah t, patel m, nath s, menon sk. a model for construction of height and sex from shoulder width, arm length and foot length by regression method. j forensic sci criminol. 2015;3:102. 18. forman mr, zhu y, hernandez lm, himes jh, dong y, danish rk, et al. arm span and ulnar length are reliable and accurate estimates of recumbent length and height in a multiethnic population of infants and children under 6 years of age. j nutr. 2014;144:1480–1487. 19. kumar s, shetty p. estimation of stature from middle finger length-in salem region. indian j forensic med toxicol. 2014;8:30–33. 20. krishan k, kanchan t, sharma a. multiplication factor versus regression analysis in stature estimation from hand and foot dimensions. j forensic leg med. 2012;19:211–214. 21. srivastava a, sahai m. estimation of stature by anthropometric examination of forearm and hand. j indian acad forensic med. 2010;32:62–65. 22. ilayperuma i, nanayakkara g, palahepitiya n. a model for the estimation of personal stature from the length of forearm. int j morphol. 2010;28:1081–1086. 23. ilayperuma i, nanayakkara b, palahepitiya k. a model for reconstruction of personal stature based on the measurements of foot length. galle med j. 2008;13:6–9. 24. singh b, kaur m, kaur j, singh m, batra a. estimation of stature from forearm length in north indians–an anthropometric study. int j basic appl med sci. 2013;3:201–204. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201705 229j contemp med sci | vol. 3, no. 10, spring 2017: 229–233 research molecular and conventional methods for detection of candida species isolated from a sample of immunocompromised iraqi patients with pulmonary symptoms azhar af al-attraqchi,a jabbar salman hassan,a haider n dawood,b marwa a hadabc amedical microbiology/ college of medicine, al-nahrain university, iraq. bal-imammian al-kadhmain city teaching hospital, baghdad, medicine department, ministry of health, iraq. cmedical microbiology, college of medicine, al-nahrain university, iraq. correspondence to jabbar salman hassan (email: jabbarsalman30@yahoo.com) (submitted: 25 december 2016 – revised version received: 18 january 2017 – accepted: 22 february 2017 – published online: 26 june 2017 ) objectives candida species has emerged as a potentially pathogenic fungus rather than benefit mucosal commensal in patients with pulmonary diseases. therefore, our study was carried out to detect candida species in sputum samples from patients with pulmonary diseases using conventional and molecular methods. methods a total of 100 sputum samples obtained from patients with pulmonary symptoms such as chronic productive cough, shortness of breath, wheezing and fever were included in this study. sputum samples were dispensed into three specimen parts; the first one was applicated for cultured on sabouraud dextrose agar at 37°c for 48 h and then the purified colony of candida underwent biochemical tests including api, candida strips, and germ tube. the second part was undergone direct gram stain, while the third part was applicated for dna extraction and then molecular diagnosis with pcr technique using specific primers. results culture result revealed 43 positive samples for candida species out of 100 samples. among these positive samples, 23 (53.5%) were positive for c. albicans in each of culture and germ tube. api 20 candida found that (40) samples were positive for candida species as, 23 (57.5%) represent candida albicans, 8 (20.0%) candida glabrata, 4 (10.0%) candida parapslosis, 4(10.0%) candida tropicalis and only one (2.5%) as candida krusei. molecular test revealed that forty one samples out of forty three culture isolates of candida species were positive as follow twenty three (53.48%), belong candida albicans, nine (20.93%) belong candida glabrata, six (13.95%) candida parapslosis, four (9.30%) belong candida tropicalis. conclusion candida albicans is highly prevalent among patients suffering from bronchopulmonary symptoms. the molecular and conventional methods gave concomitant results as detection tools for the diagnosis of such microorganisms. keywords candida albicans, phospholipase b gene, omnigene, api 20 aux introduction candidiasis is a mycotic infection caused by members of the genus candida. chiefly, candida albicans is responsible for about (70–80%) of all candida infection. the candida as an opportunistic yeast pathogen which increases predominantly in patients with predisposing condition, including immunodeficiency such as hiv infections, prolong used of broad-spectrum antibiotics, corticosteroids, diabetic patients and infections with other debilitating disease.1 in immunocompromised patients, the clinical appearance of the c. albicans infection is often very complex and identification of the organism is difficult. therefore, speedy diagnosis and management of candidiasis are crucial for these patients.2 candida was frequently isolated from the mucosal surface of normal individuals, is capable of initiating a variety of recurring diseases especially in the vagina, oral and gastrointestinal mucosa. it also can affect different organs of the body, as systemic candidiasis involves major organs including, heart, kidneys, liver, spleen, lungs, brain, peritoneum, joint, and skeletal muscles, and was referred to generalized dissemination of the pathogen.3,4 candida pneumonia is one of the most challenging of all the candida infections. pneumonia due to infection with candida spp. is extremely rare, but because of contamination with oral flora, these organisms are frequently cultured from respiratory secretions.5 candida species are the fourth common cause of lung infections in hospitalized patients, and the most commonly isolated species include c. albicans, c. glabrata, c. tropicalis, and c. parapsilosis.6 methods sputum samples have been collected from 100 patients of age group ranged from 10 to 90 years old, with a mean age 47.23 ± 19.51. some of these patients were suffering from systematic diseases such as tuberculosis, diabetes mellitus, leukemia, while others were with immunocompromised status. those patients were attending and admitting to al-yarmouk teaching hospital, al-emamain al-kadhemain teaching hospital and chest and respiratory diseases institute/baghdad medical city during the period from september 2015 to february 2016. each sputum sample was dispensed into three specimen parts. the first one was applied for culture on sabouraud dextrose agar at 37°c for 48 hrs. purified colonies from this culture had undergone biochemical tests including api candida strips and germ tube. the second part was used in direct gram stain while the third one was applied for molecular method. standard strains of c. albicans atcc 10231, was obtained from the national institute of health in baghdad which was used as a positive control. isolation and identification of candida species gram stain method was applied to each fresh sputum specimen and examined microscopically for detecting candida species. issn 2413-0516 230 j contemp med sci | vol. 3, no. 10, spring 2017: 229–233 detection of candida species in patients with pulmonary symptoms research azhar a. f. al-attraqchi et al. sputum samples were streaked on sabouraud’s dextrose agar (sda) and incubated at 37°c for 24–48 hrs. the isolates were re-identified by using api 20 c aux and germ tube production. api 20 c aux was performed according to the manufacturer’s instructions. (biomuriex, france) for the confirmatory identification of the c. albicans and other species. germ tube production is a diagnostic characteristic method for c. albicans. a small part of yeast colony to be tested was emulsified with 0.5 ml of mammalian serum in a small test tube. the tube was incubated aerobically at 37°c in an incubator for 2 hrs. a drop of the serum was removed to a slide and examined microscopically using the ×10 and ×40 objective lenses. a cylindrical filament originating from the blastoconidium without any constriction at the point of origin and without obvious swelling along the length of the filaments indicates a germ tube positive yeast.7 molecular method for diagnosis of candida species the extraction of dna was applied from each sample using sporelyse, dna genotek, purification kit (canada) with modification by mixing 200 μl of the sputum sediment with the 40 μl of lysis buffer the suspension underwent a freezing– thawing technique by subjecting the samples to liquid nitrogen for 5 min; followed by boiling for 3 minute for five cycles.8 (freezing–thawing technique was added to the protocol as an efficient step and enhance the cell lysis)*. the primer sequences were used for the amplification for plb genes of candida species were selected according to nabil s. harmal et al.,9 (table 1). an internal control has been used to measure the efficiency of the dna extraction process fluids, as well as the impact of external and internal factors on gene amplification process. since dna extracted from fluids can be variably degraded and may contain pcr inhibitors. the human beta-globin primers was taken from saiki et al.,10 and synthesized in alpha dna® (canada). the thermocycling conditions with a cleaver scientific thermal cyclers (tc 32/80-uk) were as follows: after initial denaturation at 94°c for 5 min, the 30-cycle amplification profile consisted of 95°c for 30 s, 63°c for 35 s and 72°c for 1 min. final elongation was occurred at 72°c for 10 min. pcr products were processed into a 2% (wt/vol) agarose gel (merck-germany) at 7 v/cm for 1.5 hr. a molecular marker (1-kb dna ladder; bioneer) was run concurrently. dna bands were visualized and photographed under uv light after the gel was stained with ethidium bromide. *modifications step statistical analysis statistical analysis system (sas) software was used for all statistical analysis continuous variables were expressed in mean ± standard deviation (sd). the pearson’s chi-square test or fisher exact test was used for comparing the categorical variable. a two-sided significant level of 0.05 was considered to indicate a statistically significant difference. results a total of 100 patients suffering from pulmonary diseases were enrolled in this study. 64 (64%) were males and 36 (36%) were females with a ratio of 1.8:1, fig. 1. the principal findings were the ages ranged between 10 and 90 years with mean (47.23 ± 19.51) years. table 1. sequence and product size of plb genes primers candida species primer name primer sequence (5’ → 3’) annealing temperature product size (bp) c. albicans ca f ttgtgttgctacatcaccaac 63°c 538 bp ca r tttgctggcaacttgattacc c. glabrata cg f tctcacactccattgtctca 50°c 404 bp cg r agcaggtttaccatcaga c. parapsilosis cpf tccatcgacgaattgattg 60°c 252 bp cpr accgttttgagacctcaag c. tropicalis ctf cccatacgatttatggaat 53°c 501 bp ctr ccattgacacaagcatttac table 2. correlation between candida species and gender pcr total candida albicans candida glabrata candida parapslosis candida tropicalis negative sex female count 8 4 3 1 20 36 % 34.8% 44.4% 50.0% 25.0% 34.5% 36.0% male count 15 5 3 3 38 64 % 65.2% 55.6% 50.0% 75.0% 65.5% 64.0% total count 23 9 6 4 58 100 % 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% p-value 0.899 fig. 1 gender distribution of patients with broncho pulmonary diseases. azhar a. f. al-attraqchi et al. 231j contemp med sci | vol. 3, no. 10, spring 2017: 229–233 research detection of candida species in patients with pulmonary symptoms the correlations between candida species infections and gender are shown in table 2. out of 100 patients suffering from pulmonary diseases enrolled in this study, they were categorized according to their underlying diseases as follows; 34 patients with tuberculosis, 26 patients with diabetes mellitus, 14 patients with acute lymphoblastic leukemia, 12 patients with lung cancer, 10 patients with lymphoma, 8 patients with acute myelogenic leukemia, 5 patients with asthma, 3 patients with heart failure, 3 patients with renal failure, 2 patients with liver cancer, 2 patients with ovarian cancer, 2 patients with prostate cancer, 1 patient with chronic bronchitis, 1 patient with chronic myelogenic leukemia and only 1 patient with rheumatoid arthritis, fig. 2. percentages of candida species from patients with pulmonary manifestations and other underlying diseases this relationship between candida species isolated from patients with pulmonary manifestations and other underlying diseases are summarized in table 3, which showed a statistical significant difference (p < 0.01). cultivation and gram stain a total of 100 sputum samples were cultivated on sabouraud's dextrose agar and incubated for 2 days at 37°c. forty three samples (43%) were positive for candida species the colonies were mucoid and have a creamy color. gram stain confirmed this result in that the 43 samples were gram positive. table 3. percentages of isolated candida species from patients with pulmonary manifestations with different underlying diseases pcr total p-value candida albicans candida glabrata candida parapslosis candida tropicalis negative hematological malignancies 5 2 2 2 11 22 --% 21.7% 22.2% 33.3% 50.0% 19.0% 22.0% 0.0014** solid tumor 9 5 1 1 12 28 --% 39.1% 55.6% 16.7% 25.0% 20.7% 28.0% 0.0031** ashmatic patients 1 0 1 0 3 5 --% 4.3% 0.0% 16.7% 0.0% 5.2% 5.0% 0.0149** dm 7 4 1 1 13 26 --% 30.4% 44.4% 16.7% 25.0% 22.4% 26.0% 0.0035** tb 4 1 2 1 25 33 --% 17.4% 11.1% 33.3% 25.0% 43.1% 33.0% 0.0049** fig. 2 distribution of patients with pulmonary manifestations and other underlying diseases. table 4. comparison between molecular and culture methods for detection of candida species pcr total positive negative culture positive count 41 2 43 % 95.3% 3.4% 43.0% negative count 1 56 57 % 1.8% 96.6% 57.0% total count 42 58 100 % 42.0% 58.0% 100.0% p-value < 0.001 sensitivity 97.6% specificity 96.6% positive predictive value 95.4% negative predictive value 98.3% comparison between molecular and culture as detection methods for candida species both methods gave positive results for 41 samples. separately, one sample was negative by culture and positive by pcr method, and two out of 43 samples were negative by pcr and positive by culture method. the sensitivity of culture test was 97.6% with a specificity of 96.6%, while the positive predictive value was 95.4% and negative predictive value was 98.3% with a p value of < 0.001, table 4. germ tube formation a total of 43 culture samples were examined for germ tube. the result revealed that 23 (53%) were positive for candida albicans, as shown in fig. 3 and table 5. api 20 candida kit a total of 43 culture samples were examined by api 20 aux candida strips, it was found that (40) samples were positive for candida species, 23 (57.5%) represent candida albicans, 8 (20.0%) candida glabrata, 4 (10.0%) positive cases of candida parapslosis, 4 (10.0%) candida tropicalis and only 1 (2.5%) as candida krusei (table 6). 232 j contemp med sci | vol. 3, no. 10, spring 2017: 229–233 detection of candida species in patients with pulmonary symptoms research azhar a. f. al-attraqchi et al. molecular detection conventional pcr was done for the amplification of plb gene by using a specific set of primer sequences. the results showed that, this gene (plb gene) was present in 23 out of 100 sputum samples, pcr product of this gene was 538 bp which represent candida albicans. nine candida glabrata, pcr product of this gene was 404 bp. six candida parapslosis pcr product of this gene was 252 bp. four candida tropicalis pcr product of this gene was 501 bp (figs. 4 and 5). discussion candida infections are still an important problem, especially for immunosuppressed individuals.11 inability or delay in diagnosing fungal infection defers the administration of appropriate therapy. this has grave implications for the prognosis of the patient: reliable and rapid diagnostic tests for systemic mycoses are imperative to improve rates of patient survival.12 in the present study, results indicated that there is no relationship between the infection rate with candida species and gender. the percentage of infected males were 26 out of 43 (60.4%) and females were 17 out of 43 (39.5%), hence there is no significant difference between male and female infection rate upon existing both in the same environment. this result was disagreed with other study done by saba sabeeh,13 who found that candida infection was more frequent among females than males, and disagreed with that done by ibrahim,14 who found that infection was more frequent in males than in females. the possible explanation for such discrepancy may be due to nature of the societies and duration of time for sample collection. in the present study, a relatively high percentage of candida species infection was found among patients with hematological malignancies, solid tumor, asthma, diabetes mellitus and from patients with tuberculosis. these results are in accordance with those obtained by lindau et al.15 and ansari et al 16 who proved that a fungal infection represents a growing problem in patients with hematologic malignancies particularly during chemotherapy induced neutropenia and other chronic debilitating diseases. microscopic examination of sputum using staining methods, remain popular in the diagnosis of pulmonary infection especially in low-income countries, due to its rapidity, low cost, relatively easy to perform and high positive predictive value.15 on the other hand, culture is considered to be the “gold standard” method for the diagnosis of pulmonary infections but require 20 to 100 viable organisms per sample, and this is a cumbersome in partially treated patients. culture also labor intensive and time consuming.16 in this study, positive cultures were tested by germ tube and biochemical api 20. results of germ tube revealed that 53.4% of positive culture were c. albicans. all germ tube samples were positive for c. albicans by api 20 aux. regarding other candida species tested by biochemical api 20, results revealed that 8 (20.0%) candida glabrata, 4 (10.0%) positive cases of candida parapslosis, 4 (10.0%) candida tropicalis and only 1 (2.5%) as candida krusei. that’s mean api 20 candida and germ tube technique provides a convenient and reliable method for identification of candida species. molecular method for the detection of candida species the plb gene of candida species is a novel target which shows a high variability of sequences among candida species. the nucleotide sequence variability between the different species of candida can reach 95%.17 thus, it is possible through designing a specific set of primers to target the unique sequence of plb gene. table 5. percentages of germ tube formed by candida albicans germ tube number percentage (%) negative 77 77.00 positive 23 23.00 total 100 100% chi-square (c 2) --12.792** p-value --0.0001 **(p < 0.01). fig. 4 agarose gel electrophoresis (2% agarose, 7v/cm2, and 1.5 hrs) of the pcr products of plb gene 538 pb of candida albicans lane 8: 100 bp dna ladder, lane (1–4 and 6) positive sample for candida albicans, lane 5 negative control, lane 7 positive control. fig. 5 agarose gel electrophoresis (2% agarose, 7v/cm2 and 1.5 hrs) of the pcr products of plb gene of candida glabrata. lane 13: 100 bp dna ladder, lane (1 and 9) positive sample for candida glabrata, pcr product of this gene was 404 bp. fig. 3 germ tube formation test of candida albicans. azhar a. f. al-attraqchi et al. 233j contemp med sci | vol. 3, no. 10, spring 2017: 229–233 research detection of candida species in patients with pulmonary symptoms being used the same set of primers as in the current study harmal et al9 proved that species-specific pcr assay could identify and differentiate between the four most common candida species isolated from clinical specimens namely, c. albicans, c. glabrata, c. parapsilosis and c. tropicalis. distinctive product size for each of these 4 species allow specific identification directly from the gel e ectrophoresis without the need for further genotyping. based on the molecular weight of the amplicon product from that pcr product of this gene, it was 538 bp in candida albicans, 404 bp in candida glabrata, 252 bp in candida parapslosis and 501 bp in candida tropicalis.18,19 in this study, 23 samples were positive for plb gene which is specific for candia albicans, nine belong to candida glabrata, six belong candida parapslosis, and four belong candida tropicalis. a study done by cheang pey shyuan (19) strongly suggest that plb is a significant virulence determinant of albicans species. however, the data generated here would provide the vital groundwork for elucidating the intrinsic functional role of plbs in the virulence and pathogenesis of the candida albicans and non-albicans candida species. the results of the current study showed that the plb gene provides a novel target that could be used for the identification and detection of medically important candida species from the clinical samples. from this study, we concluded that candida albicans is the most dominated isolates from patients suffering from pulmonary manifestations. culture method is still the gold standard one in comparison with the molecular method. acknowledgement the authors are grateful to all staff member of medical microbiology department college of medicine al-nahrain university for their help and cooperation. dna genotek kindly provided sporelyse dna extraction kits free of charge for evaluation. conflict of interest the authors declare that they have no competing interests. n references 1. estrada-mata e, navarro-arias mj, pérez-garcía la, mellado-mojica e, lópez mg, csonka k, et al. members of the candida parapsilosis complex and candida albicans are differentially recognized by human peripheral blood mononuclear cells. front microbiol j. 2016;6:1527. 2. ryan kj, ray cg. an introduction to infectious diseases 4th ed. new york, sherris medical microbiology. 2004;pp. 661–663. 3. ruchel r. cleavage of immunoglobulines by pathogenic yeast of genus candida, microbial sci. 1986;3:316–319. 4. greenwood d, richard c, slack s, et al. medical microbiol 5th ed. churchill livingstone, london. 1997. 5. d'eça júnior a, silva af, rosa fc, monteiro sg, de maria silva figueiredo p, de andrade monteiro c. in vitro differential activity of phospholipases and acid proteinases of clinical isolates of candida. rev soc bras med trop. 2011;44:334–338. 6. mohandas v. ballal m. distribution of candida species in different clinical samples and their virulence: biofilm formation, proteinase and phospholipase production: a study on hospitalized patients in southern india. j global infect dis. 2011;3:4–8. doi: 10.4103/0974-777x.77288. 7. donghwa k, woon s, kyoung h. rapid differentiation of candida albicans from other candida species using its unique germ tube formation. yeast j. 2002;19:957–962. doi.org/10.1002/yea.891. 8. reischl u, pulz m, ehret w, wolf h. pcr-based detection of mycobacteria in sputum samples using a simple and reliable dna extraction protocol. biotechniques. 1994;5:844–5. 9. nabil s, harmal, alireza kh, mohammed a, alshawsh fj, zamberi sk. simplex and triplex polymerase chain reaction (pcr) for identification of three medically important candida species. african j biotechnol. 2012;11:12895–12902. doi: 10.5897/ajb12.1708. 10. saiki rk, gelfand dh, stoffel s, et al. primer-directed enzymatic amplification of dna with a thermostable dna polymerase. science. 1988;239:487–491. 11. shokohi t, hashemi soteh mb, saltanat pouri z, hedayati mt, mayahi s. identification of candida species using pcr-rflp in cancer patients in iran. indian j med microbiol. 2010;28:147–151. 12. pfaller ma, diekema dj. epidemiology of invasive candidiasis: a persistent public health problem. clin microb rev. 2007;20:133–163. 13. saba sabeeh. molecular detection of candidemia in iraqi acute leukemic patients. 2011 (thesis). 14. ibrahim aa. biochemical and immunological studies on candida albicans proteinase. 2002 (thesis). 15. lindau s, nadermann m, ackermann h, et al. antifungal therapy in patients with pulmonary candida spp. colonization may have no beneficial effects. j inten care. 2015;3:31. 16. ansari sh, shirzadi e, elahi m. the prevalence of fungal infections in children with hematologic malignancy in ali-asghar children hospital between 2005 and 2010. iran j. ped hematol oncol. 2015;5:1–10. 17. nabil s, harmal, alireza khodavandi, mohammed a, alshawsh, et al. identification and differentiation of candida species using specific polymerase chain reaction (pcr) amplification of the phospholipase b gene. african j microbiol res. 2013;7:2159–2166. 18. lau a, halliday c, chen sca, playford eg, stanley k. comparison of whole blood, serum, and plasma for early detection of candidemia by multiplextandem pcr. j clin microbiol. 2010;48:811–816. 19. cheang pey shyuan. molecular cloning and gene expression analysis of the phospholipase b genes of non-albicans candida species. 2005 (thesis). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201706 24 j contemp med sci | vol. 1, no. 3, summer 2015: 24–26 research objective coronary artery disease (cad) risk factors are increasing in our country. the present study was performed to determine the prevalence of new (conditioned) risk factors among patients with cad. methods in 85 patients with ischemic heart disease (ihd) we study the presence of risk factors including plasma fibrinogen, serum ferritin, serum iron and serum uric acid. the diagnosis of ihd in these patients depends on clinical history and examination, ecg, cardiac echo, and certain investigations including cardiac enzymes. of these 85 patients, there are 23 females and 62 males, and also there were 50 persons selected as control group. we divided the patients into two groups: one group who had acute coronary heart disease (55 patients) and the second group with chronic ihd (30 patients). results the result of the study showed that there were statistically significant differences in the level of plasma fibrinogen between the two groups which were higher in the acute group, similar was the case with serum ferritin and serum iron but the level of serum uric acid was the same in both the groups. conclusion we conclude that plasma fibrinogen and serum ferritin can be used as a marker for the prediction of the presence of acute ihd. keywords ischemic heart disease , plasma fibrinogen, serum ferritin, uric acid conditioned risk factors in patients with coronary heart disease monem makki alshoka & kadhim jawad al-hamdanyb introduction traditional cardiovascular disease (cvd) risk factors include dyslipidemia, elevated blood pressure, cigarette smoking and diabetes mellitus. lifestyle factors are very important in the atherothrombotic disease process and therapeutic lifestyle changes – including a diet low in saturated fat, cholesterol and trans fats; regular physical activity; attainment and maintenance of a healthy body weight; and smoking cessation – remain central to preventive efforts.1,2 numerous novel and emerging risk factors are under study. improved understanding of the roles of these factors in the atherothrombotic disease process may aid in risk stratification and/or in identifying and testing novel targets for therapy. presently, the greatest utility of nontraditional risk factors such as inflammatory markers or measures of subclinical cvd are for identifying those individuals with at least moderate risk for a cvd event for whom aggressive therapy may be warranted.3 coronary atherosclerosis as a cause of coronary heart disease is more likely to develop with the presence of certain risk factors including elevated serum homocysteine; elevated serum triglyceride; elevated serum lipoprotein (a); elevated inflammatory markers e.g. crp and elevated prothrombotic factors e.g. fibrinogen.4 screening studies have shown that high blood pressure (bp), hyperlipidemia, smoking, family hx and diabetes mellitus (dm) are predictive of less than half of all future cardiovascular events.5 also the predictive value of these traditional risk factors is limited among patients with premature atherosclerosis. we have noticed that many patients with few traditional risk factors will develop an acute coronary syndrome (acs) without prior symptoms of the disease. many proteins, novel protein biomarkers, if externally validated may improve risk assessment for myocardial infarction (mi) and atherosclerotic cvd.6,7 new risk factors have been identified which enhanced the risk for coronary artery disease (cad), and these include: lipoprotein(a) [lp(a)], homocysteine and fibrinogen, and these may be a marker in patients who may not have the conventional risk factors. lp(a) is considered as a marker of thrombosis, although several prospective studies have found if any association between lp(a) and cad risk and cardiovascular risk tends to increase with lp(a) value over 30 mg/dl.8,9 there is unclear mechanism of how it happens. homocysteine promotes vascular diseases, but it may be related to deficiency of other factors including vit. b12 and folates, especially among the elderly.10 additional markers in patients with cad, which is associated with adverse outcome includes infectious and inflammatory markers (crp, tnf α, il1, il6) and infectious agents like cmv, chlamydia, and helicobacter pylori.11–13 fibrinogen is a large glycoprotein synthesised mostly in the liver. it is a clotting factor that activates thrombin, aggregates platelets, and stimulates smooth muscle proliferation. it is important in the development of premature atherosclerosis. several studies have shown an impressive relation between plasma fibrinogen level and occurrence of cad and stroke. also fibrinogen level may be a risk factor for sequelae of cad. determinant of high fibrinogen level includes age, female sex, smoking, obesity, stress, use of oral pills, pregnancy, and consumption of large amount of dietary fat.14 serum ferritin is positively correlated with serum crp concentrations. many solid conclusions come to the finding that serum ferritin is a positive acute phase reactant and is strongly associated with inflammatory processes including heart diseases and diabetes. this role is thought to be due to prooxidant properties. in patients with serum ferritin concentrations >200 ng/ml, the risk of mi was 2.2 times greater than the patients with serum ferritin levels <200 ng/ml. this indicated that serum ferritin indirectly enhances the role of ldl-cholesterol in the induction of cvds.15–17 patients and methods we made a study on 85 patients with chd for the presence of risk factors that include plasma fibrinogen, serum ferritin, professor of clinical medicine, departments of amedicine and bbiochemistry, college of medicine, university of babylon, babylon, iraq. correspondence to monem makki alshok (email: dr_monem_alshok@yahoo.com). (submitted: 14 may 2015 – revised version received: 27 june 2015 – accepted: 5 july 2015 – published online: summer 2015) issn 2413-0516 25j contemp med sci | vol. 1, no. 3, summer 2015: 24–26 research risk factors in patients with ihdmonem makki alshok & kadhim jawad al-hamdany serum iron and serum uric acid; and excluding patients with other traditional risk factors. we measured plasma fibrinogen, serum ferritin, serum iron and serum uric acid using biofibri kit (biolabo-sa, 02160, maizy, france), immunoassay kit (biocheck, inc., ca, usa; catalog number: bc – 1025), iron ferrozine kit (linear chemicals, s.l., barcelona, spain) and acid eurique enzymatique (pap150; a u pap 150 kit; france), respectively. twenty-three females, 62 males and 50 persons were chosen as control. the dx. (diagnosis) of cad was based on clinical history, ecg, certain laboratory investigations and cardiac echo. we divided the patients into two groups: one group with 55 patients who had acute cad and another with 30 patients who had chronic cad. statistical methods used include measurement of mean and standard deviation (sd), p value, and measurement of correlation coefficient between values. results and discussion the results of the study are shown in tables 1–4. the measurement of correlation coefficient values from tables 1–4 shows that plasma fibrinogen level is significantly higher than control in patients with acute cad and it is normal in those patients with chronic cad, and the level of fibrinogen is higher in females. the results indicate that the association between high concentration of fibrinogen and risk of cvd is well established. the relation was first reported in preliminary results from the northwick park heart study in 1980.18 the prospective cardiovascular munster (procam) study found that individuals who had low-density lipoprotein (ldl) and fibrinogen levels in the highest tertile had a 6.1-fold increase in coronary risk compared with those in the lowest tertile.19 only 13 patients with acute cad showed elevated level of serum ferritin and only 6 patients showed elevated level of serum iron which was statistically significant and there is positive correlation with plasma fibrinogen and most of these patients are males. serum uric acid increased in 17 patients with acute cad, but normal value in all patients with chronic cad and there is a negative correlation with plasma fibrinogen. it was reported that 2.2 times greater levels of cvd were observed in the group with high serum iron (indicative of elevated serum ferritin) compared to the group with low serum iron. serum ferritin was reported to be associated with cvd and cardiovascular mortality. salonen et al. also reported that increase in serum ferritin accelerates the oxidation of ldl cholesterol.9,20 in contrast to our study, it was reported in a prospective study performed in a french population; however, galan et al. failed to find a positive association between serum ferritin and ischemic heart disease (ihd).21 these findings matched well with the suggestions of sempos et al.22 5 years earlier, as the results from the two studies did not support the hypothesis that positive body iron stores, as measured by serum ferritin, are associated with an increased risk of cvd, chd or mi death. dominguez-rodriguez et al. suggested even more extreme findings that major adverse cardiovascular events is associated with lower serum ferritin levels in a study on a total of 196 and 30 days followed-up patients with a first non-st elevation acs.23 their observation was supported by an in vitro study that iron deficiency enhances atheroma inflammation through p38 mitogen activated protein kinase nuclear factor-κb-extracellular matrix metalloproteinase inducer/matrix metalloproteinase-9 pathway.24 serum uric acid had been shown mild elevation in about a quarter of patients with cad. by contrast with table 1. mean plasma fibrinogen in cad patients and control group mean plasma fibrinogen in mg/dl sd mg/dl p value acute cad 450 91.4 ± 0.002 chronic cad 365.5 59.2 ± 0.16 control 306.3 58.8 ± cad: coronary artery disease. table 2. mean of serum ferritin in cad patients and control group mean serum ferritin in ng/ml sd ng/ml p value acute cad 126.6 121.3 ± 0.002 in male chronic cad 48.3 39.7 ± 0.2 control 49.2 39.9 ± cad: coronary artery disease. table 3. mean of serum iron in cad patients and control group mean serum fe in µmol/l sd µmol/l p value acute cad 30 24.5 ± 0.02 in male chronic cad 20.5 14.6 ± 0.26 control 18.8 6.7 ± cad: coronary artery disease. table 4. mean of serum uric acid in cad patients and control group mean serum uric acid in mg/dl sd mg/dl p value acute cad 5.25 1.7 ± 0.02 chronic cad 3.75 1.02 ± 0.27 control 3.58 1.02 ± cad: coronary artery disease. 26 j contemp med sci | vol. 1, no. 3, summer 2015: 24–26 risk factors in patients with ihd research monem makki alshok & kadhim jawad al-hamdany references 1. khot un, khot mb, bajzer ct, sapp sk, ohman em, brener sj, et al. prevalence of conventional risk factors in patients with coronary heart disease. jama. 2003 aug 20;290(7):898–904. doi: http://dx.doi.org/10.1001/ jama.290.7.898 pmid: 12928466 2. ramachandran s. vasan, lisa m. sullivan, peter wf. wilson, christopher t. sempos, johan sundström, william b. kannel, et al. relative importance of borderline & elevated level of cad risk factors. ann intern med. 2005 mar 15;142(6):393–402. doi: http://dx.10.7326/0003-4819-142-6-20050315000005 3. paolo g. camici, filippo crea. coronary microvascular dysfunction. n engl j med. 2007 feb 22;356:830–40. doi: http://dx.10.1056/nejmra061889 4. yusuf s, hawken s, ounpuu s, dans t, avezum a, lanas f, mcqueen m, et al. effects of potentially modifiable risk factors associated with mi in 52 countries (the interheart study): case-control study. lancet. 2004 sept 11–17;364 (9438):937–52. doi: http://dx.doi.org/10.1016/s0140-6736(04)17018-9 pmid: 15364185 5. steven p maro, brian p griffin, eric j topal. cardiovascular risk factors in manual of cardiovascular medicine 2000. lippincott williams & wilkins; 2000, 478–81. 6. xiaoyan yin, et al. protein biomarkers of new-onset cardiovascular disease. circ cardiovasc genet. 2015;8:8–10. 7. khot un, khot mb, bajzer ct, sapp sk, ohman em, brener sj, et al. prevalence of conventional risk factors in patients with coronary heart disease. jama. 2003 aug 20;290(7):898–904. doi: http://dx.doi.org/10.1001/jama.290.7.898 pmid: 12928466 8. down jr, beere pa, whitney e, clearfield m, weis s, rochen j, et al. design & rationale of the air force/texas coronary atherosclerosis preventive study (afcaps/texcaps). am j cardiol. 1997 aug 1;80(3):287–93. doi: http:// dx.doi.org/10.1016/s0002-9149(97)00347-0 pmid: 9264420 9. kannel wb. lipids, diabetes and cad insight from framingham study. am heart j. 1985 nov;110(5):1100–7. doi: http://dx.doi.org/10.1016/00028703(85)90224-8 10. marinou k, antoniades c, tousoulis d, pitsavos c, goumas g, stefanadis c. homocysteine: a risk factor for coronary artery disease? hellenic j cardiol. 2005 jan–feb;46(1):59–67. 11. mehta jl, saldeen tg, rand k. interactive role of infection, inflammation and traditional risk factors in atherosclerosis and coronary artery disease. j am coll cardiol. 1998 may;31(6):1217–25. doi: http://dx.doi.org/10.1016/ s0735-1097(98)00093-x pmid: 9581711 12. douglas ps, hoffmann u, patel mr, mark db, al-khalidi hr, cavanaugh b, et al. outcomes of anatomical versus functional testing for coronary artery disease. n engl j med. 2015 apr 2;372(14):1291–300. doi: http://dx.10.1056/ nejmoa1415516 pmid: 25773919 13. hansson g.k. mechanisms of disease: inflammation, atherosclerosis, and coronary artery disease. n engl j med. 2005;352:1685–95 doi: http:// dx.10.1056/nejmra043430 14. farchi g, fidanza f, mariotti s, menotti a. is diet an independent risk factor for mortality? 20 years mortality in the italian rural cohorts of the seven countries study. eur j clin nutr. 1994 jan;48(1):19–29. pmid: 8200326 15. sung kc, kang jh, shin hs. relationship of cardiovascular risk factors and serum ferritin with c-reactive protein. arch med res. 2007 jan;38(1):121–5. doi: http://dx.doi.org/10.1016/j.arcmed.2006.08.008 pmid: 17174735 16. wood rj. the iron-heart disease connection: is it dead or just hiding? ageing res rev. 2004 jul;3(3):355–67. doi: http://dx.doi.org/10.1016/j. arr.2004.04.002 pmid: 15231242 17. chen x, scholl to, stein tp. association of elevated serum ferritin levels and the risk of gestational diabetes mellitus in pregnant women: the camden study. diabetes care. 2006 may 1;29(5):1077–82. doi: http://dx.doi. org/10.2337/diacare.2951077 pmid: 16644640 18. heinrich j, balleisen l, schulte h, assmann g, van de loo j. fibrinogen and factor vii in the prediction of coronary risk. results from the procam study in healthy men. arterioscler thromb. 1994 jan 1;14(1):54–9. doi: http:// dx.doi.org/10.1161/01.atv.14.1.54 pmid: 8274478 19. salonen jt, nyyssönen k, korpela h, tuomilehto j, seppänen r, salonen r. high stored iron levels are associated with excess risk of myocardial infarction in eastern finnish men. circulation 1992 sep 1;86(3):803–11. doi: http://dx.doi.org/10.1161/01.cir.86.3.803 pmid: 1516192 20. kiechl s, willeit j, egger g, poewe w, oberhollenzer f. body iron stores and the risk of carotid atherosclerosis: prospective results from the bruneck study. circulation. 1997 nov 18;96(10):3300–7. doi: http://dx.doi. org/10.1161/01.cir.96.10.3300 pmid: 9396420 21. galan p, noisette n, estaquio c, czernichow s, mennen l, renversez jc, et al. serum ferritin, cardiovascular risk factors and ischaemic heart diseases: a prospective analysis in the su.vi.max (supplementation en vitamines et minéraux antioxydants) cohort. public health nutr. 2006 feb;9(1):70–4. doi: http://dx.doi.org/10.1079/phn2005826 pmid: 16480536 22. sempos ct, looker ac, gillum rf, mcgee dl, vuong cv, johnson cl. serum ferritin and death from all causes and cardiovascular disease: the nhanes ii mortality study. national health and nutrition examination. ann epidemiol. 2000 oct;10(7):441–8. pmid: 11023623 23. dominguez-rodriguez a, carrillo-perez tome m, hernandez-garcia c, arroyo-ucar e, juarez-prera r, blanco-palacios g, et al. serum ferritin and acute coronary syndrome: a strong prognostic factor? int j cardiol. 2011 oct 6;152(1): 129–30. doi: http://dx.10.1016/j.ijcard.2011.07.052 pmid: 21856027 24. fan y, wang j, wei l, he b, wang c, wang b. iron deficiency activates pro-inflammatory signaling in macrophages and foam cells via the p38 mapk-nf-κb pathway. int j cardiol. 2010 oct 6;152(1):49–55. doi: http:// dx.10.1016/j.ijcard.2010.07.005 pmid: 20674992 25. palmer tm, nordestgaard bg, benn m, tybjærg-hansen a, davey smith g, lawlor da, et al. association of plasma uric acid with ischaemic heart disease and blood pressure: mendelian randomisation analysis of two large cohorts. bmj. 2013 jul 18;347:f4262. doi: http://dx.10.1136/bmj.f4262 pmid: 23869090 26. kawada t. serum uric acid and ischemic heart disease incidence. int j cardiol. 2012 feb 9;154(3):381. doi: http://dx.10.1016/j.ijcard.2011.11.040 pmid: 22192295 observational findings, there is no strong evidence for causal associations between uric acid and ischemic heart disease or blood pressure.25 kwada et al. recently overviewed and conducted meta-analysis on this association precisely, and concluded that hyperuricemia may increase the risk of ihd events, independently of traditional ihd risk factors.26 in conclusion, plasma fibrinogen, serum ferritin and to a lesser extent serum uric acid could be regarded as a marker and predictor of cad and its sequelae.  384 j contemp med sci | vol. 7, no. 6, november-december 2021: 384–391 original angiotensin-converting enzyme receptor genotype and its activity level as potential predictors of the severity covid-19 among iraqi patients suzan haleem kamel1 , fadhil jawad al-tu’ma1*, riyadh mohi al-saegh2 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 2section of nephrology, department of internal medicine, college of medicine, university of kerbala, kerbala, iraq. *correspondence to: fadhil jawad al-tu’ma (e-mail: fadhil.jawad@uokerbala.edu.iq) introduction more than 140 million cases of infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) have been reported worldwide by april 19, 2021, with over 3 million deaths due to the virus1 coronavirus disease situation dashboard. coronavirus disease 2019 (covid-19) which emerged in wuhan, china, has spread to almost all countries and regions of the world, becoming one of the most lethal pandemic after the spanish flu in 1918–1920. it is caused by an rna virus (2019 novel coronavirus or 2019-ncov or sars-cov-2). as of july 13, 2020, a total of 12,768,307 confirmed covid-19 cases and 566,654 related deaths have been reported. beyond its important morbidity and mortality and the huge burden of health care systems, covid-19 has a massive societal and economic impact globally.1,2 this pathogen was later renamed as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the coronavirus study group (gorbalenya, 2020),3 and the disease was named coronavirus disease 2019 (covid-19) by the who. covid-19 can be either silent (asymptomatic) or associated with many symptoms, such as familiar cold symptoms (fever, stuffy nose, cough, weakness) bronchitis and pneumonia.4 covid-19 is moderately infectious with a relatively high mortality rate, but the information available in public reports and published literature is rapidly increasing. many risk factors have been described for this coronavirus such as elderly age, male gender, race, obesity, hypertension, diabetes and geographic region.5,6 clinically, most of covid-19 cases (80%) are either asymptomatic or have mild forms.7 however, about 13.8 and 6.1% have severe and critical life-threatening disease that requires admission to hospital and sometimes in the intensive care unit. in the context of outstretched heath care systems and limited resources, risk stratification is pivotal to identify patients who the most need in-hospital and intensive management. biomarkers along with some clinical factors might help to predict adverse outcomes among covid-19 patients. hence, we conducted this systematic review and meta-analysis to summarize available data on the association between some common hematological, inflammatory, biochemical parameters and the severity of covid-19.7,8 to date, there is no established curative treatment for covid-19. although some drugs such as hydroxychloroquine are integrated in treatment guidelines or under investigation in interventional studies, the management of covid-19 is mostly supportive.7,9 identifying biological abnormalities induced by covid-19 may contribute to a better understanding of the pathophysiology of the disease and ultimately guide the development of targeted adjuvant therapies besides antivirals drugs. furthermore, such information on the biological profile of covid-19 can guide clinicians in the assessment and treatment of these patients.10 genetic factors also play a major role in covid19 infection. inter-individual inherited differences in susceptibility to sars-cov-2 infection is linked to the presence of genetic polymorphisms (variants) in many abstract objectives: the purpose of this study is to determine whether the angiotensin-converting enzyme activity and various biomarkers are used to investigate the severity of covid-19 and to study the genetic variation occurs in angiotensin-converting enzyme-2 (ace-2) receptor in severe covid-19-related genes in the iraqi population of kerbala province. methods: this case-control study was conducted on 176 subjects who survived hospitalization and diagnosed by physician. various biomarkers including ferritin, c-reactive protein, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, angiotensinconverting enzyme-2 activity levels were determined. accordingly, they were divided into three groups: 59 of them were infected with severe covid-19, 54 of them were infected with moderate covid-19 and 63 of them were checked and obtained as apparently healthy control. severe and moderate patients were collected from al-hayat tertiary center at al-hussein medical city, kerbala health directorates, kerbala – iraq during oct., 2020-july, 2021 with matched age ranged between (23–88) years. blood samples of apparently healthy and covid-19 samples were subjected to genomic dna extraction within 24–48 hours of aspiration. the genomic dna extracted was subjected to electrophoresis through 1.5% of agarose gels which was detected by staining with the fluorescent dye ethidium bromide and then visualized by illumination with uv light to confirm the presence and integrity of the extracted dna. results: genotyping of ace-2 (i/d) polymorphism (rs4646994), which has a high prevalence, was performed by polymerase chain reaction assay. the amplification of an alu repetitive element in an intron of the ace-2 has shown three potential genotypes of i/i and d/d as homozygous, and i/d as heterozygous. individuals with normal homozygous (dd) revealed band of (190 bp), while individuals with normal (ii) revealed band of (490 bp), and the individuals with heterozygous (id) revealed two bands (190, 490 bp) respectively. every severe covid-19 group carried (dd) allele genotype, moderate group carried (id and ii) alleles and finally the control group carried (dd, id, ii) alleles genotype. conclusion: in the ace-2 polymorphism, the d/d genotype allele is implicated as a risk factor for severe covid-19 patients, in iraqi population. keywords: covid-19, homozygote, ferritins, genotype issn 2413-0516 (submitted: 09 september 2021 – revised version received: 16 september 2021 – accepted: 12 october 2021 – published online: 26 december 2021) 385j contemp med sci | vol. 7, no. 6, november-december 2021: 384–391 s.h. kamel et al. original ace receptor genotype and its activity level as potential predictors of the severity covid-19 among iraqi patients genes especially in those that code for the host receptors involved in viral entry process. these dna changes are transmissible from one generation to another, detectable in at least 1% of individuals in a population and could explain the differences between individuals in the susceptibility to some multigenic, complex diseases like covid19.11,12 two main approaches can be used in genetic epidemiology to establish a link between genetic variations and the risk of developing a disease: genetic linkage analysis and association studies (candidate gene and genome-wide association studies).13 angiotensin-converting enzyme (chromosome xp22.2) is the enzyme responsible for converting angiotensin-2 to angiotensin (1–7) form,14 it is expressed in most organs such as, thyroid and lungs, heart, esophagus, kidney, adipose tissue, liver, retina, the vascular system, the small intestine, nasal and bronchial tissue, and alveolar type ii epithelial cells.15,16 ace-2 is also known as a host cell receptor that contributes to the viral infection by corona viruses. the covid-19 virus binds to the target cells through ace-2 receptor which makes the covid-19 attachment, invasion and penetration processes easier.17 there are other receptors that can be used but, the virus has greater affinity for ace-2 and weaker affinities for two other receptors, cd147 and grp78 (glucose-regulate protein 78).16 the ace-2 expression level has been reported to be significantly increased among men than women, which could explain the male predominance of covid-19.18,19 however, in another study ace-2 expression was not significantly associated with gender/disease severity bias among covid-19 italian patients.20 similar results were obtained by another author, who didn’t observe a disparity between age groups and gender groups (male vs female) in ace-2 gene expression.21 ace-2 is very polymorphic gene with about 1700 polymorphisms and their frequencies vary between populations, some of these polymorphisms were correlated with increased expression of ace-2 protein and were more frequent among the east-asian populations.17,22,23 not only that, ace deletion allele that is linked to alterations of ace expression, also influences the spread of the virus and outcomes of infection with covid-19 especially in the asian populations.24,25 angiotensin converting enzyme-2 receptor (ace-2) is a protein on the surface of many cell types. it is an enzyme that generates small proteins – by cutting up the larger protein angiotensinogen – that then go on to regulate functions in the cell. using the spike-like protein on its surface, the sars-cov-2 virus binds to ace-2 – like a key being inserted into a lock – prior to entry and infection of cells. hence, ace-2 acts as a cellular doorway – a receptor – for the virus that causes covid-19. ace-2 acts as the receptor for the sars-cov-2 virus and allows it to infect the cell. ace-2 receptor is present in many cell types and tissues including the lungs, heart, blood vessels, kidneys, liver and gastro-intestinal tract. it is present in epithelial cells, which line certain tissues and create protective barriers. the purpose of the presented study is to investigate the activity levels of angiotensin-converting enzyme and various biomarker levels in adults infected with covid-19 and study the molecular basis of angiotensin-converting enzyme-2 (ace-2) receptor in covid-19-related gene polymorphism in iraqi populations of kerbala province. materials and methods the current case-control study was conducted on 176 subjects who survived hospitalization and diagnosed by physician. various biomarkers including ferritin, c-reactive protein, lactate dehydrogenase (ldh), alanine aminotransferase (alt), aspartate aminotransferase (ast), angiotensin-converting enzyme-2 (ace-2) activity were determined. accordingly, they were divided into three groups: 59 of them were infected with severe covid-19, 54 of them were infected with moderate covid-19 and 63 of them were checked and obtained as apparently healthy control. severe and moderate patients were collected from al-hayat tertiary center at al-hussein medical city, kerbala health directorates, kerbala–iraq during oct., 2020-july, 2021 with matched age ranged between (23–88) years. blood samples of apparently healthy and covid-19 samples were obtained from each case and used for some biomarkers determination (ferritin, alt, ast and alp activity levels) and molecular studies. one ml of whole blood was collected in edta containing tube and subjected for genomic dna extraction within 24–48 hours of aspiration. then dna concentration and purity were measured by uv absorption at 260 and 280 nm (bio drop, u.k.). the genomic dna extracted was subjected to electrophoresis through 1.5% of agarose gels electrophoresis in 1x (tbe) buffer at 100 volts for 75 minutes or until dye markers have migrated an appropriate distance, depending on the size of the dna to be visualized. the percentage of agarose used depends on the size of fragments to be resolved, where an agarose gels percentage are normally in the range of 0.5% to 2%; the ethidum bromide staining was done according to robinson and lafleche method.26 dna ladder 100 bp bands were used as standard for comparison with bands that resulted for allelic gene migration through gel electrophoresis and then detected at staining with the fluorescent dye ethidium bromide and then visualized by illumination with uv light to confirm the presence and integrity of the extracted dna. genotyping for ace-2 receptor gene polymorphism was performed by the polymerase chain reactionamplification refractory mutation system (pcr-arms) method using thermocycler (biometra, germany). the primers were taken in a lyophilized state; their units are known as a mass in picomoles then mixed by suitable vortex and their sequence of ace-2 receptor gene was listed in table 1. the subsequent steps were done for the reconstitution and dilution of the primers: the tube was centrifuged at 10,000 rpm for 5–10 min before de-capping. the chosen volumes from nuclease free water were added according to the manufacturer to obtain a 100 p-moles/μl (master stock). ten microliters of the master stock were transported to a 0.5 ml eppendorf tube that contained 90 μl of nuclease free water to obtain a 10 pmoles/μl a working primer stock solution. the master stock and working stock were kept at –20°c. the working stock was warmed up and kept on ice for use in pcr and then stored at –20°c after each use. table 1. primers used in molecular study of angiotensin converting enzyme receptor-2 gene polymorphism primer name sequence annealing temp. (°c) product size (bp) ace-rs4646994f 5`-ctggagacca ctcccatcctttct-3` 58 190 ace-rs4646994-r 5`-gatgtggccatca cattcgtcagat-3` 58 490 386 j contemp med sci | vol. 7, no. 6, november-december 2021: 384–391 ace receptor genotype and its activity level as potential predictors of the severity covid-19 among iraqi patients original s.h. kamel et al. the reaction setup and thermal cycling protocol of the pcr components and program for ace-2 receptor gene show in table 2. electrophoresis involves running a current through a gel loaded with the molecules of interest. the movement of the samples is directed based on the charge that the molecule carries. based on the size and charge, the molecules will travel through the gel at different speeds, allowing them to be separated from one another as shown in figure 1. since, all the dna molecules possess same amount of charge per mass, the gel electrophoresis separates them on the basis of size only. body mass index (bmi) was used to define obesity. the range of bmi (18.5–24.99 kg/m2) that set it who but it does table 2. program of polymerase chain reaction of ace-2 receptor gene in covid-19 and healthy control steps temp., °c time cycle initial denaturation 95 5 min. 1 denaturation 95 30 sec. 30 annealing 58 30 sec. extension 72 30 sec. final extension 72 5 min 1 table 3. comparison of age and body mass index and angiotensin-converting enzyme between moderate and severe covid-19 groups as compared with control group parameters moderate covid-19 n = 54 mean ± sd control group n = 63 mean ± sd p-value control group n = 63 mean ± sd severe covid-19 n = 59 mean ± sd p-value age, year 52.06 ± 14.35 40.03 ± 11.86 <0.001 40.03 ± 11.86 59.1 ± 12.64 <0.001 bmi, kg/m2 32.59 ± 5.52 33.31 ± 5.83 0.495 33.31 ± 5.83 33.07 ± 5.72 0.817 ace-2 activity, ng/ml 3.83 ± 0.82 3.03 ± 0.82 0.721 3.03 ± 0.82 4.74 ± 0.85 0.001 bmi, body mass index; n, number; significant p < 0.05; sd, standard deviation. not accurately indicate the degree of fatness. the body mass index was measure by dividing weight in kilograms by length of individual in square meter. results main features of 176 individuals included in this study were divided into 63 cases as apparently health control and the other 113 cases of them were infected with covid-19 as summarized in table 3. the covid-19 patients were sub-divided into two groups depending on the severity of the disease and some biomarker levels changes. mild covid-19 includes 54 patients and the remaining 59 patients were infected with severe covid-19 and its complications. the mean ± sd of age and bmi of each groups were indicated in table 3. in this table a significant differences between each of severe and moderate covid-19 group as compared with control group was observed with respect to the age (p < 0.001), while bmi data show a non-significant results (p = 0.817 and p = 0.495) respectively. the mean ± sd values of angiotensin converting enzyme-2 (ace-2) activity levels was higher in the severe and moderate covid-19 as compared with that found in healthy control (4.74 ± 0.85, 3.83 ± 0.82, and 3.03 ± 0.82 ng/ml) respectively. a non-significant differences between moderate and control groups (p = 0.721) was obtained and fig. 1 the amplification of rs4646994 region of human samples were fractionated on 1.5% agarose gel electrophoresis stained with ethidium bromoide. m: dna marker (ladder 100 bp). lanes 1-19 resemble 190, 490 bp of pcr products. 387j contemp med sci | vol. 7, no. 6, november-december 2021: 384–391 s.h. kamel et al. original ace receptor genotype and its activity level as potential predictors of the severity covid-19 among iraqi patients table 4. comparison of age and body mass index in severe covid-19 cases according to ace-2 gene polymorphism allele, id and ii obtained by polymerase chain reaction amplification as compared with control dd allele parameters moderate covid-19, n = 54 p-valueid allele n = 46 mean ± sd ii allele n = 8 mean ± sd age, year 52.91 ± 14.62 47.13 ± 12.32 0.297 bmi, (kg/m2) 33.07 ± 5.6 29.83 ± 4.38 0.126 n, number; bmi, body mass index; sd, standard deviation; p-value, prober value; id, insertion/deletion; ii, insertion/insertion. significant differences between severe covid-19 and apparently health control group (p < 0.001). the angiotensin-converting enzyme gene (ace-2) was located on chromosome 17q23.3, spansv21 kb, and comprises 26 exons and 25 introns. exon 26 encodes for the functionally important membrane-anchoring domain of the ace-2 protein. insertion (i allele) polymorphism had band in (490 bp) of an alu repetitive element in an intron of the ace-2 receptor gene that called homozygotes. deletion genotype (d allele) had band in (190 bp) that lack the repetitive element also called homozygotes, while (id) genotype had two band i and d in the same gene that mean in the same gene had insertion and deletion of an alu repetitive element in intron of the ace-2 receptor gene and it is called heterozygotes, so the two band in the same location differ in speed of migration as shown in figure 1. the (dd) genotype showed band at 190 bp, (ii) genotype showed band at 490 bp while (id) genotype showed both bands at 190 and 490 bp. the size of each of (dd, ii and id) genotypes was determined as indicated in table 4. therefore, individuals with normal homozygous deletion/ deletion (dd) revealed band size of (190 bp), while individuals with normal insertion/insertion (ii) revealed band size of (490 bp), and the individuals with heterozygous insertion/ deletion (id) revealed two bands size at each of (190, 490 bp) respectively, figure 1. table 4 shows the comparison of age and body mass index in moderate covid-19 group with the ace-2 receptor gene allele types by unpaired t test. in this table show age and bmi are non-significant correlated with moderate covid-19 group according to the allele (id) and (ii). table 5 shows a significant result for mean ± sd of age between severe covid-19 as compared with apparently control groups according to the ace-2 receptor gene polymorphism allele deletion/deletion (dd), but the relation with bmi was non-significant. table 6 show the results obtained for the relation between hypertension and t2dm associated with covid-19 are significant as compared with control groups according to the ace-2 receptor gene polymorphism allele (dd) but for moderate covid-19 group the relation was non-significant with ace-2 receptor gene polymorphism alleles (id) and (ii). as shown in table 6 the effect of smoking nicotine on the renin-angiotensin system was observed which indicated that 8.7% of angiotensin-converting enzyme receptor gene polymorphism allele (id) in moderate covid-19 and 8.5% of (dd) allele in severe covid-19 infection were smokers respectively table 5. shows a comparison between severe covid-19 and control groups according to the type of ace-2 receptor gene allele deletion/deletion for age and bmi parameters severe covid-19 dd allele n = 59 mean ± sd control dd allele n = 27 mean ± sd p-value age, year 59.1 ± 12.64 41.96 ± 12.62 <0.001 bmi, (kg/m2) 33.07 ± 5.72 33.93 ± 6.41 0.537 n, number; bmi, body mass index; sd, standard deviation; p-value, prober value; dd, deletion/deletion allele found in sever covid-19 and control group. table 6. comparison of gene polymorphism of ace-2 alleles (id and ii) with hypertension, type 2 diabetes mellitus and smoking in moderate covid-19 cases and compared with severe and control alleles (dd) parameters moderate covid-19 p-value severe covid-19 control p-valueid allele n = 46 (%) ii allele n = 8 (%) dd allele n = 59 (%) dd allele n = 27 (%) hypertension with 25 (54.3%) 5 (62.5%) 0.720 26 (44.1%) 0 (0.0) <0.001 without 21 (45.7%) 3 (37.5%) 33 (55.9%) 27 (100%) t2dm with 16 (34.8%) 4 (50.0%) 0.450 28 (47.5%) 0 (0.0) <0.001 without 30 (65.2%) 4 (50.0%) 31 (52.5%) 27 (100%) smoking yes 4 (8.7%) 0 (0.0) 1.000 5 (8.5%) 0 (0.0) 0.320 no 42 (91.3%) 8 (100%) 54 (91.5%) 27 (100%) n, number; t2dm, type 2 diabetes mellitus; id, insertion/deletion; ii, insertion/insertion; p value, prober value. as compared with control group. nicotine can impact the angiotensin-converting enzyme (ace-2), which is relevant because coronaviruses bind to ace-2. current and past tobacco smoking is associated with changes in ace-2 receptor expression. table 6 indicated that most of patients whether it is severe or moderate infection with covid-19 nonsmokers, the reason is due to nicotine may bind with the ace-2 receptor and decrease levels of ace-2 in multiple organs.27 smoking is associated with increased susceptibility and mortality in mers‐cov infection, potentially due to up-regulation of dipeptidyl peptidase‐iv, the host receptor for mers‐cov, in smokers. in the context of respiratory viruses, smoking has been reported to cause increased hospital and icu admissions with influenza infection, greater severity with respiratory syncytial virus bronchiolitis and increased mortality with viral pneumonia.28 in table 7 all parameters (ferritin, ldh, crp, ace2, alt and ast) are significantly elevated in moderate group according to the allele id compared with control group expect alp, while they are significantly elevated in severe covid-19 group as compared with control according to the dd allele expect ace-2 which was non-significantly changes. 388 j contemp med sci | vol. 7, no. 6, november-december 2021: 384–391 ace receptor genotype and its activity level as potential predictors of the severity covid-19 among iraqi patients original s.h. kamel et al. table 7. comparison of biochemical in ace-2 alleles, id allele in moderate covid-19 group as compared to id allele in control and also the dd allele in severe covid-19 as compared with control dd allele parameters id allele in moderate covid-19 group as compared with control dd allele in severe covid-19 group as compared with control group mean ± sd p-value group mean ± sd p-value ferritin, (ng/ml) moderate 602.24 ± 509.41 <0.001 severe 765.04 ± 359.37 <0.001 control 81.36 ± 47.31 control 70.08 ± 26.96 ldh activity, (u/l) moderate 395.78 ± 240.34 <0.001 severe 432.9 ± 211.86 <0.001 control 132.93 ± 37.95 control 140.98 ± 39.41 crp, (mg/dl) moderate 9.56 ± 3.26 <0.001 severe 15.02 ± 23.53 <0.001 control 0.34 ± 0.15 control 0.36 ± 0.13 ace-2 activity, (ng/ml) moderate 3.14 ± 0.83 0.001 severe 3.74 ± 0.85 0.345 control 2.55 ± 0.62 control 3.66 ± 0.64 alt activity, (u/l) moderate 60.81 ± 19.25 <0.001 severe 101.11 ± 266.69 <0.001 control 29.56 ± 11.71 control 26.54 ± 9.7 ast activity, (u/l) moderate 41.28 ± 12.32 <0.001 severe 51.6 ± 53.43 <0.001 control 22.77 ± 8.33 control 23.54 ± 8.78 alp activity, (u/l) moderate 89.87 ± 30.73 0.063 severe 109.48 ± 39.34 <0.001 control 77.12 ± 23.52 control 73.66 ± 24.41 ldh: lactate dehydrogenase; crp: creactive protein; ace-2: angiotensin converting enzyme; alt: alanine aminotransferase; alp: alkaline phosphatase; ast: aspartate aminotransferase; s.d: standard deviation; dd: deletion/deletion; id: insertion/deletion. table 8 show the comparison of biochemical in control group according to allele. in this table all parameters in control group are non-significant according to the allele dd and id expect ace-2 is significant. discussion as indicated previously, age and bmi are non-significantly in moderate covid-19 group according to the (id) and (ii) allele as shown in table 2, whereas age is significant between severe and control groups according to the deletion/deletion allele (dd), as shown in table 3 but bmi was non-significantly. the dd allele found in severe and control group but was absent in moderate group. the ace-2 receptor gene polymorphism allele (dd) in severe covid-19 was significantly associated with hypertension and t2dm as compared with control groups p < 0.001, but the (id) and (ii) alleles in moderate covid-19 group was association non-significantly, p = 0.720 and p = 0.450 respectively as shown in table 4. the total 113 cases of covid-19 patients were selected with demographic and clinical characteristics. we observed that high frequency of hypertensive patients was (54.3%) as compared to diabetic (34.8%) as shown in table 4. in contrast, de abajo et al., found higher prevalence of diabetes in 1339 covid-19 cases as compared with 13,390 matched controls (27.2% vs 20.3%; crude odds ratio, or, 1.50).29 similarity, singh et al., pooled different studies (n = 2209) and found higher percentage of hypertension (21%) as compared to diabetes (11%) and cvd (7%).30 in contrast, a meta-analysis of covid-19 patients (n = 1576 patients) reported percentage of different comorbid conditions such as hypertension (17%) > diabetes (8%).31 to date, few studies have been published that investigate the relationship between ace-2 gene polymorphism and covid-19 severity, but we are table 8. comparison of biochemical in control group according to allele parameters group mean ± sd p-value ferritin, (ng/ml) dd allele 70.08 ± 26.96 0.581 id allele 81.36 ± 47.31 ldh activity, (u/l) dd allele 140.98 ± 39.41 0.441 id allele 132.93 ± 37.95 crp, (mg/dl) dd allele 0.36 ± 0.13 0.278 id allele 0.34 ± 0.15 ace-2 activity, (ng/ml) dd allele 3.66 ± 0.64 <0.001 id allele 2.55 ± 0.62 alt activity, (u/l) dd allele 26.54 ± 9.7 0.305 id allele 29.56 ± 11.71 ast activity, (u/l) dd allele 23.54 ± 8.78 0.994 id allele 22.77 ± 8.33 alp activity, (u/l) dd allele 73.66 ± 24.41 0.420 id allele 77.12 ± 23.52 ldh, lactate dehydrogenase; crp, creactive protein; ace-2, angiotensin converting enzyme-2; alt, alanine aminotransferase; alp, alkaline phosphatase; ast, aspartate aminotransferase; sd, standard deviation; dd, deletion/deletion; id, insertion/deletion. still lacking definite results.17,32–34 in present study, we observed that individual with ‘dd’ genotype showed significantly 3.69fold higher risk of covid-19 severity. similarly, other studies found that ace-1 d/d-genotype showed association with covid-19 related mortality.35,36 gomez et al. found d allele was significantly associated with hypoxemic as compared to non-hypoxemic patients; however, ‘dd’ genotype individuals did not show any association with covid-19 infection.37 389j contemp med sci | vol. 7, no. 6, november-december 2021: 384–391 s.h. kamel et al. original ace receptor genotype and its activity level as potential predictors of the severity covid-19 among iraqi patients some studies have reported an association of the dd genotype of ace-2 polymorphism and higher circulating levels and activity of ace-2,38 which could explain the higher susceptibility to develop severe forms of the disease in patients with the dd genotype, in addition to hypertension and t2dm. sars-cov-2 sequesters ace-2 to invade cells, decreasing the bioavailability of ace-2 which entails a reduction in the degradation of ang ii and an exacerbation of the damaging effects of ang ii.39 thus, the lung injury and inflammation caused by the reduced ace-2 levels due to the viral infection, and also by the hypertension and diabetes may be worsened by ace-2 genotypes that further increase ace-2 levels, and hence ang ii levels, such as the dd genotype of ace-2 analyzed. in our study we confirmed that ace-2 levels are associated with the dd genotype of both polymorphisms, which in turn is associated with greater severity of the disease in hypertensive and t2dm. although according to our data we could not affirm a direct association with covid-19 severity. covid-19 is a highly contagious disease characterized by high mortality, especially for patients with severe comorbidities such as diabetes, hypertension, cvd and ckd.9 the documented history of diabetes has been stated to be an independent indicator of morbidity and death in sars patients.40,41 diabetes hyperglycemia is suspected to cause immune response dysfunction, which fails to regulate the spread of invasive pathogens. therefore, diabetic individuals are considered to be more prone to infections and incidence of infectious diseases and it will increase associated comorbidities.42 a study was released by the chinese centre for disease control and prevention, which showed elevated mortality rate in people with diabetes (2.3%, total and 7.3%, diabetes patients) study was performed in 72,314 cases of covid-19.43 blood pressure homeostasis maintained by renin–angiotensin system (ras).44,45 ras system modulated by ace-1 and ace-2, angiotensin i is converted into angiotensin ii (atii) by ace1 and degraded bioactive bradykinin.46 insertion/deletion (i/d) polymorphism has been correlated with levels of circulating and tissue ace-1 and influences almost half of the variability of serum ace levels in the general population. the ‘d’ allele of acei/d is associated with higher ace activity.47 this means individuals with dd genotype showed approximately twice ace activity levels as compared to ii genotype individuals.48 the ‘d’ allele of ace-1 gene is significantly associated with hypoxemic group as compared to non-hypoxemic group.49 recently, delanghe et al. found that the prevalence of covid-19 in 33 countries has been substantially associated with ace1 i/d polymorphism.50 however, our results clearly revealed that polymorphisms of the ace-2 gene are related to the risk of developing severe covid-19 (icu admission) in hypertension and diabetic patients. our study confirms with previously reported findings of another ace polymorphism on covid-19 patients with hypertension37 however; it did not corroborate the association with covid-19 severity found by gomez et al. and other authors for the ace i/d polymorphism, regardless of comorbidities. unlike these studies, we also found an additional association of d allele with enhanced severity in hypertension and diabetic patients, and interestingly, we further found a higher prevalence of the dd, id genotypes (all containing the d allele) among deceased icu-patients, not described so far, confirming the deleterious effect of the d allele in covid-19 outcomes. precisely most of these patients were hypertensive. in table 7 all parameters (ferritin, ldh, crp, ace-2, alt and ast) are significant in moderate group according to the allele id compared with control group expect alp. all these parameters are significantly elevated in severe group according to the allele dd as compared with control group expect ace-2, because d allele responsible for activity of ace-2 and this allele found homogenous at form dd in severe and control group that carry dd genotype, so that non-significant between severe and control according to the allele dd. genetic polymorphisms in ace-2 indicate that the ace i/d polymorphism, have been shown to affect ace-2 activity levels and confer susceptibility to hypertension type 2 diabetes,38 overweight nephropathy and certain cardiovascular and autoimmune diseases. more specifically, the dd genotype of the ace i/d polymorphism has been associated to higher levels of serum ace and higher levels of circulating il-6 in patients with myocardial infarction. in contrast, the ii genotype has been associated to lower circulating ace levels. since some of these processes have been reported to be involved in the pathogenesis of covid-19, the dd genotype could predispose to complications of covid-19 due to higher baseline ace levels and its consequences.37 indeed, an association of the dd genotype of the ace i/d polymorphism with severe covid-19 has been reported in hypertensive males.37 however, analyzing the i/d polymorphism is laborious and time-consuming and some authors have described a preferential amplification of the d allele. the ace-2 gene polymorphisms are in complete linkage disequilibrium with the ace i/d polymorphism, therefore they could be better prognostic markers. table 8 show all parameter are non-significant correlated because there were healthy peoples. ace-2 was significantly to dd and id alleles because the d allele is associated with higher ace-2 activity. mean ace activity levels in dd carriers were approximately twice that in ii genotype individuals. therefore, it may be propose a hypothesis that ace-2 gene polymorphism may play an important role in patients with covid-19 who are susceptible to develop severe lung injury or ards.48 conclusions the severity of covid-19 patients may depend on age, diabetes, hypertension and ace-2 gene polymorphism. the observed results suggest that a prospective paradigm of dd genotype that has the potential help to explain the susceptibility of the host response to sars-cov-2 infection and involve in numerous pathological. thus, ace-2 i/d polymorphism may be act as a useful tool to predict the development of disease and may have an influence on the treatment outcomes against the covid -19 to establish a population-based therapeutic development. acknowledgments all authors would like to thank the participated patients and the team of covid-19 centers for their support during this study. conflicts of interest none.  390 j contemp med sci | vol. 7, no. 6, november-december 2021: 384–391 ace receptor genotype and its activity level as potential predictors of the severity covid-19 among iraqi patients original s.h. kamel et al. references 1. world health organization. (2020). global surveillance for covid-19 disease caused by human infection with novel coronavirus (covid-19): interim guidance, 27 february 2020. world health organization. 2. hui, d. s., azhar, e. i., madani, t. a., ntoumi, f., kock, r., dar, o., et al. (2020). the continuing 2019-ncov epidemic threat of novel coronaviruses to global health—the latest 2019 novel coronavirus outbreak in wuhan, china. international journal of infectious diseases, 91: 264–266. 3. calisher c, carroll d, colwell r, corley rb, daszak p, drosten c, et al. (2020). statement in support of the scientists, public health professionals, and medical professionals of china combatting covid-19. the lancet, 395(10226), e42–e43. 4. singhal, t. (2020). a review of coronavirus disease-2019 (covid-1de abajo, f. j., rodríguez-martín, s., lerma, v., mejía-abril, g., aguilar, m., garcía-luque, a., and elvira, c. (2020). use of renin–angiotensin–aldosterone system inhibitors and risk of covid-19 requiring admission to hospital: a casepopulation study. the lancet, 395(10238), 1705-1714.9). the indian journal of pediatrics, 87(4): 281–286. 5. richardson, s., hirsch, j. s., narasimhan, m., crawford, j. m., mcginn, t., davidson, k. w., ... & northwell covid-19 research consortium. (2020). presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area. jama, 323(20): 2052–2059. 6. scully e.p., haverfield j., ursin r.l., tannenbaum c., klein s.l. considering how biological sex impacts immune responses and covid-19 outcomes. nat rev immunol. 2020;20(7):442–447. 7. gandhi, m., yokoe, d. s., & havlir, d. v. (2020). asymptomatic transmission, the achilles’ heel of current strategies to control covid-19. n engl j med 2020; 2160–382:2158. 8. lu, r., zhao, x., li, j., niu, p., yang, b., wu, h., et al. (2020). genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. the lancet, 395(10224): 565–574. 9. zhou, f., yu, t., du, r., fan, g., liu, y., liu, z., et al. (2020). clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet, 395(10229): 1054–1062. 10. strafella, c., caputo, v., termine, a., barati, s., caltagirone, c., giardina, e., & cascella, r. (2020). investigation of genetic variations of il6 and il6r as potential prognostic and pharmacogenetics biomarkers: implications for covid-19 and neuroinflammatory disorders. life, 10(12), 351. 11. torres m.m., acosta c.p., sicard d.m. susceptibilidad genética y riesgo de cáncer gástrico en una población del cauca. biomedica. 2004;24:153–162. 12. sieńko j., kotowski m., bogacz a. covid-19: the influence of ace genotype and ace-i and arbs on the course of sars-cov-2 infection in elderly patients. clin interv aging. 2020;15:1231–1240. 13. tabor, h. k., risch, n. j., & myers, r. m. (2002). candidate-gene approaches for studying complex genetic traits: practical considerations. nature reviews genetics, 3(5), 391–397. 14. guo, l., ren, l., yang, s., xiao, m., chang, d., yang, f., et al. (2020). profiling early humoral response to diagnose novel coronavirus disease (covid-19). clinical infectious diseases, 71(15): 778–785. 15. gheblawi m., wang k., viveiros a. angiotensin-converting enzyme 2: sarscov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2. circ. res. 2020;126(10): 1456–1474. 16. mariappan v., balakrishna s.r.r., pillai a. angiotensin-converting enzyme 2: a protective factor in regulating disease virulence of sars-cov-2. iubmb life. 2020 dec;72(12):2533–2545. 17. delanghe, j., speeckaert, m., et al. (2020). the host’s angiotensin-converting enzyme polymorphism may explain epidemiological findings in covid-19 infections. clinica chimica acta, 505: 192–193. 18. zhao y., zhao z., wang y., zhou y., ma y., zuo w. single-cell rna ex-pression profiling of ace2, the putative receptor of wuhan 2019-ncov. biorxiv. 2020. 19. gagliardi, m. c., tieri, p., ortona, e., & ruggieri, a. (2020). ace2 expression and sex disparity in covid-19. cell death discovery, 6(1): 1–2. 20. asselta, r., paraboschi, e. m., mantovani, a., & duga, s. (2020). ace2 and tmprss2 variants and expression as candidates to sex and country differences in covid-19 severity in italy. aging (albany ny), 12(11): 10087. 21. cai g. bulk and single-cell transcriptomics identify tobacco-use dis-parity in lung gene expression of ace2, the receptor of 2019-ncov. medrxiv. 2020. 22. cao y., li l., feng z., wan s., huang p., sun x. comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations. cell discov. 2020;6:11. 23. chen, n., zhou, m., dong, x., qu, j., gong, f., han, y., et al. (2020). epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet, 395(10223), 507–513. 24. sieńko j., kotowski m., bogacz a. covid-19: the influence of ace genotype and ace-i and arbs on the course of sars-cov-2 infection in elderly patients. clin interv aging. 2020;15:1231–1240. 25. pati abhijit. ace deletion allele is associated with susceptibility to sarscov-2 infection and mortality rate: an epidemiological study in the asian population. clinica chimica acta; international journal of clinical chemistry. 2020;510:455–458. 26. robinson, d. h., and lafleche, g. j. (2000). nucleic acid electrophoresis in agarose gels. essential molecular biology: a practical approach, 1: 89–119. 27. mohamed, t. l., nguyen, h. t., abdul-hafez, a., dang, v. x., dang, m. t., gewolb, i. h., & uhal, b. d. (2016). prior hypoxia prevents downregulation of ace-2 by hyperoxia in fetal human lung fibroblasts. experimental lung research, 42(3), 121–130. 28. han, l., ran, j., mak, y. w., suen, l. k. p., lee, p. h., peiris, j. s. m., et al. (2019). smoking and influenza-associated morbidity and mortality: a systematic review and meta-analysis. epidemiology, 30(3): 405–417. 29. de abajo, f. j., rodríguez-martín, s., lerma, v., mejía-abril, g., aguilar, m., garcía-luque, a., et al. (2020). use of renin–angiotensin–aldosterone system inhibitors and risk of covid-19 requiring admission to hospital: a case-population study. the lancet, 395(10238): 1705–1714. 30. singh, a. k., gupta, r., ghosh, a., & misra, a. (2020). diabetes in covid-19: prevalence, pathophysiology, prognosis and practical considerations. diabetes & metabolic syndrome: clinical research & reviews, 14(4), 303–310. 31. yan, y., yang, y., wang, f., ren, h., zhang, s., shi, x., et al. (2020). clinical characteristics and outcomes of patients with severe covid-19 with diabetes. bmj open diabetes research and care, 8(1), e001343. 32. dević pavlić, s., nadalin, s., starčević čizmarević, n., buretić-tomljanović, a., radojčić badovinac, a., et al. (2020). could angiotensin-converting enzyme 1 i/d polymorphism be a modificator of covid-19 response in different populations, diseases, and/or conditions?. journal of the renin-angiotensin-aldosterone system, 21(3): 1470320320957157. 33. gemmati, d., bramanti, b., serino, m. l., secchiero, p., zauli, g., et al. (2020). covid-19 and individual genetic susceptibility/receptivity: role of ace1/ ace2 genes, immunity, inflammation and coagulation. might the double x-chromosome in females be protective against sars-cov-2 compared to the single x-chromosome in males?. international journal of molecular sciences, 21(10): 3474. 34. hatami, n., ahi, s., sadeghinikoo, a., foroughian, m., javdani, f., kalani, n., et al. (2020). worldwide ace (i/d) polymorphism may affect covid-19 recovery rate: an ecological meta-regression. endocrine 2020;68: 479–484. 35. annunziata, s., bauckneht, m., albano, d., argiroffi, g., calabrò, d., abenavoli, e.,young committee of the italian association of nuclear medicine (aimn) et al. (2020). impact of the covid-19 pandemic in nuclear medicine departments: preliminary report of the first international survey. european journal of nuclear medicine and molecular imaging, 47: 2090–2099. 36. yamamoto, n., nishida, n., yamamoto, r., gojobori, t., shimotohno, k., mizokami, m., & ariumi, y. (2021). angiotensin–converting enzyme (ace) 1 gene polymorphism and phenotypic expression of covid-19 symptoms. genes, 12(10), 1572. 37. gómez, j., albaiceta, g. m., garcía-clemente, m., lópez-larrea, c., amado-rodríguez, l., lopez-alonso, i., ... & coto, e. (2020). angiotensinconverting enzymes (ace, ace2) gene variants and covid-19 outcome. gene, 762: 145102. 38. schüler, r., osterhoff, m. a., frahnow, t., seltmann, a. c., busjahn, a., kabisch, s., et al. (2017). high‐saturated‐fat diet increases circulating angiotensin‐converting enzyme, which is enhanced by the rs4343 polymorphism defining persons at risk of nutrient‐dependent increases of blood pressure. journal of the american heart association, 6(1): e004465. 39. chang, d., lin, m., wei, l., xie, l., zhu, g., cruz, c. s. d. et al. (2020). epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan, china. jama, 323(11): 1092-1093. 40. li, j., wang, x., chen, j., zhang, h., et al. (2020). association of reninangiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease 2019 (covid-19) infection in wuhan, china. jama cardiology, 5(7): 825–830. 41. yang, j. k., feng, y., yuan, m. y., yuan, s. y., fu, h. j., wu, b. y., et al. (2006). plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars. diabetic medicine, 23(6), 623–628. 391j contemp med sci | vol. 7, no. 6, november-december 2021: 384–391 s.h. kamel et al. original ace receptor genotype and its activity level as potential predictors of the severity covid-19 among iraqi patients 42. berbudi, a., rahmadika, n., tjahjadi, a. i., et al. (2020). type 2 diabetes and its impact on the immune system. current diabetes reviews, 16(5): 442. 43. wu, z., & mcgoogan, j. m. (2020). characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72,314 cases from the chinese center for disease control and prevention. jama, 323(13): 1239–1242. 44. boehm, m., and nabel, e. g. (2002). angiotensin-converting enzyme 2—a new cardiac regulator. new england journal of medicine, 347(22): 1795–1797. 45. skeggs jr, l. t., kahn, j. r., et al. (1956). the preparation and function of the hypertensin-converting enzyme. the journal of experimental medicine, 103(3): 295. 46. baudin, b. (2002). new aspects on angiotensin-converting enzyme: from gene to disease, walter de gruyter 40 (3), 256–265. 47. tiret, l., rigat, b., visvikis, s., breda, c., corvol, p., cambien, f., et al. (1992). evidence, from combined segregation and linkage analysis, that a variant of the angiotensin i-converting enzyme (ace) gene controls plasma ace levels. american journal of human genetics, 51(1): 197. 48. rigat, b., hubert, c., alhenc-gelas, f., cambien, f., corvol, et al. (1990). an insertion/deletion polymorphism in the angiotensin i-converting enzyme gene accounting for half the variance of serum enzyme levels. the journal of clinical investigation, 86(4): 1343–1346. 49. itoyama, s., keicho, n., quy, t., phi, n. c., long, h. t., van ban, v., et al. (2004). ace1 polymorphism and progression of sars. biochemical and biophysical research communications, 323(3): 1124–1129. 50. delanghe, j., speeckaert, m., & de buyzere, m. (2020). the host's angiotensinconverting enzyme polymorphism may explain epidemiological findings in covid-19 infections. clinica chimica acta, 505, 192–193. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1134 250 j contemp med sci | vol. 8, no. 4, july-august 2022: 250–253 original a study of relationship between sars-cov-2 antibodies levels and host factors among general population in zakho city, iraq nawfal r hussein1, ibrahim a naqid1*, shakir a jamal1, amer balatay2, nashwan ibrahim3, dildar h musa3, zana s m. saleem4 1department of biomedical sciences, college of medicine, university of zakho, kurdistan region, iraq. 2department of pharmacology and clinical pharmacy, college of pharmacy, university of duhok, kurdistan region, iraq. 3department of surgery, college of medicine, university of duhok, kurdistan region, iraq. 4department of internal medicine, college of medicine, university of duhok, kurdistan region, iraq. *correspondence to: ibrahim a. naqid (e-mail: ibrahim.naqid@uoz.edu.krd) (submitted: 04 april 2022 – revised version received: 22 april 2022 – accepted: 06 may 2022 – published online: 26 august 2022) abstract objectives: the aim of this cross-sectional study is to evaluate the seroprevalence of total antibodies to sars-cov-2 and associated risk factors in zakho city, kurdistan region of iraq. methods: this cross-sectional study was performed at zako city, northern, iraq. blood samples were collected from different districts of zakho city between january and march 2021. immunoassays were performed to detect the total antibodies against sars-cov-2, and the associations between several variables were investigated. results: a total of 315 participants were agreed and enrolled in the study. the average age of the participants was 32.87 ± 13.25 years. among the participants, 214/315 (67.93%) were found to have antibodies against sars-cov-2. among those who had positive antibodies, 169/214 (78.97%) were asymptomatic and 45/214 (21.03%) had a history of covid-19 related symptoms. the most common symptom was fever (95.56%), followed by loss of smell (84.44%) and myalgia (80.0%). we found that antibody levels significantly associated with age (p = 0.021). the presence of symptoms was significantly higher among subjects with a history of comorbidity diseases (p = 0.038), and older people (p = 0.015). a significant association was found between antibody levels and the marital status (p = 0.014), chronic diseases (p = 0.001), presence (p = 0.015) and duration of symptoms (p = 0.028). conclusion: a significant association between antibody levels to sars-cov-2 and age, chronic diseases, presence and duration of symptoms were found in this study. seroprevalence studies of antibodies to sars-cov-2 among general population are of utmost importance to develop infection prevention programs in our society. keywords: sars-cov-2, antibodies, risk factors, zakho city, iraq issn 2413-0516 introduction severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic is considered the most important healthcare crisis around the world since the discovery. this virus causes coronavirus infection (covid-19) which was first discovered as an outbreak in december 2019 in wuhan city, china and then rapidly spread throughout the world.1 the common clinical symptoms of the infection are fever, cough, shortness of breath, diarrheal, fatigue and myalgia.2-6 the infection may cause a clinical syndrome that may include pneumonia, respiratory failure, thromboembolic phenomena and eventually death in severe cases.7 in february 2020, the first cases of covid-19 infection were reported in kurdistan region, northern iraq. with the appearance of the first covid-19 cases in iraq, strict control measures were implemented in the kurdistan region to stop the spreading of such infection.8 after the measures were relaxed, a sharp increase in morbidity and mortality was recorded in the region. it was also reported that the re-infection rate of this virus in the region is very low.9 this report aimed to evaluate seroprevalence rate of total antibodies to sars-cov-2 and association between host factors and the levels of antibodies among general people in zakho city, iraq. materials and methods study setting and sample collection this cross-sectional study was performed at zakho city, duhok province, kurdistan region, northern iraq. samples were collected randomly from different districts of zakho city between january 10, 2021, and february 10, 2021. five millilitres of venous blood samples were collected using aseptic condition using disposable syringes. the blood samples were immediately transported to the laboratory, and sera were then separated from the blood at 3500 rpm for 10 min and kept frozen at –20°c until required. inclusion criteria a people more than 16 years old and resident at zakho city was included in the study, and agreed to participate in the study. the people who did not agree to participate and those who were absent on the day of sample collection were excluded in this study. measurement of covid-19 igg antibody elecsys anti-sars-cov-2 is used for the detection of total antibodies including igg to covid-19 virus in human serum. the assay procedure was performed according to the manufacturer’s instructions (roche diagnostics international ltd, switzerland). following the manufacturer, a cut-off index ≥ 1.0 indicates a positive result and <1.0 indicates a negative result. ethics the study design and procedure were approved by the ethics and scientific committee of the university of zakho’s college of medicine. informed consent was obtained from all subjects. 251j contemp med sci | vol. 8, no. 4, july-august 2022: 250–253 n.r. hussein et al. original a study of relationship between sars-cov-2 antibodies levels and host factors statistics a chi-square test and fisher’s exact tests was used to analyze categorical data. when variables were continuous, unpaired student t test was used to study the relationship between the variables and antibody levels. the relationship between the studied variables and igg levels was reported as mean ± standard deviation (sd). statistical analysis was performed using the graphpad prism version 8. p value < 0.05 was considered significant. results characteristics of subjects a total of 315 subjects were interviewed and recruited in present study. the average age of the subjects was 32.87 ± 13.25 years (range: 10–70 years), 190/315 (60.31%) were female. among the studied subjects, 249/315 (79.05%) were married and 60/315 (19.05%) had a history of comorbidity diseases such as hypertension and diabetics (table 1). forty three (13.65%) of the participants had a positive confirmed covid-19 using rtpcr (table 1). among the subjects, 214/315 (67.93%) tested positive for antibodies and 169/214 (78.97%) were asymptomatic and 45/214 (21.03%) had a history of covid-19 symptoms (table 2). the average duration of symptomatic subjects was 10.29 ± 5.21 days and the major symptom was fever 43/45 (95.56%), followed by loss of smell 38/45 (84.44%) and myalgia 36/45 (80.0%) and the minor symptom 6/45 (13.33) (table 1). the presence of symptoms was significantly higher among subjects with a history of comorbidity diseases (p = 0.038), and older people (p = 0.015), but no significant associations were reported between antibody positivity and gender (p = 0.49) or marital status (p = 0.56) (table 2). association between antibody levels and host factors the association between antibody levels and the age of the recruited participants was determined using pearson’s correlation coefficient. it was found that antibody levels significantly associated with age (pearson’s correlation coefficient; r = 0.156; r squared = 0.024; p = 0.021) (figure 1). unpaired student t test was used to investigate the relationship between antibody levels and the studied factors including gender, marital status, chronic diseases, history of confirmed covid-19, presence of symptoms and duration of symptoms (table 3). we found a significant association between antibody levels and the marital status (p = 0.014), chronic diseases (p = 0.001), presence of symptoms (p = 0.015) and duration of symptoms (p = 0.028). however, no significant associations were found between antibody levels and gender (p = 0.83) (table 3). table 1. characteristics of the participants recruited in this study (n = 315) characteristics of participants frequency percent (%) age (years ± std) 32.87 ± 13.25 gender: female 190 60.32 marital status: married 249 79.05 positive confirmed covid-19 43 13.65 covid-19 antibody: positive 214 67.93 comorbidity diseases (yes) 60 19.05 symptoms 45 14.28 fever 43 95.56 loss of smell 38 84.44 covid-19 symptoms myalgia 36 80.0 loss of appetite 28 62.22 shortness of breath 28 62.22 diarrhea 6 13.33 duration of symptoms (days ± std) 10.29 ± 5.21 table 2. associations between variables and total antibody positivity among 214 subjects variables asymptomatic positive (n = 169) symptomatic positive (n = 45) p-value no. (%) no. (%) gender: female 108 (63.91) 26 (57.78) 0.49 marital status (married) 127 (75.15) 36 (80) 0.56 chronic disease (yes) 30 (17.75) 15 (33.33) 0.038 age (year ± sd) 31.47 ± 12.57 36.73 ± 13.67 0.015 252 j contemp med sci | vol. 8, no. 4, july-august 2022: 250–253 a study of relationship between sars-cov-2 antibodies levels and host factors original n.r. hussein et al. discussion up to date, sars cov-2 has infected more than 380 million people, globally. in kurdistan region of iraq, more than 409 thousand people have been infected and over 7 thousand people have died.10 in this project we aimed at studying the prevalence of anti-sars-cov-2 in zakho city. we found that 214/315 (63.88%) of the participants tested positive for antisars-cov-2 antibodies. it is important to mention that our samples were not random and a population-based study is needed to determine the prevalence of infection. however, this study gave an insight into the infection rate in the city. recalling that reinfection is rare, the infection rate may start to decrease in the city. nevertheless, it is important to note that it is unclear whether post infection immunity, specifically that provided by humoral immunity, protects people or generates strong immunity for long term, and further studies are required to explain this. the most common clinical features associated with covid-19 infection have previously been reported to be fever, dry cough, shortness of breath, loss of appetite and smell.3,11,12 in our study, the most common symptoms were fever, loss of smell, myalgia, loss of appetite and mild shortness of breath. among those who tested positive for sars-cov-2 antibodies, 77% denied any history of symptoms. this indicates that the majority of infection was asymptomatic and did not require medical help. this might be due to that the majority of recruited samples were young and without comorbid diseases. in support of this, we found a significant association between the presence of symptomatic infection with age and comorbidities. our results are in agreement with a study conducted in iraq, they found that the antibody positivity levels increases gradually with the age.13 several studies have also confirmed that old patients with chronic diseases are at increased risk for more severe disease and adverse outcomes associated to covid-19.14-16 this might be explained by that the immune system is waned in elderly and with the presence of comorbidities allowing severer infection. in agreement with another study,16 we found a significant association between the presence of symptoms and long duration of symptoms with higher antibody levels. this might be due to that severe long symptomatic infection induces stronger immune reaction and hence higher antibody levels. more studies are required to investigate the effects of different host factors and their association with antibody positivity levels in our locality. we found no significant relationship between marital status and sex with the levels of antibody positivity, which is in line with the results of our previous study.13 however, a study conducted in iran reported that seropositivity was significantly associated with the female gender.17 the discrepancy between the two studies could be due to their specific study population and relatively small number size of recruited in our study. more studies are needed to determine the reason for gender-related differences in the seroprevalence rate of infection. our study has limitations. first, our sample was not random therefore the result should be interpreted with fig. 1 the association between antibody levels and age. p value was calculated using pearson’s correlation coefficient. (r = 0.156; 95% ci, 0.023–0.285, r squared = 0.024; p = 0.021). table 3. the association between various factors and antibodies level variables levels of antibody positivity (average ± std) p-value gender female (65.103 ± 61.65) male (66.91 ± 56.58) 0.83 marital status married (71.42 ± 61.32) single (48.18 ± 50.14) 0.014 chronic diseases yes (99.58 ± 65.49) no (58.25 ± 55.76) 0.001 symptoms yes (74.92 ± 69.44) no (60.76 ± 56.11) 0.015 duration of symptoms <7 days (46.49 ± 46.54) >7 days (89.97 ± 71.47) 0.028 253j contemp med sci | vol. 8, no. 4, july-august 2022: 250–253 n.r. hussein et al. original a study of relationship between sars-cov-2 antibodies levels and host factors caution. random sample collection was extremely difficult due to the restriction and pandemic control measures. second, the sample size was relatively small. however, our results give an insight into the situation in the city until further research is performed. in conclusion, a significant relationship between seropositivity rate of covid-19 infection and age, chronic diseases, presence of symptoms and duration of symptoms were observed in this study. seroprevalence studies about covid-19 infection among population are of utmost importance to develop infection control programs in our locality. a population-based study is needed to determine the accurate prevalence rate of the infection using large number of samples. references 1. zhu, n., et al., a novel coronavirus from patients with pneumonia in china, 2019. n engl j med, 2020. 382(8): p. 727–733. 2. saleem, z.s.m., severity of covid-19 infection among kidney transplant recipients in duhok city, kurdistan region, iraq. journal of contemporary medical sciences, 2022. 8(1). 3. hussein, n.r., i.a. naqid, and z.s.m. saleem, a retrospective descriptive study characterizing coronavirus disease epidemiology among people in the kurdistan region, iraq. mediterr j hematol infect dis, 2020. 12(1): p. e2020061. 4. hussein, n.r., et al., home management scheme for patients with severe covid-19 in duhok city, kurdistan region of iraq: a possible role for family physicians. journal of family medicine and primary care, 2021. 10(11). 5. ren, x., zhou, j., guo, j. et al. reinfection in patients with covid-19: a systematic review. glob health res policy 7, 12 (2022). 6. hussein, n.r., et al., the first case of covid-19 reinfection in duhok city, kurdistan region of iraq: a case report. j kermanshah univ med sci, 2020. 24(4): p. e111454. 7. mousavi, m., et al., prevalence of adverse effects of covid19 vaccines among a sample of iranian healthcare workers; a comparison between the three available vaccines in iran. journal of contemporary medical sciences, 2021. 7(6). 8. hussein, n.r., et al., the impact of breaching lockdown on the spread of covid-19 in kurdistan region, iraq. avicenna j clin microbiol infect, 2020. 7(1): p. 34–35. 9. hussein, n.r., et al., the risk of sars-cov-2 reinfection in duhok city, kurdistan region of iraq. mediterranean journal of hematology and infectious diseases, 2021. 13(1). 10. moh., latest information about coronavirus (covid-19). https://gov.krd/ coronavirus-en/dashboard/ 2022 11. sgery, a.s.h., y.m. hadi, and a.a. goreal, seasonal theory of covid-19; one-year observation. journal of contemporary medical sciences, 2021. 7(6). 12. jiang, f., et al., review of the clinical characteristics of coronavirus disease 2019 (covid-19). j gen intern med, 2020. 35(5): p. 1545-1549. 13. hussein, n.r., et al., covid-19 antibody seroprevalence in duhok, kurdistan region, iraq: a population-based study. medrxiv, 2021: p. 2021.03.23.21254169. 14. dai, m., et al., patients with cancer appear more vulnerable to sarscov-2: a multicenter study during the covid-19 outbreak. 2020. 10(6): p. 783–791. 15. liang, w., et al., cancer patients in sars-cov-2 infection: a nationwide analysis in china. lancet oncol, 2020. 21(3): p. 335–337. 16. klein, s.l., et al., sex, age, and hospitalization drive antibody responses in a covid-19 convalescent plasma donor population. j clin invest, 2020. 130(11): p. 6141-6150. 17. javadinia, s.a. and m. ariamanesh, multicenter study of antibody seroprevalence against covid-19 in patients presenting to iranian cancer centers after one year of the covid-19 pandemic. 2022. 40(2): p. 115–123. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i4.1256 175j contemp med sci | vol. 9, no. 3, may-june 2023: 175–178 original glyceryl trinitrate, a vasodilating drug acts as an antibiofilm agent in serratia marcescens ziyad hameed al-fayyadh1, ahmed mohammed turki1, harith jabbar fahad al-mathkhury2* 1department of biology, college of science, university of anbar, al-anbar, iraq. 2department of biology, college of science, university of baghdad, baghdad, iraq. *correspondence to: harith jabbar fahad al-mathkhury (e-mail: harith.fahad@sc.uobaghdad.edu.iq) (submitted: 17 march 2023 – revised version received: 05 april 2023 – accepted: 27 april 2023 – published online: 26 june 2023) abstract objectives: serratia marcescens is a gram-negative pathogen of many species. the ability of s. marcescens to form biofilms and its potent innate resistance to antimicrobials and cleaning solutions are both essential for its pathogenicity and survival. the present study was conducted to investigate the effect of glyceryl trinitrate (gtn) on the biofilm of s. marcescens, as an alternative for antibiotic therapy. methods: different specimens, including ear swabs, burns, mid-stream urine, wound swabs, and sputum, were collected from patients who were brought to al-ramadi hospital, iraq. all samples were cultured, and the colonies that were obtained were detected using the vitek® 2 compact. the ability of biofilms to develop was examined using the microtiter plate technique. the bactericidal effectiveness of gtn was estimated by the broth microdilution technique. the presence of fima and fimc in s. marcescens isolates was detected using the polymerase chain reaction (pcr) method. quantitative real-time polymerase chain reaction (qrt-pcr) was used to assess the effect of gtn on fima and fimc gene expression. results: the results demonstrated that gtn has no effect on s. marcescens growth; while its biofilm was significantly (p < 0.05) influenced. moreover, all s. marcescens isolates had fima and fimc, and the presence of gtn reduced the expression of these genes. conclusion: the findings of this study reveal that gtn can act as a promising antibiofilm agent in reference to s. marcescens. keywords: fima, fimc, glyceryl trinitrate, serratia marcescens issn 2413-0516 introduction serratia marcescens is a gram-negative, opportunistic, and motile nosocomial pathogen of the enterobacteriaceae family. it produces the characteristic red pigment known as prodigiosin.1 fimbria or pili, the synthesis of which is reportedly associated with the development of biofilm in s. marcescens, are used by bacteria to adhere to tissues in order to cause infections. the fimabcd operon encodes type i pili. the primary fimbrial subunits of s. marcescens are known to be fima and fimc.2 the biofilms produced by s. marcescens are distinct from those produced by other species of traditional biofilm-forming bacteria, such as p. aeruginosa and e. coli, which are composed of undifferentiated cells arranged in microcolonies.3 according to labbate et al.4 the development of s. marcescens biofilms is a genetically controlled process. this results in cell and structure differentiation, as evidenced by the development of extended threadlike cells, bacterial cell assemblages alongside the threadlike cells, and interconnected cell aggregates as the biofilm matures. these bacteria exhibit both intrinsic and multidrug resistance to many drugs. treatment success for s. marcescens as a nosocomial causative agent depends heavily on the timing of medication depending on the results of antibiotic susceptibility tests.5 according to a local investigation by zahraa and saad,6 34 (68%) of the s. marcescens isolates produced extended-range beta-lactamases. glyceryl trinitrate (gtn) or nitroglycerin (1, 2, 3 propanetriol trinitrate) is an organic nitrate that acts as a vasodilator.7 glycerol trinitrate possesses anti-microbial characteristics in addition to its anti-hypertensive effect. it has the potential to suppress candida albicans and p. aeruginosa planktonic growth.8,9 glycerol trinitrate significantly inhibited biofilm, staphyloxanthin, and oxidative stress tolerance in s. aureus.10 the present study was aimed at investigating the role of gtn in the biofilm of s. marcescens, as a replacement for antibiotic therapy. materials and methods isolation and identification of bacterial isolates from february to june 2021, 316 different specimens, including ear swabs, burns, mid-stream urine, wound swabs, and sputum, were collected from patients who were brought to al-ramadi hospital, iraq. an informed consent according to the declaration of helsinki was obtained from all participants. a sterile cotton swab was used to collect the swabs, and they were then taken to the laboratory in a sterile tube containing normal saline solution. all samples were cultured on blood agar and aerobically incubated at 37°c for 24 hours. the colonies that formed were grown on macconkey agar and incubated under the same conditions. using the sophisticated colorimetric approach of vitek 2 compact (biomérieux, france), the dark red colonies (lactose fermenters) were chosen for the identification of s. marcescens. a gram-negative (gn) card was used to identify s. marcescens. determination of glyceryl trinitrate’s minimal inhibitory concentration to determine the minimal inhibitory concentration (mic) of gtn, a broth microdilution test was used,11 with progressive twofold concentrations (0.01 to 0.175 mg/ ml) of gtn. biofilm assay biofilm formation was quantified according to the method described by.12 all the isolates were grown in brain heart infusion broth for 18 hours at 37°c. each isolate was diluted in https://orcid.org/0000-0002-5414-9834 mailto:harith.fahad@sc.uobaghdad.edu.iq 176 j contemp med sci | vol. 9, no. 3, may-june 2023: 175–178 glyceryl trinitrate, a vasodilating drug acts as an antibiofilm agent in serratia marcescens original h. j. f. al-mathkhury et al. tryptic soy broth (tsb), which contains 1% glucose, and pipetted thoroughly. a bacterial isolate suspension was adjusted to the 0.5 mcfarland standard. an aliquot of 200 µl of an isolate culture was added to three wells of 96-well polystyrene microplates with a u-shaped bottom. the plate was then covered and incubated for 24 hours at 37°c. following incubation, the microplates were twice cleaned to get rid of loose bacteria, tapped on paper towels (filter paper) to get rid of any remaining water, and then air-dried. each well was fixed for 20 minutes at room temperature with 200 μl of absolute methanol. an aliquot of 200 μl of 0.1% crystal violet was applied for 15 minutes. after the staining reaction was completed, excess dye was removed by repeatedly washing (2–3 washes) with distilled water. the plates were then dried by leaving them at room temperature for around 30 minutes to ensure they were completely dry. following that, 200 μl of 95% ethanol was placed into each well and left there for 10 minutes. ethanol was used to dissolve the crystal violet dye that was attached to the adherent cells. the experiment was performed in triplicate, with the absorbance of bacteria-free tsb-containing wells representing the negative control. quantification was carried out using a microplate reader at od600. the classification of bacterial adherence presented in table 1 was utilized for data simplification and computation based on od600 values obtained for individual isolates of s. marcescens. the cut-off value (odc) was determined as follows: odc = od600 of the negative control + 3 (standard deviation) (1) all experiments followed the same procedure to assess the effect of gtn on biofilm, with one exception. tryptic soy broth contained gtn at a concentration of 0.175 mg/ml. detection of fima and fimc genes using the polymerase chain reaction the genomic dna of the bacterial isolates was extracted using the abiopuretm total dna (usa) kit. pcr reaction tubes containing the mixture were placed in the thermocycler, and dna was amplified using the primers grouped in table 2 using the reactants indicated in table 3. the pcr conditions were optimized by using gradient pcr (table 4). thereafter, amplicons were resolved on a 1.5% agarose gel. measurement of gene expression using quantitative real-time polymerase chain reaction in addition to examining the effect of gtn on target gene expression, a biofilm investigation for five isolates (whose biofilm was strongly influenced by the gtn) was carried out in microtiter plates. a similar technique was used with gtn, including mueller hinton broth at 0.175 mg/ml. rna was extracted from the selected isolates with and without gtn treatment using trizoltm reagent following the manufacturer’s instructions. the primers listed in table 2 were used in the real-time polymerase chain reaction (qrt-pcr). table 5 summarizes the reaction combinations. also, the final procedure is displayed in table 4. relative quantitation was used to determine gene expression levels. the fold changes were assessed between the treated groups and each gene’s calibrators.13 these values were normalized to rplu as shown below: folding =2-δδct (1) table 1. calculation of biofilm intensity mean od 600 biofilm intensity od ≤ odc* non-biofilm producer odc < od ≤ 2odc weak 2odc < od ≤ 4odc moderate od > 4odc strong *cut off value source: khayyat et al. (2021) table 2. primers used in this study primer name sequence (5´–3´) product size (bp) fima f actacaccctgcgtttcgac 259 r gcgttagagtttgcctgacc fimc f aagatcgcaccgtacaaacc 259 r tttgcaccgcatagttcaag rplu f gcttggaaaagctggacatc 192 r tacggtggtgtttacgacga source: srinivasan et al. (2017) table 3. components of conventional polymerase chain reaction component volume (µl) master mix 10 µl forward primer (10 μm) 1 µl reverse primer (10 μm) 1 µl nuclease free water 6 µl template dna 2 µl final volume 20 µl table 4. polymerase chain reaction amplification program step temperature (°c) minute:second cycles initial denaturation 95 10:00 1 denaturation 95 00:45 40 annealing 57 00:45 extension 72 00:50 source: srinivasan et al. (2017) table 5. components of the quantitative real-time polymerase chain reaction (qrt-pcr) master mix components volume (μl) qpcr master mix 5 rt mix 0.25 mgcl 2 0.25 forward primer 0.5 reverse primer 0.5 nuclease free water 1.5 rna 2 total volume 10 177j contemp med sci | vol. 9, no. 3, may-june 2023: 175–178 h. j. f. al-mathkhury et al. original glyceryl trinitrate, a vasodilating drug acts as an antibiofilm agent in serratia marcescens δδct =δct treated (t) δct untreated (c) (2) δct = ct of target gene – ct of housekeeping gene (3) a fold change of less than twofold was considered insignificant.14 a melting curve was obtained with temperatures ranging from 72 to 95°c at 0.3°c/s. statistical analysis each experiment was replicated three times (n = 3). data were analyzed using graphpad prism 9 software using a two-tailed student’s t-test and a one-way anova. a p-value of < 0.05 was considered significant. results occurrence of bacterial isolates in specimens out of 360 specimens, 305 showed positive bacterial growth on blood agar and macconkey agar. using the vitek 2 compact system, 50 (16.3%) isolates were identified as s. marcescens. the highest isolation percentage was from wound infections (42%, n = 21), followed by burns (34%, n = 17). values of 10% (n = 5) and 14% (n = 7) were observed for midstream urine and sputum specimens, respectively. nevertheless, the bacterium was not isolated from ear swabs. biofilm formation by serratia marcescens all s. marcescens isolates produced biofilm. however, a one-way anova showed no differences between the biofilms that were produced (p > 0.05). in addition, 15 and 35 isolates, respectively, formed weak and moderate biofilms. none of the isolates could develop a strong biofilm. on the other hand, gtn significantly (p < 0.05) reduced the biofilm intensity of s. marcescens isolates, as shown in figure 1. fima and fimc detection in bacterial isolates the pcr amplification revealed the presence of fima and fimc genes in all the s. marcescens isolates, as presented in figure 2. effect of glyceryl trinitrate on fima and fimc gene expression figure 3 showed that gtn reduced the expression of the fima and fimc genes in three isolates, while the gene expression in the other two isolates was unaffected. discussion serratia marcescens is a significant human opportunistic pathogen that has been linked to a variety of nosocomial infections, including bacteremia, respiratory tract infections, eye infections, and most significantly, urinary tract infections. it produces biofilm and releases a range of virulence factors through a signal-mediated qs mechanism.15 in mosul, iraq, ali16 reported that 150 different specimens were isolated, including 3 from blood (15%), 2 from throat swabs (20%), 3 from urine (6%), and 4 from wounds (8%). in basra, iraq, mahdi17 stated that out of 160 blood samples from neonatal patients in the neonatal intensive care unit, 5 isolates were identified as s. marcescens. regardless of the reservoir, s. marcescens has a rather wide distribution among patients with a variety of clinical cases, and hospital staff is thought to be the main source of s. marcescens infection spread by direct contact.18 patients who have severe clinical problems, long hospital stays, and repeated medical interventions are more likely to contract an infection since these factors require more frequent and intense direct contact with staff hands.19 the presence of type 1 fimbriae (figure 2) and the downregulation of the fima and fimc genes are evidence that all s. marcescens formed biofilm, as can be observed from the current results. in spite of that, gtn had no impact on s. marcescens’ development. a search for an alternative therapy option besides antibiotics is essential given the significant antibiotic resistance of s. marcescens. the findings of this study revealed that gtn can serve this function because it has no effect on cell viability. instead, it damaged the pathogen’s biofilm, which is an essential component of its pathogenicity. fig. 3 effect of glyceryl trinitrate on the fima and fimc expression. fig. 1 effect of glyceryl trinitrate on serratia marcescens biofilms. asterisks: significant difference over the control; horizontal lines: mean ± standard deviation. fig. 2 fima, fimc, and rplu amplicons of selected serratia marcescens isolates run on ethidium bromide-containing agarose gel (1.5%) at 5 v/cm. 178 j contemp med sci | vol. 9, no. 3, may-june 2023: 175–178 glyceryl trinitrate, a vasodilating drug acts as an antibiofilm agent in serratia marcescens original h. j. f. al-mathkhury et al. conflicts of interest the authors have no potential conflicts of interest to disclose.  references 1. nelson g, greene m. enterobacteriaceae. in: john e. bennett, raphael dolin, martin j. blaser, editors. mandell, douglas, and bennett’s principles and practice of infectious diseases. 9th ed: elsevier; 2020. 2. shanks rm, stella na, brothers km, polaski dm. exploitation of a “hockey-puck” phenotype to identify pilus and biofilm regulators in serratia marcescens through genetic analysis. can j microbiol. 2016;62(1):83–93. 3. reisner a, haagensen ja, schembri ma, zechner el, molin s. development and maturation of escherichia coli k-12 biofilms. mol microbiol. 2003;48(4):933–46. 4. labbate m, queck sy, koh ks, rice sa, givskov m, kjelleberg s. quorum sensing-controlled biofilm development in serratia liquefaciens mg1. j bacteriol. 2004;186(3):692–8. 5. şimşek m. determination of the antibiotic resistance rates of serratia marcescens isolates obtained from various clinical specimens. niger j clin pract. 2019;22(1):125–30. 6. zahraa as, saad lh. molecular detection of extended-spectrum β-lactamasesproducer serratia marcescens causing neonatal sepsis in iraq. international journal of research in pharmaceutical sciences. 2020;11(4):5803–8. 7. kim kh, kerndt cc, adnan g, schaller dj. nitroglycerin: statpearls publishing; 2022. 8. palmeira-de-oliveira a, ramos ar, gaspar c, palmeira-de-oliveira r, gouveia p, martinez-de-oliveira j. in vitro anti-candida activity of lidocaine and nitroglycerin: alone and combined. infect dis obstet gynecol. 2012;2012:727248. 9. rosenblatt j, reitzel ra, raad i. caprylic acid and glyceryl trinitrate combination for eradication of biofilm. antimicrob agents chemother. 2015;59(3):1786–8. 10. abbas ha, elsherbini am, shaldam ma. glyceryl trinitrate blocks staphyloxanthin and biofilm formation in staphylococcus aureus. afr health sci. 2019;19(1):1376–84. 11. andrews jm. bsac standardized disc susceptibility testing method. j antimicrob chemother. 2001;48:43–57. 12. khayyat an, hegazy wah, shaldam ma, mosbah r, almalki aj, ibrahim ts, et al. xylitol inhibits growth and blocks virulence in serratia marcescens. microorganisms. 2021;9(5). 13. livak kj, schmittgen td. analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method. methods. 2001;25(4):402–8. 14. rasigade jp, moulay a, lhoste y, tristan a, bes m, vandenesch f, et al. impact of sub-inhibitory antibiotics on fibronectin-mediated host cell adhesion and invasion by staphylococcus aureus. bmc microbiol. 2011;11:263. 15. srinivasan r, mohankumar r, kannappan a, karthick raja v, archunan g, karutha pandian s, et al. exploring the anti-quorum sensing and antibiofilm efficacy of phytol against serratia marcescens associated acute pyelonephritis infection in wistar rats. front cell infect microbiol. 2017;7:498. 16. ali t. antibiomicrobial susceptibility testing of serratia species isolated from hospitalized patients in two hospitals in al-mosul, iraq. jordan m j. 2007;41:121–8. 17. mahdi s. isolation and molecular identification of a serratia spp. from suspected neonatal sepsis in intensive care unit (icu) of basra province, iraq int j inn res sci eng technol. 2016;5:4619–24. 18. mahlen sd. serratia infections: from military experiments to current practice. clin microbiol rev. 2011;24(4):755–91. 19. passaro dj, waring l, armstrong r, bolding f, bouvier b, rosenberg j, et al. postoperative serratia marcescens wound infections traced to an out-ofhospital source. j infect dis. 1997;175(4):992–5. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1329 218 j contemp med sci | vol. 3, no. 10, spring 2017: 218–223 research antimicrobial effect of probiotic lactobacillus spp. on pseudomonas aeruginosa maysaa kadhim al-malkey,a munira ch. ismeeal,a fahema jabbar abo al-hur,a sinaa w. mohammed,a hanan j. nayyefa atropical-biological research unit, college of science, university of baghdad, iraq. correspondence to maysaa kadhim al-malkey (email: maysakadhim@uobaghdad.edu.iq). (submitted: 05 january 2017 – revised version received: 17 january 2017 – accepted: 22 february 2017 – published online: 26 june 2017) objectives study the antimicrobial effect of probiotics produced from lactobacillus rhamnosus gg and lactobacillus acidophilus on pseudomonas aeruginosa isolated from burn and wound infection and their ability of protease production. methods swab samples were collected from 70 patients admitted at burns center/al-yarmouk teaching hospital. primary bacterial identification cultured on differential selective media and biochemical tests were done. the vitek2 compact system (biomerieux, france) was used to confirm the p. aeruginosa isolates by gram negative identification card and the antimicrobial susceptibility test card to each isolate was performed. protease production using skimmed milk agar 1% was performed. crud bacteriocin produced from l. acidophilus (holland & barrett, usa) and l. rhamnosus (health gensis, usa) was extracted during log phase using mrs broth (24 h/ 37ºc/ 5–10% co 2 ), then cool centrifugation was done (6000 rpm at 4ºc for 10 min). protein concentration of bacteriocin was estimated using bradford assay using bovine serum albumin as standard. results only 31 out of 48 isolates were identified as p. aeruginosa; 9 (45%) from wound and 22 (79%) from burn swabs. antimicrobial susceptibility tests included 16 antibiotics; p. aeruginosa isolates showed multi-drug resistance for antibiotics. all p. aeruginosa isolates were having the ability for protease enzyme production. antimicrobial effect of bacteriocin produced by l. acidophilus and l. rhamnosus on protease using plate diffusion method showed positive results. protein concentration of bacteriocin produced by l. rhamnosus and l. acidophilus were 74 mg/ml, 44 mg/ml, respectively. the highest zone of antimicrobial effect by l. rhamnosus was 32 mm and by l. acidophilus was 25 mm using well diffusion method. conclusions p. aeruginosa showed a multi-drug resistance and had the ability to produce protease enzyme. bacteriocin produced by l. acidophilus and l. rhamnosus showed an acceptable positive results to be used as potential alternative bio-remedy to overcome the multi-drug resistance dilemma. keywords antimicrobial effect, lactobacillus spp., bacteriocin, pseudomonas aeruginosa, protease introduction pseudomonas aeruginosa is a gram-negative aerobic bacilli and the natural flora of the skin and intestinal tract that is also found in water and soil. it is an opportunistic pathogen and one of the main causes of nosocomial infections causes severe diseases like cystic fibrosis, urinary tract infections, acute purulent meningitis, otitis media, otitis external, eye infections, wound and burn infections, septicaemia and infantile diarrhea.1 the multiple resistant to the most commonly used antibiotics is quite common in p. aeruginosa due to possession of high number of virulence factors, which is attributable to a concerted action of multidrug efflux pumps with a chromosomally encoded antibiotic resistance genes and the low permeability of bacterial cellular envelopes as well as biofilm formation phenomenon.2 wound infections due to p. aeruginosa are especially difficult in burn patients, high percentage of wound infections will lead to sepsis with significant mortality rates.3 protease-deficient strains are generally less virulent than protease producers in burned mouse models.4 proteases are enzymes that can hydrolyze peptide bonds within peptides and proteins. p. aeruginosa secretes several proteases (protease iv, elastase b, elastase a, and alkaline protease), which play an important role during infection with p. aeruginosa and are a characteristic for invasiveness as determined in clinical strains.5 these proteases are able to degrade a whole range of biological important host proteins such as fibrinogen, elastin, collagen and plasminogen as well as immunoglobulin g (igg) and the complement components 3 and c1q, which belong to the immune defense system.6 so, there is a considerable interest in developing low cost large-scale alternative remedies to prevent or reduce the multi-drug resistance of p. aeruginosa. in this regard, probiotics may close the therapeutic gap. probiotics are living microbial species, when administered in adequate amounts confer a health benefit to the host.7 probiotics have been proven to be useful in the treatment of several infections and gastrointestinal diseases such as acute diarrhea.8 multiple mechanisms have been proposed to justify the protective and therapeutic role of probiotics including lactose digestion, production of antimicrobial agents, pathogen exclusion and immunomodulation.9–11 commercially available probiotic preparations including lactic acid bacilli (lactobacillus rhamnosus, lactobacillus acidophilus, l. plantarum, l. cassei, etc.) alone or in combination with streptococcus and saccharomyces species have shown the beneficial effects.12 lactobacilli are known to produce a variety of metabolic by-products in addition to biosurfactants. some of which have antimicrobial activity including lactic acid, hydrogen peroxide, bacteriocins, and bacteriocin-like substances which has imperative biomedical advantages.13 bacteriocins are ribosomally synthesized low-molecular weight peptides or proteins with potential use in food preservation due to their bactericidal effects on food spoilage and pathogenic organisms.14 bacteriocins have unique applications in food processing and food safety because of their heat stability and sensitivity to proteolytic enzymes.15 issn 2413-0516 maysaa kadhim al-malkey et al. 219j contemp med sci | vol. 3, no. 10, spring 2017: 218–223 research antimicrobial effect of probiotic lactobacillus spp. on pseudomonas aeruginosa the current study aimed to investigate the antimicrobial effect of the probiotic strains l. rhamnosus gg and l. acidophilus on p. aeruginosa isolated from wound and burn infection in vitro. patients and methods bacterial isolation, identification and antimicrobial resistance swab samples were collected from 70 patients (both males and female with age range 1–64 years) admitted at burns center / al-yarmouk teaching hospital from the period november 2013 till february 2014. primary bacterial identification cultured on differential and selective media then biochemical tests were done. the automated microbial identification using vitek2 compact system (biomerieux, france) was used to confirm the p. aeruginosa isolates by gram negative identification card (gn id), which accommodates colorimetric reagent cards that are incubated and interpreted automatically and the antimicrobial susceptibility test card (ast) to each isolate was performed according to manufacturer’s instructions. protease production protease production by p. aeruginosa isolates using skimmed milk agar 1% (himedia, india) was performed to determine the proteolytic potency of the isolates using agar well diffusion assay.16 probiotic preparation probiotic strains from commercially available capsule l. acidophilus (holland & barrett, usa) and l. rhamnosus gg (health gensis, usa) were isolated by suspending in each capsule in 10 ml of mrs broth (himedia, india) then incubated anaerobically at 37ºc for 48 hr.17 antimicrobial assay the antimicrobial spectrum from lactobacilli spp. was determined using a loopful of each of the lactobacilli isolates from the mrs agar slants was inoculated into tubes containing 10 ml of sterile mrs broth. these broth cultures were incubated anaerobically at 37°c for 48 hr. the following assays conducted as triplicate: well diffusion method: sterile cotton swabs were dipped into the cultures of p. aeruginosa previously propagated in brain heart infusion (bhi) broth (difco, usa) for 24 hr at 37°c. the turbidity of the suspension was 0.5 mcfarland and contained more than 108 bacteria. the inoculated with 100 µl of (1.5 × 108 cfu/ml) by swabbing over the entire surface of the nutrient agar (himedia, india) plates was made. wells (6mm diameter) were made on the cultured plates. then 50 µl of lactobacilli spp. bacterial suspension was inoculated into wells. after 24 hr of incubation at 37°c, each plate was examined for the zone of inhibition. control for each zone was prepared using un-inoculated sterile mrs broth as negative control and acetic acid (33%) as positive control.18 agar disc method: the above bacterial suspension of lactobacillus spp. was incubated anaerobically on mrs agar at 37ºc for 48 hr, agar discs with (6 mm) were made, then the agar discs were seeded on plates of nutrient agar cultured with 100 µl of (1.5 × 108 cfu/ml) of p. aeruginosa. the plates were incubated for 37ºc for 24 hr. the diameters of the inhibitory zones were measured including the diameters of the discs to the nearest whole number. control was prepared using agar disc free of bacterial growth.19 extraction of crude bacteriocin crud bacteriocin produced from l. acidophilus and l. rhamnosus was extracted during log phase using mrs broth incubated anaerobically at 37ºc for 24 hr. after incubation, the cultures were centrifuged (6000 rpm at 4ºc for 10 min) to obtain culture-free supernatant which was filtered using 0.22 µm pore sterilized filter. the ph was adjusted to ph 7 with 1 m naoh.20 determination of protein content protein content was determined using colorimetric at maximum absorption at 600 nm, using brilliant blue g-250 and bovine serum albumin.21 antimicrobial activity of bacteriocin on protease production the antimicrobial spectrum from bacteriocin was determined using a loopful of p. aeruginosa isolates from the bhi agar slants that was inoculated into tubes containing 5 ml of sterile bhi broth. these broth cultures were incubated at 37°c for 24 hr. the well diffusion assay conducted as triplicate. they inoculated with 10 µl of (1 × 108 cfu/ml) by swabbing over the entire surface of the skimmed milk agar plates. wells (6 mm diameter) were made on the cultured plates. then 10 µl of bacteriocin was inoculated into wells. after 24 hr of incubation at 37°c, each plate was examined for the zone of inhibition. control for each zone was prepared using un-inoculated sterile bhi broth as negative control and phosphate saline (0.1 m/ph 7.0) as positive control. statistical analysis the data are shown as the mean ± standard deviation (sd, n = 5). the results obtained were analyzed using spss 18.0 program for windows and by analysis of variance (anova) with significance level set at p = 0.05. results from 70 swabs obtained from contaminated burn and wound infection, only 48 (69%) isolates were positive for primary bacterial isolation. only 31 (65%) isolates out of 48 were identified as p. aeruginosa (9 wound infection and 22 burn infection) (p ≤ 0.01). antimicrobial susceptibility tests included 16 antibiotics (table 1). out of 31 p. aeruginosa isolates, 20 isolates showed multi drug resistant for antibiotics (table 2) (p ≤ 0.01). all the 20 multi-drug resistant p. aeruginosa isolates were having the ability for protease production with potency (table 3); (p ≤ 0/05). figure 1 shows protease production for the isolate-11 and isolate-19 with duplicates. antimicrobial effect of lactobacilli spp. on p. aeruginosa using agar disc and well diffusion method, (isolate-8, 11 and 19) were selected due to their high protease production and multi-drug resistance to many antibiotic. the inhibition zone was measured by millimeters as in table 4. statistically, the results were significant (p ≤ 0.05). the highest inhibition zone was by l. rhamnosus than l. acidophilus in all isolates in both methods. the highest inhibition zone by l. rhamnosus was 20 mm on isolate-19, on isolate-11 was 25 mm, respectively; meanwhile, 220 j contemp med sci | vol. 3, no. 10, spring 2017: 218–223 antimicrobial effect of probiotic lactobacillus spp. on pseudomonas aeruginosa research maysaa kadhim al-malkey et al. highest inhibition zone by l. acidophilus was 18 mm on isolate-19 using agar disc method as shown in fig. 2. the highest inhibition zone of bacterial suspension by l. rhamnosus was 27 mm on both isolate-11 and isolate-19; meanwhile, highest inhibition zone by l. acidophilus was 18 mm on isolate-19 using well diffusion method as shown in fig. 3. the highest inhibition zone of bacterial supernatant on isolate-19 by both l. rhamnosus and l. acidophilus was 32 mm, 25 mm, respectively, using well diffusion method as shown in fig. 4. protein concentration of crud bacteriocin produced by l. rhamnosus gg and l. acidophilus were 74 mg/ml, 44 mg/m/l, respectively. fig. 5 shows the inhibitory effect of crud bacteriocin extracted from l. rhamnosus gg and l. acidophilus on protease production by p. aeruginosa (isolates-11 and 19) cultured on skimmed milk agar 1% after treated with crud bacteriocin comparing to untreated isolates. discussions p. aeruginosa is one of the important bacteria that can cause huge burdens for public health today due to its ability to adapt its genome and physiology during chronic infections. major features making it a very successful opportunistic pathogen includes: virulence factors, biofilm formation, motility and quorum sensing.22 according to arqués et al. (2015) determining the antagonistic effect of probiotics on the growth of p. aeruginosa and the effectiveness of various bacteriocins of probiotics may be hindered by the proteolytic activity of microbial enzymes that are secreted only during active fermentation.23 the present study basically focused on the bacteriocin, which is produced by a commercially available of l. rhamnosus gg and l. acidophilus. bacteriocins, in general, share a narrow spectrum of antimicrobial activity; however, there are certain bacteriocins that exhibit a broad spectrum of antibacterial activity and are also capable of targeting viruses, protozoa and even fungi.24 the results revealed that, the probiotics of l. rhamnosus showed enhancement of inhibitory zone diameters in agar disc and well diffusion method (bacterial suspension and supernatant) rather than l. acidophilus. the narrow inhibitory zone using the agar disc method may be due to the limited number of the lactobacilli cultured on mrs disc leading to its limited antimicrobial activity which come in accordance with table 1. antimicrobial susceptibility tests with resistant percentage of pseudomonas aeruginosa isolates no. antimicrobial resistant percentage (%) 1 piperacillin 100 2 ticarcillin 100 3 ticarcillin clavoulanic acid 100 4 cefazolin 100 5 ceftriaxone 100 6 tigecycline 100 7 piperacillin tazobactam 80 8 amikacin 65 9 gentamycin 80 10 tobramycin 85 11 imipenem 70 12 meropenem 70 13 cefepime 60 14 ceftazidime 50 15 ciprofloxacin 75 16 levaofloxacin 75 table 3. pseudomonas aeruginosa protease production potency no. of p. aeruginosa isolates protease production no. of p. aeruginosa isolates protease production isolate-1 ++ isolate-11 ++ isolate-2 + isolate-12 + isolate-3 + isolate-13 ++ isolate-4 + isolate-14 + isolate-5 +++ isolate-15 ++ isolate-6 + isolate-16 + isolate-7 + isolate-17 +++ isolate-8 +++ isolate-18 + isolate-9 ++ isolate-19 ++ isolate-10 + isolate-20 + +: mild protease production; ++: moderate protease production; +++: high protease production. fig. 1 pseudomonas aeruginosa on skimmed milk agar 1%. (1 and 2) = duplicate of isolate-11; (3 and 4) = duplicate of isolate-19 using well diffusion method. table 2. multi antimicrobial resistant by pseudomonas aeruginosa isolates number of antimicrobial resistant p. aeruginosa isolates no. % 4 2 10 5 2 10 8 2 10 9 1 5 12 1 5 13 5 25 14 7 35 total 20 100 chi square test (c 2) — 9.017** **p ≤ 0.01. maysaa kadhim al-malkey et al. 221j contemp med sci | vol. 3, no. 10, spring 2017: 218–223 research antimicrobial effect of probiotic lactobacillus spp. on pseudomonas aeruginosa table 4. antimicrobial effect of lactobacilli spp. in millimeters using agar disc method and well diffusion method isolate inhibition zones (mm) lsd value agar disc method well diffusion method co− suspensionsupernatant l. acidophilusl. rhmanosusl. acidophilusl. rhmanosusco+l. acidophilusl. rhamnosusco+ p8162412243015253057.02* p11162516273118303359.13* p19182020273125323057.53* lsd value6.59*5.83*5.19*5.22*6.72 ns6.38*5.71*6.02 ns0.00 ns— *p ≤ 0.05; ns: non significant; co+: control positive (33% acetic acid); co−: control negative (un-cultivated mrs broth). fig. 2 antimicrobial effect of lactobacilli spp. on p. aeruginosa (isolate-11, 19) using agar disc method. (1) l. acidophilus; (2 and 3) duplicate of l. rhamnosus gg; (4) negative control (un-cultivated mrs broth) using agar diffusion method. fig. 3 antimicrobial effect of bacterial suspension (lactobacilli spp.) on ps. aeruginosa (isolate-11, 19) using well diffusion method. (1) positive control (33% acetic acid); (2) l. acidophilus; (3 and 4) l. rhamnosus gg (duplicate); (5) negative control (un-cultivated mrs broth). fig. 4 antimicrobial effect of bacterial supernatant (lactobacilli spp.) on p. aeruginosa (isolate-19) using well diffusion method. (1 and 2) l. rhamnosus gg (duplicate); (3) l. acidophilus; (4); positive control (33% acetic acid); (5) negative control (un-cultivated mrs broth). fig. 5 inhibitory effect of crud bacteriocin produced by lactobacilli on protease production from ps. aeruginosa. (1) positive control (p. aeruginosa, isolate-19); (2) negative control (un-cultivated brain heart broth); (3) p. aeruginosa isolate-11 treated with crud bacteriocin from l. rhmanosus; (4) p. aeruginosa isolate-19 treated with crud bacteriocin from l. rhmanosus; (5) p. aeruginosa isolate-19 treated with crud bacteriocin from l. acidophilus. 222 j contemp med sci | vol. 3, no. 10, spring 2017: 218–223 antimicrobial effect of probiotic lactobacillus spp. on pseudomonas aeruginosa research maysaa kadhim al-malkey et al. paluszak et al. (2007).19 the antimicrobial effect of bacterial suspension using well diffusion method showed more clearly inhibitory zone in diameters, the competitive exclusion between the pathogenic bacteria as well as to the presence of other secondary metabolites by lactobacilli spp. such as the lactic acid, biosurfactant, and other fermentation product as well as bacteriocin may play a major role.25 the highest inhibitory zone was recorded using free cell supernatant which have remarkable potential for their antimicrobial activities which comes in compatible with a study by daba and saidi (2015) which study the inhibitory activity of bacteriocin producing lactic acid bacteria (lab) that isolated from raw milk against p. aeruginosa and escherichia coli using free cell supernatant and cell diffusion method.26 wala’a and nibras (2013) found that bacteriocins from l. acidophilus exhibited activity against serratia marcescens and that bacteriocin of l. acidophilus was stable at ph 4, 7 half of its activity was lost at ph 8 and whole activity was lost at other ph values.27 gho and philip (2015) focus on isolate and purify the bacteriocin of weissella confusa a3 from cow milk was shown to have inhibitory activity towards pathogenic bacteria as bacillus cereus, e. coli, p. aeruginosa and micrococcus luteus. the bacteriocin was shown to be heat stable and functioned well at low ph (2 to 6). reduction of activity was shown after treatment with proteinase k, trypsin and peptidase that confirmed the proteinaceous nature of the compound.28 ismaeel et al. (2013) investigated the biological applications of surlactin derived from l. acidophilus using different pathogenic strains and toward to in vitro (contact lenses) and in vivo (rabbits’ eyes). their results demonstrated the capability of surlactin to inhibit the adhesion of pathogens up to 60% without any antibacterial activity against staphylococcus auerus using well diffusion method. the surlactin proved to be effective for treating the infection in rabbits’ eyes with p. aeruginosa and that infection with p. aeruginosa (administrated to rabbits’ eyes) can be prevented by using surlactin.29 a study by zho et al., (2015) revealed that bacteriocin from l. acidophilus xh1 inhibited e. coli, s. aureus and b. anthracis. it showed a wide range of antimicrobial activity at ph 1.0–5.0 while at 37–120°c, it was sensitive to trypsin, pepsin and papain, but insensitive to proteinase k and neutral protease.30 many antibiotics, antimicrobial agents do possess protein as one of the major functioning fractions of the entire molecule. the proteinaceous nature or peptides do contribute toward antimicrobial activity and have tremendous potential for treating and/or preventing the infectious diseases. risk of microbial resistance can be reduced certainly with the help of such proteinaceous molecules. protein rich with and without polysaccharide, phosphate fractions in cell-bound or cell-associated biosurfactant originated from lactobacillus spp. have undoubtedly fulfilled this expectation proving to combat pathogens. brzozowski et al. (2011) reported biosurfactant production by l. fermenti 126 and l. rhamnosus ccm 1825 having proteinaceous biosurfactant with an existence of polysaccharide and phosphates biosurfactant obtained l. rhamnosus ccm 1825 possessed more proteins and phosphates as compared with l. fermenti 126.31 antimicrobial effect of bacteriocin production may contribute to probiotic functionality through three different mechanisms: firstly, as colonising peptides; secondly, bacteriocins function through direct inhibition of the growth of pathogens9; and finally, bacteriocins may serve as signalling peptides/quorum-sensing molecules in the intestinal environment.32 bacteriocin can interfere with the bacterial cell wall enzyme production leading to inhibit their virulence factors such as potency of protease production.33 conclusions in an effort to establish a new antimicrobial agent from lactic acid bacteria, novel strains capable of utilizing cheaper, renewable substrates, greater yields, and novel applications, which may act as bacteriostatic or bactericidal agents. using probiotic bacteria toward therapeutic approaches can be highly significant in preventing and/or dealing with hospital-acquired infections may be an important undertaking. acknowledgements the author would acknowledge all the patients whom participate in this study and donate the samples in spite of their suffering. conflict of interest the authors declare no conflicts of interest. n references 1. driscoll ja, brody sl, kollef mh. the epidemiology, pathogenesis and treatment of pseudomonas aeruginosa infections. drugs. 2007; 67:351–368. 2. drenkard e. antimicrobial resistance of pseudomonas aeruginosa biofilms. microbes infect. 2003;5:1213–1219. 3. murray pr, baron ej, pfaller ma, et al. manual of clinical microbiology. 2nd ed. washington, md: american society microbiology; 1999. 4. holder ia, haidaris cg. experimental studies of the pathogenesis of infections due to pseudomonas aeruginosa: extracellular protease and elastase as in vivo virulence factors. can j microbiol. 1979;25:593–599. 5. janda jm, bottone ej. pseudomonas aeruginosa enzyme profiling: predictor of potential invasiveness and use as an epidemiological tool. j clin microbiol. 1981;14:55–60. 6. engel ls, hill jm, caballero ar, et al. protease iv, a unique extracellular protease and virulence factor from pseudomonas aeruginosa. j biol chem. 1998;273:16792–16797. 7. fuller r. probiotics in human medicine. gut. 1991;32:439–442. 8. mazmanian sk, round jl, kasper dl. a microbial symbiosis factor prevents intestinal inflammatory disease. nature. 2008;453:620–625. 9. gibson gr, probert hm, van loo j, and rastall ra. dietary modulation of the human colonic microbiota: updating the concept of prebiotics. nut res rev. 2004;17:259–275. 10. syukur s, bisping b, noli za, purwati e. antimicrobial properties and lactase activities from selected probiotic lactobacillus brevis associated with green cacao fermentation in west sumatra, indonesia. j prob health. 2013;1:113. 11. jamalifar h, rahimi hr, samadi n, et al. antimicrobial activity of different lactobacillus species against multi-drug resistant clinical isolates of pseudomonas aeruginosa. iran j microbio. 2011;3:21–25. 12. saggioro a. probiotics in the treatment of irritable bowel syndrome. j clin gastroenterol. 2005;38:s104–106. 13. choi ea, chang hc. cholesterol-lowering effects of a putative probiotic strain lactobacillus plantarum em isolated from kimchi. lwt – food sci technol. 2015;62:210–217. 14. cheikhyoussef a, pogori n, chen hq, et al. antimicrobial activity and partial characterization of bacteriocin-like inhibitory substances (blis) produced by bifidobacterium infantis bcrc 14602. food control. 2009;20:553–559. maysaa kadhim al-malkey et al. 223j contemp med sci | vol. 3, no. 10, spring 2017: 218–223 research antimicrobial effect of probiotic lactobacillus spp. on pseudomonas aeruginosa 15. jiang j. shi b, zhu d, et al. characterization of a novel bacteriocin produced by lactobacillus sakei lsj618 isolated from traditional chinese fermented radish. food control. 2012;23:338–344. 16. kannan n. laboratory manual in general microbiology. new delhi: panima publishing corporation; p.135. 2002 17. forbes ba, daniel fs, alice sw. bailey and scott’s diagnostic microbiology. 12th ed., mosby elsevier company, usa. 2007. 18. clinical and laboratory standards institute (clsi). performance standards for antimicrobial discs susceptibility tests: approved standards (m02-a12), informational supplements (m100-s25); 25th ed., 2015. 19. paluszak z, kaszewska je, szala b. inhibitory effect of lactic acid bacteria of genus lactobacillus on the survival of proteus and shigella rod in mixed culture. bull vet inst pulawy. 2007; 50:335–40. 20. maqsood s, hasan f, masud t, imran m. preliminary characterisation of bacteriocin produced by lactobacillus acidophilus ts1 isolated from traditional dahi. ann microbiol. 2008;58:617–622. 21. bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. ann biochem. 1976;72:248–254. 22. cai z, liu y, chen y, yam jk. rpon regulates virulence factors of pseudomonas aeruginosa via modulating the pqsr quorum sensing regulator. int j mol sci. 2015;16:28311–28319. 23. arqués jl, rodríguez e, langa s, et al. antimicrobial activity of lactic acid bacteria in dairy products and gut: effect on pathogens. biomed res int. 2015;9. http://dx.doi.org/10.1155/2015/584183. 24. rea mc, ross rp, cotter pd, et al. classification of bacteriocins from gram positive bacteria. in: prokaryotic antimicrobial peptides. from genes to application. drider, d. and rebuffat, s. (eds). springer, berlin, pp. 29–54. 2011 25. holzapfel w, wood bj. the genera of lactic acid bacteria: springer science & business media. 2012 26. daba h, saidi s. detection of bacteriocin-producing lactic acid bacteria from milk in various farms in north-east algeria by a new procedure. agron resear. 2015;13:907–18. 27. wala’a sha, nibras nm. partial purification and characterization of bacteriocin produced by lactobacillus acidophilus. j biotech resear cent. 2013;7:80–86. 28. gho hf, philip k. purification and characterization of bacteriocin produced by weissella confusa a3 of dairy origin. plos one. 2015;10:1–17. 29. ismeeal mch, ibrahim km, and al-malikey mk. the effect of surlactin produced by lactobacillus acidophilus on eye infectious bacteria in rabbits. j baghdad sci. 2013;10:133–142. 30. zhao r, duan g, yang t, et al. purification, characterization and antibacterial mechanism of bacteriocin from lactobacillus acidophilus xh1. tropi j pharm res. 2015;14:989–95. 31. brzozowski b, bednarski w, gołek p. the adhesive capability of two lactobacillus strains and physicochemical properties of their synthesized biosurfactants. food technol biotechnol. 2011;49:177–186. 32. dobson a, cotter pd, ross rp, et al. bacteriocin production: a probiotic trait? appl environ microbiol. 2012;78:1–6. 33. wiedemann i, breukink e, van kraaij c, et al. specific binding of nisin to the peptidoglycan precursor lipid ii combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. j biol chem. 2001;276:1772–1779. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201704 163j contemp med sci | vol. 9, no. 3, may-june 2023: 163–166 original study of the genetic polymorphisms of abcb1 3435g>a in postmenopausal women breast cancer on paclitaxel chemotherapy doaa alaa mohammed hasan1* , ahmed salih sahib1, ahmed abbas hasan al-rokan2, karrar kadhim mohsin2 1department of pharmacology and toxicology, college of pharmacy, university of kerbala, karbala, iraq. 2imam al-hussein hematology and oncology center, ministry of health, karbala, iraq. *correspondence to: doaa mohammed hasan (e-mail: douaa.alaa@s.uokerbala.edu.iq) (submitted: 13 february 2023 – revised version received: 28 february 2023 – accepted: 22 april 2023 – published online: 26 june 2023) abstract objectives: the aim of study was to determine the relationship between this snps in postmenopausal women with breast cancer 3435g>a (rs1045642) polymorphism of the abcb1 gene, and serum concentrations of paclitaxel chemotherapy in iraq. methods: this study included 100 patients (45–75 years) with a clinical diagnosis of breast cancer who were going to start (paclitaxel) chemotherapy. patients at the oncology center at imam al-hussain medical city in kerbala iraq, were sorted into groups based on their postmenopausal age, disease duration, and treatment length for this study, which was conducted between july 2022 and october 2022. serum concentrations of estradiol and ca 15.3 were measured in breast cancer patients who had been taking paclitaxel. allele-specific pcr assay determined gene polymorphisms 3435g>a (rs1045642) and hplc measured paclitaxel drug concentration. results: the results found there is an inverse relationship between ca15.3 concentration and patient response to paclitaxel chemotherapy, and when compared to the genotyping frequency of abcb1 3435g>a (rs1045642) genetic polymorphism, patients with allele aa in their chromosomes respond better (15.56 ± 6.89) than those with alleles gg and ga. conclusion: after paclitaxel chemotherapy, there were statistically significant differences (p < 0.05) in the reduction of drug concentration among serum patients with the aa genotype for abcb1 3435g>a (rs1045642). keywords: abcb1, breast neoplasms, polymorphism, chromatography, high pressure liquid, paclitaxel issn 2413-0516 introduction breast cancer is a malignant tumor that has developed from cells of the breast. the disease occurs mostly in women, but does occur rarely in men.1 some breast cancers are called insitu, because they do not spread beyond the area where they begin, while others are invasive cancer. the majority of insitu tumors will not progress to become an invasive tumor, and at this stage nearly all of these cancers can be cured.2 among women, breast cancer has remained the commonest tumor in iraq, it forms 22.3% of all malignant tumor and 37% of the registered female cancers with a sharp increase in the incidence of this tumor in younger age group. it forms 5% of those under 30 years and 75% of those older than 40 years and highest number of cases is between 40 and 50 years, and the incidence looks to be increasing.3 delay in the diagnosis of breast cancer is an area of increasing litigation for medical practitioners, both in primary care and in hospital practice. whether delay in the diagnosis of breast cancer affects the outcome has been uncertain, but some studies have found evidences of reduced survival.4 chemotherapy is a cancer treatment where medicine is used to kill cancer cells. there are many different types of chemotherapy medicine, but they all work in a similar way. they stop cancer cells reproducing, which prevents them from growing and spreading in the body.5 paclitaxel belongs to the family of cytoskeletal drugs that target tubulin. as a result, paclitaxel treatment leads to abnormality of the mitotic spindle assembly, chromosome segregation, and consequently defects of cell division. by stabilizing the microtubule polymer and preventing microtubules from disassembly, paclitaxel arrests cell cycle in the g0/g1 and g2/m phases and induces cell death in cancer. it has been known that inhibition of mitotic spindle using paclitaxel usually depends on its suppression of microtubule dynamics.6 atp binding cassette (abc) multidrug transporters such as p-glycoprotein (p-gp, abcb1) and bcrp (abcg2) confer resistance against anticancer drugs and can limit their oral availability, thus contributing to failure of chemotherapy. like p-gp and bcrp, another abc transporter, mrp2 (abcc2), is found in apical membranes of pharmacologically important epithelial barriers and in a variety of tumors. mrp2 transports several anticancer drugs and might thus have a similar impact on chemotherapy as p-gp and bcrp.7 the most widely studied variant of abcb1 is a commonly synonymous c to t transition at nucleotide position 3435 in exon 26 (3435 g > a), an exon 26 of the mdr1 gene is responsible for the up regulation of this gene’s activity, which in turn effluxes a wide variety of chemicals throughout the plasma membrane (ozen et al. 2011).8 the study aimed to detect the genetic polymorphism of abcb1 3435g>a (rs1045642) in participated breast cancer women and to investigate the effect of genetic polymorphism on paclitaxel drug concentration. materials and methods a cross-sectional study was carried out at the imam al-hussein oncology center in kerbala city in iraq. the sample collection was carried out between july and december of 2022 and in the university of kerbala’s college of pharmacy laboratories. patients were randomly assigned to receive paclitaxel intravenously once a week for 1 or 3 hours. postmenopausal women having a histologically proven diagnosis of breast cancer were the patients seen at imam hussein hospital. they were admitted for endocrine, radiation, or https://orcid.org/0000-0003-0452-0962 mailto:douaa.alaa@s.uokerbala.edu.iq 164 j contemp med sci | vol. 9, no. 3, may-june 2023: 163–166 genetic polymorphisms of abcb1 3435g>a in postmenopausal women breast cancer on paclitaxel chemotherapy original d. a. m. hasan et al. cytotoxic treatments. while controls consisted of agematched, breast-cancer-free women. inclusion criteria women with histologically confirmed breast cancer and post menopause, they were required to be non-pregnant, all patients were required to have clinically or radiographically measurable disease and to have adequate renal and hepatic function normal, and postmenopausal women aged range (45–75 years). exclusion criteria patients were ineligible if they had diabetic mellitus or any other underlying medical condition that would hinder study participation, those with child-bearing potential who did not implement adequate contraceptive measures were also ineligible. genotyping for abcb1 polymorphisms detection in our research, we have shown that allele-specific amplification using polymerase chain reaction (pcr) is widely utilized for genotyping single-nucleotide polymorphisms (snps). however, the manufacturer (add bio/korea) reports that the isolation of genomic dna from whole blood that is pcr compatible is the standard method of use. primer 3 plus creates primer pairs that are specific to the template, and the specificity testing software looks for primer-target matches in databases containing abcb1 3435g>a (rs1045642) polymorphisms using blast websites (table 1). the quantities of nuclease-free water added to each primer to achieve a concentration of 100 pmol/l (represent a stock solution). lyophilized primers were dissolved with the appropriate volume of nuclease-free water per manufacturer instructions. premixed pcr tubes (accupower® pcr premix/korea) were made with a final volume of 20 µl for pcr sample collection. pcr protocol was consist of 1.5 µl of forward primer, 1.5 µl of reverse primer, 4 µl of dna template, and 12 µl of distal water, the pcr amplification program consist of initial denaturation at 95°c for 5 minutes, the 35 cycles include a 30-second denaturation at 95°c, a 45-second anneal at 60°c, a 30-second extension at 72°c, and a 5-minute extension at 72˚c. detected pcr products were run on a dna gel electrophoresis as follows. on a 1.5% agarose gel (1.5 g/100 ml 1x tbe support), 3 µl of loading buffer and 5 µl of product were loaded and the gel was run at 100 volt for 35 minutes. gel was recolored by adding ethidium bromide (0.5 g/ml). dna bands were photographed using a uv trans illuminator, and the molecular weights of the bands were determined using a dna ladder (100–1500 bp). measurement of drug concentration of paclitaxel in patient serum to prepare the sample for hplc analysis of paclitaxel concentration, 0.1 ml of human plasma was added to the extraction tube. next, i combined 4 ml of acetonitrile with 1 ml of ethyl acetate (v/v ratio of 4:1). a 3500 rpm freeze centrifuge was used. after then, the organic layer inside the glass tube was evaporated by the nitrogen stream. after centrifuging at 3500 rpm, 200 ul of 50% meoh is used to dissolve the dry residue, which is then transferred to an auto sampler vial. injecting a 100 ul aliquot of material into the hplc. the mobile phase was a 45:55 (v/v) mixture of formic acid and meoh (0.1%). the rate of the mobile phase was 1.1 ml/min. the column was c18-ods (25 cm × 4.6 mm), and the detector was a uv-vis at 305 nm (ankit jian, 2013).9 statistical analysis this study employed mean ± standard error, anova, and t tests to significantly compare means. two-tailed fisher’s exact probability (p) tests were used to compare alleles genotyping percentage frequencies in breast cancer patients and controls, various statistical methods were used to analyze data, chi-square (χ2) test for contingency tables to find the statistical difference between cases and controls about the risk factors. results the studied population included 100 female patients with breast cancer. based on the findings of the study. the majority of women diagnosed with breast cancer were recorded at 85% of the total number of postmenopausal women having surgical involvement. the results also found that all women with breast cancer have taken chemotherapy (100%) as well as 5% of women who have breast cancer have received radiotherapy treatment. the results also found that rare cases of postmenopausal women (6% of the total) had recurrences of breast cancer (table 2). amplification reaction of a bcb1 3435g>a (rs1045642) polymorphism the results of genotype abcb1 3435g>a (rs1045642) genetic polymorphism was a clear band with a molecular size 400 bps (figure 1). the size of amplicon was determined by compare table 1 primers sequences of abcb1 3435g>a (rs1045642) polymorphisms allele specific primer sequence (5’->3’) product size reverse allele a 5-gggtggtgtcacaggaagagatt-3 400 bpreverse allele g 5-gggtggtgtcacaggaagagatc-3 forward common 5-taaggctgacaaaggtggagcc-3 table 2. external affect of status for women with breast cancer percentage (%) surgery yes 85% no. 15% chemotherapy yes 100% no. 0% radiotherpy yes 5% no 95% recurrence yes 6% no 94% 165j contemp med sci | vol. 9, no. 3, may-june 2023: 163–166 d. a. m. hasan et al. original genetic polymorphisms of abcb1 3435g>a in postmenopausal women breast cancer on paclitaxel chemotherapy with dna ladder 100–1000 bp, genotype of rs1045642 which were classified into three genotypes: 1. the major genotype group (gg) for the allele g. 2. the homozygous genotype group (aa) for the allele a. 3. heterozygous (ga). the result of comparison between observed and anticipated value for abcb1 3435g>a (rs1045642) tested population were shown in figure 1 and table 3. the distribution and percentage of individuals having rs1045642 differ from those expected under hardy–weinberg equilibrium {number of observed vs expected were: gg (28); ga (36); aa (36) (goodness-of-fit χ2 for rs1045642; 7.5824, p < 0.001) and therefore it was statistically significant. relationship between concentration of chemotherapy paclitaxel of chemotherapy intake and abcb1 3435g>a (rs1045642) genetic polymorphism table 4 shows that there is an inverse relationship between the concentration of drug with the therapeutic response of the patient when using paclitaxel chemotherapy and when compared with the level of genotyping frequency among abcb1 3435g>a (rs1045642) genetic polymorphism, we noticed that patients who have allele gg in their chromosomes respond to treatment (192.8 ± 16.33) better than patients who have alleles ga and aa, respectively. lsd value was recorded 11.16 among the genes and indicated the existence of significant differences (p < 0.05) between the three alleles. discussion this study included 100 women stratified by age, bmi, and paclitaxel duration intake. the clinical demographic data and laboratory parameters of patients group were, the mean age of participants which was within a mean age of (54.36 ± 4.21) years old, number of birth (3 ± 1) times. they are many adjusted other self-reported characteristics and risk factors responsible for breast cancer. these characteristics included bmi, marital status, family history, lymph node involvement, number of patients who had surgery or chemotherapy, disease duration and diagnosis, cancer location, and histochemical test results, some of studies showed the majority of breast cancers are found in women over the age of 50 years old, and some women will develop breast cancer despite having no other known risk factors. the presence of a risk factor does not guarantee the presence of the disease, and not all risk factors have the same effect.10 the results of our current study showed that 85% of women with cancer had surgical intervention, breast surgery, also known as a wide local excision, is a type of operation in which the region of cancer in the breast that needs to be removed is cut out surgically. the cancerous tissue and a margin of healthy tissue all the way around it are removed by the surgeon, they do so while preserving the maximum amount of healthy breast tissue feasible.11 a total breast removal, sometimes known as a mastectomy, may be necessary for some women. they also have the option of undergoing this surgical procedure. the breast tissue, including the skin and nipple, as well as the tissues that protect the chest muscles, are both removed by the surgeon during the procedure.12 extremely infrequently, the surgeon will also remove the muscles that make up the chest wall. this type of mastectomy is known as a radical mastectomy.13 the study observed five women with breast cancer who were users of radiotherapy, breast cancer radiation therapy involves the use of x-rays, protons, or other high-energy particles to eradicate cancer cells.14 radiation therapy is more effective against rapidly dividing cells, such as cancer cells, than it is against stationary ones. there is no discomfort or visual impact from the x-rays or particles. after treatment, you are no longer radioactive and can safely be in close proximity to others, including children (duma et al., 2019).15 breast cancer at nearly any stage may be treated with radiation treatment. after breast cancer surgery, radiation therapy is an effective method of lowering the likelihood of a recurrence. in addition, it is frequently used to alleviate the discomfort associated with metastatic breast cancer.14 fig. 1 detection o fabcb1 3435g>a (rs1045642) genetic polymorphism by allele specific pcr with three possible genotype (gg, aa, and ga); allele-specific pcr reaction yielded a specific fragment of 400 bp in those tubes where the mutant allele (1, 6 and 9 wells) was present. (2, 4 and 7 wells) indicates a wild-type homozygous. (3, 5 and 8 wells) indicates a heterozygous samples. table 4. relationship between drug concentration of chemotherapy intake and abcb1 3435g>a (rs1045642) genetic polymorphism genotype “rs1045642” mean ± sd n drug concentration no-responder drug concentration responder p-value gg 28 176.82±14.46 192.8±16.33 00.037 ga 36 168.45±16.39 184.91±13.76 00.053 aa 36 167.36±14.8 165.26±15.7 00.247 lsd 100 9.37 11.16 table 3. distribution of abcb1 3435g>a (rs1045642) genotypes in breast cancer patients variables group frequency percentage genotype gg (wild) 28 28% aa(homo) 36 36% ga (hetero) 36 36% data presented by numbers and percentage. 166 j contemp med sci | vol. 9, no. 3, may-june 2023: 163–166 genetic polymorphisms of abcb1 3435g>a in postmenopausal women breast cancer on paclitaxel chemotherapy original d. a. m. hasan et al. (rs1045642) genetic polymorphism, the study noticed that patients with allele gg respond to treatment (192.816.33) better than patients with alleles aa and ga, respectively. the lsd value was 11.16 among the genes, indicating that there were substantial differences (p < 0.05) between the three alleles, the study found that the mutant gene a allele played a role as a protective factor in order to maintain the concentration of the drug paclitaxel in the patient’s serum for a period of time, unlike the other two genetic polymorphism aa and ga. this study differed from what was explained by (su et al., 2021)18 when he stated that not all mutated genes play a role as a protective factor, as there are some mutated genes that were pathological in women with metastatic colon cancer. this study conclude that patients whose chromosomes contained the allele gg responded to treatment more favorably than patients whose chromosomes contained the alleles aa and ga for abcb1 3435g>a (rs1045642) genetic polymorphism. conflict of interest none.  according to figure 1 and table 3 display the results of a comparison between the observed value and the expected value for the abcb1 3435g>a (rs1045642) tested population. it was statistically significant since the distribution and proportion of individuals who had rs1045642 varied from those expected under hardy–weinberg equilibrium. the number of observed vs expected were: gg (28); ga (36); aa (36) (goodness-of-fit 2 for rs1045642; 7.5824, p = 0.001). in this way, mutations, gene flow, and other factors can all upset the hardy-weinberg equilibrium. for instance, rs1045642 polymorphism introduce novel alleles into a population, which might shift the balance of existing allele frequencies.16 this investigation concurred with the findings of (barliana et al., 2021),17 who discovered the hardy–weinberg equilibrium was used to conduct an investigation of the frequency of alleles for each gene. the genetic profiles of abcb1 rs1045642 were found to be significantly different from equilibrium in the population of indonesia. table 4 shows that there is an inverse relationship between drug concentration and patient therapeutic response when using paclitaxel chemotherapy, and when compared with the level of genotyping frequency among abcb1 3435g>a this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1349 references 1. feng, y., spezia, m., huang, s., yuan, c., zeng, z., zhang, l., ... & ren, g. (2018). breast cancer development and progression: risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis. genes & diseases, 5(2), 77–106. 2. ward, c., meehan, j., mullen, p., supuran, c., dixon, j. m., thomas, j. s., ... & langdon, s. p. (2015). evaluation of carbonic anhydrase ix as a therapeutic target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast cancer models. oncotarget, 6(28), 24856. 3. alwan, n. a. (2016). breast cancer among iraqi women: preliminary findings from a regional comparative breast cancer research project. journal of global oncology, 2(5), 255. 4. tremblay, d., latreille, j., bilodeau, k., samson, a., roy, l., l’italien, m. f., & mimeault, c. (2016). improving the transition from oncology to primary care teams: a case for shared leadership. journal of oncology practice, 12(11), 1012–1019. 5. chabner, b. a., & roberts jr, t. g. (2005). chemotherapy and the war on cancer. nature reviews cancer, 5(1), 65–72. 6. weaver, b. a. (2014). how taxol/paclitaxel kills cancer cells. molecular biology of the cell, 25(18), 2677–2681. 7. xiao, q., zhou, y., & lauschke, v. m. (2020). impact of variants in atp-binding cassette transporters on breast cancer treatment. pharmacogenomics, 21(18), 1299–1310. 8. ozen f, silan c, uludag a, candan f, silan f, ozdemir s, atik s, ozdemir o. association between abcb1 (mdr1) gene 3435 c> t polymorphism and colchicine unresponsiveness of fmf patients. renal failure. 2011 oct 1;33(9):899-903. 9. chaithanya sm, annapurna mm. stability indicating rp-uplc method for the simultaneous determination of atorvastatin and amlodipine. research journal of pharmacy and technology. 2019;12(10):4867-72. 10. nelson, h. d., zakher, b., cantor, a., fu, r., griffin, j., o’meara, e. s., ... & miglioretti, d. l. (2012). risk factors for breast cancer for women aged 40 to 49 years: a systematic review and meta-analysis. annals of internal medicine, 156(9), 635–648. 11. miranda-galvis, m., loveless, r., kowalski, l. p., & teng, y. (2021). impacts of environmental factors on head and neck cancer pathogenesis and progression. cells, 10(2), 389. 12. greenup, r. a., rushing, c., fish, l., campbell, b. m., tolnitch, l., hyslop, t., ... & hwang, e. s. (2019). financial costs and burden related to decisions for breast cancer surgery. journal of oncology practice, 15(8), e666–e676. 13. berhili, s., kadiri, s., bouziane, a., aissa, a., marnouche, e., ogandaga, e., ... & benjaafar, n. (2017). associated factors with psychological distress in moroccan breast cancer patients: a cross-sectional study. the breast, 31, 26–33. 14. helms, e., onate, m. k., & sherman, m. h. (2020). fibroblast heterogeneity in the pancreatic tumor microenvironment. cancer discovery, 10(5), 648–656. 15. duma, n., santana-davila, r., & molina, j. r. (2019, august). non–small cell lung cancer: epidemiology, screening, diagnosis, and treatment. in mayo clinic proceedings (vol. 94, no. 8, pp. 1623-1640). elsevier. 16. kryukov, a. v., sychev, d. a., andreev, d. a., ryzhikova, k. a., grishina, e. a., ryabova, a. v., ... & bochkov, p. o. (2018). influence of abcb1 and cyp3a5 gene polymorphisms on pharmacokinetics of apixaban in patients with atrial fibrillation and acute stroke. pharmacogenomics and personalized medicine, 43–49. 17. barliana, m. i., kusuma, a. s. w., insani, w. n., alfian, s. d., diantini, a., mutakin, m., ... & abdulah, r. (2021). genetic variation of abcb1 (rs1128503, rs1045642) and cyp2e1 rs3813867 with the duration of tuberculosis therapy: a pilot study among tuberculosis patients in indonesia. bmc research notes, 14(1), 1–7. 18. su, s. c., lin, c. w., ju, p. c., chang, l. c., chuang, c. y., liu, y. f., ... & yang, s. f. (2021). association of linc00673 genetic variants with progression of oral cancer. journal of personalized medicine, 11(6), 468. 295j contemp med sci | vol. 3, no. 12, autumn 2017: 295–299 research oxidative stress predominates apoptosis during experimental hepatocellular carcinoma nabil m. abdel-hamid,a afrah f. salama,b mostafa el-sheekh,c naglaa sarhan,d and asmaa m. gabrb abiochemistry department, faculty of pharmacy, kafrelsheikh university, egypt. bchemistry department, biochemistry section, faculty of science, tanta university, egypt. cbotany department, faculty of science, tanta university, egypt. dhistopathology department, faculty of medicine, tanta university, egypt. correspondence to, nabil mohie abdel-hamid (e-mail: nabilmohie@yahoo.com). (submitted: 18 july 2017 – revised version received: 09 august 2017 – accepted: 10 september 2017 – published online: 26 december 2017) objective hepatocellular carcinoma ranks the third among cancer deaths in the globe. despite the fact that many strategies for diagnosing liver cancer are possible, many factors, including apoptosis, arise to interfere with cell cycle control. we initiated this study to investigate the importance of free radicals during experimental hepatocarcinogenesis, triggered by diethyl nitrosamine, against an anti-apoptotic factor, belongs to a family of oncogenes (bcl-2). methods eighteen female wistar rats were classified into two equal groups, group 1 received intraperitoneal normal saline doses, group 2 was injected with one ip dose of diethyl nitrosamine (200 mg/kg bw), 2 weeks later, they were given a single ccl 4 dose (2 ml /kg b.w., ip), blood and liver tissue samples were collected after 60 days of diethyl nitrosamine dose. the graph pad prism program was used in statistical calculations. results the results revealed that treatment with diethyl nitrosamine + ccl 4 significantly up regulated the plasma liver functions tests: aspartate transaminase, alanine transaminase, gamma-glutamyl transpeptidase, total bilirubin and increased alpha fetoprotein with decreased level of bcl-2. liver tissue showed elevation of lipid peroxide, malondialdehyde, catalase enzyme activity, with significant reduction in body weight, decreased total protein content, tissue antioxidants like glutathione s-transferase, total thiol and total antioxidant capacity, compared to control. histological examination of liver showed an increased nuclear/cytoplasm (n/c) ratio, mitotic figures and nuclear polymorphism and microacinar formation in diethyl nitrosamine + ccl 4 -treated group. conclusions disturbed hepatocellular anti-oxidant mechanisms suppress apoptosis, reflecting failure in combating chemical hepatocarcinogenesis. keywords liver cancer; diethyl nitrosamine; lipid peroxidation; tumor markers; bcl-2; apoptosis. introduction globally, hepatocellular carcinoma (hcc) ranks the sixth prevalent cancer and the third cause of death related to cancer.1 the well-known risk factors of hcc include untreated viral hepatitis (hbv and hcv), some food flavors, alcohols, aflatoxins, toxic chemicals from industrial processes and environmental pollutants.2 diethyl nitrosamine (dena), a strong hepatocarcinogen, was reported induce aberrations in the nuclear enzymes engaged in dna repair and replication.3 nitrate and nitrite are used as preservatives in both meat and fish industry, color fixatives and cost effective flavors that can generate endogenous hepatotoxic nitroso byproducts, like primary amines in the stomach ph.4 metabolic activation of dena yields pro-mutagenic adducts, o6-ethyl deoxy guanosine and 04 and o6-ethyl deoxy thymidine in liver, leading to hepatocarcinogenesis.5 dena liver model cancer is one of the most commonly used experimental models in hepatocarcinogenesis.6 it is now documented that interference to apoptosis is a contributor to hcc.7 this was also proved by oxidative stress,8 as well as, the impact of oxidative stress on apoptotic pathways was reported.8,9 one approach to control liver cancer, is studying the different factors underline the progression of the disease. oxidative stress is an important risk factors contributing to hcc and other cancers.10 b cell lymphoma protein-2 (bcl-2) is a survival protein, found in outer mitochondrial membrane, known to suppress the release of cytochrome c from the mitochondria; so, it can interfere apoptosis in cancer cells, thus, it contributes in development of cancer and resistance to anticancer compounds.11 the aim of the current work was to assess the ability of reactive oxygen metabolites in disturbing apoptosis, through studying the impact on an oncogene(bcl-2), during experimental hcc. this may lead to a more efficient strategy for treatment success via adjuvant therapeutics with a more selective antioxidant efficacy with the common or new therapeutic protocols of hcc. in addition to liver functions, we will estimate oxidative stress markers, as well as, an anti-apoptotic mediator (oncogenic protein bcl-2), with afp and histopathology of liver tissue. materials and methods chemicals dena and the other fine chemicals were obtained from sigmaahlrich chemical co., usa. rat afp and rat bcl2 elisa chemicals from tsz scientific llc, usa. other used chemicals were of analytical grade, purchased from local suppliers. animals we recruited 18 female rats weighing 180 g (±20 g) obtained from the faculty of veterinary medicine, in the university of cairo, egypt. they were accommodated for 1 week before issn 2413-0516 296 j contemp med sci | vol. 3, no. 12, autumn 2017: 295–299 oxidative stress predominates apoptosis during experimental hepatocellular carcinoma research nabil m. abdel-hamid et al. experimental work on standard chow and drinking water in the laboratory of department of zoology, faculty of science, university of tanta, egypt. the temperature was kept at 23 ± 2°c with a approximate humidity of 60% under 12 h/12 h light dark cycle. animals were classified as two equal groups (9 animals each). experimental design group 1(g1): control group, rats were given saline, intraperitoneally (i.p) for 60 days. group 2(g2): hcc group, rats were given one dose of diethylnitrosamine dissolved in saline (i.p) in the dose of 200 mg/kg b.w.12 and after 2 weeks, they were given an activating dose of carbon tetrachloride (ccl4) in olive oil in a dose of 2 ml /kg b.w. (i.p). euthanization of rats was performed after 60 days from dena injection. blood and tissue collection and preparation by the end of the experiment, animals were left without any chow overnight, weighted and euthanized. blood was collected retro-orbitally by capillary tubes, left for 10 minutes, centrifuged at 3000 rpm for 10 min and sera were collected and kept in clean stoppered plastic vials at -80°c right analyses of alanine transaminase (alt), ast, gamma-glutamyl transferase (ggt), t. bilirubin, afp and bcl-2. both alt and ast activities were spectrophotometrically estimated,13 ggt activity was determined 14 and bilirubin was spectrophotometrically estimated.15 quantitative measurement of afp level in serum was executed by elisa kit (wkea med supplies corp, china, code no. war-348), following the manufacturers’ instructions. bcl-2 concentration was computed by commercial kit, following the insert instructions [bcl-2 kit (biorbyt), life science (uscnk) company inc uk cat. no orb52840], by sandwich enzyme immunoassay. the absorbance was measured by elisa plate reader at 405 nm.16 liver tissue was immediately isolated, cleaned from tissues adhering matters, washed by saline solution, cold by ice, then dried on a filter paper and frozen at −80°c. the liver tissues were homogenized in potassium phosphate buffer (10% w/v, 0.01 m ph 7.4) for estimation of glutathione s-transferase (gst), catalase (cat) enzyme activities, total thiol (tt), total antioxidant capacity (tac) and total protein (tp) content. kcl solution (1.15 m) was used for estimation of malondialdehyde (mda) using homogenizer (hettich model eba 12r, germany). mda is an end product, produced by decomposition of unsaturated fatty acids attacked by free radicals. mda was measured spectrophotometrically.17 the protein content in the tissues was determined spectro photo metrically.18 tt was assayed by dtnb.19 tac was determined utilizing the ferric reducing antioxidant potential.20 gst enzyme activity was assayed after formation of adduct, through coupling of reduced glutathione (gsh) with 1-chloro-2,4-dinitrobenzene (cdnb), as described before.21 cat enzyme activity monitored depending on h2o2 decomposition at 240 nm. 22 histological evaluation histological study was applied on serial random liver sections (5-mm thick), using rotary microtome (litz, wetzlar, germany) and were stained with haematoxylin and eosin (h&e) staining.23 statistical analysis the results were shown as mean ± sem. significance of data variations were assessed by one way analysis of variance (anova), followed by computing t-test, which compare between the two groups, using graph pad prism software.24 a value of p < 0.05 was our margin of statistical significance. results table 1 depicted that the group treated with dena depicted significant up regulation of alt, ast and ggt activities in plasma (p < 0.001) compared to control. total bilirubin and afp levels of this group was significantly elevated (p < 0.001), in relation to control group showing the destructive role of dena. conversely, serum bcl-2 level was significantly depressed (p < 0.001), compared to control. table 2, which included liver tissue chemistry, showed that dena injection significantly elevated mda level (p < 0.001), in relation to control. both tp (p < 0.01), total thiol (p < 0.001) contents, tac (p < 0.01), gst activity (p < 0.001) and body weight (p < 0.001) were significantly decreased, while catalase activity was significantly upregulated (p < 0.001), compared to control. table 2. variations in the liver tissue content of mda, total protein, total thiol, tac,gst and cat activities, body weight and relative liver weight in female albino rats treated with hepatocarcinogen (hcc group), compared to control (values are expressed as mean ± sem; number of rats = 9) parameters group mda (nmol/g) total protein (mg/g) total thiol (mm/g) tac (µmol/g) gst (mol/min/g) catalase (mol/min/g) body weight (g) control 123.1 ± 11.5 91 ± 1.4 41.22 ± 0.80 13.6 ± 0.8 5.41 ± 0.069 2.07 ± 0.06 30.8 ± 1.2 hcc 230 ± 5.6*** 84 ± 0.7* 30.1 ± 0.7*** 9.7 ± 0.6** 4.0 ± 0.13*** 2.9 ± 0.05*** 12 ± 2.4*** ***, indicates significant, compared to control at p < 0.001; **, significant, compared to control at p < 0. 01; *, significant, compared to control at p < 0.05. table 1. variations in serum alt, ast, ggt enzyme activities, total bilirubin, afp and bcl 2 levels in female albino rats treated with hepatocarcinogen (hcc group), compared to control (values are expressed as mean ± sem; number of rats = 9) parameters group alt (u/l) ast (u/l) ggt (u/l) t. bilirubin (mg/dl) afp (ng/ml) bcl 2 (ng/ml) control 11.5 ± 0.3 93.7 ± 1.1 4.7 ± 0.3 0.172 ± 0.016 178 ± 8.2 458 ± 11.3 hcc 19.7 ± 0.7*** 127 ± 2.9*** 13.1 ± 0.4*** 0.3 ± 0.02*** 277 ± 10.9*** 347 ± 9.07*** ***, indicates significant, compared to control at p < 0.001; **, significant, compared to control at p < 0. 01; *, significant, compared to control at p < 0.05. nabil m. abdel-hamid et al. 297j contemp med sci | vol. 3, no. 12, autumn 2017: 295–299 research oxidative stress predominates apoptosis during experimental hepatocellular carcinoma histological evaluation of liver tissue from experimental groups after h&e staining showed that the control group had normal architecture, large polygonal cells with round nuclei and regular hepatic sinusoids arranged among hepatic cords (fig. 1a). the surrounding hepatocytes showed pyknotic small peripheral nuclei and acidophilic vacuolated cytoplasm and binuclear cells were observed (fig. 1b). stained sections of the dena-intoxicated group revealed a loss of the normal architecture of the liver, with congestion of the central veins and micronodules of varying sizes containing mononuclear inflammatory infiltration were also observed. pigmented hyper plastic kupffer cells were also seen (fig. 1b*). discussion the present observation was carried out to pursue the ability of free radicals to modulate apoptotic behavior in hepatocellular carcinogenesis in an experimental model. ast and alanine transaminase (alt) are reliable markers for liver damage assessment. hepatotoxicity disrupts hepatocytes membrane leading to spillage of transaminases into plasma.25 ggt, shows tissue specific action and is up regulated during many normal and disease conditions, as development and carcinogenesis.26 our study showed that dena-intoxicated group, had a significant elevation in alt, ast and ggt activities. this elevation was referred to the potential of dena to release free radicals which damage cell membranes.27 plasma total bilirubin is a sensitive test for the assessment of liver diseases.28 plasma total bilirubin was significantly elevated in dena -injected rats, possibly due to interference with the glucoronidation reaction and liberation of unconjugated bilirubin away from damaged liver tissue.29,30 high levels of afp are suggestive of hcc, and more than 70% of hcc holders show high plasma concentration due to tumor secretion. the up regulation of afp level observed in denatreated animals is suggestive of hcc.31 in our study, the anti-apoptotic factor, bcl2, in the group treated with dena was significantly depressed. this was reported before in dena related hepatic cancer, where, the initiation and progression of primary hcc was associated with proliferation and disturbed apoptosis linked to abnormal liberation of bcl-2 and bax genes.32 we noticed that oxidative potential resulted from injection of dena, manifested as, lipid peroxidation and perturbation of membrane unsaturated fatty acids in the cells might oppose apoptosis. oxygen-free radicals hit polyunsaturated fatty acid terminals in phospholipids. mda is the famous end product of lipid peroxidation, which affect dna forming chemical adducts. lipid peroxidation contributes to endogenous dna changes in humans leading to cancer and other related diseases.33 in this study, we found significant elevation in liver content of mda in group treated with dena. this was previously observed in liver and lung.34 reactive oxygen species (ros) generated in cells can damage cell membrane, leading to decrease the cellular protein synthetic function.35 in our results, tp content in hcc group was significantly decreased compared to control, mostly a result of the cellular damage generated by dena. this could be confirmed by previous reports.36 tt content of the body , including sulfhydryl (sh) terminals found in protein are sought as major systemic antioxidant in vivo, carried by albumin.37 they behave as reducing moieties in body compartments. biosynthesis of both cysteine and glutathione mainly occurs in hepatocytes, however, the rest tissues get thiols through sinusoidal supply into blood, thus any damage in liver tissue will affect its production.38 in this study, the significant down regulation of the total non-protein thiols and protein thiols confirmed oxidative stress referred to generated electrophiles by dena toxicity. this shows an accordance with.39 tac possesses superoxide dismutase (sod), catalase, glutathione peroxidase (gpx), macromolecules as albumin, ceruloplasmin and ferritin. tac constitutes the collective effect of all antioxidants in body compartments.40 in the current work, tac was decreased in group treated with dena. gst mediates the combination of gsh to many electrophilic compounds, as carcinogens, and native reactive species.41 these compounds become less harmful than the original phase and are excreted out. in our experiments, we have observed a striking decrease in gst activity in liver after dena treatment. this was reported earlier, where dena administration initiated renal carcinogenesis with the same effect on gst activity.36 catalase is a peroxisomal enzyme that mediates breakdown of h2o2 into o2 and water. it plays a pivotal role in cellular oxidant protection. fe-catalase is a tetrameric metallo protein with protohaem, being the major structural component of the active site and the principal determinant of enzymatic activity.42 (print our observations depicted that experimental hcc group contained a significant elevation of catalase activity. it seems that cat activity was increased, fig. 1 histological investigation of h&e stained liver sections in control and hcc groups. (a) stands for photomicrographs of control group(x400), (b) and (b*) are micrographs of hcc group(x200, x100, respectively): n.b: a. control hepatic tissue sections show normal cellular architecture. b: hepatic tissue section after dena injection, shows increased nuclear/cytoplasm (n/c) ratio. b* hepatic tissue section shows increased mitotic figures and nuclear polymorphism, increased width of cord cells, with microacinar formation. a b b* 298 j contemp med sci | vol. 3, no. 12, autumn 2017: 295–299 oxidative stress predominates apoptosis during experimental hepatocellular carcinoma research nabil m. abdel-hamid et al. since the level of h2o2 is elevated in hcc group. previous report depicted that few human cancer cell lines and tissues produced a high amount of h2o2 during cancer development.39,43,44 the results of our study indicated significant reduction in body weight gain (bwg) of the group treated with dena, in respect to control group. this was in accordance with naura et al.34 who showed that dena administration, greatly decreased anima total body weight. this was, in part, referred to the fact that, cancerous tissue and severe inflammation of hepatocytes may depress the muscle mass formation.45 conclusion we conclude that, disturbed antioxidant status, liberation of harmful-free radicals, concomitant to depressed cellular anti-oxidant potential may depress the apoptotic machinery, leaving a free tendency of hepatocytes to carcinogenesis. the elevated oxidative stress, shown by depressed antioxidant mediators plus elevated stress inducers, parallel to the decreased bcl-2 level, points for assuming that, the oxidative stress contributes priorly role in hepatocarcinogenesis than the pro oncogenic bcl-2. this promotes diagnosis and treatment of cancer, relying on the studied dilemma of variables, without ignoring oxidative stress at any stage. abbreviations: ast, aspartate aminotransferase; alt, alanine aminotransferase; tp, total protein; ros, reactive oxygen species; gst, glutathione-s-transferase; cat, catalase; sh-groups, sulfhydryl groups; dena, diethyl nitrosamine; tac, total antioxidant capacity; mda, malondialdehyde; t.bil, total bilirubin; ggt, gamma glutamyl transferase; bwg, body weight gain, afp, alpha feto protein. bcl-2, b cell lymphoma 2, tt, total thiol. author contributions nabil contributed to conception, design, critical review of the manuscript, afrah shared analysis and point concepts, mostafa shared drafting and review the manuscript, naglaa drafted the manuscript and asmaa executed the experimental work and drafted the article. declaration of conflicting interests the authors declare that no conflicts of interest regarding publication of the manuscript. funding the authors declare that the research didn’t receive any funding. ethical approval all implemented steps in this work complied with the ethical standards of the university research committee and with the helsinki declaration; no formal ethical review was required. acknowledgement we acknowledge tissue biology department of biology, faculty of science, university of tanta, egypt for their help all over the work. n references 1. theise, nd, chen cj, kew mc. liver cancer; lyon. 2014;577–593. 2. farazi pa, depinho ra. hepatocellular carcinoma pathogenesis: from genes to environment. nat rev cancer. 2006;6:674–687. 3. bhosale pml, ignle ad, gadre rv, rao kvk. protective effect of rhodotorula glutinis ncim3353 on the development of hepatic preneoplastic lesions. current science. 2002;83:303–308. 4. lin k, shen zy, lu sh, wu yn. intake of volatile n-nitrosamines and their ability to exogenously synthesize in the diet of inhabitants from highrisk area of esophageal cancer in southern china. biomed environ sci. 2002;15:277–282. 5. verna l, whysner j, williams gm. n-nitrosodiethylamine mechanistic data and risk assessment: bioactivation, dna-adduct formation, mutagenicity, and tumor initiation. pharmacol ther. 1996;71:57–81. 6. ha ws, kim ck, song sh, kang cb. study on mechanism of multistep hepatotumorigenesis in rat: development of hepatotumorigenesis. j vet sci. 2001;2:53–58. 7. abdel-hamid nm, nazmy mh, abdel-ghany mi, nazmy wh. cytokines as important playmakers of experimental hepatocarcinogenesis confounded by diabetes. ann hepatol. 2012;11:118–127. 8. bartsch h, nair j. chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, dna damage, and repair. langenbecks arch surg. 2006;391:499–510. 9. chandra j, samali a, orrenius s. triggering and modulation of apoptosis by oxidative stress. free radic biol med. 2000;29:323–333. 10. abdel-hamid nm. recent insights on risk factors of hepatocellular carcinoma. world j hepatol. 2009;1:3–7. 11. elmore s. apoptosis: a review of programmed cell death. toxicol pathol. 2007;35:495–516. 12. al-rejaie ss, aleisa am, al-yahya aa, bakheet sa, alsheikh a, fatani ag, et al. progression of diethylnitrosamine-induced hepatic carcinogenesis in carnitine-depleted rats. world j gastroenterol. 2009;15:1373–1380. 13. reitman s, frankel s. a colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. am j clin pathol. 1957;28:56–63. 14. szasz g. a kinetic photometric method for serum gamma-glutamyl transpeptidase. clin chem. 1969;15:124–136. 15. walter mgh. a colorimetric method for determination bilirubin in serum and plasma. micro chem j. 1970;15:231–236. 16. sabokrouh a, goodarzi mt, vaisi-raygani a, khatami s, taghizadeh-jahed m. effects of treatment with platinum azidothymidine and azidothymidine on telomerase activity and bcl-2 concentration in hepatocellular carcinoma induced rats. avicenna j med biotechnol. 2014;6:200–209. 17. lahouel m, boulkour s, segueni n, fillastre jp. [the flavonoids effect against vinblastine, cyclophosphamide and paracetamol toxicity by inhibition of lipid-peroxydation and increasing liver glutathione concentration]. pathol biol. 2004;52:314–322. 18. ohnishi st, barr jk. a simplified method of quantitating protein using the biuret and phenol reagents. anal biochem. 1978;86:193–200. 19. sedlak j, lindsay rh. estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with ellman’s reagent. anal biochem. 1968;25:192–205. 20. benzie if, strain jj. ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. meth enzymol. 1999;299:15–27. 21. habig wh, pabst mj, jakoby wb. glutathione s-transferases. the first enzymatic step in mercapturic acid formation. j biol chem. 1974;249: 7130–7139. 22. xu jb, yuan xf, lang pz. determination of catalase activity and catalase inhibition by ultraviolet spectrophotometry. chin environ chem. 1997;16:73–76. 23. bancroft jd, cook hc. in manual of histological techniques and their diagnostic application., churchill livingstone: edinburgh, london, new york, tokyo; 1994. pp. 23–26. 24. graph p. graph pad instate soft ware. www.graphpad.com. 25. whittby lg, perey-robb iw, smith at. enzymes tests in diagnosis. in lecture notes in clinical chemistry, 3rd eds, black well scientific publications; 1984. pp. 138–169. nabil m. abdel-hamid et al. 299j contemp med sci | vol. 3, no. 12, autumn 2017: 295–299 research oxidative stress predominates apoptosis during experimental hepatocellular carcinoma 26. yao df, dong zz, yao db, wu xh, wu w, qiu lw, et al. abnormal expression of hepatoma-derived gamma-glutamyltransferase subtyping and its early alteration for carcinogenesis of hepatocytes. hbpd int. 2004;3:564–570. 27. jayakumar s, madankumar a, asokkumar s, raghunandhakumar s, gokula dhas k, kamaraj s, et al. potential preventive effect of carvacrol against diethylnitrosamine-induced hepatocellular carcinoma in rats. mol cell biochem. 2012;360:51–60. 28. ramakrishnan g, augustine ta, jagan s, vinodhkumar r, devaki t. effect of silymarin on n-nitrosodiethylamine induced hepatocarcinogenesis in rats. exp oncol. 2007;29:39–44. 29. rajkapoor b, jayakar b, murugesh n, sakthisekaran d. chemoprevention and cytotoxic effect of bauhinia variegata against n-nitrosodiethylamine induced liver tumors and human cancer cell lines. j ethnopharmacol. 2006;104:407–409. 30. jagan s, ramakrishnan g, anandakumar p, kamaraj s, devaki t. antiproliferative potential of gallic acid against diethylnitrosamine-induced rat hepatocellular carcinoma. mol cell biochem. 2008;319:51–59. 31. endo y, kanai k, oda t, mitamura k, iino s. clinical significance of alphafetoprotein in hepatitis and liver cirrhosis. ann n y acad sci. 1975;259:234–238. 32. liu h, zhang l, chen s, wang z, huang, f, wang d. the role and significance of bcl-2 and bax in the hepatic carcinoma. int j morphol. 2012;30: 1466-1473. 33. marnett lj. oxy radicals, lipid peroxidation and dna damage. toxicology. 2002;181–182:219–222. 34. naura as, kalla nr, sharma rp, sharma r. anticarcinogenic effects of hexaamminecobalt(iii) chloride in mice initiated with diethylnitrosamine. biol trace elem res. 2007;119:147–165. 35. abdel-wahhab ma, omara ea, abdel-galil mm, hassan ns, nada sa, saeed a, et al. zizyphus spina-christi extract protects against aflatoxin b1-initiated hepatic carcinogenicity. afr j tradit complement altern med. 2007;4:248–256. 36. pracheta sv. anti-carcinogenic potential of euphorbia neriifolia leaves and isolated flavonoid against n-nitrosodiethylamine-induced renal carcinogenesis in mice. indian j biochem biophys. 2013;50:521–528. 37. prakash m, upadhya s, prabhu r. protein thiol oxidation and lipid peroxidation in patients with uraemia. scand j clin lab invest. 2004;64:599–604. 38. peters wh, van schaik a, peters jh, van goor h. oxidisedand total nonprotein bound glutathione and related thiols in gallbladder bile of patients with various gastrointestinal disorders. bmc gastroenterol. 2007;7:7. 39. gayathri r, priya kdpd, gunassekaran gr, sakthisekaran d. protective role of ursolic acid in den induced oxidative stress mediated hepatocellular carcinoma a focus on thiol status. int j pharm pharmaceut sci. 2010;140–146. 40. fiala s, mohindru a, kettering wg, fiala ae, morris hp. glutathione and gamma glutamyl transpeptidase in rat liver during chemical carcinogenesis. j natl cancer inst. 1976;57:591–598. 41. gad as, khadrawy ya, el-nekeety aa, mohamed sr, hassan ns, abdelwahhab ma. antioxidant activity and hepatoprotective effects of whey protein and spirulina in rats. nutrition. 2011;27:582–589. 42. fiala s, mohindru a, kettering wg, fiala ae, morris hp. glutathione and gamma glutamyl transpeptidase in rat liver during chemical carcinogenesis. j natl cancer inst. 1976;57:591–598. 43. fita i, rossmann mg. the active center of catalase. j mol biol. 1985;185:21–37. 44. ozdemirler erata g, kanbağli o, durlanik o, bulut t, toker g, uysal m. induced oxidative stress and decreased expression of inducible heat shock protein 70 (ihsp 70) in patients with colorectal adenocarcinomas. jpn j clin oncol. 2005;35:74–78. 45. gopčević kr, rovčanin br, tatić sb, krivokapić zv, gajić mm, dragutinović vv. activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in different stages of colorectal carcinoma. dig dis sci. 2013;58:2646–2652. 46. dai zj, wu wy, kang hf, ma xb, zhang sq, min wl, et al. protective effects of scutellaria barbata against rat liver tumorigenesis. asian pac j cancer prev. 2013;14:261–265. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201702 55j contemp med sci | vol. 4, no. 1, winter 2018: 55–58 research gold standard approach for removing sialoliths from the submandibular gland elham hazeim abdulkareem,a kamal turki aftan,a ahmed jassam alnaqeeba aoral and maxillofacial surgery department, college of dentistry, university of anbar, iraq. correspondence to elham hazeim abdulkareem (email: den.elham.h@uoanbar.edu.iq). (submitted: 19 october 2017 – revised version received: 07 november 2017 – accepted: 14 january 2018 – published online: 26 march 2018) objective sialolithiasis, the most common disease of the salivary gland, is characterized by calculi in gland and its duct. while most salivary calculi are small in size, giant calculi with diameters spanning several centimeters are also reported occasionally. the surgical removal of recurrent submandibular sialoliths. results this study presents of recurrent submandibular sialadenitis/sialadenosis and the characteristic signs and symptoms included pain, recurrent swelling, and discharge of pus, fibrotic and atrophic condition of the gland followed by cessation of secretary functions. radiological examinations revealed large and irregular structures in the reported cases. conclusion we have described the characteristic clinical and radiological features, diagnosis and treatment approach for submandibular salivary gland sialadenitis/sialadenosis. keywords chronic sialadenitis/sialadenosis, ct scan, excision of gland, sialoliths of the submandibular salivary gland introduction sialadenosis is one of the predominantly occurring conditions of the salivary glands.1–3 carbonate and phosphate forms of calcium, remains of desquamated epithelia of salivary gland, mucopolysaccharides and glycoprotein like organic components constitute a sialolith, though bacterial activity is unidentified in the existing literature.4–6 based on the majority of reported cases, it is estimated that out of 1,000 adults, 12 are afflicted with sialolithiasis annually and majority of them constitute male population.7 the frequency of rapidly setting (acute) and persistent (chronic) infections of sialolithiasis affecting submandibular salivary gland is higher by 90% while parotid gland affliction is 6%, remaining 2% affects sublingual gland but minor saliva secretion glands seldom develop calculi. the dimension of the stone ranges between scant centimeters and 1 mm, majorly less than 10 mm.8 also, only very few cases have noted giant calculi with the diameter exceeding 15 mm.9 the causative factors constitute stand still flow of the saliva in the mouth, diameter and extent of the ducts, course of salivary rush and its composition, extreme basic nature, presence of infectious organisms in the mouth or substantial injury to the gland or the ducts.10 due to high levels of calcium, phosphate, mucus and greater basicity, the sequential genesis of stones armor in submandibular gland as compared to any other salivary gland. when the components of stone separate out on the basis of their solubility, the calculus happens to be the primary hydroxyapatite cynosure.11 sialoliths are believed to be born in the ducts, which facilitates the stones to have an elaborate dimension and be submerged in saliva. calculi might take long duration to enhance their dimensions and in the meanwhile do not display any signs or symptoms.12 other potential reasons for calculus developing majorly in submandibular gland could be attributed to increased length and duct diameter, anti-gravitational course, lesser glide of the saliva, increased basicity, mucous and the concentration of calcium.13 the most noticeable feature includes infective condition of the related gland in addition to the repeated discomfort in neck and mouth.14 absolute closure gives continuous pain due to swelling and pus discharge while prolonged closure leads to glandular atrophy and fibrosis followed by cessation of saliva ejection. if the calculus is placed posteriorly, it can be felt by palpating the floor of the oral cavity.15 a careful palpation of the oral floor allows the detection of the exact position of a stone and the diagnosis can be confirmed through ultrasonography (usg), conventional radiography, computed tomography (ct), and/or cone beam computed tomography.16 a ct scan gives exact dimension of the submandibular gland calculus and helps to select the treatment modality.17 ct is the most meticulous method to locate the position of the stones.18 many surgical techniques are performed to treat the submandibular gland sialadenosis. the initial method used involves putting an incision in the oral cavity on the mucosal lining over the stone so that it is easily removed.19 chronic sialadenitis is a severe condition that leads to salivary gland hypofunction. prolonged barrier in the ductal system leads to infectious disorders and inflamed tissues; therefore, it should be excised.20 the following case report presents the condition of three patients with prolonged ductal obstruction, atrophy and fibrosis followed by loss of saliva secretion.21 the aim of this study is the surgical removal of recurrent large submandibular sialoths. materials and methods patients the present study was reported at the al ramadi teaching hospital, ramadi, iraq. a total of 30 patients were admitted in the maxillofacial department with an ailment of swelling, pain and inflammation on one side restricted to the lower jaw region. written consent for publication of their cases and images has been obtained from the respective patients. committee of ethics of al-ramadi teaching hospital has assented to this study (registration number: 1694 on 06/08/2017) and issn 2413-0516 56 j contemp med sci | vol. 4, no. 1, winter 2018: 55–58 the surgical management of recurrent submandibular gland sialoliths research elham hazeim abdulkareem et al. committee of ethics of university of anbar has assented to this study (registration number: 38 on 19/12/2017). patient a: a 47-years-old male with above-mentioned symptoms on the left side of the mandible was observed (fig. 1a). he had tender pain, fever and chills. palpation (bimanual) inside the mouth reflected swelling while pressure on the ductal orifice, revealed pus discharge. ct image showed calcified structure in the left submandibular gland that confirmed the primary diagnosis of sialolithiasis (fig. 1b, c). patient b: a 77-years-old female with aforementioned symptoms on the right side of the mouth floor having a large palpable stone was observed. on ct scan image, an opaque mass made the sialolithiasis diagnosis evident (fig. 2). patient c: a 55-years-old non-smoker female with symptoms similar to the other two patients, but the stone was observed in the left submandibular gland and beneath the tongue. palpable mass was mobile, soft and presents sensation of a nut below the tongue (left side). ct scan demonstrated radiopaque lesion reinforcing the diagnosis as sialolithiasis (fig 3). surgical treatments we elected to surgically remove sialolith along with the gland under general anesthesia. submandibulectomy and sialadenectomy were performed by an incision through the dermal layers, between two locations; exactly 2 cm beneath the mandibular lower border and above the gland. sialoadenectomy was performed around the muscle and the branch of the facial nerve. the position of the sialolithiasis leads to the curving of the skin near granulation cells and sinus drainage (fig. 1d) and the incision was closed layer by layer (figs. 1e and 1f ). all the fig 2. (a and b) the presence of the sialolith was confirmed on ct, with an opaque mass evident in the right submandibular region. (c) the patient underwent excision of the right submandibular gland. fig 1. (a) clinical examination revealed left submandibular swelling and tenderness, with a large salivary calculus palpable on the left floor of the mouth. (b and c) the presence of the sialolith was confirmed on ct, with an opaque mass evident in the left submandibular region. (d–f ) the patient underwent excision of the submandibular gland and stone via the standard extra-oral approach, without any complications. patients underwent the same extra-oral surgical technique; affected side submandibular gland was removed along with the calculus and had uneventful healing without any post operative intricacy. discussion sialolithiasis is a pathology manifested by impediment of ductal system by sialoliths (calculi).22 the growing dimensions a a c b b c d e f elham hazeim abdulkareem et al. 57j contemp med sci | vol. 4, no. 1, winter 2018: 55–58 research the surgical management of recurrent submandibular gland sialoliths of calculi enhance the blockage of saliva release, causing infections and swollen glands.22 complete obliteration causes painful enlargement.23 prolonged impediment of the ductal system of the salivary glands causes consequential bereavement from saliva.24 the secretions of submandibular gland lead to increased levels of hydroxyapatite as well as phosphates enhanced viscosity, more mucous and escalated basicity.25 therefore, it has more chances to develop calculus than all other salivary glands. this further leads to proclivity toward salt precipitation around the duct opening.25 the wharton’s duct is thin and traverses upward for its opening (shown in aforementioned image), and this anatomy tends to produce more hindrance in the saliva emancipation.26 the chief complaint of the patient includes ephemeral and discomforting pain swelling, during or after having food in the salivary glands which reduce within 2 or 3 h and most importantly curtailed saliva formation.26,27 complications of sialolithiasis include presence of secondary infections, abscess formation, stenosed saliva ducts, mucocele development, chronic sclerosingsialadenitis (kuttner’s tumor) and chronic atrophic parenchymal layer of the gland.28,29 bigger calculi are ovoid or long-drawn-out, firm, yellow and sponge like in appearance, which radiologically surface out as tooth-like structures. similarly, the cases mentioned in this article have recurrent swelling pus discharge and pain in the gland.30 observed subjects also have a fibrosis edema and palpable mass of sialoliths, felt by the examiner in the base of the mandible.31 large stones are managed through surgical removal with or without the gland excision, whereas smaller ones require conservative treatment in the form of locales high-temperature application, acupressure, and saliva promoting drugs (sialagogues).32,33 surgical removal of the damaged gland is a remedy for patients who have relapse of sialolithiasis and prove to be a gold standard cure in conditions when calculi are present within the non functioning glands. however, eliminating the gland is mandatory in relapsing cases.34,35 investigations helping in the diagnosis include radiographs, radiological sialography, usg, ct, mri and endoscopy of the salivary glands.29 though costly, tomographic investigation is the most meticulous non-surgical method for detecting the position, size of even the tiniest stones and the number of clustered sialoliths.17,29,36,37 after the successful removal of the submandibular gland and calculus, close follow up is needed.38 submandibularadenectomy, sometimes, has a side effect on nerve (marginal mandibular) causing palsy (permanent or short-lived), though none of the subjects suffered from this condition.39 conclusion the study was highlighting the first line of treatment of sialolithiasis as adenectomy. the finding of the present study suggests that any gland having fibrotic, infectiously swelled, pus releasing, loss of function with a sialolith should undergo sialadenectomy. conflict of interest the authors reveal that they have no monetary clash of interest in connection to this work. n fig 3. (a) a ct scan of the mandibular region showed the presence of a rounded radiopaque sialolith. (b) the sialolith was surgically removed under general anesthesia. references 1. gupta a, rattan d, gupta r. giant sialoliths of submandibular gland duct: report of two cases with unusual shape. contemp clin dent. 2013;4:78–80. 2. blitzer a. inflammatory and obstructive disorders of salivary glands. j dent res. 1987;66 spec no: 675–679. 3. williams mf. sialolithiasis. otolaryngol clin north am. 1999;32:819–834. 4. produl h, seema e, punnoose rs, sanjay a. deep and unusual sialolithiasis of submandibular duct and gland: a surgical dilemma. indian j otolaryngol head neck surg. 2013;65:309–313. 5. cawson ra, odell ew. essentials of oral pathology and oral medicine, 6th ed.; edinburgh: churchill livingstone, 1988; pp. 239–240. 6. taher aa. the incidence and composition of salivary stones (sialolithiasis) in iran: analysis of 95 cases—a short report. singapore dent j. 1989;14:33–35. 7. leung ak, choi mc, wagner ga. multiple sialoliths and a sialolith of unusual size in the submandibular duct: a case report. oral surg oral med oral pathol oral radiol endod. 1999;87:331–333. 8. choi ws, yoon hj, lee sh. giant sialolithiasis of the submandibular gland: a case report. j korean assoc oral maxillofacial surg. 2010;36:141–144. 9. cho sh, han jd, kim jh, lee sh, jo jb, kim ch, et al. removal of submandibular calculi by surgical method and hydraulic power with curved needle: a case report. j korean assoc oral maxillofacial surg. 2017;43:182–185. 10. raksin sz, gould sm, williams ac. submandibular duct sialolith of unusual size and shape. j oral surg. 1975;33:142–145. 11. mckenna jp, bostock dj, mcmenamin pg. sialolithiasis. am fam physician. 1987;36:119–125. 12. work wp, hecht dw. inflammatory disease of major salivary glands. in otolaryngology; paparella mm, shumrick da, eds.; w. b. saunders: philadelphia, 1980; vol. 13, pp. 2235–2243. 13. alyas f, lewis k, williams m, moody ab, wong kt, ahuja at, et al. diseases of the submandibular gland as demonstrated using high resolution ultrasound. br j radiol. 2005;78:362–369. 14. manjunath r, burman r. giant submandibular sialolith of remarkable size in the comma area of wharton’s duct: a case report. j oral maxillofacial surg. 2009;67:1329–1332. 15. tepan mg, rohiwal rl. multiple salivary calculi in wharton’s duct. j laryngol otol. 1985;99:1313–1314. 16. abdel-wahed n, amer me, abo-taleb ns. assessment of the role of cone beam computed sialography in diagnosing salivary gland lesions. imaging sci dent. 2013;43:17–23. 17. oteri g, procopio rm, cicciu m. giant salivary gland calculi (gsgc): report of two cases. open dent j. 2011;5:90–95. 18. weissman jl. imaging of the salivary gland. semin ultrasound ct mr. 1995;16:546–568. 19. juul ml, wagner n. objective and subjective outcome in 42 patients after treatment of sialolithiasis by transoral incision of warthon’s duct: a retrospective middle-term follow-up study. eur arch otorhinolaryngol. 2014;271:3059–3066. 20. markiewicz mr, margarone je iii, tapia jl, aguirre a. sialolithiasis in a residual wharton’s duct after excision of a submandibular salivary gland. j laryngol otol. 2007;121:182–185. 21. wallace e, tauzin m, hagan i. management of giant sialoliths: review of the literature and preliminary experience with interventional sialendoscopy. laryngoscope. 2010;120:1974–1978. 22. levy dm, remine wh, devine kd. salivary gland calculi. pain, swelling associated with eating. jama. 1962;181:1115–1119. a b 58 j contemp med sci | vol. 4, no. 1, winter 2018: 55–58 the surgical management of recurrent submandibular gland sialoliths research elham hazeim abdulkareem et al. 23. antoniadis d, mendonidou l, papanayotou p, trigonidis g. clinical study of sialolithiasis. findings from 100 cases. hell stomatol chron. 1989;33:245–251. 24. bodner l. giant salivary gland calculi: diagnostic imaging and surgical management. oral surg oral med oral pathol oral radiol endod. 2002;94:320–323. 25. yousem dm, kraut ma, chalian aa. major salivary gland imaging. radiology. 2000;216:19. 26. bialek ej, jakubowski w, zajkowski p, szopinski kt, osmolski a. us of the major salivary glands: anatomy and spatial relationships, pathologic conditions, and pitfalls. radiographics. 2006;26:745–763. 27. hasson o. sialoendoscopy and sialography: strategies for assessment and treatment of salivary gland obstructions. j oral maxillofac surg. 2007;65:300–304. 28. drage na, brown je, escudier mp, mcgurk m. interventional radiology in the removal of salivary calculi. radiology. 2000;214:139–142. 29. madani g, beale t. inflammatory conditions of the salivary glands. semin ultrasound ct mr. 2006;27:440–451. 30. kalia v, kalra g, kaur s, kapoor r. ct scan as an essential tool in diagnosis of non-radiopaque sialoliths. j maxillofac oral surg. 2015;14:240–244. 31. ying x, kang j, zhang f, dong h. recurrent sialoliths after excision of the bilateral submandibular glands for sialolithiasis treatment: a case report. exp ther med. 2016;11:335–337. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 32. graziani f, vano m, cei s, mario g. unusual asymptomatic giant sialolith of the submandibular gland: a clinical report. j craniofac surg. 2006;17: 549–552. 33. hazarika p, punnoose se, singh r, arora s. deep and unusual sialolithiasis of submandibular duct and gland: a surgical dilemma. indian j otolaryngol head neck surg. 2013;65:309–313. 34. fowell c, macbean a. giant salivary calculi of the submandibular gland. j surg case rep. 2012;2012:6. 35. iqbal a, gupta ak, natu ss, gupta ak. unusually large sialolith of wharton’s duct. ann maxillofac surg. 2012;2:70–73. 36. uggal, ravanelli m, pallottino aa, farina d, maroldi r. diagnostic work-up in obstructive and inflammatory salivary gland disorders. acta otorhinolaryngol ital. 2017;37:83–93. 37. rzymska-grala i, stopa z, grala b, gołębiowski m, wanyura h, zuchowska a, et al. salivary gland calculi – contemporary methods of imaging. pol j radiol. 2010;75:25–37. 38. arslan s, vuralkan e, çobanog˘lu b, arslan a, ural a. giant sialolith of submandibular gland: report of a case. j surg case rep. 2015;2015: pii. rjv043. 39. siddiqui sj. sialolithiasis: an unusually large submandibular salivary stone. br dent j. 2002;193:88–99. dx.doi.org/10.22317/jcms.03201812 1j contemp med sci | vol. 1, no. 3, summer 2015: 1–3 case report leiomyosarcoma (lms) is a rare malignant lesion of head and neck region. it usually arises from smooth muscle cells of blood vessel wall in this area. clinically, the tumour often presents as a slow-growing, painless destructive mass with a relatively firm consistency. it usually involves adults, and is rarely reported in children. here, we report a case of lms of maxilla in a 73-year-old male with a destructive behaviour and also discuss the diagnostic procedure proposed to make a definitive diagnosis in such unusual cases. keywords maxilla, leiomyosarcoma, neoplasm leiomyosarcoma of the maxilla: a rare challenging case sedigheh rahrotaban,a pouyan aminishakib,a samira derakhshana & sara mehrabib introduction soft tissue sarcomas are rare neoplasms in the head and neck as only 10% of all soft tissue tumours occur in this region.1 there are many histopathologic difficulties in the diagnosis of various subtypes of sarcomas.2 leiomyosarcoma (lms) is a malignant tumour of smooth muscle origin and is frequently seen at sites with large amount of smooth muscles such as female genital tract and gastrointestinal region.3,4 its occurrence in head and neck is rare accounting for 4% of all sarcomas in this area5–8 but head and neck lms behave more aggressive clinically and have poor prognosis.4 the clinical appearance of this neoplasm is usually like benign conditions so it can be mistaken for non-malignant lesions.5 aggressive primary treatment after early diagnosis is very important for improving prognosis.5 in this article, we represent a case of primary lms of the maxilla and describe the differential diagnosis of this tumour. case presentation a 73-year-old man was referred to the department of oral and maxillofacial pathology, tehran university of medical sciences, in april 2015 with a single ulcerated swelling of the posterior right area of upper jaw under complete denture about 1 month ago. clinical examination of the affected area revealed that alveolar bone was totally replaced by an exophytic ulcerated mass. no other intra/extra oral sign was observed following comprehensive clinical examination. also, the past medical/dental history of the patient was unremarkable. the panoramic view of the maxilla showed an ill-defined radiolucent lesion of the right, posterior area which seemed to invade into right maxillary sinus (fig. 1). therefore, a cone beam computed tomography (cbct) image was taken to determine the exact margin and destructive behaviour of the lesion. the imaging features of the lesion confirmed the destruction of the whole thickness of alveolar bone and infiltration to the maxillary sinus (fig. 2). then, an incisional biopsy was performed by an oral and maxillofacial surgeon, and hematoxylin and eosin (h&e) stained histopathologic slides were examined using light microscopy. the sections adepartment of oral and maxillofacial pathology, school of dentistry, tehran university of medical sciences, tehran, iran. bdepartment of oral and maxillofacial pathology, school of dentistry, zanjan university of medical sciences, zanjan, iran. correspondence to sara mehrabi (email: saramehr22@gmail.com). (submitted: 22 march 2015 – revised version received: 13 april 2015 – accepted: 27 april 2015 – published online: summer 2015) fig. 1 panoramic view of a radiograph showing destruction of the floor of the right maxillary sinus. fig. 2 cone beam computed tomography (cbct) showing destruction of buccal and palatal plate of the maxillary bone. issn 2413-0516 2 j contemp med sci | vol. 1, no. 3, summer 2015: 1–3 maxillary leiomyosarcoma case report sedigheh rahrotaban et al. revealed highly cellular fascicles of neoplastic spindle cells with pleormorphic features and scant cigar-shaped nuclei (fig. 3). numerous mitotic figures and also atypical tri-polar and ring-shaped mitotic figures were observed (fig. 4). also, scattered foci of necrosis were seen among tumoural cells. so, a primary diagnosis of “malignant spindle cell tumour” was rendered and the specimen was submitted for immunohistochemical (ihc) staining to recognise the origin of the neoplastic cells. positive ihc staining of vimentin (fig. 5), desmin, smooth muscle actin (sma) (fig. 6) and ki67 (more than 20%) (fig. 7) led to make a definitive diagnosis of lms. later, the patient was referred to an oncologist to begin the therapeutic process. discussion lms is a very rare entity in oral cavity.9 as far as we searched, there were only 12 case reports of primary maxilla lms presented in english literature during the past 15 years.1,5–8,10,11 lms may originate from sparse smooth muscles in wall of blood vessels, circumvallate papilla and myoepithelial cells of the salivary glands.12 scarcity of smooth muscles in oral cavity explains the low occurrence of this neoplasm in this region, but it also may be associated with misdiagnosis of lesion and delay in diagnosis.9 another origin for lms is pluripotential mesenchymal cells and metastasis from other body sites.5 there is no age predilection for lms12 but its frequency increases with age.9 it is more common in females.13 in our case the patient was a male, who was 73 years old at the time of diagnosis. the most frequent site of involvement of primary lms in oral cavity is mandible followed by maxilla, tongue, buccal mucosa, lip, floor of the mouth, hard palate, soft palate and maxillary sinus.12 computed tomography and magnetic resonance imaging (mri) are needed after clinical examination. especially, mri is useful for the evaluation of vascular involvement.14 in this case, cbct revealed an ill-defined mass in the posterior part of maxilla perforating buccal and palatal cortical plates and invading the right maxillary sinus. according to imaging, maxilla was the primary arising region of the lesion but determining the exact origin of the tumour of maxillary soft tissue or bone was not possible. most often, lms is a painless mass firmly attached to the surrounding structures and sometimes may be ulcerated.12 usually signs and symptoms are dependent to the location of the lesion where it arises, and are non-specific.5 in clinical evaluation, the lesion was about 3 cm with ulceration without pain, and was located under complete denture prosthetics. although pain and tenderness are relatively prominent features in leiomyoma, they are rare presentation in lms.5 the diagnosis of lms may be challenging in some cases and should be confirmed by ihc study.15 in our case, histopathologic evaluation revealed interlacing fascicles of spindle cells scantly having blunt-end nuclei with malignant features such as prominent nucleoli and mitotic activity. the tumour was moderately differentiated and there was a list of spindle cell sarcomas for differential diagnosis such as myofibrosarcoma, fibrosarcoma, malignant peripheral nerve sheath tumour (mpnst), malignant fibrous histiocytoma and rhabdomyosarcoma. so, we recommended a panel of ihc markers including vimentin, desmin, smooth muscle actin, s100 and ki67. vimentin and desmin are intermediate filament proteins. vimentin is related to mesenchymal origin of tumour and desmin is associated with skeletal and smooth muscle cells and is the most sensitive marker for skeletal and smooth muscle differentiation, but it is not specific and can be expressed in some other non-myogenous tumours. our case showed positivity for vimentin and desmin in cytoplasm of tumoural cells.13,16 special isoforms of actin like sma is useful for differentiation between smooth and skeletal muscle cells, although it can be expressed by myofibroblasts too, but the pattern of expression is different. sma is expressed in cytoplasm periphery in myofibroblasts, but uniformly in fig. 3 fascicles of spindle shaped tumoural cells (hematoxylin and eosin stain; 40×). fig. 7 immunohistochemical stain for ki67 showing more than 50% proliferation index in tumoural cells nuclei. fig. 4 tumoural cells showing atypia and atypical mitotic figures (hematoxylin and eosin stain 100×). fig. 5 immunohistochemical stain for desmin showing positivity in cytoplasm of tumoural cells. fig. 6 immunohistochemical stain for smooth muscle actin (sma) showing positivity in cytoplasm tumoural cells. 3j contemp med sci | vol. 1, no. 3, summer 2015: 1–3 case report maxillary leiomyosarcomasedigheh rahrotaban et al. cytoplasm of smooth muscle cells. in this case sma was uniformly positive in cytoplasm of tumoural cells.16 s100 is a neurogenic marker, and in our case it was used for rolling out mpnst13 and it was mild or focally positive. ck and ema positivity may be seen in some of the lmss.5,13 the ki67 index was more than 50% in this case. some histopathologic characteristics are suggested for diagnosis between leiomyoma and lms including cellular density, necrosis, cell atypia and nuclear mitotic figures. mild atypia may be seen in leiomyoma but it has a very low mitotic index, so for confirming the diagnosis of lms 5, mitosis/10 hpf is needed. presence of necrosis can be useful for the diagnosis of lms just as in our case.16 the usual route of metastatic spread for lms is bloodstream to the lungs.5 this patient had localized disease and no palpable neck nodes at the time of presentation. the initial method for treatment was wide surgical excision. neck dissection usually is not necessary because of rarity of metastasis to lymph nodes.5 prognosis of lms is related to the site of involvement. in the head and neck area, anatomic structures limits complete resection of tumour so some difficulties in tumour management is present.2 therefore, local recurrence is under expectation especially in the first two years of treatment. further studies and case presentations would be helpful for early detection of the lesions and accurate guide for diagnosis, which both have influences on tumour management and improving survival rate.  references 1. ikram m, ahmed i, ahmed d, ahmed yi. leiomyosarcoma of the maxilla with spinal metastasis: a case report. ear nose throat j. 2003 jun;82(6): 458–60. doi: http://dx.doi.org/10.3748/wjg.v13.i7.1135 pmid: 12861874 2. teijani ma, galloway tj, lango m, ridge ja, von mehran m. head and neck sarcomas: a comprehensive cancer center experience. cancers (basel). 2013 jul 15;5(3):890–900. doi: http://dx.doi.org/10.3390/cancers5030890 pmid: 24202325 3. mesquita ra, migliari da, de sousa so, alves mr. leiomyosarcoma of the buccal mucosa: a case report. j oral maxillofac surg. 1998;56:504–7. doi: http://dx.doi.org/10.1016/s0278-2391(98)90723-6 pmid: 9541354 4. gustafson p, willén h, baldetorp b, fernö m, akerman m, rydholm a. soft tissue leiomyosarcoma. a population-based epidemiologic and prognostic study of 48 patients, including cellular dna content. cancer 1992 jul 1;70(1):114–9. doi: http://dx.doi.org/10.1002/10970142(19920701)70:1%3c114::aid-cncr2820700119%3e3.0.co;2-u pmid: 1606532 5. yadav j, bakshi j, chouhan m, modi r. head and neck leiomyosarcoma. indian j otolaryngol head neck surg. 2013 jul;65(suppl 1):1–5. doi: http:// dx.doi.org/10.1007%2fs12070-011-0305-8 pmid: 3718933 6. wada s, yue l, furuta i, takazakura t. leiomyosarcoma in the maxilla: a case report. int j oral maxillofac surg. 2002 apr;31(2):219–21. doi: http://dx.doi. org/10.1054/ijom.2001.0149 pmid: 12102424 7. sandhu sv, sodhi sp, rai s, bansal h. primary leiomyosarcoma of the maxilla: an investigation loom-report of a challenging case and review of literature. j oral maxillofac pathol. 2014;18(3):453–9. doi: http://dx.doi. org/10.4103/0973-029x.151350 pmid: 25949006 8. taghipour zahir s, sharahjin ns. leiomyosarcoma of the maxilla in a 24-year-old man who initially presented with odontalgia, and suffered from tumour mismanagement. bmj case rep. 2013 dec;5:2013, pii.  doi: http:// dx.doi.org/10.1136/bcr-2013-200933 pmid: 24311413 9. eppsteiner rw, de young br, milhem mm, pagedar na. leiomyosarcoma of the head and neck: population-based analysis. 2011 sep;137(9):921–4. doi: http://dx.doi.org/10.1001/archoto.2011.147 pmid: 21930982 10. montgomery e, goldblum jr, fisher c. leiomyosarcoma of the head and neck: a clinicopathological study. histopathology 2002 jun;40(6):518–25. doi: http://dx.doi.org/10.1046/j.1365-2559.2002.01412.x pmid: 12047762 11. riaz n, warriach ra, aftab a. massive leiomyosarcoma of the maxilla. j ayub med coll abbottabad. 2011 apr-jun;23(2):180–3. pmid: 24800376 12. nagpal dk, prabhu pr, shah a, palaskar s. leiomyosarcoma of buccal mucosa and review of literature. j oral maxillofac pathol. 2013 jan;17(1):149.  doi: http://dx.doi.org/10.4103/0973-029x.110732 pmid: 23798856 13. weiss sw, goldblum jr. enzinger and weiss’s soft tissue tumors, 6th ed. philadelphia: mosby; 2014. 14. skoulakis c, chimona ts, tsirevelou p, papadakis ce. subcutaneus leiomyosarcoma of the neck: a case report. cases j. 2010 feb 3;3:52. doi: http://dx.doi.org/10.1186/1757-1626-3-52 pmid: 20205848 15. rodini co, pontes fs, pontes ha, santos ps, magalhães mg, pinto ds jr. oral leiomyosarcoma: report of two cases with immunohistochemical profile. oral surg oral med oral pathol oral radiol endod. 2007;104(4):e50–5. http://dx.doi.org/10.1016/j.tripleo.2007.05.005 pmid: 17706443 16. rosi j. rosai and ackerman’s surgical pathology, 10th ed. philadelphia: mosby; 2011. 116 j contemp med sci | vol. 9, no. 2, march-april 2023: 116–120 original isolation, identification and comparative analysis of 16s rrna of multidrug-resistant bacteria clinically isolated from al qassim region hospitals in saudi arabia hamad saleh alkhowaiter1, othman yahya alyahyawy1, abdullah abdulhafeez aljeddawi1, salah el-deen mohamed abo-aba1,2* 1department of biological sciences, faculty of science, king abdulaziz university, jeddah, saudi arabia. 2princess doctor najla bint saud al saud distinguished research centre in biotechnology, jeddah, saudi arabia. *correspondence to: salah el-deen mohamed abo-aba (e-mail: salah_aboaba@yahoo.com ) (submitted: 09 january 2023 – revised version received: 24 february 2023 – accepted: 05 march 2023 – published online: 26 april 2023) abstract objective: this study concluded that the antibiotic resistance and gene transfer across bacterial strains in the hospital setting are two possible explanations for the observed sequence changes in the target microorganisms. methods: the 16s rdna genes of all isolates were effectively amplified using pcr, and detailed identification results were derived from genbank databases. the blast search resulted in the classification of 28 isolates into five strains. the gc content of bacterial sequences varies greatly between single species. results: 16s rdna was utilized to identify bacterial species from isolates strains results demonstrated that identification of bacterial strains. sequences varied between and within strains, also, variation are noticed in genomic nucleotide content of isolated and identified strains. results of the present study demonstrated that the observed heterogeneity in the sequences of our target bacterial strains may be linked to antibiotic resistance and gene transfer between bacteria that evolved as a result of the hospital environment. conclusion: this study concluded that antibiotic-resistant bacteria are capable of transferring copies of their dna encoding a resistance mechanism to other bacteria, even those that are distantly related to them. the observed heterogeneity in the sequences of our target bacterial strains may be linked to antibiotic resistance and gene transfer between bacteria that evolved as a result of the hospital environment. keywords: multidrug-resistant, molecular identification, rna, ribosomal, 16s, polymerase chain reaction issn 2413-0516 introduction because of the high prevalence of infectious diseases, poor hygiene, and inadequate health systems, developing nations bear an outsized burden of antimicrobial resistance (amr). in developing nations, a large proportion of hospital-acquired infections is a significant concern since they continue to have an impact on treatment outcomes and the evolution of antimicrobial resistance (amr). if existing amr conditions persist unchecked, more than 10 million fatalities will be caused by amr by 2050. pathogens such staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa. enterobacteriaceae are the most common ones that cause amr because of their high risk of spreading among patients. the continual overand imprudent use of antimicrobials has enriched for bacteria that are innately or acquired resistant to antibiotics. more and more clinical emphasis is being placed on bacteria like pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii species because of their inherent resistance to numerous treatments as well as their capacity for developing high levels of mdr.1 the creation of a multidrug-resistant (mdr) hospital environment microbial for particular applications is a necessary component of knowing how antibiotic resistance genes are transferred across various bacterial species. mdr microorganisms are difficult to isolate and identify because they display extremely specialised traits that enable them to exist in their natural settings and so cannot be cultivated using ordinary laboratory procedures.2 competition for space and resources in the hospital environment exerts a strong selection pressure on mdr bacteria, which may result in increased resistance to multiple medications.3 the 16s rdna sequence has characteristics that make it an excellent candidate for use as a universal phylogenetic marker. additionally, 16s rdna gene sequencing is a valuable approach for bacterial classification, since it involves determining the nucleotide sequences of this region and comparing them to sequences available in databases to find homology matches, allowing for bacterial identification of target samples.4 mdr bacterial strains were identified and cultured on a variety of culture medium in this study from patient samples taken from different hospitals in al qassim region. samples were pathogenic bacterial strains such as pseudomonas aeruginosa, escherichia coli, acinetobacter baumanni and providencia stuartii. in this experiment, a total of 28 mdr bacterial strains were found using 16s rdna sequence analysis and bioinformatics analysis. the evolutionary and phylogenetic relationships between isolated strains were also examined. the effectiveness of the strategy was shown by comparing it to a database of varied bacterial populations. materials and methods collection of pathogenic bacterial strains within a two-month period, 38 clinical samples of blood, wounds, sputum, and urine were obtained from hospitalised patients at al qassim hospitals in saudi arabia. clinical samples were collected under aseptic circumstances and promptly sent to the microbial genetics’ laboratory at king abdulaziz university’s department of biology, college of science. isolates were re identified depending on morphology and 117j contemp med sci | vol. 9, no. 2, march-april 2023: 116–120 h.s. alkhowaiter et al. original isolation, identification and comparative analysis biochemical test as compared with microscanwalkaway plus system kit (beckman coulter) as confirmatory test. the liquid medium used for bacterial growth was luria bertani (lb) broth. the liquid cultures were incubated at 37°c was provided growing on a nutrient agar plate. an inoculating loop was sterilised with flame and was used to pick up a colony of each strain, to inoculate into 20 ml of lb broth. the inoculated lb broth was then incubated for 24 hours. dna extraction, pcr amplification and sequencing of 16s rdna16s gene sequencing and analysis genomic dna was isolated from the cell pellets of all multidrug-resistant bacteria isolates according to the manufacturer’s instructions using a dna extraction kit (promega, usa). a single dna fragment (about 1200 bp) encoding the 16s rdna gene was amplified for each isolate using previously published procedures.5 electrophoresis in 1.5 percent agarose gel was used to identify the amplified pcr products of the 16s rrna gene bacterial gene fragments. purification and sequencing of the amplified fragments were performed at macrogen sequencing laboratory in seoul, korea, using an automated sequencer abi 3100 (applied biosystems) equipped with the bigdye terminator kit v. 3.1. (applied biosystems). sequencing was performed using primers 518 f (5¢ c ca gcag cc gc gg taatacg 3¢) and 800 r (5¢ta cc ag gg ta tc ta at cc -3¢). the sequences were modified using the vector nti suite 9 programme and compared to the ncbi database using blast searches. in this comparison, we looked for sequences of type strains that were most closely linked to the isolates’ sequences. a similarity threshold of 97 percent was used to define operational taxonomic units (otus).5 bioinformatics analysis out of 38 samples, 28 samples were selected for the study. all the 28 sequences were subjected to ncbi blast search tool http://blast.ncbi.nlm.nih.gov to detect non-chance sequence similarity. blast search was restricted to 16s ribosomal rna database, where models (xm/xp) as well as uncultured/environmental samples were also filtered out, such that more reliable results would be attained. each individual sequence was solely blastd, where blast hit with the lowest expect-value (which indicate number of non-chance alignments) was picked. in order to ensure that blast out puts were governed by expected-value (aka e-value), blast algorithm parameter was decreased such the expected threshold was set to more stringent value of 1e−6. alignment of the 28 sequence was carried out using version 2 of clustalx.6 exploratory data and phylogenetic analyses were carried out under r project for statistical computing.7 where exploratory data analysis was done using seqinr r package.6 phylogenetic analysis was carried out by ape package.8 reconstruction of the phylogenetic tree was done using neighbour joint method.8 dnasp software was used to analyse the haplotype diversity (hd), the average number of nucleotide differences, the average number of nucleotide differences,9 the nucleotide diversity (π). the polymorphic site (s), the singleton variable sites (sp), and the parsimony informative sites (pip) for each gene, and the average number of nucleotide substitutions per site between species (dxy). 10 results the isolated dna of all 28 isolated strains is presented. the findings indicate that all isolates strains have effective dna isolation procedures. as shown in figure 1, 16s rdna was utilised to identify bacterial species from 28 isolates strains and two atcc strain no. (1, 2), on nutrient agar medium. the 16s dna bands are 500 bp in length. the 16s rrna was identified using a macrogen universal primer as described in the materials and methods. as demonstrated in figure 1, the findings of 16s gene separation are pure 16s rrna gene isolation. blast analysis and sequence variation the blast search resulted in categorizing 28 isolates into 5 strains namely 4 isolates of acinetobacter baumannii, 15 isolates of escherichia coli, 2 isolates for each of klebsiella pneumoniae and pseudomonas aeruginosa and 5 isolates of providencia stuartii (table 1). sequence length percentage of gc content for the 28 isolates are shown numerically in table 1 and graphically in figures 2 and 3. sequence length varied greatly between and within strains. in general one of the a. baumannii strains has the shortest length of (366 base), where one of e. coli has the longest sequence length (1263 base). when it comes to the length range k. pneumoniae had the narrowest length range (801–1017) base followed by e. coli (914–1263 base), where a. baumannii has the widest length range (366–1189 base). in the present study the percentage of gc content ranged from 50 to 56% among the 28 isolates (table 1). considering percentage of gc content within species k. pneumoniae has the narrowest gc% range (55–56%) where p. stuartii has the widest range (figure 3). figure 4 shows the maximum likelihood phylogenetic tree of the 28 isolates along with heat map. the phylogenetic tree consists of two large clades. the clade comprised a.baumannii, and only one of the tow p.aeruginosa isolates. the second clade consisted of two huge clusters. the first cluster composed of k. pneumoniae and e. coli, where the second cluster comprised p. stuartii and the second p. aeruginosa strain. principle component analysis (pca) was carried out for a better understanding of the diversity between and within the five species. the general idea underlying pca similar species should cluster, there for pca outlines factors basically responsible for differences between strains. as such, the more similar strains the more the closer they located. results of pca are presented graphically in figure 5. that is a. baumannii and p. aeruginosa clustered together and a bit further p. stuartii where fig. 1 agarose gel electrophoresis 16s pcr products out of 38 samples, 28 samples were selected for the study. 118 j contemp med sci | vol. 9, no. 2, march-april 2023: 116–120 isolation, identification and comparative analysis original h.s. alkhowaiter et al. table 1. 16s rdna sequence lengths and %gc values of 28 visolates from al qassim hospitals in saudi arabia strains sequence length gc% acinetobacter baumannii 1189 52 acinetobacter baumannii 774 52 acinetobacter baumannii 366 50 pseudomonas aeruginosa 544 53 acinetobacter baumannii 669 50 providencia stuartii 814 53 providencia stuartii 595 52 providencia stuartii 922 54 providencia stuartii 722 53 providencia stuartii 836 51 escherichia coli 1158 55 escherichia coli 1224 54 escherichia coli 1226 55 escherichia coli 914 55 escherichia coli 964 55 escherichia coli 937 55 escherichia coli 1036 55 escherichia coli 1191 55 escherichia coli 1227 54 escherichia coli 1234 55 escherichia coli 1259 54 escherichia coli 1236 55 escherichia coli 1202 55 escherichia coli 1202 56 klebsiella pneumoniae 801 56 klebsiella pneumonia 1017 55 pseudomonas aeruginosa 1172 53 escherichia coli 1263 54 fig. 2 boxplot displaying the distribution of sequence length for the strains, where top and bottom lines represent the maximum and minimum values, the top and bottom of each box represent the first quartile (q1) and third quartile (q3) where the line inside each box is the median (second quartile q2). fig. 3 boxplot displaying the distribution of percentage of gc content of the strains where top and bottom lines represent the maximum and minimum values, the top and bottom of each box represent the first quartile (q1) and third quartile (q3) where the line inside each box is the median (second quartile q2). fig. 4 maximum likelihood phylogenetic tree of the 28 isolates along with heat map. fig. 5 principal component analysis of the five strains, x-axis is the first principal component and y-axis is the second. 119j contemp med sci | vol. 9, no. 2, march-april 2023: 116–120 h.s. alkhowaiter et al. original isolation, identification and comparative analysis table 4 shows the conserved regions along the 28 antibiotic sequences and measurements of conservation (c), homosigoisty and p-value. conservation (c) is calculated as the proportion of conserved sites in the alignment region, where homosigosity is measured as 1-heterzygosity. only 3 conserved regions were observed. the length of the 3 conserved regions was not the constant, ranged from 109 bases to 251 bases. the p-values of the three conserved regions were less than 0.01. discussion a considerable variation are noticed in genomic nucleotide content of bacteria, with gc content (number of the same strand guanine +cytosine sites divided by dna sequence length) ranging from less than 13% to more than 75% between sole species.11 albeit the particular reasons for these gc varieties, wither within and between species, are still not known, it is largely thought that a number of variables in connection with evolutionary history as well as the environment are responsible.12 dna sequence analysis introduces an efficient tool for understanding the evolutionary forces that shaped nucleotide variations as well as bring insight into the significance of specific genomic regions.13 haplotype diversity (aka gene diversity) is the probability that two arbitrary sampled alleles are different.14 this study builds on the published evidence that, antibiotic-resistant bacteria are capable of transferring copies of their dna encoding a resistance mechanism to other bacteria, even those that are distantly related to them. these bacteria are then capable of passing on the resistance genes, resulting in the production of generations antibiotic-resistant bacteria. vertical gene transfer, which happens by cell-to-cell conjugation, is referred to as “horizontal gene transfer”.15 conclusion results of the present study demonstrated that the observed heterogeneity in the sequences of our target bacterial strains may be linked to antibiotic resistance and gene transfer between bacteria that evolved as a result of the hospital environment. consequently, hospital environments are considered “hot spots” for antibiotic resistant bacteria, and this suggests that significant precaution should be taken at all stages of the health-care system to minimize. conflict of interest none.  table 2. estimated parameters of the polymorphic sites for 28 isolates from al qassim hospitals in saudi arabia number of sites monomorphic sites polymorphic sites singleton sites parsimony informative 238 109 42 87 table 3. estimated parameters of dna polymorphism 28 isolates from al qassim hospitals in saudi arabia no. haplotype haplotype diversity +sd nucleotide diversity o average number of nucleotide differences 14 0.8+0.1 0.13 30 table 4. length of conserved regions, conservation, homzigosity and p-values of the 28 isolates sequence from al qassim hospitals in saudi arabia region start-end conservation homozigosity p-value 256–420 0.56 0.92 0.001 464–572 0.56 0.92 0.01 829–1079 0.62 0.93 <0.0001 p. aeruginosa and 2 e. coli constituted a different component. this pattern is somehow similar to phylogenetic tree figure 4. dna sequences analysis polymorphic sites analyses of polymorphic sites for the 28 isolates are shown in table 2. the total number of aligned sites was 1301 sites, the number of sites without alignment gaps or missing data was 238 of which 109 invariable (monomorphic) sites. the number of variable (polymorphic) site was 129, of which 42 singleton variable sites and 87 parsimony informative sites. haplotype & nucleotide analyses number of haplotypes, haplotypes diversity and standard deviation (sd) of haplotypes diversity are shown in table 3. number of haplotypes was 14. hapotype (gene) diversity was 0.8. nucleotide diversity (π) was 0.13. 1 ± 0.8, where average number of nucleotide differences was 30. references 1. poole k. multidrug efflux pumps and antimicrobial resistance in pseudomonas aeruginosa and related organisms. j mol microbiol biotechnol. 3:255–264. 2. aslam b, wang w, arshad mi, et al. antibiotic resistance: a rundown of a global crisis. infect drug resist. 2018;11:1645–1658. 3. andersson di, balaban nq, baquero f, et al. antibiotic resistance: turning evolutionary principles into clinical reality. fems microbiol rev. 2020;44(2):171–188. 4. clarridge je. impact of 16s rrna gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. clin microbiol rev. 2004;17(4):840–862. 5. sabir js, salah said aa, hussein mm. rayah al-saud, mutwakil mohammed n. isolation, identification and rapd-pcr analysis of new isolated bacillus thuringensis. life sci j. 2013;10:1352–1361. 6. larkin ma, blackshields g, brown np, et al. clustal w and clustal x version 2.0. bioinformatics. 2007;23(21):2947–2948. 120 j contemp med sci | vol. 9, no. 2, march-april 2023: 116–120 isolation, identification and comparative analysis original h.s. alkhowaiter et al. 7. wallace bc, lajeunesse mj, dietz g, et al. open mee: intuitive, open‐source software for meta‐analysis in ecology and evolutionary biology. methods ecol evol. 2017;8(8):941–947. 8. alsanie wf, felemban em, farid ma, hassan mm, sabry a, gaber a. molecular identification and phylogenetic analysis of multidrug-resistant bacteria using 16s rdna sequencing. j pure appl microbiol. 2018;12(2):489–496. 9. zhang j, jiao t, zhao s. genetic diversity in the mitochondrial dna d-loop region of global swine (sus scrofa) populations. biochem biophys res commun. 2016;473(4):814–820. 10. lynch m, crease tj. the analysis of population survey data on dna sequence variation. mol biol evol. 1990;7(4):377–394. 11. hildebrand f, meyer a, eyre-walker a. evidence of selection upon genomic gc-content in bacteria. plos genet. 2010;6(9): e1001107. 12. mann s, chen y-pp. bacterial genomic g+ c composition-eliciting environmental adaptation. genomics. 2010;95(1):7–15. 13. hutter s, vilella aj, rozas j. genome-wide dna polymorphism analyses using variscan. bmc bioinformatics. 2006;7(1):1–10. 14. nei m. molecular evolutionary genetics. columbia university press; 1987. 15. hussain t. pakistan at the verge of potential epidemics by multi-drug resistant pathogenic bacteria. adv life sci. 2015;2(2):46–47. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1337 295j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 original biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review saad majeed al-tamimi* consultant internist and cardiologist, college of dentistry, al-farahidi university, baghdad, iraq. *correspondence to: saad majeed al-tamimi (e-mail: saad65altamimy@yahoo.com) (submitted: 19 may 2022 – revised version received: 22 june 2022 – accepted: 07 july 2022 – published online: 26 october 2022) abstract background: hrvb (heart rate variability biofeedback) is a non-pharmacological method for chronic diseases evaluation. methods: adults chronic sufferers, hrvb as the primary therapy with or without controlled circumstances, and psycho physiological results as regression analysis were all included in a systematic search. results: there were 21 publications in overall. hrvb was found to be feasible in chronic patients with no adverse reactions, according to the findings. significant favourable impacts on hypertension and cardiovascular prognostic, inflammation condition, asthma issues, depression and anxiety, sleeping disruptions, cognitive function, and pain were reported in diverse patient characteristics that could be linked to enhanced quality of life. increases in treatment practice were accompanied by increases in heart rate variability, implying that hrvb may have a regulatory influence on autonomic function. conclusions: hrvb has the potential to help individuals with chronic conditions. more research is needed to reinforce these findings as well as identify the most efficient strategy. keywords: biofeedback, psychology, heart rate, chronic disease issn 2413-0516 introduction no communicable chronic conditions, such as cardiovascular diseases, malignancy, chronic lung disease, obesity, and mental health problems, had been accountable for about 70% of all mortality globally in 2016, according to the world health organization. their worldwide incidence is rising, and the resulting socially and economically implications are becoming more severe.1 as a result, a fundamental priority for transforming healthcare and lowering health-care expenditures is the efficiency and profitability of diseases control. chronic disorders are often caused by disturbances in the autonomic nervous systems (ans) balancing, which result in sympathetic sensory overload and a shortage of vasodilatation.2 this dysautonomia can be viewed as a result of disease, but it can also be viewed as a key potential cause in the onset and progression of chronic diseases. physiological changes such as stress hormone production and secretion (e.g., cortisol, norepinephrine), sleeping disturbances, pro-inflammatory cytokine production (e.g., il-6), hypertensions, or immunological malfunction can all lead to health decline and the formation of comorbidity.3 furthermore, a modelling depending on various epidemiological research found a relationship between reduced vagus nerve function and the aetio-pathogenesis of cardiovascular disorders, cancer, and alzheimer’s disease. emphasis is being placed on therapies that could boost vagal activity and re-establish independent balancing in this context.4 heart rate variability (hrv) is an indicator of health that is used to estimate parasympathetic performance and is evaluated at resting. low hrv is a predictor of cardiovascular illness and death risks, while elevated hrv represents the ability of the heart system to respond to internal and extrinsic alterations (e.g., anxiety, activity). short-term autonomic modulation by the sympathetic nervous system (sns) and the vagus nerve of the parasympathetic nervous system (pns) causes cardiac variability.5 each of those interconnected systems govern heart rate (hr) by increasing or shrinking it in response to physiological processes underlying in short-term hrv management, including baroreflex control and respiratory sinus arrhythmia (rsa). the first enhances hr once bp drops and reduces hr whenever bp rises; the latter enhances hr during intake and reduces hr during expiration.6 physiological elements (e.g., hormones, inflammatory condition), neuropsychiatric elements (e.g., feelings, anxiety, cognitively regulations), and ecological or health behaviours all have a role in the long-term regulatory frameworks of hrv (e.g., physical exercises, tobacco, alcohol).7 hrv is defined by time fluctuations among each heartbeat and is connected to the electrocardiogram’s rr interval (ecg). ecg or pulse wave measurements are used to determine hrv levels in both the time and frequency domains.8 the root mean squared of consecutive rr interval disparities shows largely parasympathetic activity in the temporal domain, while the standard deviations of normal-to-normal rr intervals (sdnn) indicates both sympathetic and parasympathetic variations on hr. short-term hrv assessment is mostly focused on the hrv power spectrum, which is separated into high frequency (hf; 0.15–0.4 hz) and low frequency (lf; 0.04–0.15 hz) regions that tend to correspond with various physiological systems in the frequency response.9 the hf-band represents respiratory impacts on hr modulation (rsa), which are caused by parasympathetic cardio vagal output, which causes rapid variations in hr. aside from that, the lf-band correlates to bar reflex activities, which would be a virtuous cycle between sensory receptors and the brainstem that regulates blood pressure through both sympathetic and parasympathetic output, resulting in significantly slower fluctuations in hr.10 lf-band, in particular, must be regarded as a representation of the baroreflex activity generated by both sympathetic and parasympathetic hr frequency modulation, rather than as the sole representation of sympathetic stimulation. the mailto:saad65altamimy@yahoo.com 296 j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review original s.m. al-tamimi intricacy of the physiological systems included in autonomic cardiovascular responses such as rsa and baroreflex activities should therefore be taken into account when interpreting the hrv power spectral density.11 hrv is controlled by the brainstem, cortical, and sub cortical regions, and mental function may be altered by hrv due to neuronal interactions between the central autonomic networks and heart activities. according to current studies, the amygdala, insula, and anterior cingulate are all implicated in emotion regulation, implying that emotion and hrv are linked.12 because vagal outflow prevails during rest due to substantial cardio modulator impacts, the authors proposed a neurovisceral implementation strategy in which vagal activities promote reciprocal heart-brain connection, implying that hrv may affect cerebral activities.13 following that, according to mccraty and coworkers’ psychophysiological theory, a particular cardiac rhythm pattern emerges once hr synchronises with other oscillatory components including rsa and baroreflex at a particular resonant frequencies equivalent to 6 breaths/min. sine wave oscillations of respiration, hr, and bp represent synchronisation of these oscillatory processes and indicate a “coherence condition.” hrv is considerably boosted under these situations, according to the authors, due to increased vagal activation, which could have a good impact on brain activities and, in particular, emotional control. these heart-brain connections caused by vagal afferents and afferents indicate that vagal nerve stimulation has a role in the pathophysiology of chronic disorders and that vagal-activating therapies may be problematic.14 hrvb (heart rate variability biofeedback) is a non-pharmacological method that improves emotional self-regulation and autonomic cardiac modulation by boosting hrv and recovering cardiac vagal function.15 once breathing is around 6 breaths per minute, the baroreflex and the breath synchronise, resulting in a unique hrv signal sequence. this cardiac synchronization condition arises at a resonant frequencies of about 0.1 hz, resulting in large amplitudes in hrv sine wave oscillations and a noticeable peak in the hrv power spectrum’s lf-band. various researches have looked into the impacts of hrvb on different psychophysiological complaints associated with chronic diseases since the late 1990s, and lehrer has recommended a standardised technique of practice. hrvb has been shown to have good effects on stress in a meta-analysis, while a systematic review found that hrvb may have advantages for athletic performance.16 the goal of this systematic review was to see if hrvb may be an efficient and realistic non-pharmacological strategy for managing chronic illness sufferers. as a result, we conducted a review of all research involving elderly patients that looked at the impacts of hrvb training on psychophysiological results connected to chronic disorders. method search strategy publications from the bibliographic resources pubmed/medline, springer link, and sciencedirect/elsevier, that were submitted between 2010 and 2020, were reviewed. eligibility criteria all publications that matched the relevant particular needs of the established picos criteria relating to demographic, interventions, comparisons, outcomes, as well as research designs have been included in the systematic review: affected individuals (over the age of 18) with chronic diseases; accounting the impacts of hrvb as a destined therapies for psychophysiological diagnoses as regression model; assessing learning outcomes of hrvb from minimally two sessions with guidelines for regulate frequencies respirations at roughly 6 breaths/min; and using a biofeedback equipment showing the hrv in actual time. we included all research strategies and comparisons methodologies with or without a control group to introduce a comprehensive assessment of hrvb interventions for outcome measures. the study involved investigations that used hrvb lonely, hrvb in conjunction with standard treatment, or hrvb in conjunction with another non-pharmacological interference, but only if the procedure would include a control group that received the same standard care or non-pharmacological interference, in order to assess the hrvb’s real benefit. due to potential confounder’s issues in the interpretation of the data, we omitted investigations that coupled hrvb instruction with another non-pharmacological treatment when the procedure did not provide a control group that permitted us to separate the hrvb different impacts. data processing and study collection to eliminate unintentional inclusion and exclusion, research screening was performed manually reviewing abstracts and then making revisions depending on the contents of each publication. publications were initially categorised based on whether or not they fulfilled picos requirements; subsequently, publications that matched our eligibility requirements were documented, with information on procedure, measures, and outcomes. results our screening approach turned up 626 papers (pubmed: 95; sciencedirect: 23; springer link: 508), plus three more papers found through sourced citations. numerous entries were deleted using the selection approach shown in figure 1 due to duplication (repeated: 39) or because they were not relevant research articles. by implementing the previous reported qualifying criteria in this sequence to the final 463 publications, 434 were exempted: adult people with chronic illnesses, excluding those medical settings such as substance abuse problems (due to the complicated matter concerning behavioural problems) and pregnant women (and wasn’t a chronic condition); interventional research of hrvb exercise (2 or more discussions with particular respiration guidelines); and the use of a biofeedback instrument (heartbeat detector or ecg) with real-time hrv monitor. a collection of 21 investigations had been considered, encompassing 883 participants and sample sizes ranging from 13 to 210, with the primary goal of analysing psychophysiological results. there were 11 randomised controlled trials, 4 unregulated investigations, and the rest were pilot, feasibility, or laboratory investigations that included a wait-list comparison group, an apparently healthy comparison group, a 297j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 s.m. al-tamimi original biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review conventional treatment control group, or another interventional control subjects. just trials examining the impacts of hrvb separately have been included in the randomized investigations. the only difference between the intervention and control groups in randomized research was the hrvb treatment: hrvb vs. no treatment; hrvb vs. other treatments; hrvb + standard care vs. identical standard care; hrvb + alternative treatment vs. same other interference; hrvb + other interventions vs. same other intervention table 1 summarises them and categorises them by kind of chronic condition. feasibility in chronic patients adhesion hrvb was evaluated in people with a diagnosis of chronic conditions and in a number of clinical settings. in a one-year longitudinal research, the maximal attrition incidence for hrvb respondents was observed to be about 25%, while assessed employment levels for hrvb everyday routine were over 70%.17 time limits, transportation challenges, and other factors were mentioned in investigations as factors for fig. 1 flow chart for the literature search. 298 j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review original s.m. al-tamimi ta bl e 1. t he b as el in e ch ar ac te ris tic s o f e ac h in cl ud ed st ud y st ud y sa m pl e st ud y de si gn in g de pe nd en t f as th m a co nt ro l t es to rs a nd fu lfi lm en t t im e he ar t r at e va ria bi lit y bi of ee db ac k in te rv en tio n vi ab ili ty si gn ifi ca nt ch an ge s i n he ar t r at e va ria bi lit y bi of ee db ac k co ho rt he ar t r at e va ria bi lit y in ec k di sa bi lit y in de xe s no nsi gn ifi ca nt ou tc om es ca rd io va sc ul ar d is ea se s cl im ov e t a l. (2 01 4) co ro na ry a rt er y di se as es ag e ra ng ed fr om 45 − 80 • r an do m iz ed co nt ro l t ria l • h ea rt r at e va ria bi lit y bi of ee db ac k + st an da rd c ar e (n = 1 3) st an da rd c ar e (n = 1 1) h o sp it a l a n xi et y a n d d ep re ss io n sc a le ; t yp e d pe rs on al ity ; b lo o d pr es su re ; h rv in te rv en tio ns be fo re a nd a fte r 10 tr ai ni ng s (4 5− 60 m in ut es ) t w ic e a w ee k + d ai ly • 7 in di vi du al s w ho ha ve s lip pe d ou t o f th e ne ck im pa irm en t in de x (c ar ee rs a nd ti m e lim ita tio ns ) n ot d et er m in ed • i nc re as e in th e av er ag e co ns is te nc y ra tin g ** co rr el at ed w ith s d n n i • h o sp it a l a n xi et y a n d d ep re ss io n sc a le ; t yp e d pe rs on al ity ; bl o o d pr es su re n ol an e t a l. (2 01 0; 2 01 2) h yp er te ns io n ag e ra ng ed fr om 3 5 to 64 y ea rs • r an do m iz ed co nt ro l t ria l • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 3 5) • a ut og en ic re lie f (n = 3 0) bl o o d p re ss u re ; ba ro re fle x se ns iti vity ; h ig hse ns iti vi ty cre ac tiv e pr ot ei n; il 6; h rv • i nt er ve nt io ns b ef or e an d af te r o ve r t he c ou rs e of e ig ht w ee ks , t he re w ill b e si x 60 -m in ut e tr ai ni ng se ss io ns a nd a 2 0m in ut e da ily a st hm a co nt ro l t es t. n ot d et er m in ed • d ay tim e bl o o d p re ssu re ** a nd 2 4 h sy st ol ic bl o o d p re ss u re *, a nd pu ls e pr es su re * • i nc re as e of h f po w er ** an d in te rb ea t in te rva l* * du rin g co gn iti ve ta sk • c ha ng es in h f po w er ar e ne ga tiv el y re la te d to in cr ea se s in h ig h se ns iti vi ty c -r ea ct iv e pr ot ei ns . * , r es po ns iv ene ss to b ar or efl ex es * , an d in te rb ea t in te rv a l* • s en si tiv e to th e ba ro re fle x • c ha ng es in in te rl eu ki n -6 h ad n o eff ec t o n h rv pa ra m et er s or b ar or efl ex re sp on si ve ne ss . pa tr on e t a l. (2 01 3) fo llo w in g he ar t su rg er y, th e pa tie nt s’ ag es ra ng ed fr om 5 2 to 6 9. • r an do m iz ed co nt ro l t ria l • h ea rt r at e va ri a bi li ty bi o fe ed ba ck + st re ss m an ag em en ts (n = 1 3) • s tr es s m an ag em en ts (n = 1 3) sp ie lb er g er s ta te a n xi et y in ve n to ry ; c en te r fo r ep id em io lo g ic a l st u d ie sd ep re ssi o n s ca le ; h rv • i nt er ve nt io ns b ef or e an d af te r o ve r t he c ou rs e of tw o w ee ks , t he re w ill b e fiv e 45 -m in ut e tr ai ni ng se ss io ns a nd a 1 5m in ut e da ily a st hm a co nt ro l as se ss m en t. n ot d et er m in ed • d ep re ss io n (c en te r fo r ep id em io lo g ic a l st u d ie sd ep re ss io n s ca le *) • o ve ra ll po w er is in cr ea si ng * *; • c ha ng es in h rv w er e fo un d to b e in ve rs el y re la te d to c ha ng es in de pr es si on * • s pi el be rg er st at e a n xi et y in ve n to ry (c on tin ue d) 299j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 s.m. al-tamimi original biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review ta bl e 1. t he b as el in e ch ar ac te ris tic s o f e ac h in cl ud ed st ud y— co nt in ue d st ud y sa m pl e st ud y de si gn in g de pe nd en t f as th m a co nt ro l t es to rs a nd fu lfi lm en t t im e he ar t r at e va ria bi lit y bi of ee db ac k in te rv en tio n vi ab ili ty si gn ifi ca nt ch an ge s i n he ar t r at e va ria bi lit y bi of ee db ac k co ho rt he ar t r at e va ria bi lit y in ec k di sa bi lit y in de xe s no nsi gn ifi ca nt ou tc om es yu e t a l. (2 01 8) co ro na ry a rt er y di se as e ag e ra ng e 35 − 70 y ea rs • r an do m iz ed co nt ro l t ria l • h ea rt r at e va ria bi li ty b io fe ed ba ck + s ta nd ar d ca re (n = 1 05 ) • s ta nd ar d ca re (n = 1 05 ) be ck d ep re ss io n in ve n to ry ; c h in es e h o st il it y in ve n to ry -s h o rt f o rm ; h rv ; c ar di ov as cu la r pr og no si s • i nt er ve nt io ns b ef or e an d af te r • f ol lo w -u p af te r a ye ar si x w ee kl y tr ai ni ng se ss io ns • d ro po ut ra te o f 26 .4 7% in th e h ea rt ra te v a ri a bi li ty b io fe ed ba ck g ro up a nd 34 .4 4% in th e co nt ro l gr ou p • d ep re ss io n (b ec k d ep re ss io n in ve n to ry to ta l s co re ** ; c og ni tiv e de pr es si on s ub sc al e* *) ; • h os til ity ** (c h in es e h o sti li ty in ve n to ry -s h o rt fo rm ); • a t t he fo llo w -u p, th e re su lts re m ai ne d th e sa m e; • r ea dm is si on s ar e re du ce d. * an d at fo llo w -u p, e m er ge nt v in so m ni a in te ns ity in de xt s ** • r es pi ra to ry ra te w as de cr ea se d ** ; • a t t he fo llo w -u p, th e ris e in l f po w er ** w as su st ai ne d • a t t he fo llo w -u p, th er e w as n o di ffe ren ce b et w ee n th e gr ou ps in te rm s of re du cin g al l-c au se re ad m is si on s an d al l-c au se am bu la to ry ca re . ob es ity m ey er e t a l. (2 01 8) in di vi du al s w ith a bm i o f 3 0 or h ig he r ar e co ns id er ed ob es e. t he p ar tic ipa nt s ra ng ed in a ge fro m 1 8 to 4 5 ye ar s ol d. • p ilo t s tu dy • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 1 0) • w ai tlis t c on tr ol (n = 1 0) pe rc ei ve d s tr es s sc a le ; p at ie n t h ea lt h q u es ti o n n a ir ed ep re ss io n a n d a n xi et y; sh o rt f o rm g en er a l h ea lt h su rv ey ; s el fef fi ca cy ; h rv • i nt er ve nt io ns b ef or e an d af te r • 3 -m on th s fo llo w -u p 6 tr ai ni ng s w ee kl y • e ig ht p eo pl e dr op pe d ou t o f t he in te rv en tio n. • d ep re ss io n (p at ie n t h ea lt h q u es ti o n n a ir ed ep re ss io n a n d a n xi et y* ); • s tr es s (p er ce iv ed st re ss s ca le *) ; • s el feffi ca cy (s el f ef fi ca cy s um s co re *) ; • q ua lit y of li fe (b od ily to ta l r an ks * an d m en ta l to ta l g oa ls s co re d* * w er e pr es er ve d at fo llo w -u p on s h o rt f o rm g en er a l h ea lt h su rv ey ) • p oo le d re su lts • s d n n ** p er fo rm an ce im pr ov em en t, to ta l po w er * *; • t he re du ct io n in re sp ira to ry ra te * w as su st ai ne d at th e fo llo w -u p. n ot d et er m in ed (c on tin ue d) 300 j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review original s.m. al-tamimi ta bl e 1. t he b as el in e ch ar ac te ris tic s o f e ac h in cl ud ed st ud y— co nt in ue d st ud y sa m pl e st ud y de si gn in g de pe nd en t f as th m a co nt ro l t es to rs a nd fu lfi lm en t t im e he ar t r at e va ria bi lit y bi of ee db ac k in te rv en tio n vi ab ili ty si gn ifi ca nt ch an ge s i n he ar t r at e va ria bi lit y bi of ee db ac k co ho rt he ar t r at e va ria bi lit y in ec k di sa bi lit y in de xe s no nsi gn ifi ca nt ou tc om es as th m a le hr er e t a l. (2 01 8) as th m a th e pa rt ic ipa nt s ra ng ed in a ge fro m 2 7 to 4 7 ye ar s ol d. • r a n d o m iz ed co n tr o l tr ia l • h ea rt r at e va ri a bi li ty b io fe ed ba ck g ro up (n = 3 1) el ec tr o en ce ph a lo g ra m b io fe ed ba ck + s oo th in g m us ic + a g ro up o f pe op le e xh al in g at a ra te o f 1 5 br ea th s pe r m in ut e (n = 3 3) m et ac h o li n e ch a ll en g e te st ; a st h m a c o n tr o l te st ; a st h m a q u a li ty o f li fe ; sp iro m et ry a nd im pu ls iv e os ci llo m et ry ; e ve ry da y co m pl ai nt s an d m ax im um o ut flo w s; ex pe lle d ni tr ic o xi de • i nt er ve nt io ns b ef or e an d af te r sh or t p ro to co l ( n = 2 0) : 6 h ea rt r at e va ri a bi li ty bi o fe ed ba ck tr ai ni ng s ov er 1 0 w ee ks + 2 0m in ut es p er d ay p ra st hm a co nt ro l t es tic e lo ng pr ot oc ol (n = 1 1) : 1 0 h ea rt r at e va ri a bi li ty bi o fe ed ba ck te n w ee ks of p ra ct ic e pl us a d ai ly pr as th m a co nt ro l t es t o f tw en ty m in ut es • 1 9 pe rc en t o f t he to ta l of p at ie nt s  le av e ou t. • a st hm a co m pl ai nt s (a st h m a c o n tr o l te st ** ; a st h m a q u a li ty o f li fe ** ); • s en si tiv ity o f a irw ay s (m et ac h o li n e ch a lle n g e te st ** ); • f un ct io ni ng o f t he lu ng s (m ax im um v el oc ity * ); • d ys fu nc tio n of th e ai rw ay s w ith in te rv al s of p oo r as th m a sy m pt om s* (n itr oge n di ox id e re le as ed * ) n ot d et er m in ed • j us t a fte r t he rap y, th er e w as no d iff er en ce in a st h m a co n tr o l te st , a st h m a q u a lit y o f li fe , m et ac h o li n e ch a ll en g e te st , o r m ax im al c irc ul at io n ac ro ss g ro up in gs ; • t he re is n o di ffe re nc e be tw ee n a qu ic k an d a le ng th y tr ea tm en t. ch ro ni c b ra in in ju ry ki m e t a l. (2 01 3) ch ro ni c br ai n in ju ry ag e = 2 3− 63 • p ilo t s tu dy • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 1 3) • n o co nt ro l g ro up in te g ra te d v is u a l a n d a u d it o ry ; co n ti n u o u s pe rf o rm a n ce t es t; be h av io r ra ti n g in ve n to ry o f ex ec u ti ve f u n cti o n -a d u lt ; h rv • i nt er ve nt io ns b ef or e an d af te r w ee kl y tr ea tm en t o f t en se ss io ns a nd a re si de nt ia l as th m a co nt ro l a ss es sm en t f ro m s es si on fo ur . • n ot d et er m in ed • n ot d et er m in ed • i m pr ov em en t i n th e co ns is te nc y pr op or tio n* an d th e lf /h f pr op or tio n ** ; • t he c oh er en t r at io s* an d th e lf /h f ra tio w er e co nn ec te d w ith em ot io na l s ta bi lit y an d ve rb al m em or ie s (b eh av io r ra ti n g in ve n to ry o f ex ec u ti ve f u n cti o n -a d u lt ) * *; • a tt en tio n (in te g ra te d vi su a l a n d a u d it o ry + co n ti n u o u s pe rf o rm a n ce t es t) c or re la te d w ith l f/ h f* * • n ot d et er m in ed (c on tin ue d) 301j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 s.m. al-tamimi original biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review ta bl e 1. t he b as el in e ch ar ac te ris tic s o f e ac h in cl ud ed st ud y— co nt in ue d st ud y sa m pl e st ud y de si gn in g de pe nd en t f as th m a co nt ro l t es to rs a nd fu lfi lm en t t im e he ar t r at e va ria bi lit y bi of ee db ac k in te rv en tio n vi ab ili ty si gn ifi ca nt ch an ge s i n he ar t r at e va ria bi lit y bi of ee db ac k co ho rt he ar t r at e va ria bi lit y in ec k di sa bi lit y in de xe s no nsi gn ifi ca nt ou tc om es ch an g et a l. (2 02 0) ac ut e is ch em ic st ro ke • r a n d o m iz ed co n tr o l tr ia l h ea rt r at e va ri a bi li ty bi o fe ed ba ck + st an da rd c ar e (n = 1 9) • s ta nd ar d ca re (n = 1 9) m in i-m en ta l st a tu s ex am in at io n ; h o sp it a l a n xi et y a n d d ep re ss io n sc a le ; h rv • i nt er ve nt io ns b ef or e an d af te r • o ne a nd th re e m on th s 4 da ys o f t ra in in g pr oc es s + 2 0 m in ut es o f d ai ly as th m a m an ag em en t te st in g ov er th re e m on th s. • t hr ee p eo pl e ha d to dr op o ut . • o nth ebe ds id e tr ai ni ng s es si on s ar e m on ito re d. • a nx ie ty a nd d ep re ss io n (h o sp it a l a n xi et y a n d d ep re ss io n s ca le *) a t 1 an d 3 m on th s • c og ni tiv e fu nc tio ns (m in i-m en ta l st at u s ex am in at io n ** ) a t 1 a nd 3 m on th s • d ec re as e of h ea rt ra te * at 1 a nd 3 m on th s; • i nc re as e of s d n n *, rm ss d *, l f* a nd to ta l p ow er * at 1 a nd 3 m on th s n ot d et er m in ed ch ro ni c p ai n d ob bi n et a l. (2 01 3) re fra st hm a co nt ro l te st or y irr ita bl e bo w el s yn dr om e ag e = 1 8− 60 • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 3 1) • h yp no th er ap y (n = 3 0) • r a n d o m iz ed co n tr o l tr ia l • i nt er ve nt io ns b ef or e an d af te r • f ol lo w -u p af te r th re e m on th s ir ri ta bl e bo w el sy n d ro m e sy m pt o m se ve ri ty s co re s; h o sp it a l a n xi et y a n d d ep re ss io n s ca le o ve r t he c ou rs e of tw el ve w ee ks , t he re w ill b e th re e 60 -m in ut e tr ai ni ng c ou rs es a nd a 20 -m in ut e da ily a st hm a co nt ro l a ss es sm en t. • 1 5 dr op ou ts (7 in h ea rt r at e va ri a bi li ty b io fe ed ba ck g ro up ) • s ym pt om s (ib ss ss *) a t po st -in te rv en tio n; • a nx ie ty a nd d ep re ss io n (h o sp it a l a n xi et y a n d d ep re ss io n s ca le *) • a t t he fo llo w -u p, th e re su lts re m ai ne d th e sa m e. n ot d et er m in ed • t he re h as be en n o di st in ct io n on th e h o sp it a l a n xi et y a n d d ep re ss io n sc a le a fte r t he tr ea tm en t. w ee ks e t a l. (2 01 5) ≥ 3 m on th s of ch ro ni c di sc om fo rt (fi br om ya lg ia , he ad ac he s, ne ur op at hy , e tc .) 45 to 6 8 ye ar s ol d • r a n d o m iz ed co n tr o l tr ia l h ea rt r at e va ri a bi li ty b io fe ed ba ck (n = 1 0) • f ee db ac k fa de (n = 1 0) : f ee db ac k le ve ls w er e sl ow ly re du ce d fro m 90 % to 0 % . 10 -c m v is ua l a na lo g sc al e (v a s) ; p a in d is a bi li ty q u es ti o n n a ir e; 1 1it em ta m pa s ca le o f ki n es io ph o bi a • i nt er ve nt io ns b ef or e an d af te r • f ol lo w -u p af te r th re e m on th s o ve r t he c ou rs e of th re e w ee ks , t he re w ill b e ni ne tr ai ni ng s es si on s. • 6 d ro pp ed o ut o f t he in te rv en tio ns ; • a t t he fo llo w -u p ex am in at io n, th re e w er e m is si ng . • n ot d et er m in ed • n ot d et er m in ed pa in in te ns ity on v a s, t sk 1 1, pa in d is a bi li ty q u es ti o n n a ir e (c on tin ue d) 302 j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review original s.m. al-tamimi ta bl e 1. t he b as el in e ch ar ac te ris tic s o f e ac h in cl ud ed st ud y— co nt in ue d st ud y sa m pl e st ud y de si gn in g de pe nd en t f as th m a co nt ro l t es to rs a nd fu lfi lm en t t im e he ar t r at e va ria bi lit y bi of ee db ac k in te rv en tio n vi ab ili ty si gn ifi ca nt ch an ge s i n he ar t r at e va ria bi lit y bi of ee db ac k co ho rt he ar t r at e va ria bi lit y in ec k di sa bi lit y in de xe s no nsi gn ifi ca nt ou tc om es ca nc er g re en be rg et a l. (2 01 5) n on -s m al l c el l l un g ca nc er (n sc lc ) i s a ty pe o f l un g ca nc er .  th e pa rt ic ip an ts ’ ag es ra ng ed fr om 46 to 7 1. • i nt er be at in te rva lli ty fe as ib ili ty an al ys is • h ea rt r at e va ri a bi li ty b io fe ed ba ck g ro up (n = 1 6) • t he re is n o co nt ro l gr ou p. h o sp it a l a n xi et y a n d d ep re ss io n sc a le ; p at ie n t h ea lt h q u es ti o n n a ir e; f a st h m a co n tr o l te st l; d is tr es s t he rm om et er an d pr ob le m a re as • i nt er ve nt io ns b ef or e an d af te r si x ex er ci se s es si on s (3 0– 45 m in ut es ) th ro ug ho ut c he m ot he rap y pl us a d ai ly a st hm a m an ag em en t a ss es sm en t o f 2 0 m in ut es • t he re w er e ei gh t ca se s in to ta l; • 1 h ad fi ni sh ed th e pr og ra m ; • h ea rt r at e va ri a bi lit y bi o fe ed ba ck d on e du rin g ch em ot he ra py ; • t hr ou gh ou t t ra in in g se ss io ns , t he p ot en tia l to re du ce re sp ira to ry ra te , h ea rt ra te , a nd an xi et y • t he re a re n o st at is tic al an al ys is a va ila bl e. n ot d et er m in ed n ot d et er m in ed de pr es si on ca ld w el l e t a l. (2 01 8) m aj or d ep re ss iv e di so rd er (m d d ) i s a ty pe o f d ep re ss io n th at 1 8 to 2 5 ye ar s ol d • r a n d o m iz ed co n tr o l tr ia l • h ea rt r at e va ri a bi li ty bi o fe ed ba ck + ps yc ho th er ap y (n = 1 0) • p sy ch ot he ra py (n = 1 0) • n on -d ep re ss ed co nt ro l g ro up (n = 1 1) be ck d ep re ss io n in ve n to ry ; h rv • i nt er ve nt io ns b ef or e an d af te r fi ve s es si on s + 15 –2 0 m in ut es o f a st hm a sy m pt om s at h om e. o ve r t he c ou rs e of s ix w ee ks , 4 –5 in st an ce s ea ch w ee k n ot d et er m in ed • d ep re ss io n (b d iii ** ) • s d n n ** n ot d et er m in ed h ar to gs e t a l. (2 01 7) m aj or d ep re ss iv e di so rd er (m d d ) t he pa rt ic ip an ts ra ng ed in a ge fr om 2 3 to 6 2 • e xp er im en ta l st ud y • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 1 0) • t he re is n o co nt ro l gr ou p be ck d ep re ss io n in ve n to ry ; p o si ti ve o u tc o m e li st ; h rv • i nt er ve nt io ns b ef or e an d af te r ei gh t w ee kl y tr ai ni ng se ss io ns (4 5– 60 m in ut es ) + a d ai ly p ra st hm a co nt ro l t es t o f t w en ty m in ut es • t hr ee fa ilu re s du e to a la ck o f m ot ili ze d vi su al an d au ra l i nt eg ra tio n; • s ev en p eo pl e fin is he d th e en tir e pr og ra m ; • o ne d ep re ss iv e w or se ni ng fi ve p ar tic ip an ts  h av e ha d cl in ic al b en efi ts : • d ep re ss io n (b d i) • e le m en ts o f r es ili en cy (p o si ti ve o u tc o m e li st au to no m y ra tin gs ); (h am d to ta l s co re ** ) • d ur in g th e pr og ra m , th e de gr ee o f c on si st en cy o f fi ve p ar tic ip an ts im pr ov ed . • t he a ve ra ge h r ha s de cr ea se d * n ot d et er m in ed (c on tin ue d) 303j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 s.m. al-tamimi original biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review ta bl e 1. t he b as el in e ch ar ac te ris tic s o f e ac h in cl ud ed st ud y— co nt in ue d st ud y sa m pl e st ud y de si gn in g de pe nd en t f as th m a co nt ro l t es to rs a nd fu lfi lm en t t im e he ar t r at e va ria bi lit y bi of ee db ac k in te rv en tio n vi ab ili ty si gn ifi ca nt ch an ge s i n he ar t r at e va ria bi lit y bi of ee db ac k co ho rt he ar t r at e va ria bi lit y in ec k di sa bi lit y in de xe s no nsi gn ifi ca nt ou tc om es li n et a l. (2 01 9) m aj or d ep re ss iv e di so rd er a ge ra ng ed fro m 2 0 to 7 5 ye ar s. • c as eco nt ro l st ud y • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 2 4) ; • c on tr ol o f t he w ai tin g lis t ( n = 2 4) be ck a n xi et y in ve n to ry ; b ec k d ep re ss io n in ve n to ry ; p it ts bu rg h sl ee p q u a li ty in d ex ; p re -s le ep a ro u sa l sc a le ; h rv • i nt er ve nt io ns b ef or e an d af te r • f ol lo w -u p af te r o ne m on th si x w ee kl y tr ai ni ng s es si on s (6 0 m in ut es ) p lu s da ily p ra st hm a co nt ro l te st ic e of 1 0 m in ut es • t he re w er e fiv e w ith dr aw al s. • d ep re ss io n (b ec k d ep re ss io n in ve n to ry to ta l s co re ** , c og ni tiv e de pr es si on *, s om at ic de pr es si on *) ; • a nx ie ty (b ec k a n xi et y in ve n to ry to ta l s co re ** ); • s le ep (p re -s le ep a ro u sa l sc a le to ta l s co re *, pi tt sb u rg h s le ep q u a li ty in d ex to ta l s co re ** , a nd co gn iti ve a ro us al o f p re sl ee p a ro u sa l sc a le ** ) • a t t he fo llo w -u p, th e re su lts re m ai ne d th e sa m e. • r es pi ra to ry ra te de cr ea se s ** ; • s d n n ** , l f po w er *, l f/ h f* , a nd o ve ra ll po w er * al l i nc re as ed ** ; at th e fo llo w -u p, th e re su lts re m ai ne d th e sa m e. • n o di ffe re nc e be tw ee n gr ou ps fo r pi tt sb u rg h sl ee p q u a li ty in d ex a nd pr esl ee p a ro u sa l sc a le to ta l sc or es ch ro ni c s tr es s d e br ui n et a l. (2 01 6) ch ro ni c st re ss e va lu at ed fr om p er ce iv ed st re ss s ca le s co re ag e ra ng ed fr om 1 8 to 4 0 • r a n d o m iz ed co n tr o l tr ia l • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 2 5) • m ed ita tio n fo r aw ar en es s (n = 2 7) • p hy si ca l a ct iv ity (n = 2 3) ac s; b eh av io r ra ti n g in ve n to ry o f ex ec u ti ve fu n c ti o n -a d u lt ; ff m q sf ; s el fc o m pa ss io n s ca le sh o rt f o rm ; pe n n s ta te w o rr y q u es ti o n n a ir e • i nt er ve nt io ns b ef or e an d af te r • f ol lo w u p af te r 6 m on th s. o ve r t he c ou rs e of fi ve w ee ks : 1 st w ee k: 1 0 m in ut es p er d ay 2 nd w ee k = 1 5 m in ut es p er d ay w ee ks 3 –5 = 2 0 m in ut es pe r d ay • t he re w er e 19 w ith dr aw al s in th e h ea rt r at e va ri a bi li ty b io fe ed ba ck ca te go ry , i nc lu di ng o ne in th e h ea rt r at e va ria bi li ty b io fe ed ba ck co ho rt (o cc up at io ns an d tim e co ns tr ai nt ); • p ar tic ip an ts w ith an a tt en da nc e ra te of m or e th an 7 0% sh ow ed g re at er g ai ns . • a tt en tio n co nt ro l* (a cs ); • e xe cu tiv e fu nc tio nin g* (b eh av io r ra ti n g in ve n to ry o f ex ec u ti ve fu n c ti o n -a d u lt ); • m in df ul a w ar en es s* (f iv e fa ce t m in d fu ln es s q u es ti o n n a ir esh o rt fo rm ); • s el fco m pa ss io n* (s el fc o m pa ss io n s ca le sh o rt f o rm ); • w or ry in g* (p en n s ta te w o rr y q u es ti o n n a ir e) • w he n co m pa re d to it s pe er s, at te nt io n co nt ro l an d ex ec ut iv e fu nc tio ni ng ha d sm al l i m pa ct v al ue s at fo llo w -u p. n ot d et er m in ed at th e po st in te rv en tio n an d fo llo w -u p, th er e w as n o su bs ta nt ia l di st in ct io n. (c on tin ue d) 304 j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review original s.m. al-tamimi ta bl e 1. t he b as el in e ch ar ac te ris tic s o f e ac h in cl ud ed st ud y— co nt in ue d st ud y sa m pl e st ud y de si gn in g de pe nd en t f as th m a co nt ro l t es to rs a nd fu lfi lm en t t im e he ar t r at e va ria bi lit y bi of ee db ac k in te rv en tio n vi ab ili ty si gn ifi ca nt ch an ge s i n he ar t r at e va ria bi lit y bi of ee db ac k co ho rt he ar t r at e va ria bi lit y in ec k di sa bi lit y in de xe s no nsi gn ifi ca nt ou tc om es h al lm an e t a l. (2 01 1) st re ss re la te d ch ro ni c ne ck s ho ul de r p ai n ag e ra ng ed fr om 2 5 to 5 0 ye ar s • p ilo t s tu dy • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 1 2) • t he c on tr ol g ro up (n = 1 2) p ar tic ip at ed in s es si on s 1 an d 10 w ith ou t r ec ei vin g an y gu id an ce in be tw ee n. bo rg c r1 0; s tr es s m ed ic in e sy m pt o m sc a le ; h o sp it a l a n xi et y a n d d ep re ss io n s ca le ; sh o rt f o rm g en er a l h ea lt h su rv ey ; n ec k d is a bi li ty in d ex ; h rv • i nt er ve nt io ns b ef or e an d af te r te n tr ai ni ng s es si on s ev er y w ee k. n ot d et er m in ed • q ua lit y of li fe (o n th e sh o rt f o rm g en er a l h ea lt h s u rv ey , b od ily pa in *, s oc ia l f un ct io n* , a nd vi ta lit y* *) • l f po w er ** in cr ea se s; lf p ow er *, p n n 50 *, a nd in te rb ea t in te rv a l * im pr ov e th ro ug ho ut st re ss re co ve ry . • s tr es s m ed ici n e sy m pt o m sc a le , b or g cr 10 , a nd n ec k d is a bi li ty in d ex , h o sp it a l a n xi et y a n d d ep re ss io n sc a le va n de r z w an et a l. (2 01 5) ch ro ni c st re ss w as as se ss ed u si ng sc a le o f pe rc ei ve d st re ss t he p ar tic ipa nt s ra ng ed in a ge fro m 1 8 to 4 0 ye ar s ol d. • r a n d o m iz ed co n tr o l tr ia l • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 2 6) ph ys ic al e xe rc is e (n = 2 3) m ed ita tio n fo r aw ar en es s (n = 2 7) d ep re ss io n a n xi et y st re ss sc a le s; p it ts bu rg h sl ee p q u a li ty in d ex ; s ca le s o f ps yc h o lo g ic a l w el lbe in g • i nt er ve nt io ns b ef or e an d af te r • f ol lo w u p af te r s ix m on th s o ve r t he c ou rs e of fi ve w ee ks : 1 st w ee k: 1 0 m in ut es p er d ay 2 nd w ee k = 1 5 m in ut es p er d ay w ee ks 3 –5 = 2 0 m in ut es pe r d ay • n in e po st -t es t a nd / or fo llo w -u p ex am in atio ns w er e m is si ng ; • i nd iv id ua ls w ith a p ar tic ip at io n ra te o f m or e th an 7 0% s ho w ed gr ea te r g ai ns . • s tr es s* *, a nx ie ty ** , a nd de pr es si on ** (d ep re ss io n a n xi et y st re ss s ca le s) ; • w el l-b ei ng ** (s ca le s o f ps yc h o lo g ic a l w el l be in g ); • s le ep q ua lit y* (p it ts bu rg h s le ep q u a li ty in d ex ); • a t t he fo llo w -u p, th e re su lts re m ai ne d th e sa m e. n ot d et er m in ed • a t p os tin te rve nt io n an d fo llo w -u p, th er e w er e no d iff er en ce s be tw ee n gr ou ps . ps yc hi at ric d is or de rs je st er e t a l. (2 01 8) in di ca tio ns o f ps yc hi at ric d is ea se s (d ep re ss iv e, a nx iet y, a nd b ip os iti ve ou tc om e lis ta r di so rd er s) t he a ge s of th e pa rt ic ip an ts ra ng ed b et w ee n 63 to 9 6 ye ar s ol d. • e xp er im en ta l st ud y • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 2 0) • n o co nt ro l g ro up sp ie lb er g er s ta te a n xi et y in ve n to ry ; be ck d ep re ss io n in ve n to ry ; t ra il m a ki n g t es t pa rt a a n d b in te rv en tio ns b ef or e an d af te r o ve r t he c ou rs e of th re e w ee ks , t he re w ill b e si x 30 -m in ut e tr ai ni ng s es si on s pl us a h om e as th m a co nt ro l a ss es sm en t tw o tim es p er w ee k. • p ar tic ip an ts h ig hlig ht ed th e im pa ct o f h ea rt r at e va ri a bi li ty b io fe ed ba ck on s tr es s or c on ce rn (6 7% ), de pr es si on o r so rr ow (5 6% ), an d st re ss (4 4% ); or n o be ne fit s of h ea rt r at e va ri a bi li ty b io fe ed ba ck o n te ns io n (5 0% ); • a nx ie ty s ym pt om s ar e sl ig ht ly w or se ne d (c om pe tit iv e na tu re o f bf s of tw ar e) o ne p ar tic ip an t h as c on fir m ed th is. • d ep re ss io n* * (b d iii ); • s ta te a nx ie ty ** a nd tr ai t a nx ie ty ** a re tw o ty pe s of a nx ie ty (s pi el be rg er s ta te a n xi et y in ve n to ry ); • a tt en tio n sk ill s (t m ta ** ) n ot d et er m in ed n ot d et er m in ed (c on tin ue d) 305j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 s.m. al-tamimi original biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review ta bl e 1. t he b as el in e ch ar ac te ris tic s o f e ac h in cl ud ed st ud y— co nt in ue d st ud y sa m pl e st ud y de si gn in g de pe nd en t f as th m a co nt ro l t es to rs a nd fu lfi lm en t t im e he ar t r at e va ria bi lit y bi of ee db ac k in te rv en tio n vi ab ili ty si gn ifi ca nt ch an ge s i n he ar t r at e va ria bi lit y bi of ee db ac k co ho rt he ar t r at e va ria bi lit y in ec k di sa bi lit y in de xe s no nsi gn ifi ca nt ou tc om es sc hu m an e t a l. (2 01 8) po st -t ra um at ic s tr es s d is or de r ( pt sd ).  t he pa rt ic ip an ts ra ng ed in a ge b et w ee n 26 un til 5 0 ye ar s ol d. • p ilo t s tu dy • h ea rt r at e va ri a bi li ty b io fe ed ba ck (n = 6 ) co nt ro lli ng th e w ai tli st w ith br ea th in g te ch ni qu es (n = 6 ) pt sd c h ec kl is t fo r th e d sm 5; h rv • i nt er ve nt io ns b ef or e an d af te r • f ol lo w u p ra ng ed fro m fo ur to 1 6 w ee ks . o ve r t he c ou rs e of 4 w ee ks , o ne s es si on + 10 –1 5 m in ut es tw ic e da ily p ra st hm a co nt ro l te st ic e • t he p ro ce du re w as pe rf or m ed b y te n pe op le ; • d ai ly p ra st hm a m on ito rin g te st ic e pa rt ic ip at io n ra te o f > 70 % ; • s ym pt om s in cl ud e a de cr ea se in in di vi du al s’ irr ita bi lit y, a nx ie ty , a nd sl ee p di st ur ba nc es . • p ts d -s pe ci fic s ym pt om s (p ts d c h ec kl is t fo r th e d sm 5* ) m ai nt ai ne d at fo llo w sup • t he re su lts h av e be en co m bi ne d. • r m ss d * in cr ea se d at th e 16 -w ee k fo llo w -u p n ot d et er m in ed ta n et a l. (2 01 1) po st -t ra um at ic s tr es s d is or de r ( pt sd ).  th e pa rt ic ip an ts ra ng ed in a ge fr om 2 4 to 6 2 ye ar s ol d. • p ilo t s tu dy • h ea rt r at e va ria bi li ty b io fe ed ba ck + s ta nd ar d ca re (n = 1 0) • s ta nd ar d ca re (n = 1 0) cl in ic ia n -a d m in is te re d p ts d s ca le ; pt sd c h ec kli st -s pe ci fi c in te rv en tio ns b ef or e an d af te r o ve r t he c ou rs e of 8 w ee ks , t he re w ill b e 8 tr ai ni ng s es si on s (h al f a n ho ur  e ac h) a nd a 2 0m in ut e as th m a co nt ro l t es t tw o tim es p er d ay . • 1 d ro po ut (t ra ns po rt pr ob le m ); • p ar tic ip an ts ra te d th e h ea rt r at e va ri a bi li ty b io fe ed ba ck in te rv en tio n as a n 8/ 10 fo r s at is fa st hm a co nt ro l an d th at th ey h ad a pl ea sa nt ti m e. • p ts d -s pe ci fic s ym pt om s (c li n ic ia n -a d m in is te re d pt sd s ca le ** ; p ts d ch ec kl is tsp ec if ic *) n ot d et er m in ed • a t t he e nd o f th e in te rv en tio n, th er e w as no d iff er en ce be tw ee n th e gr ou ps . tr ou ss el ar d et a l. (2 01 6) re m itt ed s ch iz oph re ni a ag e ra ng ed fro m 2 5 to 4 6 ye ar s. • p ilo t s tu dy • h ea rt r at e va ria bi li ty b io fe ed ba ck (n = 1 0) • n o co nt ro l g ro up sp ie lb er g er s ta te a n xi et y in ve n to ry ; d er o g at is s tr es s pr o fi le ; p o si ti ve a n d n eg at iv e sy n d ro m e sc a le ; w a rw ic k– ed in bu rg h m en ta l w el lbe in g s ca le ; fr ei bu rg m in d fu ln es s in ve n to ry in te rv en tio ns b ef or e an d af te r w ee kl y tr ai ni ng s es si on s of 8 –1 2 ho ur s (o ne h ou r) pl us d ai ly re la xa tio n te ch ni qu es . • n o dr op ou t; • m os t p eo pl e w an t t o re m ai n af te r q ui tt in g; • i n in di vi du al s w ith a hi gh le ve l o f c om pl ai nt s, th er e ar e m or e be ne fit s. • m in df ul ne ss * (f re ib u rg m in d fu ln es s in ve n to ry ); • a nx ie ty s tr es so rs * an d em ot io na l s tr es so r* (d er o g at is s tr es s pr o fi le ) n ot d et er m in ed w a rw ic k– ed in bu rg h m en ta l w el l be in g s ca le , po si ti ve a n d n eg at iv e sy n d ro m e sc a le , sp ie lb er g er st at e a n xi et y in ve n to ry *< 0. 05 ; * *≤ 0. 01 ; “ n ot d et er m in ed ” i m pl ie s th at th is d at a w as n ot n ot ed in th e st ud y. 306 j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review original s.m. al-tamimi withdrawals,18–20 and medical defects.21 because to significant dropout rates in lung cancer sufferers owing to disease-related health and lifestyle decline or mortality, one procedure was early discontinued.21 satisfaction generally, participants were pleased with the stress reduction and positive feeling improvement they experienced while biofeedback, and the advantages lasted for a long time.17,20,22,23 none of the respondents in any of the research looked at expressed displeasure. participants with remitted schizophrenia readily finished the intervention with no participation requirement, and the majority expressed an incentive to keep afterward due to claimed psychological advantages.23 there was a 67 percent acceptance rating for beneficial effects on anxiety or worry amongst elderly patients with psychiatric conditions, as well as 56 percent satisfaction for state of depression or sorrow and 50 percent satisfaction for stress.22 adverse effects hrvb was found to have no major side effects, indicating that it is safe to use in patients with chronic conditions. other minor side effects were noted, including such anxiety, due to the intrinsic pressure felt by sufferers to fulfil the biofeedback device’s predefined respiratory objectives.22 a familiarisation phase was adopted in a procedure to gradually eliminate the respiratory rating from a natural rhythm of ~14 breaths/min to a goal rate of ~6 breaths/minute to minimise any frustrations linked to sluggish breath and hyperventilation.24 efficacy in terms of psychophysiological results cardiovascular disorders and hypertensive disorders hrvb was helpful in lowering 24-hour systolic blood pressure (–2.1 0.9 mmhg, p = 0.03) and 24-hour pulse pressure (–1.40.6 mmhg, p = 0.02) following 8 weeks of regular practise, according to a randomised controlled trial (rct) involving 65 participants (autogenic relaxation).25 a trial of 24 patients with atrial fibrillation found no change in blood pressure.18 since the participants were already on beta-blocker medications when the trial began, all initial systolic and diastolic bp readings were within normal ranges through the latter example, which indicates a substantial restriction of the outcomes. as a consequence, hrvb appears to have a beneficial effect on blood pressure in hypertensive individuals and cardiovascular prognosis in heart sufferers. inflammatory condition in a research of 65 hypertension individuals, a negative relationship between alterations in the inflammatory condition (evaluated by highly sensitive c-reactive proteins and interleukin-6) and efferent vagal activity (evaluated by hf power, rr interval, and baroreflex activities) was discovered.26 the researchers hypothesized that increasing efferent vagal action would reduce pro-inflammatory mediators, implying that hrvb could have anti-inflammatory properties. asthma disorders all asthma attacks and lung capacity increased in two randomized trials with 94 and 64 participants, respectively, and airway inflammation decreased. when compared to the control group, food and medicine intake was decreased after 10 weeks of daily hrvb practise, indicating that hrvb has a lot of potential in the particular treatment of asthma attacks.27 hrvb was more successful in lowering prescription usage 30 and airways inflammations than electroencephalogram (eeg) biofeedback and standard treatment,27 it was also just as efficient as active controls at alleviating asthma attacks,27 bronchial permeability and lung capacity.27 anxiety, depression, and psychological response hrvb was found to have substantial favourable impact in 12 of the 15 research that looked at depression as a predictor variables; similarly, hrvb was found to have significant positive impacts in 9 of the 12 studies that looked at stress and anxiety. in 12 investigations involving 326 different patient features who suffered from mental illness, depressed mood, tension, and anxiety were considerably reduced,28–30 persistent discomfort,31 chronic stress,32 psychiatric disorders22,23 and obesity.33 anxiety and despair levels were reduced over many weeks to a year after hrvb treatment.30–32 other beneficial psychological consequences were assessed, including greater mindfulness practice, self-compassion, and well-being. in two investigations with a total of 151 patients suffering from chronic stress19,32 and one research with ten participants in remission from schizophrenia, there was a reduction in worry or anger.23 disruptions in sleep three of the four studies that looked at sleep problems found that hrvb improved sleeping patterns in individuals with severe depressed disorders,30 and anxiety symptoms (totalling 162 participants). increases in sleeping were linked to lower levels of depression30 and stress.32 characteristics of post-traumatic stress disorder (ptsd) in three investigations involving 60 patients, ptsd-specific indicators decreased considerably following 4–8 weeks of hrvb therapy. in a research with a small sample size of 20 participants, hrvb was found to be no more efficient than the traditional therapy.20 cognitive performances massive gains in attention skills and executive functioning in individuals afflicted by chronic stress,19 executive function in individuals with mental signs, 38 have been implemented to enhance cognitive capacities. in addition, individuals who had an acute ischemic stroke improved their cognitive function significantly.23,24 nevertheless, no improvement in cognitive abilities were identified in 13 individuals with persistent brain damage, which is likely owing to the kind of neurological problems.34 pain 50 participants with irritable bowel syndrome and 24 individuals with stress-related chronic neck-shoulder pain improved following hrvb exercise.35 improvements were sustained three months following hrvb exercise, according to one research.31 a further research, nevertheless, contradicts these 307j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 s.m. al-tamimi original biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review findings, finding no substantial pain decrease in patients with diverse chronic pain characteristics.36 there was no information about the prescriptions and/or use of painkillers. lifestyle advancements in quality of life, particularly a rather more active lifestyle and a rise in both social and physical performance, were found to be linked to a reduction in pain 50 and ptsd-related indicators.17 discussion modifications in hrv may mitigate the relationship of interventions the hrvb benefits on hrv were documented in 11 of the 21 investigations in the literature review. the lowered mean hr25 and breathing percentage at rest30,33 all over meetings, as well as the enhanced cohesion proportion throughout sessions,18 showed highest synchronisation between respiratory and cardiovascular processes, recommend that individuals correctly performed hrvb activities and demonstrate the impacts of routine practice. hrv indicators that increased in time, such as sdnn,24,29,30,33 pnn31 or rmssd,24 and in frequencies, such as overall power,24,30,33 suggested an improvement in cardiac autonomic regulation. 9 numerous studies have found that higher hrv values are linked to better outcome measures. in patients with chronic brain damage, a higher consistency proportion was linked to enhanced affective and psychological functioning.37 in cardiovascular events, greater hf power was linked to lower stress and anxiety levels, as well as lower inflammation condition in hypertensive individuals.26 as a consequence, the authors suggest that by boosting overall hrv and completely overwhelming cardiac vagus nerve activity, hrvb could have inhibitory action on autonomic function implicated in physiological control systems. the central-autonomic combination of vagal afferents may help to improve psycho physiological performance in a more cohesive and effective process in this manner.38 furthermore, by optimising and enabling interrelated biological processes, 0.1 hz oscillations as a resonant frequencies may play a prominent part in physical and mental health.39 hrvb could be a potential strategy for managing a broad variety of ailments conditions and their effects by increasing vagal heart activity.40 troublesome explanation of hrv the hf power reflects pns arousal while spontaneous breathing at resting (about 10–15 breaths/min), while the lf power mostly indicates baroreflex action influenced by both sns and pns. whereas an elevation in hf strength indicates an elevation in pns arousal, a rise in lf power may result in a rise in baroreceptor regulation controlled by ans control and cannot clearly differentiate synchronous from parasympathetic involvement. the findings of hrv in the spectral domain in the research used in this study were characterized as the reinstitution of cardiac vagal regulation, expressed in either hf or lf regions. furthermore, the effect of respiratory rate on hrv radio spectrum demonstrates that for a respiratory rate under 9 breaths/min, vagal activities may pass over into the lf-band.41-43 latest discoveries reveal that parasympathetic inhibition can remove hrv power in the lf-band in healthy persons beneath slow breathing conditions, showing the importance of cardiac vagal activity throughout a limited range of frequencies of 4–9 breaths/minute.44 in this respect, some studies propose increasing the frequencies limitation between the hf and lf bands from 0.15 hz to 0.1 hz to account for the particular effects of slow breathing on the hrv frequency distribution as well as the complicated interactions among pns and sns signalling heart-brain connections.39 numerous technical features of hrv signal acquisition, including such monitoring length, instrument employed (ecg or pulse sensor), hrv variables evaluated, and respiratory circumstances, vary between procedures. ventilation was not measured in any of the procedures, therefore not determined on breathing rate, tidal volume, or intake to exhalation ratios was supplied, although their recognised impacts on hrv.45 respiratory variables should be observed to interpret the data more precisely because variations in rhythms of respiration may alter the hrv power spectrum as a potential mediator.43 other variables that may play a role in hrv regulation, such as medicine and physically activities,46 must also be taken into account. as a consequence, studies relating to the increase of hrv indices must be regarded with caution, as they do not show a direct cause-and-effect connection among hrvb and ans control. hrvb versus other interventions the known methods implementations were also explored as component of this systematic review. hrvb was largely effective when opposed to normal care or waiting listing controlled trials47,29,30,33,35 but not when contrasted to active controls that included various non-pharmacological treatments.19,27,31,32 autogenic relaxing, progressive muscle relaxation, electroencephalogram biofeedback,27 hypnotherapy, 50 mindfulness training, and physical activity19,32 have all been shown to improve psycho physiological results. hrv indicators were not enhanced by any of the other non-pharmacological treatments utilised as a controlled group, indicating hrvb’s distinct abilities on autonomic cardiac modulation (table 1). whenever hrvb is used in conjunction with regular care, the findings demonstrate that it can be used as an adjunctive therapy for clinical patients. nevertheless, since of potential confounders, we eliminated some of the studies that show promising prospects for integrated non-pharmacological therapies in chronic illness treatment, such as physically exercises, healthcare management,48 or relaxation techniques.49 guidelines for hrvb training regimes the majority of the regimens we looked at gave 4–12 monitored workshops with ongoing home practise. home practise was created to strengthen diaphragmatic breathing instructions and enhance hrv responsiveness, whereas monitored training were offered to ensure that hrvb activities were completed. the authors discovered a dose-response relationship between hrvb practise and symptomatology decrease19,32 implying the need of consistent practise and the presence of a practise barrier at which hrvb can deliver the desired results. we may estimate that optimum practise should comprise at least one monitored workshop accompanied by consistent home practise of at least 10 minutes daily for four weeks depending on the examined publications. this finding is consistent with previously reported hrvb protocol guidelines, which suggest 5 monitored sessions with 20-minute daily 308 j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review original s.m. al-tamimi practises.50 patients may choose shorter practise hours if they are concerned about dropping out due to time constraints. hrvb practise, on the other hand, is likely to be helpful when tailored to the patient’s profiles and skills, with the option of providing more monitored sessions and extended procedures as needed. guidelines for hrvb training programs the researchers of the examined procedures have offered a series of findings: to prevent minor side effects, provide a familiarisation phase of slow breathing activities at the start of the treatment (anxiety or breathlessness);47,24 practice the slow abdomen breathing exercises by introducing pursed-lips abdominal breathing with slightly delayed exhale;24 and, all through the session, gradually reduce the amount of time subjected to optical biofeedback in order to develop full independence in hrvb practise.36 lehrer’s methodology includes a brief anti-hyperventilation warning (“in hopes of avoiding hyperventilation during the timed respiration activity, kindly eliminate exceedingly breathing techniques.”).50 participants who are new to 0.1 hz respiration must be given instructions to “breathe superficially and spontaneously.” even though it has been demonstrated that an participant’s hr frequencies range makes it easier to enter cardiac coherence phase,51 hrvb has often been established on roughly 6 breaths/min. in terms of the inhalation/exhalation proportion (i/e), a lower i/e ratio appears to result in enhanced relaxing, reducing stress, mindfulness, and good energy in participants 68, and a 1/2 proportion could boost baroreflex responsiveness.52 others’ findings, on the other hand, demonstrate that a 1/1 ratio is more beneficial than extended exhalation (40 percent intake and 60 percent expiration) in increasing hrv.53 as a result, more research is required to explain these various issues and to find the best beneficial breathing technique. future studies possibilities our findings are consistent with earlier studies that show hrvb has a favourable impact on clinical results and demonstrate that hrvb is a viable and prospective treatment option for people with chronic conditions.40,54,55 the researchers concluded that hrvb could help restore autonomic heart control and emotional self-control, as evidenced by the positive association between clinical results and hrv indicators.26,37,34,56 given the role of the autonomic nervous system in pathogenesis6 and the fact that hrv is a measure of cardiac morbidity, a potential regulating impact of hrvb on functional status provides attractive alternative therapy possibilities. our research is hampered by the lack of risk-of-bias evaluation of the included research, despite the fact that it gives a qualitative summary of hrvb outcomes and methods. given the diversity of procedures employed in hrvb studies, subsequent papers should emphasise analysing risk of bias, evaluating the significance of every report’s results, and doing meta-analyses to get more firm conclusions on the possible impact of hrvb. further controlled trials are needed to much more precisely assess the efficacy of hrvb in comparison to standard treatment and effective control circumstances (e.g., relaxation, mindfulness meditation, physical exercise). as potential confounders, respiratory rhythm (incidence, peak flow, and inspiration to exhalation ratio), physical activity, and medications should all be observed.46 as according respiratory rate and hrv signals collection, the various time and frequency parameters of hrv should be properly analysed.42 investigators may be inspired by a beautiful study whose findings were released after the comprehensive study’s eligibility deadline.57 it was carried out on depressed patients, and the procedure was based on a very thorough medication regimen that included a 5-week hrvb intervention during psychiatric inpatient recovery. the findings demonstrated an increase in hrv-lf amplitude and consistency ratios, as well as a reduction in depression scores and resting breathing rate, indicating that physicians have great potential. different analysis techniques to enhance the extracting included in hrv must be prepared and implemented in the future for a reliable estimation of functional connectivity utilising hrv. quantitative evaluations of hrvb’s impact on sympathetic cardiac control could possibly be more useful.58 lastly, in future investigations, established protocols for both treatment procedures and data collection should be observed to improve the effect of meta-analyses and review articles.43 conclusion the efficacy of hrvb as an adjunctive therapy in patients with chronic conditions is highlighted in this comprehensive review. because of the wide range of individuals and results, it’s hard to draw mechanical generalizations about how hrvb affects intervention effects. hrvb may have a regulatory influence on autonomic heart control by enhancing hrv and recovering vagal functionality, according to our findings. the enhanced vagal flow may therefore impact brain activities and improve emotional self-regulation, implying that hrvb could be useful as a supplemental remedy for various of chronic conditions. considering the excellent benefits of hrvb on psychophysiological results across a variety of patient characteristics, it’s apparent that hrvb has a bright future in the treatment of chronic disorders. confirming these findings, clarifying the understanding of the hrv power spectrum, and determining the most effective strategy in chronic disease management will require more research. conflict of interest none.  references 1. lehrer p, kaur k, sharma a, shah k, huseby r, bhavsar j, et al. heart rate variability biofeedback improves emotional and physical health and performance: a systematic review and meta analysis. applied psychophysiology and biofeedback. 2020;45(3):109–29. 2. reneau m. heart rate variability biofeedback to treat fibromyalgia: an integrative literature review. pain management nursing. 2020;21(3):225–32. 3. economides m, lehrer p, ranta k, nazander a, hilgert o, raevuori a, et al. feasibility and efficacy of the addition of heart rate variability biofeedback to a remote digital health intervention for depression. applied psychophysiology and biofeedback. 2020;45(2):75–86. 4. pizzoli sf, marzorati c, gatti d, monzani d, mazzocco k, pravettoni g. a meta-analysis on heart rate variability 309j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 s.m. al-tamimi original biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review biofeedback and depressive symptoms. scientific reports. 2021; 11(1):1–10. 5. prinsloo ge, rauch hl, derman we. a brief review and clinical application of heart rate variability biofeedback in sports, exercise, and rehabilitation medicine. the physician and sportsmedicine. 2014;42(2):88–99. 6. burch jb, ginsberg j, mclain ac, franco r, stokes s, susko k, et al. symptom management among cancer survivors: randomized pilot intervention trial of heart rate variability biofeedback. applied psychophysiology and biofeedback. 2020;45(2):99–108. 7. park sm, jung hy. respiratory sinus arrhythmia biofeedback alters heart rate variability and default mode network connectivity in major depressive disorder: a preliminary study. international journal of psychophysiology. 2020;158:225–37. 8. amjadian m, bahrami ehsan h, saboni k, vahedi s, rostami r, roshani d. a pilot randomized controlled trial to assess the effect of islamic spiritual intervention and of breathing technique with heart rate variability feedback on anxiety, depression and psycho-physiologic coherence in patients after coronary artery bypass surgery. annals of general psychiatry. 2020;19(1):1–10. 9. dell’acqua c, dal bò e, benvenuti sm, palomba d. reduced heart rate variability is associated with vulnerability to depression. journal of affective disorders reports. 2020;1:100006. 10. wang s-z, li s, xu x-y, lin g-p, shao l, zhao y, et al. effect of slow abdominal breathing combined with biofeedback on blood pressure and heart rate variability in prehypertension. the journal of alternative and complementary medicine. 2010;16(10):1039–45. 11. hasuo h, kanbara k, fukunaga m. effect of heart rate variability biofeedback sessions with resonant frequency breathing on sleep: a pilot study among family caregivers of patients with cancer. frontiers in medicine. 2020;7:61. 12. pagaduan jc, chen y-s, fell jw, wu ssx. can heart rate variability biofeedback improve athletic performance? a systematic review. journal of human kinetics. 2020;73:103. 13. lin i-m, lin p-y, fan s-y. the effects of heart rate variability (hrv) biofeedback on hrv reactivity and recovery during and after anger recall task for patients with coronary artery disease. applied psychophysiology and biofeedback. 2022:1–12. 14. pinna t, edwards dj. a systematic review of associations between interoception, vagal tone, and emotional regulation: potential applications for mental health, wellbeing, psychological flexibility, and chronic conditions. frontiers in psychology. 2020;11:1792. 15. reneau m. feasibility and acceptability of heart rate variability biofeedback in a group of veterans with fibromyalgia. the journal of alternative and complementary medicine. 2020;26(11):1025–31. 16. karavidas mk, lehrer pm, vaschillo e, vaschillo b, marin h, buyske s, et al. preliminary results of an open label study of heart rate variability biofeedback for the treatment of major depression. applied psychophysiology and biofeedback. 2007;32(1):19–30. 17. schuman dl, killian mo. pilot study of a single session heart rate variability biofeedback intervention on veterans’ posttraumatic stress symptoms. applied psychophysiology and biofeedback. 2019;44(1):9–20. 18. climov d, lysy c, berteau s, dutrannois j, dereppe h, brohet c, et al. biofeedback on heart rate variability in cardiac rehabilitation: practical feasibility and psycho-physiological effects. acta cardiologica. 2014;69(3):299–307. 19. de bruin ei, van der zwan j, bögels sm. a rct comparing daily mindfulness meditations, biofeedback exercises, and daily physical exercise on attention control, executive functioning, mindful awareness, self-compassion, and worrying in stressed young adults. mindfulness. 2016;7(5):1182–92. 20. tan g, dao tk, farmer l, sutherland rj, gevirtz r. heart rate variability (hrv) and posttraumatic stress disorder (ptsd): a pilot study. applied psychophysiology and biofeedback. 2011;36(1):27–35. 21. greenberg br, grossman ef, bolwell g, reynard ak, pennell na, moravec cs, et al. biofeedback assisted stress management in patients with lung cancer: a feasibility study. applied psychophysiology and biofeedback. 2015;40(3):201–8. 22. jester dj, rozek ek, mckelley ra. heart rate variability biofeedback: implications for cognitive and psychiatric effects in older adults. aging & mental health. 2019;23(5):574–80. 23. trousselard m, canini f, claverie d, cungi c, putois b, franck n. cardiac coherence training to reduce anxiety in remitted schizophrenia, a pilot study. applied psychophysiology and biofeedback. 2016;41(1):61–9. 24. chang w-l, lee j-t, li c-r, davis ah, yang c-c, chen y-j. effects of heart rate variability biofeedback in patients with acute ischemic stroke: a randomized controlled trial. biological research for nursing. 2020;22(1):34–44. 25. nolan rp, floras js, harvey pj, kamath mv, picton pe, chessex c, et al. behavioral neurocardiac training in hypertension: a randomized, controlled trial. hypertension. 2010;55(4):1033–9. 26. nolan r, floras j, ahmed l, harvey p, hiscock n, hendrickx h, et al. behavioural modification of the cholinergic anti‐inflammatory response to c‐reactive protein in patients with hypertension. journal of internal medicine. 2012;272(2):161–9. 27. lehrer pm, irvin cg, lu s-e, scardella a, roehmheld-hamm b, aviles-velez m, et al. heart rate variability biofeedback does not substitute for asthma steroid controller medication. applied psychophysiology and biofeedback. 2018;43(1):57–73. 28. hartogs bm, bartels-velthuis aa, van der ploeg k, bos eh. heart rate variability biofeedback stress relief program for depression. methods of information in medicine. 2017;56(06):419–26. 29. caldwell yt, steffen pr. adding hrv biofeedback to psychotherapy increases heart rate variability and improves the treatment of major depressive disorder. international journal of psychophysiology. 2018; 131:96–101. 30. lin i-m, fan s-y, yen c-f, yeh y-c, tang t-c, huang m-f, et al. heart rate variability biofeedback increased autonomic activation and improved symptoms of depression and insomnia among patients with major depression disorder. clinical psychopharmacology and neuroscience. 2019;17(2):222. 31. dobbin a, dobbin j, ross s, graham c, ford m. randomised controlled trial of brief intervention with biofeedback and hypnotherapy in patients with refractory irritable bowel syndrome. the journal of the royal college of physicians of edinburgh. 2013;43(1):15–23. 32. van der zwan je, de vente w, huizink ac, bögels sm, de bruin ei. physical activity, mindfulness meditation, or heart rate variability biofeedback for stress reduction: a randomized controlled trial. applied psychophysiology and biofeedback. 2015;40(4):257–68. 33. meyer p-w, friederich h-c, zastrow a. breathe to ease-respiratory biofeedback to improve heart rate variability and coping with stress in obese patients: a pilot study. mental health & prevention. 2018;11:41–6. 34. kim s, zemon v, cavallo mm, rath jf, mccraty r, foley fw. heart rate variability biofeedback, executive functioning and chronic brain injury. brain injury. 2013;27(2):209–22. 35. hallman dm, olsson em, von schéele b, melin l, lyskov e. effects of heart rate variability biofeedback in subjects with stress-related chronic neck pain: a pilot study. applied psychophysiology and biofeedback. 2011;36(2):71–80. 36. weeks dl, whitney aa, tindall ag, carter gt. pilot randomized trial comparing intersession scheduling of biofeedback results to individuals with chronic pain: influence on psychologic function and pain intensity. american journal of physical medicine & rehabilitation. 2015;94(10s):869–78. 37. ginsberg jp, berry me, powell da. cardiac coherence and posttraumatic stress disorder in combat veterans. alternative therapies in health & medicine. 2010;16(4). 38. mccraty r, childre d. coherence: bridging personal, social, and global health. altern ther health med. 2010;16(4):10–24. 39. schwerdtfeger ar, schwarz g, pfurtscheller k, thayer jf, jarczok mn, pfurtscheller g. heart rate variability (hrv): from brain death to resonance breathing at 6 breaths per minute. clinical neurophysiology. 2020;131(3):676–93. 40. richard gevirtz phd b. the promise of heart rate variability biofeedback: evidence-based applications. biofeedback (online). 2013;41(3):110. 41. shaffer f, mccraty r. zerr ch.(2014) a healthy heart is not a metronome: an integrative review of the heart’s anatomy and heart rate variability. frontiers in psychology. 5. 42. shaffer f, ginsberg j. an overview of heart rate variability metrics and norms. front public health. 2017; 5: 258. epub 2017/10/17. https://doi. org/10.3389/fpubh. 2017.00258 pmid: 29034226; 2017. 43. laborde s, mosley e, thayer jf. heart rate variability and cardiac vagal tone in psychophysiological research–recommendations for experiment planning, data analysis, and data reporting. frontiers in psychology. 2017;8:213. 44. kromenacker bw, sanova aa, marcus fi, allen jj, lane rd. vagal mediation of low-frequency heart rate variability during slow yogic breathing. psychosomatic medicine. 2018;80(6):581–7. 45. larsen p, tzeng y, sin p, galletly d. respiratory sinus arrhythmia in conscious humans during spontaneous respiration. respiratory physiology & neurobiology. 2010;174(1-2):111–8. 46. fatisson j, oswald v, lalonde f. influence diagram of physiological and environmental factors affecting heart rate variability: an extended literature overview. heart international. 2016;11(1):heartint. 5000232. 47. yu l-c, lin i-m, fan s-y, chien c-l, lin t-h. one-year cardiovascular prognosis of the randomized, controlled, short-term heart rate variability biofeedback among patients with coronary artery disease. international journal of behavioral medicine. 2018;25(3):271–82. https://doi 310 j contemp med sci | vol. 8, no. 5, september-october 2022: 295–310 biofeedback of heart rate variability in the treatment of chronic diseases: a systematic review original s.m. al-tamimi 48. feldman jm, matte l, interian a, lehrer pm, lu s-e, scheckner b, et al. psychological treatment of comorbid asthma and panic disorder in latino adults: results from a randomized controlled trial. behaviour research and therapy. 2016;87:142–54. 49. lin i-m, ko j-m, fan s-y, yen c-f. heart rate variability and the efficacy of biofeedback in heroin users with depressive symptoms. clinical psychopharmacology and neuroscience. 2016;14(2):168. 50. lehrer p, vaschillo b, zucker t, graves j, katsamanis m, aviles m, et al. protocol for heart rate variability biofeedback training. biofeedback. 2013;41(3). 51. szulczewski mt. an anti-hyperventilation instruction decreases the drop in end-tidal co2 and symptoms of hyperventilation during breathing at 0.1 hz. applied psychophysiology and biofeedback. 2019;44(3):247–56. 52. modesti pa, ferrari a, bazzini c, boddi m. time sequence of autonomic changes induced by daily slow-breathing sessions. clinical autonomic research. 2015;25(2):95–104. 53. lin i-m, tai l, fan s-y. breathing at a rate of 5.5 breaths per minute with equal inhalation-to-exhalation ratio increases heart rate variability. international journal of psychophysiology. 2014;91(3):206–11. 54. wheat al, larkin kt. biofeedback of heart rate variability and related physiology: a critical review. applied psychophysiology and biofeedback. 2010;35(3):229–42. 55. goessl vc, curtiss je, hofmann sg. the effect of heart rate variability biofeedback training on stress and anxiety: a meta-analysis. psychological medicine. 2017;47(15):2578–86. 56. patron e, messerotti benvenuti s, favretto g, valfre c, bonfa c, gasparotto r, et al. biofeedback assisted control of respiratory sinus arrhythmia as a biobehavioral intervention for depressive symptoms in patients after cardiac surgery: a preliminary study. applied psychophysiology and biofeedback. 2013;38(1):1–9. 57. tatschl jm, hochfellner sm, schwerdtfeger ar. implementing mobile hrv biofeedback as adjunctive therapy during inpatient psychiatric rehabilitation facilitates recovery of depressive symptoms and enhances autonomic functioning short-term: a 1-year pre–post-intervention followup pilot study. frontiers in neuroscience. 2020;14:738. 58. wu jk, huang z, zhang z, xiao w, jiang h. quantitative assessment of autonomic regulation of the cardiac system. journal of healthcare engineering. 2019;2019. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i5.1289 88 j contemp med sci | vol. 4, no. 2, spring 2018: 88–91 neonatal mortality rate at al-elwaya maternity hospital in baghdad city: retrospective study hawraa hussein ghafel maternal and neonate nursing department, college of nursing, university of baghdad, baghdad, iraq. correspondence to hawraa hussein ghafel (email: hawraa_2004@yahoo.com). (submitted: 24 february 2018 – revised version received: 10 march 2018 – accepted: 22 april 2018 – published online: 26 june 2018) objective this study was conducted to assess the neonatal mortality rate in baghdad city during the last 10 years (2008–2017). this study was conducted at al-elwaya maternity hospital in baghdad city: the retrospective study last 10 years ago (2008– 2017) regarding neonatal mortality rate. this study was started in october 2017 to february 2018. the data regarding neonatal mortality rate was achieved from the data recorded in the statistical department in the hospital. results the result shows meaningful s-shaped auto-regression model tested in two-tailed alternative statistical hypothesis for two factors, time sequence per (years) factor as (independent), and number of dead birth factor as (dependent). slope value was estimated and indicated that increasing with inverse transformation of one lagged period factor (1 year), occurring with a positive increment on natural logarithm transformation of the unit of dead birth number, and that estimated by (−0.95284), and the increment was recorded as highly significant and positive effectiveness at p < 0.01, as well as strong and highly significant correlation ship at p < 0.01 between studied factors and accounted (0.69863), with meaning determination coefficient of r-square (48.808%), which explanation disturbances among the numbers of dead birth along with the studied periods. conclusion the results of this study show that the neonatal mortality rate increased with the time sequence for the last 10 years (2008–2017) in al-elwaya maternity hospital in baghdad city. keywords retrospective study, neonatal mortality rate, prediction of neonatal mortality, time sequence introduction the first 28 days of life during the neonatal period are the most vulnerable period for a newborn’s survival. newborns face the highest risk of dying in their first month of their life, at a global rate of 19 deaths per 1000 live births. globally, 2.6 million newborns died in the first month of their life in 2016, approximately 7,000 newborn deaths every day most of which happened in the first week, with about 1 million dying on the first day and approximately to 1 million dying within the next 6 days.1 the large number of the neonatal deaths are occurred in the first day and week, with about 1 million dying on the first day and approximately 1 million dying within the next 6 days. more than 60 countries will miss the reducing neonatal mortality to at least as low as 12 deaths per 1000 live births by 2030. about half of them will not reach the target by 2050. these countries carry about 80% of the burden of neonatal deaths in 2016.2 neonatal mortality accounts for a large proportion of child deaths in many countries, especially in low-income settings. mortality during the neonatal period is considered as an indicator of both maternal and newborn health and care.3 the newborn mortality rate (nmr) is the number of deaths of newborns under 1 year old per 1000 live births.4 the nmr has often been used as an indicator of the standard of a country’s social, educational and healthcare systems. strategies, which address inequalities both within a country and between countries, are necessary if there is going to be further improvement in global perinatal health.5 in 2015, in the united kingdom, 1360 babies who were born after 24 weeks’ gestation died in their first 28 days of life. overall, neonatal mortality rates are declining. this means that generally fewer babies per 1000 births are dying each year. in 2014, 31% of babies who died in their first 28 days of life died related to complications after birth; this was the largest cause of death. about 28% of babies died due to congenital anomalies, and 18% of deaths were due to babies being born extremely early, how early a baby is born has an effect on their chance of survival, and a high proportion of neonatal deaths in the united kingdom are due to complications caused by prematurity. the following survival rates have been calculated from the number of live births and deaths in 2013.6 about 0.75 million neonates die every year in india, the highest rate of any country in the world. the neonatal mortality rate (nmr) declined from 52 per 1000 live births in 1990 to 28 in 2013, but the rate of decline has been slow and lags behind that of newborn mortality rates. among neonatal deaths, the rate of decline in early neonatal mortality rate (enmr) is much lower than that of late nmr. the high level and slow decline in early nmr are also reflected in a high and stagnant perinatal mortality rate. the rate of decline in nmr, and to an extent enmr, has accelerated with the introduction of national rural health mission in mid-2005. almost all states have witnessed this phenomenon, but there is still a huge disparity in nmr between and even within the states. the disparity is further compounded by rural–urban, poor–rich and gender differentials. there is an interplay of different demographic, educational, socioeconomic, biological and care-seeking factors, which are responsible for the differentials and the high burden of neonatal mortality. addressing inequity in india is an important cross-cutting action that will reduce newborn mortality.7 methodology this study conducted at al-elwaya maternity hospital in baghdad city: retrospective study for the last 10 years (2008– 2017) regarding the neonatal mortality rate. this study was started in october 2017 to february 2018. the data regarding neonatal mortality rate was achieved from the data recorded issn 2413-0516 research methods hawraa hussein ghafel et al. 89j contemp med sci | vol. 4, no. 2, spring 2018: 88–91 neonatal mortality rate in baghdad: retrospective study research in the statistical department in the hospital. the following statistical data analysis approaches were used to analyze and assess the results of the study under application of the statistical package (spss) version 18.0: fitted of long-term trends for simple auto-linear and auto-nonlinear regression equations, such that [inverse, (polynomial autoregression quadratic, cubic), power, compound, s-shaped, logistic, compound, growth and exponential] predicated equations are applicable for newborn death rate, incidence rates of congenital delivery, and neonatal mortality rates, as dependent variables affected by lagged sequence of times as an independent variable (per year). for the abbreviations of the comparisons significant (cs), used the followings: ns, non-significant at p > 0.05, s, significant at p < 0.05, hs, highly significant at p < 0.01. the neonatal mortality rate calculated with the number of deaths during the first 28 days* of life per 1000 live births in a given year.9–13 results table 1 shows that the neonatal mortality rate in general increases with the sequences of the years for the last 10 years from 2008–2017, according to the data recorded in the statistical department in al-elwaya maternity hospital except in 2015, due to the loss of data related to migration. table 2 shows meaningful s-shaped auto-regression model tested in two-tailed alternative statistical hypothesis for two factors, time sequence per (year) factor as (independent), and number of dead birth factor as (dependent). slope value was estimated and indicating that increasing with inverse transformation of one lagged period factor (1 year), occurring with a positive increment on natural logarithm transformation of the unit of dead birth number, and that estimated by (−0.95284), and the increment was recorded as highly significant and positive effectiveness at p < 0.01, as well as strong and highly significant correlation ship at p < 0.01 between studied factors, and accounted (0.69863), with meaning determination coefficient of r-square (48.808%), which explanation disturbances among the numbers of dead birth along with the studied periods. figure 1 shows the long-term trend of time sequence (years) factor on the number of neonatal mortality rate factor. table 3 shows meaningful power-shaped auto-regression model tested in two-tailed alternative statistical hypothesis for two factors, time sequence per (year) factor as (independent), and nmr factor as (dependent). slope value was estimated and indicating that increasing with natural logarithm transformation one lagged of period factor (1 year), occurring with a positive increment on natural logarithm transformation unit of (nmr), and that estimated by (0.491195), and the increment was recorded as highly significant and positive effectiveness at p < 0.01, as well as strong and highly significant correlation ship at p < 0.01 between studied factors, and accounted (0.74364), with meaning determination coefficient of r-square (55.301%), which explanation disturbances among the numbers of dead birth along with the studied periods. figure 2 shows the long-term trend of time sequence (years) factor on the (nmr) factor. table 1. distribution of the neonatal mortality rate (2008–2017) years the total number of deliveries the total number of a life deliveries a life males a life females the total number of neonatal death neonatal mortality rate 2008 16,912 16,653 8564 8589 0.259 15.55 2009 18,369 18,107 8972 9135 0.262 14.46 2010 20,232 20,129 10,072 10,057 0.303 15.05 2011 18,596 18,297 8192 10,105 0.299 16.34 2012 16,315 15,791 8504 7287 0.278 17.60 2013 17,579 17,279 8656 8673 0.300 17.36 2014 22,648 22,275 12,136 10,139 0.390 17.50 2015 22,001 21,675 8872 12,803 0.326 15.04 2016 19,752 19,499 9653 9846 0.353 18.10 2017 19,342 19,192 9657 9535 0.356 18.54 table 2. correlation of time sequence (per year) on the neonatal mortality rate dependent variable method ... s-shaped model no of dead birth simple correlation coefficient 0.69863 meaningful linear regression tested in two-tailed alternative statistical hypothesisr-square 0.48808 f (statistic) 8.58095 sig. level 0.0168 (s)* variables in the equation variable b se.b beta t-test sig. of (t) sequence per year −0.95284 0.325278 −0.69863 −2.929 0.0168 (constant) 5.769005 0.122418 – 47.125 0.0000 predicted equation is y = e^(b0 + (b1/t)) or ln(y) = b0 + (b1/t) *s: significance at p < 0.05, t: time sequence (per year). 90 j contemp med sci | vol. 4, no. 2, spring 2018: 88–91 neonatal mortality rate in baghdad: retrospective study research hawraa hussein ghafel et al. discussion of the results in this study, the results show that the neonatal mortality rate increased with the time sequence for the last 10 years (2008– 2017) in al-elwaya maternity hospital in baghdad city. the majority of the neonatal deaths are concentrated in the first day and within a week, neonatal mortality accounts for a large proportion of child deaths in many countries, especially in low-income settings like iraq. mortality during neonatal period is considered as an indicator of both maternal and newborn health and care. most of the pregnant woman does not receive the primary health care during pregnancy related to several causes, such as the emigration, country’s social, educational and healthcare systems and strategies. other factors such as duration of hospitalization, birth weight, and gestational age have effect on newborns mortality and gestational blood pressure has direct effect on newborns mortality. overall neonatal mortality rates have declined over the past several decades in the under developing countries; the southeastern states have remained the leading states in high infant death in the united states. in the previous study, the researcher studied the differences in infant mortality in the united states from 2005 to 2009 according to mother’s characteristics (age of mother, marital status, maternal race, maternal education), birth characteristics (month when fig. 1 long-term trend scatter diagram correlation of time sequence factor on neonatal mortality rate factor. table 3. correlation of time sequence (per year) on the nmr dependent variable method ... power – s-shaped model no of dead birth simple correlation coefficient 0.74364 meaningful linear regression tested in two-tailed alternative statistical hypothesisr-square 0.55301 f (statistic) 11.13457 sig. level 0.0087 (hs)* variables in the equation variable b se.b beta t-test sig. of (t) sequence per year 0.491195 0.147203 0.743645 3.337 0.0087 (constant) 6.502163 1.667833 – 3.899 0.0036 predicted equation is y = b0 × (t^b1) or ln(y) = ln(b0) + (b1 × ln(t)) *hs, highly significance at p < 0.01, t: time sequence (per year). fig. 2 long-term trend scatter diagram correlation of time sequence factor on the nmr. maternal prenatal care began, birth weight), and infant’s characteristics (age of infant at death). in the previous study, data suggested that mothers with no prenatal care had a very high overall neonatal death rate. it is suggested that better education and living quality should be available and improved for the mothers.8 in uganda, nmr remains high at 27 deaths per 1000 live births. there is paucity of data on factors associated with nmr in rural communities in uganda. the objective of this study was to determine nmr as well as factors associated with neonatal mortality in the rural communities of three districts from eastern uganda. the neonatal mortality was found to be 34 per 1000 live births. neonatal mortality in rural communities is higher than the national average. the use of chw’s to mobilize and sensitize households on appropriate maternal and newborn care practices could play a key role in reducing neonatal mortality.14 variables such as duration of hospitalization, birth weight, gestational age have negative effect on infant mortality, and gestational blood pressure has positive direct effect on infant mortality that at whole represented 66.5% of infant mortality.15 results of another study suggest that the current community based newborn survival intervention should provide an even greater focus to essential newborn care practices, low birth weight newborns, and female education.16–18 hawraa hussein ghafel et al. 91j contemp med sci | vol. 4, no. 2, spring 2018: 88–91 research neonatal mortality rate in baghdad: retrospective study conclusion the results of this study show that the neonatal mortality rate increased with the time sequence for the last 10 years (2008– 2017) in al-elwaya maternity hospital in baghdad city. recommendations 1. decrease incidence of infection and injuries during delivery. 2. enhance the services that are provided in neonatal intensive care unit. 3. enhance the primary health care services. 4. increase the awareness of pregnant women about the important of prenatal care. 5. further studies about the causes of neonatal death in all governorate in iraq. n references 1. unicef data: monitoring the situation of children and women, the neonatal period is the most vulnerable time for a child, 2017 available at https://data.unicef.org/topic/child-survival/neonatal-mortality/ 2. who: global health observatory (gho) data, 2016. available at http://www. who.int/gho/child_health/mortality/neonatal_text/en/ 3. chartbin: current worldwide neonatal mortality rate (per 1000 live births), 2011. available at http://chartsbin.com/view/1451 4. who: list of countries by infant mortality rate, 2017. available at http:// www.who.int/gho/child_health/mortality/mortality_under_five_text/en/ 5. yu vy. global, regional and national perinatal and neonatal mortality. j perinat med. 2003;31:376–379. 6. mbrrace-uk, 2017. perinatal mortality surveillance report. retrievedfrom:https://www.npeu.ox.ac.uk/downloads/files/mbrrace uk/reports/mbrrace-uk-pms-report-2015%20final%20full%20 report.pdf in bliss: http://www.bliss.org.uk/references-for-neonatalmortality. 7. sankar mj, neogi sb, sharma j, chauhan m, srivastava r, prabhakar pk, et al. state of newborn health in india. j perinatol. 2016;36:s3–s8. 8. he x, akil l, aker wg, hwang hm, ahmad ha. trends in infant mortality in united states: a brief study of the southeastern states from 2005–2009. int j environ res public health. 2015;12:4908–4920. 9. neonatal mortality rate (per 1000 live births) http://www.who.int/whosis/ whostat2006neonatalmortalityrate.pdf 10. neonatal mortality rate: http://www.who.int/healthinfo/indicators/2015/ chi_2015_28_mortality_neonatal.pdf 11. neonatal mortality rate: https://naphsis-web.sharepoint.com/about/ documents/neonatal_mortality_rate-jerry_edit.pdf 12. the center for health statistics, statistical analysis division: formulas, http:// adph.org/healthstats/assets/formulas.pdf 13. measures of mortality. indicators of child mortality, 2012. http://www. medicalbiostatistics.com/childmortality.pdf 14. kananura rm, tetui m, mutebi a, bua jn, waiswa p, kiwanuka sn, et al. the neonatal mortality and its determinants in rural communities of eastern uganda, reprod health. 2016;13:13. 15. ghojazadeh m, velayati a, mallah f, azami-aghdash s, mirnia k, piri r, et al. contributing death factors in very low-birth-weight infants by path method analysis. niger med j. 2014;55:389–393. 16. shah r, sharma b, khanal v, pandey uk, vishwokarma a, malla dk. factors associated with neonatal deaths in chitwan district of nepal. bmc res notes. 2015;8:818 17. who: neonatal mortality, risk factors and causes: a prospective populationbased cohort study in urban pakistan (submitted: 09 january 2008 – revised version received: 21 june 2008 – accepted: 25 june 2008 – published online: 06 january 2009) 18. bulletin of the world health organization. 2009;87:130–138. doi: 10.2471/ blt.08.050963. http://www.who.int/bulletin/volumes/87/2/08-050963/en/. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms. 06201807 102 j contemp med sci | vol. 4, no. 2, spring 2018: 102–105 case report lichen planus pemphigoides limited to the oral cavity: a case report and literature review maryam koopaiea,b,c and mahnaz fatahzadehd adepartment of oral medicine, school of dentistry, tehran university of medical sciences, tehran, iran. bresearch center for caries prevention, dentistry research institute, tehran university of medical sciences, tehran, iran. cschool of dentistry, international campus, tehran university of medical sciences, tehran, iran. drutgers school of dental medicine, diagnostic sciences, newark, nj, usa. correspondence to maryam koopaie (email: m-koopaie@tums.ac.ir). ( ) introduction lichen planus pemphigoides (lpp) is a rare mucocutaneous condition of autoimmune etiology which is characterized by a combination of clinical, histopathological, and immunological features of both lichen planus (lp) and bullous/mucous membrane pemphigoid (bp/mmp).1–4 to date, only about 90 cases of cutaneous llp and 35 cases of oral lpp have been reported.1,2,5 lichen planus pemphigoides has a female predilection and often occurs in the fourth decade of life. occurrence of lpp in children is very rare with fewer than 20 cases in the literature.2 cutaneous manifestations include pruritic erythematous patches with or without bullae and erosions often affecting the extremities.3 oral lesions of lpp are variable and include white reticular plaques to erosive, ulcerative components, vesicular eruptions, and desquamative gingivitis.4 the most commonly affected site in the oral cavity is buccal mucosa.6 histopathologic features of lpp resembles lp and when bullae are present, also bullous pemphigoid (bp).4 microscopic features include hyperkeratosis, acanthosis, band-like lymphocytic infiltration, colloid bodies typical of lp, as well as subepidermal blisters expected in bp.6 direct immunofluorescent (dif) studies show linear deposits of igg and/or c3 along the basement membrane. in lp, dif shows granular deposition of fibrinogen and c3 along the basement membrane zone (bmz).1 nature and pathogenesis of lpp have been subject to controversy. while previous studies considered lpp as simultaneous presentation of lp with bp or mmp, more recent studies suggest lpp as a variant of bp.3,7 the latter consideration is supported by the difference in mean age of affected patients and severity of manifestations between lp and bp. the mean age of patients with lp (40–50) is lower than those affected by bp (70–80).1,8 in addition, patients affected by bp generally experience more severe manifestations and have less favorable response to therapy. furthermore, circulating auto-antibodies in lpp and bullous pemphigoid share similarities,4 and are directed against bp180 domain.4 more specifically, anti collagen xvii auto-antibodies in lpp react to region 4 within bp180 noncollogenous-16a domain whereas in bp auto antibodies are directed against regions 2 and 3 within the same domain.4 some authors postulate that pathogenesis of lpp and development of bp/mmp from an immunologically unrelated disorder oral lichen planus (olp) may be explained by epitope spreading.3,9,10 it is thought that lichen planus-induced disruption of bmz may lead to exposure of bmz proteins, production of auto-antibodies against them and clinical presentations of pemphigoid.3 there are also reports of an association between lpp and phototherapy, hepatitis b infection and internal malignancies such as lymphoma, colon cancer and castleman’s disease in the literature.11 in addition, a number of medications such as calcium channel blocker, nsaids, interferon, ribavirin, therapeutic hormones, infertility medications12,13 and weight reduction drugs have been implicated in the onset of lpp.14,15 therefore, a through medical and medication history is a necessary part of work up process. case history an 85-year-old woman was presented to the oral medicine department of the tehran university of medical sciences with the complaint of oral erosions of 5-year duration. she reported pain and burning sensation in the soft palate and inability to use her maxillary prosthesis. her past medical history was significant for hypertension (htn) controlled with atenolol. review of systems was negative for cutaneous lesions. intraoral exam revealed a superficial ulcer of about 2 cm on soft palatal midline where patient reported prior vesicular eruptions (fig. 1). there were also white reticular striations with associated erythema on her buccal mucosa bilaterally. histopathological examination of an incisional specimen from the buccal issn 2413-0516 lichen planus pemphigoides (lpp) is a rare autoimmune vesiculobullous disorder and its exclusive presentation in the oral cavity is an even more remote occurrence. we describe an 85-year-old woman with a symptomatic soft palatal erosion, which compromised her ability to wear a maxillary prosthesis. she had been diagnosed with and treated for lichen planus affecting her buccal mucosa 3 years prior to the onset of the lesion on palate. persistence of the palatal lesion and its lack of response to local steroid therapy prompted a repeat biopsy for histopathological and direct immunofluorescence examination both of which confirmed lpp diagnosis. in oral lichen planus pemphigoides treatment is empirical. in this patient, a combination of systemic and topical steroids was effective in resolving the palatal ulceration. this case report highlights the importance of monitoring patient’s response to therapy and appropriate diagnostic workup when signs and symptoms persist or change character. keywords oral lichen planus pemphigoid, lichen planus, direct immunofluorescence, bullous lichen planus, mucous membrane pemphigoid submitted: 19 november 2017 – revised version received: 11 january 2018 – accepted:17 february 2018– published online: 26 june 2018 maryam koopaie and mahnaz fatahzadeh 103j contemp med sci | vol. 4, no. 2, spring 2018: 102–105 lichen planus pemphigoides limited to the oral cavity mucosa, the initial site of her symptomatic oral erosions 3 years prior to onset of soft palatal lesion, had revealed stratified squamous mucosa with mild epithelial acanthosis, diffuse vacuolar degeneration of basal layer and individual cell apoptosis. microscopic features were consistent with lichen planus and administration of topical steroids (triamcinolone acetonide in orabase) alleviated her symptoms until palatal erosion developed. persistence of palatal erosion and its failure to respond to topical therapy prompted tissue sampling. histopathological examination demonstrated subepithelial separation and infiltration of inflammatory cells specially eosinophils in the area (fig. 2a). direct immunofluorescence showed linear deposits of igg and c3 in the bmz (fig. 2b). both microscopic features and dif were compatible with mmp. following consultation with the physician regarding the status of her htn, she was started on 5 mg of prednisone daily.2 we opted to use systemic steroids because topical therapy was ineffective and patient could not tolerate intralesional injections. one month later, the lesion had improved significantly and she was able to use her denture. the lesion was fully resolved on 3 month follow-up and systemic steroid was tapered. the timeline for development of lpp from lp in this patient is illustrated (fig 3a). in this patient, combination of systemic and topical steroids was effective in resolving the palatal ulceration (fig. 3b). discussion pathogenesis of lpp is controversial. while some authors consider lpp and pemphigoid as two distinct conditions,3 others suggest lpp as a variant of pemphigoid16,17 because clinically blisters develop in association with lichen planus lesions. evolution of lp to bp or mmp caused by epitope spreading has also been suggested.5,3 table 1 summarizes the features of lpp with oral involvement in six patients one of whom is our patient. all six patients were female and the time line for development of lichen planus to bp or mmp ranged from simultaneous to 17 years.3,18–21 development of lpp in the context of olp within 3 years as seen in our patient resembles the case reported by maceyko et al.18 both microscopic and dif findings in the palatal specimen confirmed mmp, but indirect immunofluorescence (iif) was negative. these findings are compatible with other studies4,5,7,16 as only 10% of mmp patients demonstrate positive iif for circulating anti-basement membrane zone antibodies.22 exclusive presentation of lpp in the oral cavity as seen in this patient is rare and only a few cases are reported in the literature.20,21 fig. 1 clinical presentation of soft palatal erosion at the initial presentation. fig. 2 (a) low power histopathological photomicrograph of the palatal specimen demonstrating subepithelial separation at the bmz and infiltration of inflammatory cells including lymphocytes and eosinophils. (h and e, ×10). (b) direct immunofluorescent photomicrograph of the palatal specimen illustrating linear deposition of igg autoantibodies along the bmz. fig. 3 (a) timeline diagram of the patient’s diseases. (b) clinical presentation of the healed soft palatal lesion during 3 months follow-up. a b ba case report 104 j contemp med sci | vol. 4, no. 2, spring 2018: 102–105 lichen planus pemphigoides limited to the oral cavity maryam koopaie and mahnaz fatahzadeh involvement of lpp limited to oral mucosa is difficult to differentiate from erosive lichen planus or subepithelial vesiculating mucositis such as pemphigoid or linear iga disease. correct diagnosis of lpp relies on biopsy for histopathological examination and dif studies and the latter helps differentiate between lp and subepithelial diseases one of which is lpp.21 when warranted, presence of lichenoid changes and additional tests can help separate lpp from other subepithelial conditions. this patient had been diagnosed with buccal mucosa lesions of lp and later developed lpp lesions on her soft palate. she could have had lpp without bullae affecting buccal mucosa but since dif study was not performed, it was not diagnosed until soft palatal lesion appeared. this highlights the importance of proper diagnostic approach. this scenario could also depict occurrence of lpp in an anatomical site different from the initial lp as previously reported in the literature.23,24 differentiation of lpp from bullous lichen planus (blp) can be challenging. in blp, bullae arise on pre-existing lichen planus lesions whereas in lpp, bullae appear before, during or after lichenoid lesions. also, in blp, subepithelial bullae are main features while in lpp, bullae may or may not be present and histopathological features of bp together with lichenoid lesions and eosinophilic spongiosis are prominent findings. in addition, unlike blp, degeneration of basal cells is absent in lpp. moreover, dif study of blp shows patchy or globular deposition of igm, igg, iga, c3 and fibrinogen but in lpp there is linear deposition of igg, iga and c3 that is similar to bp. furthermore, iif study for blp is negative but in lpp it is positive in <50% of cases. lpp is usually less severe than bp and the course of blp is similar to lichen planus.23 a number of triggers for the onset of lpp eruptions are reported in the literature. these include hepatitis b infection or drugs such as simvastatin, ramipril, captopril, furosemide, ibuprofen and paracetamol. formation of auto-antibodies against bmz components has been suggested as the underlying mechanism for medication-induced bullous eruptions.13 this patient was not taking any of the medications reported to induce lpp11,16 and the likelihood of an etiological role for atenolol is very low. management of lpp involves using topical and systemic corticosteroids with or without steroid-sparing agents such as dapsone, azathioprine, tetracycline or isotretinoin.4,25 recommended dosage of systemic steroids, the most commonly used agent for cutaneous lpp in adults is 0.5 mg/kg daily or 40– 60 mg daily.3 dapsone, an anti-microbial agent used in management of many immunologic conditions has also proven effective for skin lesions of lpp although high rate of recurrence have been noted.24 furthermore, tetracycline and combination of nicotinamide and erythromycin are reported to be efficacious in treatment of lpp in adults and children, respectively.3 moreover, severe cases of cutaneous lpp have been managed with immunosuppressive agents such as cyclosporine or methotrexate.3,24 oral lesions of lpp tend to improve with treatment directed at the skin disease.21 however, treatment of oral lesions, depending on severity of involvement, may be individualized.21 in mild cases treatment include topical corticosteroid and in more severe cases systemic corticosteroid with or without steroid sparing agents is indicated.2,24 conclusion lichen planus pemphigoides is a rare immunobullous disease that has clinical and histopathologic manifestations of mmp or bp and olp. the interval for development of lpp in the context of lp is variable. when the nature and character of lesions change and lesions do not respond to routine treatment, it is imperative to suspect lpp and repeat tissue biopsy for confirmation. since there are cases of oral lichen planus pemphigoides (olpp) which do not present with intact bullae, immunofluorescent studies are essential for differentiating between olpp and olp when warranted by clinical findings. lpp is a systemic disease that may affect larynx, conjunctivae, skin and esophagus causing severe complications such as dysphagia, respiratory distress and blindness with significant impact on quality of life. this case report highlights the importance of close monitoring of patient’s response to therapy and appropriate workup when signs and symptoms persist or change character. this approach would help facilitate timely diagnosis, improve outcome and reduce likelihood of complications. table 1. clinical, histopathology and therapeutic features of six patients with llp author age and sex time lp to lpp histopathology dif description of lesions 1 dense band-like lymphohistiocytic inflammatory infiltrate, 2 colloid bodies (linear c3 and igg deposition along bmz) skin oral skin oral skin oral maceyko et al.18 65 f 3 years pruritic papules and blisters on trunk, extremities oral erosions and bullae 3, + subepidermal blister – 3 – zhu et al.19 69 f 3 weeks pruritic rash on the entire body superficial oral ulcers 3 – 3 – mignogna et al.3 72 f 1 year no skin lesion erosive, vesicular and keratotic oral lesions – 3 – 3 zaraa et al.20 51 f 17 years erythematous plaques, bullae and lichenoid lesions combination of white and erosive lesions 3 – 3 – solomon et al.21 63 f simultaneous no skin lesion desquamative gingivitis and bullae – 3 – 3 present case 85 f 2 years no skin lesion oral erosions and ulcers – 3 – 3 case report maryam koopaie and mahnaz fatahzadeh 105j contemp med sci | vol. 4, no. 2, spring 2018: 102–105 lichen planus pemphigoides limited to the oral cavity references 1. sultan a, stojanov ij, lerman ma, kabani s, haber j, freedman j, et al. oral lichen planus pemphigoides: a series of four cases. oral surg oral med oral pathol oral radiol. 2015;120:58–68. doi:10.1016/j.oooo.2015.03.012 2. sami n. autoimmune bullous diseases: approach and management; springer, new york, 2016. 3. mignogna md, fortuna g, leuci s, stasio l, mezza e, ruoppo e. lichen planus pemphigoides, a possible example of epitope spreading. oral surg oral med oral pathol oral radiol endod. 2010;109:837–843. doi:10.1016/j. tripleo.2009.12.044 4. jonkman mf. autoimmune bullous diseases: text and review; springer, 2015. 5. bavarian r, sultan as, mignogna md. comments regarding “four cases of mucous membrane pemphigoid with clinical features of oral lichen planus” and on the utility of immunofluorescence. int j dermatol. 2017;56:1515. doi:10.1111/ijd.13663 6. kabuto m, fujimoto n, yamaguchi a, tanaka t. evaluation of mononuclear cells in lichen planus pemphigoides. acta derm venereol. 2016;96:276–278. doi:10.2340/00015555-2191 7. yamada h, ishii h, seto k, kuwashima y. oral mucous membrane pemphigoid associated with lichen planus: a subtype of lichen planus pemphigoides? asian j oral maxillofac surg. 2004;16:135–138. doi:10.1016/ s0915-6992(04)80023-2 8. skvara h, stingl g. lichenoid eruption with single plantar blisters: a very rare case of lichen planus pemphigoides. j eur acad dermatol venereol. 2009;23:596–597. doi:10.1111/j.1468-3083.2008.02982.x 9. powell am, black mm. epitope spreading: protection from pathogens, but propagation of autoimmunity? clin exp dermatol. 2001;26:427–433. doi:10.1046/j.1365-2230.2001.00852.x 10. fujii m, takahashi i, honma m, ishida‐yamamoto a. bullous lichen planus accompanied by elevation of serum anti‐bp180 autoantibody: a possible transitional mechanism to lichen planus pemphigoides. j dermatol. 2017;44:e124–e125. doi:10.1111/1346-8138.13732 11. anand d, bernardin r, rubin ai. blisters and plaques on the extremities. what is your diagnosis? lichen planus pemphigoides. int j dermatol. 2011;50:147–149. doi:10.1111/j.1365-4632.2010.04722.x 12. laureano a, rafael m, pinto gm, cardoso j. lichen planus pemphigoides possibly induced by hormone therapy. eur j dermatol. 2013;23:903–904. doi:10.1684/ejd.2013.2180 13. maoz kb, brenner s. lichen planus pemphigoides triggered by narrowband uvb, paracetamol, and ibuprofen, with autoantibodies to 130 kda antigen. skinmed. 2008;7:33–36. doi:10.1111/j.1540-9740.2007.07667.x 14. rosmaninho a, sanches m, oliveira a, alves r, selores m. lichen planus pemphigoides induced by a weight reduction drug. cutan ocul toxicol. 2011;30:306–308. doi:10.3109/15569527.2011.566234 15. stoebner p, michot c, ligeron c, durand l, meynadier j, meunier l. simvastatin-induced lichen planus pemphigoides. ann dermatol venereol. 2003;130:187–190. 16. harting ms, hsu s. lichen planus pemphigoides: a case report and review of the literature. dermatol online j. 2006;12. 17. bhuiyan i, hossain ms, khan msa, alam m, haque m. lichen planus pemphigoides: a case report. j shaheed suhrawardy med coll. 2012;4: 35–37. doi:10.3329/jssmc.v4i1.12002 18. maceyko rf, camisa c, bergfeld wf, valenzuela r. oral and cutaneous lichen planus pemphigoides. j am acad dermatol. 1992;27:889–892. doi:10.1016/0190-9622(92)70275-k 19. zhu yi, fitzpatrick je, kornfeld bw. lichen planus pemphigoides associated with ramipril. int j dermatol. 2006;45:1453–1455. doi:10.1111/j.13654632.2006.02711.x 20. zaraa i, mahfoudh a, sellami mk, chelly i, el euch d, zitouna m, et al. lichen planus pemphigoides: four new cases and a review of the literature. int j dermatol. 2013;52:406–412. doi:10.1111/j.1365-4632.2012.05693.x 21. solomon lw, helm tn, stevens c, neiders me, kumar v. clinical and immunopathologic findings in oral lichen planus pemphigoides. oral surg oral med oral pathol oral radiol endod. 2007;103:808–813. doi:10.1016/j. tripleo.2006.03.020 22. glick m. burket’s oral medicine; 12th ed.; pmph-usa, 2015. 23. huang s-y, hsu rc-c, yu h-s, chen g-s. lichen planus pemphigoides: a case report. dermatol sinica. 2001;19:210–216. 24. schmidgen mi, butsch f, schadmand‐fischer s, steinbrink k, grabbe s, weidenthaler‐barth b, et al. pembrolizumab‐induced lichen planus pemphigoides in a patient with metastatic melanoma. j dtsch dermatol ges. 2017;15:742–745. doi:10.1111/ddg.13272 25. washio k, nakamura a, fukuda s, hashimoto t, horikawa t. a case of lichen planus pemphigoides successfully treated with a combination of cyclosporine a and prednisolone. case rep dermatol. 2013;5:84–57. doi:10.1159/000350285 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms. 062018010 case report 253j contemp med sci | vol. 3, no. 11, summer 2017: 253–259 review methodological heterogeneity in dental fluorosis investigations: a critical review mohammad hossein khoshnevisan,a,b mohammad reza nokhostin,c behrooz namvar,b seyed hadi sajjadi,d mina pakkhesala,e apreventive dentistry research center, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran. bcommunity oral health department school of dentistry, shahid beheshti university of medical sciences, tehran, iran. crestorative dentistry department, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. dorthodontics department, dental branch, islamic azad university of medical sciences, tehran, iran. ecommunity oral health department, school of dentistry, guilan university of medical sciences, rasht, iran. correspondence to mina pakkhesal (email: m_pakkhesal@yahoo.com). (submitted: 25 february 2017 – revised version received: 22 march 2017 – accepted: 13 may 2017 – published online: 26 september 2017) objectives the purpose of this investigation was to conduct a comprehensive and critical review of pertinent literature concerning dental fluorosis in order to examine the methodology used in different studies and report the potential sources of heterogeneity among identified reports. methods the pubmed database was searched using medical subject headings (mesh) for articles in english language, published between january 2000 and december 2016. the search was limited to human studies and having abstract available. subsequently, potential sources of heterogeneity were categorized. results through pubmed search, a total of 56 articles were selected out of 2369 initially identified papers. subsequently, 17 out of the 56 articles were excluded due to being irrelevant or no full text available. likewise, genetic studies, in-vitro reports and those studies with no evidence of association were excluded as well. therefore, 39 remaining articles were critically analyzed in order to identify the potential sources of heterogeneity in dental fluorosis studies. quality assessment of the reviewed papers demonstrated the following categories as potential sources of heterogeneity in dental fluorosis investigations: “different methods of fluoride exposure by children”, “different characteristics of study samples under investigation”, “methodological flaws in the fluorosis research design”. conclusion given the existing methodological heterogeneity, a consensus development is highly crucial for the standard diagnosis of fluorosis and improvement in homogeneity in future fluorosis investigations globally. keywords dental, enamel, fluorosis, dental fluorosis, methodology introduction tooth decay is a major public health problem affecting a great number of adults and children worldwide. untreated decay may cause pain, infection, leading mostly to extensive tooth loss in many lowand middle-income countries or disadvantaged communities in high-income countries. fluoride is an essential nutrient involved in mineralization of teeth and bones. it especially plays important role in tooth remineralization and prevention of tooth decay.1 fluoride is still considered the best protection against dental caries. water fluoridation was named as 1 of the 10 most important public health measures of the 20th century by the u.s. centers for disease control and prevention (cdc).2 in 2007, who world health assembly passed resolution wha 60/16 to enforce universal fluoride access for caries prevention as part of the basic right for human health.3 there are three basic fluoride delivery methods for caries prevention; community wide (salt, milk and water fluoridation); professionally applied (fluoride gel, varnish) as well as self-administered (toothpastes and mouth-rinses) methods.1 although, there are some anti fluoridation activities, most claims were unsubstantiated. more than 3000 studies or research papers have been published on fluoride or fluoridation subjects. based on available reports, the overwhelming weight of the evidence supports the safety and effectiveness of this preventive public health practice.4 some studies have reported that, caries prevention effect of fluoride has been accompanied with very mild to mild fluorosis due to steady exposure to fluoride. this change in the appearance of tooth enamel, is caused by subsurface porosity as a result of hypoplasia or hypo-mineralization that take place during tooth development period.1 although, fluoride’s effect may be different in various stages of enamel formation, its effect is maximized when exposure occurs during all stages of tooth formation.5 the severity of dental fluorosis depends on dosage of fluoride, duration and timing of fluoride exposure.6 teeth are most susceptible to developmental disturbances during the mineralization phase of tooth formation. in general, the permanent dentition is more susceptible to disturbances in mineralization by environmental toxicants and drugs than the primary dentition, most likely as a consequence of its later development.7 fluorosis is less prevalent and less apparent in primary than in permanent teeth. the fluorosis of primary teeth has only shortrather than long-term consequences. therefore, the major concern about fluorosis is related to the permanent dentition and particularly the maxillary incisors, because they have great cosmetic importance.8 primary-tooth fluorosis may be used as a prognostic factor in the occurrence of fluorosis in the permanent dentition. therefore, its recognition by the clinician should raise awareness of possible increased risk in permanent dentition.9 the occurrence of fluorosis (which was known as mottled enamel) in the primary dentition was first described in 1935 among children in arizona.9 studies of primary tooth fluorosis have been conducted in certain high water containing fluoride issn 2413-0516 254 j contemp med sci | vol. 3, no. 11, summer 2017: 253–259 methodological heterogeneity in dental fluorosis investigations review mina pakkhesal et al. areas in africa and europe. the results of these studies have demonstrated the widespread occurrence of primary tooth fluorosis in these locations. the primary molars, particularly the primary second molars were most frequently and severely affected.10 the goal of any public health administration is to provide optimum amount of fluoride to public in order to maximize caries prevention at the community level. at the same time, according to best practice guidelines, any sources of overexposure should be recognized and eliminated. recognition of such sources through scientific research is the best logical method in order to identify and control potential sources of fluoride overexposure. through the initial review of current literature, we recognized several methodological issues in need of special attention by the research community. therefore, our aim was to get the most out of published papers using a systematic review methodology. in this article, the results of our findings are reported. methods the pubmed database was searched using the medical subject headings (mesh) for studies published between the year 2000 and dec 2016. the search was limited to review articles, english language, and human studies with available abstract. by using mesh search, we used the range of keywords found in relevant articles. for example, “dental fluorosis” includes the following keywords: “fluoroses, dental”, “dental fluoroses”, “dental fluorosis”, “mottled enamel” and “enamel, mottled”. after initial evaluation, some articles were excluded for being irrelevant, in vitro animal (mice) studies, reports or unavailable full texts. the remaining articles were critically evaluated in order to synthesize the contemporary understanding on clinical diagnosis of fluorosis, in order to better control the main sources of disorder while, making the best use out of optimum fluoride exposure. the full texts of included studies were independently evaluated by two review authors. any disagreements were discussed and a third review author consulted to achieve consensus where necessary. results the initial electronic search in pubmed identified 56 articles from a total of 2369 studies. (table 1) subsequently, 17 out of the 56 articles were excluded due to irrelevance or full text not being accessible, genetic studies and in-vitro reports. finally, data extraction was conducted from 39 remaining articles. (fig. 1) qualitative analyses of the reviewed papers demonstrated the following categories as potential sources of heterogeneity in dental fluorosis investigations: difference in methods of fluoride exposure by children, different characteristics of study samples under investigation, methodological flaws in the fluorosis research design. (table 2) discussion the purpose of this investigation was to conduct a comprehensive and critical review of pertinent literature concerning dental fluorosis in order to examine the methodology used in different studies and report the potential sources of heterogeneity among identified reports. table 1. pubmed search strategy search step inclusion & exclusion criteria no. of publications 1 search dental fluorosis [mesh terms] 2369 2 search dental fluorosis [mesh terms] filters: review 194 3 search dental fluorosis [mesh terms] filters: review; abstract 135 4 search dental fluorosis [mesh terms] filters: review; abstract; publication date from 2000/01/01 to 2016/12/31 64 5 search dental fluorosis [mesh terms] filters: review; abstract; humans 63 6 search dental fluorosis [mesh terms] filters: review; abstract; humans; english 56 different methods of fluoride exposure by children researchers have identified 4 sources of increasing the risk of dental fluorosis as follows: 1) the natural or fluoridated drinking water, 2) fluoride supplements, 3) topical fluoride (especially fluoride toothpastes when ingested during brushing), and formula prescribed for children under 2-years of old. furthermore, using multiple sources of fluoride such as can-foods, soda, etc., have been found to be an important contribution to daily over-consumption of fluoride.11,12 many studies did not take into account the effect of all risk factors involved or could affect children’s risk of tooth decay or dental fluorosis. there was also substantial variation between the results of the studies, many of which took place before the introduction of fluoride toothpaste.1 water fluoridation can be regarded as a low-cost method of fluoride delivery for those communities where oral health care and particularly fluoride dentifrices are not widely available and/or is not affordable, causing reduction in socioeconomic related dental inequalities.13–15 around the world, dental fluorosis has always been regarded as a public health problem in those areas where natural fluoride in the community drinking water exceeds optimal levels. however, residents of optimally fluoridated areas have not been considered to be at risk for dental fluorosis.16 the ‘‘optimal’’ water fluoride concentrations can minimize the risk for both dental fluorosis and dental caries vary between 0.7 ppm and 1.2 ppm, depending on mean temperature of any given geographical area.17 enamel fluorosis occurs at fluoride levels (1.8–2.2 mg/l) which is much lower than the skeletal fluorosis becomes clinically evident (20–80 mg per day).14 based on current literature, the fluorosis caused by water fluoridation (40%) is being less than that attributable to other fluoride sources (60%). by the 1990s, fluoride toothpaste accounted for ≥90% of the toothpaste market in most economically developed countries, and accidental or intentional ingestion of fluoridated toothpaste especially in children has become a potentially important risk factor for excessive fluoride exposure.18 when recent data are compared to historical data, the results seem to indicate a trend toward a higher prevalence of fluorosis. however, due to widespread use of fluoridated products (such as fluoride supplements, fluoride toothpaste, and fluoride in the food and beverages), concern has been expressed in mina pakkhesal et al. 255j contemp med sci | vol. 3, no. 11, summer 2017: 253–259 review methodological heterogeneity in dental fluorosis investigations id en ti fic at io n sc re en in g el ig ib ili ty in cl u d ed excluded not relevant & not available full text of articles n = 8 excluded genetic studies in vitro, reports, no evidence of association was detected n = 9 pub med n = 2369 initial search in electronic databases n = 56 included eligible articles n = 48 included eligible articles for full-text assessment n = 39 fig. 1 flow chart showing search strategy and numbers of included and excluded articles. table 2. potential sources of heterogeneity in dental fluorosis investigations different methods of fluoride exposure by children • topical (fluoride toothpastes/ fluoride mouth rinses/ fluoride varnish/ fluoride gels) • systemic (fluoridated drinking water/ fluoride supplements/ infant formula/ fluoride containing foods and beverages) different characteristics of study sample under investigation • demographic factors (age/ timing of fluoride exposure related to enamel formation, gender, race, place of residence, geographical location/environments, climate, socio-economic status, parent’s income and education levels, major changes in infant feeding practices, healthy vs systematic disorders, etc ….) • behavioral factors (oral health behaviors, parents’/caregivers’ attitude, access to fluoride, life style, diet/food content) • other related factors (coal heating effects) methodological flaws in the fluorosis research design • use of not ideal study design • differences in the sampling method/selection of participant • variety diagnostic methods of dental fluorosis (clinical examinations, conventional photographs, digital images) • problems in clinical examination (teeth dried or not; duration of drying; cleaning of teeth and lighting used), problems in images (position of the flash, angle of the light) • using narrower standardization of examiners (interand intra examiner reliability) • distinguish fluorosis from other kinds of developmental defects of enamel (ddes), non-fluoride-induced enamel opacities, and dental hypoplasia lesions • using different fluoride indices (dean index, thylstrup and fejerskov index, tooth surface index of fluorosis, fluorosis risk index, chronological fluorosis assessment (cfa-index) • using different types of outcomes (d(m)fs/d(m)fs, tooth discoloration, prevalence of fluorosis for each tooth type, quality-of-life scores/ohrqol, societal tolerance level and perceptions of fluorosis and caries, acceptability and/or aesthetic perceptions/cosmetic disorder, public perception of enamel fluorosis as an aesthetic problem) recent years over a possible increase in the prevalence of dental fluorosis worldwide in optimally fluoridated and even sub optimally fluoridated areas. also, concurrent use of multiple sources of fluoride mainly fluoride intake from meals prepared with fluoridated salt; living at a high altitude; living in a hot climate have been reported. dental fluorosis was association has been reported with: bottled water, soft drinks, and juices; excessive fluoride in deep well water; boiled water as well.16 a higher risk of developing dental fluorosis is expected when excess exposure to ingested topical fluoride in young children occurs during permanent tooth development. mild dental fluorosis is demonstrated by white lines or streaks visible only to dentists under good lighting in the clinic.1 moreover, infant formula reconstituted with fluoridated water has been reported to be a risk factor for enamel fluorosis when consumed during the period of 13–24 months.10,19 the shortest duration of breast-feeding usually corresponded to the longest duration of infant formula use.20 many studies have reported a clear association between fluoride supplements use by children under age 6 and enamel fluorosis. subjects who used fluoride supplements during the first 6 years of life had a 28-fold increase in the risk of fluorosis when compared to unexposed subjects.10 it is important that, individual water sources be tested for fluoride level before prescribing or recommending fluoride products and suppliant for young children in order to control their fluoride ingestion.17 consistent evidence is available that shows fluoride tablets used during the first 3 years of life increased the risk of developing fluorosis; the first year of life appears to be the high risk period for incisors.21,22 it is possible that slow dissolution fluoride lozenges may start to play a role in caries control in older children and 256 j contemp med sci | vol. 3, no. 11, summer 2017: 253–259 methodological heterogeneity in dental fluorosis investigations review mina pakkhesal et al. adults, those who are no longer at risk of enamel fluorosis. therefore, topically “applied” fluoride had dramatic caries reductions (approximately 80%) when compared to systemic fluoride users. thus, chewable fluoride supplements can provide caries protective benefit in children and adults when the overall conditions are considered. the decision-making on the fluoride supplements prescription to children ought to be delegated on dentists and primary care pediatricians and by no means it should be taken as a common preventive measure in the pediatric population.23 different characteristics of study samples under investigation individuals, families and communities may differ in their fluoride exposure level.1 demographic factors the most critical period for fluorosis in the permanent dentition is considered to be during the latter stages of pre-eruptive tooth development; for the anterior permanent teeth which is between age 22 and 25 months old.8,24–26 the fluorosis can be used as a biomarker for the level of fluoride exposure in populations during the time of enamel formation.22,27 moreover, subjects living in a middle income households who had used fluoride supplements through the first six years of life demonstrated 28-fold increase in the risk of fluorosis compared with unexposed subjects in the lower median income households.23 interestingly, children from high socioeconomic group place approximately 24% more dentifrice on their toothbrush than their counterparts from lower socioeconomic classes.24 when considering socio-environmental, health context and especial demographic characteristics of a given population group, the production of epidemiological data on fluorosis should allow an understanding of the health and disease situation, even though offering limited conditions for the comparability of findings.28 the relationship between water fluoridation and social inequalities can be evaluated by the human development index (hdi). this is particularly important for developing countries where water fluoridation is feasible.29,30 other variables that have been associated with an increased risk of fluorosis are: socio-demographic variables such as the child’s age, gender, and race; parent’s income and education levels; feeding practices such as weaning before 9 months of age and breast feeding period; and fluoride mouth-rinse. it has been reported that, male caucasian children, with high income and educational level parents were more likely to have fluorosis.27 furthermore, the increased prevalence of fluorosis in black non-hispanics may suggest a genetic influence on fluorosis susceptibility.12 the severity of dental fluorosis depends on when and for how long the overexposure to fluoride occurs, the individual response, weight, degree of physical activity, nutritional factors and bone growth, suggesting that similar dose of fluoride may lead to different levels of dental fluorosis. other factors that may increase the individual susceptibility to dental fluorosis are altitude, malnutrition and renal insufficiency.11,27 fluorosis begins with the exposure of the tooth bud to high concentrations of fluoride ion during its formation. other factors such as low body weight, skeletal growth rate, and periods of bone remodeling also affect the severity of this condition.28 epidemiological studies performed in the countries of kenya and tanzania have indicated that higher prevalence and severity of fluorosis may be related to high altitude, even when suboptimal concentrations of fluoride are present in the drinking water.16 behavioral factors the increase in level of enamel fluorosis in permanent incisors was attributed to increased use and swallowing of fluoride toothpaste by infants and children. in some countries like australia, canada, and republic of ireland, it has led in reduction of fluoride levels in drinking water.22 fluoride ingested from toothpaste was correlated with tooth-brushing habits, frequency of brushing, frequency of rinsing, post brushing rinsing behavior, fluoride concentration in dentifrices, amount of toothpaste dispensed (the diameter of the orifice of the tubes, the length of the head of the toothbrush, the flavor and attractive visual characteristics of the dentifrice, weight of toothpaste used), as well as child’s body weight of the child.10,12,18,24,31,32 parental supervision is necessary for placement of toothpaste and every time children should be reminded to limit the amount of toothpaste used until age 6–7 years old. this may be due to parental brushing for their babies with toothpaste at too young age (before 24 months) or when children have not learned how to adequately rinse out their mouths and they, ingest much of toothpaste while brushing their teeth or rinsing their mouth.10 healthcare professionals play an important role in explaining to parents and/or caregivers about the risks and benefits of fluoridated dentifrices used for children. moreover, if the risk of fluorosis is of concern, the fluoride level of toothpaste for young children is recommended to be not lower than 1000 ppm.25 however, improper use of fluoride toothpaste in children under 6 can increase the risk of fluorosis.33 another factor that may contribute to the amount of dentifrice ingested could be the quantity of detergent present in toothpaste formulation. because dentifrices with a low detergent, generate less foam during brushing, and as a result, it poses smaller risk of ingestion.24 besides, motivated parents would probably be more involved in controlling the oral hygiene habits of their children, which may lead to increased fluoride exposure.18 also, “preventionism” behavior, as defined by the irrational use of fluoride in populations with more access to goods and services may be implicit in the genesis of some iatrogeny.28 the majority of foods assessed for their fluoride level are those meant for infants and young children. seafoods have high concentration of fluoride. also, processing foods with fluoridated water typically have higher fluoride concentrations than foods processed with non-fluoridated water. likewise, processing method of infant cereals has been shown to affect their fluoride concentration.17 the ingestion of fluoride may be delayed depending on the type of food present in the stomach.24 the major inhibitor of fluoride absorption is calcium. the presented data indicate that, now the calcium intakes by infants are less than in the 1930s to 1960s period. in addition, the considerable increase in consumption of soft drinks and fruit juices by children during the past 20 years has been associated with mina pakkhesal et al. 257j contemp med sci | vol. 3, no. 11, summer 2017: 253–259 review methodological heterogeneity in dental fluorosis investigations decreased milk consumption and a consequent decrease in calcium intake. thus, bioavailability of fluoride in the diets of infants and children is probably greater now than before.8 the increase in percentage of communities with fluoridated water has resulted in an increase in the mean content of fluoride consumed, not only in soft drinks and fruit juices, but in canned goods, leading to increased intake of fluoride by individuals and communities with non-fluoridated water.8 furthermore, tea is regularly consumed by a minority of children in the united states, and because of its high fluoride concentration (commonly 3 mg/l), can contribute substantially to the fluoride intakes of these children.8 other related factors endemic fluorosis is a public health concern in china due to excessive consumption of fluoride in drinking water, brick tea, food that was contaminated by indoor coal burning, and inhalation of coal burning air. also, the high-fluoride spring water was a possible source of fluorosis.34 additionally, due to the mobilization of fluoride by the burning of mineralized coals in unvented or poorly vented stoves, millions of people in guizhou suffer from dental and skeletal fluorosis. unhygienic living environments and unhealthy lifestyles can promote endemic fluorosis. therefore, multiple factors, including geographical location, humid climate, the geological and geochemical environment, economics, educational level, drying and storing methods of foodstuffs, unhealthy use of coal in the household as well as traditional lifestyles may contribute to incidence in endemic fluorosis.34 methodological flaws in the fluorosis research design study design and control of bias concern for the increase in the prevalence of dental fluorosis led to studies designed to identify the various risk factors for fluorosis. while a few studies were case-control, most others were cross-sectional in design. this (cross-sectional) study design is not an ideal method for assessing risk indicators or risk factors. hence, this design has been used in some fluorosis literature. another major criticism of most fluorosis studies is the use of retrospective assessment of fluoride exposures, due to inherent recall bias in such design. although, the recall bias should be assumed as random, and therefore not overly affecting the results.27 sampling method the brazilian literature shows differences in the sample planning, sample size, age bracket, inter-examiner reliability, reproducibility, health surveillance data accuracy, and territorial base.28 furthermore, the ages of the study participants in some of the studies were not appropriate to assess the research question. some studies used children between 6 and 13 years old. six-year-old children do not have many erupted permanent teeth yet. this sample may have underestimated the prevalence of fluorosis.27 the dental fluorosis prevalence study in mexico showed two issues: (1) researchers did not record the place of residence for their participants at the time of oral examination. (2) also, researchers failed to verify the duration of stay in place of residence from birth to age 6 years for the study participants. as a result, it is not possible to determine if the fluorosis observed in these samples was a true reflection of the communities where they lived. bias could have been introduced if some participants were not permanent residents of those communities under investigation. also, adding to the question about the representativeness of the samples due to the fact that many of the studies were used convenient samples.16 another limitation was related to the small sample size as a consequence of limited funding; adequate number of subjects could be recruited for evaluation, if there was no financial constraints.18 diagnostic methods of dental fluorosis one major factor that limits the comparability of epidemiologic studies on enamel defects is related to a variety of diagnostic methods used. dental fluorosis has several fluorosis-specific criteria used for its diagnosis. studies of the diagnosis of dental fluorosis can be affected by a large number of factors, such as examiner bias, intra-examiner and inter-examiner reliability, examiner drift, index validity due to varying methodologies (teeth dried or not; duration of drying; cleaning of teeth and lighting used).35 in epidemiological studies of dental fluorosis, the photographic method offers the following advantages: images can be read by trained examiners, blinded to the fluoridation status of the subject; images can be double-scored and cross-checked with the development of consensus in divergent scores. images are permanent record of the enamel, and are useful for measuring changes in enamel over time.35 the use of a standard photographic technique may be helpful in controlling potential discrepancies and can better reflect the reality.36 a technique employing standardized film, handling procedures, camera equipment, exposure time, lighting conditions, drying time of teeth, camera angulation, lip retraction and processing procedures has been developed and employed successfully in seven eu countries and in the national survey of children’s oral health in ireland.37 therefore, an experienced and/or trained examiner is necessary when taking standardized photos in order to minimize both specular reflection and lip shadow. the only potential disadvantage of using photography method is the increase in the cost of the study. despite the advances in digitalized imaging, conventional photographs are more often used in epidemiological investigations. the digital camera offers the following advantages: it maintains confidentiality, as it can photograph the teeth alone; measurements of variation in density are possible with digital images; greater resolution increases the definition of the image; and images can be easily stored in digital systems. for using digital camera, one should be well versed and experienced with the equipment. in this case, the investigators have to account for the cost of equipment.35 the results suggest inter-examiner reliability is greater and fluorosis scores higher when using the photographic method compared to clinical examinations.38–40 examiners training has been reported in many collected studies on dental fluorosis prevalence in mexico. however, the measurements of calibration were rarely reported. fluorosis is a difficult condition to diagnose, even for the experienced examiner. the lack of information on accuracy of the examiners, who were determining fluorosis status, makes it difficult to know whether differences 258 j contemp med sci | vol. 3, no. 11, summer 2017: 253–259 methodological heterogeneity in dental fluorosis investigations review mina pakkhesal et al. reported among regions or in time trends as were a true reflection of the population differences in fluorosis prevalence or they were instead a reflection of differences in the examiners’ assessments.16 distinguish fluorosis from other kinds of developmental enamel defects it should be noted that fluorosis is not the only type of disturbance found in dental enamel; enamel opacities can result from number of causes unrelated to fluoride use. the differential diagnosis between fluorosis and non-fluoride induced opacities should be established based on symmetrical and asymmetrical and/or discrete patterns of opaque defects. these criteria imply that all symmetrically distributed and non-discrete opaque conditions of enamel are considered fluorosis. it’s important to emphasize that non-fluoride enamel opacities include all categories of opacities not defined as fluorosis, i.e. dental hypoplasia lesions that are commonly characterized as discrete, demarcated white or discolored opacities often affecting a single tooth and, less frequently, multiple teeth, with a symmetrical distribution, and result from a wide variety of systemic or local factors.11 using different fluoride indices in relation to fluorosis publications, the first major difficulty relates to the comparability of the studies, due to the diversity of indices used. in 1942, h.t. dean developed an index for description and diagnosis of enamel fluorosis. this classification is still the ‘gold standard’, though other indices have been developed including the widely used thylstrup and fejerskov fluorosis index (tfi).12,41 dean’s index scores the two teeth that are most affected. the dean index even though describing the severity of fluorosis with less variation, is widely used, and is the index recommended by the world health organization (who), since it can be used safely in public health studies. thylstrup et al. proposed a modification of dean’s index known as the tf index. this classifies clinical features of fluorosis that reflect histopathological changes following histological examination using ordinary and polarized light on affected enamel. the index requires the teeth to be dried before examination, which gives better precision, so it is recommended for populations with a higher prevalence of the disease. other indices are used less frequently, since they are not conducive to comparative approaches.28 tooth surface index of fluorosis (tsif) described by horowitz et al. provides an analysis based on aesthetic concerns and examines teeth when wet. the fluorosis risk index (fri), developed by pendrys, is designed to produce an accurate association between agespecific exposures to fluoride and the development of fluorosis. it divides the enamel surface of the permanent teeth into two developmentally related groups of surface zones. code 1 began formation during the first year of life and code 2 began during the third to sixth years of life. the scores are recorded for each zone.10 in addition; it must be kept in mind that fluorosis prevalence is directly influenced by the case definition used to calculate it. for example, the case definition for the tooth surface index of fluorosis is based on the tooth surface unit, while dean’s index defines cases of fluorosis on the basis of individual teeth.16 in 1993, evans developed the chronological fluorosis assessment (cfa) index to investigate the chronological development of enamel fluorosis. evans et al. refined the estimated time for enamel fluorosis to occur.10 using different types of outcomes fluorosis as intrinsic discoloration occurs following a change to the structural composition of the dental hard tissues.42 the primary outcome was caries increment in the permanent or primary dentition, as measured by the change in decayed, (missing), and filled tooth surfaces [d(m) fs/d(m)fs] from baseline. the other primary outcome measure was the percentage prevalence of fluorosis in the permanent dentition. the timing of the outcome measurement should have been taken when most of the permanent teeth of interest were erupted in the study participants. if available, the prevalence of fluorosis for each tooth type is recorded.25 do and spencer found reported that, children who had mild fluorosis demonstrated higher quality-of-life scores than those children who had caries or more advanced fluorosis. this research should be expanded to define the societal tolerance level and perceptions of fluorosis and caries. evidence, rather than our professional perceptions, should guide us to decide what is acceptable by society.43,44 dental fluorosis is not a condition that causes pain or has clinical symptoms. the effects of mild fluorosis are subjective; thus, reports of dental fluorosis prevalence and severity alone do not give enough information to understand the effects at the public health level. to examine the effects of dental fluorosis, the early studies assessed acceptability and/or aesthetic perceptions concerning photographs of cases and/or subjects’ teeth with and without dental fluorosis in interested populations.45 studies have shown that the oral region is of primary importance in determining overall facial attractiveness. also, dental appearance is an important contributor to one’s self perceived body image. mild fluorosis is seen by dental professionals to be of little cosmetic consequence but to the person involved it may be an aesthetic problem.37 numerous studies have addressed the public perception of enamel fluorosis as an aesthetic problem. although, these studies were conducted in various countries (australia, canada, uk, usa) using different indices of enamel fluorosis, the findings generally from all these studies reflect that, both parents and children are less concerned about low levels of fluorosis than dentists. children with such low-level fluorosis are less likely to have experienced tooth decay. however, aesthetically objectionable fluorosis is a rare outcome, observed in only about 2 percent of children.14 dental fluorosis has not been identified as a public health problem in north america. however, given the trend toward whitening teeth and the increased demand for cosmetic dentistry, public rejection of even the mildest form of fluorosis could pose problems for dentistry’s timetested reliance on this proven and cost-effective caries preventive agent.7 since dental fluorosis in the united states and other nations without high levels of naturally-occurring fluoride is mild or very mild, with little impact on oral health related quality of life (ohrqol), dental professionals should emphasize on the mina pakkhesal et al. 259j contemp med sci | vol. 3, no. 11, summer 2017: 253–259 review methodological heterogeneity in dental fluorosis investigations appropriate use of fluoride for its caries prevention benefits as well as preventing moderate/severe fluorosis.45 one review study in identifying the risk factors for dental fluorosis has reported that, the reasons for increase in the prevalence of dental fluorosis as reported by different investigations are due to employing various study designs, using different sample populations, many exposed to multiple sources of fluoride, and using different indices to measure fluorosis. as a result not only the conclusions of some of these studies are not similar, but in some cases is confusing and even contradictory.27 conclusion given the existing methodological heterogeneity, a consensus development is highly crucial for the standard diagnosis of fluorosis and improvement in homogeneity in future fluorosis investigations globally. conflicts of interest there are no conflicts of interest. n references 1. iheozor-ejiofor z, worthington hv, walsh t, o’malley l, clarkson je, macey r, et al. water fluoridation for the prevention of dental caries. cochrane database syst rev. 2015;(6):cd010856. 2. levy sm. an update on fluorides and fluorosis. j can dent assoc. 2003; 69:286–291. 3. petersen pe. world health organization global policy for improvement of oral health—world health assembly 2007. int dent j. 2008;58:115–121. 4. clark mb, slayton rl. section on oral health. fluoride use in caries prevention in the primary care setting. pediatrics. 2014;134:626–633. 5. bronckers al, lyaruu dm, denbesten pk. the impact of fluoride on ameloblasts and the mechanisms of enamel fluorosis. j dent res. 2009;88:877–893. 6. clarkson jj, mcloughlin j. role of fluoride in oral health promotion. int dent j. 2000;50:119–128. 7. billings rj, berkowitz rj, watson g. teeth. pediatrics. 2004;113:1120–1127. 8. fomon sj, ekstrand j, ziegler ee. fluoride intake and prevalence of dental fluorosis: trends in fluoride intake with special attention to infants. j public health dent. 2000;60:131–139. 9. warren jj, kanellis mj, levy sm. fluorosis of the primary dentition: what does it mean for permanent teeth?. j am dent assoc. 1999;130:347–356. 10. browne d, whelton h, o’mullane d. fluoride metabolism and fluorosis. j dent. 2005;33:177–186. 11. abanto alvarez j, rezende km, marocho sm, alves fb, celiberti p, ciamponi al. dental fluorosis: exposure, prevention and management. med oral patol oral cir bucal. 2009;14:103–107. 12. denbesten p, li w. chronic fluoride toxicity: dental fluorosis. monogr oral sci. 2011;22:81–96. 13. sampaio fc, levy sm. systemic fluoride. monogr oral sci. 2011;22:133–145. 14. newbrun e. what we know and do not know about fluoride. j public health dent. 2010;70:227–233. 15. harding ma, o’mullane dm. water fluoridation and oral health. acta med acad. 2013;42:131–139. 16. soto-rojas ae, ureña-cirett jl, martínez-mier ea. a review of the prevalence of dental fluorosis in mexico. rev panam salud publica. 2004;15:9–18. 17. warren jj, levy sm. current and future role of fluoride in nutrition. dent clin north am. 2003;47:225–243. 18. siew tan b, razak ia. fluoride exposure from ingested toothpaste in 4-5-year-old malaysian children. community dent oral epidemiol. 2005;33:317–325. 19. pendrys dg. risk of enamel fluorosis in nonfluoridated and optimally fluoridated populations: considerations for the dental professional. j am dent assoc. 2000;131:746–755. 20. hujoel pp, zina lg, moimaz sa, cunha-cruz j. infant formula and enamel fluorosis: a systematic review. j am dent assoc. 2009;140:841–854. 21. buzalaf ma, levy sm. fluoride intake of children: considerations for dental caries and dental fluorosis. monogr oral sci. 2011;22:1–19. 22. o’mullane dm, baez rj, jones s, lennon ma, petersen pe, rugg-gunn aj, et al. fluoride and oral health. community dent health. 2016;33:69–99. 23. oganessian e, lencová e, broukal z. is systemic fluoride supplementation for dental caries prevention in children still justifiable?. prague med rep. 2007;108:306–314. 24. ekambaram m, itthagarun a, king nm. ingestion of fluoride from dentifrices by young children and fluorosis of the teeth—a literature review. j clin pediatr dent. 2011;36:111–121. 25. wong mc, glenny am, tsang bw, lo ec, worthington hv, marinho vc. topical fluoride as a cause of dental fluorosis in children. cochrane database syst rev. 2010;(1):cd007693. 26. denbesten pk. biological mechanisms of dental fluorosis relevant to the use of fluoride supplements. community dent oral epidemiol. 1999;27:41–47. 27. mascarenhas ak. risk factors for dental fluorosis: a review of the recent literature. pediatr dent. 2000;22:269–277. 28. da cunha lf, tomita ne. dental fluorosis in brazil: a systematic review from 1993 to 2004. cad saude publica. 2006;22:1809–1816. 29. warren jj, levy sm. systemic fluoride. sources, amounts, and effects of ingestion. dent clin north am. 1999;43:695–711. 30) nona attaran, mohammad h khoshnevisan, zahra ghorbani, mina pakkhesal, danoosh dehghanian. dental caries predictors in countries with different human development index: a review of articles. j int oral health. 2016;8:182–190. 31. davies rm, ellwood rp, davies gm. the rational use of fluoride toothpaste. int j dent hyg. 2003;1:3–8. 32. ellwood rp, cury ja. how much toothpaste should a child under the age of 6 years use?. eur arch paediatr dent. 2009;10:168–174. 33. wong mc, clarkson j, glenny am, lo ec, marinho vc, tsang bw, et al. cochrane reviews on the benefits/risks of fluoride toothpastes. j dent res. 2011;90:573–579. 34. chen j, liu g, kang y, wu b, sun r, zhou c, et al. coal utilization in china: environmental impacts and human health. environ geochem health. 2014;36:735–753. 35. martins cc, chalub l, lima-arsati yb, pordeus ia, paiva sm. agreement in the diagnosis of dental fluorosis in central incisors performed by a standardized photographic method and clinical examination. cad saude publica. 2009;25:1017–1024. 36. sajjadi sh, khosravanifard b, moazzami f, rakhshan v, esmaeilpour m. effects of three types of digital camera sensors on dental specialists’ perception of smile esthetics: a preliminary double-blind clinical trial. j prosthodont. 2016;25:675–681. 37. whelton hp, ketley ce, mcsweeney f, o’mullane dm. a review of fluorosis in the european union: prevalence, risk factors and aesthetic issues. community dent oral epidemiol. 2004;32:9–18. 38. cruz-orcutt n, warren jj, broffitt b, levy sm, weber-gasparoni k. examiner reliability of fluorosis scoring: a comparison of photographic and clinical examination findings. j public health dent. 2012; 72:172–175. 39. soto-rojas ae, martínez-mier ea, ureña-cirett j, jackson rd, stookey gk. development of a standardisation device for photographic assessment of dental fluorosis in field studies. oral health prev dent. 2008;6:29–36. 40. mohd nor na, chestnuttl ig, chadwick bl. examiner reliability in fluorosis scoring: a comparison of photographic and clinical methods. community dent health. 2016;33:145–150. 41. sabokseir a, golkari a, sheiham a. distinguishing between enamel fluorosis and other enamel defects in permanent teeth of children. peer j. 2016;4:e1745. 42. watts a, addy m. tooth discolouration and staining: a review of the literature. br dent j. 2001;190:309–316. 43. do lg, spencer a. oral health-related quality of life of children by dental caries and fluorosis experience. j public health dent. 2007;67:132–139. 44. ismail ai, hasson h. fluoride supplements, dental caries and fluorosis: a systematic review. j am dent assoc. 2008;139:1457–1468. 45. chankanka o, levy sm, warren jj, chalmers jm. a literature review of aesthetic perceptions of dental fluorosis and relationships with psychosocial aspects/oral health-related quality of life. community dent oral epidemiol. 2010;38:97–109. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201702 74 j contemp med sci | vol. 4, no. 2, spring 2018: 74–77 research impact of transcutaneous electrical nerve stimulation (tens) on endometrial thickness in the healthy women, a step to improve the female fertility ihsan m ajeena,a nada al-haris,b huda n al-kefae,a shehab a goloom,a khalid al-abudic amiddle euphrate neuroscience center, kufa medical college, university of kufa, najaf, iraq. bradiological department, kufa medical college, university of kufa, najaf, iraq. cal-sader medical city, ministry of health, najaf, iraq. correspondence to ihsan m ajeena (email: ihsan.ajeena@uokufa.edu.iq). (submitted: 19 november 2017 – revised version received: 11 january 2018 – accepted:17 february 2018– published online: 26 june 2018) objective to determine the physiotherapeutic effect of transcutaneous electrical nerve stimulation (tens) on endometrial lining thickness in healthy women at their childbearing age. methods this is a prospective randomized controlled clinical trial including 46 healthy women within their childbearing age during the first year of their marriage. uterine blood flow and endometrial thickness (et) are measured at day 13 of mc before tens application and at the same day of the next cycle after six sessions of tens were applied. results tens significantly increases endometrial thickness from 5.80 ± 0.24 to 7.60 ± 1.57 mm. this has a beneficial effect to increase the rate of pregnancy as it properly prepares of the uterine lining for implantation of fertilizing ova mediated by the reduction of both resistance and pulsatility indices of uterine arteries. these findings were the regardless age and bmi of the participants. conclusion there are beneficial effects of tens to increase endometrial thickness in healthy women at their childbearing age. keywords transcutaneous electrical nerve stimulation, female fertility, endometrial thickness, pulsatility index, resistance index. introduction the technique of transcutaneous electrical nerve stimulation (tens) is easy, safe and noninvasive.1 at the same time, it is inexpensive, its application has no side effects as it is free of any pharmacological element and do not interfere with consciousness.2 it emits mild electrical impulses that might be felt as a tingling sensation at the site. as it is widely applied in different therapeutic modality used in clinical practice, parameters such as intensity, rate, and duration of these electrical impulses can be adjusted according to the required purpose.3 one of the main uses for tens is those related to analgesic techniques where electrical pulses emitted primarily aim to provide a degree of symptomatic pain relief by stimulating sensory nerves and thereby exciting either the pain gate mechanism and/or endogenous opioid system.4 in addition to its analgesic effects, tens can modify the skin temperature and enhance blood flow, this observation led many studies to the impact of tens on the peripheral vasculature and reproductive system.5,6 the human endometrium is a plastic tissue with a lot of blood vessels as spiral arteries, which are terminal branches of uterine arteries.7 it is known that normal function of the reproductive system mostly depends on the organization of the blood flow in the endometrium and on its proper thickness.8,9 normally, these blood flow patterns changes during the menstrual cycle (mc), as new blood vessels are formed in the endometrium.10 these changes in the vasculature pattern and in the endometrial thickness are variable as the endometrium is thin immediately after menstruation and gradually grows thicker then after.11 materials and methods this study was conducted at the college of the medicine\ university of kufa and licensed private clinics as a prospective randomized controlled clinical trial including 46 healthy women within their childbearing age. it lasted for 14 months from april 2016 to may 2017. the study was approved by the ethical committee at the college and the participants were informed about the aim of the study and briefed about the techniques used, and their verbal approvals were collected. the device used for tens consists of a battery-powered simple stimulator along with one or two pairs of adhesive electrodes and their connecting leads. the parameters of the stimulation used in this study were 2 hz for the frequency, 10–15 ma for the intensity and 0.6 ms for the pulse width.12 the role that was strictly followed throughout the study is not to increase the intensity to a level that hurts (always stay under the point of discomfort).5,10 all participants have undergone for six sessions of tens, each lasted for 30 min. the applications were done on a day of 2, 4, 6, 8 12 and 13 of the mc. the electrodes of tens were applied to specific points over the skin of participants after being properly prepared.13,14 these points were selected according to dermatosomatic segments of sympathetic outflow that innervate the uterus (t12–l2 and s2–s3). 15,16 these points are five; three of them on both sides of body (sanyinjiao sp-6, zusanli st-36, and guilai st-29) and two of them on midline of the body (zhoncji ren-3 and guanyuan ren4) (figure 1). endometrial thickness (et) measurement was done using transabdominal ultrasonography that measures the thickest echogenic area from one basal endometrial border across the endometrial canal to the other basal surface.17 on the other hand, the pulsed doppler curves measures both pulsatility index (pi) and resistance index (ri) of uterine arteries on right and left sides. the pi value was calculated according to formula; pi = (a − b)/mean, and resistance index formula; ri = (a − b/a), where a is the peak systolic doppler shift, b is the end diastolic shift frequency and mean is the maximum doppler shifted frequency over the cardiac cycle.18 these measurements (et, pi and ri) were done at day 13 of the mc before the use of tens technique (pre-teens) and at the same issn 2413-0516 ihsan m ajeena et al. 75j contemp med sci | vol. 4, no. 2, spring 2018: 74–77 research impact of transcutaneous electrical nerve stimulation ( tens) on endometrial thickness day of the next mc after the application of the six sessions of tens. in general, the normal et that is important to prepare the uterus for a pregnancy state ranges from more than 6 to less than or equal to 10 mm. additionally, the participants were subdivided into two groups according to their age and into three subgroups according to their body mass index (bmi).19 the correlation of these two variables were considered for the different parameters tested. results the results of this study showed the range of age was between 15 and 42 years, bmi was 16.9–40.16, hemoglobin was 11–14 and rbs was 90–158 (table 1). tens application shows the significant increase of et and the significant decrease of both ri and pi of uterine arteries (table 2). participants with age less than 25 years have none significant increase in their et, ri, and pi (table 3). the impact of the bmi of the participants on the effect of tens application is not significant on et, ri, and pi of both left and right side uterine arteries (table 4). there is no significant correlation between changes in the et and the change in ri and pi of both uterine arteries in the post-tens application that measured by pearson correlation (table 5). discussion previous studies have been focused on using tens for pain relief mechanism especially postoperatively or during labor.1,2 however, in the last few years, some studies have been performed to test the effect of tens in reproductive field. among these are studies related to the uterine blood flow and endometrial thickness and subsequent pregnancy rate in infertile women.3,5 hence, the idea of this study is to study the effect of tens on healthy women within childbearing age immediately after the first year of their marriage, before being infertile. infertility is defined as the inability of couples to conceive after the end of the first year of their marriage.20 it focused on using tens to prepare the uterus for implantation of fertilized ova by its effect on increase growing and thickness of endometrium due to increasing uterine blood flow.3,6,21 the et significantly increases along with a significant decrease in ri and pi of the uterine arteries in the post-tens fig 1. sites of the five tens points: sp-6 (sanyinjiao), st-36 (zusanli), st-29 (guilai), ren -3 (zhoncji) and ren -4 (guanyuan). table 1. the characteristic of participants subjects’ characteristics (n = 46) mean ± sd range age (years) 22.63 ± 6.41 15–42 body mass index (kg/m2) 26.87 ± 4.95 16.9–40.16 hemoglobin (g/dl) 12.56 ± 1.08 11–14 random blood sugar (mg/dl) 103.23 ± 15.86 90–158 table 2. effect of tens on endometrial thickness and resistance and pusatility indices for uterine arteries on both sides variables pre-tens (n = 46) post-tens (n = 46) p-value et (mm) 5.80 ± 0.24 7.60 ± 1.57 <0.05 ri of uterine arteries left 0.91 ± 0.04 0.80 ± 0.01 <0.05 right 0.88 ± 0.02 0.80 ± 0.01 <0.05 pi of uterine arteries left 3.27 ± 0.24 2.33 ± 0.07 <0.05 right 3.12 ± 1.52 2.40 ± 0.59 <0.05 et, endometrial thickness; ri, resistance index; pi, pulsatility index; tens, transcutaneous nerve stimulation. table 3. the effect of age on post-tens measurements of endometrial thickness and resistance and pusatility indices for uterine arteries on both sides variables age (years) p-value <25 (n = 32) ≥25 (n = 14) et (mm) 7.62 ± 1.56 7.57 ± 1.65 >0.05 ri of uterine arteries left 2.38 ± 0.57 2.24 ± 0.39 >0.05 right 2.40 ± 0.60 2.39 ± 0.57 >0.05 pi of uterine arteries left 0.80 ± 0.05 0.78 ± 0.05 >0.05 right 0.80 ± 0.05 0.79 ± 0.03 >0.05 et, endometrial thickness; ri, resistance index; pi, pulsatility index; tens, transcutaneous nerve stimulation. table 4. the effect of body mass index on post-tens measurements of endometrial thickness and resistance and pusatility indices for uterine arteries on both sides variables body mass index (kg/m2) p valuenormal (n = 17) overweight (n = 17) obese (n = 12) et (mm) 7.70 ± 1.86 7.00 ± 1.22 8.33 ± 1.30 >0.05 ri of uterine arteries left 0.82 ± 0.06 0.78 ± 0.05 0.78 ± 0.04 >0.05 right 0.80 ± 0.05 0.78 ± 0.04 0.80 ± 0.04 >0.05 pi of uterine arteries left 2.49 ± 0.62 2.27 ± 0.49 2.17 ± 0.29 >0.05 right 2.48 ± 0.75 2.42 ± 0.59 2.25 ± 0.21 >0.05 et, endometrial thickness; ri, resistance index; pi, pulsatility index; tens, transcutaneous nerve stimulation. application. the mechanism underlying these effects is through their action on increasing production and secretion of endogenous opioid peptides, particularly b-endorphin in the central nervous system (cns), that has inhibitory signal on vasomotor center. this will inhibit uterine sympathetic 76 j contemp med sci | vol. 4, no. 2, spring 2018: 74–77 impact of transcutaneous electrical nerve stimulation (tens) on endometrial thickness research ihsan m ajeena et al. tone as a result of inhibiting excessive α motor neuron activity and stimulating the ia afferent nerve of the muscle that is the antagonist to the spastic muscle, thereby decreasing blood flow impedance by reducing uterine arteries spasticity. this inhibition is accomplished by peripheral stimulating of large diameter afferent fibers (aβ) fibers which activate local inhibitory mechanisms in the dorsal horn of the spinal cord that lead to the presynaptic inhibition of nociceptive afferent fibers (aδ) and c fibers, all these will result in increasing the uterine blood flow and subsequently, improve growth and thickness of the endometrium.22 these results are agreed with previous studies.3,23 this study showed that uterine arteries on both sides are affected to the same extent by tens application. this is because both have the same origin from the internal iliac artery.24 subsequently, a decrease in the ri and pi will increase uterine blood flow leading to optimal endometrial circulation which in turn will improve the growth and thickness of the endometrium.12,23,25 this will increase the rate of conception by increasing chances of the fertilized ova to be implanted in the uterus.26 this in consistence with other studies like that of randolph et al.14,27 concluded a positive correlation between high endometrial thickness >8 mm and a higher rate of conception. some researches stated that endometrial thickness of >7 mm was a predictive factor for pregnancy while thickness of <6 mm is associated with no pregnancy.28,29 some studies stated that the ideal et for conception is 5–8 mm, while isaksson et al.30 identified the ideal range of et is 9–11 mm. however, the majority of researches suggested the correlation of specific values of et during ovulation and the likelihood of pregnancy. at the same time, our results were consistent with that of many other studies in that a reduced ri and pi of uterine arteries after tens will increase uterine receptivity and lining thickness of endometrium.13,24,31 on contrary, tsai et al.32 found that endometrial thickness does not have useful prediction value and kolibiianahis33 showed that endometrial thickness cannot predict ongoing pregnancy. going along with these findings were that of ng et al.34 reported no relationship between endometrial thickness, morphology and pregnancy outcomes. furthermore, the findings of schild et al.35 disagreed with ours as they stated no association between uterine arterial blood flow and endometrial thickness. the results of this study showed no significant impact of the participants’ age on tens effect for all the et, ri and pi. these could be explained if we know that the uterus is affected less than ovaries with increasing age of women. so endometrial aging has a negative effect on changing et. this is consistent with the findings of previous studies that endometrial aging was negatively associated with pregnancy outcome, while et was positively associated with pregnancy outcome.36–38 this study showed that the change in bmi has no significant role in modulating the effect of tens application on et, ri, and pi. this seems logical as the effect of tens is mediated by the cns mechanisms mainly that mediated by endorphin (nevertheless, the variety of responses such as neurological, histochemical and neuropharmacological).39 what further explains these findings is fact that the endometrium represents the vascular mucosal lining of the uterus. that is why the effect of obesity on the endometrium had received less attention.40 what further minimizes the effect of bmi in this study is that the participants preand post-tens application was the same. many other studies had concluded same results regarding the impact of the bmi.41,42 the results of this study confirmed the r-pearson correlation between changes that occur in endometrial thickness after tens application and pi, ri of both sides uterine arteries with no significant correlation at p > 0.05, this result does not mean that there was no relationship between two variables but the relationship does not reach significantly effective. therefore, this result passed with the suggestion that increasing uterine arteries blood flow cause increasing the endometrial growth and thickness, not vice versa, that means the uterine blood flow increasing are the causes and increase endometrial thickness, however, this results of increasing endometrial thickness in agreeing with the previous study.43 limitation of the study we hoped to use eight points of stimulation instead of five, but the two-channel tens device can only provide four electrodes. considering that three of the five stimulation points were bilaterally located, two tens units had been used simultaneously in this study. here, another limitation is issued regarding the output pulses. although the two units were started at the same time, their outputs signals cannot be guaranteed to be absolutely synchronous, to guarantee the uniformity of stimulation. in future studies, this could be managed using a tens device with four channels instead of two which was not available at the time of his research. conclusion this provides evidence-based findings for the use of tens in assisted reproductive technique (art), program to decrease time, cost and efforts. furthermore, tens application could be used to adjunct known medication like clomiphene and hcg administration that induced ovulation to decrease their side effects and to increase the chance of pregnancy. tens technique is easy to perform, safe with low cost. it has the ability to prepare the uterus for pregnancy by increasing both the uterine blood flow and the endometrial thickness. hence, it has a positive influence to increase the endometrial receptivity for implantation of fertilizing ova. conflict of interest the authors declare that there is no conflict of interests for this article.  table 5. post-tens pearson correlation between endometrial thickness and resistance and pusatility indices for uterine arteries on both sides parameter post-tens r (pearson correlation) p-value ri of uterine arteries left −0.116 >0.05 right −0.129 >0.05 pi of uterine arteries left −0.224 >0.05 right −0.223 >0.05 tens, transcutaneous nerve stimulation; ri, resistance index; pi, pulsatility index, r = pearson correlation. ihsan m ajeena et al. 77j contemp med sci | vol. 4, no. 2, spring 2018: 74–77 research impact of transcutaneous electrical nerve stimulation ( tens) on endometrial thickness references 1. jarzem pf, harvey ej, arcaro n, kaczorowski j. transcutaneous electrical nerve stimulation [tens] for chronic low back pain. j musculoskelet pain. 2005;13:3–9. 2. desantana jm, sluka ka, lauretti gr. high and low frequency tens reduce postoperative pain intensity after laparoscopic tubal ligation:a randomized controlled trial. clin j pain. 2009;25:12–19. 3. zheng ch, zhang j, wu j, zhang mm. the effect of transcutaneous electrical acupoint stimulation on pregnancy rates in women undergoing in vitro fertilization: a study protocol for a randomized controlled trial. trials. 2014;15:162. 4. khadilkar a, milne s, brosseau l, robinson v, saginur m, shea b, et al. transcutaneous electrical nerve stimulation (tens) for chronic low-back. cochrane database syst rev. 2006;4:cd003008. 5. madaschi c, braga dp, figueira rc, iaconelli a jr. effect of acupuncture on assisted reproduction treatment outcomes. acupunct med. 2010;28:180–184. 6. salsabili n, ansari nn, berjis k, sedighi a, salsabili h. effect of physiotherapeutic tens in a woman with un explained infertility. physiother theory pract. 2011;27:155–159. 7. ng eh, yeung ws, ho pc. endometrial and subendometrial vascularity are significantly lower in patients with endometrial volume 2.5 ml or less. reprod biomed online. 2009;18:262–268. 8. gomez o, figueras f, martinez jm, del río m, palacio m, eixarch e, et al. sequential changes in uterine artery blood flow pattern between the first and second trimesters of gestation in relation to pregnancy outcome. ultrasound obstet gynecol. 2006;28:802–808. 9. world health organization/department of reproductive health and research (who/rhr 2008 and johns hopkins bloomberg school of public health/center for communications programs (ccp), knowledge for health project. family planning: a global handbook for providers. baltimore and geneva: ccp and who/rhr. 10. dominguez f, remohi j, pellecer, simon c. human endometrial receptivity: a genomic approach. reprod biomed online. 2003;6:332–338. 11. raine-fenning nj, campbell bk, kendall nr, clewes js, johnson ir. quantifying the changes in endometrial vascularity throughout the normal menstrual cycle with three-dimensional power doppler angiography. hum reprod. 2004;19:330–338. 12. zheng ch, zhang mm, huang gy, wang w. the role of acupuncture in assisted reproductive technology. evid based complement alternat med. 2012;2012:15. 13. stener-victorin e, fujisawa s, kurosawa m. ovarian blood flow responses to electroacupuncture stimulation depend on estrous cycle and on site and frequency of stimulation in anesthetized rats. j appl physiol. 2006;101:84–91. 14. ho m, huang lc, chang yy, chen hy. electroacupuncture reduces uterine artery blood flow impedance in infertile women. taiwan j obstet gynecol. 2009;48:148–151. 15. sharma kn. atlas of acupuncture points. usa, 2012. isbn:13 9781480205420. 16. stener-victorin e, wu x. effects and mechanisms of acupuncture in the reproductive system. auton neurosci. 2010;157:46–51. 17. fong k, kung r, lytwyn a. endometrial evaluation with transvaginal us and hysterosonography in asymptomatic postmenopausal women with breast cancer receiving tamoxifen. radiology. 2001;220:765–773. 18. jayaprakasan k, campbell bk, clewes js, johnson ir, rainefenning nj. three-dimensional ultrasound improves the interobserver reliability of antral follicle counts and facilitates increased clinical work flow. ultrasound obstet gynecol. 2008;31:439–444. 19. world health organization expert consultation. appropriate body mass index for asian population and its implication for policy and intervention strategies. lancet. 2004;363:157–163. 20. sutter pd. rational diagnosis and treatment in infertility. best pract res clin obstet gynaecol. 2006;20:647–664. 21. engmann l, diluigi a, schmidt d, nulsen j. the use of gonadotropinreleasing hormone (gnrh) agonist to induce oocyte maturation after co treatment with gnrh antagonist in high-risk patients undergoing in vitro fertilization. fertile steril. 2008;89:84–91. 22. salsabili n, salsabili h, berjis k, akbariasbagh f. the effect of transcutaneous electrical nerve stimulation (tens) on the pregnancy rate in women undergoing assisted reproduction techniques and embryo transfer. gynecol obstet. 2011;s5:001. 23. zheng ch, huang gy, zhang mm. effects of acupuncture on pregnancy rates in women undergoing in vitro fertilization: asystemstic review and meta-analysis. fertil steril. 2012;97:599–611. 24. gong x, li q, zhang q, zhu g. predicting endometrium receptivity with parameters of spiral artery blood flow. j huazhong univ sci technol med sci. 2005;25:335–338. 25. mercé lt, barco mj, bau s, troyano j. are endometrial parameters by threedimensional ultrasound and power doppler angiography related to in-vitro fertilization/embryo transfer outcome? fertil steril. 2008;89:111–117. 26. chesterton ls. effects of tens frequency, intensity and stimulation site parameter manipulation on pressure pain thresholds in healthy human subjects. pain. 2013;106:73–80. 27. randolph jf, sowers jrm. reproductive hormones in the early menopausal transition: relationship to ethnicity, body size, and menopausal status. j clin endocrinol metab. 2003;88:1516–1522. 28. merviel p, heraud mh, grenier n, lourdel e, sanguinet p, copin h. predictive factors for pregnancy after iui: an analysis of 1038 cycles and a review of the literature. fertil steril. 2010;93:79–88. 29. meduri g, bausero p, perrot-applanat m. expression of vascular endothelial growth factor receptors in the human endometrium: modulation during the menstrual cycle. biol reprod. 2000;62:439–447. 30. isaksson r, tiitinen a, reinikainen lm, cacciatore b. comparison of uterine and spiral artery blood flow in women with unexplained and tubal infertility. ultrasound obstet gynecol. 2003;21:174–180. 31. franconi g, manni l, aloe l, mazzilli f, giambalvo dal ben g, lenzi a, et al. acupuncture in clinical and experimental reproductive medicine. a review. j endocrinol invest. 2011;34:307–311. 32. tsai hd, change cc, hsieh yy, lee cc, lo hy. artificial insemination: role of et and pattern, of vascular impedance of the spiral and uterine arteries, and of the dominant follicle. j reprod med. 2000;45:195–200. 33. kolibiianahis em, zikopoulos ka, fatemi hm, osmanagaoglu k, evenpoel j, van steirteghem a, et al. endometrial thickness cannot predict ongoing pregnancy achievement in cycles stimulated with clomiphene citrate for iui. reprod biomed online. 2004;8:115–118. 34. ng eh, chan cc, tang os, yeung ws, ho pc. the role of endometrial and subendometrial blood flows measured by three-dimensional power doppler ultrasound in the prediction of pregnancy during ivf treatment. human reprod. 2006;21:164–170. 35. schild rl, knobloch c, dorn c, fimmers r, van der ven h, hansmann m. endometrial receptivity in-vitro fertilization program assessed by spiral artery blood flow, endometrial thickness, endometrial volume and uterine artery blood flow. fertil steril. 2001;7:361–366. 36. smith-bindman r, weiss e, feldstein v. how thick is too thick? when endometrial thickness should prompt biopsy in postmenopausal women without vaginal bleeding. ultrasound obstet gynecol. 2004;24:558–565. 37. amir w, micha b, ariel h, liat l-g, jehoshua d, adrian s. predicting factors for et during treatment with art. fertil steril. 2007;87:799–804. 38. esmailzadeh s, faramarzi m. endometrial thickness and pregnancy outcome after iui. fertil steril. 2007;88:432–437. 39. gesink law dc, maclehose rf, longnecker mp. obesity and time to pregnancy. hum reprod. 2006;22:414–420. 40. dechaud h, bessueille e, bousquet pj, reyftmann l, hamamah s, hedon b. optimal timing of ultrasonographic and doppler evaluation of uterine receptivity to implantation. reprod biomed online. 2008;16:368–375. 41. chui s, chow fc, szeto yt, chan k, lam cw. a case series on acupuncture treatment for female infertility with some cases supplemented with chinese medicines. eur j integr med. 2014;6:337–341. 42. wang w, check jh, liss jr, choe jk. a matched, controlled study to evaluate the efficacy of acupuncture for improving pregnancy rates following in vitro fertilization-embryo transfer. clin exp obstet gynecol. 2007;34:137–138. 43. ng eh, chan cc, tang os, yeung ws, ho pc. the role of endometria blood flow measured by three-dimensional power doppler ultrasound in the prediction of pregnancy during in vitro fertilization treatment. eur j obstet gynecol reprod biol. 2007;135:8–16. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms. 06201804 30 j contemp med sci | vol. 4, no. 1, winter 2018: 30–32 research evaluation of impact teeth prevalence and related pathologic lesions in patients in northern part of iran (2014-2016) amir hosein pakravan,a mohammad mehdi nabizadeh,b shima nafarzadeh,c* sina jafari,d atena shivae tahmineh bamdadianf adepartment of oral and maxillofacial surgery, dental school, sari university of medical sciences, sari, iran. b dental school, sari university of medical sciences, sari, iran. ccellular and molecular research center, babol university of medical sciences, babol, iran. ddepartment of prosthodontics, dental school, university of shahed, tehran, iran. edepartment of oral and maxillofacial pathology, dental school, sari university of medical sciences, sari, iran. fdental school, dental school, sari university of medical sciences, sari, iran. correspondence to shima nafarzadeh (email: shima.nafar2004@yahoo.com). (submitted: 05 october 2017 – revised version received: 07 november 2017 – accepted: 20 november 2017 – published online: 26 march 2018) objective in this research, our purpose was to evaluate how prevalent impacted tooth is. we also evaluated the type of third molar impaction and pathologic lesions related to impacted teeth in patients reffered to sari and babol dental school in 2014-2016. methods the study was cross-sectional, and was carried out on 2109 panoramic radiographs of patients with age over 20 years referring to sari and babol dental faculties during 2014-2016. results among the patients, 392 (18.5%) presented with at least one impacted tooth. 243 women (20.6%) and 149 men (16%) had impacted teeth. conclusion the most common encountered impact tooth was found to be the third molar of mandible and dentigerous cyst was the most detected lesion associated with tooth impaction. keywords impact teeth, lesions, prevalence lesions introduction impaction is defined as a pathological condition in which the tooth by cannot erupt into the oral cavity in the appropriate time and considering physiological limits as a normal eruption process.1 impaction prevalence is affected by factors such as age, dental eruption timing, genetic and environmental factors. local mechanical factors involving during the time of tooth development and eruption are critical.2–5 many patients do not know about their impacted tooth and it is discovered during routine examinations. therefore, it is very important to inform dental practitioners about this frequently occurring phenomenon in everyday clinical practice and to emphasize the importance of early detection and intervention to prevent possible harmful consequences6. several radiographs are available for examination of impacted teeth. panoramic radiographs, are often the first prescribed radiographs because they can provide information about all the teeth in upper and lower jaw and the surrounding structures. the panoramic radiograph is used as the basic method in epidemic research due to its economic and practical characteristics.4,7 however, in many cases, a diagnosis based on 2d radiography is not sufficient, because it is very difficult to assess the buccolingual aspects of the relation between the canine crown and the roots of the incisors. the usually impacted permanent teeth are the third molars, maxillary canines or central incisors, and mandibular second premolars.8,9 tooth impaction ranges from 0.8– 3.6% of the general population.6,10 some authors reported that third molar impaction rate is 16.7% to 68.6%.11,12 carter et al. in a meta-analysis study found 24.4% worldwide third molar tooth impaction and they showed that impaction of third molars in mandible was greater than maxilla, but its prevalence was not significantly different between men or women.5 extraction of impacted third molars is controversial in dentistry.13 problems seen associated to the tooth impaction varies from simple to complicated life threatening problems. such as caries, pulp disease, periapical and periodontal disease, temporomandibular joint disorder, infection of the facial area, resorption of rooh and the adjacent tooth, and even oral and head and neck tumors. hyperplastic dental follicule, dentigerous cyst or odontogenic keratocyst are among the most common simple problems observed in tooth impaction.11,13-18 for this reason, prophylactic extraction of third molar teeth is prescribed for future disease prevention. however, there is limited evidence about risk of caries and periodontitis in a second molar in adjacent place to a retained third molar.19 most studies found that pericoronal radiolucency greater than 2.5 mm around the crown of impacted teeth is suggestive of a pathologic lesion 20–22 in this study we evaluated how prevalent impacted teeth are, the type of impaction of third molars and pathologic lesions related to impacted teeth in patients reffered to sari and babol dental school during 2014-2016. materials and methods this study was a cross-sectional research and was performed on 2109 panoramic radiographs of patients with age over 20 years referring to sari and babol dental faculties during 2014-2016. all radiographs that were of good quality were selected regardless of the reason for prescribing, and in terms of the presence of an impact tooth , its radiographic features were reviewed by an expert oral and maxillofacial surgeon. all panoramic radiographs were taken with the soredex in both colleges. the classification designed by pell and gregory’s was used in order to compare the issn 2413-0516 amir hosein pakravan et al. 31j contemp med sci | vol. 4, no. 1, winter 2018: 30–32 research prevalence of impact teeth in iran position of mandibular third molar with the anterior edge of the mandibular ramus:23 class 1when the total mesiodistal diameter of the crown is in front of the anterior border of the mandibular ramus; class 2when nearly half of the tooth is is covered by the ramus; class 3when the tooth is entirely placed in the mandibular ramus. also comparison between the third molar place to the occlusal plane of the 2nd molar is categorized as below:23 level awhen the level of occlusal surface of the third molar is the same or higher than the 2nd molar; level bwhen the level of occlusal surface of the third molar is between the occlusal and the cervical level of 2nd molar; and level cwhen the level of occlusal surface of the third molar is lower than the cervical line of the 2nd molar. in the second part of the study, all histopathological reports of impacted teeth were evaluated. patient information, radiographic findings and histopathological findings were recorded in a form. data were analyzed using the spss16 software. the p-value less than 5% was considered statistically significant. results the panoramic radiographs of 2109 patients aged 20–68 years (934 men and 1175 women) were examined. among the patients, 392 (18.5%) presented with at least one impacted tooth. about 243 women (20.6%) and 149 men (16%) had impacted teeth. the most frequent position for third molar was mesioangular position and the lowest prevalence was for horizontal (table 1). the highest frequency was found in position a and the least frequent was in position c (table 2). in addition, the highest and lowest percentages were for class 1 and 3, respectively (table 3). in the second part of the study, 206 samples (107 women and 99 men) of the lesions associated with impacted teeth were studied. the most lesions associated with impacted tooth was dentigerous cyst, hyperplastic follicle and okc, respectively (table 5). discussion the impacted teeth are one of the most important and challenging issues in dentistry. the impacted teeth can be accompanied by several complications that some have a life-threatening risk.11,13,18 various factors including the high density of the bone on the tooth, the prevention of growth by the adjacent tooth, tooth angle, the thickness of the mucosa covering the tooth or the genetic factors lead to latency.11 in our study, 2109 radiographs were surveyed in both centers, including 1175 women and 934 men. also on the histopathologic evaluation of the cases, 206 patients with impacted teeth found to have lesions. in this research, the impacted teeth prevalence was 18.5% and was higher in women than men. saglam et al. reported impacted teeth prevalence to be 11% in turkey.24 in addition, nagahara et al in japan reported a prevalence of 4.9% among 3979 patients.25 aitasalo et al. did a study in japan in 1972. they examined 4063 panoramic radiographs and found 14.1% of patients with impacted teeth. the third molar was the most common impacted tooth in both jaws.26 another similar study by chu et al. was done among 7486 hong kong patients. they found that prevalence of impacted tooth was 28.3%. the difference in table 1. the angle of third molar placement relative to the longitudinal axis of second molar teeth orientation frequency percentile (%) mesioangular 81 45 distoangular 24 33.3 vertical 60 13.3 horizontal 15 8.4 table 2. distribution of third molars based on pell and gregory’s classification level frequency percentile (%) a 109 61 b 61 33.9 c 10 5.1 table 3. position of the mandibular wisdom teeth relative to the anterior edge of ramus classification frequency percentile (%) 1 107 59.4 2 49 27.2 3 24 13.4 table 4. frequency of distribution of impacted teeth in both jaws tooth frequency percentile (%) third mandibular molar 180 45/9 third maxillary molar 123 31.4 maxillary canine 59 15 second mandibular premolar 19 4.9 mandibular canine 5 1.3 second mandibular molar 4 1 second maxillary premolar 2 0.5 table 5. type and frequency of dental lesions of patients participating in the study lesions frequency percentile (%) dentigerous cyst 68 33 hyperplastic follicle 51 24.7 okc 49 23.7 odontoma 11 5.4 ameloblastoma 11 5.4 ossifying fibroma 8 3.9 complex odontoma 5 2.4 fibroodontoma 3 1.5 the dimensions of jaws between women and men was one of the reasons for more prevalent tooth impaction in women.10 in the recent study, mesioangular angulation was the most seen type of impacted mandibular third molar, which was followed by distoangular, vertical and horizontal angulations. eshghpour et al.,12 hashemipour et al.,11 quek et al.,27 moris and jerman,28 and hassan29 found out the most prevalent type of impaction was mesioangular impaction in the mandibular third molars in iranian, african american, singaporean, american, and arabian populations respectively. also in our study, level a was the most common impaction level. findings 32 j contemp med sci | vol. 4, no. 1, winter 2018: 30–32 prevalence of impact teeth in iran research amir hosein pakravan et al. of monaco et al.,30 obiechina et al.,31 hugoson and kugelberg,32 and hashemipour et al. was in agreement with our research result.11 in our study, the classification had been done according to the relationship of occlusal surfaces of the third molar and the adjacent second molar. however, in some studies, the impaction level were assessed based on the position of cej to the alveolar bone leve.12 in present study, the most common impaction depth was class one. in agreement with this finding, haghanifar et al 33 founded that level a and class 2 were the most common type of impaction, but obiechina et al.,31 and eshghpour et al.12 founded that class 2 pell and gregory’s classification was the most prevalent type of impaction. differences may be because of the classification or age of patients. in the present study all patients were older than 20 years. hashemipour et al.11 found that the third molar impaction prevalence was 44.3% in the southeast region of iran. moreover, a lower prevalence has been seen in some studies, such as research from eliasson et al.34 30.3%, hattab et al.35 33%, and hassan29 40.8%. however, quek et al. 68.6% showed a higher prevalence for impaction in a study done in singapore.27 acknowledgment the research was a result of a dental student thesis and was supported financially by sari university of medical sciences. conflicts of interest the authors disclose no conflicts of interest. n references 1. sajnani ak, king nm. prevalence and characteristics of impacted maxillary canines in southern chinese children and adolescents. j investig clin dent. 2014;5:38–44. 2. bishara se. impacted maxillary canines: a review. am j orthod dentofacial orthop. 1992;101:159–171. 3. yamaoka m, furusawa k, yamamoto m. influence of adjacent teeth on impacted third molars in the upper and lower jaws. aust dent j. 1995;40:233–235. 4. gisakis ig, palamidakis fd, farmakis et, kamberos g, kamberos s. prevalence of impacted teeth in a greek population. j invest clin dent. 2011;2:102–109. 5. carter k, worthington s. predictors of third molar impaction: a systematic review and meta-analysis. j dent res. 2016;95:267–276. 6. kaczor-urbanowicz k, zadurska m, czochrowska e. impacted teeth: an interdisciplinary perspective. adv clin exp med. 2016;25:575–585. 7. haney e, gansky sa, lee js, johnson e, maki k, miller aj, et al. comparative analysis of traditional radiographs and cone-beam computed tomography volumetric images in the diagnosis and treatment planning of maxillary impacted canines. am j orthod dentofacial orthop. 2010;137:590–597. 8. bedoya mm, park jh. a review of the diagnosis and management of impacted maxillary canines. j am dent assoc. 2009;140:1485–1493. 9. santosh p. impacted mandibular third molars: review of literature and a proposal of a combined clinical and radiological classification. ann med health sci res. 2015;5:229–234. 10. chu fc, li tk, lui vk, newsome pr, chow rl, cheung lk. prevalence of impacted teeth and associated pathologies—a radiographic study of the hong kong chinese population. hong kong med j. 2003;9:158–163. 11. hashemipour ma, tahmasbi-arashlow m, fahimi-hanzaei f. incidence of impacted mandibular and maxillary third molars: a radiographic study in a southeast iran population. med oral patol oral cir bucal. 2013;18:e140–e145. 12. eshghpour m, nezadi a, moradi a, shamsabadi rm, rezaei nm, nejat a. pattern of mandibular third molar impaction: a cross-sectional study in northeast of iran. niger j clin pract. 2014;17:673–677. 13. almendros-marques n, alaejos-algarra e, quinteros-borgarello m, berini-aytes l, gay-escoda c. factors influencing the prophylactic removal of asymptomatic impacted lower third molars. int j oral maxillofac surg. 2008;37:29–35. 14. planinic d, bodina i, peric b. prevalence of odontogenic keratocysts associated with impacted third molars. coll antropol. 2010;34:221–224. 15. vigneswaran at, shilpa s. the incidence of cysts and tumors associated with impacted third molars. j pharm bioallied sci. 2015;7:s251–s254. 16. gaddipati r, ramisetty s, vura n, kanduri rr, gunda vk. impacted mandibular third molars and their influence on mandibular angle and condyle fractures—a retrospective study. j craniomaxillofac surg. 2014;42:1102–1105. 17. ma’aita j, alwrikat a. is the mandibular third molar a risk factor for mandibular angle fracture? oral surg oral med oral pathol oral radiol endod. 2000;89:143–146. 18. mikic im, zore if, crcic vf, matijevic j, plancak d, katunaric m, et al. prevalence of third molars and pathological changes related to them in dental medicine. coll antropol. 2013;37:877–884. 19. nunn me, fish md, garcia ri, kaye ek, figueroa r, gohel a, et al. retained asymptomatic third molars and risk for second molar pathology. j dent res. 2013;92:1095–1099. 20. edamatsu m, kumamoto h, ooya k, echigo s. apoptosis-related factors in the epithelial components of dental follicles and dentigerous cysts associated with impacted third molars of the mandible. oral surg oral med oral pathol oral radiol endod. 2005;99:17–23. 21. rakprasitkul s. pathologic changes in the pericoronal tissues of unerupted third molars. quintessence int. 2001;32:633–638. 22. saravana gh, subhashraj k. cystic changes in dental follicle associated with radiographically normal impacted mandibular third molar. br j oral maxillofac surg. 2008;46:552–553. 23. pell gj, gregory bt. impacted mandibular third molars: classification and modified techniques for removal. dent digest. 1933;39:330–338. 24. saglam aa, tuzum ms. clinical and radiologic investigation of the incidence, complications, and suitable removal times for fully impacted teeth in the turkish population. quintessence int. 2003;34:53–59. 25. nagahara k, yuasa s, yamada a, ito k, watanabe o, iizuka t, et al. etiological study of relationship between impacted permanent teeth and malocclusion. aichi gakuin daigaku shigakkai shi. 1989;27:913–924. 26. aitasalo k, lehtinen r, oksala e. an orthopantomographic study of prevalence of impacted teeth. int j oral surg. 1972;1:117–120. 27. quek sl, tay ck, tay kh, toh sl, lim kc. pattern of third molar impaction in a singapore chinese population: a retrospective radiographic survey. int j oral maxillofac surg. 2003;32:548–552. 28. morris cr, jerman ac. panoramic radiographic survey: a study of embedded third molars. j oral surg. 1971;29:122–125. 29. hassan ah. pattern of third molar impaction in a saudi population. clin cosmet investig dent. 2010;2:109–113. 30. monaco g, montevecchi m, bonetti ga, gatto mr, checchi l. reliability of panoramic radiography in evaluating the topographic relationship between the mandibular canal and impacted third molars. j am dent assoc. 2004;135:312–318. 31. obiechina ae, arotiba jt, fasola ao. third molar impaction: evaluation of the symptoms and pattern of impaction of mandibular third molar teeth in nigerians. odontostomatol trop. 2001;24:22–25. 32. hugoson a, kugelberg cf. the prevalence of third molars in a swedish popu lation. an epidemiological study. community dent health. 1988;5:121–138. 33. haghanifar s, moudi e, seyedmajidi m, mehdizadeh m, nosrati k, abbaszadeh n, et al. can the follicle-crown ratio of the impacted third molars be a reliable indicator of pathologic problem?. j dent (shiraz). 2014;15:187–191. 34. eliasson s, heimdahl a, nordenram a. pathological changes related to longterm impaction of third molars. a radiographic study. int j oral maxillofac surg. 1989;18:210–212. 35. hattab fn, rawashdeh ma, fahmy ms. impaction status of third molars in jordanian students. oral surg oral med oral pathol oral radiol endod. 1995;79:24–29. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.03201807 189j contemp med sci | vol. 3, no. 9, winter 2017: 189–192 research role of apo e and superoxide dismutase in patients with obstructive lung diseases anwar j almzaiel,a ali mansoor jasim al-ameri,b rafad tariqc adepartment of biochemistry, college of medicine, university of alqadisiyah, iraq. bdepartment of microbiology and immunology college of medicine, university of kerbala,kerbala, iraq. bmaster biochemistry student, department of biochemistry, college of medicine, university of kerbala, kerbala, iraq. correspondence to: anwar.almzaiel@qu.edu.iq. (submitted: 09 december 2017 – revised version received: 11 january 2017 – accepted: 16 february 2017 – published online: 26 march 2017 ) objective obstructive lung diseases (old) are chronic inflammatory disorders of the respiratory tract including asthma and chronic obstructive pulmonary disease (copd). apo lipoprotein e (apo e), is a multifunctional protein as it intervenes the binding of lipoproteins or lipid complexes to specific cell-surface receptors. experimental studies referred to the function of apo e as an endogenous negative regulator of airway hyper responsiveness and goblet cell hyperplasia. the protective role of apo e pathways primarily in respiratory disease was explained in human studies and research utilizing experimental murine model systems. literature data reveal a strong association between redox status, including the enzyme superoxide dismutase (sod) with both the development a severity of old. this study aims to investigate the relation between sod antioxidant enzyme activity in addition to investigating the level of apo e and the development of obstructive lung diseases (old). methods patients with old (n = 40) and 40 age-matched healthy controls were enrolled in this study. serum samples were collected to test the role of apo e and to test the effect of antioxidant enzyme sod, and their influence on old, all measured by elisa. result the results showed a significant decrease in the level of serum sod activity in patients with old when compared with control group (p < 0.05). however, the levels of apo e did not show a significant difference between the two groups. conclusion decreased level of antioxidant sod suggests the presence of an oxidative stress in asthmatic airways favoring a more oxidative state is present in the airway inflammation. the level of apo e was non significantly increased in serum of patient, this suggests that protein level of apo e does not change but may be apo e gene expression is altered. keywords apo e, superoxide dismutase, obstructive lung diseases introduction chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (copd) are common problems and are regarded as the major public health burdens.1,2 they are characterized by reversible and irreversible airway obstruction, respectively. although remain the primary reason of these diseases unknown, progressive and permanent pulmonary tissue damage (airway remodeling) that leads to the total loss of lung function as a result of inflammation which is considered a central feature for them. in fact, the inflammatory mechanisms and other biological pathways involved in asthma and copd pathogenesis must be explained, in order to find new possible diagnostic/prognostic biomarkers and for the validation of new drug targets.3 there are significant differences in the patterns of fundamental inflammation, in spite of the similarity in clinical symptoms of both diseases, they are caused by airway narrowing as a result of inflammation.4 in copd, the inflammatory cell infiltrate in small airways include mainly neutrophils and cytotoxic t cells (cluster of differentiation-8 (cd8) positive lymphocytes). parenchymal destruction in copd is associated with loss of lung tissue elasticity, and small airways collapse during exhalation. asthma has been characterized mainly by type 2 helper t cell (th2) cytokine-mediated eosinophilic airway inflammation associated with airway hyperresponsiveness.2 recent studies suggest that 13%–20% of patients with copd have an overlap phenotype with asthma; this is as high as 50% in patients over 50 years, because of 30% of people with asthma smoke, a proportion of these will develop chronic airflow limitation, which is likely to be indistinguishable from copd.5–7 antioxidants are one of chemical substances can inhibit the oxidation of a molecule. in the living organisms, antioxidants can nullify the pathology effects of oxidation caused by free radicals.8 superoxide dismutase superoxide dismutase is considered one of oxidoreductases , which catalyze the dismutation of o2 •− into oxygen and h2o2, therefor it is classified as a major cellular defense against o2 •− and peroxynitrite. thus, cu or mn will be important modulator of sod activity of sod1/sod3 or sod2, respectively.9 apo lipoproteine e apo lipoproteine e (apo e) is a protein presents in the interstitial fluid and lymph, as well as in the plasma and is synthesized and secreted from a variety of tissues and different kinds of cells.10–12 it has three major isoforms (apo e2, apo e3, and apo e4) with different effects on lipid and neuronal homeostasis.13 intracellular apo e may regulate various cellular processes physiologically or pathophysiologically. apo lipoprotein (apo) e is a multifunctional protein as it intervenes the binding of lipoproteins or lipid complexes in the plasma or interstitial fluids to specific cell-surface receptors as a major function of apo e.13 issn 2413-0516 190 j contemp med sci | vol. 3, no. 9, winter 2017: 189–192 role of apo e and superoxide dismutase in patients with obstructive lung diseases research ali mansoor jasim al-ameri et al. many respiratory diseases, including asthma, acute lung injury, cancer, emphysema, pulmonary fibrosis, and pulmonary hypertension are associated with apo e which is expressed by lung cells, which allows apo e/ low density lipoprotein receptor (ldlr) dependent pathways to modulate normal lung health, as well as the pathogenesis of these diseases. the protective roles of apo e and apo lipoproteine a-i (apo a-i) pathways primarily in lung biology and respiratory disease were explained in human studies and research utilizing experimental murine model systems.14 materials and methods assay procedure serum levels of superoxide dismutase were determined by classic competitive-elisa using elisa minikits (elabscience, china). whereas serum levels of apo e were determined by classic sandwich-elisa using elisa minikits (elabscience, china) according to the instructions enclosed with the kits. calculation of results average the duplicate readings for each standard and sample. create a standard curve by plotting the mean od value for each standard on the y-axis against the concentration on the x-axis and draw a best-fit curve through the points on the graph. by using professional microsoft excel to do this calculation, a best fitting equation of standard curve will be calculated using od values and concentrations of standard sample. the software will calculate the concentration of samples after entering the od value of samples. results the antioxidant status which represented in the present study was reduced sod which is the master antioxidant in the body. this was achieved by evaluating the changes according to the activity of the disease. a brief illustration in (table 1) which summarize the overall findings regarding the studied parameters of the current study. superoxide dismutase the results in fig. 1 show a significant decrease (p < 0.05) in sod concentration in serum of patient with old group compared to the control group. the data show that the mean ± sd of sod in patients with old and control group were 445.702 ± 148.217 and 520.757 ± 179.902 pg/ml, respectively. apo lipoproteine e the results in fig. 2 showed non-significant change (p > 0.05) in serum apo e concentration in patients with old group compared to the control group. the data show that the mean ± sd of apo e in old and control group were 727.47 ± 189.31 and 697.51 ± 207.32 pg/ml, respectively. discussion the results of the present study revealed a significant decrease in sod levels in patients with old. an enzymatic antioxidant system is critical for the redox status homeostasis and for protecting human cells. in fact, when this homeostasis is disrupted and there are more reactive species than antioxidants several diseases can develop.15 recent studies indicate that an increased oxidative stress is related to the pathogenesis of copd,1,16–18 and contribute this to the fact that copd patients produce more h2o2 compared to “healthy”.19 another study hypothesized that loss of asthmatic sod activity is due to greater susceptibility to oxidative inactivation.20 an increase in ros generation in asthma and/or copd patients is confirmed by the changes in the antioxidant table 1. summarizes parameters studied represented by serum (sod and apo e) for control and patients with old parameters group mean ± sd p-value sod; pg/ml patient 445.702 ± 148.217 < 0.05 control 520.757 ± 179.902 apo e; ng/ml patient 727.47 ± 189.31 > 0.05 control 697.51 ± 207.32 sod, superoxide dismutase; apo e, apo lipoprotein e; old, obstructive lung disease; student’s t-test p-value of < 0.05 was considered to be statistically significant. fig. 1 serum superoxide dismutase (sod) activity in patients with obstructive lung disease (old) and control. serum samples were isolated from the blood of patients with old. sod level was assessed by elisa. data are expressed as means ± sd, for 40 patients, with duplicate measurements. *indicates significant differences compared to the control n = 40, (student’s t-test, p < 0.05). fig. 2 serum apo lipoprotein e (apo e) levels in patients with obstructive lung disease (old) and control. serum samples were isolated from the blood of patients with old. apo e level was assessed by elisa. data are expressed as means ± sd, for 40 patients, with duplicate measurements, compared to the control group n = 40, (student’s t-test, p > 0.05). ali mansoor jasim al-ameri et al. 191j contemp med sci | vol. 3, no. 9, winter 2017: 189–192 research role of apo e and superoxide dismutase in patients with obstructive lung diseases enzymes activity. these results support the hypothesis that an oxidant-antioxidant imbalance, associated with oxidative stress in copd patients, plays an important role in the progression of disease severity.21 previous studies also observed the same fact and concluded that oxidative stress remains increased among asthmatics with low levels of antioxidant activity.22 there is a strong evidence that an imbalance between the reducing and oxidizing systems favoring a more oxidative state is present in the airway inflammation and a deficiency in the amount of antioxidants exists in the asthmatic airway.23 superoxide dismutases (sods) are the major antioxidant defense systems against o2 radical. 24 vulnerability of cuznsod influenced by redox likely amplifies injury and inflammation during acute asthma attacks when reactive oxygen species are explosively generated. overall, this study identifies a new paradigm for understanding the chemical basis of inflammation.20 the difference in mean sod level was found to significantly decrease among asthmatic patients.25 antioxidants not only protect against the direct injurious effects of oxidants, but also alter the inflammatory events that play an important role in the pathogenesis of copd.26 the outcome of the present investigation confirms the role of an oxidant-antioxidant imbalance in the etiology of copd and/or asthma. the data from the current study have shown non-significant increase in the apo e levels in patients when compared with control group (fig. 2). this finding is consistent with the results of many previous studies as shown below. although the primary function of apo e is to facilitate lipid transport into cells by receptor-mediated endocytosis mediated by the ldl receptor, it is has been recognized that apo e modulates a variety of additional important biological functions. recent studies suggested that apo e may be involved in therapy strategy for old. experiments utilizing humanized apo e knock-in mice have demonstrated that human apo eε3 had a protective effect on the severity of hdm-induced airway disease.27 the ability of apo e, which is expressed by lung macrophages, to attenuate ahr, and goblet cell hyperplasia is mediated by low density lipoprotein (ldl)receptors expressed by airway epithelial cells. so, the administration of an apo e mimetic peptide, corresponding to amino acids 130–149 of the ldl receptor-binding domain of the holo-apo e protein, significantly reduced ahr and goblet cell hyperplasia in hdm-challenged apo e−/−mice. these findings identified the apo e-ldl receptor pathway as a new drug able target for asthma that can be activated by administration of apo e-mimetic peptides.28 recommendation further studies are to be conducted to predict the causal association of other markers such as lipid profile, apo a and non-enzymatic antioxidants with the development of old; asthma and copd. it is worthy to investigate apo e in cell culture and study the different effect of polymorphic apo e alleles by using gene expression. conflicts of interest none. n references 1. lundgren-nilsson å, jonsdottir ih, pallant j, ahlborg g. internal construct validity of the shirom-melamed burnout questionnaire (smbq). bmc public health. 2012;12:1. 2. abramson mj, perret jl, dharmage sc, mcdonald vm, mcdonald cf. distinguishing adult-onset asthma from copd: a review and a new approach. int j chron obstruct pulmon dis. 2014;9:945-962. 3. rossi r, de palma a, benazzi l, riccio am, canonica gw, mauri p. biomarker discovery in asthma and copd by proteomic approaches. proteomicsclinical applications. 2014;8:901–915. 4. barnes pj. immunology of asthma and chronic obstructive pulmonary disease. nat rev immunol, 2008a;8:183–192. 5. thomson nc, chaudhuri r, livingston e. asthma and cigarette smoking. eur respir j. 2004;24:822–833. 6. broekema m, timens w, vonk jm, volbeda f, lodewijk me, hylkema mn, et al. persisting remodeling and less airway wall eosinophil activation in complete remission of asthma. am journal of respiratory and critical care medicine. 2011;183:310–316. 7. piras b, miravitlles m. the overlap phenotype: the (missing) link between asthma and copd. multidisciplinary respiratory medicine. 2012;7:1. 8. singh pp, chandra a, mahdi f, roy a, sharma p. reconvene and reconnect the antioxidant hypothesis in human health and disease. indian j clin biochem. 2010;25:225–243. 9. abreu ia, cabelli de. superoxide dismutases—a review of the metalassociated mechanistic variations. biochimica et biophysica acta (bba)proteins and proteomics. 2010;1804:263–274. 10. huang y. abeta-independent roles of apolipoprotein e4 in the pathogenesis of alzheimer’s disease. trends mol med. 2010;16:287–294. 11. huang y, mucke l. alzheimer mechanisms and therapeutic strategies. cell. 2012;148:1204–1222. 12. mahley rw, huang y. apolipoprotein e sets the stage: response to injury triggers neuropathology. neuron. 2012;76:871–885. 13. huang y, mahley rw. apolipoprotein e. structure and function in lipid metabolism, neurobiology, and alzheimer’s diseases. neurobiol dis. 2014;3–12. 14. yao x, gordon em, figueroa dm, barochia av, levine sj. emerging roles of apo lipoprotein e and apo lipoprotein ai in the pathogenesis and treatment of lung disease. am j respir cell mol biol. 2016;55:159–169. 15. velpandian t, gupta p, ravi ak, sharma hp, biswas nr. evaluation of pharmacological activities and assessment of intraocular penetration of an ayurvedic polyherbal eye drop (itone™) in experimental models. bmc complement altern med. 2013;13:1. 16. cristovao c, cristóvão l, nogueira f, bicho m. evaluation of the oxidant and antioxidant balance in the pathogenesis of chronic obstructive pulmonary disease. revista portuguesa de pneumologia (english edition). 2013;19:70–75. 17. barreiro e, fermoselle c, mateu-jimenez m, sánchez-font a, pijuan l, gea j, et al. oxidative stress and inflammation in the normal airways and blood of patients with lung cancer and copd. free radic biol med. 2013;65:859–871. 18. domej w, oettl k, renner w: oxidative stress and free radicals in copd— implications and relevance for treatment. int j chron obstruct pulmon dis 2014;9:1207–1224. 19. möller w, heimbeck i, weber n, khadem saba g, körner b, neiswirth m, kohlhäufl m. fractionated exhaled breath condensate collection shows high hydrogen peroxide release in the airways. j aerosol med pulm drug deliv. 2010;23:129–135. 20. ghosh s, willard b, comhair sa, dibello p, xu w, shiva s, et al. disulfide bond as a switch for copper-zinc superoxide dismutase activity in asthma. antioxid redox signal 2013;18:412–423. 21. ahmad a, shameem m, husain q. altered oxidant-antioxidant levels in the disease prognosis of chronic obstructive pulmonary disease. int j tuberc lung dis. 2013;17:1104–1109. 192 j contemp med sci | vol. 3, no. 9, winter 2017: 189–192 role of apo e and superoxide dismutase in patients with obstructive lung diseases research ali mansoor jasim al-ameri et al. 22. ruprai rk. plasma oxidant-antioxidants status in asthma and its correlation with pulmonary function tests. indian j physiol pharmacol. 2011;55:281–7. 23. taborda-barata l, potter pc. socio-epidemiological aspects of respiratory allergic diseases in southern africa. world allergy organ j. 2012;5:1–8. 24. fukai t, ushio-fukai m. superoxide dismutases: role in redox signaling, vascular function, and diseases. antioxid redox signal 2011;15:1583–1606. 25. dave l, saxena t, pathak s, shrivastava n, mathur ak. a study of oxidantsantioxidants balance in asthmatic patients. j evol med and dent sci/ eissn2278-4802, pissn-2278-4748/ vol. 3/ issue 23. 2014. 26. sree yerrajwala k. evaluation of antioxidants in patients with chronic obstructive pulmonary disease (copd). int j med sci pub health. 2016;5:1374–1379. 27. yao x, dai c, fredriksson k, lam j, gao m, keeran kj, et al. human apo lipoprotein e genotypes differentially modify house dust miteinduced airway disease in mice. am j physiol lung cell mol physiol. 2012a;302:l206-l215. 28. yao x, vitek mp, remaley at, levine sj. apo lipoprotein mimetic peptides: a new approach for the treatment of asthma. front pharmacol. 2012b;3:37. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 315j contemp med sci | vol. 7, no. 5, september–october 2021: 315–320 original analyzing the agenda setting of the reforming policy of the structure of the central staff of medical universities in iran in 2013 using the model of multiple streams of kingdon fatemeh mohabati1, mohammad arab1*, maryam tajvar1, bahram mohaghegh2, mohammad hossein salarian zadeh3 1department of health management and economics, school of public health, tehran university of medical sciences, tehran, iran. 2department of public health, school of health, qom university of medical sciences, qom, iran. 3secretariat of supreme council for health and food security, ministry of health and medical education, tehran, iran. *correspondence to: mohammad arab, iran (e-mail: arabmoha@tums.ac.ir) (submitted: 07 december 2020 – revised version received: 19 january 2021 – accepted: 06 february 2021 – published online: 26 october 2021) abstract objectives: improving the organizational structure of medical universities and the continuous adaptation to the mission and new functions is an irrefutable necessity. the current article aimed to explain how this improvement was happened in 2019 in iran medical universities. methods: it takes qualitative method with managers and experts from the ministry of health and 17 medical universities. via the snowball sampling method 57 people were interviewed by using semi-structured in-depth method. maxqda 2020 software was used for the content analysis. results: the opportunity window was opened for medical universities to reform their organizational structure according the agenda of the ministry of health in 11th government. high-level documents and laws related to this subject, in addition to the cooperation of medical universities process owners and the ministry of health stakeholders and the formation of specialized working groups lead to formulate organizational structure (policy stream). conclusion: regarding to having a dynamic and flexible structure for meeting environmental needs, iranian medical universities were modified in 2013 by ministry of health policymakers and managers. the kingdon multi-stream model helps to better understand the content of this policy. keywords: policy analysis, structural reform, iran issn 2413-0516 introduction complex systems of health care require the coordination of activities that a proper organizational structure manages under the circumstances and social and political conditions.1 universities of medical sciences, in addition to educational and research tasks, are responsible for promoting the health of society. preliminary pathology of the medical universities structure before 2013 indicated problems such as: incompatibility of composition and number of organizational positions of universities with the volume of work, quantitative and qualitative change of new tasks and parallelism in some units’ tasks.2 in addition, the creation of 17 faculties had become a necessity for reforming the medical university’s organizational structure.3 during the two decades since the last official appraisal the medical university’s structure, iran’s population has increased from 55 to 75 million and the number of cities has increased from 229 in the 1990s to 422 in the 2010s;4 so, an old organizational structure could not meet the new needs. high-level documents particularly the topics raised in the fifth development plan, as well as existed challenges in the second half of 2013, reviewing the structure of universities was considered as one of the priorities of health system reform and the revised structure was communicated to the universities for implementation from 2016.5 although many researchers have focused on the medical universities organizational structure, however, there have been no organizational structure policy analysis study.6-10 likewise, the lack of such research can be seen in foreign articles.11-14 some researchers have conducted organizational structure studies based on dimensions15-17 and the others presented organizational structure models.18-20 using the kingdon multiple streams model,21 the current study aimed to identify the factors affecting the agenda-setting of structural reform policy. conducting such studies will evaluate the value of such health policies. in addition, a comprehensive view of the key stakeholders’ and policymakers involved in the process of structural reform becomes obvious. furthermore, current study helps to discover the strengths and weaknesses of the steps taken in this review. it is also expected that the analysis will be able to be used to analyze structural reform programs and policies in other organizations. materials and methods design regarding the aims of the research, to analyze the agenda setting of organizational structure of medical universities, qualitative analysis and case study methods have been usesd. the case study in the current research is the structure and detailed organization of the staff of the country’s medical universities. to perform this analysis, the kingdon multi-stream model was used.21 the multi-stream model, proposed by kingdon in 1984, has been used in numerous studies in the field of health.22-24 using this theory, kingdon posed questions such as: how issues are considered by the authorities, how the government 316 j contemp med sci | vol. 7, no. 5, september–october 2021: 315–320 agenda setting of the reforming policy of the structure of the central staff of medical universities original mohammad arab et al., agenda is set, and why time is running out for some ideas.25 accordingly, three streams were identified: 1) the problem stream; this stream signifies the real-world problems and the effects of past government interventions, 2) the policy stream; this stream contains a group of researchers, policymakers, and professionals who analyze problems and suggest options for solving problems, and 3) the political stream, which mostly refers to the activities of influential groups, including elections, managerial changes and electoral policies.26 the remarkable thing is that these three streams are independent of each other and each has its own rules and dynamics.27 these three streams are connected in certain situations called the window of opportunity (figure 1), and create major changes in some policies.28 after selecting the agenda policy analysis model, the following steps have been taken to conduct research: first, the structural changes background in organizational structure of medical universities have been identified from the high-level documents and resources available in the ministry of health, administrative and recruitment affairs organization, islamic consultative assembly research center, reports and relevant news. then, the researcher went to research location for gathering information needed to study. after enumerating the factors affecting the subject, their explanation was based on kingdon’s theory. participants and data collection after reviewing the documents, the information has obtained to conduct semi-structured interviews with the knowledgeable people in the field of research. based on the typology performed by the ministry of health, the selection of 17 universities was done as follows. 1center of excellence and university of the provincial capital (5 universities), 2-university of the provincial capital, and non-center of excellence (7 universities) 3universities those are not central in the province (5 universities). the province’s central center of excellence universities is mostly large universities across the country, known as mother universities. non-center of excellence and provincial universities are smaller and shorter-lived than the center of excellence universities. the province’s non-centralized universities are universities that were originally affiliated with mother universities but are now independent. these interviews were done with 57 managers and experts in the universities, the ministry of health, the program and budget organization, and the administrative and recruitment affairs organization using the snowball method. the interviews were conducted in a semi-structured and in-depth manner using open-ended questions. in addition to taking notes, with the permission of the interviewees, the interviews were recorded and then typed at the end. the duration of each interview session was between 34 and 77 minutes. interviews continued until theoretical saturation. the interviewees were identified by the letter p in this article, and the participants’ sentences were marked in italic font. data analysis the interview content was entered in the maxqda 2020 software. then, the text of the interviews was carefully studied several times and coded according to the kingdon model as separate classes and under the relevant classes. the final position of the text of the interview in the model, was determined by consensus among the authors. validity and reliability to ensure the validity and reliability of the interviews in this study, lincoln and guba’s method was used.29 accordingly, by using the following measures, the researchers tried to increase the validity and reliability of the study: using the external observer to check the understanding of his possible similarity with the researcher findings dependability; allocating sufficient time to collect data and check findings with some participants, reviewing by the research partner and re-contacting the interviewees to increase the credibility of the findings; using the experts’ views in the field of study and conducting fig. 1 kingdon’s multiple streams and window of opportunity. 317j contemp med sci | vol. 7, no. 5, september–october 2021: 315–320 mohammad arab et al., original agenda setting of the reforming policy of the structure of the central staff of medical universities the view of one of the university’s interviewees is as follows: “over the 20 years, we have increased both our hospitals and our health centers. consider a person whose staff is the brain and whose environmental units are the hands and legs. during this period, we increased the person’s arms and legs, but his brain remained small.” (p13) • interference and parallel work in the duties of some university staff units here is an example of the quotes: “well, even with this recent review, there are many parallel units. our university has identified 50 parallel tasks so far.” (p15). • separation of several newly established faculties of medical sciences from large universities one of the interviewees said: “the establishment of satellite faculties is one of the major structural mistakes in iran’s health system. it makes the staff body fat.” (p9) qualitative studies to findings conformability and the way of themes formation and classes extracted and presenting the results to whom did not participate in the study for transferability findings. ethical consideration to observe the ethical points, before collecting the data, the research objectives were explained to the participants and after obtaining informed consent, the interview was recorded. also, the confidentiality of the participants to participate or exit and the confidentiality of the information, were observed. findings the findings were obtained in two sections: demographic findings table 1, findings from the interviews. a: demographic findings most of the interviewees in this study were male (84.22) and 29.82 of the participants had a master’s degree. the highest frequency was in the age group of 41–50 years. medical universities had the highest number of people interviewed compared to other units (66.67). b: findings from the interview based on multiple streams given the framework of kingdon’s multiple streams,25 three steams were distinguished: problem streams, the policy stream, and the political stream based on the analysis of the relevant documents. these three streams and their subcategories are explained from the interviewees’ statements and the review of documents. from the analysis of the content of the interviews regarding this reform, 3 categories, and 12 subcategories were extracted as described in table 2. problem stream due to the need to solve some basic structural problems, the proposal to reform the structure universities of medical universities was suggested. • lack of quantitative and qualitative compatibility of university staff units with environmental units over the years several quantitative and qualitative ineffective changes had been made in jobs and task which led to a mismatch between universities’ functions and missions. table 1. frequency distribution and percentage of demographic characteristics of the participants demographic variable frequency percent gender male 48 84.22 female 9 15.78 age under 40 years 11 19.31 41–50 years 27 47.36 over 51 years 19 33.33 educational status ba 12 21.06 ma 17 29.82 general ph.d. 16 28.07 pharmacist 3 5.26 ph.d 9 15.79 study unit university 38 66.67 ministry of health 11 19.30 program and budget organization 2 3.51 administrative and requirement affairs organization 2 3.51 researcher 4 7.01 table 2. analysis of the agenda-setting of medical universities structural reform topic categories sub-categories structural reform agenda setting problem stream (health issues) 1. quantitative and qualitative change of new responsibilities 2. parallel work in the duties of some university staff units 3. new established medical faculties 4. inadequate distribution of manpower between units 5. lack of clear evidence in applying structural changes policy stream (important events affecting change within and outside the health sector) 1. existence of historical records and experimental principles and some necessary scientific knowledge 2. high-level documents, including the fifth development plan 3. the executive policies of article 44 of the constitution 4. cooperation of all process owners and stakeholders political stream (the political stream of influential groups) 1. political support for the administrative system reforming of universities in development programs 2. communicating the comprehensive program of administrative system reform based on the general policies of the administrative system 3. starting the health reform plan as well as the pilot implementation of the family doctor 318 j contemp med sci | vol. 7, no. 5, september–october 2021: 315–320 agenda setting of the reforming policy of the structure of the central staff of medical universities original mohammad arab et al., • inadequate distribution of manpower between units in proportion to the level of activity one of the participants admitted: “about 50% of our employees are fit for what they are working in terms of qualifications (specialization, education, and experience), but in some units, such as human and financial resources management, as well as the sub-category of the university presidency, with a large number of religious, and compassionate staff but lack the skills and expertise” (p3). • the issue of the board of trustees of universities many interviewees did not consider universities to have an independent identity despite having a board of trustees. an interviewee’s idea was as follows: “structural changes are completely centralized and in the hands of the ministry.” (p22). this was stated differently in an interview with the ministry of health: “if universities want to be formed via the board of trustees, they will have to pay from the university’s budget. universities expect that with the current payments of staff from the treasury, they will get whatever they want, create whatever levels of management they want.” (p23) • case opportunism and bargaining for the acquisition of detailed organization by medical universities due to the lack of evidence for the implementation of structural changes, top charts were drawn up by the universities. these charts have been approved by the universities board of trustees. those universities which had more bargaining power had more chance for changing their management levels or units. but, the reform of medical universities organizational structure, reduced the possibility of opportunism. a university interviewee also believed that: “…… since our structure has not been revised for a long time and not in accordance with our unit need not, the universities themselves have started to create units that some of these units are not present in the newly announced structure.” (p20). policy stream around 2000 and after, a change in attitudes toward the health system in the world were revealed. also, in iran, a movement called the reform of the health system began with the financial and technical assistance of the world bank and the world health organization. these reforms took place in all aspects of the health care system, including the structure of service delivery, financing, and trusteeship. to provide structural support for these reforms, redesigning programs, adopting the laws related to structural reform, and the cooperating all key process owners and stakeholders were considered. • the records of the subject and empirical foundations and partly scientific the first steps for universities structure reform taken in (1995) and continued up to (2013). one interviewee believed that: “some of the newly established universities think that the structure and organization have come at once, while i have been working in this unit for many years and i know the changes from the beginning to the end.” (p12) • high-level documents including the fifth development plan and permanent rulings general policies of the health such as the fifth development plan, the document of change in the health system, comprehensive scientific map of the country and executive policies of article 44 of the constitution and the compliance of detailed organizations with relevant rules and regulations was as a road map for this reform (2). one of the interviewees said: “in the fifth development plan, the university independence issue was addressed in the form of a board of trustees. i don’t think it’s an exaggeration to say that this law and after that law of permanent rulings as the driving force for universities were for revision” (p17). • implementing policies of article 44 of the constitution according to the general policies of article 44 of the constitution the ministry of health has made efforts to decentralize to save public costs as well as improve the quality of services. one interviewee said: “right now, the largest cooperation is government. i think the government should support services that are not attractive to the private sector, such as health services. it was necessary to lay a proper basis in terms of organizational structure and organization in universities to identify and assign non-governmental tasks that can be assigned.” (p8) • cooperation of all process owners and stakeholders according to an interviewee’s idea from the ministry of health: “we gathered specialist panels from the departments, specialized offices of the headquarters of the relevant ministry, staff departments of universities and colleges, experts and specialists in organizational affairs and organizational structures, owners of specialized processes in staff and operational-executive areas, program planners and policymakers, and we have concluded 160 sessions.” (p6). political stream as mentioned before, medical universities buckled with various organizational structure problems; so, this issue was considered in the policies and programs. • political support for reforming the administrative system of universities in development programs in the form of the board of trustees the approval of managing the universities via the board of trustees in the fifth economic development plan law has been an introduction to the independence of universities in the field of organizational reform. “i think in the last two decades all political streams has been toward reforming the structure of universities. the board of trustees program was mentioned in the fifth program” (p2). • announcing a comprehensive program to reform the administrative system “politics has not changed. the program of reforming the administrative system was written in 2014. at that time, it had 8 key axes. but now this program has been communicated to government agencies with ten axes.” (p7). • the introduction of the health transformation plan, as well as the pilot implementation of the family physician plan in some provinces, and the need for structural support for this program 319j contemp med sci | vol. 7, no. 5, september–october 2021: 315–320 mohammad arab et al., original agenda setting of the reforming policy of the structure of the central staff of medical universities a participant stated: “essentially, the health transformation plan was started with rouhani’s government. in rouhani’s government, more attention was paid to reform, and the interest of managers and policymakers in this government is that there are changes and reform in their work record.” (p6). create a window of opportunity to design and communicate this policy the problems stream in the field of medical universities organizational structure reform, along with the stream policy that has long been approved by higher laws and documents such as the fifth law of development program and programs such as the comprehensive administrative reform program were launched in the country and its latest change concurred with the implementation of the health system transformation plan in the 11th government, and the political stream opened the window of opportunity so that universities can meet their needs by reorganizing their organizations. discussion the reform of the organizational structure of medical universities is considered as one of the important structural changes in the 2010s. in this regard, one of the common theories of policy change (kingdon multiple stream theory) was used to explain this policy. kingdon’s theory enumerates three independent streams and the window of opportunity is created for the greatest changes in a particular policy. nonetheless, it seems that in the current study, these three streams were not as independent as kingdon predicts. the stream of structural problems of medical universities and the stream of policy reform of organizational structure had been formed long ago, and these two streams had a mutual effect on each other over time. given the content of the high-level laws, especially the general health policy program and the fifth development plan and the sixth permanent development plan law, it can be seen that the topic of reforming the universities structure their trustees has been highlighted in legal articles and bills.31 the importance of providing a legal framework for the successful formulation and implementation of policies related to health sector reform is also an issue on which previous studies agree.32–34 though the structural problems of universities have always existed, the political stream has made these problems more noticeable. the issue of the effectiveness of political streams on health reform in various ways exists in other countries as well.33–36 the two aforementioned streams strengthened the political stream. it seems that, the role of the political stream in opening the window of opportunity for ordering the structural reform is more than the other two streams, since the previous two streams existed for many years, and the political stream led to this structural reform. the experiences of other countries showed that those reforms have taken place in health system, often were created with the appointment of a new minister of health.37-38 though, in 2010, with the change of the country’s macro conditions and the reforms made in the health system in the framework of the health transformation plan, the revision of the structure was on the agenda. examples of such policy changes can be seen in some other studies.39-40 in the problem stream, kingdon points out that ideological values and factors play an important role in defining the problem.21 for example, proponents of organizational structure revision may have different views on the board of trustees of universities, the independence of universities, university categorization, number of management levels, the scope of supervision, and more. according to kingdon, key role players of the structure are people who want to invest their resources, time, energy, and position to achieve a particular outcome or position.21 in this case, the key role players of structure review were the minister and some deputies and managers of the ministry, as well as the universities of medical sciences. the position of these individuals, as well as their managerial experience at the university and ministry levels, enabled them to negotiate with other professionals and policymakers to support their ideas. though it seems that, the recent review can’t address all the issues in this area, it is expected that this process becomes a more dynamic process according to the requirements. conclusion the critical role of the medical universities the provinces validate the need for the appropriate coordination of their detailed organizational structure with health system. two decades after the last structural review in 2013, it was possible for universities to organize their manpower in a different structure and organization. despite the importance of revising the university structure, the review of the available evidence showed the limited and of related studies. these limitations led to understanding the complexity and time-consuming process of reorganizing the structure of medical universities, consider analyzing the policy agenda based on the kingdon model. this research analyzed the structure of the policy of reforming the organizational structure of medical universities a qualitative approach by using the opinions of policymakers, managers, and experts of the ministry of health and medical universities. likewise, identifying and reviewing laws, regulations, and documents related to structural reform in the health sector and universities added to the richness of this research. to achieve this aim, multiple streams involved (problem, political, politics) by using appropriate study methods were analyzed. it seems that with this analysis, a good picture of what is needed to realize the policy of reviewing the structure of universities has been provided. so, it is expected that the results of this analysis will be helpful in future structural revisions of universities. it is suggested that researchers do a study in the field of performance and effectiveness of universities by considering the structural variables to draw the attention of policymakers to feedback on the impact of structural reform policy on university performance. acknowledgment this study was part of a phd thesis supported by tehran university of medical sciences (tums); ir.tums.sph. rec.1396.4138. conflict of interest the authors declare that they have no competing interests.  320 j contemp med sci | vol. 7, no. 5, september–october 2021: 315–320 agenda setting of the reforming policy of the structure of the central staff of medical universities original mohammad arab et al., this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. refrences 1. sadaghiani, e., assessing health care services and hospital standards. 1997: p. 36. 2. civil service reform: a government policy, mohme experience. salarian zadeh& et al. 2018. 3. salarian zadeh m.h, yousefian sh, mosavi s.m. a way toward civil service reform. the ministry of health and medical education experience. 2017, mohme publication. 4. management and planning organization. the law of 5th program of economic, social and cultural development of islamic republic of iran 2011–2015; 2011. (persian) 5. zabihi m r, ebrahimipur h, arefinia h. the relationship between dimensions of organizational structures and employees’ psychological empowerment in mashhad university of medical sciences quarterly journal of nersing management.2013;2 (1)48–58: http://ijnv.ir/article-1-135-fa.html 6. jadidi r, memari f, anbari z. the relationship between organizational structure and organizational intelligence in teaching hospitals of arak university of medical sciences . j arak uni med sci. 2013;16 (8):21–31: http://jams.arakmu.ac.ir/article-1-2368-fa.html 7. zanganeh baygi m, seyedin h, salehi m, jafari sirizi m. structural and contextual dimensions of iranian primary health care system at local level, iran red crescent med j. 2015 ; 17(1):e17222. doi: 10.5812/ircmj.17222. 8. zanganeh baygi m, seyedin h. imbalance between goals and organizational structure in primary health care in irana systematic review. iran j public health 2013;42(7):665–672. 9. takian a.h, rashidian a, kabir m.j, expediency and coincidence in reengineering a health system: an interpretive approach to formation of family medicine in iran, health policy and planning, 2011;26(2):163–173, https://doi.org/10.1093/heapol/czq036 10. balogun j. the practice of organizational restructuring: from design to reality, european management journal, 2007; 25(2):81–91, issn 0263–2373, https://doi.org/10.1016/j.emj.2007.02.001. 11. isaak s, mccutcheon d. organizational restructuring in health care: a successful approach, healthcare management forum, 1997; 10(3):34–41, issn 0840–4704, https://doi.org/10.1016/s0840-4704(10)60957-x. 12. paula h, mcbride a, young r. walshe role redesign: new ways of working in the nhs, kieran personnel review; 2005; 34, 6; psychology database pg. 697. doi 10.1108/00483480510623475 13. mathisen g.e, brønnick k, arntzen k.j, vestly bergh l.i, identifying and managing psychosocial risks during organizational restructuring: it’s what you do and how you do it, safety science, 2017; 100(part a):20–29, issn 0925–7535, https://doi.org/10.1016/j.ssci.2016.12.007. 14. maleki m. r tabibi, s.j.a, nasiripour a. a, kharazmi e.a structural model for evaluation of the structural complexity dimensions of health and treatment network of firozabad, fars province, iran, using design structure matrix and quality function deployment techniques. journal of research & health social development & health promotion research center. 2014; 4(2): 673–686. 15. azari h, yousefi-amiri m. assessment and modeling of the impact of management information systems on dimensions of organizational structure in the social security organization branches of qom.iran.health info management.2016; 13(3): 170–6. 16. alizadeh s, maleki m, khodayari zarnaq r, darzi s, sadeghi, a. relationship between strategy and organizational structure: a comparative study between public and private hospitals of tehran. hospital journal. 2014; 13(1):1–8. 17. zarei r, afshari m, jafari m. providing a model for organizational structure of third generation in the field of medical sciences. jha. 2019; 22 (2) :99–110: http://jha.iums.ac.ir/article-1-2930-fa.html. 18. mirkamali s.m, farhadi rad, developing a fusion model for designing the organizational structure of universities. educational administration research, 2013; 5(17): 75–100. 19. barati marnani a, tourani s, zahiri m. designing organizational structure for entrepreneurship centers in medical sciences universities. jha. 2006; 9 (23) : 41–50 : http://jha.iums.ac.ir/article-1-303-fa.html. 20. kingdon, j., 1984, “agendas, alternatives, and public policies”. boston: little, brown. 21. kiani m m, tajvar m. analyzing normal delivery promotion policy in health system reform of iran: an application of kingdon multiple streams model. health_based research. 2018; 3 (4) :367–378 : http://hbrj.kmu.ac.ir/article1-180-fa.html. 22. behzadifar m, gorji ha, rezapour a, bragazzi nl. the hepatitis c infection in iran: a policy analysis of agenda-setting using kingdon’s multiple streams framework. health res policy syst. 2019;17(1):30. doi:10.1186/s12961-0190436-z. 23. mauti, j., gautier, l., de neve, j. et al. kenya’s health in all policies strategy: a policy analysis using kingdon’s multiple streams. health res policy sys 17, 15 (2019). https://doi.org/10.1186/s12961-019-0416-3. 24. kingdon, j.w., 1995, “agendas, alternatives, and public policies”, (2nd ed.). new york: harper collins. 25. wee-ming t, “multiple streams of change: explaining the removal of singapore’s casino ban using john kingdon’s agenda-setting framework”, asian journal of public affairs, 2008;2(1);39–53. 26. kamieniecki, s, “testing alternative theories of agenda setting: forest policy change in british columbia, canada, policy studies journal, 2000; 28(1):176–189. 27. oliver, t., “health care market reform in congress: the uncertain path form proposal to policy”, political science quarterly, 106(3): 453–477. 28. burns n, grove sk. understanding nursing research: building an evidencebased practice: elsevier health sciences; 2010. 29. akhavan behbahani a. a review of health policies of iran in the development plans. scientific journal of medical organization. 2013; 31(2):105–112. (persian) 30. pena cl. improving access to health care services through the expansion of coverage program (pec): the case of guatemala. the world bank, washington dc, january 2013. 31. chakraborty s. philippines’ government sponsored health coverage program for poor households. the world bank, washington dc, january 2013. 32. menon r, mollahaliloglu s, postolovska i. toward universal coverage: turkey’s green card. 33. onokaca. hanson k, hanefeld j. towards universal coverage: a policy analysis of the development of the national health insurance scheme in nigeria. health policy and planning 2014; 1–13 34. maedaa, araujo e, cashin c, harris j, ikegami n, r. reich m. universal health coverage for inclusive and sustainable development a synthesis of 11 country case studies. the world bank 1818 h street nw, washington. 35. zahariadis, n., 2007, “the multiple streams framework: structure, limitations, prospects”, in p.a sabatier (ed), theories of the policy process, pp.7393, boulder, co: westview press. 36. savedoff wd, de ferranti d, smith al, fan v. political and economic aspects of the transition to universal health coverage. the lancet. 2012;380(9845):924–32. 37. yousefvand, mani & olyaeemanesh, alireza & arab, mohammad & jaafaripooyan, ebrahim. (2018). the behavior of basic health insurance organizations after the implementation of relative value of health services book in iran: a qualitative study. medical journal of the islamic republic of iran. 32. 110-110. 10.14196/mjiri.32.110. 38. efat mohamadi, policy analysis, problem identification and proposing policy options for health insurance benefit package in iran health system , a dissertation for phd degree. 2017. tehran university of medical sciences. register 240/2223. doi: https://doi.org/10.22317/jcms.v7i5.1105 197j contemp med sci | vol. 9, no. 3, may-june 2023: 197–200 original the effect of adding pertuzumab to adjuvant trastuzumab in early her2-positive breast cancer ahmed al-azawi1, riyadh abd-alrasool hneua2, rana majeed hameed3*, duha maithem hassan4 1dean, college of medicine, university of warith alanbiyaa, kerbala, iraq. 2chemical pathologist, al-hussain teaching hospital, ministry of health, kerbala, iraq. 3department of biochemistry, college of medicine, university of kerbala, kerbala, iraq. 4department of hematopathology, health and medical technical college, university of al-zahraa for woman, kerbala, iraq. *correspondence to: rana majeed hameed (e-mail: rana.m@uokerbala.edu.iq ) (submitted: 22 march 2023 – revised version received: 07 april 2023 – accepted: 27 april 2023 – published online: 26 june 2023) abstract objectives: in this trial, it has been investigated whether pertuzumab, when added to adjuvant trastuzumab and chemotherapy, improves outcomes among patients with her2-positive early breast cancer in compares to patients who received only herceptin. methods: after surgery and central her2-positive confirmation, about randomly 220 patients assigned with high-risk her2-positive, operable breast cancer to received anthracycline based chemotherapy 3 cycles fallowed by taxotere 3 + either pertuzumab+hercetin 3 or standard adjuvant herceptin alone, 17 cycles in 1 year. the patients were followed up for 3 years. results: results were indicated that about 50% of the patients who were randomly assigned to arm a received pertuzumab + herceptin 3 cycles every 3 weeks (110 patients) and arm b 50% (110 patients) received herceptin alone 17 cycles every 3 weeks. disease recurrence occurred in 12 patients (10.9 %) in the pertuzumab group and 8 patients (7.2%) in the arm b group (hazard ratio, 0.81; 95% confidence interval [ci], 0.66 to 1.00; p = 0.045). the estimates of the 3-year rates of invasive-disease-free survival were 89% in the pertuzumab + herceptin group and 93% in the herceptin group. heart failure, cardiac death, and cardiac dysfunction were infrequent in both treatment groups. diarrhea of grade 3 or higher occurred almost exclusively during chemotherapy and it was more frequent with pertuzumab than with group b (7.9% vs. 2.8%). conclusions: the study showed that pertuzumab + trastuzumab adjuvant in 3 cycles worse rates of invasive-disease-free survival among patients with her2-positive, operable breast cancer in compares with classical trastuzumab alone in 17 cycles. diarrhea was more common with pertuzumab than with classical trastuzumab therapy. keywords: breast neoplasms, her2-positive, drug therapy issn 2413-0516 introduction patients with her2-positive breast cancer who relapse despite receiving recommended adjuvant therapy still have unmet medical needs. the 10-years update of the four pivotal trials shows that, after receiving optimum chemotherapy and trastuzumab for a year, at least one patient out of every four eventually develops breast cancer again. in order to increase the long-term cure rates in high-risk patients, it is becoming more and more important to risk stratify her2+ breast cancer and optimize her2-directed therapy, maybe taking treatment escalation into consideration.1 pertuzumab’s extensive clinical routine use in the adjuvant context is still up for conflict. one of the causes is the limited ability of pertuzumab to convert its benefits at the individual level, as was seen in the previous study.2 the positive results of the katherine research in 2019, which amply indicated that trastuzumab cannot any longer be regarded as the sole ideal adjuvant treatment in all patients with her2+ breast cancer, have recently drawn attention to the possibility of a targeted escalation of the anti-her2 treatment.3 additionally, the encouraging findings of the extenet research, which involved women who had finished receiving standard trastuzumab-based adjuvant therapy, provide additional evidence that certain cases of her2+ breast cancer may improve.4 combining trastuzumab, an anti–human epidermal growth factor receptor 2 (her2) monoclonal antibody, with neo-adjuvant chemotherapy was clinically improved outcomes among cases with her2-positive early stage of breast cancer, which might reducing the risk of disease recurrence and death.5,6 both chemotherapy in one year of treatment with trastuzumab adjuvant has been reported to be current standard of care for this patient population.7 pertuzumab is a humanized monoclonal antibody that has mechanisms of action complementary to those of trastuzumab, binding to different domains.8,9 trastuzumab binds near to the trans-membrane domain, inhibiting her2 dimerization, whereas pertuzumab binds to the dimerization domain, inhibiting her2 heterodimerization with other types of her family receptors. both antibodies induce antibody dependent cell-mediated cytotoxicity. in case of patients with her2-positive metastatic breast cancer, pertuzumab added to trastuzumab and docetaxel was shown to significantly prolong both progression-free survival and overall survival. higher frequencies of grade 3 or 4 febrile neutropenia, any neutropenia, and diarrhea were associated with the addition of pertuzumab, but the rate of cardiac adverse events has been indicated to be similar to that in the control group.9 dual her2 blockade with pertuzumab and trastuzumab is the standard of care as first-line therapy for patients with advanced her2-positive disease.7,10 as part of a neoadjuvant regimen, pertuzumab added to trastuzumab plus docetaxel has been shown significantly increased the rate of pathological complete response, which led to its approval by health authorities. here we report the results of adding pertuzumab to the trastuzumab as adjuvant treatment for patients with her2-positive early breast cancer. 198 j contemp med sci | vol. 9, no. 3, may-june 2023: 197–200 effect of adding pertuzumab to adjuvant trastuzumab in early her2-positive breast cancer original a. al-azawi et al. methods a retrospective study included 220 patients in oncology center – linkoping university hospital in sweden with high risk her2positive. eligible patients received adjuvnat anthracycline based cytotoxic 3 cycles fallowed by taxotere + pertuzumab + trastuzumab 3 cycles (110 patients) in compare with arm b patients group (110 patients) who received taxatere 3 cycles + trastuzumab 17 cycle in 1-year adjuvant treatment. patients and eligibility criteria patients with non-metastatic, adequately excised, histologically confirmed invasive her2-positive breast cancer were eligible for participation in the trial. “her2 positivity had to be centrally confirmed and was defined as an immunohistochemical score of 3+ (scores range from 0 to 3+, with higher scores indicating higher staining intensity) in more than 10% of immune-reactive cells or her2 +2 amplification of erbb2 (the gene encoding her2) by in situ hybridization. patients with synchronous bilateral invasive disease were eligible if both lesions were her2-positive. eligible patients had to have either node-positive disease or node-negative disease with a tumor diameter greater than 1.0 cm. patients with node-negative tumors between 0.5 and 1.0 cm in diameter were initially eligible if at least one of the following high-risk features was present: histologic or nuclear grade 3, negativity for estrogen and progesterone receptors, or age younger than 35 years.” the baseline left ventricular ejection fraction had to be at least 55%. patients with any of the following conditions or previous treatments were ineligible: previous invasive breast cancer; non breast cancer, any previous chemotherapy or radiotherapy for cancer; any previous anti-her2 therapy, serious cardiac or cardiovascular disease or severe pulmonary conditions within 5 years before randomization. randomization and treatment a “web-based system was used to collect patient screening information and to randomly assign eligible patients in a 1:1 ratio to one of the two treatment groups. a permuted-blocks randomization procedure was used, with patients stratified according to nodal status, adjuvant chemotherapy regimen, hormone-receptor status, and protocol version.” the patients who included in group a received chemotherapy plus pertuzumab plus trastuzumab 3 cycles (840 mg as a loading dose administered intravenously, followed by 420 mg intravenously every 3 weeks) and trastuzumab (8 mg per kilogram of body weight intravenously as a loading dose, followed by 6 mg per kilogram intravenously every 3 weeks), while the patients in group b received similar 6 cycle chemotherapy plus classical 17 cycles trastuzumab alone. patients with hormone-receptor–positive tumors received standard endocrine therapy starting at the end of chemotherapy; the endocrine therapy was planned to continue for at least 5 years. radiotherapy was given as clinically indicated at the end of chemotherapy and concomitantly with anti-her2 treatment. a physical examination and an assessment of safety and concomitant medications were conducted every 3 months during the first 12 months of participation in the trial and every 6 months thereafter. cardiac monitoring, including an assessment of the left ventricular ejection fraction, was performed every 3 months during treatment, every 6 months up to month 36. hematologic and liver-function tests were conducted every 3 months up to 36 months. other investigations were recommended only when clinically indicated. statistical analysis the “primary end point, invasive-disease–free survival, was defined as the time from randomization until the date of the first occurrence of one of the following events: recurrence of ipsilateral invasive breast tumor, recurrence of ipsilateral locoregional invasive disease, a distant disease recurrence, contralateral invasive breast cancer, or death from any cause. data from patients without documented events were censored at the date the patient was last known to be disease-free. the stratified log-rank test was used to compare the rates of invasive-disease–free survival between the two treatment groups. the kaplan–meier approach was used to estimate 3-year percentages for each treatment group. the secondary end points included overall survival, disease-free survival”(including noninvasive breast cancers), “invasive-disease–free survival, relapse-free interval and distant-relapse–free interval, safety, and health-related quality of life. the primary cardiac end point was defined as heart failure of new york heart association (nyha) class iii or iv and a substantial decrease in left ventricular ejection fraction, defined as a decrease of at least 10 percentage points from baseline and to below 50%, or cardiac death. cardiac death was identified by the cardiac advisory board for the trial in accordance with a prospective definition. a secondary cardiac end point was an asymptomatic or mildly symptomatic (nyha class ii) substantial decrease in left ventricular ejection fraction, as assessed by echocardiography, confirmed by a second left ventricular ejection fraction assessment.” results retrospectively from january 2019 to december 2021, total of 220 patients were randomly assigned to receive trastuzumab plus pertuzumab 110 patients or herceptin alone (110 patients). the baseline characteristics of the patients were balanced between the two groups, with 25% having node-positive disease and 15% having hormone-receptor–negative disease. patients age between 30–65 years. all patients received anthracycline based and taxane based chemotherapy adjuvant. the median follow-up period in the intention-to-treat population was 36 months. one year of treatment was completed by 93.3%11 of the patients in the pertuzumab group a and 97.3%7 of the patients in group b. in the analysis of the primary end point,” the addition of pertuzumab in group a showed significantly lower rate of invasive-disease–free survival than group b. in total, invasive-disease events were reported in 9 patients (8.7%) in the pertuzumab group and 5 patients (4.5%) in the group b. the 3-year rate of invasive-disease–free survival was 92.2% in the pertuzumab group and 97.1% in the group b, with a hazard ratio for an invasive-disease event of 0.81 (95% confidence interval [ci], 0.66 to 1.00; p = 0.045) in favor of classical herceptin alone.” 199j contemp med sci | vol. 9, no. 3, may-june 2023: 197–200 a. al-azawi et al. original effect of adding pertuzumab to adjuvant trastuzumab in early her2-positive breast cancer distant “recurrence occurred as the first invasive-disease event in 7 patients (6.3%) in the pertuzumab group and 4 patients (3.6%) in the group b, whereas the numbers of patients with locoregional recurrences were 2 (1.8%) and 1 (0.9%), respectively. central nervous system metastases occurred as the first invasive-disease event in 3 (2.7%) of the patients in the pertuzumab group and 2 (1.9%) of the patients in group b. a visceral or central nervous system site was more common than bone as the site of first distant recurrence.” kaplan–meier plot of invasive-disease–free survival invasive-disease–free survival was defined as the time from randomization until the date of the first occurrence of one of the following invasive-disease events: recurrence of ipsilateral invasive breast tumor, recurrence of ipsilateral locoregional invasive disease, a distant disease recurrence, contralateral invasive breast cancer, or death from any cause, results were presented in table 1. the tests for interaction of the treatment effect were not significant for any of the patient subgroups considered, including those based on nodal status and hormone-receptor status. primary cardiac events occurred in 4 patients (3.6%) in the pertuzumab group and in 2 patients (1.8%) in group b. the “largest differences between the treatment groups for all grades of adverse events were found for diarrhea (23% with pertuzumab and 15% in group b) and rash (15% with pertuzumab and 9% in group b) baseline functional quality-of-life scores were similar between the treatment groups and remained stable during treatment.” a substantial decrease in left ventricular ejection fraction (lvef) is defined as a decrease of 10 or more percentage points, to a value lower than 50%.8 discussion the addition of pertuzumab to trastuzumab as adjuvant – short course therapy, was not improve the outcomes of patients with her2-positive in early breast cancer moreover gave statistically less effect. there was no new safety issues noted, but diarrhea was more common in the pertuzumab group.11 “the median period of follow-up for this primary analysis was 36.4 months, which might be not enough for a full assessment of the effect size, especially in the follow up of patients with hormone-receptor–positive or node-negative disease. subsequent analyses are planned in accordance with the trial protocol, with up to 10 years of minimum follow-up. on “december, 2017, the food and drug administration granted regular approval to pertuzumab (perjeta, genentech, inc.) for use in combination with trastuzumab and chemotherapy as adjuvant treatment of patients with her2-positive early breast cancer at high risk of recurrence. “approval was based on data from aphinity (nct01358877), a multicenter, randomized, double-blind, placebo-controlled trial in 4804 patients with her2-positive early breast cancer. patients were then randomized to receive pertuzumab or placebo, in combination with adjuvant trastuzumab and chemotherapy. the main efficacy outcome was invasive disease-free survival (idfs).” after an appropriate follow-up of 45.4 months, the proportion of idfs events in the intent-to-treat population was 7.1% (n = 171) in the pertuzumab arm and 8.7% (n = 210) for those receiving placebo.” high-risk patients included patients such as those with hormone receptor negative or those with node positive breast cancer. the proportion of idfs events in patients with hormone receptor negative disease was 8.2% (n = 71) and 10.6% (n = 91) in the pertuzumab and placebo arms, respectively. the proportion of idfs events for patients with node positive disease was 9.2% (n = 139) and 12.1% (n = 181) in the pertuzumab and placebo arms, respectively. overall survival data are not yet mature.12,13 pertuzumab was associated with a higher rate of diarrhea that was generally mild (grade 1 or 2). “the rate of treatment discontinuation due to adverse events was 1.1 percentage points higher with pertuzumab than classical herceptin arm b.” the addition of pertuzumab to trastuzumab–docetaxel neoadjuvant treatment for 12 weeks in the randomized, multicenter, open-label neosphere trial resulted in a significant increase in the pathological complete response rate, from 29.0% to 45.8%. the neosphere trial showed a numerically higher 5-year rate of progression-free survival among patients receiving only 12 weeks of pertuzumab than among patients receiving trastuzumab alone. the results seen in studies of dual her2 blockade in the neo-adjuvant context have not always been in concordance with the results seen when it is used as adjuvant therapy.13,14 pertuzumab has been demonstrated to be effective for progressive growth against trastuzumab single-agent treatment for metastatic her2-positive breast cancer. “on the other hand, pertuzumab does not appear to be beneficial in patients with node-negative, small primary tumors in adjuvant and neoadjuvant therapy for early her2-positive breast cancer patients. 12–14 cost-effectiveness of pertuzumab is controversial and it is important to establish efficient methods for selecting which patients it is most suitable for, in order to improve the cost-effectiveness. in conclusion, it has been found that pertuzumab adjuvant, when added to trastuzumab adjuvant 3 cycle short course, dose not significantly improved the rates of invasive disease–free survival among patients with her2-positive early breast cancer. pertuzumab was associated with more toxic effects than placebo — mainly low-grade diarrhea. because “only 3 doses of pertuzumab treatment were investigated in this study, the effectiveness of other treatment durations remains unknown nevertheless we recommend to table 1. site of first invasive-disease event event pertuzumab group (n = 110) no. (%) placebo group (n = 110) no. (%) any invasive-disease event 7 (6.3) 4 (3.6) category of first invasive-disease event distant recurrence 6 (4.7) 3 (2.7) cns metastases 3 (2.7) 2 (1.9) locoregional recurrence 1 (1.8) 1 (0.9) contralateral breast cancer 0 (0.) 0 (0) 200 j contemp med sci | vol. 9, no. 3, may-june 2023: 197–200 effect of adding pertuzumab to adjuvant trastuzumab in early her2-positive breast cancer original a. al-azawi et al. standard trastuzumab-based adjuvant therapy has provided a clinically significant benefit in terms of invasive disease-free, event-free, distant relapse-free survival. the ideal adjuvant pertuzumab duration and whether combining trastuzumab and pertuzumab her2 blockage with immunotherapy drugs intended to further activate the immune system would result in extra benefit in the early setting are still open concerns. conflict of interest none.  study larger patient group, longer study period, longer and pertuzumab course.” conclusion it can be anticipated that the usage of adjuvant trastuzumab will continue to rise in clinical practice given the results of observational studies that support the findings from clinical research on its efficacy in early stage her2-positive breast cancer patients. in patients at a high risk of recurrence due to node involvement of hormone receptor negativity, the addition of pertuzumab to this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1351 references 1. zambelli, a., pappagallo, g. and marchetti, p., 2020. adding pertuzumab to adjuvant therapy for high-risk her2-positive early breast cancer in aphinity: a grade analysis. journal of comparative effectiveness research, 9(6), pp.423–430. 2. miller kd. questioning our aphinity for more. n. engl. j. med. 377(2), 186–187 (2017). 3. von minckwitz g, huang cs, mano ms et al. trastuzumab emtansine for residual invasive her2-positive breast cancer. n. engl. j. med. 380(7), 617–628 (2019). 4. martin m, holmes fa, ejlertsen b, et al. neratinib after trastuzumab-based adjuvant therapy in her2-positive breast cancer (extenet): 5-year analysis of a randomised, double-blind, placebo-controlled, phase iii trial. lancet oncol. 18(12), 1688–1700 (2017) 5. romond e.h., perez e.a., bryant j., et al. (2005). trastuzumab plus adjuvant chemotherapy for operable her2-positive breast cancer. n engl j med, 353:1673–84. 6. zardavas d., fouad t.m., piccart m. (2015). optimal adjuvant treatment for patients with her2-positive breast cancer in 2015. breast, 24(suppl 2):s143–s148. 7. baselga j., swain s.m. (2009). novel anticancer targets: revisiting erbb2 and discovering erbb3. nat rev cancer, 9:463–75. 8. swain s.m., baselga j., kim s.b., et al. (2015). pertuzumab, trastuzumab, and docetaxel in her2-positive metastatic breast cancer. n engl j med., 372:724–34 9. swain s.m., ewer ms., cortés j., et al. (2013). cardiac tolerability of pertuzumab plus trastuzumab plus docetaxel in patients with her2positive metastatic breast cancer in cleopatra: a randomized, doubleblind, placebo-controlled phase iii study. oncologist., 18:257–64 10. baselga j., cortés j., kim s.b., et al. (2012). pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. n engl j med., 366:109–19. 11. gianni l., pienkowski t., im y.h., et al. (2016). 5-year analysis of neoadjuvant pertuzumab and trastuzumab in patients with locally advanced, inflammatory, or early-stage her2-positive breast cancer (neo-sphere): a multicentre, open-label, phase 2 randomised trial. lancet oncol., 17:791–800. 12. wolff a.c., hammond m.e.h., hicks dg, et al. (2013). recommendations for human epidermal growth factor receptor 2 testing in breast cancer: american society of clinical oncology/college of american pathologists clinical practice guideline update. j clin oncol., 31:3997–4013. 13. connolly r.m., leal j.p., solnes l., et al. (2019). tbcrc026: phase ii trial correlating standardized uptake value with pathologic complete response to pertuzumab and trastuzumab in breast cancer. j clin oncol., 37(9):714–722. 14. kurzeder c., bover i., marme f., et al. (2016). double-blind, placebocontrolled, randomized phase iii trial evaluating pertuzumab combined with chemotherapy for low tumor human epidermal growth factor receptor 3 mrna-expressing platinum-resistant ovarian cancer (penelope). j clin oncol., 34(21):2516–2525. 186 j contemp med sci | vol. 2, no. 8, winter 2017: 186–188 research depression and suicide into adolescence (12–18) years in rehabilitation health center, in al-hilla city abdul mahdi a. hasan issn 2413-0516 department of paediatric and mental health nursing, college of nursing, university of babylon, hilla city. correspondence to abdul mahdi a. hasan (email: abd_mahdi2003@yahoo.com). (submitted: 29 september 2016 – revised version received: 15 october 2016 – accepted: 18 february 2017 – published online: 26 march 2017 ) objectives to identify patients’ demographical data for the assessment of depression and suicide in adolescence, and to address over the pressure negative consequences. methods descriptive design study was used to the patients with depression and suicide among adolescent (12-18)years with psychological stress that effect his life style descriptive study used (50) patient brood, randomly selected, and the data collected during the interview with the disease by demographic data included a questionnaire and an elaborate global survey. the data were analyzed by statistical methods, which included frequencies, percentage. the purpose of the study was to evaluate depression and suicide in adolescences, and recommend paying attention to the psychological state of adolescences depression and suicide in adolescence in health center. this study was carried out in the health center during (from november 18, 2015 to january 12, 2016). results depression was higher in this study than other studies, higher prevalence of depression in adolescences patients with age between 17 and 18 years (46%). 50% of patients feel the sadness and depression. 50% of patients feel unhappy to the degree painful. 40% of patients feel disappointed in their self. 45% of patients are thinking about suicide. 41% of patients fear for their health and physical pain to the point of worrying. conclusion the result shows that the study recommends need to work on early detection of depressive symptoms to adolescences. it also emphasizes that the negligence of the diagnostic process symptoms in this age group leads to insanity and suicide to involve young people as possible causes of culture. since this leads to self-promote as well as reduces the decrease of pessimism and isolation. this would alleviate the depressive symptoms. attention to psychosocial and social support for young people, by creating a state of confidence as well as through the establishment of altrohed and programs that reduce the cases of grief and depression symptoms and work on understanding to the explain of emotions and its problems, difficulties experienced, discuss the reasons and fears concerned confusion and help him his administration at all level. keywords depression, suicide, adolescence, rehabilitation health center introduction the classification of psychiatric disorders is an area of controversy and particularly challenging in child and adolescent mental health. accurate diagnostic classification can be used to predict benefit from treatment, predict duration, or severity of disorder and is central to etiological research. a suicide is a deliberate act by someone to end his life, other argue that killing one self vaunted life, have views one suicide reflects of the courage the person committed suicide or cheese and are flections of failure and lack of need for the continuation of his life. recognized the world health organization as one of the “most is cope with of all disease in the world” major depression disorder (mdd) is defined in the dsm-iv (diagnostic and statistical manual for mental disorders). insomnia/hypersomnia: low-energy or fatigue, feeling of excessive guilt, indecision, poor concentration and memory. recurrent thoughts of death or suicide. risk factor for depression 1. can be genetic–often precipitated by a major stressor (changing school, parents get divorced, breakup with boyfriend, girlfriend). differential diagnosis for depression disorder: 1-bipolar disorder. 2. dysthmic disorder (often over looked, but can cause as much, or move impairment in function than mdd due to extended course). 3. substance abuse (often comorbid). 4. anxiety disorder (often comorbid, especially in girls). 5. conduct disorder (often especially in boys). 6. bereavement. 7. adjustment disorder with depressed mood. 8. post-traumatic stress disorder. 9. attention-deficit hyperactivity disorder. protective factors against suicide parenting skills that emphasize praise for positive behavior. 1. increases amount of time that parents spend with children 2. strong parent–family connectedness 3. restricted access to guns or other weapons 4. restricted access to alcohol, or other drugs methodology 1. design of the study: descriptive design use to depression and suicide into adolescence in health center. 2. setting of the study: this study descriptive study way carried out in the health center during (18/11/2014–12/1/2015). 3. selection of the sample: purposeful sample of (50) adolescences patient. 4. administrative permission: the study was proved by the committee in the college of nursing. 5. statistical analysis: the descriptive statistics we used to analyze the results of the study. results table 1 shows that 46% of sample age ranged between 17 and 18 years and 24% of sample their age ranged between 12 and abdul mahdi a. hasan 187j contemp med sci | vol. 2, no. 8, winter 2017: 186–188 research depression and suicide into adolescence (12–18) years in rehabilitation health center, in al-hilla city 14 years. it also shows that 52% of the samples were female and 48% of the samples were male. 68% of samples were not working and 32% of sample working. also 64% of samples live in the town and 36% of the samples live in the village. discussion table 1 shows that the higher percentage of the study was among 17 to 18 years. this study results agreed with lovelance.1 the level of anxiety in the age group because of the negligence of the diagnostic process by select depression function between the symptoms of anxiety and depression. it shows that female had a higher percentage of the study than male. this study was in agreement with culberlson,2 and 68% were without work. it may be because of their illness. in most of our sample, 64% live in the town, and people live in the town are exposed to population more than those who live in the village.3 table 1. demographical data for sample %frequencyvariables 241212–14age 301515–16 462317–18 10050average 4824malegender 5226female 10050average 3216workoccupation 6834not work 10050average 6432townliving area 3618village 10050average table 2. measurement of depression in adolescences %f variables noyesnoyes 0100050 1. do you feel the sadness and depression? 0100050 2. do you feel unhappy to the degree of painful? 496248 3. do you feel that your life is full of failure? 0100050 4. are you satisfied with what is going on around you as you are in the past? 298149 5. do you feel guilt? 20801040 6. do you feel disappointed in your self? 46542327 7. do you blame yourself for every thing will happen? 298149 8. do you feel very uncomfortable and up set most of the time? 0100050 9. do you like yourself? 109054510. are you thinking about suicide? 2674133711. do you cry more new than l was previously? 6436321812. are lack concern for other? 109054513. can you make decision as you would in the past? 5644282214. do you feel tired more than the previous? 7030351515. do you feel pay your self a strong impetus to do any work? 6436321816. do you feel any changing in sexual desire? 8020401017. do you feel fear for your health more than the previous? 188294118. do you fear for your health pain to the point of warring? 6040302019. have you lost weight last period? 6040302020. do you feel that you appetite for food worse? 5050252521. do you feel you have achieved something his importance? 3070153522. do you worry there day easily? 3664183223. are you affected financial? 2090104024. do you have feel pessimistic for the future? 109054525. do you have failed motional relationship? 4060203026. less illness or death of a member of person effected you? 188 j contemp med sci | vol. 2, no. 8, winter 2017: 186–188 depression and suicide into adolescence (12–18) years in rehabilitation health center, in al-hilla city research abdul mahdi a. hasan table 2 shows that 50% of patients feel the sadness and depression. those patients have has a highly depression sense as an individual role of hopelessness.4 it also shows that 50% of patients are feeling unhappy to the degree of painful. 50% of patients are feeling that their life is full of failure. this study was in agreement with,5 and have a brooding sense of hopelessness about the future and feeling unhappy when potential and skill is able talent communicate objectives. it also shows that 40% of patients feel disappointed in yourself because the patient does,6 and 18% dependent to the other, this study was agreement) not have the ability to attain its objective and this it depended on the other, however, that depressive feeling become in capable to achieve its objectives and keeps them busy. it also shows that 45% of patients are thinking about suicide. this study was in agreement with wilson. there is a certain relationship between depression and suicide as the ratio between the proportion of suicide between depressed than others to reach,7 and shows that 41% of patients fear for their health physical pain to the point of worrying. this study was in agreement with8, and the presence of a significant relationship between social isolation and pain dysfunction. the study shows that the necessity of therapeutics to normalize the overlap style that differs from the study other group.9 conclusion the result of this study concluded that: 1. depression was high in study than other studies, higher prevalence of depression in adolescent patients with age between 17 and 18 years (46%). 2. 50% of patients feel the sadness and depression. 3. 50% of patients feel unhappy to the degree painful. 4. 40% of patients feel disappointed in their self. 5. 45% of patients are thinking about suicide. 6. 41% of patients fear for their physical health pain to the point of worrying. recommendation through the final result, this study recommends the following: 1. need to work on early detection of depressive symptoms to adolescences so that studies can emphasize about the negligence of the diagnostic process symptoms in this age group that leads to insanity and suicide. 2. to involve young people as possible of cultural experiences and benefit from their expertise. since this leads to self-promote as well as reduces the decrease of pessimism and isolation. this would alleviate the depressive symptoms. 3. attention to psychosocial and social support for young people, by creating a state of confidence as well as through the establishment of altrohed and programs that reduce the cases of grief and depression symptoms. 4. work on understanding to explain emotions and its problems, difficulties experienced, discuss the reasons and fears concerned, confusion and help him his administration at all levels. conflict of interest none. n references 1. american psychiatric association. diagnostic and statistical manual of mental disorders: dsm-5. 5th ed. washington, dc: american psychiatric association; 2013. 2. committee on psychosocial aspects of child and family health and task force on mental health. policy statement—the future of pediatrics: mental health competencies for pediatric primary care. pediatrics. 2009;124(1):410–421. 3. borowsky lw, ireland m, resnick md. adolescent suicide attempts: risks and protectors. pediatrics. 2001;107:485–493. 4. friendman ra. uncovering and epidemic-screening for mental illness in teens. n engl j med. 2006;355:2717–2719. 5. bell jl. the impact of social isolation, pain and physical dysfunction on depression among three elderly ethnic minority groups experiencing joint symptoms, umi dissertation services. 2007. 6. ibrahim as, lbrahim r. behavior in health and illness. new york, ny heart stone book; carlton press. 2009. 7. lovelance gc. anxiety among recently a admitted nursing how resistants, precursor to clinical depression mai. 2007;35:30. 8. seigmar ep, klein dc, miller wr. depression. in h. leitenberg (ed.) hand book of behavior modification and behavior therapy. 2006. new jersey: prentice hall. 9. walsin lr. depression discorders, in. jl jacobson and ah jacobson: (eds), psychiatric secrets philadelphia, pa: mosby, 2008. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 12 j contemp med sci | vol. 4, no. 1, winter 2018: 12–16 research knowledge and attitude of health-network physicians toward pediatric oral health in tehran in 2016 m. r. khami,a,b m. bonabi,b s. m. mohebbi,a,b aresearch center for caries prevention, dentistry research institute, tehran university of medical sciences, tehran, iran. bdepartment of community oral health, school of dentistry, tehran university of medical sciences. tehran, iran. correspondence to m. bonabi (email: maede.b@gmail.com). (submitted: 03 october 2017 – revised version received: 27 october 2017 – accepted: 06 january 2018 – published online: 26 march 2018) introduction children’s oral health is still a major public health concern. oral health problems in childhood can cause such problems as severe pain and discomfort, and if not controlled, lead to more serious consequences, such as malnutrition and mental health problems.1,2 the american academy of pediatric dentistry (aapd) has recommended that the first dental visit occur within 6 months of the eruption of the first tooth and no later than 12 months of age,2 but the american academy of pediatrics (aap) previously recommended the first dental visit by age three. however, the guideline was changed in 2003 to establish a dental home visit by age one for children with caries risk.3 these disc repancies resulted in variation in the practices of family practice physicians, pediatricians, general dentists and pediatric dentists. anyway, pediatric oral health care should be addressed by a multi-professional approach and sharing the responsibilities for the target population.4,5 an effective strategy can be integrating pediatric oral health advices into general health messages in public health care systems and implementation of a common risk factor approach.6 on the other hand, this approach will benefit non-affluent people who commonly get their health needs from public sector. for this strategy, training physicians in pediatric oral health is of great importance.7 nevertheless, previous studies have shown the lack of oral health content in medical school programs and residency curricula in many countries.8,9 moreover, physicians have reported to have insufficient information on oral health care.3,4,8–14 therefore, the aim of our study is to investigate knowledge and attitude of pediatric oral health care among public health networks’ physicians in non-affluent areas of tehran, as well as their self-perceived need to gain oral health information. materials and methods sampling the study population was a sample of general practitioners (n = 107) working in primary health centers in non-affluent areas of tehran, which were selected by census sampling method. questionnaire and variables the data collection tool was a self-administered anonymous questionnaire, content validity and reliability of which had been evaluated in former studies.15,16 the questionnaire requested information on demographic characteristics (age, gender), as well as the following items: practice background practice background questions comprised years of practical experience, practice type (private or public), weekly hours of clinical practice and number of daily patients’ visits. knowledge and attitude toward pediatric oral health knowledge part contained four multiple choice questions and 10 five-item likert scale questions, which were ranged from strongly agree to strongly disagree, and were scored from 1 to 5. by summing the scores, the final scores, with the range of 0–14 were calculated and sub-grouped into quartiles for knowledge. respondents with a final score in the highest quartile were considered as those with high knowledge. questions included the timing of primary and permanent tooth eruption, the time/age to begin tooth cleaning and brushing for children and use of fluoride (toothpaste and sealant) for them, transmission of the bacteria that causes dental issn 2413-0516 objective previous studies have shown the lack of oral health content in medical school programs. the aim of our study is to investigate knowledge and attitude of pediatric oral health care among public health physicians in non-affluent areas in 2016. methods our sample was public health physicians in non-affluent areas of tehran (n = 107) and the data collection tool was a selfadministered questionnaire. knowledge and attitude toward pediatric oral health, tendency and perceived necessity to gain oral health information, demographic characteristics and practice background were sections of our questionnaire. results the response rate was 79%. knowledge scores of 19.5% and attitude scores of 45.3% of the participants were in the highest quartile while the correspondent highest quartile scores for the necessity and tendency of gaining oral health information were 74.1 and 13%, respectively. those with more knowledge of pediatric oral health, feeling more necessity and reporting more tendency to gain oral health information were more likely to have positive attitude toward pediatric oral health. conclusion the irrefutable lack of pediatric oral health care knowledge was seen among public health physicians of non-affluent areas of tehran. if an understanding of preventive care in children become more relevant among public health physicians, they will show better dental health. keywords attitude, health-network, knowledge, pediatric oral health, physician m. r. khami et al. 13j contemp med sci | vol. 4, no. 1, winter 2018: 12–16 research knowledge and attitude of health-network physicians decay, the effects of pacifier sucking and mouth breathing, and the advantages of sealant therapy. in attitude part, there were seven five-item likert scale questions, which ranged from strongly agree to strongly disagree and scored from 1 to 5. calculating final scores, with the range of 0–40, and setting quartiles were done as explained above. questions included physicians’ opinions about oral health care and enquired preventability of dental caries and periodontitis, the duty of physicians about examining children’s oral cavity, effectiveness of routine dental visits in preventing dental disease, importance of physicians’ role in preventing oral diseases, association of oral health problems and general health problems, and tendency to implement preventive oral health activities. tendency and necessity to gain oral health information physicians’ opinion about “tendency” and “necessity” to gain oral health care information was measured in the questionnaire by three multiple choice questions. tendency to gain oral health information among physicians was assessed through a five-point likert scale question which was scored from 0 (not intended) to 4 (highly intended). necessity to gain oral health information was assessed by two questions, each scored from 1 to 5. the necessity score was calculated by summing up the scores of these two questions, ranging from 1 to 10. statistical analysis all numerical data were entered and analyzed using the statistical package for social sciences (spss version 21). initial descriptive statistics were frequency, mean and standard deviation. chi-square test served to assess the statistical significance of differences in frequencies between subgroups. also, logistic regression models were fitted to the data to assess the association of outcome measures with interpretive factors and to compute odds ratios (or) and 95% confidence intervals (ci). ethical considerations the study was considered by research ethic committee in tehran university of medical sciences (tums). the questionnaires were anonymous. there was no obligation to participate in the study. results background from 107 physicians, 85 returned the questionnaire and the overall response rate was 79%. the mean age of the physicians was 41.5 (std = 7.8, range 27–61), 64 (75.3%) were female and 67 (79%) just worked in public health sector (table 1). knowledge and attitude toward pediatric oral health a great majority (89.2%) of participants identified dental plaque as the strongest predictor of dental caries in future and 87.1% of them mentioned fluoride as caries-preventive component of tooth-pastes. however, just 10.6% agreed that pacifier sucking in children under 4-years old is not a risk factor for maxillofacial anomalies; and one third (32%) identified transmission of the bacteria from mother to child as a cause of tooth decay. the mean knowledge score was 7.8 (std = 1.9, range: 3–12). knowledge scores of 19.5% of the participants were in the highest quartile. regarding attitude, the highest mean score (4.69) referred to the statement “prevention is more important than other activities”; and the lowest mean score (3.69) referred to the statement “as physician i would like to implement preventive oral health activities.” the mean attitude score was 32.47 (std = 5.6, range: 10–40). attitude scores of 45.3% of the participants were in the highest quartile. figure 1 shows the percentages of respondents with favorable answers to attitude questions. as it can be seen in table 2, older physicians were more likely to acquire an attitude score in the highest quartile (p = 0.015). tendency and necessity to gain oral health information the mean of tendency scores was 2.52 (std = 0.9, range: 0–4) and the mean of necessity scores was 8.11 (std = 1.08, range: 5–10). the correspondent highest quartile scores for the necessity and tendency of gaining oral health information were 74.1 and 13% respectively. table 3 shows attitude and knowledge status of respondents according to their tendency and necessity to gain oral health information. table 4 shows factors associated with attitude of respondents though a logistic regression model. fig 1. the percentages of respondents with favorable answers to attitude questions. table 1. characteristics of non-affluent health networks’ physicians of tehran (n = 85) n* (%) gender male 21 (24.7) female 64 (75.3) working sector public (only) 67 (78.8) public and private 17 (20) working experience (years) 0–5 21 (24.7) 6–14 26 (30.6) 15–30 30 (35) age 22–36 18 (21.2) 36–57 61 (71.8) *working sector, working experience and age had some missing. the total number of respondents was 85. 14 j contemp med sci | vol. 4, no. 1, winter 2018: 12–16 knowledge and attitude of health-network physicians research m. r. khami et al. table 2. knowledge and attitude of non-affluent health networks’ physicians of tehran (n = 85) according to their background characteristics highest knowledge score (quartile 4) n (%) p-value* highest attitude score (quartile 4) n (%) p-value* age 22–36 3 (3.8) 1.00 5 (6.3) 0.015 36–57 12 (15.2) 38 (48.1) gender male 3 (3.5) 0.75 11 (12.9) 1.00 female 13 (15.3) 35 (41.2) working sector public + private 4 (4.8) 0.73 12 (14.3) 0.095 public only 12 (14.3) 33 (39.3) working experience (years) 0–5 5 (6.1) 0.77 8 (9.8) 0.2 6–14 4 (4.9) 14 (17.1) 15–30 7 (8.5) 22 (26.8) *chi–chi-square test was used to evaluate statistical differences between subgroups. table 3. knowledge and attitude of non-affluent health networks’ physicians of tehran (n = 85) according to their tendency and necessity to gain oral health information highest knowledge score (quartile 4) n (%) p-value* highest attitude score (quartile 4) n (%) p-value* necessity to gain oral health information high (quartile 4) 12 (14) 0.92 41 (48.2) 0.001 low (quartiles 1–3) 4 (4.7) 5 (5.9) tendency to gain oral health information high (quartile 4) 13 (15) 0.44 9 (10.6) 0.058 low (quartiles 1–3) 3 (3.5) 37 (43.5) total (%) 16 (18.8) 46 (54.1) *respondents reporting high necessity to gain oral health information were more likely to acquire an attitude score in the highest quartile (p = 0.001). table 4. factors associated with attitude of non-influent tehran health networks’ physicians (n = 85) by a logistic regression model b se p-value exp (b) 95% ci for exp (b) lower upper age 0.082 0.108 0.445 1.086 0.879 1.341 gender (male) −0.904 0.776 0.244 0.405 0.088 1.853 working sector (private and public) −0.792 0.871 0.364 0.453 0.082 2.499 working experience −0.006 0.107 0.955 0.994 0.807 1.225 knowledge 0.381 0.180 0.034 1.464 1.028 2.083 tendency for oral health information 0.839 0.383 0.028 2.315 1.093 4.902 necessity to get oral health information 0.856 0.347 0.014 2.354 1.192 4.648 m. r. khami et al. 15j contemp med sci | vol. 4, no. 1, winter 2018: 12–16 research knowledge and attitude of health-network physicians those with more knowledge of pediatric oral health (or = 1.464, 95% ci: 1.028–2.083), feeling more necessity (or = 2.35, 95% ci: 1.192–4.64) and reporting more tendency (or = 2.31, 95% ci: 1.093–4.9) to gain oral health information were more likely to have positive attitude toward pediatric oral health. discussion the present study investigates knowledge and attitude of pediatric oral health care among public health networks’ physicians in non-affluent areas of tehran. according to the results, attitude toward pediatric oral health among participants was associated to their age, their knowledge, and their motivation toward the subject. although, this is not the first study on health network physicians in iran to assess their knowledge and attitudes toward oral health care, our results can provide perspectives toward the formulation of at least, pediatric oral health care programs for medical students or continuing educational programs for physicians. although, our relatively high response rate (79%) speaks for representativeness of the sample, our results are subject to cautious interpretation as we used self-administered questionnaire for data collection. this is due to social desirability bias.17 on the other hand, our participants were all professionals, which decreases the probability of misconceptions and errors occurring in questionnaire surveys on lay populations.18 in agreement to rabiei et al.15 study on physicians and primary health nurses,16 the present study showed lack of knowledge in the field of pediatric oral health care among the primary care physicians working in the non-affluent health centers in tehran, as just 19.5% of the participants were in the highest quartile of knowledge score. results highlighted that most of our respondents (72%) consider their role as important in preventing oral diseases among children. the corresponding percentage among nurses in rabiei et al.15 study was 40%. on the other hand, this finding was in concurrence with that of di giuseppe et al.16 study in italy in which, 68% of physicians mentioned that they should examine oral cavity and teeth through their routine patients visit. in concordance to rabiei et al.19 study, a great majority of our respondents (85%), believed that prevention is prior to treatment in pediatric dentistry. necessity and tendency to gain oral health information had a considerable difference among respondents (74% vs. 13%) which was similar to rabiei et al.15 study in the field of necessity that reported majority of physicians were aware of their lack of information on oral health and reported being willing to receive more education. although, physicians knew about the lack of their oral health knowledge, they may think if they learn more in this field their duties will be extended. in the present study, being older (p = 0.015) was in association with higher scores of oral health attitude. this may reflect changes in practice and training of physicians in iran. moreover, higher attitude scores among participants working just in public sector compared to the others were near to significant (p = 0.095). this may relate to preventive mission of public health centers and the effect of socio-economic status of referring patients. the present study was conducted in non-affluent parts of tehran and most of their patients cannot afford private dental services which will increase physicians’ exposure to oral health problems and consequently improve their attitude toward oral health. in rabiei et al.15 study, physicians who worked in both public and private sector had better knowledge. moreover, higher attitude scores were connected to higher “necessity to gain information” which may be a reflection of this fact that more understanding leads to more temperament for learning. as last tables showed, “higher knowledge”, “necessity and tendency to gain oral health information” were significantly connected to higher levels of pediatric oral health attitude among physicians of our study. in rabiei et al.’s15 study, attitude was connected to working area, willingness to receive information and oral health care knowledge among nurses of health networks of tehran in 2012. conclusion the irrefutable lack of pediatric oral health care knowledge was seen among health network physicians of non-affluent areas of tehran. beside they demonstrated positive attitude and tendency to gain oral health care information. if an understanding of preventive care in infants and children become more relevant among primary care physicians, children and adults will show better dental health. acknowledgment this study has been supported by research center for caries prevention, dentistry research institute, tehran, iran. conflicts of interest disclosure all authors state any actual or potential conflicts of interest with regard to the present study. n references 1. the american academy of pediatric dentistry. available from: http://www. aapd.org/. 2. american academy of pediatric dentistry: aapd reference manual, 2003–2004. pediatr dent. 2003;25. 3. hale kj. oral health risk assessment timing and establishment of the dental home. pediatrics. 2003;111:1113–1116. 4. petersen pe. global policy for improvement of oral health in the 21st century—implications to oral health research of world health assembly 2007, world health organization. community dent oral epidemiol. 2009;37:1–8. 5. seymour ra, rudralingham m. oral and dental adverse drug reactions. periodontol 2000. 2008;46:9–26. 6. sheiham a. oral health, general health and quality of life. bull world health organ. 2005;83:644. 7. mouradian we. the face of a child: children’s oral health and dental education. j dent educ. 2001;65:821–831. 8. krol dm. educating pediatricians on children’s oral health: past, present, and future. pediatrics. 2004;113:e487–e492. 9. lewis cw, grossman dc, domoto pk, deyo ra. the role of the pediatrician in the oral health of children: a national survey. pediatrics. 2000;106:e84. 10. dela cruz gg, rozier rg, slade g. dental screening and referral of young children by pediatric primary care providers. pediatrics. 2004;114:e642–e652. 11. ismail ai, nainar s, sohn w. children’s first dental visit: attitudes and practices of us pediatricians and family physicians. pediatr dent. 2003;25:425–430. 12. sanchez om, childers n, fox l, bradley e. physicians’ views on pediatric preventive dental care. pediatr dent. 1996;19:377–383. 16 j contemp med sci | vol. 4, no. 1, winter 2018: 12–16 knowledge and attitude of health-network physicians research m. r. khami et al. 13. tsamtsouris a, gavris v. survey of pediatrician’s attitudes towards pediatric dental health. j pedod. 1990;14:152. 14. koranyi k, rasnake l, tarnowski k. nursing bottle weaning and prevention of dental caries: a survey of pediatricians. pediatr dent. 1991;13:32–34. 15. rabiei s, mohebbi sz, patja k, virtanen ji. physicians’ knowledge of and adherence to improving oral health. bmc public health. 2012;12:855. 16. di giuseppe g, nobile cg, marinelli a, angelillo if. knowledge, attitude and practices of pediatricians regarding the prevention of oral diseases in italy. bmc public health. 2006;6:176. 17. sjöström o, holst d. validity of a questionnaire survey: response patterns in different subgroups and the effect of social desirability. acta odontol scand. 2002;60:136–140. 18. helöe la. comparison of dental health data obtained from questionnaires, interviews and clinical examination. eur j oral sci. 1972;80:495–499. 19. rabiei s, mohebbi sz, yazdani r, virtanen ji. primary care nurses’ awareness of and willingness to perform children’s oral health care. bmc oral health. 2014;14:26. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.03201803 237j contemp med sci | vol. 3, no. 10, spring 2017:237–238 letter to editor dear editor-in-cheif this paper helps to find best methods for improving and rehabilitating health services in iraq. it is not meant to be comprehensive but to stimulate the reader to consider the importance of allocation of resources appropriately, to encourage the development of research programs, data collection, computerisation, and data analyses and to implement best methods to allocate financial and human resources. having stated the aims of this letter one needs to consider the: 1. background of this work, 2. strategy and 3. tactics to be implemented to achieve our goals. if we take the definition of health by the world health organisation in its broader sense in its 1948 constitution as ‘a state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity’, and the definition of health resources allocation, which states that all materials, personnel both teaching and care providers, medical facilities, funds, and anything else that can be used for providing health care and services, then we would understand the importance of resource allocation.1 background of health services in iraq since the opening of the first medical school, the royal college of medicine “al-albeit university of iraq” in the 20s of last century by the british, occupiers of iraq, and followed many years later by mosul medical school as well as the school of nursing. iraqi health services, which is a mix of national health and private medical services. iraqi hospitals continued to improve from the 20s until 1958. unfortunately, following the revolution of 1958, the health services went between development and decay. the latter was prominent in the late 70s when the iraqi government interfered with the teachings of medicine by expelling high calibre teachers from the medical schools. in addition, the other degrading factor was recruiting the newly graduate doctors led to many wars as privates. these untrained junior doctors suffered a lot of psychological and health ailments. the level of the medical schools and training deteriorated to unacceptable levels. the national health hospitals due to lack of appropriate management and corruption paid heavily. after the 2003 us occupation of iraq, the iraqi health services received its final knock out with unprecedented corruption in all aspects of health services.2 there was also destruction to major pharmaceutical industries in samara and mosul. the damage to infrastructure, training of jaffar allawi consultant endocrinologist diabetologist – queen mary’s hospital – st georges hospital teaching, roehampton lane, london sw15 5pn, united kingdom. (email: jaffar.allawi@stgeorges.nhs.uk). doctors and nurses at the under and postgraduate levels, the standards of hospitals, has led to serious health care issues. health care resources in iraq all national health government hospitals are built and financed by the iraqi government. individual/s investors are the main financers for the private sector hospitals. the latter has mushroomed in numbers before the american occupation in 2003. following the occupation, many private hospitals were either sold to other investors or closed. the iraqi government has an allocation of funds for new hospitals together with their supply of equipment and maintainance. also, there are many religious organisations like al attabh al hussainiah and al abbassia, which have invested in nonprofit making hospitals in different parts of iraq. then, trained personnel required for these hospitals were appointed. unfortunately, the latter has not been adequately addressed, i.e. the level of experience and training. an important fact should be mentioned here is the number of doctors, dentists, pharmacists who are highly qualified, well trained who work in europe, us, and england has increased. the number of these medical personnel is estimated to be over 30,000. a serious improvement to health care is to consider repatriation but off course at a cost.3 the strategy to improve health care services in iraq from the definition stated earlier, one needs to allocate the right sum of funds to the needs of the health care service whether teaching or otherwise. the funds and requirements are almost always not clear in iraq due to insufficient data provided by the ministry of health. therefore, it is paramount that data should be collected and analysed to prepare the application for funds correctly. it is well known that well-informed people make better decision: 1. collecting data, entering a capable data base, storing data preferably in cloud storage, securing the data with passwords and antiviral software. 2. the data should be cleaned and entered using a statistical package like bmdp, sas, spss, and stata. 3. high calibre and well-trained statisticians should analyse the data to be informative to give idea of i) number of population, ii) male–female ratios, iii) age quartiles, iv) disease prevalence, v) population of urban rural or both and finally vi) what type of medical care services can be provided. allocating resources in health care services journal of contemporary medical sciences 238 j contemp med sci | vol. 3, no. 10, spring 2017:237–238 jaffar allawiletter to editor preparation for application for funds for a medical service/s one needs to study populations and their needs for care by arranging epidemiologic survey methods, such as home to home data collection, school pupils, private companies, and government offices including police and military. a detailed analysis on the collected data can be done as pilot studies to start with the help of medical students and nurses. following pilot studies, one can start a more comprehensive data collection for larger cohort. prevalence of disease and its management can help to establish costs of doctors and nurses point of care and follow up care as well as costs of medications. this would be ideal for chronic conditions like hypertension, diabetes, rheumatoid arthritis and chronic kidney disease as an example.4 teaching doctors, dentists, pharmacists, and nurses on repeated visits to iraq and meeting the new generation nurses and doctors, one feels the big gap between older education and training. the recent graduates training and level of education are not adequate that can be easily noticed. also, the management of medicines in private hospitals is not appropriate. most medicines have unknown source of manufacturing and expiration is doubtful. in this setting, it is very important to establish recertification, appraisal, and revalidation annual exams for all personnels in medical practice including dentists, pharmacists, nurses and tetanisations. funds should be allocated for this purpose. hospitals and medical institutions almost all hospitals in iraq need extreme rehabilitation and maintenance. these institutions lack modernisation and proper setting for managing patients. they are also very poorly equipped and standard of cleanliness and hygiene is again questionable. here, again the need of improvement is paramount. allocation of fig. 1 the university insignia. funds to improve may require employing european or american management organisations and part of these contracts should aim at training iraqi personnel. allocating resources from the above, the iraqi government and the ministry of health with the help of ministry of finance and banks should prepare holistic feasibility studies, project management and time and allocation of resources. extreme penalties on contractors should be implemented on non-performing contractors. the help of many european and us government is available if the will of the iraqi’s to help themselves is established. conclusion iraq health services continued to deteriorate for many years now it has reached its nadir. efforts of doctors, banks, ministry of health, universities with active teams should plan a strategy to improve the ailing health services in iraq. allocation of funds towards effective service is the responsibility of all iraqis and not just the government of iraq. n fig. 2 al majediah hospital 1930 later the royal hospital. references 1. iglehart, john k. revisiting the canadian health care. new engl j med. 2000;342:2007–2012. 2. albujeer an, taher a. dental education and oral health service in iraq. iranian j pub health. 2017;46: 713–714. 3. resource allocation in healthcare: implications of models of medicine as profession. eileen-henner w. kluge, phd @ www.medscape.com 4. detsky as, stricker sc, mulley ag, thibault ge. prognosis, survival and the expenditure of hospital resources for patients in an intensive care unit. n eng j med. 1981;305:667–672. dx.doi.org/10.22317/jcms.06201708 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 273j contemp med sci | vol. 3, no. 11, summer 2017: 273–277 original adepartment of pharmacology & therapeutics, faculty of medicine, university of kufa, iraq. bal-najaf center for cardiothoracic surgery, najaf, iraq. cministry of health & environment, al-najaf health directorate, iraq. ddiwaniya teaching hospital, diwaniya, iraq. eschool of medicine, university of tasmania, tas, australia. correspondence to hayder al-aubaidy (email: h.alaubaidy@utas.edu.au). (submitted: 13 may 2017 – revised version received: 16 june 2017 – accepted: 26 june 2017 – published online: 26 september 2017) objective this study highlights the protective effects of montelukast on myocardial ischemic reperfusion injury induced by cardiopulmonary bypass during valve replacement surgery. methods a total of 60 patients with valvular disease undergoing elective valve surgery were enrolled in this randomized single-blinded study. participants were divided into two main groups: montelukast-treated group consisted of 30 patients who were given 10 mg montelukast sodium (singulair®, msd, usa) tablet, once daily at bedtime for 3 days before valve surgery. control group consisted of 30 patients who underwent valve surgery without taking montelukast tablets. blood samples were collected at following times (t 0 ; t 1 before aortic cross clamp; t 2 after aortic cross clamp; and t 3 24 h after the surgery), for measuring several inflammatory markers. ejection fraction (ef) was measured before surgery and three months after surgery. pulmonary functions were measured before and after the surgery in both study groups. results there were significant increase in the levels of tnf-a, il-6, a2 macroglobulin/creatinine ratio and ctni, in the control group compared to the montelukast-treated group among different study times, (p < 0.05). in addition, the ef was significantly higher in the montelukast-treated group after the valve surgery, (p < 0.05). levels of forced vital capacity (fvc), forced expiratory volume 1 (fev 1 ), and fev 1 /fvc ratio where significantly higher in the montelukast-treated group than the control group, (p < 0.05). conclusion this study shows the benefits of using pre-surgical montelukast supplement in ameliorating the inflammatory process in patients undergoing cardiopulmonary bypass during valve replacement surgery. keywords montelukast, mitral and aortic valve replacement surgery, ischemia reperfusion injury, interleukin-6, cardiac troponin 1, tumor necrotic factor-alpha, alpha 2 macroglobulin/creatinine, ejection fraction, forced vital capacity (fvc), forced expiratory volume in one second (fev 1 ), fev 1 /fvc ratio introduction cardiopulmonary bypass (cpb) is an important procedure, which is routinely performed as part of a classical cardiac surgery and support of life,1 but it is associated with injury that may induce pathological changes in the form of systemic inflammatory response syndrome (sirs) or multiple organ dysfunction syndrome (mods).2 sirs remains as a distress in open-heart surgery, and lack of adequate patient screening is an ongoing problem.3,4 it is particularly noteworthy that sirs is initiated by many factors including surgical trauma, reperfusion of ischemic organ and cpb.4 the cpb is the major factor for initiating sirs since off-pump cardiac surgery has been shown to significantly reduce inflammatory response.5,6 other related factors triggering the inflammatory response including hemodilution; electrolyte imbalance; pharmacological agents which are used during surgery; myocardial cardioplegic arrest; formation of heparin– protamine complex; and the release of endothelin and the expression of adhesion molecule on leukocyte and endothelium.5,6 there are evidences supporting that cpb can induce the activation of most of the body’s major host defensive processes, include the activation of various complements, coagulation factors, kinins, fibrinolysis, leukocytes, platelets and inflammatory cytokines.7 this study aims to evaluate the possible protective effects of montelukast as a selective cystienyl leukotriene-1 receptor antagonist in the myocardial ischemic reperfusion injury induced by heart valve replacement surgery. patients and methods sixty patients (23 males and 37 females) with valvular heart disease undergoing elective valve surgery were randomly included in this single-blinded clinical trial. the study was conducted at the cardiothoracic center, al sadir medical city, najaf, iraq between april 2015 and december 2016. participants were classified into two main study groups: group 1 montelukast-treated group (mk group) included 30 patients who underwent valve surgery (14 patients had a mitral valve replacement, mvr) and (16 patients had an aortic valve replacement, avr). participants in this group were given 10 mg montelukast sodium (singulair®, msd, usa) tablet, once daily at bed time for 3 days before valve surgery.8,9 group 2 (control group, c group) included 30 participants who underwent valve replacement surgery (17 patients had a mitral valve replacement, mvr) and (13 patients had an aortic valve replacement, avr). participants in this group had no montelukast supplement, and they were considered as controls. blood samples were collected at following times, (t0 after anesthesia; t1 before aortic cross clamp; t2 after aortic cross clamp; t3 24 h after the valve replacement issn 2413-0516 attenuation of acute systemic inflammatory response after valve surgery najah r. hadi,a fadhil g. al-amran,b alaa a. naeem,c ali f. abd alsaheb,d mohammed a. alturfy,b waleed k. fakher,b yaser q. majeed,b nada r. alharis,a hayder a. al-aubaidy e 274 j contemp med sci | vol. 3, no. 11, summer 2017: 273–277 montelukast in myocardial ischemia original hayder al-aubaidy et al. surgery), for the measurements of the following inflammatory biomarkers (tumer necrotic factor alpha (tnf-a); interleukin 6 (il-6); alpha 2 macroglobumin over creatinine (a2 macroglobulin/creatinine) ratio and cardiac troponin 1 (ctni). for all participants, serum samples were separated and stored at −80°c until analysis. the determination of cytokines was formed using an elisa assay (cloud-clone corp., usa). bronchial wash was collected from each participants before the valve replacement surgery and after the completion of the operation in order to determine the levels of il6 and tnfa. echocardiography was also performed to determine the ejection fraction (ef) before the surgery and was repeated three months after the surgery. pulmonary function test was undertaken 2 days before the surgery and 10 days after the surgery in order to evaluate the lung protective effects of montelukast in non-asthmatic participants to determine the levels of the forced vital capacity (fvc); forced expiratory volume 1 (fev 1); and the fev 1/fvc ratio as measured by spirometer. statistical analysis data were analyzed to determine the mean and standard error of the mean using statistical package for social sciences (spss) version 20 software for windows. the study used independent sample t-test, paired t-test and chi-squared test for categorical variables. a p-value of ≤0.05 was considered significant. results participants of the two study groups were comparable for age, gender and comorbidities (table 1). tumor necrotic factor alpha (tnf-`) concentration serum levels of tnf-a were significantly lower in the montelukast-treated group compared to the control group, (p < 0.05), at different study times: before anesthesia; before aortic cross clamp; after aortic cross clamp and 24 h after surgery as shown (fig. 1). interleukine 6 (il6) concentration the il-6 levels were significantly (p < 0.05) lower in montelukast-treated group as compared to the control group at all study times (fig. 2). cardiac troponin i (ctni) the concentrations of ctni were significantly lower in the montelukast-treated group compared to the control group at the all selected study times, (p < 0.05), (fig. 3). alpha 2 macroglobulin/creatinine ratio (a2m/cr) similarly, the levels of the a2m/cr ratio were significantly (p < 0.05) lower in the montelukast-treated groupy times (fig. 4). ejection fraction (ef) the ef was significantly higher in the montelukast-treated group compared to the control group after the valve replacement surgery, (p < 0.05), (fig. 5). fev 1 /fvc ratio this study showed a non-significant difference in fev1/fvc ratio between the two study groups before the valve replacement surgery. however, the ratio became statistically significant, (p < 0.05), in the montelukast-treated group as compared to the control group (fig. 6). correlation between il-6 and fev 1 /fvc ratio there was a significant negative correlation between the il-6 in the bronchial wash and fev1/fvc ratio, (r = −0.675, p < 0.001), for all participants (fig. 7). correlation between tnf-` and fev 1 /fvc ratio similarly, these was a significant negative correlation between the levels of tnf-a in bronchial wash and the fev 1/fvc ratio, (r = −0.708, p < 0.001), (fig. 8). table 1. demographic characteristics of the participants included in this study. data are represented as mean ± standard error of mean. p considered to be significant at or below 0.05. variable mk group (n = 30) c group (n = 30) age/years 48.93 ± 1.68 48.43 ± 1.94 gender (male/female) 13 (43.3%)/17 (56.7%) 10 (33.3%)/20 (66.7%) body mass index (kg/m2) 27.1 ± 3.9 26.1 ± 2.6 smoking/non smoking 20/10 21/9 fasting plasma glucose (mmol/l) 5.2 ± 1.7 5.7 ± 2.3 hypertensive 6 7 systolic blood pressure (mm hg) 145.5 ± 15 143.7 ± 16.8 diastolic blood pressure (mmhg) 86.8 ± 14 86.5 ± 11.4 heart rate (beats/min) 85.6 ± 17.5 84.3 ± 12.6 blood urea (mg/dl) 26.2 ± 8.5 27.5 ± 9 serum creatinine (mg/dl) 0.78 ± 0.09 0.72 ± 0.1 *significant difference between the two study groups at any time at p equal or less than 0.050. fig. 1 tnf-` concentration at different study times into two groups; montelukast-treated group and control group. hayder al-aubaidy et al. 275j contemp med sci | vol. 3, no. 11, summer 2017: 273–277 original montelukast in myocardial ischemia fig. 2 il-6 concentration at different study times into two groups; montelukast-treated group and control group. fig. 3 ctni concentration at different study times into two groups; montelukast-treated group and control group. fig. 4 `2m/cr concentration at different study times into two groups; montelukast-treated group and control group. fig. 5 ejection fraction before and after valve surgery in montelukast-treated group and control group. fig. 6 fev 1 /fvc before and after valve surgery in montelukast treated group and control group. discussion leukotrienes (lts) are defined as bioactive proinflammatory molecules that are produced by the 5-lipoxygenase pathway from arachidonic acid metabolism in many cells, including epithelial cells, fibroblasts, myoblasts, smooth muscle cells, basophils, eosinophils, neutrophils, macrophages, and lymphocytes. they play potent inflammatory roles in human body response.10 these cytokines, particularly tnf-a, are early regulators of the immune response and can induce the release of secondary cytokines, such as il-6 and tnf-a, which provokes neutrophil-mediated tissue injury by acting on endothelial cells and other neutrophils.10 this study focussed on the anti-inflammatory effects of specific cytokines, (tnf-a and il-6), in ameliorating the inflammatory response after elective heart valve surgery. both levels of these cytokines were significantly lower, (at all times), in the montelukast-treated groups as compared to the control group, p < 0.05, (figs 1 and 2). this proves the importance of using anti-inflammatory 276 j contemp med sci | vol. 3, no. 11, summer 2017: 273–277 montelukast in myocardial ischemia original hayder al-aubaidy et al. fig. 7 correlation between il-6 and fev 1 /fvc. fig. 8 correlation between tnf-` and fev 1 /fvc. supplements prior to any invasive heart surgery to reduce the inflamatory result and improves the outcomes.11,12 our findings also indicated that montelukast is able to reduce the levels of ctni (fig. 3). this is in agreement with the previous studies and confirm that montelukast, at high doses, might have cardioprotective effects during endotoxemia attributed to its antioxidant and anti-inflammatory properties.13,14 it is also effective in reducing levels of lipopolysaccharides (lps) induced heart injury and decreasing malondialdehyde (mda) levels, one of lps-end products.13 malondialdehyde (mda) also has a potent role in inflammation, and it can increase the antioxidant glutathione (gsh) contents in heart tissue.13 medical treatment with montelukast supplements was also beneficial in increasing the percentage of the ef among the montelukast-treated group as compareed to the control group (fig. 5). this is because montelukast has the ability of reducing the oxidative stress and apoptosis and providing beneficial effects on myocardial remodeling.15,16 this study also showed significant negative correlations between the ratio of fev1/fvc and the il6, tnfa (figs 7 and 8), respectively. this proves the pulmonary protective effects of montelukast in reducing the inflammatory process and improving overall lung functions.17,18 conclusion this study shows the benefits of using pre-surgical montelukast supplement in ameliorating the inflammatory process in patients undergoing cardiopulmonary bypass during valve replacement surgery. conflict of interest authors wish to declare that there is no conflict of interest, including specific financial interests and relationships and affiliations relevant to the subject of the manuscript, exist with this study. n references 1. charbonney e, wilcox e, shan y, d’empaire pp, duggal a, rubenfeld gd, et al. systemic angiopoietin-1/2 dysregulation following cardiopulmonary bypass in adults. future sci oa. 2017;3:fso166. 2. xie xj, tao ky, tang ml, du l, an q, lin k, et al. [establishment and evaluation of extracorporeal circulation model in rats]. sichuan da xue xue bao yi xue ban. 2012;43:770–774. 3. sablotzki a, friedrich i, muhling j, dehne mg, spillner j, silber re, et al. the systemic inflammatory response syndrome following cardiac surgery: different expression of proinflammatory cytokines and procalcitonin in patients with and without multiorgan dysfunctions. perfusion. 2002;17:103–109. 4. al-rashid f, kahlert p, selge f, hildebrandt h, patsalis pc, totzeck m, et al. risk assessment of patients undergoing transfemoral aortic valve implantation upon admission for post-interventional intensive care and surveillance: implications on shortand midterm outcomes. plos one. 2016;11:e0167072. 5. turagam mk, mirza m, werner ph, sra j, kress dc, tajik aj, et al. circulating biomarkers predictive of postoperative atrial fibrillation. cardiol rev. 2016;24:76–87. 6. zakkar m, ascione r, james af, angelini gd, suleiman ms. inflammation, oxidative stress and postoperative atrial fibrillation in cardiac surgery. pharmacol ther. 2015;154:13–20. 7. landis rc, brown jr, fitzgerald d, likosky ds, shore-lesserson l, baker ra, et al. attenuating the systemic inflammatory response to adult cardiopulmonary bypass: a critical review of the evidence base. j extra corpor technol. 2014;46:197–211. 8. balani sk, xu x, pratha v, koss ma, amin rd, dufresne c, et al. metabolic profiles of montelukast sodium (singulair), a potent cysteinyl leukotriene1 receptor antagonist, in human plasma and bile. drug metabol dispos. 1997;25:1282–1287. 9. zhao jj, rogers jd, holland sd, larson p, amin rd, haesen r, et al. pharmacokinetics and bioavailability of montelukast sodium (mk-0476) in healthy young and elderly volunteers. biopharm drug dispos. 1997;18:769–777. 10. kuru s, kismet k, barlas am, tuncal s, celepli p, surer h, et al. the effect of montelukast on liver damage in an experimental obstructive jaundice model. viszeralmedizin. 2015;31:131–138. 11. peng j, zhou h, kuang g, xie l, tian t, liu r. the selective cysteinyl leukotriene receptor 1 (cyslt1r) antagonist montelukast regulates extracellular matrix remodeling. biochem biophys res commun. 2017;484:474–479. 12. said mm, bosland mc. the anti-inflammatory effect of montelukast, a cysteinyl leukotriene receptor-1 antagonist, against estradiol-induced nonbacterial inflammation in the rat prostate. naunyn schmiedebergs arch pharmacol. 2017;390:197–205. 13. khodir ae, ghoneim ha, rahim ma, suddek gm. montelukast attenuates lipopolysaccharide-induced cardiac injury in rats. hum exp toxicol. 2016;35:388–397. 14. wang l, he y, zhang y, zhou h, yu l, yang j, et al. effects of active components of fuzi and gancao compatibility on bax, bcl-2, and caspase-3 in chronic heart failure rats. evid based complement alternat med. 2016;2016:7686045. 15. becher um, ghanem a, tiyerili v, furst do, nickenig g, mueller cf. inhibition of leukotriene c4 action reduces oxidative stress and apoptosis in hayder al-aubaidy et al. 277j contemp med sci | vol. 3, no. 11, summer 2017: 273–277 original montelukast in myocardial ischemia cardiomyocytes and impedes remodeling after myocardial injury. j mol cell cardiol. 2011;50:570–577. 16. mueller cf, becher mu, zimmer s, wassmann s, keuler b, nickenig g. angiotensin ii triggers release of leukotriene c4 in vascular smooth muscle cells via the multidrug resistance-related protein 1. mol cell biochem. 2010;333:261–267. 17. barbosa js, almeida paz fa, braga ss. montelukast medicines of today and tomorrow: from molecular pharmaceutics to technological formulations. drug deliv. 2016;23:3257–3265. 18. lajqi n, ilazi a, kastrati b, islami h. comparison of glucocorticoid (budesonide) and antileukotriene (montelukast) effect in patients with bronchial asthma determined with body plethysmography. acta inform med. 2015;23:347–351. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201706 28 j contemp med sci | vol. 9, no. 1, january-february 2023: 28–34 original comparison of rectal suppository and intramuscular morphine for management of patients with renal colic referred to the emergency department: a randomized double-blinded controlled trial arash ardestani zadeh1, mohammadreza moonesan2, fatemeh taheri1, davood arab1*, tahmineh mokhtari3,4* 1clinical research development unit, kowsar educational, research and therapeutic hospital, semnan university of medical sciences, semnan, iran. 2department of emergency medicine, kosar hospital, semnan university of medical sciences, semnan, iran. 3hubei key laboratory of embryonic stem cell research, hubei university of medicine, shiyan, china. 4department of histology and embryology, school of basic medical sciences, hubei university of medicine, shiyan, china. *correspondence to: tahmineh mokhtari (email: mokhtari.tmn@gmail.com), davood arab (email: drdavoodarab@semums.ac.ir) abstract objectives: to compare the analgesic effects of rectal suppository morphine (rsm) with intramuscular morphine (imm) in patients suffered from renal colic referred to emergency ward (ew). methods: in a controlled, randomized, clinical trial, 74/90 patients with renal colic referred to the ew between march 2016 and march 2017 were randomly enrolled into two groups of rsm (10 mg) and imm (10 mg/ml). vital signs and severity of pain were recorded at admission time (0), 15, 30 and 60 min after treatment. results: the results showed that there was a significant decrease in vas score of rsm group compared to imm group after 30 and 60 min of administration (p < 0.05). furthermore, no significant difference was recorded in vital signs, except there was a significant decrease in heart rate (15 and 60 min) and respiratory rate (60 min) of rsm group compared to imm group (p < 0.05) and no side effects were recorded during the investigation. conclusion: in conclusion, the use of rectal route of morphine had higher efficiency compared to the im route of morphine in relieving pain of patients with renal colic. although, decreased heart and respiratory rates were recorded, the values were in normal range. as well, no major complications were recorded for each method. keywords: renal colic, morphine; pain management, suppositories, injections, intramuscular issn 2413-0516 introduction urolithiasis has been shown to be a common disease with incidence of 8–15% in north americans and europeans for centuries.1 approximately 2.4/1000 people in iran suffer from renal colic, whereas it differs from 0.5 to 2 in every 1000 ones in other countries.2 the mean cost of urolithiasis and renal colic was estimated about 2.1 billion dollars in 2000.3 recently, it has been reported that changes in individuals’ diet and lifestyle leads to increase in incidence of renal colic worldwide.4 renal colic as a frequent disorder usually presents as an acute and severe pain in the flanks due to obstruction of the urinary flow via a stone or the passage of a stone from the urinary tract, which can radiate to the abdomen and genitalia.5,6 this type of pain is often defined as the worst pain which patient has ever experienced.7,8 the mechanism of pain is associated with the enhanced pressure in the urinary tract along with construction of urethral smooth muscles and enhanced pressure in the regional blood flow.9 a wide range of analgesics are used to manage the pain. in emergency ward (ew), opioids and non-steroidal anti-inflammatory drugs (nsaids) are most frequent to relieve the pain.10 according to the literature, stable doses of opioids can provide analgesic impacts for weeks or years and efficiency, low cost, and titration possibility make them popular for managing pain.3,11 the high doses of opiates administered systemically has been shown to be related to different side effects e.g., respiratory depression, vomiting, nausea, sedation, and pruritus.12 on the other hand, the human rectum is a body cavity in which drugs can be retained and absorbed easily and is effective rather than orally, especially in cases with nausea and vomiting.13 as a most common type of opioids, morphine is an analgesic with a direct impact on the central nervous system (cns) and the most powerful analgesic drugs in managing and curing severe, acute and chronic pains.14 different routes of morphine have been used to control pain in patients following operation or in emergency department.15-17 morphine suppositories administered via rectal route are one of the newly released morphine forms. different doses of 5, 10, 20, and 30 mg of morphine can be contained in each rectal suppository. it has been shown that about two-thirds of rectal morphine can be absorbed via the gastrointestinal tract and then, it paases through portal vein to be metabolized in the liver. the maximum effects of rectal route has been recorded 20–60 min following administration.12 the administration of suppository morphine is often forgotten by clinicians seeking to use the oral routs18 and to the best of our knowledge, there was no previous study to compare the analgesic effect of rectal suppository morphine (rsm) with intramuscular morphine (imm) to relieve pain in patients with renal colic. materials and methods study population this study was a randomized, controlled, clinical trial was performed between march 2016 and march 2017. ninety patients with renal colic (18 to 55 years) referred to ew of kosar hospital (semnan university of medical sciences, semnan, iran) were randomly selected, but 74 patients were eligible to be enrolled. this trial was approved by ethics committee of (submitted: 01 december 2022 – revised version received: 22 december 2022 – accepted: 02 january 2023 – published online: 26 february 2023) mailto:mokhtari.tmn@gmail.com mailto:drdavoodarab@semums.ac.ir 29j contemp med sci | vol. 9, no. 1, january-february 2023: 28–34 a.a. zadeh et al. original suppository or intramuscular morphine for pain control in renal colic semnan university of medical sciences (5/1/2016). written informed consent form was signed by all subjects to accept that they had adequate information about the investigation. the study was registered at iranian registry of clinical trial (irct number: irct2015111825098n1). inclusion and exclusion criteria in this study, 90 patients referred to ew with main complaint of flank pain, and the patients who clinically diagnosed for renal colic were selected. the inclusion criteria were defined as all cases with acute severe flank pain that radiated to ipsilateral groin or abdomen. then, the urinary stone was confirmed through ultrasonography, computed tomography (ct) scan, or intravenous (iv) pyelography, or the patients with stone passage and the patients were selected for investigation. the exclusion criteria was described as follows: patients with history of allergy to opioids, addiction, fever ≥38ºc, unstable hemodynamic status, liver failure, cardiac or respiratory failure, evidence of peritoneal inflammation, renal failure or kidney transplantation, aortic aneurysm or dissection, pregnancy, receiving any analgesia within 6 h before the study and age <18 and >55 years. an emergency medicine specialist performed all evaluations. sixteen cases were excluded from the study due to the above exclusion criteria. randomization and intervention in this study, a convenience sampling was used to select 90 patients (74 were eligible). then, patients were randomly allocated into two groups (37 in each group) using permuted balanced block randomization in a completely random manner. for this purpose, six blocks of four were used in which the structure of each block was four-way, the combination of two methods of interference in a perfectly balanced way. to random assignment of blocks to each group a random digits table was carried out. therefore, a list of eligible participants (n = 74) was prepared and according to this list each case was randomly enrolled in the study group, respectively. no additional matching was performed and one of the investigators scheduled for randomization before the initiation of study. none of participants and data analysts knew about the groups. the treatment groups were defined as below: ss-mp group (n = 37): patients received 10 mg morphine suppository (opirec® 10 mg, aburaihan pharmaceutical co.). im-mp group (n = 37): patients received 10 mg morphine intramuscularly (im) (morphine sulfate 10 mg/ml, daou pakhsh pharmaceutical co.). procedures and evaluations data were collected using a designed checklist containing the factors as below to make the assessments: a: patient’s demographic information including age, gender and weight. b: history of similar pain, history of urinary stones and main data including vital signs (blood pressure [bp], respiratory and heart rate [hr] and axillary temperature) and severity of pain using a 10-centimeter visual analogue scale (vas). vas was used for evaluation of the pain severity was in several time points of admission time (0), 15, 30 and 60 min after administration of medications. vas is a measurement instrument that tries to measure a pain severity scored 0 (no pain) -10 clinical observation.19 clinically, the difference of vas score between 0 and 15 min (0–15), 0 and 30 min (0–30), 0 and 60 min (0–60) time points were calculated and compared in two groups of morphine routes. moreover, other varibales including hr, bp (systolic and diastolic), respiratory rate, and the side effects of drugs (secondary outcomes including drowsiness, nausea & vomiting, facial flushing, and dizziness) were investigated in defined time points. after gathering the data, the vas scores of different time points were compared. after 60 min, if the severity of pain did not relief by 50%, 5 mg/im morphine was used to relive the pain. in this study, four patients were excluded due to missing or inconsistent data (n = 3 in rsm group and n = 1 in imm group) during the study and data of 70 cases were analyzed. due to incomplete information and data in the questionnaire, patients were excluded from the study (figure 1). sample size estimation and statistical analysis using the findings of the study performed by safdar et al. (2006) reporting an average reduction of one hour of pain equal to 5.0 ± 1.6 in patients with intramuscular morphine injection compared with the average further reduction of 6.0 ± 1.6 in patients with morphine suppository in term of vas, setting the statistical power and confidence levels to be 95%, a sample size of 136 people (68 in each group) was estimated to be enough using g*power.3.1 software. but conservatively up to 7 people were added in each group to deal with possible data loss.20 data were analyzed using spss (ver. 22). mean, standard deviation, frequency and percentage were used to summarize the data in tables. the analysis of variance (anova) for repeated measures models applied to compare the two groups. fisher’s exact tests and chi-square were used to determine the differences in the qualitative data. in addition, ttest was used for evaluation of differences in the quantitative data. significant level was defined as p < 0.05. results in this study, the data of 70/74 cases (n = 3 in rsm group and n = 1 in imm group were excluded) with renal colic treated fig. 1 consort flow diagram. 30 j contemp med sci | vol. 9, no. 1, january-february 2023: 28–34 suppository or intramuscular morphine for pain control in renal colic original a.a. zadeh et al. between the two groups (p = 0.030). in addition, there were no significant differences in hr of two groups based on time points of 0 (p = 0.116) and 30 (p = 0.139), but significant decreases were seen in time point of 15 (p = 0.04) and 60 min (p = 0.014) in rsm group compared to imm group. comparing two groups showed that there was no significant difference in terms of changes in the respiratory rate (per min) between the two groups (p = 0.622). based on time points, respiratory rate was significantly decreased in 60 min (p = 0.017) in rsm group compared to the imm group. however, in the other time points no differences was observed (0: p = 0.799, 15: p = 0.291, 30: p = 0.605, and 60: p = 0.855, respectively) (table 2 and figure 1). no major complications were reported in two groups. discussion in this investigation, we aimed to compare the influence of rectal and im morphine/pain management in patients with renal colic. in ew, management of patients with colic pain is one of the most important part of caring system.21 nsaids and opioids as well as the combination of spasmolytic and anti-inflammatory drugs have been recorded to be mostly used for pain control of these patients.10,22,23 among these, narcotics such as morphine, tramadol, codeine and meperidine have long been used.20,24,25 morphine, as an opioid analgesic, has been used for pain control of patients with renal colic given by different routes.26,27 morphine administered with iv route is a drug of choice to manage pain in acute renal colic.28 nevertheless, the other routes of morphine administration, e.g., oral, im and rectal are available to relieve pain in different conditions.29,30 studies have proven that im administration of opioids has been considered to be safer than iv.31 although, oral and parenteral narcotics are used usually for pain relief in ew or following surgical procedures, these routs can exacerbate the incidence of sedation, vomiting, and nausea, which ultimately delays recovery.32-34 thus, non-parenteral route of analgesic drugs, especially their rectal route has been suggested in different studies.13,35,36 according to the literature, it has been proved that rectal route morphine results less analgesia and lower pain scores compared to its iv administration.34,37 the findings of both groups showed the similarity in the different demographic features including age, gender, weight, table 1. comparing the characteristics of patients with renal colic based on study groups groups p-value ss-mp (n = 34) im-mp (n = 36) n (%) or mean ± sd* (range) n (%) or mean ± sd (range) age (year) 37.75 ± 10.92 (18–68) 38.89 ± 10.46 (18–60) 0.329 gender (male) 24 (70.6) 29 (80.6) 0.331 weight (kg) 79.88 ± 14.17 84.3 ± 23.41 0.446 history of urinary stones 23 (67.6) 22 (61.1) 0.568 history of simmilar pain 22 (64.7) 22 (61.1) 0.756 axillary temperature ( °c) 36.76 ± 0.53 36.76 ± 0.32 0.543 opioids for pain relief 3 (8.8%) 9 (25) 0.73 sd: standard deviation; upj: ureteropelvic junction. with morphine administered via im or rectal routes were considered. the demographic features of cases with renal colic in two groups were compared (table 1). the mean age of patients in rsm group was 37.75 ± 10.92 year (18 to 55) and imm group was 38.89 ± 10.46 year (18 to 55). no significant difference was reported in the mean age of two groups (table 1, p = 0.329). as well, 24 patients (70.6%) in rsm group and 29 patients (80.6%) in imm group were male and no significant differences were seen in the distribution of sex in two groups (table 1, p = 0.331). the mean weight of patients in rsm group was 79.88 ± 14.17 kg and in imm group was 84.3 ± 23.41 kg and no differences was recorded in the weight of patients in two groups (table 1, p = 0.446). the history of urinary stones and similar pains were compared between two groups as was shown in table 1. no significant differences were reported in the distribution of history of urinary stones (table 1, p = 0.568) and similar pains (table 1, p = .756) of patients in two treatment groups. furthermore, frequency of extra doses of opioids was not significant in two groups (table 1, p = 0.073). using chi-square test, no significant differences were observed in the distribution of side effects in both groups (table 1, p = .599). all these results proved that two groups were similar in these characteristics before the treatment period. the mean vas score, systolic bp (mmhg), diastolic bp (mmhg), hr (bpm), and respiratory rate (per min) were compared in two groups (table 2, figure 2). a significant difference was observed in terms of pain intensity between two groups (p = .017). also, there were significant differences in vas score of two treatment groups according to time points of 30 (p = .037) and 60 (p = 0.027) after treatment; but no differences were recorded in time point of 0 (p = 0.58) and 15 (p = 0.083). there was a significant difference in the mean vas score difference of 0–30 (p = 0.001) and 0–60 (p = 0.001) time points; but no significant difference was recorded for mean vas score difference of 0–15 min (p = 0.083). based on the results, no significant difference was also observed between groups for systolic (p = 0.201) and diastolic pressure (p = 0.350). as well, no significant difference was seen in the mean systolic pressure (p = 0.762, p = 0.068, p = 0.072 and p = 0.232, respectively), diastolic pressure (p = 0.345, p = 0.506, p = 0.222, and p = 0.105, respectively) on different time points of 0, 15, 30, and 60. in addition, significant difference was observed in terms of changes in the hr (per min) 31j contemp med sci | vol. 9, no. 1, january-february 2023: 28–34 a.a. zadeh et al. original suppository or intramuscular morphine for pain control in renal colic table 2. comparing the vital signs and pain severity (vas) between two groups based on different time points, including admission time (0), 15, 30 and 60 min after drugs administration groups ss-mp (n = 34) im-mp (n = 36) p-value mean ± sd* mean ± sd vas score 0 8.26 ± 1.79 9 ± 1.26 0.116 15 min after treatment 5.95 ± 1.78 6.73 ± 1.97 0.139 30 min after treatment 4.32 ± 2.58 5.58 ± 2.38 0.018 60 min after treatment 2.58 ± 2.95 4.19 ± 3.03 0.081 systolic pressure (mmhg) 0 124.38 ± 10.31 127.27 ± 16.38 0.762 15 min after treatment 116.56 ± 10.44 123.64 ± 14.32 0.068 30 min after treatment 113.75 ± 11.33 120 ± 12.82 0.073 60 min after treatment 115.31 ± 9.91 119.55 ± 12.14 0.232 diastolic pressure (mmhg) 0 79.38 ± 7.0 79.32 ± 11.37 0.848 15 min after treatment 76.88 ± 6.02 77.14 ± 11.37 0.988 30 min after treatment 72.5 ± 8.37 77.5 ± 11.73 0.889 60 min after treatment 72.5 ± 7.53 77.73 ± 10.99 0.888 heart rate (bpm) 0 78.4 ± 7.59 82.55 ± 8.23 0.115 15 min after treatment 76.4 ± 6.67 81.59 ± 7.08 0.04 30 min after treatment 76.6 ± 6.6 81.86 ± 7.32 0.073 60 min after treatment 75.23 ± 6.42 81.55 ± 8.15 0.017 respiratory rate (per min) 0 18.21 ± 2.11 18.48 ± 2.6 0.779 15 min after treatment 17.14 ± 0.81 17.76 ± 2.23 0.291 30 min after treatment 16.79 ± 1.76 17.19 ± 1.91 0.073 60 min after treatment 16.93 ± 1.77 17.05 ± 2.01 0.017 *sd: standard deviation. and also history of urinary stones and similar pain. based on the findings of present study, rectal route of morphine could decrease the vas score significantly compared to the imm group. additionally, the score differences of 0–30 and 0–60 showed significant improvement in rvs group. taken together, administration of rectal route of morphine could be successful in the management of pain in patients with renal colic. although the mean heart and respiratory rate decreased in rectal route group compared to im group, these criteria were within normal range in this group, which were not a cause for concern. as well, no serious complications have been reported in these two groups. based on the literature, efficiency of different routes of morphine have been compared in a wide range of painful conditions. to show the efficiency and safety of morphine administered via rectal route in relieving pain, rahimi et al. (2016) used preemptive suppository morphine after laparoscopic cholecystectomy in a placebo-controlled study. the results from vas score proved that administration of morphine suppository was effective in analgesic requirements following laparoscopic cholecystectomy.12 in a randomized controlled trial, butler et al. (2017) evaluated the effects of belladonna and opium suppositories for reduction of pain in vaginal surgery and showed that these drugs are safe to use following this surgery.37 as well, cole et al. (1990) demonstrated that morphine hydrogel suppository appears to be effective in management of postoperative pain.38 studies to compare the different routes of morphine especially the rectal and intramuscular routes of morphine are so limited. guldbrand and mellstrom (1995) compared the rectal rout of morphine-scopolamine with im route as a premedication in healthy children scheduled for minor ent surgery. their findings indicated that administration of rectal route of drug worked better and resulted in slightly less post-operative pain and nausea. they suggested to use rectal route of morphine-scopolamine as a premedication for minor ent surgery on children as a good alternative compared to the im route.39 additionally, in a study by wilkinson et al. (1992), the effectiveness of rectal vs. oral sustained-release morphine were compared in the patients with cancer. they found no significant difference between the oral and the rectal route in measurements based on vas score or side effects. as well, they recommended to use the rectal route of morphine, when the oral route is not accessible for long time.40 the bioavailability of im morphine is roughly complete (100%), whereas the bioavailability of rectal morphine is only 50–60%.41,42 therefore, the poorer rectal bioavailability leads to lower plasma concentrations of morphine compared with the im application. however, the evidence showed that the rectal and im morphine reached the peak plasma concentration after 30 min and 1 hour, respectively, indicating that the absorption was rapid in the rectal route.43 according to the results, no major complications were recorded for two forms of administrations. the safety and 32 j contemp med sci | vol. 9, no. 1, january-february 2023: 28–34 suppository or intramuscular morphine for pain control in renal colic original a.a. zadeh et al. fig. 2 comparing the vital signs and pain severity between two groups based on different time points including admission time (0), 15 min, 30 min, and 60 min after drug administration. effectiveness of morphine suppository in relieving pain have been proven in different studies. westerling et al. evaluated the bioavailability and absorption of rectally administered morphine in 21 healthy women undergoing gynaecological operations and demonstrated that the mean bioavailability was 31% (range 12%–61%) and none of the cases showed any clinical sign of respiratory depression.43 furthermore, babul et al. (2013) compared the safety and effectiveness of controlled‐release morphine tablets and suppositories in pain management of patients with cancer and reported that controlled‐release morphine suppositories provides pain control comparable to that provided by tablets when received every 12 h at a 1/1 dose ratio, and suggested a reliable alternative approach of pain management for patients unable to take oral opioid medications.44 in an experimental study by barnhart et al. (2000), the systemic bioavailability and therapeutic plasma levels of morphine following iv and im administration as well as respiratory, cardiovascular, and analgesic values were compared in dogs. no differences were recorded in analgesia values and vital organs e.g., respiratory and cardiovascular between control and morphine groups.45 33j contemp med sci | vol. 9, no. 1, january-february 2023: 28–34 a.a. zadeh et al. original suppository or intramuscular morphine for pain control in renal colic fig. 3 the schematic summary of the study. conclusion our findings indicated that the use of rectal morphine suppositories may be more effective in diminishing pain in cases referred to ew with renal colic compared with im route in ew. since the morphine suppositories are due to no adverse impact on the vital signs of patients and also have no other major complication, we recommend to use rectal route of morphine as a safe and more effective method in relieving pain of renal colic patients in ew (figure 3). limitation one of our study limitations was that the possibility of evaluating other variables attributed to alterations of pain severity. one of these limitations is the differences in the level of education and awareness of the patients under study that may have been effective in expressing the severity of pain based on vas. on the other hand, there are several variables that affect the pain severity, which of course has not been possible to consider all of them in one study. acknowledgments we would like to thank the clinical research development unit of kowsar educational and research and therapeutic center of semnan university of medical sciences for providing facilities to this work. funding semnan university of medical sciences (no. a-10-140-4). competing interest author declares no conflict of interest.  references 1. golzari se, soleimanpour h, rahmani f, zamani mehr n, safari s, heshmat y, et al. therapeutic approaches for renal colic in the emergency department: a review article. anesth pain med. 2014;4(1):e16222. 2. basiri a, shakhssalim n, khoshdel ar, pakmanesh h, radfar mh. drinking water composition and incidence of urinary calculus: introducing a new index. iranian journal of kidney diseases. 2011;5(1):15. 3. phillips e, hinck b, pedro r, makhlouf a, kriedberg c, hendlin k, et al. celecoxib in the management of acute renal colic: a randomized controlled clinical trial. urology. 2009;74(5):994–9. 4. romero v, akpinar h, assimos dg. kidney stones: a global picture of prevalence, incidence, and associated risk factors. reviews in urology. 2010;12(2-3):e86. 5. holdgate a, pollock t. systematic review of the relative efficacy of nonsteroidal anti-inflammatory drugs and opioids in the treatment of acute renal colic. bmj. 2004;328(7453):1401. 6. faridaalaee g, mohammadi n, merghati sz. intravenous morphine vs intravenous ketofol for treating renal colic; a randomized controlled trial. emergency. 2016;4(4):202. 7. edwards je, meseguer f, faura c, moore ra, mcquay hj. single dose dipyrone for acute renal colic pain. the cochrane database of systematic reviews. 2002(4):cd003867. 8. iguchi m, katoh y, koike h, hayashi t, nakamura m. randomized trial of trigger point injection for renal colic. international journal of urology : official journal of the japanese urological association. 2002;9(9):475–9. 9. serinken m, karcioglu o, turkcuer i, ozkan hi, keysan mk, bukiran a. analysis of clinical and demographic characteristics of patients presenting with renal colic in the emergency department. bmc research notes. 2008;1:79. 10. renal colic in adults: nsaids and morphine are effective for pain relief. prescrire international. 2009;18(103):217–21. 11. larkin gl, peacock wft, pearl sm, blair ga, d’amico f. efficacy of ketorolac tromethamine versus meperidine in the ed treatment of acute renal colic. am j emerg med. 1999;17(1):6–10. 12. rahimi m, farsani dm, naghibi k, alikiaii b. preemptive morphine suppository for postoperative pain relief after laparoscopic cholecystectomy. advanced biomedical research. 2016;5. 13. de boer ag, moolenaar f, de leede lg, breimer dd. rectal drug administration: clinical pharmacokinetic considerations. clinical pharmacokinetics. 1982;7(4):285–311. 14. morgan s. intravenous paracetamol in patients with renal colic. emergency nurse : the journal of the rcn accident and emergency nursing association. 2011;18(9):22–5. 15. blankenstein tn, gibson lm, claydon ma. is intramuscular morphine satisfying frontline medical personnels’ requirement for battlefield analgesia in helmand province, afghanistan? a questionnaire study. british journal of pain. 2015;9(2):115–21. 16. walford j. comparison of intravenous morphine and paracetamol. emergency nurse : the journal of the rcn accident and emergency nursing association. 2015;23(5):24–7. 17. poonai n, datoo n, ali s, cashin m, drendel al, zhu r, et al. oral morphine versus ibuprofen administered at home for postoperative orthopedic pain in children: a randomized controlled trial. cmaj : canadian medical association journal = journal de l’association medicale canadienne. 2017;189(40):e1252-e8. 18. cole l, hanning cd. review of the rectal use of opioids. journal of pain and symptom management. 1990;5(2):118–26. 19. ducharme j. analgesia, anesthesia, and procedural sedation. tintinalli’s emergency medicine. 2015:231–8. 20. safdar b, degutis lc, landry k, vedere sr, moscovitz hc, d’onofrio g. intravenous morphine plus ketorolac is superior to either drug alone for treatment of acute renal colic. ann emerg med. 2006;48(2):173–81, 81.e1. 21. turkcuer i, serinken m, karcioglu o, zencir m, keysan mk. hospital cost analysis of management of patients with renal colic in the emergency department. urological research. 2010;38(1):29–33. 22. holdgate a, pollock t. nonsteroidal anti-inflammatory drugs (nsaids) versus opioids for acute renal colic. cochrane database of systematic reviews. 2004(1). 23. golzari se, soleimanpour h, rahmani f, mehr nz, safari s, heshmat y, et al. therapeutic approaches for renal colic in the emergency department: a review article. anesthesiology and pain medicine. 2014;4(1). 24. portis aj, sundaram cp. diagnosis and initial management of kidney stones. american family physician. 2001;63(7):1329–38. 25. nakhaei amroodi m, reza shafiee g, mokhtari t. prevalence of the shoulder dislocation due to tramadol-induced seizure. shafa ortho j. 2015;2(1). 26. beltaief k, grissa mh, msolli ma, bzeouich n, fredj n, sakma a, et al. acupuncture versus titrated morphine in acute renal colic: a randomized controlled trial. journal of pain research. 2018;11:335. 27. mangal r, higgins d, pham t. is intravenous (iv) acetaminophen as effective as iv morphine for treatment of renal colic? evidence-based practice. 2018;21(3):6. 28. etteri m, maj m, maino c, valli r. intranasal ketorolac and opioid in treatment of acute renal colic. emergency care journal. 2018;14(1). 34 j contemp med sci | vol. 9, no. 1, january-february 2023: 28–34 suppository or intramuscular morphine for pain control in renal colic original a.a. zadeh et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 29. tveita t, thoner j, klepstad p, dale o, jystad a, borchgrevink pc. a controlled comparison between single doses of intravenous and intramuscular morphine with respect to analgesic effects and patient safety. acta anaesthesiol scand. 2008;52(7):920–5. 30. borracci t, cappellini i, campiglia l, picciafuochi f, berti j, consales g, et al. preoperative medication with oral morphine sulphate and postoperative pain. minerva anestesiologica. 2013;79(5):525–33. 31. australian and new zealand college of anaesthetists and faculty of pain medicine: acute pain management: scientific evidence. 2005. 32. thomas sh. management of pain in the emergency department. isrn emergency medicine. 2013;2013. 33. rogers e, mehta s, shengelia r, reid mc. four strategies for managing opioid-induced side effects in older adults. clinical geriatrics. 2013;21(4):http://www.consultant360.com/articles/four-strategiesmanaging-opioid-induced-side-effects-older-adults. 34. butler ka, yi j, klauschie j, ryan dl, hentz jg, cornella jl, et al. 7: randomized clinical trial of postoperative belladonna and opium (b&amp;o) suppositories in vaginal surgery. american journal of obstetrics & gynecology. 2016;214(4):s459. 35. hanning cd, vickers ap, smith g, graham nb, mcneil me. the morphine hydrogel suppository: a new sustained release rectal preparation. british journal of anaesthesia. 1988;61(2):221–7. 36. butler k, yi j, wasson m, klauschie j, ryan d, hentz j, et al. randomized controlled trial of postoperative belladonna and opium rectal suppositories in vaginal surgery. american journal of obstetrics and gynecology. 2017;216(5):491.e1–.e6. 37. butler k, yi j, wasson m, klauschie j, ryan d, hentz j, et al. randomized controlled trial of postoperative belladonna and opium rectal suppositories https://doi.org/10.22317/jcms.v9i1.1316 in vaginal surgery. american journal of obstetrics & gynecology. 2017;216(5):491.e1–.e6. 38. cole l, hanning cd, robertson s, quinn k. further development of a morphine hydrogel suppository. british journal of clinical pharmacology. 1990;30(6):781–6. 39. guldbrand p, mellstrom a. rectal versus intramuscular morphinescopolamine as premedication in children. acta anaesthesiologica scandinavica. 1995;39(2):224–7. 40. wilkinson tj, robinson ba, begg ej, duffull sb, ravenscroft pj, schneider jj. pharmacokinetics and efficacy of rectal versus oral sustained-release morphine in cancer patients. cancer chemotherapy and pharmacology. 1992;31(3):251–4. 41. stanski dr, greenblatt dj, lowenstein e. kinetics of intravenous and intramuscular morphine. clinical pharmacology and therapeutics. 1978;24(1):52–9. 42. jonsson t, christensen cb, jordening h, frølund c. the bioavailability of rectally administered morphine. pharmacology & toxicology. 1988;62(4):203–5. 43. westerling d, lindahl s, andersson ke, andersson a. absorption and bioavailability of rectally administered morphine in women. european journal of clinical pharmacology. 1982;23(1):59–64. 44. babul n, provencher l, laberge f, harsanyi z, moulin d. comparative efficacy and safety of controlled-release morphine suppositories and tablets in cancer pain. the journal of clinical pharmacology. 1998;38(1):74–81. 45. barnhart md, hubbell ja, muir ww, sams ra, bednarski rm. pharmacokinetics, pharmacodynamics, and analgesic effects of morphine after rectal, intramuscular, and intravenous administration in dogs. american journal of veterinary research. 2000;61(1):24–8. http://www.consultant360.com/articles/four-strategies-managing-opioid-induced-side-effects-older-adults http://www.consultant360.com/articles/four-strategies-managing-opioid-induced-side-effects-older-adults 176 j contemp med sci | vol. 4, no. 3, summer 2018: 176–178 short communication introduction head and neck cancer including oral cavity, pharynx, hypopharynx, and larynx is one of the most common malignancies around the world.1 moreover, 90% of head and neck cancers have been diagnosed as squamous cell carcinoma (scc).2 cancer is a major cause of mortality worldwide and a lot of new cases occur every year.3 the process of oral carcinogenesis is multifactorial and multistep, and the exact sequence is almost unknown.4,5 in spite of presence of risk factors such as tobacco and alcohol in some patients, most of them ultimately are not diagnosed as malignancies. this implies that genetic has an important role in susceptibility to cancers.6 unfortunately, despite the advanced methods in surgery, radiotherapy and chemotherapy, the prognosis of oral scc (oscc) is not good yet. but in case of early detection, the prognosis could be better. biopsy followed by histopathological evaluation is gold standard for diagnosis of oscc, but it is more helpful in late stages of disease. biomarkers are measurable indicators which can be useful for early diagnosis of some lesions.7 the most common laboratory diagnostic procedures involve the chemical and cellular analysis of blood. other biologic fluids like saliva are also used in diagnostic tests.8 salivary biomarkers in oropharyngeal carcinoma are cost-effective. saliva contains a wide range of components and is easy access, so patients feel more comfortable. the method is also non-invasive and handling is safe. there are also other advantages for using saliva instead of blood.7 therefore, nowadays there is a tendency to use of salivary biomarkers as diagnostic and prognostic factors. the matrix metalloproteinase (mmp) family involves diverse substrates.9 they are large family of zinc-dependent endopeptidase, and they have ability to digest extracellular matrix.10 generally, mmps are made up of a prodomain, a catalytic domain, a hinge region, and a hemopexin domain. they are secreted from the cell or anchored to the plasma membrane. mmps have six separated groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type mmps and others.11 matrix metalloproteinase-3 (stromelysin-1) is a secretory enzyme which several growth factors regulate its expression and the process of wound healing can stimulate its secretion.12 it is expressed by keratinocytes, fibroblasts, and chondrocytes.13 it has found that overexpression of mmp3 is in association with developing malignancies including head and neck carcinomas.14,15 the p53 protein is activated after dna damage or oncogenic signals. it has an important role in cell cycle control, dna repair, and apoptosis.16 altered expression and functional loss of p53 are common genetic changes in human malignancies.17,18 until now, conflicting results have been obtained from the study on mmp-3 and p53 markers in oscc patients, which could be due to differences and limitations in the immunohistochemical and rt-pcr methods in semi quantitative cytokine assessments. in addition, these two markers have not been evaluated simultaneously in patients with oscc. therefore, in this study, we decided to use a completely quantitative method to examine the levels of salivary p53 and mmp-3 in oscc patients to find their real changes in oscc affected patients. among the available methods, studies can perform on the tissue blocks, blood, and other biologic fluids. saliva is believed to be a reliable tool for diagnosis of oscc because it is in direct contact with cancerous tissue.7 is salivary evaluation of p53 and mmp-3 a good tool for early detection of oral squamous cell carcinoma? shima nafarzadeh,a mohsen emamgholipour,b fatimah bijani,c hamed hosseinkazemi,a amrollah mostafazadeh,d oveis khakbaz,a fatemeh baladi,d and soraya khafrie objective oral squamous cell carcinoma (oscc) is one of the most common malignancies around the world. despite the advancement in treatment methods, the prognosis is still not good. based on clinicians’ idea, the early diagnosis of the lesion can lead to better prognosis. some salivary biomarkers such as matrix metalloproteinase-3 and p53 may detect oscc in early stages. in this study, we wanted to compare salivary mmp-3 and p53 levels in oscc patients and control group. methods fifteen patients with oscc (9 male and 6 female) were selected from oral pathology department, babol, iran. salivary mmp-3 and p53 were measured by elisa and compared with control group. data was analyzed by anova, t-test, and mann–whitney. result there was no significant differences between salivary mmp-3 and p53 concentration in patients with oscc and healthy individuals. conclusion based on our findings and other similar studies, salivary mmp-3 and p53 levels might not be accurate enough to detect early stages of oscc. but there are controversial statements about these questions. so, supplementary studies are needed to be done in future. keywords oscc, saliva, p53, mmp3, elisa9 aoral health research center, institute of health, babol university of medical sciences, babol, iran. bstudent research committee, babol university of medical sciences, babol, iran. cdepartment of oral and maxillofacial pathology, dentistry school, babol university of medical sciences, babol, iran. dbabol university of medical sciences, babol, iran. edepartment of biostatistics and epidemiology, faculty member of biostatistics sciences, babol university of medical sciences, babol, iran. correspondence to mohsen emamgholipour (email:mohsenemamgholipour@gmail.com). (submitted: 24 june 2018 – revised version received: 02 july 2018 – accepted: 05 august 2018 – published online: 26 september 2018) issn 2413-0516 shima nafarzadeh et al. 177j contemp med sci | vol. 4, no. 3, summer 2018: 176–178 short communication p53 and mmp-3 in saliva of oscc this study was designed based on the evaluation of p53 and mmp-3 in whole unstimulated saliva of patients with oscc using elisa method. materials and methods fifteen patients with oscc were selected from oral and maxillofacial pathology department of dentistry faculty of babol university of medical sciences, babol, iran. a total of 9 patients were male and the rest of them were female. their age range was between 51 and 89. their diagnosis were proven by biopsy and histopathological evaluation. invasion of dysplastic tumoral epithelial cells into the connective tissue was essential for diagnosis. all slides were re-evaluated to confirm the diagnosis. all of them were in grades i and ii. we informed the patients and asked them to participate in our study. oral and written consents were obtained from participants. moreover, 30 healthy individuals without any dysplastic or malignant lesions were selected as control group. systemic diseases, smoking, chronic periodontitis, use of nsaids, corticosteroids, and antihistamines, history of chronic lung diseases or any treatment for malignancy, and pregnancy for women were our other exclusion criteria. salivary specimens were collected at the morning between 9 and 12 am. they were banned from eating, drinking, chewing gum, and smoking for at least 2 h before sampling. saliva samples were obtained in a dry and sterile tube of 50 ml (1–5 ml for each factor). all samples were immediately centrifuged at 4°c for 20 min at a speed of 6000 rpm to separate the cells and then were stored at −80°c until final analysis. matrix metalloproteinase-3 and p53 salivary concentration were measured by elisa according to the manufacturer’s instructions (e1711hu and e0907hu, shanghai crystal day biotech co., china). t-test and mann–whitney test were used for statistical analysis. results about 9 men and 6 women participated in our study with the diagnosis of oscc. the tongue was the most site of involvement. t-test showed no significant difference in saliva p53 concentration among control (mean ± sd: 1011.136 ± 655.323) and oscc (mean ± sd: 709.215 ± 454.961) groups (p = 0.171). mann–whitney analysis showed no significant differences in saliva concentration of mmp-3 among control (mean ± sd: 45.406 ± 67.349) and scc (mean ± sd: 35.706 ± 43.844) groups (p = 0.268). t-test showed no significant differences in saliva p53 concentration among men (mean ± sd: 755.808 ± 499.032) and women (mean ± sd: 639.326 ± 413.945) with scc (p = 0.654). mann–whitney analysis showed no significant differences in saliva concentration of mmp-3 among men (mean ± sd: 45.177 ± 53.002) and women (mean ± sd: 21.499 ± 22.004) with scc (p = 0.906) (fig. 1). discussion oral squamous cell carcinoma is one of the most common malignancies in head and neck region. despite the advancement in therapeutic techniques, the prognosis has not been improved significantly. to achieve the goal, early diagnosis and treatment is essential. nowadays, tendency to use biomarkers is increasing. scientists are in favor with salivary biomarkers because their stimulation is safe and simple. in recent years, many researches have been done on serum and salivary biomarkers to find diagnostic and prognostic markers in malignancies. in the present research, we compared salivary mmp-3 and p53 levels in oscc patients and control group. zhang et al.19 in a meta-analysis study on asian and european societies showed that there was no significant association between mmp-3 level and head and neck cancer risk. in contrast, in european subgroup mmp-3 polymorphism was significantly in association with hnc risk.19 our study took place in iran and our result was the same as asian subgroup in their research. about these findings we hypothesize that mmp-3 could be a diagnostic marker in some nations like europeans. on the other hand, some authors stated in their article that the concentration of salivary mmp-3 level elevated in patients with oral scc in compared to control group.20 but our examination did not show significant differences in these groups. agha-hosseini et al.21 in their study showed that serum and unstimulated salivary levels of mmp-3 are significantly correlated with each other and both of them increase in oscc patients with higher stages of tumor. we did not work on patients’ serum biomarkers but there was no relation between salivary mmp-3 and risk of malignancy. moreover, all of our patients were in low grade. taghavi et al.22 in an immunohistochemical study on esophageal squamous cell carcinoma have not found a significant association between tumor and adjacent normal tissues based on p53 positive expression, but they stated that there was significant positive expression of p53 in esophageal scc patients in comparison with healthy population. other authors also showed that p53 antibody could be detected in the saliva of patients with oscc and might be useful for early detection and screening.23 in other experiments overexpression of muted and unmuted p53 in patients with scc was detected.24 some other studies on salivary p53 level showed higher levels in oscc patients compared to control group.25,26 conflicting findings in other research showed that there is still no clear relationship found between p53 overexpression and known risk factors for esophageal and gastric cancers.27 however, we fig. 1 unstimulated whole saliva concentration of p53 and mmp-3 in patients with scc in men and women. data are expressed as mean ± sd. p < 0.05 is significant. 178 j contemp med sci | vol. 4, no. 3, summer 2018: 176–178 p53 and mmp-3 in saliva of oscc short communication shima nafarzadeh et al. also did not find significant difference in salivary p53 level in oscc patients and control group. based on our findings and other similar studies, salivary mmp-3 and p53 levels might not be accurate enough to detect early stages of oscc. but there are controversial statements in this regard, and we think that more researches with larger statistical samples are needed to investigate the p53 and mmp-3 expression in oscc. n references 1. jemal a, bray f, center mm, ferlay j, ward e, forman d. global cancer statistics. ca cancer j clin. 2011;61:69–90. 2. agha-hosseini f, mirzaii-dizgah i, farmanbar n, abdollahi m. oxidative stress status and dna damage in saliva of human subjects with oral lichen planus and oral squamous cell carcinoma. j oral pathol med. 2012;41:736–740. 3. jemal a, siegel r, xu j, ward e. cancer statistics, 2010. ca cancer j clin. 2010;60:277–300. 4. califano j, van der riet p, westra w, nawroz h, clayman g, piantadosi s, et al. genetic progression model for head and neck cancer: implications for field cancerization. cancer res. 1996;56:2488–2492. 5. rosin mp, cheng x, poh c, lam wl, huang y, lovas j, et al. use of allelic loss to predict malignant risk for low-grade oral epithelial dysplasia. clin cancer res. 2000;6:357–362. 6. sturgis em, wei q. genetic susceptibility–molecular epidemiology of head and neck cancer. curr opin oncol. 2002;14:310–317. 7. radhika t, jeddy n, nithya s, muthumeenakshi rm. salivary biomarkers in oral squamous cell carcinoma an insight. j oral biol craniofac res. 2016;6:s51–s54. 8. kaufman e, lamster ib. the diagnostic applications of saliva–a review. crit rev oral biol med. 2002;13:197–212. 9. egeblad m, werb z. new functions for the matrix metalloproteinases in cancer progression. nat. rev. cancer. 2002;2:161–174. 10. chen y, zhang w, geng n, tian k, jack windsor lj. mmps, timp-2, and tgf-b1 in the cancerization of oral lichen planus. head neck. 2008;30:1237–1245. 11. visse r, nagase h. matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. circ res. 2003;92:827–839. 12. liu sy, liu yc, huang wt, huang gc, su hj, lin mh. requirement of mmp-3 in anchorage-independent growth of oral squamous cell carcinomas. j oral pathol med. 2007;36:430–435. 13. varun br, nair bj, sivakumar tt, joseph ap. matrix metalloproteinases and their role in oral diseases: a review. oral maxillofac pathol j. 2012;3: 186–191. 14. sorsa t, tjäderhane l, salo t. matrix metalloproteinases (mmps) in oral diseases. oral dis. 2004;10:311–318. 15. stokes a, joutsa j, ala-aho r, pitchers m, pennington cj, martin c, et al. expression profiles and clinical correlations of degradome components in the tumor microenvironment of head and neck squamous cell carcinoma. clin cancer res. 2010;16:2022–2035. 16. levine aj, momand j, finlay ca. the p53 tumour suppressor gene. nature. 1991;351:453–456. 17. lee jj, kuo my, cheng sj, chiang cp, jeng jh, chang hh, et al. higher expressions of p53 and proliferating cell nuclear antigen (pcna) in atrophic oral lichen planus and patients with areca quid chewing. oral surg oral med oral pathol oral radiol endod. 2005;99:471–478. 18. biramijamal f, allameh a, mirbod p, groene hj, koomagi r, hollstein m. unusual profile and high prevalence of p53 mutations in esophageal squamous cell carcinomas from northern iran. cancer res. 2001;61:3119–3123. 19. zhang c, li c, zhu m, zhang q, xie z, niu g, et al. meta-analysis of mmp2, mmp3, andmmp9 promoter polymorphisms and head and neck cancer risk. plos one. 2013;8:e62023. 20. stott-miller m, houck jr, lohavanichbutr p, méndez e, upton mp, futran nd, et al. tumor and salivary matrix metalloproteinase levels are strong diagnostic markers of oral squamous cell carcinoma. cancer epidemiol biomarkers prev. 2011;20:2628–2636. 21. agha-hosseini f, mirzaii-dizgah i, mahboobi n, shiorazian s, harirchi i. serum and saliva mmp-3 in patients with olp and oral scc. j contemp dent pract. 2015;16:107–111. 22. taghavi n, biramijamal f, sotoudeh m, moaven o, khademi h, abbaszadegan mr, et al. association of p53/p21 expression with cigarette smoking and prognosis in esophageal squamous cell carcinoma patients. world j gastroenterol. 2010;16:4958–4967. 23. tavassoli m, brunel n, maher r, johnson nw, soussi t. p53 antibodies in the saliva of patients with squamous cell carcinoma of the oral cavity. int j cancer. 1998;78:390–391. 24. agha-hosseini f, mirzaii-dizgah i, miri-zarandi n. unstimulated salivary p53 in patients with oral lichen planus and squamous cell carcinoma. acta med iran. 2015;53:439–443. 25. schoelch ml, regezi ja, dekker np, ng io, mcmillan a, ziober bl, et al. cell cycle proteins and the development of oral squamous cell carcinoma. oral oncol. 1999;35:333–342. 26. kuropkat c, venkatesan tk, caldarelli dd, panje wr, hutchinson j, preisler hd, et al. abnormalities of molecular regulators of proliferation and apoptosis in carcinoma of the oral cavity and oropharynx. auris nasus larynx. 2002;29:165–174. 27. figueroa jd, terry mb, gammon md, vaughan tl, risch ha, zhang ff, et al. cigarette smoking, body mass index, gastro-esophageal reflux disease, and non-steroidal anti-inflammatory drug use and risk of subtypes of esophageal and gastric cancers by p53 overexpression. cancer causes control. 2009;20:361–368. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201812 18 j cont med sci | vol. 1, no. 2, spring 2015: 18–22 research objective clinical evidence suggests oxidative stress and inflammation linked to reactive oxygen species (ros) over generation may be a key in the pathogenesis of certain disease. cardiovascular disease is one example of this process. ros have been proposed to be the mediators of inflammation in experimental cardiovascular pathology. till now, areas with regard to the effects of ros in the ischemic heart disease (ihd) are not completely understood. the present study was conducted to investigate the involvement of inflammation as a consequence of oxidative stress in the pathology of ihd. methods serum lipid profile, trace element, antioxidant enzyme activities [catalase (cat), superoxide dismutase (sod)], hydrogen peroxide (h 2 o 2 ) and the inflammation marker (tnf-α and il-6) were measured in 50 patients with ihd and 50 healthy subjects as control group. all results were statistically analysed. results a highly significant decrease was found in the serum level of both zinc (zn) and copper (cu) in patients with ihd compared to control (p < 0.05). serum levels of antioxidants (sod, cat) were also significantly decreased in the patient group (p < 0.05) compared to control. however, h 2 o 2 and cytokines (tnf-α and il-6) levels demonstrated a significant increase in ihd patients (p < 0.01). conclusion the results of the present study provide evidences that oxidative stress, inflammation and trace element are closely linked and involved in ihd that may have future role in developing new therapeutic strategies. keywords oxidative stress, inflammation, ischemic heart disease, antioxidants, trace elements  oxidative stress and inflammation in ischemic heart disease: role of trace elements, oxidants and antioxidants anwar jasib thaaban almzaiel introduction heart disease is one of the major health problems of developing countries of the world. recent research has shown that free radicals, particularly, reactive oxygen species (ros) play an important role in the pathogenesis of oxidative myocardial damage with consequential cardiac malfunction.1 oxidative stress describes the condition where an excessive production of ros overwhelms endogenous anti-oxidant defense mechanisms. the resultant elevation in ros levels has a detrimental effect on cellular function, a consequence of ros-induced damage to lipid membranes, enzymes and nucleic acids. generation of ros has been involved in various cardiovascular disorders, including ischemia/reperfusion (i/r), atherosclerosis and cardiotoxicity induced by drugs.2 these ros caused an injury to vascular cells and cardiac myocytes directly, and can initiate a series of local chemical reactions that result in an amplification of the initial ros-mediated cardiomyocyte dysfunction.3 oxidative stress is now thought to play an important role in the pathogenesis of coronary heart disease through oxidation of low-density lipoprotein (ldl)-cholesterol and free radical formation.4 oxidised ldl-cholesterol aids the evolution of early arterial wall lesions into atherosclerotic plaques by promoting the formation of foam cells from macrophages as well as the recruitment and retention of monocytes in the arterial wall.5 ros may contribute to atherogenesis by damaging the arterial endothelium, promoting thrombosis and interfering with the normal vasomotor regulation. it is assumed that antioxidant enzymes, superoxide dismutase (sod), glutathione peroxidase (gsh-px) and catalase (cat) constitute a first line of defense against oxidative stress by removing ros.6 sod, catalyses the dismutation of the superoxide anion (o2 •-) into hydrogen peroxide (h2o2) within cells and in the extracellular matrix, while cat and gsh-px remove h2o2, and gsh-px can also convert lipid peroxyl radicals to nontoxic alcohols.6 low levels of activity of these antioxidant enzymes have been shown to associate with increased risk of chd.3 recently, inflammation has been linked, both experimentally and clinically, to cardiovascular disease.7 during inflammation ros are generated, which can be due to immune cells, such as [dendritic cells (dcs), lymphocytes, neutrophils and macrophages] or interleukins and other inflammatory cytokines, such as tumor necrosis factor (tnfα).8,9 because metals can cause oxidative modification of ldl-cholesterol and the synthesis of ros, the effect of several prooxidant metals, including cu on cardiovascular disease has come under investigation. recent researches demonstrate the importance of certain elements in the pathogenesis of cardiovascular disorders. statistically significant positive correlations have been found between trace element concentrations (cu, zn, se) of heart tissue with physiological parameters (co: cardiac output, ef: ejection fraction) of the heart. it is probable that free oxygen radicals and oxidatively modified particles of ldl participate in the development of atherosclerotic lesions and the potential role of natural antioxidants (vit. c ascorbic acid; vit. e tocopherol) is inhibition of this process.10 however, understanding of inflammation and ros, especially with their pathophysiological role in cardiovascular dysfunction, is still unclear and further investigations will facilitate the development and/or delivery of selective department of medical biochemistry, college of medicine, university of al-qadisiyah, qadisiyyah, iraq. correspondence to anwar jasib thaaban almzaiel (email: vipvip128@yahoo.com) (submitted: 13 january 2015 – revised version received: 10 april 2015 – accepted: 25 april 2015 – published online: spring 2015) 19j cont med sci | vol. 1, no. 2, spring 2015: 18–22 research oxidative stress and inflammation in ihd patientsanwar jasib thaaban almzaiel anti-inflammatory agents (antioxidants) that provide better management of cardiovascular diseases. materials and methods subjects serum zn and cu, antioxidant enzymes and inflammatory marker levels were measured in 50 (30 males and 20 females) patients with ischemic heart disease (ihd), and 50 healthy subjects (32 males and 18 females) as a control group. the mean age of the control group (58 ± 19.4 y) and in the patient group (60 ± 20.1 y) were randomly selected from patients ihd from march to august 2014. information regarding the medical history of each subject was obtained, including age, sex, diseases suffered and duration of illness with their daily diet and occupation. none of the patients had consumed alcohol, nor was there any history of surgery. methods all groups were subjected to thorough clinical history, examination and specific cardiac investigation. venous blood samples (5 ml) were collected from the patient and control groups. serum was separated by centrifugation (gallenkamp, germany) at 3000 rpm for 10 min and stored in capped plastic tubes at –20°c until analysis. total cholesterol, ldl-cholesterol, high-density lipoprotein (hdl)-cholesterol and triglyceride were measured by commercial kits (randox laboratories, san francisco, ca, usa). trace elements like cu and zn were determined by using a pye unicam model sp9 atomic absorption spectrophotometer. activities of antioxidant enzymes like cat and sod in the serum were determined by the method of mccord and fridovich11 and mueller et al.,12 respectively. horseradish peroxidase (hrp)-linked assay using homovanillic acid was used to measure the production of h2o2. 13 tnf-α and il-6 were measured by elisa. statistical analysis data are expressed as means ± sem. statistical analysis was carried out using statgraphics centurion xvi (statpoint technologies, inc., va, usa). the significant difference between control and experimental subjects were determined by student’s t-test. in order to explore significant differences between control and different patient groups, one-way anova or a non-parametric ranking (kruskal-wallis) were carried out as appropriate. after anova, post hoc analysis using tukey’s test was carried out. a p value of <0.05 is considered significant throughout. results the levels of total cholesterol, ldl cholesterol and triglyceride were significantly higher and that of hdl-cholesterol was significantly lower than those of controls (p < 0.05, table 1). there was a clear relationship between serum trace element (cu and zn) concentrations and ihd. serum cu and zn levels were found to be significantly lower in ihd patients compared to control (p < 0.05; table 2). female patient results showed a significant difference in serum cu, hdl-cholesterol and triglyceride levels (p < 0.05) than those found in males (table 3). table 4 shows serum cu and zn levels from patients grouped according to the type of disease with ihd. there was a significant change in cu and zn concentrations in patients with ihd only or ihd and hypertension compared to healthy controls (p < 0.05). a significant decrease was found in the activities of antioxidant enzymes cat and sod (p < 0.01, figs. 1 & 2, respectively) in the ihd patients compared to the control, whereas h2o2 levels were significantly increased in patients compared to control (p < 0.01, fig. 3). serum levels of tnf-α and il-6 were measured in patients with ihd using elisa. tnf-α and il-6 were significantly increased in the serum of ihd patients compared to control (p < 0.01, fig. 4). discussion it is well known that ros is of great importance in a number of biological processes, mostly through their action as signalling molecules that mediate different intracellular pathways, by influencing the permeability of cell membranes or through other mechanisms. therefore, it is reasonable to assume that ros would also exert an action, either directly or indirectly, on the cardiac cell or on other systems related to cardiovascular function e.g. the lipid and carbohydrate metabolism. ihd patients in this study showed a significant decrease in serum cu (table 2), which is consistent with the findings observed by other investigators.14,15 also lipid profile was significantly different from control lipid profile (table 1). our findings support the argument that decreased serum levels of cu was associated with increased oxidative stress and develop ihd risk factors due to cu is an antioxidant nutrient. cu plays an important role in the regulation of oxidative free radicals and its deficiency lead to increase the ability for peroxidation of lipoprotein,16 oxidation and free radical formation are two components of atherogenesis. in addition, cu table 2. serum cu and zn levels in both ihd patients and healthy control groups. p valuecontrol group n = 50 patient group n = 50 parameter <0.05*15.49 ± 1.299.49 ± 1.01serum copper (µ mol/l) <0.05*13.35 ± 1.147.59 ± 0.66serum zinc (µ mol/l) *the mean difference is significant at p < 0.05. table 1. serum total cholesterol, ldl, hdl and triglyceride levels in both ihd patients and healthy control groups. parameter patient group n = 50 control group n = 50 p value total cholesterol (mmol/l) 16.22 ± 0.66 5.45 ± 1.17 <0.05* ldl-cholesterol (mmol/l) 14.93 ± 1.81 9.63 ± 0.87 <0.05* hdl-cholesterol (mmol/l) 0.73 ± 0.19 1.05 ± 0.39 <0.05* triglycerides (mmol/l) 1.72 ± 1.68 <0.05* *the mean difference is significant at p < 0.05. 20 j cont med sci | vol. 1, no. 2, spring 2015: 18–22 oxidative stress and inflammation in ihd patients research anwar jasib thaaban almzaiel deficiency leads to an increase in plasma cholesterol and oxidises ldl-cholesterol, increasing its atherogenicity.17 alternatively, copper may be a risk marker for inflammation rather than a risk factor for coronary heart disease directly involved in the pathogenesis of atherosclerosis. there is more than one explanation for the mechanism of copper table 3. serum cu, zn levels and lipid profile in ihd patients and healthy controls classified by sex. p < 0.05male, n = 30female, n = 20parameter <0.05*12.2 ± 1.0815.74 ± 0.98copper (µmol/l) ns10.75 ± 0.6910.3 ± 0.51zinc (µmol/l) ns15.93 ± 0.6914.49 ± 0.59total cholesterol (mmol/l) ns12.48 ± 0.6210.5 ± 0.73ldlcholesterol (mmol/l) <0.05*0.66 ± 0.150.86 ± 0.18hdl-cholesterol (mmol/l) <0.05*2.3 ± 0.581.92 ± 0.41triglycerides (mmol/l) *the mean difference is significant at p < 0.05. ns (non-significant) p > 0.05. table 4. serum cu, zn levels in ihd patients and healthy control classified depending on the disease. p valuecu (µmol/l) p valuezn (µmol/l) noparameter ns13.96 ± 2.8ns10.35 ± 1.6311ihd with diabetes mellitus < 0.05*11.82 ± 0.94<0.0510.95 ± 0.839ihd with hypertension ns15.44 ± 2.26ns14.73 ± 1.134ihd with asthma <0.05*11.12 ± 1.05<0.0510.56 ± 0.5726ihd only 15.49 ± 1.2913.35 ± 1.1450control *the mean difference is significant at p < 0.05. fig. 1 serum cat activity in patients with ihd. serum samples were isolated from the blood of patients with ihd. cat activity was assessed in the presence of amplex red and hrp. data are expressed as means ± sem, for 50 patients, n = 50 with triplicate measurements. **indicates significant differences compared to the control (student’s t-test, p < 0.01). fig. 2 serum sod activity in patients with ihd. serum samples were isolated from the blood of patients with ihd. sod activity was assessed in the presence of cytochrome c and xo. data are expressed as means ± sem, for 50 patients, n = 50 with triplicate measurements. **indicates significant differences compared to the control (student’s t-test, p < 0.01). fig. 3 h 2 o 2 production in patients with ihd. serum samples were isolated from the blood of patients with ihd and control. h 2 o 2 levels in the serum were determined using hrp/hva. data are expressed as means ± sem, for 50 patients, n = 50 with triplicate measurements. **indicates significant differences compared to the control (student’s t-test, p < 0.01). fig. 4 serum pro-inflammatory cytokine levels in the patients with ihd. plasma serums were isolated from blood of patients with ihd and control. tnf-α and il-6 levels were analysed by elisa. data are expressed as the means ± sem, for 50 patients, n = 50 with triplicate measurements. **indicates significant differences compared to the control (student’s t-test, p < 0.01). deficiency in enhancing heart diseases. copper decreased cardiac myocyte functional capacity with subsequent impaired pumping capacity of the heart and finally heart failure. the abnormal levels of copper in patient with chd are probably due to changes in the concentration of ceruloplasmin in the plasma, an acute phase protein that was synthesised by the liver in response to tissue damage and inflammation.18 the present study demonstrates a significant difference in serum cu between females and males with ihd (table 3). these findings were consistent with the findings by another researcher.19 however, a number of factors influence circulating concentrations of copper. circulating concentrations of copper are positively associated with age, white blood cell count, cigarette smoking, serum ldl-cholesterol, systolic blood pressure, body mass index and oral contraceptive use or estrogen replacement 21j cont med sci | vol. 1, no. 2, spring 2015: 18–22 research oxidative stress and inflammation in ihd patientsanwar jasib thaaban almzaiel therapy.20,21 in a couple of studies, copper was inversely associated with hdl-cholesterol.20,22 the results of the present study provide additional evidence of the importance of zinc in ihd. as expected, serum zinc levels were significantly decreased in patients with ihd (table 2). these results which are consistent with recent studies suggest that low serum concentrations of zinc are associated with coronary artery disease.23,24 low serum zn levels in the ihd patient group may be related to excess release of steroids due to the release of leukocyte endogenous mediators which redistributes the body zn from serum and may cause a drop in serum zn and also due to elevated levels of α2 macroglobulin which is a transport protein containing large amounts of zn.25 recent characterization, suggest that zinc may share absorptive mechanisms with a variety of divalent cations, including cadmium, copper, iron and lead.26 metallothionein has a greater binding capacity for cu than for zn27 thus causing elevation of serum cu level and lowering serum zn level. recent studies also indicate that deficiency in zn is also a risk factor for the development of atherosclerosis caused by lipid peroxidation by oxidative stress,28 also zn may modulate atherosclerosis through its effects on gene stabilization, transcription factor levels and apoptosis.29,30 only a significant decrease was found in cu and zn levels in patients with ihd only or ihd and hypertension patients (table 4). this may be contributing to a pathological condition associated with this disease. in the present study, cat and sod activities were significantly decreased in ihd patients (figs. 1 & 2). the results of this study are in agreement with those reported by kayyum et al.31 thus, low levels of antioxidant enzyme activity could reflect either a possible accumulation of h2o2 (an ros) which has the potential to bring about oxidative damage of the bio-macromolecules and the consequential tissue damage or low levels of defense against it, a high oxidative stress status has been reported in ihd patients, even in stable cases.32,33 for instance, circulating oxidised ldl level is positively associated with severity of acute coronary syndromes33 and with subclinical chd.34 in contrast, the serum h2o2 level was significantly increased in ihd patients (fig. 3), this increase indicates an intensification of ros production due to neutrophil activation and/or derangement within the mitochondrial electron transfer.35 our findings also have established that tnf-α and il-6 significantly increase in patients with ihd (fig. 4). this reveals that ihd enhances the immune system and releases pro inflammatory modulators that result in ihd necrosis. it has been demonstrated that increased secretion of tnf-α and ifn-gamma by circulating mononuclear cells from patients with ihd may relate to the propagation of atherosclerosis by promoting intravascular coagulation and cell adhesion. therefore, tnf-α may be involved in the evolution of atheroma after the initial ischemic event.36 our findings of increased tnf-α and il-6 levels in patients with ihd are consistent with previous studies.37,38 increased plasma concentration of tnf-α has been found in patients with coronary artery disease, stressed myocardium activates pro-inflammatory cytokines, such as tnf-α, which produce abnormalities in the myocyte contractile function and soluble tnf receptors that bind to tnf-α which may prevent and even reverse tnf-α damage. pre-treatment with tnf-α antibodies reduces myocardial infarct size. tnf-α is a key molecule among the proinflammatory cytokines, which activates a transcriptional factor (nf-κb) in the nucleus39 which triggers and mediates the inflammatory response such as activation of neutrophil migration, production of proinflammatory cytokines and activation of metalloproteinase. furthermore, oxidative stress can activate a variety of transcription factors such as nf-κb.40 conclusion the findings of the present study provide evidence that oxidative stress and inflammation are intimately linked with ihd. it is likely that ihd pathology is mediated by the generation of ros and induction of inflammation. therefore, future studies are needed to understand the interaction of inflammation, immune cells and ros with cardiovascular disease which may prove useful in developing a therapeutic approach to the resolution of inflammation in ihd.  references 1. bandyopadhyay d, chattopadhyay a, ghosh g, datta ag. oxidative stressinduced ischemic heart disease: protection by antioxidants. curr med chem. 2004 feb;11(3):369–87. doi: http://dx.doi.org/10.2174/0929867043456016 pmid: 14965238 2. scolletta s, carlucci f, biagioli b, marchetti l, maccherini m, carlucci g, et al. nt-probnp changes, oxidative stress, and energy status of hypertrophic myocardium following ischemia/reperfusion injury. biomed pharmacother. 2007 feb–apr;61(2–3):160–6. doi: http://dx.doi. org/10.1016/j.biopha.2006.10.007 pmid: 17350221 3. blankenberg s, rupprecht hj, bickel c, torzewski m, hafner g, tiret l, et al. glutathione peroxidase 1 activity and cardiovascular events in patients with coronary artery disease. n engl j med. 2003 oct 23;349(17):1605–13. doi: http://dx.doi.org/10.1056/nejmoa030535 pmid: 14573732 4. steinberg d, parthasarathy s, carew te, khoo jc, witztum jl. beyond cholesterol. modifications of low-density lipoprotein that increase its atherogenicity. n engl j med. 1989 apr 6;320 (14): 915–24. pmid: 2648148 5. fuster v, badimon l, badimon jj, chesebro jh. the pathogenesis of coronary artery disease and the acute coronary syndromes (1). n engl j med. 1992 jan 23;326(4):242–50. doi: http://dx.doi.org/10.1056/ nejm199201233260406 pmid: 1727977 6. stocker r, keaney jf jr. role of oxidative modifications in atherosclerosis. physiol rev. 2004 oct 1;84(4):1381–478. doi: http://dx.doi.org/10.1152/ physrev.00047.2003 7. pearson ta, mensah ga, alexander rw, anderson jl, cannon ro 3rd, criqui m, et al. markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the centers for disease control and prevention and the american heart association. circulation 2003 jan 28;107(3):499–511. doi: http:// dx.doi.org/10.1161/01.cir.0000052939.59093.45 pmid: 12551878 8. galkina e, ley k. immune and inflammatory mechanisms of atherosclerosis. annu rev immunol. 2009 apr;27(1):165–97. doi: http://dx.doi.org/10.5551/ jat1994.1.supplemment1_s6 9. gilmont rr, dardano a, engle js, adamson bs, welsh mj, li t, et al. tnfalpha potentiates oxidant and reperfusion-induced endothelial cell injury. j surg res. 1996 feb 15;61(1):175–82. doi: http://dx.doi.org/10.1006/ jsre.1996.0101 pmid: 8769963 10. belosjorow s, bolle i, duschin a, heusch g, schulz r. tnf-alpha antibodies are as effective as ischemic preconditioning in reducing infarct size in rabbits. am j physiol heart circ physiol. 2003 mar;284(3):h927–30. doi: http://dx.doi.org/10.1152/ajpheart.00374.2002 pmid: 12578818  11. mccord jm, fridovich i. superoxide dismutase. an enzymic function for erythrocuprein (hemocuprein). j biol chem. 1969 nov 25;244(22):6049–55. pmid: 5389100 12. mueller s, riedel hd, stremmel w. determination of catalase activity at physiological hydrogen peroxide concentrations. anal biochem. 1997 feb 22 j cont med sci | vol. 1, no. 2, spring 2015: 18–22 oxidative stress and inflammation in ihd patients research anwar jasib thaaban almzaiel 1;245(1):55–60. doi: http://dx.doi.org/10.1006/abio.1996.9939 pmid: 9025968 13. baggiolini m, ruch w, cooper ph. measurement of hydrogen peroxide production by phagocytes using homovanillic acid and horseradish peroxidase. methods enzymol. 1986;132: 395–400. doi: 10.1016/s0076 6879 (86)32024-x. pmid:3547021 14. centers for disease control and prevention. plan and operation of the nhanes ii mortality study,1992. hyattsville, md: national center for health statistics, dhhs publication (phs); 1999. pp. 99–1314. 15. centers for disease control. laboratory procedures used by the clinical chemistry division, centers for disease control, for the second national health and nutrition examination survey (nhanes ii), 1976–1980. atlanta, ga: centers for disease control; 1981. 16. prohaska jr. biochemical changes in copper deficiency. j nutr biochem. 1990 sept;1(9):452–61. doi: http://dx.doi.org/10.1016/0955-2863(90)90080-5 17. klevay lm, inman l, johnson lk, lawler m, mahalko jr, milne db, et al. increased cholesterol in plasma in a young man during experimental copper depletion. metabolism 1984 dec;33(12):1112–8. doi: http://dx.doi. org/10.1016/0026-0495(84)90096-9 18. suciu, a., z. chirulescu, zeana c, pîrvulescu r. study of serum ceruloplasmin and of the copper/zinc ratio in cardiovascular diseases. rom j intern med. 1992 jul–sep;30(3):193–200. doi: pmid: 1475597 19. johnson pe., milne db, lykken gi. effects of age and sex on copper absorption, biological half-life, and status in humans. am j clin nutr. 1992 nov;56(5):917–25. pmid: 1329483 20. salonen jt, salonen r, korpela h, suntioinen s, tuomilehto j. serum copper and the risk of acute myocardial infarction: a prospective population study in men in eastern finland. am j epidemiol. 1991 aug 1;134(3):268–76. pmid: 1877585 21. fischer pwf, l’abbé mr, giroux mr. effects of age, smoking, drinking, exercise, and estrogen use on indices of copper status in healthy adults. nutr res. 1990 oct;10(10):1081–90. doi: http://dx.doi.org/10.1016/s02715317(05)80330-6 22. kromhout d, wibowo aa, herber rf, dalderup lm, heerdink h, de lezenne coulander c, et al. trace metals and coronary heart disease risk indicators in 152 elderly men (the zutphen study). am j epidemiol. 1985 sep;122(3):378–85. pmid: 4025288 23. vlad m, caseanu e, uza g, petrescu m. concentration of copper, zinc, chromium, iron and nickel in the abdominal aorta of patients deceased with coronary heart disease. j trace elem electrolytes health dis. 1994 jun;8(2):111–4. pmid: 7881275 24. oster o. trace element concentrations (cu, zn, fe) in sera from patients with dilated cardiomyopathy. clin chim acta. 1993 feb 28;214(2):209–18. pmid: 8472386 25. waalkes mp, perez-olle r. role of metallothionein transport and toxicity of metals. in: koropatnick dj, zalups r, editors. molecular biology and toxicology of metals. london: taylor & francis; 2000. pp. 414–55. 26. fleming re, migas mc, zhou x, jiang j, britton rs, brunt em, et al. mechanism of increased iron absorption in murine model of hereditary hemochromatosis: increase duodenal expression of the iron transport dmti. proc natl acad sci usa. 1999 mar 16;96(6):3143–8. doi: http://dx.doi. org/10.1073/pnas.96.6.3143 pmid: 10077651 27. walsh ct, h. h. sandstead, prasad as, newberne pm, fraker pj. zinc: health effects and research priorities for the 1990s. environ health perspect. 1994;102(suppl 2):5–46. doi: http://dx.doi.org/10.1289/ehp.941025 pmid: 7925188 28. hennig b, wang y, ramasamy s, mcclain cj. zinc deficiency alters barrier function of cultured porcine endothelial cells. j nutr. 1992 jun;122(6):1242–7. pmid: 1316957 29. reiterer g, toborek m, hennig b. peroxisome proliferator activated receptors alpha and gamma require zinc for their anti-inflammatory properties in porcine vascular endothelial cells. j nutr. 2004 jul;134(7):1711–5. pmid: 15226458 30. connell p, young vm, toborek m, cohen da, barve s, mcclain cj, et al. zinc attenuates tumor necrosis factor-mediated activation of transcription factors in endothelial cells. j am coll nutr. 1997 oct;16(5):411–7. doi: http:// dx.doi.org/10.1080/07315724.1997.10718706 pmid: 9322188 31. kayyum-shaikh a, suryakar an. oxidative stress and antioxidant status before and after supplementation of a-z anti-oxidant tablets in coronary artery disease. biomed res. 2009;20:136–40. 32. holvoet p, vanhaecke j, janssens s, van de werf f, collen d. oxidized ldl and malondialdehyde-modified ldl in patients with acute coronary syndromes and stable coronary artery disease. circulation 1998 oct 13;98(15):1487–94. doi: http://dx.doi.org/10.1161/01.cir.98.15.1487 pmid: 9769301 33. ehara s, ueda m, naruko t, haze k, itoh a, otsuka m, et al. elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. circulation 2001 apr 17;103(15):1955–60. doi: http://dx.doi.org/10.1161/01.cir.103.15.1955 pmid: 11306523 34. liu ml, ylitalo k, salonen r, salonen jt, taskinen mr. circulating oxidized low-density lipoprotein and its association with carotid intima-media thickness in asymptomatic members of familial combined hyperlipidemia families. arterioscler thromb vasc biol. 2004 aug 24;24(8):1492–7. doi: http://dx.doi.org/10.1161/01.atv.0000135982.60383.48 pmid: 15205217 35. wysocki h, kaźmierczak m, wykretowicz a. peroxide plasma level in patients with coronary heart disease as a possible indicator of ischemia during exercise test. coron artery dis. 1993 jul;4(7):645–7. doi: http://dx.doi. org/10.1097/00019501-199307000-00009 pmid: 8281369 36. feldman am, combes a, wagner d, kadakomi t, kubota t, li yy, et al. the role of tumor necrosis factor in the pathophysiology of heart failure. j am coll cardiol. 2000 mar 1;35(3):537–44. doi: http://dx.doi.org/10.1016/s07351097(99)00600-2 pmid: 10716453 37. pearson ta, mensah ga, alexander rw, anderson jl, cannon ro 3rd, criqui m, et al. markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the centers for disease control and prevention and the american heart association. circulation 2003 jan 28;107(3):499–511. doi: http:// dx.doi.org/10.1161/01.cir.0000052939.59093.45 pmid: 12551878 38. ferrari r, bachetti t, confortini r, opasich c, febo o, corti a, et al. tumor necrosis factor soluble receptors in patients with various degrees of congestive heart failure. circulation 1995 sep 15;92(6):1479–86. doi: http:// dx.doi.org/10.1161/01.cir.92.6.1479 pmid: 7664430 39. gordon jw, shaw ja, kirshenbaum la. multiple facets of nf-κb in the heart: to be or not to nf-κb. circ res. 2011 apr 29;108(9):1122–32. doi: http:// dx.doi.org/10.1161/circresaha.110.226928 pmid: 21527742 40. baetz d, regula km, ens k, shaw j, kothari s, yurkova n, et al. nuclear factor-kappab-mediated cell survival involves transcriptional silencing of the mitochondrial death gene bnip3 in ventricular myocytes. circulation 2005 dec 13;112(24):3777–85. doi: http://dx.doi.org/10.1161/ circulationaha.105.573899 pmid: 16344406 14 j cont med sci | vol. 1, no. 2, spring 2015:14–17 research objective to determine the cutoff of antibodies directed against salmonella enterica subspecies enterica serotype typhi, paratyphi a, and paratyphi b in normal population in karbala city, south iraq. methods widal test was performed for 90 serially diluted serum samples collected from apparently healthy volunteers. a commercial widal test kit was used. agglutination results were observed within 1 minute. results in the current study, 23.33%, 22.22%, 6.6%, 4.4%, 38.88% and 5.55% of individuals had antibody titre ≥1:20 for s. enterica subspecies enterica serotype typhi o, h, s. enterica serotype paratyphi ao, ah, s. enterica serotype paratyphi bo and bh, respectively. conclusion based on these results, antibody titres of 1:160 for o and h, 1:40 for ao, 1:80 for ah and bh and 1:320 for bo antigens were considered as baseline titre in karbala. keywords salmonella enterica, widal test, anti-o,h agglutinins determination of baseline widal titre in healthy individuals in karbala city, south of iraq suhad hadi mohammed, mohammed neama hmood, hawraa jasim mohammed, hussien saleh nasir, wejdan jabar sultan & hassan mohammed introduction iraq is considered as one of the many endemic countries with typhoid fever.1 the definitive diagnosis of typhoid fever requires the isolation of salmonella enterica serotype typhi from the blood, feces, urine or other body fluids. in developing countries, facilities for isolation and culture are often not available especially in smaller hospitals, and diagnosis relies upon the clinical features of the disease and the detection of agglutinating antibodies to s. enterica serotype typhi by the widal test, which has been used very extensively in the sero-diagnosis of typhoid fever and, in developing countries it remains the only practical test available. many studies2–6 however, have produced data which have cast serious doubts on the value of the widal test. classically, a fourfold rise of antibody in paired sera is considered diagnostic of typhoid fever.7 however, paired sera are often difficult to obtain and specific chemotherapy has to be instituted on the basis of a single widal test. determination of the baseline titre of agglutination of antibodies against salmonella typhi and salmonella paratyphi in healthy individuals in karbala has not been established before. thus, the current study concentrates on the determination of cutoff value related to this area. materials and methods the current study was conducted in the department of clinical laboratory, college of applied medical sciences, karbala, south iraq from september to november 2014. ninety healthy individuals with different age and sex who reside in various places of karbala district were enrolled. none of the volunteers had a history of recent vaccination or recorded infection with salmonella or other infectious disease. mean age of individuals was 25 ± 8.27 years. number of male volunteers were 60 while female volunteers were 30. the male:female ratio was 2/1. commercially available widal test kit (spinreact, s.a. ctra santa coloma, sant esteve de bas, spain) was used. the antigens provided within this kit include antigen suspension of s. enterica serotype typhi o, h and serotype paratyphi ao, ah, bo, bh, brucella and proteus in glycine buffer, ph 8.2 preservative. the blood was collected in gel tube containers from volunteers, and left for 15 minutes for clotting, then centrifugation was done for 5 minutes on 6000 rpm. the serum collected after centrifugation was properly labelled for widal test procedure. the widal test procedure in this study is slide agglutination method (semi quantitative). we delivered 80, 40, 20, 10 and 5 μl of undiluted serum into separate circles of the slide test. then, one drop (50 μl) of the antigen was placed on each circle. disposable stick was used for mixing and spreading of the reactants over the entire area enclosed by the circle. after 1 minute, agglutination was observed by naked eyes. statistical analysis statistical analysis was done using spss (statistical package for the social sciences; ibm) version 20. a p value 0.05 was considered significant at a confidence interval of 95% according to chi-square test. results a total of 90 healthy adult volunteers of different age, sex and socioeconomic groups were screened for the agglutination against s. enterica subspecies enterica serotypes, typhi, paratyphi a and paratyphi b (table 1). sixty were males and 30 were females, male/female ratio was 2/1. mean age was 24.9 ± 8.27 years. in the current study, 69 (76.7%), 70 (77.7%), 84 (93.3%), 87 (96.7%), 60 (66.6%) and 85 (94.4%) cases had antibody titres less than 1:20 for o, h, ao, ah, bo and bh, respectively. whereas, 21 (23.3%) had anti-o agglutinins more than or equal to 1:20. in addition, antibody titre ≥1:20 were seen in department of clinical laboratories, college of applied medical sciences, karbala university, iraq. correspondence to suhad hadi mohammed (email: shm.med.school@gmail.com). (submitted: 17 march 2015 – revised version received: 28 may 2015 – accepted: 06 june 2015 – published online: spring 2015) table 1. demographic data male female total number 60 30 90 mean age ± sd 24.2 ± 7.7 23.09 ± 8.55 24.95 ± 8.27 15j cont med sci | vol. 1, no. 2, spring 2015:14–17 research determining baseline widal titre in healthy individualssuhad hadi mohammed et al. table 2. frequencies of agglutinins agglutinin n (%) <1:20 n (%) ≥1:20 o agglutinins 69 (76.7) 21 (23.3) h agglutinins 70 (77.8) 20 (22.2) ao 84 (93.3) 6 (6.7) ah 87 (96.7) 3 (3.3) bo 60 (66.7) 30 (33.3) bh 85 (94.4) 5 (5.6) table 3. cross tabulation of agglutinins titre with sex agglutinins female male <1:20 n (%) ≥1:20 n (%) total <1:20 n (%) ≥1:20 n (%) total o 50 (83.3) 10 (16.6) 60 19 (63.3) 11 (36.6%) 30 fisher’s exact test 0.03* h 50 (83.3) 10 (16.6) 60 20 (66.66) 10 (33.3) 30 fisher’s exact test 0.06 ao 58 (96.6) 2 (3.3) 60 26 (86.6) 4 (13.3) 30 fisher’s exact test 0.09 ah 58 (96.6) 2 (3.3) 60 29 (96.6) 1 (3.3) 30 fisher’s exact test 0.7 bo 47 (78.3) 13 (21.6) 60 13 (43.3) 17 (56.6) 30 fisher’s exact test 0.001* bh 57 (95) 3 (5) 60 28 (93.3) 2 (6.6) 30 fisher’s exact test 0.5 *statistically significant difference. fig. 1 distribution of positive cases according to the titre of agglutinins. titre o .00 20.00 40.00 80.00 160.00 320.00 titre h .00 20.00 40.00 80.00 160.00 320.00 titre ao .00 20.00 80.00 160.00 titre ah .00 20.00 40.00 320.00 titre bo .00 20.00 40.00 80.00 160.00 320.00 titre bh .00 20.00 40.00 80.00 320.00 21 (23.3%), 20 (22.2%), 6 (6.7%), 3 (3.3%), 30 (33.3%) and 5 (5.6%) for o, h, ao, ah, bo and bh, respectively (table 2). fig. 1 shows the distribution of the positive cases with ≥1:20 titre among the different titres. the current study revealed that there were significant differences between the presence of o, bo agglutinins and sex, as shown in table 3. as shown in table 4, among 21 cases who had titre ≥1:20 anti-o agglutinins, 15 had titre ≥1:20 anti-h, 5 had titre ≥1:20 anti-ao, 13 had titre ≥1:20 anti-bo, 2 had titre ≥1:20 anti-bh and 4 had titre ≥1:20 anti-ah agglutinins. there were positive correlations between the presence of o agglutinins and h, ao, ah and bo agglutinins. cutoff value according to the equation (mean ± 2sd) the cutoff values for o and h is 1:160, 1:40 for ao, 1:80 for ah and bh, and 1:320 for bo, are considered as baseline titre in this region (table 5). discussion iraq is one of the developing countries where enteric fever is a major public health problem associated with significant morbidity and mortality.8,9 blood culture has remained the gold standard test in diagnosis of typhoid fever, but its utility in early diagnosis is limited and does not exceed 50% even in the best laboratory.10 the administration of antibiotic therapy before diagnosis is considered to be one of the main reasons for poor isolation rates.10 thus, diagnosis of typhoid fever largely relies upon the clinical features and serological tests like widal.10 controversies over the diagnostic utility of widal could be due to several factors like high endemicity, antigenic cross reactivity with other agents, nonavailability of paired sera for the documentation of rising tires, diverse 16 j cont med sci | vol. 1, no. 2, spring 2015:14–17 determining baseline widal titre in healthy individuals research suhad hadi mohammed et al. methods and criteria of interpretation etc.11 in areas where fever due to infectious causes is a common occurrence, the possibility exists that false positive reactions may occur as a result of nontyphoidal fever. the purpose of the present study was to reappraise the diagnostic value of the widal test by evaluating the basal antibody levels in the adult healthy population. in the current study, 69 (76.7%), 70 (77.7%), 84 (93.3%), 87 (96.7%), 60 (66.6%) and 85 (94.4%) of cases had antibody titres less than 1:20 for o, h, ao, ah, bo and bh, respectively. whereas, 21 (23.3%) had anti-o agglutinins more than or equal to 1:20. in addition, antibody titre ≥1:20 were seen in 21 (23.3%), 20 (22.2%), 6 (6.7%), 3 (3.3%), 30 (33.3%) and 5 (5.6%) for o, h, ao, ah, bo and bh, respectively. there were significant differences between the presence of o, bo agglutinins with sex; the females had antibody titre more than male. this may reflect that females more prone to infection or because of the immune system of the females which results in more antibody titres. among 21 cases which had titre ≥1:20 anti-o agglutinins, 15 had titre ≥1:20 anti-h, 5 had titre ≥1:20 anti-ao, 13 had titre ≥1:20 anti-bo, 2 had titre ≥1:20 anti-bh and 4 had titre ≥1:20 anti-ah agglutinins. there were positive correlation between the presence of o agglutinins and h, ao, ah, bo agglutinins. this means that the increase levels of o agglutinins is accompanied by the increase of other types of agglutinins. the cutoff values for o and h is 1:160, 1:40 for ao, 1:80 for ah and bh and 1:320 for bo, and thus considered to be baseline titre for these agglutinins in this region. any titre above these would be significant and indicative of enteric fever.12 our results is in agreement with study carried out in baghdad,13 the high level of agglutinins titres in our region among population might possibly be due to that the population is permanently “immunologically sensitized” due to constant exposure, the response to infection is more rapid, reaching higher levels and is less likely to be affected by antibiotic use when compared to nonendemic.14 several studies support that re-evaluation of the widal of the widal baseline titre for healthy individuals should be done at regular intervals.14 conclusion titre of 1:160 for anti-o and h agglutinins is considered as baseline titre for these table 4. correlations among agglutinins titre o total 0.00 20.00 40.00 80.00 160.00 320.00 titre h 0.00 64 2 2 2 0 0 70 20.00 1 2 1 2 0 0 6 40.00 2 0 1 0 0 0 3 80.00 0 0 0 4 2 0 6 160.00 0 0 0 0 1 0 1 320.00 2 0 0 0 0 2 4 total 69 4 4 8 3 2 90 correlation r = 0.673*, p = 0.000 titre o total 0.00 0.00 0.00 0.00 0.00 0.00 titre ao 0.00 68 2 4 6 2 2 84 20.00 0 2 0 1 0 0 3 80.00 0 0 0 0 1 0 1 160.00 1 0 0 1 0 0 2 total 69 4 4 8 3 2 90 correlation r = 0.356*, p = 0.001 titre o total 0.00 20.00 40.00 80.00 160.00 320.00 titre ah 0.00 69 4 3 7 2 1 86 20.00 0 0 0 0 0 1 1 40.00 0 0 0 1 0 0 1 320.00 0 0 1 0 1 0 2 total 69 4 4 8 3 2 90 correlation r = 0.424* , p = 0.000 titre o total 0.00 20.00 40.00 80.00 160.00 320.00 titre bo 0.00 47 2 1 4 1 0 55 20.00 3 1 1 0 0 0 5 40.00 4 0 2 2 0 0 8 80.00 2 1 0 1 2 0 6 160.00 4 0 0 1 0 0 5 320.00 9 0 0 0 0 2 11 total 69 4 4 8 3 2 90 correlation r = 0.222*, p = 0.036 titre o total 0.00 20.00 40.00 80.00 160.00 320.00 titre bh 0.00 66 3 4 7 3 2 85 20.00 1 0 0 0 0 0 1 40.00 0 1 0 0 0 0 1 80.00 1 0 0 0 0 0 1 320.00 1 0 0 1 0 0 2 total 69 4 4 8 3 2 90 correlation r = 0.079, p = 0.4 *statistically significant difference. 17j cont med sci | vol. 1, no. 2, spring 2015:14–17 research determining baseline widal titre in healthy individualssuhad hadi mohammed et al. agglutinins in this region. knowledge of endemic titre and proper interpretation of widal test are necessary for accurate diagnosis of enteric fever to avoid misuse of antibiotics, thereby preventing the occurrence of drug resistance.  table 5. cutoff value titre o titre h titre ao titre ah titre bo titre bh mean 22.2222 24.0000 5.1111 7.7778 58.0000 8.6667 sd 57.70475 69.42460 25.18446 47.56492 106.25600 48.16171 references 1. adawia f. abbas abdul-razak sh. hasan, abbas a. al-duliami. the distribution of anti-salmonella antibodies in the sera of healthy blood donors in baquba city. iraqi j comm med. 2011;24(3):241–4. 2. schroeder sa. interpretation of serologic tests for typhoid fever. jama. 1968 oct 21;206(4):839–40. doi: http://dx.doi.org/10.1001/jama.206.4.839 pmid: 5695671 3. sen a, saxena sn. a critical assessment of the conventional widal test in the diagnosis of typhoid and paratyphoid fevers. indian j med res. 1969 oct;57(10):1813–9. pmid: 4984499 4. reynolds dw, carpenter rl, simon wh. diagnostic specificity of widal’s reaction for typhoid fever. jama. 1970 dec 21;214(12):2192–3. doi: http:// dx.doi.org/10.1001/jama.214.12.2192 pmid: 4921576 5. wicks acb, holmes gs, davidson l. endemic typhoid fever. a diagnostic pitfall. q j med. 1971 jul;40(159):341–54. doi: pmid: 5564533 6. anonymous. typhoid and its serology. br med j. 1978 feb 18;1(6110):389– 90. doi: http://dx.doi.org/10.1136/bmj.1.6110.389-a  7. parker mt. enteric infection: typhoid and para typhoid fever. in: wilson gs, miles as, parker mt, editors. topley and wilson’s principles of bacteriology, virology and immunity, vol iii, 7th ed. london: edward arnold publishers limited; 2003. pp. 424–42. 8. deepak p, komal rr, bimala s, suresh rk, bishnu rt. baseline titre and diagnostic cut off value for widal test: a comparative study in healthy blood donors and clinically suspected of enteric fever. jhas. 2012;2(i):22–6. 9. crump ja, luby sp, mintz ed. the global burden of typhoid fever. bull world health organ. 2004 may;82(5):346–53. pmid: 15298225 10. madhusudhan ns, manjunath ah. determination of baseline widal titre among healthy population. int j biomed res. 2013 jan 1;2(12):437. doi: http://dx.doi.org/10.7439/ijbr.v3i12.843 11. pang t, puthucheary sd. significance and value of the widal test in the diagnosis of typhoid fever in an endemic area. j clin pathol. 1983 apr;36(4):471–5. doi: http://dx.doi.org/10.1136/jcp.36.4.471 pmid: 6833514 12. sukia s, bhavesh p, chitnis ds. 100 years of widal test and it’s reappraisal in an endemic area. indian j med res. 1997 feb;105:53–7. 13. madha m, sheet s, shayma a. mahmud. frequency of salmonella antibodies among febrile patients with suspected enteric fever. al-taqani. 2013;26(3):112–9. 14. clegg a, passey m, omena m, karigifa k, suve n. re-evaluation of the widal agglutination test in response to the changing pattern of typhoid fever in the highlands of papua new guinea. acta trop. 1994 sep;57(4):255–63. doi: http://dx.doi.org/10.1016/0001-706x(94)90071-x pmid: 7810382 9j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 research objective to determine the effect of some growth conditions in proteolytic activity of clinical isolates of trichophyton rubrum. methods isolation and identification of a dermatophyte t. rubrum from hair and skin scrapings of patients with dermatophytosis by using the morphological and cultural characteristics. optimal growth conditions of eight isolates including temperature, ph, culture media type and incubation period were studied, in addition to that the proteolytic activity and its optimal production in liquid media were tested. results the results showed that the colonies of t. rubrum on sabouraud dextrose agar (sda) were like cotton or powder-like, white or light beige, flat or elevated, with or without pigments on the reverse. the optimal conditions of growth were 30°c, ph6 and sda media. the proteolytic activity against casein as substrate showed that t. rubrum isolates have an ability to produce exocellular protease ranged from 10.5–80.1u/ml according to the source of each isolates. on the other hand, the proteolytic activity varied based on the ph value, temperature, incubation period and concentration of substrate. conclusion the present data may refer to the vital role of proteolytic activity in the invasion and pathogenesis of t. rubrum infection. keywords trichophyton rubrum, growth conditions, proteolytic activity. in vitro, determination of optimal conditions of growth and proteolytic activity of clinical isolates of trichophyton rubrum sara k kadhima jawad k al-janabia & adnan h al-hamadanib introduction the dermatophytoses are among the most common human disease. although the prevalence of those infections varies greatly, at least 10–20% of the world population may be infected with dermatophytes.¹ the highly developed host-parasite relationship is responsible for a multitude of clinical manifestation and many of these host-parasite interactions depend upon specific moieties and enzyme production, especially in trichophyton rubrum which may enhance survival in tissues by chemically or physically altering the immediate environment or they may act directly by digesting host proteins, thus providing a source of nutrition. more attention has been given to the enzymes produced by pathogenic fungi due to their role in human pathogenicity. however, exoenzymes are found to be produced by dermatophtes are keratinases, lipases, phospholipases, elastases, collagenases and proteases.2 the dermatophytes are a group of closely related fungi that have the capacity to invade keratinized tissue (skin, hair, nail, feathers, horns and hooves) of human and other animals to produce an infection, dermatophytosis. about 40 species belonging to the genera microsporum, trichophyton and epidermophyton are considered as dermatophytes. they possess two important properties, keratinophilic and keratinolytic. this mean that they have the ability to digest keratin in vitro in their saprophytic state and utilise it as a substrate, and some may invade tissues in vivo and provoke tineas.3 t. rubrum is the most common causative agent of dermatophytosis worldwide, mainly occupying the humans’ feet, skin and between fingernails. t. rubrum is known to be one of the most prominent anthrophilic species of dermatophtyes, a fungus commonly causing skin diseases.4 very little is known about the mechanism of its invasion and pathogenicity.5 though it is usually not life-threatening, infections are longlasting, recurring and incredibly difficult to cure. the fungal pathogen’s ability to produce and secrete proteolytic enzymes is a major virulence factor.6 the aim of the present study is the isolation and identification of t. rubrum from clinical specimens with dermatophytosis by conventional method (macroscopic and microscopic characteristics, biochemical and physiological tests, testing the optimal temperature, ph, culture media, substrate concentration and incubation period for growth and proteolytic activity of protease produced by t. rubrum isolates in vitro). materials and methods clinical specimens a total of 150 clinical specimens (hair, nails and skin scrapings) were collected from patients who attended the dermatology and venereal disease centre at mergan hospital and private clinic in babylon city from february 2014 to may 2014. the specimens were inoculated on sabouraud’s dextrose agar (sda) containing cycloheximide (0.5 g/l) and chloramphenicol (0.05 g/l) at ph 5.6 and incubated at 29±2°c for 14–21 days. method of isolation and identification of fungi were performed as previously published.7–9 influence of environmental factors in the growth of t. rubrum 1. temperature to examine the effect of temperature on growth of eight isolates of t. rubrum, preliminary experiments were investigated using temperature which ranged from 20°c to 40°c. then the following temperature regimes i.e. 20°c, 25°c, 30°c, 35°c and 40°c were conducted by taking a disk (0.5 cm) of fungal tissues from the edge of colony age (7–10 days) using cork borer. the centre of new petri dishes containing sda medium were inoculated using three replicates. fungal growth of t. rubrum were calculated by taking two intersecting lines from the auniversity of babylon, college of science, department of biology, babylon, iraq. buniversity of al-qadisiyah, college of medicine, department of microbiology, qadisiyah, iraq. correspondence to sara kareem kadhim (email: sarakareem217@yahoo.com). (submitted: 2 february 2015 – revised version received: 15 april 2015 – accepted: 23 april 2015 – published online: summer 2015 ) issn 2413-0516 10 j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 growth effect in proteolytic activity of clinical isolates of t. rubrum research sara k kadhim et al. centre of the dish at 2 days interval for 7 days.10 2. ph to examine the effect of ph on growth of eight isolates of t. rubrum, the following ph regimes i.e. 5, 6, 7 and 8 were conducted. all the other steps like inoculation technique, incubation time, replication and growth measurements for the fungus were applied as previously mentioned; except that the incubation temperature was 30°c.11 3. culture media the effect of four types of media i.e. sda, pda, cma, and yea on growth of eight isolates of t. rubrum were investigated, using similar steps that were applied as previously mentioned; except the incubation temperature was 30°c and the ph of growth medium was 6, replication and growth measurements were done as previously mentioned.12 protease production media the cells of t. rubrum isolates were grown on synthetic medium composed of peptone (10 g/l) and dextrose (40 g/l) with ph 5.4, and was autoclaved at 121°c for 15 min, cooled in flasks (250 ml size), and inoculated by placing 0.5 cm agar medium plugs containing active mycelium (5 days old) from the fungus growing in slant culture. flasks were incubated at 35°c on a rotary shaker at 150 rpm for 30 days.13 determination of protease activity proteolytic activity against casein substrate was measured according to the method published by hanlon and hodges (1981).14 the reaction mixture contained 0.5 ml of substrate, 0.5 ml of culture filtrate and 0.1 ml of 0.5m tris-hcl buffer, ph8. the reaction was carried out at 40°c for 30 min, stopped by addition of 2 ml of 0.67m trichloroacetic acid (tca) and then allowed to stand for 1 h. the precipitate was removed by centrifugation at 3000 g for 15 min. absorbance of the supernatant was measured at 280 nm by spectrophotometer against a reaction blank prepared as above except that tca solution was added before the addition of the culture filtrate. one unit of enzyme activity was defined as the amount of enzyme that could liberate products having an absorbance of 0.1 under described conditions. the specific activity was expressed as the number of units of activity per mg protein. absorbance was used to calculate the activity of enzyme using the formula: enzyme activity u/ml o.d time volume = × × ( ) . (crude enzyme) 0 01 effect of some culture conditions on the production of protease in this work, the activities of protease were measured under several nutritional conditions including substrate concentration (0.5, 1, 1.5, 2, 2.5, 3%), incubation intervals (3, 6, 9, 12, 15 days), ph of culture media (5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9) and the incubation temperature (20, 25, 30, 35, 40, 45, 50˚c).15 statistical analysis statistical analysis was performed by using spss computing program for the analysis of the results. a p-value under 0.05 was considered statistically significant.16 results influence of environmental factors on the growth of t. rubrum isolates 1. temperature the results of effect of different temperature (20, 25, 30, 35, 40°c) in growth rate of eight isolates of t. rubrum grown in sda during different periods of incubation (2, 5, 7 days) revealed significant differences (p ≤ 0.05) and 30°c was the optimal temperature at the seventh day of incubation for all studied fungal isolates, where the colonies diameter of isolates no. 1 to no. 8 were (3.5, 3.4, 3.7, 4, 9, 3.7, 7.3 and 6.5 cm) respectively, (fig. 1). 2. ph figure 2 shows the results of testing eight isolates of t. rubrum for growth under different ph values (5–8) that were grown on sda at 30°c and different incubation periods (2, 5, 7 days) showed that the ph = 6 was the optimal ph value for growth of all tested isolates of t. rubrum after 7 days of incubation and the statistical analysis showed a significance difference (p ≤ 0.05) among tested treatments. on the other hand, the growth of colonies diameter of fungal isolates (no. 1 to no. 8) were (4.2, 4, 4.5, 3.9, 9, 3.7, 7.3 and 6.5 cm) respectively. 3. culture media the data of effect of culture media (sda, pda, cma, yea) in growth rate of eight isolates of t. rubrum during different periods of incubation (2, 5, 7 days) and 30°c is shown in fig. 3, the optimal growth for all tested isolates (no. 1–8) were (4, 4.5, 4.3, 4.7, 9, 4.3, 7.3 and 6.5 cm), respectively, on sda rather than other used media and after 7 days of incubation. a statistical analysis showed a significance difference (p ≤ 0.05) among tested treatments. proteolytic activity of t. rubrum isolates results of present study showed that t. rubrum isolates have the ability to produce exocellular protease which was indicated by proteolytic activity against casein when added as substrate to the culture filtrates of t. rubrum. after 9 days of growth, the t. rubrum no. 1 showed the highest proteolytic activity (80.1 u/ml) and the minimum activity was showed by the t. rubrum no. 8 (10.5 u/ml). the influence of cultural conditions (substrate concentration, ph, incubation period and temperature) on the production of protease by t. rubrum isolates were investigated. the t. rubrum isolates showed high ability to produce protease 48.3 u/ml at 0.5% (fig. 4). also the result showed that high activity of the enzyme obtained at the 9th day of incubation, reached to 88.5 u/ml, and then a progressive decrease in proteolytic activity was observed when incubated for 12 and 15 days, as shown in fig. 5. on the other hand, results showed that the proteolytic activities of t. rubrum isolates were rapidly increased, reaching a maximum of 78.5 u/ml at ph7, then a progressive decrease in protease production occurred with increasing ph values (fig. 6). the highest proteolytic activity 83.6 u/ml achieved by t. rubrum isolates when incubated at 30°c, then decreased with the increase of temperature, while the isolates lost its ability to produce proteolytic activity when incubated at 50°c (fig. 7). 11j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 research growth effect in proteolytic activity of clinical isolates of t. rubrumsara k kadhim et al. discussion environmental factors play an important role in the growth of keratinophilic fungi. temperature is one of the important ecological factors that affects the growth of microorganisms and reproduction, every fungus has a definite range of temperature within which it grows and sporulates. usually most of the fungi grow at temperature ranging from 15°c to 35°c; some of the fungi require a range of higher temperature for their optimum growth.17,18 all isolates of t. rubrum grew well at temperature between 25°c and 35°c but the maximum growth varied according to the isolate type. it was found that 30°c and 35°c were the most suitable for optimum growth of most of the isolates during 7 days in sda medium in the ph = 6, but colony diameters were reduced in low temperature (2°c) and high temperature (40°c) which may inhibit the growth of fungi compared with other temperatures (25, 30, 35°c). the present results are in agreement with sharma et al.,19 who found that 33°c was most suitable for t. rubrum 0 1 2 3 4 5 20 25 30 35 40 20 25 30 35 40 20 25 30 35 40 20 25 30 35 40 te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c)   lsd(0.05) = 0.227   t. rubrum no.1 t. rubrum no.2 0 1 2 3 4 5   2   day 5   day 7   day lsd(0.05) = 0.223 0 1 2 3 4 5 2   day 5   day 7   day lsd(0.05) = 0.597 0 1 2 3 4 5   2   day 5   day 7   day lsd(0.05) = 0.235   0 1 2 3 4 5 6 7 8 9 10   2   day 5   day 7   day lsd(0.05) = 0.273   0 1 2 3 4 5 2   day 5   day 7   day 2   day 5   day 7   day lsd(0.05) = 0.632 0 1 2 3 4 5 6 7 8 9 10   2   day 5   day 7   day lsd(0.05) = 0.255   co lo ny di am et er ( cm ) co lo ny di am et er ( cm ) co lo ny di am et er ( cm ) co lo ny di am et er ( cm ) co lo ny di am et er ( cm ) co lo ny di am et er ( cm ) co lo ny di am et er ( cm ) co lo ny di am et er ( cm ) 0 1 2 3 4 5 6 7 8 9 1 0 2 0 2 5 3 0 3 5 4 0 lsd(0.05) = 0.243 t. rubrum no.3 t. rubrum no.5 t. rubrum no.7 t. rubrum no.4 t. rubrum no.6 t. rubrum no.8 20 25 30 35 40 20 25 30 35 40 20 25 30 35 40 2   day 5   day 7   day fig. 1 effect of different temperature on the growth of t. rubrum isolates on sda after 7 days of incubation. 12 j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 growth effect in proteolytic activity of clinical isolates of t. rubrum research sara k kadhim et al. in terms of dry weight of mycelium as well as in colony diameter. high temperature could lead to fungal cell rupture and loss of membrane or damage to the intra-cytoplasmic compounds and cell analyses where de maranon et al.,20 found that maximum growth was obtained at 32°c because the dermatophytes grow best in culture at temperature lower than human body. increasing or decreasing of temperature than optimum range may lead to the breakdown of enzymes.21 so increase in the rate of growth at 29–35°c may attribute to enzyme activity which reaches to the top at optimum temperature and then they use the food source in the media to build macro-molecular and then build the fungal mass. in addition, respiration process gets reduced at low temperature 0 1 2 3 4 5 5 6 7 8 ph 2   day 5   day 7   day 0 1 2 3 4 5 5 6 7 8 ph 2   day 5   day 7   day co lo ny d ia m et er (c m )   0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 6 7 8 ph 2   day 5   day 7   day 0 2 4 6 8 10 5 6 7 8 ph 2   day 5   day 7   day co lo ny d ia m et er (c m )   0 0.5 1 1.5 2 2.5 3 3.5 4 5 6 7 8 ph 2   day 5   day 7   day 0 1 2 3 4 5 6 7 5 6 7 8 ph 2   day 5   day 7   day 0 1 2 3 4 5 5 6 7 8 ph 2  day 5  day 7  day lsd(0.05) = 0.275 t. rubrum no.1 co lo ny d ia m et er (c m )   0 1 2 3 4 5 6 7 8 5 6 7 8 ph 2  day 5  day 7  day co lo ny d ia m et er (c m ) co lo ny d ia m et er (c m ) co lo ny d ia m et er (c m ) co lo ny d ia m et er (c m ) co lo ny d ia m et er (c m )   t. rubrum no.3 t. rubrum no.5 t. rubrum no.7 t. rubrum no.2 t. rubrum no.4 t. rubrum no.6 t. rubrum no.8 lsd(0.05) = 0.249 lsd(0.05) = 0.268 lsd(0.05) = 0.333 lsd(0.05) = 0.331 lsd(0.05) = 0.330 lsd(0.05) = 0.257 lsd(0.05) = 0.265 fig. 2 effect of different ph on the growth of t. rubrum isolates after 7 days of incubation. 13j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 research growth effect in proteolytic activity of clinical isolates of t. rubrumsara k kadhim et al. but at higher temperature the process will stop which leads to the death of the fungi.21 dermatophytes could grow over a wide range of ph. this result demonstrated the important role of ph in the growth of t. rubrum isolates in different ph (5, 6, 7 & 8) in sda media for 7 days at 30°c. it was found that ph 6 was the most suitable for fugal growth. fungi can tolerate a wide range of ph and change the ph as they grow, some species increase the ph of medium fig. 3 effect of different culture media on the growth of t. rubrum isolates after 7 days of incubation. 0 1 2 3 4 5   2   day 5   day 7   day 0 1 2 3 4 5   2   day 5   day 7   day co lo ny d ia m et er (c m )   0 1 2 3 4 5   2   day 5   day 7   day co lo ny d ia m et er (c m ) co lo ny d ia m et er (c m )   0 2 4 6 8 10   2   day 5   day 7   day co lo ny d ia m et er (c m )   0 1 2 3 4 5 culture media culture mediaculture media culture media culture media culture media culture media culture media   2   day 5   day 7   day 0 1 2 3 4 5 6 7   2   day 5   day 7   day co lo ny d ia m et er (c m ) co lo ny d ia m et er (c m )   0 1 2 3 4 5 6 7 8   2  day 5  day 7  day co lo ny d ia m et er (c m )   0 1 2 3 4 5 sda pda cma yea sda pda cma yea   2   day 5   day 7   day lsd(0.05) = 0.249 lsd(0.05) = 0.245 lsd(0.05) = 0.245 lsd(0.05) = 0.257 lsd(0.05) = 0.275 lsd(0.05) = 0.289 lsd(0.05) = 0.279 lsd(0.05) = 0.245 co lo ny d ia m et er (c m )   t. rubrum no.1 t. rubrum no.2 t. rubrum no.3 t. rubrum no.5 t. rubrum no.7 t. rubrum no.4 t. rubrum no.6 t. rubrum no.8 sda pda cma yea sda pda cma yea sda pda cma yea sda pda cma yea sda pda cma yea sda pda cma yea but others decrease it. dermatophytes tend to produce an alkaline ph when growing on sabouraud’s medium, this is due to the deamination of amino acids and consequent formation of ammonia.22 on the other hand, certain non-pathogenic molds such as penicillium 14 j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 growth effect in proteolytic activity of clinical isolates of t. rubrum research sara k kadhim et al. and aspergillus species, are known to shift the ph of the medium towards acidity.23 the ph of the culture media is important for mineral availability, enzyme activity, membrane function, growth and sporulation of keratinophilic fungi. although the alteration that was found in medium was used for fungal growth, the cytoplasm remains conservative on the rate of hydrogen and hydroxyl ions because the plasma membrane that did not permit these ions to cross cytoplasm. ph is effective on enzymes found in cytoplasm that leads to the alteration of bioprocess of microorganisms and thus seem to be effective on either ion uptake or a loss to the nutrient medium.24 sharma et al.,19 observed that ph7 and 33°c temperature were the most suitable for the growth of t. rubrum. our results are consistent with the lsd (0.05) = 0.3908 lsd (0.05) = 0.398 lsd (0.05) = 0.4345 lsd (0.05) = 1.910 lsd (0.05) = 0.4605 en zy m e a ct ivi ty (u /m l)     0 1 0 2 0 3 0 4 0 5 0   0 1 0 2 0 3 0 4 0 5 0     0 10 20 30 40 substrate con. substrate con. substrate con. substrate con. substrate con. substrate con. substrate con.substrate con.   en zy m e a ct iv ity (u /m l) en zy m e a ct iv ity (u /m l)   0 1 0 2 0 3 0 4 0   en zy m e a ct iv ity (u /m l) en zy m e a ct iv ity (u /m l) en zy m e a ct iv ity (u /m l)   0 1 0 2 0 3 0 4 0 5 0     en zy m e ac tiv ity (u /m l) 0 1 0 2 0 3 0 4 0 5 0 6 0   en zy m e ac tiv ity (u /m l) 0 1 0 2 0 3 0 4 0     0 1 0 2 0 3 0 4 0 5 0       t. rubrum no.1 t. rubrum no.2 t. rubrum no.3 t. rubrum no.5 t. rubrum no.7 t. rubrum no.4 t. rubrum no.6 t. rubrum no.8 lsd (0.05) = 0.8348 lsd (0.05) = 1.0678 lsd (0.05) = 0.431 0.5 1 1.5 2 2.5 3 0.5 1 1.5 2 2.5 3 0.5 1 1.5 2 2.5 3 0.5 1 1.5 2 2.5 3 0.5 1 1.5 2 2.5 30.5 1 1.5 2 2.5 3 0.5 1 1.5 2 2.5 3 0.5 1 1.5 2 2.5 3 fig. 4 effect of different substrate con. on the protease activity produced by t. rubrum isolates. 15j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 research growth effect in proteolytic activity of clinical isolates of t. rubrumsara k kadhim et al. results of danew and klossek,25 who pointed the importance of ph in the growth of fungus. majority of fungi have been found to grow well at a ph range from 4.2– 9.3. usually too alkaline and too acidic solutions are not favorable for the growth of fungi. this might be because proteins have a tendency to develop lesser viscosity and simultaneously, their colloidal behavior changes in the sense that hydrolysis of proteins tends to form simpler products that results in the formation of colloidal particles of smaller dimension. this disturbance in the proper colloidal state of the cytoplasm hinders its normal function. under such circumstances, fig. 5 effect of different incubation period (day) on the protease activity produced by t. rubrum isolates. the formation of the complex protein molecules becomes difficult resulting in the retardation of growth.19 the nature of a particular medium has a great role to play in the growth and sporulation of fungi. kaul and sambali26 reported that keratinophilic fungi grow well in media rich in nitrogen and carbon contents. maximum growth of lsd(0.05) = 1.131   0 10 20 30 40 50 60 70 80 90 100 3 6 9     0 10 20 30 40 50 60 70 80 90 100 3 6 9         0 10 20 30 40 50 60 70 3 6 9 incubation period (day) incubation period (day) incubation period (day) incubation period (day) incubation period (day) incubation period (day)     en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l)   0 10 20 30 40 50 60 70 80 3 6 9         0 10 20 30 40 50 60 70 80 90 3 6 9     en zy m e ac tiv ity (u /m l) 0 10 20 30 40 50 60 3 6 9       0 10 20 30 40 50 3 6 9     en zy m e a ct ivi ty (u /m l)  en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l)   en zy m e ac tiv ity (u /m l) en zy m e a ct ivi ty (u /m l)  0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 3 6 9       t. rubrum no.1 t. rubrum no.3 t. rubrum no.5 t. rubrum no.7 t. rubrum no.2 t. rubrum no.4 t. rubrum no.6 t. rubrum no.8 incubation period (day) incubation period (day) lsd(0.05) = 1.8698 lsd(0.05) = 2.274 lsd(0.05) = 1.5138 lsd(0.05) = 3.189 lsd(0.05) = 2.5405 lsd(0.05) = 1.9445 lsd(0.05) = 3.4355 12 15 12 15 12 15 12 15 12 15 12 15 12 15 12 15 16 j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 growth effect in proteolytic activity of clinical isolates of t. rubrum research sara k kadhim et al. t. rubrum isolates were observed on sda followed by pda. the reason for an increased rate growth of t. rubrum in the sda medium was due to the presence of nutritional requirements for the growth of fungus in this medium such as the rate of glucose in the medium was 40 gm and peptone was 10 gm. peptone contains a proportion of nitrogen which is reached to 13%.27 it was believed that glucose was the most important determinant of suitable growth of fungi.28 our results agreed with singh,29 jain30 and sharma & sharma,31 which found that sda medium showed maximum growth and sporulation for all test fungi. sharma24 also agrees with the present investigation and suggested that the sda medium is as an excellent source for almost all dermatophytic and keratinophilic fungi. the present study will help to maintain the fungus in the laboratory conditions for preparation of inocula for different studies concerning t.rubrum no.1     0 10 20 30 40 50 60 70 80 90 100 110 5 5.5 6 6.5 7 7.5 8 8.5 9 5 5.5 6 6.5 7 7.5 8 8.5 9 5 5.5 6 6.5 7 7.5 8 8.5 9 5 5.5 6 6.5 7 7.5 8 8.5 9 5 5.5 6 6.5 7 7.5 8 8.5 9 5 5.5 6 6.5 7 7.5 8 8.5 9 5 5.5 6 6.5 7 7.5 8 8.5 9 5 5.5 6 6.5 7 7.5 8 8.5 9 ph 0 10 20 30 40 50 ph en zy m e a ct ivi ty (u /m l)     0 10 20 30 40 50 60 70 80 90 ph   0 10 20 30 40 50 60 ph en zy m e a ct iv ity (u /m l)   0 10 20 30 40 ph en zy m e a ct ivi ty (u /m l)   0 10 20 30 ph en zy m e a ct iv ity (u /m l)     0 10 20 30 40 50 60 70 ph en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l)   0 10 20 30 40 50 60 ph en zy m e a ct ivi ty (u /m l)  t.rubrum no.3 t.rubrum no.5 t.rubrum no.6 t.rubrum no.4 t.rubrum no.7 t.rubrum no.8 t.rubrum no.2 lsd(0.05) = 2.2028 lsd(0.05) = 1.9518 lsd(0.05) = 1.9768 lsd(0.05) = 1.406 lsd(0.05) = 0.540 lsd(0.05) = 1.231 lsd(0.05) = 1.819 lsd(0.05) = 1.8755 fig. 6 effect of different ph on the protease activity produced by t. rubrum isolates. control of the human pathogen causing dermatophytic infections. effect of some culture conditions on the production of protease 1. incubation period after protease production, the medium was incubated for different incubation periods (3, 6, 9, 12, 15 days), the production of protease reached maximum activity of t. rubrum isolates after 9 days 17j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 research growth effect in proteolytic activity of clinical isolates of t. rubrumsara k kadhim et al. fig. 7 effect of different temperature on the protease activity produced by t. rubrum isolates. of incubation. the optimal activity for protease of 8 isolates were 90.5, 88.5, 66.2, 67.8, 83.6, 48.5, 43.3 and 85.8 u/ml then a progressive decrease in protease production was observed when incubated for 12 and 15 days. the incubation period of 9 days was maintained to study the effect of other factors. these results was in agreement with mahmood et al.32 that showed 9 days was the best incubation period for the production of enzyme. abdel-hafez et al.33 and el-said34 found that the maximum protease production by t. rubrum was observed after 8 days of incubation at 30°c. a progressive decrease in proteolytic activity occurred thereafter. studies on other species of fungi have shown that in most cases proteolytic activity increases with the beginning of autolysis.35,36 apodaca and mckerrow37 showed that during log phase growth most of the proteolytic enzyme of t. rubrum are repressible in vitro by small molecules and are likely t. rubrum no.1 lsd(0.05) = 4.265   0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 20 25 30 35 40 45 50 te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c) te mpe rature (c)   0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0     0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0     0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0   0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0   en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l) en zy m e a ct ivi ty (u /m l) 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0     0 10 20 30 40 50 60 70 80     0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0   t. rubrum no.3 t. rubrum no.5 t. rubrum no.2 t. rubrum no.4 t. rubrum no.6 t. rubrum no.7 t. rubrum no.8 lsd(0.05) = 2.488 lsd(0.05) = 2.352 lsd(0.05) = 2.074 lsd(0.05) = 2.770 lsd(0.05) = 1.180 lsd(0.05) = 3.877 lsd(0.05) = 2.354 20 25 30 35 40 45 50 20 25 30 35 40 45 50 20 25 30 35 40 45 50 20 25 30 35 40 45 50 20 25 30 35 40 45 50 20 25 30 35 40 45 50 20 25 30 35 40 45 50 18 j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 growth effect in proteolytic activity of clinical isolates of t. rubrum research sara k kadhim et al. repressed during early growth in vivo. the entire complement of proteinases in t. rubrum tends to be produced constitutively during the stationary phase of growth in vitro. reduction in the production of enzyme was due to the cell which may reach to the decline phase, accumulation of waste materials and unavailability of nutrients.38 incubation time being an important factor has been responsible for optimum enzyme formation and it varies from one organism to another due to the variation in the lag and log phase of growth.39 2. temperature temperature is a very important factor in the enzyme production since it plays a role in the structure of the enzyme and in the growth of microorganisms.40,41 the optimum temperature for protease production by t. rubrum isolates in the present study was at 30°c which reached to 86.2, 80.1, 83.6, 82.1, 73.6, 83.4, 68.1 and 84.6 u/ml for eight tested isolates then reduced with a rise in the temperature. the results in this study agreed with the results reported by el-said,34 who reported that the optimum temperature for protease production from t. rubrum isolates was 30°c. decrease or increase in incubation temperature is an essential factor because of its importance in microorganism growth, metabolite production and suppression of cell viability.42 another possible reason could be due to the breaking down of enzyme at higher temperature as enzyme denature.43 the results of the study agreed with other studies which reported that most enzymes denaturated and lost its activity when temperature exceeds 35°c, also high temperature have affected the fungus growth and protease production. also the studies indicated that the protease production of fungus decreases when incubation temperature was less or high than 30°c. recent investigations have shown that proteases secreted by dermatophytes are similar to those of other fungi such as aspergillus spp.44–46 3. ph acidity of the culture media is one of the most critical parameter that affected the production of proteases. ph is affected in the ionic state of amino acids that is responsible for the primary and secondary structure of enzyme and leads to affect the enzyme activity.47 the results in this study demonstrated that the optimum ph for proteases production was neutral range. similarly for this work, abdel-hafez et al.33 and el-said34 found that the maximum protease production by t. rubrum was within the range of ph 6–8. asahi et al.48 showed that the extra cellular proteinases of t. rubrum also have optimal proteolytic activity at neutral ph. but in other studies with t. rubrum, proteinase with a ph optimum of 4.5 was detected.49 t. rubrum has been extensively studied by apodaca and mckerrow37 that showed to produce two strongly keratinolytic proteinases, as well as a poorly keratinolytic, trypsinor chemotrypsin-like general proteinase. all these proteinases have a ph optimum of approximately 8. in a study of protease production during autolysis in different species of filamentous fungi, it was observed that autolysis occurred at ph values between 6.5–8.50 human skin has a weak acidic ph and it is noteworthy that proteinases with an optimal activity under acidic conditions are reported to be important virulence factors in t. mentagrophytes.51 dermatophyte proteolysis results in the liberation of excess ammonium ion, raising the ph of the growth medium.52 this reaction, an attribute relatively uncommon in fungi isolated clinically, has been used as the basis of screening media such as dermatophyte test medium. 4. substrate concentration the concentration of casein is an important factor for protease production. enzyme production was found to influence with casein concentration. the results in this study agreed with reddy and saritha53 who found that the suitable substrate concentration was 0.6 gram per 100 ml of production medium and rajendran et al.54 reported an initial substrate concentration of 0.5% of pectinase, this may be due to that in high substrate concentration no more enzymes are available free for carrying the reaction since this enzyme is constant.55 further increase in substrate concentration resulted in drastic reduction in enzyme production, probably due to catabolic repression of biosynthesis56 or a result of reduced mass transfer of oxygen by higher amount of solid substrate.57 the increase of substrate concentration leads to decrease in enzyme activity, this is probably due to the disintegration of some substrate compounds that play role as inhibitors for enzymes and decreased enzyme activity. while the small amounts of substrate concentration does not have any activity because of increase enzyme production, their results was in agreement with teixeira et al.,58 who showed the best pectin concentration for pectinase production from aspergilus japonica was 0.5%.  references 1. hryncewicz-gwóźdź a, jagielski t, dobrowolska a, szepietowski jc, baran e. identification and differentiation of trichophyton rubrum clinical isolates using pcr-rflp and rapd methods. eur j clin microbiol infect dis. 2011 jun;30(6):727–31. doi: http://dx.doi.org/10.1007/s10096-010-1144-3 pmid: 21416216 2. liu t, xu x, leng w, xue y, dong j, jin q. analysis of gene expression changes in trichophyton rubrum after skin interaction. j med microbiol. 2014 may;63(pt 5):642–8. doi: http://dx.doi.org/10.1099/jmm.0.059386-0 pmid: 24586032 3. kalinowska k, hryncewicz-gwóźdź a, plomer-niezgoda e. differentiation of dermatophytes belonging to the genus trichophyton. mikol lek. 2009;16:171–7. 4. white t, henn m. genomic determinants of infection in dermatophyte fungi. the fungal genome initiative. ma, usa: dermatophyte genome steering committee; mar 2012. available at: http://www.genome.gov/ pages/research/sequencing/seqproposals/dermatophyte_wp_seq.pdf 5. maranhão fc, paião fg, martinez-rossi nm. isolation of transcripts over-expressed in human pathogen trichophyton rubrum during growth in keratin. microb pathog. 2007 oct;43(4):166–72. doi: http://dx.doi. org/10.1016/j.micpath.2007.05.006 pmid: 17590307 6. chen j, yi j, liu l, yin s, chen r, li m, et al. substrate adaptation of trichophyton rubrum. microb pathog. 2010 feb;48(2):57–61. doi: http:// dx.doi.org/10.1016/j.micpath.2009.12.001 pmid: 20005286 7. kwon-chung kj, bennet je. medical mycology. philadelphia and london: lea & febiger; 1992. 8. irene w, richard cs. clinical microbiology reviews: the dermatophytes. american society for microbiology. 1998;8:240–59. 9. burns t, breathnach s, cox n, griffiths c. rook’s textbook of dermatology, vol 2, 8th ed. blackwell publishing; 2010. pp. 1814–29. 10. aubaid ah. enzymatic activity, purification of keratinase and proteinase and their roles in the pathogenicity and immunogenicity; 1997. 11. chmel l,  hasilíková a, hrasko j, vlácílíková a. the influence of some ecological factors on keratinophilic fungi in the soil. sabouraudia. 1972 mar;10(1):26–34. doi: http://dx.doi.org/10.1080/00362177285190071 pmid: 5063162 12. zhao s, shamoun sf. the effects of culture media, solid substrates, and relative humidity on growth, sporulation and conidial discharge of 19j contemp med sci | vol. 1, no. 3, summer 2015: 9–19 research growth effect in proteolytic activity of clinical isolates of t. rubrumsara k kadhim et al. valdensinia heterodoxa. mycol res. 2006 nov;110(11):1340–6. doi: http:// dx.doi.org/10.1016/j.mycres.2006.08.001 pmid: 17070027 13. day wc, toncic p, stratman sl, leeman v, harmon sr. isolation and properties of an extracellular protease of trichophyton granulosum. biochimica biophysica acta. 1968;167:597–606. 14. hanlon gw, hodges na. requirement for glucose during production of extracellular serine protease by cultures of bacillus licheniformis. fems microbiol lett. 1981 may;11(1):51–4. doi: http://dx.doi. org/10.1111/j.1574-6968.1981.tb06933.x 15. joo hs, kumar cg, park cg, kim kt, paik sr, chang cs. optimization of the production of an extracellular alkaline protease from bacillus horikoshii. process biochem. 2002 oct;38(2):155–9. doi: http://dx.doi.org/10.1016/ s0032-9592(02)00061-4 16. paulson ds. biostatistics and microbiology: a survival manual. new york: springer science and business media; 2008. 17. meier cl, rapp j, bowers rm, silman m, fierer n. fungal growth on a common wood substrate across a tropical elevation gradient: temperature sensitivity, community composition and potential for above-ground decomposition. soil biol biochem. 2010 jul;42(7):1083–90. doi: http:// dx.doi.org/10.1016/j.soilbio.2010.03.005 18. sharma a, sharma m, chandra s. influence of temperature and relative humidity on growth and sporulation of some common dermatophytes. ind j fund appl life sci. 2012;2(4):2231–6345. 19. sharma s, sharma p, agrawal r. effect of temperature and ph combinations on growth pattern of dermatophytes isolated from hiv positive patients. asian j biochem pharmaceut res. 2011;3(1):2231–560. 20. martínez de marañón, chaudanson n, joly n, gervais p. slow heat rate increase yeast thermotolerance by maintaining plasma membrane integrity. biotechnol bioeng. 1999 oct 20;65(2):176. doi: http://dx.doi.org/10.1002/ (sici)1097-0290(19991020)65:2%3c176::aid-bit7%3e3.3.co;2-x pmid: 10458738 21. rehman fu, aslam m, tariq mi, shaheen a, sami aj, naveed nh, et al. isolation of cellulolytic activities from tribolium castaneum (red flour beetle). african journal of biotechnology 2009 dec;8(23):6710–5. doi: http://dx.doi. org/10.5897/ajb09.435 22. taschdjian cl. a colorimetric assay of dermatophyte growth in broth culture. j invest dermatol. 1952 may;18(5):369–72. pmid: 14927984 23. foster jw. chemical activities of fungi. new york: academic press inc.; 1949. 24. sharma m. taxonomical, physiological and para-clinical studies of fungi causing skin and other infections in human beings [ph.d. thesis], botany department. jaipur, india: university of rajasthan; 1983. 25. danew p, klossek p. the dependence of the physiological properties of trichophyton mentagrophytes on the ph value of the culture medium. mycoses 1989 jun;32(6):303–8 pmid: 2779611 26. kaul s, sumbali g. impact of some ecological factors on the occurrence of poultry soil-inhabiting keratinophiles. mycopathologia 1998;143(3):155–9. pmid: 10353212 27. kurbanoğlu eb, algur öf. use of ram horn hydrolysate as a peptone for bacterial growth. turk j biol. 2002;26(2):115–23. 28. kunert j. physiology of keratinolytic fungi. in: kushwaha rks, guarro j, editors. revista iberoamericana de micologia. bilbao, spain: university of bilbao; 2000. p. 77–85. 29. singh kv. radial growth of soil inhabiting keratinophilic fungi and related dermatophytes on various media and at different temperatures. geobios. 1983;10(6):284–6. 30. jain n. antidermatophytic activity of some plant metabolites [ph.d. thesis], botany department. jaipur, india: university of rajasthan; 2001. 31. sharma m, sharma m. influence of culture media on mycelia growth and sporulation of some soil dermatophytes compared to their clinical isolates. j microbiol antimicrobials. 2011;3(8):196–200. 32. mahmood ar, hamada ta, ahmed ia. isolation of extracellular protease from trichophyton mentagrophytes of skin isolates. tikrit j pure sci. 2008;14(3):35–41. 33. abdel-hafez sii, el-said ahm, maghraby ta. studies on fungi isolated from skin diseases and associated fungi of students in qena and red sea governorates, egypt. bull fac sci. 1995;24(2):181–209. 34. el-said ahm. studies on fungi isolated from dermatomycoses patients in egypt. mycobiology 2002;30(3):154–9. doi: http://dx.doi.org/10.4489/ myco.2002.30.3.154 35. reyes f, lahoz r, moreno av. synthesis of 1, 3-ßglucanase and ß-n-acetylglucosaminidase during autolysis of neurospora crassa. j gen microbiol. 1981;126:347–53. 36. reyes f, calatayud j, martínez mj. chitinolytic activity in the autolysis of aspergillus nidulans. fems microbiol lett. 1988;49:239–43. 37. apodaca g, mckerrow jh. purification and characterization of a 27,000-mr extracellular proteinase from trichophyton rubrum. infect immun. 1989 oct;57(10):3072–80. pmid: 2674015 38. raju evn, divakar g. production of pectinase by using bacillus circulans isolated from dump yards of vegetable wastes. int j pharmaceut sci res. 2013;4(7):2615–22. doi: http://dx.doi.org/10.13140/2.1.2487.6488 39. bhatti hn, mustafa g, asgher m. production of enzymes by fusarium moniliforme under solid-state fermentation. chem soc pak. 2007;29(2):161–5. 40. rosés rp, guerra n. optimization of amylase production by aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material. world j microbiol biotechnol. 2009 nov;25(11):1929–39. doi: http://dx.doi.org/0.1007/s11274-009-0091-6 41. sudheer kumar y, varakumar s, reddy ovs. production and optimization of polygalacturonase from mango (mangifera indica l.) peel using fusarium moniliforme in solid state fermentation. world j microbiol biotechnol. 2010 nov;26(11):1973–80. doi: http://dx.doi.org/10.1007/s11274-010-0380-0 42. ali ma, vidhale nn. protease production by fusarium oxysporum in solidstate fermentation using rice bran. am j microbiol res. 2013;1(3):45–7. doi: http://dx.doi.org/0.12691/ajmr-1-3-2 43. darah m n, lim sh. enhancement of polygalacturonase production from enterobacter aerogenes nbo 2 by submerged fermentation. 2013;5(5):173– 89. doi: http://dx.doi.org/10.12988/asb.2013.313 44. brouta f, descamps f, monod m, vermout s, losson b, mignon b. secreted metalloprotease gene family of microsporum canis. infect immun. 2002 oct;70(10):5676–83. doi: http://dx.doi.org/10.1128/iai.70.10.56765683.2002 pmid: 12228297 45. descamps f, brouta f, monod m, zaugg c, baar d, losson b, mignon b. isolation of a microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo. j invest dermatol. 2002 oct;119(4):830–5. doi: http://dx.doi.org/10.1046/j.1523-1747.2002.01784.x pmid: 12406327 46. jousson o, léchenne b, bontems o, capoccia s, mignon b, barblan j, et al. multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes trichophyton and microsporum. microbiology 2004 feb;150(pt 2):301–10. doi: http://dx.doi.org/10.1099/ mic.0.26690-0 pmid: 14766908 47. griffin dh. fungal physiology. new york: wiley-liss; 1994. p. 458. 48. asahi m, lindquist r, fukuyama k, apodaca g, epstein wl, mckerrow jh. purification and characterization of major extracellular proteinases from trichophyton rubrum. biochem j. 1985 nov;232(1):139–44. doi: http://dx.doi. org/10.1042/bj2320139 pmid: 3910025 49. tanaka s, summerbell rc, tsuboi r, kaaman t, sohnle pg, matsumoto t, ray tl. advances in dermatophytes and dermatophytosis. j med vet mycol. 1992;30(1):29–39. 50. santamaria f, reyes f. proteases produced during autolysis of filamentous fungi. trans br mycol soc. 1988;91:217–20. doi: http://dx.doi.org/10.1016/ s0007-1536(88)80207-9 51. tsuboi r, sekiguchi k, ogawa h. the properties and biological role of an acidic proteinase from trichophyton mentagrophytes. jpn j med mycol. 1992;33:147–51. 52. gilberto ul, braga, ricardo hrd, messias cl. protease production during growth and autolysis of submerged metarhizium anisopliae cultures. rev microbiol. 1999 apr/jun;30(2). doi: http://dx.doi.org/10.1590/s000137141999000200004 53. reddy mp, saritha kv. bio-catalysis of mango industrial waste by newly isolated fusarium sp. (pstf1) for pectinase production. biotech. 2015 mar 22. doi: 10.1007/s13205-015-0288-3 54. rajendran r, karthik s, radhai r, rajapriya p, balakumal c. production and optimization of fungal pectinase from fusarium. int j curr res. 2011;3(4):254–8. 55. dhital r, panta op, karki tb. optimization of cultural conditions for the production of pectinase from selected fungal strain. j food sci technol nepal 2013;8(1):65–70. 56. omojasola pf, jilani op. cellulase production by trichoderma longi, aspergillus niger and saccharomyces cervisiae cultured on waste materials from orange. pakistan j biol sci. 2008;11(20):2382–8. doi: http://dx.doi. org/10.3923/pjbs.2008.2382.2388 57. ghosh b, ray rr. current commercial perspective of rhizopus oryzae: a review. j appl sci. 2011 dec 1;11(14):2470–86. doi: http://dx.doi. org/10.3923/jas.2011.2470.2486 58. teixeira mfs, fiho jl, durán nl. carbon source effect on pectinase production from aspergillus japonicus 586. braz j microbiol. 2003;31:286–90. 162 j contemp med sci | vol. 8, no. 3, may-june 2022: 162–168 original influential factors on oral health status of the elderly iranians: a path analysis azadeh fartash tolou1, , mohammad pooyan jadidfard1, , hadi ghasemi1, , farzaneh boroumand2, mahshid namdari1, mohammad h. khoshnevisan1,3* 1dental public health program, community oral health department, school of dentistry, shahid beheshti university of medical sciences, tehran, ir-iran. 2 3 postdoctoral research fellow, school of mathematical and physical sciences, macquarie university, sydney, australia. chair, community oral health department, dental research institute, dental research center, school of dentistry, shahid beheshti university of medical sciences tehran, ir-iran. *correspondence to: mohammad h. khoshnevisan (e-mail: khoshmh@gmail.com) (submitted: 09 may 2022 – revised version received: 24 may 2022 – accepted: 05 june 2022 – published online: 26 june 2022) abstract objectives: this study aimed at assessing the influential factors on oral health status of the elderly iranian population (65 to 74 years of age) by using path analysis. methods: this study was conducted on 7,521 elderly individuals participating in the iranian national oral health survey in 2012. the dmft and functional health (fh) indices were evaluated first. then socio-economic status (ses), insurance type (it), dental attendance pattern (dap), reason of attendance (r of a), tooth cleaning (tc), and nutritional pattern (np) were evaluated according to responses provided in the questionnaire. path analysis was applied to analyze the patterns between these variables and dmft and fh indices. results: the participants’ mean dmft was 26.6 ± 8.03, and the median fh was 0 (0.10). path analysis revealed that seniors with higher ses, and having a dental insurance with greater coverage had direct effect on more regular dap, and indirect effect on improvement of fh and dmft index. more regular dap had direct effect on improvement of fh, and dmft indices. likewise, np with low risk of caries had direct effect on improvement of fh and dmft index. conclusion: out of all evaluated factors, it, ses, dap, and np demonstrated to be particularly important and significantly affecting dmft and fh indices in the elderly population. out of all aforementioned factors, it in low ses individuals was more important for obtaining required oral healthcare services. apparently, seniors’ oral health is absolutely in need of special attention with particular focus on providing early access to preventive care. keywords: oral health, elderly, dmft, fh index, path analysis, national survey issn 2413-0516 introduction although preventable, oral diseases have a considerable prevalence, affecting over 3.5 billion people worldwide. dental caries is among the most common diseases globally, with a higher prevalence in deprived communities and developing countries.1 on the other hand, in relation to aging population, we are currently witnessing the most important demographic phenomenon in the 21st century, with respect to the increase in both census and ratio. in 2004, approximately 600 million people worldwide were ≥60 years of age. this rate is estimated to double by the year 2025, and reach 2 billion by 2050; about 80% of which residing in developing countries. in the year 2000, the ratio of the elderly population to the entire population of the world was 10%. this ratio was 6.5% in iran in the same year. in the year 2020, the ratio of the elderly population to the entire population of the world increased to 13.5%, and this ratio reached to 10% in iran. according to the available statistics, the population of iran is moving towards aging, similar to most other parts of the world.2 at old age, many individuals often suffer from multiple health problems, and usually need to take several medications on a daily basis. thus, poor oral hygiene and repeated exposure to risk factors in this age group can increase and complicate their oral health status. if left untreated, severe dental caries can lead to complete destruction of the crown, development of periodontal diseases, tooth loss, and related local and systemic complications in the elderly. all these factors can deteriorate the quality of life in this vulnerable population.3 benefitting from oral healthcare services is a multi-factorial phenomenon, depending on a number of elements such as the dentition status, oral health attitudes, as well as socioeconomic status (ses).4 in 2012, approximately 30% of the iranian elderly population, over 65 years of age had a dental visit in the past year.5 in the same year, the mean dmft index of the 65 to 74-year-old individuals in iran was 25.71, with the number of decayed (d), missing (m) and filled (f) teeth reported as 2.44, 22.56, and 0.71 (11.72%, 84.22%, and 4.05% of the total mean), respectively. the prevalence of complete edentulism was >52% in this age group.6 according to available reports, health insurance can enhance the beneficiaries’ access to healthcare services. in 2012, aside from the private and supplementary health insurance systems, four main basic insurance systems were available in iran, namely the medical services insurance fund (msif), social security organization (sso), imamkhomeini relief committee (ikrc), and armed forces medical services insurance fund (afmsif). in terms of the covered population, msif had the largest and afmsif had the smallest population under insurance coverage. the four https://orcid.org/0000-0002-9042-0046 https://orcid.org/0000-0001-7438-8376 https://orcid.org/0000-0003-3331-1094 https://orcid.org/0000-0002-7083-8816 https://scholar.google.com/citations?view_op=view_org&hl=en&org=7030147968151678976 https://scholar.google.com/citations?view_op=view_org&hl=en&org=7030147968151678976 https://scholar.google.com/citations?view_op=view_org&hl=en&org=7030147968151678976 https://scholar.google.com/citations?view_op=view_org&hl=en&org=7030147968151678976 https://scholar.google.com/citations?view_op=view_org&hl=en&org=7030147968151678976 mailto:khoshmh@gmail.com 163j contemp med sci | vol. 8, no. 3, may-june 2022: 162–168 a.f. tolou et al. original influential factors on oral health status of the elderly iranians: a path analysis aforementioned insurance organizations had wide variations in respect to dental service coverage. the franchise was 30% of the fee for dental services in all four insurance systems. in 2012, the share of dental costs paid out of all covered services was 1% for the treatment unit of sso and msif, 5% for ikrc, and 20% for afmsif. also, there was a fund under the name “therapeutic support fund” in the general council of armed forces, that would pay up to 100% of the treatment costs for the insured individuals under certain circumstances (i.e. patients with special need conditions), even covering some dental services, which were not normally covered by this organization. apparently, those individual covered by the afmsif would benefit from considerably wider range of services compared to other three insurance systems.8 this study aimed to assess the effect of influential factors on oral health status of the iranian elderly population with 65 to 74 years old. materials and methods data sources the iranian national oral health survey (inohs-2012) was conducted in 2012, by using the world health organization’s (who) standard questionnaire. subjective (self-report) and objective (clinical examination) methods were used to assess the oral health status of the participants. this study is using part of the data related to geriatric cases, including 65 to 74 years old age group. dental caries was among the oral conditions evaluated in the elderly population. also, a standard who questionnaire was used in order to assess the effect of other influential factors affecting the oral health status. aside from demographic information (age, gender, place of residence), ses (level of education, occupation, family size, having a personal vehicle, number of rooms in the house living in, as well as home ownership status), tc (tooth cleaning), np (nutritional pattern), dap (dental attendance pattern, and insurance-related information were collected. as recommended by the who, a total of 300 elderly individuals were recruited from each province. in the inohs2012, samples were collected from all provinces of iran. in case of tehran, the capital city, it was considered as two provinces due to its high population (>10 million). study participants were selected by cluster random sampling. the selected samples filled out the questionnaire if they were willing to participate in the study, and were scheduled for a clinical examination afterwards. the examiners and those recorded the information were calibrated by a who representative in order to standardize the process of clinical oral examination and data collection prior to the initiation of study. after collection of the completed questionnaires, distorted instruments and cases without clinical examination report were excluded from the data bank. through this vigorous process, a total number of 7521 samples remained in the study. more details on implementation of the inohs-2012 are available elsewhere.6,7 conceptual framework and measurements in the present study, a conceptual model was first designed in order to identify the influential factors on elderly oral health status based on the questions included in the questionnaire (figure 1). the dmft and functional health (fh) index were calculated as objective indices for assessment of the health status of the entire dentition according to the data obtained from the clinical examination forms. the dmft index was calculated by summing the number of decayed (d), missing (m), and filled (f) teeth. the fh index was calculated by summing the number of sound and caries-free restored teeth in the oral cavity for each individual using the formula [32(m+d)]. to assess the influential factors on oral health of the elderly, the conceptual model was tested by path analysis. the four insurance systems, namely the ikrc (=1), msif (=2), sso (=3), and afmsif (=4) were considered as answer choices for the question regarding the participants’ insurance type (it) (other insurance types such as supplemental insurance were not evaluated in this study). two questions were asked regarding the life style. the first question was about factors related to hygienic behaviors, such fig. 1 conceptual framework (primary model). table 1. cariogenic risk of selected cariogenic items # cariogenic items several times a day (=6) every day (=5) several times a week (=4) once a week (=3) several times a month (=2) seldom/ never (=7) 1 biscuit/cake/ cookies × 2 sugar containing syrup × 3 jam/honey × 4 sugar containing gum × 5 candy/chocolate × 6 sweetened milk × 7 tea with sugar × 9 164 j contemp med sci | vol. 8, no. 3, may-june 2022: 162–168 influential factors on oral health status of the elderly iranians: a path analysis original a.f. tolou et al. as “how often do you brush your teeth?” never (=1), once or several times a month (=2), several times a week (=3), and once or several times a day (=4). the second question was about np. the participants were asked “how often do you use the following five cariogenic items?” the lowest rate of consumption was allocated a score of 1, and the maximum rate of consumption was allocated a score of 6. accordingly, the maximum risk for the seven cariogenic items was 42 and minimum risk was 7. therefore, the participants could be anywhere between the range of 7 to 42 depending on the rate and type of cariogenic items consumed. based on the frequency of responses, the participants were categorized into three groups: those with scores between 31–42 were categorized as high risk (group 1), those with scores between 19–30 were categorized as moderate risk (group 2), and those with scores between 7–18 were categorized as low risk (group 3). two questions addressed the care seeking behavior. the first question asked about the reason for the latest dental visit, and the answer choices were: presence of a problem (=1) and check-up (=2). the second question asked for the time of participant’s last dental visit (dap). the answer choices were: had no visit (=1), over a year ago (=2), and during the past year (=3). demographics for ses categorization of participants, the related items including occupation, level of education, home ownership, family size, number of rooms, and having a personal vehicle for leisure (not for work) were weighted. the ses score was calculated separately for the rural and urban populations. because, the number of family members was not among the ses determinants in urban population; while family members in rural population are considered as human resource for work and family income. thus, the number of family members was considered as a determining factor for ses in rural populations. finally, the ses score was calculated for the entire urban and rural populations and categorized as low (=1), moderate (=2), and high (=3). statistical procedures for describing the data, the frequency (percentage) of categorical variables and mean (standard deviation) and median interquartile range (iqr = q1, q3) of the numerical variables were all reported. the mean was reported for dmft index and median was reported for fh index (since the variance was larger than the mean). the kolmogorov-smirnov test was used to assess the normal distribution of all numerical data. the kruskal-wallis test was used to compare dmft and fh based on it, ses, np, tc, and time of last dental visit. the mann-whitney test was applied to compare dmft and fh indices based on gender and reason of attendance (r of a). path analysis was used to analyze the correlation between oral health variables and other variables in a causal model. path analysis is a technique that determines to what extent a hypothetical model agrees with the existing data. it also enables testing of a causal model according to cross-sectional data. path model has maximum efficacy when only observed variables are entered in the model. by applying the model fit, it is tested to what extent the conceptual framework is supported by the actual data. in other words, it indicates the fit of the experimental model (based on data) with the theoretical model (conceptualized by the researcher). the fit indices used in the present study included the root mean square error of approximation (rmsea), comparative fit index (cfi), tucker lewis index (tli), standardized root mean squared residual (srmr), and coefficient of determination (cd).10-13 data were analyzed by spss 25 and stata 14. the ethics committee of the school of dentistry, shahid beheshti university of medical sciences approved the proposal for this study (ir.sbmu.drc.1399.021). results the study participants’ (n = 7,521) demographic information is demonstrated in table 2. the participants’ mean dmft was 26.6 ± 8.03 and the median fh index was 0 (0.10). a total of 4,292 (57.1%) participants had a dmft of 32 (diagram 1), and 4,285 (57%) had a fh index of 0 (diagram 2). the kolmogorov-smirnov test showed that, the data were not normally distributed. thus, appropriate tests were applied to analyze the association of variables with different factors. the participants’ mean dmft was higher in male (26.74 ± 7.94) than female (26.94 ± 8.10) and the median fh was higher in female 0 (0.11) compared to male 0 (0.10), but the dmft and fh index for male and female participants were not significantly different. although, significant associations detected between ses, it, tc, and r of a with dmft and fh, and also between dap and np with fh (p < 0.01). a significant association was also found between dap and dmft (p < 0.05). as shown in table 2, the lowest mean dmft was recorded in participants covered by the afmsif and the highest mean dmft was recorded in participants covered by the sso insurance. as shown in diagram 2, and considering the high frequency of fh index for zero, the median was zero in most cases (table 2). path analysis according to available literature, rmsea < 0.10 indicates reasonable fit. also, srmr < 0.05 indicates a good fit. cfi > 0.9, tli > 0.9, and cd close to 1 indicate good fit as well.14,15 in goodness of fit test conducted on present data, rmsea, srmr, and cd showed optimal fit (table 3). as shown in table 4 and figure 2, direct effect of ses on dap, r of a, tc, and np, and also direct effect of dap and np on dmft and fh were noted. moreover, ses and it had indirect effects on dmft and fh as well. the assessment of total effects, confirmed the significance of the abovementioned direct and indirect effects. figure 2, shows the estimated parameters for the final model. according to the path model, significant pathways noted from ses and it to dap, and increased ses level [β (standardized coefficient) = 0.05] and changing the it from ikrc to afmsif (β = 0.04) that resulted in a more regular dap. the path from ses to r of a indicated that, increasing the level of ses (β = 0.02) resulted in higher attendance of participants for prevention (rather than treatment). the path from ses to tc indicated that, increased ses level (β = 0.28) led to increased frequency of tooth brushing. the path from ses to np showed that increased level of ses (β = –0.05) led to higher consumption of cariogenic items. the path from dap to dmft and fh indicated that, more regular dap (β = –4.42) led to a reduction in dmft and increase in fh (β = 4.89). the path from np to dmft and fh showed that as the cariogenic risk of items decreased, dmft decreased (β = –0.46) and [table 1] 165j contemp med sci | vol. 8, no. 3, may-june 2022: 162–168 a.f. tolou et al. original influential factors on oral health status of the elderly iranians: a path analysis table 2. mean (sd) distribution of dmft and fh median (first and third quarters) and association of dmft and fh variables with other risk factors variables type/levels frequency (%) dmft mean (sd) p value fh index p value gender male 4235 (49.1%) 26.74 ± 7.94 0.721a 0 (0,10) 0.456a female 4398 (50.9%) 26.49 ± 8.10 0(0,11) low 2781 (37%) 27.14 ± 7.61 <0.001b 0 (0,9) ses <0.001b moderate 2364 (31.4%) 27.9 ± 7.84 0 (0,9) high 2376 (31.6%) 25.52 ± 8.55 0 (0,14) insurance type ikrc 174 (3.1%) 25.91 ± 8.77 0.001b 0 (0,10) 0.002b msif 2858 (50.5%) 26.44 ± 7.86 0 (0,10.25) sso 2290 (40.5%) 26.87 ± 7.95 0 (0,10) afmsif 337 (6%) 25.32 ± 9.04 0 (0,15) life style behaviors tooth cleaning (hygienic behaviors) never 1386 (22%) 26.67 ± 7.15 0.001b 0 (0,9) <0.001b once or several times a month 828 (13.2%) 26.02 ± 7.97 0 (0,12) several times a week 805 (12.8%) 25.53 ± 8.76 0 (0,13) once or several times a day 1262 (57.3%) 26.67 ± 8.25 2 (0,11) nutrition pattern high risk 5776 (88.7%) 26.42 ± 7.89 0.086b 0 (0,12) 0.003amoderate risk 368 (5.7%) 26.94 ± 7.78 0 (0,10) low risk 367 (5.6%) 26.81 ± 8.75 0 (0,11) care seeking behaviors reason of attendance problem 4721 (92.8%) 26.22 ± 8.01 0.001b 0 (0,12) 0.003a check up 369 (7.2%) 26.81 ± 8.75 0 (0,9) dental attendance pattern never (no visit) 387 (8.7%) 25.09 ± 8.43 0.047b 0 (0,12) <0.001b over a year ago 3565 (80.1%) 27.88 ± 7.44 0 (0,6) during the past year 497 (11.2%) 24.64 ± 8.52 4 (0,14) aman-whitney test, bkruskal-wallis test. fh increased (β = 0.46) accordingly. indirect significant paths included the path from ses and it to dmft and fh. increased level of ses and it had an indirect effect on reduction of dmft (β = –0.15, –0.17, respectively). also, they had an indirect effect on increasing the fh (β = 0.21, 0.19, respectively). a mutual arrow between ses and it shows the positive correlation between these two factors (covariance = 0.09). it had no effect on r of a (β = –0.000), for this reason a straight line was drawn (instead of an arrow) in the final model. discussion descriptive findings of the present study showed significant positive correlations between fh and dmft with ses, it, tc, r of a, and dap, and also between np with fh. this study revealed complex pathways between different factors such as the it, ses and dap with dmft and fh among the iranian elderly population by using path analysis. as shown in figure 2 (bilateral arrows) a positive correlation was detected between ses and it. also noted, direct correlations 166 j contemp med sci | vol. 8, no. 3, may-june 2022: 162–168 influential factors on oral health status of the elderly iranians: a path analysis original a.f. tolou et al. diagram 2. data accumulation at point 0 for fh index. diagram 1. data accumulation at point 32 for dmft. table 3. fit indices for path model for dmft and fh index population error baseline comparison size of residuals rmsea 90% ci (lower bound, upper bound) pclose cfi tli srmr cd 0.06 (0.047, 0.070) 0.10 0.72 0.35 0.03 0.8 note: bold values indicate optimal fit. table 4. the decomposition of effects into direct, indirect, and total effects between dmft, fh and the other risk factors cov. (it, ses) coef. p value [conf. interval 95%] 0.09 <0.001 0.07 0.10 direct effects dap← it ses 0.04 0.05 0.001 <0.001 0.01 0.06 0.03 0.06 r of a← it ses 0.00 0.02 0.99 <0.001 –0.01 0.011 0. 00 0. 027 tc← ses 0.28 <0.001 0.24 0.32 np← ses –0.05 <0.001 –0.06 –0.03 dmft← dap r of a tc np –4.42 0.62 0.14 –0.46 <0.001 0.22 0.09 0.02 –4.99 –3.84 –0.39 1.65 –0.02 0.31 –0.85 –0.06 fh index← dap r of a tc np 4.89 –0.58 –0.02 0.46 <0.001 0.28 0.76 0.03 4.27 5.50 –1.66 0.48 –0.20 0.20 0.04 0.88 indirect effects dmft← it ses –0.17 –0.15 <0.001 <0.001 –0.27 –0.07 –0.34 –0.06 fh index← it ses 0.19 0.21 <0.001 <0.001 0.07 0.30 0.11 0.31 total effects dmft← dap r of a tc np it ses –4.42 0.62 0.14 –0.46 –0.17 –0.15 <0.001 0.22 0.09 0.02 <0.001 <0.001 –4.99 –3.84 –0.39 1.65 –0.02 0.31 –0.85 –0.06 –0.27 –0.07 –0.24 –0.06 fh index← dap r of a tc np it ses 4.89 –0.58 –0.02 0.46 0.19 0.21 <0.001 0.28 0.76 0.03 <0.001 <0.001 4.27 5.50 –1.66 0.48 –0.20 0.15 0.04 0.88 0.07 0.30 0.11 0.31 note: abbreviations: ses, socio-economic status; it, insurance type; dap, dental attendance pattern; r of a, reason of attendance; tc, tooth cleaning; np, nutrition pattern. between ses and it with dap, and between ses with r of a, tc, and np, in such a way that, higher ses was positively correlated with better insurance coverage. likewise, it appeared that individuals with higher financial status or better insurance coverage had more regular dap. such individuals often went for a check-up without having a certain complaint. thus, despite having higher consumption rate of cariogenic items, they less commonly experienced caries and its related problems due to practicing better oral hygiene and dental care. this study detected the direct effect of dap and np on fh and dmft. those with more regular dap and better np (eating less cariogenic items) had better oral health indices. ses and it both had indirect correlations (through dap as a mediator variable) with fh and dmft, in such a way that, the frequency of dental visits significantly affected oral health status and its related indices. similarly, it (based on its coverage) can significantly affect the improvement of oral health fig. 2 experimental framework (final model) note: all significant values indicate standardized coefficients (*p < 0.05, **p < 0.01, ***p < 0.001). indices. furthermore, this study revealed that, all individuals regardless of their ses status were at high risk of dental problems, except those who could afford paying for their dental services. the goodness of fit test showed good fit for rmsea value, very good fitting for srmr, and relatively good fit for cd. although, cfi and tli did not show a good fit. overall, majority of the variables showed relatively good fit for the model (table 3). the path analysis clearly revealed the 167j contemp med sci | vol. 8, no. 3, may-june 2022: 162–168 a.f. tolou et al. original influential factors on oral health status of the elderly iranians: a path analysis correlations, causal relationships, and the pathways between the variables and data followed our conceptual model, confirming the role of dap as a mediator variable.11 the results showed that, dap had a causal relationship with oral health, confirming better oral health with more regular dap. likewise, other studies reported routine dental attenders had better oral health status.16-18 in line with the present findings regarding the relationship between dap and dmft, bottenberg et al, in 201919 demonstrated that, dental attenders with more regular dap had significantly lower number of untreated caries compared with irregular attenders. also, aarabi et al.13 indicated that the geriatric cases with a history of a dental visit in the past year had fewer carious teeth. other studies also confirmed higher perceived oral health in those who had access to oral healthcare services and had more regular dap.20,21 in this study, individuals with higher ses had high consumption of cariogenic items. however, they experienced less caries due to better tc pattern, which was in agreement with the results of sabbah et al,22 who showed higher ses individuals had better tc behaviors. the present study revealed that, it affected the oral health status, which was in line with the results of moradi et al,23 who confirmed that, in addition to ses, it had a direct correlation with better oral health status, (except the study participants were between 15 to 45 years old). this study showed the causal relationship between ses and oral health status with dap being more important mediating factor than np. in fact, according to present results, ses had an inverse correlation with np in the elderly population, because consumption of cariogenic items increased by increasing ses level. deihnelt et al.24 reported that, the consumption of sugar is lower in people with lower ses, living in developing countries, compared with those with moderate and high ses group. this finding probably confirms the elimination of np as a mediator variable in causal relationship with oral health. however, it should be noted that high sugar consumption alone is not enough to cause dental caries, especially if the consumers practice good oral hygiene and brush their teeth after consumption of sugary substances.25-27 in line with current study results regarding the causal relationship between insurance and fh; simon et al.,28 showed that, insurance status was significantly correlated with edentulism (fh = 0). those without insurance were at higher risk (1.56 times) of edentulism. those without dental insurance coverage were more interested or forced to extract their carious or even all the remaining sound teeth in order to use a denture instead of restoring their teeth. ghorbani et al.,29 evaluated 18 to 84-year-old residents in tehran and showed that individuals with low ses who did not have dental insurance had twice the number of extracted teeth compared with others. meanwhile, it should be noted that, type of dental procedures covered by the insurance system is also an important factor. those insurance systems covering preventive procedures, playing highly effective role in oral health promotion. in contrast, an insurance system that only covers tooth extraction and denture replacement, contribute to mass edentulism of insured population, and should be held responsible for tooth loss in low ses individuals. conversely, the insurance systems that cover level 2 preventive services (treatment) help preserving the natural dentition. in line with current results regarding the inverse causal relationship of it with dmft, a cross-sectional study by singh et al.30 was conducted on workers with and without sso insurance as part of a national survey. the mean dmft of uninsured workers was higher with odds of dental caries being 2 times (or = 1.94) higher than insured workers.30 overall, the findings of this study indicate that, it, ses, dap, and life style (including tc and np) affected the dmft and fh indices for all elderly individuals. thus, higher budget should be allocated to expand the basic insurance coverage for at risk elderly population. also, preventive and therapeutic interventions should be provided simultaneously for the benefit of geriatric target group. although based on present results, the insured individuals had better oral health status than the uninsured ones, the oral health status was still below the optimal level for all. therefore, further attention should be directed to major determinants of oral health such as ses and regular dap in order to promote the oral health of the insured elderly. concerning the national statistics showing that 52% of the iranian elderly are completely edentulous, obviously, any program and intervention for oral health promotion of senior citizens should be started at a younger age. unfavorable oral health status of the elderly in the present study can be the result of unhealthy life style, inappropriate oral health behaviors, and poor dap throughout their life. moreover, it should be noted that the majority of the seniors pay less attention to their oral health since they mostly need to deal with a number of underlying local and systemic diseases. nonetheless, it should be noted that in assessment of oral health status, the main limitation of dmft index is that it measures disease instead of health. to overcome this shortcoming, the fh index which was extracted from dmft index used in the present study as a harmonizing factor. this index provides some information regarding the function and quality of dentition, and is in fact representing the number of functional dentition. some individuals may have a high number of non-functional carious teeth; in such cases, dmft alone cannot indicate the actual status of the masticatory system. it should be noted that there are some organizations, providing supplemental insurance coverage to their employees with better coverage, (in addition to basic coverage provided by the four main insurance systems) such as banks, municipality, petroleum company, and communication organization.8 due to their very small covered population, a separate group was not allocated to such category. further studies are required if the effect of greater coverage under supplemental insurance is considered. also, it should be taken into consideration that, analysis of secondary population-based data often has some limitations, and this study was no exception. considering the prominent role of insurance systems in oral health promotion, future national surveys are recommended to collect more detailed information regarding the number and types of services provided by different supplementary insurance systems. likewise, longitudinal studies with respect to the parameters mentioned in this study are recommended to include cases from early adulthood to old age in order to better reveal the protective factors in elderly oral health promotion. conclusion of all parameters evaluated in this study, it, ses, dap, and np were found to be particularly important in affecting dmft and fh indices in the elderly population. out of all aforementioned factors, it in low ses individuals was more important for obtaining required oral healthcare services. apparently, 168 j contemp med sci | vol. 8, no. 3, may-june 2022: 162–168 influential factors on oral health status of the elderly iranians: a path analysis original a.f. tolou et al. seniors’ oral health is absolutely in need of special attention with particular focus on providing access to early prevention and care. acknowledgments the authors would like to thank all participants and the authorities in charge of the iranian national oral health survey conducted in 2012, which was carried out by the oral health office of the health deputy of the ministry of health and medical education in iran; with cooperation of the dental research institute, school of dentistry, shahid beheshti university of medical sciences. conflict of interest the authors have no conflict of interests regarding the authorship or publication of this paper. author contribution all authors contributed equally to study design, data analysis and data interpretation. aft was the main author, mhk, mpj and hgh provided guidance throughout the study, and revised and finalized the manuscript. mhk was responsible for the national oral health survey and data collection, and mn and fb provided consultation and reanalyzed the data.  references 1. peres ma, macpherson lm, weyant rj, daly b, venturelli r, mathur mr, listl s, celeste rk, guarnizo-herreño cc, kearns c, benzian h. oral diseases: a global public health challenge. the lancet. 2019 jul 20;394(10194):249-60. 2. fathi e. population aging in iran and its future, statistical research and training center, 2021 april. 3. petersen pe, yamamoto t. improving the oral health of older people: the approach of the who global oral health programme. community dentistry and oral epidemiology. 2005 apr;33(2):81-92. 4. jain vk, sequeira p, jain j, chancy u, maliyil mj, bhagwandas sc. barriers in utilization of oral health care services among patients attending primary and community health centres in virajpet, south karnataka. national journal of medical and dental research. 2013 apr 1;1(3):39. 5. bayat f, akbarzadeh a, monajemic f. assessment of demand for and utilization of dental services by insurance coverage in a developing oral health care system. journal dental school 2017;35(2):36-42. 6. khoshnevisan mh, ghasemianpour m, samadzadeh h, baez rj. oral health status and healthcare system in ir iran. j contemp med sci. 2018;4(3):107-8. 7. watt rg, venturelli r, daly b. understanding and tackling oral health inequalities in vulnerable adult populations: from the margins to the mainstream. br dent j. 2019;227(1):49–54. 8. jadidfard mp, yazdani s, khoshnevisan mh. social insurance for dental care in iran: a developing scheme for a developing country. oral health dent manag. 2012 dec 1;11(4):189-98. 9. shafiei s, yazdani s, jadidfard mp, zafarmand ah. measurement components of socioeconomic status in health-related studies in iran. bmc research notes. 2019 dec;12(1):1-1. 10. åström a, ekbäck g, ordell s, nasir e. long‐term routine attendance: influence on tooth loss and oral‐health related quality of life in swedish older adults. community dent and oral epidemiol. 2014; 5:460‐469. 11. norris ae. path analysis. in munro’s statistical methods for health care research: sixth edition 2011 nov 30 (pp. 399-418). wolters kluwer health. 12. furuta m, komiya‐nonaka m, akifusa s, shimazaki y, adachi m, kinoshita t, kikutani t, yamashita y. interrelationship of oral health status, swallowing function, nutritional status, and cognitive ability with activities of daily living in japanese elderly people receiving home care services due to physical disabilities. community dentistry and oral epidemiology. 2013 apr;41(2):173-81. 13. aarabi g, reissmann dr, seedorf u, becher h, heydecke g, kofahl c. oral health and access to dental care–a comparison of elderly migrants and non-migrants in germany. ethnicity & health. 2018 oct 3;23(7): 703-17. 14. norris ae. structural equation modeling. inmunro’s statistical methods for health care research: sixth edition 2011 nov 30 (pp. 419-443). wolters kluwer health. 15. gulcan f, ekbäck g, ordell s, klock k, lie s, åström a. exploring the association of dental care utilization with oral impacts on daily performances (oidp)‐a prospective study of ageing in norway and sweden. acta odontol scand. 2018;76(1):21–29. 16. åström a, ekbäck g, ordell s, gulcan f. changes in oral health related quality of life (ohrqol) related to long‐term utilization of dental care among older people. acta odontol scand. 2018;76(8):559–566. 17. thomson wm, williams sm, broadbent jm, poulton r, locker d. long‐term dental visiting patterns and adult oral health. j dent res. 2010;89(3):307‐311. 18. diehnelt de, kiyak ha. socioeconomic factors that affect international caries levels. community dentistry and oral epidemiology. 2001 jun;29(3):226-33. 19. bottenberg p, vanobbergen j, declerck d, carvalho jc. oral health and healthcare utilization in belgian dentate adults. community dentistry and oral epidemiology. 2019 oct;47(5):381-8. 20. torppa‐saarinen e, suominen al, lahti s, tolvanen m. longitudinal pathways between perceived oral health and regular service use of adult finns. community dentistry and oral epidemiology. 2019 oct;47(5):374-80. 21. warford jj. environment, health, and sustainable development: the role of economic instruments and policies. bull world health org 1995;73:387–95. 22. sabbah w, tsakos g, sheiham a, watt rg. the role of health-related behaviors in the socioeconomic disparities in oral health. soc sci med 2009;68:298–303. 23. moradi g, bolbanabad am, moinafshar a, adabi h, sharafi m, zareie b. evaluation of oral health status based on the decayed, missing and filled teeth (dmft) index. iranian journal of public health. 2019 nov;48(11):2050. 24. diehnelt de, kiyak ha. socioeconomic factors that affect international caries levels. community dentistry and oral epidemiology. 2001 jun;29(3):226-33. 25. armfield jm, spencer aj, roberts-thomson kf, plastow k. water fluoridation and the association of sugar-sweetened beverage consumption and dental caries in australian children. american journal of public health. 2013 mar;103(3):494-500. 26. van loveren c. sugar restriction for caries prevention: amount and frequency. which is more important?. caries research. 2019;53(2):168-75. 27. anderson ca, curzon me, van loveren c, tatsi c, duggal ms. sucrose and dental caries: a review of the evidence. obesity reviews. 2009 mar;10:41-54. 28. simon l, nalliah rp, seymour bb. lack of dental insurance is correlated with edentulism. journal of the massachusetts dental society 2015;63(4):28–31. 29. ghorbani z, ahmady ae, ghasemi e, zwi a. socioeconomic inequalities in oral health among adults in tehran, iran. community dent health. 2015 mar 1;32(1):26-31. 30. singh a, purohit bm, masih n, kahndelwal pk. risk factors for oral diseases among workers with and without dental insurance in a national social security scheme in i ndia. international dental journal. 2014 apr;64(2):89-95. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.1218 https://doi.org/10.22317/jcms.v8i3.xxxx 152 j contemp med sci | vol. 9, no. 3, may-june 2023: 152–157 original physiological effects of calprotectin and b cell activating factor in covid-19 patients hazhar m. balaky1* , akam jasim mustafa2, parween abdulsamad ismail3, araz muhammad yousif4 1mergasor technical institute, erbil polytechnic university, erbil, iraq. 2department of chemistry, faculty of science, soran university, soran, kurdistan region, iraq. 3department of chemistry, college of education, university of salahaddin, erbil, iraq. 4basic science department, dentistry college, hawler medical university, erbil, iraq. *correspondence to: hazhar m. balaky (e-mail: hazharbalaky86@yahoo.com) (submitted: 10 february 2023 – revised version received: 26 february 2023 – accepted: 11 march 2023 – published online: 26 june 2023) abstract objectives: this study set out to determine how calprotectin and b cell activating factor contributes to early covid-19 patient severity prediction. methods: the study included 25 healthy controls and 52 patients with sars-cov2 infection who were clinically diagnosed with covid-19 illness and were between the ages of 23 and 35. the serum levels of calp and baff were measured using the elisa method. to gauge crp levels, an immunoturbidometric assay was performed. results: variations in serum levels of calp and baff were found to be statistically insignificant in the study (p = 0.7109 & p = 0.7575, respectively). when compared to the control group (103.95 ± 36.67 ng/ml; 403.03 ± 1.03), covid-19 patients had non-significantly raised levels of calp and baff (106.5 ± 4.67 ng/ml; 436.9 ± 12.77 pg/ml, respectively). according to roc curve analysis, the area under the receiver operating characteristics curve (auc) for calp and baff was (0.5170) and (0.5259), respectively. (r = 0.6923; p = 0.0001). there was a significant positive correlation between serum calp and baff levels. the connection between serum crp levels and calp (r = 0.3010; p = 0.1271) and baff levels (r = 0.2912; p = 0.1406) was insignificantly positive. conclusion: the current study’s findings suggested that serum calp and baff concentrations were increased in covid-19 patients, suggesting that these inflammatory markers may be helpful indicators of the severity of covid-19. keywords: calprotectin, b cell activating factor, c-reactive protein, covid-19 issn 2413-0516 introduction a pneumonia outbreak was reported in wuhan (hubei province, china) in december 2019 due to infection with a new coronavirus strain that causes the severe acute respiratory syndrome known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the coronavirus disease 19 (covid-19) pandemic caused by this virus has affected millions of individuals worldwide.1 covid-19 is associated with several clinical signs and symptoms frequently seen in autoimmune illnesses, including arthralgias, myalgias, exhaustion, sicca, and rashes.2 patients with covid-19 have also been noted to exhibit thrombosis, myositis, myocarditis, arthritis, encephalopathy, and vasculitis, in addition to these less typical autoimmune disease symptoms.3 these clinical data, along with the rising number of “recovered” patients referred to as “long haulers” or “long covid” who still exhibit postcovid-19 symptoms, raise the hypothesis that inflammation in response to sars-cov-2 infection increases tissue damage during the acute phase and has some long-term consequences.4 furthermore, severe covid-19 is more likely to occur in persons with underlying conditions such as diabetes, hypertension, cardiovascular disease, and lung disease, and the age-related case fatality rate increases significantly.5 since the virus’s introduction, it has been of utmost importance to comprehend immunity to the virus, the kinetics and protective function of the immune response in the community, and the level of exposure as measured by serosurveys.6 neutrophil activation signature calprotectin has become a useful biomarker during the first wave of the pandemic to assess covid-19 patient risk.7 the self-aggravating thrombo-inflammatory storm in people with severe covid-19 may be directly attributed to the neutrophil related inflammatory marker termed calprotectin. silvin et al.8 revealed the relationship between elevated calprotectin levels and immature neutrophils and nonclassical monocytes. he also claimed that increased damage-related molecular pattern creation causes this relationship. in light of this, it has been postulated that calprotectin is a crucial mediator of the hyperinflammatory host response and the rise in inflammatory monocytes, neutrophils, and platelets that contribute to the particular coagulopathy in severe covid-19.1 additionally, doctors are adopting calprotectin increasingly frequently to aid in diagnosing and treating a range of different inflammatory illnesses due to its stability, assay repeatability, and inexpensive cost. the molecular functions of calp in health and unresolved inflammation are poorly understood by most clinicians.9 b-cell activating factor (baff), a member of the tumor necrosis factor (tnf) class, is expressed by macrophages, monocytes, dendritic cells, activated neutrophils, and stromal cells.10 first, it has been shown to be necessary for the creation of the humoral response as well as the growth and survival of b lymphocytes.11 recent research suggests that baff may also regulate innate immune responses, particularly at the level of the respiratory mucosa.12 the membrane-bound or soluble protein baff, which can induce autoimmune disorders in mice and humans,13 is synthesized excessively. the transmembrane activator and cyclophilin ligand interactor (taci), the b cell maturation antigen, and the baff receptor are the three known baff receptors (bma). all of these receptors are present in b and t lymphocytes as well as antigen-presenting https://orcid.org/0000-0002-4579-2617 mailto:hazharbalaky86@yahoo.com 153j contemp med sci | vol. 9, no. 3, may-june 2023: 152–157 hazhar m. balaky et al. original physiological effects of calprotectin and b cell activating factor in covid-19 patients cells, proving that baff action goes beyond b cell biology.14 therefore, this case-control study investigates circulating baff and calprotectin levels, function, diagnostic value, and prognostic relevance in covid-19 infected patients. materials and methods the 77 participants in the present study included 52 confirmed covid-19 patients between june 2021 to november 2022 aged (23 to 35 years) and 25 healthy volunteers as the control group. after a covid-19 clinical diagnosis was made using rt-pcr test, blood samples were taken before the study group’s condition was treated. each participant’s blood sample was taken at a volume of 5 ml and centrifuged for 15 minutes at 3500 rpm. after that, the serum was frozen and kept at 70⁰c. the research purpose elisa kits were used to measure the levels of calprotectin (calp) and b-cell activating factor (baff) following the manufacturer’s instructions (sunlong biotech, china). the crp levels were measured by immunoturbidometric assay via a fully automated biochemistry analyzer. statistical analysis statistical analysis was performed using graphpad prism version 8 computer program. the unpaired t-test (man-whitney u) was used to compare the biochemical parameters between the study groups. roc curve analysis and spearman correlation analysis were also performed for biochemical parameters. all comparisons were deemed significant if the p-value was less than 0.05. results serum level of calprotectin figure. 1 and table 1 revealed a non-significant elevation (p = 0.7109) in circulating concentration of serum calprotectin levels in covid-19 patients (106.5 ± 4.67 ng/ ml) as compared to controls (103.9 ± 5.36 ng/ml). serum level of b cell activating factor the results in figure 2 and table 2 also showed that there were non-remarkable increase (p = 0.7575) in circulating levels of baff in covid-19 patients (436.9 ± 12.77 pg/ml) as compared to healthy controls (403.0 ± 31.03 pg/ml). relationship between calp, baff, and crp in covid-19 patients correlation analysis assessed the relationships among serum crp levels, calp and baff levels. baff and serum calp levels were positively correlated (r = 0.6923; p = 0.0001) (figure 3). figure 3 also shows a non-significant correlation between serum crp levels and calp (r = 0.3010; p = 0.1271) and baff level (r = 0.2912; p = 0.1406) (figure 3). these research results may link baff activation and the immuno-inflammatory and pathogenic response, indicating disease activity and tissue damage in various chronic viral infection illness states in covid-19. roc curve based on the (receiver operating characteristic) roc curve, the area under the curve (auc) of serum calp and serum baff were (0.5170) and (0.5259) respectively. (figure 4). table 1. comparison of calp and baff levels among covid-19 patients and healthy controls parameters controls covid-19 patients p-value calprotectin (ng/ml) 103.9 ± 5.36 106.5 ± 4.67 0.7109 b-cell activating factor (pg/ml) 403.0 ± 31.03 436.9 ± 12.77 0.7575 the value expressed in mean ± se fig. 1 calprotectin levels between the sera of the studied groups. fig. 2 b cell-activating factor (baff) levels in sera of the two studied groups. 154 j contemp med sci | vol. 9, no. 3, may-june 2023: 152–157 physiological effects of calprotectin and b cell activating factor in covid-19 patients original hazhar m. balaky et al. discussion serum level of calprotectin numerous recent studies have discovered that patients with coronavirus disease-19 illness had higher calprotectin levels.1,7 additionally, these investigations have demonstrated that calprotectin can predict the need for mechanical breathing, identify death, and differentiate between mild and severe illness states.8,13 calprotectin is widely distributed in neutrophils, making up around two-thirds of the cytosol’s soluble protein composition. the strong relationship between serum calprotectin levels and coronavirus’s present and potential severity indicates that neutrophils are implicated as active promoters of inflammation and respiratory impairment in coronavirus disease. the patients who needed mechanical ventilation while in the hospital also had much greater levels of calprotectin. this data shows a direct link between the severe acute respiratory syndrome caused by covid-19, elevated serum calprotectin levels, and neutrophil activation.13 two studies from medical schools in michigan, shanghai, and washington dc, on the role of calprotectin as an early indicator of neutrophil activation in covid-19 disease. the levels of calprotectin were noticeably elevated in hospitalized individuals with coronavirus illness. calprotectin levels also correlate with the severity of respiratory failure and the demand for mechanical ventilation. this favours using calprotectin as a biomarker to estimate the severity of a condition and the likelihood that it will result in mortality. higher levels of calprotectin were also associated with an increased risk of dying from thrombotic issues.13,15 according to recently released research in the academic journal cell, calprotectin may be able to differentiate between severe and mild covid-19 disease. the circulating biomarker most obviously elevated in patients with advanced illness was calprotectin. this study offers future therapeutic strategies explicitly targeting calprotectin to treat the severe form of covid-19 and shows its potential use in the prognosis of severe disease.8 similar to the current study, two inflammation-related biomarkers, growth differentiation factor-15 and calprotectin, have been researched for their potential role in predicting mortality and disease severity in sars-cov-2 infected patients. the study results show that calprotectin levels are markedly higher in people with covid-19, and they suggest that calprotectin may help assess the severity of the illness and forecast in hospital mortality.1 calprotectin overexpression may be a direct source of the self-amplifying thrombo-inflammatory storm observed in patients with severe covid-19. silvin et al.8 investigation discovered a connection between elevated calprotectin levels and immature neutrophils and nonclassical monocytes, supporting their hypothesis that this correlation originates from the excessive production of fig. 3 correlation of serum calp with baff (a), serum crp with calp (b), serum crp with baff (c). fig. 4 receiver operating characteristic (roc) curve analysis of serum calp levels (a) and baff levels (b). 155j contemp med sci | vol. 9, no. 3, may-june 2023: 152–157 hazhar m. balaky et al. original physiological effects of calprotectin and b cell activating factor in covid-19 patients damage-associated molecular patterns. due to the host’s hyperinflammatory response and the rise in inflammatory monocytes, neutrophils, and platelets, it has been proposed that calprotectin is a crucial mediator of specific coagulopathy in severe covid-19 patients.16 according to a recent investigation, people with confirmed sars-cov-2 infection exhibited increased plasma calprotectin levels compared to suspected patients with negative rt-pcr.17 numerous researchers have confirmed the role of calprotectin in evaluating illness severity, including studies by chen et al.,18 kaya et al.,19 garcia de guadiana-romualdo et al.,1 mahler et al.,20 and bauer et al.21 neutrophils are crucial to the immunopathology of covid-19, according to a recent study by tomar et al.22 as a result, it has been discovered that measuring blood calprotectin levels is a trustworthy indication of covid-19 severity. studies proved that neutrophils mainly secrete calprotectin in response to inflammation. recently, it was shown that calprotectin is a biomarker of inflammation that can be used to track the progression of numerous inflammatory diseases.23–28 limited research studies look at the connection between the severity and predicator value of serum calprotectin in covid-19. patients brought to the icu had significantly higher serum levels of calprotectin than non-icu patients, and those who died had much higher levels, according to chen et al.,18 who conducted a study with 121 covid-19 patients (41 icu, 81 non-icu). additionally, that study discovered a correlation between higher mortality in covid-19 patients with a substantial rise in serum calprotectin. in their trial with 94 covid-19 patients, shi et al. study13 found that patients needing mechanical breathing had higher serum calprotectin levels than those not. based on the available literature and the present study results, we can conclude that serum calprotectin may be employed as a predictive biomarker for covid-19 disease severity and prognosis. serum level of b cell activating factor the greater b-cell activation in covid-19 patients may be responsible for the many autoantibodies and immune complexes frequently found in these individuals’ blood. our research offered the first conclusive evidence that circulating baff levels in covid-19 patients were higher than in healthy controls. these findings suggest that baff participates in persistent viral infection and aids in disease progression. specifically, baff impacts b cell development, maturation, survival, and activation.29,30 studies have shown that the process of viral infection caused by covid-19 and others induces contact between t cells and activated b cells with t cells.31-33 moreover, it has been shown that viral infections, including infections with the respiratory syncytial virus, might cause baff release.34,35 consequently, even though the fundamental mechanisms may change, it is believed that baff is frequently produced by viral infection.36 according to previous research, our findings provide new evidence that covid-19 infection may induce the biosynthesis and release of baff, which may affect how b cells react to viral infection. the tumor necrosis factor family member b-cell activating factor (baff), also known as b lymphocyte stimulator (blys) or b cell activating factor, has a unique role in regulating peripheral b-cell survival, homeostasis, and the antibody response.29,37 its believed that baff could reduce apoptosis and essentially enhances t cell-independent and t cell-dependent humoral immune responses. the study led by sutherland et al.38 found that baff increased tand b-cell responses, particularly th1-type responses. numerous cells, particularly those connected to the immuno-inflammatory response, such as monocytes, macrophages, neutrophils, dendritic cells (dcs), t lymphocytes, and b lymphocytes, are able to produce baff.39,40 interleukin-10-producing b cells were enriched and more frequent in chronic hepatitis b patients during hepatic flare-ups, according to a study by das et al.32 a different study showed that individuals with chronic hepatitis b have frequently activated b cells, with an abnormally high proportion of these individuals’ b cells exhibiting activation markers. this demonstrates that this subset of b cells may control t-cell immunity in chronic hepatitis.31 the intrinsic b-cell activation molecule fc receptor like1, as well as the b-cell activation markers cd69 and cd86, were also shown to be present in increased amounts in acute and chronic hepatitis b patients.33 these results indicate that b cells are essential for the progression of hbv infection, as well as hbv viral antigens and their interactions with t lymphocytes. additionally, it was thought that baff had a negative impact on the microenvironments of solid and hematological malignancies. so far, it has been demonstrated that baff encourages invasive migration in hypoxic breast cancer cell lines.41 blood baff levels, generated by neutrophils, have been linked to oral cavity cancers.42 it has been observed that the prognosis and circulating baff levels in multiple myeloma are related.43 according to koizumi et al. report.44 it was found that baff promoted tumor invasion and dissemination in human pancreatic cancer cases. however, no research has yet been done to determine how baff contributes to the growth of hepatocellular carcinoma. therefore, based on the available literature data and the results of the present study, serum b cell activating factor contributes to early patient severity of covid-19 and may be a good predictive marker for the disease prognosis. diagnostic performance of serum calp and baff in covid-19 patients due to the non-significantly increased blood levels of calp and baff found in covid-19 patients in the present study, the ability of blood calp and baff to predict covid-19 was assessed using the roc curves. according to a presentation by chen et al.,18 extremely high levels of calprotectin were connected to the poor overall survival of covid-19 patients, supporting the predictability of calprotectin revealed by earlier investigations. recent studies by shi et al.13 and zuo et al.,15 and others13,15 demonstrated high serum calprotectin is closely connected to the likelihood of dying in covid-19, usually from thrombotic problems, which lends credence to the verdicts of the current study. calprotectin is said to have a higher predictive accuracy when compared to the covid-gram risk score created for prediction by liang et al.45 and chen et al.18 calprotectin was thought to have the highest prediction accuracy of all the predictors, according to chen et al.18 they examined the receiver operating characteristic curve (roc) analysis of calprotectin, hmgb1, covid-gram risk score, and calprotectin/hmgb1 combo for predicting icu admission and possible mortality to illustrate results similar to the present investigation. 156 j contemp med sci | vol. 9, no. 3, may-june 2023: 152–157 physiological effects of calprotectin and b cell activating factor in covid-19 patients original hazhar m. balaky et al. conclusion this study is the first to demonstrate that covid-19 patients had greater serum levels of the potent inflammatory markers calp, baff, and crp. the clinical illness condition of covid-19 and increased baff levels are correlated. the sars-cov-2 infection risk and severity correlated with serum calp and baff levels. the study results suggest a connection between serum baff levels and the manifestation of covid-19, and assessing serum baff levels may be used as an inflammatory biomarker to identify and classify clinical problems related to covid-19. according to the available evidence, calprotectin may be a valuable inflammatory biomarker to evaluate the risk and severity of covid-19. the relationship between calprotectin and the inflammatory process may present fresh opportunities for managing and improving covid-19. the combined use of serum calp, baff and crp levels may be helpful to evaluate and understand how inflammation-related factors such as viral load, sars-cov2 antibodies, corticosteroid use, anticoagulants, and pharmaceutical agents would affect neutrophil function. a comprehensive prospective investigation is required to comprehend the progression of the covid-19 infection, the potential role of blood baff levels in determining and monitoring the condition’s prognosis, and the treatment response to immunomodulatory medicine. additional study is also required to completely comprehend the roles played by baff in the emergence of covid-19-associated diseases and the development of the infection. conflict of interests all authors declare that they have no conflicts of interest.  references 1. garcía de guadiana-romualdo, l. et al. circulating levels of calprotectin, a signature of neutrophil activation in prediction of severe respiratory failure in covid-19 patients: a multicenter, prospective study (calcov study). inflammation research 71, 57–67 (2022). 2. guan, w.-j. et al. clinical characteristics of coronavirus disease 2019 in china. new england journal of medicine 382, 1708–1720 (2020). 3. machhi, j. et al. the natural history, pathobiology, and clinical manifestations of sars-cov-2 infections. journal of neuroimmune pharmacology 15, 359–386 (2020). 4. chang, s. e. et al. new-onset igg autoantibodies in hospitalized patients with covid-19. nature communications 12, 1–15 (2021). 5. van kampen, j. j. et al. duration and key determinants of infectious virus shedding in hospitalized patients with coronavirus disease-2019 (covid-19). nature communications 12, 1–6 (2021). 6. ortega, n. et al. seven-month kinetics of sars-cov-2 antibodies and role of pre-existing antibodies to human coronaviruses. nature communications 12, 1–10 (2021). 7. udeh, r., advani, s., de guadiana romualdo, l. g. & dolja-gore, x. calprotectin, an emerging biomarker of interest in covid-19: a systematic review and meta-analysis. journal of clinical medicine 10, 775 (2021). 8. silvin, a. et al. elevated calprotectin and abnormal myeloid cell subsets discriminate severe from mild covid-19. cell 182, 1401–1418. e1418 (2020). 9. jukic, a., bakiri, l., wagner, e. f., tilg, h. & adolph, t. e. calprotectin: from biomarker to biological function. gut 70, 1978-1988 (2021). 10. wang, l. et al. b cell activating factor regulates periodontitis development by suppressing inflammatory responses in macrophages. bmc oral health 21, 1–15 (2021). 11. yang, s.-c., tsai, y.-f., pan, y.-l. & hwang, t.-l. understanding the role of neutrophils in acute respiratory distress syndrome. biomedical journal 44, 439–446 (2021). 12. veras, f. p. et al. sars-cov-2–triggered neutrophil extracellular traps mediate covid-19 pathology. journal of experimental medicine 217 (2020). 13. shi, h. et al. neutrophil calprotectin identifies severe pulmonary disease in covid‐19. journal of leukocyte biology 109, 67–72 (2021). 14. nascimento, m. et al. b-cell activating factor secreted by neutrophils is a critical player in lung inflammation to cigarette smoke exposure. frontiers in immunology 11, 1622 (2020). 15. zuo, y. et al. neutrophil extracellular traps in covid-19. jci insight 5 (2020). 16. hanssen, n. m., spaetgens, b., nagareddy, p. r. & murphy, a. j. dampening mortality in covid-19: therapeutic insights from basic cardiometabolic studies on s100a8/a9. circulation 143, 971–973 (2021). 17. cherubini, f., cristiano, a., valentini, a., bernardini, s. & nuccetelli, m. circulating calprotectin as a supporting inflammatory marker in discriminating sars-cov-2 infection: an observational study. inflammation research 70, 687–694 (2021). 18. chen, l. et al. elevated serum levels of s100a8/a9 and hmgb1 at hospital admission are correlated with inferior clinical outcomes in covid-19 patients. cellular & molecular immunology 17, 992–994 (2020). 19. kaya, t. et al. serum calprotectin as a novel biomarker for severity of covid-19 disease. irish journal of medical science (1971-) 191, 59–64 (2022). 20. mahler, m., meroni, p.-l., infantino, m., buhler, k. a. & fritzler, m. j. circulating calprotectin as a biomarker of covid-19 severity. expert review of clinical immunology 17, 431–443 (2021). 21. bauer, w. et al. outcome prediction by serum calprotectin in patients with covid-19 in the emergency department. journal of infection 82, 84–123 (2021). 22. tomar, b., anders, h.-j., desai, j. & mulay, s. r. neutrophils and neutrophil extracellular traps drive necroinflammation in covid-19. cells 9, 1383 (2020). 23. romand, x. et al. systemic calprotectin and chronic inflammatory rheumatic diseases. joint bone spine 86, 691–698 (2019). 24. wirtz, t. h. et al. association of serum calprotectin concentrations with mortality in critically ill and septic patients. diagnostics 10, 990 (2020). 25. kunutsor, s. k. et al. plasma calprotectin and risk of cardiovascular disease: findings from the prevend prospective cohort study. atherosclerosis 275, 205–213 (2018). 26. wang, q., chen, w. & lin, j. the role of calprotectin in rheumatoid arthritis. journal of translational internal medicine 7, 126–131 (2019). 27. yurtsever kum, n. et al. elevated serum calprotectin as an inflammatory marker in obstructive sleep apnea. cranio®, 1–7 (2020). 28. candar, t., baklacı, d., kuzucu, i̇. & kayabaşı, s. a proinflammatory marker in chronic rhinosinusitis: serum calprotectin. acta biochimica polonica (2020). 29. do, r. k. g. & chen-kiang, s. mechanism of blys action in b cell immunity. cytokine & growth factor reviews 13, 19–25 (2002). 30. schneider, p. & tschopp, j. baff and the regulation of b cell survival. immunology letters 88, 57–62 (2003). 31. oliviero, b. et al. enhanced b-cell differentiation and reduced proliferative capacity in chronic hepatitis c and chronic hepatitis b virus infections. journal of hepatology 55, 53–60 (2011). 32. das, a. et al. il-10–producing regulatory b cells in the pathogenesis of chronic hepatitis b virus infection. the journal of immunology 189, 3925–3935 (2012). 33. wang, k. et al. overexpression of fc receptor-like 1 associated with b-cell activation during hepatitis b virus infection. brazilian journal of medical and biological research 45, 1112–1118 (2012). 34. toubi, e. et al. elevated serum b-lymphocyte activating factor (baff) in chronic hepatitis c virus infection: association with autoimmunity. journal of autoimmunity 27, 134–139 (2006). 35. mcnamara, p. et al. respiratory syncytial virus infection of airway epithelial cells, in vivo and in vitro, supports pulmonary antibody responses by inducing expression of the b cell differentiation factor baff. thorax 68, 76–81 (2013). 157j contemp med sci | vol. 9, no. 3, may-june 2023: 152–157 hazhar m. balaky et al. original physiological effects of calprotectin and b cell activating factor in covid-19 patients 41. zhu, j. et al. blys is up-regulated by hypoxia and promotes migration of human breast cancer cells. journal of experimental & clinical cancer research 31, 1–7 (2012). 42. jablonska, e. et al. overexpression of b cell-activating factor (baff) in neutrophils of oral cavity cancer patients—preliminary study. neoplasma 58, 211 (2011). 43. fragioudaki, m. et al. serum baff levels are related to angiogenesis and prognosis in patients with multiple myeloma. leukemia research 36, 1004–1008 (2012). 44. koizumi, m. et al. increased b cell-activating factor promotes tumor invasion and metastasis in human pancreatic cancer. plos one 8, e71367 (2013). 45. liang, w. et al. development and validation of a clinical risk score to predict the occurrence of critical illness in hospitalized patients with covid-19. jama internal medicine 180, 1081–1089 (2020). 36. ittah, m. et al. induction of b cell-activating factor by viral infection is a general phenomenon, but the types of viruses and mechanisms depend on cell type. journal of innate immunity 3, 200–207 (2011). 37. rahman, z. s. & manser, t. b cells expressing bcl-2 and a signaling-impaired baff-specific receptor fail to mature and are deficient in the formation of lymphoid follicles and germinal centers. the journal of immunology 173, 6179–6188 (2004). 38. sutherland, a. p. et al. baff augments certain th1-associated inflammatory responses. the journal of immunology 174, 5537–5544 (2005). 39. nardelli, b. et al. synthesis and release of b-lymphocyte stimulator from myeloid cells. blood, the journal of the american society of hematology 97, 198–204 (2001). 40. scapini, p. et al. g-csf–stimulated neutrophils are a prominent source of functional blys. the journal of experimental medicine 197, 297–302 (2003). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1347 27j contemp med sci | vol. 1, no. 3, summer 2015: 27–30 research objective this study focuses on the relationship between calcium ion concentration in human body and thyroid hormones with liver enzymes to determine the effect of some physiological and biochemical parameter of liver [glutamic oxaloacetic transaminase (got), glutamic pyruvic transaminase (gpt), bilirubin] and thyroid stimulating hormone (tsh; t3, t4), on the levels of calcium ion in the blood. methods the current study included 40 patients from two different hospitals in karbala city. this study has been investigated to find the effect of thyroid hormones (t3, t4, tsh) and liver enzymes (got, gpt, bilirubin) on calcium ion concentration. results the results revealed there is no significant difference (p > 0.01) between calcium ion concentration and thyroid hormones (t4, t3, tsh) in each groups of different ages, respectively. therefore, weak correlation between calcium and hormones pointed to an increase or decrease of calcium ion concentration effect by these hormones. conclusion there is no significant difference (p > 0.01) between liver enzymes (got, gpt, bilirubin) and calcium ion concentration in each groups of different ages respectively, although weak correlation between calcium and hormones that pointed to an increase or decrease of calcium ion concentration is also affected by these enzymes. keywords calcium ion, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, bilirubin, t3, t4, tsh study of the relationship between calcium ion and thyroid hormones, liver enzymes in some patients of hypocalcaemia and hypercalcaemia jasim a abdullah, hanen abdulkalek, sura haider & aliaa aamer introduction calcium is a body’s abundant divalent cation and more than 99% of the body calcium is concentrated in the skeletal system and approximately 1% is rapidly exchangeable with blood calcium. small amounts of calcium located outside the bone circulates in the serum partly bound to protein and partly ionized. calcium has a major role in the transmission of nerve that include normal heart beat and has vital role in cardiac action potential and is essential for cardiac pacemaker automaticity, and this ion is also involved in blood clotting and hormone secretion.1,2 intracellular calcium is a key of intracellular second messenger that play a pivotal role in controlling various cellular processes such secretion, differentiation, proliferation, motility and cell death. in contrast, extracellular calcium is crucial for a spectrum of physiological phenomena including blood coagulation, muscle function and maintenance of skeletal integrity.3 calcium balance is tightly regulated by the interplay between gastrointestinal absorption, renal excretion, bone reabsorption, vitamin d and parathyroid hormone (pth) system.4 the thyroid gland plays a role in calcium metabolism and regulation, by secreting when needed and the thyroid stimulating hormones (tshs) are key to regulate the metabolism of thyroid gland to produce both t4 (thyroxine) and t3 (tri-iodothyronine). t4 is the main product and is converted in the periphery via de iodination to t3 which is the main biological active of thyroid hormones. the production of tshs is regulated by thyrotrophic; therefore, thyroid stimulating hormone in response is regulated by a negative feedback mechanism related to serum level of free t3 and t4.5 the liver enzyme has an important role in amino acid metabolism. aspartate aminotransferase (ast) and alanine aminotransferase (alt) are enzymes found mainly in the liver. they are also found in red blood cells, heart muscle tissues and other organs such as the pancreas and kidney.6 ast and alt formerly was called serum glutamic oxaloacetate transaminase (sgot) and serum glutamic pyruvic transaminase (sgpt), respectively. ast or alt levels are valuable aid primarily in the diagnosis of liver diseases but not specific for diagnosis of liver disease.7 materials and methods 1. collection of sample the specimens were collected from two hospitals, al-hussainy hospital and al-hindia hospital in karbala city. venous bloods were collected in a laboratory, as specimens, from 40 patients. the average age was 10–45 years. 2. separating serum from blood first, blood taken from patients by syringe was placed in the plane tubes (not anticoagulant tube). then, these tubes were centrifuged at 5000 rpm for 5 min. the serum thus separated from blood was transferred to another tube and gave a special number and name to the tube to avoid its loss. 3. measurement of calcium the first test is the measurement of calcium ion in the body via electrolytes instrument in both hospitals, al-hussainy and al-hindia. 4. hormonal test procedure (tsh-t3-t4) by elisa i. prepare both the samples (serum) and reagents in room temperature. ii. format the microplate wells for each serum reference, control and patient specimen to be assayed in duplicate. iii. pipette 50 µl of the appropriate serum reference, control or specimen into the assigned well. department of clinical laboratories, college of applied medical sciences, karbala university, iraq. correspondence to jasim a abdullah (email: jasim.abdulabbas@gmail.com). (submitted: 23 march 2015 – revised version received: 26 may 2015 – accepted: 2 june 2015 – published online: summer 2015) issn 2413-0516 28 j contemp med sci | vol. 1, no. 3, summer 2015: 27–30 calcium ion and thyroid hormones research jasim a abdullah et al. iv. add 100 µl of working reagent a (t3, t4, tsh), enzyme conjugated solution to all the wells. v. swirl the microplate gently for 20–30 sec to mix and cover it. vi. incubate 60 min at room temperature. vii. discard the contents of the microplate by decantation or aspiration. if decanting, blot the plate dry with absorbent paper. viii. we used automatic plate washer and washed plates for 3 times. ix. add 100 µl of working signal reagent solution to all wells. don’t shake the plate after substrate addition. x. incubate at room temperature for 15 min. xi. add 50 µl of stop solution to each well and gently mix for 15–20 sec. xii. read the absorbance in each well at specific wave length compatible with hormones that we tested (t3, t4, tsh). xiii. the result should be read within 30 min of adding the stop solution. 5. biochemical test procedure (include got, gpt, bilirubin) we used reflatrone device to determine this parameter. this procedure includes: i. working the device (reflatrone). ii. take specific strip for test that you need (got, gpt, bilirubin) and add in the dedicated space one drop or 32 µl from the patient serum. iii. put the strip in the device and wait for some minutes and read the result. 6. statistical analysis we used the correlation test to deduce correlation factor between calcium ion concentration and thyroid hormones and liver enzymes at probability level 0.01 and degree freedom, n–2.8 results and discussion the relationship between calcium ion concentration and thyroid hormones the present study included 40 patients and tested the relationship between thyroid hormones (t3, t4), tsh and calcium ion concentration; in other words, this experiment studied the effect of thyroid hormones on calcium ion concentration. through this study we observed the relationship between calcium ion concentration and thyroid hormones (t4, t3), tsh. there is no significant difference when p > 0.01 in all groups of different ages. weak correlation between calcium ion concentration and thyroid hormones (t4, t3, tsh) pointed to an increase of calcium ion concentration depending on an increase of these hormones, but it was weak. this result is shown in table 1 and fig. 1. the calcium sensing receptor (casr) represents the molecular mechanism by which parathyroid cell detect change in blood ionized calcium concentration and modulate parathyroid hormone (pth) secretion to maintain serum calcium levels within a narrow physiological range.9 depending on some studies that observed the blood ionized calcium concentration are remarkably stable in healthy individual because of the hemostatic system involving the action of the three calciotropic hormone on the organ of bone. therefore, the secretion of pth is highly dependent on the ionized calcium concentration and represent a simple negative feed-back loop. the serum pth concentration decreases as the serum pth secretion is not entirely suppressible; however, there is relatively narrow range of regulation of pth secretion by extracellular calcium.10 the major effect of pth is to maintain normal ionized serum calcium concentration. pth stimulates the bone reabsorbing releasing calcium into extracellular fluid. the rise in calcium concentration is caused principally by two effects, first is the effect of pth to increase calcium and phosphate absorption from the bone and second is a rapid effect of pth to decrease the excretion of calcium by the kidneys.11 effect of exercises on thyroid hormone t3 demonstrated that this effect can lead to increase t3 hormone.12 renal calcium influenced by thyroid hormone was decrease in hyperthyroidism and increase in hypothyroidism.3 other studies showed the elevated level of total and free t4 hormone also have been reported in patient with acute psychiatric illness, iodinated contrast agent also elevate t4 hormone level by inhibiting peripheral conversion of t4 to t3. the decrease of t4 hormone occurs in patient with most severe monothyroid illness.13 the serum calcium level decreased significantly in patient with high tsh concentration in contrast with normal tsh. also, renal calcium influenced by thyroid hormones have been decreased in hypothyroidism and increased in hypothyroidism.9 the inhibitory action of calcium on the table 1. relationship between calcium and tsh, t3 and t4 parameters age ca+2 – tsh ca+2 – t4 ca+2 – t3 10–20 years (n = 12) 0.4117 0.4105 0.1566 20–30 years (n = 10) 0.10502 0.00604 0.20412 30–40 years (n = 18) 0.08956 0.17519 0.00602 *the number refers to correlation factor (r value). 0 5 10 15 20 25 30 10–20 year       co nc en tr at io n m g/ dl a nd p g/ l age relationship between calcium and hormones        ca tsh t3 t4 20–30 year 30–40 year fig. 1 mean value of calcium and thyroid hormones. 29j contemp med sci | vol. 1, no. 3, summer 2015: 27–30 research calcium ion and thyroid hormonesjasim a abdullah et al. thyroid gland has been suspected since the last century but more recent studies have confirmed its effect. it is known that calcium decreases thyroid activity and that its absorption increases thyroid insufficiency.14 the thyroid gland normally enlarge in response to an increase demand for thyroid hormone that occur in puberty, pregnancy, iodine deficiency and immunologic viral or genetic disorder in this increase in thyroid follicles and thyroid hormone synthesis. therefore, elevation of thyroid hormone leads to graves’ disease or other thyroid abnormalities. the primary characteristics of graves’ disease are diffuse thyroid enlargement (as much as two to three times the normal size, 40–60 grams), then the tumor release tsh that induce table 2. relationship between calcium and (got, gpt and bilirubin) parameters age ca+2 – gpt ca+2 – got ca+2 – bilirubin 10–20 years (n = 12) 0.2273 0.5427 0.0660 20–30 years (n = 10) 0.2424 0.03136 0.02636 30–40 years (n = 18) 0.01288 0.0263 0.0942 *the number refers to correlation factor (r value). elevated thyroid synthesis and release and are not responsive to normal hormonal feedback control. such patients present with many symptoms of hyperthyroidism.15 however, peripheral metabolism of thyroid hormone can be changed significantly by condition, which can alter the deionization pathway and lead to change in the circulating level of thyroid hormones. the biological effect of short-term change in the thyroid hormone level are not currently completely understood but are potentially important in the body adjustment to stressful or catabolic state. low dietary calcium intake and hypocalcaemia are major stimuli for pth secretion leading to parathyroid gland hyperplasia.16 relationship between calcium ion concentration and the liver enzyme got, gpt and bilirubin in the present study, we tested the relationship between calcium ion concentration and liver enzymes (got, gpt and bilirubin). we observed there is no significant difference (p ≥ 0.01) between calcium ion concentration and liver enzymes (got, gpt and bilirubin) in all groups of different ages respectively. weak correlation between calcium and liver enzyme pointed to the increase or decrease parameter effects on calcium ion concentration. this result is shown in fig. 2 and table 2. a significant decrease in total and ionized calcium phosphorus and chloride and an increase in sodium were also observed. significant increase was found in most enzymes such as ast, gk and ggt.17 a diet supplement by calcium leads to an increase in serum glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. addition of calcium ion significantly reduced liver gst and elevated liver gpx activities.18 human erythrocytes that incubated with different concentration of calcium chloride (0.17–1.67 mm) showed hemolytic after addition of bilirubin. hemolysis was observed only when cell were incubated first with calcium followed by bilirubin and not vice versa. this hemolytic was found to be dependent upon several factors such as concentration of bilirubin, time of incubation of rbcs with calcium and time of incubation of bilirubin with the calcium-loaded rbc19 and difference in the calcium-induced bilirubin dependent hemolytic phenomenon have been found among different mammalian erythrocytes.20  fig. 2 mean value of calcium and liver enzymes. 0 5 10 15 20 25 30 35 10–20 year co nc en tra tio n m g/ dl   age relationship between calcium and liver enzyme          ca gpt got bilirubin 20–30 year 30–40 year references 1. bongard fs, sue dy, vintch jr. current diagnosis and treatment critical care, 3rd ed. new york: mcgraw hill/lange; 2008. p. 51. 2. nix s, william s. basic nutrition and diet therapy, 13th ed. st. louis: mosby/ elsevier; 2009. p. 126. 3. sharan kj, siddiqui a, swarnkar gn, chattopadhyay n. role of calcium sensing receptor in bone biology. indian j med res. 2008;127(3):247–86. pmid: 18497443 4. kumar r. calcium metabolism in principle and practice of nephrology. j clin chem. 1995;19:964–71. 5. aaron rm, robert bs. thyroid hormone in cardiac surgery. j pharmacol. 2009;11(9):49–60. 6. hafkenscheid jc, dijt cc. determination of serum aminotransferases: activation by pyridoxal-5-phosphate in relation to substrate concentration. clin chem. 1979;25(1):55–9. pmid: 761381 7. sampson ej, whitner vs, burtis ia, mckneally ss, fast dm, bayse dd. an inter laboratory evaluation of the ifcc method for aspartate to aminotransferase with use of purified enzyme materials. clin chem. 1980;26(8):1156–64. pmid: 7389087 8. geller nl. advances in clinical trial biostatistics, 3rd ed. usa: marcel dekker; 2004. p. 66. 9. chen ar, goodman wg. role of the calcium-sensing and extracellular calcium signalling. am j physiol renal physiol. 2004;286:1005–11. 10. mundy gr, guise ta. hormonal control of calcium homeostasis. clin chem. 1999;45(8 pt 2):1347–52. pmid: 10430817 11. swathi k, haseena s, shaik hs. effect of tsh suppression therapy on levels of tsh, t4 and t3. j pharm sci res. 2014;6(2):115–20. 12. ciloglu f, peker i, pehlivan a, karacabey k, ilhan n, saygin o, et al. exercise intensity and its effect on thyroid hormones. neuro endocrinol lett. 2005 dec;26:172–780. 13. deruiter j. thyroid hormone tutorial: the thyroid and thyroid hormones. endocrine pharmacotherapy module 2002:5260. 14. watts dl. the nutritional relationships of the thyroid. j orthomolecular med. 1989;4(3):165–9. 30 j contemp med sci | vol. 1, no. 3, summer 2015: 27–30 calcium ion and thyroid hormones research jasim a abdullah et al. 15. chen h, hayakawa d, emura s, ozawa y, taguchi h, yano r, et al. effect of low calcium diet on the ultra-structure of the rat parathyroid gland. okajimas folia anat jpn. 2001 dec;78(5):153–9. doi: http://dx.doi. org/10.2535/ofaj1936.78.5_153 pmid: 11915356 16. brandi ml. molecular mechanisms of parathyroid hyperplasia and neoplasia hormones. hormone res. 1997;47(4–6):194–98. doi: http://dx.doi. org/10.1159/000185464 17. bauer pj. affinity and stoichiometry of calcium binding by arsenazo iii. j anal biochem. 1981;110(1): 61-72. doi: http://dx.doi.org/10.1016/0003-2697 (81)90112-3 pmid: 7212270 18. bergmeyer h, walefeld m. méthode cinétique pour la détermination du tgo et tgp sans phosphate de pyridoxal. clin chem acta. 1978;24–58. 19. ali mk, tayyab s. calcium induced bilirubin-dependent hemolysis of human erythrocytes. biochim biophys acta. 1997 may 22;1326(1): 124–30. doi: http://dx.doi.org/10.1016/s0005-2736(97)00020-5 pmid: 9188807 20. ali mk, tayyab s. differential resistance to calcium-induced bilirubin dependent hemolysis in mammalian erythrocytes. comp biochem physiol c pharmacol toxicol endocrinol. 1999 jan;122(1):109–13. doi: http://dx.doi. org/10.1016/s0742-8413(98)10088-9 pmid: 10190034 letter to editor dear editor the arbaeen pilgrimage is one of the biggest pilgrimages in the world. its size is twice as the size of hajj pilgrimage in mecca. arbaeen visitors are estimated at 15–17 millions, in which 2–3 million of them are non-iraqis migrated from 22 nations and 5 continents. this event takes almost 10 days to travel on feet from different iraqi provinces to kerbala with the distance from minimum 50 to maximum 300 miles.1 there are many problems that may threaten the national and global health due to many communicable and infectious diseases happened during this pilgrimage. the first noted infectious diseases are food-borne illness and dermatological diseases due to sharing rest places (population rest stations), such as gal, gastrointestinal disease, and the most common is respiratory infections like influenza.2 the number of visitors increasing each year, long distance of the pilgrim, low sanitation, and the weakness of iraqi health care system worsen the problem.3,4 the iraqi ministry of health in collaboration with iraqi red crescent, health care ngos, and health professions as volunteers provide primary health care and treatments by placing health stations and mobile hospitals on the visitors’ way, but these stations do not cover the whole needs. ammar n.h. albujeera,b,c, alya almahafdhad anab’a al-hayat foundation for medical sciences and health care, najaf, iraq. bdepartment of community oral health, school of dentistry, tehran university of medical sciences, tehran, iran. cschool of medicine and dentistry, university of santiago de compostela, santiago de compostela, spain dschool of medicine, international campus, tehran university of medical sciences, tehran, iran. correspondence to ammar n.h. albujeer (email: ammar.dent@yahoo.com). (submitted: 02 april 2018 – revised version received: 25 march 2018 – accepted: 04 april 2018 – published online: 26 june 2018) this event may lead to epidemics if not well organized and supported by who and another global health organizations in addition to ministries of health of visitors’ countries through providing obvious plans and health programs. as well as, by training and educating people about hygiene especially those who provide food and rest places for visitors. although things mentioned above as preventive methods are important, the most critical point is what can be done through media by increasing the awareness about these disease using native languages of the visitors5. conflicts of interest authors declare that they have no conflicts of interest. references 1. sim d. arbaeen. world’s largest annual pilgrimage as millions of shia muslims gather in karbala. international business times. 2016. 2. abubakar i, gautret p, brunette gw, blumberg l, johnson d, poumerol g, et al. global perspectives for prevention of infectious diseases associated with mass gatherings. the lancet infect dis. 2012;12:66–74. 3. webster pc. lraq’s health system yet to heal from ravages of war. the lancet. 2011;378:863–866. 4. allawi j. allocating resources in health care services. j. contem med sci. 2017;3:237–238. 5. field v, gautret p, schlagenhauf p, burchard gd, caumes e, jensenius m, et al. travel and migration associated infectious diseases morbidity in europe, 2008. bmc infect dis. 2010;10:330. does arbaeen event is taking its place in global mass gathering programs? journal of contemporary medical sciences 16 j contemp med sci | vol. 1, no. 4, autumn 2015: 16–19 research objectives this study focus on the preparation of the compound nanoscale layers of zinc oxide (zno) with naproxen and cephalexin. methods ion exchange technique via sol-gel method synthesised under aqueous environment, resulted in the formation of inorganic organic characteristics of nanohybrid by using x-ray diffraction (xrd) method and study antibacterial activity of hybrid and free compound against some bacteria. results the powder x-ray diffraction showed new level for nanohybrid compounds differ from free compounds. the antibacterial results show more nanohybrid compounds concentration increased its effectiveness against bacteria, the greater the concentration of the compound greater is the increase in its effectiveness against bacteria inhibitory. conclusions the possibility of the bind of drugs (cephalexin and naproxen) in the preparation of nanohybrid-zno compound and the use of nanocompounds as antibacterial agent in better treatment of pathogenic bacteria. and this nanocompound is more efficient from free compounds. keywords nanohybrid, biological activities, zinc oxide, naproxin, cephalexin preparation of nanohybrid compound from the drugs (naproxen and cephalexin) with zinc oxide and studying biological activities against aeromonas bacteria maryam sabah & mohammed abdulla jabor introduction metal oxide nanoparticles and composite materials are widely applied in the field of research and development and diverse applications in industries including surface coatings, optoelectronics, bioengineering, biodiagnostics, and agriculture. the emergence of bacterial resistance to antibiotics and its dissemination, however, are major health problems leading to treatment drawbacks for a large number of drugs.3,4 consequently there has been increasing interest in the use of inhibitors of antibiotic resistance for combination therapy.5,6 investigations have been carried out on the biological activities of zinc nanoparticles; however, the effects of nanoparticles on the activities of antibiotics have not been demonstrated. their intrinsic properties are mainly determined by size, shape, composition, crystallinity, and morphology. highly ionic zno nanoparticles are unique in that they can be produced with high surface area, unusual crystal structures, and size. the main advantages of using zno nanoparticles are its excellent stability or long shelf life with organic antimicrobial agents.2 in particular, the vigorous antimicrobial properties of nanoscale zno particles have been the focus of industrial applications in biocides coating in water treatment, paints, and cosmetic products.7 the scope of zno nanoparticles has been a keen area of interest for biologist due to their distinguished antimicrobial and distinct activity which have opened new frontiers to biological sciences.8 especially its nanoscale form has a strong toxicity towards a wide range of micro organisms including bacteria,9 fungi,10 fish,11 algae,12 and plants.13 materials and methods synthesis of zinc nanoparticles following the method described by bashi et al.14 with some modification in the composite nanoscale hybrid preparation issn 2413-0516 by adding 100 ml of each of the drug (separately) of the prepared zno solution (by melting1 g of zno in 100 ml of distilled water after removing the ions). the mixture is stirred magnetically at 40°c for 6 hours and then placed in an incubator at room temperature for 18 hours, followed by separation of sludge mediated centrifuge at 3,000 r/min for 20 minutes and then it is washed with distilled water ions removed him several times and then dried sludge at a temperature of 50°c. it was then crushed in a ceramic mortar and finally stored, as explained in fig. 1. fig. 1 flowchart showing details of synthesis of nano zno. department of biotechnology, college of science, correspondence to maryam sabah (email: maryam.alhasnawy@yahoo.com). (submitted: 16 october 2015 – revised version received: 27 october 2015 – accepted: 9 november 2015 – published online: autumn 2015) university of babylon, babylon, iraq 17j contemp med sci | vol. 1, no. 4, autumn 2015: 16–19 research nanohybrid compound preparation from naproxen and cephalexinmaryam sabah & mohammed abdulla jabor characterisation of nanohybridisation compounds the composite nanoscale hybrid characteristic (cephalexin-zno and naproxen-zno) is found through spectroscopic methods and microscopic examinations scanner (scanning electron microscope, sem) and atomic force (atomic force microscope, afm), as follows: 1. characteristic using x-ray diffraction (xrd) the nanohybrid compound (naproxen-zno and cephalexin zno) characteristic by use xrd. the xrd between zno and cephalexin shows diffraction level at (111) at 2θ = 9.466 with crystalline distance (d) = 0.933 and another diffraction level (222) at 2θ = 18.9285 with crystalline distance (d) = 0.469 and (333) at 2θ = 28.3907 with crystalline distance (d) = 0.314. the xrd between zno and naproxen shows diffraction level at (111) at 2θ = 4.7661 with crystalline distance (d) = 1.90651 and another diffraction level (200) at 2θ = 56.7084 with crystalline distance (d) = 1.62195 this result provide the squeeze drugs in the layers of zno. 2. antibacterial activity the study of the effectiveness of inhibitory monohybrid compounds (zno-ceph and zno-napro) and free compounds (napro, ceph and zno) against aeromonas spp by using diffusion method.16 the concentrations using in the study range is between 0.1 and 15 mg/ml. results and discussion spectrum xrd xrd pattern of the prepared zno nanoparticles is shown in fig. 2. the observed diffraction peaks of zno at 2-theta = 31.72°, 34.38°, and 36.26° are associated with 100, 002, and 110; all the reflections can be assigned to the standard powder pattern of zno.14 spectrum xrd for cephalexin-zno xrd pattern of the prepared ceph-zno nanohybridisation is shown in fig. 3. the xrd between zno and cephalexin shows diffraction level 111 at 2θ = 9.466 with crystalline distance (d) = 0.933 and another diffraction level 222 at 2θ = 18.9285 with crystalline distance (d) = 0.469 and (333) at 2θ = 28.3907 with crystalline distance (d) = 0.314. in addition, the presence of the diffraction level related to zno indicates that the level of zno still keep of their natural structure and the cephalexin may squeeze in the layers of zno. effectiveness of the two nanocompounds inhibitory (zno-ceph and zno-napro) and free compounds (napro, ceph, and zno) against bacteria results of statistical analysis in table 1 shows that there are significant differences p < 0.05 between the tested nanohybrid compounds and free compounds on one hand and between the concentrations used for the same compound on the other hand. when comparing the three free compounds first used (napro, ceph, zno) with concentrations of these compounds, it did not show any inhibitory effect against bacteria at any of the concentrations studied. this indicates that there was no significant difference p > 0.05. as for the nanohybrids compounds ceph and napro showed no effective inhibitory at three concentrations 0.05, 0.1, 0.25 mg/ml while the nanohybrid compounds (napro-zno) 0.05 if given the inhibition rate of 20 mm followed by nano hybrid composite (ceph-zno) at a rate of inhibition of 16.66 mm at the same concentration and thus differed in terms of efficiency free compounds did not show any effect against bacteria. but when comparing all compounds at the first three concentrations 0.05, 0.1, 0.25 mg/ml found that there was no significant difference p > 0.05. while the results showed significant differences clear of nanohybrid compounds and superiority on free compounds at the same concentrations as superiority nanohybrids (napro-zno) if given the inhibition rate 20 mm followed by ceph-zno at a rate of 16 mm inhibitory 16.66 mm. this indicates the efficiency of nanohybrid compounds compared to free compounds. the result show the nanoparticle compound affected the bacteria for several reasons. many studies provide nanoparticles have larger surface area available for interactions, which enhances bactericidal effect than the large-sized particles; hence they impart cytotoxicity to the microorganisms.17 the mechanism by which the nanoparticles are able to penetrate the bacteria is not understood completely, but studies suggest that when bacteria were treated with zno nanoparticles, changes took place in its membrane morphology that produced a significant increase in its permeability affecting proper transport through the plasma membrane18 leaving the bacterial cells incapable of properly regulating transport through the plasma membrane, resulting in cell death.19 experimental observations of this study have fig. 2 spectrum xrd for zno. fig. 3 xrd for cephalexin-zno nanohybridisation. 18 j contemp med sci | vol. 1, no. 4, autumn 2015: 16–19 nanohybrid compound preparation from naproxen and cephalexin research maryam sabah & mohammed abdulla jabor table 1. inhibition zone (mm) of naproxen and cephalexin and its nanocompounds against aeromona concentration (mg/ml) zno naproxen cephalexin cephalexin-zno naproxen-zno lsd 0.05 compound 0.05 a 0.0 ± 0.0 a a 0.0 ± 0.0 a a 0.0 ± 0.0 a d 0.0 ± 0.0 a e 0.0 ± 0.0 a 0.322 0.1 a 0.0 ± 0.0 a a 0.0 ± 0.0 a a 0.0 ± 0.0 a d 0.0 ± 0.0 a e 0.0 ± 0.0 a 0.25 a 0.0 ± 0.0 a a 0.0 ± 0.0 a a 0.0 ± 0.0 a d 0.0 ± 0.0 a e 0.0 ± 0.0 a 0.5 a 0.0 ± 0.0 b a 0.0 ± 0.0 b a 0.0 ± 0.0 b d 0.0 ± 0.0 b d 8.0 ± 0.57 a 1 a 0.0 ± 0.0 c a 0.0 ± 0.0 c a 0.0 ± 0.0 c c 6.33 ± 0.33 b d 8.0 ± 0.52 a 5 a 0.0 ± 0.0 c a 0.0 ± 0.0 c a 0.0 ± 0.0 c b 10.0 ± 0.57 a c 9.0 ± 0.55 b 10 a 0.0 ± 0.0 c a 0.0 ± 0.0 c a 0.0 ± 0.0 c b 10.0 ± 1.15 b b 11.0 ± 0.59 a 15 a 0.0 ± 0.0 c a 0.0 ± 0.0 c a 0.0 ± 0.0 c a 16.66 ± 0.88 b a 20.0 ± 1.15 a lsd 0.05 concentration 0.373 lsd 0.05 interference 0.843 the numbers refer to mean ± standard error; capital letters indicate significant differences (p < 0.05) between the concentrations of each compound; small letters indicate significant differences (p < 0.05) between compounds of each concentration. explained significantly the antibacterial behaviour of zno nanoparticles. it is clear that zno nanoparticles have high preventive effect on life of listeria monocytogenes. nanoparticles have a relatively large surface area and have more contact with bacteria.20 as a final result the nonspecific mode of action of nanoparticles against bacteria makes them ideal candidates as antimicrobial agents with less risk of development of bacterial resistance. the results have shown that the particles possess antibacterial properties against bacterial pathogens.21 references  1. soosen sm, bose l, george kc. optical properties of zno nanoparticles. sb academic review. 2009; 57–65.  2. kolodziejczak-radzimska a, jesionowski t. zinc oxide—from synthesis to application: a review. materials 2014 apr;7(4):2833–81. doi: http://dx.doi. org/10.3390/ma7042833 3. braga lc, leite aa, xavier kg, takahashi ja, bemquerer mp, chartone-souza e, et al. synergic interaction between pomegranate extract and antibiotics against staphylococcus aureus. can j microbiol. 2005 jul;51(7):541–7. doi: http://dx.doi.org/10.1139/w05-022 pmid: 16175202  4. schito gc. the importance of the development of antibiotic resistance in staphylococcus aureus. clin microbiol infect. 2006 mar;12(suppl 1):3–8. doi: http://dx.doi.org/10.1111/j.1469-0691.2006.01343.x pmid: 16445718  5. gibbons s. phytochemicals for bacterial resistance—strengths, weaknesses and opportunities. planta med. 2008 may;74(6):594–602. doi: 10.1055/s-20081074518 pmid: 18446673  6. wright gd. bacterial resistance to antibiotics: enzymatic degradation and modification. adv drug deliv rev. 2005 jul;57(10):1451–70. doi: http:// dx.doi.org/10.1016/j.addr.2005.04.002 pmid: 15950313  7. ravishankar rv, jamuna ba. nanoparticles and their potential application as antimicrobials. in: a méndez-vilas a, editor. science against microbial pathogens: communicating current research and technological advances. spain: formatex research center, 2011; 197–209.  8. allahverdiyev am, kon kv, abamor es, bagirova m, rafailovich m. coping with antibiotic resistance: combining nanoparticles with antibiotics and other antimicrobial agents. expert rev anti infect ther. 2011 nov;9(11):1035–52. doi: 10.1586/eri.11.121 pmid: 22029522  9. huang z, zheng x, yan d, yin g, liao x, kang y, et al. toxicological effect of zno nanoparticles based on bacteria. langmuir. 2008 apr;24(8):4140–4. doi: 10.1021/la7035949 pmid: 18341364 10. he l, liu y, mustapha a, lin m. antifungal activity of zinc oxide nanoparticles against botrytis cinerea and penicillium expansum. microbiol res. 2011 mar;166(3):207–15. doi: 10.1016/j.micres.2010.03.003 pmid: 20630731 11. lin s, zhao y, xia t, meng h, ji z, liu r, et al. high content screening in zebrafish speeds up hazard ranking of transition metal oxide nanoparticles. acs nano. 2011 sep;5(9):7284–95. doi: 10.1021/nn202116p pmid: 21851096 12. wong sw, leung pt, djurisić ab, leung km. toxicities of nano zinc oxide to five marine organisms: influences of aggregate size and ion solubility. anal bioanal chem. 2010 jan;396(2):609–618. doi: 10.1007/s00216-0093249-z pmid: 19902187 13. lin d, xing b. root uptake and phytotoxicity of zno nanoparticles. environ sci technol. 2008 aug;42(15):5580–5585. doi: http://dx.doi.org/10.1021/ es800422x pmid: 18754479 14. bashi am, hussein mz, zainal z, tichit d. synthesis and controlled release properties of 2,4-dichlorophenoxy acetate–zinc layered hydroxide nanohybrid. journal of solid state chemistry. 2013 jul; 203:19–24. doi: http://dx.doi.org/10.1016/j.jssc.2013.03.047 15. pan x, medina-ramirez i, mernaugh r, liu j. nanocharacterization and bactericidal performance of silver modified titania photocatalyst. colloids surf 19j contemp med sci | vol. 1, no. 4, autumn 2015: 16–19 research nanohybrid compound preparation from naproxen and cephalexinmaryam sabah & mohammed abdulla jabor b biointerfaces. 2010 may;77(1):82–89. doi: 10.1016/j.colsurfb.2010.01.010 pmid: 20153152 16. egorove ns. mir publishers, moscow;1985. 17. adams lk, lyon dy, alvarez pj. comparative eco-toxicity of nanoscale tio2, sio2, and zno water suspensions. water res. 2006 nov;40(19):3527 –3532. doi: http://dx.doi.org/10.1016/j.watres.2006.08.004 pmid: 17011015 18. auffan m, rose j, bottero jy, lowry gv, jolivet jp, wiesner mr. towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. nat nanotechnol. 2009 oct;4(10):634–641. doi: 10.1038/ nnano.2009.242 pmid: 19809453 19. dumas em, ozenne v, mielke re, nadeau jl. toxicity of cdte quantum dots in bacterial strains. ieee trans nanobioscience. 2009 mar;8(1):58–64. doi: http://dx.doi.org/10.1109/tnb.2009.2017313 pmid: 19304497 20. rezaei-zarchi s, javed a, javeed gm, mirjalili h, moghaddam b. comparative study of antimicrobial activities of tio 2 and cdo nano particles against the pathogenic strain of escherichia coli. ir j pathol. 2010;5(2): 83–89. 21. khani ph, zand am, imani s, rezayi m, rezaei-zarchi s. determining the antibacterial effect of zno nanoparticle against the pathogenic bacterium, shigella dysenteriae (type 1). int j nano dim. 2010;1(4):279–285. j contemp med sci | vol. 3, no. 12, autumn 2017: 326–330 research adepartment of community oral health, school of dentistry, tehran university of medical sciences, tehran, iran. bresearch center for caries prevention, dental research institute, tehran university of medical sciences, iran. cdepartment of periodontics, school of dental medicine, university of pennsylvania, philadelphia, pennsylvania, usa. correspondence to, simin zahra mohebbi (e-mail: smohebbi@sina.tums.ac.ir). (submitted: 09 july 2017 – revised version received: 29 july 2017 – accepted: 08 september 2017 – published online: 26 december 2017) objective we aimed to evaluate knowledge, attitude, and practice of senior dental students by providing a course entitled “geriatric oral health care” at tehran university of medical sciences. the increasing population of the elders in both developed and developing countries highlights the importance of geriatric dental education. methods the intervention group (n = 68) filled in a self-administered questionnaire about their background, knowledge, attitude, and practice of geriatric oral health care before and after the course. the course consisted of lectures, group discussions, and oral health examination and instructions for elderly patients. the control group completed the same questionnaire without receiving any courses on geriatric oral health care. statistical analysis was performed using t-test, one-way anova, chi-square test, and linear regression modeling. results most of the participants (88.1%) had previously treated elderly patients and 35.1% had the experience of living with the elders. a considerable improvement was observed in both knowledge and self-reported practice of the students following the course (p < 0.05), while the students’ attitude did not improve (p > 0.05). conclusions a short-term training program on geriatric oral health care has a great impact on the students’ knowledge and practice, supporting the necessity of incorporating a geriatric dentistry course in the dental curriculum. keywords geriatric dentistry, oral health, dental education, dental students introduction the aging population is increasing rapidly in all parts of the world. over the next decades, the elderly population is expected to grow much more quickly than the total population due to advances in medical technology and improvements in health status.1–3 the annual rate of global population growth is estimated to be 1.2–2.4%, while the fastest growing group is the age group 60 years old and older.4–6 it has been estimated that there will be 1.2 and 2 billion elderly people by 2025 and 2050, respectively.5 iran is facing a rapid expansion of the older population. the recent census has shown a remarkable increase in the percentage of aged people from 7.22% in 2006 to 8.20% in 2011.3 as people grow old, systemic diseases can negatively influence their oral health.7 consequently, the treatment deci sions for older patients become more complex and challenge able for dentists.8 today, older people wish to retain most of their teeth and they look for extensive and complicated dental treatments such as implants and complex restorations.9,10 so, there will be a growing demand for geriatric dental 11treatments. several reports have investigated different barriers which could interfere with providing oral health care to older patients.12–14 saunders et al. reported that the primary barriers to the expansion of geriatric dentistry at dental schools were the lack of trained faculty members, a crowded curriculum, and fiscal concerns.12 multiple medical conditions, complexity of dental treatments in older patients, and insufficient training are other barriers, which result in dental practitioners’ indeci sion to treat older patients.13,14 in 2005, the world health organization (who) recommended to provide oral health care professionals with 326 educational courses on geriatric oral health.5 some studies have demonstrated the efficacy of training courses on geriatric dentistry in dental practitioners. for example, entwistle showed that the dentists’ willingness to provide care for older people could be improved by adding geriatric care to the dental curriculum.15 waldrop reported that dental students’ knowledge of aging improved after one year of education in this subject.16 another study emphasized the necessity of geriatric dentistry training courses at dental schools to address the needs of the vast elderly population in india.17 also, findings of a recent investigation in iran demonstrated that most of the dental students were unwilling to treat the elders and that they did not receive any specific training on geriatric dentistry.14 therefore, it is important to provide training courses on geriatric oral health care for dental students in iran. unfortunately, the national curriculum of dentistry does not provide the students with sufficient knowledge and competency based education on geriatric dentistry.18 the aim of the present study was to assess the effect of implementing a short training course on “geriatric oral health care” on knowledge, attitude, and practice of senior dental students in tehran. methods ethics approval ethics approval was obtained from the ethics committee of school of dentistry, tehran university of medical sciences. participation in the study was voluntary and the questionnaires were anonymous. participants were informed about the objectives and protocol of the study. issn 2413-0516 senior dental students’ training on oral health care for geriatric patients: an interventional study katayoun sargeran,a,b simin zahra mohebbi,a,b zahra chinipardaz,c and yaser gurania katayoun sargeran et al. 327j contemp med sci | vol. 3, no. 12, autumn 2017: 326–330 research senior dental students’ training on oral health care for geriatric patients study population convenient sampling was used to include all senior dental students of tehran university of medical sciences (tums) and shahid beheshti university of medical sciences (sbmu) during the 2015 academic year in this interventional study. the tums dental students were considered as the intervention group (total number = 68, number of participants = 62, response rate 91%) and participated in a course entitled “geriatric oral health care”, while the sbmu dental students (control group, total number = 73, number of participants = 50, response rate 68.5%) did not take any courses in this regard. variables of the questionnaire we developed a self-administered questionnaire based on previously validated questionnaires.19,20 the questionnaire was evaluated for content validity by five faculty members from the department of community oral health at tums. a pilot study was performed amongst ten sixth-year dental students in order to check the reliability of the questionnaire. a cronbach’s alpha coefficient greater than 0.7 confirmed good internal consistency. the questionnaire was composed of the following sections: background information, general knowledge and attitude towards aging and the elders, knowledge and attitude towards providing oral health care for elderly patients, and the students’ self-reported geriatric oral health care practice. background questions included age, gender, marital status, grade point average (gpa), living place (with parents/ in the dormitory/ living independently), prior experience of living with the elders (yes/no), and prior experience of treating elderly patients (yes/no). the first section consisted of 14 questions in order to assess the students’ knowledge of geriatrics. the following statements are examples of this section: less time is available to plan for the elderly population in poor countries, women have a longer life-expectancy, clinical decision making for elderly patients should be based on their health and physical ability rather than their chronological age, aging causes an increase in heart beats and a decrease in respiratory capacity, etc. the second section, knowledge of oral health care for elderly patients, included 16 question which targeted the students’ knowledge about various subjects such as the growing trend of aging in iran, the necessity of being familiar with common geriatric diseases for successful dental practice, reducing oral cancer in the elders through preventive measures, comprehensive oral health instructions, special considerations during dental treatment, and appropriate cooperation between dental and medical team. in the knowledge sections, the provided answers were ‘yes’, ‘no’ and ‘i don’t know’. the third section, attitude towards aging, contained 13 questions such as “i prefer to treat young patients rather than old ones”, “taking care of the older people is a social responsibility”, “geriatric medical care needs a large amount of financial resources”’, “the elderly generally engage less in social activities”, “it is interesting for me to listen to the elder’s experiences”, and “i understand older patients more than younger ones”. four questions measured the students’ attitude towards providing oral health care for geriatric patients as follows: “more attention should be paid to treatment needs in the elderly population”, “tooth loss is a consequence of aging”, “providing preventive oral care for geriatric patients is not profitable for dentists” and “providing preventive oral care for geriatric patients is economically beneficial to the society”. the responses in the attitude sections were categorized on a likert scale from “strongly agree” to “strongly disagree”, including “i don’t know”. the last section measured the students’ practice of oral health care in the elderly patients through four questions with multiple-choice answers: “how often should elderly patients with xerostomia visit a dentist?”, “what would you do to deal with patients who take multiple drugs for their systemic disease?”, “do you check the blood pressure of the elderly patients before starting treatment?” and “what do you consider in treating patients with arthritis?”. study design and intervention description “geriatric oral health care” training was conducted in three sessions during a one-week period for students in the intervention group, as part of their community oral health practical course. in the first session, the educational content was delivered using lecture and problem based learning (pbl) methods by two lecturers from the department of community oral health, tums. the students were asked to fill in the self administrated questionnaire prior to the intervention. educational topics were related to geriatrics and geriatric oral health (45 minutes each). afterward, a paper-patient was introduced to the class by a power point presentation. all the students discussed the case with each other in small groups (5 students in each group). at the end of the first session, the groups presented their answers and discussed them with their classmates and teachers. in the second session, the students attended a municipal health center in tehran, where the elders were invited to receive oral health examination and education. the students examined the participants in order to determine their oral health status and oral treatment needs. meanwhile, each student explained oral hygiene and denture care instructions to the patient. during the session, the students’ performance and skills were evaluated with a “direct observation of procedural skills” (dops) checklist by two faculty members and a general dentist. in the last session, the students gave presentations on their field observations and experiences and had discussions with classmates and teachers. the session was ended with final conclusions and the students were asked to fill in the same self-administrated questionnaire and the course satisfaction form. the control group completed the same questionnaire twice within a week without receiving any education regarding geriatric oral health care. statistical analysis we dichotomized answers to all the questions. a positive response in attitude sections and a correct response in knowledge and practice sections received a score of 1 while false/ negative/i don’t know the answers were assigned a score of 0. the scores were calculated on a scale of 0-100 in each section of the questionnaire. then, the total score was calculated for knowledge, attitude, and practice sections in the pre-test and post-test for each participant in intervention and control groups. the obtained data were analyzed using spss version 18. t-test and one-way anova were used to compare mean values, and the chi-square test was employed to compare frequency between the two groups. linear regression models were conducted for the multivariate assessment to control the effects of confounders. p values less than 0.05 were considered significant. 328 j contemp med sci | vol. 3, no. 12, autumn 2017: 326–330 senior dental students’ training on oral health care for geriatric patients research katayoun sargeran et al. results sample description the study population comprised 112 students, 69 (61.6%) female students and 43 (38.4%) male students. the mean age of the students was 24.2 years (range: 22–28) in the intervention group and 25.42 years (range: 23–40) in the control group. out of 68 students in the intervention group, 62 students completed the questionnaire (response rate = 91%) while the response rate was 68.8% in the control group (of 73 distributed questionnaires, 50 questionnaires were returned). most of the participants (88.1%) reported that they had prior experience in treating elderly patients, whereas the rest of them (11.9%) did not have any experience in this regard. in terms of living with the elders, only 35.1% of the students had ever lived with. except for age, the intervention and control groups were similar in background variables (table 1). educational intervention findings the mean score of general knowledge towards aging and elders was 73.5 and 86.1 in the intervention and control group at baseline, respectively. these scores increased to 91.7 and 87.6 after the intervention. the baseline score of the students’ knowledge about providing geriatric oral health care in the intervention group was 55 which increased to 96.1 after the intervention. in the control group, this score increased from 83.2 to 84.4. there was a significant difference in the knowledge score in both knowledge sections between the two groups after the intervention (p < 0.05). the mean score of the students’ attitude towards geriatrics was 68.3 and 67.8 in the intervention and control group, respectively, which increased to 69.2 and 69.3 after the intervention. the mean baseline score of the students’ attitude towards providing geriatric oral health care was 76.4 and 73.6 in the intervention and the control group respectively which increased to 80.3 in the intervention group and 75.6 in the control group after the intervention. the groups showed no significant differences in the attitude towards geriatrics (p = 0.55) and attitude regarding geriatric oral health care (p = 0.33) after the intervention. the baseline score of the students’ self-reported oral health care practice for the elderly patients was 31.4 and 45 in the intervention and control group, respectively. after the intervention, the score significantly increased to 89.5 in the intervention group and to 47 in the control group after the intervention (p = 0.00). linear regression analysis was used to assess the effect of background variables on the changes of the students’ knowledge, attitude and practice. no significant relationship was detected between changes in the students’ practice and background variables (p > 0.05). the key factor in changing the students’ knowledge was the intervention (table 2). another model revealed that having the experience of treating elderly patients was effective in changing the attitude of the participants (table 3). discussion the growth of the elderly population is inevitable.3 this demographic transition stimulates dental schools to train dental students in geriatric oral health care21 so that the students feel competent in treating elderly patients.22 the current study evaluated the effect of a short-term training program on “geriatric oral health care” on senior dental students in iran. table 1. demographic characteristics of participants (n = 112) in intervention and control groups intervention control mean age 24.02 25.42 gender female male 37 (59.7%) 25 (40.3%) 32 (64%) 18 (36%) marital status single married 47 (75.8%) 15 (24.2%) 38 (76%) 12 (24%) place of residence with parents independent dormitory 27 (44.3%) 14 (23%) 20 (32.8%) 28 (57.1%) 10 (20.4%) 11 (22.4%) prior experience of living with the elders yes no 20 (32.3%) 42 (67.7%) 19 (38.8%) 30 (61.2%) prior experience of treating elderly patients yes no 53 (85.5%) 9 (14.5%) 43 (91.5%) 4 (8.5) gpa* 16.30 16.36 *grade point average. table 2. association between changes in knowledge and background factors in participants (n = 112) after the educational intervention b std. error beta p-value ·  intervention/control groups 2.0 0.29 0.61 0.00 ·  gender (male/female) 0.67 0.43 0.16 0.12 ·  age 0.22 0.09 0.02 0.81 ·  marital status (single/married) 0.71 0.51 0.14 0.16 ·  prior experience of living with the elders (yes/no) 0.35 0.50 0.08 0.47 ·   prior experience of treating elderly patients (yes/no) 0.79 0.65 0.12 0.22 ·  gpa 0.09 0.19 0.05 0.61 ·   place of residence (with parents/dormitory/independent) 0.42 0.23 0.18 0.07 katayoun sargeran et al. 329j contemp med sci | vol. 3, no. 12, autumn 2017: 326–330 research senior dental students’ training on oral health care for geriatric patients our results revealed that such training interventions were significantly effective in enhancing the students’ knowledge about and practice of geriatric oral health care despite the students’ attitude did not improve significantly. in the present study, the students’ knowledge was evaluated in two parts: general knowledge about geriatrics, and knowledge about providing oral health care for elderly patients. the students presented a good level of knowledge in the first part and a moderate level in the second part. we found no study evaluating the second part for comparison. however, the results of studies by fabiano et al.19 and waldrop et al.16 demonstrated the low level of students’ knowledge about aging. friedman and brecknock reported that dental and dental hygiene students’ knowledge was inadequate and did not significantly change as reported by similar studies in the 1980s and 1990s.23 moreover, wood and millgan reported that the students’ knowledge of aging did not change and seemed to have even declined in comparison with the past years.24 the poor knowledge of dental students regarding aging was reported by visschere et al.25 they mentioned it could be probably due to the lack of a special course in geriatric dentistry at most dental schools. our finding on the improvement of the students’ knowledge after educational intervention is in agreement with some other studies.26,27 teasdale and shaikh conducted a cd-based educational tool to train students in geriatric oral health. they found that a self-instructional e-learning tool was effective in enhancing the students’ knowledge.28 on the contrary, fabiano et al. pointed out that dental students’ knowledge was not improved following the educational intervention; however, they recognized issues that prevented the patients’ compliance and affected the oral health of the elderly patients.19 in the present study, the participants showed a moderate level of positive attitude towards aging and geriatric oral health care. this finding is in agreement with the results of a study conducted by gupta et al. that showed students at the university of california, los angeles (ucla) had a relatively positive attitude towards the geriatric population.29 furthermore nochajski et al. showed that in general, dental students displayed a modestly positive attitude towards older adults in three of the four scales (integrity, autonomy, acceptability) and a relatively more negative attitude in the fourth (instrumentality).30 however, the findings of an investigation in belgium showed that the attitude of recently graduated dentists towards the institutionalized elderly people was rather negative.25 on the other hand, sadaf & yazdanie showed that the dental students’ attitudes were very positive towards elderly patients and they showed a strong desire to treat them.31 they concluded that geriatric dentistry should be introduced as a separate subject in the curriculum to properly utilize the students’ positive attitudes and improve the quality of life of the elders. there was no significant difference between preand post-test scores in attitude sections, which showed that educational intervention did not have any impacts on the students’ general attitude towards aging and geriatric oral health care. this result is in accordance with the findings of prior studies. claus reported that training programs in geriatric dentistry failed to make a significant change in the dental students’ attitude towards the elders.32 according to de visschere et al., geriatric dentistry education in the undergraduate curriculum did not appear to influence the recently graduated dentists’ attitude towards the institutionalized elderly people.25 although the majority of the participants had the experience of treating elderly patients, most of them did not prefer to provide oral health care for them. this may be due to the fact that senior dental students are often under pressure to meet their clinical requirements for graduation, so they prefer to select patients with minimal dental needs. this point has been mentioned in other reports, as well.27,33 the participants’ score of self-reported oral health care practice for the elders was low. following the educational intervention, the score improved markedly in the intervention group. unfortunately, we found no study in this regard for comparison. however, our finding indicates the great impact of educational intervention on the students’ perceived practice for elderly patients. the current study was an educational trial with a control group, which is the most powerful study design. the intervention and control groups were not different in demographic factors except for the mean age, which was slightly higher in the control group. additionally, the national dentistry curriculum is similar in all dental schools in iran and no courses were available on geriatric oral health care at the time of the study. the measurement tool for attitude assessment was developed based on previous validated questionnaires; however, we tested the validity and reliability of the questionnaire once more. we asked the students to use a code instead of their personal information in order to improve the validity of the answers and decrease the possibility for errors. this study had some limitations. the loss of participation for getting post-test was occurred in the control group, may be as a result of lack of motivation to take the post-test. also, the questionnaire was self-administrated and some questions were occasionally left unanswered. finally, there was a short table 3. association between changes in attitudes and background factors in participants (n = 112) after the educational intervention b std. error beta p-value ·  intervention/control groups 0.002 0.90 0.01 0.98 ·  gender (male/female) 1.48 0.87 0.17 0.09 ·  age 0.09 0.19 0.05 0.63 ·  marital status (single/married) 1.43 1.04 0.14 0.17 ·  prior experience of living with the elders (yes/no) 0.92 1.01 0.10 0.36 ·  prior experience of treating elderly patients (yes/no) 2.66 1.31 0.20 0.04 ·  gpa* 0.29 0.38 0.07 0.44 ·  place of residence (with parents/dormitory/independent) 0.39 0.47 0.08 0.41 330 j contemp med sci | vol. 3, no. 12, autumn 2017: 326–330 senior dental students’ training on oral health care for geriatric patients research katayoun sargeran et al. period of time between preand post-tests. since the students were in their final semester, long-term evaluation of the educational intervention was impossible. nonetheless, it could be the topic of further researches in this field. conclusions while the attitude of dental students did not change following a short-term training program on geriatric oral health care, their knowledge and self-reported practice improved markedly. it seems that long-term training and other educational methods should be used to form a positive attitude towards geriatric oral care in dental students. acknowledgements we would like to express our gratitude to head and staff of the municipal health centers in tehran contributed to this work. conflict of interest none. n references 1. grigsby js. paths for future population aging. gerontologist. 1991;31: 195–203. 2. treas j, logue b. economic development and the older population. popul dev rev. 1986;12:645–673. 3. noroozian m. the elderly population in iran: an ever growing concern in the health system. iran j psychiatry behav sci. 2012;15:6:1–6. 4. united nations, department of economic and social affairs, population division (2015). world population ageing 2015 (st/esa/ser.a/390). http:// www.un.org/en/development/desa/population/publications/pdf/ageing/ wpa2015_report.pdf. accessed 13 december 2016. 5. petersen pe, yamamoto t. improving the oral health of older people: the approach of the who global oral health programme. community dent oral epidemiol. 2005;33:81–92. 6. holm-pedersen p, walls a, ship j. text book of geriatric dentistry. 3rd ed. wiley blackwell, 2015. 7. ghezzi em, ship ja. systemic diseases and their treatments in the elderly: impact on oral health. j public health dent. 2000;60:289–296. 8. kotzer rd, lawrence hp, clovis jb, matthews dc. oral health-related quality of life in an aging canadian population. health qual life outcomes. 2012;10:50. 9. ettinger rl, mulligan r. the future of dental care for the elderly population. j calif dent assoc. 1999;27:687–692. 10. eklund sa. changing treatment patterns. j am dent assoc. 1999;130: 1707–1712. 11. issrani r, ammanagi r, keluskar v. geriatric dentistry–meet the need. gerodontology. 2012;29:e1–e5. 12. saunders rh, yellowitz ja, dolan ta, smith bj. trends in predoctoral education in geriatric dentistry. j dent educ.1998;62:314–318. 13. patil manashvini s, patil sanjayagouda b. geriatric patient – psychological and emotional considerations during dental treatment. gerodontology. 2009;26:72–77. 14. hatami b, ahmady ae, khoshnevisan mh, lando ha. dental students perceived barriers in geriatric dental care active involvement. oral health dent manag. 2014;13:675–679. 15. entwistle ba. oral health promotion for the older adult: implications for dental and dental hygiene practitioners. j dent educ. 1992;56:636–639. 16. waldrop dp, fabiano ja, nochajski th, zittel-palamara k, davis el, goldberg lj. more than a set of teeth: assessing and enhancing dental students’ perceptions of older adults. gerontol geriatr educ. 2006;27:37–56. 17. shah n. need for gerodontology education in india. gerodontology. 2005;22:104–105. 18. dental education program in dental schools of the islamic republic of iran (1st ed) tehran: ministry of health and medical education, council for dental and sub-dental eduation, 2000. 19. fabiano ja, waldrop dp, nochajski th, davis el, goldberg lj. understanding dental students’ knowledge and perceptions of older people: toward a new model of geriatric dental education. j dent educ. 2005;69:419–433. 20. stewart tj, roberts e, eleazer p, boland r, wieland d. reliability and validity issues for two common measures of medical students’ attitudes toward older adults. educ gerontol. 2006;32:409–421. 21. mendoza-núñez vm, de la luz martínez-maldonado m, correa-muñoz e. perceptions on the importance of gerontological education by teachers and students of undergraduate health sciences. bmc med educ. 2007;7:1. 22. mohammad ar, preshaw pm, ettinger rl. current status of predoctoral geriatric education in us dental schools. j dent educ. 2003;67:509–514. 23. friedman pk, brecknock sa. comparison of dental and dental hygiene students’ performance on “facts of aging quiz”. j mass dent soc. 2003;52:36–39. 24. wood gj, mulligan r. cross-sectional comparison of dental students’ knowledge and attitudes before geriatric training, 1984–99. j dent educ. 2000;64:763–771. 25. de visschere l, van der putten gj, de baat c, schols jg, vanobbergen j. the impact of undergraduate geriatric dental education on the attitudes of recently graduated dentists towards institutionalised elderly people. eur j dent educ. 2009;13:154–161. 26. broder h, block mj. effects of geriatric education on the knowledge of dental students. spec care dentist. 1986;6:177–179. 27. anehosur gv, nadiger rk. evaluation of understanding levels of indian dental students’ knowledge and perceptions regarding older adults. gerodontology. 2012;29:e1215–e1221. 28. teasdale ta, shaikh m. efficacy of a geriatric oral health cd as a learning tool. j dent educ. 2006;70:1366–1369. 29. gupta s, venkatraman s, kamarthi n, goel s, goel s, keswani t. assessment of the attitude of undergraduate dental students toward the geriatric population. trop j med res. 2014;17:104–108. 30. nochajski th, waldrop dp, davis el, fabiano ja, goldberg lj. factors that influence dental students’ attitudes about older adults. j dent educ. 2009;73:95–104. 31. sadaf a, yazdanie n. attitude of dental students towards elderly. pakistan oral dent j. 2012;32:176–179. 32. claus lm. dental student attitudes towards the elderly and training in geriatric dentistry. int dent j. 1982;32:371–378. 33. chowdhry n, aleksejuniene j, wyatt c, bryant r. dentists’ perceptions of providing care in long-term care facilities. j can dent assoc. 2011;77:b21. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201707 208 j contemp med sci | vol. 3, no. 10, spring 2017: 208–212 research effect of serum cystatin c in early diabetic nephropathy in type 2 iraqi diabetic patients ahmed abed kadhem al-saedy,a kismet m. turki,b shaymaa z. nadaa adepartment of biochemistry, college of medicine, university of karbala, holy karbala, iraq. bdepartment of biochemistry, college of medicine, university of baghdad, baghdad, iraq. correspondence to ahmed abed kadhem (email: ahmed_albarkh@yahoo.com). (submitted: 17 december 2016 – revised version received: 28 december 2016 – accepted: 22 january 2017 – published online: 26 june 2017) objective this study aimed to find the effect of serum cystatin c in early diabetic nephropathy. method this study was conducted in al kindy teaching hospital during the period from december, 2015 to june, 2016. the study included 90 subjects (30 males and 30 females) with diabetic type 2 and 30 healthy control. age is between 30 and 70 years. patients were with no history of liver disease, thyroid or other endocrine diseases through clinical interviewing. they were divided in to three groups. 30 healthy controls, 30 patients with type 2 diabetes mellitus with no albuminuria (albumin excretion in urine <30 µg/ml ) and 30 patients with type 2 diabetes mellitus with micro albuminuria (albumin excretion in urine >30 µg/ml ). results there were no significant differences in age, body mass index (bmi) among the studied groups (p > 0.05). serum cystatin c levels showed significant difference (p < 0.001) among studied groups, it was significantly higher in micro albuminuria than the other two groups. there was highly significant positive correlation between cystatin c and serum creatinine (r = 0.697, p < 0.001), and a significant negative correlation between cystatin c and gfr (r = − 0.455, p = 0.011) and showed significant positive correlation between cystatin c and urine albumin (r = 0.526, p = 0.003 ) in type 2 diabetic patients with micro albuminuria. conclusion cystatin c is negatively correlated with the amount of gfr, so that cystatin c considered reliable and sensitive marker for identifying changes in gfr. in type 2 diabetes mellitus with micro albuminuria serum cystatin c level considered predict marker for renal failure. keywords diabetic nephropathy, micro albuminuria, serum cystatin c introduction diabetic nephropathy is a common complication of diabetes mellitus type 2. the mechanism involved includes changes in blood vessels that supply peripheral nerves and metabolic disorder.1 diabetic nephropathy characterized by an increased excretion of protein, particularly albumin in urine, decline of the glomerular filtration rate (gfr) and elevated blood pressure leading to end stage renal failure.2 diabetic nephropathy has been reported to occur in 25–40% of people with type 1 or type 2 diabetes.3 early diagnosis of diabetic nephropathy is important, and early therapy decreases the progression of renal disease.4 micro albuminuria was measured in people with diabetes mellitus and people with gfr less than 60 (ml/min/1.73 m2) and people with strong chronic kidney disease.5 micro albuminuria is used as screening and diagnostic diabetic nephropathy. it is a marker of early diagnosis of kidney disease and also used as a predictor for development coronary heart disease and mortality.6 the macro albuminuria increase of albumin and large amount other protein can pass through the urine.7 screening for diabetic nephropathy must be initiated at the time of diagnosis in patient with type 2 diabetes. first screening has been recommended at 5 years after diagnosis in patient with poor glycemic and high normal blood pressure levels.8 cystatin c is a 13 kda protein with low molecular weight and produced by all nucleated cells and freely filtered by the renal glomeruli and reabsorbed in the proximal tubule. cystatin c is not affected by age or muscle mass in healthy person. increased urinary cystatin c is a marker of renal tubular dysfunction.9 cystatin c has multiple biological functions including modulation of the immune system and controlling extracellular proteolysis. cystatin c is reabsorbed by proximal tubule epithelial cells and is not returned to the circulation.10 cystatin c is highly correlated with gfr and not influenced by inflammatory conditions, muscle mass, age, body composition and gender. increased cystatin c is associated with increase cardiovascular morbidity risk and atherosclerosis progression in diabetes and severity obese children.11 cystatin c is not normally detected in the urine and found in the urine of patients with tubular disease suggesting role as a marker of renal tubular damage.12 it is abundant in the serum and less dependent on extra renal factor compared to creatinine and greater sensitivity for revealing mild renal dysfunction even in presumably healthy individuals compared to conventional renal indictor.13 cystatin c levels have been used most commonly to assess kidney function. serum creatinine and serum cystatin c are endogenous markers of kidney function. serum creatinine levels are associated positively with greater muscle mass and dietary meat intake, while, the serum cystatin c level is less sensitive to inter-individual differences in muscle mass. the levels of these markers are increased in person with higher body mass index, inflammation, and diabetes.14 many studies show the cystatin c as it believes as a marker of tubular and glomerular dysfunction for early diabetic nephropathy, and the urine level in cystatin c is high in micro albuminuria in diabetic patients compared with normal albuminuria.15 this study aimed to test the effect of serum cystatin c in early diabetic nephropathy in type 2 diabetes mellitus of diabetic iraqi patients. materials and methods this study was conducted in al kindy teaching hospital during the period from december 2015 to june 2016. the study included 90 subjects (30 males and 30 females) with issn 2413-0516 ahmed abed kadhem al-saedy et al. 209j contemp med sci | vol. 3, no. 10, spring 2017: 208–212 research effect of serum cystatin c in early diabetic nephropathy in type 2 iraqi diabetic patients diabetic type 2 and 30 healthy controls. age is between 30 and 70 years. patients with no history of liver disease, thyroid or other endocrine disease through clinical interviewing were divided into three groups of 30 healthy controls, 30 type 2 diabetes mellitus with no albuminuria (albumin excretion in urine <30 µg/ml) and 30 type 2 diabetes mellitus with micro albuminuria (albumin excretion in urine >30 µg/ml). urine sampling was performed by giving each subject a suitable disposable container with immediate freezing in the deep freeze at −20oc. the general physical, chemical and microscopic examinations of urine were performed on another part of urine specimen to check for the final selection or exclusion of a subject in the study. blood sampling was performed at 8.00−11.00 am in the fasting state for all the patients and controls, and about 7 ml of venous blood was obtained by antecubital venipuncture using disposable syringes with g 21 needles in the sitting position. blood was divided into edta tube for haemoglobin and plain tube for hemoglobina1c test (2.0 ml). the blood was allowed to clot for about 20−30 minutes at room temperature, and the serum was recovered by centrifugation at 3000 rpm for 10 minutes and transferred into plain plastic tubes and stored at −20oc. all patients’ height and weight were measured. body mass index (bmi) was calculated as a ratio of the weight to the height (kg/m2). blood pressure was measured three times, and the average value was considered for data analysis. fasting blood glucose (fbg) concentration was determined by using glucose oxidase method. hba1c% assay using the siemens dimension clinical chemistry system is an in vitro diagnostic assay for the quantitative determination of hba1c in human. albumin excretion in urine was measured by the fully-auto chemiluminescence immunoassay (clia) analyzer maglumi1000). serum urea and serum creatinine levels were determined using enzymatic colorimetric methods. gfr was estimated using the modification of diet in renal disease (mdrd) abbreviated equation: {gfr = 186 × (serum creatinin)−1.154 × (age)−.203 × (0.742 if female )} and serum cystatin c was measured using cosbio kit, enzyme-linked immunosorbent assay (elisa). results the result expressed as mean ± sd difference between the groups was analyzed by one-way analysis of variance (anova). the correlation between various variables was calculated using the person correlation coefficient. receiver operating characteristic (roc) analysis was employed to calculate the area under the curve (auc) and to find the best cut off values to diagnostic specificity and sensitivity. p <0.05 was accepted as statistically significant. all statistical calculations were done using statistical package for the social science version 20 (spss). the result (table 1) showed no statistically significant difference in terms of age, bmi (p > 0.05) between the studied groups. systolic blood pressure showed a statistically significant difference (p < 0.001) among study groups. diastolic blood pressure also showed statistically significant difference (p < 0.001) among study groups. t-test showed statistically significant difference in the duration of diabetes among no albuminuria and micro albuminuria diabetic group. the blood glucose levels showed a statically significant difference (p < 0.001) among study groups. it was significantly higher in micro albuminuria than any albuminuria and healthy control. glycated hemoglobin (hba1c%) levels showed a statically significant difference (p < 0.05) among studied groups. it was significantly higher in micro albuminuria than any albuminuria and healthy control group. urine albumin levels showed a significant higher in type 2 diabetic patients with micro albuminuria than type 2 diabetic patients with no albuminuria and healthy control group, which is highly increased significantly (p < 0.001). the levels of blood urea were significantly higher in micro albuminuria (p < 0.05) when compared with no albuminuria and healthy control group. serum creatinine levels was significantly higher in micro albuminuria than no albuminuria and healthy group (p < 0.001). the level of glomerular filtration rate (gfr) was significantly higher in micro albuminuria than any albuminuria and healthy group (p < 0.05). serum cystatin c levels showed significantly increased (p < 0.001) among studied groups. it was significantly higher in micro albuminuria than any albuminuria. the correlation analyses revealed the relationship between serum table 1. clinical characteristics and baseline investigation of study groups p-anovamicro albuminuriano albuminuriahealthy control characteristics > 0.0555.2 ± 9.753.7 ± 11.553.5 ± 11.7age (year) > 0.0529.4 ± 5.529.21 ± 4.827.5 ± 4.1bmi (kg/m2) < 0.001143.0 ± 16.2133.0 ± 17.1124.2 ± 12.7bp (systolic) mm/hg < 0.00192.5 ± 5.087.0 ± 5.879.5 ± 8.1bp (diastolic) mm/hg t-test (< 0.05)7.9 ± 5.65.9 ± 2.0—duration dm (year) < 0.001236 ± 83.1197.6 ± 63.098.6 ± 12.4fbs (mg/dl) < 0.0018.2 ± 1.67.5 ± 0.74.8 ± 0.4hba1c (%) < 0.00190.9 ± 69.018.7 ± 4.95.3 ± 1.7urine albumin (µg/ml) < 0.00143.2 ± 17.032.8 ± 7.134.6 ± 8.3serum urea (mg/dl) < 0.0011.1 ± 26.30.8 ± 21.70.7 ± 18.2serum creatinine (mg/dl) < 0.00175.1 ± 26.385.6 ± 21.791.6 ± 18.2gfr (ml/min/1.73 m2) < 0.00120.4 ± 16.08.3 ± 4.34.6 ± 3.4serum cystatin c (ng/ml) anova f-test p < 0.05 is considered as significant (s); p < 0.001 is considered as highly significant (hs); and p > 0.05 considered as non-significant; sd: standard deviation (mean ± sd); bmi: body mass index; bp: blood pressure; dm: diabetes mellitus; gfr: glomerular filtration rate. 210 j contemp med sci | vol. 3, no. 10, spring 2017: 208–212 effect of serum cystatin c in early diabetic nephropathy in type 2 iraqi diabetic patients research ahmed abed kadhem al-saedy et al. cystatin c and serum creatinine amount gfr, and urine albumin excretion in diabetic patients with micro albuminuria. there was a highly significant positive correlation between cystatin c and serum creatinine (r = 0.697, p < 0.001) as shown in fig. 1, and significant negative correlation between cystatin c and gfr (r = − 0.455, p = 0.011) as shown fig. 2 and shows significant cystatin c and urine albumin (r = 0.526, p = 0.003) in type 2 diabetic patients with microalbuminuria as shown in fig. 3. fig. 1 relationship between serum cystatin c and serum creatinine. fig. 2 relationship between serum cystatin c and gfr. table 2. area under operator characteristic (roc) curve for prediction of micro albuminuria in diabetic patients p-valuerocparameter < 0.00011.0urine albumin (µg/ml) < 0.00010.758blood urea (mg/dl) 0.0040.706 s. cystatin c (ng/ml) 0.0050.689serum creatinine (mg/dl) 0.060.638fbs (mg/dl) 0.110.616gfr ml/min/1.73 m2 0.250.586hba1c% 0.710.528bmi (kg/m2) roc: reactive operator characteristics, p < 0.001 considered high significant. and p > 0.05 considered non-significant, fbs (fasting blood sugar), gfr (glomerular filtration rate), hba1c% (hemoglobin a1c), and bmi (body mass index). fig. 4 roc figure showing the tradeoff between sensitivity (rate true positive result) and 1specificity (rate of false positive results) for selected measurements when used as a test to predict micro albuminuria in diabetic patients differentiating them from diabetics with normal albuminuria. fig. 3 relationship between serum cystatin c and micro albumin (1, c). anova f-test p <0.05 is considered as significant (s). p < 0.001 is considered as highly significant (hs). and p > 0.05 considered as non-significant, sd: standard deviation (mean ± sd), bmi: body mass index, bp: blood pressure, dm (diabetes mellitus), gfr (glomerular filtration rate). the area under the receiver operator characteristic (roc) curve was used to discriminate between diabetic patients with micro albuminuria and diabetic patients with no albuminuria depending on levels of analytes of this study. the area under the curve was high for urine albumin (roc = 1.0), serum urea (roc = 0.758), serum cystatin c (roc area = 0.706), serum creatinine (roc area = 0.689), fasting blood sugar (roc area = 0.638), gfr (roc area = 0.616), hba1c% (roc area = 0.586), and bmi (roc area = 0.528) as shown in table 2 and fig. 4. the best discriminative cut-off value for each of those analytes was selected, and the sensitivity and specificity of each analytes were calculated to predict diabetic patients with micro albuminuria and to define those with no albuminuria. ahmed abed kadhem al-saedy et al. 211j contemp med sci | vol. 3, no. 10, spring 2017: 208–212 research effect of serum cystatin c in early diabetic nephropathy in type 2 iraqi diabetic patients the cut-off value of urine albumin level at (28.0 µg/ml) had a sensitivity 100 % and a specificity 100%. serum urea level at (31 mg/dl) had a sensitivity of 90% and a specificity of 56%. serum cys-c level at cut-off value of (15.2 ng/ml) had a sensitivity of 53% and a specificity 96%. serum creatinine cut-off level at (1.0 mg/dl) had a sensitivity 43% and specificity 86%. fasting blood sugar level at a cut-off value (216 mg/dl) had a sensitivity 66 % and a specificity 66%. gfr level at a cut-off value (93) had a sensitivity 80% and a specificity 46%. as shown in table 3. roc : reactive operator characteristics, p < 0.001 considered high significant. and p > 0.05 is considered as non significant, fbs (fasting blood sugar), gfr (glomerular filtration rate), hba1c% (hemoglobin a1c), and bmi (body mass index) discussion various parameters have been recently investigated in serum and urine that play an important role as early detection markers of acute kidney diseases and its complications such as cystatin c, kidney injury molecule-1.16 diabetic nephropathy is the leading cause end stage renal disease, and hyperglycemia plays a role in the development of diabetic nephropathy. hyperglycemia causes increase hyper filtration and renal injury. in general, the hyperglycemia is considered as the key initiator for kidney damage by activation of other metabolic pathways and formation or increase in oxidative stress.17 the presence of micro albuminuria is considered as indicator or warning signal to renal and cardiovascular disease in patients with type 2 diabetes mellitus, and also considered as the earliest marker of diabetic nephropathy and associated with significant glomerular damage. studies showed that reduced risk factors lead to decrease level of micro albuminuria and decrease in renal and cardiovascular diseases.18 recent studies showed that the micro albuminuria does not necessary reflect renal impairment in addition to the early structure damage in both tubular structure and glomerular that may be present in normal albuminuria.11 so, there is a need to find biomarkers that help in identification of the patients’ risk of the disease and monitoring preventive and therapeutic effects. in this study, there is an increased level of cys-c in diabetic patients with micro albuminuria is highly significant when compared to diabetic patients with no albuminuria and control group. this is in agreement with the results of other study.19 serum cystatin c performed well as a marker to detect acute renal failure and used to detect development of acute renal failure earlier than serum creatinine.20 other study reported that serum cystatin c is a better early predictor of progression of esrd in person with type 2 diabetes, and the ability of serum cystatin c to detect pathologies changes in renal function is very high.21 other studies reported that the increase in serum cystatin c is more than serum creatinin in kidney damage. serum cystatin c is not influenced by race, muscle mass, gender and age. this may suggest serum cystatin c equation to calculate gfr that is more important than serum creatinin equation in calculation of gfr.22 the present study shows a statistically significant positive correlation between serum cys-c and serum creatinine of diabetic patients with micro albuminuria .this is in agreement with the finding of many studies.23 there are negative correlations between serum cys-c and the amount of gfr. serum cys-c shows significant positive correlation with urine albumin. these results are in agreement with other study who stated that there was a significant positive correlation between serum cys-c and urine albumin exertion.24 the micro albuminuria is used as the earliest marker of diabetic nephropathy. however, other study showed that the micro albuminuria is not necessary to reflect renal impairment and suggested that the early structure damage in glomeruli and tubular structure may be present in the normal albuminuria. so, it becomes necessary to find marker to evaluate early tubular damage indecently of albuminuria development and progression in diabetic patients with early nephropathy.11 the cys-c filtered freely by renal glomeruli and reabsorbed in the proximal tubule and has molecular weight of approximately 120 times greater than serum creatinine. however, increased concentration cystatin c in the blood indictor effected the kidney or mild renal impairment.23 the area under a roc curve quantifies the ability of the statistics to correctly discriminate between patients into abnormal and normal micro albuminuria. the classification of patients depended on to define urine albumin excretion. generally, a test in roc curve with range from 0.5 to 1.0, however, the area if it was 0.5, the test did not identify the ability into positive and negative roc evaluation by reporting p value that tested the null hypotheses. the area under curve really equals 0.5 in others words the p value answers this question. if the p value small that means test dose actually discriminates between abnormal and normal. if the p value is large that means the diagnostic test is not better than flipping a coin to diagnostic patients. the best test to diagnosis disease will be that one with sensitivity and specificity 100%. however , roc curve is designed for tuning sensitivity and specificity to have comparison between highest sensitivity and highest specificity.17 in the study, the urine albumin excretion more active diagnosis test for microalbuminuric patients which goes in line with the patients with higher glucose level and long duration of disease in addition to the serum level cys-c, serum creatinine, gfr, and serum urea followed with micro albuminuria in diagnostic diabetic nephropathy. this is a useful biomarker to prognosis and detection in patients with micro albuminuria in early stage of diabetic nephropathy. also in early stage can guide early intervention for the patient’s own healthy and prevent disease progression, according to the results of this study all the patients with type 2 diabetes mellitus female and male having high serum cys-c in early stage of diabetic nephropathy. this analytes showed higher predictive ability to detection and progression of the diseases. table 3. the cut-off values of some study analytes that best predict micro albuminuria in diabetic patients specificity (%)sensitivity (%)cut-off parameter 10010028urine albumin 569031blood urea 965315.2s. cystatin c 86431.0serum creatinine 6666216fasting blood sugar 468093gfr 212 j contemp med sci | vol. 3, no. 10, spring 2017: 208–212 effect of serum cystatin c in early diabetic nephropathy in type 2 iraqi diabetic patients research ahmed abed kadhem al-saedy et al. conclusion poor glycemic control is characteristic of diabetes patients with micro albuminuria. in the study result show cys-c level increase with development and progression of diabetic nephropathy. and show cys-c negative correlated with amount gfr, so that cys-c considered a reliable and sensitive marker for identifying changes in gfr. cys-c used full detection marker of acute renal failure earlier than creatinine .cystatin c in diabetic patients with micro albuminuria considered to predict practically well renal events. conflict of interest none. n references 1. zychowska m, rojewska e, przewlocka b, mika j. mechanisms and pharmacology of diabetic neuropathy–experimental and clinical studies. pharmacol rep. 2013;65:1601–1610. 2. mise k, hoshino j, ueno t, sumida k, hiramatsu r, hasegawa e, et al. clinical implications of linear immunofluorescent staining for immunoglobulin g in patients with diabetic nephropathy. diabet res clin pract. 2014;106: 522–530. 3. macisaac rj, ekinci ei, jerums g. markers of and risk factors for the development and progression of diabetic kidney disease. am j kidney dis. 2014;63:s39–s62. 4. josipović j, katičić d, pavlović d. diabetic nephropathy: diagnosis, prevention and treatment. medix. 19(107/108), 2013. 5. viberti g, wheeldon nm. microalbuminuria reduction with valsartan (marval) study investigators. microalbuminuria reduction with valsartan in patients with type 2 diabetes mellitus a blood pressure–independent effect. circulation. 2002;106:672–678. 6. xia f, liu g, shi y, zhang y. impact of microalbuminuria on incident coronary heart disease, cardiovascular and all-cause mortality: a meta-analysis of prospective studies. int j clin exp med. 2015;8:1. 7. jafar th, stark pc, schmid ch, landa m, maschio g, de jong pe, et al. progression of chronic kidney disease: the role of blood pressure control, proteinuria, and angiotensin-converting enzyme inhibition: a patient-level meta-analysis. ann intern med. 2003;139:244–252. 8. kim ss, song sh, kim ij, jeon yk, kim bh, kwak is, et al. urinary cystatin c and tubular proteinuria predict progression of diabetic nephropathy. diabetes care. 2013;36:656–661. 9. waheed hj. a comparative study for cystatin c and some biochemical markers for predicting diabetic nephropathy in iraqi patients. int j curr microbiol app sci. 2015;4:108. 10. papadopoulou-marketou n, skevaki c, kosteria i, peppa m, chrousos gp, papassotiriou i, et al. ngal and cystatin c: two possible early markers of diabetic nephropathy in young patients with type 1 diabetes mellitus: one year follow up. hormones (athens). 2015;14:232–40. 11. oh my, lee h, kim js, ryu ws, lee sh, ko sb, et al. cystatin c, a novel indicator of renal function, reflects severity of cerebral microbleeds. bmc neurol. 2014;14:1. 12. fiseha t. urinary biomarkers for early diabetic nephropathy in type 2 diabetic patients. biomar res. 2015;3: p. 1. 13. bhavsar na, appel lj, kusek jw, contreras g, bakris g, coresh j, et al. aask study group, comparison of measured gfr, serum creatinine, cystatin c, and beta-trace protein to predict esrd in african americans with hypertensive ckd. am j kidney dis. 2011;58:886–893. 14. sun ym, su y, li j, wang lf. recent advances in understanding the biochemical and molecular mechanism of diabetic nephropathy. biochem biophys res commun. 2013;433:359–361. 15. hawazin youssef khalaf. association between micro albuminuria and glycosylated hemoglobin, some oxidative stress biomarkers and atherogenicity in type 2 diabetes women. college of medicine and committed of postgraduate studies at al mustansiriya university. 2015. 16. al-tu’ma fj, dheyauldeen mh, al-saegh rm. measurement of urinary kidney injury molecule-1 as a predictive biomarker of contrast-induced acute kidney injury. jcms. 2017;3:178–181. 17. wang c, li j, xue h, li y, huang j, mai j, et al. type 2 diabetes mellitus incidence in chinese: contributions of overweight and obesity. diabet res clin prac. 2015; 107:424–432. 18. gray n, picone g, sloan f, yashkin a. relation between bmi and diabetes mellitus and its complications among us older adults. southern med j. 2015;108:29–36. 19. pavkov me, knowler wc, hanson rl, williams de, lemley kv, myers bd, et al. comparison of serum cystatin c, serum creatinine, measured gfr, and estimated gfr to assess the risk of kidney failure in american indians with diabetic nephropathy. am j kidney dis. 2013;62:33–41. 20. sit d, basturk t, yildirim s, karagoz f, bozkurt n, gunes a. evaluation of the serum cystatin c values in prediction of indications for hemodialysis in patients with chronic renal failure. int urol nephrol. 2014;46: 57–62. 21. gompou a, perrea d, karatzas t, bellos jk, kastania an, boletis i, et al. relationship of changes in cystatin-c with serum creatinine and estimated glomerular filtration rate in kidney transplantation. transplant proc. 2015;47:1662–1674. 22. wang t, wang q, wang z, xiao z, liu l. diagnostic value of the combined measurement of serum hcy, serum cys c, and urinary microalbumin in type 2 diabetes mellitus with early complicating diabetic nephropathy. isrn endocrinol. 2013. 23. suzuki y, matsushita k, seimiya m, yoshida t, sawabe y, ogawa m, et al. serum cystatin c as a marker for early detection of chronic kidney disease and grade 2 nephropathy in japanese patients with type 2 diabetes. clin chem laborat med. 2012;50:1833–1839. 24. sur a. cystatin c, a better predictor of renal impairment in essential hypertensive patients. hypertension. 2015;120:80–89. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201702 222 j contemp med sci | vol. 4, no. 4, autumn 2018: 222–226 the atheroprotective effects of genistein in hypercholestrolemic male rabbit adnan w al mudhafar,a najah r hadi,b rafid m.a wasfi,c and zaid m mohammeda* adepartment of food science, college of agriculture, kufa university, iraq. b department of pharmacology and therapeutics, college of medicine, kufa university, iraq. c department of clinical pharmacy, college of pharmacy, kufa university, iraq. *correspondence to zaid m mohammed (email: zaidmahdi13@yahoo.com). (submitted: 10 september 2018 – revised version received: 18 september 2018 – accepted: – published online: objective the occurrence of this disease is related to different risk factors such as cardiovascular issues and elevated levels of plasma cholesterol, hypertension, diabetes mellitus and many others. methods three groups of domestic male rabbits, six in each group, were studied. each group constituted a different diet condition where group i had a normal chow diet; group ii a 1% cholesterol-diet, group iii a 1% cholesterol-diet and genistein. the level of serum total cholesterol (tc), triglycerides (tg) and high density lipoprotein hdl-c serum interleukin-6 (il-6), serum high sensitive c-reactive protein (hs-crp), serum monocyte chemo-attractant protein type 1 high mobility group box 1 (mcp-1) and hmg-box1 was monitored by collecting blood samples at the start of the study, 28 and 56 days. then, the aorta was removed to be examined (histopathology) for atherosclerosis lesion and thickening in the aortic intima-media. results in comparison to the control group (i), levels of tc, tg, low-density lipoprotein (ldl) cholestrol, very ldl cholestrol, hs-crp, il-6, mcp-1 and hmg-box1 had increased while serum hdl-c had reduced in the animals that followed a high-fat diet. histologically, the aortic intimal thickening and atherosclerosis lesions has increased in the induced-untreated animals. the genistein treated group showed a substantial decrease of lipid parameters in comparison with the induced-untreated group. genistein counteracted the changes in hs-crp, il-6, mcp-1 and hmg-box1 in compared with the induced-untreated group (p < 0.05). histomorphometric measurements indicated that genistein significantly minimizes the thickness of the aortic intima-media and atherosclerosis lesions in comparison to the animals on a high-fat diet. conclusion the outcomes of this investigation show that genistein significantly decrease the progression of atherosclerosis in hypercholesterolemic animals via inhibition of inflammatory markers and reduced levels of lipid parameters. keywords genistein, atherosclerosis, inflammation introduction atherosclerosis is a serious, chronic inflammatory disease where arteries become clogged with lipids (plaques or atheroma).1 it begins with the accumulation of fatty substances in the arteries, this leading to the hardening and narrowing of said arteries.2 the initial step in the pathogenesis of atherosclerosis is the lipid retention followed by chronic inflammation at sensitive sites in the major walls of arteries, this resulting in fatty streaks, which then develop into fibroatheromas.3,4 endothelial dysfunction indicates the start of this process. a variety of mediators induce endothelial dysfunction, some of which are currently unknown, others known and related to established cardiovascular risk factors such as dyslipidemia, smoking, diabetes mellitus, aging and hypertension.5 other risk factors also contribute, for example lipids in the blood, low-density lipoprotein (ldl) and very ldl (vldl) bind to endothelial cells and oxidize in the subendothelial space. t cells and monocytes bind to the endothelial cells and migrate to the subendothelial space, where monocytes engulf the oxidized ldl and transformed into foam cells. this process represents the first stage after which macrophages further elaborate proinflammatory cytokines that recruit smooth muscle cells, this producing an increase in smooth muscle cells and replication in dense extracellular matrix. the resulting atherosclerosis lesion is a subendothelial fibrous plaque composed of a lipid core surrounded by connective tissue fibers and smooth muscle cells.6 normal adaptive vascular responses, such as the release of endothelium-derived no, are subsequently impaired. endothelial activation results in inflammatory cells and the adhesion of platelets that can release proatherogenic growth factors. these factors result in the migration and proliferation of vascular smooth muscle cells (vsmcs) and the increased generation of extracellular matrix. lymphocytes and monocytes invade the vessel walls, contributing to the processes of inflammation that results in atherosclerosis lesion formation. forms of (ldl) cholesterol that accumulate in the vessel walls, may be taken up by macrophages and other vascular cells, resulting in foam cell formation.7,8 inflammation plays an important role in plaque initiation, disruption and progression. levels of c-reactive protein (crp), an acute phase protein released from the liver in response to infection or inflammation, is a predictor of cardiovascular events.9,10 various forms of vascular injury, including elevated levels of modified (ldl), stimulate proinflammatory effects resulting in the recruitment of t lymphocytes and monocytes, the earliest feature of atherosclerosis lesion formation.11 this infiltration by inflammatory cells is mediated by various chemokines including interleukins (il) and monocyte chemo-attractant protein type 1 (mcp-1).12 these monocytes change into macrophages and up-regulate their scavenger receptor expression, allowing them to accumulate lipids which results in the formation of fatty streaks and foam cells. these activated macrophages release growth factors and numerous cytokines including tumor necrosis factor-a, interleukins (il, especially il-1b, il-6 and il-8), and mcp-1. these factors, which can stimulate adhesion molecule expression, macrophage issn 2413-0516 26 december 2018)06 december 2018 research a.w. al mudhafar et al. 223j contemp med sci | vol. 4, no. 4, autumn 2018: 222–226 the atheroprotective effects of genistein in hypercholestrolemic male rabbit activation, leukocyte migration, vsmc proliferation and vascular permeability, are normally expressed in the vessel wall, but their concentrations are significantly higher in atherosclerosis plaque.13 a wide body of literature showed that genistein, which is an isoflavone phytoestrogen, has a central role in the regulation of different biological activities, acting as an inhibitor for tyrosine kinase proteins.14 with this in mind, many researchers are investigating its potential action in the treatment of diseases such as the cancer, and its effect on skeletal and cardiovascular health. these investigations have focused on the potential hypolipidemic, anti-inflammatory, antioxidative and estrogenic effects of the genistein.15 materials and methods three groups of domestic male rabbits, six in each group, were studied, each under different conditions. group i was the control group kept on a normal chow diet; group ii was given a 1% of cholesterol-diet, group iii a 1% cholesterol-diet and genistein. levels of serum total cholesterol (tc), triglycerides (tg) and hdl-c serum il-6, serum high sensitive crp (hs-crp), serum mcp-1 and serum hmg-box1 was monitored by collecting blood samples at the start of the study, 28 and 56 days. at the end of the experiment, the aorta was removed to be examined (histopathology) for atherosclerotic changes or changes in the thickness of aortic intima-media. blood sampling the sampling process involved taking 5 ml of blood from the central ear artery of the rabbits following overnight fasting. blood sampling was carried out at zero time, 28 and 56 days. the samples of blood were permitted to clot at 37°c, then cenotrifuged for 12 min at 6000 rpm. the isolated serum was analyzed to measure serums tc, tg, hdl-c, ldl-c, vldl-c, il-6 and crp, hmg-box1, mcp-1. tissue sampling for histopathology at the end of the experiment (56 days), the rabbits were euthanized using chloroform. their chest walls were dissected for resection of the aorta. the removed connective tissue samples, after removal of adherent fat, were immediately fixed in a 10% xylene solution. following fixation tissue sampling, the samples were processed in the usual method. samples were observed with a microscope under a magnification power of 10× and 40×, the histopathological alterations determined. atherosclecrosis lesions were categorized into different phases as outlined by the american heart association classifications.16 results influence of genistein on serum lipid profiles after 28 days, the results revealed a substantial increase in the lipid profile of the rabbits that followed a high cholesterol diet. over the same period, the genistein-treated rabbits showed a substantial decrease in serum lipids in comparison to the untreated rabbits (table 1). influence of genistein on hs-crp, il-6 and mcp1 according to the statistical analyses, at the beginning of the study, the groups of rabbits showed non-significant differences in levels of serum hs-crp, il-6 and mcp-1. however, substantial differences (p < 0.05) in levels of hs-crp, il-6 and mcp-1 were recorded after 28 days where a significant increase was noticed in the high cholesterol diet animals. after 56 days, the genistein-treated rabbits showed a substantial decrease (p < 0.05) in the levels of hs-crp, il-6 and mcp-1 (table 2). influence of genistein on hmg-box1 prior to the experiment, the levels of serum hmg-box1 in the rabbits, were statistically analyzed, the results showing non-significant differences. after 28 days, the high cholesterol diet group showed a substantial increase in hmg-box1 levels. after 56 days, the genistein-treated rabbits showed a clear increase in the level of hmg-box1 (table 3). influence of genistein on aortic intima-media thickness after 56 days, the high cholesterol diet rabbits showed a clear increase in the thickness of the aortic intima-media, as shown in figs. 1 and 2. the genistein-treated rabbits had a lower thickening in the aortic intima-media (figure 3 and table 4). table 1. influence of cholesterol-enriched diet and genistein 10 mg/kg/day on serum lipid profile. values are represented as means ± sem (six rabbits in each group) tc (mg/dl) tg (mg/dl) hdl (mg/dl) normal control zero time 48.8 ± 3.0 49.5 ± 2.1 21.0 ± 1.7 28 days 47.0 ± 3.8 50.8 ± 3.4 20.1 ± 1.5 8 weeks 46.5 ± 2.4 44.3 ± 0.8 21.5 ± 0.8 induced-untreated zero time 46.3 ± 3.7 57.3 ± 1.8 20.2 ± 1.4 28 days 625.0 ± 12.1* 123.7 ± 17.8* 13.9 ± 0.5* 8 weeks 859.0 ± 73.8† 172.0 ± 6.7† 12.0 ± 0.2† genistein 10 mg/kg zero time 51.0 ± 2.7 51.5 ± 1.8 21.8 ± 1.1 28 days 663.8 ± 17.8* 138.5 ± 17.0* 12.3 ± 0.4* 8 weeks 271.8 ± 50.6† 85.8 ± 12.3† 17.1 ± 0.9† *means at 28 days significant to means at 0 days; †means at 56 days means to averages at 28 days. research 224 j contemp med sci | vol. 4, no. 4, autumn 2018: 222–226 the atheroprotective effects of genistein in hypercholestrolemic male rabbit a.w. al mudhafar et al. table 2. influence of cholesterol-enriched diet and genistein 10 mg/kg/day on serum inflammatory marker (hs-crp, il-6, mcp-1). values are represented as means ± sem (six rabbits in each group) hs-crp mg/l il-6 pg/l mcp-1 normal control zero time 3.0 ± 0.2 1.3 ± 0.1 0.7 ± 0.1 28 days 3.4 ± 0.1 1.3 ± 0.1 1.0 ± 0.1 8 weeks 3.3 ± 0.2 1.2 ± 0.2 0.7 ± 0.1 induced-untreated zero time 3.0 ± 0.2 1.5 ± 0.3 1.0 ± 0.2 28 days 5.5 ± 0.2* 4.7 ± 0.3* 4.2 ± 0.3* 8 weeks 7.5 ± 0.2† 6.6 ± 0.3† 5.6 ± 0.2† genistein 10 mg/kg zero time 3.1 ± 0.1 1.3 ± 0.3 1.2 ± 0.2 28 days 6.1 ± 0.3* 4.8 ± 0.3* 4.6 ± 0.2* 8 weeks 5.3 ± 0.3† 2.5 ± 0.4† 1.6 ± 0.1† *means at 28 days significant to means at 0 days, †means at 56 days significant to means at 28 days. table 3. influence of cholesterol-enriched diet and genistein 10 mg/kg/day on serum inflammatory marker (hmg-box1). values are represented as means ± sem (six rabbits in each group) hmg-box1 normal control zero time 0.7 ± 0.03 28 days 0.8 ± 0.1 8 weeks 0.7 ± 0.2 induced-untreated zero time 0.8 ± 0.1 28 days 2.9 ± 0.04* 8 weeks 4.1 ± 0.09† genistein 10 mg/kg zero time 0.7 ± 0.6 28 days 3.8 ± 0.08* 8 weeks 1.9 ± 0.4† *means at 28 days significant to means at 0 days; †means at 56 days significant to means at 28 days. fig. 1 photomicrograph of histophotometric section in aortic arch of rabbit fed on normal diet for 8 weeks (normal control) show the normal intimal thickness and intact continuous endothelium. stained with haematoxylin and eosin (10×), where, i: intima of the aorta, m: media of the aorta, a: adventitia of the aorta and l: the lumen of the aorta. fig. 2 photomicrograph of histomorphometric section in aortic arch of rabbits on hypercholeterolemic diet for 8 weeks (induceduntreated) show diffuse intimal thickening and in completely coalesced extracellular lipid underneath a layers of macrophages and smooth muscle cells. the section stained with hematoxylin and eosin (10×), where, i: intima of the aorta, m: media of the aorta, a: adventitia of the aorta and l: the lumen of the aorta. fig. 3 photomicrograph of histomorphometric section in aortic arch of genistein hyperlipidemic rabbits. the section stained with hematoxylin and eosin (10×), where, i: intima of the aorta, m: media of the aorta, a: adventitia of the aorta and l: the lumen of the aorta. discussion the outcome of the current study reveals a direct relationship between a cholesterol-enriched diet and serum tc, tg, ldl-c, vldl levels, where a clear increase has been noted in these serums after 56 days. levels of hdl-c decreased significantly. similar results were found by prasad and lee17 and nigrisa and paolo.18 additionally, the outcomes of this investigation indicate a direct relationship between genistein and the levels of serums tc, tg, hdl-c, ldl-c, vldl-c, which agrees with the results research a.w. al mudhafar et al. 225j contemp med sci | vol. 4, no. 4, autumn 2018: 222–226 the atheroprotective effects of genistein in hypercholestrolemic male rabbit table 4. thickening values in aortic intima-media (px) after 56 days. values are represented as means ± sem (six rabbits in each group) group aortic intimal thickness (µm) normal control 36.5 ± 2.8 dietary induced-untreated 454.6 ± 38.3* genistein group 135.0 ± 11.0† *means for induced-untreated animals significant to control group. †means for genistein-treated animals significant to induced-untreated group. of eftekhari et al.19 and tang et al.20 these results demonstrate that cholesterol levels lower when increasing ldl receptor activity, this increasing the absorption of ldl from the blood into the liver. genistein reduces the activity of enzymes involved in fatty acid synthesis, such as fatty acid synthase, resulting in decreased serum triglyceride and vldl. in terms of levels of hs-crp, the results here agree with the results of zhao and wu,21 in that a remarkable increase was noted in levels of hs-crp in comparison to rabbits on a normal diet. however, genistein additives substantially decrease the hs-crp plasma level in comparison to normal animals, these findings in line with kim and lim.22 cholesterol-enriched diet caused significant increased levels of mcp-1 in comparison to control group, this in agreement with chen et al.23 who showed that mcp-1 expression increased after a 3-week, high cholesterol diet in comparison to a control group. it has been proved that the mcp-1 plays an essential role in atherosclerosis disease, mcp-1 is expressed mainly through endothelial and inflammatory cells. the expression level is upregulated after tissue injury and proinflammatory stimuli, both correlating with atherosclerotic disease.24 the generation of mcp-1 substantially decreased in the genistein treated animals, in comparison with the induced-untreated rabbits, this also in agreement with kim and lim22 who showed that a comparatively low amount of genistein might decrease mcp-1 through suppression of nf-κb activation. in the current investigation, a clear increase was noted in levels of il-6 in the hypercholesterolemic rabbits. this could be attributed to the fact that hypercholesterolaemia causes endothelial microinflammation, which increases the proinflammatory cytokine il-6. these results are in agreement with those of uich et al.25 and elwakkad et al.26 in the current study, genistein treatment caused a significant reduction of il-6 levels, this supported by ibrahim et al.27 and palanisamy et al.28 these collective results demonstrate that genistein caused a substantial reduction in serum il-6 in nonalcoholic steatohepatitis rats induced by a high fat diet (hfd). genistein presents anti-inflammatory activity by reducing ikb-a phosphorylation in a nuclear factor kappa-light-chainenhancer of activated b cells (nf-κb) and inhibitor of kappa light polypeptide gene enhancer in b-cells (ikkb). in these studies, it was found that aortic intimal thickening and atherosclerosis area in hypercholesterolemic animals was substantially decreased by treatment with genistein. comparable outcomes were found by wang et al.29 conclusion the findings of this investigation demonstrated that genistein significantly decreased atherosclerosis progression in hypercholesterolemic rabbits via inhibition of inflammatory markers, reduced levels of lipid parameters. conflict of interest none. n references 1. libby p, ridker pm, hansson gk. progress and challenges in translating the biology of atherosclerosis. nature. 2011;473:317–325. 2. alexander rw, dzau vj. vascular biology: the past 50 years. circulation. 2000;102:iv112–iv116. 3. banach m, serban c, sahebkar a, mikhailidis dp, ursoniu s, ray kk et al. impact of statin therapy on coronary plaque composition: a systematic review and meta-analysis of virtual histology intravascular ultrasound studies. bmc med. 2015;13:229. 4. collins r, armitage j, parish s, sleigh p, peto r, hear t protection study collaborative group. mrc/bhf hear t protection study of cholesterol-lowering with simvastatin in 5963 people with diabetes: a randomised placebo-controlled trial. lancet. 2003 jun 14;361:2005–2016. 5. steffel j, lüscher tf. predicting the development of atherosclerosis. circulation. 2009;119:919–921. 6. aziz m, yadav ks. pathogenesis of atherosclerosis. med clin rev. 2016; 2:22. 7. taketomi y, murakami m. phospholipase a2 as a potential drug target for airway disorders. obstructive airway diseases: role of lipid mediators. 2016; apr 19:41. 8. corrado e, rizzo m, coppola g, fattouch k, novo g, marturana i, et al. an update on the role of markers of inflammation in atherosclerosis. j atheroscler thromb. 2010;17:1–11. 9. shafi dar m, pandith aa, sameer as, sultan m, yousuf a, mudassar s. hs-crp: a potential marker for hypertension in kashmiri population. indian j clin biochem. 2010;25:208–212. 10. rutter mk, meigs jb, sullivan lm, d’agostino rb, wilson pw. c-reactive protein, the metabolic syndrome, and prediction of cardiovascular events in the framingham offspring study. circulation. 2004;110:380–385. 11. munro jm, cotran rs. the pathogenesis of atherosclerosis: atherogenesis and inflammation. lab invest. 1988;58:249–261. 12. gerszten re, garcia-zepeda ea, lim yc, yoshida m, ding ha, gimbrone ma et al. mcp-1 and il-8 trigger firm 184 adhesion of monocytes to vascular endothelium under flow conditions. nature. 1999;398: 718–723. 13. dewberry r, holden h, crossman d, francis s. interleukin-1 receptor antagonist expression in human endothelial cells and atherosclerosis. arterioscler thromb vasc biol. 2000;20:2394–2400. 14. garcía dc, valdecantos pa, miceli dc, roldán-olarte m. genistein affects proliferation and migration of bovine oviductal epithelial cells. res vet sci. 2017;114:59–63. 15. incir s, bolayirli im, inan o, aydın ms, bilgin ia, sayan i, et al. the effects of genistein supplementation on fructose induced insulin resistance, oxidative stress and inflammation. life sci. 2016;158:57–62. 16. stary hc. natural history and histological classification of atherosclerotic lesions: an update. arterioscler thromb vasc biol. 2000;20:1177–1178. 17. prasad k, lee p. suppression of hypercholesterolemic atherosclerosis by pentoxifylline and its mechanism. atherosclerosis. 2007;192:313–322. 18. de nigris f, mancini fp, balestrieri ml, byrns r, fiorito c, williams-ignarro s, et al. therapeutic dose of nebivolol, a nitric oxide-releasing betablocker, reduce atherosclerosis in cholesterol-fed rabbits. nitric oxide. 2008;19:57–63. 19. eftekhari mh, honarvar nm, rajaeefard a, owji a. effect of daidzein and genistein on serum glucose, lipid profile and paroxonase activity in diabetic rats. curr top nutraceutical res. 2014;12:85–90. 20. tang c, zhang k, zhao q, zhang j, effects of dietary genistein on plasma and liver lipids, hepatic gene expression, and plasma metabolic profiles of hamsters with diet-induced hyperlipidemia. j agric food chem. 2015;63:7929–7936. 21. zhao sp, wu zh. atorvastatin reduces serum leptin concentration in hypercholesterolemic rabbits. clin chim acta. 2005;360:133–140. research 226 j contemp med sci | vol. 4, no. 4, autumn 2018: 222–226 the atheroprotective effects of genistein in hypercholestrolemic male rabbit a.w. al mudhafar et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 22. kim mj, lim y. protective effect of short-term genistein supplementation on the early stage in diabetes-induced renal damage. mediators inflamm. 2013;2013:510212. 23. chen yl, chang yj, jiang mj. monocyte chemotactic protein-1 gene and protein expression in atherogenesis of hypercholesterolemic rabbits. atherosclerosis. 1999;143:115–123. 24. lin j, kakkar v, lu x. impact of mcp-1 in atherosclerosis. curr pharm des. 2014;20:4580–4588. 25. ikeda u, ikeda m, seino y, takahashi m, kano s, shimada k. interleukin 6 gene transcripts are expressed in atherosclerosis lesion of genetically hyperlipidemic rabbits. atherosclerosis. 1992;92:213–218. 26. elwakkad as, mohamed si, fathalla m. relation between hypercholesterolaemia and vascular endothelial microinflammation. east mediterr health j. 2007;13:515–521. 27. ibrahim as, el-shishtawy mm, peña a, liou gi. genistein attenuates retinal inflammation associated with diabetes by targeting of microglial activation. mol vis. 2010;16:2033–2042. 28. palanisamy n, kannappan s, anuradha cv. genistein modulates nf-κbassociated renal inflammation, fibrosis and podocyte abnormalities in fructose-fed rats. eur j pharmacol. 2011;667:355–364. 29. wang y, chun ok, song wo. plasma and dietary antioxidant status as cardiovascular disease risk factors: a review of human studies. nutrients. 2013;5:2969–3004. research 388 j contemp med sci | vol. 8, no. 6, november-december 2022: 388–394 original circulating levels of interferon regulatory factor-5 correlates with disease activity in systemic lupus erythematosus iraqi patients zainab m. dahham*, namir i. a. haddad department of chemistry, college of science, university of baghdad, baghdad, iraq. *correspondence to: zainab m. dahham (email: zainab@sc.uobaghdad.edu.iq) (submitted: 11 august 2022 – revised version received: 10 september 2022 – accepted: 17 october 2022 – published online: 26 december 2022) abstract objectives: systemic lupus erythematosus (sle) is a chronic autoimmune disease characterized by a diversity of the phenotypes among the patients. sle is still one of the great challenges due to the lacking of specific biomarkers for diagnosis, assessing disease activity, and prediction of response to therapy. this study aimed to investigate the role of circulating levels of irf5 protein in sample of sle iraqi patients and its correlation with disease activity, to identify a potential immunological biomarker to mirror disease activity. methods: blood samples were taken from 59 participants diagnosed with sle cases classified according to the american college of rheumatology (acr) criteria. they were scored through the sle disease activity index 2000 (sledai-2k) to estimate the disease activity, and according to it they were subdivided into “sle-1 group” (sledai-2k ≤5), and “sle-2 group” (sledai-2k >5), as well as age and gender matched healthy control group. circulating levels of irf5 protein were measured in sera samples by elisa method. results: our result revealed that the circulating levels of irf5 protein were significantly higher in the sle-2 group rather than control group (p < 0.01), while there was a non-significant difference between sle-1 group and control group (p > 0.05), as well as between both sle patient groups. moreover, the circulating irf5 protein levels were found to be correlated positively and significantly with disease activity index in both sle patient groups. the correlation between the circulating levels of irf5 protein with other parameters revealed that a significant positive correlation was found in sle-1 group with esr and globulins, and negative correlation with hb and (albumin/globulin) ratio, while in sle-2 group were positively correlated with urea, creatinine, and uric acid. the analysis of receiver operator curves (roc) for circulating levels of irf5 protein in sle-1 and sle-2 groups showed a good accuracy to distinguish sle patients from healthy individuals (auc = 0.758, sensitivity = 65.5%, and specificity = 69%,), and (auc = 0.788, sensitivity = 77.3%, and specificity = 72.0%,), respectively. conclusion: the circulating levels of irf5 protein correlate with disease activity in sle patients reflects the possibility of using it as a potential immunological biomarker for diagnosis, and monitoring the disease activity. keywords: irf5 protein, lupus erythematosus, systemic, iraqi patients issn 2413-0516 introduction systemic lupus erythematosus (sle) is a chronic multi-system autoimmune disease. it is characterized by the production of excessive auto-antibodies due to break of immune system tolerance to self-antigen.1-3 this subsequently leads to formation of circulating immune complexes, and immunologically mediated tissues injury.4-6 sle is a heterogenous disease with a wide range of clinical manifestations and immunological disorders. although the etiology and pathogenesis of sle are still obscure, several lines of evidence documented that immune disorders may be caused by genetic susceptibility and/or environmental factors.7-9 the incidence of sle primarily afflicts women in the reproductive years rather than men, with female to male ratio of approximately 9:1.10,11 the diversity in the clinical manifestations among the patients is a great obstacle and might reflect the differences in underlying pathogenesis. sle is still one of the great challenging for physicians and investigators. for more accurate diagnosis it is necessary to find a new reliable and specific biomarker for sle.12-14 interferon regulatory factor-5 (irf5) is a member of the irf family of the transcription factors. it encodes a 60-63-kda polypeptides, acts as a regulator of the production of numerous pro-inflammatory cytokines, including type i interferon ifns (alpha and beta), il-12, il-6, il-23, and tumor necrosis factor alpha (tnf-α).15,16 it has been shown that the immune response against viral, fungal, and bacterial infections is regulated by irf5.17 several recent studies revealed important roles of irf5 in innate and adaptive immunity, cell growth regulation, apoptosis, and macrophage polarization.18 many genome-wide association studies document that there is a robust correlation between irf5 snps and sle, and that irf5 high-risk variants play an important role in sle pathogenesis.19 numerous studies reported that irf5 risk variants generally correlated with elevated irf5 expression levels in sle blood cells, and with interferon-alpha activity in sle patients.20 to date, there are few studies about endogenous and extracellular irf5 protein. wang and his colleagues (2018), they determined the level of irf5 protein in wbcs of community-acquired pneumonia (cap) patients and healthy donors using flowcytometry techniques.21 idborg et al. (2019), who confirm the presence of extracellular irf5 protein in circulation. they measured the concentration of it in plasma of sle patients and age matched healthy controls. the techniques that used are immunoprecipitation followed by mass spectrometry (ip-ms) and elisa method.22 this study aimed to determine the concentration of circulating irf5 protein in sera of sample of iraqi sle patients, and examine its correlation with disease activity to identify its predictive value using roc analysis for sle diagnosis as a potential immunological biomarker reflects disease severity. materials and methods subjects a total of 59 patients diagnosed with sle disease (56 females and 3 males) were recruited from the rheumatology unit of baghdad teaching hospital between november 2020 and february 2021. the sle patients had met the 1997 american mailto:zainab@sc.uobaghdad.edu.iq 389j contemp med sci | vol. 8, no. 6, november-december 2022: 388–394 z.m. dahham et al. original circulating levels of interferon regulatory factor-5 correlates with disease activity in systemic lupus college rheumatology (acr) revised criteria for the classification of sle.23 patients with other autoimmune disease, malignant disease, kidney disease, liver disease, alcohol intake, cigarette smoking, and other acute infections were excluded. full history was taking, clinical examination and all the required routine laboratory tests were performed for all patients in order to assessment the disease activity score using sle disease activity index 2000 (sledai-2k) score,24 and according to it they were subdivided into “sle-1 group” (sledai-2k ≤5) and “sle-2 group” (sledai-2k >5). a total of 29 age matched healthy controls were also recruited with no evidence of any chronic medical illness. samples collections venous blood specimens of 5 ml were drawn after overnight fasting from each participant, then the blood was immediately divided into two portions. the first one (2 ml) was transferred into tube containing k3edta, and they were stirred gently for a few seconds to avoid blood clotting and they were used for hematological parameters determination. the reminder blood was transferred into a glass tube with a gel separator. the blood samples were allowed to clot for 10 minutes at 37oc in a water bath, then they were centrifuged at 3000 × g for 10 minutes. the obtained clear serum was dispensed in several aliquots, and stored frozen at –20oc until being used to estimate the different parameters included in the study. hemolyzed sera were excluded. determination of circulating levels of irf5 protein irf5 protein levels were estimated in sera samples using human ifr5 (interferon regulatory factor5) elisa kit was supplied by (my biosource company, usa) according to manufacturer’s protocol. laboratory testing complete blood count (cbc) was done by abbott hematology auto-analyzer (cell-dyn-ruby, usa). erythrocyte sedimentation rate (esr) was determined by the westergren method. general urine examination (gue) was determined by routine techniques. protein urea was quantified by 24-h urine collections. anti-nuclear antibodies (ana) and ds-dna antibodies (ds-dna) were determined using elisa method by naissa immuno auto-analyzer (neomedica, europe). while c3 and c4 concentrations were determined using turbidimetry method by hipro immuno auto-analyzer (hipro, china). other biochemical tests were performed by colorimetric methods according to the manufacturer’s instructions using commercial kits. statistical analysis the results were presented as mean ± standard deviation (mean ± sd). the differences among the studied groups were calculated by applying analysis of variance one-way (anova), and followed by post hoc tukey analysis to test the differences between every two groups within anova. the degree of correlation between parameters was calculated by pearson’s correlation test. the percentage of significance was obtained by r and p values. the p-value is considered significant if it is < 0.05, and highly significant if it ≤0.01. receiver operator characteristics curve (roc) analysis was constructed for circulating levels of irf5 protein to estimate its diagnostic yield for sle disease, the area under the curve were considered exceptional (1–0.9), excellent (0.9–0.81), good (0.8–0.71), fair (0.7–0.61), and poor (0.6–0.5). the statistical analyses were performed using software statistical package for social science (spss) version 26.0 (im spss, chicago, il, usa) and graphpad prism, version. 9.3.1 (san diego, california, usa). results the demographic data, clinical and immunological characteristics of the 88 participants, 59 patients diagnosed as sle according to the american college rheumatology (acr) criteria, and 29 healthy controls are described in table 1. the treatment with medication that used at time of enrollment were also mentioned. it is clear from the results in table 1 that among the 59 sle patients, 95% (m:f = 3:56) are females. as for organ involvement, 45 patients (76.2%) had immunological disorders, and 31 patients (52.5%) had hematological disorders. the next common manifestations were arthritis in 25 patients (42.3%) and oral ulcers in 22 patients (37%), followed by renal disorders in 16 patients (27.1%). as for medications, most of the sle patients under therapy, 48 patients (81.3%) with prednisolone treatment, 43 patients (72.8%) with hydroxychloroquine treatment, and 15 patients (25.4%) with azathioprine treatment. there were no statistical differences in age, gender, and bmi among the three studied groups (sle-1, sle-2, and control), as well as in disease duration between the sle patient groups (p > 0.05). however, there is a significant difference in sledai–2k score between the sle patient groups (p < 0.05). the number of sle patients with family history in sle-1 group and sle-2 group are 4 (14%) and 8 (27%), respectively. on the other hand, all controls with no family history. to investigate the role of circulating levels of irf5 protein in sle pathogenesis, sera samples of sle patients and controls were analyzed. the results showed that circulating levels of irf5 protein were significantly increased in sle-2 group with a mean of 1.75 ± 0.65 ng/ml as compared with control group with a mean of 1.24 ± 0.19 ng/ml (p < 0.01). upon comparison between sle-1 group and control group the results show a slight increase of circulating levels of irf5 protein in sle-1 group with a mean of 1.48 ± 0.65 ng/ml than control group. but this increase was statistically non-significant, as well as non-significant difference was observed between sle patient groups as shown in figure 1. moreover, the circulating levels of irf5 protein in sle-1 and sle-2 groups were significantly and positively correlated with disease activity index (sledai-2k), (r = 0.441, p = 0.017) and (r = 0.502, p = 0.005), respectively as shown in (figure 2a and 2b). additionally, we found a significant positive correlation between circulating levels of irf5 protein and relative expression of irf5 mrna levels in both sle-1 and sle-2 groups, (r = 0.887, p < 0.0001) and (r = 0.847, p < 0.0001), respectively. data not shown as it is another part of our project and it is under publication elsewhere.25 the correlation of circulating levels of irf5 protein with other parameters in both sle patient groups were summarized in table 2. circulating levels of irf5 protein in sle-1 group were positively and significantly correlated with esr (r = 0.452, p = 0.014) and globulins (r = 0.463, p = 0.011), and negative correlation with hb (r = –0.459, p = 0.012) and (albumin/globulin) ratio (r = –0.484, 390 j contemp med sci | vol. 8, no. 6, november-december 2022: 388–394 circulating levels of interferon regulatory factor-5 correlates with disease activity in systemic lupus original z.m. dahham et al. table 1. baseline characteristics of sle patients and healthy controls characteristic total sle patients sle-1 group sle-2 group control group p-value demographic data samples number 59 29 30 29 age (year), mean ± sd (range) 34.59 ± 10.96 (14–55) 34.53 ± 10.8 (18–55) 34.66 ± 11.2 (14–53) 33.03 ± 9.6 (18–52) 0.81 bmi (kg/m2), mean ± sd 27.17 ± 6.1 26.06 ± 6.3 28.32 ± 5.5 26.35 ± 5.6 0.285 gender female, n (%) male, n (%) 56 (95%) 3 (5%) 27 (93%) 2 (7%) 29 (97%) 1 (3%) 26 (90%) 3 (10%) 0.574 disease duration (year), mean (range) 4.85 (0.1–33) 4.64 (0.3–23) 5.04 (0.1–33) 0.956 family history with sle 12 (20%) 47 (80%) 4 (14%) 25 (86%) 8 (27%) 22 (73%) -yes, n (%) no, n (%) clinical and immunological manifestations vasculitis, n (%) 2 (3.3%) 0 (0%) 2 (6.6%) arthritis, n (%) 25 (42.3%) 10 (34.5%) 15 (50%) myositis, n (%) 0 (0%) 0 (0%) 0 (0%) pleurisy, n (%) 3 (5%) 0 (0%) 3 (10%) proteinuria, n (%) 12 (20.3%) 0 (0%) 12 (40%) hematuria, n (%) 2 (3.3%) 0 (0%) 2 (6.6%) urinary casts, n (%) 2 (3.3%) 0 (0%) 2 (6.6%) oral ulcers n (%) 22 (37.2%) 10 (0%) 12 (43.3%) alopecia, n (%) 11 (18.6%) 4 (13.8%) 7 (33.3%) fever, n (%) 10 (16.9%) 3 (10.3%) 7 (33.3%) new rash, n (%) 11 (18.6%) 5 (17.2%) 6 (36.6%) thrombocytopenia, n (%) 12 (20.3%) 3 (10.3%) 9 (30%) leucopenia, n (%) 4 (6.7%) 3 (10.3%) 1 (3.3%) anemia, n (%) 29 (49.1%) 11 (37.9%) 18 (60%) low complement n (%) 18 (30.5%) 3 (10.3%) 15 (50%) (+) ana, n (%) 40 (67.7%) 15 25 (+) ds-dna antibodies, n (%) 36 (61%) 15 (51.7%) 21 (70%) sledai-2k, mean ± sd (range) 7.37 ± 3.94 (2–18) 4.1 ± 0.72 (2–5) 10.53 ± 3.08 (6–18) <0.01 medications prednisolone yes, n (%) 48 (81%) 11 (19%) 22 (76%) 7 (24%) 26 (87%) 4 (13%) treatment no, n (%) hydroxychloroquine yes, n (%) 43 (73%) 16 (27%) 19 (66%) 19 (34%) 24 (80%) 6 (20%) treatment no, n (%) azathioprine yes, n (%) 15 (25%) 44 (75%) 8 (28%) 21 (72%) 7 (23%) 23 (77%) treatment no, n (%) methotrexate yes, n (%) 3 (5%) 56 (95%) 1 (3%) 28 (97%) 2 (7%) 28 (93%) treatment no, n (%) the collected data was analyzed by mean ± sd (mean ± standard deviation), range (minimum-maximum), or number (percentage). p-value was used for the comparison among the three studied groups (sle-1, sle-2, and control). p > 0.05 = non-significant differences, p < 0.05 = significant differences, p ≤ 0.01 = high significant differences. bmi, body mass index; ana, anti-nuclear antibodies; ds-dna antibodies, double strand deoxy nucleic acid antibodies; sledai-2k, systemic lupus erythematosus disease activity index 2000. 391j contemp med sci | vol. 8, no. 6, november-december 2022: 388–394 z.m. dahham et al. original circulating levels of interferon regulatory factor-5 correlates with disease activity in systemic lupus p = 0.008). while circulating levels of irf5 protein in sle-2 group were positively correlated with urea (r = 0.632, p < 0.0001), creatinine (r = 0.751, p < 0.0001), and uric acid (r = 0.595, p = 0.001). in order to estimate the ability of circulating levels of irf5 protein to distinguish the active sle patient from healthy subjects as a diagnostic biomarker. we analyzed it using roc curve analysis. the result showed that the circulating levels of irf5 protein in sle-1 and sle-2 groups had a good ability to discriminate sle patients from healthy persons. by which the auc, sensitivity and specificity were (0.758, 65.5%, 69.0%), and (0.778, 72.4%, 70.0%) respectively, at the cut off value of 1.35 (ng/ml), and 1.365 (ng/ml) respectively, which was the good value of sle correct prediction, as shown in (fig 3a and 3b). discussion this study has been prepared to evaluate the circulating levels of irf5 protein in a sample of sle iraqi patients, and to examine the association with disease activity index and other parameters. moreover, applying the roc curve analysis in fig. 1 circulating levels of irf5 protein among the studied groups. the results were expressed as mean ± sd (mean ± standard deviation), p > 0.05 = statistically non-significant differences, * statistically significant differences at p < 0.05, ** statistically significant differences at p ≤ 0.01. fig. 2 (a and b), the correlation between circulating levels of irf5 protein (ng/ml) and sledai-2k score in sle-1 group and sle-2 group, respectively. r, pearson coefficient. p > 0.05 = statistically non-significant correlation, *statistically significant correlation at p < 0.05, ** statistically significant correlation at p ≤ 0.01. table 2. the correlation between circulating levels of irf5 protein (ng/ml) with other biochemical parameters parameter circulating levels of irf5 protein (ng/ml) sle-1 group n = 29 sle-2 group n = 30 r p-value r p-value demographic data age (year) 0.353 0.06 0.124 0.515 gender (f/m) –0.259 0.175 –0.76 0.689 bmi (kg/m2) 0.218 0.255 0.084 0.659 disease duration (year) 0.098 0.612 -0.001 0.997 family history with sle –0.222 0.309 0.409 0.058 hematological parameters wbc x 103 /ul –0.297 0.118 0.353 0.060 rbc x 106 /ul –0.245 0.201 –0.212 0.261 hb (gm/dl) –0.459 0.012* –0.269 0.151 plt x 103 /ul 0.153 0.429 0.366 0.051 esr (mm/1 hr) 0.452 0.014* 0.106 0.546 biochemical parameters urea (mg/dl) 0.145 0.452 0.632 <0.0001** creatinine (mg/dl) 0.04 0.838 0.751 <0.0001** uric acid (mg/dl) 0.353 0.060 0.595 <0.001** got (u/l) -0.069 0.723 0.352 0.057 gpt (u/l) 0.224 0.242 0.184 0.0331 alp (u/l) 0.366 0.051 -0.51 0.79 total serum protein (g/l) 0.207 0.281 -0.172 0.364 serum albumin (g/l) -0.394 0.063 -0.180 0.340 globulins (g/l) 0.463 0.011* 0.011 0.954 albumin /globulins -0.484 0.008** -0.098 0.608 total cholesterol (mg/dl) -0.052 0.787 0.328 0.077 tri glyceride (mg/dl) 0.065 0.737 0.272 0.145 vldl (mg/dl) 0.063 0.747 0.272 0.145 hdl (mg/dl) -0.214 0.264 0.098 0.607 ldl (mg/dl) -0.029 0.879 0.302 0.105 r, pearson coefficient. p > 0.05 = statistically non-significant correlation, *statistically significant correlation at p < 0.05, **statistically significant correlation at p ≤ 0.01. bmi, body mass index; wbc, white blood cells; rbc, red blood cells; hb, hemoglobin; plt, platelets; esr, erythrocyte sedimentation rate; got, glutamate oxaloacetate transaminase; gpt, glutamate pyruvate transaminase; alp, alkaline phosphatase; vldl, very low-density lipopro tein; hdl, high-density lipoprotein; ldl, low-density lipoprotein. 392 j contemp med sci | vol. 8, no. 6, november-december 2022: 388–394 circulating levels of interferon regulatory factor-5 correlates with disease activity in systemic lupus original z.m. dahham et al. order to predict the ability of it for diagnosis of sle patients as an immunological biomarker. our result revealed that circulating levels of irf5 protein were found to be increased in both sle-1 and sle-2 groups as compared with healthy controls, and correlated positively and significantly with disease activity index. irf5 is a transcription factor plays an important role in inflammatory response. it is likely a key regulator of the toll like receptors (tlrs). in the case of the unstimulated cell, irf5 is generally localized in the cytoplasm as a monomer. activation of the above receptors leads to cascading signals. irf5 undergoes posttranslational modification, which eventually leads to homodimerization, a critical event prior to nuclear translocation.17 the extracellular protein of irf5 still has an unknown function in the circulation and away from complete transcription factors and other nuclear molecules. it can have an unknown function or may regardless of the function.22 the functions of extracellular and intracellular protein might be different and unusual secretion is also probable.26 our finding is in agreement with idbord et al., who found that circulating levels of irf5 protein were significantly higher in sle patients compared with control individuals. they reported that the high level of irf5 protein in plasma samples of sle patients may be reflected the increase of cell death during apoptosis clearance in sle patients.22 in other hands, we cannot only be clarified by this reason because the reports of transcription factor in circulation are limited.27,28 there is no information about the function of extracellular of irf5 protein. however, the fact that irf5 may be present in microparticles, known to mediate cell-cell signaling thus further studies are needed about circulating irf5 protein.22 the expression of irf5 gene is significantly raised in peripheral mononuclear cells (pbmc) from sle patients as compared to age-matched healthy individuals, and this can stimulate the expression of type i interferon.29 the elevated serum levels of ifnalpha have been shown to be associated with the activity and severity of sle disease. these findings support to explain our results about the positive correlation between circulating levels of irf5 protein and disease activity.30 numerous studies in different countries population have showed that the hematological abnormalities are present in most of sle patients. the common hematological syndrome in sle patients is anemia, which is most often owing to the anemia of chronic disease. esr levels were significantly increase in sle patients during sle flare and infections as compared to healthy controls.31-33 many studies indicated that esr considered an important factor in assessing sle disease activity, as esr elevates when disease activity increase. it is believed that the reason for the high rate of erythrocyte sedimentation is the decrease in the concentration of proteins in the blood plasma, as well as the change in the shape of the surface of the erythrocytes and their adhesion to each other.34 these findings may be explaining our results about the negative correlation between hb and circulating irf5 protein, as well as the positive correlation between it and esr level. sle is characterized by raised levels of autoreactive antibodies and gamma globulin. the production of autoantibodies requires the synthesis of gamma globulin and this led to increase the globulin levels in blood sle patients.35,36 our results about positive proteinuria in sle patients as shown in table 1 are in agreement with previous studies that documented the presence of protein in urine in sle patients.37,38 serum albumin was determined in sle patients as a part of routine biochemical tests. the decrease of serum albumin levels in sle patients may be caused by elevating albumin catabolism as a result of chronic inflammation and/or because of poor diet from proteins content and calories. moreover, the common manifestation in sle patients is nephritis which characterized by attack the kidney membranes due to the presence of auto-antibodies against these membranes. consequently, membranes are disrupted and impaired its group auc se p-value 95% ic cut-off value sensitivity specificity ppv npv accuracy sle-1 0.758 0.064 0.001 0.633-0.883 1.350 65.5% 69.0% 67.9% 66.7% 67% sle-2 0.778 0.06 <0.001 0.659-0.896 1.365 72.4% 70.0% 72.4% 70.0% 71.1% fig. 3 (a&b) receiver operator curve (roc) analysis for the predictive value of circulating levels of irf5 protein in sle-1 (n = 29) and sle-2 (n = 30) versus healthy controls (n = 29), respectively. auc = area under the curve, se = standard error, ci = confidence interval, ppv = positive predictive value, npv = negative predictive value. 393j contemp med sci | vol. 8, no. 6, november-december 2022: 388–394 z.m. dahham et al. original circulating levels of interferon regulatory factor-5 correlates with disease activity in systemic lupus filtration ability. in normal condition the filtering membranes do not allow albumin and another blood proteins to be missing in the urine. however, in lupus nephritis the protein loss in urine which in turn lowers serum albumin concentrations,39,40 and this lead to decrease the ratio of albumin/globulin. kwon et al. (2018) found that the (albumin/globulin) ratio in sle with nephritis was lower than in sle without nephritis.36 all above findings supported our results about the positive correlation between circulating level of irf5 protein and globulins, as well as a negative correlation with the ratio of (albumin/ globulin). our results of the positive correlation with urea, creatinine, and uric acid could be explained by the existence of some patients with lupus nephritis as shown in table 1. consequently, the existence of any criterion correlated with kidney disorder for example proteinuria and hematuria lead to raise the score of disease activity. therefore, patients with lupus nephritis graded the high score of disease activity among the patients, subsequently increasing their levels of circulating irf5 protein. roc curve analysis, it showed that circulating levels of irf5 protein in sle-1 and sle-2 groups could represent a good predictor for sle diagnosis. conclusion our study results suggests that the irf5 may play an important role in sle pathogenesis, and irf5 may be useful in diagnosis of sle. the circulating levels of irf5 protein associated with disease flare in sle patients reflect the possibility of using it as a potential biomarkers for diagnosis, monitoring the disease course and response to therapy. moreover, the elisa method is more rapid and inexpensive rather than real time pcr for determination of irf5 gene expression. conflict of interest none.  references 1. la paglia, g.m.c., leone, m.c., lepri, g., vagelli, r., valentini, e., alunno, a. and tani, c., 2017. one year in review 2017: systemic lupus erythematosus. clin exp rheumatol, 35(4), pp. 551–561. 2. idborg, h. and oke, v., 2021. cytokines as biomarkers in systemic lupus erythematosus: value for diagnosis and drug therapy. international journal of molecular sciences, 22(21), p.11327. 3. abdulridha, r.h., saud, a.m. and alosami, m.h., 2022. evaluation of interferon alpha (ifn-α) in women with systemic lupus erythematosus in iraq. iraqi journal of science, pp. 4225–4233. 4. hellquist, a., järvinen, t.m., koskenmies, s., zucchelli, m., orsmark-pietras, c., berglind, l., panelius, j., hasan, t., julkunen, h., d’amato, m.a.u.r.o. and saarialho-kere, u., 2009. evidence for genetic association and interaction between the tyk2 and irf5 genes in systemic lupus erythematosus. the journal of rheumatology, 36(8), pp. 1631–1638. 5. trentin, f., zucchi, d., signorini, v., elefante, e., bortoluzzi, a. and tani, c., 2021. one year in review 2021: systemic lupus erythematosus. clin exp rheumatol, 39(2), pp. 231–241. 6. hiba s. ahmed, hind s. ahmed, ali ad’hiah, 2020. interleukin-1 single nucleotide polymorphisms and risk of systemic lupus erythematosus among iraqi patients. meta gene 23 (100640). 7. quan, w., an, j., li, g., qian, g., jin, m., feng, c., li, s., li, x., xu, y. and hu, x., 2021. th cytokine profile in childhood-onset systemic lupus erythematosus. bmc pediatrics, 21(1), pp. 1–10. 8. katsuyama, t., tsokos, g.c. and moulton, v.r., 2018. aberrant t cell signaling and subsets in systemic lupus erythematosus. frontiers in immunology, 9, p. 1088. 9. jasem, m.a., 2007. hyperprolactenemia in women with systemic lupus erythematusus. baghdad science journal, 4(4). 10. urrego, t., ortiz-reyes, b., vanegas-garcía, a.l., muñoz, c.h., gonzález, l.a., vásquez, g. and gómez-puerta, j.a., 2020. transferrina y ceruloplasmina en orina de pacientes con lupus eritematoso sistemico.? son utiles para diferenciar pacientes con nefritis lupica? reumatología clínica, 16(1), pp. 17–23. 11. tselios, k., gladman, d.d., touma, z., su, j., anderson, n. and urowitz, m.b., 2019. disease course patterns in systemic lupus erythematosus. lupus, 28(1), pp. 114–122. 12. liu, c.c., kao, a.h., manzi, s. and ahearn, j.m., 2013. biomarkers in systemic lupus erythematosus: challenges and prospects for the future. therapeutic advances in musculoskeletal disease, 5(4), pp. 210–233. 13. capecchi, r., puxeddu, i., pratesi, f. and migliorini, p., 2020. new biomarkers in sle: from bench to bedside. rheumatology, 59(supplement_5), pp. v12-v18. 14. rekvig, o.p., 2018. systemic lupus erythematosus: definitions, contexts, conflicts, enigmas. frontiers in immunology, 9, p. 387. 15. mancl, m.e., hu, g., sangster-guity, n., olshalsky, s.l., hoops, k., fitzgeraldbocarsly, p., pitha, p.m., pinder, k. and barnes, b.j., 2005. two discrete promoters regulate the alternatively spliced human interferon regulatory factor-5 isoforms: multiple isoforms with distinct cell type-specific expression, localization, regulation, and function. journal of biological chemistry, 280(22), pp. 21078–21090. 16. cevik, o., li, d., baljinnyam, e., manvar, d., pimenta, e.m., waris, g., barnes, b.j. and kaushik-basu, n., 2017. interferon regulatory factor 5 (irf5) suppresses hepatitis c virus (hcv) replication and hcv-associated hepatocellular carcinoma. journal of biological chemistry, 292(52), pp. 21676–21689. 17. wang, m., lim, k.h. and chow, k.t., 2019. native polyacrylamide gel electrophoresis immunoblot analysis of endogenous irf5 dimerization. jove (journal of visualized experiments), (152), p. e60393. 18. banga, j., srinivasan, d., sun, c.c., thompson, c.d., milletti, f., huang, k.s., hamilton, s., song, s., hoffman, a.f., qin, y.g. and matta, b., 2020. inhibition of irf5 cellular activity with cell-penetrating peptides that target homodimerization. science advances, 6(20), p. eaay1057. 19. ebrahimiyan, h., bagheri-hosseinabadi, z. and abbasifard, m., 2021. interferon regulatory factor 5 in rheumatoid arthritis and systemic lupus erythematosus. rheumatology research, 6(1), pp.1–13. 20. li, d., matta, b., song, s., nelson, v., diggins, k., simpfendorfer, k.r., gregersen, p.k., linsley, p. and barnes, b.j., 2020. irf5 genetic risk variants drive myeloidspecific irf5 hyperactivation and presymptomatic sle. jci insight, 5(2). 21. wang, x., guo, j., wang, y., xiao, y., wang, l. and hua, s., 2018. expression levels of interferon regulatory factor 5 (irf5) and related inflammatory cytokines associated with severity, prognosis, and causative pathogen in patients with community-acquired pneumonia. medical science monitor: international medical journal of experimental and clinical research, 24, p. 3620. 22. idborg, h., zandian, a., ossipova, e., wigren, e., preger, c., mobarrez, f., checa, a., sohrabian, a., pucholt, p., sandling, j.k. and fernandescerqueira, c., 2019. circulating levels of interferon regulatory factor-5 associates with subgroups of systemic lupus erythematosus patients. frontiers in immunology, 10, p. 1029. 23. hochberg, m.c., 1997. updating the american college of rheumatology revised criteria for the classification of systemic lupus erythematosus. arthritis and rheumatism, 40(9), pp. 1725–1725. 24. gladman, d.d., ibanez, d. and urowitz, m.b., 2002. systemic lupus erythematosus disease activity index 2000. the journal of rheumatology, 29(2), pp. 288–291. 25. zainab m. dahham, namir i. a. haddad, correlation between gene expression of interferon regulatory factor-5 and disease activity index in systemic lupus erythematosus iraqi patients. iraqi journal of science (under publication). 26. radisky, d.c., stallings-mann, m., hirai, y. and bissell, m.j., 2009. single proteins might have dual but related functions in intracellular and extracellular microenvironments. nature reviews molecular cell biology, 10(3), pp. 228–234. 27. raghavan, s., manzanillo, p., chan, k., dovey, c. and cox, j.s., 2008. secreted transcription factor controls mycobacterium tuberculosis virulence. nature, 454(7205), pp. 717–721. 28. lomnytska, m., dubrovska, a., hellman, u., volodko, n. and souchelnytskyi, s., 2006. increased expression of cshmt, tbx3 and utrophin in plasma of ovarian and breast cancer patients. international journal of cancer, 118(2), pp. 412–421. 394 j contemp med sci | vol. 8, no. 6, november-december 2022: 388–394 circulating levels of interferon regulatory factor-5 correlates with disease activity in systemic lupus original z.m. dahham et al. 29. feng, d., stone, r.c., eloranta, m.l., sangster‐guity, n., nordmark, g., sigurdsson, s., wang, c., alm, g., syvänen, a.c., rönnblom, l. and barnes, b.j., genetic variants and disease‐associated factors contribute to enhanced interferon regulatory factor 5 expression in blood cells of patients with systemic lupus erythematosus,” arthritis & rheumatism: official journal of the american college of rheumatology, vol. 62, no. 2 pp. 562–573, 2010. 30. jefferies, c.a., 2019. regulating irfs in ifn driven disease. frontiers in immunology, 10, p. 325. 31. sasidharan, p.k., bindya, m. and sajeeth kumar, k.g., 2012. hematological manifestations of sle at initial presentation: is it underestimated? international scholarly research notices, 2012. 32. newman, k., owlia, m.b., el-hemaidi, i. and akhtari, m., 2013. management of immune cytopenias in patients with systemic lupus erythematosus— old and new. autoimmunity reviews, 12(7), pp. 784–791. 33. ismail zoair, m., zaki el-ghannam, m., gaber, a.e.a. and mohammed soliman, a., 2021. assessment the role of urine osteoprotegerin as a biomarker in lupus nephritis. al-azhar medical journal, 50(3), pp. 2201–2212. 34. majeed, w.a., jasim, h.m., farhan, a.a., ali, a.s. and gorial, f.i., 2017. detection of hematological biomarkers associated with lupus nephritis in a sample of iraqi patients. journal of biotechnology research center, 11(2). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1299 35. baum, c.g., chiorazzi, n., frankel, s. and shepherd, g.m., 1989. conversion of systemic lupus erythematosus to common variable hypogammaglobulinemia. the american journal of medicine, 87(4), pp. 449–456. 36. kwon, o.c., lee, j.s., ghang, b., kim, y.g., lee, c.k., yoo, b. and hong, s., 2018, december. predicting eventual development of lupus nephritis at the time of diagnosis of systemic lupus erythematosus. in seminars in arthritis and rheumatism (vol. 48, no. 3, pp. 462–466). wb saunders. 37. nazri, s.k.s., wong, k.k. and hamid, w.z.w., 2018. pediatric systemic lupus erythematosus: retrospective analysis of clinico-laboratory parameters and their association with systemic lupus erythematosus disease activity index score. saudi medical journal, 39(6), p. 627. 38. al-sarray, z.a., al-rayahi, i.a., al-hafidh, a.h. and dwayyikh, a.t., 2020. serum protein electrophoresis in iraqi systemic lupus erythematous patient. al-nisour journal for medical sciences, 2(1). 39. yip, j., aghdassi, e., su, j., lou, w., reich, h., bargman, j., scholey, j., gladman, d.d., urowitz, m.b. and fortin, p.r., 2010. serum albumin as a marker for disease activity in patients with systemic lupus erythematosus. the journal of rheumatology, 37(8), pp. 1667–1672. 40. cojocaru, m., cojocaru, m.i., silosi, i. and vrabie, d.c., 2010. kidney damage in autoimmune diseases. journal of medical biochemistry, 29(2), pp. 61–65. 284 j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 original explaining the conceptual considerations of virtual university in medical education: a systematic review soleiman ahmady1 , amin habibi1* , zohreh koshgoftar2 1department of medical education, virtual school of medical education and management, shahid beheshti university of medical sciences, tehran, iran. *correspondence to: amin habibi (email: amin28@gmail.com) (submitted: 23 july 2022 – revised version received: 02 august 2022 – accepted: 16 august 2022 – published online: 26 october 2022) abstract aim: most of existing literature on the concept of virtual university of medical sciences have been collected in a non-systematic manner. this issue highlights the need to conduct research on virtual university-related topics through systematic research evidence and empirical evaluations. the aim of the present study is to investigate the real complexity and diversity of the concept of virtual university of medical sciences. methods: this was a systematic review with the behemoth approach and aimed to identify, review, analyze and integrate models, theories and frameworks related to the concept of virtual university of medical sciences. eligible articles were searched in pubmed, eric, ieee, isi, scopus, ecampus research unit from 2001 to 2022 using related keywords in three stages. results: a total of 13 articles were finally identified according to inclusion and exclusion criteria. pedagogical, technology, managerial, educational design, technology implementation, and educational-administrative management components were referred to in 5, 5, 4, 7, 6 and 4 articles, respectively. thematic analysis of the models was carried out in two theoretical dimensions (pedagogical component, managerial component and technological component) and operational dimension (educational design, administrative-educational management and technology implementation). conclusion: the results of the present study emphasize the need to pay attention to these six components in the establishment and development of virtual universities of medical sciences. in order to improve the education process in virtual universities, the process quality evaluation framework can be used. keywords: virtual university, medical education, systematic review, models issn 2413-0516 introduction the use of online education as a means of delivering education has grown dramatically over the past decade. online learning environments have become an important part of online training and evidence shows that this trend will continue with the technological advancement. organizations and educational institutions have welcomed this movement and are using the advantages of using computer and communication technology as a tool to transfer learning to learners.1,2 the virtual learning is a broad and multifaceted term that includes different methods of special presentation. although virtual learning has different specific architectures, the physical separation of the teacher and the learner is the basis of all types of this phenomenon. besides, it is assumed in virtual learning that the educational materials are presented using technology to facilitate learning.3 the terms e-learning, distance education, virtual education, online education and other similar descriptions are synonymous and are all used interchangeably in the present research. decades ago, virtual education could be thought of as a video recorded lecture by a teacher that was listened to by students using a videocassette player. today, this term means using a computer and usually the internet to provide this content.5 modern virtual education uses methods such as e-mail, chat rooms, online forums and discussions with the guidance of instructors. it can be stated that the virtual university is a multimedia network learning environment that is different from traditional learning environments in terms of the possibility to personalize and customize the teaching environment.2 harasim et al. state that the virtual university supports the design and delivery of courses and programs for any type of graduate education, which can include the provision of academic degrees, staff training services, professional development and workplace training.6 leidner & jaronpa suggest a similar definition. they state that the mission and vision of any type of virtual learning environment (such as a virtual university) is to redefine the physical boundaries of the classroom; enable more teamwork; transformation of learning into a continuous and time-independent process and the possibility of creating multi-level and multi-speed knowledge using information technology.7 a critical review of virtual university literature showed that there are many different terms related to virtual universities. virtual universities are also known as virtual teaching/learning environments, online teaching/learning, web-based teaching/learning environments, virtual learning communities and flexible learning environments.8 benatallah et al. explain that “the online or virtual university has emerged as a powerful vision for the future of higher education by using new information and communication technologies to fundamentally restructure higher education and re-equip the university institution in the face of new environmental changes.”9 such a scenario generally includes a university without walls, consisting of elements including learners and professors (students and employees), employers (job performance) and graduates, where the entire process of teaching and learning, research is implemented by information technology-based programs.10 despite the validity of the previously mentioned definitions, virtual university is as much related to a method of distance education as it is different from it. the main difference is related to the totality and individuality of its function. this means that the virtual university continues to work and exist https://orcid.org/0000-0003-0551-6068 https://orcid.org/0000-0002-0184-6716 https://orcid.org/0000-0002-8178-2619 mailto:amin28@gmail.com 285j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 s. ahmady et al. original explaining the conceptual considerations of virtual university in medical education without the need for a geographic environment to represent its existence (for example, traditional academic campuses). the existing literature on the virtual university has identified some research issues associated with the transformation of a traditional classroom-based university into a virtual university. however, it is important to note that the current literature does not provide a deep understanding of these issues, but tends to only acknowledge their existence.2 dealing with the advantages and disadvantages of virtual universities, as well as providing guidelines for a shift towards virtual universities, should be one of the most basic priorities of any research in this field. this is important because universities should be aware of the positive and negative aspects of virtual universities before converting from a traditional educational environment to a virtual educational environment. it is also important for instructional designers to be aware of these issues. they need to follow such guidelines in order to reduce the potentially harmful effects and increase the advantages of virtual universities. most of existing literature on the concept of virtual university of medical sciences have been collected in a non-systematic manner. this issue highlights the need to conduct research on virtual university-related topics through systematic research evidence and empirical evaluations.11 the aim of the present study is to investigate the real complexity and diversity of the concept of virtual university of medical sciences. methods this was a systematic review with the behemoth approach and aimed to identify, review, analyze and integrate models, theories and frameworks related to the concept of virtual university of medical sciences. in recent years, the medical science research community has increasingly recognized the potential contribution for educational theories and models in systematic reviews. however, the identification of educational theories and models during a systematic review is generally opportunistic or even incidental in many cases. therefore, there is a need for systematic, formal and predetermined methods in a systematic review to identify sources that identify theories and models in the literature.12 such findings have led to the development of the behemoth framework as a search method. this approach is a process to identify theories, models and frameworks for developing and testing complex interventions using systematic reviews. considering that the goal of researchers is to combine frameworks; models and theories instead of examining interventions, therefore, with such a presumption in mind, the behemoth approach was used in the present study in order to search for theories, models and educational frameworks related to the concept of virtual university. in this approach, four main elements of behavior of interest (be), health context (h), exclusions (e) and models or theories (moth) are determined. then various related theories are identified and the practical application of each theory is identified subsequently.13 to define and guide the final search strategy, the behemoth model was used to systematically identify models, define be, h, e, and moth (table 1). search strategy: in this systematic study, all published and peer-reviewed articles and related theses published table 1. definition of behemoth framework elements in the present study be: behavior of interest virtual university, open university, cyber university h: health context healthcare, medical education e: exclusion no reference to the model, framework and theory no studies in the field of medical sciences articles published before 2001 non-english articles articles published in newspapers, general journals, reports and letters to the editor moth: models or theories: model* or theory* or concept* or framework* between 2001 and 2022 were searched in pubmed, eric, ieee, isi, scopus, ecampus research unit using keywords including (virtual university) or (open university) or (cyber university)) and ((health care) or (medical education))) and ((((theor*) or (model*)) or (concept*)) or (framework*). the search process was guided in three different stages (table 2). inclusion and exclusion criteria the inclusion criteria included peer-reviewed english articles published from 2001 until now that were related to the concept of virtual university of medical sciences. in order to achieve the research objective, the following exclusion criteria were taken into account: no reference to the model, framework and theory no studies in the field of medical sciences, articles published before 2001, non-english articles. articles published in newspapers, general journals, reports and letters to the editor. selection of articles to avoid selection bias, the search was conducted by two researchers independently, and then similar and repeated searches were excluded from the study. at baseline, the titles of the articles that were searched by the research team using the above-mentioned keywords were equal to 64,571 titles. finally, 66 articles were fully reviewed and 13 related articles were included in the final analysis phase after the searching process (figure 1). a checklist was designed based on the study objectives and literature review, which investigates 6 pedagogical, technological, managerial, educational design, technology implementation and educational-management components (table 3). quality assessment to assess the quality of theoretical studies, there are no comparable tools like other types of studies such as the prisma checklist, pico for clinical evidence, or casp and spider for qualitative studies.14 on the other hand, theoretical evidence cannot be evaluated using a variety of tools that have been developed for routine systematic reviews, most of which focus 286 j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 explaining the conceptual considerations of virtual university in medical education original s. ahmady et al. table 2. steps to conducting a systematic search based on the behemoth approach steps search strategy database all articles found number of articles after removing duplicates step 1a be and h ((virtual university) or (open university) or (cyber university)) and ((health care) or (medical education)) 199023 jul 2021 pubmed, eric, ieee, isi, scopus, ecampus research unit sid, magiran 64571 138 step 1b be and h and models, theories, concepts or framework (((virtual university) or (open university) or (cyber university)) and ((health care) or (medical education))) and ((((theor*) or (model*)) or (concept*)) or (framework*)) 20831 53 step 1c be or h and models, theories, concepts or framework (((virtual university) or (open university) or (cyber university)) and ((((theor*) or (model*)) or (concept*)) or (framework*))) or (((health care) or (medical education)) and ((((theor*) or (model*)) or (concept*)) or (framework*))) 123477 168 step 2 step 1(a,b,c) with list of most common theories step 1(a and b and c) with number of lists of most common theories step 1(a and b and c) :359 step 1(a and b and c) :64 number of lists of most common theories:4 step3 step 2 and key original model citation(s) step 2 and key original model citation(s) step 2(68) and key original model citation (5) 73 287j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 s. ahmady et al. original explaining the conceptual considerations of virtual university in medical education ta bl e 3. c he ck lis t d es ig ne d ba se d on st ud y ob je ct iv es th em es ex tr ac te d in th e fra m ew or k bu ild in g a v irt ua l u ni ve rs ity fo r o rt ho pa ed ic s (4 6) a n io m t ba se d cy be r tr ai ni ng fra m ew or k (4 5) vi rt ua l u ni ve rs ity ed uc at io n in th e co nt ex t of th e he al th em er ge nc y du e to co vi d -1 9 (4 4) to w ar ds g rid s er vi ce s fo r a v irt ua l re se ar ch en vi ro nm en t (4 3) th e ev ol ut io n of p ed ag og ic m od el s f or w or kba se d le ar ni ng w ith in a vi rt ua l un iv er sit y (4 2) m et ho do lo gy fo r d es ig n of v irt ua l le ar ni ng en vi ro nm en ts – vi rt ua l u ni ve rs iti es (4 1) a d yn am ic m od el fo r a cy be r ph ys ic al h ea lth ca re (4 0) a s tu dy o f pe da go gi ca l a sp ec ts of a v irt ua l un iv er si ty (3 9) a fr am ew or k pr op os al fo r bl oc kc ha in ba se d sc ie nt ifi c pu bl ish in g u sin g sh ar ed g ov er na nc e (3 8) th e vi rt ua l h ea lth u ni ve rs ity (3 7) m od el lin g of w eb ba se d vi rt ua l u ni ve rs ity ad m in is tr at io n fo r n ig er ia n u ni ve rs itie s (3 6) in no va tio n m od el in h um an re so ur ce s ca pa ci ty d ev el op m en t in s up po rt in g u ni ve rs ita s te rb uk a as a c yb er u ni ve rs ity (3 5) a st ra te gi c m od el of v irt ua l un iv er si ty (3 4) pe da go gi ca l co m po ne nt te ch no lo gy co m po ne nt m an ag em en t co m po ne nt ed uc at io na l de si gn te ch no lo gy im pl em en ta tio n ad m in is tr at iv e ed uc at io na l m an ag em en t 288 j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 explaining the conceptual considerations of virtual university in medical education original s. ahmady et al. on internal validity and study design. in fact, study methodology and theoretical development are different areas of research that require different skills.15 therefore, the research team adopted a recommended inductive and subjective approach instead of using checklist-like tools. “applied quality assessment prompts” were used as the most appropriate framework to ensure clarity of objectives, having a specific and appropriate research design, providing a clear report of findings and sufficient data to support the interpretations and appropriate analysis.15 methodological quality assessment was carried out with data simultaneously. table 4 shows the five steps to quality assessment guidelines as a framework that is used in research to assess the quality of articles and perform the final synthesis. results & discussion the aim of the present research was to identify, investigate, analyze and integrate models, theories and frameworks related to the concept of virtual university in the field of medical sciences and health system. a total of 13 articles were identified in the data extraction stage. the articles were reviewed using on a checklist. pedagogical, technology, managerial, educational design, technology implementation, and educational-administrative management components were referred to in 5, 5, 4, 7, 6 and 4 articles, respectively. the research team performed the thematic analysis of the extracted models based on two theoretical and operational dimensions (figure 2), which include table 4. qualitative assessment of articles review stage criteria inclusion criteria to include a model/theory or framework in a study, a mechanism should be defined in which the main components of a virtual university in the health system and the relationship between them were clearly shown. literature search the review included multiple forms of formal electronic and manual searches and citation tracking to follow the development and impact of known theories on subsequent relevant literature. data extraction spreadsheets were created from the data extracted from the models. quality control the review of models and theories did not involve conducting a standardized critical evaluation. synthesis they were graded according to the review question as well as by checking the details or originality. 1. theoretical dimension: this dimension includes three main components: • pedagogical component. • technology component. • management component. 2. operational dimension: this dimension considers the strategies, actions and procedures based on which subsystems of a university operate and includes three main components: • educational design. • technology implementation. • administrative-educational management. all these dimensions and the relationship between them will be explained separately. it is also necessary to mention that the operational components of this framework are semantically placed among the theoretical dimensions of the framework considering their operational nature, and therefore, each of these components will be explained in combination with the theoretical components. theoretical dimension pedagogy and educational design pedagogy is defined as guiding the child and is known as the art and science of teaching children. pedagogic techniques for effective content transfer include learning content, learning style, time of learning, learning with the help of the instructor, and responsible instructor.16 fig. 2 components of the virtual university. 289j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 s. ahmady et al. original explaining the conceptual considerations of virtual university in medical education such definition is based on the perspective of theorists in the field of education and is thus consistent with the “teacher centered approach” at the school level. developments in the field of education and learner-centered and learning-centered approaches, emphasis on the facilitating role of the teacher, as well as the development of pedagogy in higher education have led to the redefinition of pedagogy. also, the current understanding of pedagogy is not limited to child education, but is considered as a basic teaching concept for in all academic courses. considering such a definition of the pedagogy and looking at the extensive changes made in different pedagogic concepts, the change of the educational paradigm is reflected in this component of the proposed framework. here, the curriculum is seen as a continuum where the main importance is acquired competencies, not just completed assignments. besides, assessment becomes an important element in the teaching and learning process and is not merely a punishment or a factor for determining grades. the main focus of this component is on learning modules, communication, interaction and cooperation between learners, and the use of the latest information and communication technology is also emphasized. technologies that provide the possibility of academic networking between students and professors. in this component, a “learning unit” as a wide conceptual area includes three basic elements, namely “content”, “interaction” and “evaluation”.17 as these elements play an important role in the learning process and the type of their evaluation based on the theory of active learning. the content, level of interaction and types of communication activities are determined by the learning objectives. then, the educational design layer is placed as one of the operational components of the framework at a higher level where the basic planning, design and implementation of teaching-learning processes, especially the learning units and related activities are determined.17–19 educational design as a basic component of the framework is carried out in six separate but related steps: 1. specifying and explaining the available educational and technological options: in this step, the learning needs are identified, the target audience is defined, the human and financial resources and the existing technological infrastructure are evaluated. in other words, possible options are identified and selected for a successful educational design. 2. general design of the course (main program): in this step, goals, background, prerequisites, themes, content and keywords are determined for each educational course. in a general view, this step includes deciding on the structure of the course and summarizing the anticipated activities and strategies, as well as compiling the course timetable and relevant bibliography. 3. designing course units: in this step, teaching and learning units are designed along with units related to modular learning. the educational strategies for each unit, as well as the content and calendar of activities are determined according to the duration of the course. 4. interactive system design: in this step, learning activities, their dynamic level, the sequence, the resources and tools used, as well as the writing of practical guides and other teaching strategy elements are designed to ensure their proper implementation. 5. designing a feedback system: here, while emphasizing the educational effect of the concept of feedback, the aim is self-evaluation design, systematic and final evaluation of the course, for continuous and interactive feedback during the teaching-learning process. 6. final design and presentation of general information: upon reaching the final step, the final evaluation of the entire process will be implemented. in this step, there is the possibility of final adjustment of the general program and its reorganization according to individual activities (second and third steps) as well as the evaluation program (fourth step).17-20 after expanding the concept of the pedagogical component and identifying some of its most important elements, the present framework proposes the following schematic view as the pedagogical components of the virtual university in the health system (figure 3). fig. 3 pedagogical components of the virtual university in the health system. 290 j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 explaining the conceptual considerations of virtual university in medical education original s. ahmady et al. technology components and technology implementation today, all users of virtual networks have become active producers of knowledge by using open-source software as well as a variety of information technology tools with different levels of complexity. from this point of view, the most important features of the virtual university technology component in the health education system include “interactivity” and the possibility of reusing technology. in this component, the technology implementation element has a strong presence as one of the elements of the operational component of the framework. the implementation of information technology generally consists of a number of successive steps including requirements analysis, project design, testing, establishment and maintenance. although documentation and training are often added to these steps.36,38,41,43 in these components, three basic parts that work together were identified: part 1: technological support online and technical support services are very necessary to speed up the education process for all users, teachers and students. on the other hand, information technology support has an important effect on knowledge process capabilities; because it allows knowledge creation and sharing at lower costs and is considered a key component in knowledge management. therefore, the concept of support in a virtual university goes beyond mere provision of different principles and guides and has a direct impact on the process of knowledge management in a virtual university. in the current framework, this part is considered as a basis for providing other services, and its components and tools are shown separately in figure 4.19-21-22 part 2: data this part focuses on accepted international protocols and standards, which, in addition to validating university activities, provide the possibility of cooperation between educational and service institutions, as well as information retrieval. e-learning standards are a set of laws that not only ensure coordinated provision of education everywhere; but also, it provides a common language for the learning management system and e-learning courses, so that they can share information with each other and work together. in fact, components of the training course or training objects are identified and defined using these rules.23 fig. 4 technological support. e-learning standards are divided in different ways from the meaning point of view. here, first, they are divided from the perspective of content coverage they provide, and then the most important e-learning standards for use in a virtual university will be discussed. 1. meaning: this field focuses on the general concepts of understanding: semantics, pragmatics, etc. 2. quality: this field includes all aspects of quality management, including development, assurance of results, processes and potentials. 3. didactics: this area focuses on issues related to educational questions: methods, learners and learning environments. 4. educational technology: this area focuses on all technological solutions that have been created for educational purposes: such as information exchange, interfaces and accessible questions. 5. learning content: this area includes all aspects related to e-learning objects and collects resources and packaging. 6. context: the main purpose of this area includes all disciplines and information related to electronic education, including laws and regulations (49). table 5 shows the most common standards in electronic education. our conceptual advancement in the technology component and its expansion gives a more complete picture of the framework (figure 5). part 3: services this part of the technological component indicates the services provided by a virtual university to users, learners and faculty members. these educational services should promote and support an open and collaborative environment. the portal is known as the most basic service for providing virtual university services, but according to the proposed framework, the university portal should act in such a way as to provide access to various resources and services and communication and collaboration tools for all users. it also allows interaction between students, faculty members and users who are interested in the field of healthcare and health sciences as a whole. such a portal facilitates interaction, communication, study and discussion about clinical cases. services are also provided by electronic learning management system. a learning management system creates a communication channel between instructors and learners and helps instructors manage online learning. software are able to manage all types of content, including videos, courses, documents, etc., and allow students to communicate with learning 291j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 s. ahmady et al. original explaining the conceptual considerations of virtual university in medical education fig. 5 adding the data part to the part 1. table 5. e-learning standards standard developer important component website ims ims global learning consortium standard packaging (www.imsproject.org) scorm adl a combination of several standards (scorm; www.adlnet.org/scorm/history/2004/ index.cfm) ltsc ieee content relevance (ltsa; http://ltsc.ieee.org/wg1/files/ ieee_1484_01_d09_ltsa.pdf ) lom ieee metadata (lom; http://ltsc.ieee.org/wg12) papi ieee learner profile (www.edutool.com/papi/drafts/08/) aicc aicc supply, control, presentation and follow-up of results between educational management systems and online courses www.aicc.org management system software through any device. instructors can also evaluate learners by using features such as receiving their assignment reports, progress status and similar cases. such a system will be the backbone of a virtual university. here we are dealing with a web-based software and program that manages, documents, follows up, reports, and provides educational courses and educational programs. this software allows the instructor to provide the content of the educational course as easily as possible to the participants and students, to hold quizzes, tests and exams offline or in person (online in class), assign related assignments to students in a web-based system, follow up student status, and access other such facilities in an internet-based system. online classes are one of the other educational facilities that can be held with the help of the university’s learning management system. the virtual classroom is an educational tool that is based on communication networks and can replace the traditional classroom and solve the associated problems. although the virtual classroom does not change the channels of information transmission, it tries to use appropriate tools to carry out this information transfer in the best possible way.17 in addition to increasing the educational quality, such optimization also reduces educational costs. the virtual classroom is, in fact, a discussion group whose topic is the same as the lesson subject. a discussion group whose title is the same as the lesson title is created on the network, and the students become members of the discussion group. then, the faculty member who is also a member of the group, will present the related course materials at regular intervals. they should also be designed as an interactive learning space that simultaneously pursues formal and informal educational activities. rather being related to educational content, interaction in this class means interaction and mutual effect of educational activities and communication. the virtual clinic is another interactive space that is identified in the proposed framework, and all learners and faculty members will be able to use its services from the moment they enter the university. education is provided in this space based on diagnostic discussions (a virtual space in which people discuss the cases of interest to make a definitive diagnosis online), clinical pathology topics (topics related to the postmortem findings of pathologists) and presentation of clinical cases. education through the presentation of clinical cases and scenarios is currently recognized as one of the leading and effective methods in the field of medical sciences. the use of the case-based learning method prepares students with diverse experiences such as problem solving, building knowledge 292 j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 explaining the conceptual considerations of virtual university in medical education original s. ahmady et al. in the presence of each other, communication and group participation. just like traditional universities, another element of the framework that guarantees the success of virtual universities is a virtual library. the virtual library makes it possible to provide all the appropriate information that are supposed to be exchanged between students and faculty members of the virtual university and to support the researches of the virtual university. such a library helps users to find the required information through the resources available in the library databases and performs the tasks of information collection, accessibility and dissemination. the virtual attribute mostly refers to the lack of spatial dimension of this type of library. this type of library does not exist physically, is created on computer networks and enables the access of virtual university users to electronic and network resources.24 in this framework, it is recommended to use blockchain technology and launch the digital library of the virtual university of medical sciences in such a network.25 by definition, blockchain refers to a database consisting of a list of transaction records, which are always growing and increasing in number. these records are called blocks, which are connected to each other through cryptography. blocks are placed in a chain of nodes with a peer-to-peer network, and the resulting storage is called a digital ledger.26 this meaning fits well with what librarians have always done, that is to collect, keep, and share authoritative information. blockchain can help librarians achieve this goal, especially in the world of scientific publications. one of the potential applications of blockchain is to create verifiable and scheduled copies of journal articles. another potential application of blockchain in libraries is a digital rights management tool.25 digital resources are inherently reproducible, which in turn creates issues for libraries and publishers. because blockchain creates a unique verifiable record that anyone can access, it can be attached to digital material and used as a way to show the “provable scarcity” of that resource. this enables digital materials to be uniquely identified, controlled and transferred. publishers can be also assured that there have been no copies of them. web 2.0 tools web 2.0 is a set of programs and digital technologies that enable users to interact and collaborate with each other and share their content and information. unlike web 1.0, which is called a static and read-only network, web 2.0 is a dynamic network that can read and write content. this technology allows the user to add, play, evaluate and change information. although these technologies have not been formed with the approach of educational application, they have formed new conceptualizations regarding the dynamics of virtual university education (figure 6). web 2.0 challenges the existing models of designing web-based curricula, entails new epistemological considerations, and proposes a different theoretical basis. in this type of learning, the way educational content is produced and published is similar to web 2.0. in other words, the content does not follow the traditional method of production, organization and distribution, instead it is a collaborative effort (table 6).19-21-22 finally, the technology component is displayed in the form of a conceptual pyramid after putting different parts together (figure 7). management components and administrative-educational management these components are identified with the following four basic elements in the current framework: 1. vision: vision, which is called perspective, view, imagined future, or ideal and desirable future, is a description of future conditions, in other words, it is an vision of the future state of a group, when goals and strategies are achieved. the vision of the organization should be brief, memorable, desirable and ideal and should take into account all levels (17-26-27). furthermore, such a university is recognized as a virtual campus with a networked, open-source learning method based on real health care processes that are approved by the ministry of health and medical education. fig. 6 types of web and its applications. table 6. classification of web 2.0-based technologies based on the educational design parts of the framework subcategories technologies related to each subcategory teaching planning and design 1. mihanblog 2. blogfa 3. crocodile 4. huppa 5. rubistar 6. irubric 7. ning 8. edmodo 9. moodle 10. google 11. sketchup 12. gliffy 13. google 14. sites 15. salam.ir 16. parsijoo.ir 17. audacity presentation and teaching methods 1. flashcardexchange 2. wordnik 3. del.icio.us 4. flickr 5. google 6. salam.ir 7. parsijoo.ir 8. webspiration 9. slideshare 10. gliffy 11. paint 12. audacity 13. evernote 14. community 15. walk 16. footnote 17. googlemap 18. create a graph 19. movie maker 20. wordle 21. viber 22. telegtam 23. youtube 24. surveymonke 25. polldaddy 26. polleverywhere 27. prezi 28. protagonize 29. glogster 30. blogfa 31. mihanblog 32. powerpoint assessment edmodo, ning, moodle, polleverywhere google trends, crocodile, huppa, viber, bubble.us quizstar telegram, paint, footnote, microsoft office word 293j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 s. ahmady et al. original explaining the conceptual considerations of virtual university in medical education fig. 7 technology component and its elements. 2. planning: the implementation strategies of a virtual university in the health education system are considered in this element: • building and maintaining an educational virtual space for online interaction, learning and work in order to develop the professional competencies and qualifications of medical students. • training of human resources for the efficient application of information and communication technology in the fields of education and health. • strengthening the educational capacities of academic institutions and health service providers. • integration and systematization of national health information and knowledge management. • using information and communication technology for online education, based on the technical capacity available in academic health services institutions. • methodological development of clinical training related to the health needs of iran. 3. operational management: project management is a goalbased management model that defines the demarcation of different decisions, allocation of resources and assignment of responsibilities based on different components (courses, clinical topics, etc.) in different academic and service institutions. in this sense, management takes place in two different parts. • the first case is pedagogical management, which is related to the teaching-learning process and basic functions of faculty members such as educational design and creation of educational resources. • the second case is institutional management, which deals with administrative and executive processes, where leaders and managers act at institutional and local levels, including the creation of logistics coordination and training regarding virtual learning environments (42-40-34). 4. process quality evaluation: in this proposed framework, follow-up, control and evaluation are carried out to improve the quality of the program process through process analysis, academic results and the quality of services provided by the university. in this evaluation, managers’ self-criticism has been identified as a fundamental pillar of process improvement. also, continuous surveying of different university groups (faculty members, students, users) makes it possible to improve the quality of curricula and services at both institutional and curriculum levels. conclusion the results of the present study emphasize that the six components of pedagogy, technology, management, educational design, technology implementation and administrative-educational management are essential in the establishment and development of virtual universities of medical sciences. virtual university of medical sciences plays an important role in the development of electronic learning, the realization of educational justice and the creation of various educational opportunities in virtual form. in this regard, the process quality assessment framework can be used to improve the education process in these universities. conflict of interest none.  294 j contemp med sci | vol. 8, no. 5, september-october 2022: 284–294 explaining the conceptual considerations of virtual university in medical education original s. ahmady et al. references 1. barnett r. realizing the university (london, institute of education, university of london). blake, r. smith, and p. standish (1998) the universities we need: higher education after dearing. 1997. 2. anderson m. virtual universities: future implications for students and academics. in 16th annual conference of the australasian society for computers in learning in tertiary education (ascilite), brisbane, australia. retrieved september 1999 (vol. 17, p. 2003). 3. robins k, webster f, editors. the virtual university? knowledge, markets, and management. oxford university press; 2002 nov 7. 4. rada r. understanding virtual universities. intellect books; 2001. 5. ahmad r, piccoli g. virtual learning environments: an information technology basic skills course on the web. amcis 1998 proceedings. 348. 6. harasim lm, hiltz sr, teles l, turoff m. learning networks: a field guide to teaching and learning online. mit press; 1995. 7. leidner de, jarvenpaa sl. the use of information technology to enhance management school education: a theoretical view. mis quarterly. 1995 sep 1:265–91. 8. anastasiades ps. virtual universities: a critical approach. ininternational conference on computers in education, 2002. proceedings. 2002 dec 3 (pp. 1170-1171). ieee. 9. dabous ft, rabhi fa, ray pk, benatallah b. middleware technologies for b2b integration. annual review of communications. 2003 nov;56(3). 10. slaughter s, leslie ll. academic capitalism: politics, policies, and the entrepreneurial university. 1999. 11. cornford j. the virtual university is... the university made concrete? information, communication & society. 2000 jan 1;3(4):508–25. 12. hean s, o’halloran c, pitt r, green c, temple j. a systematic review of the contribution of theory to the development and delivery of effective interprofessional curricula in health professional education. hentet fra https://www. bemecollaboration.org/downloads/1243/hean_protocol_ dec2012. pdf. 2013. 13. booth a, harris j, croot e, springett j, campbell f, wilkins e. towards a methodology for cluster searching to provide conceptual and contextual “richness” for systematic reviews of complex interventions: case study (cluster). bmc medical research methodology. 2013 dec;13(1):1–4. 14. votruba n, ziemann a, grant j, thornicroft g. a systematic review of frameworks for the interrelationships of mental health evidence and policy in low-and middle-income countries. health research policy and systems. 2018 dec;16(1):1–7. 15. campbell m, egan m, lorenc t, bond l, popham f, fenton c, benzeval m. considering methodological options for reviews of theory: illustrated by a review of theories linking income and health. systematic reviews. 2014 dec;3(1):1–1. 16. pedagogical perspectives, inspirational horizons in the higher education curriculum. bi-quarterly journal of higher education curriculum studies.; 10 (19): 171–93. 17. méndez jb. the virtual health university: an elearning model within the cuban health system. medicc review. 2008 mar 28;10(1):22–8. 18. ivanova g, smrikarov a. methodology for design of virtual learning environments–virtual universities. in e-learning ii conference, berlin, germany 2005. 19. conole g, wills g, carr l, vadcard l, hall w, grange ss. building a virtual university for orthopaedics. inedmedia+ innovate learning 2003 (pp. 2227). association for the advancement of computing in education (aace). 20. abdollahi d. a study of pedagogical aspects of a virtual university. international journal of educational and psychological researches. 2018 jan 1;4(1):12. 21. mbam bc, odachi gn. modelling of web-based virtual university administration for nigerian universities. afrrev stech: an international journal of science and technology. 2014 jul 3;3(2):86–107. 22. cecil j, gupta a, pirela-cruz m, ramanathan p. an iomt based cyber training framework for orthopedic surgery using next generation internet technologies. informatics in medicine unlocked. 2018 jan 1; 12:128–37. 23. fallon c, brown s. e-learning standards: a guide to purchasing, developing, and deploying standards-conformant e-learning. crc press; 2002 nov 25. 24. van drie jh, lajiness ms. approaches to virtual library design. drug discovery today. 1998 jun 1;3(6):274–83. 25. mackey tk, shah n, miyachi k, short j, clauson k. a framework proposal for blockchain-based scientific publishing using shared governance. frontiers in blockchain. 2019 nov 15; 2:19. 26. gupta ss. blockchain. ibm online (http://www. ibm. com). 2017. 27. flores rr, vértiz-osores ri, ochoa gl, romero aa. virtual university education in the context of the health emergency due to covid-19: challenges in the evaluation processes. international journal of early childhood special education (int-jecse). 2020;12(1):467–77. 28. shahtalebi s, shatalebi b, shatalebi f. a strategic model of virtual university. procedia-social and behavioral sciences. 2011 jan 1; 28:909-13. 29. bradley c, oliver m. the evolution of pedagogic models for work-based learning within a virtual university. computers & education. 2002 jan 1;38(1-3):37–52. 30. khayal is, farid am. a dynamic model for a cyber-physical healthcare delivery system with human agents. in2017 ieee international conference on systems, man, and cybernetics (smc) 2017 oct 5 (pp. 3624-3629). ieee. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i5.1253 363j contemp med sci | vol. 8, no. 6, november-december 2022: 363–369 original link of gut microbiome with risk of type 2 diabetes mellitus alhanouf m. alkhammash1, wafa a. alshehri2*, , amani m. alhozali3, , ahmed bahieldin1 ¹department of biological sciences, faculty of science, king abdulaziz university, jeddah, saudi arabia. ²department of biology, college of science, university of jeddah, jeddah, saudi arabia. ³department of medicine, faculty of medicine, king abdulaziz university, jeddah, saudi arabia. *correspondence to: wafa a. alshehri (e-mail: waalsheri@uj.edu.sa) (submitted: 15 february 2022 – revised version received: 11 march 2022 – accepted: 12 april 2022 – published online: 26 december 2022) abstract the gut microbiota presented in the human digestive system plays an active role in maintaining host health. recent studies that rely on the use of high throughput metagenomic sequencing (mgs) revealed the association of dysbiosis in the intestinal microbial community and many chronic diseases, specifically type 2 diabetes (t2d). this disease is considered one of the most prevalent metabolic diseases worldwide comprising up to 90%. this article presents the most prominent research in this field and summarizes the method used to study the diversity of the gut microbiome through the use bioinformatics tools. we highlight the role of gut metabolic products in the development of t2d, as many studies have proven the influence of microbial metabolite products, such as lps and scfas, on blood glucose levels through the microbial-host cross-talk. in addition, this article underlines the interaction of epigenetic mechanisms and the gut microbiome in regulating and developing t2d. the latter can be an appropriate preventative or therapeutic approach for several human metabolic diseases. therefore, future research is required to build new type of t2d therapy based on epigenetic mechanisms, microbial composition, and microbial metabolites. keywords: gastrointestinal microbiome, diabetes mellitus, type 2, microbial metabolites, epigenomics issn 2413-0516 introduction one of the alarming human health issues in the world is diabetes mellitus, which is a metabolic disorder through which patients are unable to regulate glucose absorption. the most common diabetes type is type 2 diabetes (t2d), representing about 90% of cases globally, while type 1 diabetes (t1d) represent only 10%.1 in developing countries, 85% of the early death is related to metabolic disorders, of which deaths due to diabetes constitute about 80%.2 according to the internation diabetes federation (2019), 463 million people have diabetes worldwide, of which 55 million people live in the middle east and north africa region. these numbers are speculated to rise to 108 million in year 2045. in 2019, saudia arabia became one of the top five countries for diabetes with 4.3 million patients, and the diabetes prevalence reached 18.3 (internation diabetes federation, 2019).3 patients with t2d commonly suffer from damage or lack of insulin secretion.4 the disease is recognized by hyperglycemia caused by a deficiency of insulin production, which exhaustion results of pancreatic beta cells function after establishing insulin resistance.5 several studies indicated that obesity and unhealthy lifestyles, such as the lack of exercise and poor dietary habits, are linked to t2d.6 previously, it was believed that only genetic and environmental factors can influence the pathogenicity of t2d.7 however, recent studies on the human microbiome have found evidence that supports the effect of the intestinal microbiome in stimulating inflammation, insulin resistance, obesity, and t2d.8 findings of the latter report indicated differences in the gut microbiome in t2d patients compared with healthy individuals. these results have led to the investigation of gut microbiota as an essential factor in diabetes risk development.9 gut microbiota has a wide range of functions that impact human physiology, e.g., vitamin synthesis, regulation of the intestinal mucosal barrier, protection against pathogens, host nutrition alteration, energy harvest modulation, fermentation of indigestible carbohydrates, and development of the host immune system.10 in humans, the gastrointestinal (gi) tract contains bacteria, viruses, archaea, and eukaryotes. this dynamic population has more than 1014 microbial cells with 35,000 species and over 10 million non-redundant genes.11 these collective genomes of gut microbiota present enormous metabolic properties to the host because the gut microbiome has at least 100 times more encoded genes than those of the human genome.12 researchers found that the human gut microbiome contains six prime phyla, e.g., firmicutes, bacteroidetes, actinobacteria, fusobacteria, verrucomicrobia, and proteobacteria.13 moreover, most bacterial species in the adult gut belong to firmicutes and bacteroidetes, constituting more than 90% of the gut microbial community and related to obesity and t2d.13 barengolts (2016) reported that a healthy gut microbiome consists of 64% firmicutes, 23% bacteroidetes, 8% proteobacteria, and 3% actinobacteria.14 furthermore, the alteration in the bacterial diversity of the gut microbiome leads to the overgrowth of proteobacteria, resulting in dysbiosis. metabolic diseases such as diabetes, obesity, multiple sclerosis, crohn’s disease, rheumatoid arthritis, and inflammatory bowel disease are correlated with gut microbiome dysbiosis.15,16 the development of gut microbiota can be impacted by many factors, as illustrated in figure 1. the maternal nutrition of the mother during pregnancy, embryonic environment, and mode of birth are the first elements in obtaining the gut microbiota. then, it is completely formed during the age of 2–3 years, similarly to the adult microbiome, and remains relatively stable in adulthood.17 additionally, dietary patterns such as breast feeding, diet, and diet components play a critical role in maintaining the gut community and maturity. all these factors, along with an individual’s genetic and environmental factors (such as drug or antibiotic intake, infections, lifestyle patterns, and migration to a different location), contribute to shaping the microbial gut population.18 http://orcid.org/0000-0001-5686-7437 http://orcid.org/0000-0003-4622-5902 mailto:waalsheri@uj.edu.sa 364 j contemp med sci | vol. 8, no. 6, november-december 2022: 363–369 link of gut microbiome with risk of type 2 diabetes mellitus original a.m. alkhammash et al. diversity in the human gut microbiome during the last two decades, the gut microbiome received considerable attention from researchers because of its participation in several medical and psychological disorders.19 scientists considered it as another body organ that interacts with the host to health promotion and, in some cases, triggers the disease. in this context, it is noted that the microorganisms that inhabit the gi tract are referred to as gut microbiota, while the genomes of the microbiota is known as the gut microbiome. in microbiome research, the diversity of microorganisms within a specific body area is used as a measurable outcome and defined based on species richness and eveness as well as microbial relative abundance and distribution among different organs.20-22 microbiome studies aim currently to understand the mechanical role of microbial diversity in disease etiology and public health. to achieve these objectives, scientists focused on identifying the factors that lead to differences in microbiome environments. age is one of the factors that shape or effect the microbiome, where microbiota appears to be volatile in the infancy stage and then becomes more stable after the initial three years.13 also, environment, genetic factors, and lifestyle factors such as diet and smoking seem to impact the composition of human gut microbiome.23,24 understanding human gut microbiome and factors that drive its variability composition could lead to important insights into mental and physical health. in this context, many studies proposed a cross-talk between the brain and the gi tract allowing each to influence the other.25 this interaction, called the gut-brain axis, may cause the symptom of the disease health disorders.26 besides, the gut microbiome composition has a crucial role in the gut-brain axis.27 consequently, understanding the etiology and consequences of mental and physical health disorders and the development of efficient treatments depends on studying the gut microbiome composition. recently, the new developments in the technologies of high throughput metagenomic sequencing (mgs) enhanced the understanding of the symbiotic correlation between the host and the gut microbiome.28 mgs allows researchers to recognize the pathogenic mechanisms of the microbiome that associate with the host’s disease and thus provide ways to modify the microbial community in the gut for therapeutic and even preventive objectives. metagenomics and metatranscriptomics are nucleic acid sequencing-based methods used for microbiota characterization. sequencing portions or whole gene of bacterial 16s ribosomal rna subunit is one of these methods. it is utilized as a phylogenetic marker to predict the bacterial population structure, support microbiota preliminary investigation, and determine variations in the diversity and abundances of individual bacterial species between microbiota samples.29 in living organisms, mrna translating to proteins through the complex called the polysome that is made of several ribosomes. ribosomes are composed of several proteins besides several rnas called ribosomal rnas (rrna). in prokaryotes, rrna is identified as a 16s rrna subunit, while in eukaryotes is known as the 18s rrna subunit.30 both rrna subunit types have evolutionarily conserved regions surrounding nine variable regions.31 usually, the conserved regions are used to design the universal primers for pcr to amplify the targeted variable regions; and the resulting amplicons are sequenced for phylogenetic analysis. finally, the sequences of the variable regions are compared to a set of reference sequences with pre-assigned taxonomy to conduct the phylogenetic analysis, which allows estimating the composition of taxonomic units in the community. indeed, data is used to assess the composition of the bacterial population when completing the sequencing of partialor full-length 16s rrna gene. after the sequences is completed, the data quality filtering steps apply, and the bioinformatics tooles (the qiime (caporaso et al., 2010))32 or mothur (schloss et al., 2009)33 are used to assign the taxonomy by mapping sequences to the reference databases greengene (desantis et al., 2006)34 or silva (quast et al., 2013).35 subsequently, several statistics are applied to the resulting operational taxonomic unit (otu) table, which evaluates the diversity of bacterial communities and shows the similarities or differences between these communities. numerous studies have indicated the clinical relevance of estimating microbiota diversity within the intestinal tract, where the decrease of microbiome diversity is often associated with alterations in the gut microbiota composition, called dysbiosis. alpha and beta diversity are two types of measures used to analyze the diversity of the microbiome. the number of unique taxa (richness) and their distribution (evenness) in a specific sample is defined as alpha diversity, which evaluates by many indices involving chao1, shannon index, and the simpson index. in addition, the number of species that are different among the groups, known as beta diversity, evaluates the differences in species composition between two groups of samples.36 eubiosis and dysbiosis in the gut microbiome eubiosis is a term that refers to a healthy composition of the intestinal microbiome that maintains host health. in contrast, dysbiosis refers to an unhealthy microbiome composition that induces the risk for certain diseases. the gut microbiome in a state of eubiosis has various roles in producing metabolic products such as short-chain fatty acids (scfas), branchedchain amino acids (bcaas), and impacting the metabolism of lipid. moreover, the gut in eubiosis consists of 95% bacteroidetes and 5% firmicutes, which is considered a standard b/f ratio in the gut bacterial community, thus, enhances intestinal health, regulates opportunistic pathogens, and participates in the whole body’s good health.37 changes in the desirable fig. 1 factors influencing the development of gut microbiome. 365j contemp med sci | vol. 8, no. 6, november-december 2022: 363–369 a.m. alkhammash et al. original link of gut microbiome with risk of type 2 diabetes mellitus microflora in a healthy gut can lead to a dysbiosis state (petersen and round, 2014), which force a negative effect that causing obesity and multiple diseases such as t2dm and subsequent cvd (figure 2).37 for instance, turnbaugh et al. (2007) carried out his piouneering experiment on germ-free (gf) mice in which they colonized gut microbiota from conventionally raised (conv) mice. then, they observed changes in body weight.21 regardless of the reduction in food intake, the gf mice exhibited increased body fat within 10–14 days. this increase in body fat has been attributed to microbial fermentation of undigestible polysaccharides, absorption of monosaccharides, and the microbiome genes that stimulate the growth of adipocytes. these discoveries guided the researchers to support the claim that obese persons are usually more efficient at energy harvest because of their microbiome than thin counterparts, explaining the weight gain. the influence of gut microbiome in type 2 diabetes recent studies found that the development risk of obesity and t2d is connected with the composition imbalance in gut microbiome populations (moreno-indias et al., 2014),38 in addition to increasing insulin resistance, lipid levels, adiposity, and inflammation linked to decreasing gastric microbial diversity, known as the bacterial species number or richness.39 amar et al. (2011) found that proteobacteria (gram-negative bacteria) translocate out of the intestinal lumen preceding the advent of abdominal adiposity and t2d.40 besides, microbial metabolites could contribute to the risk of obesity through slowing intestinal transit and boosting energy absorption.38 various studies have been performed in microbiome profiling of t2d cases and controls in humans. in 2010, larsen et al. compared the gut microbiome between diabetic and non-diabetic individuals by using principal component analysis, finding that t2d is associated with the changes in the composition of gut microbiota at the phylum level (larsen et al., 2010).41 they reported that t2dm shows a reduced abundance of firmicutes phylum and clostridia class contrasted with the control group, while increasing abundance of betaproteobacteria class, which correlates with plasma glucose concentration. in the human gi tract, several bacteria producing butyrate belong to the firmicutes phylum, specifically class clostridia.42,43 the massive metagenomic species (mgs) research in chinese and european cohorts showed that butyrate producing bacteria, such as faecalibacterium prausnitzii and roseburia intestinalis known for their anti-inflammatory properties (vrieze et al., 2012),44 were less abundant in patients with t2d disease.45,46 moreover, the combined analysis of these two datasets showed that butyrate-producing taxa were reduced in t2d and remained after controlling microbiome-modifying impacts of metformin.47 another study found that increasing f. prausnitzii is associated with an anti-diabetic impact, which was remarked instantly after gastric-bypass surgery, regardless of significant weight reduction.48 also, in a danish study that included 277 non-diabetic individuals, scientists found an increase in serum bcaas levels in people who had insulin resistance, caused by an increase in the production of bcaa and a decrease in the transportation into bacterial cells.49 the most important species that leading the biosynthesis of bcaas are prevotella copri and bacteroides vulgatus. in mice, the researchers noted that p. copri can stimulate insulin resistance and promote bcaas circulating levels. they also indicated that gut microbiota plays a crucial role in insulin resistance and might be a significant resource for increasing bcaas levels. radwan et al. (2020) examined the bacterial microbiome composition and abundance in patients with t1d and t2d and characterized the gut microbiota features to find the possible differences associated with this population.50 the results emphasized a substantial increase in abundance in gram-negative bacteria, possibly opportunistic pathogenic taxa such as pseudomonas and prevotella in all patients with diabetes compared to healthy individuals. they also found that the gemella (gram-positive bacteria) related to increased the risk of diabetes and significantly had an increase in abundance in all groups of people with diabetes. the control group has more abundant commensal bacterial taxa as turicibacter, terrisporobacter, and clostridium than the t1d group. in 2013, zhang et al. used 16s ribosomal dna (rdna)– based sequencing and discovered that prediabetes people who were recently diagnosed with t2d had a lower abundance of akkermansia muciniphila, which may be considered as a biomarker of glucose intolerance.51 in obese adults, dao et al. (2016) proved that a healthier metabolic condition associate with the presence of a. muciniphila in a high abundance.52 a. muciniphila is a human gut mucin-degrading bacterium that represents 3–5% of the human intestinal microbial population. moreover, the study showed that optimal clinical outcomes as glucose homeostasis, blood lipids, and body composition had linked to the presence of a. muciniphila in higher abundance in the baseline after caloric restriction. in addition, increasing a. muciniphila levels were observed due to treatment with metformin, which could intercede with many of its metabolic benefits.53 the latest research found that patients with t2d have a significantly higher firmicutes/bacteroidetes ratio than healthy controls.54 on the contrary, the chinese, european, and danish cohort studies observed that patients with t2d have a lower firmicutes/bacteroidetes ratio.45,46 the contradiction among these studies is perhaps because of perplexing influences such as different techniques of sequencing, study populations, using a medication, and diet. additional controlled studies are needed to study these confusing variables. these unrelated disease variables can substantially influence the result of the microbiome.55 overall, research outcomes to date recognized a relationship between the gut microbiome and t2d. fig. 2 influence of diet on the gut microbiome and risk of t2d. 366 j contemp med sci | vol. 8, no. 6, november-december 2022: 363–369 link of gut microbiome with risk of type 2 diabetes mellitus original a.m. alkhammash et al. type 2 diabetes and microbial metabolites microbial metabolites are responsible for regulating host metabolism and gut integrity. therefore, they can be considered as a vital link connecting the gut microbiome to insulin resistance, which can result into t2d. some of the metabolites are beneficial, e.g., scfa, indole derivatives, sulfur-containing amino acids, bile acids, and vitamins, while others are harmful, e.g., lipopolysaccharide (lps), methane, bcaa, phenol, amines, p-cresol, and ammonia.56 the essential metabolites of interest are mentioned below. short chain fatty acids (scfas) the most metabolites produced by the gut microbiota are short-chain fatty acids (scfas) such as butyrate, propionate and acetate representing in the human intestinal lumen at the ratio of 1:1:3.57,58 nevertheless, the ratio of these types of scfas depends on both the composition of microbes and the diet. the bacteria-derived scfas have a different destiny, where most butyrate is used locally as their primary energy source through colonocytes. also, propionate is metabolized by the liver upon absorption, while acetate is easily absorbed and distributed to peripheral tissues through the blood circulation.59 a recent study documented that metabolites have interacted with host metabolism. gut microbiota ferment the indigestible carbohydrates such as oligosaccharides or polysaccharides to generate scfas by various metabolic pathways.60 insulin sensitivity and energy metabolism are effected by scfas. previous reports supposed that scfas can change the concentrations of several gut peptides involved in glucose metabolism, gut barrier function, and energy homeostasis.61,62 while, more recent reports have documented that butyrate and propionate suppress the weight gain in mice with high fat diet-induced obesity, and acetate has been proven to reduce food intake in healthy mice.63,64 also, perry et al. (2016) reported that acetate plays a vital role in parasympathetic activity to increase food intake and support glucose-stimulated insulin secretion in a rodent model.64 bindels et al. (2013) have recognized a g protein-coupled receptor family, which is responsible for the effects of scfas.65 examples of gprs include g protein-coupled receptors 43 (gpr43) and g protein-coupled receptors 41 (gpr41). when scfas bind to gpr43 and gpr41, this leads to increased plasma levels of glucagon-like peptide-1 (glp-1) and peptide yy (pyy), improved glucose homeostasis, and reduced appetite due to their central nervous system interventions.66 gut microbiota can also produce indole from tryptophan, which contributes to the secretion of glp-1 by intestinal entero-endocrine cells.67 secondary bile acids (sbas) bile acids are amphipathic molecules produced from cholesterol by hepatocytes and released into the gastrointestinal lumen to facilitate dietary lipids solubilization and absorption.68 gut microbiota is implicated directly in the host metabolism of bile acids. the bile salts are efficient in enterohepatic recycling, which depends on bile acid deconjugation and dihydroxylation by microbial-derived bile salt hydrolases (bsh), causing a rise in the secondary bile acids.69 the latter are more hydrophobic and can, therefore, be reabsorbed by passive diffusion, thus limiting the loss of bile acid via the feces. in the human gut microbiota, the specific activity of bsh varies in different phyla, where bsh of firmicutes and actinobacteria can metabolize all conjugated bile salts, while its activity in bacteroidetes is restricted to tauro-conjugated bile acids.69 additionally, bile acids are signal molecules are involved in peripheral metabolism and play significant roles via their action on both bile acid receptors, e.g., the g-protein coupled receptor tgr5 (gpbar1), and the nuclear receptor fxr, both of which express in enteroendocrine cells.68 moreover, the affinity and potency of the receptor vary considerably among the different bile acids. in this way, the microbiome in the intestinal tract can profoundly impact the host metabolism by modifying bile acid pool composition across modified synthesis and reuptake of bile acid.68 lipopolysaccharide (lps) membrane-bound lipopolysaccharide (lps) is a structural component of the gram-negative bacterial membrane. lps acts as a signaling molecule via numerous cellular pattern recognition receptors (prrs).70 furthermore, the first step in the cascading pro-inflammatory response is the translocation of lps. one of the most prrs reviewed are the toll-like receptors (tlrs).71 tlr-4 invigorated by bacterial lipopolysaccharides (lps), resulting in inflammatory responses, cytokine production, and chemokine-mediated recruitment of inflammatory cells.61 previous reports suggested that plasma concentrations of gut microbiota-derived lipopolysaccharide (lps) are increased due to the gut microbiota alterations which triggers the creation of the abovementioned inflammatory factors, by means of the cd14-dependent mechanism.72,73 several other studies found that lps elevation levels (endotoxemia) are clinically correlating with insulin resistance and obesity.74 epigenetics, gut microbiome, and type 2 diabetes mellitus newly different researches proved a direct link between the gut microbiome and epigenetic mechanisms in the pathogenesis of all diabetes types.75 epigenetics is identified as a heritable phenotype modifications that control gene expression without altering the dna sequence. the epigenetic mechanisms in eukaryotes include mainly dna methylation, fig. 3 microbes and host metabolism in t2d, modified from (kristine et al., 2015).77 367j contemp med sci | vol. 8, no. 6, november-december 2022: 363–369 a.m. alkhammash et al. original link of gut microbiome with risk of type 2 diabetes mellitus chromatin restructuring, post-transcriptional histone modifications, and gene expression regulation through non-coding rnas.76 many factors are involved in regulating epigenetic mechanisms like the cross-talk of microbial metabolites, environmental influences including ph, oxygen, and temperature, and external influences such as antibiotics and diet, thus causing alteration of many metabolic diseases in humans.78 the last findings provide new opportunities for using epigenetic regulations in altering the gut microbiota and thus their use in treating or delaying autoimmunity in patients. as mentioned earlier, evidence has emerged confirming that the community of gut microbiota is connected with type 2 diabetes as a chinese study indicated a reduction in species of roseburia and faecalibacterium besides the rise in escherichia coli in patients with type 2 diabetes than non-diabetic people (qin et al., 2012)45 which possess anti-inflammatory attributes.51,79 the microbial-produced scfa metabolites, like butyrate, play a crucial role in epigenetic modifications, such as hdac inhibition.80 furthermore, butyrate has a vital role in the differentiation of t cells (tregs) through acetylation of non-coding regions of the foxp3 locus.81 newly, experiments have emerged looking at the relationship between changes in the gut microbial community in people with type 2 diabetes and their association with metabolic and epigenetic mechanisms have undoubtedly demonstrated that epigenetic modulations such as dna methylation are linked to type 2 diabetes.6 genome-wide association studies (gwas) have shown an association between single nucleotide polymorphisms (snps) in type 2 diabetes and the defect in insulin secretion, which links with the defect in the cells of the pancreatic islet.82 in 2014, dayeh et al. suggested contributing epigenetic mechanisms with insulin resistance regulation and developmental process in type 2 diabetes through pancreatic cells. this study has analyzed the levels of dna methylation of cpg sites and transcriptome in pancreatic islets in both patients with type 2 diabetes and healthy individuals. the authors have analyzed the levels of dna methylation of cpg sites and transcriptome in pancreatic islets in both patients with type 2 diabetes and healthy individuals. therefore, they identified 1,649 cpg sites and 853 genes involving kcnq1, tcf7l2, and fto, with differential dna methylation in islets from type 2 diabetes patients. their findings also involve 102 genes exhibiting differential dna methylation and differential gene expressions such as cdkn1a, pde7b, sept9, and exoc3l2 genes in islets from type 2 diabetes. those genes are crucial for regulating insulin secretion in b-cells and glucagon secretion in pancreatic a cells.83 conclusion gut bacteria have an essential role in developing many diseases, especially type 2 diabetes, as available evidence indicated the association between microbiome dysbiosis and t2d. several studies have demonstrated that gut microbial composition differs significantly between type 2 diabetics and healthy individuals. host glucose metabolism was shown to be affected by microbial metabolites produced by gut microbes, which are involved in different metabolic pathways. also, several studies have revealed that people with type 2 diabetes have a less abundant gut microbial community of scfas butyrate-producing bacteria, which reflects their dietary pattern that depends on consuming a high-fat high-glycemic index, and low-fiber dietary compared to a healthy diet. future efforts should integrate microbial metabolites with metagenomic sequencing (mgs) studies, search for causes affecting their production, and identify possible pathogenic metabolites. in addition, various studies have implicated the gut microbiome’s interactions with the epigenome of the host, which indicates the possible role of gut microbiome in controlling host metabolism. modulation of gut microbiome to the host epigenome might result from the direct and repeated contact with the host and various metabolites derived from microbes that are generated in the intestine. moreover, many studies have a focus on the gut microbiome’s epigenetic mechanisms and its impact on t2d. this new field may hold a promising future for discovering new therapeutic solutions that can convert gut microbiome dysbiosis to a healthy and balanced state and thus provide new insights towards preventing and treating t2d in the future. conflict of interest none.  references 1. mohammad, s. & ahmad, j. 2016. management of obesity in patients with type 2 diabetes mellitus in primary care. diabetes metab syndr, 10, 171–81. 2. upadhyaya, s. & banerjee, g. 2015. type 2 diabetes and gut microbiome: at the intersection of known and unknown. gut microbes, 6, 85–92. 3. internation diabetes federation. 2019. mena members [online]. available: https://www.idf.org/our-network/regions-members/middle-east-andnorth-africa/members/46-saudi-arabia.html [accessed feb 26, 2020]. 4. langenberg, c. & lotta, l. a. 2018. genomic insights into the causes of type 2 diabetes. the lancet, 391, 2463–2474. 5. stumvoll, m., goldstein, b. j. & van haeften, t. w. 2005. type 2 diabetes: principles of pathogenesis and therapy. lancet, 365, 1333–46. 6. sharma, m., li, y., stoll, m. l. & tollefsbol, t. o. 2020. the epigenetic connection between the gut microbiome in obesity and diabetes. frontiers in genetics, 10. 7. shoelson, s. e., lee, j. & goldfine, a. b. 2006. inflammation and insulin resistance. the journal of clinical investigation, 116, 1793–1801. 8. sikalidis, a. k. & maykish, a. 2020. the gut microbiome and type 2 diabetes mellitus: discussing a complex relationship. biomedicines, 8, 8. 9. burcelin, r., serino, m., chabo, c., blasco-baque, v. & amar, j. 2011. gut microbiota and diabetes: from pathogenesis to therapeutic perspective. acta diabetologica, 48, 257–273. 10. nicholson, j. k., holmes, e., kinross, j., burcelin, r., gibson, g., jia, w. & pettersson, s. 2012. host-gut microbiota metabolic interactions. science, 336, 1262–7. 11. jandhyala, s. m., talukdar, r., subramanyam, c., vuyyuru, h., sasikala, m. & nageshwar reddy, d. 2015. role of the normal gut microbiota. world j gastroenterol, 21, 8787–803. 12. qin, j., li, r., raes, j., arumugam, m., burgdorf, k. s., manichanh, c., nielsen, t., pons, n., levenez, f., yamada, t., mende, d. r., li, j., xu, j., li, s., li, d., cao, j., wang, b., liang, h., zheng, h., xie, y., tap, j., lepage, p., bertalan, m., batto, j. m., hansen, t., le paslier, d., linneberg, a., nielsen, h. b., pelletier, e., renault, p., sicheritz-ponten, t., turner, k., zhu, h., yu, c., li, s., jian, m., zhou, y., li, y., zhang, x., li, s., qin, n., yang, h., wang, j., brunak, s., dore, j., guarner, f., kristiansen, k., pedersen, o., parkhill, j., weissenbach, j., bork, p., ehrlich, s. d. & wang, j. 2010. a human gut microbial gene catalogue established by metagenomic sequencing. nature, 464, 59–65. 13. huttenhower, c., gevers, d., knight, r., abubucker, s., badger, j. h., chinwalla, a. t., creasy, h. h., earl, a. m., fitzgerald, m. g., fulton, r. s., giglio, m. g., hallsworth-pepin, k., lobos, e. a., madupu, r., magrini, v., martin, j. c., mitreva, m., muzny, d. m., sodergren, e. j., versalovic, j., wollam, a. m., worley, k. c., wortman, j. r., young, s. k., zeng, q., aagaard, k. m., abolude, o. o., allen-vercoe, e., alm, e. j., alvarado, l., andersen, g. l., anderson, s., https://www.idf.org/our-network/regions-members/middle-east-and-north-africa/members/46-saudi-arabia.html https://www.idf.org/our-network/regions-members/middle-east-and-north-africa/members/46-saudi-arabia.html 368 j contemp med sci | vol. 8, no. 6, november-december 2022: 363–369 link of gut microbiome with risk of type 2 diabetes mellitus original a.m. alkhammash et al. appelbaum, e., arachchi, h. m., armitage, g., arze, c. a., ayvaz, t., baker, c. c., begg, l., belachew, t., bhonagiri, v., bihan, m., blaser, m. j., bloom, t., bonazzi, v., paul brooks, j., buck, g. a., buhay, c. j., busam, d. a., campbell, j. l., canon, s. r., cantarel, b. l., chain, p. s. g., chen, i. m. a., chen, l., chhibba, s., chu, k., ciulla, d. m., clemente, j. c., clifton, s. w., conlan, s., crabtree, j., cutting, m. a., davidovics, n. j., davis, c. c., desantis, t. z., deal, c., delehaunty, k. d., dewhirst, f. e., deych, e., ding, y., dooling, d. j., dugan, s. p., michael dunne, w., scott durkin, a., edgar, r. c., erlich, r. l., farmer, c. n., farrell, r. m., faust, k., feldgarden, m., felix, v. m., fisher, s., fodor, a. a., forney, l. j., foster, l., di francesco, v., friedman, j., friedrich, d. c., fronick, c. c., fulton, l. l., gao, h., garcia, n., giannoukos, g., giblin, c., giovanni, m. y., goldberg, j. m., goll, j., gonzalez, a., griggs, a., et al. 2012. structure, function and diversity of the healthy human microbiome. nature, 486, 207–214. 14. barengolts, e. 2016. gut microbiota, prebiotics, probiotics, and synbiotics in management of obesity and prediabetes: review of randomized controlled trials. endocr pract, 22, 1224–1234. 15. clemente, j. c., ursell, l. k., parfrey, l. w. & knight, r. 2012. the impact of the gut microbiota on human health: an integrative view. cell, 148, 1258–70. 16. fujimura, k. e., slusher, n. a., cabana, m. d. & lynch, s. v. 2010. role of the gut microbiota in defining human health. expert review of anti-infective therapy, 8, 435–454. 17. faith, j. j., guruge, j. l., charbonneau, m., subramanian, s., seedorf, h., goodman, a. l., clemente, j. c., knight, r., heath, a. c., leibel, r. l., rosenbaum, m. & gordon, j. i. 2013. the long-term stability of the human gut microbiota. science, 341, 1237439. 18. sharma, r. & prajapati, p. k. 2015. rising risk of type 2 diabetes among inhabitants of jamnagar, gujarat: a cross-sectional survey. ayu, 36, 10–7. 19. hagerty, s. l., ellingson, j. m., helmuth, t. b., bidwell, l. c., hutchison, k. e. & bryan, a. d. 2019. an overview and proposed research framework for studying co-occurring mentaland physical-health dysfunction. perspectives on psychological science, 14, 633–645. 20. gevers, d., knight, r., petrosino, j. f., huang, k., mcguire, a. l., birren, b. w., nelson, k. e., white, o., methe, b. a. & huttenhower, c. 2012. the human microbiome project: a community resource for the healthy human microbiome. plos biology, 10, e1001377. 21. turnbaugh, p. j., ley, r. e., hamady, m., fraser-liggett, c. m., knight, r. & gordon, j. i. 2007. the human microbiome project. nature, 449, 804–810. 22. ley, r. e., turnbaugh, p. j., klein, s. & gordon, j. i. 2006. human gut microbes associated with obesity. nature, 444, 1022–1023. 23. lozupone, c. a., stombaugh, j. i., gordon, j. i., jansson, j. k. & knight, r. 2012. diversity, stability and resilience of the human gut microbiota. nature, 489, 220–230. 24. david, l. a., maurice, c. f., carmody, r. n., gootenberg, d. b., button, j. e., wolfe, b. e., ling, a. v., devlin, a. s., varma, y., fischbach, m. a., biddinger, s. b., dutton, r. j. & turnbaugh, p. j. 2014. diet rapidly and reproducibly alters the human gut microbiome. nature, 505, 559–563. 25. carabotti, m., scirocco, a., maselli, m. a. & severi, c. 2015. the gut-brain axis: interactions between enteric microbiota, central and enteric nervous systems. annals of gastroenterology, 28, 203–209. 26. grenham, s., clarke, g., cryan, j. f. & dinan, t. g. 2011. brain-gut-microbe communication in health and disease. frontiers in physiology, 2, 94–94. 27. chen, x., d’souza, r. & hong, s.-t. 2013. the role of gut microbiota in the gut-brain axis: current challenges and perspectives. protein & cell, 4, 403–414. 28. bäckhed, f., ley, r. e., sonnenburg, j. l., peterson, d. a. & gordon, j. i. 2005. host-bacterial mutualism in the human intestine. science, 307, 1915–20. 29. rapin, a., pattaroni, c., marsland, b. j. & harris, n. l. 2017. microbiota analysis using an illumina miseq platform to sequence 16s rrna genes. curr protoc mouse biol, 7, 100–129. 30. woese, c. r. & fox, g. e. 1977. phylogenetic structure of the prokaryotic domain: the primary kingdoms. proceedings of the national academy of sciences, 74, 5088–5090. 31. van de peer, y., chapelle, s. & de wachter, r. 1996. a quantitative map of nucleotide substitution rates in bacterial rrna. nucleic acids research, 24, 3381–3391. 32. caporaso, j. g., kuczynski, j., stombaugh, j., bittinger, k., bushman, f. d., costello, e. k., fierer, n., peña, a. g., goodrich, j. k., gordon, j. i., huttley, g. a., kelley, s. t., knights, d., koenig, j. e., ley, r. e., lozupone, c. a., mcdonald, d., muegge, b. d., pirrung, m., reeder, j., sevinsky, j. r., turnbaugh, p. j., walters, w. a., widmann, j., yatsunenko, t., zaneveld, j. & knight, r. 2010. qiime allows analysis of high-throughput community sequencing data. nature methods, 7, 335–336. 33. schloss, p. d., westcott, s. l., ryabin, t., hall, j. r., hartmann, m., hollister, e. b., lesniewski, r. a., oakley, b. b., parks, d. h., robinson, c. j., sahl, j. w., stres, b., thallinger, g. g., van horn, d. j. & weber, c. f. 2009. introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. applied and environmental microbiology, 75, 7537. 34. desantis, t. z., hugenholtz, p., larsen, n., rojas, m., brodie, e. l., keller, k., huber, t., dalevi, d., hu, p. & andersen, g. l. 2006. greengenes, a chimerachecked 16s rrna gene database and workbench compatible with arb. applied and environmental microbiology, 72, 5069. 35. quast, c., pruesse, e., yilmaz, p., gerken, j., schweer, t., yarza, p., peplies, j. & glöckner, f. o. 2013. the silva ribosomal rna gene database project: improved data processing and web-based tools. nucleic acids research, 41, d590–d596. 36. gotelli, n. & chao, a. 2013. measuring and estimating species richness, species diversity, and biotic similarity from sampling data. 37. petersen, c. & round, j. l. 2014. defining dysbiosis and its influence on host immunity and disease. cell microbiol, 16, 1024–33. 38. moreno-indias, i., cardona, f., tinahones, f. j. & queipo-ortuño, m. i. 2014. impact of the gut microbiota on the development of obesity and type 2 diabetes mellitus. frontiers in microbiology, 5. 39. le chatelier, e., nielsen, t., qin, j., prifti, e., hildebrand, f., falony, g., almeida, m., arumugam, m., batto, j. m., kennedy, s., leonard, p., li, j., burgdorf, k., grarup, n., jørgensen, t., brandslund, i., nielsen, h. b., juncker, a. s., bertalan, m., levenez, f., pons, n., rasmussen, s., sunagawa, s., tap, j., tims, s., zoetendal, e. g., brunak, s., clement, k., dore, j., kleerebezem, m., kristiansen, k., renault, p., sicheritz-ponten, t., de vos, w. m., zucker, j. d., raes, j., hansen, t., bork, p., wang, j., ehrlich, s. d. & pedersen, o. 2013. richness of human gut microbiome correlates with metabolic markers. nature, 500, 541–6. 40. amar, j., serino, m., lange, c., chabo, c., iacovoni, j., mondot, s., lepage, p., klopp, c., mariette, j., bouchez, o., perez, l., courtney, m., marre, m., klopp, p., lantieri, o., dore, j., charles, m., balkau, b. & burcelin, r. 2011. involvement of tissue bacteria in the onset of diabetes in humans: evidence for a concept. diabetologia, 54, 3055–61. 41. larsen, n., vogensen, f. k., van den berg, f. w. j., nielsen, d. s., andreasen, a. s., pedersen, b. k., al-soud, w. a., sørensen, s. j., hansen, l. h. & jakobsen, m. 2010. gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. plos one, 5, e9085. 42. louis, p. & flint, h. j. 2009. diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. fems microbiology letters, 294, 1–8. 43. van den abbeele, p., belzer, c., goossens, m., kleerebezem, m., de vos, w. m., thas, o., de weirdt, r., kerckhof, f.-m. & van de wiele, t. 2013. butyrateproducing clostridium cluster xiva species specifically colonize mucins in an in vitro gut model. the isme journal, 7, 949–961. 44. vrieze, a., van nood, e., holleman, f., salojärvi, j., kootte, r. s., bartelsman, j. f., dallinga-thie, g. m., ackermans, m. t., serlie, m. j., oozeer, r., derrien, m., druesne, a., van hylckama vlieg, j. e., bloks, v. w., groen, a. k., heilig, h. g., zoetendal, e. g., stroes, e. s., de vos, w. m., hoekstra, j. b. & nieuwdorp, m. 2012. transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome. gastroenterology, 143, 913-6.e7. 45. qin, j., li, y., cai, z., li, s., zhu, j., zhang, f., liang, s., zhang, w., guan, y., shen, d., peng, y., zhang, d., jie, z., wu, w., qin, y., xue, w., li, j., han, l., lu, d., wu, p., dai, y., sun, x., li, z., tang, a., zhong, s., li, x., chen, w., xu, r., wang, m., feng, q., gong, m., yu, j., zhang, y., zhang, m., hansen, t., sanchez, g., raes, j., falony, g., okuda, s., almeida, m., lechatelier, e., renault, p., pons, n., batto, j. m., zhang, z., chen, h., yang, r., zheng, w., li, s., yang, h., wang, j., ehrlich, s. d., nielsen, r., pedersen, o., kristiansen, k. & wang, j. 2012. a metagenome-wide association study of gut microbiota in type 2 diabetes. nature, 490, 55–60. 46. karlsson, f. h., tremaroli, v., nookaew, i., bergström, g., behre, c. j., fagerberg, b., nielsen, j. & bäckhed, f. 2013. gut metagenome in european women with normal, impaired and diabetic glucose control. nature, 498, 99–103. 47. forslund, k., hildebrand, f., nielsen, t., falony, g., le chatelier, e., sunagawa, s., prifti, e., vieira-silva, s., gudmundsdottir, v., pedersen, h. k., arumugam, m., kristiansen, k., voigt, a. y., vestergaard, h., hercog, r., costea, p. i., kultima, j. r., li, j., jørgensen, t., levenez, f., dore, j., nielsen, h. b., brunak, s., raes, j., hansen, t., wang, j., ehrlich, s. d., bork, p. & pedersen, o. 2015. disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota. nature, 528, 262–266. 48. furet, j.-p., kong, l.-c., tap, j., poitou, c., basdevant, a., bouillot, j.-l., mariat, d., corthier, g., dore, j., henegar, c., rizkalla, s. & clement, k. 2010. differential adaptation of human gut microbiota to bariatric surgery– induced weight loss. diabetes, 59, 3049. 369j contemp med sci | vol. 8, no. 6, november-december 2022: 363–369 a.m. alkhammash et al. original link of gut microbiome with risk of type 2 diabetes mellitus 49. pedersen, h. k., gudmundsdottir, v., nielsen, h. b., hyotylainen, t., nielsen, t., jensen, b. a. h., forslund, k., hildebrand, f., prifti, e., falony, g., le chatelier, e., levenez, f., dore, j., mattila, i., plichta, d. r., pöhö, p., hellgren, l. i., arumugam, m., sunagawa, s., vieira-silva, s., jørgensen, t., holm, j. b., trošt, k., consortium, m., kristiansen, k., brix, s., raes, j., wang, j., hansen, t., bork, p., brunak, s., oresic, m., ehrlich, s. d. & pedersen, o. 2016. human gut microbes impact host serum metabolome and insulin sensitivity. nature, 535, 376–381. 50. radwan, s., gilfillan, d., eklund, b., radwan, h. m., el menofy, n. g., lee, j., kapuscinski, m. & abdo, z. 2020. a comparative study of the gut microbiome in egyptian patients with type i and type ii diabetes. plos one, 15, e0238764. 51. zhang, x., shen, d., fang, z., jie, z., qiu, x., zhang, c., chen, y. & ji, l. 2013. human gut microbiota changes reveal the progression of glucose intolerance. plos one, 8, e71108. 52. dao, m. c., everard, a., aron-wisnewsky, j., sokolovska, n., prifti, e., verger, e. o., kayser, b. d., levenez, f., chilloux, j., hoyles, l., dumas, m.-e., rizkalla, s. w., dore, j., cani, p. d. & clement, k. 2016. akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology. gut, 65, 426. 53. shin, n. r., lee, j. c., lee, h. y., kim, m. s., whon, t. w., lee, m. s. & bae, j. w. 2014. an increase in the akkermansia spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice. gut, 63, 727–35. 54. zhao, l., lou, h., peng, y., chen, s., zhang, y. & li, x. 2019. comprehensive relationships between gut microbiome and faecal metabolome in individuals with type 2 diabetes and its complications. endocrine, 66, 526–537. 55. falony, g., joossens, m., vieira-silva, s., wang, j., darzi, y., faust, k., kurilshikov, a., bonder, m. j., valles-colomer, m., vandeputte, d., tito, r. y., chaffron, s., rymenans, l., verspecht, c., de sutter, l., lima-mendez, g., d’hoe, k., jonckheere, k., homola, d., garcia, r., tigchelaar, e. f., eeckhaudt, l., fu, j., henckaerts, l., zhernakova, a., wijmenga, c. & raes, j. 2016. population-level analysis of gut microbiome variation. science, 352, 560–4. 56. khan, muhammad t., nieuwdorp, m. & bäckhed, f. 2014. microbial modulation of insulin sensitivity. cell metabolism, 20, 753–760. 57. cummings, j. h., pomare, e. w., branch, w. j., naylor, c. p. & macfarlane, g. t. 1987. short chain fatty acids in human large intestine, portal, hepatic and venous blood. gut, 28, 1221–7. 58. mowat, a. m. & agace, w. w. 2014. regional specialization within the intestinal immune system. nature reviews immunology, 14, 667–685. 59. koh, a., de vadder, f., kovatcheva-datchary, p. & bäckhed, f. 2016. from dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites. cell, 165, 1332–1345. 60. reichardt, n., duncan, s. h., young, p., belenguer, a., mcwilliam leitch, c., scott, k. p., flint, h. j. & louis, p. 2014. phylogenetic distribution of three pathways for propionate production within the human gut microbiota. isme j, 8, 1323–35. 61. reimann, f., tolhurst, g. & gribble, f. m. 2012. g-protein-coupled receptors in intestinal chemosensation. cell metab, 15, 421–31. 62. lin, h. v., frassetto, a., kowalik, e. j., jr., nawrocki, a. r., lu, m. m., kosinski, j. r., hubert, j. a., szeto, d., yao, x., forrest, g. & marsh, d. j. 2012. butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms. plos one, 7, e35240. 63. frost, g., sleeth, m. l., sahuri-arisoylu, m., lizarbe, b., cerdan, s., brody, l., anastasovska, j., ghourab, s., hankir, m., zhang, s., carling, d., swann, j. r., gibson, g., viardot, a., morrison, d., louise thomas, e. & bell, j. d. 2014. the short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism. nat commun, 5, 3611. 64. perry, r. j., peng, l., barry, n. a., cline, g. w., zhang, d., cardone, r. l., petersen, k. f., kibbey, r. g., goodman, a. l. & shulman, g. i. 2016. acetate mediates a microbiome-brain-β-cell axis to promote metabolic syndrome. nature, 534, 213–7. 65. bindels, l. b., dewulf, e. m. & delzenne, n. m. 2013. gpr43/ffa2: physiopathological relevance and therapeutic prospects. trends in pharmacological sciences, 34, 226–232. 66. everard, a. & cani, p. d. 2014. gut microbiota and glp-1. rev endocr metab disord, 15, 189–96. 67. chimerel, c., emery, e., summers, david k., keyser, u., gribble, fiona m. & reimann, f. 2014. bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine l cells. cell reports, 9, 1202–1208. 68. martin, a. m., sun, e. w., rogers, g. b. & keating, d. j. 2019. the influence of the gut microbiome on host metabolism through the regulation of gut hormone release. frontiers in physiology, 10. 69. jones, b. v., begley, m., hill, c., gahan, c. g. m. & marchesi, j. r. 2008. functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome. proceedings of the national academy of sciences, 105, 13580–13585. 70. gordon, s. 2002. pattern recognition receptors: doubling up for the innate immune response. cell, 111, 927–930. 71. delzenne, n. m. & cani, p. d. 2011. gut microbiota and the pathogenesis of insulin resistance. current diabetes reports, 11, 154. 72. cani, p. d., bibiloni, r., knauf, c., waget, a., neyrinck, a. m., delzenne, n. m. & burcelin, r. 2008. changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet–induced obesity and diabetes in mice. diabetes, 57, 1470–1481. 73. pendyala, s., walker, j. m. & holt, p. r. 2012. a high-fat diet is associated with endotoxemia that originates from the gut. gastroenterology, 142, 1100–1101.e2. 74. cani, p. d., amar, j., iglesias, m. a., poggi, m., knauf, c., bastelica, d., neyrinck, a. m., fava, f., tuohy, k. m., chabo, c., waget, a., delmee, e., cousin, b., sulpice, t., chamontin, b., ferrieres, j., tanti, j.-f., gibson, g. r., casteilla, l., delzenne, n. m., alessi, m. c. & burcelin, r. 2007. metabolic endotoxemia initiates obesity and insulin resistance. diabetes, 56, 1761–1772. 75. allin, k. h., nielsen, t. & pedersen, o. 2015. mechanisms in endocrinology: gut microbiota in patients with type 2 diabetes mellitus. eur j endocrinol, 172, r167–77. 76. paul, b., barnes, s., demark-wahnefried, w., morrow, c., salvador, c., skibola, c. & tollefsbol, t. o. 2015. influences of diet and the gut microbiome on epigenetic modulation in cancer and other diseases. clinical epigenetics [online], 7. available: http://europepmc.org/abstract/ med/26478753 https://doi.org/10.1186/s13148-015-0144-7 https:// europepmc.org/articles/pmc4609101 https://europepmc.org/articles/ pmc4609101?pdf=render [accessed 2015]. 77. bu, p., wang, l., chen, k. y., rakhilin, n., sun, j., closa, a., et al. 2015. mir1269 promotes metastasis and forms a positive feedback loop with tgf-β. nature communications, 6(1), 1–12. 78. romano, k. a. & rey, f. e. 2018. is maternal microbial metabolism an earlylife determinant of health? lab animal, 47, 239–243. 79. remely, m., aumueller, e., jahn, d., hippe, b., brath, h. & haslberger, a. g. 2014. microbiota and epigenetic regulation of inflammatory mediators in type 2 diabetes and obesity. benef microbes, 5, 33–43. 80. donohoe, d. r., holley, d., collins, l. b., montgomery, s. a., whitmore, a. c., hillhouse, a., curry, k. p., renner, s. w., greenwalt, a., ryan, e. p., godfrey, v., heise, m. t., threadgill, d. s., han, a., swenberg, j. a., threadgill, d. w. & bultman, s. j. 2014. a gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiotaand butyrate-dependent manner. cancer discovery, 4, 1387–1397. 81. furusawa, y., obata, y., fukuda, s., endo, t. a., nakato, g., takahashi, d., nakanishi, y., uetake, c., kato, k., kato, t., takahashi, m., fukuda, n. n., murakami, s., miyauchi, e., hino, s., atarashi, k., onawa, s., fujimura, y., lockett, t., clarke, j. m., topping, d. l., tomita, m., hori, s., ohara, o., morita, t., koseki, h., kikuchi, j., honda, k., hase, k. & ohno, h. 2013. commensal microbe-derived butyrate induces the differentiation of colonic regulatory t cells. nature, 504, 446–50. 82. ruchat, s.-m., elks, c. e., loos, r. j. f., vohl, m.-c., weisnagel, s. j., rankinen, t., bouchard, c. & perusse, l. 2008. association between insulin secretion, insulin sensitivity and type 2 diabetes susceptibility variants identified in genome-wide association studies. acta diabetologica, 46, 217. 83. dayeh, t., volkov, p., salö, s., hall, e., nilsson, e., olsson, a. h., kirkpatrick, c. l., wollheim, c. b., eliasson, l., rönn, t., bacos, k. & ling, c. 2014. genome-wide dna methylation analysis of human pancreatic islets from type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion. plos genetics, 10, e1004160. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1305 http://europepmc.org/abstract/med/26478753 http://europepmc.org/abstract/med/26478753 https://doi.org/10.1186/s13148-015-0144-7 https://europepmc.org/articles/pmc4609101 https://europepmc.org/articles/pmc4609101 https://europepmc.org/articles/pmc4609101?pdf=render https://europepmc.org/articles/pmc4609101?pdf=render 45j contemp med sci | vol. 4, no. 1, winter 2018: 45–50 research correlation between live attenuated measles viral load and growth inhibition percentage in non-small cell lung cancer cell line rasha fadhel obaid,a younis a. al-khafaji,b hamid n. obied,c salman a. al-jibouri,a salam j. mohammed,d and rana f. al-kilabia adepartment of medical microbiology, college of medicine, university of kufa, najaf, iraq. bdepartment of medical microbiology, college of dentistry, university of babylon, babylon, iraq. cdepartment of pharmacology, college of medicine, university of babylon, babylon, iraq. ddepartment of community medicine, college of medicine, university of kufa, najaf, iraq. correspondence to rasha fadhel obaid (email: rasha20_12@yahoo.com). (submitted: 06 october 2017 – revised version received: 17 october 2017 – accepted: 13 november 2017 – published online: 26 march 2018) objective this study was performed to evaluate the anticancer effect of live attenuated measles virus (edmonston strain) on human lung cancer cell line in vitro. methods human lung cancer cell line (a549) was treated with different titers of the propagated live attenuated measles virus on vero cells, including 10−1, 10−2, 10−3, 10−4, 10−5 of virus stock (8.860 log 10 rna copies/µl). the virus cytopathic effect (cpe) was studied at 24, 48, 72, and 96 hr. oncolytic effects of the virus were assessed by cytotoxicity assay, apoptosis analysis, and proliferation marker ki-67. the growth inhibition percentage was determined by enzyme-linked immunosorbent assay (elisa) reader using crystal violet staining assay and compared with the viral load measured by quantitative real time-pcr. results the results of the study showed that measles virus had an inhibitory effect on the growth of human lung cancer cell line compared with untreated control group. there was highly significant positive correlation between viral load and pgi at 24, 48, 72, 96 hr (p < 0.001). the growth inhibition rate of a cell was associated with a decrement in the expression of the ki-67 protein and increase in the ratio of early and late apoptotic cells in comparison with untreated control group. conclusion live attenuated measles virus strain induced cytotoxic effect against human lung cancer cell line (a549) by induction of apoptosis as an important mechanism of anti-tumor activity, in addition, it indicates a correlation between the quantity of mv genomes and percentage of growth inhibition. this relation has proved that measles virus had anticancer effect. keywords annexin-v, cytotoxicity, ki-67 protein, lung cancer cell line (a549), measles virus, qrt-pcr introduction viral therapy is one of the most promising branches of medicine which detect viruses that can kill cancer cells. the detected viruses can be used as a method to treat malignant tumors, and this is done through the proliferation of the virus inside malignant tumor cells.1,2 cancer is one of the leading worldwide causes of death, and despite the considerable progress made by researchers in the diversity of cancer treatment methods, the mortality rate from malignant tumors are still alarmingly high, so discouraging cancer growth and reducing its spread is one of the major challenges facing modern medicine.3 the current goal for developing new therapies for the treatment of cancer is to design or select therapeutic agents that have a high therapeutic index (i.e. high potency against malignant cells) with little or no toxicities to normal cells.4 an oncolytic virus, is a type of viruses that are highly selective to tumor cells: it can replicate, spread and proliferated within the tumor cells without affecting normal tissue,5 and genetically programmed to replicate within cancer cells and directly induce cytotoxic effect through cell lysis or apoptosis.6 the use of replicating viruses for cancer therapy is attracting increasing interest. the advantages of virotherapy include the potential lack of cross-resistance with standard therapies and the ability to cause tumor destruction by numerous mechanisms.7 a variety of viruses have been developed as oncolytic therapies, including adenovirus, vaccinia virus, herpesvirus, coxsackie a virus, newcastle disease virus, and reovirus.8 during the past decades, measles virus vaccine strains have emerged oncolytic platform that may not be compromised by pre existing mv immunity.9 measles virus vaccine edmonston or schwarz strains uses mainly cd46 molecules to infect cells, during cancer development, tumor cells are often selected to express high levels of cd46 molecules, which inhibit the complement system.10 this cd46 overexpression makes the tumor cells less sensitive to lysis by the complement but highly sensitive to measles vaccine virus infection.11 this study was performed to evaluate the antitumor effect of live attenuated measles virus (edmonton) vaccine strain on human lung tumor cell line in vitro, and determination of the correlation between the load of measles virus genomes and percentage of growth inhibition at different time intervals. materials and methods cell culture the human lung cancer cell line (a549) and vero cell line were purchased from national cell bank of iran-pasteur institute (ncbi). cell line was maintained in roswell park memorial institute medium 1640 (rpmi-1640, gibco-brl), supplemented with 10 % fetal bovine serum (gibco) and 100 µl/ml penicillin/streptomycin (sigma). cells were incubated at 37°c in 5% co2 incubator for 24 hr or until a confluent monolayer formed. the cells were subcultured for several times and then seeded into 96-well microplates at a concentration of 4 × 105 cells per well with complete rpmi-1640 growth medium.12 issn 2413-0516 46 j contemp med sci | vol. 4, no. 1, winter 2018: 45–50 correlation between live attenuated measles viral load and growth inhibition percentage research rasha fadhel obaid et al. virus preparation live attenuated measles virus (edmonston) were obtained from vaccine and sera institute in baghdad, iraq. each vial vaccine contains 10 doses of lyophilized hyper attenuated measles virus, each dose contains at least 1000 ccid50. virus prepared as recommended by manufacturing company by adding 5 ml of sterile deionized distilled water (ddw) to dissolve the lyophilized powder and shake well before use. propagation of measles virus vaccine on vero cell line in this study, lives attenuated measles virus was propagated on vero cell line after 90% confluence. growth medium was decanted, cells washed with sterile pbs (ph 7.2) then inoculated with 2 ml of (diluted hyper attenuated measles virus strain), and incubated at 37°c for 2 hr with mild rolling of the flask every 10 min. after the end of incubation period, the remaining virus inoculum was discarded and the cells were maintained with 7 ml of serum-free rpmi-1640 medium. the cells were re-incubated at 37°c for 3–7 days, when syncytia reached 80–90% of infected culture. the supernatant medium from each flask was collected aseptically in sterile tubes and the remaining attached cells were scraped then underwent freezing and thawing for three times, and centrifugation of all harvested culture at 3000 rpm for 30 min at 4°c in cold centrifuge, and virus was propagated on vero cell line from passage 1 till passage 8 and virus titer was assayed by real-time polymerase chain reaction (rt-pcr) in each viral passage.13–15 exposure stage human lung cancer cell line (a549) were cultured in 96-wellflat bottom microtitration plates and incubated with serial titer dilutions of measles virus (concentrated inoculums = 8.860 log10). the plated cells were divided into two groups: group-1 was the control group (untreated); group-2 cell line was treated with measles virus. three replicates were used for each virus titer dilution, as well as the control group which was treated with serum-free medium only. the plates were incubated at 37°c in 5% co2 incubator for 24, 48, 72, and 96 hr. 16 then viral load, ki-67 expression, apoptosis, and cytotoxicity of measles virus were estimated. assessment of measles virus cytotoxicity by crystal violet assay crystal violet (cv) assay was used to determine the optical density of the cell growth in each well of the microtiter plate by using elisa reader. it is a technique used in cell culture laboratories to detect remaining adherent cells that staining with crystal violet dye, which binds to proteins and dna. this technique was performed according to method described by castro-garza et al.12 assessment of apoptosis by annexin v-fitc annexin-v concentrations was determinated in cell culture supernatant by quantitative elisa kit (us biological): the test was performed according to the instructions of the kit manual of manufacturing company. estimation human ki-67 protein ki-67 protein concentrations was determinated in cell culture supernatant by quantitative elisa kit (elabscience): the test was performed according to instructions of the kit manual of manufacturing company. molecular test for detection viral load of measles virus in cell culture (vero and lung cell lines) supernatant by qrt-pcr primers and probe the primers and probe were designed by using the complete sequence of nucleoprotein in measles virus using ncbi genebank and primer3 online and these primers were provided by bioneer company, korea as viral rna extraction viral rna was extracted from frozen cell culture samples by using accuzol tm total rna extraction kit (bioneer, korea) the extraction process was proceeded according to the instructions of the manufacturing company. reverse transcription real-time pcr real-time-pcr was performed for detection and quantitation of measles virus in cell culture experimental samples based on specific primers and probe for nucleoprotein gene in measles virus and this technique was performed according to the method described by hummel et al.17 statistical analyses statistical analysis was done by using spss version 20 in which, mean and standard deviation were used as descriptive statistics and analysis of variance with lsd for comparison between groups. p value ≤ 0.05 regarded significant. results detection of measles virus load in supernatant of infected vero line by qrt-pcr the viral titer was determined by the quantitative rt-pcr (qrt-pcr) assay. this assay was used to detect the mv n gene of rna genome, starting from the first passage up to eighth passage. the titer of propagated human measles virus on vero cell was increased gradually with the subsequent passage number until it reached maximum titer at the eighth passage which it was 8.860 log10. table 1 and fig. 1 reflected a significant increase in mv viral load as detected by qrt-pcr, which reached its maximum viral load at passage 8 (p < 0.05). figure 1 revealed that the threshold cycle ct value of rt-pcr test as an indicator of the viral load detected in supernatant infected vero cell culture. the lowest the threshold cycle ct values indicated the highest mv rna load were obtained. table 1. the primers and probe primer sequence product size measles n primer *f agacattgacactg-catcgg 89 bp *r aagattcctgccat-ggcttg measles n probe fam-aggtcagctgacgccctgct-bhq1 ncbi reference code: measles virus n gene for nucleoprotein, partial cds, strain: mvi/tokyo.jpn/38.00(ko) genbank: ab095426.1. *f, forward primer; *r, reverse primer. rasha fadhel obaid et al. 47j contemp med sci | vol. 4, no. 1, winter 2018: 45–50 research correlation between live attenuated measles viral load and growth inhibition percentage fig 2. real-time pcr standard curve of mvv nucleoprotein gene in supernatant vero cell culture at different passages, the log starting quantity curve was shown at rt-pcr efficiency 99.7%. fig 3. cytotoxic effect of measles virus vaccine on lung a549 cell line after 24, 48, 72, and 96 hr of exposure as evidenced by crystal violet assay (p < 0.001). correlation between the number of viral loads and percentage growth inhibition in lung cancer cell line to determine the effect of measles virus vaccine on cancer cells, we studied correlation between the number of viral genomes load and percentage growth inhibition. the growth inhibition percentage in lung (a549) cancer cell line was determined by elisa reader and compared with the viral load measured by qrt-pcr. cells were exposed to different titer of measles virus (10−1, 10−2, 10−3, 10−4, and 10−5) for 24, 48, 72, and 96 hr. standard curve shows that the threshold cycle (ct) on the y-axis and the starting quantity of rna target on the x-axis. slope, y-intercept, and correlation coefficient values are used to provide information about the performance of the reaction. the standard curve demonstrated a high linear correlation (r2) = 0.989, y-intercept = 42.675, the regression slope was −3.329, and efficiency was 99.7% (fig. 2). oncolytic effect of measles virus on human lung a549 cytotoxic effect of measles virus was investigated in human lung adenocarcinoma cell line exposed to different titers of measles virus (10−1, 10−2, 10−3, 10−4, and 10−5) for 24, 48, 72, 96 hr, and control group (untreated), then stained with crystal violet dye. the optical density for cell line cell was measured at 459 nm by elisa reader. the result of this study showed that measles virus had a growth inhibition effect on lung cell line in comparison with control group and the cytotoxic effect was increased proportionally with the viral load and time. figure 3 showed that the growth inhibition percentage of measles virus on human lung cell line at high titer (10−1) was 39.015, 70.642, 77.076, and 90.959% for 24, 48, 72, and 96 hr, respectively. while the growth inhibition percentage of measles virus at low titer (10−5) was 11.20, 19.843, 26.678, and 52.827% after 24, 48, 72, and 96 hr, respectively. however, fig. 3 reflected high significant differences in growth inhibition percentage between cell that was treated with measles virus and untreated control group (p < 0.001). fig 1. real-time pcr amplification plot of measles virus vaccine (mvv) nucleoprotein gene in supernatant of vero cell culture at different passages. the amplification plots were shown as red plot (p8), blue plot (p7), green plot (p6), yellow plot (p5), pink plot (p4), black plot (p3), orange plot (p2), and the gray plot (p1). fig 4a. correlation between viral load and percentage growth inhibition in lung cell line at 24, 48, 72, and 96 hr. viral load— , pgi— . 48 j contemp med sci | vol. 4, no. 1, winter 2018: 45–50 correlation between live attenuated measles viral load and growth inhibition percentage research rasha fadhel obaid et al. table 2. viral load detection by qrt-pcr. viral rna copies/µl in supernatant of vero cell culture infected with measles virus vaccine strain at different passages. mean ± sd for three replicates (p < 0.05) passage number viral load log 10 (rna copies/µl) mean ± sd p1 4.3300 ± 0.24434 p2 4.5967 ± 0.24007 p3 5.2067 ± 0.15044 p4 5.7467 ± 0.09292 p5 6.2600 ± 0.14177 p6 7.6967 ± 0.13868 p7 8.5067 ± 0.07024 p8 8.8600 ± 0.13528 figure 4a shows measles virus load in lung cell line treated with (10−1) virus was (6.94 log10) at 24 hr, and increased gradually until it reached maximum titer (10.336) at 96 hr. also, the percentage of growth inhibition of lung cancer cell line treated with (10−5) measles virus, was (39.015) at 24 hr, then increased gradually reaching it’s high mean value (90.959 log10) at 96 hr. fig. 4b revealed that the threshold cycle ct value of rt-pcr test as an indicator of the viral load which was detected in supernatant of infected lung cancer cell line culture at different durations of exposure. the lowest the threshold cycle ct values, indicate that higher mv rna load were obtained at dilution (10−1). growth inhibition rate at various time durations was increased proportionally with viral load in lung cell lines culture. the result demonstrated that there was a significant correlation between viral load measured by qrt-pcr and percentage of growth inhibition determined by elisa reader. this correlation proved that measles virus had anticancer cytotoxic effect. there is a significant strong positive correlation between viral load and pgi at 24, 48, 72, and 96 hr (p < 0.001). effect of measles virus on proliferation and apoptosis in lung cancer cell the result showed that measles virus-induced apoptosis and growth inhibition of cancer cells. the inhibition of cell growth was associated with a decrement in the expression of ki-67 protein and increased in the ratio of early and late apoptosis compared with control group. induction of cell apoptosis is an important mechanism of anti-tumor activity of measles virus. fig 4b. real-time pcr amplification plot of nucleoprotein gene of mvv in supernatant lung cell line at 24, 48, 72, 96 hr incubation. the positive viral amplification was shown to cross-up the threshold cycle line. the amplification plots were shown as red plot for virus dilution (10−1), blue plot for virus dilution (10−2), green plot for virus dilution (10−3), yellow plot for virus dilution (10−4), black plot for virus dilution (10−5), and the line plot under threshold line as negative control (untreated). fig 5. (a) mean value of ki-67 expression after treatment with measles virus at different durations (p < 0.001). (b) mean value of annexin-v after treatment with measles virus at different durations (p < 0.001). however, fig. 5a and b reflected a significant difference between cell that was treated with measles virus in comparison with control group (p < 0.001). discussion live attenuated measles virus edmonston strain was propagated on vero cell line and used as oncolytic virus for human nonsmall cell lung cancer cell line. qrt-pcr technique was used as rapid quantitative method for estimating of the titer of propagated measles virus vaccine. it is a highly sensitive and specific diagnostic tool. the titer of propagated measles virus vaccine gradually increased with subsequent passage number due to rapid adaptation of the virus on vero cell line, started with 4.33 log10 copies/µl, with mild increment in the second passage to 4.596 copies/µl and reached its maximum titer in eighth passage which was 8.86 (table 2 and fig. 1). the cycle threshold value measured by rt-pcr which was used as an indicator of the viral load found in the supernatant of cell culture. these results are in agreement with many other previous studies el mubarak et al.18 who documented that qrt-pcr is efficient rapid technique for detecting measles infection or vaccination. also, ammour et al.,19 reported that qrt-pcr was successfully used for estimating the titers of measles, a b rasha fadhel obaid et al. 49j contemp med sci | vol. 4, no. 1, winter 2018: 45–50 research correlation between live attenuated measles viral load and growth inhibition percentage mumps and rubella viruses in infected cell culture supernatants, and this technique is cheaper and faster than titration methods that used in cell culture techniques. the present study indicated that both tissue culture and rt-pcr-technique are efficient technique for estimation of successful propagation of measles virus on vero cell. this study showed that measles virus had greatest growth inhibition effect on human lung cancer cell line in comparison to a control group and the virus cytotoxic effect was increased according to virus load increment, in parallel with time. figure. 3 explained that there was significant difference in inhibitory effect of measles virus on human lung a549 compared with control group after 24, 48, 72, and 96 hr post exposure (p < 0.001). the percentage of growth inhibition was correlated positively and significantly with the viral load in cancer cell line if considered together which means that a higher viral load will lead to high percentage of growth inhibition, because a high titer of virus destroy a large number of cancer cells, while a lower viral load would destroy lower percentage of cell number. the result consistent with previous studies, documented that measles virus has antitumor activity. blechacz et al.20 reported that mv-edmonton, successfully infected human hepatocellular carcinoma cell, resulted in transgene expression and syncytium formation in dependent on expression of cd46 receptor, and tumor cell killing was assessed by crystal violet assay. also, mcdonald et al.21 who found that mv has potent antitumor activity against breast cancer lines and xenografts, resulting in significant cytopathic effects consisting of extensive syncytia formation and massive cell death after 72–96 hr from infection. induction of cancer cell apoptosis is the key to its treatment. the annexin-v affinity assay was a widely used as method for apoptosis analysis, as well as for the discrimination between apoptotic and viable cell. this test is based on the changes in the cell membrane caused by apoptotic processes, loss of plasma membrane asymmetry is an early step in apoptosis. the rate of apoptotic cell is increased significantly with the duration intervals and the dose of measles virus, associating with a decrease in expression of ki-67 protein. the present results consistent with other previous studies that documented that oncolytic virus induces apoptosis. liu et al.22 revealed that the measles vaccine virus-induced apoptosis and leading to cytotoxicity against glioma cell line in vitro. on other hand, li et al.23 found that adenovirus could inhibit ki-67 expression and cellular proliferation and induce apoptosis of prostate carcinoma in vivo. the present study revealed that measles virus can replicate and destroy cancer cells, and yield high load of new progeny viruses, which spread, infect and kill more cancer cells around it. also, a high load of measles virus increased inhibition of cancerous cells as compared with low-virus titer. conclusion live attenuated measles virus strain induced cytotoxic effect against human lung cancer cell line (a549) by induction of apoptosis as an important mechanism of anti-tumor activity, in addition, it indicated a correlation between the quantity of mv genomes and percentage of growth inhibition. this relation has proved that measles virus had anticancer effect. acknowledgment we would like to thank the staff of cancer research laboratory, in pharmacology department, college of medicine, babylon university for their assistance of this research work. n references 1. russell sj, peng kw, bell jc. oncolytic virotherapy. nature biotechnol. 2012;30:658–670. 2. achard c, boisgerault n, delaunay t, tangy f, grégoire m, fonteneau jf. induction of immunogenic tumor cell death by attenuated oncolytic measles virus. j clin cell immunol. 2015;6. http-omicsonline. 3. singh pk, doley j, kumar gr, sahoo ap, tiwari ak. oncolytic viruses & their specific targeting to tumour cells. indian j med res. 2012;136: 571–584. 4. dorer de, nettelbeck dm. targeting cancer by transcriptional control in cancer gene therapy and viral oncolysis. adv drug delivery rev. 2009;61:554–571. 5. he s, li p, chen ch, bakst rl, chernichenko n, yong ay, et al. effective oncolytic vaccinia therapy for human sarcomas. j surg res. 2012;175: e53–e60. 6. takano m, takahashi, agarikuchi t, kurebayashi y, minami a, otsubo t, et al. histochemical fluorescent staining of sendai virus-infected cells with a novel sialidase substrate. virology. 2014;464:206–212. 7. goldufsky j, sivendran s, harcharik s, pan m, bernardo s, stern rh, et al. oncolytic virus therapy for cancer. oncolytic virother. 2013;2: 31–46. 8. zhao d, chen p, yang h, wu y, zeng x, zhao y, et al. live attenuated measles virus vaccine induces apoptosis and promotes tumor regression in lung cancer. oncol rep. 2013;29:199–204. 9. knuchel mc, marty rr, morin tna, ilter o, zuniga a, naim hy. relevance of a pre-existing measles immunity prior immunization with a recombinant measles virus vector. hum vaccin immunother. 2013;9: 599–606. 10. fishelson z, donin, n zell s, schultz s, kirschfink m. obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mcrps) in tumors. mol immunol. 2003;40:109–123. 11. guillerme jb, gregoire m, tangy f, fonteneau jf. antitumor virotherapy by attenuated measles virus (mv). biology. 2013;2:587–602. 12. castro-garza j, barrios-garcía hb, cruz-vega d, said-fernández s, carranzarosales p, molina-torres ca, et al. use of a colorimetric assay to measure differences in cytotoxicity of mycobacterium tuberculosis strains. j med microbiol. 2007;56:733–737. 13. msaouel p, iankov id, allen c, morris jc, von messling v, cattaneo r, galanis, e. engineered measles virus as a novel oncolytic therapy against prostate cancer. the prostate. 2009;69:82–91. 14. haas c, ertel c, gerhards r, schirrmacher, v. introduction of adhesive and co-stimulatory immune functions in to tumor cells by infection with newcastle disease virus. int j oncol. 1998;13:1105–1115. 15. anne g, samantha b, carole s. measles virus induces oncolysis of mesothelioma cells and allows dendritic cells to cross-prime tumor-specific cd8 response. j aacr. 2008;68:4882–4892. 16. madlum kn, galib ra, ghazi fm, ali zh. the effects of zizyphus stem bark and termite shelter tubes on hela cell growth. rjpbc. 2015;6:620. 17. hummel kb, lowe l, bellini wj, rota pa. development of quantitative genespecific real-time rt-pcr assays for the detection of measles virus in clinical specimens. j virol methods. 2006;132:166–173. 18. el mubarak hs, de swart rl, osterhaus ad, schutten m. development of a semi-quantitative real-time rt-pcr for the detection of measles virus. j clin virol. 2005;32:313–317. 19. ammour y, faizuloev e, borisova t, nikonova a, dmitriev g, lobodanov s, et al. quantification of measles, mumps and rubella viruses using real-time quantitative taqman-based rt-pcr assay. j virol methods. 2013;187:57–64. 20. blechacz b, splinter pl, greiner s, myers r, peng kw, federspiel mj, et al. engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma. hepatology. 2006;44:1465–1477. 50 j contemp med sci | vol. 4, no. 1, winter 2018: 45–50 correlation between live attenuated measles viral load and growth inhibition percentage research rasha fadhel obaid et al. 21. mcdonald cj, erlichman c, ingle jn, rosales ga, allen c, greiner sm, et al. a measles virus vaccine strain derivative as a novel oncolytic agent against breast cancer. breast cancer res treat. 2006;99:177–184. 22. liu c, sarkaria jn, petell ca, paraskevakou g, zollman pj, schroeder m, et al. combination of measles virus virotherapy and radiation therapy has synergistic activity in the treatment of glioblastoma multiforme. clin cancer res. 2007;13:7155–7165. 23. li j, luo j, gu d, jie f, pei n, li a, et al. adenovirus-mediated angiotensin ii type 2 receptor overexpression inhibits tumor growth of prostate cancer in vivo. j cancer. 2016;7:184–191. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.03201810 48 j contemp med sci | vol. 9, no. 1, january-february 2023: 48–55 original the relation of autonomic biomarkers to the patient’s outcome with acute coronary syndrome zana lazgeen abdi1, hishyar ms garmavy2*, suzan rasool omer3 1directorate of general health, duhok, ministry of health, kurdistan region, iraq. 2department of pharmacology, college of pharmacy, university of duhok, duhok, iraq. 3department of clinical pharmacy, college of pharmacy, university of duhok, duhok, iraq. *correspondence to: hishyar ms garmavy (e-mail: hishyar.mohammed@uod.ac) abstract objectives: this study aimed to evaluate the neurohormonal activity in patients with acute coronary syndrome and their relation to clinical outcomes by measuring plasma chromogranin a and plasma and rbc cholinesterase activity. methods: in this case-control study, cardiac and neurohormonal parameters were compared between fifty-one patients with acute coronary syndrome admitted to the cardiac center in azadi teaching hospital in duhok-iraq, and thirty comparable gender and agehealthy subjects. results: a significant increase in sympathetic activity was reported in patients with the acute coronary syndrome, reflected by the rise in the plasma chromogranin a compared to the control group (994.47 vs. 1203.95 ng/l, respectively, p < 0.05). meanwhile, cardiac troponin was significantly elevated in those patients compared to healthy subjects (0.17 vs. 4.82 ng/ml, respectively). however, the parasympathetic biomarkers (plasma and rbc cholinesterase activity) did not differ substantially between patients and healthy controls (0.83 vs. 0.92, p > 0.05 and 1.36 vs. 1.37, p > 0.05, respectively). serum troponin i was more valid than chromogranin a in differentiating acute coronary syndrome from healthy subjects. the area under the curve for troponin i was (0.989) compared to (0.724) for chromogranin a. furthermore, plasma chromogranin a but not plasma and rbc cholinesterase activity was significantly increased in fatal cases compared to nonfatal patients (1166.68 vs. 3435.64 ng/l). conclusion: plasma chromogranin a was less effective than troponin i in detecting acute coronary cases; however, it can be helpful as a prognostic marker in those patients. parasympathetic biomarkers were not appreciable in diagnosing and detecting risky patients. keywords: autonomic biomarkers, chromogranin a, plasma and rbc cholinesterase activity, acute coronary syndrome issn 2413-0516 introduction despite the scientific community’s remarkable efforts, cardiovascular diseases account for over one-third of all mortality globally.1 furthermore, according to world health organization statistics, acute myocardial infarction is the human’s most important cause of death.2 acute coronary syndrome (acs) is the leading cause of death, disability, and human misery worldwide. unfortunately, their incidence rises with age; however, this happens much earlier in men than in women.3,4 acs is often diagnosed by clinical presentation, electrocardiogram, elevated cardiac enzymes, and echocardiography.5 in recent years, breakthroughs in the sensitivity and precision of cardiac troponin testing and advances in imaging modalities have helped to diagnose acute chest pain.6 although recent advancements in medical and interventional therapy options have improved the diagnosis and earlier treatment of individuals suffering from acs, acs is still one of the world’s top causes of morbidity and death. therefore, identifying the high-risk patient population is crucial in averting such unfavourable scenarios and controlling the aggressiveness of therapeutic options.7 in the last decade, it has become apparent that autonomic dysregulation has played a significant role in developing and progressing severe cardiovascular disorders such as myocardial infarction, heart failure, and sudden cardiac death.8 in addition, cardiac disease can lead to sympathovagal imbalances, which are essential in triggering and maintaining atrial and ventricular cardiac arrhythmias.9 arrhythmias, particularly ventricular arrhythmias, are the primary cause of death in individuals with myocardial infarction. researchers previously reported that several factors, such as sympathetic neuronal remodelling, gap junction remodelling, and cardiac fibrosis, can establish ventricular arrhythmias following myocardial infarction.10 during acute myocardial ischemia, pain, anxiety, and a drop in cardiac output or systemic blood pressure might contribute to increased sympathetic activity and generation of norepinephrine. the secreted norepinephrine stimulates sympathetic nerve regeneration and nerve remodelling, ultimately leading to arrhythmogenesis.11 assessment of cardiovascular autonomic nerves is not straightforward since various variables, like body posture, emotional state, consumed food, and medications, impact autonomic function. furthermore, no single cardiovascular autonomic test is sufficiently reliable; thus, combining different approaches is necessary.12 chromogranin a measurement has gained interest in cardiovascular disease because increased plasma concentrations are associated with the increased risk of clinical deterioration and death in patients with acute coronary syndromes or chronic heart failure.13 chromogranin a is primarily produced by adrenal medulla cells and adrenergic terminals, although it may be made by myocardial cells under stress conditions. after secretion, it cleaves into several biologically active fragments, including vasostatins and catestatin.14 physiologically chromogranin a is necessary for blood pressure regulation and cardiac inotropic and chronotropic function. in addition, chromogranin a was a sensitive marker of myocardial dysfunction, with a high predictive value in heart failure and ischemic heart disease clinical outcomes.15 (submitted: 25 october 2022 – revised version received: 19 november 2022 – accepted: 28 november 2022 – published online: 26 february 2023) 49j contemp med sci | vol. 9, no. 1, january-february 2023: 48–55 z.l. abdi et al. original the relation of autonomic biomarkers to the patient’s outcome with acs in contrast to the sympathetic branch of the autonomic nervous system, the parasympathetic branch has anti-inflammatory effects. nicotinic receptors stimulation mediates this anti-inflammatory response by reducing the release of inflammatory markers il 6 and tnf alpha from tissue macrophages.16 as a result of its link to systemic inflammation, parasympathetic dysfunction may increase the risk of cardiovascular disease.17 unfortunately, until now, no clinically reliable test exists for direct measurement of the parasympathetic activity in the body; because acetylcholine is exceptionally labile, and its quantification in circulation is not dependable and unreliable. furthermore, traditional non-invasive markers of cardiac autonomic dysfunction, such as heart rate variability and blood pressure during exercise recovery, are poor predictors of cardiovascular disease.18 therefore, an indirect method of cholinergic activity may be used by measuring serum cholinesterase activities as parasympathetic biomarkers (acetylcholinesterase, butyrylcholinesterase activity, or both). disturbances in the levels of ache have been linked to inflammation and metabolic dysfunction in otherwise healthy adults.19 however, a study on coronary angiography showed low levels of blood ache activity in patients who developed significant adverse cardiovascular events over two years of follow-up.20 moreover, serum bche has been implicated in the development of coronary artery diseases (cad),21 demonstrating that individuals in the lowest quintile of bche activity had significantly higher rates of all-cause and cardiovascular mortality. in addition, goliasch and a co-worker (2012) showed a strong association between decreased serum cholinesterase and long-term adverse outcomes in patients with known cad, which was more robust in stable patients with cad than in those with acs.22 impaired cardiac parasympathetic responsiveness, enhanced sympathetic activity, and their induced inflammatory responses are negative prognostic indicators for morbidity and mortality associated with arrhythmias and sudden death.23 therefore, interventions that reduce sympathetic tone and increase parasympathetic activity may reduce the risk of sudden cardiac death and ventricular tachyarrhythmias. de ferrari and colleagues observed that weak sympathetic reflexes characterize approximately 75% of animals resistant to ventricular fibrillation in response to acute myocardial ischemia. in the remaining 25%, powerful vagal reflexes counteract concomitant reflex sympathetic hyperactivity, decrease heart rate, and are essential for survival.24,25 the study aimed to assess autonomic nervous system activity in patients with acute coronary syndrome by measuring plasma chromogranin a and plasma and rbc cholinesterase activity. in addition, to identify the relationship between autonomic activity and clinical outcomes in patients with the acute coronary syndrome. methodology and study design this case-control study was conducted at the azadi teaching hospital in duhok and college of pharmacy, university of duhok, from september 2021 to march 2022. after taking ethical committee approval (24102021-10-32r1), two groups of subjects of both genders, ages 35 and 75 years, were included in this study; however, pregnant women, patients taking cholinesterase antagonists, and patients with chronic inflammatory and neoplastic diseases were excluded from this study. the first group comprised 51 patients with acute coronary syndrome confirmed by a cardiologist and admitted to the cardiac center or cardiac care unit (ccu) within 24 hours of the onset of symptoms. they were recruited sequentially for this study; patients with acs comprised 34 males and 17 females. the second group consisted of 30 healthy subjects (18 males and 12 females) of comparable age and gender, randomly selected from the azadi teaching hospital staff in duhok-iraq. data acquired at study entry included age, gender, chief complaint and duration, past medical history, family history, and drug history. in addition, a blood sample was taken from all patients (3 ml of venous blood) within 24 hours from the acute attacks. and the control subjects fasted overnight (10–12 hours). the blood samples were collected in an edta tube and, for less than one hour, centrifuged at 3000 (rpm) for 10 min at –4°c. then the plasma and rbc were separated and deeply frozen at –20°c until the analysis. plasma chromogranin a concentration was measured using human chromogranin-a, cga elisa kit, 2021 shanghai korean biotech co. (cat no. e1730hu). while serum troponin i was measured using cobas®6000 (cobas e 601) roche hitachi, elecsys troponin i stat kit, germany. the diagnostic value for acs in males and females was > 0.3 ng/ml. in addition, plasma and rbc cholinesterase activity were measured using the electrometric method.26,27 the statistical calculations were conducted in jmp pro version 14.3.0. the general and medical information of patients and control was introduced in mean and standard deviation (sd), or %, and number. the sympathetic and parasympathetic factor comparisons among patients and healthy subjects were determined in an independent t-test. comparisons of sympathetic and parasympathetic factors in patients with different characteristics were evaluated in an independent t-test or one-way anova. the area under the curve for the acute coronary syndrome diagnosis from healthy controls for chromogranin a and troponin i level was examined in the roc curve. the cut-offs were explored from the roc table and presented in sensitivity and specificity. statistically, the significance of the difference level was determined by a p-value of < 0.05. results this study recruited eighty-one participants. thirty healthy individuals served as a control group, and fifty-one were patients diagnosed with acute coronary syndrome. the mean age of the participants was 57.06 years (control = 55.06 years vs. patients = 58.53), and nearly two-thirds of the participants were male, 18 (60%) from the control group, and 34 (66.7%) were patients. statistically, there was no difference between the patient and control group regarding age and gender. more than two-thirds of the patients were diagnosed with stemi 35 (68.63%), and the others were diagnosed with nstemi 16 (31.37%). meanwhile, around one-third of the patients, 17 patients (33.33%), had a positive family history of ischemic heart diseases. statistically, there was a highly significant difference among the patients regarding family history of ischemic heart diseases and prevalence of acs, as shown in table 1. 50 j contemp med sci | vol. 9, no. 1, january-february 2023: 48–55 the relation of autonomic biomarkers to the patient’s outcome with acs original z.l. abdi et al. table 1. comparisons of general information between patients with acute coronary syndrome and healthy controls participants information study groups no (%) p-value (two-sided)control (n = 30) patients (n = 51) age 55.60 (10.66) 58.53 (11.77) 0.2665 gender male female 18 (60.00) 12 (40.00) 34 (66.67) 17 (33.33) 0.5456 final diagnosis stemi nstemi 35 (68.63) 16 (31.37) na history of ihd no yes 30 (100) 0 (0.00) 34 (66.67) 17 (33.33) 0.0004 pearson chi-squared test was performed for statistical analyses. table 2 illustrates the biochemical parameters of the studied participants. these biochemical markers reflect autonomic nervous system activity and cardiac biomarker. in this study, chromogranin a, which reflects the overall sympathetic activity in the blood, was significantly increased in patients with acs compared to the control group (994.47 vs. 1203.95 ng/l, respectively) figure 1. in addition, troponin i, which reflects myocardial cell damage, was highly significantly different in acs patients compared to healthy subjects (0.17 vs. 4.82 ng/ml, respectively) figure 2. in contrast to the sympathetic system, parasympathetic biomarkers such as plasma and rbc cholinesterase activity did not differ substantially between acs patients and healthy controls (0.83 vs. 0.92 and 1.36 vs. 1.37, respectively). table 3 shows the influence of gender, age, readmission, past medical history, and family history on blood autonomic markers. no statistically significant differences were reported in these biomarkers between males and females as well as among the different age groups of the patients. similarly, no statistically significant difference in these markers was reported concerning the duration of readmission to the hospital. however, there was a highly significant increase in the level of plasma chromogranin a in patients with a previous history of ischemic heart disease compared to those who did not have ischemic heart disease (1854.80 vs 1206.12 ng/l). in comparison, there were no statistically significant differences in plasma and rbc cholinesterase (p > 0.05). meanwhile, there was a significant difference in plasma chromogranin a table 2. comparisons of autonomic and cardiac biomarkers between patients and healthy controls chemical factors study groups mean (sd) p-value (two-sided)control (n = 30) patients (n = 51) plasma chromogranin a range 994.47 (127019) 583.98–1447.27 1203.95 (293.48) 611.69–6970.28 0.0007 plasma cholinesterase activity range 0.92 (0.13) 0.27–1.58 0.83 (0.23) 0.35–1.66 0.1016 rbc cholinesterase activity range 1.37 (0.12) 0.81–1.82 1.36 (0.13) 0.03–1.62 0.7268 troponin i 0.17 (0.03) 4.82 (2.81) <0.0001 an independent t-test was performed for statistical analyses. fig. 1 box plot of comparisons of plasma chromogranin a between acs cases and healthy controls. fig. 2 box plot of comparisons of troponin i between acs cases and healthy controls. levels between individuals with a family history of myocardial infarction (mi) and those without a family history of mi (1763.52 vs. 1255.32 ng/l). however, there was no statistically significant difference in plasma or rbc cholinesterase levels between the two groups (p > 0.05). furthermore, patients admitted to the hospital within the first 24 hours from symptoms had a statistically significant lower plasma chromogranin a level (1199.04 vs 1939.63 ng/l) than those admitted after 24 hours. however, there were no statistically significant changes in plasma and rbc cholinesterase activity (p > 0.05). table 4 demonstrated that patients who had previously taken beta-blockers, statins, or antiplatelets were not statistically different from those who had previously been naive to these medications in terms of plasma concentration of chromogranin a, rbc, and plasma, cholinesterase activity (p > 0.05). 51j contemp med sci | vol. 9, no. 1, january-february 2023: 48–55 z.l. abdi et al. original the relation of autonomic biomarkers to the patient’s outcome with acs table 3. comparisons of autonomic factors in patients with different characteristics (n = 51) characteristics of participants sympathetic and parasympathetic factors mean (sd) chromogranin a plasma cholinesterase rbc cholinesterase gendera male female p-value 1176.27 (312.43) 1265.72 (246.01) 0.3676 0.82 (0.24) 0.86 (0.21) 0.5227 1.35 (0.13) 1.38 (0.14) 0.4843 age categoryb 36–45 46–60 61 and older p-value 1090.70 (272.77) 1176.45 (283.36) 1278.74 (305.89) 0.2931 0.87 (0.16) 0.89 (0.27) 0.76 (0.18) 0.1536 1.40 (0.10) 1.32 (0.18) 1.36 (0.12) 0.3931 past medical historya ihd no ihd p-value 1854.80 (1065.28) 1206.12 (273.30) 0.0024 0.83 (0.19) 0.84 (0.25) 0.9176 1.33 (0.12) 1.38 (0.14) 0.1946 family historya mi or ihd no mi or ihd p-value 1763.52 (1142.59) 1255.32 (259.69) 0.0324 0.84 (0.21) 0.83 (0.25) 0.8769 1.35 (0.14) 1.37 (0.13 0.6406 readmission timea within one week within one month within three months p-value 2599.70 (1930.37) 1248.24 (291.33) 1256.29 (0.0608) 0.91 (0.24) 0.79 (0.22) 0.66 (0.3757) 1.31 (0.15) 1.39 (0.12) 1.57 (0.1431) admission durationb within 24 hrs. after 24 hrs. 1199.04 (294.98) 1939.63 (1095.33) 0.0008 0.83 (0.23) 0.82 (0.27) 0.8993 1.35 (0.14) 1.43 (0.09) 0.1943 aan independent t-test and banova one-way were performed for statistical analyses. table 4. comparisons of autonomic factors in patients with a drug history drug history sympathetic and parasympathetic factors mean (sd) chromogranin a plasma cholinesterase rbc cholinesterase statin no statin statin p-value 1216.88 (293.81) 1149.04 (305.36) 0.5629 0.84 (0.22) 0.78 (0.27) 0.4515 1.36 (0.14) 1.31 (0.21) 0.4086 antiplatelet not received received p-value 1189.23 (274.83) 1251.07 (359.12) 0.5673 0.84 (0.25) 0.81 (0.18) 0.7168 1.37 (0.14) 1.34 (0.12) 0.5287 beta-blockers not received received p-value 1212.33 (287.78) 1180.36 (322.25) 0.7605 0.81 (0.21) 0.91 (0.29) 0.2068 1.34 (0.11) 1.42 (0.17) 0.0687 an independent t-test was performed for statistical analyses. although patients with st-elevation myocardial infarction (stemi) showed a lower level of chromogranin a than those with non-st-elevation myocardial infarction (nstemi); however, there was no statistical difference between the two groups. meanwhile, plasma and rbc cholinesterase activity were also lower in stemi than in those with nstemi; however, this difference was not statistically different between the two groups (p > 0.05), as shown in table 5. patients who were readmitted to the hospital after an acute coronary syndrome had higher chromogranin a levels than those who were not (1292.25 (286.95 vs. 1131.01 (284.34, respectively); nonetheless, there was no significant difference between the two groups (p > 0.05). however, there was a significant rise in plasma chromogranin a levels in fatal instances (death cases) compared to nonfatal cases (stable patients) (1166.68 vs. 3435.64 ng/l). in contrast, there was no statistically significant difference in plasma and rbc cholinesterase activity between the fatal and nonfatal outcome patients (p > 0.05). the validity parameters of plasma chromogranin a and troponin i in differentiating acs from healthy control are shown in tables 6 and 7. serum troponin i was of the highest validity in determining acs from healthy subjects with an area under the curve (auc) of 0.989 (figure 3), while chromogranin a was of the lower validity (auc = 0.724) for differentiating acs cases from controls (figure 4). 52 j contemp med sci | vol. 9, no. 1, january-february 2023: 48–55 the relation of autonomic biomarkers to the patient’s outcome with acs original z.l. abdi et al. table 5. comparisons of autonomic factors in patients with diagnosis and clinical outcomes diagnosis and clinical outcomes sympathetic and parasympathetic factors mean (sd) chromogranin a plasma cholinesterase rbc cholinesterase final diagnosisa nstemi stemi p-value 1282.74 (268.43) 1172.44 (301.40) 0.2765 0.89 (0.36) 0.83 (0.20) 0.4467 1.39 (0.12) 1.33 (0.16) 0.2475 follow-upa not admitted readmitted p-value 1131.01 (284.34) 1292.25 (286.95) 0.0760 0.84 (0.24) 0.82 (0.22) 0.7301 1.35 (0.13) 1.38 (0.13) 0.4312 patient outcomea died stable p-value 3435.64 (2552.09) 1166.68 (285.83) <0.0001 0.73 (0.17) 0.84 (0.23) 0.1969 1.37 (0.15) 1.36 (0.13) 0.7918 aan independent t-test and banova one-way were performed for statistical analyses. table 6. roc area for plasma chromogranin a (ng/l) in differentiating acs cases from healthy controls prob specificity (%) sensitivity (%) ppv (%) npv (%) accuracy (%) 1038.915 0.6507 96.43 52.38 95.7 57.4 70.00 1032.583 0.6479 96.43 54.76 optimum * 95.8 58.7 71.43 1021.419 0.6448 92.86 54.76 92.0 57.8 70.00 1019.573 0.6421 92.86 57.14 92.3 59.1 71.43 1017.321 0.6419 89.29 57.14 88.9 58.1 70.00 1016.976 0.6398 89.29 59.52 89.3 59.5 71.43 the cut-off is between 1016.976 and 1038.915 for the diagnosis of acute coronary syndrome. therefore, the optimum concentration for diagnosing acute coronary syndrome is 1032.58. table 7. roc area for troponin i (ng/ml) in differentiating acs cases from healthy controls prob specificity sensitivity sens-(1-spec) ppv npv accuracy 0.6000000 1 100.00 90.48 0.9048 100.0 87.9 94.37 0.2400000 0.6856 100.00 92.86 0.9286 * 100.0 90.6 95.77 0.2300000 0.55 93.10 92.86 0.8596 95.1 90.0 92.96 0.2200000 0.4064 93.10 95.24 0.8834 95.2 93.1 94.37 0.2100000 0.2773 89.66 95.24 0.8489 93.0 92.9 92.96 0.2000000 0.1769 82.76 97.62 0.8038 89.1 96.0 91.55 0.1900000 0.1075 72.41 100 0.7241 84.0 100.0 88.73 0.1800000 0.0632 58.62 100 0.5862 77.8 100.0 83.10 the cut-off is between 0.18 and 0.6 for the diagnosis of acute coronary syndrome. (the optimum number for the diagnosis of the acute coronary syndrome is 0.24). regarding plasma chromogranin a, the cut-off value was between 1016.976 and 1038.915 for the diagnosis of the acute coronary syndrome. the optimum number for the diagnosis of the acute coronary syndrome is 1016.976 (table 6). regarding serum troponin i, the cut-off value was between 0.18 and 0.6 for the diagnosis of the acute coronary syndrome. the optimum number for the diagnosis of the acute coronary syndrome is 0.24 (table 7). discussion acute coronary syndrome (acs) primarily affects patients over the age of 50 years. however, younger patients can also be concerned with a higher prevalence of stemi, even with less severe coronary artery disease.28 the average age of the acs patient in this study was 58.53 years which is nearly similar to another study done in duhok city.29 huffman and colleagues reported 60.6 years as the mean age for acs patients in their study conducted in india.30 however, in the global registry of acute coronary events (grace) across 14 countries, the mean age of patients with stemi was 64 years, nstemi was 68 years, and unstable angina was 66 years. this disparity in the mean age of acs patients between low and high-income countries probably reflects socio-economic burdens, unhealthy lifestyles, and poor health education in low and medium-income countries.31 53j contemp med sci | vol. 9, no. 1, january-february 2023: 48–55 z.l. abdi et al. original the relation of autonomic biomarkers to the patient’s outcome with acs in recent years, high-sensitivity cardiac troponin (hs-ctn) assays have emerged as the preferred biomarker in the universal definition of myocardial infarction. it dramatically helped to diagnose acute chest pain. this study also confirms cardiac troponin’s role in diagnosing acs. a dramatically higher troponin level was observed in the blood in almost all cases of acs compared to the control group. furthermore, elevated cardiac troponins levels have been linked to poor outcomes in acs in elderly patients.38 however, the predictive value of cardiac troponin is not absolute for all cases of acs. for example, high-risk patients with unstable angina usually do not have cardiac troponin elevation. furthermore, cardiac troponin levels do not account for all pathological processes associated with acs, such as left ventricular (lv) dysfunction, hemodynamic stress, inflammation, or renal dysfunction.39 several studies have found that measuring chromogranin a in the blood can reflect sympathetic activity in the body.40-42 as a result, this study employs plasma chromogranin a levels to assess sympathetic activation following acs. chromogranin a is preferred over other modalities in evaluating adrenergic activity because of its long half-life in the plasma.43 the potential role of chromogranin a as a cardiac biomarker is not an innovative topic. for more than ten years, scientific committees have observed that an increase in the concentration of chromogranin a and its metabolites in the circulation are associated with the risk of clinical worsening and death in patients with acute coronary syndromes or chronic heart failure.13,44,45 angelone and colleagues (2012) reported a high plasma chromogranin a level in patients with acute coronary syndromes.43,46 this elevation correlates with conventional cardiovascular biomarkers such as bnp and endothelin-1 (et-1).47 ji and colleagues (2012) also reported that catestatin, a chromogranin a metabolite, is an independent predictor of in-hospital complications in stemi patients like heart failure and malignant arrhythmia. furthermore, patients with angina had significantly higher plasma catestatin levels than those who did not have ihd.48 others, however, are skeptical of the significance of chromogranin a as a prognostic indicator of acs and heart failure. this biological molecule, they believe, is not a specific cardiac biomarker, and its elevation can be seen in chronic inflammatory diseases, autoimmune diseases, renal failure, and hepatic insufficiency.49 similar to the finding of angelone and colleagues, this study also showed a significantly increased plasma chromogranin a in patients with acs compared to normal individuals. qasim and colleagues reported a similar finding in patients with acute myocardial infarction in duhok, iraq.50 this study also reported a higher plasma level of chromogranin a in patients with a history of ihd and a positive family history of ihd, which contradicts the study by qasim and co-workers. the elevation in plasma chromogranin a level in patients with acs is not unexpected, given that its release increases under stress conditions like acs cases.51 and their metabolites are generated by proteolytic processing in the heart to protect the heart from ischemia/reperfusion damage through inhibiting cardiac contraction and relaxation, counter-regulating beta adrenergic, and endothelinrgic stimulation.46,47 furthermore, this study also reported that patients who were delayed for more than 24 hours to the point of definitive care had significantly higher plasma chromogranin a levels. fig. 3 roc curve showing the trade-off between sensitivity (true positive) and 1specificity (false positive) for troponin i when used in differentiating acs patients from healthy controls. fig. 4 roc curve showing the trade-off between sensitivity (true positive) and 1specificity (false positive) for plasma chromogranin a when used in differentiating acs patients from healthy controls. the prevalence of stemi and nstemi varies in different studies conducted on acs cases; some researchers reported a higher prevalence of nstemi than stemi among their studied individuals.32,33 in this study, however, more than two-thirds of the patients (68.63%) were diagnosed with stemi, similar to ricci and co-workers (2017), who reported a higher prevalence of stemi in their survey, particularly in younger patients, which contradict the previous studies done by bhatt and co-workers and others. the higher prevalence of stemi among study participants could be attributed to gender, with males being affected more than females. furthermore, at younger ages, males were more susceptible to stemi than females.34 the family history of ihd is a non-modifiable long-term independent risk factor for acs. it represents a genetic predisposition to coronary artery disease, particularly in younger people.35 this observation was evident in a study conducted in pakistan, where researchers looked for risk factors for acs in patients aged 18 to 40; nearly one-third of their participants had a positive family history of ihd.36 in concordance with that study, almost two-thirds of the participants recruited in this study had a positive family history of ihd. webring and co-workers (2016) also reported that cas more frequently occurred in patients with a positive family history of ischemic heart disease.37 54 j contemp med sci | vol. 9, no. 1, january-february 2023: 48–55 the relation of autonomic biomarkers to the patient’s outcome with acs original z.l. abdi et al. this elevation could be due to the release of chromogranin a from the myocardium after a prolonged ischemic period.52 in addition, chromogranin a levels have risen in proportion to clinical severity and are associated with prognosis in patients with chronic and post-infarction heart failure. as a result, the level of chromogranin a in the blood may indicate the degree of cardiac damage.53 meanwhile, vasostatin i, a derivative of chromogranin a, showed an antiadrenergic activity in the heart.54 after three months of follow-up of all cases of acs, nearly 17% of patients have died. a higher level of chromogranin a was reported in those fatal cases compared to nonfatal ones, and the difference was statistically significant.43 anna and co-workers also reported that circulating chromogranin a levels would be predictive of the incidence of death in acs.43 meanwhile, high chromogranin a levels predict mortality in severely septic patients without prior cardiovascular disease.55 furthermore, d’amico and colleagues reported that high chromogranin a plasma levels are strictly associated with the risk of mortality after myocardial infarction or acute coronary syndrome, as well as heart failure.56 although there were some differences between plasma and rbc cholinesterase activity; however, no statistically significant difference was reported in these enzymes between the acs cases and the healthy control group in this study, as well as among patients with acs. however, a noticeable reduction in plasma but not rbc cholinesterase activity was observed among patients. this reduction was primarily seen in stemi cases, patients not taking statin lipid-lowering drugs, and unstable patients who required readmission to the hospital, especially after one and three months; meanwhile, it was reported in fatal cases. ackland and co-workers found that parasympathetic dysfunction may increase the risk of cardiovascular disease. this association was related to immunological dysregulation induced by cholinergic dysfunction.17 arbel and colleagues also reported a low level of blood ache activity in cases of coronary angiography who developed significant adverse cardiovascular events over two years of follow-up.20 calderon and co-workers also noticed that low serum levels of bche have been linked to a higher cardiovascular mortality rate.21 however, goliasch and co-workers demonstrated a strong association between low serum cholinesterase activity and long-term adverse outcomes in stable cases of coronary artery diseases.22 conclusion plasma chromogranin a was less effective than troponin i in detecting acute coronary cases; however, it can be helpful as a prognostic marker in those patients. the role of parasympathetic biomarkers was not appreciable in the early diagnosis and detection of risky patients with acute coronary syndrome.  references 1. nichols m, townsend n, scarborough p, rayner m. cardiovascular disease in europe 2014: epidemiological update. european heart journal. 2014;35(42):2950–9. 2. jiang w, chen c, huo j, lu d, jiang z, geng j, et al. comparison between renal denervation and metoprolol on the susceptibility of ventricular arrhythmias in rats with myocardial infarction. scientific reports. 2018;8(1):10206. 3. bueno hjec. epidemiology of acute coronary syndromes. esc cardiomed. 2018. 4. pagidipati nj, peterson ed. acute coronary syndromes in women and men. nature reviews cardiology. 2016;13(8):471–80. 5. garg p, morris p, fazlanie al, vijayan s, dancso b, dastidar ag, et al. cardiac biomarkers of acute coronary syndrome: from history to high-sensitivity cardiac troponin. internal and emergency medicine. 2017;12(2):147–55. 6. katus h, ziegler a, ekinci o, giannitsis e, stough wg, achenbach s, et al. early diagnosis of acute coronary syndrome. european heart journal. 2017;38(41):3049–55. 7. yilmaz as, çinier g, kahraman f, çetin m, çirakoglu öfjjoarim. the pr interval predicted major adverse cardiovascular events in patients with acute coronary syndrome who underwent percutaneous coronary intervention: 3 years follow-up results/primer perkütan koroner girisim uygulanan akut koroner sendrom hastalarinda pr mesafesi majör istenmeyen olaylari öngörmüstür: 3 yillik takip sonuçlari. journal of academic research in medicine. 2021;11(3):241–9. 8. ardell jl, rajendran ps, nier ha, kenknight bh, armour ja. centralperipheral neural network interactions evoked by vagus nerve stimulation: functional consequences on control of cardiac function. american journal of physiology heart and circulatory physiology. 2015;309(10):h1740–52. 9. aço jrlc. role of the autonomic nervous system in the genesis of cardiac arrhythmias: pathophysiology and therapy implications. 2021. 10. fukuda k, kanazawa h, aizawa y, ardell jl, shivkumar k. cardiac innervation and sudden cardiac death. circulation research. 2015;116(12):2005–19. 11. franciosi s, perry fkg, roston tm, armstrong kr, claydon ve, sanatani s. the role of the autonomic nervous system in arrhythmias and sudden cardiac death. autonomic neuroscience: basic & clinical. 2017;205:1–11. 12. mosqueda-garcia r. evaluation of autonomic failure. disorders of the autonomic nervous system. london: crc press; 2019. p. 25–60. 13. goetze jp, alehagen u, flyvbjerg a, rehfeld jf. making sense of chromogranin a in heart disease. the lancet diabetes & endocrinology. 2013;1(1):7–8. 14. bauer mb, currie kp. adrenal medulla hormones. in: litwack g, editor. hormonal signaling in biology and medicine. united states: elsevier; 2020. p. 635–53. 15. di comite g, morganti a. chromogranin a: a novel factor acting at the cross road between the neuroendocrine and the cardiovascular systems. journal of hypertension. 2011;29(3):409–14. 16. cooper tm, mckinley ps, seeman te, choo th, lee s, sloan rp. heart rate variability predicts levels of inflammatory markers: evidence for the vagal anti-inflammatory pathway. brain, behavior, and immunity. 2015;49:94–100. 17. ackland gl, minto g, clark m, whittle j, stephens rcm, owen t, et al. autonomic regulation of systemic inflammation in humans: a multicenter, blinded observational cohort study. brain, behavior, and immunity. 2018;67:47–53. 18. shenhar-tsarfaty s, brzezinski ry, waiskopf n, finkelstein a, halkin a, berliner s, et al. blood acetylcholinesterase activity is associated with increased 10 year all-cause mortality following coronary angiography. atherosclerosis. 2020;313:144–9. 19. mushtaq g, greig nh, khan ja, kamal ma. status of acetylcholinesterase and butyrylcholinesterase in alzheimer’s disease and type 2 diabetes mellitus. cns & neurological disorders drug targets. 2014;13(8):1432–9. 20. arbel y, shenhar-tsarfaty s, waiskopf n, finkelstein a, halkin a, revivo m, et al. decline in serum cholinesterase activities predicts 2-year major adverse cardiac events. molecular medicine (cambridge, mass). 2014;20(1):38–45. 21. calderon-margalit r, adler b, abramson jh, gofin j, kark jd. butyrylcholinesterase activity, cardiovascular risk factors, and mortality in middle-aged and elderly men and women in jerusalem. clinical chemistry. 2006;52(5):845–52. 22. goliasch g, haschemi a, marculescu r, endler g, maurer g, wagner o, et al. butyrylcholinesterase activity predicts long-term survival in patients with coronary artery disease. clinical chemistry. 2012;58(6):1055–8. 23. shen mj, zipes dp. role of the autonomic nervous system in modulating cardiac arrhythmias. circulation research. 2014;114(6):1004–21. 24. de ferrari gm, vanoli e, stramba-badiale m, hull ss, jr., foreman rd, schwartz pj. vagal reflexes and survival during acute myocardial ischemia in 55j contemp med sci | vol. 9, no. 1, january-february 2023: 48–55 z.l. abdi et al. original the relation of autonomic biomarkers to the patient’s outcome with acs conscious dogs with healed myocardial infarction. the american journal of physiology. 1991;261(1 pt 2):h63–9. 25. heusch g. vagal cardioprotection in reperfused acute myocardial infarction. jacc cardiovascular interventions. 2017;10(15):1521–2. 26. mohammad fk, faris ga, al-kassim na. a modified electrometric method for measurement of erythrocyte acetylcholinesterase activity in sheep. veterinary and human toxicology. 1997;39(6):337–9. 27. mohammad fk, alias as, ahmed oa. electrometric measurement of plasma, erythrocyte, and whole blood cholinesterase activities in healthy human volunteers. journal of medical toxicology : official journal of the american college of medical toxicology. 2007;3(1):25–30. 28. ricci b, cenko e, vasiljevic z, stankovic g, kedev s, kalpak o, et al. acute coronary syndrome: the risk to young women. journal of the american heart association. 2017;6(12). 29. mohammad am, rashad hh, habeeb qs, rashad bh, saeed sy. demographic, clinical and angiographic profile of coronary artery disease in kurdistan region of iraq. american journal of cardiovascular disease. 2021;11(1):39–45. 30. huffman md, mohanan pp, devarajan r, baldridge as, kondal d, zhao l, et al. effect of a quality improvement intervention on clinical outcomes in patients in india with acute myocardial infarction: the acs quik randomized clinical trial. jama. 2018;319(6):567–78. 31. nowbar an, gitto m, howard jp, francis dp, al-lamee r. mortality from ischemic heart disease. circulation cardiovascular quality and outcomes. 2019;12(6):e005375. 32. bhatt dl, lopes rd, harrington ra. diagnosis and treatment of acute coronary syndromes: a review. jama. 2022;327(7):662–75. 33. alkhouli m, alqahtani f, jneid h, al hajji m, boubas w, lerman a. agestratified sex-related differences in the incidence, management, and outcomes of acute myocardial infarction. mayo clinic proceedings. 2021;96(2):332–41. 34. ralapanawa u, kumarasiri pvr, jayawickreme kp, kumarihamy p, wijeratne y, ekanayake m, et al. epidemiology and risk factors of patients with types of acute coronary syndrome presenting to a tertiary care hospital in sri lanka. bmc cardiovascular disorders. 2019;19(1):1–9. 35. wahrenberg a, kuja-halkola r, magnusson pke, häbel h, warnqvist a, hambraeus k, et al. cardiovascular family history increases the risk of disease recurrence after a first myocardial infarction. journal of the american heart association. 2021;10(23):e022264. 36. cheema fm, cheema hm, akram z. identification of risk factors of acute coronary syndrome in young patients between 18-40 years of age at a teaching hospital. pakistan journal of medical sciences. 2020;36(4):821–4. 37. wibring k, herlitz j, christensson l, lingman m, bång a. prehospital factors associated with an acute life-threatening condition in non-traumatic chest pain patients a systematic review. international journal of cardiology. 2016;219:373–9. 38. sedighi sm, nguyen m, khalil a, fülöp t. the impact of cardiac troponin in elderly patients in the absence of acute coronary syndrome: a systematic review. international journal of cardiology heart & vasculature. 2020;31:100629. 39. eggers km, lindahl b. prognostic biomarkers in acute coronary syndromes: risk stratification beyond cardiac troponins. current cardiology reports. 2017;19(4):29. 40. dang h, li j, liu c, xu f. chromogranin a provides additional prognostic information in children with severe hand, foot, and mouth disease: a prospective observational study. international journal of infectious diseases: ijid : official publication of the international society for infectious diseases. 2020;93:367–74. 41. dev nb, gayen jr, o’connor dt, mahata sk. chromogranin a and the autonomic system: decomposition of heart rate variability and rescue by its catestatin fragment. endocrinology. 2010;151(6):2760–8. 42. capellino s, lowin t, angele p, falk w, grifka j, straub rh. increased chromogranin a levels indicate sympathetic hyperactivity in patients with rheumatoid arthritis and systemic lupus erythematosus. the journal of rheumatology. 2008;35(1):91–9. 43. jansson am, røsjø h, omland t, karlsson t, hartford m, flyvbjerg a, et al. prognostic value of circulating chromogranin a levels in acute coronary syndromes. european heart journal. 2009;30(1):25–32. 44. corti a, marcucci f, bachetti t. circulating chromogranin a and its fragments as diagnostic and prognostic disease markers. pflugers archiv : european journal of physiology. 2018;470(1):199–210. 45. goetze jp, alehagen u, flyvbjerg a, rehfeld jf. chromogranin a as a biomarker in cardiovascular disease. biomarkers in medicine. 2014;8(1): 133–40. 46. angelone t, mazza r, cerra mc. chromogranin-a: a multifaceted cardiovascular role in health and disease. current medicinal chemistry. 2012;19(24):4042–50. 47. mahata sk, corti a. chromogranin a and its fragments in cardiovascular, immunometabolic, and cancer regulation. annals of the new york academy of sciences. 2019;1455(1):34–58. 48. ji l, pei zq, ma df, zhang j, su js, gao xd, et al. [prognostic value of circulating catestatin levels for in-hospital heart failure in patients with acute myocardial infarction]. zhonghua xin xue guan bing za zhi. 2012;40(11):914–9. 49. jeske w, glinicki p. prognostic value of circulating chromogranin a levels in acute coronary syndrome. european heart journal. 2010;31(1):128; author reply -9. 50. abdullah qh, shaikho sk, ali ihjdmj. role of chromogranin a in the assessment of sympathetic activity in acute myocardial infarction. duhok medical journal. 2011;5(2). 51. tota b, angelone t, cerra mc. the surging role of chromogranin a in cardiovascular homeostasis. frontiers in chemistry. 2014;2:64. 52. herlitz j, thuresson m, svensson l, lindqvist j, lindahl b, zedigh c, et al. factors of importance for patients’ decision time in acute coronary syndrome. international journal of cardiology. 2010;141(3):236–42. 53. pieroni m, corti a, tota b, curnis f, angelone t, colombo b, et al. myocardial production of chromogranin a in human heart: a new regulatory peptide of cardiac function. european heart journal. 2007;28(9):1117–27. 54. gallo mp, levi r, ramella r, brero a, boero o, tota b, et al. endotheliumderived nitric oxide mediates the antiadrenergic effect of human vasostatin-1 in rat ventricular myocardium. american journal of physiology heart and circulatory physiology. 2007;292(6):h2906–12. 55. hsu ch, reyes lf, orihuela cj, buitrago r, anzueto a, soni nj, et al. chromogranin a levels and mortality in patients with severe sepsis. biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals. 2015;20(3):171–6. 56. d’amico m a, ghinassi b, izzicupo p, manzoli l, di baldassarre a. biological function and clinical relevance of chromogranin a and derived peptides. endocrine connections. 2014;3(2):r45–54. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1264 170 j contemp med sci | vol. 4, no. 3, summer 2018: 170–175 original introduction nowadays, polymers is extensively used as coating on medical implants.1 in addition to polymer properties, coating technique has remarkable effects on its surface and bulk properties. there are different implant coating techniques such as: spin coatings, dip coating,2 plasma spraying3 and electrohydrodynamic atomization (ehda).4,5 however, there are currently few techniques that can produce polymeric coating with desired surface properties on medical devices. electrohydrodynamic atomization is a technique that has recently attracted much attention for biomedical application. ehda have been used for several decades in different field of industry such as ink-jet printers and agriculture treatment. however, many new applications have been investigated. examples of biomedical applications include: mass spectrometry, nanoparticles production for pharmaceutical application and targeted drug delivery.4,6,7 this is because of simplicity, easy controllability, low cost and large scale production potential. it is possible to provide homogenous coating on both side of highly complex and fine structures like drug eluting stents by ehda technique. in an electrospray set up a syringe pump push out a solution to get out of a capillary nozzle that connected to high voltage power supply. applied voltage produces high density of charge on exiting liquid droplets. this droplets are unstable and break up into very fine charged droplet that move toward a grounded or opposite charged collector. the collector should be conductive to electrospray can be continued. this is a unique advantage for coating implants with conductive metallic substrate like stents and increase coating efficiency and reduce solution waste. this is due to the deposition of sprayed droplets preferably occurring on metallic substrate rather than deposition onto any parts of the opposite surface.5,6 in this study, ehda and spinning technique was used to create micro/nanostructured polymeric coatings on stainless steel plates. poly(lactic acid) (pla) with dexamethasone was used for coating. then, the effects of resultant micro/nanostructures on coating properties coating was studied. furthermore, safety and drug release kinetics of prepared coatings were evaluated. materials and methods materials poly(lactic acid) (mw = 260 kda) and dapi were obtained from sigma. dichloromethane (dcm), tetrahydrofuran (thf) were purchased from merck (germany). all cell culture materials were prepared from gibco company (ireland). ficcole opaque and red blood cell (rbc) lysis buffer was from biosera. coating preparation for polymer-drug coating, a 1% w/v solution of pla and dex with a ratio of 85/15 was used in thf and dcm (50:50 v/v). ehda instrument (fnm co., tehran, iran) was used for coating. to gain a uniform coating, samples were placed on rotating collector. ehda condition was as follows: applied voltage 7 v, flow rate 2 ml/h, tip to collector distance 7 cm and ef f icacy and safety of micro/nanostructured polymeric coatings for drug eluting stents mostafa rahvar,a gholamreza ahmadi lakalayeh,a reza faridi majidi,a ismaeil haririan,b bahereh t. marouf,c and hossein ghanbaria,d objectives eff icacy of a polymeric coating for drug eluting stents (des) depends on its safety and structural properties. today, it is well known that factors such as surface texture, morphology and drug release kinetics are among the most critical factors that determines the ultimate destiny and success of des. therefore, the ability to design and control these critical properties guarantee the success of des in the body. methods in this study, two different micro/nanostructured coatings was prepared using poly(lactic acid) and dexamethasone by electrohydrodynamic atomization (ehda) and spinning as coating methods. to analyze structural properties of coatings different techniques was used including: x-ray diffraction, scanning electron microscopy and confocal microscopy. platelet, neutrophil and peripheral blood mononuclear cell adhesion was studied to evaluate safety of coatings. then, antibacterial properties of coatings were considered. finally, drug release profile was evaluated for 15 days. results the results showed that suitable surface properties of micro/nanostructured coatings led to very low platelet, neutrophil, pbmn on the surfaces. micro/nanostructured coatings showed two drug release kinetics that are applicable for different drug delivery systems. conclusion based on the results, ehda method have great potential as a coating method for des. keywords drug eluting stents, surface micro/nanotopography, drug release kinetics, electrospraying adepartment of medical nanotechnology, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. bmedical biomaterial research center, tehran university of medical sciences, tehran, iran. cdepartment of materials science and engineering, faculty of engineering, urmia university, urmia, iran. dresearch center for advanced technologies in cardiovascular medicine, tehran heart center, tehran university of medical sciences, tehran, iran. correspondence to hossein ghanbari, (email: hghanbari@tums.ac.ir). (submitted: 07 july 2018 – revised version received: 16 july 2018 – accepted: 02 august 2018 – published online: 26 september 2018) issn 2413-0516 mostafa rahvar et al. 171j contemp med sci | vol. 4, no. 3, summer 2018: 170–175 original efficacy and safety of micro/nanostructured polymeric coatings collector speed 15 rpm. spin-coating condition was done using the same ehda instrument with the following parameters: flow rate 8 ml/h, tip to collector distance 1 cm and rotator speed 15 rpm. different groups shown in paper are as follows: ehda– pla–dex, spin–pla–dex. x-ray diffraction x-ray diffraction (xrd) were used for analysis of coating structures. xrd was performed using a stoe-stadv diffractometer (darmstadt, germany) with cu kα radiation and λ = 1.54060 å. the scans were recorded at 2q from 1° to 20°. coating morphology the morphology of coatings were investigated using philips xl30 scanning electron microscope (sem, the netherlands). a thin nanometric layer of au (10 nm thickness) sputter was coated on samples before scanning. coating topography the topography of samples was considered by confocal microscope and µsoft premium software (nanofocus, germany). root mean square roughness (sa) of surface was reported as average surface roughness. wettability of coatings water contact angle measurement was performed to determine wettability of surfaces using goniometer (oca 15 plus, usa). thus, 4 µl droplets of water were placed on the surface and their images were captured. for each sample water contact angle of three different area on the surface were calculated and reported. platelet adhesion on the coatings scanning electron microscopy was used to study platelet adhesion on coatings. anticoagulated human blood was obtained from healthy volunteers and centrifuged at 250 g at 25°c for 15 min to separate platelet rich plasma (prp) from blood. cell blood counter instrument was used to gain number of platelet. about 1 ml of prp (containing 1,50,000 cells) was added to samples and incubated (2 h at 37°c). then, samples were washed using phosphate buffered saline. for sem, coatings were f ixed with glutaraldehyde for 60 min and dehydrated with ethanol series (30–100%) and observed by sem. since collagen have specif ic domain for platelet adhesion, ehda coated collagen was used as positive control. peripheral blood mononuclear cell and neutrophil adhesion on the coatings peripheral blood mononuclear cell (pbmc) and neutrophil adhesion were studied by dapi staining. pmnc and neutrophil was separated from human blood by ficoll-paque. centrifugation was performed for 30 min at 150 g. then, buffy coat layer which contain pmnc was removed and the remaining rbcs were omitted using rbc lysis buffer. about 1 ml of solution containing of 1,50,000 pbmc was added to coatings in a 24-well plate and incubated (3 h, 37o). finally, dapi staining was performed using following protocols: fixation for 30 min with paraformaldehyde (4%), staining with dapi for 1 min and then washing with pbs. finally, 10 images were captured by fluorescence microscope for each coating and cell counting were performed using imagej software. for neutrophil adhesion, the lower phase after centrifugation that contain rbcs and neutrophils was incubated with rbc lysis buffer and neutrophil were separated. all the other stages was similar to pbmc adhesion studies. antibacterial potential of the coatings agar test of samples against escherichia coli atcc25922 was used to evaluate bactericidal potential of samples based on 121clsi protocol and using 2 × 108 cfu/ml of bacteria. dexamethasone release for drug release studies, f irst a series of standards of dexamethasone was prepared (0–50 µg/ml) and a standard curve was drawn (maximum absorption wavelength of dexamethasone is 242 nm). samples were placed separately in capped tubes containing 3 ml phosphate buffered saline. then, samples were incubated at 37°c for 15 days. at determined time periods, 500 μl of solu tion was collected and replaced by 500 μl of fresh phosphate buffered saline. then, dexamethasone absorbance in collected solutions was measured at 242 nm using spectrophotometry. statistical analysis data were reported as mean ± sem. result analysis was done using t-tests. signif icance of difference between samples was compared at p-value < 0.05. results fig. 1 shows x-ray diffractogram of samples. pla have two sharp peaks at 2q about 17° and 19°. these peaks are seen in spin–pla–dex diffractogram. thus, spin coating has no significant influence on pla crystallinity. however, these peaks was not observed in ehda–pla–dex coating. therefore, ehda samples had lower degree of crystallinity. dex have a strongest crystallographic peak at 2q about 13.54° that was obviously observed in spin–pla–dex, but it was not seen in ehda–pla–dex diffractogram. thus, dex mainly preserved its crystalline form in spin–pla–dex. scanning electron microscopic images of coating is observed in fig. 2a and b. ehda coatings had microbead and-nanofiber morphology, but, spin coatings had micro/ nanoporous morphology. three-dimensional images of coating is shown in fig. 3. the values for surface roughness (sa) is shown in fig. 4. ehda led to surface with higher significantly higher roughness compared with spin coating method (1.734 ± 0.09 µm versus 0.908 ± 0.01 µm). fig. 5 showed the results of wettability measurements. water contact angle for es–pla–dex and spin– pla–dex was 130° and °96, respectively. scanning electron microscope images of platelet adhesion on coatings is observed in fig. 6. there were many activated and aggregated platelets on collagen as positive control. while, very few number of platelets attached on coating surfaces is in non-activated form. furthermore, very low number of pbmc was observed on the surfaces of both samples that may be related to abovementioned factors for platelet adhesion. since neutrophil compose the main part of white blood cells and they are components of innate immunity system that immediately respond to foreign material, there was rather higher number of these cells on the coating surface (fig. 7a–d). fig. 8 shows the 172 j contemp med sci | vol. 4, no. 3, summer 2018: 170–175 efficacy and safety of micro/nanostructured polymeric coatings original mostafa rahvar et al. a b fig. 2 sem images of the morphology of coating that show microbead-and-nanof`iber structure of ehda (a) and micro/ nanoporous (b) structure of spin coatings. fig. 1 x-ray diffractograms of neat pla, dexamethasone, spin– pla–dex and ehda–pla–dex. fig. 3 three-dimensional images of coating surface using confocal microscopy. a b fig. 4 root mean square roughness (ra) of samples. numerical data of pbmc and neutrophil adhesion acquired by imagej analysis software. the results of bactericidal effects of the coatings is observed in fig. 9. the results of agar test showed that both samples had no bactericidal properties. this is related to lack of any antibacterial agents in the coatings.8–10 fig. 5 the images (a and b) and numerical values (c) of water contact angles of samples. a b c fig. 6 sem images of platelet adhesion and activation on ehda-collagen (a and b), ehda–pla–dex(c) and spin-pla (d) samples. a b c d fig. 7 fluorescence microscope images of pbmc and neutrophil adhesion on ehda (a and c) and spin coatings (b and d), respectively. a b c d mostafa rahvar et al. 173j contemp med sci | vol. 4, no. 3, summer 2018: 170–175 original efficacy and safety of micro/nanostructured polymeric coatings obtained for dexamethasone absorption in the range of 0–50 µg/ml. the release profile of dexamethasone from coatings is observed in fig. 11. ehda–pla–dex had a triphasic pattern of dex release. the initial burst release of dexamethasone was 22% during first 6 h of study. then, a second phase with nearly constant rate (lasted to 7 days) and third phase that had lower release rate compared to second phase (until to 15 days). the cumulative release was near 64% after 15 days for these samples. spin–pla–dex coatings showed bi-phasic release pattern with 10% release after 6 h and a near linear release profile until the end of study. where, only 30% dex was released after 15 days. discussion polymer crystallinity is degree of crystalline regions in polymer structure and is related to alignment of polymer molecules. in this study, the results showed that ehda cause lower degree of crystallinity for both pla and dex. these observation is due to rapid evaporation of solution in ehda process due to low flow rate of solution, high distance between tip and nozzle and electrostatic repulsion. thus, both polymer and drug does not have sufficient time to crystalize.11 by controlling the determining factors of ehda such as type of solvent, concentration and flow rates of solution, different product may be obtained including f ibers,12 particles7 and their composites6,13 in the range of micrometer to nanometer. in this study, we used condition to create a microbead-and-nanofiber morphology. surface wettability is depended on both surface chemistry and micro/nanotexture. ehda led to surface with significantly higher hydrophobicity. this is mainly related to higher roughness of micro/nanotextured coating of ehda samples because of similar surface chemistry on both surfaces.14,15 results of platelet adhesion study showed very low number of un-activated platelets on coated surfaces compared with control collagen. platelet adhesion depends on different coating properties. previously, it has been shown that micro/ nanotextured structures reduce protein and platelet adhesion on the surface.16 thus, hemocompatible nature of pla, specific micro/nanotexture of samples and highly hydrophobic nature of coating possibly are the main factors that cause low platelet adhesion on the surface.17–19 the results of drug release studies showed that ehda coatings had triphasic release pattern with fig. 8 numerical data of pbmc and neutrophil adhesion on the surface analyzed by image j software. fig. 9 agar test of bactericidal effects of coatings. fig. 10 standard curve of dexamethasone in the range of 0–50 µg/ml fig. 11 dexamethasone release from different micro/nanotextured coatings. dexamethasone release the standard curve for dexamethasone is observed in fig. 10. a linear relation with correlation coefficient of 0.999 was 174 j contemp med sci | vol. 4, no. 3, summer 2018: 170–175 efficacy and safety of micro/nanostructured polymeric coatings original mostafa rahvar et al. a high burst effect. however, spin coated samples had a biphasic pattern with very low burst release. drug delivery systems such as spin–pla–dex are favorable for sustained drug release. while delivery systems such as ehda–pla–dex are suitable for applications like drug eluting stents. because of physical damage to arterial wall during stent implantation, there is need to rather a high amount of anti-inflammatory drug during first hours and days after implantation.20,21 for example, endeavor is among the most efficient drug eluting stents which release about 80% of its anti-inflammatory during first 10 days after implantation.22 the burst release from samples is mainly related to drug that is on the surface or near the surface.23,24 the second phase is probably related to diffusion of dexamethasone from coating matrix and also very little degradation of pla. the third phase is due to diffusion and further pla degradation and erosion.25 various parameters have an important role in drug release behavior such as type of polymer and drug, morphology of coating and degree of crystallinity of polymer, molecular weight and hydrophobicity of polymer.26,27 the polymer and drug was similar for both micro/nanotextured coatings. but, crystallinity of coating was significantly different. higher amorphous regions in the ehda–pla–dex coatings led to higher drug release due to higher penetration of water into amorphous regions.28 but, higher crystallinity in spin–pla–dex samples limits mobility of polymer chains that decrease dex release.29 also, higher roughness of ehda samples lead to higher surface contact area for these coating that in turn increase drug release rate specially from nanof ibrous parts of ehda–pla–dex samples.30,31 conclusion in this study, ehda and spinning technique were used to produce two different micro/nanotextured coatings on stainless steel plates. the impact of resultant micro/nanostructures on safety and efficacy of coating for drug eluting stents application showed higher efficacy and safety profiles of ehda coating due to their specific microbead-and-nanofiber structures. acknowledgment this research was supported by tehran university of medical sciences (grant number 93–03-87–27070) and national institute for medical research development (nimad: grant number 957310). conflict of interest none. n references 1. hutmacher d, hürzeler mb, schliephake h. a review of material properties of biodegradable and bioresorbable polymers and devices for gtr and gbr applications. int j oral maxillofac implants. 1996;11:667–678. 2. aksakal b, hanyaloglu c. bioceramic dip-coating on ti–6al–4v and 316l ss implant materials. j mater sci mater med. 2008;19:2097–2104. 3. de groot k, geesink r, klein cp, serekian p. plasma sprayed coatings of hydroxylapatite. j biomed mater res. 1987;21:1375–1381. 4. johnson cd, d’amato ar, puhl dl, wich dm, vesperman a, gilbert rj. electrospun fiber surface nanotopography influences astrocyte-mediated neurite outgrowth. biomed mater. 2018;13:054101. 5. guo q, mather jp, yang p, boden m, mather pt. fabrication of polymeric coatings with controlled microtopographies using an electrospraying technique. plos one. 2015;10:e0129960. 6. bock n, dargaville tr, woodruff ma. electrospraying of polymers with therapeutic molecules: state of the art. prog polym sci. 2012;37:1510–1551. 7. zhang l, huang j, si t, xu rx. coaxial electrospray of microparticles and nanoparticles for biomedical applications. expert rev med devices. 2012;9:595–612. 8. zhang x, wang l, levänen e. superhydrophobic surfaces for the reduction of bacterial adhesion. rsc adv. 2013;3:12003–12020. 9. anselme k, davidson p, popa am, giazzon m, liley m, ploux l. the interaction of cells and bacteria with surfaces structured at the nanometre scale. acta biomater. 2010;6:3824–3846. 10. xu lc, siedlecki ca. submicron-textured biomaterial surface reduces staphylococcal bacterial adhesion and biof ilm formation. acta biomater. 2012;8:72–81. 11. zilberman m, schwade nd, rs meidell rs, eberhaet rc. structured drugloaded bioresorbable f ilms for support structures. j biomater sci polym ed. 2001;12:875–892. 12. ma m, hill rm, rutledge gc. a review of recent results on superhydrophobic materials based on micro-and nanofibers. j adhes sci technol. 2008;22:1799–1817. 13. jiang l, zhao y, zhai j. a lotus-leaf-like superhydrophobic surface: a porous microsphere/nanof iber composite f ilm prepared by electrohydrodynamics. angew chem int ed engl. 2004;43:4338–4341. 14. zhang j, han y. a topography/chemical composition gradient polystyrene surface: toward the investigation of the relationship between surface wettability and surface structure and chemical composition. langmuir. 2008;24:796–801. 15. huang q, lin l, yang y, hu r, vogler ea, lin c. role of trapped air in the formation of cell-and-protein micropatterns on superhydrophobic/ superhydrophilic microtemplated surfaces. biomaterials. 2012;33: 8213–8220. 16. roach p, farrar d, perry cc. surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry. j am chem soc. 2006;128:3939–3945. 17. moradi s, hadjesfandiari n, toosi sf, kizhakkedathu jn, hatzikiriakos sg. effect of extreme wettability on platelet adhesion on metallic implants: from superhydrophilicity to superhydrophobicity. acs appl mater interfaces. 2016;8:17631–17641. 18. lima ac, mano jf. micro-/nano-structured superhydrophobic surfaces in the biomedical field: part i: basic concepts and biomimetic approaches. nanomedicine (lond). 2015;10:103–119. 19. milner kr, snyder aj, siedlecki ca. sub-micron texturing for reducing platelet adhesion to polyurethane biomaterials. j biomed mater res a. 2006;76:561–570. 20. venkatraman s1, boey f. release profiles in drug-eluting stents: issues and uncertainties. j control release. 2007;120:149–160. 21. bozsak f, gonzalez-rodriguez d, sternberger z, belitz p, bewley t, chomaz jm, et al. optimization of drug delivery by drug-eluting stents. plos one. 2015;10:e0130182. 22. stefanini gg, holmes dr jr. drug-eluting coronary-artery stents. n engl j med. 2013;368:254–265. 23. langer rs, peppas na. peppas, present and future applications of biomaterials in controlled drug delivery systems. biomaterials. 1981;2:201–214. 24. wang x, venkatraman ss, boey fy, loo js, tan lp. controlled release of sirolimus from a multilayered plga stent matrix. biomaterials. 2006;27:5588–5595. 25. fredenberg s, wahlgren m, reslow m, axelsson a. the mechanisms of drug release in poly(lactic-co-glycolic acid)-based drug delivery systems—a review. int j pharm. 2011;415:34–52. 26. kamaly, n., et al., degradable controlled-release polymers and polymeric nanoparticles: mechanisms of controlling drug release. chem rev. 2016;116:2602–2663. 27. liechty wb, kryscio dr, slaughter b, peppas na. polymers for drug delivery systems. ann rev chem biomol eng. 2010;1:149–173. 28. odian g. principles of polymerization, john wiley & sons, 2004. 29. karavelidis v, karavas e, giliopoulos d, papadimitriou s, bikiaris d. evaluating the effects of crystallinity in new biocompatible mostafa rahvar et al. 175j contemp med sci | vol. 4, no. 3, summer 2018: 170–175 original efficacy and safety of micro/nanostructured polymeric coatings polyester nanocarriers on drug release behavior. int j nanomedicine. 2011;6:3021–3032. 30. sill tj, von recum ha. electrospinning: applications in drug delivery and tissue engineering. biomaterials. 2008;29:1989–2006. 31. kenawy er, abdel-hay fi, el-newehy mh, wnek ge. processing of polymer nanofibers through electrospinning as drug delivery systems. mater chem phys. 2009;113:296–302. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201811 182 j contemp med sci | vol. 3, no. 9, winter 2017: 182–185 research assessment of cox2 and cmv in patients with chronic hcv infection heba f. hassan,a basim m. khashman,b omer a. abdul qader,c alaa w. izzatd adepartment of basic sciences. college of dentistry. university of baghdad, iraq. biraqi national cancer research center (incrc), university of baghdad, iraq. ckulliyyah of dentistry oral medicine and oral pathology department, international islamic university malaysia, malaysia. dcollege of dentistry, university of baghdad, iraq. correspondence to heba f. hassan (email: heba_micro08@yahoo.com). (submitted: 19 december 2017 – revised version received: 11 january 2017 – accepted: 20 february 2017 – published online: 27 march 2017 ) objective this study was established to determine the expression of cox-2 and cmv in patients with chronic c infection. methods a total of 30 formalin-fixed of paraffin-embedded liver samples obtained from patients with chronic hepatitis c infection. in addition, 30 apparently normal liver autopsies were used as control group used for immunohistochemistry technique (ihc) to study the expression of cyclooxygenase (cox-2) and cytomegalovirus (cmv) in these samples. results in current study, the results of expression of cox-2 were found in 25 cases (83.3%) of chronic hcv were strongly positive with a significant increase at p < 0.001, while the result of cmv expression was detected as brown cytoplasmic membranous staining of cells with positive cmv expression demonstrated in 21 (70.0%) out of 30 cases of chronic hcv infection cases. all control groups were negative for the expression of cox-2 and cmv. conclusion this study concludes that cox-2 and cmv participates in the pathogenesis of chronic hcv infection. keywords chronic hcv infection, cyclooxygenase (cox-2), cytomegalovirus (cmv) introduction hepatitis c virus (hcv) is a serious global health trouble that affects 130-170 million people worldwide.1,2 data from who estimate that 3–4 million individuals are infected with hcv every year.3 its complex disease persistent infection leading to cirrhosis, fibrosis, hepatocellular carcinoma (hcc) and liver transplantation.4 hcv is classified as flaviviridae family and genus hepacivirus. a single-stranded positive-sense rna genome enveloped virus with a ~ 9.6 kb,5 there are seven genotypes of hcv, each with many subtypes depend on antigenic variability and geographical differences in distribution, transmission, disease progression and respond differently to treatment.6 hcv genome encodes large polyprotein of 3010 amino acids which is processed co-translationally into three structural and seven nonstructural (ns) polypeptides.7,8 cyclooxygenase (cox) is the rate limiting enzymes in the production of prostanoids from arachidonic acid.9 there are two cox isoforms present in human that share more than 60% identity at the amino acid level identified. cox-1 is constitutively expressed both in normal cell types and in cirrhotic livers and is involved in the homeostatic functions of pgs.10 whereas cox-2 is induced by different stimuli such as hormones, cytokines, growth factor and mitogens, cox-2 was not seen in normal liver, but showed de novo synthesis and pronounced upregulation in liver cirrhosis.11 high cox-2 expression has been reported to be relationship with liver damage and the liver cell pathologies such as the degree of inflammation, the viral infections, the development of fibrosis and hcc in human.12,13 this was illustrated by the effect of cox-2 on the secretion of matrix metalloproteinases (mmps) by liver cells that be implicated in carcinogenesis and fibrinogenesis occurring in hcv-induced liver disease.14 cmv is the largest member of the virus family herpesviridae belong to human herpes virus-5 and is ubiquitous virus that infects most all humans at some time in their lifetime,15 is a double-strand dna virus 235-kb that encodes more than 200 proteins.16 approximately70-100% of the world’s populations are carriers to this virus,17 the prevalence of infection are estimated 100% in asia and africa, and approximately 80% in usa and europe depending on socioeconomic status.18 it has become the most common cause of severe disease with high morbidity and mortality in immune compromised individuals.19 primary human cmv infection is occurred in early life and the virus persistence in a latent state, and reactivation may occur later in life.20 therefore, reactivation of the virus is due to periods of down-regulation of the immune system, such as illness-related stress drug treatment, or during on-going activation of the immune system such as infection with other pathogens and inflammatory diseases.21 cmv can infect all organ and tissues, but manifestations of organ involvement generally include symptoms from the liver, the intestine, cns and lung.22 materials and methods patients and control samples: this study was carried on 30 liver biopsy specimens that diagnosed with chronic hcv infection in 22 males and 8 females were collected at random from the pathology archive at the liver and digestive system technical hospital in baghdad during the period from february 2010 to november 2012. the liver biopsy samples were estimated by a single pathologist, and the stage of fibrosis was depend on ishak scores fibrosis from 0 to 6 where a score of 2 is defined as fibrous expansion of most portal areas, with or without short fibrous septa; three fibrous expansion of most portal areas with occasional portal-to-portal bridging; four fibrous expansion of most portal areas with marked bridging; five incomplete cirrhosis and 6 definite cirrhosis.23 all patients were confirmed to hcv infection by elisa test. control group of liver specimens were obtained from 30 autopsies (21 males and 9 females). from each tissue block, a series of 4 μm sections were cut and stained with hematoxylin and eosin for pathological evaluation and immune histochemistry. issn 2413-0516 heba f. hassan et al. 183j contemp med sci | vol. 3, no. 9, winter 2017: 182–185 research assessment of cox2 and cmv in patients with chronic hcv infection immunohistochemical staining the antibodies used in this study included anti-cox-2 antibody and anti-cmv antibody. the sections were dewaxed in xylene then rehydrated in graded alcohol. endogenous peroxidase activity and non-specific binding were blocked by incubation with 3% hydrogen peroxide and protein block, respectively. peroxidase blocking reagent for 10 min. slides were pre-treated in microwave oven in citrate buffer (ph 6). subsequently, slides were incubated with mouse monoclonal anti-cox-2 and anti-cmv antibody and slides were then incubated sequentially with primary antibodies for 1hour at 37°c and secondary antibody for 10 minutes at room temperature followed by incubation with the streptavidine-hrp antibodies for 10 minutes at 37°c. diaminobenzidinehydrochloride (dab) was used as the chromogen to visualize peroxidase activity. the sections were counterstained with hematoxylin and overlaid with cover slips. assessment of immunohistochemistry results positive reading was detected when the cells display a brown cytoplasmic pigmentation of immunostaining, while negative reading was detected by the absence of immunostaining. assessment of anti-cox2 antibody and anti-cmv antibody immunoreactivity. the scoring system of immunostaining antibodies was assessed the positively stained cells, which counted at five representative random fields (40x) by using light microscope, the scoring of the antibodies with the criteria combined intensity with the rate of positive cell. the percentage of positive cells for the protein of interest were scored as 1 = (0–25%), 2 = (26–50%), 3 = (51–75%) and 4 = (76–100%)[8]. statistical analysis: evaluation of the statistical significance of the data was performed by using the t-test and chi-square test. results distribution of patients and control group according to gender distribution of patients and control group according to gender was listed in table 1. the results of this study showed that chronic hcv infection in the male was more predominance among patients, where 22 males (73.4%) and 8 females (26.6 %) out of total cases. there was no statistically significant difference (p > 0.05) in gender between both studied groups, the mean age of patients was 44.9 years compared with control group was 46.9. results of immunohistochemical cox2 expression the current study showed strong brown staining was seen in most cells with positive cox2 expression by immunohistochemical (ihc) staining was found in 25 cases (83.3%) of chronic hcv were strongly positive with a significant increased at p < 0.001 (table 2, fig. 1). on the other hand there was no positive result among control group. cmv expression the result of this study showed cmv expression was detected as brown cytoplasmic membranous staining of cells with table 1. distribution of patients and control group according to gender studied groups total chronic hcv control gender type male count 22 21 43 % 73.4% 70.0% 71.7% female count 8 9 17 % 26.6 % 30.0% 28.3% total count 30 30 60 % 100.0% 100.0% 100.0% mean of age 44.9 46.9 range of age 14-65 29-68 median of age 45.5 42.5 p value 0.984ns table 2. expression of cox2 in patients with chronic hcv infection and healthy control group studied groups chronic hcv control cox2 negative count 5 30 % 16.7% 100.0% positive count 25 0 % 83.3 % 0.0% total count 30 10 % 100.0% 100.0% p value < 0.001** positive cmv expression by immunohistochemical (ihc) staining demonstrated in 21 (70.0%) out of 30 cases of chronic hcv infection cases. there was highly significant statistically differences found between the patients and healthy control group at p < 0.001 (table 3 and fig. 2). discussion the present study showed that high over expression of cox-2 (83.3%) in liver tissue in patients with chronic hcv infection with different stage of fibrosis. cox-2 is concerned in anti-apoptosis, inflammation and carcinogenesis. this result agreed with nunez et al.24 was found cox-2 is over expressed in the fig 1. (a) human liver tissue stained by immunohistochemistry for cox2 in patients with chronic hcv infection stained by dab chromagen is showing as brown in positive cases at magnification 200x (b) negative expression. a b 184 j contemp med sci | vol. 3, no. 9, winter 2017: 182–185 assessment of cox2 and cmv in patients with chronic hcv infection research heba f. hassan et al. liver tissue in patients with chronic hcv infection and that the cox-2 hepatic upregulation was present especially at areas of active inflammation. these results were explained by the major action of cox-2 as factor in inflammation with the final induction of cox-2 in necro-inflammatory injury of the liver25 bassiouny et al.26 reported that the up-regulation of cox-2 expression was apparent in patients with chronic hcv infection regardless of the presence of cirrhosis while 80% of cirrhotic cases showed marked cox-2 expression. cheng and hada27 was proved that up-regulation of cox-2 expression was present in cirrhotic tissues relative to hcc as well as in well-differentiated hcc. over-expression of cox-2 in cirrhosis was apparent in a study that assess in patients with cirrhosis due to hbv and hcv infection while our series inclusive hcv infection patients mohamed et al.28 another study, we were able to express a significant relation found between overall cox-2 expression and viral load of the hcv-rna.29 while waris and siddiqui30 was found the increase in cox-2 mrna expression in cells containing the hcv replicon, indicate that hcv gene expression control cox-2 activity at the stage of transcription. holt and adams31 who indicated that cox-2-derived mediators have special functions at various times in the pathogenesis of chronic hepatitis. yosry et al.29 suggest that cox-2 expression seems to be correlated between inflammatory activity of the liver of patients with chronic hcv infection and the response to antiviral treatment which was clear by the reduction of cox-2 expression. also manning et al.32 indicated that reduction of liver cox-2 level after giving inf as treatment. gomaa et al.33 show upregulation of cox-2 in hcv infection, which is related with poor virological response. cmv is more widespread in developing countries and can affect 70–100% of the human population. in the current study showed high expression of cmv (70.0%) in the liver tissue in patients with hcv infection in different with stage of fibrosis. the infection by cmv was depended on the detection of cmv replication in the blood. reactivation of cmv infection was occurred in the absence of an effective immune response.34 cmv reactivated in immunosuppression35,36 and by directly increasing hcv replication is unknown. this is the result of explosion of high levels of cmv replication.37 bayram et al.38 was shown that cmv was diagnosed in hbv or hcv patients as a dual infection and that it can increase the risk of the disease. in egypt, tabll et al.39 were examined the potential role thatcmv plays in chronic hcv progression. also bader el din et al.,40 demonstrate higher rate of cmv co-infection in chronic hcv patients in egypt than other patient populations. whether cmv infection predisposes patients to hcv or hcv infection predisposes patients to cmv is not known. various studies indicated that cmv causes hepatitis with fibrosis of liver cells and inflammation that means cmv influence the liver and overall immunological condition of the body.38,40,41 high level of liver enzymes and remarkable histological changes in the liver of cmv-hcv co-infected patients.38 the serum levels of ast and alt enzymes showed elevated in cmv, thus indicating a role of cmv in pathogenesis of the liver. rafael et al.,42 indicated that cmv infection interacted with hcv and raised the effect on the liver enzymes and cause hepatitis. considering the fact that hcmv viruses contribute an immunomodulatory effect resulting in developed of immunosuppression43,44 and dysregulation of cytokine which could fast hcv pathogenesis of patients in critical condition.39 cmv infection promotes hcv pathogenesis by prohibition the normal mechanisms responsible for hcv elimination, thus playing vital role in hcv existence and pathogenicity.45 conclusion cox-2 and cmv seem to be related to the inflammatory activity and participate in the pathogenesis in patient with chronic hcv infection. acknowledgment i would like to express my thanks and my gratitude to all who contributed in this study. conflicts of interest there are no conflicts of interest. n table 3. expression of cmv in patients with chronic hcv infection and healthy control group studied groups chronic hcv control cmv negative count 9 30 % 30.0% 100.0% positive count 21 0 % 70.0 % 0.0% total count 30 32 % 100.0% 100.0% p value < 0.001** references 1. mccombs j, matsuda t, tonnu-mihara i, et al. the risk of long-term morbidity and mortality in patients with chronic hepatitis c results from an analysis of data from a department of veterans affairs clinical registry. jama intern med. 2013;12505. 2. hassan h: the correlation of hcv, hla-dr typing and endothelin level with oral lichen planus, phd thesis. medical microbiology. al-nahrain university, 2015. 3. world health organisation. available at: http://www.who.int. accessed september. 1,2012. 4. kwon yc, ray rb, ray r: hepatitis c virus infection: establishment of chronicity and liver disease progression. excli j. 2014;13:977–996. 5. leftkowitz e, king a, adams m, et al. virus taxonomy ninth report of the international committee on taxonomy of viruses. 2011; london: elsevier. fig 2. (a) human liver tissue stained by immunohistochemistry for cmv in patients with chronic hcv infection stained by dab chromagen is showing as brown in positive cases at magnification 200x (b) negative expression. a b heba f. hassan et al. 185j contemp med sci | vol. 3, no. 9, winter 2017: 182–185 research assessment of cox2 and cmv in patients with chronic hcv infection 6. simmonds p, becher p, collett ms, et al. flaviviridae. in: king amq, adams mj, carstens eb, lefkowitz ej (eds) virus taxonomy. elsevier, oxford. 2011; pp 1003–20. 7. kwon yc, ray rb, ray r. hepatitis c virus infection: establishment of chronicity and liver disease progression. excli journal. 2014;13:1611–2156. 8. hussein a, khashman b, hussain s: role of tgf-β1and gremlin-1in the pathogenesis of chronic hcv infection and hepatocellular carcinoma. indian j appl res. 2013;3. 9. little d, samuel lj, anthony tb. cyclooxygenase (cox) inhibitors and the intestine. j vet intern. 2007;21:367–377. 10. cheng as, chan hl, leung wk, wong n, johnson pj, sung jj: specific cox-2 inhibitor, ns-398, suppresses cellular proliferation and induces apoptosis in human hepatocellular carcinoma cells. int j oncol. 2003;23:113–119. 11. giannitrapani l, soresi m, ingraob s, et al. response to antiviral therapy and hepatic expression of cyclooxygenases in chronic hepatitis c. eur j gastroenterol hepatol. 2007;19:927–933. 12. martinez n, ricote c, manzon c, et al. role of cox-2 in the pathogenesis of chronic liver disease. med clin. 2003;29:743–748. 13. nu´n˜ezo, ferna´ndez-martı´nez a pl, majano pl, et al. increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2, and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis c virus infection: role of viral core and ns5a proteins. gut. 2004;53:1665–1672. 14. ching jl, chun wc, fang sw, et al. anti-hepatitis c virus activity of acacia confusa extract via suppressing cyclooxygenase-2. antiviral research. 2011;89:35–42. 15. ross s, novak z, pati s, boppana s. diagnosis of cytomegalovirus infections infect disord drug targets. 2011;11:466–474. 16. boeckh m, geballe ap. cytomegalovirus: pathogen, paradigm, and puzzle. j clin invest. 2011;121:1673y80. 17. scholz m, doerr hw, cinatl j. human cytomegalovirus retinitis: pathogenicity, immune evasion and persistence. trends microbiol. 2003;11:171–178. 18. cannon mj, schmid ds, hyde tb. review of cytomegalovirus seroprevalence and demographic characteristics associated with infection. rev med virol. 2010;20:202–213. 19. gandhi mk, khanna r. human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments. lancet infect dis. 2004;4:725–738. 20. görzer i, kerschner h, redlberger-fritz m, puchhammer-stöckl e. human cytomegalovirus (hcmv) genotype populations in immunocompetent individuals during primary hcmv infection. j clin virol. 2010;48:100–103. 21. gredmark s, jonasson l, van gosliga d, ernerudh j, söderberg-nauclér c. active cytomegalovirus replication in patients with coronary disease. scand cardiovasc j. 2007;41:230–234. 22. cecilia s, jay a nelson. human cytomegalovirus latency and reactivation a delicate balance between the virus and its host’s immune system. intervirology. 1999;42:314–321. 23. brunt em. grading and staging the histopathological lesions of chronic hepatitis: the knodell histology activity index and beyond. hepatology. 2000;31:241–246. 24. nunez o, fernandez-martýnez a, majano pl, et al. increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2 and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis c virus infection: role of viral core and ns5a proteins. gut. 2003;53:1665–1672. 25. cheng as, chan hl, leung wk, et al. specific cox-2 inhibitor, ns398, suppresses cellular proliferation and induces apoptosis in human hepatocellular carcinoma cells. int j oncol. 2003;23:113–119. 26. el-bassiouny ae, zoheiry mm, nosseir mm, et al. expression of cyclooxygenase-2 and transforming growth factor-beta1 in hcv-induced chronic liver disease and hepatocellular carcinoma. med gen med. 2007;9:45–51. 27. cheng j, hada t. the significance of cox-2 and cox-2 inhibitors in liver fibrosis and liver cancer current medicinal chemistry. 2005:4:199–206. 28. mohammed na, el-aleem sa, el-hafiz ha, et al. distribution of constitutive (cox-1) and inducible (cox-2) cyclooxygenase in postviral human liver cirrhosis: a possible role for cox-2 in the pathogenesis of liver cirrhosis j clin pathol. 2004;57:350–354. 29. yosry a, el-hendawy a, el-sahhar m, esmat g. et al. over-expression of hepatocyte cyclooxygenase-2 in egyptian chronic hepatitis c-infected patients and the effect of interferon-based therapy. kasr el aini med j. 2012;18. 30. waris g, siddiqui a. hepatitis c virus stimulates the expression of cyclooxygenase-2 via oxidative stress: role of prostaglandin e2 in rna replication. j virol. 2005;79:9725–9734. 31. holt ap, adams dh. complex roles of cyclo-oxygenase 2 in hepatitis. gut. 2007;56:903–904. 32. manning ds, sheehan km, byrne mf, kay ew, murray fe. cyclooxygenase-2 expression in chronic hepatitis c and the effect of interferon alpha treatment. j gastroenterol hepatol. 2007;22:1633–1637. 33. gomaa wm, ibrahim ma, shatat me: overexpression of cyclooxygenase-2 and transforming growth factor-beta 1 is an independent predictor of poor virological response to interferon therapy in chronic hcv genotype 4 patients. saudi j gastroenterol. 2014;20:59–65. 34. gandhi mk, khanna r. human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments. lancet infect dis. 2004;4:725–738. 35. noraz n, lathey jl, spector sa: human cytomegalovirus-associated immunosuppression is mediated through interferon-alpha. blood. 1997;89:2443–2452. 36. rook ah. interactions of cytomegalovirus with the human immune system. rev infect dis. 1988;10:s460–s467. 37. sia ig, wilson ja, groettum cm, espy mj, smith tf, paya cv: cytomegalovirus (cmv) dna load predicts relapsing cmv infection after solid organ transplantation. j infect dis. 2000;181:717–720. 38. bayram a, ozkur a, erkilic s: prevalence of human cytomegalovirus coinfection in patients with chronic viral hepatitis b and c: a comparison of clinical and histological aspects. j clin virol. 2009;45:212–217. 39. tabll a, shoman s, ghanem h, et al. assessment of human cytomegalovirus coinfection in egyptian chronic hcv patients. virol j. 2011;8:343. 40. bader el-din ng, abd el-meguid m, tabll aa, et al. human cytomegalovirus infection inhibits response of chronic hepatitis-c-virusinfected patients to interferon-based therapy. j gastroenterol hepatol. 2011;26:55–62. 41. humar a, kumar d, raboud j, caliendo am, moussa g, levy g, et al: interactions between cytomegalovirus, human herpesvirus-6, and the recurrence of hepatitis c after liver transplantation. am j transplant. 2002;2:461–466. 42. rafael e, de la hoz a, stephens g, christopher s: diagnosis and treatment approaches to cmv infections in adult patients. j clin virol. 2002;25:s1–s12. 43. lee so, razonable rr: current concepts on cytomegalovirus infection after liver transplantation. world j hepatol. 2010:27;325–336. 44. varani s, landini mp: cytomegalovirus-induced immunopathology and its clinical consequences. herpesviridae. 2011;7:2:6. 45. chakraborty a, patil k, dasgupta s, et al. incidence of cmv-hcv coinfection in renal transplant recipient. bmj. 2012; case reports. doi:10.1136/bcr.12.2011.5314. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 9j cont med sci | vol. 1, no. 2, spring 2015:9–13 research objective the polymorphic human liver enzyme alcohol dehydrogenase (adh) is responsible for the oxidative metabolism of ethanol. an allele encoding active form of cytosolic enzyme is known to reduce the likelihood of alcoholism in iraqi men. the polymorphisms of adh modifies the predisposition to the development of alcoholism. determination of genotypes adh2 and adh3 loci in alcoholic and nonalcoholic iraqi men. method using leukocyte dna extraction and then amplifying by polymerase chain reaction (pcr) by using specified primers, then allele specific primer extension and pcr-restriction fragment length polymorphism (rflp) methods with another set of primers is employed in order to determine the variants of adh2 and adh3, respectively. results the iraqi alcoholics had significantly lower frequencies of adh2*2 and adh3*1 alleles than did the non-alcoholic as compared with the general population of east asians but more than in caucasians population, suggesting that genetic variation in adh enzyme modulating the rate of metabolism of ethanol to acetaldehyde influences drinking and the risk of developing alcoholism. the simplest explanation of the significant lower frequency of adh2*2 and adh3*1 alleles among iraqi alcoholic men is that each can produce higher transient level of acetaldehyde, which trigger aversive reactions; these alleles are less likely to become alcoholic. conclusion this study suggests that both adh2 and adh3 genotypes exert an influence on alcohol metabolic rate, alcohol-flush reaction and susceptibility to develop alcoholism. adh2 and adh3 genotypes may have a protective role in the risk for alcoholism in iraqi alcoholic population. keywords alcohol dehydrogenase, genotype, alcoholism, cytosolic enzyme alcohol dehydrogenase genotype (adh2 and adh3) in alcoholism of iraqi people fadhil jawad al-tu’ma,a hedef dafer el-yassinb & kammela hadi shammamc introduction ethanol is a kind of drug affecting the mind, mood and other mental processes. consumption of ethanol is very common throughout the world. uncontrolled or excessive intake of alcoholic beverages is defined as alcoholism. alcoholism is a public concern for many countries, because it affects physical or mental health, social and familial relationships, and occupational responsibilities.1 individuals give different responses when exposed to comparable amount of alcohol. alcoholism must be accepted as a chronic disorder with a complex origin and outcome.2 alcoholism is an important cause of chronic liver diseases, but only 10%–20% of alcoholics develop cirrhosis.3 while a group of alcoholics do not develop cirrhosis or other chronic liver diseases, other groups who possibly consume less alcohol can have considerable liver damage. individual-based genetic variations in the genes encoding the enzymes playing active role in ethanol metabolism are considered to be responsible for this difference.4 most ethanol elimination occurs by oxidation to acetaldehyde and acetate, catalyzed principally by alcohol dehydrogenase (adh) and aldehyde dehydrogenase (aldh). there are multiple isozymes of adh in human liver. the adh isozymes primarily involve in hepatic ethanol metabolism are homo and hetero dimeric molecules whose subunits are encoded by adh1, adh2 and adh3 genes5 and are closely linked to chromosome 4.6,7 polymorphic alleles at the adh2 (b-subunit) and adh3 (γ-subunit) loci encode isozymes that differ strikingly in catalytic properties5 these differences are thought to underlie apart of the threefold variation in alcohol elimination rates among individuals (wagner et al, 1976) of which 50% is thought to be genetic in origin.8,9 humans have seven adh genes tightly clustered on chromosome 4q22 in a head-to-tail array extending over 365 kb. the order of the genes (from 5’ to 3’) is adh7-adh1cadh1badh1a-adh6-adh4-adh5, running as shown in fig. 1.10,11 all the adh enzymes are broad substrate oxidoreductases that use nad+/nadh as cofactors.10 adh1a, adh1b and adh1c encode α, β and γ subunits, respectively; these can form heterodimers, and are defined as class i adh forms. class i adhs have km value for ethanol ranged between 0.05–34 mm.10 adh4 encodes π-adh, a class ii adh with km value for ethanol of 34.0 mm. adh5 encodes x-adh, which is also a glutathione-dependent formaldehyde dehydrogenase, χ-adh has very low affinity for ethanol. adh7 encodes σ-adh (also known as μ-adh); it is the most efficient of these enzymes at oxidising retinol. the protein encoded by adh6 has not been purified from tissue (table 1). the primary site of ethanol oxidation is the liver, in which there are high concentrations of most of the adhs, except σ-adh.10 consideration of the enzyme concentrations in liver and the kinetic properties of the adhs suggest that class i enzymes (encoded by adh1a, adh1b and adh1c) and class ii enzyme (encoded by adh4) make the most significant contribution to ethanol metabolism.10,12 (hurley, 2002 ; lee, 2004). it has been calculated that class i enzyme contributes ~70% of the total ethanol oxidising capacity of the liver at an ethanol concentration of 22 mm (0.1%; 0.08% is defined as a department of biochemistry, college of medicine, university of karbala, karbala, iraq. b department of biochemistry, college of medicine, baghdad university, baghdad, iraq. c institute of forensic medicine, ministry of health, baghdad, iraq. correspondence to fadhil jawad al-tu’ma (email: biochemistry.medker@gmail.com; f_altoma_56@yahoo.com). (submitted: 30 april 2015 – revised version received: 02 june 2015 – accepted: 29 june 2015 – published online: spring 2015) 10 j cont med sci | vol. 1, no. 2, spring 2015:9–13 adh2 and adh3 in iraqi people research fadhil jawad al-tu’ma et al. legally intoxicated in iraq), and class ii enzyme contributes ~30% (table 2).10,12 the pharmacokinetics of ethanol metabolism influences the risk for alcohol dependence. various studies have shown that coding variations in the genes encoding two alcohol-metabolising enzymes, adh1b and adh1c are associated with risk for alcoholism.10,13 the adh1b*2 allele in which arginine 48 is replaced with histidine encodes the b2 subunit, which has a 40-fold higher vmax than the b1 subunit encoded by adh1b1 (48 arg370 reference allele).12 adh1b*2 is relatively common among asians, where it has been shown to be protective against alcoholism10,14; although rarer in europeans, it has also been shown to be protective in that group.15,16 a different allele, adh1b*3, encodes the b3 subunit in which arginine 370 is replaced by cysteine; the b3 subunit has a 30-fold higher vmax than the b1 subunit. adh1b*3 is relatively common among individuals of african ancestry, and individuals carrying this polymorphism have a higher rate of metabolising alcohol.17 adh1c, which encodes the γ subunit, has polymorphisms at amino acids 272 and 350; these are in high linkage dis-equilibrium (ld), with the 272arg–350 ile form called γ1 (encoded by adh1c1) and the 272gln–350val called γ2 (encoded by adh1c*2) (hoog,1986).18 the vmax of γ1 is about twice that of γ2.10,12,19 the aim of the present study is to identify the adh variants in alcoholic and non-alcoholic iraqi people, and also to investigate the relationship between adh gene polymorphism with the tendency of iraqi people to develop alcohol tolerance, and then to compare with other ethnic groups and races in world. materials and methods seventy alcoholic male iraqi people with the mean ± sd (40.35 ± 11.01 years old) who were alcohol dependent by dsm-iii criteria (american psychiatric association 1980),20 were selected from ibn-rushed teaching hospital/baghdad, iraq from jan. to sep. 2013. another 70 non-alcoholic subjects were selected as control group from the male students, hospital staff (physicians, laboratory staffs, pharmacists and others) with mean ± sd (33.02 ± 7.83 years old). informed consent was obtained, and 10 ml of blood samples was drawn from each subject. genomic dna kit from bioneer, inc. company, south korea, was used to extract dna and then polymerase chain reaction (pcr) was performed to amplify target dna. genotyping of adh3 we chose two primers (5’-aatctacctctttccagagc-3’) and (5’-gc t t ta a g a g ta a ata at c g t c c cc-3’) to amplify a fragment of 146 bp for alleles detection, aliquots of amplified dna products were digested with ssp1 (restriction enzyme) at 37°c for 16 hours (overnight). ssp1 was provided by takara bio inc., japan. genotyping of adh2 allele-specific primer extension – pcr method was chosen, adh2 gene located in 4q22 has a functional polymorphism arg48his. two pairs of allele specific primers were used for adh2 genotyping. fig. 1 relative sizes and position of the seven human adh gene on the long arm of chromosome 4 (i.e. chromosome 4q). table 1. adh genes and proteins10 official gene name* old name† non-standard name‡ protein class¶ adh1a adh1 adh1a α i adh1b adh2 adh1b β i adh1c adh3 adh1c γ i adh4 adh4 adh2 π ii adh5 adh5 adh3 χ iii adh6 adh6 adh5 adh6 v adh7 adh7 adh4 σ iv adh: alcohol dehydrogenase. table 2. kinetic properties of adh proteins official gene name* amino acid differences between alleles protein name k m (ethanol) mm turnover (min–1) adh1a a 4.0 30 adh1b*1 arg48, arg370 β 1 0.05 4.0 adh1b*2 his48, arg370 β 2 0.9 350 adh1b*3 arg48, cys370 β 3 40 300 adh1c*1 arg272, ile350 γ 1 1.0 90 adh1c*2 gln272, val350 γ 2 0.6 40 adh1c*352thr thr 352† ---------adh4 π 30 20 adh5 χ >1,000 100 adh6 adh6 ? ? adh7 σ 30 1800 adh: alcohol dehydrogenase. adh7 adh1c adh1a adh6 adh4 adh5 adh1b 75 14 15 43 61 35 365 kb 11j cont med sci | vol. 1, no. 2, spring 2015:9–133 research adh2 and adh3 in iraqi peoplefadhil jawad al-tu’ma et al. for 48 arg allele: adh 2.1, 538 bp. forward primer: (5’-tctgtagatggtggctgtaggaatctgac g-3’). this primer is arginine specific (cgc). the third base from 3’-end is changed from t→a. this primer binds on exon iii. reverse primer: (5’-tactttttttccc tcctcccgt t tctact tcta-3’). this is sequence specific primer binding on intron iii. for 48 his allele: adh 2.2, 538 bp. forward primer: (5’-tctgtagatggtggctgtaggaatct gcca-3’). this primer is histidine specific (cac). the third base from 3’-end is changed from t→c. this binds on exon iii. reverse primer: (5’-tactttttttccct cctcccgtttctacttcta-3’). this is sequence specific primer binding on intron iii.1 pcr primers were provided by bioneer inc. company. results both the pcr products were evaluated on 2% of agarose gel electrophoresis. the three alleles included in adh3 variant which appear in the regions 63 and 67 bp (polymorphic type) which is also known as adh3.2, 146 bp (wild type) (adh3.1) and (63,67,146) (heterozygosity) (adh3.1/3.2) were detected as shown in fig. 2. for adh2 variant, two alleles appear, one in the region 538bp (adh2*1) and no band for adh2*2 allele, see fig. 3. we use two statistical analysis programs in this study, spss-ibm and minitab 17 to compare between groups. there were striking differences between the alcoholics and the non alcoholics in both the genotype and allele frequencies, at two loci examined see (tables 3 and 4). the adh2*2 and adh3*1 alleles were all significantly less frequent among alcoholics than among non-alcoholics. both of these alleles encode the higher activity forms of b2 and γ1, respectively. the allele ratio adh3*2/adh3*1 is most common in alcoholic and non-alcoholic subjects (p = 0.002). adh3*2 and adh2*1 were higher in alcoholic than in non-alcoholic, both of these alleles encoded the low activity forms. the difference in adh2 and adh3 allele frequencies was still significant between the two groups. it appears that theadh2*2 and the adh3*1 alleles are protective against the development of alcoholism. discussion the present article is the first report of a significant difference in adh2 and adh3 genotypes between alcoholic and non-alcoholic iraqi men. alcoholics have significantly lower frequencies of both adh2*2 and adh3*1 alleles than that found in non-alcoholics from the same population (table 4). this indicates that the adh2 and adh3 alleles affect the propensity for alcoholism. adh2 and adh3 are closely linked7 to chromosome 4 (tsukahara and yoshida 1989). among the alcoholics homozygous for adh2*2, the adh3*1 allele frequency is significantly different than that among the total population of non-alcoholics. among the alcoholics homozygous for adh2*1 and adh3*2, allele frequency is significantly higher (p < 0.041) than that found in the non-alcoholic population. thus, theadh3*2 allele appears to be accompanying the adh2*1 allele. from table 2, we conclude that individuals possessing adh2*2 and adh3*1 alleles should, therefore, generate acetaldehyde more rapidly after ethanol consumption than do individuals with only adh2*1 and adh3*2 alleles. as they do not experience the side effects of acetaldehyde, they fig. 2 screening of adh3 genotyping on agarose gel electrophoresis. fig. 3 screening of adh2 genotyping on agarose gel electrophoresis. table 3. distribution of adh2 genotypes for both alcoholics and control group gene no. adh2.1 adh2.2 allele1* allele2* adh2 (alcoholics) 70 47(67%) 23(33%) 62 38 adh2 (control) 70 35(50%) 35(50%) 50 50 p value 0.05 0.075 0.049 0.06 table 4. distribution of adh3 genotypes for both alcoholics and control group gene no. adh3.1 adh3.2 adh3.1/3.2 allele1* allele2* adh3 (alcoholics) 70 17 (24%) 23 (33%) 30 (43%) 40.5 59.5 adh3 (control) 70 26 (37%) 18(26%) 26 (37%) 50 50 p value 0.082 0.041 0.002 0.03 0.036 12 j cont med sci | vol. 1, no. 2, spring 2015:9–13 adh2 and adh3 in iraqi people research fadhil jawad al-tu’ma et al. references 1. chinnaswamy p, vijayalakshmi v. subtypes of adh2 gene in alcoholics. indian j clin biochem. 2005;jul;20(2):104–9. doi: http://dx.10.1007/ bf02867408 pmid: 23105541 2. gemma s, vichi s, testai e. individual susceptibility and alcohol effects: biochemical and genetic aspects. ann ist super sanita. 2006;42(1):8–16. pmid: 16801720 3. li tk, yin sj, crabb dw, o’connor s, ramchandani va. genetic and environmental influences on alcohol metabolism in humans. alcohol clin exp res. 2001 jan;25(1):136–44. doi: http://dx.doi. org/10.1111/j.1530-0277.2001.tb02138.x pmid: 11198709 4. tekin f, lter t. alkol metabolizması. güncel gastroenteroloji. 2005; 58–62. 5. bosron wf, li tk. genetic polymorphism of human liver alcohol and aldehyde dehydrogenases, and their relationship to alcohol metabolism and alcoholism. hepatology. 1986;may–june;6(3):502–10. doi: http://dx.doi. org/10.1002/hep.1840060330 pmid: 3519419 6. hellgren m, strömberg p, gallego o, martras s, farrés j, persson b, et al. alcohol dehydrogenase 2 is a major hepatic enzyme for human retinol metabolism. cell mol life sci. 2007 feb;64(4):498–505. doi: http://dx.doi. org/10.1007/s00018-007-6449-8 pmid: 17279314 7. yasunami m, kikuchi d, sarapata d, yoshida a. the human class i alcohol dehydrogenase gene cluster: three genes are tandem organized in an 80-kb-long segment of the genome. genomics. 1990 jun;7(2):152–8. doi: http://dx.doi.org/10.1016/0888-7543(90)90535-3 pmid: 2347582 8. luo x, kranzler hr, zuo l, wang s, schork nj, gelernter j. multiple adh genes modulate risk for drug dependence in both africanand europeanamericans. hum mol genet. 2007 feb 15;16(4):380–90. doi: http://dx.doi. org/10.1093/hmg/ddl460 pmid: 17185388 9. martin ng, perl j, oakeshott jg, gibson jb, starmer ga, wilks av. a twin study of ethanol metabolism. behav genet. 1985 mar;15(2):93–109. doi: http://dx.doi.org/10.1007/bf01065891 10. hurley td, edenberg hj, li, tk. the pharmacogenomics of alcoholism. pharmacogenomics: the search for individualized therapies. weinheim, germany: wiley-vch; 2002. pp. 417–41. 11. lee sl, chau gy, yao ct, et al. functional assessment of human alcohol dehydrogenase family in ethanol metabolism: significance of first-pass metabolism. alcohol clin exp res. 2006 jul;30(7):1132–42. doi: http://dx.doi. org/10.1111/j.1530-0277.2006.00139.x pmid: 16792560 consume more ethanol. the simplest explanation of the significantly lower frequency of adh2*2 and adh3*1 alleles among alcoholic men in iraqi is that each can produce higher transient levels of acetaldehyde, through faster production, and that even transient elevation of acetaldehyde may trigger aversive reactions. these aversive reactions may make people with these alleles less likely to become alcoholics. our results have suggested that adh2*2 has the major effects on alcoholics than the minor effects obtained from adh3*1. table 5. distribution of adh2 and adh3 genotypes in caucasian race and oriental people genotype distribution in caucasian race (%) distribution in oriental people (%) results of the present work (%) adh3.1 50–60 90 40 adh3.2 40–50 10 59 adh2. 1 90 30 62 adh2.2 0–10 70 38 table 5 shows that the distribution of adh2 and adh3 genotypes in caucasian race are strongly different from oriental people. adh2*2 and adh3*1 were high in orient people and low in caucasian while adh3*2 and adh2*1 were low in orient people and high in caucasian. our findings lie in between them, which suggested that iraqi people have a considerable chance to avoid or become alcohol tolerance. conclusion 1. this study suggests that both adh2 and adh3 genotypes exert an influence on alcohol metabolic rate, alcohol-flush reaction and susceptibility to develop alcoholism. adh2 and adh3 genotypes may have protective roles in the risk for alcoholism in iraqi alcoholic population. 2. higher acetaldehyde generated by more active adh isozymes should deter heavy drinkers. since the kinetic differences among adh2 encoded b isozymes are much more striking than those between adh3 encoded γ1 isozymes. we expected that the differences arising from the adh2 alleles play larger role in affecting the risk of alcoholism. the simplest explanation of the significant lower frequency of adh2*2 and adh3*1 alleles among iraqi alcoholic men is that each can produce higher transient level of acetaldehyde, which trigger a aversive reactions; these alleles are less likely to become alcoholic.  12. lee sl, höög jo, yin sj. functionality of allelic variations in human alcohol dehydrogenase gene family: assessment of a functional window for protection against alcoholism. pharmacogenetics. 2004 nov;14(1):725–32. doi: http://dx.doi.org/10.1097/00008571-200411000-00003 pmid: 15564879 13. li tk. pharmacogenetics of responses to alcohol and genes that influence alcohol drinking. j stud alcohol. 2000 jan;61(1):5–12. doi: http://dx.doi. org/10.15288/jsa.2000.61.5 pmid: 10627090 14. thomasson hr, crabb dw, edenberg hj, li tk, hwu hg, chen cc, et al. low frequency of the adh2*2 allele among atayal natives of taiwan with alcohol use disorders. alcohol clin exp res. 1994 jun;18(3):640–3. doi: http://dx.doi.org/10.1111/j.1530-0277.1994.tb00923.x pmid: 7943668 15. whitfield jb. alcohol dehydrogenase and alcohol dependence: variation in genotype-associated risk between populations. am j hum genet. 2002 nov;71(5):1247–50; author reply 1250–1. doi: http://dx.doi. org/10.1086/344287 pmid: 12452180 16. borràs e, coutelle c, rosell a, fernández-muixi f, broch m, crosas b, et al. genetic polymorphism of alcohol dehydrogenase in europeans: the adh22 allele decreases the risk for alcoholism and is associated with adh31. hepatology. 2000 apr;31(4):984–9. doi: http://dx.doi.org/10.1053/ he.2000.5978 pmid: 10733556 17. thomasson hr, beard jd, li tk. adh2 gene polymorphisms are determinants of alcohol pharmacokinetics. alcohol clin exp res. 1995 dec;19(6):1494–9. doi: http://dx.doi.org/10.1111/j.1530-0277.1995. tb01013.x pmid: 8749816 18. höög jo, hedén lo, larsson k, jörnvall h, von bahr-lindström h. the gamma 1 and gamma 2 subunits of human liver alcohol dehydrogenase. cdna structures, two amino acid replacements, and compatibility with changes in the enzymatic properties. eur j biochem. 1986 sep 1;159(2): 215–8. doi: http://dx.doi.org/10.1111/j.1432-1033.1986.tb09856.x pmid: 3758060 19. crabb dw, matsumoto m, chang d, you m. overview of the role of alcohol dehydrogenase and aldehyde dehydrogenase and their variants in the genesis of alcohol-related pathology. proc nutr soc. 2004 feb;63(1):49–63. doi: http://dx.doi.org/10.1079/pns2003327 pmid: 15099407 20. american psychiatric association. diagnostic and statistical manual of mental disorders, 3rd ed. washington, dc: american psychiatric association; 1980. pp. 169–70. 13j cont med sci | vol. 1, no. 2, spring 2015:9–133 research adh2 and adh3 in iraqi peoplefadhil jawad al-tu’ma et al. 21. kopun m, propping p. the kinetics of ethanol absorption and elimination in twins and supplementary repetitive experiments in singleton subjects. eur j clin pharmacol. 1977;11:337–433. 22. sorensen tia, bentsen kd, eghoje k, orholm m, hoybye g, christoffersen p. prospective evaluation of alcohol abuse and alcoholic liver injury in men as predictors of development of cirrhosis. lancet 1984;2:241–4. 23. tsukamoto s, muto t, nagoya t, shimamura m, saito m, wagner jg, et al. elimination of alcohol from human blood. j pharm sci. 1976;65: 152–4. 24. maly ip, toranelli m, sasse, d. distribution of alcohol dehydrogenase isoenzymes in the human liver acinus. histochem. cell biol. 1999;111: 391–7. 1j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 review chronic psychosocial stress reveals alzheimer’s disease in a novel at-risk rat model karim a. alkadhi* department of pharmacological and pharmaceutical sciences, college of pharmacy, university of houston, houston, tx 77204, usa *correspondence to: dr. karim a. alkadhi (e-mail: kalkadhi@uh.edu) abstract extensive individual variations in the time of onset and severity of the sporadic type of alzheimer’s disease (ad), may be due to some patient-related external factors. stress is increasingly recognized as an external factor in the development of ad. several labs, including mine, have demonstrated that chronic stress or corticosterone administration aggravates the disease in both transgenic and non-transgenic animal models. we have developed a novel rat model that simulates seemingly normal individuals who are predisposed to develop ad. this review summarizes the findings we have reported on the effect of chronic psychosocial stress in this at-risk model of ad. behavioral (learning and memory tests), electrophysiological (evoked long-term potentiation) and molecular (protein levels of memory-related signaling molecules a well as ad-related molecules. our findings suggest that even mild psychosocial stress noticeably transforms this seemingly normal rat model to a full-fledge ad phenotype. keywords: rat ad model, amyloid-beta, learning and memory, signaling molecules issn 2413-0516 introduction alzheimer’s disease (ad) is an insidious gradual decline of intellectual and emotional wellbeing. the disease is characterized by extraneuronal accumulation of high levels of the neurotoxic amyloid-beta (aβ) peptides, followed later by intracellular accumulation of abnormal tau, a necessary protein for the functioning of intracellular microtubules. hyperphosphorylation of tau protein can lead to neuronal death and gradual loss of mental abilities.1,2 the early symptoms of ad develop slowly and include loss of memory and cognitive skills sometimes with menacing personality changes. the two forms of ad are the early-onset familial type and the sporadic type. the early-onset familial form is rare, representing less than 1% of all cases of ad, and may start as early as age 40, and is due to mutations in the genes for amyloid precursor protein (app). the second type of ad, the late-onset sporadic, which generally starts after age 60, constitutes the vast majority of ad cases. the sporadic nature of the late-onset disease indicates an environmental link that may hasten the emergence of ad symptoms. in addition to its late onset, the variation in susceptibility to and time of onset of the disease suggests that, aside from possible genetic factors, environmental determinants, including chronic stress, may play a critical role in the severity of sporadic ad.3–6 it is commonly believed that the cognitive deficits in ad result from progressive synaptic dysfunction and irreversible neurodegeneration, which are probably initiated by soluble small oligomeric aβ peptides, in particular the aβ1-42 form. progressive malfunction of synaptic transmission occurs during development of ad begins as a localized reduction in synaptic function and gradually progresses to large-scale impairment of neurotransmission in the brain.7–9 the earlier amyloid cascade hypothesis has been substantially revised to explain the weak correlation of the severity of dementia with the amount of neuronal loss and extent of the amount of the toxic aβ in the ad brain. the new idea is that soluble aβ can cause cognitive impairment in the absence of neurodegeneration.10 soluble toxic aβ can disrupt all forms of synaptic plasticity, including long-term potentiation (ltp), which is widely accepted as the cellular correlate of learning and memory.11 chronic stress is known to intensify the destructive changes seen with various brain disorders including schizophrenia,12 cushing’s disease (whitworth et al., 2000),13 hypothyroidism,14–16 and ad.3–5 reports of hypercorticostroidism in ad patients17,18 and from animal model studies19,20 infer that glucocorticoids may contribute to the regulation of app levels suggesting involvement of the stress hormones in the pathogenesis of ad. in fact, epidemiological studies show that individuals under chronic stress are more prone to get mild cognitive deficits, or even ad, than non-stressed individuals.21,22 dysregulation of the hypothalamic-pituitary-adrenal (hpa) axis can be a result of exposure to severe and/or prolonged mental stress, which leads to harmful changes in the brain morphology and function.23,24 the hippocampal formation is a brain region particularly vulnerable to the detrimental effects of stress. it is among the first brain areas to succumb to the assault of ad as indicated by substantial impairment of the hippocampus-dependent cognitive capacities. my laboratory has reported that chronic intracerebroventricular infusion of pathogenic doses of aβ peptides in chronically stressed rats impairs learning and memory and negatively impacts long-term potentiation (ltp) in the hippocampal area ca1.3,4,25 the combination is more harmful than either chronic stress or aβ infusion alone. in this project, we wanted to establish if chronic stress accelerates the emergence of ad symptoms in normal individuals who are at-risk for developing this disease. we have generated a novel rat model meant to represent normal individuals who are vulnerable to develop ad but are not yet showing ad symptoms. the symptom-free at-risk aβ rat model of ad exogenous aβ administration models of ad as well as transgenic models have limitations in that both models do not fully reproduce the complexity of the human ad pathology. injection of aβ peptides into the brain may produce some injury at (submitted: 18 december 2022 – revised version received: 14 january 2023 – accepted: 25 january 2023 – published online: 26 february 2023) mailto:kalkadhi@uh.edu 2 j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 chronic stress and alzheimer’s disease review k.a. alkadhi the site of injection, which could contribute to the inflammatory processes. administration of aβ may also cause neurodegeneration, inflammation and microglial activation.26,27 these unwanted effects of the procedure can be rectified to a significant extent by adjusting the infusion rate, the volume of injection and the recovery time. the aβ effect is dose-dependent both in vitro28–31 and in vivo.32–34 this indicates that the neurotoxic effects of aβ appear only after it achieves a threshold concentration. we have developed a unique at-risk ad rat model by intracerebroventricular (icv) infusion of a sub-pathogenic concentration of aβ (sub aβ). we carried out several various tests with results indicating that unstressed, this model of ad is not significantly different than control rats. this is a new non-transgenic rat ad model that reproduces a human condition where there is vulnerability to ad with no noticeable cognitive impairment. however, when this at-risk ad rat model is generated in rats undergoing chronic stress, these rats show ad symptoms similar to those previously reported in a full-fledged model of ad, where rats received full toxic dose of aβ.3 this model represents individuals who appear normal but can develop ad dementia when exposed to stressful conditions. animal groups and treatments to analyze the effects of chronic psychosocial stress on our at-risk ad model, wistar rats were assigned into four experimental groups: control, stress, sub aβ, and stress/subaβ. the stress and stress/subaβ groups underwent psychosocial stress for 6 weeks. then, the subaβ and stress/subaβ groups were infused, icv, with sub-pathogenic concentration (160 pmol/day) during the fifth and sixth weeks using 2-week osmotic pumps. this dose was established by constructing a dose-response relationship (aβ doses-performance in the memory test maze, the rawm). the control and stress groups were infused with the inactive reverse peptide, aβ42-1 (160 pmol/day).35,36 for chronic stress, an ‘‘intruder’’ form of psychosocial stress was chosen.37 it was carried out as follows: rats in the stress groups (2 cages with 6 rats/cage) were kept together undisturbed for one week to allow rats to acclimate and establish social hierarchy. then chronic stress was initiated by daily random switching of two rats from each cage to other cage for a period of six weeks. we showed that this type of chronic psychosocial stress markedly increased blood corticosterone level38 and elevated blood pressure.39 behavioral tests: the radial arm water maze task the radial arm water maze (rawm: figure 1a) is a black circular pool (diameter: 1.5 meter) filled with water at room temperature with six v-shaped stainless-steel structures arranged to create six swim paths (arms) radiating from one open central area (see full description in gerges et al., 2004a).14 the rawm is a reliable behavioral test for analyzing hippocampus dependent spatial learning and memory. it can be viewed as a hybrid of the radial arm maze and the morris water maze (see figure 1a). the rawm combines the spatial complexity of the radial arm maze with the motivated fast learning of the morris water maze but avoids shortcomings in these two mazes.40–42 the training protocol for each rat in the rawm involves a memory acquisition (learning) phase consisting of four oneminute successive learning trials. this is then followed 20 min later by a short-term memory (stm) test, and 24 hr later by a long-term memory (ltm) test. the rat will have to find a platform hidden 1 cm below the water level at the far end of one of the six swim arms. each time the rat entered an arm other than that containing the hidden platform an error was scored. this procedure was repeated for a minimum of 8 consecutive days.3–6,35,36 learning, short-term memory and long-term memory throughout the initial days of testing, the ability of rats in all 4 groups (control, stress, subaβ and stress/subaβ) to learn the location of the survival platform during the four acquisition phase trials is initially similar as rats learned the location of the hidden platform. however, particularly in the fourth trial, the number of errors committed by the stress/subaβ rat group is significantly increased. this indicates that the learning ability of the stress/subaβ group is markedly impaired as days went by35 (figure 2a). the short-term memory (stm) is tested 20 min after the last learning trial. as expected, stress alone impairs stm in the fig. 1 the methods: (a) diagrams of the construction of the radial arm water maze (rawm) in relation to morris water maze (mwm) and radial arm maze (ram). in the rawm, the light blue regions are the swimming central and arms field. the opaque oval in arm 4 is the submerged platform. (b) the mini-osmotic pump assembly. the stainless-steel short cannula is inserted through a burr hole in the skull into the cerebral ventricle. (c) positioning of the mini-osmotic pump in the lateral cerebral ventricle of the rat. 3j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 k.a. alkadhi review chronic stress and alzheimer’s disease fig. 2 the radial arm water maze (inset at panel a) tests show diminished learning and memory in the stress/subaβ rats. (a) the number of errors made by all groups in the last learning (trial number 4. days 6–8 averaged). more errors are committed by the stress/subaβ group than any other group. (b) the shortterm memory test (stm, measured 20 min after the last learning trial) is more severely impaired in the stress/subaβ rats than the other three groups. (c) the long-term memory (ltm) test was conducted 24 h after the last learning trial, shows severe impairment in rats of the stress/subaβ group. in all tests, the unstressed subaβ rats are cognitively normal and are not significantly different from control rats. (*= p <0.05, **= p <0.001, n = 12 rats/group; mean ± sem). chronically stressed rat group by making significantly higher number of errors compared to both control and subaβ groups (figure 2b). even more interesting; the stress/subaβ group has impaired stm by making significantly more errors than all other groups, including the stress group. these findings are further confirmed by a second test; the days to criterion (dtc), which reveals that the stress/subaβ rats need significantly (p < 0.05) more days to reach a criterion than the other three experimental groups. (this criterion is defined as the number of days in which the rat makes no more than one error in three consecutive days).35 in the long-term (24 hr) memory test, the stress/subaβ animals have made significantly more errors than the control, stress, and subaβ groups (figure 2c). notably, the 6-week chronic psychosocial stress did not affect long-term memory in the stress alone group, confirming our earlier reports.37,43 in support, the dtc test (not shown) reveals that the stress/ subaβ rats need significantly more days to reach the criterion than the other three experimental groups.35 levels of cognition-related signaling molecules to understand how stress hastens the appearance of ad symptoms in the at-risk (subaβ) rats and to correlate these findings with the results from the learning and memory studies. we examined the underlying molecular mechanism. we used western blot (immunoblot) analysis to measure the levels of principal cognition-related signaling molecules in this preclinical ad model and the effects of chronic psychosocial stress. calcium calmodulin kinase ii (camkii) stress generally decreases the levels of camkii; however, certain forms of stress may upregulate camkii expression. for instance, stress caused by postnatal maternal deprivation and pubertal immobilization upregulates camkii.44 the reason for this differential response is not well understood. the critical role of camkii in the memory processes and synaptic plasticity is well studied. long-term potentiation (ltp), a type of synaptic plasticity, is believed to be the cellular correlate of learning and memory. the accepted molecular cascade of events leading to the formation of the active phosphorylated camkii (p-camkii) and eventually expression of ltp can be experimentally initiated by high frequency stimulation of the synaptic pathway. it is generally recognized that high frequency stimulation (hfs) initiates presynaptic release of the neurotransmitter glutamate, which activates glutamate receptors on the postsynaptic membrane, causing ca2+ entry through n-methyl-d-aspartate (nmda) glutamate receptor.45–47 this transient increase of intracellular ca2+ leads to activation of protein kinase c gamma (pkcg), which phosphorylates neurogranin molecule, causing separation of calmodulin from the neurogranin-calmodulin complex.48,49 the free calmodulin forms a calcium/calmodulin complex, which binds to and activates camkii. activation of camkii enables it to self-phosphorylate (autophosphorylation).50,51 autophosphorylation makes camkii intrinsically active (p-camkii), where it activates alpha-amino-3-hydroxy-5methyl-4-isoxazole (ampa) glutamate receptors and the synaptic vesicle-specific protein, synapsin, both of which are 4 j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 chronic stress and alzheimer’s disease review k.a. alkadhi important for ltp expression and memory formation.47,52–54 the activation of these protein molecules by p-camkii continues even when intracellular ca2+ concentration returns to normal levels and ends only when it is dephosphorylated by protein phosphatases including calcineurin.52,55,56 it has been suggested that p-camkii may be acting as a molecular switch that changes temporary ca2+ signals into long-term biochemical changes that creates synaptic plasticity leading to memory formation.57 pathogenic doses of aβ peptides or chronic psychosocial stress significantly reduced the levels of p-camkii without changing the levels of total camkii.3 the levels of both p-camkii and total camkii in the subaβ rats were not significantly different than those in control rats.35 however, in both the stress/subaβ and stress groups, the p-camkii levels were significantly down regulated compared to control and subaβ groups (figure 3a), with no significant change in the levels of total camkii.35 these findings suggest impairment of the mechanism of camkii phosphorylation and/or enhanced dephosphorylation. the finding that the levels of the phosphatase (dephosphorylating) enzyme, calcineurin are markedly increased in the stress/subaβ and stress groups compared to control and subaβ groups (figure 3c)35 strongly reinforce the proposition of enhanced dephosphorylation. fig. 3 the effects of chronic stress and at-risk model on memory-related major signaling molecules in the hippocampal area ca1. (a) levels of p-camkii in stress and stress/subaβ groups are significantly lower (*p < 0.05–0.01, n = 6–7 rats/group) than in the control and subaβ groups. (b) p-creb levels are significantly decreased (*p < 0.05, n = 5–6 rats/group) only in the stress/subaβ group. (c) shows a significant increase in the levels of calcineurin (*p < 0.05–0.01, n = 7–8 rats/group) in stress and stress/subaβ groups. (d) there is a significant decrease (*p < 0.05, n = 6–7 rats/group) in the levels of bdnf only in stress/subaβ group. all results are expressed as mean ± sem. insets in all panels are representative western blots. (from alkadhi, 2023). 5j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 k.a. alkadhi review chronic stress and alzheimer’s disease calcineurin phosphorylated signaling molecules, including p-camkii, are typically dephosphorylated (inactivated) by protein phosphatases, primarily calcineurin. calcineurin dephosphorylates inhibitor 1 protein, which regulates protein phosphatase 1 (pp1).58 this enzyme is efficient dephosphorylator of p-camkii.59 the hippocampus region of the brain contains several phosphatases; of which calcineurin is the principal phosphatase engaged in the regulation of synaptic plasticity. calcineurin is implicated in the induction and maintenance of long-term depression (ltd),60 which diminishes postsynaptic activity in hippocampal neurons.61 moreover, increased levels of calcineurin in the hippocampus block ltp62 and diminish hippocampus-dependent memory formation in mice.63 chronic psychosocial stress triggered upregulation of calcineurin in area ca1 of the hippocampus.3,14,37 different stressors may produce different effects on the levels of calcineurin. for instance, stress produced by predator exposure has no significant effect on the expression of calcineurin in various regions of rat brain.64 remarkably, calcineurin expression in the hippocampus of rats depressed by maternal deprivation, is gender-specific; in that it is decreased in male but not female animals.65 astonishingly, in the hippocampal formation, the dentate gyrus (dg) area seems to be resistant to the impact of moderately chronic stress, which produces a significant decrease, rather than an increase, in calcineurin levels. thus, the dg area seems to have a defense mechanism against chronic stress whereby calcineurin levels are reduced in order to maintain normal p-camkii levels. this quality may be responsible for the normal early ltp of the dg area in chronically stressed rats.66 it is reasonable to assume that this quality is there to preserve neurogenesis, which is a vital function of the dg. elevated calcineurin has a negative effect on cognition and appears to be involved in ad pathogenesis.67,68 examination of ad brains shows that mrna of calcineurin is increased in pyramidal neurons of the hippocampus, suggesting that calcineurin has an important role in the pathogenesis of ad.69 moreover, treatment of mice (ad model: tg2576) with the calcineurin inhibitor tacrolimus reverses cognitive impairment.70 experiments on cultured cortical neurons showed that disruption of amyloid precursor protein (app) function was due to a calcineurin-dependent breakdown of another calcium calmodulin kinase, camkiv, and its nuclear target cyclic amp response element binding (creb) protein.71 the disruption of app results in neurite degeneration, oxidative stress, nuclear condensation and cell death. furthermore, calcineurin activation in astrocytes is associated with the formation of reactive inflammatory processes related to ad.72 in the subaβ rats, calcineurin levels in area ca1 of hippocampus were normal and not significantly different than those in the control group. however, ca1 areas of both the stress and the stress/subaβ groups showed significantly increased levels of calcineurin (figure 3c). similar increases in calcineurin levels were seen in the ca1 area of rats treated with a full pathogenic dose of aβ peptides.3 cyclic amp response element binding (creb) protein active creb signaling pathway performs an important function in mediating the effects of chronic stress on calcium current, ltp and neurogenesis in the dg area.73 results from my lab consistently revealed that moderate chronic psychosocial stress in rats did not significantly influence the expression of p-creb or total-creb.5,6,25 phosphorylated creb (pcreb) is essential for long-term memory formation in mammals.74 the role of creb in the impairment of memory and synaptic plasticity in ad has been suggested by several studies. for example, nuclear translocation of pcreb is inhibited by aβ leading to a decreased in the cre-mediated responses.75 in addition, creb phosphorylation is decreased by aβ through reduction of activation of nmda glutamate receptor.76 the levels of p-creb and total creb in brain area ca1 were significantly reduced in the stress/subaβ group but were normal in control, stress and subaβ rat groups (figure 3b). infusion of full pathogenic dose of aβ caused a significant reduction of p-creb levels in area ca1 of rats.5,6 creb plays an essential role in the production of proteins required for long-term memory and synaptic plasticity in the brain.77 therefore, the reduced levels of creb seen may explain the impaired long-term memory in the stress/subaβ animals. these results further ascertain that chronic psychosocial stress hastens the emergence of ad symptoms in at-risk individuals. brain derived neurotropic factor (bdnf) brain-derived neurotrophic factor (bdnf) is a member of a family of neurotrophic factors that includes nerve growth factor, neurotrophin-3, and neurotrophin-4/5. these factors activate various isoforms of tropomyosin kinase (trk) receptors. bdnf exists at high concentration in hippocampal neurons, where its expression is controlled by neural activity. bdnf sustains synaptic plasticity and supports survival of existing and new neurons in the central nervous system. bdnf supports survival of neurons in the cortex,78 hippocampus79 and basal forebrain.80 it is released after tetanic stimulation to modulate the induction and maintenance phases of ltp in the hippocampus.36,81,82 the role of bdnf in memory processes is recognized when expression of its mrna is correlated with performance in various cognition tests.83,84 the essential role of bdnf in synaptic plasticity and memory, supports the assumption that its loss may add to memory dysfunctions associated with neurodegenerative diseases including ad. additionally, it has been reported that exposure to various stressors both acute85 and chronic can significantly down regulate both bdnf mrna expression and protein levels in the hippocampus. in fact, autopsied ad brains show marked reduction of bdnf mrna and protein in the hippocampus and temporal cortex of.86 however, there are conflicting reports concerning the levels of bdnf in the brains of animal models of ad. the reason for this discrepancy is unclear; it may be due to an assortment of factors including the type of ad model used, the dose level of aβ administered, different phases of the disease at which the levels of bdnf was determined etc. we showed a significant decrease in bdnf levels in the brains of rats of the stress/subaβ group compared to its levels in stress and subaβ animal groups, which are not significantly changed.6,87 similar findings have been reported on the effect of sub-lethal dose of aβ in cultured cortical neurons.88 my lab reported significantly higher levels of bdnf in rats infused with a full pathogenic dose of aβ.5 this may sound inconsistent but can be interpreted as an attempt by the brain cells to achieve repair at this stage of the disease. 6 j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 chronic stress and alzheimer’s disease review k.a. alkadhi alzheimer’s disease-relate proteins the source of aβ is a transmembrane protein called amyloid precursor protein (app). the domain of app is partly embedded in the plasma membrane where it can be cleaved. cleaving and processing of app can occur through two major pathways: a non-amyloidogenic pathway, which generates non-toxic aβ peptides, and an amyloidogenic pathway that produces the pathogenic peptides.89 the cleaving of app can occur through cutting by three enzymes: α, β, and γ secretases. the major non-amyloidogenic pathway is started by α-secretase slicing of app within the aβ domain, thereby preventing the creation of pathogenic aβ forming a harmless large soluble app fragment (soluble alpha-amyloid precursor protein; α-sapp).89 in the amyloidogenic pathway, the making of aβ peptides begins with the proteolytic cleaving of app just above the aβ domain by β-secretase discharging the soluble beta-amyloid precursor protein (β-sapp) fragment and generating the c-terminal fragment 99 (c99). the β-sapp is then cleaved by γ-secretase to release the pathogenic aβ peptides.89 the majority of β-secretase activity originates from a membrane aspartyl protease called β-site app cleaving enzyme 1 (bace1), which serves as the rate limiting step in the production of pathogenic peptides. we studied the levels of app in area ca1 of the four experimental groups using immunoblot analysis. no significant change in the app levels is seen in the stress, subaβ or stress/subaβ compared with those of the control group (figure 4a). the levels of bace in area ca1 are normal in stress or subaβ rats, however, chronic stress significantly increases the levels of bace in stress/subaβ group compared with control, stress, and subaβ rats (figure 4b). the elevated levels of bace in stress/subaβ rats suggests that the processing of app though the pathogenic pathway is enhanced in these animals. consistent with these finding, we also have reported that full pathogenic aβ dose (300 pmol/day) caused a significant elevation of bace in area ca1.4 possible mechanisms of the negative effects of stress abnormal levels of aβ peptides in the brain interrupt phosphorylation of camkii and interfere with ltp induction.4,90,91 previously, we have shown that chronic stress decreases the levels of p-camkii and reduces the magnitude of ltp in hippocampal area ca1.3,14 as mentioned earlier, camkii has an essential role in the development of learning and memory. it is possible that the stress-induced disruption of the camkii dependent protein phosphorylation may enhance the loss of p-camkii already caused by aβ, whereby contributing to the mechanism by which chronic stress impairs memory in this model of ad. high levels of the stress hormones, corticosteroids, during stressful conditions activates type-ii glucocorticoid receptors boosting ca2+ influx, which results in inhibition of ca1 pyramidal neuronal excitability.92,93 earlier reports have suggested that aβ peptides disrupt intracellular ca2+ signaling94–96 and inhibit ca2+-dependent post-translational protein phosphorylation. additionally, acute application of aβ in the dentate gyrus during repetitive stimulation causes inhibition of ltp together with a reduction in p-camkii levels.91 therefore, it is understandable that stress worsens ca2+ -dependent signaling processes in aβ rats. fig. 4 levels of ad-related proteins. (a) no significant changes in the levels of amyloid precursor protein (app) of area ca1 were observed among the four experimental groups. (b) chronic stressing of subaβ group (stress/ subaβ) significantly increased the basal level of bace in these rats. results are expressed as mean ± sem. (*) significant difference from other groups (p < 0.05, n = 6–7 rats/group). insets in both panels are representative blots. (from tran et al. 2011). as an attempt by the brain to reduce injury, the levels of neurotrophic factors, including bdnf, are elevated in specific brain areas in reaction to various types of disorders, including traumatic brain injury, seizure, ischemia and neurotoxins.97,98 bdnf plays a major role in neuronal health and survival.99 a protective mechanism may be activated in the early stages of ad to counter the aβ-induced neurotoxicity. this is suggested by previous reports that show an enhanced mrna of bdnf in the hippocampus100 as well as increased protein levels of bdnf in the forebrain in appsw mice model of ad101 and in area ca1 in the full dose aβ-treated rats.4 in contrast, we have shown that chronic stress significantly decreases bdnf levels in area ca1 of the hippocampus.37 therefore, by reducing the availability of bdnf, stress hinders the repair process and consequently boosts the effect of aβ. at the subcellular level, mental stress and ad seem to interfere with mitochondrial function by a similar mechanism. the mitochondrial enzyme that binds to aβ peptide directly is 17β-hydroxysteroid dehydrogenase type 10 (17β-hsd) or the 7j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 k.a. alkadhi review chronic stress and alzheimer’s disease aβ-binding alcohol dehydrogenase (abad). it has been shown that mental stress impaired mitochondrial function and increased abad expression in the brain therefore, it is possible that increasing abad by chronic stress brings mitochondrial dysfunction to a critical threshold level required to show clear ad phenotype. stress modifies the processing of various proteins involved in the expression of ad. glucocorticoids and stress exposure are shown to increase the concentrations of app and bace proteins, signifying that stress may be forcing the processing of app toward the pathogenic pathway. such effect explains the enhanced levels of pathogenic aβ protein3,4,102 and the increased plaque formation85 that have been reported with stress. in summary, chronic stress has a profound effect on the course of development of ad. in several studies with various experimental approaches, my lab has shown the negative impact of chronic psychosocial stress on the development of dementia in a novel rat model that simulates seemingly normal individuals susceptible to developing ad (at-risk for ad). the findings of these studies have shown that exposure to chronic stress in these individuals accelerates the progress of ad symptoms. acknowledgments the author appreciates the technical support for this review from the department of pps. conflicts of interest disclosure the author declares no actual or potential conflict of interest related to this review.  references 1. tanzi re, bertram l. 2005. twenty years of the alzheimer’s disease amyloid hypothesis: a genetic perspective. cell 120:545–555. 2. castellani rj, lee hg, zhu x, perry g, smith ma. 2008. alzheimer disease pathology as a host response. j neuropathol exp neurol 67:523–531. 3. srivareerat m, tran tt, alzoubi kh, alkadhi ka. 2009. chronic psychosocial stress exacerbates impairment of cognition and long-term potentiation in beta-amyloid rat model of alzheimer’s disease. biol psychiatry 65:918–926. 4. srivareerat m, tran tt, salim s, aleisa am, alkadhi ka. 2011. chronic nicotine restores normal abeta levels and prevents short-term memory and e-ltp impairment in abeta rat model of alzheimer’s disease. neurobiol aging 32:834–844. 5. alkadhi ka, srivareerat m, tran tt. 2010. intensification of long-term memory deficit by chronic stress and prevention by nicotine in a rat model of alzheimer’s disease. molecular cell neurosci 45:289–296. 6. alkadhi ka. 2012. chronic psychosocial stress exposes alzheimer’s disease phenotype in a novel at-risk model. front biosci (elite ed) (invited review)) 4:214–229. 7. mesulam mm (1999) neuroplasticity failure in alzheimer’s disease: bridging the gap between plaques and tangles. neuron 24:521–529. 8. rowan mj, klyubin i, cullen wk, anwyl r. 2003. synaptic plasticity in animal models of early alzheimer’s disease. philos trans r soc lond b biol sci 358:821–828. 9. selkoe dj, 2004. alzheimer disease: mechanistic understanding predicts novel therapies. ann intern med 140:627–638. 10. shankar gm, li s, mehta th, garcia-munoz a, shepardson ne, smith i, brett fm, farrell ma, rowan mj, lemere ca, regan cm, walsh dm, sabatini bl and selkoe dj. 2008. amyloid-beta protein dimers isolated directly from alzheimer’s brains impair synaptic plasticity and memory. nat med 14: 837–842. 11. sakono m, zako t. 2010. amyloid oligomers: formation and toxicity of abeta oligomers. febs j. 277(6):1348–1358. 12. walker e, mittal v, tessner k. 2008. stress and the hypothalamic pituitary adrenal axis in the developmental course of schizophrenia. annu rev clin psychol 4:189–216. 13. whitworth ja, mangos gj, kelly jj. 2000. cushing, cortisol, and cardiovascular disease. hypertension 36:912–916. 14. gerges nz, aleisa am, schwarz la, alkadhi ka. 2004a. reduced basal camkii levels in hippocampal ca1 region: possible cause of stress-induced impairment of ltp in chronically stressed rats. hippocampus 14:402–410. 15. gerges nz, alzoubi kh, park cr, diamond dm, alkadhi ka. 2004b. adverse effect of the combination of hypothyroidism and chronic psychosocial stress on hippocampus-dependent memory in rats. behav brain res 155:77–84. 16. alzoubi kh, aleisa am, and alkadhi ka. 2008. effect of chronic stress or nicotine on hypothyroidism–induced enhancement of ltd: electrophysiological and molecular studies. neurobiol. disease 32(1):81–87. 17. elgh e, lindqvist astot a, fagerlund m, eriksson s, olsson t, näsman b. 2006. cognitive dysfunction, hippocampal atrophy and glucocorticoid feedback in alzheimer’s disease. biol psychiatry.;59(2):155–161. 18. hartmann a, veldhuis jd, deuschle m, standhardt h, heuser i. 1997. twenty-four hour cortisol release profiles in patients with alzheimer’s and parkinson’s disease compared to normal controls: ultradian secretory pulsatility and diurnal variation. neurobiol aging 18:285–289. 19. budas g, coughlan cm, seckl jr, breen kc. 1999. the effect of corticosteroids on amyloid beta precursor protein/amyloid precursor-like protein expression and processing in vivo. neurosci lett; 276(1):61–64. 20. islam a, kalaria rn, winblad b, adem a. 1998. enhanced localization of amyloid beta precursor protein in the rat hippocampus following long-term adrenalectomy. brain res; 806: 108–112. 21. wilson rs, evans da, bienias jl, mendes de leon cf, schneider ja, bennett da. 2003. proneness to psychological distress is associated with risk of alzheimer’s disease. neurology 61:1479–1485. 22. wilson rs, schneider ja, boyle pa, arnold se, tang y, bennett da. 2007. chronic distress and incidence of mild cognitive impairment. neurology 68:2085–2092. 24. mcewen bs. 2008. central effects of stress hormones in health and disease: understanding the protective and damaging effects of stress and stress mediators. eur j pharmacol 583:174–185. 25. alkadhi ka. 2011. chronic stress and alzheimer’s disease-like pathogenesis in a rat model: prevention by nicotine. current neuropharmacology (alkadhi ka and eriksen j : issue eds): neurodegeneration “hot topic” issue) 9:587–597. 26. pepeu g, giovannelli l, casamenti f, scali c, bartolini l. 1996. amyloid beta-peptides injection into the cholinergic nuclei: morphological, neurochemical and behavioral effects. prog brain res 109:273–282. 27. nabeshima t, itoh a. 1998. toxicity of beta-amyloid peptide. j toxicol sci 23 suppl 2:177–180. 28. goodman y, mattson mp. 1994. secreted forms of beta-amyloid precursor protein protect hippocampal neurons against amyloid beta-peptideinduced oxidative injury. exp neurol 128:1–12. 29. green ps, gridley ke, simpkins jw. 1996. estradiol protects against betaamyloid (25-35)-induced toxicity in sk-n-sh human neuroblastoma cells. neurosci lett 218:165–168. 30. lambert mp, barlow ak, chromy ba, edwards c, freed r, liosatos m, morgan te, rozovsky i, trommer b, viola kl, wals p, zhang c, finch ce, krafft ga, klein wl. 1998. diffusible, nonfibrillar ligands derived from abeta1-42 are potent central nervous system neurotoxins. proc natl acad sci u s a 95:6448–6453. 31. assis-nascimento p, jarvis km, montague jr, mudd lm. 2007. beta-amyloid toxicity in embryonic rat astrocytes. neurochem res 32:1476–1482. 32. lue lf, kuo ym, roher ae, brachova l, shen y, sue l, beach t, kurth jh, rydel re, rogers j. 1999. soluble amyloid beta peptide concentration as a predictor of synaptic change in alzheimer’s disease. am j pathol 155:853–862. 33. ingelsson m, fukumoto h, newell kl, growdon jh, hedley-whyte et, frosch mp, albert ms, hyman bt, irizarry mc. 2004. early abeta accumulation and progressive synaptic loss, gliosis, and tangle formation in ad brain. neurology 62:925–931. 34. carrotta r, di carlo m, manno m, montana g, picone p, romancino d, san biagio pl. 2006. toxicity of recombinant beta-amyloid prefibrillar oligomers on the morphogenesis of the sea urchin paracentrotus lividus. faseb j 20:1916–1917. 8 j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 chronic stress and alzheimer’s disease review k.a. alkadhi 35. tran tt, srivareerat m, alkadhi ka. 2010. chronic psychosocial stress triggers cognitive impairment in a novel at-risk model of alzheimer’s disease. neurobiol dis 37:756–763. 36. tran tt, srivareerat m, alkadhi ka. 2011. chronic psychosocial stress accelerates impairment of long-term memory and late-phase long-term potentiation in an at-risk model of alzheimer’s disease. hippocampus. 21:724–732. 37. aleisa am, alzoubi kh, gerges nz, alkadhi ka. 2006c. chronic psychosocial stress-induced impairment of hippocampal ltp: possible role of bdnf. neurobiol dis 22:453–462. 38. gerges nz, stringer jl, alkadhi ka. 2001. combination of hypothyroidism and stress abolishes early ltp in the ca1 but not dentate gyrus of hippocampus of adult rats. brain res 922(2):250–260. 39. alkadhi ka, alzoubi kh, aleisa am, tanner fl, nimer as. 2005. psychosocial stress-induced hypertension results from in vivo expression of long-term potentiation in rat sympathetic ganglia. neurobiol dis. dec;20(3):849–857. 40. alamed, j., wilcock, d. m., diamond, d. m., gordon, m. n., & morgan, d. 2006. two-day radial-arm water maze learning and memory task; robust resolution of amyloid-related memory deficits in transgenic mice. nature protocols, 1, 1671–1679. 41. hodges h. 1996. maze procedures: the radial-arm and water maze compared. brain res cogn brain res 3:167–181. 42. diamond dm, park cr, heman kl, rose gm. 1999. exposing rats to a predator impairs spatial working memory in the radial arm water maze. hippocampus 9:542–552. 43. aleisa am, alzoubi kh, alkadhi ka. 2006b. nicotine prevents stressinduced enhancement of long-term depression in hippocampal area ca1: electrophysiological and molecular studies. j neurosci res 83:309–317. 44. novak g, fan t, o’dowd bf, george sr. 2013. postnatal maternal deprivation and pubertal stress have additive effects on dopamine d2 receptor and camkii beta expression in the striatum. int j dev neurosci. may;31(3):189–195. 45. malenka rc, kauer ja, perkel dj, mauk md, kelly pt, nicoll ra, waxham mn. 1989. an essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation. nature. 17;340(6234):554–557. 46. nicoll ra, malenka rc, kauer ja. 1989. the role of calcium in long-term potentiation. ann n y acad sci. 568:166–170. 47. fukunaga k, stoppini l, miyamoto e, muller d. 1993. long-term potentiation is associated with an increased activity of ca2+/calmodulin-dependent protein kinase ii. j biol chem 268:7863–7867. 48. kim jj, yoon ks. 1998. stress: metaplastic effects in the hippocampus. trends neurosci 21:505–509. 49. gerendasy dd, sutcliffe jg. 1997. rc3/neurogranin, a postsynaptic calpacitin for setting the response threshold to calcium influxes. mol neurobiol 15:131–163. 50. wang jh, kelly pt. 1995. postsynaptic injection of ca2+/cam induces synaptic potentiation requiring camkii and pkc activity. neuron 15:443–452. 51. holmes, wr. 2000. models of calmodulin trapping and cam kinase ii activation in a dendritic spine. j comput neurosci 8(1): 65–85. 52. fukunaga k, muller d, miyamoto e. 1996. camkinase ii in long-term potentiation. neurochem int 28:343-358. 53. nayak as, moore ci, browning md. 1996. ca2+/calmodulin-dependent protein kinase ii phosphorylation of the presynaptic protein synapsin i is persistently increased during long-term potentiation. proc natl acad sci u s a 93:15451–15456. 54. barria a, derkach v, soderling t. 1997. identification of the ca2+/ calmodulin-dependent protein kinase ii regulatory phosphorylation site in the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-type glutamate receptor. j biol chem 272:32727–32730. 55. wang jh, kelly pt. 1996. regulation of synaptic facilitation by postsynaptic ca2+/cam pathways in hippocampal ca1 neurons. j neurophysiol. 76(1):276–286. 56. fukunaga k, miyamoto e. 2000. a working model of cam kinase ii activity in hippocampal long-term potentiation and memory. neurosci res 38:3–17. 57. lisman j, schulman h, cline h. 2002. the molecular basis of camkii function in synaptic and behavioural memory. nat rev neurosci 3:175–190. 58. mulkey rm, endo s, shenolikar s, malenka rc. 1994. involvement of a calcineurin/inhibitor-1 phosphatase cascade in hippocampal long-term depression. nature 369:486–488. 59. strack s, barban ma, wadzinski be, colbran rj. 1997. differential inactivation of postsynaptic density-associated and soluble ca2+/calmodulindependent protein kinase ii by protein phosphatases 1 and 2a. j neurochem. 68(5):2119–2128. 60. thiels e, kanterewicz bi, knapp lt, barrionuevo g, klann e. 2000. protein phosphatase-mediated regulation of protein kinase c during long-term depression in the adult hippocampus in vivo. j neurosci. 20(19): 7199–7207. 61. wang jh, kelly pt. 1997. postsynaptic calcineurin activity downregulates synaptic transmission by weakening intracellular ca2+ signaling mechanisms in hippocampal ca1 neurons. j neurosci. 17(12):4600–4611. 62. winder, dg.,. mansuy im, osman m, t. moallem m and kandel er. 1998. “genetic and pharmacological evidence for a novel, intermediate phase of long-term potentiation suppressed by calcineurin.” cell 92(1): 25–37. 63. mansuy im, winder dg, moallem tm, osman m, mayford m, hawkins rd, kandel er. 1998. inducible and reversible gene expression with the rtta system for the study of memory. neuron 21:257–265. 64. zoladz pr, park cr, halonen jd, salim s, alzoubi kh, srivareerat m, fleshner m, alkadhi ka, diamond dm. 2012. differential expression of molecular markers of synaptic plasticity in the hippocampus, prefrontal cortex, and amygdala in response to spatial learning, predator exposure, and stress-induced amnesia. hippocampus 22 (3):577–589. 65. takase k, yamamoto y, yagami t. 2012. maternal deprivation in the middle of a stress hyporesponsive period decreases hippocampal calcineurin expression and causes abnormal social and cognitive behaviours in adult male wistar rats: relevance to negative symptoms of schizophrenia. behav brain res 232:306–315. 66. gerges, nz, aleisa, am; schwarz la and alkadhi ka. 2003. chronic psychosocial stress decreases calcineurin in the dentate gyrus: a possible mechanism for preservation of early ltp. neuroscience 117:869–874. 67. gong cx, singh tj, grundke-iqbal i, iqbal k. 1994. alzheimer’sdisease abnormally phosphorylated tau is dephosphorylated by protein phosphatase-2b (calcineurin). j neurochem. 62(2):803–806. 68. berridge mj. 2010. calcium hypothesis of alzheimer’s disease. pflugers arch 459: 441–449. 69. hata r, masumura m, akatsu h, li f, fujita h, nagai y, et al. 2001. upregulation of calcineurin abeta mrna in the alzheimer’s disease brain: assessment by cdna microarray. biochem biophys res commun 284: 310–316. 70. taglialatela g, hogan d, zhang wr, dineley kt. 2009. intermediateand long-term recognition memory deficits in tg2576 mice are reversed with acute calcineurin inhibition. behav brain res 200(1):95–99. 71. mbebi c, see v, mercken l, pradier l, muller u, loeffler jp. 2002. amyloid precursor protein family-induced neuronal death is mediated by impairment of the neuroprotective calcium/calmodulin protein kinase ivdependent signaling pathway. j biol chem 277: 20979–20990. 72. norris, c. m., kadish, i., blalock, e. m., chen, k. c., thibault, v., porter, n. m., et al. 2005. calcineurin triggers reactive/inflammatory processes in astrocytes and is upregulated in aging and alzheimer’s models. the journal of neuroscience, 25, 4649–4658. 73. datson na, speksnijder n, mayer jl, steenbergen pj, korobko o, goeman j, de kloet er, joe¨ls m, lucassen pj. 2012. the transcriptional response to chronic stress and glucocorticoid receptor blockade in the hippocampal dentate gyrus. hippocampus 22(2):359–371. 74. alberini cm. 2009. transcription factors in long-term memory and synaptic plasticity. physiol rev 89(1):121–145. 75. arvanitis dn, ducatenzeiler a, ou jn, grodstein e, andrews sd, tendulkar sr, ribeiro-da-silva a, szyf m, cuello ac. 2007. high intracellular concentrations of amyloid-beta block nuclear translocation of phosphorylated creb. j neurochem 103:216–228. 76. snyder em, nong y, almeida cg, paul s, moran t, choi ey, nairn ac, salter mw, lombroso pj, gouras gk, greengard p. 2005. regulation of nmda receptor trafficking by amyloid-beta. nat neurosci 8:1051–1058. 77. lonze be, ginty dd. 2002. function and regulation of creb family transcription factors in the nervous system. neuron 35:605–623. 78. ghosh a, carnahan j, greenberg me. 1994. requirement for bdnf in activity-dependent survival of cortical neurons. science 263:1618–1623. 79. lindholm d, carroll p, tzimagiogis g, thoenen h. 1996. jul autocrineparacrine regulation of hippocampal neuron survival by igf-1 and the neurotrophins bdnf, nt-3 and nt-4. eur j neurosci.;8(7):1452–1460. 80. knusel b, beck kd, winslow jw, rosenthal a, burton le, widmer hr, nikolics k, hefti f. 1992. brain-derived neurotrophic factor administration protects basal forebrain cholinergic but not nigral dopaminergic neurons from degenerative changes after axotomy in the adult rat brain. j neurosci 12:4391–4402. 81. bramham cr, messaoudi e. 2005. bdnf function in adult synaptic plasticity: the synaptic consolidation hypothesis. prog neurobiol 76:99–125. 82. soule j, messaoudi e, bramham cr. 2006. brain-derived neurotrophic factor and control of synaptic consolidation in the adult brain. biochem soc trans 34:600–604. https://pubmed.ncbi.nlm.nih.gov/16005635/ https://pubmed.ncbi.nlm.nih.gov/16005635/ https://pubmed.ncbi.nlm.nih.gov/16005635/ https://pubmed.ncbi.nlm.nih.gov/23313435/ https://pubmed.ncbi.nlm.nih.gov/23313435/ https://pubmed.ncbi.nlm.nih.gov/23313435/ 9j contemp med sci | vol. 9, no. 1, january-february 2023: 1–9 k.a. alkadhi review chronic stress and alzheimer’s disease 83. huang ej, reichardt lf. 2001. neurotrophins: roles in neuronal development and function. annu rev neurosci 24:677–736. 84. yamada k, mizuno m, nabeshima t. 2002. role for brain-derived neurotrophic factor in learning and memory. life sci 70:735–744. 85. lee kw, kim jb, seo js, kim tk, im jy, baek is, kim ks, lee jk, han pl. 2009. behavioral stress accelerates plaque pathogenesis in the brain of tg2576 mice via generation of metabolic oxidative stress. j neurochem 108:165–175. 86. connor b, young d, yan q, faull rl, synek b, dragunow m. 1997. brainderived neurotrophic factor is reduced in alzheimer’s disease. brain res mol brain res 49:71–81. 87. alkadhi ka. 2023. a rat model of pre-clinical alzheimer’s disease. chapter 4 in handbook of animal models in neurological disorders. martin, patel and preedy (eds). elsevier inc. 88. tong l, balazs r, thornton pl, cotman cw. 2004. beta-amyloid peptide at sublethal concentrations downregulates brain-derived neurotrophic factor functions in cultured cortical neurons. j neurosci. 24(30):6799–6809. 89. querfurth, h. w., & laferla, f. m. 2010. alzheimer’s disease. the new england journal of medicine, 362, 329–344. 90. townsend m, mehta t, selkoe dj. 2007. soluble abeta inhibits specific signal transduction cascades common to the insulin receptor pathway. j biol chem 282:33305–33312. 91. zhao d, watson jb, xie cw. 2004. amyloid beta prevents activation of calcium/calmodulin-dependent protein kinase ii and ampa receptor phosphorylation during hippocampal long-term potentiation. j neurophysiol 92:2853–2858. 92. conrad cd, lupien sj, mcewen bs. 1999. support for a bimodal role for type ii adrenal steroid receptors in spatial memory. neurobiol learn mem 72:39–46. 93. pavlides c, watanabe y, magarinos am, mcewen bs. 1995. opposing roles of type i and type ii adrenal steroid receptors in hippocampal long-term potentiation. neuroscience 68:387–394. 94. dong h, goico b, martin m, csernansky ca, bertchume a, csernansky jg. 2004. modulation of hippocampal cell proliferation, memory, and amyloid plaque deposition in appsw (tg2576) mutant mice by isolation stress. neuroscience 127:601–609. 95. liang z, liu f, grundke-iqbal i, iqbal k, gong cx. 2007. down-regulation of camp-dependent protein kinase by over-activated calpain in alzheimer disease brain. j neurochem 103:2462–2470. 96. liu f, grundke-iqbal i, iqbal k, oda y, tomizawa k, gong cx. 2005. truncation and activation of calcineurin a by calpain i in alzheimer disease brain. j biol chem 280:37755–37762. 97. durany n, michel t, kurt j, cruz-sanchez ff, cervas-navarro j, riederer p. 2000. brain-derived neurotrophic factor and neurotrophin-3 levels in alzheimer’s disease brains. int j dev neurosci 18:807–813. 98. lindvall o, ernfors p, bengzon j, kokaia z, smith ml, siesjo bk, persson h. 1992. differential regulation of mrnas for nerve growth factor, brainderived neurotrophic factor, and neurotrophin 3 in the adult rat brain following cerebral ischemia and hypoglycemic coma. proc natl acad sci u s a 89:648–652. 99. zuccato c, cattaneo e. 2009. brain-derived neurotrophic factor in neurodegenerative diseases. nat rev neurol 5(6):311–322. 100. tang y, yamada k, kanou y, miyazaki t, xiong x, kambe f, murata y, seo h, nabeshima t. 2000. spatiotemporal expression of bdnf in the hippocampus induced by the continuous intracerebroventricular infusion of beta-amyloid in rats. brain res mol brain res 80:188–197. 101. hellstrom-lindahl e, court j, keverne j, svedberg m, lee m, marutle a, thomas a, perry e, bednar i, nordberg a. 2004. nicotine reduces a beta in the brain and cerebral vessels of appsw mice. eur j neurosci 19:2703–2710. 102. catania c, sotiropoulos i, silva r, onofri c, breen kc, sousa n, almeida of. 2009. the amyloidogenic potential and behavioral correlates of stress. mol psychiatry 14:95–105. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i1.1319 144 j contemp med sci | vol. 4, no. 3, summer 2018: 144–147 original evaluation of salivary enzymes and oral lesions among gas station workers compared to nearby shopkeepers in tehran arghavan tonkaboni,a katayoun sargaran,b yalda ahmadi,c abdolreza mohammadnia,d,e reihaneh eghbali-zarch,f majid faridpoor,g and ahmad reza shamshirih adepartment of oral medicine, school of dentistry, tehran university of medical sciences, tehran, iran. bdepartment of community oral health, school of dentistry, tehran university of medical sciences, tehran, iran. cinternational campus, tehran university of medical sciences, tehran, iran. dchronic respiratory diseases research center, national research institute of tuberculosis and lung diseases, shahid beheshti university of medical sciences, tehran, iran. edepartment of biotechnology, school of advanced technologies in medicine, shahid beheshti university of medical sciences, tehran, iran. fprivate clinic, tehran, iran. gprivate laboratory, tehran, iran. hresearch center for caries prevention, dentistry research institute, department of community oral health, school of dentistry, tehran university of medical sciences, tehran, iran. correspondence to katayoun sargaran (email: k-sargeran@tums.ac.ir). (submitted: 17 march 2018 – revised version received: 03 april 2018 – accepted: 14 may 2018 – published online: 26 september 2018) objective studies on the association of air pollution and oral and dental health status are limited. this study was aimed to evaluate salivary enzymes and oral lesions among gas station workers compared to nearby shopkeepers, in relation to air pollution. methods in this study, we compared the level of total antioxidant capacity, superoxide dismutase and glutathione peroxidase in the saliva and oral lesions between gas station workers and shopkeepers. fifty-two participants including 26 gas station workers and 26 shopkeepers were evaluated. these participants had at least 1 year of working experience and worked for 5 days a week. the inclusion criteria for the two groups were as follows: absence of systemic diseases, no history of using immunosuppressive medications or oral spray, no history of radiotherapy or chemotherapy, absence of xerostomia and age range of 20–60 years. shopkeepers were recruited from the nearby shops (less than 1 km distance from the gas stations). saliva samples were collected from all 52 participants and evaluated. results the mean age was 33.08 ± 8.82 years for the gas station workers and 34.19 ± 12.28 years for the shopkeepers. the mean work experience was 8.38 ± 6.25 years and 8.38 ± 9.17 years for the gas station workers and shopkeepers, respectively. level of salivary enzymes were not different between the two groups. no difference was observed about oral lesions among the two groups. conclusion this study showed that no difference existed between gas station workers and nearby shopkeepers regarding salivary enzymes and the appearance of oral lesions. further studies are required to assess the effect of environmental pollutants on the saliva. keywords total antioxidant capacity, superoxide dismutase, glutathione peroxidase, oral lesions, air pollution. introduction air pollution can have hazardous effects on human health. it negatively affects the nervous, respiratory, cardiovascular, renal, gastrointestinal system, pregnancy and cell function. according to recent statistics, about 3 million people lose their life annually due to air pollution, 90% of which, live in developed countries.1 environmental and occupational factors play an important role in health status of individuals. occupational and environmental exposures may be related to disease symptoms. environmental exposures in a specific patient may necessitate some interventions to prevent future side effects and minimize the risk of occupational diseases.2 environmental lead contamination is a global health dilemma. lead is one of the most important environmental pollutants.3–5 it is mainly produced by the exhaustion of fossil fuels. extensive use of lead in industries has resulted in air pollution and water and food contamination. thus, the serum and salivary level of lead and its accumulation in different tissues in the human body have significantly increased in the recent years.6 as mentioned earlier, long-term deposition of lead in hard tissues is much higher than that in soft tissues.7 heavy metals such as lead and cadmium have no physiological activity and are toxic even in low concentrations.8 some studies have shown that dental hard tissue can uptake and store lead and other heavy metals from the environment. dental hard tissue can become carious or may undergo abrasion, erosion or attrition. in the recent years, many studies have evaluated the effect of heavy metals on dental caries. moreover, some authors believe that acidic vapors can cause non-carious dental lesions. extensive dental problems can compromise the quality of mastication, mental power and work quality and result in gastrointestinal and other systemic problems. they can also cause serious economic and social problems.9 antioxidants play an important role in inhibition of synthesis of reactive oxygen species such as o2, h2o2 and alkyl peroxyl and repairing the damage caused by their activity.10 the oral cavity is the main route of entry of foods, drinks and inhalation substances. saliva is the first protective barrier against environmental factors. saliva possesses many protective mechanisms such as secretory immunoglobulin a, enzymatic-protein defense system, histatins, lysozyme and lactoferrin. the salivary antioxidant system is another defense mechanism of the saliva that includes uric acid, superoxide dismutase (sod), total antioxidant capacity (tac) and glutathione peroxidase (gpx).10 antioxidants comprise a great issn 2413-0516 arghavan tonkaboni et al. 145j contemp med sci | vol. 4, no. 3, summer 2018: 144–147 original gas pollution and antioxidant portion of our nutritional regimen. these antioxidants along with intracellular antioxidants and the enzymatic system protect the human body against inflammation, infections and tumoral processes.11 tabrizizadeh et al.12 showed that lead can cause acute and chronic toxicity with a wide range of oral and systemic manifestations. they evaluated mine workers and workers of a textile company in yazd city and reported that the observed signs and symptoms were not related to systemic intoxication with lead. instead, they were most probably due to direct exposure of oral mucosa to lead during respiration. those occupationally exposed to lead vapor show gingival margin pigmentation, a blue line along the gingival margin due to deposition of lead sulfide as the result of the activity of bacteria in the gingival sulcus, grayish areas on the buccal and tongue mucosa, decreased salivary flow, periodontal disease, metallic taste in the mouth, and tongue tremor when compressed.12 winkler et al.13 evaluated and compared mine workers working at two different depths in a coal mine and found no significant difference in the saliva of the two groups. however, they proved that excessive contamination of soil in the working environment decreased the saliva flow and consequently decreased the salivary level of antioxidants.13 air pollution adversely affects the human health. on the other hand, no previous study has evaluated the effect of air pollution on. thus, we aimed to assess oral lesions and level of tac, sod and gpx in the saliva and compared them between gas station workers and nearby shopkeepers. materials and methods study population this study was conducted on 52 individuals out of which, 26 were gas station workers and 26 were shopkeepers. all participants signed written informed consent forms prior to participation in the study. gas station workers were chosen from three gas stations in tehran city and had a minimum of 1 year of work experience in gas station and work for 5 days a week. the inclusion criteria for the two groups were as follows: absence of systemic diseases, no history of using immunosuppressive medications or oral spray, no history of radiotherapy or chemotherapy, absence of xerostomia and age range of 20–60 years. shopkeepers were recruited from the nearby shops (less than 1 km distance from the gas stations). sample size calculation the following equation was used to calculate the sample size for the comparison of tac, sod and gpx salivary levels between the two groups: n d = + ( ) ( ) / z z sd sd 1 2 1 1 2 2 2 2 2 a b the standard deviation for the level of tac, sod and gpx was calculated to be 0.13 according to ceretti et al.14 minimum sample size was calculated to be 26 in each group to compare a difference with a magnitude of 0.1. data collection 1. data were collected through clinical examination and mucosal status. the results of laboratory tests were also recorded. 2. spectrophotometry and chromatography were used for the assessment of tac, sod and gpx. 3. disposable dental mirrors and dental explorers were used for clinical examination. also, 15 ml falcon tubes were used for saliva collection. saliva collection patients were instructed to spit into falcon tubes when comfortably seated straight to collect unstimulated saliva. they were requested to spit into the tubes until 5 mm of the tube was filled with saliva. the tubes were then capped, placed on dry ice and stored at −20°c. using saliva kits, the salivary level of the three enzymes was measured. oral lesions oral mucosa was evaluated with a dental mirror and a spotlight to determine presence/absence of lichen planus, white and red lesions, ulcers, or aphthous lesions. the history of aphthous lesions was also evaluated. ethical considerations this study was conducted on patients who signed written informed consent forms. the study was approved in the ethics committee of tehran university of medical sciences, international campus (ir.tums vcr.rec.1395.1252). results a total of 26 gas station workers and 26 shopkeepers were evaluated in this study. the mean age was 33.08 ± 8.82 years for the gas station workers and 34.19 ± 12.28 years for the shopkeepers. the mean work experience was 8.38 ± 6.25 and 8.38 ± 9.17 years for the gas station workers and shopkeepers, respectively. the difference in this regard was not significant between the two groups (table 1). assessment and comparison of tac, sod and gpx salivary levels between the two groups are shown in table 2. comparisons were made using t-test. the difference between the two groups was not significant in any of these values (p > 0.05, table 2). oral lesions were also compared between the two groups. the results revealed that 7.7% of shopkeepers had oral lesions while no gas station worker had oral lesions. however, this difference was not statistically significant between the two groups (p > 0.05, table 3). discussion by the growing population, number of motor vehicles has significantly increased. also, by the increase in industries and factories, demand for fossil fuels has increased, resulting in significant and irreparable environmental destruction. this also has resulted in increased air pollution. air pollution is due table 1. demographic information of participants (n = 52) mean standard deviation age shopkeepers 34.19 12.28 gas station workers 33.08 8.82 work experience shopkeepers 8.38 9.17 gas station workers 8.38 6.25 146 j contemp med sci | vol. 4, no. 3, summer 2018: 144–147 gas pollution and antioxidant original arghavan tonkaboni et al. to the presence of solid and liquid pollutant particles as well as carbon dioxide, sulfur dioxide, nitrogen dioxide and ozone in the air. high level of pollutants in the air can cause significant health problems for children and the elderly and result in irreversible long-term health complications.1 polluted air includes 10 µ or smaller particles, sulfur dioxide, nitrogen dioxide, photochemical oxidants such as ozone, carbon monoxide and lead.15 ozone and solid particles produced by the exhaustion of motor vehicles have created serious public health concerns.16 gas station workers are the true representatives of occupational groups constantly exposed to gases and vapors of fossil fuels. this exposure has created some concerns due to probable long-term effects on human health.17 when entered into the blood stream, these materials exert their cytotoxic and genotoxic effects.17–19 exposure to genotoxic materials can occur through environmental exposure, non-specific contamination, occupational exposure or accidental industrial exposure.20 evidence shows that gases produced by the exhaustion of gasoline and diesel are mutagenic and carcinogenic for laboratory animals and probably humans.21 we evaluated the level of tac, sod and gpx in the saliva and compared them between the two groups of gas station workers and shopkeepers. we found no significant difference between the two groups in this respect. however, hallare et al.22 in a similar study reported that gas station workers compared to controls that had higher number of micronucleated cells.22 also, santos-mello and cavalcante17 cytologically evaluated gas station workers in brazil and reported a significant increase in chromosomal deletions in the metaphase in gas station workers (0.829%) compared to controls (0.126%).17 similarly, a study by benites et al.23 showed that gas station workers experienced a significant increase in micronucleated cells. however, fredga et al. did not notice any increase in number of chromosomal disorders in gas station workers in sweden.23 similar to gas station workers, police officers are also exposed to air pollutants. such occupational exposures may cause mutagenic damage to epithelial cells of the buccal mucosa.24 a previous study showed that the tac of the saliva decreases by an increase in number of carious teeth and advanced age.25 our study indicated no difference in level of antioxidants between the two groups. thus, it may be concluded that the level of exposure of gas station workers to pollutants was not high enough to affect tac, sod and gpx, or the level of air pollution was the same up to 1 km distance from the gas station. oral lesions were also evaluated in our study. the results showed that the two groups were not different in this regard. to our knowledge, this is the first study of its kind in iran, which is considered as a strength of our study. the present study had some limitations. it was difficult to encourage people to participate in the study. also, it was hard to find adequate number of eligible participants to reach the required sample size. we offered free dental clinical examination in order to persuade them to participate in the study. acknowledgement the authors would like to thank the research deputy of international campus, tehran university of medical sciences. conclusion this study revealed no significant difference in salivary level of antioxidants and oral lesions between gas station workers and shopkeepers. further studies are required to assess the effect of environmental pollution on the saliva. conflict of interest none n table 2. level of tac, sod and gpx enzymes in the gas station workers and shopkeepers (n = 52) enzyme mean standard deviation p-value tac shopkeepers 9.37 6.23 0.69gas station workers 9.08 5.04 sod shopkeepers 96.63 46.91 0.91gas station workers 93.86 44.14 gpx shopkeepers 21.74 61.57 0.59gas station workers 62.04 23.80 table 3. oral lesions in gas station workers and shopkeepers (n = 52) oral lesions present absent shopkeepers 2 (7.7%) 24 (92.3%) gas station workers 0 26 (100%) references 1. kampa m, castanas e. human health effects of air pollution. environ pollut. 2008;151:362–367. 2. kales sn, christiani dc. acute chemical emergencies. n engl j med. 2004; 350:800–808. 3. frank rm, sargentini-maier ml, turlot jc, leroy mj. comparison of lead levels in human permanent teeth from strasbourg, mexico city, and rural zones of alsace. j dent res. 1990;69:90–93. 4. cleymaet r, bottenberg p, slop d, clara r, coomans d. study of lead and cadmium content of surface enamel of schoolchildren from an industrial area in belgium. community dent oral epidemiol. 1991;19:107–111. 5. cleymaet r, bottenberg p, retief dh, slop d, michotte y, coomans d. in vivo use of a dual acid etch biopsy for the evaluation of lead profiles in human surface enamel. caries res. 1991;25:256–263. 6. karahalil b, aykanat b, ertas n. dental lead levels in children from two different urban and suburban areas of turkey. int j hyg environ health. 2007;210:107–112. 7. hu h, rabinowitz m, smith d. bone lead as a biological marker in epidemiologic studies of chronic toxicity: conceptual paradigms. environ health perspect. 1998;106:1–8. 8. alomary a, al-momani if, massadeh am. lead and cadmium in human teeth from jordan by atomic absorption spectrometry: some factors influencing their concentrations. sci total environ. 2006;369:69–75. 9. cenić-milosević d, mileusnić i, kolak v, pejanović d, ristić t, jakovljević a, et al. environmental lead pollution and its possible influence on tooth loss and hard dental tissue lesions. vojnosanit pregl. 2013;70:751–756. arghavan tonkaboni et al. 147j contemp med sci | vol. 4, no. 3, summer 2018: 144–147 original gas pollution and antioxidant 10. raju pk, vasanti d, kumar jr, niranjani k, kumar ms. oral hygiene levels in children of tribal population of eastern ghats: an epidemiological study. j int oral health. 2015;7:108–110. 11. goldie mp. antioxidants in oral health care: making the connection. int j dent hyg. 2005;3:93–95. 12. tabrizizadeh m, boozarjomehri f, akhavan karbasi m, maziar f. evaluation of the relationship between blood lead level and prevalence of oral complication in koushk lead mine workers, yazd province. j dent tehran univ med sci. 2006;19:91–98. 13. winkler o, hadnagy w, idel h. cytokines detectable in saliva of children as appropriate markers of local immunity of the oral cavity–an approach for the use in air pollution studies. int j hyg environ health. 2001;204: 181–184. 14. ceretti e, feretti d, viola gc, zerbini i, limina rm, zani c, et al. dna damage in buccal mucosa cells of pre-school children exposed to high levels of urban air pollutants. plos one. 2014;9:e96524. 15. cruz ddl, siador jc, peralta t, aguilar p, barlis e, socorro jd, et al. national air quality status report (2003-2004). philippines: environmental management bureau, department of environment and natural resources; 2005. 16. torres e, subida r, gapas j, sarol j, villarin j, vinluan r, et al. public health monitoring of the metro manila air quality improvement sector development program. manila, philippines: world health organization (who), asian development bank (adb), philippines department of health (doh). 2004;156. 17. santos-mello r, cavalcante b. cytogenetic studies on gas station attendants. mutat res. 1992;280:285–290. 18. hadnagy w, seemayer nh. cytotoxic and genotoxic effects of extract of particulate emission from a gasoline-powered engine. environ mol mutagen. 1988;12:385–396. 19. crebelli r, tomei f, zijno a, ghittori s, imbriani m, gamberale d, et al. exposure to benzene in urban workers: environmental and biological monitoring of traffic police in rome. occup environ med. 2001;58:165–171. 20. anderson d. factors contributing to biomarker responses in exposed workers. mutat res. 1999;428:197–202. 21. crebelli r, conti l, crochi b, carere a, bertoli c, del giacomo n. the effect of fuel composition on the mutagenicity of diesel engine exhaust. mutat res. 1995;346:167–172. 22. hallare av, gervasio mk, gervasio pl, acacio-claro pj. monitoring genotoxicity among gasoline station attendants and traffic enforcers in the city of manila using the micronucleus assay with exfoliated epithelial cells. environ monit assess. 2009;156:331–341. 23. benites ci, amado ll, vianna ra, martino-roth mda g. micronucleus test on gas station attendants. genet mol res. 2006;5:45–54. 24. burgaz s, demircigil gc, karahalil b, karakaya ae. chromosomal damage in peripheral blood lymphocytes of traffic policemen and taxi drivers exposed to urban air pollution. chemosphere. 2002;47:57–64. 25. hershkovich o, shafat i, nagler rm. age-related changes in salivary antioxidant profile: possible implications for oral cancer. j gerontol a biol sci med sci. 2007;62:361–366. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201806 1j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 review genetic causes, clinical manifestations and diagnosis of central nervous system malformations with emphasis on corpus callosum basmah alomrani1, murid javed2, hani s. h. mohammed ali 1,4*, salah e. m. abo-aba1,4*, albandary albakheet3, hanan alqudairy3, namik kaya3 1department of biological science, king abdul-aziz university, jeddah, saudi arabia. 2originelle fertility clinic and women’s health centre, ottawa, canada. 3department of genetics, king faisal specialists hospital and research center, riyadh, saudi arabia. 4princess dr. najla bent saud al-saud center for excellence research in biotechnology, king abdul-aziz university, jeddah, saudi arabia. *correspondence to: hani s. h. mohammed ali (e-mail: hmohammedali@ksa.edu.sa); salah e. m. abo-aba (e-mail: salah_aboaba@yahoo.com) (submitted: 11 december 2021 – revised version received: 04 january 2022 – accepted: 25 january 2022 – published online: 26 february 2022) abstract the pathological processes of the central nervous system, intrinsic or extrinsic, occurring during the embryonic period are the most common cause of pregnancy termination. the defects of the neural tube which is primitive form of brain and spinal cord are the most common central nervous system (cns) malformations. in some defects the fetus continues to develop and results in the birth of a child with sever mental and physical deficiencies. the corpus callosum is the largest white matter tract connecting the two cerebral hemispheres and permits the transfer of information between the two cerebral hemispheres. its partial or complete agenesis is caused by abnormalities during embryonic development. the severity of symptoms varies widely depending on the degree of dysgenesis. hypotonia, visual impairment, seizures, spasticity, motor co-ordination issues, and atypical facial features are common symptoms. in this review we focus on genetic causes, clinical manifestations and diagnosis of various malformations of central nervous system with special emphasis on corpus callosum. keywords: central nervous system, corpus callosum, diagnosis issn 2413-0516 introduction malformations result from pathological processes occurring during the embryonic period. these pathological processes could be intrinsic or extrinsic. fetal brain malformations occur at a rate of 2–3 per 1000 pregnancies and are considered the most common cause of pregnancy termination.1 the fetal brain begins to develop shortly after conception and continues to grow throughout pregnancy. the symptoms and prognosis of congenital brain malformations vary, depending on the type and severity. neural tube defects (ntds) are the most common central nervous system (cns) malformations. the neural tube closure takes place at the fourth week post conception. the prevalence of ntds at birth has decreased over the past few years due to the introduction of antenatal folic acid use, especially during the 1st trimester.2 the brain develops from the cranial part (rostral part) of the neural tube. by the end of the fourth-week of gestation three primary cerebral vesicles are differentiated from the neural tube; namely the prosencephalon (forebrain), mesencephalon (midbrain), rhombencephalon (hindbrain).3 by the fifth week of gestation, the three primary vesicles further subdivide into five secondary vesicles; telencephalon, that differentiates to the cerebral hemispheres; diencephalon, that differentiates to thalamus, hypothalamus, posterior pituitary and the optic vesicles; mesencephalon, that forms the midbrain; metencephalon, that forms pons and cerebellum; and myelencephalon, that differentiates to medulla oblongata.4 primary and secondary cerebral vesicles in early embryonic development are illustrated in figure 1. epigenetic and epigenomic focus on mechanisms governing brain and behavior development, genetic evolution, neural resilience and homeostasis. epigenetic studies tackle the etiology of neurological conditions in addition to normal neural processes.5 the four main epigenetic mechanisms involved in the dynamic identification of nuclear structure and genomic landscape are dna methylation, chromatin modification, non-coding rna editing of nuclear acids, and modification of histones. environmental epigenomic science explores the environmental determinants that modulate the initiation and progression of certain neurological conditions in addition to the identification of response of treatment interventions.6 furthermore, pharmaco-epigenomics is a new science that is interested in therapies improving recovery of cognitive, behavioral, and senso-motor functions.7 dna methylation is the most common mechanism which is catalyzed by dna methyltransferase (dnmts) that transfers a methyl group to dna sequence and suppresses gene-transcription. a similar rna counterpart (dmt2) is responsible for gene-silencing in the developmental process. the dna methylation shows interactions with various items of epigenetic machinery and contributes to the expression of epigenetic marks essential for multigenerational inheritance.9 many studies found that individual-specific dna methylation is related to neuronal identity and specific functional development. the de novo methylation of dna was found associated with the prevention of early premature development of stem cell neurons. moreover, dnmt level is high in the nervous system of the embryo to preserve dna methylation during the division of neural precursors. different forms of dnmt are found in the adult brain where they enhance brain cell repair and improve cell turnover. dna methylation organizes the time of astrocyte development by mediating the mailto:hmohammedali@ksa.edu.sa mailto:salah_aboaba@yahoo.com 2 j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 genetic causes, clinical manifestations and diagnosis of central nervous system review b. alomrani et al. methylenation of certain factors in the process of glial maturational genes.10 epigenetics plays an important role in the identification of neural dysfunction associated with some conditions such as rett syndrome where the study of mecp2 mutant mice provides substantial insight into the pathological mechanisms of the syndrome.11 dynamic remodeling of the chromatin network is mediated by many enzymes and signal transportation pathways. chromatin is formed of dna units with proteins, histone and non-histone, responsible for 3d folding of dna and functional regulation of the nucleus. furthermore, the nucleus contains molecular platforms for organizing genome-wide chromatin remodeling. histone alteration and chromatin remodeling are related to neural stem cells repair which have been linked to many cognitive and behavioral disorders.12 in the eukaryotic nucleus, the areas of non-proteincoding have been remarkably increased, which reflected the functional development during evolution. however, the quantity of protein-coded areas has been unchanged. human accelerated genes (hars) which are responsible for the evolution of humans from ancestors are identified in non-protein-coding regions of the genome, most of them adjacent to the essential neurodevelopmental genes. the most important genetic loci are har1, which is expressed with reelin, which participate in cortical neurons development.13 two sub-types of rna editing enzymes can modify the magnitude of expression and the functional differentiation of rna. during neural development, rna editing enzyme expressed changing subcellular localization within neurological development. the rna editing can change biogenesis, maturation properties, immense gene products which play a broad variety of neurodevelopmental functions enhancing adult neural homeostasis.14 animal studies showed that rna editing is important for cognitive and behavioral responses of the nervous system. many neurological diseases as well as brain cancer were associated with deregulation of rna editing.15 several neurodevelopmental disorders, such as autism spectrum disorders (asds), are linked to defects in epigenetic mechanisms. epigenetic defects such as dna methylation at certain imprinted gene loci were found associated with neurological disorders like prader-willi and angelman syndromes.16 there is a considerable amount of evidence about the relation between neurodegenerative diseases and abnormalities in epigenetic mechanisms. the evidence suggested the lack of a simple mendelian inherited pattern, dysregulation of transcriptional mechanisms as well as many pathological rna alterations. mechanisms such as microsatellite sequence were found important in regulating cognitive functions, circadian rhythm, and social skills.12 cancer was found related to some genomes-wide epigenetic changes that enhance tumor induction, progression, invisibility, metastatic ability, and response to the treatment. cancer epigenetic etiology is a result of multiple inter-related mechanisms such as dna methylation, non-gene protein coding, chromatin modifications, and editing of nucleic acids. the abnormalities in these epigenetic mechanisms enhance the silencing of tumor-suppressing genes and the activation of oncogenes. some cns cancers such as glioblastomas were found significantly associated with alteration in gene expression profiles that inhibit tumor-suppressing genes.17 in this review, we highlight the genetic causes, clinical manifestations and diagnosis of different cns malformations. agenesis of corpus callosum (acc) the corpus callosum (cc) is the largest white matter tract connecting the two cerebral hemispheres. it comprises 4190 million topographically organized axons. functionally, it permits the transfer of information between the two cerebral hemispheres, and inhibits any concurrent activity in the contralateral hemisphere.18 it contributes to cognition and behavior. thickness of cc, fiber cross-section and bundle size increases through childhood and adolescence with slight differences among sexes. fetal development of cc may be affected by various genetic factors and maternal alcohol abuse. figure 2 illustrates the normal appearance of the corpus callosum in the mid-sagittal. the cc starts to develop at sixth week of gestation, and continues to differentiate to attain its shape by the eighteenth to twentieth weeks of gestation; therefore imaging of this structure before 20 weeks may not be optimal (figure 3).19 in mid-sagittal views of the fetal brain (two-dimensional (2d) ultrasound) the cc appears as a thin anechoic space, lined superiorly and inferiorly by two echogenic lines. sonographic demonstration of the cc requires careful imaging in a center with a high level of expertise to make a full assessment and exclude co-existing brain abnormalities.20 among infants, 2–3% will have major cc malformations that will be detected at birth or a few weeks after birth. agenesis of corpus callosum (acc) is the most common abnormality affecting the cc. it can be partial or complete, isolated or complex (associated with other malformations).21 other abnormalities of cc are hypoplasia of cc (thinning of cc) and hyperplasia of cc (thickening of cc).20 the most common causes of acc are gene mutations.22 the overall incidence of acc is 0.5–70 in 10,000 livebirths.19 generally, the outcome of isolated acc is favorable. patients showed normal intelligence and neuropsychological fig. 2 brain magnetic resonance imaging (mri) t 1 weighted mid-sagittal view showing the normal appearance of corpus callosum (red arrow). fig. 1 primary and secondary cerebral vesicles (a) three primary brain vesicles in 3–4 weeks embryo and (b) five secondary brain vesicles in 5th-week embryo. printed with permission.8 3j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 b. alomrani et al. review genetic causes, clinical manifestations and diagnosis of central nervous system development in up to 70% of cases.23 in the other individuals, deficits in language comprehension, humor, theory of mind and social reasoning are noted. the overall rate of chromosomal abnormality in fetuses with acc is 18%. careful imaging in a center with a high level of expertise is required to make a full assessment and to exclude coexisting abnormalities. a wide range of neurological disorders and tumors were associated with epigenetic abnormalities. defects in chromatin remodeling have been linked to agenesis of the corpus callosum, mainly chr.22q11.2 microdeletion or duplication. a little data is available about the effect of nuclear signaling and cellular mechanisms in disorders associated with commissural phenotypes such as callosal agenesis. however, a lack of c11orf46 in reference to microdeletion of ch.11p13-14.1 is strongly linked to neurodevelopmental abnormalities which are related to agenesis of the corpus callosum.24 the c11orf46 is a nuclear protein that is expressed in an age-related pattern with a larger amount expressed at pre and post-natal periods in comparison to adulthood. the cellular expression pattern of c11orf46 in the developmental stage of the brain is a nuclear punctate type in neurod2-positive cells. the role of c11orf46 in cortical development was demonstrated in animal studies where knockdown of c11orf46 lead to a reduction in corpus callosum thickness. inhibition of c11orf46 reduced callosal development and commissural connection between cerebral hemispheres.25 the c11orf46 plays an important role in axonal connectivity and it is linked to regulators repressive chromatin which is related to trans-callosal connectivity. moreover, c11orf46 affinity to kmt-rc is a determinant to orderly brain development. an epigenomic editing trial, working on hek293 cells, found that c11orf46 could effectively suppress the genes responsible for corpus callosum development.26 genetic defect: genetic causes of acc are heterogeneous. it could be a part of monogenic syndromes or complex chromosomal abnormalities.19 the overall rate of chromosomal abnormalities in fetuses with acc is 18%; nevertheless, recent studies suggest that chromosomal abnormalities are rare in isolated cases.20 the increased use and resolution of comparative genomic hybridization (cgh) have implicated many more genes and genomic loci in corpus callosum development,27 and have revealed a great diversity of genetic causes for acc syndromes (table 1a–1c). at present, however, the table 1a. different autosomal dominant gene syndromes and their responsible genes that may be associated with acc syndromes gene gene locus apert syndrome fgfr2 10q26 greig cephalopolysyndaktyly syndrome gli3 7p14.1 miller–dieker syndrome microdeletion 17p13.3 mowat–wilson syndrome zeb2 2q22 opitz gbbb syndrome microdeletion 22q11.2 coffin-siris / fifth digit syndrome arid1a 1p36.11 arid1b 6q25.3 smarca4 19p13.2 smarcb1 22q11.23 smarce1 17q21.2 sox11 2p25.2 rubinstein–taybi syndrome microdeletion 16p13.3 ep300 22q12 basal cell nevus syndrome ptch 9q22.3 sufu 10q24.32 table 1b. different autosomal recessive gene syndromes and their responsible genes that may be associated with acc syndrome gene gene locus acrocallosal syndrome kif7 15q26.1 gli3 7p13 andermann syndrome slc12a6 15q13-q14 dincsoy syndrome fryns syndrome pign 18q21.33 fukuyama congenital muscular dystrophy fktn 9q31.2 hydrolethalus syndrome kif7 15q26.1 joubert syndrome 33 autosomal recessive genes lowry wood syndrome rnu4atac 2q14.2 meckel-gruber syndrome 11 autosomal recessive genes microcephalic osteodysplastic primordial dwarfism type (mopd) і/ііі rnu4atac 2q14.2 muscle eye brain disease neu-laxova syndrome phgdh 1p12 psat1 9q21.2 psph 7p11.2 peters-plus syndrome b3glct 13q12.3 septo-optic dysplasia toriello-carey syndrome vici syndrome epg5 18q12.3-q21.1 walker-warburg syndrome pomt1 pomt2 fktn 9q31.2 warburg-mikro syndrome fig. 3 prenatal ultrasound of fetal brain midsagittal view showing the normal appearance of corpus callosum at 20 weeks of gestation.20 4 j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 genetic causes, clinical manifestations and diagnosis of central nervous system review b. alomrani et al. exonic deletions and duplications, small in-frame deletions, and missense, frameshift/nonsense, and splicing mutations in gli3 gene.37 debeer et al.38 2003 found a missense mutation in the gli3 gene resulting in the replacement of arginine amino acid with tryptophan at position 625 of the resultant protein; arg625trp (r625w). this mutation was partially penetrant. another mutation reported by kalff-suske et al.,39 1999 was g-to-t transversion at nucleotide 1627 of the gli3 gene, resulting in a glu543-to-ter mutation. the gli3 gene mutations may result in another genetic syndrome called pallister-hall syndrome (phs); but the site and type of mutation totally different from that occurring in gcps cases. mutations typically involve exons spanning nucleotides 1813-2103 of the gli3 cdna; and most of them were deletions resulting in a frameshift and premature protein termination codon with the final production of truncated proteins.40 miller–dieker syndrome (mds) it is a rare genetic disorder that comprises severe lissencephaly (smooth brain) and characteristic facial features; including a high and prominent forehead, bitemporal narrowing, short upturned nares, protuberant upper lip with downturned vermilion border, and small jaw (figure 5). manifestations include hypotonia and feeding difficulties. affected individuals suffer from severe mental retardation, seizures, and developmental delay. other malformations reported in these individuals include omphalocele and congenital heart defects.41 hypoplasia or complete agenesis of cc is described in most cases of mds.42 genetic defect: approximately 80% of individuals with mds have a de novo deletion involving the short arm of chromosome 17 (17p13.3) in the area involving pafah1b1 gene (formerly lis1); whereas 20% have inherited a deletion from a parent who carries a balanced chromosome rearrangement.43 most deletions are microdeletions that can be detected in 70% of cases by either high resolution karyotyping or fish technique (flourescent in situ hybridization).43 pafah1b1 gene abnormalities may cause mds, isolated lissencephaly or subcortical band heterotopia (sbh).45 mowat–wilson syndrome (mws) it is a very rare disorder that its prevalence has been estimated to be 1:50,000 – 1:70,000 live births.46 the mws is characterized by distinctive facial features (widely spaced eyes, broad eyebrows with a medial flare, low-hanging columella, prominent chin, and uplifted earlobes with a central depression (figure 6). the facial features evolve and become more pronounced with age.47 hirschsprung disease (in 44% of cases) or chronic constipation (in 30% of cases) had been described table 1c. different x-linked gene syndromes and their responsible genes that may be associated with acc syndrome gene gene locus aicardi syndrome not known xp22 atr-x syndrome cranial frontonasal syndrome efnb1 xq13.1 fg syndrome cask xp11.4 hydrocephalus masa syndrome lujan-fryns syndrome mls syndrome oral-facial digital syndrome type 1 proud syndrome proud syndrome x-linked lissencephaly fig. 4 a = a patient with apert syndrome, b = face, c = hoofshaped hand, d = syndactyly shape of a rosebud, and e = syndactyly of feet (printed with permission from rathore et al.,33 2017). cause of 55–70% of cases with acc cannot be identified by clinical evaluation.28,19 non-genetic causes of acc include maternal alcohol use during pregnancy29 or maternal phenylketonuria.30 apert syndrome apert syndrome is characterized by the presence of multi-suture craniosynostosis and syndactyly of the hands (fusion of the digits and nails) (figure 4). affected individuals have large anteriorly displaced “anterior fontanelle” and cloverleaf skull. the ocular abnormalities, hearing loss, and nonprogressive ventriculomegaly had been reported in most cases. cervical vertebral fusions are found in 68% of affected individuals. most individuals with apert syndrome have normal intelligence or mild intellectual disability. abnormalities of the corpus callosum; namely acc, were described in 23% of cases. genetic defect: fibroblast growth factor receptor (fgfr2) gene mutations are linked to many genetic disorders, one of them is apert syndrome. heterozygous mutation in fgfr2 mostly reported small intragenic deletions/insertions finally resulting in replacement of serine amino acid with tryptophan amino acid at protein position 252 (written as ser252trp). another mutation replaces proline amino acid with arginine amino acid at position 253 (written as pro253arg). these changes are described as “gain-of-function” because they increase the activity of the resultant protein, leading to stronger signaling.31,32 greig cephalopolysyndaktyly syndrome (gcps) it is a rare genetic disorder characterized by macrocephaly, widely spaced eyes, preaxial polydactyly, and cutaneous syndactyly. affected individuals have developmental delay, intellectual disability, and/or seizures. approximately 200 cases are reported worldwide.34 the acc is reported in approximately 20% of affected individuals.35 genetic defect: the gcps is caused by heterozygous mutation in gli family zinc finger 3 (gli3) in 80% of cases, whereas 20% of cases are caused by deletion of the short arm of chromosome 7 (7p14.1) in the area involving gli3 gene.36 reported mutations causing gcps were large deletions, 5j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 b. alomrani et al. review genetic causes, clinical manifestations and diagnosis of central nervous system fig. 5 typical facial features of miller–dieker syndrome (mds), including a prominent forehead, bitemporal hollowing, short nose with upturned nares, prominent upper lip, and micrognathia. (printed with permission from kim et al.,44 2011). fig. 6 main common features of the mowat-wilson syndrome: large eyebrows, medially flaring and sparse in the middle part, and widely spaced eyes (printed with permission from garavelli et al.,57 2007). table 2. genes and percentage of css caused by a pathogenic variant gene gene locus percentage of css caused by pathogenic variants in this gene arid1a 1p36.11 5% arid1b 6q25.3, 37% smarca2 9p24.3 2% smarca4 19p13.2 7% smarcb1 22q11.23 7% smarce1 17q21.2 2% sox11 2p25.2 2% unknown 40% with mws.48 congenital heart defects; with a predilection for abnormalities of the pulmonary arteries and/or valves, and genitourinary anomalies (particularly hypospadias and cryptorchidism in males) are frequently described manifestations with mws.48 hypoplasia or complete agenesis of cc is described in most cases of mws.48 genetic defect: mws is caused by hetero-zygous mutation in zeb2 gene in 84% of affected individuals.50 15% of mws patients are caused by deletions in zeb2 gene.51 1% of cases are caused by chromosome rearrangements at the long arm of chromosome 2; which disrupt zeb2 gene51 missense, splice site, or in-frame variants in zeb2 represent fewer than 2% of cases with typical mws.49 yoneda et al.52 2002, reported 3-bp in-frame deletion affecting zeb2 in an adult with mild intellectual disability, atypical facial features, and megacolon. zweier et al.53 2006, found a novel splice site variant in the utr in a patient with facial features and speech delay. opitz gbbb syndrome it is a genetic disorder that affects structures along the midline of the body. patients have characteristic facial features including widely spaced eyes and a cleft lip. other manifestations include laryngotracheoesophageal abnormalities causing breathing problems and difficulty swallowing. affected individuals usually suffer from intellectual disability, heart defects, and/or imperforate anus.54 acc and/or cerebellar vermis and dandy-walker malformations were identified in 50% of affected individuals.54 genetic defect: opitz gbbb syndrome has two genetic forms one is autosomal dominant (ados; type ii) and the other is x-linked (osx; type i) form.55 the autosomal dominant form is caused by microdeletion (submicroscopic) at the long arm of chromosome 22 (22q11.2). coffin-siris syndrome (css)/fifth digit syndrome it is a rare genetic disorder characterized by aplasia or hypoplasia of the distal phalanx/nail of the fifth and additional digits, developmental delay, intellectual disability, distinctive facial features, hypotonia, hypertrichosis, and hearing impairment. congenital anomalies were also reported in individuals affected including malformations of the heart, gastrointestinal, genitourinary, and central nervous systems.56 genetic defect: css is caused by a heterozygous pathogenic variant in one of the genes listed in table 2. rubinstein–taybi syndrome (rsts) (broad thumb-allux syndrome) the rsts is characterized by distinctive facial features, broad and often angulated thumbs and halluces, short stature, and moderate-to-severe intellectual disability. the characteristic facial features include downward slanting of palpebral fissures, high palate, grimacing smile, and talon cusps. additional features include ocular abnormalities, hearing loss, respiratory difficulties, congenital heart defects, renal abnormalities, cryptorchidism, feeding problems, recurrent infections, and severe constipation.58 rarely, rsts is associated with acc;59 however, it had been reported in some cases.60 genetic defect: the rsts is caused by heterozygous mutations in either crebbp gene (50%-60% of cases)61 or ep300 gene (8%-10% of cases).62 rusconi et al.63 2015, described 14 patients with rsts having crebbp deletions of different sizes; ranging from single exons to whole gene deletions, showing no difference in their phenotype. somatic mosaicism and mosaic microdeletions have been noted in some individuals with rsts. they tend to have a mild phenotype.64 6 j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 genetic causes, clinical manifestations and diagnosis of central nervous system review b. alomrani et al. basal cell nevus syndrome (bcns)/ nevoid basal cell carcinoma syndrome (nbccs)/ (gorlin syndrome) bcns is a rare autosomal dominant disorder characterized by the development of multiple jaw keratocysts, and/or basal cell carcinomas; manifesting from the third decade onward, skeletal anomalies, and ectopic calcifications (especially the falx).65,66 only 5% of children with bcns develop medulloblastoma with a peak incidence at one to two years of age.67 more than 100 clinical features that are variable within and among families have been associated with bcns 68 the prevalence of bcns had been reported in some studies to be 1:30,827.69 the acc in cases with bcns was estimated in some studies to be 10%.66 genetic defect: identification of a heterozygous germline pathogenic variant in either ptch1 gene or sufu gene establishes the diagnosis of bcns. the ptch1 gene is located on the short arm of chromosome 9 (9q22.3). the ptch1 gene provides instructions for producing a protein (patched-1), which functions as a receptor for sonic hedgehog protein. together, patched-1 and sonic hedgehog trigger signals that affect cell development and function that is essential for early development.70 the ptch1 is seen as a tumor suppressor gene.71 sim et al.,72 2018, reported frameshift mutations in ptch1 gene (c.817_818ins(t), c.1226_1227ins(a), and c.2748del(c)), splicing (c.1504-2a>t), and missense (c.385t>c) mutation. mutations were found in exon 1, 6, 9, 17, and intron 10. the sufu-related bcns is associated with a high risk for medulloblastoma of up to 33% compared to that in ptch1-related bcns which was less than 2%.65 acrocallosal syndrome (acls) acls is a rare autosomal recessive disorder characterized by preor postaxial polydactyly, cutaneous syndactyly, hallux duplication, agenesis of the corpus callosum, widely spaced eyes, macrocephaly, moderate-to-severe id, intracerebral cysts, seizures, umbilical & inguinal hernias.73 genetic defect: the acls is caused by homozygous mutations (or compound heterozygous) in kif7 gene. kif7 is located on the long arm of chromosome 15 (15q26.1) and comprise 19 exons. kif7 is a 1343 amino acid protein with a kinesin motor, coiled coil, and gli-binding domains. the acls may also result from a heterozygous mutation in gli3 (7p14.1); the causative gene for greig cephalopolysyndactyly syndrome (gcps). the clinical manifestations of acls and gcps overlap significantly; that’s why acls is considered the severe form of gcps.73 the proteins produced from the kif7 and gli3 gene are part of the sonic hedgehog (shh) signaling pathway (figure 7). this pathway is involved in cell growth, cell specialization, and the regulation of microtubular dynamics necessary for the proper function of the primary fig. 7 schematic overview of sonic hedgehog signaling pathway (cervello et al.,80 2017). 7j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 b. alomrani et al. review genetic causes, clinical manifestations and diagnosis of central nervous system cilia, and hence genetic disorders involving genes regulating this pathway are called ciliopathies. the resultant proteins in shh signaling pathway are responsible for patterning of the brain and limbs.74 the kif7 gene mutations had been linked also to joubert syndrome, a rare genetic disorder characterized by cerebellar hypoplasia, ataxia, psychomotor delay, and an altered respiratory pattern in the neonatal period. retinal degeneration, renal cysts, liver fibrosis, and skeletal involvement were also described with joubert syndrome.75 pathogenic variants in kif7 reported with acls include; in-frame deletion mutation (p.218-221del),76 nonsense, missense, and splice site mutations were also described. ibisler et al,.77 2015 reported a nonsense mutation in exon 11 of the kif7 gene (c.2335g>t, p.glu779). left side: in the absence of hh ligand, ptch inhibits smo allowing gli to form a complex with kif7 and sufu, promoting gli phosphorylation by pka, ck-1, and gsk-3b. upon phosphorylation, gli proteins are processed into transcriptional repressors (gli-r) or are targeted to the proteasome. right side: the hh ligand binding to ptch and to co-receptors cdon and boc and relieves repression of smo, triggering its interaction with kif7. this facilitates the release of gli from sufu/fu complex, bypassing proteolytic cleavage. full-length gli factors lead to the expression of hh target genes. andermann syndrome/agenesis of corpus callosum with peripheral neuropathy (accpn) it is an autosomal recessive disorder characterized by progressive sensorimotor neuropathy with areflexia, developmental delay.78 sensory nerve action potentials cannot be recorded at the median, ulnar, or sural nerves as early as the first year of life. the 86% of affected individuals develop scoliosis.79 the acc is present in 60% of affected individuals. genetic defect: the accpn is caused by biallelic pathogenic variants in slc12a6. the slc12a6 gene provides instructions for making a protein called k-cl cotransporter (electroneutral cotransporter); which is critical for the development and maintenance of nerve tissue. the slc12a6 gene is located on the long arm of chromosome 15 (15q13-q14). small intragenic deletions/insertions and missense, nonsense, and splice-site variants in slc12a6 were reported in cases with accpn. howard et al.81 2002, identified 4 truncating mutations in the slc12a6 gene. fryns syndrome it is a rare autosomal recessive genetic disorder characterized by diaphragmatic defects including diaphragmatic hernia, characteristic facial features, short distal phalanges of the fingers and toes, pulmonary hypoplasia, microphthalmia, brain malformations including agenesis of the corpus callosum, and cardiovascular anomalies.82 the prognosis in fryns syndrome is influenced by the malformations present. survival beyond the neonatal period is rare.83 genetic defect: the molecular diagnosis of fryns syndrome is established when biallelic pathogenic variants (homozygous/compound heterozygous mutations) in pign gene are present in a proband with suggestive clinical findings i.e. diaphragmatic hernia.84 the pign gene is located on the long arm of chromosome 18 (18q21.33). this gene encodes glycosylphosphatidylinositol (gpi) ethanolamine phosphate transferase-1, an enzyme responsible for gpi anchor to cell surface proteins. maydan et al.85 2011 determined that the pign gene contains 31 exons spanning 142.8 kb. missense, nonsense, and splice-site variants had been associated with pign associated fryns syndrome e.g. c.2620-1g>a.86 fukuyama congenital muscular dystrophy (fcmd) fcmd is a rare autosomal recessive congenital muscular dystrophy characterized by early-infantile hypotonia and weakness with contractures of the joints. affected individuals suffer from intellectual disability and speech delays. the condition is seen primarily in children of japanese ancestry.87 ophthalmologic abnormalities, including visual impairment in 53% and retinal abnormalities in 32% were also reported in patients suffering from fcmd.88 cobblestone lissencephaly and white matter abnormalities can be detected in the mri images of the brain of individuals with fcmd89 (figure 8). cobblestone fig. 8 cobblestone lissencephaly in fcmd (barkovich,90 2005). (a) axial t 2 -weighted image showing occipital cobblestone with a thick cortex and smooth outer surface (b) axial t2-weighted image at a higher level showing polymicrogyria of the frontal lobes. (c) coronal t 1 -weighted image showing the characteristic cortical cysts in the cerebellar hemispheres (smaller arrows). https://www.omim.org/genemap/18/207?start=-3&limit=10&highlight=207 8 j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 genetic causes, clinical manifestations and diagnosis of central nervous system review b. alomrani et al. cerebral cortex malformations are associated with congenital muscular dystrophies as in fcmd, walker–warburg syndrome, and muscle–eye–disease of santavuori.90 the elevated serum creatinine and interstitial fibrosis without muscle degeneration and regeneration seen on histopathological examination of muscle biopsies are consistent features of fcmd.91 genetic defect: the molecular diagnosis of fcmd is confirmed when biallelic pathogenic variants in fktn gene are detected in a proband with clinical findings suggestive of fcmd. the fktn gene is located on the long arm of chromosome 9 (9q31.2). this gene encodes for a 461-amino acid fukutin protein, which is expressed in various tissues in normal individuals. it is believed that fukutin play an important role in neuronal migration and glycosylation of alpha-dystroglycan (dag1) of skeletal and cardiac muscle fibers.92,93 kobayashi et al.87 2017, analyzed fktn gene in 107 affected individuals that showed the prevalence of the homozygous japanese founder variant (c.*4392_*4393insab185332.1) among patients; where there is insertion of retrotransposal sequence into 3’-utr (untranslated region) of fktn. the second common mutation in fktn gene associated with fcmd reported in korea by lim et al.,94 2010 is (c.647+2084g>t) (p.arg216serfster10); an intronic mutation that activates a pseudoexon between exons 5 and 6 resulting in a truncated fukutin protein. saredi et al.95 2009, found 1 bp deletion mutation in fktn gene in an italian patient with muscular dystrophy. the mutation (c.42del) (p.thr14_leu15inster) results in a premature termination of protein synthesis producing a truncated protein with loss of its function. joubert syndrome (jbts) and joubert syndrome and related disorders (jsrd) jbts is characterized by three primary findings: (1) a distinctive cerebellar and brain stem malformation called the molar tooth sign (mts) (figure 9), (2) hypotonia and (3) developmental delays. affected individuals may also suffer from breathing abnormalities (tachypnea or apnea), abnormal eye movements, renal disease, ocular colobomas, occipital encephalocele, hepatic fibrosis or polydactyly.96,97 the acc is common in 80% of individuals with jbts.98 callosal abnormalities are relatively frequent in those with biallelic kif7 pathogenic variants suggesting overlap with acrocallosal syndrome.99 genetic defect: the jbts is diagnosed genetically by the presence of biallelic pathogenic variants in one of the 33 autosomal recessive js-related genes or heterozygous mutation in one x-linked gene. table 3a and 3b show js related genes and examples of the most common pathologic variants occurring in each gene. 15. lowry wood syndrome (lws) a rare genetic disorder characterized by epiphyseal dysplasia, short stature, microcephaly and retinal dystrophy.100 other skeletal manifestations of this disorder included a bilateral absence of radial heads, lateral dislocation of both patellae, and dislocation of both hips.120 clinical features of microcephalic osteodysplastic primordial dwarfism type i (mopd1) fig. 9 brain mri t 1 weighted axial view showing “molar tooth” sign shown by the arrow in a patient with jbts (embiruçu et al., 101 2009) overlap with those of lowry wood syndrome; which is also caused by a biallelic mutation in the rnu4atac gene. genetic defect: the lws is caused by biallelic pathologic variants in the rnu4atac gene encoding a small nuclear rna (snrna); located on the long arm of chromosome 2 (2q14.2).100 shelihan et al.121 2018, identified compound heterozygosity in the rnu4atac gene, one was for an r.53c-t transition and the other r.8c-a transversion. other mutations that had been associated with rnu4atac gene in cases of lowry wood syndrome were n.46g>a transition, r.5a-c transversion,100 r.120t-g transversion, and r.114g-c transversion.121 meckel-gruber syndrome (mks)/ dysencephalia splanchnocystica the mks is a rare autosomal recessive lethal ciliopathy characterized by bilateral polycystic kidneys, occipital encephalocele and postaxial polydactyly122 (figure 10). other abnormalities that could be found in mks are cleft lip and palate, congenital heart defects, and pulmonary hypoplasia.123 the worldwide incidence of mks has been estimated to be 1 in 135,000 live births,124 however, it has a higher incidence in some populations where the consanguineous marriage rate is high, for example, hutterites,125 finland,126 kuwaiti bedouin tribes,127 and in saudi arabia.128 the mks phenotype overlap with other ciliopathies such as joubert syndrome (jbts), coach syndrome (cerebellar vermis hypo/aplasia, oligophrenia, congenital ataxia, ocular coloboma, and hepatic fibrosis), oro-facio-digital syndrome (ofd), nephronophthisis (nphp), and bardet–biedl syndrome (bbs); that forms a challenge in the diagnosis of mks.109,129,130 genetic defect: the molecular diagnosis of mks is established when biallelic pathologic variants are found in one of the mks causing genes. to date, there are 11 autosomal 9j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 b. alomrani et al. review genetic causes, clinical manifestations and diagnosis of central nervous system table 3a. joubert syndrome related genes and pathogenic variants gene gene locus pathogenic variants phenotypic variant (other than molar tooth sign) ethnic group references 1-ahi1 6q23.3 missense and frameshift mutations c.2988del (p.val997fs) retinal dystrophy, renal disease bachmann-gagescu et al., 2015a99 2-cplane1 5p13.2 p.arg2904ter tongue hamartomas +/cleft lip/ palate +/polydactyly dutch, french canadian vilboux et al., 2017102 3-cc2d2a 4p15.32 c.100c>t (p.arg34ter) renal disease, hepatic disease french canadian doherty et al., 2010103 4-cep290 12q21.32 c.6012-12t>a retinal dystrophy, renal disease, hepatic disease japanese population suzuki et al., 2016104 5-cspp1 8q13.1-q13.2 c.363_364delta skeletal dysplasia hutterite tuz et al., 2014105 6-inpp5e 9q34.3 missense and frameshift mutations c.1897_1898del (p.gln633fs) retinal dystrophy, hepatic disease vilboux et al., 2017102 7-kiaa0586 14q23.1 c.392del (p.arg131fs) skeletal dysplasia alby et al., 2015106 8-mks1 17q22 retinal dystrophy slaats et al., 2016107 9-nphp1 2q13 renal disease french canadian vilboux et al., 2017102 10-rpgrip1l 16q12.2 renal disease, hepatic disease delous et al., 2007108 11-tctn2 12q24.31 tongue hamartomas +/cleft lip/ palate +/polydactyly bachmann-gagescu et al., 2015a99 12-tmem67 8q22.1 hepatic disease brancati et al., 2009109 13-tmem216 11q12.2 p.arg73leu retinal dystrophy, renal disease, tongue hamartomas +/cleft lip/ palate +/polydactyly ashkenazi jewish valente et al., 2010110 14-arl13b 3q11.1-q11.2 cantagrel et al., 2008111 15-b9d1 17p11.2 romani et al., 2014112 16-b9d2 19q13.2 tongue hamartomas +/cleft lip/ palate +/polydactyly bachmann-gagescu et al., 2015a99 17-c2cd3 11q13.4 tongue hamartomas +/cleft lip/ palate +/polydactyly bachmann-gagescu et al., 2015a99 18-cep41 7q32.2 retinal dystrophy lee et al., 2012a113 table 3b. joubert syndrome related genes and pathogenic variants gene gene locus pathogenic variants phenotypic variant (other than molar tooth sign) ethnic group references 19-cep104 1p36.32 srour et al., 2015114 20-cep120 5q23.2 tongue hamartomas +/cleft lip/palate +/polydactyly 2-skeletal dysplasia shaheen et al., 2015b115 21-ift172 2p23.3 skeletal dysplasia halbritter et al., 2013116 22-katnip (kiaa0556) 16p12.1 sanders et al., 2015117 23-kif7 15q26.1 tongue hamartomas +/cleft lip/palate +/polydactyly, acrocallosal syndrome dafinger et al., 201175 24-ofd1 xp22.2 renal disease, tongue hamartomas +/cleft lip/palate +/polydactyly coene et al., 2009118 25-pde6d 2q37.1 thomas et al., 2014119 26-poc1b 12q21.33 27-tctn1 12q24.11 28-tctn3 10q24.1 tongue hamartomas +/cleft lip/palate +/polydactyly (continued) 10 j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 genetic causes, clinical manifestations and diagnosis of central nervous system review b. alomrani et al. recessive genes associated with mks. table 4 shows the 11 mks causative genes with examples of the most common pathologic variant occurring in each gene. the mks1 gene (609883) is located on the long arm of chromosome 17 (17q22). the mks1 gene encodes a protein responsible for centriole migration to the apical membrane and the formation of the primary cilium during embryonic life. it is 21,170 bp in length, comprises 18 exons, and encodes a 559-amino acid protein.132 kyttala et al.133 2006; identified a 29-bp deletion in intron 15 of mks1 gene (c.1408-35_14087del29) (609883.0001) in 3 finnish families. the same intronic deletion was found in other finnish families and families of european descent; it was then denoted as the finnish founder variant (campomelic variant).124 microcephalic osteodysplastic primordial dwarfism type (mopd) і/ііі the mopd is a rare autosomal recessive developmental disorder characterized by extreme intrauterine growth retardation, severe microcephaly, central nervous system abnormalities, dysmorphic facial features, skin abnormalities, skeletal changes, limb deformations144 (figure 11). the syndrome was characterized by majewski as mopd types i and iii; which were considered parts of the same clinical spectrum table 3b. joubert syndrome related genes and pathogenic variants—continued gene gene locus pathogenic variants phenotypic variant (other than molar tooth sign) ethnic group references 29-tmem107 17p13.1 retinal dystrophy, tongue hamartomas +/cleft lip/palate +/polydactyly 30-tmem138 11q12.2 retinal dystrophy, renal disease 31-tmem231 16q23.1 french canadian srour et al., 2015114 32-tmem237 2q33.1 p.arg18ter renal disease hutterite 33-ttc21b 2q24.3 34-znf423 16q12.1 renal disease fig. 10 showing a fetus aged 16 weeks gestational age (a) occipital encephalocele and bilateral postaxial polydactyly and (b) bilateral polycystic kidneys (hartill et al., 131 2017). and since that time they are combined and denoted as the same disorder.146 the acc had been reported in patients with mopd і, whether partial or complete.147,148 genetic defect: the mopd і is caused by homozygous or compound heterozygous mutation in the rnu4atac gene (601428); the gene that is also associated with lowry wood syndrome. this gene as mentioned above is located on the long arm of chromosome 2 (2q14.2); and is responsible for the production of small nuclear rna (snrna) u4atac (a nonprotein-coding gene); which is a component of the minor spliceosome.149 the spliceosome is a large ribonucleoprotein (rnp) complex that catalyzes the removal of introns from a transcribed pre-mrna, and the ligation of the flanking exons.150 spliceosomes are either major or minor that differ in their splicing processes and snrna components. the minor spliceosome is assembled within the nucleus from 5 subunits; u11, u12, u4atac, and u6atac, together with u5. this type of spliceosome splices a rare class of pre-mrna introns, denoted u12type. figure 12 shows the detailed structure of the minor spliceosome.151 he et al.149 2011, identified 4 different mutations in the rnu4atac gene associated with mopd і, all these mutations ended up in defective u12-dependent splicing (reduced by greater than 90% compared to wildtype u4atac). the mutations were genomic 51g-a, genomic 55g-a, genomic 30g-a, 111g-a. edery et al.152 2011, identified another mutation in rnu4atac gene; 51g-a. abdel-salam et al.153 2012, identified a homozygous mutation for the genomic 55g-a in rnu4atac gene (previously reported by he et al. 149 2011), in 2 sibs with mild mopd1 phenotype. vici syndrome vici syndrome (omim242840) is an autosomal recessive disorder characterized by agenesis of the corpus callosum, cataracts, oculocutaneous hypopigmentation, progressive cardiomyopathy, and combined immunodeficiency154 (figure 13). affected individuals suffer also from severe developmental delay, intellectual disability and acquired microcephaly.155 an additional finding is muscle myopathy manifested as progressive hypotonia and elevated serum creatinine kinase. histopathological examination of skeletal muscle fibers by light microscopy reveals wide variation in fiber size, centralized nuclei, and small fibers with high glycogen content.156 11j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 b. alomrani et al. review genetic causes, clinical manifestations and diagnosis of central nervous system table 4. mks causative genes and the most common pathologic variant gene gene locus pathogenic variant references mks1 17q22 finnish c.1408-35_1408-7del29 kyttälä et al., 2006133 mks2 (tmem216) 11q12.2 ashkenazi c.218g > t (p.r73l) edvardson et al., 2010134 mks3 (tmem67) 8q22.1 pakistani c.1575 + 1g > a smith et al., 2006135 mks4 (cep290) 12q21.32 4-bp del, c.384_387taga, (p.asp128glufster34) baala et al., 2007b136 mks5 (rpgrip1l) 16q12.2 european c.1843a > c (p.t625p) arts et al., 2007137 mks6 (cc2d2a) 4p15.32 finnish c.1762c > t (p.?) tallila et al., 2008138 mks7 (nphp3) 3q22.1 ivs19as, 2-bp del, (c.2694-1_2 del) bergmann et al., 2008139 mks8 (tctn2) 12q24.31 ivs13as, a-g, -2 shaheen et al., 2011140 mks9 (b9d1) 17p11.2 505, t-c, +2, (p.thr82cysfster44), splice site donor mutation) hopp et al., 2011141 mks10 (b9d2) 19q13.2 301a-c transversion, (p. ser101arg) dowdle et al., 2011142 mks11 (tmem231) 16q23.1 c.751g-a transition, (p.val251serfster9) shaheen et al., 2013143 fig 11. a photo of a patient diagnosed with mopd і at 4 weeks (left) and 6 months of age (right). note typical facial features with sloping forehead, beaked nose with downturned nasal tip, small low-set ears, sparse hair and joint contractures (both wrists, both elbows, and both knees) (nagy et al.,145 2012). patients with vici syndrome may develop by time progressive reduction of absolute lymphocyte count and immunoglobulin level that may necessitate intravenous immunoglobulin (ivig) therapy.154 patients usually present with recurrent chest infections, particularly pseudomonas and klebsiella associated with lymphopenia.157 genetic defect: vici syndrome is caused by a biallelic pathogenic variant in epg5 gene that is located on the long arm of chromosome 18 (18q12.3-q21.1). epg5 gene encodes a protein that has a role in the autophagy pathway (a conserved lysosomal degradation pathway).158 cullup et al.158 2013, identified compound heterozygosity (2 different mutations in the same patient) in the epg5 gene: one was (4588c-t transition), resulting in a (gln1530-to-ter; 615068.0001), and the other one was 1-bp duplication (5704dupt), resulting in a frameshift and premature termination (tyr1902leufster2; 615068.0002). ehmke et al.159 2014, identified a homozygous mutation (2 similar mutations in the same patient) in exon 43 of the epg5 gene; which was (c.7447c-t transition) resulting in an (arg2483-to-ter; 615068.0006). maillard et al.155 2017, identified compound heterozygosity in the epg5 gene; one was a (c.4007g-a transition), resulting in a (gly1336-to-glu substitution; 615068.0007), and the other one was 1-bp insertion (c.2352_2353insg), resulting in a frameshift and premature termination (ala785glyfster20; 615068.0008). primary microcephaly with or without cortical malformation (mcph) the mcph is an autosomal recessive neurodevelopmental disorder in which an individual has a head circumference more than 3 standard deviations (sd) below the ageand sexmatched population mean.161 motor development may be normal or delayed, whereas mental retardation may vary from mild to severely delayed speech acquisition.162,163 at least 17 genetic loci (mcph1–17) have been implicated in mcph, all of which have now been connected to single genes: mcph1, wdr62, cdk5rap2, knl1, aspm, cenpj, stil, cep135, cep152, znf335, phc1, cdk6, cenpe, sass6, mfsd2a, ankle2, and cit.164 mutations in wdr62, encoding wd repeat-containing protein 62, are responsible for mcph2, which is the second most frequent form of mcph after mcph5 caused by aspm mutations. over 40 pathogenic mutations in wdr62 have already been reported. cortical malformations may be associated in addition to microcephaly in these patients.165,166 genetic defect: the mcph2 is caused by homozygous or compound heterozygous mutation in wdr62 gene (mim 613583). the wdr62 gene is located on the long arm of chromosome 19 (19q13.12). wdr62 gene contains 32 exons and encodes a 1,523-amino acid protein.167 these researchers identified a homozygous mutation in exon 31 of the wdr62 gene in 2 turkish sibs affected with mcph2. the mutation was a 4-basepair deletion (tgcc) that resulted in a frameshift and premature termination of the protein (val1402glyfster12; 613583.0001). nicholas et al.163 2010, identified a homozygous 1313g-a transition in exon 10 of the wdr62 gene in 2 pakistani patients, resulting in an (arg438-to-his substitution; 613583.0006) at a highly conserved residue. zombor et al.,168 2019, identified a homozygous missense mutation in exon 6 of the wdr62 gene, c.668t>c, p.phe223ser. mutations were detected by whole exome sequencing (wes). 12 j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 genetic causes, clinical manifestations and diagnosis of central nervous system review b. alomrani et al. fig. 13 brain mri t 1 weighted mid-sagittal view showing complete agenesis of the corpus callosum at the age of 2 months in a patient diagnosed with vici syndrome (del campo et al.,160 1999). fig. 14 l-serine biosynthesis pathway. the first and limiting step of this pathway is the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate by phgdh (acuna-hidalgo et al.,172 2014). fig. 12 the structure of the minor spliceosome (chen and moore, 2015).151 neu-laxova syndrome (nls) the nls is a rare autosomal-recessive disorder characterized by characteristic facial features with shortened eyelids, proptosis, round gaping mouth, microcephaly, intrauterine growth restriction (iugr), ichthyosis (skin hyperkeratosis), and flexion deformities169 (figure 14). the cns malformations reported with nls were variable such as abnormal gyration, lissencephaly, agenesis of the corpus callosum, dandy-walker anomaly, choroid plexus cysts, or neural-tube defects.170-172 the nls is caused by mutations in the genes encoding enzymes required for the l-serine biosynthesis pathway; hence it is considered also an inborn error of metabolism (iem).173 genetic defect: the molecular diagnosis of nls is confirmed by the identification of biallelic pathogenic variants in one of the 3 causative genes; phgdh gene (phosphoglycerate dehydrogenase gene [mim 606879]), psat1 gene (phosphoserine aminotransferase 1 gene [mim 610936]), or psph gene (phosphoserine phosphatase gene [mim 172480]). the phgdh gene is located on the short arm of chromosome 1 (1p12); and encodes 3-phosphoglycerate dehydrogenase enzyme (the first and rate-limiting enzyme in the l-serine biosynthesis pathway). shaheen et al.174 2014, identified 2 mutations in the phgdh gene in saudi patients clinically diagnosed with nls. both mutations were at a region of highly conserved residue within the nad(p)binding domain and at the phgdh dimer interface. the first mutation was c.418g-a transition resulting in a gly140to-arg substitution; 606879.0007. the second mutation reported by shaheen et al.174 2014, was c.488g-a transition resulting in a arg163-to-gln substitution; 606879.0008. both mutations were detected by a combination of autozygosity mapping and exome sequencing and confirmed by sanger sequencing. the psat1 gene is located on the long arm of chromosome 9 (9q21.2); and encodes phosphoserine aminotransferase enzyme, the second enzyme in l-serine biosynthesis pathway. the psat gene contains 9 exons and spans 56 kb.175 acuna-hidalgo et al.173 (2014) identified a homozygous complex insertion/deletion mutation in the last exon of the psat1 gene in a fetus with neu-laxova syndrome; (c.1023_1027delinsagacct) resulting in a frameshift and premature termination (arg342aspfster6; 610936.0003). the mutation was detected by detailed reanalysis of exome sequencing. acuna-hidalgo et al.173 (2014), identified another mutation in the highly conserved residue of the psat1 gene in 2 stillborn fetuses and a preterm newborn who died within the first week 13j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 b. alomrani et al. review genetic causes, clinical manifestations and diagnosis of central nervous system of life, who were all affected by nls. the mutation was c.296c-t transition resulting in an ala99-to-val substitution; 610936.0004. conclusion many malformations of the central nervous system can occur during fetal development. the prevalence of neural tube defects has decreased over the past few years due to the introduction of antenatal folic acid use, especially during the first trimester. in this review we enlisted many malformations with genetic links. with advancement in the prenatal genetic testing, these abnormalities can be detected using pre-implantation genetic testing on the day-5 embryo or via non-invasive prenatal genetic testing of pregnant women to avoid devastating lifelong sufferings.  references 1. groen h, bouman k, pierini a, rankin j, rissman a, haeuser m, yevtushok l, loanem, erwich j, de walle hek. stillbirth and neonatal mortality in ppregnancies complicated by major congenital anomalies: findings from a large european cohort. prenat diagn. 2017; 37(11): 1100–1111. 2. seidahmed, m z, abdelbasit o b, and salih m a. epidemiology of neural tube defects. saudi med j. 2014; 35 (sup 1): s29-s35. 3. sadler tw: langman’s medical embryology, ed 12. lippincott, williams & wilkins, philadelphia, 2012. 4. bruce m.carlson. human embryology and developmental biology 6th edition. elsevier. 2018: 205–207. 5. mehler, m. f. 2008. epigenetic principles and mechanisms underlying nervous system functions in health and disease. progress in neurobiology, 86, 305–341. 6. jaenisch, r. & bird, a. 2003. epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. nature genetics, 33, 245–254. 7. claes, b., buysschaert, i. & lambrechts, d. 2010. pharmaco-epigenomics: discovering therapeutic approaches and biomarkers for cancer therapy. heredity, 105, 152–160. 8. ishikawa, y; yamamoto, n; yoshimoto, m; ito, h (2012). “the primary brain vesicles revisited: are the three primary vesicles (forebrain/midbrain/ hindbrain) universal in vertebrates?” brain, behavior and evolution. 79 (2): 75–83. 9. portela, a. & esteller, m. 2010. epigenetic modifications and human disease. nature biotechnology, 28, 1057. 10. cariaga-martínez, a. e., gutiérrez, k. j. & alelú-paz, r. 2018. the vast complexity of the epigenetic landscape during neurodevelopment: an open frame to understanding brain function. international journal of molecular sciences, 19, 1333. 11. moretti, p. & zoghbi, h. y. 2006. mecp2 dysfunction in rett syndrome and related disorders. current opinion in genetics & development, 16, 276-281. 12. gräff, j. & mansuy, i. m. 2009. epigenetic dysregulation in cognitive disorders. european journal of neuroscience, 30, 1–8. 13. silva kr, costa r, rached ra, martinelli filho m, caldas jg, carnevale fc, moreira lf, stolf na. warfarin prevents venous obstruction after cardiac devices implantation in high-risk patients: partial analysis. brazilian journal of cardiovascular surgery. 2008;23:542–9. 14. kawahara y, ito k, sun h, aizawa h, kanazawa i, kwak s. glutamate receptors: rna editing and death of motor neurons. nature. 2004 feb 26;427(6977):801–2. 15. gautam vk, singh r, khurana s. transient behavior abnormality following injury to splenium of corpus callosum during ventriculoperitoneal shunt surgery. iosr journal of dental and medical sciences (iosr-jdms). 2013;5(1):26-9. 16. nicholls rd, knepper jl. genome organization, function, and imprinting in prader-willi and angelman syndromes. annual review of genomics and human genetics. 2001 sep;2(1):153–75. 17. van den boom, j., wolter, m., kuick, r., misek, d. e., youkilis, a. s., wechsler, d. s., sommer, c., reifenberger, g. & hanash, s. m. 2003. characterization of gene expression profiles associated with glioma progression using oligonucleotidebased microarray analysis and real-time reverse transcription-polymerase chain reaction. the american journal of pathology, 163, 1033-1043. 18. vasudevan c, mckechnie l, levene m. long-termoutcome of antenatally diagnosed agenesis of corpus callosum and cerebellar malformations. semin fetal neonatal. 2012;17:295–300. 19. schell-apacik, c. c., wagner, k., bihler, m., ertl-wagner, b., heinrich, u., klopocki, e., kalscheuer, v. m., muenke, m., von voss, h. agenesis and dysgenesis of the corpus callosum: clinical, genetic and neuroimaging findings in a series of 41 patients. am. j. med. genet. 146a: 2501-2511, 2008. 20. santo s, d’antonio f, homfray t, rich p, pilu g, bhide a, thilaganathan b and papageorghiou a t. counseling in fetal medicine: agenesis of the corpus callosum. ultrasound obstet gynecol. 2012; 40: 513–521. 21. yeh hr, park hk, kim hj, ko ts, won hs, lee my, shim jy, yum ms. brain dev. neurodevelopmental outcomes in children with prenatally diagnosed corpus callosal abnormalities. 2018; 40(8):634-641. 22. bartha-doering l, schwartz e, kollndorfer k, fischmeister fps, novak a, langs g, werneck h, prayer d, seidl r, kasprian g. effect of corpus callosum agenesis on the language network in children and adolescents. brain struct funct. 2021 apr;226(3):701-713. doi: 10.1007/s00429-020-02203-6. epub 2021 jan 26. pmid: 33496825; pmcid: pmc7981296. 23. moutard ml, kieffer v, feingold j, lewin f, baron jm, adamsbaum c, gélot a, isapof a, kieffer f, de villemeur tb. isolated corpus callosum agenesis: a ten-year follow-up after prenatal diagnosis (how are the children without corpus callosum at 10 years of age?). prenat diagn. 2012; 32(3):277-83. 24. peter, c. j., saito, a., hasegawa, y., tanaka, y., nagpal, m., perez, g., alway, e., espeso-gil, s., fayyad, t. & ratner, c. 2019. in vivo epigenetic editing of sema6a promoter reverses transcallosal dysconnectivity caused by c11orf46/arl14ep risk gene. nature communications, 10, 1-14. 25. peter, c. j., saito, a., hasegawa, y., tanaka, y., perez, g., alway, e., espesogil, s., fayyad, t., ratner, c. & dincer, a. 2018. in vivo epigenetic editing of sema6a promoter reverses impaired transcallosal connectivity caused by c11orf46/arl14ep neurodevelopmental risk gene. biorxiv, 491779. 26. yim, y. y., teague, c. d. & nestler, e. j. 2020. in vivo locus-specific editing of the neuroepigenome. nature reviews neuroscience, 21, 471-484. 27. poon as, o'driscoll s, meng th. optimal frequency for wireless power transmission into dispersive tissue. ieee transactions on antennas and propagation. 2010 mar 1;58(5):1739-50. 28. bedeschi mf, bonaglia mc, grasso r, pellegri a, garghentino rr, battaglia ma, panarisi am, di rocco m, balottin u, bresolin n, bassi mt. agenesis of the corpus callosum: clinical and genetic study in 63 young patients. pediatric neurology. 2006 mar 1;34(3):186-93. 29. sowell er, mattson sn, thompson pm, jernigan tl, riley ep, toga aw. mapping callosal morphology and cognitive correlates. effects of heavy prenatal alcohol exposure. neurology. 2001; 57:235–244. 30. levy hl, lobbregt d, barnes pd, poussaint ty. maternal phenylketonuria: magnetic resonance imaging of the brain in offspring. j pediatr. 1996; 128:770–775. 31. park wj, theda c, maestri ne, meyers ga, fryburg js, dufresne c, cohen mm, jabs ew. analysis of phenotypic features and fgfr2 mutations in apert syndrome. am j hum genet. 1995; 57:321–8. 32. fenwick al, bowdin sc, klatt re, wilkie ao. a deletion of fgfr2 creating a chimeric iiib/iiic exon in a child with apert syndrome. bmc med genet. 2011;12:122. 33. rathore e, rathore ah. apert syndrome: report of a rare congenital malformation. pak j med sci. 2017 may-jun;33(3):773-775. doi: 10.12669/ pjms.333.12878. pmid: 28811814; pmcid: pmc5510146. 34. biesecker lg. the greig cephalopolysyndactyly syndrome. orphanet j rare dis. 2008;3:10. 35. démurger f, ichkou a, mougou-zerelli s, le merrer m, goudefroye g, delezoide al, quelin c, manouvrier s, baujat g, fradin m, pasquier l, megarbane a, faivre l, baumann c, nampoothiri s, roume j, isidor b, lacombe d, delrue ma, mercier s, philip n, schaefer e, holder m, krause a, laffargue f, sinico m, amram d, andre g, liquier a, rossi m, amiel j, giuliano f, boute o, dieux-coeslier a, jacquemont ml, afenjar a, van maldergem l, lackmy-port-lis m, vincent-delorme c, chauvet ml, cormierdaire v, devisme l, genevieve d, munnich a, viot g, raoul o, romana s, gonzales m, encha-razavi f, odent s, vekemans m, attie-bitach t. new insights into genotype-phenotype correlation for gli3 mutations. eur j hum genet. 2015; 23:92–102. 36. johnston j j, sapp j c, turner j t, amor d, aftimos s, aleck k a, bocian m, bodurtha j n, cox gf, curry c j, day r, donnai d, field m, fujiwara i, gabbett m, gal m, graham jm, hedera p, hennekam rc, hersh jh, hopkin rj, kayserili h, kidd am, kimonis v, lin a e, lynch s a, maisenbacher m, mansour s, mcgaughran j, mehta l, murphy h, raygada m, robin n h, 14 j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 genetic causes, clinical manifestations and diagnosis of central nervous system review b. alomrani et al. rope a f, rosenbaum k n, schaefer g b, shealy a, smith w, soller m, sommer a, stalker h j, steiner b, stephan m j, tilstra d, tomkins s, trapane p, tsai ac, van allen m i, vasudevan pc, zabel b, zunich j, black g c, biesecker lg. molecular analysis expands the spectrum of phenotypes associated with gli3 mutations. hum mutat. 2010; 31:1142–54. 37. johnston, j. j., olivos-glander, i., killoran, c., elson, e., turner, j. t., peters, k. f., abbott, m. h., aughton, d. j., aylsworth, a. s., bamshad, m. j., booth, c., curry, c. j., and 36 others. molecular and clinical analyses of greig cephalopolysyndactyly and pallister-hall syndromes: robust phenotype prediction from the type and position of gli3 mutations. am. j. hum. genet. 2005; 76: 609–622. 38. debeer, p., peeters, h., driess, s., de smet, l., freese, k., matthijs, g., bornholdt, d., devriendt, k., grzeschik, k.-h., fryns, j.-p., kalff-suske, m. variable phenotype in greig cephalopolysyndactyly syndrome: clinical and radiological findings in 4 independent families and 3 sporadic cases with identified gli3 mutations. am. j. med. genet. 120a: 49-58, 2003. 39. kalff-suske, m., wild, a., topp, j., wessling, m., jacobsen, e.-m., bornholdt, d., engel, h., heuer, h., aalfs, c. m., ausems, m. g. e. m., barone, r., herzog, a., and 11 others. point mutations throughout the gli3 gene cause greig cephalopolysyndactyly syndrome. hum. molec. genet. 8: 1769-1777, 1999. 40. kang, s., graham, j. m., jr., olney, a. h., biesecker, l. g. gli3 frameshift mutations cause autosomal dominant pallister-hall syndrome. nature genet. 15: 266-268, 1997. 41. cardoso c, leventer rj, ward hl, toyo-oka k, chung j, gross a, martin cl, allanson j, pilz dt, olney ah, mutchinick om, hirotsune s, wynshaw-boris a, dobyns wb, ledbetter dh. refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, miller-dieker syndrome, and other phenotypes secondary to deletions of 17p13.3. am j hum genet. 2003; 72:918–30. 42. barkovich aj, koch tk, carrol cl. the spectrum of lissencephaly: report of ten patients analyzed by magnetic resonance imaging. ann neurol. 1991; 30:139–46. 43. dobyns wb, curry cj, hoyme he, turlington l, ledbetter dh. clinical and molecular diagnosis of miller-dieker syndrome. am j hum genet. 1991; 48:584–94. 44. kim yj, byun sy, jo sa, shin yb, cho eh, lee ey, hwang sh. miller-dieker syndrome with der(17)t(12;17)(q24.33;p13.3)pat presenting with a potential risk of mis-identification as a de novo submicroscopic deletion of 17p13.3. korean j lab med. 2011 jan;31(1):49-53. doi: 10.3343/kjlm.2011.31.1.49. pmid: 21239872; pmcid: pmc3111033. 45. sicca f, kelemen a, genton p, das s, mei d, moro f, dobyns wb, guerrini r. mosaic mutations of the lis1 gene cause subcortical band heterotopia. neurology. 2003; 61:1042–6. 46. mowat dr, wilson mj. mowat-wilson syndrome. in: cassidy sb, allanson je, eds. management of genetic syndromes. new york, ny: john wiley and sons; 2010:517-29. 47. wenger tl, harr m, ricciardi s, bhoj e, santani a, adam mp, barnett ss, ganetzky r, mcdonald-mcginn dm, battaglia d, bigoni s, selicorni a, sorge g, monica md, mari f, andreucci e, romano s, cocchi g, savasta s, malbora b, marangi g, garavelli l, zollino m, zackai eh. charge-like presentation, craniosynostosis and mild mowat-wilson syndrome diagnosed by recognition of the distinctive facial gestalt in a cohort of 28 new cases. am j med genet a. 2014; 164a:2557–66. 48. ivanovski i, djuric o, caraffi sg, santodirocco d, pollazzon m, rosato s, cordelli dm, et al. phenotype and genotype of 87 patients with mowat-wilson syndrome and recommendations for care. genet med. 2018; 20:965–75. 49. garavelli l, ivanovski i, caraffi sg, santodirocco d, pollazzon m, cordelli dm, abdalla e, accorsi p, adam mp, baldo c, bayat a, belligni e, bonvicini f, breckpot j, callewaert b, cocchi g, cuturilo g, devriendt k, dinulos mb, djuric o, epifanio r, faravelli f, formisano d, giordano l, grasso m, grønborg s, iodice a, iughetti l, lacombe d, maggi m, malbora b, mammi i, moutton s, møller r, muschke p, napoli m, pantaleoni c, pascarella r, pellicciari a, poch-olive ml, raviglione f, rivieri f, russo c, savasta s, scarano g, selicorni a, silengo m, sorge g, tarani l, tone lg, toutain a, trimouille a, valera et, vergano ss, zanotta n, zollino m, dobyns wb, paciorkowski ar. neuroimaging findings in mowat-wilson syndrome: a study of 54 patients. genet med. 2017; 19:691–700. 50. saunders cj, zhao w, ardinger hh. comprehensive zeb2 gene analysis for mowat-wilson syndrome in a north american cohort: a suggested approach to molecular diagnostics. am j med genet a. 2009; 149a:2527–31. 51. dastot-le moal f, wilson m, mowat d, collot n, niel f, goossens m. zfhx1b mutations in patients with mowat-wilson syndrome. hum mutat. 2007; 28:313–21. 52. yoneda m, fujita t, yamada y, yamada k, fujii a, inagaki t, nakagawa h, shimada a, kishikawa m, nagaya m, azuma t, kuriyama m, wakamatsu n. late infantile hirschsprung disease-mental retardation syndrome with a 3-bp deletion in zfhx1b. neurology. 2002; 59:1637–40. 53. zweier c, horn d, kraus c, rauch a. atypical zfhx1b mutation associated with a mild mowat-wilson syndrome phenotype. am j med genet a. 2006; 140:869–72. 54. fontanella b, russolillo g, meroni g. mid1 mutations in patients with x-linked opitz g/bbb syndrome. hum mutat. 2008; 29:584–94. 55. mcdonald-mcginn dm, emanuel bs, zackai eh. 22q11.2 deletion syndrome. genereviews. 2013;https://www.ncbi.nlm.nih.gov/books/nbk1523. 56. kosho t, miyake n, carey jc. coffin–siris syndrome and related disorders involving components of the baf (mswi/snf) complex: historical review and recent advances using next generation sequencing. in american journal of medical genetics part c: seminars in medical genetics 2014 sep (vol. 166, no. 3, pp. 241-251). 57. garavelli l, mainardi pc. mowat-wilson syndrome. orphanet j rare dis. 2007 oct 24;2:42. doi: 10.1186/1750-1172-2-42. pmid: 17958891; pmcid: pmc2174447. 58. fergelot p, van belzen m, van gils j, afenjar a, armour cm, arveiler b, beets l, burglen l, busa t, collet m, deforges j, de vries bba, dominguez garrido e, dorison n, dupont j, francannet c, garcia-minaur s, gabau vila e, gebre-medhin s, gener querol b, genevieve d, gerard m, gervanisi cg, goldenberg a, josifova d, lachlan k, mas s, maranda b, moilanen js, nordgren a, parent p, rankin j, reardon w, rio m, roume j, shaw a, smigiel r, sojo a, solomon b, stembalska a, stumpel c, suarez f, terhal p, thomas s, touraine r, verloes a, vincent-delorme c, wincent j, peters djm, bartsch o, larizza l, lacombe d, hennekam rc. phenotype and genotype in 52 patients with rubinstein-taybi syndrome caused by ep300 mutations. am j med genet a. 2016; 170:3069–82. 59. paul lk, brown ws, adolphs r, tyszka jm, richards lj, mukherjee p, et al. agenesis of the corpus callosum: genetic, developmental and functional aspects of connectivity. nat rev neurosci. 2007; 8:287–99. 60. shubhankar mishra, sunil kumar agarwalla, dnyaneshwar ramesh potpalle, and nishant nilotpal dash. rubenstein-taybi syndrome with agenesis of corpus callosum. j pediatr neurosci. 2015; 10(2): 175–177. 61. bartsch o, schmidt s, richter m, morlot s, seemanova e, wiebe g, rasi s. dna sequencing of crebbp demonstrates mutations in 56% of patients with rubinstein-taybi syndrome (rsts) and in another patient with incomplete rsts. hum genet. 2005; 117:485–93. 62. negri g, milani d, colapietro p, forzano f, della monica m, rusconi d, consonni l, caffi lg, finelli p, scarano g, magnani c, selicorni a, spena s, larizza l, gervasini c. clinical and molecular characterization of rubinsteintaybi syndrome patients carrying distinct novel mutations of the ep300 gene. clin genet. 2015; 87:148–54. 63. rusconi d, negri g, colapietro p, picinelli c, milani d, spena s, magnani c, silengo mc, sorasio l, curtisova v, cavaliere ml, prontera p, stangoni g, ferrero gb, biamino e, fischetto r, piccione m, gasparini p, salviati l, selicorni a, finelli p, larizza l, gervasini c. characterization of 14 novel deletions underlying rubinstein-taybi syndrome: an update of the crebbp deletion repertoire. hum genet. 2015; 134:613–26. 64. gervasini c, castronovo p, bentivegna a, mottadelli f, faravelli f, giovannucci-uzielli ml, pessagno a, lucci-cordisco e, pinto am, salviati l, selicorni a. high frequency of mosaic crebbp deletions in rubinstein–taybi syndrome patients and mapping of somatic and germ-line breakpoints. genomics. 2007 nov 1;90(5):567-73. 65. smith mj, beetz c, williams sg, bhaskar ss, o’sullivan j, anderson b, daly sb, urquhart je, bholah z, oudit d, cheesman e, kelsey a, mccabe mg, newman wg, evans dg. germline mutations in sufu cause gorlin syndrome-associated childhood medulloblastoma and redefine the risk associated with ptch1 mutations. j clin oncol. 2014; 32:4155–61. 66. kimonis ve, mehta sg, digiovanna jj, bale sj, pastakia b. radiological features in 82 patients with nevoid basal cell carcinoma (nbcc or gorlin) syndrome. genet med. 2004; 6:495–502. 67. amlashi sf, riffaud l, brassier g, morandi x. nevoid basal cell carcinoma syndrome: relation with desmoplastic medulloblastoma in infancy. a population-based study and review of the literature. cancer. 2003; 98:618–24. 68. farndon pa. gorlin (naevoid basal cell carcinoma) syndrome. in: eeles r, easton df, ponder baj, eng c, eds. genetic predisposition to cancer. london, uk: hodder arnold; 2004:193–213. 69. evans dg, howard e, giblin c, clancy t, spencer h, huson sm, lalloo f. birth incidence and prevalence of tumor-prone syndromes: estimates from a uk family genetic register service. am j med genet a. 2010; 152a:327–32. 70. reinders m g, van hout a f, cosgun b, and gille j j. new mutations and an updated database for the patched‐1 (ptch1) gene. mol genet genomic med. 2018; 6:409–415. 15j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 b. alomrani et al. review genetic causes, clinical manifestations and diagnosis of central nervous system 71. kogerman, p., krause, d., rahnama, f., kogerman, l., unden, a. b., zaphiropoulos, p. g., & toftgård, r.. alternative first exons of ptch1 are differentially regulated in vivo and may confer different functions to the ptch1 protein. oncogene. 2002; 21, 6007–6016. 72. sim y c, kim g h, choi s w, ahn k m. novel ptch1 gene mutation in nevoid basal cell carcinoma syndrome. j craniofac surg. 2018; 29(3):e252-e255. 73. elson e, perveen r, donnai d, wall s, black gc. de novo gli3 mutation in acrocallosal syndrome: broadening the phenotypic spectrum of gli3 defects and overlap with murine models. j. med. genet. 2002: 39 (11): 804–6. 74. mu he, radhika subramanian, fiona bangs, tatiana omelchenko, karel f. liem jr, tarun m. kapoor & kathryn v. anderson. the kinesin-4 protein kif7 regulates mammalian hedgehog signalling by organizing the cilium tip compartment. nature cell biology. 2014;16: 663–672. 75. dafinger c, liebau mc, elsayed sm, hellenbroich y, boltshauser e, korenke gc, fabretti f, janecke ar, ebermann i, nürnberg g, nürnberg p, zentgraf h, koerber f, addicks k, elsobky e, benzing t, schermer b, bolz hj. mutations in kif7 link joubert syndrome with sonic hedgehog signaling and microtubule dynamics. j clin invest. 2011; 121:2662–7. 76. walsh d m, shalev s a, simpson m a, morgana n v, gelman-kohan z, chemke j, trembath r c, maher e r. acrocallosal syndrome: identification of a novel kif7 mutation and evidence for oligogenic inheritance. european journal of medical genetics. 2013; 56 (1): 39-42. 77. ibisler a, hehr u, barth a, koch m, epplen j t, and hoffjan s. novel kif7 mutation in a tunisian boy with acrocallosal syndrome: case report and review of the literature. mol syndromol. 2015; 6 (4): 173–80. 78. dupré n, howard hc, mathieu j, karpati g, vanasse m, bouchard jp, carpenter s, rouleau ga. hereditary motor and sensory neuropathy with agenesis of the corpus callosum. ann neurol. 2003; 54:9–18. 79. mathieu j, bédard f, prévost c, langevin p. motor and sensory neuropathies with or without agenesis of the corpus callosum: a radiological study of 64 cases. can j neurol sci. 1990; 17:103–8. 80. cervello m, augello g, cusimano a, rita emma m, balasus d, azzolina a, mccubrey j a, montalto g. pivotal roles of glycogen synthase-3 in hepatocellular carcinoma. advances in biological regulation. 2017; 65: 59–76. 81. howard, h. c., mount, d. b., rochefort, d., byun, n., dupre, n., lu, j., fan, x., song, l., riviere, j.-b., prevost, c., horst, j., simonati, a., and 12 others. the k-cl cotransporter kcc3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum. nature genet. 32: 384-392, 2002. note: erratum: nature genet. 2002; 32: 681 only. 82. lin ae, pober br, mullen mp, slavotinek am. cardiovascular malformations in fryns syndrome: is there a pathogenic role for neural crest cells? am j med genet a. 2005; 139:186–93. 83. alessandri jl, gordon ct, jacquemont ml, gruchy n, ajeawung nf, benoist g, oufadem m, chebil a, duffourd y, dumont c, gérard m, kuentz p, jouan t, filippini f, nguyen ttm, alibeu o, bole-feysot c, nitschké p, omarjee a, ramful d, randrianaivo h, doray b, faivre l, amiel j, campeau pm, thevenon j. recessive loss of function pign alleles, including an intragenic deletion with founder effect in la réunion island, in patients with fryns syndrome. eur j hum genet. 2018; 26:340–9. 84. mcinerney-leo am, harris je, gattas m, peach ee, sinnott s, dudding-byth t, rajagopalan s, barnett cp, anderson lk, wheeler l, brown ma, leo pj, wicking c, duncan el. fryns syndrome associated with recessive mutations in pign in two separate families. hum mutat. 2016; 37:695–702. 85. maydan, g., noyman, i., har-zahav, a., neriah, z. b., pasmanik-chor, m., yeheskel, a., albin-kaplanski, a., maya, i., magal, n., birk, e., simon, a. j., halevy, a., rechavi, g., shohat, m., straussberg, r., basel-vanagaite, l. multiple congenital anomalies-hypotonia-seizures syndrome is caused by a mutation in pign. j. med. genet. 2011; 48: 383-389. 86. brady pd, moerman p, de catte l, deprest j, devriendt k, vermeesch jr. exome sequencing identifies a recessive pign splice site mutation as a cause of syndromic congenital diaphragmatic hernia. eur j med genet. 2014; 57:487–93. 87. kobayashi k, kato r, kondo-iida e, taniguchi-ikeda m, osawa m, saito k, toda t. deep-intronic variant of fukutin is the most prevalent point mutation of fukuyama congenital muscular dystrophy in japan. j hum genet. 2017; 62:945–8. 88. saito k, kobayashi m. fukuyama congenital muscular dystrophy. in: emery aeh, ed. muscular dystrophies. oxford, uk: oxford university press; 2001: 39-54. 89. kato z, morimoto m, orii ke, kato t, kondo n. developmental changes of radiological findings in fukuyama-type congenital muscular dystrophy. pediatr radiol. 2010; 40:s127–9. 90. barkovich j, in magnetic resonance in epilepsy (second edition), 2005. isbn 978-0-12-431152-7. 91. taniguchi m, kurahashi h, noguchi s, sese j, okinaga t, tsukahara t, guicheney p, ozono k, nishino i, morishita s, toda t. expression profiling of muscles from fukuyama-type congenital muscular dystrophy and lamininalpha-2 deficient congenital muscular dystrophy; is congenital muscular dystrophy a primary fibrotic disease? biochem biophys res commun. 2006;342:489–502. 92. kobayashi, k., nakahori, y., miyake, m., matsumura, k., kondo-iida, e., nomura, y., segawa, m., yoshioka, m., saito, k., osawa, m., hamano, k., sakakihara, y., nonaka, i., nakagome, y., kanazawa, i., nakamura, y., tokunaga, k., toda, t. an ancient retrotransposal insertion causes fukuyamatype congenital muscular dystrophy. nature. 1998; 394: 388-392. 93. hayashi, y. k., ogawa, m., tagawa, k., noguchi, s., ishihara, t., nonaka, i., arahata, k. selective deficiency of alpha-dystroglycan in fukuyama-type congenital muscular dystrophy. neurology. 2001; 57: 115–121. 94. lim bc, ki c-s, kim j-w, cho a, kim mj, hwang h, kim kj, hwang ys, park wy, lim y-j, kim io, lee js, chae jh. fukutin mutations in congenital muscular dystrophies with defective glycosylation of dystroglycan in korea. neuromuscul disord. 2010; 20:524–30. 95. saredi s, ruggieri a, mottarelli e, ardissone a, zanotti s, farina l, morandi l, mora m, moroni i. fukutin gene mutations in an italian patient with early onset muscular dystrophy but no central nervous system involvement. muscle nerve. 2009; 39(6):845-8. 96. steinlin m, schmid m, landau k, boltshauser e. follow-up in children with joubert syndrome. neuropediatrics. 1997; 28:204–11. 97. tusa rj, hove mt. ocular and oculomotor signs in joubert syndrome. j child neurol. 1999; 14:621–7. 98. senocak eu, oğuz kk, haliloğlu g, topçu m, cila a. structural abnormalities of the brain other than molar tooth sign in joubert syndrome-related disorders. diagn interv radiol. 2010; 16:3–6. 99. bachmann-gagescu r, dempsey jc, phelps ig, o’roak bj, knutzen dm, rue tc, ishak ge, isabella cr, gorden n, adkins j, boyle ea, de lacy n, o’day d, alswaid a, ramadevi a r, lingappa l, lourenço c, martorell l, garcia-cazorla à, ozyürek h, haliloğlu g, tuysuz b, topçu m, chance p, parisi ma, glass ia, shendure j, doherty d, et al. joubert syndrome: a model for untangling recessive disorders with extreme genetic heterogeneity. j med genet. 2015a; 52:514–22. 100. farach, l. s., little, m. e., duker, a. l., logan, c. v., jackson, a., hecht, j. t., bober, m. the expanding phenotype of rnu4atac pathogenic variants to lowry wood syndrome. am. j. med. genet. 2018; 176a: 465–469. 101. embiruçu e k, martyn m, schlesinger d, kok f. autosomal recessive ataxias: 20 types, and counting. arquivos de neuro-psiquiatria. 2009; 67(4):1143-56. 102. vilboux t, doherty da, glass ia, parisi ma, phelps ig, cullinane ar, zein w, brooks bp, heller t, soldatos a, oden nl, yildirimli d, vemulapalli m, mullikin jc, malicdan mc, gahl wa, gunay-aygun m, et al. molecular genetic findings and clinical correlations in 100 patients with joubert syndrome and related disorders prospectively evaluated at a single center. genet med. 2017; 19:875–82. 103. doherty d, parisi ma, finn ls, gunay-aygun m, al-mateen m, bates d, clericuzio c, demir h, dorschner m, van essen aj, gahl w, gentile m, gorden nt, hikida a, knutzen d, özyurek h, phelps i, rosenthal p, verloes a, weigand h, chance pf, dobyns wb, glass ia. mutations in 3 genes (mks3, rpgrip1l, and cc2d2a) cause coach syndrome/joubert syndrome with congenital hepatic fibrosis. j med genet. 2010; 47:8–21. 104. suzuki t, miyake n, trusaki y, okamoto n, alkindy a, inaba a, sato m, ito s, muramatsu k, kimura s, ieda d, saitoh s, hiyane m, suzumura h, yagyu k, shiraishi h, nakajima m, fueki n, habata y, ueda y, komatsu y, yan k, shimoda k, shitara y, mizuno s, ichinomiya k, sameshima k, tsuyusaki y, kurosawa k, sakai y, haginoya k, kobayashi y, yoshizawa c, hisano m, nakashima m, saitsu h, takeda s, matsumoto n. molecular genetic analysis of 30 families with joubert syndrome. clin genet. 2016; 90:526–35. 105. tuz k, bachmann-gagescu r, o’day dr, hua k, isabella cr, phelps ig, stolarski ae, o’roak bj, dempsey jc, lourenco c, alswaid a, bönnemann cg, medne l, nampoothiri s, stark z, leventer rj, topçu m, cansu a, jagadeesh s, done s, ishak ge, glass ia, shendure j, neuhauss sc, haldeman-englert cr, doherty d, ferland rj. mutations in cspp1 cause primary cilia abnormalities and joubert syndrome with or without jeune asphyxiating thoracic dystrophy. am j hum genet. 2014; 94:62–72. 106. alby c, piquand k, huber c, megarbané a, ichkou a, legendre m, pelluard f, encha-ravazi f, abi-tayeh g, bessières b, el chehadeh-djebbar s, laurent n, faivre l, sztriha l, zombor m, szabó h, failler m, garfa-traore m, bole c, nitschké p, nizon m, elkhartoufi n, clerget-darpoux f, munnich a, lyonnet s, vekemans m, saunier s, cormier-daire v, attié-bitach t, thomas s. mutations in kiaa0586 cause lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly syndrome. am j hum genet. 2015; 97:311–8. https://www.researchgate.net/profile/emilia-embirucu https://www.researchgate.net/profile/marcilia-martyn 16 j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 genetic causes, clinical manifestations and diagnosis of central nervous system review b. alomrani et al. 107. slaats gg, isabella cr, kroes hy, dempsey jc, gremmels h, monroe gr, phelps ig, duran k, adkins j, kumar sa, knutzen dm, knoers nv, mendelsohn nj, neubauer d, mastroyianni sd, vogt j, worgan l, karp n, bowdin s, glass ia, parisi ma, otto ea, johnson ca, hildebrandt f, van haaften g, giles rh, doherty d. mks1 regulates ciliary inpp5e levels in joubert syndrome. j med genet. 2016; 53:62–72. 108. delous m, baala l, salomon r, laclef c, vierkotten j, tory k, golzio c, lacoste t, besse l, ozilou c, moutkine i, hellman ne, anselme i, silbermann f, vesque c, gerhardt c, rattenberry e, wolf mt, gubler mc, martinovic j, encha-razavi f, boddaert n, gonzales m, macher ma, nivet h, champion g, berthélémé jp, niaudet p, mcdonald f, hildebrandt f, johnson ca, vekemans m, antignac c, rüther u, schneider-maunoury s, attié-bitach t, saunier s. the ciliary gene rpgrip1l is mutated in cerebello-oculo-renal syndrome (joubert syndrome type b) and meckel syndrome. nat genet. 2007; 39:875–81. 109. brancati f, iannicelli m, travaglini l, mazzotta a, bertini e, boltshauser e, et al. mks3/tmem67 mutations are a major cause of coach syndrome, a joubert syndrome related disorder with liver involvement. hum mutat. 2009; 30:e432–42. 110. valente em, logan cv, mougou-zerelli s, lee jh, silhavy jl, brancati f, iannicelli m, travaglini l, romani s, illi b, adams m, szymanska k, mazzotta a, lee je, tolentino jc, swistun d, salpietro cd, fede c, gabriel s, russ c, cibulskis k, sougnez c, hildebrandt f, otto ea, held s, diplas bh, davis ee, mikula m, strom cm, ben-zeev b, lev d, sagie tl, michelson m, yaron y, krause a, boltshauser e, elkhartoufi n, roume j, shalev s, munnich a, saunier s, inglehearn c, saad a, alkindy a, thomas s, vekemans m, dallapiccola b, katsanis n, johnson ca, attié-bitach t, gleeson jg. mutations in tmem216 perturb ciliogenesis and cause joubert, meckel and related syndromes. nat genet. 2010; 42:619–25. 111. cantagrel v, silhavy jl, bielas sl, swistun d, marsh se, bertrand jy, audollent s, attié-bitach t, holden kr, dobyns wb, traver d, al-gazali l, ali br, lindner th, caspary t, otto ea, hildebrandt f, glass ia, logan cv, johnson ca, bennett c, brancati f, valente em, woods cg, gleeson jg, et al. mutations in the cilia gene arl13b lead to the classical form of joubert syndrome. am j hum genet. 2008; 83:170–9. 112. romani m, micalizzi a, kraoua i, dotti mt, cavallin m, sztriha l, ruta r, mancini f, mazza t, castellana s, hanene b, carluccio ma, darra f, máté a, zimmermann a, gouider-khouja n, valente em. mutations in b9d1 and mks1 cause mild joubert syndrome: expanding the genetic overlap with the lethal ciliopathy meckel syndrome. orphanet j rare dis. 2014; 9:72. 113. lee je, silhavy jl, zaki ms, schroth j, bielas sl, marsh se, olvera j, brancati f, iannicelli m, ikegami k, schlossman am, merriman b, attié-bitach t, logan cv, glass ia, cluckey a, louie cm, lee jh, raynes hr, rapin i, castroviejo ip, setou m, barbot c, boltshauser e, nelson sf, hildebrandt f, johnson ca, doherty da, valente em, gleeson jg. cep41 is mutated in joubert syndrome and is required for tubulin glutamylation at the cilium. nat genet. 2012a; 44:193–9. 114. srour m, hamdan ff, mcknight d, davis e, mandel h, schwartzentruber j, martin b, patry l, nassif c, dionne-laporte a, ospina lh, lemyre e, massicotte c, laframboise r, maranda b, labuda d, décarie jc, rypens f, goldsher d, fallet-bianco c, soucy jf, laberge am, maftei c, boycott k, brais b, boucher rm, rouleau ga, katsanis n, majewski j, elpeleg o, kukolich mk, shalev s, michaud jl, et al. joubert syndrome in french canadians and identification of mutations in cep104. am j hum genet. 2015; 97:744–53. 115. shaheen r, schmidts m, faqeih e, hashem a, lausch e, holder i, supertifurga a, mitchison hm, almoisheer a, alamro r, alshiddi t, alzahrani f, beales pl, alkuraya fs, et al. a founder cep120 mutation in jeune asphyxiating thoracic dystrophy expands the role of centriolar proteins in skeletal ciliopathies. hum mol genet. 2015b; 24:1410–9. 116. halbritter j, bizet aa, schmidts m, porath jd, braun da, gee hy, mcinerneyleo am, krug p, filhol e, davis ee, airik r, czarnecki pg, lehman am, trnka p, nitschké p, bole-feysot c, schueler m, knebelmann b, burtey s, szabó aj, tory k, leo pj, gardiner b, mckenzie fa, zankl a, brown ma, hartley jl, maher er, li c, leroux mr, scambler pj, zhan sh, jones sj, kayserili h, tuysuz b, moorani kn, constantinescu a, krantz id, kaplan bs, shah jv, hurd tw, doherty d, katsanis n, duncan el, otto ea, beales pl, mitchison hm, saunier s, hildebrandt f, et al. defects in the ift-b component ift172 cause jeune and mainzer-saldino syndromes in humans. am j hum genet. 2013; 93:915–25. 117. sanders aawm, de vrieze e, alazami am, alzahrani f, malarkey eb, sorusch n, tebbe l, kuhns s, van dam tjp, alhashem a, tabarki b, lu q, et al. kiaa0556 is a novel ciliary basal body component mutated in joubert syndrome. genome biol. 2015; 16:293. 118. coene kl, roepman r, doherty d, afroze b, kroes hy, letteboer sj, ngu lh, budny b, van wijk e, gorden nt, azhimi m, thauvin-robinet c, veltman ja, boink m, kleefstra t, cremers fp, van bokhoven h, de brouwer ap. ofd1 is mutated in x-linked joubert syndrome and interacts with lca5-encoded lebercilin. am j hum genet. 2009; 85:465–81. 119. thomas s, wright kj, le corre s, micalizzi a, romani m, abhyankar a, saada j, perrault i, amiel j, litzler j, filhol e, elkhartoufi n, kwong m, casanova jl, boddaert n, baehr w, lyonnet s, munnich a, burglen l, chassaing n, encharavazi f, vekemans m, gleeson jg, valente em, jackson pk, drummond ia, saunier s, attié-bitach t. a homozygous pde6d mutation in joubert syndrome impairs targeting of farnesylated inpp5e protein to the primary cilium. hum mutat. 2014; 35:137–46. 120. magnani, c., tedesco, s. a., dallaglio, s., sommi, m., bacchini, e., vetro, a., zuffardi, o., bevilacqua, g. multiple joint dislocations: an additional skeletal finding in lowry-wood syndrome? am. j. med. genet. 2009; 149a: 737-741. 121. shelihan, i., ehresmann, s., magnani, c., forzano, f., baldo, c., brunetti-pierri, n., campeau, p. m. lowry-wood syndrome: further evidence of association with rnu4atac, and correlation between genotype and phenotype. hum. genet. 2018; 137: 905-909. 122. barisic i, boban l, loane m, garne e, wellesley d, calzolari e, dolk h, addor m-c, bergman j eh, braz p, draper e s, haeusler m, khoshnood b, klungsoyr k, pierini a, queisser-luft a, rankin j, rissmann a and verellen-dumoulin c. meckel–gruber syndrome: a population-based study on prevalence, prenatal diagnosis, clinical features, and survival in europe. european journal of human genetics. 2015; 23, 746–752. 123. salonen r. the meckel syndrome – clinicopathological findings in 67 patients. am j med genet. 1984; 18:671–89. 124. auber b, burfeind p, herold s, schoner k, simson g, rauskolb r, et al. a disease causing deletion of 29 base pairs in intron 15 in the mks1 gene is highly associated with the campomelic variant of the meckel-gruber syndrome. clin genet. 2007; 72:454–9. 125. schurig v, bowen p, harley f, schiff d. the meckel syndrome in the hutterites. am j med genet. 1980; 5:373–81. 126. salonen r, norio r. the meckel syndrome in finland: epidemiologic and genetic aspects. am j med genet. 1984; 18:691–8. 127. teebi as, alsaleh qa, odeh h. meckel syndrome and neural-tube defects in kuwait. j med genet. 1992; 29:140. 128. teebi as, teebi sa. genetic diversity among the arabs. community genet. 2005; 8:21–6. 129. baala l, romano s, khaddour r, saunier s, smith um, audollent s, et al. the meckel-gruber syndrome gene, mks3, is mutated in joubert syndrome. am j hum genet. 2007a; 80:186–94. 130. leitch cc, zaghloul na, davis ee, stoetzel c, diaz-font a, rix s, et al. hypomorphic mutations in syndromic encephalocele genes are associated with bardet-biedl syndrome. nat genet. 2008; 40:443–8. 131. hartill v, szymanska k, sharif s m, wheway g and johnson c a. meckel– gruber syndrome: an update on diagnosis, clinical management, and research advances. front. pediatr. 2017; 5 (244):1–9. 132. dawe h r, smith u m, cullinane a r, gerrelli d, cox p, badano j l, blair-reid s, sriram n, katsanis n, attie-bitach t, afford s c, copp a j, kelly d a, gull k, johnson c a. the meckel-gruber syndrome proteins mks1 and meckelin interact and are required for primary cilium formation. hum. molec. genet. 2007; 16: 173–186. 133. kyttälä m, tallila j, salonen r, kopra o, kohlschmidt n, paavola-sakki p, et al. mks1, encoding a component of the flagellar apparatus basal body proteome, is mutated in meckel syndrome. nat genet. 2006; 38:155–7. 134. edvardson s, shaag a, zenvirt s, erlich y, hannon gj, shanske al, et al. joubert syndrome 2 (jbts2) in ashkenazi jews is associated with a tmem216 mutation. am j hum genet. 2010; 86:93–7. 135. smith um, consugar m, tee lj, mckee bm, maina en, whelan s, et al. the transmembrane protein meckelin (mks3) is mutated in meckelgruber syndrome and the wpk rat. nat genet. 2006; 38:191–6. 136. baala, l., audollent, s., martinovic, j., ozilou, c., babron, m.-c., sivanandamoorthy, s., saunier, s., salomon, r., gonzales, m., rattenberry, e., esculpavit, c., toutain, a., and 23 others. pleiotropic effects of cep290 (nphp6) mutations extend to meckel syndrome. am. j. hum. genet. 2007b; 81: 170–179. 137. arts hh, doherty d, van beersum se, parisi ma, letteboer sj, gorden nt, et al. mutations in the gene encoding the basal body protein rpgrip1l, a nephrocystin-4 interactor, cause joubert syndrome. nat genet. 2007; 39:882–8. 138. tallila j, jakkula e, peltonen l, salonen r, kestilä m. identification of cc2d2a as a meckel syndrome gene adds an important piece to the ciliopathy puzzle. am j hum genet. 2008; 82:1361–7. 139. bergmann, c., fleigauf, m., bruchle, n. o., frank, v., olbrich, h., kirschner, j., schermer, b., schmedding, i., kispert, a., kranzlin, b., nurnberg, g., becker, c., and 17 others. loss of nephrocystin-3 function can cause embryonic 17j contemp med sci | vol. 8, no. 1, january-february 2022: 1–17 b. alomrani et al. review genetic causes, clinical manifestations and diagnosis of central nervous system lethality, meckel-gruber-like syndrome, situs inversus, and renal-hepaticpancreatic dysplasia. am. j. hum. genet. 2008; 82: 959–970. 140. shaheen, r., faqeih, e., seidahmed, m. z., sunker, a., alali, f. e., alqahtani, k., alkuraya, f. s. a tctn2 mutation defines a novel meckel gruber syndrome locus. hum. mutat. 2011; 32: 573-578. 141. hopp, k., heyer, c. m., hommerding, c. j., henke, s. a., sundsbak, j. l., patel, s., patel, p., consugar, m. b., czarnecki, p. g., gliem, t. j., torres, v. e., rossetti, s., harris, p. c. b9d1 is revealed as a novel meckel syndrome (mks) gene by targeted exon-enriched next-generation sequencing and deletion analysis. hum. molec. genet. 2011; 20: 2524–2534. 142. dowdle, w. e., robinson, j. f., kneist, a., sirerol-piquer, m. s., frints, s. g. m., corbit, k. c., zaghloul, n. a., van lijnschoten, g., mulders, l., verver, d. e., zerres, k., reed, r. r., attie-bitach, t., johnson, c. a., garcia-verdugo, j. m., katsanis, n., bergmann, c., reiter, j. f. disruption of a ciliary b9 protein complex causes meckel syndrome. am. j. hum. genet. 89: 94-110, 2011. note: erratum: am. j. hum. genet. 2011; 89: 589 only. 143. shaheen, r., ansari, s., al mardawi, e., alshammari, m. j., alkuraya, f. s. mutations in tmem231 cause meckel-gruber syndrome. j. med. genet. 2013; 50: 160-162. 144. taybi h, linder d. congenital familial dwarfism with cephaloskeletal dysplasia. radiology. 1967;89:275–281. 145. nagy r, wang h, albrecht b, wieczorek d, kaesbach g g, haan eric, meinecke p, de la chapelle a, and westman j a. microcephalic osteodysplastic primordial dwarfism type i with biallelic mutations in the rnu4atac gene. clin genet. 2012; 82(2): 1-12. 146. majewski f, stoeckenius m, kemperdick h. studies of microcephalic primordial dwarfism iii: an intrauterine dwarf with platyspondyly and anomalies of pelvis and clavicles – osteodysplastic primordial dwarfism type iii. am j med genet. 1982; 12:37–42. 147. ferrell s, johnson a, pearson w. microcephalic osteodysplastic primordial dwarfism type 1. bmj case rep. 2016. doi:10.1136/bcr-2016-215502. 148. wang y, wu x, du l, zheng j, deng s, bi x, chen q, xie h, férec c, cooper d n, luo y, fang q & chen j-m. identification of compound heterozygous variants in the noncoding rnu4atac gene in a chinese family with two successive foetuses with severe microcephaly. human genomics 2018; 12(3): 1-8. doi: 10.1186/s40246-018-0135-9. 149. he, h., liyanarachchi, s., akagi, k., nagy, r., li, j., dietrich, r. c., li, w., sebastian, n., wen, b., xin, b., singh, j., yan, p., and 10 others. mutations in u4atac snrna, a component of the minor spliceosome, in the developmental disorder mopd i. science. 2011; 332: 238–240. 150. will cl, lührmann r. “spliceosome structure and function”. cold spring harbor perspectives in biology. 2011; 3 (7): a003707. doi:10.1101/ cshperspect.a003707. 151. chen w and moore m j. spliceosomes. current biology. 2015; 25(5):r182. 152. edery, p., marcaillou, c., sahbatou, m., labalme, a., chastang, j., touraine, r., tubacher, e., senni, f., bober, m. b., nampoothiri, s., jouk, p.-s., steichen, e., berland, s., toutain, a., wise, c. a., sanlaville, d., rousseau, f., clerget-darpoux, f., leutenegger, a.-l. association of tals developmental disorder with defect in minor splicing component u4atac snrna. science. 2011; 332: 240–243. 153. abdel-salam g m h, abdel-hamid m s, issa, m, magdy a, el-kotoury a, amr k. expanding the phenotypic and mutational spectrum in microcephalic osteodysplastic primordial dwarfism type i. am. j. med. genet. 2012; 158a: 1455–1461. 154. finocchi a, angelino g, cantarutti n, corbari m, bevivino e, cascioli s, randisi f, bertini e, dionisi-vici c. immunodeficiency in vici syndrome: a heterogeneous phenotype. am. j. med. genet. 2012; 158a: 434–439. 155. maillard c, cavallin m, piquand k, philbert m, bault j p, millischer a e, moshous d, rio m, gitiaux c, boddaert n, masson c, thomas s, bahi-buisson n. prenatal and postnatal presentations of corpus callosum agenesis with polymicrogyria caused by egp5 (sic) mutation. am. j. med. genet. 2017; 173a: 706-711. 156. mcclelland v, cullup t, bodi i, ruddy d, buj-bello a, biancalana v, boehm j, bitoun m, miller o, jan w, menson e, amaya l, trounce j, laporte j, mohammed s, sewry c, raiman j, jungbluth h. vici syndrome associated with sensorineural hearing loss and evidence of neuromuscular involvement on muscle biopsy. am. j. med. genet. 2010; 152a: 741-747. 157. al-owain m, al-hashem a, al-muhaizea, m, humaidan h, al-hindi h, al-homoud i, al-mogarri i. vici syndrome associated with unilateral lung hypoplasia and myopathy. (letter) am. j. med. genet. 2010; 152a: 1849–1853. 158. cullup, t., kho, a. l., dionisi-vici, c., brandmeier, b., smith, f., urry, z., simpson, m. a., yau, s., bertini, e., mcclelland, v., al-owain, m., koelker, s., and 25 others. recessive mutations in epg5 cause vici syndrome, a multisystem disorder with defective autophagy. nature genet. 2013; 45: 83–87. 159. ehmke, n., parvaneh, n., krawitz, p., ashrafi, m.-r., karimi, p., mehdizadeh, m., kruger, u., hecht, j., mundlos, s., robinson, p. n. first description of a patient with vici syndrome due to a mutation affecting the penultimate exon of epg5 and review of the literature. am. j. med. genet. 2014; 164a: 3170–3175. 160. del campo, m., hall, b. d., aeby, a., nassogne, m.-c., verloes, a., roche, c., gonzalez, c., sanchez, h., garcia-alix, a., cabanas, f., escudero, r. m., hernandez, r., quero, j. albinism and agenesis of the corpus callosum with profound developmental delay: vici syndrome, evidence for autosomal recessive inheritance. am. j. med. genet. 1999; 85: 479–485. 161. hofman m a. a biometric analysis of brain size in micrencephalics. j. neurol. 1984; 231: 87–93. 162. roberts e, jackson a p, carradice a c, deeble v j, mannan j, rashid y, jafri h, mchale d p, markham a f, lench n j, woods c g. the second locus for autosomal recessive primary microcephaly (mcph2) maps to chromosome 19q13.1-13.2. europ. j. hum. genet. 1999; 7: 815-820. 163. nicholas a k, khurshid m, desir j, carvalho o p, cox j j, thornton g, kausar r, ansar m, ahmad w, verloes a, passemard s, misson j p, lindsay s, gergely f, dobyns w b, roberts e, abramowicz m, woods c g. wdr62 is associated with the spindle pole and is mutated in human microcephaly. nature genet. 2010; 42: 1010-1014. 164. zaqout s, morris-rosendahl d, kaindl am. autosomal recessive primary microcephaly (mcph): an update. neuropediatrics 2017; 48:135–142. 165. banerjee s, chen h, huang h, wu j, yang z, deng w, chen d, deng j, su y, li y, wu c, wang y, zeng h, wang y, li x. novel mutations c.28g>t (p.ala10ser) and c.189g>t (p.glu63asp) in wdr62 associated with early onset acanthosis and hyperkeratosis in a patient with autosomal recessive microcephaly type 2. oncotarget. 2016; 7:78363–78371. 166. bastaki f, mohamed m, nair p, saif f, tawfiq n, aithala g, el-halik m, al-alim, hamzeh ar. novel splice-site mutation inwdr62 revealed by whole-exome sequencing in a sudanese family with primary microcephaly. congenit anom. 2016; 56:135–137. 167. bilguvar k, ozturk a k, louvi a, kwan k y, choi m, tatli b, yalnizoglu d, tuysuz b, caglayan a o, gokben s, kaymakcalan h, barak t, and 21 others. wholeexome sequencing identifies recessive wdr62 mutations in severe brain malformations. nature. 2010; 467: 207–210. 168. zombor m, kalmár t, nagy n, berényi m, telcs b, maróti z, brandau o, and sztriha l. a novel wdr62 missense mutation in microcephaly with abnormal cortical architecture and review of the literature. journal of applied genetics. 2019; https://doi.org/10.1007/s13353-019-00486-y. 169. manning m a, cunniff c m, colby c e, el-sayed y y, hoyme h e. neu-laxova syndrome: detailed prenatal diagnostic and post-mortem findings and literature review. am. j. med. genet. a. 2004; 125a:240–249. 170. ostrovskaya t i, lazjuk g i. cerebral abnormalities in the neu-laxova syndrome. am. j. med. genet. 1988; 30: 747-756. 171. shapiro i, borochowitz z, degani s, dar h, ibschitz i, sharf m. neu-laxova syndrome: prenatal ultrasonographic diagnosis, clinical and pathological studies, and new manifestations. am. j. med. genet. 1992; 43: 602-605. 172. manar a l, asma b. neu-laxova syndrome: a new patient with detailed antenatal and post-natal findings. (letter) am. j. med. genet. 152a: 31933196, 2010. 173. acuna-hidalgo r, schanze d, kariminejad a, nordgren a, kariminejad m h, conner p, grigelioniene g, nilsson d, nordenskjöld m, wedell a, freyer c, wredenberg a, wieczorek d, gillessen-kaesbach g, kayserili h, elcioglu n, ghaderi-sohi s, goodarzi p, setayesh h, van de vorst m, steehouwer m, pfundt r, krabichler b, curry c, mackenzie m g , boycott k m , gilissen c, janecke a r, hoischen a, zenker m. neu-laxova syndrome is a heterogeneous metabolic disorder caused by defects in enzymes of the l-serine biosynthesis pathway. am j hum genet. 2014; 95(3):285–93. 174. shaheen r, rahbeeni z, alhashem a, faqeih e, zhao q, xiong y, almoisheer a, al-qattan s m, almadani h a, al-onazi n, al-baqawi b s, saleh m a, alkuraya f s. neu-laxova syndrome, an inborn error of serine metabolism, is caused by mutations in phgdh. am. j. hum. genet. 2014; 94: 898-904. 175. baek j y, jun d y, taub d, kim y h. characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of l-serine biosynthesis. biochem. j. 2003; 373: 191–200. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1167 153j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 diversity and colonization of endophytic actinomycetes in some medicinal plants: review kholoud alzahrani1,2, samyah jastaniah1, reda amasha1, magda m. aly1,3,* 1department of biology, faculty of sciences, king abdulaziz university, jeddah, saudi arabia. 2department of biology, university college of umluj, university of tabuk, saudi arabia. 3botany and microbiology department, faculty of sciences, kafrelsheikh university, kafr el-sheikh, egypt. *correspondence to: magda m. aly (e-mail: magdammali@hotmail.com) (submitted: 19 december 2021 – revised version received: 04 january 2022 – accepted: 25 january 2022 – published online: 26 june 2022) abstract the diversity of culturable endophytic actinomycetes related to plants and especially medicinal plants are isolated and characterized. the increased colonization and distribution of endophytic actinomycetes in plants have been reported in many studies. endophytic actinomycetes belong to different genera are distributed in plants species such as streptomyces, which is the dominant genus, micromonospora, microbispora, nocardiopsis, rhodococcus and nocardia. moreover, there are many novel strains had been described belong to these genera. endophytic actinomycetes seem to contribute with plant development and displaying beneficial traits that can be exploited in plant maintenance, substances supplements, protection and defense. colonization of endophytic actinomycetes in plant tissues are largely influenced by plant species and the environmental factors surrounding the host plants such as soil ph, water content, rainfall, soil salinity, and temperatures. some endophytic actinomycetes may occur in low numbers and sometimes in localized positions within the plant. thus, this review summarizes an aspect of the diversity and colonization of endophytic actinomycetes, including their mechanism of action, importance, and isolation and identification methods. keywords: endophytic, actinobacteria, plants, streptomyces, colonization issn 2413-0516 introduction actinomycetes are high guanine and cytosine ratio (>50%) of their dna content, gram-positive and filamentous bacteria that form non-septate branching mycelia similar to those of fungi.1 they had prokaryotic nucleus and their colonies are powdery consistency and stick firmly to agar surface, producing hyphae with conidia or sporangia.2 actinomycetes are distributed in various natural habitats, including soil, extreme environments, plants, lichens and fresh and marine water.3 the endophytic microorganisms colonize the interior of all plant parts, viz: root, stem, or seeds, regardless of their place of origin, without causing any harmful effects on host plant.4 their ability to access and thrive in the host tissues make them unique by many specific interactions within the host plants.5 there are increasing reports for the existence of new endophytic actinomycetes within various tissues of crops and medicinal plants. in the early stages of endophytic colonization, endophytic actinomycetes are firstly observed in subsequently in the root cortex, and root hairs.6,7 from their point of entry, endophytic actinomycetes may systemically colonize plants from roots to shoots, shoots to flowers or fruits, and/or from flowers to fruits and seeds, and they may also cause localized colonization inside/outside plant organs.8 the diversity aspects of endophytic actinomycetes have addressed by few research groups includes thorough collections and sampling of plant specimens (herbs, shrubs, trees, and woody climbers) and subjecting the specimens to surface sterilization and isolations of endophytes.9 in last twenty years, many studies have focused on endophytic actinomycetes by investigating their existence into the medicinal plants’ organs. also, they have developed many methods to sterilize the plants organs surfaces and modified many culture media. these studies help understanding the colonizing and diversity of endophytic actinomycetes in medicinal plants.10,11 therefore, according to these studies and reviews as well, this review highlights the endophytic actinomycetes colonizing and their diversity in different medicinal plants organs from multiple habitats in many countries. colonization of endophytic actinomycetes in plants the word endophyte is derived from the greek words endon (within) and phyton (plant), which means “in plant”. this term is used as broad as its literal definition and spectrum of potential hosts and inhabitants, e.g., endophytic actinomycetes.10 many studies indicated that almost all the plants are colonized by endophytes microorganisms.12 the diversity of endophytic microorganisms in plants has been reported from different types such as archaea, eubacteria, and fungi. among bacteria, endophytic bacteria were isolated from different phylum mainly: actinobacteria, firmicutes, proteobacteria and bacteroidetes. distribution of endophytic actinomycetes varied in all bacterial phyla. however, proteobacteria were most dominant phyla followed by actinobacteria.4 gohain et al.13 (2015) investigated six selective medicinal plants, emblica officinalis, terminalia chebula, t. arjuna, murraya koenigii, rauwolfia serpentina and azadirachta indica for isolation of endophytic actinomycetes which gained specific niches in plants by colonizing stems, roots, petioles, leaves, fruit, buds and seeds. the ability to enter and thrive in the host plant tissues, make endophytic actinomycetes unique, and showing multidimensional interactions.11 furthermore, endophytic actinomycetes can play myriad crucial roles in hosted plants. some of these roles have been discovered and linked to many enhancements of growth and defense mechanisms towards pathogens infections while others still unknown till now. review mailto:magdammali@hotmail.com 154 j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 diversity and colonization of endophytic actinomycetes in some medicinal plants: review review k. alzahrani et al. colonizing mechanisms in plant organs the colonization of endophytic actinomycetes in different plants is multivariate depending on many factors. thus, understanding these factors approach a better explanation of the relationships between endophytic actinomycetes and hosted plants. multiple plant host activities are influenced by the presence of endophytic actinomycetes, which can promote plant host growth, evoke defense responses against pathogens attacks.5 flourishing colonization by endophytic actinomycetes is affected by different factors including the plant tissue type, the plant genotype, the microbial taxon and strain type, and biotic and abiotic environmental conditions.14 the communities of endophytic actinomycetes have been categorized into three groups, which are “obligate” or “facultative” and “passive”. obligate actinomycetes endophytes are depend basically on plants metabolism for survival, and whose transmission amongst plants take place by the action of various vectors or by vertical transmission.15 facultative actinomycetes endophytes spend definite stages of their life cycle in host plant independently. indirectly, they are associated with hosted plants through soil environment and atmosphere.16 actinomycetes endophytes which are lacking the capability to colonize and infect, can enter plant via wounds and cracks on the plant. they are documented as a passive mode of endophytic colonization.17 in hosted plants, emerging lateral roots build naturally forming a ‘highway’ for actinomycetes endophytes to enter by breaking through the endodermis, cortex, epidermis casparian strip (band around endodermis) and pericycle. from these cells, endophytes can further enter the phloem and xylem vessels that transport photosynthates, nutrients and water.8 actinomycetes endophytes are beneficial guests to their plant hosts. they have albitites to produce chemical diversity of secondary metabolites. much of the natural products by actinomycetes endophytes have likely evolved due to their interactions with other microorganisms in highly diverse environments.18,19 for instance, root exudates such as proteins, amino acids, organic acids, abundance of carbohydrates and inorganic nutrients may be involved in recruiting actinomycetes endophytes from the rhizosphere.14,20 endophytic actinomycetes have to compete with plant cells for iron, and therefore, siderophore production is increasing availability of minerals in addition to iron chelation which is highly important for endophytic growth.21 defense mechanisms in plant tissues plants have a variety of defense mechanisms against pathogens. the response of the host plant drastically differs to the colonization of endophytic actinomycetes and pathogens. on the other hand, endophytic actinomycetes enhance the disease resistance through the mechanism of induced systematic resistance and systemic acquired resistance.22 pathogenic microorganisms, which include bacteria, fungi or viruses, are associated molecular patterns. these are essential structures that are conserved and necessary for pathogens survival, but plants have developed multiple families of receptor proteins to recognize them and encourage the plant immune system.23 pattern-recognition receptors have enhanced to recognize common pathogens compounds, such as bacterial flagellin or fungal chitin or microbial/pathogen-associated molecular patterns. pattern recognition is translated into a first line of defense which keeps the most potential invaders under control.24 endophytic actinomycetes have been equipped with necessary traits that enable them to invade, colonize and translocate in the plant’s interior.25 plants may subsequently influence the biocontrol expression of endophytic actinomycetes against the pathogen.26 colonizing organs in plant tissues diversity of endophytic actinomycetes from plant species are documented from various regions around the world. as a matter of fact, they used morphological and microscopic identification methods in addition to methods based on sequencing of highly conserved macromolecules, notably 16s rrna genes, which has provided valuable data for constructing phylogenies above the genus level.27 thus, studying the diversity of endophytic actinomycetes by isolating them from inner plant organs, give a supportive hypothesis about their roles in plants. they are able to colonize both intracellularly and extracellularly the interior of plants after inoculation the soil with these microorganisms.25,26,28 matsumoto and takahashi29 (2017) revealed that specific strains belonging to various genera could be isolated from plant roots but not from soil. endophytes inside a plant may either become localized at the point of entry or spread throughout the plant.4 the maximum endophytic actinomycetes have been gained from roots followed by stems and least isolates were in leaves and they have been detected in plant reproductive organs, such as flowers, fruits and seeds, but in small numbers (table 1). specifically, in a leaf, endophytic actinomycetes can colonize in xylem vessels, palisade mesophyll cells, upper epidermis cells as well as spaces between spongy cells of mesophyll layer.30 alternatively, these endophytic actinomycetes can get into phyllosphere epiphytes through natural openings (e.g., stomata, hydathodes), wounds and cracks generated by wind, insect and pathogen attacks.31 endophytic actinomycetes genera distribution even thought, there are many reports demonstrated the distribution of endophytic actinomycetes in several organs in plants mainly in roots, stems or leaves. the isolation of endophytic actinomycetes from herbaceous and woody plants is a matter to speculate, as no consensus exists between the research groups.9 the total number of endophytic cultured, uncultured or rare isolates in addition to their genera and families were recorded. chen et al.32 (2011) reported 8 endophytic actinomycetes from root of elaeagnus angustifolia while 47 isolates were detected in roots of four different plants, phyllanthus niruri, withania somnifera, catharanthus roseus and hemidesmus indicus.33 goudjal et al.34 (2013) identified 27 isolates from roots of cleome arabica, solanum nigrum, astragallus armatus, aristida pungens and panicum turgidum while rahayu et al.35 (2019) isolated 7 endophytic actinomycetes from roots of zingiber officinale. endophytes actinomycetes importance actinomycetes are widespread in nature and represent the largest taxonomic group within the domain bacteria. they are 155j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 k. alzahrani et al. diversity and colonization of endophytic actinomycetes in some medicinal plants: review table 1. number of endophytic actinomycetes isolates from different plant parts country plant species organ no. of isolates references india brassica juncea root stem leaf 13 4 3 chaudhry et al., 202036 vietnam cinnamomum cassia presl root stem leaf 29 67 15 vu et al., 202037 thailand andrographis paniculata asystasia gangetica berleria lupulina clinacanthus nutans justicia subcoriacea ruellia squarrosa root stem leaf 49 1 2 phongsopitanuna et al., 202038 china thymus roseus schipcz root stem leaf 54 35 37 musa et al., 202039 india ageratum houstonianum scoparia dulcis (l.) phyllanthus niruri (l.) discorea bulbifera (l.) root stem leaf flower 7 5 2 2 momin et al., 201940 china camellia sinensis root stem leaf 18 9 19 shan et al., 201841 indonesia ficus deltoidea root stem leaf fruit 6 7 24 2 janatiningrum et al., 201842 china dracaena cochinchinensis lour root stem leaf 117 113 74 salam et al., 201743 india catharanthus roseus root leaf 6 5 ranjan and jadeja, 201744 brazil vochysia divergens stem leaf 5 5 gos et al., 201745 india rauvolfia serpentine gymnema sylvestre stevia crenata bacopa monnieri andrographis paniculata withania somnifera root stem leaf 27 17 24 singh and gaur, 201646 india syzygium cumini root stem leaf 28 21 1 saini et al., 201647 india azadirachta indica a. juss. root stem leaf 15 13 7 kaur, 201648 india schima wallichii root stem leaf fruit 9 6 4 3 passari et al., 201649 thailand centella asiatica (l.) root stem leaf fruit 9 19 4 4 phuakjaiphaeo and kunasakdakul, 201550 india combretum latifolium blume root stem leaf 64 31 22 rao et al., 201551 india emblica officinalis gaertn root stem leaf 18 9 9 gangwar et al., 201552 (continued) review 156 j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 diversity and colonization of endophytic actinomycetes in some medicinal plants: review review k. alzahrani et al. table 1. number of endophytic actinomycetes isolates from different plant parts country plant species organ no. of isolates references india aloe vera mentha arvensis ocimum sanctum root stem leaf 28 7 5 gangwar et al., 201453 algeria triticum durum root leaf 16 7 sadrati et al., 201354 australia callitris preissii root stem leaf 189 3 2 kaewkla and franco, 201355 eucalyptus camaldulensis root stem leaf 9 8 2 eucalyptus microcarpa root stem leaf 169 21 1 pittosporum phylliraeoides root stem leaf 91 28 33 abundant in soil and into plant tissues and have been extensively explored for their therapeutic applications.56 targeting a compound for a particular biological activity involves the screening of a number of strains against wide targets, the resulting positive is designated as the “lead”.9 symbiotic actinomycetes residing as endophytes within the plant tissues have generated immense interest as potential source of novel compounds, which may find applications in medicine, agriculture, and environment such as antibacterial, antifungal, antiviral and anticancer.56 the antimicrobial compounds produced by endophytic bacteria represent a promising alternative protection to plants against phytopathogens.57 the genera streptomyces and micromonospora are the potential producers of antibiotics. newer antibiotics are being discovered from actinomycetes and the endophytes are the better choices for antibiotics. actinomycetes are the potent producers of enzyme inhibitors. enzyme inhibitors are specific biochemical tools that are potential in the treatment of diseases.9 cell-wall degrading enzymes are important for plants to break plant cell walls and translocate compounds to the apoplast. genes encoding cell-wall degrading enzymes widely exist in the genomes of endophytic bacteria. for example, genes encoding plant polymer-degrading cellulases, xylanases, cellobiohydrolases, endoglucanase, cellulose-binding proteins, pectinase and chitinase, are found in endophytic actinomycetes.58,59 enzymes produced from actinomycetes play an important role in food, fermentation, textile and paper industries.60 the use of actinomycetes endophytes in agriculture has immense potential to reduce the environmental impacts caused by chemical fertilizers, especially n fertilizers.61 common characteristics of actinomycetes endophytes include the ability to synthesize plant hormones such as indole-3acetic acid, solubilize phosphate, secrete siderophores, and confer plant tolerance to biotic and abiotic stresses.62–64 some actinomycetes endophytes carry genes necessary for biological nitrogen fixation (bnf), potentially enabling them to convert dinitrogen gas (n2) into usable forms of nitrogen such as ammonium and nitrate within the host plant.65 endophytic streptomyces as a dominant genus streptomyces accounts for the major dominant genus in many studies, which is the most commonly isolated endophytic actinomycetes. they were targeted for the first time for endophytic actinomycetes isolates. streptomyces isolates were about 66%, which is closely to a study for kaur48 (2016) who reported that 65.7% of all endophytic isolates belong to genus streptomyces. also, salam et al.43 (2017) found that 86.8% of all isolates from roots, stems and leaves of dracaena cochinchinensis were identified as streptomyces species. from tea plants (camellia sinensis), shan et al.41 (2018) detected that 51.1% of the isolates were identified as streptomyces. priya33 (2012) studied four medicinal plants, phyllanthus niruri, withania somnifera, catharanthus roseus and hemidesmus indicus and genus streptomyces was the dominant endophytic actinomycetes with 56.8% while it was recorded by 37.2% in 15 plant samples, obtained from taklamakan desert.66 rare and alternative genera of endophytic actinomycete even though there are many studies support the hypothesis that the genus streptomyces is the most abundant genera in endophytic actinomycetes, there are a wide range of other genera were recorded that are difficult to be isolated or rare.67 the criteria of isolation are important to find novel endophytes.68 furthermore, some studies showed that the diversity of uncultured endophytic actinomycetes is comparable to those of the cultured endophytic actinomycetes.56 gohain et al.13 (2015) isolated 76 endophytic actinomycetes belong to 16 genera which were verrucosispora, isoptericola, kytococcus, streptomyces, micromonospora, saccharopolyspora, kocuria, micrococcus, brevibacterium, amycolatopsis, timonella, leifsonia, microbacterium, mycobacterium and nocardia. another study by gos et al.45 (2017) isolated 10 isolates belong to 8 genera from stems and leaves of vochysia divergens, which were microbispora, actinomadura, microbacterium, 157j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 k. alzahrani et al. diversity and colonization of endophytic actinomycetes in some medicinal plants: review aeromicrobium, micrococcus, streptomyces, sphaerisporangium and williamsia. even though, salam et al.43 (2017) reported 2 major genera of endophytic actinomycetes streptomyces and nocardiopsis in addition to ten rare endophytic actinomycetes, arthrobacter, brevibacterium, kocuria, microbacterium, pseudonocardia, rhodococcus, nocardioides, nocardia, nocardiopsis, and tsukamurella. moreover, 13 different genera of endophytic actinomycetes were detected by shan et al.41 (2018). they included the two rare endophytic actinomycetes, piscicoccus and mobilicoccus. although janatiningrum et al.42 (2018) observed only 1 genera was uncultured endophytic actinomycete in addition to 6 genera, streptomyces, verrucosispora, rhodococcus, kineospora, intrasporangium and actinomadura, that were identified from roots, stems, leaves and fruits from ficus deltoidea. musa et al.39 (2020) obtained 126 endophytic actinomycete isolates belonging to 24 genera from thymus roseus. the genera agromyces, alloactinosynnema, labedella, microbacterium, mycobacterium, williamsia, blastococcus, dietzia, micromonospora, pseudarthrobacter and solirubrobacter belong to rare genera. similarly, out of 23 genera were obtained by wang et al.66 (2021), 6 genera were identified as rare endophytic actinomycetes, labedella, rathayibacter, leucobacter, frigoribacterium, aeromicrobium and kineococcus. qin et al.69 (2009) reported that medicinal plants of tropical rain forests were the richest source of novel endophytic actinomycetes. evidently, endophytic actinomycetes which investigated from medicinal plants and tropical rain forests have a considerable attention of the scientific research community (table 2). additionally, roots are a major organ that had been studied to its novel strains. isolation of endophytes actinomycetes understanding the colonization of endophytic actinomycetes can be gained by studying their characteristics, and explain their interactions and applications into plant tissues. the most commonly used isolation procedures start with surface sterilization of the plant organs. ordinary and specific modified cultures and table 2. novel endophytic actinomycete species isolated from plants plant species isolation region plant organ family novel strain references typha angustifolia l. yunnan province, southwest china root streptomycetaceae streptomyces typhae sp. nov. p1417t peng et al., 202170 peganum harmala l. xinjiang uygur autonomous region of china root pseudonocardiaceae actinokineospora pegani sp. nov. trm 65233t lei et al., 202071 anabasis elatior (c.a.mey.) schischk. xinjiang, north-west china root pseudonocardiaceae amycolatopsis anabasis sp. nov. egi 650086t wang et al., 202072 excoecaria agallocha linn guangxi zhuang autonomous region, pr china stem microbacteriaceae microbacterium excoecariae sp. nov. cbs5p-1t chen et al., 202073 mentha haplocalyx briq. guizhou, pr china stem nakamurellaceae nakamurella flava sp. nov. n5bh11t yan et al., 202074 phragmites australis taklamakan desert in xinjiang uygur autonomous region, china leaf nocardioidaceae aeromicrobium endophyticum sp. nov. 9w16y-2t li et al., 201975 podochilus microphyllus lindl. trat province, thailand. root pseudonocardiaceae actinomycetospora endophytica sp. nov. a-t 8314t sakdapetsiri et al., 201876 triticum aestivum l. langfang, hebei province, china root glycomycetaceae glycomyces rhizosphaerae sp. nov. neau-c8 li et al., 201877 anabasis aphylla l. xinjiang, northwest pr china root glycomycetaceae glycomyces anabasis sp. nov. egi 6500139t zhang et al., 201878 glycine max l. harbin, heilongjiang province, china root streptosporangiaceae nonomuraea glycinis sp. nov. neaubb2c19t li et al., 201779 capparis spinosa urumqi city, xinjiang, north-west china fruit streptomycetaceae streptomyces capparidis sp. nov. egi 6500195t wang et al., 201780 grosourdya appendiculata (blume) rchb.f. nakhorn ratchasima province, thailand. root micromonosporaceae verrucosispora endophytica sp. nov. a-t 7972t ngaemthao et al., 201781 glycine max (l.) merr. harbin, heilongjiang province, china root micromonosporaceae plantactinospora soyae sp. nov. neau-gxj3t guo et al., 201682 (continued) review 158 j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 diversity and colonization of endophytic actinomycetes in some medicinal plants: review review k. alzahrani et al. table 2. novel endophytic actinomycete species isolated from plants plant species isolation region plant organ family novel strain references bruguiera gymnorhiza zhanjiang mangrove forest national nature reserve in guangdong, china stem intrasporangiaceae phycicoccus endophyticus sp. nov. ip6sc6t liu et al., 201683 bruguiera sexangular dongzhaigang national nature reserve in hainan, china stem propionibacteriaceae microlunatus endophyticus sp. nov. s3af-1t tuo et al., 201684 prosopis laegivata san luis potosi, mexico root micrococcaceae kocuria arsenatis sp. nov. cm1e1t román-ponce et al., 201685 anabasis elatior (c.a.mey.) schischk. urumqi, xinjiang province, north-west china root microbacteriaceae frigoribacterium endophyticum sp. nov. egi 6500707t wang et al., 2015b86 veratrum nigrum l. wuchang, heilongjiangprovince, northern china root micromonosporaceae plantactinospora veratri sp. nov. neaufhs4t xing et al., 201587 salsola affinis c. a. mey urumqi, xinjiang province, north-west china root micrococcaceae arthrobacter endophyticus sp. nov. egi 6500322t wang et al., 2015a88 aloe barbadensis pune, maharashtra, india leaf micrococcaceae micrococcus aloeverae sp. nov. ae-6t prakash et al., 201489 artemisia argyi yesanpo located in laishui county, hebei province, china root glycomycetaceae glycomyces artemisiae sp. nov. ixs4t zhang et al., 201490 artemisia annua l. yunnan province, southwest china root streptomycetaceae streptomyces endophyticus sp. nov. yim 65594t li et al., 201391 isolation procedures are critical steps in working with endophytic actinomycetes. surface sterilization of the used part must be carried out. the sterilizing agent should kill any microbe on the plant surface without affecting the host tissue and the endophytic microorganisms. qin et al.69 (2009) described the methods of cleaning and sterilization of plant organs. initially, plant organs samples were air dried, washed to remove the surface soils and adherent epiphytes completely. after that, the samples were subjected to a five-step surface sterilization procedure with 5% sodium hypochlorite for 4–10 min., 2.5% sodium thiosulfate for 10 min., 75% ethanol for 5 minutes, sterile water and finally in 10% sodium bicarbonate for 10 min. after being thoroughly dried under sterile conditions, the surfacesterilized tissues were subjected to continuous drying at 100°c for 15 min. it is very important that sterility is guaranteed for all tools and steps of this procedure. optimination of the procedures for each plant tissue, since sensitivity varies with species, age and surface properties must be considered. the choice of the isolation culture media is crucial as it directly affects the number and type of endophytic actinomycetes that can be isolated from the plant organs. international streptomyces protocol media were also used for isolation as well as some other commonly used media include starch casein agar, starch casein nitrate agar, tryptic soya-yeast extract agar and glycerol aspargine agar.13,33,38,40,45 major identification methods culture characteristics should be determined on international streptomyces protocol (isp) media.80 the colors of aerial and substrate mycelia in addition to color of the soluble pigments are important for actinomycete identification. morphological characteristics are observed by light and scanning electron microscopes and growth at different temperatures (5–60°c) and different ph values are recorded.81 some physiological and biochemical characteristics like utilization of different carbon and nitrogen sources by the actinomycetes are important for identification.82 cell chemistry was determined by detecting diagnostic sugars in whole-cell hydrolysates, amino acids in the cell wall and cell total content of fatty acids and phospholipids. the g+c content of the dna, and 16s rrna gene were performed to confirm the identification of the actinomycetes.78 conclusion and future perspective actinomycetes endophytes are gained importance due to their ability to colonization mechanisms and distribution in all plant organs. many studies had investigated endophytic actinomycetes which associated with different plant species and their organs such as roots, stems, leaves, fruits and flowers. they are considered as useful microorganisms due to their roles as a defense barrier against some pathogenic microorganisms. many medicinal plant hosted many novel taxa, thus, there is a need to specific focusing on isolation of rare endophytic actinomycetes to determine their potent useful and benefit effects on plant growth and production as well as their applications in medical and industrial fields. finally, it is clear that endophytic actinomycetes play a key role in maintaining plant health by contributing to all biotic and a biotic stress tolerance. conflict of interest the authors declare that there is no conflict of interest involved in this study.  159j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 k. alzahrani et al. diversity and colonization of endophytic actinomycetes in some medicinal plants: review references 1. hogg, s. (2013). essential microbiology. (2nd ed.), wiley-blackwell. 2. anandan, r. (2016). an introduction to actinobacteria. in d. dharumadurai and g. manogaran (edt.) basics and biotechnological applications, intechopen, pp. 3 – 37. 3. jiang, y. (2016). isolation and cultivation methods of actinobacteria. in q. li, x. chen and c. jiang (eds), basics and biotechnological applications. (pp. 39 – 57), intechopen. 4. suman, a., yadav, a. and verma, p. (2016). endophytic microbes in crops: diversity and beneficial impact for sustainable agriculture. in: singh d., singh h., prabha r. (eds), microbial inoculants in sustainable agricultural productivity. springer, new delhi. 5. khare, e., mishra, j., & arora, n. k. (2018). multifaceted interactions between endophytes and plant: developments and prospects. frontiers in microbiology, 9, 2732. 6. rangjaroen, c., sungthong, r., rerkasem, b., teaumroong, n., noisangiam, r. and lumyong, s. (2017). untapped endophytic colonization and plant growth-promoting potential of the genus novosphingobium to optimize rice cultivation. microbes environ. vol. 32: 84–87. 7. castanheira, n., dourado, a., pais, i., semedo, j., scotti-campos, p., borges, n. and fareleira, p. (2017). colonization and beneficial effects on annual ryegrass by mixed inoculation with plant growth promoting bacteria. microbiol. res. vol. 198: 47–55. 8. compant, s., clément, c. and sessitsch, a. (2010). plant growth-promoting bacteria in the rhizo-and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. soil biol. biochem., vol. 42: 669–678. 9. nalini, m. and prakash, h. (2017). diversity and bioprospecting of actinomycete endophytes from the medicinal plants. letters in applied microbiology, vol. 64: 261-270. 10. kobayashi, d. and palumbo, j. (2000). bacterial endophytes and their effects on plants and uses in agriculture. c.w. bacon and j.f. white (eds), marcel dekker, new york. 11. mengistu, a. “endophytes: colonization, behaviour, and their role in defense mechanism”, international journal of microbiology, vol. 2020, article id 6927219, 8 pages. 12. strobel, g. and daisy, b. (2003). bioprospecting for microbial endophytes and their natural products. microbiol. mol. biol. rev. vol. 67: 491–502. 13. gohain, a., gogoi, a., debnath, r., yadav, a., singh, b., gupta, v., sharma, r. and saikia, r. (2015). antimicrobial biosynthetic potential and genetic diversity of endophytic actinomycetes associated with medicinal plants. fems. microbiol. lett. vol. 362(19): 1121-1129. 14. hardoim, p., van overbeek, l., berg, g., pirttilä, a., compant, s., campisano, a., döring, m. and sessitsch, a. (2015) the hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. microbiol. mol. biol. rev. vol. 79(3): 293-320. 15. hardoim, p., van-overbeek, l., and van-elsas, j. (2008). properties of bacterial endophytes and their proposed role in plant growth. trends microbiol. vol. 16: 463–471. 16. abreu-tarazi, m., navarrete, a., andreote, f., almeida, c., tsai, s. and almeida, m. (2010). endophytic bacteria in long-term in vitro cultivated axenic pineapple microplants revealed by pcr dgge. world j. microbiol. biotechnol. vol. 26: pp 555–560. 17. christina, a., christapher, v., and bhore, s. (2013). endophytic bacteria as a source of novel antibiotics: an overview. pharmacogn. rev. vol. 7: 11–16. 18. seipke, r., kaltenpoth, m. and hutchings, m. (2012) streptomyces as symbionts: an emerging and widespread theme. fems microbiol. rev. vol. 36: 862–876. 19. van der meij, a., worsley, s., hutchings, m. and van wezel, g. (2017). chemical ecology of antibiotic production by actinomycetes. fems microbiol. rev., vol. 41: 392–416. 20. kawasaki, a., donn, s., ryan, p. r., mathesius, u., devilla, r., jones, a., & watt, m. (2016). microbiome and exudates of the root and rhizosphere of brachypodium distachyon, a model for wheat. plos one, 11(10), e0164533. 21. patle, p., navnage, n. and ramteke, p. (2018). endophytes in plant system: roles in growth promotion, mechanism and their potentiality in achieving agriculture sustainability. international journal of chemical studies. vol. 6: 270–274. 22. kumar, a. and verma, j. (2018). “does plant-microbe interaction confer stress tolerance in plants: a review?” microbiological research. vol. 207: 41–52. 23. plett, j. and f. martin, m. (2018). know your enemy, embrace your friend: using omics to understand how plants respond differently to pathogenic and mutualistic microorganisms differently to pathogenic and mutualistic microorganisms. the plant journal, vol. 93(4): 729–746. 24. brader, g., compant, s. and vescioetal, k. (2017). ecology and genomic insights into plant-pathogenic and plant-nonpathogenic endophytes. annual review of phytopathology, vol. 55(1): 61–83. 25. liu, h., carvalhais, l. c., crawford, m., singh, e., dennis, p. g., pieterse, c., & schenk, p. m. (2017). inner plant values: diversity, colonization and benefits from endophytic bacteria. frontiers in microbiology, 8, 2552. 26. chow, y., rahman, s. and ting, a. (2017). understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction, and ergosterol assay. j. adv. res. vol. 8: 13–21. 27. ludwig, w., euzéby, j., schumann, p., busse, h., trujillo, m. e., kämpfer, p., et al. (2015). “road map of the phylum actinobacteria,” in bergey’s manual of systematics of archaea and bacteria, eds w. b. whitman, f. rainey, p. kämpfer, m. trujillo, j. chun, p. devos, et al. (hoboken, nj: wiley). 28. stepniewska, z. and kuzniar, a. (2013). endophytic microorganismspromising applications in bioremediation of greenhouse gases. appl. microbiol. biotechnol. vol. 97: 9589–9596. 29. matsumoto, a. and takahashi, y. (2017). endophytic actinomycetes: promising source of novel bioactive compounds. the journal of antibiotics. vol. 70: 514–519. 30. olivares, f., james, e., baldani, j. and döbereiner, j. (1997). infection of mottled stripe disease-susceptible and resistant sugar cane varieties by the endophytic diazotroph herbaspirillum. new phytol., vol. 135: 723–737. 31. vorholt, j. (2012). microbial life in the phyllosphere. nat. rev. microbiol., vol. 10: 828–840. 32. chen, m., zhang, l. and zhang, x. (2011). isolation and inoculation of endophytic actinomycetes in root nodules of elaeagnus angustifolia. modern applied science, vol. 5, 2: 264 267. 33. priya, m. (2012). endophytic actinomycetes from indian medicinal plants as antagonists to some phytopathogenic fungi. vol. 1, 259. 34. goudjal, y., toumatia, o., sabaou, n., barakate, m., mathieu, f. and zitouni, a. (2013) endophytic actinomycetes from spontaneous plants of algerian sahara: indole-3-acetic acid production and tomato plants growth promoting activity. world j. microbiol. biotechnol., vol. 29: 1821–1829. 35. rahayu, s., fitri, l. and ismail, y. (2019). endophytic actinobacteria isolated from ginger (zingiber officinale) and its potential as a pancreatic lipase inhibitor and its toxicity. biodiversitas vol. 20(5): 1312-1317. 36. chaudhry, h., nisar, n., mehmood, s., iqbal, m., nazir, a., & yasir, m. (2020). indian mustard brassica juncea efficiency for the accumulation, tolerance and translocation of zinc from metal contaminated soil. biocatalysis and agricultural biotechnology, 23, 101489. 37. vu, t., nguyen, q., dinh, t., quach, n., khieu, t., hoang, h., chu-ky, s., vu, t., chu, h., lee, j., kang, h., li, w. and phi, q. (2020). endophytic actinomycetes associated with cinnamomum cassia presl in hoa binh province, vietnam: distribution, antimicrobial activity and, genetic features. j. gen. appl. microbiol. vol. 66(1): 24-31. 38. phongsopitanuna, w., sripreechasakc, p., rueangsawang, k., panyawut, r., pittayakhajonwut, p. and tanasupawat, s. (2020). diversity and antimicrobial activity of culturable endophytic actinobacteria associated with acanthaceae plants. science asia, vol. 46: 288-296. 39. musa, z., ma, j., egamberdieva, d., mohamad, o., abaydulla, g., liu, y., li, w. and li, l. (2020). diversity and antimicrobial potential of cultivable endophytic actinobacteria associated with the medicinal plant thymus roseus. frontiers in microbiology. vol. 11, 191. 40. momin, m., passari, a.,singh, b. and tripathi, s. (2019). isolation and morphological identification of endophytic actinomycetes from medicinal plants of mizoram, northeast, india. medicinal plants of india: conservation and sustainable use, 343-351. 41. shan, w., zhou, y., liu, h. and xiaomin, y. (2018). endophytic actinomycetes from tea plants (camellia sinensis): isolation, abundance, antimicrobial, and plant-growth-promoting activities. biomed research international, vol. 2018, article id 1470305, 12 pages. 42. janatiningrum, i., solihin, d., andini, a. and lestari, y. (2018). comparative study on the diversity of endophytic actinobacteria communities from ficus deltoidea using metagenomic and culture-dependent approaches. biodiversitas. vol. 19(4): 1514-1520. 43. salam, p., pandey, v., shrestha, s. and anal, a. (2017). the need for the nexus approach, in water-energy-food nexus: principles and practices. washington, dc: john wiley and sons, inc.,1–10. 44. ranjan, r. and jadeja, v. (2017). isolation, characterization and chromatography-based purification of antibacterial compound isolated from rare endophytic actinomycetes micrococcus yunnanensis. j. pharm anal. vol. 7(5): 343-347. review 160 j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 diversity and colonization of endophytic actinomycetes in some medicinal plants: review review k. alzahrani et al. 45. gos, f., savi, d., shaaban, k., thorson, j., aluizio, r., possiede, y., rohr, j. and glienke, c. (2017). antibacterial activity of endophytic actinomycetes isolated from the medicinal plant vochysia divergens (pantanal, brazil). front. microbiol., vol. 8, 1642. 46. singh, s. and gaur, r. (2016). evaluation of antagonistic and plant growth promoting activities of chitinolytic endophytic actinomycetes associated with medicinal plants against sclerotium rolfsii in chickpea. j. appl. microbiol. vol. 121: 506–518. 47. saini, p., gangwar, m., kalia, a., singh, n. and narang, d. (2016). isolation of endophytic actinomycetes from syzygiumcumini and their antimicrobial activity against human pathogens. j. app. nat. sci. vol. 8: 416–422. 48. kaur, n. (2016). endophytic actinomycetes from azadirachta indica a. juss.: characterization and antimicrobial activity. international journal of advanced research, vol. 4(6): 676-684. 49. passari, a., chandra, p., zothanpuia, mishra, v., leo, v., gupta, v., kumar, b. and singh, b. (2016). detection of biosynthetic gene and phytohormone production by endophytic actinobacteria associated with solanum lycopersicum and their plant-growth-promoting effect. res. microbiol. vol. 167: 692–705. 50. phuakjaiphaeo, c., and kunasakdakul, k. (2015). isolation and screening for inhibitory activity on alternaria brassicicola of endophytic actinomycetes from centella asiatica (l.) urban. j. agri. technol. vol. 11: 903–912. 51. rao, h., rakshith, d. and satish, s. (2015). antimicrobial properties of endophytic actinomycetes isolated from combretum latifolium blume, a medicinal shrub from western ghats of india. frontiers in biology. vol. 10(6): 528–536. 52. gangwar, m., kaur, n., saini, p. and kalia, a. (2015). the diversity, plant growth promoting and antimicrobial activities of endophytic actinomycetes isolated from emblica officinalis gaertn. international journal of advance research. vol. 3: 1062-1071. 53. gangwar, m., dogra, s., gupta, u. and kharwar, r. (2014). diversity and biopotential of endophytic actinomycetes from three medicinal plants in india. african. j. microbiol. res. vol. 8: 184–191. 54. sadrati, n., daoud, h., zerroug, a., dahamna, s. and bouharati, s. (2013). screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated from wheat (triticum durum). journal of plant protection research. vol. 53(2): 128-136. 55. kaewkla, o. and franco, c. (2013) rational approaches to improving the isolation of endophytic actinobacteria from australian native trees. microb. ecol. vol. 65: 384–393. 56. singh, r. and dubey, a. (2018). diversity and applications of endophytic actinobacteria of plants in special and other ecological niches. frontiers in microbiology. vol. 9, 1767. 57. brader, g., compant, s., mitter, b., trognitz, f. and sessitsch, a. (2014). metabolic potential of endophytic bacteria. curr. opin. biotechnol. vol. 27: 30–37. 58. straub, d., rothballer, m., hartmann, a. and ludewig, u. (2013). the genome of the endophytic bacterium h. frisingense gsf30(t) identifies diverse strategies in the herbaspirillum genus to interact with plants. front. microbiol., vol. 4(168). 59. ashkan, m., aly, m., aldhebiani, a. and al-shehri, w. (2020). molecular identification and enzymatic activities of endophytic bacteria of ammannia baccifera grown at hot spring area, laith, saudi arabia. prensa med. argent, s2: 010. 60. sharma, m., dangi, p. and choudhary, m. (2014). actinomycetes: source, identification, and their applications. int. j. curr. microbiol. app. sci., vol. 3(2): 801-832. 61. kandel, s., joubert, p. and doty, s. (2017). “bacterial endophyte colonization and distribution within plants,” microorganisms, vol. 5: 1–26. 62. gaiero, j., mccall, c., thompson, k., day, n., best, a. and dunfield, k. (2013). inside the root microbiome: bacterial root endophytes and plant growth promotion. am. j. bot. vol. 100: 1738–1750. 63. lebeis, s. (2014). the potential for give and take in plant-microbiome relationships. front. plant sci. vol. 5, 287. 64. ashkan, m., aly, m. and aldhebiani, a. (2021). screening and characterization of endophytic bacteria from heliotropium pterocarpum growing at hot spring for their biological impacts. bbiosc. biotech. res. comm. vol. 14, 1. 65. santi, c., bogusz, d. and franche, c. (2013). biological nitrogen fixation in non-legume plants. ann. bot. vol. 111: 743–767. 66. wang, t., li, f., lu, q., wu, g., jiang, z., liu, s., habden, x., razumova, e., osterman, i., sergiev, p., dontsova, o., hu, x., you, x. and sun, c. (2021). studies on diversity, novelty, antimicrobial activity, and new antibiotics of cultivable endophytic actinobacteria isolated from psammophytes collected in taklamakan desert. journal of pharmaceutical analysis. vol. 11 (2): 241-250. 67. chaurasia, a., meena, b., tripathi, a., pandey, k., rai, a. and singh, b. (2018). actinomycetes: an unexplored microorganisms for plant growth promotion and biocontrol in vegetable crops. world j. microbiol. biotechnol., vol. 34, 9: 132. 68. tiwari, k. (2015). the future products: endophytic fungal metabolites. j. biodivers. biopros. dev., vol. 2(145). 69. qin, s., li, j., chen, h., zhao, g., zhu, w., jiang, c. (2009). isolation, diversity, and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain forests in xishuangbanna, china. appl. environ. microbiol. vol. 75: 6176-6186. 70. peng c, zhuang x, gao c, wang z, zhao j, huang sx, liu c, xiang w. (2021). streptomyces typhae sp. nov., a novel endophytic actinomycete with antifungal activity isolated the root of cattail (typha angustifolia l.). antonie van leeuwenhoek, 114(6):823-833. 71. lei, y., xia, z., luo, x. and zhang, l. (2020). actinokineospora pegani sp. nov., an endophytic actinomycete isolated from the surface-sterilized root of peganum harmala l. int. j. syst. evol. microbiol., vol. 70(7): 4358-4363. 72. wang, h., li, x., gao, r., xie, y., xiao, m., li, q. and li w. (2020). amycolatopsis anabasis sp. nov., a novel endophytic actinobacterium isolated from roots of anabasis elatior. int. j. syst. evol. microbiol. vol. 70(5): 3391-3398. 73. chen, m., li, f., yan, x. and tuo, l. (2020) microbacterium excoecariae sp. nov., a novel endophytic actinobacterium isolated from bark of excoecaria agallocha linn. int. j. syst. evol. microbiol. vol. 70(12): 6235-6239. 74. yan, x., chen, m., yang, c., an, m., li, h., shi, h. and tuo, l. (2020). nakamurella flava sp. nov., a novel endophytic actinobacterium isolated from mentha haplocalyx briq. int. j. syst. evol. microbiol., vol 70(2): 835-840. 75. li, f., liao, s., liu, s., jin, t. and sun, c. (2019). aeromicrobium endophyticum sp. nov., an endophytic actinobacterium isolated from reed (phragmites australis). j. microbiol. vol. 57(9): 725-731. 76. sakdapetsiri, c., ngaemthao, w., suriyachadkun, c., duangmal, k. and kitpreechavanich, v. (2018). actinomycetospora endophytica sp. nov., isolated from wild orchid (podochilus microphyllus lindl.) in thailand. int. j. syst. evol. microbiol. vol. 68(9): 3017-3021. 77. li, w., zhao, j., shi, l., wang, j., wang, h., wang, x. and xiang, w. (2018). glycomyces rhizosphaerae sp. nov., isolated from the root and rhizosphere soil of wheat (triticum aestivum l.). int. j. syst. evol. microbiol. vol. 68(1): 223-227. 78. zhang, y., wang, h., alkhalifah, d., xiao, m., zhou, x., liu, y., hozzein, w. and li, w. (2018). glycomyces anabasis sp. nov., a novel endophytic actinobacterium isolated from roots of anabasis aphylla l. int. j. syst. evol. microbiol. vol. 68(4): 1285-1290. 79. li, z., song, w., zhao, j., zhuang, x., zhao, y., wang, x. and xiang, w. (2017). nonomuraea glycinis sp. nov., a novel actinomycete isolated from the root of black soya bean [glycine max (l.) merr]. int. j. syst. evol. microbiol. vol. 67(12): 5026-5031. 80. wang, h., li, q., xiao, m., zhang, y., zhou, x., narsing, r., duan, y. and li, w. (2017). streptomyces capparidis sp. nov., a novel endophytic actinobacterium isolated from fruits of capparis spinosa l. int. j. syst. evol. microbiol. vol. 67(1): 133-137. 81. ngaemthao, w., pujchakarn, t., chunhametha, s. and suriyachadkun, c. (2017). verrucosispora endophytica sp. nov., isolated from the root of wild orchid (grosourdya appendiculata (blume) rchb. f.). int. j. syst. evol. microbiol., 67(12): 5114-5119. 82. guo, x., guan, x., liu, c., jia, f., li, j., li, j., jin, p., li, w., wang, x. and xiang, w. (2016). plantactinosporasoyae sp. nov., an endophytic actinomycete isolated from soybean root [glycine max (l.) merr]. int. j. syst. evol. microbiol. vol. 66(7): 2578-2584. 83. liu, s., xu, m., tuo, l., li, x., hu, l., chen, l., li, r. and sun, c. (2016). phycicoccus endophyticus sp. nov., an endophytic actinobacterium isolated from bruguiera gymnorhiza. int. j. syst. evol. microbiol. vol. 66(3): 1105-1111. 84. tuo, l., li, j., liu, s., liu, y., hu, l., chen, l., jiang, m. and sun, c. (2016) microlunatus endophyticus sp. nov., an endophytic actinobacterium isolated from bark of bruguiera sexangula. int. j. syst. evol. microbiol. vol. 66(1): 481-486. 85. román-ponce, b., wang, d., vásquez-murrieta, s., chen, w., santos, p., sui, x. and wang, e. (2016). kocuria arsenatis sp. nov., an arsenic-resistant endophytic actinobacterium associated with prosopis laegivata grown on high-arsenic-polluted mine tailing. int. j. syst. evol. microbiol. vol. 66(2): 1027-1033. 86. wang, h., zhang, y., chen, j., guo, j., li, l., hozzein, w., zhang, y., wadaan, m. and li, w. (2015b). frigoribacterium endophyticum sp. nov., an endophytic 161j contemp med sci | vol. 8, no. 3, may-june 2022: 153–161 k. alzahrani et al. diversity and colonization of endophytic actinomycetes in some medicinal plants: review actinobacterium isolated from the root of anabasis elatior (c. a. mey.) schischk. int. j. syst. evol. microbiol. vol. 65(4): 1207-1212. 87. xing, h., liu, c., zhang, y., zhao, j., li, c., liu, h., li, l., wang, x. and xiang, w. (2015). plantactinospora veratri sp. nov., an actinomycete isolated from black false hellebore root (veratrum nigrum l.). int. j. syst. evol. microbiol. vol. 65(6): 1799-1804. 88. wang, h., li, l., zhang, y., hozzein, w., zhou, x., liu, w., duan, y. and li, w. (2015a). arthrobacter endophyticus sp. nov., an endophytic actinobacterium isolated from root of salsola affinis c. a. mey. int. j. syst. evol. microbiol. vol. 65(7): 2154-2160. 89. prakash, o., nimonkar, y., munot, h., sharma, a., vemuluri, v., chavadar, m. and shouche, y. (2014). description of micrococcus aloeverae sp. nov., an endophytic actinobacterium isolated from aloe vera. int. j. syst. evol. microbiol. vol. 64(10): 3427-3433. 90. zhang, x., ren, k., du, j., liu, h. and zhang, l. (2014). glycomyces artemisiae sp. nov., an endophytic actinomycete isolated from the roots of artemisia argyi. int. j. syst. evol. microbiol. vol. 64(10): 3492-3495. 91. li, j., zhao, g., zhu, w., huang, h., xu, l., zhang, s. and li, w. (2013). streptomyces endophyticus sp. nov., an endophytic actinomycete isolated from artemisia annua l. int. j. syst. evol. microbiol. vol. 63(1): 224-229. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i3.1222 review 202 j contemp med sci | vol. 4, no. 4, autumn 2018: 202–206 research analysis of condylar volume in relation to craniofacial morphology using cone beam computed tomography maryam mostafavi,a arman saeedi vahdat,b laleh javadian,c and aisan ghaznavia* adepartment of oral and maxillofacial radiology, dentistry faculty, urmia university of medical sciences, urmia, iran. b department of oral and maxillofacial radiology, dentistry faculty, tabriz university of medical sciences, tabriz, iran. cdepartment of oral and maxillofacial radiology, general dentist,dentistry faculty, urmia university of medical sciences, urmia, iran. *correspondence to aisan ghaznavi (email: aisanghaznavi@yahoo.com). (submitted: 14 september 2018 – revised version received: 28 september 2018 – accepted: 17 november 2018 – published online: 26 december 2018) objectives this study aimed at comparing the size and shape of mandibular condyle in mature adult population with different skeletal patterns. methods a total of 198 patients within the age range of 15–64 years, including 68 males and 130 females, were allocated into three groups based on the a point–nasion–b point angle anb angle: skeletal classes i (n = 65), ii (n = 69), and iii (n = 64). the cone beam computed tomography (cbct) was used to evaluate right and left temporomandibular joint (tmj) in each patient. tmj evaluation was comprised of size of condyle, area of condyle, and morphology index. the mimics software was used to calculate the size and area of the condyle. the size, area, and morphology index were compared between the study groups using parametric tests. results based on the results of paired t-test, there was a significant difference between the sizes of right and left condyles, in favor of the right condyle (p = 0.02). in addition, the mean size of the condyle in class iii subjects was higher than those of classes i and ii; however, the difference between the classes i and ii was insignificant. the size and area of condyle were higher in males compared with females. conclusion based on the results of this study, there might be a correlation between the facial skeletal morphology and area of condyle. keywords cone beam computed tomography, mandibular condyle, malocclusion, morphology introduction the temporomandibular joint (tmj) is one of the complex joints in the human body, which plays an essential role in many important functions, such as chewing, swallowing, and speaking. the coordination and balance of tmj are extremely important for a normal rodent. condyle, as an integral part of tmj, has the capacity of remodeling in response to mandibular rotation and displacement in different directions. the morphology and volume of condyle represent the level of adaptability to functional stimuli and influence the long-term stability of orthodontic and orthognathic treatments. moreover, condyle is important in determining the mandibular size, and condylar morphology can be associated with the position of maxillary and mandibular bases.1 previous studies have examined the relationship between tmj morphology and malocclusion using two-dimensional (2d) images.2,3 considering the limitations of these images, it is difficult to conduct such analyses and thoroughly examine all dimensions. however, with the advent of three-dimesional (3d) imaging modalities, such as computed tomography (ct) and cone beam computed tomography (cbct), evaluation of condylar morphology is now possible. krisjane et al.4,5 morphologically evaluated tmj in patients with classes ii and iii malocclusions using ct images. the results showed that condylar size was smaller in class ii patients, compared with class iii patients. considering the high efficacy of cbct in presenting 3d images without superimposition, magnification, or distortion (with limited exposure dose), researchers started to use this imaging modality for the analysis of tmj only 2 years after its introduction to jaw and facial imaging.6 research has been conducted on the relationship between skeletal morphology and condylar volume and morphology using cbct. clinicians can assess abnormal condyle morphology and potential tmj disorders with respect to the normal range of condyle morphological parameters.7 in a previous study, saccucci et al.8 reported a correlation between skeletal morphology and condylar volume and surface, based on the cbct images in a caucasian population and suggested further studies on different populations considering the racial and regional differences. since no study has been carried out in this area in the country where authors live, the aim of this study was to evaluate the relationship between the morphological index and condylar volume and surface in patients with classes i–iii orthodontics in the northwest of that country. materials and methods in this study, a total of 198 3d cbct images, acquired for the evaluation of orthodontic problems, were examined in patients, aged between 15 and 64 years. since clinical examinations had been already performed on these patients, those with clinical signs and symptoms of tmj disorders were not recruited in the study. all images were acquired using newtom vgi (qr, verona, italy) with a cone-shaped x-ray beam, a 360° rotation, and a flat-panel detector (pixel size, 0.5 mm). the imaging features included intermittent exposure with a maximum voltage of 120 kvp, current of 3–8 ma, and scan time of 18 s. the images were evaluated by two observers (a student of dentistry and a radiologist) and observed on a 17-inch lcd vaio screen (f-series, japan) at a resolution of 1024 × 1208 in a room with medium lighting. the images were recorded in dicom format using nnt viewer 2.21, and then entered into planmeca in order to determine the anb angle. the lateral cephalometric view was derived from cbct images and issn 2413-0516 m. mostafavi et al. 203j contemp med sci | vol. 4, no. 4, autumn 2018: 202–206 research cbct in condylar volume analysis categorized into classes i–iii, based on the steiner’s analysis. the patients were classified with respect to the anb angle: class i: 2°–4°; class ii: >4°; and class iii: <2° (fig. 1). the images were entered into mimics 10.01 for volumetric measurements after determining the anb angles. each condyle was reconstructed three-dimensionally using the mentioned software. first, a mask was constructed from one side of the patient, which was used to parallelize the axial planes with the frankfurt plane (fig. 2). using axial sections, the upper limit of the condyle was defined where the first radiopaque area was viewed in the synovial area, while the lower limit was determined using the plane crossing the sigmoid notch. another mask was constructed by separating the condyle and adjacent tissues (fig. 3). in all coronal sections with thickness of 3 mm, areas unrelated to condyle (e.g. cortical bone, cartilage, etc.) were removed. in the axial sections, the limits of the constructed mask were determined. finally, 3d reconstruction was done, and volumetric measurements (in mm2) were performed using the software (fig. 4). to evaluate intraoperator errors, the cbct images of 10% of subjects were reevaluated (with at least a 1-week interval). moreover, for determining interoperator errors, 10% of cbct images were reevaluated by another observer. in this study, statistical analysis was performed using spss version 16 (chicago, spss inc). parametric tests, including one-way anova: analysis of variance (is a statistical technique for testing if 3(+) population means are all equal). tukey’s multiple comparison test, independent sample t-test, and paired t-test, were used to determine differences in condylar volume among classes i–iii patients and to evaluate gender differences. results a total of 198 patients were recruited in this study, including 130 (65.7%) females and 68 (34.3%) males. the mean age of the patients was 33.01 ± 0.66 years. based on these findings, 65 (32.8%) patients had class i malocclusions, 69 (34.8%) patients had class ii malocclusions, and 64 (32.3%) patients had class iii malocclusions. kolmogorov–smirnov test was used to analyze the normal distribution of data. considering the significant levels of variables (>0.05), the zero assumption that data were not normally distributed was rejected; it was concluded that all variables have a normal distribution. therefore, parametric tests, including one-way anova, tukey’s multiple comparison test, independent sample t-test, and paired t-test, were applied. according to one-way anova, condylar volume and surface were significantly different among classes i–iii patients in both right and left sides (p = 0.00). moreover, the results of tukey’s multiple comparison test showed that condylar volume was significantly different on the left and right sides between classes i and iii patients (p = 0.00), as well as classes ii and iii (p= 0.00) patients. the results of one-way anova showed that the condylar morphological index was significantly different on the left (p = 0.03) and right (p = 0.04) sides in classes i–iii patients. fig. 1 the lateral cephalometric view in a cbct image. fig. 2 the mask from one side of the patient’s face. fig. 3 the mask from the condyle and adjacent tissues. fig. 4 the 3d image of the condyle. 204 j contemp med sci | vol. 4, no. 4, autumn 2018: 202–206 cbct in condylar volume analysis research m. mostafavi et al. moreover, the results of tukey’s multiple comparison test showed that the condylar morphological index was significantly different between the left (p = 0.03) and right (p = 0.02) sides in classes i and iii patients, as well as classes ii and iii patients (left and right sides, p = 0.01) (table 1). the mean condylar volume and surface, as well as the morphological index, were higher in class iii patients, compared with classes i and ii patients (tables 2 and 3). nevertheless, the difference was not significant between classes i and ii patients. according to the intraclass correlation, or the interclass correlation coefficient (icc), index, the interobserver agreement, as well as the agreement between the first and second observations, was very good (>93%). based on the independent sample t-test, condylar volume and surface were greater in men, compared with women (p = 0.00); however, the morphological index was not significantly different. the results of paired t-test showed that the left and right condylar volumes were significantly different, with a higher right condylar volume (p = 0.02); however, the condylar surface was not significantly different. discussion radiological examination of tmj is quite challenging, since thorough evaluation is not possible with conventional radiography, and accurate analysis requires advanced techniques. with the advent of cbct technique, complete evaluation of the bone structure of tmj is now possible. according to the review of literature, only one study has examined the relationship between condylar volume and surface and skeletal classes i–iii, using cbct images in a caucasian population.8 therefore, the aim of this study was to evaluate condylar volume and surface, as well as the morphological index in classes i–iii patients in the northwest of the country where the authors live. in this study, the mean age of the subjects was 15–64 years due to the limited number of samples under 30 years. since the condyle undergoes remodeling and degenerative changes with age, affecting the condylar surface and volume, only samples with a normal condylar bone structure were recruited in the study. a total of 198 3d cbct images, acquired for orthodontic purposes, were collected from patients. of all patients, 65 patients with the mean age of 32.5 years were classified as class i, 69 patients with the mean age of 34.8 years were classified as class ii, and 64 patients with the mean age of 31.5 years were classified as class iii. the left and right condylar volume and surface were significantly higher in class iii patients, compared with classes i and ii patients. furthermore, class iii patients showed a significantly different morphological index, compared with classes i and ii patients. however, the condylar morphological index, surface, and volume were not significant between classes i and ii patients. saccucci et al. conducted a study to evaluate condylar volume and surface in classes i–iii patients, using cbct images from 200 subjects, aged 15–30 years. according to the results, condylar volume and surface in skeletal class iii were significantly higher than skeletal classes i and ii; however, these parameters were significantly lower in skeletal class ii, compared with classes i and iii. moreover, the morphological index was significantly different between the skeletal classes on both sides.8 these results are similar to this study for the skeletal class iii and different for classes i and ii. considering table 1. the post-hoc evaluation (tukey’s test) volume (l) volume (r) surface (l) surface (r) mi (l) mi (r) class 1 p = 0.73 p = 0.65 p = 0.65 p = 0.94 p = 0.80 p = 0.16 class 2 class 1 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.03 p = 0.02 class 3 class 2 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.01 p = 0.01 class 3 table 2. the mean, standard deviation, range, minimum, and maximum values of the morphological index, condylar surface, and condylar volume in men and women n mean std. deviation range minimum maximum female volume (left) 130 1482.12 301.87 1374.13 929.87 2304.00 surface (left) 130 883.41 125.95 753.95 613.12 1367.07 mi (left) 130 0.6 0.06 0.41 0.5 0.91 volume (right) 130 1504.66 286.39 1387.07 922.97 2310.04 surface (right) 130 883.17 110.48 596.4 607.7 1204.1 mi (right) 130 0.59 0.05 0.32 0.48 0.80 male volume (left) 68 1859.94 498.81 2743.45 913.83 3657.28 surface (left) 68 1068.83 210.05 1190.13 644.45 1834.58 mi (left) 68 0.58 0.07 0.5 0.46 0.96 volume (right) 68 1874.18 491.43 2732.15 867.97 3600.12 surface (right) 68 1066.10 217.58 1157.5 643.1 1800.6 mi (right) 68 0.57 0.05 0.031 0.46 0.77 for volume/surface (independent sample t-test), p = 0; for right im (independent sample t-test), p = 0.59; for left im (independent sample t-test), p = 0.22. m. mostafavi et al. 205j contemp med sci | vol. 4, no. 4, autumn 2018: 202–206 research cbct in condylar volume analysis the similar sample size and methods, the differences might be due to racial differences. in this study, the mean condylar volume in classes i–iii patients were respectively 1545.48, 1515.44, and 1859.62 mm² on the right side and 1524.45, 1500.95, and 1835.58 mm² on the left side. however, in the study by saccucci et al., the condylar volume in classes i–iii patients were respectively 2693.09, 2350.64, and 2672.80 mm² on the right side and 2675.09, 2352.02, and 2792.78 mm² on the left side. despite the use of similar methods, the difference in the mean condylar volume and surface was significant between these populations. in a previous study, al-koshab et al. aimed to assess condylar morphology and glenoid fossa in a southeast asian population. the mean condylar volume, regardless of the skeletal class, was 1460.69 and 1450.89 on the right and left sides, respectively. the overall difference in condylar volume in these studies may indicate the effects of regional and racial factors, which can be evaluated in various studies on different populations.9 moreover, katsavrias et al. reviewed 189 patients using submentovertex radiography. the results showed that the main components of condylar shape were different among skeletal classes, and the condylar head was longer in class iii.10 in another study performed in 2016, alhammadi et al. reviewed the 3d cbct images of tmj from 60 patients (aged 18–25 years) with skeletal classes i–iii. overall, 20 patients were assigned to class i, 20 patients were assigned to class ii, and 20 patients were assigned to class iii. according to the results, the lowest condylar width and the highest condylar height were reported in class ii patients.11 according to the results of this study, condylar volume and surface were greater in class iii patients, compared with the classes i and ii. this finding may be attributed to two major factors. first, according to the moss’s functional matrix theory, growth of each skeletal unit in the maxillofacial surface is defined based on the functional requirements of the corresponding tissue. since class iii patients show larger mandibular volume, the functional requirements of the mandibular unit, and consequently condylar volume, are higher in these patients.12 a study by enomoto et al.13 on mice showed that type of diet is effective in condylar volume. however, this finding cannot be fully accepted, as the biting force is not applied continuously, and the parafunctional habits are probably more effective than diet in condylar volume in classes i and ii malocclusions. second, growth hormones play a very important role in the linear and angular size of craniofacial structures. abundance table 3. the mean ± sd of morphological index, condylar surface, and condylar volume in men and women in the left and right condyles left right mean std. deviation mean std. deviation volume 1609.99 419.05 1629.76 407.15 surface 946.18 181.63 945.09 177.33 mi 0.6 0.06 0.59 0.059 for volume (paired t-test), p = 0.02; for surface (paired t-test), p = 0.83. or shortage of growth hormones can cause disproportionate growth in the base of skull and jaws and decrease the anterior face height, compared with the posterior face height. considering the effects of growth hormone receptors on the growth of condyle, it can be concluded that higher condylar volume in class iii patients can be due to its excessive response to growth hormones. moreover, in studies conducted in east asia on patients with mandibular prognathism, there were mutations on some loci of chromosomes 1, 6, and 19. according to these findings, despite the irrefutable effects of environment, genetics play an undeniable role in mandibular growth and morphology, i.e., mandibular size, length, and height. in this study, condylar volume and surface on both the sides were significantly higher in men than women. however, the morphological index was not significantly different on either sides between males and females. in 2010, tecco et al. conducted a study on a caucasian population, using cbct images and mimics software. the results regarding the difference in condylar morphological index and volume between men and women are consistent with the findings of the present study, which showed significantly higher values in men, compared with women. however, the difference in terms of condylar surface was not significant among men and women.14 in this regard, al-koshab et al.9 showed that condylar volume, width, height, and shared space were significantly higher in men than women from east asia. according to a study by saccucci et al.,8 the condylar surface and volume were larger in men, compared with women. moreover, gomes et al.15 showed that condylar dimensions were larger in men; the results of these studies are consistent with the present study. in addition, in our study, the right and left condylar surfaces were not significantly different. however, there was a significant difference between the right and left condylar volumes (the left condylar volume was larger). on the other hand, the morphological index was not significantly different between the right and left sides. krisjane et al.4 showed a significant difference in condylar height between the right and left condyles in patients with condylar asymmetry. consistent with the present study, tecco et al.14 showed that the right condylar volume was larger than the left condylar volume, which can be due to the similar sample size and methods of these studies. on the other hand, in the study by saccucci et al.,8 the difference between the right and left condylar volume was not significant, which is contrary to the present results, as well as studies by krisjane and tecco. conclusion the results of this study showed differences in condylar volume and surface among classes i–iii patients. therefore, by performing similar studies on different populations, these findings can be applicable in the assessment of orthodontic problems. conflict of interest none. n 206 j contemp med sci | vol. 4, no. 4, autumn 2018: 202–206 cbct in condylar volume analysis research m. mostafavi et al. references 1. saccucci m, polimeni a, festa f, tecco s. do skeletal cephalometric characteristics correlate with condylar volume, surface and shape? a 3d analysis. head face med. 2012;8:15. 2. gianelly aa, petras jc, boffa j. condylar position and class ii deep-bite, nooverjet malocclusions. am j orthod dentofacial orthop. 1989;96:428–432. 3. widman dj. functional and morphologic considerations of the articular eminence. angle orthod. 1988;58:221–236. 4. krisjane z, urtane i, krumina g, bieza a, zepa k, rogovska i. condylar and mandibular morphological criteria in the 2d and 3d msct imaging for patients with class ii division 1 subdivision malocclusion. stomatologija. 2007;9:67–71. 5. krisjane z, urtane i, krumina g, zepa k. three-dimensional evaluation of tmj parameters in class ii and class iii patients. stomatologija. 2009;11:32–36. 6. larheim ta, abrahamsson ak, kristensen m, arvidsson lz. temporomandibular joint diagnostics using cbct. dentomaxillofac radiol. 2014;44:20140235. 7. song y, zhang x, gao y, hou f, yu y. the condylar morphology in adult females of skeletal class ii division 1 malocclusion with various vertical skeletal features: a study by cone beam computed tomography. int j clin exp med. 2016;9:8304–8311. 8. saccucci m, d’attilio m, rodolfino d, festa f, polimeni a, tecco s. condylar volume and condylar surface in class i, class ii and class iii young adult subjects. head face med. 2012;8:34. 9. al-koshab m, nambiar p, john j. assessment of condyle and glenoid fossa morphology using cbct in south-east asians. plos one. 2015;10:e0121682. 10. katsavrias eg, halazonetis dj. condyle and fossa shape in class ii and class iii skeletal patterns: a morphometric tomographic study. am j orthod dentofacial orthop. 2005;128:337–346. doi:10.1016/j. ajodo.2004.05.024 11. alhammadi ms, fayed ms, labib a. three-dimensional assessment of temporomandibular joints in skeletal class i, class ii, and class iii malocclusions: cone beam computed tomography analysis. j world fed orthod. 2016;5:80–86. 12. moss ml, rankow rm. the role of the functional matrix in mandibular growth. angle orthod. 1968;38:95–103. 13. enomoto a, watahiki j, yamaguchi t, irie t, tachikawa t, maki k. effects of mastication on mandibular growth evaluated by microcomputed tomography. eur j orthod. 2010;32:66–70. 14. tecco s, saccucci m, nucera r, polimeni a, pagnoni m, cordasco g, et al. condylar volume and surface in caucasian young adult subjects. bmc med imaging. 2010;10:28. 15. gomes af, nejaim y, brasil dm, groppo fc, ferreira caria ph, haiter neto f. assessment of volume and height of the coronoid process in patients with different facial types and skeletal classes: a cone-beam computed tomography study. j oral maxillofac surg. 2015;73:1395.e1–e5. doi:10.1016/j.joms.2015.02.020 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 323j contemp med sci | vol. 7, no. 6, november-december 2021: 323–329 review explaining the concept of learning based on reflection in clinical education: scoping review soleiman ahmady1,2, zahra ayazi3*, masomeh kalantarion3, nasrin khajeali4 1head of department of medical education, virtual school of medical education and management, shahid beheshti university of medical sciences, tehran, iran; 2research affiliated faculty at department of lime, karolinska institute, sweden. 3department of medical education, virtual school of medical education and management, shahid beheshti university of medical sciences, tehran, iran. 4department of medical education, jundishapur university of medical sciences, ahvaz, iran. *correspondence to: zahra ayazi (e-mail: ayazi_z56@yahoo.com) abstract objective: this study was conducted with the aim of theoretically explaining the concept of reflection in medical science education in texts and identifying the dimensions and characteristics of education based on reflection and their application in clinical nursing education and existing models in 2021. methods: this scoping review was conducted in five phases following the method developed by denyer and tranfield. the scope of the review was limited to reflection and reflective in nursing education. using the keywords in combination and separately, they were searched in databases of eric/ www.iranmedex.com/ www.magiran.com/ www.irandoc.ac.ir/ www.sid.ir/ www.medlib.ir/ all sciences/ web of science/search engines google/google scholar/scopus/ovid/eric/wiley/pubmed and science direct from1970s to 2021. results: based on inclusion and exclusion criteria, 24 articles and documents were selected. during the theoretical explanation of the concepts and components of reflection in clinical nursing education, the model is abstracted. in this model: the clinical education triangle has 3 main components in its three angles: patient, teacher, or tutor, and student. it is necessary that the sides of this triangle including teaching-learning, teaching-caring, and caring-learning to be formed in the best way and to serve the student and the patient. conclusion: the results of this study emphasize the practical and constructive nature of knowledge in learning based on reflection and its special position so that by eliminating the gap between theory and practice in nursing education, one can see the excellence of the operational part of education and promotion of the level of health of the society by providing an appropriate performance of the nursing staff. keywords: reflection, clinical education, scoping review, nursing, theoretical model issn 2413-0516 introduction “reflection in the field of learning” is a general term for mental and emotional activities in which people engage to discover their experiences which leads to a new understanding. learning based on reflection is the process of exploring and discovering the subject matter that has been considered through experience, where the individual generates and clarifies the meaning by himself, and the conceptual view of the individual is modified. learning based on reflection requires learners to review their previous interpretations of knowledge and, as a group, create a new common sense of position and commitment to action based on this new learning.1 therefore, reflection is addressed with terms like reflective training, reflective teaching, reflective learners, reflective skills, or reflection. approaches used to promote learning based on reflection include action research, case write-ups, microteaching, and supervised practicals.2,3 nursing is a professional knowledge that is dependent on practice along with science. in a way that in many studies, it is known as practice-oriented professional. in the field of testing science and nursing, art is in the domain of clinical, so in addition to learning knowledge, a nursing student needs to learn skills. clinical education is undoubtedly the most important and fundamental part of the nursing education program.7,8 it is close to half of the undergraduate education and therefore clinical education is referred to as the heart of nursing education.9 the value of clinical education is accepted by all; and all experts in the field of nursing education believe that the development of scientific and professional nursing education is impossible without improving clinical education. in this regard, many nursing schools consider the evaluation of clinical education as the main pillar of educational planning.10 it is important for “both the nurse and the patient” to perform along with a reflective approach in nursing practices. in holistic caregiving, nurses in the task of taking care of mankind, should always consider mankind with a holistic nature and provide care considering all his dimensions; and in this area, perform their caregiving actions with the least mistakes. therefore, it is necessary to reflect on the desired action while performing specific measures or to reflect after performing them, so that the distance between theory and practice is reduced and the necessary points are learned from specific experiences.11 the importance of a reflective approach for a nurse is because one can avoid making mistakes or substitute the right things. so it can help to increase the development of his abilities; in a way that when performing reflection, nurses become able to provide their care with better understanding and more competent knowledge and have motivation in changing and improving the quality of holistic care.11 since clinical education and the need to reduce and eliminate the gap between theoretical and clinical education in nursing education have a special position, so it seems that the use of models such as reflection, is effective in this regard. therefore, this study was conducted with the aim of theoretically 4,5,6 (submitted: 02 september 2021 – revised version received: 21 september 2021 – accepted: 11 october 2021 – published online: 26 december 2021) 324 j contemp med sci | vol. 7, no. 6, november-december 2021: 323–329 explaining the concept of learning based on reflection in clinical education: scoping review review s. ahmady et al. explaining the concept of reflection in medical science education in texts and identifying the dimensions and characteristics of education based on reflection and their application in clinical nursing education and existing models in 2021. materials and methods the concept of reflection, despite its relatively broad application, does not have clear definitions and there is some degree of ambiguity and lack of transparency around its characteristics. therefore, in order to distinguish this concept in medical education from similar concepts and also because of lack of the provision of a specific model of reflection in the clinical education of nursing in the world and iran, we decided to use the method of “arksey & o’malley scoping review”,12 with a general overview, so that while clarifying the concept of reflection and examining the existing models, a comprehensive model of their integration is provided. scoping review is used to quickly provide a concept map, main sources, and key evidence of research background and can be used as an independent background review project in cases that are both a broad field of study and are not been comprehensively studied in the past.13 the research methodology is defined by denyer and tranfield based on five steps of structuring the research, determining the scope of the study, selecting and reviewing articles, data analysis, and conclusions.14 in the first step, research questions were determined as: “what are the concepts and components of reflection in medical education, especially clinical education?” then, in the second step, to answer the questions of the previous step, the following different electronic databases were used systematically: eric/ www.iranmedex.com/ www.magiran.com/ www. irandoc.ac.ir/ www.sid.ir/ www.medlib.ir/ all sciences/ web of science/ search engines google/ google scholar/ scopus/ ovid/ eric/ wiley/ pubmed and science direct. given that there was no other equivalent for the word reflection in the mesh dictionary, the following keywords were used to search for a wide range of articles: “reflection, reflective learning, reflective training, reflective teaching, reflective thinking, reflective practice” or “levels of reflectivity, reflective learning model, reflective practice in nursing.” the above keywords were searched in english and farsi during the 1970s (the introduction of the term reflection and reflective in education by burton) till 2021 in the mentioned databases. in order to select the main studies in the third step: inclusion and exclusion criteria were modified and adjusted based on the research question and scoping review objectives during the searches and study of documents during an interactive process. articles that are published in english or farsi and in a scientific-research journal (approved) and the term reflection or performance-based on reflection is in the title or abstract and their full text is available were included in the study. criteria for excluding sources from the study were: lack of access to the full text of the article, scientific-promotional articles, letter to the editor, lecture, critique report, or articles not related to the field of medical education. in the initial search, several articles were found. in the first stage, after reviewing the titles of the articles (1414 items), a large number of sources were removed due to lack of thematic relevance (1308 items), and 110 articles, books, and documents were selected for further review. then the summary of the articles was studied and those relevant to the concept of reflection in clinical education particularly nursing were chosen for full text studying. at this stage, 52 articles and documents of which full texts were available were read. finally, based on inclusion and exclusion criteria, 24 articles and documents which had stated complete details regarding the desired concept, were selected as a base for scoping review and chosen for inclusion in the analysis (figure 1). in the fourth step, after the final sources were selected, the definitions and frameworks provided in these sources were reviewed and the details of each of these definitions, frameworks, and models were embedded in the excel software for better matching and comparison in the next stage.12 the purpose of collecting studies in the fifth step was to compile and summarize the results and provide a report. analysis and combining of results is part of any review study, but the depth and type of analysis in this method was different from the systematic review because, in this type of study, the results of a scoping study are usually displayed in tabular form with some narrative interpretations by the researcher15 wherein this study, by matching different findings in relevant researches and relevant, a summary is given in tables 1 and 2. ethical considerations the data were collected after obtaining the approval of the research proposal from the ethics committee of the shahid beheshti university of medical sciences (ir.sbmu.sme. rec.1400.014). results definitions and various components of reflection in the researched models and the consequences obtained in the models are summarized (table 1); then the articles related to the relevant topics in the world are presented (table 2); therefore, ultimately we are able to present a conceptual model of fig. 1 selection process of studies to include in the scoping review. 325j contemp med sci | vol. 7, no. 6, november-december 2021: 323–329 s. ahmady et al. review explaining the concept of learning based on reflection in clinical education: scoping review ta bl e 1. d efi ni tio ns a nd co m po ne nt s o f a pp lie d re fle ct io n m od el s i n cl in ic al e du ca tio n row m od el /d es ig ne r/ ye ar m od el co m po ne nt s describing the experience feelings/mental preparation evaluation/ combination analysis conclusion action plan self-awareness/ self-assessment detection/ change negotiation discovering new options obtaining knowledge and skills by reflection testing new behavioral method substitution of the new method self-confidence enhancing professional behavior inter professional role 1. g ib bs ’ m o d el o f re fl ec ti o n (g ib bs /s r1 98 8) 16 ,1 7 2. sm yt h ’s m o d el o f re fl ec ti o n (s m yt h 19 89 )18 3. m ez ir o w ’s r ef le c ti ve m o d el (m ez ir o w 20 02 )19 4. bo rt o n ’s m o d el o f re fl ec ti o n (b o rt o n /2 00 3) 20 ,2 1 5. a r ef le c ti ve m o d el f o r in te rp ro fe ss io n a l le a rn in g (z ar ez ad eh et a l. 2 00 9) 22 6. jo h n ’s r ef le c ti ve f ra m ew o rk (j oh n 2 00 9) 23 ,2 4 7. th e co re m o d el f o r cr it ic a l re fl ec ti o n c ot tr el l ( 20 10 )25 ,2 6 326 j contemp med sci | vol. 7, no. 6, november-december 2021: 323–329 explaining the concept of learning based on reflection in clinical education: scoping review review s. ahmady et al. ta bl e 2. r es ea rc h re la te d to le ar ni ng b as ed o n re fle ct io n in cl in ic al e du ca tio n (n ur si ng ) au th or /y ea r o f pu bl ic at io n re se ar ch o bj ec tiv e sa m pl e re se ar ch m et ho d ke y fin di ng s pl at ze r, et a l. 20 00 d et er m in in g ba rr ie rs to re fle ct io n le ar ni ng a g ro up o f u nd er gr ad ua te n ur se s q ua lit at iv e re se ar ch th e ex is tin g cu ltu re o r t he e nv iro nm en t w he re n ur se s w or k, is th e la rg es t b ar rie r t o re fle ct io n an d le ar ni ng th ro ug h ex pe rie nc e. 27 ru th -s ah d 20 03 cr iti ca l a na ly si s of re fle ct io n pe rf or m an ce w ith a pp lic at io n in n ur si ng n ur si ng s tu de nt s an d pr of es so rs q ua lit at iv e re se ar ch pe rf or m an ce b as ed o n re fle ct io n, b eg in s w ith b eg in ne r s tu de nt s an d th e va lu e an d im po rt an ce o f re fle ct io n ca n be ta ug ht to b eg in ne rs . i n nu rs in g m ea su re s, re fle ct io n ne ed s to b e ex ec ut ed s o th at w e ca n pe rf or m m or e eff ec tiv e m ea su re s fo r p at ie nt s.2 8 ri ch ar ds on 2 00 4 d et er m in in g th e st ra te gi es to le ar ne rs ’ le ar ni ng in c la ss e nv iro nm en t st ud en ts q ua nt ita tiv e re se ar ch th e us e of q ue st io nn ai re s ho ul d be c on si de re d in o rd er to fu lfi l t he ta sk s as a p re ci se re fle ct io n of co gn iti ve p ro ce ss in g of p eo pl e an d pe op le a ct d iff er en tly in re fle ct iv e le ar ni ng .29 g us ta fs so n, e t a l. 20 07 d et er m in in g th e eff ec t o f r efl ec tiv e pe rf or m an ce in n ur si ng c ar e n ur se s in th e ar ea of c lin ic al c ar e q ua lit at iv e re se ar ch pe rf or m an ce b as ed o n re fle ct io n is a v al ua bl e to ol fo r n ur si ng c ar e an d in th is m et ho d, n ur se s fin d th e ab ili ty to p er fo rm h ol is tic n ur si ng c ar e an d ha ve lo gi ca l t hi nk in g an d ex pa nd th ei r k no w le dg e an d it’ s a w ay to in te gr at e th e ne w n ur si ng k no w le dg e w ith th e pr ev io us o ne in th e di re ct io n of ch an gi ng n ur si ng p er fo rm an ce .30 ch on g 20 09 ex pl ai ni ng n ur si ng s tu de nt s’ pe rc ep tio n of re fle ct iv e pe rf or m an ce n ur si ng s tu de nt s q ua lit at iv e re se ar ch n ur si ng s tu de nt s ha ve a p os iti ve p er ce pt io n of re fle ct iv e pe rf or m an ce in c lin ic al n ur si ng w hi ch ra is es le ar ni ng a ct iv iti es a nd s el fdi re ct ed m ot iv at io n of s tu de nt s.3 1 m et tiä in en , e t a l. 20 13 d et er m in in g th e ap pl ic at io n of th e le ve ls of a w ar en es s an d th e le ve ls o f c rit ic al a w ar ene ss o f r efl ec tiv e le ar ni ng fo r a dj us te d us e n ur si ng s tu de nt s q ua lit at iv e re se ar ch n ur si ng s tu de nt s in th ei r w or ki ng e nv iro nm en t c re at ed c on ce pt ua l s tr uc tu re s th ro ug h pe rc ep tio n an d th in ki ng , a nd re fle ct ed o n its th eo re tic al p er ce pt io ns a nd b ac kg ro un ds . i n th e le ve l o f c rit ic al kn ow le dg e, th e st ud en ts b ec am e aw ar e to w ar ds th ei r k no w le dg e an d le ar ne d to c rit ic iz e th is kn ow le dg e an d th ei r o w n as su m pt io ns .32 n ai ck er 2 01 3 ex am in in g an d de sc rib in g th e ro le o f n ur sin g tu to rs in fa ci lit at in g re fle ct iv e le ar ni ng in s tu de nt s n ur si ng tu to rs q ua lit at iv e re se ar ch in p ra ct ic e, n ur si ng tu to rs , d id n’ t h av e m uc h su cc es s in te ac hi ng re fle ct io n. l ea rn in g ba se d on th is, is n ot c on si de re d as th e offi ci al a pp ro ac h in th ei r p ro gr am s, ho w ev er , t ut or s tr ie d to e xp lo it th es e m ea su re s in th ei r t ea ch in g ac tiv iti es .33 ry an , e t a l. 20 13 th eo riz in g a m od el fo r t ea ch in g an d ev al ua tin g of re fle ct iv e le ar ni ng in h ig he r ed uc at io n st ud en ts a nd pr of es so rs q ua lit at iv e re se ar ch h ig he r e du ca tio n is a m ul tidi m en si on al s pa ce w hi ch ju st ifi es th e ne ce ss ity o f p ay in g at te nt io n to a ll di ffe re nt d im en si on s of s tu de nt s’ le ar ni ng .34 m un cy 2 01 4 d et er m in in g th e ty pe o f l ea rn in g of st ud en ts w he n us in g an d ed iti ng th ei r d ai ly w rit in gs in b lo gs st ud en ts q ua lit at iv e re se ar ch bl og gi ng is a v al ua bl e to ol to le ar n re fle ct io n am on g st ud en ts .35 g aš ev ić , m irr ia hi & d aw so n 20 14 d et er m in in g an d an al yz in g th e eff ec ts o f us in g vi de o an d ed uc at io n fo r s up po rt in g le ar ni ng b as ed o n re fle ct io n in s tu de nt s st ud en ts q ua lit at iv e re se ar ch th e us e of v id eo a nd e du ca tio n im pr ov es re fle ct iv e le ar ni ng p ro ce ss es in s tu de nt s.3 6 u ga ld e 20 15 d et er m in in g th e re la tio ns hi p be tw ee n re fle ct iv e le ar ni ng , i nt er es ts a nd m ot iv at io ns in a ch ie vi ng s uc ce ss in s ta nd ar d re ad in g in in te rm ed ia te s ch oo ls st ud en ts o f in te rm ed ia te sc ho ol s (e ur op ea n st ud en ts ) q ua lit at iv e re se ar ch m ot iv at io na l a ct iv iti es o f s tu de nt s le ad to a d iff er en ce in th ei r l ea rn in g an d fo r e ac h of th em , o ne or s om e le ar ni ng a ct iv ity w as c on si de re d as v al ua bl e. t he n ee d fo r p re pa rin g an d ex is te nc e of c le ar st an da rd s be tw ee n re fle ct iv e le ar ni ng a nd s tu de nt s’ su cc es s w as e vi de nt .37 sa pe rs te in , e t a l. 20 15 pr es en tin g a m od el o f r efl ec tiv e pe rf or m an ce e du ca tio n w ith th e pr ep ar at io n an d im pl em en ta tio n of c ur ric ul um d ur in g th e 4 ye ar s of m ed ic al e du ca tio n in g ra du at e st ud en ts a nd p ro fe ss or s of e dw ar d h eb er t s ch oo l o f m ed ic in e u su q ua lit at iv e re se ar ch pr ac tic e ba se d on re fle ct io n lo ng itu di na lly in 3 s ta ge s of e du ca tio n an d by p ro vi di ng a s am pl e of re fle ct iv e w rit in g to s tu de nt s sh ow th at , b y re pl ac in g su ch a c ur ric ul um , h ow w e ca n ob se rv e its po te nt ia l b en efi ts s uc h as im pr ov ed in te rpr of es si on e du ca tio n an d im pr ov ed s el faw ar en es s an d cl in ic al p er fo rm an ce .38 327j contemp med sci | vol. 7, no. 6, november-december 2021: 323–329 s. ahmady et al. review explaining the concept of learning based on reflection in clinical education: scoping review reflection and learning based on it from the scoping review (figure 2) with the use of the potential of this kind of learning for further application in clinical education specifically for nurses for the study objectives. conceptual model of reflection and learning based on it from scoping review during the theoretical explanation of the concepts and components of reflection in medical education in the text and identifying the dimensions and characteristics of education based on reflection and their application in clinical nursing education, the model is schematically abstracted (figure 2). in this model: the clinical education triangle has 3 main components in its three angles: patient, teacher, or tutor, and student. the purpose of these components in the clinic is to achieve patient health. in this way, it is necessary that the sides of this triangle including teaching-learning, teaching-caring, and caring-learning to be formed in the best way and to serve the student and the patient. in order for the student to learn better, in the clinic, benefiting from his experience, the teacher and patient enter a cycle that focuses on reflection in education and has 4 cyclic stages of do/review/plan/experience or practice. in the case of implementing these stages, the student enters the phase of thinking which, by thinking about experiences, manifests as reflective thinking, critical reflective, or reflective learning. teachers use different teaching methods and teaching strategies to teach better. in case of implementing the stages of review/do/experience or practice, the student enters the phase of doing/experiencing and tries to take proper clinical measures for the patient by achieving reflective (professional) practice and it’s in this stage that along with self-monitoring, the teacher also gives feedback. the key point of this model is that a clinical student, including nursing, after the theory training process and reaching this point of clinical training, still has a gap between theory and practice, which with the help of professors, this gap must be repaired and compensated based on reflection so that a nursing student can better provide clinical services and bring the health system and patient closer to the lofty goal of recovery and promotion of health. discussion as freshwater addressed it in 2002, “being a good therapeutic staff and physician” requires appropriate knowledge and skills, but it also requires establishing and maintaining a therapeutic relationship between patients and their caregivers.39 this concept implies that a good relationship with the patient has a beneficial effect on the well-being and improvement of the patient. the importance of a therapeutic relationship has long been recognized in psychotherapy, but recent attention to patient-centered care suggests improvements in outcomes such as patient satisfaction and improved chronic patient care.40 an essential aspect of relationship therapy is recognizing and understanding the personal beliefs and value systems of the people involved, both physicians and patients. there may be differences between these systems and this can cause an acute emotional reaction in the physician which in turn may affect his next decisions and measures. recent neurological research shows that effective reasoning is a largely unconscious process in which there is a modulation of the logical processing of information by emotions. for example, anger towards a patient may lead to a response that would be different if it was empathy. establishing a therapeutic relationship is an essential component of professional practice and a key characteristic of professionalism. reflection directed by a supervisor or advisor is very beneficial for this approach because the beliefs and main and essential presumptions of the individual are identified and challenged. reflection is important for establishing a therapeutic relationship, especially for postgraduate education and continuing medical education, but it is also used for general medical education, especially during the clinical period.40 reflection is effective in developing professional practice. therapeutic staff and physicians often have to respond to a wide range of conditions that are both complex and not well defined. this “disorder” of professional practice is at the heart of professional expertise.41 experts are quick to make decisions that are appropriate to these complex situations, and they are able to create a set of mental models through an action-on-reflection process that mobilizes them quickly and can effectively resolve the situation through action-in-reflection. developing professional expertise requires nothing more than a set of knowledge and skills.42 the performance of an expert is a complex combination of knowledge and skills tailored to the unique situation he or she faces. frequent confrontation with the complexities of professional life is an essential issue and directed reflection can maximize the opportunity to learn in this approach. reflection is essential in developing professional acts for postgraduate education and continuous education. although these three approaches have different outcomes, they all have an essential aspect. a conscious process to develop perception and create meaning from one situation to future measures done consciously is the essence of reflection. in this study, only one study compared different methods. this study was conducted to identify whether writing an important incident report, two-person interview, or a combination of the two, is more effective in creating a reflection. the results of this study indicate that interview with a tutor is the most effective way to reflect on professionalism. during the experience of medical school, there is no longitudinal study and evidence of the benefits of reflection in long-term development, especially in subsequent clinical care. however, reflection in general medicine students has increased self report measures about self-awareness, professional thinking skills, and skills needed for close examination of patients.43 fig. 2 conceptual model of reflection and learning based on it. 328 j contemp med sci | vol. 7, no. 6, november-december 2021: 323–329 explaining the concept of learning based on reflection in clinical education: scoping review review s. ahmady et al. four studies that reported positively targeted results in reflection were: increased skill in reflection and diagnostic thinking (sobral, 2000), professional identity (niemi, 1997), medical-humanistic skills score (wiecha et al. 2002), and final examination results for obstetrics (lonka et al., 2001). as a result, students found that reflection was helpful, and the application of reflection led to improved self-report outcomes and objective learning and professional development.44-47 grant et al. in 2006, by a study of first-year medical students, showed that instead of text-based reflection writing methods, students prefer to pursue group-based and creative reflective activities.48 it seems that the experience of the use of multimedia, voice, image, and video and creatively exploiting them for reflection, like digital storytelling, not only increases the students’ involvement but also increases the depth of their reflection.49 individuals have a variety of preferred methods for presenting their thoughts and feelings, including painting, photography, and sculpture.50 conclusion what emerges from the study of relevant research in the field of reflective learning indicates that the idea of this model entered the field of learning and education from burton in 1970 and gradually became more apparent in clinical education. of course, clinical education of students needs a lot of effort so that by reflection, one can expect the better performance of the students, especially the big group of nursing students. the results of reviewing various researches in parallel have confirmed the efficiency and positive effect of this type of learning in acquiring skills, student motivation, and the ability of collaborative learning and subsequently improving skills in clinical and working environments. but in order to separate from the traditional texture of education and learning, much effort is required in the implementation of this method and the result of combining the data of studied researches also emphasizes the practical and constructive nature of knowledge in learning based on reflection and its special position so that by eliminating the gap between theory and practice in clinical education, one can see the excellence of the operational part of education and promotion of the level of health of the society by providing an appropriate performance of the nursing staff. acknowledgments this study is the result of a phd thesis at shahid beheshti university of medical sciences by the second author. conflicts of interest disclosure the authors declare no conflicts of interest.  references 1. vachon b, leblanc j. effectiveness of past and current critical incident analysis on reflective learning and practice change. medical education. 2011;45(9):894–904. 2. mubuuke ag, kiguli-malwadde e, kiguli s, businge f. a student portfolio: the golden key to reflective, experiential, and evidence-based learning. journal of medical imaging and radiation sciences. 2010;41(2):72–8. 3. yasin rm, rahman s, ahmad ar. framework for reflective learning using portfolios in pre-service teacher training. procedia-social and behavioral sciences. 2012;46:3837–41. 4. elena s. reflective capability–a specific goal of a teachers professional ethics course. procedia-social and behavioral sciences. 2011;11:145–9. 5. park jy, son j-b. expression and connection: the integration of the reflective learning process and the writing process into social network sites. journal of online learning and teaching. 2011;7(1):170–8. 6. pollard c, stringer e, cockayne d. clinical clinical education: a review of the literature nurse education in practice.nurse educpract.2007;7(5):315–22. 7. nahas vl, nour v, alnobani m. jordanian undergraduate nursing students’ perceptions of effective clinical teachers. nurse educ today.1999; 19(8):639–48. 8. wong j, wong s. towards effective clinical teaching in nursing. j adv nurse.1987;12(4):505–13. 9. benor de, leviyof i. the the development of students’ perceptions of effective teaching: the ideal, best and poorest clinical teacher in nursing. j nurse educ.1997;36(5):206–11. 10. hosseiny n, karimi z, malek zadeh j. the situation of clinical education based on nursing students’ opinion in yasuj nursing and midwifery school. iranian journal of medical education. 2005;5(2)171–5(persian). 11. haghani f, jafari mianaei s, ehsani m. reflective learning and teaching: a review. iranian journal of medical education. 2014;13(11):989–98. 12. arksey h, and o malley l. (2005). scoping studies: towards a methodological framework. international journal of social research methodology, 8(1), 19–32. 13. mays s, arksey h, o’malley l. scoping studies: towards a methodological framework. international j social res methodol: theory & pratice 2005; 8: 5. 14. colquhoun hl, levac d, o’brien kk, straus s, tricco ac, perrier l, et al. scoping reviews: time for clarity in definition, methods, and reporting. j clin epidemiol 2014; 67(12): 1291–4. 15. moradi m, miralmasi a. pragmatic research method (quantitative and qualitative research), first). tehran: iranian academy of statistical analysis (school of quantitative and qualitative research). retrieved from www. analysisacademy.com. 2020. 16. gibbs g. learning by doing: a guide to teaching and learning methods, oxford, further education unit, oxford polytechnic,1988. 17. questions taken from: jasper, m. beginning reflective practice, cheltenham, nelson thornes, 2003. 18. smyth j. developing and sustaining critical reflection in teacher education, journal of teacher education, 40(2), pp 2–9, 1989. 19. mezirow j. learning as transformation: critical perspectives on a theory in progress. san francisco: jossey-bass, 2000. 20. borton t. (1970) reach touch and teach, london: hutchinson cited in jasper m, 2003. beginning reflective practice, heltenham: nelson thornes.:link download: borton, t, 1970. reach touch and teach, https://archive.org/ details/reachtouchteachs0000bort, 2003. 21. borton (2014). reach touch and teach. nurse education today 34, 488–489, 2014. 22. zarezadeh y, pearson p, and dickinson, c. a model for using reflection to enhance inter professional education, international journal of education, 1(1) available from www.macrothink.org/ije, 2009. 23. johns c. becoming a reflective practitioner, 3rd ed. oxford: wiley blackwell, 2009. 24. palmer a, burns s, and bulman c. reflective practice in nursing.-oxford: blackwell scientific publications,1994. 25. cottrell s. skills for success: the personal development planning handbook 2nd ed, 2010. 26. basingstoke: palgrave mcmillan cited in cottrell, s critical thinking skills: developing effective analysis and argument 2nd ed basingstoke: palgrave mcmillan, 2011. 27. platzer h, blake d, ashford d. barriers to learning from reflection: a study of the use of groupwork with post‐registration nurses. journal of advanced nursing. 2000;31(5):1001–8. 28. ruth-sahd la. reflective practice: a critical analysis of data-based studies and implications for nursing education. journal of nursing education. 2003;42(11):488–97. 29. richardson jt. methodological issues in questionnaire-based research on student learning in higher education. educational psychology review. 2004;16(4):347–58. 30. gustafsson c, asp m, fagerberg i. reflective practice in nursing care: embedded assumptions in qualitative studies. international journal of nursing practice. 2007;13(3):151–60. 31. chong mc. is reflective practice a useful task for student nurses? asian nursing research. 2009;3(3):111–20. 329j contemp med sci | vol. 7, no. 6, november-december 2021: 323–329 s. ahmady et al. review explaining the concept of learning based on reflection in clinical education: scoping review 32. mettiäinen s, vähämaa k. does reflective web-based discussion strengthen nursing students’ learning experiences during clinical training? nurse education in practice. 2013;13(5):344–9. 33. naicker k. the role of educators in facilitating reflective learning in students 2013. 34. ryan m, ryan m. theorising a model for teaching and assessing reflective learning in higher education. higher education research & development. 2013;32(2):244–57. 35. muncy ja. blogging for reflection: the use of online journals to engage students in reflective learning. marketing education review. 2014;24(2):101–14. 36. gašević d, mirriahi n, dawson s, editors. analytics of the effects of video use and instruction to support reflective learning. proceedings of the fourth international conference on learning analytics and knowledge; 2014: acm. 37. ugalde j. the relationship between reflective learning, interest and motivation and achievement toward reading standards in middle school. 2015. 38. saperstein ak, lilje t, seibert d. a model for teaching reflective practice. mil med. 2015;180(4 suppl):142–6. 39. freshwater d (ed). therapeutic nursing: improving patient care through self awareness and reflection, london: sage, 2002. 40. stewart m, brown jb, donner a, mcwhinney ir, oates j, weston ww, jordan j. 2000. the impact of patient-centered care on outcomes. j fam pract 49(9):805–807. 41. schon da. the reflective practitioner. temple smith: london (1983). 42. eraut m. developing professional knowledge and competence. london: falmer press (1994). 43. baernstein a, fryer-edwards k. 2003. promoting reflection on professionalism: a comparison trial of educational interventions for medical students. acad med 78:742–747. 44. sobral dt. 2000. an appraisal of medical students’ reflection-in-learning. med educ 34:182–187. 45. niemi pm. 1997. medical students’ professional identity: self-reflection during the pre-clinical years. med educ 31:408–415. 46. wiecha jm, vanderschmidt h, schilling k. 2002. heal: an instructional design model applied to an online clerkship in family medicine. acad med 77:925–926. 47. lonka k, slotte v, halttunen m, kurki t, tiitinen a, vaara l, paavonen j. 2001. portfolios as a learning tool in obstetrics and gynaecology undergraduate training. med educ 35:1125–1130. 48. grant a, kinnersley p, metcalf e, pill r, houston h. 2006. students’ views of reflective learning techniques: an efficacy study at a uk medical school. med educ 40(4):379–388. 49. sandars j, homer m. reflective learning and the net generation. med teach 30:877–879, 2008. 50. gauntlett d. creative explorations. abingdon: routledge, 2007. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i6.1109 193j contemp med sci | vol. 3, no. 9, winter 2017: 193–196 research comparison and evaluation of diagnostic techniques of mycobacterium tuberculosis in iraq abdul-sattar a. k. al-seedi,a jawad k. al-khafaji,b hadi m. a. al-mosawic adepartment of microbiology, college of medicine, babylon university, iraq. bdepartment of microbiology, college of medicine, babylon university, iraq. cdepartment of pathology, college of medicine, babylon university, iraq. correspondence to (email: abdalstare1@gmail.com) (submitted: 10 december 2016 – revised version received: 05 january 2017 – accepted: 20 january 2017 – published online: 26 march 2017 ) objective diagnostic tests devoted to the rapid, sensitive, and specific identification of the causative agent are key components of successful wellness plans directed at tuberculosis control. this study focusses on rapid and accurate detection of tuberculosis cases among babylon population. methods the sputum samples were collected from 60 patients suspected have suffering from tuberculosis infection, in the specialized chest and respiratory center, hilla city and department of medical microbiology, college of medicine, babylon university, hilla-iraq during the period from february to june 2015. molecular detection of mycobacterium tuberculosis in patients’ sputum samples using real-time pcr, and gene x-pert for suspected tb-infected patients. the clinical signs were recorded for each patient, including night sweating, fever, loss of weight, and history of cough. results gene x-pert mtb/rif technique, real-time pcr recording the high sensitivity for afb positive smear (100%) for both, afb negative smear (66%, and 58%), respectively. afb sensitivity was (16.6%), and the specificity was (100%) for all in the present study. conclusion the comparison between advance technique (gene x-pert and real-time pcr) and classical technique (afb) for the diagnosis of mtb, shows that genetic technique is the best with high sensitivity and specificity. keywords tuberculosis, real-time pcr, gene-x-pert introduction tuberculosis (tb) has been one of the oldest infectious diseases affecting human. it was identified 4000 years ago, from the middle east and europe, as the cause of death, suggesting that this disease has been already a widespread health problem back then. in a detailed history, hippocrates wrote about patients with wasting away associated with coughing and chest pain, often with blood in sputum. these symptoms had been allowed hippocrates to diagnose tb, which at that fourth dimension has been called “consumption”. the occurrence of descriptions of patients with these signs indicated that the disease was already well entrenched in early times.1,2 tb is a communicable and deadly infectious disease caused by mycobacteria, essentially mycobacterium tuberculosis.3 tb affects 8.8 million people each year, most of whom dwell in low economic society.4 additionally, an estimated 2 billion people are believed to be latently infected, providing a great reservoir.5 tuberculosis typically attacking the lungs (as pulmonary tb) but can affect the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, the gastrointestinal system, bones, joints, and even the skin (extra pulmonary tb).6 although in that respect other mycobacteria such as mycobacterium bovis, mycobacterium africanum, mycobacterium canetti, and mycobacterium microti also can cause tuberculosis, these species are less common. the usual symptoms of pulmonary tuberculosis are a chronic cough with blood-tinged sputum, fever, night sweats and weight loss. tuberculosis considers an immunological disease and the pathology of disease mediates by the host immune response.7 diagnostic tests devoted to the rapid, sensitive, and specific identification of the causative agent are key components of successful wellness plans directed at disease control. moreover, the precise determination of mycobacterial burden might be beneficial for fast assessment of patient response to standard therapy, particularly in those patients suspected of harboring antibiotic resistant m. tuberculosis strains.8 chest radiographs may be applied to exclude pulmonary tb disease in a person with a normal immune system who has a positive tst (tuberculin skin test) reaction or igra (ifn-γ release assay) and has no symptoms or signs of tb disease.9 gain techniques for the diagnosis of tuberculosis have attracted considerable interest in diagnosis, particularly with the hope of reducing the time taken to find and identify mycobacterium tuberculosis in respiratory and non-respiratory specimens.10 real-time pcr has further advantages, including precision, reproducibility, accuracy, quality control procedures and reduced pollution. in addition, real-time pcr eliminates the need for electrophoresis after the cycling reaction. furthermore, this technique cuts the analysis time for 3 or 4 days, which is important in lengthy microbiological diagnoses, such as those needed for tb.11 the gene x-pert system, a realtime pcr that simultaneously detects both mycobacterium tuberculosis complex (mtbc) and rifampin resistance, was developed.12 in contrast to some real-time pcr instruments, the x-pert mtb/rif is an on-demand assay described as a simple method that can be performed by personnel with minimal grooming and can provide answers within 2 h.13 the detection of rifampin resistance, as a surrogate for multidrug-resistant tb (mdr-tb), directly from smear-positive respiratory specimens from patients bearing a high risk of mdr-tb has recently been advocated by the world health organization.14 chemotherapy of drug susceptible tb consists of three or four drug regimen, administered for 6 months. the long duration of therapy solutions in poor compliance leading to the emergence of multi-drug resistant forms of m. tuberculosis.15 issn 2413-0516 194 j contemp med sci | vol. 3, no. 9, winter 2017: 193–196 comparison and evaluation of diagnostic techniques of mycobacterium tuberculosis in iraq research abdul-sattar a. k. al-seedi et al. amplification mix preparation in the new sterile tube each sample was prepared 10* (n+1) μl of pcr-mix-1, 5* (n+1) μl of pcr buffer flu, 0,5* (n+1) μl taq dna polymerase and 0,5* (n+1) μl of udg enzyme. vortex rinsed gently and centrifuged briefly. fifteen microliter of reaction mix was added to each tube. ten microliter of extracting dna was added to appropriate tube. two controls were prepared for each panel: 10 μl of dna-buffer was added to the tube labeled amplification negative control. 10 μl of c-mtb&ic was added to the tube labeled amplification positive control. the tubes were ready to insert in to the thermalcycler. data analysis: the fluorescent single intensity was detected in two channels: mycobacterium tuberculosis was detected on the fam (green) channel, ic dna on the joe (yellow) / hex/cy3 channel. gene x-pert mtb/rif assay preparing the sputum samples each x-pert mtb/rif cartridge was labelled with the sample id. in a leak-proof sputum collection container, a. the lid of the sputum collection container was carefully opened. b. approximately 2 times the volume of the sr was poured to the sputum (2:1 dilution, sr:sputum). c. the lid was replaced and secured. d. shaked vigorously 10 to 20 times by vortex for at least 10 s. the sample was incubated for 10 minutes at room temperature, and then shaken the specimen vigorously 10 to 20 times or vortex for at least 10 seconds. the sample was incubated at room temperature for an additional 5 minutes. the test procedure the computer was turned on, farther turn on the gene x-pert instrument. open the software shortcut icon of the gene x-pert in the windows® desktop by double-click on it. user name and password have been used to log on the gene-x-pert dx system software. click create, test, in the gene x-pert dx system window. the scan sample id dialog box appeared. in the sample id box, scan or type the sample id. make sure you type the correct sample id (sample id is associated with the test results and is shown in the view results window and all the reports). the scan cartridge barcode dialog box appears. the barcode on the x-pert mtb/ rif cartridge has been scanned. the create test window appears. using the barcode information, the software automatically fills the boxes for the following fields: select assay, reagent lot id, cartridge sn, and expiration date. click start test. enter your password if requested. the instrument module door with the blinking green light has been opened, and loaded the cartridge. the door was closed. the test starts and the green light stop-blinking. when the test is finished, the light turns off. wait until the system releases the door lock at the end of the run, then open the module door and remove the cartridge. reading the results: the gene x-pert instrument system generates the results from measured fluorescent signals and embedded calculation algorithms. the results can be seen in the view results window. results among 60 samples, only 10 samples which give a positive afb, 50 samples give a negative afb result, 39 samples positive, materials and methods study population a total of 60 patients suspected with tuberculosis infection, in the specialized chest and respiratory center, hilla city during the period from february to june 2015. the clinical signs were recorded for each patient, including the night sweating, fever, loss of weight, and history of dry cough. checklist sheets were drawn out for each patient including age, gender, radiological finding, and incidence area. sputum samples were collected from tb according to the stated procedure. afb smear the slides were flooded with carbol fuchsin dye. the slides are heated slowly until it is steaming with direct flame for at least 5 minutes. the smear was decolorized with a decolorizing agent and left until the color of the solution is gone. then it was washed with tap water. methylene blue was applied to slides by which the dye covered the entire slides surface and left for maximum 60 seconds than washing with water. microscopically examination: the positive acid fast bacilli smear appears as pink or red rods in a blue background.16 the number of afb was calculated by country number of cell in smear according to standard method.17 real time pcr assay extraction of dna the required number of 1.5 ml disposable polypropylene micro centrifuge tubes was developed, including single tube for negative control of extraction (negative control, c-). ten microliter of the mtb ic (internal control) and 300 μl of lysis solution was added to each pipe. one hundred microliter of samples was added to the appropriate tubes using pipette tips with aerosol barriers. the control was prepared as follows: add 100 μl of negative control cto the labeled cneg. vortex the tubes and incubated for 5 min at 65˚c. centrifuged for 10 s. four hundred microliter of prec solution was added and mixed by vortex. centrifuged all tubes at 13,000 rpm for 5 min and using a micropipette with a plugged aerosol barrier tip, carefully removed and discarded supernatant from each tube without disturbing the pellet. changed tips between the tubes. five hundred microliter of wash solution was added into each tube. vortex vigorously rinsed to ensure pellet washing. centrifuged all tubes at 13,000 rpm for 60 s using a micropipette with a plugged aerosol barrier tip. carefully removed and discarded supernatant from each tube without disturbing the pellet. changed tips between the tube. two hundred microliter of wash solution 4 was added to each tube. vortex vigorously rinsed to ensure the pellet washing. centrifuge all tubes at 13,000 rpm/min for 60 s and using a micropipette with a plugged aerosol barrier tip. carefully removed and discarded supernatant from each tube without disturbing the pellet. the tubes were incubated with open caps at 65˚ c for 5 min. resuspended the pellet in 50 μl of rebuffer (the elution volume can be increased upwards to 90 μl). incubate for 5 min at 65˚c and vortex periodically. the tubes were centrifuged at 13000 g for 60 s. the supernatant contains rna/dna ready for amplification. if amplification is not performed on the same day of extraction, the processed samples can be stored at 4˚c for a maximum period of 5 days or frozen at –20˚c for a long time. abdul-sattar a. k. al-seedi et al. 195j contemp med sci | vol. 3, no. 9, winter 2017: 193–196 research comparison and evaluation of diagnostic techniques of mycobacterium tuberculosis in iraq 21 samples negative results for real-time pcr, 43 samples positive, 17 negative results for gene x-pert respectively as shows in table-1. discussion gene x-pert mtb/rif technique, real-time pcr recording the high sensitivity for afb positive (100%) for both, for afb negative (66%, and 58%) respectively, afb sensitivity was (16.6%), and the specificity was (100%) as shown in table 1. the sensitivity of real-time pcr and x-pert mtb/rif are given similar results with smears positive, but it is low in real-time technique for smears negative. these are matching with armand et al.,18 who suggested that both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens, but does not match in the sensitivity of the x-pert mtb/rif test with the smear-negative specimens were more reduced than that of the real-time pcr assay (48 versus 69%, p < 0.005). american thoracic society workshop,19 who estimated that the gene x-pert assay was introduced lately. its advantage lies in higher sensitivity and specificity for the diagnosis of tb, together with the detection of drug resistance, and in smear-positive specimens. the sensitivity and specificity of pcr are in the range 90–100%, with a positive prognostic value of > 95%, whereas in smear-negative specimens, the sensitivity of pcr was reduced to < 50%. our study found that the sensitivity of gene x-pert for afb smearnegative was (66%). these results agreed with the work reported by zeka et al.,20 who suggested that the x-pert mtb/rif assay have reported test sensitivities of 57 to 76.9% in cases of smear-negative. in our study, real-time pcr has a second value of sensitivity (69%) for smear negative after the gene x-pert. these results were in accordance with those results reported by raviglion,21 who proposed that the real-time pcr assay with internal control achieved a sensitivity of 96.2 % and specificity of 99.2%. templeton et al.,22 explains that internal control reaction has been included to determine the robustness of the pcr resulting by monitoring the nucleic acid extraction as well as the presence of inhibitors. rajpal et al.,23 stated that direct microscopy may give negative results if the number of afb less than 5000 bacilli/ml, consistently positive specimens would have to contain 105 bacilli per ml varying with the extent of the wound or the presence citations. the scanty or numerous afb smear test, which differs according to the number of bacilli in microscopy filed. in our study, the sensitivity for the diagnosis of m. tuberculosis of gene x-pert and real-time pcr higher than afb because the gene x-pert and real-time pcr depends on diagnosis on dna amplification and have ability to detect less than 10 of acid fast bacilli per ml in a sputum sample while afb smear depending on the ability of m. tuberculosis to take stain and, may give a negative result if there few number of acid fast bacilli less than 5000 bacilli per ml in the field. conclusions the comparison between advance technique (gene x-pert and real time pcr) and classical technique (afb) for the diagnosis of mtb, show that genetic technique is the best with high sensitivity, and specificity. consent all authors declare that written informed consent was obtained from the patients for publication of this research article. competing interests authors have declared that no competing interests exist. n table 1. results of diagnostic techniques for tb test no. of patients total sensitivity specificitytrue positive false negative afb 10 (16.7%) 50 (83.3%) 60 16.6 % 100% real-time pcr 39 (65%) 21(35%) 60 afb +ve afb –ve 100% 100% 58% gene x-pert 43 (71.7%) 17(28.3%) 60 afb +ve afb –ve 100% 100% 66% the chi-square statistic is 43.2658. the p-value is < 0.00001. the result is significant at p < 0.005. references 1. shah ns, wright a, bai gh. world wide emergence of extensively drugresistant tuberculosis. emerg infect dis. 2007;13: pp. 380–387. 2. who. pathways to better diagnostics for tuberculosis: a blueprint for the development of tb diagnostics. geneva no. 121, 2010. 3. kumar v, robbins sl. robbins basic pathology. 8th edn., saunders/elsevier, philadelphia. isbn:10: 2007; pp: 946. 4. who. the stop tb strategy, case reports, treatment outcomes and estimates of tb burden. global tuberculosis control: epidemiology, strategy, financing. 2009; pp. 187–300. 5. gagneux s. host pathogen coevolution in human tuberculosis. phil trans r soc b. 2012;367:850–859. 6. reddy jr, kwang j, lechtenberg kf, khan nc, prasad rb, chengappa mm. an immunochromatographic serological assay for the diagnosis of mycobacterium tuberculosis. comp immunol microbiol infect dis. 2002;25:21–7. 7. shiloh mu, champion pa. to catch a killer: what can mycobacterium models teach us about mycobacterium tuberculosis pathogenesis?. curr opin microbiol. 2010;13:86–92. 8. ramaswamy s, musser jm. molecular genetic basis of antimicrobial agent resistance in mycobacterium tuberculosis: update. tuber. lung dis. 1998;79:3–29. 9. cdc. centers for disease conterol and prevention (2013): tuberculosis. department of health and families, northern territory government. 10. hajia m, rahbar m, alkhani y. efficiency of pcr method for screening pulmonary tuberculosis patients under dots protocol therapy. iranian j public health. 2005;34:9–13. 11. sales ml, fonseca, aa, orzil alencar ap, silva mr, issa ma, filho pms, et al. validation of a real-time pcr assay for the molecular identification of mycobacterium tuberculosis. braz j microbiol. 2014;45:1363–1369. 12. blakemore r, story e, helb d, kop j, banada p, owens mr, et al. evaluation of the analytical performance of the xpert mtb/rif assay. j clin microbiol. 2010;48:2495–2501. 13. boehme cc, et al. rapid molecular detection of tuberculosis and rifampin resistance. n engl j med. 2010;363:1005–1015. 14. world health organization (who): stop tb partnership retrieved on october 2016. 196 j contemp med sci | vol. 3, no. 9, winter 2017: 193–196 comparison and evaluation of diagnostic techniques of mycobacterium tuberculosis in iraq research abdul-sattar a. k. al-seedi et al. 15. jhamb ss, singh rp, singh pp. a comparison of conventional and radiometric methods for the assessment of antitubercular activity of drugs against mycobacterium tuberculosis in mice and macrophabe models. indian j tuberc. 2008;55:70–76. 16. de kantor in, kim sj, frieden t, laszlo a, luelmo f, norval py, et al. laboratory services in tuberculosis control: microscop part ii, culture part iii. global tuberculosis program, who. geneva 1998. 17. who (1998). laboratory services in tuberculosis control culture part iii. who/tb/98.258. geneva. 18. armand s, et al. comparison of the xpert mtb/rif test with an is6110-taqman real-time pcr assay for direct detection of mycobacterium tuberculosis in respiratory and nonrespiratory specimens. j clin microbiol. 2011;49:1772–1776. 19. american thoracic society workshop. rapid diagnosis tests for tuberculosis. what is the appropriate use?. am j respir crit care med. 1997;155:1804–1814. 20. zeka an, tasbakan s, cavusoglu c. evaluation of the genexpert mtb/rif assay for the rapid diagnosis of tuberculosis and detection of rif-resistance in pulmonary and extrapulmonary specimens. j clin microbiol. 2011;49:4138–4141. 21. raviglione m. tuberculosis the essentials. 4th ed. informa ealthcare usa. inc. 2010;81–113. 22. templeton k, scheltinga sa, crielaard j, van schie j, graffelman aw, sillekens p, et al. comparison and evaluation of real-time pcr, real-time nucleic acid sequence-based amplification, conventional pcr, and serology for diagnosis of mycoplasma pneumoniae. j clin microbiol. 2003;41: 4366–4371. 23. rajpal s, dhingra vk, aggarwal jk. sputum grading as predictor of treatment outcome in pulmonary tuberculosis. ind j tub. 2002;49: 139–141. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 400 j contemp med sci | vol. 8, no. 6, november-december 2022: 400–407 original new strategies for inhibition of listeria monocytogenes and klebsiella pneumoniae biofilm formation and persistence samyah d. jastaniah1, taghreed yasir jamal1, reda h. amashah1, magda m. aly1,2,* 1department of biology, college of science, king abdulaziz university, jeddah, saudi arabia. 2botany and microbiology department, faculty of science, kafrelsheikh university, kafr el-sheikh, egypt. *correspondence to: magda m. aly (e-mail: mmmohammad@kau.edu.sa) (submitted: 14 august 2022 – revised version received: 29 august 2022 – accepted: 10 september 2022 – published online: 26 december 2022) abstract objectives: this study aimed to find new strategies for the prevention of bacterial biofilms and investigate the effect of some plant extracts on the biofilm formation by certain pathogenic bacterial strains in vitro. methods: fourteen different biofilm forming bacterial isolates were collected and their biofilm were quantitatively measured under different temperature, ph and growth medium using crystal violet staining method. exopolysaccharides (eps) produced by the isolates were estimated and a comparison between the tested isolates was made. the effect of some plant extracts on bacterial growth, biofilm formation and exopolysaccharide quantity was determined. results: the two isolates, listeria monocytogenes (atcc13932) and klebsiella pneumoniae (atcc700613) were among the most active biofilm forming bacterial isolates. the optimum temperature, ph and media for eps production and biofilm formation were determined. the effect of some plant extracts of cranberry; pomegranate peel and, arak on growth and formation of biofilm and eps were recorded. moreover, minimum inhibition concentration of each plant extract was performed. conclusion: eps quantity produced from the tested isolates depends on some effective factors such temperature, media contents and ph. aqueous-cranberry, aqueous-pomegranate peel and methanolic-arak extracts have antibacterial and antibiofilm activities on l. monocytogenes and k. pneumonia. thus, application of new natural approaches for inhibiting bacterial biofilm is important to prevent persistent and recurrent biofilm related infections. keywords: biofilms, plant extracts, exopolymeric, listeria monocytogenes, klebsiella pneumonia issn 2413-0516 introduction bacterial biofilm is a structured community of bacterial cells attached to a surface and surrounded in a self-produced exopolymeric substance (eps) matrix made up primarily of polysaccharides and other biomolecules such as proteins, lipids, and nucleic acids. bacterial biofilm identified as a significant virulence mechanism in the pathogenesis of many medically important bacterial pathogens, causing serious life-threatening infections. bacteria within the biofilm can persist, causing chronic and recurrent infections and developing antibacterial and immunological resistance. the phenomenon of biofilm recalcitrance makes them extremely difficult to treat and eradicate effectively.1 protection from the hostile environment, nutritional availability, metabolic cooperation, and the acquisition of new genetic material are all functional advantages of bacterial biofilm. many pathogenic bacteria, including listeria monocytogenes, streptococcus mutans, staphylococcus aureus, staphylococcus epidermidis, escherichia coli, klebsiella pneumoniae, and pseudomonas aeruginosa, have been shown to form biofilms.2 the main concern brought on by biofilms is a development in antibiotic resistance that makes it difficult to cure infections caused by biofilms. globally, public health is at risk due to the rise in microbial resistance to antibiotics, which decreases the efficacy of treatments and raises morbidity, mortality, and medical expense rates.3 due to the enhanced resistance provided by sessile cells, many of the control strategies for biofilm control appear to be almost worthless. consequently, biofilms are not easily eliminated by managing elements like heating, drying, cleaners, and detergents and persist on surfaces, especially in hospitals, where they cause contamination and the spread of infections. therefore, new strategies other than the traditional ones are urgently required, and a suitable solution for the “biofilm problem” is anticipated in near future.4 it has been demonstrated that natural compounds made from numerous plant extracts inhibit adhesion, quorum sensing (qs) and subsequently the development of biofilms. the strategy of quorum sensing inhibition (qsi), which targets autoinducers, can be used to prevent the growth of biofilms and represent a novel, natural, widespread, antibacterial approach utilized by plants with significant impact on biofilm formation and consequent inhibition of its related illnesses.5–8 numerous workers have recently discovered that many therapeutic and food-related herbal plants have anti-qs properties. moreover, qs obstruction will reduce the virulence of invasive pathogens, making them more vulnerable to the applied mode of therapy and facilitating simple clearance by host defense mechanism. for example, garlic, chamomile, vanilla, and burdock leaf can block the quorum sensing process.9 however, research into the development of therapeutically useful and secured anti-qs chemicals is still in its early stages, necessitating more investigation of natural materials.10 the ability of several plants to prevent the growth of biofilm in pathogens such l. monocytogenes, k. pneumoniae, p. aeruginosa, s. pyogenes, s. mutans, and s. aureus has also been observed. certain plant extracts are efficient at delaying the growth of multi-species biofilm.5 the majority of investigations to date have concentrated on observing the anti-biofilm activity of plant extracts taken singly rather than in combination.3,11 therefore, it is necessary to investigate natural medicinal alternatives with minimal side effects for the treatment and prevention of various biofilm-related infections. this study aimed to investigate the bacterial biofilm formation of some certain isolated pathogenic strains and the effect of some mailto:mmmohammad@kau.edu.sa 401j contemp med sci | vol. 8, no. 6, november-december 2022: 400–407 s.d. jastaniah et al. original new strategies for inhibition of l. monocytogenes and k. pneumoniae plant extracts on the in-vitro biofilm formation of the isolates. materials and methods the used plants three plants, fresh samples of cranberry (vaccinium macrocarpon) and pomegranate peel (punica granatum) were collected from local markets of jeddah while arak; (salvadora persica) was collected from al bahah region. all plants were collected in sterile plastic bags, washed and extracted. agar well diffusion method was used to determine the inhibitory activity of each plant extract. mic of the plant extracts was performed. the effect of the plant extract on the inhibition of biofilm formation and on eps formation were accomplished by a spectrophotometric method. study setting this study was conducted during the period march 2021march 2022 in the central laboratories of biological sciences department, at faculty of science in king abdulaziz university, jeddah, saudi arabia. the ethical approval (reference no 138-21) was obtained from the unit of biomedical ethics research committee of king abdulaziz university hospital (kauh) in jeddah for permitting sample collection. sample collection different fourteen biofilm forming bacterial strains were collected in agar culture plates including gram-positive and gram-negative pathogenic strains from the clinical and molecular microbiology laboratory of king abdulaziz university hospital; staphylococcus aureus (atcc 29213), methicillin-resistant staphylococcus aureus (mrsa) (atcc 43300), streptococcus pyogenes (atcc 12344), streptococcus agalactiae (atcc 12386), streptococcus mutans (atcc 25175), streptococcus mitis (clinical strain), enterococcus faecalis (atcc 27270), listeria monocytogenes (atcc 13932), e. coli (atcc 25922), klebsiella pneumoniae (esbl) (atcc 700613), pseudomonas aeruginosa (atcc 27853), proteus mirabilis (clinical strain), acinetobacter baumannii (atcc19606), and serratia marcescens (atcc13880). culture conditions and standardization each collected strain was routinely cultured aerobically on blood agar plates at 37°c for 24 hrs. and maintained at 4°c until required for the study. the isolates were then subcultured in tryptic soy broth at same conditions with continuous agitation in a shaking incubator prior to each experiment. the tested strains broth cultures were standardized and adjusted with sterile saline to turbidity equivalent to 0.5 on the mcfarland standard scale which contains approximately 1.5 × 108 cfu/ml and the diluted 1:10 in sterile broth to obtain the final suitable concentration for final inoculation used for the in-vitro experiments. screening of all isolates for biofilm formation all collected bacterial isolates were screened and assayed for biofilm formation using crystal violet staining (cvs) method described by christensen et al. (1995)12 to determine the ability to form biofilm and to measure the biofilm if formed. studying the growth curve and biofilm curve for the selected isolates after inoculating nutrient broth medium with certain isolate, growth was estimated by measuring turbidity using spectrophotometer through recording optical density. previous step was repeated at 2 hrs. intervals until the absorbance no longer increase. after that, readings were plotted on a graph with time and optical density as absorbance and the changes in growth phases were tracked. eps extraction and estimation the biofilm was quantitatively estimated in terms of the quantity of eps produced by the strain. the eps was extracted from the overnight broth culture of the tested strains by centrifugation13. the phenol-sulphuric acid method (dubois et al., 1956)14 was used to estimate the total carbohydrate content in the eps by spectrophotometric method and comparing with a standard of glucose solutions for confirmation. the comparison between the tested bacterial strains was made. biofilm and eps estimation under different conditions biofilm formation by the selected bacteria was estimated after growing bacteria in different conditions. also, the total carbohydrate content in the eps of the selected isolate was extracted and estimated by the assay method as mentioned before but the broth culture was incubated under different temperatures; 25, 30, 35, 37, 40 and 45°c. the optimum temperature for eps and biofilm was determined. also, eps of the selected isolate was extracted and estimated as mentioned before but the selected isolate was cultured in different broth media; nutrient broth, muller-hinton broth, tryptic soy broth, lb broth and brain heart infusion broth. the biofilm was measured in each media and the optimum broth media for eps and biofilm was determined. similarly, the selected bacterium was grown in broth culture medium with different ph; 5, 5.5, 6, 6.5, 7 and 7.5. at the end, the biofilm was measured in each ph and the optimum ph for eps and biofilm was determined. plant extract preparation certain plants; cranberry, pomegranate peel, arak were collected from the markets according to their importance in the scientific research articles. to obtain the extracts, the fresh plant was initially air-dried at room temperature and ground to a fine powder in the blender. grinded powder from each plant were soaked in methanol solvent separately. this step was repeated but with soaking in distilled water instead of methanol under shaking condition for one day, then filtered in whatman filter paper no. 1. then the methanol-solved filtrates were evaporated using the rotary evaporator and the water-solved filtrates were lyophilized. finally, dried extracts were stored at 4°c in an airtight screw-cap bottles. before applying the extract in any experiment, it was dissolved in distilled water or dmso depending on the solvent used and then filtered by 0.22 µm syringe filter and stored in amber bottles at 4°c. the inhibitory activity plant extracts agar well diffusion method (heatley, 1944)15 was used to determine the inhibitory activity of both aqueous and organic (methanolic) extracts from each plant (cranberry, 402 j contemp med sci | vol. 8, no. 6, november-december 2022: 400–407 new strategies for inhibition of l. monocytogenes and k. pneumoniae original s.d. jastaniah et al. pomegranate peel and arak). each plate well was filled with the extract and then the plates were incubated at 37°c for 24 h. inhibition of bacterial growth was measured as inhibition zone diameters and the average value was calculated. determination of minimum inhibition concentration of the plant extracts minimum inhibition concentration (mic) of the plant extracts was performed in 96well microtiter plates against the tested isolates. each selected plant extract was diluted to two-fold with mhb in each well. afterward, bacterial suspensions and the indicator phenol red were added into the wells and the plates were then incubated at 37°c for 24 h.16 microplates were read using a microplate reader. mic was recorded as the lowest concentration of the plant extract and absolute inhibition of observable growth. the plates were incubated following reading for another more 24 hrs. and the biofilm was stained and assessed as mentioned before by crystal violet staining assay to detect the mic that inhibit the biofilm also. antibiofilm assay of plant extracts the effect of the plant extract on the inhibition of biofilm formation was accomplished by a spectrophotometric method as stated by plyuta et al. (2013).17 broth cultures and the plant extracts with the mic were incubated in 96-well microplates under a standard condition. the biofilm biomass was assayed as described before and the percentage of inhibition was estimated. the effect of plant extracts on eps formation the broth culture of the selected isolate was incubated with the sub-inhibitory concentration of the extract in the optimum medium and ph at the optimum temperature for 48 hrs. the eps was extracted and estimated as described before and the values were compared with that recorded before treating by the plant extracts. statistical analysis all the experiments were performed in triplicate. all values are expressed as the mean. the significance of the results was analyzed by one way analysis of variance (anova), through the statistical package for social science (spss) program (version 25.0) to analyze the quantitative data collected; this allowed identification of the relationships between several variables, as well as testing of the assumptions of the research. the main data analysis consisted of descriptive statistics in the form of indicators of central tendencies (means) and variability (standard deviations). otherwise, we use alternative non-parametric tests such as kruskal–wallis tests and results with p-value <0.05 were considered significant. results from the clinical and molecular microbiology laboratory of king abdulaziz university hospital, different fourteen biofilm forming bacterial strains were collected in agar culture plates. the samples include gram-positive and gram-negative pathogenic strains. the study was conducted in the central laboratory of biological sciences department in the faculty of science in the period from march 2021 to march 2022. screening of the bacterial biofilm forming isolates and biofilm measurements all collected bacterial strains were assayed to test the ability to form biofilm and to measure the biofilm if formed. all isolates were confirmed as biofilm forming strains and their biofilms are ranged according to the strength. table 1 showed that the scale range of the mean of the biofilm formation is between 0.1777 and 0.6427. mrsa was the least biofilm forming isolates (0.1777) and a. baumannii was the most biofilm forming isolates (0.6427) within the group. two strains among the most active biofilm forming bacterial isolates were selected to be tested in the further experiments; listeria monocytogenes (atcc 13932) and klebsiella pneumoniae (esbl) (atcc 700613) according to their importance. growth curve for the selected bacterial isolates the growth for the selected isolates was estimated by spectrophotometer and growth curve was made according to the time intervals (0, 2, 4, 6, 8, 24, 36, 48, 60 hrs.) starting with certain concentration for each isolate. figure 1 showed that number of cells of the two bacterial isolates were increased gradually until reach to stationary phase after 24 hours and the growth gradually deceased until constant while the cells remaining metabolically active. it was noticed from the curve that the mean growth rate of klebsiella pneumoniae is much higher than that of listeria monocytogenes within the same time interval. after 8 hours, the growth rate of l. monocytogenes is increasing suddenly at variance with the growth rate of klebsiella pneumoniae which is increasing gradually within all intervals. biofilm formation for the selected isolates at different incubation periods biofilm formation for the selected isolates was estimated by spectrophotometer after staining with crystal violet and biofilm curve was made according to the time intervals (0, 2, 4, 6, 8, 24, 36, 48, 60 hrs), starting from the zero point before table 1. screening of the bacterial biofilm forming isolates and measuring the formed biofilm bacterial isolate biofilm (mean ± sd) biofilm type bacterial isolate biofilm (mean ± sd) biofilm type s. aureus 0.23 ±. 0.68 moderate e. coli 0.24 ± .0.02 moderate mrsa 0.17 ±. 0.13 weak k. pneuomoniae 0.64 ± .0.03 strong s. pyogenes 0.29 ± .0.14 moderate p. aeruginosa 0.60 ± .0.06 strong s. agalactiae 0.24 ± .0.15 moderate s. marcescens 0.54 ± .003 strong s. mutans 0.27 ± .0.06 moderate p. mirabilis 0.21 ± .016 moderate s. mitis 0.19 ± .0.22 weak e. faecalis 0.19 ± .002 weak l. monocytogenes 0.65 ± .0.01 strong a. baumannii 0.64 ± .005 strong 403j contemp med sci | vol. 8, no. 6, november-december 2022: 400–407 s.d. jastaniah et al. original new strategies for inhibition of l. monocytogenes and k. pneumoniae biofilm formed. figure 2 showed that both isolates bacterial cells were increased gradually until reach to stationary phase after 24 hours and the biofilm became matured and stable with metabolically active cells. it was noticed from the curve that the biofilm formation rate of klebsiella pneumoniae is higher than that of listeria monocytogenes within short time intervals. 4antimicrobial sensitivity testing for the selected isolates the selected isolates were examined for sensitivity to several important commercial antibiotics by discdiffusion method according to kirby-baur technique described by baur et al. (wayne, 2017).18 using clinical and laboratory standards institute guidelines to interpret diameter of growth inhibition zone. table 2 showed that both isolates were resistant to about half of the antibiotics and they were slightly different from each other in the sensitivity results. exopolysaccharides extraction and estimation for the selected isolates biofilm was quantitatively estimated in terms of the quantity of exopolysaccharides (eps) produced by the strains by estimation of the total carbohydrate content in the eps of the selected isolates by spectrophotometric method and comparing with a standard of glucose solutions for confirmation l. monocytogense has more carbohydrate content in the eps layers (od: 1.523) than k. pneumoniae (od:1.351) where l. monocytogense carbohydrate content is equal to 0.21 ± 0.02 µg/mg of cells while k. pneumoniae carbohydrate content is approximately equal to 0.18 ± 0.03 µg/mg of cells (table 3). exopolysaccharides (eps) estimation for the selected isolates under different temperatures the total carbohydrate content in the eps of the selected isolate was estimated under different temperatures; 25°c, 30°c, 35°c, 37°c, 40°c and 45°c. figure 3 showed that both isolates were similar in the optimum temperature for eps and the sequent biofilm formation which was 37°c. it was clear that the two temperatures 35°c and 37°c were very approximate in the effect on eps measurements for both isolates and they were both optimum temperatures. temperature 25°c was the minimum for both isolates epss. as shown in figure 3, k. pneumoniae has mean of exopolysaccharides less than l. monocytogenes under different temperatures. exopolysaccharides estimation for the selected isolates under different media the total carbohydrate content in the eps of the selected isolate was estimated under different media. in figure 4, it was shown that k. pneumoniae has mean of exopolysaccharides less than l. monocytogenes under different media including tryptic soy broth, luria-bertani broth and brain heart infusion broth. bacteria grown in tryptic soy broth and brain heart infusion have the largest values of the exopolysaccharides for both isolates and they are considered as the optimum media while nutrient broth and muller-hinton broth have the lowest values of eps for both isolates. exopolysaccharides (eps) estimation for the selected isolates under different ph measures the total carbohydrate content in the eps of the selected isolate was estimated under different ph measures. as shown in fig. 1 bacterial growth curves estimation during definite time intervals for the two selected isolates, l. monocytogenes and k. pneumoniae. fig. 2 biofilm formation progress estimation during definite time intervals for the selected isolates; l. monocytogenes and k. pneumoniae. table 2. antimicrobial sensitivity testing for l. monocytogenes and k. pneumoniae antimicrobial agents dose inhibition zones (mm) antimicrobial agents dose inhibition zones (mm) l. monocytogenes k. pneumoniae l. monocytogenes k. pneumonia bacitracin 10 units r r amikacin 30 mcg 18 16 chloramphenicol 30 mcg r r ceftazidime 30 mcg r r penicillin g 10 units r r aztreonam 30 mcg r r polymyxin b 300 units r 10 piperacillin 100 mcg 17 r gentamicin 10 mcg 17 8 imipenem 10 mcg 35 30 neomycin 30 mcg 14 9 ciprofloxacin 5 mcg r 19 404 j contemp med sci | vol. 8, no. 6, november-december 2022: 400–407 new strategies for inhibition of l. monocytogenes and k. pneumoniae original s.d. jastaniah et al. table 3. the quantity of exopolysaccharide in the selected isolates; l. monocytogenes and k. pneumoniae bacteria absorbance (490 nm) epss (µg/mg of cells) l. monocytogenes 1.523 0.21 ± 0.02 k. pneumoniae 1.351 0.18 ± 0.03 fig. 3 exopolysaccharides (eps) measurements for listeria monocytogenes and klebsiella pneumoniae under different temperatures. fig. 4 exopolysaccharides (eps) measurements for listeria monocytogenes and klebsiella pneumoniae under different media. fig. 5 exopolysaccharides (eps) measurements for listeria monocytogenes and klebsiella pneumoniae under different ph measures. antibacterial effect of the plant extracts on the selected isolates with minimum inhibitory concentrations (mic) and sub-inhibitory concentrations (sub-mic) it was reported from the test that not all solved plant extract were effective with all isolates. both isolates were resistant to aqueous solved of arak extract (aa) and less sensitive to organic-solved cranberry (co) and pomegranate peel (po) extracts. thus, organic-solved arak (ao), aqueous-solved cranberry (ca) and pomegranate peel (pa) extracts were chosen. table 4 showed the antibacterial effect of the plant extracts on the selected isolates, the minimum inhibitory concentrations and the sub-inhibitory concentrations. the effect of plant extracts on l. monocytogense growth is more than on k. pneumoniae growth. both isolates were affected by the extract of arak and cranberry respectively. k. pneumoniae growth was not affected by pomegranate peel extract and was resistant. arak extract made the biggest inhibition zone on l. monocytogenes and the smallest inhibition zone on k. pneumoniae comparing to other selected plant extracts. the effect of the plant extracts on bacterial growth, eps production and biofilm formation figures 6–8 showed that there is an effect of the plant extracts; cranberry, pomegranate peel and arak respectively on bacterial growth, eps production and biofilm for both selected isolates. it was clear in figures 9 and 10 that the effect of plant extracts varies from one extract to another in the two different types of bacteria where eps and biofilm were the least possible with the extract of arak and cranberry respectively. table 5 provides all the descriptive measures (mean, sd, se, 95% ci, etc.) table 4. antibacterial effect of the plant extracts on the selected isolates with minimum inhibitory concentrations (mic) and sub inhibitory concentrations (sub-mic) isolate plant extract cranberry (aqueous) pomegranate peel (aqueous) arak (organic) iz (mm) mic (mg/ml) sic (mg/ml) iz (mm) mic (mg/ml) sic (mg/ml) iz (mm) mic (mg/ml) sic (mg/ml) l. monocytogenes 18.0 12.5 6.0 17 25 12.5 20 29 14 k. pneumoniae 16.0 25.0 12.5 0.0 nd nd 19 29 14 difference sig at p ≤ 0.05 sig at p ≤ 0.05 not sig at p ≤ 0.05 iz, inhibition zone; mic, minimum inhibitory concentration; sic, sub-inhibitory concentration; nd, not detected. figure 5, k. pneumoniae has mean of exopolysaccharides less than l. monocytogenes under different measures of ph. it was found that ph 7 and ph 7.6 have the largest values of the exopolysaccharides for both isolates and they are considered as the optimum ph while the cells grown at ph 6 and ph 8 have the lowest values of eps for both isolates. 405j contemp med sci | vol. 8, no. 6, november-december 2022: 400–407 s.d. jastaniah et al. original new strategies for inhibition of l. monocytogenes and k. pneumoniae significance difference between them. the value of f-statistic by anova for the effect of plant extracts on exopolysaccharides is 44.766 and the p-value is 0.002. since the p-value is <0.05, this means that not all means of exopolysaccharides for all groups are same and there was significant difference between them. discussion l. monocytogenes and k. pneumonia produced biofilms which is influenced by physical factors such as nutrient media composition, ph, temperature, and biological factors. bacterial biofilms are strongly linked to and controlled by bacterial quorum sensing (qs), a cell-to-cell communication system that allows bacteria to monitor population density and control physiological processes by releasing and receiving small signal molecules called autoinducers to trigger the expression of specific genes for biofilm formation.19 the significance of biofilm-related infections caused by indwelling medical implants and appliances, such as contact lenses, artificial joints, and catheters, has been emphasized. bacteria within the biofilm can endure, leading to repeated, chronic infections and the development of immunological and antibacterial resistance. compared to planktonic cells, bacteria that are in biofilms are more resistant to host defenses and conventional antibiotics. in fact, when cells are embedded in biofilms, they can become 10–1000 times more resistant to the actions of antimicrobial drugs.20 biofilm also enables gene transfer among bacteria which can lead to increase in the number of virulent strains2 that means transfer resistance genes within members of the biofilm micro-community.20 the pharmaceutical industry is actively researching plant-derived substances such as crude extracts of leaves, roots, and stems, as well as individual compounds isolated from these, essential oils and essential oil constituents for potential applications in the treatment. although there is now a lot of study on plants and their active ingredients, the main emphasis is on the antibacterial qualities against bacteria that form biofilms.21 plant extracts are increasingly being studied as alternative antimicrobial agents since they are safe, accessible, affordable, effective, and have a variety of phytochemicals with little to zero chance of building resistance.11 due to their nature-based methodology and affordability, products like these that combine conventional therapy with the most recent research find wider consumers.22 a large variety of phytochemicals with antibacterial activities against therapeutically relevant pathogens are produced by numerous plant species.21 literature has already documented experimental evidence of the use of the plant fig. 6 the effect of the plant extracts cranberry on bacterial growth, eps production and biofilm for both selected isolates; listeria monocytogenes and klebsiella pneumoniae. fig. 7 the effect of the plant extracts pomegranate peel on bacterial growth, eps production and biofilm for both selected isolates; listeria monocytogenes and klebsiella pneumonia. fig. 8 the effect of the plant extracts arak on bacterial growth, eps production and biofilm for both selected isolates; listeria monocytogenes and klebsiella pneumoniae. table 5. the effect of the plant extracts on biofilm and exopolysaccharides biofilm sum of squares df mean square f sig. between groups 0.155 3 0.052 19.516 0.008* within groups 0.011 4 0.003 total 0.166 7 exopolysaccharides between groups 1.613 3 0.538 44.766 0.002* within groups 0.048 4 0.012 total 1.661 7 *: significant difference at p ≤ 0.05. of biofilm and exopolysaccharides of the bacterial isolates with all plant extracts. the value of f-statistic by anova for the effect of plant extracts on biofilm is 19.516 and the p-value is 0.008. since the p-value is <0.05, this means that not all means of the biofilm for all groups are the same and thus there was 406 j contemp med sci | vol. 8, no. 6, november-december 2022: 400–407 new strategies for inhibition of l. monocytogenes and k. pneumoniae original s.d. jastaniah et al. phytochemicals such as peptides, unsaturated long-chain aldehydes, phenols or secondary plant metabolites, such as alkaloids, flavonoids, tannins, terpenes, and terpenoids, separated from essential oils, water extracts, or methanol and butanol soluble compounds for their potential antibacterial significance in the biomedical, and food industries.23 the main chemical components of the plant including polyphenolics, flavonoids, and triterpenoids10 can penetrate the bacterial cell wall and cytoplasmic membrane as typical lipophiles, disrupt the structure of the different layers of polysaccharides, fatty acids, and phospholipids and permeabilize them.24 plant extracts’ ability to dissolve biofilms relies on the type of solvent utilized, specifically whether it is methanolor ethanol derived extracts.25 in earlier investigations, the ability of plant extracts to reduce biofilm adherence and development on the surfaces was demonstrated.26 in some studies, it was reported that cinnamon essential oils can reduce or eliminate bacterial biofilms on a stainless-steel surface.24 lemon essential oils can eliminate biofilm on polypropylene surfaces.27 neem, cranberry extracts, peppermint and coriander essential oils can prevent bacterial cell adhesion.28 ginger extract can lower the signal molecules that bacteria use to communicate with each other, lower the formation of eps, and make it easier to separate biofilm from any surface.26 due to the emergence of bacteria strains that cause urinary tract diseases that are resistant to antibiotics, some specialized medicinal plant extracts may offer an alternative to shortterm coating for preventing biofilm formation on urinary catheters.29 pharmacological investigations revealed that ‘s arak stem extract (salvadora persica l.) has potent antibacterial and antiplaque effects on oral microorganisms.30 green tea polyphenols have been shown by schneider-rayman et al. (2021)31 to have an inhibitory effect on s. mutans dental biofilm by preventing the development of q.s and eps. additionally, one study demonstrated that plant extracts from moringa, curry, and guava were the most efficient in delaying the formation of dental multi-species biofilms by focusing on growth, adhesion, and coaggregation without interfering with the oral cavity’s homeostasis.22 according to quelemes et al. (2015),32 neem leaf extract had an inhibitory effect on the growth of mrsa biofilm and planktonic aggregations. with a lower dosage and no risk of resistance developing, myrrh oil has the potential to be a commercially viable antibiotic that eliminates persistent cells in the biofilm.33 cranberries exhibit anti-biofilm characteristics against p. aeruginosa and escherichia coli by inhibiting motility and blocking adhesion.34 according to one study, it was showed that pomegranate peel extract prevented biofilm formation as a result of preventing quorum sensing and swimming motility of y. enterocolitica.35 acinetobacter baumannii-related nosocomial infections require an alternative therapy because carbapenem resistance has increased. norwogonin, a component from the plant scutellaria baicalensis, appears to exhibit antibacterial properties against a. baumannii and methanol extract of the plant actinidia deliciosa reduced production of bacterial biofilm components.36 conclusion eps quantity produced from the bacteria depends on some effective factors such temperature, media contents and ph. aqueous-cranberry, aqueous-pomegranate peel and methanolic-arak extracts have antibacterial and antibiofilm activities. aqueous-cranberry and methanolic-arak extracts have antibiofilm activities on l. monocytogenes and k. pneumonia much higher than aqueous-pomegranate peel. although aqueous-pomegranate peel doesn’t have antibacterial effect on k. pneumonia, but it has antibiofilm effect on the same strain. thus, application of new natural approaches for inhibiting bacterial biofilm is urgently needed and important to prevent persistent and recurrent biofilm related infections. conflict of interest none.  references 1. mashhady, m.a., abkhoo, j., jahani, s., abyar, s., & khosravani, f. (2016). inhibitory effects of plant extracts on pseudomonas aeruginosa biofilm formation. international journal of infection, 3(4). 2. satpathy, s., sen, s.k., pattanaik, s., & raut, s. (2016). review on bacterial biofilm: a universal cause of contamination. biocatalysis and agricultural biotechnology, 7, 56–66. 3. quave, c.l., plano, l.r., pantuso, t., & bennett, b.c. (2008). effects of extracts from italian medicinal plants on planktonic growth, biofilm formation and adherence of methicillin-resistant staphylococcus aureus. journal of ethnopharmacology, 118(3), 418–428. 4. sadekuzzaman, m., yang, s., mizan, m., & ha, s. (2015). current and recent advanced strategies for combating biofilms. comprehensive reviews in food science and food safety, 14(4), 491–509. 5. borges, a., abreu, a., malheiro, j., saavedra, m.j., & simõe, m. (2013). biofilm prevention and control by dietary phytochemicals. cecav-centro de ciência animal e veterinária. 6. lou, z., hong, y., liu, y., song, x., ai, l., wang, h., . . . tang, y. (2014). effect of ethanol fraction of burdock leaf on biofilm formation and bacteria growth. european food research and technology, 239(2), 305–311. 7. guzman, j.p.m.d., de las alas, t.p.l., lucban, m.c., & sevilla, c.e.c. (2020). green tea (camellia sinensis) extract inhibits biofilm formation in acyl homoserine lactone-producing, antibiotic-resistant morganella morganii isolated from pasig river, philippines. heliyon, 6(10), e05284. 8. mclean, r.j., pierson iii, l.s., & fuqua, c. (2004). a simple screening protocol for the identification of quorum signal antagonists. journal of microbiological methods, 58(3), 351–360. 9. kazemian, h., ghafourian, s., heidari, h., amiri, p., yamchi, j.k., shavalipour, a., sadeghifard, n. (2015). antibacterial, anti-swarming and anti-biofilm formation activities of chamaemelum nobile against pseudomonas aeruginosa. revista da sociedade brasileira de medicina tropical, 48(4), 432–436. 10. husain, f.m., ahmad, i., al-thubiani, a.s., abulreesh, h.h., alhazza, i.m., & aqil, f. (2017). leaf extracts of mangifera indica l. inhibit quorum sensing– regulated production of virulence factors and biofilm in test bacteria. frontiers in microbiology, 8, 727. 11. chusri, s., sompetch, k., mukdee, s., jansrisewangwong, s., srichai, t., maneenoon, k., voravuthikunchai, s. (2012). inhibition of staphylococcus epidermidis biofilm formation by traditional thai herbal recipes used for wound treatment. evidence-based complementary and alternative medicine, 2012. 12. christensen, g.d., baldassarri, l., & simpson, w.a. (1995). [38] methods for studying microbial colonization of plastics. in methods in enzymology (vol. 253, pp. 477–500). academic press. 13. smitinont, t., tansakul, c., tanasupawat, s., keeratipibul, s., navarini, l., bosco, m., & cescutti, p. (1999). exopolysaccharide-producing lactic acid bacteria strains from traditional thai fermented foods: isolation, 407j contemp med sci | vol. 8, no. 6, november-december 2022: 400–407 s.d. jastaniah et al. original new strategies for inhibition of l. monocytogenes and k. pneumoniae identification and exopolysaccharide characterization. international journal of food microbiology, 51(2–3), 105–111. 14. dubois, m., gilles, k.a., hamilton, j.k., rebers, p. t., & smith, f. (1956). colorimetric method for determination of sugars and related substances. analytical chemistry, 28(3), 350–356. 15. heatley, n.g. (1944). a method for the assay of penicillin. biochemical journal, 38(1), 61. 16. semeniuc, c.a., pop, c.r., & rotar, a.m. (2017). antibacterial activity and interactions of plant essential oil combinations against gram-positive and gram-negative bacteria. journal of food and drug analysis, 25(2), 403–408. 17. plyuta, v.a., andreenko, j.v., kuznetsov, a.e. et al. (2013). formation of pseudomonas aeruginosa pao1 biofilms in the presence of hydrogen peroxide. the effect of the aiia gene. mol. genet. microbiol. virol, 28, 141–146. 18. wayne, pa. (2017). clinical and laboratory standards institute. performance standards for 205 antimicrobial susceptibility testing: 27th informational supplement. m100-s27. clinical 206 and laboratory standards institute. 19. sandasi, m., leonard, c., van vuuren, s., & viljoen, a. (2011). peppermint (mentha piperita) inhibits microbial biofilms in vitro. south african journal of botany, 77(1), 80–85. 20. famuyide, i.m., aro, a.o., fasina, f.o., eloff, j.n., & mcgaw, l.j. (2019). antibacterial and antibiofilm activity of acetone leaf extracts of nine underinvestigated south african eugenia and syzygium (myrtaceae) species and their selectivity indices. bmc complementary and alternative medicine, 19(1), 141. 21. namasivayam, s.k.r., & roy, e.a. (2013). anti-biofilm effect of medicinal plant extracts against clinical isolate of biofilm of escherichia coli. int. j. pharm. pharm. sci, 5(2), 486–489. 22. john, n.r., gala, v.c., & sawant, c.s. (2013). inhibitory effects of plant extracts on multi-species dental biofilm formation in-vitro. int j pharm bio sci, 4(2), 487–495. 23. harjai, k., bala, a., gupta, r.k., & sharma, r. (2013). leaf extract of azadirachta indica (neem): a potential antibiofilm agent for pseudomonas aeruginosa. pathogens and disease, 69(1), 62–65. 24. de oliveira, m.m.m., brugnera, d.f., do nascimento, j. a., batista, n. n., & piccoli, r. h. (2012). cinnamon essential oil and cinnamaldehyde in the control of bacterial biofilms formed on stainless steel surfaces. european food research and technology, 234(5), 821–832. 25. wojnicz, d., kucharska, a.z., sokół-łętowska, a., kicia, m., & tichaczek-goska, d. (2012). medicinal plants extracts affect virulence factors expression and biofilm formation by the uropathogenic escherichia coli. urological research, 40(6), 683–697. 26. nikolić, m., vasić, s., đurđević, j., stefanović, o., & čomić, l. (2014). antibacterial and anti-biofilm activity of ginger (zingiber officinale (roscoe)) ethanolic extract. kragujevac journal of science (36), 129–136. 27. millezi, f., pereira, m.o., batista, n., camargos, n., auad, i., cardoso, m., & piccoli, r. (2012). susceptibility of monospecies and dual‐species biofilms of staphylococcus aureus and escherichia coli to essential oils. journal of food safety, 32(3), 351–359. 28. bazargani, m.m., & rohloff, j. (2016). antibiofilm activity of essential oils and plant extracts against staphylococcus aureus and escherichia coli biofilms. food control, 61, 156–164. 29. adesina, t., nwinyi, o., & olugbuyiro, j. (2015). prevention of bacterial biofilms formation on urinary catheter by selected plant extracts. pak. j. biol. sci, 18(2), 67–73. 30. abou-zaid, a.a., abd-elmaguid, n.m., abd el-hafez, a., & amer, m.m. (2015). the effect of arak stems extracts on chemical characteristics, bacterial activity and sensory evaluation of beef sausage products. international journal of advances in agricultural and environmental engg (ijaaee), vol 2. 31. schneider-rayman, m., steinberg, d., sionov, r.v., friedman, m., & shalish, m. (2021). effect of epigallocatechin gallate on dental biofilm of streptococcus mutans: an in vitro study. bmc oral health, 21(1), 1–11. 32. quelemes, p.v., perfeito, m.l., guimarães, m.a., dos santos, r.c., lima, d.f., nascimento, c., eaton, p. (2015). effect of neem (azadirachta indica a. juss) leaf extract on resistant staphylococcus aureus biofilm formation and schistosoma mansoni worms. journal of ethnopharmacology, 175, 287–294. 33. bhattacharjee, m.k., & alenezi, t. (2020). antibiotic in myrrh from commiphora molmol preferentially kills nongrowing bacteria. future science oa, 6(4), fso458. 34. ulrey, r.k., barksdale, s.m., zhou, w., & van hoek, m.l. (2014). cranberry proanthocyanidins have anti-biofilm properties against pseudomonas aeruginosa. bmc complementary and alternative medicine, 14(1), 1–12. 35. oh, s.k., chang, h.j., chun, h.s., kim, h.j., & lee, n. (2015). pomegranate (punica granatum l.) peel extract inhibits quorum sensing and biofilm formation potential in yersinia enterocolitica. microbiology and biotechnology letters, 43(4), 357–366. 36. hickl, j., argyropoulou, a., sakavitsi, m.e., halabalaki, m., al-ahmad, a., hellwig, e., vach, k. (2018). mediterranean herb extracts inhibit microbial growth of representative oral microorganisms and biofilm formation of streptococcus mutans. plos one, 13(12), e0207574. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1302 78 j contemp med sci | vol. 4, no. 2, spring 2018: 78–81 research simple and fast development and validation of high-performance liquid chromatography method with uv determination of thiopental in rat plasma saeed rezaee,a ali asghar hemmati,b,c alireza malayeri,b,c,d and nima bakhtiarib,c 1department of pharmaceutics, school of pharmacy, zanjan university of medical science, zanjan, iran. bdepartment of pharmacology, school of pharmacy, ahvaz jundishapur university of medical science, khozestan, ahvaz, iran. cmedicinal planet research center, ahvaz jundishapur university of medical science, khozestan, ahvaz, iran. dnab’a al-hayat health research center, nab’a al-hayat foundation for medical sciences and health care, najaf, iraq. correspondence to alireza malayeri (email: armalayeri@yahoo.com). (submitted: 19 january 2018 – revised version received: 28 january 2018 – accepted: 22 march 2018 – published online: 26 june 2018) introduction thiopental (5-ethyl-5-(1-methylbutyl)-2-thiobarbituric acid)1 shown in fig. 1 is an ultra-short-acting agent of anesthetic, which was used from 1934 to till now.2 it is also indicated for hypoxic-ischemic injuries and status epilepticus in human.1 the anesthetic effect of thiopental according to structural activity analysis study is back to malonyl urea ring.1 there is a controversy about analgesic effect of this agent in pain model in laboratory animals. the recent study is accepting thiopental as cause hyperalgesia.3 but there was no any pharmacokinetic– pharmacodynamics analysis modeling (pk–pd) that explains this issue with focus of effect by concentration in effect compartment. for a new study, need acceptable method of thiopental determination is important. this finding causes the old agent of anesthetic back to one of hot topic of pharmacology study. although this drug is not new, but there is no method that is adjusted for determination of this agent in laboratory animal especially rat. materials and methods chemicals and reagents thiopental (t120000 sigma-aldrich european pharmacopeia reference standard, usa) was used as internal standards. 1-naphtylamine (8222910100 catalogue num, merck, germany) was used as an internal standard. chromatography grade of methanol and acetonitrile (merck, germany) were used as mobile phase. blood sample collection for blood sample collection, 10 male wistar rats were moved to pharmacology animal house 1 week before the study. animals were kept in a room with 12 cycles of light and dark. rats were fed with special food that was adjusted in nutrition for lab animal (khorak-dam pars, tehran, iran). the water was available for animal ad libidum.4 rats were taken to surgery in department of pharmacology. the surgery was catheterized with silicone catheter (sil-c35 solomon, usa). after the surgery, the blood sample was collected. the amount of blood collection was under ahwaz ethical committee protocol. all types of study on animal parts were done under supervision of ahwaz lab animal ethical committee with registry code ir.ajums.rec.1395.137. preparation of standards samples thiopental and internal standard stocks solution were prepared separately by dissolving the accurately weighted standard compounds in methanol to give final concentrations of 1,000 and 70 µg/ml, respectively. the stock solutions were further diluted with mobile phase that mentioned below to achieve the spiking working standards. the spiking standard solutions (20 μl) were used to spike blank rat plasma samples (200 μl), for both calibration curves and qc samples during method validation study. all the working standards were kept at 4°c until the time of analysis. chromatography conditions the chromatography system consisted of a knaur® smartline series hplc pump, smartline® series photo diode array wave issn 2413-0516 objective nowadays old agent thiopental back to hot-topic of research as a new model of neuropharmacology study. because of its simple, fast, and cost-benefit method of determination, this agent comes with an issue in pharmacokinetic and pharmacodynamics (pk–pd) area of research. previous study was focused on human plasma and pilot study showed that there is differentiation between human and rat plasma. methods separation was performed on a nuleodur c18 ec hplc column (250 × 4.0 × 5 µm), using a mixture of acetonitrile; potassium; dihydrogen phosphate buffer (10 mm, ph 2.7) as the mobile phase delivered at a flow rate of 1.2 ml/min, 280 nm and room temperature was selected for detection of thiopental and 1-naphtylamine as an internal standard. plasma sample (100 µl) were treated with 180 µl precipitation solution and 20 µl of internal standard. after the mixture were vortexed and centrifuged at 1,000g, 40 µl of clear supernatant was directly injected into a 20-µl loop of hplc apparatus. calibration curve was fitted by peak area ratio of thiopental to internal standard. all the stages of blood collection were under supervision of ahvaz ethical committee. results thiopental and internal standard retention time were at 8.3 and 14.5 min, respectively. about 0.5 µg/ml was the level of limit of quantification of our method. the range of 99.4–100.3 was accuracy of the methods. interand intra-day precisions were 4–19% and 6–8%, respectively. a good relationship in the form of the power was found (r2 = 0.999). conclusion the presented simple, fast, and cost-benefit method was a great accurate, precise, and sensitive for determination of thiopental in plasma of rat. keywords chromatography, liquid, thiopental, plasma, rats saeed rezaee. 79j contemp med sci | vol. 4, no. 2, spring 2018: 78–81 research simple and fast development and validation of hplc method length uv–visible detector and chromegate® software for data acquisition and integration (knaur® corporation, germany). chromatography was performed on a nucleodur® c18 ec column (250 × 4.0 mm2 i.d., 5 μm particle size). thiopental and internal standard were detected at 280 nm. the mobile phase was an isocratic mixture of acetonitrile:methanol:potassium di-hydrogen phosphate buffer (10 mm, ph 2.7) (40:10:50). flow rate was set at 1.2 ml/min. chromatography was performed at ambient temperature. sample preparation plasma samples (100 µl) were treated by addition of 20 µl internal standard (70 µg/ml) and 180 µl modified precipitation solution. after vortex mixing and centrifugation at 10,000 g, 50 µl of the clear supernatant was directly injected into the chromatography column. results photo diode array analysis of sample showed 280 nm was optimized and high sensitive for detection of thiopental and naphtylamine. for better separation between spike wave of plasma and naphtylamine, and gained sharped and suitable shape of 25 cm c18 column was selected. the selected internal standard (naphtylamine) is well separated from spike wave of plasma of rat and thiopental. in fig. 2, rat blank plasma was shown and there was not any remarkable interface between thiopental and internal standard as shown in the figure. in fig. 3, the final optimized condition was shown. cause retention time of internal standard and thiopental recorded as 8.3 and 14.5, respectively. for clean-up of plasma, direct precipitation method was used. the optimized solution for plasma clean-up in this technique was combination of methanol, tca 10%, and zinc sulphate. our pilot study showed our optimized solution result in good recovery in comparison each of: methanol, tca 10%, and acetonitrile. regression analysis was shown that calibration curve was fit with power equation. the equation was c = par 12 2 1 1 9889 . . that from peak area ratio the amount of concentration is calculated. in the above equation, par is the peak area ratio of the external to internal standard, c is the concentration of the standard samples of thiopental sodium. the limit of detection and quantification was 0.001 and 0.05, respectively. accuracy was in the range of 99.4–100.3 that is shown in table 2. during the stability studies at different conditions (like three cycles of freeze and defrost) were within 15% of the initial nominal concentration. the method was stable and also instability was seen. discussion all the reports are about to determination and quantification of thiopental in human plasma.5–8 gas chromatography (gc) is the main method for detection of thiopental in plasma.9–11 although pervious methods were differ in the level limit of quantification (loq) in human. level of loq is important in pharmacokinetic (pk) study but in lab animal pk study, this importance is more. by accounting inter-individual variability in rat that cause different dose administration, the amount of dose was not transgression 300 µg (as bolus) per rat. disposition of this drug is very fast in rats.12 in previous gc method, the loq was in the range of 5,13 10,14 25,11 and 200 µg/l.10,15. in previous hplc uv detection method by organic agent protein precipitation, the loq was 0.025–1 mg/l.16–19 in our study, loq was 0.5 µg/ml and have many reasons, such as modified protein precipitation, using internal standard, etc. fig. 1 thiopental and isomer structure formulae. fig. 2 rat blank plasma chromatogram. table 1. parameters of the standard curve equations (par = a ×c b) parameter par value se p-value a 12.2 0.04883 <0.0001 b 0.9889 0.0009 <0.0001 r2 0.999 – sy.x 2.054 – rmse 2.027 – aicc 60.77 – 80 j contemp med sci | vol. 4, no. 2, spring 2018: 78–81 simple and fast development and validation of hplc method research saeed rezaee. in human, the major problem is shape of the peak and clean-up procedure for report of determination of thiopental and modification is a major need. uv detection was used based on the presence of malonyl rings in the structure of the tested compound. uv detection is suitable but range of detection changeable, and it seem our methods is most suitable in rat determination of thiopental for pharmacologic study in recent days. the thiopental is present in many model of study in rat and mice. the range of study is more important in basic study because the dose that is administrated in rat or mice is low and pharmacokinetic of this is show fast disposition. a c18 column were used in pervious detection of thiopental in human as stationary phases. high lipophilicity of the thiopental led to either long retention times or broad and bad-shaped peaks on c18 columns. use of long column and the optimized mobile phase resulted in elution of both the thiopental and the internal standard at reasonable retention times with good shaped peak. different extraction procedures such as liquid–liquid extraction with ethyl acetate, dichloromethane, diethyl ether and direct protein precipitation with either methanol, tca or acetonitrile were assessed. among these methods of sample clean up, protein precipitation with methanol, zinc sulphate, and tca was selected because of its simplicity, good shaped peak, and acceptable recovery of the analytes. it has been reported that meoh alone in the ratio of 2:1 of meoh to plasma sample could result in protein precipitation up to 91.4% of total plasma proteins.20. several barbiturate such as phenobarbital and so on were tested to be used as internal standard and none of them was not suitable and finally naphtylamine [(naphthalen-2-yl)amine] was selected due to well absorption at 280 nm. multiphasic disposition kinetics was detected following the administration of thiopental to rats, which could be related to fast distribution nature of the thiopental. acknowledgements this paper was extracted from part of ph.d thesis no. aprc9512 authors’ contribution all authors participated in the design, interpretation of the study, analysis of the data, and review of the manuscript; hplc experiments were conducted by nima bakhtiari. alireza malayeri was the coordinator of animal studies and saeed rezaee is the principal investigator and statistical analyst. financial disclosure there is no conflict of interest. funding/support this work was supported by a grant from vice chancellor for research affairs of ahvaz jundishapur university of medical sciences. references 1. russo h, bressolle f. pharmacodynamics and pharmacokinetics of thiopental. clinical pharmacokinetics. 1998;35:95–134. 2. lundy j, tovell r. some of the newer local and general anesthetic agents. methods of their administration. northwest med (seattle). 1934;33: 308–311. 3. steeds ce. the anatomy and physiology of pain. surgery (oxford). 2009;27:507–511. 4. council nr. guide for the care and use of laboratory animals: national academies press; 2010. 5. christensen jh, andreasen f. determination of thiopental by high pressure liquid chromatography. basic & clinical pharmacology & toxicology. 1979;44:260–263. 6. meier p, thormann w. determination of thiopental in human serum and plasma by high-performance capillary electrophoresis—micellar electrokinetic chromatography. journal of chromatography a. 1991;559:505–513. 7. shiu gk, nemoto em. simple, rapid and sensitive reversed-phase highperformance liquid chromatographic method for thiopental and pentobarbital determination in plasma and brain tissue. journal of chromatography b: biomedical sciences and applications. 1982;227:207–212. 8. sennello lt, kohn fe. gas chromatographic determination of thiopental in plasma using an alkali flame ionization detector. analytical chemistry. 1974;46:752–755. 9. carroll f, smith d, mark l, brand l, perel j. determination of optically active thiopental, thiamylal, and their metabolites in human urine. drug metabolism and disposition. 1977;5:343–354. 10. becker ke. plasma levels of thiopental necessary for anesthesia. anesthesiology. 1978;49:192–196. 11. jung d, mayersohn m, perrier d. gas-chromatographic assay for thiopental in plasma, with use of a nitrogen-specific detector. clinical chemistry. 1981;27:113–115. 12. gustafsson ll, ebling wf, osaki e, stanski dr. quantitation of depth of thiopental anesthesia in the rat. anesthesiology: the journal of the american society of anesthesiologists. 1996;84:415–427. 13. braddock li, marec n. the gas chromatographic analysis of sub-microgram quantities of barbiturates using a flame ionization detector. journal of chromatographic science. 1965;3:274–277. 14. schepens p, heyndrickx a. placental transfer of thiopental. eur j toxicol. 1975;8:87–93. 15. becker jr k. gas chromatographic assay for free and total plasma levels of thiopental. anesthesiology. 1976;45:656–660. 16. stanski dr, burch pg, harapat s, richards rk. pharmacokinetics and anesthetic potency of a thiopental isomer. journal of pharmaceutical sciences. 1983;72:937–940. 17. burch pg, stanski dr. decreased protein binding and thiopental kinetics. clinical pharmacology & therapeutics. 1982;32:212–217. table 2. recovery, intraand inter-day accuracy and precision results (n = 5) nominal concentration (µg/ml) percision (%) accuracy (% ± sd) recovery (%±sd) intra-day inter-day intra-day inter-day 0.05 37 67 99.41 ± 0.54 99.11 ± 0.67 64 ± 7 5 16 11 100.30 ± 0.50 100.10 ± 0.11 85 ± 13 10 2 21 101 ± 0.42 100.30 ± 0.34 89 ± 21 70 9 5 100 ± 0.19 100.00 ± 0.05 88 ± 8 100 2 5 100.2 ± 0.19 100.20 ± 0.05 83 ± 6 internal standard (µg/ml) – – – – 96 ± 6 saeed rezaee. 81j contemp med sci | vol. 4, no. 2, spring 2018: 78–81 research simple and fast development and validation of hplc method 18. christensen jh, andreasen f. individual variation in response to thiopental. acta anaesthesiologica scandinavica. 1978;22:303–313. 19. burch pg, stanski dr. the role of metabolism and protein binding in thiopental anesthesia. anesthesiology. 1983;58:146–152. 20. polson c, sarkar p, incledon b, raguvaran v, grant r. optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography–tandem mass spectrometry. journal of chromatography b. 2003;785:263–275. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms. 06201805 300 j contemp med sci | vol. 3, no. 12, autumn 2017: 300–305 research department of molecular and medical biotechnology, college of biotechnology, al-nahrain university, aljadriya, baghdad, iraq. correspondence to: hassan mohammad naif (e-mail: drnaifhassan@gmail.com). (submitted: 27 august 2017 – revised version received: 02 september 2017 – accepted: 11 october 2017 – published online: 26 december 2017) objective non-hodgkin lymphomas are a diverse lymphoid neoplasms and it is the sixth most common cancer in the world, but it is well established that immune dysregulation especially by cytokines is one of major risk factors. this study investigates the association of genetic polymorphisms of il-12p401188a/c and its circulating serum measurement in patients with advanced stage of nhls in iraq. methods fifty-five confirmed patients with advanced stages of nhls and 40 apparently healthy individuals were used. genetic polymorphism and circulating levels of il-12p40 were examined by allele-specific polymerase chain reaction (as-pcr) using specific primers and elisa, respectively. results the results of snp il-12p40 1188a>c had three genotypes, which were aa, ac and cc. these genotypes represented 32.73%, 38.18% and 29.09%, respectively, among nhl patients and 52.5%, 37.5% and 10%, respectively, among controls with a significant difference for the homozygous mutant genotype (or = 4.667, 95% ci = 1.319–16.512, p = 0.017). serum levels of il-12p40 were significantly increased in an allele-dependent manner, which was linked to the cc homozygosity as being the highest. conclusions this study has revealed a significant correlation between the gene polymorphisms of il-12p40 and the induction of serum il-12 with the risk to poor prognosis in patients with nhl. the correlation with the nhl grades and the prognostic value warrant further progressive investigation of developing prognostic biomarkers for nhl using patients at various disease stages. keywords non-hodgkin’s lymphoma, single nucleotide polymorphism, il-12, allele-specific pcr introduction non-hodgkin lymphomas are a diverse group of mature lymphoid neoplasms with a wide range of cellular, histologic presentations, cells of origin and etiologies.1 numerous environmental and genetic factors have been documented to be associated with the incidence of nhls, however the exact causes are beyond the current knowledge.2 regardless of causes, the integrity of the immune system represents the cornerstone in the resistance or progression of the disease. many disorders of this system such as immune deficiency and autoimmune disease like rheumatoid arthritis and systemic lupus erythematosus are reported to predispose to nhls.3 interleukin-12 (il-12) is among the main proinflammatory cyto kines of the immune system. il-12 is a multifunctional cytokine acting as a key regulator by inducing and maintaining t helper 1, which bridges innate and adaptive immunity,4 and mediates immune response in several types of cancers,5,6 and its p70 subunit is associated with nhl.7 it is a heterodimeric proinflammatory cytokine formed by a 35,000-dalton light (p35) and a 40,000-dalton heavy chain (p40), which are encoded by the il-12a and il-12b genes, respectively, and mapped to human chromosomes 3p12-q13.2 and 5q31-33.8 il-p40 (il-12b) encodes the p40 subunit of both pathways of il-12 and il-23.9 the inherited variations in il-12p40 and il-12p70 genes that could modulate cancer susceptibility, and as a consequence possess predictive, therapeutic or prognostic implications.10 following the discovery of il-12, three other members (il-23, il-27, and il-35) have been added to the il-12 family and shown to play critical roles in th1 cell functions.11,12 several single nucleotide polymorphisms have been identified in the il-12 gene such as 3’utr 1188 a/c polymorphism, which is associated with different diseases. several polymorphisms have been described in the promoter region,13 and in the 3’ untranslated region (utr) of il-12 p40 gene, il-12b.14 in this study, we hypothesized that potential functional polymorphisms il-12p40 may contribute to risk of nhl in population from iraq. hence, snps in il-12p40 gene was examined to assess its effect on the susceptibility to nhls. materials and methods study subjects this case control study included 55 patients with confirmed nhls during the period from january 2015 to december 2015 from six teaching hospitals and medical centers in baghdad medical city (teaching hospital of pediatric, baghdad teaching hospital and hematology center, al-mustansiriya university, al-kadhimyia teaching hospital). family unrelated, apparently healthy 35 individuals were randomly selected to represent the control group. the mean ages of patients and control were 33.45 and 36.49 years, respectively. the stage of nhl disease of those recruited patients was mainly at the advanced stage of disease, so results will indicate this stage as such with no treatment history. this research was approved by the ethics committee of scientific research of the university. an informed consent to take part in the study was obtained from each participant after receiving approval of the experimental protocol by the ethics committee, which was essentially in line with the principles of the declaration of helsinki. issn 2413-0516 association of gene polymorphism and serum levels of il-12p40 with the susceptibility to non-hodgkin’s lymphoma in iraq hassan mohammad naif hassan mohammad naif 301j contemp med sci | vol. 3, no. 12, autumn 2017: 300–305 research association of gene polymorphism and serum levels dna extraction and genotyping three ml of venous blood was collected from each participant in edta tube. dna was extracted from blood samples using ready kit (gsynctm dna mini kit whole blood protocol, geneaid, korea) according to the manufacturer’s instructions. primers used for both genes are shown in table 1. pcr conditions for il-12p40 gene were an initial denaturation for 5 min at 95°c, followed by 30 cycles of denaturation at 94°c for 30 s, annealing at 61°c for 30 s and extension at 72°c for 1 min, and final extension at 72°c for 7 min. the primers of toll-like receptor (tlr2) were used as an internal control in the amplification of il-12p40 gene. a ready 50 µl pcr master mix (bioneer, korea) was used for amplification for il-12p40 gene. template dna (10 µl) from each sample and primers (5 µl from each) was added to each master mix tube. after mixing, the master mix tubes were transferred to the thermocycler (mygenie 32 thermal block, bioneer, korea) which is previously programmed with the above protocol according to the gene to be amplified. the amplified products were determined by comparison with a commercial 1000 bp ladder (kappa biosystem, usa). statistical analysis the statistical package for the social sciences (spss, version 14) was used for statistical analysis. the risk association between the genotype and nhls susceptibility was estimated by the calculation of the adjusted odd ratio and 95% confidence intervals using bivariate logistic regression. for this analysis, subjects who were homozygous for the wild type allele were considered as a reference, and polymorphisms as dependent variables. chi square test (χ2) was used to determine the significant difference between each two alleles. a p-value < 0.05 was considered statistically significant. results detection of il-12p40 polymorphism by using allele-specific pcr this study investigated the association of il-12p40 1188a/c polymorphism with an incidence of nhl in patients and controls. a total of 55 nhl patients and 40 apparently healthy control individuals were recruited for this purpose. results of allele-specific pcr for the snp il-12p40 1188a/c in nhl patients and controls revealed specific amplification of 780 bp together with 254 bp of the internal control (fig. 1). il-12p40 1188a/c had three genotypes which were aa, ac and cc. these genotypes represented 32.37%, 38.18% and 29.09%, respectively, in nhl patients and 52.5%, 37.5% and 10%, respectively, among controls. a significant difference for the homozygous mutant genotype cc (or = 4.667, 95% ci = 1.319–16.512, p = 0.017). at allelic level, the frequency of c allele in nhl patient was 48.18% compared with 28.75% in controls with significant difference (or = 2.304, 95% ci = 1.250–4.249) (table 2). il-12p40 levels in nhl patients and controls in regard to il-12 production in this study, figure 2 shows the mean serum levels of il-12p40 in nhl patients and controls. nhl patients produced higher levels of il-12p40 table 1. nucleotide sequences of primer sets and their corresponding genes gene primers 5’→3’ amplicon reference il-12p40 consensus f: atcttggagcgaatgggcat r1: ttgtttcaatgagcatttagcatct r2: gtttcaatgagcatttagcatcg 780 bp [15] tlr2 (internal control) f: cctggcaagtggaccattgac r: ggccactccaggtaggtctt 254 bp [16] f, forward primers set; and r, reverse primer pairs; tlr, toll-like receptor. fig. 1 genotyping of il-12p40 1188a/c allele’s distribution in patients with nhl by using allele-specific (as)-pcr. gel electrophoresis and detection method were as described for figure 3-1. m: 100 bp dna marker. the 780 bp represents the amplification of il-12p40 1188a/c, while the 254 bp represents the amplification of tlr2 gene as an internal control. the il-12p40 a/c run side-by-side on the gel and the lanes order for il-12p40 a are 1, 3, 5, 7, and 9 whereas il-12p40 c alleles are run in lanes 2, 4, 6, 8, and 10. 302 j contemp med sci | vol. 3, no. 12, autumn 2017: 300–305 association of gene polymorphism and serum levels research hassan mohammad naif (156.44 ± 54.188 pg/ml) than the control group (107.34 ± 56.957 pg/ml) with a significant difference (t = 4.136, p = 0.001 (fig. 2). analysis of the correlation of serum levels of il-12p40 among the three genotypes (aa, ac and cc) in nhl patients revealed that cc genotype carriers had higher levels of il-12p40 (119.51 ± 55.678 pg/ml) than either ac genotype carriers (138.68 ± 63.102 pg/ml) or aa genotype carriers (155.35 ± 61.877 pg/ml) with significant differences between cc and aa genotype (p = 0.04) (fig. 3). table 2. genotypes and alleles of snp of il-12p40 1188a/c in patients and controls variables cases n = 55 control n = 40 p-value or (95% ci) il-12p40 1188a/c aa ac cc 18(32.73%) 21(38.18%) 16(29.09%) 21(52.5%) 15(37.5%) 4(10%) 0.056 0.293 0.017 1.0 1.633(0.655–4.074) 4.667(1.319–16.512) alleles a c 57(51.82%) 53(48.18%) 57(71.25%) 23(28.75%) 0.007 1.0 2.304(1.250–4.249) n, total number; or, odds ratio; ci, confidence interval; p, probability. fig. 2 levels of il-12p40 in the sera of nhl patients and controls as measured by an elisa kit. fig. 3 the relationship between the levels of il-12p40 and the three genotypes: aa, ac and cc of the snp il-12p40 1188a/c. hassan mohammad naif 303j contemp med sci | vol. 3, no. 12, autumn 2017: 300–305 research association of gene polymorphism and serum levels discussion the current cross-sectional study revealed that the snp of il-12p40 1188a/c (rs3212227) had three genotypes: aa, ac and cc. both ac and cc genotypes were associated with a significant increased risk to nhl in studied patients at advanced stage of disease compared to healthy controls. the mean circulating serum levels of il-12p40 in nhl patients had significantly higher levels than the control group. this elevation was linked with the cc homozygosity as being the highest inducer, and aa was the lowest. il-12 has a pleotropic activities where it plays an important role to induce th1 immune response,17 and cancer immunotherapy.18,19 it also can stimulate ifn-gamma production, activation of cytotoxic t and natural killer cells, and impairs angiogenesis in tumours.5 it can suppress tumour development due to induction of apoptosis in cancerous cells.20 il-12 therapy increased the median number of circulating cd8 t lymphocytes in patients with relapsed nhl.21 in the present study, the snp il-12p40 1188ac/cc genotypes were associated significantly with an increased risk of nhl patients in iraq population. there is a general consensus that ac/cc genotypes are associated with an increased risk to overall cancer development. this was exemplified by similar association between the c allele of il-12p40 1188a/c polymorphism and bladder cancer,22 cervical cancer,23 nasopharangeal carcinoma,24,25 and esophageal cancer.26 notably, the 1188a/c polymorphism is also implicated in an increased risk for psoriasis and soriatic arthritis as well as type 2 diabetes susceptibility.27 it has been found that il-12p40 of the circulating serum cytokines were significantly associated with fl and/or dlbcl.28 on the contrary, the serum il-12p40 levels were significantly higher in controls than those in osteosarcoma patients with genotype cc and ac/cc were associated with the risk of osteosarcoma.29 furthermore, 1188a variant was shown to be correlated with reduced levels of il-12p40 subunit of il-12, while 1188c variant associated with increase this subunit of the cytokine,30,31 which is similar to the finding of the present study. however, several analyses indicated that the c allele could lead to a decrease in the expression of il-12p40, which consequently results in a lack of il-12 protein.22,32,33 on the other hand, the results of this study are inconsistent with a previous study,34 where the a allele was found but not c to be associated with different cancers including the nhl and in tb infected patients,35 that resulted in lower plasma levels of il-12 in normal control rather than in tb infected patients. thus, it is expected there will be an increase of il-12 associated cc genotype resulting in robust cell-mediated immune response, as with the current result that revealed a strong relationship c allele with high levels of il-12 which was more prevalent in nhls patients than in healthy controls. the significance of il-12p40 subunit in cc genotype carriers compared with either ac genotype carriers or aa genotype carriers indicates that the variant il-12p40 1188c causes an increase in the production of this subunit in an allele-dependent manner, as clearly demonstrated by this study where the nhl patients with homozygous mutant genotype (cc) had the highest level of il-12p40 subunit and have graded down with the heterozygous genotype (ac), to the lowest with the homozygous wild genotype (aa). whether this elevation can be attributed to the effective role of il-12 alone as anti-tumour therapeutic,10,36 is still not well understood considering the complex nature of other activities of the immune system in cancers in general and nhls in particular. with respect to the elevation of il-12 and its association with c allele, this issue is still controversial. it gets more complicated when the elevation of total il-12 levels was seen in lymph nodes tissue microenvironment,37 whether this overproduction includes il12p40 is not yet clear. the discrepancy is that the increased production of il-12 does not mean that il-12p70 is overproduced. the induction rather involves only the il-p40 subunit. the homodimers of this subunit antagonizes il-12p70 activity by binding to the β1 subunit of the il-12 receptor.38 therefore, the increased production of this subunit, in fact, causes reduction in the activity of il-12 and hence reduces the efficiency of cell-mediated response and increases the susceptibility to the malignancy. the snp il-12p40 1188a/c is located in the 3’utr of the gene.39 this region although does not encode for a protein, it can influence the amount of translated protein through other mechanisms including effects on mrna stability or on transcriptional activity.40 thus the snp affects the gene silences and could regulate the level of il-12p40 mrna expression.41 moreover, there was no association between the il-12p40 promoter genotypes and the severity of psoriasis was found.42 this snp is linked to the susceptibility to psoriasis and specifically accompanied by the overproduction of il-12p40, which suggests that il-12p40 may act as a proinflammatory mediator. however, such effect is more likely not to be attributed only to mutant il-12p40 rather than to be multifactorial and is warranted further analysis. there are other heterodimeric cytokines in addition to il-12p40, such as il-23, il-27, and il-35, whose subunits consist of either or both the il-12 p35 and p40 chains.43 its high affinity to il-23 receptor and hence abolish il-23 role in immune response.44,45 the main role of il-23 involves the stimulation of th17 cells to produce il-17,38 which has an important role in the immunity against nhl.46 therefore, specific therapeutic antibodies for either il-12 subunits may lead to the dysfunction of several cytokines during therapy.21 similarly, inherited variations of il-12 genes (particularly, functional snps) may dramatically alter the expression and/or protein structure of more than one cytokine. however, il-12 therapy in nhl patients warrants further careful investigation of the drug candidates. it is important to note that the high levels of il-12p40 were commonly seen in patients with a good prognosis,47–49 therefore it is suggested to serve as a tumour progression and/or prognostic marker. the latter aligned well the current study where the level of circulating il-12p40 combined with its genetic polymorphisms can efficiently serve as a good prognostic marker. this conclusion needs further analysis after addressing the limitations of this study. there are two caveats in this study. first, patients recruited were from an advanced stage of nhl, so results won’t reflect disease stages/progression. second, there were no results available on the pre-diagnosis cytokine levels of those patients. therefore, these limitations will be warranted by designing a larger scale additional investigations to precisely define the correlation between genetic polymorphism, cytokine levels with nhl outcomes and subtyping. on the other hand, the genetic variants of il-12p40 may or may not 304 j contemp med sci | vol. 3, no. 12, autumn 2017: 300–305 association of gene polymorphism and serum levels research hassan mohammad naif have relationship with the fact that the tumour develops metastasis which may suggest its role, if any, in initiation the cause rather than a consequence of metastasis.50 conclusion the snp of il-12p40 1188a/c had three genotypes: aa, ac and cc. both ac and cc genotypes of the snp il-12p40 1188a/c were associated with a significantly increased risk to nhls among iraqi patients. the allele frequency distribution was also correlated with an overproduction of circulating il-12p40 reaching its maximum levels with the cc mutant homozygosity in patients with advanced stage of nhl. taken together, the results strongly suggest that the allele c of the snp il-12p40 1188a/c may serve as a contributing risk factor for poor prognosis in patients with nhl. the correlation with the nhl grades and the prognostic value warrant further progressive investigation of developing prognostic biomarkers for nhl using patients at various disease stages. acknowledgements the author would like to thank ms. mayasah ali for her technical assistance and dr. qasim sherhan for manuscript comments and data analyses. n references 1. kim as. molecular aspects of non-hodgkin lymphomas. in: greer jp, arber da, glader b, list af, means rt, paraskevas f, rodgers gm and foerster j. (eds.). wintrobe’s clinical hematology. 13th ed. lippincott williams and wilkins, philadelphia. 2014;1801. 2. hartge p, smith mt. environmental and behavioral factors and the risk of non-hodgkin lymphoma. cancer epidemiol biomarkers prev. 2007;16: 367–368. 3. grulich ae, vajdic cm, cozen w. altered immunity as a risk factor for nonhodgkin lymphoma. cancer epidemiol biomarkers prev. 2007;16:405–408. 4. trinchieri g. interleukin-12 and the regulation of innate resistance and adaptive immunity. nat rev immunol. 2003;3:133–146. 5. del vecchio m, bajetta e, canova s, lotze mt, wesa a, parmiani g, et al. interleukin-12: biological properties and clinical application. clin cancer res. 2007;13:4677–4685. 6. chang sw, xu gq, fan yl. association of interleukin-12 gene polymorphisms with cancer susceptibility: a meta-analysis. int j clin exp med. 2015;8:5317–5322. 7. rabkin cs, engels ea, landgren o, schuurman r, camargo mc, ruth pfeiffer, et al. circulating cytokine levels, epstein-barr viremia, and risk of acquired immunodeficiency syndrome-related non-hodgkin lymphoma. am j hematol. 2011;86:875–878. 8. gately mk, renzetti lm, magram j, stern as, luciano adorini l, gubler u, et al. the interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. annual review immunol. 1998;16:495–521. 9. zheng hf, zuo xb, lu ws, li y, cheng h, zhu kj, et al. variants in mhc, lce and il12b have epistatic effects on psoriasis risk in chinese population. j dermatol sci. 2011;61:124–128. 10. lasek w, zagożdżon r, jakobisiak m. interleukin 12: still a promising candidate for tumor immunotherapy? cancer immunol immunother. 2014;63:419–435. 11. hamza t, barnett jb, li b. interleukin 12 a key immunoregulatory cytokine in infection applications. int j mol sci 2010;11:789–806. 12. vignali da, kuchroo vk. il-12 family cytokines: immunological playmakers. nat immunol. 2012;13:722–728. 13. morahan g, huang d, wu m, holt bj, white gp, kendall ge, et al. association of il12b promoter polymorphism with severity of atopic and non-atopic asthma in children. lancet. 2002;360:455–459. 14. huang d, cancilla mr, morahan g. complete primary structure, chromosomal localisation, and definition of polymorphisms of the gene encoding the human interleukin-12 p40 subunit. genes immun. 2000;1:515–520. 15. latsi p, pantelidis p, vassilakis d, sato h, welsh ki, du bois rm. analysis of il-12 p40 subunit gene and ifn-gamma g5644a polymorphisms in idiopathic pulmonary fibrosis. respir res. 2003;4:6. 16. chen l, lin mj, zhan ll, lv xp. analysis of tlr4 and tlr2 polymorphisms in inflammatory bowel disease in a guangxi zhuang population. world j gastroenterol. 2012;18:6856–6860. 17. becskei a, grusby mj. contribution of il-12r mediated feedback loop to th1 cell differentiation. febs lett. 2007;581:5199–5206. 18. zabala m, lasarte jj, perret c, sola j, berraondo p, alfaro m, et al. induction of immunosuppressive molecules and regulatory t cells counteracts the antitumor effect of interleukin-12-based gene therapy in a transgenic mouse model of liver cancer. j hepatology. 2007;47:807–815. 19. yuzhalin ae, kutikhin ag. interleukin-12: clinical usage and molecular markers of cancer susceptibility. growth factors. 2012;30:176–191. 20. yuzhalin ae, kutikhin ag. interleukin-12: clinical usage and molecular markers of cancer susceptibility. growth factors. 2012;30:176–191. 21. younes a, pro b, robertson mj, flinn iw, romaguera je, hagemeister f, et al. phase ii clinical trial of interleukin-12 in patients with relapsed and refractory non-hodgkin’s lymphoma and hodgkin’s disease. clin cancer res. 2004;10:5432–5438. 22. jaiswal pk, singh v, srivastava p, mittal rd. association of il-12, il-18 variants and serum il-18 with bladder cancer susceptibility in north indian population. gene. 2013;19:128–134. 23. chen x, jiang j, shen h, hu z. genetic susceptibility of cervical cancer. j biomed res. 2011;25:155–164. 24. wei ys, lan y, luo b, lu d, nong hb. association of variants in the interleukin-27 and interleukin-12 gene with nasopharyngeal carcinoma. mol carcinog. 2009;48:751–757. 25. ben chaaben a, busson m, douik h, boukouaci w, mamoghli t, chaouch l, et al. association of il-12p40 +1188 a/c polymorphism with nasopharyngeal cancer risk and tumor extension. tissue antigens. 2011;78:148–151. 26. tao yp, wang wl, li sy, zhang j, shi qz, zhao f, et al. associations between polymorphisms in il-12a, il-12b, il-12rβ1, il-27 gene and serum levels of il-12p40, il-27p28 with esophageal cancer. j cancer res clin oncol. 2012;138:1891–1900. 27. eirís n, gonzález-lara l, santos-juanes j, queiro r, coto e, coto-segura p. genetic variation at il12b, il23r and il23a is associated with psoriasis severity, psoriatic arthritis and type 2 diabetes mellitus. j dermatol sci. 2014;75:167–172. 28. charbonneau b, maurer mj, ansell sm, slager sl, fredericksen zs, ziesmer sc, et al. pretreatment circulating serum cytokines associated with follicular and diffuse large b-cell lymphoma: a clinic-based case-control study. cytokine. 2012;60:882–889. 29. wang j, nong l, wei y, qin s, zhou y, tang y. association of interleukin-12 polymorphisms and serum il-12p40 levels with osteosarcoma risk. dna cell biol. 2013;32:1–6. 30. ling p, gately mk, gubler u, stern as, lin p, hollfelder k, et al. human il-12 p40 homodimer binds to the il-12 receptor but does not mediate biologic activity. j immunol. 1995;154:116–127. 31. wong rh, wei jc, huang ch, lee hs, chiou sy, lin sh, et al. association of il-12b genetic polymorphism with the susceptibility and disease severity of ankylosing spondylitis. j rheumatol. 2012;39:135–140. 32. ognjanovic s, yuan jm, chaptman ak, fan y, yu mc. genetic polymorphisms in the cytokine genes and risk of hepatocellular carcinoma in low-risk nonasians of usa. carcinogenesis. 2009;30:758–762. 33. ma x, yan w, zheng h, du q, zhang l, ban y, et al. regulation of il-10 and il-12 production and function in macrophages and dendritic cells. f1000 faculty rev. 2015;4:1465. 34. yang z, liang y, qin b, zhong r. meta-analysis of the association between the il-12b +1188 a/c polymorphism and cancer risk. onkologie. 2013;36:470–475. 35. peresi e, oliveira lr, da siva wl, da costa eap, araujo jp, ayres ja, et al. cytokine polymorphisms: their influence and levels in brazilian patients with pulmonary tuberculosis during antituberculosis treatment. tuberc res treat. 2013; 2013:285094. 36. tugues s, burkhard sh, ohs i, vrohlings m, nussbaum k, vom berg j, et al. new insights into il-12-mediated tumor suppression. cell death differ. 2015;22:237–246. hassan mohammad naif j contemp med sci | vol. 3, no. 12, autumn 2017: 300–305 research association of gene polymorphism and serum levels 37. spachacz r, kasprzak a, stefańska k, trejster e, seidel j, zabel m. tissue expression of cytokines (il-1alpha, il-2, il-6, il-12, tnf-alpha) in b-cell lymphomas in children. folia morphol. 2003;62:483–484. 38. teng mw, bowman ep, mcelwee jj, smyth mj, casanova jl, cooper am, et al. il-12 and il-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases. nat med. 2015;21:719–729. 39. seegers d, zwiers a, strober w, pena as, bouma ga. taqi polymorphism in the 3’utr of the il-12 p40 gene correlates with increased il-12 secretion. genes immunol. 2002;3:419–423. 40. matoulkova e, michalova e, vojtesek b, hrstka r. the role of the 3’ untranslated region in post-transcriptional regulation of protein expression in mammalian cells. rna biol. 2012;9:563–576. 41. kaarvatn mh, vrbanec j, kulic a, knezevic j, petricevic b, balen s, et al. single nucleotide polymorphism in the interleukin 12b gene is associated with risk for breast cancer development. scand j immunol. 2012;76:329–335. 42. litjens nh, van der plas mj, ravensbergen b, numan-ruberg sc, van assen y, thio hb, et al. psoriasis is not associated with il-12p70/il-12p40 production and il12b promoter polymorphism. j invest dermatol. 2004;122:923–926. 43. abbas, ak, lichtman ah, pillai s. cellular and molecular immunology. philadelphia, pa: elsevier. 2011. 44. shimozato si, ugai, m, chiyo m, takenobu h, nagakawa h, wada a, et al. the secreted form of the p40 subunit of interleukin (il)-12 inhibits il-23 functions and abrogates il-23-mediated antitumour effects. immunol. 2006;117:22–28. 45. uhlig hh, mckenzie bs, hue s, thompson c, joyce-shaikh b, stepankova r, et al. differential activity of il-12 and il-23 in mucosal and systemic innate immune pathology. immunity. 2006;25:309–318. 46. lu t, yu s, liu y, yin c, ye j, liu z, et al. aberrant circulating th17 cells in patients with b-cell non-hodgkin’s lymphoma. plos one. 2016;11:e0148044. 47. jones ea, pringle jh, angel ca, rees rc. th1/th2 cytokine expression and its relationship with tumor growth in b cell non-hodgkin’s lymphoma (nhl). leuk lymphoma. 2002;43:1313–1321. 48. klinke dj. a multiscale systems perspective on cancer, immunotherapy, and interleukin-12. mol cancer. 2010;9:242. 49. youssef ss, mohammad mm, ezz-el-arab lr. clinical significance of serum il-12 level in patients with early breast carcinoma and its correlation with other tumor markers. macedonian j med sci. 2015;3:640–644. 50. zhu kj, zhu cy, shi g, fan ym. meta-analysis of il-12b polymorphisms (rs3212227, rs6887695) with psoriasis and psoriatic arthritis. rheumatol int. 2013;33:1785–1790. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201703 305 260 j contemp med sci | vol. 3, no. 11, summer 2017: 260–263 original dental education in iraq: issues, challenges and future mohammad hossein khoshnevisan,1,2 ammar n.h. albujeer,3,4,5 abbas taher,5 alya almahafdha5,6 1preventive dentistry research center, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran. 2community oral health department, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 3department of stomatology, school of medicine and dentistry, santiago de compostela university, santiago de compostela, spain. 4school of dentistry, tehran university of medical sciences, tehran, iran. 5nab’a al-hayat foundation for medical sciences and health care, najaf, iraq. 6school of medicine, international campus, tehran university of medical sciences, tehran, iran. correspondence to ammar n. h. albujeer (email: ammar.dent@yahoo.com). (submitted: 15 may 2017 – revised version received: 28 may 2017 – accepted: 05 june 2017 – published online: 26 september 2017 ) objective medical sciences are the substance for appreciation of prevention and treatment of human diseases and improvement of one’s quality of life. dentistry on the other hand, is a highly demanding profession that requires standard and evidence-based training, so that upon graduation, dentists would be well prepared and qualified for challenging career. therefore, using a dental curriculum with updated clinical and scientific foundation can help meeting the above objectives and many other requirements. likewise, a dental curriculum is required to develop and improve skills, maintain comprehensive professional standards to provide more effective and efficient services to individuals and communities. given that the dental education can have a direct effect on oral health as well as overall health status at the national level, the aim of this paper was to review the current status of dental education system in iraq. there are thirty dental faculties in iraq currently. however, the oral health situation is in need of major improvement. this recognizes the importance of dental education reevaluation starting from curriculum structure, content, learning and assessment methods as well as administration and governance of dental education. there is a need for urgent attention in order to enable effective contribution of new dental graduates in improvement contribute in improvement and promotion of oral health for all citizens living in all communities at the national level. keywords dental curriculum, dental education system, oral health promotion, dmft index, iraq introduction geographically, iraq is located in strategically important area with multicultural population and abundant natural resources. historically, the iraqi civilization goes back to 7000 years ago. sumerians’ contribution to the field of education is well acknowledged. they had a great knowledge of medicine and other fields. they are also the ones who divided the circle into 360 degrees for the first time. back in nineteenth century, when modern education started, a few available faculties were used exclusively by small wealthy families. at the time, religious faculties as the principal centers, provided elementary and religious education to majority of children nationwide.1 it was not until 1970s that free compulsory education was established at all levels from primary to higher education. among others, baghdad and najaf were the two cities that were famous for having the outstanding scientific and religious centers of the time for several decades. the number of very famous physicians and scientists are attributed to universities in this country. however, numerous political unrests have disturbed the progress and advancement of science in this country. the first dental school started in 1953 as part of medical university in baghdad which was comparable with modern dental faculties of the time.2 subsequently, four other faculties were established as reported in table 1. the dental education was initially a 5-year program, using british curriculum. aside from local graduates, there are some foreign-trained dentists who are practicing dentistry in iraq as well.2 similar to some dental programs around the world, the training of dental students is mainly focused on treatment instead of prevention in iraq.3 this policy has resulted in oral health deterioration of iraqi population as expected. it seems that global standardization of dental education is inevitable for further improvement of oral health in iraq. dental education system by 2003, there were four dental faculties at the national level, with two of them had limited postgraduate programs. currently, there are 36 dental faculties (table 2), 16 public and 20 private, offering a 5-year program with b.d.s degree. the number of dental students in iraq is incredibly rising due to increasing number of admissions each year, especially by private dental faculties. it should also be noted that the number of iraqis studying dentistry abroad is increasing as well. there is an urgent need for the three ministries of health, higher education, and ministry of planning to re-address the total number of admissions for public and private faculties of dentistry as soon as possible. after admission, students take the basic medical and dental science courses during the first 2 years, and then focus on didactic and clinical dentistry preclinical training for the last 3 years. although, dental curriculum has been revised many times since 1953 to 2016, and revisions followed the new educational methodologies, such as evidence-based approaches, problem-based learning and case presentations through comprehensive patient treatment. however, the skills and competencies of new dental graduates are not translated into daily preventive practice yet. dental education program the curriculum for b.d.s. program in iraq has 46 subjects to be taken in 5 years. the teaching language is english, and subjects are taken in two stages as shown in table 3. issn 2413-0516 ammar n.h. albujeer et al. 261j contemp med sci | vol. 3, no. 11, summer 2017: 260–263 original dental education in iraq in stage i (first and second year), students have to complete applied and non-applied medical and dental basic sciences, passing 16 subjects. the courses for these 2 years include: medical physics (4 units), medical chemistry (4 units), medical biology (4 units), dental anatomy (3 units), computer (4 units), human rights (2 units), arabic literature language (2 units), embryology (3 units), dental materials (3 units), prosthodontics (5 units), oral histology (3 units), general histology (4 units), general anatomy (4 units), physiology (4 units) and biochemistry (4 units). the total hours of stage i are 750 hours for theory and 840 hours for practical training (demonstration and laboratory work). after successful completion and passing of these subjects, students will enter the second stage of the training program. in year 3–5 students, they take preclinical and clinical courses. the preclinical and clinical part in the stage ii has 118 units for the following subjects: prosthodontics (14 units), preclinical fixed prosthodontics and operative dentistry (8 units), oral surgery (13 units), community dentistry (3 units), pharmacology (4 units), general pathology (4 units), dental radiology (3 units), microbiology (4 units), infection control (1 unit), operative/ endodontics (8 units), periodontology (10 units), orthodontics (9 units), oral pathology (5 units), general medicine (4 units), general surgery (3 units), pedodontics (5 unit), clinical endodontics, clinical fixed prosthodontics (7 units), oral medicine (3 units), preventive dentistry (4 units), dental implantology (1 unit), forensic dentistry (1 unit), dental occlusion (1 unit) and research project (4 units). the total hours for stage ii is 4410 hours, with 1140 hours for lectures (theory) and 3270 hours for practical training.4 in addition, there are obligatory summer courses, which are about 20% of the total program score.5 in summary, students shall successfully complete and pass a total of 171 units in 5-year program as shown in table 3. since 2016, the ministry of higher education and scientific research added national exam under the name of “quality accreditation exam” for all graduated dental students take place after finishing the 5-year dental program. this is the mechanism that the government approved the quality of dental education for new graduates both in public and private institutions in the country. after passing the national exam, dental graduates will be eligible for the title of b.d.s, but should still fulfill 2 years of obligatory practice in rural primary health care centers and 1 year in hospital spatiality dental clinic in cities, before being table 1. profile of iraqi dental colleges dental schools information 2003 2016 dental colleges 4 30 dental students (undergraduate) 1,000 13,000 dental students (postgraduate) 10 150 total admission/year (undergraduate and postgraduate) 200 2,760 graduates/year 250 1,000–1,500 postgraduate programs 10 10–11 postgraduate disciplines 5 10–11 academic staff 50 350 schools for laboratory technicians 1 3 schools for hygienists 0 0 table 2. dental faculties in iraq no. university name founded 1 university of baghdad 1953 2 university of al mosul 1982 3 al-mustansiriya university 2000 4 babylon university 2002 5 university of tikrit 2004 6 basrah university 2005 7 university of kufa 2006 8 alyarmouk university college 2006 9 ibn hayyan university college 2009 10 al rafidain university college 2009 11 university of anbar 2010 12 university of karbala 2011 13 al-kafeel university college 2011 14 university of wasit 2012 15 university of maysan 2012 16 university of qadisiyah 2013 17 university of iraq 2013 18 university of muthanna 2013 19 al-israa university college 2013 20 dejlah university college 2013 21 al-hussein university college 2013 22 asul-dien university college 2013 23 university of kirkuk 2014 24 al-rashid university college 2014 25 national university of science and technology college 2015 26 uruk university college 2015 27 university of dhi qar 2016 28 al-safwah university college 2016 29 al-kitab university college 2016 30 al-hilla university college 2017 31 belad al-rafdein university college under construction 32 qurtuba university college under construction 33 al-mustafa al-amen university college under construction 34 al-bayyan university college under construction 35 al-mustaqbal university college under construction 36 al-ammed university college under construction able to obtain the license from the ministry of health to practice dentistry in a private clinic. oral health status the iraqi health care delivery system is largely hospital-based and capital-intensive model with less emphasis on prevention. public dental services are mainly provided by the ministry of health (moh) rather than the dental faculties’ affiliated hospitals that are operated by the ministry of higher education. 262 j contemp med sci | vol. 3, no. 11, summer 2017: 260–263 dental education in iraq original ammar n.h. albujeer et al. in addition, the private sector is actively providing dental services to the general population. the private sector is supervised and regulated by the moh as well. although, the moh provides curative and prevention care, the private sector is mainly focused on emergency and curative services. in addition to oral health status, aside from a few sporadic small surveys, there are two major national surveys of children conducted by the iraqi ministry of health over 2001 and 2012. the first survey was conducted in baghdad, ninevah and basrah provinces using 2040 samples of 15-year-old children. the last survey was conducted in five provinces (baghdad, erbil, najaf, salahdien and maysan) in 2012 using 3260 samples of 12-year-old children. the 2001 national survey of 15-year-old children in the three provinces demonstrated a dmft index of 1.35 in the 2040 participating students. the 2012 national survey was conducted by the iraqi moh in collaboration with the who and using the who recommended oral health assessment form (1997) for clinical assessment of 12-year-old children in five provinces. the dmft index for the 3260 participating students was 1.57 as shown in figure 1. oral health manpower before 2003, the estimated number of registered dentists was less than 4000 according to iraqi dental association records. in 2010, there were 4863 dentists with population ratio of 1.7/10,000.6 in 2015, there were 7277 dentists with population ratio of 2.3/10,000.7 currently, there are close to 8500 registered dentists, with dentist population ratio of 2.6 dentists for every 10,000 citizens. this number is growing rapidly with increasing number of dental graduates. a new objective is to achieve a new dentist population ratio of 6.4/10,000 by 2020.3 most of these dentists working in public sector in the morning and private clinics in afternoon, no more than 10% of them are working as a university staff as demonstrated in table 4. the number of dental technician is required to increase in order to better serve the dentists and the public. discussion several decades of sanction and war have completely disrupted the socio-political and economic structure in iraq, hampering progress in educational and scientific developments. as a result, most of the educational institutions including dental faculties require overall rehabilitation, including physical facilities, curriculum and dental education administration and governance as part of post-conflict reconstruction to name a few.1 the scarce public resources for oral health in iraq can never be sufficient to cover the cost of treatment for oral and dental diseases.7 therefore, the oral health needs of the iraqi population should be reflected in its dental curriculum. preventive dentistry should be a major focus of dental education. with the hopes that, through competent teaching and learning methods, graduating dentists would have acquired the necessary skills and competencies that can enable them to contribute to oral health improvement of the population at the national level. such curriculum should at the same time allow for student reflection, creativity and scientific inquiry. yet any dental curriculum must be reviewed and updated regularly. aside from learning science, and developing clinical skills, a dental graduate must learn ethics and the skills to become critical lifelong learner through continuing education and professional development. also, we must appreciate that education in general, is tremendously vital for social change. however, if the oral health status in iraq is not acceptable, there is a need for changing dental education system to benefit all citizens. faculty development is also very important. the way instructors teach dental students, has a profound effect on the direction of the profession in the future. the faculty can carefully evaluate and consider what role is expected of the fig.1 dmft index for 12-years-old children (n = 3260). table 3. dental colleges’ curriculum in iraq stage 1 year 1 applied and non-applied medical basic sciences. year 2 applied medical basic sciences and preclinical dental technique courses stage 2 year 3 preclinical dental technique courses and medical sciences year 4 completion of preclinical dental courses and start of clinical courses – with obligatory summer preclinical training courses. year 5 clinical dental course (practical & theory) – with obligatory summer clinical training courses. table 4. number of academic staff members in various departments of iraqi dental colleges department staff numbers oral and maxillofacial surgery 60 oral and maxillofacial pathology 10 oral and maxillofacial radiology 10 oral medicine and diagnosis 10 pediatric dentistry 30 restorative and aesthetic dentistry 70 periodontics 20 prosthodontics (removable & fixed) 70 orthodontics 40 endodontics 30 total academic staff members in dental colleges 350 ammar n.h. albujeer et al. 263j contemp med sci | vol. 3, no. 11, summer 2017: 260–263 original dental education in iraq this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dental professional to play in the community for both short and long term needs. we need to decide whether we want to “train” or “educate” our dental students in iraq? by training, we teach our students to do a list of things, the same way as others did in the past, while by educating we teach our students to think critically and independently, to do things when justified and question or refer when indicated. by education we can develop innovative dental graduates who are insightful, thoughtful and not simply a passive consumer. these dental professionals are the real agents for social and scientific change.3 on the other hand, a clear strategy should be made available for the number of dental faculties needed for iraq. given the current economic constraints of the country, establishment of any new standards in dental faculties requires enormous amounts of money as well as significant annual budgets to keep the faculties running. in absence of clear strategy, dental education would not be cost-effective; whether the capital is invested by public or private parties. even the quality of dental education is especially vulnerable and will likely deteriorate in the face of annual budget shortages in existing faculties. conclusion and recommendation in conclusion, dental education in iraq face many challenges. there are many approaches to meet these challenges, it is necessary to consider the local needs of the country, develop new strategies and define new governance for dental education to deal with acute problems and pave the way for educating a dentist with global standards for further improvement in oral health of the iraqi population at the national level. the dental education expanded considerably after 2003 in iraq, in spite of all challenges and shortcomings that occurred after 2003. the number of dental faculties increased by almost nine folds (from 4 to 36) with increased number of dental student enrollment by six folds (from 250 to 1500) annually, without marked changes in postgraduate programs and positions. in relation to dental technician faculties, the expansion does not match with the increasing number of new dental faculties. likewise, for the dental hygienist and dental nurses (assistants), there is extreme shortage of both, and there is a great need to incorporate these allied dental trainings to the existing dental faculties. dental hygienists can play extremely important role in oral disease prevention and oral health promotion at the local, national and international levels. in this regard, the global goals of oral health 2020 strategy can be used as a guide for national policy development.9 acknowledgement the authors are thankful to dr. ibtisam aboud, the former director of oral health center in the iraqi ministry of health for her assistance in providing the dmft data for this article. conflict of interest none. n references 1. ranjan rk, jain pc. the decline of the educational system in iraq. j peace stud. 2009;16(1-2). 2. university of baghdad cod. history of college of dentistry 2017 [available from: http://www.codental.uobaghdad.edu.iq/pageviewer. aspx?id=2. 3. albujeer an, taher a. dental education and oral health service in iraq. iranian j pub health. 2017;46:713–714. 4. research moheas. college of dentistry curriculum. 2016. 5. research moheas. university of baghdad, college of dentistry, curriculum. unpublished. 2016. 6. health mo. annual statistical report 2010. 292. 7. health mo. annual statistical report 2015. 2015. 8. allawi j. allocating resources in health care services. journal of contemporary medical sciences. 2017;3:237–238. 9 hobdell m, petersen pe, clarkson j, johnson n. global goals for oral health 2020. international dental journal. 2003;53(5):285–258. dx.doi.org/10.22317/jcms.09201703 59j contemp med sci | vol. 5, no. 1, january–february 2019: 59–63 short communication health-related quality of life in children with sickle cell disease: a concept analysis yusra al nasiria* and adhra al mawalib aoman college of health sciences, nursing program, ministry of health, muscat, oman. bcenter of studies & research, ministry of health, muscat, oman. *correspondence to dr. yusra al nasiri (email: yusra444@hotmail.com). (submitted: 05 november 2018 – revised version received: 22 november 2018 – accepted: 06 december 2018 – published online: 26 february 2019) objective the purpose of this concept analysis is to examine the health-related quality of life in children with sickle cell disease. methods meleis’s concept analysis methodology was followed for analyzing health related quality of life concept. this includes defining the concept, clarifying ambiguities, describing the critical attributes, identifying the observable and measurable empirical referent, delineating the antecedents, the consequences, and modeling to illustrate the concept in its entirety. articles focusing on quality of life, health-related quality of life (hrqol) in children with sickle cell disease (scd) were examined. results the concept appears well developed and the internal characteristic of hrqol is delineated. to ensure that its characteristics maintained contextual relevance, a concept called ‘fine tune’ should be done. since hrqol concept is a multidimensional concept, a factor analysis test may be needed to explore the dimensionality and the structure of its meaning. also, internal consistency reliability needs to be done to assess the items independence of one another. conclusion successful patient outcomes is an important goal for nursing and it is critical to health care decision making for practical, ethical, and financial. the analysis of hrqol concept provided more clarity to the concept definition and also to the underlying outcome measures. naming the variables of the concepts clearly facilitates comparing and contrasting the healthcare interventions on various patients’ outcomes. in addition, by examining the concept in children with scd, pediatric nurses become more cognizant of the concept’s meaning that would easily facilitate the understanding of the children’s and their parents’ perception of hrqol. keywords concept analysis, health related quality of life, sickle cell disease introduction sickle cell disease (scd) is a chronic, inherited hematological disorder that is associated with life-threatening complications that affect all major systems (stuart and nagel, 2004). frequent painful crises, infections, acute chest syndrome, priapism, splenic sequestrations stroke, and organ failure are the most common complications affecting children with scd.1,2 sickle cell disease has many complications that impact all aspects of children’s life, which include the physical, psychological, social, and mental. determining health-related quality of life (hrqol) provides an understanding of scd burden on those children.3,4 it helps providing information to families, and health care providers regarding the impact of the disease on children with scd. measuring hrqol is considered as an important indicator to evaluate health care interventions and treatments.4 this in turn, will provide opportunities to identify patients’ responses to treatment, and tailor appropriate therapies based on the patients’ perspective of their hrqol.5 in clinical practice, hrqol information can be useful in identifying and prioritizing health problems for individual scd cases as well as facilitates identifying any hidden or unexpected health problems. thus, it aids to decision-making, and in monitoring the health status of the patients.4 it was also found that, measuring hrqol has shown to improve communication between patients and providers as well as create a patient-centered environment. in addition, hrqol is considered as an important predictor for morbidity and mortality outcomes.1 therefore, understanding hrqol concept and clarifying its theoretical and practical aspects is very important for nursing practice. concept analysis method will help clarifying the concept and define its meaning that could be clearly understood by pediatric nurses when dealing with children having scd. also, analyzing hrqol concept would lead to the development of disease specific measurement tools for the concept to be utilized specifically for children with scd. the concept “qol” is a commonly used concept across different disciplines. initially, qol was applied in sociology but later it was applied to other disciplines. although qol is used in everyday language, it is viewed as a multifaceted concept.6 qol and hrqol have been used inter-changeably and remain vague and unclear concepts. the concept hrqol referred and used widely in healthcare literature. it is considered as a fundamental concept in health care because one of the nursing goals is to enhance the health outcomes and strive to provide quality of care for the patients. therefore, focusing on qol and the issues that might affect it can lead to improve nursing care through recognizing the effect of the diseases, evaluating treatments, and facilitating resource decision.7 although hrqol concept is frequently used, it has still remained unclear and not well defined and often confused with qol concept. concept analysis is considered as a means to clarify vague and overused concept and also to distinguish the concept from other similar concepts. therefore, the purpose of this analysis is to clarify the meaning of hrqol through reviewing the literature. the concept analysis in this paper will be guided by the guidelines presented in meleis model for the concept analysis.8 meleis model has been used widely in nursing and found to be clear and easy to follow. first, a literature was reviewed to examine the uses, define the concept, clarify ambiguities and describe the critical attributes of the concept. second, the observable and measurable issn 2413-0516 60 j contemp med sci | vol. 5, no. 1, january–february 2019: 59–63 hrqol: a concept analysis short communication y. al nasiri and a. al mawali empirical referent of the concept was identified. third, the concept was differentiated from other concepts. then, the antecedents and the consequences that may result from hrqol was delineated. at last, modeling was used to illustrate the concept in its entirety.27, 28 search strategy different databases were searched which include pubmed, cinhal, psychinfo, google scholar, and eric using the key term “hrqol” and “scd”. there were many articles found on hrqol; therefore, the search was limited to include articles published between 2000 and 2017. only research articles were included in this review. studies done on children with scd were included. the studies that did not give a clear definition of the concept were excluded. also, one article was chosen from the articles written by the same author. a total of 16 articles were included in this review. use of the concept (hrqol) health-related quality of life emerged as an important and distinct perspective after the appraisal of qol concept. therefore, hrqol concept was narrowed as a measurable concept because it was found that, most of the outcomes of qol have no corresponding interventions.6 therefore, qol was given a new label as hrqol to facilitate the concept measurement. based on the reviewed literature, the concept tends to be widely used in the healthcare setting; more specifically for patients with chronic illness. definition of the concept in the dictionary, life has been defined as “capacity for growth, existence, and functional activity”, quality is referred as “the standard of something when compared with other things like it”.7 the term quality can have both positive or negative features but mostly it is viewed as something superior. world health organization defined qol as “individuals” perceptions of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards, and concerns”.9 according to centers for disease control and prevention (cdc), qol is a broad, multidimensional concept that includes subjective evaluations of the positive and the negative aspects of life.10 on the other hand, hrqol-based (cdc) refers to the individual health’ perception of the physical, mental functional status, social, and socioeconomic status. in the reviewed literature, hrqol is being defined as a multidimensional concept which encompasses domains related to physical, emotional, social, and cognitive functioning.1,3,11–17 some literature specified that, hrqol goes beyond the direct measures of health, their life expectancy, and the causes of death.2,5 it focuses on health impact on the qol. characteristics of hrqol exploring the literature revealed that the concept of hrqol was viewed hrqol in patients with scd as a multidimensional concept and identified the aspects of it that includes physical, cognitive, social, and emotional.2,3,11–13,15–19 most of the literature cited the subjective nature of hrqol as the core attribute for this concept and is viewed as the subjective evaluation of the individuals for their health and lives as well as the subjective perception of personal thoughts, feelings, and the meaning of one’s life.2,3,11–13,15–19 in addition, hrqol concept is viewed as the well-being of the individual. satisfaction in life is also seen to be associated with hrqol.2,3,5,17,20–22 the last important characteristic for hrqol concept is that it is a dynamic concept, it is changing and depends on the context in which it is measured. dimensions of the hrqol (critical attributes) the critical attributes are the recurrent characteristics of the concept.8 the critical attributes of hrqol are as follows: multidimensional this includes the physical, psychological, social functioning. some researchers included the cognitive functioning or the mental capacity to evaluate ones’ health or life.5,12–15,17,19,22 the physical functioning domain includes the effect of the disease on the physical aspect of health such as walking, running, lifting heaving items, doing exercises, feeling pain, and energy status. the emotional functioning domain includes the effect of the disease on the children’s emotions and feelings such as fear, sadness, worry, angry, and sleep disturbance. the social domain includes the individual’s interaction and also the relationship with families, peers, and others. the cognitive domain includes the children’s memory, attention, and ability to keep up with the school requirement and attendance. subjective nature all of the literature indicated that hrqol is a subjective evaluation and assessment of one’s qol.1–4,11–13,15–19,23 it depends on the person’s perception, satisfaction, and well-being. as in chronic illness like scd, pain is subjective and can be best determined by the children and their parents. contextual health-related quality of life is a dynamic concept, and it varies based on the context in which it is measured and varies according to the changes in the circumstances. it depends on the situation and the time. intangible health-related quality of life is all about personal perception of well-being, satisfaction, and the perception of the effect of the illness on aspects of qol. health-related this concept is viewed as related to health and it is an important goal of nursing toward patients with chronic illness.2–4,6,8,15–18 empirical referents empirical referents are the categories in the phenomena; which demonstrate the existence of the concept and that it is really present. for hrqol, the empirical referents are the measures of the attributes. it is the statement of the patients; for example, statement about their satisfaction or dissatisfaction. the measurement is dependent on the researchers’ conceptualization of hrqol and also how it is operationalized. y. al nasiri and a. al mawali 61j contemp med sci | vol. 5, no. 1, january–february 2019: 59–63 short communication hrqol: a concept analysis therefore, the way it is defined determines what will be included in the assessment measurement. generally, hrqol measurement it is a subjective assessment of the individual’s well-being and also about one’s perception to what degree be able to perform different facets of life. the measurements include the perception of the physical functioning and to which extent one is able to perform activities and if the roles in daily activities are impeded; so the qol is influenced by the individual’s physical status or condition. the second measurement for hrqol is assessing to which extent the emotional status of the individuals limits the daily functioning and the ability to perform their roles. for the social functioning, the social activities, the interactions, and relationship with each other are assessed. at last, the individual’s perception of general health is measured. there are many instruments developed to measure hrqol, which may reflect the ambiguity of the concept. some instruments are so generic that is suitable for all population. others are specific tools, either disease specific or specific to certain population such as children or elderly, it is important to identify the rationale behind the choice of the tool.2–4,11–13,15–19,24 differentiating hrqol concept from other concepts the concept hrqol is often confused with other concepts such as the general status of health, functional ability, utility, mood, and symptoms. health is being defined by who as “a state of physical, mental, social well-being but not merely the absence of the diseases”. however, there is a recent conceptualization of health that makes it so definite as a state that including both the wellness and illness. functional ability is also confused with qol. it is considered as a health-related variable and was defined as the ability of the individuals to meet their basic needs, fulfill their roles, and maintain their well-being.7 a distinguish was made between the functional capacity and functional performance to make the concept more one’s perception of abnormal physical, emotional, and cognitive status. hrqol is often negatively correlated with the physiological abnormality which is an objective measure.4 mood is the affective domain of the individual which includes happiness, sadness, depression, and anxiety. utility is a concept that is not often used in the health care, but it is applied to health care decision-making to measure the preference for health status. the decision of the individual is thought to be based on the anticipated satisfaction of the individual or qol. qol is a multidimensional concept that includes physical, psychological, social, and cognitive domains of health, general well-being, satisfaction, and role functioning.2,3,13,15,17–19,22 however, the term qol found to be a very broad concept, and the confusion occurred when many outcomes of this concept were broad and had no corresponding interventions. therefore, when confusion occurred and the same variables are measured every time by the researchers (functional performance, symptoms, physiological dysfunction), the new concept “health-related quality of life” or “hrqol” has emerged later in the literature. in response to the confusion of the measurement related to qol outcome, a new concept was developed and narrowed to the qol that is related to health status and health care to link some parts of life that is influenced by health. a new label was given which is “hrqol” that mainly focuses on the physical, psychosocial, and cognitive health dimensions.25 delineating antecedents of hrqol concept the contextual condition under which hrqol is perceived and is expected to occur is “life”. the concept “qol” is always present, but it can occur only when life begins. since this concept is multidimensional and it is value-based and subjective in nature, then the ability to evaluate one’s self and make a decision in regards to life or may be treatments can be considered another antecedent for the concept hrqol. therefore, the ability of the individual to perform the cognitive evaluation of self or and to be conscious about his status is important antecedent; for example, sometimes the adolescents with chronic illness may make a decision regarding their treatments without the willingness of their parents to find a way to cope with their illness. also, they find their own ways of coping to go on with living.7 delineating consequences of hrqol concept consequences are the conditions that preceded by the evaluation of hrqol concept. the consequence of hrqol concept can be related to the consequences that occur after individual’s evaluation or individual perception of hrqol (such as satisfaction) and the decision that might result in making a change in the individual’s circumstances; for example, when the individual evaluates hrqol as high, it means he is satisfied and happy in life. it may also mean that he is empowered because he has high self-esteem about his life his physical and psychosocial health is good. however, the negative consequence of individual’s perception or evaluation of hrqol as poor is that, it might result in, dissatisfaction, low self-esteem, low coping abilities, changes in self-concept, depressed, isolated and it may even have extended to risk-taking as in adolescents with chronic illness. modeling hrqol concept the author illustrates two exemplars for hrqol using a model case and a contrary case. a model case maya is a 12-year-old child diagnosed with scd. maya seldom gets pain and looks physically healthy. she has many friends and looks satisfied with life. a contrary case dana is a 12-year-old child diagnosed with scd. dana gets frequent pain crisis and required frequent admissions, she is isolated and does not have friends. she wrote a storybook recently and won, but she did not want the gift and planned not to go and get the gift. she is dissatisfied with her life. analogizing health-related quality of life can be described as the “perception of well-being or ill-being”. so, when the individual perceive himself as being well it indicates that he remained healthy (physically, mentally, socially and emotionally) and therefore, he is seen to have a good hrqol and when the person perceives himself being ill, it indicates that he thinks that his health is interrupted either (physically, mentally, socially and emotionally) and, therefore, he can be seen as having poor hrqol. 62 j contemp med sci | vol. 5, no. 1, january–february 2019: 59–63 hrqol: a concept analysis short communication y. al nasiri and a. al mawali synthesizing health-related quality of life is a multidimensional concept that describes the physical, emotional, social, and cognitive health functions of the patients with chronic illness. assumptions in the analysis of this paper, two assumptions have been made. the first assumption is that the conceptual clarity is essential to guide high-quality research and for patient care. lack of conceptual clarity may lead to chaotic studies on patient care outcomes.26 therefore, if hrqol demonstrates to be a clear concept then the patient outcomes that the author’s research will focus on in the future will be accurately identified and, therefore, appropriate interventions can be designed responding to those outcomes. the second assumption is that in the measurement tool, the operationalization of the concept must very closely represent the conceptual definitions. it is essential that the instrument items must reflect the whole entirety of the concept emotional.26 in the literature, there are different instruments developed to measure hrqol; therefore, this may affect the coherence of the concept’s meaning if all these instruments, in fact, measure hrqol. failure to define hrqol concept may lead to chaos in the health care literature and may result in the development of different tools. considering these two assumptions, this paper analysis attempted to capture the richness of hrqol concept as it is defined and distinguish hrqol concept from other common variables that are measured under the rubric of hrqol. conclusion conducting a concept analysis enhanced the understanding of hrqol concept. the examination of its usage, critical attributes, antecedents, consequences and cases illustration provided insight into the essence of hrqol concept. the concept hrqol is considered as a very important phenomena in nursing practice. it is significant for nurses working in pediatric settings. by examining the concept in children with scd, pediatric nurses become more cognizant of the concept’s meaning that would easily facilitate the understanding of the children’s and their parents’ perception of hrqol. successful patient outcomes is an important goal for nursing and it is critical to health care decision making for practical, ethical, and financial. the analysis of hrqol concept provided more clarity to the concept definition and also to the underlying outcome measures. naming the variables of the concepts clearly facilitates comparing and contrasting the healthcare interventions on various patients’ outcomes. in addition, identifying the variables of hrqol and understanding the relationship between them provides clarity about the effect of the intervention on the outcomes. hrqol is an important concept in nursing because patients’ qol always matters; so being responsive and sensitive to patients’ perception of their qol helps to intervene to obtain a positive impact. health-related quality of life concept is helpful for generating nursing theory because by understanding the nature of the phenomena and anticipating its occurrence, identifying the concept antecedents, consequences, variables or attributes and understanding the relationship between them, this lead to link the concepts of the phenomena together and serve as a building blocks to generate a theory. there are many attributes identified from the concept hrqol and the relationship between them can be easily described and explained. therefore, linking the variables of hrqol, the antecedents, and the consequences and identifying the relationship among them can set a building blocks for a model or a theory that can serve as a guideline that influences nursing care. health-related quality of life unlike the qol concept, hrqol can be considered matured and a well-developed concept because there is a consistency present across the health care literature about its conceptualization of definition. also, the conceptual characteristics and attributes of hrqol are present and agreed upon. in addition, hrqol dimensions, boundaries as well as the potential indicators are identified. however, there are many instruments developed to measure hrqol and this in fact suggests that a refinement of the conceptualization is needed to be done as the validity of the conceptualization across the population has not been established yet. although the concept appears well developed, the internal characteristics of hrqol was not clearly delineated. therefore, to ensure that its characteristics maintained contextual relevance, a concept called “fine tune” should be done. therefore, a further study on this concept would be refining the hrqol instrument and test its psychometric properties using a quantitative approach. since hrqol concept is a mutli-dimensional concept, a factor analysis test may be needed to explore the dimensionality and the structure of its meaning. also, internal consistency reliability needs to be done to assess the items independence of one another. acknowledgment i would like to thank professor betty chang, ucla, for the valuable input provided in this paper. conflicts of interest disclosure none.  references 1. brousscau dc, panepinto j, nimmer m, hoffmann rg. the number of people with sickle cell disease in the united states: national and state estimates. am j hematol. 2010;85:77–78. 2. sawyer mg, reynolds ke, couper j, french d, kennedy d, martin j, et al. a two-year prospective study of the health-related quality of life of children with chronic illness--the parents’ perspective. qual life res. 2005;14:395–405. 3. strine tw, chapman dp, balluz ls, moriarty dg, mokdad ah. the associations between life satisfaction and health-related quality of life, chronic illness, and health behaviors among u.s. community-dwelling adults. j community health. 2008;33:40–50. 4. taylor rm, gibson f, franck ls. a concept analysis of health-related quality of life in young people with chronic illness. j clin nurs. 2008;17:1823–1833. 5. palermo tm, schwartz l, drotar d, mcgowan k. parental report of health-related quality of life in children with sickle cell disease. j behav med. 2002;25:269–283. 6. hijmans ct, fijnvandraat k, oosterlaan j. double disadvantage: a casecontrol study on health-related quality of life in children with sickle cell disease. health qual life outcomes. 2010;8:121. doi:10.1186/1477-7525-8121. pubmed pmid: 20977722. pubmed central pmcid: pmc2988059. 7. wrotniak bh, schall j, brault m, balmer d, stallings v. health-related quality of life in children with sickle cell disease using the child health questionnaire. j pediatr health care. 2014;28:14–22. y. al nasiri and a. al mawali 63j contemp med sci | vol. 5, no. 1, january–february 2019: 59–63 short communication hrqol: a concept analysis 8. morse jm, mitcham c, hupcey je, tason m c. criteria for concept evaluation. j adv nurs. 1996;24:385–390. 9. world health organization. 2010. available from: http://www.who.int/ about/definition/en/print.html (accessed february 2015). 10. centre for disease and control prevention (cdc). available from: http:// www.cdc.gov/hrqol/ (accessed february 2015). 11. ameringer s, elswick rk, smith w. fatigue in adolescents and young adults with sickle cell disease: biological and behavioral correlates and healthrelated quality of life. j pediatr oncol nurs. 2014;31:6–17. available from: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3982311/. 12. beverung lm, varni jw, panepinto ja. clinically meaningful interpretation of pediatric health-related quality of life in sickle cell disease. j pediatr hematol oncol. 2015;37:128–133. doi:10.1097/mph.0000000000000177 13. fisak b, belkin mh, lehe ac, bansal mm. the relationship between healthrelated quality of life, treatment adherence and disease severity in pediatric sickle cell disease sample. child care health dev. 2010;38:204–210. doi:10.1111/j.1365-2214.2011.01223.x. pubmed pmid: 21434965. 14. garratt a, schmidt l, mackintosh a, fitzpatrick r. quality of life measurement: bibliographic study of patient assessed health outcome measures. bmj. 2002;324:1417. 15. jackson jl, lemanek kl, clough-paabo e, rhodes m. predictors of health-related quality of life over time among adolescents and young adults with sickle cell disease. j clin psychol med settings. 2014;21: 313–319. 16. limbers ca, skipper s. health-related quality of life measurement in siblings of children with physical chronic illness: a systematic review. fam syst health. 2014;32:408–415. 17. schlenz am, schatz j, mcclellan cb, roberts cw. responsiveness of the pedsql to pain-related changes in health-related quality of life in pediatric sickle cell disease. j pediatr psychol. 2012;37:798–807. 18. dampier c, lebeau p, rhee s, lieff s, kesler k, ballas s. health related quality of life in adults with sickle cell disease (scd): a report from the comprehensive sickle cell centers clinical trial consortium. am j hematol. 2011;86:203–205. doi:10.1002/ajh.21905. pubmed pmid: 21264908. pubmed central pmcid: pmc355439. 19. lowry tj, pakenham ki. health-related quality of life in chronic fatigue syndrome: predictors of physical functioning and psychological distress. psychol health med. 2008;13:222–238. 20. the oxford english dictionary. clarendon press, uk, london, 1989. 21. wilson ib, cleary pd. linking clinical variables with health-related quality of life: a conceptual model of patient outcomes. jama. 1995;273:59–65. 22. ziadni m, patterson c, pulgaron e, robinson m, barakat l. health-related quality of life and adaptive behaviors of adolescents with sickle cell disease: stress processing moderators. j clin psychol med settings. 2011;18:335–344. doi:10.1007/s10880-011-9254-3. pubmed pmid: 21681659. 23. dale jc, cochran cj, lmswap ej, buchanan gr. health-related quality of life in children with sickle cell disease. j ped health care. 2011;25:208–210. 24. gill tm, feinstein ar. a critical appraisal of the quality of quality-of-life measurements. jama. 1994;272:619–626. 25. haas bk. a multidisciplinary concept analysis of quality of life. west j nurs res. 1999;21:728–742. 26. meleis a. theoretical nursing: development and process. 5th ed.; j. b. lippincott, williams & wilkins, philadelphia, 2012. 27. muszalik m, kędziora-kornatowska k, kornatowski t. functional assessment and health-related quality of life (hrqol) of elderly patients on the basis of the functional assessment of chronic illness therapy (facit)-f questionnaire. arch gerontol geriatr. 2009;49:404–408. 28. panipento ja, o’mahar km, debaun mr, loberiza fr, scott jp. healthrelated quality of life in children with sickle cell disease: child and parents perception. br j haematol. 2005;130:437–444. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 28 j contemp med sci | vol. 5, no. 1, january–february 2019: 28–34 original article osteocacin favours the expression of synaptonemal complex protein 3 in azoospermic mouse model akanji omotosho dhulqarnain,a nasrin takzaree,b gholamreza hassanzadeh,b somayeh solhjo,b heidar tooli,b mahsa yaaghoobi nejad,b pedram shafaat,b and tayebeh rastegarb* ainternational campus, tehran university of medical science, tehran, iran. banatomy department, school of medicine, tehran university of medical science, tehran, iran. *correspondence to tayebeh rastegar (email: trastegar@tums.ac.ir). (submitted: 11 june 2018 – revised version received: 25 june 2018 – accepted: 07 july 2018 – published online: 26 february 2018) objective infertility is the inability to conceive after regular unprotected sex for more than 1 year without the use of contraceptive. azoospermia is defined as an absence of fertile sperm in seminal fluid. men with azoospermia will guide the formulation of a therapeutic plan. uncarboxylated form of the osteocalcin (ocn) modulates fertility. in this study, we investigated the role of osteocalcin on sycp3 expression in azoospermic mouse model. methods male mice (nmri) ranging in age from 4 to 6 weeks (25 ± 5 g) were randomly divided into five groups (in all groups n = 5), control, sham i, sham ii, azoospermic model and azoospermic experimental ocn (3 ng/g/day) treated groups. at the end of the treatment period, (15th week age) the mice were sacrificed, left testes removed, weighted and put in fixative for morphology and ihc technique. results testis weight was reduced in azoospermic and azoospermic ocn treated group compared with control and sham groups, but in azoospermic ocn treated group is more than azoospermic mice (p ≤ 0.05). daily injections of ocn improved spermatogenesis and sycp3 expression in azoospermic ocn treated mice but not in azoospermic model. conclusion our results suggests that the osteocalcin overexpressed sycp3 expression and improved spermatogenesis. keywords azoospermia, osteocalcin, sycp3, spermatogenesis introduction spermatogenesis is the process of production of male gametes known as spermatozoa that occurs in seminiferous tubules which are located within the testes. it is the process by which an animal produces spermatozoa from spermatogonial stem cells by the way of mitosis and meiosis. due to a number of different factors, the sperm analysis in an ejaculate may fail to reach the minimum required for fertilization to take place. thus, it can render such individual infertile.1 infertility is the inability to conceive after regular unprotected sex for more than 1 year without the use of contraceptive. it is a reproductive problem that affects approximately 15% of couples in general human population. male factor of infertility accounts for about 50%2 and about 7% of all men are affected by fertility problem.3 azoospermia is diagnosed when no spermatozoa are detected upon microscopic evaluation of two centrifuged semen samples. azoospermia is found in approximately 1% of all men and up to 15% of infertile men, depending upon the demographic nature of the infertile platoon.4 azoospermia and oligospermia has been attributed to the effects of procedures like radiotherapy and chemotherapy in high dose such as busulfan5 which may leads to a prolong period of infertility in patients.6 sex steroid hormone are important regulation of bone remodeling which help to maintain bone integrity.7–9 based on physiological and clinical observations, it is hypothesized that bone mass, energy metabolism and reproduction might be coordinately regulated.10 subsequent researches revealed that bone is an endocrine organ favoring whole-body glucose homeostasis, energy metabolism, and fertility. these functions of bone are mediated by an osteoblast-specific secreted molecule, osteocalcin (ocn), that when undercarboxylated acts as a hormone favoring β-cell proliferation, insulin secretion, and fertility.11 it has now been discovered that osteocalcin, in addition to its endocrine role as a regulator of energy homeostasis, favors male fertility by promoting synthesis of testosterone, a steroid hormone required for many aspects of testicular function from leydig cells of the testes. also, a receptor was identified for osteocalcin that is expressed and transduces its signal in leydig cells. in view of this, osteocalcin can be said to affect male fertility by regulating the level of testosterone production from the testes.12 synaptonemal complex protein 3 (sycp3) is an important marker of meiotic germ cell division during the process of spermatogenesis.13 abnormalities in human synaptonemal complex formation are considered to be responsible for a proportion of unexplained azoospermic cases. therefore, sycp3 could be a tool for the prediction of human spermatogenesis progression, especially in infertile men.14 testosterone hormone secretion in leydig cell is under the influence of hypothalamus–hypophysis–testis axis. hypothalamus secretes gonadotropin releasing hormone which stimulate the pituitary gland for the production of follicle stimulating hormone and leutinizing hormone (lh). this suggests that hypothalamus–testis axis regulates testosterone production in leydig cell and ultimately affect reproduction. also, considering the role of osteocalcin in testosterone production which affects reproduction, then we suspect a relationship between osteocalcin and sycp3 expression in germ cells during spermatogenesis as it may affect reproduction in men. in light of this, osteocalcin, through its own pathway may be an important factor in regulating the expression of sycp3 during meiotic division of germ cells.15 thus, in this study, we have supposed that osteocalcin improve spermatogenesis in azoospermic mice by the expression of scycp3. issn 2413-0516 a.o. dhulqarnain et al. 29j contemp med sci | vol. 5, no. 1, january–february 2019: 28–34 original article expression of sycp3 by osteocalcin materials and methods animal preparation male mice (nmri) ranging in age from 4 to 6 weeks (25 ± 5 g) were obtained from the pasteur institute of iran. animals were housed in wire cages at 23 ± 1°c under a 12 h light–dark cycle with 70% humidity and fed a standard diet and water. animals were maintained and experiments were conducted in accordance with the principles of laboratory animal care of tehran university of medical sciences, iran. all animals were randomly allocated into five groups, control group (n = 5), azoospermic experimental group (n = 5) that received a single intraperitoneal injection of busulfan (40 mg/kg body weight) diluted in dimethyl sulfoxide (dmso, usa) at 5 weeks old,16 sham i group (n = 5) that received a single intraperitoneal injection of dmso (busulfan solvent) at 5 weeks old, sham ii group (n = 5) that received a single intraperitoneal injection of dmso at 5 weeks old and after 5 weeks they received phosphate buffer solution (pbs) intraperitoneal injection for 1 month and azoospermic experimental treated group (n = 5) that received a single intraperitoneal injection of busulfan (40 mg/kg body weight) diluted in dmso at 5 weeks old and after 5 weeks they received recombinant osteocalcin (h00000632, novus, usa) intraperitoneal injection 3 ng/g/day for 1 month.12 drugs preparation busulfan (b2635, sigma, usa) was first dissolved in dmso (p8340, sigma, usa), then an equal volume of sterile water was added to obtain a final busulfan concentration of 20 mg/ml.17 osteocalcin was dissolved in pbs (p4417, sigma, usa).18 surgical procedure at the end of the treatment period (15th week), the mice were weighed and anesthetized using intraperitoneal ketamine (50 mg/kg) and xylazine (20 mg/kg), and were killed, the peritoneal cavity was opened through a lower transverse abdominal incision. as well as, left testes in all groups were immediately removed, weighed and put in fixative for staining.17 preparation of busulfan-induced azoospermic model the busulfan-treated infertile mouse model was prepared as described by brinster with some modification. mice were received a single dose intraperitoneal injection of busulfan (40 mg/kg) at 4–6 weeks of age. hematoxylin–eosin stain of testicular cross section, also eosin–nigrosine staining of seminal fluid was performed to evaluate the azoospermic model 5 weeks after busulfan injection.16,19 confirmation of busulfan-induced azoosperic model hematoxylin and eosin staining technique hematoxylin and eosin (h&e) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes. testes tissues of control and azoospermic mouse were fixed in buines solution, embedded in paraffin, sectioned at 5 μm and sections were stained with h&e, then the morphological aspect of seminiferous tubules was studied.20 eosin–nigrosin staining technique eosin–nigrosin is a staining technique that assesses the vitality of a sperm sample. briefly 20 μl of semen were gently stirred (30 s at 37°c) in 60 μl of eosin–nigrosin stain (5%-nigrosin and 4%-eosin-y at ratio 3:1). then, smears were prepared and dried at room temperature. the slides were examined by a single observer with phase-contrast microscope (magnification: 1000×) after preparation. a total of 200 spermatozoa per slide were scored for dead/live spermatozoa. this method is based on the degree of membrane permeability of dead spermatozoa which heads show pink or red coloration, whereas the low permeability of live gametes excludes eosin and therefore their head maintains whitish.19 immunohistochemistry for sycp3 testes tissues were fixed in formaldehyde solution, embedded in paraffin, sectioned at 5 μm and sections were incubated with primary rabbit polyclonal anti-scp3 antibody (ab15093, abcam, 1/100/overnight) and then secondary goat antirabbit hrp conjugated antibody (ab6721, abcam, 1/100/2 h) was added. counterstain nucleus staining was done by hematoxylin. statistics the results were expressed as mean ± se. the statistical significance between the mean values was determined by one-way analysis of variance (anova), tukey and duncan post-test. p ≤ 0.05 was considered significant. results osteocalcin increased testes weight of azoospermic mice the obtained results in this study were illustrated in fig. 1. testis weight was reduced in azoospermic and sham ii group compared with control and sham i groups, but in azoospermic ocn treated group is more than azoospermic mice (p ≤ 0.05). confirmation of azoopermia in mice five weeks after the injection of mice with busulfan, in histopathological study, most of the endogenous sperm cells were removed while the interstitial tissue and sertoli cells remained; some seminiferous tubules appeared as sertoli-cell-only structures compared with control group (fig. 2). the results of eosin–nigrosin staining which determine the survival rate of sperm and confirm the azoospermic model as shown by the phase-contrast microscope indicates that the acidophilic content is introduced into the head of the dead sperm and is therefore visible in pink, while the sperm head vivid live colors were observed (fig. 3). osteocalcin improved spermatogenesis in azoospermic but does not affect somatic cells of the testes histological study of seminiferous tubules of control group was with normal spermatogenesis of thickening germ cells; also in sham i group that received solvents (dmso and pbs. in azoospermic and sham ii groups which tubules are relatively evacuated and absence of germ cell line. osteocalcin-treated 30 j contemp med sci | vol. 5, no. 1, january–february 2019: 28–34 expression of sycp3 by osteocalcin original article a.o. dhulqarnain et al. group shows a thick germinal layer and the presence of various germ cells indicative of presence and the improvement of spermatogenesis (fig. 4). using the one-way anova, spss 16 software and tukey test for statistical analysis of germ cell, the number of spermatonia, spermatocytes, round and elongated spermatids increase in experimental group, no significant change in control and sham i and the number significantly decrease in azoospermic and sham ii compared with the control, sham i and experimental group (p < 0.05). also, there is no significant difference in the number of germ cells in azoospermic and sham ii group (p < 0.05). the diameter of the germinal layer of the seminiferous tubule is increased in experimental group compared with the azoospermic group. there is no significant difference in the diameter of the germinal layer of control and sham i. no significant difference in azoospermic and sham ii but there is significant difference in azoospermic and sham ii when compared with control, sham i and experimental group. this result indicates that osteocalcin increase the number of germ cells and thereby, increase in the thickness of the germinal layer of seminiferous tubule (p < 0.05). we also discovered that in all the groups, there is no significant differences in the diameter of the whole length of seminiferous tubule (p < 0.05). however, there is no significant difference in the number of sertoli, leydig and myhoid cells all using the anova test (p < 0.05) (fig. 5). osteocalcin favours the expression of sycp3 in azoozspermic mouse we performed immunohistochemistry to access the presence of sycp3 protein in all the groups which is positive for control, sham i and experimental group but none in azoospermic and sham ii groups as observed with light microscope. this suggest that busulfan caused the apoptosis of germ cells which result in disruption of process of spermatogenesis and therefore, no sycp3 protein expression (figs. 6 and 7). discussion the aim of this research is to determine the role of osteocalcin in spermatogenesis progression in azoospermic mice model fig. 1 testis weight in different groups. testis weight was reduced in azoospermic and sham ii groups compared with control and sham i group, but in azoospermic ocn treated group is more than azoospermic and sham ii mice (p ≤ 0.05). # significant with control group. * significant with sham i group. • significant with azoospermic group. ° significant with sham ii group. fig. 2 h&e staining in busulfan induced azoospermic model preparation. (a) histological morphology of untreated testicle, as normal control. (b) histological morphology of testicular tissue 5 weeks after injection with 40 mg/kg busulfan. most of the endogenous sperm cells had been removed, while interstitial tissue and sertoli cells remained. scale bar: 50 µm. a b fig. 3 the eosin–nigrosin-stain produces a dark background on which the sperm stand out as lightly colored objects. (a) dead sperm take up eosin and appear pinkish in color (b) whereas normal live sperm exclude the eosin stain and appear white in color. ba a.o. dhulqarnain et al. 31j contemp med sci | vol. 5, no. 1, january–february 2019: 28–34 original article expression of sycp3 by osteocalcin fig. 4 h&e staining transverse cross section of testis seminiferous tubule. (a) spermatocyte (s) display on the tubules. (b) control group with normal spermatogenesis of thickening germ cells. (c) sham i group that received solvents (dmso and pbs). (d) azoospermic group which tubules are relatively evacuated and absence of germ cell line. (e) sham ii group azoospermic mice which received osteocalcin solvent (pbs) for 30 days, presence of vacuoles and loss of germ cells. (f) osteocalcin-treated group shows a thick germinal layer and the presence of various germ cells indicative of presence and the improvement of spermatogenesis. scale bar: 50 µm. a b c d e f fig. 5 data of the variables of the testis seminiferous tubule. (a) number of spermatogonia cells. (b) spermatocytes. (c) round spermatid cells. (d) elongated spermatid cells. (e) sertoli cells. (f) leydig cells. (g) myoid cells. (h) germinal layer thickness. (i) seminiferous tubules diameter (p < 0.05). # significant with control group. * significant with sham i group. • significant with azoospermic group. ° significant with sham ii group. control: control group; sham i: which received solvents (dmso and pbs). azospermia: azoospermiac animals that received busulfan 40 mg/kg/i.p. sham ii: azoospermiac mice received pbs for 1 month. experimental: azoospermic mice that received osteocalcin. a b c d e f g h i 32 j contemp med sci | vol. 5, no. 1, january–february 2019: 28–34 expression of sycp3 by osteocalcin original article a.o. dhulqarnain et al. fig. 7 mean and standard error (mean ± se) of sycp3 positive spermatocytes cells. control group kept in normal condition. sham i which received busulfan solvent (dmso) and after 5 weeks, animals received osteocalcin solvent (pbs) for 30 days. azoospermia group contains animals that received single dose of busulfan (40 mg/kg) by intraperitoneal injection. sham ii group animals received 40 mg/kg dose of busulfan in single dose by intraperitoneal injection. animals in this group received osteocalcin solvent (pbs) for 30 days after 5 weeks. experimental group (azoospermia– ocn) includes azoospermic mice that received osteocalcin dose of 3 ng/g/ day after 5 weeks for 1 month (p < 0.05). #significant with control group. * significant with sham i group. • significant with azoospermic group. ° significant with sham ii group. which can be used to provide insight into treatment of infertility. our results showed the depletion of germinal layer of seminiferous tubules in azoospermic mice which confirm the general belief about busulfan as causative agent for apoptosis in germ cells seminiferous tubules. our main concern is to find the expression of sycp3 which signals the progression of spermatogenesis in azoospermic mice after treatment with osteocalcin. recent researches have directly linked osteocalcin to energy metabolism and reproduction in men but not in women. it perform these roles when it is in its undercarboxylated form. the increasing evident of the role of gonad in bone metabolism provide a suitable ground for this doubts, and raises the possibility that bone, through a feedback mechanism in its endocrine function could affect reproduction in either gender.21 fig. 6 immunohistochemistry of cross section testis seminiferous tubule for detection of sycp3. (a) showing spermatocyte cells with positive sycp3 in the testicular germinal layer. (b) control group with normal spermatogenesis and thickening of the germinal layer. (c) sham i group that received solvents (dmso and pbs). (d) azoospermic group, absence of positive sycp3 cells. (e) sham ii group azoospermic mice that received osteocalcin solvent (pbs) for 30 days without any sycp3 positive cells. (f) osteocalcin-treated group shows thick germinal layer and the presence positive sycp3 cells. scale bar: 50 µm. a b c d e f a.o. dhulqarnain et al. 33j contemp med sci | vol. 5, no. 1, january–february 2019: 28–34 original article expression of sycp3 by osteocalcin leydig cells of the testes are responsible for the secretion of testosterone which also determines the development of male reproductive organs, germinal cells and estrogen production. the main regulation of testosterone of the testes is the pituitary hormone, luteinizing hormone (lh).13,14 with this, we least expect that there will be another hormone that plays an important role in regulating men fertility, and this hormone, osteocalcin, was made in bone. the fact that osteocalcin−/− male mice are subfertile with a marked decrease in circulating testosterone and low sperm count established the biological relevance of this regulation in vivo in the mouse.22 having said that, it is widely believed that ostelcalin favors testosterone production which ultimately leads to improved spermatogenesis. in this research, we confirmed the role of osteocalcin in the progression of spermatogenesis through the expression of a meiotic protein marker (sycp3) in spermatocytes. consequently, there is significant increase in the thickness of the germinal layer and reduction of the lumen as a result of increase in germ cells which progress the process of spermatogenesis. however, schwetz et al.23 showed that osteocalcin is not a strong determinant of serum testosterone and sperm count in men from infertile couples. we also found out that the amount of leydig cells of the testes is independent of the level of undercarboxylated osteocalcin with insignificant changes in all the groups investigated. we also found out that the number of sertoli and myhoid cells in the seminiferous tubules are independent of the circulating level of osteocalcin. it has been shown that osteocalcin favours testosterone biosynthesis through the pancreas–bone–testes axis by binding to its receptor gpr6ca in leydig cells of the testes.22 they demonstrated that testes size and weight, epididymides and seminal vesicles weights, sperm count and circulating testosterone levels were all reduced in 12 weeks old ocnosb −/− mice22 which support the idea that osteocalcin expression in osteoblast improve fertility in men. however, different researches show that osteocalcin dependent and insulin dependent reproduction have different pathways. also, it is believed that sycp3 is expressed only in germ cells of testes and ovary.13 in light of this, we observed the expression of this protein in germ cells. in this research, we observed positive correlation between the expression of sycp3 and osteocalcin. this protein expression is absent in azoospermic group. in azoospermic group, there is apoptotic germ cell line which disrupt the progression of spermatogenesis. recent researches show that sycp3 protein expression in mice occur at the level of prophase 1 in meiotic division.13,24,25 this is in-line with the discovery of mahmoud et al.26 in human that the expression of sycp3 begins at the level of primary spermatocytes. the disruption of the sycp3 gene has been found to obstruct formation of axial elements and to induce cell death of mutant spermatocytes during an extended zygotene stage.24 truly, we discovered the expression of this protein in spermatocytes which are actively dividing cells in the process of spermatogenesis. we conclude that the lack of expression of sycp3 protein during meiotic division causes germ cell apoptosis and consequently, no progression of spermatogenesis which leads to infertility. however, osteocalcin hormone can help restore spermatogenesis by favoring the expression of this protein. to shed more light on the role of osteocalcin hormone in progression of germ cell meiotic division, we encourage more research on interaction between osteocalcin hormone and sycp3 gene in animal model which could give insight and provide important breakthrough in addressing the genetic causes of infertility in human. acknowledgment the authors are grateful for tehran university of medical sciences. this work was supported by grant no. 34910 from tehran university of medical sciences. compliance with ethical standards this work was supported by grant no. 34910. all authors received research grants from tehran university of medical sciences. all authors declares that they have no conflict of interest. animals were maintained and experiments were conducted in accordance with the principles of laboratory animal care of tehran university of medical sciences, iran. conflict of interest none. n references 1. clermont y. the cycle of the seminiferous epithelium in man. am j anat. 1963;112:35–51. 2. seshagiri pb. molecular insights into the causes of male infertility. j biosci. 2001;26:429–435. 3. krausz c. male infertility: pathogenesis and clinical diagnosis. best pract res clin endocrinol metab. 2011;25:271–285. 4. hammoud ao, gibson m, peterson cm, meikle aw, carrell dt. impact of male obesity on infertility: a critical review of the current literature. fertil steril. 2008;90:897–904. 5. de rooij dg, van de kant hj, dol r, wagemaker g, van buul pp, van duijn-goedhart a, et al. long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey. biol reprod. 2002;66:486–494. 6. jackson h, fox bw, craig aw. the effect of alkylating agents on male rat fertility. br j pharmacol chemother. 1959;14:149–157. 7. khosla s, melton lj 3rd, atkinson ej, o’fallon wm. relationship of serum sex steroid levels to longitudinal changes in bone density in young versus elderly men. j clin endocrinol metab. 2001;86:3555–3561. 8. nakamura t, imai y, matsumoto t, sato s, takeuchi k, igarashi k, et al. estrogen prevents bone loss via estrogen receptor alpha and induction of fas ligand in osteoclasts. cell. 2007;130:811–823. 9. riggs bl, o’fallon wm, muhs j, o’connor mk, kumar r, melton lj 3rd. longterm effects of calcium supplementation on serum parathyroid hormone level, bone turnover, and bone loss in elderly women. j bone miner res. 1998;13:168–174. 10. ducy p, amling m, takeda s, priemel m, schilling af, beil ft, et al. leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. cell. 2000;100:197–207. 11. oury f. a crosstalk between bone and gonads. ann n y acad sci. 2012;1260:1–7. 12. pi m, wu y, quarles ld. gprc6a mediates responses to osteocalcin in β-cells in vitro and pancreas in vivo. j bone miner res. 2011;26:1680–1683. 13. parra mt, viera a, gomez r, page j, benavente r, santos jl, et al. involvement of the cohesin rad21 and scp3 in monopolar attachment of sister kinetochores during mouse meiosis i. j cell sci. 2004;117:1221–1234. 14. judis l, chan er, schwartz s, seftel a, hassold t. meiosis i arrest and azoospermia in an infertile male explained by failure of formation of a component of the synaptonemal complex. fertil steril. 2004;81:205–209. 15. oury f, sumara g, sumara o, ferron m, chang h, smith ce, et al. endocrine regulation of male fertility by the skeleton. cell. 2011;144:796–809. 16. brinster cj, ryu by, avarbock mr, karagenc l, brinster rl, orwig ke. restoration of fertility by germ cell transplantation requires effective recipient preparation 1. biol reprod. 2003;69:412–420. 34 j contemp med sci | vol. 5, no. 1, january–february 2019: 28–34 expression of sycp3 by osteocalcin original article a.o. dhulqarnain et al. 17. ahar nh, khaki a, akbari g, novin mg. the effect of busulfan on body weight, testis weight and mda enzymes in male rats. int j women’s heal reprod sci. 2014;2:316–319. 18. zhou b, li h, liu j, xu l, zang w, wu s, et al. intermittent injections of osteocalcin reverse autophagic dysfunction and endoplasmic reticulum stress resulting from diet-induced obesity in the vascular tissue via the nfκb-p65-dependent mechanism. cell cycle. 2013;12: 1901–1913. 19. björndahl l, söderlund i, kvist u. evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment. hum reprod. 2003;18:813–816. 20. bancroft jd, layton c. the hematoxylin and eosin. theory & practice of histological techniques. 7th ed.; churchill livingstone of elsevier, philadelphia, 2013, p. 173–214. 21. schuh-huerta sm, pera rar. reproductive biology: bone returns the favour. nature. 2011;472:46–47. 22. oury f, sumara g, sumara o, ferron m, chang h, smith ce, et al. endocrine regulation of male fertility by the skeleton. cell. 2011;144:796–809. 23. schwetz v, gumpold r, graupp m, hacker n, schweighofer n, trummer o, et al. osteocalcin is not a strong determinant of serum testosterone and sperm count in men from infertile couples. andrology. 2013;1:590–594. 24. yuan l, liu jg, zhao j, brundell e, daneholt b, höög c. the murine scp3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. mol cell. 2000;5:73–83. 25. liebe b, alsheimer m, hoog c, benavente r, scherthan h. telomere attachment, meiotic chromosome condensation, pairing, and bouquet stage duration are modified in spermatocytes lacking axial elements. mol biol cell. 2004;15:827–837. 26. aarabi m, modarressi mh, soltanghoraee h, behjati r, amirjannati n, akhondi mm. testicular expression of synaptonemal complex protein 3 (sycp3) messenger ribonucleic acid in 110 patients with nonobstructive azoospermia. fertil steril. 2006;86:325–331. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 134 j contemp med sci | vol. 4, no. 3, summer 2018: 134–139 original intracerebroventricular injection of wharton’s jelly mesenchymal stem cells attenuates brain damage in rat model of hypoxia: optimization of vascular endothelial growth factor and downregulation of inflammatory factors kobra mehrannia,a tahmineh mokhtari,b,c seyed mohammad hossein noori mogehi,a mohammad akbari,a javad tavakkoly bazzaz,d simin mahakizeh,a yousef mohammai,a sahar ijaz,a and gholamreza hassanzadeha,e adepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. bnervous system stem cells research center, semnan university of medical sciences, semnan, iran. ddepartment of genetic, school of medicine, tehran university of medical sciences, tehran, iran. edepartment of neuroscience, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. correspondence to gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir). (submitted: 19 may 2018 – revised version received: 24 june 2018 – accepted: 29 june 2018 – published online: 26 september 2018) objective in this study, we investigated the effects of intracerebroventricular (icv) wharton’s jelly mesenchymal stem cells (wj-mscs) injection in rat model of hypoxic brain injury by evaluating the amount of vascular endothelial growth factor (vegf) and proinflammatory factors in hippocampus. methods about 24 rats were allocated to four groups of study: (1) control and intact animals (co), (2) sham group (sh): animals were placed in the hypoxia chamber without inducing hypoxia and injected pbs, (3) hypoxia (h), (4) h + wj-msc. hypoxia was induced by placing animals in the hypoxia chamber for 30 days (4 h a day). after 3 days of vehicle or wj-mscs injection, the rats were sacrificed and brain tissues were prepared for molecular and histopathological studies. results despite a decrease in the gene expression of interleukin 1 beta, tumor necrosis factor-α, il18, and the number of dark neurons in ca1 region of hippocampus in h + wj-msc groups compared to h (p < 0.05). there is an increase in all these factors in both h and h + wj-msc groups compared to co and sh groups (p < 0.05). the gene expression and protein concentration of vegf increased in both h and h + wj-msc groups compared to co and sh groups (p < 0.05). conclusion based on the findings, wj-mscs could reduce the number of dark neurons in hippocampus by increasing the vegf synthesis and reducing inflammation in hypoxic condition. keywords hypoxia, wharton’s jelly mesenchymal stem cells, proinflammatory factors, vascular endothelial growth factor, hippocampus, rat introduction an environment with stable condition and high nutrient supply is required for neurons to have proper functions.1 so, the well-established teamwork of the cells at the blood–brain barrier (bbb), first explained by paul ehrlich in 1885, is indispensable to form the barrier between central nervous system and peripheral environment.2 in bbb, endothelial cells of line barrier vessels are supported by pericytes and vascular feet of astrocytes.3 the bbb disturbance is a mechanism defined in the pathogenesis of different neurodegenerative disorders.4,5 hypoxia is the reduction of oxygen that impaired the bbb in vivo and in vitro6 as it caused the changes like vascular leakage and up-regulation of inflammatory factor and vascular endothelial growth factor (vegf).7 oxidative stress has an important role in the extension of hypoxic–ischemic brain damage. brain neurons for their qualified function require suitable environment, blood supply and homeostatic balance.8 all of the brain injuries such as hypoxia, stroke, and ischemia are mediated by physiopathological events, which destroy bbb. accordingly, enhanced reactive oxygen species (ros) trigger inflammation by releasing proinflammatory factors including interleukin 1 beta (il1β), tumor necrosis factor-α (tnfα) and il18. during hypoxia, pericytes and astrocytes prevent disruption of bbb by keeping tight junction and preventing death of the endothelial cells.9,10 disruption of the tight junction, proteins lead to vasogenic edema.11–13 vegf is responsible for the vascular leakage and edema in the brain that occurs during hypoxia.6 in recent years mesenchymal stem cells (mscs) have been used in cell therapy.14–16 these cells can be cultured in vitro while retaining the potential to give rise to osteoblasts, chondrocytes, astrocytes, neurons and skeletal muscles.17 some evidences have indicated that stem cells do their therapeutic actions by secretion of some molecules like growth factors (gfs). wharton’s jelly mscs (wj-mscs) could be effective in treating ischemic stroke18 and hypoxia.17 in vitro, mscs release angiogenic factors like vegf which can stimulate endothelial cells to migrate and proliferate.19 in this regard, we hypothesized that hypoxia induction might upregulate vegf and inflammatory factors while single dose intracerebroventricular (icv) injection of wj-mscs after 72 h as a therapeutic approach might optimize expression of vegf and decrease inflammatory factors and dark neurons in the hypoxia rat model. materials and methods animals in this study all of the experiments have been approved by the tehran university of medical sciences. about 24 male wistar issn 2413-0516 kobra mehrannia et al. 135j contemp med sci | vol. 4, no. 3, summer 2018: 134–139 original icv injection of wj-mscs in rat model of hypoxia albino rats, weighing 150–200 g were used in this study. experiments were performed between 8 am and 12 am in 12 h period of light/dark, 23 ± °2c temperature and with free availability of food and water to the animals. all the steps were performed according to the instructions of iranian council and confirmed by the ethical committee of tehran university of medical sciences. animal grouping the animals were randomly divided into four groups as listed below: 1. control group (co): intact rats without any intervention. 2. sham group (sh): animals were kept in the chamber without induction of hypoxia but with icv injection of pbs. 3. hypoxic group (h): animals were kept in the chamber with induction of hypoxia and icv injection of pbs. 4. hypoxia + injection of wj-mscs group (h + wj-mscs): animals were kept in hypoxia chamber and 24 h after hypoxia, 10⁵ wj-mscs were injected into both the cerebral ventricles using hamilton syringe (ap = −0.9 mm, ml = −1.8 mm, and dv = 3.5 mm). induction of hypoxia hypoxia was induced by placing six rats in one hypoxia plexiglass chamber (30 × 20 × 20) and revealing them to a gas mixture of 8% oxygen and 92% nitrogen for 4 h from 8 am to 12 pm for 30 days.14 the gas composition was evaluated by an oximeter (lutron do-5510 o2 meter, taipei, taiwan). all experiments were designed to minimize animal suffering in vivo techniques. isolation and cultivation of wj-mscs umbilical cords were isolated by explant method. warton’s jelly was chopped into small pieces (2 mm). then, they were cultured in dulbecco’s modified eagle’s medium (gibco, usa) for a week and were supplemented with 15% fetal bovine serum (fbs, gibco, usa), 1 μg/ml amphotericin b (gibco, usa), 100 u/ml penicillin (gibco, usa) and 100 μg/mg streptomycin (gibco, usa). the culture medium was placed in a co2 (5%) incubator at 37°c. after 2 weeks, the pieces were removed, and the medium was renewed. this process was performed several times. after getting 90% confluence, wj-mscs were collected with 0.25% trypsin ethylenediaminetetraacetate (gibco, usa) and the cells in the third passage were used for injection. flow cytometry after isolation, wj-mscs were incubated for 20 min with fluorescein isothiocyanate-conjugated monoclonal antibodies against cd45 and cd90 (ebioscience, usa); then, the cells were suspended in pbs (gibco, usa) to carry out facs calibur flow cytometry (bdbiosciences, usa) analysis. rna extraction and quantitative real-time pcr to check the expression of vegf, il1β, tnfα, and il18 genes in each group, tissues were examined using real-time pcr technique. first, the primers were designed as shown below, based on the reference sequences provided by the accession numbers of genbank: vegfa: gtgacctgaggttcttttctgtt, il18: atgtctaccctctcctgt, tnfα: caccacgctcttctgtct, il1β: aggcagggagggaaacaca and gapdh: ccatgttctgatgggtgtgaacca. then, the entire rna was extracted from the tissues and converted to cdna. then the cdna was amplified by pcr method and examined for expression of the genes mentioned. this technique has four basic steps: 1. the total rna from the cells was collected from each group. 2. using the reverse transcription enzyme, the cdna was converted. 3. the resultant cdna was treated to remove genomic dna with dnase i enzyme. 4. repeated pcr in real time. the qrt-pcr technique was used quantitatively to confirm the expression of the genes. for this purpose, the kinetics of the whole cell was first extracted using a solution of kyazol according to the synagen protocol, and exposed to dnase i (fermentas) to ensure whether genomic dna was contaminated. then, the quality of the extracted rnas was evaluated by spectrophotometry (dpi-1, kiagen). single-strand cdna of oligo dt primer (mwg-biotech, germany) and reverse transcription enzyme (fermentas) were prepared based on the protocol. each pcr reaction was performed using pcr master mix (applied biosystems) and syber green in the abi step one (applied biosystems, sequences detection systems, foster city, ca), according to the manufacturer’s protocol. western blotting lysis of hippocampus tissues was done by protein lysis buffer (sigma, st. louis, mo, usa). protein concentration was determined by loading onto 8–15% sodium dodecyl sulfate polyacrylamide gels. then these were transferred to membrane (pvdf) and incubated overnight at 4°c for binding of primary antibodies and 1 h for secondary antibodies to detect proteins (chemiluminescence reagent; santa cruz biotechnology, santa cruz, usa). histopathological study for histopathological study, the rats were anesthetized with ketamine (80 mg/kg) and xylazine (15 mg/kg) (razico, iran), and perfused using 0.9% saline and then 4% paraformaldehyde (sigma, usa). hippocampus was dissected and maintained in 10% formalin, and the fixative was renewed after 12 h. the tissue samples were examined under light field microscope (olympus, cx31, tokyo, japan) in ×400 magnification after nissl staining for counting dark neurons in the ca1. furthermore, in prepared images, the thickness of ca1 layer was measured using image processing and analysis program of imagej software version 1.32j (nih, bethesda, md, usa). statistical analysis results were presented as mean ± sd. data analysis was performed using one-way analysis of variance and the post-hoc tukey’s and tamhane’s t2 tests (spss-16 software) and p < 0.05 was considered significant. 136 j contemp med sci | vol. 4, no. 3, summer 2018: 134–139 icv injection of wj-mscs in rat model of hypoxia original kobra mehrannia et al. results flow cytometry analysis for immunophenotypic characteristics of wj-mscs based on the findings obtained from at least three independent samples, there was an up-regulation trend in the expression of cd90 (98.9%, fig. 1a) and down-regulation trend in the expression of cd45 (1.32%, fig. 1b) in the cultured wj-mscs (fig. 1b). effects of icv injection of wj-mscs on gene expression of proinflammatory factors of hippocampal region in hypoxic rats a significant increase was observed in the gene expression of proinflammatory factors (including tnfα, il1β and il18) of hippocampus region in h and h + wj-msc groups in comparison to the co and sh groups (p < 0.05, fig. 2a–c). moreover, there was a significant decrease in the gene expression of proinflammatory factors in h + wj-msc group in comparison to h group (p < 0.05, fig. 2a–c). effects of icv injection of wj-mscs on gene expression and protein levels of vegf of hippocampal region in hypoxic rats the results of this study showed that there was a significant increase in the gene expression of vegf in h and h + wj-msc groups compared to the co and sh groups (p < 0.05, fig. 3a). furthermore, there was a significant decrease in the gene expression of vegf in h + wj-msc group compared to h group (p < 0.05, fig. 3a). based on the western blot analysis, a significant enhance was seen in protein levels of vegf in h group compared with co and sh groups and also significant decrease in h + wj-msc group compared with h group (p < 0.05, fig. 3b, c). effects of icv injection of wj-mscs on the number of dark neurons of ca1 region of hippocampus in hypoxic rats in h and h + wj-msc groups, the number of dark neurons and the thickness of ca1 layer was significantly increased in the ca1 region of hippocampus compared to co and sh groups (p < 0.05, fig. 4a–c). in h + wj-msc group, the number of dark neurons and the thickness of ca1 layer in the hippocampus was significantly decreased compared to h group after one dose injection of wj-mscs (p < 0.05, fig. 4a–c). discussion in the present study, we evaluated the effects of one dose icv injection of wj-mscs in chronic hypoxia induced brain injury in rats. in hypoxia group, the number of dark neurons, the thickness of ca1 layer and the levels of inflammatory factor increased in the ca1 region of hippocampus following 30 day hypoxia through decreasing the oxygen supply of brain in animals using a hypoxic chamber which confirmed our model. fig. 1 evaluation of cd surface marker profiles of wj-mscs using flow cytometry. (a) cultured wj-mscs were labelled with the indicated antibodies and analyzed by wj-mscs with down regulation trend in the expressions of cd45 and up regulation trend in the expressions of cd90 by flow cytometry assay. fpo fpo fig. 2 effects of icv injection of wj-mscs on gene expression of proinflammatory factors of hippocampal region in hypoxic rats. (a) tnfα, (b) il1β and (c) il18. a: p < 0.05 compared to co group. b: p < 0.05 compared to sh group, c: p < 0.05 compared to h group. co: control group, sh: sham group, h: hypoxia group, h + wj-msc: hypoxia + injection of wharton’s jelly derived mesenchymal stem cells group. fpo fig. 3 effects of icv injection of wj-mscs on gene expression of vegf of hippocampal region in hypoxic rats. (a) gene expression of vegf in different group using real time pcr, b, c) protein concentration of vegf in different group using western blot analysis. a: p < 0.05 compared to co group. b: p < 0.05 compared to sh group, c: p < 0.05 compared to h group. co: control group, sh: sham group, h: hypoxia group, h + wj-msc: hypoxia + injection of wharton’s jelly derived mesenchymal stem cells group. kobra mehrannia et al. 137j contemp med sci | vol. 4, no. 3, summer 2018: 134–139 original icv injection of wj-mscs in rat model of hypoxia dark neurons are defined as neurons with pyknotic, hyper-electron density and hyperbasophilia characteristics are recognized in the several pathological conditions such as traumatic brain injury, stroke and hypoxia.16,20,21 cognitive impairments are common in the brain injury due to hypoxia,22–24 related to extending cell death in pyramidal neurons of hippocampus as a most vulnerable region to hypoxia.25,26 it should be noticed that these neurons play an important role in learning and memory functions.27 as well, increased the number of hypertrophic neurons induced by hypoxia increased the thickness of ca1 region. inflammation is a critical factor in production of proinflammatory cytokines such as tnfα, il1β and il18 and, development of injury throughout the brain.28,29 lievre et al.30 reported an increased in amount of ros after transient hypoxia. according to the literature, it has been confirmed that leukocytes are activated and migrated into the injured area of brain and cytokines are secreted in high amounts within hours after injury in adult models of hypoxic–ischemic brain injury.31–33 il-1, il-6, il-10, tnf-α and transforming growth factor-β (tgf-β) are the most studied cytokines associated with the inflammatory responses following hypoxic– ischemic brain injury.34 hedtjärn et al.28 demonstrated that hypoxic–ischemic brain injury markedly enhanced the following il-18.28 in ischemic stroke, t lymphocytes infiltrate into the brain and release cytotoxic molecules and proinflammatory chemokines which are responsible for early inflammation in the injured region of the brain.35 according to our results, vegf increases in the hippocampus area following chronic hypoxic condition. different studies have been performed to define the enhanced vegf in hypoxia condition. lennmyr et al.36 confirmed the expression of vegf following permanent and transient occlusion of the middle cerebral artery in the rat. zhang et al.37 stated that vegf enhances angiogenesis and promotes bbb leakage in the brain of ischemic rats. schoch et al.6 has proved that hypoxia induced vegf expression and this factor is responsible for vascular leakage and formed edema in the brain. yeh et al.38 investigated the effects of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (yc-1) against hypoxia induced bbb hyperpermeability in endothelial cell culture of adult rat brain. their findings indicated that yc-1 may protect the bbb against hypoxia induced damage by inhibition of hif-1alpha accumulation and vegf production.38 in contrast, sun et al.39 demonstrated that vegf induced neurogenesis, neuroprotection and angiogenesis after focal cerebral ischemia. as well, within 24 h after hypoxic condition induced by ischemia, the endothelial cells of vessels around the injured regions begin to proliferate under the induction of several known proangiogenic mediators gene and protein expression, e.g. erythropoietin,40 angiopoietins and tie receptors,40 vegf41 and tgf-β,42 etc. it was suggested that angiogenesis mediated by vegf may protect brain against reduced blood supply and restore blood stream and promote neurogenesis in the injured regions.40 in this study, we used one dose injection of wj-mscs to manage the hypoxia in animals. according to the results of this study, gene expression and protein concentration of vegf optimized after hypoxia whereas, the gene expression of proinflammatory cytokines including tnfα, il1β and il18 decreased in the treated group compared to the hypoxic group. mesenchymal stem cells have been found to facilitate the regeneration of injured tissues by neurogenesis or releasing cytokines which reduce the inflammation43 and enhance the protection of neurons against hypoxic condition. mscs release bioactive agents, gfs and anti-apoptotic molecules with direct or endogenous mechanisms.11 munoz et al.44 showed that implanting the bone marrow mscs into the mouse hippocampus enhanced neurogenesis in this region. nam et al.45 investigated the effects of mscs expression of matrix metalloproteinases and angiogenesis in the permanent model of rodent middle cerebral artery occlusion (mcao). decreased infarction volume and neurological repair were observed which relate to the increased vascular density after mscs administration.45 quittet et al.46 evaluated the effect of mscs released vegf in an ischemic stroke model of rat and declared that using these kind of cells to deliver gfs such as vegf can reduce the impairment induced by stroke and could be introduced as an effective strategy to repair the brain injury following stroke. zhou et al.47 stated that of bone marrow mscs which co-overexpress both bdnf and vegf improved neuroprotection in a rat model of cardiac arrest-induced global cerebral ischemia. these differences between the results of various studies and our results can be defined by time-dependent manner of vegf expression. the source of mscs is important in which different studies used bone marrow mscs in their investigations. as enhanced vegf levels increase the permeability of bbb following hypoxia, it can be suggested that wj-mscs control the levels of vegf in different pathologic conditions like hypoxia. the optimized levels of vegf are required to increase angiogenesis, however, the mechanism of controlled and time dependent manner under the regulation of mscs is unknown. immunosuppressive and anti-inflammatory effects of mscs are proven in different studies. mscs can release different inhibitory agents to suppress the inflammation such as indoleamine 2,3-dioxygenase, inducible nitric oxide synthase and tnf-α stimulating gene/protein 6 (tsg-6), fpo fig. 4 effects of icv injection of wj-mscs on the number of dark neurons of ca1 region of hippocampus in hypoxic rats. (a) nissl staining showed the distribution of dark neurons in several groups (×400). (b) comparing the number of dark neurons in different groups. a: p < 0.05 compared to co group. b: p < 0.05 compared to sh group, c: p < 0.05 compared to h group. co: control group, sh: sham group, h: hypoxia group, h + wj-msc: hypoxia + injection of wharton’s jelly derived mesenchymal stem cells group. 138 j contemp med sci | vol. 4, no. 3, summer 2018: 134–139 icv injection of wj-mscs in rat model of hypoxia original kobra mehrannia et al. prostaglandin e2 (48). to show the anti-inflammatory effects of mscs, vendrame et al.49 evaluated the impacts of human cord blood cells on a rat model of stroke. as well, alizamir et al.18 demonstrated that icv injection of wj-mscs following mcao reduced the number of dark neurons in cortical region through reducing bax gene expression which agrees with the results of our study. conclusion based on the result of our present study, wj-mscs could protect the pyramidal neurons of hippocampus against hypoxic condition by optimizing the vegf synthesis and reducing inflammation. acknowledgments this study was a part of the phd thesis supported by tehran university of medical sciences, tehran, iran (grant no. 95-1-101-30-31780). conflict of interest none. n references 1. engelhardt s, huang sf, patkar s, gassmann m, ogunshola oo. differential responses of blood-brain barrier associated cells to hypoxia and ischemia: a comparative study. fluids barriers cns. 2015;12:4. 2. he y, yao y, tsirka se, cao y. cell-culture models of the blood–brain barrier. stroke. 2014;45:2514–2526. 3. engelhardt s, al‐ahmad aj, gassmann m, ogunshola oo. hypoxia selectively disrupts brain microvascular endothelial tight junction complexes through a hypoxia‐inducible factor‐1 (hif‐1) dependent mechanism. j cell physiol. 2014;229:1096–1105. 4. hassanzadeh g, hosseini quchani s, sahraian ma, abolhassani f, sadighi gilani ma, dehghan tarzjani m, et al. leukocyte gene expression and plasma concentration in multiple sclerosis: alteration of transforming growth factor-βs, claudin-11, and matrix metalloproteinase-2. cell mol neurobiol. 2016;36:865–872. 5. zlokovic bv. the blood-brain barrier in health and chronic neurodegenerative disorders. neuron. 2008;57:178–201. 6. schoch hj, fischer s, marti hh. hypoxia‐induced vascular endothelial growth factor expression causes vascular leakage in the brain. brain. 2002;125:2549–2557. 7. tang yl, tang y, zhang yc, qian k, shen l, phillips mi. improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated heme oxygenase-1 vector. j am coll cardiol. 2005;46:1339–1350. 8. roy cs, sherrington cs. on the regulation of the blood‐supply of the brain. j physiol. 1890;11:85–158. 9. al ahmad a, taboada cb, gassmann m, ogunshola oo. astrocytes and pericytes differentially modulate blood–brain barrier characteristics during development and hypoxic insult. j cereb blood flow metab. 2011;31:693–705. 10. nakagawa s, deli ma, kawaguchi h, shimizudani t, shimono t, kittel a, et al. a new blood–brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. neurochem int. 2009;54:253–263. 11. sorby-adams aj, marcoionni am, dempsey er, woenig ja, turner rj. the role of neurogenic inflammation in blood-brain barrier disruption and development of cerebral oedema following acute central nervous system (cns) injury. int j mol sci. 2017;18. pii:e1788. 12. bazrafkan m, nikmehr b, shahverdi a, hosseini sr, hassani f, poorhassan m, et al. lipid peroxidation and its role in the expression of nlrp1a and nlrp3 genes in testicular tissue of male rats: a model of spinal cord injury. iran biomed j. 2018;22:151–159. 13. nikmehr b, bazrafkan m, hassanzadeh g, shahverdi a, sadighi gilani ma, kiani s, et al. the correlation of gene expression of inflammasome indicators and impaired fertility in rat model of spinal cord injury: a time course study. urol j. 2017;14:5057–5063. 14. mahakizadeh s, akbari m, sharifzadeh m, rastegar t, abolhassani f, hassanzadeh g. wharton’ jelly mesenchymal stem cells and insulin effect on bdnf expression in ca1 and ca3 regions of rats’ hippocampus after chronic hypoxia. j contemp med sci. 2018;4:63–69. 15. trounson a, mcdonald c. stem cell therapies in clinical trials: progress and challenges. cell stem cell. 2015;17:11–22. 16. bang oy. clinical trials of adult stem cell therapy in patients with ischemic stroke. j clin neurol. 2016;12:14–20. 17. zhu w, chen j, cong x, hu s, chen x. hypoxia and serum deprivation‐induced apoptosis in mesenchymal stem cells. stem cells. 2006;24:416–425. 18. alizamir t, akbari m, mokhtari t, khaksarian m, hassanzadeh g. associated functional motor recovery induced by intracerebroventricular (icv) microinjection of wharton’s jelly mesenchymal stem cells following brain ischemia/reperfusion injury in rat: decreased dark neurons and bax gene expression in the cerebral corte. j contemp med sci. 2017;3. 19. huang nf, li s. mesenchymal stem cells for vascular regeneration. regen med. 2008;3:877–892. 20. ping z, liu w, kang z, cai j, wang q, cheng n, et al. sulforaphane protects brains against hypoxic-ischemic injury through induction of nrf2dependent phase 2 enzyme. brain res. 2010;1343:178–185. 21. krysko dv, berghe tv, d’herde k, vandenabeele p. apoptosis and necrosis: detection, discrimination and phagocytosis. methods. 2008;44:205–221. 22. back sa. cerebral white and gray matter injury in newborns: new insights into pathophysiology and management. clin perinatol. 2014;41:1–24. 23. logitharajah p, rutherford ma, cowan fm. hypoxic–ischemic encephalopathy in preterm infants: antecedent factors, brain imaging, and outcome. pediatr res. 2009;66:222–229. 24. perez a, ritter s, brotschi b, werner h, caflisch j, martin e, et al. long-term neurodevelopmental outcome with hypoxic-ischemic encephalopathy. j pediatr. 2013;163:454–459. 25. muttikkal tj, wintermark m. mri patterns of global hypoxic-ischemic injury in adults. j neuroradiol. 2013;40:164–171. 26. maurya vk, ravikumar r, bhatia m, rai r. hypoxic-ischemic brain injury in an adult: magnetic resonance imaging findings. med j armed forces india. 2016;72:75–77. 27. mokhtari t, akbari m, malek f, kashani ir, rastegar t, noorbakhsh f, et al. improvement of memory and learning by intracerebroventricular microinjection of t3 in rat model of ischemic brain stroke mediated by upregulation of bdnf and gdnf in ca1 hippocampal region. daru. 2017;25:4. 28. hedtjärn m, leverin al, eriksson k, blomgren k, mallard c, hagberg h. interleukin-18 involvement in hypoxic-ischemic brain injury. j neurosci. 2002;22:5910–5919. 29. farahabadi a, akbari m, amini pishva a, zendedel a, arabkheradmand a, beyer c, et al. effect of progesterone therapy on tnf-α and inos gene expression in spinal cord injury model. acta med iran. 2016;54:345–351. 30. lièvre v, becuwe p, bianchi a, bossenmeyer-pouriè c, koziel v, franck p, et al. intracellular generation of free radicals and modifications of detoxifying enzymes in cultured neurons from the developing rat forebrain in response to transient hypoxia. neuroscience. 2001;105:287–297. 31. perego c, fumagalli s, de simoni mg. temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. j neuroinflammation. 2011;8:174. 32. garcia jh, liu kf, yoshida y, chen s, lian j. brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (wistar rat). am j pathol. 1994;145:728–740. 33. liu f, mccullough ld. inflammatory responses in hypoxic ischemic encephalopathy. acta pharmacol sin. 2013;34:1121–1130. 34. han hs, yenari ma. cellular targets of brain inflammation in stroke. curr opin invest drugs. 2003;4:522–529. 35. brait vh, arumugam tv, drummond gr, sobey cg. importance of t lymphocytes in brain injury, immunodeficiency, and recovery after cerebral ischemia. j cereb blood flow metab. 2012;32:598–611. 36. lennmyr f, ata ka, funa k, olsson y, terént a. expression of vascular endothelial growth factor (vegf) and its receptors (flt-1 and flk-1) following permanent and transient occlusion of the middle cerebral artery in the rat. j neuropathol exp neurol. 1998;57:874–882. 37. zhang zg, zhang l, jiang q, zhang r, davies k, powers c, et al. vegf enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. j clin invest. 2000;106:829–838. 38. yeh wl, lu dy, lin cj, liou hc, fu wm. inhibition of hypoxia-induced increase of blood–brain barrier permeability by yc-1 through the kobra mehrannia et al. 139j contemp med sci | vol. 4, no. 3, summer 2018: 134–139 original icv injection of wj-mscs in rat model of hypoxia antagonism of hif-1α accumulation and vegf expression. mol pharmacol. 2007;72:440–449. 39. sun y, jin k, xie l, childs j, mao xo, logvinova a, et al. vegf-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. j clin invest. 2003;111:1843–1851. 40. fernández-lópez d, faustino j, derugin n, vexler zs. acute and chronic vascular responses to experimental focal arterial stroke in the neonate rat. transl stroke res. 2013;4:179–188. 41. cobbs cs, chen j, greenberg da, graham sh. vascular endothelial growth factor expression in transient focal cerebral ischemia in the rat. neurosci lett. 1998;249:79–82. 42. haqqani as, nesic m, preston e, baumann e, kelly j, stanimirovic d. characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and icat nanolc-ms/ms. faseb j. 2005;19:1809–1821. 43. uccelli a, moretta l, pistoia v. mesenchymal stem cells in health and disease. nat rev immunol. 2008;8:726–736. 44. munoz jr, stoutenger br, robinson ap, spees jl, prockop dj. human stem/ progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. proc natl acad sci usa. 2005;102:18171–18176. 45. nam hs, kwon i, lee bh, kim h, kim j, an s, et al. effects of mesenchymal stem cell treatment on the expression of matrix metalloproteinases and angiogenesis during ischemic stroke recovery. plos one. 2015;10:e0144218. 46. quittet ms, touzani o, sindji l, cayon j, fillesoye f, toutain j, et al. effects of mesenchymal stem cell therapy, in association with pharmacologically active microcarriers releasing vegf, in an ischaemic stroke model in the rat. acta biomater. 2015;15:77–88. 47. zhou l, lin q, wang p, yao l, leong k, tan z, et al. enhanced neuroprotective efficacy of bone marrow mesenchymal stem cells co-overexpressing bdnf and vegf in a rat model of cardiac arrest-induced global cerebral ischemia. cell death dis. 2017;8:e2774. 48. shi y, su j, roberts ai, shou p, rabson ab, ren g. how mesenchymal stem cells interact with tissue immune responses. trends immunol. 2012;33: 136–143. 49. vendrame m, gemma c, de mesquita d collier l, bickford pc, sanberg cd, et al. anti-inflammatory effects of human cord blood cells in a rat model of stroke. stem cells dev. 2005;14:595–604. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201804 8 j contemp med sci | vol. 5, no. 1, january–february 2019: 8–13 original article fibrin-collagen hydrogel as a scaffold for dermoepidermal skin substitute, preparation and characterization mahsa mollapour sisakht,a,b mohammad ali nilforoushzadeh,a* javad verdi,a,c* hamid reza banafshe,a,d* zahra safaei naraghi,e,f and seyed abdolreza mortazavi-tabatabaeig aapplied cell sciences department, kashan university of medical science, kashan, iran. bskin and stem cell research center, tehran university of medical sciences, tehran, iran. cdepartment of tissue engineering and applied cell sciences, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. dphysiology research center, kashan university of medical sciences, kashan, iran. edepartment of dermatology, tehran university of medical sciences, razi hospital, tehran, iran. fdepartment of pathology, tehran university of medical sciences, razi hospital, tehran, iran. gproteomics research center, shahid beheshti university of medical sciences, tehran, iran. *correspondence to mahsa mollapour sisakht (email: mahsamollapur@yahoo.com). (submitted: 02 october 2018 – revised version received: 17 october 2018 – accepted: 23 november 2018 – published online: 26 february 2019) objective bioengineered skin substitutes were created to address wound healing problems. skin substitutes contains live human cells seeded onto a matrix to provide cytokine, growth factor and other proteins from ecm required to decrease healing time. these products are classified based on their durability, the cells seeded on them, and their originality. in this study, we aimed to investigate fibrin-collagen hydrogel as a new scaffold to design a bilayer temporary skin equivalent. methods fibrin gel was prepared by cross-linking fibrinogen with thrombin and mixing it with collagen type i. human fibroblasts and keratinocytes were isolated from skin biopsies of healthy donors and foreskin and the cells were seeded onto three-dimensional hydrogel layer-by-layer. morphological assessment, histological analysis, immunocytochemistry were performed to characterize the scaffold properties. results the results of scaffold characterization demonstrated good porosity, cell viability, and biocompatibility of the scaffold. conclusion fibrin-collagen as a natural material in organotypic cell culture modeling demonstrates that hydrogel scaffolds can be properly designed to generate bilayer or composite temporary skin grafts. keywords tissue engineering, fibrin-collagen, hydrogel, skin substitute introduction tissue engineering as a major component of regenerative medicine is now being marketed. different types of product, especially in dermatology, are found globally. despite its high cost, market studies show that the use of advanced wound dressing containing live skin cells has increased.1 among the different types of skin substitutes, acellular dermal matrices have the biggest market share.2 composite matrices derived from human keratinocytes, fibroblasts and bovine or porcine collagen developed by organogenesis are marketed as apligraf. orcel, tissuetech, and theraskin composite products.3 skin substitutes can be synthetic or natural.4 of the synthetic and natural materials used to prepare skin substitute, hydrogels, and gels have advantages such as high exudate capacity, non-adherence, ease of removal from wounds, accelerated healing, pain and inflammation reduction, low cost, and easy development and handling5 for wound management. hydrogels are composites which are crosslinked by chemical or physical methods in water. they are characterized by remarkable water-absorbing swellability.6 significant efforts have been made in research and development to commercialize hydrogels. some hydrogel dressings synthesized by chemical cross-linking have appeared under the brand names of geliperm, curasol, and tegagel.5 the major drawback of hydrogels is their low mechanical stiffness, which can be improved by mixing or crosslinking with other polymers.7 this study presents the preparation and characterization of a combination of collagen type i and fibrin as a natural composite polymer and assesses the ability of skin-cell seeding onto a scaffold to generate a permanent full-thickness or dermal– epidermal skin equivalent. we hypothesize that the addition of type i collagen to fibrin will increase the mechanical features of fibrin. it is shown that this scaffold is suitable for three-dimensional (3d) cell cultures and it is possible for clinical application as a skin substitute. materials and methods isolation and culture of fibroblast human dermal fibroblasts were isolated from neonatal foreskin obtained at the time of circumcision, after receiving written informed consent from their parents. samples were carried to the laboratory in dmem medium containing 10% (v/v) fbs and penicillin (100 iu/ml)-streptomycin (100 μg/ml) (all of them from gibco, thermo fisher scientific). before working with samples they were washed three times in washing solution, pbs (gibco, thermo fisher scientific) containing penicillin-streptomycin to remove of probably infection and any blood. skin was cut into strips 1 cm wide using scalpel and this sample was incubated overnight in 4°c in dispase 0.1% (gibco, thermo fisher scientific) for easy separation of epidermal layer from dermal layer. the dermis was cut into very small pieces (5 mm2) using curved scissors and these pieces were transferred into a collagenase type ii (200 iu/ml) (gibco, thermo fisher scientific) solution in an incubator at 37°c for 2 h. then, the suspension was centrifuged at 500g for 5 min. the pellet was resuspended in culture medium. issn 2413-0516 m.m. sisakht et al. 9j contemp med sci | vol. 5, no. 1, january–february 2019: 8–13 original article fibrin-collagen hydrogel for dermoepidermal skin substitute the isolated cells were counted using hemocytometer (cell viability determined using trypan blue exclusion) and then seeded into culture dishes at cell density of 2 × 105 cells/cm2 (fig. 1a–1c). the cells were cultured in a co2 incubator at 37°c and the culture medium was changed twice a week. the cells were subcultured before they reached confluence by treating them with trypsin-edta (gibco, thermo fisher scientific) for 5 min and a ratio of 1:3 was split into new dishes.8 isolation and culture of keratinocyte for separation of keratinocytes, the side up of skin was placed in 0.25% trypsin and then incubated at 37°c for 30 min. then, it was transferred into a 15-ml centrifuge tube and was vortexed gently for 2 min, and then the same volume of fbs was added to inhibit the action of the trypsin. the suspension was centrifuged at 500g for 5 min, the supernatant was discarded and the cell pellet was resuspended in keratinocyte growth fig. 1 cells isolation and characterization. (a and b) foreskin preparation for keratinocyte and fibroblast isolation. (c) white arrow shows cell pellet after centrifuge. (d) expanded confluent keratinocyte from cornified layer of epidermis cultured in kgm. all of these cells were stained for ck14 and epcam and analyzed by flow cytometry. representative phase-contrast images of these cells seeded on tissue culture plastic (right) (scale bar: 100 µm). (e) keratinocytes from basal layer of epidermis were stained for cd14 and epcam. (f) fibroblasts were stained for vimentin and cd90 and analyzed by flow cytometry. phase-contrast micrograph of cells at passage three expanded on cell culture plastic (scale bar: 100 µm). kgm, keratinocyte growth medium. a d e f b c 10 j contemp med sci | vol. 5, no. 1, january–february 2019: 8–13 fibrin-collagen hydrogel for dermoepidermal skin substitute original article m.m. sisakht et al. with cells.12 (fig. 2f). sections of paraffin-embedded hydrogel which contain keratinocyte and fibroblast cells were deparaffinized (xylol, 5 min), dehydrated (ethanol 100-96-80-7050%, 2 min each), and washed in pbs (5 min). the sections were incubated for 30 min in 0.3% triton x-100 (sigma-aldrich, usa), and blocked in 10% bsa in pbs (sigma-aldrich, usa) for 30 min. immunostaining was performed using a primary antibody, ck14 (bd bioscience, usa) for keratinocytes, vimentin (abcam, usa) for fibroblasts cells seeded into hydrogel followed by a fitc-conjugated secondary antibody (dako, switzerland). pictures of immunofluorescence staining were taken with the fitc-filter sets in the fluorescent microscope (olympus, japan)10 (fig. 3g–l). results figure 1 shows the morphology of fibroblast cells from the dermis and keratinocytes from the epidermis. expression of the cell markers was analyzed by flow cytometry for ck14 and epcam fitc-conjugated antibodies for keratinocyte cells and cd90 and vimentin for fibroblast cells. as shown, the expression of ck14 and epcam differ in different culture media at 75.2% and 0.341%, respectively, for cells cultured with kgmgold keratinocyte growth medium singlequot (lonza; sweden). the ck14 and epcam markers measured by flow cytometry for keratinocyte cells cultured with keratinocyte growth medium (epilife medium; thermo fisher scientific) were 0.087% and 98.5%, respectively. cd90 and vimentin for fibroblast cells were 99.2% and 99.6%, respectively. we isolated and cultured keratinocyte from the basal layer of the epidermis and cornified layer. as expected, the length of viability were different for fibrin-collagen hydrogels. cells from the basal layer contain proliferative keratinocytes which have unlimited self-renewal. unlike keratinocytes from the cornified layer, basal layer keratinocyte cells were able to proliferate for more than one month in a hydrogel scaffold. sem images of freeze dried hydrogel without cells are shown in fig. 2d. in this image, high porosity and open interconnected geometry of the fig. 2 scaffold preparation and characterization. (a) typical view of fibrin-collagen hydrogel. (b) trilaminar fibrin network after gelation. (c) freeze dried hydrogel. (d) representative sem image of fibrin-collagen hydrogel (scale bars: 50 µm). (e) imagej analysis of sem image to analysis pore size and area. a b c d e medium (epilife medium) (gibco, thermo fisher scientific). in addition, we used another cell culture media for keratinocyte cell culture. the cell pellet was resuspended in kgm-gold keratinocyte growth medium singlequot (lonza, sweden). epidermal cells for both of them were attached within 24–48 h and the cells began to spread out on dish. the culture medium were changed daily and the dishes were confluent by 10–15 days (fig. 1a–1c). characterization of fibroblast and keratinocyte for characterization of keratinocytes and fibroblast, common types of monoclonal antibodies were used consequently: antihuman epcam produced in mouse and cytokeratin 14 (ck14) for keratinocyte and cd90, vimentin for fibroblast (all from bd bioscience, usa). the cells were fixed using 4% formaldehyde (sigma-aldrich, usa) and after three times of washing with pbs, antibodies were incubated on the cells for 1 h at 4°c. the positive cells were detected using fluorescence microscope (zeiss, germany) (fig. 1d–1f). scaffold preparation fibrin hydrogel were made using human type i plasma fibrinogen at a concentration of 10 mg/ml in the presence of thrombin at a concentration of 100 u/ml (both from sigma-aldrich, usa) were used in a 3:1 ratio, respectively. the collagen gel mixture was prepared using rat tail type i gel (sigma-aldrich, usa), then collagen gel combined with 0.2 ml neutralization buffer containing 0.15m sodium hydroxide (naoh) to obtain a ph of about 7.49,10 (fig. 2a and 2b). freeze drying and scanning electron microscopy the obtained hydrogel was dried under freeze dryer (christ alpha 1-4 ld plus, germany) at −50°c and 150 µmhg for 24 h followed by freezing at 70°c for 5 h (fig. 2c). all samples were kept in room temperature for further analyses. freeze dried samples were analyzed for morphological assessment and pore size evaluation by scanning electron microscopy (sem, zeiss) observations. samples were frozen in liquid nitrogen, fractured and gold coated before scanning.11 (fig. 2d and 2e). fibroblast and keratinocyte culture onto hydrogel fibroblasts (5 × 104 cells/gel) were centrifuged and resuspended in 100 µl dmem (gibco, thermo fisher scientific) and mixed with 3 ml of prepared hydrogel. the combination of the cell and fibrinogen solution was placed in a 6-well cell culture plate. then, thrombin was added to each well and kept at room temperature for 10 min followed by 1 h at 37°c in an incubator containing 5% co2. 10 in same procedure, keratinocytes (5 × 104 cells/gel) was prepared separately and added to the top of fibroblast layer in the same process. kgm medium (gibco, thermo fisher scientific) added to the well for 1 day and then processed, and paraffin-embedded for histological analysis (fig. 3a–3c). histological staining and ihc the cell seeded scaffold was fixed using 10% formalin, processed, and paraffin-embedded. paraffin sections (3 µm) were stained with hematoxylin and eosin (sigma-aldrich, usa) to assess the histological morphology of the fibrin-collagen hydrogel (fig. 2d and 2e). masson’s trichrome staining was performed to distinguish the collagen content in the hydrogel m.m. sisakht et al. 11j contemp med sci | vol. 5, no. 1, january–february 2019: 8–13 original article fibrin-collagen hydrogel for dermoepidermal skin substitute pores were observed which allowed cells to become trapped in the scaffold and migrate through it successfully. imagej analysis of the sem image is shown in fig. 2e, where 51.086% of the area is composed of pores and the mean ± sd of the size of the pore is 42.38 feret diameter. despite the advantages of fibrin as a bioscaffold for skin engineering, the application of fibrin-based scaffolds is limited due to their poor mechanical properties and fast biodegradability.13 figs. 3a and 3d shows cells at the time of seeding and at 3 and 7 days after seeding onto hydrogel (figs. 3b and 3c). these images reveal that cells can proliferate after encapsulation and have suitable migration during 3d culture. the collagen is added to the fibrin to address this problem. fibrin-collagen hydrogels were thus prepared as described above and masson trichrome staining confirmed the presence of collagen in the scaffold (fig. 3f). immunofluorescence staining for fibroblasts and keratinocyte cells stained with ck14 and vimentin (figs. 3g–3l) was present after cell seeding on day 7. discussion hydrogels were first introduced by wichterle and lim5,14 as crosslinked 2-hydroxyethyl methacrylate hydrogels and have been applied in numerous applications because of their hydrophilic features. subsequently, synthetic and natural hydrogels have been fabricated using different materials.15,16 some features of hydrogel can promote epithelialization of the wound and make hydrogel of interest as a wound dressing. exudate uptake from bed wounds promotes fibroblast proliferation and keratinocyte migration, efficient delivery (slow release) of bioactive molecules (antimicrobial agents, biomacromolecules, growth factors and other small molecule agents) to the wound bed.7,17 hydrogels are the best choice compared with other dressings because of their elasticity and flexibility, which are requirements of ideal wound dressings.7 one of the most promising areas for hydrogels is tissue engineering. three-dimensional networks of hydrogel allow desirable cellular encapsulation, proliferation and survival, fig. 3 three-dimensional skin cell culture. (a–c) phase-contrast image of fibroblasts at the time of seeding and 7 days after cell seeding into hydrogel (scale bar: 100 µm). (d and e) hematoxylin/eosin staining of hydrogel contain keratinocyte cell at the time of seeding and 7 days after cell seeding into hydrogel (scale bar: 100 µm). (f) masson’s trichrome staining to show collagen content of hydrogel. (g–l) immunofluorescence staining for human ck14 and vimentin show two layers of skin substitute at day 7 after cell seeding. dapi stains the nuclei in blue. (scale bar = 50 µm). a d g j b e h k c f i l 12 j contemp med sci | vol. 5, no. 1, january–february 2019: 8–13 fibrin-collagen hydrogel for dermoepidermal skin substitute original article m.m. sisakht et al. biodegradability, biocompatibility and similarities to the natural extracellular matrix that are unique in comparison with other scaffolds.18 despite these advantages and the weak mechanical properties, a major challenge to hydrogel scaffolds is the difficulty associated with engineering complex tissues with multiple cell types, especially as a bioengineered skin substitute. therefore, hydrogel scaffolds developed for tissue engineering should typically be assessed for their properties.18,19 biological, physicochemical, and topographical surface characteristics of scaffolds are vital parameters in controlling and affecting cellular adhesion and proliferation.20 in this study, we present a natural composition of hydrogel for skin cell culture. the engineered hydrogels have been synthesized using self-assembled fibrin-collagen which mimics the components of the native ecm. fibrin-based biomaterials as cell carriers have been widely investigated in recent decades.21 injectable fibrin gel known as fibrin glue has been investigated more than other forms.22,23 less attention has been paid to fibrin as a scaffold for skin regeneration due to its low mechanical properties.24 because of the important role of fibrin, it can be a suitable scaffold to accelerate wound healing. we investigated a method of natural crosslinking with collagen type i to enhance the mechanical features of fibrin. the trilaminar structure of fibrin after fabrication was visualized by phase-contrast microscopy in fig. 2b. we show here that the optimal concentration of hydrogel was isolated and cultivated skin cells could be seeded on it as a 3d scaffold. kretzschmar et al.25 and watt26 showed that, within the epidermis, proliferation takes place in the basal layer of keratinocytes having unlimited self-renewal capacity that expresses different cell markers in comparison with other layers. in organotypic cultures, keratinocytes from the cornified layer are cultivated at the air– liquid interface.27 in this study, keratinocytes were cultivated from different layers of the epidermis. the study shows that basal cell layer keratinocytes are more vital in a 3d cell culture onto hydrogel in comparison with the cornified cell layer. flow cytometry of select markers revealed differences between keratinocyte cells and high expression of fibroblast cell markers.28 hematoxylin and eosin staining of a hydrogel at the time of seeding and after cell seeding revealed that fibrin-collagen hydrogel is suitable as a scaffold. importantly, the potential of fibrin-collagen hydrogel for tissue-engineered dermal and epidermal skin grafts after 3d cell culture were confirmed by immunofluorescent staining. these results show improvement in maintaining skin cells in 3d hydrogels as a next generation skin equivalent for the treatment of full-thickness defects. conclusion accelerating hydrogel-based dressings have attracted considerable research interest due to their unusual properties and various applications. the combination with hydrogels show potential on full-thickness wounds with irregular margins, although further study is necessary to evaluate different synthetic and natural materials in a clinical setting for enhanced skin regeneration. further studies should be planned to address a novel method of fabrication that is cost-effective, effective for drug loading, fixing of defects in design or feature, and to improve efficacy. the fibrin-collagen hydrogel offers stability and human fibroblasts and keratinocytes provide viable components to produce dermal and epidermal skin replacement. in an ongoing study, we have recently developed a fibrin-collagen hydrogel as a scaffold for skin cell culture. we anticipate the completion of this study for the release of these novel results. we believe that, as a fibrin-collagen wound dressing, it has great potential clinical applications for wound healing. acknowledgments and funding sources this project is phd thesis belonging to the kashan university of medical sciences and skin and stem cell research center, tehran university of medical sciences. the work was supported financially by the iranian council for the development of stem cell sciences and technologies. conflict of interest the authors declare no conflict of interest. n references 1. sulivan f. medtech insight. cytomedix. 2012. available from: https://www. sec.gov/archives/edgar/data/1091596/000114420412001334/v244875_ ex99-1.htm. 2. nilforoushzadeh ma, sisakht mm, seifalian am, amirkhani ma, banafshe hr, verdi j, et al. regenerative medicine applications in wound care. curr stem cell res ther. 2017;12:658–674. 3. alrubaiy l, al-rubaiy kk. skin substitutes: a brief review of types and clinical applications. oman med j. 2009;24:4–6. 4. halim as, khoo tl, mohd yussof sj. biologic and synthetic skin substitutes: an overview. indian j plast surg. 2010;43:s23–s28. 5. kamoun ea, kenawy es, chen x. a review on polymeric hydrogel membranes for wound dressing applications: pva-based hydrogel dressings. j adv res. 2017;8:217–233. 6. akhtar mf, hanif m, ranjha nm. methods of synthesis of hydrogels … a review. saudi pharm j. 2016;24:554–559. 7. madaghiele m, demitri c, sannino a, ambrosio l. polymeric hydrogels for burn wound care: advanced skin wound dressings and regenerative templates. burns trauma. 2014;2:153–161. 8. parenteau nl, bilbo p, nolte cj, mason vs, rosenberg m. the organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function. cytotechnology. 1992;9:163–171. 9. klar as, guven s, zimoch j, zapiorkowska na, biedermann t, bottcherhaberzeth s, et al. characterization of vasculogenic potential of human adipose-derived endothelial cells in a three-dimensional vascularized skin substitute. pediatr surg int. 2016;32:17–27. 10. klar as, guven s, biedermann t, luginbuhl j, bottcher-haberzeth s, meuli-simmen c, et al. tissue-engineered dermo-epidermal skin grafts prevascularized with adipose-derived cells. biomaterials. 2014;35: 5065–5078. 11. simoni rc, lemes gf, fialho s, goncalves oh, gozzo am, chiaradia v, et al. effect of drying method on mechanical, thermal and water absorption properties of enzymatically crosslinked gelatin hydrogels. an acad bras cienc. 2017;89:745–755. 12. cohen ah. masson’s trichrome stain in the evaluation of renal biopsies. am j clin pathol. 1976;65:631–643. 13. ahmed ta, dare ev, hincke m. fibrin: a versatile scaffold for tissue engineering applications. tissue eng part b rev. 2008;14:199-215. 14. gibs i, janik h. review: synthetic polymer hydrogels for biomedical applications. chem chem technol. 2010;4:297–304. 15. bracaglia lg, messina m, winston s, kuo cy, lerman m, fisher jp. 3d printed pericardium hydrogels to promote wound healing in vascular applications. biomacromolecules. 2017;18:3802–3811. m.m. sisakht et al. 13j contemp med sci | vol. 5, no. 1, january–february 2019: 8–13 original article fibrin-collagen hydrogel for dermoepidermal skin substitute this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 16. hu y, zhang z, li y, ding x, li d, shen c, et al. dual-crosslinked amorphous polysaccharide hydrogels based on chitosan/alginate for wound healing applications. macromol rapid commun. 2018;39:e1800069. 17. annabi n, rana d, shirzaei sani e, portillo-lara r, gifford jl, fares mm, et al. engineering a sprayable and elastic hydrogel adhesive with antimicrobial properties for wound healing. biomaterials. 2017;139:229–243. 18. el-sherbiny im, yacoub mh. hydrogel scaffolds for tissue engineering: progress and challenges. glob cardiol sci pract. 2013;2013:316–342. 19. shin h, jo s, mikos ag. biomimetic materials for tissue engineering. biomaterials. 2003;24:4353–4364. 20. boyan bd, hummert tw, dean dd, schwartz z. role of material surfaces in regulating bone and cartilage cell response. biomaterials. 1996;17:137–146. 21. li y, meng h, liu y, lee bp. fibrin gel as an injectable biodegradable scaffold and cell carrier for tissue engineering. scientificworldjournal. 2015;2015:685690. 22. christman kl, fok hh, sievers re, fang q, lee rj. fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction. tissue eng. 2004;10:403–409. 23. cakmak o, babakurban st, akkuzu hg, bilgi s, ovali e, kongur m, et al. injectable tissue-engineered cartilage using commercially available fibrin glue. laryngoscope. 2013;123:2986–2992. 24. litvinov ri, weisel jw. fibrin mechanical properties and their structural origins. matrix biol. 2017;60–61:110–123. 25. kretzschmar k, watt fm. markers of epidermal stem cell subpopulations in adult mammalian skin. cold spring harb perspect med. 2014;4. pii: a013631. 26. watt fm. epidermal stem cells: markers, patterning and the control of stem cell fate. philos trans r soc lond b biol sci. 1998;353:831–837. 27. schoop vm, mirancea n, fusenig ne. epidermal organization and differentiation of hacat keratinocytes in organotypic coculture with human dermal fibroblasts. j invest dermatol. 1999;112:343–353. 28. pontiggia l, biedermann t, meuli m, widmer d, bottcher-haberzeth s, schiestl c, et al. markers to evaluate the quality and self-renewing potential of engineered human skin substitutes in vitro and after transplantation. j invest dermatol. 2009;129:480–490. 159j contemp med sci | vol. 3, no. 9, winter 2017: 159–162 research intestinal parasitic infection effect on some blood components hanaa daaj khalaf al-mozan,a yahya tomaa daoud,b khalid majeed dakhilc abiology department, science college, thi-qar university, iraq. bbiology department, science for women college, baghdad university, iraq. ctechnical institute in nassiriyah, iraq. correspondence to hanaa daaj khalaf al-mozan (email: hanaa_daaj @yahoo.com). (submitted: 14 december 2016 – revised version received: 27 december 2016 – accepted: 10 january 2017 – published online: 26 march 2017) objectives to know the effect of intestinal parasitic infection on the differential count of white blood cells and sex factor effect on some blood criteria. methods the fecal samples were examined by direct and indirect methods. blood samples were withdrawn for the testing of the differential count of white blood cells, hb, pcv, t.wbc and numbers of eosinophils. results the results showed that al-jibaish general hospital was the highest with parasitic infection (50.4%) in comparison with other hospitals. a high percentage (30%) of entamoeba histolytica was formed. when compared with the percentage of each parasite under study between those hospitals, entamoeba histolytica appeared with 12% in al-shatra and 46.6% in al-jibaish. there were significant differences between the parasite types in the percent of lymphocytes, neutrophils, monocytes and eosinophils. it ranged from increase and decrease according to parasite type. the significant difference appeared at the six-factor effect on the number of eosinophils was calculated by using the statistical analysis (χ2) in level p < 0.05. conclusion e. histolytica is the most common parasite. al-jibaish district suffers from a lack of services. there is no effect on the sex factor on blood criteria. each one parasite has a different effect on blood components. keywords intestinal parasites, white blood cells, entamoeba histolytica, haemoglobin introduction intestinal parasitic infection is a serious public health problem throughout the world particularly in developing countries. it is estimated that intestinal parasites infect more than three billion people worldwide.1,2 these parasites cause several symptoms, including: diarrhea, fever, vomiting, coughing, anorexia, gas or bloating and anemia.3 anemia in children can be caused by iron deficiency and by health factors such as parasitic infection.4 many studies have shown that hook worm causes chronic intestinal blood loss,5 blood loss can also occur by trichuris infection.6 hemoglobin is the red pigment in red cell. its loss occurs in some pathological conditions including: parasitic infection, malnutrition, blood loss and chronic infection,7 which is expressed anemia.8 packed cell volume has relation with anemia where the loss of packed cell volume occurs as a result of specific conditions such as anemia.9 white blood cells are considered as one of the basic components of blood and present in peripheral blood,10 to provide defense against germs, parasites and tumors as well as other diseases.11 there are five types of white blood cells vary in size, proportions and functions which are as follow: neutrophils, eosinophils, basophils, lymphocytes and monocytes.9 materials and methods stool samples specimens were collected in clean containers to avoid contamination with urine, water or any other disinfectants. the stool samples were examined by the naked eye for color, odor, and the presence of blood or mucus.12,13 finally, they were examined microscopically by the direct method using normal saline and lugol’s iodine,14,15 and indirect method by floatation with zinc sulfate.12,13 blood samples blood samples (1 ml) were withdrawn by intravenous injection with sterile medical device and transferred to plastic tubes that contain the anticoagulant edta (ethylene diamine tetra acetic acid) and performed the following tests: packed red blood cell volume capillary tubes were filled with blood to three quarters. they were blocked at the end by artificial mud and wiped from outside. then they were put in microhematocrit rotor with speed 13000 r/min for 5 min. packed cell volume was read by using microhematocrit reader.9,16 measurement of hb hb was measured by a reflotron plus, the product by a german company roche established in accordance with the accompanying instructions of the company as follow: a limited volume of blood was taken by micropipette that accompanying with apparatus. blood was put on the exact location of the accompanying tape. there are some tapes were provided from company with apparatus, and there certain place (limited place) in the apparatus for the tape (to insert the tape) for it in the apparatus and was left for 3 min where the result appears on the screen of the apparatus and taking into account the zero of the apparatus before each using. total leukocytes count blood was put in tube contain edta. blood was taken by specific pipette for white blood cells count to gradient 0.5 that is issn 2413-0516 160 j contemp med sci | vol. 3, no. 9, winter 2017: 159–162 intestinal parasitic infection effect on some blood components research hanaa daaj khalaf al-mozan et al. marked on the pipette tube (20 micrometers). diluted liquid was withdrawn to the mark 11 (0.4 ml) and was mixed by hand until became homogenous. the solution was put in neubaur chamber and a cover slip was put on it. the chamber was left for 2 min to settle the cells. then, the cells were calculated under the microscope by the objective lens.17 differential count of white blood cells a drop of blood was put on a clean slide and was pulled by the edge of another slide taking into account the good distribution of the cells on smear. the slide was left to dry and was stained by leishman stain and was left to dry with room temperature then 100 cells of white blood cells were calculated using oily lens. the absolute number of each type of white blood cells in millimeter per cubic was extracted from the total count of white blood cells and percent of each type of white blood cells.9,18,19 the statistical analysis the statistical analysis was performed by using t-test and chi-square test (χ2) according to for 20. results intestinal parasite infection was found in 333 from total 1001 fecal samples of children aged seven years and less than that age who visited different hospitals in thi–qar province. entamoeba histolytica appeared with percent 12% in al-shatra and 46.6% in al-jibaish, as for the rest parasites h. nana, e. vermicularis, e. coli, t. homimis and g. lamblia, the statistical analysis (χ2) in level p < 0.05 not found significant differences between the hospitals forward those parasites (table 1). as for differential count of white blood cells, the results were changed either by excess from the normal criteria of blood or by decrease from the normal criteria of blood and in both cases the results were changed from the normal level. in other word there is effect of some parasites either by increase or by decrease according to type (species) of parasite while some types of parasites haven’t effect where the percent of lymphocytes was 71% at infection with g. lamblia and 33% with (e. histolytica + g. lamblia + t. hominis), neutrophils with 23.5% when infection by g. lamblia and 64% when infection by e. histolytica + g. lamblia + t. hominis. monocytes appeared with 13% at g. lamblia + h. nana and 1% with infection by e. histolytica + h. nana, eosinophils with 6% at infection by (g. lamblia + h. nana), (e. histolytica + h. nana) and 0% when the infection of e. histolytica + g. lamblia + t. hominis, and the differences were significant. and 3% represented the highest rate for basophils at infection with g. lamblia + h. nana. when statistical analysis compared between the percentages of white blood cells for the infected children with the percentages of white blood cells for non-infected children found some of them near and other far according to parasite type (table 2). as for the effect of sex, a number of eosinophils were 154.3 cells/mm3 in an infected male and 218.0 cell/mm3 in an infected female. no significant differences for the effect sex on criteria hb, pcv and wbc in those infected children (table 3). discussion e. histolytica with 30% represented the most common intestinal parasite in this study, which is in agreement with previous studies.21,22 e. histolytica is the highest prevalence in the world specially in tropical and sub tropical countries, and iraq is represented as one of them.23 the humid climate provides favourable environmental conditions for the maturity cyst of entamoeba,24 and then transmit it to human25 in addition to e. histolytica that can transmit directly (without need intermediate host).23 the presence high percentage of infection in al-jibaish may due to marginalization of the district from all services, such as healthy or environmentally, culturally and educationally also. dryness marshes and assemblage brackish water and breeding table 1. percent of infection with different type of parasites between hospitals of thi–qar province total infectional-jibaish general hospital al-rifaai general hospital al-shatra general hospital suq-al-shuyukh general hospital nassiriyah maternity and children hospitaltype of parasite %no. of infected %no. of infected %no. of infected %no. of infected %no. of infected %no. of infected 30.030046.66130.565123130.15040.193e. histolytica 3.6362.332.35257.2124.811g.lamblia 0.220.000.000.001.220.00t. hominis 0.110.810.000.000.000.00e. coli 0.550.000.000.820.001.33e. vermicularis 0.330.810.000.410.610.00h. nana 34.734750.46632.97015.13939.16546.1107total h. nanae. vermicularise. colit. hominisg. lambliae. histolyticax2 calculated 0.000.000.000.005.8821.68 1.001.00 0.200.001*sig. hospital hanaa daaj khalaf al-mozan et al. 161j contemp med sci | vol. 3, no. 9, winter 2017: 159–162 research intestinal parasitic infection effect on some blood components table 2. parasite's type effect on the differential count of white blood cells baso. % mean ± sd eosino.% mean ± sd mono. % mean ± sd neutro.% mean ± sd lymph % mean ± sd type of parasite 0.2 ± 0.151.3 ± 0.64.4 ± 253.3 ± 18.8140.8 ± 16.49e. histolytica n = 288 0 ± 0.003 ± 2.42.5 ± 0.723.5 ± 2.1271 ± 26.87g.lamblia n = 24 0 ± 0.002 ± 1.74 ± 1.549.5 ± 7.7744.5 ± 2.82e. vermicularis n = 5 2 ± 0.005 ± 0.004 ± 0.0042 ± 0.0047 ± 0.00h. nana n = 1 1 ± 0.003 ± 0.005 ± 0.0056 ± 0.0035 ± 0.00t . hominis n = 1 2 ± 0.004 ± 0.005 ± 0.0047 ± 0.0042 ± 0.00e. coli n = 1 1 ± 0.41 ± 0.73 ± 1.1546 ± 8.6249 ± 9.1e. histolytica + g. lamblia n = 10 0 ± 0.006 ± 0.001 ± 0.0042 ± 0.0051 ± 0.00e.histolytica + h. nana n = 1 3 ± 0.006 ± 0.0013 ± 0.0033 ± 0.0045 ± 0.00g.lamblia + h. nana n = 1 0 ± 0.000 ± 0.003 ± 0.0064 ± 0.0033 ± 0.00e. histolytica + g. lamblia + t. hominis n = 1 2.684.714.3912.5113.77t calculated 0.2 ± 0.262.3 ± 0.926 ± 2.1954 ± 15.4337.5 ± 12.64non infected n = 100 table 3. effect of sex factor on some blood components in children that infected with intestinal parasites sig.calculated x 2no. of infectedtests female n = 158 mean ± se male n = 175 mean ± se 0.830.0411.8 ± 0.4411.1 ± 0.32hb g/100 ml 0.900.9534.1 ± 0.8833 ± 0.85pcv cell/mm3 0.142.088385.7 ± 998.818573.5 ± 908.74wbc cell/mm3 0.001*11.01218.0 ± 37.57154.3 ± 33.92number of eosinophile cell/mm3 n, number; x 2 tabulated , 3.84; p < 0.05. animals in the houses led to attract huge number of insects that transfer diseases as well as vegetable cultivation by the population of that region and frequent eating it certainly does not help them to avoid the danger of its contamination, moreover drinking water of rivers and ponds without hesitation. as for differential count of white blood cells , the reason of presence significant differences between parasites, s types in percent of lymphocytes, neutrophils, monocytes eosinophils except basophils may be due to variation of damage nature that caused by each type of parasites (where worms usually more effect than protozoa on blood criteria) and variation of importance and work each cell of white bloods cells and immunity of patient. there is no significant difference for sex effect on hb and pcv which is in agreement with the past research26 and disagreement with other research.27 there were no significant differences on the total count of white blood cells between males and females which is in agreement with the already published research.26,27 it was found that sex factor effect on the number of eosinophils which is disagreement with refs. 26 and 27. the reason may belong to that both double infections with (e. histolytica + h. nana) and (h. nana + g. lamblia) in which the number of eosinophils increased with them. they have occurred in females excluding males. conclusion e. histolytica is the most common parasite in thi-qar province. al-jibaish district was the highest with intestinal parasitic infection as a result of lack of services. there is no effect for sex factor on blood criteria of infected children with intestinal parasitic where variations that occurred on blood criteria depend on parasite, s type. recommendations the study must be performed on parasites that resides in the blood. pay attention with hygiene and aren’t eaten fruits and vegetables unless after being washed. increased of attention with elimination of the vectors such as insects. increased of attention with al-jibaish district from all aspects and progress in it to the level that deserve it for being represent rural identity of iraq. conflict of interest none. n 162 j contemp med sci | vol. 3, no. 9, winter 2017: 159–162 intestinal parasitic infection effect on some blood components research hanaa daaj khalaf al-mozan et al. references 1. markell ek, john dt, krotoski wa. markell and voge’s medical parasitology. 8th ed. philadelphia: w.b. saunders 1999. 2. who intestinal parasites. available at: http://apps.who.int/ctd/intpara/ burdens.htm 2010. 3. dariel j. cleanse and purify thyself book one. medford, oregon. christobe publishing, u.s.a 2007. 4. i.n.a.c.g. guidelines for eradication of iron deficiency anemia. a report of the international nutritional anemia consultative group (inacg). new york, washington, nutrion foundation 1997. 5. crompton d. the public health importance of hook worm disease. parasitology. 2000;121:39–50. 6. stephenon ls, holland cd, cooper es. the public health significance of trichuris trichiura. parasitology. 2000;121:73–95. 7. al-asady har. epidemiological study for some pathogenic intestinal parasites with focus on viability of entamoeba histolytica in basrah city. phd thesis, college of sciences, al-basrah university 2007. 8. al-quraishy aas. study on anemia realty in al-qadisiya province during economic resource period. phd thesis, college of education, al-qadisiya university 2002. 9. harmening dm. clinical hematology and fundamentals of hemostasis. fifth edition. united states of america f.a. davis company. philadelphia 2009. 10. goldsby t, thomas j, osborne ba. immunology 4th ed. w.h. freeman comp., new york, 2000;25. 11. junqueira lc, carneiro j. basic histology. 10th ed., prentice – hall international inc. u.s.a 2003. 12. ichhpujani rl, bhatia r. medical parasitology, 1st ed. jaypee bros. med. publ., new delhi 1994. 13. paniker cj. textbook of medical parasitology. 6th ed. new delhi. jaypee brothers medical publishers 2007. 14. lumsden wh, burns s, mcmillan a. protozoa in practical medical microbiology. by collee jg, marmion bp, fraser ag and simmons a. churchill livingstone, tokyo 1996. 15. zeibig ea. clinical parasitology: a practical approach. w.b. saunders company, philadelphia 1997. 16. jubouri, talal hamad; ali musa. epidemiological study of patients with clinical and giardia giardiasis in children shirqat city 2008. 17. ahamid as, salhi em. practical physiology for community health departments. ministry of higher educ. & scientific research, foundation of technical institutes. al-mosul univ. press 1994. 18. talib vh, khurana sr. a handbook of medical laboratory technology . 5th ed., c.b.s. publ., new delhi. philadelphia 1996. 19. lewis sm, bain bj, bates i. dacie and lewis practical hematology. 9th ed. churchill livingstone 2001. 20. albeldawi aa. manners of statistics. first edition. jordan, d. w. for publishing and distribution 2009. 21. al-rekabi, nuha jabbar. a study of some intestinal parasites that cause diarrhea in children in the city of nasiriyah. master thesis. faculty of education university of thi-qar. 2006;92. 22. wadood ua, bari a, rhman au, qasim kf. frequency of intestinal parasite infestation in children hospital quetta. pakistan j med res. 2005;44. 23. razmjou e, rezaian m, haghighi a, kazemi b, farzami b, kobayashi s, et al. comparison of the recombinant glucose phosphate isomerase from different zymodems of entamoeba histolytica with their natural counterparts by isoenzyme electrophoresis. iranian j publ health. 2005;34:35–40. 24. egwunyenga a, ataikiru dp. soil – transmitted helminthiasis among school age children in ethiope east local government area of delta state, nigeria. afri j biotech. 2005;4:938–941. 25. garba cmg, mbofung cmf. relationship between malnutrition and parasitic infection among school children in the adamawa region of cameroon. pakistan j nutr. 2010;9:1094–1099. 26. al-zubaidi, zainab hadi. worm hookworm ancylostoma duodenale and its impact on the image mhimh blood in the village in the province of babylon. master thesis. faculty of science university of babylon. 2002. 27. al-khalifawi, mohammad jubair. study of the parasites of the gastrointestinal tract in children under five and their impact on some of the blood levels: master – faculty of science university of mustansiriya: 2006;92. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 213j contemp med sci | vol. 3, no. 10, spring 2017: 213–217 research anti-oxidative and neuroprotective effects of flaxseed on experimental unilateral spinal cord injury in rat morteza gholaminejhad,a somayeh arabzadeh,b mohammad akbari,a yousef mohamadi,a gholamreza hassanzadeh,a* adepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. bdepartment of animal physiology, developmental biology laboratory, school of biology, college of science, university of tehran, tehran, iran. correspondence to gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir) (submitted: 17 april 2017 – revised version received: 12 may 2017 – accepted: 28 may 2017 – published online: 26 june 2017 ) objectives investigation of anti-oxidative and neuroprotective effects of flaxseed on rats with unilateral sci. methods 30 male wistar rats were randomly assigned to five groups: control (ctrl), laminectomy, flaxseed, spinal cord injured (sci) and sci + flaxseed (treatment) groups. sci model was induced by placing a 50 g weight for 5 min on to a 2.2 mm × 2.5 mm platform applied to the dura at vertebral level t10. after 4 weeks, the blood of rats in different groups was collected and the influence of flaxseed on antioxidant enzymes and oxidative stress marker level, histologic alterations and locomotion score were assessed. results our results showed that in the sci group, the mean level of superoxide dismutase (sod), glutathione peroxidase (gpx) and catalase (cat) were significantly decreased (p < 0.01), whereas the mean malondialdehyde (mda) content was significantly increased (p < 0.001) in comparison to the laminectomy group. furthermore, the mean levels of sod, gpx and cat in the treatment group was higher than the sci group (p < 0.001), while, the mean mda content in the treatment group was significantly less than the sci group (p < 0.001). in addition, comparison between sci and treatment groups determined a significant decrease in tissue degeneration and volume of cavities in the treatment group. however, basso, beattie and bresnahan (bbb) scores were significantly increased in flaxseed-treated rats on days 14 (p < 0.05), 21 and 28 (p < 0.01) after surgery compared with the sci group. conclusions our study for the first time showed the anti-oxidative and neuroprotective effects of flaxseed on experimental unilateral spinal cord injury in rat. keywords spinal cord injury, flaxseed, oxidative stress, neuroprotective, anti-oxidative introduction spinal cord injury (sci) is a serious health problem which is followed by great psychological disruption and financial burden on patients’ families.1 the sci includes two pathological phases that lead finally to the loss of neurons that can have bad consequences.2,3 in the primary phase a direct mechanical trauma on the spine causes tearing in tissue and then during secondary phase a cascade of fatal molecular events such as oxidative stress, excitotoxicity and inflammation induces a wide cell death in the injured site.4 another important factors accelerate post-traumatic degeneration due to secondary destruction are oxidative stress and apoptosis.5 disruption of the balance between the production of oxidant compounds and antioxidant defense system induces oxidative stress. superoxide dismutase (sod), glutathione peroxidase (gpx) and catalase (cat) are antioxidant enzymes, which are important in the regulation of oxidant/antioxidant balance.6–8 in addition, during lipid peroxidation several products such as malondialdehyde (mda) and nitric oxide (no) are generated, which can provide valuable information related to the severity of cell damage caused by free radicals. the changes in the activities of antioxidant enzymes in biological systems reflect the level of oxidative stress.6 although the precise molecular pathway of secondary injury is still controversial, therapeutic strategies that inhibit oxidative stress may lead to sci improvement. flaxseed (linum usitatissimum) is a seed from flax plant, an annual herb, which is a member of the linaceae family.9 it is a rich source of omega-3 fatty acid a-linolenic acid, lignan phytoestrogen, and soluble fibers.10,11 flaxseed has a remarkable antioxidant effect12 and antiinflammatory effect due to high levels of free radical scavenger properties and omega-3 fatty acids,13 respectively. furthermore, vitamin e, a fat-soluble vitamin, is present in flaxseed. vitamin e has anti-carcinogenic and anti-oxidative effect with the protection of cell constituents from the damaging effects of free radicals that might contribute to cancer development.14 since flaxseed has introduced as an antioxidant herb, the aim of this study was to investigate the anti-oxidative and neuroprotective effects of flaxseed in unilateral sci model. materials and methods animal preparations male adult wistar rats weighing between 250 and 300 g were used in this study (n = 30). animals were kept under constant laboratory conditions, room temperature (20–22°c), a 12-h light–dark cycle, and they had free access to food and water. animal handling was performed following the guidelines of the iranian animal ethics committee, tehran university of medical sciences rules. rats were randomly divided into five groups. each group consists of 6 rats with following description: 1) control (crtl) group: without any manipulation. 2) laminectomy group: with laminectomy without sci. issn 2413-0516 214 j contemp med sci | vol. 3, no. 10, spring 2017: 213–217 anti-oxidative and neuroprotective effects of flaxseed on rats with sci research gholamreza hassanzadeh et al. 3) flaxseed group: without any surgery and feed by 10% flaxseed for 4 weeks. 4) sci group: with sci without any treatment. 5) sci + flaxseed (treatment) group: with sci and feed by 10% flaxseed for four weeks. spinal cord injury the surgical manner was performed under general anaesthesia induced by intraperitoneal ketamine (80 mg/kg) (sigma, taufkrichen, germany) and xylazine (15 mg/kg) (sigma, taufkrichen, germany). following t8–t12 midline skin incision, paravertebral muscles were dissected, and laminectomy was performed at t10 vertebrae as the dura mater was to remain intact. the spinal cord was unilaterally pressed by a 50 g weight for 5 min using a rectangular plate, which was longitudinally oriented over the spinal cord; the plate had an area of 2.2 mm × 2.5 mm. rats’ body temperatures during the surgery were kept constant between 36 and 37°c during surgery. then, paravertebral fascia and skin were sutured separately. upon awakening, rats were neurologically appraised. food, water and urine uptake were monitored. after 4 weeks, the rats were anaesthetized according to mentioned protocol to collect their blood for further analysis. in this study, we tried to use the minimum number of animals and reduce their suffering. the basso, beattie and bresnahan locomotion rating scale scores the basso, beattie and bresnahan (bbb) were used for the assessment of motor function of rats in different groups.15 two observers who were blind to the treatment group evaluated outcomes. in brief, the bbb scores range from 0 to 21 points, which indicates complete paralysis to normal motor function. the assessments were performed for 24 h and 7, 14, 21, 28 days after surgery. rats with a score more than 3 were excluded from the study and with the score less than one selected for the following analysis for 24 h post-sci. drug administration the flaxseed and normal food were grounded and with a little distilled water mixed. finally, the plates were prepared by adding 10% flaxseed to the normal food. evaluation of stress oxidative activity on week 4 postoperatively, the blood was obtained from the each group. centrifuged at 1500 r.p.m. for 10 min. supernatant solution was collected to determine the following biochemical indices. superoxide dismutase (sod) (cat # zb-sod48), glutathione peroxidase (gpx) (cat # zb-gpx48), catalase (cat) (cat # zb-cat48) and malondialdehyde (mda) (cat # zb-mda48) testing kits (zellbio, deutschland, germany) were used to detect the oxidative stress of the injured spinal cord according to their manufacture protocols. hematoxylin–eosin staining for histological analysis, animals were anaesthetized by intraperitoneal ketamine (80 mg/kg) and xylazine (15 mg/kg) and perfused with normal saline, 4 weeks after sci. then, they were received transcardial perfusion of 0.01 m phosphate-buffered saline (pbs) followed by 4% paraformaldehyde (400 ml/rat) for fixation. after perfusion, a 2-cm-long piece of the spinal cord was dissected out from each rat and fixed for 48 h with mentioned fixation buffer. a 3-mm-thick transverse section was made in the epicentre and 3 mm cranially and caudally. afterwards, samples in each group were embedded in paraffin for transverse paraffin sections. the paraffin sections (5 μm thick) mounted on poly-l-lysine-coated slides for histopathological examination by hematoxylin and eosin staining. in the end, slides were observed under light microscopy (olympus, tokyo, japan). statistical analysis data were analyzed using the statistical software package spss.20 statistical software (spss, inc., chicago, il, usa). the normality of nominal variables was analyzed by using the kolmogorov–smirnov test. for comparing data among groups, one-way anova tests were used. the results were expressed as mean+sd. p-values <0.05 were considered to indicate statistical significance. result flaxseed attenuated oxidative stress in rats following sci the mean level of sod, gpx, cat and the contents of mda were measured in each group to verify the antioxidant effects of flaxseed treatment 4 weeks after injury. in the sci group, the mean level of sod, gpx and cat were significantly decreased (p < 0.01), whereas the mean mda content was significantly increased (p < 0.001) in comparison to the laminectomy group, at 4 weeks after injury (fig. 1). the treatment group had a 1.5 fold higher mean level of sod, gpx and cat compared with the sci group (p < 0.001), however, sod and gpx activity in the treatment group were significantly higher than the laminectomy group (p < 0.001) (fig. 1a–c). fig. 1 flaxseed attenuated oxidative stress in rats following sci. the mean level of sod (a), gpx (b) and cat (c) decreased in the sci group in comparison with a laminectomy group (p < 0.01). however, in the treatment group, sod (a), gpx (b) and cat (c) levels significantly increased compared with the sci group (p < 0.001). the mean mda (d) content in the sci group significantly increased in comparison with laminectomy group (p < 0.001). the mda content in the treatment group was significantly less than the sci group (p < 0.001), but it was still higher than the laminectomy group (p < 0.01). data were presented as mean + sd. ctrl: control. *p < 0.05; **p < 0.001; ***p < 0.001. a b dc gholamreza hassanzadeh et al. 215j contemp med sci | vol. 3, no. 10, spring 2017: 213–217 research anti-oxidative and neuroprotective effects of flaxseed on rats with sci in addition, the mean mda content in the treatment group was significantly less than the sci group (p < 0.001), but it was still higher than the laminectomy group (p < 0.01) (fig. 1d). it showed that flaxseed treatment couldn’t restore the effect of sci on mda content to the control level. moreover, the sod, gpx and cat level in the flaxseed group were significantly higher than the laminectomy group (p < 0.001) (fig. 1a–c). flaxseed reduced histological damage after sci hematoxylin and eosin staining of spinal cord tissue 4 weeks after sci showed differences between the control, sci and treatment groups. progressive destruction of the dorsal white matter and central grey matter was obvious in the sci group in comparison to the control group. in the sci group, following injury, bleeding and recruitment of polymorphonuclear cells, induced cavity in damage location was completely obvious. however, in the treatment group density of polymorphonuclear cells decreased and bleeding wasn’t seen. comparison between sci and treatment groups determined significant beneficial effects of flaxseed as decreased the volume of contusion-induced cavities and improved tissue structure in the treatment group (fig. 2). flaxseed improved iocomotor functional recovery of rats following sci the effects of flaxseed on locomotor functional recovery in rats after sci, were measured by bbb test on days 1, 3, 7, 14, 21 and 28 post sci. as shown in figure 3, there isn’t any significant difference in bbb scores between the sci and the treatment group on days 3 and 7 after sci. however, bbb scores were significantly increased in flaxseed-treated rats on days 14 (p < 0.05), 21 and 28 (p < 0.01) after surgery compared with the sci group. discussion sci is a cause of major neurological disability which is followed by great psychological disruption that includes both primary and secondary injuries.16 the primary injury of the spinal cord is followed by a long period of secondary injury. one of the most important factors involved in post traumatic degeneration is the level of oxidative stress.5 oxidative stress could lead to lipid peroxidation, dna and protein oxidation and other disruptions.17,18 oxidative and anti-oxidative parameters such as mda, gpx, sod and cat have been studied in experimental sci model. the increased level of mda, sod and decreased level of cat, gpx have been reported in sci rat model.19 liu et al. showed that oxidative stress level increased during first weeks after injury.20 mda is the end product of lipid peroxidation, and the content of mda is an indicator of free radical levels.21 sod with clearing o2 and preventing tissue injury by toxic oxygen-free radicals can be an indicator of antioxidant activity.22 therefore, the level of sod and mda are important biomarkers of oxidative stress status. in this study, we showed that the mean level of sod, gpx and cat decreased in the sci group, while the mda content increased in comparison to the laminectomy group. our results confirm that the stress oxidative level will increase as secondary bad consequences of sci, which may lead to following neurons disruption. up to now, researchers reported the influences of bilateral sci on stress oxidative level, while in this study we represented that even unilateral sci can disrupt the balance of stress oxidative condition. therefore, reduction of oxidation stress level can be a key factor in the therapeutic schedule of sci. flaxseed (linum usitatissimum) is the seed from the flax plant, an annual herb, which is a rich source of plant omega-3 fatty acid a-linolenic acid, lignan phytoestrogen, and soluble fibres.10,11 there are some studies that have indicated the remarkable antioxidant12 and anti-inflammatory effects of flaxseed due to high levels of free radical scavenger properties and omega-3 fatty acids, respectively.13 lignan has been shown to provide neuroprotective and fig. 3 flaxseeds improve locomotor functional recovery of rats following sci. bbb test represented the significant increase in flaxseed-treated rats on days 14 (p < 0.05), 21 and 28 (p < 001) after surgery compared with the sci group. while, there are not any significant differences in bbb scores between the sci and the treatment group on days 3 and 7 after sci. data were presented as mean+sd. ctrl: control. *p < 0.05; **p < 0.001. fig. 2 flaxseed reduced histological damage after sci. hematoxylin and eosin staining showed progressive destruction of the dorsal white matter and central grey matter in sci rats compared with the control group. in the treatment group, the volume of induced cavity significantly reduced in comparison with the sci group. a (4x), b (10x), c (40x): control. d (40x): 3 days after sci. e (40x): sci. f (40x): treatment. scale bar = 100μm. a d b e fc 216 j contemp med sci | vol. 3, no. 10, spring 2017: 213–217 anti-oxidative and neuroprotective effects of flaxseed on rats with sci research gholamreza hassanzadeh et al. anti-oxidative effect against neurodegenerative diseases such as sci.23 in 2006, king et al. reported that omega-3 fatty acid can improve sci.24 in vitro study showed that flaxseed not only acts as a scavenger of hydroxyl radicals, but also inhibits the lipid peroxidation. scientific evidences support the role of flaxseed in the prevention of some chronic diseases such as many types of cancer, diabetes, cardiovascular diseases and cerebrovascular stroke due to their biological active components.25–28 moreover, it has been reported that flaxseed improves biochemical and oxidative stress parameters in patients with metabolic syndrome (ms).29 in 2016, one research group reported that flaxseed oil (fso) has a neuroprotective effect against ccl4-induced brain injury in gamma-irradiated rats. this effect is interrelated to the ability of fso to scavenge the free radicals, enhances the antioxidant enzyme activity, increases glutathione contents and down-regulates the inflammatory responses.30 based on previous studies, flaxseed has a significant therapeutic potential to decrease the level of oxidative stress. in the present study, for the first time to our knowledge, we assessed the effect of flaxseed in the unilateral sci model. we found that 4 weeks after sci induction gpx, sod and cat were markedly increased in the treatment group compared with control, whereas the mda level was decreased. therefore, we verified that flaxseed can suppress the oxidative stress in sci. in addition, our investigation demonstrated that flaxseed decreased tissue degeneration and the size of contusioninduced cavities in the treatment group, which resulted in improvement of motor activities. to evaluate the effect of flaxseed on locomotor function, animals were exposed to bbb test. animals in the treatment group had significantly more recovery of motor function compared to sci animals. it indicates that locomotor recovery after sci in rats showed significant improvements with flaxseed treatment, as was confirmed by decreases in oxidative stress as a secondary injury. in summary, our results showed that treatment with flaxseed diminished tissue degeneration and improved locomotor function, which can be explained by a reduction in the oxidative stress level after unilateral sci. according to our results, flaxseed introduces as a new neuroprotective factor in sci, and it needs to be more investigated in the future. acknowledgments this work was supported by the tehran university of medical sciences [grant number: 95-04-30-33618]. conflicts of interest authors have declared that no competing interests exist. n references 1. pickett ge, campos-benitez m, keller jl, duggal n. epidemiology of traumatic spinal cord injury in canada. spine. 2006;31:799–805. 2. balentine jd. pathology of experimental spinal cord trauma. i. the necrotic lesion as a function of vascular injury. lab invest. 1978;39: 236–253. 3. cortez sc, mcintosh tk, noble lj. experimental fluid percussion brain injury: vascular disruption and neuronal and glial alterations. brain res. 1989; 482:271–282. 4. zhang hy, wang zg, wu fz, kong xx, yang j, lin bb, et al. regulation of autophagy and ubiquitinated protein accumulation by bfgf promotes functional recovery and neural protection in a rat model of spinal cord injury. mol neurobiol. 2013;48:452–464. 5. chen hc, hsu pw, tzaan wc, lee aw. effects of the combined administration of vitamins c and e on the oxidative stress status and programmed cell death pathways after experimental spinal cord injury. spinal cord. 2014;52:24–28. 6. guéraud f, atalay m, bresgen n, cipak a, eckl pm, huc l, et al. chemistry and biochemistry of lipid peroxidation products. free radic res. 2010;44: 1098–1124. 7. nazıroğlu m. trpm2 cation channels, oxidative stress and neurological diseases: where are we now?. neurochem res. 2011;36:355–366. 8. somogyi a, rosta k, pusztai p, tulassay z, nagy g. antioxidant measurements. physiol meas. 2007;28:r41–55. 9. rubilar m, gutiérrez c, verdugo m, shene c, sineiro j. flaxseed as a source of functional ingredients. j soil sci plant nutr. 2010;10:373–377. 10. oomah bd. flaxseed as a functional food source. j sci food agri. 2001;81:889–894. 11. pengilly nl. 13 traditional food and medicinal uses of flaxseed. flax: the genus linum. 2003:252. 12. touré a, xueming x. flaxseed lignans: source, biosynthesis, metabolism, antioxidant activity, bio‐active components, and health benefits. comp rev food sci food saf. 2010;9:261–269. 13. calder pc. polyunsaturated fatty acids, inflammation, and immunity. lipids. 2001;36:1007–1024. 14. winter r. vitamin e: your protection against exercise fatigue, weakened immunity, heart disease, cancer, aging, diabetic damage, environmental toxins: three rivers press. 2013. 15. basso dm, beattie ms, bresnahan jc. a sensitive and reliable locomotor rating scale for open field testing in rats. j neurotrauma. 1995;12:1–21. 16. hong z, chen h, hong h, lin l, wang z. tsp-1 expression changes in diabetic rats with spinal cord injury. neurol res. 2009;31:878–882. 17. dumont rj, okonkwo do, verma s, hurlbert rj, boulos pt, ellegala db, et al. acute spinal cord injury, part i: pathophysiologic mechanisms. clin neuropharmacol. 2001;24:254–264. 18. fatima g, sharma vp, das sk, mahdi aa. oxidative stress and antioxidative parameters in patients with spinal cord injury: implications in the pathogenesis of disease. spinal cord. 2015;53:3–6. 19. varija d, kumar k, reddy k, reddy v. prolonged constriction of sciatic nerve affecting oxidative stressors & antioxidant enzymes in rat. indian j med res. 2009;129:587–592. 20. liu d, liu j, wen j. elevation of hydrogen peroxide after spinal cord injury detected by using the fenton reaction. free radic biol med. 1999;27:478–482. 21. yue c, chen j, hou r, liu j, li x, gao z, et al. effects of selenylation modification on antioxidative activities of schisandra chinensis polysaccharide. plos one. 2015;10:e0134363. 22. carda ap, marchi kc, rizzi e, mecawi as, antunes-rodrigues j, padovan cm, et al. acute restraint stress induces endothelial dysfunction: role of vasoconstrictor prostanoids and oxidative stress. stress. 2015;18:233–243. 23. khalatbary ar. natural polyphenols and spinal cord injury. iran biomed j. 2014;18:120–129. 24. king vr, huang wl, dyall sc, curran oe, priestley jv, michael-titus at. omega-3 fatty acids improve recovery, whereas omega-6 fatty acids worsen outcome, after spinal cord injury in the adult rat. j neurosci. 2006;26: 4672–4680. 25. adolphe jl, whiting sj, juurlink bh, thorpe lu, alcorn j. health effects with consumption of the flax lignan secoisolariciresinol diglucoside. br j nutr. 2010;103:929–938. 26. kinniry p, amrani y, vachani a, solomides cc, arguiri e, workman a, et al. dietary flaxseed supplementation ameliorates inflammation and oxidative tissue damage in experimental models of acute lung injury in mice. j nutr. 2006;136:1545–1551. gholamreza hassanzadeh et al. 217j contemp med sci | vol. 3, no. 10, spring 2017: 213–217 research anti-oxidative and neuroprotective effects of flaxseed on rats with sci this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 27. lee jc, bhora f, sun j, cheng g, arguiri e, solomides cc, et al. dietary flaxseed enhances antioxidant defenses and is protective in a mouse model of lung ischemia-reperfusion injury. am j physiol lung cell mol physiol. 2008;294:l255–265. 28. lee jc, krochak r, blouin a, kanterakis s, chatterjee s, arguiri e, et al. dietary flaxseed prevents radiation-induced oxidative lung damage, inflammation and fibrosis in a mouse model of thoracic radiation injury. cancer biol ther. 2009;8:47–53. 29. pilar bc, da costa güllich aa, ströher dj, zuravski l, mezzomo j, coelho rp, et al. 28-days dietary supplementation with golden flaxseed improves biochemical and oxidative parameters in patients with metabolic syndrome. j functional foods. 2014;10:232–242. 30. ismail af, salem aa, eassawy mm. modulation of gamma-irradiation and carbon tetrachloride induced oxidative stress in the brain of female rats by flaxseed oil. j photochem photobiol b. 2016; 161:91–99. dx.doi.org/10.22317/jcms.06201703 234 j contemp med sci | vol. 3, no. 10, spring 2017: 234–236 research report of a case of surgical ciliated cyst in the left maxillary sinus amirhossein pakravan,a shima nafarzadehb* aoral and maxillofacial surgery department, mazandaran university of medical sciences, sari, iran. boral and maxillofacial pathology department, babol university of medical sciences, babol, iran. correspondence to shima nafarzadeh (email: shima.nafar2004a2yahoo.com). (submitted: 28 march 2017 – revised version received: 16 april 2017 – accepted: 22 april 2017 – published online: 26 june 2017 ) surgical ciliated cyst happens as a delayed complication in the maxillary sinus, and is more frequent in asia in comparison with western countries. we report a case of surgical ciliated cyst in maxillary sinus in a male patient after 30 years of surgery for sinusitis treatment. the patient had swelling and pain in the region, and his radiographic views showed a cystic lesion. incisional biopsy was performed and surgical ciliated cyst was reported as diagnosis. then, enucleation with curettage was performed for him as treatment. keywords surgical ciliated cyst, maxilla, sinus introduction surgical ciliated cyst is a type of true epithelial lined antral cysts, which arises from sinonasal mucosa and gland. it is believed to occur after sinus trauma and surgery because of a separation, which occurs in the sinus lining that forms a cystic cavity lined by epithelium. even a difficult extraction can damage the floor of sinus and lead to the cyst formation.1 surgical ciliated cyst was first described in 1927 in japan by kubo.2 very limited cases with this entity have been reported in english articles.3 the lesion is more frequent in japan.4 its prevalence in patients with 5 to 49 years of age who had a previous history of maxillary sinus surgery was estimated to be more than 20% in japan. postoperative maxillary cyst, traumatic ciliated cyst, and paranasal cyst were also used as synonyms for the lesion.1,3 surgical ciliated cyst is considered as an inflammatory and destructive lesion in sinus region, which is developed after orthognathic and caldwell-luc radical antrostomy.5 clinical view of the lesion resembles tumoral lesion and causes vestibular and palatal expansion. moreover, fistula formation was noted in some cases.6 few research reported the lesion in mandible after orthognathic surgery, and some research reported its occurrence in mandible after genioplasty and osteocartilaginous graft.1,7 if a non-vital tooth is seen in the area, the lesion resembles a radicular cyst.8 here, we report a case of surgical ciliated cyst occurred in the left maxillary sinus after 30 years of sinus surgery. case report a 49-year-old male patient came to the oral and maxillofacial surgeon clinic in sari, iran presenting with the chief complaint of a swelling and pain in the left side of his upper jaw. the patient’s past medical history revealed a surgery in his maxillary sinuses 30 years ago for treatment of sinusitis. his familial and medical history was not noticeable. he also did not show smoking or alcohol consumption habit in past years. on extra-oral examination, tenderness of the area was detected. radiographic examination showed a well-defined radiolucency in his left maxillary sinus, extending to the maxillary ridge and between premolar and molar roots, measuring about 4 × 1.5 cm in diameter. swelling of buccal cortex with destruction in some areas was seen. in addition to that, a similar lesion with smaller size was obvious in his right maxillary sinus (figs. 1 and 2). root canal therapy for premolar teeth and re-treatment for involved molar were done to eliminate dental inflammation source. his pain was relieved but swelling was persistent after 1 month. incisional biopsy for the left sinus mass and excisional biopsy for the right sinus were performed. histopathologic examination demonstated a cystic lesion covered by ciliated pseudo-stratified columnar epithelium (figs. 3 and 4). mucus cells in the lining and mucus in the lumen space were detected. chronic inflammatory cells infiltration was found in the cyst connective tissue wall. based on the clinical and microscopic findings, surgical ciliated cyst was made as diagnosis (figs. 3 and 4). the patient underwent excisional surgery for the left side lesion with complete curettage. he was asked for follow up sessions, and no recurrence was evident after 3 months. discussion surgical ciliated cyst is believed to develop after a mucosal epithelial remaining during a bone-healing phase after an osteotomy in the area.3 the exact prevalence of this cyst is unknown in iran, but it is estimated to have a 3–20% incidence in japan, perhaps due to the high prevalence of sinusitis in this country and also the preference of surgery to antibiotic therapy there.9 the cyst is detected as a unilocular or multilocular radiolucency with the capacity to expand into the canine fossa and also to the nasal wall of sinus. aggressive behavior of the cyst has been occasionally obsereved.10 in cases that involved teeth are nonvital, it can mimic a radicular cyst.8 it is important to differentiate a surgical ciliated cyst from sinus pseudocysts or mucoceles, which have similar linings, and sometimes are related to trauma.11 cyst formation duration varies from 6 month to 49 years.12 a surgical ciliated cyst that developed at 5 years after maxillary advancement surgery, with significant upper displacement of the sinus floor, communication with nasal fossa, and palatine bone perforation was reported by amin et al.4 our patient mentioned a history of surgery 30 years ago. this period may show the slow process of cystic changes and its inaggressive nature. issn 2413-0516 shima nafarzadeh et al. 235j contemp med sci | vol. 3, no. 10, spring 2017: 234–236 research asurgical ciliated cystin maxilla fig 1. ct scan shows a well-defined radiolucent lesion in the left maxillary sinus. fig 2. periapical radiograph shows a destructive lesion involving the apical area of premolar and molar teeth. fig 3. histopathologic view shows a cystic lesion lined by ciliated pseudo-stratified columnar epithelium (× 40). fig 4. microscopic view shows ciliated pseudo-stratified columnar epithelium (× 100). the histopathologic feature of the cyst showed typical characteristics of the cyst consist of a connective tissue wall lined by ciliated pseudo-stratified columnar epithelium and areas of mucous cells. maruyama et al. evaluated 360 ciliated cysts histologically and found that 66% of them showed pseudo-stratified ciliated epithelium, 28% transition epithelium, and 6% squamous epithelium. goblet cells were present in all cysts except in areas with squamous epithelium. the number of goblet cells was observed to be related to the inflammatory cells infiltration.13 ciliated cells can be seen in other cysts such as odontogenic keratocyst, however, the epithelial characteristics of the latter will help to be distinguished.14 the treatment was discussed by yoshikawa et al. in 1982. they proposed enucleation or marsupialisation of the cyst. the marsupialisation was the preferred method for unilocular cysts with a thin wall or large cysts with extensive bone perforation.15 precise studies are necessary to determine the incidence of surgical ciliated cyst in iran. since surgical ciliated cyst of the maxilla is believed to result from entrapment of the epithelium of sinus in the maxilla during surgical procedure, a preventive measure such as careful suturing and patient followup is highly recommended.3,16 n references 1. neville bw, damm dd, allen cm, bouquat je. oral & maxillofacial pathology, 4th ed. india: saunders; 2016. pp. 295–296. 2. kubo i. a buccal cyst occurred after a radical operation of the maxillary sinus. z f otol tokyo. 1927;3:896–897. 3. cano j, campo j, alobera ma, baca r. surgical ciliated cyst of the maxilla. clinical case. med oral patol oral cir bucal. 2009;14: e361–4. 4. bartnik w, bartnik-krystalska a. postoperative maxillary cyst. otolaryngol pol. 2004;58:641–643. 5. amin m, witherow h, lee r, blenkinsopp p. surgical ciliated cyst after maxillary orthognathic surgery: report of a case. j oral maxillofac surg. 2003;61:138–141. 6. sugar aw, walker dm, bounds ga. surgical ciliated (postoperative maxillary) cysts following mid-face osteotomies. br j oral maxillofac surg. 1990;28:264–267. 7. anastassov ge, lee h. respiratory mucocele formation after augmentation genioplasty with nasal osteocartilaginous graft. j oral maxillofac surg. 1999;57:1263–1265. 236 j contemp med sci | vol. 3, no. 10, spring 2017: 234–236 asurgical ciliated cystin maxilla research shima nafarzadeh et al. 8. leung yy, wong wy, cheung lk. surgical ciliated cyst may mimic radicular cysts or residual cysts of maxilla: report of 3 cases. j oral maxillofac surg. 2012;70:264–269. 9. kaneshiro s, nakajima t, yoshikawa y, iwasaki h, tokiwa n. the postoperative maxillary cyst: report of 71 cases. j oral surg. 1981;39:191–198. 10. pe mb, sano k, kitamura a, inokuchi t. computed tomography in the evaluation of postoperative maxillary cysts. j oral maxillofac surg. 1990;48:679–684. 11. thio d, phelps pd, bath ap. maxillary sinus mucocele presenting as a late complication of a maxillary advancement procedure. j laryngol otol. 2003;117:402–403. 12. lockhart r, ceccaldi j, bertrand jc. postoperative maxillary cyst following sinus bone graft: report of a case. int j oral maxillofac implants. 2000;15:583–586. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 13. maruyama m, onodera k, ooya k. a histopathological and lectinhistochemical study of the lining epithelium in postoperative maxillary cysts. oral dis. 2002;8:241–248. 14. yamazaki m, cheng j, nomura t, saito c, hayashi t, saku t. maxillary odontogenic keratocyst with respiratory epithelium: a case report. j oral pathol med. 2003;32:496–498. 15. yoshikawa y, nakajima t, kaneshiro s, sakaguchi m. effective treatment of the postoperative maxillary cyst by marsupialization. j oral maxillofac surg. 1982;40:487–491. 16. fernandes ks, gallottini mhc, felix vb, santos pss, nunes fd. surgical ciliated cyst of the maxilla after maxillary sinus surgery: a case report. oral surg. 2013;6:229–233. dx.doi.org/10.22317/jcms.06201707 216 j contemp med sci | vol. 4, no. 4, autumn 2018: 216–221 research an electrochemical sensing platform for sensitive detection dna methylation using fe 3 o 4 /tmc/au nanocomposite and poly(i-arginine)/ reduced graphene oxide modified screen-printed carbon electrode leila syedmoradi,a hassan hajghassem,b gholamreza tavoosidana,c seyed mahdi rezayat,a reza faridi-majidi,a* and kobra omidfard,e* adepartment of medical nanotechnology, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. b department of microelectromechanical systems, faculty of new sciences and technologies, university of tehran, tehran, iran. cdepartment of molecular medicine, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. dbiosensor research center, endocrinology and metabolism molecular-cellular sciences institute, tehran university of medical sciences, tehran, iran. eendocrinology and metabolism research center, endocrinology and metabolism research institute, tehran university of medical sciences, tehran, iran. correspondence to reza faridi-majidi and kobra omidfar (email: refaridi@sina.tums.ac.ir, omidfar@tums.ac.ir). (submitted: 16 july 2018 – revised version received: xx xxxx xxxx – accepted: xx xxxx xxxx – published online: xx xxxx xxxx) objective this study, a simple electrochemical nanogenosensor has been developed for the rapid and sensitive detection of methylated septin 9 dna as a useful biomarker for early colorectal cancer detection or screening. methods the process consists of three main steps: (i) the surface modification of screen-printed carbon electrode (spces) with a poly(l-arg)/ reduced graphene oxide composite film followed by immobilizing anti-5-methylcytosine antibody, (ii) preparation of probe-modified fe3o4/n-trimethylchitosan/au nanocomposites for the hybridization with complementary dna sequences, (iii) capturing methylated dna target by antibody-modified spces and subsequent electrochemical detection through redox peak currents of gold nanoparticles which generated a concentration-dependent response. results the surface modification of the electrode and hybridization with the methylated target were confirmed by cyclic voltammetry method and differential pulse voltammetry was used for quantitative evaluation of methylated target dna. conclusion the assay showed a wide linear range from 0.01 to 1000 pm with a low detection limit of 0.01 pm. keywords fe 3 o 4 /tmc/au nanoparticle, polyarginine, dna methylation, sept9, anti-5-methylcytosine antibody introduction dna methylation, best-known epigenetic modifications, provides a useful source of biomarkers for cancer classification and prognosis.1,2 the first evidence on link between dna methylation and cancer was reported by feinberg and vogelstein3 and afterward, the analysis of dna methylation biomarkers, has become one of the attractive field in early cancer diagnosis, prognosis and therapeutic monitoring.4,5 to date, the huge number of dna methylation biomarkers have been discovered and analyzed in various malignancies including prostate cancer, breast cancer, lung and colorectal cancers.5,6 methylated septin 9 (msept9) dna has been reported as a biomarker for screening and early detection of colorectal cancer (crc).7,8 a number of clinical trials have evaluated the performance of msept9 in human plasma for crc detection. the sensitivity and specificity of msept9 in these studies varied from 50–90% to 88–91%, respectively, depending on the experimental assays.7–9 up to now, several methods have been developed to detect methylated dna biomarkers including methylation-specific polymerase chain reaction (pcr). (ms-pcr), pyrosequencing, bisulfate sequencing, methylation specific microarray and methylation-specific fluorescence resonant energy transfer.10,11 however, these methods are complicated and time consuming, requiring a large amount of purified pcr samples and costly instrumentation which limit the use of them in clinical laboratories. during the past decade, the different models of biosensor technologies including electrochemical, electrical, optical, and mass based methods have been developed and reported for the detection of methylated dna allowing rapid, easier to use and cost effective diagnostic than conventional methods.12,13 among these biosensors, electrochemical biosensors have been widely applied in biosensing due to their unique features such as high sensitivity, rapid and simple implementation, low cost, and compatibility with miniaturization technologies.14,15 furthermore, the incorporation of nanomaterials and conductive polymers into electrochemical biosensors can efficiently improve the assay sensitivity as well as enhance signal-to background ratio and stability of biosensors.16,17 in this study, spces modified with reduced graphene oxide (rgo) and poly(l-arginine) [poly(l-arg)] was used as sensing platform. graphene oxide (go) and rgo show great potential for electrochemical biosensing due to their large surface area, good electrical properties, ease of synthesis and functionalization, good water dispersity, and excellent biocompatibility. these properties make graphene-based nanomaterials excellent scaffolds for the construction of sensitive electrochemical biosensors for a variety of analytes.18–20 recent research reported the electrodes covered with rgo showed better electrochemical biosensing performance as compared to bare electrode or electrodes modified with carbon nanotubes.21 electrochemical reduction is commonly used for the subsequent reduction of predeposition go to rgo on electrode surface due to its simplicity, fast, low-cost, and greater efficiency rather than other approaches such as chemical and thermal reduction.21–23 issn 2413-0516 l. syedmoradi et al. 217j contemp med sci | vol. 4, no. 4, autumn 2018: 216–221 research electrochemical biosensor for detection of dna methylation in addition to graphene based nanomaterials, poly(amino acid)-modified electrodes such as poly(l-arg) have also been used in electrochemical sensing due to their excellent electrocatalytic ability. poly(l-arg) possesses a protein-like structure containing a positively guanidyl group which able to interact with the negatively charged groups of go through electrostatic interactions.24,25 thus, the poly(l-arg)/rgo composite film can take advantage of the good electronic properties of graphene and excellent electrocatalytic activity of l-arginine, leading to the excellent performance of sensing platform.24,26 here, we report an immuno-dna based technique for the simple and fast detection of sept9 methylated dna target, considered a useful biomarker for screening and early detection of crc. in this work, rgo-modified spces were prepared by the electrochemical reduction of go suspension predeposited on electrode surfaces. the rgo-modified electrodes were then coated with the poly(l-arg), followed by the simple entrapment of anti-5-methylcytosine (anti-5mc) antibody on the polymer-modified surfaces. next, fe3o4/ntrimethylchitosan (tmc)/au nanocomposites modified with biotinylated single-strand dna (ssdna). probe are used for the subsequent hybridization between dna target sequence and nanoparticle-dna probes. finally, the nanoprobe–dna hybrid was captured by the antibody-modified spces, which in turn produce a signal current proportional to the amount of dna target. experimental section materials and reagents chemicals ferric chloride hexahydrate (fecl3×6h2o), ferrous chloride tetrahydrate (fecl2×4h2o), naoh, and acetic acid were obtained from acros organics (usa). chloroauric acid (haucl4), sodium dodecyl sulfate (sds), low molecular weight chitosan, dialysis tube with molecular cutoff 12,000 da, sodium azide, streptavidin, monoclonal anti-5mc antibody, go and l-arginine were purchased from sigma chemical company (st. louis, mo, usa). the analytical hcl, nacl, d-glucose, glutaraldehyde, n-methyl pyrrolidone (nmp), iodomethane, sodium hydroxide, sodium iodide and acetone were purchased from merck (darmstadt, germany). all other chemicals used were of analytical grade and deionized water was used in all the experiments. the spces (3.4 cm length × 1.0 cm width × 0.05 cm height) were purchased from dropsens (spain). oligonucleotides biotinylated ssdna probes and target methylated dna sequences used in this study were purchased from tag copenhagen a/s (denmark); the dna sequences are listed as follows: methylated sequence (target): 5¢m c g g m c g c c c c a g c c a g m c g m c g c a g g g c cmcgggcccmcgcmcgggggmcgc-3¢ probe sequence: 5¢-ggcccgggccctgcgcgctg-biotin-3¢ procedures preparation of au/tmc/fe3o4 nanocomposites the au/tmc/fe3o4 nanocomposites is composed of three layers (magnetic core, intermediate tmc polymer, and gold nps coating shell) were separately prepared according to the previous literature. briefly, fe(ii) and fe(iii) chloride were dissolved in distilled water at a ratio of 0.5 and fe3o4 nanoparticles were then precipitated in the presence of naoh.27 the tmc polymer was prepared using iodomethane in the presence of nmp and naoh according to previous publication. colloidal au nanoparticles were synthesized by the reduction of aqueous haucl4 with d-glucose in the presence of naoh at 60°c. in the following, a thin layer of previously produced tmc polymer was coated on magnetic nanoparticle (mnp). by use of the cross-linker glutaraldehyde. for this purpose, the 1:1m ratio of fe3o4 to tmc, were dissolved in a solution containing glutaraldehyde, nacl, and sds. the mixture was incubated at 50°c for 5 h under stirring to obtain tmc coated nanoparticles. next, 200 mg of tmc@fe3o4 nps were exposed to 25 ml of colloidal gold nps at room temperature for 1 h under stirring condition. the product, fe3o4/tmc/au nanocomposites, was magnetically pelleted, washed with pbs and kept away from light at 4°c after adding sodium azide (0.01%).27,28 characterization of produced nanomaterials the size and morphology of fe3o4 nanoparticles, fe3o4/tmc nanocomplexes, and au/tmc/fe3o4 were evaluated by transmission electron microscopy (tem) (zeiss, em10c, 80 kv, germany). the chemical interactions in the product formulation was confirmed by fourier transform infrared spectroscopy (ftir). in addition to above-mentioned characterization, the crystallinity of the nanocomposites and magnetic properties of magnetic nanomaterials were studied by x-ray diffraction and vibrating sample magnetometer respectively as previously reported by our group.16,29 preparation of nanocomposite-conjugated probes the biotin-labeled probe was attached onto the au/fe3o4 nps through a two steps process: (1) coating the au/fe3o4 nps with streptavidin; (2) immobilization of biotinylated dna probe onto the streptavidin-coated nps. first, to prepare the streptavidin-coated nps, 20 μg streptavidin dissolved in 500 μl pbs was added to the 20 mg of au/tmc/fe3o4 nps and incubated at 37°c for 1 h under shaking condition. the streptavidin was electrostatically conjugated with the gold magnetic particles. the nanocomposites were washed and magnetically separated. the products were then magnetically separated and washed. the coupling efficacy was then evaluated by measuring the od280 values of streptavidin suspension before and after conjugation process. next, the biotinylated ssdna probe which could specifically detect sept9 was immobilized onto the streptavidin-coated nps. 250 microliters of pbs buffer containing 400 pmol of biotinylated dna probe was added to streptavidin-coated nanocomposites and incubated at 37°c under shaking condition. finally, the particles were rinsed with pbs buffer and the immobilization efficiency was estimated from the od260 values of the oligonucleotide solutions preand post-immobilization process. fabrication of poly(l-arg)/rgo-modified electrode the spces consist of a carbon working electrode (4 mm diameter), carbon counter electrode, and a silver pseudo-reference electrode. first, 5 μl of 1 mg ml−1 go in water was 218 j contemp med sci | vol. 4, no. 4, autumn 2018: 216–221 electrochemical biosensor for detection of dna methylation research l. syedmoradi et al. drop-casted onto the working electrode and a thin film of rgo was formed by cv method from −1.5 to 0 v vs. ag/agcl for 10 cycles at a scan rate of 100 mv s−1.22,30 the modified electrode was then washed with pbs solution to remove remaining go particles on the electrode surface. next, 50 μl of l-arg was electrodeposited onto rgo-modified spce surface by cv method from −2 to 2 v at 100 mv s−1 for seven cycles.24,25 after a washing step, 5 μl of pbs solution containing 100 ng of anti-5mc antibody was added to the working zone of each spce and kept overnight at 4°c. the ab-modified spces were washed with pbs buffer to remove the excess ab and unreacted surface sites were then blocked using bsa (3%) for 15 min. quantification of methylated dna by using fabricated nanobiosensor hybridization process the hybridization was performed by stirring at room temperature. first, various concentrations of methylated dna target were prepared in pbs buffer and then, 4 mg of probe-modified nps was added to each concentration and incubated for 2 h under gently shaking. after hybridization process, the nps were magnetically separated, washed, and re-suspended in pbs buffer. in the following, 20 µl of the resulting suspensions were added to the ab-modified spces and kept at 37°c for 40 min under gently shaking. after a washing step with pbs, the electrodes were assessed by electrochemical measurements.16 electrochemical measurements: electrochemical characteristics of stepwise modifies electrodes were performed cv mode and differential pulse voltammetry (dpv) method using 50 µl of hcl (1m) on the surface of modified electrodes. all electrochemical measurements, performed at room temperature, involved oxidoreduction of gold nanoparticles in the nano-complex. the basic analysis of the spces was obtained through cv mode, at the potential range of −0.7 to +1.1 v and the scan rate of 50 mv s−1. the performance of the biosensor was evaluated by dpv mode.16,28 the specificity of the constructed biosensor was evaluated by using the pcr-amplified unmethylated dna sequence as a negative control in this sensing platform. the accelerated stability of the modified electrodes was assessed every 7 h for over 3 days at 37°c, and was compared with newly prepared electrodes. furthermore, the reproducibility of assay was evaluated using detection of two concentrations in independent measurements.31 results and discussion strategy of analysis the goal of this study was to define the valuable properties of nanomaterial and conductive polymer for development of an ultrasensitive dna-based electrochemical nanobiosensor. in this work, poly(l-arg)/rgo composite film and fe3o4/tmc/ au nanocomposite were applied to make a highly sensitive assay that can detect dna methylation. to increase the sensitivity of the assay, fe3o4/tmc/au nanocomposite was used as a tracing tag to label dna probe.32 besides, the composite film made of conducting rgo/arg was electrochemically polymerized on the spces. fe3o4/tmc/au nanocomposite-labeled dna probe was used to capture the target sequence, and then on rgo/arg/anti-5mc modifies spce recognition was performed between methylated dna and anti-5mc antibody. an overview of the detection process is proposed in fig. 1. characterization of fe3o4/tmc/au nanoparticles transmission electron microscopy was used to investigate the size and shape of the fe3o4, fe3o4/tmc and fe3o4/tmc/au nanoparticles. the monodisperse fe3o4 nanoparticles with a diameter of about 10 nm can be seen in fig. 2a. a core–shell structures with a diameter of 12 nm was observed for the magnetic fe3o4/tmc nanocomposite (fig. 2b). after the assembly of gold nps on the surface of fe3o4/tmc nanocomposite, the diameter of the final nanocomposites has significantly enhanced and reached 20 nm (fig. 2c). ftir analysis was used in order to study the positive assembly of tmc-coated fe3o4 nps and final fe3o4/tmc/au nanocomposites. as seen in fig. 3, in ftir spectra of tmc four typical peaks were observed at about 3430 cm−1 (overlap of o–h and intermolecular hydrogen bonding vibrations), 2900 cm−1 (assign to the c–h bond), 1650 cm−1 (ascribe the n–h bending vibration) and 1070 cm−1 (c–o–c stretching vibration). furthermore, two specific peaks at about 1470 and 1600 cm−1 can be used for differentiating tmc from chitosan. in ftir spectra of fe3o4/tmc fig. 1 schematic of the principal mechanism for methylated dna detection by the genosensor. l. syedmoradi et al. 219j contemp med sci | vol. 4, no. 4, autumn 2018: 216–221 research electrochemical biosensor for detection of dna methylation immobilization of biotinylated dna probe onto the streptavidin fe3o4/tmc/au nanocomposite streptavidin–fe3o4/tmc/au particles was bound to the biotin labeled probe through the interaction between streptavidin and biotin. the non-covalent interaction between streptavidin and biotin is exceptionally fast and consistent. fe3o4/tmc/au nanocomposite was not only used as a label in this study, but also applied as the immobilization substrate and separation system that is actually essential in biosensors development. we detected an immobilization efficacy of about 87% by measuring the od260 values before and after immobilization. characterizations of poly(l-arg)/rgo composite film the surface modification of the spces with poly(l-arg)/rgo composite film were assessed with electrochemical characterization. the surface of bare spces were coated with a thin layer of rgo through electro-deposition of go solution. a cathodic peak appeared at −0.8 v which is attributed to the chemical reduction of the oxygencontaining groups in go. meanwhile, the cathodic peak slightly shifted toward a more positive potential and the peak current decreased with each cycle (data not shown). this results confirm depositing go and its reduction to rgo on the spce surface. the second step of electrode modification was performed by the polymerization of l-arg, the potential was cycled between −0.2 and +2 v vs. spce. resulted voltammogram of poly(l-arg)/rgo/spce shows the current peak of arginine increased with cv scan rates indicating that arginine was electropolymerized on the spce surface (data not shown). electrochemical characterization of the genosensor to confirm the accuracy of the genosensor preparation procedure, each step of surface modification was investigated using cv. figure 4 presents the cv obtained at bare spce, rgo-modified spce, arg-modified spce and rgo/ arg-modified spce in the presence of 5.0 mm [fe(cn)6] 3−/4− containing 0.1m kcl. comparing with the bare spce, redox peak currents were obviously increased with the formed layers, demonstrating electrical conductivity of the composite film is good which in fig. 3 ftir spectra of tmc (a), fe 3 o 4 /tmc (b) and fe 3 o 4 /tmc/au (c) nanoparticles. nanocomposite we found a detectable peak at about 1502 cm−1 that can relate to the shifted n–h bond. there is an additional peak at about 1630 cm−1 which was made due to the interaction between the n–h groups of the tmc and the cho group of glutaraldehyde. lastly, a strong peak at about 585 cm−1 (curves b and c) is assigned to the fe–o bonds and obtained from naked fe3o4 particles. 16,27 characterization of streptavidin coupled fe3o4/ tmc/au nanocomposite streptavidin was immobilized on the surface of au nanoparticle in fe3o4/tmc/au nanostructure by physical adsorption. uv spectroscopy analysis of the reaction solution, both before and after immobilization of streptavidin was applied to evaluate the amount of streptavidin conjugated onto the surface of fe3o4/tmc/au nanocomposite. the freshly prepared streptavidin solution before coupling demonstrates a maximum absorption at 280 nm. the od280 values of preand post-immobilization defined the efficiency of 83% for the binding of streptavidin to the whole nanocomposite. fig. 2 tem images of the fe 3 o 4 (a), fe 3 o 4 /tmc (b) and fe 3 o 4 /tmc/ au (c) nanoparticles. fig 4. cyclic voltammograms of the modified electrodes in 5.0 mm [fe(cn) 6 ]3−/4− containing 0.1m kcl after each step of modification: bare electrode (a), rgo-modified electrode (b), arg-modified electrode (c), rgo/arg-modified electrode (d). 220 j contemp med sci | vol. 4, no. 4, autumn 2018: 216–221 electrochemical biosensor for detection of dna methylation research l. syedmoradi et al. fig 5. (a) differential pulse voltammograms for the electrochemical detection of methylation upon serial dilutions of target between 0.01 and 1000 pm in 1m hcl. the concentrations of target methylated dna are: 0.01, 0.1, 1, 10, 100, 500, and 1000 pm. (b): the calibration curve of methylated dna as the relationship between current and methylated dna concentration. each data point is the average of three replicates. table 1. spike and recovery results obtained from methylation-nanobiosensor in plasma. data are presented as the means of three replicates added methylated dna (pm) detected methylated dna (pm) recovery (%) rsd% 10 10.43 ± 0.6 104.3 5.25 100 101.67 ± 2.32 101.67 2.03 1000 998.10 ± 6.17 99.84 0.63 turn lead to the enhancement of electron transfer between the surface of the composite layer and the electrode. analytical performance for quantitative detection of methylated dna the quantitative analysis of methylated dna target was investigated by dpv because this method can provide high current sensitivity and peak resolution. the standard solutions with concentration ranging from 0.01 to 1000 pm methylated dna target were prepared by diluting the stock solution with pbs. analyses were performed in triplicates for each concentration. differential pulse voltammetry measurements were performed between +0.05 and +0.35 v at scan rate of 50 mv/s by holding the working electrode at a condition potential of +1.3 v for 70 s in hcl. as shown in fig. 5a, the reduction peak current of gold nanoparticles gradually enhanced with increasing the concentration of methylated dna from 0.01 to 1000 pm. the increased values of current for methylated dna concentration are shown in fig. 5b. the limit of detection of this method was calculated to be 0.01 pm. reproducibility and stability of the developed genosensor in order to define the reproducibility of proposed genosensor, the mean intra-assay coefficient of variation coefficient of variation (cv). for 10 and 100 pm of target methylated dna estimated to be 3.0–2.1% respectively, and the inter-assay was 5.2 and 3.8%. furthermore, the performance of nanogenosensor in the biological sample was examined by adding two concentrations of target methylated dna (10, 100, and 1000 pm) to plasma samples. the obtained recovery results are presented in table 1, showing suitable data with relative standard deviation (rsd) values ranging between 0.63% and 5.25%. the recovery is between 99.84% and 104.3%, that clearly demonstrate the capability of the presented method for detection of methylated dna in real samples. the accelerated stability of the modified electrodes was defined. various spces were prepared under the same procedure as explained in the method section, stored and assessed in 3 days (data not shown) our prior publication.16 the response of the nangenosensor stayed stable within the limits up to 21 h. then, the developed nanogenosensor showed an average of 22% reduction in electrochemical sensing performance. conclusion in this study, we have developed immuno-dna electrochemical sensing platform using poly(l-arg)/rgo composite film and fe3o4/tmc/au nanocomposite as labeling tags for the rapid, simple, and sensitive detection of methylated biomarker. the fabricated nanogenosensor exhibited good linear range with a low detection limit of 0.01 pm, providing a useful alternative technology to the conventional methodologies. moreover, the synergistic influence of rgo and poly(l-arg) led to a great analytical performance of the sensing platform which can be applied to determine methylated dna biomarker with suitable results. acknowledgments this research has been supported by tehran university of medical sciences (tums), grant no. 26027. conflict of interest the authors declare no competing financial interest. n ba l. syedmoradi et al. 221j contemp med sci | vol. 4, no. 4, autumn 2018: 216–221 research electrochemical biosensor for detection of dna methylation this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1. hao x, luo h, krawczyk m, wei w, wang w, wang j, et al. dna methylation markers for diagnosis and prognosis of common cancers. proc natl acad sci usa. 2017;114:7414–7419. 2. paska av, hudler p. aberrant methylation patterns in cancer: a clinical view. biochem med (zagreb). 2015;25:161–176. 3. feinberg ap, vogelstein b. hypomethylation distinguishes genes of some human cancers from their normal counterparts. nature. 1983;301:89–92. 4. mikeska t, craig jm. dna methylation biomarkers: cancer and beyond. genes (basel). 2014;5:821–864. 5. mikeska t, bock c, do h, dobrovic a. dna methylation biomarkers in cancer: progress towards clinical implementation. expert rev mol diagn. 2012;12:473–487. 6. warton k, samimi g. methylation of cell-free circulating dna in the diagnosis of cancer. front mol biosci. 2015;2:13. 7. li y, song l, gong y, he b. detection of colorectal cancer by dna methylation biomarker sept9: past, present and future. biomark med. 2014;8:755–769. 8. song l, jia j, peng x, xiao w, li y. the performance of the sept9 gene methylation assay and a comparison with other crc screening tests: a meta-analysis. sci rep. 2017;7:3032. 9. nian j, sun x, ming s, yan c, ma y, feng y, et al. diagnostic accuracy of methylated sept9 for blood-based colorectal cancer detection: a systematic review and meta-analysis. clin transl gastroenterol. 2017;8:e216. 10. kurdyukov s, bullock m. dna methylation analysis: choosing the right method. biology (basel). 2016;5. pii: e3. 11. noehammer c, pulverer w, hassler mr, hofner m, wielscher m, vierlinger k, et al. strategies for validation and testing of dna methylation biomarkers. epigenomics. 2014;6:603–622. 12. syedmoradi l, esmaeili f, norton ml. towards dna methylation detection using biosensors. analyst. 2016;141:5922–5943. 13. taleat z, mathwig k, sudhölter ejr, rassaei l. detection strategies for methylated and hypermethylated dna. trac, trends anal chem. 2015;66:80–89. 14. krejcova l, richtera l, hynek d, labuda j, adam v. current trends in electrochemical sensing and biosensing of dna methylation. biosens bioelectron. 2017;97:384–399. 15. hossain t, mahmudunnabi g, masud mk, islam mn, ooi l, konstantinov k, et al. electrochemical biosensing strategies for dna methylation analysis. biosens bioelectron. 2017;94:63–73. 16. daneshpour m, moradi ls, izadi p, omidfar k. femtomolar level detection of rassf1a tumor suppressor gene methylation by electrochemical nano-genosensor based on fe 3 o 4 /tmc/au nanocomposite and pt-modified electrode. biosens bioelectron. 2016;77:1095–1103. 17. rahman mm, li xb, lopa ns, ahn sj, lee jj. electrochemical dna hybridization sensors based on conducting polymers. sensors (basel). 2015;15:3801–3829. 18. liu z, forsyth h, khaper n, chen a. sensitive electrochemical detection of nitric oxide based on aupt and reduced graphene oxide nanocomposites. analyst. 2016;141:4074–4083. 19. li d, yang xl, xiao bl, geng fy, hong j, sheibani n, et al. detection of guanine and adenine using an aminated reduced graphene oxide functional membrane-modified glassy carbon electrode. sensors. 2017;17. pii: e1652. 20. kanyong p, rawlinson s, davis j. a voltammetric sensor based on chemically reduced graphene oxide-modified screen-printed carbon electrode for the simultaneous analysis of uric acid, ascorbic acid and dopamine. chemosensors. 2016;4:25. 21. haque am, park h, sung d, jon s, choi sy, kim k. an electrochemically reduced graphene oxide-based electrochemical immunosensing platform for ultrasensitive antigen detection. anal chem. 2012;84:1871–1878. 22. jian jm, liu yy, zhang yl, guo xs, cai q. fast and sensitive detection of pb2+ in foods using disposable screen-printed electrode modified by reduced graphene oxide. sensors (basel). 2013;13:13063–13075. 23. khoshfetrat sm, mehrgardi ma. amplified electrochemical genotyping of single-nucleotide polymorphisms using a graphene–gold nanoparticles modified glassy carbon platform. rsc advances. 2015;5:29285–29293. 24. devadas b, cheemalapati s, chen s-m, ali ma, al-hemaid fm. highly sensing graphene oxide/poly-arginine-modified electrode for the simultaneous electrochemical determination of buspirone, isoniazid and pyrazinamide drugs. ionics. 2015;21:547–555. 25. li y, feng x, zhao l, pu q. on-surface formation of polyarginine/reduced graphene oxide film and its application in measuring puerarin in healthcare products. int. j. electrochem. sci. 2018;13:3948–3957. 26. zhang f, wang z, zhang y, zheng z, wang c, du y, et al. simultaneous electrochemical determination of uric acid, xanthine and hypoxanthine based on poly (l-arginine)/graphene composite film modified electrode. talanta. 2012;93:320–325. 27. shirazi h, daneshpour m, kashanian s, omidfar k. synthesis, characterization and in vitro biocompatibility study of au/tmc/fe3o4 nanocomposites as a promising, nontoxic system for biomedical applications. beilstein j nanotechnol. 2015;6:1677–1689. 28. daneshpour m, omidfar k, ghanbarian h. a novel electrochemical nanobiosensor for the ultrasensitive and specific detection of femtomolarlevel gastric cancer biomarker mirna-106a. beilstein j nanotechnol. 2016;7:2023–2036. 29. daneshpour m, karimi b, omidfar k. simultaneous detection of gastric cancer-involved mir-106a and let-7a through a dual-signal-marked electrochemical nanobiosensor. biosens bioelectron. 2018;109:197–205. 30. mousavi mf, amiri m, noori a, khoshfetrat sm. a prostate specific antigen immunosensor based on biotinylated-antibody/cyclodextrin inclusion complex: fabrication and electrochemical studies. electroanalysis. 2017;29:2818–2831. 31. omidfar k, rasaee mj, zaraee ab, amir mp, rahbarizadeh f. stabilization of penicillinase-hapten conjugate for enzyme immunoassay. j immunoassay immunochem. 2002;23:385–398. 32. shirazi h, ahmadi a, darzianiazizi m, kashanian s, kashanian s, omidfar k. signal amplification strategy using gold/n-trimethyl chitosan/iron oxide magnetic composite nanoparticles as a tracer tag for high-sensitive electrochemical detection. iet nanobiotechnol. 2016;10:20–27. 191j contemp med sci | vol. 4, no. 4, autumn 2018: 191–194 research model disability survey: a pilot study in the north batinah governorate adhra al-mawali,a* magdi morsi,a hilal al-kharusi,a waleed al-shekaili,a carla sabariego,b and lindsay leec acentre of studies & research, ministry of health, muscat, sultanate of oman.. b health systems, policy & communication unit, disability policy group, swiss paraplegic centre, nottwil, switzerland. cblindness and deafness prevention, disability and rehabilitation unit, world health organization, geneva, switzerland. *correspondence to adhra al-mawali (email: adhra.almawali@gmail.com). (submitted: 14 august 2018 – revised version received: 23 september 2018 – accepted: 29 september 2018 – published online: 26 december 2018) objective the continuous shift to non-communicable diseases in oman require a more consistent inclusion of functioning and disability information in health monitoring and evaluation frameworks. oman currently relies on the census to generate data on disability which is usually very limited in scope, and provides only rough prevalence estimates of persons with specific impairments. the lack of accurate, comprehensive and up-to-date information on disability is a major obstacle for planning services and allocating resources. thus, there was a need to conduct a comprehensive population-based survey to fill this information gap. the pilot aimed to examine the feasibility of the arabic version of the model disability survey (mds) in the cultural context of oman, identify potential problems with the survey, and develop strategies to deal with them before a large national implementation is launched. methods the mds is the tool recommended by the world health organization(who) to collect comprehensive data about functioning and disability to quantify both the impact of health conditions or impairments as well as the impact of the environment on disability. to determine the applicability of measuring disability, a pilot study was carried out in the north batinah governorate of oman with a convenience sample of 288 adults aged 18 year or older in collaboration with who. results the overall disability prevalence was estimated to be 14.6% (n = 42) in the mds pilot study calculated by determining the proportion of people in the population who experience severe performance problems. the overall results corroborated that the arabic translation of the mds survey tool works well in the field and is suitable for a large scale implementation, after minor revisions. conclusion since the results of this pilot study in oman show that the disability measurement survey tool has been successfully tested, we recommend that the mds be extended and implemented nationally. it should also be integrated to existing routine household surveys to allow continuous monitoring of disability in countries. keywords disability, physical functioning, cognitive functioning, disability survey, oman introduction oman is facing a continuous rise in non-communicable diseases (ncds). ncds – including hypertension, heart disease, asthma, diabetes, stroke, depression, back pain and hearing and vision disorders – are frequently associated with moderate to high levels of disability.1 additionally, life expectancy is steadily increasing due to improvements in health care, and ageing is also associated with disability. increased life expectancy and the high prevalence of ncds require a more consistent inclusion of functioning and disability information in health monitoring and evaluation frameworks. in recognition of its importance for complementing information on mortality and morbidity, the international classification of diseases, eleventh revision will include information on functioning and disability.2 information at population level collected through a dedicated disability survey is relevant for evaluating the effect of public health interventions targeting functioning and disability. such interventions can be directed at improving or optimizing functioning of individuals, for instance through provision of rehabilitation services, or at reducing environmental barriers, for instance through dedicated transportation, employment or accessible health care policies. information on functioning and disability is also relevant for developing evidence-based plans of action and policies, for planning and allocating resources to health and social services, and to monitor and report on the implementation of the convention on the rights of persons with disabilities and the indicators of the sustainable development goals. in the who international classification of functioning, disability and health (icf), disability encompasses impairments, such as problems with breathing, memory or pain, limitations of activities, such as problems with mobility and self-care, and participation restrictions, such as problems at work or school. although associated with health conditions, disability is not understood as the direct consequence of a disease. in line with the icf, disability is the outcome of the interaction between a health condition (e.g. arthritis, back problems, hearing loss or depression) and environmental barriers (e.g. limited access to health care, negative attitudes of others, inaccessible transportation and public buildings, or lack of inclusive employment laws).3 disability is also understood in the icf as a continuum, ranging from no disability (or full functioning) to very high levels of disability. disability is therefore a matter of degree and universal, since any person with health conditions or age-related decrements in health will experience disability to some extent at some point in his or her life.3 patterns and the level of disability in countries are influenced therefore by trends in health conditions and by trends in environmental barriers and other factors – such as road traffic crashes and accidents.4 the sultanate of oman relies currently on the census to generate data on disability. based on the census in 1993, the disability rate was 1.9% among omani nationals. this rate rose to 2.4% in 2003 and to 3.2% in 2010.5,6 such increase could be partially attributed to an actual increase chronic health conditions associated with disability coupled with issn 2413-0516 192 j contemp med sci | vol. 4, no. 4, autumn 2018: 191–194 model disability survey: a pilot study in the north batinah governorate research a. al-mawali et al. better care resulting in higher life expectancy. however, census data is usually very limited in scope, and provides only rough prevalence estimates of persons with specific impairments. the lack of accurate, comprehensive and up-to-date information on disability is a major obstacle for planning services and allocating resources for the care and rehabilitation of persons with different levels of disability. thus, there is a need to conduct a comprehensive population-based survey to fill this information gap. choosing the model disability survey tool to measure disability in oman the model disability survey (mds) was developed by the world bank and who in 2012 and is the tool recommended by who to collect comprehensive data about functioning and disability, in line with the icf. the mds is implemented to estimate the distribution of disability in a country as well as how many people have severe, moderate and mild disability. the mds also identifies their unmet needs as well as the barriers and inequalities faced by these persons in daily life. thus, data generated by the mds is being used by countries to quantify both the effect of health conditions or impairments as well as the effect of the environment on disability. this allows countries to determine which interventions and policies will likely produce the most benefit for the population.7 to determine the applicability of measuring disability, a pilot study was carried out in oman by the ministry of health in 2016, in collaboration with who. the pilot aimed to examine the feasibility of the arabic version of the mds in the cultural context of oman; identify potential problems with the survey, and; develop strategies to deal with them before a large national implementation is launched. methodology a convenience sample of 288 adults aged 18 year or older was selected in this pilot study. the selection was done to include men and women of varied ages as well as to include both healthy respondents and persons with impairments and health conditions. it is to be noted that the sample used is not representative of the omani population. data collection was done in the morning only and the individual who was at home during the visit was selected for the interview. for this reason, the sample includes more women, who were not working or not studying. their socio-demographic characteristics are shown in table 1. a household questionnaire was used which was answered by the head of the household and included a household roster and a children module. the individual questionnaire was answered by a randomly selected adult member of the household. the questionnaire contained eight modules: socio-demographic characteristics; work history and benefits; environmental factors; functioning; health conditions and capacity; health care utilization; well-being; and empowerment.8 results and discussion the overall disability prevalence estimated in the mds is calculated by determining the proportion of people in the population who experience severe performance problems. for the table 1. demographic characteristics of sample used in the pilot study characteristic mds sample n percentage (%) gender male 102 35.4 female 186 64.6 total 288 100 age (years) 18–39 150 52.1 40–59 96 33.3 >60 42 14.6 total 288 100 marital status single 48 16.7 married 219 76 separated or divorced 4 1.4 widowed 17 5.9 total 288 100 present pilot study this rate is 14.6% (n = 42). approximately 21% of included households had a person in need of extra support for health care and 18% had at least a person in need of extra physical or emotional care or support. persons experiencing severe capacity difficulties are generally at high risk of experiencing severe performance problems if no accommodations are in place. it is therefore of interest to know the percentage of persons experiencing severe capacity difficulties and severe performance problems, which in the case of this pilot study are 14.2% of the whole sample. however, in this sample 3.1% experience severe capacity difficulties but do not experience severe performance problems, suggesting that the environment is facilitating to some extent for these individuals. the proportion of persons in the sample experiencing severe performance problems without severe capacity difficulties is very low: 0.3%. table 2 presents the socio-demographic characteristics of these three distinct groups. it is important to note, however, that the sample size is too small to allow for interpretations. capacity is the synthesis of all of the intrinsic physical and mental capacities of a person, determined by their health state. one of the most important objectives for carrying out the pilot study in oman is to confirm that metric scales of capacity and performance can be created for the country using item response theory. the higher the scores are, the worse the difficulties in capacity. figure 1 shows the distribution of the convenience sample on the capacity continuum; the scale ranges from 0 no capacity difficulties to 100 (very severe capacity difficulties). the mds enables decision-makers to go beyond to identify the factors that are responsible for inequalities which allows them to identify appropriate and effective interventions and policies, whilst also distribute outcomes of interest along the disability continuum for those who experience severe, moderate and mild disability, as identified by suitable thresholds.9 a. al-mawali et al. 193j contemp med sci | vol. 4, no. 4, autumn 2018: 191–194 research model disability survey: a pilot study in the north batinah governorate in several countries, the proportion of severe capacity difficulties is analogous to previous prevalence estimates of disability calculated from the census; however, in the mds this proportion is usually much higher than previous estimates. in the case of this pilot study, the severe capacity difficulties proportion is 17.4% of the included population. this larger estimate is mostly due to the broader definition of disability used in mds. historically, censuses identify people with a narrow set of conditions or impairments as “disabled”, while in the mds the capacity estimate is a composite score of difficulties in 17 functioning domains. in this pilot sample, 27.8% experience moderate capacity difficulties while 12.5% experience mild capacity difficulties. interviewers rated that approximately 96% of respondents were highly or very highly cooperative. additionally, the answers of approximately 96% of the respondents were rated as highly or very highly accurate by interviewers. regarding the questions, following problems were observed. questions such as ‘how many weeks do you work during the year?’ were perceived as difficult due to problems to calculate the number of weeks accurately. questions about attitudes of others posed some problems as the majority of table 2. demographic characteristics of disability groups characteristic severe disability group 1: performance is better than capacity (n = 9) group 2: performance and capacity equal (n = 41) group 3: capacity is better than performance (n = 1) n percentage (%) n percentage (%) n percentage (%) gender male 3 33.3 14 34.1 1 100 female 6 66.7 27 65.9 0 0 age group (years) 18-39 3 33.3 13 31.7 0 0 40-59 5 55.6 10 24.4 0 0 >60 1 11.1 18 43.9 1 100 work status not working 0 0 4 66.7 1 100 working 2 100 2 33.3 0 0 education no education, illiterate 4 44.4 31 75.6 1 100 no education, literate 0 0 1 2.4 0 0 less than primary school 1 11.1 4 9.8 0 0 prep school not completed 1 11.1 0 0 0 0 prep school 2 22.2 3 7.3 0 0 secondary 1 11.1 2 4.9 0 0 higher 0 0 0 0 0 0 marital status single 0 0 13 31.7 0 0 married or legal cohabitation 8 88.9 20 48.8 1 100 separated or divorced 0 0 1 2.4 0 0 widowed 1 11.1 7 17.1 0 0 fig. 1 capacity continuum and distribution of the population on the continuum. 194 j contemp med sci | vol. 4, no. 4, autumn 2018: 191–194 model disability survey: a pilot study in the north batinah governorate research a. al-mawali et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. respondents asked about the relationship of these questions to health and disability. especially questions such as ‘do people around you tend to become impatient with you?’ and ‘is living with dignity a problem for you because of the attitudes and actions of others?’ caused problems. the introduction of the “attitudes of others” questions would need a revision that stresses why these questions are relevant. finally, question ‘to what extent would you agree with the statement that you are a person who tends to find fault with others?’ was perceived as embarrassing by respondents, and questions ‘do you think that the problems you have told me about have made you a stronger person? & do you think that the problems you have told me about have made you more determined to reach your goals?’ would need a response option “does not have any problem” for respondents who reported having no problems at all. these issues need revisions before a full implementation can be started. conclusion our overall results above corroborate that the arabic translation of the mds survey tool works well in the field and is suitable for a large scale implementation, after minor revisions. it is recommended by who that a stand-alone version of the mds is implemented every 5–10 years while a brief version – a module developed to be integrated to existing household surveys – should be implemented routinely to allow a continuous monitoring of disability in countries.10 since the results of this pilot study in oman show that the disability measurement survey tool has been successfully tested, we recommend that this is extended and implemented nationally. conflict of interest none. n references 1. al-mawali a. non-communicable diseases: shining a light on cardiovascular disease, oman’s biggest killer. oman med j. 2015;30:227–228. 2. escorpizo r, kostanjsek n, kennedy c, nicol mm, stucki g, ustün tb. functioning topic advisory group (ftag) of the icd-11 revision: harmonizing who’s international classification of diseases (icd) and international classification of functioning, disability and health (icf): importance and methods to link disease and functioning. bmc public health. 2013;13:742. 3. world health organization. international classification of functioning, disability and health (icf). 2018. available from: http://www.who.int/ classifications/icf/en/ [cited 2018 september 5]. 4. world health organization. who global disability action plan 2014–2021: better health for all people with disability. world health organization, geneva, 2015. 5. ministry of national economy oman. disability statistics in the sultanate of oman: the experience of data collection during 3 censuses (1993, 2003, 2010). 11th meeting of the washington group on disability statistics. southampton, bermuda. 2011. available from: https:// www.cdc.gov/nchs/ppt/citygroup/meeting11/wg11_session6_2_aldagheishi.pdf ) 6. al-balushi t, al-badi ah, ali s. prevalence of disability in oman: statistics and challenges. can j appl sci. 2011;1:81–96. 7. world health organization. model disability survey: providing evidence for accountability and decision-making. available from: http://www.who.int/ disabilities/data/mds_v4.pdf?ua=1 [cited 2018 september 6]. 8. world health organization. model disability survey. [cited 2018 september 6]; available from: http://www.who.int/disabilities/data/model-disability-survey4. pdf?ua=1. 9. cieza a, sabariego c, bickenbach j, chatterji s. rethinking disability. bmc med. 2018;16:14. 10. kostanjsek n. use of the international classification of functioning, disability and health (icf) as a conceptual framework and common language for disability statistics and health information systems. bmc public health. 2011;11:s3 (biomed central). 153j contemp med sci | vol. 4, no. 3, summer 2018: 153–157 original anatomical relationship of optic nerve canal to the posterior paranasal sinuses on computerized tomography in iraqi patients haider najim al-tameemia and haider abdul kadum hassanb adepartment of radiology, college of medicine, university of kufa, najaf, iraq. bdepartment of radiology, al-sadder medical city, najaf, iraq. correspondence to haider najim al-tameemi (email: haidern.altameemi@uokufa.edu.iq). (submitted: 14 march 2018 – revised version received: 19 may 2018 – accepted: 12 june 2018 – published online: 26 september 2018) objective to determine the patterns of relationship between optic nerve canal and posterior paranasal sinuses (pns) in iraqi patients from middle euphrates region using computerized tomography (ct). methods a cross-sectional observational study was conducted on 100 patients (52 male, 48 female) referred to the ct unit for evaluation of pns. patients were examined by the same ct device without intravenous contrast medium. patients with sphenoid/ethmoid tumors, trauma or history of surgical intervention and those less than 13 years old were excluded. the relationship between optic canal and posterior pns was evaluated using criteria of delano’s classification. pneumatization of the anterior and posterior clinoid processes (pacp and ppcp) and any bony dehiscence in the bony wall were observed. results the most frequent type of optic canal according to delano’s classification was type i (77.5). there was no statistically significant age or gender difference. there was statistically significant higher frequency of dehiscence in higher delano’s types iii (4 of 7 cases, 57.1%) and iv (6 of 8, 75%). pacp and ppcp showed statistically significant association with type iv and to lesser extent in type iii. all cases of ppcp were seen only in types iii and iv with statistically significant difference. conclusion the relationship between optic nerve and sphenoid sinuses was slightly different in our patients from other populations. therefore, iraqi radiologists and ear–nose–throat surgeons need to be aware of this anatomical difference to avoid serious damage to the optic nerve. during pre-operative ct assessment, it is important for radiologist to pay attention to the presence of optic canal bony dehiscence in higher delano’s types. presence of pacp and ppcp may alert to the presence of higher delano’s types, hence a serious optic nerve canal course. keywords paranasal sinus, computerized tomography, optic nerve introduction anatomically, the sphenoid sinuses are located at the skull base at the junction of the anterior and middle cerebral fossae. the lateral wall of the sphenoid sinus can show various prominences, the most important being the carotid canal and the optic canal. the optic canal is found in the posterosuperior angle between the lateral, posterior and superior walls of the sinus. pneumatization of the sphenoid above and below the optic canal can result, respectively, in a supraoptic recess and an infraoptic recess.1,2 in some cases, dehiscence in the bony margin can also be present.3 ethmoid sinus consists of multiple obliquely oriented parallel groups of air cells between the lateral nasal wall and the medial orbital wall.4,5 the sphenoethmoid cells, or onodi cells, are important group of ethmoid cells, where the posterior ethmoid cells extend superiorly or laterally to the sphenoid sinus, and the pneumatization can reach the clinoid processes, and become closely related to the optic nerve. therefore, accurate identification of these structures and possibly onodi cells on a preoperative computerized tomography (ct) scan is the best way to avoid such severe complications.6 functional endoscopic sinus surgery (fess) is a minimally invasive and effective surgical technique7 for treating chronic inflammatory sinus disease. moreover, during last two decades and with the significant technical improvement in surgical and diagnostic tools, fess has also become one of the treatments of choice in the orbital and skull base problems.8,9 however, fess is not totally safe as many complications have been reported10 with blindness secondary to the optic nerve damage being one of the most catastrophic ones.11 therefore, for the fess to be successful and to minimize complications, it is mandatory to clearly understand and accurately depict the sinonasal anatomy. this depiction is routinely done in a plane corresponding to the surgical orientation and is elegantly accomplished by the ct scan, which has been established as the imaging technique of choice for demonstration of the sinonasal anatomy before fess.12 considering the documented ethnic variations in sinonasal anatomy,13 this study was conducted to assess the patterns of the relationship between optic nerve canal and posterior paranasal sinuses in iraqi patients so that iraqi radiologists and ear– nose–throat (ent) surgeons will have an idea about regional anatomical variations regarding this critical relationship. the study assumes to include a sample that represents iraqi population from the middle euphrates territory because patients visiting our ct unit are not only from najaf governorate’s cities, but also from nearby governorates. up to our best knowledge and search, there is no published article specifically assessing the patterns of the optic nerve canal in relation to posterior paranasal sinuses (pns) in iraqi population. materials and methods this hospital-based cross-sectional observational study was conducted on 100 patients (52 male, 48 female) in the ct unit of middle euphrates neurocenter, al-sadder medical city, najaf, iraq. issn 2413-0516 154 j contemp med sci | vol. 4, no. 3, summer 2018: 153–157 optic nerve canal and posterior paranasal sinuses on ct original haider najim al-tameemi and haider abdul kadum hassan inclusion criteria all patients referred from ent specialist for evaluation of sinonasal diseases and were examined by ct scan using paranasal sinuses protocol. exclusion criteria patients with known or obvious sphenoid/ethmoid tumor, trauma or history of surgical intervention were excluded. children less than 13 years old (when the sphenoid sinus is not yet well developed) were also excluded. data acquisition relevant demographic and clinical data were taken. the radiological, coronal, axial and sagittal ct sections of the pns for each patient were evaluated by a board-certified radiologist in the following approach: 1. general evaluation to assess for any exclusion criteria. 2. specific evaluation of the relationship between optic nerve and sphenoid and ethmoid sinuses on both right and left sides using criteria of 4-grades delano’s system,14 which classifies the course of the optic nerve canal into: type i, adjacent to sphenoid sinus; type ii, indentation on sphenoid sinus; type iii, optic nerve traversing sphenoid sinus; and type iv, adjacent to sphenoid and posterior ethmoid sinuses. 3. further evaluation of any other significant variation in the sphenoid sinus that can affect the optic nerve (like pneumatization of the anterior and posterior clinoid processes) as well as any bony dehiscence. statistical analysis of the data was done using spss, version 2.0. sample size was calculated by the standard equation of sample size for cross-sectional study with confidence level of 95%. n = z × p(1 − p)\d, where: n = sample size; z = 1.96 for confidence 95%; p = prevalence of variation (for grade 3 which is the rarest type) which equal to 0.007 (7%); d = 0.005 allowed error (chance error). n = (1.96) × 0.07(1 − 0.07)\(0.05) = 99.9. hundred patients were included in this study (corresponding to 200 optic nerves). the frequency of each of delano’s type was calculated. the correlation of different delano’s types with the pneumatization of anterior and posterior clinoid processes and with bony dehiscence was assessed using pearson correlation coefficient. p-value of <0.05 was considered as statistically significant. no patient consent was obtained because of the non interventional nature of the study, with the confidentiality of patient’s personal data being preserved. results this study included 100 patients (48 female and 52 male) with mean age of 33.94 ± 12.39 years. collectively, a total of 200 optic nerve canals were studied. generally, all delano’s types were more frequent in young age groups (15–35 years). however, this predominance was not statistically significant (p values were 0.780 for the right side and 0.694 for the left side). types of optic nerve canals according to delano’s classification the most frequent type of optic nerve canal according to delano’s classification (figs. 1–4) was type i (77.5%), followed by type ii (15%), type iv (4%) and least type iii (3.5%) with almost approximate right to left prevalence (table 1). fig. 1 coronal ct image of a 44-year-old male showing type i delano’s classification of optic nerve relation to the sphenoid sinus. fig. 2 coronal ct image of a 50-year-old male showing type ii delano’s classification of optic nerve relation to the sphenoid sinus. fig. 3 coronal ct image of a 45-year-old female showing type iii delano’s classification of optic. haider najim al-tameemi and haider abdul kadum hassan 155j contemp med sci | vol. 4, no. 3, summer 2018: 153–157 original optic nerve canal and posterior paranasal sinuses on ct table 1. frequency of the optic nerve canal according to delano’s classification type right left total p-value i 79*(79%) 76 (76%) 155 (77.5%) 0.929 ii 13 (13%) 17 (17%) 30 (15%) iii 4 (4%) 3 (3%) 7 (3.5%) iv 4 (4%) 4 (4%) 8 (4%) total 100 (100%) 100 (100%) 200 (100%) *values between brackets are percentages. table 2. relationship between gender and delano’s types of optic nerve canal type side male female p-value i right 41 38 0.559 left 43 33 ii right 8 5 0.153 left 6 11 iii right 1 3 0.809 left 1 2 iv right 2 2 1 left 2 2 table 4. relation between delano’s optic nerve canal type and pneumatization of the anterior clinoid process type pacp total p-value (+) (−) i 0 155 (100%) 155 (100%) 0.771 ii 2 (6.7%) 28 (93.3%) 30 (100%) 0.718 iii 2 (28.5%) 5 (71.5%) 7 (100%) 0.04 iv 5 (62.5%) 3 (37.5%) 8 (100%) 0.001 total 9 (4.5%) 191 (95.5%) 200 (100%) fig. 4 coronal ct image of a 25-year-old male showing type iv delano’s classification of optic nerve relation to the sphenoid sinus. relation between delano’s and gender there was a male predominance in type i on both sides and type ii only on right. female predominance was present in type iii on both sides and only in left in type ii. type iv was equally seen in both genders and on both side. however, no type showed statistically significant difference with all p-value of <0.005 (table 2). relationship between delano’s types and optic nerve canal dehiscence there was a statistically significant high frequency of dehiscence in higher delano’s types iii (4 of 7 cases, 57.1%) and iv (6 of 8, 75%) as demonstrated in table 3, fig. 5. delano’s types and pneumatization of the anterior clinoid process among the 200 optic nerve canals studied, 22 were associated with pneumatization of the anterior clinoid processes (pacp), however, was only statistically significant in type iv (5 out of 8, 62%, p-value 0.001) and to lesser extent in type iii (2 out of 7, 28.5%, p-value 0.04) as shown in table 4, fig. 6. delano’s types and pneumatization of the posterior clinoid process all four cases of pneumatization of the posterior clinoid processes (ppcp) seen in our study were present only in types iii (2 out of 6, 28.5%) and iv (2 out of 8, 25%) with statistically significant difference. no ppcp was seen in either type i or type ii (table 5). table 3. relation between delano’s optic nerve canal types and the presence of canal bony dehiscence type total bony dehiscence of optic nerves p-value (+) (−) i 155 (77.5%) 5 (3.2%) 150 (96.8%) 0.371 ii 30 (15%) 2 (6.6%) 28 (93.4%) 0.718 iii 7 (3.5%) 4 (57.1%) 3 (42.9%) 0.001 iv 8 (4%) 6 (75%) 2 (25%) <0.001 total 200 (100%) 17 (8.5%) 183 (91.5%) fig. 5 coronal ct image of a 33-year-old male showing bilateral bony dehiscence (white arrows). 156 j contemp med sci | vol. 4, no. 3, summer 2018: 153–157 optic nerve canal and posterior paranasal sinuses on ct original haider najim al-tameemi and haider abdul kadum hassan dx.doi.org/10.22317/jcms.03201801 fig. 6 coronal ct image of a 46-year-old male showing pneumatization of the anterior clinoid processes (white arrow). table 5. relation between delano’s optic nerve canal type and pneumatization of the posterior clinoid process type ppcp total p-value (+) (−) i 0 155 (100%) 155 (100%) 0.999 ii 0 30 (100%) 30 (100%) 0.998 iii 2 (28.5%) 5 (71.5%) 7 (100%) 0.046 iv 2 (25%) 6 (75%) 8 (100%) 0.007 total 4 (2%) 196 (98%) 200 (100%) discussion the analysis of the optic canals in relation to the sphenoid and posterior ethmoid sinuses in our sample of 100 iraqi patients (200 canals) of middle euphrates territory was as follows: prevalence of delano’s types overall, type i was the most commonly encountered relationship (77.5%), followed by type ii with nearly equal prevalence of types iii and iv. up to our best knowledge, we found only three published studies conducted in neighboring and arabic countries.15–17 compared to turkish study,15 type i was higher (77.5% versus 64%) while types ii–iv were lower in our study. this difference between our patients and those populations is related to the ethnic variation as seen by badia et al study.13 although larger sample and wide multicentric anatomical surveys within different territories of iraq may be more accurate, our current results alert iraqi surgeons to be aware of this anatomical difference and not to rely on what is published in the anatomical textbooks or foreign literatures, to avoid serious damage to the optic nerve. delano’s types and age although all types were generally more common in younger age groups, this was not statistically significant which has probably occurred because the majority of the examined patients with chronic rhinosinusitis was of young age group. no statistically significant gender difference regarding types of optic nerve canals was seen in our patients and we could not compare this with other studies as they did not mention the gender distribution of delano’s types. one of the important anatomical features that should be evaluated when interpreting a sinus ct scan is the optic canal bony dehiscence because this renders optic nerve more vulnerable to injury during surgical intervention. the turkish study by spaci et al15 found that bony dehiscence was more common in types ii and iii, while in our study, it was more common and statistically significant in types iii (57.1%) and iv (75%). therefore, bony dehiscence should be carefully looked for when optic nerve canal found to be in iii or iv configuration, because the higher the type of delano’s, the higher the prevalence of bony dehiscence is. the 4.5% prevalence of pacp (9 out of 200 optic nerve canals) was lesser than that in turkey (22 cases, 11%),15 libyans (46, 15.3%)16 and sudan (28, 13.9%).17 furthermore, because pacp was highest in types iii and iv, radiologists can use them as indirect indicator of a higher delano’s type, hence a higher vulnerability of the optic nerve to surgical damage. similarly, because all four cases of ppcp were seen in types iii (28.5%) and iv (25%) and no ppcp was seen in other types with statistically significant difference, ppcp (in addition to the pacp) could also be a warning sign for the presence of a serious optic nerve canal course. none of the other published studies included ppcp in their evaluations. limitations although the sample size of this study was according to the standardized statistical equation, we think that for our results to be generalizable, a larger sample from different regions of iraq are required. apart from few locations, there are no much published studies about the delano’s pattern of the optic canal in nearby counties to thoroughly compare our results with. in conclusion, delano’s type i was the most common optic nerve canal course with no statistically significant age or gender difference. we think that to minimize complications and avoid risk of serious damage to the optic nerve, it is essential for iraqi radiologists and ent surgeons to have proper knowledge of this anatomical relationship and awareness of the current results regarding the slight anatomical difference between iraqi and non-iraqi populations. because optic canal bony wall dehiscence was more frequent in delano’s types iii and iv, the study recommends that the radiologists should meticulously look for the dehiscence when a higher delano’s type is detected during pre-operative ct scan evaluation of pns. furthermore, presence of pacp and ppcp may alert for the presence of a serious optic nerve canal course. conflict of interest authors wish to declare that there is no conflict of interest, including specific financial interests and relationships and affiliations relevant to the subject of the manuscript, exist with this study. n haider najim al-tameemi and haider abdul kadum hassan 157j contemp med sci | vol. 4, no. 3, summer 2018: 153–157 original optic nerve canal and posterior paranasal sinuses on ct references 1. rice dh, schaefer sd, anatomy of the paranasal sinuses. in: rice dh, schaefer sd. endoscopic paranasal sinus surgery. lippincott williams & wilkins, 3rd ed., 2004. 2. van cauwenberge p, sys l, de belder t, watelet jb. anatomy and physiology of the nose and the paranasal sinuses. immunol allergy clin north am. 2004;24:1–17. 3. sethi ds, stanley re, pillay pk. endoscopic anatomy of the sphenoid sinus and sella turcica. j laryngol otol. 1995;109:951–955. 4. bolger we. anatomy of the paranasal sinuses. in: kennedy dw, bolger we, zinreich j. diseases of the sinuses, diagnosis and management. b.c. decker, 2001. 5. stammberger hr, kennedy dw. anatomic terminology group. paranasal sinuses: anatomic terminology and nomenclature. ann otol rhinol laryngol suppl. 1995;167:7–16. 6. kantarci m, karasen rm, alper f, onbas o, okur a, karaman a. remarkable anatomic variations in paranasal sinus region and their clinical importance. eur j radiol. 2004;50:296–302. 7. stammberger h, kopp w. functional endoscopic sinus surgery: the messerklinger technique. philadelphia, b.c. decker, 1991, p. 283. 8. backous dd, esquivel cr. skull base medical and surgical issues commonly encountered in the practice of otolaryngology, vol. 38, 2005, pp. 13–14. 9. sellari-franceschini s, berrettini s, santoro a, nardi m, mazzeo s, bartalena l, et al. orbital decompression in graves‘ ophthalmopathy by medial and lateral wall removal. otolaryngol head neck surg. 2005;133:185–189. 10. fokkens w, lund v, mullol j. european position paper on rhinosinusitis and nasal polyps group. zhonghua er bi yan hou tou jing wai ke za zhi. 2008;43:317–320. 11. cumberworth vl, sudderick rm, mackay is. major complications of functional endoscopic sinus surgery. clin otolaryngol allied sci. 1994;19:248–253. 12. zinreich sj. functional anatomy and computed tomography imaging of the paranasal sinuses. am j med sci. 1998;316:2–12. 13. badia l, lund vj, wei w, ho wk. ethnic variation in sinonasal anatomy on ct-scanning. rhinology. 2005;43:210–214. 14. delano mc, fun fy, zinreich sj. relationship of the optic nerve to the posterior paranasal sinuses: a ct anatomic study. ajnr am j neuroradiol. 1996;17:669–675. 15. sapçı t, derin e, almaç s, cumali r, saydam b, karavuş m. the relationship between the sphenoid and the posterior ethmoid sinuses and the optic nerves in turkish patients. rhinology. 2004;42:30–34. 16. hewaidi g, omami g. anatomic variation of sphenoid sinus and related structures in libyan population: ct scan study. libyan j med. 2008;3: 128–133. 17. kajoak sa, ayad ce, najmeldeen m and abdalla ea. computerized tomography morphometric analysis of the sphenoid sinus and related structures in sudanese population. glob adv res j med med sci. 2014;3:160–167. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201808 201j contemp med sci | vol. 9, no. 3, may-june 2023: 201–205 original characteristics of vitamin d3 receptor genotypes in t2dm of iraqi obese women baraa abdul-kareem mutar1, thikra ali allwsh2, ammar gany yassin1, fadhil jawad al-tu’ma1*, hassanien s. taghi3 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 2department of chemistry, college of science, university of mosul, mosul, iraq. 3department of pharmaceutics, college of pharmacy, al-bayan university, iraq. *correspondence to: fadhil jawad al-tu’ma (e-mail: fadhil.jawad@uokerbala.edu.iq) (submitted: 10 april 2023 – revised version received: 29 april 2023 – accepted: 12 may 2023 – published online: 26 june 2023) abstract objectives: this study aims to examine the association between the (rs1544410) polymorphism of the vdr gene with the pathogenesis of t2dm in iraqi obese women. methods: a case-control study was performed on 50 patients with t2dm and 50 apparently healthy subjects who were admitted to al-hussein teaching hospital and al-hassan center of diabetes and endocrinology unit/kerbala health directorate—iraq during (april 2022–march 2023). the t2dm groups were divided into two groups, 25 obese and 25 non-obese; the control group was divided into 25 obese and 25 non-obese as apparently healthy groups. the elisa kit was used to measure serum 25(oh)d3, heat shock protein-70, vdbp, insulin and c-peptide. also hba1c% and insulin resistance (homa-ir) were evaluated. the vitamin d3 receptor gene (vdr) variant and the snp (rs1544410) polymorphism was determined using allele specific polymerase chain reaction, 1.5% agarose gel electrophoresis and then visualized by gel photo-documentation system. results: the result of vitamin d3 variants genotype (rs 1544410) was a clear band with a molecular size of 200 bps. the size of the amplicon was determined by compare with dna ladder 100–1500 bp. the result of the comparison between observed and anticipated values for snip with (rs 1544410) in the tested population was statistically significant, p = < 0.001 and the difference between demographic characteristics and (rs 1544410) snp, age and bmi shows non-significant difference among all groups. the difference between biomarkers and (rs1544410) snp was performed using one-way anova test to compare the mean levels of hsp-70, vdbp, c-peptide, rbc and hba1c% which shown a non-significant difference among the variants of vdbp genotype (rs1544410) in obese women (patients and control) studied groups, p value > 0.05. conclusion: the logistic analysis of the (rs1544410) snp of the patients concluded that hsp-70, vdbp, and c-peptide level was no significantly related to the also c-peptide, was shown to be a related risk factor to both ct and ct alleles (1.003, p > 0.05) in comparison with cc alleles. furthermore, hba1c% level was demonstrated to be related as a risk factor for the cg allele in comparison with cc and gg alleles (1.009, p < 0.05). keywords: diabetes mellitus, type 2, receptors, calcitriol, vitamin d-binding protein, hsp-70, obesity issn 2413-0516 introduction diabetes mellitus (dm) is a chronic metabolic condition that causes blood glucose levels to rise as a result of either reduced insulin production or body cells that do not react to the effects of insulin (insulin resistance).1 although there are various forms of diabetes, type 2 diabetes mellitus (t2dm) is the most frequent type and accounts for 90–95% of cases of diagnosed (dm). t2dm is more common in adults and elderly2 resulting in anomalies in the metabolism of carbohydrates, lipids, and proteins, as well as disturbance of the regulatory systems that control the storage and mobilization of metabolic fuel.3 obesity is mostly measured by means of body mass index (bmi ), but anthropometric classification systems do not reflect the presence or severity of comorbidities.4 vitamin d3 receptor gene factor nuclear transcription by the mediation of 1,25(oh)2d3, vdr influences calcium absorption, bone remodeling, and the rate of mineralization. there are 11 exons in the 3q11 region on the long arm of chromosome 12, and 2 to 9 of them are actively transcribed.5 numerous investigations have revealed that the nuclear receptor superfamily’s vdr gene, which is found on chromosome 12’s long arm (12q13.11), plays a significant role in the etiology of osteoporosis.6 the majority of research on vdr polymorphisms has been done in caucasian populations and has concentrated on five snps: (1) rs10735810 or foki in exon 2; (2) rs1544410 or bsmi in intron 8; (3) rs731236 or taqi in exon 9; (4) rs7975232 or apai in intron 8 and (5) rs757343.7 vitamin d receptor gene sigle nucleotide polymorphism of bsmi variant modulate glucose intolerance, insulin secretion and sensitivity.8 vdr gene’s variants can alter insulin secretion, leading to insulin resistance, as well as vitamin d3 biosynthesis, transportation, and action.9 reported about t2dm patients have a considerably higher prevalence of the bsmi snp. several studies showed a similar connection between bsmi polymorphism and t2dm in other groups.10 bsmi snp and risk of t2dm in various ethnic groups are thus not conclusively linked. snp is connected to therapeutic responsiveness and illness susceptibility.11 t2dm) and vdr polymorphisms are still not clearly linked. many genetic vdr polymorphisms have been identified in studies carried out in different places with varying populations of people. just one research has examined the relationship between the vdr bsmi (rs1544410) polymorphism and vitamin d3 insufficiency, obesity, and insulin resistance among non-diabetic subjects across various age groups to date, and it was conducted in the central area of malaysia.12 accordingly, the aforementioned study had found that the bsmi (rs1544410) polymorphism was associated with increased risk for vitamin d deficiency and insulin resistance among the malaysian population.12 the risk of metabolic mailto:fadhil.jawad@uokerbala.edu.iq 202 j contemp med sci | vol. 9, no. 3, may-june 2023: 201–205 characteristics of vitamin d3 receptor genotypes in t2dm of iraqi obese women original f. j. al-tu’ma et al. syndrome elements including abdominal obesity has also been linked to vitamin d insufficiency.13 the control of hormone sensitive genes and the modulation of vitamin d pathways are both important functions of the vdr gene. it’s interesting to note that the vdr gene is expressed in both pancreatic beta cells and adipocytes. as a result, it may affect body composition either directly by controlling adipocyte development and metabolism or indirectly by modulating insulin levels.14 the objective of the presented work was to examine the association between the (rs1544410) polymorphism of the vdr gene with the pathogenesis of t2dm in iraqi obese women. materials and methods a case-control study was performed on 50 patients with t2dm and 50 apparently healthy subjects who were admitted to al-hussein teaching hospital and al-hassan center of diabetes and endocrinology unit/kerbala health directorate—iraq during (april 2022–march 2023). the t2dm groups were divided into two groups, 25 obese and 25 nonobese; the control group was divided into 25 obese and 25 non-obese as apparently healthy groups. seven ml of blood was drawn from the vein of all subjects by using a disposable syringe and then divided into two parts: the first part (3 ml) was placed in a gel tube and left at room temperature for about (30 min) for clotting, then put in the centrifuge at 4000 x g to obtain serum which was used for the determination of biomarker levels, including blood glucose by using the enzymatic colorimetric method and heat shock protein-70 (hsp-70), vitamin d3 binding protein (vdbp), 25(oh)d3, insulin, and c-peptide levels by using elisa specialized kits. the remaining blood (4 ml) was put in tow (edta) containing. the first edta tube containing (2 ml) of blood used to determine the hba1c% level and the second edta tube was stored by freezing at –20˚c until using for genomic dna extraction, and then performing various molecular analyses. the (rs1544410) polymorphism of the vdr gene was determined by using genomic dna extracted by (promega kit), and then by allele specific polymerase chain reaction. maximum absorbance for proteins and nucleic acids occurs at 260 and 280 nm, respectively. the ratio of absorbance at these wavelengths has been used as a measure of purity in both nucleic acid and protein extractions. when the ratio is between 1.8 and 2.0, dna is commonly considered to be pure. the target gene (vdr gene) was amplified using allele-specific pcr with a specific primer indicated below which were [purchased from bioneer, korea]. fc 5-agaaccatctctcaggctcc-3 ft 5-agaaccatctctcaggctct-3 r 5-cctcactgcccttagctctg-3 the reactions were conducted in pcr tubes under sterile conditions; the total volume of the reaction mixture was brought to 25 liter using deionized distilled h2o, and the master mix containing optimal concentrations of reaction requirements (mgcl2 , 1.5 mm, each dntps 200 m) was used. different volumes of primer (0.5 μl,1 μl, 1.5 μl) with different volumes of template dna (1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl) and different temperatures of the reaction conditions were trailed to optimize the conditions of the reaction. pcr tube was centrifuged for 30 seconds at 2000 x g in a micro-centrifuge to mix solutions well at room temperature then tubes were placed in the thermocycler to start the reaction. programs of the pcr protocol reaction for vdr gene polymorphism for bsmi (rs1544410) was employed by gel electrophoresis by using 1.5% agarose in 1x tbe buffer (tris borate edta) was prepared by diluting 10x tbe buffer with deionized water. the comb was removed and the glass plates were placed in a vertical electrophoresis chamber, 1 x tbe buffer was added inside the chamber until reaching above the level of wells, and then the samples were loaded into the gel wells by using 10 μl micropipettes. the electrophoretic conditions include negatively charged nucleic acids move across the gel toward the positive (+) electrode as a result of an electric field being given to the system (60 v, 45 ma for 35 min.) after sample loading (anode). ethidium bromide was used to dye the agarose gel. agarose gel was placed above the uv transilluminator device and exposed to uv light. the photos were captured using a digital camera and visualized by a pc connected to the transilluminator. the uv transilluminator device was covered with a protective shield to avoid exposure to uv light when the light was on. the study protocol has been approved by the research ethics committee of the college of medicine at the university of kerbala, as well as a committee from al-hussein teaching hospital and al-hassan centre of diabetes and endocrinology unit, under the kerbala health directorates. this ensures that the research is conducted with the highest ethical standards and guidelines. results the results of variants genotype (rs1544410) indicate a clear band with a molecular size 200 bps (figure 1). the size of amplicon was determined by compare with dna ladder 100–1500 bp. gene polymorphisms. the distribution of genotyping groups of patients shows in table 1. result of comparison between observed and anticipated values for snip with (rs 1544410) in the tested population were shown in figure 2, and table 2. the distribution and percentage of individuals having (rs 1544410) differ from those expected under hardy–weinberg equilibrium [number of fig. 1 genotyping of gene variants of vdr (rs 1544410). genotype variants of vdr of gene (rs 1544410) snp which were classified into three genotypes. 1. the major genotype group (cc) homozygous for the allele c. 2. the minor genotype group (tt) homozygous for the allele t. 3. heterozygous (ct). 203j contemp med sci | vol. 9, no. 3, may-june 2023: 201–205 f. j. al-tu’ma et al. original characteristics of vitamin d3 receptor genotypes in t2dm of iraqi obese women observed vs expected], which were: cc (36%, 25.6); tt (30%,19.6); ct (24%, 44.8) (goodness-of-fit χ2 for (rs 1544410), 19.397, p = < 0.001 and therefore it was statistically significant. the difference between demographic characteristics and (rs 1544410) snp (table 3), was performed using one-way anova test to compare the age, bmi and groups of study. no significant difference was found between all groups. the difference between biomarkers and rs (1544410) snp (table 4) was performed using one-way anova test to compare the mean levels of hsp-70, vdr, cpeptide, rbc and while blood test, hba1c was shown all no significant difference among the variants of vdr genotype (rs 1544410) in obese (patients & control) studied groups, p value > 0.05. the odds ratios of the detected genotypes of the (rs 1544410) of the patients with levels of biomarkers. the logistic analysis of the (rs 1544410) snp of the patients concluded that hsp70, vdr, c. peptide level was no significantly related to the also c. peptide, was shown to be related risk factor to the both ct and ct alleles (1.003, p = 0.338) in comparison with cc alleles. furthermore, hba1c level was demonstrated to be related risk factor for the cg allele in comparison with cc & gg alleles (1.009, p = 0.917). table 2. hardy–weinberg equilibrium for (rs 1544410) genotype in studied groups genotypes alleles hardy– weinberg equilibrium x 2 testc t genotype n = 100 frequency % 0.533 0.467 19.397 p < 0.001 [s] cc (wild type) 36 40 cg ( heterozygous mutant type ) 24 26.7 gg (homozygous mutant type) 30 33.3 table 3. difference between demographic characteristic in (rs 1544410) snp in studied groups demographic parameters rs 1544410 (n = 90 ) p-valuecc (n = 36) ct (n = 24) tt (n = 30) age 42.33 ± 8.89 46.75 ± 12.32 47.07 ± 12.08 0.156 [ns] bmi 29.35 ± 6.39 28.45 ± 6.19 27.18 ± 5.74 0.363 [ns] group patient 18 12 15 0.903 [ns] control 18 12 15 study group patient with obese 10 6 6 0.953 [ns] patient without obese 8 6 9 control with obese 10 6 6 control without obese 8 6 9 bp yes 12 11 13 0.563 [ns] no 24 13 17 smoking yes 1 1 0 0.564 [ns] no 35 23 30 results are presented as mean ± sd, or n = number of subjects and percentage, p < 0.05 considered significantly different, [s] = significant, [ns] = non-significant table 1. distribution of gene variants of vdr genotype (rs 1544410) different genotypes in studied groups variable group frequency percentage genotype cc (wild) 36 40 ct (hetero) 24 26.7 tt (homo) 30 33.3 data presented by numbers and percentage. fig. 2. variants of vdr observed (obs.) vs expected (exp.) genotype frequencies % of rs 1544410 among individuals’ sample. discussion the number of people with diabetes is expected to skyrocket from 382 million (8.3% of the population) in 2013 to 592 million (10.3%) in 2035. previous research has established a causal https://www.google.com/search?rlz=1c1gcea_eniq1040iq1040&sxsrf=apwxede6qempecohmvhpalhb-f0gg-e9jq:1683617465216&q=discussion&spell=1&sa=x&ved=2ahukewjmos7n2-f-ahxqr_edhrzwdqqqkeeckab6bagheae 204 j contemp med sci | vol. 9, no. 3, may-june 2023: 201–205 characteristics of vitamin d3 receptor genotypes in t2dm of iraqi obese women original f. j. al-tu’ma et al. link between vdr polymorphisms and metabolic parameters related to type 2 diabetes.15 the human vdr gene is found on chromosome 12q13.1 to give a rapid rundown. there are both coding and non-coding exons in the vdr gene, which undergo alternative splicing.16 increased secretion from cells, which may be caused by a genetic variation in the vdr gene, is linked to type 1 and type 2 diabetes.17 homozygous dominant model analysis was used to verify the role of the critical allele in the vdr gene’s connection to type 2 diabetes. there were 40% of the groups were homozygous for cc, 26% for ct, and 33.3% for tt. analysis using a recessive model also helped shed light on how the minor allele contributed to the link between the vdr gene and type 2 diabetes. specifically, the current study found that the a-allele of the vdr gene’s rs1544410 polymorphism was linked to higher insulin secretion, as evaluated by disposition index, in women with a history of dm. vdr belongs to the nuclear receptor family of transcriptional regulators. including beta cells in the pancreas, it is found ubiquitously and forms a heterodimer with a retinoid x receptor (rxr).18 consistent with previous research, this study found that vitamin d may play a role in the prevention and treatment of various health problems.19 potentially beneficial effects on insulin secretion and sensitivity have been postulated. possible processes include modulation of calcium homeostasis and activation of vitamin d receptor (vdr) on pancreatic beta cells and insulin-sensitive organs.20 the risk of diabetes, metabolic syndrome, insulin secretion, and insulin resistance has been shown to be inversely related to circulating concentrations of vitamin d.21 polymorphisms in the vdr gene have been linked to diabetes and insulin resistance in multiple studies, including a meta-analysis and those conducted by malik et al.22 there are currently about 25 distinct polymorphisms associated with the vdr locus. these vdr polymorphisms have been linked to type 2 diabetes and altered insulin secretion in a number of studies.23 metabolic alterations associated with obesity are known as metabolic syndrome, and they are linked to vdr polymorphisms.24 according to the results of the current study, those with the rs1544410 genotype of the vdr gene are more likely to develop type 2 diabetes and, by extension, obesity. in people with vitamin d3 deficiency, the (rs10735810) polymorphism of the vdr gene was discovered to be an additional independent driver of insulin secretion. in addition, the level of vdr mrna expression has been linked to insulin secretion.25 the likelihood of cluster disease has been linked to polymorphisms in the vitamin d receptor gene, according to a number of studies.26 but there’s a lack of consistency in findings, which may be because study populations are of different races. it is not yet understood how vdr polymorphisms contribute to the pathogenic landscape of mets at the molecular level. furthermore, linkage disequilibrium (ld) and haplotypes for the four previously indicated vdr snps and their relationships with metabolic syndrome in the thai population have not been documented.27 several other researchers agreed that the underlying pathophysiological processes of these correlations are still poorly understood. because vdr is found in pre-adipocytes, it is plausible that vitamin d has a direct effect on adipocyte development and metabolism.28 conclusion vdr polymorphisms could have a pivotal role in the presence of t2dm. vitamin d3 binding protein (vdbp) has gained attention as a new target to generate a drug for the treatment of endocrine nutritional and metabolic disorders like obesity and type 2 diabetes mellitus. the logistic analysis of the (rs1544410) snp of the patients concluded that hsp-70, vdbp, and c-peptide level was no significantly related to the also c-peptide, was shown to be a related risk factor to both ct and ct alleles (1.003, p > 0.05) in comparison with cc alleles. furthermore, hba1c% level was demonstrated to be related as a risk factor for the cg allele in comparison with cc and gg alleles (1.009, p < 0.05). conflict of interest none.  table 4. difference between alleles of variants of vdr genotype (rs 1544410) with mean levels of biomarkers in obese studied groups biomarkers rs 1544410 (n = 50) p-value cc (n = 10) cg (n = 12) gg (n = 28) hsp-70 30.22 ± 6.28 28.88 ± 7.06 29.63 ± 7.88 0.769 [ns] vdr 73.16 ± 21.42 72.34 ± 16.58 68.94 ± 17.75 0.669 [ns] c. peptide 139.72 ± 68.61 163.72 ± 126.68 130.73 ± 46.30 0.334 [ns] rbs 184.19 ± 96.81 197.46 ± 113.47 173.23 ± 92.75 0.678 [ns] hba1c 7.19 ± 3.06 7.28 ± 3.69 6.45 ± 2.40 0.521 [ns] w.h.r 0.97 ± 0.11 0.94 ± 0.11 0.93 ± 0.12 0.337 [ns] results are presented as mean ± sd, p < 0.05 considered significantly different, [s] = significant, [ns] = non-significant. table 5. the odds ratios of vdr genotype (rs 1544410) with biomarkers level variables snip or (95% ci) p-value hsp-70 cc 1a – ct 0.972(0.901 – 1.048) 0.464 [ns] tt 0.988(0.921 – 1.059) 0.732 [ns] vdr cc 1a – ct 0.998(0.969 – 1.027) 0.875 [ns] tt 0.988(0.961 – 1.015) 0.384 [ns] c-peptide cc 1a – ct 1.003(0.997 – 1.009) 0.338 [ns] tt 0.998(0.990 – 1.006) 0.593 [ns] hba1c% cc 1a – ct 1.009 (0.853 – 1.193) 0.917 [ns] tt 0.918 (0.778 – 1.084) 0.315 [ns] rbs cc 1a – ct 1.001 (0.996 – 1.006) 0.618 [ns] tt 0.999 (0.994 – 1.004) 0.648 [ns] results are presented as numbers and percentage, p < 0.05 considered significantly different, [s]; significant, [ns]; non significant, or: odds ratio, ci; confidence interval, a; reference category 205j contemp med sci | vol. 9, no. 3, may-june 2023: 201–205 f. j. al-tu’ma et al. original characteristics of vitamin d3 receptor genotypes in t2dm of iraqi obese women 16. gyapay, g., schmitt, k., fizames, c., jones, h., vega-czarny, n., spillett, d., muselet, d., prud’homme, j.-f., dib, c., & auffray, c. (1996). a radiation hybrid map of the human genome. human molecular genetics, 5(3), 339–346. 17. mackawy, a. m., & badawi, m. e. (2014). association of vitamin d and vitamin d receptor gene polymorphisms with chronic inflammation, insulin resistance and metabolic syndrome components in type 2 diabetic egyptian patients. meta gene, 2, 540–556. 18. bouillon, r., carmeliet, g., verlinden, l., van etten, e., verstuyf, a., luderer, h. f., lieben, l., mathieu, c., & demay, m. (2008). vitamin d and human health: lessons from vitamin d receptor null mice. endocrine reviews, 29(6), 726–776. 19. wang, h., chen, w., li, d., yin, x., zhang, x., olsen, n., & zheng, s. g. (2017). vitamin d and chronic diseases. aging and disease, 8(3), 346. 20. chiu, k. c., chu, a., go, v. l. w., & saad, m. f. (2004). hypovitaminosis d is associated with insulin resistance and β cell dysfunction. the american journal of clinical nutrition, 79(5), 820–825. 21. knekt, p., laaksonen, m., mattila, c., härkänen, t., marniemi, j., heliövaara, m., rissanen, h., montonen, j., & reunanen, a. (2008). serum vitamin d and subsequent occurrence of type 2 diabetes. epidemiology, 666–671. 22. malik, r., farooq, r., mehta, p., ishaq, s., din, i., shah, p., & majid, s. (2018). association of vitamin d receptor gene polymorphism in adults with type 2 diabetes in the kashmir valley. canadian journal of diabetes, 42(3), 251–256. 23. shaat, n., ignell, c., katsarou, a., & berntorp, k. (2017). glucose homeostasis, beta cell function, and insulin resistance in relation to vitamin d status after gestational diabetes mellitus. acta obstetricia et gynecologica scandinavica, 96(7), 821–827. 24. ignell, c., shaat, n., ekelund, m., & berntorp, k. (2013). the impact of ethnicity on glucose homeostasis after gestational diabetes mellitus. acta diabetologica, 50, 927–934. 25. ogunkolade, b.-w., boucher, b. j., prahl, j. m., bustin, s. a., burrin, j. m., noonan, k., north, b. v., mannan, n., mcdermott, m. f., & deluca, h. f. (2002). vitamin d receptor (vdr) mrna and vdr protein levels in relation to vitamin d status, insulin secretory capacity, and vdr genotype in bangladeshi asians. diabetes, 51(7), 2294–2300. 26. al-daghri, n. m., al-attas, o. s., alkharfy, k. m., khan, n., mohammed, a. k., vinodson, b., ansari, m. g. a., alenad, a., & alokail, m. s. (2014). association of vdr-gene variants with factors related to the metabolic syndrome, type 2 diabetes and vitamin d deficiency. gene, 542(2), 129–133. 27. karuwanarint, p., phonrat, b., tungtrongchitr, a., suriyaprom, k., chuengsamarn, s., & tungtronchitr, r. (2018). genetic variations of vitamin d receptor gene in metabolic syndrome and related diseases in the thai population. asia pacific journal of clinical nutrition, 27(4), 935–944. 28. kamei, y., kawada, t., kazuki, r., ono, t., kato, s., & sugimoto, e. (1993). vitamin d receptor gene expression is up-regulated by 1, 25-dihydroxyvitamin d3 in 3t3-l1 preadipocytes. biochemical and biophysical research communications, 193(3), 948–955. references 1. zou, q., qu, k., luo, y., yin, d., ju, y., & tang, h. (2018). predicting diabetes mellitus with machine learning techniques. frontiers in genetics, 9, 515. 2. zheng, y., ley, s. h., & hu, f. b. (2018). global aetiology and epidemiology of type 2 diabetes mellitus and its complications. nature reviews endocrinology, 14(2), 88–98. 3. padhi, s., nayak, a. k., & behera, a. (2020). type ii diabetes mellitus: a review on recent drug based therapeutics. biomedicine & pharmacotherapy, 131, 110708. 4. pischon, t., boeing, h., hoffmann, k., bergmann, m., schulze, m. b., overvad, k., van der schouw, y., spencer, e., moons, k., & tjønneland, a. (2008). general and abdominal adiposity and risk of death in europe. new england journal of medicine, 359(20), 2105–2120. 5. horst-sikorska, w., dytfeld, j., wawrzyniak, a., marcinkowska, m., michalak, m., franek, e., napiórkowska, l., drwęska, n., & słomski, r. (2013). vitamin d receptor gene polymorphisms, bone mineral density and fractures in postmenopausal women with osteoporosis. molecular biology reports, 40, 383–390. 6. li y, xi b, li k, wang c. association between vitamin d receptor gene polymorphisms and bone mineral density in chinese women. molecular biology reports. 2012 may;39:5709–17. 7. mosaad, y. m., hammad, e. m., fawzy, z., aal, i. a. a., youssef, h. m., elsaid, t. o., monir, r., & el-deek, b. s. (2014). vitamin d receptor gene polymorphism as possible risk factor in rheumatoid arthritis and rheumatoid related osteoporosis. human immunology, 75(5), 452–461. 8. manchanda, p., & bid, h. (2012). vitamin d receptor and type 2 diabetes mellitus: growing therapeutic opportunities. indian journal of human genetics, 18(3), 274. 9. sung, c.-c., liao, m.-t., lu, k.-c., & wu, c.-c. (2012). role of vitamin d in insulin resistance. journal of biomedicine and biotechnology, 2012. 10. ortlepp, j., metrikat, j., albrecht, m., von korff, a., hanrath, p., & hoffmann, r. (2003). the vitamin d receptor gene variant and physical activity predicts fasting glucose levels in healthy young men. diabetic medicine, 20(6), 451–454. 11. anuradha, c. v. (2013). phytochemicals targeting genes relevant for type 2 diabetes. canadian journal of physiology and pharmacology, 91(6), 397–411. 12. rahmadhani, r., zaharan, n. l., mohamed, z., moy, f. m., & jalaludin, m. y. (2017). the associations between vdr bsmi polymorphisms and risk of vitamin d deficiency, obesity and insulin resistance in adolescents residing in a tropical country. plos one, 12(6), e0178695. 13. karonova, t., grineva, e., belyaeva, o., bystrova, a., jude, e. b., andreeva, a., kostareva, a., & pludowski, p. (2018). relationship between vitamin d status and vitamin d receptor gene polymorphisms with markers of metabolic syndrome among adults. frontiers in endocrinology, 9, 448. 14. pike, j. w., meyer, m. b., benkusky, n. a., lee, s. m., john, h. s., carlson, a., onal, m., & shamsuzzaman, s. (2016). genomic determinants of vitamin d-regulated gene expression. vitamins & hormones, 100, 21–44. 15. zaki, m., kamal, s., basha, w. a., youness, e., ezzat, w., el-bassyouni, h., & amr, k. (2017). association of vitamin d receptor gene polymorphism (vdr) with vitamin d deficiency, metabolic and inflammatory markers in egyptian obese women. genes & diseases, 4(3), 176–182. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1352 106 j contemp med sci | vol. 5, no. 2, march–april 2019: 106–111 original cord blood sex hormones concentration: relation to birth weight and pregnancy complications hanan l. al-omary and zainab m. alawad* physiology department, college of medicine, university of baghdad, baghdad, iraq. *correspondence to zainab m. alawad (email: zainabm.alawad@gmail.com). (submitted: 06 november 2018 – revised version received: 20 december 2018 – accepted: 14 january 2019 – published online: 26 april 2019) objectives umbilical cord blood can be taken at birth and largely gives indication of fetal and maternal conditions. the aim of the study was to investigate the relation between sex hormones in cord blood and birth weight of newborns and pregnancy complications. methods fifty cord blood samples were collected from newborns at labor room of baghdad teaching hospital between may and october 2018. blood was withdrawn from their mothers for lead analysis. five milliliters (ml) of cord blood was taken, 3 ml was used for testosterone and estradiol analysis (using enzyme-linked immunosorbent assay) and 2 ml for lead measurement by lead care analyzer. newborns weight and head circumference were measured. delivered women were divided into four groups: women with normal pregnancy, women with pre-eclampsia, diabetic women and polycystic ovary syndrome (pcos) women. results there was no significant difference in age between women in all groups (p > 0.05). birth weights, estradiol, and testosterone were significantly different between groups. estradiol was higher in cord blood of newborns of pcos women (p < 0.05) than others. testosterone was higher in cord blood of babies of pcos and pre-eclampsia women compared with those of diabetes (p < 0.05). there were no significant differences between male and female neonates regarding cord estradiol (3596.27 ± 1934.69, 3714.57 ± 1581.47 pg/ml respectively), and testosterone (393.18 ± 87.14, 361.43 ± 102.14 ng/ml respectively) (p > 0.05). maternal lead levels correlated positively with cord lead (r = 0.905, p < 0.05), which correlated negatively with head circumference (r = −0.766, p < 0.05). birth weight correlated negatively with estradiol (r = −0.295), but positively with testosterone (r = 0.006) (p > 0.05). conclusion cord blood estradiol and testosterone levels do not differ between males and females. estradiol was high in cord blood of pcos mothers. testosterone was high in cord blood of pcos and pre-eclampsia mothers. the increase in cord lead causes decrease in babies head circumference. keywords cord blood, pregnancy, birth weight, estradiol, testosterone introduction pregnancy is a special stage of life in which circulating hormone levels are obtained from maternal, placental, and fetal origins. the placenta is a steroidogenic organ which has a role in the synthesis of great quantities of free androgens and estrogens from the adrenals of the fetus and gonadal sources.1,2 steroids are lipophilic substances and can pass placental barrier in both directions.3 fetal blood goes out of the placenta (full of steroids) through the umbilical vein and goes back from the fetus to the placenta through the umbilical artery. measuring the hormones in maternal blood does not actually reflect the concentrations in fetal circulation.4 in addition, measuring hormone concentration through amniocentesis does not give exact indication and it is invasive procedure. for these reasons cord blood is currently the practical way to measure fetal hormones.5 cord blood (both serum and plasma) is a specially challenging medium to measure because it has unique steroid concentrations due to the mix of placental and fetal steroid production and metabolism.6 umbilical cord blood is usually obtained after delivery, so the hormone levels in the cord plasma or serum are said to represent the levels in the fetal circulation near the end of pregnancy.7 umbilical cord blood has about equal quantities of venous and arterial contents, in spite of the fact that the relative amounts are not yet estimated exactly. it was noted that the estimation of umbilical cord hormones is influenced by several obstetric and maternal factors.1 sex hormone levels in fetal blood have been the subject of interest over many years because of their relation to maternal metabolic disorders, cancer risk later in life, reproductive, and behavioral/neurodevelopmental disorders.7 the aim of this study was to investigate the relation between sex hormones in cord blood and birth weight of the baby and pregnancy complications. patients and methods in this prospective study, 50 cord blood samples had been collected from the labor room of baghdad teaching hospital in the period between may 2018 and october 2018. an informed consent was taken from all pregnant women. the work was approved by the ethical committee of college of medicine/ university of baghdad. it was in agreement with the helsinki declaration of 1975 that was revised in 2000. all pregnancies were singleton. immediately after birth, the umbilical cord was clamped and cut. another clamp was put 20–25 cm from the first one. the part between the clamps was cut and a mixed arterial and venous blood sample (5 ml) was collected into a plastic tube. about 2 ml of each sample was analyzed for lead using lead care analyzer device and 3 ml of each sample was centrifuged and kept in −20°c till the time of analysis of estradiol and tesstosterone by enzyme-linked immunosorbent assay (elisa). at the same time whole blood (2 ml) was drawn from the pregnant women for lead measurement. there is no normal level of lead in blood since it does not constitute the human body. little exposure to lead in adults are not thought to cause poisoning [blood lead <10 micrograms (µg) per deciliter (dl)], but it can harm on the long term.8 issn 2413-0516 107j contemp med sci | vol. 5, no. 2, march–april 2019: 106–111 original sex hormones in cord blood and pregnancy complicationshanan l. al-omary and zainab m. alawad testosterone and estradiol concentrations were measured by elisa (cloud–clone corp., cea461ge, using immulite 2000 xpi, siemens) as showed in fig.1. full medical and obstetrical history was taken from the pregnant women, including age, pregnancy complications like diabetes (already having or gestational diabetes confirmed by fasting blood sugar), pre-eclampsia (systolic blood pressure ≥ 140 mmhg or diastolic blood pressure ≥90 mmhg, measured after 20 weeks of pregnancy, proteinuria ≥ 0.3 g or more in 24 h urine),9 or having polycystic ovary syndrome (pcos) (diagnosed according to rotterdam criteria).10 gestational age was determined by directly counting days since the start of the last menstrual period or by early pregnancy ultrasound. normal pregnancy period ranges from completed 37 to 42 weeks.11 gender of the baby was also recorded. the weight of the placenta and the baby were measured using digital balance in the place. normal range of birth weight is between 2.5 and 5 kg,12 and placenta ideally weighs about 500 g. it was weighed after removing the membranes and cutting the cord.13 head circumference was recorded using measuring tape (normal head circumference of newborn is about 35 cm).14 maternal and cord blood lead measurements were achieved by lead care analyzer, that contains the lead care kit, and the lead care analyzer device version 3.3 (esa, inc., usa). a total of 50 µl of whole blood was put in the edta tubes to be mixed with treatment reagent in the tubes provided by the kit. the blood was mixed with the reagent, so the color of the mixture became brown. then the tubes were left to stand up for a minute giving the chance for the mixture to drain down to the bottom. by a pipette, 35 µl of the mixture was drawn and put on the device sensor after calibration and the process of analysis began.15 statistical analysis data were analyzed by spss version 20. analysis of variance (anova) test was applied for comparing parameters between groups, results were expressed as mean ± standard deviation. correlations were analyzed by pearson’s correlation test. a p-value of <0.05 was considered significant in this study. results descriptive data concerning all pregnant females was shown in table 1.the mean age was 29.66 ± 4.94 ranging between 22 and 40 years. mean body mass index (bmi) was 24.8 ± 2.08. the study included 22 males and 28 females. there were 18 normal pregnancies, 13 with pre-eclampsia, 8 with diabetes and 11 with pcos. a comparison between normal pregnancy and those pregnancies of women with pre-eclampsia, diabetes and pcos was done (table 2). there was no significant difference between the groups regarding age. bmi was significantly different between the groups. birth weights were significantly higher in babies of diabetic patients compared with other groups (table 2). estradiol was significantly higher in cord table 1. descriptive data for all patients. parameter mean ± sd age (years) 29.66 ± 4.94 bmi (kg/m2) 24.8 ± 2.08 birth weight (kg) 3.25 ± 0.56 gender males 22 females 28 estradiol (pg/ml) 3794.52 ± 1775.62 testosterone (ng/ml) 359.60 ± 102.34 diseases normal pregnancy 18 pre-eclampsia 13 diabetes 8 pcos 11 blood lead (mg/dl) maternal 3.03 ± 1.06 cord 0.38 ± 0.30 gestational age (weeks) 38.35 ± 1.08 head circumference (cm) 37.28 ± 0.56 placental weight (g) 529.46 ± 29.57 pcos: polycystic ovary syndrome, bmi: body mass index, sd: standard deviation. fig. 1 the device used to assess estradiol and testosterone concentration. table 2. comparison of age, bmi, birth weight, estradiol and testosterone levels in cord blood and gestational age between groups normal pregnancy (n = 18) pre-eclampsia (n = 13) diabetes (n = 8) pcos (n = 11) p age (years) 30.83 ± 3.07 28.15 ± 3.69 29.75 ± 5.78 27.55 ± 3.17 >0.05 bmi (kg/m2) 24.76 ± 2.10 25.34 ± 1.99 23.50 ± 1.60 25.50 ± 2.23 <0.05 birth weight (kg) 3.80 ± 0.46 3.29 ± 0.39 4.77 ± 7.20 3.26 ± 0.23 <0.05 estradiol (pg/ml) 4014.56 ± 1902.29 3904.92 ± 1806.48 1800 ± 840.52 4154.55 ± 1103 <0.05 testosterone (ng/ml) 351.28 ± 64.87 364.92 ± 89.46 257 ± 36.42 477.27 ± 46.92 <0.05 gestational age (weeks) 38.66 ± 0.99 38.09 ± 1.17 37 ± 0.1 39.13 ± 0.1 <0.05 pcos: polycystic ovary syndrome, bmi: body mass index. 108 j contemp med sci | vol. 5, no. 2, march–april 2019: 106–111 sex hormones in cord blood and pregnancy complications original hanan l. al-omary and zainab m. alawad group statistics diseases n mean std. deviation std. error mean gestational age pcos 11 39.1273 0.10090 0.03042 no 18 38.6556 0.99186 0.23378 group statistics diseases n mean std. deviation std. error mean gestational age diabetes 8 37.0000 0.00000 0.00000 no 18 38.6556 0.99186 0.23378 group statistics diseases n mean std. deviation std. error mean gestational age pre-eclampsia 13 38.0923 1.16795 0.32393 no 18 38.6556 0.99186 0.23378 group statistics diseases n mean std. deviation std. error mean testosterone pcos 11 477.2727 46.92354 14.14798 no 18 351.2778 64.86530 15.28890 group statistics diseases n mean std. deviation std. error mean testosterone diabetes 8 257.0000 36.42213 12.87717 no 18 351.2778 64.86530 15.28890 group statistics diseases n mean std. deviation std. error mean testosterone pre-eclampsia 13 364.9231 89.46364 24.81275 no 18 351.2778 64.86530 15.28890 group statistics diseases n mean std. deviation std. error mean estradiol pcos 11 4154.5455 1103.05361 332.58318 no 18 4014.5556 1902.29262 448.37467 group statistics diseases n mean std. deviation std. error mean estradiol diabetes 8 1800.0000 84.51543 29.88072 no 18 4014.5556 1902.29262 448.37467 group statistics diseases n mean std. deviation std. error mean estradiol pre-eclampsia 13 3904.9231 1806.47513 501.02606 no 18 4014.5556 1902.29262 448.37467 group statistics diseases n mean std. deviation std. error mean bwt pcos 11 3.2636 0.22923 0.06911 no 18 4.7694 7.20651 1.69859 group statistics diseases n mean std. deviation std. error mean bwt diabetes 8 3.8000 0.45981 0.16257 no 18 4.7694 7.20651 1.69859 group statistics diseases n mean std. deviation std. error mean bwt pre-eclampsia 13 3.2923 0.39256 0.10888 no 18 4.7694 7.20651 1.69859 group statistics diseases n mean std. deviation std. error mean bmi diabetes 8 23.5000 1.60357 0.56695 no 18 24.7588 2.10284 0.49564 group statistics diseases n mean std. deviation std. error mean bmi pre-eclampsia 13 25.3374 1.99897 0.55441 no 18 24.7588 2.10284 0.49564 group statistics diseases n mean std. deviation std. error mean bmi pcos 11 25.5009 2.23358 0.67345 no 18 24.7588 2.10284 0.49564 group statistics diseases n mean std. deviation std. error mean age pre-eclampsia 13 28.1538 3.69338 1.02436 no 18 30.8333 3.07265 0.72423 not group statistics diseases n mean std. deviation std. error mean age diabetes 8 29.7500 5.77556 2.04197 no 18 30.8333 3.07265 0.72423 e2 anova diseases sum of squares df mean square f sig. between groups 65.453 15 4.364 89.017 0.000 within groups 1.667 34 0.049 total 67.120 49 109j contemp med sci | vol. 5, no. 2, march–april 2019: 106–111 original sex hormones in cord blood and pregnancy complicationshanan l. al-omary and zainab m. alawad test anova diseases sum of squares df mean square f sig. between groups 54.231 16 3.389 8.678 0.000 within groups 12.889 33 0.391 total 67.120 49 blood of fetuses of pcos mothers than those of pre-eclampsia, diabetes and normal pregnancy (4154.55 ± 1103, 3904.92 ± 1806.48, 1800 ± 840.52, 4014.56 ± 1902.29 respectively). on the other hand, testosterone was significantly higher in babies of pcos patients and pre-eclampsia compared with those of diabetes and normal pregnancy (477.27 ± 46.92, 364.92 ± 89.46, 257 ± 36.42, 351.28 ± 64.87 respectively). males and females cord blood were compared for estradiol and testosterone levels and nonsignificant differences was found between them although testosterone was higher in male newborns (table 3). cord blood lead correlated positively and significantly with maternal lead, but negatively and significantly with babies’ head circumference (table 4). birth weight correlated negatively with estradiol and positively with testosterone in the cord blood, however both correlations were not significant (table 5). gestational age had a nonsignificant positive correlation with estradiol, but a significant negative correlation with testosterone (table 6). discussion cord blood sex hormones measurements and their relations to maternal complications and fetal birth weight were studied in this work. birth weight was higher in babies of diabetic mothers, this agrees with other studies that reported high birth weight in babies born to diabetic mothers.16,17 it was reported that infants of diabetic women are at high risk of being overweight and they may become obese at a young age.18 polycystic ovary syndrome women had their fetuses with a cord blood significantly higher in estradiol and testosterone. this result goes with that found by daan et al.19 who suggested that high androgen environment in pregnancy of pcos women may increase androgen in their fetuses. the high insulin levels in pregnant women who have pcos predispose to the increase in fetal androgen and this is due to the inhibition of placental aromatase action that makes the conversion of the androgen in maternal and fetal circulation to estrogens low.20 other studies had also reported increase androgen in cord blood of babies born to pcos mothers.21,22 on the other hand some studies found a decrease in androgen concentrations23,24 or observed no differences in comparison to controls.25 the explanation for the decreased androgens in cord blood may indicate a modification of fetal steroid metabolism,23 and the differences in the amount of testosterone may be affected by variations in the tissue activity of placenta in pcos. in addition, there are differences in the diagnostic criteria of pcos, differences in ethnicity and in the applied statistical tests. cord blood testosterone was higher in fetuses of mothers with pre-eclampsia than in those born for normal mothers, this was in accordance with the results of chinnathambi et al. who reported a high testosterone in plasma of women with pre-eclampsia and in cord blood of their fetuses and they attributed that to the effect of testosterone on vascular endothelia since they changed vascular adaptation throughout pregnancy by decreasing nitric oxide effect as a vasodilator, in addition to the fact that testosterone increases vascular resistance in these patients. also, testosterone suppresses the relaxation of blood vessels induced by acetylcholine in mesenteric arteries.26 other studies also demonstrated positive correlation between cord blood testosterone and high blood pressure in the mother.27,28 estradiol was high in cord blood of fetuses of pcos mothers, this goes with caanen et al.’s29 study, they reported a disturbance in the function of placental enzymes in pcos, especially aromatase which catalyze the conversion of 16-hydroxytestosterone to estradiol. on the other hand, kallen30 showed that estradiol is low in cord blood of pcos women. estrogen levels in cord blood are affected by fetal adrenal and placental steroidogenesis, and decreased estradiol levels in cord blood of babies of pcos women was due to abnormal placental steroidogenesis.31 there were no differences in cord blood estradiol and testosterone between male and female fetuses, this agrees with other studies which reported that estrogen in umbilical cord is not significantly different between males and females,24,32–34 while other studies found higher estradiol levels in females cord blood,35 and higher testosterone table 5. correlations between cord blood estradiol and testosterone with birth weight birth weight (kg) r p cord blood estradiol (pg/ml) −0.295 >0.05 cord blood testosterone (ng/ml) 0.006 >0.05 table 6. correlation between cord blood estradiol, testosterone and gestational age gestational age (weeks) r p cord blood estradiol (pg/ml) 0.830 >0.05 cord blood testosterone (ng/ml) −0.703 <0.05 table 3. comparison of estradiol and testosterone concentrations in cord blood between males and females males (n = 22) females (n = 28 ) p estradiol (pg/ml) 3596.27 ± 1934.69 3714.57 ± 1581.47 >0.05 testosterone (ng/ml) 393.18 ± 87.14 361.43 ± 102.14 >0.05 table 4. correlations between cord blood lead and maternal lead, cord blood lead and newborns’ head circumference cord blood lead (mg/dl) r p maternal lead (mg/dl) 0.905 <0.05 head circumference (cm) −0.766 <0.05 110 j contemp med sci | vol. 5, no. 2, march–april 2019: 106–111 sex hormones in cord blood and pregnancy complications original hanan l. al-omary and zainab m. alawad concentrations in males.35,36 these findings were in assumption that they are reflection of their levels in utero in the early pregnancy stages, which refers to the exposure to high androgen during fetal development that occurs in male more than female fetuses.37 exposure of pregnant females to lead is one of the important factors affecting pregnancy outcome. in this study there was significant increase in cord lead with the increase in maternal lead levels, this agrees with a study that reported a significant positive correlation between maternal and cord lead.38 lead level correlated negatively and significantly with neonates’ head circumference which was also found by akbari-nassaji et al.39 on the other hand some studies found no significant relationship between cord blood lead and head circumference.40,41 cord blood estradiol correlated negatively but not significantly with birth weight, this was found in another study which suggested that this correlation is modified by some adjustment of insulin like growth factor level in these fetuses.42 whereas other studies documented that the greater estradiol levels were obtained from cord blood of high birth weight babies.31,43 testosterone in cord blood correlated positively with birth weight which was found by another study that assumed testosterone effect from early in utero period.44 keelan et al.,7 reached the same result in their work that studied the metabolic changes in pcos women who had increased testosterone levels in cord blood of their babies and a corresponding increase in their birth weights. another study found no relation between birth weight and androgen levels.22 however, factors affecting fetal growth and birth weight that is related to sex hormones are complicated and have many aspects, which makes it difficult to determine. the negative correlation between cord blood testosterone and gestational age had met results of other studies who emphasizes on testosterone and other androgen types.21,45 this correlation is related partly to the amount of sex hormone binding globulins (shbg) that have positive correlation with gestational age, and their increase will cause the decrease in testosterone which explains our results.45 the correlation of estradiol with gestational age had not been demonstrated clearly in other studies 43 thus more work to investigate this correlation is needed. using elisa for analyzing sex steroids could be considered as a limitation since it was suggested that mass spectrometry method is more accurate and can detect lower androgen concentrations than elisa.7 also, determination of biologically active fractions of sex steroids by adjusting albumin and shbg concentrations during gestation was not done in this study, nonetheless, it is important in order to have more valid conclusions.44 conclusion cord blood estradiol and testosterone levels do not differ significantly between male and female fetuses. estradiol was significantly high in cord blood of neonates of pcos mothers. testosterone was significantly high in cord blood of newborns of pcos and pre-eclampsia women. the increase in cord lead causes decrease in neonates’ head circumference. conflict of interest none.  references 1. albrecht ed, pepe gj. placental steroid hormone biosynthesis in primate pregnancy. endocr rev. 1990;11:124–150. 2. pašková a1, pařízek a, hill m, velíková m, kubátová j, dušková m, et al. steroid metabolome in the umbilical cord: is it necessary to differentiate between arterial and venous blood? physiol res. 2014;63:115–126. 3. reis fm1, florio p, cobellis l, luisi s, severi fm, bocchi c, et al. human placenta as a source of neuroendocrine factors. biol neonate. 2001;79:150–156. 4. cohen-bendahan cc, van goozen sh, buitelaar jk, cohen-kettenis pt. maternal serum steroid levels are unrelated to fetal sex: a study in twin pregnancies. twin res hum genet. 2005;8:173–177. 5. sloboda dm, hickey m, hart r. reproduction in females: the role of the early life environment. hum reprod update. 2010;17:210–227. 6. hill m1, parízek a, kancheva r, dusková m, velíková m, kríz l, et al. steroid metabolome in plasma from the umbilical artery, umbilical vein, maternal cubital vein and in amniotic fluid in normal and preterm labor. j steroid biochem mol biol. 2010;121:594–610. 7. keelan ja, mattes e, tan h, dinan a, newnham jp, whitehouse aj, et al. androgen concentrations in umbilical cord blood and their association with maternal, fetal and obstetric factors. plos one 2012;7:e42827. 8. ahamed m, siddiqui mk. low level lead exposure and oxidative stress: current opinions. clin chim acta. 2007;383:57–64. 9. stella cl, sibai bm. preeclampsia: diagnosis and management of the atypical presentation. j matern fetal neonatal med. 2006;19:381–386. 10. wang r, mol bw. the rotterdam criteria for polycystic ovary syndrome: evidence-based criteria? hum reprod. 2017;32:261–264. 11. tunón k, eik-nes sh, grøttum p, von düring v, kahn ja. gestational age in pregnancies conceived after in vitro fertilization: a comparison between age assessed from oocyte retrieval, crown-rump length and biparietal diameter. ultrasound obstet gynecol. 2000;15:41–46. 12. janssen pa, thiessen p, klein mc, whitfield mf, macnab yc, cullis-kuhl sc. standards for the measurement of birth weight, length and head circumference at term in neonates of european, chinese and south asian ancestry. open med. 2007;1:e74–e88. 13. mercer js, vohr br, erickson-owens da, padbury jf, oh w. seven-month developmental outcomes of very low birth weight infants enrolled in a randomized controlled trial of delayed versus immediate cord clamping. j perinatol. 2010;30:11–16. 14. lindley aa, benson je, grimes c, cole tm 3rd, herman aa. the relationship in neonates between clinically measured head circumference and brain volume estimated from head ct-scans. early hum dev. 1999;56:17–29. 15. al-omary hl, alawad zm, hussei sy. lymphocyte apoptosis in third trimester of pregnancy. j clin diagn res. 2018;12:cc05–cc08. 16. touger l, looker hc, krakoff j, lindsay rs, cook v, knowler wc. early growth in offspring of diabetic mothers. diabetes care. 2005;28:585–589. 17. morgan k, rahman m, atkinson m, zhou sm, hill r, khanom a, et al. association of diabetes in pregnancy with child weight at birth, age 12 months and 5 years-a population-based electronic cohort study. plos one 2013;8:e79803. 18. kc k, shakya s, zhang h. gestational diabetes mellitus and macrosomia: a literature review. ann nutr metab. 2015;66:14–20. 19. daan nm, koster mp, steegers-theunissen rp, eijkemans mj, fauser bc. endocrine and cardiometabolic cord blood characteristics of offspring born to mothers with and without polycystic ovary syndrome. fertil steril. 2017;107:261–268.e3. 20. xita n, tsatsoulis a. fetal programming of polycystic ovary syndrome by androgen excess: evidence from experimental, clinical, and genetic association studies. j clin endocrinol metab. 2006;91:1660–1666. 21. barry ja, kay ar, navaratnarajah r, iqbal s, bamfo je, david al, et al. umbilical vein testosterone in female infants born to mothers with polycystic ovary syndrome is elevated to male levels. j obstet gynaecol. 2010;30:444–446. 22. mehrabian f, kelishadi r. comparison of the metabolic parameters and androgen level of umbilical cord blood in newborns of mothers with polycystic ovary syndrome and controls. j res med sci. 2012;17:207–211. 23. maliqueo m, lara he, sánchez f, echiburú b, crisosto n, sir-petermann t. placental steroidogenesis in pregnant women with polycystic ovary syndrome. eur j obstet gynecol reprod biol. 2013;166:151–155. 111j contemp med sci | vol. 5, no. 2, march–april 2019: 106–111 original sex hormones in cord blood and pregnancy complicationshanan l. al-omary and zainab m. alawad this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 24. anderson h, fogel n, grebe sk, singh rj, taylor rl, dunaif a. infants of women with polycystic ovary syndrome have lower cord blood androstenedione and estradiol levels. j clin endocrinol metab. 2010;95:2180–2186. 25. steegers-theunissen rp, verheijden-paulissen jj, van uitert em, wildhagen mf, exalto n, koning ah, et al. cohort profile: the rotterdam periconceptional cohort (predict study). int j epidemiol. 2016;45:374–381. 26. chinnathambi v, balakrishnan m, ramadoss j, yallampalli c, sathishkumar k. testosterone alters maternal vascular adaptations: role of the endothelial no system. hypertension. 2013;61:647–654. 27. troisi r, potischman n, roberts jm, ness r, crombleholme w, lykins d, et al. maternal serum oestrogen and androgen concentrations in preeclamptic and uncomplicated pregnancies. int j epidemiol. 2003;32:455–460. 28. rohrmann s, sutcliffe cg, bienstock jl, monsegue d, akereyeni f, bradwin g, et al. racial variation in sex steroid hormones and the insulinlike growth factor axis in umbilical cord blood of male neonates. cancer epidemiol biomarkers prev. 2009;18:1484–1491. 29. caanen mr, kuijper ea, hompes pg, kushnir mm, rockwood al, meikle wa, et al. mass spectrometry methods measured androgen and estrogen concentrations during pregnancy and in newborns of mothers with polycystic ovary syndrome. eur j endocrinol. 2016;174:25–32. 30. kallen cb. steroid hormone synthesis in pregnancy. obstet gynecol clin north am. 2004;31:795–816.. 31. troisi r, potischman n, roberts j, siiteri p, daftary a, sims c, et al. associations of maternal and umbilical cord hormone concentrations with maternal, gestational and neonatal factors (united states). cancer causes control. 2003;14:347–355. 32. troisi r, potischman n, roberts jm, harger g, markovic n, cole b, et al. correlation of serum hormone concentrations in maternal and umbilical cord samples. cancer epidemiol biomarkers prev. 2003;12:452–456. 33. hickey m, hart r, keelan ja. the relationship between umbilical cord estrogens and perinatal characteristics: implications for early life origins of reproductive cancers. cancer epidemiol biomarkers prev. 2014;23:946–952. 34. hill m, pašková a, kančeva r, velikova m, kubatova j, kancheva l, et al. steroid profiling in pregnancy: a focus on the human fetus. j steroid biochem mol biol. 2014;139:201–222. 35. simmons d, france jt, keelan ja, song l, knox bs. sex differences in umbilical cord serum levels of inhibin, testosterone, oestradiol dehydroepiandrosterone sulphate, and sex hormone-binding globulin in human term neonates. biol neonate. 1994;65:287–294. 36. van de beek c, thijssen jh, cohen-kettenis pt, van goozen sh, buitelaar jk. relationships between sex hormones assessed in amniotic fluid, and maternal and umbilical cord serum: what is the best source of information to investigate the effects of fetal hormonal exposure? horm behav. 2004;46:663–669. 37. krogh c, cohen as, basit s, hougaard dm, biggar rj, wohlfahrt j, et al. testosterone levels in umbilical-cord blood and risk of pyloric stenosis. pediatrics 2011;127:e197–e201. 38. reddy ys, aparna y, ramalaksmi ba, kumar bd. lead and trace element levels in placenta, maternal and cord blood: a cross-sectional pilot study. j obstet gynaecol res. 2014;40:2184–2190. 39. neda an, fahimeh s, tahereh zk, leila f, zahra n, bahman c, et al. lead level in umbilical cord blood and its effects on newborns anthropometry. j clin diagn res. 2017;11:sc01–sc04. 40. falcón m, viñas p, luna a. placental lead and outcome of pregnancy. toxicology 2003;185:59–66. 41. west wl, knight em, edwards ch, manning m, spurlock b, james h, et al. maternal low level lead and pregnancy outcomes. j nutr. 1994;124: 981s–986s. 42. nagata c, iwasa s, shiraki m, shimizu h. estrogen and α-fetoprotein levels in maternal and umbilical cord blood samples in relation to birth weight. cancer epidemiol biomarkers prev. 2006;15:1469–1472. 43. lagiou p, samoli e, hsieh cc, lagiou a, xu b, yu gp, et al. maternal and cord blood hormones in relation to birth size. eur j epidemiol. 2014;29:343–351. 44. hollier lp, keelan ja, hickey m, maybery mt, whitehouse aj. measurement of androgen and estrogen concentrations in cord blood: accuracy, biological interpretation, and applications to understanding human behavioral development. front endocrinol (lausanne). 2014;5:64. 45. zlotkin sh, casselman cw. percentile estimates of reference values for total protein and albumin in sera of premature infants (less than 37 weeks of gestation). clin chem. 1987;33:411–413. dx.doi.org/10.22317/jcms.04201908 51j contemp med sci | vol. 8, no. 1, january-february 2022: 51–58 original synthesis, characterization, and antimicrobial evaluation for new azo-linked derivative of heterocyclic compounds ahlam adnan shafeeq1 , zaid mohammed ali hamandi2, mohammed hassan mohammed3, ghassan al-shamma4 1department of pharmaceutical chemistry, college of pharmacy, al ameed university, iraq. 2hematology and oncology centre, medical city, ministry of health, baghdad, iraq. 3department of pharmaceutical chemistry, college of pharmacy, baghdad university, baghdad, iraq. 4department of chemistry and biochemistry, college of medicine, al-nahrain university, baghdad, iraq. *correspondence to: ahlam adnan shafeeq (e-mail: a.aljubori@alameed.edu.iq) (submitted: 11 december 2021 – revised version received: 16 december 2021 – accepted: 15 january 2022 – published online: 26 february 2022) abstract objectives: using different activated heterocyclic amines to prepare diazonium salts to be coupled with ornidazole (heterocyclic agent) to form the azo linkage compounds (a1, a2) as possible antimicrobial agent. methods: the synthetic process involves the preparation of diazonium salts by diazotization reaction of sulfamethoxazole and 5-amino1,3,4-thiadiazole-2-thiol that contain primary amino groups with (hno 2 ) acid in the presence of hcl at about 0˚c. then, azo compounds (a1, a2) were synthesized, by the reaction of nitroimidazole derivative (ornidazole) with the prepared diazonium salts at 0–5˚c, and the chemical structure of these compounds were characterized and confirmed by measuring its fourier transform infrared (ft-ir) spectrum and proton nuclear magnetic resonance (h1-nmr) spectrum. results: thin layer chromatography and melting point measurements were used to establish the product’s purity. this compound was tested for antimicrobial activity using the broth microdilution spectrometric method on five different strains of bacteria and one strain of fungi, as well as the bactec mgit 960 system for mycobacterium tuberculosis bacilli. conclusion: using levofloxacin and nystatin as reference drugs, the compounds (a1, a2) showed moderate to good action against tested gram-positive and gram-negative bacteria, and fungi, but no activity against mycobacterium tuberculosis. keywords: heterocyclic compound, azo compound, antimicrobial agents, ornidazole issn 2413-0516 introduction a heterocyclic compound (aromatic and non-aromatic) is a cyclic structure with at least one heteroatom in the ring. heterocyclic compounds have five and six-membered rings and have attracted the attention of the pharmaceutical community because of their therapeutic values.1,2 heterocycles bearing nitrogen, sulfur, oxygen and thiazole moieties constitute the core structure of a number of biologically interesting compounds. some of these compounds are tetrazoles, fused thiazoles, thiadiazoles, oxadiazoles, imidazole, and triazoles, which are structural subunits of several biologically active compounds.3 aromatic azo compounds have been extensively studied because of their broad-spectrum pharmaceutical applicability mainly because azo drugs act as prodrug agents as well as building blocks of various polymers and natural products.4 azo compounds are characterized by the presence of the azo moiety (–n=n–) in their structure conjugated with two distinct or identical, mono or polycyclic aromatic systems.5 these compounds are medically important because of their antibiotic, antifungal and anti-human immunodeficiency virus (hiv) properties. the presence of a heterocyclic ring in conjugation with azo pharmacophore enhances the bio-potency of azo compounds. the 1,3,4-thiadiazole derivative is a very important class of nitrogen-containing aromatic heterocyclic compounds which has gained increasing attention because of its diverse antibacterial, anti-inflammatory, and antifungal properties.6 the use of azo compounds as drugs dates back to the first commercially available antibiotic prontosil, a sulfonamide prodrug, which was identified in the 1930 and was later used to identify the first azoreductase.7 azoreductases are a group of nadph dependent flavoenzymes identified in a range of bacterial species found in the gut including escherichia coli and pseudomonas aeruginosa. members of this family are also found in humans where they are known as nadph quinone oxidoreductases.8 by the end of february 2020 and the beginning of march 2020, sars-cov-2 (coronavirus) infection spread worldwide and the world health organization (who) declared it a pandemic on march 11th, 2020. as a result, designing and formulating new therapeutic agents became highly important. imidazole rings are ubiquitous in natural products and possess unique structural features with a wide spectrum of biological activities. therefore, the incorporation of imidazole ring and t. ana et al.31 t. tehreem et al.30 http://orcid.org/0000-0003-2507-6725 mailto:a.aljubori@alameed.edu.iq 52 j contemp med sci | vol. 8, no. 1, january-february 2022: 51–58 synthesis, characterization, and antimicrobial evaluation for new azo-linked derivative of heterocyclic compounds original a.a. shafeeq et al. diazo (n=n) moiety in a single molecule could result in the formulation of compounds with interesting properties and diverse biological activity. the main protease (mpro) or (3clpro) of the coronavirus is conserved among the coronaviruses and it is mainly responsible for the viral replication. novel azo imidazole derivatives have been made for the computational study of the inhibitory potential of these novel azo imidazole derivatives against the main protease mpro: (6lu7) of sars-cov-2.9 the threat posed by multiple drug resistant pathogens such as methicillin-resistant staphylococcus aureus (mrsa) indicates the need to develop antibiotics with novel mechanisms of action. analogues of nitroaromatic compound (pt638) shown in figure 1 were synthesized to explore the importance of the sulfonamide linker and the impact of altering the functionalization of the phenyl ring. these structure–activity relationship studies revealed that the nitro substituent was essential for antimicrobial activity.10,11 this raises the possibility that bio-reduction of newly developed anti-tuberculosis (tb) drugs (delamanid and (r) pa-824) may be mediated by nitroreductase reduction.12 the antimicrobial mechanism of sulfonamides involves competitive inhibition of folic acid synthesis which prevents the growth and reproduction of micro-organisms.13 yet, few studies have investigated the activity of trimethoprim and sulfamethoxazole against mycobacterium tb (mtb). one study suggests that the folate biosynthesis pathway is a good mtb target for drug development.14 sulfonamides are the potent agents that monitor growth and proliferation of mtb by inhibiting the activity of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) enzymes, this could explain the mechanism of sulfonamides effect on mtb.14 some studies investigated 3-nitrotriazole or 3-nitroimiazole based amides and sulfonamides as potential antitubercular agents.15 considering the importance of sulfonamide-based aromatic heterocycles, new research has been designed to synthesize novel azaheterocyclic sulfonamide schiff bases synthesized as carbonic anhydrase inhibitors.16 recently, carbonic anhydrases (cas) have emerged as potential drug targets in mycobacteria. several in vitro studies have shown that all the mtb-cas could be efficiently inhibited by sulfonamides/sulfamates. to increase the inhibitory properties, new molecules were synthesized by diazotization of aminosulfonamide and by coupling with phenols or amines and prontosil was found to be the best inhibitor of this enzyme.17 ornidazole has a heterocyclic structure consisting of a nitroimidazole nucleus with a 2-hydroxy-3-chloro-propyl group in position 1 and a methyl group in position 2. it is used in the treatment of susceptible protozoal infections and also in anaerobic bacterial infections.18 it is a third-generation 5-nitroimidazole antibiotic, and compared with other antibiotics, it has a longer elimination half-life and greater capacity to penetrate into lipid tissues, which makes it a good choice in dental and gastrointestinal surgeries.19 we aimed to synthesize and characterize new azo-linked derivatives as antimicrobial agents and study their biological activity using the broth microdilution spectrometric method for selected bacteria and fungi and the bactec mgit 960 system for mycobacterium tuberculosis bacilli (mtb). materials and methods materials all reagents and solvents were purchased from commercial sources and utilized without any further purification. for analytical thin layer chromatography (tlc), merck silica gel plates (germany) covered in aluminum sheets (silica gel 60 f254) were utilized. the melting points were calculated without correction using a barnstead/electrothermal device (usa). agilan varian 400 mhz spectrometer (usa) was used to record the 1 h-nmr spectra and shimadzu ft-ir spectrometer (japan) was used to record the ft-ir spectrum. chemical synthesis nitroimidazole derivatives were synthesized following procedures listed below and the steps are summarized in scheme 1.20,21 synthesis of compound (a1): 1-chloro-3-(4-((5-mercapto1,3,4-thiadiazol-2-yl) diazenyl)-2-methyl-5-nitro-1h-imidazol-1-yl) propanol the diazotization of 5-amino-1,3,4-thiadiazole-2-thiol was accomplished by dissolving (0.399 g, 3 mmol) in 2 ml ethanol, to which 3 ml concentrated hydrochloric acid and 3 ml water were added in a suitable beaker. the resulting solution was agitated for 30 mins and cooled in an ice bath. during the reaction, the temperature of the reactants was kept below 5°c. a solution of sodium nitrite (0.28 g, 4 mmol) in 2 ml water was chilled with crushed ice before being dropped into a solution of amino-1,3,4-thiadiazole-2-thiol, which was held in an ice bath with constant stirring and kept below 10°c. in a 2m sodium hydroxide solution, the resulting product was combined with (0.658 g, 3 mmol) ornidazole. in an ice bath, the mixture was agitated for 6 hours at a temperature of 0–5°c. the precipitate was then filtered, washed thoroughly with fig. 1 enzymatic activation of nitroaromatic compound (pt638). . oh n n n+o o cl r nh2 nano2 hcl 0-5 c0 r n+ ncl r n n ohn n n+ o o ch3 cl diazonium salt 10% naoh compound a1, a2 where:s h n n o o o h3c r in compound a1 = r in compound a 2 = nn shs scheme 1. the general synthetic pathway of compound (a1, a2). 53j contemp med sci | vol. 8, no. 1, january-february 2022: 51–58 a.a. shafeeq et al. original synthesis, characterization, and antimicrobial evaluation for new azo-linked derivative of heterocyclic compounds water, recrystallized from absolute ethanol, dried, and collected as compound a1. synthesis of compound (a2): 4-((1-(3-chloro-2 hydroxypropyl)-2-methyl-5-nitro-1h-imidazol-4-yl)diazenyl)-n-(5-methylisoxazol-3-yl)benzenesulfonamide the diazotization of sulfamethoxazole was done by dissolving (0.759 g, 3 mmol) in 1 ml ethanol, to which 3 ml of concentrated hcl and 3 ml of water were added in a suitable beaker, and the resulting solution was stirred for 20 minute and cooled by immersing it in a bath of crushed ice. the same procedure was done as in the synthesis of compound (a1). the mixture was stirred for 15 hours at 0–5°c in an ice bath. then, this mixture was filtered, washed well with water, recrystallized from absolute ethanol, dried, and collected as compound a2. methods of characterization and identification thin layer chromatography (tlc) the tlc was done on silica gel (f-254 type 60) pre-coated aluminum sheets (merk, germany) to check the purity of the product and track the progress of the reaction. by interacting with iodine vapor or exposing it to uv light, the final compound was identified. acetone:methanol (5:5) was used as the solvent system to elute the chromatogram. melting point the melting points of the synthesized compounds were determined using an electro-thermal melting point apparatus (barnstead/electrothermal, usa), and the uncorrected results were recorded. infrared spectra infrared spectra were determined and recorded using shimadzu ftir-8400 at the chemistry department, college of science, mustansiriyah university. proton nuclear magnetic resonance (h1-nmr) the h1nmr spectra for each synthesized compound were determined at the college of science, university of tehran, iran. antimicrobial activity determination of inhibition percent the minimum inhibitor concentration for the finally synthesized compound was compared to levofloxacin, which was used as a reference drug against five bacteria strains: gram positive (methicillin resistant staphylococcus aureus mrsa, enterococcus faecalis) and gram-negative bacteria (salmonella, escherichia coli, pseudomonas aeruginosa). moreover, nystatin was used against candida albicans for the antifungal activity test.22 for each one of tested compounds, a broth microdilution method was used on a 96-well microtiter plate; this approach was done according to the (clsi.2018) references.23 determination of inhibition percent 24,25 a spectrophotometer set to a wavelength of 600–630 nm was used to detect the turbidity or growth (also known as optical density [od]) of the tested substance in the well. the absorbance (a) of each specified well was recorded which is proportional to the microbe’s growth (turbidity) and the percentage of inhibition was calculated using the following formula: % of inhibition = 1 – (od test/od control) × 100 where, od test: microbes in culture media and tested compound; od control: od of control (microbes in culture media). determination of antitubercular susceptibility testing (ast)26 the synthesized compound’s anti-tb activity in vitro was investigated at the national tb program’s reference center in iraq’s ministry of health. the mgit960 method was used to test the sensitivity of mtb to the produced compound in liquid media using the mycobacteria growth indicator tube (mgit). ast was interpreted in two different categories; the in vitro results are categorized by either “critical concentration” or “minimal inhibitory concentration”, however, the first is used internationally. the critical concentration of an anti-tb medication is the lowest concentration that prevents 99% of growth. the critical concentration for the wild strain of mtb is the 90% inhibition produced by pyrazinamide. as a result, in this study, the standard solution was prepared accordingly.26 the isolate was prepared using both liquid and solid medium. the bactec mgit 960 liquid medium (7 ml modified middlebrook 7h9 broth) and lowenstein-jensen (lj) solid medium were procured from the national tb program’s reference facility in baghdad, iraq. because both were nitroimidazole derivatives, ornidazole (10 mg/2.5 ml dmso) was manufactured in the same way as the critical concentration (84 g/ml) of the second-line anti-tb delamanid medication.27 the doses of synthesized compounds were 16.564 mg and 22.014 mg for compounds a1 and a2, respectively, and were calculated according to the following equation.28 molecular weight of ornidazole (219.6 g/mol) dose of tested compound = dose of ornidazole * molecular weight of compound molecular weight of ornidazole (219.6 g/mol) dose of tested compound = dose of ornidazole * molecular weight of compound results and discussion results of chemical syntheses the compound (m) was successfully synthesized, and table 1 summarizes its physical appearance, molecular weight (m.wt.), percentage yield, melting point, and retardation factor (rf) value. results of characterization and identification of the synthesized compounds ft-ir characterization: with respect to the final compounds (a1, a2), fourier transform infrared (ft-ir) spectra revealed distinct bands of absorption that allowed recognition. not only were ir data useful in identifying the final chemical, they were also useful in following up on reactions based on the emergence or absence of specific group frequencies. ft-ir characterization of compound (m): 3200– 3600cm–1 (o-h stretching vibration broad band of alcohol), 3066.92 cm–1 (c-h stretching vibration of heterocyclic ring), 54 j contemp med sci | vol. 8, no. 1, january-february 2022: 51–58 synthesis, characterization, and antimicrobial evaluation for new azo-linked derivative of heterocyclic compounds original a.a. shafeeq et al. 2630.99 cm–1 (s-h stretching vibration of thiol), 1614.47 cm–1 (c=n stretching vibration of heterocyclic ring), 1500.67 cm–1 (n-o stretching vibration of no2), 1388.79 cm –1 (n=n stretching vibration), 1134.18, 1033.88 cm–1 ( c-o stretching vibration of alcohol), 750.33 cm–1 (c-cl stretching vibration). ft-ir characterization of compound (a2): 3300 cm–1 (n-h stretching vibration of sulfonamide), 3200–3600 cm–1 (o-h stretching vibration broad band of alcohol), 1645.33 cm–1 (c=n stretching vibration of heterocyclic ring), 1467.88 cm–1 (n-o stretching vibration of no2), 1427.37 cm –1 (n=n stretching vibration), 1365.00 cm–1 (s=o stretching vibration of sulfonamide), 1166.97, 1138.04 cm–1 (c-o stretching vibration of alcohol), 742.62 cm–1 (c-cl stretching vibration). h1-nmr characterization: h1nmr spectroscopy can be used to identify hydrogen atoms (protons) in organic molecules. these spectra were created for the synthesized compounds (a1, a2) and listed in tables 2 and 3, showing a distinct signal corresponding to their structures. antimicrobial activity result determining percent of growth inhibition:24,29 the broth dilution method was used in this work to determine the percent of growth inhibition for gram-positive bacteria (enterococcus faecalis, methicillin resistance staphylococcus aureus mrsa), gram-negative bacteria (escherichia coli, pseudomonas aeruginosa, salmonella typhi), and fungi (candida albicans) except for the negative control (dmso and bacterial suspension). this was done by filling wells with serial dilutions of the tested compounds (a1, a2) and a known amount of bacterial suspension. after a 24-hour incubation period at 37°c, the od was determined depending on the absorbance in a spectrophotometer at 620 nm and the resulting value showed a negative inhibition value (growth promotion); this was recorded as stimulation using the following formula: percentage of negative inhibition = (od test/od control) × 100 while the growth inhibition for the test was determined using the formula % of inhibition = 1 – (od test/od control) × 100 the effects on % of inhibition of the selected bacteria and fungi for the tested synthesized compound (a1, a2) was calculated according to the mentioned formula (figure 2). the formula (a = 2– log %) was used to find the relationship and regression between the percent of inhibition logarithm and absorbance of each tested compound (figures 3 and 4) for the selected six microbes which gave a linear regression. we found acceptable values since the absorbance decreased as the % of inhibition increased indicating a decrease in the number of microbes absorbing light so the amount of the cell reduction (% inhibition) was calculated (tables 4 and 5). the effects on the microbial colony count for table 2. h1nmr data and their interpretations of compound a1 compound chemical shift (ppm) group no. of protons interpretations a1 m 4* 2.46 imidazole –ch 3 3 singlet 3.43 –ch of propanol 1 multipalate 7.05 -nh of thiadiazole ring 1 singlet (tautomerism of thiol to thione) 4.01 –ch 2 cl 2 doublet 4.21 imidazole –ch 2 2 doublet 13.16 – sh 1 singlet table 3. h1nmr data and their interpretations of compound a2 compound chemical shift (ppm) group no. of protons interpretations a2 2.76 oxazole-ch 3 3 singlet 2.84 imidazole –ch 3 3 singlet 3.21 –ch of propanol 1 multipalate 3.36 –oh of propanol 1 doublet 4.06 –ch 2 cl 2 doublet 4.23 imidazole –ch 2 2 doublet 4.30 –nh 1 singlet 6.11 oxazole-ch 1 singlet 7.62 7.77 aromatic-h 4 multipalate signals result from overlapping of nonequivalent aromatic protons table 1. the physical appearance, molecular weight, percentage of yield, melting points, and retardation factor (r f ) values of final compounds r f valuesobserved melting point (˚c)% yieldmolecular weight (g/mol)physical appearancecompound no. 0.75218-220 18.40363.80yellow powdera1 0.78118313.66483.5orange powdera2 55j contemp med sci | vol. 8, no. 1, january-february 2022: 51–58 a.a. shafeeq et al. original synthesis, characterization, and antimicrobial evaluation for new azo-linked derivative of heterocyclic compounds fig. 2 effect of tested synthesized compounds (a1, a2) on growth of selected bacteria and fungi compared with levofloxacin and nystatin. table 4. the antimicrobial effect of compound (a1) on selected bacteria and fungi microbes absorbance = (2–log % g.l.) log % of growth inhibition (g.l.) s. typhi 0.029 1.97 e. coli 0.015 1.984 p. aeruginosa 0.571 1.428 e. faecalis 0.0452 1.954 s. aureus mrsa 0.0329 1.967 c. albicans 0.134 1.8656 fig. 3 the regression (r) and linear plot of absorbance (light absorbed by microbes) versus log % of growth inhibition of compound (a1) for selected bacteria and fungi. table 5. the antimicrobial effect of compound a2 on selected bacteria and fungi microbes absorbance = (2–log % g.l.) log % of growth inhibition (g.l.) s. typhi 0.0328 1.9671 e. coli 0.158 1.841 p. aeruginosa 0.551 1.448 e. faecalis 0.0877 1.9122 s. aureus mrsa 0.0467 1.953 c. albicans 0.14 1.859 each one of tested synthesized compounds were recorded and shown in (figures 5 and 6). anti-tb drug susceptibility testing:26 the anti-tb activity of the final synthesized compounds was determined by comparing growth in the agent-containing tube fig. 4 the regression (r) and linear plot of absorbance (light absorbed by microbes) versus log % of growth inhibition of compound (a2) for selected bacteria and fungi. (compound a1, a2) to growth in the mgit tube without the agents (the control tube). a fluorescent chemical is inserted in a silicone round-bottom tube in the mgit tube. the fluorescent chemical becomes sensitive in the presence of oxygen dissolved in the broth. during bacterial growth in the mgit tube, free oxygen is consumed and replenished by co2. this decrease in free o2 causes the sensor within this tube to glow. as a result, o2 depletion is exactly proportional to fluorescence intensity, which is detected automatically by the bactec mgit 960 equipment (table 6). if the test agent is successful against mycobacteria, it will prevent the bacteria from developing, resulting in reduced fluorescence, but growth in the control tube will be unaffected, resulting in increased fluorescence. ast results are indicated as resistant when the control’s growth unit (gu) value is 400 or higher and the gu value of the tube containing the agent being tested is 100 or higher. table 6. ast report for inh, ornidazole (reference) and the synthesized compounds (a1, a2) on mycobacterium growth patient no. g. unit inh ornidazole a1 a2 ast 1 400 r* r r r 2 400 r r r r 3 400 r r r r 4 400 r r r r 5 x 200* x 200* x 200* x 200* x 200* g.c. 400 c c c c r*, resistance of mycobacterium; c*, control of growth control tube (g.c.); g. unit, growth unit; x 200*, error occurs if the inoculum contains only a low number of viable organisms.[26] 56 j contemp med sci | vol. 8, no. 1, january-february 2022: 51–58 synthesis, characterization, and antimicrobial evaluation for new azo-linked derivative of heterocyclic compounds original a.a. shafeeq et al. fig. 5 the effects on the microbial colony count for compound a1. and/or through incorporation of different biologically active heterocycle rings such as thiadiazole. they have important applications in many pharmaceutical, biological and analytical fields, affecting the orientation of the tested compounds in binding to the active site of the target enzymes, leading to different affinities and biological activities. also, the synthesized compound did not have any anti-tb effect. although there was an error in reading the ast report in patient conclusion the results of this study indicate that the diazotization and coupling reaction between nitroimidazole (ornidazole) and sulfa drugs (sulfamethoxazole) in addition to 5-amino 1,3,4-thiadiazole -5-thiol will give superior antimicrobial activity compared with levofloxacin and nystatin, when they are linked through an azo bond which has biological activity 57j contemp med sci | vol. 8, no. 1, january-february 2022: 51–58 a.a. shafeeq et al. original synthesis, characterization, and antimicrobial evaluation for new azo-linked derivative of heterocyclic compounds fig. 6 the effects on the microbial colony count for compound a2. deazaflavin-dependent nitroreductase (ddn) which is the only identified enzyme within mtb that activates nitroimidazole prodrugs such as pretomanid and delamanid. conflicts of interest the authors confirm that there is no conflict of interest regarding the publication of this research paper. number 5, still all of them were clinically classified as resistant. the resistance to nitroimidazole (ornidazole) may be attributed to the effect of various strains of mutation in this bacteria, as a result of one or more of the mechanisms of nitroimidazole, or as alterations in the drug target, alterations in the permeation of the drug to reach its target by changing the affinity of the drug to its receptor and also the effect on enzyme 58 j contemp med sci | vol. 8, no. 1, january-february 2022: 51–58 synthesis, characterization, and antimicrobial evaluation for new azo-linked derivative of heterocyclic compounds original a.a. shafeeq et al. acknowledgment the authors would like to acknowledge the college of medicine al-nahrain university, college of science mustansiriyah university, and the reference center of the national tb program in baghdad, for their valuable support, and great assistance to accomplish this work.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1153 references 1. yadav p., devprakash a., senthilkumar g, benzothiazole. different methods of synthesis and diverse biological activities. ijpsdr, 2011;3(1): 7 pages. 2. shinde d., aaglawe m., dhule s., bahekar s., and wakte ps. synthesis & antibacterial activity of some oxazolone derivaties. j. kor chem sty, 2003;47:133–136. 3. weerasek s., and vajragupta o.: antifungal. cytotoxic activities and docking studies of 2,5-dimercapto-1,3,4-thiadiazole derivatives. afr. j. ph. pharm. 2011;5(4):477–485. 4. ashok r. p., geetika p., gurupada m., shiv p. p., sumantra b., anjaneyulu p., and subhash b. direct oxidative azo coupling of anilines using a self-assembled flower-like cuco2o4 material as a catalyst under aerobic conditions . acs omega., 2020;5:30416−30424. 5. ikbal a., tanmay m., a. k. m. m. i., jasper r. p., pietro p., sabu th. and jayanta k. b. structure and elastic properties of an unsymmetrical bi-heterocyclic azo compound in the langmuir monolayer and langmuir−blodgett film. acs omega., 2020;5:21538−21549. 6. ravi bn, j k, m mn, et al. synthesis, characterization and pharmacological evaluation of 2-aminothiazole incorporated azo dyes. j mol struct 2020;1204:127493. 7. ali ryan: review article azoreductases in drug metabolism. british journal of pharmacology. 2017;174:2161–2173. 8. ali r., elise k., nicola l., edward l. and edith s.: activation of nitrofurazone by azoreductases: multiple activities in one enzyme. scientific reports, 2011;1, 63:1–5. 9. abhijit ch., sailesh ch., pranesh r., dipu k. m., biswajit s., dhiraj b. synthesis, characterization and computational study on potential inhibitory action of novel azo imidazole derivatives against covid-19 main protease (mpro: 6lu7). journal of molecular structure, 2021, 129230(1225):1–14. 10. susan w., adam j. r., suzanne n., stephen p., and alan h. f. activation of bicyclic nitro-drugs by a novel nitroreductase (ntr2) in leishmania. plos pathogens, 2016 | doi:10.1371/journal.ppat.1005971. 11. evelyn b., mariano m., rafael c. and javier j. e. cyp1a1 and cnr nitroreductase bioactivated niclosamide in vitro. mutagenesis, 2013;28(6):645–651. 12. dibyarupa p., sulagna b., jike c., aaron s., sudip k. and john s. giardia, entamoeba, and trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases). antimicrob. agents chemother., 2009;53(2):458–464. 13. ana t., vesna n., ljubiša n., ivan s. antimicrobial sulfonamide drugs. advanced technologies. 2017;6(1):58–71. 14. catherine vilchèze and william r. jacobs: the combination of sulfamethoxazole, trimethoprim, and isoniazid or rifampin is bactericidal and prevents the emergence of drug resistance in mycobacterium tuberculosis j. acc, 2012;56(10):5142–5148. 15. maria v. p., william d. b., howard s. r., alexander a. and and diane k. s. et al. nitrotriazoleand imidazole-based amides and sulfonamides as antitubercular agents. antimicrob agents chemother. 2014:58(11): 6828–6836. 16. mujahid a., hummera r., shazia sh., sadia r. and muhammad h. h. et al., sulfonamide-based azaheterocyclic schiff base derivatives as potential carbonic anhydrase inhibitors: synthesis, cytotoxicity,and enzyme inhibitory kinetics. biomed research international. 2020, article id 8104107, 9 pages. 17. ashok a., jean-yves w., fabrizio c., claudiu t., and seppo p., et al. carbonic anhydrase inhibitors as novel drugs against mycobacterial β-carbonic anhydrases: an update on in vitro and in vivo studies. molecules. 2018;23(11):2911. 18. merdjan h., baumelou a., diquet b., chick o.and singles’ s. pharmacokinetics of ornidazole in patients with renal insufficiency; influence of haemodialysis and peritoneal dialysis. br. j. clin pharmac.,1985, 19, pp 211–217. 19. yanchang z., lin z., yongkui y. and peizhe s. fenton-like oxidation of antibiotic ornidazole using biochar-supported nanoscale zero-valent iron as heterogeneous hydrogen peroxide activator. int. j. environ. res., 2020;17: 1324. 1–7. 20. rajeev k. sh., satheesh m., and sharma a. k., “synthesis and characterization of ornidazole, 5-asa azo adduct for colon targeting.” j. chem. pharm. res., 2014;6 (6):75–78,. 21. sevil ş., necla k. et.al., “synthesis and anticancer and antimicrobial evaluation of novel ether-linked derivatives of ornidazole.” turk j pharm sci, 2020;17(1):81–93. 22. sakineh o., vida kh., ali k., motaleb gh. and farideh a. synthesis, characterization, spectroscopy and biological activity of 4-((3-formyl-4hydroxyphenyl)azo)-1-alkylpyridinium salts. j. chem. sci., 2018;130:114,9 pages. 23. clsi. 2018. performance standards for antimicrobial susceptibility testing. 28th ed. wayne, pa: clinical and laboratory standards institute. 24. c.o. akujobi c.o. and njoku h.o.” bioassay for the determination of microbial sensitivity to nigerian honey .global j. pharmacol., 2010; 4 (1):36–40. 25. akinduti pa., motayo b., idowu om., isibor p., olasehinde g., obafemi yd., ugboko h., oyewale jo1., oluwadun., adeyemi g. suitability of spectrophotometric assay for determination of honey microbial inhibition. journal of physics: conf. series 1299, 2019;012131:8 pages. 26. world health organization 2018. technical manual for drug susceptibility testing of medicines used in the treatment of tuberculosis. isbn 978-92-4151484–2. 27. mamoru f., masanori k., norimitsu h., yongge l., makoto m.: mechanisms of resistance to delamanid, a drug for mycobacterium tuberculosis. tuberculosis, 2018;108:186–194. 28. noor h. aldabagh and mohammed h.m. “design, synthesis and antituberculosis evaluation of ciprofloxacin-sulfonamide conjugate using hybridization approach.” ph.d. thesis, collage of pharmacy, baghdad university; pp. 72, 2015. 29. akinduti pa., motayo b., idowu om., isibor p., olasehinde g., obafemi yd., ugboko h., oyewale jo., oluwadun, adeyemi g. suitability of spectrophotometric assay for determination of honey microbial inhibition. journal of physics: conf. series 1299, 2019;012131:8 pages. 30. tehreem t., mirza i. sh., rukhsana t., muhammad r., muhammad a. and mariusz m, et al. diaryl azo derivatives as anti-diabetic and antimicrobial agents: synthesis, in vitro, kinetic and docking studies. journal of enzyme inhibition and medicinal chemistry. 2021;36(1):1509–1520. 31. ana t., vesna n., ljubiša n., ivan s. antimicrobial sulfonamide drugs. advanced technologies. 2017;6(1):58–71. 119j contemp med sci | vol. 4, no. 3, summer 2018: 119–125 review introduction in the domain of research, deceit, fraud, forgery, piracy, plagiarism, bias, fabrication, falsification, negligence, fudging, faking, forging, deceit, misconduct, cooking and trimming, epistemological heteronomy or whatever name one may attribute to such inappropriate acts, are not uncommon and the same holds true in health research as well. although the commonly used terms are fraud and misconduct and these are often used interchangeably, the term fraud would be defined in legal terms as a crime of knowingly cheating or deceiving another person. “fabrication” “falsification”, and “plagiarism” are the common classification of fraud but these terms have to be distinguished from “error” which may be unavoidable in spite of sincere effort on the part of the researcher. does all fraudulent clinical research, when applied in clinical practice, always endanger the safety and well-being of patients? if it did not, should it still be considered as fraud? the question is not whether it had any adverse impact on patients but whether an act of fraud was committed at any stage of proposing, performing, reviewing or reporting of research. committing a fraud is a crime just as attempting to rob a bank is a crime, whether or not the money in the bank was actually stolen or not. clinical practitioners rely on research evidence to update their practice. if apparent evidence is from a fraudulent research, the patient may gain no benefit from that treatment at best, and at worst, suffer ill effects and even death or disability. if a fraudulent research is proven to have caused the death or serious injury to a participant, it would constitute a criminal offence in the justice system of the country than just a civil offence and the perpetrator would be held liable accordingly. bias, however is distinct from fraud in that bias is inherent in clinical research and every researcher is obliged to identify potential bias and avoid them or at least reduce them as much as possible. if a researcher does not take the effort to reduce avoidable bias or does not disclose the potential bias in a research, it may tantamount to misconduct but not fraud. but if a misleading or incorrect declaration is made, for example, stating that study participants were randomly selected when actually it was not, that would amount to falsification which should be considered as a fraudulent act. good clinical research practice (gcp) provides a “framework for the scientific and ethical integrity of research on human participants for generating valid observations and documentations.”1 it serves the interest of both those “actively involved in conducting clinical research, and the rights, safety and well-being of participants, which are compliant with the principles stated in the declaration of helsinki, and other international ethical guidelines.”1 for a developing country where innovative clinical research is still in its infancy, it would be reliant on international standards and guidelines. hence, it is important that a clear set of locally specific guideline should be laid out which is clear, concise, and easily accessible for review by researchers and yet compliant on all matters of ethics and etiquette including maintaining the autonomy and sanctity of the individual study participant. the objective of this paper is to present an overview of fraud and misconduct as they have happened in clinical research internationally, to draw attention to that malady, which should be appreciated by all researchers and to point to the step that we have taken in the ministry of health in bringing out our guideline for the responsible conduct of clinical studies and trials. “two cardinal rules of biomedical research are that scientists pursue absolute truthfulness and objectivity and that they report only honest data.”2 but these ethical rules are often forfeited for various reasons. monetary consideration including job opportunities, desire for personal fame, professional, academic, and scientific ambition are the common reasons for committing fraud in research.3 in the scientific community, the concept of “publish or perish” has only made the situation worse,4 and this is equally evident in clinical research. “are such events just the tip of the iceberg?” “are they on the increase?” “why do such errant practices happen?” “and what role should the biomedical establishment, including the editors of medical journals and of information retrieval systems, have in this”?2 “what are the consequences of misconduct in research?” “how can research misconduct be curtailed?” these are the questions that not only the scientific community but also even the society wants to know. fraud and misconduct in clinical research: a step to improve ethical practice in research adhra al-mawali,a john idikula,a avinash daniel pintoa strict compliance to ethical principles in conducting clinical research is mandatory to ensure safety and well-being of study participants and legal protection of researchers. many instances of fraud and misconduct are reported in world literature that has harmed clients and also eroded the trust that society had on bioscience researchers. in the light of a review of international experience on ethical practice in clinical research, we prepared the ‘guideline for responsible conduct of clinical studies and trials’ which covers important aspects of clinical research including protection of the rights, safety, well-being, and autonomy of study participants; role and responsibilities of the researchers, supervisors, and sponsors including pharmaceutical agencies; documentation required; reporting of research findings and related issues. clinical researchers must acquire and apply knowledge, attitude, and skill in conducting ethically sound high-quality clinical research as prescribed in this guideline. this review and our guideline will hopefully be useful resources to all researchers worldwide. keywords clinical research, bioethics, guideline, bias, scientific misconduct acentre of studies and research, ministry of health, oman correspondence to adhra al-mawali (email: adhra.almawali@gmail.com). issn 2413-0516 (submitted: 07 august 2018 – revised version received: 16 august 2018 – accepted: 22 august 2018 – published online: 26 september 2018) 120 j contemp med sci | vol. 4, no. 3, summer 2018: 119–125 fraud and misconduct in clinical research review adhra al-mawali et al. there was a time when most societies respected health professionals as inerrant, dependable, and honest but that honorable reputation is withering because of bad publicity generated by news of unbecoming practices, although by a few “bad apples” in the professional basket. international experience related to erosion of ethical and moral standards in research in order to appreciate and understand the nature and magnitude of the problem, the questions posed above can be further explored. misconduct in research is just the tip of the iceberg in a north american study on doctoral and post-doctoral students, 36% were aware of misconduct while a surprising 15% would consider committing fraud to obtain a research grant or get a paper published.3 a meta-analysis published in 2009 revealed that at least 2% of scientists admitted to having committed some form of scientific fraud at least once5 but as many as 72% were aware of their colleagues of having committed some questionable malpractice. in a nonsystematic survey of 80 professors of medical institutions in the uk, over 50% of the responders were aware of some instance of medical misconduct.6 in another study, as many as 18% even admitted that they may do possible misconduct in the future7—a worrying finding indeed. from all the available evidence in such published studies, one has to conclude that fraud and misconduct in research are not a relatively minor and isolated practice but is quite widespread.3 what is even more surprising is that even the intension to commit fraud to achieve personal gain is rampant. fraud and misconduct in research are on the increase worldwide accurately estimating the number of cases of fraud can be difficult but once investigation of a case starts, it could have multiple ramifications as more such instances of malpractice may get uncovered as seen in the investigation of dr. darsee, at harvard medical school in 1981.2 between the years 2000 and 2010, 742 english language research papers were retracted from the pubmed database and of these, nearly 27% were retracted for fraud.8 the number of papers retracted per year have increased sharply and so has the retractions of published papers for fraud.8 this is not only due to improved detection rate by using sophisticated software programs and physical monitoring but an actual increase in the absolute number.5 how much and why would people who are generally held in high esteem by the society, stoop so low just to have a few more publications or add some glory to their name? when will it stop or will it? the answer we hope is not just blowing in the wind! role of the biomedical establishment on fraud in research many north american and european countries have governmental agencies that are empowered to regulate, investigate, moderate, litigate, and convict persons committing scientific fraud, while most other countries rely on this role to be played by the universities, sponsors or professional institutions.9 sadly, many countries do not have legislations that are specific to manage fraud in clinical research.9 even though prescribed standards are not available, every institution and organization that facilitates research should have a research and ethics committee that not only prescribes the required standards of research but ensure compliance to it in all phases of the research.9 unfortunately, most of these boards or committees do not have the mandate to investigate and suitably reprimand offenders. although there are guidelines on authorship,10 they are not strictly followed or enforced. although most journals require all authors to acknowledge their contribution to the paper by submitting a signed document, their actual contribution or their role in the paper to justify the order in the listing of authors are often not ascertained by the publishers. it is the responsibility of journal editors and their referees to be constantly aware of the possibility of bias and fraud and to take all possible steps to prevent publication of papers suspected of these offences.11 when an incident of misconduct is discovered in a manuscript, editors have the responsibility to avoid publishing it.12 but there is a limit to how much the editor of a journal or the reviewers can pick up as fraud. in response to the increasing incidence of fraud in research and the consequent demand on the publishers to be more vigilant, a small group of journal editors in the uk joined forces to establish the committee on publication ethics (cope) in 1997.13 it’s membership is open to journal editors and others interested in publication ethics and now has a worldwide membership of over 10,000.13 cope provides advice on publication ethics and the management of research and publication misconduct.13 although cope provides a forum for members to discuss individual cases of misconduct, it does not get involved in the actual investigation of such cases but encourages editors to ensure that suspected cases are investigated by the local authorities.13 a consensus statement on research misconduct in the uk was also put out by bmj/ cope.14 adverse consequences of misconduct in research many patients may be put to risk in fraudulent studies. in a 10-year period from 2000 to 2010, 9189 patients are known to have been treated in the 180 primary studies that were eventually retracted and 70,501 were treated in 851 secondary studies, which cited one of the retracted paper.15 this clearly indicates the magnitude of the problem; including receiving no therapeutic benefit or even an undue risk the patients may be put to if they are subjected to the recommendation of such fraudulent studies. historically, several surgical, medical, or other therapeutic interventions that were promoted by well-meaning researchers did not stand the test of time because of improper or flawed research design and poorly reviewed publications. superficial temporal artery to middle cerebral artery (ec-ic) anastomosis for cerebral ischemic symptoms and stroke prevention which was popularized in the 1960s and 1970s is one such example.16,17 when vitamin e was commercially introduced into the market in the 1980s, drug companies began to promote it as the treatment for many chronic or nonspecific clinical conditions based on low-quality or biased studies 18. later, the therapeutic benefit for most of it was disproved.19-22 although the participants in these studies may not have been harmed by taking vitamin e, they would obviously have been denied the benefit of a better therapeutic intervention for their clinical condition for which they were enrolled in this study. adhra al-mawali et al. 121j contemp med sci | vol. 4, no. 3, summer 2018: 119–125 review fraud and misconduct in clinical research history is replete with many such examples: gastric cooling for peptic ulcer, mental transposition for intractable lower limb edema, tonsillectomy for children with recurrent tonsillitis, antibiotics for childhood diarrhea are just a few such examples, none of which have withstood the test of time or rigorous scientific scrutiny. there is equally a danger that beneficial interventions from the good quality research could be delayed or even gone unrecognized due to researcher bias or even publication bias curtailing research misconduct fraud in any form in any research is condemnable. it is important that individuals who are proven guilty should be suitably reprimanded which may require them to resign from the institution where the fraud took place, reimburse grants already issued and forfeit future research awards.2 should health professionals who commit fraud in research be allowed to practice their profession without review by licensing officials? although severity of punishment may serve as a deterrent to commit fraud, there is always a possibility that fraudsters will come up with more ingenious ways to avoid getting caught rather than improve their ways. plagiarism detecting software and other programs that can evaluate data using algorithms to suspect fraudulent data are helpful to suspect some frauds. “central statistical monitoring” using statistical programs are becoming more efficient especially for evaluating data in multicenter studies for fraud or bias.23 it is generally accepted that plagiarism and duplicate publication are among the most common research misconduct and the most pervasive in all areas of academic activities.12 whistle-blowing by colleagues involved in the same or similar research or in the same facility is a reliable and credible way to initiate an enquiry about the originality and truthfulness of a research. of course, the integrity, honesty and sincerity of the whistle-blower also matters in this regard. every research facility has to have a clear set of guidelines on research ethics and etiquette. they may use a standard international or national guideline, which can be suitably adapted to its loco-regional specifics. many countries have clearly defined national level regulations like the federal register in the usa that is updated regularly.24 list of individuals convicted of fraud and the action taken on them are even publicized which could act as a deterrent.25 the declaration of helsinki, which was drafted in response to the atrocities committed on victims in the name of medical research that came to light after the second world war and the belmont report are among the original guidelines of the 20th century. strict adherence to these principles must be ensured by the respective institutions and research supervisors. researchers should practice research ethics for their intrinsic goodness and also as an example to others and they should also have the courage to courteously report any suspected misconduct to the appropriate authority.26 whistle-blowers who raise any questions in good faith should not be penalized in any way. teaching research ethics to bioscience students in high schools and universities have not received the high priority it should have. increasing incidence of reported misconduct reinforces the need for research students to be exposed to and be trained in the professional ethical standards of their specific discipline.27 it should also include senior scientists and faculty members. continuing education in research ethics should be made compulsory and an integral part of the professional requisite of the practicing bio-scientist.28 it is reasonable to recommend that accreditation of biomedical institutions should include verification of strict adherence to bioethical practices including periodic training in ethics of its personnel. so also, biomedical researchers should have sufficient knowledge, attitude, and skills in applying concepts of bioethics in their professional practice to obtain professional licensing and periodic renewal of such license to conduct clinical research. in a publication titled “repairing research integrity” from the office of research integrity in the usa, titus et al. have suggested a list of strategies to improve research integrity.29 these are: adopt zero tolerance to suspected misconduct, protect and encourage whistle-blowers, have clearly defined fraud reporting process, training of mentors and supervisors to monitor research more effectively, improve auditing process of research, and encourage, promote and role model ethical behavior among researchers.29 health research and research ethics in developing countries although there have been rapid advances in health care services and health delivery in most developing countries, health research was not a high priority endeavor till about the 1970s or 1980s. before this, there was significant reliance on clinical research and innovation taking place in advanced countries. since then, in most developing countries the quantity and quality of research have increased although it is still not at par with many developed countries. attempts to catch up on innovation and research will undoubtedly create a competitive environment in which inadvertent compromise on research ethics and etiquette may take place. that makes it all the more imperative that young researchers become wellgrounded on concepts of research ethics the centre of studies & research (csr) in the directorate general of planning and studies of the ministry of health (moh) in oman has the responsibility of co coordinating, facilitating, monitoring, training, supervising, and encouraging research in the moh.30 the csr has developed a website and one of its main activities is to register and process all research proposals arising in the moh, which are then put up for approval by the research and ethical review & approval committee (rerac), the equivalent of the institutional review board (irb). the guidelines for responsible conduct of clinical studies and trials is available online at the website of csr (http://mohcsr.gov.om) so that researchers have an opportunity to go over it and incorporate necessary ethical requisites in their research. this guideline provides concepts of ethics and etiquette necessary for conducting clinical research.31 the ministry of health in oman has mandated that all research conducted in ministry of health facilities must strictly adhere to the guidelines set out here and no research proposal can be approved if they do not meet all applicable criteria specified in it. this is the first of such guidelines originating among the gulf co-operation countries (gcc) and has been considered as a model for preparing a standard guideline for gcc and probably even the middle east. this guideline is adapted from well-documented and published information on the subject from reputed agencies. among them are: (1)“the current revision of the declaration of helsinki”32 which is the accepted basis for clinical trial dx.doi.org/10.22317/jcms.09201812 122 j contemp med sci | vol. 4, no. 3, summer 2018: 119–125 fraud and misconduct in clinical research review adhra al-mawali et al. ethics, and must be fully followed and respected by all parties involved in the conduct of such trials, (2)“the belmont report: ethical principles and guidelines for the protection of human subjects of research by the national commission for the protection of human subjects of biomedical and behavioral research,”33 (3)“ethics of clinical research: an islamic perspective,”34 (4)publications related to ethics by the world health organization (who) like the handbook for gcp1, and (5)several other guidelines from countries known to have advanced and innovative research capacity are also used. guidelines for responsible conduct of clinical studies and trials we share here some of the ethical concepts brought out in our guideline in a sequential order through which the progression of a clinical research takes place. at every stage in conducting a research, there are issues pertaining to ethics and etiquette that becomes relevant. the researcher needs to be fully aware of these to appreciate and apply these concepts at every stage.31 the relevance of many of these concepts can be easily appreciated in the light of the examples and situations mentioned earlier. identifying a clinical problem becoming aware of a clinical problem or observing an unexpected or unusual event in clinical practice is what should initiate an enquiry to develop a greater understanding of the clinical event or situation.31 “whether such an observation was an adverse event or a favorable event, it should not be ignored but further investigated through appropriate enquiry or research.”31 not acting on it is like walking away without doing anything from the scene of a road traffic accident which would be morally, socially and ethically inappropriate. information retrieval and critical appraisal “prior to conducting clinical research, the researcher is obliged to carry out an exhaustive search to identify and critically evaluate all available and accessible information on the topic to be researched. if a critical review of available literature on the topic or situation clarifies the answer to the problem, there is no further need to address that issue unless there ars some lacunae in the understanding of the problem or some new information has since become available that warrants a review of the problem. it would be unethical to spend time, effort and resources to repeat research that has already been done for the sake of doing a research.”31 the researcher must review all available literature on the topic and not just the ones that he agrees with. a biased literature search has often led to biased conclusion, which is detrimental to the progress of science.31 it is also the responsibility of the researcher to critique the publications and to take up those that have a high quality as well to avoid inherent flaws in earlier studies. framing an answerable research question “in order to conduct research, a potentially answerable question has to be framed. answers that are sought should attempt to resolve the identified clinical problem or provide more knowledge and information on it.”31 formulating a research hypothesis every clinical research is intrinsically testing a hypothesis, whether such hypothesis is overtly stated or inherently implicit. every scientific hypothesis should be testable and preferably, even refutable but should not contain any moral, spiritual or other unprovable or emotively sensitive component.31 utilizing an appropriate study design “every researcher must employ the best possible research design to answer the identified research problem as there are many instances where inferior study design has provided the wrong answer.”31 “historically, there are many instances of useless or even harmful treatment that has been promoted and propagated by well-meaning clinicians on patients because published research that recommended such treatment was the result of poor study design.”31 hence, every researcher is ethically obliged to employ the most appropriate study design so that the findings of the research can provide the basis for evidence-based practice.31 informed consent obtaining a properly informed consent from each of the study participants (or participant’s legal representative) is of critical importance in all clinical studies. the researcher should comply with the local rules and guidelines in addition to ensuring that all aspects of good clinical research practice are adhered to.1 except under special circumstances, consent should be taken in writing and preferably in the presence of a neutral witness. all rights, duties, and privileges of the participants, as well as that of the researchers, should be clearly explained. consent should be freely given without fear or favor, including the right to withdraw from the study at any time without discrimination, forfeit of any rights or privileges, or bear any penalty. participant’s rights and privileges must be honored including the right to privacy, confidentiality, and anonymity. also, the participant’s willingness to consent must not forfeit the right to seeking any future legal recourse. observational studies it is a common malady in observational studies that a lot of data is collected from the participants. one should avoid collecting data that are not directly relevant to the study or as part of the hypothesis being tested. the common excuse is that some interesting associations may emerge. such “fishing expeditions” should be avoided.31 interventional studies all interventional studies are fraught with serious ethical issues. the researcher is directed to thoroughly consider all aspects of such experimental clinical studies to ensure that no ethical issues are overlooked.31 “the ethical concepts of beneficence, nonmaleficence, justice, and equity are most applicable in this situation and of equal importance ethically is the control group, especially on matters of the use of placebo.”31 in a surgical trial, can a placebo surgery ever be justified? even in the use of a placebo surgery, ethical analysis and guidelines are available.35 the system and process of reporting adverse events including management of participants who have any adverse event should be specified in the study protocol. also, clinical management such as management of any associated adhra al-mawali et al. 123j contemp med sci | vol. 4, no. 3, summer 2018: 119–125 review fraud and misconduct in clinical research conditions should be clearly specified. the indications for breaking an allocation code to clinically manage a study participant or to perform an interim analysis have to be specified in the protocol. these safety measures are critical to ensure safety of the participants in the trial and also to avoid potential legal implications. patient safety and well-being are of paramount concern and must not be sacrificed for any gains in the study. the researcher must be thorough with local rules and regulations and also international guidelines on ethics. selecting the study population the choice of the study population depends on the prevalence, nature, and manifestation of the clinical problem; and based on the nature of the study and the level of existing knowledge, the study population would vary.31 it is well documented historically that vulnerable population groups have been sometimes subjected to therapeutic trials in an unethical manner. such instances, although much less likely because oo strict control and monitoring, could still happen even in developed countries.36 there is incriminating evidence that, loopholes in the rules and regulations of some developing countries have been exploited to run therapeutic trials in populations of those countries while the same research would not have been possible in developed countries.37–39 such practice is certainly unethical and researchers should be aware of such situations and avoid them.40 proper sampling most clinical studies deal with a sample drawn from the population and hence it is important to make a proper choice of the study sample such that it truly represents the study population, failing which the outcome of the study cannot be generalized to the reference population.31 many published articles do not clearly mention the sampling process there are also many instances of wrongly reporting the sampling process (such as stating that the sample was random when in fact, it was not truly so) which are well known and such declarations amount to fraud in research and not just a breach of research ethics.31 incorrect sampling technique is a common malady and it leads to selection bias which affects the external validity of the study. maintaining and following up after intervention “inadequate follow-up after intervention, use of an unproven or inappropriate “proxy” outcome measure, incomplete follow-up, loss to follow-up, etc. are common problems that happen in longitudinal and interventional studies.”30 truthfully documenting all such problems and analyzing and interpreting them appropriately are important requirements in every study. methods such as “intention to treat analysis” “per protocol” or any other should be clearly indicated. eliminating inconvenient results and pretending they never existed is a fraud—whether or not such an action had an influence on the interpretation of the study. avoiding/reducing bias “bias is defined as a systematic deviation from truth and hence, the researcher must take every effort to avoid or at least reduce all potential bias in the study.”31 “bias may occur at various stages in the study process from selective review of literature all the way to interpretation of results.”30 selection bias, intervention bias, confounding bias, recall bias, measurement bias, etc. are among the most common in clinical studies. analyzing and managing data ensuring integrity of data from its collection all the way through to data maintenance, analysis, and interpretation are important in making the correct conclusion in any study while any form of fabrication and falsification of research results are serious forms of misconduct.31 so also are failing to report data that contradicts conclusions or excluding information without justification. all raw and analyzed data should be preserved for a period of at least 3 years after publication for any verification if required.31 it is also important to know that the ownership of the data lies with the organization or the institution in which the research was conducted and not with the research team. powerful statistical software that are available enable researchers to manage, analyze, and interpret research data with relative ease.31 however, appropriate use of statistical tests is important to make reasonable and justifiable conclusions. data “dredging,” data “tweaking,” data “trimming,” data “torturing” and such manipulations on the data are often resorted to by researchers to “prove a point.” such manipulation on the data, as long as it is not “falsified” or “fabricated,” may not amount to fraud, but still is an act of misconduct. conclusion and interpretation of a clinical trial “the conclusion section of the research presentation should confine to what has been achieved by the study and not extrapolate it to what could be achieved by making unproven recommendations or state speculative opinions that are not substantiated by the study results.”31 the stated conclusion in many research publications often exceeds the research objectives or the interpretation of the actual results obtained. such inflated claims may mislead the eventual user, the clinician, to overestimate the applicability of the study outcome in routine clinical practice.31 research output it is an ethical obligation for a researcher to publish research results in a timely manner through an appropriate forum to the scientific community. the researcher must disclose sufficient information to replicate such studies by other researchers. it is ethically and socially wrong to leak research findings to the public through any medium before it has been peer-reviewed and published in a scientific journal. all persons who contributed to the study should be duly credited as authors if there is scientific contribution or acknowledged if contribution is nonscientific or nonprofessional. the role and responsibility of each of the researchers and the order of listing the authors in the forthcoming publication should be decided at the planning stage of the study. our guideline titled guidelines for responsible conduct of clinical studies and trials31 gives more details on issues pertaining to informed consent of trial participants including special reference to vulnerable groups, privacy and confidentiality, plagiarism, misuse of privileged information, data management, authorship, publication issues like self citation, duplicate publication, early release of information, reporting of suspected misconduct, obligation to report, conflict of interest, qualification and practice privilege, medical care of trial participants, record keeping, safety reporting including notification of adverse events, premature termination or suspension of trial, role and responsibility of sponsor, international collaborative research and several such related issues, all of which can have ethical overtones.31 124 j contemp med sci | vol. 4, no. 3, summer 2018: 119–125 fraud and misconduct in clinical research review adhra al-mawali et al. all potential researchers are encouraged to read these guidelines and most of all, practice ethical principles in all facets of clinical research, for the good of all and the glory of clinical research.40 conclusion the scientific community all over the world has to accept the reality that various academic, professional, and personal pressures have led to compromises on applying ethical principles and hence led to increasing fraud and misconduct in clinical research. a review of the literature indicates that the situation is only getting worse. unfortunately, such incidents of professional or research misconduct get bad publicity in the social media and that further erodes the trust and respect that the clinical professionals and researchers expect from the society. hence, a good system of imparting knowledge in all aspects of ethics and etiquette in clinical research and ensuring its diligent practice by all researchers is important, particularly among young researchers in developing countries. this does not imply that senior researchers and faculty are above board. in fact, they should not only continue to be updated themselves but even be competent to teach, supervise, and guide their apprentices on bioethics as well as explicitly practice the same in their own practice of research. the guidelines for responsible conduct of clinical studies and trials is available online at the csr website and is designed to be a reference for researchers. it gives details on various aspects related to research ethics and etiquette that needs to be applied by every researcher at every stage in the planning and conduct of clinical research. it is hoped that this guideline will help researchers to conduct their research with full consideration to ethical issues and avoid any form of research misconduct. conflict of interest disclosure the authors declare no competing or conflict of interest. n references 1. world health organization. handbook for good clinical research practice: guidance for implementatio; 2005. 2. altman l, melcher l. fraud in science. br med j (clin res ed). 1983;286: 2003–2006. 3. jaffer u, cameron ae. deceit and fraud in medical research. int j surg. 2006;4:122–126. 4. al-adawi s, ali bh, al-zakwani i. research misconduct: the peril of publish or perish. oman med j. 2016;31:5–11. 5. fanelli d. how many scientists fabricate and falsify research? a systematic review and meta-analysis of survey data. pols one. 2009;4:e5738. 6. lock s. misconduct in medical research: does it exist in britain? br med j. 1988;297:1531–1535. 7. geggie d. a survey of newly appointed consultants’ attitudes towards research fraud. j med ethics. 2001;27:344–346. 8. steen rg. retractions in the scientific literature: is the incidence of research fraud increasing? j med ethics. 37:249–253. 9. gupta a. fraud and misconduct in clinical research: a concern. perspect clin res. 2013;4:144 –147. 10. international committee of medical journal editors. defining the role of authors and contributors. [cited 2017 november]; available from: http:// www.icmje.org/recommendations/browse/roles-and-responsibilities/ defining-the-role-of-authors-and-contributors.html. 11. pollock a, evans m. bias and fraud in medical research: a review. j r soc med. 1985;78:937–940. 12. al-lamki l. plagiarism and other types of publication misconduct: a case for teaching publication ethics in medical schools. sultan qaboos univ med j. 2009;9:1–4. 13. the committee on publication ethics (cope). (available at http:// publicationethics.org/about). [accessed on march 15, 2017]. 14. bmj. a consensus statement on research misconduct in the uk. br med j. 2012;344. 15. steen rg. retractions in the medical literature: how many patients are put at risk by flawed research? j med ethics. 2011;37:688–692. 16. weinstein pr, rodriguez y, baena r, chater nl. results of extracranialintracranial arterial bypass for intracranial internal carotid artery stenosis: review of 105 cases. neurosurgery. 1984;15:787–794. 17. yarsergill m. anastomosis between the superficial temporal artery and a branch of the middle cerebral artery. microsurgery applied to neurosurgery stuttgart, frg, georg thieme verlag. 1969. 18. machlin l. clinical uses of vitamin e. acta vitaminol enzymol. 1984;7: suppl:33–43. 19. ernster vl, goodson wh 3rd, hunt tk, petrakis nl, sickles ea, miike r. vitamin e and benign breast” disease”: a double-blind, randomized clinical trial. surgery. 1985;97:490–494. 20. huang h, he m, liu l, huang l. vitamin e does not decrease the incidence of chemotherapy-induced peripheral neuropathy: a meta-analysis. contemp oncol. 2016;20:237–241. 21. london rs, sundaram gs, murphy l, manimekalai s, reynolds m, goldstein pj. the effect of vitamin e on mammary dysplasia: a double-blind study. obstet gynecol. 1985;65:104–106. 22. mcneil jj, robman l, tikellis g, sinclair mi, mccarty ca, taylor hr. vitamin e supplementation and cataract: randomized controlled trial. ophthalmology. 2004;111:75–84. 23. george sl, buyse m. data fraud in clinical trials. clin investig (lond). 2015; 5:161–173. 24. electronic code of federal regulations. title 42: public health. part 93 public health service policies on research misconduct. 2017 [cited 2017 november]; available from: https://www.ecfr.gov/cgi-bin/text-idx?%20 sid=0b07ed68cf889962cae6c2b45d89150b&node=pt42.1.93&rgn=div5. 25. us department of health and human services. the office of research integrity. case summaries. [cited 2017 november]; available from: https:// ori.hhs.gov/case_summary. 26. lock s. fraud in medical research. j res nurs. 1997;2:161–163. 27. anestidou l. research ethics education: the view from below. am j bioeth. 2002;2. 28. eisen a, berry rm. the absent professor: why we don’t teach research ethics and what to do about it. am j bioeth. 2002;2:38–49. 29. titus sl, wells ja, rhoades lj. repairing research integrity. nature. 2008;453:980–982. 30. al mawali ah, al qasmi am, al sabahi sm, idikula j, elaty ma, morsi m, et al. oman vision 2050 for health research: a strategic plan for the future based on the past and present experience. oman med j. 2017;32:86. 31. centre of studies & research ministry of health oman. guidelines for responsible conduct of clinical studies and trials, 2016. available from: http://mohcsr.gov.om/clinical-trials-and-studiesguideline/. 32. world medical association. declaration of helsinki. ethical principles for medical research involving human subjects. bull world health organ. 2001;79:373–374. 33. national commission for the protection of human subjects of biomedical and behavioural research, ryan kjp. the belmont report: ethical principles and guidelines for the protection of human subjects of research-the national commission for the protection of human subjects of biomedical and behavioral research: us government printing office; 1978. 34. fadel he. ethics of clinical research: an islamic perspective. j ima. 2010;42. 35. savulescu j, wartolowska k, carr a. randomised placebo-controlled trials of surgery: ethical analysis and guidelines. j med ethics. 42: 776–783; 2016. 36. sharav vh. children in clinical research: a conflict of moral values. am j bioeth. 2003;3:1–59. 37. glickman sw, mchutchison jg, peterson ed, cairns cb, harrington ra, califf rm, et al. ethical and scientific implications of the globalization of clinical research. n engl j med. 2009;360:816–823. adhra al-mawali et al. 125j contemp med sci | vol. 4, no. 3, summer 2018: 119–125 review fraud and misconduct in clinical research this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 38. macklin r. double standards in medical research in developing countries: cambridge, ma: university press; 2004. 39. shapiro ht, meslin em. ethical issues in the design and conduct of clinical trials in developing countries. n engl j med. 2001;345: 139–142. 40. emanuel ej, wendler d, killen j, grady c. what makes clinical research in developing countries ethical? the benchmarks of ethical research. j infect dis. 2004;189:930–937. 41. al-mawali a. leukemic stem cells shows the way for novel target of acute myeloid leukemia therapy. j stem cell res ther. 2013;3:151. dx.doi.org/10.22317/jcms.09201802 13j contemp med sci | vol. 1, no. 1, winter 2015:13–16 research objective carriage of staphylococcus aureus, especially methicillin-resistant s. aureus (mrsa) is a problem within healthcare organizations and in the community. the aim of the study was to screen s. aureus carriage and their susceptibility to cefoxitin and oxacillin among medical students. methods a total of 100 nasal swabs were collected. isolation and identification of the isolate as s. aureus was done using gram stain, coagulase test and catalase test. s. aureus isolates were confirmed as mrsa using cefoxitin (30 μg) disc and oxacillin (30 μg) disc by kirbybauer disc diffusion method on mueller-hinton agar. from 100 nasal swabs, 76 were coagulase negative staphylococci and 20 were coagulase positive staphylococci. results from 100 nasal swabs, 76 were coagulase negative staphylococci and 20 were coagulase positive staphylococci. from these, 60% and 40% were oxacillinand cefoxitin-resistant isolates, respectively. the data obtained from this study revealed that there were carriers of mrsa among the medical students. keywords mrsa, coagulase, oxacillin, cefoxitin screening of nasal carriage for staphylococcus aureus and their resistance to oxacillin and cefoxitin among medical students in karbala university suhad hadi mohammed, mohammed neama hmood, areej abbas abd, seham aaid obaid, baraa ahmed fahad & fatemah hadi kadhem introduction staphylococcus aureus has long been recognized as an important human pathogen.1 the anterior nares represent the primary ecological reservoir of s. aureus in humans, and nasal carriage is a major risk factor for a variety of infections.2 three patterns of nasal carriages are known (persistent carrier, intermittent, and non-carrier). approximately 20% of the individuals almost always carry one type of strain and they are called persistent carriers. a large proportion of the population (60%) harbours s. aureus intermittently, and the strains change with varying frequencies. such persons are called intermittent carriers. finally, a minority of the people (20%) almost never carry s. aureus and they are called noncarriers. a persistent carriage is more common in children than in adults, and many people change their pattern of carriage between the age of 10 and 20 years. the reasons for these differences in the colonization patterns are unknown.3 healthcare workers (hcws) constitute an important reservoir of s. aureus. several studies have reported that the rate of the nasal carriage of s. aureus among the hcws ranges from 16.8% to 56.1%.4–7 treatment of infection caused by s. aureus has become more problematic since the development of antimicrobialresistant s. aureus (mrsa). as the mrsa strains are resistant to all β-lactam antibiotics, the treatment options are limited significantly. the incidence of nosocomial infection caused by mrsa continues to increase worldwide. infections caused by mrsa strains are associated with longer hospital stay, prolonged antibiotic administration and higher costs than infections caused by methicillin-susceptible s. aureus (mssa) strains.8 by definition, mrsa strains harbours the meca gene, which encodes the low-affinity penicillin-binding protein (pbp) designated as pbp2a.9,10 the clinical and laboratory standards institute (clsi) states that the oxacillin and cefoxitin disk tests are equivalent in sensitivity and specificity for the detection of mecamediated resistance in s. aureus.11 the screening of the nasal carriage in hcws is an important component in the control of mrsa in any healthcare facility. the identification of the colonized staff members allows an appropriate management of these persons to prevent the spread of organism within hospitals or in communities. because medical students belong to the hcw in future, the aim of our study is to screen s. aureus nasal carriage of these individuals, and to identify the prevalence of oxacillin and cefoxitin resistance among the isolated s. aureus. materials and methods specimen collections anterior nasal swabs were taken from 100 healthy students from third and fourth stage whom they had internship programme in hospitals during summer from october 2014 to march 2015. sterile swab was moistened with sterile normal saline and was rotated at least 5 times in both nares, then placed in the transport media, using standard methods.12–14 specimens processing all specimens were directly inoculated from transport media into plates of mannitol salt agar (msa) and blood agar and incubated at 37°c for 24 h (fig. 1). all colonies from primary cultures were subcultured onto msa and incubated at 37°c for 24 h.15 s. aureus were identified depending on the morphological features on culture media (beta-hemolytic on blood agar and mannitol fermentation on msa) and biochemical tests (catalase positive test and coagulase positive department of clinical laboratories, college of applied medical sciences, karbala university, karbala, iraq. correspondence to suhad hadi mohammed (email: shm.med.school@gmail.com). (submitted: 03 january 2015 – revised version received: 26 february 2015 – accepted: 01 march 2015 – published online: winter 2015) issn 2413-0516 14 j contemp med sci | vol. 1, no. 1, winter 2015:13–16 detection of oxacillin and cefoxitin resistance in staphylococcus aureus research suhad hadi mohammed et al. were resistant to cefoxitin and 12 (60%) were resistant to oxacillin. there is positive significant correlation between the two drugs (table 2). distribution of the resistant strains among sex and age the prevalence of s. aureus nasal carriage was higher among the older age group individuals (70%) than the younger age group (30%). concerning the resistance profile, there were 62.5% (5/8) of cefoxitin-resistant isolates, and 66.6% (8/12) of oxacillin-resistant isolates found among the older than the younger volunteers (table 3). the current study revealed that 62.5% (5/ 8) of cefoxitin-resistant isolates and 50% (6/12) of oxacillin-resistant isolates were found in females. discussion the primary reservoir of s. aureus in humans is the anterior nares. nasal carriage is recognized as a major risk factor for the development of both community-acquired and nosocomial infections.17,18 this appears to play a key role in the epidemiology and pathogenesis of infection.17,18 the factors that distinguish between a carrier and a non-carrier are still unknown. enhanced adhesion of s. aureus, to cell associated and cell-free secretions, along with the induction of reduced mucociliary activity, could well explain the nasal colonization by s. aureus. it is imperative that nasal carriage due to s. aureus strains should be prevented in order to stem the rate of infection, and in preventing the transmission of resistant strains of the organism.8 although nasal carriage of s. aureus is harmless in healthy individuals, they can become carriers who could pose the risk of spreading infections to the community at large, and since the section of individuals under this study were medical students, their interaction and exposure to hospital environment could cause major risks in transmitting to patients fig. 1 a: blood agar plate; b: beta hemolytic isolates; c: mannitol salt agar; d: mannitol salt agar plate with s. aureus isolates; e: catalase test; f: coagulase test. a d b e c f fig. 2 a: mueller-hinton agar without antibiotic disc with bacterial colonies (control); b: mueller-hinton agar with antibiotic disc. a b table 1. culture results of nasal swabs culture results isolates n (%) culture positive s. aureus 20 (%) coagulase negative staphylococci 76 (%) culture positive micrococcus 3 other bacteria bacillus 1 table 2. screening for mrsa antibiotic resistant n (%) sensitive n (%) total n (%) oxacillin 12 (60) 8 (40) 20 (100) cefoxitin 8 (40) 12 (60) 20 (100) correlation r = 0.667 p value = 0.001* mrsa: methicillin-resistant s. aureus. *correlation significant at 0.01 level. test) also gram staining showed grampositive grape-like clusters (fig. 1). antibiotic susceptibility test all s. aureus isolates were tested for cefoxitin and oxacillin susceptibility by kirby-bauer method on mueller-hinton agar (mha) (hi-media). plates were incubated at 37°c for 24 h. following the incubation, the inhibition zone diameter was measured. identification of mrsas were done by following clsi.16 isolates were considered susceptible to oxacillin and cefoxitin if the zone of inhibition around the disks was ≥22 mm, and resistant if the zone was ≤21 mm (fig. 2). statistical analysis ibs, spss version 20 was used in the analysis of the present data. results a total of 100 nasal swab samples were collected and screened during this study. from these, a total of 20 (20%) were identified as coagulase positive s. aureus isolates and 80 (80%) were identified as coagulase negative staphylococci isolates (table 1). the coagulase positive isolates were then tested to demonstrate their resistances to methicillin by using oxacillin and cefoxitin discs, 8 (40%) 15j contemp med sci | vol. 1, no. 1, winter 2015:13–16 research detection of oxacillin and cefoxitin resistance in staphylococcus aureussuhad hadi mohammed et al. and spreading nosocomial infections. therefore, it is necessary to detect s. aureus carriage in medical students. the current study revealed that out of the 100 samples collected from medical students, 96 were identified as staphylococcus while 4 specimens showed no growth of staphylococcal colonies, instead they showed growth of micrococcus sp. and bacillus. twenty samples out of 96 (20.8%) were coagulase positive s. aureus isolates and 76 (79.1%) were coagulase negative staphylococci. the prevalence of s. aureus carriage has been reported in healthy populations in several countries; 43.2% of s. aureus in nasal cavity of adults in iraq, 17.3% in nasal cavity of turkish children, 36% in nares of japanese adults and 32.4% in nasal cavity of adults in the united states.19–22 pant and rai’s (2007)23 findings revealed higher s. aureus nasal colonization rate (43.8%) in staffs of teaching hospital in nepal. also, in abia state of nigeria, chigbu and ezeronye (2003)24 reported 50% nasal colonization in both hospital and nonhospital subjects. chatterjee et al. (2009)25 showed that the overall prevalence of s. aureus nasal colonization was 52.3%. whereas onanuga and temedie (2011)26 showed that 33.3% s. aureus isolates were obtained from 120 nares specimens screened. whilst, adesida et al. (2007)27 reported a much lower (14.0%) nasal colonization in medical students in lagos, nigeria. these variations may be attributed to the characteristics of the population under study. a population that is on antibiotics at the time of sampling may yield a much lower prevalence of s. aureus while a population from hospital settings may yield a much higher prevalence because of the high prevalence of infectious patients in that environment. other factors that can cause variations may be sampling and culture techniques. table 3. distribution of s. aureus isolates among age group s. aureus n (%) oxacillin sensitivity cefoxitin sensitivity sensitive resistance sensitive resistance age groups younger 6 (30) 2 4 3 3 older 14 (70) 6 8 9 5 sex male 10 (50) 4 6 7 3 female 10 (50) 4 6 5 5 total 20 (100) 8 12 12 8 concerning the detection of mrsa, all the 20 s. aureus isolates were tested for cefoxitin and oxacillin resistance using a disk diffusion method and 60% and 40% were found to be susceptible to oxacillin and cefoxitin, respectively. higher incidence of mrsa was found in another study in iraq/baghdad among hcws and patients in hospital.28 in another study, resistant percentage was found to be of 90.9%.29 fey et al. (2003)30 stated that the resistance to methicillin was 81%, while jain et al. (2008)31 observed about 75.26% of isolates were methicillin-resistant. these observed differences may due to the variation in the geographic area, sources of clinical specimens, genetic background and the collection site of the isolates.31 conclusion the data obtained from this study revealed that there were reservoirs or carriers of mrsa in medical students. screening for resistant strains of staphylococci in medical students should be adopted as a protocol in medical colleges, in order to curb the spread of drug-resistant staphylococci from the hospital to the community. this will also help in monitoring the hcws population who might pose a risk to patients and hospital personnel and the community, at large.  references 1. rongpharpi sr, hazarika nk, kalita h. the prevalence of nasal carriage of staphylococcus aureus among healthcare workers at a tertiary care hospital in assam with special reference to mrsa. j clin diagn res. 2013 feb;7(2):257–60. doi: http://dx.doi.org/10.7860/jcdr/2013/4320.2741 pmid: 23543837 2. van den akker el, nouwen jl, melles dc, van rossum ef, koper jw, uitterlinden ag, et al. staphylococcus aureus nasal carriage is associated with glucocorticoid receptor gene polymorphisms. j infect dis. 2006 sept 15; 194(6):814–8. doi: http://dx.doi.org/10.1086/506367 pmid: 16941349 3. kluytmans j, van belkum a, verbrugh h. nasal carriage of staphylococcus aureus: epidemiology, underlying mechanisms and associated risks. clin microbiol rev. 1997 july;10(3):505–20. pmid: 9227864 4. dan m, moses y, poch f, asherov j, gutman r. carriage of methicillinresistant staphylococcus aureus by non-hospitalized subjects in israel. infection. 1992 nov;20(6):332–35. doi: http://dx.doi.org/10.1007/ bf01710678 pmid: 1293052 5. duncan ib, collins am, neelin em, roy te. nasal carriage of staphylococcus pyogenes by student nurses. can med assoc j. 1957 dec 1;77(11):1001–9. pmid: 13489588 6. mcanally tp, lewis mr, brown dr. effect of rifampin and bacitracin on nasal carriers of staphylococcus aureus. antimicrob agents chemother. 1984 april; 25(4):22–6. pmid: 6732212 7. paul mo, lamikanra a, aderibigbe da. nasal carriers of coagulase-positive staphylococci in a nigerian hospital community. trans r soc trop med hyg. 1982 jan;76(3):319–23. doi: http://dx.doi.org/10.1016/0035-9203(82)901808 pmid: 7112654 8. kumar p, shukla i, varshney s. nasal screening of healthcare workers for nasal carriage of coagulase positive mrsa and prevalence of nasal colonization with staphylococcus aureus. biology and medicine. 2011;3(2) special issue: 182–6. 9. clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing. eighteenth informational supplement. clsi document m100-s22. wayne, pa: clinical and laboratory standards institute; 2012. 10. chambers hf, sachdeva m. binding of beta-lactam antibiotics to penicillinbinding proteins in methicillin-resistant staphylococcus aureus. j infect dis. 1990 jun;161(6):1170–6. 11. clinical and laboratory standards institute: performance standards for antimicrobial susceptibility testing. fifteenth informational supplement january 2005. approved standard, m100-s15, vol. 25, no. 1. wayne, pa. 12. kloos we, bannerman tl. staphylococcus and micrococcus. in: murry pr, baron ej, pfaller m, tenover afc, yolken rk, editors. manual of clinical microbiology, 7th ed. washington, dc: american society for microbiology; 1999. pp. 264–82. 13. monsen t, rönnmark m, olofsson c, wiström, j. an inexpensive and reliable method for routine identification of staphylococcal species. eur j clin microbiol infect dis. 1998 may;17(5):327–35. doi: http://dx.doi.org/10.1007/bf01709455 pmid: 9721961 14. jain a, agarwal a, verma rk. cefoxitin disc diffusion test for detection of meticillin-resistant staphylococci. j med microbiol. 2008 aug;57 (pt 8):957–61. doi: http://dx.doi.org/10.1099/jmm.0.47152-0 pmid: 18628495 15. talan da, staatz d, staatz a, overturf gd. frequency of staphylococcus intermedius as human nasopharyngeal flora. j clin microbiol. 1989 oct; 27(10):2393 pmid: 2584388 16 j contemp med sci | vol. 1, no. 1, winter 2015:13–16 detection of oxacillin and cefoxitin resistance in staphylococcus aureus research suhad hadi mohammed et al. 16. clinical laboratory standards institute. performance standard for antimicrobial susceptibility testing: seventeenth informational supplement m100-s17. clinical laboratory standards institute, wayne, pa, usa; 2007. 17. boelaert jr, van landuyt hw, de baere ya, deruyter mm, daneels rf, schurgers ml, et al. staphylococcus aureus infections in hemodialysis patients: pathophysiology and use of nasal mupirocin for prevention. j chemother. 1995 jul;7 suppl 3:49–53. pmid: 8609538 18. koziol-montewka m, chudnicka a, ksiazek a, majdan m. rate of staphylococcus aureus nasal carriage in immunocompromised patients receiving hemodialysis treatment. int j antimicrob agents. 2001 aug;18(2):193–6. doi: http://dx.doi. org/10.1016/s0924-8579(01)00350-8 pmid: 11516945 19. moellering rc jr. problems with antimicrobial resistance in gram-positive cocci. clin infect dis. 1998 may;26(5):1177–8. pmid: 9597248 20. uemura e, kakinohana s, higa n, toma c, nakasone n. comparative characterization of staphylococcus aureus isolates from throats and nose of healthy volunteers. jpn j infect dis. 2004 feb;57(1):21–4. pmid: 14985632 21. soysal a, sahin h, yagci a, barlan i, bakir m. the low rate of methicillinresistant staphylococcus aureus in turkish children. jpn j infect dis. 2006 jun;59(3):195–6. pmid: 16785704 22. onanuga a, onaolapo j.a. antimicrobial susceptibility of community associated staphylococcus aureus isolates from healthy women in zaria, nigeria. trop j pharmaceutical res. 2008 apr 3;7(1):929–39. doi: http:// dx.doi.org/10.4314/tjpr.v7i1.14679 23. pant j, rai sk. occurrence of staphyloccous aureus in hospital environment and staffs in teaching hospital in katmandu, nepal. j nepal assoc. medi lab sci. 2007;8:72–3. 24. chigbu co, ezeronye ou. antibiotic resistant staphylococcus aureus in abia state of nigeria. afr j biotech. 2003 oct 31;2(10):374–8. doi: http://dx.doi. org/10.5897/ajb2003.000-1077 25. chatterjee ss, ray p, aggarwal a, das a, sharma m. a community-based study on nasal carriage of staphylococcus aureus. indian j med res. 2009 dec;130(6):742–8. pmid: 20090137 26. onanuga a, temedie tc. nasal carriage of multi-drug resistant staphylococcus aureus in healthy inhabitants of amassoma in niger delta region of nigeria. afr health sci. 2011 jun;11(2):176–81. pmid: 21857847 27. adesida sa, abioye oa, bamiro bs, brai bic, smith si, amisu ko, et al. associated risk factors and pulsed field gel electrophoresis of nasal isolates of staphylococcus aureus from medical students in a tertiary hospital in lagos, nigeria. braz j infect dis. 2007 feb;11(1):63–9. doi: http://dx.doi. org/10.1590/s1413-86702007000100016 pmid: 17625730 28. ali m. al-dahbi, harith j. al-mathkhury. distribution of methicillin resistant staphylococcus aureus in iraqi patients and healthcare workers. iraqi j sci. 2013;54(2):293–300. 29. al-geobory ha. comparative study between methicillin resistant staphylococcus aureus (mrsa) and methicillin sensitive staphylococcus aureus (mssa), and detect the antimicrobial effects of some plant extracts on them [msc thesis]. baghdad, iraq: college of science, baghdad university; 2011. 30. fey pd, saïd-salim b, rupp me, hinrichs sh, boxrud dj, davis cc, et al. comparative molecular analysis of community or hospital-acquired methicillin-resistant staphylococcus aureus. antimicrob agents chemother. 2003 jan;47(1):196–203. 31. jain a, agarwal a, verma rk. cefoxitin disc diffusion test for detection of meticillin-resistant staphylococci. j med microbiol. 2008 aug;57(pt 8): 957–61. doi: http://dx.doi.org/10.1099/jmm.0.47152-0 pmid: 18628495 1j contemp med sci | vol. 5, no. 1, january–february 2019: 1–7 review article incidence and mortality rate of cervix cancer in iran from 1990 to 2016: a systematic review and meta-analysis shirin riahi,a ali mohammad mokhtari,b mouhebat vali,c elham abdzadeh,d shokrollah mohseni,e hamid salehiniya,f,g and soheil hassanipourh* anon-communicable diseases research center, alborz university of medical sciences, karaj, iran. bsocial determinants of health research center, mashhad university of medical sciences, mashhad, iran. cstudent research committee, shiraz university of medical sciences, shiraz, iran. ddepartment of biology, faculty of science, university of guilan, rasht, iran. esocial determinants in health promotion research center, hormozgan health institute, hormozgan university of medical sciences, bandar abbas, iran. fzabol university of medical sciences, zabol, iran. gdepartment of epidemiology and biostatistics, tehran university of medical sciences, tehran, iran. hgastrointestinal and liver diseases research center, guilan university of medical sciences, rasht, iran. *correspondence to soheil hassanipour (email: soheil.epid@gmail.com). (submitted: 21 october 2018 – revised version received: 29 october 2018 – accepted: 11 november 2018 – published online: 26 february 2019) objective cervix cancer (cc) is one of the major health problems among iranian women and elsewhere around the world. considering the importance of this cancer, this study was conducted to determine the age-standardized incidence rate (asir) and age-standardized mortality rate (asmr) of cc in iran. methods a search was conducted using international databases (medline/pubmed, proquest, scopus, embase and isi/web of knowledge), and national databases (scientific information database, magiran, iranmedex, and irandoc). this systematic review was carried out according to the preferred reporting items for systematic reviews and meta-analyses check list in 2018. thereafter, persian and english language papers referring to the asir and asmr of cc in iran were included. data were independently extracted by two reviewers. the joanna briggs checklist was carried out to evaluate the quality of studies. result a total of 522 papers were obtained in the initial search of the databases, and 25 articles were included to the review by further refinement and screening. based on the random-effect model, the asir and asmr of cc in iran was 2.14 per 100,000, 95% ci (1.89–2.38) and 0.93 per 100,000, 95% ci (0.81–1.05), respectively. conclusion the incidence and mortality of cc in iran was lower than other parts of the world. it should be noted that because of the high heterogeneity of the results of this study, we must judge with caution regarding the results. keywords incidence, mortality, cervical cancers, iran, systematic review introduction today, cancer is one of the major health problems in iran and in the rest of the world.1 the findings revealed that female genital cancers are the fourth most common malignancy in women.2,3 the incidence rate and prevalence of female genital cancers in various parts of the world is different. according to globocan 2012, the incidence of age-related cervical cancer is 13.1 per 100 000 and the standardized death rate is 6.9 per 100 000 population.1 cervix cancer (cc) is the seventh common among all other one, although in some parts of the world, such as africa and south asia, it is the first cause of cancer deaths.4,5 among women in developing countries, this malignancy is one of the most important causes of female mortality and after breast cancer, it is the most frequent malignancy.6,7 cc is not only the most common and prevalent malignancy among women in many developing countries, but also the social significance of the disease becomes more pronounced because of their younger age at death.8 certainly, for some reasons, such as having a long period before the invasion, the existence of a screening program and the availability of appropriate treatment for primary lesions, this cancer can be prevented.9 in epidemiology and demography, most rates, such as the incidence, prevalence, and morbidity are strongly related to age. this is also true for many types of cancers. for different purposes, age-specific comparisons may be helpful. however, the comparison of crude age-specific rates over time and between populations may be highly misleading if the age composition of the compared population is different.10 for this reason, standardization is important when comparing disease rates between regions or countries and is widely used in cancer research. age-standardized rate is a summary amount that will be observed for a population, provided that the age-specific rate of the population and the age composition of the population should be considered the same as the reference population (standard population).11 generally, genital cancers are one of the most common causes of female mortality. detection and timely treatment of these cancers can increase women’s longevity.12 the first step is to control the burden of disease related to cancers in any community, recognizing their status in the population, as well as collecting information about the incidence, type, and location of cancers.13 although organized review studies have been conducted on some of the risk factors for these cancers14 a comprehensive, structured review of asir and asmr of cc, has not yet been conducted in iran. therefore, considering the above-mentioned points and the importance of this cancer, this study was conducted with the aim of estimating the standardized incidence and mortality rate of cervix cancer in iran. materials and methods this study is a systematic and meta-analysis of the incidence and mortality of cc in iran which was designed in 2018. issn 2413-0516 2 j contemp med sci | vol. 5, no. 1, january–february 2019: 1–7 cervix cancer in iran review article s. riahi et al. the methodology of this study is based on the preferred reporting items for systematic reviews and meta-analysis (prisma) checklist.15 search strategy in september 2018, the researchers examined six international databases medline/pubmed, proquest, scopus, embase, sciencedirect and google scholar, and four iranian sites sid, magiran, iranmedex and irandoc. selected keywords for international databases include “gynecological cancer”, “female genital cancer”, “cervix”, “cervix cancer”, cervix neoplasms, “cervix tumor”, “cancer of cervix”, “neoplasms of the cervix”, “incidence”, “epidemiology”, “occurrence”, and “iran”. the collected data entered the endnote x7 software, and duplicate articles were automatically deleted. it is worth to mention here that two researchers explored articles individually. inclusion and exclusion criteria studies that clearly reported the incidence and mortality rate of cc in iran were analyzed. on the other hand, the studies that reported the incidence with insufficient sample sizes, as well as posters presented in the conferences were excluded. quality assessment to check and control the quality of the articles, the checklist used by the joanna briggs institute was used. this tool consists of nine questions that are categorized as “yes, no, unclear, and not used.” the purpose of this tool is to assess the methodological quality of studies, and ways to achieve and identify the errors in studies, design, implementation, and analysis of data. the quality assessment results were presented in table 1. screening studies initial research was conducted by two people (first and second authors). screening of studies, extraction of results, and also the quality control of articles was evaluated separately by two individuals (first and second authors). if there was no match between the two, the supervisor of the team (responsible author) would announce the final comment on that article. statistical analysis the heterogeneity of the studies was evaluated by cochran test (with significance <0.1) and its composition using statistics i2. in the case of heterogeneity, the random effects model was used with the inverse-variance method and in the absence of table 1. jbi critical appraisal checklist applied for included studies in the systematic review author name (year) q. 1 q. 2 q. 3 q. 4 q. 5 q. 6 q.7 q. 8 q. 9 saalabian (1990) yes yes yes yes yes yes yes no yes sadjadi (2003) no yes yes unclear yes yes yes yes no babai (2005) yes yes yes yes yes no yes no yes sadjadi (2005) yes yes yes yes yes yes yes yes no fallah (2007) yes no no unclear yes yes no yes yes sadjadi (2007) yes yes yes unclear yes yes no no yes mehrabani (2008) yes yes yes yes yes no yes no unclear somi (2008) yes yes yes yes yes no yes no unclear mohagheghi (2009) yes yes yes yes yes no yes no unclear mousavi (2009) yes yes yes yes yes no yes no yes babaei (2009) yes yes yes yes yes no yes yes yes masoompour (2011) yes yes yes yes yes no yes no yes almasi (2012) yes yes yes yes yes no yes no yes fateh (2013) yes yes yes yes no no yes no yes khorasanizadeh (2013) yes yes yes yes no no yes no yes arab (2014) yes yes yes yes no no yes no yes arab (2014) yes yes yes yes yes no yes no unclear taheri (2014) yes yes yes yes yes no yes no unclear sharifian (2014) yes yes no yes yes no yes no unclear rahimi (2015) yes yes no yes yes no yes yes no masoompour (2016) yes yes yes yes yes no yes yes no almasi (2016) yes yes yes yes yes no yes yes no singh (2012) yes yes yes yes yes no yes yes unclear khorasanizadeh (2013) yes yes yes yes yes no yes no unclear q. 1: samples were representative? q. 2: participants were appropriately recruited? q. 3: sample size was adequate? q. 4: study subjects and the setting were described? q. 5: data analysis was conducted? q. 6: objective, standard criteria, and reliably were used? q. 7: appropriate statistical analyses were used? q. 8: confounding factors, subgroups, and differences were identified and accounted? q. 9: subpopulations were identified using objective criteria? s. riahi et al. 3j contemp med sci | vol. 5, no. 1, january–february 2019: 1–7 review article cervix cancer in iran heterogeneity, a fixed-effect model was used. all analyses were performed by the stata version 12 software (stata corp lp, college station, tx, usa). results description of literature search after searching for all international and domestic databases, 564 articles were found that, after removing repetitive articles, 489 articles were entered into the review phase in terms of title and abstract. after reviewing the titles and abstracts of articles, 57 articles joined to the next stage. at this stage, the full text of the articles was examined and 27 articles considered for the final analysis. in the screening stages of studies, some articles were excluded for a variety of reasons, which included an unrelated topic (327 cases), an unrelated population (31 cases), and repeated results (four cases). the flowchart of the studies was presented in fig. 1. description of included studies the included studies16–40 were published from 1990 to 2016. the main characteristics of the selected studies have been represented in table 2. asir of cervix cancer the highest asir of cc (4.97 per 100,000) from golestan province between 2004 and 2010 and the lowest asir (0.4 per 100,000) from ardebil province between 1996 and 1999 was reported. asmr of cervix cancer the highest asmr of cc (0.9.9 per 100,000) from southern khorasan province between 2003 and 2005 and the lowest asmr (0.4 per 100,000) from sistan and baluchistan province between 2003 and 2005 was reported. results of meta-analysis due to the high heterogeneity of the studies, the random effects model was used. thus, the asir and asmr of cc in iran was fig. 1 flowchart of the included eligible studies in systematic review. table 2. characteristics of the included articles in the study author name (year) study region time period asir asmr saalabian (1990) fars 1985–1989 2.30 sadjadi (2003) ardabil 1996–1999 0.4 babai (2005) semnan 1998–2002 1.08 sadjadi (2005) iran (all area) 2002 4.5 fallah (2007) ardabil 1996–2000 0.93 gilan 1.84 mazandaran 2.33 golestan 2.76 kerman 3.02 sadjadi (2007) kerman 1996–2000 1.4 mehrabani (2008) fars 1990–2005 1.13 somi (2008) east azerbaijan 2006–2007 1.87 mohagheghi (2009) tehran 1998–2001 4.8 mousavi (2009) ardabil 2003–2004 1.64 esfahan 2004–2005 1.90 kerman 2005–2006 1.90 golestan lorestan mousavi (2009) iran (all area) 2003–2006 1.0 babaei (2009) ardabil 2004–2006 1.4 masoompour (2011) fars 1998–2002 1.5 almasi (2012) fars 2003–2009 0.87 1.07 1.2 1.1 3.55 3.66 fateh (2013) shahroud 2002–2010 1.80 khorasanizadeh (2013) iran (all area) 2007 2.47 arab (2014) iran (all area) 2004 and 2008 1.71 and 2.2 arab (2014) iran (all area) 2005 1.4 taheri (2014) golestan 2004–2010 4.97 sharifian (2014) iran (all area) 2003–2009 rahimi (2015) tehran 1998–2001 1.87 golestan 2004–2008 2.56 east azerbaijan 2006–2007 1.8 khuzestan 2002–2009 1.08 shahroud 2001–2010 semnan 1998–2002 masoompour (2016) fars 2007–2010 2.55 almasi (2016) iran (all area) 2012 2.8 1.2 singh (2012) iran (all area) 2012 1.0 khorasanizadeh (2013) iran (all area) 2003–2005 1.4 east azarbaijan 1.8 west azarbaijan 1.6 ardabile 0.5 isfahan 0.9 ilam 0.9 (continued) 4 j contemp med sci | vol. 5, no. 1, january–february 2019: 1–7 cervix cancer in iran review article s. riahi et al. 2.14 per 100,000, 95% ci (1.89–2.38) and 0.93 per 100,000, 95% ci (0.81–1.05), respectively. the results of the forest plot for incidence and mortality rate are shown in figs. 2 and 3. subgroup analysis due to the heterogeneity of the results, subgroups were analyzed based on the geographical region. according to the results of the analysis of subgroups, the highest asir of cc in the northern region was 2.97 (95% ci; 2.28–4.14) and the lowest in the northwestern region of the country was 1.28(95% ci; 0.83–1.73). the highest asmr in northwest provinces was 1.39 (95% ci; 0.74–2.04) and the lowest in the southwest was 0.64 (95% ci; 0.46–0.81). meta-regression results of meta-regression manifested a significant association between publication year and asir of cc. thus, year of study is a cause of variability in results (regression coefficient = 0.038, p = 0.011). according to the results, an increasing survival rate across the study period was observed. due to the limitation of the number of reported years for asmr, no meta-regression analysis was possible. results of meta-regression has been shown in fig. 4. publication bias the publication bias among the studies was evaluated using the egger test. based on the results, there was no publication bias among the studies (bias: 6.62, 95% ci = −1.79 to 15.06; p = 0.107 for asmr, bias: 3.65, 95% ci = −5.09 to 13.91; p = 0.443 for asir). discussion cancer is the third leading cause of death in iran (41). there are few studies on the epidemiology of cancer among population of developing countries including iran.6,10,42–47 based on the findings of this study, the asir of cc in iran was 2.14 and asmr was 0.93 per 100 000. in a study in korea, the incidence of cc in the years 1993–2002 has declined from 19.0 to 15.1 per 100,000.48 a research conducted in india represented that the asir of cc was 22.9 per 100,000.49 therefore, as it is observable, the incidence of cc in iran differs from that of the world as well as in other countries and is at a lower level. one of the possible reasons for this is that the incidence of cc is related to sexual factors, fertility, as well as human papillomavirus, which has a lower prevalence in islamic countries.20 as an example, the overall incidence of female genital cancers in qatar was 0.9 per 100,000.50 in a study on female genital cancers in one of the major provinces of iran, it was found that the relative frequency of cc in the period of 1391–1394 was declining, and one of the reasons is to perform a screening for early diagnosis of cervical cancer by testing pap smears in the country.6,51 in this study, the highest incidence of cc in the provinces of golestan and tehran and its lowest incidence was reported in ardebil province between 1996 and 1999. according to the studies, changes in the age of marriage, sexual activity at an early age, the number of pregnancies and individual health associated with reproductive organs can be a factor in the incidence of cc.3,51 in our country, women are more likely to be married at an early age and have used contraceptive pill for a longer time. on the other hand, the prevalence of hepatitis b infection, which has regional varieties, may affect the incidence of cc. therefore, the differences between regions can be due to the above-mentioned issues.52 the probability of higher diagnostic errors in smaller cities due to less facilities and the possibility of referring patients with ovarian cancer from other provinces to tehran (for more advanced diagnostic and therapeutic measures) can be effective in increasing the incidence rate in tehran province. based on the results of this study, the mortality rate of this cancer at the age of 45–75 has a steady increase and after age 60 there is a sharp increase, suggesting that people with a higher average age may have a higher incidence of cc.53 accordingly, one of the reasons for the higher mortality of cc in tehran than other provinces can be attributed to the difference in the mean age of women in these districts.54 another possible contributing factor is the contamination of air. there is a close relationship between air pollution and some of the cancers of women, including breast and cervical cancer.55 the results of the study manifest that jobs with low sitting time or low energy consumption are associated with an increase in the incidence of breast, uterine, and ovarian cancer.56 strengths and limitation of study the strengths of this study is that it is the first study with a wide range on the incidence and mortality of cc in iran. its limitations include lack of examination of dissertations, non-electronic, and non-printed sources, as well as the type of studies available in iran. it should be mentioned that in the previous studies, table 2. characteristics of the included articles in the study— (continued) author name (year) study region time period asir asmr bushehr 0.7 charmahalbakhtiari 0.7 north khorasan 1.0 khorasan razavi 0.5 south khorasan 1.9 khuzestan 1.1 zanjan 0.8 semnan 0.6 sistanbaluchestan 0.3 fars 1.1 ghazvin 1.1 ghom 1.2 kohgiluyeboírahmad 0.5 kordestan 1.1 kermanshah 1.6 kerman 0.5 golestan 0.6 gilan 1.2 lorestan 0.5 mazandaran 0.8 markazi 0.8 hormozgan 1.2 hamedan 0.6 yazd 0.8 s. riahi et al. 5j contemp med sci | vol. 5, no. 1, january–february 2019: 1–7 review article cervix cancer in iran fig. 2 forest plot of the random-effect meta-analysis for age standardized incidence rates of cervix cancer in iran. fig. 3 forest plot of the random-effect meta-analysis for age standardized mortality rates of cervix cancer in iran. cc were diverse, in other words, different studies classified this malignancy in a special way and eventually reported the standardized incidence of age. to give an instance, in some studies, cc has been reported in general, some have been divided into two or three distinct categories, and in some, only the uterine body cancer has been examined. therefore, it was not possible to compare the standardized incidence rates reported in all existing studies and it was decided to investigate studies that reported only cancer of the uterus. another major limitation of our study was the lack of sufficient information on age composition and other information such as sample size and confidence interval that could be used to analyze. 6 j contemp med sci | vol. 5, no. 1, january–february 2019: 1–7 cervix cancer in iran review article s. riahi et al. conclusion overall, according to the findings of this study, the asir and asmr of cc in iran is lower than in other countries of the world. on the other hand, given the heterogeneity observed in studies in different regions, the interpretation of the results should be more cautious. acknowledgments this research was supported by the alborz university of medical sciences with the code number 1719-25-02-1396 and the code of ethics abzums.rec.1396.221. conflicts of interest none. nfig. 4 meta-regression plots of change in asir of cervix cancer according to changes in continuous study moderator’s year. references 1. bray f, ferlay j, soerjomataram i, siegel rl, torre la, jemal a. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin. 2018;68:394–424. 2. weiderpass e, labrèche f. malignant tumors of the female reproductive system. saf health work. 2012;3:166–180. 3. maheshwari a, kumar n, mahantshetty u. gynecological cancers: a summary of published indian data. south asian j cancer. 2016;5:112–120. 4. pedersen k, fogelberg s, thamsborg lh, clements m, nygard m, kristiansen is, et al. an overview of cervical cancer epidemiology and prevention in scandinavia. acta obstet gynecol scand. 2018;97:795–807. 5. shrestha ad, neupane d, vedsted p, kallestrup p. cervical cancer prevalence, incidence and mortality in low and middle income countries: a systematic review. asian pac j cancer prev. 2018;19:319–324. 6. mokhtari am, riahi s, fathalipour m, delam h, hashemnejad m, hassanipour s. the age-standardized rate of female genital cancers in iran: a systematic review and meta-analysis. j hayat. 2018;24:204–219. 7. torre la, islami f, siegel rl, ward em, jemal a. global cancer in women: burden and trends. cancer epidemiol biomarkers prev. 2017;26:444–457. 8. shanta v, krishnamurthi s, gajalakshmi ck, swaminathan r, ravichandran k. epidemiology of cancer of the cervix: global and national perspective. j indian med assoc. 2000;98:49–52. 9. mcgraw sl, ferrante jm. update on prevention and screening of cervical cancer. world j clin oncol. 2014;5:744–752. 10. rezaianzadeh a, mokhtari am, hassanipour s, maghsoudi a, dehghani sl, nazarzadeh m, et al. the age-standardized incidence rate of ovarian cancer in iranian women: a systematic review and meta-analysis. middle east j cancer. 2018;9:171–178. 11. naing nn. easy way to learn standardization : direct and indirect methods. malays j med sci. 2000;7:10–15. 12. funston g, o’flynn h, ryan naj, hamilton w, crosbie ej. recognizing gynecological cancer in primary care: risk factors, red flags, and referrals. adv ther. 2018;35:577–589. 13. platz ea. reducing cancer burden in the population: an overview of epidemiologic evidence to support policies, systems, and environmental changes. epidemiol rev. 2017;39:1–10. 14. momenimovahed z, salehiniya h. cervical cancer in iran: integrative insights of epidemiological analysis. biomedicine (taipei). 2018;8:18. 15. liberati a, altman dg, tetzlaff j, mulrow c, gotzsche pc, ioannidis jp, et al. the prisma statement for reporting systematic reviews and metaanalyses of studies that evaluate healthcare interventions: explanation and elaboration. bmj. 2009;339:b2700. 16. almasi-hashiani a. farahmand m. trend of incidence rate for female genital cancers based on cancer registry data in fars province during 2003–2009. feyz. 2012;16:353–360 (in persian). 17. arab m, noghabaei g. comparison of age-standard incidence rate trends of gynecologic and breast cancer in iran and other countries. iran j public health. 2014;43:1372–1379. 18. arab m, noghabaei g, kazemi sn. comparison of crude and age-specific incidence rates of breast, ovary, endometrium and cervix cancers in iran, 2005. asian pac j cancer prev. 2014;15:2461–2464. 19. kiadaliri aa. social disparity in breast and ovarian cancer incidence in iran, 2003– 2009: a time trend province-level study. j breast cancer. 2013;16:372–377. 20. mohagheghi ma, mosavi-jarrahi a, malekzadeh r, parkin m. cancer incidence in tehran metropolis: the first report from the tehran populationbased cancer registry, 1998–2001. arch iran med. 2009;12:15–23. 21. mousavi sm, gouya mm, ramazani r, davanlou m, hajsadeghi n, seddighi z. cancer incidence and mortality in iran. ann oncol. 2009;20:556–563. 22. rahimi z, kasraei r, najafi f, tanhapoor m, abdi h, rahimi z, et al. cancer notification at a referral hospital of kermanshah, western iran (2006–2009). asian pac j cancer prev. 2015;16:133–137. 23. sadjadi a, malekzadeh r, derakhshan mh, sepehr a, nouraie m, sotoudeh m, et al. cancer occurrence in ardabil: results of a population-based cancer registry from iran. int j cancer. 2003;107:113–118. 24. sharifian a, pourhoseingholi ma, norouzinia m, vahedi m. ovarian cancer in iranian women, a trend analysis of mortality and incidence. asian pac j cancer prev. 2014;15:10787–10790. 25. taheri n, fazel a, mahmoodzadeh h, omranpour r, roshandel g, gharahjeh s, et al. epidemiology of female reproductive cancers in iran: results of the gholestan population-based cancer registry. asian pac j cancer prev. 2014;15:8779–8782. 26. zendehdel k, sedighi z, hassanloo j, nahvijou a. audit of a nationwide pathology-based cancer registry in iran. basic clin cancer res. 2011;3:7–13. 27. babai m, mousavi s, malek m, danaie n, jandaghi j, tousi j, et al. survey of cancer incidence during a 5-year (1998–2002) period in semnan province. koomesh. 2005;6:237–244. 28. sadjadi a, nouraie m, mohagheghi ma, mousavi-jarrahi a, malekezadeh r, parkin dm. cancer occurrence in iran in 2002, an international perspective. asian pac j cancer prev. 2005;6:359–363. 29. sadiadi a, zahedi mj, moghadam sd, nouraie m, alimohammadian m, ghorbani a, et al. the first population-based cancer survey in kerman province of iran. iranian j publ health. 2007;36:26–34. 30. somi mh, farhang s, mirinezhad sk, naghashi s, seif-farshad m, golzari m. cancer in east azerbaijan, iran: results of a population-based cancer registry. asian pac j cancer prev. 2008;9:327–330. 31. babaei m, jaafarzadeh h, sadjadi ar, samadi f, yazdanbod a, fallah m, et al. cancer incidence and mortality in ardabil: report of an ongoing population-based cancer registry in iran, 2004–2006. iranian j publ health. 2009;38:35–45. 32. masoompour sm, yarmohammadi h, rezaianzadeh a, lankarani kb. cancer incidence in southern iran, 1998–2002: results of population-based cancer registry. cancer epidemiol. 2011;35:e42–e47. 33. fateh m, emamian mh. cancer incidence and trend analysis in shahroud, iran, 2000–2010. iran j cancer prev. 2013;6:85–94. 34. masoompour sm, lankarani kb, honarvar b, tabatabaee sh, moghadami m, khosravizadegan z. changing epidemiology of common cancers in southern iran, 2007–2010: a cross sectional study. plos one. 2016;11:e0155669. 35. singh gk, azuine re, siahpush m. global inequalities in cervical cancer incidence and mortality are linked to deprivation, low socioeconomic status, and human development. int j mch aids. 2012;1:17–30. s. riahi et al. 7j contemp med sci | vol. 5, no. 1, january–february 2019: 1–7 review article cervix cancer in iran 36. khorasanizadeh f, hassanloo j, khaksar n, taheri sm, marzaban m, rashidi bh, et al. epidemiology of cervical cancer and human papilloma virus infection among iranian women analyses of national data and systematic review of the literature. gynecol oncol. 2013;128:277–281. 37. mehrabani d, tabei sz, heydari st, shamsina sj, shokrpour n, amini m, et al. cancer occurrence in fars province, southern iran. iran red crescent med j. 2008;10:314–322. 38. fallah m. cancer incidence in five provinces of iran: ardebil, gilan, mazandaran, golestan and kerman, 1996–2000, tampere university press, 2007. 39. roshandel g, boreiri m, sadjadi a, malekzadeh r. a diversity of cancer incidence and mortality in west asian populations. ann glob health. 2014;80:346–357. 40. salehiniya h, dashdebi sg, rafiemanesh h, mohammadian-hafshejani a, enayatrad m. time trend analysis of cancer incidence in caspian sea, 2004–2009: a population-based cancer registries study (northern iran). caspian j intern med. 2016;7:25–30. 41. saadat s, yousefifard m, asady h, moghadas jafari a, fayaz m, hosseini m. the most important causes of death in iranian population; a retrospective cohort study. emerg (tehran). 2015;3:16–21. 42. rezaianzadeh a, hassanipour azgomi s, mokhtari am, maghsoudi a, nazarzadeh m, dehghani sl, et al. the incidence of breast cancer in iran: a systematic review and meta-analysis. j anal oncol. 2016;5:139–145. 43. hassanipour s, mokhtari a, fathalipour m, salehiniya h. the incidence of lung cancer in iran: a systematic review and meta-analysis. world cancer res j. 2017;4:e980. 44. rezaianzadeh a, jalali m, maghsoudi a, mokhtari am, azgomi sh, dehghani sl. the overall 5-year survival rate of breast cancer among iranian women: a systematic review and meta-analysis of published studies. breast dis. 2017;37:63–68. 45. hassanipour s, fathalipour m, salehiniya h. the incidence of prostate cancer in iran: a systematic review and meta-analysis. prostate int. 2018;6:41–45. 46. hassanipour s, namvar g, fathalipour m, salehiniya h. the incidence of kidney cancer in iran: a systematic review and meta-analysis. biomedicine (france). 2018;8:22–27. 47. salehiniya h, hassanipour s, mansour-ghanaei f, mohseni s, joukar f, abdzadeh e, et al. the incidence of esophageal cancer in iran: a systematic review and meta-analysis. biomed res ther. 2018;5:2493–2503. 48. chung hh, jang mj, jung kw, won yj, shin hr, kim jw, et al. cervical cancer incidence and survival in korea: 1993–2002. int j gynecol cancer. 2006;16:1833–1838. 49. yeole bb. trends in cancer incidence in female breast, cervix uteri, corpus uteri, and ovary in india. asian pac j cancer prev. 2008;9:119–122. 50. bener a, ayub h, kakil r, ibrahim w. patterns of cancer incidence among the population of qatar: a worldwide comparative study. asian pac j cancer prev. 2008;9:19–24. 51. esmaeili r, ahmadi f, mohammadi e, tirgari seraj a. life threatening: the most important concern of patients confronting cancer diagnosis. j hayat. 2013;18:12–22. 52. yang x, cheng y, li c. the role of tlrs in cervical cancer with hpv infection: a review. signal transduct target ther. 2017;2:17055. 53. chaichian s, khateri s, moradi y, shadmani fk, mansori k, khazaei z, et al. trends in cervical cancer incidence in iran from 2003 to 2009. middle east j cancer. 2018;9:57–63. 54. refaei m, dehghan nayeri n, khakbazan z, pakgohar m. cervical cancer screening in iranian women: healthcare practitioner perceptions and views. asian pac j cancer prev. 2017;18:357–363. 55. keramatinia a, hassanipour s, nazarzadeh m, wurtz m, monfared ab, khayyamzadeh m, et al. correlation between nitrogen dioxide as an air pollution indicator and breast cancer: a systematic review and metaanalysis. asian pac j cancer prev. 2016;17:419–424. 56. zheng w, shu xo, mclaughlin jk, chow wh, gao yt, blot wj. occupational physical activity and the incidence of cancer of the breast, corpus uteri, and ovary in shanghai. cancer. 1993;71:3620–3624. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 33j contemp med sci | vol. 4, no. 1, winter 2018: 33–38 research extraction, purification and therapeutic use of bacteriophage endolysin against multi-drug resistant staphylococcus aureus: in vivo and in vitro study mohammed r. ali,a ahmed s.abdulamir,a and shurooq r. kadhimb adepartment of microbiology, college of medicine, al-nahrain university, baghdad, iraq. bcollege of pharmacy, al-mustansiriya university, baghdad, iraq. correspondence to mohammed r. ali (email: dr_mohamadrazak@colmed-alnahrain.edu.iq). (submitted: 12 january 2018 – revised version received: 24 january 2018 – accepted: 10 february 2018 – published online: 26 march 2018) introduction bacterial infections are responsible for significant morbidity and mortality in clinical settings.1 many infections that would have been cured easily by antibiotics in the past now are resistant, resulting in sicker patients and longer hospitalizations. the endolysins of the bacteriophages are highly evolved molecules that have been specifically developed by phages to quickly and efficiently allow their progeny to be released from the host bacterium. these enzymes damage the bacterial cell wall’s integrity by hydrolyzing the four major bonds in its peptidoglycan component.2 the endolysins that have been characterized are amidases; usually do not have signal sequences to translocate them through the cytoplasmic membrane and cleave their substrate in the peptidoglycan. instead, the endolysins’ translocation is controlled by a second phage gene products called holins.3 the bacteriophages during development in the infected bacterium, endolysin accumulates in the cytoplasm in until bacteriophage maturation. during a specific time controlled genetically, holin molecules inserted in the cytoplasmic membrane are activated, resulting in the formation of pores so the preformed endolysin in the cytoplasm can access the peptidoglycan, thereby causing cell lysis and the release of the new bacteriophages.3 bacteriophages are able to lyse their targeted bacterial hosts, and this has been known for almost a century, and since the late 1910s bacteriophages have been used to prevent and treat human and animal diseases of bacterial origin. but bacteriophage-encoded enzymes have only recently begun to be used for various applications; e.g.: reducing bacterial contamination in dairy products and the preparation of bacterial vaccines.4,5 materials and methods sampling and processing of bacteria samples of bacteria were collected in al-imamein al kadhymain medical city hospital. a total of 50 different staphylococcus aureus isolates were collected from bacteriology laboratory. the specimens were collected from hospitalized patients and outpatients suffering from severe urinary tract infection, otitis media, skin infection, and septicemia. the specimens were cultured in screw universal tubes containing nutrient broth wrapped by parafilm or by using sterile swabs; both were put in ice bags and were transferred at the same day to the laboratory of medical microbiology department in the college of medicine, al-nahrain university to subculture on nutrient agar or to be stored in refrigerator at 4°c for 24 h.6 after storing the samples, under aseptic conditions, for 24 h at 4°c in a refrigerator, specimens were plated on nutrient agar in streaking methods. staphylococcus aureus formed smooth round colonies with a fluorescent golden color in nutrient agar. single colonies of s. aureus were isolated from a growing stock by abc streaking on nutrient agar plates to isolate single discrete colonies. the media were prepared according to the manufacturing instructions and were sterilized by autoclaving at 121°c for 15 min. the bacteria were stocked in luria–bertani broth (lb) containing glycerol (30% v/v), preserved at −20°c. diagnosis of the isolated bacteria relied first on conducting catalase test to distinguish from streptococci. then coagulase test was done and positive results confirm the diagnosis of issn 2413-0516 background resistant infection with multidrug-resistant staphylococcus aureus representing a real problem for health care providers. bacteriophage lytic enzymes or lysins are highly evolved molecules that have been specifically developed by phages to quickly and efficiently allow their progeny to be released from the host bacterium while destructing that bacterium. objectives isolation of endolysin from s. aureus bacteriophages, and administering them systemic in vivo lab animal and measure the therapeutic efficacy, as well as evaluation of their biosafety. method this study was performed from march 2015 to august 2017, 50 bacteriological samples of s. aureus were collected, and examined with their anti-biogram, then bacteriophage cocktails were done for five resistant strains of them. endolysins were extracted from their corresponding bacteriophages, they were characterized and the enzymatic and antibacterial activities assays were performed on them in addition to in vivo trial on animals regarding the therapeutic effect of these extracted endolysins on laboratory mice. results this study showed that the extracted endolysin from these bacteriophages was effective in treating laboratory mice from bacteremia with s. aureus and saving their lives when injected intraperitoneal. conclusion endolysin can be extracted directly from their bacteriophages and used by injection of mice with bacteremia with the proper dose of the extracted endolysin of the corresponding bacteriophages which was effective in all of them. keywords bacteriophage, endolysin, resistant infection, s. aureus abbreviations edta, ethylenediaminetetraacetic acid; lb, luria–bertani broth; mdr, multi-drug resistant; pbs, phosphate buffer saline; psa, phage to staphylococcus aureus 34 j contemp med sci | vol. 4, no. 1, winter 2018: 33–38 extract and therapeutic use of endolysin against s. aureus research mohammed r. ali et al. s. aureus with gram staining, which shows gram-positive cocci in clusters. hence, the identification of s. aureus was confirmed. sampling and processing of bacteriophages different crude samples for phage isolation were obtained from different regions in baghdad including sewage (30–40 ml), waste water (30–40 ml), feces of sheep (20 g), chicken litter (15–20 g), swab from surgical lounge in al imamein al-kadhymain medical city hospital during the period from january 2015 to june 2015. the samples were put in clean test tubes wrapped by parafilm, put in ice bag and transported to the laboratory in the same day. part of the samples was stored in the refrigerator at 4°c until used, other part was worked in the same day. primary phages are those phages that were isolated from environmental specimens when were mixed with target bacteria. the procedure of isolating and propagating primary phages was done according to the methodology conducted in a recent patent.7 (1) bacterial stocks were prepared by growing bacteria overnight on nutrient broth. about 100 µl of 10 bacterial isolates were mixed together in a sterile 50 ml test tube. then, 2–3 ml of crude samples, which were derived from sewage, cattle feces, chicken litter, mastitis discharge swabs that might contain s. aureus specific phages were added to the mixture. then, 2–3 ml, equal volume, of nutrient broth and 2 ml of lambda buffer were added to the mixture as well. then, the mixture was incubated overnight at 37°c. (2) next day, 5 ml of the crude mixture were dispensed into a sterile 15 ml test tube, centrifuged at 1,000 g for 3 min at room temperature. (3) one ml of supernatant was transferred to 1.5 ml eppendorf tube. then, 1:10 v/v chloroform was added to the supernatant with gentle shaking for 7–10 min at room temperature to lyse the remaining bacteria. (4) centrifugation of the eppendorf tubes at 1,000 g for 3 min; the supernatant was transferred into new eppendorf tube and equal volume of lambda buffer was added. thus, the primary phage suspension, if any, was produced. virulent phages were screened by phage spotting test on a nutrient-agar. phage spotting can be used to provide a first approximation of the ability of a phage to lyse certain bacterial isolates. the formation of clear zones suggested the presence of lytic phages.8 at first, the target bacteria were refreshed in nutrient broth at 37°c for 24 h. after overnight incubation, 200 µl of the bacterial broth were poured on to the nutrient agar plate to make bacterial lawn. after 2–3 min, the lawn should have been dried. using a mechanical pipette, 10 µl of primary phage suspension were dropped on to the surface of the bacterial lawn and were allowed to dry before incubating at 37°c for 24 h. on the next day, a lytic and specific phage can be discovered for the target bacteria if zone of lysis was developed at the spot where the primary phage suspension was applied. then, specific lytic phages to mdr s. aureus were picked up by sterile loop and put into 1 ml of lambda buffer in 1.5 ml sterile eppendorf tubes with gentle shaking for 5 min. about 1:10 v/v chloroform was added to the lysate with gentle shaking for 5–7 min at room temperature. host cell debris was pelleted by centrifugation at 1,000g for 3 min, and the supernatant containing phages was transferred to 1.5 ml sterile eppendorf tubes and stored at 4°c. the supernatant was called transient phage stock suspension.9 extraction of endolysin up to 100 ml of broth containing bacteria (s. aureus) was incubated for 18 h at 37°c. next day, the bacteria was put in 1 l of broth for 3 h (1 × 1012). a total of 300 ml of the bacteriophage at titer 1 × 1013 pfu/ml was added for 20 min (1:10 moi) after dividing the total volume in 50 ml tubes and then were put directly in ice. they were centrifuged at 10,000 rpm for 15 min and the sediment was taken. the sediment was put in 6 ml of 0.05 m phosphate buffer + deoxyribonuclease (5 mg). then it was incubated for 60 min at 37°c. ethylenediaminetetraacetic acid (edta) (0.005 m) was added and centrifuged at 10,000 rpm for 1 h and the supernatant was taken. disodium tetrathionate (0.3 m) was added and mixed for 1 h at 4°c. ammonium sulfate was added to 85% saturation and incubated for 18 h at 4°c. next day, this was centrifuged at 10,000 rpm for 1 h. then the mixture was resuspended in 5 ml of 0.05 m phosphate buffer (ph 6.1). dialysis against 200 ml of the phosphate buffer saline (pbs) with (2×) conc. and ph = 6.1 at 4°c overnight was done. then it was added to column chromatography sephadex g100 in 0.1 m phosphate buffer (nacl = 8 g/l, kcl = 0.2 g/l, na2hpo4 = 1.4 g/l, kh2po4 = 0.2 g/l) ph 6.1, in 18 × 0.5 cm column. they were collected in 0.5 ml eppendorf tubes at 10 min intervals. from each eppendorf tube, 10 µl was dropped by using automatic pipette to bacterial lawns of the specific bacteria to see which eppendorf tube contain the endolysin, if any. in vivo therapeutic challenge of the extracted phage endolysin albino rattus norvegicus males of mean body weight 25 ± 1.5 g per mouse; the age of these mice was 2 months. the therapeutic effect of extracted endolysin was evaluated by using three groups of mice for each bacterium (s. aureus) each group was composed of five mice. the first two groups of mice received intraperitoneal (ip) injections of 400 µl aliquots of bacterial suspension at concentration 108 cfu/ml. one of these groups, 0.4 ml of (80 µg/ml for staphylococcus and 20 µg/ml for pseudomonas) of a specific endolysin was injected 3 h after the bacterial challenge to evaluate the ability of administered endolysin to rescue the tested bacteremic mice from the inevitable fate of death by a bacterial infection (220). on the other hand, the third group received only ip injection of 0.4 ml of (80 µg/ml for staphylococcus and 20 µg/ml for pseudomonas) of a specific endolysin. after disinfecting the area of injection by 70% alcohol, the first group, control group, mice were injected ip with 108 cfu/ml of the bacterial isolate alone. then, every hour, the mice of control, test, and endolysin only groups were monitored for their health and physical activities and timely health score was recorded. according to biswas,10 certain health scoring system was used as shown in table 1. mohammed r. ali et al. 35j contemp med sci | vol. 4, no. 1, winter 2018: 33–38 research extract and therapeutic use of endolysin against s. aureus table 4. changes of optical density of bacterial broth with endolysin addition time (min) od of sa1* od of sa2 od of sa3 0 1.34 1.34 1.34 5 1.3 1.32 1.29 10 1.25 1.27 1.24 15 1.21 1.24 1.16 20 1.12 1.19 1.09 25 1.05 1.14 1.02 30 0.96 1.07 0.94 35 0.85 0.98 0.85 40 0.76 0.92 0.74 45 0.64 0.83 0.63 50 0.56 0.74 0.5 55 0.52 0.65 0.46 60 0.49 0.56 0.43 δ od/min 0.014 0.013 0.015 *net od of sa1–3 after deducting od of the broth only (0.46). od, optical density; sa, staphylococcus aureus. results bacteriophage cocktails in this study, three phages active against s. aureus were isolated and purified. all of the isolated phages formed visible plaques in the early stage when tested on bacterial lawn of specific mdrs s. aureus. they were isolated directly from the environment by showing lysis on bacterial lawns of mdr s. aureus as shown in fig. 1. characteristics of these phages were determined by the diameter, clarity/turbidity, margin cut, and shape of their plaques. the size of plaques ranged between 1.5 and 2.6 mm with a mean of 1.97 mm. the plaques morphology of the three phages ranged between oval 1/3(33.33%) and circular 2/3 (66.66%). plaques clarity is different among the three phages; it ranged between clear 1/3 (33.33%), and semi-clear 2/3 (66.66%). margin cut of the four phages were irregular 3/3 (100%) as shown in table 2. endolysin extraction endolysin was successfully extracted from all the three s. aureus bacteriophages by using sephadex g100 column chromatography. table 1. the health scoring system of bacteremic mice health signs health level normal and unremarkable condition 5 slight illness (lethargy and ruffled fur) 4 moderate illness (severe lethargy, ruffled fur, and hunched back) 3 severe illness (severe lethargy, ruffled fur, hunched back, and exudative accumulation around partially closed eyes) 2 moribund state 1 death 0 fig 1. phage spot assay of bacteriophage to staphylococcus aureus. after sephadex g100 chromatography, each bacteriophage lysate gave eight eppendorf tubes of 0.5 ml eluted fluid, one of them showed positive result on corresponding bacterial lawns as shown in table 3. the optical density of bacterial broth for each bacteria was measured initially once at zero time, just before the addition of corresponding endolysin. after the addition of the purified endolysin after 3 days of their extraction with the concentrations (80, 70, and 95 µg/ml), the optical density was measured every 5 min till 1 h. the optical density of the tested bacterial broth was obviously decreasing with time as shown in tables 4 and 5. endolysin therapy fifteen mice were used, ten of them were injected ip with 400 µl aliquots of bacterial suspension of concentration 108 cfu/ml, five of these (group two) injected with 0.5 ml of 80 µg/ml of a specific endolysin after 2 h. while the last five mice (group three) were injected with 0.5 ml of 80 µg/ml of a specific endolysin alone. the result of this experiment showed that the health score of the first group (five mice injected with bacteria alone) started to decrease after 2 h, afterward the health score declined progressively. they died after 8 h. on the other hand the second group table 2. the plaque characteristics of the isolated phages to mdr staphylococcus aureus bacteria bacteriophage isolates plaques size (mm) margin cut plaques clarity plaques shape psa1 1.5 irregular clear circular psa2 2.6 irregular semi-clear oval psa3 1.8 irregular semi-clear circular psa, phage to staphylococcus aureus. table 3. the fraction that endolysin like activity was found for each bacteriophage bacteriophage endolysin positive tube psa1 4th psa2 3rd psa3 4th psa, phage to staphylococcus aureus. 36 j contemp med sci | vol. 4, no. 1, winter 2018: 33–38 extract and therapeutic use of endolysin against s. aureus research mohammed r. ali et al. (five mice injected with bacteria and endolysin) lived for more than 4 weeks with full physical activity and the third group (five mice injected with endolysin alone) also lived for more than 4 weeks with full physical activity as shown in tables 6 and 7. discussion endolysins have been proven as efficient antibacterial agents against major gram-positive pathogens such as s. aureus,11 bacillus anthracis,12 streptococcus agalactiae,13 and streptococcus pneumonia,14 but their potential to combat gram-negative infections remains undemonstrated.15 phage lysin therapy is a possible alternative to antibiotics for the treatment of bacterial infections. indeed, it has proven to be medically superior to antibiotic therapy.16,17 our results support this notion, as phage lysin was shown to be highly efficacious against infections caused by inoculation with antibiotic-resistant s. aureus. in this study, three phages for different s. aureus were isolated and characterized. the phages demonstrated high lytic activity underscoring its great potential to treat s. aureus infections. fig 2. the decline of od values for bacterial growth of sa1 due to lysis by (80 µg/ml) endolysin. fig 3. rate of od change in every 5 min for bacterial growth of sa1 due to lysis by (80 µg/ml) endolysin. fig 4. the decline of od values for bacterial growth of sa2 due to lysis by (70 µg/ml) endolysin. fig 5. rate of od change in every 5 min for bacterial growth of sa2 due to lysis by (70 µg/ml) endolysin. fig 6. the decline of od values for bacterial growth of sa3 due to lysis by (95 µg/ml) endolysin. table 5. pace of od change in every 5 min time (min) δ od of sa1 δ od of sa2 δ od of sa3 5 0.04 0.02 0.05 10 0.05 0.05 0.05 15 0.04 0.03 0.08 20 0.09 0.05 0.07 25 0.07 0.05 0.07 30 0.09 0.07 0.08 35 0.11 0.09 0.09 40 0.09 0.06 0.11 45 0.12 0.09 0.11 50 0.08 0.09 0.13 55 0.04 0.09 0.04 60 0.03 0.09 0.03 od, optical density; sa, staphylococcus aureus. mohammed r. ali et al. 37j contemp med sci | vol. 4, no. 1, winter 2018: 33–38 research extract and therapeutic use of endolysin against s. aureus the experiments presented here revealed that a single intraperitoneal injection of the corresponding lysin rescued mice from death due to s. aureus even when bacteremia was already well-established. we observed that effective protection fig 7. rate of od change in every 5 min for bacterial growth of sa3 due to lysis by (95 µg/ml) endolysin. table 6. health score of mice injected with bacteria alone or with endolysin hours health scores bacteremic group 1 median of health scores of bacteremic group 1 health scores of test group 2 (bacteria + endolysin) median of health scores of test group 2 (bacteria + endolysin) p-value (mann whitney test) 0 5,5,5,5,5 5 5,5,5,5,5 5 0.920 2 3,3,4,3,2 3 3,4,3,3,4 3 0.465 4 3,2,3,3,1 3 5,5,4,4,5 5 0.012 6 1,1,1,1,0 1 5,5,5,5,5 5 0.012 8 0,0,0,0,0 0 5,5,5,5,5 5 0.012 table 7. health score of mice injected with endolysin alone or nothing hours health scores endolysin only group 3 median of health scores of endolysin only group 3 health scores of healthy group 4 median of health scores of healthy group 4 p-value (mann whitney test) 0 5,5,5,5,5 5 5,5,5,5,5 5 0.920 2 5,5,5,5,5 5 5,5,5,4,5 5 0.674 4 5,5,5,5,5 5 5,5,4,4,5 5 0.347 6 5,5,5,5,5 5 5,5,5,5,5 5 0.920 8 5,5,5,5,5 5 5,5,5,5,5 5 0.920 was achieved in mice when a single dose of 0.5 ml of 80 µg/ml of a specific endolysin was administered 2 h after inoculation of s. aureus. the results obtained in this experiment is encouraging, since our findings and those of others18 support the development in a near future of modified procedures to improve the use of lysins in a more efficient way. for example, intravenous administration of the enzymes may confer a superior protection to animals when used later after the challenge. most interestingly, a single intraperitoneal injection of endolysin was sufficient for a complete cure of mice. it also provides a rapid and specific lytic activity, making these proteins very promising candidates in current antimicrobial therapies. we have shown here that bacteriophage lysins from s. aureus can be used to reduce the bacterial burden of these bacteria efficiently which are multidrug resistant, both in vitro and in vivo. our study emphasize the potential therapeutic role of phage lysins for treatment of gram positive bacterial infections. animal studies have generally supported the utility and safety of endolysin phage therapy against bacterial pathogens, like s. aureus.19,20 in this study, endolysin phage therapy of septicemic laboratory mice, through ip injection with mdr s. aureus, showed that administration of endolysin resolved the infection dramatically. it was found that the treatment with endolysin provided significant protection against mortality in mice infected with inocula of 108 cfu of highly virulent s. aureus. the current results demonstrated the in vitro efficacy of endolysin phage therapy and a proof for the concept of successful in vivo endolysin phage therapy. the route of endolysin phage administration was particularly important to the efficacy of the treatment. intraperitoneal route provides significant protection similar to that of intravenous route.21 it was stated that endolysin for s. aureus phages administered by the ip route are distributed, in high titers, more rapidly and delivered for a more sustained period of time to all of the tissues.21 in these experiments, the control group of mice, which were bacterially infected without endolysin phage therapy, showed that all the mice died within just 8 h. actually using a severe model of sepsis that leads to 100% lethality of mice in less than 24 h is a difficult challenge for every imaginable antibacterial agent. two hours after bacterial injection, health score of mice began to decline progressively. a single injection of endolysin, rescued 100% of mdr s. aureus septicemic mice. thus, this study showed that endolysin administered in a single dose 2 h post bacterial infection resulted in 100% survival of treated mice when compared to the control group. n references 1. wise r. the relentless rise of resistance? j antimicrob chemother. 2004;54:306–310. 2. young r. bacteriophage lysis: mechanism and regulation. microbiol rev. 1992;56:430–481. 3. wang i, smithand d, young r. the protein clocks of bacteriophage infections. annu rev microbiol. 2000;54:799–825. 4. szostak m, hensel a, eko f, klein r, auer t, mader h, et al. bacterial ghosts: non-living candidate vaccines. j biotechnol. 1996;44: 161–170. 5. gaeng s, scherer s, neve h, loessner m. gene cloning and expression and secretion of listeria monocytogenes bacteriophage-lytic enzymes in lactococcus lactis. appl environ microbiol. 2000;66:2951–2958. 6. bauer a, kirby w, scherris jc, turck m. antibiotic susceptibility testing by a standardized single disc method. am j clin pathol. 1966;45:493–496. 7. budzik jm, rosche wa, rietsch a, o’toole ga. isolation and p. aeruginosa crispr/cas and bacteriophage resistance a generalized transducing phage for pseudomonas aeruginosa strains pao1 and pa14. j bacteriol. 2004;186:3270–3273. 38 j contemp med sci | vol. 4, no. 1, winter 2018: 33–38 extract and therapeutic use of endolysin against s. aureus research mohammed r. ali et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 8. jassim sa, abdualamir as, abu baker f. methods for bacteriophage design in “international application published under the patent cooperation (pct)” (w.i.p. organization, ed.), 2010. vol.wo2010064044 a1. 9. hall-stoodley l, costerton j, stoodley p. bacterial biofilms: from the natural environment to infectious diseases. nat rev microbiol. 2004;2:95–108. 10. biswas b, adhya s, washart p, paul b, trostel an, powell b. bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycinresistant enterococcus faecium. infect immun. 2002;70:204–210. 11. rashel m, uchiyama j, ujihara t, uehara y, kuramoto s, sughihara s, et al. efficient elimination of multidrug-resistant staphylococcus aureus by cloned lysin derived from bacteriophage umr11. j infect dis. 2007;196:1237–1247. 12. schuch r, nelson d, fischetti va. a bacteriolytic enzyme that detects and kills bacillus anthracis. nature. 2002;418:884–889. 13. cheng q, nelson d, zhu s, fischetti va. removal of group b streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme. antimicrob agents chemother. 2005;49:111–117. 14. loeffler jm, djurkovic s, fischetti va. phage lytic enzyme cpl-1 as a novel antimicrobial for pneumococcal pneumonia. infect immun. 2003;71:6199–6204. 15. briers y, walmagh m, lavigne r. use of bacteriophage endolysin el188 and outer membrane permeabilizers against pseudomonas aeruginosa. j appl microbiol. 2011;110:778–785 (issn 1364-5072). 16. donovan dm. bacteriophage and peptidoglycan degrading enzymes with antimicrobial applications. recent pat biotechnol. 2007;1: 113–122. 17. jingmin g, wei x, liancheng l, jing h, xin f, changjiang s, et al. lysgh15, a novel bacteriophage lysin, protects a murine bacteremia model efficiently against lethal methicillin-resistant staphylococcus aureus infection. j clin microbiol. 2011;49:111–117. 18. schuch r, nelson d, fischetti va. a bacteriolytic agent that detects and kills bacillus anthracis. nature. 2002;418:884–889. 19. mcvay cs, velasquez m, fralick ja. phage therapy of pseudomonas aeruginosa infection in a mouse burn wound model. antimicrob agents chemother. 2007;51:1934–1938. 20. soothill js. bacteriophage prevents destruction of skin grafts by pseudomonas aeruginosa. burns. 1994;20:209–211. 21. catherine sm, velásquez m, fralick ja. doi: antimicrob agents chemother. 2007;51:1934. dx.doi.org/10.22317/jcms.03201808 20 j contemp med sci | vol. 1, no. 3, summer 2015: 20–23 research objective interleukin (il)-15 is highly expressed in skeletal muscles, where it exerts anabolic effects, increase protein content in muscle fibres and promotes muscle growth. alcoholics frequently suffer myopathy. therefore, we analyse the level of il-15 [and other myokines, such as tumor necrosis factor-α (tnf-α)] in alcoholics. follow-up of skeletal muscle cytokines (myokines) such as il-15 and tnf-α level in alcoholism, in an attempt to reveal if a certain level of myokines can be considered as a risk factor for short-term motility. methods il-15 and tnf-α were determined by enzyme-linked immunoassay analytic techniques in blood samples of 70 chronic alcoholics and 70 ageand sex-matched controls, and then the levels of myokines were correlated with liver enzymes aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), gamma glutamate transferase (ggt), amount of ethanol consumed, duration and creatine kinase (ck) activity levels. results all the alcoholic patients were heavy drinkers (217.04 ± 149.93 g/day), who started at an early age (13.97 ± 8.96 years). il-15, tnf-α levels and liver enzyme activity were significantly higher in these patients than in controls. significant relationship was found between il-15, quantity of ethanol consumption, tnf-α, ck, ast/alt and between tnf-α and daily ethanol consumption (quantity) and ggt. conclusion a certain level of myokines such as il-15 and tnf-α can be considered as a risk factor of alcoholics for short-term motility. keywords il-15, tnf-α, alcoholic myopathy, liver impairment myokines in alcoholic myopathy kammela hadi shammam,a fadhil jawad al-tu’mab & hedef dafer el-yassinc introduction interleukin (il)-15 is a relatively newly described cytokine, highly expressed in skeletal muscle. il-15 mrna levels are highly up-regulated in response to strength training, and it seems to exert anabolic effects, increasing protein content in muscle fibres1 and promoting myogenic differentiation and muscle growth. it would exert an opposite effect to that of tumor necrosis factor α (tnf-α), since it is able to antagonize muscle protein break-down in a cancer cachexia model.2 since its discovery in 1994,3 it has become clear that it is secreted by many tissues, such as muscle, kidney, heart, lung, dendritic cells, monocytes and macrophages and also enterocytes,4 but muscle is the major site of il-15 mrna transcription and probably secretion.3 in addition to its actions on muscle, it also exerts many other functions on memory t cells, natural killer (nk) cells, eosinophils, neutrophils, monocytes and macrophages5 and shares stimulatory actions on t-cells with il-2, partly due to the similarity of their respective receptors. indeed, the heterotrimeric il-15 receptor is composed of beta and gamma chains identical to those of the il-2 receptor, together with a specific alpha chain. it is, therefore, not surprising that it acts as a potent activator of t and b lymphocytes. it is also involved in the maintenance of t cell memory and in the activation of other immune cells, such as neutrophils and nk cells.6 it may be also related to the pathogenesis of autoimmune diseases.7 regarding its metabolic function, it probably participates in the cross-talk between muscle and fat, leading to a reduction of the latter. in chronic alcoholism, muscle wasting is a prominent feature. chronic alcoholic myopathy has been found in nearly 50–60% alcoholics,8–10 and it is defined by muscle atrophy, predominantly affecting type iib fibres11,12 and leading, sometimes, to incapacitating weakness. il-15 increased after exercises and physical activity that occurs in alcoholics, due to encourage aggression or violence by disrupting normal brain function. according to the disinhibition hypothesis, for example, alcohol weakens brain mechanisms that normally restrain impulsive behaviours, including inappropriate aggression.13 by impairing information processing alcohol can also lead a person to misjudge social cues. tnf-α, a pro-inflammatory cytokine, also increases in acute or chronic alcoholics due to the impairment of liver. studies have found that alcohol may increase the liver’s sensitivity to inflammatory cytokines, such as tnf-α, in two ways. first, alcohol may directly or indirectly stimulate kupffer cells to produce and release tnf-α into small channels (i.e. sinusoids) in which the blood flows through the liver. one indirect mechanism is that the alcohol induces an increase in the levels of a bacterial endotoxin in the blood. second, alcohol may enhance the sensitivity of hepatocytes to tnf-α.14 the processes by which alcohol is broken down in the hepatocytes generate a variety of molecules that can be toxic to the liver, or it may interfere with normal physiological processes. for example, alcohol breakdown through the enzyme known as cytochrome p450 2e1(cyp2e1) leads to the formation of small oxygen-containing molecules called reactive oxygen species (ros). unless they are rapidly eliminated or converted into harmless molecules by antioxidants, it can interact with and damage complex molecules in the cells (e.g. proteins and dna).15 both increased and decreased levels of ros can lead to apoptosis of hepatocytes. materials and methods seventy alcoholic patients were included (patient group), who were admitted to ibn-rushd teaching hospital, baghdad, a institute of forensic medicine, ministry of health, baghdad, iraq. b department of biochemistry, college of medicine, university of karbala, karbala, iraq. c department of biochemistry, college of medicine, university of baghdad, baghdad, iraq. correspondence to fadhil jawad al-tu’ma (email: f_altoma_56@yahoo.com). (submitted: 17 april 2015 – revised version received: 21 may 2015 – accepted: 23 may 2015 – published online: summer 2015) issn 2413-0516 21j contemp med sci | vol. 1, no. 3, summer 2015: 20–23 research myokines in alcoholic myopathykammela hadi shammam et al. iraq. their mean [± standard deviation (sd)] age was 40.35 ± 11.01 years. all of them were heavy drinkers of ethanol (>217 g/day) during a prolonged period (>13 years). in the control group (70 subjects), the mean (± sd) age was 33.0 ± 7.83 years, and they were nonalcoholic. some clinical and biochemical parameters of patients and controls are shown in table 1. cytokines and biochemical parameters blood samples were taken at 8.00 am in fasting conditions, and were immediately frozen at −80°c. the following parameters were determined: tnf-α by enzyme linked immune sorbent assay (elisa; range of detection 32–2000 pg/ml); and il-15 by elisa (sensitivity <3 pg/ml; range of detection 15.6–1000 pg/ml). in addition, patients underwent liver enzyme assays, including aspartate and alanine aminotransferases (ast and alt), alkaline phosphatase alp, gamma-glutamyl transferase (ggt) and creatine kinase (ck). biostatistics spearman’s correlation coefficient was used to compare quantitative parameters. when variables did show a normal distribution, student’s t-test and pearson’s test were employed. results as shown in table 1, all the cytokines were determined, including il-15 and tnf-α levels; liver enzyme activity were significantly higher in alcoholic patients than in controls. all the patients were heavy drinkers (217.04 ± 149.93 g/day) with many years of consuption (13.97 ± 8.96 years). significant relationship was found between il-15, quantity of ethanol consumption, tnf-α, ck, ast/ alt and between tnf-α and daily ethanol consumption (quantity) and ggt (see figs. 1–6), respectively. significant correlations were found between some studied parameters, as presented in figs 1–6. discussion the high value of il-15 levels may be due to muscle contraction and violence associated with convulsion and cramps happened to the alcoholic patients, resulted from many alteration in electrolyte, calcium and sodium ions, caused table 1. mean ± sd of serum ast, alt, alp, ck, ggt activity levels, ast/alt activity ratio, il-15 and tnf-α level for studied groups parameter patient group n = 70 mean ± sd control group n = 70 mean ± sd p value ast (u/l) 20.01 ± 9.80 8.68 ± 3.79 0.08 alt (u/l) 13.84 ± 6.27 7.51 ± 2.57 0.030 alp (u/l) 62.98 ± 22.90 50.87 ± 17.81 0.001 ast/alt* 1.68 ± 0.89 0.83 ± 0.24 0.001 ggt (u/l) 97.45 ± 15.58 28.68 ± 13.55 0.061 ck (u/l) 133.68 ± 44.43 50.40 ± 24.49 0.001 il-15 (pg/ml) 59.01 ± 11.09 34.92 ± 9.03 0.053 tnf-α (pg/ml) 83.42 ± 10.12 41.68 ± 15.74 0.072 alt: alanine aminotransferase; ast: aspartate aminotransferase; alp: alkaline phosphate; ck: creatine kinase; ggt: gamma glutamate transferase; il-15: interleukin-15; tnf-α: tumor necrosis factor-alpha. fig. 1 correlation between il-15 level and quantity of ethanol consumed per day in alcoholic patients group. fig. 2 correlation between il-15 level and tnf-α in alcoholic patients group. 22 j contemp med sci | vol. 1, no. 3, summer 2015: 20–23 myokines in alcoholic myopathy research kammela hadi shammam et al. by changes in the membrane permeability, due to lipid peroxidation which occurred from reaction of acetaldehyde and phospholipids of muscle cells wall.16 il-15 mrna levels were up-regulated in human skeletal muscle following strength training, suggesting that il-15 may accumulate within the muscle as a consequence of regular training or physical activity as happened to alcoholics.17 oxidative stress produced from alcohol consumption is associated with numerous deleterious consequences for the cell (e.g. lipid peroxidation or even cell apoptosis or necrosis) and leads to different enzyme leakage to outside the cells, causing an increase in the level of these enzymes in the blood. so there were appositive correlation between il-15, quantity of ethanol consumed, ck and ast/alt ratio as shown in figs. 1, 3 and 4, respectively. the study found positive correlation between il-15 and tnf-α (fig. 2) due to liver impairment that happened in chronic alcohol due to damaging its cells (kupffer cells) which converted to macrophages when activated by viruses or any foreign substances like alcohol, and endotoxins which were secreted from intestine bacteria due to alcohol effects travel to blood stream, then to kupffer cells which activate them and secrete numerous cytokines including tnf-α.18 tnf-α was higher in alcoholics than in nonalcoholics (table 1), this result agree with what was mentioned about the effects of alcohol consumption on different body organs. one of the factors that can enhance the production of tnf-α is alcohol, endotoxin is released from the bacteria (gram-negative) normally living in the intestine when those bacteria die from alcohol and acetaldehyde (toxic substance). if endotoxin enters the blood stream and reaches the liver, it interacts with kupffer cells, activating the cells to produce cytokines. in a healthy person, endotoxin interacts primarily with kupffer cells, and this interaction is considered crucial to secretion of tnf-α, which then interacts with receptors on both kupffer cells and hepatocytes. endotoxins seem to be important in the development of early alcoholic liver disease. thus tnf-α was correlated positively with quantity and ggt, figs. 5 and 6, respectively. alcohol fig. 3 correlation between il-15 and ck activity level in alcoholic patient group. fig. 4 correlation between il-15 level and ast/alt ratio in alcoholic patient group. fig. 5 correlation between tnf-α and quantity of ethanol consumed per day in alcoholic patient group. 23j contemp med sci | vol. 1, no. 3, summer 2015: 20–23 research myokines in alcoholic myopathykammela hadi shammam et al. references 1. furmanczyk ps, quinn ls. interleukin 15 increases myosin accretion in human skeletal myogenic cultures. cell biol int. 2003;27:845–51. doi: http:// dx.doi.org/10.1016/s1065-6995(03)00172-0 pmid: 14499665 2. carbó n, lópez soriano j, costelli p, alvarez b, busquets s, baccino fm, et al. interleukin 15 mediates reciprocal regulation of adipose and muscle mass: a potential role in body weight control. br j cancer 2000;83:526–31. 3. grabstein kh, eisenman j, shanebeck k, rauch c, srinivasan s, fung v, et al. cloning of a t cell growth factor that interacts with the beta chain of the interleukin-2 receptor. science 1994 may;264(5161):965–8. doi: http:// dx.doi.org/10.1126/science.8178155 pmid: 8178155 4. van heel da. interleukin 15: its role in intestinal inflammation. gut. 2006 apr;55(4):444–5. doi: http://dx.doi.org/10.1136/gut.2005.079335 pmid: 16531523 5. quinn ls. interleukin 15: a muscle-derived cytokine regulating fat-to-lean body composition. j anim sci. 2008 apr;86(14 suppl):e75–e83. doi: http:// dx.doi.org/10.2527/jas.2007-0458 pmid: 17709786 6. fehniger ta, caliguri ma. interleukin 15: biology and relevance to human disease. blood 2001 jan 1;97(1):14–32. doi: http://dx.doi.org/10.1182/blood. v97.1.14 pmid: 11133738 7. waldmann ta. targeting the interleukin-15/interleukin 15 receptor system in inflammatory autoimmune diseases. arthritis res ther. 2004;6(4):174–7. pmid: 15225362 8. peters tj, martin f, ward k. chronic alcoholic myopathy—common and reversible. alcohol 1985 may;2(3):485–9. doi: http://dx.doi. org/10.1016/0741-8329(85)90120-x 9. urbano-márquez a, estruch r, navarro-lopez f, grau jm, mont l, rubin e. the effects of alcoholism on skeletal and cardiac muscle. n engl j med. 1989 feb 16;320(7):409–15. doi: http://dx.doi.org/10.1056/ nejm198902163200701 pmid: 2913506 also increased tnf-α by another way, it oxidises to acetaldehyde, which reacts with proteins and dna and to form adducts. these adducts induce certain immune cells (recognize these adducts as foreign bodies) to produce many deafens cells like interleukins, interferon’s and pro-inflammatory molecules like tnf-α. conclusion il-15 and tnf-α levels were higher in iraqi alcoholics than in nonalcoholic. both of them exert dangerous effects on the body and can consider as a risk factor for short-term motility.  fig. 6 correlation between tnf-α level and ggt activity levels in alcoholic patient group. 10. preedy vr, paice a, mantle d, dhillon as, palmer tn, peters tj. alcoholic myopathy: biochemical mechanisms. drug alcohol depend. 2001 aug;63(3):199–205. doi: http://dx.doi.org/10.1016/s0376-8716(00)00219-2 11. preedy vr, marway js, macpherson ajs, peters tj. ethanol-induced smooth and skeletal muscle myopathy: use of animal studies. drug alcohol depend. 1990 aug;26:1–8. doi: http://dx.doi.org/10.1016/0376-8716(90)90076-q pmid: 1698602 12. conde-martel a, gonzález-reimers e, gonzález-hernández t, santolaria f, martinez-riera a, romero-perez jc, et al. relative and combined roles of ethanol and protein malnutrition on skeletal muscle. alcohol alcohol. 1992 mar;27(2):159–63. pmid: 1524607 13. gustafson r. alcohol and aggression. j offender rehabil. 1994;21(3/4):41–80. 14. nanj aa, jokelainen k, fotouhinia m, rahemtulla a, thomas p, tipoe gl, et al. increased severity of alcoholic liver injury in female rats: role of oxidative stress, endotoxin, and chemokines. am j physiol gastrointest liver physiol. 2001 dec;281(6):g1348–g1356. pmid: 11705739 15. wu d, cederbaum ai. oxidative stress and alcoholic liver disease. semin liver dis. 2009 apr 22;29(2):141–54. doi: http://dx.doi. org/10.1055/s-0029-1214370 16. wickramasinghe sn, marjot dh, rosalki sb, fink rs. correlations between serum proteins modified by acetaldehyde and biochemical variables in heavy drinkers. j clin pathol. 1989 mar;42(3):295–9. doi: http://dx.doi. org/10.1136/jcp.42.3.295 pmid: 2703546 17. nielsen ar, hojman p, erikstrup c, fischer cp, plomgaard p, mounier r, et al. association between il-15 and obesity: il-15 as a potential regulator of fat mass. j clin endocrinol metab. 2008 nov;93(11):4486–93. doi: http://dx.doi. org/10.1210/jc.2007-2561 pmid: 18697873 18. neuman mg. apoptosis in diseases of the liver. critic rev clin lab sci. 2001;38:109–66. 35j contemp med sci | vol. 5, no. 1, january–february 2019: 35–40 original article pomegranate protects renal proximal tubules during gentamicin induced-nephrotoxicity in rats hayder m. al-kuraishy,* ali i. al-gareeb, and marwa s. al-naimi department of clinical pharmacology, medicine and therapeutic, medical faculty, college of medicine, al-mustansiriya university, baghdad, iraq. *correspondence to hayder m. alkuraishy (email: hayderm36@yahoo.com) (submitted: 14 october 2018 – revised version received: 19 november 2018 – accepted: 10 december 2018 – published online: 26 february 2019) objective to evaluate the nephroprotective effect of pomegranate in gentamicin induced-nephrotoxicity in rats. methods thirty sprague-dawley male rat were used, which randomly divided into three groups, 10 rats in each group. group 1 (n = 10): rats treated with distilled water for 12 days. group 2 (n = 10): rats treated with distilled water + gentamicin. group 3 (n = 10): rats treated with pomegranate + gentamicin. blood urea, serum creatinine, serum malondialdehyde (mda), superoxide dismutase (sod), glutathione reductase (gsh) , neutrophil gelatinase associated lipocalin (ngal), kidney injury molecules (kim-1), and cystatin c were measured. results blood urea and serum creatinine were significantly elevated in gentamicin group compared with the control group (p = 0.007 and 0.04) respectively. also, estimated gfr was significantly decreased in gentamicin group compared with the control group (p = 0.04). cystatin-c and mda serum level were increased but sod and gsh were decreased insignificantly in gentamicin group compared with the control group (p > 0.05). pomegranate reduced blood urea significantly (p = 0.002). pomegranate improves endogenous anti-oxidant capacity, since it increase gsh significantly compared with gentamicin group (p = 0.02). there were insignificant effects on mda and sod. moreover, pomegranate reduce serum levels of kim-1and ngal in gentamicin group (p < 0.05). conclusion pomegranate attenuates gentamicin induced-nephrotoxicity through potentiation of endogenous anti-oxidant capacity and inhibition of inflammatory pathway. keywords nephrotoxicity, pomegranate, gentamicin, anti-oxidant capacity introduction nephrotoxicity is a renal-specific condition in which the excretions of toxic metabolites are accumulated due to toxic agents and drugs. in relation 20% of nephrotoxicity is induced and caused by drugs; this percentage is augmented in the elderly due to rise in the life span and poly-medications.1 there are diverse mechanisms lead to nephrotoxicity, including renal tubular toxicity, inflammation, glomerular damage, crystal nephropathy, and thrombotic microangiopathy.2 proximal renal tubular cells are in prolonging contact with drugs due to tubular reabsorption and concentration processes. toxic agents and drugs cause potential damage to the tubular transport system through induction of free radical formation and oxidative stress which lead to tubular cell mitochondrial damage.3 the drugs that cause tubular damage are aminoglycoside, amphotercin b, antiviral like adefovir, foscarnet and cytotoxic agents like cisplatin.4 gentamicin is an antibiotic belongs to aminoglycoside group, used for treatment of different bacterial infections, it is bactericidal acts as protein synthesis inhibitor through binding to the 30s subunit of bacterial ribosome.5 actually, 90% of administrated gentamicin is not metabolized by the liver and it is mainly excreted unchanged in the proximal renal tubules leading to extensive necrosis at a higher dose.6 gentamicin enters the cell by specific ion channels and by endocytosis. most of the cells clear gentamicin deposition by efflux it but remains concentrated at renal cortical cells.7 overproduction of reactive oxygen species and free radicals are the main mechanism beyond the gentamicin induced nephrotoxicity via over-expression of cation transporter proteins (megalin and cubilin) at proximal renal tubules that augment the accumulation of gentamicin and free radical generations.8,9 pomegranate (punica granatum l.) is a potent nutraceutical herb used in prevention and treatment of different diseases. it is wealthy in phytochemicals which are related to steroid hormones in their structures that bind estrogenic receptors which called phyto-estrogens.10,11 these compounds are activated by gastro-intestinal enzyme which act as a partial agonist on the estrogenic receptors.12 pomegranate peel contains 50% of bioactive constituents including flavonoids, phenolics, proanthocyanide, and ellagitannins with different minerals. pomegranate’s edible part contains 10% seeds and 40% arils, arils mainly enclose anthocyanins while; the seeds contain mainly anthocyanin and glucosides. punicic acid is the chief component of pomegranate oil.13 pomegranate inhibits lipid peroxidation and scavenge oxidative free radicals via augmentation of tissue catalase, peroxidase and superoxide dismutase in renal and other organs.14 keenly, pomegranate inhibits gene expression of pro-inflammatory cytokines via suppression of nf-κb activation. ellagic acid leads to significant inhibition of prostaglandin e2 through inhibition of cox-2.15,16 regarding the effect of pomegranate on the kidney function, it chiefly affects renal glomeruli through induction of endothelial no by flavonoids which enhance glomerular blood flow. in addition, pomegranate showed less effect on renal medulla with mild increase in the interstitial volume.17 therefore, the aim of this study was to evaluate the nephroprotective effect of pomegranate in gentamicin induced-nephrotoxicity in rats. materials and methods thirty sprague–dawley male rat were used, these animals were gained from the national center for drug control and research. rats age ranged from 3 to 4 months and their body weight ranged from 200 to 400 g. the animals were isolated as three rats in each sterilized cage and placed with suitable issn 2413-0516 36 j contemp med sci | vol. 5, no. 1, january–february 2019: 35–40 pomegranate protects renal proximal tubules during gentamicin induced-nephrotoxicity in rats original article h.m. alkuraishy et al. temperature (22–25°c) with artificial 12/12 light cycle. they were left for 1 week for adaptation without any intervention with free access to normal chow pellets and water. human care for animals was according to the guide to the care and use of laboratory animal. after acclimatization period, weights of rats were taken and rat with wound infection were excluded then rats were randomly divided into three groups, 10 rats in each group. the study protocol and method for induction of aki was according to singh et al.’s18 method. control group (n = 10): rats treated with distilled water (5 ml/kg, p.o.) for 12 days and on days 6–12 received an intraperitoneal (i.p.) injection of normal saline (5 ml/kg) daily. gentamicin group (n = 10): rats treated with distilled water (5 ml/kg, p.o.) for 12 days and on days 6–12 received gentamicin 100 mg/kg, i.p. pomegranate group (n =10): rats treated with pomegranate (50 mg/kg, p.o.) for 12 days and on days 6–12 received gentamicin 100 mg/kg, i.p. at an interval of 1 h. anthropometric measurements length was measured by using graduated tape from nose to the anus (naso-anal length in cm). rat body weight was measured by using specific digital balance in grams. body mass index equal body weight in grams over the square of length in cm, bmi = bw (g)/length (cm)2. estimated glomerular filtration rate (egfr) was measured according to schwartz formula, egfr = k × height (cm)/serum creatinine (mg/dl), k = 0.55.19 sample collection on 13th day, chloroform was used to anesthetize the rats and sharp scissors were used to exudate the rats. the blood sample was allowed to drain in sterile gel tube, and centrifuged for 10 min at 5000 rpm, so the formed supernatant layer was isolated as serum sample and kept in freezer at −20 to be assessed later. assessment of biochemical variables blood urea and serum creatinine were estimated by using an auto-analyzer (ilab-300-biomerieux diagnostic, milano, italy) expressed as mg/dl. serum malondialdehyde (mda), superoxide dismutase (sod), glutathione reductase (gsh), neutrophil gelatinase associated lipocalin (ngal), kidney injury molecules (kim-1) and cystatin c were measured by elisa kit methods according to the instruction of the kit manufacture (mybiosource, usa). statistical analysis statistical package for the social sciences software (spss inc., chicago, il, usa) was used for data analysis. data of this study was presented as mean ± sd and the variables were tested by using unpaired student t-test between control and treated groups. one-way anova test with post-hoc test was used to investigate the significance of differences among different groups. the levels of significance was regarded when p < 0.05. results effects of gentamicin on the renal biomarkers comparison of different variables between controls and gentamicin group showed that there was statistically insignificant difference in the weight and height, of the two groups, while bmi was significantly high in gentamicin group compared with the control group (p = 0.001). both blood urea and serum creatinine were significantly elevated in gentamicin group compared with the control group (p = 0.007 and 0.04), respectively. also, estimated gfr was significantly decreased to 11.19 ± 5.16 ml/min/1.73 in gentamicin group compared with the control group (16.89 ± 4.21 ml/min/1.73, p = 0.04). regarding the oxidative stress and anti-oxidant biomarkers, mda serum level was increased but sod and gsh were decreased insignificantly in gentamicin group compared with the control group (p > 0.05). as well, kim-1 and ngal were significantly elevated in gentamicin group compared with the control group (p = 0.02 for ngal and p < 0.01 for kim-1) (table 1). indeed, cystatin-c serum level was significantly increased during induction of nephrotoxicity by gentamicin from 0.024 ± 0.0005 ng/ml in the control group to 0.0280 ± 0.0016 ng/ml in the experimental group (p = 0.01) (fig. 1). effects of pomegranate on the renal biomarkers concerning the effect of pomegranate on gentamicin induce nephrotoxicity it causes insignificant effect on the weight, height, and bmi of rats (p > 0.05). pomegranate reduced blood table 1. effect of gentamicin on the anthropometric variables, biochemical and inflammatory biomarkers in gentamicin induced-nephrotoxicity variables control (n = 10) gentamicin (n = 10) p weight (g) 268.00 ± 25.01 288.37 ± 34.02 0.24 height (cm) 21.50 ± 0.83 21.99 ± 0.88 0.31 bmi (g/cm2) 0.57 ± 0.02 0.59 ± 0.04 0.0001* blood urea (mg/dl) 41.83 ± 7.46 56.87 ± 9.33 0.007* serum creatinine (mg/dl) 0.70 ± 0.14 1.08 ± 0.40 0.04* estimated gfr (ml/min/1.37) 16.89 ± 4.21 11.19 ± 5.16 0.04* mda (ng/ml) 289.85 ± 44.18 408.11 ± 145.8 0.08 sod (pg/ml) 48.12 ± 32.92 26.39 ± 16.86 0.13 gsh (μg/ml) 15.94 ± 2.39 13.89 ± 2.94 0.18 kim-1 (pg/ml) 73.78 ± 16.29 354.98 ± 46.38 0.0001* ngal (pg/ml) 15.78 ± 3.07 20.04 ± 2.88 0.02* *p < 0.05, unpaired t-test. bmi: body mass index, gfr: glomerular filtration rate, mda: malondialdehyde, sod: superoxide dismutase, gsh: glutathione reductase, kim-1: kidney injury molecule-1, ngal: neutrophil gelatinase associated lipocalin. fig. 1 cystatin-c serum levels in gentamicin induced-nephrotoxicity (*p < 0.05). h.m. alkuraishy et al. 37j contemp med sci | vol. 5, no. 1, january–february 2019: 35–40 original article pomegranate protects renal proximal tubules during gentamicin induced-nephrotoxicity in rats urea significantly from 56.87 ± 9.33 to 40.25 ± 8.95 mg/dl (p = 0.002) without significant reduction of serum creatinine (p > 0.05) or improvement of estimated gfr. pomegranate improves endogenous anti-oxidant capacity, since it increase gsh significantly up to 17.91 ± 3.34 µg/ml compared with 13.89 ± 2.94 µg/ml in gentamicin group (p = 0.02). there were insignificant effects on mda and sod. moreover, pomegranate attenuated renal injury significantly, since it reduces serum levels of kim-1 from 354.98 ± 46.38 pg/ml in gentamicin group to 102.71 ± 57.20 pg/ml (p = 0.0001) (table 2). whereas, no significant change in cystatin-c in pomegranate group (0.026 ± 0.007 ng/ml) compared with gentamicin group 0.0280 ± 0.0016 ng/ml (p = 0.67) (fig. 2). intergroup variations illustrated significant changes in bmi, biochemical data, biomarkers of inflammatory and pro-inflammatory mediators (table 3). discussion this study illustrated that gentamicin was proficient to induce experimental nephrotoxicity in rats via significant rise of blood urea and serum creatinine with significant decline in the estimated gfr. these findings were corresponded with different recent studies.20,21 since there were two groups of rats with minimal differences in the weight and length, control group showed low weight compared with high weight in gentamicin group which might explain high bmi in gentamicin group. ibraheem et al.’s22 study showed that co-adminstration of gentamicin with fructose for 8 weeks encourage metabolic syndrome and increasing in the body weight and bmi which to a degree explain high body weight in gentamicin group. it has been recognized by diverse studies that the production of free radicals and induction of oxidative stress are the most imperative pathway of gentamicin induced-nephrotoxicity. so, overproduction of reactive oxygen species is connected with depletion of proximal renal tubules anti-oxidant potential which afterward developed into lipid peroxidation and tubular damages.23 table 2. effect of pomegranate on the anthropometric variables, biochemical and inflammatory biomarkers in gentamicin induced-nephrotoxicity variables gentamicin (n = 10) pomegranate (n = 10) p weight (g) 288.37 ± 34.02 276.50 ± 29.22 0.46 height (cm) 21.99 ± 0.88 21.67 ± 0.78 0.45 bmi (g/cm2) 0.59 ± 0.04 0.59 ± 0.45 1.0 blood urea (mg/dl) 56.87 ± 9.33 40.25 ± 8.95 0.002* serum creatinine (mg/dl) 1.08 ± 0.40 0.87 ± 0.18 0.15 estimated gfr (ml/min/1.73) 11.19 ± 5.16 13.69 ± 4.97 0.34 mda (ng/ml) 408.11 ± 145.8 371.25 ± 66.81 0.52 sod (pg/ml) 26.39 ± 16.86 32.17 ± 14.90 0.47 gsh (μg/ml) 13.89 ± 2.94 17.91 ± 3.34 0.02** kim-1 (pg/ml) 354.98 ± 46.38 102.71 ± 57.20 0.0001* ngal (pg/ml) 20.04 ± 2.88 15.29 ± 1.54 0.0011* *p < 0.01. **p < 0.05, unpaired t-test. bmi: body mass index, gfr: glomerular filtration rate, mda: malondialdehyde, sod: superoxide dismutase, gsh: glutathione reductase, kim-1: kidney injury molecule-1, ngal: neutrophil gelatinase associated lipocalin. fig. 2 pomegranate produced insignificant reduction of cystatin-c serum levels in gentamicin induced-nephrotoxicity. table 3. intergroup variations in the anthropometric, biochemical and renal biomarkers in gentamicin induced-nephrotoxicity regarding the effect of pomegranate compared with the control variables group і (n = 10) group іі (n = 10) group ііі (n = 10) anova weight (g) 268.00 ± 25.01 288.37 ± 34.02 276.50 ± 29.22 0.74 height (cm) 21.50 ± 0.83 21.99 ± 0.88 21.67 ± 0.78 0.29 bmi (g/cm2) 0.57 ± 0.02 0.59 ± 0.04 0.59 ± 0.45 0.001* blood urea (mg/dl) 41.83 ± 7.46 56.87 ± 9.33 40.25 ± 8.95 0.0001* serum creatinine (mg/dl) 0.70 ± 0.14 1.08 ± 0.40 0.87 ± 0.18 0.001* gfr (ml/min/1.73) 16.89 ± 4.21 11.19 ± 5.16 13.69 ± 4.97 0.04** mda(ng/ml) 289.85 ± 44.18 408.11 ± 145.8 371.25 ± 66.81 0.005* sod (pg/ml) 48.12 ± 32.92 26.39 ± 16.86 32.17 ± 14.90 0.05 gsh (μg/ml) 15.94 ± 2.39 13.89 ± 2.94 17.91 ± 3.34 0.0001* kim-1 (pg/ml) 73.78 ± 16.29 354.98 ± 46.38 102.71 ± 57.20 0.0001* cys-c (ng/ml) 0.024 ± 0.0005 0.028 ± 0.0016 0.026 ± 0.0031 0.0002* ngal (pg/ml) 15.78 ± 3.07 20.04 ± 2.88 15.29 ± 1.54 0.05 *p < 0.01. **p < 0.05, one-way anova test. bmi: body mass index, gfr: glomerular filtration rate, mda: malondialdehyde, sod: superoxide dismutase, gsh: glutathione reductase, kim-1: kidney injury molecule-1, cys-c: cystatin-c, ngal: neutrophil gelatinase associated lipocalin. 38 j contemp med sci | vol. 5, no. 1, january–february 2019: 35–40 pomegranate protects renal proximal tubules during gentamicin induced-nephrotoxicity in rats original article h.m. alkuraishy et al. therefore, serum level of mda is elevated while; sod and gsh are reduced in different models of gentamicin induced-nephrotoxicity as illustrated by hajihashemi et al.’s24 study that confirmed the protective effect of hydroalcoholic extract of zataria multiflora in reduction of mda with significant effect in rise of anti-oxidant enzyme activities. despite these findings, gentamicin in this study elevates mda serum levels, reduced sod and gsh but not significantly which might due to insufficient gentamicin dose, small sample size or short duration of the experimental study. this study also illustrated significant effect of gentamicin in rise kim-1 and ngal sera levels as correspond with luo et al.’s study that showed both kim-1 and ngal sera level are sensitive and specific biomarkers and correlated with renal histopathological changes during gentamicin induced-nephrotoxicity within 7 days. the increment in those biomarkers is time and dose dependent due to progressive gene expression of kim-1 and ngal.25 usually, renal tissues expresse a low level of kim-1 but subsequent to the renal injury, kim-1 gene expression is noticeably up-regulated at renal proximal convoluted tubules. moreover, kim-1 serum levels are more sensitive than ngal for development of acute renal injury.26 neutrophil gelatinase associated lipocalin is a protein expressed at renal proximal convoluted tubules which also up-regulated after renal ischemia and injury. it detects in vitro and in vivo tubular damage, since it rise within 2 h of renal ischemia thus it regarded as a reliable biomarker for detection early acute renal injury as it correlated with the severity of renal damage. indeed, urinary ngal can differentiate between intrinsic and prerenal causes of acute renal injury as urinary ngal >104 μg/l indicate intrinsic cause while level <47 μg/l indicate a prerenal cause of acute renal injury.27 but in this study urinary ngal levels were not measured due to difficulties in the collection of rat’s urine. these studies are without doubt corresponding with findings of this study. cystatin-c serum level was significantly increased in gentamicin group compared with the control group as supported by kader et al.’s28 study that confirmed significant elevation in cystatin-c serum level during gentamicin induced-nephrotoxicity. cystatin-c is a surrogate biomarker of gfr, not affected by muscle mass, age, gender and food type, it superior to serum creatinin since; it detect earlier renal injury 2 days prior to elevation of blood urea and serum creatinine . normally, cystatin-c is filtered by glomeruli and reabsorbed by proximal convoluted tubules thus; elevation of serum cystatin-c indicate a glomerular damage while elevation of urinary cystatin-c indicating renal tubular damage.29,30 therefore, gentamicin in this study led to full prone nephrotoxicity and acute renal injury through reduction of gfr and elevation of glomerular and tubular damage biomarkers in the experimental rats. co-administration of pomegranate with gentamicin in this study leads to significant reduction of gentamicin induced-nephrotoxicity through reduction of blood urea significantly and significant amelioration of estimated gfr as observed in alimordan et al.’s31 study that showed the nephro-protective effect of pomegranate in attenuation of gentamicin nephrotoxicity. also, body weight and bmi changes were insignificant improved compared with the control group as demonstrated by primarizky et al.’s32 study which pointed out that pomegranate is effective in preservation of normal body weight in rats during gentamicin induced-nephrotoxicity. regarding the effect of pomegranate on the oxidative stress, lipid peroxidation and anti-oxidant potential, pomegranate led to insignificant reduction of lipid peroxidation marker (mda) and significant elevation of anti-oxidant marker (gsh). these findings are inconsistent with different studies that confirmed the anti-oxidant effect of pomegranate in attenuation of nephrotoxicity.33,34 insignificant effect of pomegranate on lipid peroxidation in this study may be due to short duration of the experiment study, small sample size which may affect the statistical results or due to mda serum level variability. also, el arabey35 confirmed the insignificant effect of high dose of pomegranate in prevention of cisplatin induced nephrotoxicity. certainly, there are different studies regarding the dose dependent and the anti-oxidant potential in attenuation of drug induced-nephrotoxicity. low dose of pomegranate illustrated a protective effect in male rats, while high dose does not showed this defending effect. as well, gender differences in the protective effect of pomegranate was observed in nematbakhsh et al.’s36,37 studies that showed male rats were more prone to the nephrotoxicity. these factors were excluded in this study, since we used male rats and fixed dose of pomegranate. furthermore, pomegranate significantly reduced inflammatory and renal tubular injury biomarkers (kim-1 and ngal) due to significant nephroprotective effect and attenuation of gentamicin induced-nephrotoxicity. boroushaki et al.’s33 experimental study showed that pre-treatment with pomegranate lead to dose-dependent nephroprotective effect. likewise, pomegranate led to reduction of cystatin-c but not to insignificant level. reduction of cystatin-c serum level indicates a protective effect on the glomeruli, since cystatin-c serum level is correlated with gfr. therefore, pomegranate reduces renal tubular injury and improves glomerular function in different models of drug induced-nephrotoxicity.34 the nephroprotective effect of pomegranate is due to different constituents including gallic acid, punicallin, punicalgin, and ellagic acid which exhibit potent free radicals scavenging and anti-oxidant effects.38 moreover, gallic acid exhibits significant nephroprotective effect in attenuation of vancomycin nephrotoxicity.39 but in this study active constituents of pomegranate juice were not determined as it is well evaluated in different studies. it has been shown that anti oxidants ameliorate oxidative stress marker (mda) but a higher dose of these anti-oxidants may induced oxidative stress causing augmentation of renal ischemic-reperfusion injury.40 this finding might explain insignificant effect of pomegranate on the mda levels in this study, since we used a high dose of pomegranate (100 mg/kg). pomegranate therapy leads to significant weight reduction due to inhibition of digestive enzymes by tannis that lead to appetite suppression and reduction of nutrients absorption41 which might clarify the reduction in rat body weight in this study. h.m. alkuraishy et al. 39j contemp med sci | vol. 5, no. 1, january–february 2019: 35–40 original article pomegranate protects renal proximal tubules during gentamicin induced-nephrotoxicity in rats conclusion pomegranate attenuates gentamicin induced-nephrotoxicity through potentiation of endogenous anti-oxidant capacity and inhibition of inflammatory pathway. acknowledgment the authors would like to thank the research deputy of international campus, al-mustansiriyia university college of medicine. conflict of interest none. funding and sponsorship none.  references 1. tiong hy, huang p, xiong s, li y, vathsala a, zink d. drug-induced nephrotoxicity: clinical impact and preclinical in vitro models. mol pharm. 2014;11:1933–1948. 2. ennulat d, ringenberg m, frazier ks. toxicologic pathology forum opinion paper: recommendations for a tiered approach to nonclinical mechanistic nephrotoxicity evaluation. toxicol pathol. 2018;46:636–646. 3. brocca a, virzì gm, pasqualin c, pastori s, marcante s, de cal m, et al. cardiorenal syndrome type 5: in vitro cytotoxicity effects on renal tubular cells and inflammatory profile. anal cell pathol (amst). 2015;2015:469461. 4. kim sy, moon a. drug-induced nephrotoxicity and its biomarkers. biomol ther (seoul). 2012;20:268–272. 5. a v, s a, kuriakose j, midhun sj, jyothis m, latha ms. protective effect of rotula aquatica lour against gentamicin induced oxidative stress and nephrotoxicity in wistar rats. biomed pharmacother. 2018;106:1188–1194. 6. moffett bs, morris j, galati m, munoz fm, arikan aa. population pharmacokinetic analysis of gentamicin in pediatric extracorporeal membrane oxygenation. ther drug monit. 2018;40:581–588. 7. ge s, beechinor rj, hornik cp, standing jf, zimmerman k, cohenwolkowiez m, et al. external evaluation of a gentamicin infant population pharmacokinetic model using data from a national electronic health record database. antimicrob agents chemother. 2018;62. pii: 00669-18. 8. aly haa, hassan mh. potential testicular toxicity of gentamicin in adult rats. biochem biophys res commun. 2018;497:362–367. 9. moreira ma, nascimento ma, bozzo ta, cintra a, da silva sm, dalboni ma, et al. ascorbic acid reduces gentamicin-induced nephrotoxicity in rats through the control of reactive oxygen species. clin nutr. 2014;33:296–301. 10. magee pj, rowland ir. phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer. br j nutr. 2004;91:513–531. 11. al-kuraishy hm. central additive effect of ginkgo biloba and rhodiola rosea on psychomotor vigilance task and short-term working memory accuracy. journal of intercultural ethnopharmacology. 2016 ;5:7. 12. huber r, gminski r, tang t, weinert t, schulz s, linke-cordes m, et al. pomegranate (punica granatum) seed oil for treating menopausal symptoms: an individually controlled cohort study. altern ther health med. 2017;23:28–34. 13. akhtar s, ismail t, fraternale d, sestili p. pomegranate peel and peel extracts: chemistry and food features. food chem. 2015;174:417–425. 14. al-kuraishy hm, al-gareeb ai. potential effects of pomegranate on lipid peroxidation and pro-inflammatory changes in daunorubicin-induced cardiotoxicity in rats. int j prev med. 2016;7:85. 15. rasheed z, akhtar n, anbazhagan an, ramamurthy s, shukla m, haqqi tm. polyphenol-rich pomegranate fruit extract (pomx) suppresses pmaciinduced expression of pro-inflammatory cytokines by inhibiting the activation of map kinases and nf-kappab in human ku812 cells. j inflamm (lond). 2009;6:1. 16. kim ye, hwang cj, lee hp, kim cs, son dj, ham yw, et al. inhibitory effect of punicalagin on lipopolysaccharide-induced neuroinflammation, oxidative stress and memory impairment via inhibition of nuclear factor-kappab. neuropharmacology. 2017;117:21–32. 17. mansouri e, basgen j, saremy s. the effects of pomegranate extract on normal adult rat kidney: a stereological study. vet res forum. 2016;7:1–6. 18. singh ap, junemann a, muthuraman a, jaggi as, singh n, grover k, et al. animal models of acute renal failure. pharmacol rep. 2012;64:31–44. 19. gacka e, życzkowski m, bogacki r, paradysz a, hyla-klekot l. the usefulness of determining neutrophil gelatinase-associated lipocalin concentration excreted in the urine in the evaluation of cyclosporine a nephrotoxicity in children with nephrotic syndrome. dis markers. 2016;2016:6872149. 20. abd-elhamid th, elgamal da, ali ss, ali fem, hassanein ehm, el-shoura eam, et al. reno-protective effects of ursodeoxycholic acid against gentamicin-induced nephrotoxicitythrough modulation of nf-κb, enos and caspase-3 expressions. cell tissue res. 2018;374:367–387. 21. helal mg, zaki mmaf, said e. nephroprotective effect of saxagliptin against gentamicin-induced nephrotoxicity, emphasis on anti-oxidant, antiinflammatory and anti-apoptic effects. life sci. 2018;208:64–71. 22. ibraheem zo, basir r, aljobory akh, ibrahim oe, alsumaidaee a, yam mf. impact of gentamicin coadministration along with high fructose feeding on progression of renal failure and metabolic syndrome in sprague–dawley rats. biomed res int. 2014;2014:823879. 23. al-kuraishy hm, al-gareeb ai, al-maiahy tj. concept and connotation of oxidative stress in preeclampsia. journal of laboratory physicians. 2018;10:276. 24. hajihashemi s, jafarian t, ahmadi m, rahbari a, ghanbari f. ameliorative effects of zataria multiflora hydro-alcoholic extract on gentamicin induced nephrotoxicity in rats. drug res (stuttg). 2018;68(7):387–394. 25. luo qh, chen ml, sun fj, chen zl, li my, zeng w, et al. kim-1 and ngal as biomarkers of nephrotoxicity induced by gentamicin in rats. mol cell biochem. 2014;397:53–60. 26. szeto cc, kwan bc, lai kb, lai fm, chow km, wang g, et al. urinary expression of kidney injury markers in renal transplant recipients. clin j am soc nephrol. 2010;5:2329–2337. 27. makris k, markou n, evodia e, dimopoulou e, drakopoulos i, ntetsika k, et al. urinary neutrophil gelatinase-associated lipocalin (ngal) as an early marker of acute kidney injury in critically ill multiple trauma patients. clin chem lab med. 2009;47:79–82. 28. de geus hr, bakker j, lesaffre em, le noble jl. neutrophil gelatinaseassociated lipocalin at icu admission predicts for acute kidney injury in adult patients. am j respir crit care med. 2011;183:907–914. 29. kader c, sunbul m, das yk, yarim m, bedir a, karaca e, et al. telbivudine attenuates gentamicin-induced kidney injury in rats. int j antimicrob agents. 2017;49:595–602. 30. al suleimani ym, abdelrahman am, karaca t, manoj p, ashique m, nemmar a, et al. the effect of the dipeptidyl peptidase-4 inhibitor sitagliptin on gentamicin nephrotoxicity in mice. biomed pharmacother. 2018;97:1102– 1108. 31. alimoradian a, changizi-ashtiyani s, ghiasabadi farahani a, kheder l, rajabi r, sharifi a. protective effects of pomegranate juice on nephrotoxicity induced by captopril and gentamicin in rats. iran j kidney dis. 2017;11:422– 429. 32. primarizky h, yuniarti wm, lukiswanto bs. benefits of pomegranate (punica granatum linn) fruit extracts to weight changes, total protein, and uric acid in white rats (rattus norvegicus) as an animal model of acute renal failure. vet world. 2016;9:1269–1274. 33. boroushaki mt, asadpour e, sadeghnia hr, dolati k. effect of pomegranate seed oil against gentamicin-induced nephrotoxicity in rat. j food sci technol. 2014;51:3510–3514. 34. cekmen m, otunctemur a, ozbek e, cakir ss, dursun m, polat ec, et al. pomegranate extract attenuates gentamicin-induced nephrotoxicity in rats by reducing oxidative stress. ren fail. 2013;35:268–274. 35. el-arabey aa. negative response of phytoestrogens of pomegranate flower extract against cisplatin-induced nephrotoxicity in female rats. int j prev med. 2016;7:89. 40 j contemp med sci | vol. 5, no. 1, january–february 2019: 35–40 pomegranate protects renal proximal tubules during gentamicin induced-nephrotoxicity in rats original article h.m. alkuraishy et al. 36. motamedi f, nematbakhsh m, monajemi r, pezeshki z, talebi a, zolfaghari b, et al. effect of pomegranate flower extract on cisplatin-induced nephrotoxicity in rats. j nephropathol. 2014;3:133–138. 37. nematbakhsh m, ebrahimian s, tooyserkani m, eshraghi-jazi f, talebi a, ashrafi f. gender difference in cisplatin-induced nephrotoxicity in a rat model: greater intensity of damage in male than female. nephrourol mon. 2013;5:818–821. 38. zhang l, yang x, zhang y, wang l, zhang r. in vitro antioxidant properties of different parts of pomegranate flowers. food bioprod process. 2011;89: 234–240. 39. jang a, srinivasan p, lee ny, song hp, lee jw, lee m, et al. comparison of hypolipidemic activity of synthetic gallic acid–linoleic acid ester with mixture of gallic acid and linoleic acid, gallic acid, and linoleic acid on high-fat diet induced obesity in c57bl/6 cr slc mice. chem biol interact. 2008;174:109–117. 40. al-kuraishy hm, al-gareeb ai. eustress and malondialdehyde (mda): role of panax ginseng: randomized placebo controlled study. iranian journal of psychiatry. 2017;12:194. 41. al-gareeb ai, aljubory kd, alkuraishy hm. niclosamide as an anti-obesity drug: an experimental study. eating and weight disorders-studies on anorexia, bulimia and obesity. 2017;22:339-44. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 97j contemp med sci | vol. 4, no. 2, spring 2018: 97–101 moral skills of iranian general dentists reza yazdani,a mohamadjavad kharazifard,b negin morafeghc acommunity oral health department, tehran university of medical sciences, international campus, tehran, iran. bdental research center, tehran university of medical sciences, tehran, iran. cdentist, tehran, iran. correspondence to reza yazdani (email: ryazdani@tums.ac.ir). (submitted: 09 february 2018 – revised version received: 14 march 2018 – accepted: 17 april 2018 – published online: 26 june 2018) objective this study aimed to assess the moral skills of iranian general dentists. methods this cross-sectional study was performed on 384 general dentists selected in national congresses, in 2016. volunteers were asked to fill out the moral skills inventory with four domains of integrity, courage, reasoning and sensitivity. demographic factors were also assessed and their correlation with moral skills was evaluated using the backward linear regression model. results the mean acquired score of sensitivity, reasoning, integrity and courage score was 2.96, 5.71, 4.11 and 4.50 out of 8, respectively and the mean percentage of total moral skills of dentists was 54%. males and females were not significantly different in terms of scores acquired in the four domains (p > 0.05). the scores obtained in the four moral domains were higher in younger graduates (by increase in the graduation year) (p = 0.02). a significant inverse correlation was found between moral integrity and reasoning with father’s level of education (p < 0.05). the correlation between moral sensitivity and mother’s level of education did not reach statistical significance (p > 0.05). conclusion the mean percentage of moral skills of iranian dentists was moderate. thus, especial emphasis should be placed on moral skills particularly moral sensitivity in medical ethics educational programs. keywords moral courage, moral integrity, moral reasoning, moral sensitivity introduction recent advances in health domains and technology have also altered health-related ethical aspects. public concerns regarding ethical issues in the medical field are increasing. ethics in dentistry is a fundamental topic in dental education. adherence of dentists to dental ethics principles increases the patients’ trust in receipt of dental care. moral and communication skills improve the patient– dentist relationship and increase the cooperation of patients, their adherence to preventive instructions and their satisfaction with dental care. in addition to cognitive skills, efficient communication with patients and adherence to professional ethical principles and respecting the patients’ rights are among the prerequisites for provision of dental care services. instruction of professional ethics has a theoretical background. rest designed a four-component model for moral development based on a theory by lawrence kolberg who described moral development using a classified model such that during growth and development of each individual, moral systems are replaced with more complete systems.1,2 rest followed two related research paths. the moral reasoning was his main topic of research.1–3 rest’s approach included three phases of pre-conventional, conventional and post-conventional. these attempts resulted in development, accreditation and use of defining issues test (dit) to measure the moral reasoning component.4–6 also, rest designed a four-component model of moral behavior with moral reasoning being one component of it. bebeau et al.5 extensively assessed the efficacy of rest’s four-component model and dit for dental education and profession and published many studies on this topic in accredited journals. this model, also used in the current study, includes four components of moral sensitivity, moral reasoning, moral integrity and moral courage. however, different terms have been used for each of these concepts in different studies even those by rest, which indicates the experimental, rather than theoretical nature of this model. also, different measurement instruments may particularly focus on some specific aspects. the moral skills inventory by chambers is an instrument to analyze the moral behavior.7 this inventory was used by khosravi et al.8 on three groups of dental students in tehran university of medical sciences to assess their moral behavior. they showed that dental students gained the highest score in moral reasoning and the lowest score in moral integrity. also, first year dental students gained a higher score than third year and senior dental students. chambers assessed the moral skills inventory in three groups of dental students, faculty members of dental school and members of the american college of dentists.7 they acquired the lowest score in moral integrity and the highest mean score in moral reasoning. bebeau et al. developed a professional role orientation inventory with 40 items for assessment of perception of dentists of their professional role. this test included four attitudinal dimensions each with 10 items covering authority, responsibility, agency, and autonomy. they assessed the differences between different professions and dental students. the results showed that professionals had different attitudes toward their professional role, which were somewhat different from the models described in the literature regarding professionalism.5 moral skills of iranian general dentists have not been evaluated before. this study aimed to determine the moral skills of iranian general dentists. the effect of demographic variables such as age, gender, level of education and occupation of the parents of dentists on their moral behavior was also assessed. methods this descriptive, cross-sectional study was performed on general dentists selected in two national congresses, in 2016. the instrument used for data collection in this study was the 16-question moral skills inventory designed by chambers in issn 2413-0516 research 98 j contemp med sci | vol. 4, no. 2, spring 2018: 97–101 moral skills dentists research reza yazdani et al. 2011.7 this inventory has been translated to farsi by khosravi et al.8 in 2012 and its validity and reliability have been confirmed.8 the questionnaire was administered among 450 iranian general dentists who attended the 56th congress of iranian dental association and 10th congress of iranian general dentists’ association from all over the country. a total of 384 complete questionnaires were returned. the questions were divided into four domains of sensitivity, integrity, reasoning and courage. moral sensitivity is defined as the knowledge about the fact that ethical issues are part of the situation, the ability to recognize when a moral response is appropriate and the knowledge about the fact that how our actions affect others. moral reasoning, also known as moral judgment or moral development is defined as thinking about ethical challenges.1,5 this component of the rest model has been well known. moral reasoning is not equal to coping with a known challenge or a law or theory that determines the best approach. some levels have been described for moral maturity. the higher the level of an action theoretically, the higher the level of moral development would be. moral integrity determines the level of adherence to moral and ethical standards. for instance, a dentist becomes aware of the malpractice of his colleague. if he believes that his colleague did not follow the ethical principles but at the same time he thinks that it is not his problem, then he has low level of moral integrity. moral courage was defined by rest1 as the power of ego, perseverance and bravery. duckett and ryden9 reported the 4th component to be character while bebeau et al.5,6 reported the 4th component to be character and competence to emphasize on active control of situations. each domain had four questions and each question had three answer choices. only one answer choice could be selected as response. each choice was allocated a score from 0 to 2. the highest score in each domain was 8 (four questions in each domain and maximum score of 2 for each question). maximum total score was 32 and minimum total score was 0. demographic factors including father’s occupation, mother’s occupation, father’s level of education, mother’s level of education, gender and age were also evaluated and their correlation with moral skills was assessed. the data obtained from the questionnaire were analyzed using the backward linear regression model. p < 0.05 was considered statistically significant. results the results are reported in two domains. first, we report the frequency of data and then assess the correlation of variables with moral components. regarding the father’s occupation, 15.63% were businessman (highest frequency) and 1.30% was nurse (lowest frequency). among mother’s, 30.99% were housewife (highest frequency) and 0.52% were midwife (least frequency). in terms of level of education, most parents (20.57% of fathers and 26.82% of mothers) had bachelor’s degree. the highest acquired score by both males and females belonged to moral reasoning (5.65 in males and 5.75 in females). the lowest score acquired by males and females belonged to moral sensitivity 2.97 in males and 2.94 in females; table 1. tables 2–5 show the questions of moral sensitivity, moral reasoning, moral integrity and moral courage and the response rate to each question. the backward linear regression model was used to assess the correlation of moral skills with level of education of mothers and fathers, age, gender and graduation year. the results showed a significant inverse correlation between moral integrity and moral reasoning and fathers’ level of education (p < 0.05). other moral skills had no significant association with fathers’ level of education (p > 0.05). also, the results showed an association between mother’s level of education and moral sensitivity; however, this association did not reach statistical significance (p > 0.05). the mean scores were not significantly different between males and females (p > 0.05). assessment of the relationship of moral skills and graduation year revealed that the mean score of moral courage, moral sensitivity, moral reasoning and moral integrity was significantly higher in younger graduates (increased by an increase in graduation year) (p = 0.023). the mean total score acquired by dentists was 54%. discussion adherence to ethical and moral principles in work is one criterion of professionalism. the moral and ethical principles in educational centers and universities are particularly important. a total of 384 general dentists were enrolled in this study and the association of demographic factors with moral skills was assessed in them. the questions of moral skills were divided into four components: moral sensitivity, reasoning, courage and integrity. the highest score in both males and females belonged to moral reasoning and the lowest score belonged to moral sensitivity. in line with our results, chambers et al.7 reported the score of moral sensitivity in the three groups to be 4.92–5.23; moral sensitivity ranked the lowest after moral integrity. also, table 1. mean and standard deviation of scores acquired by the iranian general dentists with regard to moral skills moral skills male female total mean mean standard deviation minimum maximum mean standard deviation minimum maximum sensitivity 2.97 1.90 0 7 2.94 1.94 0 11 5.71 reasoning 5.65 1.24 3 8 5.78 1.21 3 12 4.11 integrity 4.09 1.38 2 7 4.15 1.47 2 11 4.50 courage 4.55 1.446 2 7 4.44 1.40 2 11 2.96 reza yazdani et al. 99j contemp med sci | vol. 4, no. 2, spring 2018: 97–101 research moral skills dentists table 2. questions in moral sensitivity domain and frequency and percentage of responses by general dentists questions first choice second choice third choice -i tend to see almost every aspect of dentistry as involving an ethical dimension. -i am pretty sensitive to ethical matters. -when ethical issues are clear, i am prepared to do my part. (49.2%) 189 (20.3%) 78 (%30.5%) 117 question 2. technical quality of dental procedures is -an ethical issue in every single case. -an ethical issue when the average level of skill is close enough to the minimal standard that it might cause a problem. -clearly an ethical issue if below standard. (67.7%) 250 (24.2%) 93 (8.1%) 31 question 3. one of your practice partners teaches part-time at a dental school and wants to participate in a multisite research project. there is a handsome “finder’s fee.” you would consider enrolling some of your patients in the study if -the university and its ethics review board have approved the study. -you can present the study to patients in a way they can agree to. -your review of available information shows that the product is potentially very helpful in practice. (33.6%) 129 (33.9%) 130 (32.6%) 125 question 4. access to care is -clearly an ethical issue. -a complex issue with some ethical components. -not really an ethical issue. (39.3%) 151 (46.45%) 178 (14.35%) 55 table 3. questions in moral reasoning domain and frequency and percentage of responses by general dentists questions first choice second choice third choice question 5. i tend to decide problematic situations that arise in dentistry -based on what gives me the best outcome in each case. -with a pretty careful eye to what others are doing. -on principle. (61.25%) 235 (4.95%) 19 (33.9%) 130 question 6. a colleague calls requesting the chart of a patient on whom you have completed a fair bit of work but have yet to receive about $3000 in payment. you consider informing the patient that you cannot release a copy of the chart until you receive at least some of the payment owed. eventually you decide against this approach because -it might be illegal. -it casts a shadow of “commercial interest” on the entire profession. -it stands in the way of the patient receiving oral health care. (22.45%) 86 (8.3%) 32 (69.3%) 266 question 7. restricting procedures performed to those you like and can do well is perhaps unethical if -market segmentation draws the attention of the federal trade commission. -that is not the custom in the community where you practice. -all patients count on you to provide comprehensive care. 0 (6.8%) 26 (93.2%) 358 question 8. on a state or national policy level, the most appropriate approach to medicaid is as -an economic matter, especially considering “no show” rates. -a political issue involving understanding between the ada and government groups. -a matter of distributive justice (people getting their fair share). (8.1%) 31 (25.3%) 97 (66.7%) 256 table 4. questions in moral integrity domain and frequency and percentage of responses by general dentists questions first choice second choice third choice question 9. i place high value on projecting my (ethical) character into what i do on all occasions. -learning from each ethical situation and taking a broad perspective. -making certain i am within my rights. (42.7%) 164 (40.9%) 157 (16.4%) 63 question 10. because dentistry necessarily involves conflicting circumstances and multiple goals, the best policy is usually to -be guided by a single standard of integrity in all situations. -match each action to the particular situation. -respond to others’ expectations of you. (19.3%) 74 (60.9%) 234 (19.8%) 76 question 11. i admire dentists who -place principle above success always. -blend principle and success. -selectively succeed as long as this does not compromise principle. (14.8%) 57 (26.8%) 103 (58.3%) 224 question 12. the reason i value my professional standards is that they -anchor my identity and focus my action. -ensure my standing in the professional community. -create realistic expectations among colleagues and patients. (36.7%) 141 (32.8%) 126 (30.5%) 117 100 j contemp med sci | vol. 4, no. 2, spring 2018: 97–101 moral skills dentists research reza yazdani et al. participants acquired a higher score in moral reasoning (5.72) compared to other components. our results showed a significant inverse correlation between fathers’ level of education and moral integrity and reasoning (p = 0.023 and 0.033, respectively). however, these components had no significant correlation with other demographic variables (p > 0.05). in the study by khosravi et al.8 no significant association was found between moral skills and fathers’ level of education. moreover, associations were noted between moral sensitivity and mother’s level of education and graduation year but these correlations were not statistically significant (p > 0.05). khosravi et al.8 did not find a significant association between mothers’ level of education and moral sensitivity either (p = 0.746). data analysis revealed no significant difference between males and females in any moral component (p > 0.05) and the mean scores were relatively the same in males and females. similarly, bebeau et al.10 assessed the role of gender in moral skills of dental students and found no significant difference between males and females with regard to moral sensitivity. however, differences were noted between males and females in taking responsibility, moral integrity, moral reasoning and moral courage. these differences can be explained by the effect of culture and social beliefs in each community on moral skills. in our study, the mean score of all four components of moral skills significantly increased by an increase in graduation year (p < 0.05). this indicates that in the recent years, more emphasis has been placed on instruction of professional ethics in dental curricula in universities. al-zain et al.11 reported that the mean score of senior dental students was significantly higher than that of junior dental students. moreover, in the study by chambers level of moral skills increased by an increase in age; this finding was different from our results but in line with that of khosravi et al.7,8 dental students in academic year 2013 studying in tehran university acquired higher scores in all components except for moral integrity compared to dental students in academic years 2008 and 2010; however, this difference was not statistically significant. conclusion considering the low level of moral skills of general dentists, special emphasis must be placed on moral skills, particularly moral sensitivity in educational curricula of dental students and continuing education courses. acknowledgments we would like to express our gratitude to all dentists who participated in this study. the present study was based on a research plan approved in tehran university of medical sciences, international campus under the ethics code ir. tums.vcr.rec.1395.1131. conflict of interest none.  5. bebeau mj, born do, ozar dt. the development of a professional role orientation inventory. j am coll dent. 1992;60:27–33. 6. bebeau mj. influencing the moral dimensions of dental practice. in moral development in the professions: psychology and applied ethics; rest j, narvaez d, eds.; 1994; pp. 121–146. 7. chambers dw. developing a self-scoring comprehensive instrument to measure rest’s four-component model of moral behavior: the moral skills inventory. j dent educ. 2011;75:23–35. table 5. questions in moral courage domain and frequency and percentage of responses by general dentists questions first choice second choice third choice question 13. when i see something that does not look fair, -i am reluctant to get involved because i know i should not stick my nose in other people’s business. -i may speak up if the case is obvious and straight forward. -i tend to take the initiative to find out what is going on and to try to set things straight. (32.3%) 124 (47.1%) 181 (20.6%) 79 question 14. two separate patients have come to you with questionable work performed by a colleague in your community. you do not disparage your colleague to your patients but you would be willing to talk with your colleague -if you could be certain there are no extenuating circumstances. -if a few more similar patients appear. -right now. (24.7%) 95 (54.4%) 209 (20.8%) 80 question 15. you and a good friend have been talking for years about taking medicaid patients. you are willing to do so -when the numbers seem good enough to sustain the viability of this change. -if your friend does so as well. -because you are convinced that the service is needed in your community. (23.46%) 90 (15.4%) 59 (61.2%) 235 question 16. debating anti-fluoridation is -a waste of time, generally. -appropriate for those who have such skills. -a professional responsibility. (12.2%) 47 (48.2%) 158 (39.6%) 152 references 1. rest jr. moral development in the professions: psychology and applied ethics; psychology press, 1994. 2. rest jr, bebeau mj, thoma sj. postconventional moral thinking: a neokohlbergian approach; psychology press, 1999. 3. rest jr. the major components of morality. in morality, moral development, and moral behavior; kurtines w, gewortz j, eds. wiley: new york, 1984; pp. 24–38. 4. bebeau mj, rest jr, yamoor cm. measuring dental students’ ethical sensitivity. j dent educ. 1985;49:225–235. reza yazdani et al. 101j contemp med sci | vol. 4, no. 2, spring 2018: 97–101 research moral skills dentists this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 8. yazdani r, khosravi a. evaluation of moral skills in dental students first, third and last year of dental school, tehran, tehran university of medical sciences; 2013. 9. bebeau mj, monson ve. guided by theory, grounded in evidence: a way forward for professional ethics education. in handbook of moral and character education; nucci l, narvaez d, eds.; routledge: new york, ny, 2008; pp. 557–582. 10. you d, bebeau mj. gender difference in ethical abilities of dental students. j dent educ. 2012;76:1137–1149. 11. al-zain sa, al-sadhan sar, ahmedani ms. perception of bds students and fresh graduates about significance of professional ethics in dentistry. j pak med assoc. 2014;64:118–123. dx.doi.org/10.22317/jcms.06201809 278 j contemp med sci | vol. 3, no. 11, summer 2017: 278–283 original 1 department of anatomy, school of medicine, tehran university of medical sciences, tehran, iran 2department of molecular medicine, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran 3department of anatomical sciences, medical sciences faculty, tarbiat modares university, tehran, iran 4faculty of medicine, institute of neuroanatomy, rwth aachen university, aachen, germany 5department of anatomical sciences, faculty of medicine, gilan university of medical sciences, rasht, iran *correspondence to iraj ragerdi kashani (email: ragerdi@tums.ac.ir). (submitted: 10 june 2017 – revised version received: 02 july 2017 – accepted: 10 july 2017 – published online: 26 september 2017) objective experimental and clinical studies suggest that oxidative stress plays an important role in the pathogenesis of multiple sclerosis. multiple studies have shown that non-psychoactive cannabinoid, cannabidiol (cbd) exerts antioxidant effects, and has recently been approved for treatment of inflammation, pain, and spasticity associated with ms patients. the aim of this study is to determine the effectiveness of cbd in a multiple sclerosis mouse model, i.e. cuprizone-induced demyelination. methods adult male bl/6 mice were fed with 0.2% cuprizone for 5 weeks which caused severe demyelination of the corpus callosum (cc). animals were simultaneously treated with 5 mg·kg−1 cbd by daily intra-peritoneal injections. using immunohistochemistry and transmission electron microscope, we evaluated the effects of cbd on demyelination, malondialdehyde levels and the activity of reduced glutathione, catalase and superoxide dismutase was evaluated by biochemical analysis. results cbd ameliorate the cuprizone-induced demyelination and microglia accumulation. biochemical analysis showed that oxidative stress induced by cuprizone was reduced by cbd. conclusion our data implicate that cbd attenuates destructive effects of cuprizone in the cc by decreasing oxidative stress and microglia repletion. keywords cannabidiol, demyelination, oxidative stress, microglia, cuprizone introduction multiple sclerosis (ms) is a severe demyelinating, chronic inflammatory disease characterized by multifocal damage of myelin in the central nervous system (cns).1 a key study by lucchinetti et al.2 described 4 different lesion subtypes: pattern 1 and 2 lesions are autoimmune mediated, while pattern 3 and 4 lesions are presumed to be caused by a primary oligodendrogliapathy. pattern 3 lesions in particular display many pathological similarities with cuprizone-induced demyelination: (i) actively demyelinating lesions with little involvement of t-cells but microglia accumulation and (ii) hypoxia-like tissue damage with signs of metabolic stress and mitochondrial dysfunction which eventually leads to pronounced oligodendrocyte apoptosis.3 ms has long been considered as t-cell-mediated autoimmune disease of the cns characterized by microglia activation, recruitment of systemic immune-competent cells and production of cytotoxic mediators and pro-inflammatory cytokines, which lead to neuronal tissue damage (inflammatory cascade).4 although controlled clinical studies published evidence for the effectiveness of immune-modulatory, anti inflammatory or immunosuppressive therapies, however this effect is mainly seen in patients at early stages of the disease, and the effects are limited, especially in improvement of the accumulation of constant clinical deficits.5, 6 therefore, there is a high need for new effective drugs that limit demyelination and axonal degeneration, thus complementing the currently available therapies.7 a number of studies suggest the importance of primary neurodegenerative mechanisms that are evident in chronically demyelinating plaques and are believed to be the result of oxidative stress.4,8 oxidative stress basically reflects a condition in which the pro-oxidant–anti-oxidant balance in a cell is disturbed; cellular biomolecules undergo severe oxidative damage, ultimately impairing cell viability.9 the cns is particularly sensitive to oxidative stress because of high oxygen consumption, relatively small amounts of conventional anti-oxidants and anti-oxidative enzymes, and large amounts of polyunsaturated lipids which are highly susceptible to oxidation.10 the discovery of the psychoactive effects of cannabis sativa marked the beginning of a new field of research into the pharmacological and physiological role of the cannabinoids.11 cannabis sativa contains a class of phytocannabinoids that include tetrahydrocannabinol (thc), the major psychoactive constituent, and cannabidiol (cbd), a non-psychoactive constituent.12 cbd exerts multiple pharmacological actions in the cns via no-cb1 and no-cb2 receptors.13 in addition to its beneficial effects on pain and spasticity associated with ms, cbd may influence the pathogenesis of ms by exerting anti-inflammatory, anti-oxidant and neuroprotective effects.12 it has been shown, that cbd can control microglia functions. furthermore, it appears neuroprotective in a mouse model of alzheimer disease14 and ameliorates immune-regulatory actions in both the theiler’s murine encephalitis virus (tmev)15 and experimental autoimmune encephalomyelitis (eae) models of ms.16 previous studies demonstrate that cannabinoids have safety and efficacy in reducing the symptoms of multiple issn 2413-0516 protective effects of cannabidiol on cuprizone-induced demyelination in c57bl/6 mice maryam sajjadian,1 iraj ragerdi kashani,1* parichehr pasbakhsh,1 mahmoud hassani,2 ameneh omidi,3 nasrin takzare,1 tim clarner,4 cordian beyer,4 adib zendedel4,5 iraj ragerdi kashani et al. 279j contemp med sci | vol. 3, no. 11, summer 2017: 278–283 original amelioration of cpz-induced demyelization by cbd sclerosis in animal models17 and also in humans.18 to further investigate the myeloprotective prospective of cbd, we used the potential of non-immune driven cuprizone intoxication.19 demyelination in the cuprizone model is an early pathological event mainly affecting oligodendrocytes and axons without the breakdown of the blood brain barrier and concomitant leukocyte/blood monocyte infiltration.15 histopathological features of the cuprizone-induced demyelination closely resemble type iii ms lesions as defined by lucchinetti et al.2 and oxidative stress was shown to be a key factor in cuprizone pathophysiology.20 the goal of this study was to evaluate the anti-oxidant effects of cbd during experimentallyinduced demyelination by cuprizone in the corpus callosum (cc) of young male mice. materials and methods animals research and animal care were approved by the review board for the care of animal subjects of the district government (tehran, iran). in vivo experiments were performed with 8 weeks old male c57bl/6 mice (approx. 20 g, pasteur, iran). animals were housed under temperature-controlled conditions with a 12/12-hour light–dark cycle and ad libitum access to water and food. cuprizone-induced demyelination and cbd treatment in this study cuprizone as an oral agent was used that causes acute, fast recovering demyelinated lesions.21 eight-week-old (20 g) male c57bl/6 mice were randomly divided into four groups (n = 10 per group). one group was fed normal diet and served as controls. the three other groups received diets containing 0.2% (w/w) cuprizone (sigma, usa) to induce demyelination. cbd (sigma, usa) was dissolved in pbs which served as vehicle. the vehicle plus cbd complex (5 mg·kg−1) was injected intraperitoneally into cuprizone-treated mice (cpz+c group) every other day for 5 weeks. the chosen cbd dosage closely resembled that used in another study to treat cpz model of ms in mice.22 the 4 groups were as follows: control group that was fed normal diet, cuprizone group which was given only cpz, vehicle-treated cuprizone group (cpz+v) and cannabidiol-treated cuprizone group (cpz+c). mice were anaesthetized with ketamine (115 mg/kg) (sigma, usa) and xylazine (10 mg/kg) (sigma, usa) and killed after 5 weeks. animal handling and treatment protocols have been previously described in detail.4 for ihc and biochemical studies, the numbers of animals in the experimental groups were as follows: control (n = 4), cpz (n = 5), cpz plus cbd (n = 6). luxol fast blue staining myelination was analyzed in sections by luxol fast blue staining (lfb, sigma, usa). sections were placed overnight in lfb at 56°c and washed in 95% ethanol and distilled water to remove excess blue stain. the color was then differentiated (until white matter was easily distinguishable from gray matter) in a lithium carbonate solution for 15 s, followed by distilled water and three 80% alcohol washing steps. slices were further passed through fresh xylene twice, mounted with entellan (merck, germany). in order to evaluate sections based upon demyelination, lfb stained sections were scored between zero and three, by three independent, blinded readers. a score of three is equivalent to a normal myelin status, whereas zero is referred to maximum demyelinated cc. a score of one or two corresponds to one-third or two-third fiber myelination of the cc, respectively. transmission electron microscopy for electron microscopic examination, three animals per group were transcardially perfused with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 m pbs. brains were quickly removed and placed on ice. the cc was dissected, and the samples were placed in a tube. samples were then transferred immediately after extraction in 2.5% glutaraldehyde (fluka) in 0.1 m cacodylate buffer (ph 7.4) at 4°c overnight and transferred to 1% osmium tetroxide in the same buffer for 1 h at room temperature. tissue was transversely cut into 1 mm blocks which were further fixed in osmium tetroxide at 4°c overnight, dehydrated through ascending ethanol washes, and embedded in epoxy resin (taab laboratories). 1 µm sections were cut, stained with toluidine blue, and examined by light microscopy to identify demyelinated areas. selected areas were subsequently examined by tem (ieo 906 germany, 100 kv). according to the method of zambonin and et al.23, sagittal sections of the cc were submitted to a quantitative evaluation to determine the percentage of correctly myelinated axons per field. cc images were taken from 4 sections with an intersectional distance of 50 µm. photographs of axons orientated in cross-section were taken, and quantification of myelinated axons was performed on 4 images per animal and treatment at a magnification of x 3500 using the image j software. immunohistochemistry after anesthesia, the animals were transcardially perfused with pbs followed by 4% paraformaldehyde (pfa, sigma, germany) in 0.1 m pbs, ph 7.4. brains were removed, postfixed in 4% pfa overnight and rinsed with pbs. after overnight post-fixation, brains were processed, embedded, and sectioned into 5 µm sections from the levels 215–275 as stated in the mouse brain atlas by sidman et al. (http:// www.hms.harvard.edu/research/brain/atlas.html). for ihc, sections were placed on silane-coated slides, de-paraffinized, rehydrated, heat-unmasked and blocked with pbs containing 5% normal serum. afterwards, sections were exposed to antiionized calcium binding adaptor molecule (anti-iba-1, 1:4.000; wako, germany) for overnight at 4°c. the next day, sections were treated with h2o2/pbs (0.3%, roth, germany) to block the endogenous peroxidase. then, sections were incubated with the appropriate secondary antibodies followed by the avidin-biotin complex (abc) method. diaminobenzidine (dab) was used as chromogen. finally, sections were dehydrated in graded alcohols and mounted. biochemical analysis in order to recognize the effects of free radical-mediated following cuprizone-induced demyelization with or without cbd treatment, determination of reduced glutathione (gsh), lipid peroxidation (lpo), catalase (cat) and superoxide dismutase (sod) tissue levels were carried out in the cc. to this end, mice were transcardially perfused with 0.1 m pbs (pbs, sigma, usa). following dissection of the brains, the ccs were 280 j contemp med sci | vol. 3, no. 11, summer 2017: 278–283 amelioration of cpz-induced demyelization by cbd original iraj ragerdi kashani et al. isolated. after that, tissue samples were homogenized on ice by application of a tissue homogenizer (remi, india). lpo products (malondialdehyde; mda) in the cc were measured according to kashani et al.22 briefly, tissues homogenates were produce in 0.15 m kcl (5% w/v homogenate). microcentrifuge tubes each containing 0.6 ml were incubated 1 h at 37 °c. following that, 1.2 ml of 28% w/v trichloroacetic acid (tca) solution (5%) was added, and by adding 1.2 ml of water, the final volume was reached to 3 ml. subsequently, centrifugation at 3000 xg for 10 min was performed and 2.5 ml of the supernatant was collected. after that, the color was developed by addition of 0.5 ml of 1% w/v thiobarbituric acid dissolved in 0.05 n naoh keeping the solution in boiling water bath for 15 min until the appearance of pink color. finally, the absorbance was read in a spectrophotometer at 532 nm. mda contents were declared as nmol/g wet tissue. the gsh content of cc was also determined by spectrophotometer. briefly, proteins from homogenized tissues (10% w/v in pbs, ph 7.4) were removed, and after adding an equal volume of 10% tca, incubated at 4°c for 2 h. then, the samples were centrifuged at 2000 xg for 15 min, and the supernatant was added to 2 ml of 0.4 m tris buffer (ph 8.9) including 0.02 m edta (ph 8.9) (sigma, usa). after that, 0.01 m 5,5’-dithio-bis 2-nitrobenzoic acid was added. eventually, the mixture was diluted with 0.5 ml dw, and absorbance was read in a spectrophotometer at 412 nm. results are shown as µg gsh/g wet tissue sample. total sod activity in the cc homogenate was measured based on the ability of sod to inhibit the production of formazan dye resulting from the reaction of wst-1 (water-soluble tetrazolium salt) and superoxide anion. briefly, samples were mixed with wst-1 solution and enzyme solution (xanthine oxidase) and incubated at 37°c for 30 min. then the absorbance of the solution at 450 nm was measured. the activity of total sod in the brain tissue was calculated by referring to the standard curve and expressed as u/mg protein. data analysis all data are given as means ± sd. statistical differences between various groups were analyzed by one-way analysis of variance (anova) followed by tukey’s post hoc test using graphpad prism 7 (graphpad software inc., usa). results cbd treatment delays demyelination standard lfb staining revealed a normal myelin structure in the control group (fig. 1a). the animals treated with cuprizone and pbs (cpz+v group) or without pbs (cpz) exhibited a significant decrease in lfb staining, characterized by bright areas indicative of myelin disorganization (fig. 1b, c and e). in the animals treated with cuprizone plus cbd (cpz+c group), lfb staining was significantly stronger than in the cpz group but the myelin aspect was not uniform and displayed vacuoles (fig. 1d and e, p ≤ 0.05). cannabidiol effects on the myelination index tem photographs were used to determine the percentage of myelinated axons in the cc. under control conditions, we found nearly the whole axon population as densely packed axons with a typical range in diameter and homogeneous regular myelin sheets (fig. 2a). cuprizone treatment with or fig. 1 demyelination in the corpus callosum (cc) after 5 weeks of cuprizone-induced demyelination with or without cannabidiol treatment. myelination index was evaluated in lfbstained sections. photomicrographs were taken from coronal sections of the cc. (a) control, (b) shows cuprizone-induced demyelination, (c) cuprizone with vehicle, (d) cuprizone-induced demyelination in the cannabidiol-treated group. quantification of the myelination index in the center of the cc reveals a significant myelin loss in the cuprizone group and a restoration of myelin in the group co-treated with cannabidiol. data represented means ± sd. *p ≤ 0.05 cpz vs. control, †p ≤ 0.05 cpz+v vs. control, #p ≤ 0.05 cpz+c vs. cpz+v. scale bar 100 µm. without pbs resulted in a massive reduction in myelinated axons numbers up to 90% (fig. 2b and c). we also observed very small myelinated axons with frazzled myelin layers. the co-administration of cbd partially inhibited the mentioned decline (fig. 2d and e, p ≤ 0.05 cuprizone versus cuprizone and cbd). cbd effect on microglia accumulation microglia accumulation was evaluated by ihc against iba-1. quantification of iba-1 staining intensity in the cc showed that in control animals, few microglia and low staining intensity can be detected (fig. 3a and e). in contrast, the area of iba-1 staining and its intensity significantly increased in the cpz and cpz plus vehicle animals (fig. 3b–e, p ≤ 0.05). staining values for iba-1 were significantly lower in cpz plus cbd animals (fig. 3d and e, p ≤ 0.05). mda and levels of anti-oxidants in order to analyze the effects of cuprizone and cbd exposure on parameters distinctive for oxidative stress, we determined of mda, gsh, cat and sod levels in homogenates of the cc. as shown in fig. 4, the activities of antioxidant enzymes (gsh, cat and sod) decreased significantly (after a c b d e iraj ragerdi kashani et al. 281j contemp med sci | vol. 3, no. 11, summer 2017: 278–283 original amelioration of cpz-induced demyelization by cbd fig. 2 representative tem micrographs of axons in the corpus callosum (cc). for details of abbreviations of the groups see legend of figure 1. micrographs show a massive decrease in the number of axons in the cc after cuprizone and a partial restoration of axon numbers and myelination after cannabidol. data represent means ± sd, *p ≤ 0.05. scale bar 1 µm. fig. 4 effect of cuprizone and cannabidiol on the levels of malondialdehyde (mda), reduced glutathione (gsh), catalase (cat) and superoxide dismutase (sod) in the corpus callosum (cc). cuprizone reduced gsh, cat and sod but increased mda compared with control. the application of cannabidiol reversed the effect on gsh, cat and sod. cannabidiol exposure itself lowered mda levels compared with the control group. data represented mean ± sd. *p ≤ 0.05 cpz vs. control, †p ≤ 0.05 cpz+v vs. control, #p ≤ 0.05 cpz+c vs. cpz+v. cuprizone-induced demyelination without cbd treatment compared with the control group (fig. 4b–d). the level of the anti-oxidant enzymes (gsh, cat and sod) was significantly increased (p ≤ 0.05) after cuprizone-induced demyelination with cbd treatment (cpz+c group) compared with the cuprizone-induced demyelination with pbs as a vehicle (cpz+v group). in contrast, mda levels were significantly increased after cuprizone-induced demyelination without cbd treatment (22.1 ± 2.9) compared with control mice (fig. 4a). interestingly, cuprizone-induced demyelination with cbd treatment (cpz+c group) significantly (p ≤ 0.05) decreased mda levels compared with cuprizone-induced demyelination without cbd (fig. 4a). discussion in the present study, we demonstrated that the intraperitoneal administration of cbd significantly ameliorate demyelination in the cc, decreased microglia accumulation and alleviated oxidative stress after cuprizone-intoxication. recent years have provided evidence that oxidative stress plays an important role in the pathogenesis of ms.1,2,5 here, we used the cuprizone mouse model which is known to cause oxidative damage of oligodendrocytes and dystrophic axons in the brain which closely mimics to some extent the so-called pattern iii lesions in ms patients.22,24 our results show that fig. 3 effect of cuprizone and cannabidiol treatment on microglial accumulation in the midline of the corpus callosum (cc). for details of abbreviations of the groups see legend of fig. 1. ihc revealed that in control animals, only few iba-1+ cells can be found (a, e), whereas cuprizone-induced demyelination without cannabidiol treatment (cpz) significantly increased the number of iba-1-positive staining intensity (b, c, and e). cannabidiol treatment significantly attenuated the cuprizone-induced microglial accumulation (d, e). data represented means ± sd. **p ≤ 0.001 cpz vs. control, ††p ≤ 0.05 cpz+v vs. control, #p ≤ 0.05 cpz+c vs. cpz+v. scale bar, 100 µm. a c b d e 282 j contemp med sci | vol. 3, no. 11, summer 2017: 278–283 amelioration of cpz-induced demyelization by cbd original iraj ragerdi kashani et al. cuprizone feeding significantly increased mda and decreased the activities of a set of anti-oxidant enzymes/factors such as sod, gsh and cat. these results are in good agreement with observations from other authors.15,22,24–26 cuprizone is able to increase levels of mda, a routine index of lipid peroxidation, and simultaneously decrease gsh levels and antioxidant enzyme activity (cat, sod).3 although the underlying mechanisms of ros-dependent myelin loss are not yet clear, there are several plausible explanations. first, ros can directly induce the apoptosis of oligodendrocytes, thereby leading to demyelination in ms.23 second, oligodendrocyte precursor cells may be more sensitive to oxidative stress compared to mature oligodendrocytes and thus limit cns repair and remyelination. 28 third, ros has direct effects on the lipid and protein components of myelin through peroxidation, and degrades myelin basic protein (mbp) through the production of matrix metalloproteinases.27,28 we demonstrate that cbd partially reverses these effects. it is assumed that cbd is able to restore the normal balance between oxidative stress markers and anti-oxidant endogenous mechanisms that is often disrupted in neurodegenerative disorders.29 anti-oxidant effects of cbd may involve intracellular mechanisms that enhance the ability of endogenous anti-oxidant enzymes to control oxidative stress, in particular the signaling triggered by the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2).29,30 nrf2 (also known as nfe2l2) is a critical regulator of genes involved in the detoxification of reactive oxygen species.30 cbd is thought to bind to an intracellular target which plays a major role in the control of anti-oxidant-response elements located in genes encoding for different anti-oxidant enzymes of the so-called phase ii-anti-oxidant response and is involved in the regulation of nrf2.29 cuprizone treatment causes a significantly increase in the number of iba-1 positive cells in the cc, thus being indicative for local neuroinflammation.4 another relevant finding of our study was that cbd administration attenuated the microglia accumulation in the cc of cuprizone-treated mice. this result is consistent with other data which shown that cbd administration reduced microglia accumulation in different brain injury models.31–34 it seems safe to conclude that microglia plays a detrimental role in the demyelinated lesion.32,34 microglia are the resident immune cells of the cns, participating in the regulation of immune responses due to their ability to present antigens and to secrete immuneregulatory factors such as neurotrophins, chemokines, cytokines, and ros.31,34 microglia can recruit and reactivate t cells in the cns and release detrimental molecules such as free radicals, inflammatory cytokines, and proteases, which then contribute to demyelination through a variety of different pathogenic processes.33 wu et al. showed that the production of ros by microglia occurred primarily by nox activation and required the hv1 proton channel.35 ablation of microglia or impairment of their function decreased disease progression in experimental ms animal models.34,36 thus, suppression of microglia will potentially reduce inflammatory lesions and limit demyelination within the cns. in addition, mecha et al. found that cbd induced apoptosis of microglial cells through lipid raft involvement.31,37 lipid rafts are regions of pla sma membranes with a distinct, characteristic structural composition, that participate in various cellular functions, including protein trafficking, transcytosis, endocytosis, cell survival, and cell death.37,38 cbd induces lipid raft coalescence, which has been linked to critical signaling pathways, such as apoptosis.37–39 conclusion our data implicate that cbd can attenuate destructive cuprizone effects in the brain by decreasing oxidative stress and reducing microglia accumulation. it remains to be analyzed in further studies whether such protective effects are also operant in human ms patients and are suited as therapeutic options. acknowledgments the study was supported by a grant from the tehran university of medical sciences and health services, tehran, iran (grant no. 21616-30-02-92). conflict of interest there is no conflict of interest. n references 1. suneetha a. role of dimethyl fumarate in oxidative stress of multiple sclerosis: a review. journal of chromatography b. 2016;1019:15–20. 2. lucchinetti c, bruck w, parisi j, scheithauer b, rodriguez m, lassman h. heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. annals of neurology. 2000;47(6):707–17. 3. praet j, guglielmetti c, berneman z, van der linden a, ponsaerts p. cellular and molecular neuropathology of the cuprizone mouse model: clinical relevance for multiple sclerosis. neuroscience & biobehavioral reviews. 2014;47:485–505. 4. zendedel a, kashani ir, azimzadeh m, pasbakhsh p, omidi n, golestani a, et al. regulatory effect of triiodothyronine on brain myelination and astrogliosis after cuprizone-induced demyelination in mice. metabolic brain disease. 2016;31(2):425–33. 5. lassmann h. what drives disease in multiple sclerosis: inflammation or neurodegeneration? clinical and experimental neuroimmunology. 2010;1(1):2–11. 6. wiendl h, hohlfeld r. multiple sclerosis therapeutics unexpected outcomes clouding undisputed successes. neurology. 2009;72(11):1008–15. 7. janssens k, maheshwari a, van den haute c, baekelandt v, stinissen p, hendriks jj, et al. oncostatin m protects against demyelination by inducing a protective microglial phenotype. glia. 2015;63(10):1729–37. 8. mahad dh, trapp bd, lassmann h. pathological mechanisms in progressive multiple sclerosis. the lancet neurology. 2015;14(2):183–93. 9. sinha k, das j, pal pb, sil pc. oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. archives of toxicology. 2013;87(7):1157–80. 10. carvalho an, lim jl, nijland pg, witte me, van horssen j. glutathione in multiple sclerosis: more than just an antioxidant? multiple sclerosis journal. 2014;20(11):1425–31. 11. gomez o, sanchez‐rodriguez a, le m, sanchez‐caro c, molina‐holgado f, molina‐holgado e. cannabinoid receptor agonists modulate oligodendrocyte differentiation by activating pi3k/akt and the mammalian target of rapamycin (mtor) pathways. british journal of pharmacology. 2011;163(7):1520–32. 12. cabral ga, jamerson m. marijuana use and brain immune mechanisms. int rev neurobiol. 2014;118:199–230. 13. giacoppo s, galuppo m, pollastro f, grassi g, bramanti p, mazzon e. a new formulation of cannabidiol in cream shows therapeutic effects in a mouse model of experimental autoimmune encephalomyelitis. daru journal of pharmaceutical sciences. 2015;23(1):48. 14. martín-moreno am, reigada d, ramírez bg, mechoulam r, innamorato n, cuadrado a, et al. cannabidiol and other cannabinoids reduce microglial iraj ragerdi kashani et al. 283j contemp med sci | vol. 3, no. 11, summer 2017: 278–283 original amelioration of cpz-induced demyelization by cbd activation in vitro and in vivo: relevance to alzheimer’s disease. molecular pharmacology. 2011;79(6):964-73. 15. acs p, kipp m, norkute a, johann s, clarner t, braun a, et al. 17β‐estradiol and progesterone prevent cuprizone provoked demyelination of corpus callosum in male mice. glia. 2009;57(8):807–14. 16. kozela e, lev n, kaushansky n, eilam r, rimmerman n, levy r, et al. cannabidiol inhibits pathogenic t cells, decreases spinal microglial activation and ameliorates multiple sclerosis‐like disease in c57bl/6 mice. british journal of pharmacology. 2011;163(7):1507–19. 17. baker d, pryce g, croxford jl, brown p. cannabinoids control spasticity and tremor in a multiple sclerosis model. nature. 2000;404(6773):84. 18. wade dt, makela p, robson p, house h, bateman c. do cannabis-based medicinal extracts have general or specific effects on symptoms in multiple sclerosis? a double-blind, randomized, placebo-controlled study on 160 patients. multiple sclerosis journal. 2004;10(4):434–41. 19. gudi v, gingele s, skripuletz t, stangel m. glial response during cuprizoneinduced de-and remyelination in the cns: lessons learned. frontiers in cellular neuroscience. 2014;8:73. 20. draheim t, liessem a, scheld m, wilms f, weißflog m, denecke b, et al. activation of the astrocytic nrf2/are system ameliorates the formation of demyelinating lesions in a multiple sclerosis animal model. glia. 2016. 21. zendedel a, beyer c, kipp m. cuprizone-induced demyelination as a tool to study remyelination and axonal protection. journal of molecular neuroscience. 2013;51(2):567–72. 22. kashani ir, rajabi z, akbari m, hassanzadeh g, mohseni a, eramsadati mk, et al. protective effects of melatonin against mitochondrial injury in a mouse model of multiple sclerosis. experimental brain research. 2014;232(9):2835–46. 23. zambonin jl, zhao c, ohno n, campbell gr, engeham s, ziabreva i, et al. increased mitochondrial content in remyelinated axons: implications for multiple sclerosis. brain. 2011;134(7):1901–13. 24. ljubisavljevic s. oxidative stress and neurobiology of demyelination. molecular neurobiology. 2016;53(1):744–58. 25. gilgun-sherki y, melamed e, offen d. the role of oxidative stress in the pathogenesis of multiple sclerosis: the need for effective antioxidant therapy. journal of neurology. 2004;251(3):261–8. 26. miljkovi’ d, bla’evski j, petkovi’ f, djedovi’ n, mom’ilovi’ m, stanisavljevi’ s, jevti’ b, stojkovi’ mm, spasojevi’ i. a comparative analysis of multiple sclerosis–relevant anti-inflammatory properties of ethyl pyruvate and dimethyl fumarate. the journal of immunology. 2015;194(6):2493–503. 27. witherick j, wilkins a, scolding n, kemp k. mechanisms of oxidative damage in multiple sclerosis and a cell therapy approach to treatment. autoimmune diseases. 2010;2011. 28. liu j, tian d, murugan m, eyo ub, dreyfus cf, wang w, et al. microglial hv1 proton channel promotes cuprizone‐induced demyelination through oxidative damage. journal of neurochemistry. 2015;135(2):347–56. 29. fernández‐ruiz j, sagredo o, pazos mr, garcía c, pertwee r, mechoulam r, et al. cannabidiol for neurodegenerative disorders: important new clinical applications for this phytocannabinoid? british journal of clinical pharmacology. 2013;75(2):323–33. 30. schneider k, valdez j, nguyen j, vawter m, galke b, kurtz tw, et al. increased energy expenditure, ucp1 expression, and resistance to diet-induced obesity in mice lacking nuclear factor-erythroid-2-related transcription factor-2 (nrf2). journal of biological chemistry. 2016;291(14):7754–66. 31. toth cc, jedrzejewski nm, ellis cl, frey wh. cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type i diabetic peripheral neuropathic pain. molecular pain. 2010;6(1):1. 32. gomes fv, llorente r, del bel ea, viveros m-p, lópez-gallardo m, guimarães fs. decreased glial reactivity could be involved in the antipsychotic-like effect of cannabidiol. schizophrenia research. 2015;164(1):155–63. 33. rawji ks, yong vw. the benefits and detriments of macrophages/microglia in models of multiple sclerosis. clinical and developmental immunology. 2013;2013. 34. heppner fl, greter m, marino d, falsig j, raivich g, hövelmeyer n, et al. experimental autoimmune encephalomyelitis repressed by microglial paralysis. nature medicine. 2005;11(2):146–52. 35. wu lj. microglial voltage-gated proton channel hv1 in ischemic stroke. translational stroke research. 2014;5(1):99–108. 36. huitinga i, ruuls s, jung s, rooijen nv, hartung h, dijkstra c. macrophages in t cell line‐mediated, demyelinating, and chronic relapsing experimental autoimmune encephalomyelitis in lewis rats. clinical & experimental immunology. 1995;100(2):344–51. 37. chen yc, kung fl, tsai il, chou th, chen is, guh jh. cryptocaryone, a natural dihydrochalcone, induces apoptosis in human androgen independent prostate cancer cells by death receptor clustering in lipid raft and nonraft compartments. the journal of urology. 2010;183(6):2409–18. 38. wu hy, goble k, mecha m, wang cc, huang ch, guaza c, et al. cannabidiol‐ induced apoptosis in murine microglial cells through lipid raft. glia. 2012;60(7):1182–90. 39. mecha m, feliú a, carrillo-salinas f, rueda-zubiaurre a, ortegagutiérrez s, de sola rg, et al. endocannabinoids drive the acquisition of an alternative phenotype in microglia. brain, behavior, and immunity. 2015;49:233–45. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201707 7j contemp med sci | vol. 1, no. 1, winter 2015:7–12 research objective to identify the effect of exercise and nutrition on breast cancer occurrence and to find the relationship between life style and some demographic and reproductive variables. methods a retrospective study (case control study) of a purposive sample of 400 women; 200 women diagnosed with breast cancer visited the merjan teaching hospital oncology cancer center in babylon governorate as a study group and 200 women free of breast cancer as a control group. an assessment tool was constructed for the purpose of the study. it comprised of socio-demographic data, reproductive data, information related to breast cancer (stage, side, period, family history) and information related to their life style: exercise (5 items) and nutrition (14 items) of 10 years previous breast cancer occurrence, physical measurements for measuring obesity and overweight (bmi) as well as medical records to explain in the stage of breast cancer and other details which may be assisting this study data were collected from 4 may 2013 to 4 july 2013. analysis of data was performed through the application of descriptive and inferential statistical data analysis approach. results the study demonstrates that the highest percentage (39.5%) of study sample was in age group 50–54 years in comparison with the other age groups for both the study and control groups. there was a high significant difference at p < 0.01 between the life aspects (exercise and nutrition) and breast cancer in women while no relationship between life aspects and demographical and reproductive variables except with interval between pregnancies and duration of contraception of study group was noted. conclusion establishing an activation media and ministry of health role for guiding women and their families about the importance of exercise and healthy nutrition which contributes to reducing the incidence of breast cancer occurrence and the importance of early detection of breast cancer. keywords life style, breast cancer, women, retrospective study effect of life style (exercise and nutrition) on occurrence of breast cancer in women: a retrospective study in babylon governorate najat hamza hassana & rabea m alib introduction the two most important parts of physical health involve exercise and nutrition. these two go hand-in-hand. not fueling the body with proper nutrition negates many of the benefits of exercise and vice versa.1 diet and exercise have a direct impact on the quality of life of people in their later years. according to the national institute on ageing, regular physical exercise and proper nutrition can help people remain independent for long. this healthy lifestyle can keep a variety of age-related conditions and diseases at bay permanently. while genes play a significant role in the life span of people, proper nutrition and regular exercise directly affects how well they live. according to the american geriatrics society, a lifetime of healthy living may be the most beneficial to healthy ageing, but it is never too late to begin eating well and exercising regularly. eat foods of a variety of colors and those rich in omega-3 fatty acids to remain strong. stay physically fit with regular exercise and reduce the risk of falling.2 cancer is a disease that is caused by both genetic and environmental factors. some women are born with a genetic mutation that dramatically increases their risk of developing breast cancer. but for all women, including women with a genetic mutation, environmental factors such as diet, exercise and chemical exposures affect genes in ways that determine whether cancer actually develops.3 researchers found more than 3000 women with and without breast cancer, those who exercised during their childbearing years were less likely to develop cancer after menopause. the effects of physical activity were strong among postmenopausal women. women may significantly reduce their risk for breast cancer by exercising a couple hours each day. a new study found that even mild physical activity like walking reduced risk for the disease that strikes 227 000 new women each year.4 diet is thought to be partly responsible for about 30%–40% of all cancers. but diet alone is unlikely to be the “cause” or “cure” of cancer. although more research needs to be done on diet and breast cancer, findings suggest that physical activity, a healthy diet (particularly one low in fat and high in vegetables and fiber) and a healthy weight can help reduce the risk of breast cancer or its reoccurrence.5 materials and methods a retrospective study (a case control study) was conducted on the effect of life style on occurrence of breast cancer in women. a purposive sample of 400 women; 200 diagnosed with breast cancer as a study group and 200 the control group which was free from breast cancer or any other types of cancer past and present, were collected from different districts within babylon governorate and looks back retrospectively for previous 10 years. the questionnaire was designed for the purpose of the study. it comprised of four parts: socio-demographic data such as mother’s age, body mass index (bmi), marital status, family income through previous 10 years; reproductive data such as gravidity, parity, age at menarche, age at first pregnancy, pregnancy interval, menopausal age, age at last menstrual period, reproductive age, breast feeding, duration of contraceptive pills; a technical institute, al-furat al-awsat technical university, karbala, iraq. b maternity and child health nursing, college of nursing, university of baghdad, baghdad, iraq. correspondence to najat hamza hassan (noorhuda4@yahoo.com) (submitted: 08 december 2014 – revised version received: 24 january 2015 – accepted: 12 february 2015 – published online: winter 2015) issn 2413-0516 8 j contemp med sci | vol. 1, no. 1, winter 2015:7–12 effect of life style on occurrence of breast cancer research najat hamza hassan & rabea m ali information related to woman’s healthy life style exercise which includes 5 items, and the nutrition aspect includes 14 items. these items were rated according to scale (always, sometimes and never), and information related to breast cancer were rated based on stage, side, period and family history of breast cancer (previous 1–10 years). bmi was indicated for measuring obesity and overweight. the investigator measures the current bmi according to who categories of bmi in 2002 which are: underweight = <18.5 kg/m2; normal weight = 18.5–24.9 kg/m2; overweight = 25–29.9 kg/m2; obesity = 30 kg/m2 or greater. medical records explain in which stage of breast cancer they are in and other details which may assist this study. for pilot study and reliability of the questionnaire a convenient sample of 20 breast cancer women who attended merjan teaching hospital oncology cancer center in babylon governorate for this preliminary study was conducted from 4 may 2013 to 10 july 2013. the reliability coefficients of the pilot study are 0.973. the time required for each interview ranged from 20 to 30 min for each women and 5–10 min for measuring the weight and height to estimate the bmi. statistical data analysis approaches were used in order to analyse and assess the results of the study depended on descriptive data analysis, and inferential data analysis. results table 1 shows the distribution of the two samples (study and control) according to their demographic characteristics variables (age groups, marital status, family income) it reported a non significant difference at p > 0.05, which indicates that the two independent groups seems from the same population. in addition, bmi reported a highly significant difference at p < 0.01, with bad assessment at the study group compared with the control. the age group, 50–54 years, was shown to be the larger group (39.5%) in comparison with the other age groups for both the study and control groups. the highest percentages (44.5%) of study group were overweight, while (48.5%) the control group had normal weight. about 70.5% of study and 66.5% of control group were married, 42.5% and 47%, respectively, in both groups had sufficient family income. table 1. distribution of the studied demographical characteristics variables in the study and control samples with comparisons significant demographical characteristics samples groups no. % asymp. sig. (*) (2-tailed) age groups study 20–24 2 1.0 z = 0.000 p = 1.000 ns 25–29 1 0.5 30–34 3 1.5 35–39 4 2.0 40–44 9 4.5 45–49 30 15.0 50–54 79 39.5 55–59 59 29.5 60 ≥ 13 6.5 control 20–24 2 1.0 25–29 1 0.5 30–34 3 1.5 35–39 4 2.0 40–44 9 4.5 45–49 30 15.0 50–54 79 39.5 55–59 59 29.5 60 ≥ 13 6.5 bmi (kg/m²) study underweight 35 17.5 z = 2.611 p = 0.009 hs normal weight 68 34 over weight 89 44.5 obesity 8 4 control underweight 40 20 normal weight 97 48.5 over weight 53 26.5 obesity 10 5 marital status study married 141 70.5 z = 0.900 p = 0.368 ns single 16 8 widow 27 13.5 divorced 14 7 separated 2 1 control married 133 66.5 single 18 9 widow 29 14.5 divorced 16 8 separated 4 2 family income study sufficient 85 42.5 z = 0.984 p = 0.325 ns barely sufficient 48 24 not sufficient 67 33.5 control sufficient 94 47 barely sufficient 47 23.5 not sufficient 59 29.5 * c.s.: comparisons significant; ns: non sig. at p > 0.05; hs: highly sig. at p < 0.01. table 2 shows that the highest percentage of study and control group 38% and 39%, respectively, were having 3–4 gravida. regarding parity, 39% of study group were having 1–2 para, while for control group 33% of them were having 3–4 para. the highest percentage of both groups were in post-rep. age. a 62.5% and 75.5% of study group and the highest percentage of the users, 33.5% and 23%, respectively, were using pills. table 3 demonstrates the mean of some reproductive variables. the mean age at menarche for study sample was 12.48 ± 0.81 years which was lower than 9j contemp med sci | vol. 1, no. 1, winter 2015:7–12 research effect of life style on occurrence of breast cancernajat hamza hassan & rabea m ali table 2. distribution of the studied reproductive variables in the study and control samples with comparisons significant reproductive characteristics samples groups no. percent c.s p–value gravidity study 0 30 15 χ2 = 1.985 p = 0.921 ns 1–2 54 27 3–4 76 38 5–6 37 17.5 7 and more 3 1.5 control 0 32 16 1–2 44 22 3–4 78 39 5–6 41 20.5 7 and more 5 2.5 parity study 0 30 15 χ2 = 2.588 p = 0.858 ns 1–2 78 39 3–4 62 31 5–6 28 14 7 and more 2 1 control 0 32 16 1–2 64 32 3–4 66 33 5–6 34 17 7 and more 4 2 reproductive age study rep. age 37 18.5 fept(**) p = 0.704 ns post-rep. age 163 81.5 control rep. age 40 20 post-rep. age 160 80 breast feeding study yes 125 62.5 fept p = 0.000 hs no 45 22.5 control yes 151 75.5 no 17 8.5 the use of contraception study not use 76 38 χ2 = 7.179 p = 0.066 ns pills 67 33.5 helix 28 14 surgery 20 10 control not use 93 46.5 pills 46 23 helix 41 20.5 surgery 20 10 c.s.: comparisons significant; fept: fisher exact probability test. the age at menarche for control group, 12.59 ± 0.83 years. the mean age at first pregnancy 27.01 ± 11.81 years was noted for study sample, while 23.53 ± 11.52 years for the control group. pregnancy interval mean was 1.32 ± 0.72 years for study sample, and 1.20 ± 0.69 years for control. age at menopause was 52.90 ± 1.97 years for study sample and 52.54 ± 1.92 years for control. the mean age at last menstrual cycle was 56.81 ± 42.30 years for study sample and 56.54 ± 42.32 years for control, and regarding the duration of contraception use, 7.44 ± 1.70 years for the study group and 7.03 ± 2.00 years for the control. table 4 shows that the majority (60%) of cases had third stage of breast cancer, 52% of cases had left breast cancer, 93% of cases had no family history, and 51% of cases had 3–4 years period of breast cancer. high significant differences were found between breast cancer stages, breast cancer side and period of breast cancer, while no significant differences were found in family history. table 5 shows the results of testing coincidence’s responding between the difference of the studied groups according to sub and main domains of “life style to breast cancer in women” through equality of variances and equality of mean value parameters. the results of testing indicated that highly significant differences at p < 0.001 were obtained (fig. 1). table 6 shows significant differences between study and control group in items (do exercise at least 20 sec or more 3 times weekly, and i walk whenever possible). regarding nutritional aspects the results shows low mean scores in majority of items and low rs and assessed failure for both study and control groups, except items 3, 4 ,5, 6 and 12 for study group, and the items 3, 4, 5, 6, 11 & 12 for control group with high rs, and assessed pass. there are significant differences between the study and control group in items 1, 5, 6, 7 and 11. the results from table 7 has reported that the distribution of the life style to breast cancer in women through the two dichotomous of responding had no relationship with their demographical characteristics variables and as well as of some related variables (reproductive characteristics) with the overall assessments at the study group except with interval between pregnancies and duration of contraception only and we can table 3. descriptive statistics related to some reproductive characteristics in both groups reproductive characteristics groups no. m.s. std. dev. std. error mean age at menarche study 200 12.48 0.81 0.06 control 200 12.59 0.83 0.06 age at first pregnancy study 170 27.01 11.81 0.08 control 168 23.53 11.52 0.08 pregnancy interval study 170 1.32 0.72 5.10 control 168 1.20 0.69 4.92 age at menopause study 112 52.90 1.97 0.19 control 112 52.54 1.92 0.18 age at last menstrual cycle study 112 56.81 42.30 4.00 control 112 56.54 42.32 4.00 duration of pills contraception study 130 7.44 1.70 0.15 control 125 7.03 2.00 0.18 c.s.: comparisons significant; m.s.: mean of score. 10 j contemp med sci | vol. 1, no. 1, winter 2015:7–12 effect of life style on occurrence of breast cancer research najat hamza hassan & rabea m ali table 4. distribution of the study sample according to breast cancer history about breast cancer stage freq. percent cum. percent c.s. breast cancer stage stage 1 8 4 4 χ2 = 187.36 p = 0.000 hs stage 2 70 35 39 stage 3 120 60 99 stage 4 2 1 100 breast cancer side right 94 47 47 χ2 = 94.840 p = 0.000 hs left 104 52 99 both of them 2 1 100 family history non applicable 186 93 93 χ2 = 3.143 p = 0.534 ns mother 5 2.5 95.5 sister 3 1.5 97 daughter 1 0.5 97.5 aunt 3 1.5 99 grandma 2 1 100 period of disease in years 1–2 45 22.5 22.5 χ2 = 94.070 p = 0.000 hs 3–4 102 51 73.5 5–6 51 25.5 99 7 and more 2 1 100 c.s.: comparisons significant; ns: non sig. at p > 0.05; hs: highly sig. at p < 0.01. table 5. comparisons significant for the studied sub and main domains of “life style to breast cancer in women” between study and control groups sub and main domains levene’s test for equality of variances t-test for equality of means c.s. (f) statistic sig. (t) statistic d.f. sig. (2-tailed) exercise 4.385 0.037 –4.878 390.5 0.000 hs nutrition 0.161 0.688 –4.272 398.0 0.000 hs life style for exercise and nutrition 0.692 0.406 –6.405 398.0 0.000 hs c.s.: comparisons significant; ns: non sig. at p > 0.05; hs: highly sig. at p < 0.01. conclude that the studied questionnaire can be amended for all individuals of the population concerning with breast cancer irrespective of the differences in their demographical and reproductive characteristic variables. discussion the age group, 50–54 years, was the larger group (39.5%) in comparison with the other age groups for both study and control groups. their ages ranged between 22 and 83 years with mean ± sd of 53.19 ± 6.48 years for study group, while the mean ± sd of control group was 50.99 ± 9.05 years. this study was in agreement with study conducted by benz 20096 who stated that up to 80% of breast cancers occur after 50 years. the national cancer institute (2010)7 reports that more than three-quarters of breast cancer cases occur in women over age 50 years and stated that the risk of getting breast cancer increases with age. regarding bmi, the majority (44.5%) of study had overweight (25– 29.9 kg/m2), while 48.5% of control group had normal weight (18.5–24.9 kg/ m2). in addition to that, bmi reported a highly significant difference at p < 0.01, with bad assessment at the study group compared with the control. buist and adiana (2003)8 have indicated that their study looked at the relationship between tumor growth and bmi, an indicator of obesity that is based on a person’s height and weight. understanding how bmi is related to tumor growth could provide study 1.489 3 2.5 2 1.5 1 1.639 2.008 2.091 1.749 1.865 exercise nutrition life aspects for exercise and nutrition control study control study control fig. 1 bar chart for mean of score at the sub and main domain of life aspects (exercise and nutrition) at the study and control groups. prince pencil 11j contemp med sci | vol. 1, no. 1, winter 2015:7–12 research effect of life style on occurrence of breast cancernajat hamza hassan & rabea m ali critical tools to assist clinicians with pre and post-diagnostic breast cancer surveillance and would help clarify the role bmi plays as a predictor of recurrence and death from breast cancer. the findings of the current study was in agreement with the findings of perez (2010)9 who indicated that a mid-life increase in bmi may substantially increase postmenopausal breast cancer risk, according to research by investigators from nci and columbia university’s mailman school of public health. in a recent analysis, women who reported a table 6. descriptive statistics (mean of score, standard deviation, relative sufficiency and assessment of items according to the scale’s cutoff point) for the studied questionnaire’s life style to breast cancer in women in the two groups items study control p value c.s.(*) m.s. std. dev. r.s. % m.s. std. dev. r.s. % exercise 1. do exercise at least 20 sec or more, 3 times weekly 1.06 0.23 35 1.14 0.40 38 0.029 s 2. stop exercise when you feel fatigue 1.11 0.46 37 1.18 0.54 39 0.055 ns 3. my job needs for walking 1.31 0.70 44 1.34 0.71 45 0.563 ns 4. my work is to exercise inside the house 2.42 0.76 81 2.56 0.71 85 0.052 ns 5. i walk wherever possible 1.55 0.69 52 1.98 0.87 66 0.000 hs nutrition 1. when i choose animal protein i choose fish 1.72 0.80 57 1.95 0.88 65 0.010 s 2. i eat the chest of a chicken without skin 1.73 0.87 58 1.79 0.88 60 0.391 ns 3. i eat red meat free of fat once a week 2.00 0.73 67 2.05 0.72 68 0.494 ns 4. decreasing the use of solid animal fats or butter when cooking 2.24 0.73 75 2.24 0.68 75 0.829 ns 5. use vegetable oils such as corn oil, olive oil, etc. 2.50 0.62 83 2.64 0.53 88 0.019 s 6. when i choose vegetarian protein i choose legumes 3.00 0.00 100 2.98 0.14 99 0.045 s 7. i eat non-cooked vegetables, 2.5 cups a day 1.32 0.53 44 1.47 0.61 49 0.007 hs 8. i eat 5 pieces of fresh fruit per day 1.20 0.43 40 1.27 0.50 42 0.166 ns 9. i eat sweets and sugars per day 1.96 0.91 65 1.88 0.87 63 0.392 ns 10. i drink adequate fluids (8 glasses of water or 2.5 litres per day) 1.86 0.77 62 1.93 0.81 64 0.401 ns 11. i use yogurts low or free in fat 1.89 0.85 63 2.08 0.88 69 0.029 s 12. refrained from eating irregular meals 2.17 0.90 72 2.10 0.92 70 0.458 ns 13. eating a daily multivitamin 1.87 0.88 62 1.97 0.91 66 0.230 ns 14. i adopted canned foods 1.40 0.69 47 1.31 0.59 44 0.313 ns c.s.: comparisons significant; ns: non sig. at p > 0.05; hs: highly sig. at p < 0.01. table 7. association among demographical and reproductive variables with overall assessments in life style among women with breast cancer predicted variables study control c.c. p value c.c. p value age 0.135 0.881 ns 0.251 0.098 ns bmi: kg/m² 0.155 0.176 ns 0.111 0.475 ns marital status 0.078 0.873 ns 0.210 0.056 ns family income 0.147 0.111 ns 0.201 0.015 s gravidity 0.164 0.583 ns 0.281 0.025 s parity 0.128 0.831 ns 0.266 0.047 s age at menarche 0.075 0.887 ns 0.170 0.204 ns age at first pregnancy 0.132 0.698 ns 0.158 0.507 ns interval between pregnancies 0.169 0.025 s 0.025 0.744 ns age at menopause 0.328 0.096 ns 0.280 0.393 ns age at last menstrual cycle 0.326 0.205 ns 0.265 0.582 ns reproductive age 0.021 0.765 ns 0.079 0.263 ns breast feeding 0.076 0.317 ns 0.038 0.620 ns the use of contraception 0.081 0.838 ns 0.157 0.369 ns duration of contraception use 0.355 0.028 s 0.360 0.029 s gain in bmi of five points (5 kg/m2) or more between age 20 and postmenopausal age (55–74 years) had nearly twice the risk of developing postmenopausal breast cancer compared to women who maintained their bmi during the same time period. in addition, researchers found independent positive associations between postmenopausal breast cancer and bmi gain both before and after age 50 years. regarding marital status, 70.5% of study and 66.5% of control group were married. khan et al. (2002)10 said that the previous studies have found that married women are more likely to be diagnosed at an earlier stage of cancer than those who are unmarried. but randi et al. (2004)11 said that our study suggests that marital status is not materially associated with cancer risk. thus, the evidence that married subjects are at lower risk of several other major diseases may not be applicable to cancer. regarding family income, 42.5% and 47%, respectively for both groups have sufficient income, while the highest proportion was with their insufficient income. this result refers to the fact that the sample is from the lowand 12 j contemp med sci | vol. 1, no. 1, winter 2015:7–12 effect of life style on occurrence of breast cancer research najat hamza hassan & rabea m ali middle-income class. march (2002)12 said that a new study adds to mounting evidence that women with lower incomes and less education are more likely to be diagnosed with advanced breast cancer than women with higher socioeconomic standard. curado and others (2007)13 stated that the recent media reports have highlighted the increasing incidence of breast cancer in lowand middle-income countries. the relative risk of breast cancer diagnosis associated with current and recent use of hormonal contraceptives did not appear to vary with family history of breast cancer.14 some studies have suggested that women who began using hormonal contraceptives before the age of 20 or before their first full-term pregnancy are at increased risk for breast cancer, but it is not clear how much of the risk stems from early age at first use, and how much stems from use before the first full-term pregnancy.15 regarding the effect of exercise and nutrition on breast cancer occurrence the results of testing indicated that highly significant differences at p < 0.001 were obtained. american cancer society stated that many studies have shown that moderate to vigorous physical activity is linked with lower breast cancer risk.16 a diet that is rich in vegetables, fruits, poultry, fish and low-fat dairy products has also been linked with a lower risk of breast cancer in some studies. but it is not clear if specific vegetables, fruits or other foods can lower risk. research has shown that poor diet and not being active are two key factors that can increase a person’s cancer risk.17 conclusion the present study shows the highest percentage of women having breast cancer was of age group 50–54 years, overweight 25–29.9 kg/m2, secondary school graduated, married and having sufficient family income. the results indicated that the individuals of the study groups reported no significant differences were found in family history while highly significant differences of women were breast feeding their babies. the study also showed that the sub and main domain of life style for exercise and nutrition reported a highly significant difference at p < 0.01. the results of testing indicated that a significant difference at p < 0.05 was obtained at the related variables (reproductive characteristics) with the overall assessments at the study group except with interval between pregnancies and duration of contraception only. recommendations 1. activation of media and ministry of health role for increasing the awareness of women and their families about the importance of reducing the risk factors which contributing on breast cancer occurrence through. a. monthly breast self-examination and regular mammography for age ≥ 40 years are the recommended methods of early detection of breast cancer. b. at least 8 hours sleep daily. c. exercising regularly for least 1 hour 3 times a week. d. encourage the women about healthy diet and nutrition. e. encourage breast feeding and maintaining a healthy weight. 2. further study on large population.  references 1. simms k. exercise and nutrition: coping mechanisms for life. fully living. april 16: 2011. 2. ray l. nutrition and exercise for the elderly. national institute on aging: exercise & physical activity. sep 30;2010. 3. breast cancer research program. environmental influences and breast cancer. university of california; 2002. 4. lauren e. mccullough, sybil m. eng, patrick t. bradshaw, rebecca j. cleveland, susan l. teitelbaum, alfred i. neugut, et al. fat or fit: the joint effects of physical activity, weight gain, and body size on breast cancer risk. cancer. 2012 jun 25;118(19):4860–8. doi: http://dx.doi.org/10.1002/cncr.27433 5. breastcancer.org: nutrition and breast cancer risk reduction. 17 sept 2012. 6. benz cc. impact of age on the biology of breast cancer. cancer and developmental therapeutics program buck institute for age research adjunct professor. san francisco: university of california; 2009. pp 9–10. 7. national cancer institute: what you need to know about breast cancer? 2010. 8. buist d, diana s. optimizing breast cancer outcomes: bmi, tumor markers, and quality of care. american cancer society; 2003. 9. perez v. gain in body mass index increases postmenopausal breast cancer risk. 20 april, 2010. 10. khan s, et al. association between marital status and stage at diagnosis of breast cancer: what role does screening play? university of texas medical branch, 11 nov 2002. 11. randi g, altieri a, gallus s, chatenoud l, montella m, franceschi s, et al. marital status and cancer risk in italy. prev med. 2004 may;38(5):523-8. 12. march d. researcher find educational level/income increase risk of advanced breast cancer diagnosis; 2002. 13. curado mp, edwards b, shin hr, et al. cancer incidence in five continents. france: international agency for research on cancer, vol. ix. lyon, 27 dec 2007. 14. national cancer institute: “hormone therapy”. genetics of breast and ovarian cancer. 12 aug 2006. 15. world health organization international agency for research on cancer (1999). “hormonal contraception and post-menopausal hormonal therapy,” retrieved 12 mar 2011; 1999. 16. american cancer society: diet and activity factors that affect risks for certain cancers. breast cancer; 2014. 17. american cancer society. diet and physical activity: what’s the cancer connection? 2014. 59j contemp med sci | vol. 4, no. 2, spring 2018: 59–62 research preoperative upper endoscopy and bariatric surgeries abdolreza pazouki, vahid rezaei, adnan tizmaghz, ali kabir minimally invasive surgery research center, center of excellence, iran university of medical sciences, tehran, ir iran. correspondence to ali kabir (email: aikabir@yahoo.com). (submitted: 04 july 2017 – revised version received: 12 august 2017 – accepted: 02 october 2017 – published online: 26 june 2018 ) objectives in this study, the frequency rate of incidental findings in esophageal and gastric endoscopy of morbid obese subjects’ candidate for bariatric surgery was determined. methods and materials in this observational study, 1663 consecutive patients candidate for bariatric obesity in rasool-akram hospital were enrolled and the upper endoscopy was done and the frequency rate of incidental findings was determined. results in this study, 41, 31, 27.3, 6, 0.8, 0.8% respectively had helicobacter pylori infection, hiatal hernia, esophagitis, esophageal ulcer, gastric ulcer and polyp. esophagitis and esophageal ulcer were significantly more common among male subjects (9% males, 5% females and 33% males, 25% females) and the mean age was higher among those with esophageal ulcer and h. pylori infection (p = 0.01 and 0.007 respectively). in general it can be concluded that about 1/3 of patients (35%) with morbid obesity have incidental findings on endoscopy. conclusion totally, according to the obtained results, it may be concluded that nearly 1/3 of morbid obese subjects candidate for bariatric surgery have incidental findings in preoperative upper endoscopy. keywords bariatric surgeries, esophagogastroduodenoscopy, incidental findings introduction obesity and overweight is correlated with some chronic diseases such as coronary artery, diabetes mellitus, most of cancers and musculoskeletal problems.1 the fat stored in a body acts as an energy source. when the body intake a lot of energy, additional energy accumulates in fats and contribute with weight gain. obesity is as the result of sedentary life and intake of a lot of energy. the obesity -associated diseases include type ii mellitus diabetes, dyslipidemia, obstructive sleep apnea and the increased risk of coronary artery diseases.2 the obesity could lead to increasing pressure on the spine and also the thermo-regulation disorder in warm weather condition. the treatment of obesity depends upon the alternation of life style and mostly emphasis on diet and exercise. obese people (bmi = 35–45) with associated disease are those that just changing the life style is not effective, thus fda recommended to administrate drugs or do operation for these kinds of patients.3 the annual cost of weight loss is estimated to be more than 117 billion dollars each year. weight loss was a major goal of the world in 2010 to promote the quality of people’s health.4 the medicine and sport collage of usa had proposed various methods such as diet, exercise, administration of different drugs and behavioral strategies, to lose weight. weight loss leads to reduction of ldl, tg and increase of hdl, improvement of glucose tolerance, decrease of insulin resistance and reduce fast glucose rates and inflammatory markers like crp that is correlated with coronary artery diseases. heart, lung and blood institute guideline recommended at least 10% of weight loss in reducing the risk of vcd.5 bariatric surgery is one of the most effective procedure for immediate weight loss in patients and mostly this surgery is performed in stomach and esophagus, so the endoscopic evaluation of these organs become impossible.3 for example, hiatal hernia can be diagnosed through endoscopy and in-patients with this problem, gastric banding is contraindicated. gastric ulcers in remnant stomach would be ignored otherwise the effect of obesity on incidence of different gastro-esophageal disorders is not clearly understood. it is important to treat most of these disorders before the bariatric surgery.3 so the aim of this study was to find the prevalence of gastro-esophageal disorders in morbid obese patients. the bariatric surgery is the most effective and long lasting remedy for obese patients which depends on sequential exercise after surgery.2 however, research demonstrate that about 80% of patients undergoing surgery do not take the recommendation of doctors to do an exercise for more than 150 minutes in a week.1 because in bariatric surgeries, large part of stomach is involved these parts always become inaccessible to endoscopy. so the possible finding from endoscopy of upper gi tracts before surgery could be helpful to make the right decision for patients.2,3 for instance, the gastric band surgery is a contraindicated in the presence of hiatus hernia that could be easily diagnosed by endoscopy before the surgery. also ulcers will be inaccessible in endoscopy after gastric bypass, that they should be diagnosed and treated before the surgery. thus given the importance of this issue, this study evaluated the frequency of accidental findings of esophagi and gastric endoscopy before the bariatric surgery. material and methods in this cross-sectional study, all 1663 patients with obesity (defined as bmi ≥ 40 or bmi ≥ 35 along with underlying risk factors like diabetes) that referred to obesity clinic of rasool-akram hospital, a referral who were candidate for bariatric surgery during 2012–2013 were enrolled in this study. people asked to fill the consent form before upper gi endoscopy and the frequency of accidental findings in esophagus and stomach were determined. all data were registered in iran national obesity surgery database (www. obesitysurgery.ir). after collection of information, data were analyzed with spss version 13 software. the frequency for qualitative variables issn 2413-0516 60 j contemp med sci | preoperative upper endoscopy and bariatric surgeries research abdolreza pazouki et al. and means and standard deviations were calculated for quantitative variables. chi-square and fisher’s exact test and independent t-test were used for interpretation of results and the p-value less than 0.05 consider significant. results of 1663 patients, 82.7% were females and 17.3% were males. the mean age of people were 40.8 + 10.5 years. the average of bmi was 45.7% + 5.33 kg/m2. among patients who underwent the preoperative upper endoscopy, 41.2% had helicobacter pylori infection, 31% hiatal hernia, 27.3% esophagitis, 6% esophageal ulcer, 0.8% peptic ulcer, and 0.8% polyp (figure 1). there was statistically relation with mean age of patients with esophageal ulcer (p = 0.002) and h. pylori infection (p = 0.001), also the frequency of esophageal ulcer was higher in female than male (9.1% vs 5.3%) and this difference was statistically relevant (p = 0.014). on the other hand, the frequency of hiatus hernia, peptic ulcer, and bmi had not any statistical relation with mean age of patients. also the rest esophagogastroduodenoscopy findings had not any statistical correlation with sex, too (p = 0.007). peptic ulcer, polyp and h. pylori infection had no correlation with bmi of patients. discussion unfortunately routine endoscopy examination of upper gi tract before bariatric surgery in asymptomatic patients is not recommended in reference books but type of bariatric surgery and treatment of patients can be influenced by most of the endoscopy findings such as peptic ulcers, hiatal hernia, esophagitis and reflux. thus, in current study we aimed to evaluate prebariatric surgery endoscopy incidental findings and its effects on choosing appropriate type of bariatric surgery for the patients. also treatment of some gastric disorders that accidentally diagnosed may have a great impact on outcomes of operation. for example, in-patients with hiatal hernia, gastric banding is not recommended. in gastric bypass surgery, a part of the stomach is permanently unavailable from endoscopic evaluations and this is important to have preoperative screening endoscopy, especially in patients with a family history of stomach cancer or esophageal metaplasia.3 in this study 41.2% of patients had been infected with h. pylori, 31% of people hiatal, 27.3% had esophagitis hernia, 6% esophagitis ulcer, 0.8% peptic ulcer, and 0.8% had polyp. the incidence of esophagitis and ulcers of esophagus among men was significantly higher and also the mean age of patients was significantly higher in patients with esophagitis, ulcer of esophagus and infection of h. pylori. one study conducted in spain showed that 48.7% of people from 194 candidate for bariatric surgery treatment, had a gastric disorders in upper gi tract. three patients (1.5%) had a peptic ulcer and 69.3% had h. pylori infection.4 however, the frequency of positive results of endoscopy and existence of h. pylori in our study was less than current study; the frequency of ulcer was somewhat similar. because of the importance of gastric ulcers endoscopy appears to be reasonable. in one study in brazil, 57.9% of 126 candidate for bariatric surgery had a one positive finding in endoscopy. in overall 3.2% of patients had an ulcer and 53.2% were positive for h. pylori infection.5 in our study the frequency of positive cases was similar, however, the overall frequency of endoscopic findings and positive h. pylori infection was less than this study. in our study frequency of gastric ulcers was twice and this can show the importance of endoscopy. table 2. the distribution of endoscopy symptoms with age and bmi disease age bmi positive negative positive negative mean sd mean sd p-value mean sd mean sd p-value hiatus hernia 40.89 10.68 40.87 10.55 >0.05 45.36 5.93 45.67 6.51 >0.05 esophageal ulcer 44.06 10.95 40.68 10.53 0.002 45.73 6.89 45.55 6.29 >0.05 peptic ulcer 41.71 10.24 40.87 10.59 >0.05 45.26 8.43 45.57 6.31 >0.05 h. pylori 42.73 10.06 45.77 6.21 0.0001 45.77 45.41 6.21 6.42 >0.05 polyp 39.15 9.69 46.63 7.74 >0.05 46.63 7.74 45.56 6.32 >0.05 esophagitis 41.79 10.72 40.54 10.52 0.032 45.37 5.88 45.65 6.51 >0.05 table 1. percentage of endoscopic finding in relation with sex* gender disease hiatal hernia esophagitis esophageal ulcer peptic ulcer h. pylori polyp pos. neg. pos. neg. pos. neg. pos. neg. pos. neg. pos. neg. male 32.80 67.20 33.80 66.20 9.10 90.90 1.70 98.30 42.50 57.50 1 99 female 42.20 69.30 25.90 74.10 5.30 94.70 7 99.30 40.90 59.10 10 99.30 p-value >0.05 0.007 0.0014 >0.05 >0.05 >0.05 pos.: positive, neg.: negative. *values are percentages. vol. 4, no. 2, spring 2018: 59–62 abdolreza pazouki et al. 61j contemp med sci | research preoperative upper endoscopy and bariatric surgeries table 3. comparison of our results with similar studies* present study spain study, 2006 chili study, 2007 brazil study, 2009 uae study, 2010 belgium study, 2013 h. pylori infection 41 69 53 53 85 17 hiatal hernia 31 13 24 esophagitis 27.3 30 esophageal ulcer 6 peptic ulcer 0.8 1 2 3 7 gastric polyp 0.8 gastritis 67 36 *all values are in percentages. in one study done by al-akwaa et al.6 in uae in 2010, 62 patients were enrolled in that study that 85.5% were positive for h. pylori infection and 67.7% had gastritis and 13% has hiatus hernia.6 in current study, 31% of patients had hiatus hernia and 41% were positive for h. pylori infection that were almost half of the rate of infection in comparison with current study. hiatal hernia in our study was higher and due to its role in the type of surgery, endoscopy can be useful. de palma et al. in italia published a review article in 2012 which proclaim that it cannot be definitely recommend to do upper gi endoscopy before bariatric surgery and the need to conduct further studies in this field indicates the importance of our survey.7 in retrospective study that conducted by praveenraj et al.8 from india in 2015, 283 patients were enrolled that 54 patients (54.4%) had an abnormal findings in endoscopy and hiatus hernia in 2 patients was observed. in current research, the incidence of positive cases were less but the hiatus hernia was more prevalent. the high prevalence of hiatal hernia in our study increases the need for endoscopy. csendes et al. from chili in 2007 evaluated 426 candidate for bariatric surgery in pre-operative endoscopy, 55% had a positive criteria 2.6% had peptic ulcer. h. pylori infection was 53% in the patients.9 peptic ulcer was high incidence in current study, whereas h. pylori positive cases has higher than chili’s survey. in retro prospective study conducted by d’hondt et al.10 from belgium in 2013, 652 patients were enrolled in that survey, 68.1% had an abnormal finding in endoscopy results, 24.3% had hiatus hernia, esophagitis was observed in 30.8%, and gastritis in 36.2% and ulcer in 7.5% of patients; and 17.6% were positive for h. pylori infection. all cases in this study was higher than our research but the incidence of h. pylori infection due to low level of health condition in iran was higher than this study. our study has some limitation that just conducted in one obesity center and the endoscopy performed by multi-specialist; but this cannot affect our result because the pathologic findings are objective and can be detected by each specialist. but overall for the first time, the large sample, about 1663 cases, were examined during 1 year. conclusion in conclusion, this is inferred that about 1/3 of obese patients that were candidate for bariatric surgery had an accidental findings in esophagus and stomach endoscopy (35%), so administration of upper gi tract endoscopy before bariatric surgery is may be useful for selecting the type of appropriate procedure for the obese patients. at last, it is proposed that to achieve more accurate results that could be cited or generalized, conduct multicenter studies with larger sample sizes. acknowledgments this study has been funded and supported by iran university of medical sciences (iums); grant no 91-01-140-17375. we used data from national obesity surgery database, iran, tehran. we would like to express our thanks from national obesity surgery database team who prepared us useful data. conflict of interest none.  references 1. donnelly je, blair sn, jakicic jm, manore mm, rankin jw, smith bk. american college of sports medicine position stand. appropriate physical activity intervention strategies for weight loss and prevention of weight regain for adults. med sci sports exerc. 2009;41:459–471. 2. aldana sg, greenlaw rl, diehl ha, salberg a, merrill rm, ohmine s, et al. effects of an intensive diet and physical activity modification program on the health risks of adults. j am diet assoc. 2005;105:371–381. 3. andersen re, wadden ta, bartlett sj, zemel b, verde tj, franckowiak sc. effects of lifestyle activity vs structured aerobic exercise in obese women: a randomized trial. jama. 1999;281:335–340. 4. diez-rodriguez r, ballesteros-pomar md, vivas-alegre s, barrientoscastaneda a, gonzalez-de francisco t, olcoz-goni jl. [upper endoscopy findings in obese morbid patients candidates for bariatric surgery]. gastroenterol hepatol. 2015;38:426–430. 5. dietz j, ulbrich-kulcynski jm, souto ke, meinhardt ng. prevalence of upper digestive endoscopy and gastric histopathology findings in morbidly obese patients. arq gastroenterol. 2012;49:52–55. 6. al-akwaa am. prevalence of helicobacter pylori infection in a group of morbidly obese saudi patients undergoing bariatric surgery: a preliminary report. saudi j gastroenterol. 2010;16:264–267. 7. de palma gd, forestieri p. role of endoscopy in the bariatric surgery of patients. world j gastroenterol. 2014;20:7777–7784. 8. praveenraj p, gomes rm, kumar s, senthilnathan p, parathasarathi r, rajapandian s, et al. diagnostic yield and clinical implications of preoperative vol. 4, no. 2, spring 2018: 59–62 62 j contemp med sci | preoperative upper endoscopy and bariatric surgeries research abdolreza pazouki et al. upper gastrointestinal endoscopy in morbidly obese patients undergoing bariatric surgery. j laparoendosc adv surg tech a. 2015;25:465–469. 9. csendes a, burgos am, smok g, beltran m. endoscopic and histologic findings of the foregut in 426 patients with morbid obesity. obes surg. 2007;17:28–34. 10. d’hondt m, steverlynck m, pottel h, elewaut a, george c, vansteenkiste f, et al. value of preoperative esophagogastroduodenoscopy in morbidly obese patients undergoing laparoscopic roux-en-y gastric bypass. acta chir belg. 2013;113:249–253. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201801 vol. 4, no. 2, spring 2018: 59–62 1j contemp med sci | vol. 4, no. 1, winter 2018: 1–6 review on teucrium polium biological activities and medical characteristics against different pathologic situations mozafar khazaei,a seyed noureddin nematollahi-mahani,b tahmineh mokhtari,c fatemeh sheikhbahaeib afertility and infertility research center, kermanshah university of medical sciences, kermanshah, iran. b department of anatomy, afzalipour school of medicine, kerman university of medical sciences, kerman, iran. cresearch center of nervous system stem cells, department of anatomy, school of medicine, semnan university of medical sciences, semnan, iran. correspondence to fatemeh sheikhbahaei (email: am23384@gmail.com). (submitted: 08 september 2017 – revised version received: 27 september 2017 – accepted: 13 october 2017 – published online: 26 march 2018) objective herbal medicines and the use of these drugs in treating and preventing of certain diseases in the world and iran have increased dramatically. teucrium polium, one of the plants that has been used in traditional medicine nearly 2,000 years ago belongs to labiatae family and is used as a diuretic, tonic, antipyretic, anti-fungal, anti-spasmodic, anti-rheumatic, carminative and an antibacterial agent. methods information of this article has collected from the database of pubmed, sciencedirect and scopus. results the effects of t. polium on the liver, kidney, stomach, brain has investigated and antidiabetic, antioxidant, antimicrobial, and anticancer effects of this agent, has introduced. several studies revealed that t. polium has a hypoglycemic effect and can help to control blood sugar. in addition, due to the undeniable effects of this plant against cancer cells, it can be considered as a natural resource, for the treatment of cancer. conclusion this review investigates multiple properties of t. polium. keywords cytotoxicity, medicinal properties, teucrium polium introduction treatment of various diseases using medicinal plants traced back to ancient times. teucrium polium, the herb that has used in traditional medicine, almost 2,000 years ago, is found mainly in the mediterranean region and different areas of iran especially in semi-arid parts of the mountains and plains.1,2 this herbaceous plant, belonging to the lamiaceae family and flowering time is between june and august.3 (fig. 1). this plant includes 300 species around the world, and has identified in 12 species in iran. among them, t. polium, has been used in traditional medicine.4 this plant has diuretic, tonic, antipyretic, anti-fungal, anti-spasmodic, anti-rheumatic, carminative and antibacterial properties.5,6 moreover, it has hypoglycemic effects and has been used in diabetic patients as a hypoglycemic agent. daily consumption of this plant helps to maintain normal levels of blood sugar and can be appropriate for conventional medications to control blood sugar.7 in addition, t. polium has anti inflammatory activity,8 as well as reduce high body weight and high blood pressure9 and has antioxidant and lipid lowering properties. numerous studies investigated the effects of t. polium against different pathological situations in different organs. so, in this review, we tried to demonstrate the different biological activates and medical properties of t. polium as a traditional plant. particularly, we focused on the mechanisms underlying t. polium’s antioxidant, antibacterial and anticancer properties. source and chemical compounds of t. polium pheochromocytoma teucrium polium is perennial, herbaceous, with almost woody plants, to a height of 30 cm and has a white appearance and cotton. flowers can be seen in white, yellow and white to yellow. this variability is seen not only in color, but also in flower stems that are branched or lying.10 t. polium distributes in rocky and sandy areas of mediterranean, different parts of europe, north of africa and southwest of asia, including iran (in various regions of north, west, south and central arid mountains).2 some compounds of t. polium have been introduced in different investigations including tannin, terpenoid, saponin, flavonoid, sterol, β-caryophyllene, diterpenoids, caryophyllene oxide, asparagine, ditryne and resinous substances.11,12 vokou and bessiere13 reported that t. polium contain tannin, terpenoid, saponin, sterol, and flavonoid. it is used in the treatment of gastrointestinal diseases. several compounds extracted from different parts of this plant have been structurally characterized: including iridoid, flavonoid, eudesman and clerodane. the first returns to the years 1974–1979. in egypt, α-pinene, menthofuran, monoterpenes (myrcene, ocimene, pulegone have presented as the main of t. polium components). β-eudesmol has detected in t. polium in tunisia.14 in the region of saudi arabia (subspecies not listed), monoterpenes (β-pinene, limonene, α-phellandrene, linalool, and terpinen-4) and terpenes (γ-cadinenes, cedrenol, guaiol) have detected.6 in greece (subspecies not reported), t. polium contains terpenes: αand τ-cadinols and β-caryophyllene.13 it has been reported that this agent in spain is rich of monoterpenes that often consisting of αand β-pinenes, limonene, terpinen-4 and pinocarveol-18 (table 1).15 antioxidant capacity of t. polium therapeutic benefits of medicinal plants are often attributed to their antioxidant properties.16,17 different researches in recent years demonstrated that t. polium has high antioxidant properties. many species of medicinal plants, especially those belonging to the mint family, such as sage, tucrium, pennyroyal and thyme have strong antioxidant activity.18 number of phenolic compounds with strong antioxidant activity in extracts of these plants have been identified.19 hplc analysis issn 2413-0516 review 2 j contemp med sci | vol. 4, no. 1, winter 2018: 1–6 review on teucrium polium biological activities and medical characteristics khazaei m et al. showed that the highest levels of flavonoid in t. polium species are in chamaedrys and t. polium. in vitro research showed that t. polium has the antioxidant activity and free radical scavenging activity.19,20 in several research, the effect of t. polium on oxidation of various tissues have studied and the inhibitory effects of the plant extract against peroxidation, have proved. in this regard, the methanolic extract of t. polium protects red blood cells against lipid peroxidation induced by hydrogen peroxide.21 kadifkova panovska et al.20 reported that different factions of t. polium (diethyl ether, ethyl acetate and n-butanol) have inhibitory effect against oxidation. aqueous extracts prepared from leaves of t. polium, suppresses iron-induced lipid peroxide oxidation in rat liver as well as trolox, i.e., analog of vitamin e. anti-diabetes effects of t. polium some plants are used as hypoglycemic agents and their extracts have studied in the treatment of diabetic patients.22,23 several animal studies were carried out to evaluate the effect of t. polium on blood glucose concentrations. in a study by jemal et al. (2012), it was shown that intravenous injection of boiled t. polium caused a significant reduction in blood glucose in diabetic group, compared with a control group that received normal saline.7 hydro-alcoholic extract of t. polium can reduce blood cholesterol and glucose and also improves insulin secretion in diabetic rats.24 esmaeili et al. (2009) reported that flavonoid compounds of t. polium, increase insulin secretion from pancreatic beta cells (56%). this finding was related to flavonoid antioxidant effects of t. polium.25 zal et al.26 demonstrated that t. polium decreased blood glucose in diabetic mice and it has been normal within 8 days. other studies have shown that aqueous extract of t. polium reduces blood sugar in diabetes rat with single dose; it increases insulin secretion and thereby decreases blood glucose in diabetic rats.27 but, the extracts of this plant did not have a significant effect on blood sugar of rabbits.28 according to studies that evaluated the effect of t. polium on blood sugar levels, it seems that the plant extract may improve insulin function in an animal model. but according to the conflicting reports and deficit of human studies on the effects of this herb on blood sugar level, currently commenting on proven effects of t. polium as hypoglycemic agent is difficult and clinical research seems is necessary. anti-cancer effects of t. polium cancer has remained a major cause of disability and mortality in worldwide.29 despite recent advances in early detection and treatment of cancer, continues to be unstoppable, threatening health and quality of human life. traditionally, herbal compounds have long been considered in the treatment of cancer and drugs such as doxorubicin and paclitaxel that are derived from plants are also used in the treatment of cancer. despite the abundance of different plant species in the world, only 1–10% of plant species composition around the world, have been studied.30 in recent years, anti-cancer properties of t. polium have evaluated in several experiments and cytotoxic effects of total extracts and derivatives of this plant against cancer cells has been reported. anti-cancer properties of this plant know more about to terpenoid and flavonoid compounds. for example, in a study, anti-cancer effect of methanolic extract of t. polium and vincristine, vinblastine and doxorubicin against cell lines is shown: skmel -3 (melanoma), saos-2 (osteosarcoma), sw480 (colon cancer), mcf-7 (breast cancer), kb (oral epidermal cell line), el (bladder carcinoma) and a431 (epidermoid carcinoma). administration of t. polium and anti-cancer drugs such as vincristine, vinblastine or doxorubicin can enhance the ability of chemotherapy drugs to cancer and also minimizes side effects.31 flavonoids have been considered as the most potent inducer of apoptosis.32 anti-cancer and cytotoxic effects of t. polium in different types of human cell lines is tested such as cervical cancer (hela), chronic myeloid (k562), colon cancer (caco-2, hct-116, lovo, sw480), glioblastoma multiforme (reyf-1), hepatoblastoma (hepg2), carcinoma of the larynx (hep-2), lung cancer (cor-l23), prostate cancer (du145, pc3).33–36 additionally, the cytotoxic effects of ethanolic extract of t. polium in four cell lines were examined to evaluate the formation of colonies: a549 (adenocarcinoma of the lung), bt20 (cancer of the ducts of the breast), mcf7 (breast adenocarcinoma) and pc12 (pheochromocytomafv mice). as well, they compared cytotoxicity of t. polium with taxol, an anti-cancer agent. the results demonstrated that ethanolic extract of t. polium, inhibits growth of all cell lines effectively. fig 1. teucrium polium. table 1. isolated chemical compounds of t. polium in different studies chemical compound country year author (reference) β-pinene, limonene, α-phellandrene, linalool, terpinen-4-ol, γand δ-cadinenes, cedrol, cedrenol, guaiol saudi arabia 1979 hassan et al.(6) tannin, terpenoid, saponin, sterol, and flavonoid greece 1985 vokou et al.(13) myrcene, α-pinene, menthofuran, ocimene, pulegone egypt 1974 wassel et al.(14) αand β-pinenes, sabinene, terpinen-4-ol and pinocarveol spain 1993 pérez-alonso(14) review khazaei m et al. 3j contemp med sci | vol. 4, no. 1, winter 2018: 1–6 review on teucrium polium biological activities and medical characteristics table 2. t. polium biological activities and medical characteristics according to the different studies authors groups t. polium twaij (1987) rat recovery of stomach ulcer munir (1988) rat a significant reduction in blood glucose concentration lalibert and villeneuve (1996) human lethargy, jaundice, elevated liver enzymes (using t. polium 6 months) zal et al. (2001) rat glucose reduction within 8 days zal et al. (2001) rat degenerative changes in liver lobules rasekh (2001) hyperlipidemic rat lower cholesterol and triglycerides afifi et al. (2005) rabbit no significant effect on blood sugar baluchnejadmojarad t et al. (2005) rat analgesic effect shahraki et al. (2006) rat lower blood sugar levels of diabetic mice shahraki et al. (2006) rat analgesic effect of visceral and somatic panovska et al. (2007) rat repair and regeneration of liver mehrabani et al. (2009) rat reducing gastric ulcer index by 90% mousavi (2011) rat lower obesity parameters ayoubi et al. (2013) rat lower glucose in diabetic mice belmekki et al. (2013) in vitro the inhibitory effects on bacteria tabatabaei et al. (2014) in vitro antimicrobial effect against gram positive bacteria mousavi et al. (2015) rat improving the devastating effects of diabetes on memory nematollahi (2007) in vitro inhibit the growth of cell lines: ic 50 : bt20:106 µg/ml ic 50 : mcf-7:140 µg/ml ic 50 : pc12:120 µg/ml ic 50 : a549:90 µg/ml eskandary (2007) in vitro reduce the formation of clonal cell lines reyf-1 methanolic extract: ic 50 : 95 µg/ml mahdinia (2012) in vitro cytotoxicity on (u87) ic 50 : 64.47 µg/ml rajabalian et al. (2008) in vitro cytotoxicity on cell lines: ic 50 : saos-2:109 µg/ml ic 50 : skmel:3–83 µg/ml ic 50 : mcf-7:174 µg/ml ic 50 : sw480:139 µg/ml ic 50 : kb:174 µg/ml ic 50 : ej:108 µg/ml ic 50 : a431:93 µg/ml kundaković (2011) in vitro cytotoxicity on cell lines: ic 50 : mda-mb-361:130 µg/ml ic 50 : mda-mb-453:367 µg/ml menichin (2009) in vitro cytotoxicity on cell lines: ic 50 : raw264/7:29/4 µg/ml ic 50 : caco-2:52/7 µg/ml ic 50 : 2c32:91 µg/ml ic 50 : 23cor-l:104 µg/ml review 4 j contemp med sci | vol. 4, no. 1, winter 2018: 1–6 review on teucrium polium biological activities and medical characteristics khazaei m et al. among the cell lines, a549 was more sensitive to pc12.37 cytotoxic and anti-tumor effects of the aqueous and methanolic extract of t. polium on human glioblastoma cell line (reyf-1) has been studied and proved that methanolic extract of t. polium decreases colony formation in a dose-dependent. the methanolic extract was more effective than aqueous extract, due to the better solubility of the active chemical components in methanol.34 it has been reported (2012) that terpenoids and antioxidants compounds derived from petroleum ether fraction of t. polium accelerate cytotoxic effects against u87.38 antimicrobial effects of t. polium teucrium polium aqueous extract has anti-bacterial properties, but anti-fungal effect has not been confirmed.39 the results demonstrated that t. polium extract significantly has antimicrobial activity in vitro, especially on the strains of gram-positive bacteria.40 oil extracted from the t. polium with a minimum concentration of 3–5 μl/ml, has the inhibitory effect on bacillus cereus bacteria, enterococcus faecalis and escherichia coli.41 raei et al.42 confirmed the antibacterial activity of t. polium essential oil against urinary-isolated klebsiella pneumoniae. motamedi et al.43 evaluated the antibacterial effects of t. polium on staphylococcus aureus strains and they suggested that t. polium was an effective medicinal plant for treatment of infections caused by s. aureus. sevindik et al.44 determined the antimicrobial activity and chemical composition of t. polium essential oils provided from north anatolian. they showed that this plant had an inhibition effect on resistant micro-organisms including methicillin-resistant pseudomonas aeruginosa, s. aureus atcc 6538, s. aureus (mrsa), e. coli q157:h7 and b. cereus ccm 99.44 so, this natural antibacterial source can be carried out to produce new drugs against different bacteria. antinociceptive and analgesic effects of t. polium oral administration of this plan is used as a visceral analgesic agent. in one study, use of t. polium extract, increased the time of tail reaction to painful stimulus in rats and it can be concluded that this plant extract may has a visceral analgesic in addition to somatic analgesic. although the exact mechanism, needs further investigation. t. polium extract at a dose of 200 mg/kg for a period of 2 weeks, has been created a significant analgesic effect.69 verdi et al.45 proved that aqueous extract of t. polium leaves induced antinociceptive effects through the central mechanisms in rat model of pain. zendehdel et al.46 showed that the analgesic effect of t. polium mediated by opioidergic and histaminergic h1 and h2 receptors in mice model of visceral pain. effects of t. polium on nervous system related disorders teucrium polium extract had positive effects on mice that were suffering from amnesia with scopolamine. it has been reported that t. polium extract can significantly reduce lipid peroxidation in the hippocampus and cerebral cortex. different doses of this agent were able to prevent learning difficulties.47 in another study, results revealed t. polium (200 and 400 mg/kg), prevents the damaging effects of diabetes on memory, but a dose of 100 mg/kg t. polium does not has any positive effect on memory disorders.48 as well, mousavi et al. (2015) investigated the beneficial effects of t. polium on diabetes-induced brain tissue oxidative damage and memory deficits in rats. their findings of proved that t. polium protected the rat against the memory impairments induced diabetes by reducing the brain tissues oxidative damage in rats.47 simonyan et al.49 demonstrated that hydroponic t. polium had protective effects on hippocampal neurodegeneration by modulating neurotransmitters activates and network plasticity in ovariectomized rats. effects of t. polium on gastric disorders teucrium polium extract inhibits the movement of the stomach and in traditional medicine; it is used as an antispasmodic. in a study, rats were treated with indomethacin to induce gastric ulcer. t. polium reduced the scarring at a rate of 50% after a week, 80% after 2 weeks and 90% after 4 weeks. the mucosal healing, and the reduction of proliferation, mucosal hyperplasia, migration of inflammatory cells were observed.50 this agent at a dose of 150 mg/kg promoted wound healing at the rate of 50% in mice that their stomach ulcers were induced by starvation. whereas, oral consumption of the extract improves gastric ulcers at the rate of 85%.51 vascular effects of t. polium teucrium polium decreases aortic smooth muscle contraction induced by kcl and phenylephrine. endothelium can inhibit contraction of smooth muscle of vessels through the synthesis and secretion of substances such as nitric oxide (no) and can induce the contraction through the synthesis and secretion of endothelin. the difference in the effect of the contraction induced by kcl and phenylephrine in the presence or without endothelium indicates that part of the effect, mediates by endothelium. it seems that factors secreted by the endothelium that effect vessels, are important due to relaxant properties of this plant that effect through the endothelium, in addition to the smooth muscle. this effect mainly applies via the inhibition of calcium influx.52 effects of t. polium on renal disorders some herbs may lead to kidney damage and should be used with caution. in a study (2013), the effect of ethanolic extract of t. polium on rat kidney was evaluated. treatment of mice with doses of 50, 100, 150, 200 mg/kg for 28 days, did not increase the kidney damage in-group receiving t. polium compared to the control group. however, 28 days after stopping the drug, kidney damage, emerged including degeneration, degradation and vacuolization compared to the control group.53 effects of t. polium on hepatic disorders in recent years, extensive studies have been done to evaluate the effect of t. polium extract on the liver. in most of these studies, t. polium had been caused degenerative changes, necrosis, hepatic toxicity and general side effects. zal et al.26 observed degenerative changes in hepatic lobuls in diabetic rats and it was concluded that although t. polium has hypoglycemic effect, but should be cautious about taking it. in another study, mehdinia et al.54 review khazaei m et al. 5j contemp med sci | vol. 4, no. 1, winter 2018: 1–6 review on teucrium polium biological activities and medical characteristics evaluated t. polium fractions of petroleum ether on the mice liver toxicity and their results presented that administration of the extract significantly decreased body weight and changes some liver enzymes. in humans, several cases about liver damage have been reported and in one case after eating t. polium, hepatitis created that need liver transplantation.55 in another report, following 5–6 months after taking t. polium, patients getting lethargy, jaundice with elevated liver enzymes and jaundice disappeared after 8 weeks of discontinuation.56 in five patients who were given t. polium, at least 1 month, liver biopsy showed acute hepatitis in these patients. liver function tests, 10–30 days after use improved and liver function was normal after 2–6 months.57 in another report, 62 years old man, has used tea containing t. polium once daily, due to hypercholesterolemia and diabetes. then after taking drug for 4 months, getting jaundice and liver biopsy revealed acute hepatitis and necrosis.58 injection of t. polium in doses of 50–150 mg/kg for 10 days, significantly reduced serum cholesterol and triglyceride levels in rats with hyperlipidemia.12 a number of studies have also reported that t. polium consumption has cholestatic effects and causes hepatitis symptoms and reduction of the nerve cells conduct.59,60 hepatocyte necrosis, following t. polium, has reported in several studies, as well as elevated liver enzymes such as alp, alt, ast.52,61 panovska et al. assessed protective activity of ethyl acetate extract of t. polium against carbon tetrachloride induced liver damage. in this study, injection of t. polium extract induced liver regeneration for 7 days.62 although, t. polium as the herbal medicine, can be useful agent, but according to the studies on the liver, it seems to have toxic effects on this tissue and its use in humans should be done with caution. conclusion due to the effect of different species of t. polium on cancer cell lines, this plant can be considered as a natural source of powerful anti-cancer drugs in the future. in addition, due to the important role of angiogenesis in tumor growth, it seems that it is necessary to study anti-angiogenesis effect of t. polium, as a confirmation of cancer prevention. although, t. polium can reduce blood sugar by increasing insulin secretion or increase hepatic metabolism or glucose, but since the aqueous and alcoholic extracts of this plant have hepatotoxic and necrotic properties in liver cells, has limited the therapeutic application of this plant and recommendation to consume in humans requires more studies and surveys. n references 1. galati e, mondello m, d’aquino a, miceli n, sanogo r, tzakou o, et al. effects of teucrium divaricatum heldr. ssp. divaricatum decoction on experimental ulcer in rats. j ethnopharmacol. 2000;72:337–342. 2. naghibi f, mosaddegh m, mohammadi motamed m, ghorbani a. labiatae family in folk medicine in iran: from ethnobotany to pharmacology. iran j pharm res. 2010;4:63–79. 3. capasso f, de fusco r, fasulo m, lembo m, mascolo n, menghini a. antipyretic and antibacterial actions of teucrium polium (l.). pharm res commun. 1984;16:21–29. 4. akin m, oguz d, saracoglu h. antibacterial activity of essential oil from thymbra spicata var. spicata l. and teucrium polium (stapf brig.). interventions. 2010;8:53–58. 5. said o, khalil k, fulder s, azaizeh h. ethnopharmacological survey of medicinal herbs in israel, the golan heights and the west bank region. j ethnopharmacol. 2002;83:251–265. 6. hassan m, muhtadi f, al‐badr a. glc‐mass spectrometry of teucrium polium oil. j pharm sci. 1979;68:800–801. 7. gharaibeh mn, elayan hh, salhab as. hypoglycemic effects of teucrium polium. j ethnopharmacol. 1988;24:93–99. 8. tariq m, ageel a, al-yahya m, mossa j, al-said m. anti-inflammatory activity of teucrium polium. int j tissue react. 1988;11:185–188. 9. gharaibeh m, elayan h, salhab a. anorexic effect of teucrium polium in rats. int j crude drug res. 1989;27:201–207. 10. ricci d, fraternale d, giamperi l, bucchini a, epifano f, burini g, et al. chemical composition, antimicrobial and antioxidant activity of the essential oil of teucrium marum (lamiaceae). j ethnopharmacol. 2005;98:195–200. 11. niazmand s, ahmadpour e, mousavian m, saberi z. the inotropic and chronotropic effects of aqueous ethanolic extract from teucrium polium l. on guinea pig isolated heart. j babol univ med sci. 2008;10:7–13. 12. niazmand s, hajizade m, keshavarzi-poortafti, z. the effects of aqueous extract from teucrium polium l. on rat gastric motility in basal and vagalstimulated conditions. iran j basic med sci. 2007;10:60–65. 13. vokou d, bessiere j-m. volatile constituents of teucrium polium. j nat prod. 1985;48:498–499. 14. wassel g, ahmed s. chemical composition of the wild egyptian plant teucrium polium l. pharmazie. 1974;29:540–541. 15. ljubuncic p, azaizeh h, portnaya i, cogan u, said o, saleh ka, et al. antioxidant activity and cytotoxicity of eight plants used in traditional arab medicine in israel. j thnopharmacol. 2005;99:43–47. 16. hertog mg, feskens ej, kromhout d, hollman p, katan m. dietary antioxidant flavonoids and risk of coronary heart disease: the zutphen elderly study. lancet. 1993;342:1007–1011. 17. zhang z, chang q, zhu m, huang y, ho wk, chen z-y. characterization of antioxidants present in hawthorn fruits. j nutr biochem. 2001;12:144–152. 18. hirasa k, takemasa m. spice science and technology: crc press; new york, usa, 1998. 19. shahidi f. natural antioxidants: chemistry, health effects, and applications: the american oil chemists society; illinois, usa, 1997. 20. kadifkova panovska t, kulevanova s, stefova m. in vitro antioxidant activity of some teucrium species (lamiaceae). acta pharm. 2005;55:207–214. 21. suboh s, bilto y, aburjai t. protective effects of selected medicinal plants against protein degradation, lipid peroxidation and deformability loss of oxidatively stressed human erythrocytes. phytother res. 2004;18:280–284. 22. torres ic, suarez jc. a preliminary study of hypoglycemic activity of lythrum salicaria. j nat prod. 1980;43:559–563. 23. jimenez j, risco s, ruiz t, zarzuelo a. hypoglycemic activity of salvia lavandulifolia. planta med. 1986;52:260–262. 24. ayoubi a, omidi a, valizade r, mousaei a. effect of hydroalcoholic extract of aloe vera and teucrium on serum glucose and lipid profile in streptozotocin diabetic male rats. j birj univ med sci. 2013;20:144–152. 25. esmaeili ma, zohari f, sadeghi h. antioxidant and protective effects of major flavonoids from teucrium polium on β-cell destruction in a model of streptozotocin-induced diabetes. planta med. 2009;75:1418–1420. 26. zal f, vasei m, rasti m, vessal m. hepatotoxicity associated with hypoglycemic effects of teucrium polium in diabetic rats. arch iran med. 2001;4:188–192. 27. shahraki m, miri me, palan m, mirshekari h, shahraki e. the survey of teucrium polium toxicity effect on liver and serum lipoproteins in normoglycemic male rats. zahedan j res med sci. 2006;8:227–232. 28. afifi f, al-khalidi b, khalil e. studies on the in vivo hypoglycemic activities of two medicinal plants used in the treatment of diabetes in jordanian traditional medicine following intranasal administration. j ethnopharmacol. 2005;100:314–318. 29. jemal a. global burden of cancer: opportunities for prevention. lancet. 2012;380:1797–1799. 30. verpoorte r. pharmacognosy in the new millennium: leadfinding and biotechnology. j pharm pharmacol. 2000;52:253–262. 31. rajabalian s. methanolic extract of teucrium polium l. potentiates the cytotoxic and apoptotic effects of anticancer drugs of vincristine, vinblastine and doxorubicin against a panel of cancerous cell lines. exp oncol. 2008;30:133–138. 32. haïdara k, alachkar a, al moustafa a-e. teucrium polium plant extract provokes significant cell death in human lung cancer cells. health. 2011;3:366. review 6 j contemp med sci | vol. 4, no. 1, winter 2018: 1–6 review on teucrium polium biological activities and medical characteristics khazaei m et al. 33. abu-dahab r, afifi f. antiproliferative activity of selected medicinal plants of jordan against a breast adenocarcinoma cell line (mcf7). sci pharm. 2007;75:121–146. 34. line mc. evaluation of cytotoxic effect of teuerium polium on a new glioblastoma multiforme cell line (reyf-1) using mtt and soft agar clonogenic assays. int j pharmacol. 2007;3:435–437. 35. kandouz m, alachkar a, zhang l, dekhil h, chehna f, yasmeen a, et al. teucrium polium plant extract inhibits cell invasion and motility of human prostate cancer cells via the restoration of the e-cadherin/catenin complex. j ethnopharmacol. 2010;129:410–415. 36. stankovic ms, curcic mg, zizic jb, topuzovic md, solujic sr, markovic sd. teucrium plant species as natural sources of novel anticancer compounds: antiproliferative, proapoptotic and antioxidant properties. int j mol sci. 2011;12:4190–4205. 37. nematollahi-mahani s, rezazadeh-kermani m, mehrabani m, nakhaee n. cytotoxic effects of teucrium polium on some established cell lines. pharm biol. 2007;45:295–298. 38. mahdinia z, eftekharvaghefi r, nabipour f. in vitro inhibition of the growth of glioblastoma by teucrium polium crude extract and fractions. int j phytomed. 2013;4:582–588. 39. mosadegh m, dehmoubed sharifabadi a, nasiri p, esmaeili s, naghibi f. the study of phytochemical, antifungal and antibacterial effects of teucrium polium and cichourium intybus. sci j kurdistan univ med sci. 2002;7:1–6. 40. tabatabaei yf, alizadeh bb, heidari sm, mortazavi s. the in vitro study of antimicrobial effect of teucrium polium extract on infectious microorganisms. sci j hamadan univ med sci. 2014;21:16–24. 41. belmekki n, bendimerad n, bekhechi c. chemical analysis and antimicrobial activity of teucrium polium l. essential oil from western algeria. j med plant res. 2013;7:897–902. 42. raei f, ashoori n, eftekhar f, yousefzadi m. chemical composition and antibacterial activity of teucrium polium essential oil against urinary isolates of klebsiella pneumoniae. j essent oil res. 2014;26:65–69. 43. motamedi h, alivand s, ebrahimian m, moosavian s. the antibacterial properties of methanolic extract of teucrium polium against mrsa. j kermanshah univ med sci. 2015;18:557–562. 44. sevindik e, abacı zt, yamaner c, ayvaz m. determination of the chemical composition and antimicrobial activity of the essential oils of teucrium polium and achillea millefolium grown under north anatolian ecological conditions. biotechnol biotechnol equip. 2016;30:375–380. 45. verdi j, komeilizadeh h, kamalinejad m, sh s. analgesic effects of aqueous extract of teucriun polium l. in experimental models of pain in male rats. iran j pharm res. 2010:62. 46. zendehdel m, taati m, jadidoleslami m, bashiri a. evaluation of pharmacological mechanisms of antinociceptive effect of teucrium polium on visceral pain in mice. iran j vet res. 2011;12:292–297. 47. mousavi sm, niazmand s, hosseini m, hassanzadeh z, sadeghnia hr, vafaee f, et al. beneficial effects of teucrium polium and metformin on diabetes-induced memory impairments and brain tissue oxidative damage in rats. int j alzheimer’s dis. 2015;2015:493729. 48. hasanein p, shahidi s. preventive effect of teucrium polium on learning and memory deficits in diabetic rats. med sci monit. 2012;18:br41. 49. simonyan k, chavushyan v. protective effects of hydroponic teucrium polium on hippocampal neurodegeneration in ovariectomized rats. bmc complement altern med. 2016;16:415. 50. mehrabani d, rezaee a, azarpira n, fattahi mr, amini m, tanideh n, et al. the healing effects of teucrium polium in the repair of indomethacin-induced gastric ulcer in rats. saudi med j. 2009;30:494–499. 51. twaij ha, albadr aa, abul-khail a. anti-ulcer activity of teucrium polium. int j crude drug res. 1987;25:125–128. 52. feridoni e, niazmand s, harandizadeh f, hosseini s, mahmodabadi m. vasorelaxant effect of hydroalcoholic extract of teucrium polium l. on isolated rat aorta. 2012;1:35–44. 53. rafieian-kopaei m, baradaran a. teucrium polium and kidney. j renal inj prev. 2013;2:3. 54. mehdinia z, eftekhar-vaghefi r, mehrabani m, nabipour f, mehdinia n, nematollahi-mahani sn. the effect of petroleum ether fraction of teucrium polium extract on laboratory mouse liver. j kerman univ med sci. 2013;20:279–291. 55. mattéi a, pierre r, didier s, cyril f, michel r, henri b. liver transplantation for severe acute liver failure after herbal medicine. j hepatol. 1995;22:597. 56. laliberte l, villeneuve j-p. hepatitis after the use of germander, a herbal remedy. cmaj. 1996;154:1689. 57. savvidou s, goulis j, giavazis i, patsiaoura k, hytiroglou p, arvanitakis c. herb-induced hepatitis by teucrium polium l.: report of two cases and review of the literature. eur j gastroenterol hepatol. 2007;19:507–511. 58. mazokopakis e, lazaridou s, tzardi m, mixaki j, diamantis i, ganotakis e. acute cholestatic hepatitis caused by teucrium polium l. phytomedicine. 2004;11:83–84. 59. polymeros d, kamberoglou d, tzias v. acute cholestatic hepatitis caused by teucrium polium (golden germander) with transient appearance of antimitochondrial antibody. j clin gastroenterol. 2002;34:100–101. 60. shahraki mr, arab mr, mirimokaddam e, palan mj. the effect of teucrium polium (calpoureh) on liver function, serum lipids and glucose in diabetic male rats. iran biomed j. 2007;11:65–68. 61. forouzandeh h, azemi me, rashidi i, goudarzi m, kalantari h. study of the protective effect of teucrium polium l. extract on acetaminophen-induced hepatotoxicity in mice. iran j pharm res. 2013;12:123–129. 62. panovska t, kulevanova s, gjorgoski i, bogdanova m, petrushevska g. hepatoprotective effect of the ethyl acetate extract of teucrium polium l. against carbontetrachloride-induced hepatic injury in rats. acta pharm. 2007;57: 241–248. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.03201801 review 22 j contemp med sci | vol. 4, no. 1, winter 2018: 22–25 research anti-diabetic effects of s. aegyptiaca extract on streptozotocinnicotinamide induced type 2 diabetes rats fazel albosof,a seyed ahmad hoseini,b amir siahpoush,c,d ali reza malayeri,c,d and mohammad hosein haghighizadehe adepartment of nutrition, school of paramedicine, ahvaz jundishapur university of medical science, ahvaz, iran. bnutrition and metabolic disease research center, ahvaz jundishapur university of medical science, ahvaz, iran. cdepartment of pharmacogenosy, school of pharmacy, ahvaz jundishapur university of medical science, ahvaz, iran. dmedicinal planet research center, ahvaz jundishapur university of medical science, ahvaz, iran. edepartment of biostatistic, school of health, ahvaz jundishapur university of medical science, ahvaz, iran. correspondence to (submitted: 18 august 2017 – revised version received: 21 october 2017 – accepted: 10 november 2017 – published online: 26 march 2018) issn 2413-0516 objective type 2 diabetes mellitus is one the most prevalent disorders worldwide which is associated with unhealthy lifestyle and puts great burden on affected individuals and societies. in addition to chemical drugs and treatment options, using natural products including different types of plant extracts have been in use for curing type 2 diabetes for many years. in this research article, we have examined the effects of administrating suaeda aegyptiaca extract on streptozotocin-induced diabetic rats. methods streptozotocin-induced diabetic rats were administrated with 100, 200 and 400 mg/kg of s. aegyptiaca extract or metformin for a period of 4 weeks. blood was taken from animals, and levels of factors including blood urea, cholesterol, creatinine, hdl, ldl, sgot, sgpt and triglyceride were evaluated. results using s. aegyptiaca extract was associated with dramatic changes in lipid profile. it also decreased serum levels of urea, cholesterol and altered activity of sgot and sgpt enzymes close to normal in treated rats. in comparison with metformin, s. aegyptiaca extract showed more significant results. conclusion results of this study implies that s. aegyptiaca extract can be a good candidate for subsequent studies to define a new natural treatment option for type 2 diabetes mellitus. introduction type 2 diabetes mellitus is a complicated metabolic disease which affects energy balance of the cell and consequently the body. diabetes results in complications like hyperglycaemia and altered lipid metabolism. among the main and most important complications associated with diabetes are cardiovascular problems.1 these manifestations are the results of islet β-cells inability to secrete sufficient levels of insulin in response to varying degrees of situation including over nutrition, inactivity, obesity, and insulin resistance.2 with 422 million affected individuals diabetes is one of the most prevalent disorders.3 it affects different organs of the body and due to this complicated nature puts a heavy burden, both financially and emotionally on patients, their families and also on the society and the healthcare system. the estimated worldwide prevalence of diabetes among adults has increased from 108 million in 1980 to 422 million in 2014. this statistics are predicted to rise to around 439 million by 2030.4 about 90% of cases of diabetes are diagnosed as type 2 diabetes mellitus.5 due to the dependency of this disorder on lifestyle, increase of prevalence in developing countries is predicted to be much more than in developed ones with 69% versus 20%.4 this increase is related to westernization of developing countries. consuming high energy diet and reduction in daily physical activity are the two determining factors in this westernization, and changes in lifestyle is a great risk factor.6,7 statistics show that in developing countries people within the range of working age are affected most, in comparison with those older than 60 years in developed countries.4 diabetes is the result of interaction between genetics and environment. although the main metabolic defects of type 2 diabetes are present to some degree in most patients, this disorder is highly heterogeneous.8 many different susceptibility genes have been identified that interacts with environmental factors in different stages of life.9 there are various chemical treatment options available for curing diabetes or decreasing its manifestations. most of these drugs cause problems including drug resistance (reduction of efficiency), side effects, and even toxicity.10 therefore, need for new less harmful drugs is high especially due to high prevalence of this disorder. most of the plants contain carotenoids, flavonoids, terpenoids, alkaloids, glycosides which can have anti-diabetic effects.11,12 hence, study of plants is a logical approach for finding new treatment options for diabetes. the anti-hyperglycemic effects that results from treatment with plants are often due to their ability to improve the performance of pancreatic tissue, which is done by increasing insulin secretions or reducing the intestinal absorption of glucose.13 suaeda aegyptiaca is a halophile plant which belongs to the family amaranthaceae and grows in the northern parts of iran and specifically in khozestan province.14 in this article we discuss the effects of administrating different doses of s. aegyptiaca extract on diabetic rats and compare it with the results of using metformin on the same animals. materials and methods plant material and preparation fresh leaves of s. aegyptiaca were collected in khozestan province, iran and confirmed scientifically by the department of botany of ahvaz jundishapur university of medical keywords , streptozotocin-nicotinamide, diabetessuaeda aegyptiaca ali reza malayeri (email: amalayeri@gmail.com). fazel albosof et al. 23j contemp med sci | vol. 4, no. 1, winter 2018: 22–25 research anti-diabetic effects of s. aegyptiaca extract on streptozotocin-nicotinamide sciences, ahvaz, iran. leaves were air dried and then milled using mechanical grinders. three hundred grams of s. aegyptiaca leafs powder was dissolved in 1,200 ml of distilled water and ethanol mixture (70:30) and kept for 72 h at room temperature. the mixture was filtered with whatman paper and then centrifuged for 20 min with speed of 3,500 rpm. the supernatant was removed and dried at room temperature. finally, the obtained semisolid mass was freshly used.15 experimental animals thirty six adult male wistar rats (150–250 g) were purchased from animal house of ahvaz jundishapur university of medical science (ajums) and were kept in cages with standard laboratory conditions (temperature 22 ± 2°c with a 12/12 h light–dark cycle).16 rats were allowed ad libitum access to normal laboratory diet and tap water. all animals’ procedures were in accordance with standards guide for the care and use of laboratory animals, established by the national research council of the national academic. induction of non-insulin dependent diabetes mellitus for experimental induction of type 2 diabetes mellitus in adult male wistar rats, after they were fasted overnight, firstly nicotinamide (120 mg/kg body weight for each rat) (merck, germany) was dissolved in normal saline and then was administrated intraperitonealy to them. after 15 min the rats received an ip injection of stz (60 mg/kg bw) (sigma-aldrich, usa) dissolved in citrate buffer (ph 4.5). development of diabetes was confirmed by the assessment of glucose level in blood before and 72 h after stz injection. animals with glucose level of more than 126 mg/dl or more were included in the study.17 experimental design animals were randomly divided into six groups (n = 6) and treated daily for 4 weeks as follows: group i: intact control rats were administered normal saline daily for 4 weeks; group ii: diabetic control rats; group iii, iv and v: diabetic rats which received s. aegyptiaca leaves hydro-alcoholic extract orally by gastric tube in doses of 100, 200 and 400 mg/kg body weight respectively daily for 4 weeks; group vi: diabetic rats received metformin (100 mg/kg body weight, sigma-aldrich, usa) orally for 4 weeks as standard medication. by the end of the experiment, animals were deprived of food overnight (24 h) and after mild anesthesia by ether, blood sample was directly collected from their hearts and were centrifuged at 3,500 rpm for about 15 min to obtain blood serum. serum samples were refrigerated at −70°c until evaluation of insulin and serum biochemical parameters.18 lipid profiling (including measurements of triglycerides, hdl and ldl) was done using spectrophotometer instrument. lipid profile, sgot and sgpt levels measurement the activities of pathophysiological enzymes such as serum alp, sgot and sgpt, as well as serum. triglyceride (tg) total cholesterol (tc), ldl-cholesterol (ldl-c) and hdl-cholesterol (hdl-c) were estimated with pars azmoon kits using auto-analyzer. vldl-cholesterol (vldl-c) concentration according to norbert formula, equals one fifth of tg content.20 serum leptin concentration was measured by elisa kit (labor diagnostika nord gmbh, germany). interand intra-assay coefficients of variation was 5.8 and 4.3% respectively. low-end sensitivity of this kit was 0.5 ng/ml. statistical analysis data was analyzed using spss software version 16 by the equation mean ± sem. one-way anova test and student’s t-test were used for statistical analysis followed by significant difference (lsd) test. values of p < 0.05 were considered to be statistically significant.19 results effects of s. aegyptiaca extract on blood urea results of this study shows that administration of s. aegyptiaca extract in 400 mg/kg decreases the amount of blood urea to a comparable level to negative control rats. administration of s. aegyptiaca extract in 100 and 200 mg/kg also decreases the amount of blood urea to a comparable level with metformin. effects of s. aegyptiaca extract on cholesterol level our findings showed that the level of cholesterol increases in diabetic rats in comparison with control animals (fig. 1). using s. aegyptiaca extract has reversed this phenomenon and the highest decrease in level of cholesterol as seen in the group of rats that used 400 mg/kg of body weight of the extract (p < 0.000). our results showed that the administration of metformin does not affect levels of cholesterol in the blood samples of rats in comparison with positive control group (p > 0.615). effects of s. aegyptiaca extract on creatinine in this study by induction of diabetes in rats using stz, level of creatinine in the blood samples of affected rats shows a slight decrease in comparison with healthy animals, although this difference was not statistically significant (p > 0.677). fig 1. variations in cholesterol level in different studied groups. control n: positive control. control p: negative control. t1: s. aegyptiaca extract (100 mg/kg). t2: s. aegyptiaca extract (200 mg/kg). t3: s. aegyptiaca extract (400 mg/kg). metformin: metformin (100 mg/kg). 24 j contemp med sci | vol. 4, no. 1, winter 2018: 22–25 anti-diabetic effects of s. aegyptiaca extract on streptozotocin-nicotinamide research fazel albosof et al. same difference was observable in changes of the levels of creatinine between control group, the group that has administered s. aegyptiaca extract and the group that has used metformin (p < 0.933, 0.945, 0.100 and 990 for s. aegyptiaca extract 100, 200, 400 mg/kg and metformin respectively). effects of s. aegyptiaca extract on hdl and ldl levels results of this study showed increase in levels of ldl and decrease in levels of hdl in diabetic rats in comparison with healthy animals. in case of ldl, administration of different doses of s. aegyptiaca extract was associated in attenuation of effects of diabetic condition in affected rats. this decrease in levels of ldl using s. aegyptiaca extract was higher than the effect that administration of metformin causes. in case of hdl, using s. aegyptiaca extract has resulted in increase in level of this substance in a comparable level to healthy animals (p < 0.001). in this case using metformin did not significantly changed the level of hdl in blood samples of studied animals. effects of s. aegyptiaca extract on sgot and sgpt as demonstrated in table 1, administration of stz increased levels of sgot and sgpt enzymes in serum of animals. for sgot, using s. aegyptiaca extract was associated with increase in levels of the enzyme in the serum of treated animals (p < 0.050, 0.006 and 0.001 for 100, 200 and 400 mg/kg of s. aegyptiaca extract respectively). in comparison with administration of metformin, s. aegyptiaca extract (especially with 400 mg/kg dose) was much more effective in lowering levels of sgot in the blood (p < 0.037). for sgpt, our results showed a similar trend for all four drugs, meaning that all of them have similar effect on decreasing levels of the enzyme in the serum samples of studied animals (table 1). effects of s. aegyptiaca extract on triglyceride administration of stz increased the level of triglyceride in diabetic rats in comparison to healthy animals. this increase has been meaningfully lowered by the administration of 100, 200 and 400 mg/kg of s. aegyptiaca extract (p < 0.000 for all three doses). in case of using metformin the decrease in amount of triglyceride was lesser in comparison with s. aegyptiaca extract concentration. based on our results, using metformin does not make any significant difference in levels of triglyceride between affected rats and healthy animals. discussion with estimated prevalence of 6.4% in 2010, diabetes mellitus is one of the disorders that puts a heavy burden not only on affected individuals and their families, but also on the healthcare system and society. therefore, finding new ways for curing this disorder is of immense importance. in this study, we examined the effects of using s. aegyptiaca extract as a substance for reversing effects of diabetes in rats that have been under administration of stz for induction of diabetes. suaeda egyptiaca belongs to halophytes, a group of plants comprising about 1% of the flora of the world.14 our findings indicate drastic changes in levels of triglycerides, blood urea, ldl, hdl and cholesterol. our results also implied on the positive effects of s. aegyptiaca extract on the levels of sgot and sgpt enzymes in the studied diabetic rats. there are numerous studies that examined effects of using plant extracts on diabetic rats. eidi and colleagues observed similar results by administrating garlic (allium sativum l.) on streptozotocin induced diabetic rats. in their study oral administrations of the garlic extract significantly decreased serum glucose, total cholesterol, triglycerides, urea, uric acid, creatinine, sgot and sgpt levels, while increased serum insulin in diabetic rats but not in normal rats (p < 0.05).20 in another study, ahangarpour and colleagues evaluated ant diabetic effects of dorema aucheri hydroalcoholic leave extract. they found that d. aucheri has highly significant blood glucose lowering effect. it also has a dramatic effect on serum lipid profiles, insulin and leptin levels. similar to our study, they found that sgpt and sgot levels will dramatically change using different dosages of d. aucheri in streptozotocin-induced diabetic rats.21 patel and colleagues have used dihar, a polyherbal formulation consisting of extracts from eight different herbs to measure anti-hyperglycemic, anti-hyperlipidemic and antioxidant activities of it. results of this study demonstrated that administration of dihar for streptozotocin-induced diabetic rats significantly lowers the serum creatinine and urea levels in studied animals.22 table 1. effect of s. aegyptiaca extract on enzymatic profile of rats mean std. deviation std. error 95% confidence interval for mean minimum maximum lower bound lower bound sgot control n 40.1429 1.57359 0.59476 38.6875 41.5982 38.00 42.00 (ast) control p 62.4286 1.98806 0.75142 60.5899 64.2672 59.00 65.00 t1 59.2857 1.25357 0.47380 58.1264 60.4451 58.00 61.00 t2 56.1429 5.17779 1.95702 51.3542 60.9315 50.00 63.00 t3 54.4286 5.28700 1.99830 49.5389 59.3182 48.00 62.00 metformin 60.4286 1.51186 0.57143 59.0303 61.8268 59.00 63.00 sgpt control n 56.4286 4.92805 1.86263 51.8709 60.9863 49.00 62.00 (alt) control p 87.2857 7.82548 2.95775 80.0484 94.5231 77.00 98.00 t1 85.4286 4.68534 1.77089 81.0954 89.7618 81.00 95.00 t2 84.8571 2.91139 1.10040 82.1646 87.5497 82.00 89.00 t3 83.5714 4.64963 1.75739 79.2712 87.8716 75.00 88.00 metformin 83.4286 3.95209 1.49375 79.7735 87.0836 78.00 90.00 fazel albosof et al. 25j contemp med sci | vol. 4, no. 1, winter 2018: 22–25 research anti-diabetic effects of s. aegyptiaca extract on streptozotocin-nicotinamide this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. all in all we showed that the administration of different doses of s. aegyptiaca extract on streptozotocin-induced diabetic rats is associated with changes of different factors in serum of the animals. this difference was particularly emphasized for creatinine, blood urea, hdl, ldl and also sgot and sgpt enzymes. moreover, results of the present study showed for the first time that s. aegyptiaca extract has comparable effects with metformin in reducing complications of diabetes in diabetic rats. therefore, subsequent studies can be particularly helpful in providing more details about mode of action of this substance which consequently might result in introduction of an effective and natural treatment option for diabetes. conclusion in this study we examined anti-diabetic effects of s. aegyptiaca extract on streptozotocin-induced diabetic rats. our data showed that s. aegyptiaca extract in different concentrations has a positive effect on diabetic profile of rats. our data also showed that this extract has comparable, and in some cases better, anti-diabetic effects with a routinely used drug metformin. therefore, s. aegyptiaca extract can be a suitable candidate for subsequent studies to define a new therapeutic agent for treatment of diabetes. n references 1. holmes d. diabetes: new marker to predict risk of t2dm. nat rev endocrinol. 2017;13:625. 2. aslibekyan s, garvey wt. obesity: obesity and cardiometabolic disease more than meets the eye. nat rev endocrinol. 2017;13:566–568. 3. global report on diabetes. who reports, 2016. 4. shaw je, sicree ra, zimmet pz. global estimates of the prevalence of diabetes for 2010 and 2030. diabetes res clin pract. 2010;87:4–14. 5. gonzalez el, johansson s, wallander ma, rodríguez la. trends in the prevalence and incidence of diabetes in the uk: 1996–2005. j epidemiol community health. 2009;63:332–336. 6. chan jc, malik v, jia w, kadowaki t, yajnik cs, yoon kh, et al. diabetes in asia: epidemiology, risk factors, and pathophysiology. jama. 2009;301:2129–2140. 7. colagiuri s. diabesity: therapeutic options. diabetes obes metab. 2010;12:463–473. 8. nolan cj, damm p, prentki m. type 2 diabetes across generations: from pathophysiology to prevention and management. lancet. 2011;378:169–181. 9. hochberg z, feil r, constancia m, fraga m, junien c, carel jc. child health, developmental plasticity, and epigenetic programming. endocr rev. 2011;32:159–224. 10. miller br, nguyen h, hu cj, lin c, nguyen qt. new and emerging drugs and targets for type 2 diabetes: reviewing the evidence. am health drug benefits. 2014;7:452–463. 11. kooti w, farokhipour m, asadzadeh z, ashtary-larky d, asadi-samani m. the role of medicinal plants in the treatment of diabetes: a systematic review. electron physician. 2016;8:1832–1842. 12. hafidh rr. a comprehensive anticancer molecular study for genistein the promising anticancer drug. j contemp med sci. 2017;3:264–269. 13. afrisham r, aberomand m, ali ghaffari m, siahpoosh a, jamalan m. inhibitory effect of heracleum persicum and ziziphus jujuba on activity of alpha-amylase. j botany. 2015;2015:8. 14. qi xy, chen wj, zhang lq, xie bj. mogrosides extract from siraitia grosvenori scavenges free radicals in vitro and lowers oxidative stress, serum glucose, and lipid levels in alloxan-induced diabetic mice. nutr res. 2008;28: 278–284. 15. ahangarpour a, mohammadian m, dianat m. antidiabetic effect of hydroalcholic urticadioica leaf extract in male rats with fructose-induced insulin resistance. iran j med sci. 2012;37:181–186. 16. al-hasan akj. effects of low-and high-level pulsed nd: yag laser irradiation on red blood cells and platelets indices of albino rats in vitro. iraq med j. 2017;1:10–19. 17. shirwaikar a, rajendran k, punitha is. antidiabetic activity of alcoholic stem extract of coscinium fenestratum in streptozotocin-nicotinamide induced type 2 diabetic rats. j ethnopharmacol. 2005;97:369–374. 18. zamami y, takatori s, goda m, koyama t, iwatani y, jin x. royal jelly ameliorates insulin resistance in fructose-drinking rats. biol pharm bull. 2008;31:2103–2107. 19. nada sz, neopterin, interleukin-6, and non hdl-c as predictors for cardiac disease among type 2 diabetic women with and without renal complications. iraq med j. 2017;1:79–82. 20. eidi a, eidi m, esmaeili e. antidiabetic effect of garlic (allium sativum l.) in normal and streptozotocin-induced diabetic rats. phytomedicine. 2006;13:624–629. 21. ahangarpour a, zamaneh ht, jabari a, nia hm, heidari h. antidiabetic and hypolipidemic effects of dorema aucheri hydroalcoholic leave extract in streptozotocin-nicotinamide induced type 2 diabetes in male rats. iran j basic med sci. 2014;17:808–814. 22. patel ss, shah rs, goyal rk. antihyperglycemic, antihyperlipidemic and antioxidant effects of dihar, a polyherbal ayurvedic formulation in streptozotocin induced diabetic rats. indian j exp biol. 2009;47: 564–570. dx.doi.org/10.22317/jcms.03201805 313j contemp med sci | vol. 3, no. 12, autumn 2017: 313–318 research investigation of role magnetized water used in supplementary feeding for honeybees to modulate the genotoxic side effects induced by cyclophosphamide in mice bone marrow cells ekhlas. m. farhan,a rukaibaa a. chechan,b lina q. al-kinanic aministry of sciences and technology, baghdad, iraq. bdeptartment of food science, college of agriculture, university of baghdad, iraq. cdeptartment of plant protection, college of agriculture, university of karbala, iraq. correspondence to: ekhlas mohammed farhan (e-mail: sunlife88201@yahoo.com). objective to evaluate the possible protective role of honey formed through innovative way by using magnetized water instead of tab water in supplementary feeding for bees (hw) and compared with two honey types formed by two different feeding solutions; sugar syrup (hs) and nectar of flowers (hf) against cyclophosphamide (cp) genotoxicity in mice bone marrow cells, by using the micronucleus assay (mn). methods mice were divided into 8 groups from 3 animals each. g1 as control. g2 were exposed to cp (40 mg/kg), g3, g4, and g5 received two dose of three types of honey at doses (300, 600 mg/kg) respectively. the other groups g6, g7 and g8 were supplemented with three types honey (300 mg/kg) in three different experimental protocols, as pre 2 h, post 2 h, and concomitant treatment for 7 and 14 days. results examination and analysis of mn showed no mutagenic effect of three types honey per se doses, especially in low dose (300 mg/kg). meanwhile, cp induced a significant (p < 0.01) increase in mn frequency. while dual treatment with groups of honey hw caused a significant reduction in mn induced by cp in bone marrow cells in a time-dependant manner. also, the results confirmed the protective efficacy of hw group and/or hf group as compared with hs group, against cp-toxicity. conclusion our study suggests using magnetized water in supplementary feeding of bee, that could give the honey protective effect against genotoxicity induced by cp, it is also fosters antioxidant activity of honey constituents. therefore, honey hw can be used as an adjuvant with chemotherapeutic agents for minimizing the genotoxic side effects of the anticancer drug cp. keywords genoprotective, supplementary feeding, magnetized water, cyclophosphamide (cp), micronucleus test introduction in the rapidly changing lifestyles of our present times, the use of synthetic chemicals, chemotherapeutic agents, and carcinogenic substances, as well as the widespread environmental pollutants, have negatively impacted human and other living organisms at the cellular and genetic levels. carcinogenic and mutagenic agents to which the human is continuously exposed leads to accumulation of free radicals in the body, compromising the ability of the biological system to remove toxins and repair the damage they cause, hence the oxidative phenomenon, which plays a major role in the development of many diseases.1,2 natural products were known since the most ancient ages for their therapeutic values due to their varied chemical content, which helps preserve and develop self defense mechanisms, represented by the natural cellular resistance, which protects the cells from toxic products and other environmental stresses. many studies have indicated the usability of naturally occurring chemical substances as effective antioxidants that improve human health and protect against cancer and heart diseases.3–4 it is noteworthy here that antioxidants are both anti-mutagenic, i.e., protect against potential nucleic acid damage and anti-carcinogenic, i.e., prevent the development of secondary tumors without interfering with the therapeutic action of chemotherapeutic agents.5,6 since ancient times, natural honey has been widely used as a conventional medicine, scientific research has proven the therapeutic benefits of honey in treating several human diseases. such as anti inflammatory, gastroprotective, antioxidant, antitumor and anticancer effects.7,8 honey contains about 200 substances including fructose, glucose, amino acids, vitamins, minerals, water and enzymes.9 this protective effect of honey may be attributed to the biologically active compounds such as vitamins, flavonoids, and antioxidants that work together to scavenge free radicals. composition of natural honey varies, depending on many factors such as the geographical areas, source of honeybee food, climate, environmental conditions.10 sidr honey is a “monofloral honey”, a type of honey, that is made from bees who feed only on the nectar of the sidr tree, which has been used as a natural medicine for centuries. sidr honey has strong antioxidant properties, and has wide medicinal applications and uses.11 due to the absence of adequate natural food to provide winter food supplies, beekeepers use supplemental feeding of bees, mainly by sugar syrup, oftentimes, general water in supplementary feeding is used for dissolve the sugar.12 this present study throws light for the first time about used magnetized water instead of general water as an supplementary feeding. water magnetization has been used for many years and was found that magnetization of water alters the properties of water.13 several studies have demonstrated a lot of microscopic and macroscopic differences between magnetized water and normal water, which include the surface tension, contact angel, viscosity, electrical conductivity, ph, etc.14 raymond-whish et al.15 reported the effect of commercial magnetic water conditioners on the total dissolved salts and ph on different solutions. more hydroxyl (oh-) ions are created to form alkaline molecules, and reduce acidity, for this reason cancer cells do not survive well in an alkaline environment.16 numerous animal studies have proved that electromagnetic fields have potential effects on cellular mechanism, development issn 2413-0516 (submitted: 02 july 2017 – revised version received: 19 july 2017 – accepted: 22 september 2017 – published online: 26 december 2017) 314 j contemp med sci | vol. 3, no. 12, autumn 2017: 313–318 investigation of role magnetized water used in supplementary feeding for honeybees research ekhlas. m. farhan et al. and growth especially in the reproductive system.17 previously, it has been demonstrated that exposure to extremely low-frequency electromagnetic field (elf-emf) can increase height of fallopian tube epithelial cells. although the exact mechanism of increasing epithelial cell height is not clear yet, it has been hypothesized that it could be due to increased permeability of cellular membrane to small molecules, especially ca2+ current, free radical chemical reactions and increase in superoxide dismutase activity in the liver.18 previous studies have investigated the effects of magnetized water on the height of endometrial epithelial cells, fallopian tube epithelium and the number of corpinstance it could decrease the amounts of malondialdehyde (mda), increase the superoxide dismutase activity in the heart, kidney and liver and also decrease the amounts of nitric oxide which all result in decreasing oxidative stress. therefore, it could be assumed that this potential reduction of oxidative stress will result in improving the reproductive system.15,19 also, it has been demonstrated that intake of magnetized water will result in reduced dna damage in stz-induced diabetic rats.20 the other study showed that administration of magnetized water for at least 6 weeks suppressed the lymphocyte dna damages in animals with diethyl nitrosamine (den)-induced cancer.21 material and methods cyclophosphamide (cp) drug cyclophosphamide (cp) was purchased from sigma chemical company (st. louis, mo, usa), 40 mg/kg. b.w. of cp were dissolved in sterilized distilled water. distilled water to prepare the required dose and concentration, which is equivalent to (1 mg cp/animal). were used and injected intraperitoneally, according to method of premkumar et al.22 feeding colonies (bees) six colonies were selected as a container of bees belonging to the local l. apismellifera l. the colonies were fed every three days and from 30/4 to 15/6 ·   the first group (3 colonies): colonies were feeding on dissolved sugar in magnetized water (water magnetizer/ crylomag mw) the sugar to water ratio in the groups (1:2) hw. ·   the second group (3 colonies): the colonies fed by dissolved sugar in conventional water (tap water). the sugar to water ratio in the groups (1:2) (hs). ·   third group (3 colonies): feeding the bees naturally on the flowers of sidr trees. they are prescribed for the treatment of many diseases hf. animals and treatments the present study was carried out using mice at 12–9 weeks’ age and 25–30 g, in weight which were purchased from national center for drug control and research/ministry of health/ baghdad. they were housed in plastic cages containing hardwood chips, in the animal house laboratory in (biotechnology research center), al-nahrain. the animals were given water and fed with a suitable quantity of water and complete diet. experience design: the animals were divided into 14 groups ·   group 1: negative control (3 mice) : treated with (0.1 ml) pbs. ·   group 2: positive control (3 mice) the animals were treated with 0.2 ml cp 40 mg/kg for 24 hr ·   groups 3, 4 and 5: the animals were treated with two doses (300 and 600 mg/kg) respectively, of each type of honey under study (9 mice). ·   pre-drug treatment with cp: the animals group (3 mice) were orally given (0.5 ml) hf (group 6), hw (group 7) and hs (group 8) respectively, per day for 7 and 14 days, before injected cp (0.2 ml). ·   post-drug treatment with cp: the animals group (3 mice) were orally given (0.2 ml) cp (40 mg/kg) once (1 day), then followed by honeys (300 mg/kg b.w), hf (group 9). hwr (group 10), and hs (group 11) respectively, per day for 7 and 14 days. ·   co-drug treatment with cp: the animals group (3 mice) were orally given (0.2 ml) cp (40 mg/kg) with (0.5 ml) of honeys (300 mg/kg b.w), hf (group 12). hw (group 13) and hs (group 14) respectively, per day for 7 and 14 days. mn assay the mn test was performed as previously described.23,24 the bone marrow mn test is a well-known in vivo assay for the assessment of genotoxicity in animals such as mice and rats. all groups treated with cp had the drug injected intraperitoneally,25 while three type of honey was administered by oral intubation (o.i.) via an orogastric tube.26 each group was treated daily for 7 and 14 consecutive days.27 24 hours after the last treatment. both femurs were dissected and bone marrow was flushed from the femoral cavity with fetal calf serum. the cells were dispersed by gentle pipetting and collected by centrifugation at 1500 rpm for 10 min the pellet was resuspended in a small volume of fetal calf serum and used for smear preparation. after air-drying, the smears were stained by giemsa.28 from each animal, 1000 pces were examined for mn-pces under 1000 magnification using light microscope. in addition, the number of pces among 500 total erythrocytes (pce + nce) per animal was recorded to evaluate bone marrow toxicity.29,30 to calculate the protective (anti-mutagenic) rate of each honey against cp) mutagenic effect, as reflected by induction of micronucleus formation the following equation was applied.4,30 100% (mn) in (cp + honey) groups × 100% (mn) in (cp) groups statistical analysis the statistical analysis system-sas (2012) program was used to effect of difference factors in study parameters. least significant difference-lsd test (anova) was used to significant compare between means in this study.31 results and discussion this study aimed at evaluating the potential genetic, cellular, and preventive impact of treatment with three type of honey per se, combined, precedent, simultaneous, and subsequent treatment with three types of honey for 7 and 14 consecutive days. and cp and treatment with cp per se. results of the micronuclear test showed a highly significant ability of cp (positive control) to produce mn, 10.03 ± 1.04 in ekhlas. m. farhan et al. 315j contemp med sci | vol. 3, no. 12, autumn 2017: 313–318 research investigation of role magnetized water used in supplementary feeding for honeybees bone marrow cells of mice, corresponding controls (negative control), 1.12 ± 0.07 as shown in fig. 1, table 1. this is consistent with results arrived at by many previous researchers, who treated bone marrow cells using members of the group of alkylating anticancer drugs of which the drug under study cp, is also a member.22,32,33 micronuclei originate from acentric chromatid or chromosomal splinters or from whole chromosomes that do not join the nucleus proper at the end of the final phase of cell division (telophase), due to a failure of proper connection to spindle fibers during the anaphase of cell division. these chromosomes or chromosomal splinters are joined together by means of a nuclear membrane and are small in size, compared with the size of the nucleus proper.1 micronuclei form as a result of treatment with antineoplastic drugs that either cause direct damage to the nucleic acid or inhibit the cell cycle or damage the spindle fibers of mitotic division.34 the micronuclear test being the most sensitive and the most powerful statistical tool to evaluate dna breaks, which indicate potential mutations.35 meanwhile, results showed that three types of honey, which used in this study formed by different feed sources (hs, hw and hf, had no significant effect on the induction of micronuclei in bone marrow cells, which showed nearly the same values of control as shown in table 1. significantly (p < 0.01) as compared with negative control, especially in the low dose, although, the high dose (600 mg/kg) of honey (hs) showed significant differences (p < 0.01) of frequencies micronuclei as compared with negative control, but no statistically significant in micronucleus in comparison with positive group. micronucleus test is good evidence of cytogenetic toxicity, and features a quick and simple test short-term test for this evaluation as it is inexpensive.36 it is clear that the emergence of micronucleus in the cells (pecs) in mice treated with cp, obtained from the current study is strong evidence on the impact of toxic cellular genetic property and an indicator of its ability mutagens.37 as noted also that treatment with hf combine, precedent, simultaneous, and subsequent, treatment with hf and cp, caused a highly significant reduction in the numbers of micronuclei in micronucleated polychromatic erythrocytes (mn-pces) by 2.80 ± 0.08, 4.46 ± 0.15 and 2.35 ± 0.08, for 7 day respectively, however, the numbers of micronuclei decreased significantly (p < 0.01) by 1.76 ± 0.02, 2.15 ± 0.05 and 1.68 ± 0.03, for 14 days respectively, as compared with the positive control 9.26 ± 0.62 (table 2). this result is consistent with findings of previous studies that suggested the possibility of prevention of genotoxic effects resulting from chemotherapy, by using hf.37,38 several explanations have been offered for anti-mutagenic activity of honey, the mechanisms of the protective of honey against cp genotoxicity may be due to one or more of the following: antioxidant action39,40 and to stimulate the antioxidative enzyme glutathione peroxidas,41 leading to an increase in antioxidant capacity of the cells which might fortify the efficiency of protective pathways against cytogenetic damage in cp exposure,42 therefore, it has been used as a compared group with other types of honey’s used in this investigation. it is clear that there were high significant differences in frequencies of mn between the groups which treated with both honey formed by supplementary feeding (hs and/or hw) respectively, in three interactions used (before, after, with treatment) (tables 3 and 4). furthermore; it has been shown that hw achievement best efficacy in a reduction frequencies of micronucleus significantly (p < 0.01) by 3.12 ± 0.07, 3.32 ± 0.07 and 1.63 ± 0.03 for 7 day respectively, the numbers of micronuclei decreased significantly (p < 0.01) by 1.42 ± 0.3, 2.02 ± 0.06 and 0.87 ± 0.02, for 14 days respectively, as compared with the corresponding controls 10.08 ± 0.52 (table 4). furthermore, the data obtained when treatment with hs was 6.47 ± 0.37, 7.13 ± 0.28 and 5.75 ± 0.22 for 7 days respectively, the numbers of micronuclei increased significantly (p < 0.01) by 7.39 ± 0.36, 8.25 ± 0.51 and 7.15 ± 0.33 for 14 days respectively, as compared with the corresponding controls 10.12 ± 0.72, table 3. the results of honey hw came similar with the results of comparison group (honey hf) and was more effective than hs, at any treatment or at any time intervals used. and the fact that this is the first study in the world, which throws light of cytogenetic effect of honey formed by supplementary feeding, therefore, no literature available for the purpose of comparing table 1. frequency of mn in bone marrow cells of mice treated with different doses of types honey (hf, hs and hw) micronuclei (mn)%dose (mg/kg)experimental groups 1.12 ± 0.07c0negative control 10.03 ± 1.04a40positive control (cp only) 1.67 ± 0.08c300honey formed by (floral resources) hf 2.15 ± 0.08c600 3.90 ± 0.11bc300honey formed by sugar syrup (hs) 4.76 ± 0.17b600 1.18 ± 0.04c300honey formed by (sugar with magnetized water) (hw) 2.32 ± 0.08c600 0.00026**–p-value means having with the different letters in same column differed significantly. **(p < 0.01). differences a, b, c and d, are significant (p ≤ 0.01 ) to comparison rows. fig. 1 giemsa-stained binucleated in bone marrow cells of mice treated with cyclophosphamide (cp) a: polychromatic erythrocytes “normal”; b: polychromatic erythrocytes with “micronucleus” (x1000). a b 316 j contemp med sci | vol. 3, no. 12, autumn 2017: 313–318 investigation of role magnetized water used in supplementary feeding for honeybees research ekhlas. m. farhan et al. table 2. frequency of mn in bone marrow cells of mice treated with honey formed by-nectar of flowers (sidr), and cyclophosphamide groups treatment dose mg/kg the calculated number of polychromatic erythrocytes total no. of mn% the rate of antimutagens effects negative control 0.00 1000 1.45 ± 0.07c – positive control treated with cp only 40 1000 9.26 ± 0.62a – 7 day honey (hf) 300 1000 1.67 ± 0.04c – pre-honey (hf) 1000 2.80 ± 0.08bc 69 post-honey (hf) 1000 4.46 ± 0.15b 54 co-treatment honey (hf) + cp 1000 2.35 ± 0.08bc 76 p-value – – 0.0001** – negative control 0.00 1000 1.45 ± 0.07b – positive control treated with cp only 40 1000 9.26 ± 0.62a – 14 day honey ( hf) 300 1000 1.32 ± 0.02b – pre-honey (hf) 1000 1.76 ± 0.02b 80 post-honey (hf) 1000 2.15 ± 0.05b 78 co-treatment honey (hf) + cp 1000 1.68 ± 0.03b 83 p-value – – 0.0001** – means having with the different letters in same column differed significantly. **(p < 0.01). differences a, b, c and d, are significant (p ≤ 0.01 ) to comparison rows. table 3. frequency of mn in bone marrow cells of mice treated with honey formed by sugar syrup (hs), and cyclophosphamide groups treatment dose mg/kg the calculated number of polychromatic erythrocytes total no. of mn% the rate of antimutagens effects negative control 0.00 1000 1.64 ± 0.08d – positive control treated with cp only 40 1000 10.12 ± 0.72a – 7 day honey (hs) 300 1000 3.90 ± 0.13cd – pre-honey (hs) 1000 6.47 ± 0.37b 42 post-honey ( hs) 1000 7.13 ± 0.28b 29 co-treatment honey (hs) + cp 1000 5.75 ± 0.22bc 45 p-value – – 0.0001** – negative control 0.00 1000 1.64 ± 0.08c – positive control treated with cp only 40 1000 10.12 ± 0.72a – 14 day honey ( hs) 300 1000 3.90 ± 0.13c – pre-honey (hs) 1000 7.39 ± 0.36b 34 post-honey (hs) 1000 8.25 ± 0.51ab 18 co-treatment honey (hs) + cp 1000 7.15 ± 0.33b 31 p-value – – 0.0001** – means having with the different letters in same column differed significantly. **(p < 0.01). differences a, b, c and d, are significant (p ≤ 0.01 ) to comparison rows. results. may be attributed that the magnetized water could influence effectively on the oxidant-antioxidant balance, for instance, it could decrease the amounts of malondialdehyde (mda), increase the superoxide dismutase activity in the heart, kidney and liver and also decrease the amounts of nitric oxide which all result in decreasing oxidative stress.18,19 can ameliorate the deleterious effect of free radicals by decreasing the chemical reactions that caused damage to dna, proteins and lipids. water magnetization changes water properties which becomes more energized, active, soft and high ph toward slight alkaline and free of germs,43 also al-mufarrej et al.44 mentioned that, water solution increases the fluidity, dissolving capability for various constituents like minerals and vitamins and consequently improves the biological activity of solutions, affecting positively the performance of animals and plants. physics shows that water changes its weight under the influence of magnetic fields. more hydroxyl (oh-) ions are created to form alkaline molecules, and reduce acidity, for this reason cancer cells do not survive well in an alkaline environment.45 some animal studies have concluded that the magnetization of water increased the permeability through cell membranes,46 and that the magnetic field directly affected intracellular fluid and intracellular substances to activate enzymes inside the cells and to accelerate biochemical reactions in the body.47 previous studies with magnetized water, reported that the long-term intake of magnetized water (over 8 weeks) may be beneficial in both prevention and treatment of complications in diabetic, treatment effect of magnetized water not only decreased the blood ekhlas. m. farhan et al. 317j contemp med sci | vol. 3, no. 12, autumn 2017: 313–318 research investigation of role magnetized water used in supplementary feeding for honeybees table 4. frequency of mn in bone marrow cells of mice treated with honey formed by sugar with magnetized water (hw), and cyclophosphamide groups treatment dose mg/kg the calculated number of polychromatic erythrocytes total no. of mn % the rate of antimutagens effects negative control 0.00 1000 0.78 ± 0.06c – positive control treated with cp only 40 1000 10.08 ± 0.52a – honey (hw) 300 1000 1.18 ± 0.03c – 7 day pre-honey (hw) 1000 3.12 ± 0.07b 69 post-honey (hw) 1000 3.32 ± 0.07b 67 co-treatment honey (hw) + cp 1000 1.63 ± 0.03c 85 p-value – – 0.0001 – negative control 0.00 1000 0.78 ± 0.06c – positive control treated with cp only 40 1000 10.08 ± 0.52a – 14 day honey (hw) 300 1000 1.18 ± 0.03bc pre-honey (hw) 1000 1.42 ± 0.3bc 85 post-honey (hw) 1000 2.02 ± 0.06b 80 co-treatment honey (hw) + cp 1000 0.87 ± 0.02c 92 p-value – – 0.0001** – means having with the different letters in same column differed significantly. **(p < 0.01). differences a, b, c and d, are significant (p ≤ 0.01 ) to comparison rows. glucose and glycated hemoglobin levels but also reduced blood and liver dna damages in stz-induced diabetic rats.20 ma et al.48 presented the possibility that magnetic water can prevent aging and fatigue by increasing the cell membrane permeability. also, the other study showed that administration of magnetized water for at least 6 weeks suppressed the lymphocyte dna damages in animals with den)-induced cancer.21 conclusion in conclusion, the present study demonstrated dangerous effect of anticancer drug cp could be avoided by using the treatment of honey formed by the innovative way (sugar with magnetized water). additionally, our results indicate the use magnetized water in honey formation may be beneficial to increase efficacy honey for modulating the genotoxicity induced by cp in mice, diminishing the deleterious side effects of anticancer drug with preservation of its chemotherapeutic efficacy. this is the first study in the world, no literature available for using the magnetic water to attract honey bees for the purpose of honey formation. therefore, further studies are required to confirm these results, especially about using the magnetized water in the formation of honey and to investigate the possible mechanism from honey (hw) responsible for such effect. the present study, therefore, recommends further studies that show the relationship between used magnetized water on supplementary feeding and the stimulation of these antioxidants in the body and its ability to reduce the toxic effects of cancer drugs. n references 1. fenech m, kirsch-volders m, natarajan at, surralles j, crott jw, parry j, et al. molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. mutagenesis. 2011;26: 125–132. 2. mena s, ortega a, estrela jm. oxidative stress in environmental-induced carcinogenesis. mutat res. 2009;674:36-44 3. kumar r, kumar t, kamboj v, chander h. pharmacological evaluation of ethanolic extract of kigelia pinnata fruit against ethylene glycol induced urolithiasis in rats. asian j plant sci res. 2012;2:63–72. 4. ekhlas mohammed f, al–taai. protective effects of sweet orange peel (citrus sinensis l.) the induction of micronuclei induced by cyclophosphamide in human peripheral lymphocytes. j food technol res. 2016;3:28–35. 5. lina abdul-fk, fatimah aa. reduction of dacarbazine cytogenetic effects on somatic cells in male mice using bee glue (propolis) to manifest the scientific miracles in the quran. electron physician. 2016;8:3015–3023. 6. shen dw, pouliot lm, hall md, gottesman mm. cisplatin resistance: a cellular self -defense mechanism resulting from multiple epigenetic and genetic changes. pharmacol rev. 2012;64:706–721. 7. ahmed s, othman nh. honey as a potential natural anticancer agent: a review of its mechanisms. evid based complement alternat med. 2013;2013:829070. 8. erejuwa oo, sulaiman sa, wahab ms. effects of honey and its mechanisms of action on the development and progression of cancer. molecules. 2014;19:2497–2522. 9. chechan ra. separation of monosaccharides (glucose and fructose) from date juice by crystallization. thesis, college of agriculture baghdad university. 2001. 10. aljadi am, kamaruddin my. evaluation of the phenolic contents and antioxidant capacities of two malaysian floral honeys. food chem. 2004;85:513–518. 11. abdel-latif mm. chemoprevention of gastrointestinal cancers by natural honey. world j pharmacol. 2015;4:160–167. 12. dustmann jh, wehling mw, von der ohe. conversion of specific sugar solutions after their intake by honeybees”, the xxxvth int. apicultural congress of apimondia, antwerpen, 354, 1997. 13. hafizi l, gholizadeh m, karimi m, hosseini g, mostafavi-toroghi h, haddadi m, et al. effects of magnetized water on ovary, pre-implantation stage endometrial and fallopian tube epithelial cells in mice. iran j reprod med. 2014. 14. sueda m, katsuki a, nonomura m, kobayashi r, tanimoto y. effects of high magnetic field on water surface phenomena. j phys chem. 2007;111:14389–14393. 15. verma ss. magnetic water treatment. chem business j. 2011;13–16. 16. iorio r, delle monache s, bennato f, di bartolomeo c, scrimaglio r, cinque b, et al. involvement of mitochondrial activity in mediating elf318 j contemp med sci | vol. 3, no. 12, autumn 2017: 313–318 investigation of role magnetized water used in supplementary feeding for honeybees research ekhlas. m. farhan et al. emf stimulatory effect on human sperm motility. bioelectromagnetics. 2011;32:15–27. 17. gholizadeh m, arabshahi h, saeidi mr, mahdavi b. the effect of magnetic water on growth and quality improvement of poultry. middle-east j sci res. 2008;3:140–144. 18. wang d, cheng x, yan x. effect of magnetized liquor on free radical metabolism in the heart of mice. chinese j med phys. 2002;19:243–244. 19. raymond-whish s, mayer lp, o’neal t, martinez a, sellers ma, christian pj, et al. drinking water with uranium below the u.s. epa water standard causes estrogen receptor-dependent responses in female mice. environ health perspect. 2007;115:1711–1716. 20. lee hj, kang mh. effect of the magnetized water supplementation on blood glucose, lymphocyte dna damage, antioxidant status, and lipid profiles in stz induced rats. nutr res pract. 2013;7:34–42. 21. lee hj, jo hr, jeon ej, kang mh. effect of the magnetized water supplementation on lymphocyte dna damage in mice treated with diethylnitrosamine. korean j nutr. 2010;43:570–577. 22. premkumar k, kavitha s, santhiya st, ramesh ar. interactive effects of saffron with garlic and curcumin against cyclophosphamide induced genotoxicity in mice. asia pac j clin nutr. 2004;13:292–294. 23. ahmadi a, hosseinimehr sj, naghshvar f, hajir e, ghahremani m. chemoprotective effects of hesperidin againstgenotoxicity induced by cyclophosphamide in mice bone marrowcells. arch pharm res. 2008;31:794–797. 24. hosseinimehr sj, ahmadashrafi s, naghshvar f, ahmadi a, ehasnalavi, tanha m. chemoprotective effects of zataria multiflora against genotoxicity induced by cyclophosphamide in mice bone marrow cells. integrat cancer therap. 2010;9:219–223. 25. anton e. ultrastructural changes of stromal cells of bone marrow and liver after cyclophosphamide treatment in mice. tissue cell. 1997;29:1–9. 26. sakr sa, zoil ms, el-shafey ss. ameliorative effect of grapefruit juice on amiodarone-induced cytogenetic and testicular damage in albino rats. asian pac j trop biomed. 2013;3:573–579. 27. eroğlu he. the cytogenetic effects of black tea and green tea on cultured human lymphocytes. braz arch biol technol. 2011;54:1159–1165. 28. schmid w. chemical mutagen testing on in vivo somatic mammalian cells. agents actions. 1973;3:77–85. 29. naghshvar f, abianeh sm, ahmadashrafi s, hosseinimehr sj. chemoprotective effects of carnosine against genotoxicity induced by cyclophosphamide in mice bone marrow cells. cell biochem funct. 2012;30:569–573. 30. serpeloni jm, bisarro dos reis m, rodrigues j, campaner dos santos l, vilegas w, varanda ea, et al. in vivo assessment of dna damage and protective effects of extracts from miconia species using the comet assay and micronucleus test. mutagenesis. 2008;23:501–507. 31. sas. statistical analysis system, user’s guide. statistical. version 9.1th ed. sas. inst. inc. cary. n.c. usa. 2012. 32. vijayalaxmi kk, d’souza mp. studies on the genotoxic effects of anticancer drug carboplatin in vivo mouse. int j hum genet. 2004;4:249–255. 33. asita ao, molise t. antimutageic effects of red apple and watermelon juices on cyclophosphamide-induced genotoxicity in mice. african journal of biotechnology. 2011;10:17763–17768. 34. samanta s, dey p. micronucleus and its applications. diagn cytopathol. 2012;40:84–90. 35. araldi rp, de melo tc, mendes tb, de sá júnior pl, nozima bh, ito et, et al. using the comet and micronucleus assays for genotoxicity studies: a review. biomed pharmacother. 2015;72:74–82. 36. kirsch-volders m, vanhauwaert a, eichenlaub-ritter u, decordier i. indirect mechanisms of genotoxicity. toxicol lett. 2003;140–141. 37. el-fiky sa, farag im, zoheir kma, hassan nha, elalfy hgm. the protective role of honey-bee products against the genotoxic effects of cyclophosphamide in male mice. j appl sci res. 2013;9:4745–4758. 38. shahrour a, zowail m, sharafeldin k. the protective role of honey against cytotoxicity of cadmium chloride in mice. afr j biotechnol. 2016;15: 2620–2626. 39. silva tms, santos fp, evangelista-rodrigues a, et al. phenolic compounds, melissopalynological, physicochemical analysis and antioxidant activity of jandaı’ra (melipona subnitida) honey. j food comp anal. 2013;29:10–18. 40. el sohaimy sa, masry shd, shehata mg. physicochemical characteristics of honey from different origins. ann agricult sci. 2015;60:279–287. 41. tomás-zapico c, coto-montes a. a proposed mechanism to explain the stimulatory effect of melatonin on antioxidative enzymes. j pineal res. 2005;39:99–104. 42. ustundag a, duydu y. the influence of melatonin and nacetylcysteine in delta-aminolevulinic acid and lead induced genotoxicity in lymphocytes in vitro. biol trace elem res. 2007;117:53–64. 43. rajaei f, borhani n, sabbagh-ziarani f, mashayekhi f. effects of extremely low-frequency electromagnetic field on fertility and heights of epithelial cells in pre-implantation stage endometrium and fallopian tube in mice. zhong xi yi jie he xue bao. 2010;8:56–60. 44. al-mufarrej s, al-batshan ha, shalaby mi, shafey tm. the effects of magnetically treated water on the performance and immune system of broiler chickens. inte j poul sci. 2005;4:96–102. 45. park ks, kim jh, kim ms, kim jm, kim sk, choi jy, et al. effects of insulin and antioxidant on plasma 8-hydroxyguanine and tissue 8-hydroxydeoxyguanosine in streptozotocin-induced diabetic rats. diabetes. 2001;50 2837–2841. 46. lednev vv. possible mechanism for the influence of weak magnetic fields on biological systems. bioelectromagnetics. 1991;12:71–75. 47. liboff ar, cherng s, jenrow ka, bull a. calmodulin-dependent cyclic nucleotide phosphodiesterase activity is altered by 20 μt magnetostatic fields. bioelectromagnetics. 2003;24:32–38. 48. ma yl, ren h, ren s, zhen ek, hao g, zhao yw. a study of the effect of magnetized water on enzyme activities by potentiometric enzyme electrode method. j tongji med univ. 1992;12:193. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201705 7j contemp med sci | vol. 1, no. 4, autumn 2015: 7–12 research objective this study includes the investigation of antibacterial and antivirulence activities of three types of pomegranate peel extracts and then determines the interaction between the extracts and antibiotic in vitro. methods the ability of most common isolated bacteria from urinary tract infection (uti) to produce different virulence factors were tested and the effect of plant extracts on virulence factors were determined; in addition the correlation between extracts and antibiotics were evaluated by using fractional inhibitory concentrations. results the inhibition zones diameters of the pomegranate peel extracts against most common isolated bacteria (staphylococcus aureus and escherichia coli) increase significantly with increase in concentrations. there is no effect of the extracts on the ability of studied bacteria to produce hemolysin and protease enzymes, while both studied bacteria lost its ability to produce β-lactamase enzyme after treating with mic. in addition, extracts were affected largely on adherence activity and biofilm forming ability of tested bacteria. the results found that the pomegranate peel extracts effect alone against pathogenic bacteria was good than they interacted with antibiotics, in most of the results. conclusion the alcohol extract was the best solvent in its effects on bacterial pathogen and its effect was largely on the ability of the studied bacteria to form biofilm and adhesion on the epithelial cell. the pomegranate peel extracts were high synergism with some antibiotics against pathogenic bacteria. keywords pomegranate peel extract, antivirulence effect, interaction with antibiotic antivirulence effects of pomegranate peel extracts on most common urinary tract infection pathogens in pregnant women wafaa sadeq al-wazni & bashair sami hadi introduction urinary tract infection (utis) is an injury resulting from the presence and the growth of microorganism in different parts of the urinary tract, so it could be defined as the colonization of and invasion of the structures in the urinary tract by micro-organisms.1 in human, the urinary tract is one of the most common sites of bacterial infection and most case of uti caused by bacteria which ascend from the perineum, and the reason of the ascent of bacteria raised by conditions like pregnancy.2,3 utis are the most common problem during pregnancy as result of physiologic changes which are related to pregnancy that make healthy women more susceptible to acquired various injury.4 utis are caused by different type of gram positive and negative bacteria like escherichia coli, klebsiella species, proteus mirabilis, pseudomonas aeruginosa, enterococcus, staphylococcus, and streptococcus agalactiae.5 the isolate bacteria produce deferent virulence factors which play essential roles in pathogenicity of these bacteria such as hemolysin (is an important virulence factor attached with especial receptors on the erythrocytes wall then making pours in the cell wall so the erythrocytes will lyse), β-lactamase (enzyme that works to destroy the β-lactam ring of antibiotic and causing the loss of effectiveness and contain serine amino acid in their hydroxyl group which represented the active site of this enzyme).6 pathogen adhesion to the host tissue is regarded as an important initiating step in many types of infection because it helps the bacteria to resist the defense mechanism in the body,7 and biofilm formation (a slimy layer with embedded micro colonies) is most important and widespread mode for increase pathogenicity of the microorganism and helps bacteria to resist the surrounding environment condition and antibiotic concentration.8 some pathogens which are responsible for uti are resistance to different types of antibiotic therefore relatively limited choice of antimicrobial agents can eliminate from the body so has become necessary to work to find a new techniques more effective for the treatment of infection caused by drug-resistant microorganism. many types of medicinal plants contain various components some of which can operate in synergy with antibiotic but others are able to sensitize the pathogen to antibiotic.9 pomegranate is one of the medicinal plants used in medicine for treatment of several disease, which was one of the oldest fruits that have not changed much through the history of man and regarded as an important source of phenolic compounds, including hydrolysable tannins, which possess high antioxidant activity.10 the screening of medicinal plant extracts for interactions with antibiotics is expected to provide chance to determine inhibitors that may benefit medicine.11 the purpose of this study was to find a safety method to reduce pathogenicity of high virulence pathogenic bacteria responsible for uti by using natural material and attempt to find a safety method to solve the problem of multi-drug resistance pathogen. materials and methods 1. isolation and diagnosis of bacteria one hundred morning midstream urine samples were collected from pregnant women attending maternity and women’s hospital in karbala province during december 2011 to march 2012. the isolated bacteria were diagnosed biochemically according to methods described by collee et al.12 and baron et al.13 then the diagnosed bacteria were confirmed by api20e and api staph system accomplished according to manufacturer’s instructions. the isolated bacteria used in the study were chosen according to their ability to give deep violet color on the wall and bottom of the test tubes, which was used to determine the biofilm forming as described by mathur et al.14 issn 2413-0516 karbala university, college of science, department of biology, iraq. (submitted: 20 september 2015 – revised version received: 23 september 2015 – accepted: 3 october 2015 – published online: autumn 2015) 8 j contemp med sci | vol. 1, no. 4, autumn 2015: 7–12 interaction between the extracts of pomegranate peels and antibacterial and antivirulence activities research wafaa sadeq al-wazni et al. 2. ability of isolated bacteria to produce virulence factors blood agar plates and skim milk agar plates were used to determine the ability of isolated bacteria to produce hemolysin and protease enzyme, respectively, as described by collee et al.12 and baron et al.13 the β-lactamase production was prepared according to who.15 the adherence activity for studied bacteria was carried out according to svanborg et al.16 in addition we screwed isolates for their ability to form biofilm by tube and tissue culture plant methods as described by mathur et al.14 and maldonado et al.17 3. plants extracts plant extracts (aqueous, alcohol, and acetone) were prepared according to ahmed et al.18 and al-jboriy et al.19 then stock solution was prepared for each extract by dissolving 1 g of dry extract with 10 ml of distilled water, so the final concentration of extract would be 0.1 g/ml, from this solution other concentrations were prepared (0.01–0.1) g/ml, which was used to determine the antibacterial activity of peel extracts against s. aureus and e. coli bacteria by agar well diffusion method as mentioned by egharevba et al.20 but agar dilution method was used to detect minimum inhibitory concentration (mic) of the plant extracts according to nccls.21 the extracts were subjected to phytochemical screening according to ling et al.22 4. effect of pomegranate peel extracts on the bacterial virulence factors mic of each extract was added to the bacterial suspension, and all tests were made as mentioned in step 2. 5. combination studies the combined antimicrobial activity of the pomegranate peel extracts and antibiotics were done by evaluating the fractional inhibitory concentrations (fic) as described by manda et al.23 fic (antibiotic) = mic of antibiotic in combination/mic of antibiotic alone fic (extract) = mic of extract in combination/mic of extract alone then the interactions between the antibiotics and the peel extracts were evaluated by using the fic indices as described by pankey et al.24 and kamatou et al.25 which were calculated by using the formula: fic index = σfic = fic (antibiotic) + fic (plant extract) the combinations were classified as synergistic (fic indices were <1), additive (fic indices were 1), indifferent (fic indices were between 1 and 2), and antagonistic (fic indices were >2 ). 6. statistical analysis data analysis of variance was carried out by sas. lsd was used to compare mean at 0.0001.26 result and discussion 1. frequency of urinary tract pathogens in pregnant women fifty-two bacterial isolates were isolated in this study, gram-positive bacteria 43(83%) occurred more frequently than gram-negative bacteria 9(18%) where s. aureus 20(39%) and e. coli 6(11%) were the commonest offending isolated as shown in fig. 1. first, this might be due to environment, the socioeconomic conditions of the pregnant and the reinfection, when infection happened in the first trimester (some time it is possible in the third trimester) of pregnancy which was ensured by the patients’ information. one isolate of both s. aureus and e. coli had been chosen to continue and complete other steps of the study. these two isolates were subjected to standard tests for determination of their ability to produce different virulence factors such as hemolysin, protease, β-lactamase, adherence, and biofilm formation ability. from the result, it appeared that these two isolates gave positive results for the previous tests. many studies indicate the relationship between the bacterial virulence factors and their pathogenicity such as muder et al.27 who found that the bacteria which had the ability to produce hemolysin and protease enzymes in some way were showed to increase its invasion activity and ability to resist host immune system. while alchalabi28 reported that several virulence factors such as hemolysin, cytotoxic necrotizing factor, aerobactin, biofilm, and different types of adhesion have been responsible for e. coli pathogenesis, because of the relationship between the bacterial ability to produce virulence factors and their infectivity or pathogenicity. 2. plant extracts pomegranate peel extracts screening alkaloid, saponins, flavonoids, tannins, phenolic, glycosides, and resins as a phytochemical. the flavonoids and saponins were absent in aqueous extract, but during ethanol extraction, only saponins were not extracted. while in the acetone extraction this solvent succeeded to extract nearly all the studied active material. the antibacterial activity of plant extracts depends on the extraction conditions such as type and concentration of the solvent, time and temperature for the extraction process, all these factors effect on the type and the amount of the active material that extracted and found large amount of phytochemical material increase antibacterial activity of extract against pathogenic bacteria.29 3. the antibacterial activity of pomegranate peel extracts the results showed clearly that pomegranate peel extracts were active against s. aureus and e. coli bacteria in comparison to ciprofloxacin as a positive control and the distilled water as a negative control. the alcohol solvent could be considered as the best one among the three solvents which were used in this study. acetone follow alcohol and distilled water might be the last good solvent with respect to their activity against the chosen isolates as shown in tables 1 and 2. the result showed a relationship between the value of inhibition zone diameter for each one of the studied bacteria and type of solvents used in the extraction process. when the three solvents were used fig. 1 frequency of urinary tract pathogens in pregnant women. 9j contemp med sci | vol. 1, no. 4, autumn 2015: 7–12 research interaction between the extracts of pomegranate peels and antibacterial and antivirulence activitieswafaa sadeq al-wazni et al. at different concentrations against s. aureus bacteria, alcohol solvent gave the widest inhibition zone, when used at 0.01, 0.05, 0.075 and 0.1 g/ml and it was followed by the value of inhibition zone diameter for the acetone extract, the aqueous extract had the lowest effect when used at the concentrations above and when 0.025 g/ml concentration of these three types of extracts were used against s. aureus, the acetone extract was the best one as the highest effect then followed by the alcohol and the aqueous extracts. among the solvent and according to their effects on e. coli isolate, acetone extract was considered as the best one when it used at 0.01 g/ml and 0.075 g/ml, while the alcohol extract had the highest effect on e. coli bacteria at 0.025 g/ml, 0.05 g/ml, and 0.1 g/ml concentration in contrast with the other extracts. when the pomegranate peel extracts were used as an antibacterial the best solvent chosen as extractor could be the polar solvents especially the ethanol solvent due to the best effect on both selected isolates. the results agree with rathinamoorthy et al.30 who attributed the antibacterial activity of pomegranate peel extracts to the presence of the broad spectrum antimicrobial compounds that act against both selected isolates. 4. antivirulence activity of pomegranate peel extracts the studied bacteria which had the ability to produce a number of virulence factors (hemolysin, protease, β-lactamase) were treated with the mic of each one of the plant extracts (which reached to 0.006 g/ml in aqueous extract, 0.004 in both alcohol and acetone extracts). the results explained that capacity of s. aureus and e. coli bacteria to produce hemolysin toxin and protease enzyme had not been affected and remained without any alteration after incubation period with these extracts compared to control. these bacteria completely lost their ability to produce β-lactamase enzymes after their treatment with the mic of each extract, although these bacteria were active producer for this enzyme before they have been treated with the extracts, as shown in table 3. forty cells of s. aureus bacteria adhered on the assayed epithelial cell, while only 20 cells of e. coli adhered to the epithelial cells. these data were regarded as control for detection the effects of the mic of pomegranate peel extracts on adhesion ability of studied bacteria. as shown in fig. 2, the number of adhered s. aureus bacterial cells on the epithelial cell was clearly declined when the bacteria was treated with mic of extracts. the aqueous extract reduced the number of the adherence bacterial to only 10 bacteria/cell, but the number of the adherence cells reached to 3 and 1 bacteria/cell in the presence of the acetone and the alcohol extracts, respectively. the adherence of e. coli cell reached to one bacteria/cell when the acetone extract was added to bacterial suspension, but only three bacterial cells were seen to be attached to table 1. antibacterial activity of extracts against s. aureus bacteria concentration g/ml inhibition zone rate (cm) concentration rangeaqueous pomegranate peel extract alcohol pomegranate peel extract acetone pomegranate peel extract 0.01 1.25* 1.65* 1.45* 1.45* 0.025 1.6* 1.73* 1.9* 1.74* 0.05 1.61 1.95* 1.91 1.82 0.075 1.81* 2.21* 2.00 2.01* 0.1 1.88 2.26 2.5* 1.96* extraction range 1.63* 1.96* 2.03* control (ciprofloxacin) antibiotic 5 μg/disc 1.2 lsd 0.05 (concentration = 0.064, extraction solvent = 0.58, interaction = 1.296). p ≤ 0.0001* = significant different. table 2. antibacterial activity of extracts against e. coli bacteria concentration g/ml inhibition zone rate (cm) concentration rangeaqueous pomegranate peel extract alcohol pomegranate peel extract acetone pomegranate peel extract 0.01 1.38* 1.50* 1.68* 1.52* 0.025 1.51* 1.90* 1.76* 1.72* 0.05 1.63* 1.93 1.91* 1.82* 0.075 1.73* 2.08* 2.21* 2.01* 0.1 1.78 2.36* 2.33 2.15* extraction range 1.64* 1.97* 1.98* control (ciprofloxacin) antibiotic 5 μg/disc 2.5 lsd 0.05 (concentration = 0.086, extraction solvent = 0.077, interaction = 1.737). p ≤ 0.0001* = significant different. 10 j contemp med sci | vol. 1, no. 4, autumn 2015: 7–12 interaction between the extracts of pomegranate peels and antibacterial and antivirulence activities research wafaa sadeq al-wazni et al. epithelial cells after the bacterial suspension was incubated with alcohol extract, while only 5 bacteria/cell were attached after treatment with the aqueous extract, in contrast with control (e. coli without the extracts), as shown in fig. 3. as a comparison, ethanol extract was the best anti-adhesive factor, followed by acetone, while the aqueous extract had the least effect. this may return to the weak ability of distilled water to extract the active materials from plants peel in affected amounts compared to acetone and alcohol solvents. the pomegranate peel extracts have been worked as anti-adhesive, because of the large amounts of the saponins, flavonoids, alkaloids, tannins, phenolic, glycosides, and resins, which were directly responsible for the anti-adhesive activity against the pathogen.31 the effect of the plant extracts is return to its ability to inhibiting cell attachment; therefore pretreatment of the body surface with plant extracts produced an unfavorable film that prevent and reduce the surface adhesion of pathogenic bacteria.32 figure 4 illustrates that s. aureus was a high producer for biofilm formation; this was shown through its optical density that reaches to 1.7 when it was measured for bacterial suspension without any types of the extracts (regarded as control). but, the optical density came to be reduced largely when the mic of each one of the extracts was added to s. aureus suspension before reading the optical density. the optical density (od) of s. aureus suspension with the mic of the aqueous and the acetone extracts almost the same, and this result was different from the results of the other studies which found that the acetone extract had more effect than the aqueous extract. whereas the alcohol extracts, still the best solvent in its effects on studied bacteria e. coli, was regarded as high producer biofilm because the od of its suspension without extracts reached to 1.5. but when the suspension of e. coli bacteria was treated with mic of the aqueous and acetone extracts, the od of it reduced, which means the biofilm formation activity were declined clearly compared to the control, as shown in fig. 5. table 3. ability of isolated bacteria to produce virulence factors with and without extracts virulence factors s. aureus s. aureus with pomegranate peel extracts by e .coli e. coli with pomegranate peel extracts by distill water 96% ethanol 70% acetone distill water 96% ethanol 70% acetone hemolysin + + + + + + + + protease + + + + + + + + β-lactamase production + − − − + − − − +: indicates present, −: indicates absent. fig. 2 effects of extracts on adherence activity of s. aureus bacteria. fig. 3 effects of extracts on adherence activity of e. coli bacteria. fig. 4 effects of extracts on biofilm formation activity of s. aureus bacteria. fig. 5 effects of extracts on biofilm formation activity of e. coli bacteria. 11j contemp med sci | vol. 1, no. 4, autumn 2015: 7–12 research interaction between the extracts of pomegranate peels and antibacterial and antivirulence activitieswafaa sadeq al-wazni et al. the biofilm formation activity of bacteria were declined after treatment with extracts but this decline do not transfer bacteria from high producer (the control) to the poor producer (od lower than 0.1) after treatment with extracts. s. aureus and e. coli bacteria were treated with the aqueous and the acetone extracts remained as a high producer (od high than 0.5), but when bacteria was treated with alcohol extract it came to be transferred from high producer to producer only (od between 0.5–0.1). this proved that alcohol solvent represented as the best solvent in extraction and preserves the activity of active compounds in pomegranate peel extracts compared with acetone and aqueous extracts. these results may be due to the formation of variety of biological properties and the activity of the extracts of chemical composition when different solvents were used in preparing these extracts. many searchers were emphasizing by many trials to find some materials that inhibit the activity of virulence factors of pathogenic bacteria such as chemical material that was extracted from plants.33 the pomegranate peel extracts contain compound such as tannins that can interact with macromolecules, including carbohydrates and proteins, which made these compounds as promising anti-adhesive and antibiofilm.34 5. interactions between the extracts and antibiotics in vitro the effect of the combination of the peel extracts and the antibiotics on the susceptibility of the s. aureus bacteria shown in table 4 illustrated that the interaction of the ethanol extracts table 4. effect of the combination between pomegranate peel extracts and antibiotics on s. aureus bacteria interactionfic indexmean fic (extract)mean fic (antibiotic)pomegranate extractantibiotics synergy0.20.10.1ethanol 96% chlo antagonism2.0251.1250.9acetone 70% indifference1.330.8330.5d.w. synergy0.20.10.1ethanol 96% cip antagonism2.730.9371.8acetone 70% indifference1.930.8331.2d.w. indifference1.1250.750.375ethanol 96% tet antagonism1.310.9370.375acetone 70% antagonism1.951.50.45d.w. chlo: chloramphenicol, cip: ciprofloxacin, tet: tetracycline. table 5. effect of the combination between pomegranate peel extracts and antibiotics on e. coli bacteria interactionfic indexmean fic (extract)mean fic (antibiotic)pomegranate extractantibiotics additive10.50.5ethanol 96% chlo indifference1.680.9370.75acetone 70% antagonism21.250.75d.w. antagonism2.0250.91.125ethanol 96% cip indifference1.250.6250.625acetone 70% antagonism2.1871.250.937d.w. indifference1.1250.750.375ethanol 96% tet synergy0.8750.6250.25acetone 70% indifference1.6251.250.375d.w. chlo: chloramphenicol, cip: ciprofloxacin, tet: tetracycline. with the chloramphenicol and ciprofloxacin antibiotics were synergy. but with tetracycline it’s been indifferent. while at the combination of the acetone extract with the three types of the antibiotics had been antagonism. the fic value illustrated that the combination was indifference in the aqueous extracts with the chloramphenicol and ciprofloxacin antibiotics and came to be antagonism with the tetracycline antibiotic. while in the study of e. coli bacteria, susceptibility found that the synergy was present only in the interaction between the tetracycline and the acetone extract and the other results were divided between antagonism and indifference effect, but in the case of ethanol and the chloramphenicol it was additive (the effect which is less than synergistic but not antagonism), as shown in table 5. the effect of chloramphenicol and ciprofloxacin antibiotics against s. aureus bacteria was marginally improved in the presence of the alcohol extract and the effect of the tetracycline antibiotic against e. coli was improved in the presence of the acetone extract, while any other combination between studied antibiotic and the extracts had not any beneficial effects against studied s. aureus and e. coli bacteria. so the results found that the effect of pomegranate peel extracts alone was well than they interact with antibiotics in most of the results. while other studies found that the efficacy of antimicrobial agents could be improved by combining antibiotics with crude plant extracts against different pathogens 12 j contemp med sci | vol. 1, no. 4, autumn 2015: 7–12 interaction between the extracts of pomegranate peels and antibacterial and antivirulence activities research wafaa sadeq al-wazni et al. in vitro and found that it may reduce mics of antibiotics against resistant organisms. the combination of the plants extracts and the antibiotics could be useful in fight emergency drug resistance pathogens.35 the antimicrobial compounds extracted from plants have been found to be synergistic enhancers in that though they may not explain any antimicrobial properties alone, but when used with antibiotic they enhance the activity of the drug. the synergistic effect of the association of antibiotic and plant extracts against resistant pathogens leads to new choices for the treatment of infectious diseases. also synergy between plant product and drug will solve problems of toxicity and overdose since when they combine a little concentration of two agents is required. therefore, there is an urgent need to find source of natural compound to solve the problem of multiple drug resistance.36  references 1. haider g, zehra n, munir aa, haider a. risk factors of urinary tract infection in pregnancy. j pak med assoc. 2010 mar;60(3):213–6. pmid: 20225781 2. nahar sj, khanum h, shimasaki k. occurrence of escherichia coli infection among the women of dhaka city. arpn j agricultural bio sci. 2010 nov;5(6):68–73. 3. sawalha rmh. prevalence of urinary tract infection among children of primary schools in nablus, m.sc. thesis. an-najah national univ. of nablus, palestine; 2009. 4. umar n, basavaraj s. sirwar. prevalence of urinary tract infection in pregnant women. j evolution of med dent sci. 2012;1(4):315–20 5. ali mm. evaluation of antimicrobial susceptibility & rapid urine screening tests in asymptomatic urinary tract infection in pregnant women in karbala. k j pharm sci. 2011;2:22–34. 6. akindele aa, adewuyi ik, adefioye oa, adedokun sa, olaolu ao. antibiogram and beta-lactamase production of staphylococcus aureus isolates from different human clinical specimens in a tertiary health institution in ile-ife, nigeria. american-eurasian j sci res. 2010;5(4):230–3. 7. atabek a. investigating bacterial outer membrane polymers and bacterial interactions with organic molecules using atomic force microscopy, master’s thesis. worcester polytechnic institute, ma, usa. 8. hola v, ruzicka f, votava m. the dynamics of staphylococcus epidermis biofilm formation in relation to nutrition, temperature and time. scripta medica (brno) 2006;79(3):169–74. 9. aiyegoro o, adewusi a, oyedemi s, akinpelu d, okoh, a. interactions of antibiotics and methanolic crude extracts of afzelia africana (smith.) against drug resistance bacterial isolates. int j mol sci. 2011;12:4477–503. 10. dahham ss, ali mn, tabassum h, khan m. studies on antibacterial and antifungal activity of pomegranate (punica granatum l.). americaneurasian j agric & environ sci. 2010;9(3):273–81. 11. endo eh, ueda-nakamura t, nakamura cv, filho bpf. activity of spraydried micro particles containing pomegranate peel extract against candida albicans. molecules. 2012 aug;17:10094–107. doi: http://dx.doi. org/10.3390/molecules170910094 pmid: 22922280 12. collee jg, fraser ag, marmion bp, simmons a. mackie and mccartney. practical medical microbiology, 14th ed. uk: the churchill livingstone. inc.; 1996. pp. 329–41. 13. baron ej, peterson lr, finegold sm. bailey & scott’s diagnostic microbiology, 9th ed. usa: mosby-year book; 1994. 14. mathur t, singhal s, khan s, upadhyay dj, fatma t, rattan a. detection of biofilm formation among the clinical isolates of staphylococci: an evaluation of three different screening methods. indian j med microbiol. 2006;24(1):25–30. doi: http://dx.doi.org/10.4103/0255-0857.19890 pmid: 16505551 15. who. techniques for the detection of β-lactamase producing strains of neisseria gonorrhoeae. 1978;616:137–43. 16. eden cs, eriksson b, hanson la. adhesion of escherichia coli to human uroepithelial cells in vitro. infect immun. 1977 dec;18(3):767–74. 17. maldonado nc, silva de ruiz c, cecilia m, nader-macias me. a simple technique to detect klebsiella biofilm-forming-strains. inhibitory potential of lactobacillus fermentum crl 1058 whole cells and products. communicating current research and educational topics and tends in applied microbiology a. mendez vilas (ed); 2007. 18. ahmad i, mehmood z, mohammad f. screening of some indian medical plants for their antimicrobial properties. j enthnophar. 1998;62(2):183–93. doi: http://dx.doi.org/10.1016/s0378-8741(98)00055-5 19. al-jboriy ka, al-anasary bs, ali hsa. study of the sensitivity of some pathogenic isolated from respiratory infection in human against same plant extracts. anbar j veterinary sci. 2010;3(2):103–8. 20. egharevba ho, kunle of, iliya i, orji pn, abdullahi ms, okwute sk, et al. phytochemical analysis and antimicrobial activity of punica granatum l. (fruit bark and leaves). new york sci j. 2010;3(12):91–8. 21. clsi. methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—ninth edition. clsi document m07-a9. wayne, pa: clinical and laboratory standards institute; 2012. 22. ling yc, feng xs, xia g. preliminary phytochemical analysis of acanthopanan trifoliatus (l.) merr. j med plants res. 2011 sept 9;5(17):4059–64. 23. mandal s, mandal md, pal n. evaluation of combination effect of ciprofloxacin and cefazolin against salmonella enterica serovar typhi isolates by in vitro methods. calicut med j. 2004;2(2):e2. 24. kamatou gpp, viljoen am, van vuuren sf, van zyl rl. in vitro evidence of antimicrobial synergy between salvia chamelaeagnea and leonotis leonurus. sa j bot. nov 2006;72(4):634–6. doi: http://dx.doi.org/10.1016/j. sajb.2006.03.011 25. pankey g, ashcraft d, patel n. in vitro synergy of daptomycin plus rifampin against enterococcus faecium resistant to both linezolid and vancomycin. antimicrob agents chemother. 2005 dec;49(12):5166–8. doi: http://dx.doi. org/10.1128/aac.49.12.5166-5168.2005 pmid: 16304195 26. sas. sas/stat user’s guide for personal computers. release 6.12. nc, usa: sas inst. inc.; 2012. 27. muder rr, brennen c, rihs jd, wagener mm, obman a, stout je, yu vl. isolation of staphylococcus aureus from the urinary tract: association of isolation with symptomatic urinary tract infection and subsequent staphylococcal bacteremia. clin infect dis. 2012 jan 1;42(1):46–50. doi: http://dx.doi.org/10.1086/498518 pmid: 16323090 28. al-chalabi r, al-ubaidy a, al-ibadi m. detection of urovirulence genes (eae, e-hly, α-hly) of uropathogenic escherichia coli by specific pcr. j biot res center. 2010;4(1):44–54. 29. kdhim zr. extraction and purification of flavonoids from camellia sinesis leaves and punica granatum peels and determination of their antioxidant activity, m.sc. thesis. karbala univ; 2012. 30. rathinamoorthy r, udayakumar s, thilagavathi g. antibacterial efficacy analysis of punica granatum l. leaf, rind and terminalia chebula fruit extract treated cotton fabric against five most common human pathogenic bacteria. int j pharm & life sci. 2011 oct;2(10):1147–53. 31. o’mahony r, al-khtheeri h, weerasekera d, fernando n, vaira d, holton j, et al. bactericidal and anti-adhesive properties of culinary and medicinal plants against helicobacter pylori. world j gastroenterol. 2005 dec 21;11(47): 7499–507. pmid : 16437723 32. sandasi m, leonard cm, viljoen am. the in vitro antibiofilm activity of selected culinary herbs and medicinal plants against listeria monocytogenes. lett appl microbiol. 2010 jan;50(1):30–5. doi: http:// dx.doi.org/10.1111/j.1472-765x.2009.02747.x pmid: 19874481 33. jiang p, li j, han f, duan g, lu x, gu y, yu w. antibiofilm activity of an exopolysaccharide from marine bacterium vibrio sp. qy101. plos one. 2011 apr 7;6(4):e18514. doi: http://dx.doi.org/10.1371/journal.pone.0018514 pmid: 21490923 34. janecki a, kolodziej h. anti-adhesive activities of flavan-3-ols and proanthocyanidins in the interaction of group a-streptococci and human epithelial cells. molecules 210 oct 15;15(10):7139–52. doi: http://dx.doi. org/10.3390/molecules15107139 pmid: 20953158 35. adwan g, mohammad mhanna m. synergistic effects of plant extracts and antibiotics on staphylococcus aureus strains isolated from clinical specimens. middle-east j scientific res. 2008;3(3):134–9. 36. aiyegoro oa, okoh ai. use of bioactive plant products in combination with standard antibiotics: implications in antimicrobial chemotherapy. j med plants res. 2009 dec;3(13):1147–52. 224 j contemp med sci | vol. 3, no. 10, spring 2017: 224–228 research role of vitamin d3 level and apo-b/apo-a1 ratio in patients with unstable angina in kerbala province, iraq fadhil jawad al-tu’ma,a zahraa mohsen mohammeda and haidar hussein al-sarrafb adepartment of biochemistry, college of medicine, university of kerbala, iraq. bdepartment of internal medicine, al-hussein teaching hospital, al-hussein medical city, kerbala health directorate, iraq. correspondence to zahraa mohsen mohammed (email: zahraa.alhadi1988@gmail.com). (submitted: 05 january 2017 – revised version received: 29 january 2017 – accepted: 22 february 2017 – published online: 26 june 2017 ) objectives vitamin d deficiency may be responsible for endothelial dysfunction which in turn affects the onset and progression of coronary artery disease and its risk factors. the apo b/apo ai ratio indicates the balance between atherogenic and anti atherogenic particles, the higher the value, the higher is the cardiovascular risk. the aim of study is to find a possible association between unstable angina and vitamin d3, apo a1, apo b, and apo b/apo a1 ratio and other risk factors (age, body mass index and smoking). methods this case-control study was conducted during the period from nov. 2015 to sep. 2016. a total of 40 patients of unstable angina presented with typical chest pain to the coronary care unit in al-hussein teaching hospital, al-hussein medical city/ kerbala. the diagnosis was based on the clinical history and electrocardiography. a total of 50 persons were matched in age, gender and bmi as a control group. the procedures were measured using auto immunoassay analyzer. results vitamin d3 deficiency (< 30 ng/ml) was prevalent in unstable angina (ua) compared with controls (vitamin d3 ≥ 30 ng/ml) (odds ratio [or], 21.93; 95%, confidence interval [ci], 5.91–81.31; p < 0.001). the results obtained that serum 25(oh) d3 was highly significant in smoker compared with non-smoker in both groups (p < 0.001, p < 0.05, respectively). the serum apo b, apo a1 and apo b/apo a1 ratio were highly significant between both groups (p < 0.01, p < 0.001, p < 0.001, respectively). on the other hand, vitamin d3 recorded significant correlation with each of the age and bmi in control group (all p < 0.01). conclusion the present study showed a highly significant association between vitamin d3, apo b, apo a1 levels, apo b/apo a1 ratio and unstable angina as compared with the control group, and significant correlation between vitamin d3 and age, bmi and smoking. keywords unstable angina, vitamin d3, apoa1, apo b, apo b/apo a1 ratio issn 2413-0516 introduction unstable angina (ua) is a type of angina pectoris that is irregular. it is also classified as a type of acute coronary syndrome. the term ua has been conventionally applied to patients with myocardial ischaemia without myocardial necrosis. the ua is differentiated from stable angina in that the pain is often of more recent onset with increasing frequency and severity, and unlike stable angina is often not relieved by rest, and can occur at rest and on minimal exertion.1 the ua is considered to be present in patients with ischaemic symptoms suggestive of an acute coronary syndrome and no elevation in troponin, with or without ecg changes indicative of ischaemia (st segment depression or transient elevation or new t wave inversion). since an elevation in troponin may not be detectable for up to 12 hours after presentation, ua and non st-segment elevation myocardial infraction are frequently indistinguishable at initial evaluation.2 unstable angina is caused by disruption of an atherosclerotic plaque with partial thrombosis and possibly embolization or vasospasm.3 the diagnosis of ua is characterized by at least one of the following: 1. occurs at rest or minimal exertion and usually lasts more than 20 minutes (if nitroglycerin is not administered) 2. being severe and of new onset (within 1 month) 3. occurs with a crescendo pattern (brought on by less activity, more severe, more prolonged or increased frequency than previously).4 nitroglycerin can be used immediately to widen the coronary arteries and help increase blood flow to the heart. in addition, nitroglycerin causes peripheral venous and artery dilation reducing cardiac preload and afterload. these reductions allow for decreased stress on the heart and therefore lower the oxygen demand of the heart’s muscle cells. antiplatelet drugs such as aspirin and clopidogrel can help to reduce the progression of atherosclerotic plaque formation, as well as combining these with an anticoagulant such as a low molecular weight heparin.2 traditionally, vitamin d3 has been a well-known vital nutrient for calcium homeostasis and healthy bones. however, recent researches reveal that vitamin d3 is associated with numerous outcomes including not only rickets or osteomalacia but also as a potential influencing factor in the pathogenesis of several chronic non-skeletal diseases, such as cancer, immunity disorders, cvd and problems in pregnancy, because the discovery that the vitamin d receptor (vdr) is ubiquitously expressed in almost all body cells.5 since then, several, but not all, observational studies that have been published indicated that low vitamin d3 levels are associated with higher incidence of cardiovascular events and mortality.6–9 there is strong observational epidemiologic evidence that suggests that low levels of serum 25(oh)d3 are a novel risk factor for all-cause mortality, cardiovascular mortality, chd events, stroke, and heart failure. suboptimal vitamin d3 is thought to influence cvd risk by acting on established risk factors such as hypertension, diabetes, and inflammation.10 the apo b and apo ai are the two major apolipoproteins involved in lipid transport and independent predictors of cvd in the general population. the apo b/apo ai ratio fadhil jawad al-tu’ma et al. 225j contemp med sci | vol. 3, no. 10, spring 2017: 224–228 research role of vitamin d3 level and apo-b/apo-a1 ratio indicates the balance between atherogenic and anti atherogenic particles, the higher the value, the higher is the cardiovascular risk. it may be a better marker of the cvd risk than conventional lipid measurements.11–13 previous reports have shown that apo b/apo ai ratio for men and women, respectively, < 0, 7 and < 0, 6 is associated with low risk for cvd. the advantage of calculating this index is that the concentration of apolipoproteins does not change after the meal and do not change in different times of day. in addition, with regards to the tests performed after ami have occurred there is no matter how much time has elapsed since the in the blood collection.14 the aim of the study is to find a possible association between unstable angina and vitamin d3, apo a1, apo b, and apo b/apo a1 ratio and other risk factors (age, body mass index (bmi) and smoking) in kerbala province. materials and methods this case-control study was conducted during the period from nov. 2015 to sep. 2016. a total of 40 male patients of unstable angina had been selected from coronary care unit (ccu) in al-hussein teaching hospital/al-hussein medical city kerbala health directorate/holy kerbala iraq. the (mean ± sd) of age is (52.85 ± 11.38) ranged between 28 and 72 years old. a total of 50 male individuals as control were matched with patients group in their age and body mass index, and the mean ± sd of age is 52.72 ± 8.74 and ranged between 40 and 72 years old. approvals of the study program were taken from the administration of al-hussein general hospital, and from the patients and the controls groups. serum 25(oh)d3, apo a1, apo b, troponin i, random blood sugar (rbs), urea and creatinine. serum 25(oh)d3 and troponin i levels were measured using auto immunoassay analyzer [maglumi clia (chemiluminescence immunoassay)]. serum apo a1, apo b, urea, creatinine and random blood sugar were measured using auto analyzer biochemistry (cormay). statistical analysis statistical analysis was done by statistical package for the social sciences (spss 21). descriptive statistics were presented as mean ± standard deviation (sd) for continuous variables (age, bmi, glucose, apo a1, apo b, apo b/a1 ratio, urea, creatinine, troponin i and 25(oh) d3). independent samples student’s t-test, chi square, fisher’s exact test, correlation coefficient (r) test were used to compare, assess and describe the association between the different studied variables; p values < 0.05 were considered to indicate statistical significance and highly significant if it is < 0.01. results the characteristics of population of this study are presented in table 1. the results show the evaluation of vitamin d3, apo b, apo a1, apo b/apo a1 ratio, troponin i and urea. it has significant association between patient and control group. and no significant association in creatinine and sugar between patients and control groups. there was highly significant association between patient and control groups when classified vitamin d3 levels to sufficiency (≥ 30) and deficiency (< 30), and the results are shown in table 2. the odds ratio of this study of unstable angina was 21.93 (95% confidence interval 5.91–81.31; p < 0.001). table 3 presents the correlation between vitamin d3 and the age in two groups, and there was a significant correlation in patient group as shown in fig. 1 and control group as shown in fig. 2, and significant correlation between vitamin d3 and bmi in the control group as shown in fig. 3. a significant association was shown between smokers and non-smokers in patient group (p < 0.01). additionally, it showed significant association between smokers and nonsmokers in control group (p < 0.05), as shown in table 4. discussion table 1. associated between patient and control groups in all biomarkers parameters mean ± sd p valuecontrol (n = 50) patient (n = 40) 25(oh)d3 (ng/ml) 35.15 ± 11.79 16.93 ± 4.85 < 0.001** apo b (mg/dl) 98.05 ± 37.73 115.75 ± 43.16 0.041* apo a1 (mg/dl) 144.49 ± 28.16 117.74 ± 25.48 < 0.001** apo b/a1 0.68 ± 0.21 1.01 ± 0.39 < 0.001** troponin i (pg/ml) 6.37 ± 1.78 25.62 ± 40.16 0.001** urea (mg/dl) 30.34 ± 5.86 33.90 ± 5.08 0.003** creatinine (mg/dl) 0.91 ± 0.17 1.01 ± 0.16 0.39 sugar (mg/dl) 108.40 ± 13.15 109.60 ± 14.19 0.68 *significant association p < 0.05; **high significant association p < 0.01. table 2. comparison between vitamin d3 in patient and control groups case type patient control 25(oh)d3 category < 30 37 18 92.5% 36.0% ≥ 30 3 32 7.5% 64.0% p value < 0.001** total 40 50 100% 100% **highly significant association p < 0.01. table 3. correlation between vitamin d3 and the age, bmi in patient and control groups case type n mean ± sd r p value 25(oh)d3 (ng/ml) age (year) age patient 40 16.93 ± 4.85 52.85 ± 11.38 −0.53 0.001** control 50 35.15 ± 11.79 52.72 ± 8.74 −0.47 0.001** 25(oh)d3 (ng/ml) bmi (kg/m2) bmi patient 40 16.93 ± 4.85 28.36 ± 1.89 −0.1 0.52 control 50 35.15 ± 11.79 29.55 ± 2.01 −0.42 0.002** **highly significant correlation p < 0.01. 226 j contemp med sci | vol. 3, no. 10, spring 2017: 224–228 role of vitamin d3 level and apo-b/apo-a1 ratio research fadhil jawad al-tu’ma et al. in agreement with other case-control study in india. the database contained 100 patients (chronic stable angina) and 100 controls showed the same results, which reported that 75% of the ihd cases were vitamin d3 deficient (< 20 ng/ml); while among the controls, 10% were vitamin d3 deficient. the deficiency vitamin d3 levels were highly significant with coronary artery disease, chronic stable angina (p ≤ 0.0001)16. the presented data were also in agreement with previous study in an eastern european country observed an association between low serum concentration of vitamin d3 and acs. the study included 60 acs patients and 60 matched controls without acs during a three-year follow up period. vitamin d determination was performed in all study patients. the results of patients with acs had a statistically significant mean vitamin d3 (low level of vitamin d3) as compared with control group (14.08 ng/ml vs. 23.23 ng/ml, p = 0.001).17 brøndum-jacobsen et al. and ng et al. observed the relationship between vitamin d3 and ischaemic heart disease, myocardial infraction and early death.18,19 the odds ratio of this study of risk unstable angina events was about 22 times higher in men with vitamin d3 levels < 30 ng/ml compared with those with levels ≥ 30 ng/ml (95% confidence interval 5.91–81.31; p < 0.001). this result agrees with wang et al., which concluded that vitamin d deficiency is associated with incident cvd. the study contains 1739 participants without prior cvd. vitamin d status was assessed by measuring vitamin d3 levels. prespecified thresholds were used to characterize varying degrees of vitamin d3 deficiency (< 15 ng/ml, < 10 ng/ml). during a mean follow-up of 5.4 years, 120 personal developed a first cardiovascular event. the risk of cardiovascular events was 1.62 times higher in those with vitamin d3 levels < 15 ng/ml versus the individuals with vitamin d3 ≥ 15 ng/ml (95% confidence interval 1.11 to 2.36, p = 0.01) and 1.80 (95% confidence interval 1.05 to 3.08, p = 0.01) for levels < 10 ng/ml.20 several mechanisms proposed to explain the relation between vitamin d3 deficiency and cvd is that chronic vitamin d3 deficiency causes secondary hyperparathyroidism, acting through pathogenic pathways associated with pth excess: increased pancreatic cell dysfunction and insulin resistance, predisposing to the metabolic syndrome and diabetes; increasing blood pressure (by activation of the renin angiotensin system), and leading to left ventricular hypertrophy (with subsequent apoptosis and fibrosis); and stimulation of systemic and vascular inflammation, augmenting atherogenesis.20,21 other studies suggest that parathyroid hormone has a proinflammatory effect, stimulating vascular smooth muscle cells to release cytokines.22 known risk factors for cvd, including smoking, obesity, advanced age and inactivity (reduced sun exposure), are associated with lower vitamin d3, which make the dissection of the causal role of fig. 1 correlation between vitamin d3 and the age in unstable angina group. fig. 2 correlation between vitamin d3 and the age in control group. fig. 3 correlation between vitamin d3 and body mass index in control group. in recent years, the researchers focused on vitamin d3 due to its effects on several organs such as cardiovascular system, in addition to its important role in the mineralization of bone. most studies observed that low vitamin d3 levels were associated with a high risk of cardiovascular disease.15 the results of this study showed that the concentration of vitamin d3 was highly significant in the patient group as compared with the normal control group. this results were table 4. comparison between vitamin d3 and smoking in patients and control groups parameter case type smokers n mean ± sd p value 25(oh)d3 (ng/ml) patient positive 22 14.68 ± 2.50 0.001* negative 18 19.68 ± 5.62 control positive 16 29.88 ± 4.76 0.029 negative 34 37.63 ± 13.28 *significant association p < 0.05; **highly significant association p < 0.01. fadhil jawad al-tu’ma et al. 227j contemp med sci | vol. 3, no. 10, spring 2017: 224–228 research role of vitamin d3 level and apo-b/apo-a1 ratio low vitamin d3 status in cvd difficult. finally, recent evidence has suggested that vitamin d3 may be a negative acute phase reactant; so, chronic disease may lead to low vitamin d3 even in the presymptomatic phases of cvd.23 the results of this study showed that vitamin d3 deficiency was highly significant with age in the control group, and in agreement with a previous study, which indicates that the odds for vitamin d3 deficiency increased with age, vitamin d3 level wall be less. they noticed that aging decreases the capacity of the human skin to produce vitamin d. they found that a 70-year old individual makes four times less vitamin d3 from the sun than a 20 year old personal.16 in this study, it was noticed that there a significant inverse association between vitamin d3 and bmi in the control group, increase of bmi leading to decrease vitamin d3, it’s in line with other research.24 people who are obese, a (bmi ≥ 30), are associated with lower serum vitamin d3 levels compared with non-obese individuals; people who are obese may need larger than usual intakes of vitamin d3 to achieve vitamin d3 levels comparable to those of normal weight. obesity does not affect skin’s capacity to synthesize vitamin d3, but greater amounts of subcutaneous fat sequester more of the vitamin and alter its release into the circulation.25 some other studies indicate significant association between vitamin d3 and smokers, where the smoking associated with decrease vitamin d3, as shown in this study. in this study among adults, it was observed that lower vitamin d3 levels and higher prevalence of vitamin d3 insufficiency and deficiency in the adult male subjects with unstable angina in smokers rather than nonsmoker. similar results have been seen in many other studies.26,27 studies revealed the relationship between apolipoprotein (b and a1) and coronary artery disease, which is considered a strong indicator to detect the risk of coronary artery disease.28 in this study, it was observed a highly significant association between unstable angina and apo b, apo a1 and apo b/apo a1 ratio. it was found the significant association between apo b and unstable angina when compared with the control group, the greater apo b the likelihood of increased risk of cardiovascular disease. this is in line with other study,29 and it was inversely associated significantly between apo a1 and unstable angina when compared with control group, decrease apo a1 leading to increase the risk of ischaemic heart disease. this agrees with another study.30 in addition to increase of the apo b/apo a1 ratio leading to high risk of unstable angina, these data were in agreement with other study. database contains 45 participants with cvd and 44 healthy participants. there were significant differences in apo b/apo ai ratio (p < 0.05).31 in this study, the results were not significantly associated between vitamin d3 and apo b, apo a1, apo b/apo a1 ratio. this result was in agreement with other study. it was observed that no significant association between vitamin d3 and (apo b, apo a1).32 vitamin d3 has been proposed to modulate the transcription activity of a group of genes known to be involved in lipid metabolism. in addition, it was suggested that vitamin d3 may upregulate lipoprotein lipase activity in adipocytes, and this will decrease circulating triglyceride levels.32 conclusions according to the results of the study, it can be concluded that there was a highly significant association for level of vitamin d3 between patient and control groups. there was prevalent level of low vitamin d3 among patients. there was a significant inverse correlation between vitamin d3 level and the ages, bmi in the control group. there was a significant association between vitamin d3 level and the smoking. there was a significant correlation between apo b, apo a1 levels and apo b/apo a1 ratio with each of unstable angina and control groups. there was no significant effect of vitamin d3 on apo b, apo a1 levels and apo b/apo a1 ratio. recommendation 1. a screening program for vitamin d3 might be adopted to help in the prevention and control of ischaemic heart disease. 2. further studies with larger sample size, study the effect correction of vitamin d3 deficiency on secondary prevention of ischaemic heart disease. 3. study the correlation between vitamin d3 level and atrial natriuretic peptide (anp) in ischaemic heart disease and control groups. conflict of interest none. n references 1. sandoval y, apple fs, smith sw. high-sensitivity cardiac troponin assays and unstable angina. eur heart j acute cardiovasc care. 2016 jul 7:2048872616658591. 2. yeghiazarians y, braunstein jb, askari a, stone ph. unstable angina pectoris. new england journal of medicine. 2000;342:101–14. 3. maitra a, abbas ak. the endocrine system in robbins and cotran pathologic basis of disease 7th edition. 2005 chapter 24 pp.1218–1221. edited by kumar v. abbas ak, fausto n. 4. braunwald e, antman em, beasley jw, califf rm, cheitlin md, hochman js, et al. acc/aha guideline update for the management of patients with unstable angina and non–st-segment elevation myocardial infarction—2002: summary article. circulation. 2002;106:1893–900. 5. kwon hj. vitamin d receptor signaling is required for heart development in zebrafish embryo. biochemical and biophysical research communications. 2016;470:575–8. 6. duranton f, rodriguez-ortiz me, duny y, rodriguez m, daurès jp, argilés a. vitamin d treatment and mortality in chronic kidney disease: a systematic review and meta-analysis. am j nephrol. 2013;37:239–48. 7. zittermann a, iodice s, pilz s, grant wb, bagnardi v, gandini s. vitamin d deficiency and mortality risk in the general population: a meta-analysis of prospective cohort studies. am j clin nutr. 2012;95:91–100. 8. bittner v, wenger nk, waters dd, demicco da, messig m, larosa jc. retracted: vitamin d levels do not predict cardiovascular events in statintreated patients with stable coronary disease. am heart j. 2012;164:387–393. 9. pilz s, kienreich k, tomaschitz a, lerchbaum e, meinitzer a, märz w, et al. vitamin d and cardiovascular disease: update and outlook. scand j clin lab invest. 2012;72:83–91. 10. michos ed, lutsey pl. 25-hydroxyvitamin d levels and coronary heart disease risk reclassification in hypertension—is it worth the "hype"?. atherosclerosis. 2016;245:237–9. 11. lamprea-montealegre ja, sharrett ar, matsushita k, selvin e, szklo m, astor bc. chronic kidney disease, lipids and apolipoproteins, and coronary heart disease: the aric study. atherosclerosis. 2014;234:42–6. 12. jing f, mao y, guo j, zhang z, li y, ye z, et al. the value of apolipoprotein b/apolipoprotein a1 ratio for metabolic syndrome diagnosis in a chinese population: a cross-sectional study. lipids health dis. 2014;13:81. 228 j contemp med sci | vol. 3, no. 10, spring 2017: 224–228 role of vitamin d3 level and apo-b/apo-a1 ratio research fadhil jawad al-tu’ma et al. 13. walldius g. the apob/apoa-i ratio is a strong predictor of cardiovascular risk. lipoproteins in health and diseases. 2012;95–148. 14. walldius g, jungner i. the apob/apoa‐i ratio: a strong, new risk factor for cardiovascular disease and a target for lipid‐lowering therapy–a review of the evidence. j intern med. 2006;259:493–519. 15. christakos s, dhawan p, verstuyf a, verlinden l, carmeliet g. vitamin d: metabolism, molecular mechanism of action, and pleiotropic effects. physiol rev. 2016;96:365–408. 16. ab hameed raina ms, shah za, changal kh, raina ma, bhat fa. association of low levels of vitamin d with chronic stable angina: a prospective casecontrol study. north american j med sci. 2016;8:143. 17. knežević praveček m, hadžibegović i, cvitkušić lukenda k, raguž a, dunđer i, gabaldo k, et al. vitamin d deficiency in patients with acute coronary syndrome: clinically relevant or just a bystander?. cardiologia croatica. 2015;10. 18. ng ll, sandhu jk, squire ib, davies je, jones dj. vitamin d and prognosis in acute myocardial infarction. int j cardiol. 2013;168:2341–2346. 19. brøndum-jacobsen p, benn m, jensen gb, nordestgaard bg. 25-hydroxyvitamin d levels and risk of ischemic heart disease, myocardial infarction, and early death. arterioscler thromb vasc biol. 2012;32:2794–802. 20. wang tj, pencina mj, booth sl, jacques pf, ingelsson e, lanier k, et al. vitamin d deficiency and risk of cardiovascular disease. circulation. 2008;117:503–11. 21. anderson jl, may ht, horne bd, bair tl, hall nl, carlquist jf, et al. relation of vitamin d deficiency to cardiovascular risk factors, disease status, and incident events in a general healthcare population. am j cardiol. 2010;106:963–8. 22. martín-ventura jl, ortego m, esbrit p, hernández-presa ma, ortega l, egido j. possible role of parathyroid hormone–related protein as a proinflammatory cytokine in atherosclerosis. stroke. 2003;34:1783–9. 23. ford ja, maclennan gs, avenell a, bolland m, grey a, witham m, record trial group. cardiovascular disease and vitamin d supplementation: trial analysis, systematic review, and meta-analysis. am j clin nutr. 2014;100:746–55. 24. vimaleswaran ks, berry dj, lu c, tikkanen e, pilz s, hiraki lt, et al. causal relationship between obesity and vitamin d status: bi-directional mendelian randomization analysis of multiple cohorts. plos med. 2013;10:e1001383. 25. malone m. recommended nutritional supplements for bariatric surgery patients. ann pharmacother. 2008;42:1851–8. 26. jiang cq, chan yh, xu l, jin yl, zhu t, zhang ws, et al. smoking and serum vitamin d in older chinese people: cross-sectional analysis based on the guangzhou biobank cohort study. bmj open. 2016;6:e010946. 27. shinkov a, borissova am, dakovska l, vlahov j, kassabova l, svinarov d. winter 25-hydroxyvitamin d levels in young urban adults are affected by smoking, body mass index and educational level. eur j clin nutr. 2015;69:355–60. 28. khadem-ansari mh, rasmi y, rahimi-pour a, jafarzadeh m. the association between serum apolipoprotein ai and apolipoprotein b and the severity of angiographical coronary artery disease. singapore med j. 2009;50:610. 29. mashayekhi nr, sadrnia s, chehrei a, javaheri j. the correlation between serum apoa1 and b and coronary artery disease as well as its severity. int cardiovasc res j. 2014;8:1. 30. manson je, bassuk ss. biomarkers of cardiovascular disease risk in women. metabolism. 2015;64:s33–9. 31. tamang hk, timilsina u, singh kp, shrestha s, raman rk, panta p, et al. apo b/apo ai ratio is statistically a better predictor of cardiovascular disease (cvd) than conventional lipid profile: a study from kathmandu valley, nepal. j clin diagn res. 2014;8:34–6. 32. garcía-bailo b. plasma vitamin d and biomarkers of cardiometabolic disease risk in adult canadians, 2007–2009. prevent chronic dis. 2013;10. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201705 17j contemp med sci | vol. 4, no. 1, winter 2018: 17–21 research investigating the relationship between fetus heart rate in the first trimester screening with fetus sex elham keshavarz,a elham tavakkol,a amin momeni moghaddam,a mohammad jalali,b leila molaeipoor,c ali kabird adepartment of radiology, mahdieh hospital, shahid beheshti university of medical sciences, tehran, iran. b department of cardiology, school of medicine, alborz university of medical sciences, karaj, iran. cdepartment of epidemiology, pasteur institute of iran, tehran, iran. dminimally invasive surgery research center, iran university of medical sciences, tehran, iran. correspondence to ali kabir (email: aikabir@yahoo.com). (submitted: 17 august 2017 – revised version received: 05 september 2017 – accepted: 13 november 2017 – published online: 26 march 2018) objective there is a common belief that sex of the fetus is predictable by assessing fetal heart rate (fhr) in the early weeks of pregnancy. we aimed to assess the relationship between fhr in the first trimester screening with fetus sex. methods this mixed prospective–retrospective cohort study is done on 374 fetus samples of pregnant women who referred to evaluate nuchal translucency (nt) in a hospital in tehran, iran between october 2013 and march 2015. adjusted multivariable linear regression model, receiver operating characteristic (roc) curve and youden index were used in analysis. results there were 52.9% male among fetuses. the median and interquartile range (iqr) of fhr in male and female fetus were 155.5 (152–160) and 155 (150–160) beats per minute (bpm), respectively (p = 0.213). the crude regression results showed that the average fhr of the male fetuses was 1.27 bpm higher than females but it was not statistically significant (p = 0.131). the adjusted regression results showed that fetus sex was not significantly associated with fhr in terms of similar age and mother’s gravid and parity. area under the roc curve was 0.54 (95% confidence interval: 0.48, 0.59) which shows very low fhr power to determine sex of the fetus in the first trimester of pregnancy. the best cut off point was equal to 152 pulse bpm, sensitivity and specificity were 82.81 and 30.11, respectively. conclusion the results showed that fhr is not a good factor to determine sex of the fetus. keywords fetal, first, heart rate, maternal age, pregnancy trimester, sex, ultrasonography introduction increasing gestational age, pre-pregnancy weight gain, inactivity and major changes in maternal nutrition are among important factors in increasing high-risk pregnancies in recent decades. hypertension disorders, obesity and gestational diabetes are among important risk factors that affect health of the fetus and newborn in addition to maternal health threatening.1 studies in recent decades have shown that the fetus sex during pregnancy is associated with pregnancy and childbirth complications.2 a male fetus is exposed with higher complication of catching disease and mortality during pregnancy and infant period.3 in many studies, male gender is known as an independent risk factor associated with pregnancy and childbirth complications.4,5 the male fetus is associated with a higher risk of developing gestational diabetes, preeclampsia during pregnancy, congenital anomalies and x-dependent genetic diseases.4,6,7 on the other hand, male sex is accompanied with a higher risk of preterm delivery, prolapsed umbilical cord and placenta previa.5 also the likelihood of cesarean delivery and use of assistive devices for delivery of a male is more than female fetuses. the prevalence of being underweight during birth also has been reported more in male than female infants.4,8 hence, it seems that awareness about sex of the fetus in the early weeks of pregnancy and following diagnostic, treatment and appropriate supportive interventions are effective in improving health of the fetus and newborn. on the other hand, recognizing sex of the fetus in the first trimester of pregnancy, can resolve parents prompt request to know their child’s gender and subsequently prepares them a perfect opportunity to provide cloths and other needs. various methods are used to detect fetus sex during pregnancies, which are mainly based on ultrasound techniques. using ultrasound method to determine sex of the fetus in the second and third trimester is highly efficient. however, the diagnostic power of this tool in the first trimester of pregnancy is weak and unreliable.9 there is a common belief among pregnant women and some doctors that sex of the fetus in the early weeks of pregnancy is predictable by assessing fetal heart rate (fhr). based on this view, the girls fhr is more than boys.10 however, in previous studies very contradictory results have been reported regarding the predictive power of this method.11,12 till now, few citable studies have been performed around the world and iran about the predictor role of fhr in determining fetus gender in early pregnancy.12–14 different factors are important to measure fhr that have not been mentioned in most previous studies including how to measure fhr, fhr changes, gestational age, maternal age and mother’s gravid and parity.15–17 due to the above-mentioned evidence, it seems that early detection of fetus sex in the first trimester of pregnancy can effectively reduce morbidity and mortality in fetus and newborn by providing supportive measures by doctors and parents. hence, with applying same conditions to measure fhr and considering possible confounding factors listed above, we tried to evaluate the association between fhr in the first trimester of pregnancy screening with the baby’s gender. materials and methods this is a mixed prospective–retrospective cohort study. this study is done with the approval of shahid beheshti university of medical science’s research council on sample of the fetuses of pregnant women who referred to mahdieh hospital, which is a third level referral hospital, to evaluate nuchal translucency in tehran province, iran in between october 2013 and march 2015. issn 2413-0516 18 j contemp med sci | vol. 4, no. 1, winter 2018: 17–21 fetus heart rate and sex research elham keshavarz et al. the sample size was estimated 374 fetuses considering type one error (α) equal to 0.01, mean and standard deviation of fhr 167.3 ± 10.1 in boys and 167 ± 9.1 in girls,14 and by using the formula of comparing the mean of two independent community. but with considering the loss of 20% of our cases during follow-up, 451 cases were considered. after receiving informed written consent from parents, singleton pregnant mother’s fhr who had pregnancy ultrasound between 11 and 14 weeks and 2 days of pregnancy with crown-rump length (crl) length of 45–84 mm, was measured by m-mode ultrasound (ge e6) and recorded in the first seconds of the exam when the fetus was in rest. other fetuses with structural abnormalities, the uncertainty of the exact date of pregnancy, abortion, bleeding, multiple pregnancy, congenital diseases, invalid last menstrual period (lmp), decreased amniotic fluid and uncertain sex of the fetus (with any reason in next referrals) were excluded from the study. ultrasound results were measured by different people and then to reduce bias between observers, all ultrasound results were reviewed by researchers. fetus sex was determined through a phone call or in person after the birth. other required information including week of pregnancy, maternal age, mother’s gravid and parity, abortion, bleeding during pregnancy, decreased amniotic fluid, the exact date of pregnancy (valid lmp) and congenital anomalies detected in fetus ultrasound screening were fully extracted from available records using checklist. to describe the data, descriptive statistics [frequency, median, and interquartile range (iqr)] were used. to investigate the relationship between qualitative and quantitative variables, chi-square, mann–whitney and the median test were used. to investigate the role of predictor variables in determining fhr, we used crude and adjusted multivariable linear regression model. also to eliminate confounding factor’s effect, the backward stepwise model was used. in the backward stepwise model by hosmer and lemshow method, only variables with p-value <0.2 were included in final model. for estimating fhr accuracy in predicting fetus sex in the first trimester of pregnancy, the receiver operating characteristic (roc) curve was drawn. the youden index was used for detecting the optimal cut off point. data analysis was done using stata 11.2 software (stata corp. lp) and a confidence interval level of 99%. results in this study, 451 patients were studied in which 60 cases were excluded for various reasons including abortion, bleeding, multiple pregnancy, congenital diseases and other associated conditions with exclusion criteria. also, 17 cases were excluded because of uncertainty about the newborn’s gender despite our telephone follow-up. the average fhr of 17 eliminated fetuses was not significantly different from those remained in the study (p = 0.316). finally, a total number of 374 fetuses between 11 and 14 weeks and 2 days of pregnancy were enrolled in the study according to the study criteria. from the total number of 374 samples, 198 cases (52.9%) were male. the most male and female fetuses were at 12th and 13th week of pregnancy, respectively. the median (iqr) of fhr in male and female fetuses were 155.5 (152–160) and 155 (150– 160) beats per minute (bpm), respectively. the average of mother’s ages in both male and female sexes was about 24 years with a range of 15–37 years. more than 70% of mothers of both boys (74.75%) and girls (72.73%) had one or two previous pregnancy history. chi-square test showed that variable levels including table 1. demographic and obstetric characteristics in the first trimester screening by fetal gender variables male (number = 198) female (number = 176) p-value number percentage number percentage gestational age (week) 0.072 11 20 10.10 17 9.66 12 90 45.45 90 51.14 13 84 42.42 58 32.95 14 4 2.02 11 6.25 gravidity 0.110 1 75 37.88 57 32.39 2 73 36.87 71 40.34 3 46 23.23 48 27.27 4 4 2.02 0 0.00 parity 0.154 1 108 54.55 80 45.45 2 70 35.35 81 46.02 3 20 10.10 15 8.52 maternal age (years) 0.778 15–20 39 19.70 35 19.89 21–25 80 40.40 65 36.93 26–30 55 27.78 57 32.39 30< 24 12.12 19 10.80 elham keshavarz et al. 19j contemp med sci | vol. 4, no. 1, winter 2018: 17–21 research fetus heart rate and sex weeks of pregnancy, gravid, parity and maternal age had no significant difference between both sexes (table 1). overall, the median fhr showed no significant difference between both sexes (p = 0.579). the median test showed that the median of both sex’s fhr due to different levels of age, mother’s parity and gravidity and weeks of pregnancy had no significant difference. to predict changes in fhr average, the effect of each variable including sex of the fetus, week of pregnancy, gravid, parity and maternal age were examined using crude linear regression. the result of our analysis showed that the average fhr of male fetuses was 1.27 times per minute higher compared to female fetuses but it was not statistically significant (p = 0.131, β = 1.27). age, mother’s gravid and parity did not show a significant relationship with fhr. also, the adjusted regression results showed that none of the mother’s gravid levels had significant relationship with fhr (gravid 2 and 3 compared to gravid 1 had β = −0.97 and 0.19, respectively) (table 2). mean fhr of 13th and 14th week of pregnancy was significantly 3.8 and 7.9 beats which was lower than 11th week, respectively, after adjustment for gravidity (p = 0.01 and 0.001, respectively) (table 2). predicting fetus sex in the first trimester of pregnancy by fhr was drawn by roc curve (fig. 1). for drawing roc curve, two vertical and horizontal axes were defined as sensitivity and specificity. considering youden index, the best cut off point was equal to 152 pulse bpm (sensitivity of 82.81 and specificity of 30.11). in roc curve, the auc is between 0 and 1 and whatever the number is closer to one (slope of the curve is upward and to the left), it indicates the higher strength of the fhr in predicting the baby’s gender. area under the roc curve was estimated 0.54 with a confidence interval of 0.48, 0.59 which shows very low fhr power to determine sex of the fetus in the first trimester of pregnancy. it covers 50% and shows that the probability of choosing fhr for predicting fetal sex is “50–50” and is not different with chance. discussion this study investigated the relationship between fhr in the first trimester and fetus sex.we controlled the possible confounding role of fhr changes during different periods of pregnancy in the design phase of this study. to do so, only table 2. effect of variables on fetal heart rate in the first trimester using the linear regression model variables unadjusted regression coefficient %95 ci§ p-value adjusted regression coefficient* %95 ci p-value gender female ref# male 1.27 −0.380, 2.92 0.131 gestational age (week) 11 ref# ref# 12 −2.05 −4.89, 0.78 0.156 −2.46 −5.27, 0.36 0.087 13 −3.70 −6.60, 0.80 0.013 −3.84 −6.75, −0.93 0.01 gravidity 1 ref# ref# 2 −0.44 −2.33, 1.45 0.646 0.19 −1.71, 2.10 0.841 3 −1.33 −3.43, 0.80 0.222 −0.97 −3.07, 1.12 0.362 parity 1 ref# 2 0.34 −1.41, 2.08 0.703 3 1.58 −1.35, 4.52 0.290 maternal age (years) 15–20 ref# 21–25 1.70 −0.57, 3.98 0.143 26–30 1.77 −0.62, 4.16 0.146 30< 1.46 −1.60, 4.52 0.347 §: confidence interval. fig 1. the predicted fetus sex, according to fetal heart rate in the first trimester of pregnancy by using receiver operating characteristic curve. 20 j contemp med sci | vol. 4, no. 1, winter 2018: 17–21 fetus heart rate and sex research elham keshavarz et al. embryos who had minimum amount of fhr fluctuations were studied. in other words, only fetuses between 11 and 14 weeks and 2 days of pregnancy were enrolled in the study.18 on the other hand, to eliminate confounding factor effects, such as maternal bleeding, multiple pregnancy, fetal abnormalities and other things, a total of 77 cases were excluded according to exclusion criteria. to unify data collection tool, the fhr was measured by ultrasound device in a state of immobility and to reduce bias between the observers, ultrasound results were read and checked by the researcher. also to adjust the gravid, parity and maternal age roles, the adjusted linear regression was used in analyzing data. our findings showed that despite moderating many possible factors influencing fhr (mother’s parity, gravid and age), any significant correlation was not seen between heart rate in the first trimester of pregnancy with fetal sex. in a study in 1964 on 95 fetuses with weekly measuring of the fhr during the final 2 months of pregnancy, the average of girls and boys fhr was 141.7 and 140.0 bpm, respectively. this result is consistent with ours and shows no relationship between fhr with fetal sex. moreover, this study showed that fhr pattern is different for boys and girls. so that often boy’s heart rhythm is based on linear pattern and girls one is more based on complex pattern. so it is possible to determine the sex of the fetus by fhr pattern help. however, it should be noted that this study is associated with fhr in the third trimester of pregnancy and its comparability to the first trimester is less. other limitations of this study include small sample size.19 in other study, fhr of 220 pregnant women at 14–41 weeks of pregnancy was recorded. no significant correlation between fhr and the baby’s gender was observed at any stage of pregnancy.11 this study was done in a big range of pregnancy with high fhr fluctuation and people during the first, second and third trimesters of pregnancy were entered into the study. in fact, fluctuations of the fhr were not controlled in this study. another study on 477 pregnant women with fetuses under 14 weeks, which sonography was done on them to check factors such as bleeding in the first trimester and uncertainty of the exact date of pregnancy were entered into the study. the sonography samples were repeated at 18–24 weeks by the same team and fetal sex was determined, 51% were boys. like our study, average maternal age, gravity, parity and gestational age did not have significant difference between boys and girls. boys and girls fhr mean was 154.9 ± 22.8 and 151.7 ± 22.7 bpm, respectively, without significant difference (p = 0.13). among important points in generalization a study, is to select sample in a way that can be the representative of the studied population. mckenna enrolled the people into the study who mainly did sonography due to disease or complication of pregnancy. so it seems that these people are not the representative of healthy mothers and fetuses. while in our study, only mothers who referred for routine screening ultrasound were examined and pregnancies with complications were excluded.12 also these patients entered the study under 14th week of pregnancy and it is possible that fhr be slower in 6–7 weeks of pregnancy compared with 8–14 weeks.20 our study was conducted at 11th and 14th week of pregnancy. during this period, the minimum fhr fluctuations occur in pregnancy.20 one study on 655 fetuses (female: 50.7%) in 8–13 weeks of pregnancy showed that the boys and girls fhr mean in the first trimester of pregnancy was 167 ± 9.1 and 167.3 ± 10.1 bpm (p = 0.62).14 in other similar studies, it was shown that contrary to public perception about faster fhr in girls, there was no difference between boys and girls in this point.12,21–23 up to now, the studies done about the relationship between fhr and fetal sex often had very low sample size.19,24,25 on the other hand, most of the studies are done in a big range of pregnancy and are often performed in the second and third trimester of pregnancy.11,22,24,26 therefore, fluctuations that occur in fhr in different phases of pregnancy, are not considered in many of the studies. also, lot of different tools are used to auscultate fhr in previous studies and because the accuracy of diagnostic tools and their power is not evaluated in these studies; thus, the comparison chance between studies is less.14 however, this study was done with appropriate sample size and high statistical power and with a short distance from pregnancy in 11–14 weeks and 2 days that in this period fhr has lower fluctuation.12 also, an ultrasound device with high diagnostic power is used. on the other hand, the role of important effective variables, such as maternal age, mother’s gravid and parity were adjusted in this study so it seems that the results of this study can be reliable. we were faced with some limitations in this study. some families refused to cooperate with the researcher to determine sex of the baby in the follow-up period. also, a limited number of patients’ data were collected through telephone calls that may cause recall bias. despite all these limitations, studying fhr in a period with more uniform situation, adjusting for possible confounding factors are among our strengths. moreover, although sonographies were done by different people, but all sonographies results were reviewed by the researchers. so the observer bias reduced to its minimum level. median of fhr was not different between various weeks of pregnancy. however mean fhr, adjusted for gravidity, had a decreasing trend with increase in gestational week which was not different with 12th and 11th week, but significantly different between 13th or 14th week and 11th week. however, this difference (4 or 8 bpm) was neither big nor clinically significant nor measurable by doppler sonography. so, despite the common believe that fhr has minimum fluctuations during 11th to 14th gestational week, 20 our study showed that there are some small changes during these weeks after controlling the confounder effect of gravidity. conclusion the results of this study showed that fhr is not a good predictive factor to determine sex of the fetus. due to the importance of health point of early fetal sex diagnosis in identifying disorders related to gender especially at the same time with the first trimester screening, it is recommended to pay more attention to that in next studies. and further investigation of this hypothesis should be done with taking into account other unknown variables that may be effective in relation to gender and fhr. conflict of interest none. n elham keshavarz et al. 21j contemp med sci | vol. 4, no. 1, winter 2018: 17–21 research fetus heart rate and sex this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1. bernardes j, gonçalves h, ayres-de-campos d, rocha ap. linear and complex heart rate dynamics vary with sex in relation to fetal behavioural states. early hum dev. 2008;84:433–439. 2. goncalves h, ayres-de-campos d, bernardes j (eds.). the effect of gender, gestational age and behavioral states on fetal heart rate variability, 8th conference of the european study group on cardiovascular oscillations (esgco), 2014; ieee, trento, italy. 3. saadia z, farrukh r. association between fetal sex ratio and maternal eclampsia—a descriptive study in pakistani population. internet j gynecol obstet. 2009;12. 4. graatsma m. monitoring of fetal heart rate and uterine activity, 1st ed., amsterdam; 2010. 5. odeh m, grinin v, kais m, ophir e, bornstein j. sonographic fetal sex determination. obstet gynecol surv. 2009;64:50–57. 6. ogueh o, steer p. gender does not affect fetal heart rate variation. br j obstet gynaecol. 1998;105:1312–1314. 7. dawes nw, dawes gs, redman cw. fetal heart rate patterns in term labor vary with sex, gestational age, epidural analgesia, and fetal weight. am j obstet gynecol. 1999;180:181–187. 8. hanprasertpong t, phupong v. first trimester embryonic/fetal heart rate in normal pregnant women. arch gynecol obstet. 2006;274:257–260. 9. druzin ml, hutson jm, edersheim tg. relationship of baseline fetal heart rate to gestational age and fetal sex. am j obstet gynecol. 1986;154:1102–1103. 10. barcroft j. fetal circulation and respiration. physiol rev. 1936;16:103–128. 11. genuis sk, genuis s, chang wc. antenatal fetal heart rate and “maternal intuition” as predictors of fetal sex. j reprod med. 1996;41:447–449. 12. bernard j. prediction from human fetal measures. child dev. 1964;35:1243–1248. 13. aibar l, puertas a, valverde m, carrillo mp, montoya f. fetal sex and perinatal outcomes. j perinat med. 2012;40:271–276. 14. sheiner e, levy a, katz m, hershkovitz r, leron e, mazor m. gender does matter in perinatal medicine. fetal diagn ther. 2004;19:366–369. 15. jimenez df, tarantal af. fetal gender determination in early first trimester pregnancies of rhesus monkeys (macaca mulatta) by fluorescent pcr analysis of maternal serum. j med primatol. 2003;32:315–319. 16. bracero la, seybold dj, witsberger s, rincon l, modak a, baxi lv. first trimester fetal heart rate as a predictor of newborn sex. j matern fetal neonatal med. 2016;29:803–806. 17. jones j, weerakkody y. fetal heart rate: trikeapps 2015 [updated 2015]. available from: http://radiopaedia.org/articles/fetal-heart-rate. 18. akbarzade m, rafiee b, asadi n, zare n. the effect of maternal relaxation training on reactivity of non-stress test, basal fetal heart rate, and number of fetal heart accelerations: a randomized controlled trial. int j commun based nurs midwifery. 2015;3:51. 19. di renzo gc, rosati a, sarti rd, cruciani l, cutuli am. does fetal sex affect pregnancy outcome? gender med. 2007;4:19–30. 20. lange s, van leeuwen p, geue d, hatzmann w, grönemeyer d. influence of gestational age, heart rate, gender and time of day on fetal heart rate variability. med biol eng comput. 2005;43:481–486. 21. pink? blue? you’re pregnant. now you want to know the sex of your baby 2014 [updated oct, 2014]. available from: http://www.babybpm. com/. 22. mckenna d, ventolini g, neiger r, downing c. gender-related differences in fetal heart rate during first trimester. fetal diagn ther. 2006;21:144–147. 23. petrie b, segalowitz sj. use of fetal heart rate, other perinatal and maternal factors as predictors of sex. percept mot skills. 1980;50:871–874. 24. cuestas e, bas j, pautasso j. sex differences in intraventricular hemorrhage rates among very low birth weight newborns. gender med. 2009;6:376–382. 25. dubose tj, dickey d, butschek c, porter l, hill lw, poole ek. fetal heart rate (fhr) is not an indicator of the baby’s sex. j ultrasound med. 1988;7:237. 26. naeye rl, burt ls, wright dl, blanc wa, tatter d. neonatal mortality, the male disadvantage. pediatrics. 1971;48:902–906. dx.doi.org/10.22317/jcms.03201804 145j contemp med sci | vol. 5, no. 3, may–june 2019: 145–148 original advantage of day 3 over day 2 embryo transfer hind abdulkadim* adepartment of urosurgery and infertility, college of medicine, university of kufa, najaf, iraq. bfertility center, al sader teaching hospital, najaf, iraq. *correspondence to hind abdulkadim (email: dr.hindabdulkadimd@yahoo.com). (submitted: 15 october 2018 – revised version received: 06 november 2018 – accepted: 18 february 2019 – published online: 26 june 2019) introduction working in vitro associate with stressors that present in the culture system, which are absent in vivo within the female reproductive tract. stressors evident in the embryology laboratory that have a negative influence on embryos include: frequent temperature fluctuation during manipulation, changes to the concentration of carbon dioxide with subsequent changes in ph, changes in atmospheric oxygen; and the accumulation of ammonium from amino acids.1 during early stages of embryonic life prior to activation of the embryonic genome, the embryo have only limited capacity at a molecular level to respond to a stress which can apply a major hazard on subsequent viability. in addition it is well known that the effects of a stress can be masked at the level of morphology and may only become evident at a subcellular level with the embryo having reduced metabolic activity, high rate of apoptosis, and lowering in pregnancy rate. some of these stressors are unavoidable sequel of in vitro culture even in the best ivf laboratory and earlier embryo transfer is adopted to minimize this hazard.2 to maximize the chance of pregnancy, selection of human embryos with a good developmental competence for transfer is mandatory. embryos are usually graded and selected depending on their morphology and progress rate. transfer of human embryo (et) in cleavage stage is commonly done on the 3rd day after oocyte pickup. the main reasons why et conducted when embryos are at the morula stage is that the human embryo is normally located in the endometrial cavity on days 4–5 after fertilization.3 so, the intrauterine environment is physiologically more suitable for developing morula on the 3rd day than it is on the 2nd day.4 also, extending the time of embryonic culture until activation of the embryonic genome at 4–8 cell stage might optimize the selection of viable embryos for transfer. the authors found that an extra 24 h of observation for embryonic development in vitro was possible by postponing and shifting the transfer until day 3 to recognize and discard the embryos that are developmentally arrested or retarded.5 generally, delaying et may therefore be appropriate, if embryo growth is satisfactory during in vitro culture and can increase the chance of successful implantation and facilitate the election of highest quality embryo for transfer.6 previous researches found that day 3 transfer associate with higher pregnancy and implantation rate than day 2 et.7–9 however, delaying et and keeping embryos in in vitro culture conditions could have adverse effects on embryo development and could reduce the number of viable embryos available for transfer.10 a lot of studies found an elevation in the percentage of growth-retarded embryos on day 3 and there were morphological similarities between days 2 and 3 embryos.11 in a study by laverge et al.,12 the implantation and pregnancy rates were identical between transfers done on day 2 vs. day 3; however, the overall quality score of the embryo decreased when the embryos were cultured up to day 3. further studies have shown the benefit of et in patients with poor response on day 2 compared with day 3. there have been an increase in clinical and ongoing pregnancy rates after et on day 2 than on day 3 in poor responders, indicating that the miscarriage rate can be reduced by limiting embryo culture to only 2 days which could also provide an alternative management for poor responders.13 in addition, racowsky et al.14 found that earlier embryo transfer can improve pregnancy rate for poorly progressed embryos suggesting that the in vivo environment can rescue less healthy embryos. however, the optimal day for et of human cleavage-stage embryos remains a matter of debate and some recent studies have found no difference in ivf outcome between days 2 and 3 et. materials and methods this study was a cohort retrospective study performed on infertile couples attending infertility treatment center in objectives this study was done to compare embryo quality and pregnancy rate between days 2 and 3 embryo transfer, the effect of extended culture on embryo development potential also analyzed. methods in an 18-month period extending from january 2014 to june 2015, all couples were undergoing infertility treatment in the form of intracytoplasmic sperm injection (icsi) whatever the cause and in whom fresh embryo transfer were done (258 cycle) were included in this prospective study. the patients were classified into two groups according to the day of embryo transfer. the policy of transfer was uniform in both groups and the transfer was done according to patient criteria or the number of embryos available. results the main outcome measures were embryo quality, embryo development potential and pregnancy rate. our data suggest no significant statistical difference in pregnancy rate between days 2 and 3 embryo transfer (42.9% vs. 42.0% respectively, p > 0.05). the percentage of good quality embryos was slightly insignificantly higher in day 2 group than day 3 (86.03% vs. 84.7%, p > 0.05). the percentage of slow growing embryos was significantly higher in those cultured for 3 days than those remain for just 2 days in vitro (15.9% vs. 23.3%, p < 0.05). conclusion a similar pregnancy rate was obtained by doing embryo transfer on days 2 and 3 after icsi in spite of slight regression in embryo quality and higher rate of developmental delay in cases of extended culture. so, no advantage for day 3 over day 2 embryo transfer. keywords embryo transfer, embryo quality, intracytoplasmic sperm injection issn 2413-0516 146 j contemp med sci | vol. 5, no. 3, may–june 2019: 145–148 advantage of day 3 over day 2 embryo transfer original h. abdulkadim al-sadr medical city in najaf, from january 2014 to june 2015. no written/verbal informed consent was provided from the patients. they underwent intracytoplasmic sperm injection (icsi) according to the standard protocols. demographic, clinical and laboratory informations about patients were collected. the subjects were couples undergoing icsi with fresh embryo transfer as a treatment option for various fertility problems (258 cycle) female partners was <42 years old and had normal endometrial thickness (7–12 mm) on et day, no apparent endometrial pathology and less than three failed previous cycles. controlled ovarian hyper stimulation was done either by pituitary down regulation in the early follicular phase with a gnrh agonist (decapeptyl 0.1 mcg/day) or mid follicular pituitary down regulation with a gnrh antagonist (cetrorelix, 250 mcg/day). 150–300 iu/l of recombinant fsh (gonal-f, 75 iu/l) were used to induce multiple follicle growth and the response was evaluated with serial ultrasound monitoring. when two or more follicles grow up to 17 mm or more, 10 000 iu/ml of human chorionic gonadotropin was taken as an ovulation trigger and oocytes were collected 34–36 h later through transvaginal ultrasound guided aspiration under general anesthesia. denudation of the oocytes was achieved both mechanically and enzymatically. after that all mature metaphase ii oocytes are injected with husband sperm (intracytoplasmic sperm injection). oocytes were examined for signs of fertilization 16–18 h post injection, the two pronuclei zygotes were cultured for 24–48 h in fertipro cleavage medium supplemented with 10% human serum albumin. two to three embryos at the four or eight cell stage were transferred to the uterus depending on the patient conditions and prognosis (particularly female age and previous unsuccessful attempts) on the 2nd or 3rd day after insemination. et performed using soft catheter (cook ob/gyn) on day 2 or 3 after oocyte retrieval. luteal phase support was started on the day of ovum pick up and patients used vaginal progesterone, 400 mg every 12 h. a positive pregnancy test was considered when β-hcg levels above 25 miu/ml, 2 weeks after et, fertilization rate, cleavage rate embryo quality and their developmental potential with the average number of embryos transferred were assessed. embryo grading depended on degree of fragmentation and morphology of blastomer. grades 1 and 2 embryos described as good quality embryos while grades 3 and 4 described as bad quality embryos. rate of embryo development also evaluated, embryo less than four cells on day 2 or less than eight cells on day 3 was regarded as slow growing embryos (days were calculated from time of oocyte injection). embryos with normal fertilization and of good quality (grades 1–2) were chosen for transfer. day 3 transfer was done on 1, day two fall on a holiday or 2. the patient cannot undergo day 2 transfer. embryo quality, embryo progression and pregnancy rate were compared between days 2 and 3 et. statistical analysis statistical analysis was done using statistical package for social science, version 20.0. numerical data expressed as mean ± sem. categorical data are expressed as frequencies and percentages. to compare between the parameters of two groups, independent samples, student’s t-test was used and chi-square was used for the comparison of categorical variables. the difference between the values were considered statistically significant at p < 0.05. results about 258 intracytoplasmic sperm injection cycle were included in this study. in 189 cycle embryo transfer was done in day 2 post-injection while day 3 transfer was done in 69 cycle. table 1 shows the main criteria of patients represented by their age, body mass index, duration of infertility, number of attempts, number of oocyte collected, number of mature injected oocyte and number of transferred embryos. no significant statistical difference in these parameters between days 2 and 3 groups (p > 0.05). table 2 shows fertilization rate of injected oocytes (number of zygotes/total number of injected oocytes), percentage of good quality embryos, percentage of bad quality and slow growing embryos. no significant statistical differences in fertilization rate and embryo quality between both groups (p > 0.05), while the ratio of slow growing embryos is significantly higher in group of day 3 et (p < 0.05). in table 3, pregnancy rate was calculated and compared between patients included in days 2 and 3 embryo transfer. no significant statistical difference in positive pregnancy rate was found between both groups (p > 0.05). table 1. the main criteria of patient included in the study variable day 2 (no. = 189) mean ± sem day 3 (no. = 69) mean ± sem p age (years) 29.6 ± 0.5 31.37 ± 0.8 0.07 bmi (kg/m2) 27.5 ± 5.2 27.7 ± 4.2 0.9 infertility period 7.4 ± 5.7 5.1 ± 6.6 0.3 no. of attempts 1.3 ± 0.7 1.4 ± 0.8 0.5 no. of oocyte 9.4 ± 0.4 9.04 ± 0.7 0.6 mii 7.8 ± 0.3 7.39 ± 0.6 0.5 no. of zygote 5.2 ± 0.2 4.75 ± 0.4 0.3 no. of embryo 4.9 ± 0.2 4.51 ± 0.4 0.4 transferred embryo 2.94 ± 0.81 2.77 ± 0.85 0.5 bmi, body mass index. table 2. fertilization rate, percentage of good and bad quality, and slow growing embryos variable (%) day 2 (no. = 189) mean ± sem day 3 (no. = 69) mean ± sem p fr 70.3 ± 1.6 68.5 ± 2.7 0.6 good 86.03 ± 1.8 84.7 ± 3.2 0.7 bad 14.8 ± 1.9 15.3 ± 3.2 0.9 slow growing embryos 15.9 ± 1.7 23.3 ± 4.01 0.049 table 3. pregnancy rate in day 2 vs. day 3 embryo transfer day 2, no. (%) day 3, no. (%) total p pt positive 81 (42.9) 29 (42.0) 110 (42.6) 0.9 negative 108 (57.1) 40 (58.0) 148 (57.4) total 189 (100) 69 (100) 258 (100) 147j contemp med sci | vol. 5, no. 3, may–june 2019: 145–148 original advantage of day 3 over day 2 embryo transferh. abdulkadim discussion a lot of research has documented that the implantation of the zygote was comparable to that of cleaved embryo.15,16 quinn et al.17 reported that the culture conditions used for the fertilized oocytes affected pregnancy rate. that is, in presence of suboptimal culture conditions, pregnancy rates can be increased by earlier transfer. the authors found that if culture environment is optimized, pregnancy rate would be the same after et on day 1 or 2. further studies, mentioned that embryos transferred on day 2 were comparable to day 3.5,18,19 however, in these studies, patients with good prognostic factor, fewer embryos were transferred, while for us the number of embryos transferred remained fixed. in a study done by laverge et al.,12 to compare pregnancy outcome between days 2 and 3 et, they observed that the clinical pregnancy and implantation rates was the same between days 2 and 3, but overall embryo quality scores were lower on day 3. shen et al.8 found that ongoing pregnancy rate were increased and abortion rate were decreased on day 2 et in cycles with low numbers of embryos in poor responders younger than 40 years. findings of these studies encourage earlier embryo transfer suggesting that in vivo environment will be healthier for early growing embryo. regarding our study, we found no differences in clinical outcomes regarding pregnancy rate and overall embryo quality. while, slow growing or arrested embryos were significantly higher in day 3 group. bahceci et al.4 found that pregnancy rate per transfer was significantly higher in day 2 vs. day 3 et for poor responder. a cochrane meta-analysis failed to prove any improvement in clinical outcome on day 3 transfer. shifting et from days 2 to 3, associated with an elevation in clinical pregnancy rates but due to a higher miscarriage rate with the day 3 et, the live birth rate remained the same. this may be due to unavoidable adverse effects of culture system on early stages of embryonic development causing elevation of miscarriage rate on day 3 transfer.18 however, in another study it was suggested that extended culture to day 3 may lead to selection of best quality embryos for transfer.20 in a retrospective study, dawson et al.6 showed that the pregnancy rate was insignificantly higher with day 3 et and on postponing et from days 2 to 3, 16% of embryos stopped growing. so, waiting until day 3 allowed us to recognize these growth retarded or arrested embryos and avoid their transfer thus lowering miscarriage rate. while certain studies suppose that earlier embryo transfer can rescue less healthy embryos. in our study, pregnancy rate was the same but slow growing embryos was significantly higher in day 3 group. this confirming dawson finding and suggesting that extended culture can be used as a non-invasive method to identify embryos with poor developmental potential. under most ideal conditions, the culture media could exert a negative impact on the developing embryos and led to a higher miscarriage rate with poor embryo quality. in our study, overall embryo quality was identical for both groups, which may be due to the restricted control on culture condition and laboratory environment. many studies have shown that in vitro cultured blastocysts have higher implantation rates, so may be more effective for those who produce enough number of high quality embryos at the cleavage stage, promoting the selection of the top quality embryos21,22 and improving pregnancy rate and offer the opportunity of single embryo transfer. dar et al.23 found a considerably elevated risk of preterm delivery (<37 weeks) in singletons following blastocyst culture compared with day 3 transfer. they supposed that extended culture may have adverse effects on the future placentation. the retrospective design of this study and random population are considered limitations which may have affected the outcomes. conclusion we conclude that the same outcomes was obtained in days 2 and 3 embryo transfer. so we suggest that there is no need to extend in vitro culture an extra 24 h and depending day 2 transfer as a routine instead of traditional day 3 et. this conclusion also offer to our patients the opportunity to select which day would be suitable for her to do et. further studies are recommended to confirm the influence of earlier embryo transfer on live birth and miscarriage rate in comparison with the extended culture. conflicts of interest none.  references 1. gardner dk, lane m, culture system for human embryo. in: gardner dk, weissman a, howles cm, shoham z (eds.). text book of assisted reproduction. taylor & francis group, 2018, pp. 200–210. 2. modares sz, zamaniyan n, baheiraei n, saharkhiz n, abed f, malih n, et al. a comparative analysis between day 2 and day 3 embryo transfer in ivf/ icsi: a retrospective cross-sectional study. int j women’s health reprod sci. 2016;3:119–124. 3. bastu e, celik c, keskin g, buyru f. evaluation of embryo transfer time (day 2 vs day 3) after imposed single embryo transfer legislation: when to transfer? j obstet gynaecol. 2013;33:387–390. 4. bahceci m, ulug u, ciray hn, akman ma, erden hf. efficiency of changing the embryo transfer time from day 3 to day 2 among women with poor ovarian response: a prospective randomized trial. fertil steril. 2006;86:81–85. 5. oatway c, gunby j, daya s. day three versus day two embryo transfer following in vitro fertilization or intracytoplasmic sperm injection. cochrane database syst rev. 2004:cd004378. 6. dawson kj, conaghan j, ostera gr, winston rm, hardy k. delaying transfer to the third day post-insemination, to select non-arrested embryos, increases development to the fetal heart stage. hum reprod. 1995;10:177–182. 7. carrillo aj, lane b, pridman dd, risch pp, pool tb, silverman ih, et al. improved clinical outcomes for in vitro fertilization with delay of embryo transfer from 48 to 72 hours after oocyte retrieval: use of glucoseand phosphate-free media. fertil steril. 1998;69:329–334. 8. shen s, rosen mp, dobson at, fujimoto vy, mcculloch ce, cedars mi. day 2 transfer improves pregnancy outcome in in vitro fertilization cycles with few available embryos. fertil steril. 2006;86:44–50. 9. frankfurter d, keefe dl, trimarchi jb. day 2 embryo transfer improves ivf-et outcome in the poor responder. fertil steril. 2003;80:61. 10. ashrafi m, kiani k, mirzaagha e, shabani f. the pregnancy outcomes of day 2 versus day 3 embryo transfer: a cross-sectional study. int j fertil steril. 2007;1:47–54. 11. dayal mb, frankfurter d, athanasiadis i, peak d, dubey a, gindoff pr. day 2 embryo transfer (et) and day 3 et afford similar reproductive outcomes in the poor responder. fertil steril. 2011;95:1130–1132. 12. laverge h, de sutter p, van der elst j, dhont ma. a prospective, randomized study comparing day 2 and day 3 embryo transfer in human ivf. hum reprod. 2001;16:476–480. 13. ertzeid g, dale po, tanbo t, storeng r, kjekshus e, abyholm t. clinical outcome of day 2 versus day 3 embryo transfer using serum-free 148 j contemp med sci | vol. 5, no. 3, may–june 2019: 145–148 advantage of day 3 over day 2 embryo transfer original h. abdulkadim culture media: a prospective randomized study. j assist reprod genet. 1999;16:529–534. 14. racowsky c, jackson kv, cekleniak na, fox jh, hornstein md, ginsburg es. the number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. fertil steril. 2000;73:558–564. 15. scott la, smith s. the successful use of pronuclear embryo transfers the day following oocyte retrieval. hum reprod. 1998;13:1003–1013. 16. dale b, fiorentino a, de simone ml, di matteo l, di frega as, wilding m, et al. zygote versus embryo transfer: a prospective randomized multicenter trial. j assist reprod genet. 2002;19:456–461. 17. quinn p, stone ba, marrs rp. suboptimal laboratory conditions can affect pregnancy outcome after embryo transfer on day 1 or 2 after insemination in vitro. fertil steril. 1990;53:168–170. 18. shahine lk, milki aa, westphal lm, baker vl, behr b, lathi rb. day 2 versus day 3 embryo transfer in poor responders: a prospective randomized trial. fertil steril. 2011;95:330–332. 19. de los santos mj, mercader a, galán a, albert c, romero jl, pellicer a. implantation rates after two, three, or five days of embryo culture. placenta. 2003;24:s13–s19. 20. dobson at, raja r, abeyta mj, taylor t, shen s, haqq c, et al. the unique transcriptome through day 3 of human preimplantation development. hum mol genet. 2004;13:1461–1470. 21. kaur p, swarankar ml, maheshwari m, acharya v. a comparative study between cleavage stage embryo transfer at day 3 and blastocyst stage transfer at day 5 in in-vitro fertilization/intra-cytoplasmic sperm injection on clinical pregnancy rates. j hum reprod sci. 2014;7:194–197. 22. van der auwera i, debrock s, spiessens c, afschrift h, bakelants e, meuleman c, et al. a prospective randomized study: day 2 versus day 5 embryo transfer. hum reprod. 2002;17:1507–1512. 23. dar s, librach cl, gunby j, bissonnette f, cowan l; ivf directors group of canadian fertility and andrology society. increased risk of preterm birth in singleton pregnancies after blastocyst versus day 3 embryo transfer: canadian art register (cartr) analysis. hum reprod. 2013;28:924–928. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201905 1j contemp med sci | vol. 1, no. 1, winter 2015:1–6 research objective although a huge number of experimental works may be observed in the literature for microemulsions, modelling reports on these nano-systems are rare. additionally, no comprehensive work so far has detailed the changes occurring to a micellar droplet when oil molecules are introduced (i.e. obtaining microemulsions from micelles). this work aimed to simulate a micelle and a microemulsion system containing polysorbate 80 as surfactant and study the structural changes in the two systems. methods molecular dynamics simulation employing a coarse-grained model was used. results the results showed that the oil molecules located in the hydrophobic core of the micelle make its size larger, while the co-surfactant molecules distribute throughout the system and enter the hydrophilic shell of the micelle. conclusion it was shown that insertion of oil molecules makes the droplets more spherical, while co-surfactants tend to enlarge them. keywords microemulsion, polysorbate 80, molecular dynamics, structural changes from micelle to microemulsion: an investigation of structural changes using molecular dynamics amir amania,b & milad amanic introduction “microemulsion” was first defined in 1981 as “a system of water, oil, and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution.”1 when preparing a mixture of oil and water, in the absence of surfactant(s), a twophase system is formed. by addition of a surface active agent (surfactant), the surfactant molecules move to the interface of oil and water and lower the interfacial tension. the surfactant molecules then try to trap some oil (water) molecules within their aggregates and make a nanodispersion of oil (water) in the water (oil) phase. addition of more surfactant molecules will eventually “solubilize” the whole oil (water) phase, in continuous phase.2,3 this forms the microemulsion particles. structurally, microemulsions are similar to micelle droplets (i.e. aggregates of surfactant molecules,4 while an internal phase is located within the droplet. the internal phase should be immiscible with the continuous phase. usually, in addition to the surfactant molecules, a short chain alcohol is added to the preparation to provide flexibility of interface and promote the stability of the microemulsions. such alcohols are called co-surfactants.5 it is a common practice to employ molecular simulation approaches in order to fill the gaps left by experimentalist and provide insight into dynamics and thermodynamic quantities at molecular levels. considering the literature, an ocean of experiments can be found on microemulsions. also, numerous simulation works have been reported on micelles. however, limited number of molecular simulation reports is available for microemulsions. in works by baaden et al., the role and behavior of tri-n-butyl phosphate (tbp) as a surfactant, a modifier of interface, or a solute in a mixture of water and oil was studied. the results of this molecular dynamics (md) simulation showed that hydrogen bonding at high tbp concentrations may lead to the formation of microemulsions.6,7 a microemulsion system was simulated employing mesoscale md simulations, by use of single site representation of water and oil.8 chen et al. used a similar method to investigate the structural properties of the microemulsion droplets.9 the density profile of the microemulsion beads have also been given in an oil/water/ctab system.10 more recent works have focused on the structure and flexibility of interfacial surface in aerosol ot (docusate sodium) micelles.11,12 our previous work detailed coarse grained md simulation of a micelle of polysorbate 80 along with a self-assembly study of the micelle.13 in brief, the report detailed dynamics and structural properties of the micelle as well as a single polysorbate 80 molecule. in this work, we tend to extend our work to a microemulsion system containing glyceryl tri octanoate as oil phase, ethanol as co-surfactant, and polysorbate 80 as surfactant in water, a system which has been experimentally investigated too.14 since the ingredients that have been used in this work are pharmaceutically approved, this microemulsion has the potential to be used in various delivery routes. we aim to investigate the possible structural changes occurred by introducing the oil molecules into the micelle. to do so, a micellar system with and without co-surfactant was compared with the microemulsion droplet. to avoid enlarging, detailing some parameters such as diffusion constant, which are not directly related to structural changes, have been excluded from this article. the obtained data provides insight into the structural differences between a micelle and a microemulsion droplet and may be considered as a complementary work to our previous experiments.15,16 methodology molecular model the molecular model of the polysorbate 80 molecule was considered, as detailed previously,13 with modifications adopted from mapping polyethylene glycol.17 briefly, five bead types were employed including –chn–ch2–ch2–ch2–, –chn–ch2– ch=ch–, –ch2–co–o–, –chn–chn–o–, and –chn–chn– oh for the surfactant molecule. the oil molecule consisted of a department of medical nanotechnology, school of advanced technologies in medicine, bmedical biomaterials research center, tehran university of medical sciences, tehran, iran. c department of computer science, school of mathematics, statistics, and computer sciences, university of tehran, tehran, iran. correspondence to amir amani (email: aamani@sina.tums.ac.ir). (submitted: 29 september 2014 – revised version received: 27 december 2014 – accepted: 02 january 2015 – published online: winter 2015) issn 2413-0516 2 j contemp med sci | vol. 1, no. 1, winter 2015:1–6 investigation of structural changes in micelle using md research amir amani & milad amani –chn–ch2–ch2–ch2– and –ch2–co– o– bead types and ethanol was taken as a single bead. the parameters of bonded and non-bonded interactions were taken from previous reports of martini force field.18,19 md simulations were performed by dlpoly_2.1920 in the npt ensemble, with pressure and temperature set at 1 bar and 310°k, respectively. the berendsen’s algorithm was employed with barostat and thermostat relaxation times fixed at 1.0 ps. the van der waals interactions had a cutoff value of 12.0 å and the timestep was 40fs. our previous experimental results have shown that in the most stable form, the intended microemulsion system contains 170 molecules of ethanol and 17 oil molecules per 60 molecules of polysorbate 80.14 the phase diagram of the microemulsion has also been given previously.16 to study the structural changes when moving from a micelle to a microemulsion (i.e. from having surfactant only molecules in water to having a mixture of surfactant, co-surfactant and oil molecules, dispersed in water) the following four systems were simulated: 1. a single micelle system which was previously self assembled13 with 60 surfactant molecules (i.e. aggregation number of polysorbate 8021 in 10323 water beads (mi), 2. a micelle plus co-surfactant system (i.e. random replacement of 170 water beads in mi with ethanol to yield mi-a), 3. a microemulsion system containing 60 surfactant molecules, 170 co-surfactants, and 8 oil molecules (i.e. having half the oil molecules of the ultimate micro emulsion, me-0.5), and 4. a microemulsion with 60 surfactant molecules, 170 co-surfactants, and 17 oil molecules to represent a whole droplet of microemulsion (me-whole). in other words, mi, mi-a, me-0.5, and me-whole represent systems containing surfactant/water, surfactant/co-surfactant/water, surfactant/co-surfactant/ oil/water, and surfactant/co-surfactant/ oil/water, respectively. in me-0.5 and me-whole, oil molecules were initially positioned randomly. to obtain the true simulation time, the overall time was scaled by 4, as suggested by the martini force field. the simulations were performed for 100.0 ns, of which the first 5.0 ns was taken as equilibration and the remaining was used to extract the averages. during the equilibration period, parameters such as potential energy and eccentricity of formed droplets were monitored to confirm reaching the equilibrium, beyond which no substantial change is observed in these parameters. results and discussion figures 1 and 2 illustrate variation of potential energy of the system as well as eccentricity of the assembled droplets during simulation period. as the details show, a fall in the potential energy is observed during the first ~1.0 ns, of which the first half is associated with a sharp fall, followed by a gradual decrease in the second half. it is arguable that during the first ~0.5 ns, some self assembly processes occur, while in the time range 0.5–1.0 ns, only rearrangement of surfactant/co-surfactant/oil molecules happen to obtain the most stable droplet. a similar pattern is observed for the eccentricity of the formed droplets. therefore, an approximate equilibration period of 1.0 ns appears to be sufficient for stabilizing the droplets. fig. 3 shows snapshots of the equilibrated systems under study. as shown in fig. 3a, the micelle forms a nearly spherical shape, as reported previously.13 in addition, polar groups of the micelle are laid in a hydrophilic shell, covering the hydrophobic moieties of the surfactants to form a typical micelle droplet. in fig. 3b, where co-surfactant is added to the micellar system, the ethanol molecules appear to distribute throughout the system with few diffusing to the droplet. these molecules remain in the hydrophilic parts of the surfactant molecule and may contribute to form a shell around the hydrophobic core. oil molecules then move to the centre of the droplets to form the oil-inwater microemulsion droplets and lie between the surfactant hydrophobic and hydrophilic chains (see fig. 3c, d). these findings are verified by the details given in fig. 4 and tables 1 and 2, as will be discussed later. the radii of gyration (rg) of the whole droplets as well as the hydrophobic core, consisting of hydrophobic tails of the surfactant and the oil molecules, for the four systems under study have been given in table 1. from the results, by introducing oil molecules, no fig. 1 change in potential energy of the systems during simulation time. fig. 2 eccentricity of four systems as a function of simulation time. 3j contemp med sci | vol. 1, no. 1, winter 2015:1–6 research investigation of structural changes in micelle using mdamir amani & milad amani important change in the size of the whole droplet is observed (26.59 å vs. 26.32 å). this is not consistent with the general understanding about microemulsions as they are commonly known db ca fig. 3 snapshots of the equilibrated droplets for the mi (a), mi-a (b), me-0.5 (c), and me-whole (d). the hydrophilic, hydrophobic, ethanol, and oil moieties are represented by red, black, blue, and yellow colours, respectively. a core-shell system has been formed in all the system, having oil and hydrophobic parts of surfactant in the core and hydrophilic parts in the shell, wherever applicable. distance from centre of mass (å) distance from centre of mass (å) distance from centre of mass (å) distance from centre of mass (å) a b c d fig. 4 density profile of different systems as regards with the droplets centre of mass (a) mi, (b) mi-a, (c) me-0.5, and (d) me-whole. the centre of droplets mainly consisted of oil, covered by hydrophobic parts of the surfactant. the shell mostly contains the hydrophilic parts of the surfactant and the co-surfactant, wherever applicable. table 1. radius of gyration of the whole droplet as well as the hydrophobic core in the systems under study, averaged over the trajectory whole droplet (å) hydrophobic core (å) mi 26.32 16.00 mi-a 26.55 15.87 me-0.5 26.45 16.58 me-whole 26.59 17.74 me: microemulsion; mi: micelle. table 2. the obtained ellipsoidal semiaxes (a, b, and c) and the eccentricity (e) of the systems under study a (å) b (å) c (å) <a/c> e mi 38.65 35.93 26.08 1.48 0.73 mi-a 39.22 36.25 25.92 1.51 0.75 me-0.5 39.04 34.88 27.52 1.42 0.71 me-whole 38.74 35.65 27.62 1.40 0.70 me: microemulsion; mi: micelle. as swollen micelles.22 this is probably due to fact that in a typical microemulsion, concentrations of oil and surfactant are more or less similar. thus, a considerable volume of nanoemulsion droplet is expected to be occupied by oil molecules. however, in the system understudy, the total number of oil beads is 153 which is substantially smaller than the number of surfactant beads – 1680. 4 j contemp med sci | vol. 1, no. 1, winter 2015:1–6 investigation of structural changes in micelle using md research amir amani & milad amani on the other hand, oil molecules have penetrated into the hydrophobic core and enlarged it (i.e. from 16.00 å to 17.74 å). the effect of this increase in the core size on the size of whole droplet is neutralized by stretching (i.e. thinning) the hydrophilic shell, thus, only a small change (i.e. <0.3 å) is observed in the whole droplet size. the lack of a considerable change in the radius of gyration of micelle (mi) compared with microemulsion (me-whole) has been experimentally reported in a similar system.14 additionally, comparing the rg values of mi with mi-a in table 1 shows that when ethanol molecules are introduced to the system, a small decrease in radius of gyration of the core is observed while the whole droplets becomes somewhat larger. this is due to fact that some of the co-surfactant molecules go into the hydrophilic part of the micelle (i.e. micelle shell) which leads to expanding the shell. simultaneously, the ethanol molecules “push” the hydrophobic parts of the surfactants together, making a degree of packing the core part of the micelle. using the equation r rg= 5 3/ , the radius of the droplets in each system may be estimated as 33.98, 34.28, 34.15, and 34.33 å for mi, mi-a, ne-.5, and me-whole, respectively. the deviation of the radius and rg of the micelle in current work from that of our previous report (i.e. 27.55 å) is most probably due to the modifications performed on the mapping polyethylene glycol and alkyl parts of the surfactant. to characterize the droplets shape, an ellipsoid was assumed for each droplet, having three semiaxes, a, b, and c. the semiaxes were computed using principal axes of inertia in the droplet i1 > i2 > i3 by: i m a b i m a c i m b c 1 2 2 2 2 2 3 2 2 1 5 1 5 1 5 = + = + = + ( ) ( ) ( ) where total mass of droplet is shown by m and the eccentricity of the droplets was computed by the following equation: e c a = −1 2 2 table 2 lists the shape parameters of the systems. considering the details in table 2, which lists the shape parameters of the droplets, a prolate ellipsoid is obtained for all the systems. similar shapes have been documented for micelles with different surfactant types.13,23,24 furthermore, comparing mi with mi-a systems, a slight increase in the eccentricity is observed. this shows that the ethanol molecules which have entered the hydrophilic shell have made the droplets more elongated. while, addition of oil molecules has made the droplet slightly more spherical (i.e. less eccentricity and a/c values). to investigate this effect, one may consider the packing parameter, p, as a useful approach in predicting the geometry of micelles: p v a lc = 0 , where v, a0, and lc represent the volume of hydrophobic tail, the headgroup area, and the maximum length of the tail.25 spherical micelles have p values <1/3, while non-spherical ones are characterized by higher values for p. in our previous work, a packing parameter of ~0.12 was determined for the surfactants in the micelle.13 it is arguable that when oil molecules are introduced to the system, they enter the hydrophobic core and squeeze the hydrophobic tails of the surfactants. therefore, the volume occupied by the hydrophobic tails decreases (i.e. v) and the packing parameter decreases too. consequently, a more spherical micelle may be expected from the system. similarly, ethanol molecules contribute to making smaller values for the headgroup area and larger values for p. as a result, deviation from sphere could be expected for the droplet. undoubtedly, such changes in the micelle shape may be associated with changes in the thermodynamic stability of the system26 and further studies are required to examine it. fig. 2 shows the average density profiles of the systems. as described previously,13 the effect of the deviation of the droplets shape from sphere was ignored. in our previous work, the density profiles of the following moieties were reported for the polysorbate 80: the hydrophobic tails, the core parts of the molecule, the terminal polar droplets having hydroxyl groups, and the hydrophilic parts. in this work, to make the comparison between the four systems easier, the molecular structure of polysorbate 80 was divided into two distinct groups: a hydrophilic part (containing –ch2–co–o–, –chn–chn–o–, and –chn–chn–oh) and a hydrophobic part (having –chn–ch2–ch2– ch2– and –chn–ch2–ch=ch–). therefore, the density distribution of hydrophobic part, hydrophilic part, oil, and ethanol were computed. from fig. 4a, the core of the micelle consists merely of hydrophobic beads and the hydrophilic particles come into sight at ~8 å from the centre of mass. comparing the data from fig. 4a and b, no considerable change in the distribution of surfactant droplets may be observed by the addition of ethanol molecules. the closest ethanol molecules to the centre of mass lie ~18 å from centre. their density then starts to increase to some extent and a peak in concentration appears to be ~32 å off centre. this peak could be due to interactions between the ethanol molecules and the hydrophilic parts of the polysorbate 80, especially the beads having hydroxyl groups. the interaction level for these beads with ethanol is defined as level ii in the forcefield,19 showing strong attractions between them, probably due to the existence of hydrogen bonds. remembering the important effect of co-surfactants in forming microemulsion droplets, this peak could be associated with forming stable microemulsion droplets. moving on to the fig. 4c and d, the oil molecules appear to push the hydrophobic parts and locate into the centre of the droplet. their density then starts to decrease and the oil molecules disappear at ~25 å and ~27 å from centre of mass for the me-0.5 and me-whole, respectively. interestingly, at 12-15 å from centre, a transient increase in the density of oil is observed, at which the density of oil and hydrophilic parts of the polysorbate 80 are more or less equal. considering the two bead types present in the oil, the alkyl group (i.e. –chn–ch2–ch2–ch2–) may not be the cause as the group has a strong hydrophobic nature, thus, cannot make suitable interactions with the hydrophilic parts of the surfactant. we believe that the increase in concentration of oil is due to its –ch2–co–o– bead types which could make suitable interactions with the hydrophilic parts of the surfactant (i.e. interaction level iv). 5j contemp med sci | vol. 1, no. 1, winter 2015:1–6 research investigation of structural changes in micelle using mdamir amani & milad amani furthermore, by adding oil to the system, the ethanol molecules are driven from the droplet: they start to show up at ~21å from centre of mass in me-0.5 and me-whole, compared with ~18å in mi-a. moreover, the radial distribution function, g(r), of each of the moieties was computed and reported in fig. 5. as the details show, the hydration shells do not appear to be different in the four systems, showing the importance of the fig. 5 averaged radial distribution functions for the four systems with water beads. shells, which are composed of surfactant molecules, in interaction with water molecules. conclusion the md simulations of a nano-system containing polysorbate 80 as surfactant with or without ethanol (i.e. co-surfactant) and oil were performed in this study to identify the possible structural changes. in general, oil molecules tend to move to the central region of the droplets, thus, making the hydrophobic core of the droplets larger. interestingly, insertion of oil/ethanol does not influence the overall size substantially. it was also found that addition of oil molecules make the droplets more spherical, while ethanol molecules deviate the aggregate from a spherical shape. acknowledgment the study has been funded by tehran university of medical sciences grant number 88-01-87-8624.  references 1. danielsson i, lindman b. the definition of microemulsion. colloids surf. 1981 dec;3(4):391–2. doi: http://dx.doi.org/10.1016/0166-6622(81)80064-9 2. lawrence mj, rees gd. microemulsion-based media as novel drug delivery systems. adv drug deliv rev. 2000 dec 6;45(1):89–121. doi: http://dx.doi. org/10.1016/s0169-409x(00)00103-4 pmid: 11104900 3. tenjarla s. microemulsions: an overview and pharmaceutical applications. crit rev ther drug carrier syst. 1999;16(5):461–521. doi http://dx.doi. org/10.1615/critrevtherdrugcarriersyst.v16.i5.20 pmid: 10635455 4. aulton me. aulton’s pharmaceutics: the design and manufacture of medicines. edinburgh: churchill livingstone; 2007. 5. bagwe rp, kanicky jr, palla bj, patanjali pk, shah do. improved drug delivery using microemulsions: rationale, recent progress, and new horizons. crit rev ther drug carrier syst. 2001;18(1):77–140. doi: http:// dx.doi.org/10.1615/critrevtherdrugcarriersyst.v18.i1.20 6. baaden m, burgard m, wipff g. tbp at the water−oil interface: the effect of tbp concentration and water acidity investigated by molecular dynamics simulations. j phys chem b. 2001 nov;105(45):11131–41. doi: http://dx.doi. org/10.1021/jp011890n 7. baaden m, schurhammer r, wipff g. molecular dynamics study of the uranyl extraction by tri-n-butylphosphate (tbp): demixing of water/”oil”/ tbp solutions with a comparison of supercritical co 2 and chloroform. j phys chem b. 2002 jan;106(2):434–41. doi: http://dx.doi.org/10.1021/jp0123785 8. bearchell ca, heyes dm. mesoscale modelling studies of microemulsions. phys chem chem phys. 2001 nov 26;3(23):5255–65. doi: http://dx.doi. org/10.1039/b105096f 9. chen z, cheng x, cui h, cheng p, wang h. dissipative particle dynamics simulation of the phase behavior and microstructure of ctab/octane/1butanol/water microemulsion. colloids surf a physicochem eng asp. 2007 jul;301(1–2):437–43. doi: http://dx.doi.org/10.1016/j.colsurfa.2007.01.022 10. li y, guo y, bao m, gao x. investigation of interfacial and structural properties of ctab at the oil/water interface using dissipative particle dynamics simulations. j colloid interface sci. 2011 sept;361(2):573–80. doi: http://dx.doi.org/10.1016/j.jcis.2011.05.078 11. pieniazek pa, lin y-s, chowdhary j, ladanyi bm, skinner jl. vibrational spectroscopy and dynamics of water confined inside reverse micelles. j phys chem b. 2009 nov 12;113(45):15017–28. doi: http://dx.doi. org/10.1021/jp906784t 12. nevidimov av, razumov vf. molecular dynamics simulations of aot reverse micelles’ self-assembly. mol phys. 2009 oct 20;107(20):2169–80. doi: http:// dx.doi.org/10.1080/00268970903203736 13. amani a, york p, de waard h, anwar j. molecular dynamics simulation of a polysorbate 80 micelle in water. soft matter. 2011;7(6):2900–8. doi: http:// dx.doi.org/10.1039/c0sm00965b 14. amani a. design, characterisation and in vitro aerosolisation performance of nanoemulsion for nebulisation of hydrophobic drugs (phd thesis). university of bradford; 2008. 15. amani a, york p, chrystyn h, clark bj. factors affecting the stability of nanoemulsions—use of artificial neural networks. pharm res. 2010 jan;27(1):37–45. doi: http://dx.doi.org/10.1007/s11095-009-0004-2 pmid: 19908130 16. amani a, york p, chrystyn h, clark bj, do dq. determination of factors controlling the particle size in nanoemulsions using artificial neural networks. eur j pharm sci. 2008 sep 2;35(1–2):42–51. doi: http://dx.doi. org/10.1016/j.ejps.2008.06.002 pmid: 18617002 17. lee h, de vries ah, marrink sj, pastor rw. a coarse-grained model for polyethylene oxide and polyethylene glycol: conformation and hydrodynamics. j phys chem b. 2009 oct 8;113(40):13186–94. doi: http://dx.doi.org/10.1021/jp9058966 pmid: 19754083 18. lee h, pastor rw. coarse-grained model for pegylated lipids: effect of pegylation on the size and shape of self-assembled structures. j phys chem b. 2011 jun 23;15(24):7830–7. doi: http://dx.doi.org/10.1021/jp2020148 pmid: 21618987 19. marrink sj, risselada hj, yefimov s, tieleman dp, de vries ah. the martini force field: coarse grained model for biomolecular simulations. j phys chem b. 2007 jul 12;111(27):7812–24. doi: http://dx.doi.org/10.1021/ jp071097fpmid: 17569554 20. forester tr, smith w. dl_poly_2.0-a general-purpose parallel molecular dynamics package. j mol graph. 1996 jun;14(3):136–41. doi: http://dx.doi. org/10.1016/s0263-7855(96)00043-4 pmid: 8901641 21. de campo, l, yaghmur a, garti n, leser me, folmer b, glatter o. fivecomponent food-grade microemulsions: structural characterization by sans. j colloid interface sci. 2004 jun 1;274(1):251–67. doi: http://dx.doi. org/10.1016/j.jcis.2004.02.027 pmid: 15120300 6 j contemp med sci | vol. 1, no. 1, winter 2015:1–6 investigation of structural changes in micelle using md research amir amani & milad amani 22. zulauf m, eicke hf. inverted micelles and microemulsions in the ternary system water/aerosol-ot/isooctane as studied by photon correlation spectroscopy. j phys chem. 1979 feb;83(4):480–6. doi: http://dx.doi. org/10.1021/j100467a011 23. abel s, sterpone f, bandyopadhyay s, marchi m. molecular modeling and simulations of aot-water reverse micelles in isooctane: structural and dynamic properties. j phys chem b. 2004 dec;108(50):19458–466. doi: http://dx.doi.org/10.1021/jp047138e 24. senapati s, keiper js, desimone jm, wignall gd, melnichenko yb, frielinghaus h, berkowitz ml. structure of phosphate fluorosurfactant based reverse micelles in supercritical carbon dioxide. langmuir. 2002 oct;18(20):7371–6. doi: http://dx.doi.org/10.1021/la025952s 25. tanford c. micelle shape and size. j phys chem. 1972 oct;76(21):3020–4. doi: http://dx.doi.org/10.1021/j100665a018 26. leitmannova liu a. advances in planar lipid bilayers and liposomes. london: academic press; 2011. 51j contemp med sci | vol. 4, no. 1, winter 2018: 51–54 research pmsg and hcg hormones effect on the development and growth of ovarian follicles masoumeh majidi zolbin,a sepideh nematian,b faezeh daghigh,c fariba namdar,d soosan tak zaree,e mahmoodreza madadian,f mahdi abbasi,a seyed mohammad hosein noori mogahi,a and nasrin takzareea adepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. bdepartment of obstetrics and gynecology, yale school of medicine, new haven, usa. cdepartment of physiology, school of medicine, tabriz university of medical sciences, tabriz, iran. ddepartment of physiology, school of medicine, baqiyatollah university of medical sciences, tehran, iran. edepartment of obstetrics and gynecology, iran university of medical sciences, tehran, iran. fpharmaceutical sciences branch, islamic azad university, tehran, iran. correspondence to nasrin takzaree (email: takzaree@gmail.com). (submitted: 07 september 2017 – revised version received: 21 october 2017 – accepted: 01 november 2017 – published online: 26 march 2018) objective gonadotropins are generally used in animals and humans to stimulate ovulation and increase the number of available oocytes for techniques such as in vitro fertilization. ovulation-inducing drugs are used to achieve multiple oocytes in one cycle, thereby increasing the chances of pregnancy in patients with infertility. the aim of this study was to evaluate the effect of gonadotropins on stimulation of ovulation and their inductive role on the growth and development of follicles in the ovary. methods to determine the effect of human chronic gonadotrophin (hcg) and pregnant mare serum gonadotrophin (pmsg) on ovary, this study used 20 rats that were randomly divided to experimental and control groups. rats were subsequently euthanized 48 h after injection and the ovaries were fixed and stained with h&e. data were analyzed with tukey’s multiple comparisons with one-way anova test in graphpad prism software. result our analysis demonstrated that hcg and pmsg hormones will significantly increase the number of stimulated oocyte in the ovary but it does not have any significant role on the ovary weight and volume (p < 0.05). conclusion this study’s results confirmed the inductive role of pmsg and hcg hormones on folliculogenesis. keywords hcg, follicle, ovulation, pmsg introduction ovulation-stimulating medications are used to increase the chances of pregnancy in patients with infertility by achieving multiple oocytes in one cycle.1 in animals and humans, there is an emerging use of gonadotropin to induce ovulation and increase the number of oocytes accessible for research and developmental reproduction techniques such as in vitro fertilization (ivf). in individuals with poor ovarian response, it is essential to administer ovulation-stimulating drugs to get an adequate amount of oocytes in one cycle, leading to enhanced fertility. full competence of oocytes to develop the term is acquired depending on the proper timing of hormonal activation. the decrease in follicle numbers because of failure ensures aberrant hormonal regulation as a result of incomplete feedback mechanisms. this hormonal dysregulation will, in turn, accelerate the loss of follicles at the advanced age.2 the aim of this study was to increase the number of oocyte in each cycle.3,4 ovulation is one of the most fundamental processes for successful reproduction. induction of ovulation would expand the number of healthy follicles, and extracting oocytes is one of the most important reproductive tools that can be used to produce embryo in laboratory from eggs and sperm.5,6 maturation and resultant release of oocytes from the ovary are directly influenced by secreted hormones from the anterior pituitary specifically the effects of lh and fsh on the ovary. gonadotropins are commonly used to stimulate the ovary and increase ovulation induction in human and animal fertilization programs. in laboratory methods, pregnant mare serum gonadotrophin (pmsg) and human chronic gonadotrophin (hcg) can induce similar effects on ovarian hormones as well as triggering a new era of ovulation and stimulation of reproductive behaviors. in the ovulation stimulation process, ovarian steroid hormones (estrogen and progesterone) are secreted more physiologically and can have varied effects on different cells with hormones receptor in one period.7,8 follicle growth and ovulation are under the control of fsh and lh, which are secreted directly from the adenohypophysis and prompted by releasing hormones (gnrh) from the hypothalamus. in the early stages of the natural cycle, the secretion of gnrh results in a greater release of fsh and lower levels of lh from the pituitary, which will incite follicular growth.9 disruption in any of the above sections results in infertility. nowadays, various methods are used to remedy infertility worldwide. the use of gonadotropins is a well-known method for increasing the number of follicles and ovulation in animals and humans.10 worthington’s experiences showed that the use of pmsg during 6-week intervals will increase the weight of the uterus, ovary, stimulating the conversion of follicles to the corpus luteum and increased the number of follicles.11 the application of pmsg in immature rats increases ovulation and prevents the atresia of periantral and antral follicles.12 wang examined the effects of lh and fsh on the growth of ovarian follicles in the mice that had previous pituitary gland removal. they found that fsh increases the number of antral and periantral follicles, while lh increases the primary and secondary follicles.13 another study by popova et al.14 on immature rats found that pmsg and fsh had similar effects issn 2413-0516 52 j contemp med sci | vol. 4, no. 1, winter 2018: 51–54 pmsg and hcg hormones effect research masoumeh majidi zolbin et al. on ovarian stimulation.14 the objective of this method is to stimulate folliculogenesis and increase the number of oocytes in a cycle. following hormonal stimulation of ovulation, the levels of ovarian hormones, especially estradiol and progesterone, extend beyond physiological limits. increasing ovarian hormones can have various effects on reproductive organs and fetal development. in line with the importance of ovarian stimulation in infertility treatment, most hmg and r-fsh hormones are used to stimulate ovarian follicles and increase their counts. as well as considering the effect of these hormones on in vivo these studies did not occur to figure out the development ovarian follicles due to legal and ethical barriers.7 in existing study, the influences of these two hormones were evaluated on follicle development of rat’s ovary. materials and methods animals this experimental study was performed on 20 adult female wistar rats weighing 200–220 g and ranging from 2 to 3 months of age. they were placed in individual cages during the study period, with 12 h of darkness and 12 h of light available. rats had access to water and food ad libitum. the housing temperature range was kept between 25 and 30°c. the mice were kept at the animal facility center for 2 weeks to adapt to the new environment prior to the study. ovulation stimulation in ovaries to stimulate the ovulation, rats were randomly divided into two major groups: control, experimental. pmsg and hcg (sigma) were used to induce ovulation. in experimental rats group 100 iu/kg pmsg and 48 h later 100 iu/kg hcg intraperitoneally were injected then 100 iu pbs were injected to the control group.14 histological study after completion of treatment, animals were sacrificed by inhalation of ether. ovarian tissue samples were collected and excess fat tissues were removed from the ovary. their size was measured and recorded by a stereomicroscope (olympus, tokyo, japan) and weighed by a digital scale (sartorius, germany). subsequently, the number of primary follicles, secondary follicles, growing follicles, grown mature follicles and primary antral follicles were investigated in intermediate sections. to avoid any errors in counting, image j software (digital image processing for medical applications image j 1.46 r, java 1.6.0-20, cambridge university) was used. all of the above items were also performed for control group samples. statistical analysis to compare the percentage of produced follicle in stimulated ovary vs. control group data were analyzed by tukey’s multiple comparisons in one-way anova test. also, each type of follicle compared with control group with student t-test. all statistical tests were executed on graphpad prism and p < 0.05 were considered as significant. results histologic study of ovarian follicle in both group demonstrate that the number of follicles in the stimulated group with pmsg and hcg hormones are significantly higher than the control group (p < 0.05) (table 1). there was no significant difference in weight and volume of both treated and control group’s ovaries (p > 0.05) (figs. 1–3). discussion gonadotropins application is an approach for increasing the number of follicles and ovulation in animals and humans. in the present study, the effect of hcg and pmsg on the growth and development of ovarian follicles in adult female rats was assessed. the aim of this study was to evaluate the effects of gonadotropins on ovulation induction. there was a significant difference in the number of follicles in the experimental and control groups secondary to ovarian stimulation (p < 0.05) as shown in table 1. fig 1. the average number of follicle in hcg and pmsg stimulated group compared to control group. table 1. histological comparison of developed follicle in experimental group tukey’s multiple comparisons test mean diff. 95.00% ci of diff. significant? summary p-value primary follicle vs. secondary follicle −3.7 −5.561 to −1.839 yes **** <0.0001 primary follicle vs. vesicular follicle −5.6 −7.461 to −3.739 yes **** <0.0001 primary follicle vs. mature follicle −18.8 −20.66 to −16.94 yes **** <0.0001 primary follicle vs. atretic follicle −21.4 −23.26 to −19.54 yes **** <0.0001 secondary follicle vs. vesicular follicle −1.9 −3.761 to −0.03915 yes * 0.0433 secondary follicle vs. mature follicle −15.1 −16.96 to −13.24 yes **** <0.0001 secondary follicle vs. atretic follicle −17.7 −19.56 to −15.84 yes **** <0.0001 vesicular follicle vs. mature follicle −13.2 −15.06 to −11.34 yes **** <0.0001 vesicular follicle vs. atretic follicle −15.8 −17.66 to −13.94 yes **** <0.0001 mature follicle vs. atretic follicle −2.6 −4.461 to −0.7391 yes ** 0.0023 masoumeh majidi zolbin et al. 53j contemp med sci | vol. 4, no. 1, winter 2018: 51–54 research pmsg and hcg hormones effect treatment with gonadotropin is the common way for improving the number of follicle and ovulation stimulation.10,16,17 the primary goal of this method is to stimulate folliculogenesis and increase the number of oocytes in a cycle. following hormonal stimulation of ovulation, the levels of ovarian secretion hormones, especially estradiol and progesterone, go beyond physiological levels. increasing ovarian hormones can have different effects on reproductive organs and fetal development. angiogenesis is associated with follicular development and is regulated independently within each follicle potentially making the function of its vasculature critically important in determining its fate. ovarian follicles require an adequate blood supply of oxygen, nutrients and hormones, in addition to eliminate co2 and other metabolites. acquisition of an adequate vascular supply is probably a limiting step in the selection and maturation of the dominant follicle. the degeneration of the capillary network is a relevant phenomenon that causes follicular atresia through the interruption of the metabolic supply for follicular cells. in addition, an increase in vascular density around antral follicles contributes to the inhibition of atresia.15 the somatic cells of the follicles (granulosa and cumulus cells) recognize ovulation stimulation, leading to the resumption of miosis and the release of oocytes, as well as the change in follicular structure. ovulation is controlled by several factors including endocrine hormones, metabolic signals, vasculogenesis, angiogenesis and intrafollicular paracrine factors that are secreted by theca, cumulus granulosa cells, and the oocyte itself. increasing evidence suggests that physiological angiogenesis in ovarian follicles and corpus luteum are fundamental features of mammalian reproduction. failures in vascular development in these structures may be the reason for several ovarian dysfunctions observed during the estrous cycle and pregnancy. therefore, it is necessary to evaluate both in vivo and in vitro influences of the angiogenic factors, alone or in association, on the survival (anti-apoptotic effects) of ovarian cells in different species.16 maturation and oocyte release from the ovary are directly influenced by the secreted hormone from the anterior lobe of pituitary gland. this results from lh and fsh secretion and follicle development in the ovary. one of the signs for ovarian function is dependent on the establishment and continual remodeling of a complex vascular system. the controlled, physiological angiogenesis that accompanies folliculogenesis, ovulation and luteal development requires the coordinated activity of multiple cells and hormone interaction and different angiogenic factors.16,17 our studies finding confirms that follicle development is accompanied by blood vessel growth to provide nutrient for growing follicle and move them toward maturation.17 gonadotropins are commonly used to stimulate the ovary as well as increase and induce ovulation in animals and humans in order. this brings about the increase in number of oocytes and the chance of getting pregnant. injection of these hormones in adult mice can trigger a new round of ovulation and stimulate animal reproductive behaviors.18,19 in the present study, the effect of hcg and pmsg hormones on the weight, growth and development of ovarian follicles in female rats were investigated. the results of this study showed an increase in the size of the ovary in the hcg group and a small increase in ovarian weight in the pmsg group, which was not significant. granulosa cells have receptor for gnrh, these hormones can have an immediate effect on the hypothalamic-pituitary gonadal axis, as well as the binding to granulosa cells receptors, which our data are reasonable with this explanation. however, the results of wang reported ovarian weight gain, also they mentioned that fsh plays an important role in regulating estradiol secretion and antral follicles growth.20 fsh in combination with lh stimulates the growth of follicles before ovulation. indeed, the direct response to increased lh during ovulation is related to granulosa cells are due to its more receptors than cumulus cells. fig 3. histological section of blood vessel distribution in rat ovary in control and experimental group (hcg and pmsg). a and b (10×, 40×) showing natural development of blood vessel in ovaries stroma but with hormonal stimulation of ovary the number of follicle in different stages will increase and these progressive growth demands higher number of blood vessel to provide nutrient in the stroma and close to newly formed follicle. vasculogenesis is a positive sign for follicle regeneration (c and d) 10×, 40×. blood vessel is demonstrated by arrow head in each section. a b dc fig 2. histological section of ovary in control and experimental group (hcg, pmsg). follicle development in the ovary of control group (a) (4×), (b) (10×). follicle development photomicrograph in the treated group with hcg and pmsg. (c) (4×), (d) (10×). a b dc 54 j contemp med sci | vol. 4, no. 1, winter 2018: 51–54 pmsg and hcg hormones effect research masoumeh majidi zolbin et al. conclusion the results of this study showed that stimulation of ovulation with pmsg and hcg can increase the number of follicles and decrease the size of graph and corpus luteum follicles. as the follicle continues to develop, endothelial cells are recruited to the thecal layer from the blood vessels in the adjacent ovarian stroma. hormonal stimulation in ovary will increase the number of follicles and follicular growth also associated with the development of an individual capillary network and continued angiogenesis to nourish the rapidly expanding follicle. n references 1. kim ss, soules mr, battaglia de. follicular development, ovulation, and corpus luteum formation in cryopreserved human ovarian tissue after xenotransplantation. fertil steril. 2002;78:77–82. 2. shanbhag s, aucott l, bhattacharya s, hamilton ma, mctavish ar. interventions for ‘poor responders’ to controlled ovarian hyperstimulation (coh) in in-vitro fertilisation (ivf). cochrane database syst rev. 2007;20:cd004379. 3. fauser bc, devroey p. reproductive biology and ivf: ovarian stimulation and luteal phase consequences. trends endocrinol metab. 2003;14:236–242. 4. tanbo t, dale po, lunde o, moe n, abyholm t. obstetric outcome in singleton pregnancies after assisted reproduction. obstet gynecol. 1995;86:188–192. 5. uslu b, dioguardi cc, haynes m, miao dq, kurus m, hoffman g, et al. quantifying growing versus non-growing ovarian follicles in the mouse. j ovarian res. 2017;10:3. 6. ertzeid g, storeng r. the impact of ovarian stimulation on implantation and fetal development in mice. hum reprod. 2001;16:221–225. 7. wei s, gong z, guo h, zhang t, ma z. fsh and ecg impact follicles development and expression of ovarian fshr and caspase-9 in mice. iran j vet res. 2017;18:79–85. 8. van der auwera i, d’hooghe t. superovulation of female mice delays embryonic and fetal development. hum reprod. 2001;16:1237–1243. 9. fritz ma, speroff l. clinical gynecologic endocrinology and infertility; wolters kluwer health/lippincott williams & wilkins, 2011. 10. wide l, bakos o. more basic forms of both human follicle-stimulating hormone and luteinizing hormone in serum at midcycle compared with the follicular or luteal phase. j clin endocrinol metab. 1993;76:885–889. 11. worthington ca, kennedy j. ovarian response to exogenous hormones in six-week-old lambs. aust j biol sci. 1979;32:91–96. 12. braw rh, tsafriri a. effect of pmsg on follicular atresia in the immature rat ovary. j reprod fertil. 1980;59:267–272. 13. wang x-n, greenwald gs. hypophysectomy of the cyclic mouse. ii. effects of follicle-stimulating hormone (fsh) and luteinizing hormone on folliculogenesis, fsh and human chorionic gonadotropin receptors, and steroidogenesis. biol reprod. 1993;48:595–605. 14. popova e, krivokharchenko a, ganten d, bader m. comparison between pmsg‐and fsh‐induced superovulation for the generation of transgenic rats. mol reprod dev. 2002;63:177–182. 15. kon h, tohei a, hokao r, shinoda m. estrous cycle stage-independent treatment of pmsg and hcg can induce superovulation in adult wistarimamichi rats. exp anim. 2005;54:185–187. 16. lamraoui r, afri-bouzebda f, bouzebda z, frank m, gherissi de. effect of repeated administration of hcg on ovarian response in pmsg superovulated ouled djellal ewes (algeria). j tropicultura. 2014;32:10–15. 17. mester b, ritter lj, pitman jl, bibby ah, gilchrist rb, mcnatty kp, et al. oocyte expression, secretion and somatic cell interaction of mouse bone morphogenetic protein 15 during the peri-ovulatory period. reprod fertil dev. 2015;27:801–811. 18. daghash h, et al. impacts of gnrh, pmsg and hcg treatments on follicular diameter, conception and lambing rates of egyptian ewe lambs using intravaginal sponges. egypt j sheep goat sci. 2016;11:132. 19. stone bj, steele kh, fath-goodin a. a rapid and effective nonsurgical artificial insemination protocol using the nset™ device for sperm transfer in mice without anesthesia. transgenic res. 2015;24:775–781. 20. hannon pr, flaws ja. the effects of phthalates on the ovary. front endocrinol. 2015;6:8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.03201811 224 j contemp med sci | vol. 4, no. 4, july-august 2019: 224–226 comparison of the effect of dexmedmotidine and ketamine on controlling pain after cesarean section via intra-peritoneal method alireza kamali,1 maryam maktabi,2 zoha khademi,1 and taha fereidooni1 1department of anesthesiology and critical care, arak university of medical sciences, arak, iran. 2department of gynecology, arak university of medical sciences, arak, iran. correspondence to maryam maktabi (email: dralirezakamalianesthesiology@gmail.com). (submitted: 26 april 2019 – revised version received: 12 may 2019 – accepted: 23 june 2019 – published online: 26 august 2019) objective the present study aimed to compare the effect of dexmedmotidine and ketamine on controlling pain after cesarean section via intra-peritoneal method. methods in this clinical and double-blind clinical trial, patients were randomly divided into two groups (dexmedmotidine and ketamine). in the first group, 5 mg/kg ketamine and 1 mg/kg dexmedetomidine were injected in 100 mg normal saline. pain score was measured on the basis of the visual analog pain scale during the recovery at 4, 6 and 12 h after the surgery. the data were then analyzed by spss (version 20). results a total of 70 patients participated in the study. the results showed that the mean pain scores were the same in different postoperative hours in patients (p ≥ 0.05). the mean opioid use in the ketamine group was lower than inter-peritoneal dexmedmotidine (p = 0.03). moreover, the mean postoperative analgesia in the ketamine group was higher than inter-peritoneal dexmedmotidine (p = 0.04). conclusion according to the results, the mean opioid consumed in the ketamine group was less than inter-peritoneal dexmedmotidine. additionally, the mean postoperative analgesia in the ketamine group was higher than that of inter-peritoneal dimethomidine. therefore, it can be concluded that ketamine has a better effect on reducing pain after cesarean section. keywords intra-peritoneal, dexmedmotidine, cesarean section, ketamine, pain control introduction pain is a completely mental experience, which produces adverse hemodynamic and metabolic responses in patients.1,2 millions of people around the world undergo surgery and then experience post-surgical pain. postoperative pain leads to harmful effects, such as atleticas, thrombosis, ischemic myocardium, cardiac arrhythmias, water and electrolyte disorders, urinary retention and ileus.3,4 one of the most commonly used surgical procedures is cesarean section. caesarean section is used in cases where delivery is impossible or if there is a risk to the mother and the baby. this procedure has played an important role in reducing maternal and fetal deaths and complications over the last century.5,6 cesarean section is performed in two ways, including general anesthesia or regional anesthesia.7 one of the problems of cesarean section is acute postoperative pain that causes unpleasant psychological responses such as anxiety, sadness, aggression, insomnia, and lack of logical connection with the physician and nurse. it may also reduce breastfeeding and mother’s tolerance for breastfeeding.8,9 one of the most important issues in cesarean section is to reduce postoperative pain. by reducing the pain, mothers will be able to perform their motherhood duties well, leading to earlier and more appropriate lactation. opioid drugs such as morphine and pethidine, which are the most commonly used drugs to relieve postoperative pain, are associated with unpleasant complications such as addiction, respiratory arrest, resistance, nausea, vomiting, etc. therefore, the use of alternative opioid drugs to prevent postoperative pain has always been pursued by surgeons and researchers.10 genealogy is one of the widely used methods that reduces the effect of opioid drugs on the fetus.11 first identified in 1951 by griffin, intra-perotone anesthesia is a new method for reducing postoperative pain. various studies have focused on pain relief after laparoscopy, hysterectomy, and laparoscopic surgery via intra-peritoneal method. the systemic effect of this method begins two minutes after the intraperitoneal injection.12 various adjuvants can be used, including dexmedmotidine and ketamine. ketamine is an nmda receptor antagonist and an anesthetic drug.13 ketamine is also an intravenous anesthetic with an analgesic effect which stimulates the cardiovascular system with minimum respiratory suppression.14 dexmedmotidine is analgesic, sedative, and antihypertensive.15 dexmedmotidine and ketamine alone increase the duration of analgesia and reduce the use of narcotics. on the other hand, the intra-peritoneal method during childbirth has a significant effect on the health of the fetus because it reduces the likelihood of anesthetic drug transfer from mother to fetus. given that all studies conducted on abdominal surgery have only investigated the effect of a single drug, we aimed to compare the effect of dexmedmotidine and ketamine on controlling pain after cesarean section via intra-peritoneal method. materials and methods this double-blind clinical trial was carried out on all patients who underwent cesarean section. all candidates for cesarean section referred to taleghani hospital in arak, iran. the candidates for cesarean section who entered the study were randomly divided into two groups (dexmedmotidine and ketamine). inclusion criteria 1. aged 18–35 years. 2. asa (american society of anesthesiologists). 3. female candidates for cesarean section. issn 2413-0516 original a. kamali et al. 225j contemp med sci | vol. 4, no. 4, july-august 2019: 224–226 comparison of the effect of dexmedmotidine and ketamine exclusion criteria 1. having a history of allergy to dexmedmotidine and ketamine. 2. having heart and respiratory disease. 3. emergency cesarean section. at first, all patients provided informed consent. then, 5 mg/kg ketamine and 1 mg/kg dexmedmotidine were injected in the ketamine and dexmedmotidine group, respectively. in both groups, the normal saline solution mixed with drugs was injected into the patient’s peritoneum. the pain was measured according to the visual analog scale (vas) at 4, 6 and 12 h after the operation. in this scale, zero expresses the lowest value and the 10 represents the highest value. it should be noted that the data were measured and recorded by a gynecologist who was unaware of the groupings. the drugs in each group were prepared by an anesthetist. the sample size and number were calculated as follows: n z z = + æ è çç ö ø ÷÷ + + = 1 2 1 2 2 2 70 a b d d m m ( ) ( ) 1 2 1 2 patients were divided into two groups (n = 35). data analysis the data were analyzed using spss (version 20), while descriptive statistics and t-test were used to analyze parametric and nonparametric data. ethical considerations 1. obtaining a letter of introduction from the university’s authorities to be introduced to the research centers. 2. obtaining a letter of introduction from the authorities of the researcher centers. 3. the purpose of the study was described for all research units and written consent was obtained from them. results a total of 70 patients were included in the study, who were classified into two groups. they were evaluated in terms of mean age. according to the results, the mean age in the dexmedmotidine group and ketamine group was 34.4 ± 3.1 and 35.1 ± 4.7 years, respectively (p = 0.6). there was no significant difference between the two groups in terms of mean age. it can be said that the mean age of patients was similar in the two groups. table 1 shows the comparison of pain scores between the two groups. given the fact that p < 0.05, there was no significant difference between the groups in scores of pain 4, 6, and 12 h after the recovery. the mean pain score was almost similar in patients at different hours after the surgery. according to table 2, there was a significant difference between the two groups in terms of drug abuse in 12 h after the surgery (p = 0.03), indicating that the mean drug in the ketamine group was lower than that of dexmedmotidine. in table 3, the mean duration of analgesia was evaluated and p = 0.04 was significant in two both. this indicates that the table 1 comparison of pain scores group dexmedmotidine ketamine p pain mean ± sd mean ± sd vas 0.0 ± 0.0 0.0 ± 0.0 ≥0.05 vas 4 h after the surgery 1.1 ± 0.65 0.98 ± 0.23 0.2 vas 6 h after the surgery 1.6 ± 3.4 1.7 ± 3.1 0.4 vas 12 h after the surgery 1.1 ± 3.7 0.98 ± 3.8 0.6 table 2. comparison of drug abuse 12 h after the surgery group dexmedmotidine ketamine p variable mean ± sd mean ± sd drug use 12 h after the surgery (mg) 35 ± 4.4 20.4 ± 3 0.03 table 3. comparison of the mean postoperative analgesia group dexmedmotidine ketamine p variable mean ± sd mean ± sd postoperative mean pain (h) 12.6 ± 2.8 5.1 ± 13.3 0.04 duration of analgesia in the ketamine group was greater than that of dexmedmotidine. discussion cesarean section refers to the removal of the embryo from the abdominal wall and the uterus. this is done to ensure the health of the mother and the baby. 16 cesarean section is characterized by acute post-operative pain. effective management of post-operative pain is part of the surgical process and involves a multifaceted approach in which different drugs are used with different mechanisms and prescriptions. the administration of non-opioid analgesics is an essential component of multi-faceted pain management programs.17 therefore, the present study aimed to investigate the effect of dexmedmotidine and ketamine, using intra-peritoneal method, in order to control postoperative pain in patients who underwent cesarean section. in this study, there was no significant difference between the two groups in terms of the pain scores at different hours after surgery (p ≥ 0.05). furthermore, the mean pain scores were similar in patients at different hours after surgery. in this study, there was a significant difference between the two groups in terms of drug abuse 12 h after the surgery (p = 0.03). moreover, the mean opioid use in the ketamine group was lower than that of dexmedmotidine. similarly, shariat moharari et al. investigated the effect of ketamine and bupivacaine on analgesia after laparoscopic cholecystectomy. shariat moharari et al. found that the duration of extubation in the ketamine group was higher. moreover, pain was significantly lower in the ketamine group 6 h after surgery. the total amount of meperidine consumed within the 24-h period was lower in the ketamine group. in addition, the drug demand decreased. there was an increase in the duration of extubation.18 in this study, there was a significant difference between the two groups (dexmedmotidine and ketamine) in terms of mean postoperative anesthesia. moreover, the mean postoperative original 226 j contemp med sci | vol. 4, no. 4, july-august 2019: 224–226 comparison of the effect of dexmedmotidine and ketamine a. kamali et al. analgesia in the ketamine group was significantly higher than that of dexmedmotidine (p = 0.04). oza et al. aimed at comparing the effects of intraperitoneal anesthesia of bupivacaine and dexmedmotidine only after laparoscopic surgery. oza et al. investigated the duration of analgesia in the dexmedmotidine group. they found that the rate of intake of analgesics in the dexmedmotidine and bupivacaine was 1.76 and 2.56, respectively. this difference was significant (p ˂ 0.05). the mean analgesia was lower in the 24-h period in the dexmedmotidine group (p-value of −0.05). there was a significant difference the two groups in terms of pain up to 12 h after the surgery, and the pain was lower in the dexmedmotidine group (p < 0.05). bupivacaine and dexmedmotidine increased the duration of analgesia. they also reduced the number of applications for postoperative analgesia.19 chiruvella et al. used dexmedmotidine and rupivacaine to manage the postoperative pain after hysterectomy. the pain was lower in the rupivacaine and dexmedmotidine groups. the duration of analgesia was higher in this group. they stated that interferon dexmedotomidine can reduce pain and drug use.20 their results are not consistent with those of our study. in our study, the mean duration of analgesia was lower in the intraperitoneal ketamine group. in our study, ketamine and dexmedmotidine were given intraperitoneally. conclusion according to the results, the mean opioid use in the ketamine group was lower than that of dexmedmotidine. moreover, the mean postoperative analgesia in the ketamine group was higher than that of dexmedmotidine. therefore, it can be concluded that ketamine has a better effect on pain relief after cesarean section. conflicts of interest none.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1 shang ab, gan tj. optimising postoperative pain management in the ambulatory patient. drugs 2003;6:855–867. 2 agah m, dabbagh a, hashemi m. evaluation of the effect of intravenous magnesium on acute postoperative pain in elective orthopedic surgeries of the lower limb. pajoohandeh 2006;1:149–152. 3 al-mustafa mm, abu-halaweh sa, aloweidi as, murshidi mm, ammari ba, awwad zm, et al. effect of dexmedetomidine added to spinal bupivacaine for urological procedures. saudi med j. 2009;30:365–370. 4 festin mr, laopaiboon m, eriksson c, rubertsson c, astrid nystedt a, hildingsson i. cesarean section. birt. 2011;4:12–18. 5 eltzschig hk, lieberman es, camann wr. regional anesthesia and analgesia for labor and delivery. n engl j med. 2003;34:319–332. 6 ghadamgahi hb. prevalence and causes of cesarean in semnan. arch j. 2007;4:52–58. 7 wallace dh, leveno kj, cunningham fg, giesecke ah, shearer ve, sidawi je. randomized comparison of general and regional anesthesia for cesarean delivery in pregnancies complicated by severe preeclampisia. obsetet gynecol. 1995;8:193–199. 8 mcvey jd, tobias jd. dexmedetomidine and ketamine for sedation during spinal anesthesia in children. j clin. 2010;22:538–545. 9 gramke hf, de rijke jm, van kleef m, raps f, kessels ag, peters ml, et al. the prevalence of postoperative pain in a cross-sectional group of patients after day case surgery in a university hospital. clin j pain. 2007;23:543–548. 10 madineh h, rajaei esfahani m, ghaherei h, akhlaghi m, gangi f. the effect of intravenous low dose ketamine on postoperative pain. j shahrekord univ med sci. 2005;7:29–34. 11 khan m, raza r, zafar s, shamim f, raza sa, pal km, et al. intraperitoneal lignocain versus bupivacain after laparoscopic cholecystectomy. j surg res. 2012;178:62–69. 12 kahokehr a. intraperitoneal local anesthetic for postoperative pain. saudi j anaesth. 2013;7:55–60. 13 argiriadou h, himmelseher s, papagiannopoulou p, georgiou m, kanakoudis f, giala m, et al. improvement of pain treatment after major abdominal surgery by intravenous s+-ketamine. anesth analg. 2004;98:1413–1418. 14 selim mf, elnabtity am, hasan am. comparative evaluation of epidural bupivacaine dexmedetomidine and bupivacaine -fentanyl on doppler velocimetry of uterine and umbilical arteries during labor. j prenat med. 2012;6:47–54. 15 kamali a, azadfar r, pazuki s, shokrpour m. comparison of dexmedetomidine and fentanyl as an adjuvant to lidocaine 5% for spinal anesthesia in women candidate for elective caesarean. open access maced j med sci. 2018;6:1862–1867. 16 martin tc, bell p, ogunbiyi i. comparison of general anesthesia and spinal anesthesia for cesarean section in antigua and barbuda. west indian med j. 2007;56:330–333. 17 shohani m, rasoli m, maleki f. comparison study of pain after cesarean section by general and spinal anesthesia. sci j ilam univ med sci. 2012;21:17–25. 18 shariat moharari r, hadavi m, pourfakhr p, najafi a, etezadi f, khajavi mr. evaluation of the postoperative analgesic efficacy of intraperitoneal ketamine compared with bupivacain in laparascopic cholecystectomy. arch anesth crit care 2016;2:146–149. 19 oza vp, parmar v, badheka j, nanavati ds, taur p, rajyaguru am. comparative study of postoperative analgesic effect of intraperitoneal instillation of dexmedetomidine with bupivacaine and bupivacaine alone after laparoscopic surgery. j minim access surg. 2016;12:260–264. 20 chiruvella s, nallam s. interaperitoneal insillation of ropivacain plus dexmedetomidine for pain relief after laparoscopic hysterectomy: a comparison with ropivacaine alone. j dr ntr univ health sci. 2016;5:93–97. dx.doi.org/10.22317/jcms.08201908 original 202 j contemp med sci | vol. 4, no. 4, july-august 2019: 202–207 prevalence of dental trauma in 5to 6-, 12-, and 15-year-old children in iran muna al-shuhayeb,1 dara ghaznavi,2 hoda parandeh shirvan,1 ghasemianpour m.,3 and khoshnevisan m.h.3,4 1department of periodontics, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 2department of periodontics, dental faculty, tabriz university of medical sciences, tabriz, iran. 3preventive dentistry research center, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran. 4dental public health phd program, community oral health department, school of dentistry, shahid beheshti university of medical sciences (sbmu), tehran, iran. correspondence to khoshnevisan mh (email: mh.khoshnevisan@sbmu.ac.ir). (submitted: 26 may 2019 – revised version received: 09 june 2019 – accepted: 23 june 2019 – published online: 26 august 2019) objectives this study assessed the prevalence of dental trauma among the 5to 6-, 12and 15-year-old iranian children. age, sex and place of residence of children with dental trauma and the correlation between parental level of education and prevalence of dental trauma were also evaluated. materials and methods in this descriptive, cross-sectional study, we used the 2012-national survey data on oral health, which included a sample of iranian individuals recruited from all provinces of iran. a cluster random sample of 26,000 children in three age groups were included in this study. spss software was used for statistical analysis after quality control and data clean up. results the overall prevalence of dental trauma was 4.1% for the total sample. this value was 5.4% in boys and 3.1% in girls. the most common age of occurrence was 15 years (6%) followed by 12 (4.9%) and 5–6 years (1.5%) of age. the highest percentage of dental trauma in 12and 15-year-old children was noted in “kohkiloye and boyerahmad” province. higher level of parental education was associated with lower frequency of trauma in all age groups. the prevalence of dental trauma was lower among those residing in rural compared with urban areas. conclusion this study demonstrated relatively high prevalence of dental traumatic injuries among 5to 15-year-old iranian children (4.1%). based on the reported associations, more effort is necessary to educate all parents and children on trauma prevention with priority in urban areas. keywords dental trauma, prevalence, education, oral health, iran introduction usually dental trauma occurs as a result of falls or various kinds of accidents. the maxillary anterior teeth are most vulnerable to dental trauma. the prevalence of dental trauma in boys is often twice the rate in girls due to their higher outdoor and sport activities.1 dental trauma may vary in severity from a small enamel crack to complete avulsion of involved teeth. in addition to pain and bleeding, dental trauma may result in tooth loss and create an unaesthetic facial appearance, which can negatively affect the children and their social interactions, and can leave psychological sequel.2 dental trauma has a higher incidence rate in children with attention-deficit hyperactivity disorder. based on a review study by glendor et al.3 most adhd patients are predisposed to dental trauma when they are between 8 and 17 years of age. several classifications have been proposed for dental trauma based on its causes.4 oral and dental problems in children may adversely affect their growth and development as well as their quality of life.5 given the importance of oral health-related quality of life in these children, and high prevalence of dental trauma in these age groups, attempts must be made to prevent and control the occurrence of dental trauma in young children.6,7 evidence shows that dental trauma accounts for 5% of all traumas. it has also been reported that the risk of dental trauma is the highest before the age of 12 years with the annual incidence rate of 18/1000 population.2,8 oral and dental trauma has been considered the third most common type of trauma.2,8 the prevalence of dental trauma widely varies in different countries and in different age groups. previous studies on this topic were not large-scale and therefore were not suitable for national decision making and strategy planning. also, different methodologies and different classifications have been used in previous studies which highlight the need for largescale, comprehensive studies to determine the national prevalence of dental trauma and related factors in iran. therefore, this study aimed to assess the prevalence of dental trauma by using a large representative national sample of iranian children of 5–6, 12 and 15 years old recruited from all provinces of iran. this epidemiological study provides baseline information regarding the prevalence of dental trauma and selected factors involved. such information is highly valuable for strategic planning to control dental trauma. materials and methods a world health organization (who) designed and recommended questionnaire (2013) for children were used for data collection.9 this questionnaire was translated to persian language and standardized prior to use. a dental team (dentist and hygienist) was calibrated prior to perform oral examinations of children and completing the questionnaires. a calibrated dentist performed the oral examination and calibrated dental hygienists assisted the dentist in data entry.10 the national survey proposal was approved by the ethics committee of the research institute of dental sciences, school of dentistry, shahid beheshti university of medical sciences with the reference number: (inohs-2012). after obtaining informed consent from parents, a sample of 5to 6-, 12and 15-year-old children was recruited for this study. the exclusion criteria were oral and systemic diseases and any other issn 2413-0516 original m. al-shuhayeb et al. 203j contemp med sci | vol. 4, no. 4, july-august 2019: 202–207 prevalence of dental trauma in 5to 6-, 12-, and 15-year-old children in iran condition that could compromise the patient cooperation, unwillingness for participation in the study and children with non-iranian ethnic background. children were selected by cluster random sampling of urban and rural, high and low socioeconomic classes of both sexes, in order to have a representative sample at the provisional national level. the sample size was calculated according to the who protocol. a total of 20 clusters (n = 15) from each province (n = 300) were recruited for each age group (5–6, 12 and 15 years) yielding a total sample of 28,800 children at the national level. this study was performed in accordance with the methodology suggested by the who.9 a standard protocol was used for clinical examination and data collection. the clinical examination was carried out by calibrated dentists and recorded by calibrated hygienists. the interview data were collected and recorded by calibrated hygienists as well.9 the questionnaire was translated to persian language and standardized prior to its administration. the collected data on oral health status of subjects were analyzed after proper quality control and data clean up using spss version 22.0 (spss inc., il, usa). the absolute and relative frequency of dental trauma in different age groups were reported for each province. chi-square test was used to compare urban/rural and male to female ratios in each age group of children with dental trauma. the mann–whitney and chisquare linear-by-linear association test were used to compare the level of education in parents and age distribution of subjects with and without dental trauma. descriptive statistics on distribution of age, sex and place of residence of patients with dental trauma and the correlation between the parental level of education and the prevalence of dental trauma were also evaluated. results a total of 28,800 subjects from 31 provinces of iran were recruited for this study. after elimination of incomplete questionnaires, a total of 26,407 subjects remained in the study. table 1 shows the frequency distribution of subjects from each province. table 2 shows the frequency distribution of males/ females and urban/rural areas. as demonstrated in table 2, the male/female distribution was almost the same in all three age groups. in relation to place of residence, about 60% of the sample were living in urban areas and about 40% were living in rural areas for all age groups. table 3 presents the percentage of subjects with dental trauma in different provinces with notable variations. the national prevalence of dental trauma was 4.1%. the percentage of dental trauma in all three age groups was higher than the national mean in 13 provinces. the overall percentage of dental trauma was 1.5%, 4.9% and 6% in 5to 6-, 12and 15-year-old children, respectively. the comparison of dental trauma at the provincial level revealed that the highest prevalence of dental trauma was reported from “kohkiloye and boyerahmad” province (7.6%) followed by “fars” province (7%). the highest percentage of dental trauma in the entire country was observed in 15-year-old age group (6%), while the lowest percentage was reported for 5to 6-year-old (1.5%) children. the mean prevalence at the national level for 12-year-old children was 4.9%. based on these data, 13 provinces had a higher prevalence of dental trauma than the national mean value. the national mean was 6% in 15-year-old children. a higher mean prevalence rate of dental trauma in 15-year-old children was shown in 13 provinces when compared with the national mean value. the highest prevalence of dental trauma in 15-year-old children was noted in “kohkiloye and boyerahmad” province (11.2%). the prevalence of dental trauma in 5to 6-year-old children was 1.5%. also, 10 provinces had a higher prevalence rate than the mean national value in this age group. the highest number of 5to 6-year-old children with dental trauma was noted in “fars” province (3.4%) while the lowest prevalence was detected in “west azerbaijan” (0%) province. the national mean prevalence of dental trauma in 12-year-old children was 4.9%. the mean was higher than the national mean value for 12-year-old children in 13 provinces. the highest prevalence of dental trauma in 12-year-old children was noted in “kohkiloye and boyerahmad” (10.4%) while the lowest prevalence was noted in “kermanshah” province (1.3%). the lowest prevalence was noted in “east azerbaijan” province (1.0%). table 4 shows the distribution of children with dental trauma by sex. the prevalence of dental trauma was higher among boys (5.2%) when compared with girls (3.1%) of the same age with 68:1 ratio (p < 0.0001). the results showed that urban living 5to 6-year-old children experienced significantly higher dental trauma than rural living counterparts (1.7% vs. 1.2%, p = 0.045). also, 5to 6-year-old boys had significantly higher prevalence of dental trauma than girls of the same age group (1.91% vs. 0.99%, p < 0.0003). the national prevalence of dental trauma in 12-year-old children was 4.9%. the urban living 12-year-old children had higher prevalence of dental trauma than rural living counterparts (5.1% vs. 4.6%, p = 0.28). also, the prevalence of dental trauma in 12-year-old boys was significantly higher than girls of the same age group (6.45% vs. 3.39%, p < 0.0001). the urban living 15-year-old children had a higher prevalence of dental trauma in comparison with rural living 15-year-old children (6.1% vs. 5.7%, p = 0.35). also, the prevalence of dental trauma in 15-year-old boys was significantly higher than that of 15-year-old girls (7.37% vs. 4.58%, p < 0.0001). as demonstrated in table 5, the prevalence of dental trauma decreased significantly as parental level of education increased (p = 0.06). likewise, by increase in the maternal level of education, the prevalence of dental trauma decreased significantly (p = 0.01). the prevalence of dental trauma significantly decreased (p = 0.03), when considering the highest level of education for either parents (table 5). regarding the prevalence of different types of trauma, the results showed that enamel fracture had the highest prevalence (58.4%) followed by enamel and dentin fracture (19.4%) and pulp involvement (5.2%). discussion this epidemiological investigation provides the latest prevalence data on who recommended three age groups of iranian children (5-6, 12, and 15 years) using a large representative sample. a total of 26,407 children were recruited at the national level. the overall prevalence of dental trauma was 4.1% for the three age groups. this rate was reported to be 8% in 7–14 years old in a study conducted by faghih nasiri et al.11 in 1995–1996. the rate reported by ansari and mobini12 in 1996–1998 was 8.96% in patients presenting to a pediatric department in tehran. our reported value for the prevalence of dental trauma in tehran was original 204 j contemp med sci | vol. 4, no. 4, july-august 2019: 202–207 prevalence of dental trauma in 5to 6-, 12-, and 15-year-old children in iran m. al-shuhayeb et al. table 1 frequency distribution of subjects recruited from each province province age group total 5–6 years old 12 years old 15 years old ardebil 280 289 290 859 isfahan 301 299 300 900 ilam 293 287 289 869 east azarbaijan 301 300 299 900 west azarbaijan 285 264 233 782 alborz 300 300 300 900 boushehr 297 299 297 893 tehran* 593 569 569 1731 chaharmahal and bakhtiari 301 297 300 898 south khorasan 275 281 267 823 khorasan razavi 300 296 299 895 north khorasan 304 297 297 898 khouzestan 286 299 275 880 zanjan 299 288 291 878 semnan 304 251 279 834 sistan and balouchestan 231 210 209 650 fars 294 284 296 874 qazvin 297 296 292 885 qom 194 172 147 513 kordestan 296 292 284 872 kerman 247 250 250 747 kermanshah 301 299 291 891 kohkiloye and boyerahmad 301 297 299 897 golestan 271 262 224 757 gilan 194 182 190 566 lorestan 304 304 298 906 mazandaran 251 243 249 743 markazi 298 300 294 892 hormozgan 165 157 176 498 hamadan 299 300 300 899 yazd 300 297 300 897 total 8962 8761 8684 26407 *sample size in tehran province was considered double the rate in other provinces due to its population size. table 2 frequency distribution of male and female participants age groups gender place of residence total male (%) female (%) urban (%) rural (%) 5–6 years 4549 (50.8) 4413 (49.2) 5608 (62.6) 3354 (37.4) 8962 12 years 4403 (50.3) 4358 (49.7) 5402 (61.7) 3359 (38.3) 8761 15 years 4079 (47.0) 4605 (53.0) 5448 (62.7) 3236 (37.3) 8684 total 13031 13376 16458 9949 26407 original m. al-shuhayeb et al. 205j contemp med sci | vol. 4, no. 4, july-august 2019: 202–207 prevalence of dental trauma in 5to 6-, 12-, and 15-year-old children in iran table 3 percentage of subjects with dental trauma in different provinces province 5–6 years old 12 years old 15 years old percentage of children with dental trauma† mean number of traumatized teeth percentage of children with dental trauma† mean number of traumatized teeth percentage of children with dental trauma mean number of traumatized teeth ardebil 0.7 1.00 3.5 1.00 5.6 1.07 isfahan 1.3 1.25 4.7 1.29 4.3 1.13 ilam 2.0 1.33 1.7 1.25 1.4 1.46 east azarbaijan 0.7 1.00 2.7 1.14 1.0 1.32 west azarbaijan 0.0 – 1.9 2.50 2.6 1.03 alborz 1.7 1.40 5.3 1.56 7.0 1.27 boushehr 1.3 3.00 3.7 1.30 5.2 1.24 tehran* 2.4 1.64 6.5 1.32 10.8 1.13 chaharmahal and bakhtiari 1.3 1.00 7.1 1.24 9.1 1.50 south khorasan 1.5 2.25 3.2 1.33 4.9 1.22 khorasan razavi 1.0 0.67 2.7 1.20 4.3 1.21 north khorasan 0.7 1.50 7.7 1.30 8.1 1.20 khouzestan 1.7 1.20 5.0 1.57 8.8 1.49 zanjan 1.4 1.50 5.9 1.29 3.5 1.29 semnan 1.0 1.00 2.9 1.14 2.6 1.14 sistan and balouchestan 1.7 1.75 1.9 1.25 6.7 1.37 fars 3.4 1.70 8.3 1.30 9.5 1.17 qazvin 2.4 1.29 5.2 1.36 4.2 1.33 qom 1.0 2.50 4.7 1.43 8.3 1.39 kordestan 1.0 7.50 2.4 1.17 3.5 1.58 kerman 1.6 1.33 5.2 1.33 10.8 1.20 kermanshah 0.3 1.00 1.3 1.00 1.7 0.82 kohkiloye and boyerahmad 1.3 1.25 10.4 1.13 11.2 1.07 golestan 1.5 1.00 5.0 1.31 2.7 1.49 gilan 1.0 2.50 2.8 1.20 8.8 1.22 lorestan 1.3 1.00 7.9 1.38 6.4 1.20 mazandaran 0.8 1.00 2.9 1.29 2.8 2.22 markazi 2.7 2.13 5.4 1.31 8.1 1.44 hormozgan 1.8 1.00 1.9 1.00 4.1 0.63 hamadan 1.4 1.67 8.4 1.12 6.1 1.30 yazd 3.0 1.33 9.1 1.15 7.7 1.26 total 1.50 1.60 4.9 1.29 6.0 1.2 table 4 distribution of males and females with dental trauma based on their place of residence in different age groups age group gender place of residence total (%) urban (%) rural (%) 5–6 years male 2.2 (61) 1.5 (26) 1.91 (87) female 1.3 (36) 0.9 (14) 1.13 (50) 12 years male 7.1 (194) 5.3 (90) 6.45 (284) female 3.1 (83) 3.9 (65) 3.39 (148) 15 years male 7.3 (194) 7.5 (107) 7.37 (301) female 5.1 (141) 4.2 (76) 4.71 (217) total male 5.47 (449) 4.61 (223) 5.2 (672) female 3 (254) 2.91 (149) 3.1 (415) original 206 j contemp med sci | vol. 4, no. 4, july-august 2019: 202–207 prevalence of dental trauma in 5to 6-, 12-, and 15-year-old children in iran m. al-shuhayeb et al. table 5 level of education of parents of subjects with dental trauma level of education elementary school or illiterate, n (%) middle school and high school, n (%) college or university education, n (%) father 4.9 (344) 4.5 (392) 4.1 (122) mother 4.9 (437) 4.4 (353) 3.7 (77) highest level of education for either parents 4.9 (299) 4.6 (431) 3.9 (145) 6.5%, which is lower than the rate reported by ansari and mobini,12 which may be related to the population type. one being the pediatric department which is a referral center for children with specific problems, and the current study was a population based study on mostly disease-free subjects. the prevalence of dental trauma was reported to be 34% in north ireland in 1998.13 21% in brazil in 200914 and 10.2% in india in 2014.15 these values are all higher than the rate reported in the current investigation. this controversy may be due to methodologic differences, sampling distribution and sample size, geographical size and location or differences in trauma classifications, diagnoses as well as participants’ age groups. moreover, cultural, social, geographical, educational and behavioral differences of individuals living in different communities may play an important role in prevalence of dental trauma. for instance, aside from accidents, participation of children in specific high-risk sports such as american football, rugby, etc. may be higher in some countries resulting in different rate of traumatic injuries due to level of engagement in such sports activities.16 it has been reported that age 10 is a common period for occurrence of the most dental traumas.11,12 however, marcenes et al.5 from syria reported the highest prevalence of trauma (11.7%) at age 12. al‐majed et al.17 in saudi arabia reported the highest rate of trauma in 12to 14-year-old (34%) children. differences in the reported rates are due to using different age groups in addition to the level of involvement and participation of children in different sport activities in different countries.1 although faghih nasiri et al.11 from iran, traebert et al.7 from brazil and marcenec et al.5 from syria reported equal or found no difference in prevalence of trauma in the two genders, the rate of dental trauma in our study was significantly higher (5.4%) in boys when compared with girls (3.1%). this is in line with the percentages reported by ansari and mobini12 with 60% dental trauma in boys and 40% in girls. asnaashari et al.18 reported the 2.1:1 ratio for the prevalence of trauma in boys to girls. also, afshar and mozaffari19 reported that the prevalence of trauma in boys was 3.4 times the rate in girls. navabazam and farahani20 reported that the prevalence of trauma in boys was 5.1 times the rate in girls. a report from taiwan shows the prevalence data 1.4:1 on trauma in boys compared with girls in 1999.21 in another study from brazil, the prevalence of trauma in boys was higher than that in girls in 2009.14 chopra et al.15 from india reported that the prevalence of trauma in boys was higher than that in girls. the higher prevalence of dental trauma in boys was probably due to their greater participation in either sport activities, fighting or accidents. the prevalence of dental trauma in boys is often twice the rate in girls due to their higher activities.1 adolescent boys are more susceptible to trauma than girls due to their increased outdoor activities. cultural differences in different countries may also explain the prevalence rates of dental trauma in girls. for example, in iran, girls are less involved in outdoor sport activities, which can decrease the rate of dental trauma in them. some studies have shown increasing prevalence of dental trauma in girls compared with previous years. for instance, burden22 reported that at present, girls are more interested in sports and outdoor activities; thus, the difference in prevalence rates of dental trauma in boys and girls is decreasing. however, it remains for other countries to report such differences. given the high prevalence of dental trauma, extensive educational programs are recommended at the national level to enhance the public knowledge regarding the risks of dental trauma and its adverse consequences. this can be done by the media and internet as well. these programs should also include proper management of injured patients. also, continuing education courses on this topic are required to provide up-to-date information for dental clinicians, which is imperative to improve the children’s health status and quality of life, increase their satisfaction rate and decrease treatment costs by appropriate management. conclusion the prevalence of dental trauma was relatively high in the three selected age groups. there should be a priority to enhance public knowledge to better prevent and control this condition. media as well as educational programs of other types involving parents, children and school teachers can be instrumental in decreasing the prevalence of dental trauma at the local, national, and international levels. conflicts of interest none. ■ references 1. bastone eb, freer tj, mcnamara jr. epidemiology of dental trauma: a review of the literature. aust dent j. 2000;45:2–9. 2. petersson ee, andersson l, sörensen s. traumatic oral vs non-oral injuries. swed dent j. 1996;21:55–68. 3. glendor u. aetiology and risk factors related to traumatic dental injuries--a review of the literature. dent traumatol. 2009;25:19–31. 4. bezroukov v. the application of the international classification of diseases to dentistry and stomatology. community dent oral epidemiol. 1979;7:21–24. 5. marcenes w, al beiruti n, tayfour d, issa s. epidemiology of traumatic injuries to the permanent incisors of 9–12‐year‐old school children in damascus, syria. endod dent traumatol. 1999;15:117–123. 6. locker d. prevalence of traumatic dental injury in grade 8 children in six ontario communities. can j public health. 2005;96:73–76. 7. traebert j, peres ma, blank v, böell rd, pietruza ja. prevalence of traumatic dental injury and associated factors among 12‐year‐old school children in florianópolis, brazil. dent traumatol. 2003;19:15–18. original m. al-shuhayeb et al. 207j contemp med sci | vol. 4, no. 4, july-august 2019: 202–207 prevalence of dental trauma in 5to 6-, 12-, and 15-year-old children in iran 8. andreasen jo, andreasen fm, andersson l, editors. textbook and color atlas of traumatic injuries to the teeth. wiley-blackwell; 2018. 9. world health organization. oral health surveys: basic methods. geneva: world health organization; 2013. 10. khoshnevisan mh, ghasemianpour m, samadzadeh h, baez rj. oral health status and healthcare system in ir iran. j contemp med sci. 2018;4:107–118. 11. faghih nasiri a, mahmoodian j, kosari a. prevalence of traumatic crown fracture in 7-14 year-old students of tehran in 1375 [dissertation]. dental college of tehran university (article in persian). 12. ansari g, mobini m. epidemiologic assessment of traumatic injuries to the teeth in children referred to paedodontic dept. of tehran and beheshti universities, 1997-1999. j dent school shahid beheshti univ med sci. 2001;18:287–296. 13. hamilton fa, hill fj, holloway pj. an investigation of dento-alveolar trauma and its treatment in an adolescent population. part 1: the prevalence and incidence of injuries and the extent and adequacy of treatment received. br dent j. 1997;182:91–95. 14. robson f, ramos‐jorge ml, bendo cb, vale mp, paiva sm, pordeus ia. prevalence and determining factors of traumatic injuries to primary teeth in preschool children. dent traumatol. 2009;25:118–122. 15. chopra a, lakhanpal m, rao n, gupta n, vashisth s. traumatic dental injuries among 12to 15-year-old school children in panchkula. arch trauma res. 2014;3:e18127. 16. glendor u. epidemiology of traumatic dental injuries–a 12 year review of the literature. dent traumatol. 2008;24:603–611. 17. al‐majed i, murray jj, maguire a. prevalence of dental trauma in 5-6‐ and 12-14‐year‐old boys in riyadh, saudi arabia. dent traumatol. 2001;17:153–158. 18. asnaashari m, tavakkoli ma, shafiei ardestani s. prognosis of traumatic injuries to the anterior teeth (treated in shahid beheshti and tehran dental schools during 1996-2001). iran endod j. 2006;1:37–42. 19. afshar h, mozaffari kogidi m. the prevalence of dental injuries (luxations and floctures) and related factors, in children who accepted in the research pediatric in the faculty of dentisrty of tehran university of medical sciences in 1379. 20. navabazam a, farahani ss. prevalence of traumatic injuries to maxillary permanent teeth in 9‐to 14‐year‐old school children in yazd, iran. dent traumatol. 2010;26:154–157. 21. chen yl, tsai tp, see lc. survey of incisor trauma in second grade students of central taiwan. changgeng yi xue za zhi. 1999;22:212–219. 22. burden dj. an investigation of the association between overjet size, lip coverage, and traumatic injury to maxillary incisors. eur j orthod. 1995;17:513–517. dx.doi.org/10.22317/jcms.08201904 original 8 j contemp med sci | vol. 6, no. 1, january–february 2020: 8–12 review issn 2413-0516 introduction today, mobile phone has become a technology of modern communication in the social and individual life of human beings. smart phone is an integral part of the daily activities and connections of human beings. the influence of mobile applications in various aspects of life is a universal phenomenon.1 today, the use of mobile phones and their effects on our daily life, and particularly its effectiveness in the health care, is dramatically rising.2,3 healthcare applications have made the smartphones as a useful tool in the traditional medicine for self-care and clinical interactions. also, mobile phones have the significant role in education and monitoring of patients.4 mobile health apps are widely used to diagnose chronic diseases such as cardiovascular and diabetes disease.5 moreover, those have focused on interventional research of specific medical issues such as: skin cancer,6 weight management,7 and cardiovascular.8 these apps are not limited to diagnosis and evaluation of the patient’s condition, but also include beyond the clinical aspects such as patient appointment, counseling, patient management, and medical training.9 the main operating systems for smartphones are google android and ios apple. apple itunes and google play stores offer many applications for mobile health.10 intelligent methods as the learning-based interactive models have been introduced in medical fields such as stroke imaging,11,12 medical imaging,13 medical decision support,14 and medical data analysis.15 patients with stroke can receive medical care information by the interactive models, which provide them with the prognosis and required treatments.11 artificial intelligence is becoming more advanced in interventional applications such as weight control and daily activities. those apps with cognitive and scientific models can enhance human performance.16 the aim of this review research is to systematic investigation of mobile smart applications to provide the digital methods for monitoring the nutritional health of diabetic patients through self-care education and scientific consideration. the outcomes of this review are used to determine the scientific basis of diabetes nutrition applications to help advance, research, and development of future applications. methods and materials google play store (google play store (https://play.google. com) and the apple ios store (https://itunes.apple.com) were used to find the applications with specific healthcare functionalities. the bluestacks 4.1.14.1460 software was used to access these two stores. all applications were extracted and evaluated through the bluestacks. according to the study requirements, the apps in a systematic evaluation on two operating systems were considered, independently. sample searches were found in may 2018. the selected apps were reviewed in healthcare settings. this search was done on a set of free apps. free programs were accurately and systematically considered for the domain of health and medical care. the input criteria for this study were: monitoring nutrition in diabetic patients, offering diet to diabetics, exercise and physical activity, and analyzing tools for activities and assessing the diet of diabetic patients. initial selection and evaluation of the apps were carried out with review of the title and summary of the apps. exclusions were those that were not specifically dedicated to diabetes for diabetics and health services and were not developed as a game or not accessible in english. the apps entered into this study were evaluated and information was collected to create consideration of intelligent applications to support diabetic patients: a scoping review for nutrition mobile phone apps zahra koohmareh,1 amir jamshidnezhad,1 and ali pazahr2 1department of health information technology, faculty of allied medical sciences, ahvaz jundishapur university of medical sciences, ahvaz, iran 2department of computer engineering, islamic azad university, ahvaz branch, ahvaz, iran correspondence to: amir jamshidnezhad, ahvaz jundishapur university of medical sciences, iran (email: jamshidnejad-a@ajums.ac.ir). (submitted: 09 september 2019 – revised version received: 04 october 2019 – accepted: 22 october 2019 – published online: 26 february 2020) objectives the main objective is to identify the functionality of smart applications in mobile platform for controlling, teaching, and healthy lifestyles in the field of nutrition for diabetic patients. methods current review was conducted on mobile platform to consider the functionalities of available applications to support the diabetic patients with the intelligent tools. the results of inclusion criteria (such as nutrition, weight control, blood pressure, education, and interactive analysis tools) were evaluated and organized in the form of a scoping review. all applications in the systematic search were independent of both operating systems. the search samples were found in may 2018 by experts in medical informatics. results 273 potential applications were identified according to the results of the inclusion criteria with evidence-based strategies and systematic evaluations to find the apps for diabetic patients. approximately, 29% of apps in both stores (79/273) met primary assessment criteria. of these, 40% of apps (32/79) excluded from more consideration due to lack in most criteria of this study. the remaining apps are described as follows: 8% of total apps (22/273) and 4% of the apps (11/273) met general criteria and all criteria (including general and special) in the google play store, respectively. only 2 apps met general criteria with the apple ios while none of them covered with special criteria according to the objectives of this study. conclusion there are various applications for control, education, and self-care in diabetes. google is an acceptable operating system for designers interested in examining interactive apps and techniques in disease control, education, and healthy lifestyle programs. keywords mobile health, smart apps, diet, diabetes, self care 9 review consideration of intelligent applicationszahra koohmareh amir jamshidnezhad and ali pazahr j contemp med sci | vol. 6, no. 1, january–february 2020: 8–12 observations through the text available in app stores and on the website of the applications (if present). the collected data were reviewed, collected, summarized, considered, and compared. fig. 1 shows the flowchart and selection process. application analysis tool applications were studied, considered and analyzed (see table 1 measuring tools for apps). the table was created with the factors used in the present and other studies.3,17,18 table 2 shows the general criteria chosen for the study. primary assessment criteria were administrated based on the identified and released apps in the google android market and the ios. then, the selected apps were determined based on inclusion criteria as the key factors. at first, apps related to diabetic patients were identified, then apps were limited to nutrition applications for diabetic patients. finally, apps including the interactive features for analyzing data and interaction with the patients were the final goal. the study population of app users in this research involved patients with various types of diabetes, those affiliated with diabetic patients, such as family, doctors, nutritionists, and non-patients using the healthcare applications. results this review is now for considering the features of the available apps on the mobile platform for consulting, educating, and nurturing diabetic patients. by using all inclusion criteria, finally 20 apps in google play and 2 applications in apple itunes were considered. exclusion criteria for both app stores were games, non-english language, and duplicate applications. most apps in the investigation outcomes provided one of the following services: cooking recipes for diabetics, nutritional information for patients, patient health records, self-care information, alert and reminders for insulin injections, weight control, and daily activity control for patients. fig. 2 shows the diagram of the screening process of this study. the results showed two hundred and fifty apps in android market and twenty three apps in the ios stores appeared in the review results (fig. 2). to filter more search results, non-english programs, games, and unrelated apps were removed in further evaluations. the programs that were left were considered for review based on inclusion factors. we found that among the selected programs, a lot of the apps were designed to control insulin, weight control, selfcare in patients, and a list of dietary orders. therefore, these apps were not included in the entry criteria such as dietary control, education, blood pressure control, and weight control, according to the status of diabetic patients. the results showed that 20 apps covered the general criteria, and of these, 10 apps with special criteria in google play have been equipped. table 2 presents the apps with initial and secondary criteria. in addition, table 2 shows that there were not apps including all general and special criteria of this study in the ios apple store. out of the 23 search apps with ios, only 2 apps were found with the general criteria, while none were eligible according to special criteria in terms of the use of clinical interactive surface in nutrition for diabetic patients. fig. 2 shows the results of the inclusion criteria with evidence-based strategies and systematic evaluations to find the apps for diabetic patients. according to fig. 2, approximately 29% of apps in both stores (79/273) met primary assessment criteria. of these, 40% of apps (32/79) excluded more consideration due to lack in most criteria of this study. the remaining apps are described as follows: 7% of total apps (20/273) and 4% of the apps (10/273) met general criteria and all criteria (including general and special) in the google play store, respectively. only 2 apps met general criteria with the apple ios while none of them covered with special criteria according to the objectives of this study.fig. 1 flowchart of systematic investigation process. table 1. measurment tools. factors for diabetesrelated apps in the initial search selected general criteria for diabetesrelated apps special criteria for diabetes-related apps insulin education nutrition plans communication diet interactive data analysis environment diet blood pressure exercise and physical activity body activity body mass control weight weight blood pressure personal health record education social media decision support alternative medicine conference calculator monitoring/export 10 review consideration of intelligent applications zahra koohmareh amir jamshidnezhad and ali pazahr j contemp med sci | vol. 6, no. 1, january–february 2020: 8–12 discussion diabetes mellitus or diabetes is one of the most commonly diagnosed diseases in the world. today, many efforts have been made to improve the diabetic patients care. in recent years, common approaches to using mobile technologies play a crucial role in medical care and patient self-management in a variety of diseases such as diabetes. mobile health has emerged as the main part of the electronic health. the use of wireless technologies such as bluetooth, wi-max, wi-fi, short message services, and data transmissions assist the users to access to the electronic health services.19 mobile health apps collect and provide the healthcare data. these apps present the dynamic services for active participation of patients and providers in the healthcare and a new tool for improving health outcomes.20 this study presents an investigation of literature on a wide space of health apps for smart phones in the self-care and education for diabetics’ patients. according to this review results, we found a lack of scientific information measuring the diagnostic value of health apps present in medical literature. the information about the diagnostic accuracy of currently available health apps on apple’s and google’s app stores is almost absent21. as a result, app users and healthcare professionals should be aware of the limitations when recommending specific programs. most of the apps that appeared in this review results included a manual of dietary instructions for diabetic patients, only for patient monitoring, weight control, and glucose monitoring. the aim of this research was to systematically review the dietary apps for diabetics using the interactive environment for data and physical activity analyzing of patients. the methodologic consideration of the features in the apps showed the limitations of the ios platform for developing the diabetic apps using the intelligent techniques. blood test grapher and blood pressure grapher were the only 2 apps with the ios in which the diabetic features were used while lacking in terms of the interactive tools. however, the android google supported several apps used for nutrition services including the intelligent techniques for analyzing of patients activities and control the diabetes effectively. therefore, android is a preferred platform for apps makers interested in gusing interactive tools in health and medical care applications. conclusion as conclusion, today, due to increase in the incidence of diabetes as a worldwide disease, the need to address the applications created in this area has been considered. diabetes education and self-care, especially food training, are the important fig. 2 flowchart of the screeining process. 11 review consideration of intelligent applicationszahra koohmareh amir jamshidnezhad and ali pazahr j contemp med sci | vol. 6, no. 1, january–february 2020: 8–12 issues for the human healthcare. given the widespread nature of this disease and the need for communities to educate and self-manage diabetes, there were a limited number of applications available that were designed and developed for nutrition with the patients’ interactive tools for diabetic patients. in general, patients’ interactive surface was used for a limited diabetes nutritional apps due to hardware and software constraints on mobile phones. the review results showed that google is an acceptable platform for developers interested with inclusion criteria as well as in examining interactive apps and techniques in disease control, education and healthy lifestyle programs. limitations this study was intended to find a review of the existing applications in the medical scope, in particular, the apps developed in the field of education and dietary orientation for diabetic patients. therefore, the limitations of this study were: access to a large number of diabetes-related apps according to a wide range of criteria in the search program was a challenging issue, therefore, the criteria selected as the inclusion criteria were restricted in this study. second, this investigation is meant to prepare a picture of applications existing in the medical industry instead of each specific specialization. third, apps were considered in the field of medical care rather than apps with the scope of general healthcare. as a result, a big part of the applications were excluded from the final analysis. acknowledgment many thanks to research center of nutrition and metabolism at ahvaz jundishapur university of medical sciences, iran to support this research. table 2. list of the apps with inclusion criteria. applications rating platform scope inclusion criteria general specialgoogle android apple app store sugar sense-diabetes app21 4.2  diabetic patient  diabetic diet & symptoms of diabetes22 4.9  diabetic patient and normal patient   diabetic diet plan23 4.3  diabetic patient   sugar free diet medicine reminder24 4.9  diabetic patient   diabetic calculator25 4.1  type 1 diabetes  diabetes treatment26 4.0  everyone  low carb program27 3.4  type 2 diabetes and prediabetes  how to reverse type 2 diabetes28 3.7  type 2 diabetes  beat-diabetes care and management app29 4.7  diabetic patient   sidiary diabetes management30 4.4  diabetic patient  one drop-diabetes management31 4.3  diabetic patient   diabetes pal32 3.8  diabetic patient  gather health family diabetes33 4.4  diabetic family members   is your diabetes under control34 3.9  diabetic patient and everyone  diabetometer35 4.5  diabetic patient and diabetic family members   diabetes and symptoms36 4.1  diabetic patient  life in control diabetes coach37 4.5  diabetic patient and doctor   glucosio: diabetes tracker38 4.2  diabetic patient  nutrition diabetes39 3.7  diabetic patient and doctor   diabetes mellitus from zero to hero40 4.9  diabetic patient, doctor, nurse, health worker   blood test grapher41 no ratings  diabetic patient  blood pressure grapher42 no ratings  diabetic patient  12 review consideration of intelligent applications zahra koohmareh amir jamshidnezhad and ali pazahr j contemp med sci | vol. 6, no. 1, january–february 2020: 8–12 declaration of conflicting interest the author(s) declared there is not conflicts of interest in this study. references 1. mm. rahmati, s. bakhshi. “lifestyle and consuming pattern case study: cellphone”. j. iran. cult. res. 8(4):119-42;2015. 2. m. goodarzi, i. ebrahimzadeh. “impact of distance education via short message service of mobile phone on metabolic control of patients with type 2 diabetes mellitus in karaj-iran”. quart. horizon med. sci.19(4):22434;2014. 3. hj. seabrook, jn. stromer, c. shevkenek, a. bharwani, j. de grood, wa. ghali. “medical applications: a database and characterization of apps in apple ios and android platforms”. bmc res. notes. 7(1):573;2014. 4. asm. mosa, i. yoo, l. sheets.” a systematic review of healthcare applications for smartphones”. bmc med. inform. decision making. 12(1):67;2012. 5. mnk. boulos, s. wheeler, c. tavares, r. jones. “how smartphones are changing the face of mobile and participatory healthcare: an overview, with example from ecaalyx”. biomed. eng. online. 10(1):24; 2011. 6. a. karargyris, o. karargyris, a. pantelopoulos, editors. “derma/care: an advanced image-processing mobile application for monitoring skin cancer”. tools with artificial intelligence (ictai), 2012 ieee 24th international conference on; 2012: ieee. 7. j. rivera, a. mcpherson, j. hamilton, c. birken, m. coons, s. iyer, et al. “mobile apps for weight management: a scoping review”. jmir mhealth and uhealth. 4(3);2016. 8. b. martínez-pérez, i. de la torre-díez, m. lópez-coronado, j. herrerosgonzález. “mobile apps in cardiology”. jmir mhealth and uhealth. 1(2);2013. 9. cl. ventola. “mobile devices and apps for health care professionals: uses and benefits”. pharm. therap. 39(5):356;2014. 10. t. dehling, f. gao, s. schneider, a. sunyaev. “exploring the far side of mobile health: information security and privacy of mobile health apps on ios and android”. jmir mhealth and uhealth. 3(1);2015. 11. e-j. lee, y-h. kim, n. kim, d-w. kang.” deep into the brain: artificial intelligence in stroke imaging”. j. stroke. 19(3):277; 2017. 12. d. shanthi, g. sahoo, n. saravanan.” designing an artificial neural network model for the prediction of thrombo-embolic stroke”. int. j. biomet. bioinform. (ijbb). 3(1):10-8;2009. 13. n. mirderikvand, m. naderan, a. jamshidnezhad. “accurate automatic localisation of lung nodules using graph cut and snakes algorithms”. 2016 6th international conference on computer and knowledge engineering (iccke). 194–199; 2016. 14. l. kabootarizadeh, a. jamshidnezhad, z. koohmareh, a. “ differential diagnosis of iron-deficiency anemia from β-thalassemia trait using an intelligent model in comparison with discriminant indexes”. acta inform med. 27(2): 78–84; 2019. 15. r. altman. “artificial intelligence (ai) systems for interpreting complex medical datasets”. clin. pharmacol. therap. 101(5):585-6;2017. 16. h. vainio, f. bianchini. “weight control and physical activity”: iarc; 2002. 17. t. chomutare, l. fernandez-luque, e. årsand, g. hartvigsen. “features of mobile diabetes applications: review of the literature and analysis of current applications compared against evidence-based guidelines”. j. med. internet res. 13(3);2011. 18. o. el-gayar, p. timsina, n. nawar, w. eid.” mobile applications for diabetes self-management: status and potential. j. diab. sci. technol”. 7(1):247-62; 2013. 19. r. ershad sarabi, f. sadoughi, r. jamshidi orak, k. bahaadinbeigy. “role of mobile technology in iran healthcare system: a review study”. j. health biomed. inform. 4(4):313-26;2018. 20. mhm. jazayeri, a. jamshidnezhad, “top mobile applications in pediatrics and children’s health: assessment and intelligent analysis tools for a systematic investigation”. mjms 26(1):5–14; 2019. 21. buechi r, faes l, bachmann lm, thiel ma, bodmer ns, schmid mk, et al. “evidence assessing the diagnostic performance of medical smartphone apps: a systematic review and exploratory meta-analysis”. bmj open. 2017;7(12):e018280. 22. lazer apps inc. diabetic diet & symptoms of diabetes. google play [download]. available at: https://play.google.com/store/apps/ details?id=com.lazerapps.android.diabetesgo&hl=en_us. 23. gato apps. diabetic diet plan. google play [download]. available at https:// play.google.com/store/apps/details?id=com.gatoapps.diabeticdiet&hl=en_ us. 24. manafi group. sugar free diet medicine reminder. google play [download]. available at https://play.google.com/store/apps/details?id=sugarfree.diet. medicinereminder&hl=en_us. 25. jake2701. diabetic calculator. google play [download]. available at https:// play.google.com/store/apps/details?id=mine.mydiabeticcalculator&hl=en_ us. 26. xtell technologies. diabetes treatment. google play [download]. available at https://play.google.com/store/apps/details?id=com.xtelltechnologies. diabetestreatment. 27. diabetes digital media. low carb program. google play [download]. available at https://play.google.com/store/apps/details?id=uk.co.diabetes. lowcarb&hl=en. 28. healthsensei101. how to reverse type 2 diabetes. google play [download]. available at https://play.google.com/store/apps/details?id=com. diabetes60system.type2diabetes&hl=en. 29. beato. beat0-diabetes care and management app. google play [download]. available at https://play.google.com/store/apps/ details?id=com.healtharx.beato&hl=en. 30. sinovo gmbh & co. kg. sidiary diabetes management. google play [download]. available at https://play.google.com/store/apps/ details?id=com.sidiary.app&hl=en. 31. one drop. one dropdiabetes management. google play [download]. available at https://play.google.com/store/apps/details?id=today.onedrop. android&hl=en. 32. telcare llc. diabetes pal. google play [download]. available at https://play. google.com/store/apps/details?id=com.telcare.android.client&hl=en. 33. gather health. gather health family diabetes. google play [download]. available at https://play.google.com/store/apps/details?id=com. gatherhealth.gatherdm&hl=en. 34. twayesh projects. is your diabetes under control. google play [download]. available at https://play.google.com/store/apps/details?id=anace.com. audiobooks.diabetes&hl=en_us. 35. ghrian technologies pvt. ltd. diabetometer. google play [download]. available at https://play.google.com/store/apps/details?id=com. ghriantech.diabetometer&hl=en_us. 36. appz inventors. diabetes and symptoms. google play [download]. available at https://play.google.com/store/apps/details?id=com.appz. diabetes&hl=en. 37. life in control. life in control diabetes coach. google play [download]. available at https://play.google.com/store/apps/details?id=com. lifeincontrol&hl=en. 38. glucosio. glucosio: diabetes tracker. google play [download]. available at https://play.google.com/store/apps/details?id=org.glucosio. android&hl=en_us. 39. built by doctors world ltd. nutrition diabetes. google play [download]. available at https://play.google.com/store/apps/details?id=com. builtbydoctors.ntdiabetes&hl=en. 40. utopia production. diabetes mellitus(dm) from zero to hero. google play [download]. available at https://play.google.com/store/apps/ details?id=com.utopia.diabetes777&hl=en. 41. ai imai. blood test grapher. itunes [download]. available at https://itunes. apple.com/us/developer/ai-imai/id1133573418?mt=8. 42. ai imai. blood pressure grapher. itunes [download]. available at https://itunes.apple.com/ro/app/blood-pressure-grapher/ id1171409283?mt=8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.02202002 15j contemp med sci | vol. 2, no. 5, winter 2016: 15–19 research background the commonest form of life-long treatment for individuals with β-thalassaemia major (tm) is blood transfusion; however, regular and multiple transfusion can result in iron overload as well as serious infections. objectives this study was designed to evaluate clinical manifestations in β-tm patients according to the blood transfusion rate. methods forty-two patients of homozygous β-tm in the thalassaemia division/children’s teaching hospital, kerbela in the middle part of iraq, during march–june 2015. all patients (17 males and 25 females) with age range 3–31 years were blood transfusion-dependent and on iron chelation therapy. they were divided into three groups according to the yearly average of blood transfusion received during the last 3 years; those who received less than 16 times/year (g1, n = 16), between 16 and 21 times/year (g2, n = 14) and more than 21 times/year (g3, n = 12). several biochemical tests were carried out to estimate the serum level of ferritin, total serum bilirubin (tsb), gpt, got, alkaline phosphatase (alp), albumin and total protein. results the results showed that half of patients were presented either with splenomegaly (28.6%) or splenectomy (21.4%), about one quarter of them (26.2%) were presented with hepatomegaly, more than half of patients (54.8%) were represented with hcv infection and the majority (97.6%) of patients have normal bmi. however, results of biochemical markers revealed that all of patients (100%) have albumin and total protein concentrations respectively, while all of them (100%) and the majority of them (76.2%) have elevated serum level more than the upper limit of normal reference of ferritin and tsb respectively. additionally, liver enzymes levels (gpt, got and alp) also recorded an elevation in about 31, 42.9 and 21.4% of thalassaemic patients. on the other hand, results demonstrated that increasing in the rate of blood transfusion revealed an association with the reduction in the frequency of patients with normal liver, spleen and negative hcv infection, and the three different groups of thalassaemic patients have significant differences in their age, bmi, rate of transfusion and total protein. however, the rest of biochemical tests (ferritin, tsb, gpt, got, alp and alb) revealed non-significant differences in their serum levels among the three groups of patients, but the percentage of patients that have elevated alp were significantly different (0, 35.7 and 33.3%) (p = 0,029) among the g1, g2 and g3 respectively. conclusion although blood transfusion is the commonest therapy to improve the life-span of β-thalassaemic patients worldwide, its rate should be reduced as less as possible to avoid the serious complications by searching for another criteria to indicate transfusion rather than haemoglobin concentration such as the antioxidant status of thalassaemic patients. keywords thalassaemia major, iron overload, liver enzymes, ferritin influence of blood transfusion rate on some clinical manifestations in β-thalassaemia major patients khalid mahdi salih,a wafaa fawzi. al-mosawyb issn 2413-0516 adepartment of biology, college of sciences, al-mustansiriyiah university, baghdad, iraq. bdepartment of pharmacology, college of pharmacy, kerbela university, kerbela, iraq. correspondence to khalid mahdi (email: khalid.salih11@yahoo.com). (submitted: 7 november 2015 – revised version received: 11 december 2015 – accepted: 4 january 2016 – published online: 26 march 2016) introduction thalassaemia major was firstly reported by dr. thomas cooley in 1925 as classical, fatal and mediterranean anaemia termed as cooley’s anaemia, now termed as β-thalassaemia major (tm).1 β-thalassaemia is found in arabic countries especially those which are located on the mediterranean, saudi arabia, jordan, syria and yemen.2 survey in iraq showed that β-thalassaemia trait is carried by 4.5–5% of the population,3 and about 6–10% of the population have haemoglobinopathies of which thalassaemia is a major part,4 specifically in the northern part of iraq due to the high rate of consanguineous marriages in this region.5,6 tm presents as a progressive anaemia during 6–24 months of age.7 the anaemia is severe (hb 2–7 g/dl) and has detrimental effects on most organ systems; in some patients, death would result without chronic blood transfusions.7–9 according to the british committee for standards in haematology, it has been recommended that the volume of transfusion is between 15 and 20 ml/kg depending on the pretransfusion hb and haematocrit of packed cells provided by the blood bank.10,11 however, current practice for transfusion therapy in arabian gulf countries recommended that regular blood transfusions is usually administered for every 2–5 weeks to maintain the pre-transfusion hb level above 9.5–10.5 g/dl.12 the present study was designed to evaluate the status of spleen, liver, body mass index, hepatitis c infection and some biochemical markers in β-tm patients according to the frequency of blood transfusion. material and methods patients descriptive cross-sectional study was conducted in the thalassaemia division/children’s teaching hospital, kerbela in the middle part of iraq, during march–june 2015. forty-two patients of homozygous β-tm (17 males and 25 females) with age range of 3–31 years were included. all patients are blood transfusion-dependent (15–20 ml packed rbcs/kg, at 2–3 weeks interval) to maintain a pretransfusion haemoglobin concentration above 8 g/l, also all of them were on iron chelation therapy. from a written consent form, medical histories for all patients were recorded including: sex, age, blood groups, blood transfusion rate and complications in liver, spleen, and hepatitis c virus (hcv) infection. patients were divided into three groups according to the yearly average of blood transfusion received during the last 3 years; those who received less mailto:khalid.salih11@yahoo.com 16 j contemp med sci | vol. 2, no. 5, winter 2016: 15–19 evaluation of clinical manifestations in β-thalassaemia major patients research khalid mahdi salih et al. than 16 times/year (g1, n = 16), between 16 and 21 times/year (g2, n = 14), and more than 21 times/year (g3, n = 12). methods the weight and height of all patients were measured to calculate body mass index (bmi). for adults (>20 years aged), a bmi of <18.5 is considered underweight, while a bmi >25 is considered overweight and above 30 is considered obese. for children and adolescents (2–20 years aged), a bmi that is less than the 5th percentile is considered underweight and above the 95th percentile is considered obese, while those with a bmi between the 85th and 95th percentile are considered to be overweight.13 the blood samples were collected at morning, 2 weeks after previous transfusion. three millilitres of venous blood were aspirated from cubital vein, and sera were obtained and stored at −20°c until to be used in the biochemical tests. a commercial reagents were used for the determination of serum ferritin level (vidas® ferritin, biomerieux®, lyon, france) based on an immunoenzymatic method with a final reading inflorescence (enzyme-linked fluorescent assay), according to procedures validated in our laboratory. total serum bilirubin (tsb) concentration was determined by using a diagnostic kit (accent-200, tsb, poland) based on chemical oxidation, utilising vanadate as an oxidising agent and measuring the absorbance before and after the vanadate oxidation.14 the concentrations of albumin (alb) and total protein (tp) as well as three enzymes: alanine aminotransferase (alt), aspartate aminotransferase (ast) and alkaline phosphatase (alp) were determined according to the methods recommended by international federation of clinical chemistry (ifcc) by using diagnostic kits from accent-200, poland.15,16 statistical analysis statistical analyses were carried out by using vassar stats web site for statistical computation.17 values were reported as the mean (m) ± standard error (se) and one-way anova test was used to compare study groups. comparison of categorical data between the different groups was carried out with chi square test. all statistical tests were 2-tailed, and a p value of <0.05 was considered as statistically significant. results the general characteristics extracted from the clinical profile of all patients under the permission of the thalassaemia centre were summarised in table 1. the status of all patients in respect to their spleen, liver as well as hcv infection showed that half of patients (50%) have normal spleen, and the rest half of them were presented either with splenomegaly (28.6%) or splenectomy (21.4%). while liver status showed that about three quarter of patients (73.8%) have normal liver and about one quarter of them (26.2%) were presented with hepatomegaly. on the other hand, more than half of patients (54.8%) were represented with hcv infection positive. the results of bmi values and other biochemical tests were demonstrated in table 2. since the normal range references of these parameters are variable according to sex and age of individual, therefore the frequency of normal and abnormal cases were calculated to reflect the actual impact of this disease on the health status of patients. this table revealed that the majority (97.6%) of patients and all of them (100%) have normal bmi, albumin and total protein concentrations respectively. in contrast, all of the patients (100%) and the majority of them (76.2%) have elevated serum level more than the upper limit of normal reference of ferritin and tsb respectively. additionally, liver enzymes levels (gpt, got and alp) also recorded an elevation in about 31, 42.9 and 21.4% of thalassaemic patients respectively. the gender occurrence and blood groups distribution, status of liver and spleen, as well as hcv infection among the different groups of tm patients (g1, g2 and g3) were summarised in table 3. although statistical analysis by using chi-square test (χ2) showed no significant table 2. the average values of bmi and other biochemical parameters and their abnormal frequency of overall patients parameters m ± sd cases (n) (%) normal abnormal bmi (kg/m2) 20.11 ± 3.72 41 (97.6%) 1 (2.4%) ↓ ferritin (ng/ml) 3162.7 ± 2081.9 0 (0%) 42 (100%) ↑ tsb (mg/dl) 2.01 ± 1.35 10 (23.8%) 32 (76.2%) ↑ gpt (u/l) 37.6 ± 40.5 29 (69%) 13 (31%) ↑ got (u/l) 43.4 ± 33.8 24 (57.1%) 18 (42.9%) ↑ alp (u/l) 148.1 ± 71.1 33 (78.6%) 9 (21.4%) ↑ albumin (g/dl) 4.49 ± 0.23 42 (100%) 0 (0%) tp (g/dl) 7.29 ± 0.62 42 (100%) 0 (0%) (↓) less than the lower limit of normal reference (↑) more than the upper limit of normal reference table 1. general characteristics of patients character values gender (n) (%) female 25 (59.5%) male 17 (40.5%) total 42 (100%) age (years) min. 3 max. 31 average 18.3 blood groups (n) (%) a+ 15 (35.7%) b+ 9 (21.4%) ab+ 7 (16.7%) o+ 11 (26.2%) blood transfusion rate (times/year) min. 7.3 max. 28.4 m ± sd 18.7 ± 4.8 spleen normal 21 (50%) splenomegaly 12 (28.6%) splenectomy 9 (21.4%) liver normal 31 (73.8%) hepatomegaly 11 (26.2%) hcv infection positive 23 (54.8%) negative 19 (45.2%) 17j contemp med sci | vol. 2, no. 5, winter 2016: 15–19 research evaluation of clinical manifestations in β-thalassaemia major patientskhalid mahdi salih et al. association in all of these characters among the different groups, the cases of patients with normal liver, spleen and negative hcv infection were dramatically decreased as the transfusion rate increased (fig. 1). by using one-way analysis of variance, the significance of differences in age, bmi, transfusion rate and biochemical tests among the different groups of thalassaemic patients were demonstrated in table 4. the results showed highly significant differences among the three groups in their age, bmi and transfusion rate; however, biochemical tests revealed non-significant differences (ferritin, tsb, gpt, got, alp and alb) except tp showed significant differences among different groups. the frequencies of cases that have elevated serum level of certain marker more than its upper limit of table 4. analysis of variance of all measurements among different groups of thalassaemic patients param eters (m ± se) patients group anova p value g1(n = 16) g2(n = 14) g3 (n = 12) age (years) 12.8 ± 1.7 22.9 ± 1.5 20.2 ± 1.08 <0.0001 bmi (kg/m2) 17.2 ± 0.73 21.8 ± 0.68 21.9 ± 1.07 0.00011 transfusion rate (times/ year) 13.7 ± 0.54 19.4 ± 0.35 24.4 ± 0.69 <0.0001 ferritin (ng/ml) 2951 ± 500 3368 ± 663 3204 ± 554 0.869 tsb (mg/dl) 2.03 ± 0.43 1.97 ± 0.36 2.05 ± 0.22 0.990 gpt (u/l) 28.3 ± 3.8 58 ± 16.9 26.4 ± 5.9 0.071 got (u/l) 40.2 ± 3.3 59 ± 14.2 29.4 ± 4.4 0.077 alp (u/l) 170.6 ± 17.1 119.7 ± 12.6 151.3 ± 26.5 0.151 alb (g/dl) 4.47 ± 0.06 4.44 ± 0.04 4.57 ± 0.07 0.366 tp (g/dl) 7 ± 0.16 7.6 ± 0.11 7.2 ± 0.18 0.046 table 3. distribution of gender and blood groups among patients groups and their statistical significance character g1 (n = 16) g2 (n = 14) g3 (n = 12) chi-square p value gender male 8 (50%) 5 (35.7%) 4 (33.3%) χ2 = 0.988 p = 0.610female 8 (50%) 9 (64.3%) 8 (66.7%) blood groups a+ 5 (31.3%) 6 (42.9%) 4 (33.4%) χ2 = 8.126 p = 0.228 b+ 6 (37.5%) 2 (14.3%) 1 (8.3%) ab+ 3 (18.7%) 3 (21.4%) 1 (8.3%) o+ 2 (12.5%) 3 (21.4%) 6 (50%) liver status normal 14 (87.5%) 10 (71.4%) 7 (58.3%) χ2 = 3.079 p = 0.214hepatomegaly 2 (12.5%) 4 (28.6%) 5 (41.7%) spleen status normal 9 (56.2%) 7 (50%) 5 (41.7%) χ2 = 5.638 p = 0.227splenomegaly 4 (25%) 2 (14.3%) 6 (50%) splenectomy 3 (18.8%) 5 (35.7%) 1 (8.3%) hcv infection positive 6 (37.5%) 10 (71.4%) 7 (58.3%) χ2 = 3.556 p = 0.168negative 10 (62.5%) 4 (28.6%) 5 (41.7%) fig. 1 frequency of cases with normal liver, spleen and absence of hcv infection among different groups of thalassaemic patients. fig. 2 frequency of cases with elevated serum level of biochemical markers among different groups of thalassaemic patients. normal range were calculated. the results showed that 62.5, 85.7 and 83.3% of patients in g1, g2 and g3 respectively, have elevated tsb but without significant differences among them according to chi-square test (fig. 2). similarly, those for gpt (25, 42.8 and 25%) and got (56.2, 50 and 16.6%) are non-significant. while those for alp (0, 35.7 and 33.3%) revealed significant difference (p = 0.029) among the three groups of thalassaemic patients. 18 j contemp med sci | vol. 2, no. 5, winter 2016: 15–19 evaluation of clinical manifestations in β-thalassaemia major patients research khalid mahdi salih et al. discussion this study was limited to one thalassaemia centre in kerbela district/iraq because it was difficult to recruit patients from other centres in other iraqi cities, also β-tm seems to be more prevalent in kerbela district which is mainly due to the high rate of consanguineous marriage among its population. although an increasing number of tm patients are now treated with bone marrow transplantation, the majority of the patients still depend on regular transfusions.18 all tm patients in this study were managed by blood transfusion approach at a rate of 18.7 times/ year (table 1) which is compatible with current practice for transfusion therapy in arabian gulf countries.12 the patients of this study presented with several complications including splenomegaly or splenectomised (50% of all patients), hepatomegaly (26.2%) and hcv infection (54.8%) (table 1). several studies have been demonstrated that patients with β-tm may go through several complications as the transfusion-related infections like hbv, hcv and hiv.19 iron overload complications are also noticed that includes endocrinopathy, heart and liver diseases and chelation therapy complications.20 similarly, our results found that all patients (100%) were undergone iron overload with an average of ferritin level in about 3162.7 ng/ml, in addition to elevation in serum level of tsb, gpt, got and alp in about 76.2, 31, 42.9 and 21.4% of total patients respectively, but those for albumin and total protein remain within their normal limits (table 2). these results are compatible with those found by other researchers, some of them observed liver dysfunction in β-tm patients with a significant elevation in the activities of both aminotransferases enzymes (ast and alt) in the sera of thalassaemic patient groups compared with control,21,22 but no significant differences in the albumin levels of all patient groups compared to control was noticed.22 however, other researchers found that iron over load and jaundice is common finding of thalassaemia and both serum ferritin and bilirubin parameters of iron over load and jaundice are correlated. but no statistical correlation was found between these two parameters.23 on the other hand, patients with haematological disorders such as tm have a high risk of exposure to hcv.24 after the discovery of hcv in 1989, it was found to be the major cause of transfusion-associated hepatitis in the world.25 approximately, the prevalence of hcv in iraq among tm patients was 66.6%,26 which is nearly comparative to the prevalence among saudi arabia tm patients which was 70%.27 as it is known, the commplications of repeated blood transfusion were mainly developed from iron overload since every unit of blood contains ~200 mg of iron28 that can be manifested by a high serum ferritin levels from transfusions29 and may be exacerbated in some patients due to increased absorption of iron from the diet in response to ineffective erythropoiesis.30 in contrary to these findings, our results found that the rate of blood transfusion does not revealed significant association in the distribution of sex, blood groups, status of liver and spleen as well as hcv infection among the three different groups (table 3), although the cases of patients with normal liver, spleen and negative hcv infection were dramatically decreased as the transfusion rate increased (fig. 1). additionally, the most of biochemical markers (tsb, gpt, got, alp and alb) showed non-significant differences among the three groups (table 4). these findings may be due to non-significant difference in the serum ferritin level in the different groups and all patients in these groups undergone iron overload, also the three different groups consist of patients with significantly different age and bmi (table 4). also our results revealed that all patients in g1 who received the lowest blood transfusion rate have normal level of alp which is significantly differ from patients in g2 and g3 with further transfusion rate, whose alp level were elevated in 37.5 and 33.3% of their patients (fig. 2). this elevation of alp in g2 and g3 may be due to two factors, the first one is hepatic toxicity due to serum ferritin as an index of iron overload which was high in g2 (3368 ± 663 ng/ml) and g3 (3204 ± 554 ng/ml) in comparison with that in g1 (2951 ± 500 ng/ml). some researchers found that severe haemosiderosis and hepatic fibrosis were common in patients with tm despite the use of chelation therapy,31 while others reported that patients with tm in iraq are poorly managed of iron overload, though iron chelation is used, and clinical signs of iron overload appear in young thalassaemic patients due to poor control.32 however, the second factor is the prevalence of hcv infection because the frequency of cases that have serum negative for hcv was dramatically decreased from 62.5% in g1 down to 41.7 and 28.6% in g3 and g2 respectively. the key problem with hcv infection is it’s propensity to produce chronic liver disease, cirrhosis and hepatocellular carcinoma occurring after a number of years.31 since liver disease is a leading cause of death in patients with transfusion-dependent thalassaemia,33 transfusion-associated hepatotropic infections, especially hcv infection, and hepatic siderosis can act either synergistically or independently in promoting chronic liver disease, and they may induce cellular damage through similar oxidative pathways.34 finally, this study demonstrated that the level of total protein was the only marker showed significant difference among the different groups, but its value was still within normal limits. this finding needs further investigation to search for protein–nature components in the blood of thalassaemic patients rather than those investigated in this study.  references 1. weatherall dj. phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias. nat rev genet. 2001;2(4):245–255. pmid: 11283697 2. awad mh. homozygous β-thalassemia in mosul. phd thesis, mosul univ, iraq; 1999. 3. al-awqati n, angastiniotis m. international news, help needed in iraq. tif news. 1998; 23:15. 4. rasheed ne, ahmed sa. effect of β-thalassemia on some biochemical parameters. mejfm. 2009;7(2):1–6. 5. al-allawi na, jubrael jm, hughson m. molecular characterization of betathalassemia in the dohuk region of iraq. hemoglobin. 2006;30(4):479–86. pmid: 16987803 6. adaay mh, al-anzy mm, al-samarrai a-mh, al-tikriti ka, al-samarrai fa. some observations on the occurrence of β-thalassemia in mosul. iraqi j med sci. 2011;9(3):270–274. 7. galanello r, origa r. beta-thalassemia. orphanet j rare dis. 2010;5:11. doi: 10.1186/1750-1172-5-11 pmid: 20492708 8. olivieri n. the β-thalassemias. n engl j med. 1999;341(2):99–109. pmid: 10395635 9. thein sl. genetic modifiers of beta-thalassemia. haematologica. 2005; 90(5):649–60. pmid: 15921380 10. piomelli s. the management of patients with cooley’s anemia: transfusions and splenectomy. semin hematol. 1995;32(4):262–268. pmid: 8560283 19j contemp med sci | vol. 2, no. 5, winter 2016: 15–19 research evaluation of clinical manifestations in β-thalassaemia major patientskhalid mahdi salih et al. 11. cappellini md, cohen a, eleftheriou a, piga a, porter j, taher ali. guidelines for the clinical management of thalassaemia, 2nd rev edn. thalassaemia international federation, isbn-13: cyprus; 2008. 12. goodnough lt, brecher me, kanter mh, aubuchon jp. transfusion medicine: first of two parts–blood transfusion. n engl j med. 1999;340:438–447. pmid: 9971869 13. who expert consultation. appropriate body-mass index for asian populations and its implications for policy and intervention strategies. lancet. 2004;363:157–163. pmid: 14726171 14. tokuda k, tanimoto k. new method of measuring serum bilirubin using vanadic acid. jpn j clin chem. 1993; 22(2):116–122. 15. tietz nw, rinker ad, shaw lm. international federation of clinical chemistry: ifcc methods for the measurement of catalytic concentration of enzymes, part 5. clin chim acta. 1983;135(3):339f–367f. pwid: 6661822 16. burtis ca, ashwood er. tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. pa: wb saunders; 2009. 17. lowry r. concepts & applications of inferential statistics. 2013. available at www.vassarstats.net. 18. ghavamzadeh a, jahani m, baybordi e. bone marrow transplantation in iran. bone marrow transplant. 1994;13:743–744. pmid: 7920308 19. chen ac, peng ct, wu sf, wu kh, chiang ip, tsai ch. effect of deferiprone on liver iron overload and fibrosis in hepatitis-c-virus-infected thalassemia. hemoglobin. 2006;30:209–14. pmid: 16798645 20. ferrara m, matarese sm, borrelli b, perrotta a, simeone g, greco n, et al. cardiac involvement in beta-thalassemia major and beta-thalassemia intermedia. hemoglobin. 2004;28:123–9. pmid: 15182054 21. shams s, ashtiani mth, monajemzadeh m, koochakzadeh l, irani h, jafari f, et al. evaluation of serum insulin, glucose, lipid profile, and liver function in β-thalassemia major patients and their correlation with iron overload. lab med. 2010;41(8):486–489. 22. sabri ms. biochemical study on splenectomy and non-splenectomy iraqi major thalassemic patients. ibn al-haitham j pure appl sci. 2010;23(1):254–259. 23. sultana n, sadiya s, rahman mh. correlation between serum bilirubin and serum ferritin level in thalassaemia patients. bangladesh j med biochem. 2011;4(2):6–12. 24. ross rs, viazov s, gross t, hofmann f, seipp h-m, roggandorf m. transmission of hcv from patients to an anesthesiology assistant to five patients. n eng j med. 2000;343:1851–4. pmid: 11117977 25. sibal a, mirsha d, arara m. hepatitis c in childhood. indian med assoc. 2002;100:630–685. 26. al-hilli ha, ghadhban jm. prevalence of serological markers of hepatitis b virus (hbs ag) and hepatitis c virus (hcv ab) among blood donors and certain risk groups. j fac med baghdad. 2000;42(1):45–52. 27. al-faleh f, ramia s. hepatitis c virus infection in saudia, review article. ann saudi med. 1997;17(1):77–82. pmid: 17377468 28. porter jb. practical management of iron overload. brit j haematol. 2001;115(2):239–53. pmid: 11703317 29. ikram n, hassan k, younas m. ferritin levels in patients of beta thalassaemia major. internat j pathol. 2004;2(2):71–4. 30. taher a, cappellini md. update on the use of deferasirox in the management of iron overload. therapeut clin risk manag. 2009;5: 857–68. 31. li ck, chik kw, lam cwk, to kf, yu sch, lee v, et al. liver disease in transfusion dependent thalassaemia major. arch dis child. 2002;86:344– 347. doi: 10.1136/adc.86.5.344 32. al-kataan ma, al-rasheed sm, ahmed fa. serum iron status in betathalassemic patients with clinical signs of iron overload. tikrit med j. 2009;15(1):9–12. 33. zurlo mg, de stefano p, borgna-pignatti c, di palma a, piga a, melevendi c, et al. survival and causes of death in thalassaemia major. lancet. 1989;2:27–30. pmid: 2567801 34. bonkovsky hl, banner bf, rothman al. iron and chronic viral hepatitis. hepatology. 1997;25:759–768. pmid: 9049232 http://www.ncbi.nlm.nih.gov/pubmed/?term=tietz nw%5bauthor%5d&cauthor=true&cauthor_uid=6661822 http://www.ncbi.nlm.nih.gov/pubmed/?term=rinker ad%5bauthor%5d&cauthor=true&cauthor_uid=6661822 http://www.ncbi.nlm.nih.gov/pubmed/?term=shaw lm%5bauthor%5d&cauthor=true&cauthor_uid=6661822 http://www.ncbi.nlm.nih.gov/pubmed/6661822 http://www.ncbi.nlm.nih.gov/pubmed/?term=wu kh%5bauthor%5d&cauthor=true&cauthor_uid=16798645 http://www.ncbi.nlm.nih.gov/pubmed/?term=chiang ip%5bauthor%5d&cauthor=true&cauthor_uid=16798645 http://www.ncbi.nlm.nih.gov/pubmed/?term=tsai ch%5bauthor%5d&cauthor=true&cauthor_uid=16798645 http://www.ncbi.nlm.nih.gov/pubmed/?term=perrotta a%5bauthor%5d&cauthor=true&cauthor_uid=15182054 http://www.ncbi.nlm.nih.gov/pubmed/?term=simeone g%5bauthor%5d&cauthor=true&cauthor_uid=15182054 http://www.ncbi.nlm.nih.gov/pubmed/?term=greco n%5bauthor%5d&cauthor=true&cauthor_uid=15182054 http://dx.doi.org/10.1136%2fadc.86.5.344 82 j contemp med sci | vol. 4, no. 2, spring 2018: 82–87 research senior dental student’s utilization and self-reported skills of information technology (it) simin z mohebbi,a reza yazdani,a mohammad mofatteh,b maedeh bonabia aresearch center for caries prevention, dental research institute, tehran, iran department of community oral health, school of dentistry, tehran university of medical sciences, tehran, iran. bdepartment of orthodontics, ahwaz university of medical sciences, ahvaz, iran. correspondence to mohammad mofatteh (email: dr.mohamadmofateh@yahoo.com). (submitted: 19 november 2017 – revised version received: 11 january 2018 – accepted:17 february 2018– published online: 26 june 2018) background given that one of the targets of global oral health care goals in 2020 is to increase the proportion of the population covered by health care information systems, the future dentists should be more oriented about information technology (it) to help the health systems in reaching this goal. the aim of this study was to evaluate the utilization of it among senior dental students and to evaluate the association of self-reported skills with utilization of it. methods the target population comprised senior dental students of all four dental schools of tehran city in 2015. an anonymous selfadministered questionnaire queried their utilization of it in four areas: general use of computers, general use of internet, professional use of computers, and professional use of internet. in addition, the students self-reported it skill was obtained in two areas: internet searching skills and computer using skills. background questions were also asked. results totally, 218 questionnaires out of 250 were returned for an overall response rate of 85%. the students’ mean score for professional usage of computer was 15.3 (± 4.2) out of 30 and for professional usage of internet 10.8 (± 3.1) out of 20. the mean score for general usage of computers was 22.6 (± 5.4) out of 40 and for general usage of internet 46.1 (± 13.2) of 80. about computer skills, a mean of 18.6 (±4) out of 28 and internet searching skills a mean of 15.3 (±4) out of 24 was acquired. the linear regression model revealed that older student (regression coefficients = 0.325, p = 0.001) and those with higher computer using skills (regression coefficients = 0.223, p = 0.031) and internet searching skills (regression coefficients = 0.220, p = 0.027) obtained higher scores in general usage of it. also higher research experience gained was associated with higher scores in professional usage of it (regression coefficients = −0.378, p =0.02). conclusion it utilization and skills were not desirable in present students. the fact that it skills were associated with a higher it utilization regardless of other background factors casts light on the importance of training to facilitate full utilization. keywords information technology, senior dental students, internet searching skill, computer using skill, it utilization introduction world has witnessed rapid progress and expansion of information technology (it) in dentistry in the last few years.1 nowadays, internet and social networks have found their place among young doctors and this penetration of computers and internet has revolutionized the study and practice of dentistry.2 along with general usage of computer and internet for communication and entertainments, dental education and research, office management, digital imaging, electronic health records, video conferencing, remote communication with patients and other dentists and, continuous distance and patient education are some examples of professional it usages.1 several studies of it usage reveal that about 60–95% of dental students use internet for personal and educational purposes and most of them own personal computers or laptops.3–7 a study in finland represented that all dental students use internet for both personal and educational purposes but senior students are more likely to use it.3 another study in jordan showed that more than 91% of students use internet for educational purposes.8 nevertheless, the students’ computer literacy and skills were insufficient as less than half the students had been exposed to some form of computer education in the university making full use of it inaccessible.9 it has been suggested that sufficient training and skill has an important role in student attachment to it while other background factors have lesser impact on it usage.4 a previous study in iran revealed the lesser dentists are familiar with it, the more management and financial errors happen in office. this may lead to miscommunication between doctor and patient.2 lack of time, unavailability and high costs may be also named as barriers of it usage.8,10 because of such barriers, despite great spread of dental webpages, their usage and effectiveness in dental training is still unclear and as a result, some of dental students are not enthusiastic to them.2 however, it has undeniable role in training students and actually can be addressed as one of the main components in new methods which increases the training speed.1 the aim of this study is to evaluate the orientation and utilization of it among senior dental students and to evaluate the association of self-reported skills with usage of it. methods the target population comprised senior dental students of all four dental schools of tehran city in 2015 in a census way (n = 250). the questionnaire was constructed from previous literature1,2,4,11 and some researchers made questions. it was assessed by five community oral health experts and one epidemiologist for content validity. after minor revisions, we distributed the questionnaire two times within a 2-week-interval among 20 dental students, out of the main target population, to check the reliability (minimum agreement 0.9). the survey was voluntary with no identifiable information issn 2413-0516 simin z. mohebbi et al. 83j contemp med sci | vol. 4, no. 2, spring 2018: 82–87 research dental students’ adherence to it collected. the ethics committee of tehran university of medical sciences (tums) approved the survey. no honorarium was offered to the participants. the self-reported questionnaire collected data on the utilization of it in four areas as follows: general usage of computers (8 questions), general usage of internet (16 questions), professional usage of computers (6 questions), and professional usage of internet (4 questions). it also included 13 questions on it self-reported skills in two areas: internet searching skills (6 questions) and computer using skills (7 questions). background questions were also asked about age, sex, marital status, living place (dormitory or not), name of university, grade point average, self-estimated family economic status, paternal and maternal educational level, research experience, owning personal computers, emails and website. the questions in utilization section, had five-point likert scale for response alternatives from daily use to never used. the self-reported skill section questions had four-point scale as i don’t know how to use it, i have some primary experiences, i know how to use it but i want to learn more, my skills are sufficient. the answers were scored and totals calculated in each section. for further analysis, we summed general usage of computer and internet scores to reach a total general it usage. also professional usage of computer and internet scores were summed to reach a total professional it usage. all numerical data were entered and analyzed using the statistical package for social sciences (spss version 18). initial descriptive statistics were expressed as frequency, mean and standard deviation. analyses were performed using pearson correlation test and linear regression. results background information totally, 218 questionnaires out of 250 were returned for an overall response rate of 85%. most participants were between 24 and 26-year-old (59%), 144 were females (67%) and 40% has got a− as last grade point average. among the participants, 29 (14%) had middle economic status and 37 (17%) were in high level while others were categorized in the very high level of self-reported economic status (69%). less than 1% (n = 1) of fathers were illiterate, 3% of them had primary education, 96% had academic degrees while 3% of mothers were illiterate (n = 3), 5% had elementary education and 92% had academic degrees. among all, 31 (15%) lived in university dormitories and 165 (77%) were single. from all 218 respondents 209 (97%) had personal laptops, 214 (99%) had personal emails and 71 (33%) had research experiences other than their thesis. utilization of it among students professional usage of computers the students’ score for professional usage of computers was between 26.6 and 100 with a mean of 51 (±14) out of 100 (table 1). among all, 96% used computers for writing and file saving, 67% benefited from digital graphic editing, 23% for designing webpages and 93% for creating multimedia through computers. moreover, 69% of the students used computer for creating and editing scientific audio and video files. professional usage of internet the students’ score for professional usage of internet ranged between 20 and 95 with a mean of 54 (±15.5) out of 100 (table 2). among all, 91% of students used internet to access faculty portal, 91% to search among references, 91% downloaded educational videos and 74% downloaded educational books and articles. general usage of computers the students’ score for general usage of computers ranged between 25 and 100 with a mean of 56.5 (±13.5) out of 100 (table 3). among all, 97% used computers for writing and file saving while 78% benefited from digital graph editing. also, table 1. professional usage of computer (with no internet connection) for academic education and research among senior dental students of tehran (%) daily weakly monthly less than once in a month never for saving data 29 (14%) 72 (34%) 47 (22%) 53 (26%) 9 (4%) digital graphic editing (e.g. photoshop) 4 (2%) 20 (10%) 66 (31%) 87 (41%) 33 (16%) designing websites 3 (2%) 7 (3%) 4 (2%) 33 (16%) 157 (77%) creating multimedia (e.g. powerpoint) 9 (4%) 32 (15%) 107 (51%) 57 (27%) 6 (3%) creating and editing audio and video files 4 (2%) 17 (8%) 39 (19%) 80 (39%) 64 (31%) table 2. professional usage of internet for academic education and research among senior dental students of tehran (%) daily weakly monthly less than once in a month never access to faculty portal 11 (5%) 46 (22%) 60 (28%) 78 (36%) 18 (9%) search among references 24 (11%) 39 (19%) 61 (29%) 68 (32%) 18 (9%) download educational videos 12 (5%) 17 (7%) 49 (22%) 83 (40%) 53 (26%) download educational books and articles 12 (6%) 43 (20%) 71 (34%) 77 (36%) 9 (4%) 84 j contemp med sci | vol. 4, no. 2, spring 2018: 82–87 dental students’ adherence to it research simin z. mohebbi et al. 28% of students designed webpages and 86% created multimedia through computers. moreover, 71% of them created and edited entertainment audio and video files, 96% used computers for saving general files and 91% for playing films and music and finally 73% for games. general usage of internet the students’ score for general usage of internet was between 25 and 100 with a mean of 57.6 (±16.5) of a max of 100 (table 4). among all respondents, 95% students used internet for accessing general information, 65% for online game, 94% for online music and films, 51% for online shopping, 84% for payments, 97% for emailing, 88% for chatting, 23% for designing and managing websites and weblogs, 56% for using online softwares, 69% for downloading and sharing files and music, 84% for sharing photos, 81% for international phone calls, 57% for video chatting, 70% for reading rss feeds, 81% for following weblogs and commenting on their posts and finally 91% for using social networks. self-reported it skills among students computer using skills among students the students’ score for computer using skills ranging between 25 and 100 with a mean of 66.4 (±14.2) out of a max of 100 (table 5). among all, 93% of students had the ability to use windows for their tasks, 95% could do advanced web search, 89% could use painting software, 97% knew how to use microsoft office word, 97% could use microsoft office powerpoint, 75% had the ability to use microsoft office excel and 43% were able to use spss. internet searching skills among students the students’ score for internet searching skills ranging between 25 and 100 with a mean of 63.7 (±16.6) out of 100 (table 6). among all students, 92% could search in medline, 57% of students could use cochrane and 90% were able to read full text articles, 83% were able to use educational e-books, 78% could access resources of e-learning, 82% could do advanced search in search engines such as google. table 3. general usage of computer (with no internet connection) among senior dental students of tehran (%) daily weakly monthly less than once in a month never for saving data 53 (25%) 69 (32%) 43 (20%) 41 (20%) 7 (3%) digital graphic editing (e.g. photoshop) 8 (4%) 27 (13%) 43 (21%) 84 (40%) 46 (22%) designing websites 5 (3%) 10 (5%) 9 (5%) 33 (16%) 144 (72%) creating multimedia (e.g. powerpoint) 9 (4%) 32 (16%) 80 (39%) 56 (27%) 29 (14%) creating and editing audio and video files 18 (9%) 32 (15%) 31 (15%) 66 (32%) 61 (29%) using offline saved files 48 (23%) 63 (30%) 46 (22%) 46 (22%) 8 (4%) listening to music and watching films 111 (52%) 53 (25%) 28 (13%) 18 (9%) 2 (9%) games 30 (15%) 39 (19%) 41 (20%) 39 (19%) 54 (27%) table 4. general usage of internet connection among senior dental students of tehran (%) daily weakly monthly less than once in a month never accessing general information 108 (51%) 67 (31%) 19 (9%) 19 (9%) 1 (5%) online games 24 (11%) 30 (14%) 34 (16%) 50 (24%) 73 (35%) listening to music or watching films 82 (39%) 48 (23%) 27 (13%) 42 (20%) 12 (6%) online shopping 12 (6%) 23 (11%) 33 (16%) 40 (19%) 102 (49%) online payments 19 (9%) 39 (18%) 75 (35%) 46 (22%) 35 (16%) using email 66 (31%) 65 (31%) 36 (17%) 37 (18%) 7 (3%) chatting 88 (42%) 38 (18%) 33 (16%) 28 (13%) 25 (12%) managing a website or weblog 8 (4%) 12 (6%) 10 (5%) 26 (13%) 152 (73%) using online softwares 21 (10%) 26 (13%) 22 (11%) 46 (23%) 89 (44%) downloading, sharing and uploading files 33 (16%) 43 (21%) 30 (15%) 37 (18%) 63 (31%) sharing photos 37 (18%) 53 (25%) 40 (19%) 46 (22%) 34 (16%) making international phone calls 59 (28%) 41 (20%) 30 (14%) 41 (20%) 39 (19%) video chatting 14 (7%) 28 (13%) 24 (11%) 54 (26%) 91 (43%) reading rss feeds 40 (20%) 34 (17%) 23 (11%) 47 (23%) 61 (30%) following weblogs and commenting 33 (16%) 31 (15%) 40 (19%) 63 (30%) 40 (19%) using social networks 80 (38%) 45 (21%) 37 (17%) 33 (16%) 18 (9%) simin z. mohebbi et al. 85j contemp med sci | vol. 4, no. 2, spring 2018: 82–87 research dental students’ adherence to it table 5. computer literacy among senior dental students, tehran, iran (%) computer using skill i cannot use it. i have elementary experiences. i can use it but i want to learn more. i can use it perfectly. basic use of windows 15 (7) 25 (12) 99 (46) 77 (36) advanced web search 10 (5) 48 (22) 97 (45) 60 (28) advanced use of printer 24 (11) 68 (32) 87 (41) 36 (17) microsoft word 3 (1) 50 (23) 101 (47) 61 (28) microsoft powerpoint 6 (3) 34 (16) 93 (44) 80 (38) microsoft excel 54 (25) 92 (43) 55 (26) 14 (7) statistical softwares like spss 122 (57) 59 (27) 31 (14) 4 (2) table 6. internet literacy among senior dental students, tehran, iran (%) internet searching skill i cannot use it. i have elementary experiences. i can use it but i want to learn more. i can use it perfectly. searching medline (pubmed) 16 (8) 48 (23) 71 (34) 74 (35) using cochrane library 92 (43) 65 (31) 44 (21) 12 (6) using full text articles 20 (10) 74 (36) 82 (40) 31 (15) using dental e-books 36 (17) 73 (35) 72 (34) 30 (14) using digital references 46 (22) 77 (36) 59 (28) 31 (15) advanced use of search engines such as google 17 (8) 37 (17) 70 (33) 90 (42) utilization in relation to background and self-reported skills the linear regression model was controlled for owning personal computer, other background information, computer using skills and internet searching skills (table 7) revealed that older student (regression coefficient = 0.325, p = 0.001) and those with higher computer using skills (regression coefficient = 0.223, p = 0.031) and internet searching skills (regression coefficient = 0.220, p = 0.027) obtained higher scores in general usage of it. another regression model revealed students with higher research experience gained higher scores in professional usage of it (regression coefficient = −0.378, p = 0.02). discussion our study showed insufficient utilization and self-reported it skills related to it usage among senior dental students in tehran based on their mean scores acquired from the total maximum possible. despite the introduction of it course as two credits in the second year of dentistry curriculum in iran,12 the present students could almost get about the half of what should be achieved in professional and general it fields, the students’ skill in it was also just a little better (65%) of the maximum possible. the fact that more skillful students had higher it usage may be a key finding to focus in improving the current situation. one of the targets of global oral health care goals in 2020, was mentioned to increase the proportion of the population covered by health care information systems in different countries, and this means that the future dentists should be more oriented about it. our study declared no differences among male and female students in using computers and internet and their attachment to it while mariño et al.1 study from australia showed that male students generally use internet more than female students. on the other hand, mohebbi et al.2 stated that in their study, iranian male dentists demonstrated higher professional usage of internet in 2014. two other studies in jordan and turkey reported higher frequency and skill in using internet and computers among male students in 2005.8,13 rahman6 also stated that in saudi arabia men had more access to internet and computers than women in 2011. one of the reasons of difference in our results with the others might be that almost all mentioned studies are older than ours. it seems that this gender difference of technology and it access may be on the way of fading. in a previous study conducted in 2012 among dentists,2 about half of the dentists reported to use computer and internet for general or professional usage. they had stated that 12.5% of dentists did not have any personal e-mail while it was just 1% in our study. it can all be justified according to time and generation gaps. in comparison of the trend of it usage from the previous study on dentists and the present dental students, we understand that in using social networks, there has been a difference of about two-fold between dental students and dentists in iran; while 21.5% of dentists used internet for social networks daily, 38% of students did so which can also be a pathway for the increase of rape in it, used among everyone including our respondents. in our study, 97% owned personal computer and 99% had personal e-mail. in mentioned jordanian study 74% of students owned personal computer and 90% had personal 86 j contemp med sci | vol. 4, no. 2, spring 2018: 82–87 dental students’ adherence to it research simin z. mohebbi et al. e-mail which is a high rate but less than our study.8 rahman et al.6 from saudi arabia mentioned that 92% of their students had personal laptops. uribe et al.7 from chile stated that 92.2% of students had personal e-mails which was the nearest to ours.7 a total of 26% for owning personal computer was mentioned in bello et al.5 study from nigeria. virtanen stated that in 2002, 60% of dental students had personal e-mail in finland which is not a high rate. about 18% used medline and full text articles [3]. in romanov’s4 study in finland, 24% of searched medline twice a month, 10% seeked full text articles twice a month and 12% never search medline while 40% never used full text articles. it seems that insufficient professional use of computer and searching skills are even found among developed countries such as finland and training are not enough for meeting their needs because 1/3 of them also proclaimed for more training needs. in this finnish study, general usage of computers reported higher than professional which was similar to ours.4 odusanya14 stated that 19% of nigerian students used computer for games, 18% for translation and 58% of them had personal e-mail which is a low proportion, while in our study 63% of students used computers for games, which is much more than odusanya study and almost everyone had personal e-mail which was also significantly more than odusanya study. in professional usage of computers like making powerpoint slides in rahman’s6 study in saudi arabia, the rate was 61% while ours was 97% which is a high one. skill and literacy among dental students in saudi arabia, 35% had proper skills in using pubmed and 42% had proper skills in using search engines like google. in bello et al.5 study (nigeria), 54% were trained to use computers, 18.9% had appropriate computer literacy and skill, 58.8% had moderate and 22.3% had mild computer skills. in our study, it skills was assessed in two field: computer using skills and internet searching skills among students. both skills were insufficient and students could gain about half of the maximum possible which maybe comparable with those of the nigerian students. therefore, emphasis should be placed on training it skills and increasing related literacy by interactive continuous workshops and by it-enabled teaching and learning. strengths and limitations of the study the study included all dental students living in tehran, and the high response rate speaks for the representativeness of the present study and increases its value. our cross-sectional design permits investigation of potential associations between one’s it utilization and level of skill; causality clearance, however, would require a longitudinal design. surveys with a self-administered questionnaire may also have some typical shortcomings, such overand under-reporting due to social acceptance, a phenomenon that the anonymous character of this study may have minimized. the other limitation of our study was poor accessibility to senior dental students due to their educational schedules and sometimes they do not have enough time to answer all questions. this limitation was dealt with by explanations about the importance of their participation in planning for future intervention. conclusion information technology utilization and skills were comparable to developing countries and in some aspects with developed ones but was skill insufficient. especially in searching skills which might play a key role for learning and research purposes, most of students expressed a need to achieve more skills. the fact that it skills were associated to a higher it utilization regardless of other background factors casts light on the table 7. the association between general and professional usage of computer and internet and background factor and computer and searching skills; linear regression general usage of it professional usage of it p-value regression coefficient 95% of ci of β p-value regression coefficient 95% of ci of β independent variables owning personal computer 0.165 −0.119 −25.57 4.40 0.751 0.056 −7.01 9.64 owning website or weblog 0.238 −0.96 −12.07 3.02 0.647 −0.069 −5.20 3.27 age 0.001 0.325 1.054 4.10 0.350 0.174 −1.00 2.75 gender 0.295 −0.099 −10.57 3.23 0.675 0.079 −3.81 5.82 marital status 0.472 0.064 −4.52 9.71 0.577 0.088 −3.49 6.18 last score 0.636 0.044 −2.35 3.84 0.102 0.279 −0.36 3.77 socioeconomic status (ses)* 0.346 0.085 −6.95 2.45 0.423 −0.155 −4.27 1.83 paternal educational* 0.596 −0.69 −6.14 3.54 0.138 0.417 −0.79 5.44 maternal education* 0.277 0.147 −2.11 7.30 0.074 −0.455 −5.78 0.28 living in dormitory 0.583 0.055 −6.23 11.03 0.843 0.037 −4.831 5.88 research experience 0.083 −0.148 −11.99 0.75 0.020 −0.378 −9.00 −0.82 computer using skills 0.31 0.223 −2.03 −0.098 0.746 0.069 −0.51 0.71 internet searching skills 0.027 0.230 0.12 1.91 0.973 −0.007 −0.61 0.59 *these variables are used in continuous forms. simin z. mohebbi et al. 87j contemp med sci | vol. 4, no. 2, spring 2018: 82–87 research dental students’ adherence to it importance of training as the main factor to provide full utilization. acknowledgments this study was a part of thesis no. 6071 that has been supported by school of dentistry, tehran university of medical sciences. we would like to thank students for their active participation. competing interest the authors declare that they have no competing interests. authors’ contributions the authors sr, ry, mm, mb have contributed equally to this work in making substantial contributions to conception and design, acquisition of data, and analysis and interpretation of data and being involved in drafting the manuscript or revising it critically for important intellectual content. all authors read and approved the final manuscript. references 1. mariño r, habibi e, morgan m, au-yeung w. information and communication technology use among victorian and south australian oral health professions students. j dent educ. 2012;76:12. 2. mohebbi sz, sahebjamei m, kharazifard mj, ebrahimpour mr, bonabi m. application of information technology by iranian dentists: assessment of knowledge and performance. j islam dent assoc iran. 2014;26:1. 3. virtanen ji, nieminen p. information and communication technology among undergraduate dental students in finland. eur j dent educ. 2002;6:147–152. 4. romanov k, aarnio m. a survey of the use of electronic scientific information resources among medical and dental students. bmc med educ. 2006;6:28. 5. bello i, arogundade f, sanusi a, ezeoma i, abioye-kuteyi e, akinsola a. knowledge and utilization of information technology among health care professionals and students in ile-ife, nigeria: a case study of a university teaching hospital. j med internet res. 2004;6:4. 6. rahman g. use of computers among students of dental college in saudi arabia. j educ ethics dent. 2011;1:1. 7. uribe s, mariño rj. internet and informa tion technology use by dental students in chile. eur j dent educ. 2006;10:3. 8. rajab ld, baqain zh. use of information and communication technology among dental students at the university of jordan. j dent educ. 2005;69:3. 9. mattheos n, nattestad a, schittek m, attström r. computer literacy and attitudes among students in 16 european dental schools: current aspects, regional differences and future trends. eur j dent educ. 2002;6:1. 10. walmsley ad, white da, eynon r, somerfield l. the use of the internet within a dental school. eur j dent educ. 2003;7:27–33. 11. flores-mir c, palmer, n g. northcott hc, huston c, computer and internet usage by canadian dentists. j can dent assoc. 2006;72:3. 12. deputy ministry for education. 2016. http://gpde.behdasht.gov.ir/uploads/ omoomi_dandan91.pdf [accessed 10 august 2016]. 13. komerik n. use of the internet among dental students in turkey. journal dent educ. 2005;69:4. 14. odusanya o, bamgbala oa. computing and information technology skills of final year medical and dental students at the college of medicine university of lagos. niger postgrad med j. 2002;9:4. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms. 06201806 4 j contemp med sci | vol. 1, no. 3, summer 2015: 4–8 research objective this study includes the investigation of antifungal activity of the local propolis against dermatophytes and yeast. methods two fungi belonging to dermatophytes, trichophyton mentagrophytes and t. tonsurans, isolated from patients were tested. six concentrations (0, 5, 10, 15, 20, and 25 mg/ml) of propolis extract were tested against these two fungal pathogens. clotrimazole was used as control with 2 mg/ml concentration. so, five oral candida albicans isolates were extracted from patients, these isolates were given numbers 1, 2, 3, 4 and 5. seven concentrations (0.5, 1, 5, 10, 15, 20 and 25 mg/ml) of propolis extract were tested against c. albicans isolates. results the results revealed presence of significant difference (p < 0.05) in the effect of propolis extract against two dermatophytes of the current study. t. tonsurans was more sensitive to propolis extract terms, the percentage of inhibition being 100% in each of the concentrations (10, 15, 20, 25 mg/ml), whereas it showed 72.4% at 5 mg/ml concentration. t. mentagrophytes did not show complete inhibition percentage which gave a high percentage, 94.2% with 25 mg/ml concentration. additionally, results revealed presence of significant difference (p < 0.05) in the effect of propolis extract against c. albicans isolates of this study. c. albicans 1 isolate was the most effective isolate among the others toward propolis extract. results of this study also showed increasing inhibition zone if the concentration of propolis extract was increased. conclusion the local propolis that was collected from the apiaries of holy karbala province has antifungal activity against t. mentagrophytes, t. tonsurans and c. albicans isolates. keywords antifungal activity, dermatophytes, trichophyton mentagrophytes, trichophyton tonsurans, candida albicans, propolis antifungal activity of propolis against dermatophytes and candida albicans isolated from human mouth alaa abdul-hussein al-daamy, haider abd-al ameer, hasan zuher, hussein monather, bashaer ahmmed & niesreen kadhim introduction mycoses cause a wide range of diseases in human. the diseases are divided into three groups depending on where they occur on our body.1 mycosis (plural: mycoses) is a fungal infection of animals, including humans.2 mycoses are common and a variety of environmental and physiological conditions can contribute to the development of fungal diseases. inhalation of fungal spores or localized colonization of the skin may initiate persistent infections; therefore, mycoses often start in the lungs or on the skin.3 dermatophytes can be divided into superficial, subcutaneous and systemic.4 trichophyton is known as a dermatophyte; a part or group of three genera of fungi cause skin disease in people and animals. in many parts of the world trichophyton mentagrophytes is isolated most frequently. t. mentagrophytes is typically found in moist, carbon-rich environments. it is characterized by flat suede-like colonies, white to cream colour and with distinctive odour. the colour on the underside of the colonies is usually yellow to reddish brown. the granular colony formed typically has a powdery appearance due to the large amount of microconidia spores formed. the macroconidia are smooth, cigar shaped and thin walled with 4–5 cells separated by parallel cross-walls. in comparison to other fungi, t. mentagrophytes grows fairly rapidly.5,6 t. tonsurans is an anthropophilic fungus with a worldwide distribution which causes inflammatory or chronic non-inflammatory finely scaled lesions of skin, nails and scalp. it is a common cause of tinea capitis among the australian aboriginals and american negros.7 candida albicans is the most common fungal human pathogen and is found naturally in human digestive and reproductive organs; as it prefers moist area.8 it causes disease called candidiasis. oral candidiasis (thrush) is the most common oral infection, which is characterized by extensive white pseudomembrane consisting of desquamated epithelial cell, fibrin and fungal hyphae. it is most often seen in patients with diabetes, aids and those using steroid aerosol inhalers.9 propolis is a mixture of bees wax and resins collected by the honeybees from plant buds, leaves and exudates.10 bees use propolis not only as a building material but also as a means of maintaining low levels of bacterial and fungal concentrations in the hive.11 propolis has long been used in oriental folk medicine for curing infections12 and in european ethno-pharmacology as an antiseptic and anti-inflammatory agent for healing wounds and burns.10 there are many pharmaceutical properties of propolis including antibacterial,13 antifungal,14 antiviral,15 antiprotozoal,16 anti-inflammatory,17 antioxidant,18 hepato protective,19 immunostimulating20 and antitumour.21 more than 150 components such as polyphenols, phenolic aldehydes, sesquiterpene quinines, coumarins, amino acids, steroids and inorganic components have been identified in propolis samples.22 the properties and chemical composition of propolis vary with geographical origin23 and the differences in chemical composition are basically due to differences in the bearing plants.24 the solvent that is mostly used for propolis preparation is aqueous ethanol, followed by others, such as ethyl ether, water, methanol and chloroform.25 materials and methods microorganisms: two fungi due to dermatophytes are obtained from clinical laboratories, department of applied medical sciences college, karbala university. these fungi are trichophyton mentagrophytes and trichophyton tonsurans. department of clinical laboratories, college of applied medical sciences, karbala university, iraq. correspondence to alaa abdul-hussein al-daamy (email: aakm7789@gmail.com). (submitted: 27 august – revised version received: 4 september – accepted: – published online: summer 2015) issn 2413-0516 5j contemp med sci | vol. 1, no. 3, summer 2015: 4–8 research antifungal activity of the local propolis against dermatophytes and yeastalaa abdul-hussein al-daamy et al. five isolates of c. albicans obtained from medical microbiology of al-hindiyya hospital were isolated from the oral cavities of the patients and given numbers 1, 2, 3, 4 and 5. propolis extraction and preparation of the concentrations: the method described by ahmed et al. (1998)26 was followed to obtain propolis extract. preparation of propolis extract concentrations have been worked on a series of concentrations including 0.5, 1, 5, 10, 15, 20 and 25 mg/ml by the method described by alaa et al. (2015)27 to test these concentrations against c. albicans. while six concentrations (0, 5, 10, 15, 20, 25 mg/ml) were tested against dermatophytes. determination of antifungal activity against dermatophytes: sabouraud’s dextrose agar (sda) was used in this test and it was prepared as per the manufacturer’s instruction (himedia, india). the method of el-kady et al.,28 with some modification, was followed to determine antifungal activity, as it is blended with concentrations of propolis extract with sabouraud dextrose agar before sterilization, and five concentrations (5, 10, 15, 20, 25 mg/ml) of propolis extract were obtained from it by weighing 0.2, 0.4, 0.6, 0.8, 1 g of the dry propolis extract. these were placed in five conical flasks separately containing 40 ml of sabouraud’s dextrose agar to obtain the final concentration of the above concentrations, respectively. also two conical flasks of media as controls were used, in one of them no quantity of the extract was not added. in other words, we get 0 mg/ml concentration of propolis extract, and the second control added was clotrimazole with concentration of 2 mg/ml as standard antifungal agent. ph of each conical flask was adjusted to 5 by using 1% hcl and naoh. then all flasks were sterilized with autoclave at 121°c for 15 minutes. after the end of the sterilization process, flasks were left to cool to a temperature up to 45–50°c and added 50 µg/ml chloramphenicol (candidtm, india) and 0.5 g/l cyclohexamide and poured in petri dishes and left to cool. wells (6 mm) were worked in the centre of these media. after cultivation of two fungi isolates on sda for 2 weeks, inoculation volume (6 mm) were translated from cultivation cultures to each well. all dishes were incubated at 28°c for 10 days. growth diameter was measured (two diameters perpendicular rate), and the results were recorded. inhibition percentage was calculated by using the following equation: inhibition percentage = control growth-test growth control growth × 100% determination of antifungal activity against c. albicans: the antifungal activity was determined by using the agar diffusion technique. nutrient broth was used as an activation media and was prepared according to the manufacturer’s instruction (himedia, india). potato dextrose agar (pda) was used to determine the antifungal activity of propolis against c. albicans. this agar was prepared according to the manufacturer’s instruction (himedia, india). the media was sterilized in autoclave (121°c for 15 min), after autoclavation it was left to cool and then added an antibiotic (chloramphenicol 50 mg/l). then, the media was poured in petri dishes and left to set; three wells of 5 mm in diameter were bored equidistantly to each petri dish media using a sterile cork borer. then, 50 µl of appropriate dilution of c. albicans isolates were spread on it which activated in nutrient broth at 37°c for 24 hours. the wells were completely filled with the tested concentrations of propolis extract, and the cultures were left in incubator for 2 days at 37°c. the antifungal activity of propolis extract was determined by measuring the diameters of the inhibition zone and compared with control to which 1 mg/ml of nystatin was added as antifungal agent. statistical analysis: according to c. albicans, statistical analysis included factorial experiences analysis 8 × 5 with three replicates. while, statistical analysis of experiment of dermatophytes included factorial experiences analysis, 7 × 2 with two replicates. the factors analysed are the concentration of propolis extract and types of fungi. the significant differences between the averages were also tested by using the test less significant difference (lsd) at the level of probability of 0.05.29 results antifungal activity against dermatophytes: the results showed significant differences (p < 0.05) between two species of genus trichophyton sp. in the current study. in addition, there were significant differences (p < 0.05) between concentrations of propolis extract used in this study. depending on the results shown in table 1 and fig. 1, it is seen that t. tonsurans was more sensitive to propolis extract in terms of table 1. antifungal activity of propolis extract against dermatophytes dermatophytes concentration of propolis extract (mg/ml) clotrimazole 2 mg/ml lsd 0.05 of concentration 0 5 10 15 20 25 diameter growth (mm) 2.379 trichophyton mentagrophytes a 43.5 a b 7 a bc 5.5 a bcd 4.75 a cd 4.5 a de 2.5 a e 0 a trichophyton tonsurans a 29 b b 8 a c 0 b c 0 b c 0 b c 0 b c 0 a lsd 0.05 of fungus 1.271 lsd0.05 of interference 3.365 the numbers refer to mean of diameter growth (mm) ± stander error. various horizontally capital letters refer to present significant differences (p < 0.05) between the concentrations of propolis extract for each fungus separately. various vertical small alphabets refer to present significant differences (p < 0.05) between two fungi for each concentration separately. 6 j contemp med sci | vol. 1, no. 3, summer 2015: 4–8 antifungal activity of the local propolis against dermatophytes and yeast research alaa abdul-hussein al-daamy et al. differences (p < 0.05) between them, also there was an increasing inhibition zone if the concentration was increased, i.e. the concentration 25 mg/ml has the high antifungal activity against all isolates of c. albicans. in contrast, the concentration 0.5 mg/ml has no antifungal activity. according to the results in table 2 and results of statistical analysis, it showed that c. albicans 1 was more sensitive towards propolis extract, because it showed high inhibition zone compared with other c. albicans isolates in all concentration except the concentration 1 mg/ml which showed c. albicans 3 was the most sensitive from the others. conversely, c. albicans 4 was the most resistant to propolis extract from the other isolates, as it gave less inhibition rate. discussion inhibition activity of propolis was studied by many authors, these studies were conducted to an inhibition activity of propolis depending on two factors, these factors were the area geographic and concentration of the collected propolis in these studies.30,31 some differences in results were found among different researchers depending on several factors; propolis composition varies from region to region and therefore, the difference in the composition fig. 1 inhibition percentage of propolis extract against dermatophytes. table 2. inhibition zone (mm) of propolis extract on c. albicans isolates from human mouth no. of c. albicans isolates inhibition diameter (mm) lsd 0.05 concentration nystatin concentration of propolis extract (mg/ml) 1 (mg/ml) 0.5 1 5 10 15 20 25 1 f 2.7 ± 0.62 a h 0 ± 0.0 a g 1.2 ± 0.0 b e 9.8 ± 0.34 a d 12.8 ± 0.5 a c 14.9 ± 0.4 a b 17.6 ± 0.7 a a 22.6 ± 1.9 a 0.388 2 f 2.4 ± 0.13 ab g 0 ± 0.0 a g 0 ± 0.0 c e 5.7 ± 0.19 b d 7.7 ± 0.41 b c 9.8 ± 0.21 b b 12.9 ± 0.8 b a 15.5 ± 0.7 c 3 f 2.6 ± 0.18 ab h 0 ± 0.0 a g 1.82 ± 0.1 a e 5.1 ± 0.26 c d 6.3 ± 0.32 c c 7.6 ± 0.18 d b 9.8 ± 0.43 c a 12.8 ± 0.4 d 4 d 2.3 ± 0.26 ab f 0 ± 0.0 a f 0 ± 0.0 c f 0 ± 0.0 e e 1.19 ± 0.2 d c 3.9 ± 0.23 e b 6.7 ± 0.35 d a 10.9 ± 0.2 e 5 f 2.11 ± 0.25 b g 0 ± 0.0 a g 0 ± 0.0 c e 3.95 ± 0.2 d d 5.8 ± 0.37 c c 9.2 ± 0.55 c b 13.3 ± 0.8 b a 17.2 ± 0.9 b lsd 0.05 isolates 0.524 the numbers refer to mean of inhibition diameter (mm) ± standard error. various horizontally capital letters refer to present significant differences (p < 0.05) between the concentrations of propolis extract for each isolate separately. various vertically small letters refer to present significant differences (p < 0.05) between isolates of c. albicans for each concentration separately. between the concentrations of the propolis extract to each isolates of c. albicans when compared with control (nystatin). in concentration 0.5 mg/ml, we showed no inhibition zone against all the isolates of yeast but in the concentration of 1 mg/ml, inhibition zones were present only against isolates 1 and 3, while in the concentration of 5 mg/ml showed no inhibition zone only against c. albicans 4; the other concentrations showed inhibition zone against all isolates with significant differences (p < 0.05). when we compared the inhibition zone of each concentration to each separately isolated nystatin we found significant percentage of inhibition with 100% in each of the concentrations (10, 15, 20, 25 mg/ml) compared with clotrimazole which also gave the 100% inhibition 100% as well. but the percentage of inhibition was 72.4% at a concentration 5 mg/ml. the results also showed that the percentage of inhibition of t. mentagrophytes were 0.0, 83.9, 87.3, 89.0, 94.2% of the concentrations 0, 5, 10, 15, 20, 25 mg/ml, respectively. antifungal activity against c. albicans: in the current study, we observed that local propolis has antifungal activity against c. albicans (1, 2, 3, 4 and 5). we found significant differences (p < 0.05) 7j contemp med sci | vol. 1, no. 3, summer 2015: 4–8 research antifungal activity of the local propolis against dermatophytes and yeastalaa abdul-hussein al-daamy et al. of the extract may lead to the inhibition of a difference in percentage from propolis extracted from one area to another.32,33 inhibition percentage is also different from one study to other according to the concentration of extracts, temperature and duration of cuddling. in addition, the method that is used to extract propolis may lead to variation in the results. in one study; that used serial concentrations of iranian propolis against t. tonsurans, found a certain inhibition of growth depending on the used concentration of extract propolis.34 in other studies, the antifungal activities of 70% of ethanolic extract of propolis (eep) against fungi were in the following order: c. crusei, c. albicans, c. glabrata, c. parapsilosis and c. tropicalis strains. analysis among the tested yeasts showed that c. crusei was the most sensitive in 70% of eep and the sensitivity of the yeasts decreased in the order: c. albicans, c. parapsilosis, c. tropicalis and c. glabrata.35 other studies showed commercial propolis products had a more pronounced activity against gram-positive bacteria and c. albicans ft2010, and a less evident action against gram-negative bacilli and c. albicans atcc 10231. the controversial result concerning c. albicans ft2010 and c. albicans atcc 10231 could be explained by the inherent virulence of each strain. that is one reason to employ different microbial strains of a same species. among the yeasts, this study showed that c. albicans was more susceptible to propolis than other species. this result is supported by rezende et al. (2006),36 who found the following order of susceptibility to hydroalcoholic propolis: c. albicans, c. tropicalis, c. krusei and c. guilliermondii. another study has shown that a commercial 20% ethanol propolis extract inhibited c. albicans strains collected from hiv-positive patients with oral candidiasis.37 another study showed that the propolis volatiles were also active against non-pathogenic fungi and fungal human pathogens aspergillus niger, saccharomyces cerevisiae, c. albicans, c. tropicalis, c. glabrata, cladosporium cladosporioides, cladosporium sphaerospermum.38 in another study it was seen that the ethanolic extract showed higher zone of inhibition against c. albicans (13.2 mm) at 200 mg/ml concentration.39 the results of one study showed that amphotericin b and thyme essential oil had inhibitory effects on the growth of different candida species, therefore, after performing controlled studies and experimenting with different types of honey formulation, it was concluded that thyme essential oil may be used in treating empirical candidiasis. in conclusion, these essential oils have anti-candida activity, in vitro and related to their concentration on paper disc.40 the properties and chemical composition of propolis vary with geographical origin30 and the differences in chemical composition are basically due to the differences in the bearing plants, although many active components have been identified in propolis.31,32 one iranian study showed that the total flavonoid and phenolic contents were 7.3% and 36%, respectively, which suggests that the strong antimicrobial activity of iranian propolis against gram-positive and c. albicans may be due to the high levels of phenolic and flavonoid compounds.33 the difference between our study and other studies may be due to the type of propolis, the type of strain and the concentration of the extract that was used in the present study. results of the current study are similar to the results of one new study about the activity of propolis extract against bacteria,27 and the results in both the studies provide the activity of the local propolis extract towards microorganisms. conclusion the local propolis that was collected from the apiaries of holy karbala province has antifungal activity against t. mentagrophytes, t. tonsurans and oral c. albicans isolates from patients.  references 1. carris lm, little cr stiles cm. introduction to fungi. the plant health instructor. 2012. doi: 10.1094/phi-i-2012-0426-01 2. kazemi a. an overview on the global frequency of superficial/cutaneous mycoses and deep mycoses. jundishapur j microbiol. 2013 jun 1;6(3):202–4. doi: http://dx.doi.org/10.5812/jjm.10725 3. bench m. fungal infections. woman’s health. front shop pharmacy magazine. 15 february 2013. 4. malcolm d, richardson d, warnock w. fungal infection: diagnosis and management, 4th ed. london: john wiley & sons; 2012. 5. brooks gf. jawetz, melnick, & adelbergs medical microbiology, 25th ed. new york: mcgraw hill; 2010. pp. 630–2. 6. trichophyton spp. (https://en.wikipedia.org/wiki/trichophyton), jan 2015. 7. julius k. laboratory handbook of dermatophytes: a clinical guide and laboratory handbook of dermatophytes and other filamentous fungi from skin, hair, and nails. belmont, ca: star publishing co.; 1997. 8. kim s, francoeur t. thrush in the mouth. webmd. retrieved from http:// www.webmed.com/oral-health/thrush-in-the-mouth.; 2011. 9. akpan a, morgan r. oral candidiasis. postgrad med j. 2002;78(922): 455–9. doi: http://dx.doi.org/10.1136/pmj.78.922.455 pmid: 12185216 10. ghisalberti el. propolis: a review. bee world j. 1979;60:59–84. 11. popova m, silici s, kaftanoglu o, bankova v. antibacterial activity of turkish propolis and its qualitative and quantitative chemical composition. phytomedicine 2005 mar;12(3):221–8. doi: http://dx.doi.org/10.1016/j. phymed.2003.09.007 pmid: 15830845 12. cheng pc, wong g. honey bee propolis: prospects in medicine. bee world 1996;77(1):8–15. doi: http://dx.doi.org/10.1080/0005772x.1996.11099278 13. lu lc, chen yw, chou cc. antibacterial and dpph free radical-scavenging activities of the ethanol extract of propolis collected in taiwan. j food drug analysis 2003;11(4):277–82. 14. kujumgiev a , tsvetkova i , serkedjieva y , bankova v , christov r , popov s. antibacterial, antifungal and antiviral activity of propolis of different geographic origin. j ethnopharmacol. 1999 mar;64(3):235–40. doi: http:// dx.doi.org/10.1016/s0378-8741(98)00131-7 pmid: 10363838 15. amoros m, lurton e, boustie j, girre l, sauvager f, cormier m. comparison of the anti-herpes simplex virus activities of propolis and 3-methyl-butyl2-enyl caffeate. j nat prod. 1994 may;57(5):644–7. doi: http://dx.doi. org/10.1021/np50107a013 pmid: 8064297 16. scheller s, szaflarski j, tustanowski j, nolewajka e, stojko a. biological properties and clinical application of propolis i. some physic-chemical properties of propolis. arzeneimittelforschung 1977;27(4):889–90. pmid: 577469 17. dobrowolski jw, vohora sb, sharma k, shah sa, naqvi sa, dandiya pc. antibacterial, antifungal, antiamoebic, antiinflammatory and antipyretic studies on propolis bee products. j. ethnopharmacol. 1991 oct;35(1):77–82. doi: http://dx.doi.org/10.1016/0378-8741(91)90135-z pmid: 1753797 18. isla mi, moreno min, sampietro ar, vattuone ma. antioxidant activity of argentine propolis extracts. j ethnopharmacol. 2001 jul;76(2):165–70. doi: http://dx.doi.org/10.1016/s0378-8741(01)00231-8 19. gonzales r, corcho i, remirez d, rodriguez s, ancheta o, merino n, gonzales a, pascual c. hepato protective effects of propolis extract on carbon tetrachloride-induced liver injury in rats. phytother res. 1995 mar;9(2):114–7. doi: http://dx.doi.org/10.1002/ptr.2650090207 20. dimov v, ivanovska n, bankova v, nikolov n, popov s. immunomodulatory action of propolis: iv. prophylatic activity against gram-negative infections and adjuvant effect of water-soluble derivative. vaccine 1992 jan;10(12):817–23. doi: http://dx.doi.org/10.1016/0264-410x(92)90043-j pmid: 1455907 21. orsolić n, sver l, terzić s, tadić z, basić i. inhibitory effect of water-soluble derivative of propolis and its polyphenolic compounds on tumor growth 8 j contemp med sci | vol. 1, no. 3, summer 2015: 4–8 antifungal activity of the local propolis against dermatophytes and yeast research alaa abdul-hussein al-daamy et al. and metastasizing ability: a possible mode of antitumor action. nutr cancer 2003 nov;47(2):156–63. doi: http://dx.doi.org/10.1207/s15327914nc4702_8 pmid: 15087268 22. frenkel k, wei h, bhimani r, ye j, zadunaisky ja, huang mt, ferraro t, conney ah, grunberger d. inhibit of tumor promoter-mediated processes in mouse skin and bovine lens by caffeic acidphenethyl ester. cancer res. 1993 mar;53(6):1255–61. pmid: 7680281 23. marcucci mc. propolis: chemical composition, biological properties and the therapeutic activity. apidologie 1995;26(2):83–99. doi: http://dx.doi. org/10.1051/apido:19950202 24. markham ke, mitchell ka, wilkins al, daldy ja, lu y. hplc and gc-ms identification of the major organic constituents in new zealand propolis. phytochemistry 1996;42(1):205–11. doi: http://dx.doi. org/10.1016/0031-9422(96)83286-9 25. silva kr, francielle tm, kelly ad, shirley dk, herta sds. antimicrobial activity from a brazilian propolis oily extract compared with other propolis extract. revista ciencias eatase naturais. 2010;12(2):327–38. 26. ahmed i, mehmood z, mohammad f. screening of some indian medicinal plants for their antimicrobial properties. j ethnopharmacol. 1998 sept;62(2):183–93. doi: http://dx.doi.org/10.1016/s0378-8741(98)00055-5 pmid: 9741890 27. alaa ak, noor ya, raghad ms, murtadha km, ali jd, rawaa sa. study of antibacterial activity in the local iraqi propolis. j cont med sci. 2015;1(2): 6–8. 28. el-kady ia, mohamed ss, mostafa em. antibacterial and antidermatophyte activities of some essential oils from spices. qatar university sci j. 1993;13(1):63–9. 29. al-emam mmt. design and analysis of experiments. riyadh: mars publishing house, kingdom of saudi arabia; 2007. 30. kujumgiev a, tsvetkova i, serkedjieva y, bankova v, christov r, popov s. antibacterial, antifungal and antiviral activity of propolis of different geographic origin. j ethnopharmacol. 1999 mar;64(3):235–40. doi: http://dx.doi.org/10.1016/s0378-8741(98)00131-7 pmid: 10363838 31. marcucci mc. propolis: chemical composition, biological properties and the therapeutic activity. apidologie 1995;26(2):83–99. doi: http://dx.doi. org/10.1051/apido:19950202 32. burdock ga. review of the biological properties and toxicity of bee propolis (propolis). food chem toxicol. 1998 apr;36(4):347–63. doi: http://dx.doi.org/10.1016/s0278-6915(97)00145-2 pmid: 9651052 33. yaghoubi smj, ghorbani gr, soleimanian zad s, satari r. antimicrobial activity of iranian propolis and its chemical composition. daru. 2007;15(1):45–8. 34. gavanji s, khorsand ma, malakootikah j, asgarii mj, gavanji j, vaezi r. antimicrobial effects of propolis bee as green product. international conference on environment 2010. 35. kačániová m, melich m, kačániová v, felšöciová s, sudzinova j. the antimicrobial activity of honey and propolis against yeasts candida species. lucrari stiintifice: zootehnie si biotehnologii 2013;42(2):167–73. 36. rezende g, pimenta f, costa d. antimicrobial activity of two brazilian commercial propolis extracts. braz j oral sci. 2006 jan–mar;5(16): 32–33. 37. martins rs, péreira es, lima sm, senna mi, mesquita ra, santos vr. effect of commercial ethanol propolis extract on the in vitro growth of candida albicans collected from hiv-seropositive and hiv-seronegative brazilian patients with oral candidiasis. j oral sci. 2002 mar;44(1):41–8. doi: http:// dx.doi.org/10.2334/josnusd.44.41 pmid: 12058869 38. bankova v, popova m, trusheva b. propolis volatile compounds: chemical diversity and biological activity: a review. chem cent j. 2014 may 2;8:28. doi: http://dx.doi.org/10.1186/1752-153x-8-28 pmid 24812573 39. kumar n, ahmad mk, dang r, husain a. antioxidant and antimicrobial activity of propolis from tamil nadu zone. j med plants res. 2008 dec;2(12): 361–4. doi: http://dx.doi.org/10.5897/jmpr 40. omran sm, esmailzadeh s. comparison of anti-candida activity of thyme, pennyroyal, and lemon essential oils versus antifungal drugs against candida species. jundishapur journal of microbiology 2009;2(2):53–60. 131j contemp med sci | vol. 5, no. 3, may–june 2019: 131–135 original mammographic, ultrasonographic and pathologic correlations of focal asymmetric breast densities among a sample of iraqi women hiba m. abdulwahid,a* enam a. khalel,b and nada a.s. alwana anational cancer research center, university of baghdad, baghdad, iraq. bradiology department, oncology teaching hospital, baghdad, iraq. *correspondence to hiba m. abdul wahid (email: dr.hiba.mohammed85@gmail.com). (submitted: 25 december 2018– revised version received: 20 january 2019 – accepted: 25 april 2019 – published online: 26 june 2019) introduction breast cancer is the most common malignancy in women over the age of 40 and the most reported cause of cancer death in women worldwide.1,2 it has been shown that the early detection and screening followed by appropriate management reduces the associated morbidity and mortality rates of the disease.3 previous surveys from iraq have reported that breast cancer is the most frequent registered malignancy4 and that most of the cases are often detected among middle aged women in relatively advanced stages.5–7 mammography is an important screening tool in the early detection of breast cancers.8 it can detect about 75% of breast cancers at least a year before they become symptomatic.9 mammogram interpretation is a time-consuming and sometimes considered as a difficult task due to a wide variability of the detected breast abnormalities and overlapping dense fibroglandular tissues.10 although there is a great difference in the parenchymal patterns and sizes of breasts, the internal structures are nearly symmetrical with similar densities and architectures at mammography. asymmetric breast densities are repeatedly seen at screening or diagnostic mammography and are usually nonspecific and common finding in healthy women, however; these findings are sometimes due to a hidden malignancy.11,12 thus, it has been recommended that when asymmetric finding is detected on mammogram, it should be assessed carefully and further evaluation is needed to decide whether it reflects a normal variant or something that is more significant.13 the american college of radiology-breast imaging reporting and data system (acr-bi-rads) has developed a lexicon for asymmetric breast densities.14 an asymmetric finding represents an area of breast tissue that has a fibroglandular density that is more extensive in one breast in comparison with the corresponding region in the contralateral breast while a mass is a three-dimensional lesion with convex outward margins and is usually seen on two orthogonal views.15 asymmetric findings on mammogram were classified into four types according to the fourth edition of bi-rads including: (1) asymmetry as an area of fibroglandular tissue seen on one mammographic view frequently caused by overlapping of normal parenchymal tissue of breast; (2) global asymmetry which is seen over at least one quarter of the breast and is usually a normal variant; (3) developing asymmetry is a new larger and more conspicuous than on a previous examination16,17; and (4) focal asymmetry, the core of this study, which is defined as asymmetry of tissue density with similar shape on two views but has no borders and should be distinguished from a mass. it may represent true abnormality rather than superimposition.14 the aim of the study is to evaluate the mammographic focal asymmetric breast densities (fabd) in order to highlight which fabd might need further workup through detailed ultrasonographic characterization and comparison with the pathological results in indicated cases. patients and methods a cross-sectional analytic study was performed in the oncology teaching hospital, medical city from march 2018 objective the aim of the study is to evaluate the mammographic focal asymmetric breast densities (fabd) in order to highlight which fabd might need further workup through detailed ultrasonographic characterization and comparison with the pathological results in indicated cases. methods a cross-sectional analytic study was performed in the oncology teaching hospital, medical city from march 2018 to november 2018. the study included 70 women who attended the main referral center for early detection of breast tumors with fabd were detected by mammography. the focal asymmetry was analyzed and the other associated findings were assessed and registered. while breast ultrasound was performed for all patients, fine needle aspiration (fna) was carried out for cases with suspicious findings on ultrasound and any fna suspicious or malignant lesion was subsequently biopsied and the results were recorded. results mammographic fabd was found in the right breast in 37 cases (52.9%) and in the left in 33 cases (47.1%). the upper outer quadrant was the most common location of fabd (43 cases). in 25 cases (34.2%) with fabd, there was no abnormality found on ultrasound, but normal looking breast parenchymal tissue. ultrasound had shown benign findings in 30 cases (44.2%). on the other hand, suspicious and/ or malignant features were observed in 15 cases (22.4%) which subsequently proved to be malignant by fna and biopsy. architectural distortion or grouped microcalcifications and a clinically palpable fabd were associated with malignant fabd; no benign fabd revealed to be associated with these findings. conclusion the finding of fabd is common on mammography and mostly represents a benign entity of normal breast parenchymal tissue. however, it may indicate an underlying hidden malignancy especially in the presence of superadded mammographic findings as ill-defined mass, architectural distortion or grouped microcalcification. keywords mammography, ultrasonography, asymmetric breast density, iraq issn 2413-0516 132 j contemp med sci | vol. 5, no. 3, may–june 2019: 131–135 mammographic, ultrasonographic and pathologic correlations of focal asymmetric breast densities original h.m. abdulwahid et al. to november 2018. the study included 70 females who attended the main referral center for early detection of breast tumors either for screening or diagnostic mammogram examinations, and fabd (as defined by the acr-bi-rads lexicon) had been detected on their mammograms. all mammograms were performed using the analog mammogram machine (siemens). two mammographic views were taken for each breast; mediolateral oblique and craniocaudal views. the mammographic images were interpreted by an experienced specialist radiologist. the focal asymmetry was analyzed and compared with previous mammograms (when available) searching for any change. other associated findings were assessed and registered, these include: architectural distortion, grouped microcalcification and any associated mass lesion. breast ultrasound was performed for all patients by using ge machine (voluson e6 with linear transducer of frequency of 7.5–12 mhz) and ultrasound findings were also recorded (any mass or underlying suspicious area or only prominent fibroglandular tissue) and classified according to the ultrasound bi-rads lexicon. fine needle aspiration was done for any suspicious finding on ultrasound, any fine needle aspiration (fna) suspicious or malignant lesion was subsequently biopsied and the corresponding cytological and histopathological results were recorded. statistical analysis the collected data were tabulated and analyzed using microsoft excel 2010. the categorical data were presented as frequency and percentage tables. p-value <0.05 were considered as statistically significant. results: during the study period, fabd were found in the mammograms of 70 female patients. breast ultrasound was performed for all those patients. fabd was found in the right breast in 37 cases (52.9%) and in the left in 33 cases (47.1%). the upper outer quadrant (uoq) was the most common location of fabd, it was found in 43 cases (61.4%) followed by the retroareolar region in 15 cases (21.4%). other locations are summarized in table 1. in 25 cases (35.7%) with fabd on mammogram, there was no abnormality found on ultrasound, but normal looking breast parenchymal tissue. ultrasound had shown benign findings in 30 cases (42.8 %). suspicious and/or malignant features were the sonographic findings in 15 cases (21.4%) which subsequently proved to be malignant by fna and biopsy. this was demonstrated in table 2. ductal dilatation was the ultrasound finding in 12 cases of fabd (17%) and was located in the retroareolar region with significant relation between retroareolar fabd and ductal dilatation (p < 0.05). ultrasound showed cystic lesions in nine cases (12.8%), seven of them (10%) appeared as simple breast cysts, one (1.4%) was a complicated cyst, and the other one (1.4%) was a complex cystic lesion with a solid component invading the cyst wall (i.e. suspicious for malignancy; bi-rads iv lesion). fna and biopsy subsequently revealed breast cancer in the last complex lesion described. solid masses were the ultrasound findings in nine cases (12.8%), four of them (5.7%) appeared well defined and table 2. final diagnostic outcome according to ultrasound and pathologic results final diagnostic outcome no. percentage (%) note normal appearing breast tissue by ultrasound 25 35.7 depend on us bi-rads classification benign findings 30 42.8 depend on us bi-rads classification malignant findings 15 21.4 diagnosed by fna and biopsy total 70 100 bi-rads, breast imaging reporting and data system; fna, fine needle aspiration. table 1 location of mammographic fabd in each breast location in either breast no. percentage (%) uoq 43 61.4 retroareolar 15 21.4 uiq 5 7.14 midaspect 4 5.7 liq 3 4.28 total 70 100 fabd, focal asymmetric breast densities; uoq, upper outer quadrant; uiq, upper inner quadrant. diagnosed sonographically as probably benign lesion (bi-rads iii) while five cases (7.14%) with fabd were irregular hypoechoic masses on us and had spiculated outlines (highly suggestive of malignancy; bi-rads v lesions), they were actually proved to be malignant on fna and biopsy later on. in 14 cases (20%) with fabd, ultrasound showed heterogeneous ill-defined areas seen in locations corresponding to their fabd locations on mammogram. pathological results showed fibrocystic changes in 8.5 % (six cases), ductal carcinoma in situ (dcis) in 2.85% (two cases), and invasive ductal carcinoma in 8.5 % (six cases). in one case (1.4%), the ultrasound showed thickened skin with edematous breast parenchymal tissue and inflammatory breast cancer was the histopathological result in this case. the summary was illustrated in table 3. an associated mass on mammogram was seen in six cases (two appeared benign and four proved to be malignant). in the malignant cases, the mass was irregular with ill-defined margin while in the benign cases, the mass was relatively well defined. grouped microcalcification was observed in only two fabd and architectural distortion was observed in nine fabd areas. all these were proved to be malignant on subsequent pathological results. in this study, two cases with proved malignant fabd were palpable on clinical examination (figs. 1–5). discussion many previous studies have reported that both malignant and benign breast lesions might be the cause of fabd on mammograms; indicating that it is challenging to decide which lesions need supplementary assessment with fna or biopsy.15 there 133j contemp med sci | vol. 5, no. 3, may–june 2019: 131–135 original mammographic, ultrasonographic and pathologic correlations of focal asymmetric breast densitiesh.m. abdulwahid et al. table 3. ultrasound findings of fabd lesions correlated with the corresponding us bi-rads classification and pathological results ultrasound findings us bi-rads no. percentage (%) pathological results (biopsy) when indicated normal fibroglandular tissue i 25 35.7 ductal dilatation ii 12 17 cyst simple cyst ii 7 10 complicated cyst iii 1 1.4 complex cyst (with solid component) iv 1 1.4 breast cancer solid mass probably benign mass iii 4 5.7 irregular hypoechoic mass v 5 7.14 invasive ductal carcinoma heterogeneous ill-defined area iv 6 8.5 fibrocystic changes 2 2.8 ductal carcinoma in situ 6 8.5 invasive ductal carcinoma thick skin, edema, no mass, suspicious axillary ln iv-c 1 1.4 inflammatory breast cancer total 70 100 fig. 1 a 52-year-old female presented with focal asymmetry at the upper outer quadrant of left breast at mediolateral and craniocaudal mammographic views (a and b). subsequent ultrasound showed irregular hypoechoic lesion with ill-defined margin (c). fna and biopsy showed invasive breast carcinoma. a b c fig. 2 a 56-year-old female referred for screening mammography. there is focal asymmetry at the upper outer quadrant of right breast in craniocaudal and mediolateral oblique views (a). ultrasound showed heterogeneous ill-defined area (suspicious) (bi-rads iv). biopsy revealed fibrocystic changes. a b c fig. 4 a 43-year-old female presented for mammography. there is focal asymmetry at the retroareolar region of the left breast associated with relatively well circumscribed mass (a and b). ultrasound (c) showed well circumscribed oval shaped hypoechoic mass (typical benign ultrasound features) (bi-rads iii) and the patient was recommended to follow up. a b c fig. 3 a 60-year-old female presented with focal asymmetry at the retroareolar region of right breast in mediolateral oblique and craniocaudal views (a and b). ultrasound revealed ductal dilatation with echogenic content inside (c). fna result was consistent thick intraductal secretion. a b c fig. 5 a 44-year-old female presented with heterogeneously dense breast tissue on mammography. there is focal asymmetry at the upper outer quadrant of left breast on mediolateral oblique view (a) (craniocaudal view not available). ultrasound at corresponding region showed heterogeneous ill-defined area (b). biopsy revealed ductal carcinoma in situ (dcis). a b were many reported benign pathologies that cause fabd such as focal fibrotic changes of the breast, pseudoangiomatous hyperplasia and breast infections (tuberculous or pyogenic).18–20 these were consistent with the results of this study which revealed about 42.8% of fabd were actually caused by benign findings such as ductal dilatation, fibrocystic changes and probably benign lesions. some literatures reported that breast cancer was found in about 0–14% of asymmetric breast tissue.21 others showed that invasive lobular carcinoma of breast was found in 15% of 134 j contemp med sci | vol. 5, no. 3, may–june 2019: 131–135 mammographic, ultrasonographic and pathologic correlations of focal asymmetric breast densities original h.m. abdulwahid et al. mammographic focal asymmetry,22 mucinous breast carcinoma in 4% of focal asymmetry,23 and tubular breast carcinoma in 5%.24 similarly, in this study, the results show that 21.4% of fabd were caused by malignant breast pathology and were: invasive ductal carcinoma in 15.6%, dcis in 2.8%, inflammatory breast cancer in 1.4% and papillary breast cancer in 1.4% of cases. about 35.7% of fabd cases included in this study had shown no abnormality on ultrasound. this was lower than those reported by rissanen et al.25 which showed that 53% of the fabd cases had normal ultrasound in their study which included 15 patients. other researchers documented that all patients with fabd had normal ultrasounds; nevertheless the number of examined cases in their studies was too limited to detect abnormal ultrasonographic findings.19 all cases with fabd that were located in the upper inner quadrant (uiq) in this study had revealed normal ultrasonographic findings except one case which showed heterogeneous ill-defined area and proved to be fibrocystic changes on fna and biopsy. this was in agreement with the study of zare and langroudi26 which showed normal ultrasound in all fabd cases located in the uiq. in the current study, 80% of cases with retroareolar fabd showed ductal dilatation on ultrasound with significant relationship between retroareolar fabd and ductal dilatation (p < 0.05). these results were also consistent with the latter study which reported that 80% of retroareolar asymmetry was actually due to ductal dilatation.26 architectural distortion was found in nine cases (12.8% of total cases studied) and all were proved to be malignant on biopsy. grouped microcalcifications were detected in two cases with fabd; both of which were also malignant as confirmed histopathologically by subsequent biopsy results. these findings were in accordance with the results recorded by sperber et al.27 four cases of fabd in this study were associated with an irregular mass on mammogram and another two cases of fabd were palpable on clinical examination. all these were histopathologically confirmed to be invasive ductal carcinoma by the corresponding excised biopsies; hence further evaluation of such cases would be mandatory. that was in agreement with the findings reported by sperber et al.27 who believed that biopsy was absolutely necessary when focal asymmetry on mammography was palpable on clinical examination. on the other hand, our results contradict those presented by dennis et al.28 who proposed that a palpable abnormality with normal mammogram should not be biopsied. in such manner, many cases of breast cancer would be missed. many previous literatures had reported that focal asymmetry that was not present in the previous mammogram or became larger in size when compared with the previous mammogram (i.e. new or growing fabd) should have a special consideration as it would be more suspicious than focal asymmetry that was stable in size and appearance.13 in this study, all patients attended the breast unit for the first time (either for screening or diagnostic mammogram) and no previous mammograms were available for comparison. if a focal asymmetry had its central part more dense than its periphery, then it should be regarded as suspicious because this concentrated density might be due to small hidden malignancy,29 whereas if the focal asymmetry had subtle lucencies within, it would be mostly a benign finding due to the superimposed fibroglandular tissue with the lucencies representing areas of interspersed fat. conclusion the finding of fabd is common on mammography and is mostly a benign entity representing normal breast parenchymal tissue. fabd may indicate underlying hidden malignancy in the presence of superadded mammographic findings as ill-defined mass, architectural distortion or clustered microcalcification. mammographic fabd that are not associated with the latter findings nor with a clinically palpable mass are most likely indicating a benign finding and could be followed safely. acknowledgments the authors would like to acknowledge the support of the staff working in the radiology and pathology departments of the oncology teaching hospital and the main referral center for early detection of breast tumors. conflicts of interest the authors disclose that there are no conflicts of interest.  references 1. bray f, ferlay j, soerjomataram i, siegel rl, torre la, jemal a. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin. 2018;68:394–424. 2. kelly km, dean j, comulada ws, lee sj. breast cancer detection using automated whole breast ultrasound and mammography in radiographically dense breasts. eur radiol. 2010;20:734–742. 3. smith ra, cokkindes v, brooks d, saslow d, shah m, brawley ow. cancer screening in the united states, 2011: a review of current american cancer society guidelines and issues in cancer screening. ca cancer j clin. 2011;61:8–30. 4. annual report. iraqi cancer registry 2015. iraqi cancer board, ministry of health and environment, republic of iraq, 2018. 5. alwan nas. breast cancer among iraqi women: preliminary findings from a regional comparative breast cancer research project. j glob oncol. 2016;2:255–258. 6. alwan nas, tawfeeq f, maallah m, sattar s. the stage of breast cancer at the time of diagnosis: correlation with the clinicopathological findings among iraqi patients. j neoplasm 2017;2:1–10. 7. alwan nas, kerr d, al-okati d, pezella f, tawfeeq fn. comparative study on the clinicopathological profiles of breast cancer among iraqi and british patients. open public health j. 2018;11:177–191. 8kopans db, smith ra, duffy sw. mammographic screening and “overdiagnosis”. radiology. 2011;260:616–620. 9. schonberg ma, ramanan ra, mccarthy ep, marcantonio er. decision making and counseling around mammography screening for women aged 80 or older. j gen intern med. 2006;21:979–985. 10. fenton jj, egger j, carney pa, cutter g, d’orsi c, sickles ea, et al. reality check: perceived versus actual performance of community mammographers. ajr am j roentgenol. 2006;187:42–46. 11. samardar p, de paredes es, grimes mm, wilson jd. focal asymmetric densities seen at mammography: us and pathologic correlation. radiographics. 2002;22:19–33. 12. kopans db. suspicious lesions and lesions with a high probability of malignancy. in: mcallister l, ed. breast imaging. 3rd ed.; lippincott williams & wilkins, philadelphia, pa, 2007, pp. 513–553. 135j contemp med sci | vol. 5, no. 3, may–june 2019: 131–135 original mammographic, ultrasonographic and pathologic correlations of focal asymmetric breast densitiesh.m. abdulwahid et al. 13. youk jh, kim ek, ko kh, kim mj. asymmetric mammographic findings based on the fourth edition of bi-rads: types, evaluation, and management. radiographics. 2008;10:11. 14. sickles ea, d’orsi cj, bassett lw, et al. acr bi-rads: mammography. in: acr bi-rads atlas, breast imaging reporting and data system. 4th ed.; american college of radiology, reston, virginia, 2013. 15. sickles ea. the spectrum of breast asymmetries: imaging features, work-up, management. radiol clin north am. 2007;45:765–771, v. 16. leung jw, sickles ea. developing asymmetry identified on mammography: correlation with imaging outcome and pathologic findings. ajr am j roentgenol. 2007;188:667–675. 17. brenner rj. asymmetric densities of the breast: strategies for imaging evaluation. semin roentgenol. 2001;36:201–216. 18. venta la, wiley el, gabriel h, adler yt. imaging features of focal breast fibrosis: mammographic-pathologic correlation of non-calcified breast lesions. ajr am j roentgenol. 1999;173:309–316. 19. piccoli cw, feig sa, palzaao jp. developing asymmetric breast tissue. radiology. 1999;211:111–117. 20. crowe dj, helvie ma, wilson te. breast infection. mammographic and sonographic findings with clinical correlation. invest radiol. 1995;30:582–587. 21. dawson js, wilson ar. short-term recall for ‘probably benign’ mammographic lesions detected in a three yearly screening programme. clin radiol. 1994;49:391–395. 22. skaane p, skjørten f. ultrasonographic evaluation of invasive lobular carcinoma. acta radiol. 1999;40:369–375. 23. goodman dn, boutross-tadross o, jong ra. mammographic features of pure mucinous carcinoma of the breast with pathological correlation. can assoc radiol j. 1995;46:296–301. 24. elson bc, helvie ma, frank ts, wilson te, adler dd. tubular carcinoma of the breast: mode of presentation, mammographic appearance and frequency of nodal metastases. ajr am j roentgenol. 1993;161:1173–1176. 25. rissanen t, pamilo m, suramo i. ultrasonography as a guidance method in the evaluation of mammographically detected nonpalpable breast lesions of suspected malignancy. acta radiol. 1998;39:292–297. 26. zare z, faghihi langroudi t. findings of breast sonography in patients with focal asymmetric breast density on mammography. iran red crescent med j. 2011;13:404–406. 27. sperber f, metser u, gat a, shalmon a, yaal-hahoshen n. focal asymmetric breast density: mammographic, sonographic and pathological correlation in 97 lesions--a call to restrain biopsies. isr med assoc j. 2007;9:720–723. 28. dennis ma, parker sh, klaus aj, stavros at, kaske ti, clark sb. breast biopsy avoidance: the value of normal mammograms and normal sonograms in the setting of a palpable lump. radiology. 2001;219:186–191. 29. kopans db. interpreting the mammogram. in: mcallister l, ed. breast imaging. 3rd ed.; lippincott williams & wilkins, philadelphia, pa, 2007, pp. 365–479. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201902 214 j contemp med sci | vol. 4, no. 4, july-august 2019: 214–217 immunohistochemical assessment of estrogen, progesterone receptors and her2/neu overexpression by core needle biopsy in a sample of iraqi breast cancer patients dahlia raouf mohammed,1 nada a.s. alwan,2 and raji hussein1 1medical city, ministry of health, baghdad, iraq. 2national cancer research center, university of baghdad, baghdad, iraq. correspondence to nada a.s. alwan (email: nadalwan@yahoo.com). (submitted: 18 april 2019 – revised version received: 04 may 2019 – accepted: 25 may 2019 – published online: 26 august 2019) objective the aim of this study was to assess er, pr and human epidermal growth factor receptor 2 (her2)/neu expression in the specimens obtained by core needle biopsies (cnb) and to correlate the findings with some clinicopathological parameters such as (age, family history, side of breast lump, histological type and grade). methods this cross-sectional study was conducted on 62 female patients complaining of palpable breast lumps. all were subjected to the triple assessment test. patients with mammography or ultrasonography results signifying bi-rads 4 or 5 were selected for cnb using freehand automated gun instrument. twenty-three out of 52 malignant cases (44.2%) were with positive scores for er. twenty-six (50%) of cases were positive regarding pr expression. her2/neu overexpression revealed positive scores in 22 cases (42.3%). results the correlation between expression of er, pr and her2/neu with family history, the side of breast lump and pathological type of carcinoma were statistically not significant. on the other hand, a significant correlation was noted between her2/neu expression and women’s age; the positive expression was more demonstrated among younger aged women (mean age 45 year), nevertheless, this correlation was not significant with er, pr expression. significant correlation was also found between her2/neu overexpression in breast cancer specimens and the histopathological grading, while that association was not maintained for er and pr expression. conclusion immunohistochemical assessment of hormone receptors and her2/neu expression in specimens of breast cancer tissues obtained by cnb technique can indicate tumor aggressiveness and further need for neoadjuvant therapy before surgical intervention. keywords core needle biopsy, estrogen, progesterone, her2/neu, immunohistochemistry introduction breast cancer is the leading cause of cancer death among females in developing countries.1 in iraq, it is the most common registered cancer among the society and the main cause of cancer related death among women.2 previous iraqi studies showed clearly that the disease usually affect middle age females and present relatively at late stages.3–6 reproductive hormones are thought to influence breast cancer risk by increasing cell proliferation, thereby increasing the likelihood of dna damage, as well as promotion of cancer growth.7 earlier studies suggest that reproductive patterns are more strongly associated with risk of hormone receptor-positive breast cancer compared with triple-negative breast cancer.8,9 human epidermal growth factor receptor 2 (her2) is a member of the epidermal growth factor receptor (egfr/erbb) family. amplification or over-expression of the erbb2 gene occurs in approximately 30% of breast cancers and is associated with increased disease recurrence and a worse prognosis.10 signaling through the erbb family of receptors promotes cell proliferation and opposes apoptosis, and therefore must be tightly regulated to prevent uncontrolled cell growth.11 core needle biopsy (cnb), also called tru-cut biopsy, is widely accepted in routine assessment for the diagnosis of breast cancer. it is a reliable method for histological diagnosis and it provides enough material to allow determination for additional markers such er, pr and her2 status. those analyses are critical due to their implications in the guidance of clinical adjuvant proposal. the aim of this study is to assess er, pr and her2/neu expression in specimens obtained by cnb from a sample of iraqi breast cancer patients and to correlate the findings with some clinicopathological parameters including age, family history, side of breast lump, histological type and grade. patients, materials and methods this cross-sectional study was conducted at the main referral centre for early detection of breast tumors, medical city, baghdad and the iraqi national cancer research centre, baghdad university. sixty-two female patients complaining of palpable breast lumps were included in this study. all were subjected to the triple assessment test with physical examination, imaging (mammography and/or ultrasonography) and fna. patient with mammography or ultrasonography results signifying bi-rads 4 or 5 were selected for cnb using freehand automated gun instrument (bard mgnum biopsy instrument). at least 2–6 cores were obtained and the specimens were processed and stained with h&e stain for histopathological evaluation. that was followed by further staining with ihc markers er, pr and her2/neu. scoring was based on the examination of all tumor cells on the slide according to allred scoring guideline for er and pr. a proportion score (ps) was estimated through assessment of proportion of tumor cells with positive nuclear staining and includes five grades. an intensity score (is) was estimated by average staining intensity of all positive tumor cells and includes four grades. a total issn 2413-0516 original d.r. mohammed et al. 215j contemp med sci | vol. 4, no. 4, july-august 2019: 214–217 immunohistochemical assessment of estrogen, progesterone score (ts) equal to sum of ps and is. a positive result for both er and pr is defined as ts ≥ 3. weak positivity (score 3–4). moderate positivity (score 5–6). strong positivity (score 7–8). scoring of the ihc staining for her2/neu overexpression was according to asco/cap (hercep test). statistical analyses of all results were performed utilizing spss software statistical package (version 18) using t-test and chi-square. p-value at level of significance <0.05 was used to assess the correlation with some clinicopathological parameters. results this study showed that the peak frequency of breast carcinoma occurred in the age group of 40–49 years. the mean age was 50+ years old. there was an even distribution of breast carcinoma masses in right and left breast (48% and 52%) respectively. about 20% showed positive family history while 80% were with negative family history for breast cancer. out of total 62 patients, malignant histopathological changes (b5) were displayed in 52 (83.9%) patients, while the remaining 10 (16.1%) patients where non-malignant (i.e., b1, b2, b3 and b4) as diagnosed by cnb technique. lobular type has been identified in (17.8%) while forty-one cases (78.8%) were idc in cnb. more than two-third of cases (68%) of idc by cnb were of grade ii. twenty-three out of 52 malignant cases (44.2%) were with positive scores for er, 29 cases (55.8%) were with negative scores. equal numbers 26 (50%) of cases for positive and negative scores regarding pr expression. her2/neu overexpression revealed positive scores in 22 cases (42.3%) while negative scores in the remaining 30 cases (57.7%). regarding the strength of expression for estrogen and progesterone receptors, the highest number of cases with positive scores for er receptors showed weak positivity while the highest number with pr receptor score revealed strong positivity. the correlation between expression of er, pr and her2/ neu with family history, the side of breast lump and pathological type of carcinoma were statistically not significant. a significant correlation was noted between her2/neu expression and women’s age; the positive expression was more among younger aged women (mean age 45 years), on the other hand this correlation was not significant with er, pr expression (table 1). regarding the correlation with histological type of malignancy; er pr and her2/neu receptors expression showed a non-significant statistical correlation with both types (i.e., ductal and lobular carcinoma) for 50 out of 52 malignant cases (as it was not possible to classify two cases by core biopsy diagnosis) as illustrated in table 2. while this correlation was not significant with er and pr expression, as in table 3. discussion this iraqi study focuses on immunohistochemical staining for cnb specimens and their correlation with clinicopathological data. previous studies from the western societies and iraq showed that the positive expression of er and pr was increasing with age.12–14 in an iraqi survey, the rates of the positive er and pr and her2 breast cancers were 67.8%, 65.3% and 29.4% respectively.14 table 1. distribution of expression of er, pr, and her2/neu with respect to age of the patients receptor expression mean age sd* t-test df** p-value er +ve 49.5 11.7 0.51 50 0.61 er −ve 51.1 11.6 pr +ve 51.2 11.02 0.44 50 0.66 pr −ve 49.8 12.2 her2/neu +ve 45.7 10.1 2.72 50 0.009*** her2/neu −ve 54 11.4 *sd = standard deviation. **df = degree of freedom. ***highly significant. table 2. distribution of receptors according to the type of mammary carcinoma receptor expression ductal ca lobular ca odds ratio confidence interval p-value er +ve 18 5 0.25 0.05–1.39 0.4 er −ve 23 4 pr +ve 19 5 0.76 0.18–3.26 0.71 pr −ve 20 4 her2/neu +ve 19 2 3.02 0.56–16.33 0.18 her2/neu −ve 22 7 significant correlation was also found between her2/neu overexpression in breast cancer specimens and the histopathological grading. table 3. distribution of er, pr, and her2/neu according to histopathological grading receptor expression grade i (n = 3) (%) grade ii (n = 33) (%) grade iii (n = 14) (%) p-value er +ve 3 (100) 14 (42.4) 4 (28.6) 0.3 er −ve 0 (0) 19 (57.6) 10 (64.3) pr +ve 3 (100) 16 (48.5) 5 (35.7) 0.28 pr −ve 0 (0) 17 (51.5) 9 (64.3) her2/neu +ve 0 (0) 12 (36.4) 9 (64.3) 0.047* her2/neu −ve 3(100) 21 (63.6) 5 (35.7) *significant. the difference in the results of the ihc staining among different studies from the same region might be due to technical variations due to the use of different manufacturers and various kits. in the current study, however, no significant association between er, pr and age was displayed, probably due to the lower number of cases included. on the other hand, a significant correlation was found between her2/neu overexpression and women’s age; the positive expression was more demonstrated among younger aged women (i.e., below 50 year), reflecting the more aggressive behavior of mammary carcinoma in young women. the correlation of family history with the expression of er, pr, and her2/neu was not statistically significant. studies original 216 j contemp med sci | vol. 4, no. 4, july-august 2019: 214–217 immunohistochemical assessment of estrogen, progesterone d.r. mohammed et al. fig. 1 immunohistochemical stain for estrogen receptors in invasive ductal carcinoma. nuclear brown positive staining of tumor cells against a negative cytoplasmic and stromal background. the total score is 5 (moderate positivity) (40× + 3.0×). fig. 2 immunohistochemical stain for progesterone receptors in invasive ductal carcinoma. strong nuclear positivity of tumor cells against a negative cytoplasmic and stromal background. total score is 8 (strong positivity) (40×). fig. 3 immunohistochemical stain for progesterone receptors in invasive ductal carcinoma. strong nuclear positivity of tumor cells against a negative cytoplasmic and stromal background. total score is 8 (strong positivity) (40× + 3.0×). fig. 4 strong (3+) membrane immunoreactivity for her2/neu. a complete membrane staining and strong intensity of more than 30% of tumor cells (40× + 3.0×). fig. 5 her2/neu score 1+ (negative) incomplete membrane immunoreactivity and faint staining in more than 10% of tumor cells (40× +3.0×). fig. 6 infiltrative lobular carcinoma showed strong nuclear positivity for progesterone receptors (40× + 3.0×). suggest an ethnic and racial difference between communities regarding the impact of family history of breast cancer on the expression of hormonal receptors.15,16 an iraqi study carried out to illustrate the clinicopathological features of patients with positive family history of breast cancer revealed that the peak age frequency at the time of diagnosing breast cancer among those patients was in the fifth decade of life; illustrating no distinct correlation for their identification.17 in this study as well no significant correlation was noted regarding the correlation of er, pr or her2/neu with the type of mammary carcinoma. while both progesterone and estrogen receptors did not show any significant correlation with histopathological grading of breast cancer, her2/neu receptors over expression revealed a significant association. the more the aggressive the tumor was, the more likelihood her2/neu expression being positive. similar findings were noted in other local studies.12–14 a recent meta-analysis has shown that the cnb tissue could replace open excision biopsy for determining er, pr, and her2 status.18 the 2015 european society of medical oncology breast cancer clinical practice guideline recommends a preoperative pathological examination of the cnb, with a report on er, pr, and her2 status by ihc or fluorescence in situ hybridization.19 similar to previous single center studies,20 the results is subject for selection bias. some patients were initially diagnosed in other institutes and referred to the surgical department. in those cases, cnb was not reexamined, except in cases of vague diagnoses. original d.r. mohammed et al. 217j contemp med sci | vol. 4, no. 4, july-august 2019: 214–217 immunohistochemical assessment of estrogen, progesterone conclusion immunohistochemical staining are readily assessed for hormone receptors and her2/neu expression in specimens of breast cancer tissues obtained by cnb technique to document tumor aggressiveness and the further need for neoadjuvant therapy. significant correlations were demonstrated between her2/neu expression and the age of the patients; higher positive scores were displayed among younger women. likewise, a statistical association was noted between her2/neu over expression in breast cancer specimens and the histopathological grading. nevertheless, no significant correlation was shown between the expressions of er, pr and her2/neu and the side of the affected breast, the family history and the histopathological type. recommendations further study with larger sample size is recommended to correlate the ihc expression as prognostic and predictive markers in cnb and their significance with clinicopathological parameters and to follow up the patients response to adjuvant therapy. acknowledgments the authors would like to thank the staff at the main referral centre for early detection of breast tumors, medical city teaching hospital, baghdad) and those working in the national cancer research center of baghdad university or their kind help and cooperation. references 1. bray f, ferlay j, soerjomataram i, siegel rl, torre la, jemal a. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. cancer j clin. 2018;68:394–424. 2. annual report. iraqi cancer registry 2016. iraqi cancer board, ministry of health and environment, republic of iraq; 2018. 3. alwan na. breast cancer: demographic characteristics and clinicopathological presentation of patients in iraq. east mediterr health j. 2010;16:1159–1164. 4. alwan nas, tawfeeq f, maallah m, sattar sa, saleh wa. the stage of breast cancer at the time of diagnosis: correlation with the clinicopathological findings among iraqi patients. j neoplasm 2017;2:1–10. 5. alwan nas. breast cancer among iraqi women: preliminary findings from a regional comparative breast cancer research project. j glob oncol. 2016;2:255–258. 6. alwan nas. tumor characteristics of female breast cancer: pathological review of mastectomy specimens belonging to iraqi patients. world j breast cancer res. 2018;1:1–3. 7. hulka bs, moorman pg. breast cancer: hormones and other risk factors. maturitas 2001;38:103–113; discussion 113–116. 8. eliassen ah, missmer sa, tworoger ss, spiegelman d, barbieri rl, dowsett m, et al. endogenous steroid hormone concentrations and risk of breast cancer among premeno-pausal women. j natl cancer inst. 2006;98:1406– 1415. 9. kaaks r, berrino f, key t, rinaldi s, dossus l, biessy c, et al. serum sex steroids in premeno-pausal women and breast cancer risk within the european prospective investigation into cancer and nutrition (epic). j natl cancer inst. 2005;97:755–765. 10. tan m, yu d. molecular mechanisms of erbb2-mediated breast cancer chemoresistance. adv exp med biol. 2007;608:119–129. 11. roy v, perez ea. beyond trastuzumab: small molecule tyrosine kinase inhibitors in her-2-positive breast cancer. oncologist 2009;14:1061–1069. 12. alwan nas, mualla f, naqash m, kathum s, tawfiq fn, nadhir s. clinical and pathological characteristics of triple positive breast cancer among iraqi patients. gulf j oncol. 2017;25:6–15. 13. alwan nas, kerr d, al-okati d, pezella f, tawfeeq f. comparative study on the clinicopathological profiles of breast cancer among iraqi and british patients. open public health j. 2018;11:177–191. 14. alwan nas, tawfeeq fn, muallah fh. breast cancer subtypes among iraqi patients: identified by their er, pr and her2 status. j fac med baghdad 2017;59:304–307. 15. hines lm, risendal b, slattery ml, baumgartner kb, giuliano ar, byers t. differences in estrogen receptor subtype according to family history of breast cancer among hispanic, but not non-hispanic white women. cancer epidemiol biomarkers prev. 2008;17:2700–2706. 16. jiang x, castelao je, chavez-uribe e, fernandez rodriguez b, celeiro muñoz c, redondo cm, et al. family history and breast cancer hormone receptor status in a spanish cohort. plos one 2012;7:e29459. 17. alwan nas. family history among iraqi patents diagnosed with breast cancer. int j sci res. 2017;6:868–872. 18. chen x, yuan y, gu z, shen k. accuracy of estrogen receptor, progesterone receptor, and her2 status between core needle and open excision biopsy in breast cancer: a meta-analysis. breast cancer res treat. 2012;134:957–967. 19. senkus e, kyriakides s, ohno s, penault-llorca f, poortmans p, rutgers e et al. primary breast cancer: esmo clinical practice guidelines for diagnosis, treatment and follow-up. ann oncol. 2015;26:v8–v30. 20. you k, park s, ryu jm, kim i, lee sk, yu j, et al. comparison of core needle biopsy and surgical specimens in determining intrinsic biological subtypes of breast cancer with immunohistochemistry. j breast cancer 2017;20:297–303. dx.doi.org/10.22317/jcms.08201906 original 27j contemp med sci | vol. 1, no. 4, autumn 2015: 27–30 research objective coronary heart disease (cad) is the most prevalent chronic disease and the main leading cause of death in the world, with more than half a million newly diagnosed cad patients each year. the development of atherosclerosis involves the interaction of multiple metabolic and cellular processes. central to this are disorders of lipoprotein metabolism. apolipoprotein a-i is the major protein component of high-density lipoprotein (hdl) in plasma. chylomicrons secreted from the intestinal enterocyte also contain apo a-i, but it is quickly transferred to hdl in the blood stream. the aim of this study is to determine if apo-a1 can be used as indicator to severity and extent of cad. methods this study was conducted in cardiac catheterisation unit at al hussein medical city from november 2014 to september 2015. it included 76 patients (49 males and 27 females) who presented with signs and symptoms of cad and have undergone angiography. control group consisted of 20 healthy subjects (14 males and 6 females) matched for age and bmi. serum levels of apo-a1 were measured in both groups. after coronary angiography was done for all patients, the extent and severity of cad was correlated with serum levels of apo-a1. the extent of cad was determined by angiography, according to number of coronary arteries involved and degree of narrowing in coronary artery diameter. results the angiographic finding in patient group was normal in 22 patients (28.9%), single vessel involvement in 15 patients (19.7%), two vessels disease in 15 patients (19.7%) and three vessels disease in 18 patients (23.8%). the left main stem disease (lmd) was found in 6 patients (7.9%). there was no significant difference in the serum level of apo-a1 between patient with cad and control group (p = 0.147). there was no significant correlation between serum level of apo-a1 and the extent cad (r = 0.004, p = 0.975). conclusion there was no significant difference in serum level of apo-a1 between the patients group and the control group. also, there was no significant correlation between serum level of apo-a1 and the extent of cad. keywords coronary artery disease (cad), apolipoprotein a1, left main stem disease (lmd) role of apo-lipoprotein a1 as cardiac biomarker for severity of coronary atherosclerosis fadhil jawad al-tu’ma,a zainab abdul-hussein abd-yasera & karim obais al-naffib introduction globally, cardiovascular diseases (cvds) which include coronary heart disease (chd), strokes, rheumatic heart disease (rhd), cardiomyopathy and other heart diseases represent the leading cause to death.1 cvds refer to any disease which affects the cardiovascular system, especial chd, vascular disease of brain, kidney and peripheral arterial disease.2 beside endothelial dysfunction leading to inflammatory reaction, lipid metabolism disorders represent the second key event in the initiation and rapid development of atherogenesis.3 the development of atherosclerosis involves the interaction of multiple metabolic and cellular processes.4 central to this are disorders of lipoprotein metabolism.5 apolipoprotein a-i is the major protein component of high-density lipoprotein (hdl) in plasma. chylomicrons secreted from the intestinal enterocyte also contain apo a-i, but it is quickly transferred to hdl in the bloodstream.6 there is considerable interest in the potential value of measuring circulating concentrations of apolipoproteins to assist in the assessment of the risk of cad, as well as in their potential aetiological relevance to the disease.7 apolipoproteins are important components of lipoprotein particles, and there is accumulating evidence that the measurement of various forms of apolipoproteins may improve the prediction of the risk of cvd.8–10 there is abundant evidence that the risk of coronary atherosclerotic cvd is directly related to plasma lipid and apolipoprotein levels, but the relationships between the serum apoa-i levels and the extent of cad have not been consistently shown. to investigate the possible relationship of the serum levels of apoa-i, lipids and other lipoproteins with the severity of coronary lesions and number of vessels diseased, these parameters were examined in angiographically defined cad patients and in a group of control subjects with no angiographical or clinical evidence of cad. increased ldl-c concentration levels are a well-established risk factor for cad and are currently recommended as the primary target for lipid-lowering therapy for the prevention and treatment of cvd, although its unique superiority over other circulating predictors of cad is unclear.11 our aim in this study is show if can use the serum level of apo-a1 as indicator to severity and extent of coronary atherosclerosis disease. methods this project was conducted at department of medicine (angiographic department) in al hussein teaching hospital / holy karbala city-iraq and in the department of biochemistry, college of medicine, university of karbala from november 2014 to september 2015. in this cross-sectional study, 76 patients (49 males and 27 females) were studied who had undergone angiography and were found to have cad. control group consisted of 20 healthy subjects (14 males and 6 females) matched for age and bmi. the demographic data, family history and results of the coronary angiography were obtained from patients’ files and filled in specially designed data collection form. the classification of atherosclerotic patient depended on the extent of cad. there are 4 main coronary arteries: left main adepartment of biochemistry, college of medicine, university of kerbala/holy kerbala, iraq. bdepartment of internal medicine, college of medicine, university of kerbala/holy kerbala, iraq. correspondence to fadhil jawad al-tu’ma (email: f_altoma_56@yahoo.com). (submitted: 23 august 2015 – revised version received: 14 september 2015 – accepted: 28 september 2015 – published online: autumn 2015) issn 2413-0516 28 j contemp med sci | vol. 1, no. 4, autumn 2015: 27–30 role of apo-lipoprotein a1 as cardiac biomarker for severity of coronary atherosclerosis research fadhil jawad al-tu’ma et al. coronary artery, left anterior descending artery (lad), left circumflex (lcx) and right coronary arteries (rca) were assessed. all patients underwent coronary angiography and the result collected from the catheterisation laboratory according to the patient’s name and file number. the angiographic results concern the presence of significant (lesions more than or equal to 70% diameter stenosis for coronary arteries and more than or equal to 50% diameter stenosis for left main coronary artery by visual estimation)12 coronary artery lesion and the numbers of arteries involved by a significant lesion and classified as follows: • normal (no significant lesion). • lms (left main stem) disease. • one vessel involvement. • two vessels involvement. • three vessels involvement. none of the patients had any contraindication for coronary angiography. the angiographic result subscribed by authorised interventional cardiologists at the iraqi center for heart diseases. coronary angiography is performed under local anaesthesia. as the procedure was sterile, all potential access sites had to be disinfected, shaved and sterilised. the patient was asked to lie down in supine position on the cardioangiograph table at the beginning of the procedure, and prepared for the procedure in sterile conditions. coronary angiography was performed with the patient in the fasting state. blood samples were collected after overnight fasting, about 5 ml venous blood was withdrawn and placed in plain tube and serum was separated after 15 min at room temperature by centrifugation at 300 rpm for 15 min. the apo-a1 measured by turbidimetric monoreagent for the quantitative determination of apo-a1 wavelength: (fig. 1) 340 nm, hg 334/365 nm. the apo-a1 concentration in the sample is calculated by interpolation of its absorbance (a) from the calibration curve. the result measured by turbidimetry with absorbance reading at 340 nm shown on the y axis against apo-a1 and concentration shown on the x axis was obtained by excel program 2013. results there were 76 patients included in this study, with a mean age of 57.76 ± 9.69 years (range 32–79 years), of which 49 patients were males (64.4%) and 27 patients were females (35.6%) and a control group of 20 healthy people with no significant disease (presence of disease was ruled out by history, physical examination and biochemical tests) with mean age of 43.9 ± 4.03 years were selected. regarding the angiographic finding in patient group, it was normal in 22 patients (28.9%), single vessel involvement in 15 patients (19.7%), two vessels disease in 15 patients (19.7%) and three vessels disease in 18 patients (23.8%). lmd was found in 6 patients (7.9%), while the remaining 70 patients (92.1%) were free of lmd. these findings are shown in table 1. the apo-a1 had non-significant differences between coronary atherosclerosis disease patient and control group (p = 0.147). the lipid profile compared between coronary atherosclerosis disease patient and control group is shown in table 2. the coronary atherosclerosis disease patients had significantly higher s.tg (p = 0.007) and significantly lower s.hdl (p = 0.0001) levels compared with control people. also there was significant difference in serum level of vldl (p = 0.008) between two groups. regarding other lipid, there are no significant difference in serum cholesterol and serum ldl-c between coronary atherosclerosis disease patients and control group. all mention above shows in table 2. table 3 shows the correlations between extent of coronary atherosclerosis (angiographic finding), apo-a1 and lipid profile in patients group. no significant differences were observed between the serum levels of lipids or routinely measured lipoproteins a1 (fig. 2). there was significant relation between ldl-c and the number of involved coronary artery vessels or the severity of coronary lesions (r = 0.264) (p = 0.021). discussion the prevention of coronary vessel disease has become one of the most important healthy challenges of recent times. several y = 0.0043x + 0.027 r² = 0.9796 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 50 100 150 200 250 300 350 ab so rb an ce   apo ‐ a1  concentration   standard curve of apo-a1        fig. 1 standard curve of apo-a1 obtained by spectrometer at 340 nm by exel program 2013. table 1. angiographic findings of the studied group (n = 76) angiographic findings no. % normal 22 28.95 one vessel 15 19.74 two vessel 15 19.74 three vessel 18 23.68 lmd 6 7.89 total 76 100 table 2. comparison between atherosclerotic patient and control group in the measured parameters variables control n = 20 atherosclerotic patient n = 76 p values apo_a1 167.89 ± 20.26 160.38 ± 40.10 ns s. tc 171.70 ± 34.33 155.07 ± 42.46 ns s.tg 137.65 ± 39.34 177.97 ± 70.29 0.007 s.hdl-c 43.20 ± 10.62 36.01 ± 6.45 0.0001 s.ldl-c 100.97 ± 34.21 83.45 ± 36.59 ns s.vldl-c 27.52 ± 7.86 35.48 ± 14.13 0.008 bmi 27.11 ± 1.83 28.99 ± 5.00 ns 29j contemp med sci | vol. 1, no. 4, autumn 2015: 27–30 research role of apo-lipoprotein a1 as cardiac biomarker for severity of coronary atherosclerosifadhil jawad al-tu’ma et al. factors (modifiable and non-modifiable) such as ht, dm, smoking etc. are recognised as risk factors for coronary vessel disease and aggressive correction of these play vital role in coronary vessel disease prevention. however, measuring apo-a1 and apo b/apo-a1 ratio cannot be useful in laboratory tests. the findings of the current study indicated significant correlation between the ldl level and extent of coronary atherosclerosis disease. no significant correlation was observed between apo-a1 and the severity of coronary atherosclerosis disease. the analysis of ldl showed that this variable increased with the number of vessels affected. several studies agree that high levels of ldl are correlated with the presence and severity of coronary atherosclerosis disease13–15 korhonen et al.’s16 study adds that high levels of ldl are associated with an increased risk of coronary atherosclerosis disease, and the more vessels obstructed, the higher the serum ldl level in the group, thus corroborating the findings of the present study. the results of the present study are in agreement with the study conducted by pischon et al. in harvard university, demonstrating that serum apo-a1 level is not associated with severity of coronary atherosclerosis disease.17 references 1. mathers cd, salomon ja, ezzati m, begg s, vander-hoorn s ,lopez ad. (2006): global burden of disease and risk factors. new york, ny: oxford university press. doi: 10.1596/978-0-8213-6262-4 2. tarnow l, groop ph, hadjadj s, kazeem g, cambien f, marre m, et al. european rational approach for the genetics of diabetic complication (euragedic): patient populations and strategy. nephrol dial transplant. 2007;13(1):161–8. doi: 10.1093/ndt/gfm501. pmid: 17704113 3. ross r, epstein fh. atherosclerosis—an inflammatory disease. n eng j med. 1999; 340(2):115–26. doi: 10.1056/nejm199901143400207. pmid: 9887164 4. steinberg d. atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime. nat med. 2002;8:1211–7. doi: 10.1038/ nm1102-1211. pmid: 12411947 5. krauss rm, kesaniemi ya. cardiovascular disease and hyperlipidaemia. curr opin lipidol. 1994;5:249–51. doi: 10.1097/00041433-199408000-00001. pmid: 7981954 6. wasan km, brocks dr, lee sd, sachs-barrable k, thornton sj. impact of lipoproteins on the biological activity and disposition of hydrophobic drugs: implications for drug discovery. nat rev drug discov. 2008;7(1):84–99. doi: 10.1038/nrd2353. pmid: 18079757 7. thompson a, danesh j. associations between apolipoprotein b, apolipoprotein ai, the apolipoprotein b/ai ratio and coronary heart disease: a literature-based sabin et al. obtained similar results. they found that after adjusting the role of gender, age, smoking and hypertension, apo-a level and apo b/apo-a ratio were independently correlated to peripheral atherosclerosis and brain stroke.18 in a prospective study by sweetnam et al., after adjusting the results for cardiovascular risk factors, regardless of plasma lipids, a strong correlation was found between the incidence of ischemic heart disease and low level of apo-lipoprotein a1.19 the results of a study performed by yazici et al. in turkey revealed a significant correlation between the apo-a serum level and cardiac troponins in the patients with angina pectoris. however, no significant relationship was found between the severity of coronary atherosclerosis disease and the number of diseased vessels.20 these results are in agreement with our study regarding the correlation between apo-a1 and the severity of coronary atherosclerosis. in a study by habib et al., including 140 patients in saudi arabia, a significant correlation was observed between apo-a1 and severity and extension of coronary arteries stenosis in the patients but not in the control group.21 however, our study did not show any such result. conclusion we conclude that there is no difference in apo-a1 level in patients with coronary atherosclerosis and in healthy people. also, the level of apo-a1 cannot be used as an indicator to determine the extent of cad.  fig. 2 correlation between extent of cad and level of apo-a1. table 3. correlations of apo-a1 and lipid profile with coronary atherosclerosis disease severity parameters correlated extent of cad apo-a1 s. tc s.tg s.hdl-c s.ldl-c apo-a 1 r p 0.004 0.975 s. tc r p 0.149 0.200 0.106 0.302 s.tg r p 0. 2030.078 0.113 0.274 0.484 0.0001 s.hdl -c r p 0.0770.506 0.148 0.150 0.086 0.407 –0.066 0.522 s.ldl -c r p 0. 264 0.021 0.046 0.653 0.931 0.0001 0.195 0.057 –0.098 0.344 s.vldl -c r p 0. 2000.083 0.117 0.256 0.487 0.0001 0.997 0.000 –0.056 0.590 0.197 0.054 meta-analysis of prospective studies. j intern med. 2006;259:481–92. doi: 10.1111/j.1365-2796.2006.01644.x. pmid: 16629854 8. talmud pj, hawe e, miller gj, humphries se. nonfasting apolipoprotein b and triglyceride levels as a useful predictor of coronary heart disease risk in middle-aged uk men. arterioscler thromb vasc biol. 2002;22:1918–23. doi: 10.1161/01.atv.0000035521.22199.c7. pmid: 12426225 9. walldius g, jungner i, holme i, aastveit ah, kolar w, steiner e. high apolipoprotein b, low apolipoprotein a-i, and improvement in the prediction of fatal myocardial infarction (amoris study): a prospective study. lancet. 2001;358:2026–33. doi: 10.1016/s0140-6736(01)07098-2. pmid: 11755609 10. lamarche b, moorjani s, lupien pj, cantin b, bernard pm, dagenais gr, et al. apolipoprotein a-i and b levels and the risk of ischemic heart disease during a five-year follow-up of men in the québec cardiovascular study. circulation. 1996;94:273–8. doi: 10.1161/01.cir.94.3.273. pmid: 8759066 11. third report of the national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel iii) final report. circulation. 2002;106:3143– 421. doi: 10.3410/f.718113553.793483968. pmid: 12485966 12. american college of cardiology foundation/ american heart association task force on practice guidelines and the society for cardiovascular angiography and interventions. circulation. 2011;124:e574–651. doi: 10.1161/cir.0b013e31823ba622. pmid: 22064601 30 j contemp med sci | vol. 1, no. 4, autumn 2015: 27–30 role of apo-lipoprotein a1 as cardiac biomarker for severity of coronary atherosclerosi research fadhil jawad al-tu’ma et al. 13. watts gf, mandalia s, brunt jnh, slavin bm, coltart dj, lewis b. independent associations between plasma lipoprotein subfraction levels and the course of coronary artery disease in the st. thomas' atherosclerosis regression study (stars). metabolism. 1993;42:1461–7. doi: 10.1016/00260495(93)90199-x. pmid: 8231842 14. ehara s, ueda m, naruko t, haze k, itoh a, otsuka m, et al. elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. circulation. 2001;103:1955–60. doi: 10.1161/01.cir.103.15.1955. pmid: 11306523 15. schwertner há, fischer jr. comparison of various lipid, lipoprotein and bilirubin combinations as risk factors for predicting coronary artery disease. atherosclerosis. 2000;150:381–7. doi: 10.1016/s0021-9150(99)00387-1. pmid: 10856530 16. korhonen t, savolainen mj, koistinen mj, ikäheimo m, linnaluoto mk, kervinen k, et al. association of lipoprotein cholesterol and triglycerides with the severity of coronary artery disease in men and women. atherosclerosis. 1996;127:213–20. doi: 10.1016/s0021-9150(96)05958-8. pmid: 9125311 17. pischon t, girman cj, sacks fm, rifai n, stampfer mj, rimm eb. non-highdensity lipoprotein cholesterol and apolipoprotein b in the prediction of coronary heart disease in men. circulation. 2005;112(22):3375–83. doi: 10.1161/circulationaha.104.532499. pmid: 16316964 18. sabino ap, de oliveira sousa m, moreira lima l, dias ribeiro d, sant’ana dusse lm, das gracas carvalho m, et al. apob/apoa-i ratio in young patients with ischemic cerebral stroke or peripheral arterial disease. transl res. 2008;152(3):113–8. doi: 10.1016/j.trsl.2008.06.005. pmid: 18774540 19. sweetnam pm, bolton ch, downs lg, durrington pn, mackness mi, elwood pc, et al. apolipoproteins a-i, a-ii and b, lipoprotein(a) ( and the risk of ischaemic heart disease: the caerphilly study. eur jm clin invest. 2000; 30(11):947–56. doi: 10.1046/j.1365-2362.2000.00725.x. pmid: 11114956 20. andrikoula m, mcdowell if. the contribution of apob and apoa1 measurements to cardiovascular risk assessment. diabetes obes metab. 2008;10(4):271–8. doi: 10.1111/j.1463-1326.2007.00714.x. pmid: 18333887 21. habib ss, abdel-gader am, kurdi mi, al-aseri z, soliman mm. lipoproteina(a) is a feature of the presence, diffuseness, and severity of coronary artery disease in saudi population. saudi med j. 2009;30(3):346–52. pmid: 19271061 23j contemp med sci | vol. 1, no. 4, autumn 2015: 23–26 research objective to detect the genes that play a role in resistance to cyclohximide by some pathogenic fungi. methods isolates of dermatophytic fungi (trichophyton rubrum, microsporum canis, candida albicans and aspergillus spp.) were isolated from patients with fungal infection attending the al-diwaniya teaching hospital and identified based on the microscopic and macroscopic characteristics. the effect of cycloheximide and ammonium hydroxide in isolation of these fungi in sabourauds dextrose agar were tested by the quantative transcrptive assay of gene expression (cyh1, cyh2, cdr1, cdr2 and abc gene ) using real-time pcr. results the result of this estimation revealed that the amplified dna (pcr product) were 463 bp for cyh1; 265 bp for cdr1 ; 160 bp for cdr2; 359 bp for cyh2 in the pathogenic fungi c. albicans , t. rubrum, m. canis and a. niger. conclusion the tested pathogens had resistance genes to cycloheximide such as (chy1, chy2, cdr1, cdr2 and hkg). keywords cycloheximide, cycloheximide resistant fungi, antifungal activity, cycloheximide resistance genes detection of cyclohximide resistant gene in selected pathogenic fungi gasak falah ali,a adnan h. al-hamadania & adnan w mohammadb introduction the present work aims to detect the genes that play a role in resistance to cyclohximide in some pathogenic fungi. cycloheximide is used in a range of media for the isolation of pathogenic fungi to inhibit certain non-pathogenic fungi similar to saprophytic moulds and yeasts. it is especially useful in the isolation of dermatophytes. since the pathogenicity of fungi and the immune status of patients vary, care should be taken when a medium with cyclohximide is used for the isolation of fungi because certain opportunistic fungi might be missed. chloramphenicol is a broad-spectrum antibiotic, which is inhibitory to a wide range of gram-negative and gram-positive bacteria but may have an inhibitory effect on several pathogenic fungi.1 the cycloheximide (actidione), which is an antibiotic that is produced by streptomyces griseous, is highly active against a large number of yeast. but have no marked antibacterial activity.2 the use of cycloheximide in culture media is to facilitate the isolation of numerous pathogenic fungi from medical samples and the environment.3 atp-binding cassette (abc) transporters are integral membrane proteins that can be found in all kingdoms of life. some members of the super family are involved in processes like dna repair, translation or regulation of gene expression, but most couple the binding and hydrolysis of ntps, usually atp, to transport a large variety of substrates across cellular membranes.4 the drug efflux pump encoded by candida drug resistance genes (cdrl) of candida albicans was the first abc efflux pump implicated in conferring resistance to cycloheximide in a pdrs disruptant hypersensitive strain of saccharomyces cerevisiae.5 the present work was designed to evaluate the efficacy of ammonium hydroxide in isolation of pathogenic fungi instead of cyclohexamide and study the molecular basis of the resistant of these fungi to cyclohexamide. methods patients a total of 103 patients [males (71) and females (31)] were clinically diagnosed as cases of dermatophytosis by the dermatologist after attended the outpatient clinic of the department of dermatology at al-diwaniya teaching hospital from the beginning of december 2014 to the end of february 2015. mycological examination the evidence of infection established on demonstration of fungal elements [branching septate hyphae and spore (arthrospores)] by the direct microscopically examination of hair, scales from the lesion by koh mounting. enough materials of sample was taken to permit repeated direct examination, then, a single preparation may not provide enough evidence for the existence of an infecting fungus. potassium hydroxide (koh) mounts skin scrapings and hair fragments were placed on a surface of clean slide swamped with drops of 10% koh, heated mildly (30°c) for about 5–10 min, after which the slide was allowed to cool. after cooling, the cover slip was applied and the slide was examined under the low (10×) and high (40×) microscope lens. gentle warming of the preparation speeded up the reaction.6 fungal genomic dna extraction fungal genomic dna from of c. albicans isolates were extracted using ez-10 spin column fungal genomic dna mini-preps kit, and done according to company instructions. estimation of dna yield and quality: the extracted genomic dna was checked using nanodrop spectrophotometer (thermo. usa), that checks and measures the purity of dna by reading the absorbance at 260/280 nm. preparation of primers the primers were prepared according to the manufacturer’s instructions by dissolving the lyophilised primers with te buffer to stock solution with counteraction of 100 pmol/μl, after spinning-down and allowing it to stay overnight at 4°c, primers working solution with te buffer, using the equation c1v1 = c2v2 (concentration versus volume) to gel final working solution (20 pmol/μl) for each primer. primers the pcr detection primers were designed using ncbi-gene bank database and primer 3 design online, and supported by bioneer, korea. adepartment of microbiology , college of medicine, al-qadissiyah university, iraq. bdepartment of anatomy, college of medicine, university of al-qadisiyah, qadisiyah, iraq. correspondence to gasak falah ali (email: nemo.ali@mail.ru). (submitted: 23 july 2015 – revised version received: 11 august 2015 – accepted: 19 august 2015 – published online: autumn 2015) issn 2413-0516 24 j contemp med sci | vol. 1, no. 4, autumn 2015: 23–26 detection of cyclohximide resistant gene in selected pathogenic fungi research gasak falah ali et al. fig. 2 agarose gel electrophoresis image that show the pcr product 265 bp analysis of crd1. where m: marker (100–2000 bp), lane 1: c. albicans; lane 2: t. rubrum; lane 3: m. canis; lane 4 and 5: a. niger; lane 5: c. albicans standard strain (atcc 1032). polymerase chain reaction (pcr) pcr technique was performed for detection multidrug resistance-related abc transporter genes groups of study fungus. the method was carried out according to method described in a study by walker et al.7 pcr master mix reaction preparation pcr master mix reaction was prepared using accupower pcr premix kit according to the manufacturer’s instructions. the pcr master mix reaction components (dna template volume 5 µl, f. primer 10 pmol volume 1.5 µl, r. primer 10 pmol volume 1.5 µl, pcr water volume 12 µl and total volume 20 µl) were placed in standard pcr tubes containing the pcr premix, as lyophilised materials containing all other components needed pcr reaction, such as taq dna polymerase, dntps, tris–hcl ph: 9.0, kcl, mgcl2, stabilizer and tracking dye. the tube was then placed in exispin vortex and centrifuged for 3 min and transferred to the mygene pcr thermocycler. pcr thermo cycler conditions pcr thermocycler conditions for each gene were done using conventional pcr thermocycler system initial denaturation: repeat1, temp. 95°c, time 5 min, denaturation: repeat 30, temp 95°c, time 30 s, annealing: repeat 30, temp 30°c, time 30 s, extension: repeat 30, temp 72°c, time 45 s, final exten0sion: repeat 1, temp. 72°c, time 7 min, hold: -,4c, forever. gel electrophoresis pcr products of each genes were analysed using agarose gel electrophoresis method. results dna quality and purity the results of evaluating and estimating the dna extraction were measured using nano drop spectrophotometer at a wavelength of 260–280 nm. it gave an optimal concentration of dna for amplification ranging from 10.7 to 155.9 ng/μl and purity ranged from 1.4 to 1.99 nm (table 1). based on the standard values of dna concentration for amplification, the values of the present study are considered of efficient value and suitable for the establishment of the dna extracted with target primers or sequences amplification (applied biosystems, 2008). dna amplification the result of amplification was performed on the dna extracted from all the studied specimens and confirmed by electrophoresis analysis. in this analysis, the strands of dna resulted from the successful binding between specific primers of target gene and specimen extracted dna. the successful binding appeared as single compact band under the uv light using ethidium bromide as a specific dna stain. the electrophoresis was also used to estimate dna molecular size depending on dna marker (100-bp dna ladder) and the result of this estimation revealed that the amplified dna marker (100-bp dna ladder) and the result of this estimation revealed that the amplified dna (pcr product) were 463 bp for cyh1 (fig. 1); 265 bp for cdr1 (fig. 2); 160 bp for cdr2 table 1. values of dna concentration and purity of selected sample of extracted dna sample no concentration ng/μl purity 260/280 nm 1 39.4 1.76 2 25.1 1.61 3 24.1 1.7 4 17.8 1.40 5 10.7 1.23 6 31.6 1.47 7 18.1 1.42 8 155.9 1.99 9 40.1 1.77 10 54.1 1.75 11 141.7 1.68 12 72.4 1.4 pcr primers primer sequence amplicon (bp) pcrchy1 f cggtttcggtggtcaaacc 463 r tgtcaccacccaattcgaaatg pcrchy2 f agggtgtcgtaaacataccattc 359 r accttgagcaaataaggaagctc pcrcdr1 f tgctgccatgttctttgctg 265 r tggtctggcttcgaaaagtg pcrcdr2 f acacgtctttgtcgcaacag 160 fig. 1 agarose gel electrophoresis image that show the pcr product (463 bp) analysis of chy1. where m: marker (100–2000 bp), lane 1: c. albicans; lane 2: t. rubrum; lane 3: m. canis; lane 4 and 5: a. niger; lane 5: c. albicans standard strain (atcc 1032). 25j contemp med sci | vol. 1, no. 4, autumn 2015: 23–26 research detection of cyclohximide resistant gene in selected pathogenic fungigasak falah ali et al. (fig. 3); 359 bp for cyh2 (fig. 4) in the pathogenic fungi c. albicans, t. rubrum, m. canis and a. niger. light-cycler pcr was set up with fast start dna master sybr green including heat activatable taq polymerase with each primer and extracted dna template. the product of pcr is measures once each cycle immediately. after the 72°c incubation (extension step) by detection of fluorescence related with the binding of sybr green to the amplification product. fluorescence curves were established and analysed with the cycler software, version exicycler realtime pcr. the analysis of melting curve is performed immediately after the amplification protocol. the peak melting temperature achieved represented the specific amplification product. to guarantee the reliability, the test is considers to have positive results if the signal from the amplified product is clearly positive in both specimens. discussion the resistance mechanism of drugs in microorganisms conventionally may take the pathway of either identifying a cellular determinant that prevents entry of the drug or removes the drug from the cell or inactivates the drug or prevents the drug from inhibiting the target of various combinations of the mentioned pathways. in fungi, mutation in gene encoding target proteins, upregulations of expression of multidrug efflux pumps and drug target themselves, altering the stoichiometry of the inhibitor target ratio in favour of fungus are possible mechanisms.8 the mechanism of the resistance to the cycloheximide in yeast cells happens by either alteration of ribosomal proteins or expression of certain precise transport systems responsible for a multidrug resistance usually linked to the expression of mdr genes. the results of the pcr is conformational showed that the resistance gene to cycloheximide is found in the pathogenic fungi and this gene responsible for the cycloheximide resistance and several studies show that the first resistance mechanism to cycloheximide, is caused by a single change at ribosomal proteins (rps) l29 or l42 (previously l41) of the amino acid which promotes the cycloheximide cyh (cycloheximide) sensitivity (cyhs)/resistance(cyhr) is a property of the 80s ribosome from candida maltosa9, cryptococcus neoformans,10,11 schwanniomyces occidentalis12 or the phaffia rhodozyma13 and these results agree that the mutations in cyh2 decrease the affinity of cycloheximide for the ribosome,14 there is no proof that cycloheximide really binds to l29. the mutation can alter the conformation of an adjacent site. without a doubt, studies find that cells carrying x2 together with a mutation which alters protein l3, causing resistance to trichodermin, another inhibitor of elongation, are far less resistant to cycloheximide than cells carrying. the second resistance mechanism to the cycloheximide, several yeast mdr genes has been. they are usually connected with either transmembrane proteins, which consist of the abc or members of mfs (major facilitator system) families. the abundant happening and close similarity of the identified proteins reveal the importance of these protein families from s. cerevisiae the genome sequence has revealed the presence of as several as 28 orfs which is homologous to either the abc or the major facilitator system.15 the multidrug transporters of yeast include the abc’s protein cdr1-3 from c. albicans,16 and the msf (major facilitator system) benr (camdr1) that renders cells resistant to cycloheximide, benomyl, methotrexate, 4-nitro-quinoline-n-oxide or terbinafine.17 the c. maltose cyrr and the c. dubliniensis cdmdr1, homologous to camdr1 (c. albicans multidrug resistance), converse resistance to cycloheximide or to fluconazole, cycloheximide, benomyl or sulphometuronmethyl, respectively,18 this result agrees with the studies that a second possible mechanism for drug resistance is overexpression of the candida drug resistance (cdr)1 and cdr2 genes, which code transporters of the abc family, and the multidrug resistance (mdr)1 gene, which codes for a main facilitator transporter. later it was found that the resistant to 7-aminocholesterol (rta2) gene might also be involved in the development of azole resistance in a mutant c. albicans strain with deletions of cdr1, cdr2 and mdr1.19 conclusion the tested pathogens had resistance genes to cycloheximide such as chy1, chy2, cdr1, cdr2 and hkg.  fig. 3 agarose gel electrophoresis image that show the pcr product 160 bp analysis of crd2. m: marker (100–2000 bp), lane 1: c. albicans; lane 2: t. rubrum; lane 3: m. canis; lane 4 and 5: a. niger; lane 5: c. albicans standard strain (atcc 1032). fig. 4 agarose gel electrophoresis image that show the pcr product 359-bp analysis of chy2. where m: marker (100–2000 bp), lane 1: c. albicans; lane 2: t. rubrum; lane 3: m. canis; lane 4 and 5: a. niger; lane 5: c. albicans standard strain (atcc 1032). references 1. sutton da. specimen collection, transport, and processing: mycology. in: murray pr, baron ej, jorgensen jh, pfaller ma, yolken rh, editors. manual of clinical microbiology, 8th ed. washington dc: american society for microbiology; 2003. 2. martin sj, reutelingsperger cpm, mcgahon aj, rader ja, van schie rcaa, laface dm, et al. early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of bcl-2 and abl. j exp med. 1995;182:1545–6. doi: 10.1084/jem.182.5.1545 3. filipello marchisio v, cassinelli c, piscozzi a, tullio v, mischiati p. a preliminary survey of cycloheximide resistant airborne fungi inturin, italy. mycopathologia. 1993;123:1–8. pmid: 8247094 26 j contemp med sci | vol. 1, no. 4, autumn 2015: 23–26 detection of cyclohximide resistant gene in selected pathogenic fungi research gasak falah ali et al. 4. ambudkar sv, dey s, hrycyna ca, ramachandra m, pastan i, gottesman mm. biochemical, cellular, and pharmacological aspects of the multidrug transporter. annu rev pharmacol toxicol. 1999;39:361–98. pmid: 1033108 5. prasad r, wergifosse pd, goffeau a, balzi e. molecular cloning and characterization of a novel gene of c. albicans, cdr1, conferring multiple resistance to drugs and antifungals. curr genet. 1995;27:320–9. pmid: 7614555 6. hainer bl. dermatophyte infections. am family physician. 2003;67(1):101–8. pmid: 12537173 7. walker je, saraste m, runswick mj, gay nj. distantly related sequences in the alphaand beta – subunits of atp synthase, myosin, kinase and other atp-requiring enzymes and a common nucleotide binding fold. embo j. 1982;1:945–51. pmid: 6329717 8. kanafani za, perfect jr. antimicrobial resistance: resistance to antifungal agents: mechanisms and clinical impact. clin infect dis. 2008;46(1):120–8. doi: 10.1086/524071 pmid: 18171227 9. mutoh e, mochizuki m, ohta a, takagi m. inducible expression of a gene encoding an l41 ribosomal protein responsible for the cycloheximideresistant phenotype in the yeast candida maltosa. j bacteriol. 1995;177:5383–6. pmid: 7665534 10. varma a, kwon-chung kj. 2000. construction of stable episomes in cryptococcus neoformans. curr genet. 34:60–66. pmid: 9683676 11. warner jr. the economics of ribosome biosynthesis in yeast. trends biochem sci. 1999;24:437–40. pmid: 10542411 12. del pozo l, abarca d, hoenicka j, lmenez a. two different genes from schwanniomyces occidentalis determine ribosomal resistance to cycloheximide. eur j biochem. 1993;213:849–57. 13. kim ig, nam sk, sohn jh, rhee sk, an gh, lee sh, et al. cloning of the ribosomal protein l41 gene of phaffia rhodozyma and its use a drug resistance marker for transformation. appl environ microbiol. 1998;64:1947–9. pmid: 9572978 14. stocklein w, piepersberg w. binding of cycloheximide to ribosomes from wild-type and mutant strains of saccharomyces cerevisiae. antimicrob agents chemother. 1980;18:863–7. doi: 10.1128/aac.18.6.863 pmid: 7016025 15. goffeau a, park j, paulsen it, jonniaux jl, dinh t, mordant p, et al. multidrug resistant transport proteins in yeast: complete inventory and phylogenetic characterization of yeast open reading frames with the major facilitator superfamily. yeast. 1997;13:43–54. doi: 10.1002/(sici)10970061(199701)13:1<43::aid-yea56>3.0.co;2-j pmid: 9046086 16. gupta v, kohli a, krishnamurthy s, puri n, aalamgeer sa, panwar s, et al. identification of polymorphic mutant alleles of camdr1, a major facilitator of candida albicans which confers multidrug resistance and it in vitro transcriptional activation. curr genet. 1998;34:192–9. pmid: 9745021 17. sasnauskas k, jomantiene r, lebediene e, lebedys j, januskaa, janulaitis a. cloning and sequence analysis of a candida maltosa gene which confers resistance to cycloheximide. gene. 1992;116:105–8. 18. moran gp, sanglard d, donnelly sm, shanley db, sullivan dj, coleman dc. identification and expression of multidrug transporters responsible for fluconazole resistance in candida dubliniensis. antimicrob agents chemother. 1998;42:1819–1830. pmid: 9661028 19. jia xm, ma zp, jia y, gao ph, zhang jd, wang y, et al. rta2, a novel gene involved in azole resistance in candida albicans. biochem biophys res commun. 2008;373:631–6. doi: 10.1016/j.bbrc.2008.06.093 pmid: 18601908 207j contemp med sci | vol. 4, no. 4, autumn 2018: 207–210 research treatment of resistant acral vitiligo with fractional er:yag laser muhsin al-dhalimi,a* haider al-sabak,a and aula hussainb adepartment of dermatology, faculty of medicine, university of kufa, iraq. b department of dermatology, karbala health directorate, ministry of health, iraq. *correspondence to muhsin al-dhalimi (email: muhsin.aldhalimi@uokufa.edu.iq). (submitted: 12 july 2018 – revised version received: 08 september 2018 – accepted: 15 september 2018 – published online: 26 december 2018) objectives to detect the results of treatment of refractory acral vitiligo by combination of fractional erbium-yag (er:yag) 2940 nm laser and narrow band-ultraviolet b (nb-uvb) in comparison with nb-uvb alone. methods thirty four patients who had generalized, stable (6 months or more) with resistant acral vitiligo were enrolled. the predominantly affected limbs, lower or upper, were treated with both laser and nb-uvb, while the less predominantly affected limbs were treated with nb-uvb alone. during the treatment period, the patients were seen every 4 weeks regularly. results the response rate in areas treated with both fractional er:yag laser and nb-uvb was as follows: four patients (13.3%) had good response, six patients (20%) had moderate response and seven patients (23.3%) had poor response and 13 patients (43.3%) had no response. the statistical analysis showed a significant response (p-value <0.001). conclusion combination therapy is safe and more effective in the treatment of acral vitiligo than nb-uvb monotherapy. keywords acral vitiligo, erbium-yag laser, nb-uvb introduction vitiligo is an autoimmune, acquired, multifactorial, depigmenting disorder characterized by the appearance of well demarcated white macules or patches in the skin, due to progressive loss of functional melanocytes in the epidermis.1 it has a great cosmetic and psychological effects on the life of affected patients.2 among many clinical types and variants, acral vitiligo is characterized by a high resistance to many treatment modalities. this is probably due to lower hair follicle density, melanocyte density, langerhans cell density and epidermal mhcii expression.3 narrow band-ultraviolet b (nb-uvb) phototherapy is a very important method in treating vitiligo but the treatment course usually takes long duration maybe 1 year. skin ablation by erbium-yag (er:yag) laser combined with nb-uvb phototherapy and 5-fu has been tried in the treatment of nonsegmental vitiligo.4 it is found to be safe and well-tolerated technique that improves the result of short-term nb-uvb therapy and is anticipated to increase patient compliance. the er:yag laser emits light at 2940 nm, with the water affinity being nearly 15 times greater than that of the co2 laser. this allows the er:yag laser to vaporize tissue more efficient, leaving only a small residue of coagulated tissue. as a consequence of the limited photothermal effects, hemostasis and collagen contraction are significantly reduced with er:yag lasers.5 accordingly, we investigated the results of treatment of refractory acral vitiligo by combination of fractional er:yag 2940 nm laser and nb-uvb in comparison with nb-uvb alone. patients and methods this prospective comparative interventional study was conducted at the laser research unit, college of medicine, university of kufa; for the period from november 2014 to december 2015. the ethical approval was obtained from the scientific council of dermatology and venereology iraqi board for medical specializations. thirty four patients who had generalized, stable (6 months or more) with resistant acral vitiligo were enrolled. full history and physical examination, assessment of skin phenotypes were done. all patients were types iii and iv. exclusion criteria included: pregnancy, lactation, history of cutaneous photosensitivity, eye cataract or skin cancers, localized vitiligo, psychologically unstable patient and those with severe illnesses. patients who have medical or surgical intervention for vitiligo lesions within last 1 month were also excluded. the predominantly affected limbs, lower or upper, were treated with both laser and nb-uvb, while the less predominantly affected limbs were treated with nb-uvb alone. fractional er:yag laser 2940 nm (quanta system dna laser technology matisse) was used. the parameters of the laser device applied in this study were as follows: flat tip, fractional, pulse duration 0.3 ms, frequency 2 hz, and the energy 0.3 j, one pass. three sessions of fractional er:yag laser were performed at 1 month interval. no local anesthesia was needed during laser therapy. er:yag laser probe was held perpendicularly on acral part (hands or feet) with one pass of three pulses stumping distributed throughout the lesion. full body nb-uvb cabinet (waldmann, germany) was used. the treatment started 5 days after each laser session, two times per week on non-consecutive days for 24 sessions. the initial dose was (0.5 j/cm2) that modified at each session according to the degree of erythema developed during treatment. during the treatment period, the patients were seen every 4 weeks regularly; at each visit the response and adverse effects were recorded. the patients were followed up each month for 3 months after the last treatment session to look for any complications or any sign of relapse or further improvement. all vitiliginous lesions were carefully monitored and repeated evaluations were done before each session and at the end of the treatment period. patients were monitored for repigmentation; overall and per lesional and development of perifollicular pigmentation. issn 2413-0516 208 j contemp med sci | vol. 4, no. 4, autumn 2018: 207–210 treatment of resistant acral vitiligo with fractional er:yag laser research m. al-dhalimi et al. the clinical response to therapy was visually scored as the percentage of repigmentation of the vitiligenous lesions and rated as follows:6 excellent response: if >75% repigmentation of the depigmented lesions at the end of therapy. good response: if >50–75% repigmentation of the depigmented lesions at the end of therapy. moderate response: if >25–50% repigmentation of the depigmented lesions at the end of therapy. poor response: if ≤25% repigmentation of the depigmented lesions at the end of therapy. no response: if 0% repigmentation of the depigmented lesions at the end of therapy. side effects such as erythema, pruritus and burning sensation were assessed at every follow-up visit. photographic assessment color photographs for each patient were obtained at baseline, and at the end of 12 weeks treatment period. consecutive photographic documentation were taken every 4 weeks and at the end of the treatment in a good illumination and at the same site, using sony-digital, high sensitivity, 9.1 mega pixel, dsc-hx1 still camera. all the treated patients were assessed blindly by two independent board certified dermatologists by visual analogue scale (vas) of improvement by scale scoring from 0 to 10. patient satisfaction at the end of the sessions, the patients were asked about their satisfaction. the degree of satisfaction was ranged from 0 to 10. results thirty patients with vitiligo completed the study, four patients were defaulted due to inconvenience of the study protocol with their work. there were 17 females (56.7%) and 13 males (43.3%). their ages ranged from 18 to 64 years with a mean ± sd (32.9 ± 15.2) years. duration of the disease ranged from 1 to 24 years with a mean ± sd (8.7 ± 5.3). ten patients (33%) had positive family history. in this study, the upper limbs were treated in 26 patients with laser and nb-uvb and the lower limbs were treated with nb-uvb alone. in the remaining four patients, the lower limbs were treated with laser and nb-uvb and the upper limbs were treated with nb-uvb alone. the response rate in areas treated with both fractional er:yag laser and nb-uvb was as follows: four patients (13.3%) had good response, six patients (20%) had moderate response and seven patients (23.3%) had poor response and 13 patients (43.3%) had no response. the statistical analysis showed a significant response (p-value <0.001). among the 17 responding patients (56.7%), the upper limbs were treated in 13 of them and the lower limbs were treated in the remaining four patients. no response was noticed after the first session of laser. the patient’s first response was started after the second session of laser. the areas treated with nb-uvb alone showed no response at all (table 1, figs. 1 and 2). there was a significant variation in the mean of vas of improvement between limbs treated with laser and nb-uvb and limbs treated with nb-uvb alone. the average was higher in limbs with combined therapy (table 2). the mean score for patient’s satisfaction of limbs treated with combined therapy was 4.6000 ± 3.11393 while for limbs treated with nb-uvb alone was 0.2667 ± 0.44978, so there was highly significant differences between two limbs in each patient (p-value ≤0.001) (table 3). adverse effect regarding treatment of nb-uvb, one patient developed mild erythema at some stage during treatment that clear without treatment within 24–48 hours after each session and about half of the patients had transient hyperpigmentation that disappeared spontaneously at the end of follow-up period. no patient treated with laser was developed any localized effects. no patient terminates the treatment course because of side effects. no systemic side effect was reported. follow-up at the end of 3 months of follow-up period after the last sessions, no patient had recurrence or koebnerization. table 1. distribution of patients according to response groups p-valuelimbs treated with laser and nb-uvb limbs treated with nb-uvb alone response no response 13 30 <0.001 43.3% 100.0% poor response 7 0 23.3% 0% moderate response 6 0 20.0% 0% good response 4 0 13.3% 0% excellent response 0 0 0% 0% total 30 30 100.0% 100.0% m. al-dhalimi et al. 209j contemp med sci | vol. 4, no. 4, autumn 2018: 207–210 research treatment of resistant acral vitiligo with fractional er:yag laser fig. 1 a 57-year-old male with refractory acral vitiligo, (a) before treatment, (b) after treatment with fractional er:yag laser and nb-uvb showing good response. a b fig. 2 a 18-year-old female with refractory acral vitiligo, (a) before treatment, (b) after treatment with fractional er:yag laser and nb-uvb showing moderate response. a b table 2. comparison in the vas of improvement between limbs treated with combined therapy (er:yag laser and nb-uvb) and limbs treated with (nb-uvb) alone groups n mean std. deviation p-value average limbs treated with laser and nb-uvb 30 1.5500 1.48179 0.011 limbs treated with nb-uvb alone 30 0.1250 0.23146 table 3. comparison between limbs treated with combined therapy (laser and nb-uvb) and limbs treated with nb-uvb alone regarding patients satisfaction groups n mean std. deviation p-value patient satisfaction limbs treated with combined therapy 30 4.6000 3.11393 <0.001 limbs treated with nb-uvb alone 30 0.2667 0.44978 discussion vitiligo is a disfiguring condition that can cause considerable psychological and cosmetic distress to the patient and the family. the lesions on acral areas are considered as difficult to treat.4 as conventional treatment of vitiligo on hands and feet results in an unsatisfactory outcome, various ablative treatment options were tried with favorable results.7 combination therapy may produce higher rates of repigmentation compared to traditional monotherapies. so in this 210 j contemp med sci | vol. 4, no. 4, autumn 2018: 207–210 treatment of resistant acral vitiligo with fractional er:yag laser research m. al-dhalimi et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. study, a combination treatment of fractional er:yag laser (2940 nm) and nb-uvb was used. the er:yag laser cause vaporization and coagulation. as er:yag laser emits light at 2940 nm with high water affinity, this allows the er:yag laser to vaporize tissue more than coagulate, leaving only a small residue of coagulated tissue. so the photothermal effects, hemostasis and collagen contraction are significantly reduced.5 fractionated laser produce microthermal zones. each microdot of a damage is surrounded by normal, unaffected skin, which acts as a reservoir for healing and enables these microwounds to resolve quickly.4 resurfacing removes the old epidermis that would be replaced by new epidermis formation. this is probably accompanied by production of many growth factors and cytokines like il6 that cause t cells suppression and tnf, fgf, mmp which stimulate proliferation and migration of residual melanocyte as well producing repigmentation of vitiligo lesion.7 this can be regarded as reverse koebner’s phenomenon.8 in this work, the patients received three sessions of fractional er: yag laser at 1 month interval on the resistant acral lesion, then nb-uvb administrated to the entire body 5 days after each laser session twice a week, for 3 months with gradually increasing dose at each session. in one study, 5-fu was used in combination with er:yag laser in treatment of periungual vitiligo. the response rate was 47.8% in the treated group while it was 1.1% in the control group.9 this work showed better response rate (50–75%) in the limbs treated with combination therapy. in another study, nb-uvb was used in addition to 5-fu and one session er:yag laser. the results were moderate in 78.1% of patients as compared with 23.4% in the mono-therapy group (nb-uvb only).4 the results of this study were slightly better than the results of the present work. this is probably due to the addition of nb-uvb to 5-fu and laser therapy. potent topical steroids were used in combination with nb-uvb preceded by dermabrasion with of fractional er:yag. the study showed that 50% of lesions achieved 50% repigmentation in the dermabrasion side after 1 month, while only 4.2% with topical steroids and uvb alone.10 this study showed better response rate (50–75%) in the limbs treated with combination therapy in spite of not using potent topical steroid in our work. fractional co2 laser was used as ablative therapy in other studies instead of er:yag laser in difficult to treat areas.7 triple therapy was tried (laser, potent topical steroid, nb-uvb) in refractory vitiligo in two studies. the results showed good repigmentation in (23.1%) patients treated with laser, topical steroid and nb-uvb, compared with (3.9%) in patients without laser treatment.7 the results of previous study were better than the present study that showed good response in (13.3%) the patients. the difference in the results may be attributed to addition of potent topical steroids and more potent dermabrasion effect of fractional co2 laser in comparison with fractional er:yag laser. the other study showed that 44% of patients achieved over 50% repigmentation.11 the results were comparable with the result of the present work. in half-body randomized comparative study using co2 laser treatment followed by nb-uvb, good response was obtained in 10% of the patients, moderate response in 20% of patients, poor response in 20%, while in the control group (nb-uvb alone), 20% of patients showed 25% response rate.12 the sample size in this study was small (10 patients) as compared to this study sample (30 patients), not all treated sites were acral. the results of the present work were also better. in this study, the left and right sides are not compared, because it was cosmetically and psychologically unaccepted for the patients and the patients refused treatment of one side and leaving the other side without laser treatment. conflict of interest none. n references 1. sandoval-cruz m, garcía-carrasco m, sánchez-porras r, mendoza-pinto c, jiménez-hernández m, munguía-realpozo p, et al. immunopathogenesis of vitiligo. autoimmun rev. 2011;10:762–765. 2. bhatnagar a, kanwar aj, parsad d, de d. comparison of systemic puva and nb-uvb in the treatment of vitiligo: an open prospective study. j eur acad dermatol venereol. 2007;21:638–642. 3. esmat sm1, el-tawdy am, hafez ga, zeid oa, abdel halim dm, saleh ma, et al. acral lesions of vitiligo: why are they resistant to photochemotherapy? j eur acad dermatol venereol. 2012;26:1097–1104. 4. anbar ts, westerhof w, abdel-rahman at, ewis aa, el-khayyat ma. effect of one session of er:yag laser ablation plus topical 5fluorouracil on the outcome of short-term nb-uvb phototherapy in the treatment of non-segmental vitiligo: a left–right comparative study. photodermatol photoimmunol photomed. 2008;24:322–329. 5. zachary cb. modulating the er:yag laser. lasers surg med. 2000;26:223–226. 6. sami y. efficacy of puva therapy vs nb-uvb therapy. arc dermatol. 2007;143:578–584. 7. vachiramon v, chaiyabu tr c, rattanaumpawan p, kanokrungsee s. effects of a preceding fractional carbon dioxide laser on the outcome of combined local narrowband ultraviolet b and topical steroids in patients with vitiligo in difficult-to-treat areas. lasers surg med. 2016;48:197–202. 8. radmanesh m, sohrabian n, radmanesh r. repigmentation of vitiliginous facial area after q-switched nd-yag laser therapy for depigmentation: is it a case of true reverse koebner phenomenon in vitiligo? iran j dermatol. 2013;16:159–161. 9. anbar t, westerhof w, abdel-rahman a, el-khayyat m, el-metwally y. treatment of periungual vitiligo with erbium-yag-laser plus 5-flurouracil: a left to right comparative study. j cosmet dermatol. 2006 5:135–139. 10. bayoumi w, fontas e, sillard l, le duff f, ortonne jp, bahadoran p, et al. effect of a preceding laser dermabrasion on the outcome of combined therapy with narrowband ultraviolet b and potent topical steroids for treating nonsegmental vitiligo in resistant localizations. br j dermatol. 2012;166:208–211. 11. li l, wu y, li l, sun y, qiu l, gao xh, et al. triple combination treatment with fractional co2 laser plus topical betamethasone solution and narrowband ultraviolet b for refractory vitiligo: a prospective, randomized half-body, comparative study. dermatol ther. 2015;28:131–134. 12. shin j, lee js, hann sk, oh sh. combination treatment by 10,600 nm ablative fractional carbon dioxide laser and narrowband ultraviolet b in refractory non segmental vitiligo: a prospective, randomized half-body comparative study. br j dermatol. 2012;166:658–661. 179j contemp med sci | vol. 4, no. 4, autumn 2018: 179–186 review gac (momordica cochinchinensis spreng.) fruit and its potentiality and superiority in – health benefits ali abdulqader,a faisal ali,a,b amin ismail,a,c* and norhaizan mohd esaa,d adepartment of nutrition and dietetics, faculty of medicine and health sciences, universiti putra malaysia, serdang, selangor, malaysia. b biochemistry & molecular biology department, university hospital, faculty of medicine and health sciences, sana’a university, yemen. cresearch center of excellent, non-communicable diseases, faculty of medicine and health sciences, universiti putra malaysia, serdang, selangor, malaysia. dlaboratory of molecular biomedicine, institute of bioscience, universiti putra malaysia, serdang, selangor, malaysia. *correspondence to amin bin ismail (email: aminis@upm.edu.my). (submitted: 09 october 2018 – revised version received: 22 october 2018 – accepted: 14 november 2018 – published online: 26 december 2018) objective this review aimed to collect and summarise the scientific literature on the nutritional content, biological activities and nutraceutical value of gac fruit. method we searched pubmed, science direct, google scholar, biomed for all the studies published from 2003 to 2018 which related to the gac fruit. however, the literature on its biological activity against disease-causing cell damage such as diabetes, obesity, and cancer were found to be limited. results the diversity of bioactive compounds underlies the potential use and application of medicinal plants as an excellent source of dietary supplements. fruits that are found to be rich in multi-phytochemicals are seriously being considered as ‘super’ fruits due to their unique antioxidants. gac fruit (momordica cochinchinensis spreng), is one of those ‘super’ fruits found to be rich in phytonutrients because all its fractions (i.e. aril, seeds, pulp and peel) have been widely used in folk healing and ancestral medicine. but, the entire potentiality towards the health benefits of gac fruit is not well known or understood. importantly, gac fruit contains significantly higher amounts of lycopene and relatively high levels of β-carotene. conclusion the findings from this review suggest that the phytochemicals found in gac fruit especially carotenoids and their potential health benefits make it an ideal candidate for the research of hyperglycemia-related eye disorders. keywords gac fruit, phytochemicals, nutritional content, carotenoids, medicinal plants, phytomedicine. introduction the moist tropical weather in the southeast asian region (sar) is a rich source of nutrient for medicinal plants that has been used for generations for multiple purposes and applications. from among the abundance and wide variety of fruits and vegetables found across the region, particularly in vietnam, gac fruit is reputed to be an exceptional source of phytochemicals.1 “gac” is the popular name given to this tropical fruit found in vietnam as mentioned, as well as grown in other locations throughout the southeast asia region.2 the gac fruit is known by several other names such as (fak kao) in thailand, (teruah) in malaysia, (mak kao) in laos, (bhat kerala) in india, (moc niet tu) in china, and the english name is ‘spiny bitter gourd’ or ‘sweet gourd’.3 nutritionally, gac fruit contains extraordinarily elevated or high-levels of carotenoids, particularly β-carotene, lycopene, and lutein, α-tocopherol, and essential fatty acids available in its portions (i.e. peel, pulp, aril, and seeds).4 this fruit also contains relatively high levels of polyphenols and flavonoids.5 the biological activities of these bioactive compounds have been established via their ability to scavenge free radicals and act as antioxidants. recently, the food and pharmacological industry have taken a keen interest in gac fruit and producing gac fruit products such as gac powder, gac oil capsules, gac juice, and frozen gac fruit. moreover, these products have been released into the market serving as food additives, in cosmetics, and for medicinal and pharmaceutical purposes.3 figure 1 shows the morphology of gac fruit. methodology an internet online literature search was carried out to collect and compile relevant research data for this review. the following terms were used in performing the searches; momordica cochinchinensis spreng, gac fruit, carotenoids, age-related macular degeneration, diabetic retinopathy. all data related to gac fruit and other keywords were downloaded via several internet search engines: pubmed, sciencedirect, google scholar, and biomed. carotenoids in gac fruit all parts or portions of the gac fruit (i.e. the peel, pulp, and aril) were found to be an excellent source of carotenoids. indeed, the aril of the gac fruit was reported to comprise a high amount of lycopene, about 10-fold higher than that known to be in lycopene-rich fruit and vegetables.6 in a separate study, vuong et al.7 reported the lycopene and β-carotene concentrations of the edible portion of gac fruit to be 802 and 175 µg/g respectively. also, in the same year, aoki et al.6 reported 380 µg/g of lycopene and 101 µg/g of β-carotene in gac aril. in a further study, ishida et al.1 found very high levels of carotenoids (β-carotene, α-carotene, and lycopene) in gac aril, with concentrations of 718, 107, and 2227 µg/g, respectively. another study reported gac aril content of lycopene and β-carotene was 408 and 83 µg/g respectively.8 while nhung et al.9 mentioned that gac fruit had the highest amount of lycopene with a concentration of 3728 µg/g fw and that the concentration of β-carotene was issn 2413-0516 180 j contemp med sci | vol. 4, no. 4, autumn 2018: 179–186 gac fruit and its potentiality in health benefits review abdulqader a. et al. similarly very high at 379 µg/g fw. recent reports have indicated very promising and excitingly high-levels of both lycopene and β-carotene in gac aril, of 6800 and 2900 µg/g respectively.10 a further study reported concentrations of 6300 and 5700 µg/g for lycopene and β-carotene respectively.11 investigating the carotenoids in gac fruit, carrot root and tomato fruit, as to which was more digestible, determined that both carotenoids (lycopene and β-carotene) from gac fruit were found to be at least eight times higher than the carotenoids found in carrot root and tomato fruit.12 besides gac aril, the yellow pulp and the peel of the gac fruit were equally found to be a rich source of carotenoids. it was reported that the total carotenoid content of gac pulp was 283 µg/g, and this concentration is high compared with other carotenoid-rich fruits.8 the gac pulp content of carotenoids was further analysed, revealing that gac pulp contains 22 µg/g of β-carotene and 0.9 µg/g of lycopene, and in addition to β-carotene, the gac pulp was also found to contain zeaxanthin and β-cryptoxanthin.6 furthermore, in addition to lycopene and β-carotene, gac peel was found to be remarkably, if not extremely rich in lutein, and much higher than found in the other fractions of the fruit, (52,020 µg/g).5 however, at this stage, only the aril has been processed as the peel and pulp which constitute a significant quantity (15% and 42% respectively) of the total weight of the fruit, are usually discarded.3 previous in vitro digestion studies concluded that the bioavailability and bioaccessibility of carotenoids from gac fruit aril were much higher than those from carrot and tomato.12 fatty acids in gac fruit in addition to carotenoids, the fatty acid content found in gac fruit has been investigated by several researchers. fortunately, the existence of unsaturated fatty acids in gac fruit was found to be relatively high.1 it was reported that 100 g of gac seed pulp contains 852 mg of fatty acids, and of that total amount, 70% was found to be unsaturated and 50% was polyunsaturated.13 a further investigation reported that the gac seed pulp content of fatty acids was 102 mg/g of the edible portion.7 remarkably, oleic, palmitic, and linoleic acids were found to be predominant in gac aril (32.3%, 29.2%, and 28.1% respectively), while the predominant fatty acid found in gac seed was stearic acid with small levels of linoleic, oleic and palmitic acids of 60.5%, 20.3%, 9%, and 5.6% respectively.1 in a separate study by newly, the total percentage of unsaturated fatty acids in gac aril was reported to be 71.38%, which was much higher than that of saturated fatty acids, with a final percentage of 28.62%. indeed, the most dominant fatty acids were oleic acid 48.99% followed by palmitic acid 24.18% and linoleic acid 21.09%.14 phenolic and flavonoid compounds in gac fruit phenolic and flavonoid compounds were also found in gac fruit. a study reported that the concentrations of phenolics and flavonoids found in gac fruit were about 26.08 and 1.32 mg/100 g, respectively.15 another study reported that the gac fruit content of phenolics and flavonoids was investigated by several researchers, where it was determined that gac peel, pulp, and aril content of phenolics was 55, 205, and 191 µg/g fw respectively, whereas gac peel, pulp, and aril content of flavonoids was 0.118, 0.143, and 0.084 mg/g fw respectively.16 gac fruit content of the phenolic and flavonoid compound variety among the fractions based on the maturity stages determined that ferulic acid and p-hydroxybenzoic acid were most apparent in gac pulp, while apigenin, rutin, and luteolin were the main compounds found in mature pulp and aril.5 traditional and epidemiological uses gac fruit is indigenous to vietnam and southeast asian countries and is used and consumed as a dietary supplement as well as for medicinal purposes. in vietnam, gac aril is cooked with rice to provide a scrumptious glossy look, with an oily taste for the slimy traditional rice dish named as (“xoi” gac), which is served during special occasions and festivals like lunar new year, birthdays, and weddings.3 besides that, gac oil after being extracted from the aril has also been used as a tonic and given to lactating or pregnant women and children to treat dry eyes (xerophthalmia) and night-blindness vision.2 it is also reported that the application of gac oil is used to heal skin infections, wounds and burns, and has helped to stimulate cell growth and closure in wounds.2 in china, the seed of the gac fruit has been utilised for many generations. the gac seed (in mubiezi, china) has been traditionally used to treat inflammation, swelling, scrofula, tinea, diarrhoea with skin infections such as sores, carbuncles, furuncles and boils in humans and animals.17 in bangladesh, the m. cochinchinensis spreng known as ‘kakroal’ or ‘kark gach’, uses all kakroal fractions (i.e. fruit, seed, leaf, and roots) in medicinal systems for the treatment of various ailments such as cancer, diabetes, liver diseases, skin infections, itches, rheumatoid arthritis, colic, and blood purification.18 thirty days of gac fruit nourishment was found to improve β-carotene levels among 185 preschoolers who were selected based on their high risk of vitamin a deficiency.7 antioxidant activities of gac fruit the antioxidant activity of gac fruit has been examined using diphenyl-picrylhydrazyl (dpph) radical scavenging, ferric reducing antioxidant power (frap), and 2,2' -azino-bis(3-ethy lbenzothiazoline-6-sulphonic acid (abts). these assays are widely used and accepted in evaluating the antioxidant properties of different compounds in foods, plants, fruits, and herbs. moreover, different fractions of the gac fruit at different maturity stages have been assessed regarding their potential antioxidant frap, by applying dpph assays. the results showed that gac aril at the fully ripened stage had the highest frap value at 531.17 µmol/g. in the same study, the dpph assay test fig. 1 gac (m. cochinchinensis spreng) fruit morphology. abdulqader a. et al. 181j contemp med sci | vol. 4, no. 4, autumn 2018: 179–186 review gac fruit and its potentiality in health benefits showed that gac peel and pulp had the highest antioxidant activity at the immature stages of 2.56 and 2.35 µmol/g, respectively.5 a separate study reported that dpph and frap assessments of 100 g of gac fruit extract, were equivalent to 45.1 and 5.8 mg, respectively.15 moreover, gac peel extract using ethyl acetate showed the abts antioxidant capacity yield of 737 µm trolox equivalent (te) /100 g dry weight (dw) with total carotenoids of 271.1 mg/100 g dw.19 the seed of the fruit has also been found to be rich in chymotrypsin inhibitor.20 the antioxidant activities of a chymotrypsin inhibitor from gac seed have been established in rat hepatocyte culture exposed to tert-butyl hydroperoxide (t-bhp)-induced oxidative stress.21 anticancer activity of gac fruit several studies investigated the ability of gac fruit extract to inhibit and suppress the proliferation of several cancer cell lines via various mechanisms. a crude water extract obtained from the gac fruit was assessed to determine its antitumor and anti-angiogenic activities in vitro and in vivo, which resulted in the cell proliferation of two cancer cell lines (colon 26-20 and hepg2 cells) significantly inhibited when treated with the water extract. indeed, the crude water extract was able to suppress the progression of cancer by reducing the wet tumour weight by 23.6% in a ‘rat’ cancer group. moreover, histology and immunohistochemistry investigations appeared significant toward decreasing vascularisation for the gac treated group.22 it was also reported that a water extract taken from gac aril significantly reduced the cell viability of two cancer cell lines, notably mcf-7 breast cancer and melanoma with a rate of 60% and 71%, respectively.11 notwithstanding, gac aril extract has shown an anticancer effect against the human mcf-7 breast cancer cell line by both apoptosis pathways (intrinsic and extrinsic). the mixture of hexane, acetone, and ethanol was successfully used to extract carotenoids from gac aril that were then compared with lycopene and tamoxifen. cell shrinkage and chromatin condensation have also been induced by aril extract using 4' ,6-diamidino-2-phenylindole (dapi) fluorescent staining, while flow cytometry assay showed a higher percentage of mcf-7 cells in the early apoptosis stage. moreover, aril extract was found to be capable of enhancing the activity of caspase-6, -8, and -9, where the expression of the proapoptotic bax gene dramatically increased when assessed by real-time polymerase chain reaction (rt-pcr).10 furthermore, the anticancer potentiality of gac seed extract was also investigated in a study which showed that gac seed extract appeared to exhibit potent inhibitory effects against human breast cancer zr-75-30 cells. moreover, the extract significantly repressed the expression secretion of two matrix metalloproteinases mmp-2 and mmp-9 when assessed based on western blot and gelatin zymography tests.23 the anticancer activity of gac seed extract was further explored on human sgc7901 and mkn-28 gastric cancer cell lines, where the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay revealed that the survival of both cell lines had significantly decreased when compared with the control. furthermore, gac seed extracts were able to promote apoptosis by increasing the enzyme activities of caspase-3 and -9 and via parp and p53 signal pathways.24 notably, the antiproliferative activity of gac seed extract has been established again, and the ethanolic extract of gac seed was found to be capable of suppressing the proliferation of a549 lung cancer cells by inducing apoptosis via the activation of p53 and inactivation of pi3k/akt signalling.25 recently, gac seed extracts showed anticancer potential activity against two melanoma (mm418c1 and d24) cell lines.26 dna protective activity of gac fruit the dna protective effect of gac fruit against tk6 human lymphoblast cells, induced by h2o2 and ultraviolet uvc was investigated. in the previous study, it was found that tk6 exposure to 50 µm h2o2 for 5 min, produced massive oxidative dna damage, while tk6 exposure to uvc significantly enhanced dna migration, when the tk6 cells were treated with gac peel extract using ethanol 95% and 50%, thus leading to a significant decrease in dna damage.27 antimicrobial activity of gac fruit gac pulp (flesh) and seed pulp (aril) were examined concerning their potential activity against different strains of both gram-positive and gram-negative bacteria. the results of such work indicated that water and ethanolic extraction of gac pulp displayed higher antibacterial effect on grampositive strains than gram-negative strain bacteria. accordingly, the highest antibacterial activity was observed in the ethanolic extract of gac flesh against both strains, micrococcus luteus 745 (20 mm) and m. luteus 884 (18.5 mm) inhibition zone.28 another study assessed the antimicrobial activities from various parts of the gac fruit (i.e. peel, pulp, and aril) against six pathogenic bacteria (escherichia coli, staphylococcus aureus, bacillus cereus, pseudomonas aeruginosa, salmonella typhimurium, and klebsiella pneumonia). such results demonstrated that the data of this study confirmed that ethanolic extraction of various parts from gac fruit had antimicrobial activity against all pathogenic strains, as mentioned previously.29 antiulcer activity of gac fruit it has also been documented that 7 and 14 days of treatment with an ethanolic extract of gac seed, significantly accelerates the healing of acetic acid via enhancing and upregulation of the angiogenesis, and increases the expression of mrna as well as the vascular endothelial growth factor (vegf), tested by real-time pcr and western blot.29 anti-inflammatory activity of gac fruit surprisingly, gac seed is found to be a rich source of saponins, which are known for their health benefits.30 two triterpenoid saponin compounds (gypsogenin glycoside and quillaic acid glycoside) were isolated and examined regarding their anti-inflammatory activity. for example, compound 2 (quillaic acid glycoside) which is a saponin compound that isolated from gac seed demonstrated antiinflammatory effects in raw 264.7 cells via inhibiting the lipopolysaccharide-induced expression of nitric oxide and il-6 via the nf-κb pathway.31 other health benefits of gac fruit recently, two saponin compounds which were isolated from gac seed showed a promising therapeutic effect against 182 j contemp med sci | vol. 4, no. 4, autumn 2018: 179–186 gac fruit and its potentiality in health benefits review abdulqader a. et al. cisplatin-induced renal damage in llc-pk1 kidney cells in vitro via blocking mitogen-activated protein kinases (mapks) signalling cascade.32 likewise, the gastroprotective effect of momordica semen extract has also been explored in vivo. the pre-treatment with momordica saponin i as an ingredient isolated from the momordica seed decreased gastric mucosa damage, thereby reducing the progressing acute and chronic gastrointestinal disorders (e.g., gastritis and gastric ulcer).33 the adverse reproductive parameters of male rats induced by valproic acid (vpa) was investigated. the m. cochinchinensis aril was mixed with distilled water, filtered, then dried and given orally to the rats in different concentrations. the protective effect of the extract against adverse male reproductive parameters and testicular damage induced by vpa was evident; the aril extract significantly protects the decrease of the weights of the epididymis and seminal vesicle. the aril extract also increased the sperm concentration and seminiferous diameter. moreover, testicular histology tests confirmed dramatically declining testicular histopathologies as compared with the vpa group.34 table 1 explained the biological activities of gac fruit parts extracts. further discussion and integration this study aims to scientifically summarise the literature studies that have characterised the bioactive compounds in gac fruit and its portions. however, there is limited literature presently table 1. health benefits and biological activities of gac fruit gac part type of study health benefits mechanism of actions fresh gac aril clinical trial provitamin a activity improve β-carotene status which may lead to improve eye vision.7 gac peel, pulp and aril in vitro dna protective activity decrease dna damage induced by h 2 o 2 and uvc.27 water extract of gac aril in vitro and in vivo anti-tumor and anti-angiogenic activities inhibition of the growth of colon adenocarcinoma cell, reduce wet tumor, reduce the blood vessels density around the carcinoma.22 gac aril in vitro anticancer activity suppression of the proliferation of breast cancer (mcf7) and melanoma (mm418c1 and d24) cell lines.11 gac pulp and aril in vitro antimicrobial activity inhibition gram-positive and gram-negative bacteria growth.28 peel, pulp and aril in vitro antioxidant and antimicrobial scavenge radical activity and ferric reducing power, inhibition growth of different pathogenic bacteria such as (e. coli, s. aureus, and b. cereus).16 gac aril in vitro anticancer induce cell apoptosis of mcf-7 via intrinsic and extrinsic signalling pathways.10 gac seed in vitro anti-inflammatory activity reduce the production of nitric oxide (no), decrease mrna level of no synthase (inos) and cyclooxygenase (cox)-2, inhibit the translocation of p65 and p50 of raw 264.7.35 gac aril in vivo male reproductive improvement protected against decrease weights of epididymis and seminal vesicle, increased sperm concentration and seminiferous tubular diameters.34 gac seed in vitro anti-inflammatory inhibited lipopolysaccharide-induced expression of nitric oxide and il-6 via nf-κb pathway.31 gac seed in vivo anti-gastritis and wound healing activities protective effect in a helicobacter pylori-insulted gastritis model, wound healing effect on cutaneous injury and stimulation of calcitonin gene-related peptide and somatostatin receptors.33 gac seed in vitro renoprotective activity ameliorate of cisplatin-induced nephrotoxicity effect, blocking of mapks signalling cascade.32 gac seed in vivo antiulcer and wound healing activities acceleration of gastric ulcer healing via upregulation of vegf expression in an acetic acid rat model.29 gac seed in vitro anticancer activity inhibition cell survival of sgc7901 and mkn-28 gastric cancer cells, seed treatment block the cell cycle at s phase downregulation of parp and bel-2 proteins, activation of the enzymatic pathways caspase-3, -9 and -8.24 gac seed in vitro anticancer activity inhibition the a549 and h1299 lung cancer cell growth, induce apoptosis via upregulation of p53, bax and downregulation of bcl-2 and pi3k/akt signal pathways.25 gac seed in vitro anticancer activity suppression the proliferation and inhibit the invasion and migration of the breast cancer cell line (zr-75-30), inhibition the expression of mmp-2 and mmp-9.23 gac seed in vitro antioxidant activities improve hepatocytes cell damage induced by t-bhp, decreased lipid peroxidation and oxidized glutathione (gssh) that induced by t-bhp and increased glutathione (gsh) depletion.21 gac seed in vitro antioxidants and anticancer activities free radical scavenging via dpph and abts tests and ferric reducing power, suppression of the proliferation of human melanoma cells mm418c1 and d24 lines.26 abdulqader a. et al. 183j contemp med sci | vol. 4, no. 4, autumn 2018: 179–186 review gac fruit and its potentiality in health benefits available regarding the biological activity of gac fruit to fight against and prevent disease-causing cell damage such as cancer, diabetes and its complications, obesity, inflammation, atherosclerosis, cardiovascular disease, and microbial infections. moreover, a limited number of published papers have reported the abundance of multiple phytochemicals (e.g., carotenoids, phenolic compounds, flavonoids, vitamin e, and essential fatty acid) in gac fruit and its portions. the richness and abundance of the gac fruit in various bioactive compounds also make it a ‘super’ fruit possessing several health benefits. it is worth mentioning that the superiority of this fruit will undoubtedly attract the best researchers in this field in undertaking challenging and possibly lucrative investigations in the future. furthermore, most of the studies related to gac fruit have mainly emphasised the phytochemicals and/or bioactive compound contents especially carotenoid pigments. due to the presence and high concentrations of carotenoids, this also gives the gac fruit it’s bright, shiny, deep orange-red colour. indeed, β-carotene, lycopene, and lutein were the primary carotenoids that were mostly quantified.1,5–7,9,11 most of the previous studies investigated the gac fruit-rich extract, with little focus on the actual compounds. therefore, the purification of β-carotene, lycopene, and lutein and their application separately, could be more effective and conducive to various diseases. fortunately, carotenoids in gac fruit are mostly combined with oil and essential fatty acids. this characteristic enhances the priority and thereby promoting the advantages in the overall process of producing gac fruit than other fruits and vegetables. indeed, this is mainly because of the oil aiding in the absorption and digestion of carotenoids in the gastrointestinal tract in the digestive system.12,36 the phytonutrients in the parts of the gac fruit (i.e. peel, pulp, and aril) and discussed earlier in this study, have been found to be rich in carotenoids. however, from among these portions, only the aril is processed while the other parts, like the peel and pulp, are usually discarded and considered as wastage, rather than valuable carotenoids or bi-products of the fruit.3,4 surprisingly, the concentration of lutein is considerably high in gac peel compared with the other parts.5 lutein is one of the carotenoid pigments (xanthophylls) found to be uniquely concentrated in the retina of the eye.37 interestingly, several studies have reported that low levels of lutein are associated with eye impairment and disorders such as diabetic retinopathy and age related-macular degradation. prior studies have also revealed that diabetic retinopathy (dr) and age-related macular degeneration (amd) are related to having a relatively lower level of lutein and other xanthophylls.38,39 therefore, gac peel-rich lutein pigment should be utilised and processed as a significant source of lutein supplementation rather than merely being wasted and discarded. dr and amd are the leading cause of blindness in developing and developed countries as mentioned earlier and are characterised by chronic exposure of hyperglycaemia by the retina.40–42 however, the present treatment of the disease has many drawbacks such as retinal haemorrhage, retinal detachments, in addition to the treatment costs which place a considerable burden on the patients and their families.43–45 the role of carotenoid pigments in the protection and avoidance of eye disease has been well acknowledged in the literature.46–48 therefore, based upon the information presented and discussed in this review, and to the best of the author’s knowledge, there have been no studies investigating the use of gac fruit-rich carotenoid extracts for the treatment of eye disorders such as dr and amd. many pathophysiological pathways are involved in the development and progression of dr and amd diseases,49 and many of which have faced speculation toward the disruption of the balance of the ocular angiogenesis process in favour of pro-angiogenic signals.50 despite the recent progress in understanding the pathogenesis of dr and amd, including the role of growth factors, the diseases are still neither preventable nor curable. however, recent studies have indicated that regulating angiogenic markers may be an opportunistic target in the treatment of dr and amd.51 currently, attempts have been made to utilise phytotherapy such as medical herbalism, which relies on an empirical appreciation of medicinal herbs and its nutraceuticals due to the degree of safeness, efficacy and pharmacological action in the prevention and treatment of chronic diseases.52–55 for the later reasons, numerous efforts have been employed for screening and development of anti-angiogenic products from natural sources. notwithstanding, previous attempts have also been used on several phytochemicals and their derived metabolites from various medicinal plants, by testing their action and response as anti-angiogenic agents.48,56 the outcome of such studies was promising but far from perfect and acceptable, due to the low level of extracted carotenoids, particularly lutein in the extraction yield. as mentioned previously, gac fruit is abundantly rich in carotenoids and contains high quantities of lutein as compared with other medicinal plants. indeed, this advantage could enable novel, if not innovative approaches to be adapted to extract a prominent level of carotenoids from the fruit, and to examine its pharmacological effect and biological mechanisms of action. likewise, the determination of the pharmacological function using combined and individual molecules can also lead to exploring the mechanisms of action. discovering the mechanistic pathway by which the mechanism derives the molecules, and reverse or normalise the angiogenic process in diabetic patient’s eyes would be incredibly valuable. however, to date, there have been no large-scale studies to investigate the effect of the gac fruit’s rich extract against angiogenesis markers related to eye disease among all levels including in vitro, in vivo, and clinical studies. therefore, gac fruit and its multi-content of phytochemicals and especially carotenoids could be an ideal agent for a myriad of diseases, but further research studies would still be required to investigate the efficacy, the nature and role of gac fruit and its bioactive compounds by the combined and synergistic use. figure 2 shows the potential future work that can be conducted using gac fruit and its parts extracts. herefore, considering all the information discussed, discovering new sources that would be able to regress, slowdown, or delay the progression of dr and amd would be highly advantageous and recommended. finally, gac fruit-rich carotenoids may be a potential agent to control or to balance the angiogenesis process, which may open and expose new insights and knowledge for the cure and treatment of angiogenesis-related eye diseases. conclusion gac (m. cochinchinensis spreng.) fruit, is a conventional fruit found in vietnam and other southeast asian countries, which contains relatively high levels of carotenoids, especially lycopene and β-carotene. the gac fruit is a distinctive and promising phytonutrient which has been used for generations for 184 j contemp med sci | vol. 4, no. 4, autumn 2018: 179–186 gac fruit and its potentiality in health benefits review abdulqader a. et al. fig. 2 potential future research on gac fruit-rich carotenoids fruit. rpe, retinal pigmented epithelial cells; huvec, human umbilical vein endothelial cells; pedf, pigment epithelium-derived factor; fgf, fibroblast growth factor; vegf, vascular endothelial growth factor; plgf, placenta like growth factor; igf, insulin-like growth factor. abdulqader a. et al. 185j contemp med sci | vol. 4, no. 4, autumn 2018: 179–186 review gac fruit and its potentiality in health benefits both nutritional and medicinal purposes. additionally, used in the body, β-carotene is converted to vitamin a, thereby enhancing the immune system, helping to prevent eye-associated diseases. indeed, studies undertaken in recent years have exploited this conviction by revealing the high levels of antioxidants contained in the fruit. therefore, it is not surprising to observe the current research in nutritional sciences and health now being directed with utmost enthusiasm and motivation toward this unique fruit at both the cellular and molecular levels. acknowledgement we would like to acknowledge the staff in the library of the faculty of medicine and health sciences at universiti putra malaysia for their valuable assistance in the management and collection of literature and research papers. author contributions aa perfumed the work and manuscript formatting; fa conceived, supervised and edited the review; ai and ne supervised, and contributed to the structure of the manuscript and review. all authors read and approved the final manuscript. conflict of interest the authors have no conflict of interest related to this manuscript. n references 1. ishida bk, turner c, chapman mh, mckeon ta. fatty acid and carotenoid composition of gac (momordica cochinchinensis spreng) fruit. j agric food chem. 2004;52:274–279. 2. vuong lt. gac: a fruit from heaven. vietnam j. 2003. 3. chuyen h, nguyen mh, roach pd, golding jb, parks se. gac fruit (momordica cochinchinensis spreng.): a rich source of bioactive compounds and its potential health benefits. int j food sci technol. 2015;50:567–577. 4. kha tc, nguyen mh, roach pd, parks se, stathopoulos c. gac fruit: nutrient and phytochemical composition, and options for processing. food rev int. 2013;29:92–106. 5. kubola j, siriamornpun s. phytochemicals and antioxidant activity of different fruit fractions (peel, pulp, aril and seed) of thai gac (momordica cochinchinensis spreng). food chem. 2011;127:1138–1145. 6. aoki h, kieu nt, kuze n, tomisaka k, van chuyen n. carotenoid pigments in gac fruit (momordica cochinchinensis spreng). biosci biotechnol biochem. 2002;66:2479–2482. 7. vuong le, dueker sr, murphy sp. plasma β-carotene and retinol concentrations of children increase after a 30-d supplementation with the fruit momordica cochinchinensis (gac). am j clin nutr. 2002;75:872–879. 8. vuong lt, franke aa, custer lj, murphy sp. momordica cochinchinensis spreng.(gac) fruit carotenoids reevaluated. j food compos anal. 2006; 19:664–668. 9. nhung dt, bung pn, ha nt, phong tk. changes in lycopene and beta carotene contents in aril and oil of gac fruit during storage. food chem. 2010;121:326–331. 10. petchsak p, sripanidkulchai b. momordica cochinchinensis aril extract induced apoptosis in human mcf-7 breast cancer cells. asian pac j cancer prev. 2015;16:5507–5513. 11. wimalasiri d, piva t, huynh t. diversity in nutrition and bioactivity of momordica cochinchinensis. int j adv sci eng inf technol. 2016;6:378–380. 12. müller-maatsch j, sprenger j, hempel j, kreiser f, carle r, schweiggert rm. carotenoids from gac fruit aril (momordica cochinchinensis [lour.] spreng.) are more bioaccessible than those from carrot root and tomato fruit. food res int. 2017;99:928–935. 13. vuong lt. underutilized β-carotene–rich crops of vietnam. food nutr bull. 2000;21:173–181. 14. nhi tt, tuan dq. enzyme assisted extraction of gac oil (momordica cochinchinensis spreng) from dried aril. j food nutr sci. 2016;4:1–6. 15. bharathi lk, singh hs, shivashankar s, ganeshamurthy an, sureshkumar p. assay of nutritional composition and antioxidant activity of three dioecious momordica species of south east asia. proc natl acad sci india sec b. 2014;84:31–36. 16. tinrat s, akkarachaneeyakorn s, singhapol c. evaluation of antioxidant and antimicrobial activities of momordica cochinchinensis spreng (gac fruit) ethanolic extract. int j pharm sci res. 2014;5(8):3163–3169. 17. xiao cw, hu sh, rajput zi. adjuvant effect of an extract from cochinchina momordica seeds on the immune responses to ovalbumin in mice. front agric china. 2007;1:90–95. 18. rahmatullah m, biswas a, haq wm, seraj s, jahan r. an ethnomedicinal survey of cucurbitaceae family plants used in the folk medicinal practices of bangladesh 1. chron young sci. 2012; 3: 212–222. 19. chuyen hv, roach pd, golding jb, parks se, nguyen mh. optimisation of extraction conditions for recovering carotenoids and antioxidant capacity from gac peel using response surface methodology. int j food sci technol. 2017;52:972–980. 20. tsoi ay, wong rc, ng tb, fong wp. first report on a potato i family chymotrypsin inhibitor from the seeds of a cucurbitaceous plant, momordica cochinchinensis. biol chem. 2004;385:185–189. 21. tsoi ay, ng tb, fong wp. antioxidative effect of a chymotrypsin inhibitor from momordica cochinchinensis (cucurbitaceae) seeds in a primary rat hepatocyte culture. j pept sci. 2005;11:665–668. 22. tien pg, kayama f, konishi f, tamemoto h, kasono k, hung nt, et al. inhibition of tumor growth and angiogenesis by water extract of gac fruit (momordica cochinchinensis spreng). int j oncol. 2005;26:881–889. 23. zheng l, zhang ym, zhan yz, liu cx. momordica cochinchinensis seed extracts suppress migration and invasion of human breast cancer zr-75-30 cells via down-regulating mmp-2 and mmp-9. asian pac j cancer prev. 2014;15:1105–1110. 24. liu hr, meng ly, lin zy, shen y, yu yq, zhu yz. cochinchina momordica seed extract induces apoptosis and cell cycle arrest in human gastric cancer cells via parp and p53 signal pathways. nutr cancer. 2012;64:1070–1077. 25. shen y, meng l, sun h, zhu y, liu h. cochinchina momordica seed suppresses proliferation and metastasis in human lung cancer cells by regulating multiple molecular targets. am j chin med. 2015;43:149–166. 26. le a, huynh t, parks s, nguyen m, roach p. bioactive composition, antioxidant activity, and anticancer potential of freeze-dried extracts from defatted gac (momordica cochinchinensis spreng) seeds. medicines (basel). 2018;5. pii: e104. 27. klungsupya p, saenkhum j, muangman t, rerk-am u, laovitthayanggoon s, leelamanit w. non-cytotoxic property and dna protective activity against h 2 o 2 and uvc of thai gac fruit extracts in human tk6 cells. j appl pharm sci. 2012;2:4–8. 28. innun a. i-seec 2012. proceeding-science and engineering. 2013;1–6. 29. kang jm, kim n, kim b, kim jh, lee by, park jh, et al. enhancement of gastric ulcer healing and angiogenesis by cochinchina momordica seed extract in rats. j korean med sci. 2010;25:875–881. 30. lin zy, liu x, yang f, yu yq. structural characterization and identification of five triterpenoid saponins isolated from momordica cochinchinensis extracts by liquid chromatography/tandem mass spectrometry. int j mass spectrom. 2012;328–329:43–66. 31. jung k, chin yw, yoon kd, chae hs, kim cy, yoo h, et al. anti-inflammatory properties of a triterpenoidal glycoside from momordica cochinchinensis in lps-stimulated macrophages. immunopharmacol immunotoxicol. 2013;35:8–14. 32. jung k, lee d, yu js, namgung h, kang ks, kim kh. protective effect and mechanism of action of saponins isolated from the seeds of gac (momordica cochinchinensis spreng.) against cisplatin-induced damage in llc-pk1 kidney cells. bioorg med chem lett. 2016;26:1466–1470. 33. jung k, chin yw, chung yh, park yh, yoo h, min ds, et al. anti-gastritis and wound healing effects of momordicae semen extract and its active component. immunopharmacol immunotoxicol. 2013;35:126–132. 34. sukhorum w, sampannang a, sripanidkulchai b, iamsaard s. momordica cochinchinensis (l.) spreng. aril extract prevents adverse reproductive parameters of male rats induced with valproic acid. int j morphol. 2016;34:870–877. 186 j contemp med sci | vol. 4, no. 4, autumn 2018: 179–186 gac fruit and its potentiality in health benefits review abdulqader a. et al. 35. yu js, kim jh, lee s, jung k, kim kh, cho jy. src/syk-targeted anti-inflammatory actions of triterpenoidal saponins from gac (momordica cochinchinensis) seeds. am j chin med. 2017;45:459–473. 36. failla ml, chitchumronchokchai c, ferruzzi mg, goltz sr, campbell ww. unsaturated fatty acids promote bioaccessibility and basolateral secretion of carotenoids and α-tocopherol by caco-2 cells. food funct. 2014;5:1101–1112. 37. handelman gj, snodderly dm, adler aj, russett md, dratz ea. measurement of carotenoids in human and monkey retinas. meth enzymol. 1992;213:220–230. 38. hu bj, hu yn, lin s, ma wj, li xr. application of lutein and zeaxanthin in nonproliferative diabetic retinopathy. int j ophthalmol. 2011;4:303–306. 39. brazionis l, rowley k, itsiopoulos c, o’dea k. plasma carotenoids and diabetic retinopathy. br j nutr. 2009;101:270–277. 40. boscia f. current approaches to the management of diabetic retinopathy and diabetic macular oedema. drugs. 2010;70:2171–2200. 41. barot m, gokulgandhi mr, patel s, mitra ak. microvascular complications and diabetic retinopathy: recent advances and future implications. future med chem. 2013;5:301–314. 42. curcio ca. soft drusen in age-related macular degeneration: biology and targeting via the oil spill strategies. invest ophthalmol vis sci. 2018;59:amd160–amd181. 43. simó r, hernández c. advances in the medical treatment of diabetic retinopathy. diabetes care. 2009;32:1556–1562. 44. selvaraj k, gowthamarajan k, karri vv, barauah uk, ravisankar v, jojo gm. current treatment strategies and nanocarrier based approaches for the treatment and management of diabetic retinopathy. j drug target. 2017;25:386–405. 45. austeng d, morken ts, bolme s, follestad t, halsteinli v. nurse-administered intravitreal injections of anti-vegf: study protocol for noninferiority randomized controlled trial of safety, cost and patient satisfaction. bmc ophthalmol. 2016;16:169. 46. krinsky ni, johnson ej. carotenoid actions and their relation to health and disease. mol aspects med. 2005;26:459–516. 47. gorusupudi a, nelson k, bernstein ps. the age-related eye disease 2 study: micronutrients in the treatment of macular degeneration. adv nutr. 2017;8:40–53. 48. wu j, cho e, willett wc, sastry sm, schaumberg da. intakes of lutein, zeaxanthin, and other carotenoids and age-related macular degeneration during 2 decades of prospective follow-up. jama ophthalmol. 2015;133:1415–1424. 49. xu hz, song z, fu s, zhu m, le yz. rpe barrier breakdown in diabetic retinopathy: seeing is believing. j ocul biol dis infor. 2011;4:83–92. 50. babapoor-farrokhran s, jee k, puchner b, hassan sj, xin x, rodrigues m, et al. angiopoietin-like 4 is a potent angiogenic factor and a novel therapeutic target for patients with proliferative diabetic retinopathy. proc natl acad sci usa. 2015;112:e3030–e3039. 51. tolentino ms, tolentino aj, tolentino mj. current and investigational drugs for the treatment of diabetic retinopathy. expert opin investig drugs. 2016;25:1011–1022. 52. al-zuaidy mh, mumtaz mw, hamid aa, ismail a, mohamed s, razis afa. biochemical characterization and 1h nmr based metabolomics revealed melicope lunu-ankenda leaf extract a potent anti-diabetic agent in rats. bmc complement altern med. 2017;17:359. 53. abu bakar sajak a, mediani a, maulidiani, mohd dom ns, machap c, hamid m, et al. effect of ipomoea aquatica ethanolic extract in streptozotocin (stz) induced diabetic rats via 1h nmr-based metabolomics approach. phytomedicine. 2017;36:201–209. 54. khazaei mr, makalani f, ghanbari e, fayzemahdavi m, khazaei m. an overview of effective herbal and antioxidant compounds on diabetes. j contemp med sci. 2018;4:126–133. 55. albosof f, hoseini sa, siahpoush a, malayeri ar, haghighizadeh mh. anti-diabetic effects of s. aegyptiaca extract on streptozotocin-nicotinamide induced type 2 diabetes rats. j contemp med sci. 2018;4:22–25. 56. xing q, zhang g, kang l, wu j, chen h, liu g, et al. the suppression of kallistatin on high-glucose-induced proliferation of retinal endothelial cells in diabetic retinopathy. ophthalmic res. 2017;57:141–149. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 264 j contemp med sci | vol. 3, no. 11, summer 2017: 264–269 original a comprehensive anticancer molecular study for genistein the promising anticancer drug rand r. hafidh department of microbiology, college of medicine, university of baghdad, baghdad, iraq. correspondence to dr. rand r. hafidh (email: randriadh@comed.uobaghdad.edu.iq). (submitted: 25 may 2017 – revised version received: 10 june 2017 – accepted: 21 july 2017 – published online: 26 september 2017 ) objective genistein a potent isoflavonoid isolated from dietary soybean with a wide range of biological and therapeutic activities, particularly, in cancer prevention. the molecular mechanism that explains this powerful chemopreventive activity is still not fully understood. the study designed to give a clear and complete picture about this mechanism using a flow cytometry analysis and a quantitative real-time pcr to investigate 16 gene families for 1023 genes. methods human cervical cancer cells were treated with genistein ic 50 for 48 h. cell-cycle arrest and apoptosis induction were investigated using flow cytometry analysis. the high-throughput quantitative pcr array was used to explore the upand down-regulated genes affected by the treatment. results the quantitative real-time pcr analysis demonstrated the upand down-regulation to 11 cancer-related gene families due to genistein treatment. most of the upand down-regulated genes have a role in cell proliferation, dna replication and cell cycle progress. these findings were in harmony with the flow cytometry analysis in which a significant increase was manifested in the apoptotic cells (p < 0.05) of the treated cell cultures (18.34%) when compared with untreated cell cultures (5.7%). conclusion the results highly pointed on the importance of genistein as promising anticancer drug. a comprehensive map regarding the anticancer molecular mechanism of this natural compound was given and many new therapeutic targets to control cancer development were uncovered. keywords genistein, anticancer, cervical cancer, flavonoids introduction the research to explore new anticancer compounds has become crucial with the increasing level of the carcinogenic and mutagenic substances in the environment. the side effects and emergence of chemotherapy-resistant cancer cells among patients have made cancer research and finding of new anticancer drugs from natural products particularly medicinal plants pivotal.1 genistein or 4 ́,5,7-trihydroxyisoflavone, is a soybean isoflavone and a well-studied chemopreventive agent. it has been reported to potentiate the anticancer effect of some chemotherapeutics. interestingly, it is well known for its ability to prevent cancer progression.2,3 usually, the cervical cancer is one of the most diagnosed cancers and an important cause of mortality in women worldwide. once cervical cancer has metastasized, patient outcome is poor. in contrast, it is fully treatable in the early stages.4 recently, an important natural inhibitor of cancer metastasis; genistein, has emerged, which is widely distributed in beans. moreover, diet with genistein; the bioactive molecule (“nutraceutical”), has been linked to reduce the rate of cancer metastasis in a number of population-based studies.3,5 the ability of genistein to stop metastasis and prompt apoptosis in cancer cells has been proved by several studies. hence, genistein is able to lower mortality linked to solid organ cancer by important mechanisms.3,6 the ability to reduce the mortality associated with various cancer types, particularly cervical cancer is because this vital small molecule has a moderately extensive array of biological activities. recent studies have investigated the anticancer activity of genistein and found that this activity is due to its issn 2413-0516 anti-inflammatory, antioxidant, induction of apoptosis, anti-angiogenesis, and anti-metastasis effect in addition to its action as immunomodulatory.7,8 on the other hand, studies with huge cancer genomics data that could explain genistein anticancer activity still not enough. therefore, this study was tried to look into the anticancer mechanism of genistein. by using high-throughput quantitative pcr array (wafergen’s smartchip mrna array), this study has explored 1023 genes related to 16 families. the study investigated the differences in the gene expression between treated and untreated cells. the distinctive aspect of this array in comparison to other microarrays is that the detected mrnas is directly engaged in signaling pathways as well as cancer pathogenesis. thus may have a role in the anticancer effect of genistein and tried to give a complete map for the target genes which up or down regulated by this product. this map will facilitate the understanding of the anticancer mechanism used by genistein and easiest the way to produce a new and cheap anticancer drug. materials and methods preparation of the compound genistein, from glycine max (soybean), 98%, g6776-10mg, (sigma) was prepared by dissolving in absolute dimethyl sulfoxide (dmso), (bio basic inc., ny, usa) and incubated at −20°c as stock concentration 10 mg/ml. the stock solution was diluted in 10% rpmi medium to give the working concentrations. the working concentrations (15, 30, and 60 µg/ml) were prepared freshly before each test. rand r. hafidh 265j contemp med sci | vol. 3, no. 11, summer 2017: 264–269 original genistein anticancer molecular mechanism cancer cell and cytotoxicity assay the cancer cell line, cervix adenocarcinoma cells (hela; atcc ccl-2) was used to estimate the cytotoxic effects of genistein. the mts [3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2h-tetrazolium] colorimetric method (the cell titer 96 aqueous one solution cell proliferation assay, promega, usa) was used. it was used to decide the viable cells in proliferation or cytotoxicity assays. in each run, the experiment was repeated for three times with four wells per-working concentration. the absorbance was measured using a 96-well plate elisa reader (sunrise basic tecan, grödig, austria). apoptosis detection methods flow cytometry analysis the level of cell apoptosis in the treated hela cells was investigated by using the concentration (80 µg/ml) of genistein that killed 50% of the cells (ic50). dna staining solution (500 μl) containing 25 μl of propidium iodide (pi) 1 mg/ml (mp biomedicals, llc, iiikrick, france), a double-stranded nucleic acid intercalating agent, and 50 μl ribonuclease a from bovine pancrease (1 mg/ml), (sigma, germany) in pbs was used to re-suspend the ice-cold ethanol fixed pellets.9 for each sample, the assay was measured in duplicate. cyan adp apparatus (beckman coulter, usa) was used to measure the propidium iodide fluorescence of individual nuclei. the flow cytometry results were analyzed by the software summit (v4.3). smartchip real-time pcr system the gene expression profiling of 4,128 reaction wells on a single sample was enabled by smartchip human oncology gene panel version 1.5.1 from (wafergen, biosystems, fremont, ca, u.s.a.) using the smartchip nanodispensere. the pre-optimized pcr primers that have been confirmed in both microliterand nanoliter-scale sybr green real-time pcr experiments are already loaded in smartchip panels. quadruplicate primers of 1023 genes belonging to 16 functional groups was used (table 1). ten endogenous controls and six exogenous controls were used. the smartchip human oncology gene panel was used to measure differential gene expression from 0.5 µg of rna of genistein treated hela cells vs. untreated cells, only significantly upand down-regulated genes are displayed in this study. rna extraction the hela cell (1 × 105 cell/well) with roswell park memorial institute-1640 (rpmi-1640) maintenance medium with or without genistein ic50 (80 µg/ml) was incubated in duplicate for 48 h. total rna was isolated using gf-1 kit (vivantis technologies, malaysia). rna quality and quantity were determined by life science uv/vis spectrophotometer, du series 700 (beckman coulter, usa). rna concentration used in downstream experiments was 546 ng/µl. moreover, total rna quality was checked by agilent 2100 bioanalyzer (agilent, usa) and rna integrity number (rin) was measured. rin of total rna was 8.5 and passed the quality control limit for running downstream experiments. the isolated rna was stored at −80°c and was ready for use in downstream application, namely, qrt-pcr. real-time quantitative rt-pcr the reverse-transcription of 0.5 µg of the isolated hela cell’s rna was done using iscripttm cdna synthesis kit (bio-rad, hercules, canadad).the isolated cdna was stored at −80°c for qrt-pcr reaction. real-time quantitative pcr reaction was conducted using the smartchip which contains a 4128-nanowell array (72 × 72). these chips were preloaded with primer content optimized for performance with the smartchip real-time pcr system. the smartchip nanodispenser was used to dispense the sample, combined with master mix and controls, under vacuum into the 5184-nanowell smartchip. five hundred (500) ng of starting sample yields the cdna equivalent of 96 pg of sample per well. once loaded with sample, the smartchip was placed into the wafergen smartchip cycler. each prepared sample undergoes 4,128 nano-scale real time polymerase chain reactions (rt-pcr) in parallel. the smartchip real-time pcr system employs a qpcr reaction compatible with sybr green i dna-binding dye. results were reported in the form of ct (threshold cycles) and tm (melting temperatures) for amplicon-melting analyses. forward and reverse primers for each gene were designed using bioinformatics tools. primers were selected based on criteria such as specificity, insensitivity to sequence polymorphisms, amplicon size, and minimization of primer artifacts. primer specificity was determined using (a) melting curve analysis of amplicon product, (b) gel electrophoresis analysis of amplicons and (c) sequence verification of the amplicons. the up and down gene regulation was analyzed by smartchip software. the sensitivity of real-time pcr is 20–30 copies of rna per well with specificity of genes discrimination <90% homology. to keep precision of readings, standard deviation <0.25 ct between replicates was table 1. the gene families and the number of genes in each family covered by smartchip real-time pcr system from wafergen using smartchip human oncology gene panel gene family number of genes per family adme* 173 apoptosis 198 cancer 325 cell cycle/proliferation 52 cardiovascular disease 226 dna damage repair 36 drug target 43 g-protein coupled receptor 31 growth factor 12 homeostasis/metabolism 9 inflammation 143 kinase 194 proteinase 11 signal transduction 303 transcription factor 95 neurodisease/phosphatase 10 *drug absorption, distribution, metabolism and elimination genes. 266 j contemp med sci | vol. 3, no. 11, summer 2017: 264–269 genistein anticancer molecular mechanism original rand r. hafidh used. for valid runs, all primers share the same tm, amplicon size for each primer is either equal or close by, and no primer dimers or primer secondary structures are allowed. thermal cycling of primers was 95°c for 180 s once, and for 40 cycles, 95°c for 60 s, and 60°c for 70 s with amplicon size range 80–120 bp. automatic screening for low-efficiency wells turned out to be zero and pcr efficiency for all primers was measured and taken into account for calculating fold changes in gene expression. ten endogenous reference genes (tfrc-1, gapdh-2, taf-10, gusb, taf11, hprt, ppia, hmbs, actb, and b2m); all-means normalization method, where the means of all expressed genes are employed, were used in the current study. in addition, six exogenous yeast controls were used (wgbs-ycf1-6); at least four exogenous controls, yeast control, were detected and were linear in proportion. at each run, two negative tissue controls (ntc), chip no. 34565 and 35935, and two tissue positive controls, brain tissue (ptc), chip no. 34576 and 35939, were used. the fold change calculation used was based on the comparative ct method. the comparative ct method is also known as the 2-δδct method and as follows: δδct = δct treated – δct untreated, where: δct treated = ct treated – all mean treated and δct untreated = δct untreated – all mean untreated. the up-/down-regulated genes are those the genes that are at least two-fold change were considered as up-/ down-regulated. data analysis the current study showed data as mean ± sd. spss software version (12.0.0.2) was used to analyse data. the confidence intervals (95%) were used to evaluate the ability of the tested compound to inhibit the growth of the cells. the value of ic50 was calculated by linear regression index equations. regarding flow cytomteric results, r2 fraction stand for sub-g apoptotic cells. in addition, the total cells minus apoptotic cells was calculated to get the cells’ percentage at different cell cycle phases. for real-time qpcr, smartchip qpcr software exerted all data analysis. log upor down-regulation of gene expression more than 2 log was considered highly significant and was presented in this study. by using student t-test, p values less than 0.05 were considered significant for the variations among percentages of apoptotic cells. fig. 1 the histograms represent dna content frequency of hela cells after 48 h (a) untreated cultures (b) cultures treated with genistein ic50. the cell cycle distribution was affected by the treatment and apoptosis was induced. pi was used to stain the cells. cyan adp apparatus and summit (v4.3) software were used to measure the fluorescence of the pi-stained cells. the estimated percentages of cells with fractional dna content: apoptotic cells (r2), cells in g0/g1 (r3), s (r4), and g2/m (r5) phases of the cycle were provided by the software program. total cell number (r1). a b rand r. hafidh 267j contemp med sci | vol. 3, no. 11, summer 2017: 264–269 original genistein anticancer molecular mechanism results the cytotoxic effect of genistein on hela cells genistein concentration that killed 50% of the cervix adenocarcinoma cells was (80 µg/ml). later, this concentration was used to investigate the mechanism of the compound cytotoxicity by different techniques. detection of apoptosis by flow cytometry flow cytometry was used to measure apoptosis in the hela cells treated with genistein and compared with untreated cells. apoptosis was the type of cell death detected by flow cytometry after 48-h treatment time. however, apoptosis was confirmed by integrating the results of both flow cytometry and rt-qpcr. a remarkable potential of genistein to induce apoptosis in the treated cells comparing to untreated cells was revealed by the flow cytometric analysis (fig. 1). hence, hela cells treated with genistein ic50 showed rise in the percentage of the apoptotic cells. the apoptotic cells increased significantly (p < 0.05) in the treated cell cultures (18.34%) when compared with untreated cell cultures (5.7%). detection of the upand down-regulation gene expression by smartchip real-time pcr system the quantitative analysis by real-time pcr explained the anticancer mechanism of genistein. the treatment of hela cells with genistein for 48 h showed that 11 gene families from 16 total family groups integrated in the smartchip human oncology panel were affected by genistein. the treatment affected the family groups by clear upand down-regulation to their genes (table 2). the gene family adme showed two up-regulated genes and two down-regulated genes from total of 173 genes. the range was (4.05 to 2.34) with mean 3.19 ± 1.21 and (−3.19 to −5.57) mean −4.38 ± 1.68 for the upand down-regulation, respectively. apoptosis gene family revealed 2 and 3 upand down regulated genes from total of 198 genes. the range was (4.05 to 2.34), 3.19 ± 1.21 and (−3.41 to −5.57), −4.24 ± 1.17 for up and down-regulated genes, respectively. the gene family signal transduction showed 3 and 6 from 303 genes of upand down-regulated genes, respectively. with the range of (4.05 to 2.03), 2.81 ± 1.09 for up-regulated genes and (−2.46 to −5.57), −3.51 ± 1.13 for down-regulated genes. from total of 325 genes of cancer family, one gene was up-regulated and two were down-regulated. the range was (−2.17 to −2.65), −2.41 ± 0.34. in the kinase family, the treatment revealed two down-regulated genes from 194 genes. the family genes’ range was (−2.56 to −2.96), −2.76 ± 0.29. cardiovascular disease family showed one up-regulated gene and four down-regulated genes from 226 genes. the genes’ range was (−2.03 to −5.57), −3.88 ± 1.58. the up-regulated genes were two in transcription factor family while the down-regulated gene was only one from total of 95 genes. the range was (4.05 to 2.03), 3.04 ± 1.43. the treatment caused upand down-regulation to only one gene in the inflammation family genes from total of 143. table 2. the upand down-regulated genes in different gene families after 48-h treatment of hela cells with limonin versus untreated hela cells gene family differentially expressed geneslog change adme upregulated ddit34.05,cdkn1a2.34 downregulated ptgs2−3.19, il1a−5.57 apoptosis upregulated ddit34.05, cdkn1a2.34 downregulated bnip3l−3.41, bcl3−3.72, il1a−5.57 signal transduction upregulated ddit34.05, cdkn1a2.34, evi12.03 downregulated mef2c−2.46, map2k5−2.56, ptgs2−3.19, adora2b−3.59, bcl3−3.72, il1a−5.57 cancer upregulated cdkn1a2.34 downregulated mki67-2−2.17, mll3−2.65 kinase upregulated downregulated map2k−2.56, pdgfrl−2.96 cardiovascular disease upregulated chi3l12.72 downregulated col8a1−2.03, ptgs2−3.18, adamts1−4.75, il1a−5.57 transcription factor upregulated ddit34.05, evi12.03 downregulated mef2c−2.45 inflammation upregulated cdkn1a2.34 downregulated ptgs2−3.19 dna damage repair upregulated downregulated rpa1−2, atrx−2.04 cell cycle/ proliferation upregulated downregulated ccnf−2.2 g-protein coupled receptor upregulated downregulated pdgfrl−2.96 genistein treatment for 48 h resulted in two down regulated genes from 36 genes of dna damage repair family. the range of the down-regulated genes was (−2 to −2.04), −2.02 ± 0.03. one gene was down regulated in both cell cycle/proliferation family and g-protein coupled receptor family from their 52 and 31 genes, respectively. on the other hand, there were minor changes in the expression of genes belong to drug target, homeostasis/metabolism and neurodisease/phosphatase families. discussion it has been proved that natural products and their antioxidant components are the major source of human health promotion and maintenance. hence, nature is still the perfect source for health promotion and for the supplementation of safe drugs.10 a potent antiviral and anticancer effect from beans or their sprout extracts have discovered by many studies.11,12 furthermore, genistein, an isoflavone finds in many plants especially soybeans and fava beans proved to be highly effective anticancer compound.2,3,6 the results of this study have clarified the real anticancer mechanism of this highly promising anticancer drug, 268 j contemp med sci | vol. 3, no. 11, summer 2017: 264–269 genistein anticancer molecular mechanism original rand r. hafidh genistein. also, the massive cancer genomic data of genistein anticancer activity were searched to facilitate the development of molecularly targeted cancer therapies. the function of the upand down-regulated genes was synchronized with the increased percentage of apoptotic cells in treated hela cells (5.7–18.34%), which investigated by a flow cytometry analysis. even though, the dramatic changes in adme and apoptosis gene families were previously highlighted in many studies,13–15 a highlight on the genistein real mechanism of action was only given in this study. the results showed up-regulation to dna damage inducible transcript 3 gene (ddit3) by four families: adme, apoptosis, signal transduction, and transcription factor. this is known as a protein-coding gene and has a critical role in the induction of apoptosis and cell cycle arrest.16 the study revealed number of down-regulated genes in many families like: cancer (2 genes), kinase (2 genes), and transcription factor (one genes). these results enhance other findings who found that genistein targets many pathways like caspases, b cell lymphoma 2 (bcl-2, extracellular signal-regulated kinase 1/2 (erk1/2), kinesin-like protein 20a (kif20a), nuclear transcription factor kappab (nf-kappab), inhibitor of nf-kappab (ikappab), wingless and integration 1 beta-catenin (wnt/ beta-catenin), mitogen-activated protein kinase (mapk) and phosphoinositide 3 kinase/akt (pi3k/akt) that can attribute to its anticancer therapeutic effect.17,18 in addition, the up-regulation to many important genes, which induce apoptosis following caspase activation was manifested in adme, apoptosis, signal transduction, cancer and inflammation families. a gene with such function is cyclin dependent kinase inhibitor 1a gene (cdkn1a).19 similarly, the up-regulation to three genes in signal transduction family was in harmony with other previous studies.14,20 most of these up-regulated genes have a role in the stimulation of apoptosis and cell cycle arrest. the down-regulation to six genes in the signal transduction family was the highest number among all the tested families. although, the ability of genistein to down-regulate some of signal transduction family’s genes was studied,20,21 a full map regarding signal transduction genes upand down-regulation was clarified by this study. from the down-regulated genes, which manifested after genistein treatment in dna damage repair family were rpa1 (replication protein a1) and atrx (atrx, chromatin remodeler). these two protein-coding genes play an essential role in dna replication.22,23 moreover, only one gene was down-regulated in the cell cycle/proliferation family. it is an important regulator of cell cycle transitions, cyclin f (ccnf), through its ability to activate and bind to cyclin-dependent protein kinases.24 therefore, these down-regulated genes may suppress cancer development and could be a new therapeutic target for cancer control. one of the important findings for this study is the down regulation to prostaglandin-endoperoxide synthase 2 gene (ptgs2). ptgs2 is a key step in the production of prostaglandin e2 (pge2) in cancer cells, which plays important roles in mod ulating motility, proliferation and resistance to apoptosis.25 this down-regulation was found in adme, signal transduction, cardiovascular disease and inflammation families. the cardiovascular disease gene family showed one up-regulated gene and four down-regulated genes. the up regulated gene was chitinase 3-like 1, cartilage glycopro tein-39 (chi3l1), which plays a role in inflammatory cell apoptosis, and tissue remodeling (the capacity of cells to respond to and cope with changes in their environment).26 recently, chi3l 1 gene found to be a good target to overcome some highly malignant diseases.27 in turn, the up-regulation to such target gene highlights the importance of our study and promotes further studies to explore the anticancer molecular mechanism of such promising drugs. a protein-coding gene; pdgfrl (platelet-derived growth factor receptor like), which is highly expressed in human cer vical cancer and associated with colorectal cancer, and hepato cellular carcinoma was down regulated after treatment. this gene belongs to g protein-coupled receptor family, known by its ability to regulate the cell proliferation of various cancers.28 so, it could be a good therapeutic target in controlling cancer cell proliferation. conclusions genistein, the soybean isoflavone, found to be a promising anticancer agent with a cheap and inexpressive source. this study gives a complete picture about the anticancer molecular mechanism of genistein by exploring 16 gene families and can be used as a good reference for future more specific studies on each gene family. funding this research was conducted with the help of universiti putra malaysia (upm) to visiting scientists under grant no. u-1254-os; upm provided the bench work and covered research fees, while publication fees were not under financial cover. conflict of interest none n references 1. hafidh rr, abas f, abdulamir as, jahanshiri f, abu bakar f, sekawi z. a review: cancer research of natural products in asia. int j canc res. 2009;5:69–82. 2. xie x, wang ss, wong tc, fung mc. genistein promotes cell death of ethanol-stressed hela cells through the continuation of apoptosis or secondary necrosis. cancer cell int. 2013;13:63. 3. pavese jm, farmer rl, bergan rc. inhibition of cancer cell invasion and metastasis by genistein. cancer metastasis rev. 2010;29:465–482. 4. roomi mw, cha j, kalinovsky t, roomi n, niedzwiecki a, rath m. effect of a nutrient mixture on the localization of extracellular matrix proteins in hela human cervical cancer xenografts in female nude mice. exp ther med. 2015;10:901–906. 5. mcclements dj, xiao h. designing food structure and composition to enhance nutraceutical bioactivity to support cancer inhibition. semin cancer biol. 2017. 6. dhandayuthapani s, marimuthu p, hormann v, kumi-diaka j, rathinavelu a. induction of apoptosis in hela cells via caspase activation by resveratrol and genistein. j med food. 2013;16:139–146. 7. mohamed sia, jantan i, haque ma. naturally occurring immunomodulators with antitumor activity: an insight on their mechanisms of action. int immunopharmacol. 2017;50:291–304. 8. tafrihi m, nakhaei sistani r. e-cadherin/beta-catenin complex: a target for anticancer and antimetastasis plants/plant-derived compounds. nutr cancer. 2017;69:702–722. rand r. hafidh 269j contemp med sci | vol. 3, no. 11, summer 2017: 264–269 original genistein anticancer molecular mechanism 9. siddik zh. checkpoint controls and targets in cancer therapy. totowa, n.j.: humana press; 2010. xiv, p. 273. 10. hafidh rr, abdulamir as, abu bakar f, abas f, jahanshiri f, sekawi z. antioxidant research in asia in the period from 2000–2008. am j pharmacol toxicol. 2009;4:48–66. 11. hafidh rr, abdulamir as, bakar fa, jalilian fa, abas f, sekawi z. novel molecular, cytotoxical, and immunological study on promising and selective anticancer activity of mung bean sprouts. bmc complement altern med. 2012;12:208. 12. hafidh rr, abdulamir as, abu bakar f, sekawi z, jahansheri f, jalilian fa. novel antiviral activity of mung bean sprouts against respiratory syncytial virus and herpes simplex virus -1: an in vitro study on virally infected vero and mrc-5 cell lines. bmc complement altern med. 2015;15:179. 13. yang z, kulkarni k, zhu w, hu m. bioavailability and pharmacokinetics of genistein: mechanistic studies on its adme. anticancer agents med chem. 2012;12:1264–1280. 14. chen j, duan y, zhang x, ye y, ge b. genistein induces apoptosis by the inactivation of the igf-1r/p-akt signaling pathway in mcf-7 human breast cancer cells. food funct. 2015;6:995–1000. 15. zhu j, zhang c, qing y, cheng y, jiang x, li m, et al. genistein induces apoptosis by stabilizing intracellular p53 protein through an ape1-mediated pathway. free radic biol med. 2015;86:209–218. 16. liu x, liu k, qin j, hao l, li x, liu y, et al. c/ebpbeta promotes angiogenesis through secretion of il-6, which is inhibited by genistein, in egfrviiipositive glioblastoma. int j cancer. 2014;136:2524–2534. 17. spagnuolo c, russo gl, orhan ie, habtemariam s, daglia m, sureda a, et al. genistein and cancer: current status, challenges, and future directions. adv nutr. 2015;6:408–419. 18. dai w, wang f, he l, lin c, wu s, chen p, et al. genistein inhibits hepatocellular carcinoma cell migration by reversing the epithelialmesenchymal transition: partial mediation by the transcription factor nfat1. mol carcinog. 2013;54:301–311. 19. yu gp, xiao qy, shi zq, tang ls, ma xp, zhang ly, et al. genetic polymorphisms in apoptosis-related genes and the prognosis of hepatocellular carcinoma. am j cancer res. 2015;5:3249–3259. 20. song m, tian x, lu m, zhang x, ma k, lv z, et al. genistein exerts growth inhibition on human osteosarcoma mg-63 cells via ppargamma pathway. int j oncol. 2015;46:1131–1140. 21. whirledge s, senbanjo lt, cidlowski ja. genistein disrupts glucocorticoid receptor signaling in human uterine endometrial ishikawa cells. environ health perspect. 2014;123:80–87. 22. lin yl, shivji mk, chen c, kolodner r, wood rd, dutta a. the evolutionarily conserved zinc finger motif in the largest subunit of human replication protein a is required for dna replication and mismatch repair but not for nucleotide excision repair. j biol chem. 1998;273:1453–1461. 23. episkopou h, draskovic i, van beneden a, tilman g, mattiussi m, gobin m, et al. alternative lengthening of telomeres is characterized by reduced compaction of telomeric chromatin. nucleic acids res. 2014;42:4391–4405. 24. d’angiolella v, donato v, forrester fm, jeong yt, pellacani c, kudo y, et al. cyclin f-mediated degradation of ribonucleotide reductase m2 controls genome integrity and dna repair. cell. 2012;149:1023–1034. 25. kim hb, kim m, park ys, park i, kim t, yang sy, et al. prostaglandin e2 activates yap and a positive-signaling loop to promote colon regeneration after colitis but also carcinogenesis in mice. gastroenterology. 2017;152:616–630. 26. hamilton g, rath b, burghuber o. chitinase-3-like-1/ykl-40 as marker of circulating tumor cells. transl lung cancer res. 2015;4:287–291. 27. hamilton g, rath b, ulsperger e. how to target small cell lung cancer. oncoscience. 2015;2:684–692. 28. zhang q, wu yz, zhang ym, ji xh, hao q. activation of g-protein coupled estrogen receptor inhibits the proliferation of cervical cancer cells via sustained activation of erk1/2. cell biochem funct. 2015;33:134–142. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201704 279j contemp med sci | vol. 5, no. 5, september-october 2019: 279–280 ferritin level may be a major factor in young females with recurrent aphthous stomatitis najla dar-odeha,b and osama abu-hamamda,b acollege of dentistry, taibah university, al madinah al munawara, saudi arabia. bschool of dentistry, university of jordan, amman, jordan. correspondence to najla dar-odeh (email: najla_dar_odeh@yahoo.com). (submitted: 14 april 2019 – revised version received: 03 july 2019 – accepted: 13 august 2019 – published online: 26 october 2019) introduction recurrent aphthous stomatitis (ras) is considered the most common type of recurrent oral ulcers. clinically these ulcers present in three forms; the minor, major and herpetiform ulcers. the most common form of these is the minor type which affects more than 85% of ras patients.1 minor ulcers appear as shallow ulcers with a yellowish-whitish floor and a well-defined red halo that is regular in shape. occurrence and recurrence of ras will eventually affect the quality of life of patients due to the associated oral soreness and burning sensation jeopardizing oral functions like speaking and mastication. approximately 20% of the general population is affected by ras, but incidence varies from 5% to 50% according to ethnicity and socioeconomic status.2 although etiology of ras is not well-determined yet, nutritional and psychosocial factors have been suggested in a certain category of patients.3 a small percentage of ras patients (5–10%) show low serum levels of iron, folate, zinc, or vitamins b1, b2, b6 and b12 indicating that nutritional deficiency is apparently an etiological factor for ras.3 this case series aims to report the laboratory findings namely hemoglobin level, serum vitamin b12 and serum ferritin in seven ras patients who attended the oral medicine clinic at taibah university dental hospital (tudh) for treatment of their oral ulcers. it also aims to report the low serum ferritin level that was observed in these patients. this report is part of a larger project that was initiated at tudh/female section to investigate oral diseases among female patients. ethical approval was obtained from taibah university, college of dentistry research ethics committee-ethical approval #tucdrec/20171213/dar-odeh. case series patients attended the oral medicine clinic complaining of recurrent oral ulcers. all patients reported no medical illness except for the oral ulcers. they had minor ulcers that appeared exclusively in the labial and/or buccal mucosa (figs. 1 and 2). history and clinical features were suggestive of ras. topical treatment based on hyaluronic acid was prescribed to patients and they were referred to the same laboratory to perform the following tests: complete blood count, serum ferritin and serum vitamin b12. patients’ age range was 13–34 years (mean = 20.4, sd = 6.9). only one patient was anemic [hemoglobin (hb) = 9.2 g/dl], while the rest of patients had a normal hb level (mean = 13, sd = 1.74). serum ferritin level ranged from 6 to 50.8 ng/ml (mean = 26.1, sd = 15.2), and serum vitamin b12 ranged from 164 to 764 pg/ml (mean = 461.4, sd = 226.7). patients who had low serum ferritin were prescribed iron supplements, and they showed marked improvement in symptoms following therapy. discussion the report describes hematological findings in a series of young female patients who attended the oral medicine clinic or tudh. six ras patients had low serum ferritin level. there are a number of factors that need to be approached when addressing ras in young female patients, namely; serum ferritin levels, anemia, and stress initiated by school life. iron deficiency is considered the most common nutritional deficiency,4 with women at a higher predisposition to developing iron deficiency,4 and to become anemic. possible causes for iron deficiency in our patients may include reduced intake and/or increased loss through menstruation. in women, appearance of ras may coincide with menses, and studying-related stress may further explain the higher prevalence of ras in students.5 previous studies have pointed out to the poor oral health of young women in this geographic area manifested as jaw pathology in the form of remaining roots and periapical (tooth-related) bone lesions.6,7 this poor level of oral health is expected to adversely affect oral functions like mastication with resultant reduced consumption of healthy diet. anemia in women is defined by the world health organization as an hb level that is below 12.0 g/dl,8 hence, only one of the patients had anemia (hb level <12.0 g/dl). iron deficiency, on the other hand, is best evaluated by measuring serum ferritin levels.9 this may explain why ras is more prevalent among women. taking into consideration the fact that ferritin is an acute phase protein that can be elevated in a variety of inflammatory recurrent aphthous stomatitis is considered the most common type of recurrent oral ulcers that affects adversely the quality of life of patients. its etiology is not well understood; however, nutritional and psychosocial factors have been suggested in a certain category of patients. the aim of this case series is to describe the hematological picture namely hemoglobin level, serum vitamin b12 and serum ferritin in six young female patients affected by recurrent aphthous stomatitis, and to report its association of low serum ferritin. keywords ferritin, aphthous stomatitis, young, females issn 2413-0516 case report 280 j contemp med sci | vol. 5, no. 5, september-october 2019: 279–280 ferritin level as a major factor in young females with ras n. dar-odeh and o. abu-hamamd and infectious conditions, individuals may still have normal ferritin levels in spite of being iron deficient (false negatives).4 serum ferritin level was found to have no association with certain infections of the oral mucosa like candidiasis in young females;10 however, it is critical for the growth and differentiation of all cells, and its deficiency may lead to epithelial abnormality or atrophy.11 many studies have reported a highly significant reduction in the total epithelial thickness, particularly the thickness of the maturation compartment.12 iron deficiency without overt anemia can result in neuropsychological effects and it has been linked to delayed cognitive development in children and adolescents.13 interestingly, the diagnosis of nutritional deficiency and anemia from oral mucosa changes can be established in the absence of symptomatic anemia or even in the pre-anemic stage.14 patients who are not anemic but have serum ferritin levels of <15–30 ng/ml are diagnosed as having tissue iron deficiency.15 six out of seven of our patients had serum ferritin levels of ≤30 ng/ml indicating tissue iron deficiency. it had been suggested that hematologic screening of ras patients for anemia or deficiency of iron, folate, and b vitamins is appropriate for patients with major ras or cases of minor ras that worsen during adult life.1 according to the findings of this case series, another category of ras patients that require hematologic screening may also include young females. routine hematological screening for serum ferritin, folic acid and vitamin b12 should be assessed in all patients with ras, particularly young females, to treat any nutritional deficiency and to prevent more important related systemic manifestations. conflicts of interest the authors declare that they have no conflicts of interest.  fig. 1 minor aphthous ulcer (arrow) affecting the labial mucosa of a non-anemic 22-year-old woman with a serum ferritin level of 11.9 ng/ml. fig. 2 minor aphthous ulcers affecting the labial mucosa in a 13-year-old girl. references 1. akintoye so, greenberg ms. recurrent aphthous stomatitis. dent clin north am. 2005;49:31–47. 2. rogers rs. recurrent aphthous stomatitis: clinical characteristics and associated systemic disorders. semin cutan med surg. 1997;16:278–283. 3. dar-odeh ns, alsmadi om, bakri f, abu-hammour z, shehabi aa, al-omiri mk, et al. predicting recurrent aphthous ulceration using genetic algorithmsoptimized neural networks. adv appl bioinforma chem. 2010;3:7–13. 4. lu sy. perception of iron deficiency from oral mucosa alterations that show a high prevalence of candida infection. j formos med assoc. 2016;115:619–627. 5. mccann al, bonci l. maintaining women’s oral health. dent clin north am. 2001;45:571–601. 6. el khateeb sm, abu-hammad o, fadel h, dar-odeh n. a retrospective analysis of radiographic jaw findings in young women; prevalence and predictors. j int soc prev community dent. 2017;7:22–27. 7. dar-odeh ns, aleithan fa, alnazzawi aa, al-shayyab mh, abu-hammad so, abu-hammad oa. factors affecting oral health determinants in female university students: a cross-sectional survey in saudi arabia. int j adolesc med health. 2017. 8. cappellini md, motta i. anemia in clinical practice-definition and classification: does hemoglobin change with aging? semin hematol. 2015;52:261–269. 9. guyatt gh, oxman ad, ali m, willan a, mcilroy w, patterson c. laboratory diagnosis of iron-deficiency anemia an overview. j gen intern med. 1992;7:145–153. 10. dar-odeh ns, shehabi a, al-bitar zb, al-omari ik. oral candida colonization in patients with fixed orthodontic appliances: the importance of some nutritional and salivary factors. afr j microbiol res. 2011;5:2150–2154. 11. bermejo f, garcía-lópez s. a guide to diagnosis of iron deficiency and iron deficiency anemia in digestive diseases. world j gastroenterol. 2009;15:4638–4643. 12. wu yc, wang yp, chang jy, cheng sj, chen hm, sun a. oral manifestations and blood profile in patients with iron deficiency anemia. j formos med assoc. 2014;113:83–87. 13. grantham-mcgregor s, ani c. a review of studies on the effect of iron deficiency on cognitive development in children. j nutr. 2001;131:649s–666s; discussion 666s–668s. 14. lu sy, wu hc. initial diagnosis of anemia from sore mouth and improved classification of anemias by mcv and rdw in 30 patients. oral surg oral med oral pathol oral radiol endod. 2004;98:679–685. 15. pasricha srs, flecknoe-brown sc, allen kj, gibson pr, mcmahon lp, olynyk jk, et al. diagnosis and management of iron deficiency anaemia: a clinical update. med j aust. 2010;193:525–532. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201908 case report 13j contemp med sci | vol. 1, no. 4, autumn 2015: 13–15 research objectives because of the less studies in this field in iraq, this study aimed to use local streptococcus pyogenes isolates to produce hyluronic acid. methods the quantitative estimation of hyaluronic acid (ha) produced from eight local s. pyogenes isolates at different ph (6.3, 6.6, 6.9, 7.2, and 7.5) and glucose concentration (4%, 6%, 8%, and 1%) were done using the ha elisa kit. results this study showed that the maximum yield of ha was obtained at ph 7.5, and it was found that the differences in ph of the production media of ha enhanced the ha production, but the glucose concentration has no beneficial effect for ha production. conclusion there were differences in ha production among local isolates at the same ph. keywords streptococcus pyogenes, hyaluronic acid, ha elisa kit, ph, glucose concentration production and optimisation of hyaluronic acid extracted from streptococcus pyogenes kawkab abdulla al-saadiaaa, hassan fadhil najib, & ali hmood al-saadib introduction hyaluronic acid (ha), also known as hyaluronan, is a linear polysaccharide (up to 10 mda) consisting of alternating units of (1,4)-glucuronic acid and (1,3)-n-acetyl-d-glucosamine (glcnac).1 additionally to retaining and modulating the flow of water, it also helps as the backbone for proteoglycan gathering by binding aggrecan monomers via link protein.2 furthermore, ha carries numerous functions in altering cellular functions during the development of human, specifically during the development of diarthrodial joints. in developed cartilage, ha has a great significance of ligand for cell-matrix interactions with pericellular matrix via the cd44 cell receptor.3 the physical, chemical, and biological attribution of the ha includes lubricity, visco-elasticity, holding of water, biocompatibility, cell multiplication, morphogenesis, inflammation, and wound repair as well as specific signal transduction and cellular interactions through cell surface receptors.4,5 ha is highly hygroscopic, biocompatible, and decomposable biopolymer, real attractive for biomaterials fabrication. it is intensively used in cosmetics, surgery, and delivery of drugs.6 ha has been normally extracted from rooster combs and bovine vitreous humor. however, it is difficult to isolate high molecular weight ha at industrially practicable rate from these sources, because it makes a complex with proteoglycans exist in animal tissue. it is presently impractical to manipulate the molecular weight of the biopolymer while it is synthesized in animal tissue. moreover, the use of animal-derived biochemicals for human therapeutics has embossed ethical outcomes, and is met with growing resistance. to overcome these disadvantages, the recent tendency involves the usage of lancefield’s group a and group c streptococci, which naturally produce a mucoid capsule of ha.7 the requests for ha products from bacterial fermentation have fundamentally expanded because of both their increased use as medical devices and the immune issues that happened from the use of animal-based ha5. because of both the high prices of ha and the high standard requirements of its applications in medical products, high-quality ha products rather than high quantity have been the essential criteria utilized when selecting the bacterial strains utilized for ha generation. streptococci are ideally meant and adapted for studying the biosynthesis of ha due to the abundant availability of hyaluronate and since in this organism the hyaluronate is the only polymer into which glucuronic acid is comprised.8 in streptococci, ha is created as a secondary metabolite and the production is affected by different agents that involve genetic as well as nutritional. streptococci produces ha both under aerobic and anaerobic condition.9 materials and methods bacterial isolates eight local isolates of streptococcus pyogenes were isolated from ent infectious, and identified depending on traditional methods as described by macfaddin,10 in addition to use the strepto-system 9r according to the manufacture’s instructions and molecular methods by amplification of universal and specific species genes. these isolates are given numbers 1, 2, 3, 4, 5, 6, 7, and 8. isolation of ha ha creating bacteria were chosen based upon their hemolytic character. best hemolysis producing colonies were picked from every blood agar plate and streaked on todd hewitt agar plates. the plates were incubated at 37ºc in a 5% co2 atmosphere for 24 hours.9 ph effect on the production of ha the effect of ph on the production of ha was measured by the following technique. ten milliliters of tood hewitt broth (thb) medium kept up at five different ph (6.3, 6.6, 6.9, 7.2, and 7.5) were inoculated with a loopful of s. pyogenes isolates, and incubated at 37ºc for16 hours. the overnight cultures were inoculated into 10 ml of fresh thb medium and inoculated at 37ºc for 24 hours under shaking condition. the resulting suspensions were centrifuged, washed with 10 ml of 10 mm tris hcl (ph 7.5) and vortexed for 10 seconds.9 glucose concentration effect in the production of ha the effect of glucose concentration in the production of ha was measured by the following technique. four conical flasks issn 2413-0516 auniversity of karbala, college of science, department of biology, iraq. buniversity of babylon, college of science, department of biology, iraq. correspondence to kawkab abdulla al-saadi (email: kowkab_abdalla@yahoo.com). (submitted: 30 august 2015 – revised version received: 26 september 2015 – accepted: 9 october 2015 – published online: autumn 2015) 14 j contemp med sci | vol. 1, no. 4, autumn 2015: 13–15 production and optimisation of ha extracted from streptococcus pyogenes research kawkab abdulla al-saadiaa et al. containing 10 ml of thb medium, each was supplemented with different glucose concentration (0.4, 0.6, 0.8, and 1%) were inoculated with 15–30 colonies of s. pyogenes isolates, and incubated at 37ºc for 16 hours. the overnight culture were sub-cultured in to 10 ml of fresh thb media and incubated at 37ºc for 24 hours under shaking condition. the resulting suspensions were centrifuged, washed with 10 ml of 10 mm tris hcl (ph 7.5), and vortexed for 10 seconds.9 ha extraction bacterial cells cultivated under different condition were pelleted out and the cell pellets were resuspended in 1.5 ml of water and vortexed for 10 seconds. this washing sequence was repeated twice and the cell pellets were resuspended in water to a final volume of 1.5 ml. the ha capsule was extracted by adding 1.5 ml of chloroform and shaking for 1 minute. cell remained at room temperature for 1 hour and then was pelleted and the aqueous phase was used for estimation.9 quantitative estimation of ha by ha elisa kit the quantitative estimation of ha according to the manufacture’s instructions of the ha elisa kit (elabscience/china) was carried out. results quantitative estimation of ha eight different isolates of s. pyogenes with pathogenic characters were used for ha production. after the cultivation of s. pyogenes isolates in todd hewitt agar plates, the large white mucoid colonies were selected for ha production. the ha content of these isolates, which were incubated at different ph, were analyzed according to ha elsa kit protocol. based on the results obtained in fig. 1 the maximum ha yield was observed with isolated numerated one at ph 7.5 (67.9 ng/ml). while the same ph (7.5) shows no ha production in isolated number 5, 6, 7, and 8. discussion the elisa procedure is sensitive, simple, and is based on a microtitre plate format. the assay involves competition between ha absorbed to the plate and ha free in solution for binding to biotinylated cartilage proteoglycan binding region. the range of the assay is 10–2500 ng/ml with 50% inhibition at 0 10 20 30 40 50 60 70 1 2 3 4 5 6 7 8 ha concentration (ng /ml ) s. pyogenes isolates ph 7.5 ph 7.2 ph 6.9 ph 6.6 ph 6.3 fig. 1 ha yield (ng/ml) generated from s. pyogenes under different ph. about 200 ng/ml. this technique involves fewer experimental steps and is simpler to perform than other methods.11 production media were maintained at an optimised ph of some isolates estimated at four different glucose concentrations (0.4, 0.6, 0.8, and 1%) according to the elsa kit protocol. based on the results shown in fig. 2, all of selected isolates undergo an acute decline in ha production. the study by saranraj et al.9 showed gradually increase of ha yields with an increase of glucose concentration. the decline of ha yields may be due to the decrease of the ph of the medium resulted from the high consumption of the carbon sources, which led to the production of organic acid and reduce the ph of the medium.12 the other reason may be its agitation speed. the increase of agitation speed led to a significant decrease of cell growth and ha production.12 fig. 2 ha yield (ng/ml) generated from some s. pyogenes isolates under different glucose concentration. 0 2 4 6 8 10 12 ha  concentration   (ng)    0.4%          0.6%               0.8%           1%   glucose  concentration    17.2 1-7.5 2-6.6 6-7.2 7-6.6 references 1. boeriu cg, springer j, kooy fk, van den broek la, eggink g. production methods for hyaluronan. international journal of carbohydrate chemistry. 2013;2013:1–14. doi: http://dx.doi.org/10.1155/2013/624967 2. mankin h, mow v, buckwalter j, iannotti j, ratcliffe a. form function of articular cartilage. orthopaedic basic science. rosemont, ill: american academy of orthopaedic surgeons; 1994. pp. 1–44. 3. knudson cb. hyaluronan receptor-directed assembly of chondrocyte pericellular matrix. j cell biol. 1993 feb;120(3):825–834. doi: http://dx.doi. org/10.1083/jcb.120.3.825 pmid: 7678838 4. burdick ja, prestwich gd. hyaluronic acid hydrogels for biomedical applications. adv mater. 2011;23(12):41–56. doi: http://dx.doi.org/10.1002/ adma.201003963 pmid: 21394792 5. choi s, choi w, kim s, lee sy, noh i, kim cw. purification and biocompatibility of fermented hyaluronic acid for its applications to biomaterials. biomater res. 2014 jun;18(1):6. doi: 10.1186/2055-7124-18-6 pmid: 26331057 6. teodor ed, truica g, radu gl. hyaluronic acid detection from natural extract by diode array-capillary electrophoresis methods. revue roumaine de chimie. 2012;57(3):223–227. 7. reddy kj, karunakaran kt. purification and characterization of hyaluronic acid produced by streptococcus zooepidemicus strain 3523-7. j biosci biotech. 2013;2(3):173–179. 8. van de rijn i. streptococcal hyaluronic acid: proposed mechanisms of degradation and loss of synthesis during stationary phase. j bacteriol. 1983 dec;156(3):1059–1065. pmid: 6358186 15j contemp med sci | vol. 1, no. 4, autumn 2015: 13–15 research production and optimisation of ha extracted from streptococcus pyogeneskawkab abdulla al-saadiaa et al. 9. saranraj p, sivakumar s, sivasubrmanian j, geetha m. production, optimization and spectroscopic studies of hyaluronic acid extracted from streptococcus pyogenes. international journal of pharmaceutical and biological archive. 2011;2(3):954–959. 10. macfaddin jf. biochemical tests for identification of medical bacteria, 3rd ed. the baltimore, usa: williams and wilkins; 2000. 11. fosang aj, hey nj, carney sl, hardinghami te. an elisa plate based assay for hyaluronan using biotinylated proteoglycan g1 domain (ha-binding region). matrix. 1990 oct;10(5):306–313. doi: http://dx.doi.org/10.1016/ s0934-8832(11)80186-1 pmid: 2150688 (online abst.). 12. liu l, liu y, l j, du g, chen j. (2011). microbial production of hyaluronic acid: current state, challenges, and perspectives. microb cell fact. 2011 nov 16;10:99. doi: 10.1186/1475-2859-10-99. 36 j contemp med sci | vol. 1, no. 4, autumn 2015: 36–38 research background integrated assessment has become imperative especially after applying the new integrated curriculum. aim to shed the light on some strategies for incorporating integrated assessment when implementing the new integrated curriculum. conclusion integrated assessments must be applied in educational institutions. these assessments provide important ways to enhance student outcomes. several strategies could be applied which should be selected in accordance to students’ learning outcome. integrated assessment in medical education hedef d. el-yassin introduction learning is by connecting things. if you cannot connect you cannot learn “lord chesterton” integration must be considered as an educational strategy. and assessing how students use that basic science content in clinical reasoning or in the performance of a skill would provide valuable evidence for the effectiveness of a specific integration education strategy. integrated curriculum needs integrated assessment integrated assessments in medical schools provide an engaging and creative learning platform that closely links graduate doctors (day one doctor) to real life i.e., what will he/she experience in their practice. integrated assessment is a process that combines and blends the learning outcomes from multiple modules into a series of streamlined, realistic, case-oriented activities. these assessments are conducted over a period of time with numerous formative and summative components. assessments no longer take on the feared final exams i.e., no more traditional assessment model of “topic–teach–assess graduate”. lectures’ objectives and outcomes must be written in a way as to encourage a broad range of assessment strategies which will measure a student’s performance and knowledge of processes on a learning activity or project. teachers should select an assessment strategy or strategies, which are “most” relevant to their students’ learning outcome. assessment strategies assessment strategies may include: 1. authentic assessment 2. performance assessment 3. systematic observations 4. portfolios and journals authentic assessment authentic assessment is a term which has been coined to describe alternative assessment methods. these methods should authentically allow a student to demonstrate their ability to perform tasks, solve problems or express knowledge in ways which simulate situations which are found in real life (hymes, 1991).1 according to hart (1994)2 the assessment strategy which fits these criteria is a combination of: 1. performance assessment, 2. systematic observations, and 3. portfolios. issn 2413-0516 department of biochemistry, college of medicine, university of baghdad, iraq. correspondence to h.d. el-yassin (email: hedefelyassin@gmail.com). (submitted: 12 september 2015 – revised version received: 18 october 2015 – accepted: 24 october 2015 – published online: autumn 2015) the normal emphasis on the assessment of knowledge and skills in separate disciplinaries is replaced by measures of knowledge and skills blended into seamless assessment components that occur naturally (fig. 1). this creates experienced doctors ready for the workplace (healthcare institute). there is no right or wrong assessment strategy, there are only various ways of attempting to determine what a student knows and what he/she is able to do. measurement tools or strategies are only as good as their relationship to the goals and expected outcomes which have been established for a module. fig. 1 types of integrated assessment. 37j contemp med sci | vol. 1, no. 4, autumn 2015: 36–38 research integrated medical assessment hedef d. el-yassin performance assessment a. developed to “test” the ability of students to demonstrate their knowledge and skills (what they know and can do) in a variety of “realistic” situations and contexts.3 b. can be short or extended open-ended or multiple choice questions. in a more extended definition, performance assessments can be reading or writing, projects, processes, problem solving, analytical tasks or other tasks which allow the student to demonstrate their ability to meet specified outcomes and goals.4 systematic observations means that “all” students are observed regularly. observations are recorded for both typical and atypical behaviour. then these observations are reflected upon by the observer and interpreted to guide students’ to meeting the lesson outcomes and goal(s).4 the key to useful observation is that they must be systematic. observations are only useful if the “data” is recorded, evaluated and used to improve student performance. portfolios · portfolios are collections of students’ skills, ideas, interests and accomplishments that span a period of time (hart, 1994).2 a portfolio may represent one discipline or any number of disciplines. portfolios are often taken on either the physical appearance of folders, binders or notebooks. or there are electronic portfolios which use multimedia and hypermedia to display the students’ work. the following are the examples on how to implement integrated assessment in an integrated curriculum: – combinations of multiple-choice questions (self-assessment questions or saqs) with essay questions (termed concept appraisals or capps) that ask learners to provide a narrative interpretation of the mechanisms behind or reasons for the findings in a clinical scenario.5,6 – wood and colleagues7 describe a validation study of a clinical reasoning exercise in which learners are asked to write a single paragraph explaining the mechanisms behind a particular patient problem (wood et al. 2009)8; these assignments are then graded by independent raters to assess whether learners’ performance on this exercise correlates with other measures. – williams and klamen (2012)9 have described a diagnostic justification exercise used with simulated patient encounters in which learners are asked to develop a differential diagnosis and explain their rationale for including the diseases/ conditions on that differential (ila). – concept maps represent another strategy for assessing integration of knowledge. mcgaghie et al. (2000)10 demonstrated that students’ maps regarding pulmonary physiology concepts became more coherent as a result of participating in an instructional unit on respiratory physiology, and the maps became more similar to maps developed by their instructors (fig. 2). – longer essays have also been suggested as a means for assessing students’ integration of knowledge from a problembased-learning case. for example, ferguson (2006, 1997)11,12 describes a method by which individual learners are asked to write a narrative about a case that they have studied in small groups over several weeks. the learner is asked to write, in the form of a conversation with a patient, how the patient’s signs, symptoms and laboratory and imaging results relate to underlying mechanisms of disease, how the treatment recommendations are based on this understanding, and what the patient can expect from the disease and treatment. writing the narrative in the form of a conversation accomplishes an additional purpose of practicing the skill of explaining difficult concepts in understandable terms. · progress tests have been used extensively in europe to assess integration across courses. these tests are given periodically throughout the curriculum, and the items are intended to test cumulative knowledge across courses and vertically across the curriculum. progress tests provide a unique opportunity for assessing growth in students’ knowledge (williams et al. 2012),9 and can provide data on based on decisions about the curriculum as a whole as well as remediation strategies for the individual student. to accomplish these goals, however, requires significant investment of faculty and administrative time to develop item banks and ensure that exams remain relevant. swanson and case (1997)13 provide examples of multiple-choice questions based on patient scenarios that test integration of basic science and clinical knowledge. in addition, they have suggested that open-book exams, especially those that require learners to apply scientific literature, may be especially helpful in assessing higher-order thinking skills such as integration of material. an additional benefit is that such exams drive faculty to write questions that cannot be answered by turning to a page in a book. assessment in clinical education during clinical education, assessing learners’ ability to apply basic science concepts through their diagnostic reasoning fig. 2 concept map of the definition of endocrinology. 38 j contemp med sci | vol. 1, no. 4, autumn 2015: 36–38 integrated medical assessment research hedef d. el-yassin skills often occurs in the context of patient care. bowen (2006)14 identifies learner skills in six areas: 1. data acquisition and reporting, 2. problem representation, 3. generation of hypotheses, 4. identifying appropriate diagnoses on the differential, 5. having relevant experience for the case, and 6. general presentation/organisational skills. she identifies clues that will uncover deficits in each of these areas and offers educational strategies for addressing each of them during clinical education. she further suggests that clinical teachers should ‘‘. . . encourage reading that promotes conceptualization rather than memorization.” rubrics for integrated assessment a rubric is a great tool for teachers, because it is a simple way to set up grading criteria for assignments. not only is this tool useful for teachers, it is also helpful for students as well. a rubric defines in writing what is expected of the student to get a particular grade on an assignment. a good rubric describes levels of quality for each criterion. these levels of performance may be written as different ratings (e.g., excellent, good, needs improvement) or as numerical scores (e.g., 4, 3, 2, 1). why rubrics for integrated assessment the following points tell as why rubrics are preferred for integrated assessment: – measure outcomes based on real-life criteria – specify performance indicators – ensure consistent assessment – improve the quality of assessment conclusion integrated assessments must be applied in educational institutions. these assessments provide important ways to enhance student outcomes. several strategies could be applied all should be selected in accordance to students’ learning outcome. references 1. hymes dl, chafin ae, gonder p. the changing face of testing and assessment; problems and solutions. arlington, va: american association of school administrators; 1991. p. 102. 2. hart d. authentic assessment: a handbook for educators. menlo park, ca: addison-wesley pub. co.; 1994. 3. wiggins gp. assessing student performance. san francisco: jossey-bass publishers; 1993. 4. sowell ej. curriculum: an integrative introduction. new jersey: prentice-hall inc.; 1996. 5. bierer sb, dannefer ef, taylor c, hall p, hull al. methods to assess students’ acquisition, application and integration of basic science knowledge in an innovative competency-based curriculum. med teach 2008;30:e171–e177. doi: http://dx.doi.org/10.1080/01421590802139740 pmid: 18777415 6. bierer sb, taylor ca, dannefer ef. evaluation of essay questions used to assess medical students’ application and integration of basic and clinical science knowledge. teach learn med 2009;21(4):344–50. 7. woods nn, brooks lr, norman gr. the value of basic science in clinical diagnosis: creating coherence among signs and symptoms. med educ 2005;39:107–12. 8. wood tj, cunnington jpw, norman gr. assessing the measurement properties of a clinical reasoning exercise. teach learn med 2009;12: 196–200. 9. williams rg, klamen dl. examining the diagnostic justification abilities of fourth-year medical students. acad med 2012;87:1008–14. 10. mcgaghie wc, mccrimmon dr, mitchell g, thompson ja, ravitch mm. quantitative concept mapping in pulmonary physiology: comparison of student and faculty knowledge structures. adv physiol ed 2000;23:72–81. 11. ferguson kj. beyond multiple-choice questions: using case-based learning patient questions to assess clinical reasoning. med educ 2006;40:1142. 12. ferguson kj, steiner la, kreiter cd, pomrehn pr. assessment of case-based learning within a hybrid curriculum. presentation at the association of american medical colleges research in medical education annual meeting, washington, dc, 4 november 1997. 13. swanson db, case sm. assessment in basic science instruction: directions for practice and research. adv health sci educ 1997;2:71–84. 14. bowen jl. educational strategies to promote clinical diagnostic reasoning. n engl j med 2006;355:2217–25. 28 j contemp med sci | vol. 2, no. 5, winter 2016: 28–32 research background stroke is one of the leading causes of death globally. awareness of stroke modifiable risk factors and warning signs are important for stroke prevention. objectives to assess hypertensive patients’ knowledge regarding lifestyle risk factors and warning signs of stroke. method a descriptive study was conducted on 114 hypertension patients who attended chronic disease center in sulaimani city. the study was carried out for the period of february to april 2015. a questionnaire has been used to collect relative data about patients’ characteristics and knowledge regarding lifestyle risk factors and warning signs of stroke. data were analysed through spss 20, and descriptive statistics (frequency, percentage and mean) and inferential statistics (f-test and t-test), the figure of p > 0.05 was considered as the statistical significant. result patients’ knowledge regarding stroke lifestyle risk factors and stroke warning signs were low of 55.3 and 76.3% respectively. physical inactivity has lower (17.5) percentage among lifestyle risk factors. the patients’ knowledge was influenced by patients’ age, gender, education levels and duration of hypertension (p > 0.05). conclusions hypertensive patients complained from lack of knowledge regarding stroke lifestyle risk factors and warning signs. physical inactivity was less identified risk factors. young, female, high level of education and longer duration of disease tended to have higher knowledge. recommendations our study results recommended that there was a need to bring high risk group awareness about stroke risk factors and warning signs, particularly to the population with hypertension. keywords hypertensive, knowledge, lifestyle, warning signs, stroke assessment of hypertensive patients’ knowledge about lifestyle risk factors and warning signs of stroke muhammad rashid amena issn 2413-0516 aphd, chn, rehabilitation nursing, head of adult nursing branch, college of nursing, faculty of medical science, university of sulaimani, sulaimani, iraq. correspondence to muhammad rashid amen (email: muhammad.amen@univsul.edu.iq). (submitted: 21 december 2015 – revised version received: 28 january 2016 – accepted: 7 february 2016 – published online: 26 march 2016) introduction stroke is one of the leading causes of death globally and is a major cause of disability worldwide.1 stroke is a preventable and treatable disease through the control of modifiable risk factors and the early recognition of stroke warning symptoms respectively.2 according to the third national mortality retrospective sampling survey in iraq, stroke disease has become the 2nd leading cause of death in iraq and is responsible for 11.3% of all deaths.3 the percentage of stroke among individuals in the kurdish population is around 19%, especially among hypertensive and older aged patients.4 in kurdistan region, more than half (58.8%) of the hypertensive patients were uncontrolled. the factors associated with uncontrolled hypertension were smoking, lack of exercise and irregularity of treatment.5 a known modifiable risk factors of stroke include physiological and lifestyle risks.6 importantly, modifiable risk factors linked to lifestyle, such as smoking, physical inactivity, obesity, unhealthy diet and high alcohol consumption, were among the most prevalent unfavourable behavioural patterns may promotes the development of well-documented physiological risk factors such as arterial hypertension, diabetes mellitus, or hyperlipidemia, and ultimately lead to atherosclerosis and ischemic stroke.7 people with hypertension are at increased risk for experiencing a stroke; effective stroke prevention programme should be focused to improve the high risk group awareness about the early warning symptoms of stroke and modifiable risk factors.8 hence, there was a need to study the knowledge and level of awareness among the high risk group such as hypertensive regarding warning signs, and modifiable risk factors to prevent occurrence or at least detect it early.9 another important component of successful stroke prevention is improvement of public knowledge about stroke.10 in addition lack of knowledge may lead to unhealthy lifestyle behaviours, which are a major problem because healthy lifestyle behaviours are associated with a lower risk of stroke. therefore, it is important to learn more about stroke knowledge and health behaviours among hypertensive patients.11 objectives of the study the aims of this study were: • to assess the level of hypertensive patients’ knowledge regarding the influence of life style risk (low physical activity, unhealthy diet, obesity, smoking and alcohol intake) and stroke warning signs. • to find out association between knowledge and patients characteristics such as age, gender, level of education, financial status, bmi and duration of hypertension. materials and methods with the use of a descriptive design, 114 hypertensive patients were recruited from chronic diseases center in sulaimani city (is the only center in sulaimani city which provide medications for patients with chronic disease free). the study was carried out for the period of february to april 2015. the exclusion criteria were patients with history of stroke; health care professional and unwilling to participate in the study. the patients were informed that participation is voluntary. they were also informed of the confidentiality of their participation. before administering the questionnaire, researcher mailto:muhammad.amen@univsul.edu.iq 29j contemp med sci | vol. 2, no. 5, winter 2016: 28–32 research assessment of hypertensive patients’ knowledge about strokemuhammad rashid amen stressed to patients that participation or lack of participation in the study would not influence care (medications) received. the study used purposive (non-probability) sampling technique to select sample. the data were collected during the patients’ visit to the center in order to receive medications. the study was approved by ethics committee in college of nursing, university of sulaimani. the stroke knowledge instrument used in our study was a questionnaire, which was prepared after a comprehensive research of relevant literatures and references,1,6–8,10–18 pretested and modified in terms of questions clarity and cultural adaptation by experts. the questionnaire was administered to ten hypertensive patients for evaluating test retest reliability. questionnaire consisted of three sections. the first section included eight items to provide information about the demographic and clinical characteristics of the respondents (age, gender, level of education, financial status, bmi, duration of hypertension, smoking, and alcohol intake). second section included five items to determine the knowledge regarding lifestyle risk factors (physical inactivity, obesity, unhealthy diet, smoking, and unhealthy use of alcohol). the third section included five items to determine the knowledge with regard stroke warning signs. the questionnaire was used close-ended questions to assess patient knowledge in 2nd and 3rd sections, with a ‘yes’ response indicating that the patient is knowledgeable and ‘no’ is not knowledgeable. one point was given for each ‘yes’ answer and zero for each ‘no’ answer. knowledge scores were categorised for each section as follows: low knowledge identify (≤2) risk factors or warning symptoms, moderate knowledge (3) and high knowledge (≥4).14 data analysis the data were analysed with spss 20. socio-demographic, clinical characteristics and level of knowledge were analysed using frequencies and percentage. patients’ stroke prevention knowledge (which includes stroke lifestyle risk factors and warning signs) were analysed using percentage of means. f-test and t-test were used to explore the relationship between stroke prevention knowledge and patients’ characteristics and the figure of p > 0.05 was considered as the statistical significant. results a total of 130 hypertensive patients were proposed to be included in the study, 114 patients were eligible and agreed to participate, giving a response rate of 87.7%. table 1 shows the demographic and some clinical characteristics of the study participants. participants’ mean age was (58.4 ± 14.2) years, the age more than 77% was between 45 and 65 years. of the respondents, 58.8% were male, 45.7% had low education (illiterate or primary school), and 43% with self reported insufficient financial status. most of the patients were insufficient or barely sufficient financially and had received a low or moderate level of education. the mean bmi of the study population was 28.6 ± 5.1 kg/m2. the percentage of normal body weight was 20.2; while 36.8% were found to be obese and remaining were overweight according to their calculated bmi. approximately more than half 50.9% of participant indicated that they smoked and 8.8% reported that they drink alcohol. regarding duration of their hypertension, 43% of them had it for less than 1 year, 31.6% was between 1 and 3 years and other one-fourth were more than 3 years. the main finding of present study presented in figs. 1, 2 show respondents’ knowledge about lifestyle risk factors and stroke warning signs. the patients’ knowledge about lifestyle stroke risk factors was low knowledge for 53.5%, moderate knowledge for 36% and only 10.5% of participant had high knowledge. knowledge regarding stroke warning signs was lower, it was low knowledge for 75.4%, moderate knowledge for 20.2% and high knowledge for 4.4%. lifestyle risk factors and stroke warning signs are shown in table 2. the mean of knowledge for the total stroke life risk table 1. socio-demographic and clinical characteristics of study characteristics frequency % age groups <45 years 6 5.3 45–65 years 88 77.2 >65 years 20 17.5 mean ± std 58.4 ± 14.2 gender female 47 41.2 male 67 58.8 levels of education high 20 17.5 medium 42 36.8 low 52 45.7 smoking no 56 49.1 yes 58 50.9 total 114 100 body mass index normal 23 20.2 over-weight 49 43.0 obese 42 36.8 mean ± std 28.6 ± 5.1 financial status sufficient 28 24.6 barely sufficient 37 32.5 insufficient 49 43.0 duration of disease/year <1 49 43.0 1–3 36 31.6 >3 29 25.4 alcohol intake no 104 91.2 yes 10 8.8 total 114 100 30 j contemp med sci | vol. 2, no. 5, winter 2016: 28–32 assessment of hypertensive patients’ knowledge about stroke research muhammad rashid amen the identified stroke warning signs by the participant in present study were lower than identified lifestyle risk factors, 34.2% did not recognised any warning sign, 22.7% identified one warning sign, two signs were identified by 17.5%, followed three signs by 20.2%, four warning signs were recognised by 4.4% only, and no participants had knowledge to recognise all warning signs as illustrated by in fig. 4. table 3 shows the results of the association of participants’ characteristics with stroke risk factors and stroke warning signs. there were significant association between age, gender and education with knowledge regarding stroke risk factors and warning signs, younger patients, female and more educated had more correct answer, while the duration of disease was associated to risk factors, but not to warning signs knowledge (p < 0.05). association was not found between financial status and bmi with any kind of knowledge (p > 0.05). discussion identification of the major lifestyle risk factors of stroke and its warning signs have a direct implication for the prevention of stroke with the possible therapeutic measures in high risk group such as hypertensive patients. this descriptive study found that hypertensive patients lacked stroke prevention knowledge which includes stroke lifestyle risk factors and factors was 36.3 ± 22.7 (low knowledge). of patients, 17.5% only identified physical inactivity as stroke risk factors, followed by obesity and smoking and tobacco use was 39.5%, excessive alcohol intake came third was 42.1%, and unhealthy diet had greatest percentage among lifestyle stroke risk factor was 43%. the mean knowledge for total stroke warning signs was 27.3 ± 26.3 it was low knowledge. the most common stroke warning signs indicated by participants were ‘sudden numbness or weakness of the face, arm, or leg’ 29.8%, followed by ‘sudden confusion, trouble speaking or understanding others’ 28.9%, then ‘severe headache with no known cause’ 23.7%, ‘sudden trouble seeing in one or both eyes’ 22.8% and ‘sudden dizziness, trouble walking, loss of balance or coordination’ 20.2%. more than 25% of participants did not identified any stroke lifestyle risk factor, 17.5% recognise one risk factors, (18.4%) identified two risk factors followed by (28.07%) identified three risks, 9.7% identified four risks and all risks were identified by <1%, as showed in fig. 3. fig. 1 percentage of knowledge levels regarding stroke lifestyle risk factors. fig. 2 percentage of knowledge levels regarding stroke warning signs. fig. 3 number and percentage of identified stroke lifestyle risk factors. fig. 4 number and percentage of identified stroke warning signs. 31j contemp med sci | vol. 2, no. 5, winter 2016: 28–32 research assessment of hypertensive patients’ knowledge about strokemuhammad rashid amen warning signs. more than half of the respondents had low knowledge; more than one-fourth were not aware of any established lifestyle stroke risk factors. about one-ten (10.5%) of subjects had high knowledge and were aware of four risk factors and more; the percentage of participants who identified all lifestyle risk factors was less than one. the respondents’ knowledge regarding established stroke risk factors was better than that for the warning signs of stroke, more than three-quarter of participants’ knowledge were low in identifying stroke warning signs, and more than one-third were unable to identify any warning signs. few (4.4%) of them had high knowledge or were able to list four warning signs. our finding was lower than pakistani study result which reported 86.9% of the participants correctly stated at least one of stroke risk factors, 70.2% knew at least two and 53% knew at least three. furthermore it was lower than the reported by an australia study, 76.2% respondents correctly listed ≤1 established stroke risk factor, but only 49.8% respondents correctly listed ≤1 warning sign.16 ‘unhealthy diet’ was the most common and physical inactivity was less common identified risk factors of stroke in this study. it is of interest that only few 17.5% of respondents identified ‘physical inactivity’ as a risk factor for stroke. a very worrisome finding in the current study was the respondents’ low knowledge of some stroke risk factors like physical inactivity and stroke warning signs. this may reflect that the health care professional focused on diet, stopping alcohol intake, smoking cession and reduce weight, while they ignored role of physical activity in controlling hypertension, and preventing stroke. this result agreed with the finding of a china study which reported most of the participants knew that hypertensive patients should eat foods that are low in sodium, quit smoking, and reduce alcohol consumption.11 the commonest stroke warning sign recognised by the patients were ‘confusion or trouble speaking or understanding others’ 34%, followed by ‘numbness or weakness of the face, arm, or leg’ 33%, and ‘severe headache with no known cause’ 27%. these figures were similar to finding of kuwaiti study, which reported the ‘confusion, problem in speaking and understanding’ 36.4% followed by ‘weakness of arm and leg’ 34.7% as a most warning sign identified. the most common warning sign identified by the people in pakistani study was weakness of one or both sides of the face or body 71.4% followed by numbness of one or both sides of the face or body 60.6%.15 while an iranian study considered smoking as the most identified lifestyle risk factor, and most common warning sign as a pain.17 furthermore the most identified risk was smoking and ‘blurred and double vision or loss of vision in an eye’ was most established warning signs in an australian study,16 and 62% of participants in study conducted in india described paralysis of one side of the body as the most common warning signs of stroke.19 we found a positive relationship between stroke prevention knowledge and patients’ characteristics indicating that the table 3. association between stroke risk factors, warning signs knowledge with respondents’ characteristics characteristics risk factors knowledge warning signs knowledge mean std mean std age groups/year <45 73.3 20.7 56.7 19.7 45–65 33.6 26.2 25 23.3 >65 37 29.2 29 28.7 f (p-value) 4.3 (0.007) 3.2 (0.026) gender female 40.6 27.6 30.8 27.3 male 30.2 26.9 22.6 21.5 t-test (p-value) 2.08 (0.045) 1.97 (0.049) level of education high 54 22.6 48 26.3 medium 44.8 26.1 41 19.2 low 22.7 24.4 8.5 13.3 f (p-value) 10.4 (0.0001) 34.8 (0.0001) financial status sufficient 40.7 25.2 30 23.4 barely sufficient 34.6 25.7 23.8 27.4 insufficient 35.1 30.7 28.6 24.8 f (p-value) 0.33 (0.8) 0.39 (0.76) body mass index normal 41.7 23.3 27.8 28.1 overweight 35.1 28.7 29 24.9 obese 34.8 29 25.2 24.6 f (p-value) 0.39 (0.76) 0.17 (0.92) duration of hypertension/year <1 year 28.6 25.8 22.9 24.2 1–3 years 37.2 27.5 28.3 25.9 >3 years 48.3 27.5 33.8 25.7 f (p-value) 3.35 (0.022) 1.19 (0.32) table 2. frequencies and percentage of participants’ positive responses to stroke risk factors and warning signs categories frequency % lifestyle stork risk factors physical inactivity 20 17.5 obesity 45 39.5 unhealthy diet 49 43 smoking and tobacco use 45 39.5 excessive intake of alcohol 48 42.1 total (mean ± std) 36.3 ± 22.7 stroke warning signs sudden confusion, trouble speaking or understanding others 33 28.9 sudden numbness or weakness of the face, arm, or leg 34 29.8 sudden dizziness, trouble walking, loss of balance or coordination 23 20.2 severe headache with no known cause 27 23.7 sudden trouble seeing in one or both eyes 26 22.8 total (mean ± std) 27.3 ± 26.3 32 j contemp med sci | vol. 2, no. 5, winter 2016: 28–32 assessment of hypertensive patients’ knowledge about stroke research muhammad rashid amen young, female, higher educated and longer duration of hypertension had better knowledge regarding stroke lifestyle risk factors and warning signs, while the knowledge not influenced neither by financial status nor bmi. low level of education and old age may lead to limited interaction with society and therefore to less interest in following medical developments, and thereby resulting in a low level of health care knowledge about stroke. previously, better stroke knowledge was observed in women compared with men in the majority of the studies although there is a general lack of knowledge in both genders. four out of 18 studies reported better risk factor knowledge and eight out of 15 studies reported better knowledge in stroke warning signs in women compared with men. women tended to know more evidence-based stroke risk factors than men.1 these findings are consistent with previous studies; pakistani study found strong association between intermediate and-above level of education and younger age group with their correct identification of stroke risk factors. same findings reported by other study conducted in india.19 furthermore iranian study reported that knowledge and attitude towards stroke were significantly associated with age and level of education. other study stated that one in five respondents were not aware of any stroke risk factors, and almost one in three was not aware of any stroke warning signs. stroke knowledge was poorest among groups that have the highest risk of stroke.17 the results of our study have contributed to the understanding of strokes’ lifestyle risk factor and stroke warning signs in at-risk individuals. the current findings and mentioned previous studies conducted in several countries may signify that the lack of awareness about stroke among the general public and high risk groups is a worldwide problem. one implication of our results was the importance of increasing public awareness about stroke and stroke prevention, particularly in the at-risk population. these findings have significant implications for clinical practice. it was important for nurses to pay more attention to patients’ knowledge concerning physical activity and stroke warning signs, so that they can assist patients to gain knowledge and engage in behaviours that could help patients prevent stroke. conclusions depending on our findings we concluded that knowledge regarding lifestyle risk factors and warning signs was low among hypertensive patient. majority of participants were unable to identify more than a single risk factor and recognise more than one stroke warning sign. physical inactivity was less identified risk factor and recognisation of warning signs was below one third. been young, female, high educated and long duration of hypertension possess more knowledge, while financial status and bmi had no effect on patients’ knowledge. recommendations the present findings emphasise the need for effective stroke education efforts in particular regarding physical inactivity and stroke warning signs. we believed that the use of public media and school education will probably change the level of the population’s knowledge towards stroke. male hypertensive patients and those with a lower education level need targeted stroke education. considerable emphasis should therefore be placed on improving stroke risk perception among hypertensive patients.  references 1. stroebele n, müller-riemenschneider f, nolte ch, müller-nordhorn j, bockelbrink a, willich sn. knowledge of risk factors, and warning signs of stroke: a systematic review from a gender perspective. int j stroke. 2011;6(1): 60–6. doi: 10.1111/j.1747-4949.2010.00540.x pmid: 21205242 2. alzawahmah m. assessment of stroke knowledge and attitudes among riyadh medical students: a cross-sectional study. neurology. 2015;84(14):p2.303. 3. who 2014. world bank and united nation for population. the top 10 causes of death. who 2014 updates. 4. al-shimmery ek, amein sh, al-tawil ng. prevalence of silent stroke in kurdistan, iraq. neurosciences (riyadh). 2010;15(3):167–71. pmid: 20831024 5. bushra b kh, kameran hi. uncontrolled hypertension in a group of hypertensive patients in erbil. j kufa f nurs sci. 2013;3(2). 6. aycock m, kirkendoll d, coleman c, clark c, albright c, alexandrov w. family history of stroke among african americans and its association with risk factors, knowledge, perceptions, and exercise. j cardiovas nurs. 2015;30(2):p e1–e6. doi: 10.1097/jcn.0000000000000125 pmid: 24598552 7. von s, jukka p, ulrike g, beate g, ulf s, sami c et al.; investigators. lifestyle risk factors for ischemic stroke and transient ischemic attack in young adults in the stroke in young patients study. stroke. 2013;44:119–125. doi: 10.1161/strokeaha.112.665190 pmid: 23150649 8. yadav pk, shewta s, kumar vk, joshua a, krishnan s, kumar sp. survey of knowledge and awareness about cerebro-vascular stroke, its risk factors, warning signs and immediate treatment among mangalore urban population: a cross-sectional study. int j health rehabil sci. 2013;2(2):116–122. 9. mansour a. prevalence and control of hypertension in iraqi diabetic patients: a prospective cohort study. open cardiovasc med j. 2012;6:68–71. doi: 10.2174/1874192401206010068 pmid: 22654998 10. chiuve se, rexrode km, spiegelman d, logroscino g, manson je, rimm eb. primary prevention of stroke by healthy lifestyle. circulation. 2008;118(9):947–954. doi: 10.1161/circulationaha.108.781062 pmid: 18697819 11. wan lh, zhao j, zhang xp, deng sf, li l, he sz, et al. stroke prevention knowledge and pre-stroke health behaviors among hypertensive stroke patients in mainland china. j cardiovasc nurs. 2014;29(2):e1–9. doi: 10.1097/jcn.0b013e31827f0ab5 pmid: 23388703 12. zeng y, he gp, yi gh, huang yj, zhang qh, he ll. knowledge of stroke warning signs and risk factors among patients with previous stroke or tia in china; j clin nurs. 2012;21(19–20):2886–95. doi: 10.1111/j.13652702.2012.04118.x pmid: 22985321 13. sloma a, backlund lg, strender le, skånér y. knowledge of stroke risk factors among primary care patients with previous stroke or tia: a questionnaire study. bmc family practice. 2010;11:47. doi: 10.1186/14712296-11-47 pmid: 20550690 14. awad a, al-nafisi h. public knowledge of cardiovascular disease and its risk factors in kuwait: a cross-sectional survey. bmc public health. 2014;14:1131. doi: 10.1186/1471–2458-14–1131 pmid: 25367768 15. aly z, abbas k, kazim sf, taj f, aziz f, irfan a, et al. awareness of stroke risk factors, signs and treatment in a pakistani population. j pak med assoc. 2009;59(7):495–99. pmid: 19579747 16. sug ys, heller rf, levi c, wiggers j, fitzgerald pe. knowledge of stroke risk factors, warning symptoms, and treatment among an australian urban population. stroke. 2001;32:1926–1930. pmid: 11486127 17. borhani ha, karimi aa, amiri a, ghaffarpasand f. knowledge and attitude towards stroke risk factors, warning symptoms and treatment in an iranian population. med princ pract. 2010;19:468–472. doi: 10.1159/000320306 pmid: 20881415 18. reeves mj, hogan jg, rafferty ap. knowledge of stroke risk factors and warning signs among michigan adults. neurology. 2002;59(10):1547–1552. pmid: 12451195 19. pandian jd, kalra g, jaison a, deepak ss, shamsher s, singh y, et al. knowledge of stroke among stroke patients and their relatives in northwest india. neurol india. 2006;54(2):152–156. pmid: 16804258 http://www.ncbi.nlm.nih.gov/pubmed/?term=stroebele%20n%5bauthor%5d&cauthor=true&cauthor_uid=21205242 http://www.ncbi.nlm.nih.gov/pubmed/?term=m%c3%bcller-riemenschneider%20f%5bauthor%5d&cauthor=true&cauthor_uid=21205242 http://www.ncbi.nlm.nih.gov/pubmed/?term=nolte%20ch%5bauthor%5d&cauthor=true&cauthor_uid=21205242 http://www.ncbi.nlm.nih.gov/pubmed/?term=m%c3%bcller-nordhorn%20j%5bauthor%5d&cauthor=true&cauthor_uid=21205242 http://www.ncbi.nlm.nih.gov/pubmed/?term=bockelbrink%20a%5bauthor%5d&cauthor=true&cauthor_uid=21205242 http://www.ncbi.nlm.nih.gov/pubmed/?term=willich%20sn%5bauthor%5d&cauthor=true&cauthor_uid=21205242 http://www.ncbi.nlm.nih.gov/pubmed/21205242 http://www.ncbi.nlm.nih.gov/pubmed/?term=al-shimmery%20ek%5bauthor%5d&cauthor=true&cauthor_uid=20831024 http://www.ncbi.nlm.nih.gov/pubmed/?term=amein%20sh%5bauthor%5d&cauthor=true&cauthor_uid=20831024 http://www.ncbi.nlm.nih.gov/pubmed/?term=al-tawil%20ng%5bauthor%5d&cauthor=true&cauthor_uid=20831024 http://www.scopemed.org/?jid=20 http://www.scopemed.org/?jid=20&iid=2013-2-2.000 http://www.ncbi.nlm.nih.gov/pubmed/?term=mansour%20aa%5bauth%5d http://dx.doi.org/10.2174%2f1874192401206010068 http://www.ncbi.nlm.nih.gov/pubmed/?term=logroscino%20g%5bauthor%5d&cauthor=true&cauthor_uid=18697819 http://www.ncbi.nlm.nih.gov/pubmed/?term=manson%20je%5bauthor%5d&cauthor=true&cauthor_uid=18697819 http://www.ncbi.nlm.nih.gov/pubmed/?term=rimm%20eb%5bauthor%5d&cauthor=true&cauthor_uid=18697819 http://www.ncbi.nlm.nih.gov/pubmed/?term=wan%20lh%5bauthor%5d&cauthor=true&cauthor_uid=23388703 http://www.ncbi.nlm.nih.gov/pubmed/?term=zhao%20j%5bauthor%5d&cauthor=true&cauthor_uid=23388703 http://www.ncbi.nlm.nih.gov/pubmed/?term=zhang%20xp%5bauthor%5d&cauthor=true&cauthor_uid=23388703 http://www.ncbi.nlm.nih.gov/pubmed/?term=deng%20sf%5bauthor%5d&cauthor=true&cauthor_uid=23388703 http://www.ncbi.nlm.nih.gov/pubmed/?term=li%20l%5bauthor%5d&cauthor=true&cauthor_uid=23388703 http://www.ncbi.nlm.nih.gov/pubmed/?term=he%20sz%5bauthor%5d&cauthor=true&cauthor_uid=23388703 http://www.ncbi.nlm.nih.gov/pubmed/?term=zeng%20y%5bauthor%5d&cauthor=true&cauthor_uid=22985321 http://www.ncbi.nlm.nih.gov/pubmed/?term=he%20gp%5bauthor%5d&cauthor=true&cauthor_uid=22985321 http://www.ncbi.nlm.nih.gov/pubmed/?term=yi%20gh%5bauthor%5d&cauthor=true&cauthor_uid=22985321 http://www.ncbi.nlm.nih.gov/pubmed/?term=huang%20yj%5bauthor%5d&cauthor=true&cauthor_uid=22985321 http://www.ncbi.nlm.nih.gov/pubmed/?term=zhang%20qh%5bauthor%5d&cauthor=true&cauthor_uid=22985321 http://www.ncbi.nlm.nih.gov/pubmed/?term=he%20ll%5bauthor%5d&cauthor=true&cauthor_uid=22985321 http://www.ncbi.nlm.nih.gov/pubmed/22985321 http://www.ncbi.nlm.nih.gov/pubmed/?term=sloma%20a%5bauthor%5d&cauthor=true&cauthor_uid=20550690 http://www.ncbi.nlm.nih.gov/pubmed/?term=backlund%20lg%5bauthor%5d&cauthor=true&cauthor_uid=20550690 http://www.ncbi.nlm.nih.gov/pubmed/?term=strender%20le%5bauthor%5d&cauthor=true&cauthor_uid=20550690 http://www.ncbi.nlm.nih.gov/pubmed/?term=sk%c3%a5n%c3%a9r%20y%5bauthor%5d&cauthor=true&cauthor_uid=20550690 http://www.ncbi.nlm.nih.gov/pubmed/?term=aly%20z%5bauthor%5d&cauthor=true&cauthor_uid=19579747 http://www.ncbi.nlm.nih.gov/pubmed/?term=abbas%20k%5bauthor%5d&cauthor=true&cauthor_uid=19579747 http://www.ncbi.nlm.nih.gov/pubmed/?term=kazim%20sf%5bauthor%5d&cauthor=true&cauthor_uid=19579747 http://www.ncbi.nlm.nih.gov/pubmed/?term=taj%20f%5bauthor%5d&cauthor=true&cauthor_uid=19579747 http://www.ncbi.nlm.nih.gov/pubmed/?term=aziz%20f%5bauthor%5d&cauthor=true&cauthor_uid=19579747 http://www.ncbi.nlm.nih.gov/pubmed/?term=irfan%20a%5bauthor%5d&cauthor=true&cauthor_uid=19579747 http://www.ncbi.nlm.nih.gov/pubmed/?term=sug%20yoon%20s%5bauthor%5d&cauthor=true&cauthor_uid=11486127 http://www.ncbi.nlm.nih.gov/pubmed/?term=heller%20rf%5bauthor%5d&cauthor=true&cauthor_uid=11486127 http://www.ncbi.nlm.nih.gov/pubmed/?term=levi%20c%5bauthor%5d&cauthor=true&cauthor_uid=11486127 http://www.ncbi.nlm.nih.gov/pubmed/?term=wiggers%20j%5bauthor%5d&cauthor=true&cauthor_uid=11486127 http://www.ncbi.nlm.nih.gov/pubmed/?term=fitzgerald%20pe%5bauthor%5d&cauthor=true&cauthor_uid=11486127 http://www.ncbi.nlm.nih.gov/pubmed/?term=borhani%20haghighi%20a%5bauthor%5d&cauthor=true&cauthor_uid=20881415 http://www.ncbi.nlm.nih.gov/pubmed/?term=karimi%20aa%5bauthor%5d&cauthor=true&cauthor_uid=20881415 http://www.ncbi.nlm.nih.gov/pubmed/?term=amiri%20a%5bauthor%5d&cauthor=true&cauthor_uid=20881415 http://www.ncbi.nlm.nih.gov/pubmed/?term=ghaffarpasand%20f%5bauthor%5d&cauthor=true&cauthor_uid=20881415 http://www.ncbi.nlm.nih.gov/pubmed/?term=pandian%20jd%5bauthor%5d&cauthor=true&cauthor_uid=16804258 http://www.ncbi.nlm.nih.gov/pubmed/?term=kalra%20g%5bauthor%5d&cauthor=true&cauthor_uid=16804258 http://www.ncbi.nlm.nih.gov/pubmed/?term=jaison%20a%5bauthor%5d&cauthor=true&cauthor_uid=16804258 http://www.ncbi.nlm.nih.gov/pubmed/?term=deepak%20ss%5bauthor%5d&cauthor=true&cauthor_uid=16804258 http://www.ncbi.nlm.nih.gov/pubmed/?term=shamsher%20s%5bauthor%5d&cauthor=true&cauthor_uid=16804258 http://www.ncbi.nlm.nih.gov/pubmed/?term=singh%20y%5bauthor%5d&cauthor=true&cauthor_uid=16804258 148 j contemp med sci | vol. 4, no. 3, summer 2018: 148–152 original effect of grape seed on quality of life in multiple sclerosis patients amir siahpoosh,a nastaran majdinasab,b negin derakhshannezhad,a hamid reza khalilic and alireza malayeric,d amedicinal plants research center and department of pharmacognosy, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran. bdepartment of neurology, ahvaz jundishapur university of medical sciences, ahvaz, iran. cmedicinal plants research center and department of pharmacology, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran. dresearch center, nab’a al-hayat foundation for medical sciences and health care, najaf, iraq. correspondence to alireza malayeri (email: armalayeri@yahoo.com). (submitted: 25 april 2018 – revised version received: 18 may 2018 – accepted: 27 june 2018 – published online: 26 september 2018) objective multiple sclerosis (ms) is an inflammatory disease in which the myelin sheaths of the neural cells in the brain and the spinal cord are damaged. this injury affects the ability of parts of the nervous system that are responsible for communication and cause many physical and mental signs and symptoms. the grape seed which is scientifically called vitis vinifera is the native plant in southern europe and western asia and its extract has strong antioxidants, anti-inflammatory and neuron and nervous system protection properties. therefore, it is considered to control the free radicals and inflammation that contribute to the development and progression of ms. methods the effect of grape seed extract on the fatigue control in patients was investigated as a double-blinded design. about 66 patients, all treated with interferon, were randomly divided into two groups. the first group received placebo for the first month and the second group had grape seed capsules for 1 month. fatigue was calculated based on multiple sclerosis quality of life-54 before and after the study. the results were analyzed by t-test to compare the mean in both groups and also the pair t-test was applied to compare the fatigue test score before and after the treatment in the groups. results the results showed that the use of grape seed capsules significantly altered the physical and mental activity of patients with ms. conclusion the existing ms drugs reduce the number of relapses, severity of disease and prevent disease progression. they have less effect on symptoms and improving performance and the quality of life along with side effects. due to the absence of significant side effects of grape seed and its proper effect, it can be used to improve fatigue and physical and mental activity in patients. keywords grape seed, multiple sclerosis, fatigue, quality of life introduction multiple sclerosis (ms) is a relatively common disease. it is a chronic progressive disease of the nervous system that usually occurs in the third and fourth decades of life.1 there is a clear difference in ms emergence between different populations and ethnic groups. the highest known prevalence (250 per 100,000 people) is common in the north of scotland and more than 2 million people worldwide suffer from ms.2 two pathological processes occur in ms. the first process is inflammation during which the body performs an autoimmune attack against the myelin sheaths of the neural fibers in the white matter of the central nervous system. these areas that have lost myelin are seen as plaque or lesions that the recovery of inflammation also improves the disease. the second process is neurodegenerative in which the nerve axon in the white matter of the brain is degraded which is irreversible in some cases. the collected data show that oxidative stress plays an important role in the development of ms. active oxygen species produce oxidative stress. their excessive production in the body causes various damages including loss of myelin and damage to axons and consequently ms. oxidative stress causes more damage in cns cells with weak antioxidant defense. anti-oxidant therapy can theoretically and practically prevent the spread of tissue damage and increase tissue survival.3 over the past two decades the effectiveness of several immune system regulating and suppressing therapies have been proven to effective in controlling ms. most of these drugs affect the inflammatory phase of the disease, and reduce the number of relapse attacks and disability compared with the placebo.4 the phenolic compounds in the grape (seed and pulp) have a strong free radical inhibitory activity.5 fatigue is one of the most common and debilitating symptom of multiple sclerosis the main cause of which is still unknown. however, it is believed that the destruction of neurons is the central cause of fatigue and the reduction of physical activity as an environmental factor of fatigue. however, treatments are usually ineffective due to the complex nature of fatigue in patients with multiple sclerosis. fatigue treatment methods cannot be fully effective either pharmaceutically (amantadine, pamulin, modafinil) or non-pharmaceutically in patients with multiple sclerosis.6 the grape seed with the scientific name vitis vinifera is a native plant of southern europe and western asia. grape seed and skin contain many active compounds, including flavonoids, polyphenols, anthocyanins and proanthocyanidins, and acetylbene compounds like resveratrol.7,8 various studies show that grape seed extract has anti-oxidant, anti inflammatory and antimicrobial activity effects as well as cardiovascular and liver protective effects and protection of the neurons and the nervous system.9 the evidence show that proanthocyanidins in the grape seed can suppress immunosuppression caused by ultraviolet radiation which is a risk factor for skin cancer and induce interleukin-12 and stimulates the t-cytotoxic cells and dna reconstruction.10 the grape seed extract reduces the free radicals produced by lipid peroxidation in the central nervous system in old rats and hypoxic ischemic brain injury in neonatal rats.11,12 smith and lassmann13 proved that nitric oxide as a potential intermediary causes the initial demyelination of microglia cells. increased nitric oxide has a direct relationship with the incidence of autoimmune inflammatory diseases, including multiple sclerosis, rheumatoid arthritis, issn 2413-0516 amir siahpoosh et al. 149j contemp med sci | vol. 4, no. 3, summer 2018: 148–152 original effect of grape seed on quality of life in multiple sclerosis patients insulindependent diabetes and inflammatory bowel disease.14 therefore, one of the important treatments for multiple sclerosis is to use nitric oxide synthesis inhibitor. the resveratrol in the grape seed has strong anti-inflammatory and nitric oxide inhibition effects.15 regarding the key role of free radicals in the development of ms disease and the antioxidant, anti-inflammatory and immune system regulation effects of grape seed extract, its usage is considered to control fatigue in patients with multiple sclerosis.16,17 materials and methods this study was a double-blind, randomized, single centered and placebo-controlled research. patient selection at first, 100 patients with ms disease who were clinically diagnosed by mcdonald criteria and referred to the ms association in khuzestan province were selected.18 among these patients the ones who did not have relapse during the last 6 weeks included in the study and the patients who had stroke and other neurological diseases, such as depression, diabetes and other autoimmune diseases such as lupus and those with new ms attacks as well as patients who had taken corticosteroid therapy up to 6 weeks before the study or pregnant or breastfeeding were excluded. in this study, 70 patients were selected and 30 patients were excluded (10 patients with stroke, 11 with depression and 9 had received corticosteroids) were excluded from the study. all participants received written information about the study and after this study they were requested to sign a written consent form. drug preparation in this study, red grapes were first purchased from reputable centers and seeds were separated manually, and then seeds were extracted by 70% ethanol by maceration method. accordingly 70% ethanol solvent (1000 ml) was poured on the plant powder (100 g). it was then kept at the laboratory for 48 h and then shaken for 2 h. the extract was then filtered, concentrated with rotary evaporator and then completely dried by freeze dryer and kept in a freezer (−20°c) until the preparation of the product.19 the capsules were formulated and prepared by the medicinal plants research center of ahvaz jundishapur university of medical sciences. after preparing the desired formulation, the prepared powder was placed into the capsule shell by manually filling machine and packed in 60 capsules and labeled with information required by patients (such as effective ingredients, usage, warnings, etc.). the placebo group used the formulation without the extract and the capsules were uniform and packed identically. packages were encoded at the medicinal herbs research center and were not unpacked until the end of the study. treatment procedure the 60 patients were randomly separated into two groups of treatment and placebo according to a balanced 1:1 randomization in which a computer-generated randomization table was applied (each group included 30 persons). the treatment group received a capsule containing 450 mg powder of grape seed extract (plus excipients 50 mg) twice a day for 1 month while placebo group received a capsule of the same size without extract (drug preparation section). the study was approved by the medical ethics committee of ahvaz jundishapur university of medical sciences (registration number: ir. ajums. rec.1396.326) and it was registered in the iranian registry of clinical trials (irct). it is available at www.irct.ir under registration number: irct201708218013n1. measurement tools patients’ fatigue was calculated based on multiple sclerosis quality of life-54 (msqol-54) before the start of the study. after a month, msqol-54 was measured in each group again. then msqol-54 scales and physical and mental health composite score were compared in both groups.20 based on the rating of physical health factors, role limitation due to emotional problem, emotional well-being, energy, health perception, social function, cognitive function, health distress, sexual function and change in health the total score was calculated. statistical analysis to describe the data, mean and standard deviation were used for quantitative variables. the t-test was used to compare the mean in two groups and the pair t-test was applied to compare the preand post-fatigue score between the groups and a p-value less than 0.05 was considered as the level of significant difference in all tests. results patient demographics the results of the demographic data of patients who continued until the end of the trial are presented in table 1. finally, 66 patients (in the placebo and treatment group) were selected. patients were within the age range of 32.26 ± 7.12. the mean age in the treatment and placebo groups was 31.05 ± 6.25 and 33.47 ± 8.56. based on the patient’s statements mean duration of the disease in the treatment and placebo groups was 4.05 ± 1.25 and 4.56 ± 2.12. there was no difference between the placebo and treatment group in terms of demographic data (p <0.05). results of treatment efficacy the baseline of physical and mental scores (base on msqol-54) of the two groups are shown in tables 2 and 3. there was no significant difference between the two groups at baseline (p > 0.05). discussion so far no definitive treatment is discovered for ms. side effects of current commonly used drugs, such as interferon beta, glatiramer acetate, and mitoxantrone have also led researchers to seek drugs with less side effects. although most patients are hopeful to have a natural or near normal life, their quality of life is affected by their reduced function.20 the disease modifying therapies reduce the number of relapses, severity of disease and prevent disease progression but have less effect on symptoms and performance and quality of life 150 j contemp med sci | vol. 4, no. 3, summer 2018: 148–152 effect of grape seed on quality of life in multiple sclerosis patients original amir siahpoosh et al. improvement. therefore, finding new treatments to reduce the symptoms of the disease is very important.21 today, research on medicinal plants has attracted attention to identify new and safe drug molecules by screening the existing active compounds.22 but limited plant compounds have been investigated for therapeutic purposes. saffaron (crocus sativus) is among the plants that has experimental autoimmune encephalomyelitis symptoms (experimental model of ms) in mice by inhibiting oxidative stress and leukocyte penetration into the central nervous system and thus has the potential for use in the treatment of ms.23 another drug for ms treatment on which many clinical trials have been done so far is cannnabis that has a positive effect on detrusor over activity, spasticity, trembling and neuropathic pain in patients with ms.24–26 however, it is not a good drug due to the side effects of cannabis, such as addiction, drowsiness, depression, anxiety, and decreased immune function.26 the grape seed is a natural drug with strong anti-inflammatory and antioxidant effects and also prevents peroxidation in the central nervous system and inhibits harmful free radicals such as hydroxyl and superoxide. the grape seed has regulatory effects on the catalase, superoxide reductase and peroxidase enzymes. it reduces the inflammatory cytokines such as alpha-tnf, interferon gamma and interleukin 1 and 6 that cause the onset of inflammation in ms patients and increases anti-inflammatory cytokines such as il-4, il-10 and tgf-beta (transforming growth factor beta) involved in the phase of disease improvement.27–29 many compounds have been identified in grape seed the most important of which are proanthocyanidins, anthocyanins and resveratrol.9 table 1. demographic information of patients who completed the trial medicine (patient) placebo (patient) gender male 10 11 female 23 22 marital status single 11 9 married 22 23 divorced 0 1 education illiterate 1 2 high school/ primary school 18 11 high school diploma 0 3 associate’s degree 2 3 bachelor’s degree 9 7 master’s degree 0 3 job housewife 17 12 employee 5 8 unemployed 4 3 freelance 4 4 student 0 2 table 2. comparison of the mean of the measured physical sub-scales in the two groups. the results are expressed as mean ± sd, and p < 0.05 is acceptable sub-scores treatment placebo before after before after physical function 10.77 ± 1.26 12.63 ± 2.38* 10.92 ± 1.25 12.55 ± 2.48* health perceptions 11.01 ± 1.24 11.56 ± 1.89 10.19 ± 1.99 10.37 ± 1.01 energy/fatigue 5.78 ± 0.5 7.63 ± +0.96* 6.23 ± 0.16 6.74 ± 0.53 role limitations-physical 6.52 ± 0.4 8.27 ± 2.25* 7.20 ± 0.63 6.91 ± 0.68 pain 7.63 ± 0.77 8.46 ± 0.84* 7.55 ± 0.67 7.67 ± 0.91 sexual function 4.26 ± 0.50 4.72 ± 0.44 4.10 ± 0.62 4.24 ± 0. 94 social function 8.08 ± 1.08 9.45 ± 1.01* 8.70 ± 0.90 9.22 ± 1.05 health distress 4.97 ± 0.44 7.31 ± 0.94* 6.48 ± 0.10 6.68 ± 0.8 total physical score 59.72 ± 10.34 71.05 ± 9.10* 62.53 ± 9.66 65.54 ± 10.01 table 3. comparison of the mean of the measured mental sub-scales in the two groups. the results are expressed as mean ± sd, and p <0.05 is acceptable sub-scores treatment placebo before after before after health distress 6.33 ± 0.87 9.31 ± 0.74* 8.25 ± 0.94 11.48 ± 1.76 overall quality of life 5.12 ± 0.45 6.73 ± 0.80* 6.47 ± 0.85 6.66 ± 0.92 emotional well-being 15.73 ± 1.76 20.37 ± 2.19* 16.90 ± 1.65 17.06 ± 1.39 role limitations-mental 13.21 ± 1.46 14.25 ± +1.93 12.99 ± 1.67 14.32 ± 1.27 cognitive function 8.80 ± 0.95 9.55 ± 0.57 8.63 ± 0. 46 8.41 ± 0.44 total mental score 48.73 ± 4.11 61.27 ± 6.13* 53.26 ± 5.86 55.31 ± 4.09 amir siahpoosh et al. 151j contemp med sci | vol. 4, no. 3, summer 2018: 148–152 original effect of grape seed on quality of life in multiple sclerosis patients resveratrol is a very valuable compound in preventing inflammatory and neurodegenerative diseases. part of the antioxidant effects of the grape seed is associated to resveratrol in addition to the oligomeric proanthocyanidins.30–32 resveratrol plays a significant role in neuronal differentiation by inhibiting sirt1 and sirt3 (32). resveratrol effective mechanism is probably due to transcriptional regulation of the nf-κb proteins.33 resveratrol can also affect estrogen and vitamin d receptors in the brain.34 according to statistical tests, there is a significant difference in the total mental and physical score of patients in the grape seed group before and after intervention. there is also a significant difference between the sub scores of the grape seed group before and after intervention in each subscale. therefore, according to these results, the positive effects of grape seed on improving the quality of life and the subscales physical function, energy/fatigue, role limitations-physical, pain, health distress, physical health composite, health distress mental, overall quality of life, emotional well-being, mental health composite, social function are proven. according to the results in the placebo group, there is no significant difference in the total score of the patients before and after the intervention which indicates that the increase in the total score in this group has not been effective. also, patients do not differ significantly before and after intervention in all subscales and all of these results indicate neutrality placebo on patients. also, the change in the total mental and physical score of the questionnaire between the grape seed and placebo groups is statistically significant before and after the intervention. among the sub-scales of intervention the change in grape seed group sub score is not significant in physical activity, role limitations-mental and cognitive activity but the change in other sub-scores in the grape group is significant compared to the placebo. conclusion this study suggests that grape seed can improve fatigue in ms patients. due to the absence of significant side effect for grape seed and its proper effect, it can be used to improve fatigue in patients with ms. given the fact that the questionnaire is related to the quality of life, the grape seed capsule can be added as a complementary treatment to the usual treatment of these patients to improve their quality of life. acknowledgment this study is a part of pharm. d. thesis of negin derakhshannezhad and with the number b-078/94 registered at the ahvaz jundishapur university of medical sciences and medicinal plants research center, ahvaz, iran, also, it has been supported partial financially by grant number (g121-1661) from research center of nab’a al-hayat foundation for medical sciences and health care, iraq. conflict of interest none. n references 1. rao sm. neuropsychology of multiple sclerosis: a critical review. j clin exp neuropsychol. 1986;8:503–542. 2. valko m, rhodes cj, moncol j, izakovic mm, mazur m. free radicals, metals and antioxidants in oxidative stress-induced cancer. chem biol interact. 2006;160:1–40. 3. gilgun-sherki y, melamed e, offen d. the role of oxidative stress in the pathogenesis of multiple sclerosis: the need for effective antioxidant therapy. j neurol. 2004;251:261–268. 4. van horssen j, witte me, schreibelt g, de vries he. radical changes in multiple sclerosis pathogenesis. biochim biophys acta. 2011;1812: 141–150. 5. jayaprakasha gk, singh rp, sakariah kk. antioxidant activity of grape seed (vitis vinifera) extracts on peroxidation models in vitro. food chem. 2001;73:285–290. 6. pilutti la, greenlee ta, motl rw, nickrent ms, petruzzello sj. effects of exercise training on fatigue in multiple sclerosis: a meta-analysis. psychosom med. 2013;75:575–580. 7. montealegre rr, peces rr, vozmediano jc, gascueña jm, romero eg. phenolic compounds in skins and seeds of ten grape vitis vinifera varieties grown in a warm climate. j food compos anal. 2006;19:687–693. 8. yilmaz y, toledo rt. health aspects of functional grape seed constituents. trends food sci technol. 2004; 15:422–433. 9. shi j, yu j, pohorly je, kakuda y. polyphenolics in grape seeds-biochemistry and functionality. j med food. 2003;6:291–299. 10. chu h, tang q, huang h, hao w, wei x. grape-seed proanthocyanidins inhibit the lipopolysaccharide-induced inflammatory mediator expression in raw264.7 macrophages by suppressing mapk and and nf-kb signal pathways. environ toxicol pharmacol. 2016;41:159–166. 11. feng y, liu ym, fratkins jd, leblanc mh. grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rats. brain res bull. 2005;66:120–127. 12. balu m, sangeetha p, murali g, panneerselvam c. modulatory role of grape seed extract on age-related oxidative dna damage in central nervous system of rats. brain res bull. 2006;68:469–473. 13. smith kj, lassmann h. the role of nitric oxide in multiple sclerosis. lancet neurol. 2002;1:232–241. 14. kolb h, kolb-bachofen v. nitric oxide in autoimmune disease: cytotoxic or regulatory mediator? immunol today. 1998;19:556–561. 15. bi xl, yang jy, dong yx, wang jm, cui yh, ikeshima t, et al. resveratrol inhibits nitric oxide and tnf-α production by lipopolysaccharide-activated microglia. int immunopharmacol. 2005;5:185–193. 16. aleessa asaj. psycho-social and medical patterns of psychiatric disorders in multiple sclerosis patients, baghdad-iraq. iraqi j public health. 2017;1:3. 17. majdinasab n, siahpush a, mousavinejad s k, malayeri a r, sajedi s a, bizhanzadeh p. effect of boswellia serrata on cognitive impairment in multiple sclerosis patients. j herb med. 2016;6:119–127. 18. polman ch, reingold sc, banwell b, clanet m, cohen ja, filippi m, et al. diagnostic criteria for multiple sclerosis: 2010 revisions to the mcdonald criteria. ann neurol. 2011;69:292–302. 19. siahpoosh a, soleimani i. in vitro evaluation of inhibitory effect of phoenix dactylifera bark extract on rat lipid peroxidation and blood hemolysis. trop j pharm res. 2016;15:1707–1713. 20. stuifbergen ak, harrison tc. complementary and alternative therapy use in persons with multiple sclerosis. rehabil nurs. 2003;28:141–147. 21. goodin ds, frohman em, garmany gp, halper j, likosky wh, lublin fd, et al. disease modifying therapies in multiple sclerosis: report of the therapeutics and technology assessment subcommittee of the american academy of neurology and the ms council for clinical practice guidelines. neurology. 2002;58:169–178. 22. martín r, carvalho-tavares j, hernández m, arnés m, ruiz-gutiérrez v, nieto ml. beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: a potential therapeutic role. biochem pharmacol. 2010;79:198–208. 23. ghazavi a, mosayebi g, salehi h, abtahi h. effect of ethanol extract of saffron (crocus sativus l.) on the inhibition of experimental autoimmune encephalomyelitis in c57bl/6 mice. pak j biol sci. 2009;12:690–695. 152 j contemp med sci | vol. 4, no. 3, summer 2018: 148–152 effect of grape seed on quality of life in multiple sclerosis patients original amir siahpoosh et al. 24. kavia rb, de ridder d, constantinescu cs, stott cg, fowler cj. randomized controlled trial of sativex to treat detrusor overactivity in multiple sclerosis. mult scler j. 2010;16:1349–1359. 25. barnes mp. sativex: clinical efficacy and tolerability in the treatment of symptoms of multiple sclerosis and neuropathic pain. expert opin pharmacother. 2006;7:607–615. 26. hall w. the adverse health effects of cannabis use: what are they, and what are their implications for policy? int j drug policy 2009;20: 458–466. 27. terra x, montagut g, bustos m, llopiz n, ardèvol a, bladé c, et al. grapeseed procyanidins prevent low-grade inflammation by modulating cytokine expression in rats fed a high-fat diet. j nutr biochem. 2009;20:210–218. 28. wang h, xue y, zhang h, huang y, yang g, du m,et al. dietary grape seed extract ameliorates symptoms of inflammatory bowel disease in il10‐ deficient mice. mol nutr food res. 2013;57:2253–2257. 29. van oosten bw, polman ch. cytokines and multiple sclerosis. in: neurochemistry, springer, usa, 1997, pp. 121–124. 30. sun ay, wang q, simonyi a, sun gy. resveratrol as a therapeutic agent for neurodegenerative diseases. mol neurobiol. 2010;41:375–383. 31. frémont l, belguendouz l, delpal s. antioxidant activity of resveratrol and alcohol-free wine polyphenols related to ldl oxidation and polyunsaturated fatty acids. life sci. 1999;64:2511–2521. 32. schirmer h, pereira tc, rico ep, rosemberg db, bonan cd, bogo mr, souto aa. modulatory effect of resveratrol on sirt1, sirt3, sirt4, pgc1α and nampt gene expression profiles in wild-type adult zebrafish liver. mol biol rep. 2012;39:3281–3289. 33. ren z, wang l, cui j, huoc z, xue j, cui h, et al. resveratrol inhibits nf-κb signaling through suppression of p65 and ikappab kinase activities. pharmazie. 2013;68:689–694. 34. das dk, maulik n. resveratrol in cardioprotection: a therapeutic promise of alternative medicine. mol interv. 2006;6:36–47. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201807 101j contemp med sci | vol. 5, no. 2, march–april 2019: 101–105 original predictive factors of survival rate in oral squamous cell carcinoma: a retrospective study in iran narges gholizadeh,a hanieh sadat emami razavi,b gholamali jafari haftkhani,c and nafiseh sheykhbahaeia aoral & maxillofacial medicine, school of dentistry, tehran university of medical science, tehran, iran. bdepartment of operative dentistry, school of dentistry, alborz university of medical science, karaj, iran. cdepartment of ear, nose, throat and head and neck surgery, baqiyatallah university of medical science, tehran, iran. correspondence to nafiseh sheykhbahaei (email: dsheykhbahaei@gmail.com). (submitted: 12 january 2019 – revised version received: 27 february 2019 – accepted: 05 march 2019 – published online: 26 april 2019) objective oral cancer as a part of this collection plays an important role in the burden of diseases, especially in south asia. despite numerous advances in the treatment of cancers, oral cancer is one of the top 10 causes of death due to delayed detection. given the role of the environmental and climatic conditions in cancers, there is a need for epidemiological studies in different geographic areas. methods in this study, data were collected through medical records of the patients with oral cancer during 2009–2012 in the archives of the cancer department of the ministry of health of the islamic republic of iran. the follow-up of 82 cases was possible in the study. data collection were performed from questionnaire. the data were analyzed by spss version 20. the kaplan–meier survival curves were utilized and, moreover, the corresponding influential factors were examined using the cox regression test. results the overall 5-year survival rate was 40.24% (se = 5.5). the most widely used treatment method was a combination of three methods of surgery, radiation therapy, and chemotherapy. the gender of 64.63% of patients were male. the mean age of patients were 66.1 years. the age and regular periodic follow-up had a statistically significant relation with the survival rate (p = 0.02, 0.03). conclusion oral cancers require special attention due to high prevalence and overall survival rates. training oral health care providers, and periodic follow-up can be an important step in increasing the survival of these patients. keywords oral squamous cell carcinoma, delay, survival rate introduction nowadays, increasing the world’s population and the average age, promoting life expectancy, improving the health status and high exposure to carcinogenic risk factors have led to an increase in the global incidence of cancer. oral cancer as a part of this collection plays an important role in the burden of diseases, especially in south asia.1,2 the world health organization (who) in 2012 has emphasized the prevention of cancer and the promotion of quality-of-life of cancer patients. nearly 40% of deaths from cancer will be prevented by controlling risk factors, and whose one-third burden will be reduced by timely diagnosis and treatment.3 cancer is the third most common cause of death in iran with more than 30,000 deaths annually.4 about 5% of all cancers occur in the head and neck and about half of these are in the oral cavity. squamous cell carcinoma (scc) is the most common head and neck cancer whose incidence has been reported to have descending trend over the last three decades in the world.5 according to global data on the incidence of oral cancer map in 1997, iran, along with other south asian countries, is considered as one of the countries with a high rate of oral cancer with an outbreak of between 20 and 36.3 per 100,000 people.4 according to data released by the iranian ministry of health in 2003, the incidence of oral cancer is among the 10 most common cancers in men and women. the oral cancers are related to tobacco use, smoking, alcohol consumption, low socioeconomic status, and poor health, and improper diet, viral infections such as hpv, poorly fitted dentures, and chronic trauma.6 the biological behavior of scc suggests highly invasive and poor prognostic nature.3 in the industrialized countries, men are two to three times more likely to be involved than women, mainly because of further exposure to the risk factors.1 the mean age of the diagnosis is 57.1 years in males and 52.5 years in females, with the highest incidence in the sixth decade of life.6 genetic factors are now widely recognized as an etiology because the scc is developed in children and adolescents without any risk factor.1 the main anatomical areas include the tongue, the floor of mouth, the gums, the palate, and other parts of the mouth; the involvement of various oral areas is affected by geographical factors. for example, the most common involved area is the tongue between the european and american populations, and buccal mucus cancers among the asian population due to a habit of chewing tobacco such as betel quid.7 despite numerous advances in the treatment of cancers, oral cancer is one of the top 10 causes of death due to delayed detection with several causes, such as early-stage asymptomatic nature, clinical presentation similar to other lesions, and variation in clinical manifestations. given the role of the environmental and climatic conditions in cancers, there is a need for epidemiological studies in different geographic areas. materials and methods this study was conducted in retrospective and cross-sectional design, whose data were collected through the 436 medical records of the patients with oral cancer during 2009–2012 in the archives of the cancer department of the ministry of health of the islamic republic of iran. totally, the enrolled medical records in our study were 82 out of 436 cases with a histopathologic diagnosis of oral squamous cell carcinoma, and a contact information or mailing address. all new cases of cancer detection in iran refer to cancer registry centers from pathology labs through the forms prepared by the ministry of issn 2413-0516 102 j contemp med sci | vol. 5, no. 2, march–april 2019: 101–105 survival rate in oral squamous cell carcinoma original n. gholizadeh et al. health since 2003, consisting of demographic profiles, diagnostic times and pathological parameters of the disease. other items included in this form are the location of the lesion, the degree of differentiation and degree of invasion with the special codes. information of our patients was gathered by a checklist designed as two groups of questions that were completed by the information in cancer registration center forms and telephone by patients or relatives, including survival status, treatment method and delayed time. in collecting patient information, the lack of a response to three times of phone call with a 2-week interval resulted in the use of postal services, and in the absence of a repeated response, the use of the medical record available at the cancer institute of imam khomeini hospital. meanwhile, if any, the relatives were asked to explain about the causes of death in the deceased subjects, which classified into two groups of cancer complications and other causes such as accidents or heart attacks. concerning a delay studied within the two groups of <4 weeks and >4 weeks, the patient delay was defined as interval from the onset of disease symptoms to the first patient referral, and professional delay as interval from the first patient referral to diagnosis. this study was approved by the ethics committee of tehran university of medical science. the spss version 20 software was used to analyze the attained data using descriptive statistics of tables and figures, and the kaplan–meier curve to evaluate the survival, as well as the simple and multivariate cox regression test to assess the effect of the studied factors on survival rate. in all tests, p < 0.05 was considered as statistically significant difference. results our subjects included 53 (64.63%) males and 29 (35.37%) females and 97.33% of them were married. the results showed that the prevalence of oral cancer in females was twice lower than in males. according to fig. 1, 32.9% of patients were ≤60 years and 67.1% were >60 years with the mean age of 66.1 years for all patients ranging from 31 to 89 years. in fact, most of our patients were living in their 6th to 8th decades of life. on the basis of histological grade of the lesions, 67.39% of the patients suffered from completely differentiated lesions, 17.39% had relatively differentiated lesions and 15.22% of them showed less differentiated lesions. in this study, there were several diagnostic procedures, the participants found the risk of cancer symptoms by themselves (n = 42, 71.18%), a physician due to referral for other diseases (n = 13, 22.03%) and a dentist (n = 4, 6.79%). the statistical findings of the first referral in this study can be categorized according to whom the patient has referred to for the first time. the first referral of the patients was to a specialist physician (n = 41, 56.18%), a general practitioner (n = 24, 32.87%) and a dentist (n = 8, 10.95%). about delay factors in cancer management, the mean patient delay was reported to be 16.3 ± 17.7 weeks (approximately 4 months) ranging from a week to a year (52 weeks). a delay <4 weeks was 37.8% and a delay >4 weeks was 46.3%. the mean professional delay was 4.75 ± 5.29 weeks ranging from a week to 28 weeks. the patients with the oral cancers were treated in three methods, surgery, radiotherapy and chemotherapy. in many cases, the treatment of patients was a combination of two or three methods. accordingly, the most commonly used treatment (36.6%) for the patients was the combination of all three treatments. surgery 15.9%, chemotherapy 3.7%, radiotherapy 6.1%, chemoradiotherapy 11%, chemosurgery 15.9%, radio surgery 7.3% and 2.4% of patients with no treatment. the periodic follow-up was fulfilled in the hospital (n = 30, 66.66%) and in the private medical clinic (n = 15, 33.34%) and no follow-up programs (n = 0). an oral hygienist performed the periodic follow-up for 12.2% of the patients. in this study, the locations of tumor were respectively the lateral borders of tongue (20.4%), the floor of mouth (2.1%), the base of the tongue (1.7%), lips (1.3%), gums and palate (0.95%) and other parts of the mouth (7.7%). in this study, 49 (59.75%) survived and 33 (40.25%) died during the study due to the cancer complications (n = 43, 93.47%) and other causes (n = 3, 6.93%). as can be seen in table 1, the 1-year overall survival rate was 91.46% (se = 3.09), the 3-year overall survival rate was 53.7% (se = 5.5) and the 5-year overall survival rate was 40.24% (se = 5.5). the survival rate in females was more than in males, but the gender showed no statistically significant relationship with survival rate (p = 0.41). however, the age of the patients had statistically significant relationship with survival rate, meaning the reduction in the survival rate with age (p > 0.001). moreover, the survival rate was significantly improved by the regular periodic follow-up (p = 0.03). the patients experiencing periodic follow-up under the supervision of oral hygienists had no significant difference in the survival rate (p = 0.51). the survival rate showed no significant relationship with the patient delay >4 weeks (p = 0.37), and also with the professional delay (p = 0.54), as well as with the degree of tumor differentiation (p = 0.35). according to table 2, the multivariate cox regression revealed that the survival rate was not in significant relationship with the treatments of only surgery (p = 0.055), only radiotherapy (p = 0.64) and only chemotherapy (p = 0.73). discussion this study examined the association between the demographic variables of oral scc (oscc) patients, tumor-related characteristics, and the used diagnostic and therapeutic methods with survival rate of patients. data were collected from patients during 2009–2012 in cancer registry center of ministry of health and medical education and cancer institute of imam khomeini hospital, iran. based on the results, only age and periodic follow-ups showed a significant correlation with the survival rate. this study obtained 1-year survival rate of 91.46% with a standard error (se) of 3.09, 2-year overall survival rate of 69.51% (se = 5.08), 3-year overall survival rate of 53.7% (se = 5.5), and 5-year overall survival rate of 40.24% (se = 5.5). in a study by warnakulasuriya et al.8 5-year overall survival rate in most countries for oral cancer was reported to be 50–60%. in a study by silverman9 in the united states, the 5-year overall survival rate was 60%, which is 10% higher than this study. the difference in the survival rate in this research with other studies in developed countries could be due to better health infrastructures of these countries or better access to treatment centers for the patients. in a study in netherlands in 2014, the survival rate was improved in spite of the increased incidence of oscc.10 an increase in the incidence of cancer can be attributed to changes in lifestyle, and improved survival n. gholizadeh et al. 103j contemp med sci | vol. 5, no. 2, march–april 2019: 101–105 original survival rate in oral squamous cell carcinoma rate due to the development of health facilities and the promotion of public awareness. in this study, the mean age of all patients with oral cancer was 66.1 years. in addition, 32.9% of subjects under study were aged ≤60 years old and 67.1% were aged >60 years. also, the minimum and maximum age of these patients was 31 and 89 years, respectively. in the study by warnakulasuriya et al.8 in england in 2010, the mean age of patients was 62 years. in a study in 2013, the frequency of patients over the age of 40 years was about eight times more than people under 40 years.7 salian et al.6 reported a maximum outbreak in the 5th and 6th decades. in another study, the age range of affected individuals was reported between 11 and 94 years. the incidence of scc is very rare in children and syndromes associated with genetic disorders can be raised in case of any incidence.1 in a study by chamani11 in iran, the mean age of patients with oral cancer was 11 years less than this study, which was 54.5 years. bhurgri13 in india showed that only 2% of patients were over 60 years of age. in countries with high prevalence of oral cancers, it can be expected that the age of disease onset is lower than in other countries. moreover, the lower life expectancy in these countries is effective in this age ratio. in recent decades, the prevalence of scc in younger people has been considered,5 which could be due to changes in lifestyle for example the onset of tobacco and alcohol use and sexual intercourse at an earlier age. in this study, only the age variable showed a significant relationship with the survival rate of patients. aging led to a decrease in the level of body immunity and an increase in the comorbidities such as cardiovascular, respiratory and renal diseases in patients with scc, causing the reduction in patient survival.14 however, this relationship was not significant in the study of sargeran15 in iran, chandu et al.16 in australia and chen et al.17 in taiwan, as well as three other studies in 2014.18–20 this study showed a higher prevalence of oscc in men. according to the results, 64.63% of the patients were male and the rest were female. in an epidemiological study in argentina, the prevalence of oral cancer in men between 1992 and 2000 was 1.24 times higher than in women.21 in the study by sargeran et al.22 in iran, 55% of patients were male and the rest were women. in another study in australia, 56.8% of the patients were male.16 in several other studies, male involvement was 1.5, 1.9, and 1.4 times higher than women.1,7,8 meanwhile, an increase in the incidence of oral cancer in women has been noted in recent years.5,23 two studies confirmed the higher prevalence of oscc in women.24,25 further, a study reported a steady increase in the incidence of scc in middle-aged women over the past two decades, despite a lower incidence of oral cancer in women.23 changes in the gender ratio in patients, especially in recent years, can be strongly attributed to changes in lifestyle, smoking habits, non-inhaled tobacco usage, alcohol consumption, hormonal changes, and nutritional deficiencies such as iron, riboflavin, vitamins and minerals in women.5,23 since cigarette and alcohol habits are less common in iranian women, further attention should be driven toward other predisposing factors, such as nutritional deficiencies, viral agents such as hpv and chronic trauma, to prevent female involvement with scc. the relationship between gender variable and survival rate in this study was not significant, consistent with the studies by sargeran,15 chandu et al.16 and chen et al.,17 and hanemann et al.,19 fan et al.18 and fang et al.19 the responses to the therapeutic and clinical course of the disease appear not to be affected by gender factors, resulting in a lack of influence of the gender factor on the survival rate. in this study, 33 patients (40.25%) survived and the rest died. of the 49 deceased patients with scc, the cause of death was the cancer complications in 43 (93.47%) patients and other causes in three (6.53%) patients. in the study of chandu et al.,16 out of 115 patients, 88 (76.5%) survived, and 27 died of which 17 (14.8%) due to complications of cancer and 10 (8.7%) for other causes. in the study of sargeran,15 the causes for death in 470 patients were the complications of cancer in 335 (71%) and the other causes in 18 (4%). of these patients, 80 (17%) survived, and eventually 37 (8%) were undiagnosed. based on the results, it seems that other factors such as patient delay due to lack of knowledge or lack of timely access to diagnostic and therapeutic centers as well as professional delay can be considered as key factors in the high mortality rate of oral cancer. in this study, the first patient referral was to specialist physician in 41 (56.18%) out of all patients with scc, a general practitioner in 24 (32.87%) and a dentist in the rest (10.95%). in the study of kerdpon and sriplung26 in thailand, the first patient referral was to a practitioner in 133 patients (82.6%), to a dentist in 25 (15.5%) and to a public health care provider in three (1.9%), reflecting differences in care and referral systems in thailand. according to the results, it can be argued that the role of dentists has not been explained as the first referral in the diagnosis of oscc in different societies, especially in our country, and inadequate information has been provided to society in this regard. the most widely used treatment method for the patients among our statistical community was to combine all three methods of surgery, radiotherapy and chemotherapy. among patients with oscc, 37.5% experienced all three treatments, 12.5% surgery alone, 5.1% radiotherapy alone, 6.6% chemotherapy alone, 10.3% chemotherapy plus radiotherapy, 8.1% surgery plus chemotherapy and 11% radiotherapy plus surgery. in the study of sargeran,15 17% experienced only surgery, 66% surgery plus radiotherapy, and 17% radiotherapy alone. in this study, the treatment method used had a significant relationship with survival. in the study of chen et al.,17 the surgical treatment method had a significant relationship with survival rate, but not significant in this study though the p-value was very close to the significance level (0.055), indicating the importance of surgical therapy in the oscc treatment. this method, combined with other methods, leads to improved patient survival. hasegawa et al.27 also showed significantly higher loco regional control in patients treated by combining three therapies compared with surgery alone and surgery with radiotherapy. however, there was no significant difference in the overall survival rate of patients who received radiotherapy compared with the group receiving all three therapies. in this study, it has been shown that regular periodic follow-ups by physicians, in contrast to orodental health technicians, have a significant effect on increasing the survival of patients through the timely diagnosis, treatment or referral of recurrent lesions. rapid diagnosis of cancer and urgent referral of patients are important factors in improving the survival rate of patients.28 in this study, the knowledge of patients about the symptoms of oral cancer as well as the status of the treatment 104 j contemp med sci | vol. 5, no. 2, march–april 2019: 101–105 survival rate in oral squamous cell carcinoma original n. gholizadeh et al. system for early referral and treatment were investigated as similar to a study by dios et al.29 the mean patient delay was 16.3 weeks, approximately 4 months, for the patients in this study. the minimum and maximum patient delay was 1 week and 1 year (52 weeks) with a standard deviation of 17.7 weeks. in this study, 37.8 patients with oscc had a delay of <4 weeks, and 46.3 patients had a delay of >4 weeks. in the study of brouha et al.28 in the netherlands, 47% of patients had a delay of <4 weeks. in the study of kerdpon and sriplung,26 the minimum and maximum patient delay was 0 day and 102 weeks. in this study, the mean patient delay was reported to be 13 weeks. the mean patient delay for this study was 3 weeks longer. a higher patient delay and higher percentages of patients with a delay of >4 weeks can highlight the low level of awareness of cancer patients or lack of access to specialized centers for the diagnosis and treatment of cancer. a quiet clinical course in oral cancer, which is lacking in symptoms such as pain or paresthesia and bleeding until late advanced stages, can be one of the important reasons for delaying the patient referral for diagnosis. in this study, the mean professional delay was 4.75 weeks, with a minimum and maximum professional delay of 1 week and 28 weeks. the standard deviation for professional delay was 5.29 weeks. in the study of kerdpon and sriplung in thailand, the minimum and maximum professional delay was 0 day and 104 weeks. in their study, the mean professional delay was reported to be 7 weeks.26 the highly diverse clinical presentations of oscc, which can be mistaken for differential diagnosis with irritative and benign oral lesions, are among the important reasons raised in professional delay, suggesting the need for specialist retraining courses for the differentiation of clinical symptoms of oral cancer for physicians, general dentists, and specialized dentists. in this study, the most common location of tumor was the lateral borders of tongue (with a prevalence of 20.4%) and the least affected areas (0.9%) were gums and palate, in line with the findings of the study of juan-carlos as the tongue (44.7%) showed the most prevalent involved regions and the palate (0.5%) had the minimum involvement.7 in the review study of gupta et al.,5 the tongue was the most commonly involved location in the european, american and brazilian populations, while buccal mucosa and tongue were among the most commonly reported sites in southeast asia according to the patient habit of using chewing tobacco. in the study of gehani and goteti1 in libya, the tongue was reported as the most commonly involved site. the tongue and the floor of mouth are known as a premalignant condition in the oral cavity as these areas had higher loss of heterozygosity than other areas of the mouth. moreover, more contact and penetration of the carcinogens in foods and tobacco could be suggested as a cause for higher incidence of cancer in these areas. in this study, most of sccs had good degree of differentiation at diagnosis (67.39%), and the degree of tumor differentiation showed no significant relationship with survival rates. in the study of juan-carlos, the highest rate was related to the moderate degree of differentiation (61.2%). in their study, the survival rate was significantly correlated with degree of tumor differentiation, indicating a high percentage of patients with moderate degree of differentiation, poor quality of life and low survival rate.7 the degree of tumor differentiation depends on many factors at the time of diagnosis, including professional delay, patient delay, age, tumor location, and other cases. in the study of chen et al.,17 the association between histological differentiation and survival rate was significant, but not significant in this study. conclusion considering the increased percentage of people with oral cancer, it is essential to get knowledge about the epidemiologic conditions of these patients, to investigate the barriers and problems in the health care system of different societies to improve the quality-of-life of these patients, and to develop educational programs. oral cancers require special attention due to high prevalence and overall survival rates. training oral health care providers, especially dentists, on precise examination and early detection of lesions and periodic follow-up of patients after histopathological diagnosis can be an important step in increasing the survival of these patients. moreover, registration of treatment methods and response rates to these methods can be the basis for oral health decision making. conflicts of interest none.  references 1. gehani re, goteti shl. squamous cell carcinoma of oral cavity: changing trends. j dent health oral disord ther. 2015;2:00051. 2. perera m, al-hebshi nn, perera i, ipe d, ulett gc, speicher dj, et al. inflammatory bacteriome and oral squamous cell carcinoma. j dent res. 2018;97:725–732. 3. ehtesham h, safdari r, mansourian a, tahmasebian s, mohammadzadeh n, ghazisaeedi m, et al. clinical decision support system, a potential solution for diagnostic accuracy improvement in oral squamous cell carcinoma: a systematic review. j oral health oral epidemiol. 2017;6:187–195. 4. jafari a, gholizadeh n, ramezani r, razavi he, najafi s. evaluation of survival rate in patients with laryngeal cancer. j dent med. 2017;30:89–96. 5. gupta n, gupta r, acharya ak, patthi b, goud v, reddy s, et al. changing trends in oral cancer a global scenario. nepal j epidemiol. 2016;6:613–619. 6. salian v, dinakar c, shetty p, ajila v. etiological trends in oral squamous cell carcinoma: a retrospective institutional study. cancer transl med. 2016;2:33–36. 7. hernández-guerrero jc, jacinto-alemán lf, jiménez-farfán md, macariohernández a, hernández-flores f, alcántara-vázquez a. prevalence trends of oral squamous cell carcinoma. mexico city’s general hospital experience. med oral patol oral cir bucal. 2013;18:e306–e311. 8. warnakulasuriya s. living with oral cancer: epidemiology with particular reference to prevalence and life-style changes that influence survival. oral oncol. 2010;46:407–410. 9. silverman s jr, kerr ar, epstein jb. oral and pharyngeal cancer control and early detection. j cancer educ. 2010;25:279–281. 10. braakhuis bj, leemans cr, visser o. incidence and survival trends of head and neck squamous cell carcinoma in the netherlands between 1989 and 2011. oral oncol. 2014;50:670–675. 11. maleki d, ghojazadeh m, mahmoudi ss, mahmoudi sm, pournaghi azar f, torab a, piri r, azami aghdash s, naghavi behzad m. epidemiology of oral cancer in iran: a systematic review. asian pacific journal of cancer prevention. 2015;16:5427–32. 12. hwa js, kwon oj, park jj, woo sh, kim jp, ko gh, et al. the prognostic value of immunohistochemical markers for oral tongue squamous cell carcinoma. eur arch otorhinolaryngol. 2015;272:2953–2959. 13. bhurgri y. cancer of the oral cavity-trends in karachi south (1995-2002). asian pac j cancer prev. 2005;6:22–26. 14. golizadeh n, najafi sh, khayamzadeh m, afzali s, sheykhbahaei n. trend in laryngeal cancer, mortality and survival rate in iran. j contemp med sci. 2018;4:7–11. n. gholizadeh et al. 105j contemp med sci | vol. 5, no. 2, march–april 2019: 101–105 original survival rate in oral squamous cell carcinoma 15. sargeran k. oral cancer in tehran, iran: an approach for understanding disease burden. 2008. 16. chandu a, adams g, smith ac. factors affecting survival in patients with oral cancer: an australian perspective. int j oral maxillofac surg. 2005;34:514–520. 17. chen yk, huang hc, lin lm, lin cc. primary oral squamous cell carcinoma: an analysis of 703 cases in southern taiwan. oral oncol. 1999;35:173–179. 18. fan y, zheng l, mao mh, huang mw, liu sm, zhang j, et al. survival analysis of oral squamous cell carcinoma in a subgroup of young patients. asian pac j cancer prev. 2014;15:8887–8891. 19. fang qg, shi s, liu fy, sun cf. squamous cell carcinoma of the oral cavity in ever smokers: a matched-pair analysis of survival. j craniofac surg. 2014;25:934–937. 20. hanemann ja, oliveira dt, nonogaki s, nishimoto in, de carli ml, landman g, et al. expression of e-cadherin and β-catenin in basaloid and conventional squamous cell carcinoma of the oral cavity: are potential prognostic markers? bmc cancer. 2014;14:395. 21. brandizzi d, chuchurru ja, lanfranchi he, cabrini rl. analysis of the epidemiological features of oral cancer in the city of buenos aires. acta odontol latinoam. 2005;18:31–35. 22. sargeran k, murtomaa h, safavi smr, vehkalahti mm, teronen o. survival after diagnosis of cancer of the oral cavity. br j oral maxillofac surg. 2008;46:187–191. 23. la vecchia c, lucchini f, negri e, levi f. trends in oral cancer mortality in europe. oral oncol. 2004;40:433–439. 24. muscat je, richie jp jr, thompson s, wynder el. gender differences in smoking and risk for oral cancer. cancer res. 1996;56:5192–5197. 25. vatanasapt p, suwanrungruang k, kamsa-ard s, promthet s, parkin md. epidemiology of oral and pharyngeal cancers in khon kaen, thailand: a high incidence in females. asian pac j cancer prev. 2011;12:2505–2508. 26. kerdpon d, sriplung h. factors related to delay in diagnosis of oral squamous cell carcinoma in southern thailand. oral oncol. 2001;37:127–31. 27. hasegawa t, yanamoto s, otsuru m, yamada si, minamikawa t, shigeta t, et al. retrospective study of treatment outcomes after postoperative chemoradiotherapy in japanese oral squamous cell carcinoma patients with risk factors of recurrence. oral surg oral med oral pathol oral radiol. 2017;123:524–530. 28. brouha xd, tromp dm, hordijk gj, winnubst ja, de leeuw jr. oral and pharyngeal cancer: analysis of patient delay at different tumor stages. head neck. 2005;27:939–945. 29. diz dios p, padrón gonzález n, seoane lestón j, tomás carmona i, limeres posse j, varela-centelles p. “scheduling delay” in oral cancer diagnosis: a new protagonist. oral oncol. 2005;41:142–146. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.04201907 304 j contemp med sci | vol. 5, no. 6, november–december 2019: 304–308 original issn 2413-0516 introduction antenatal care (anc) has been a routine practice throughout the world since early 20th century.1 quality of anc is an important determinant of pregnancy outcome2 and has been designated one of the four pillars of safe motherhood along with clean and safe delivery, essential obstetric care, and family planning which could contribute to the reduction of maternal mortality.3 client satisfaction with the quality of care which reflects goals or preferences are met by the health care provider and or service,4 is an integral part of the quality assurance process of health delivery.5 the traditional risk approach to anc, which is based on european models assumes that better care is achieved by frequent routine visits. however, evidence-based research has found the practice of the traditional approach to anc based on the european models to be wasteful and misleading.6 women are classified by risk status to determine their chances of complications and the levels of care needed.7 focused anc recognizes that every pregnant woman is at risk for complications and should receive the same basic care and monitoring for complications.8,9 evaluating to what extent patients are satisfied with health services is clinically relevant, as satisfied patients are more likely to comply with treatment, take an active role in their own care, to continue using medical care services, and recommend center’s services to others.10 despite the fact that client satisfaction is essential for further improvement of the quality of care, little is known about the quality and associated factors of satisfaction in erbil city and kurdistan region of iraq. this study, therefore, would have a certain contribution in closing this gap. methods this cross-sectional study was conducted at two phc centers in erbil city, brayati phc center which provides focused anc services and nazdar bamerni phc center which provides risk approach anc services. the study was carried out between january, 1 and december 31, 2015. a convenience sample of 300 pregnant women (150 pregnant women from each phc centers) was collected. all pregnant women attended the phc centers for anc services in their second trimester of pregnancy (16 weeks and after) during the study period were included in the study. a designed questionnaire was constructed for data collection depending on an extensive review of relevant literature. clients satisfaction with nine items of anc “clinical” services that included: (1) explanation of complications, (2) immunization of pregnant women, (3) checking of blood pressure and body weight, (4) fixing next appointment, (5) education about importance of breastfeeding, (6) instructions about hygiene, (7)understanding of instructions, (8) full child immunization, and (9) doctor’s explanation of examination results was recorded. a score of 2 was given for every “yes” response and 0 for “no” response. satisfaction with other components of anc including clinic, attending doctors, attending nurse, waiting time, laboratory services, and pharmacy support was recorded. a score of 2 was given for “excellent” response, 1 for “good,” and 0 for “poor” and “very poor” satisfaction. therefore, the overall score was 30 (18+12). the total satisfaction score was categorized into two groups, low level of satisfaction (less than the median score of total scores) and a high level of satisfaction (equal and more than median of total scores). satisfaction with focused and risk approach antenatal care services among pregnant women attending primary health care centers in erbil city amina al-khayata, tariq salman al-hadithib acollege of medicine, university of mosul, mosul, iraq. bdepartment of community medicine, college of medicine, hawler medical university, erbil, iraq. corresponding author: tariq salman al-hadithi (email: tariq.hadithi@hmu.edu.iq) (submitted: 03 september 2019 – revised version received: 14 october 2019 – accepted: 22 october 2019 – published online: 26 december 2019) objectives client satisfaction is essential for further improvement of the quality of health care. this study aimed to assess the satisfaction with focused and risk approach antenatal care services among pregnant women in erbil city of iraq. methods this cross-sectional study was conducted at two primary health care centers in erbil city, one provides focused antenatal care services and the other provides risk approach antenatal care services. a convenience sample of 300 pregnant women (150 pregnant women from each center) participated in the study. an especially designed questionnaire was used for data collection. results around 61% of women attending focused antenatal care were very satisfied with care, while only 12% of women attending risk approach standard antenatal care were very satisfied with antenatal care (p<0.001). the perceived causes of dissatisfaction included crowding in the clinic in the morning, unfavorable waiting area, and unavailability of daily sonar exam. a significantly higher proportion (p<0.001) of the high level of satisfaction (78%) was reported among women attending focused antenatal care than risk approach standard antenatal care (38%). conclusion women attending focused antenatal care were highly satisfied with services provided to them while those attending risk approach standard antenatal care were less satisfied with services. further improvement of focused antenatal care services in erbil is required. keywords personal satisfaction, pregnancy, risk, health services 305 original focused and risk approach antenatal careamina al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019 : 304–308 verbal informed consent was obtained from each participant before being enrolled in the study. the study was approved by the research ethics committee at the author’s institute. data were analyzed using statistical package for social sciences (spss) version 19. chi-square test of association was used to compare between two proportions. when the expected count of more than 20% of cells of the table was less than 5, fisher’s exact test was used. mann–whitney test was used to compare the mean ranks of satisfaction scores of the two study groups. a p-value ≤0.05 was considered statistically significant. results sociodemographic characteristics of participants the highest proportion of pregnant women was in their third decade of life (59.3%). the mean age ± sd of women attending nazdar bamerni center was 26.39 ± 5.51 while that of women attending brayati center was 26.89 ± 6.02. with regard to occupation, 77% of pregnant women were housewives. there was no statistically significant variation in the sociodemographic characteristic of women attending the two centers with the exception of occupation (p=0.028). details are shown in table 1. satisfaction with different components of anc in general, attending women were highly satisfied with the different components of “clinical” anc services and relatively less satisfied with the other six components of anc. a statically significant variation was demonstrated between the two phc centers in the following items of anc: explanation of complication (p=0.01), immunization of pregnant women (p=0.042), education about breastfeeding (p=0.047), doctors explanation of examination results (p=0.002), clinic infrastructure (p<0.001), attending doctors (p<0.001), attending nurses (p<0.001), waiting time (p<0.001), and laboratory services (p<0.001). details are shown in table 2. perceived causes of dissatisfaction generally, relatively high proportions of women reported dissatisfaction with the presence of unfavorable waiting area and crowding in the clinic in the morning. a statistically significant variation between the two phc centers regarding crowding in the clinic in the morning (p=0.008) and unavailability of daily ultrasound examination (p<0.001) was demonstrated (table 3). suggestions for improvement of anc at phc centers a very high proportion of women in both health care centers reported the need for giving information related to pregnancy during the waiting time. a significantly higher proportion of women attending nazdar bamerni hc center reported the need for avoidance of too much talking between staffs and giving more attention to clients (p<0.001), providing appropriate waiting room (p=0.01), and the need for improvement of behavior of supporting staff (p=0.001). details are shown in table 4. the overall and level of satisfaction with antenatal care around 61% of women attending brayati phc center were very satisfied with care while only 12% of women attending nazdar bamerni phc center were very satisfied with anc (p<0.001). a significantly higher proportion (p<0.001) of the high level of satisfaction (78%) was reported among women attending brayati phc center than nazdar bamerni phc center (38%). details are shown in table 5. discussion assessment of client satisfaction offers a way of optimizing health status and prevents waste of medical resources.11 table 1. sociodemographic characteristics of participants variables nazdar phc brayati phc total no. (%) p-value no. (%) no. (%) age groups (years) <20 15 (10.0) 14 (9.33) 29 (9.7) 0.22 20–29 95 (63.33) 83 (55.33) 178 (59.3) 30–39 39 (26.0) 48 (32.0) 87 (29.0) ≥40 1 (0.67) 5 (3.33) 6 (2.0) occupation housewife 109 (72.66) 122 (81.3) 231 (77.0) 0.028 employed (public or private) 28 (18.66) 25 (16.7) 53 (17.7) others including students 13 (8.66) 3 (2.0) 16 (5.3) educational level no formal education 13 (8.7) 15 (10.0) 28 (9.3) 0.86primary school 44 (29.3) 42 (28.0) 86 (28.7) secondary school 44 (29.3) 49 (32.7) 93 (31.0) higher education* 49 (32.7) 44 (29.3) 93 (31.0) marital status married 150 (100) 149 (99.3) 299 (99.7) 1.00 widowed 0 (0.0) 1 (0.7) 1 (0.3) average monthly family income not enough 56 (37.3) 72 (48.0) 128 (42.7) 0.17 marginally enough 61 (40.7) 46 (30.7) 107 (35.7) enough 27 (18.0) 23 (15.3) 50 (16.7) more than enough 6 ( 4.0) 9 (6.0) 15 (5.0) total 150 (100.0) 150 (100.0) 300 (100.0) * diploma, bachelor & post graduate degrees. 306 original focused and risk approach antenatal care amina al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 304–308 recognition of quality shortcomings in health care in developing countries has motivated new efforts to measure and monitor health service quality via surveys of health care providers and their clients. among these tools, client survey is intended to measure user satisfaction with, or perceptions of, overall service quality or specific aspects of quality.12 anc is the key entry point for a pregnant woman to receive a broad range of health promotion and preventive services which promote the health of the mother and the baby.13 table 2. satisfaction with the different components of antenatal care services component nazdar phcc brayati phcc total no. (%) p-value no. (%) no. (%) explanation of complication yes 137 (91.3) 147 (98.0) 284 (94.7) 0.01 no 13 (8.7) 3 (2.0) 16 (5.3) immunizations of pregnant women yes 144 (96.0) 135 (90.0) 279 (93.0) 0.042 no 6 (4.0) 15 (10.0) 21 (7.0) checking blood pressure and body weight yes 150 (100.0) 150 (100.0) 300 (100.0) fixing next appointment yes 149 (99.3) 150 (100.0) 299 (99.7) 1.00 no 1 (0.7) 0 (0.0) 1 (0.3) education about importance of breast feeding yes 140 (93.3) 147 (98.0) 287 (95.7) 0.047 no 10 (6.7) 3 (2.0) 13 (4.3) instructions about hygiene yes 145 (96.7) 150 (100.0) 295 (98.3) 0.06 no 5 (3.3) 0 (0.0) 5 (1.7) understanding of instructions yes 146 (97.3) 150 (100.0) 296 (98.7) 0.122 no 4 (2.7) 0 (0.0) 4 (1.3) full child immunization yes 74 (49.3) 90 (60.0) 164 (54.7) 0.064 no 76 (50.7) 60 (40.0) 136 (45.3) doctors explanation of examination results yes 138 (92.0) 149 (99.3) 287 (95.7) 0.002 no 12 (8.0) 1 (0.7) 13 (4.3) clinic infrastructure excellent 21 (14.0) 99 (66.0) 120 (40.0) <0.001good 119 (79.3) 50 (33.3) 169 (56.3) poor 10 (6.7) 1 (0.7) 11 (3.7) attending doctors excellent 21 (14.0) 99 (66.0) 120 (40.0) <0.001 good 124 (82.7) 50 (33.3) 174 (58.0) poor 5 (3.3) 1 (0.7) 6 (2.0) attending nurse excellent 21 (14.0) 98 (65.33) 119 (39.6) <0.001 good 102 (68.0) 50 (33.33) 152 (50.6) poor 27 (18.0) 2 (1.33) 29 (9.66) waiting time excellent 20 (13.33) 92 (61.33) 112 (37.3) <0.001good 98 (65.33) 41 (27.33) 139 (46.3) poor 32 (21.33) 17 (11.33) 49 (16.33) laboratory services excellent 21 (14.0) 97 (64.7) 118 (39.3) <0.001good 111 (74.0) 51 (34.0) 162 (54.0) poor 18 (12.0) 2 (1.3) 20 (6.7) pharmacy support excellent 2 (1.33) 2 (1.3) 4 (1.3) 0.1good 89 (59.33) 106 (70.7) 195 (65.0) poor 59 (39.33) 42 (28.0) 101 (33.7) table 3. perceived causes of dissatisfaction causes of dissatisfaction nazdar phcc brayati phcc total no. (%) p-value no. (%) no. (%) poor laboratory services yes 0 (0) 0 (0) 0 (0) no 150 (100) 150 (100) 300 (100.0) crowding in the clinic in the morning yes 107 (71.3) 85 (56.7) 192 (64.0) 0.008 no 43 (28.7) 65 (43.3) 108 (36.0) long waiting time yes 22 (14.7) 16 (10.7) 38 (12.7) 0.298 no 128 (85.3) 134 (89.3) 262 (87.3) not listening to complaints of pregnant women yes 1 (0.7) 0 (0.0) 1 (0.3) 1.00* no 149 (99.3) 150 (100) 299 (99.7) unavailability of daily sonar exam. yes 81 (54.0) 2 (1.3) 83 (27.7) < 0.001 no 69 (46.0) 148 (98.7) 217 (72.3) unfavorable waiting area yes 94 (62.7) 79 (52.7) 173 (57.7) 0.08no 56 (37.3) 71 (47.3) 127 (42.3) total 150 (100.0) 150 (100.0) 300 (100.0) 307 original focused and risk approach antenatal careamina al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019 : 304–308 risk approach is not an efficient or effective strategy for reduction of maternal mortality because risk factors cannot predict the occurrence of complications.6,14 maternal and neonatal health programme promote the concept of focused anc which emphasizes quality over quantity of visits.6 the result of this study revealed that majority of women were in the age group 20–30 years. this result was supported by another study carried out in egypt.16 about satisfaction with different components of anc; in the study, more than 90% of pregnant women attending both hc centers were satisfied with an explanation about complications. this finding is much higher than that revealed in basrah study where nearly 67% of women reported that they were satisfied with an explanation about complications.16 satisfaction with immunization of pregnant women was significantly more reported (96%) in nazdar bamerni phcc. the study in basrah (standard anc) revealed that nearly 88% of women were satisfied with service of immunization of pregnant women.16 majority of women (98%) attending focused anc were satisfied with education about the importance of breastfeeding. there were significant variations between the two phccs regarding the explanation of complications, immunization of pregnant women, and education about the importance of breastfeeding, which indicate that the services provided by brayati phcc were better than nazdar bamerni phcc. a higher proportion (60%) of clients attending focused brayati phcc were satisfied with service of full child immunization. however, nearly 79% of clients in basrah reported they were satisfied with service of full child immunization.16 this means that there is a need for further improvement of child immunization in erbil. doctors explanation of examination results in brayati phcc is better than in nazdar bamerni phcc with highly statistical significant variations between the two phccs; indicating that services provided by brayati phcc are of better quality than those of nazdar bamerni phcc. excellent satisfaction with the clinic, attending doctors, attending nurse, waiting time, and laboratory was reported in brayati phcc, while good satisfaction was reported in nazdar bamerni phcc with highly statistically significant variations between the two phc centers. these findings indicate that women preferred the services provided by brayati phcc, which adopted the focused anc. regarding perceived causes of dissatisfaction, crowding in the clinic in the morning was more reported in nazdar bamerni phcc, around 71% more than in brayati phcc (56.7%) indicating that there is a need to decrease crowding in both phccs in the morning. this finding was supported by other study conducted in bangladesh, 25% of women reported dissatisfaction with crowding in the clinic in the morning.17 nearly 63% of pregnant women from nazdar bamerni phcc and 52.7% from brayati phcc reported unfavorable waiting area. these proportions are much higher than those reported in south ethiopia (4.3%).18 this means that there is a need for improvement of waiting area and increasing waiting space. more than half of women attending nazdar bamerni phcc and only 1.3% from brayati phcc reported unavailability of daily sonar exam. six percent of women from musandam region of oman reported an absence of sonar exam.19 according to suggestions for improvement of anc at phccs, nearly 15% of pregnant women from nazdar bamerni phcc and only 1.3% from brayati reported the need for avoidance of too much talking between staffs and giving more attention to the clients with significant variations between the two phccs. in ethiopia, nearly 12% of women reported avoidance of too much talking between staffs and giving more attention to the client.18 nearly 63% of pregnant women from nazdar bamerni table 4. suggestions for improvement of anc at phc centers suggestions for improvement of anc nazdar phcc brayati phcc total no. (%) p-value no. (%) no. (%) avoidance of too much talking between staffs and giving more attention to clients yes 22 (14.7) 2 (1.3) 24 (8.0) <0.001 no 128 (85.3) 148 (98.7) 276 (92.0) giving information related to pregnancy during waiting time yes 143 (95.3) 140 (93.3) 283 (94.3) 0.45 no 7 (4.7) 10 (6.7) 17 (5.7) improving ventilation system of the clinics and waiting space yes 2 (1.3) 0 (0.0) 2 (0.7) 0.49 * no 148 (98.7) 150 (100.0) 298 (99.3) providing appropriate waiting room (increasing waiting space) yes 94 (62.7) 73 (48.7) 167 (55.7) 0.01 no 56 (33.3) 77 (51.3) 133 (44.3) minimize waiting time yes 24 (16.0) 16 (10.7) 40 (13.3) 0.17 no 126 (84.0) 134 (89.3) 260 (86.7) improvement of behavior of supporting staff yes 29 (19.3) 1 (0.7) 30 (10.0) 0.001 no 121 (80.7) 149 (99.3) 270 (90.0) total 150 (100.0) 150 (100.0) 300 (100.0) table 5. overall and level of satisfaction with anc provided at both phc centers variable nazdar phcc brayati phcc total no. (%) p-value no. (%) no. (%) overall satisfaction not satisfied 20 (13.3) 3 (2.0) 23 (7.67) <0.001satisfied 112 (74.7) 55 (36.7) 167 (55.66) very satisfied 18 (12.0) 92 (61.3) 110 (36.67) satisfaction level1 low2 93 (62.0) 33 (22.0) 126 (42.0) <0.001high3 57 (38.0) 117(78.0) 174 (58.0) total 150 (100.0) 150 (100.0) 300 (100.0) 1calculation of scores depend on median of total scores (median=24) 2low level of satisfaction: <median of total scores 3high level of satisfaction: ≥median of total scores 308 original focused and risk approach antenatal care amina al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 304–308 phcc and nearly 49% from brayati asked for provision of the appropriate waiting room (increased waiting space) with significant variations between the two phccs. in ethiopia, 37% of women suggested providing an appropriate room (increase waiting space).18 around 19% of clients from nazdar bamerni phcc and only 0.7% of women from brayati phcc reported the need for improvement of behavior of support staff with higher statistical significant variations between the two phccs. nearly 9% of women suggested improvement of supporting staff in south-west ethiopia.18 the proportions of all suggestions of women were higher in nazdar bamerni phcc than brayati which mean that the services of focused anc are better than risk approach of anc. regarding the overall satisfaction with anc provided at both phccs as perceived by clients, around 61% of women attending brayati phcc were «very satisfied» with care while 74.7% of women from nazdar bamerni phcc were «satisfied» with highly statistically significant variations between the two phccs. the satisfaction with focused anc in the present study is higher than that reported in malaysia (56.7%).20 the overall satisfaction with anc provided at both phccs as perceived by clients was further incorporated by calculating the level of satisfaction by a median of total scores and mean of total scores. women attending brayati phcc reported a high rate and high level of satisfaction and high mean scores of satisfaction than nazdar bamerni phcc, which means the services provided by brayati phcc better than nazdar bamerni phcc. conclusion women attending focused anc (brayati phcc) are highly satisfied with services provided to them while those attending risk approach standard anc (nazdar bamerni phcc) are less satisfied with the services. services provided by brayati phcc were better than those provided by nazdar bamerni phcc. perceived causes of dissatisfaction, crowding in the clinic in the morning, unfavorable waiting area, and unavailability of daily sonar exam. further improvement of focused anc services in erbil is required. there should be an emphasis on child immunization in both hccs. there is a need to decrease crowding in both hccs in the morning. further improvement of waiting area and an increase in the waiting space in both phccs is needed. provision of sonar exam in all days of the week is important, avoidance of too much talking to each other, and giving more attention to the clients and behavior improvement of supporting staff in nazdar bamerni phcc. conflicts of interest the authors report no conflicts of interest. references 1. moos m. prenatal care: limitations and opportunities. j obstet gynecol neonatal nurs. 2006;35:278–285. 2. cohen jr. patient satisfaction with prenatal care provider and the risk of cesarean delivery. am j obstet gynecol. 2005;192:2029–2034. 3. turan jm, bulut a, nalbant h, ortayli n, akalin aa. the quality of hospital based antenatal care in istanbul. stud fam plann. 2006;37:49–60. 4. debono d, travaglia j. complaints and patient satisfaction: a comprehensive review of the literature. sydney (australia): university of new south wales, centre for clinical governance research in health, 2009. 5. ivanov ll, flynn bc. utilization and satisfaction with prenatal care services. west j nurs res. 1999;2:372–386. 6. rooney cif. antenatal care and maternal health: how effective is it? document who/msm/92a. geneva: who, 1992. 7. jhpiego trainer news. focused antenatal care, planning and providing care during pregnancy a maternal and neonatal health program best practice. reprod health online, 2001. 8. maine d. safe motherhood program: options and issues, new york (usa): centre for population and family health columbia university, 1991. 9. maternal neonatal health: focused antenatal care: planning and providing care during pregnancy, program brief, mnh program website; 2004 [online]. available at: www.mnh.jhpiego.org. accessed november 1, 2018. 10. pascoe gc. patient satisfaction in primary health care: a literature review and analysis. eval prog plan. 1983;6:185–210. 11. bu-alayyan s, mostafa a, al-etaibi b, sorkhou e, al-taher h, al-weqayyan a. patient satisfaction with primary health care services in kuwait. kuwait med j. 2008;40:25–30. 12. glick p. how reliable are surveys of client satisfaction with healthcare services? evidence from matched facility and household data in madagascar. soc sci med. 2009;68:368–379. 13. mgawadere fm. assessing the quality of antenatal care at lungwena health centre in rural malawi. unpublished master’s thesis, university of malawi, college of medicine, 2009. 14. maine d. safe motherhood program: options and issues, new york (usa): centre for population and family health columbia university, 1991. 15. montasser na, helal rm, megahed wm, amin sk, saad am, ibrahim tr, et al. egyptian women’s satisfaction and perception of antenatal care. int j trop dis health. 2012;2:145–156. 16. al-wafa society. client exit survey on satisfaction with primary health care services and perception of antenatal care and child care in basrah, iraq, provided through the i-help small grants program managed by abt associates inc. and funded by usaid through contract no. ran-c-00-03-00010-00, 2004. 17. hasan a: patient satisfaction with maternal and child health services. dhaka. mahidol university, 2007. 18. chemir f, alemseged f, workneh d. satisfaction with focused antenatal care service and associated factors among pregnant women attending focused antenatal care at health centers in jimma town, jimma zone, south west ethiopia. bmc res notes. 2014;7(1):164. 19. ghobashi m, khandekar r. satisfaction among expectant mothers with antenatal care services in the musandam region of oman. sultan qaboos univ med j. 2008;8:325–332. 20. pitaloka d, rizal am: patients’ satisfaction in antenatal clinic hospital university kembangaan. malay j commun health. 2006;12:8-16. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201902 journal of contemporary medical sciences in the first years of the journal life the journal of contemporary medical sciences (jcms) acquired high successful rate and more researchers to publish with it. we thank all the researchers and subscribers who supported this magazine. our journal deals with different subjects of basic and clinical medical sciences. it is an open access, peer-reviewed journal which has been accepted both nationally and internationally and encourages the researchers to publish with this journal. this journal addresses four major aspects of writing journal-style scientific papers: (1) fundamental style considerations,  (2)  a suggested strategy for efficiently writing up research results, (3) the nuts and bolts of format and content of each section of a paper (describes how to follow instructions precisely to write a scientific paper), and (4) basic information regarding peer critiques of scientific writing. editor-in-chief prof. dr. abbas matrood bashi college of applied medical sciences university of kerbala-iraq editorial note 1 286 j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 minimally invasive surgery research center, iran university of medical sciences, tehran, iran. the prevalence of non-alcoholic fatty liver disease (nafld) and non-alcoholic steatohepatitis (nash) is increasing due to increasing of morbid obesity prevalence worldwide. it has been demonstrated that weight loss is the corner stone of nafld/nash treatment. various options are available for weight loss in obese patients, such as life style modification, pharmacotherapy, endoscopic interventions and bariatric/metabolic surgery. performing bariatric/meta bolic surgery only for nafld/nash treatment is on debate, but it has been proven that bar iatric surgery is the most durable and effective treatment for weight loss and obesity-related comorbidities. keywords bariatric surgery, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, weight loss, obesity, insulin resistance, lifestyle introduction because of high fat source diet and sedentary life style which is more common recent years obesity and morbid obese population has been rapidly increased.1 according to national health and examination survey, the obesity prevalence between 20 to 74 years has been risen from 15 to 32.9%.2 visceral fat accumulation in liver tissue named non alcoholic fatty liver disease (nafld), known as a component of metabolic syndrome includes type 2 diabetes, hypertension and dyslipidemia represented insulin resistance (ir).3 nafld that is considered the liver phenotype of metabolic syndrome is the most common cause of liver dysfunction and with a prevalence of 6.3–33% worldwide.1 the first sign of nafld is accumulation of hepatic fat (steatosis) that progress to non-alcoholic steatohepatitis (nash), liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma.4 current nafld and nash treatments include weight loss obtained by life style modification and pharmaceuticals but have poor effects in patients with body mass index >35 kg/m2.5 some evidences showed bariatric surgery (bs) could alter the course of disease.6 now a days weight loss surgery considered unique method to obtain durable and permanent weight loss in morbid obesity obese could reverse metabolic syndrome consequences as well as fatty liver disease, improve liver histopathology and improve insulin resistance (ir).7 according to recent evidences we search database to approve our thesis on positive impact of bs for reversing negative metabolic syndrome effects of liver function and histopathology. methods we searched pubmed, scopus and google scholar with these at nov 1, 2016. we found 772 papers. after deleting duplicate papers in searches (429 papers), finally 518 papers remained. 346 articles were related to our subject based on their titles and abstracts. we used only papers that their full-text was related to our title. results prevalence of nafld and nash in bariatric candidates nafld, as an emerging clinical implication, is most closely associated with obesity.8 the prevalence of obesity, as a medical and societal problem, is increasing around the world, even in developing countries.8,9 morbid obesity (body mass index (bmi) > 40 kg⁄m2) is treated with the suggested weight loss interventions such as bs.9 nafld prevalence is much higher in obese (bmi > 30 kg⁄m2) patients.9 a prospective cohort study indicated that 70% of the patients who were obese and candidate for bs, presented steatosis, a liver damage pattern of nafld.10 however, results from different studies show that the prevalence of nafld varies between 16.7% and 96% in morbidly obese patients subjected to bariatric surgery.10–22 the wide range of nafld prevalence depends on the biochemical criteria and the method sensitivity used to detect nafld.8,23 furthermore, nash, as a progressive manifestation of nafld, occurs in 7.3% to 98% of such patients.10,12,13,17,18,20–22,24–30 the study which reported nash in 98% of their patients, indicated that the mean super obese state (mean bmi 52.8 kg ⁄m2) in their population is the reason of high nash prevalence.30 another study concluded that the differences in nash prevalence can be explained by the histological criteria used to diagnose nash.22 ethnicity affects nafld prevalence as well.23 in a study on morbid obese patients underwent bs, african-american patients had lower nafld and nash rate than nonhispanic whites and hispanics. non-hispanic white and hispanic patients had similar nafld prevalence. they suggested ethnic differences in fat distribution as an explanation for lower nash rate in african-americans.31 bariatric surgery and metabolic syndrome metabolic syndrome is characterized by central obesity, hypertriglyceridemia, low high density lipoprotein cholesterol (hdl-c), high blood pressure and high fasting plasma glucose.32 issn 2413-0516 review a b center of excellence of european branch of international federation for surgery of obesity, tehran, iran. c medical student research committee, iran university of medical sciences, tehran, iran. correspondence to: ali kabir (e-mail: aikabir@yahoo.com). (submitted: 04 july 2017 – revised version received: 22 july 2017 – accepted: 10 september 2017 – published online: 26 december 2017) role of bariatric surgery in treatment of non-alcoholic fatty liver disease mohammad kermansaravi,a,b mohammad ebrahimian,c delaram delbari,c simin khamoushi,c and ali kabira mohammad kermansaravi et al. 287j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 research role of bariatric surgery in treatment of non-alcoholic fatty liver disease bs is considered a safe and efficient treatment of metabolic syndrome.8 in a study of 46 morbid obese patients subjected to laparoscopic gastric bypass, metabolic syndrome and some of its components including hypertension, dysglycemia and dyslipidemia and ir were resolved after 2 years.33 similar results were observed in a study of 827 patients who underwent laparoscopic roux-en-y gastric bypass (rygb) with 4 years follow ups.34 however, improvement of metabolic syndrome is most likely associated with the amount of excess weight loss.35 insulin resistance ir is considered to be the hallmark of metabolic syndrome.36 the measure of insulin sensitivity indices has shown a reduced mean insulin sensitivity in nonobese, non-diabetic young subjects with nash in association with the different components of the metabolic syndrome. a small study of nondiabetic severely obese patients revealed that biliopancreatic diversion (bpd) improves insulin sensitivity four days after the operation. they suggested that this sharp improvement might be explained by the intramyocellular fat depletion and enteroinsular axis interruption.37 in another study, patients who underwent gastric bypass surgery (bmi approximately 30 kg/m2) had greater insulin sensitivity than weight-matched group (bmi = 25 to 35 kg/m2).38 similarly, klein et al. demonstrated that gastric bypass (gbp) induced weight loss markedly improves hepatic insulin sensitivity and metabolic abnormalities related to nafld. a decrease in intrahepatic free fatty acid (ffa) availability, caused by decreased release of ffa from intrahepatic, visceral, and subcutaneous fat is considered to be the probable explanation for marked improvement in insulin action.39 association between nafld and type 2 diabetes and the role of bariatric surgery a study on patients who underwent jejunoileal bypass (jib) found a decrease in fasting blood glucose and improvement in type 2 diabetes after jib. in their study, nafld was observed in 86.7% of patients and nash in 31.7%.40 in a retrospective study, 88.7% of patients who underwent bs and did not have diabetes and hypertension, showed liver steatosis. 7.3% of these individuals had nash and 19.3% had liver fibrosis. furthermore, they found liver steatosis in 96%, nash in 22% and liver fibrosis in 30.6% in the presence of diabetes and hypertension.20 in a study of 112 morbid obese patients underwent bariatric surgery, 57.7% of patients presented nash. in these patients, type 2 diabetes was significantly (p value = 0.018) associated with the nafld type 4 which is considered as nash.29 bariatric surgery for treatment of nafld/nash in pediatrics and adolescents a recent study on obese adolescents who underwent laparoscopic sleeve gastrectomy (sg) found complete improvement of nash in all patients and reversed hepatic fibrosis stage 2 in 90%.41 the role of obstructive sleep apnea (osa) in nafld and its improvement following bariatric surgery in a study by cottam et al., patients with fatty liver disease and comorbid conditions underwent a two stage approach; laparoscopic sg as the first stage and laparoscopic rygb as the second one. all cases of diabetes had improvement prior to the second stage and almost all patients with sleep apnea had resolution or improvement. their study demonstrates that proceeding directly with a rygb probably is not a safe option.42 a cohort study by benotti et al. showed that the severity of osa is associated with the nafld severity in severe obese patients without metabolic syndrome. they concluded that the intermittent hypoxia followed by the osa severity may be linked with the pathogenesis of nafld.43 indications of bariatric surgery in nafld/nash nash/nafld as an independent indication for bariatric/ metabolic surgery is controversial.15 based of new statements of ifso (international federation for the surgery of obesity and metabolic disorders), bariatric surgery leads to improvement and resolution of nafld and nash in morbid obese patients.44 because obesity, diabetes mellitus and hyperlipidemia (hlp) are among the risk factors for the nafld progression, so, weight loss induced surgery, improves these comorbidities together with nafld.45 actually, the indications of surgery in nafld/nash are the same as in morbid obesity and are bmi equal or more than 40, or 35 < bmi < 40 with any evidence of metabolic syndrome or related comorbidities.5,46 also bariatric surgery is indicated in overweight patients with medical-resistant severe metabolic complications.46 types of bariatric surgeries there are three different types of bariatric surgery:8,47–49 1. restrictive procedures that create a state of satiety and decrease the gastric capacity and including: a. sleeve gastrectomy (sg): creation of a lesser curve based narrow gastric tube, on a sizer bougie from 4–6 cm of pylorus up to gastroesophageal junction and resection of about 80% of stomach. b. adjustable gastric banding (agb): placement of an adjustable band around the upper part of stomach to create a small pouch. c. vertical banded gastroplasty (vbg): creation of stapled upper part of stomach in lesser curvature that is supported by an extra luminal band. 2. malabsorptive procedures, such as biliopancreatic diversion with or without duodenal switch (bpd-ds), that create a near 200 cc tube of stomach like sg, but preserves the pylorus and cut the duodenum 3–4 cm after pylorus and then create a roux-en-y dudenojejunostomy with short common limb. these procedures alter the food and calorie intake and absorption. 3. combined or mixed procedures such as roux-en-y gastric bypass (rygb), that create a stapled small proximal gastric pouch with well-nigh 30 cc capacity that is anastomosed with two limbs of jejunum in ry fashion. comparison the effectiveness of different methods of bariatric surgery in nash/nafld in general, bariatric surgery leads to weight loss, decreasing of transaminases and improvement of nafld.50 new studies show that bariatric surgery can improve the histologic features of steatosis, hepatocyte ballooning and lobular inflammation, but it effect in liver fibrosis is on debate.51 288 j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 role of bariatric surgery in treatment of non-alcoholic fatty liver disease research mohammad kermansaravi et al. bariatric surgery can also lead to significant improvement of liver function but if initial weight loss is too much and bmi loss is greater than 80%, can worsen the liver function that is usually improved after one year after surgery.25 continue to investigate the effect of two more common surgical methods throughout the world and comparing them with other relatively conventional methods described above. 1. rygb: this method of surgery stimulates glp-1 secretion that leads to significant improvement of some obesity-related syndromes, such as ir, inflammation and steatosis, that steatosis and also fibrosis improvement is due to decreasing of fat droplet accumulation in the liver and improvement of mitochondrial function.52 also rygb can correct the bile secretion derangement.53 the pyy (peptide yy) hormone secretion is increased and ghrelin hormone is decreased in this method and along with glp-1 secretion induces the satiety state.54 there are some evidences that confirm a strong association between liver fat content reduction and ir correction in rygb even before remarkable weight loss.55 víctor vargas et al. performed one liver biopsy during rygb and another biopsy in percutaneous method, 12–22 months after surgery. they found significant histopathologic improvement in liver steatosis, ballooning degeneration, portal inflammation and fibrosis that were statistically significant.48 joshua s winder et al, in a study in usa, reported nafld improvement based on computed tomography (ct) scan, in 84.2% of patients after lrygb.56 another study that is done by jeanne m. clark, et al., reported improvement of all morphologic indicators of nafld after rygb and 81% of patients had complete remission of liver steatosis and there were no worsening of these indicators.57 carlos k furuya jr, et al., in a study performed percutaneous liver biopsy, 2 years after rygb and reported significant improvement of steatosis (89%), liver fibrosis (75%) and hepatocellular ballooning (50%) after surgery.7 everton cazzo et al., found improvement of advanced liver fibrosis in 55% of patients, one year after lrygb that were statistically correlated with female gender, ewl percentage, post op bmi, post op platelet and improvement of t2dm.58 kevin b. barker et al., in their study, resulted in significant improvement of steatosis, lobular inflammation and periportal fibrosis and improvement of nash in 89% of patients after rygb. 59 the study of xiuli liu et al., liver biopsy, showed significant improvement in macro steatosis (97%), micro steatosis (87%), hepatocellular ballooning (100%) and regression of liver fibrosis, about 18 months after rygb and notified that rapid weight loss, not only not lead to worsening of hepatocellular injuries and progression of fibrosis, but also can lead to improvement of steatosis.60 soraya rodrigues de almeida, et al, in a study, notified complete improvement of nafld in 93.7% and lobular inflammation in 100% of patients after nearly 23.5 months after rygb.61 2. sleeve gastrectomy (sg): sleeve gastrectomy is another effective method for weight loss and nafld improvement. in a retrospective study, ardeshir algooneh et al., reported complete remission of nafld in 56% of patients and had a strong correlation with post op bmi, that most of nafld remissions were occurred in post op bmi: 25–30.62 melania manco et al., found 100% improvement of nash in adolescents, one year after sg and in their study, sg was most effective than lifestyle change and intragastric weight loss device.41 another study resulted in improvement of liver steatosis (66.6%), fibrosis (68%) and nafld activity score, 3 months after sg.63 comparing sg with rygb a recent study, in 35 patients with t2dm and bmi > 35, demonstrated that after 12 months after surgery, sg was significantly most effective on lfts than rygb (p: 0.007), also had better effect on nafld improvement, in spite of the fact that glycemic control were the same.64 another study displayed better effect of alanine aminotransferase (alt) improvement after sg in comparison with rygb, but more impact for rygb in liver fibrosis and nafld regression.65 comparing rygb and lagb in a study in france, who performed in 109 patients, the investigators found that rygb resulted in more statistically significant effect in weight loss and nash improvement in comparison with laparoscopic adjustable banding (lagb).46 another study in 1236 patients, between 1996 to 2012, was done with one and five years follow ups, disclosed that rygb had significantly better effects on nafld and liver histology that lagb.66 other surgical methods vbg charalabos stratopoulos, et al., in a study in 51 patients, that underwent vbg, with liver biopsy, 18 months after surgery demonstrated improvement of steatohepatitis un 86.2% patients, but unchanged liver fibrosis in 41.1% of cases and worsening of liver fibrosis in 11.7% of patients, however they didn’t see any progression to cirrhosis.30 duodenal switch (ds) ara keshishian et al., in a study showed that ds lead to improvement of liver steatosis and inflammation, but the patients had a transient worsening in aspartate aminotransferase (ast) and alt levels, in 6 month after surgery that became normal after one year. in this study after 3 years follow up, the patient had progressive improvement of nash.67 complications of bariatric surgery general complications different degrees of deficiencies in iron, folate, vitamins and trace elements are seen in all bariatric surgeries.68 gastric and anastomosis ulcers, bile reflux, dumping syndrome, adhesions, nausea and vomiting, wound infection, pulmonary emboli, abscess, inadequate weight loss and weight regain, may be present in many of bariatric procedures.69 also in some patients that have poor response to bariatric surgery, mild increasing in liver fibrosis may occur.70 mohammad kermansaravi et al. 289j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 research role of bariatric surgery in treatment of non-alcoholic fatty liver disease procedure specific complications 1. lagb: band slippage, band erosion, band migration, gastric pouch dilatation and malfunction of band’s port and tube are some of lagb specific complications.49 2. sg: bleeding, leak, gerd, antral stenosis, functional stenosis, portal thrombosis and renal failure are some of laparoscopic sleeve gastrectomy (lsg) related complications.42,49,71 3. bpd/ds: gi obstruction, leak, peptic ulcer disease, protein malnutrition, fat soluble vitamin deficiencies, nutritional derangements, transient worsening of nash, worsening of nafld and liver histology due to severe weight loss and risk of bacterial stasis and overgrowth due to blind loop syndrome are specific complications of bpd/ds.46,48,49,67,68,72 4. rygb: gi obstruction due to internal hernia, leak, malnutrition, bleeding and leak are some specific complications of rygb.49 potential mechanisms of bariatric surgery in nafld/nash treatment 1. improvement of ir: weight loss, reduction of visceral lipids, increasing of glp-1, improvement of bile acid metabolism 2. improvement of dyslipidemia: decreasing of ldl, hdl, tc and increasing of hdl 3. alterations of gut hormones: decreasing of ghrelin and increasing of glp-1, pyy, oxyntomodulin and bile acids 4. decrease of inflammation: decreasing of interleukins 1 and 8, crp and tnf-α 5. decrease of leptin and increase of adiponectin 6. decrease of hepatocellular apoptosis and oxidative stress of endoplasmic reticulum 7. weight loss and bmi loss: improvement of these major risk factors for nafld/nash 8. desirable modifications in gut microflora22,45,47,49,55,73–76 selecting the most suitable bariatric procedure in treatment of nash/nafld weight loss is a mainstay and gold standard method for nafld/nash treatment.5,62 bariatric surgery procedures as other weight loss programs are effective in improvement of nash/nafld, but are significantly most effective than non-surgical procedures such as, medical drugs and life style modifications.41,77,78 till now, the best surgical procedure for nafld/nash is not determined and meta analyses, show that all bariatric surgical procedures lead to nafld/nafld improvement in obese patients and liver steatosis in most of this patients (near 90%).47,64,70,79 recent studies demonstrated that bariatric surgery; result in improvement of biochemical and histologic parameters of liver, such as non-alcoholic steatosis and steatohepatitis, liver fibrosis, lobular inflammations, chronic portal inflammations and even hepatocellular carcinoma.47,55,62,79,80 selection of bariatric surgery procedure must be done based on other conditions and indications and also need to have regular and punctual follow up after surgery.81 in confirmed nafld or patients that are at risk of advanced nafld, as possible, must avoid of malabsorptive procedures such as bpd/ds due to rapid and severe weight loss that result in ffa rapid releasing and transfer of long chain fatty acids to liver and bacterial over growth that can lead to worsening of nafld.5,48,72 long-term effects of bariatric surgery in nafld/ nash treatment it seems that, bariatric surgery has durable and long standing effects in nafld/nash; however, it needs to perform more related studies with longer follow ups. anne-sophie schneck et al., performed a study that had a mean 55 month follow up after rygb, and resulted in durable improvement of liver injury, hepatocytes apoptosis, steatosis, inflammation, nafld activity score (nas) and liver fibrosis in 88% of patients till that time of follow up.82 swedish obese subjects (sos) study, that was done in retrospective fashion in a total of 3,570 obese participants, that were divided in two surgical(agb, vbg, bypass) and non-surgical groups, with 10 years follow ups, demonstrated improvement of transaminase levels only in surgical group that had positive correlation with weight loss. this study resulted that the short-term effects of bariatric surgery are durable and persistent in long time after surgery and have long standing protective effect against chronic liver injuries.50 non-surgical treatment (endoscopic) of nafld/ nash instead of bariatric surgery with different complications, there is an option to choose less invasive gadgets like endoscopic therapies with lower cost and risks55 including the endoscopic duodenal–jejunal bypass liner (djbl),83 intragastric balloon therapy,55 and the bioenterics intra gastric balloon (bib) are present. djbl first motivation was to treat obesity, however, it turned out that it also leads to:83 1. significant weight loss 2. improvement of type 2 diabetes 3. decrease of plasma liver parameters (normalizing of ast, alt, andg-gt and caspasecleaved ck-18) which indicates regression of nash genco a, showed that bib reduces bmi84 and improvement of metabolic profile85 even though there hasn’t been any researches done on nafld status. it could also be used in patients with lower levels of obesity.49,86 response to bariatric surgery (biopsy, mri, ct) biopsy biopsy as the gold standard10 has different complications, including bleeding, pain, hypotension, hemorrhage, bile peritonitis, pneumothorax, hemothorax, transient bacteremia, hemobilia, tumor seeding and death.87 the size of the biopsy obtained can be a better representive of liver tissue.88 computer tomography suitable for steatosis but not for visualizing inflammation of liver and early stages of fibrosis.10 since weight of scanner could be a limitation, ct might not be achievable.25 mri able to detect hepatic triglyceride (hepatic fat), concentration in nafld and monitor liver steatosis, as non-invasive 290 j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 role of bariatric surgery in treatment of non-alcoholic fatty liver disease research mohammad kermansaravi et al. technique following bariatric surgery (bs), after one year a decrease in liver fat was seen.89 limax test it can determine capacity of enzymatic liver function based on hepatic 13 c-methacetin metabolism by the cytochrome p450 1a2 system. repetitive testing will monitor disease course and response to bs.25 transient hepatic elastrography fibrosis of liver has a significant relation with elasticity that is detected by this new method.25 effectiveness of surgical and non-surgical ways of weight reduction in nafld treatment before any recommendations, since weight loss should be included as background therapy in every protocol for nafld treatment.90 bariatric surgery (bs): as average, 12 months after bs, bmi decreased to 11.9 kg/m and nash was cleared in 85% of patients. life style alteration: a healthy lifestyle results in improvement of nash and macroinflammation91 also a reduction in oxidative stress.92 in a cuban population a 5% reduction of weight and 25% resolution of nash was seen.93 in a study done by st george patients were randomized in three groups moderate (6 sessions), low (3 sessions) intensity and control, a remarkable decrease of liver enzymes were seen in moderate intensity alteration94 but the stage of fibrosis is yet to be experimented. •   dietary  limitation:  by  mri  measurement  every  5%  decre ase in bmi equals 25% in liver fat induced by hypocaloric diet.90 •    physical  activity:  day  to  day  used  calories  by  physical  activity are restricted compared with what is achieved by dietary limitation. when it is integrated with diet from the starting point of behavior treatment, it might have more favorable outcomes,90 such as reduction of hepatic steatosis, liver lipids and improvement of insulin resistance.95 •   behavioral therapy. it is burdensome for the greater part of individuals to change their life style, including their diet and physical activity.90 weight regain is not uncommon after medical treatment. lifestyle modifications when combined with pharmacotherapy have more favorable outcomes.96 probiotics as regulators for energy hemostasis are associated with better liver enzyme status and histology.97 •  statins can be used in patients with dyslipidemia and  nafld to reduce steatosis.98 •  orlistat:  it  decreases  bmi,  but  has  gastrointestinal  side effects like, flatus and diarrhea.99 •  pufa: improves both liver stestosis and biochemical  features of nafld.100 •  vitamin e: it is not recommended in diabetic patients  or those with cirrhosis, it decreases the activity of hepatic profibrogenic actions.101 what lies ahead of bariatric surgery in nafld patients? several retrospective and prospective cohort studies has been done to clarify the optimal surgical treatment, nevertheless the gap that requires filling, is the randomized clinical trial (rct) part. since nowadays bariatric surgery is used as a new treatment for nafld in obese patients, we need more longterm evidences (10 year outcomes), to find the most suitable procedure and estimation of its costs. nutrient deficiency and rapid weight loss? a negative factor of bs, how to prevent? type of surgical procedure, pre operative deficiencies, continuous post operative vomiting, modified eating habits and food intolerance could have direct relation with the risk of nutritional deficiency after bs, so, patients should be monitored for rapid weight loss after bs especially with alarm symptoms such as: nausea, vomiting, and abdominal pain.102–104 also vitamins and supplement malabsorption, can lead to nutritional deficiencies.105 anemia due to iron, folic acid and vitamins b12, c and d (50–80% of bs patients)106 deficiencies107 and iron deficiency which is common after gastric bypass.108 there are many concerns that rapid weight loss, may have a role in worsening of nafld,102,109 however mathorin reported that in 95.7% of patients fibrosis score was not higher than 1.110 to prevent the post operative effects and improve long-term outcomes of bs, it is obligatory to: 1. screening of nutritional state 2. prevention of nutrient deficiencies by prescribing appropriate supplementation, in pre and post-surgical stages.106 alternatives managing patients with nafld contains treatment of liver disease and metabolic comorbidities, that focuses on weight loss which are achieved through hypocaloric diet or increased physical activity.111 also daily usage of vitamin e improves histology of liver in non-diabetic adults with biopsy -proven nash, therefore it is not recommended for patients with nash cirrhosis, or cryptogenic cirrhosis.112 unfortunately, there is inadequate evidences to support metformin, thiazolidinediones, bile acids, or antioxidant supplements for nafld treatment. bariatric surgery and long-term effect on liver function tests •  alt,  ast,  caspase-cleaved  ck-18  and  ggt  decreased  3 months after duodenal–jejunal bypass and only permanent reduction in alt and ggt was seen in 6 months later.83 •  intragastric  balloon  and  minimum  weight  loss  of  10%  decrease alt and gamma-glutamyl transferase (ggt) in a study by ricci et al.3 •  patient  undergoing  bpd-ds  and  rygb  after  3  years  showed sustained reduction in bmi, alt, and ggt. but decrease in platelet counts only occur in bpd-ds group which reflects decrease in liver fat content related inflammation and lower secondary thrombocytosis.113 •  significant reduction in alt and ast seen in vergas et al.  study by restrictive malabsorptive procedures.48 •  preoperative ast levels and excess weight loss were associated with improvement in nash grade score. there was no significant difference in improvement in stage between the restrictive and gastric bypass group. but there was a trend for better results in the bypass group.114 •  significant  improvement  in  nash  was  detected  by  the  enzymatic capacity of cytochrome p450 1a2.25 mohammad kermansaravi et al. 291j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 research role of bariatric surgery in treatment of non-alcoholic fatty liver disease •  short-term  worsening  of  ast  and  alt  levels  at  first  6 months after duodenal switch operation seen in keshishian et al. study which normalize in 6 months later.67 changes of liver histopathology after bariatric surgery •  overall improvement in nafld aspects was reported after  bs also improvement in nash and its related parameters116 but portal abnormality remains unchanged in dixon et al. study.115 •  restrictive  mildly  malabsorptive  (gastric  bypass)  procedures caused improvement in steatosis, ballooning degeneration, mallory bodies, glycogen nuclei, lobular inflam mation, portal inflammation, and fibrosis. in a study 5 of 26 patient fibrosis persisted and 15.3% of them had nash in second biopsy.48 •  after  2  years  of  rygb  and  weight  loss  of  60%  steatosis  and fibrosis disappeared with no worsening and liver biochemical markers found within normal.7 •  rygb improves lobular inflammation, portal, and lobular  fibrosis in barker et al. study. there were no significant differences in serum aminotransferase levels. two of nineteen patients still had histopathological features of nash. one had no significant difference in portal inflammation evidence of cirrhosis and had two points improvement in steatosis and inflammation scores, the second still had evidence of mild nash. this case had weight loss of 54 and 55 kg, respectively, over 2 year period prior to followup biopsy.59 •  excess weight loss of 66% as a result of vbg leads to steatohepatitis improvement which correlated with reduction of bmi and alt. although overall decrease in fibrosis, 41% didn’t change in fibrosis and 11.7% increase in fibrosis scores.30 •  major improvements in lobular steatosis, necroinflammatory changes, and fibrosis after laparoscopic adjustable gastric band placement in dixon et al. occurred. in this study, no progression of histological parameters occurred.115 •  lrygb  significantly  improves  the  overall  centrilobular/  perisinusoidal fibrosis scores and also induces regression of centrilobular/perisinusoidal and stage of fibrosis in nash. however, no changes were noted in fibrosis or inflammation in the portal area after rapid weight loss after lrygb and no worsening of hepatocellular injury or fibrosis.22,57,61,114 •  de almedia et al. observed 1 complete remission, 1 fibrosis  improvement and 2 cases without any changes in initial liver biopsy among 4 patients presenting fibrosis preoperatively.61 •  in study of mathurin et al. with five years follow up almost  all patients had low levels of nafld. slightly increase in fibrosis and long-term improvements in steatosis and ballooning that predicted by ir levels was seen.117 •  lobular fibrosis scores had two stages improvement in 2/19  patients and one stage in 8/19 patients. only in one patient lobular fibrosis get worse.59 •  hepatic left lobe volume as hepatic fat indicator reduces  after weight loss achieved by lagb.118 •  duodenojejunal  bypass  surgery  in  rats  nourished  by  western diet normalized serum triglyceride (tg) and attenuated accumulation of tg and steatosis in the liver of.4 impact of bariatric surgery on inflammation markers •  serum  cd163  levels  increase  with  severity  of  nafld  and reduce after weight loss even in severe liver injury indicating reversibility of macrophage activation.119 •  soluble  cd14  receptor  (scd14)  and  human  neutrophil  alpha-defensins (hnds) known as participants in hepatic necro inflammation process reduce following bilio pancreatic diversion markedly correlated with improvement in nas score.120 •  fibrogenesis  regulating  factors  and  regulating  inflammation markers which observed one year after gastric bypass such as hepatic collagen-1(i), tgf-β1, α-sma, and timp-1 expression, hepatic α-sma, hepatic expression of chemokines, mcp-1, and il-8 declined. at this time marked reduction in steatosis but no change in histologic assessments of inflammation and fibrosis was noted.39 •  three protein peaks observed in obese serum belongs to  hemoglobin subunits increased significantly based on severity of liver lesions (steatosis and nash) returned to normal values after bariatric surgery. none of them was correlated with metabolic parameters or lfts.10 conclusion the pravalence of nafld is increasing along the prevalence of morbid obesity worldwide and may leads to nash in many patients. it is demonstrated that weight loss is the main treatment for nafld/nash. there are some options to achieve weight loss and nafld improvement, such as life style modification, pharmacotherapy, endoscopic management and bariatric/metabolic surgery. although it is controversial to perform bariatric/metabolic surgery only for nafld/nash, but it is proven that bariatric surgery is the most effective and durable treatment for weight loss and obesity-related comorbidities, also there are some evidences that bariatric surgery lead to decreasing of liver fat content and ir and improvement of metabolic syndrome, even before significant weight loss. other studies suggest that improvement of metabolic syndrome after bariatric surgery has correlation with amount of weight loss. also bariatric surgery with other mechanims, such as altration in gut hormones, changes in gut microflora, reduction of inflammatory state and decresing of hepatocellular apoptosis, potentially can improve the metabolic syndrome, nafld/nash and liver fibrosis. however, due to the lack of strong clinical trials and long follow-ups to evaluate the effect of bariatric surgery on nafld/nash, choose of bariatric surgery and the best surgical procedure must be selected based on other indications and it needs to perform more clinical trials and cohort studies with long-term follow-ups. 292 j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 role of bariatric surgery in treatment of non-alcoholic fatty liver disease research mohammad kermansaravi et al. 25. alizai ph, wendl j, roeth aa, klink cd, luedde t, steinhoff i, et al. functional liver recovery after bariatric surgery—a prospective cohort study with the limax test. obes surg. 2015;25:2047–2053. 26. shalhub s, parsee a, gallagher sf, haines kl, willkomm c, brantley sg, et al. the importance of routine liver biopsy in diagnosing nonalcoholic steatohepatitis in bariatric patients. obes surg. 2004;14:54–59. 27. arun j, jhala n, lazenby aj, clements r, abrams ga. influence of liver biopsy heterogeneity and diagnosis of nonalcoholic steatohepatitis in subjects undergoing gastric bypass. obes surg. 2007;17:155–161. 28. schild bz, santos ln, alves mk. [nonalcoholic fatty liver disease and its association with metabolic syndrome in the preoperative period in patients undergoing bariatric surgery]. rev assoc med bras. 2013;59:155–160. 29. lima ml, mourão sc, diniz mt, leite vh. hepatic histopathology of patients with morbid obesity submitted to gastric bypass. obes surg. 2005;15: 661–669. 30. stratopoulos c, papakonstantinou a, terzis i, spiliadi c, dimitriades g, komesidou v, et al. changes in liver histology accompanying massive weight loss after gastroplasty for morbid obesity. obes surg. 2005;15: 1154–1160. 31. kallwitz er, guzman g, tencate v, vitello j, layden-almer j, berkes j, et al. the histologic spectrum of liver disease in african-american, nonhispanic white, and hispanic obesity surgery patients. am j gastroenterol. 2009;104:64–69. 32. alberti kgm, zimmet p, shaw j. the metabolic syndrome-a new worldwide definition. the lancet. 2005;366:1059. 33. ocón bj, garcía b, benito p, gimeno s, garcía r, lópez p. effect of gastric bypass on the metabolic syndrome and on cardiovascular risk. nutricion hospitalaria. 2009;25:67–71. 34. ali mr, fuller wd, rasmussen j. detailed description of early response of metabolic syndrome after laparoscopic roux-en-y gastric bypass. surg obes relat dis. 2009;5:346–351. 35. batsis ja, romero-corral a, collazo-clavell ml, sarr mg, somers vk, lopezjimenez f, editors. effect of bariatric surgery on the metabolic syndrome:a population-based, long-term controlled study. mayo clinic proceedings; 2008: elsevier. 36. teixeira ar, bellodi-privato m, carvalheira jb, pilla vf, pareja jc, d’albuquerque la. the incapacity of the surgeon to identify nash in bariatric surgery makes biopsy mandatory. obes surg. 2009;19:1678–1684. 37. adami gf, cordera r, camerini g, marinari gm, scopinaro n. recovery of insulin sensitivity in obese patients at short term after biliopancreatic diversion. j surg res. 2003;113:217–221. 38. bikman bt, zheng d, pories wj, chapman w, pender jr, bowden rc, et al. mechanism for improved insulin sensitivity after gastric bypass surgery. j clin endocrinol metabol. 2008;93:4656–46563. 39. klein s, mittendorfer b, eagon jc, patterson b, grant l, feirt n, et al. gastric bypass surgery improves metabolic and hepatic abnormalities associated with nonalcoholic fatty liver disease. gastroenterology. 2006;130: 1564–1572. 40. meinhardt ng, souto ke, ulbrich-kulczynski jm, stein at. hepatic outcomes after jejunoileal bypass:is there a publication bias? obes surg. 2006;16: 1171–1178. 41. manco m, mosca a, de peppo f, caccamo r, cutrera r, giordano u, et al. the benefit of sleeve gastrectomy in obese adolescents on nonalcoholic steatohepatitis and hepatic fibrosis. j pediat. 2016. 42. cottam d, qureshi fg, mattar sg, sharma s, holover s, bonanomi g, et al. laparoscopic sleeve gastrectomy as an initial weight-loss procedure for high-risk patients with morbid obesity. surg endosc. 2006;20:859–863. 43. benotti p, wood gc, argyropoulos g, pack a, keenan bt, gao x, et al. the impact of obstructive sleep apnea on nonalcoholic fatty liver disease in patients with severe obesity. obesity (silver spring, md). 2016;24: 871–877. 44. de luca m, angrisani l, himpens j, busetto l, scopinaro n, weiner r, et al. indications for surgery for obesity and weight-related diseases: position statements from the international federation for the surgery of obesity and metabolic disorders (ifso). obes surg. 2016;26:1659–1696. 45. taitano aa, markow m, finan je, wheeler de, gonzalvo jp, murr mm. bariatric surgery improves histological features of nonalcoholic fatty liver disease and liver fibrosis. j gastrointest surg. 2015;19429–19436. 46. lassailly g, caiazzo r, buob d, pigeyre m, verkindt h, labreuche j, et al. bariatric surgery reduces features of nonalcoholic steatohepatitis in morbidly obese patients. gastroenterology. 2015;149:379–388. 47. holterman a, gurria j, tanpure s, disomma n. nonalcoholic fatty liver disease and bariatric surgery in adolescents. semin pediatr surg. 2014;23:49–57. references 1. sasaki a, nitta h, otsuka k, umemura a, baba s, obuchi t, et al. bariatric surgery and non-alcoholic fatty liver disease: current and potential future treatments. front endocrinol (lausanne). 2014;5:164. 2. rafiq n, younossi zm, editors. effects of weight loss on nonalcoholic fatty liver disease. seminars in liver disease; ©thieme medical publishers. 2008. 3. ricci g, bersani g, rossi a, pigò f, de fabritiis g, alvisi v. bariatric therapy with intragastric balloon improves liver dysfunction and insulin resistance in obese patients. obes surg. 2008;18:1438–1442. 4. ebertz ce, bonfleur ml, bertasso im, mendes mc, lubaczeuski c, araujo ac, et al. duodenal jejunal bypass attenuates non-alcoholic fatty liver disease in western diet-obese rats. acta cir bras. 2014;29:609–614. 5. rabl c, campos gm. the impact of bariatric surgery on nonalcoholic steatohepatitis. semin liver dis. 2012;32:80–91. 6. helling ts, helzberg jh, nachnani js, gurram k. predictors of nonalcoholic steatohepatitis in patients undergoing bariatric surgery:when is liver biopsy indicated? surg obes relat dis. 2008;4:612–617. 7. furuya ck, de oliveira cp, de mello es, faintuch j, raskovski a, matsuda m, et al. effects of bariatric surgery on nonalcoholic fatty liver disease: preliminary findings after 2 years. j gastroenterol hepatol. 2007;22:510–514. 8. shaffer ea. bariatric surgery:a promising solution for nonalcoholic steatohepatitis in the very obese. j clin gastroenterol. 2006;40 suppl 1: s44–s50. 9. de ridder rj, schoon ej, smulders jf, van hout gc, stockbrugger rw, koek gh. review article: non-alcoholic fatty liver disease in morbidly obese patients and the effect of bariatric surgery. aliment pharmacol therap. 2007;26 suppl 2:195–201. 10. trak-smayra v, dargere d, noun r, albuquerque m, yaghi c, gannagé-yared mh, et al. serum proteomic profiling of obese patients: correlation with liver pathology and evolution after bariatric surgery. gut. 2009;58:825–832. 11. karimi-sari h, mousavi-naeini sm, ramezani-binabaj m, najafizadeh-sari s, mir-jalili mh, dolatimehr f. prevalence of non-alcoholic fatty liver disease in morbidly obese patients undergoing sleeve bariatric surgery in iran and association with other comorbid conditions. jundishapur j chron dis care. 2015;4. 12. xanthakos sa, jenkins tm, kleiner de, boyce tw, mourya r, karns r, et al. high prevalence of nonalcoholic fatty liver disease in adolescents undergoing bariatric surgery. gastroenterology. 2015;149:623–634. 13. corey ke, stanley tl, misdraji j, scirica c, pratt j, hoppin a, et al. prevalence and outcome of non-alcoholic fatty liver disease in adolescents and young adults undergoing weight loss surgery. pediatr obes. 2014;9:e91–e93. 14. spaulding l, trainer t, janiec d. prevalence of non-alcoholic steatohepatitis in morbidly obese subjects undergoing gastric bypass. obes surg. 2003;13:347–349. 15. wolter s, duprée a, coelius c, el gammal a, kluwe j, sauer n, et al. influence of liver disease on perioperative outcome after bariatric surgery in a northern german cohort. obes surg. 2016:1–6. 16. morita s, neto dde s, morita fh, morita nk, lobo sm. prevalence of non-alcoholic fatty liver disease and steatohepatitis risk factors in patients undergoing bariatric surgery. obes surg. 2015;25:2335–2343. 17. beymer c, kowdley kv, larson a, edmonson p, dellinger ep, flum dr. prevalence and predictors of asymptomatic liver disease in patients undergoing gastric bypass surgery. arch surg (chicago, ill:1960). 2003;138:1240–1244. 18. kroh m, liu r, chand b. laparoscopic bariatric surgery:what else are we uncovering? liver pathology and preoperative indicators of advanced liver disease in morbidly obese patients. surg endosc. 2007;21:1957–1960. 19. ziolkowski a, wylezol m, kukla m, zwirska-korczala k, berdowska a, pardela m, et al. the comparison of scoring scales for liver biopsy assessment in morbidly obese patients undergoing bariatric surgery. obes surg. 2005;15:1309–1314. 20. lee cj, clark jm, asamoah v, schweitzer m, magnuson t, lazo m. prevalence and characteristics of individuals without diabetes and hypertension who underwent bariatric surgery:lessons learned about metabolically healthy obese. surg obes relat dis. 2015;11:142–146. 21. losekann a, weston ac, carli la, espindola mb, pioner sr, coral gp. nonalcoholic fatty liver disease in severe obese patients, subjected to bariatric surgery. arq gastroenterol. 2013;50:285–289. 22. jaskiewicz k, raczynska s, rzepko r, sledziński z. nonalcoholic fatty liver disease treated by gastroplasty. dig dis sci. 2006;51:21–26. 23. musso g, gambino r, cassader m. non-alcoholic fatty liver disease from pathogenesis to management:an update. obes rev. 2010;11:430–445. 24. harnois f, msika s, sabaté jm, mechler c, jouet p, barge j, et al. prevalence and predictive factors of non-alcoholic steatohepatitis (nash) in morbidly obese patients undergoing bariatric surgery. obes surg. 2006;16:183–188. mohammad kermansaravi et al. 293j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 research role of bariatric surgery in treatment of non-alcoholic fatty liver disease 71. boza c, salinas j, salgado n, pérez g, raddatz a, funke r, et al. laparoscopic sleeve gastrectomy as a stand-alone procedure for morbid obesity: report of 1,000 cases and 3-year follow-up. obes surg. 2012;22:866–871. 72. vander naalt sj, gurria jp, holterman al. surgical treatment of nonalcoholic fatty liver disease in severely obese patients. hepat med. 2014;6:103–112. 73. sasaki a, nitta h, otsuka k, umemura a, baba s, obuchi t, et al. bariatric surgery and non-alcoholic fatty liver disease:current and potential future treatments. front endocrinol. 2014;5. 74. hafeez s, ahmed mh. bariatric surgery as potential treatment for nonalcoholic fatty liver disease:a future treatment by choice or by chance? j obes. 2013;2013:839275. 75. mosinski jd, pagadala mr, mulya a, huang h, dan o, shimizu h, et al. gastric bypass surgery is protective from high-fat diet-induced nonalcoholic fatty liver disease and hepatic endoplasmic reticulum stress. acta physiol (oxford, england). 2016;217:141–151. 76. dietrich p, hellerbrand c. non-alcoholic fatty liver disease, obesity and the metabolic syndrome. best practice and research: clin gastroenterol. 2014;28:637–653. 77. myronovych a, kirby m, ryan kk, zhang w, jha p, setchell kd, et al. vertical sleeve gastrectomy reduces hepatic steatosis while increasing serum bile acids in a weight-loss-independent manner. obesity (silver spring, md). 2014;22:390–400. 78. milić s, lulić d, štimac d. non-alcoholic fatty liver disease and obesity:biochemical, metabolic and clinical presentations. world j gastroenterol. 2014;20:9330–9337. 79. watanabe s, hashimoto e, ikejima k, uto h, ono m, sumida y, et al; japanese society of gastroenterology; japan society of hepatology. evidence-based clinical practice guidelines for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. j gastroenterol. 2015;50:364–377. 80. neuman mg, nanau rm, cohen lb. nonmedicinal interventions in nonalcoholic fatty liver disease. can j gastroenterol hepatol. 2015;29: 241–252. 81. schneier at, citti cc, dieterich dt. management and diagnosis of fatty liver disease. expert rev gastroenterol hepatol. 2015;9:671–683. 82. schneck as, anty r, patouraux s, bonnafous s, rousseau d, lebeaupin c, et al. roux-en y gastric bypass results in long-term remission of hepatocyte apoptosis and hepatic histological features of non-alcoholic steatohepatitis. front physiol. 2016;7:344. 83. de jonge c, rensen ss, koek gh, joosten mf, buurman wa, bouvy nd, et al. endoscopic duodenal-jejunal bypass liner rapidly improves plasma parameters of nonalcoholic fatty liver disease. clin gastroenterol hepatol. 2013;11:1517–1520. 84. genco a, bruni t, doldi sb, forestieri p, marino m, busetto l, et al. bioenterics intragastric balloon: the italian experience with 2,515 patients. obes surg. 2005;15:1161–1164. 85. totté e, hendrickx l, pauwels m, van hee r. weight reduction by means of intragastric device: experience with the bioenterics intragastric balloon. obes surg. 2001;11:519–523. 86. doldi s, micheletto g, perrini m, rapetti r. intragastric balloon: another option for treatment of obesity and morbid obesity. hepato-gastroenterology. 2003;51:294–297. 87. nassif at, nagano ta, okayama s, nassif ls, branco filho a, sampaio neto j. performance of the bard scoring system in bariatric surgery patients with nonalcoholic fatty liver disease. obes surg. 2016. 88. larson sp, bowers sp, palekar na, ward ja, pulcini jp, harrison sa. histopathologic variability between the right and left lobes of the liver in morbidly obese patients undergoing roux-en-y bypass. clin gastroenterol hepatol. 2007;5:1329–1332. 89. jiménez-agüero r, emparanza ji, beguiristain a, bujanda l, alustiza jm, garcía e, et al. novel equation to determine the hepatic triglyceride concentration in humans by mri:diagnosis and monitoring of nafld in obese patients before and after bariatric surgery. bmc med. 2014;12:137. 90. marchesini g, petta s, dalle grave r. diet, weight loss, and liver health in nonalcoholic fatty liver disease:pathophysiology, evidence, and practice. hepatology (baltimore, md). 2016;63:2032–2043. 91. promrat k, kleiner de, niemeier hm, jackvony e, kearns m, wands jr, et al. randomized controlled trial testing the effects of weight loss on nonalcoholic steatohepatitis. hepatology (baltimore, md). 2010;51: 121–129. 92. angelico f, loffredo l, pignatelli p, augelletti t, carnevale r, pacella a, et al. weight loss is associated with improved endothelial dysfunction via nox 2 generated oxidative stress down-regulation in patients with the metabolic syndrome. intern emerg med. 2012;7:219–227. 48. vargas v, allende h, lecube a, salcedo mt, baena-fustegueras ja, fort jm, et al. surgically induced weight loss by gastric bypass improves non alcoholic fatty liver disease in morbid obese patients. world j hepatol. 2012;4:382–388. 49. nobili v, vajro p, dezsofi a, fischler b, hadzic n, jahnel j, et al. indications and limitations of bariatric intervention in severely obese children and adolescents with and without nonalcoholic steatohepatitis: espghan hepatology committee position statement. j pediatr gastroenterol nutr. 2015;60:550–561. 50. burza ma, romeo s, kotronen a, svensson pa, sjöholm k, torgerson js, et al. long-term effect of bariatric surgery on liver enzymes in the swedish obese subjects (sos) study. plos one. 2013;8:e60495. 51. than nn, newsome pn. non-alcoholic fatty liver disease:when to intervene and with what. clinical medicine (london, england). 2015;15:186–190. 52. baraille f, guilmeau s, postic c. gastric bypass surgery in nash: a major modulator of hepatic mitochondrial dysfunction. gut. 2015;64:524–526. 53. salgado júnior w, donadelli ca, dos santos js, nonino cb. influence of roux-en-y gastric bypass on the hepatocellular function and bile flow of obese patients assessed by scintigraphy with disida. obes surg. 2016;26:2718–2723. 54. le roux cw, aylwin sj, batterham rl, borg cm, coyle f, prasad v, et al. gut hormone profiles following bariatric surgery favor an anorectic state, facilitate weight loss, and improve metabolic parameters. ann surg. 2006;243:108–114. 55. aguilar-olivos ne, almeda-valdes p, aguilar-salinas ca, uribe m, méndezsánchez n. the role of bariatric surgery in the management of nonalcoholic fatty liver disease and metabolic syndrome. metab clin exp. 2016;65: 1196–1207. 56. winder js, dudeck bs, schock s, lyn-sue jr, haluck rs, rogers am. radiographic improvement of hepatic steatosis after laparoscopic rouxen-y gastric bypass. obes surg. 2016. 57. clark jm, alkhuraishi ar, solga sf, alli p, diehl am, magnuson th. roux-en-y gastric bypass improves liver histology in patients with non-alcoholic fatty liver disease. obes res. 2005;13:1180–1186. 58. cazzo e, jimenez ls, pareja jc, chaim ea. effect of roux-en-y gastric bypass on nonalcoholic fatty liver disease evaluated through nafld fibrosis score: a prospective study. obes surg. 2015;25:982–985. 59. barker kb, palekar na, bowers sp, goldberg je, pulcini jp, harrison sa. nonalcoholic steatohepatitis:effect of roux-en-y gastric bypass surgery. am j gastroenterol. 2006;101:368–373. 60. liu x, lazenby aj, clements rh, jhala n, abrams ga. resolution of nonalcoholic steatohepatits after gastric bypass surgery. obes surg. 2007;17:486–492. 61. de almeida sr, rocha pr, sanches md, leite vh, da silva ra, diniz mt, et al. roux-en-y gastric bypass improves the nonalcoholic steatohepatitis (nash) of morbid obesity. obes surg. 2006;16:270–278. 62. algooneh a, almazeedi s, al-sabah s, ahmed m, othman f. non-alcoholic fatty liver disease resolution following sleeve gastrectomy. surg endosc. 2016;30:1983–1987. 63. aldoheyan t, hassanain m, al-mulhim a, al-sabhan a, al-amro s, bamehriz f, et al. the effects of bariatric surgeries on nonalcoholic fatty liver disease. surg endoscopy. 2016. 64. billeter at, senft j, gotthardt d, knefeli p, nickel f, schulte t, et al. combined non-alcoholic fatty liver disease and type 2 diabetes mellitus: sleeve gastrectomy or gastric bypass?-a controlled matched pair study of 34 patients. obes surg. 2016;26:1867–1874. 65. froylich d, corcelles r, daigle c, boules m, brethauer s, schauer p. effect of roux-en-y gastric bypass and sleeve gastrectomy on nonalcoholic fatty liver disease:a comparative study. surg obes relat dis. 2016;12:127–131. 66. caiazzo r, lassailly g, leteurtre e, baud g, verkindt h, raverdy v, et al. roux-en-y gastric bypass versus adjustable gastric banding to reduce nonalcoholic fatty liver disease:a 5-year controlled longitudinal study. annals of surgery. 2014;260:893–898. 67. keshishian a, zahriya k, willes eb. duodenal switch has no detrimental effects on hepatic function and improves hepatic steatohepatitis after 6 months. obes surg. 2005;15:1418–1423. 68. miras ad, le roux cw. metabolic surgery:shifting the focus from glycaemia and weight to end-organ health. lancet diabetes endocrinol. 2014;2:141–151. 69. polymeris a. the pluses and minuses of bariatric surgery for morbid obesity:an endocrinological perspective. hormones (athens). 2012;11: 233–240. 70. lassailly g, caïazzo r, pattou f, mathurin p. bariatric surgery for curing nash in the morbidly obese? j hepatol. 2013;58:1249–1251. 294 j contemp med sci | vol. 3, no. 12, autumn 2017: 286–294 role of bariatric surgery in treatment of non-alcoholic fatty liver disease research mohammad kermansaravi et al. 93. vilar-gomez e, martinez-perez y, calzadilla-bertot l, torres-gonzalez a, gra-oramas b, gonzalez-fabian l, et al. weight loss through lifestyle modification significantly reduces features of nonalcoholic steatohepatitis. gastroenterology. 2015;149:367–378. e5. 94. st george a, bauman a, johnston a, farrell g, chey t, george j. effect of a lifestyle intervention in patients with abnormal liver enzymes and metabolic risk factors. j gastroenterol hepatol. 2009;24:399–407. 95. hallsworth k, fattakhova g, hollingsworth kg, thoma c, moore s, taylor r, et al. resistance exercise reduces liver fat and its mediators in nonalcoholic fatty liver disease independent of weight loss. gut. 2011:242073. 96. marchesini g, mazzella n, forlani g. weight loss for a healthy liver. gastroenterology. 2015;149:274–278. 97. iacono a, raso gm, canani rb, calignano a, meli r. probiotics as an emerging therapeutic strategy to treat nafld: focus on molecular and biochemical mechanisms. j nutr biochem. 2011;22:699–711. 98. ekstedt m, franzén le, mathiesen ul, holmqvist m, bodemar g, kechagias s. statins in non-alcoholic fatty liver disease and chronically elevated liver enzymes:a histopathological follow-up study. j hepatol. 2007;47:135–141. 99. ioannides-demos ll, piccenna l, mcneil jj. pharmacotherapies for obesity:past, current, and future therapies. j obesity. 2010;2011. 100. capanni m, calella f, biagini mr, genise s, raimondi l, bedogni g, et al. prolonged n-3 polyunsaturated fatty acid supplementation ameliorates hepatic steatosis in patients with non-alcoholic fatty liver disease:a pilot study. aliment pharmacol ther. 2006;23:1143–1151. 101. attar bm, van thiel dh. current concepts and management approaches in nonalcoholic fatty liver disease. scientific world journal. 2013;2013:481893. 102. younossi zm, reyes mj, mishra a, mehta r, henry l. systematic review with meta-analysis:non-alcoholic steatohepatitis—a case for personalised treatment based on pathogenic targets. aliment pharmacol ther. 2014;39:3–14. 103. dagan ss, zelber-sagi s, webb m, keidar a, raziel a, sakran n, et al. nutritional status prior to laparoscopic sleeve gastrectomy surgery. obes surg. 2016;26:2119–2126. 104. de palma gd, forestieri p. role of endoscopy in the bariatric surgery of patients. world j gastroenterol. 2014;20:7777–7784. 105. padwal r, brocks d, sharma am. a systematic review of drug absorption following bariatric surgery and its theoretical implications. obes rev. 2010;11:41–50. 106. luger m, kruschitz r, marculescu r, haslacher h, hoppichler f, kallay e, et al. the link between obesity and vitamin d in bariatric patients with omega-loop gastric bypass surgery a vitamin d supplementation trial to compare the efficacy of postoperative cholecalciferol loading (load): study protocol for a randomized controlled trial. trials. 2015;16. 107. shah m, simha v, garg a. long-term impact of bariatric surgery on body weight, comorbidities, and nutritional status. j clin endocrinol metabol. 2006;91:4223–4231. 108. fleischer j, stein e, bessler m, badia md, restuccia n, olivero-rivera l, et al. the decline in hip bone density after gastric bypass surgery is associated with extent of weight loss. j clin endocrinol metabol. 2008;93:3735–3740. 109. verna ec, berk pd. role of fatty acids in the pathogenesis of obesity and fatty liver:impact of bariatric surgery. semin liver dis. 2008;28:407–426. 110. mathurin p, hollebecque a, arnalsteen l, buob d, leteurtre e, caiazzo r, et al. prospective study of the long-term effects of bariatric surgery on liver injury in patients without advanced disease. gastroenterology. 2009;137:532–540. 111. haentjens p, massaad d, reynaert h, peeters e, van meerhaeghe a, vinken s, et al. identifying non-alcoholic fatty liver disease among asymptomatic overweight and obese individuals by clinical and biochemical characteristics. acta clin belg 2009;64:483–493. 112. prashanth m, ganesh hk, vima mv, john m, bandgar t, joshi sr, et al. prevalence of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus. j assoc physicians india. 2009;57:205–210. 113. johansson he, haenni a, zethelius b. platelet counts and liver enzymes after bariatric surgery. j obes. 2013;2013:567984. 114. mattar sg, velcu lm, rabinovitz m, demetris aj, krasinskas am, barinasmitchell e, et al. surgically-induced weight loss significantly improves nonalcoholic fatty liver disease and the metabolic syndrome. ann surg. 2005;242:610–617. 115. dixon jb, bhathal ps, hughes nr, o'brien pe. nonalcoholic fatty liver disease: improvement in liver histological analysis with weight loss. hepatology (baltimore, md). 2004;39:1647–154. 116. verbeek j, lannoo m, pirinen e, ryu d, spincemaille p, vander elst i, et al. roux-en-y gastric bypass attenuates hepatic mitochondrial dysfunction in mice with non-alcoholic steatohepatitis. gut. 2015;64:673–683. 117. mathurin p, hollebecque a, arnalsteen l, buob d, leteurtre e, caiazzo r, et al. prospective study of the long-term effects of bariatric surgery on liver injury in patients without advanced disease. gastroenterology. 2009;137:532–540. 118. giannetti m, piaggi p, ceccarini g, mazzeo s, querci g, fierabracci p, et al. hepatic left lobe volume is a sensitive index of metabolic improvement in obese women after gastric banding. int j obes (lond). 2012;36:336–341. 119. kazankov k, tordjman j, møller hj, vilstrup h, poitou c, bedossa p, et al. macrophage activation marker soluble cd163 and non-alcoholic fatty liver disease in morbidly obese patients undergoing bariatric surgery. j gastroenterol hepatol. 2015;30:1293–1300. 120. manco m, fernandez-real jm, vecchio fm, vellone v, moreno jm, tondolo v, et al. the decrease of serum levels of human neutrophil alpha-defensins parallels with the surgery-induced amelioration of nash in obesity. obes surg. 2010;20:1682–1689. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201701 187j contemp med sci | vol. 4, no. 4, autumn 2018: 187–190 review echium amoenum from viewpoint of avicenna: a brief review hossein jafari,a roshanak mokaberinejad,a* and ehsan raeis-abdollahib adepartment of traditional medicine, school of traditional medicine, shahid beheshti university of medical sciences tehran, iran. b department of physiology, school of medicine, tehran university of medical sciences, tehran, iran. *correspondence to roshanak mokaberinejad (email: rmokaberi@gmail.com). (submitted: 21 auguest 2018 – revised version received: 18 september 2018 – accepted: 27 september 2018 – published online: 26 december 2018) objectives the aim of this study was to compare the effects of echium amoenum (gol-e-gavzaban) in iranian traditional medicine (itm) under the lens of avicenna and modern medicine. results echium amoenum has many therapeutic effects in itm and modern medicine. e. amoenum is traditionally used as herbal tea in iranian traditional medicine (itm) for conditions such as common cold, bronchitis, stress and fatigue. it also possesses diaphoretic, diuretic and blood rectifying properties. some of its effects such as general tonic, cardiotonic, diaphoretic, diuretic, sedative and antitussive has been reported according to recent studies. conclusion there are many common characteristics about the pharmacological properties of e. amoenum in modern medicine and itm, but more research is needed to prove the safety and efficacy of the plant. keywords echium amoenum, iranian traditional medicine, avicenna, cardiotonic introduction echium amoenum, also named as gol-e-gavzaban in persian, from boraginaceae family is one of the most commonly used medicinal plants,1 is distributed in the northern region of iran, as well as europe and mediterranean region.2 this annual herb has more than 100 genera and 2300 species and has long been used for many different diseases in the iranian traditional medicine (itm). echium amoenum is traditionally used as herbal tea in iran for conditions such as common cold, bronchitis, stress and fatigue. it also possesses diaphoretic, diuretic and blood rectifying properties. the dried violet–blue petals of iranian borage are used as general tonic, cardiotonic, diaphoretic, diuretic, sedative and antitussive according to the itm. in addition, borago officinalis in europe has been used for hyperactive gastrointestinal, respiratory and cardiovascular disorders.3 the similarity in clinical and pharmacological aspects of e. amoenum and b. officinalis is recognized.4 so far, several pharmacological activities of constituents of e. amoenum have been thoroughly evaluated which showed anti-anxiety,5 anxiolytic,6 analgesic and antidepression,7 antiobsessive and compulsive,8 anticancer,9 anti-inflammatory,10 antimutagenic and cytotoxic,11 antioxidant and radical scavenging,12–14 antiviral,15 antibacterial,16 neonatal brain growth,17 and cardiovascular effects.18 nowadays, various metabolites of e. amoenum, such as flavonoids, saponins, terpenoids, sterols and low amounts of essential oil, have been identified through phytochemistry studies.19 uses of ancient knowledge in ethnopharmacology and related fields have prompted better understanding of progression and development of human plant utilization.20 although the fields of molecular modeling, combinatorial chemistry, and other synthetic chemistry techniques have attracted a lot of attention by pharmaceutical companies and funding organizations, medicinal plants have remained a source of inspiration for finding new drugs, new drug leads, and new chemical entities.21 in spite of notable achievements in drug discovery form medicinal plants, still many challenges await us. natural product scientists such as pharmacognosists and phytochemists have to make advancements in the quality and quantity of compounds in the drug development to compete with other drug discovery projects.22 herbal therapy in iran also have an old record and many manuscripts regarding this issue are left by great physicians such as avicenna and rhazes.23 e. amoenum is one of the important medicinal plants in the itm. the profits of e. amoenum has been first learnt by romans 300 b.c. homer, the well-known greek poet, assumed that the plant has positive effects on mood.8 in this study, we describe the traditional uses of e. amoenum and its therapeutic properties as defined by avicenna in book ii, canon of medicine, ketab al-adviah al-ghalbiah (heart drugs) or from current scientific studies. avicenna (980–1032 ad) was an outstanding iranian physician who wrote the canon of medicine (in 1025 ad). this book was taught as a chief medical reference in western and eastern countries until the 17th century.3 avicenna believed that the drug has rarefying and cardiotonic effect and also useful for stomatitis, restlessness, melanotic disorder, palpitation, cough and roughness of upper respiratory tract.24 avicenna also said in his other great book ‘al-adviah al-ghalbiah’ (heart drugs) that e. amoenum is a unique herbal drug for modulating of temperament, strengthening and exhilarating.25 also hakim khorasani, one of the most famous physician of the itm, has written in his book named makhzan al adviah about e. amoenum. he believed that this drug is useful for the treatment of cough, sore throat, pneumonia and some of pediatric febrile and eruptive disease.26 in this review article, the uses of e. amoenum as explained by avicenna' books, canon of medicine, book of al-adviah al-ghalbiah, and various databases of the latest scientific studies are discussed and compared. methods to compare the therapeutic effects of e. amoenum, an extensive search was performed through various databases such as issn 2413-0516 188 j contemp med sci | vol. 4, no. 4, autumn 2018: 187–190 echium amoenum from viewpoint of avicenna: a brief review review h. jafari et al. pubmed, scopus, sciencedirect, and google scholar. the keywords for our search were: e. amoenum, gol-e-gavzaban, avicenna and “ibn sina”. then therapeutic and pharmacologic properties of this plant were gathered from 2000 up to 2018. unrelated citations were removed. we used canon of medicine in its original language (arabic) to detect a true outcome. only the arabic versions were used because the english version did not clarify the particular effect obviously. results comparative evaluation in the canon and modern medicine cardiotonic effects avicenna: “two characteristics has been folded in this drug, so that no other medication could compete it in strengthening and exhilarating. one is its enhancing vitality properties and the other is its temperament feature” (fig. 1).25 avicenna: “it is rarefying and cardiotonic drug”.35 the itm is a medicinal system based on the humoral theory and it dates back to 10,000 years ago.27 according to the teachings of the iranian medicine, if humor of sowda (black bile), changes to abnormal form (both in terms of quantity and quality), it can cause other pathological results, among which are sadness, anxiety and concern. this condition, named as melanotic disease, means that the disease is caused by sowda. avicenna believed that e. amoenum, can remove these waste materials from the body and would indirectly lead to fright and happiness in the patient.24 melancholia has been categorized as a kind of mental disease in the itm and it forms as a result of an alteration in the quality of brain mizaj (temperament) that stops the person from rational thinking so that causes a depressed mood, terror, and mistrust without any clear cause.24,29,28 when we compare melancholia symptoms with major depression according to (dsm-4-tr) criteria, we found that this disorder have been categorized as one of the depression (mood) disorders.29 nowadays, it is shown that borage oil is effective on cardiac remodeling after myocardial infarction through an animal study on rats. based on these results, borage oil reduces development of cardiac remodeling after myocardial infarction and congestive heart failure.30 furthermore, it is largely acknowledged that emotional elements might impact physical function. this matter is lectured in comprehensive medicine as well as neuroscience. there are handy confirmations for the antagonistic effects of mental disorder on the physical health, for e.g. depressed mood and tension.31 also a positive effect on treatment of major depression of e. amoenum is emphasized and it has been suggested for mood elevation.8 avicenna says: “the burnt borage cures stomatitis in children. it relieves burning of the mouth”.35 avicenna believed that e. amoenum is useful for aphthous stomatitis of children especially when burnt.24 anti-inflammatory effects of e. amoenum examined in the j774.1a macrophage cell line with preparation of ethyl acetate, dichloromethane and hexane extracts derived from this plant and then probable cytotoxic effects were studied using mtt (a colorimetric assay for assessing cell metabolic activity) e. amoenum hexane extract revealed the maximum reduction in macrophage no secretion compared to other extracts.10 avicenna: “it is good for treating restlessness, melanotic disease and palpitation”.35 during an 8-week double-blind randomized clinical trial study on 37 patients with general anxiety disorder it was found that the aqueous extract of e. amoenum (500 mg/day) together with selective serotonin reuptake inhibitors plus fluoxetine (20 mg/day) or fluoxetine (20 mg/day) plus a placebo had positive anxiolytic effect.33 avicenna: “it is also useful in cough and roughness of wind-pipe specially when used in the form of a decoction with honey-water or sugar”.35 it may be because of anti-inflammatory and immunomodulatory and radical scavenging effect of e. amoenum.4,10 avicenna: “it is a mild purgative for melanotic humors”.35 according to the itm, repletion of redundant material in human body means “imtila” which can destroy normal function of cells and organs, thus leads to disease. purgative drugs can be effective on imtila both in prevention and treatment.35 therapeutic effect of e. amoenum as described by avicenna in canon of medicine and al-adviah al-ghalbiah are listed in table 1. other therapeutic effects antiviral activity it is shown that aqueous extract of e. amoenum is effective against herpes simplex virus type i, when it is used with a concentration of 50–1000 µg/ml during 7 days. significant activity appeared at the concentrations greater than 400 µg/ml by inhibiting virus replication.15 fig. 1 a screenshot of avicenna’s book al-adviah al-ghalbiah (courtesy of the library of the parliament of iran). h. jafari et al. 189j contemp med sci | vol. 4, no. 4, autumn 2018: 187–190 review echium amoenum from viewpoint of avicenna: a brief review antiparasitic activity the antileishmanial effects of alcoholic and aqueous extracts of e. amoenum were demonstrated in a study on balb/c mice. in this study, the level of ifn-γ were increased and parasite count decreased in the intervention group in comparison with the controls.15 antimicrobial properties the aqueous extract of e. amoenum presented a concentration dependent antimicrobial activity against staphylococcus aureus 8327 which was heat resistant.16 antioxidant and radical scavenging activity an adjacent matching in pharmacological and clinical features of e. amoenum and b. officinalis (european type) is known.4 immonomodulatory effects in one animal study on mice, it was concluded that the hydroalcoholic extract of e. amoenum can improve lymphocytic proliferation, but inhibits the proliferation of human antibodies.34 anticonvulsant effects intraperitoneal administration of the methanol extract of e. amoenum at 6.25 mg/kg to mice before the injection of picrotoxin, produced an apparent rising in the latency of seizure and delayed the death time as compared with the control group.19 positive effect on benign prostate hyperplasia it is shown that the mixed hydroalcoholic solution of e. amoenum, viola odorata and physalis alkekengi is effective on benign prostate hyperplasia. results declared that frequency of urination, intermittency, urgency, weak stream, straining and nocturia significantly decreased in the treatment group in comparison with the control group without any apparent side effect.35 toxicity and teratogenicity the existence of pyrrolizidine alkaloids in e. amoenum could be a threat for hepatotoxicity and liver damage. also, these kind of alkaloids have been displayed to have teratogenicity in pure situations. in one study with ames test, it was found that a methanol extract (0.25, 0.5, 0.75, and 1.0 mg/ml) from e. amoenum petals could be safe and non-mutagenic. however, during this study it was revealed that this plant has no mutagenic effects in usual daily doses.36 another rat model study showed that aqueous extract of e. amoenum (100, 200, 400, mg/kg/day) had no toxicity on liver during 1 and 2 weeks of treatment.19 conclusion as a common herbal plant, e. amoenum is widely used by traditional and conventional healers in iran and its adjoining countries. avicenna believed that no other medication could compete it in strengthening and exhilarating. there are many traditional and modern reports that confirm anti-inflammatory effects, antioxidant effects and antianxiolytic properties of this drug. it is probably because of active constituents of the plant, such as flavonoids, saponins, terpenoids, and essential oils. therefore it may be probably a good option for prevention or treatment of a spectrum of inflammatory disorders specially heart diseases. it is apparent that more pharmacological and toxicological experiments and clinical trials are still required for the use of this herbal drug as a certified medicinal plant in clinical setting. acknowledgement this article was based on a part of phd thesis (number 192) of traditional medicine, (hossein jafari) granted by school of traditional medicine, shahid beheshti university of medical sciences, tehran, iran (grant number 194). the authors would like to acknowledge shahid beheshti university of medical sciences cooperation and arrangements. conflict of interest there are no conflict of interest. n table 1. therapeutic effect of e. amoenum as described by avicenna in canon of medicine and al-adviah al-ghalbiah. we used closest translation for each word effect or condition noted in avicenna’s two books current name of condition mofarreh e-ghalb (exhilarating effect for heart) cardiotonic tawahhosh (useful for) a disease like panic disorder sudawi disease (useful for) melancholic disease moshele-sowda ye-raghigh (purgative) expellant of diluted black bile mofarreh (enhancing vitality properties, useful for) mood elevator ghola (useful for) effective on aphtosostomatitis lahib –efam (useful for) oral cavity and tongue inflammation so –al (useful for) cough khoshonat-e ghazib (useful for) hoarsness references 1. zarshenas mm, dabaghian f, moein m. an overview on phytochemical and pharmacological aspects of echium amoenum. nat prod j. 2016;6:285–291. available from: https://www.ingentaconnect.com/ contentone/ben/npj/2016/00000006/00000004/art00005 () [cited 2018 aug 19] 2. abolhassani m. antiviral activity of borage (echium amoenum). arch med sci. 2010;6:366–369. available from: http://www.ncbi.nlm.nih.gov/ pubmed/22371772 [cited 2018 apr 14]. 3. setayesh m, zargaran a, sadeghifar ar, salehi m, rezaeizadeh h. new candidates for treatment and management of carpal tunnel syndrome 190 j contemp med sci | vol. 4, no. 4, autumn 2018: 187–190 echium amoenum from viewpoint of avicenna: a brief review review h. jafari et al. (cts) based on avicenna’s teachings in the canon of medicine. integr med res. 2018;7:126–135. available from: https://www.sciencedirect.com/ science/article/pii/s2213422017302196 [cited 2018 mar 12]. 4. zamansoltani f, nassiri-asl m, karimi r, mamaghani-rad p. hepatotoxicity effects of aqueous extract of echium amoenum in rats. pharmacologyonline. 2008;1:432–438. available from: http://pharmacologyonline.silae.it/files/ archives/2008/vol1/42_farzaneh.pdf [cited 2018 feb 16]. 5. bakhshaei s. phyto-pharmacological effect of nine medicinal plants as a traditional treatment of depression. j appl pharm. 2017;8:76–81. available from: http://www.iioab.org/iioabj_8.s2_76-81.pdf (supplement issue: biological science) [cited 2018 feb 15]. 6. gholamzadeh s, zare s, of mi-rj. anxiolytic effect of echium amoenum during different treatment courses. res j biol sci. 2008;3:176–178 available from: http://docsdrive.com/pdfs/medwelljournals/rjbsci/2008/176-178.pdf (docsdrive.com [internet]) [cited 2018 feb 11]. 7. mansouri s. inhibition of staphylococcus aureus mediated by extracts from iranian plants. pharm biol. 1999;37:375–377. available from: http://www. tandfonline.com/doi/full/10.1076/phbi.37.5.375.6058 [cited 2018 apr 15]. 8. sayyah m, boostani h, pakseresht s, malaieri a. efficacy of aqueous extract of echium amoenum in treatment of obsessive– compulsive disorder. prog neuropsychopharmacol biol psychiatry. 2009;33:1513–1516. available from: https://ac.els-cdn.com/ s0278584609002814/1-s2.0-s0278584609002814-main.pdf?_tid=spdffa3842cd-88ff-49c7-ba3f-1dca1466fdc7&acdnat=1519620589_ e0b956d3a117e42f21ac5d3e77295cb6 [cited 2018 feb 26]. 9. gonzalez c, sanz j, marcos g, pita s, brullet e, saigi e. borage consumption as a possible gastric cancer protective factor. cancer epidemiol biomarkers prev. 1993;2:157–158. available from: https://www.researchgate.net/profile/jose_ miguel_sanz-anquela/publication/14812828_borage_consumption_as_a_ possible_gastric_cancer_protective_factor/links/00b49517ae5226ff35000000. pdf (researchgate.net [internet]) [cited 2018 apr 15]. 10. naseri n, kalantar k, amirghofran z. anti-inflammatory activity of echium amoenum extract on macrophages mediated by inhibition of inflammatory mediators and cytokines expression. res pharm sci. 2018;13:73–81. available from: http://www.ncbi.nlm.nih.gov/pubmed/29387114 [cited 2018 mar 5]. 11. uysal h, kızılet h, ayar a, taheri a. the use of endemic iranian plant, echium amoenum, against the ethyl methanesulfonate and the recovery of mutagenic effects. toxicol ind health. 2015;31:44–51. available from: http:// journals.sagepub.com/doi/10.1177/0748233712468019 [cited 2018 apr 15]. 12. bekhradnia s, ebrahimzadeh ma. antioxidant activity of echium amoenum. rev chim. 2016;67:223–226. available from: http://www.revistadechimie.ro 13. safaeian l, javanmard sh, ghanadian m, seifabadi s. cytoprotective and antioxidant effects of echium amoenum anthocyanin-rich extract in human endothelial cells (huvecs). avicenna j phytomed. 2015;5:157–166. available from: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4418065/ (ncbi.nlm. nih.gov [internet]) [cited 2017 dec 25]. 14. adel pilerood s, prakash j. evaluation of nutritional composition and antioxidant activity of borage (echium amoenum) and valerian (valerian officinalis). j food sci technol. 2014;51:845–854. available from: http:// link.springer.com/10.1007/s13197-011-0573-z [cited 2018 feb 16]. 15. farahani m. antiviral effect assay of aqueous extract of echium amoenum-l against hsv-1. zahedan j res med sci. 2013;15:46–48. available from: http://zjrms.ir/browse.php?a_code=a-10-1602-1&slc_ lang=en&sid=1&sw=antiviral+effect [cited 2018 apr 15]. 16. abolhassani m. antibacterial effect of borage (echium amoenum) on staphylococcus aureus. braz j infect dis. 2004;8:382–385. available from: www.bjid.com.br [cited 2018 mar 7]. 17. wainwright pe, huang ys, demichele sj, xing h, liu jw, chuang lt, et al. effects of high-gamma-linolenic acid canola oil compared with borage oil on reproduction, growth, and brain and behavioral development in mice. lipids. 2003;38:171–178. available from: http://doi.wiley.com/10.1007/ s11745-003-1048-2 [cited 2018 apr 15]. 18. hamidi em, khaksari m, hojabri k. the effects of aqueous extracts of echium amoenum and citrus aurantiflia on blood pressure and heart rate before and after phynelephrine injection in rat. j. kerman univ. med. sci. 2014;18:349–357. available from: http://eprints.kmu.ac.ir/22754/ (eprints. kmu.ac.ir [internet]) [cited 2018 apr 15]. 19. mehrabani m, ghannadi a, sajjadi e, ghassemi n, shams-ardakani m. toxic pyrrolizidine alkaloids of echium amoenum fisch. & mey. daru. 2006;14(3):122–128. 20. heinrich m, kufer j, leonti m, pardo-de-santayana m. ethnobotany and ethnopharmacology—interdisciplinary links with the historical sciences. 2006;107:157–160. available from: https://www.sciencedirect.com/science/ article/pii/s0378874106002959 (elsevier [internet]) [cited 2018 apr 15]. 21. balunas mj, kinghorn ad. drug discovery from medicinal plants. life sci. 2005;78:431–441. available from: http://www.sciencedirect.com/science/ article/pii/s0024320505008799 [cited 2018 jan 10]. 22. butler ms. the role of natural product chemistry in drug discovery. j nat prod. 2004;67:2141–2153. available from: http://pubs.acs.org/doi/ abs/10.1021/np040106y [cited 2018 apr 15]. 23. changizi ashtiyani s, shamsi m, cyrus a, bastani b, tabatabayei sm. a critical review of the works of pioneer physicians on kidney diseases in ancient iran: avicenna, rhazes, al-akhawayni, and jorjani. iran j kidney dis. 2011;5:300– 308. available from: http://search.proquest.com/openview/e33d4e5dc7 4738409889d5f2b81f673b/1?pq-origsite=gscholar&cbl=105769 (search. proquest.com [internet]) [cited 2018 apr 15]. 24. sina i. al-qanun fi al-tibb [the canon of medicine], vol. 437; alaalami library, beirut, 2005, p. 48–51. 25. borghei hr. the book on drugs for cardiovascular diseases. nashre ney, tehran, 2009 (imenshahidi et al., no title. 2010). 26. khorasani mha. makhzan al advieh. iran bavardaran press, tehran, 2001 (res inst islam complement med iran univ med sci). 27. khodaei ma, noorbala aa, parsian z, targhi st, emadi f, alijaniha f. avicenna (980-1032ce): the pioneer in treatment of depression. transylvanian rev. 2017;25:4376–4389. available from: https://scholar.google.com/ scholar?hl=en&as_sdt=0,5&q=6.+araj+khodaei+m,+noorbala+aa,+parsi an+z,+taheri+tarighi+s,+emadi+f,+alijaniha+f,+naseri+m,+zargaran+a. +avicenna+(980-1032ce)%3a+the+pioneer+in+treatment+of+de-+ pression.+transylvanian+review+2017%3b+ [cited 2018 apr 16]. 28. kermani n. sharhe asbaab-o-alaamaat of samarghandi. mohamad-bagher minaei, mansour keshavarz. in: describe the etiology and symptoms. jalal al-din, qom, 2008. 29. kaplan v, sadock b. pocket handbook of clinical psychiatry. in: arjmand m, editor., 5th ed.; arjmand publication, tehran, 2010, pp. 210–215. 30. maldonado-menetti jdos s, vitor t, edelmuth rc, ferrante fa, souza pr, koike mk. borage oil attenuates progression of cardiac remodeling in rats after myocardial infarction. acta cir bras. 2016;31:190–197. 31. veenhoven r. healthy happiness: effects of happiness on physical health and the consequences for preventive health. j happiness stud. 2008;9:449–469. available from: https://link.springer.com/content/ pdf/10.1007%2fs10902-006-9042-1.pdf [cited 2018 mar 4]. 32. sayyah m, siahpoosh a, khalili h, malayeri a, samaee h. a double-blind, placebo-controlled study of the aqueous extract of echium amoenum for patients with general anxiety disorder. iran j pharm res. 2012;11:697–701. available from: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3832167/ (ncbi.nlm.nih.gov [internet]) [cited 2018 apr 16]. 33. anonymous. al-qanun fil-tibb (english translation) (original authoravicenna), vol. 2. department of islamic studies, new delhi, 1998 (jamia hamdard;415_417). 34. ghods r, gharooni m, amin g, nazem e, nikbakht nasrabadi a. hypertension from the perspective of iranian traditional medicine. 2014;16:e16449. available from: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4005451/ (ncbi.nlm.nih.gov [internet]) [cited 2018 apr 16]. 35. beiraghdar f, einollahi b, ghadyani a, panahi y, hadjiakhoondi a, vazirian m, et al. a two-week, double-blind, placebo-controlled trial of viola odorata, echium amoenum and physalis alkekengi mixture in symptomatic benign prostate hyperplasia (bph) men. pharm biol. 2017;55:1800–1805. 36. moosavi m, jalali a, kianipour f, siahpoosh a, farajzadeh-shikh a. assessing mutagenicity of methanolic exteract of borage flower (echium amuenum) using ames bioassay. iran south med j 2014;17: 307–317. available from: https://ismj.bpums.ac.ir/browse.php?a_id=543&slc_ lang=en&sid=1&printcase=1&hbnr=1&hmb=1 (ismj.bpums.ac.ir [internet]) [cited 2018 apr 16]. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 154 j contemp med sci | vol. 5, no. 3, may–june 2019: 154–159 original the prevalence of dental caries among students of dentistry colleges in holy karbala governorate in iraq in 2017 ali al mousawi,a* basheer al ali,a and zainab al mousawib acollege of medicine, university of kerbala, kerbala, iraq. bcollege of dentistry, university of kerbala, kerbala, iraq. *corresponding author: ali al mousawi (e-mail: aalmousawi1@hotmail.com) (submitted: 25 october 2018 – revised version received: 28 november 2018 – accepted: 20 february 2019 – published online: 26 june 2019) introduction oral health is an integral component of general health and carries a great impact on the quality-of-life.1,2 dental caries is the most common preventable oral diseases affecting all age groups all over the world.3 it was discovered at a prevalence rate of 2–48% in an archaeological populations.4 the global burden of dental caries i of 187 countries between 1990 and 2010 was estimated in a systematic review which concluded the incidence and prevalence of dental caries remained static during this period. in addition, the most prevalent condition worldwide was untreated caries in permanent teeth affecting 2.4 billion people, while untreated caries in deciduous teeth was the tenth-most prevalent condition, affecting 621 million children, worldwide.1 however, dental decay is preventable and there has been a steady decline in its prevalence since decades in the developed countries.5 experts believe, that this decline was primarily due to the introduction of fluoride therapies after the 1960s that had a huge impact on dental decay rates.6–9 on the other hand, the prevalence in the developing countries is on rise, because of absence or poor quality of oral public health services. a major part of population in these countries has bad and neglected oral health caused by many different risk factors related to socio-economic and educational levels and difficult or no access to professional dental care.10 while a small part of population, with higher socio economic state and good home care and better access for professional services since childhood, shows low caries index.11 the main problem in dental caries is that its initial stages are asymptomatic; with symptoms appearing after the carious lesion has progressed into dentine.12 for this reason, the reported prevalence rates of tooth decay might represent only the tip of the iceberg and the actual size of the problem might be profoundly greater that shown rates. the decayed missing filled surfaces/teeth (dmft) index is well established and most commonly used measure of caries experience in dental epidemiology.13 it was developed by klein et al.14 the world health organization recommended its use for oral health surveys,15 and had introduced some modification on the index calculation to improve its sensitivity and practical use in epidemiological studies.larmas16 counted more than 7000dmft index publications in pubmed, which indicated that it is the most commonly used oral health index. the advantages of dmft index are too many and include: being simple and clear in addition to its high validity and reliability and acceptable sensitivity and specificity.17 however, dmft index fails to provide information on the extent and clinical consequences of untreated dental caries, such as pulpal involvement and dental abscess, which may be more serious than the caries lesions themselves.13 it also describes both past and present caries experience, which is not the same as caries prevalence. the index carries some limitations such as: being not indicating the number of teeth that are at risk; it might be invalid in older patients because some teeth are lost for the reasons other than caries; it might be misleading in children because teeth may be lost for orthodontic reason and finally being not significant in the root caries.17 the targeted population in this study was a sample of individuals with good dental status socio-economical level and access to dental care. the results of this study and similar studieswill provide a data base for oral health among a group with lower caries incidence. gathered information might objectives this study was done to determine the prevalence of dental caries and treatment conducted among undergraduate students (18–24 years) in holy karbalagovernorate in iraq. additionally, the selected sample was from two dentistry colleges (public and private) students who will be responsible for managing public oral health services in the country. methods a total number of 288 dentistry colleges (holy kerbalapublic dentistry college and ibin hayan private dentistry college) students were asked through self-administered questionnaire about dental caries, filling and extracted tooth. dental caries index was calculated by application of decayed missing filled surfaces/teeth (dmft)/dmfs index, following the criteria of the world health organization. analysis depended on statistical package for the social sciences-23 and amos statistical packages at a significance level of 0.05. results the mean age of the students was 21.42 ± 136 years. females formed about two-thirds of the sample (61.1%). the prevalence of dental caries was 72.9% with a mean dmft, dmfs values 3.30 ± se 0.091, 4.94 ± se 0.161, respectively. the prevalence of dental caries reported by cdc in the usa in 2011/2012 among adolescents aged 16–19 (67%). the mean caries, filling and extracted tooth values were 0.597 ± 0.96 for extracted tooth, 1.180 ± 1.4043 for carried tooth and 1.735 ± 1.9942 filled tooth, while the mean dmf was 3.15 ± 2.85. the proportion of students with caries, filling and extracted tooth were 46.5%, 54.07% and 31.6%, respectively. no statistically significant difference was found for dmf and for each component prevalence rate between the public and private dentistry college students, or among different genders. conclusion a relatively high caries – experience was present among undergraduate students in the dentistry colleges (public and private) in holy karbalagovernorate indicating the need for efficient preventive programs early in school age and through university students. keywords dental caries, undergraduate students, dentistry colleges, dmft, iraq issn 2413-0516 155j contemp med sci | vol. 5, no. 3, may–june 2019: 154–159 original the prevalence of dental caries among students of dentistry colleges in holy karbala governorate in iraq in 2017a. al mousawi et al. reflect the oral health condition of the whole population and what could be achieved by systemic care and prevention of dental caries in whole population.10 the annual report of the iraqi ministry of health in 2017 reported dental caries among the top 10causes of outpatient visits in iraq, with a percentage of 2.1% of total visitors.18 most published studies about the prevalence of dental caries in iraq were targeting school age children,19–21 and this is similar to other developing countries,22 and only few researches involved adult population,19,23–27 and only few studies investigated dental students.28–31 a study among 12-year-oldschool children in baghdad/iraq in 2009 reported a dmft value of 1.5. the researcher compared his findings to previous reported data reporting dmft among school children in baghdad at 1.23 in 1985 and then was greatly decreased to 1.1 in 1990 and to 0.6 in 1998 and 0.63 in 2003. these years were during the period of economic blockade or sanction imposed on iraq between 1990 and 2003 where sugar consumption was very low.21 materials and methods a cross-sectional descriptive study using self-administered questionnaire about oral health and knowledge and practice about interdental cleaning measures (icm) was used among a total number of 288 students in two dentistry colleges in holy karbala in iraq in april 2017. the university of kerbala was established since 16 years and include 16 colleges,32 while ibin hayan college was established since 2010. ethical approval and permission to conduct the survey was obtained before starting the study and the students were informed about the voluntary choice of participation in the study. the questionnaire consisted of 21 questions designed to evaluate the oral health, knowledge and practice about icm among the bds students. the questionnaire was piloted before conducting the definitive study among 10students and minor changes were introduced. the students were invited from kerbala dentistry college (kdc) and dental section in ibin hayan private dentistry college (ihc) in holy karbala in iraq, to participate in this survey using a self-administered structured questionnaire. the voluntary participation and anonymous-confidential nature of the questionnaire was made clear to the students. introductory talk to all the students and explanation regarding the nature and purpose of the study was given by the researcher. the questionnaire was written in arabic and organized into four parts: the first part elicited information on the demographic attributes of students including age, gender, college, and study year. the second part assessed students’ oral health by asking about possessing carried tooth, extracted tooth or filled tooth due to dental decay and also asked about the number of these teeth. the third and fourth parts assessed the participant’s oral health knowledge about icm and one closed and one open question, while the last part explored the use of these measures. the second part was used to elicit their dmft index oral while the next two parts were used to determine students’ knowledge and practice scores through summation of positive answers. the students were asked to respond to each item according to the response provided in the questionnaire. responses included multiple-choice (closed end) questions, in which the students were instructed to choose only one appropriate response from a provided list of options. whilst, open end questions allowed participants to enumerate their answers without any list of correct answer, to eliminate false positive answers. about 288 completely filled questionnaires from dental students were collected and analyzed. the obtained data were analyzed using the statistical package for the social sciences (spss) software for windows version 23.0 (ibm spss, spss inc., chicago, il, usa). excel data base was used to calculate dmft and caries prevalence rates and these dependent variables were assessed in terms of different independent variables in the study. the process of analysis used chi-square test of indifference; t-test;correlation and regression analysis for demographic, oral health indices, and knowledge and practices scores among dental students. a p-value of <0.05 was used as a cut-off level for statistical significance. correlation between knowledge and practice were examined by karl pearson’s correlation coefficient method. in addition simultaneous predictors effects was explored through structural equation model analysis using the mean percentage scores. results the students in the public college (kdc) formed three quarters (213 students, 74%) of the sample and the remaining were from the private college (ihc, 75 students, 26%). females formed about two-thirds of the sample (176 students, 61.1%). the main bulk (44%) of the students was from the third study year (table 1). females formed a significantly higher proportion in kdc compared with ihc (fig. 1). fig. 1 the gender distribution of undergraduate dentistry college students in holy karbala in iraq in 2017 (n = 288). table 1. the demographic characteristics of the undergraduate dentistry college students in holy karbala governorate in iraq in 2017 (n = 288) variable category frequency percentage (%) college holy kerbala dentistry college 213 73.96 ibin hayan dentistry college 75 26.04 study year second 14 4.86 third 127 44.10 fourth 81 28.13 fifth 66 22.92 gender male 112 38.89 female 176 61.11 total 288 100.00 156 j contemp med sci | vol. 5, no. 3, may–june 2019: 154–159 the prevalence of dental caries among students of dentistry colleges in holy karbala governorate in iraq in 2017 original a. al mousawi et al. the mean age of the students was 21.42 ± 1.36 years and males had a significantly (p = 0.008) higher mean age than females (21.68 ± 1.48 years and 21.26 ± 1.25 years, respectively). similarly, the mean age of students in the private college (ihc) was significantly higher than the mean age of students at the public college (22.24 ± 1.39 years, 21.14 ± 1.22 years, respectively, p < 0.001).the proportion of students with caries, filling and extracted tooth among the total sample were 48.0%, 54.1% and 31.7%, respectively (table 2). the prevalence of dental caries among the total sample was 69.9%, while the proportion of the students free from lifetime caries was 30.1%. gender differences in these proportions were not significant. no significant gender difference was discovered in the prevalence of tooth caries, filling or extraction (table 3). comparison of the prevalence of tooth caries, filling or extraction between the two colleges showed no significant differences between the students in the two colleges (table 4). the mean numbers of teeth with caries, filling and extracted tooth among the total sample were 1.19 ± 1.41, 1.74 ± 1.99 and 0.4860 ± 0.96 teeth, respectively. comparison of the detailed numbers of these pathological findings among males and females showed no significant differences (table 2). on the other hand, comparison of these indices between the two colleges showed highly significant differences (table 5). the mean dmft value for the total sample was 2.65 ± 2.70 and there was a highly significant difference between kdc and ihc (2.74 vs. 2.27, p < 0.001). however, no statistically significant gender difference was discovered (2.71 vs. 2.93 for males and females, respectively, p = 0.587). while a significant difference was discovered between the study years through anova test (p = 0.004). the mean dmft value for the second, third, fourth and fifth study years were 0.50, 1.48, 2.36 and 2.40, respectively. further analysis tried to assess the situation of the mostly affected proportion of the students using the significant caries (sic) index which is concerned with the worst third of the total population. the mean sic was 4.69 ± 2.41 tooth and there was no significant difference between kdc and ihc (4.4056 vs. 6.00, p = 0.072). additionally, no significant gender difference was found (4.56 vs. 4.84, p = 0.413); nor there was any significant difference between the study years (p = 0.104). this study included a section exploring student knowledge and use of interdental cleaning measures (idm) which showed significant differences across the genders, colleges and study years. the mean knowledge score for kdc students was significantly higher than ihc students (0.47 ± 0.89 point vs. 0.91 ± 1.12 point, p = 0.003). the gender difference was also significant toward female students vs. male students (0.92 ± 1.11 point vs. 0.60 ± 1.00 point, p = 0.013). however, the correlation between the knowledge score and dmft was very weak and not significant (r = 0.046, p = 0.437). on the other hand, other indicators were included to determine the use of idm where the mean use score was 0.41 ± 0.65. on comparing the use score between the colleges and genders, no significant differences were discovered. the mean use score for kdc students was significantly higher than ihc students (0.42 ± 0.63 point vs. 0.36 ± 0.71 point, p = 0.513), while mean male use score was 0.35 ± 0.60 point vs. 0.45 ± 0.68 for females, p = 0.217. similarly very weak correlation was found between the use score and dmft and was not significant (r = 0.065, p = 0.271). a further step was undertaken in the analysis to determine the instantaneous impact of knowledge and use of idm on dmft through the use of structural equation model (sem) by amos software. the model showed that the highest impact as shown by the highest regression coefficient, was for the use of idm (0.94), while knowledge imposed a minor effect of less than one half of use effect (0.41). the impact of gender and age were almost similar top knowledge, while the colleges impact was very small (fig. 2). table 4. the prevalence of tooth extraction, caries and filling of undergraduate dentistry college students in holy karbala in iraq in 2017 (n = 28, frequency and percentage -in brackets-) college have extracted tooth have carried tooth have filled tooth total holy kerbala dentistry college 68 (31.9) 105 (51.0) 112 (57.1) 285 ibin hayan dentistry college 23 (31.5) 30 (40.5) 33 (45.8) 86 total 91 (31.7) 135 (48.0) 145 (54.1) 371 significance 0.974 0.142 0.199 index college mean std. deviation significance number of extracted teeth holy kerbala dentistry college 0.46 0.84 <0.001 ibin hayan dentistry college 1.86 1.17 number of tooth with caries holy kerbala dentistry college 1.03 1.28 <0.001 ibin hayan dentistry college 2.21 1.77 number of tooth with filling holy kerbala dentistry college 1.52 1.97 <0.001 ibin hayan dentistry college 2.91 1.74 table 2. the prevalence of tooth caries, filling or extraction among undergraduate students in holy karbala governorate in iraq in 2017 (n = 288) variable frequency percentage (%) have carried tooth 135 48.0 have filled tooth 146 54.1 have extracted tooth 91 31.7 table 3. the gender distribution of the prevalence of tooth extraction, caries and filling of undergraduate dentistry college students in holy karbala in iraq in 2017 (n = 288 frequency and percentage -in brackets-) gender have extracted tooth have carried tooth have filled tooth male 34 (30.4) 55 (51.4) 59 (54.6) 285 female 57 (32.6) 80 (46.0) 86 (53.8) 86 total 91 (31.7) 135 (48.0) 145 (54.1) 371 significance 0.694 0.377 0.887 157j contemp med sci | vol. 5, no. 3, may–june 2019: 154–159 original the prevalence of dental caries among students of dentistry colleges in holy karbala governorate in iraq in 2017a. al mousawi et al. however, when the use score was removed, the sem model showed a greater impact (2.23) inthe difference between college students in the sample (fig. 3). discussion the problem of tooth decay is not a local oral health problem but is a systemic disease with impact on physical built self esteem and mental health.2 it carries a heavy burden on human being health and a great economic and sociocultural impact. dental health professionals have an important role in the improvement of the public’s health education level. for this reason, the acquisition of knowledge and attitudes relating to dental health and the prevention, control, and treatment of dental problems during the future dentists’ training period is very important.3 at present, it is assumed that the decrease in the prevalence of dental caries in many population groups is also related to a reduction in the activity and the speed of progression of the carious lesions.4 this has led to a change in the dentists’ approach to dental care, which is more oriented toward prevention rather than restoration to avoid or postpone invasive treatment. this study has indicated a serious finding regarding the high lifetime prevalence of caries (69%) among dental students in holy karbala governorate. the important issue is that these students represent a group characterized by good dental status and knowledge of etiology and prevention of dental caries. however, knowledge alone is not enough unless it is adopted as a behavior. the process of adopting any change in dental care attitudes about personal dental care must be learned and practiced throughout study years in the dentists’ learning process, especially during their undergraduate training in the dental college.6 for this reason, a study of how dental students adopt this knowledge and convert it to their own oral health care during their study could, in fact, be of a great importance since the students are the ones who will apply these same behavior patterns to their patients in their practices in the future. on the other hand, the findings in this study might indicate that the problem is much worse among students in other colleges and this might need to explore in further studies in the future. fig. 2 the results of structural equation model of the impact of gender, age, study year knowledge and use of interdental cleaning measures and college on dmft index among undergraduate dentistry college students in holy karbala in iraq in 2017. dmft, decayed missing filled surfaces/teeth. fig. 3 the results of structural equation model of the impact of gender, age, study year knowledge about interdental cleaning measures and college on dmft index among undergraduate dentistry college students in holy karbala in iraq in 2017.dmft, decayed missing filled surfaces/teeth. another noteworthy finding was the difference between the private and public college, in the benefit of the later. the students in the private colleges in iraq are usually from families of higher socioeconomic level as they could offer payment of around 10000 us$/year to these colleges. from these results, one can conclude that the worthy people might be worse than lower social class population in protecting their children’s oral health. similar four previous studies among dental students were published previously. in the first and most recent, the oral health indices were compared between fifth and first study year students in the dental college in mustansiria university. the sample included 50 students in the first and 60 students in the sixth study year (55 males and 55 females). the dmft means were significantly higher among females than males (6.48 for males and 7.08 for females) in first year. while in the fifth year students the means were 8.73 for males and 9.16 for females. the prevalence of tooth brushing, mouthwash, dental floss, and tooth picks using for the fifth year students were higher than first year students.30 whilst a previous similar study in the same college examined 30 students from each of the five study year students (75 males and 75 males) and reported almost similar findings. dental caries prevalence was 100% (none of the examined students were caries free). however, dmfs value was decreasing with advancing study year and was attributed by the researcher for better knowledge and awareness about dental health among senior students in comparison with freshmen. there was no significant difference between all the study years, and increased in filling score values with a significant difference between females in all study years.33 the gender difference found in this study was not significant and is consistent with most reviewed findings; however, the above two mentioned results reported two contradictory findings. the third study year showed reversed gender prevalence among 250 dental students (150 females and 100 males) in the college of dentistry in baghdad university in 2011. the students answered a self-administered questionnaire; where 75 students (30%) reported having dental caries and 70 students (25%) reported having tooth filling. the study found that females had better oral hygiene practices, significantly less 158 j contemp med sci | vol. 5, no. 3, may–june 2019: 154–159 the prevalence of dental caries among students of dentistry colleges in holy karbala governorate in iraq in 2017 original a. al mousawi et al. self-reported oral bad breath (40%vs.70%). it was found that smoking and presence of dental caries had statistically highly significant correlation with halitosis.31 the fourth study year surveyed 450 students from nine colleges in mosul university (50 students from each college) in 2004. the study concluded that the students have acceptable dental health knowledge. however, more than half of the students (54.6%) had gingival bleeding but they did not know the cause of bleeding (75.1%) and how to avoid it (75.5%). a large percent of them had no ideas about the causes of dental caries (75.5%), and how to avoid it (76%). their knowledge was poor regarding the age at which the primary and permanent teeth erupted (23.8%, 22.8%) and completed (18.6%, 27.3%).34 few other studies reported adult dental caries indices from outpatient clinics in iraq. a study at mustansiriyah university outpatient clinic showed that dental caries was the main cause behind teeth loss among 584 adult patients visiting the outpatient clinic in the dental college in al-mustansiriyah university in baghdad in 2013. teeth loss was more prevalent among males, however, no significant gender difference was found but significant association with age was discovered.27 a survey among 300 dental outpatient clinic visitors in najaf city (central part of iraq) showed that two thirds of them have acceptable dental health knowledge, and a great majority (91%) have good dental behavior as teeth brushing of at least once a week.26 a study in tikrit city found that the mean dmft scores were 7.5 (8.3 for female and 6.7 for male). mean dmft for both sex increases with age. the percentage of decayed teeth dmft percentage was the highest among younger age group (60.7% for female and 63.1% for male). there was statistically a highly significant difference between age, sex, dental visit type and brushing behavior and dmft.19 for similar studies in other parts of the world, a meta analysis study in saudi arabia estimated dmft in saudi arabia at 3.34 and the prevalence of dental caries was reported to be rising with age (91% for age group 12–19 years; and dmft of 7.35 while, it was 98% for age group 30–45 years; and dmft of 14.5).35 while, a study among 320 medical and dental students in serbia in 2007 reported a mean dmft value of 12.8 ± 4.7 with only one student with a value of zero.36 a second study in northern brazil city said to be not served by restorative dental services, among 889 students aged 15–19 years showed that the dmft index was 4.65 ± 0.12, and the prevalence of dental caries was 87.4%.22 conclusion this study revealed high dental caries prevalence among dental students in karbala governorate with no gender difference. however, the private college students showed higher prevalence rates and indices. as these students represent important dental health care providers in the near future; at most attention need to be given to improve future dentist knowledge and practice in iraq. conflict of interest none.  references 1. kassebaum nj, bernabé e, dahiya m, bhandari b, murray cj, marcenes w. global burden of untreated caries: a systematic review and metaregression. j dent res. 2015;94:650–658. 2. li lw, wong hm, gandhi a, mcgrath cp. caries-related risk factors of obesity among 18-year-old adolescents in hong kong: a cross-sectional study nested in a cohort study. bmc oral health. 2018;18:188. 3. frencken je, sharma p, stenhouse l, green d, laverty d, dietrich t. global epidemiology of dental caries and severe periodontitis a comprehensive review. j clin periodontol. 2017;44:s94–s105. 4. humphrey lt, de groote i, morales j, barton n, collcutt s, ramsey cb, et al. earliest evidence for caries and exploitation of starchy plant foods in pleistocene hunter-gatherers from morocco. proc natl acad sci u s a. 2014;111:954–959. 5. marthaler tm. changes in dental caries 1953-2003. caries res. 2004;38: 173–181. 6. cury ja, tenuta lm, ribeiro cc, paes leme af. the importance of fluoride dentifrices to the current dental caries prevalence in brazil. braz dent j. 2004;15:167–174. 7. bratthall d, hänsel-petersson g, sundberg h. reasons for the caries decline: what do the experts believe? eur j oral sci. 1996;104:416–422. 8. petersen pe, lennon ma. effective use of fluorides for the prevention of dental caries in the 21st century: the who approach. community dent oral epidemiol. 2004;32:319–321. 9. cortes fj, nevot c, ramon jm, cuenca e. the evolution of dental health in dental students at the university of barcelona. j dent educ. 2002;66: 1203–1208. 10. pavleova g, vesela s, stanko p. prevalence of dental caries in dentistry students. bratisl lek listy. 2015;116:93–95. 11. birkeland jm, haugejorden o. caries decline before fluoride toothpaste was available: earlier and greater decline in the rural north than in southwestern norway. acta odontol scand. 2001;59:7–13. 12. selwitz rh, ismail ai, pitts nb. dental caries. lancet. 2007;369:51–59. 13. broadbent jm, thomson wm. for debate: problems with the dmf index pertinent to dental caries data analysis. community dent oral epidemiol. 2005;33:400–409. 14. klein h, palmer ce, knutson jw. studies on dental caries: i. dental status and dental needs of elementary school children. public health rep. (1896–1970). 1938;53:751–765. 15. world health organization. oral health surveys: basic methods. world health organization, geneva, switzerland, 2013. 16. larmas m. has dental caries prevalence some connection with caries index values in adults? caries res. 2010;44:81–84. 17. jakobsen jr, hunt rj. validation of oral status indicators. community dent health. 1990;7:279–284. 18. annual statistical report, iraqi ministry of health. ministry of health, iraq, baghdad, 2017. 19. abduallah ha. experience of dental caries of adult patients in relation to the characteristic of dental visit and brushing behavior in tikrit city. mustansiria dent j. 2013;10:17–27. 20. al-ghalebi sn, el-samarrai sk. oral health status and treatment needs in relation to nutritional status among 9-10 year-old school children in nassiryia city/iraq. j baghdad coll dent. 2012;24:133–137. 21. aljourane ts, rabee mk, alwan am. evaluation of dmf in baghdad after years 2003. mustansiria dent j. 2009;6:129–133. 22. rebelo ma, lopes mc, vieira jm, parente rc. dental caries and gingivitis among 15 to 19 year-old students in manaus, am, brazil. braz oral res. 2009;23:248–254. 23. al-nuaimy kmt. dental health status among adult population in mosul city. tikrit j dent sci. 2015;3:105–111. 24. khamis mh, al-huwaizi r. severity and prevalence of caries experience in najaf city. j baghdad coll dent. 2010;22:129–132. 25. al-ani rs. tooth loss in adult urban population in ramadi city, iraq. alanbar med j. 2009;7:118–123. 26. ibraheem sa-r. dental health knowledge and behavior in al –najaf city. kufa j nurs sci. 2012;2:116–122. 27. al kotobe mf. tooth loss, prosthodontic treatment need and association factors in a sample of adults attending college of dentistry,al mustansiriya university. al-rafidain univ coll sci. 2013:125–136. 28. jazrawi kh. evaluation of the sequelae of untreated dental caries using pufa index. al-rafidain dent j. 2014;14:101–110. 159j contemp med sci | vol. 5, no. 3, may–june 2019: 154–159 original the prevalence of dental caries among students of dentistry colleges in holy karbala governorate in iraq in 2017a. al mousawi et al. 29. almas k, al-hawish a, al-khamis w. oral hygiene practices, smoking habit, and self-perceived oral malodor among dental students. j contemp dent pract. 2003;4:77–90. 30. mahmood aa. comparison of oral health status and behaviorbetween first and fifth years of al-mustansiriyah dental students. j baghdad coll dent. 2017;29:71–77. 31. hasan ga. oral hygiene practices and self-perceived halitosis among dental students. j baghdad coll dent. 2014;26:58–62. 32. al mousawi a. war-related trauma and post-traumatic stress disorder prevalence among undergraduate students in iraq in 2010. iraqi j public health. 2017;1:35–41. 33. mahmoud mk, al-ubaidi rs. dental caries severity between students in al-mustansiria university / college of dentistry. mustansiria dent j. 2011;8:24–28. 34. gasgoos ss, jazrawi kh, al-ajrab mg. dental health knowledge, attitude and behavior among first year university students, mosul. al-rafidain dent j. 2007;7:138–152. 35. al agili de. a systematic review of population-based dental caries studies among children in saudi arabia. saudi dent j. 2013;25:3–11. 36. stojanović n, krunić j. caries prevalence in medical and dental students in foca municipality. stomatol glas srb. 2007;54:89–96. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201907 332 j contemp med sci | vol. 5, no. 6, november–december 2019: 332–335 original issn 2413-0516 introduction gallstones are one of the most common diseases of the gastrointestinal tract. most of these stones remain asymptomatic throughout the patient’s life. for unknown reasons, some patients progress to a symptomatic stage that develops biliary colic following cystic duct obstruction by gallstones. this may lead to complications including cholecystitis, chronic cholecystitis, cholangitis, pancreatitis, fistula between the gallbladder and part of the intestine, and eventually ileus caused by gallstones and gallbladder carcinoma.1, 2 therefore, if bile stones develop or are symptomatic or in conditions that are more likely to cause these complications, cholecystectomy is required. this can be done in an open procedure or in a so-called laparoscopic procedure.3 the use of this method has been growing increasingly so that it has been replaced by the open method of choice4 due to it its many benefits and fewer side effects such as reduced post-operative pain, faster return to work, reduced hormonal neurotoxicity, patients’ satisfaction with the appearance of the scar and the less invasive nature of laparoscopic surgery than open surgery. the use of this method has been growing increasingly so that it has been replaced as an alternative choice method. in this way, it is necessary to dilate the space inside the abdomen to create the proper space for working with the necessary devices and tools. therefore, various gases such as nitrogen, helium and co2 are used for dilution, each with its advantages and disadvantages. the most common gas used in laparoscopy is co2 because it is non-combustible, absorbs rapidly in dissolved blood and is excreted through the respiratory tract.5, 6 arterial carbon dioxide (paco2) pressure is one of the most important determinants of blood ph, so its changes can cause many disorders for patients. since the probability of this change was high during anesthesia and it’s not possible to monitor paco2 directly, during anesthesia, et-co2 expiratory co2 pressure monitoring is used to estimate paco2. it is nowadays one of the standard monitoring methods during anesthesia and is often used as a non-invasive procedure for patients during anesthesia as well as in recovery and intensive care units.7 according to the astm international (formerly known as american society for testing and materials), the measurement of co2 pressure is now mandatory monitoring and capnography is a standard anesthesia monitoring.8, 9 continuous measurement of exhaled co2 is one of the methods that are used in the operating room for evaluation during anesthesia and in patients intubated in the tracheal intubation. but this approach can even be a non-invasive, rapid, and reliable method for predicting paco2 in non-intubated patients. 10 this measurement enables the estimation of paco2 pressure without the need for arterial blood sampling. if there is a consistent relationship between the co2 pressure and the arterial end, this method is reliable and there will be no need for repeated arterial blood sampling.11, 12 so, the aim of this paper is to determining the end-expiratory dioxide pressure in gallbladder laparoscopic surgery and compares it with the paco2 pressure. methods and materials this cross-sectional study was performed on 30 patients undergoing laparoscopic cholecystectomy. they were randomly assigned to kowsar hospital of semnan, iran in 2018– 2019. all stages of the study were approved by the research investigating the partial pressure of carbon dioxide (co 2 ) in the respiratory gases in laparoscopic gallbladder surgery and comparing it with arterial partial pressure of carbon dioxide babak hosseinzadeh zoroufchi,a setareh soltany,b abolfazl abdolahpoura adepartment of anesthesiology, kowsar hospital, semnan university of medical sciences, semnan, iran bcancer research center, kowsar hospital, semnan university of medical sciences, semnan, iran corresponding author: abolfazl abdollahpour (email: abolfazlabdollahpoor@semums.ac.ir) (submitted: 19 september 2019 – revised version received: 13 october 2019 – accepted: 25 october 2019 – published online: 26 december 2019) objective the aim of this paper is determining the end-expiratory dioxide pressure in gallbladder laparoscopic surgery and compares it with the arterial carbon dioxide pressure. methods this cross-sectional study was performed on 30 patients undergoing laparoscopic cholecystectomy. at the beginning of operation, arterial blood gas (abg) sample was taken from the patient’s radial artery before co 2 was injected into the abdomen. at the same time, co 2 was measured by a capnography device. at the end of surgery, abg sample was prepared for the second time before co 2 was removed from the abdomen and co 2 was recorded simultaneously by capnography device. after collecting data from abg samples, arterial paco 2 was compared with those obtained from capnography device results and spss 16 software was used for data analysis. results the mean pre-operative paco 2 for laparoscopic (paco 2-1 ) was 34.343 and the mean pre-operative etco 2 for laparoscopic (etco 2-1 ) was 31.37. these values after laparoscopic surgery were 34.813 for paco 2 , 34.813 (paco 2-2 ) and 33.13 (etco 2-2 ). there was also a correlation between paco 2-1 and etco 2-1 results between paco 2-2 and etco 2-2 , which was stronger between paco 2-2 and etco 2-2 . conclusion there was a strong correlation between etco 2 results from capnography and paco 2 from abg and to monitor carbon dioxide retention, capnography can be used as an alternative to abg for laparoscopic gallbladder surgery patients. key words capnography, end-expiratory pressure, arterial carbon dioxide pressure, cholecystectomy, laparoscopy 333 original investigating the partial pressure of carbon dioxidebabak hosseinzadeh zoroufchi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 332–335 and ethics committee of kowsar hospital in semnan. all patients underwent the implementation of the plan before entering the study and a consent form was obtained from all patients. the inclusion and exclusion criteria were evaluated in this study. • inclusion criteria: cholecystitis patients candidate for laparoscopic surgery. • exclusion criteria: patients with lung obstruction diseases such as asthma, emphysema, copd, and pulmonary embolism. demographic data including age, sex, height, weight, and smoking were recorded. after visiting patients at the clinic, pre-op and recording heart rate, respiratory rate, and blood pressure were measured. patients underwent laparoscopic cholecystectomy under general anesthesia with intravenous induction and maintenance of general anesthesia with inhaled and evaporated anesthetics. at the beginning of surgery before the co2 gas is pumped into the abdomen, sampling site was sterilized with 70% alcohol in order to obtain the arterial blood gas (abg) sample after the allen test to ensure proper flow of the sample in the hand. it was then prepared using a heparinized g20 syringe from the abg patient’s radial artery. at the same time, co2 was measured by a capnography and to facilitate laparoscopic surgery, the intraperitoneal space was filled with co2 up to a pressure of up to 20 cm. exposure to co2 was monitored by capnography at all stages of surgery. at the end of surgery, abg sample was prepared for the second time before co2 removal from the abdomen and at the same time, co2 was measured and recorded by the capnotrue® asp co2/ spo2 monitor capnography. abg samples were immediately sent to the laboratory and analyzed by a blood gas analyzer (avl995 blood gas analyzer) and paco2 levels were measured and recorded. after collecting data from abg samples, arterial paco2 levels were compared with those obtained from capnography results. at the end of the operation, the patient was awakened by neostigmine at the rate of 40 mg/kg after discontinuation of the anesthetic and reversal of the relaxant and transferred to recovery. data analysis descriptive findings were reported in subgroups using mean and standard deviation. multivariate regression models were used to investigate the relationship between arterial co2 pressure and co2 pressure with and without underlying variables and the final analysis was performed on the reduced model. to analyze the difference between the two aforementioned values, one-sample t-test was used and compared with 0. the significance level for all tests was 0.05. spss 16 software was used for data analysis. ethical considerations obtain informed consent to adhere to ethical principles and ensure confidentiality of research information. result twenty-four patients participated in this study which 80% were female and 20% were male. the mean age of the subjects was 41.77 ± 13, 13.89 years. the youngest was 26 years and the oldest was 80 years old. the mean height, weight, and bmi of the patients were 163.5, 70.5 and 26.35, respectively. also, 28 (93.3%) were non-smokers and 2 (6.7%) were smokers. in this study, systolic blood pressure (sbp) and diastolic blood pressure (dbp) and mean arterial blood pressure (map) were reported as 18, 76, and 90, respectively. the mean pre-operative paco2 for laparoscopic surgery was 34.343 paco2-1 and the mean pre-operative etco2 for laparoscopic surgery was 31.37. these values for post-operative laparoscopy were 34.813 for paco2, paco2-2 and 33.13 for etco2, etco2-2. the mean and standard deviation of the difference between paco2-1 and etco2-1 were 2.9 and 4.11, respectively. these values were 1.6 and 3.72, respectively, for the difference between paco2-2 and etco2-2, with a p-value of less than 0.05 for both cases. according to table 1, there was a correlation between paco2-1 and etco2-1 results as well as between paco2-2 and etco2-2. this correlation was stronger between the values of paco2-2 and etco2-2. the regression equation based on the final model is as follows: paco2–2 = – 34.676 + 0.885 etco2 – 2 + 6.030 sex 7.088 smoker + 0.267pr table 1. correlation between paco 2 -2 and etco 2 -2 results before laparoscopic surgery pearson correlation coefficient 0.423 p-value 0.020 quantity 30 table 3. correlation between paco 2 -2 and etco 2 -2 results after laparoscopic surgery pearson correlation coefficient 0.720 p-value 0.000 quantity 30 table 2. distribution graph and its fitted line based on the regression equation for predicting paco 2-1 using etco 2-1 squared r f degree of freedom 1 degree of freedom 2 p constant number the regression coefficient 0.179 6.095 1 28 0.020 19.451 0.475 table 4. distribution graph and its fitted line based on the regression equation for predicting paco 2-2 using etco 2-2 squared r f degree of freedom 1 degree of freedom 2 p constant number the regression coefficient 0.519 30.180 1 28 0.000 6.430 0.857 334 original investigating the partial pressure of carbon dioxide babak hosseinzadeh zoroufchi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 332–335 discussion co2 gas is one of the most common gases used in laparoscopic surgery and its monitoring during surgery is very important. the importance of monitoring co2 pressure is that changes in blood can cause changes in the blood ph of the patient and cause problems for the patient.13 abg measurement is used as the gold-standard for monitoring oxygenation and also checks the co2 retention rate. 14, 15 the aim of this paper is determining the end-expiratory dioxide pressure in gallbladder laparoscopic surgery and compares it with the arterial carbon dioxide pressure. in this study, there was a strong correlation between paco2 and etco2 results before and after surgery in gallbladder laparoscopic patients. in the studies that have been done, such as the study by husaini and cho16 in which 35 patients underwent intraoperative craniotomy in different surgical stages, the evidence suggests that the results of capnography agree with abg at all stages of craniotomy, both before general anesthesia and after skull opening and at the beginning of dural closure. and, there was a strong correlation between etco2 and paco2 results at all stages of craniotomy (correlation coefficients were 0.571, 0.559, and 0.629). the results of their study were consistent with our study. in another study by yazdani and tohidi, 75 copd patients were included. they found that there was a strong correlation between etco2 values from capnography and paco2 in arterial blood gases at both the initial admission and 30 min after oxygen and bronchodilator treatment (correlation coefficient r = 0.782 and p = 0.005). further evaluation by bland–altman analysis indicated the agreement of the results of the two methods of capnography and abg for measuring the relative pressure of co2 in both stages of the study. the results of their study were in line with ours.17 hasani et al.11 conducted a study comparing end-expiratory and arterial co2 in patients undergoing coronary artery bypass grafting. they found that there was no statistical difference between the co2 and arterial end points at the time before and after cardiopulmonary bypass. as a result, capnography is a non-invasive, healthy monitoring to estimate arterial blood co2 levels. end-expiratory and arterial co2 have a direct relationship with patients with no underlying disease in coronary artery bypass graft surgery. they noted that measurement of etco2 in healthy patients may eliminate the need for arterial blood to determine paco2. warner et al.18 found that etco2 results in patients with severe trauma requiring tracheal intubation were poorly correlated with paco2 outcomes. therefore, capnography should not be used to monitor this group of patients, which seems to be due to the patient’s poor hemodynamic status and its effect on arterial blood gases, which were inconsistent with our study. conclusion this study indicated that although there was a significant average 3 and 1.6 unit of difference between the mean arterial co2 pressure and co2 pressure at the beginning and end of the operation but there was a strong correlation between etco2 results from capnography and paco2 from abg. and to monitor co2 retention in gallbladder laparoscopic surgery, patients can use capnography as an alternative to abg. finally, it is recommended that further studies be conducted with a larger sample size. references 1. shanker k b, steinbrook ra, brooks dc. arterial to end tidal carbon dioxide pressure difference during laparoscopic surgery in pregnancy. anesthesiology. 2000;93:370–3. 2. ferber j, juniewicz hm, lechowicz-głogowska eb, pieniek r, wroński j. arterial to end-tidal carbon dioxide difference during craniotomy in severely head-injured patients. folia med cracov. 2001;42(4):141–52. 3. szabo g, miko i, nagy p, brath e, peto k, furka i, gamal e. adhesion formation with open versus laparoscopic cholecystectomy: an immunologic and histologic study. surg endosc. 2007;21(2):253–7. 4. schietroma m, carlei f, cappelli s, amicucci g. intestinal permeability and systemic endotoxemia after laparotomic or laparoscopic cholecystectomy. ann surg. 2006;243(3):359–369. table 5. multiple linear regression model of initial and final decrease to predict arterial dioxide pressure after intervention based on explanatory variables model  primary model final reduced model the regression coefficient the standard error p the regression coefficient the standard error p constant number –74.474 48.263 0.134 34.676 11.704 0.007 etco 2-2 0.971 0.157 0 0.885 0.127 0 gender 5.021 2.405 0.05 6.03 1.666 0.01 age 0.024 0.059 0.691 bmi 0.042 0.182 0.819 smoker 5.677 3.778 0.149 7.088 2.875 0.021 number of breaths –0.194 0.234 0.417 heart beat 0.258 0.137 0.073 0.267 0.09 0.007 mean arterial pressure –0.003 0.074 0.973 arterial oxygen saturation percentage 0.446 0.492 0.375       335 original investigating the partial pressure of carbon dioxidebabak hosseinzadeh zoroufchi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 332–335 5. moazeni-bistgani m, mohammad ali-beigi f,shahrjerdi s. assessment of oral acetazolamide on postoperative pain after laparoscopic cholecystectomy. j shahrekord univ med sci. 2010;12(2):21–6. 6. liu c, fan s-t, lai ec, lo c-m, chu k-m. factors affecting conversion of laparoscopic cholecystectomy to open surgery. arch surg. 1996;13(1):98–101. 7. takano y, sakamoto o, kiyofuji c, ito k. a comparison of the end-tidal co2 measured by portable capnometer and the arterial p co2 in spontaneously breathing patients. resp med. 2003;97(5):476–81. 8. khan fa, khan m, abbasi s. arterial to end-tidal carbon dioxide difference in neurosurgical patients undergoing craniotomy: a review of practice. j pak med assoc. 2007;57(9):446–8. 9. belpomme v, ricard-hibon a, devoir c, dileseigres s, devaud ml, chollet c, et al. correlation of arterial pco2 and petco2 in prehospital controlled ventilation. am j emerg med. 2005;23(7):852–9. 10. yosefy c, hay e, naseri y, magen e, reisin l. end tidal carbon dioxide as a predictor of the arterial pco2 in the emergency department setting. emerg med j. 2004;21(5):557–9. 11. hassani e, farasatkish r, heydarpour e, totoonchi m, mahoori a. end tidal co2 versus arterial co2 monitoring in patients undergoing coronary artery bypass graft. tehran univ med j tums publ. 2009;67(9):650–4. 12. grenier b, verchere e, mesli abdelghani. capnography monitoring during neurosurgery: reliability in relation to various intra-operative positions. anesth analg. 1999;88(21):43–8. 13. bhavani shankar k, moseley h, kumar a, delph y. capnometry and anesthesia. can j anesth/j can anesth. 1992;39(6):617–32. 14. mcmahon a, baxter j, kenny g, o dwyer p. ventilatory and blood gas changes during laparoscopic and open cholecystectomy. br j surg. 1993;80(10):1252–4. 15. casati a, gallioli g, scandroglio m, passaretta r, borghi b, torri g. accuracy of end-tidal carbon dioxide monitoring using the nbp-75 microstream capnometer. a study in intubated ventilated and spontaneously breathing nonintubated patients. eur j anaesthesiol. 2000;17(10):622–6. 16. husaini j, choy yc. end-tidal to arterial carbon dioxide partial pressure difference during craniotomy in anaesthetized patients. med j malaysia. 2008;63(5):384–7. 17. yazdani r, touhidi mh. the correlation and level of agreement between arterial blood gas pco2 and end-tidal co2 in patients with chronic obstructive pulmonary disease exacerbation. razi j med sci. 2013;19(103):48–54. 18. warner kj, cuschieri j, garland b, carlbom d, baker d, copass mk, jurkovich gj, bulger em. the utility of early end-tidal capnography in monitoring ventilation status after severe injury. j trauma acute care surg. 2009;66(1):26–31. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201907 189 review j contemp med sci | vol. 5, no. 4, july-august 2019: 189–196 the application of salivary gland scintigraphy in quantitative analysis of xerostomia as a frequent salivary gland dysfunction: a review article hanieh kaviani and mina khayamzadeh* department of oral and maxillofacial radiology, faculty of dentistry, tehran university of medical sciences, international campus, tehran, iran. *correspondence to mina khayamzadeh (email: mkhayamzadeh@yahoo.com). (submitted: 14 march 2019 – revised version received: 11 april 2019 – accepted: 29 may 2019 – published online: 26 august 2019) introduction the decrement of salvia flow could cause the dryness of mouth that also called xerostomia. as mentioned by various researcher’s xerostomia might be a symptom of different medical situations, a secondary effect of applying different kind of remedies or may also be a side effect of neck or/and head radiation. additionally, as cited by them, it is not a dangerous disease. on the other hand, it might be associated with reduced function of salivary gland. however, radiation could influence the quality and quantity of saliva produced and could reduce the ph and also increase the viscosity.1 patients who suffer from xerostomia would experience some clinical incidence of difficulty in speaking, chewing and swallowing somehow this disorders could be occured with burning mouth syndrome (bms) prevalence, altered taste, halitosis, dryness in the mouth, inflammation of the tongue, peeled and cracked lips, oral thrush and tooth decay despite adequate health care of the mouth mucosa (table 1).2–4 so, xerostomia could decrease the life quality of patients who are affected with this disorder. xerostomia causing factors the fundamental xerostomia etiology could be divided into two categories that are mentioned in table 2: local factors and systemic disorders. systemic disorders that could cause xerostomia are such as infectious, autoimmune diseases and granuloma inflammation. on the other hand, local factors that could cause xerostomia are such as radiation therapy of neck and head, some drugs and also various factors that affect lifestyle. on the other hand, xerostomia could cause tooth decay, the mandible osteoradionecrosis and also ulceration of soft tissue.5,6 anyway, it could be seen that the xerostomia intensity is related to the applied radiotherapy dose and also the volume and size of gland irradiation. newly oral pilocarpine that are known as cholinergic agonists has been widely applied in post-radiation xerostomia treatment.2,7,8 it is a cholinomimetic drug which acts mainly as a muscarinic with beta adrenergic agonists activity. it also motivates exocrine glands, sweating, lacrimation and on the other hand could increase secretions of stomach and pancreas.9 the effectiveness of this remedy has been shown in various trials somehow about half of the patients signified satisfactory responses.2,7,8 radiotherapy as well as chemotherapy and surgery are the initial modalities in treatment of neck and head cancers. in spite of radiotherapy that could affect tumors, ionizing irradiation may damage the normal tissues that is placed in the radiation side. radiation by causing morphological and functional changes in oral mucosa and salivary glands could create xerostomia disorder.1 xerostomia in general is recognized as the subjective sense of unsatisfactory of oral dryness, which is associated with objective experiences of dysfunction of salivary gland. caglar et al.10 by working on scintigraphic evaluation of salivary gland dysfunction in patients with thyroid cancer after radioiodine treatment reported that oral mucosa dryness could be known as a symptom systemic disorder like sjögren’s syndrome, a secondary effect of anticholinergic agent, antiadrenergic agents that prevent the sympathetic nervous system activity, cytotoxic chemotherapy or/and it could be created as a side effect of neck and head region radiation therapy. radioiodine therapy is a helpful treatment that accomplished under salivary gland motivation to increase salivary gland function. anyway, under a condition when salivary gland motivated, damages of parenchyma could be seen after radioiodine therapy by using of quantitative salivary salivary gland scintigraphy (sgs) that is known as the most frequent non-invasive imaging test could be used for distinguishing dysfunction of salivary gland in patients who suffer from sjögren’s syndrome or/and in patients with thyroid cancer after applying radioactive iodine therapy. one of the main side effects of salivary gland scintigraphy (sgs) in patients with differentiated thyroid cancer, and also radiation therapy in patients who suffer from neck and head cancers is oral dryness that is called xerostomia. anyway, comprehensive futuristic information around conversions in function of salivary gland after sgs are rare. accordingly, the initial purpose of this study was to distinguish the side effects of sgs on motivated flow rate of oral saliva. as a result, patients after being treated may experience correlated syndromes like xerostomia that could cause oral dryness, sore throat, dental decay, variation in voice quality, bad functions of swallowing and chewing and also altered taste. in this comprehensive study, the author tried to review the published studies characterizing sgs technique in patients who suffer from xerostomia and also aimed to discuss around progresses made in the treatment of this disorder. additionally, for determining the ability of undertaking salivary gland scintigraphy for evaluation of salivary gland health in patients who suffer from xerostomia disorder, some new methods should be developed. keywords salivary glands, xerostomia, salivary gland dysfunction, review issn 2413-0516 190 j contemp med sci | vol. 5, no. 4, july-august 2019: 189–196 the application of salivary gland scintigraphy in quantitative review h. kaviani and m. khayamzadeh table 1 xerostomia clinical appearances appeared functional problems consequences of morphologic findings swallowing problems burning mouth syndrome chewing problems halitosis dry buccal mucosa inflammation of the tongue cracked and peeled lips speaking problems oral candidiasis taste changes tooth caries table 2 the fundamental xerostomia etiology and its differential diagnosis systemic factors local factors endocrinologic causes diabetes mellitus (dm) head and neck radiation n.a autoimmune diseases of thyroid autoimmune causes syndrome of sjögren rheumatoid arthritis systemic lupus erythematosus scleroderma initial cirrhosis of bile lifestyle factors tobacco use alcohol use dehydration heavy snoring mouth breathing upper respiratory tract infections infectious causes actinomycosis human immunodeficiency virus hepatitis c virus human t-lymphotropic virus type 1 virus cytomegalovirus epsteinbarr virus most frequent categories of drugs anticholinergic drugs antiparkinsonian drugs antidepressants drugs antipsychotics drugs antihistamines drugs antihypertensives drugs sedative agents antihiv drugs cytotoxic drugs granulomatous causes tuberculosis sarcoidosis antineoplastic drugs other systemic causes end-stage disease of kidneys hemochromatosis amyloidosis parkinson disorder ectodermal dysplasia the process of aging opioids gland scintigraphy. the dysfunction of salivary gland is one of the most frequent advents of sjogren’s syndrome (ss) patients that often occur after radioactive iodine (rai) therapy. ss is a persistent disease of immune system specified by lymphoid influence of salivary glands which could cause dryness of mouth mucosa.11,12 on the other hand, the dysfunction of salivary gland is one of the main difficulties of rai therapy in patients with thyroid cancer whose tumor is welldifferentiated. according to solans et al.13 researches around salivary and lacrimal gland dysfunction it could be seen that about 30–50% of patients who suffer from thyroid cancer and have treated with rai therapy may experience xerostomia syndrome after a long period of time. as cited by nishiyama et al.14 tc-99m pertechnetate as one of the main technetium radiopharmaceuticals being applied in thyroid imaging and salivary gland scintigraphy (sgs) for evaluating dysfunction of salivary gland in patients who are affected with ss disorder. on the other hand, at a similar study by caglar et al.10 have studied scintigraphic evaluation of salivary gland dysfunction in patients with thyroid cancer and reported that tc-99m pertechnetate is widely used after rai therapy in patients with thyroid cancer. additionally, they mentioned that, there in no any comparison between the sgs exploration in patients with thyroid cancer and ss patients after rai. etiology of xerostomia xerostomia that caused from radiation and known as oral mucosa dryness is a frequent complaint from patients who are treated for neck and head malignant tumor. the fundamental etiology of xerostomia contains a diversity of systemic disorders and some other positional factors. xerostomia has many negative concepts such as decreasing the quality of life, a lot of oral complications and pain like, altered taste, sore throat, tooth decay, voice quality changes, disability of swallowing and chewing function.1,2,5,6,8 for the reason that the risk of xerostomia caused by radiation depends on the anticancer remedy type and irradiation dose delivered to the tissue of gland, xerostomia could be considered to be dependent on various factors.7 after the first week of applying radiotherapy, the flow rate of saliva could decrease rapidly and then progressively decrease more and more with increasing radiation dose under 10% of its baseline border.15 by referencing the literature, when the irradiation dose increases up to 40 gy, a considerable decrease in the flow rate of saliva would occur.16,17 in spite of the fact that the prevalence of xerostomia prevalent in about 90% of patients, management and inhibition of salivary dysfunction caused via radiation are critical challenging matters. as reported by some previous researches, none of remedial and preventive agents that have been suggested did not furnished adequate satisfactory outcomes. some of these agents are: cholinergic stimulants, oral mucosal 191j contemp med sci | vol. 5, no. 4, july-august 2019: 189–196 review the application of salivary gland scintigraphy in quantitativeh. kaviani and m. khayamzadeh lubricant formulas, hyperbaric oxygen, vitamin c/e complex supplementation, chewing gums, mesenchymal stem cell, salivary stimulants, salivary substitutes.18–24 the strategical results of fundamental trials that examine different pharmacological and non-pharmacological remedy methods for the administration of xerostomia caused by radiation are outlined in table 3. it is proved that pilocarpine medication could moderately affect xerostomia caused by radiation but the efficiency of this method on prevention xerostomia is shown to be limited. furthermore, its extensive clinical application might be restricted because of potential of unwanted symptoms caused by medic remedies particularly, headaches, vasodilation, sweating and urinary urgency.36 lovelace et al.37 by studying around management of radiotherapy-induced salivary hypofunction and consequent xerostomia in patients with oral or head and neck cancer showed that although cholinergic agonists like cevimeline and pilocarpine are more impressive remedies for xerostomia syndrome caused by radiation than artificial saliva substitute spray, acupuncture medicine and hyperbaric oxygen therapy are also the second remedies for decreasing subjective intensity of this syndrome. pathophysiology of xerostomia the pharynx and cavity of the mouth are rubbed with oil and protected by saliva. a healthy oral mucosa could produce about 1.5 l/day. this microbial secretor of the mouth replenishes teeth, prepare the initial bolus of food and also helps in lubricating the mucosa of the mouth.17 saliva and salivary glands are main parts of the immune system of oral mucosa. saliva have antimicrobial materials such as lactoferrin, lysozyme, oxidoreductases and antifungal peptide histatin.10 human body have three pairs of major salivary glands: the sublingual, the submandibular and parotid glands. the parotid is a predominantly serous gland, while the sublingual and submandibular have both serous and mucous cells together.17 the three pairs of mentioned glands provide the major part of saliva in replication to an exogenous motivate. any of the mentioned glands is contained of a well angiogenesis system of ducts and acini. additionally, these glands are divided into lobules and lobes by linked septa of tissue which include ducts, nerves, lymphatics and vessels. as mentioned by ortholan et al.17 secretory cells cluster like acini, configure functional tissue the saliva. they convey liquid via solute material in solvent liquid combined with sodium chloride. the acini purvey is the major part of protein to saliva. o’sullivan and higginson38 have worked around oral outcomes resulting from head and neck radiotherapy and cited that, due to the position of the initial tumor and local lymph glands, the cavity of the mouth and salivary glands of patients with neck and head cancer are always in the scope of radiation.cconsequently, changes that comes after radiation will happen in tissues which are in scope of radiation during the tumor radiotherapeutic treatment.38 mamais et al.39 by working on the relative susceptibility of the human parotid gland to the harmful effect of ionizing table 3 remedy selection in treatment of xerostomia caused via radiation7,25 method patients (n) author (year) outcomes surgically transfer of submandibular gland versus control 24 versus 11 rieger et al.24 surgically transfer of submandibular gland was useful in advancing impressive swallowing surgically transfer of submandibular gland 8 al-qahtani et al.26 moderate symptoms of xerostomia was seen in about 37.5% of patients surgically transfer of submandibular gland 44 jha et al.27 about 75% of patients were survived from being affecting via xerostomia amifostine cytoprotective adjuvant versus control 67 veerasarn et al.28 amifostine cytoprotective adjuvant decreased severe xerostomia from 80% to 40% 500 mg amifostine cytoprotective adjuvant before chemotherapy 54 anne et al.29 about half of patients had severe xerostomia and others affected via xerostomia after one year amifostine cytoprotective adjuvant versus control 519 versus 332 buntzel et al.30 in those patients who remedied with amifostine the occurrence of xerostomia was more gentle during the first year of treatment 500 mg amifostine before chemotherapy 20 law et al.31 g2 xerostomia occurrence during first year was about 40% and during 1.5 year was about 30% 30–45 mg of cevimeline agonist three times per day 570 chambers et al.32 cevimeline agonist could enhance unmotivated flow rate of saliva pilocarpine 84 almeida and kowalski33 pilocarpine could cause salivary flow flavourless chewing gum 25 kaae et al.21 chewing gum could cause salivary flow acupuncture medicine versus control 6 versus 6 cho et al.34 there was not any significant difference among acupuncture medicine group and the control group acupuncture medicine versus control 40 versus 46 meng et al.19 remarkable diversity among groups was discovered (in favor of acupuncture medicine) hypnotherapy 12 schiff et al.35 the extent of saliva flow could be increased in about 75% of patients by hypnotherapy auriculotherapy as an alternative medicine versus placebo drug 60 versus 65 alimi23 auriculotherapy could remedy oral dryness in 66% of patients versus 4% from placebo drug group 192 j contemp med sci | vol. 5, no. 4, july-august 2019: 189–196 the application of salivary gland scintigraphy in quantitative review h. kaviani and m. khayamzadeh radiation stated that the salivary glands tissues are permanent and do not have high index of mitotic. moreover, the salivary glands cells do not have fat rates of movement which mention that the damages of radiation therapy could not become obvious long time after the remedy.39 on this regard, it could be derived that salivary glands cells are somewhat radioresistant. however, as reported by mamais et al.39 changes in saliva composition and quality will occur soon after irradiation that known as initial effects which stating that the gland tissue is hardly radiosensitive in approximately one functional aspect. it is while by placement of major salivary glands within scope of radiation, dysfunction of saliva predictably and instantly develops in dependent on dose of radiation. tanasiewicz et al.8 by studying around salivary dysfunction caused via radiation reported that about 55% of salivary flow will decrease after the first week of radiation. additionally, as a consequence the saliva viscosity will increase, denoting that some small sac like cavity of mucosa is still operable. however, at this stage most of the patients will experience the initial signs of xerostomia. anyway, by continuing radiation therapy and increasing total dose of radiation, the function of saliva will decrease more. the extent of dysfunction of saliva caused by radiation depends on some various factors such as dose of radiation, radiation scope, initial function of salivary gland and the volume of saliva.40,41 the radiation scope, especially the volume of gland tissue that exposed to radiation is the main determinative factor of xerostomia development and salivary dysfunction.42 teguh et al.43 have investigated the effects of radiotherapy on human parotid saliva and reported that the major glands are mostly within the radiation scope, so intensive dysfunction could be seen among them. it is while, in patients whose location of tumor is somehow that makes the separation of major glands easier, the development of xerostomia and dysfunction of glands is less. according to their studies, almost all patients who affected via neck and head cancer receive about 60 gy as a medicinal dose. however, traditional treatments are mostly specified by fractionalized radiotherapy over a 6-week period of time, once per day, 5 days per week, for a tumor remedied with a 2-gy dose. xerostomia clinical course oral consequence of radiotherapy in neck and head region are the result of irradiation disadvantageous effects on mucosa of the mouth, salivary glands, dentition, bone, muscles of mastication and the two joints connecting the jawbone to the skull. the extent and the incidence of this secondary effects depends more on the total dose of radiation, ionizing irradiation type, dose fractionation and the irradiated tissue volume. xerostomia is an intellectual complaint of mouth dryness that could be caused via dysfunction of salivary gland. the xerostomia consequences are such as discomfort and dysfunction of mouth mucosa and also in some cases speech difficulties.44 the consequences of salivary gland hypofunction also include periodontal disorder, a change in microflora in oral cavity, oral soft tissues variations and also tooth caries caused via salivary gland hypofunction.45 additionally, turner45 cited that some alterations of mucosa like ulceration, atrophy and inflammation are common. variation in microbial populations of the mouth mucosa could enhance the tooth decay and also cause frequent occurrence of oral thrush.46 however, patients may experience unusual swallowing patterns, somehow the boluses movement to the throat is not smooth. dysfunction of saliva could reduce general health besides. symptoms of oral mucosa can change the diet and also lead patients to nutritional deficits.47 newly, chronic inflammation of the oesophagus in patients with xerostomia caused by radiation was recognized.48 as reported by kam et al.49 the salivary flow loss and reduced ph of oesophagus would contribute to expansion of gastroesophageal reflux disorder. xerostomia assessment via sgs procedure dugonji et al.50 have worked on diagnostic validity of dynamic salivary gland scintigraphy with ascorbic acid stimulation in patients with sjoegren’s syndrome and introduced salivary gland scintigraphy as a helpful high accurate diagnostic test for detecting syndrome of sjögren’s. additionally, as cited by vinagre et al.,51 the imaging technique is extensively known as a useful tool for salivary gland function assessment and also diagnostic specified method for detecting initial sjögren’s syndrome. however, salivary gland scintigraphy enables the quantitative assessment of salivary glands’ parenchymal function after radioiodine therapy in differentiated thyroid cancer management. it is specified by its capability in variability of intraindividual observer, also by reproducibility that makes possible the detection of variations in function of parenchyma in a little scope of 5–10%.52 this provides the initial detection of the syndrome of sjögren’s and impaired parenchyma of salivary glands that are caused due to radioiodine therapy.53 consequently, salivary gland scintigraphy could be applied as an adequate imaging technique for quantitative assessment of parenchymal function of salivary gland. as cited by hsiung et al.54 the evaluation of the salivary glands residual function could be performed by sgs and measuring flow rate of salivary gland. the main application of sgs is to evaluate the function of salivary gland in patients who recognized to complaint from oral dryness. so, sgs is a fundamental practical method that makes possible the concurrent assessment of function and parenchyma of major salivary gland. in this method after injection of 99mtc-sodium pertechnetate, consecutive anterior projection images of head are needed during a variable period of time, generally between 20 and 40 min. after that the images are stored and interest glandular regions in the skull are drawn by using hand.55 by using computer software, time–activity curves could be produced for any of the major salivary glands. these curves are divided into two phases, the excretion phase and the uptake phase. excretion phase curves that are set up by using of salivary motivation administration like lemon juice, which correlates with elimination of tracer via the oral cavity, providing information on the salivary ducts patency and the system overall functional integrity. uptake phase curves corresponding to the tracer accumulation by the glandular functional tissue and the protocol depended duration (fig. 1). although, sgs procedure is widely applied for the assessment of patients with xerostomic, but is not powerfully systematized for the evaluation of such syndromes like sjögren.56 there has been an extensive attention to sgs as a useful method in the assessment of oral mucosa dryness symptoms from decades ago. nishiyama et al.14 have worked on a study to standardize quantitative evaluation of parotid gland scintigraphy in patients with sjögren’s syndrome and cited that although sgs method that is generally based on schall’s 193j contemp med sci | vol. 5, no. 4, july-august 2019: 189–196 review the application of salivary gland scintigraphy in quantitativeh. kaviani and m. khayamzadeh fig. 1 an illustration of an ordinary salivary gland scintigraphy with normal tracer uptake and excretion phases and well-defined accumulation in time–activity curves. parot-esq (left parotid gland); submxlar-esq (left submandibular gland); parot-dta (right parotid gland); counts (tracer accumulation); boca (mouth); sec (time in seconds); submxlar-dta (right submandibular gland); fundo (background). classification57 is easy and simple to perform and also qualitative and depends on the observer, but its boundary related results may be classified incorrectly by the evaluator subjective judgement. based on this classification, dysfunction of salivary gland is categorized into four grades, in according with uptake severity and mouth activity after administration of excretory motivation, the normal state is grade 1 and the whole lack of uptake mouth activity is grade 4. this comprehensive categorization is taking into consideration as the standard method for interpretation of salivary scintigram. in the main schall’s classification, every gland was categorized independently. for purposes of simplification, some of the authors take into account high values of both submandibular and parotids glands altogether or consider just the maximum glandular value. shiboski et al.58 for determining the cut-off value have used grade 3 of schall’s classification, somehow in their study by including all of the patients they reached to a general specificity and sensitivity of sgs for the assessment of ss disorder of 98% and 54% respectively. ramos et al.59 by studying the accuracy of assessment of each diagnostic test for involvement of oral cavity in patients with ss, achieved an overall specificity and sensitivity of sgs, clarified according to the schall’s classification of 80% and 87% respectively. in order to purify the interpretation of salivary gland scintigram and enhancing diagnostic precision, there is a comprehensive attention to glandular function quantification. various parameters have been suggested by some researchers that little agreement exists about the most trustworthy one for ss recognition and also their portion to improvement of scintigraphy accuracy.52,60 quantitative scores development in sgs the evolution of powerful gamma cameras and improvement of related software’s could improve the quantitative sgs assessment, with the aim of purifying its explanation. according to aung et al.53 and chung et al.61 reported that quantitative sgs is delicate sufficient to discover abnormalities of only 25% destruction of gland parenchyma permitting to recognize smooth glandular dysfunction in early ss. during the last few decades, various parameters are derived according to time– activity curves that are presented in table 4 and are considered as accurate measures for recognition of ss.14,60,62,63 these computer-aided quantitative indicators are objective measures and numeral that easily mirror glandular function more exactly than qualitative assessment. for improving the diagnostic performance of the quantitative assessment in ss, various authors suggested an organized analysis of the parameters associated with salivary gland and oral activity.10,63 ss: sjögren’s syndrome, iss: initial ss, sss: secondary ss, sgs: salivary gland scintigraphy, isc: isolated sicca complaints, ma: maximum accumulation, ur: uptake ratio, msgb: minor salivary gland biopsy, ms: maximum secretion, e%: percentage of stimulated excretion, c%: percentage of peak tracer distribution, pri: prestimulatory oral index, poi: poststimulatory oral activity index, us: uptake speed, ef: excretion fraction, sv: secretion velocity, es: excretion speed, u4 and u14: percentage of background corrected counts at 4 and 14 min, ti: time interval among ascular perfusion peak and maximum oral activity point, tmin: time interval among stimulation to minimum count, tmax: time interval required to get peak uptake. 194 j contemp med sci | vol. 5, no. 4, july-august 2019: 189–196 the application of salivary gland scintigraphy in quantitative review h. kaviani and m. khayamzadeh table 4 various parameters of quantitative sgs derived in according to time–activity curves methods and patients restrictions outcomes authorships tmax, tmin, ur, msgb, ma, ms, sv, pri, poi, ti and quantitative sgc (control group: 21, primary ss: 29, progressive ss: 41) ss stages were categorized according to the result of msgb, ss could not be reflected based on classification criteria poi, ti, ma and ur of the sm glands will decrease with disorder progress. aung et al.53 the oral indicators correlation will decrease with msgb focus scores quantitative sgs, tmax, ur, ma (%), ms (%), pri (%), u14, u4 (control group: 15, ss patients: 17, autoimmune disorders without sc: 18) group a recognized with primary and secondary ss patients. left submandibular and parotid glands urs decreased. adams et al.60 oral tmax decreased in contrast with control group. little amount of patients with initial ss. u4 decreased. special involvement of sm, significant overlap of quantitative indicators among groups. msgb, us, es, ef, saxon test, quantitative sgs peak count (autoimmune disorder without ss: 23, iss: 34 and sss: 11) no one were incorporated as control group. es and peak count decreased. nishiyama et al.14 peak count correlation decreased on msgb. es decreased in saxon trial. the percentage of c, e and tmax; quantitative sgs (control group: 8, ss: 8, isc: 16) little ss patients number (8); no distinction among initial and secondary ss. high tmax in ss patients. henriksen and nossent56low parotid c% in ss and isc patients. low e% in ss patients conducted studies assessed various scintigraphic parameters and incorporated controls and inharmonious populations of patients. the major purpose of gaining quantitative scores is the distinction of normal from abnormal results, where qualitative examination has some restrictions and becomes subjective. anyway, loutfi et al.52 by working around the use of semi quantitative analysis for uptake and clearance of salivary gland scintigraphy established a wide scattering of a lot of quantitative indicators. dugonji et al.50 proved that to incorporate a broad range of normal values, the setting of cutout scopes to preserve the favorable specific condition and negative prognostic values may seriously compromise the positive prognostic values and sensitivity. now no any clear agreement on which quantitative indicator are more suitable and reliable for assessment of ss. the obvious discrepancy in literature about the dependability and true value of the parameters developed from the broad scattering of normality values, perhaps as a result of the studied populations incongruity, the patient’s liability in various stages of the disorder and also the absence of a normal procedure of salivary scintigraphy, both in terms of attainment as in parameters that should be processed.62 so, for accepting these indicators and comprising them in explanation of sgs, a broad attempt must be constructed in both term of systematizing procedure of sgs and carrying out multicenter trials. this could allow the designation of general normality and cutout values, plus its sensitivity assessment, specificity and resultful progressive value for assessment of ss. by knowing this fact that sgs is a comprehensive diagnostic tool, it may be beneficial for remedial decision, somehow the possibly functioning glandular tissue demonstration is crucial for secretagogues application. additionally, in follow-up stage of patient, sgs provide the assessment of remedial response and evaluation of disorder over time.64 conclusion salivary gland scintigraphy is known as a reliable non-invasive procedure for assessment of function of salivary glands in patients with xerostomia symptoms. in addition, sgs is proved to be helpful in objective assessment of xerostomia as dysfunction of salivary gland that is established by international classification criteria. the accuracy of sgs method help us to discover moderate abnormalities of glandular parenchyma. on the other hand, the outcomes of sgs is associated with ss clinicopathologic characteristics, with production of non motivated saliva, radiosialography and focus scores in biopsy of minor salivary gland. the most frequent applicable method for clarification of salivary scintigraphy is the qualitative assessment by using of schall’s classification that is a subjective method and somehow bear form restricted capability to differentiate boundary results. the application of quantitative indicators might purify the sgs interpretation and may enhance the validity of this strategy for ss assessment, while the sgs standardization is essential for making it to be capable for daily practice. moreover, as proved in according to the outcomes of this comprehensive research, sgs is a beneficial method for remedial decisions and also appropriate follow-up of patients. conflicts of interest none. ■ 195j contemp med sci | vol. 5, no. 4, july-august 2019: 189–196 review the application of salivary gland scintigraphy in quantitativeh. kaviani and m. khayamzadeh references 1. bhide sa, miah ab, harrington kj, newbold kl, nutting cm. radiationinduced xerostomia: pathophysiology, prevention and treatment. clin oncol (r coll radiol). 2009;21:737–744. 2. dirix p, nuyts s, vander poorten v, delaere p, van den bogaert w. the influence of xerostomia after radiotherapy on quality of life: results of a questionnaire in head and neck cancer. support care cancer. 2008;16:171–179. 3. mortazavi h, baharvand m, movahhedian a, mohammadi m, khodadoustan a. xerostomia due to systemic disease: a review of 20 conditions and mechanisms. ann med health sci res. 2014;4:503–510. 4. villa a, polimeni a, strohmenger l, cicciù d, gherlone e, abati s. dental patients’ self-reports of xerostomia and associated risk factors. j am dent assoc. 2011;142:811–816. 5. napeñas jj, brennan mt, fox pc. diagnosis and treatment of xerostomia (dry mouth). odontology 2009;97:76–83. 6. porter sr, fedele s, habbab km. xerostomia in head and neck malignancy. oral oncol. 2010;46:460–463. 7. pinna r, campus g, cumbo e, mura i, milia e. xerostomia induced by radiotherapy: an overview of the physiopathology, clinical evidence, and management of the oral damage. ther clin risk manag. 2015;11:171–188. 8. tanasiewicz m, hildebrandt t, obersztyn i. xerostomia of various etiologies: a review of the literature. adv clin exp med. 2016;25:199–206. 9. berk l. systemic pilocarpine for treatment of xerostomia. expert opin drug metab toxicol. 2008;4:1333–1340. 10. caglar m, tuncel m, alpar r. scintigraphic evaluation of salivary gland dysfunction in patients with thyroid cancer after radioiodine treatment. clin nucl med. 2002;27:767–771. 11. manthorpe r. sjogren’s syndrome criteria. ann rheum dis. 2002;61: 482–484. 12. vitali c, bombardieri s, jonsson r, moutsopoulos hm, alexander el, carsons se, et al. classification criteria for sjogren’s syndrome: a revised version of the european criteria proposed by the americaneuropean consensus group. ann rheum dis. 2002;61:554–558. 13. solans r, bosch ja, galofré p, porta f, roselló j, selva-o’callagan a, et al. salivary and lacrimal gland dysfunction (sicca syndrome) after radioiodine therapy. j nucl med. 2001;42:738–743. 14. nishiyama s, miyawaki s, yoshinaga y. a study to standardize quantitative evaluation of parotid gland scintigraphy in patients with sjögren’s syndrome. j rheumatol. 2006;33:2470–2474. 15. burlage fr, coppes rp, meertens h, stokman ma, vissink a. parotid and submandibular/sublingual salivary flow during high dose radiotherapy. radiother oncol. 2001;61:271–274. 16. murdoch-kinch ca, kim hm, vineberg ka, ship ja, eisbruch a. dose-effect relationships for the submandibular salivary glands and implications for their sparing by intensity modulated radiotherapy. int j radiat oncol biol phys. 2008;72:373–382. 17. ortholan c, chamorey e, benezery k, thariat j, dassonville o, poissonnet g, et al. modeling of salivary production recovery after radiotherapy using mixed models: determination of optimal dose constraint for imrt planning and construction of convenient tools to predict salivary function. int j radiat oncol biol phys. 2009;73:178–186. 18. jensen sb, pedersen am, vissink a, andersen e, brown cg, davies an, et al. a systematic review of salivary gland hypofunction and xerostomia induced by cancer therapies: management strategies and economic impact. support care cancer 2010;18:1061–1079. 19. meng z, garcia mk, hu c, chiang j, chambers m, rosenthal di, et al. randomized controlled trial of acupuncture for prevention of radiationinduced xerostomia among patients with nasopharyngeal carcinoma. cancer. 2012;118:3337–3344. 20. nevens d, nuyts s. the role of stem cells in the prevention and treatment of radiation-induced xerostomia in patients with head and neck cancer. cancer med. 2016;5:1147–1153. 21. kaae jk, stenfeldt l, eriksen jg. xerostomia after radiotherapy for oral andoropharyngeal cancer: increasing salivary flow with tasteless sugarfree chewing gum. front oncol. 2016;6:111. 22. spiegelberg l, djasim um, van neck hw, wolvius eb, van der wal kg. hyperbaric oxygen therapy in the management of radiation-induced injury in the head and neck region: a review of the literature. j oral maxillofac surg. 2010;68:1732–1739. 23. alimi d. xerostomia induced by radiotherapy. ther clin risk manag. 2015;11:1149–1152. 24. rieger j, seikaly h, jha n, harris j, williams d, liu r, et al. submandibular gland transfer for prevention of xerostomia after radiation therapy: swallowing outcomes. arch otolaryngol head neck surg. 2005;131:140–145. 25. zhuang l, yang z, zeng x, zhua x, chen z, liu l, et al. the preventive and therapeutic effect of acupuncture for radiation-induced xerostomia in patients with head and neck cancer: a systematic review. integr cancer ther. 2013;12:197–205. 26. al-qahtani k, hier mp, sultanum k, black mj. the role of submandibular salivary gland transfers in preventing xerostomia in the chemoradiotherapy patient. oral surg oral med oral pathol oral radiol endod. 2006;101:753– 756. 27. jha n, harris j, seikaly h, jacobs jr, mcewan aj, robbins kt, et al. a phase ii study of submandibular gland transfer prior to radiation for prevention of radiation-induced xerostomia in head-and-neck cancer (rtog 0244). int j radiat biol phys. 2012;84:437–442. 28. veerasarn v, phromratanapongse p, suntornpong n, lorvidhaya v, sukthomya v, chitapanarux i, et al. effect of amifostine to prevent radiotherapy-induced acute and late toxicity in head and neck cancer patients who had normal or mild impaired salivary gland function. j med assoc thai. 2006;89:2056–2067. 29. anné pr, machtay m, rosenthal di, brizel dm, morrison wh, irwin dh, et al. a phase ii trial of subcutaneous amifostine and radiation therapy in patients with head-and-neck cancer. int j radiat biol phys. 2007;67:445–452. 30. buntzel j, glatzel m, mücke r, micke o, bruns f. influence of amifostine on late radiation-toxicity in head and neck cancer—a follow-up study. anticancer res. 2007;27:1953–1956. 31. law a, kennedy t, pellitteri p, wood c, christie d, yumen o. efficacy and safety of subcutaneous amifostine in minimizing radiation-induced toxicities in patients receiving combined-modality treatment for squamous cell carcinoma of the head and neck. int j radiat biol phys. 2007;69: 1361–1368. 32. chambers ms, posner m, jones cu, biel ma, hodge km, vitti r, et al. cevimeline for the treatment of postirradiation xerostomia in patients with head and neck cancer. int j radiat oncol biol phys. 2007;68:1102–1109. 33. almeida jp, kowalski lp. pilocarpine used to treat xerostomia in patients submitted to radioactive iodine therapy: a pilot study. braz j otorhinolarynol. 2010;76:659–662 [article in english and portuguese]. 34. cho jh, chung wk, kang w, choi sm, cho ck, son cg. manual acupuncture improved quality of life in cancer patients with radiation-induced xerostomia. j altern complement med. 2008;14:523–526. 35. schiff e, mogilner jg, sella e, doweck i, hershko o, ben-arye e, et al. hypnosis for postradiation xerostomia in head and neck cancer patients: a pilot study. j pain symptom manage. 2009;37:1086.e1–1092.e1. 36. cheng cq, xu h, liu l, wang rn, liu yt, li j, et al. efficacy and safety of pilocarpine for radiation-induced xerostomia in patients with head and neck cancer: a systematic review and meta-analysis. j am dent assoc. 2016;147:236–243. 37. lovelace tl, fox nf, sood aj, nguyen sa, day ta. management of radiotherapy-induced salivary hypofunction and consequent xerostomia in patients with oral or head and neck cancer: meta-analysis and literature review. oral surg oral med oral pathol oral radiol. 2014;117:595–607. 38. o’sullivan em, higginson ij. clinical effectiveness and safety of acupuncture in the treatment of irradiation-induced xerostomia in patients with head and neck cancer: a systematic review. acupunct med. 2010;28:191–199. 39. mamais c, dias a, walker j, vydianath sr. parotid actinomycosis mimicking metastatic lymphadenopathy. west indian med j. 2011;60:349–350. 40. teymoortash a, muller f, juricko j, bieker m, mandic r, librizzi d, et al. botulinum toxin prevents radiotherapy-induced salivary gland damage. oral oncol. 2009;45:737–739. 41. aghemo a, rumi mg, monico s, banderali m, russo a, ottaviani f, et al. ribavirin impairs salivary gland function during combination treatment with pegylated interferon alfa-2a in hepatitis c patients. hepat mon. 2011;11:918–924. 42. sánchez-pablo ma, gonzález-garcía v, del castillo-rueda a. study of total stimulated saliva flow and hyperpigmentation in the oral mucosa of patients diagnosed with hereditary hemochromatosis. series of 25 cases. med oral patol oral cir bucal. 2012;17:e45–e49. 43. teguh dn, levendag pc, noever i, et al. early hyperbaric oxygen therapy for reducing radiotherapy side effects: early results of a randomized trial in oropharyngeal and nasopharyngeal cancer. int j radiat oncol biol phys. 2009;75:711–716. 44. wick jy. xerostomia: causes and treatment. consult pharm. 2007;22:985–992. 45. turner md. hyposalivation and xerostomia: etiology, complications, and medical management. dent clin n am. 2016;60:435–443. 46. pinheiro a, marcenes w, zakrzewska jm, robinson pg. dental and oral lesions in hiv infected patients: a study in brazil. int dent j. 2004;54: 131–137. 196 j contemp med sci | vol. 5, no. 4, july-august 2019: 189–196 the application of salivary gland scintigraphy in quantitative review h. kaviani and m. khayamzadeh this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.08201902 47. hammerlid e, taft c. health-related quality of life in long-term head and neck cancer survivors: a comparison with general population norms. br j cancer 2001;84:149–156. 48. rosenbluth bd, serrano v, happersett l, shaha ar, tuttle rm, narayana a, et al. intensity-modulated radiation therapy for the treatment of nonanaplastic thyroid cancer. int j radiat oncol biol phys. 2005;63:1419–1426. 49. kam mk, leung sf, zee b, chau rm, suen jj, mo f, et al. prospective randomized study of intensity-modulated radiotherapy on salivary gland function in early-stage nasopharyngeal carcinoma patients. j clin oncol. 2007;25:4873–4879. 50. dugonjić s1, ajdinović b, stefanović d, jauković l. [diagnostic validity of dynamic salivary gland scintigraphy with ascorbic acid stimulation in patients with sjoegren’s syndrome: comparation with unstimulated whole sialometry]. vojnosanit pregl. 2008;65:41–46 [article in serbian]. 51. vinagre f, santos a, santos m, prata a, oliveira a, silva jc. [salivary gland scintigraphy in the evaluation of patients with sicca complaints]. acta reumatol port 2008;33:422–428 [article in portuguese]. 52. loutfi i, nair mk, ebrahim ak. salivary gland scintigraphy: the use of semiquan titative analysis for uptake and clearance. j nucl med technol. 2003;31:81–85. 53. aung w, yamada i, umehara i, ohbayashi n, yoshino n, shibuya h. sjögren’s syndrome: comparison of assessments with quantitative salivary gland scintigraphy and contrast sialography. j nucl med. 2000;41:257–262. 54. hsiung cy, ting hm, huang hy, lee ch, huang ey, hsu hc. parotid-sparing intensity-modulated radiotherapy (imrt) for nasopharyngeal carcinoma: preserved parotid function after imrt on quantitative salivary scintigraphy, and comparison with historical data after conventional radiotherapy. int j radiat oncol biol phys. 2006;66:454–461. 55. bagesund m, richter s, agren b, dahllöf g. correlation between quantitative salivary gland scintigraphy and salivary secretion rates in children and young adults treated for hematological, malignant and metabolic diseases. dentomaxillofac radiol. 2000;29:264–271. 56. henriksen am, nossent hc. quantitative salivary gland scintigraphy can distinguish patients with primary sjögren’s syndrome during the evaluation of sicca symptoms. clin rheumatol. 2007;26:1837–1841. 57. schall gl, anderson lg, wolf ro, herdt jr, tarpley tm jr, cummings na, et al. xerostomia in sjögren’s syndrome: evaluation by sequential scintigraphy. jama 1971;216:2109–2116. 58. shiboski ch, shiboski sc, seror r, criswell la, labetoulle m, lietman tm, et al. 2016 american college of rheumatology/european league against rheumatism classification criteria for primary sjögren’s syndrome: a consensus and data-driven methodology involving three international patient cohorts. ann rheum dis. 2017;76:9–16. 59. ramos-casals m, tzioufas ag, font j. primary sjögren’s syndrome: new clinical and therapeutic concepts. ann rheum dis. 2005;64:347–354. 60. adams bk, al attia hm, parkar s. salivary gland scintigraphy in sjögren’s syndrome: are quantitative indices the answer? nucl med commun. 2003;24:1011–1016. 61. chung mk, kim dh, ahn yc, choi jy, kim eh, son yi. randomized trial of vitamin c/e complex for prevention of radiation-induced xerostomia in patients with head and neck cancer. otolaryngol head neck surg. 2016;155:423–430. 62. demangeat r, didon-poncelet a, cherfan j, demangeat jl. stimulated salivary pertechnetate clearance revised: correlation with dynamic scintigraphic indices in sicca syndrome. clin nucl med. 2000;25:888–894. 63. aung w, murata y, ishida r, takahashi y, okada n, shibuya h. study of quantitative oral radioactivity in salivary gland scintigraphy and determination of the clinical stage of sjögren’s syndrome. j nucl med. 2001;42:38–43. 64. hermann ga, vivino fb. abnormal scintigraphic patterns of uptake and secretion in sjögren’s syndrome. arthritis rheum. 2000;43:s306. 165j contemp med sci | vol. 5, no. 3, may–june 2019: 165–169 original decision support detection system for lung nodule abnormalities based on machine learning algorithms muna alsallal,a* mhd saeed sharif,b bydaa hadi,a and ruwaida albadrya asamawa technical institute, alfurat alawsat technical university, iraq. bschool of architecture, computing and engineering, university of east london (uel) london, uk *correspondence to muna alsallal (e-mail: mona27793@gmail.com) (submitted: 14 october 2018 – revised version received: 28 november 2018 – accepted: 19 january 2019 – published online: 26 june 2019) introduction lung nodules growth which is so-called a “abnormal pulmonary nodules” represents an important factor for lung cancer.1,2 it is considered to be the foremost reason of human cancerous deaths worldwide among the two genders.1 the pulmonary nodules has taken an oval shaped growth in the infected lung which sometimes called lung’s spots. patients with cancerous lung are mostly were detected at advanced stage due to difficulties of detection at an early phases.1 the most important reason for late detection of this disease is inefficiency of x-rays technique in detecting such cases.1 the only efficient method is the using of the ct-scan device. another reason for detection difficulties is the need for extensive expertise from the radiologists to be able in categorizing the lung nodules as normal or abnormal. from the radiologists point-of-view the size of normal lung nodules naturally ranges from 2 to 5 mm in thickness and from 1.5 to 3 cm in diameter. however, if the growth in thickness or in area is larger, then the case needs to be considered as critical. it is also highlighted that the radiologists time is limited when compared with the number of patients that they have to see on daily basis. so designing a technique that can recognize the abnormal nodules at an early stage is important and can be assumed as a proactive step to prevent the case aggravation. nowadays, machine learning is being a well-known method for improving robust automatic techniques to analyze wide-range of biomedical data.3 sajda in 2006 in his paper has reviewed several state-of-the-art of machine learning techniques that have demonstrated their effectiveness in many tasks such as disease detection, diagnosis and treatment monitoring. the review also defined the expansions in machine learning techniques, concentrating on the main two types of learning techniques; supervised and unsupervised. it also went through linear and non-linear approaches and show advantages and disadvantages of each. such systems were called computer aided diagnosis (cad) systems, which are widely used in healthcare research area and mostly based on machine-learning algorithms. the standard detection techniques relied on a reference datasets that can work as a foundation for developing robust systems. the zero-change dataset was chosen to measure the systems’ performance on nodule growth. the chosen dataset was used by krishnan et al.2 in their open source system for detecting the progress in lesion sizing. they have evaluated their proposed system on a proven clinical dataset (zero-change images dataset). this dataset comprises of 12 pairs of images, each pair contains one for the whole lung and the other is for just the region around the nodules. one more thing that needs to be noticed is the factors that emphasizes the efficiency of classification tasks in such designed systems. this efficiency can be influenced by two factors; chosen techniques and the set of features. current scholars highlighted that machine-learning techniques demonstrated their ability to enhance the performance of detection systems.3 bengio et al.4 stated that learning procedures can significantly enhance by combining several types of algorithms such as linear and non-linear approaches in order to gain featured deceptions of data. another paper that was written by xu et al.5 was highlighted the usefulness of using non-linear machinelearning approaches for such systems and high-weight the value of the feature extraction phase. objective investigates the possibility of the early detection in the case of lung infection. most cases of lung cancer are detected in advance stages as this type is hard to be detected in premature phases. the zero-change dataset was chosen to measure the systems’ performance on nodule growth. the chosen dataset is assumed as a proven clinical dataset and was used by several researchers in their proposed systems. the designed detection technique has been considered to be used as a decision support tool. this technique is based on using two machine learning algorithms for classification purposes. methods machine learning techniques was applied to detect interesting patterns and manipulate the dataset images in order to enhance the classification task. pre-processing procedures also have been applied using different matlab functions. in addition two well-known techniques that related to the support vector machines (svms); the radial basis function kernel-based svms and the polynomial kernelbased svms have also applied using matlab© package named prtools. results the performance of this paper proposed technique was evaluated based on several values of both chosen techniques. the procedure was implemented on the basis of leave-one-out-cross-validation procedure in order to generate unbiased outcomes. the results of cross-validation procedure is averaged and presented as a classifier outcome. the misclassification error, sensitivity, specificity and accuracy are calculated to show a clear image about the two classifiers. conclusion the experimental results have shown that the proposed system has scored high accuracy by polynomial kernel svm. a set of distinguishable representative features are correlated together by a statistics association. also, this designed system can be considered as a benchmark for developing of other tissues abnormalities signs detection systems. keywords machine learning, lung cancer, lung nodule, detection system, classification, image processing, svm issn 2413-0516 166 j contemp med sci | vol. 5, no. 3, may–june 2019: 165–169 detection system for lung nodule abnormalities based on machine learning algorithms original m. alsallal et al. this research has proposed an automatic detection technique relied on non-linear machine-learning algorithms to classify if the nodule size is normal or abnormal as a second opinion to support the radiologist decision. the system was based on four main techniques; dataset acquisition, pre-processing technique, image features extraction and finally applying a machine-learning technique for classification process. the proposed system trained the nodules sizes after extracting the required features. the next section explores the related work that was done in detecting nodules abnormalities using machine-learning approaches. the following section clarifies the experimental design which includes the system phases. then, the results will be discussed in section 4. finally, we conclude this paper content, method and outcomes in conclusion section. background many studies were conducted using several machine-learning approaches in order to detect lung nodules abnormality. bellotti et al.6 has used contour-based model to detect nodules abnormalities. they scored high performance by gaining 88.5% for detection accuracy. another group of researchers riccardi et al.7 has proposed a new system using 3d radial transforms to detect nodules with overall 71% of detection accuracy. group of researchers used three different algorithms to determine the pulmonary nodules in ct scan as described in camarlinghi et al.8 an advanced technique that based on feed forward neural networks used by abdulla and shaharum9 has been implemented by x-ray images. specific features set proposed for their study included the area, perimeter and shape. a study that was conducted by kuruvilla and gunavathi10 proposed six discrete features, three of them was mentioned in abdullah and shaharum.9 they add skewness in addition to the time of extraction features from segmented slices that contained two lungs. all those separated features were trained by non-linear machine-learning algorithm, and reported good outcomes. from the other side of the problem, support vector machines algorithms and their non-linear derivatives were widely used in detecting abnormalities in medical images. the support vector machines (svms) were used to detect colon abnormality as a classifier11,12 with encouraging outcomes. the same svm applications also implemented for ovarian abnormalities which has revealed good results.13 furey et al.13 used the svms technique to several kinds of abnormality data which related to different human body parts such as blood, colon and more. the application of svm scored high accuracy percentage in most classification cases. papers written by segal et al.14,15 applied the svm for classification purposes to separate two types of cells with different characteristics. they reported in their results high classification accuracy and has given new indicators to be noted by researchers. a study by statnikov et al.16 worked in assessing several machine learning algorithms for their classification performance based on wide range of gene expression to detect abnormality, recommended sv, recommended svms as a promising approach in this field. so svms has two vital characteristics that make them superior to their peers. support vector machine support vector machines are a group of correlated supervised learning algorithms used commonly for classification and regression. they are considered among the most recent, sophisticated, and high-performance algorithms in artificial intelligence. they aim to separate high-dimensional data in “hyperspace” (“space” with a dimensionality equal to the number of features derived from the training set) using a hyperplane.17 svm can be defined as a linear model and it always looks for a hyperplane to separate one class from another.18 svms technique is optimized by connecting with what so-called kernels. kernels works on a notion that change the depiction of the dot-product in the linear formulation to be non-linear. to illustrate: vapnik18 stated that svm based on dot-products for limited dimension which can be defined as equation (1) ( ) tf y x y= (1) where y represents the outcome of deploying the procedure on non-linear conversion to the proposed data, for example, yi = φ(xi), classification is performed by taking the sign of f(y). the notion of the non-linear transmute is mapping the data into a high-dimensional space, where the transmuted data is divided by a hyperplane and linearly separated. to optimize this separable process, the conversion of the data is completed by a kernel trick.19 the kernels is used to enhance the separation process by replacing the dot-products by non-linear kernel functions. in high level language we can briefly describe the main three types of svm kernels as shown in table 1 experimental design this paper proposed an automatic detection system consisted of four main components. dataset acquisition, features engineering technique which consists of image features extraction and classification component. the four components are shown in fig. 1. the same svm applications also implemented for ovarian abnormalities which has revealed good results.13 furey et al.13 table. 1 briefly reviewed three types of kernels kernel time kernel visualisation kernel description linear kernel there is just a “normal” dot-product, thus in 2d decision boundary is always line. separation of most of points correctly, but due to the “stiffness” of the hypothesis not all points can be captured.19 polynomial kernel the polynomial kernel induces space of polynomial combinations of the features, up to certain degree. consequently we can work with slightly “out of straight line” decision boundaries, such as parabolas.18 rbf kernel rbf kernel induced space is gaussian distributions space ... each point becomes a probability density function (up to scaling) of a normal distribution. in such space, dot-products are integrals and consequently the flexibility is very high.18 167j contemp med sci | vol. 5, no. 3, may–june 2019: 165–169 original detection system for lung nodule abnormalities based on machine learning algorithmsm. alsallal et al. used the svms technique to several kinds of abnormality data which related to different human body parts such as blood, colon and more. the application of svm scored high accuracy percentage in most classification cases. papers written by segal et al.14,15 applied the svm for classification purposes to separate two types of cells with different characteristics. they reported in their results high classification accuracy and has given new indicators to be noted by researchers. a study by statnikov et al.16 worked in assessing several machine learning algorithms for their classification performance based on wide range of gene expression to detect abnormality, recommended sv, two svms algorithms were used to perform classification process; radial basis function (rbf) and polynomial function kernels, respectively. these two techniques are trained using zero-change dataset to find an optimal mode to classify images into their corresponding classes. then, during the classification phase, the images are classified of whether normal or abnormal. the overall process of dime-sized damage progress detection will be described in details in the following sub-sections. for the purpose of this paper proposed system, a lung images data sets from a publicly available database the dataset comprises of twelve scan pairs from the crpf_database as the first six scan pairs of images take the similar portion thickness which normally equal to 1.25 mm. while the second six pairs scan set has different portion thickness which is equal to 2.5 mm. to describe the pair images; one of the pair scanned images shows the entire lung. the second one concentrates on the nodule region with the same scan resolution of the first. keeping in mind that the person who entitled to perform the scanning process has not change his location during the two scan processes. for researchers convenient, the access of the 12 scan pairs are publicly available for use in a single compressed file of dicom images. it can be downloaded conveniently from https://veet.via.cornell.edu/cgi-bin/datac website with all images accessories. they can be obtained from the crpf_ database homepage below the title “repeat single session”. furthermore, the scan images can also be downloaded separately in several versions using the direct function for image database download function. to prepare for implementing the proposed system phases a particular pre-processing procedure is applied. we first applied the shade correction technique, as the unbalanced lightning if the scan image needs to be modified if a specific object has to be properly spotted. the second step of pre-processing procedure is applied using morphological-opening task as a proactive step to assess the background. this task was applied using matlab© function which is called “imopen”. this function typically is applied for morphological-opening task that can be performed scan with 12 pixels structuring element. after morphological-opening is completed by applying “imtophat” matlab© function, the task of increasing the image contrast is performed. the task is completed by implementing “imadjust” matlab© function as shown in figure 2. then, “bwareaopen” another matlab© function is applied to eliminate the noise of the background. as a result the concluding output, which only displays the affected scan image region, is gained. for this paper purpose seven types of features were determined to be extracted from each image to train the classifier. the set of chosen features that needs to be extracted is as follows: definite number of pixels in nodule region (dnp), marginal length around the region feature (mlar) (perimeter of the entity) which can be clearly shown in fig. 3 as the fig. 1 this represents a block diagram of the proposed detection system for lungs nodules abnormality classifier 11,12 with encouraging outcomes. fig. 2 this represents the scan lung image after implementing the task of increasing the image contrast. fig. 3 this represents the marginal length around the region feature which perimeter of the entity. 168 j contemp med sci | vol. 5, no. 3, may–june 2019: 165–169 detection system for lung nodule abnormalities based on machine learning algorithms original m. alsallal et al. element surrounded by red color, maximum length of the major axis in the region (malr), minimum length of the minor axis in the region (milr), feature ratio [equal to (malr) divided by (milr) and finally region roundness (rr) which is calculated by ((4 * π * dnp)/(mlar^2)]. the classification procedure is implemented via a matlab© package named prtools.18 this paper proposed system based on implemented two well-known techniques that related to the svms; the rbf kernel-based svms and the polynomial kernel-based svms. results the performance of this paper proposed technique was evaluated based on several values of both chosen techniques. the procedure was implemented on the basis of leave-one out-cross-validation procedure to generate unbiased outcomes. the results of cross-validation procedure is averaged and presented as a classifier outcome as shown in table 2. the misclassification error, sensitivity, specificity and accuracy are calculated to show a clear image about the two classifiers. the experimental results show that the two classifiers are able to identify both classes; however, the polynomial kernel-based svms outperformed the rbf kernel-based svms. on the other hand, the system performance were assessed based on radiologists opinions which was compared with system performance as shown in table 3. the nodules features that characterised by radiologists and the proposed system are premeditated and then compared. the presence of nodules which sized from 1.5 to 3 cm in diameter and thickness that ranges from 2 to 5 mm represented the normal lung nodules. however, if those measures are characterised in larger forms then they should be considered as an abnormal case which needs more attention and investigation. in order for the system performance to be tested when compared with the radiologist’s opinions, a statistical test were applied. t-test was applied to measure the differences in mean between the marked images and the system output. the results are then depicted in table 3, the value of sigma that was obtained under 95% confidence interval, 0.211. this sigma value demonstrated table. 2 averaged results of two classifiers measure polynomial kernel svm rbf kernel svm misclassification error 0.0919 0.1826 accuracy 0.9191 0.7273 specificity 1 0.3224 sensitivity 0.881 0.763 table 3. the analysis of t-test between the proposed system performance and radiologists opinions mean n std. deviation pair 1 marked 1.71 12 0.419 testing 1.90 12 0.331 correlations of paired samples n correlation sig. pair 1 marked and testing 12 −0.019 0.901 paired differences mean std. deviation std. error mean 95% confidence interval of the difference t sig. (two-tailed) lower upper pair 1 marked and testing −0.101 0.525 0.081 −0.265 0.069 −1.131 0.211 n = the number of nodules images analysed by the radiologists and the proposed system that the means of the radiologist’s opinions and the system output are not pointedly varied. discussion the proposed system was based on the notion of integrating several techniques starting from choosing the dataset, features engineering to transform the original image to a set of proposed features then implementing the classifier. the pre-processing mechanism represents an important proactive step to optimise the classifier performance. the detection of such cases is assumed as a challenging task for most automated detection systems. this challenge relates to the speciality of nodules characteristics with regard to their size, thickness and location. the performance of a lung nodule abnormalities based on machine learning algorithms is measured by calculating sensitivity, specificity and accuracy. after applying the both proposed classifiers on zero-change dataset, the confusion matrix was generated using leaveone-out-cross-validation. table 2 presented the values of sensitivity, specificity and accuracy. the accuracy value refers to the total number of correct predictions that was made by each classifier. as shown the polynomial kernel svm outperformed the other algorithm. sensitivity represents the ability of the system that can detect nodules abnormalities which is matter. while specificity represents the system ability to identify the normality of nodules. in addition, the sigma value that resulted from t-test as shown in table 3 has shown that the differences between the marked images (radiologist opinions) and the system outcomes is not significant. 169j contemp med sci | vol. 5, no. 3, may–june 2019: 165–169 original detection system for lung nodule abnormalities based on machine learning algorithmsm. alsallal et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. conclusion this paper produced a detection system for categorising lung nodules as either normal or abnormal. this paper system worked on a notion of integrating several techniques; image acquisition, features engineering to accurately generate a clear depiction of nodules images. the zero-change dataset was used for the purpose of this paper proposed system. the experimental results have shown that the proposed system has scored high accuracy by polynomial kernel svm. a set of distinguishable representative features which are correlated together by a statistics association. also, this designed system can be considered as a benchmark for developing of other tissues abnormalities signs detection systems. in terms of developing the proposed system in future, the authors intend to integrate more intelligent techniques for feature extraction to facilitate the detection capabilities. furthermore deep learning techniques will be involved to compare two generation of machine learning algorithms performance. conflicts of interest none.  references 1. kumar d, wong a, clausi da. lung nodule classification using deep features in ct images. in: 2015 12th conference on computer and robot vision (crv), ieee, halifax, ns, canada, 2015, pp. 133-138. 2. krishnan k, ibanez l, turner wd, jomier j, avila rs. an open-source toolkit for the volumetric measurement of ct lung lesions. opt express. 2010;18:15256–15266. 3. sajda p. machine learning for detection and diagnosis of disease. annu. rev. biomed. eng. 2006;8:537–565. 4. bengio y, courville a, vincent p. representation learning: a review and new perspectives. ieee trans pattern anal mach intell. 2013; 35:1798–1828. 5. xu y, mo t, feng q, zhong p, lai m, chang ei. deep learning of feature representation with multiple instance learning for medical image analysis. in: 2014 ieee international conference on acoustics, speech and signal processing (icassp), ieee, florence, italy, 2014, pp. 1626–1630. 6. bellotti r, de carlo f, gargano g, tangaro s, cascio d, catanzariti e, et al. a cad system for nodule detection in low-dose lung cts based on region growing and a new active contour model. med phys. 2007; 34:4901–4910. 7. riccardi a, petkov ts, ferri g, masotti m, campanini r. computer-aided detection of lung nodules via 3d fast radial transform, scale space representation, and zernike mip classification. med phys. 2011;38:1962–1971. 8. camarlinghi n, gori i, retico a, bellotti r, bosco p, cerello p, et al. combination of computer-aided detection algorithms for automatic lung nodule identification. int j comput assist radiol surg. 2012; 7:455–464. 9. abdulla aa, shaharum sm. lung cancer cell classification method using artificial neural network. inform eng lett. 2012;2:49–59. 10. kuruvilla j, gunavathi k. lung cancer classification using neural networks for ct images. comput methods programs biomed. 2014;113:202–209. 11. moler ej, chow ml, mian is. analysis of molecular profile data using generative and discriminative methods. physiol genomics. 2000;4:109–126. 12. liu y. active learning with support vector machine applied to gene expression data for cancer classification. j chem inf comput sci. 2004;44:1936–1941. 13. furey ts, cristianini n, duffy n, bednarski dw, schummer m, haussler d. support vector machine classification and validation of cancer tissue samples using microarray expression data. bioinformatics. 2000;16:906–914. 14. segal nh, pavlidis p, antonescu cr, maki rg, noble ws, desantis d, et al. classification and subtype prediction of adult soft tissue sarcoma by functional genomics. am j pathol. 2003;163:691–700. 15. segal nh, pavlidis p, noble ws, antonescu cr, viale a, wesley uv, et al. classification of clear-cell sarcoma as a subtype of melanoma by genomic profiling. j clin oncol. 2003;21:1775–1781. 16. statnikov a, aliferis cf, tsamardinos i, hardin d, levy s. a comprehensive evaluation of multicategory classification methods for microarray gene expression cancer diagnosis. bioinformatics. 2005;21:631–643. 17. alsallal m. a machine learning approach for plagiarism detection. phd diss., coventry university, 2016. 18. vapnik v. the nature of statistical learning theory. springer science & business media, new york, 2013. 19. aizerman ma, braverman em, rozonoer li. theoretical foundations of the potential function method in pattern recognition learning. automation remote contr. 1964;25:821–837. 20. duin rpw, juszczak p, paclik p, pekalska e, de ridder d, tax dmj, et al. prtools4.1. a matlab toolbox for pattern recognition, delft university of technology. dx.doi.org/10.22317/jcms.06201909 70 j contemp med sci | vol. 4, no. 2, spring 2018: 70–73 research isolation and localization of cells expressing sca-1 in the adult mouse ovary: an evidence for presence of mesenchymal stem cells masoumeh majidi zolbin,a tahmineh mokhtari,b fardin amidi,a soraya parvari,c shahlla mirgaloybayat,d mehdi abbasia adepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. bresearch center of nervous system stem cells, department of anatomy, school of medicine, semnan university of medical sciences, semnan, iran. cdepartment of anatomy, school of medicine, alborz university of medical science, karaj, iran. dendometriosis research center, department of obstetrics and gynecology, rasul-e-akram hospital, iran university of medical sciences, iran. correspondence to mehdi abbasi (email: abbasima@sina.tums.ac.ir). (submitted: 18 december 2017 – revised version received: 10 january 2018 – accepted:15 february 2018– published online: 26 june 2018) objective recently growing evident declared that ‘neo-oogenesis’ continues in mature female life span and simultaneously another studies confirmed the presence of spermatogonial stem cells (sscs). even though there is an agreement between scientist about sscs population in male gender but on the other side ovarian stem cells have received raising challenges regarding the existence in the surface epithelium of ovary. mesenchymal stem cells (mscs) are the most applicable source of stem cells and the common marker of mscs is sca-1 so that the purpose of this study was to clarify the incidence of stem cells in the surface epithelium of ovary. methods forty c57bl6 mice were sacrificed and the ovary carefully excised from its surrounding fat tissue, after mechanical and enzymatic digestion cells were stained with sca-1 to estimate the incidence of positive stem cells (scs) population fluorescence activated cell sorting (facs). part of digested cells used for rt-pcr, also histological section prepared for immunohistochemical staining of sca-1 in ovarian surface epithelium facs. results the gene expression of sca-1 was confirmed in the ovarian tissue. as well, localization of sca-1+ cells was detected in the germinal layer of ovary and epithelial granular layer of primordial follicles. moreover, we could successfully isolate the sca-1+ cells through conclusion the present work findings confirmed an inclusive stem cell population in the ovary which can be a strong evident for regeneration of ovarian tissue in either purpose of ovulation scar and neo-oogenesis. keywords ovary, stem cell, sca-1, surface epithelium introduction the ovary is an organ which is responsible to prepare competent and mature oocytes in reproductive procedure. moreover, it involves in signaling pathways of oogenesis and folliculogenesis through secreting the different types of growth factors (gfs), hormones and cytokines.1 according to the principle (dogma), before or at birth, a finite number of oocytes develop and reserve in the ovary of female mammals which they can be determined as primordial follicles established during the neonatal period with suitable number and quality. recently, neo-oogenesis has been introduced as a restricted supply of oocytes by existence of renewable germ line stem cells (gscs) in the gonads of postnatal female mammals.2,3 based on neo-oogenesis dogma, the gscs of the peripheral blood, bone marrow (bm) or ovarian surface epithelium can differentiate into various types of cells in the ovary such as fibroblast-like cells, oocyte and cells with granulosa phenotype, under suitable stimulation and in vitro.4,5 as well, studies have identified existence of gscs in models of non-mammalian organisms like drosophila.6 however, there was enough evidence to confirm the presence of gscs in mammalian ovaries.2 in the literature, the presence of stem cell markers including oct47 and c-kit8 in the cellular subpopulations have been reported in the aged ovary. additionally, the other kinds of stem cells have been identified in the adult ovary, such as very small embryonic-like stem cells9 and a subpopulation of granulosa cells up-regulate some stemness genes related to bone marrow mesenchymal stem cells (mscs).10 in other way, more recently, stem cell antigen 1 (sca-1) have been introduced as a selective marker for mscs in mouse model.11 in this study, we tried to characterize the mscs in the ovary of mouse model by detecting the cells expressing sca-1 as a distanced marker of mscs using different techniques including fluorescence activated cell sorting (facs) analysis, immunohistochemistry (ihc) and rt-pcr. materials and methods animals this experimental study was performed on 40 adult females c57bl/6 mice 6 weeks of age. they were placed in individual cages during the study period, with 12 h of darkness and 12 h of light available. mice had access to water and food and ad libitum. the housing temperature range was kept between 25 and 30°c. the mice were kept at the animal facility center for 2 weeks to adapt to the new environment prior to the study. isolation of scs from mouse ovaries for isolation of scs, ovaries were harvested and the fat pad, bursa and oviduct were carefully excised. ovaries were then minced into very small pieces. ovarian tissue was digested in 0.83 mg/ml collagenase type 2 (worthington biochemical corporation nj, usa; #ls004174) in sterile hank’s balanced salt solution (hbss; diluted from 10× stock, containing no calcium, no magnesium, no phenol red; life technologies ca, usa; #14185-052) containing 3% bovine serum albumin (bsa), 1.23 mm calcium chloride, 1.03 mm magnesium chloride and 0.83 mm zinc chloride for 15 min in a 37°c shaking issn 2413-0516 masoumeh majidi zolbin et al. 71j contemp med sci | vol. 4, no. 2, spring 2018: 70–73 research sca-1+ cells in adult mouse ovary water bath (160–180 rpm), and interrupted incubation with vigorously shaking by hand for 10–20 s after 10 min of incubation. the supernatant was separated from the pellet by centrifugation at 400 g for 3 min. at last, the ovarian homogenate was re-suspended in 3 ml hbss, 3% bsa wash buffer and filtered through sterile 40 μm (bd biosciences ca, usa; #352340) nylon mesh filters before antibody staining. cell staining and facs digested ovarian homogenate which stained for sca-1 stem cell marker were sorted and characterized by facs analysis, ly-6a/e pe/cy5 (sca-1) (biolegend#108109) (1:500) stained the positive population. antibody were diluted according to the manufacturer’s recommendations in wash buffer and the ovary pellet was re-suspended in antibody staining solution and placed on ice in the dark for 30 min. an excess of wash buffer was then added and the stained cell suspension was centrifuged at 300 g for 3 min. the supernatant was carefully removed and the pellet was re-suspended in wash buffer and subsequently filtered through a 40-μm nylon filter prior to facs. isotype-matched negative controls were used to define background staining. cell sorting was performed using bd facs aria ii software (bd biosciences). immunohistochemical staining of tissue sections to confirm the presence of sca-1+ population in the ovary, ovaries were dissected, fixed in 4% pfa, and paraffin blocks were prepared and cut using a microtome into 6 µm sections. sections were deparaffinized by heating to 60°c for 1 hour, followed by three passages in xylenes for 10 min and subsequent rehydration with a graded alcohol series and finally into pbs. this was followed by boiling sections for 10 min in sodium citrate (ph = 6) for antigen retrieval. endogenous peroxidase activity was blocked by treatment with 3% h2o2 in 50% methanol for 10 min at room temperature. sections were blocked using 10% horse serum and rabbit serum (vector laboratories). slides were then incubated overnight with primary antibody (rabbit anti sca-1 ab; 109211, diluted 1:50), at 4°c. the next day 3 pbs washes were followed by incubation for 1 hour at room temperature in biotinylated horse-anti-rabbit and rabbit-anti-goat igg (1:200; vector laboratories), and colorimetric detection was performed using abc vectastain elite reagents with dab (vector laboratories). sections were counterstained with hematoxylin (sigma-aldrich). images of stained sections were captured using an olympus bx-51 microscope (olympus). ovarian stem cell culture after sorting, the cells were re-suspended in primary culture medium consisting of 40 ml dmem-f12 (gibco#11330-032) supplemented with 10% fetal bovine serum (fbs; gibco#10437028) and 103 u/ml leukaemia inhibitory factor (lif; amsbio#ams-263) were plated in 12 well plates at a density of 2 × 105 cells per well. plates were incubated at 37°c in an atmosphere of 5% co2 in air. results gene expression of sca-1 in the ovary using rt-pcr to find out gene expression of sca-1 in the ovarian homogenate, rt-pcr was performed. according to figure 1, the expression of sca-1 gene was confirmed. expression of β-actin was considered as a housekeeping gene. localization of sca-1-expressing cells in ovary tissue using ihc analysis to determine the location of ovarian mscs, we analyzed paraffin sections of normal ovary in normal mice by ihc staining with sca-1 antibody. in the analysis, we could detect the sca-1+ cells in the germinal layer of ovary and epithelial granular layer of primordial follicles (figure 2). potency of individual cells to form cells with spherical morphology to evaluate the potency of individual cells to form cells with spherical morphology, the sorted cells were cultured in media and the sca-1+ showed the potency of colony formation after 1 week of culture (figure 3). discussion the existence and confirmation of oscs are of importance for the field of reproductive science; as a result, some scientists focused more concern to figure out whether or not oogenesis occurs from these localized stem cells in the adult mammalian ovary in vivo. the current study has shown that adult mouse ovary contains a population of sca-1+ cells with stem cell characteristics. however, because of being challenging field as well fig. 1 1 stem cell gene marker expression of (a) sca-1 in ovarian homogenate. (b) expression of β-actin was considered as a housekeeping gene. a b 72 j contemp med sci | vol. 4, no. 2, spring 2018: 70–73 sca-1+ cells in adult mouse ovary research masoumeh majidi zolbin et al. fig. 2 detection of sca-1 expressing cells (sca-1+ cells) in adult mice ovary section using ihc staining with anti-sca-1 antibody. sca-1 expressing cells are found in the germinal layer of ovary and in the epithelial granular layer of primordial follicles (red arrows), magnification of 100×. fig. 3 (a) the sca-1+ cells in cell culture media showed the local colonies, (b) in 2nd week the spherical cells were floating in the media. a b as their indefinite character, derivation and function, it cannot be strongly declared that they are “stem cells” with ovarian origin and mostly have been indicated as “putative stem cells” in previous.8,12,13 it has been proven that two different types of stem cells are located in the mature organs of which the first type is represented as dynamic stem cells and the other type as quiescent stem cells. there was this concept that ovarian stem cells are located in the second category without contribution in “neo-oogenesis”.14–16 presence of mscs are almost confirmed in several organs along with bone marrow and white adipose tissue.17–19 mscs due to their multipotent characteristic are reliable therapeutic source to support regeneration and repair of injured tissues of mesenchymal basis.20 sca-1+ have recently been recognized as selective markers of mouse mscs.11 the stem cell niche is a dedicated microenvironment that sustain stem cells’ competence to self-renew and differentiate.21 here this question raises that why the stem cell niche is inactive in ovary epithelium which can be answered by unknown composition of these stem cells in the ovary. several studies conducted to isolate stem cell from the ovary with stem cell markers like masoumeh majidi zolbin et al. 73j contemp med sci | vol. 4, no. 2, spring 2018: 70–73 research sca-1+ cells in adult mouse ovary ssea-1,22,23 fragilis,24,25 and ddx416 with multiple techniques26 or based on the cell morphology.27 given the limited evidence for distinctive subpopulations of the oscs cells, in this study we were tried to separate a general fraction of cells with “msc” characteristics using cell surface expression for sca-1 with facs sorting. additionally, the presence of this fraction in the surface epithelium of ovary were confirmed by ihc staining which demonstrated distinct positive cells in the ovarian histological sections, these characteristics suggest that this cell population is fortified for progenitor cell activity. the sca-1+ cells have a high colony-forming competence with progenitor cell distinctiveness. these cells have two main characteristics: the capability to self-renewal and differentiation potential. our findings represent multiple colonies and in long time cell culture these cells progeny were able to differentiate to round and detached cells with different morphology which reflect the number of differentiated cells in culture media.28 since the expression of “mscs” in the oocyte has not been reported previously this examination raised fascinating possibility of the association between neo-oogenesis and this progenitor cells as indicated before.13,29,30 to make these cells more applicable for clinical purpose, it is necessary to differentiate the characterized cells with growth factors that they are involved in differentiation. transplantation of these cells to infertile mice also can be considered as a reliable method for evaluation of these cells function in long term. in conclusion sca-1 is a representative marker of mscs which expressing with low incidence in mouse ovary. since two types of somatic or germ stem cells defined so far in the ovary, we can assume that these cells are progenitor cells and have role in ovulatory wound healing or they are putative germ stem cells that can be consider for neo-oogenesis. studies in this area shade a light for the future of infertile patient but further study needed to overcome the challenges of stem cells in the ovary. conflict of interest none.  references 1. gheorghisan-galateanu aa, hinescu me, enciu am. ovarian adult stem cells: hope or pitfall? j ovarian res. 2014;7:71. 2. hanna cb, hennebold jd. ovarian germline stem cells: an unlimited source of oocytes? fertil steril. 2014;101:20–30. 3. liu y, wu c, lyu q, yang d, albertini df, keefe dl, et al. germline stem cells and neo-oogenesis in the adult human ovary. dev biol. 2007;306:112–120. 4. woods dc, tilly jl. isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries. nat protoc. 2013;8:966–988. 5. johnson j, bagley j, skaznik-wikiel m, lee h-j, adams gb, niikura y, et al. oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. cell. 2005;122:303–315. 6. eliazer s, buszczak m. finding a niche: studies from the drosophila ovary. stem cell res ther. 2011;2:45. 7. parte s, bhartiya d, patel h, daithankar v, chauhan a, zaveri k, et al. dynamics associated with spontaneous differentiation of ovarian stem cells in vitro. j ovarian res. 2014;7:25. 8. bui ht, van thuan n, kwon dn, choi yj, kang mh, han jw, et al. identification and characterization of putative stem cells in the adult pig ovary. development. 2014;141:2235–2244. 9. virant-klun i, skutella t. stem cells in aged mammalian ovaries. aging (albany ny). 2010;2:3. 10. dzafic e, stimpfel m, novakovic s, cerkovnik p, virant-klun i. expression of mesenchymal stem cells-related genes and plasticity of aspirated follicular cells obtained from infertile women. biomed res int. 2014;2014. 11. houlihan dd, mabuchi y, morikawa s, niibe k, araki d, suzuki s, et al. isolation of mouse mesenchymal stem cells on the basis of expression of sca-1 and pdgfr-α. nat protoc. 2012;7:2103–2111. 12. yazdekhasti h, hosseini ma, rajabi z, parvari s, salehnia m, koruji m, et al. improved isolation, proliferation, and differentiation capacity of mouse ovarian putative stem cells. cell reprogram. 2017;19:132–144. 13. virant‐klun i, zech n, rožman p, vogler a, cvjetičanin b, klemenc p, et al. putative stem cells with an embryonic character isolated from the ovarian surface epithelium of women with no naturally present follicles and oocytes. differentiation. 2008;76:843–856. 14. park es, woods dc, tilly jl. bone morphogenetic protein 4 promotes mammalian oogonial stem cell differentiation via smad1/5/8 signaling. fertil steril. 2013;100:1468–1475 (e2). 15. bhartiya d, sriraman k, parte s. stem cell interaction with somatic niche may hold the key to fertility restoration in cancer patients. obstet gynecol int. 2012;2012:11. 16. tilly jl, rueda br. stem cell contribution to ovarian development, function, and disease. endocrinology. 2008;149:4307–4311. 17. da silva meirelles l, chagastelles pc, nardi nb. mesenchymal stem cells reside in virtually all post-natal organs and tissues. j cell sci. 2006;119:2204–2213. 18. morikawa s, mabuchi y, kubota y, nagai y, niibe k, hiratsu e, et al. prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. j exp med. 2009;206:2483–2496. 19. dicker a, le blanc k, åström g, van harmelen v, götherström c, blomqvist l, et al. functional studies of mesenchymal stem cells derived from adult human adipose tissue. exp cell res. 2005;308:283–290. 20. stappenbeck ts, miyoshi h. the role of stromal stem cells in tissue regeneration and wound repair. science. 2009;324:1666–1669. 21. morrison sj, spradling ac. stem cells and niches: mechanisms that promote stem cell maintenance throughout life. cell. 2008;132:598–611. 22. khosravi-farsani s, amidi f, roudkenar mh, sobhani a. isolation and enrichment of mouse female germ line stem cells. cell j. 2015;16:406. 23. hamidabadi hg, sobhani a, bojnordi mn. multipotent ssea-1 positive cells population differentiation into primordial germ cells and subsequently progress into oocyte-like cells. arch iran med. 2015;18:404–410. 24. zou k, yuan z, yang z, luo h, sun k, zhou l, et al. production of offspring from a germline stem cell line derived from neonatal ovaries. nat cell biol. 2009;11:631–636. 25. lu z, wu m, zhang j, xiong j, cheng j, shen w, et al. improvement in isolation and identification of mouse oogonial stem cells. stem cells int. 2016;2016. 26. yazdekhasti h, rajabi z, parvari s, abbasi m. used protocols for isolation and propagation of ovarian stem cells, different cells with different traits. j ovarian res. 2016;9:68. 27. parvari s, yazdekhasti h, rajabi z, gerayeli malek v, rastegar t, abbasi m. differentiation of mouse ovarian stem cells toward oocyte-like structure by coculture with granulosa cells. cell reprogram. 2016;18:419–428. 28. gamwell lf, collins o, vanderhyden bc. the mouse ovarian surface epithelium contains a population of ly6a (sca-1) expressing progenitor cells that are regulated by ovulation-associated factors 1. biol reprod. 2012;87:1–10 (article 80). 29. johnson j, canning j, kaneko t, pru jk, tilly jl. germline stem cells and follicular renewal in the postnatal mammalian ovary. nature. 2004;428: 145–150. 30. bukovsky a, svetlikova m, caudle mr. oogenesis in cultures derived from adult human ovaries. reprod biol endocrinol. 2005;3:1. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201803 331j contemp med sci | vol. 3, no. 12, autumn 2017: 331–334 research plasma visfatin levels and insulin sensitivity or resistance relationship in type 2 diabetes shatha abdul wadood alshammaree department of chemistry, college of science, university of baghdad, baghdad, iraq. *correspondence to: shatha abdul wadood alshammaree (e-mail: shath_a@yahoo.com). (submitted: 29 august 2017 – revised version received: 07 september 2017 – accepted: 16 october 2017 – published online: 26 december 2017) objective many studies reported inconsistent results about the connection of visfatin with diabetes and insulin resistance (ir), and also the role of visfatin in the pathogenesis of diabetes are still under debate. this study aimed to evaluate the visfatin levels in patients with type 2 diabetes (t2dm) compared to non-diabetic age-matched subjects and explore the relationship between visfatin and insulin sensitivity or resistance. methods a total of 85 subjects were enrolled in this study; 60 patients with t2dm who were sub-divided into two groups according to levels of ir (ir < 2.5 and > 2.5 groups). also, 25 non-diabetic healthy subjects matched for age, body mass index, and lipid profile were included as a control group. physical and clinical examinations were done for each participant. visfatin levels were measured using enzyme-linked immune sorbent assay. ir and quantitative insulin sensitivity check index (quicki) were calculated from fasting plasma glucose and insulin. results significant higher visfatin level and ir were found in diabetic patients in comparison to control group (p < 0.05). no significant difference was found in visfatin level between diabetic patients with ir < 2.5 and control group, while, visfatin level in diabetic patients with ir > 2.5 was significantly higher in comparison to that of the group with ir < 2.5 and control group. significant negative correlation was found between visfatin levels and quicki in the diabetic patients’ group with ir < 2.5 (r = −0.324, p < 0.05). highly significant negative correlation was found between quicki and ir in control and diabetic patients with ir < 2.5, while this correlation disappears in ir > 2.5 group. conclusion an inverse relationship between visfatin levels and insulin sensitivity was found. also, the correlation between insulin sensitivity and resistance differs depending on their levels. this study suggests that visfatin may play a significant regulatory role at elevated glucose levels, spatially when ir is more than 2.5. keywords visfatin, type 2 diabetes, insulin resistance, insulin sensitivity introduction visfatin is an adipokine secreted from the adipose tissue that was formerly described nicotinamide phosphoribosyl transferase (nampt).1 it is also secreted from lymphocytes, leukocytes, muscles, and hepatocytes.2,3 it was found that visfatin is expressed as a response to inflammation from macrophages of adipose tissue rather than from adipocytes.4,5 although adipose tissue is considered an endocrine tissue, it is now believed that visfatin actions can be endocrine, paracrine, and autocrine as well. the autocrine effects of visfatin in the liver may play an important role in regulating insulin sensitivity.6 visfatin is involved in regulation of the cell cycle.7 it is secreted by activated lymphocytes and is upregulated in neutrophils and monocytes after exposure to inflammatory stimuli. it also promotes the growth of b-cell precursors and is therefore known as a pre-b-cell colony-enhancing factor.8 although, visfatin physiological role in human beings still largely unrevealed; many studies showed that visfatin has many functions such as a cytokine,19 a hormone1 and lately, as an enzyme (the rate-limiting enzyme nampt in the nad biosynthesis).10 various medical conditions were correlated with visfatin levels through its anti-diabetic,1 anti-tumor,11 antihyperlipidemia,12 and pro-inflammatory properties.9 this is due to its role in the modulation of insulin sensitivity, insulin resistance (ir) in diabetes, dyslipidemia, inflammation and atherosclerosis.13 in diabetic patients, irregular feedback mechanism in the hypothalamic-pituitary axis was found through the decrease in lh and fsh levels due to high glucose levels and ir.14 hammarstedt et al. (2006)15 reported a double increase in plasma visfatin levels in t2dm compared with controls, which is similar to other studies.12,16,17 however, others found no correlation between circulating visfatin, insulin, and glucose.18,19 lopez-bermejo et al. (2006)20 reported a significant increase in levels of visfatin in patients with t1dm compared with t2dm or non-diabetic subjects. the correlation of visfatin levels with obesity and ir was revealed by rabo et al. (2013)16 study who found that they have higher significant positive correlation in both obese and non-obese diabetic patients with a significant positive correlation with ir. this inconsistency in the results of many researches regarding the correlation of visfatin with diabetes and ir leads to more investigation. this study aimed to determine the plasma visfatin level in patients with t2dm and in control, also, explore the relationship between plasma visfatin and insulin sensitivity/resistance. materials and methods the case control study enrolled 16 patients with t2dm who attended the national diabetes center for diagnosis, treatment, and follow-up at al-mustansiyria university in baghdad, iraq. the diagnosis was based on the criteria of the world health organization (who) (fbg > 126 mg/dl).21 the duration of the disease is between 4 and 8 years, and all patients were on oral glucose lowering treatment. the mean level of age and bmi were (40 ± 6.23 years; 29.15 ± 3.21 kg/m2, respectively). twenty five healthy subjects were included as a control group who matched to patients group in age, and bmi (37.90 ± 8.60 years; 27.41 ± 4.01 kg/m2, respectively). the patients were selected with normal lipid profile to exclude the effect of lipogenesity on visfatin level. a written approval was taken from ethical and scientific committee and from each participant. any patient with any complication of diabetes or any other disease that has any effect on the studied parameters was excluded. issn 2413-0516 332 j contemp med sci | vol. 3, no. 12, autumn 2017: 331–334 plasma visfatin levels and insulin sensitivity or resistance relationship research shatha abdul wadood alshammaree samples five ml of fasting venous blood was withdrawn from each participant after physical and clinical examination. renal function was determined (uric acid and creatinine) using an autoanalyzer device (biolabo, france). fasting plasma glucose was determined using the oxidase method. fasting insulin (fi) was measured using a competitive enzyme immunoassay kit (randox; uk). ir was calculated according to the formula: ir = fasting plasma insulin (miu) × fasting plasma glucose (mm/l)]/22.5. the patient group was sub-divided according to ir levels to <2.5 and >2.5 groups.22 the determination of plasma visfatin level was done using raybio® visfatin enzyme linked immunosorbent assay (eia) kit. the sensitivity of the visfatin assay is 0.778 ng/ml, with intra-assay: cv < 10%, and interassay: cv < 15%. quantitative insulin sensitivity check index (quicki) was used to measure insulin sensitivity from fasting blood glucose and insulin using the following formula:22 quicki = 1/[log(io) + log(go)] where io is the fasting plasma insulin, and go is the fasting plasma glucose. the documented quicki level is: (0.382 ± 0.007) for non-obese, (0.331 ± 0.010) for obese and (0.304 ± 0.007) for diabetic individuals.23 statistical analysis statistical analysis was performed using a standard statistical package (spss) for windows, version 21; chicago, il, usa). the data were expressed as mean ± sd. two-way student’s t-test was used for comparison between two groups. the post has a one-way analysis of variance (anova) was used for comparison between groups. the association of visfatin with quicki was examined by pearson correlation analysis, as well as the correlation between ir and quicki. when p value less than 0.05; it is statistically significant. results the characteristics and some clinical measurements are presented in table 1. body mass index, fi, uric acid, and creatinine showed no significant differences between patients and control groups (p > 0.05) while significant increase was found in patient group when compared to control group for fpg, and ir, (p < 0.05). the plasma visfatin level was significantly higher in the diabetic patient group compared with control group (9.52 ± 1.56 vs. 8.28 ± 0.62 ng/ml, p = 0.0001, respectively). patients were sub-grouped, depending on ir levels, into ir < 2.5 and ir > 2.5 groups. the quicki level in both groups showed a significant decrease when compared to control group (0.28 ± 0.015 and 0.31 ± 0.015 vs. 0.34 ± 0.013, p = 0.042 respectively) as presented in table 2. visfatin level in a group of patients with ir > 2.5 show significant higher levels in comparison to that of patient group with ir < 2.5 and control group (9.17 ± 1.10 vs. 9.12 ± 1.02 and 8.28 ± 0.62, p = 0.017, respectively), while no significant difference was found between patient group with ir < 2.5 and control group (p > 0.05) as presented in table 2 and fig. 1. pearson correlation between visfatin and fpg, fi, ir, creatinine, and uric acid showed no significant differences, while a significant negative correlation was found with quicki (r = −0.324, p < 0.05), as presented in fig. 2. a highly negative significant correlation was found between quicki and ir for the control group (r = 0.952, p < 0.01) while, the weak negative significant correlation was observed in the patient group with ir < 2.5 (r = 0.473, p < 0.05) but this correlation disappear in the patient group with ir > 2.5 as shown in fig. 3. discussion plasma visfatin levels were higher in diabetic patients compared to control group which is consistent with other studies15,20,24 and counteract with other’s results25–27 who found lower visfatin levels in t2dm when compared with matched non-diabetic subjects. the increase in circulating visfatin levels by hyperglycemia is not clear until now, but it may be due to oxidative stress, increased apoptosis, or destruction of b lymphocytes.7 it was suggested that the elevation in levels of circulating visfatin is a compensatory mechanism for the decreased insulin sensitivity in patients with t2dm. this is in agreement with the results of other research.16,28 however, yaturu et al. (2012) measured visfatin levels in t2dm patients who were under pioglitazone treatment. they elucidate the decrease in visfatin levels, which may be due to the treatment.27 their result confirms what mcgee table 1. mean (±sd) of some characteristic and clinical measurements in patients and control groups parameters control group patients group p value no. 25 60 – age (year) 37.90 ± 8.60 40.25 ± 6.23 >0.05 sex (male/female) 11/14 35/25 – duration (year) – 6.4 ± 2.11 – uric acid (mmol/l) 341.5 ± 110.8 374.9 ± 104.1 0.196 creatinine (mg/dl) 0.99 ± 0.03 1.19 ± 0.06 0.43 fbg (mg/dl) 89.54 ± 10.93 219.91 ± 94.32 0.014 fi (μiu/ml) 10.33 ± 2.45 20.96 ± 7.92 0.067 ir 1.43 ± 0.73 5.34 ± 2.03 0.01 visfatin (ng/ml) 8.28 ± 0.62 9.52 ± 1.56 0.0001 significant difference is when p value less than 0.05 using independent student’s t-test. bmi, body mass index; fpg, fasting plasma glucose; fi, fasting insulin; ir, insulin resistance. table 2. quicki and visfatin levels (expressed as mean ± sd) in patients sub-grouped according to ir levels in comparison to control group parameters control group ir > 2.5 group ir < 2.5 group p value no. 25 30 30 – quicki 0.34 ± 0.013 0.28 ± 0.015 0.31 ± 0.015 0.042 visfatin ng/ml 8.28 ± 0.62 9.03 ± 1.02 7.96 ± 1.10 0.017 the p value less than 0.05 is considered significant using post hoc anova analysis. ir, insulin resistance; quicki, quantitative insulin sensitivity check index. shatha abdul wadood alshammaree 333j contemp med sci | vol. 3, no. 12, autumn 2017: 331–334 research plasma visfatin levels and insulin sensitivity or resistance relationship conclusion the likelihood of elevation of visfatin in diabetic people is a mechanism to compensate insulin deficiency or its inefficiency, so that it can regulate blood glucose level at certain levels. it can be concluded that increase visfatin level may be inducible to regulate elevated glucose levels spatially when ir is fig 2. the pearson correlation between visfatin and quicki in diabetic patients with ir < 2.5. fig 3. the pearson correlation between quicki and ir in: a) control group, b) diabetic patients with ir < 2.5, c) diabetic patients with ir > 2.5. fig 1. mean level and significant differences of visfatin in patients groups in comparison to control group. et al., (2011) had previously found when they used rosiglitazone for t2dm treatment.29 the plasma visfatin increase as fasting blood glucose and ir increase, but it didn’t reach significance level. previous studies documented the role of this hormone as an insulinresembling effect in lowering blood glucose through binding to the insulin receptor and initiate a signal transduction inside the cell.1,17,24 many studies failed to find a relation between visfatin and ir in t2dm30,31 while many studies reported the presence of significant association with ir. 32–34 one of the important aims of the study is to investigate the correlation of visfatin with insulin sensitivity by the use of quicki for its simplicity and inexpensive useful index for clinical studies.35 due to the documented limitation of homa ir as a tool for the estimation of ir because of the skewed distribution of fi values, the inverse logarithmic scale of homa ir; quicki is used to reflect the effect of insulin on hepatic glucose production.36 interestingly, no correlation was reported between insulin sensitivity and plasma visfatin by many researchers who used hyper-insulinemic euglycemic clamp (insulin sensitivity standard evaluation method).15,32,37,38 similarly, other study did not report correlation between visfatin and insulin sensitivity and lipid of intra-myocellular in skeletal muscle.5 therefore, persuasive facts regarding the association between visfatin and ir in t2dm are still missing. takebayashi et al., (2007) found no correlation between diabetes and levels of visfatin.39 334 j contemp med sci | vol. 3, no. 12, autumn 2017: 331–334 plasma visfatin levels and insulin sensitivity or resistance relationship research shatha abdul wadood alshammaree more than 2.5. however, more studies with a larger number of patients are needed to confirm this conclusion. acknowledgements our deep gratitude is due to all patients, participants and staff members of laboratories in the national centers for 22. gutch m, kumar s, razi sm, gupta kk, gupta a. assessment of insulin sensitivity/resistance. indian j endocrinol metabol. 2015;19:160–164. 23. katz a, nambi ss, mather k, baron ad, follmann da, sullivan g, et al. quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. j clin endocrinol metabol. 2000;85:2402–2410. 24. chen mp, chung fm, chang dm, tsai jc, huang hf, shin sj, et al. elevated plasma level of visfatin/pre-b cell colony-enhancing factor in patients with type 2 diabetes mellitus. j clin endocrinol metabol. 2006;91:295–299. 25. jian wx, luo th, gu yy, zhang hl, zheng s, dai m, et al. the visfatin gene is associated with glucose and lipid metabolism in a chinese population. diabet med. 2006;23:967–973. 26. li l, yang g, li q, tang y, yang m, yang h, et al. changes and relations of circulating visfatin, apelin, and resistin levels in normal, impaired glucose tolerance, and type 2 diabetic subjects. exp clin endocrinol diabet. 2006;114:544–548. 27. yaturu s, davis j, franklin l, shi r, venkatesh p, jain sk. visfatin levels are low in subjects with type 2 diabetes compared to age-matched controls. j diabet mellit. 2012;2:373–377. 28. saddi-rosa p, oliveira cs, giuffrida fm, reis af. visfatin, glucose metabolism and vascular disease: a review of evidence. diabetol metabol syndrome. 2010;2:21–26. 29. mcgee kc, harte al, da silva nf, al-daghri n, creely sj, kusminski cm, et al. visfatin is regulated by rosiglitazone in type 2 diabetes mellitus and influenced by nfκb and jnk in human abdominal subcutaneous adipocytes. plos one. 2011;6:e20287. 30. esteghamati a, alamdari a, zandieh a, elahi s, khalilzadeh o, nakhjavani m, et al. serum visfatin is associated with type 2 diabetes mellitus independent of insulin resistance and obesity. diabet res clin pract. 2011;91:154–158. 31. chang yh, chang dm, lin kc, shin sj, lee yj. visfatin in overweight/ obesity, type 2 diabetes mellitus, insulin resistance, metabolic syndrome and cardiovascular diseases: a meta-analysis and systemic review. diabet metabol res rev. 2011;27:515–527. 32. ognjanovic s, jacobs dr, steinberger j, moran a, sinaiko ar. relation of chemokines to bmi and insulin resistance at ages 18–21. int j obes. 2013;37:420–423. 33. wroblewski e, swidnicka-siergiejko a, hady hr, luba m, konopko m, kurek k, et al. variation in blood levels of hormones in obese patients following weight reduction induced by endoscopic and surgical bariatric therapies. cytokine. 2016;77:56–62. 34. agueda m, lasa a, simon e, ares r, larrarte e, labayen i. association of circulating visfatin concentrations with insulin resistance and low-grade inflammation after dietary energy restriction in spanish obese non-diabetic women: role of body composition changes. nutr metabol cardiovas dis. 2012;22:208–214. 35. patarrão rs, lautt ww, macedo mp. assessment of methods and indexes of insulin sensitivity. revista portuguesa de endocrinologia, diabetes e metabolismo. 2014;9:65–73. 36. abbasi f, reaven gm. evaluation of the quantitative insulin sensitivity check index as an estimate of insulin sensitivity in humans. metabol clin exp. 2002;51:235–237. 37. berndt j, klöting n, kralisch s, kovacs p, fasshauer m, schön mr, et al. plasma visfatin concentrations and fat depot–specific mrna expression in humans. diabetes. 2005;54:2911–2916. 38. brema i, hatunic m, finucane f, burns n, nolan jj, haider d, et al. plasma visfatin is reduced after aerobic exercise in early onset type 2 diabetes mellitus. diabet obes metabol. 2008;10:600–602. 39. takebayashi k, suetsugu m, wakabayashi s, aso y, inukai t. association between plasma visfatin and vascular endothelial function in patients with type 2 diabetes mellitus. metabolism. 2007;56:451–458. diabetes/al-mustansiriya university spatially dr. refif sabih alshawk for her assistant in collecting patient samples and their demographic information. conflicts of interest disclosure there are no conflicts of interest. n references 1. fukuhara a, matsuda m, nishizawa m, segawa k, tanaka m, kishimoto k, et al. visfatin: a protein secreted by visceral fat that mimics the effects of insulin. science. 2005;307:426–430. 2. garten a, petzold s, barnikol-oettler a, körner a, thasler we, kratzsch j, et al. nicotinamide phosphoribosyltransferase (nampt/pbef/visfatin) is constitutively released from human hepatocytes. biochem biophys res commun. 2010;391:376–381. 3. costford sr, bajpeyi s, pasarica m, albarado dc, thomas sc, xie h, et al. skeletal muscle nampt is induced by exercise in humans. am j physiol endocrinol metabol. 2010;298:e117–e126. 4. curat ca, wegner v, sengenes c, miranville a, tonus c, busse r, et al. macrophages in human visceral adipose tissue: increased accumulation in obesity and a source of resistin and visfatin. diabetologia. 2006;49:744. 5. varma v, yao-borengasser a, rasouli n, bodles am, phanavanh b, lee mj, et al. human visfatin expression: relationship to insulin sensitivity, intramyocellular lipids, and inflammation. j clin endocrinol metabol. 2006;92:666–672. 6. skop v, kontrová k, zídek v, pravenec m, kazdová l, mikulík k, et al. autocrine effects of visfatin on hepatocyte sensitivity to insulin action. physiol res. 2010;59:615. 7. adeghate e. visfatin: structure, function and relation to diabetes mellitus and other dysfunctions. curr med chem. 2008;15:1851–1862. 8. ognjanovic s, bao s, yamamoto sy, garibay-tupas j, samal b, bryantgreenwood gd. genomic organization of the gene coding for human preb-cell colony enhancing factor and expression in human fetal membranes. j mol endocrinol. 2001;26:107–117. 9. moschen ar, kaser a, enrich b, mosheimer b, theurl m, niederegger h, et al. visfatin, an adipocytokine with proinflammatory and immunomodulating properties. j immunol. 2007;178:1748–1758. 10. wang t, zhang x, bheda p, revollo jr, imai si, wolberger c. structure of nampt/pbef/visfatin, a mammalian nad+ biosynthetic enzyme. nat struct mol biol. 2006;13:661. 11. khan ja, xiao t, liang t. molecular basis for the inhibition of human nmprtase, a novel target for anticancer agents. nat struct mol biol. 2006;13:582. 12. rezk my. effect of visfatin on blood glucose and serum lipids in normal and streptozotocin induced diabetic rats. int j anat physiol. 2013;3:36–41. 13. rezk my. visfatin and cardiovascular protection. j drug deliv therap. 2014;4:154–166. 14. ahmed hs, tahir nt. association between diabetes mellitus and knee osteoarthritis. iraq medical journal. 2017;1:65–67. 15. hammarstedt a, pihlajamäki j, rotter sopasakis v, gogg s, jansson pa, laakso m, et al. visfatin is an adipokine, but it is not regulated by thiazolidinediones. j clin endocrinol metabol. 2006;91:1181–1184. 16. rabo sa, mohammed na, eissa ss, ali aa, ismail sm, gad rs. serum visfatin in type 2 diabetes mellitus. egyptian j int med. 2013;25:27. 17. haider dg, holzer g, schaller g, weghuber d, widhalm k, wagner o, et al. the adipokine visfatin is markedly elevated in obese children. j pediatr gastroenterol nutr. 2006;43:548–549. 18. dogru t, sonmez a, tasci i, yilmaz mi, erdem g, erturk h, et al. plasma visfatin levels in young male patients with uncomplicated and newly diagnosed hypertension. j hum hyperten. 2007;21:173–175. 19. pagano c, pilon c, olivieri m, mason p, fabris r, serra r, et al. reduced plasma visfatin/pre-b cell colony-enhancing factor in obesity is not related to insulin resistance in humans. j clin endocrinol metabol. 2006;91:3165–3170. 20. lópez-bermejo a, chico-julià b, fernàndez-balsells m, recasens m, esteve e, casamitjana r, et al. serum visfatin increases with progressive β-cell deterioration. diabetes. 2006;55:2871–2875. 21. alberti kg, zimmet pf. definition, diagnosis and classification of diabetes mellitus and its complications. part 1: diagnosis and classification of diabetes mellitus. provisional report of a who consultation. diabet med. 1998;15:539–553. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 336 j contemp med sci | vol. 5, no. 6, november–december 2019: 336–342 original issn 2413-0516 introduction leishmaniasis is a parasitic ailment brought about by the infection with blood flagellate leishmania. the disease is widespread and it is believed as a cause for a grave health dilemma in countries throughout the mediterranean regions and the middle east, including iraq.1,2 cutaneous leishmaniasis exhibit different clinical appearances depending on parasite and the host immune system.3,4,5 several studies have declared the differences between males and females regarding total mortality, vulnerability to allergic and autoimmune diseases, or particular infectious disease danger.6,7,8 possibly, males are more likely to be engaged in actions such as attacks, campaigns, traveling, and crowding, which increases the likelihood of contact with parasites.2,3 regardless of the variation in the probability of encountering different risks, immunological divergences present between both genders that may lead males to be more prone to parasitism.8,9 the current research was carried out to delineate any gender differences in the severity of cutaneous leishmaniasis lesions in regarding to parasites grading and features of the lesions (number, size, site, type) amongst patients in endemic zone. materials and methods study protocol this is a cross-sectional, descriptive study conducted during the period of january 2014 to june 2019 performed in the dermatology clinics of: rizgary hospital, hawler teaching center for skin diseases, shadi health center, hawler institute of health prevention. this study was carried out with the collaboration of prevention health department, erbil medical technical institute, erbil polytechnic university, with departments of: microbiology , anatomy & histology, college of medicine, hawler medical university, erbil, iraq. ethical considerations this study was approved by: the ethics committee of hawler medical university, erbil; the committee of erbil medical technical institute, erbil polytechnic university, iraq; health directorate of erbil; education directorate of erbil. informed consent was taken from each patient. the patients were informed about study’s objectives and they could withdraw thereof if they wished so to do. study population in the present study, the patients of age ≥18 years with cutaneous leishmaniasis were selected from outpatients attending the dermatology clinics mentioned above. patients who were inhabitants or resident of the rural districts surrounding erbil city of makhmur and kalack were involved in this study. makhmur district is situated 67 km south-west of erbil city in erbil governorate while kalack district is situated 32 km west of erbil. both districts are open land used for agriculture with large number of villages.10 exclusion criteria include patients with a suspected clinical lesion, patients who rejected to share in the study or who had received partial treatment for cl. in addition, prepubertal, children, and pregnant patients with lesions were excluded from the study to bypass any bias or misconception results which may appear due to sexual immaturity or hormonal changes during pregnancy. sample and diagnostic procedures diagnoses of the disease are based on: 1. clinical feature: this was achieved by an experienced dermatologist. examination of the patients was done to estimate the following points about the lesion: site, number, size, duration, and type as wet or dry cl. gender differences in the severity and features of lesions among cutaneous leishmaniasis patients zakarea abdullah yaseen al-khayat,a nabaz fisal shakir agha,b kawthar ibrahim fatah alharmnic adepartment of microbiology, college of medicine, hawler medical university, erbil, iraq. bprevention medicine department, medical technical institute, erbil polytechnic university, erbil, iraq. cdepartment of anatomy and histology, college of medicine, hawler medical university, erbil, iraq. (submitted: 14 august 2019 – revised version received: 01 september 2019 – accepted: 29 september 2019 – published online: 26 december 2019) objective to determine if there is any differences in the severity and features of lesions among patients complaining of cutaneous leishmaniasis in an endemic region. methods a cross-sectional, observational, descriptive study was performed (january 2014 to june 2019) in the dermatology clinics of: rizgary hospital, hawler teaching center for skin diseases, shadi health center, hawler institute of health prevention. all the patients were referred from rural districts of makhmur and kalack. the provisional diagnosis was dependent mainly on clinical examination in addition to giemsa stain. parasite (amastigote) grading and distribution of number, site, type, and size of lesions according to the gender of patients were studied. results a total of 1264 cutaneous leishmaniasis cases were diagnosed during the study period. according to stain results, 70.6% of the cases were positive to giemsa stain. parasite grading and parasite number/field were higher significantly in males. features of case severity according to the characters of the lesions (number, size, site, type) were more noted in males than females. conclusion male patients are more prone to more severe infections than females. key words cutaneous leishmaniasis, makhmur, kalack, gender, lesion 337 original gender differences in the severity and featureszakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 336–342 2. parasitological examination (giemsa stain & parasite grading): after cleaning of the lesion, a sample was obtained from the indurated margin of the lesion and examined. the sample from the cutaneous lesion was taken by fine needle aspiration as the following steps: 1. the skin around the lesion was disinfected by 70% ethanol. 2. the sterile syringe of 1 ml containing 0.2 ml of sterile normal saline was injected intradermal through intact skin in to the active red border of the lesion. 3. aspirate the injected fluid as the needle draw back till the bloody stained fluid aspirate. 4. small amount of aspirated fluid was taken and smeared on a clean glass microscope slide then left it to dry, then fixed using 100% absolute methanol for 30 s and left it to dry again. 5. stained with geimsa stain for 20 min, then rinse with tap water and dry the slide, and then examined it under oil immersion lens of the light microscope (olympus ch2, japan). 6. amastigote was diagnosed as round or spherical shape with distinctive kinetoplast. in this case was declared positive. when no amastigote was seen after 15 min of inspection, the smears was declared negative.2,4,11 7. the average parasite number on each slide was graded based on the numbers of leishmania amastigotes in high power field (hpf) using 10 × eyepiece and 100 × objective lenses on the following criteria: –, amastigotes could not be observed in the whole slide. +, 1 amastigote in the whole slide up to one amastigote per field in a total of at least 100 fields. ++, 2 to 10 amastigotes per field in a total of at least 50 fields. +++, 11–20 amastigotes per field in a total of at least 50 fields. ++++, 21 or more amastigotes per field in a total of at least 10 fields.12 statistical analysis the data analysis was performed using descriptive statistics, including mean ± standard deviation, frequency, and frequency percentage. comparisons were made using chi2 test or student’s t-test as appropriate by using standard equations. the results were reported with p ≤ 0.05 or p ≤ 0.01 or as the accepted level of significance accordingly. results in the present study, a total of 1264 patients were confirmed to have cl from january 2014 to june 2019. regarding the results of parasitological examination, table 1 denotes that giemsa stain was positive in 893 cases (70.6%). table 2 illustrates that from the total 1264 patients, males represent 54.6% of the cases. the details of the distribution of positive and negative results in addition to results of parasite grading in both genders are also clarified. from the total 893 positive cases, 506 cases (56.7%) were males and the remainder 387 (43.3%) were females. the difference in the distribution of positive and negative giemsa stain results in both gender was significant (p ≤ 0.05). table 2 also delineates the distribution of various parasite grading in both males and females in which the difference is significant (p ≤ 0.05). table 3 shows the comparison between both genders with regard to the parasite number/field in these grades (+2, +3, +4). these grades were taken into consideration because according to the instructions by ramírez et al12 which is used in this study, the mentioned grades harboring more than one parasite per field. table 3 manifests clearly by using student’s t-test that the means of the parasites number per field in above-mentioned grades varies significantly between males and females (p ≤ 0.05). table 4 demonstrates in detail the differences in the distribution of the number, site, type, and size of the lesions according to the gender of the patients. it is noted clearly from table 4 that the differences regarding the number, site were highly significant (p ≤ 0.01) while the differences regarding type and size of the lesion were significant (p ≤ 0.05). table 1. results of parasitological examination by giemsa stain result n % positive 893 70.6 negative 371 29.4 total 1264 100 table 2. distribution of results of parasitological examination (giemsa stain ) and grading according to the gender giemsa stain male female total n (%) n (%) n (%) positive 506 (56.7) 387 (43.3) 893 (100) negative 184 (49.6) 187 (50.4) 371 (100) total 690 (54.6) 574 (45.4) 1264 (100) chi-square value = 5.33 df = 1 significant (p ≤ 0.05) grading (positive cases only) +1 72 84 156 +2 137 92 229 +3 170 109 279 +4 127 102 229 total 506 387 893 chi-square value = 10.688 df = 3 significant (p ≤ 0.05) table 3. comparison between both genders regarding parasites number/ field in grades +2, +3, +4 grades male female mean ± sd mean ± sd +2 7.4 ± 1.1 6.84 ± 2.6 t = 2.241 df = 227 significant (p ≤ 0.05) +3 14.83 ± 4.16 13.34 ± 5.82 t = 2.49 df = 277 significant (p ≤ 0.05) +4 26.54 ± 5.8 24.92 ± 6.14 t = 2.047 df = 227 significant (p ≤ 0.05) 338 original gender differences in the severity and features zakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 336–342 fig. 1 shows the distribution of different lesions regarding site, size, number, gender. fig. 2 illustrates the amastigote stage obtained from cutaneous lesion stained by giemsa stain. discussion to our knowledge, this is the first study of such quality and aims on cutaneous leishmaniasis in erbil. the results which had been obtained from the present study suggest that gender may have a role in severity and pathogenesis of infection. it is clear from tables 2 and 3 that males had higher rates of infections and this was illustrated by the higher percent of male patients and the higher percent of males who yield a positive giemsa stain. in addition, significantly higher rate of parasite grading and parasites number/ field (parasite burden) in males were also noted. table 4 obviously clarified that differences in the distribution of number, site, type, and size of lesions were significant and highly significant among both gender. the present study delineate that giemsa stain has a detection rate of 70.6% in clinically diagnosed cases. this result is near to that obtained in which the rates were 73% and 69.5% respectively.2,13 the success of microscopic detection of amastigotes varies depending on the number of parasites present and duration of lesions. therefore, failure to observe amastigotes does not exclude a diagnosis of cl and such infection in endemic areas may be diagnosed on the basis of their clinical features as leishmaniasis.4,14,15 it had been claimed that amastigotes at certain time of the disease are impossible to be detected. the disappearance of such cells infected with the amastigote form in spite of the disease process is still continuous delineate that these phagocytes, giant cells, macrophages, and monocytes, at specific point of the disease process become resistant to be infected with the amastigotes.15,16 several studies have illustrated that immunological differences are noted between both sexes that may lead to increased parasitism in males.6,7 females typically have higher immune responses than males. this elevation of immunity among females is beneficial against infectious diseases, while from the other side, it may be injurious because of the increased susceptibility of females to develop autoimmune diseases.8,9 in pre-pubertal children, sex differences in response to leishmania infection were notified in which boys are more likely to develop visceral leishmaniasis than girls. during the critical period of sex differentiation, the extent to which sex steroids alter the development of the immune system and responses to infection prior to puberty and into adulthood should be considered.17,18 researches from various diverse endemic foci in the new and old worlds had concluded that regardless of cultural and occupational factors, men were noted to get cutaneous or visceral leishmaniasis more than women.17,18,19,20,21 mice infected by leishmania in experimental studies also reveal that males are more subjected to infection than females. in mice infected with leishmania major, disease evolution was found to be different in males and females according to the route of inoculation, i.e., the intradermal route was more severe in females and the intravenous route was more severe in males.22,23 other study, comparing pregnant or castrated mice to normal controls, demonstrated that susceptibility to l. major or l. mexicana strongly depended on hormone levels, which in turn regulated the expression of different cytokines.24 the relative resistance of female mice to l. mexicana infection compared to male mice was related to increased expression of gamma interferon (ifnγ). male mice castration reduces, whereas treatment of the females with testosterone increases, vulnerability to l. major.22 experimental infection of inbred age-matched male and female hamsters demonstrated that male animals were more susceptible to infection with leishmania (viannia) spp. than female animals. this difference was evident for strains of both table 4. distribution of number, site, type and size of lesion according to the gender of patients number of lesion male female total n (%) n (%) 1 313(50.2) 310(49.8) 623(100) 2 286(56.2) 223(43.8) 509(100) ≥3 91(68.9) 41(31.1) 132 (100) total 690 574 1264 chi-square value = 14.2 df = 2 highly significant (p ≤ 0.01) site of lesion limbs 366(59.3) 251(40.7) 617(100) face 219 (48.5) 233 (51.5) 452(100) abdomen & trunk 105 (53.8) 90 (46.2) 195(100) total 690 574 1264 chi-square value = 14.2 df = 2 highly significant (p ≤ 0.01) type of lesion chi-square value = 14.2 df = 2 highly significant (p ≤ 0.01) site of lesion limbs 366(59.3) 251(40.7) 617(100) face 219 (48.5) 233 (51.5) 452(100) abdomen & trunk 105 (53.8) 90 (46.2) 195(100) total 690 574 1264 wet 375 (57.6) 276(42.4) 651(100) dry 315 (51.4) 298 (48.6) 613 (100) total 690 574 1264 chi-square value = 4.82 df = 1 significant (p ≤ 0.05) diameter of lesion 0.5 × 1cm–1.5 × 2cm 171 (51.5) 161(48.5) 332 (100) 1.5 × 2cm–2.5 × 3cm 357(53) 317(47) 674(100 ) ≥2.5 × 3 cm 162(62.8) 96(37.2) 258 (100) total 690 574 1264 339 original gender differences in the severity and featureszakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 336–342 l. (v.) panamensis and l. (v.) guyanensis when either primary lesion size or severity or frequency of dissemination (cutaneous metastases) was assessed.25 in addition, the exogenous administration of the opposing sex hormone to male and female hamsters demonstrated that testosterone had a disease-promoting effect, possibly through a direct effect on the immune response or by blocking a protective effect of estrogen.26 strikingly, male hamsters had significantly more-severe disease than female animals when lesion size, lesion severity (degree of tissue necrosis), parasite burden in the draining lymph node, and rate of parasite dissemination were a b c d e f g h fig. 1 (a-h) distribution of different lesions regarding: site, size, number, gender, type of lesion. fig. 2 amastigote from cutaneous lesion, giemsa stain 100 x. 340 original gender differences in the severity and features zakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 336–342 evaluated.25 associated with the increased severity of disease in the male animals was a significantly greater intralesional production of il-4, il-10, and tgf-β cytokines known from other studies to exacerbate experimental leishmania sp.26,27 study by wilcoxen et al28 had demonstrated that there are gender-dependent differences in the secretion of il-10 and il-12 by antigen-presenting cells (apcs). apcs from male mice secreted il-10 but not il-12 during t-cell activation, and this pattern was reversed in apcs from female mice. males and females differ in their innate immune responses suggesting that some sex differences may be germline encoded. the number and activity of cells associated with innate immunity differ between the sexes. phagocytic cells, including macrophages and neutrophils, can kill parasites by generating reactive oxygen metabolites and nitric oxide, as well as by secreting enzymes.7 responses of the innate immune system play a crucial role in the initial recognition and response to parasites and may alter the expression of sex differences in parasite infection.6 pattern recognition receptors, such as toll-like receptors (tlr) are closely involved in arranging host–innate responses to infection and serve as a bridge between innate and acquired immunity. whether the sexes differ in the expression of tlr, mannose receptors, or scavenger receptors that bind to and mediate internalization of parasitic particles has not been reported, but could influence dimorphic responses to infection.17,18 among humans and lizards, the phagocytic activity of neutrophils and macrophages is higher in females than males.6 following parasitic or antigenic stimulation, the production and release of prostaglandin e2, thromboxane b2, and nitric oxide is reportedly higher in females than males.7 humoral immune responses (i.e., antibody production by b-cells) are typically greater in females than males.18 gonadal hormones exert specific effects on the male and female immunocompetence at both the cellular and the molecular level. estrogen receptors are expressed in most cells of the innate and adaptive immune system including t cells, b cells, neutrophils, macrophages, dendritic cells (dc), and natural killer (nk) cells.29 androgen receptors were identified in t and b lymphocytes. during pregnancy, activated lymphocytes also express progesterone receptors.7 estrogens affect innate immune cells. estrogens at levels of ovulatory phase or pregnancy suppress cytotoxicity of nk cells.29 notably, macrophages treated in vitro with estradiol showed decreased secretion of the proinflammatory cytokines interleukin (il)-1b, il-6, and tumor necrosis factor (tnf)-a, whereas long-term in vivo administration led to increased secretion of il-1b, il-6, and il-12p40 after toll-like receptor (tlr) 4 activation and eventually an enhanced activation status.30,31,32 estrogen receptor signaling also regulates lineage development of dcs. high estradiol levels promote the development of conventional il-12-producing dcs and the expansion of ifn--producing killer dcs. in addition, production of il6, il-8, and chemokine (c-c motif ) ligand 2 (ccl2) by immature dcs is increased. both estrogens and androgens reduce the numbers of immature t lymphocytes enhancing thymic involution during puberty and pregnancy.28,29 adaptive immunity in men is distinct from women as androgens accelerate the growth and expansion of th1 responses and trigger cd8+ t cells, while estrogens encourage th2 responses and animate antibody production.8 in comparison of women at reproductive age to age-matched men, the cd4+/ cd8+ ratio is significantly increased.7 in parallel to the low estrogen levels are the increased manifestation of the transcription factor t-bet (t-box expressed in t cells), which eventually switch the balance toward th1 immunity and ifn- expression.33 high estrogen levels constrain irf1 (interferon regulatory factor 1) supporting th2 immunity and il-4 expression.34 sex steroid hormones also modified b-cell expansion and function. estradiol reduces apoptosis of immature b cells and consequently increases the appearance of autoreactive b cells from central and peripheral checkpoints.8 however, estradiol also increases somatic hypermutation and classswitch recombination leading to high-affinity ig producing cells.34 these effects might contribute to an improved humoral response in women and explain the increased susceptibility to autoimmune diseases. in contrast to estrogens, progesterone suppresses somatic hypermutation and class-switch recombination.33 estrogens also exhibit indirect effects on the immune system by modulating the levels of growth hormone, prolactin, or thymosin.8 thus, the general paradigm on sex steroid hormones influencing the immune system stipulates that estrogens have immune-enhancing effects. in contrast, progesterone and androgens such as testosterone exert mainly immunosuppressive properties.17,18,25 the x chromosome expresses several genes implicated in immunological processes, such as toll-like receptors, multiple cytokine receptors, genes involved in t-cell and b-cell activity, and transcriptional and translational regulatory factors, while in turn the y chromosome encodes for a number of inflammatory pathway genes, which are exclusively expressed in men.37 most alleles on one x chromosome are randomly silenced during x chromosome inactivation already during embryogenesis.29 polymorphism of x-linked genes and cellular mosaicism for x-linked parental alleles may offer additional advantages to women during host responses, in particular by providing a more adaptive and balanced cellular machinery during innate immune responses.37,38 this sexual dimorphism commences already during intrauterine development, for example, a male fetus experiencing a chronic inflammatory environment primarily being induced by the maternal immune system in the male placenta via decidual sites yet also likely due to a higher gestational infection rate of male placenta.38 later in life very probably due to socioeconomic behavior, such as higher pathogen exposure during agricultural or occupational activities, men are more susceptible to many infections caused by viruses, bacteria, parasites, and fungi. they are significantly more predisposed especially to environmental and vector-borne diseases such as leptospirosis (3.5to 4-fold increased incidence), schistosomiasis (1.5-fold), brucellosis, or rabies.6,7,8 the present study concluded that there is a gender differences regarding the severity and features of lesions among patients infected with cutaneous leishmaniasis. 341 original gender differences in the severity and featureszakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 336–342 limitations & recommendations the constraints of this study were: 1. it is important to highlight that cl has very different clinical manifestations depending on the condition of the host’s immunity and the species of parasite. proper identification of the species is achieved by pcr which was not applied in this study because such a technique is time consuming and not usually available. 2. cytokines play a vital role in the host immune response to infection by initiating the healing process and/or accelerating the progression of the disease in cutaneous leishmaniasis (cl). determination of the cytokine expression pattern of ifn-γ, il-4, il-11 and il-12p40 in cl patients was not done. 3. proper studies on the effects of environmental, occupation, life style factors on the severity of infection with cl are mandatory. acknowledgement the authors would like to thank health directorate of erbil, education directorate of erbil and the members staff of health center of makhmur and kalack districts for their assistant during the study period. a special thanks are directed to the heads and staffs of rizgary hospital, hawler teaching center for skin diseases, shadi health center, hawler institute of health prevention for their great help and encouragement. conflict of interest the authors acknowledge no conflict of interest in this study. references 1. akcali c, culha g, inaloz h, savas n, onlen y, savas l. cutaneous leishmaniasis in hatay. j turk acad dermatol 2007;1(1):1–5. 2. alsamarai am, alobaidi hs. cutaneous leishmaniasis in iraq. j infect dev countr 2009;3(02):123–9. 3. gürel ms, yesilova y, ölgen mk, özbel y. cutaneous leishmaniasis in turkey. türk parazitol derg 2012;36(2):121. 4. toz so, nasereddin a, ozbel y, ertabaklar h, culha g, sevil n, ziya alkan m, jaffe cl. leishmaniasis in turkey: molecular characterization of leishmania from human and canine clinical samples. trop med int health 2009;14(11):1401–6. 5. zeyrek fy, korkmaz m, özbel y. serodiagnosis of anthroponotic cutaneous leishmaniasis (acl) caused by leishmania tropica in sanliurfa province, turkey, where acl is highly endemic. clin vacc immunol 2007;14(11): 1409–15. 6. moxley g, posthuma d, carlson p, estrada e, han j, benson ll, neale mc. sexual dimorphism in innate immunity. arthr rheumat 2002;46(1):250–8. 7. roberts cw, walker w, alexander j. sex-associated hormones and immunity to protozoan parasites. clin microbiol rev 2001;14(3):476–88. 8. bouman a, heineman mj, faas mm. sex hormones and the immune response in humans. hum reprod update 2005;11(4):411–23. 9. restif o, amos w. the evolution of sex-specific immune defences. proc r soc b biol sci 2010;277(1691):2247–55. 10. ismael ak, ngah i. rural population density effect on socio-economic characteristics: a review. j social sci 2011;7(4):655. 11. ul bari a, ber rahman s. correlation of clinical, histopathological, and microbiological findings in 60 cases of cutaneous leishmaniasis. ind j dermatol venereol leprol 2006;72(1):28. 12. ramírez jr, agudelo s, muskus c, alzate jf, berberich c, barker d, velez id. diagnosis of cutaneous leishmaniasis in colombia: the sampling site within lesions influences the sensitivity of parasitologic diagnosis. j clin microbiol 2000;38(10):3768–73. 13. abdulah sa, abul-hab j, el-deen ld. clinico-epidemiological study of cutaneous leishmaniasis in a sample of iraqi armed forces. iraqi j commun med. 2006;19(2):98–103. 14. amro a, gashout a, al-dwibe h, alam mz, annajar b, hamarsheh o, shubar h, schönian g. first molecular epidemiological study of cutaneous leishmaniasis in libya. plos negl trop dis 2012;6(6):e1700. 15. bensoussan e, nasereddin a, jonas f, schnur lf, jaffe cl. comparison of pcr assays for diagnosis of cutaneous leishmaniasis. j clin microbiol 2006;44(4):1435–9. 16. daboul mw. toward an approach for cutaneous leishmania treatment. our dermatol online 2013;4(1):46. 17. klein sl. hormonal and immunological mechanisms mediating sex differences in parasite infection. paras immunol 2004;26(6–7):247–64. 18. bernin h, lotter h. sex bias in the outcome of human tropical infectious diseases: influence of steroid hormones. j infect dis 2014;209(suppl_3):s107–13. 19. weigle ka, santrich c, martinez f, valderrama l, saravia ng. epidemiology of cutaneous leishmaniasis in colombia: environmental and behavioral risk factors for infection, clinical manifestations, and pathogenicity. j infect dis 1993;168(3):709–14. 20. ahmadi na, modiri m, mamdohi s. first survey of cutaneous leishmaniasis in borujerd county, western islamic republic of iran. east mediter health j 2013;19(10):847–53 . 21. aara n, khandelwal k, bumb ra, mehta rd, ghiya bc, jakhar r, dodd c, salotra p, satoskar ar. clinco-epidemiologic study of cutaneous leishmaniasis in bikaner, rajasthan, india. am j trop med hygiene 2013;89(1):111–115. 22. alexander j. sex differences and cross-immunity in dba/2 mice infected with l. mexicana and l. major. parasitology 1988;96(2):297–302. 23. satoskar, a., h. h. al-quassi, and j. alexander. sex-determined resistance against leishmania mexicana is associated with the preferential induction of a th1-like response and ifn-gamma production by female but not male dba/2 mice. immunol cell biol 1998;76:159–166. 24. krishnan l, guilbert lj, wegmann tg, belosevic m, mosmann tr. t helper 1 response against leishmania major in pregnant c57bl/6 mice increases implantation failure and fetal resorptions. correlation with increased ifn-gamma and tnf and reduced il-10 production by placental cells. j immunol 1996;156(2):653–62. 25. travi bl, osorio y, melby pc, chandrasekar b, arteaga l, saravia ng. gender is a major determinant of the clinical evolution and immune response in hamsters infected with leishmania spp. infect immun 2002;70:2288–96. 26. martinez je, travi bl, valencia az, saravia ng. metastatic capability of leishmania (viannia) panamensis and leishmania (viannia) guyanensis in golden hamsters. j parasitol 1991;1:762–8. 27. martínez je, valderrama l, gama v, leiby da, saravia ng. clonal diversity in the expression and stability of the metastatic capability of leishmania guyanensis in the golden hamster. j parasitol 2000;86(4):792–800. 28. wilcoxen sc, kirkman e, dowdell kc, stohlman sa. gender-dependent il-12 secretion by apc is regulated by il-10. j immunol 2000;164(12):6237–43. 29. fish en. the x-files in immunity: sex-based differences predispose immune responses. nat rev immunol 2008;8(9):737. 30. hao s, zhao j, zhou j, zhao s, hu y, hou y. modulation of 17β-estradiol on the number and cytotoxicity of nk cells in vivo related to mcm and activating receptors. int immunopharmacol 2007;7(13):1765–75. 31. kramer pr, kramer sf, guan g. 17β‐estradiol regulates cytokine release through modulation of cd16 expression in monocytes and monocyte‐ derived macrophages. arthr rheumat off j am coll rheumatol 2004;50(6):1967–75. 342 original gender differences in the severity and features zakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 336–342 32. calippe b, douin-echinard v, laffargue m, laurell h, rana-poussine v, pipy b, guéry jc, bayard f, arnal jf, gourdy p. chronic estradiol administration in vivo promotes the proinflammatory response of macrophages to tlr4 activation: involvement of the phosphatidylinositol 3-kinase pathway. j immunol 2008;180(12):7980–8. 33. pernis ab. estrogen and cd4+ t cells. curr opin rheumatol 2007;19(5): 414–20. 34. pennell lm, galligan cl, fish en. sex affects immunity. j autoimmun 2012;38(2-3):j282-91. 35. gonzález da, díaz bb, pérez md, hernández ag, chico bn, de león ac. sex hormones and autoimmunity. immunol lett 2010;133(1):6–13. 36. klein sl. immune cells have sex and so should journal articles. endocrinology 2012;153(6):2544–50. 37. sakiani s, olsen nj, kovacs wj. gonadal steroids and humoral immunity. nat rev endocrinol 2013;9(1):56. 38. libert c, dejager l, pinheiro i. the x chromosome in immune functions: when a chromosome makes the difference. nat rev immunol 2010;10(8):594. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201908 26 j contemp med sci | vol. 4, no. 1, winter 2018: 26–29 research anthropometric study of nasal index in hausa ethnic population of northwestern nigeria ibrahim mohammed,a,b tahmineh mokhtari,c,d sahar ijaz,a,b akanji omotosho d,a,b abdullahi abubakar ngaski,e maryam milanifard,a and gholamreza hassanzadeha adepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. bdepartment of anatomy, school of medicine, international campus, tehran university of medical sciences, tehran, iran. cresearch center of nervous system stem cells, department of anatomy, school of medicine, semnan university of medical sciences, semnan, iran. ddepartment of anatomy, school of medicine, semnan university of medical sciences, semnan, iran. edepartment of chemical pathology, faculty of medical laboratory sciences, usmanu danfodiyo university, sokoto, nigeria. correspondence to gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir). (submitted: 14 september 2017 – revised version received: 27 october 2017 – accepted: 02 november 2017 – published online: 26 march 2018) objective anthropometric study is very important in differentiating a true race from the local mingling of races. the nasal index has a great value in anthropological studies, because it is one of the anthropometric indices acknowledged in nasal surgery as well as management. anthropometric studies are very important area for craniofacial surgery and syndromology. this study aimed to provide baseline data of nasal index of northern nigerian people and to classify their nose type and the comparison of the data with other studies. methods the nasal width and height were measured from 200 participants of hausa ethnic group of northwestern nigeria, nasal index (ni) was calculated and analyzed statistically. results there was a significant difference in the nasal width (p = 0.0001), height (p = 0.0001) and ni (p = 0.0001) of sex groups, with nasal shapes: 120 mesorrhine (60%), 75 leptorrhine (37.5%) and 5 platyrrhine (2.55%). it shows hausa people have a mesorrhine nose. conclusion the nasal index of males is higher than females; this study presents the data for hausa people in this area, which will be of clinical, surgical interest in rhinology. we recommend a further study to compare the nasal index of hausa and other ethnic group living in northwestern nigeria. keywords anthropometry, mesorrhine, nasal index, rhinoplastic introduction anthropometric studies are scientific methods and techniques for displaying different measurement and observation on the human being as well as skeleton. anthropometric studies are very important area for craniofacial surgery and syndromology.1 variation is the great phenomena existing among human being, and it is related to different factors such as mutation and natural selection. different researches show the advantage of anthropometric measurement as a tool for studying the variation in human population and very important in forensic science for crime investigation.2 in the 20th century, the relevance of anthropometry to the study of different races was substituted by different sophisticated methods for determining racial variations. nowadays, anthropometry has got recognition in medical sciences particularly forensic medicine.2 nasal anthropometry is a study that deals with the measurements of the proportion, size as well as shape of nose of the human being.3 the study of nasal anthropology is very important in forensics as well as physical anthropology as one of the techniques used in the determination of different races, ethnicity as well as gender of an individual.4 some studies documented that bioenvironmental, geographical as well as biological factors, ethnicity, sex and age have influence in body dimensions especially in the head and neck region.5–7 in anthropology and forensic medicine, the knowledge of nasal index (ni) is relevant in distinguishing the race, ethnicity and sex of individuals whose identity is not known.2,8 ni is also useful in the analysis and classification of fossil remains as well as the study of living population.9 in clinical practice, ni is useful in rhinoplastic surgery (plastic surgery of the nose) as nasal analysis is the first step. a rhinoplastic surgeon takes to change the size or shape of the nose for a desired aesthetic effect. also, nasal analysis of a particular ethnic group can help the rhinoplastic surgeon change the shape of nose of a patient without compromising the patient’s desire to maintain his cosmetic status.10 ni measurement in healthy individuals is also useful for dysmorphologists in the early diagnosis of some dysmorphic syndromes like cleft lip and palate which are associated with nose disorders.4 the ni is determined as the percentage of the width in relation to the height of the nose. while on skeleton, the height is measured from the nasion (where the internasal suture touches the frontal bone) to a level just at the bottom of the nasal spine. the width on the skull is the maximum distance on the nasal sinuses. on living human, the height is from nasion to the subnasal (where the nasal septum touches the upper lip). the nasal width on living human is the highest distance between the two nasal wings or two alae in anatomical position. usually, the ni on living human and the ni on skeleton never correspond to each other.11 a nose is referred to leptorrhine if the ni is 69.9% or less (long and narrow nose), mesorrhine nose if the ni is between 70% and 84.9% (medium) and platyrrhine nose if the ni is above 85% (broad nose).8,12 the platyrrhine nose generally has a very prominent ala lobule with a complete and well-rounded nasal tip. the mesorrhine nose has a small prominent lobule with more clear nasal tip and the leptorrhine nose has very small prominent ala lobule with well-defined nasal tip.13 issn 2413-0516 ibrahim mohammed et al. 27j contemp med sci | vol. 4, no. 1, winter 2018: 26–29 research nasal index of hausa ethnic population of nigeria majority of caucasians have the leptorrhine type of nose characterized as long and narrow. the indo-aryan is like european people with fine nose.14 jingpo people of china have mesorrhine nose.15 indo-african14 as well as afro-american16 possessed platyrrhine nose. risley described that the ni of african descent has platyrrhine nose. sarka11 also revealed that the nose of australian aborigines varies from that of negro by more intensely depressed root. facial anthropometry is one of the most important aspects used in reconstructive surgery, forensic investigation as well as genetic counseling.10,17,18 the external nose is a part of the human nose that protrudes anteriorly from the face.19 the shape of the external nose is inconsistent,20 and is related to ethmoid bone as well as septum, which is made up of cartilage and separate nostrils apart. the aim of the study was to provide baseline data of certain nasal anthropometric measurements for male and female of northern nigerian people and to classify their nose type and the comparison of the data with other studies, so that it would be further useful as an essential tool to the researchers, clinicians, rhinoplastic and facial reconstructive surgeons and forensic experts related to this field. the results from this study will also add to the pool of the anthropological data that may be used as a reference by other health practitioners’ especially in nigeria and worldwide. materials and methods on the basis of nasal height and breadth index, martin and sallar21 categorized noses (table 1). this study is a cross-sectional study, conducted in northwestern part of nigeria on 200 volunteered participants (100 males, 100 females) adult individuals. the age range of the participant was 16–60 years. the volunteered participants of this study had no history of trauma, no physical deformities, no surgery of face or nose, and no history of cleft lip or palate. nasal width and nasal height were measured using spreading venire caliper (manual) following standard method described by martin and saller.21 nasal width was measured as a straight distance from right ala to left ala. nasal height was measured as the distance from the nasion to the subnasale. the ni was calculated as follows: ni = nasal width/nasal height × 100.22 statistical analysis the data obtained were analyzed statistically. basic descriptive statistics and independent sample t-test were carried out by computerized statistical analysis software—statistical package for social sciences (spss-22) and microsoft excel windows 2007. the p-value of less than 0.05 was considered statistically significant. results the results consisting of the statistical analysis with respect to the measurement of nasal variables such as nasal width, nasal heights as well as ni of males and females are shown in tables 2–4. in this study, 100 males (50%) and 100 females (50%) were evaluated for the ni. the descriptive analysis of data was shown in table 1. there was a significant difference in the nasal width (p = 0.0001), height (p = 0.0001) and ni (p = 0.0001) of sex groups as shown in table 3. the nasal shapes were described according to the ni and its distribution in this study was as follows: 120 mesorrhine (60%), 75 leptorrhine (37.5%) and 5 platyrrhine (2.55%) types. the distribution of nasal shapes in the sex groups was demonstrated in table 4. the most nasal shape frequency was related to mesorrhine type in male group and equally leptorrhine and mesorrhine in female group. discussion nose is very important in racial origin.23 the ni plays a vital role in anthropology and is one among the clinical anthropometric table 2. statistical analysis of nasal parameters mean sd* minimum maximum nasal width (cm) 3.91 0.42 3.01 5.37 nasal height (cm) 5.45 0.47 4.2 6.65 nasal index 71.99 7.17 54 91 table 3. descriptive statistical analysis of nasal index of males and females mean sd* minimum maximum p-value nasal width (cm) in male 4.13 0.38 3.15 5.37 0.0001 nasal width (cm) in female 3.69 0.34 3.01 4.91 nasal height (cm) in male 5.59 0.38 4.61 6.55 0.0001 nasal height (cm) in female 5.31 0.51 4.2 6.65 nasal index in male 74.08 6.92 56 89 0.0001 nasal index in female 69.9 6.83 54 91 *standard deviation. table 4. frequency (percentage) of nose shapes of males and females male frequency (%) female frequency (%) total frequency (%) leptorrhine 25 (25) 50 (50) 75 (37.5) mesorrhine 71 (71) 49 (49) 120 (60) platyrrhine 4 (4) 1 (1) 5 (2.55) table 1. nasal categories based on martin and sallar(21) method categories size of nose nasal index on living head on skull hyperleptorrhine long narrow nose 40–54.9 — leptorrhine moderately narrow nose less than 70 less than 47 mesorrhine moderate or medium size 70–84.9 47–50.9 platyrrhine moderately wide nose 85–99.9 51–57.9 hyperplatyrrhine very wide nose 100 or more 58 or more 28 j contemp med sci | vol. 4, no. 1, winter 2018: 26–29 nasal index of hausa ethnic population of nigeria research ibrahim mohammed et al. parameters to be considered in nasal surgical and medical management.24,25 ni is associated to regional and climatic variations.26 many studies have shown the racial and ethnic differences in ni between different populations.27 majority of caucasians are leptorrhine with long and narrow nose and ni of 69.9 or less. the indo-aryan is as well similar to the european with fine nose.14 the jingpo people of china are mesorrhine.15 indo-african14 as well as afro-american people have platyrrhine nose type.16 from the results of this study, the statistical analysis of nasal parameters shown in table 1, shows the mean, standard deviation, minimum and maximum range of nasal parameters, nasal width, nasal height and ni. comparing the current study with the study conducted by jovanovic on the nasal parameters of serbian population (caucasians) with mean ni of 66.78,28 it can be concluded that the hausa people living in northwestern part of nigeria had a higher mean (ni = 71.99). this shows that there are differences in the nasal parameters among different ethnic groups and races. there were differences between the nasal parameters of both men and women. this result did not corroborate with a study carried out by eliakim-ikechukwu on nasal indices and bi-alar angle between two ethnic groups igbo and yoruba in nigeria, in which he found out that no significant difference in the nasal parameters between the two ethnic groups.3 however, this present study is in conformity with the study carried out by eliakim-ikechukwu on nasal parameters of ibibio and yakurr ethnic groups in south-southern part of nigeria as there was significant difference in the nasal parameters of the two ethnic groups.29 also, a study conducted by anas and saleh revealed that anthropometric comparison of nasal indices among hausa and yoruba ethnic groups living in kano state, northern part of nigeria there was significant difference in nasal indices among the two ethnic groups.30 this result corroborates with the present study. some previous studies revealed that the mean, median, minimal and maximal width, height and the ni in males are higher than the females and there was significant difference.31,32 these are in line with the current study. ni of the hausa ethnic group living in northwestern nigeria, males were identified with mesorrhine type of nose, while females with leptorrhine and mesorrhine type of nose, this is not in conformity with the previous studies carried out by risley33 in which he revealed that african people has a platyrrhine type of nose. another study conducted by oladipo reported platyrrhine nose type in igbo, yoruba and ijaw ethnic groups of southern part of nigeria.7 esomonu4 reported a platyrrhine type of nose among bekwaras ethnic group in cross river state nigeria. nevertheless, a few studies have revealed mesorrhine type of nose in some ethnic groups in nigeria. research conducted by oladipo identified a mesorrhine nose type for andoni ethnic group of rivers state in nigeria,34 mesorrhine type in ikwerre males,35 ibibio females and yakurr males of south-southern part of nigeria,29 it was also reported that mesorrhine type in hausa people living in northern part of nigeria.30 variations in ni occur among different ethnic groups; variation can be as a result of warmer climate with higher temperature in northern part of nigeria36 compared to southern part of nigeria.37 the classification of nose into different categories is a function of ni, in anthropology as it is applied in differentiating racial and ethnic variations.8,38 the ni as well shows sexual dimorphism39 that is why it is very important in forensic science particularly in gender differentiation.15 the shape of nose of the african people, when compared to other races counterparts, are said to be affected by the climatic and environmental conditions.40 the broader nose of the african populations might be due to natural selection that affects their warm moist environmental condition.41 the current study reported the ni of hausa ethnic group living in northwestern nigeria males 71% and females 50%. the male ni was higher than the female ni. this is in conformity with research conducted by oladipo et al.,17 who reported the males from igbo, ijaw as well as yoruba ethnic groups in southern part of nigeria have higher ni than their female counterpart. also, another report by oladipo et al.,34 revealed that there is a sexual dimorphism in the ni of itsekiris and urhobos of nigeria, in which males reported to have higher ni compared to females.34 results from the present study reported that hausa ethnic group was found to be mesorrhine similar to oriental nose.42,43 moreover, negros have been reported as platyrrhine, which is not in conformity with the result obtained from this study and some previous studies amongst negros.42 this shows that not all negros are platyrrhine it might be due to interethnic genetic differences have more effect than climatic differences. conclusion this present study determined the nasal index of males and females of hausa ethnic group of northwestern nigeria. the result from this study shows hausa people have a mesorrhine type of nose. the nasal index of males is significantly higher than females, which confirmed the existence of sexual difference in nasal parameters possibly due to genetic, hormonal, nutrition and other related factors. there is no specific data for the nasal index of hausa people in northwestern nigeria; this study presents for the first time the nasal parameters for hausa people in this area, thus providing a useful baseline and an anthropometric data that will be of clinical and surgical interest in rhinology in this part of the world. we recommend a further study to compare the nasal index of hausa and other ethnic group living in northwestern nigeria. acknowledgments the authors would like to thank staffs and students of faculty of medical laboratory sciences, usmanu danfodiyo university sokoto, nigeria. we would also like to thank the anatomy department of school of medicine, tehran university of medical sciences, iran, for the success of this research. ibrahim mohammed et al. 29j contemp med sci | vol. 4, no. 1, winter 2018: 26–29 research nasal index of hausa ethnic population of nigeria references 1. choudhary a, chowdhary ds. comparative anthropometric study of nasal parameters between two ethnic groups of rajasthan state. int j med public health. 2012;2:46. 2. oladipo g, udoaka a, afolabi e, bob-manuel i. nasal parameters of itsekiris and urhobos of nigeria. int j biol anthropol. 2008;3:1–5. 3. eliakim-ikechukwu c, bassey t, ihentuge c. study of the nasal indices and bialar angle of the ibo and yoruba ethnic groups of nigeria. j biol agric healthcare. 2012;2:149–152. 4. esomonu ug, ude ra, lukpata pu, nandi em. anthropometric study of the nasal index of bekwara ethnic group of cross river state, nigeria. int res j appl basic sci. 2013;5:1262–1265. 5. satija a, kaushal s, gopichand pv, chhabra u. study of relationship between facial index and gestational age in normal newborns. nepal med coll j. 2010;12:133–136. 6. bayat pd, ghanbari a. the evaluation of craniofacial dimensions in female arak newborns (central iran) in comparison with other iranian racial subgroups. eur j anat. 2013;13:77–82. 7. hassanzadeh g, sadr m, alaghbandha n, dehbashipour a, abbas ma, heydar zeidi o. anthropometric characteristics of craniums in residents of qazvin, iran and dera ghazi khan, pakistan: a comparative study. anat sci j. 2013;10:43–49. 8. porter jp, olson kl. analysis of the african american female nose. plast reconstr surg. 2003;111:627–628. 9. alex fr, steven b, timothy gl. human body composition, 4th ed.; human kinetics publishers, 1996; pp. 167–172. 10. olotu je, eroje a, oladipo gs, ezon-ebidor e. anthropometric study of the facial and nasal length of adult igbo ethnic group in nigeria. int j biol anthropol. 2009;2:10–15. 11. sarka j. important criteria for racial identification. biology. the next generation library 2014. 12. willams p, dyson m, dussak, je, bannister lh, berry mm, collins p, et al. skeletal system. gray’s anatomy, 3rd ed.; churchill livingstone: edinburgh, 1995; pp. 609–612. 13. jimoh ro, alabi sb, kayode as, salihu am, ogidi od. rhinometry: spectrum of nasal profile among nigerian africans. braz j otorhinolaryngol. 2011;77:589–593. 14. sparks cz, jantz rl. a reassessment of human cranial plasticity: boas revisited. proc natl acad sci. 2002;99:14636–14639. 15. xu b, wang y, ma j, li m, xu l. a computer-aid study on the craniofacial features of archang race in yunnan province of china. west china journal of stomatology. 2011;19:394–396. 16. ofodile fa, bokhari f. the african american nose: part ii. ann plast surg. 1995;34:123–129. 17. oladipo gs, olabiyi ao, oremosu aa, noronha cc. nasal indices among major ethnic groups in southern nigeria. sci res essay. 2007;2:20–22. 18. krishan k. estimation of stature from cephalo-facial anthropometry in north indian population. forensic sci int. 2008;181:52e1–52e6. 19. sinnatamby cs. last’s anatomy: regional and applied, 11th ed.; churchill livingstone: edinburgh, 2006; p. 385. 20. standring s. gray’s anatomy: the anatomical basic of clinical practice, 40th ed.; elsevier: london, 2008; p. 547. 21. martin r, saller k. lehrbuch der anthropologie, 3rd ed.; gustav fischer verlag: stuttgart, 1957; p. 11. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 22. romo t, abraham mt. the ethnic nose. facial plast surg. 2003;19: 296–278. 23. madison g. the passing of the great race. part 1. language and nationality, chapter 2–4, 2004; p. 1–6. 24. hansen b, mygind n. how often do normal persons sneeze and blow the nose? rhinology. 2002;40:407–426. 25. zankl a, eberie l, schinzel a. growth chart for nose length, nasal protrusion and philtrum length from birth to 97 years. am j med genet. 2002;111: 388–391. 26. farkas lg, kolar ir, munro ir. abstract on the geography of the nose, a morfometric study. aesthet plast surg. 1986;10:191–123. 27. oladipio gs, gwunireama iu, asawa od. anthropometric comparison of nasal indices between the igbos and yorubas in nigeria. global j med res. 2006;5:37–40. 28. jovanović j, jeremić d, jovanović b, maja v, sazdanović p, maja s, et al. nasal morphological characteristics of the serbian population. arch biol sci belgrade. 2014;66(1):227–234. 29. eliakim-ikechukwu c, iro cm, ihentuge cj, bassey te. nasal parameters of ibibio and yakurr ethnic groups of south-south nigeria. j pharm biol sci. 2013;5:23–26. 30. anas iy, saleh ms. anthropometric comparison of nasal indices between hausa and yoruba ethnic groups in nigeria. j sci res rep. 2014;3: 437–444. 31. zolbin mm, hassanzadeh g, mokhtari t, arabkheradmand a, hassanzadeh s. anthropometric studies of nasal parameters of qazvin residents, iran. moj anat physiol. 2015;1:1–5. 32. tahmasebi f, khanehzad m, madadi s, hassanzadeh g. anthropometric study of nasal parameters in iranian university students. anat sci. 2015;4:167–170. 33. risley hh. the people of india, 2nd ed.; crooke w., ed., 1915; p. 20. 34. oladipo gs, eroje ma, fawehinmi hb. anthropometric comparison of the nasal indices between the adoni and okrika ethnic groups of rivers state, nigeria. int j med sci. 2009;1:135–137. 35. oladipo g s, oyakhire m o, ugboma haa. anthropometric studies of nasal indices of the ekpeye and ikwerre ethnic groups in nigeria. asian j med sci. 2010;2:167–169. 36. audu eb. an analytical view of temperature in lokoja, kogi state, nigeria. int j sci technol. 2012;2:856–859. 37. sanusi yk, abisoye sg, faluyi oo. estimation of temperature–humidity index in some selected locations in nigeria. res j eng app sci. 2013;2:70–73. 38. franciscus rg, long jc. variation in human nasal height. am j phys anthropol. 1991;85:419–427. 39. zhang xt, wang sk, zhang w. measurement and study of the nose and face and their correlations in the young adult of han nationality. plast reconstr surg. 1990;85:532–536. 40. last rj. anatomy applied and regional, 6th ed.; churchill livingstone, 1981; pp. 398–403. 41. hall rl, hall da. geographic variation of native people along the pacific coast. human biol. 1981;67:407–426. 42. farkas lg, hreczko ta, deutsch ck. objective assessment of standard nostril types—a morphometric study. ann plast surg. 1983;11:381–389. 43. aung sc, foo cl, lee st. three dimensional laser scan assessment of the oriental nose with a new classification of oriental nasal types. br j plast surg. 2000;53:109–116. dx.doi.org/10.22317/jcms.03201806 24 j contemp med sci | vol. 5, no. 1, january–february 2019: 24–27 original article evaluation of nigella sativa and honey combination for treatment of kidney stone: a randomized, placebo controlled clinical trial laleh zaheri abdehvand,a eskandar moghimipour,b ali ghorbani,c alireza malayeri,d amir siahpoosh,e mahmood khodadoost,f mehdi rajaeipour,g* and kambiz ahmadi angalih adepartment of persian pharmacy, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran. bdepartment of pharmaceutics, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran cdepartment of nephrology, faculty of medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran ddepartment of pharmacology, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran. edepartment of pharmacognosy, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran. fschool of traditional medicine, shahid beheshti university of medical sciences, tehran, iran. gdepartment of urrology, school of medicine, dezful university of medical sciences, dezful, iran. hdepartment of biostatistics, school of health, ahvaz jundishapur university of medical sciences, ahvaz, iran. *correspondence to mehdi rajaeipour (email: mehdirajaeipour@yahoo.com). (submitted: 09 november 2018 – revised version received: 25 november 2018 – accepted: 03 january 2019 – published online: 26 february 2019) objectives the aim of this study was to evaluate the efficacy of nigella sativa (ns) with honey on treatment of kidney stone. methods a clinical trial was conducted on 42 patients who were referred to the urology clinic of ganjavian hospital, dezful, iran and were identified by a sonographist that they had kidney stones smaller than 6 mm, they were without hydronephrosis, and did not require medication. they were randomly assigned to control (n = 20) and treatment (n = 22) groups. about 8 g of ns and honey combination with one glass of warm water were given daily to the treatment group for 1 month. the control group did not receive medication. both groups drank 6–8 glasses of water daily. results the rate of stone expulsion in the treatment group was significantly higher than the control group (p = 0.0001). conclusion according to our results, it seems that ns with honey may have some beneficial effects in the treatment of kidney stone. keywords nigella sativa, ranunculaceae, honey, kidney stones introduction kidney stone is one of the most common diseases of the urinary tract, with an increasing incidence (6% in white women and 12% in white men). in recent years, the occurrence of urinary stones has increased, while the age of the onset of the disease has decreased. the incidence of spontaneous recurrence after 5 years is reported between 35 and 50%. in 2005, its occurrence in iran was 147.2 per 100,000 men and 129.6 per 100,000 women and a mean recurrence rate of 16% after 1 year, 32% after 5 years, and 53% after 10 years. in america, the cost per year for kidney stones is about $ 4.5 billion.1–5 the kidney stone may cause obstruction, hydronephrosis, infection, and hemorrhage in the urinary tract. esw, pnl, and tul, and laparoscopy are widely used to remove stones; but the use of these invasive methods is not cost effective and causes severe complications, so replacing traditional therapies with medicinal plants or phytotherapy will be valued.4 in the traditional view, kidney stone is due to thick and sticky khilt (humor) which has lost its moisture through excessive heat over the time.6 in traditional, medicine kidney stone is treated in various ways, including body cleansing from akhlat (plural of khilt), diet, regulation of activity, sleep and wakefulness, and the use of different herbal, animal or mineral drugs, including nigella sativa (ns) and honey. shoniz is used in traditional medicine as a diuretic, also with warm water and honey as mofattit (lithotriptic) of stones.7–9 in traditional medicine shoniz due to be hirrif (pungent) is used as majun (mixture) to destroy rih (wind) and heat body rapidly.10,11 honey is also introduced as a mofattit, diuretic and anti osr ol-bawl (difficulty in urinating).7 it is also a basis for many traditional products. it is used as a thickener, flavoring agent and vehicle in recent years. it has an antimicrobial effect due to its high viscosity too. several studies have been carried out on herbal products that affect urinary stones, including shoniz. in 2007, a study evaluated effect of ns extract on prevention and treatment of calcium oxalate produced by ethylene glycol in 32 wistar rats. results indicated a reduced number of calcium oxalate deposits in treatment group.12 in 2008, another study examined the effect of intraperitoneal thimoquinone, the main components of ns, on 60 male wistar rats (except for the negative control group). the stones were produced by ethylene glycol. the results showed that 5 mg/kg of thymoquinone significantly reduced the number and size of calcium oxalate deposits in the kidney tubules.13 in 1997, administration of 1 g of ns (as capsules), two times per day, made significant changes in blood creatinine level in healthy individual in saudi arabia.14 in 2016, a study on ns and its main component, thymoquinone showed positive effects in prevention or curing kidney stones and renal failure through various mechanism such as antioxidative, anti-inflammatory, anti-eicosanoid and immunomodulatory effects.15 in a study, in 2016, paracetamol administration significantly increased serum creatinine (0.80 u/l) when compared with the sham group (0.31 u/l). however, serum creatinine level was reduced to 0.64, 0.57, and 0.52 u/l with 250, 500, and 1000 mg/kg doses of the extract, respectively. kidney histopathological examinations showed that ns antagonized paracetamol-induced kidney pathological damage.16 issn 2413-0516 e. moghimipour et al. 25j contemp med sci | vol. 5, no. 1, january–february 2019: 24–27 original article evaluation of nigella sativa and honey combination for treatment of kidney stone fig. 1 nigella sativa seeds and flower https://sg.carousell.com in 2016, a research showed that serum creatinine and blood urea nitrogen (bun) in ptu group was higher than the control group. a total of 400 mg/kg of ns decreased creatinine, but both 200 and 400 mg/kg dose improved bun concentration compared with ptu group. the results of this study demonstrate that the hydroalcoholic extract of ns has a protective effect on the renal tissue oxidative damage associated with ptu-induced hypothyroidism during neonatal and juvenile growth in rats.17 in the formation of kidney stones, biochemical factors may be changed.14,18 yet, no comprehensive study has been accomplished on the effect of ns with honey on the stone formation and biochemical parameters of blood and urine in human. on the other hand, this combination is used as a mofattit in the folklore. the process is available, inexpensive and not invasive unlike existing stone breaking techniques. in the presence of honey, other excipients are not required. therefore, the effect of a combination of ns and honey on the kidney stone was investigated in a clinical trial. patients and methods methods investigation was conducted in accordance with approval protocol from research ethics committee of ahvaz jundishapur university of medical sciences. code of ethics: ir.ajums. rec.1396.645. plant material nigella sativa was purchased from a local herb store in ahvaz, iran. the seeds were identified by the pharmacognosy department of ahvaz jundishapur university of medical sciences, ahvaz, iran. the seeds were powdered using an electric grinder and immediately mixed with honey in a proportion of 1:3. they were packed in disposable containers. honey honey from padena company contains 0.1% polyphenol, 520 mg/kg prolin, 35.8% fructose, 32% glucose, and 15.2% water, while its ph is 5.72.19 kidney function study calcium, phosphorus, ph, protein, specific gravity, urine volume 24 h, creatinine, bun, and uric acid levels were measured by commercial assay kits (parsazmun kit, iran) using bt 1500 automatic analyzer. oxalate and citrate levels were measured by commercial assay kits (drmankav kit, iran) using photometer. cistine was measured by colorimetric test. statistical analysis spss was used for statistical analysis. all data are presented as mean ± sd. the data obtained were tested by independent t-test, mann–whitney, or crosstab. study plan total amount of polyphenol of ns was measured by folin– ciocalteu method. antioxidant activity was evaluated by the dpph method. then a clinical trial was conducted on 42 patients (age >18 years) from 100 patients who refered to the urology clinic of ganjavian hospital, dezful, ahvaz. they did not need medication. a sonographist recognized that they had kidney stones smaller than 6 mm, without hydronephrosis. they randomly assigned to control (n = 20) and treatment (n = 22) groups. the treatment group took 8 g of the combination with one glass of warm water daily for 1 month. the control group did not receive medication. both groups drank 6–8 glasses of water per day. at the end, sonography was repeated. blood and urine biochemical test, 24 h urine volume and urine ph and specific gravity were determined before and after intervention. the rate of stone expulsion was compared in two groups with spss. exclusion criteria were pregnancy, history of the complication or sensitivity to ns, receiving drugs that affect kidney stones, use of stone breaking techniques, and diabetes. results polyphenolic content and antioxidant activity (ic50) of ns were studied. the results were 18.53 mg (based on tannic acid) and 0.2 mg/ml respectively. about 42 patients from 100 patients with kidney stones who referred to ganjavian hospital, 22 were in the treatment group and 20 in the control group; a total of 17 women and 25 men participated in the study. all participants had not cystinuria and proteinuria. there was a significant difference between age in two groups (p = 0.041), mean age in control group was 47.95 ± 9.92 and in treatment group was 56.64 ± 12.84. the rate of stone expulsion in the treatment group (90.9%) was significantly higher than the control group (40%) (p = 0.0001). the mean of stone size and stone number variables were significantly different before and after intervention in the control and treatment groups (respectively p = 0.001, 0.001). the mean difference of stone size in the control group was 1.25 ± 1.55 and in the treatment group was 3.33 ± 0.66 .the mean difference of stone numbers in the control group was 0.4 ± 0.5 and in the treatment group 0.91 ± 0.29. the odds ratio (or) for stone disposal in the treatment group was 15 (95% confidence interval: 2.7–82.7). on the other hand, the nnt was almost equal to 2. comparison of uric acid in both groups before and after intervention showed significant difference (p = 0.025). the mean of this variable was 0.78 ± 0.96 in control group and 0.19 ± 0.66 in treatment group. the results of the comparison of serum creatinine in the control and treatment groups before and after treatment indicated that the serum creatinine difference was significant in both groups (p = 0.043). the mean of serum creatinine difference in the control group was −0.015 ± 0.13 and in the treatment group was 0.07 ± 0.13. 26 j contemp med sci | vol. 5, no. 1, january–february 2019: 24–27 evaluation of nigella sativa and honey combination for treatment of kidney stone original article e. moghimipour et al. other variables did not differ significantly before and after intervention in the two groups (p >0.05 ). comparison of variables and their differences in both groups before and after intervention showed in (table 1 and 2). discussion this study showed that polyphenolic content and ic50 of ns were 18.53 mg and 0.2 mg/ml respectively, indicating higher antioxidant activity of ns than thomson and tarocco peel and equal to sanguinello peel. in this study, the ic50 for ns was 0.2 mg/ml. in a study, ic50 mentioned for tarocco peel was 2.34 mg/ml, for sanguinello 0.2 mg/ml, and for thomson peel 3.46 mg/ml. the amount of polyphenolic compounds in 1 g of dry ns extract was 0.13 mg for tarocco peel, 0.19 mg for sanguinello peel, and 0.3 mg for thomson peel.20 the results of this study showed that the mean difference in the size of the stone before and after intervention in the table 2. comparison of differences between variables before and after intervention in both groups variables differences mean ± sd p-value between groups stone size control 1.25 ± 1.55 0.0001 treatment 3.33 ± 0.66 stone number control 0.40 ± 0.50 0.001 treatment 0.91 ± 0.29 vol. 24 h control 44.5 ± 823.26 0.304 treatment −167.73 ± 397.89 urine cr 24 h control 0.16 ± 0.23 0.848 treatment 0.18 ± 0.24 urine ox 24 h control 5.42 ± 13.15 0.473 treatment 4.55 ± 13.46 urine cit 24 h control −0.54 ± 1.26 0.322 treatment −0.13 ± 1.37 urine ua 24 h control −51.47 ± 496.05 0.339 treatment −114.65 ± 406.04 urine ca 24 h control 19.63 ± 66.45 0.291 treatment 51.17 ± 115.37 urine ph control 0.25 ± 1.16 0.922 treatment 0.09 ± 0.53 urine s. g. control −3.8 ± 8.04 0.435 treatment −1.05 ± 7.00 bun control 2.63 ± 6.79 0.689 treatment 3.33 ± 3.86 serum cr control −0.015 ± 0.13 0.043 treatment 0.07 ± 0.13 serum ca control 1.87 ± 4.43 0.677 treatment 0.15 ± 0.55 serum p control −0.1 ± 0.43 0.689 treatment −0.04 ± 0.59 serum ua control 0.78 ± 0.96 0.025 treatment 0.19 ± 0.66 gfr control −28.77 ± 83.08 0.087 treatment 10.36 ± 47.54 bun: blood urea nitrogen. table 1. comparison of variables in both groups before and after intervention variables mean ± sd mean ± sd control treatment age 47.95 ± 9.92 56.64 ± 12.84 stone size before 3.45 ± 0.89 3.39 ± 0.65 after 2.20 ± 1.96 0.06 ± 0.20 stone number before 1 1 after 0.60 ± 0.50 0.09 ± 0.29 vol. 24 h before 1470 ± 712.37 1213.64 ± 639.97 after 1425.50 ± 805.71 1381.36 ± 688.78 urine cr 24 h before 1.13 ± 0.42 1.27 ± 0.39 after 0.97 ± 0.46 1.09 ± 0.34 urine ox 24 h before 16.66 ± 13.50 17.89 ± 15.81 after 11.24 ± 8.53 13.34 ± 11.07 urine cit 24 h before 2.21 ± 1.36 1.98 ± 1.48 after 2.75 ± 1.97 2.11 ± 1.85 urine ua 24 h before 521.68 ± 345.85 369.83 ± 166.54 after 573.15 ± 405.26 484.47 ± 518.99 urine ca 24 h before 98.91 ± 54.33 162.32 ± 99.68 after 79.28 ± 63.68 111.15 ± 55.29 urine ph before 5.45 ± 0.10 5.18 ± 0.39 after 5.20 ± 0.62 5.09 ± 0.29 urine s. g. before 1023 ± 8.01 1024.64 ± 7.42 after 1026.80 ± 4.88 1025.68 ± 6.74 bun before 16.88 ± 4.79 21.15 ± 3.38 after 14.25 ± 5.31 17.82 ± 5.10 serum cr before 1.04 ± 0.18 1.10 ± 0.20 after 1.05 ± 0.15 1.03 ± 0.16 serum ca before 11.08 ± 4.31 9.38 ± 0.48 after 9.21 ± 0.65 9.23 ± 0.36 serum p before 3.49 ± 0.33 3.46 ± 0.34 after 3.59 ± 0.49 3.50 ± 0.51 serum ua before 5.02 ± 1.20 5.05 ± 1.30 after 4.28 ± 1.21 4.73 ± 1.26 gfr before 117.94 ± 90.95 104.84 ± 77.60 after 89.17 ± 53.53 115.20 ± 90.70 bun: blood urea nitrogen. treatment group (3.33 ± 0.66) was more than the control group (1.25 ± 1.55). also, the mean difference of stone number before and after intervention in the treatment group (0.91 ± 0.29) was more than the control group (0.4 ± 0.5). in both of them, p-value was equal to 0.0001. a study found that the ethanolic extract of n. sativa seeds with a dose of 250 mg/kg significantly decreased the number and size of calcium oxalate deposits in different parts of wistar rat renal tubules and also prevented damages to the tubules and calyxes.12 this study confirms the views of aghili khorasani, hakim arzani, avicenna, and razi. aghili khorasani, in makhzan-uladvieh, considered honey as jali (a medicine that serves to wipe the surface of organ cleen), monaqqi (cleansing agent), moqatti (a medicine that cuts abnormal humor off the organ without altering its consistency) of sticky balgham (phlegm) and moisture, mofatit, diuretic and anti osr ol-bawl. he also introduced honey as a moqatti of hasah (stone), diuretic and anti osr ol-bawl.7 hakim arzani used ns as a diuretic; also used with warm water and honey as a stone breaker in kidney and bladder. e. moghimipour et al. 27j contemp med sci | vol. 5, no. 1, january–february 2019: 24–27 original article evaluation of nigella sativa and honey combination for treatment of kidney stone avicenna, razi, considered triturated ns with honey and warm water as an expellent of kidney stones, diuretic, qati (cutter) of digestive thick akhlat.7–9,11,21 the rate of expulsion in the treatment group (90.9%) was significantly higher than the control group (40%), which indicated a potent stone removal effect of the combination (p = 0.0001). in the formation of kidney stone, 24 h urine volume and blood or urine biochemical factors such as phosphorus, calcium, citrate, uric acid, oxalate, etc. may be changed.14,18 serum uric acid decreased in both groups after the intervention, but its decrease in the treatment group (0.19 ± 0.66) was significantly lower than the control group (0.78 ± 0.96) (p = 0.025). a study in 1997 found that taking 1 g of ns, two times a day, in healthy subjects, resulted in no significant changes in serum uric acid levels.14 serum creatinine increased in the control group and decreased in the treatment group. the mean of serum creatinine differences in the control group was −0.015 ± 0.127 and in the treatment group was 0.07 ± 0.13. mean serum creatinine before and after treatment in two groups had a significant difference (p = 0.043). a study on rats in 2017, found that the serum creatinine was reduced by dose of 400 mg/kg of ns extract.17 in another study (2016), serum creatinine levels were associated with a significant decrease following the use of 5 mg/kg of ns oil for 28 days in wistar rats (p < 0.01).22 a research (2016) showed that the administration of paracetamol significantly increased the serum creatinine level (0.80 u/l) compared with the sham group (0.31 u/l); the serum creatinine level was reduced to 0.64, 0.57, and 0.52 u/l with 250, 500, and 1000 mg/kg doses of ns extract, respectively.16 a study in saudi arabia, indicated that administration of 2 g/day of ns, made significant changes in blood creatinine level in healthy individuals.14 there were no complications following the use of combination during the study. it can be concluded that there is no renal damage due to treatment with the combination. it confirms the nephroprotectrivy of the combination. a clinical study (2010) found that ns at doses of 1–3 g/day for 3 months did not adversely affect renal and hepatic functions of diabetic patients.23 conclusion according to the odds ratio (or > 10), stone expulsion in the treatment group is 15 times more than the control group. therefore, there is a meaningful relationship between the stone expulsion and intervention type. on the other hand, the nnt is almost equal to 2; that is, clinically in the community, one person is treated from two people who receive the combination. also, a significant reduction in the stone number and size in the treatment group may be indicated to crush or dissolve the stones by the combination, which approves traditional use of ns majun in kidney sone. the mechanism of action is probably antioxidant and stone breaker. acknowledgment this research was supported by ahvaz jundishapur university of medical sciences, ahvaz, iran (grant no. mprc 9613). conflict of interest none.  references 1. moe ow. kidney stones: pathophysiology and medical management. lancet. 2006;367:333–344. 2. tiselius hg. epidemiology and medical management of stone disease. bju int. 2003;91:758–767. 3. devuyst o, pirson y. genetics of hypercalciuric stone forming diseases. kidney int. 2007;72:1065–1072. 4. fink ha, wilt tj, eidman ke, garimella ps, macdonald r, rutks ir, et al. medical management to prevent recurrent nephrolithiasis in adults: a systematic review for an american college of physicians clinical guideline. ann intern med. 2013;158:535–543. 5. safarinejad mr. adult urolithiasis in a population-based study in iran: prevalence, incidence, and associated risk factors. urol res. 2007;35:73–82. 6. shah arzani ma. mizanoteb. researcher: nasiri h, qom: sama cultural institute.1380 sh.159-160. 7. aghili shirazi mh. makhzan al-advia. ahmed kabir et al. (eds.). hindustan, 1260;557. 8. shah arzani ma. akbari medicine. 1st ed.; qom, jalaluddin, vol. 2, 2008;825–845. 9. ibn sina h. the canon of medicine. 1st ed.; beirut, arabic scientific foundation, vol. 2, 1426 ah; p. 139. 10. razi mz. taghsim al-ellal. sobhi mahmud h. (ed.). aleppo, arab scientific foundation, 1412 ah;755. 11. ibn sina h. the treatise on colic (qulanj) by avicenna. sobhi mahmud h. (ed.). aleppo, arabic scientific foundation, 1403 ah;220. 12. khoei a, hadjzadeh z, parizady m. ethanolic extract of nigella sativa l seeds on ethylene glycol-induced kidney calculi in rats. urol j. 2009;4:86–90. 13. hadjzadeh ma, mohammadian n, rahmani z, rassouli fb. effect of thymoquinone on ethylene glycol-induced kidney calculi in rats. urol j. 2008;5:149–155. 14. bamosa ao, basil a, sowayan aa, sowayan sa. effect of oral ingestion nigella sativa seeds on some blood parameters. saudi pharma j. 1997;5:126–129. 15. hayatdavoudi p, khajavi rad a, rajaei z, hadjzadeh ma. renal injury, nephrolithiasis and nigella sativa: a mini review. avicenna j phytomed. 2016;6:1–8. 16. canayakin d, bayir y, kilic baygutalp n, sezen karaoglan e, atmaca ht, kocak ozgeris fb, et al. paracetamol-induced nephrotoxicity and oxidative stress in rats: the protective role of nigella sativa. pharm biol. 2016;54:2082–2091. 17. mohebbati r, hosseini m, haghshenas m, nazariborun a, beheshti f. the eff ects of nigella sativa extract on renal tissue oxidative damage during neonatal and juvenile growth in propylthiouracil-induced hypothyroid rats. endocr regul. 2017;51:105–113. 18. ghorbani a, shahbazian h, moradi l. risk factors of renal stone in patients with recurrent nephrolithiasis: a case-control study. life sci j. 2012;9:3038–3043. 19. institute of standards and industrial research of iran. honey: specifications and test methods. 7th revision. 2013; number 92. 20. fatahi moghadam j, hy, fotohi ghazvini r, ghasem nejad m, bakhshi d. evaluation of physicochemical and antioxidant properties of some commercial citrus varieties. j hortic sci. 2011;25:211–217. 21. razi mz. the book of colic (qulanj). sobhi mahmud h (ed.). aleppo, university of aleppo, arabic scientific foundation, 1403 ad; p. 122. 22. benhelima a, kaid-omar z, hemida h, benmahdi t, addou a. nephroprotective and diuretic effect of nigella sativa l seeds oil on lithiasic wistar rats. afr j tradit complement altern med. 2016;13:204–214. 23. bamosa ao, kaatabi h, lebdaa fm, elq a, al-sultanb a. effect of nigella sativa seeds on the glycemic control of patients with type 2 diabetes mellitus. indian j physiol pharmacol. 2010;54:344–354. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 357j contemp med sci | vol. 8, no. 5, september-october 2022: 357–362 original executive function deficits associated with anxiety and depression and their impact on medical students’ academic performance in saudi arabia sulhi a. alfakeh1* , alya s. alharbi2, mohammed k. alhebshi2, abdullah f. attar2 1faculty of medicine, department of internal medicine, king abdulaziz university, jeddah, saudi arabia. 2faculty of medicine, king abdulaziz university, jeddah, saudi arabia. *correspondence to: sulhi a. alfakeh (email: salfakeh@kau.edu.sa) (submitted: 10 april 2022 – revised version received: 22 may 2022 – accepted: 17 june 2022 – published online: 26 october 2022) abstract objectives: this study aimed to determine the relationship between ef deficits associated with anxiety and depression and their impact on medical students’ academic achievement in saudi universities. methods: a cross-sectional online survey was conducted on 242 medical students. anxiety and depression were assessed using the barkley deficits in executive functioning scale (bdefs), general anxiety disorder-7 scale (gad-7), and the patient health questionnaire (phq-9). results: participants’ mean age was 22.57 (2.26) years and the mean gad-7, phq-9, and bdefs scores were 10.54 (5.83), 12.29 (6.86), and 196.78 (47.78), respectively. males, saudi students, those in their fourth academic year, and those who were not satisfied with their academic performance had significantly higher mean gad-7, phq-9, and bdefs scores. a significant negative association was found between students’ grade point average (gpa) and bdefs scores, and a significant positive association was found between gpa and gad-7 scale scores, phq-9 scores, and bdefs scores. additionally, there was a positive association between the phq-9 and bdefs scores. conclusion: a real-world application of ef scales to address ef deficits for all university students should be considered. keywords: executive function, anxiety, depression, academic performance, saudi arabia, students, medical issn 2413-0516 introduction executive functions (efs) are a widespread set of neurocognitive processes that include flexible and complex goal-directed behaviors, thoughts, and emotions. in addition, they include working memory, sustained attention, inhibition, and planning (shanmugan & satterthwaite, 2016)1 allowing for adaptation to fluctuating environmental demands.2 efs are important in many aspects, including mental and physical health, wellbeing, and educational and career development.3 they are essential in maintaining and measuring improvements in academic performance.4 memory, self-control and inhibition, flexibility, and planning are skills related to efs, which are consequential for good academic performance.5 the relationship between efs and education starts in early childhood and continues to develop over the years, with a clear impact on academic achievement.1 recently, the importance of efs and their effect on academic performance and achievement have been observed in the development of studying methods and environments. therefore, changes in these skills may alter academic achievement.6 furthermore, new academic environments challenge students’ efs, and a lack of self-control deteriorates study progress.6 additionally, failure in self-regulation also causes procrastination, which is defined as an intentional delay to initiate or complete a task, leading to severe difficulties for students in obtaining optimal educational outcomes.7 in a study conducted in the usa, 30–60% of undergraduate students complained about postponing tasks and studying for exams, writing papers, and completing assignments.7 students with ef deficits achieve lower grade point averages (gpas) than other students.6 students with cognitive or emotional disabilities are also at risk of academic failure.8 additionally, students with lower levels of multiple ef skills are more prone to encountering issues when planning and controlling their study progress.6 in 2015, a study was conducted in the netherlands on 1,332 students who were in their first academic year of applied sciences. efs with regard to behavioral ratings were assessed to measure its influence on academic achievement. results indicated that students with higher ef scores at the beginning of the first academic year gained higher grades; in contrast, students with lower ef scores attained lower grades.6 previous studies have found that medical education is a stressful and predisposing factor for anxiety.9,10 in addition, studies have also stated that first-year students are more anxious than final-year students and that their level of anxiety decreases over.11,12 however, the prevalence of anxiety symptoms was high even in patients with no cognitive impairment.13 a thorough literature review revealed that no study in the kingdom of saudi arabia (ksa) so far has aimed to assess the associations among ef deficits, anxiety, and depression among medical students. materials and methods the barkley deficits in executive functioning scale (bdefs) is used to identify areas of impairment, based on which effective student counseling can be implemented.14 it helps the counselor inform students about their strengths and weaknesses and provide the ultimate guide to successful academic life. it can also provide an indication of current or future academic performance for college students.15 this cross-sectional study was conducted in 10 saudi universities. an online survey was conducted using a self administered questionnaire. the inclusion criteria were medical students and interns in saudi arabia aged 17–25 years; non-medical students were excluded. electronic consent was obtained from all participants. regarding ethical considerations, ethical approval for the study was obtained from the research ethics committee of http://orcid.org/0000-0003-1371-876x mailto:salfakeh@kau.edu.sa 358 j contemp med sci | vol. 8, no. 5, september-october 2022: 357–362 ef associated with anxiety and depression and their impact on medical students’ academic performance original s.a. alfakeh et al. king abdulaziz university (kau), jeddah, ksa (reference number 21–22). participants were informed about the study idea and its aim at the beginning of the survey to obtain their consent for participation. students were asked about their age, gender, nationality, academic year, gpa, and satisfaction with academic performance. efs were assessed using the bdefs since it has the salient ability to predict impairments in social relationships, professional work, and everyday life.16 the bdefs is divided into five major categories: time management, organization and problem solving, self-inhibition, motivation, and emotion regulation. in addition, the scale provides a total score for efs. participants with higher scores were believed to have greater ef deficits.17–19 anxiety was assessed using the general anxiety disorder-7 (gad-7) scale, which is a valuable tool used to screen for anxiety and assess its severity in different fields.20 this scale comprises seven questions regarding whether participants have been bothered by the symptoms mentioned in the questionnaire (and how often) during the past two weeks. each symptom has four response options, scored from 0–3, where 0 represents not at all and 3 represents nearly every day. the results are interpreted according to the total score; 0–4 corresponds to minimal anxiety, 5–9 to mild anxiety, 10–14 to moderate anxiety, and 15–21 to severe anxiety.20,21 we used the patient health questionnaire (phq-9) to assess depression; it comprises 10 questions: the first includes nine items and has four response options from 0 to 3. consequently, a score of ≥10 in this questionnaire is considered a diagnostic cutoff point for depressive symptoms.22,23 data were analyzed using spss version 25 (armonk, ny: ibm corp.). qualitative data were presented as numbers and percentages. quantitative data were presented as means and standard deviations (mean [sd]). the mann–whitney u, kruskal–wallis, independent sample t-test, and anova were applied to assess the relationships between variables according to data normality. statistical significance was set at p < 0.05. results table 1 shows that the mean age of the participants was 22.57 (2.26) years, 57.9% were females, 88.8% had a saudi nationality, and 57% were from the kau university. approximately 35% (35.5%) were sixth year students, and 28.9% were satisfied with their academic performance, with a mean gpa of 4.44 (5.89). the mean gad-7, phq-9, and bdefs scores were 10.54 (5.83), 12.29 (6.86), and 196.78 (47.78), respectively. table 2 shows that male students, saudi students, those in the fourth academic year, and those who were not satisfied with their academic performance had significantly higher mean gad-7 scores (p < .05). alternatively, a non-significant relationship was found between mean gad-7 scores and participants’ university (p > .05). table 3 shows that male students, those in the fourth academic year, and those who were not satisfied with their academic performance had significantly higher mean phq-9 scores (p < .05). alternatively, a non-significant relationship was found between the mean phq-9 scores and participants’ nationality and university (p > .05). table 4 shows that students of taif university, those in the fourth academic year, and those who were not satisfied with their academic performance had significantly higher mean bdefs scores (p < .05). alternatively, a non-significant relationship was found between the mean bdefs scores and participants’ gender or age (p > .05). table 5 shows that a significant negative correlation was found between students’ gpa and bdefs scores (r = –.19, p = .003). figures 1 and 2 illustrate a highly significant positive correlation between gpa and gad-7 scores and both phq-9 (r = .647, p < .001) and bdefs (r = 0.44, p < .001) scores, table 1. participant distribution according to their demographic details, university, academic year, satisfaction with academic performance, and mean scores of gpa, gad-7, phq-9, and bdefs (n = 242) variable no. (%) age (mean ± sd) 22.57 ± 2.26 gender female 140 (57.9) male 102 (42.1) nationality non-saudi 27 (11.2) saudi 215 (88.8) university bmc 15 (6.2) fcoms 10 (4.1) isnc 29 (12) kfu 13 (5.4) kau 138 (57) ksu 10 (4.1) ksau 10 (4.1) taibah u 4 (1.7) tu 1 (0.4) uqu 12 (5) academic year second 47 (19.4) third 29 (12) fourth 31 (12.8) fifth 19 (7.9) sixth 86 (35.5) intern 30 (12.4) satisfaction with academic performance no 172 (71.1) yes 70 (28.9) gpa 4.44 ± 5.89 gad-7 10.54 ± 5.83 phq-9 12.29 ± 6.86 bdefs 196.78 ± 47.78 bmc, batarji medical college; fcoms, fakeeh college for medical sciences; isnc, ibn sena medical college; kfu, king faisal university; kau, king abdulaziz university; ksu, king saud university; ksau, king saud bin abdulaziz university; taibah u, taibah university; tu, taif university; uqu, um alqura university; gpa, grade point average; gad-7, general anxiety disorder-7 scale; phq-9, patient health questionnaire; bdefs, barkley deficits in executive functioning scale. 359j contemp med sci | vol. 8, no. 5, september-october 2022: 357–362 s.a. alfakeh et al. original ef associated with anxiety and depression and their impact on medical students’ academic performance universities; the participants were 18 to 28 years of age, and two-thirds of the population was female (64.9%). the results found that males encountered significant problems on the self-inhibition and self-motivation scales.15 this study found that students in the fourth academic year had significantly higher mean phq-9 scores than those in other academic years. the previously mentioned us study indicated that the younger the participants, the more problems they had with self-restraint and emotion regulation.15 additionally, another study found that male students and those in higher academic years may need specific attention and interventions.24 we used the phq-9 to assess depression in contrast to the beck depression inventory used in other studies.25 we used respectively. figure 3 illustrates a highly significant positive correlation between phq-9 and bdefs scores (r = .61, p < .001). discussion ef skills are essential in achieving satisfaction in academic life; although it is an important field of study, there is a significant knowledge gap regarding the assessment of ef in a representative college sample.15 thus, our study aimed to assess the relationship between ef deficits, depression, and anxiety, and the impact of these factors on academic achievement. in the present study, male students had significantly higher mean gad-7 anxiety scores. a recent study was conducted in 2019, with a sample size of 1,311 college students enrolled in five us table 2. relationship between mean gad-7 scores and participants’ demographic details, university, academic year, and satisfaction with academic performance variable gad-7 anxiety scale scores (mean ± sd) test p-value gender female 8.4 ± 5.91 4.96* <.001 male 12.09 ± 5.27 nationality non-saudi 10.24 ± 5.84 2.26* .023 saudi 12.93 ± 5.25 university bmc 12.27 ± 4.87 8** 0.0688 fcoms 9.00 ± 4.69 isnc 11.07 ± 6.27 kfu 11.85 ± 5.58 kau 10.22 ± 5.92 ksu 11.40 ± 6.41 taibah u – tu 8.83 ± 5.27 uqu 12.40 ± 6.51 ksau 9.40 ± 5.68 academic year second 11.02 ± 5.19 5** .001 third 10.97 ± 5.3 fourth 14.48 ± 5.42 fifth 9.37 ± 5.27 sixth 9.19 ± 6.44 intern 9.90 ± 4.42 satisfaction with academic performance yes 8.97 ± 5.59 2.69* .007 no 11.17 ± 5.82 bmc, batarji medical college; fcoms, fakeeh college for medical sciences; isnc, ibn sena medical college; kfu, king faisal university; kau, king abdulaziz university; ksu, king saud university; ksau, king saud bin abdulaziz university; taibah u, taibah university; tu, taif university; uqu, um alqura university; gad-7, general anxiety disorder-7 scale. *mann–whitney test; ** kruskal–wallis test. table 3. relationship between mean phq-9 scores and participants’ demographic details, university, academic year, and satisfaction with academic performance variable phq-9 depression scale scores (mean ± sd) test p-value gender female 9.8 ± 6.83 4.86* <.001 male 14.11 ± 6.3 nationality non-saudi 12.06 ± 6.95 1.58* .113 saudi 14.19 ± 5.85 university bmc 12.87± 5.3 8** .636 fcoms 9.20 ± 4.36 isnc 12.59 ± 6.25 kfu 13.15 ± 6.76 kau 12.04 ± 7.28 ksu 13.00 ± 5.53 taibah u – tu 11.50 ± 6.81 ksau 16.40 ± 7.6 uqu 112.20 ± 7.75 academic year second 12.68 ± 6.3 5** .002 third 12.24 ± 5.22 fourth 17.26 ± 7.64 fifth 9.95 ± 6.22 sixth 11.21 ± 7 intern 11.20 ± 6.18 satisfaction with academic performance yes 10.06 ± 6.93 3.16* .002 no 13.2 ± 6.63 bmc, batarji medical college; fcoms, fakeeh college for medical sciences; isnc, ibn sena medical college; kfu, king faisal university; kau, king abdulaziz university; ksu, king saud university; ksau, king saud bin abdulaziz university; taibah u, taibah university; tu, taif university; uqu, um alqura university; gphq-9, patient health questionnaire. *mann–whitney test; ** kruskal–wallis test. 360 j contemp med sci | vol. 8, no. 5, september-october 2022: 357–362 ef associated with anxiety and depression and their impact on medical students’ academic performance original s.a. alfakeh et al. prevalence of depression was found to be higher among female medical students.25 a study conducted in riyadh, saudi arabia, showed that depression was more prevalent among first-year medical students.25 this corroborates the results of the present study. additionally, a study conducted in india showed that third year medical students reported significantly increased depressive symptoms.29 table 4. relationship between mean bdefs scores and participants’ demographic details, university, academic year, and satisfaction with academic performance variable bdefs scores (mean ± sd) independent sample t-test p-value gender female 190.36 ± 46.02 1.79 .075 male 201.43 ± 48.64 nationality non-saudi 208.25 ± 52.99 1.32 .186 saudi 195.35 ± 47.03 university bmc 183.46 ± 42.8 2.24* .016 fcoms 149.5 ± 20.7 isnc 203.20 ± 38.54 kfu 200.91 ± 49.19 kau 206.84 ± 61.81 ksu 178.84 ± 47.3 taibah u 221.5 ± 48.53 tu 210.54 ± 54.09 ksau 208 ± 32.58 uqu 199.58 ± 29.8 academic year second – 5.24* .001 third 176.41 ± 32.5 fourth 228.54 ± 57.91 fifth 199.26 ± 39.22 sixth 195.41 ± 44.73 intern 194.48 ± 47.51 satisfaction with academic performance yes 192.07 ± 51.79 3.18 .002 no 198.45 ± 46.13 bmc, batarji medical college; fcoms, fakeeh college for medical sciences; isnc, ibn sena medical college; kfu, king faisal university; kau, king abdulaziz university; ksu, king saud university; ksau, king saud bin abdulaziz university; taibah u, taibah university; tu, taif university; uqu, um alqura university; bdefs, barkley deficits in executive functioning scale. *anova. table 5. spearman’s correlation analysis among participants’ age and gpa and gad-7, phq-9, and bdefs scores variable age r p-value gad-7 –.09 .162 phq-9 –.04 .457 bdefs .1 .11 gpa r p-value gad-7 .002 .97 phq-9 .09 .149 bdefs –.19 .003 gpa, grade point average; gad-7, general anxiety disorder-7 scale; phq-9, patient health questionnaire; bdefs, barkley deficits in executive functioning scale. the phq-9 because it allows us to measure the severity of the participant’s depression and assign a numerical score that can be then used to assess the relationship between depression and ef. a high prevalence of depression has been observed among medical students.26 our study supports this result. a score of ≥10 in phq-9 is considered significant for the diagnosis of depression; in our study, the mean scale score was found to be 12.29 for phq-9. this study showed that male medical students (especially those in their fourth year) were not satisfied with their academic performance and had a significantly higher mean depression score. in contrast, some studies have denied any variation in depression scores depending on sex.27,28 alternatively, the world health organization states that women are more prone to depression.25 similarly, in other studies, the fig. 1 spearman’s correlation analysis between gad-7 anxiety scores and phq-9 depression scores. n.b.: (r = 0.647, p-value = <0.001). fig. 2 spearman’s correlation analysis between gad-7 anxiety scores and bdefs scale scores. n.b.: (r = 0.44, p-value = <0.001). 361j contemp med sci | vol. 8, no. 5, september-october 2022: 357–362 s.a. alfakeh et al. original ef associated with anxiety and depression and their impact on medical students’ academic performance this study showed that 71.1% of students were not satisfied with their academic performance, and students who were not satisfied had higher mean depression scores. severe depression is a problem, and it is essential to diagnose depression and anxiety early to improve quality of life.30 in the present study, we used the gad-7, which is a valid questionnaire to measure the prevalence and severity of gad in medical students in association with ef deficits.21,31,32 the prevalence of anxiety among medical students reported in 69 studies with 40,438 students was found to be 33.8%.27 one study stated that the cognitive skills of people with anxiety are not different from those of the healthy general population.33 similarly, anxiety is frequently diagnosed in medical students and is found to negatively impact their academic performance.33 this study found that students in their fourth academic year were at the beginning of their clinical years and were found to have higher mean gad-7 anxiety scale scores. additionally, they were not satisfied with their academic performance. a similar result was found in another study that studied the importance of developing coping strategies and finding a balance between medical students’ academic and personal.9 a study conducted in singapore found that anxiety is more common in females than in males, in comparison with another study, which stated that there is no difference between the sexes.33 medical students who were not satisfied with their academic performance had a significantly higher mean gad-7 score, higher mean phq-9 depression scores, and higher mean bdefs scores. the effects of stress, anxiety, and depression on students’ academic performance have also been reported in previous.26,34–36 the present study observed a non-significant relationship between mean bdefs scores and participants’ gender or age. this result does not complement that of previous studies, as age was significantly correlated with self-inhibition and emotion regulation. additionally, younger participants reported that they had encountered more problems with inhibition.15,37,38 this variation from our results could be due to the inclusion of only medical students in our study participants. this study found that students in the fourth academic year had significantly higher mean bdefs scores. alternatively, another study did not find any association between education level and ef regarding academic progress.6 our results showed a significant negative correlation between the bdefs scores and gpa of students. the effect of ef on study process and academic performance was also revealed in a previous study, which revealed the importance of implementing clearly targeted interventions that should be combined with student-counseling facilities and policy within universities.6 limitations this study has some limitations. first, this study was conducted with a relatively small and specific population (n = 242), and all participants were medical students. second, other possible potentially important covariates that should be assessed in relation to academic performance were not included, for instance, childhood brain damage, learning disability, or any socioeconomic factors. third, all medical colleges in saudi arabia were not involved in this study, which may have affected the generalization of the results. conclusion male students, saudi students, those in the fourth academic year, and those who were not satisfied with their academic performance had significantly higher mean gad-7, phq-9, and bdefs scores. a significant negative association was found between students’ gpa and bdefs scores, and a significant positive association was found between the gpa and gad-7 scores and both phq-9 and bdefs scores. future studies including a larger sample size of saudi medical students are recommended. in addition, a real-life application of such scales should be considered to address the ef of all university students. funding none. disclosure statement there are no relevant financial or non-financial competing interests to report. data availability statement data supporting the results and analyses presented in the paper can be provided upon request.  fig. 3 spearman’s correlation analysis between phq-9 depression scores and bdefs scale scores. n.b.: (r = 0.61, p-value = <0.001). references 1. shanmugan, s., & satterthwaite, t. d. (2016). neural markers of the development of executive function: relevance for education. current opinion in behavioral sciences, 10, 7–13. 2. ahmed, s. f., tang, s., waters, n. e., & davis-kean, p. (2019). executive function and academic achievement: longitudinal relations from early childhood to adolescence. journal of educational psychology, 111(3), 446–458. 3. diamond, a. (2013). executive functions. annual review of psychology, 64, 135–168. 4. visu-petra, l., cheie, l., benga, o., & miclea, m. (2011). cognitive control goes to school: the impact of executive functions on academic performance. procedia social and behavioral sciences, 11, 240–244. 5. cortés, p. a., moyano, m. n., & quílez, r. a. (2019). the relationship between executive functions and academic performance in primary 362 j contemp med sci | vol. 8, no. 5, september-october 2022: 357–362 ef associated with anxiety and depression and their impact on medical students’ academic performance original s.a. alfakeh et al. education: review and meta-analysis. frontiers in psychology, 10(10), 1582. 6. baars, m. a., nije bijvank, m., tonnaer, g. h., & jolles, j. (2015). self-report measures of executive functioning are a determinant of academic performance in first-year students at a university of applied sciences. frontiers in psychology, 6, 1131. 7. rabin, l. a., fogel, j., & nutter-upham, k. e. (2011). academic procrastination in college students: the role of self-reported executive function. journal of clinical and experimental neuropsychology, 33(3), 344–357. 8. samuels, w. e., tournaki, n., sacks, s., sacks, j., blackman, s., byalin, k., & zilinski, c. (2019). predicting gpas with executive functioning assessed by teachers and by adolescents themselves. european educational researcher, 2(3), 173–194. 9. abdulghani, h. m., alkanhal, a. a., mahmoud, e. s., ponnamperuma, g.g., & alfaris, e. a. (2011). stress and its effects on medical students: a crosssectional study at a college of medicine in saudi arabia. journal of health, population, and nutrition, 29(5), 516–522. 10. saeed, a. a., bahnassy, a. a., al-hamdan, n. a., almudhaibery, f. s., & alyahya, a. z. (2016). perceived stress and associated factors among medical students. journal of family and community medicine, 23(3), 166–171. 11. bayram, n, & bilgel, n. (2008). the prevalence and socio-demographic correlations of depression, anxiety and stress among a group of university students. social psychiatry and psychiatric epidemiology, 43(8), 667–672. 12. beiter, r., nash, r., mccrady, m., rhoades, d., linscomb, m., clarahan, m., & sammut, s. (2015). the prevalence and correlates of depression, anxiety, and stress in a sample of college students. journal of affective disorders, 173, 90–96. 13. castaneda, a. e., suvisaari, j., marttunen, m., perälä, j., saarni, s. i., aaltosetälä, t., aro, h., koskinen, s., lönnqvist, j., & tuulio-henriksson, a. (2011). cognitive functioning in a population-based sample of young adults with anxiety disorders. european psychiatry, 26(6), 346–353. 14. prevatt, f., & yelland, s. (2015). an empirical evaluation of adhd coaching in college students. journal of attention disorders, 19(8), 666–677. 15. kamradt, j. m., nikolas, m. a., burns, g. l., garner, a. a., jarrett, m. a, luebbe, a. m., & becker, s. p. (2021). barkley deficits in executive functioning scale (bdefs): validation in a large multisite college sample. assessment, 28(3), 964–976. 16. kamradt, j. m., ullsperger, j. m., & nikolas, m. a. (2014). executive function assessment and adult attention-deficit/hyperactivity disorder: tasks versus ratings on the barkley deficits in executive functioning scale. psychological assessment, 26(4), 1095–1105. 17. barkley, r. a. (2011a). barkley adult adhd rating scale–iv (baars-iv). guilford press. 18. barkley, r. a. (2011b). barkley deficits in executive functioning scale (bdefs for adults). guilford press. 19. barkley, r. a. (2012). barkley deficits in executive functioning scale–children and adolescents (bdefs-ca). guilford press. 20. mousa, o. y., dhamoon, m. s., lander, s., & dhamoon, a.s. (2016). the md blues: under-recognized depression and anxiety in medical trainees. plos one, 11(6), e0156554. 21. spitzer, r. l., kroenke, k., williams, j. b., & löwe, b. (2006). a brief measure for assessing generalized anxiety disorder: the gad-7. archives of internal medicine, 166(10), 1092–1097. 22. alhadi, a. n., alateeq, d. a., al-sharif, e., bawazeer, h. m., alanazi, h., alshomrani a. t., shuqdar, r. m., & alowaybil, r. (2017). an arabic translation, reliability, and validation of patient health questionnaire in a saudi sample. annals of general psychiatry, 16(1), 1–9. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i5.1290 23. inoue, t., tanaka, t., nakagawa, s., nakato, y., kameyama, r., boku, s., toda, h., kurita, t., & koyama, t. (2012). utility and limitations of phq– 9 in a clinic specializing in psychiatric care. bmc psychiatry, 12(1), 1–6. 24. dekker, s., krabbendam, l., aben, a., de groot, r., & jolles, j. (2013). coding task performance in early adolescence: a large-scale controlled study into boy-girl differences. frontiers in psychology, 27(4), 550. 25. alaqeel, m. k., alowaimer, n. a., alonezan, a. f., almegbel, n.y., & alaujan, f. y. (2017). prevalence of irritable bowel syndrome and its association with anxiety among medical students at king saud bin abdulaziz university for health sciences in riyadh. pakistan journal of medical sciences, 33(1), 33–36. 26. alzahrani, a. m., hakami, a., alhadi, a., batais, m. a., alrasheed, a. a., & almigbal, t. h. (2020). the interplay between mindfulness, depression, stress and academic performance in medical students: a saudi perspective. plos one, 15(4), e0231088. 27. alharbi, h., almalki, a., alabdan, f., & haddad, b. (2018). depression among medical students in saudi medical colleges: a cross-sectional study. advances in medical education and practice, 9, 887–891. 28. al-busaidi, z., bhargava, k., al-ismaily, a., al-lawati, h., al-kindi, r., al-shafaee, m., & al-maniri, a. (2011). prevalence of depressive symptoms among university students in oman. oman medical journal, 26(4), 235–239. 29. iqbal, s., gupta, s., & venkatarao, e. (2015). stress, anxiety and depression among medical undergraduate students and their socio-demographic correlates. the indian journal of medical research, 141(3), 354–357. 30. american psychiatric association. (n.d.). what is depression? https://www. psychiatry.org/patients-families/depression/what-is-depression. 31. alatawi, a., alghamdi, a., albalwi, a., altayar, m., jalal, m., & frah, e. a. m. (2020). prevalence of generalized anxiety disorder (gad) among saudi medical students and associated risk factors. international journal of medical research and health sciences, 5(9), 1–9. 32. rutter, l. a., & brown, t. a. (2017). psychometric properties of the generalized anxiety disorder scale-7 (gad-7) in outpatients with anxiety and mood disorders. journal of psychopathology and behavioral assessment, 39(1), 140–146. 33. tian-chi quek, t., tam, w. w., tran, b. x., zhang, m., zhang, z., su-hui ho, c., & chun-man ho, r. (2019). the global prevalence of anxiety among medical students: a meta-analysis. international journal of environmental research and public health, 16(15), 2735–2753. 34. awadalla, s., davies, e. b., & glazebrook, c. a. (2020). longitudinal cohort study to explore the relationship between depression, anxiety and academic performance among emirati university students. bmc psychiatry, 20(1), 448. 35. bergmann, c., muth, t., & loerbroks, a. (2019). medical students’ perceptions of stress due to academic studies and its interrelationships with other domains of life: a qualitative study. medical education online, 24(1), 1603526. 36. moir, f., yielder, j., sanson, j., & chen, y. (2018). depression in medical students: current insights. advances in medical education and practice, 9(9), 323–333. 37. little, t. d., rhemtulla, m., gibson, k., & schoemann, a. m. (2013). why the items versus parcels controversy needn’t be one. psychological methods, 18(3), 285–300. 38. moreau, m. p. (2011). the societal construction of ‘boys’ underachievement’ in educational policies: a cross-national comparison. journal of education policy, 26(2), 161–180. https://www.psychiatry.org/patients-families/depression/what-is-depression https://www.psychiatry.org/patients-families/depression/what-is-depression 7j contemp med sci | vol. 4, no. 1, winter 2018: 7–11 research trend in laryngeal cancer, mortality and survival rate in iran narges gholizadeh,a shamsolmoulouk najafi,b mina khayam zadeh,a sajad afzali,c nafiseh sheykhbahaeid adepartment of oral & maxillofacial medicine, school of dentistry, international campus, tehran university of medical science, tehran, iran. b dental research center, tehran university of medical science, tehran, iran. cschool of dentistry, tehran university of medical science, tehran, iran. dresearch center for caries prevention, dentistry research institute, tehran university of medical sciences, tehran, iran. correspondence to nafiseh sheykhbahaei (email: dsheykhbahaei@gmail.com). (submitted: 18 october 2017 – revised version received: 27 october 2017 – accepted: 20 november 2017 – published online: 26 march 2018) objective among the noncommunicable disease, cancer is one of the highest load and charge that lead to disability and death. the goal of this study was to determine the important epidemiologic factors that effect on survival rate of patients with laryngeal cancer. methods from 307 cases, 136 (2009–2012) that their follow-up was possible by phone or postal address were included in the study. data collections were performed from the questionnaire. the data were analyzed by spss version 20. the kaplan–meier survival curves were utilized and, moreover, the corresponding influential factors were examined by using the cox regression test. results the most widely used method for the treatment was a combination of three methods of surgery, radiation therapy, and chemotherapy. the overall 5-year survival rate was 47.28%. the age and regular follow up had a statistically significant relationship with the survival rate (p = 0.02, 0.03). conclusion the survival rate was lower in older patients. patient and professional delays were high, which need more attention for improving the quality of life. keywords laryngeal neoplasms, mortality, survival rate, trends introduction among the noncommunicable diseases, cancer is one of the highest load and charge that lead to disability and death. according to who report, the rate of death caused by cancer, precede from cardiovascular diseases.1 a common feature among all cancer cells is the loss of responsiveness to the normal growth control. physical and emotional harm caused by cancer is greater than mortality rate.2 either health care condition or quality of life in cancer patients significantly affected by elevation cancer incidence rate. head and neck cancers are a group of cancers in the variant area containing the lip, oral cavity, pharynx, hypopharynx, and the larynx.1 head and neck cancer is the sixth common cancer in the world, and among these, the oral cavity is the most commonly encountered place.3 in the ranking of the common causes of men’s mortality in the worldwide, the oral cavity cancer is among the top 10 causes.4 more than 90% of the oral cavity cancers comprising squamous cell carcinoma (scc) that originates from the epithelial lining of oral cavity, including tongue, floor of mouth and lips.5 laryngeal cancer is the second most common cancer in the upper aero digestive system. according to previous studies, 30–40% of all malignant tumors of the head and neck as well as 1.5–2% of all malignancies are associated with laryngeal cancer. in addition, 85–98% of laryngeal cancers are scc. the most common sites of tumor involvement are glottic, supraglottic or both parts.6 clinical presentations can be as ulceration or exophytic mass. histological examination from biopsied specimens is mandatory to definitive diagnosis. high diversity in clinical manifestation and insufficient attention and follow up of potentially malignant lesions lead to delay in diagnosis.7 the most common risk factors for head and neck scc include individual habits such as tobacco and alcohol use. in the last 10 years, smoking has fallen by 20% in adult. this despite the fact that alcohol consumption has increased among men and women. also, association between hpv infection and head and neck cancer has been established.8 environmental factors, such as poor oral hygiene, denture use, bleeding gums, infrequent dental visits,9 inappropriate diets, especially lack of fruits and vegetables, lack of physical activity, and obesity seems that play more important role than congenital and genetical factors in etiology of cancer. contamination by carcinogenic agents, such as asbestos, polycyclic aromatic hydrocarbons, and dust, are also among the ingredients that increase the risk of these cancers.10,11 according to the who reports, the highest age-standardized rate of such head and neck cancers is found in the southeast asia region.1 in iran, 70,000 new cases of cancer occur per year and over 30,000 people die annually from cancer.12 according to recent evidence, the 5-year survival rate of 23 types of cancer has increased among 24 types of cancer over the past 25 years, but only the survival rate of laryngeal cancer has declined over these years. although the main cause of this decrease in survival rate are not well known, during this period, sharply decrease in surgical procedures and increase in the use of non surgical methods, such as chemotherapy and radiotherapy have been observed.13 in addition, due to the improvement of health care level, the average age of the general population and people with cancer are increased. other suggestive factors are dietary changes and increased alcohol use. the changes in socioeconomic status of individuals are the possible causes for reducing the survival of patients with laryngeal cancer.13,14 in recent years, changes in risk factors exposure, diagnostics, staging, and treatment methods caused to significant changes in incidence, mortality and survival rate. policy determination to improve prevention, treatment, and supervision care for these patients need to evaluate the trends in head and neck cancer.15 due to the relatively high prevalence of head and neck cancer especially laryngeal carcinoma in the southeast asian issn 2413-0516 8 j contemp med sci | vol. 4, no. 1, winter 2018: 7–11 laryngeal cancer and survival rate research narges gholizadeh et al. regions like iran, and the lack of accurate statistics about the trend of these cancers in this area, we evaluated the relationship between clinical and demographic factors and treatment type with improved survival rate among patients with laryngeal cancer, using data collected by the cancer registry center of ministry of health and medical education for patients diagnosed from 2009 to 2012. materials and methods in this retrospective cross-sectional study, for data collection, we used documents related to laryngeal cancer patients from 2009 to 2012 in cancer department of the ministry of health of the islamic republic of iran. from a total of 307 medical records with a histopathological diagnosis of laryngeal squamous cell carcinoma, 136 cases with contact information or mailing address for correspondence were included in the study. since 2003, all new cases of cancer detection in iran have been referred to cancer registry centers through the ministry of health’s designated forms from pathology labs. these forms include demographic characteristics, pathological factors of the disease and diagnostic times. the location of the lesion, the degree of differentiation, and degree of invasive are also included with the special code in these cases. in this study, a checklist containing two categories of questions was prepared to collect and categorize information about patients. the first group questions were completed based on the information in cancer registration center forms. the second part of the questions in checklist was completed by telephone conversations by patients or relatives, including survival information, types of treatments and delayed time. in the case of non-responding to a telephone call after three times of contact with a 2-week interval, we used the postal service to obtain the patient information, and if the person did not respond, their information extracted from his medical records available in the cancer institute of imam khomeini hospital. in the case of dead patients, the cause of death was asked from relatives of patients, and two death groups were classified as death due to complications of cancer and other causes such as accidents or heart attack. patient delay (time interval from the occurrence of disease symptoms to the first patient referral), and professional delay (time interval from the first patient referral to diagnosis) evaluated in the two groups, less than 4 weeks and more than 4 weeks. the study was approved by the ethics committee of tehran university of medical science. the collected data were analyzed using spss20 software and tables and figures were presented. the survival rate was investigated with kaplan–meier curve, the effect of the studied factors on the survival rate with simple and multivariate cox regression test. the statistical significance level was considered to be p < 0.05. results patient demographics and clinical data according to present study findings, the prevalence of laryngeal cancer was 20 times higher in men than in women. the participants consisted of 130 men (95.58%) and six women (4.42%), and also 98.42% of the patients were married. the mean age of the patients was 60.8, 46.3% of patients equal to or less than 60 years and 53.7% more than 60 years. the age range of the patients was 28–85 years (fig. 1). the majority of the cancer patients were in the sixth to eighth decades of their life. the patients were categorized according to the histological grade of the lesions, 41.81% with completely differentiated lesions, 38.18% with relatively differentiated lesions and 20.01% with less differentiated lesions. diagnostic process in this study, the results regarding awareness of the patients about their disease showed that 82 (74.54%) found the symptoms of cancer themselves and 16 (14.54%) were aware of their risk of developing cancer by referring to a physician due to other illnesses. the statistical findings of the first referral in this study can be categorized according to whom the patient has referred to for the first time. the first referral of 98 patients (80.34%) was to the specialist physician, five (4.09%) to the dentist and the rest (15.57%) to the general practitioner. delay in cancer management the reasons for the delay among the patients were inaccessibility to health centers (6.7%), failure to receive appointment (5%), unable to pay the costs (20%), and lack of insurance coverage (1.7%). others did not provide clear reasons. the professional delay was obtained on average 4.26 weeks. fig 1. histogram of patient’s age. narges gholizadeh et al. 9j contemp med sci | vol. 4, no. 1, winter 2018: 7–11 research laryngeal cancer and survival rate treatment outcomes the patients with laryngeal cancers were treated in three ways: surgery, radiotherapy, and chemotherapy. in many cases, the treatment of patients was a combination of two or three methods. accordingly, the most commonly used treatment (37.5%) for the patients was the combination of all three treatments. surgery 12.5%, chemotherapy 6.6%, radiotherapy 5.1%, chemo-radiotherapy 10.3%, chemo-surgery 8.1%, radio-surgery 11%, and 0.7% of patients without treatment. follow-up in the periodic follow-up, 56 (70%) patients in the hospital and 21 (26.25%) in the private medical clinic had periodic follow-up, and three (3.75%) had no follow-up programs. also, 12.1% of patients had periodic follow-up under supervision of oral hygienist. survival rate the study showed that 62 (45.59%) patients survived during the study but 74 (54.59%) died. findings related to the cause of death in the patients indicated that 67 (93.03%) died due to the cancer complications and five (6.95%) for other causes. according to statistical analysis, the overall 1-year survival rate of these patients was 88.37% with the standard error of 2.82, the overall 3-year survival rate was 58.13% with the standard error of 4.34 and the overall 5-year survival rate was 47.28% with the standard error of 4.4 (table 1). the statistical analysis showed that the survival rate was higher in women than in men, but the gender-specific had no significant relationship with survival rate (p = 0.41). in addition, the survival rate was significantly associated with the age of the patients, so that the survival rate decreased with age (p > 0.001). in addition, regular periodic follow-up leads to significant improvement in survival rate (p < 0.03). also, there was no significant difference in survival rate among patients with periodic follow-up by oral hygienists (p = 0.51). the patient’s delay rate more than 4 weeks was not significantly associated with survival rate (p = 0.37). the survival rate also had no significant relationship with the rate of professional delay (p = 0.54). the results showed that the survival rate in these patients had no significant relationship with the grade of histological differentiation of tumor (p = 0.35). according to the multivariate cox regression analysis, individual treatments had no significant relationship with survival rate (combination of three methods p = 0.055, chemo-radiotherapy p = 0.64 and radiotherapy p = 0.73) (table 2). discussion in the present study, we evaluate the effect of patient and tumor characteristics, diagnostic process, treatment outcome, and follow-up on survival rate in patients with laryngeal cancer in iran between 2009 and 2012. the results demonstrated significantly higher survival rates in patients under age 60 and regular periodic follow-up. we gathered data from cancer registry center of ministry of health and medical education and cancer institute of imam khomeini hospital. our data demonstrate that the overall 5-year survival rate was 47.28%. in the study of ramroth et al.,16 the overall 5-year survival rate of patients was 66%, about 10% higher than the current study. in the study by fararouei et al.,6 the mean survival rate of the patients was 46.86 ± 38.66. other reported 5-year survival rates were 56 and 67.6%.17,18 in another study, 1-year survival was reported to be 3.79% in early-stage patients.19 nachalon et al.20 reported the overall survival rate of 30.8 ± 4.6 years. comparing the survival rate in various studies is very challenging and different, which can be explained by the following reasons: different types of disease and patient selection in studies that may be limited based on specific therapeutic methods, site of the tumor and stage of the tumor, as well as the limited sample size is one of the main factors of varying survival rates in studies.6 this study showed a higher prevalence of laryngeal scc in males so that the number of men was 130 (95.58%) and women were six (4.42%). regarding the results of statistical analyses in this study, the gender had no significant relationship with survival rate. studies in germany16 and finland21 also revealed that men accounted for more than 90% of the patients. in the study in 2017,6 232 out of 250 patients with laryngeal cancer were males. nachalon et al.20 also reported that the ratio of men to women was 2.6/1. chen and boffetta14,22 demonstrated that survival rate was significantly lower in men than in women, while in other studies, as the results of the current study, this significant relationship was not reported.6,13,20,23,24 this increased prevalence of cancer and reduced survival rate in men may be due to higher exposure to risk factors, as well as less attention of men to individual health and more delay in referral to a physician. we find that more than half of cancer patients were over 60 years of age. the mean age of the patients in the study by ramroth et al.16 was approximately slightly higher (63 years) than the present study. however, the number of people under 60 years old in their study was 38%, which is much less than research in table 1. overall survival rate of 1–3 and 5-years in laryngeal cancer patients (n = 136) duration (year) survival rate 1 88.38% (se = 2.82) 2 68.21% (se = 4.09) 3 58.13% (se = 4.34) 5 47.28% (se = 4.4) table 2. association of studied factors with survival rate among laryngeal cancer patients (n = 136) factor p-value* 95% confidence interval age p > 0.001* 8.12–3.88 gender p = 0.41 2.58–8.38 histological grade of the tumor p = 0.35 2.82–0.09 tumor location 2.58 2.88–8.02 patient’s delay p = 0.37 2.18–8.89 professional delay p = 0.54 2.81–8.08 periodic follow-up p = 0.03* 8.23–0.89 combination three method treatment 0.055 2.11–8.28 chemo-radiotherapy 0.64 2.80–8.02 radiotherapy 0.73 2.81–8.05 *significance level was 0.05. 10 j contemp med sci | vol. 4, no. 1, winter 2018: 7–11 laryngeal cancer and survival rate research narges gholizadeh et al. tehran. this may be due to differences in people’s lifestyles. they also reported 50% increase in mortality per decade of aging. in other studies also reported that age increasing was significantly associated with a reduction in survival rate.6,14,23,24 they suggested that aging leads to increased comorbidities (such as cardiovascular, respiratory, and renal failure) in the patients. the combination of these two phenomena can be a significant predictor of the mortality rate of the patients.23 in our study, the histological grade of the tumor indicated no significant relationship with survival rate (p = 0.35). in the study of fararouei et al.,6 the tumor grade was introduced an important factor in the survival rate of patients. however, most researchers who evaluated the survival rate, have examined the relationship between tumor stage at the time of diagnosis and survival rate rather than grade.13,20,23,24 similar to the results of this study, two other studies shown that the tumor site did not lead to significant changes in the survival rate of the patients with laryngeal cancer.6,13 according to previous reports, glottic region involvement due to hoarseness is commonly diagnosed in the early stages of cancer. however, supraglottic cancer due to the lack of symptoms is usually diagnosed in the more advanced stages of the tumor, which can lead to a reduction in the survival.6 using collected data, the effect of patient and professional delay rates on survival rate of patients was measured. the mean of patient delay was 12.53 almost equivalent 3 months, ranging from 1 week to 1 year (52 weeks). the mean professional delay was about 4.26 weeks ranging from 1 to 39 weeks. one research in 2003 demonstrated that professional delay (≥12 months) was statistically significant determinant of survival rate (p-value = 0.05). while there was no correlation between patient delay and prognosis.21 alho and teppo,21 in 2009 indicated the mean patient delay, 2 months and mean professional delay, 2.1 months. their results are almost consistent with the present study, but the study in tehran shows a slightly higher delay in the mean patient and professional delay. as stated above, higher delay can indicate a low awareness of cancer patients or inadequate access to treatment centers for the diagnosis and treatment of cancer, inadequate economic status, insurance coverage or inadequate medical facilities and resources. in this research, there was no significant difference in the survival rate of patients based on the type of treatment. similarly, sabin et al.23 reported that the therapeutic method did not significantly increase the survival rate. although, our results show that in cases where surgery has been used in combination with other treatments, survival rate has increased impressively (p = 0.055). nevertheless, some studies found a significant difference in the type of treatment with survival rate. in all of these studies, it has been shown that the use of surgery alone or in combination with other therapies significantly increases survival of the patients with laryngeal cancer, as well as could lead to lower costs and subsequent problems for patients.6,13,22,24–26 the most widely used treatment method for the patients in the present study population was a combination of all three methods of surgery, radiotherapy and chemotherapy. among the patients with laryngeal cancer, 37.5% experienced all three treatments. however, in the study by ramroth et al.,16 19.2% had all three treatments. our findings also confirm that the regular follow-up of the patients by the expert can significantly improve survival rate, although these results were not seen in patients who were under the supervision of oral hygienists. this indicates that the careful examination by physician is very effective in identifying lesion recurrence and early treatment. conclusion in conclusion, early diagnosis and treatment of disease at an earlier age can be a major factor in increasing the survival rate of the patients with laryngeal cancer. hence, increasing the level of awareness of patients about warning signs and symptoms, high-quality education for health care workers and hygienists to identify normal from abnormal head and neck structures, improving the economic conditions, access to health systems to reduce costs and delay in cancer management and emphasize the proper implementation of referral and follow up systems for patients should be at the head of community-based health policies. acknowledgments thank for friendly cooperation of the center for the control and prevention of noncommunicable diseases, cancer department of the ministry of health of the islamic republic of iran, who helped us in this study. conflict of interest none. n references 1. azimi s, mortazavi h, tennant m, kruger e, rezaei b, taheri jb, et al. pattern of the head and the neck cancer in two geographically and socioeconomically different countries. j orofac sci. 2017;9:43–47. 2. sikarwar s, shrivastava jp, magnani kk, shah m, jain b. "oral cancer claws clutching the youth" a study of oral cancers at a tertiary care center in central india. ijar. 2017;3:514–517. 3. gupta a, agrawal g, tiwari s, verma k, agrawal r, choudhary v. pectoralis major myocutaneous flap in head and neck reconstruction: an interesting experience from central india regional cancer center. int j res med sci. 2015;3:3065–3068. 4. ribeiro, ip, barroso l, marques f, santos a, caramelo f, julião mj, et al. genomic and epigenetic characterization for the comparison of synchronous bilateral tongue squamous cell carcinomas—a case report. curr probl cancer. 2017;41:398–406. 5. ogmundsdóttir hm, björnsson j, holbrook wp. role of tp53 in the progression of pre-malignant and malignant oral mucosal lesions. a followup study of 144 patients. j oral pathol med. 2009;38:565–571. 6. fararouei m, daneshi n, mohammadianpanah μ, reza tabatabaei h, zarebandamiri m, dianatinasab m. factors predicting survival in patients with early stage laryngeal cancer: a cohort study between 2000 to 2015. j buon. 2017;22:996–1003. 7. hingorani dv, lemieux aj, acevedo jr, glasgow hl, kedarisetty s, whitney ma, et al. early detection of squamous cell carcinoma in carcinogen induced oral cancer rodent model by ratiometric activatable cell penetrating peptides. oral oncol. 2017;71:156–162. 8. javadi p, sharma a, zahnd we, jenkins wd. evolving disparities in the epidemiology of oral cavity and oropharyngeal cancers. cancer causes control. 2017;28:635–645. 9. hashim d, sartori s, brennan p, curado mp, wünsch-filho v, divaris k, et al. the role of oral hygiene in head and neck cancer: results from international head and neck cancer epidemiology (inhance) consortium. ann oncol. 2016;27:1619–1625. 10. ramroth h, dietz a, becher h. interaction effects and populationattributable risks for smoking and alcohol on laryngeal cancer and narges gholizadeh et al. 11 research laryngeal cancer and survival rate j contemp med sci | vol. 4, no. 1, winter 2018: 7–11 its subsists: a case-control study from germany. methods inf med. 2004;43:499–504. 11. ramroth h, ahrens w, dietz a, becher h. occupational asbestos exposure as a risk factor for laryngeal carcinoma in a population based case control study from germany. am j ind med. 2011;54:510–514. 12. education moham (ed.). iranian annual of national cancer registration report 1387. 13. petrakos i, kontzoglou k, nikolopoulos tp, papadopoulos o, kostakis a. glottic and supraglottic laryngeal cancer: epidemiology, treatment patterns and survival in 164 patients. j buon. 2012;17:700–705. 14. boffetta p, merletti f, faggiano f, migliaretti g, ferro g, zanetti r, et al. prognostic factors and survival of laryngeal cancer patients from turin, italy. a population-based study. am j epidemiol. 1997;145:1100–1105. 15. van dijk ba, brands mt, geurts sm, merkx ma, roodenburg jl. trends in oral cavity cancer incidence, mortality, survival and treatment in the netherlands. int j cancer. 2016;139:574–583. 16. ramroth h, schoeps a, rudolph e, dyckhoff g, plinkert p, lippert b, et al. factors predicting survival after diagnosis of laryngeal cancer. oral oncol. 2011;47:1154–1158. 17. chen m-f, chang jt-c, tsang nm, liao c-t, chen wc. radiotherapy of earlystage glottic cancer: analysis of factors affecting prognosis. ann otol rhinol laryngol. 2003;112:904–911. 18. preuss s, cramer k, klussmann j, eckel he, guntinas-lichius o. transoral laser surgery for laryngeal cancer: outcome, complications and prognostic factors in 275 patients. eur j surg oncol. 2009;35:235–240. 19. yu q, zhang x, ji c, yang h, gao m, hong s, et al. survival analysis of laryngeal carcinoma without laryngectomy, radiotherapy, or chemotherapy. eur arch otorhinolaryngol. 2012;269:2103–2109. 20. nachalon y, cohen o, alkan u, shvero j, popovtzer a. characteristics and outcome of laryngeal squamous cell carcinoma in young adults. oncol lett. 2017;13:1393–1397. 21. teppo h, koivunen p, hyrynkangas k, alho op. diagnostic delays in laryngeal carcinoma: professional diagnostic delay is a strong independent predictor of survival. head neck. 2003;25:389–394. 22. chen ay, halpern m. factors predictive of survival in advanced laryngeal cancer. arch otolaryngol head neck surg. 2007;133:1270–1276. 23. sabin sl, rosenfeld rm, sundaram k, har-el g, lucente fe. the impact of comorbidity and age on survival with laryngeal cancer. ear nose throat j. 1999;78:578, 581–584. 24. gourin cg, dy sm, herbert rj, blackford al, quon h, forastiere aa, et al. treatment, survival, and costs of laryngeal cancer care in the elderly. laryngoscope. 2014;124:1827–1835. 25. rudolph e, dyckhoff g, becher h, dietz a, ramroth h. effects of tumour stage, comorbidity and therapy on survival of laryngeal cancer patients: a systematic review and a meta-analysis. eur arch otorhinolaryngol. 2011;268:165–179. 26. francis e, matar n, khoueir n, nassif c, farah c, haddad a. t4a laryngeal cancer survival: retrospective institutional analysis and systematic review. laryngoscope. 2014;124:1618–1623. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.03201802 122 j contemp med sci | vol. 5, no. 2, march–april 2019: 122–124 original nephroprotective effect of curcumin (curcuma longa) in acute nephrotoxicity in sprague-dawley rats hayder m. alkuraishy,* ali i. al-gareeb, and huda abdulbaki rasheed department of clinical pharmacology, medicine and therapeutic, college of medicine, al-mustansiriya university, baghdad, iraq. *correspondence to hayder m. alkuraishy (email: hayderm36@yahoo.com). (submitted: 12 december 2018 – revised version received: 17 january 2019 – accepted: 20 february 2019 – published online: 26 april 2019) objective the objective of this study was to evaluate the nephroprotective effect of curcumin in gentamicin induced-nephrotoxicity. methods thirty male sprague-dawley rats were used which was divided into three groups, group 1 (n = 10): rats treated with distilled water plus normal saline for 12 days. group 2 (n = 10): rats treated with distilled water plus gentamicin for 12 days. group 3 (n = 10): rats treated with curcumin plus gentamicin for 12 days. blood urea, serum creatinine, malondialdehyde (mda), kidney injury molecule (kim-1) and cystatin-c were measured in both control and experimental groups. results rats treated with gentamicin showed nephrotoxicity as evidence by significant elevation in serum creatinine, blood urea, kim-1, mda, cystatin-c sera levels. conclusion curcumin produced significant nephroprotective effect in gentamicin induced-nephrotoxicity through modulation of oxidative stress and inflammatory biomarkers. keywords nephrotoxicity, gentamicin, curcumin introduction gentamicin is an antibiotic of aminoglycoside group, used for treatment of different bacterial infections, 90% of administrated gentamicin is excreted unchanged in the proximal renal tubules leading to extensive necrosis at a higher dose.1,2 overproduction of reactive oxygen species and free radicals are the main mechanism for gentamicin induced nephrotoxicity. certainly, gentamicin induces the expression of transporter proteins at proximal renal tubules causing free radical generations.3 therefore, gentamicin induced-nephrotoxicity is a multifaceted phenomenon which formerly linked to the oxidative stress only. chronic and/or high dose of gentamicin initiates in vitro and in vivo free radical productions and induction of oxidative stress. gentamicin triggers mitochondrial superoxide anions cause generation of hydroxyl radicals.4 therefore, antioxidant agents demonstrate a renoprotective outcome on gentamicin induced nephrotoxicity. one of these plants called curcumin which is member of the ginger family. curcumin is the main curcuminoids present in the turmeric which contains 77% curcumin. curcumin inhibits peroxidation by scavenging of free radicals.5 curcumin’s effect on free radicals is happened by different mechanism, it scavenges various types of free radicals such as reactive oxygen species (ros), reactive nitrogen species, and also it inhibits ros generating enzymes including cyclooxygenase, lipoxygenase and xanthine hydrogenase/oxidase.6 therefore, objective of this study was to evaluate the nephroprotective effect of curcumin in gentamicin induced nephrotoxicity. materials and methods a total number of 30 sprague-dawley male rats were used, rats age ranged from 3 to 4 months and their body weight ranged from 200 to 400 g. the animals were placed at appropriate temperature (22–25°c) with 12/12 light–dark cycle. the rats were randomly divided into three groups, 10 rats in each group. group 1 (n = 10): rats treated with distilled water (5 ml/kg, p.o.) for 12 days, on days 6–12 they received an intraperitoneal (i.p.) injection of normal saline (5 ml/kg) daily. group 2 (n = 10): rats treated with distilled water (5 ml/kg, p.o.) for 12 days and on days 6–12 they received gentamicin 100 mg/kg, i.p. group 3 (n = 10): rats treated with curcumin (100 mg/kg, p.o.) for 12 days and on days 6–12 they gentamicin 100 mg/kg, i.p. at an interval of 1 h. on 12th day rats were decapitated under light anesthesia and blood samples were obtained and serum was taken by centrifugation at 3500 rpm/15 min. the method was according to singh et al.,7 method. assessment of renal injury biomarkers blood urea and serum creatinine were assessed by auto-analyzer. biomarkers of renal injury including serum of malondialdehyde (mda), kidney injury molecules (kim-1) and cystatin-c were measured by elisa kit methods according to the instruction of the manufacture. statistical analysis data of this study was presented as mean ± sd and the variables were tested by using unpaired student t-test between control and treated groups. the p-value was regarded as significant when it is <0.05. results blood urea was increased significantly in gentamicin group up to (56.87 ± 9.33 mg/dl) compared with the control group (41.83 ± 7.46 mg/dl) (p = 0.007), while; serum creatinine in gentamicin group increased significantly (1.08 ± 0.40 mg/dl) compared with control group (0.70 ± 0.14 mg/dl) (p = 0.04). regarding the oxidative stress and endogenous anti-oxidant capacity, there were insignificant increase in the mda serum levels in gentamicin group (408.11 ± 145.8 ng/ml) compared with the control group (289.85 ± 44.18 ng/ml) (p = 0.08). issn 2413-0516 123j contemp med sci | vol. 5, no. 2, march–april 2019: 122–124 original nephroprotective effect of curcumin (curcuma longa) in acute nephrotoxicityh.m. alkuraishy et al. moreover, kim-1 was significantly raised in gentamicin group (354.98 ± 46.38 pg/ml) compared with the control group (73.78 ± 16.29) (p = 0.0001). definitely, cystatin-c serum level was significantly increased during induction of nephrotoxicity by gentamicin from 0.024 ± 0.0005 ng/ml in the control group to 0.0280 ± 0.0016 ng/ml in the experimental group (p = 0.01) (table 1). curcumin leads to significant reduction of blood urea, serum creatinine compared with gentamicin group (p < 0.05). curcumin also reduced mda, kim-1 and cystatin-c sera levels significantly compared with gentamicin group (p < 0.01) (table 2). discussion gentamicin is a bactericidal antibiotic characterized by chemical stability and rapid antibacterial activity, that is extensively used alone or in combination with β-lactam antibiotics for different and serious bacterial infections. in spite of these possessions gentamicin therapy leads to nephrotoxicity in approximately 30% of treated cases even after precise monitoring.8 this study definitely illustrated that gentamicin was talented to induced experimental nephrotoxicity in rats though significant elevation in blood urea and serum creatinine which correspond with a recent study.9 it has been well known that the production of free radicals and induction of oxidative stress are the main important pathway of gentamicin induced-nephrotoxicity. overproduction of reactive oxygen species is linked with exhaustion of proximal renal tubules anti-oxidant potential which next developed into lipid peroxidation and tubular damages.10 therefore, serum level of mda is elevated in different models of gentamicin induced-nephrotoxicity as illustrated by hajihashemi et al.,11 study that confirmed the protective effect of hydroalcoholic extract of zataria multiflora in reduction of mda with significant effect in rising of anti-oxidant enzyme activities. this study also illustrated significant effect of gentamicin in increase kim-1 levels as correspond with luo et al., study that showed both kim-1 and ngal sera level are sensitive and specific biomarkers during gentamicin induced-nephrotoxicity. the rise in those biomarkers is time and dose dependent due to gene expression of kim-1 and ngal.12 notably, inequality or imbalance in the generation of free radicals and the deficiency to detoxify these free radicals by anti-oxidants lead to induction of oxidative stress. curcumin has a higher anti-oxidant potential against free radicals due to phenolic and flavonoid compounds.13 moreover, trujillo et al.,14 study confirmed that curcumin has significant nephroprotective effect due to anti-oxidant and/or preservation of endogenous anti-oxidant capacity, anti-inflammatory, and free radical scavenging effects as well as preservation of renal mitochondrial redox balance during acute and chronic nephrotoxicity. in recent times, mercantepe et al.,15 illustrated significant nephroprotective effect of curcumin in attenuation of cisplatin induced-nephrotoxicity and acute kidney injury through modulation of reactive oxygen species and augmentation of endogenous anti-oxidant potentials. furthermore, curcumin significantly reduced renal tubular injury biomarkers due to significant nephroprotective effect and attenuation of gentamicin induced-nephrotoxicity as supported by kim et al.,16 study that demonstrated administration of 25 mg/kg/day of curcumin is able to reduce levels of kim-1 and ngal sera levels significantly in cadmium induced-nephrotoxicity due to the protective effect of curcumin on the renal tubules. as well wu et al.,17 showed important effect of curcumin in reduction of inflammatory and renal tubular damage biomarkers during glycerol induced acute nephrotoxicity. interestingly, this study proved the protective effect of curcumin on the glomerular function via reduction of cystatin-c serum levels when co-administrated with gentamicin. curcumin significantly reduce cystatin-c serum levels in both acute and chronic renal injury due to significant nephroprotective effect of curcumin through modulation of glomerular blood flow and regulation of intra-glomerular pressure and inflammations.18,19 conclusion curcumin produced significant nephroprotective effect in gentamicin induced-nephrotoxicity through modulation of oxidative stress and inflammatory biomarkers. funding and sponsorship none. conflict of interest none.  table 1. renal function and renal biomarkers in gentamicin induced-nephrotoxicity variables control (n = 10) gentamicin (n = 10) p blood urea (mg/dl) 41.83 ± 7.46 56.87 ± 9.33 0.007** serum creatinine (mg/dl) 0.70 ± 0.14 1.08 ± 0.40 0.04* mda (ng/ml) 289.85 ± 44.18 408.11 ± 145.8 0.08 kim-1 (pg/ml) 73.78 ± 16.29 354.98 ± 46.38 0.0001** cystatin-c (ng/ml) 0.024 ± 0.0005 0.028 ± 0.001 0.0001** *p < 0.05. **p < 0.01, unpaired t-test. mda: malondialdehyde, kim-1: kidney injury molecule-1, cys-c: cystatin-c. table 2. effect of curcumin on the biochemical and renal injury biomarkers in gentamicin induced-nephrotoxicity variables gentamicin (n = 10) curcumin (n = 10) p blood urea (mg/dl) 56.87 ± 9.33 46.25 ± 8.47 0.01* serum creatinine (mg/dl) 1.08 ± 0.40 0.77 ± 0.18 0.03* mda (ng/ml) 408.11 ± 145.8 208.11 ± 88.8 0.001** kim-1 (pg/ml) 354.98 ± 46.38 131.79 ± 31.22 0.0001** cystatin-c (ng/ml) 0.028 ± 0.001 0.024 ± 0.0004 0.0001** *p < 0.05. **p < 0.01, unpaired t-test. mda: malondialdehyde, kim-1: kidney injury molecule-1, cys-c: cystatin-c. 124 j contemp med sci | vol. 5, no. 2, march–april 2019: 122–124 nephroprotective effect of curcumin (curcuma longa) in acute nephrotoxicity original h.m. alkuraishy et al. references 1. tiong hy, huang p, xiong s, li y, vathsala a, zink d. drug-induced nephrotoxicity: clinical impact and preclinical in vitro models. mol pharm. 2014;11:1933–1948. 2. moffett bs, morris j, galati m, munoz fm, arikan aa. population pharmacokinetic analysis of gentamicin in pediatric extracorporeal membrane oxygenation. ther drug monit. 2018;40:581–588. 3. aly haa, hassan mh. potential testicular toxicity of gentamicin in adult rats. biochem biophys res commun. 2018;497:362–367. 4. moreira ma, nascimento ma, bozzo ta, cintra a, da silva sm, dalboni ma, et al. ascorbic acid reduces gentamicin-induced nephrotoxicity in rats through the control of reactive oxygen species. clin nutr. 2014;33:296–301. 5. nelson km, dahlin jl, bisson j, graham j, pauli gf, walters ma. the essential medicinal chemistry of curcumin. j med chem. 2017;60:1620–1637. 6. lin yg, kunnumakkara ab, nair a, merritt wm, han ly, armaiz-pena gn, et al. curcumin inhibits tumor growth and angiogenesis in ovarian carcinoma by targeting the nuclear factor-κb pathway. clin cancer res. 2007;13:3423–3430. 7. singh ap, junemann a, muthuraman a, jaggi as, singh n, grover k, et al. animal models of acute renal failure. pharmacol rep. 2012; 64:31–44. 8. teslariu o, pasca as, mititelu-tartau l, schiriac ce, gales c, saftencu pm, et al. the protective effects of zinc in experimental gentamicin induced acute renal failure in rats. j physiol pharmacol. 2016;67:751–757. 9. abd-elhamid th, elgamal da, ali ss, ali fem, hassanein ehm, el-shoura eam, et al. reno-protective effects of ursodeoxycholic acid against gentamicin-induced nephrotoxicity through modulation of nf-κb, enos and caspase-3 expressions. cell tissue res. 2018;374:367–387. 10. menon vp, sudheer ar. antioxidant and anti-inflammatory properties of curcumin. adv exp med biol. 2007;595:105–125. 11. hajihashemi s, hamidizad z, rahbari a, ghanbari f, motealeghi za. effects of cobalamin (vitamin b12) on gentamicin induced nephrotoxicity in rat. drug res (stuttg). 2017;67:710–718. 12. luo qh, chen ml, sun fj, chen zl, li my, zeng w, et al. kim-1 and ngal as biomarkers of nephrotoxicity induced by gentamicin in rats. mol cell biochem. 2014;397:53–60. 13. dudylina al, ivanova mv, shumaev kb, ruuge ek superoxide formation in cardiac mitochondria and effect of phenolic antioxidants. cell biochem biophys. 2018;77:99–107. 14. trujillo j, chirino yi, molina-jijón e, andérica-romero ac, tapia e, pedrazachaverrí j. renoprotective effect of the antioxidant curcumin: recent findings. redox biol. 2013;1:448–456. 15. mercantepe f, mercantepe t, topcu a, yılmaz a, tumkaya l. protective effects of amifostine, curcumin, and melatonin against cisplatininduced acute kidney injury. naunyn schmiedebergs arch pharmacol. 2018;391:915–931. 16. kim ks, lim hj, lim js, son jy, lee j, lee bm, et al. curcumin ameliorates cadmium-induced nephrotoxicity in sprague-dawley rats. food chem toxicol. 2018;114:34–40. 17. wu j, pan x, fu h, zheng y, dai y, yin y, et al. effect of curcumin on glycerolinduced acute kidney injury in rats. sci rep. 2017;7:10114. 18. ali bh, al-salam s, al suleimani y, al kalbani j, al bahlani s, ashique m, et al. curcumin ameliorates kidney function and oxidative stress in experimental chronic kidney disease. basic clin pharmacol toxicol. 2018;122:65–73. 19. topcu-tarladacalisir y, sapmaz-metin m, karaca t. curcumin counteracts cisplatin-induced nephrotoxicity by preventing renal tubular cell apoptosis. renal failure 2016;38:1741–1748. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.04201911 404 not found 1j contemp med sci | vol. 1, no. 4, autumn 2015: 1–6 research aim this study aimed to evaluate the association between stk39 snp rs35929607 and some cardiovascular risk factors in hypertension patients in holy kerbala city, iraq. materials and methods we included 74 hypertensive patients with no signs and symptoms of renal impairment and another 30 control subjects. the links between genotype and hypertension were examined. then, the snp related variances in the blood pressure and remaining cardiovascular risk factors were studied. results there is no significant association between stk39 rs35929607 and hypertension in current study. however allele a showed a significant association in hypertensive patient compared with control group particularly in female gender. the hypertensive patients showed a significant higher result in age, bmi, fbs, total cholesterol, and stg, ldl-c and lower level in hdl-c. conclusion the association between the snp rs35929607ofstk3 and hypertension was not significant in current study in kerbala population of iraq. furthermore, only allele a showed a significant association with hypertension in females group. further studies needed to clarify the effect of other stk39 variants on these cardiovascular risk factors. keywords hypertension, bmi, stk39 , ldl-c, polymorphism relationship between serine/threonine kinase 39 gene polymorphisms with some cardiac biomarkers in hypertensive patients fadhil jawad al-tu’ma,a zena abdul-ameer mahdia & hassan mahmood abo almaalib introduction the pathogenesis of essential hypertension is believed to be multifactorial and under the influence of multiple genetic and environmental factors.1 genes associated with blood pressure (bp) are not sufficiently known as yet.2 a recent genome wide association study examined single nucleotide polymorphisms (snps) in american old order amish and identified snps in the serine/threonine kinase 39 (stk39) that were associated with hypertension.3 the stk39 encodes a serine/threonine kinase known as a ste20-related proline rich kinase (spak), one of ste20 family. these kinases were shown to interact with a new discovered serine/threonine kinases (wnk kinases) and cation-chloride co-transporters, and it was suggested that their functional change may cause bp dysregulation.4 furthermore the spak is also related to cytoskeletal rearrangement, mitogen activated protein (map) kinases, and inflammation. the associations between the above-mentioned snps and different stk39 snps and bp were confirmed in other caucasian and chinese cohorts.5,6 however, the influence of stk39 polymorphism on bp has been inconsistent, and the effects of the variants have not been found in other large studies.7,8 although a few studies have been conducted to replicate the results in the asian population, the association between stk39 variants and hypertension is still unclear among the asians. here we aimed to examine the effect of rs35929607 of stk39 on hypertension in a population of karbala province, iraq, using 74 hypertensive and 30 control subjects. the primary purpose was to evaluate the association between the snps and the risk of hypertension in females and males. in addition, we investigated the effects of the snps on other cardiovascular risk factors. materials and methods subjects in current study, 74 hypertensive patients were studied who were all diagnosed previously and some were taking treatment along with the 30 healthy controls. and the control subjects had to have systolic bp <135 mmhg and diastolic bp <85 mmhg, without using any blood pressure lowering agents. patients were selected from the out-patient department of al-hussein teaching hospital, al-hussein medical city, holy karbala, iraq. the history, blood pressure, cardiac biomarkers and body mass index of all the subjects were investigated and measured. on the day of enrolment, clinical data including demographic variables, medical history, and antihypertensive medications were recorded for all study samples. bp was measured by trained nurses at the right brachial artery using a mercury sphygmomanometer. subjects had 10 minutes of rest in the supine position before the measurements, and the average of at least three measurements were used in the study. venous blood samples were collected after an overnight fast, and samples were analyzed within 4 hours of collection. all analyses were conducted by the hospital laboratory. genotyping genomic deoxyribonucleic acid (dna) was extracted from 2 ml of peripheral venous blood by genomic dna extraction kit, from bioneer, south korea. in order to detect allele a, tetra primer amplification refractory mutation systempolymerase chain reaction (arms-pcr) has been used. the detection of the allele g was carried out by using arms pcr reaction for stk39 rs35929607 (figs. 1, 2). the pcr was carried out using an eppendorf gradient issn 2413-0516 adeparment of biochemistry, college of medicine, university of karbala, holy karbala city, iraq. bbranch of clinical laboratory science, college of pharmacy, university of karbala, holy kerbala city, iraq. correspondence to zena abdul-ameer mahdi (email: z.al-hadedy@outlook.com). (submitted: 14 october 2015 – revised version received: 3 november 2015 – accepted: 9 november 2015 – published online: autumn 2015) 2 j contemp med sci | vol. 1, no. 4, autumn 2015: 1–6 relationship between stk39 and hypertension research fadhil jawad al-tu’ma et al. thermocycler. the reaction was carried out according to the following program: • 95°c for 7 minutes (initial denaturation), 40 cycles of the following: • 95°c for 45 seconds (denaturation) • 64°c for 45 seconds (annealing) • 72°c for 45 second (extension) • 72°c for 7 minutes (final extension) and 4°c (hold phase) the pcr products were separated by 1.5% agarose gel electrophoresis and the run at 120 v for 30–40 minutes and the gel was transferred to uv trans-illuminator for visualization under ultraviolet light. bands for the required product sizes were obtained. bands for the required product sizes of stk39 gene were obtained as outer primers 349 bp, allele-a 175 bp and allele-g 231 bp (table 1). biochemical parameters fasting blood sample of (3 ml) collected by venipuncture placed into plain tubes and allowed to clot for 10–15 minutes, table 1. four primers for stk39rs 359296079 stk-1 ctcatggaatta aaggattattaggataacg inner forward stk-2 aacactctcacaagaagagatcccagtg outer forward stk-3 cacattttggcagtgtttggacagct inner reverse stk-4 ctcccaggtcgttttcaaacaaaaataa outer reverse table 2. characteristics of the study subjects parameters hypertension mean ± sd control mean ± sd p value age (years) 51.07 ± 10.60 46.67 ± 12.62 0.07 bmi (kg/m2) 29.62 ± 5.42 24.06 ± 1.63 0.00 sbp (mmhg) 144.69 ± 21.46 117.50 ± 13.57 0.00 dbp (mmhg) 88.51 ± 10.97 71.83 ± 9.42 0.00 fbs (mg/dl) 131.07 ± 63.25 97.27 ± 9.73 0.00 serum urea (mg/dl) 29.00 ± 8.32 27.00 ± 6.32 0.23 s. creatinine (mg/dl) 0.71 ± 0.25 0.68 ± 0.24 0.49 s. albumin (g/l) 4.37 ± 0.24 4.33 ± 0.23 0.43 s. cholesterol (mg/dl) 182.88 ± 38.71 178.90 ± 35.85 0.62 s. tg (mg/dl) 181.93 ± 108.46 147.50 ± 74.87 0.11 s. hdl-c (mg/dl) 39.65 ± 23.03 41.07 ± 15.48 0.75 s. ldl-c (mg/dl) 108.79 ± 40.51 108.73 ± 36.06 0.99 s. vldl-c (mg/dl) 42.75 ± 60.47 32.33 ± 20.44 0.36 fig. 1 electrophoresis band for allele g of stk39 gene. fig. 2 electrophoresis band for allele a of stk39 gene. centrifuged, and the separated serum was used for further measurement of fasting blood sugar, urea, creatinine, albumin, and lipid profile. statistical analysis group differences in the categorical variables, genotypes, were assessed by chi-square test, and continuous variables were examined by student’s t-test. results were expressed as mean ± sd and 2-tailed value of p < 0.05 was considered statistically significant. all data were analyzed using statistical package for the social sciences (spss) 17.0 version. results the characteristics of population of this study are presented in table 2. the average age was 51.07 ± 10.60 years for the patients and 46.67 ± 12.62 years for the controls. the hypertensive patients have higher bmi, fbs, with significant p = 0.00 and high total cholesterol, triglyceride, ldl-c, and low high density lipoprotein-cholesterol (hdl-c) levels. relationship between genotype of stk39 and hypertension (fig. 3) genotype distributions of stk39 polymorphisms were almost the same between normotensive controls and hypertensive patients (table 3). fig. 3 the stk39 rs35929607 genotype in patients and control groups. 3j contemp med sci | vol. 1, no. 4, autumn 2015: 1–6 research relationship between stk39 and hypertensionfadhil jawad al-tu’ma et al. table 4. the stk39 alleles in hypertension and control groups sex alleles result hypertension n (%) control n (%) p value female allele a negative 4 (50.0) 4 (50.0) 0.05 positive 47 (82.5) 10 (17.5) male allele a negative 2 (50.0) 2 (50.0) 0.54 positive 21 (60.0) 14 (40.0) female allele g negative 47 (78.3) 13 (21.7) 0.70 positive 4 (80.0) 1 (20.0) male allele g negative 20 (58.8) 14 (41.2) 0.67 positive 3 (60.0) 2 (40.0) table 3. the stk39 rs35929607 genotypes distribution in hypertension aa n (%) ag n (%) gg n (%) p value sex female 60 (92.3) 5 (7.7) 0 (0.0) 0.38 male 34 (87.2) 4 (10.3) 1 (2.6) chronic diseases hypertension 67 (90.5) 7 (9.5) 0 (0.0) 0.27 control 27 (90.0) 2 (6.7) 1 (3.3) systolic sbp > 140 mmhg 43 (84.3) 7 (13.7) 1 (2.0) 0.11 sbp < 140 mmhg 51 (96.2) 2 (3.8) 0 (0.0) diastolic dbp > 90 mmhg 44 (84.6) 7 (13.5) 1 (1.9) 0.13 dbp < 90 mmhg 50 (96.2) 2 (3.8) 0 (0.0) relationship between studied alleles and hypertension (fig. 4) the relationship between hypertension risk and alleles of stk39 in males is presented in table 4. indicated no association between genotypes and hypertension in any group which reached statistical significance. the only significant association was in females with allele a. relation among studied variants, blood pressure, and cardiovascular risk factors to start with, we have to clarify that variants gg has been detected in one sample hence all this variants statistics depended on one sample which would have been much better having more than one case for accurate outcome. although it has not been statistically proven, all ag variants in total sample and hypertensive patients have higher systolic bp than other variants. also, an obvious relationship has been clarified for the ag variants with elevated s. urea, s. creatinine, s. total cholesterol, s. tg and s. ldl-c compared to other variants in total, hypertensive, and control groups with significant p value in the control group of serum urea. moreover, ag variant patients have lower s. hdl-c in comparison to the other variants in all groups (table 5). discussion in the current study, we found no significant association between snp stk39 rs35929607and hypertension in the case control study of 104 iraqi populations. although there was no association of this snp on adjusted bps, there was a significant association between allele a of stk39 rs35929607 and hypertension group. also, there was a significant association between stk39 genotype and age, systolic bp and albumin this relationship was more obvious in females. on the other hand, the polymorphisms of stk39 were reported to be associated with bp first in caucasian samples.3 in a meta-analysis combining four studies, wang et al.,3,4 showed the effect of snps of rs6749447 and rs3754777 on bp. another snp of stk39, rs35929607, was also related to the hypertension prevalence in women of two cohorts in swedes.5 in the chinese population, two snps of stk39 (rs6433027 and 3754777) were found to be associated with hypertension.6 in the results of current study, the associations between stk39 snps and hypertension or bp were not significant. although the reason for the difference is not clear, several possibilities may be suggested. first, all the samples were of iraqis resident in karbala holy city, and the stk39-bp relationship may be weak in this population. the influence of stk39 polymorphism on bp has not been consistently confirmed. in two large gwas, using more than 60,000 people, the effects of stk39 snps on bp were not found.7,8 in addition, in one british10 and one chinese11 study, variants of stk39 were not associated with bp. recently, rhee et al.12 reported four previously reported snps associated with salt-sensitivity in 101 koreans. however, in that study, no polymorphism of stk39 showed significant effects after adjusting of confounding factors. second, although stk39 gene encodes for a protein that plays a role in bp regulation, its final effects may be too small to be detected consistently in every cohort study. then, as the population size of this study is not big enough, it cannot be completely ruled out that some variants with impacts may not have obtained statistical significance. in this study, the influence of stk39 variants on the risk factors was clearer in women. this finding is in accordance with prior studies that have shown gender dependency of stk39 effect. in a study by fava et al.5 the association of stk39 rs35929607 variant and hypertension was evident only in women. sex differences in hypertension may be attributed to multiple factors such as lifestyle, diet, and genetic polymorphism.13 interestingly, it has been documented that spak expression is affected by both androgen and oestrogens in fig. 4 the stk39 alleles in hypertension and control groups. 4 j contemp med sci | vol. 1, no. 4, autumn 2015: 1–6 relationship between stk39 and hypertension research fadhil jawad al-tu’ma et al. table 5. genotype of the studied variants and cardiovascular risk factors risk factors alleles total hypertension control mean ± s.d. mean ± s.d. mean ± s.d. age (years) 0.35 0.11 0.91 aa 49.28 ± 11.28 50.43 ± 10.69 46.41 ± 12.36 ag 55.00 ± 11.93 57.14 ± 7.97 47.50 ± 24.75 gg 52.00 ± 0.00 0.00 52.00 ± bmi 0.87 0.93 0.1 aa 27.95 ± 5.44 29.60 ± 5.57 23.86 ± 1.55 ag 28.83 ± 4.00 29.79 ± 4.02 25.49 ± 1.53 gg 26.70 ± 0.00 0.00 26.70 ± systolic bp 0.11 0.07 0.08 aa 135.34 ± 23.06 143.24 ± 21.48 115.74 ± 13.06 ag 152.22 ± 19.22 158.57 ± 16.76 130.00 ± .00 gg 140.00 ± 0.00 0.00 140.00 ± diastolic bp 0.54 0.71 0.05 aa 83.24 ± 13.33 88.36 ± 11.39 70.56 ± 8.47 ag 87.78 ± 8.33 90.00 ± 5.77 80.00 ± 14.14 gg 90.00 ± 0.00 0.00 90.00 ± fbs 0.84 0.8 0.96 aa 120.98 ± 57.64 130.46 ± 65.75 97.44 ± 10.07 ag 127.78 ± 33.87 136.86 ± 32.87 96.00 ± 9.90 gg 95.00 ± 0.00 0.00 95.00 ± s. urea 0.19 0.37 0.04 aa 27.99 ± 7.63 28.72 ± 8.14 26.19 ± 5.95 ag 33.00 ± 9.15 31.71 ± 10.14 37.50 ±.71 gg 28.00 ± 0.00 0.00 28.00 ± s. creatinine 0.03 0.03 0.38 aa .67 ± .23 .66 ± .23 .69 ± .24 ag .89 ± .31 .87 ± .35 .94 ± .19 gg .80 ± 0.00 0.00 .80 ± s. albumin 0.08 0.24 0.03 aa 4.37 ± .23 4.38 ± .24 4.35 ± .21 ag 4.20 ± .23 4.27 ± .21 3.95 ± .07 gg 4.50 ± 0.00 0.00 4.50 ± s. cholesterol 0.31 0.18 0.8 aa 179.97 ± 36.88 180.91 ± 37.04 177.63 ± 37.08 ag 200.33 ± 46.20 201.71 ± 51.79 195.50 ± 30.41 gg 180.00 ± 0.00 0.00 180.00 ± s. tg 0.11 0.02 0.7 aa 166.67 ± 98.00 172.83 ± 104.92 151.37 ± 77.88 ag 235.67 ± 116.79 269.00 ± 110.71 119.00 ± 24.04 gg 100.00 ± 0.00 0.00 100.00 ± s. hdl-c 0.54 0.4 0.59 aa 40.75 ± 21.77 40.38 ± 23.83 41.67 ± 15.89 ag 34.52 ± 11.72 32.64 ± 12.01 41.10 ±11.03 gg 25.00 ± 0.00 0.00 25.00 ± continued 5j contemp med sci | vol. 1, no. 4, autumn 2015: 1–6 research relationship between stk39 and hypertensionfadhil jawad al-tu’ma et al. s. ldl-c 0.59 0.68 0.51 aa 107.58 ± 39.67 108.16 ± 41.14 106.14 ± 36.45 ag 118.34 ± 34.49 114.84 ± 36.03 130.60 ± 36.63 gg 135.00 ±0.00 0.00 135.00 ± s. vldl-c 0.85 0.61 0.69 aa 39.24 ± 54.49 41.59 ± 63.13 33.42 ± 21.27 ag 47.13 ± 23.36 53.80 ± 22.14 23.80 ± 4.81 gg 20.00 ± 0.00 0.00 20.00 ± table 5. continued risk factors alleles total hypertension control mean ± s.d. mean ± s.d. mean ± s.d. human prostate cancer cells.14 nevertheless, further studies are needed to clarify the underlying mechanism of gender dependency in stk39 effect on cardiovascular risk factors. the population studied in the initial gwas from wang et al.3 is american old order amish, which is a closed founder population emigrated from switzerland in the early 1700s. the study populations in articles of fava et al.5 and donner et al. are from sweden and finland. those populations are all caucasians and close in geographical location. when we checked the details of the positive snps (rs6749447, rs3754777, and rs4977950) in wang’s gwas, we found that the minor allele frequency differs greatly between europeans and asians. to determine whether stk39 is a common candidate gene for hypertension, we set to find out whether it is associated with hypertension in iraqi population or not. there were studies for two snps (rs35929607 and rs12692877) within stk39 in chinese population, which were assumed functional snps based on a multispecies sequence arrangement.3 however, neither snp was significantly associated with hypertension in the chinese population. as the result was quite different from fava’s replication study5 and these result confirmed our result. five genome-wide significant snps genotyped in this study did not show a positive association with hypertension in the chinese population. similarly, cunnington et al.10 also did not find any association between the three snps (rs6749447, rs3754777, and rs35929607) within stk39 and systolic bp or diastolic bp from a cohort of 1372 british caucasians. furthermore, in fava’s study, and after exclusion of 2398 individuals from malmo preventive project cohort, who also participated in the malmo diet and cancer study, g allele of rs35929607 was no longer associated with hypertension.5 thus, it is still questionable whether stk39 can be accepted as a common susceptibility gene for hypertension. in a european study carried out in 2009, the stk39 gene snp rs35929607 were coincidently associated significantly with systolic and diastolic blood pressure, (p < 0.05).3 in another study in tharparkar, pakistan population, they found an insignificant association of this polymorphism with ehtn (p = 0.153). they looked at the prevalence of stk39 snp and observed the prevalence of frequencies of reference and rare alleles and also observed the concordance with hwe.15 the reference allele a showed higher frequency than the rare allele g (p = 0.0001). these findings highlight the weaker association of stk39 gene with ehtn in the study population.15 conclusion it was found that a presence of significant association between the studied snp and hypertension, particularly in females, through allele a. however, the snp showed genotype-related differences in blood urea, creatinine, albumin and lipid profile levels. the current study is the first to systemically analyse the association between stk39 variants and multiple cardiovascular risk factors.  references 1. j. kuneš, j. zicha. the interaction of genetic and environmental factors in the etiology of hypertension. center for cardiovascular research and institute of physiology, academy of sciences of the czech republic, prague, czech republic. physiol res. 2009;58 (suppl. 2):s33–s41. 2. huan t, esko t, peters mj, pilling lc, schramm k. schurmann c, et al. a metaanalysis of gene expression signatures of blood pressure and hypertension. plos genet. 2015 mar;11(3):e1005035. doi: 10.1371/journal.pgen.1005035. pmid: 25785607 3. wang y, o’connell jr, mcardle pf, wade jb, dorff se, shah sj, et al. from the cover: whole-genome association study identifies stk39 as a hypertension susceptibility gene. proc natl acad sci u s a. 2009 jan;106(1):226–231. doi: 10.1073/pnas.0808358106 pmid: 19114657 4. delpire e, gagnon kb. spak and osr1: ste20 kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells. biochem j. 2008 jan;409(2):321–331. doi: http://dx.doi.org/10.1042/ bj20071324 pmid: 18092945 5. fava c, danese e, montagnana m, sjögren m, almgren p, engström g, et al. serine/threonine kinase 39 is a candidate gene for primary hypertension especially in women: results from two cohort studies in swedes. j hypertens. 2011 mar;29(3):484–491. doi: 10.1097/hjh.0b013e328342b2c1 pmid: 21178783 6. chen ly, zhao wh, tian w, guo j, jiang f, jin lj, et al. stk39 is an independent risk factor for male hypertension in han chinese. int j cardiol. 2012 jan; 154(2):122–127. doi: 10.1016/j.ijcard.2010.09.007 pmid: 20889219 7. newton-cheh c, johnson t, gateva v, tobin md, bochud m, coin l, et al. genome-wide association study identifies eight loci associated with blood pressure. nat genet. 2009 jun;41(6):666–676. doi: 10.1038/ng.361 pmid: 19430483 8. levy d, ehret gb, rice k, verwoert gc, launer lj, dehghan a, et al. genome-wide association study of blood pressure and hypertension. nat genet. 2009 jun;41(6):677–687. doi: 10.1038/ng.384 pmid: 19430479 9. sayers ew, barrett t, benson da, boltan e, bryant sh, canese k, et al. database resources of national center for biotechnology information. nucleic acids res. 2011 jan;39:d38–d51. doi: 10.1093/nar/gkq1172 pmid: 21097890 10. cunnington ms, kay c, avery pj, mayosi bm, koref ms, keavney b. stk39 polymorphisms and blood pressure: an association study in british caucasians and assessment of cis-acting influences on gene expression. bmc med genet. 2009 dec;10:135. doi: 10.1186/1471-2350-10135 pmid: 20003416 6 j contemp med sci | vol. 1, no. 4, autumn 2015: 1–6 relationship between stk39 and hypertension research fadhil jawad al-tu’ma et al. 11. niu wq, zhang y, ji kd, gao pj, zhu dl. contribution of five top whole-genome association signals to hypertension in han chinese. j hum hypertens. 2011 apr;25(4):278–280. doi: 10.1038/jhh.2010.114 pmid: 21150932 12. rhee my, yang sj, oh sw, park y, kim ci, park hk, et al. novel genetic variations associated with salt sensitivity in the korean population. hypertens res. 2011 may;34(5):606–611. doi: 10.1038/hr.2010.278 pmid: 21228780 13. ruixing y, jinzhen w, shangling p, weixiong l, dezhai y, yuming c. sex differences in environmental and genetic factors for hypertension. am j med. 2008 sep;121(9):811–819. doi: 10.1016/j.amjmed.2008.04.026 pmid: 18724972 14. qi h, labrie y, grenier j, fournier a, fillion c, labrie c. androgens induce expression of spak, a ste20/sps1-related kinase, in lncap human prostate cancer cells. mol cell endocrinol. 2001 sep;182(2):181–192. doi: http:// dx.doi.org/10.1016/s0303-7207(01)00560-3 pmid: 11514053 15. loung vasandas umedani, bushra chaudhry (department of biological and biomedical sciences, aga khan university, karachi, pakistan),vikram mehraj (unite mixte de recherche 6236, centre national de larecherchescientifique, france), muhammad ishaq (department of biochemistry, united medical and dental college, ibrahim hydri, korangi creek, karachi, pakistan) 2010. 227j contemp med sci | vol. 4, no. 4, autumn 2018: 227–232 research comparative study of the therapeutic potential of octreotide versus trimetazidine and their combination on acute pancreatitis in male rats sameer h. al-rekabi,a hassan a. al-aqooli,a and muayed s. salima* issn 2413-0516 objective acute pancreatitis continues to be associated with significant rates of mortality and morbidity, and therapeutic options are still very limited. various theories have been suggested regarding the pathophysiology of acute pancreatitis, lot of research into different medical treatments for the treatment of acute pancreatitis, but it is not clear what benefits each treatment has, or indeed if any medical treatment is beneficial apart from supportive treatment. aim to clarify the potential therapeutic effect of octreotide, trimetazidine, and their combination in acute pancreatitis. methods acute pancreatitis was induced by l-arginine and treated with octreotide subcutaneously, trimetazidine intraperitoneally and combination therapy by octreotide and trimetazidine. the rats were followed for 24 h. at the 24th hour we determined serum levels of tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), total antioxidant capacity (tac), lipase, and amylase, and the pancreatic tissues were analyzed histopathologically. results tnf-α (p < 0.001), il-1β (p < 0.001), tac (p < 0.001), lipase (p < 0.001), and amylase (p < 0.001) serum levels and scores of histopathological changes (p < 0.05) were significantly lower in combination group as compared with both octreotide and trimetazidine groups. conclusion combination treatment markedly decreases biochemical and histopathological changes in acute pancreatitis, thus ameliorate pancreatic injury in l-arginine induced acute pancreatitis. keywords acute, pancreatitis, arginine, octreotide, trimetazidine department of pharmacology and therapeutics, college of medicine, kufa university, iraq. *correspondence to muayed s. salim (email: muayedsalih66@gmail.com). (submitted: 16 august 2018 – revised version received: 02 september 2018 – accepted: 05 september 2018 – published online: 26 december 2018) introduction acute pancreatitis (ap) is defined as an acute inflammatory process of the pancreas that usually associated with a severe pain in the upper abdomen; in most instances, blood levels of pancreatic enzymes, including amylase and lipase, are increased to at least three times the upper limit of normal. the inflammatory reaction in ap results in edema of the pancreas and extensive local and systemic effects. ap may occur as a new event or as a recurrent condition. in about 85% of cases, the disease takes a mild course with complete disappearance of clinical symptoms in a few days. in a small group of about 15–20% of patients, the disease takes a severe course, necrotising pancreatitis. this may culminate in multi-organ failure and death in about 15–40% of these patients.1 acute pancreatitis continues to be associated with significant rates of mortality and morbidity, and therapeutic options are still very limited. various theories have been suggested regarding the pathophysiology of acute pancreatitis, lot of research into different medical treatments for the treatment of acute pancreatitis, but it is not clear what benefits each treatment has, or indeed if any medical treatment is beneficial apart from supportive treatment.2 materials and methods experimental animals a total of 45 sprague dawley adult male rats weighing 200–300 g, were purchased from animal resource center, veterinary medicine college, al-qassim university, the national center for drug control and researches, iraq. the animals looked healthy and they lived in the animal house of college of pharmacy, university of kufa in a temperature ranged (25 ± 2°c) at room temperature, with vacillating 12-h light/12-h dark cycles and the animals were permitted for free access to water and chow diet until the beginning of the study. study design rats were randomized into five groups (n = 9) in each group as follows: control group: injected with normal saline with two doses of 1.25 ml/100 g separated by 1-h interval, and injected with normal saline in an equivalent volume to trimetazidine and octreotide solutions. induction group: injected with larginine with two doses of 250 mg/100 g separated by 1-h interval, and injected with normal saline in an equivalent volume to trimetazidine and octreotide. octreotide treated group: injected with single octreotide dose of 4 μg/kg s.c. after induction with l-arginine. trimetazidine treated group: injected with single trimetazidine dose of 10 mg/kg i.p. after induction with l-arginine. and combination group: injected with single trimetazidine dose of 10 mg/kg i.p. and single octreotide dose of 4 μg/kg s.c. after induction with l-arginine. induction of acute pancreatitis after weighing the animal accurately, we calculated the volume of l-arginine hydrochloride solution (20%) to be injected using a dose of 250 mg/100 g body weight (1.25 ml/100 g). then filling the solution in a sterile of 2 ml syringe with 25 g 5/8” needle and inject i.p. and the animal was put in a clean cage with food and water. we do the injection in a laminar 228 j contemp med sci | vol. 4, no. 4, autumn 2018: 227–232 comparative study of the therapeutic potential of octreotide versus trimetazidine research s.h. al-rekabi et al. air flow hood with filters. after waiting for 1 h, the 2nd dose of i.p. was then delivered and the animal was again returned to its cage. rats become slow after the 2nd injection but gradually recovers.3 eliza following the induction of acute pancreatitis after 24 h, the blood was drawn using direct needle puncture of the heart. samples of the blood were left to clot at 37oc and centrifuged at 3000 rpm for 10 min; serum was pulled, and analyzed for the detection of serum lipase, amylase, interleukin-1β (il-1β), tumor necrosis factor-α (tnf-α) and total antioxidant capacity (tac). the pancreas of the rat was removed and fixed for histopathological analysis. histological examination the entire pancreas was immediately removed and fixed in 10% formalin, processed by routine histological methods and embedded in paraffin blocks. about 5 μm thick sections were cut and stained with hematoxylin–eosin (h&e) for subsequent histological examination. after fixation, evaluation of scores were carried out by a histopathologist who was evaluated blindly to the experimental treatment groups. the degree of pancreatic damage and photographs were obtained from each pancreatic section (n = 3 sections per sample) under optical microscope. the morphological criteria that was used to assess the histopathological status were: edema, acinar necrosis, hemorrhage and fat necrosis, and inflammation, according to a scoring system used by schmidt et al.4 and yenicerioglu et al.5 results at the end of this study, it is found that serum tnf-α, il-1β, interleukin-6 (il-6), tac, lipase, and amylase serum levels and scores of histopathological changes were significantly elevated in induction group as compared with that of control group. both octreotide and trimetazidine significantly counteract the increase in serum levels of tnf-α, il-1β, tac, lipase, and amylase. histological analysis revealed that both octreotide or trimetazidine markedly reduced the severity of pancreatic injury in rats underwent l-arginine induction procedure. while combination therapy of octreotide plus trimetazidine decrease these parameters significantly as compared with induction group and even with octreotide or trimetazidine groups. results are shown in figs. 1–10. discussion acute pancreatitis is an inflammatory condition of the pancreas, with varying involvement of other tissues and organs. the disease includes a spectrum of pancreatic lesions, varying from parenchymatous edema to severe hemorrhagic pancreatitis, with necrosis, infection and organ destruction.6 acute pancreatitis is a disease with high morbidity and mortality. various theories have been suggested regarding the pathophysiology of acute pancreatitis, yet the underlying mechanism is not clearly understood. oxygen free radicals (ofrs) and basic proinflammatory cytokines, such as tnf-α, fig. 1 the mean level of tnf-` (pg/ml) in the five experimental groups. *significant vs control group, #significant vs induction group, $significant vs octreotide, ¥significant vs tmz. data are expressed as mean ± sd. fig. 2 the mean level of il-1a (pg/ml) in the five experimental groups. *significant vs control group, #significant vs induction group, $significant vs octreotide, ¥significant vs tmz. data are expressed as mean ± sd. il-1β and il-6, which play a role in acute pancreatitis and other systemic inflammatory conditions, have been suggested to be responsible for the local tissue damage and multiple organ failure that occur during acute pancreatitis. the ofrs lead to membrane lipid peroxidation, changes in the main components of the cytoplasm and early activation of digestive enzymes of pancreatitis, and they initiate protein damage in acute pancreatitis.5 effect of acute pancreatitis on study parameters after l-arg induction the present study shows significant elevation (p < 0.001) in serum level of proinflammatory cytokines (tnf-α and il-1β) in induction group as compared with the control group. similar results obtained by wang et al.7 mentioning that in ap, tnf-α and il-1β may play an important role in stimulating s.h. al-rekabi et al. 229j contemp med sci | vol. 4, no. 4, autumn 2018: 227–232 research comparative study of the therapeutic potential of octreotide versus trimetazidine fig. 3 the mean level of tac (l m) in the five experimental groups. *significant vs control group, #significant vs induction group, $significant vs octreotide, ¥significant vs tmz. data are expressed as mean ± sd. fig. 4 the mean level of lipase (u/l) in the five experimental groups. *significant vs control group, #significant vs induction group, $significant vs octreotide, ¥significant vs tmz. data are expressed as mean ± sd. fig. 5 the mean level of amylase (u/l) in the five experimental groups. *significant vs control group, #significant vs induction group, $significant vs octreotide, ¥significant vs tmz. data are expressed as mean ± sd. fig. 6 histopathological changes in pancreatic tissue regarding edema, acinar necrosis, hemorrhage and fat necrosis, and inflammation. fig. 7 edema scoring in different experimental groups. fig. 8 acinar necrosis scoring in different experimental groups. the inflammatory response in the animal experimental studies, the levels of il-1β and tnf-α increased significantly in the blood and in pancreatic tissues, and associated with the severity of the disease. 230 j contemp med sci | vol. 4, no. 4, autumn 2018: 227–232 comparative study of the therapeutic potential of octreotide versus trimetazidine research s.h. al-rekabi et al. fig. 9 hemorrhage and fat necrosis scoring in different experimental groups. fig. 10 inflammation and perivascular infiltrate scoring in different experimental groups. also yılmaz et al.8 showed that there is a significant increase in il-1β and tnf-α levels in ap induction group. also there is a significant decrease (p < 0.001) in serum levels of the tac in induction group as compared with the control group. similar results obtained by cikman et al.9 regarding that there is a significant inhibition of tac in the induction group compared to the control group and this is consistent with the hypothesis that ap may generate oxidative stress. the oxidative stress is intensely involved in the inflammatory processes of ap since the ofrs are the major pro-oxidant agents and in the same time acts as inflammatory mediators through the stimulation of leukocytes activation, adhesion, and then emigration in addition to improving the expression of other inflammatory mediators, such as cellular adhesive molecules.10 furthermore, yılmaz et al.8 also shows a similar results by obtaining a low levels of tac in the induction group compared with the control group proving the low antioxidant capacity in case of ap. also this study shows a significant increase (p < 0.001) in serum levels of the lipase and amylase in induction group as compared with the control group. similar results were obtained by nader and wagih11 and aziz et al.,12 and they confirmed that there is a significant increase detected in serum levels of lipase and amylase in l-arg treated rats compared to the control group. regarding the histopathological changes, induction group had significantly (p < 0.05) higher pancreatic tissue injury scoring as compared with the control group. histopathological findings in induction group as well as control group were associated with edema, acinar cell necrosis, hemorrhage and fat necrosis, and inflammation and peri-vascular infiltration. these results were similar to that obtained by wang et al.7 showing that the pancreatic interstitial edema, leukocytes infiltrations, hemorrhage, and cellular necrosis were higher significantly in the induction group as compared with the control group. effect of treatment on study parameters after l-arg induction effect of treatment on proinflammatory cytokines (tnf-α and il-1β) after l-arg induction this study shows that octreotide treatment significantly decrease the serum levels of proinflammatory cytokines (tnf-α and il-1β) as compared with the induction group, these findings were also obtained by yang et al.13 who study the effects of octreotide on hepatic ischemic reperfusion injuries in a rabbit models and shows that the level of both tnf-α and il-1β are lower significantly in octreotide group than the disease group. also both zhang et al.14 and tian et al.15 proved that octreotide can significantly decrease serum tnf-α in acute pancreatitis. tmz treatment significantly decrease the serum level of proinflammatory cytokines (tnf-α and il-1β) as compared with the induction group, these findings were also obtained by tanoglu et al.16 who confirmed that tmz can significantly decrease serum tnf-α and il-1β in the treatment of experimental sepsis in rat model. while tanoglu et al.17 found that serum il-1β levels was significantly lower during tmz treatment and there was no significant differences in serum tnf-α level, in contrary vinokurov et al.18 show that tmz can inhibits the secretion of tnf-α and may reduce the inflammatory response as a result of the blockade of secretion of proinflammatory cytokines, which may occur due to a reduced expression of the components of the receptor for endotoxin, and that is confirmed by our study that shows a significant decrease in tnf-α level in tmz group as compared with both induction and octreotide group. combination treatment significantly decrease the serum level of proinflammatory cytokines (tnf-α and il-1β) as compared with the induction group. also there is a significant decrease in serum level of proinflammatory cytokines in combination therapy as compared with the single therapy model with either octreotide or trimetazidine alone. according to our knowledge, there are no studies on using combination therapy with octreotide and trimetazidine in acute pancreatitis in a rat model, and this study is the first. effect of treatment on total antioxidant capacity this study shows that octreotide treatment significantly increase the serum levels of tac as compared with the induction group, these findings are similar to wang et al.19 that s.h. al-rekabi et al. 231j contemp med sci | vol. 4, no. 4, autumn 2018: 227–232 research comparative study of the therapeutic potential of octreotide versus trimetazidine shows octreotide protective capacity through its ability to inhibit oxidative stress. kara et al.20 also proves the protective effect of octreotide by significantly reducing this oxidative damage in male rats. tmz treatment significantly increase the serum levels of tac as compared with the induction group, these findings were also obtained by chen et al.21 who demonstrates that tmz can decrease oxidative stress significantly. similar results proved by mahfoudh-boussaid et al.22 regarding that tmz significantly decrease the oxidative stress and improve renal function in wistar rats, and also by hassanzadeh et al.23 that concluded in their study that tmz can avoid the oxidative stress induced structural injuries in rat hippocampus and it has a neuro-protective effect that associates with its antioxidants activity. our study shows a significant increase in tac level in tmz group as compared with octreotide group proving the strong antioxidant activity of tmz. combination treatment significantly increase the serum levels of tac as compared with the induction group. also there is a significant increase in serum level of tac in combination therapy as compared with the single therapy model with either octreotide or trimetazidine alone. according to our knowledge, there are no studies on using combination therapy with octreotide and trimetazidine in acute pancreatitis in a rat model, and this study is the first. effect of treatment on pancreatic enzymes (lipase and amylase) this study shows that octreotide treatment significantly decrease the serum level of pancreatic enzymes (amylase and lipase) as compared with the induction group, these findings were also obtained by chen et al.24 that showed a significant decrease in lipase and amylase serum levels after octreotide treatment in experimentally induced pancreatitis in adult rats. woeste et al.25 also showed a significant decrease in lipase level after octreotide treatment. additionally, kafali et al.26 proved that octreotide can significantly lower amylase level in rats in early dose immediately after ap induction. zhou et al.27 proved that octreotide can decrease serum amylase level in canine model ap. our study shows a significant reduction in lipase and amylase levels in octreotide group as compared with tmz group proving the strong anti-secretory activity of octreotide on the pancreas. tmz treatment significantly decrease the serum level of the pancreatic enzymes (amylase and lipase) as compared with the induction group, these findings were also obtained by tanoglu et al.17 showing that tmz can significantly reduce serum lipase and amylase levels in rat after induction of ap. yenicerioglu et al.5 also showed that tmz significantly decreased amylase levels in l-arg induced rats. combination treatment significantly decrease the serum levels of lipase and amylase as compared with the induction group. also there is a significant decrease in serum levels of lipase and amylase in combination therapy as compared with the single therapy model with either octreotide or trimetazidine alone. according to our knowledge, there are no studies on using combination therapy with octreotide and trimetazidine in acute pancreatitis in a rat model, and this study is the first. effect of treatment on pancreatic tissue after l-arg induction this study shows that octreotide treatment significantly decrease the pancreatic tissue injury scoring in edema, acinar cell necrosis, hemorrhage and fat necrosis, and inflammation as compared with the induction group, a similar findings were obtained by zhang et al.28,29 considering edema, acinar necrosis, and hemorrhage. also considering edema, inflammation, fat necrosis, parenchymal necrosis, and hemorrhage, guler et al.30 found that octreotide significantly decrease the injury scoring of pancreatic tissue. tmz treatment significantly decrease the pancreatic tissue injury scoring in edema, acinar cell necrosis, hemorrhage and fat necrosis, and inflammation as compared with the induction group, a similar findings were obtained by yenicerioglu et al.5 proved that tmz significantly decrease histopathological criteria scoring of edema, acinar cell necrosis, hemorrhage and the level of perivascular inflammation in l-arg induced ap. additionally, tanoglu et al.17 also found that tmz reduce edema formation, inflammation, vacuolization and fatty necrosis of pancreatic tissues supporting the beneficial effects of tmz treatment in experimental ap. combination treatment significantly decrease the serum levels of pancreatic tissue injury scoring in edema, acinar cell necrosis, hemorrhage and fat necrosis, and inflammation as compared with the induction group. also there is a significant decrease in pancreatic tissue injury scoring in edema, acinar necrosis, hemorrhage and fat necrosis, and inflammation in combination therapy as compared with the single therapy model with either octreotide or tmz alone. according to our knowledge, there are no studies on using combination therapy with octreotide and tmz in acute pancreatitis in a rat model, and this study is the first. statistical analysis statistical analysis was completed by using statistical package for social sciences (spss) version 20 in which we used median, mean with standard deviation as descriptive statistics. analysis of variance with lsd and mann–whitney test for comparison between groups. p-value £0.05 was regarded as significant. conclusion as indicated by the consequences and results of this study, we concluded that: 1. this study additionally supports the role of proinflammatory cytokine (tnf-α and il-1β), and oxidative stress in the pathophysiology of l-arg induced acute pancreatitis. 2. octreotide and trimetazidine reduce inflammatory reaction associated with l-arg induced acute pancreatitis as evidenced by reducing proinflammatory cytokines (tnf-α and il-1β). 3. octreotide and trimetazidine shows antioxidant activity evidenced by increasing levels of the total antioxidant capacity after treatment with these drugs. 4. octreotide has effect on pancreatic enzymes more than tmz, while tmz has more anti-inflammatory and 232 j contemp med sci | vol. 4, no. 4, autumn 2018: 227–232 comparative study of the therapeutic potential of octreotide versus trimetazidine research s.h. al-rekabi et al. antioxidant abilities than octreotide as shown by the results. 5. however, the effects of combination therapy (octreotide plus tmz) were superior to octreotide or tmz alone. the combination therapy concentrated on numerous pathological pathways of ap, such as the extreme activated pancreatic enzymes, increased activity of inflammatory mechanism, and the microcirculatory dysfunction mechanism, and therefore, the effect of combination treatment is much better than monotherapy. furthermore, it was established that tmz can enhance the therapeutical effect of octreotide in ap in rat. thus, the treatment of tmz plus octreotide can be applied as a treatment of ap more comprehensively and more efficiently. conflict of interest there is no conflict of interest regarding the publication of this paper. n references 1. bhandari m. role of galanin and its antagonists in experimental acute pancreatitis/mayank bhandari, 1972. ph.d. thesis. flinders university, 2008. 2. felderbauer p, müller c, bulut k, belyaev o, schmitz f, uhl w, et al. pathophysiology and treatment of acute pancreatitis: new therapeutic targets–a ray of hope? basic clin pharmacol toxicol. 2005;97:342–350. 3. dawra r, saluja ak. l-arginine-induced experimental acute pancreatitis. the pancreapedia: the exocrine pancreas knowledge base. 4. schmidt j, rattner dw, lewandrowski k, compton cc, mandavilli u, knoefel wt, et al. a better model of acute pancreatitis for evaluating therapy. ann surg. 1992;215:44–56. 5. yenicerioglu a, cetinkaya z, girgin m, ustundag b, ozercan ih, ayten r, et al. effects of trimetazidine in acute pancreatitis induced by l-arginine. can j surg. 2013;56:175–179. 6. almeida jl, sampietre sn, mendonça coelho am, trindade molan na, machado mc, monteiro da cunha je, et al. statin pretreatment in experimental acute pancreatitis. jop. 2008;9:431–439. 7. wang y, guo w, li y, pan x, lv w, cui l, et al. hypothermia induced by adenosine 5’-monophosphate attenuates injury in an l-arginine-induced acute pancreatitis rat model. j gastroenterol hepatol. 2014;29:742–748. 8. yılmaz ee, bozdağ z, ibiloğlu i, arıkanoğlu z, yazgan üc, kaplan i, et al. therapeutic effects of ellagic acid on l-arginin induced acute pancreatitis. acta cir bras. 2016;31:396–401. 9. cikman o, soylemez o, ozkan of, kiraz ha, sayar i, ademoglu s, et al. antioxidant activity of syringic acid prevents oxidative stress in l-arginine induced acute pancreatitis: an experimental study on rats. int surg. 2015;100:891–896. 10. gómez-cambronero lg, sabater l, pereda j, cassinello n, camps b, viña j, et al. role of cytokines and oxidative stress in the pathophysiology of acute pancreatitis: therapeutical implications. curr drug targets inflamm allergy. 2002;1:393–403. 11. nader ma, wagih hm. nilotinib, a tyrosine kinase inhibitor exhibits protection against acute pancreatitis-induced lung and liver damage in rats. naunyn schmiedebergs arch pharmacol. 2017;390:291–300. 12. aziz nm, kamel my, rifaai ra. effects of hemin, a heme oxygenase-1 inducer in l-arginine-induced acute pancreatitis and associated lung injury in adult male albino rats. endocr regul. 2017;51:20–30. 13. yang j, sun h, takacs p, zhang y, liu j, chang y, et al. the effect of octreotide on hepatic ischemia-reperfusion injury in a rabbit model. transplant proc. 2013;45:2433–2438. 14. zhang xp, tian h, lai yh, chen l, zhang l, cheng qh, et al. protective effects and mechanisms of baicalin and octreotide on renal injury of rats with severe acute pancreatitis. world j gastroenterol. 2007;13:5079–5089. 15. tian h, zhang x, wu c, chen l, ying r, ye j, et al. effects of baicalin and octreotide on the serum tnf-alpha level and apoptosis in multiple organs of rats with severe acute pancreatitis. inflammation. 2009;32:191–201. 16. tanoglu a, yamanel l, inal v, ocal r, comert b, bilgi c. appreciation of trimetazidine treatment in experimental sepsis rat model. bratisl lek listy. 2015;116:124–127. 17. tanoglu a, yazgan y, kaplan m, berber u, kara m, demırel d, et al. trimetazidine significantly reduces cerulein-induced pancreatic apoptosis. clin res hepatol gastroenterol. 2015;39:145–150. 18. vinokurov mg, astashkin ei, yurinskaya mm, glezer mg, sobolev ke, grachev sv. trimetazidine blocks store-operated ca(2+) channels in hl-60 and thp-1 cell lines and inhibits the secretion of tumor necrosis factor. dokl biol sci. 2011;441:417–420. 19. wang j, sun z, shen j, wu d, liu f, yang r, et al. octreotide protects the mouse retina against ischemic reperfusion injury through regulation of antioxidation and activation of nf-κb. oxid med cell longev. 2015;2015:970156. 20. kara h, karatas f, tug t, canatan h, karaoglu a. protective effect of octreotide on intra-tracheal bleomycin-induced oxidative damage in rats. exp toxicol pathol. 2010;62:235–241. 21. chen a, li w, chen x, shen y, dai w, dong q, et al. trimetazidine attenuates pressure overload-induced early cardiac energy dysfunction via regulation of neuropeptide y system in a rat model of abdominal aortic constriction. bmc cardiovasc disord. 2016; 16: 225. 22. mahfoudh-boussaid a, zaouali ma, hauet t, hadj-ayed k, miled ah, ghoul-mazgar s, et al. attenuation of endoplasmic reticulum stress and mitochondrial injury in kidney with ischemic postconditioning application and trimetazidine treatment. j biomed sci. 2012;19:71. 23. hassanzadeh g, hosseini a, pasbakhsh p, akbari m, ghaffarpour m, takzare n, et al. trimetazidine prevents oxidative changes induced in a rat model of sporadic type of alzheimer’s disease. acta med iran. 2015;53:17–24. 24. chen cc, wang ss, lee fy, tsay sh, wu sl, lu rh, et al. prophylactic octreotide reduces the severity of histopathologic changes and hemodynamic shock in early taurodeoxycholate-induced experimental pancreatitis. proc natl sci counc repub china b. 1999;23:1–6. 25. woeste g, wullstein c, meyer s, usadel kh, hopt ut, bechstein wo, et al. octreotide attenuates impaired microcirculation in postischemic pancreatitis when administered before induction of ischemia. transplantation. 2008;86:961–967. 26. kafali me, gul m, alptekin h, sahin m, toy h, akoz m. is there a relationship between beginning time and efficiency of octreotide in the treatment of experimental acute pancreatitis? j korean surg soc. 2012;82:296–301. 27. zhou mt, chen bc, sun hw, jin yp, yang fj, zhang x, et al. continuous regional arterial infusion with fluorouracil and octreotide attenuates severe acute pancreatitis in a canine model. plos one. 2012;7:e37347. 28. zhang xp, zhang l, yang p, zhang rp, cheng qh. protective effects of baicalin and octreotide on multiple organ injury in severe acute pancreatitis. dig dis sci. 2008;53:581–591. 29. zhang xp, tian h, wu dj, feng gh, chen l, zhang j, et al. pathological changes in multiple organs of rats with severe acute pancreatitis treated by baicalin and octreotide. hepatobiliary pancreat dis int. 2009;8:85–92. 30. güler o, akturan s, kisli e, dolapçi i, caydere m, akova a. acute pancreatitis, bacterial translocation, and different octreotide regimens: an experimental study. surg today. 2009;39:876–883. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 140 j contemp med sci | vol. 4, no. 3, summer 2018: 140–143 original comparative study between icteric and non-icteric hepatitis a among pediatric patients admitted to babylon maternity hospital, iraq bashar sahib khalaf,a and zainab adil ghani chabuckb adepartment of pediatrics, college of medicine, university of babylon, babylon, iraq. bdepartment of microbiology, college of medicine, university of babylon, babylon, iraq. correspondence to zainab adil ghani chabuck (email: zainabibz@gmail.com). (submitted: 10 may 2018 – revised version received: 15 june 2018 – accepted: 19 june 2018 – published online: 26 september 2018) objective acute infections of viral hepatitis are considered as serious health threats of the populations around the world. there are diverse forms of the infections that exhibit wide range of epidemiology and natural histories. viral hepatitis a is the most public and common viral hepatitis throughout the childhood period, particularly in the developing countries. methods a cross-sectional sampling of (145) child who admitted into out-patients treatment center in babylon maternity hospital. for each person, different demographic parameters, were obtained. immunoglobulin g (igg) and immunoglobulin m (igm) antibodies specific for hepatitis a virus (anti-hav) were obtained in patients’ sera by enzyme-linked immunosorbent assay. results seroprevalence of hav was 100% among the tested children, with a wide range of presentation mainly as fever, hepatomegaly, choluria and jaundice, while non-jaundiced are 31%. chi-square analysis of demographic parameters showed a high significant difference with age, residence, family history and personal hygiene among jaundiced and non-jaundiced pediatric patients (p value = 0.0001) and a significant difference with nutritional status (p = 0.03), while no difference with socioeconomic status and chronic drugs usage (p = 0.054 and 0.559) respectively and with no gender differences. conclusion hepatitis a viral infection continues to be a major health problem in developing countries and it is a hyper-endemic in iraq. keywords hepatitis a, icteric, non-icteric, pediatrics and jaundice introduction hepatitis virus class a (hav), is categorized as a member of picornaviridae family, a non-enveloped ribonucleic acid (rna) virus. its route of transmission is feco-orally and associated with poor sanitation and hygiene, and primarily spreading through close contact with sick individuals leading to infection plus inflammation of the liver.1,2 hav shows a high endemic rate in the underdeveloped countries, those with deprived hygiene and sanitation, especially through drinking contaminated water and food.3 it is the most common distributed type of viral hepatitis around the world, as it is responsible for around 1.4 million new infections worldwide each year. the asymptomatic childhood infections with higher incidence are generally allied with inadequate water treatment, poor sanitation, overcrowding, and lower socioeconomic factors.4 infections of hepatitis a is usually self-limiting, acute liver illness, while the progress of the disease is not always benign; starting with a flu-like symptoms, jaundice and/or elevated liver enzymes (serum aminotransferases). initially, clinical symptoms may be like those of a viral prodrome, and with nonspecific presentations.5,6 complication of hepatitis a occurs dramatically as an acute liver failure. the elder patients especially immunosuppressed or those with underlying liver disease are at risk for this complication. in children, the course of hepatitis a usually has an asymptomatic mode while the clinical presentations are more common in adults, thus in developed populations, the symptomatic cases proportion is higher as the infection is more expected in elder patients due to routine vaccination coverage in childhood.7,8 this study aimed to compare different demographic parameters among children with hepatitis a. materials and methods this study included 145 pediatric children who were admitted to the out-patients clinic in babylon maternity hospital over a period of 1 year. they were presented with variable range of clinical signs and symptoms; 87 male and 58 female and with age range from 1 to 10 years. hepatitis is suspected, thus blood sample (2 ml) was taken from each one for preparation of serum, then immunoglobulin g (igg) and immunoglobulin m (igm) antibodies, anti-hav; specific against hepatitis a virus were identified in sera by the use of enzyme-linked immunosorbent assay. antibody levels when they are more than the determined cut-off point were considered as positive, to establish the diagnosis of hav hepatitis. in addition to that, different parameters such as age, sex, residence, family history, personal hygiene, socioeconomic status, nutritional status and history of chronic drugs usage were collected from all patients. later on the 145 patients were divided into two groups (100/45) according to their presentation with jaundice or not respectively. statistical analysis all these taken parameters were compared between these two groups by the use of chi-square through spss version 18.0. the 95% confidence interval of a proportion was used to calculate the population parameters. p-value that is obtained as less than the 0.05 the level of significance was considered statistically significant. ethical considerations this study was conducted on patients who signed written informed consent forms. the study was approved in the ethics committee of babylon university. issn 2413-0516 bashar sahib khalaf et al. 141j contemp med sci | vol. 4, no. 3, summer 2018: 140–143 original acute hepatitis a viral hepatitis among pediatric patients results the whole (145) pediatric patients who were encompassed in this study, were subjected to elisa to determine the titer of igg and igm antibodies against hepatitis a virus (anti-hav); and all of them (100%) were within the positive titer level, which indicate positivity for this viral infection. these patients were presented with variable ranges of clinical signs and symptoms, with more number presented with jaundice (100) as compared to non-jaundiced (45) individuals (fig. 1); and then children were divided into two groups according to jaundice (icteric/non-icteric) presentation as tabulated in table 1. the results of table 2 showed the distribution of hepatitis a pediatric patients in relation with different demographic parameters; as it revealed that between icteric and non-icteric viral hepatitis, there was a highly significant difference with age, residence, family history and personal hygiene (p-value = 0.0001) for each, and a significant difference with nutritional status (p-value = 0.03), while no difference with socioeconomic status and chronic drugs usage (p = 0.054 and 0.559) respectively and with no gender differences (p-value = 1). regarding managements that applied for patients with hav, according to table 3. discussion the hav viral infection that is enterically transmitted, is endemic in many of the developing countries, as its prevalence can approach 100% in children under 5 years of age.9 in the developing countries, acquirement of hav occurs very early in life and the adults 100% nearly have detectable levels of anti-hav and therefore are immunized to infection. in the more developed countries, where sanitation and hygiene is good, most individuals reaching adulthood without undergoing infection, as with a low prevalence (10%) among children, and adults are largely susceptible of being negative for anti-hav (63%).10 certain iraqi study showed that 96.4% of the population sample had positive anti-hav igg antibodies. the frequency distribution of positive anti-hav antibodies was lowermost in dahuk, al-ta’mim, diyala and al-basrah with a range of 85.2–95%.11 similar study performed in egypt with prevalence of anti-hav antibodies was 86.2%;12 in addition to other numbers of studies, as the prevalence ranged between 89.4% and 100% (in alexandria and rural areas).13–15 analogous of this high prevalence had been reported from syria (89%) and palestine (93.3%).16,17 comparable work from shiraz in iran, antibody of hepatitis a igg gave positivity in 88.2% of individuals.18 people of all ages are liable to hepatitis a virus infections, its incubation period a is approximately 28 days (range 15–50 days). it affects the liver as an acute inflammatory disease. the disease severity ranges from a mild illness for a few weeks to a severe illness that last several months.19 regarding demographic characteristics, rafeey and shoaran20 showed no differences in respect to age and sex. also, the same result was found in two studies in zanjan and isfahan (iran) on pediatric patients, as no association between sex, age and seropositivity in the first study and between sex and positive antibody in the second survey.21,22 other two studies done in iraq and egypt showed no gender differences.11,12 in developed european countries, there is a decrement in the prevalence of hav infection, specifically amongst the young age group, this is attributed to marked enhancements in hygiene and socioeconomic situation and vaccination in a widespread coverage.23 in a korean study, the female/male ratio for total seropositivity of anti-hav was not significant statistically (49.44% vs. 52.86%, p = 0.560).24,25 table 1. presentations [no. (%)] of pediatric patients with hepatitis a virus hepatitis signs and symptoms non-jaundiced (45) no. (%) jaundiced (100) no. (%) total [no. (%)] jaundice 0 100 (100) 100 (69) fever 45 (100) 95 (95) 140 (97) abdominal pain 39 (87) 95 (95) 134 (92.4) choluria 45 (100) 100 (100) 145 (100) acolia 6 (13) 15 (15) 21 (14.5) malaise 24 (53) 45 (45) 69 (47.6) vomiting 30 (66.7) 60 (60) 90 (62.1) headache 6 (13) 20 (20) 26 (17.9) diarrhea 3 (6.7) 15 (15) 18 (12.4) epistaxis 0 5 (5) 5 (3.5) hepatomegaly 45 (100) 100 (100) 145 (100) splenomegaly 3 (6.7) 10 (10) 13 (9) ascites 6 (13) 5 (5) 11 (7.6) fig. 1 ratio of jaundice to non-jaundice [no. (%)] presentation in children with hepatitis a viral hepatitis. 142 j contemp med sci | vol. 4, no. 3, summer 2018: 140–143 acute hepatitis a viral hepatitis among pediatric patients original bashar sahib khalaf et al. the likelihood of symptomatic illness from hav infection is directly related to age. young children who have hepatitis a is often asymptomatic or simply manifest signs and symptoms of viral gastroenteritis without icterus. in contrast, older children and adolescents have a sudden onset of headache, fever, and general malaise followed by the onset of jaundice, abdominal pain, nausea, vomiting and anorexia. in children under 6 years of age, most (70%) infections are asymptomatic. in adults and older children, infection is generally symptomatic, with jaundice occurring in more than 70% of patients. it occasionally yields fulminant hepatitis a.2,3,26,27 spontaneous recovery within a few weeks occurs in a majority of individuals with acute viral hepatitis, and without any remaining consequences. however, severe form of the disease may occur in some people, as a complication of illness.28 significantly, 80% of symptomatic patients get hepatomegaly, while, to a less common outcomes include cervical lymphadenopathy, splenomegaly, arthritis, a leukocytoelastic vasculitis and, rarely, evanescent rash. people those with chronic liver disease are not at increased vulnerability to infection but are at increased risk of acquiring fulminant hepatitis a.29 hav seroprevalence varies from one state to the other, this occur according to the standard of living and socioeconomic factors. this infection is distributed worldwide and is inversely proportional to the levels of personal hygiene and environmental sanitation. in the developing nations where hygiene and sanitation are unsatisfactory, especially those belonging to lower socioeconomic group, approximately 100% of the population is infected, this can be indicated by the presence of antibodies in early life and obvious infection in adults is rare.30 egypt, considered as an area of high endemicity for hav infection, with noticeable economic, sanitary and hygiene improvements have occurred in recent years, chiefly in urban regions.31 enhancements of the living parameters may lead to changes in the hav epidemiology, with a reduction in the antibody prevalence among children; accordingly a significant proportion of the adult and adolescent population will be at risk of infection.32 the disease is usually self-limited, and the treatment is supportive. there is no certain food type with a major effect on the outcomes of patients with acute hepatitis a. symptomatic treatment is targeted and needed at particular situation and symptoms. in addition to increased intake of fluid which is necessary to prevent dehydration in the case of diarrhea and emesis, also need bed rest.7 sometimes intravenous fluids may be essential, according to the severity of illness. in practice, medications that are toxic to the liver it is prudently to be limited, and usage of acetaminophen table 3. types of management needed by pediatric patients with hav managements non-jaundice (45) no. (%) jaundice (100) no. (%) total [no. (%)] pre-hospital management none 12 (26.7) 30 (30) 42 (29) antibiotics 18 (40) 50 (50) 68 (47) symptomatic 36 (80) 65 (65) 101 (70) hospital management symptomatic 36 (80) 75 (75) 111 (77) antibiotics 39 (86.7) 95 (95) 134 (92.4) vitamin k 15 (33.3) 50 (50) 65 (45) table 2. distribution of hepatitis a pediatric patients in relation with different demographic parameters variables non-jaundice (45) (no.) jaundice (100) (no.) no. (%) p-value age (years) 1–5 30 35 44.8 (65) 0.0001 5–10 15 65 55.2 (80) sex male 27 60 60 (87) 1 female 18 40 40 (58) residence rural 30 35 44.8 (65) 0.0001 urban 15 65 55.2 (80) family history yes 3 45 33.1 (48) 0.0001 no 42 55 66.9 (97) socioeconomic status low 21 30 35.2 (51) 0.054 mod-high 24 70 64.8 (94) personal hygiene bad 27 20 32.4 (47) 0.0001 good 18 80 67.6 (98) nutritional status bad 3 20 15.9 (23) 0.030 good 42 80 84.1 (122) chronic drugs usage yes 1 1 1.38 (2) 0.559 no 44 99 98.6 (143) bashar sahib khalaf et al. 143j contemp med sci | vol. 4, no. 3, summer 2018: 140–143 original acute hepatitis a viral hepatitis among pediatric patients should be thoughtfully monitored in children to limit serious potential complications; no available official guidelines at this time.33 hospitalization is rarely required except when acute hepatic failure develop. evidence of hepatic injury [inr > 1.5 and/or pt > 15 with encephalopathy, or inr > 2.0 and/or pt > 20 with or without encephalopathy]. these measures should be achieved within 8 weeks from the onset of illness, and the above described coagulopathy (prolonged prothrombin time and/or inr) should be unresponsive to therapy with vitamin k. at this time, aggressive supportive therapy is required, and should be transferred to a center for performing liver transplantation.34,35 conclusion hepatitis a viral infection still to be a major health problem in developing countries and it is a hyper-endemic in iraq. conflict of interest none. n references 1. rosenthal p. hepatitis a: a preventable threat. j pediatr gastroenterol nutr. 2002;35:595–596. 2. davidson lj, george le, kalevitch mv, rudd dp. calming the panic over hepatitis a. nursing 2004;34:45–47. 3. su cw, wu jc, huang ys, huo ti, huang yh, lin cc, et al. comparison of clinical manifestations and epidemiology between acute hepatitis a and acute hepatitis e in taiwan. j gastroenterol hepatol. 2002;17:1187–1191. 4. world health organization (who). hepatitis a surveillance and control. available from: http://www.who.int/csr/disease/hepatitis/ whocdscsredc2007/en/index4.html. accessed dec 16 2009. 5. leach ct. hepatitis a in the united states. pediatr infect dis j. 2004;23: 551–552. 6. dentinger cm. emerging infections: hepatitis a. am j nurs. 2009;109:29–33. 7. denson la. postnatal infections, part 1c: other viral infections. in: walker wa. pediatric gastroenterology disease: pathophysiology, diagnosis, management, 4th ed. hamilton, on: bc decker, 2004, pp. 1170–1178. 8. duval b, de serres g, ochnio j, scheifele d, gîlca v. nationwide canadian study of hepatitis a antibody prevalence among children eight to thirteen years old. pediatr infect dis j. 2005;24:514–519. 9. poovorawan y, chatchatee p, chongrisawat v. epidemiology and prophylaxis of viral hepatitis: a global perspective. j gastroenterol hepatol. 2002;17: s155–s166. 10. marsano ls. hepatitis. prim care clin office pract. 2003;30:81–107. 11. turky am, akram w, al-naaimi as, omer ar, alrawi jr. analysis of acute viral hepatitis (a and e) in iraq. global j health sci. 2011;3:70–76. 12. salama i, samy s, shaaban f, hassanin a, abou-ismail l. seroprevalence of hepatitis a among children of different socioeconomic status in cairo. east mediterr health j. 2007;13:1256–1264. 13. el-zimaity dm, hyams kc, imam iz, watts dm, bassily s, naffea ek, et al. acute sporadic hepatitis ein an egyptian pediatric population. am j trop med hyg. 1993;48:372–376. 14. darwish ma. high seroprevalence of hepatitis a, b, c and e virus in residents in an egyptian village in the nile delta: a pilot study. am j trop med hyg. 1996;54:554–558. 15. zaytoun ss. immunity status against hav among school children in alexandria. alexandria, department of pediatrics, university of alexandria. m sc thesis; 2003. 16. antaki n, kebbewar mk. hepatitis a seroprevalence rate in syria. trop doct. 2000;30:99–101. 17. yassin k, awad r, awad r, queder a, laaser u. the epidemiology of hepatitis a infection in palestine: a universal vaccination programme is not yet needed. epidemiol infect. 2001;127:335–339. 18. taghavi sa, hosseini asl mk, talebzadeh m, eshraghian a. seroprevalence study of hepatitis a virus in fars province, southern iran. hepat mon. 2011;11:285–288. 19. blum he. history and global burden of viral hepatitis. dig dis. 2016;34: 293–302. 20. rafeey m, shoaran m. prevalence and risk factors of hepatitis a in children in tabriz, iran. j anal res clin med. 2014;2:183–186. 21. kazemi sa, mahram m, koosha a, amirmoghaddami hr. seroprevalence of hepatitis a in 7–10 year-old children. iran j pediatr. 2007;17:47–51. 22. ataei b, javadi aa, nokhodian z, kassaeian n, shoaei p, farajzadegan z, et al. hav in isfahan province: a population-based study. trop gastroenterol. 2008;29:160–162. 23. pharm b, duval b, de serres g, gilca v, tricco ac, ochnio j, et al. seroprevalence of hepatitis a infection in a low endemicity country: a systematic review. bmc infect dis. 2005;5:56. 24. cho se, kim y. seroepidemiology of hepatitis a in south korea: a nationwide study by the eone reference laboratory. j epidemiol. 2013;23:270–274. 25. lee a, lim hs, nam cm, song sm, yoon hr, lee kr. an epidemiological analysis of hepatitis a virus serologic markers during the recent four years in korea. korean j lab med. 2009;29:563–569. 26. joshi n, yr nk, kumar a. age related seroprevalence of antibodies to hepatitis a virus in hyderabad, india. trop gastroenterol. 2000;21: 63–65. 27. rakesh p, sherin d, sankar h, shaji m, subhagan s, salila s. investigating a community-wide outbreak of hepatitis a in india. j glob infect dis. 2014;6:59–64. 28. schreiber ra, pashankar d. jaundice in older children and adolescents. pediatr rev. 2001;22:219–226. 29. jeong sh, lee hs. hepatitis a: clinical manifestations and management. intervirology. 2010;53:15–19. 30. ryan kj, ray cg. sherris medical microbiology, 4th ed., mcgraw hill, 2004, pp, 541–544. 31. el-zanaty f, way aa. egypt demographic and health survey 2000. calverton, maryland, usa, ministry of health and population, egypt, national population council and orc marco, 2001. 32. mall ml, rai rr, philip m, et al. seroepidemiology of hepatitis a infection in india: changing pattern. indian j gastroenterol. 2001;20:132–135. 33. koslap-petraco mb, shub m, judelsohn r. hepatitis a: disease burden and current childhood vaccination strategies in the united states. j pediatr health care. 2008;22:3–11. 34. narkewicz mr, dell olio d, karpen sj, murray kf, schwarz k, yazigi n, et al. pattern of diagnostic evaluation for the causes of pediatric acute liver failure: an opportunity for quality improvement. j pediatr. 2009;155:801–806. e1. 35. lee wm, stravitz rt, larson am. introduction to the revised american association for the study of liver diseases position paper on acute liver failure 2011. hepatology. 2012;55:965–967. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201805 77j contemp med sci | vol. 5, no. 2, march–april 2019: 77–81 original effects of eryngium caucasicum extract on testosterone, inflammation and oxidative status of nicotinamide-streptozotocin induced type-2 diabetes in male rats masood afshari,a ali reza malayeri,b and majid mohammadshahic* adepartment of nutrition, jundishapour university of medical sciences, ahvaz, iran. bmedical plant research center, jundishapour university of medical sciences. ahvaz, iran. cdepartment of nutrition, hyperlipidemia research center, jundishapour university of medical sciences. ahvaz, iran. *correspondence to majid mohammadshahi (email: shahi334@gmail.com). (submitted: 04 december 2018 – revised version received: 27 december 2018 – accepted: 23 january 2018 – published online: 26 april 2019) objective regarding the unfavorable side effects of anti-diabetic drugs including peripheral edema, gastrointestinal discomfort and hypoglycemia, using medicinal plants due to their useful contents such as flavonoids, alkaloids, glycopeptides, terpenoides, phenolic compounds and other constituents with antioxidant and anti-inflammatory effects, is encouraged in treatment of diabetes mellitus. thus, the hypothesis was that eryngium caucasicum extract will decrease inflammation and oxidative stress in nicotinamide-streptozotocin induced type 2 diabetes mellitus model in wistar rats. methods in this study, sixty adult male wistar rats (150–250 g) were randomly allocated into six groups (n = 10) including: (1) healthy control, (2) diabetic control, (3) diabetic rats which received sitagliptine, (4–6) diabetic rats which received 100, 200 and 300 mg/kg of e. caucasicum extract oral gavages for 30 days. eventually, total antioxidant capacity, vitamin b12, malondyaldehyde, interleukin-6, high sensitive c-reactive protein and testosterone serum levels were measured. results administration of e. caucasicum in type 2 diabetes mellitus animal model did not change serum vitamin b12 and animal weight compared with control groups. total antioxidant capacity and malondyaldehyde improved in all doses of e. caucasicum; in the highest doses, the total antioxidant capacity was higher than sitagliptine group. interleukin-6 and high sensitive c-reactive protein both decreased in all doses of e. caucasicum. administration of e. caucasicum extract improved testosterone serum level only in highest dose of e. caucasicum extract. since the highest dose showed the highest antioxidant and anti-inflammatory effects; dose-responses of antioxidant and antiinflammatory effects are suggested. conclusion in conclusion, we showed that administration of e. caucasicum in t2dm animal model has antioxidant and anti-inflammatory effects. however, in future studies other dose-escalating intervention must be performed. also toxicity in diabetes must be elucidated in future studies. keywords eryngium caucasicum extract, testosterone, inflammation, oxidative status introduction diabetes mellitus (dm) is a multi-systemic endocrine disorder with constant hyperglycemia due to either absolute or relative defective insulin secretion, insulin resistance or both.1 the prevalence of dm is rising all over the world in both developed and developing countries. about 90% of all diabetic cases are type 2 diabetes mellitus (t2dm). according to who, the global prevalence of t2dm will increase to 366 million in 2030.2 dm is reported to devote 7–13% of the healthcare budget of worldwide and a major challenge to healthcare systems.3 long-term hyperglycemia is contributed to many chronic end-organ microvascular and macrovascular damages in the eyes, kidneys and the brain. also, t2dmrelated cardiovascular diseases can increase morbidity and mortality among patients with dm.4 mild to moderate proinflammatory status has been implicated in dm. this setting is proposed as a link between disease progression and its complications. mounting evidence showed increased cytokine level and immune cell infiltration in pancreatic cells in dm.5 in addition epidemiological studies support this data that inflammatory markers like interleukin-6 (il-6) are associated with development of dm.6 il-6 reported as a mediator which act between insulin-sensitive tissues, and pancreatic islets to adapt to changes in insulin demand.7 prolonged hyperglycemia along with proinflammatory settings, disturbs normal balance of oxidant-antioxidant capacity within the cells, which results in oxidative stress and injury, beta cell death and/or de-differentiation to glucagon-producing cells.8 despite current advances in anti-diabetic drugs, their use is complicated due to the unfavorable side effects like peripheral edema, gastrointestinal discomfort and hypoglycemia.9 recently the use of medicinal plants for the sufferers of dm is encouraged because of their perceived effectiveness, fewer side effects and relatively low costs. it has been estimated that more than 1000 medicinal plants are used as anti-diabetic remedies.10 several phytoconstituents in herbal remedies reported as useful agents against dm. these include flavonoids, alkaloids, glycopeptides, terpenoides and phenolic compounds and other constituents which show fasting blood glucose (fbs) lowering effects.11 the eryngium genus, as the largest genus of the family apiaceae, consists of more than 250 flowering species all over the world.12 eryngium caucasicum which is known as eryngo has been cultivated in middle-east countries like iran and turkey.13 in northern iran it has been widely used as different foodstuffs like pickles. apiaceae have several therapeutic uses as diuretic, stone inhibitor, expectorant and antinociceptive.13 other uses in turkish folk medicine are against various inflammatory disorders, edema, sinusitis, urinary infections or inflammations etc.14 in essence, e. caucasicum reported to issn 2413-0516 78 j contemp med sci | vol. 5, no. 2, march–april 2019: 77–81 effects of eryngium caucasicum extract on testosterone, inflammation and oxidative status of nicotinamide-streptozotocin original m. afshari et al. have numerous therapeutic properties comprising strengthening generative power, diuretic, lenitive and appetizer.15 antioxidant and potent free radical scavenging activity of e. caucasicum trautv leaves have been reported. in addition it is reported to have antihypoxic and reno-protective effects.16 anti-inflammatory activities of eryngium species growing in turkey has been evaluated in vivo. aerial parts and roots reported to possess many activities.17 several bioactive compounds, mainly phenolic compounds and terpenoids have been purified from eryngium species which include triterpenoid saponins, flavonoids, coumarins and acetylenes.18 in this study we aimed to evaluate anti-inflammatory and anti-oxidative potential and possible effects of e. caucasicum extract on testosterone in nicotinamide-streptozotocin (na-stz) induced t2dm model in rats. materials and methods plant material and extract preparation fresh leaves of e. caucasicum were obtained from mazandaran province, iran and authenticated scientifically by the botany department of ahvaz jundishapur university of medical sciences (ajums), ahvaz, iran. voucher specimens are deposited in the herbarium department of biology, university of mazandaran, iran (no 1442). the plant leaves were desiccated in shade and then crushed and powdered by grinder. a total of 300 g of the powder of e. caucasicum leaves were soaked in 1200 ml of an ethanol and distilled water mixture (70–30) and stocked for 72 h at room temperature. the mixture filtered through whatman filter papers (no. 1) and then centrifuged at 3500 rpm for 20 min. condensation conducted by rotary evaporator. finally, supernatant was dried at 37°c, and the obtained semisolid mass was stocked at 4°c until injection. experimental animals sixty adult male wistar rats (150–250 g) were purchased from animal house of ajums. all experimental animals were kept in standard cages under approved conditions for animal procedures (temperature 22 ± 2°c with a 12–12 h light–dark cycle). experimental animals were allowed to access normal commercial chow and tap water. maintenance and care of experimental animals complied with the national research council of the national academic. induction of type 2 diabetes mellitus in this study, type 2 diabetes was induced by intraperitoneal (ip) injection of na (120 mg/kg body weight in normal saline) (sigma-aldrich, usa) 15 min before a single dose of stz (55 mg/kg body weight; dissolved in citrate buffer (0.1m), ph 4.5) (sigma-aldrich, usa). animals suffer from fasting for 8–12 h before t2dm induction. the development of t2dm was evaluated by fbs in animals. fbs was measured before and 72 h after injection to confirm diabetes induction. fbs more than 250 mg/dl were considered diabetic. only those animals with confirmed diabetes were used for following experiments. experimental protocol after 2 weeks of adaptation, rats were divided into the following groups (n = 10 in each group): • group 1: six healthy control (0.5 ml/kg normal saline oral gavage). • group 2: six diabetic control group (0.5 ml/kg normal saline oral gavage). • group 3: six t2dm + sitagliptine (10 mg/kg oral gavage). • group 4: six t2dm + e. caucasicum extract (100 mg/kg oral gavage). • group 5: six t2dm + e. caucasicum extract (200 mg/kg oral gavage). • group 6: six t2dm + e. caucasicum extract (300 mg/kg oral gavage). all groups treated once a day for 28 sequential days with sitagliptine or e. caucasicum extract. sitagliptine was dissolved in distilled water. body weight was recorded weekly during the experiment. at 24 h after the last injection, mice were kept fasted overnight. all animals were killed under ether anesthesia. blood samples were obtained by heart puncture. samples were poured into centrifuge tubes and centrifuged at 3500 rpm for 20 min. serum samples were refrigerated at −70°c refrigerator until biochemical analysis. biochemical assessments fasting blood glucose were assessed by an elegance glucometer (ct-x10, convergent technologies, germany) using the lateral tail vein of the animals on the first and last days of the experiment. total antioxidant capacity (tac) was assessed by spectrophotometer and commercial kits (randox, england). also malondyaldehyde (mda) serum level was assessed through thiobarbiturate method. following factors were assessed by elisa method: il-6 (behpadteb, iran), testosterone (monobiol, usa). circulating high sensitive c-reactive protein (hs-crp) was evaluated by turbidimetry method (biosystem, barcelona, spain). serum vitamin b12 was evaluated by radioimmunoassay using gamma counter. statistical analysis all data were expressed as mean ± standard error of mean (sem). results were analyzed by one-way analysis of variance (anova), followed by post-hoc test [least significant difference (lsd)]. all statistical analysis were performed by spss statistics v. 17.01 (spss inc., chicago, usa). results effects of hydro-alcoholic extract e. caucasicum on body weight, fbs and serum vitamin b12 our results indicated that stz-na-induced diabetes did not change body weight in diabetic control group (p-value = 0.634). administration of sitagliptin in diabetic rats decreased body weight significantly compared with diabetic control (p-value = 0.008) and healthy control group (p-value = 0.028). however, e. caucasicum administration in 300 mg/kg increased body weight significantly compared with sitagliptin group (p-value = 0.002). induction of diabetes significantly increased fbs in diabetic rats compared with healthy control group (p-value = 0.009). sitagliptin also decreased fbs in diabetic rats significantly (p-value = 0.004). furthermore, only high doses including 200 and 300 mg/kg decreased fbs in m. afshari et al. 79j contemp med sci | vol. 5, no. 2, march–april 2019: 77–81 original effects of eryngium caucasicum extract on testosterone, inflammation and oxidative status of nicotinamide-streptozotocin distinct period (p-value = 0.038 and 0.004 respectively). although a dose-dependent manner reported in reducing fbs following increasing doses of e. caucasicum extract as outlined in table 1. furthermore, serum vitamin b12 revealed no significant changes in any of experimental groups during intervention period. effects of hydro-alcoholic extract e. caucasicum on inflammatory biomarkers induction of diabetes using stz-na increased serum hs-crp in diabetic control group compared with healthy control group. sitagliptine significantly lowered hs-crp serum level (p-value = 0.001). treatment with plant extract also lowered serum hs-crp level in dose-dependent manner as shown in table 2. interleukin-6 serum level increased significantly after diabetes induction. administration of sitagliptine significantly improved il-6 serum level. e. caucasicum extract also lowered il-6 in a dose-dependent manner as depicted in table 2. effects of hydro-alcoholic extract e. caucasicum on tac and mda malondyaldehyde serum level increased after induction of diabetes in diabetic control group. although sitagliptine improved mda serum level, did not improve it as efficiently as e. caucasicum extract. administration of herb extract lowered serum level of mda in a dose-dependent manner. mda also in 200 and 300 mg/kg were significantly lower than sitagliptine (p-value = 0.023 and 0.006 respectively). total antioxidant capacity also decreased after t2dm induction. however, administration of sitagliptine improved tac significantly (p-value = 0.030). all three doses of e. caucasicum extract improved tac but was not as efficient as sitagliptine (p-value for all doses <0.001). effects of hydro-alcoholic extract e. caucasicum on serum testosterone level as shown in figure 1, all experimental diabetic groups showed lower testosterone serum level compared with healthy control group. sitagliptine treatment did not improve testosterone serum level in diabetic rats. but 300 mg/kg of plant extract increased serum testosterone level significantly. this observation showed dose-dependent effects of e. caucasicum extract as shown in figure. discussion the results of this study indicate that e. caucasicum exerted beneficial effects on increasing tac, decreasing mda, il-6, hs-crp and improving testosterone serum level without any changes on serum vitamin b12 and animals’ body weights in stz-na rats, t2dm animal model compared with control groups. tac improved in all doses of e. caucasicum although it was higher than sitagliptine group in the highest dose. mda also improved in all groups after administration of e. caucasicum. all of these observations showed the antioxidant effects of e. caucasicum in diabetic rats. mounting evidence has pointed out several pharmacological effects of the genus eryngium including antioxidant, anti-hypoxic and free radical scavenging effects.19 similarly, wang et al.18 and mirjana et al.20 reported the same findings. antioxidant activity of eryngium genius contributed to its aerial parts, extracted oil, roots, fruits etc.21 the antioxidant effects of eryngium extracts were evaluated using 2,2-diphenyl-l-1-picrylhydrazil and ferric reducing antioxidant power assays which showed significant antioxidant activities.22 in essence, e. caucasicum fractions demonstrated different levels of antioxidant and anti-hemolytic activities in different models. hypoxia mediates the production of nitric oxide (no) and its radicals and also ros.13 no reported to modulate iron catalyzed oxidation reactions such as fenton reaction, which generates potent oxidants such as the hydroxyl radical (oh˙). no scavenging may suggest likelihood to counteract hypoxia.23 the mechanisms by which no may block lipid peroxidation needed to be clarified, however, one plausible mechanism linked to the potential of no to cease propagation of lipid peroxidation reactions.24 eryngium table 2. effect of hydro-alcoholic extract of eryngium caucasicm (e.c.) on inflammatory and oxidative stress markers in nicotinamide-streptozotocin-induced diabetic wistar rats healthy control diabetic control diabetes + sitagliptin diabetes + 100 mg/kg e.c. diabetes + 200 mg/kg e.c. diabetes + 300 mg/kg e.c. tac (µmol/l) 0.931 + 0.135 0.371 + 0.101a 0.611 + 0.348a,b 0.776 + 0.127b 0.814 + 0.181 b 0.872 + 0.172b,c mda (µmol/l) 1.57 + 0.65 3.98 + 1.02a 2.88 + 0.83a,b 1.97 + 0.71b 1.83 + 0.96 b 1.56 + 0.71b il-6 (pg/ml) 66.68 + 11.80 124.59 + 32.09a 89.90 + 12.64a,b 101.47 + 6.16a,b 101.80 + 10.84a,b 90.25 + 11.45a,b hs-crp (µmol/l) 7047 + 1.29 14.62 + 2.49a 10.14 + 2.60a,b 11.24 + 2.79a,b 10.77 + 1.88a,b 8.94 + 1.96b ap < 0.05 compared with the control group. bp < 0.05 compared with the diabetic control group. cp < 0.05 compared with the diabetic animals received sitagliptin. results are expressed as mean + sem (standard error of mean), one-way anova and post-hoc lsd test. e.c.: eryngium caucasicm; fbs: fasting blood glucose; mda: malondialdehyde; tac: total antioxidant capacity; hs-crp: high sensitivity c-reactive protein; il-6: interleukin-6. table 1. effect of hydro-alcoholic extract of eryngium caucasicm (e.c.) on body weight, fasting blood glucose and serum vitamin b12 in nicotinamide-streptozotocin-induced diabetic wistar rats healthy control diabetic control diabetes + sitagliptin diabetes + 100 mg/kg e.c. diabetes + 200 mg/kg e.c. diabetes + 300 mg/kg e.c. weight (g) 221.08 + 6.96 216.52 + 7.04 193.67 + 6.45a,b 240.28 + 6.87c 209.30 + 6.55 211.12 + 6.35 fbs (mg/dl) 107.50 + 34.5 157.28 + 59.00a 104.57 + 22.32b 130.16 + 18.49 120.42 + 16.25b 103.85 + 17.72b serum b12 (pg/ml) 119.60 + 17.17 129.12 + 26.85 128.13 + 18.88 127.93 + 19.07 134.16 + 20.70 133.46 + 20.53 ap < 0.05 compared with the control group. bp < 0.05 compared with the diabetic control group. cp < 0.05 compared with the diabetic animals received sitagliptin. results are expressed as mean + sem (standard error of mean), one-way anova and post-hoc lsd test. e.c.: eryngium caucasicm; fbs: fasting blood glucose. 80 j contemp med sci | vol. 5, no. 2, march–april 2019: 77–81 effects of eryngium caucasicum extract on testosterone, inflammation and oxidative status of nicotinamide-streptozotocin original m. afshari et al. species reported to contain several phenolic compounds, flavonoids and coumarins which their antioxidant activities have been documented.25 because of high polyphenols and flavonoids contents of e. caucasicum extract, their efficient no scavenging activities have been reported.23 lipid peroxidation marker, mda, in our study significantly decreased in dose dependent manner; the effect on no production could be one of possible mechanisms. the correlation between total phenolic contents and antioxidant activity has been widely studied. the important point is that the no scavenging effect was mostly dose-dependent in previous studies.16 we also found almost dose-dependent effect regarding antioxidant activity of e. caucasicum in diabetic rats. we showed anti-inflammatory effects of e. caucasicum extract in diabetic rats. il-6 and hs-crp both decreased in all doses of e. caucasicum. these findings were in agreement with findings of dawilai et al.26 and jaghabir27 in which anti inflammatory effects of e. caucasicum extract were reported. alteration in serum vitamin b12 could be due to hepatic injury in diabetic rats.28 previous studies in humans reported lowered serum b12 in diabetes;29 although e. caucasicum due to its antioxidant effects could be hepato-protective, serum vitamin b12 could be lost via urine which has not examined in this study. our study did not show any significant effect in serum vitamin b12. effects of eryngium species (especially roots) against different inflammatory disorders are recognized in the traditional medicines worldwide; for instance, eryngium campestre as anti-edema30 or eryngium creticum for inflammatory wounds31 or against kidney and urinary tract inflammations and edema.32 despite widespread uses of eryngium species in the treatment of inflammatory disorders worldwide, the number of scientific papers investigating the anti-inflammatory activity potential are scarse; like eryngium yuccifolium aerial parts,33 eryngium maritimum rhizomes,34 eryngium bilardieri aerial parts and roots,35 eryngium foetidum leaves.36 ethanol and water extract of roots and aerial parts of eight eryngium species were investigated for their inhibitory effects on several inflammatory models in mice. results showed different degrees of anti-inflammatory activity for eryngium species.17 a significant anti-inflammatory activity was reported in the n-butanol extract e. billardieri, which mainly contains saponins.35 the myeloperoxidase activity as a marker of polymorphonuclear cells migration to the inflamed tissues reported to be reduced strongly after e. foetidum extract administration.37 no is also implicated in inflammation38 and as mentioned recently, e. caucasicum showed anti-scavenging effect against no. interestingly e. foetidum leaves extract showed a significant and dose-dependent anti-inflammatory activity, orally against carrageenan-induced rat paw edema.39 here, we showed dose-dependent anti-inflammatory effects of e. caucasicum in diabetic rats. additionally, hs-crp and il-6 decreased significantly in a dose-dependent manner. administration of e. caucasicum extract improved testosterone serum level only in the highest doses of e. caucasicum extract. reduced serum testosterone level reported in diabetic rats due to reduced amount and functioning of leydig cells40 and also absence of normal serum insulin levels because, insulin plays a positive role in leydig cell function and testosterone production.41 furthermore, inhibition of steroidogenesis because of elevated inflammatory biomarkers and reduction in the activity of antioxidant enzymes also has been implicated.42 we showed that e. caucasicum significantly improved serum testosterone level. since insulin acts as an anti-apoptotic factor which is able to regulate testicular apoptosis; thus reduction in insulin serum levels leads to reduced testosterone serum level. insulin secretary effects of e. caucasicum extract could improve testosterone levels in treated groups. another probable mechanism is that increasing of ros and oxidative stress can prevent production of androgens.43 the protective effects of e. caucasicum extract could be due to antioxidant and anti-inflammatory effects. some researchers also suggested that flavonoids and saponins have positive effects on androgen production and bioavailability. saponins increase testosterone generation through influencing on pituitary leuteizing hormone secretion on leydig cells.44 therefore, it can be inferred that observed effects of e. caucasicum extract on serum testosterone levels are related to the presence of these compounds. conclusion in conclusion, we showed that administration of e. caucasicum in t2dm animal model has antioxidant and antiinflammatory effects. e. caucasicum decreased serum il-6, mda and hs-crp and also increased tac. highest doses of e. caucasicum fig. 1 effect of hydro-alcoholic extract of eryngium caucasicm (e.c.) on testosterone serum level in nicotinamide-streptozotocin induced diabetic wistar rats. values outlined as mean (sem; standard error of mean), one-way anova and post-hoc lsd test. dia: diabetic, e.c.: eryngium caucasicm, sig: sitagliptin, 100: 100 mg/kg hydro-alcoholic extract e.c., 200: 200 mg/kg hydro-alcoholic extract e.c., 300: 300 mg/ kg hydro-alcoholic extract e.c. ap < 0.05 compared with the control group, bp < 0.05 compared with the diabetic control group. m. afshari et al. 81j contemp med sci | vol. 5, no. 2, march–april 2019: 77–81 original effects of eryngium caucasicum extract on testosterone, inflammation and oxidative status of nicotinamide-streptozotocin also improved testosterone serum level. on the whole, it seems that e. caucasicum influences on inflammation and oxidative stress in diabetes rat fellow a dose-dependent manner, although higher doses showed better effects. however, in future studies other doseescalating intervention must be performed; in which higher doses evaluated to clarify the exact dose-response effects of eryngo. also toxicity in diabetes must be elucidated in future studies. conflict of interest none.  references 1. defronzo ra, ferrannini e, groop l, henry rr, herman wh, holst jj, et al. type 2 diabetes mellitus. nat rev dis primers. 2015;1:15019. 2. centers for disease control and prevention. national diabetes statistics report, 2017. atlanta, ga: centers for disease control and prevention, 2017. 3. zhang p, zhang x, brown j, vistisen d, sicree r, shaw j, et al. global healthcare expenditure on diabetes for 2010 and 2030. diabetes res clin pract. 2010;87:293–301. 4. chan gc, tang sc. diabetic nephropathy: landmark clinical trials and tribulations. nephrol dial transplant. 2015;31:359–368. 5. akash ms, rehman k, chen s. role of inflammatory mechanisms in pathogenesis of type 2 diabetes mellitus. j cell biochem. 2013;114:525–531. 6. pradhan ad, manson je, rifai n, buring je, ridker pm. c-reactive protein, interleukin 6, and risk of developing type 2 diabetes mellitus. jama. 2001;286:327–334. 7. donath my, dalmas é, sauter ns, böni-schnetzler m. inflammation in obesity and diabetes: islet dysfunction and therapeutic opportunity. cell metab. 2013;17:860–872. 8. kitamura t. the role of foxo1 in β-cell failure and type 2 diabetes mellitus. nat rev endocrinol. 2013;9:615–623. 9. ghorbani a. best herbs for managing diabetes: a review of clinical studies. braz j pharm sci. 2013;49:413–422. 10. rao mu, sreenivasulu m, chengaiah b, reddy kj, chetty cm. herbal medicines for diabetes mellitus: a review. int j pharmtech res. 2010;2:1883–1892. 11. ji hf, li xj, zhang hy. natural products and drug discovery: can thousands of years of ancient medical knowledge lead us to new and powerful drug combinations in the fight against cancer and dementia? embo rep. 2009;10:194–200. 12. calviño ci, martínez sg, downie sr. the evolutionary history of eryngium (apiaceae, saniculoideae): rapid radiations, long distance dispersals, and hybridizations. mol phylogenet evol. 2008;46:1129–50. 13. nabavi sm, nabavi sf, alinezhad h, zare m, azimi r. biological activities of flavonoid-rich fraction of eryngium caucasicum trautv. eur rev med pharmacol sci. 2012;16:81–87. 14. wörz a. on the distribution and relationships of the south-west asian species of eryngium l. (apiaceae-saniculoideae). turk j bot. 2004;28:85–92. 15. trautv p. plants for a future, edible, medicinal and useful plants for a healthier world, eryngium caucasicum. 2004. 16. motallebi riekandeh s, mazandarani m, ebrahimzadeh ma, zargari m. antioxidant activities of eryngium caucasicum inflorescence. eur rev med pharmacol sci. 2016;20:946–949. 17. küpeli e, kartal m, aslan s, yesilada e. comparative evaluation of the antiinflammatory and antinociceptive activity of turkish eryngium species. j ethnopharmacol. 2006;107:32–37. 18. wang p, su z, yuan w, deng g, li s. phytochemical constituents and pharmacological activities of eryngium l. (apiaceae). 2012;3:99–120. 19. ben lajnef h, ferioli f, pasini f, politowicz j, khaldi a, filippo d’antuono l, et al. chemical composition and antioxidant activity of the volatile fraction extracted from air-dried fruits of tunisian eryngium maritimum l. ecotypes. j sci food agric. 2018;98:635–643. 20. marčetić md, petrović sd, milenković mt, niketić ms. composition, antimicrobial and antioxidant activity of the extracts of eryngium palmatum pančić and vis. (apiaceae). central eur j biol. 2014;9:149–155. 21. erdem sa, nabavi sf, orhan ie, daglia m, izadi m, nabavi sm. blessings in disguise: a review of phytochemical composition and antimicrobial activity of plants belonging to the genus eryngium. daru. 2015;23:53. 22. merghache d, boucherit-otmani z, merghache s, chikhi i, selles c, boucherit k. chemical composition, antibacterial, antifungal and antioxidant activities of algerian eryngium tricuspidatum l. essential oil. nat prod res. 2014;28:795–807. 23. khalili m, dehdar t, hamedi f, ebrahimzadeh ma, karami m. antihypoxic activities of eryngium caucasicum and urtica dioica. eur rev med pharmacol sci. 2015;19:3282–3285. 24. ebrahimzadeh ma, nabavi sf, nabavi sm, pourmorad f. nitric oxide radical scavenging potential of some elburz medicinal plants. afr j biotechnol. 2010;9:5212–5217. 25. rjeibi i, saad ab, ncib s, souid s. phenolic composition and antioxidant properties of eryngium maritimum (sea holly). j coast life med. 2017;5:212–215. 26. dawilai s, muangnoi c, praengamthanachoti p, tuntipopipat s. antiinflammatory activity of bioaccessible fraction from eryngium foetidum leaves. biomed res int. 2013;2013:958567. 27. jaghabir m. hypoglycemic effects of eryngium creticum. arch pharm res. 1991;14:295–297. 28. afrin r, arumugam s, soetikno v, thandavarayan r, pitchaimani v, karuppagounder v, et al. curcumin ameliorates streptozotocin-induced liver damage through modulation of endoplasmic reticulum stress-mediated apoptosis in diabetic rats. free radic res. 2015;49:279–289. 29. akinlade ks, agbebaku so, rahamon sk, balogun wo. vitamin b12 levels in patients with type 2 diabetes mellitus on metformin. ann ib postgrad med. 2015;13:79–83. 30. leporatti ml, ivancheva s. preliminary comparative analysis of medicinal plants used in the traditional medicine of bulgaria and italy. j ethnopharmacol. 2003;87:123–142. 31. ali-shtayeh ms, yaghmour rm, faidi yr, salem k, al-nuri ma. antimicrobial activity of 20 plants used in folkloric medicine in the palestinian area. j ethnopharmacol. 1998;60:265–271. 32. gruenwald j, brendler t, jaenicke c. pdr for herbal medicines. thomson reuters corporation. 2007. 33. benoit ps, fong hh, svoboda gh, farmsworth nr. biological and phytochemical evaluation of plants. xiv. antiinflammatory evaluation of 163 species of plants. lloydia. 1976;39:160–171. 34. lisciani r, fattorusso e, surano v, cozzolino s, giannattasio m, sorrentino l. anti-inflammatory activity of eryngium maritimum l. rhizome extracts in intact rats. j ethnopharmacol. 1984;12:263–270. 35. yesilada e, tanaka s, tabata m, sezik e. the antiinflammatory activity of the fractions from eryngium billardieri in mice. phytother res. 1989;3:38–40. 36. garcía md, sáenz mt, gómez ma, fernández ma. topical antiinflammatory activity of phytosterols isolated from eryngium foetidum on chronic and acute inflammation models. phytother res. 1999;13:78–80. 37. mekhora c, muangnoi c, chingsuwanrote p, dawilai s, svasti s, chasri k, et al. eryngium foetidum suppresses inflammatory mediators produced by macrophages. asian pac j cancer prev. 2012;13:653–664. 38. nabavi sm, ebrahimzadeh ma, nabavi sf, bahramian f. in vitro antioxidant activity of phytolacca americana berries. pharmacologyonline. 2009;1:81–88. 39. sáenz mt, fernández ma, garcia md. antiinflammatory and analgesic properties from leaves of eryngium foetidum l. (apiaceae). phytother res. 1997;11:380–383. 40. khaneshi f, nasrolahi o, azizi s, nejati v. sesame effects on testicular damage in streptozotocin-induced diabetes rats. avicenna j phytomed. 2013;3:347–355. 41. lin t, haskell j, vinson n, terracio l. characterization of insulin and insulinlike growth factor i receptors of purified leydig cells and their role in steroidogenesis in primary culture: a comparative study. endocrinology. 1986;119:1641–1647. 42. kalyani rr, dobs as. androgen deficiency, diabetes, and the metabolic syndrome in men. curr opin endocrinol diabetes obes. 2007;14:226–234. 43. zhao yt, qi yw, hu cy, chen sh, liu y. advanced glycation end products inhibit testosterone secretion by rat leydig cells by inducing oxidative stress and endoplasmic reticulum stress. int j mol med. 2016;38:659–665. 44. rojas dp, pandey ak. natural compounds to counteract testosterone depletion in aging. j steroids horm sci. 2014;5:e112. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.04201903 319j contemp med sci | vol. 3, no. 12, autumn 2017: 319–325 research associated functional motor recovery induced by intracerebroventricular (icv) microinjection of wharton’s jelly mesenchymal stem cells following brain ischemia/reperfusion injury in rat: decreased dark neurons and bax gene expression in the cerebral cortex tahereh alizamir,a mohammad akbari,a tahmineh mokhtari,b,c mojtaba khaksarian,d and gholamreza hassanzadeha,e adepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. bresearch center of nervous system stem cells, department of anatomy, school of medicine, semnan university of medical sciences, semnan, iran. cdepartment of anatomy, school of medicine, semnan university of medical sciences, semnan, iran. ddepartment of physiology, faculty of medicine, lorestan university of medical sciences, khorramabad, iran. edepartment of neuroscience, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. correspondence to: gholamreza hassanzadeh (e-mail: hassanzadeh@tums.ac.ir). objectives stroke is a situation caused activation of some events leading to neuronal damage and death. the proteins of bcl-2-family are important in regulation of cell death and life. bax, as a bcl-2-interacting protein, is a member of this family which promotes apoptosis and cell death. some therapeutic approaches have been introduced for the treatment of ischemic brain injury. neuroprotection is one of the approaches for diminishing neurological deficits. we investigated the effects of intracerebroventricular (icv) injection of wharton’s jelly mesenchymal stem cells (wj-mscs) on the cortex of the brain in ischemic rats. methods in this study, we occlude the middle cerebral artery (mca) for induction of ischemic stroke in the brain. rats were classified in four groups of co, sh, mcao and mcao + wj-mscs. single dose of intraventricular microinjection of wj-mscs was injected by a hamilton syringe. for detecting behavioral outcomes in the rats, neurological examination was carried out. after 21 days, the animals were sacrificed and their brain tissues were removed for histopathological and molecular analysis. results icv microinjection of wj-mscs significantly prevented apoptosis and cell death compared with mcao group. a significant reduction in the level of bax gene expression was observed in the mcao + wj-mscs as group compared with co, sh and mcao groups (p < 0.05). h&e staining showed considerable reduction of dark neurons in mcao + wj-mscs group rather than co, sh and mcao groups (p < 0.05). conclusions the results of the current study suggest that icv microinjection of wj-mscs had neuroprotective effects on the brain cortex of ischemic rats by reduction of the bax gene expression level and the number of dark neurons. keywords wharton’s jelly mesenchymal stem cells, bax, dark neurons, cortex, stroke introduction ischemic stroke is a condition results from reduction of cerebral blood flow, which leads to reduction of oxygen and glucose levels in the involved regions.1 this event causes severe disability and mortality in all over the world.2 the procedure taking place in this situation, are related to some biochemical and molecular pathways.3–6 these events include excitotoxicity, inflammation, edema, and apoptosis.7 after stroke the neurons of cortex and hippocampus region is going to death.8 the occlusion of middle cerebral artery (mca) is the most common approach to model brain ischemia/reperfusion (i/r) injury in animals mimicking human subjects.9 mitochondria play a critical role in inducing apoptosis. bcl-2 family proteins are one of the key regulators in the controlling the apoptosis via the mitochondrial pathway. these proteins effect the mitochondrial membrane permeability pore.10 bax were reported as an accelerator of programed cell death in 1993.11 several studies on animal models have detected the physiological roles of bax gene and protein on nervous system.12,13 bax forms oligomers which directly effect on the release of some proteins such as cytochrome c from the mitochondrial.14,15 bax function as a gateway to the intrinsic cell death, affect mitochondria by different mechanisms.16,17 as well, bax channel inhibitors prevent mitochondria activity to release cytochrome c and protect cells against apoptosis.18 it has been shown that the absence of bax prevents more than 80% of the neuronal death after axotomy. also, by knocking out of the bax gene in the embryonic neurons derived from mice, an obvious resistance to cell death was seen which demonstrate a significant role for bax in the neurons fate.19,20 stem cells are immature cells with self-renewal capacity. they can differentiate into variety of cell types of the body.21 within the last years, the importance of stem cells application in cell therapy has begun to be determined.22,23 also, some documents has indicated that stem cells do their therapeutic actions by secretion of some molecules, such as growth factors.24 wj-mscs could be effective factors in targeting ischemic stroke.25,26 taking all, we focused about this hypothesis whether application a single dose of wj-mscs intraventricularly which injected after 24 hours of ischemia reperfusion, could decrease bax gene expression and the dark neuron numbers in the rat model of mcao as a therapeutic approach. because the significance of cell regeneration in cortical neurons, we investigated this region. the examination carried out in this study include: 1) neurological examination, 2) gene expression of the pro apoptotic protein, bax, and 3) dark neurons in histological studies (h&e staining). issn 2413-0516 (submitted: 11 july 2017 – revised version received: 28 july 2017 – accepted: 16 september 2017 – published online: 26 december 2017) 320 j contemp med sci | vol. 3, no. 12, autumn 2017: 319–325 icv injection of wj-mscs following brain ischemia/reperfusion injury in rat research tahereh alizamir et al. materials and methods animals a total number of 32 male wistar albino rats (270–300 g, 12-week-old) were purchased from the pharmacology department of tehran university of medical sciences, tehran, iran. animals could have a good accessory to food and water, 12 h periods of light/darkness and 23 ± 2°c temperature. all steps of examination were carried out according to the instruction of iranian council and confirmed by the ethical committee of tehran university of medical sciences. occlusion of mca in order to induction of cerebral ischemia in the left hemisphere, we occluded the left mca. briefly, animals were anesthetized with ketamine/xylazine (razico, iran), and the rat’s neck was dissected for ligation of common carotid (proximal part) and external carotid arteries. in order to expose the left common carotid artery (cca), we applied a midline neck incision. then, cca was isolated from its adjacent nerve, vagus. then, according to find its bifurcation, we dissected this artery. after finding the internal carotid artery, an intraluminal 4–0 nylon monofilament (doccol corporation, usa) was passed from common and internal carotid arteries to go to the mca, then this monofilament was removed after 60 min. this procedure induced brain ischemia.27 one day after the induction of ischemia, wj-mscs (1 × 106) were injected cerebroventricularly. for analyzing gene expression and histological studies, the left cortex was dissected and removed at 21th day in both fresh and fixative form. animal grouping rats were randomly allocated to the following groups: control group (co): the normal rats without any procedure sham group (sh): sham-operated rats with ligation of left common carotid mcao group: rats were subjected to occlusion for 1 h followed by 24 h reperfusion mcao + wj-mscs: rats were subjected to occlusion for 1 h and a single dose icv of wj-mscs (1 × 106) was administered 24 after reperfusion. it was injected into the left cerebral ventricle by using hamilton syringe (bregma: ap = −0.9 mm, ml = −1.8 mm (midline), and dv = 3.5 mm deep from the dura).28 neurologic examination the neurologic examination was performed for each rat 24 h after procedure according to bederson et al. study.29 this neurological assessment included a grading system of 0–3. in this system, the flexion of forelimb contralateral to the injured hemisphere after suspending each rat by the tail and circling behavior of rats toward the paretic side were the criteria for evaluation. rats with normal neurological function extend both forelimbs toward the floor. grade 0 (normal): there is no observable deficit; grade 1 (moderate): we could see forelimb flexion; grade 2 (severe): we can see decreasion in resistance to lateral push and the flexion of forelimb without circling; grade 3 (severe): all features taking place in grade 2 besides circling (table 1). isolation and cultivation of wj-mscs umbilical cords were isolated by explant method. warton`s jelly was chopped into small pieces (2-mm). then, they were cultured in dulbecco’s modified eagle’s medium (dmem, gibco, usa) for a week and supplemented with 15% fetal bovine serum (fbs, gibco, usa), 1 μg/ml amphotericin b (gibco, usa), 100 u/ml penicillin (gibco, usa) and 100 μg/ ml streptomycin (gibco, usa). the culture medium placed in a co2 (5%) incubator at 37°c. after two weeks, the pieces were removed, and medium was renewed. this process was carried out for several times. after achieving 90% confluence, wj-mscs were collected with 0.25% trypsin ethylenediaminetetraacetate (edta) (gibco, usa) and the cells in third passage were used for injection. flow cytometry after isolation, wj-mscs were incubated for 20 min with fluorescein isothiocyanate (fitc)-conjugated monoclonal antibodies against cd45 and cd90 (ebioscience, usa); then the cells were suspended in pbs (pbs, gibco, usa) in order to facs calibur flow cytometry (bdbiosciences, usa) analysis. rna extraction and quantitative real-time pcr (qrt-pcr) expression of bax gene was carried out by quantitative realtime pcr. all of the rna was collected from cortex by using trizolâ reagent (qiagen, germany). by using the kit for reverse transcription (first strand cdna synthesis kit, fermentas, usa), mrna (1 μg) converted to cdna. specific primer was placed on the three-color real-time pcr machine (applied biosystems step one, usa) for further analysis. at first, incubation of samples was carried out at 95°c for 15 min for initial polymerase activation. in the next step, the samples tolerated the three subsequent phases: denaturation, at 94°c for 20 s; annealing, at 58–60°c for 30 s; and elongation, at 72°c for 30 s. at last, δδct technique was used for relative table 1. neurologic examination grading system normal grade 0 no observable deficit moderate grade 1 forelimb flexion severe grade 2 decreased resistance to lateral push and forelimb flexion without circling grade 3 same behavior as grade 2, with circling table 2. primer sequence and length: reverse (r); forward (f); base pair (bp) primers sequence length (bp) bax r: cag cca caa aga tgg tca 18 f: gca aac tgg tgc tca agg 18 gapdh r: gtgccgttgaatttgccgtg 20 f: ggagagtgtttcctcgtccc 20 tahereh alizamir et al. 321j contemp med sci | vol. 3, no. 12, autumn 2017: 319–325 research icv injection of wj-mscs following brain ischemia/reperfusion injury in rat quantification of data and further normalization to gapdh and fold change for mcao + wj-mscs group comparison to the control, sham and mcao groups. the nucleotide sequences of bax and gapdh primer is listed in table 2. histopathological study for the light microscopy study, the rats were anesthetized with ketamine/xylazine (razico, iran), and perfused by 0.9% saline and 4% paraformaldehyde (pfa, sigma), respectively. the brains were dissected and cut into the sections with 3–5 mm thickness. then the sections were post-fixed in 10% formalin 72 h at 4°c. in order to light microscopy analysis, the samples were embedded in paraffin and 5 μm coronal sections were prepared by using a rotary microtome (leica biosystems, milan, italy). one section from each five section was selected and the tissue sections stained with hematoxylin and eosin (h&e). afterward, graded alcohol (70, 80, 90, and 100% [2 times]) was used to dehydrate the sections. finally, they were mounted in canada balsam and prepared for analysis. study and survey of sections were performed by using a light field microscope (olympus, cx31, tokyo, japan). in cortex field, the intact and ischemic cells considered as dark neurons, were counted in the ×400 images by using a connected camera to the microscope.30 statistical analysis results were represented as mean ± sd. data analysis was carried out using one-way analysis of variance (anova) and the post hoc tukey’s and tamhane’s t2 tests (spss-16 software)., and p < 0.05 was considered significant. results morphologic features and flow cytometry analysis for immunophenotypic characteristics of wj-mscs based on the findings, after two weeks of expansion in medium, the cells had features of the fibroblastic spindle like cells with up to 90% confluency (fig. 1a). there was a downregulation trend in the expressions of cd45 and upward trend in the expression of cd90 for wj-mscs. findings are representative of at least three independent samples (fig. 1b). effects of wj-mscs icv injection on the functional motor recovery in rats with mcao according to the figure 2, there was a significant difference in the mean grade of neurological score. considerable decreasion was recorded in the mean grade of neurological score in mcao group compared to co and sh groups (p < 0.05, fig. 2). also, there was remarkably increase in the mean grade of neurological score in mcao+ wj-mscs group in comparison to mcao groups (p < 0.05, fig. 2). effects of wj-mscs icv injection on the bax gene expression in cerebral cortex of rat brains with mcao qrt-pcr was performed to analyze bax gene expression. significant differences were considered in the expression level of bax gene. based on turkey test, this signification was between mcao + wj-mscs and mcao (p < 0.05, fig. 3). fig. 1 evaluation of morphology and cd surface marker profile of wj-mscs. (a) fibroblastic-like morphology of wj-mscs. (b) cultured wj-mscs were labelled with the indicated antibodies and analyzed by flow cytometry. wj-mscs with down regulation trend in the expressions of cd45 and up regulation trend in the expressions of cd90 by flow cytometry assay. fig. 2 effects of wj-mscs icv injection on the functional motor recovery in rats with mcao. bars represent means ± se; (a) p < 0.05 as compared to co group; (b) p < 0.05 as compared to sh groups, (c) p < 0.05 as compared to mcao group. co: normal group with pbs icv injection, sh: sham-operated group with pbs icv injection; mcao: ischemia induction group with pbs icv injection, mcao + wj-mscs: ischemia induction group with wj-mscs icv injection. a b 322 j contemp med sci | vol. 3, no. 12, autumn 2017: 319–325 icv injection of wj-mscs following brain ischemia/reperfusion injury in rat research tahereh alizamir et al. also, significant differences were observed between mcao + wj-mscs and co (p < 0.05, fig. 3) and as well sh groups (p < 0.05, fig. 3). effects of wj-mscs icv injection on the percentage of dark neurons in cerebral cortex of rat brains with mcao h&e staining showed the extensive neuronal death (dark neuros: being shrunken with an enhanced affinity for histologic dyes) in the cortex of ischemic rats (fig. 4a). the percentage of dark neurons was determined in the prepared sections and compared in the studied groups. based on the results, there were significant differences in the percentage of dark neurons among the groups (p < 0.05, fig. 4b). according to the tukey test, the significant differences were seen in the percentage of dark neurons of mcao and mcao + wj-mscs groups compared to co and sh groups (p < 0.05, fig. 4b). the correlation between quantitative data including bax gene expression, the percentage of dark neurons and neurological grading system there was a significant correlation between quantitative data based on bivariant correlation test; bax gene expression and percentage of dark neurons (r = 0.827, p = 0.0001, fig. 5a), bax gene expression and neurological grading system (r = 0.653, p = 0.004, fig. 5b) and the percentage of dark neurons and neurological grading system (r = 0.91, p = 0.0001, fig. 5c). discussion in this study, the effects of wj-mscs injection on the treatment and recovery of ischemic rat brains was performed. in this way, the occlusion of mca was carried out due to experimental evaluations. according to the result of study, the great number of cell death (dark neurons) recorded in the cerebral cortex which confirmed our model. the occlusion of mca, as a model of stroke, is widely used in order to analyze the mechanisms involved in ischemic stroke.31,32 cerebral ischemia starts up some physiologic and biochemical events.33 despite the advances in clinical management, stroke is a major therapeutic challenges and considered as the second cause of death in the worldwide, and increasing investigations now suggest that cell transplantation could considerably enhance recovery.34,35 the findings of present study showed that the expression of bax increased in the mcao group. some investigations carried out on animal models; suggest that the apoptosis pathway which mediated by mitochondria, is involved in stroke and associated with cell death. one of this pathway is triggered by bax activity.18 bax activity causes cell death, but neutralized by binding of bcl-2.36 also, some studies found that knocking out of bax, cause decreasion ischemic insults and neurological deficits.16,17 moreover krajewski et al. found that within 0.5 to 3 hours after ischemia, there is significant increase in bax expression within neurons in different regions of the brain.37 according to the results, a single dose icv of wj-mscs (1 × 106) was administered 24 after reperfusion. based on neurological assessments, we observed an improvement in the functional outcome of treated animals. moreover, the amount of dark neurons in the cortical region decreased in this group that was strongly related to reduction of bax gene expression and the number of dark neurons in the ischemic region in the wj-mscs groups in comparison with mcao group. thus, wj-mscs transplantation has an anti-apoptotic activity and can improve the neurological function. as a therapeutic agent, stem cells or their derivatives within can transplant into the adult brain for treatment of brain diseases.21 by developing in regenerative medicine field, it seems that application of stem cells in the treatment of ischemic diseases, is a good effective approach.38 stem cells release growth factors, cytokines and chemokines into their surroundings and cause cell regeneration.39 different sources of stem cells have been tested for treatment of stroke. between them, wj-mscs are good candidates for their immunomodulatory and neuroprotective features.25,40 treatment with wj-mscs on ischemic stroke models can survive and release suitable neurotransmitters in addition to restore some lost functions.41–47 calió et al. demonstrated that transplantation of bone marrow-mscs reduced the cell apoptosis in the hippocampus at the stroke model. their finding was confirmed by high level of anti-apoptotic bcl-2 gene expression in the treated group.48 li et al. investigated the effects of adipose-derived mscs transplantation and found that this source decreased the cerebral ischemich/reperfusion injury through the suppressing apoptosis. they showed that there is an increased ratio of bax/bcl-2 at the protein level of ischemic region.49 additionally, the association of reduced i/r injury and regulation of some apoptotic markers levels such as bax and bcl-2 after mscs therapy have been proven in a study by chen et al. (2011).50 fig. 3 effects of wj-mscs icv injection on the bax gene expression in cerebral cortex of rat brains with mcao. bars represent means ± se; (a) p < 0.05 as compared to co group; (b) p < 0.05 as compared to sh groups, (c) p < 0.05 as compared to mcao group. co: normal group with pbs icv injection, sh: sham-operated group with pbs icv injection; mcao: ischemia induction group with pbs icv injection, mcao + wj-mscs: ischemia induction group with wj-mscs icv injection. tahereh alizamir et al. 323j contemp med sci | vol. 3, no. 12, autumn 2017: 319–325 research icv injection of wj-mscs following brain ischemia/reperfusion injury in rat fig. 4 effects of icv injection of wj-mscs on the percentage of dark neurons in cerebral cortex of rats with mcao. bars represent means ± se; (a) p < 0.05 as compared to co group; (b) p < 0.05 as compared to sh groups, (c) p < 0.05 as compared to mcao group. co: normal group with pbs icv injection, sh: sham-operated group with pbs icv injection; mcao: ischemia induction group with pbs icv injection, mcao + wj-mscs: ischemia induction group with wj-mscs icv injection. (h&e staining, magnifications: ×40, ×100 & ×400). 324 j contemp med sci | vol. 3, no. 12, autumn 2017: 319–325 icv injection of wj-mscs following brain ischemia/reperfusion injury in rat research tahereh alizamir et al. fig. 5 the correlation between quantitative data: (a) bax gene expression and percentage of dark neurons (r = 0.827, p = 0.0001), (b) bax gene expression and neurological grading system (r = 0.653, p = 0.004) and (c) the percentage of dark neurons and neurological grading system (r = 0.91, p = 0.0001). conclusion it seems that the injection of wj-mscs could be a good candidate for treatment, improvement and recovery of brain ischemia via effecting on the pro-apoptotic gene expression level, bax, and the number of dark neurons. conflict of interest all authors declare no conflicts of interest. n references 1. koh sh, park hh. neurogenesis in stroke recovery. transl stroke res. 2017;8:3–13. 2. attari f, sharifi zn, movassaghi s, aligholi h, alizamir t, hassanzadeh g. neuroprotective effects of curcumin against transient global ischemia are dose and area dependent. arch neurosci. 2016;3. 3. bayat m, azami tameh a, hossein ghahremani m, akbari m, mehr se, khanavi m, et al. neuroprotective properties of melissa officinalis after hypoxic-ischemic injury both in vitro and in vivo. daru. 2012;20:42. 4. chan a, yan j, csurhes p, greer j, mccombe p. circulating brain derived neurotrophic factor (bdnf) and frequency of bdnf positive t cells in peripheral blood in human ischemic stroke: effect on outcome. j neuroimmunol. 2015;286:42–47. 5. hogan ma, black jm, tashiro js, sullins e. medical surgical nursing: clinical management for positive outcomes: wb saunders company; 2002. 6. murray v, norrving b, sandercock pa, terént a, wardlaw jm, wester p. the molecular basis of thrombolysis and its clinical application in stroke. j intern med. 2010;267:191–208. 7. kuroda s, siesjö b. reperfusion damage following focal ischemia: pathophysiology and therapeutic windows. clin neurosci (new york, ny). 1997;4:199–212. 8. northington fj, ferriero dm, graham em, traystman rj, martin lj. early neurodegeneration after hypoxia-ischemia in neonatal rat is necrosis while delayed neuronal death is apoptosis. neurobiol dis. 2001;8:207–219. 9. casals jb, pieri nc, feitosa ml, ercolin ac, roballo kc, barreto rs, et al. the use of animal models for stroke research: a review. comp med. 2011;61:305–313. 10. rolland sg, conradt b. new role of the bcl2 family of proteins in the regulation of mitochondrial dynamics. curr opin cell biol. 2010;22:852–858. 11. oltvai zn, milliman cl, korsmeyer sj. bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programmed cell death. cell. 1993;74:609–619. 12. eischen cm, rehg je, korsmeyer sj, cleveland jl. loss of bax alters tumor spectrum and tumor numbers in arf-deficient mice. cancer res. 2002;62:2184–2191. 13. knudson cm, tung ks, tourtellotte wg, brown ga, korsmeyer sj. baxdeficient mice with lymphoid hyperplasia and male germ cell death. science. 1995;270:96–99. 14. antonsson b, montessuit s, sanchez b, martinou jc. bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. j biol chem. 2001;276:11615–11623. 15. hsu yt, wolter kg, youle rj. cytosol-to-membrane redistribution of bax and bcl-xl during apoptosis. proc nat acad sci. 1997;94:668–672. 16. gibson me, han bh, choi j, knudson cm, korsmeyer sj, parsadanian m, et al. bax contributes to apoptotic-like death following neonatal hypoxiaischemia:evidence for distinct apoptosis pathways. mol med. 2001;7:644–655. 17. martinou jc, dubois-dauphin m, staple jk, rodriguez i, frankowski h, missotten m, et al. overexpression of bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. neuron. 1994;13:1017–1030. 18. hetz c, vitte pa, bombrun a, rostovtseva tk, montessuit s, hiver a, et al. bax channel inhibitors prevent mitochondrion-mediated apoptosis and protect neurons in a model of global brain ischemia. j biol chem. 2005;280: 42960–42970. 19. deckwerth tl, elliott jl, knudson cm, johnson em, snider wd, korsmeyer sj. bax is required for neuronal death after trophic factor deprivation and during development. neuron. 1996;17:401–411. tahereh alizamir et al. 325j contemp med sci | vol. 3, no. 12, autumn 2017: 319–325 research icv injection of wj-mscs following brain ischemia/reperfusion injury in rat 20. mokhtari t, akbari m, malek f, kashani ir, rastegar t, noorbakhsh f, et al. improvement of memory and learning by intracerebroventricular microinjection of t3 in rat model of ischemic brain stroke mediated by upregulation of bdnf and gdnf in ca1 hippocampal region. daru. 2017;25:4. 21. lindvall o, kokaia z, martinez-serrano a. stem cell therapy for human neurodegenerative disorders-how to make it work. nat med. 2004;10. 22. abdelwahid e, siminiak t, guarita-souza lc, teixeira de carvalho ka, gallo p, shim w, et al. stem cell therapy in heart diseases:a review of selected new perspectives, practical considerations and clinical applications. curr cardiol rev. 2011;7:201–212. 23. choi sh, jung sy, kwon sm, baek sh. perspectives on stem cell therapy for cardiac regeneration. advances and challenges. circ j. 2012; 76:1307–1312. 24. ratajczak mz, jadczyk t, pędziwiatr d, wojakowski w. new advances in stem cell research:practical implications for regenerative medicine. pol arch med wewn. 2014;124:417–426. 25. drela k, lech w, figiel-dabrowska a, zychowicz m, mikula m, sarnowska a, et al. enhanced neuro-therapeutic potential of wharton’s jelly-derived mesenchymal stem cells in comparison with bone marrow mesenchymal stem cells culture. cytotherapy. 2016;18:497–509. 26. pires ao, neves-carvalho a, sousa n, salgado aj. the secretome of bone marrow and wharton jelly derived mesenchymal stem cells induces differentiation and neurite outgrowth in sh-sy5y cells. stem cells int. 2014;2014. 27. zendedel a, habib p, dang j, lammerding l, hoffmann s, beyer c, et al. omega-3 polyunsaturated fatty acids ameliorate neuroinflammation and mitigate ischemic stroke damage through interactions with astrocytes and microglia. j neuroimmunol. 2015;278:200–211. 28. paxinos g, watson c. the rat brain in stereotaxic coordinates:hard cover edition: access online via elsevier. 2006. 29. bederson jb, pitts lh, tsuji m, nishimura mc, davis rl, bartkowski h. rat middle cerebral artery occlusion:evaluation of the model and development of a neurologic examination. stroke. 1986;17:472–476. 30. atlasi ma, naderian h, noureddini m, fakharian e, azami a. morphology of rat hippocampal ca1 neurons following modified two and four-vessels global ischemia models. arch trauma res. 2013;2:124–128. 31. shahjouei s, cai py, ansari s, sharififar s, azari h, ganji s, et al. middle cerebral artery occlusion model of stroke in rodents:a step-by-step approach. j vasc interv neurol. 2016;8:1–8. 32. sutherland ba, neuhaus aa, couch y, balami js, deluca gc, hadley g, et al. the transient intraluminal filament middle cerebral artery occlusion model as a model of endovascular thrombectomy in stroke. j cereb blood flow metabol. 2016;36:363–369. 33. jeyaseelan k, lim ky, armugam a. microrna expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. stroke. 2008;39:959–966. 34. bang oy, lee js, lee ph, lee g. autologous mesenchymal stem cell transplantation in stroke patients. ann neurol. 2005;57:874–882. 35. donnan ga, fisher m, macleod m, davis sm. stroke. lancet. 2008;371: 1612–1623. 36. sato t, hanada m, bodrug s, irie s, iwama n, boise lh, et al. interactions among members of the bcl-2 protein family analyzed with a yeast twohybrid system. proc natl acad sci usa. 1994;91:9238–9242. 37. krajewski s, mai jk, krajewska m, sikorska m, mossakowski mj, reed jc. upregulation of bax protein levels in neurons following cerebral ischemia. j neurosci. 1995;15:6364–6376. 38. hu gw, li q, niu x, hu b, liu j, zhou sm, et al. exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice. stem cell res ther. 2015;6:10. 39. kwon hm, hur sm, park ky, kim ck, kim ym, kim hs, et al. multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis. vascul pharmacol. 2014;63:19–28. 40. sabbaghziarani f, mortezaee k, akbari m, kashani ir, soleimani m, moini a, et al. retinoic acid-pretreated wharton’s jelly mesenchymal stem cells in combination with triiodothyronine improve expression of neurotrophic factors in the subventricular zone of the rat ischemic brain injury. metab brain dis. 2017;32:185–193. 41. björklund a, lindvall o. cell replacement therapies for central nervous system disorders. nat neurosci. 2000;3:537–544. 42. johansson bb, grabowski m. functional recovery after brain infarction: plasticity and neural transplantation. brain pathol. 1994;4:85–95. 43. kondziolka d, wechsler l, achim c. neural transplantation for stroke. j clin neurosci. 2002;9:225–230. 44. nishino h, borlongan cv. restoration of function by neural transplantation in the ischemic brain. prog brain res. 2000;127:461–476. 45. savitz si, rosenbaum dm, dinsmore jh, wechsler lr, caplan lr. cell transplantation for stroke. ann neurol. 2002;52:266–275. 46. taran r, mamidi mk, singh g, dutta s, parhar is, john jp, et al. in vitro and in vivo neurogenic potential of mesenchymal stem cells isolated from different sources. j biosci. 2014;39:157–169. 47. uchida k, okano h, hayashi t, mine y, tanioka y, nomura t, et al. grafted swine neuroepithelial stem cells can form myelinated axons and both efferent and afferent synapses with xenogeneic rat neurons. j neurosci res. 2003;72:661–669. 48. calió ml, marinho ds, ko gm, ribeiro rr, carbonel af, oyama lm, et al. transplantation of bone marrow mesenchymal stem cells decreases oxidative stress, apoptosis, and hippocampal damage in brain of a spontaneous stroke model. free radic biol med. 2014;70:141–154. 49. li d, fang y, wang p, shan w, zuo z, xie l. autologous transplantation of adipose-derived mesenchymal stem cells attenuates cerebral ischemia and reperfusion injury through suppressing apoptosis and inducible nitric oxide synthase. int j mol med. 2012;29:848–854. 50. chen yt, sun ck, lin yc, chang lt, chen yl, tsai th, et al. adipose-derived mesenchymal stem cell protects kidneys against ischemia-reperfusion injury through suppressing oxidative stress and inflammatory reaction. j transl med. 2011;9:51. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201706 33j contemp med sci | vol. 2, no. 5, winter 2016: 33–35 research nevoid basal cell carcinoma syndrome (gorlin syndrome) is an autosomal dominant inherited condition that exhibits high penetrance and variable expressivity. the syndrome is caused by mutations in patched (ptch), a tumor suppressor gene that has been mapped to chromosome 9q22.3-q31. gorlin-goltz syndrome (ggs) is characterised by the presence of multiple odontogenic keratocysts in the jaws, basal cell carcinomas, palmar and plantar pits and intracranial calcifications. here, we present a case of familial ggs, characterised by multiple odontogenic keratocysts, broad nasal ridge, hypertelorism, enlarged head circumference and dermoid cysts. keywords dermoid cyst, gorlin-goltz syndrome, odontogenic cysts a case report of gorlin-goltz syndrome shima nafarzadeha*, fatemeh mozaffarib, oveis khakbazc introduction gorlin-goltz syndrome (ggs), also known as nevoid basal cell carcinoma syndrome (nbccs), is an autosomal dominant inherited disorder.1 this syndrome is characterised by multiple odontogenic keratocysts, basal cell carcinomas of skin, palmar and plantar pits. ggs has a wide spectrum of neurological, ocular, skeletal, genitourinary disorders.2 the dentist has the privilege of diagnosing the syndrome at first, as multiple odontogenic keratocysts are the first manifestations of this syndrome.3 this article reports a case of a 59-year-old patient with ggs, emphasising its clinical and radiographic manifestations. case report a 59-year-old male patient admitted to the school of dentistry, babol university of medical sciences with a compliant of bilateral, painless swelling of lower jaw with paresthesia in left mental foramen area. a history of okc in mandible and dermoid cysts was found in the patient,s brother. on extraoral examination, broad nasal ridge, hypertelorism and enlarged head circumference were inspected. dermoid cyst was seen on the patient’s hand (fig. 1). no palpable lymph nodes were found. intraoral examination revealed a non-tender swelling with bony hard consistency. there was no tooth mobility. the radiographic examination and ct scan from both sides of the mandible showed extensive radiolucent lesions which were then diagnosed as okc. his first okc was diagnosed in 2010. incisional biopsy was performed and then surgical drain was placed on each side of the lower jaw. in december 2010, the patient went to a dental clinic due to purulent discharge from his mandible. the patient’s last visit in january 2014 was due to bilateral facial swelling. radiographs revealed bilateral lesions in the body and ramus of the mandible which were well defined as radiolucency with corticated margins resembling soap bubble appearance (fig. 2). unicystic ameloblastoma, okc and multiple myeloma were considered as differential diagnosis. incisional biopsy was done for histopathological examination which showed a cystic lesion, lined by parakeratinised stratified squamous epithelium with surface corrugation (fig. 3). pallisading pattern in basal cells and columnar basal cell layer exhibiting reversal of polarity were seen. the epithelium and connective tissue interface was flat. detachment of the cyst lining from the fibrous wall was observed (fig. 4). the connective tissue was fibrotic with blood vessels. odontogenic epithelial islands in the connective tissue were visible (fig. 5). the diagnosis was okc and considering the clinical findings the patient was found to be a case of ggs. decompression tubes were used after performing bony windows to reduce the cysts size, and the patient was told to maintain oral hygiene and irrigate the cavity every day. the patient attended follow up sessions for 6 months, but refused to undergo enucleation surgery. discussion nevoid basal cell carcinoma syndrome (gorlin-goltz syndrome) is an autosomal dominant inherited condition that exhibits high penetrance and variable expressivity.4 it is caused by mutations in ptch, a tumor suppressor gene, a human homologue of a drosophila segment polarity gene ptch located in long arm of chromosome 9q22.3.4–7 the prevalence of gorlin syndrome is estimated to be about 1 in 60,000.4 in 1894, jarisch and white made the first descriptions of patients with this syndrome,5,8 bettley and ward were the first who related the presence of palmar and plantar pits with the syndrome in 1950–1960.5,9,10 jaw cysts are one of the most constant features of the syndrome and are present in at least 75% of the patients.4 in ggs, okc is a common lesion in the jaws.11 the cysts are frequently multiple; some patients have had as many as 10 separate cysts.4 less than 10% of patients with multiple okcs have other manifestations of this syndrome; however, it has been suggested that multiple okcs alone may be the confirmatory of the syndrome.12 these may be bilateral, may involve both jaws and most commonly arise in the lower third molar and maxillary canine regions.11 in patients with ggs, okcs are more frequent, recur faster and more commonly, consistent with a more “aggressive” phenotype.11,13 the keratocysts in patients with this syndrome tend to have more satellite cysts, solid islands of epithelial proliferation and odontogenic epithelial rests within the fibrous capsule than isolated keratocysts.4 the increased recurrence rate of okc in syndromic patients compared to non-syndromic patients is because of a familial tendency in this syndrome, s006f, early family detection and genetic counseling are critical.14,15 okc associated with nbccs have occasionally been reported to transform into aggressive neoplasms such as ameloblastomas and squamous cell carcinoma.14,16 issn 2413-0516 aassistant professor, department of oral and maxillofacial pathology, dentistry school, babol university of medical sciences, babol, iran. bfaculty, department of oral and maxillofacial pathology, dentistry school, mazandaran university of medical sciences, sari, iran. cassistant professor, department of oral and maxillofacial surgery, dentistry school, babol university of medical sciences, babol, iran. correspondence to: shima nafarzadeh (email: shima.nafar2004@yahoo.com). (submitted: 10 june 2015 – revised version received: 03 july 2015 – accepted: 11 july 2015 – published online: 26 march 2016) http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3883343/#ref1 mailto:shima.nafar2004@yahoo.com 34 j contemp med sci | vol. 2, no. 5, winter 2016: 33–35 gorlin-goltz syndrome: case report research shima nafarzadeh et al. fig. 1 similar dermoid cyst on hands. fig. 2 orthopantmograph showing multiple lesions in the body and ramus of the mandible. fig. 3 low power showing a cystic cavity lined by parakeratinised epithelium with surface corrugation. fig. 4 detachment of portions of the epithelium from the fibrous wall. fig. 5 histopathologic view shows odontogenic epithelial islands in the cyst connective tissue wall. in who 2005 classification of odontogenic tumors, the name of the cyst has been changed to “keratocystic odontogenic tumor” owing to its aggressive nature and recur rence rate.11 in 1994, evans et al., first established major and minor diagnostic criteria for this syndrome which was modified later by kimonos et al., in 2004,5,17,18 the major and minor criteria are as follows: major criteria •   multiple basal cell carcinomas or one occurring under the  age of 20 years. •  bifid, fused or markedly splayed ribs. •  okcs of the jaws confirmed by histopathology. •  palmar or plantar pits (three or more). •  first degree relative with nbccs. •  bilamellar calcifications of the falx cerebri. minor criteria •  macrocephaly (adjusted for height). •   skeletal abnormalities: sprengel deformity, marked pectus  deformity and marked syndactyly of the digits. 35j contemp med sci | vol. 2, no. 5, winter 2016: 33–35 research gorlin-goltz syndrome: case reportshima nafarzadeh et al. references 1. sabbia t, bovone s, camera a, gambini c, balbi p. [gorlin-goltz syndrome with odontogenic keratosis. report on a patient followed for 10 years]. minerva stomatol. 1994 jul–aug;43(7-8):359–63. 2. gorlin rj, goltz rw. multiple nevoid basal-cell epithelioma, jaw cysts and bifid rib. a syndrome. n engl j med. 1960 may 5;262:908–12. 3. karthiga ks, sivapatha sundharam b, manikandan r. nevoid basal cell carcinoma syndrome. indian j dent res. 2006 jan-mar;17(1):50–3. 4. neville b, damm d, allen c, bouquot j. oral and maxillofacial pathology. 3rd ed. philadelphia: wb saunders; 2009. 5. sunder s, babburi s, guduguntla s, raju p. gorlin-goltz syndrome: a rare case report. j dr ntr univ health sci. 2013;2(2):150. 6. casaroto ar, loures d, moreschi e, veltrini vc, trento cl, gottardo vd, et al. early diagnosis of gorlin-goltz syndrome: case report. head face med. 2011;7:2. 7. shakya h, mubeen k. gorlin-goltz syndrome with situs inversus: a rare case report; síndrome de gorlin-goltz com situs inversus: relato de um caso raro. rev clín pesq odontol. 2009;5(2):175–84. 8. jarisch w. zur lehre von den hautgeschwulsten. arch dermatol syph. 1894;28:163–222. 9. bettley fr. two cases of multiple naevoid basal cell epitheliomata? porokeratosis of mantoux. br j dermatol. 1953 jun;65(6):219–21. 10. ward w. nævoid basal celled caecinoma associated with a dyskeratosis of the palms and soles. a new entity. australasian j dermatoly. 1960;5(4):204–8. •   congenital  malformation:  cleft  lip  or  cleft  palate,  frontal  bossing, coarse face moderate, or severe hypertelorism. •   radiological abnormalities: bridging of sella turcica, vertebral anomalies such as hemi vertebrae, fusion or elongation of vertebral bodies, modeling defects of the hands and feet, or flame-shaped hands or feet. •  medulloblastoma, seizures, mental retardation, eningioma. •  ovarian fibroma. two major criteria or one major and two minor criteria are necessary to diagnose this syndrome.5 our patient had broad nasal ridge, hypertelorism, enlarged head circumference, dermoid cyst on hands and multiple okc in the mandible. in a case report pol et al.19 had three major features, namely palmar pits, multiple okcs in the jaw and lamellar calcification of the falx cerebri and minor features such as macrocephaly, fontal bossing and hypertelorism, thus suggesting it to be a case of the ggs. in their patient, the lining of the okcs revealed the presence of parakeratinised uniform squamous epithelial lining with multiple satellite and daughter cysts in the connective tissue wall, thus indicating the association with ggs. in a case report syyam et al.5 the present case fulfills the above three major criteria features like multiple okcs, positive family history, calcification of falx cereberi and tentorial cerebella and with two minor criteria features that includes hypertelorism and neurologic disorder as the patient had episodes of seizures. most of the anomalies in nevoid basal cell carcinoma syndrome are minor and usually not life threatening.4 the prognosis generally depends on the behaviour of the skin tumors.4 the jaw cysts are treated by enucleation. infection of the cysts in patients with this syndrome is also relatively common.4  11. kalia v, kaushal n, kalra g. the syndromic multiple odontogenic keratocyst in siblings: a familial study. ann maxillofac surg. 2011;1(1):77. 12. agrawal a, murari a, vutukuri s, singh a. gorlin-goltz syndrome: case report of a rare hereditarydisorder. case rep dent. 2012;2012:475439. 13. zedan w, robinson p, markham a, high a. expression of the sonic hedgehog receptor ‘patched’ in basal cell carcinomas and odontogenic keratocysts. j pathol. 2001;194(4):473–7. 14. tarakji b, baroudi k, hanouneh s, azzeghaiby s, nassani m. possible recurrence of keratocyst in nevoid basal cell carcinoma syndrome: a review of literature. eur j dent. 2013;7(5):126. 15. wang xx, zhang j, wei fc. familial multiple odontogenic keratocysts. j dent child. (chic). 2006;74(2):140–2. 16. reisner kr, riva rd, cobb rj, magidson jg, goldman hs, sordill wc. treating nevoid basal cell carcinoma syndrome. j am dent assoc. 1994;125(7):1007–11. 17. rayner c, towers j, wilson j. what is gorlin’s syndrome? the diagnosis and management of the basal cell naevus syndrome, based on a study of thirtyseven patients. br j plastic surgery. 1977;30(1):62–7. 18. evans d, ladusans e, rimmer s, burnell l, thakker n, farndon p. complications of the naevoid basal cell carcinoma syndrome: results of a population based study. j med genet. 1993;30(6):460–4. 19. pol ca, ghige sk, kalaskar rr, gosavi sr. gorlin-goltz syndrome: a rare case report. contemp clin dent. 2013;4(4):547. 239j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 review new modalities of treatment for hepatocellular carcinoma arman karimi behnagh,a negar rezaei,b,c mohadeseh jahannia,a zeynab noorimotlagh,a ali kabird astudent research committee, faculty of medicine branch, iran university of medical sciences, tehran, iran. bdepartment of epidemiology, school of public health, iran university of medical sciences, tehran, iran. cnon-communicable diseases research center, endocrinology and metabolism population sciences institute, tehran university of medical sciences, tehran, iran. dminimally invasive surgery research center; iran university of medical sciences, tehran, iran. correspondence to ali kabir (email: aikabir@yahoo.com). (submitted: 12 may 2017 – revised version received: 29 may 2017 – accepted: 10 june 2017 – published online: 26 september 2017) hepatocellular carcinoma (hcc) is the second most common cause of death from cancer worldwide. managing hcc is difficult. however, there are many treatment options available such as liver transplantation, radiotherapy, different ablative techniques, surgery, trans-arterial chemoembolization (tace) and systemic therapy. these treatments did not show the promising responses and the recurrence rate is still high. on the other hand, there are some new treatments such as immunotherapy, gene therapy, combination of different therapies, chinese traditional therapies and new targeted therapies. the aim of this study is to review both the recent changes in the common therapies and newly developed therapies of hcc. keywords carcinoma, hepatocellular, treatment, combined modality therapy introduction hepatocellular carcinoma (hcc) is the second most common cause of death from cancer worldwide. it is the fifth most common cancer in men and the ninth in women. in addition, the incidence rate is almost equal to the mortality rate.1 many risk factors are known for hcc, such as age, gender, alcohol, hcv, hbv, and non-viral chronic liver disease. moreover, currently among these risk factors hbv and hcv are the most important ones.2 managing hcc is difficult. however, there are many treatment options available such as liver transplantation, radiotherapy, different ablative techniques, surgery, trans-arterial chemoembolization (tace) and systemic therapy.3 these treatments did not show the promising responses and the recurrence rate is still high.4 on the other hand, there are some new treatments such as immunotherapy,5 gene therapy,6 combination of different therapies, chinese traditional therapies and new targeted therapies.7 the aim of this study is to review both the recent changes in the common therapies and newly developed therapies of hcc. methods and materials a focused literature review was performed in medline, embase and cochrane library to find the recent studies on the treatment of hcc. we limited our search to recent 5 years, although we bring a few numbers of older studies while we do forward-backward citation. in addition, we only evaluate the studies which were mainly about the human species and were published in english. for including studies in our review, we performed a double phase assessment. in the first phase, we considered only titles and abstract and in the second we assessed the full text to find out the relevance of the studies to our work. based on this explanation, we included 4622 references. after removing duplicates, 3788 references were assessed in the first phase. then, 1665 studies were excluded. as of the remaining studies, 387 of them were removed in the second phase . among 1349 remaining ones, 173 studies were selected to include in this study. an adapted prisma flow diagram shows the process (fig. 1). our exclusion criteria for both phases were: complications, biomarkers, prediction and progression factors, case reports, imaging, preclinical studies (in vitro and animal studies), cost and economy, risk factor and some other non related subjects to the therapy of hcc. below, we briefly explain different therapies of the hcc considering new changes and recent modalities and treatment options. chemotherapy one of the chemotherapy multi resistance tumours is hcc. it has a very low survival and despite the huge amount of scientific work in this matter, it appears that systemic chemotherapy is unsuccessful in hcc treatment.8 doxorubicin is the chemotherapy of choice from past, but the effect of this drug on survival rate was equal or inferior, comparing with other chemotherapy drugs in several clinical trials.9,10 there were also a systematic review of 800 patients and 13 trials applying doxorubicin chemotherapy compare to other chemotherapies. the results showed chemotherapy in advanced hcc have poor effects. doxorubicin monotherapy is not inferior to comparing with combination multiple drug chemotherapy or biological agents.10 using platinum-based chemotherapy with oxaliplatin like piaf schedule (cisplatin + interferon + doxorubicin + 5-fu), minimizing toxicity and is better to be used for older patients. though the overall survival (os) is not significantly different from doxorubicin.10 nonetheless, there are not randomized trials of platinum agents in hcc systemic treatment, folfox have uncertain benefits comparing with doxorubicin.11 sorafenib therapy is an alternative palliative therapy that will be discussed later in this article. tace tace is a standard palliative, down staging therapy in patients with progressive inoperable hcc since 1970. it has been used in intermediate hcc that we have few choices of treatment.12,13 the main concept of chemoembolization is to emboli the hepatic artery which is the main hepatic tumour vessel. this procedure may cause tumour death without affecting normal tissue.14 in addition, a chemotherapy agent delivered to hepatic vessel. there are not a standard protocol for the choice of chemotherapy issn 2413-0516 240 j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 hepatocellular treatment review ali kabir et al. drug or dosage, or rate of usage.15 anthracyclines (daunorubicin, doxorubicin, epirubicin), platinum-based agents (cisplatin, lobaplatin, miriplatin), mitomycinc, and 5-fluorouracil is the chemotherapy drugs used in tace. there are not noteworthy superiority reported on using specific chemotherapy agent in tace.13 a systematic review of 15 studies comparing tace with conservative management had reported that the patients with portal vein branch invasion and well liver function may achieve optimal advantage from treatment with tace.16 another systematic review on five randomized trials compared tace with and without chemotherapy treatment. the results revealed no superiority of using chemotherapy with chemoembolization and the adverse effect of treatment is even higher in the tace group compared to tae.17 there are also alternative choices like balloon-occluded transarterial chemoembolization (b-tace) using miriplatin (a lipo-philic anticancer drug) and gelatin particles. a significant higher local node treatment in b-tace with the miriplatin group in comparison with routine tace was reported in a non-randomized trial.18 in addition, no significant difference was reported, between tace-miriplatin plus epirubicin and tace-miriplatin group in the efficacy of therapeutic and adverse effects.19 although lack of a randomized controlled trial (rct) is noticeable in this issue. deb-tace doxorubicin-eluting beads (debs) is an embolization device with tace treatment. it is used in hypervascular tumours and emboli vessels with simultaneous administration of tumour located, controlled, sustain released dosage of chemotherapy which is mainly doxorubicin. these beads consist of vinyl alcohol and sulfonic acid groups with chemotherapy agents in different sizes.20 this method is widely available since 2006, and the treatment effects are assumed to be superior compared to tace.21 there are two systematic reviews on our search which compare the deb-tace with tace. these studies, approximately include same studies with very inconsistent results. in addition, there are just few rcts in this issue. these studies are not reporting a significant difference on survival rate.22,23 other studies are non-randomized trials or retrospective researches assessing the registries or have a low sample size or could not evaluate survival rate, which makes the interpretation and decision of the reported results imprecise.24,25 however, a recent non-randomized controlled trial reported non-significant difference in survival rate between tace and deb-tace in non-operable hcc patients. as the number of tace treatment needed is higher than deb-tace with the same survival rate, deb-tace is more preferred by both clinicians and patients.26a meta-analysis in 2013 with seven studies (693 participants) reported no significant difference between the two procedures in hard outcomes. it has been also mentioned the need of running more rct on this issue for better decisions,27 while a meta-analysis in 2014 included seven studies (700 participants). it has reported a better tumour response with deb-tace. although the survival rate was reported to not change significantly after 3 years and adverse side effects are also similarities between the two procedures.21 gelatin sponge particle tace gelatin sponge is an embolic agent generally used for embolization therapies of various diseases over 30 years. the size of embolic agent has direct influence on embolic effects. gelatin sponge particles are around 1–1.5 × 1–1.5 × 2 mm. it has been made from either spongel or gelfoam sheets.28 there are three embolization effect classifications named “short term”, “medium term” and “long term”. gelatin sponge micro particles (gsms) have medium effect.29 using the embolization component in tace procedure increases the rate of necrosis of the main tumour and gelatine sponge particles is used frequently in embolization procedures.30 gelatin sponge microparticles in a rabbit liver tumour were assessed in 2014. the results suggested the usage of gsms-tace comparing to c-tace group and hepatic arterial infusion (hai) group have higher concentration of chemotherapy drug with slow drug release.31 human research also showed good tolerance on gsms and the os rate was 100% in six months and 87.5%, in one year.32 other parallel studies confirmed the safety and effectiveness of gelatin sponge particle tace for treatment of the barcelona-clinic liver cancer (bclc) stage b hcc patients.32 hepatic resection vs. tace up to now, the curative choices of hcc are liver transplant, hepatic resection and radiofrequency ablation. liver transplant appears to be gold standard treatment, but it is restricted because of the low number of donors. hepatic resection is a better choice than radiofrequency ablation for the os. in addition, for palliative treatment options we have tace and sorafenib. tace is generally used for hcc patients further than the bclc stage a classification.33 two systematic reviews and meta-analysis compared the hepatic resection with tace in os of patients further than the bclc stage a classification. the results of both are parallel and reported a significant overall oneand three-year better survival in hepatic resection group.33,34 although, the lack of rct in this issue was mentioned in both systematic reviews. cytokine induced killer cell recently, immunotherapy is a new modality in cancer treatment. cytokine-induced killer (cik) cells are effective adoptive cell-based immunotherapy treatment. the concept of this treatment is to stimulate body immune response against tumour cells. so, the adverse side effects of chemotherapy and surgical procedures are minimized. cik cells are natural killer like t cells and express both the t cell marker cd3 and the nk-cell marker cd56. the idea of applying cik cells in cancer therapy was first reported by schmidt-wolf et al in 1991 on an animal model. in 2010, stanford medical university established an international registry on cik cells (ircc) for better application of this treatment based on clinical evidence;35 particularly, researchers from china performed rcts on combination cik with tace for hcc treatment. it is reported that the combination therapy improves quality of life and prevent cancer recurrence.36 there is a systematic review comparing cik-tace with tace alone in the hcc cancer treatment. two rcts and four non-rct studies were included with 428 patients. the os and progression-free survival (pfs) rates are better in the group treated with cik-tace.35 liver transplantation in the category of curative treatments for hcc and cirrhosis, liver transplant is a choice. in this procedure, liver tumour and underling cirrhosis and possible risk of recurrence after resecting tumour will be removed. hcc patients will be ali kabir et al. 241j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 review hepatocellular treatment candidates for liver transplant based on tumour size and nodule numbers. milan criteria are particularly used for choosing hcc patients for liver transplant (a solitary tumour nodule up to 5 cm or three nodules less than 3 cm). because of the limited number of liver grafts, hcc patients must be selected. vascular invasion, high alpha fetoprotein concentration, aggressive biology of tumour, multifocal large size tumour and having other comorbidities are the other exclusion criteria for this treatment.37–40 fei teng et al had described one-, threeand five-year survival rate of hcc patients after liver transplant: 78.9%, 53.2% and 46.4%, respectively.41 however, the lethal nature of hcc in addition to low source of liver graft makes it difficult to run rcts and the lack of evidence is tangible in this concern. there are different criteria available for selecting hcc patients for liver transplant apart from milan criteria. the most popular one is university of california, san francisco (ucsf). no significant difference was reported between applying these two criteria. other criteria are the united network for organ sharing (unos), tokyo, kyoto, kyushu, hangzhou and up-to-seven. the detail on these criteria are available elsewhere.10,42 down staging of hcc patient’s candidate for liver transplant is unavoidable because of the high dropout rate for the duration of waiting for liver transplant. the main interventions for down staging are tace, percutaneous ethanol injection (pei), and radiofrequency ablation (rfa) and sorafenib therapy in addition to tace or deb-tace. however the lack of evidence in this area is obvious.42 however, current guidelines do not recommend a liver transplant for patients outside milan criteria.43 salvage liver transplantation (slt) to minimize the waiting list dropout rate, alternative treatments are used in this period, such as local therapies and surgery. it has been proposed for patients with small hcc and preserved liver function. the efficacy of salvage transplantation that is primary resection before transplantation is assessed by poon rt et al in 2002. salvage transplantation reduces recurrence or worsening of liver function in waiting time for liver transplant.45 the positive point of surgery specially laparoscopic surgery is the curative concentrating of resection, pathological analysis of tumour for vascular invasion, tumour differentiation, number of nodules affected and improving quality of life. the negative point of laparotomy is adhesion in the surgical site which makes next liver transplantation operation difficult. however, laparoscopic or robotic approaches result in lower adhesions in surgical site. although liver function and size of tumour of patients do not let clinicians to use this approach in all patients.46 a systematic review with 1508 patients was conducted in 2013 on short and long term outcomes of slt. although the short outcomes like duration of operation is significantly higher in slt than primary liver transplant, the os rate after 1, 3 and five years were not different.47 surgical resection liver resection surgery is performed for nearly six decades.47 surgical liver resection is a curative resection of the entire tumour in microscopic level with free of tumour cutting surface. surgical resection is the treatment of choice in non cirrhosis hcc in early stages. the frequency of non-cirrhosis hcc in asia is about 8 times higher than western countries. in other hcc patients’ surgical resection may cause liver failure. so, these patients must be selected carefully for surgical treatment.48 prognostic factors for liver resection are tumour size, high level of alpha fetoprotein, micro and macro vascular invasion, and underlying liver disease such as cirrhosis.49 there are two types of resection; anatomical resection, which is wide liver resection beyond the borders of tumour and non-anatomical resection which is used in hepatic dysfunction and remains the most possible liver residue. there are some controversies reported on the type of resection. some studies suggested survival benefits from anatomic resection,50 while the others reported no significant benefit.49 a systematic review suggested anatomic resection of liver approves survival and diminish recurrence comparing to non-anatomical but the studies included in this meta-analysis are not randomized trials.51 liu ph in 2014 showed surgical resection with tace in hcc patients beyond milan criteria. they had reported a noteworthy improved long term survival with surgical resection.52 laparoscopy the first laparoscopic liver surgery was performed four decades after open surgical liver resection. currently, with more technical improvement in laparoscopic surgery, more malignant liver tumour resection is carried out with this procedure.53 there are few observational studies and a systematic review that compare open surgery and laparoscopic short-term and longterm outcomes on hcc and there are not noticeable differences between two surgical methods.54 the systematic review was conducted in 2012 and included 10 observational studies. the overall complication rate was lower in the laparoscopic group. the recurrence rate of hcc, 5-year survival, and 3-year survival was not significantly changed between two procedures.47 radiotherapy the role of radiotherapy (rt) is limited in patients with hcc due to low tolerance of normal liver tissue to radiation. however, new techniques in image guidance, breathing motion reduction strategies and appropriate dosimetry have been introduced for better local control and decreasing liver toxicity. radioembolization radioembolization (re) delivers a high dose radiation through hepatic artery. small microspheres loaded yttrium-90, a b-emitter isotope is used in the case of re. two common 90y devices are thera-sphere (btg international, london, united kingdom) which is a glass microsphere, and sirsphere (sirtexmedical, sydney, australia) which is a resin microsphere. according to the literature, both microspheres have similar outcomes.55 radiation segmentectomy and boosted radioembolization are considered two novelties in the field of re. radiation segmentectomy the combination of microcatheter technology and the localized radiation emission properties of 90y microspheres introduce a novel treatment called radiation segmentectomy. since ablation and resection are not suitable for the lesion located near critical structure, radiation segmentectomy is an appropriate way to overcome this drawback.56 michel et al conducted a follow up, multicenter study to assess the efficacy of radiation segmentectomy. radiation segmentectomy was defined as 90y microsphere infusion limited to 2≥ couinaud 242 j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 hepatocellular treatment review ali kabir et al. segments. dosing was achieved by infusing a calculated lobar dose into a segmental tumour-feeding vessel. therefore, segmental doses are higher than lobar doses by the ratio of lobar/ segmental volumes. the results show, complete response (cr) in 47%, and partial response (pr) in 39% of patients.57 a retrospective follow up study showed a cr of 95% of patients who received radiation segmentectomy.58 boosted radioembolization boosted radioembolization is a novel concept for hcc. it is a personalized therapy in which the priority is given to tumour dosimetry rather than liver dosimetry. in this method, a predictive dosimetry model, based on technetium-99m microaggregated albumin (maa), single-photon emission computed tomography/computed tomography (spect/ct), is utilized to identify tumours that need boosted microsphere dosing for better response rate.59,60 in another retrospective cohort study, garin et al showed that boosted radioembolization based on maa spect/ct caused prolonged os for hcc portal vein thrombosis (pvt) patients, without increasing liver toxicity.61 intensified modulated radiotherapy (imrt) imrt is an advanced three-dimensional conformal radiotherapy (3d-crt) that allows greater control of dose distribution. since, it can change the intensity of individual rays within each beam, it can cover the treatment volume to concave tumour shape.62 helical tomotherapy (ht) and volumetric-modulated arc therapy (vmrt) are two advanced forms of imrt. ht it is a highly advanced radiotherapy, in which a gantry 6-mv linear accelerator is rotated continuously through 360° around the patient using tens of thousands of narrow beams. it integrates both imrt and image guided radiotherapy (igrt). igrt system of ht is a daily mega-voltage computed tomography image guidance.63 three cohort studies showed a survival benefit and efficacy in utilizing ht for hcc patients.64–66 in a recent retrospective comparative study of 118 hcc patients, conducted in 2016, median survival in patients treated with ht was significantly more than with 3d-crt.67 vmrt it is an advanced form of imrt that unlike fixed-field radiation methods treat the tumour from all angles by rotating the beam around the patient. rapid arc tm (varian medical system, palo alto, ca) is a variation of vmrt.68 in radiotherapy of hcc patients, targeted volume is greatly affected by respiratory motion. active breathing coordinator (abc) is a solution for gaining a better target volume. in order to determine the feasibility of rapid arc (ra) in association with abc, gong et al conducted a study of 12 hcc patients. 3d-crt, imrt and ra plans were designed and abc was used for better assessment of target volume. the report resulted in better dose delivery and an accurate target volume besides shortening treatment time for ra.69 4d-ct is another solution to achieve better target coverage. 4d-ct scanning synchronizes ct image with respiratory cycle and is able to predict tumour movement. another study involved 10 patients showed ra with 4d-ct or 3d-ct associated with abc can effectively deliver accurate target volume compared with 3d-ct with free breathing.70 stereotactic body radiotherapy (sbrt) when ablation or tace fails, sbrt can be considered as an alternative therapy. sbrt delivers high radiation to focal hcc and decreases radiation induced liver toxicity by sparing other tissues.71 sbrt by cyberknife® is a new technology for treatment of liver lesions that delivers 100 to 200 photon beams of 6 mv.72 for appropriate detection it requires gold fiducial markers in the periphery of tumour. a recent study, evaluated the possibility of implantation of gold fiducial markers and the imaging technique it requires. the important role of radiologist in implantation and applying sonographic guidance were concluded.73 helical intensity-modulated radiotherapy-based stereotactic body radiotherapy ht is an alternative for delivering sbrt, in a recent paper published in 2016, the phase i trial was conducted by jun et al. to evaluate the feasibility and toxicity of helical intensity modulated radiotherapy (himrt) based stereotactic body radiotherapy (sbrt) in eighteen hcc patients. at a median follow up of 28 months for living patients, an os rate of 69.3% and a well-tolerated toxicity was concluded.74 differential hepatic avoidance rt (dhart) in hcc patients with a high degree in the liver function heterogeneity as for cirrhosis patients, conventional rt methods cannot spare radiation to functional liver regions and it may lead to radiation induced liver disease (rild) to overcome this drawback. stephen et al. introduced a new modality for rt that is called differential hepatic avoidance rt (dhart). in this technique sc spect (sulphur colloid single photon emission tomography) images were used to spare region of functional liver through the use of dose painting techniques in proton pencil beam scanning (pbs) and photon volumetrically modulated arc therapy (vmat) rt. this initial study indicated that dhart is achievable with either photon vmat or proton pbs therapy in hcc patients.75 varian trilogy™ varian trilogy™ is an rt delivery system that deliver a highly conformal radiation beam to a mobile target. it utilizes realtime position management™ (rpm) for respiratory control and on-board imager (obi) for image guidance.76 a prospective cohort study showed an excellent treatment outcome with minimal toxicity for this rt delivery system.77 ablation ablative therapy is considered a treatment with high safety profile and local control. radiofrequency ablation (rfa), microwave ablation (mw), laser induced tumour therapy (litt) all parts of thermal ablationethanol or acetic acid injection as part of chemical ablationcryoablation, brachytherapy, irreversible electro-poration (ire), and high intensity focused ultrasound (hifu) are all considered as ablative therapies. here we discussed technical advances in utilizing rfa, mwa, brachytherapy and hifu with a major focus on rfa. rfa poor visualization is one of the complications of ultrasound (us)-guide rfa. contrast-enhanced ultrasound (ceus), contrast-enhanced computed tomography (cect), and magnetic resonance imaging (mri) are alternatives for the guidance of ali kabir et al. 243j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 review hepatocellular treatment rfa. however, the short durations of the different vascular phases, absence of real-time imaging guidance and increased radiation exposure are the disadvantages of using these imaging techniques.78,79 to overcome these problems a method called “fusion imaging’ or “real time virtual sonography”, that synchronizes real-time us and ct or mri, is introduced.80 two cohort studies showed an excellent technical success rate, safety and effectiveness for rfa using fusion imaging.81,82 reduced energy diffusion is a complication observed in rfa using conventional electrodes. utilizing internally cold (ic) electrodes and saline-enhanced rfa are two techniques to overcome this drawback. however, these two techniques have their own problems, including the limitation of overheating and the risk of heating and ablation of non-tumour containing area, respectively.83,84 internally cooled wet (icw) electrodes that have the ability of both techniques (ic electrode and saline enhanced rfa) are developed as a solution to compensate the limitations of these techniques. in a study, utilizing modified icw electrode showed the safety and effectiveness of the technique and a successful local control.85 in a retrospective comparative study, including 165 patients, mean ablation volume was significantly greater and local tumour progression was significantly lower in patients treated with icw electrodes compared with patients treated with ic electrodes.86 conventional monopolar rfa devices have several problems including; systemic symptoms because of exposure to a wide area of the body, long ablation time and causing burns. in contrast to a monopolar device in which the electrical current flows between the electrodes and the grounding pad, in a bipolar device the current flows between two electrodes. besides compensation for monopolar device complications, the bipolar device is more sufficient and makes a large thermocoagulation volume in a single ablation procedure. a multicenter open-label trial confirmed the efficacy and safety of the celonpower system, which is a bipolar rfa device.87 incomplete peripheral ablation of the tumour is another drawback of utilizing monopolar device;88 while, multipolar technique provides a probe inserted outside the parameters of target lesion (no touch technique) and allows ablation of the tumours from margin to center. this causes complete margin ablation.89 the pathological examination of 59 nodules in a retrospective study indicates an improvement in the rate of complete necrosis in no-touch multipolar radiofrequency ablation compared with monopolar technique.90 placement of sodium hyaluronate solution onto the liver surface can be used as a procedure for rf ablation of hccs located on the liver surface. it causes separating of liver from other organs during the procedure and decreases the damage to adjacent organs. a phase one study was conducted to assess the safety of intraperitoneal injection of the sodium hyaluronate solution. complete ablation and observation of tumour recurrence in one of 28 patients indicated the safety and efficacy of the technique.91 microwave ablation (mwa) mwa is a locoregional therapy that can be conducted with the guidance of us, ct or mri. in a follow-up study, mwa was performed by using a real-time virtual navigation system. the technique effectiveness was 94.44%.92 low power output and small-diameter ablation is one of the complications of mwa.93 water or gas antenna cooling has introduced a solution for making higher power microwave system. a retrospective cohort study reported that after utilizing a gas-cold system for mwa, overall primary technique effectiveness rate and os rate were 91.6 and 76%, respectively. the results showed the safety and efficacy of the technique.94 risk of tumour seeding is considered for subcapsular tumours achieving thermal ablation.56,95 in order to reduce the probable risk of tumour seeding and local progression after thermal ablation for subcapsular hcc, a technique called no-touch wedge ablation, was described by premal et al. the technique involved probe placement at multiple oblique sites tangential and adjacent to the tumour, to create a sufficient ablation zone that is inclusive of the subcapsular tumour and the required peritumoural margins. after complete ablation of eight tumours, at an average imaging follow-up of 244 days, one case of local recurrence was observed.96 one of the limitations for us guided thermal ablation is suboptimal conspicuity of some subcapsular tumours or tumours located in hepatic dome, that may lead to causing damage to adjacent tissues.97,98 artificial ascites and plural effusion introduced a solution for this drawback.99 in a case-control study that the efficacy of mwa with artificial pleural effusion was evaluated. the analysis showed no statistical differences between case and control groups in the primary technique effectiveness and local tumour progression rates.100 brachytherapy brachytherapy is an ablative therapy that utilizes interstitial implantation of radioactive seeds and delivers high dose radiation in the target area. two types of radioactive seeds are applied, one of them is high energy gamma emitter (e.g., cobalt-60 and radium-226) and the other is low energy chemicals (e.g., iodine-125 and palladium-103). because of the high energy irradiation of the first type, low energy seeds became more common in the past decades.101 ct-guided high-doserate brachytherapy (ct-hdrbt) is a new modality in ablative technique that uses iridium-192 seed, inserted through catheters with ct guidance.102 two retrospective cohort study conducted by collettini et al. evaluated the clinical outcome of ct-hdrbt in hcc patients and showed the effectiveness of this therapy in local tumour control of lesions unsuitable for resection or thermal ablation.103,104 hifu hifu is a new ablative therapy in which a unique frequency of us wave of 0.8 to 3.5 mhz is used and can be focused at a distance from therapeutic transducer. the high focused energy is able to induce necrosis by increasing tissue temperature.105 poor visualization of hccs during hifu treatment is an obstacle. hiroyuki et al. reported a study of hifu ablation assisted using color doppler for the treatment of hcc. the usefulness of this method was concluded from the study.106 in another study conducted by michele et al. magnetic resonance-guided focused ultrasound (mrgfus) was assessed as a treatment for solid tumour in abdomen. the study resulted in the safety and feasibility of the technique.107 chinese herbal medicine chinese herbal medicine is a type of traditional medicine used in cancer therapy for a long time ago.108 in the case of hcc, studies evaluated the mechanism of action of this medicine and also this therapy has been used in vitro, in vivo, and also in recent clinical trials. 244 j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 hepatocellular treatment review ali kabir et al. ruanjianhugan oral ruanjianhugan (rjh) tablets which were made up of ten medicinal materials are kind of chinese herbal medicine used in a 22-year followed up clinical trial study. results indicated that long-term intake of rjh increases os in small hcc after resection. also, it has preventive effects on small hcc recurrence.2,102,109,110 systemic therapy antiangiogenesis agents tyrosine kinase inhibitors: sorafenib. in the chinese subset of gideon study in 2015, 338 patients were followed prospectively to assess the efficacy and safety of sorafenib. median os of child-pugh a patients were longer than child-pugh b patients. adverse effects (ae) comparison showed serious aes (25.0% vs. 23.0%) in child-pugh b patients. in overall, this study indicated that child-pugh b patients would be safely healed with sorafenib.111 adjuvant sorafenib. currently, sorafenib has been used as an adjuvant therapy after curative resection in bclc-stage three patients. it has been indicated that adjuvant sorafenib was safe and well tolerated. os and dfs had increased significantly in this study.113 sorafenib combination a. sorafenib plus cytotoxic chemotherapy agents doxorubicin: results of a phase ii study indicated that median os, pfs and time to progression (ttp) favored sorafenib.114 gemox (gemcitabine plus oxaliplatin): a randomized phase ii trial showed no significant difference of the os.115 oxaliplatin: results of a phase ii trial indicated that there is no considerable difference of os and ttp between two groups.116 b. sorafenib plus egfr inhibitors erlotinib: no survival benefit favored this combination in a phase iii trial.117 c. sorafenib plus mtor inhibitors everolimus: this combination failed to show significant advantages rather placebo in a phase iii clinical trial on 546 patients.118 temsirolimus: different phase i-ii trials resulted in no favorable benefits of this combination.119–121 d. sorafenib plus mek inhibitors refametinib: results of first line study revealed good efficacy of this combination. disease control rate (dcr) and overall response rate (orr) were 43% and 5% respectively.122 regorafenib. regorafenib is a multi-kinase inhibitor which inhibits kinases in angiogenesis and oncogenesis. it has been tried as a second line therapy after sorafenib in phase i and ii clinical trials. the orr and the dcr of regorafenib were 3% and 72% respectively. drug-related adverse effects consisted of hand–foot skin reaction, diarrhea, fatigue, hypothyroidism, anorexia, hypertension, nausea and voice changes. median os was 13.8 months. thoroughly, regorafenib was well tolerated and has antitumour activity on hcc.123 brivanib. another tki is brivanib which is a dual tki receptor of vegfr (vegf receptor) and fibroblast growth factor receptor (fgfr).110,124 brisk-fl trial of 1150 patients was performed to compare brivanib and sorafenib. results of this trial favored none of them. os, ttp, orr, dcr was the same. somehow, brivanib showed more toxicity than sorafenib. brisk-ps trial, which compared brivanib with placebo failed to give a significant increase in os.125 axitinib. it is a selective tyrosine kinase inibitor of vegfr-1,-2 and -3. results of a clinical trial of 202 patients showed no significant improve in os in comparison with sorafenib.126 sunitinib. sunitinib, a multitargeted tyrosine kinase inhibitor, have been used in the treatment of hcc. cheng et al. performed a phase iii clinical trial on 1074 patients due to compare sorafenib and sunitinib. their results showed no significant difference between os in two groups while aes of sunitinib were higher.127 linifanib. linifanib is a new atp-competitive inhibitor of all vegf and pdgf receptor tyrosine kinases, which presents no activity against representative cytosolic tyrosine kinases and serine/threonine kinase.128 a phase iii randomized clinical study on 1035 patients from 28 countries was performed to compare the efficacy and tolerability of linifanib versus sorafenib. the results showed similar os of sorafenib and linifanib. sorafenib showed more safety than linifanib but ttp and orr advocated linifanib.128 met-tki tivantinib. tivantinib, a c-met tk inhibitor, is used as a second line therapy of hcc in clinical trials. this drug is used as monotherapy and also in combination with sorafenib. in both types of monotherapy and in combination with sorafenib, safety results were acceptable.129 combination of tivantinib and sorafenib in phase ii clinical trial turned back sorafenib resistance.130 in another phase ii clinical trial of tivantinib, survival and disease control, increased and aes of the drug were manageable in addition, grade 3 neutropenia and myelo toxicity were observed as the side effects in some studies.130 cabozantinib. a randomized phase ii trial used this drug as the second-line therapy. results showed median os as 15.1 months and dcr as 68%.132 monoclonal antibodies bevacizumab. bevacizumab attaches to vegf-a which is the main form of vegf in blood. two phase ii trial studies have been done in order to assess this monoclonal antibody effect on hcc. orr was 13% and 14% interestingly, but this drug failed to develop because of safety concerns.133,134 ramucirumab. it is a human igg1 monoclonal antibody. ramucirumab targets vegfr-2. in the reach phase iii trial on 565 patients, no significant os improvement was observed except in patients with alpha fetoprotein (afp) levels more than 400 ng/ml.135 chemotherapy doxorubicin. doxorubicin has been recently compared to the oxaliplatin-flourouracil (folfox regimen) combination in a phase iii trial. os benefits extended (6.5 versus 4.9 months) in the folfex arm and orr was significantly higher (8.2% versus 2.7%).114 gemox (gemcitabine plus oxaliplatin). results of several phase ii clinical trial studies showed that orr (20%) and dcr (65%) were extended and drug safety profile was favorable.136 ali kabir et al. 245j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 review hepatocellular treatment immunotherapy tremelimumab. anti-ctla4 monoclonal antibody, tremelimumab, is a novel immune-based therapy used in hcc patients. in addition to good safety profile, this drug showed acceptable orr and dcr (18% and 76% respectively).137 pd-1 blocking antibodies viral therapy. jx-594 has currently been used in a phase ii study as an immunotherapeutic and oncolytic vaccine. high dose levels of this vaccine showed longer os in comparison of low dose levels.138 lenalidomide. a phase ii trial study has been tested lenalidomide-a thalidomide analoguein advanced hcc patients after sorafenib failure. median os and pfs were 7.6 and 3.6 months, respectively.139 tgf-b inhibitors. tgf-b inhibitor was conducted on 109 patients in a phase ii trial. median os and ttp were 9 and 3 months, respectively.140 a. personalized therapy two different strategies can be used in this approach: liquid biopsy plasma dna. we can identify cancer-associated changes by using both of these methods. detection of point mutations, detection of aberrant dna methylation, and the detection of chromosomal aberrations are detailed methods of molecular change identification.141 combination therapies and their comparison combination of minimally invasive therapies iodine-125 implantation plus radiofrequency ablation. radiation therapy needs oxygen to destroy tumour cells. however, in the center of a tumour, lack of oxygenation reduces the effects of this therapy. this would be solved by heating the tumour area which makes more blood supply and more oxygenation.142 in a clinical trial of 136 patients, combination of radiofrequency ablation and iodine-125 implantation was evaluated. this combination therapy reduced the rate of recurrence (p = 0.004). the combination group had better results in survival rate than rfa-only group. the result of this study showed that the combination of iodine-125 and rfa would be an efficient option for patient with small hcc.143 combination of rfa and cik it was reported that hyperthermia can cause stimulation and activation of the immune system.144 in a clinical trial, 62 patients with primary hcc (ranged in diameter from 2 to 8) were enrolled. this combination was compared with rfa alone. all the complications were related to rfa. examined therapy had a better pfs rate (p < 0.0001). the risk of recurrence rate was significantly lower for new therapy (hr = 0.136, 95% ci: 0.049–0.379). the function of liver in combination group did not change and had the same functionality before cit infusion. based on the results of that study, the combination of cik and rfa was a promising treatment for hcc.145 in a clinical trial by wang x et al, the combination of cik and rf hyperthermia was evaluated. thirty one patients with advanced hcc were enrolled. in this study instead of intravenous perfusion of cik, the investigators use intraperitoneal perfusion of cytokine, because it leads cik in to tumour tissue and makes the treatment more effective. median ttp was 6.1 months and median os was 8.5 months. the results showed that this combination therapy was an effective therapeutic choice for patients with advanced hcc.146 radiofrequency hyperthermia and conformal radiotherapy assessing the combination of radiotherapy and hyperthermia is a field of interest. conformal radiotherapy elevates the dose of irradiation in tumour and decrease receiving doses in normal tissue of the liver and cause low damage to normal tissue. also, hyperthermia causes more blood flow to the tumour tissue, which has an additive effect on the outcome of radiotherapy.142 in study by dong y, short and long term of radiofrequency hyperthermia combined with conformal radiotherapy was investigated. data were collected from 80 patients with primary advanced hcc. the patients were randomly assigned into two groups: experimental group and control group, each with 40 patients. bilirubin, albumin, alt and pt levels were the same between two groups before the treatments. however, after treatments bilirubin, alt and pt were reduced more significantly in the experimental group (p < 0.05). albumin was elevated in both groups but this change was significant in experimental group (p < 0.05). combined therapy was much more effective than the other therapy (p < 0.001). follow-up results demonstrated that the experimental group had significantly better os and also lower recurrence rate (p < 0.001). based on these results, the combination of radiofrequency hyperthermia and conformal radiotherapy has low damage to the liver and it is an effective treatment option for advanced hcc patients.147 percutaneous ethanol injection and radiofrequency ablation li et al, in meta-analysis compared the combination of pei + rfa with pei and rfa as monotherapy. this study analysed the results of 13 studies from different parts of the world. first of all in this study rfa and pei were compared. results showed that rfa has better function than pei in improving os. recurrence rate did not have significant difference in two therapies. on the other hand, pei shows more complete tumour necrosis than rfa. in comparison of combination of these two therapies with themselves, overall the combination therapy shows better results than rfa. in spite of good result in complete tumour necrosis for combination group, this difference is not significant.148 chemotherapy and minimally invasive ablative therapies tace and rfa. in a study effectiveness of a combined therapy of rfa and deb-tace was compared with the deb-tace procedure alone in treatment of single hcc. cr at 1 month was achieved in 80% of tumours. the group treated with the combination therapy showed a significantly lower 2-year recurrence (48.1% vs. 78.2%, p < 0.001) and significantly higher survival (91.1% vs. 60.6%, p = 0.004) than the group treated with debtace alone.149 in a retrospective analysis of 20 patients with intermediate size hcc, the combination of deb-tace and mr-guided rfa was evaluated. all of the cases had child-pugh class a and b liver function, no tumour metastasis and hcc with tumour size >3 cm. no major therapy-related complications were observed except for a sub capsular hepatoma which was seen in one patient right after the rfa. median os was 37.4 months. this combination seemed to be an effective therapy for patients with medium size hcc.150 tace and cik and rfa. retrospective analysis of patients with hcc showed that rfa + cik + tace had more 246 j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 hepatocellular treatment review ali kabir et al. id en ti fic at io n sc re en in g el ig ib ili ty in cl u d ed records excluded (n = 1665) full-text articles excluded (n = 387) records after duplicates removed (n = 3788) title/abstract screened (n = 2123) full-text assessed for eligibility (n = 1736) studies included in qualitative synthesis (n = 1349) final number of included studies in drafting manuscript (n = 173) records identified in pubmed/cochrane (n = 2471) records identified in scopus (n = 2151) fig. 1 the flowchart depicts the selection of studies for this review. acceptable effect than rfa + tace. median survival time (st) for the study group and the control group were 56 and 31 months, respectively which is statistically meaningful (p = 0.023). the results of this study demonstrated that cik lower the risk of metastasis and recurrence in the study group and can improve the outcome of tace + rfa.151 tace and pve and survival. in one rct, 62 patients were divided into two groups, a group which had a combination of tace and pve and hifu as a therapy and the other group had a combination tace and pve as a therapy. nausea and vomiting were reported as a main side effect in both groups. however, in the experimental group after hifu local pain was seen as a side effect in most of the cases. response rate became 72% after adding hifu to the therapy. this rate was higher than the response rate in control group (44%). also, controlling time of hcc in study group was higher than the control group. median survival for experimental and control group were 16 months and 10 months respectively. also, the level of afp was decreased in both groups, but in experimental group this happened in lesser scale than the control group and had significant difference (p < 0.01). this combination is a safe and effective treatment for advanced hcc.152 chemotherapy and radiotherapy one rct conducted by bush, et al. compared proton beam radiotherapy with tace in treatment for hcc. thirty six patients in tace group received at least one trace with additional trace for persistent disease and were treated with a mixture of ethiodol, carboplatin and doxorubicin with or without mitomycin. thirty three patients in proton beam treatment group received the therapy to all parts of gross disease to a dose of 70.2 gy. the 2-year os for both groups was 59%. median st was 30 months (95% ci: 20.7–39.3 months). pfs survival was more in the proton beam treatment group (48% vs. 31%, p = 0.06).153 in a meta-analysis conducted by lobo, et al. clinical outcomes of transarterial radioembolization (tare) and tace in treatment of unresectable hcc were compared. in tace group, 284 patients were injected chemotherapy into their liver tumours, while 269 patients in tare group had an injection of β-emitting yttrium-90. no significant difference was observed in survival for up to 4 years between the two groups (hr = 1.06; 95% ci: 0.81–1.46, p = 0.567). the patient treated with tace needs at least one day of hospital stay, while tare is an outpatient procedure.154 in another meta-analysis by zhang y et al, different results were observed. they believed that tare with yttrium-90 had better os, ttp and lower hospitalization time over tace .155 in a clinical trial, jun ma et al, evaluated the combination of tace and licartin on 341 patients. the major side effect of therapy in study group was thrombocytopenia, leucopenia and increased total bilirubin. all of the cases had stage iii or iv hcc. it was reported that the efficacy of this combination therapy was higher in stage iii patients. researchers suggested this combination as a treatment for hcc patients.156 in another ali kabir et al. 247j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 review hepatocellular treatment trial, this combination had acceptable outcomes for intermediate stage hcc and it was suggested as a therapeutic option for this stage.157 in another study efficacy of the combination of tace and brachytherapy was compared to tace alone. the study group had combination of loboplatin-tace and 125i seeds as radiotherapy. dcr was higher in the study group (p < 0.05). mean st was 22.9 months for study group and 19.6 months for control group. fever, pain and discomfort in liver area, nausea and vomiting and mild diarrhea were common complications in both groups. however, in the study group after 125i implantation, liver function abnormalities and varying degrees of bone marrow suppression were observed. in this study, liver damage was low in the study group. this combination has efficacy for advanced hcc.158 in another study combination of (debs)-based tace and sbrt was evaluated. os was remarkably higher in the tace + sbrt group compared with the tace-only group (33 and 20 months, respectively; p = 0.02). it can be a treatment for hcc with >3 cm tumour size.159 in a retrospective study the effect of the combination of tace + 3-dcrt + hifu was evaluated. after the combination of tace and 3-dcrt, hifu was administered. median st and ttp were 26 and 9 months, respectively. toxicity of this procedure was more common in cases with hbv and cirrhotic liver. hepatic toxicities, gastrointestinal bleeding, and leukopenia were the major therapy-related toxicity of this combination, however for hifu firstand second-degree skin burn were reported as major toxicity. this therapy is suggested for patients with unrespectable hcc.160 tace and gene therapy in two retrospective studies, the combination of adenovirus type 5 and tace was compared to tace monotherapy. combination group had a better cr, pr and stable disease rate than monotherapy. in addition, this combination causes improvement in os and pfs. it can be considered as an option for unresectable hcc.6,161 systemic therapy and chemotherapy sorafenib and chemotherapy recent studies have compared the effect of hiac and sorafenib on advanced hcc. results demonstrated that the median os was better in hiac-treated patients. hiac can be a rational choice for patients with advanced hcc.162,163 however, it was observed that in cases with micro vascular invasion, sorafenib had a better outcome.163 in a phase ii trial by cosgrove et al, safety and efficacy of sorafenib and deb-tace was evaluated. fifty patients were enrolled. most of them were male and had bclc stage c hcc. median survival for bclc stages a, b and c was 45.6, 29.7 and 8 months, respectively. cases within bclc stage c hcc who had sorafenib therapy for more than 6 months had better st. complications of this combination were mostly related to sorafenib. the results demonstrated that this combination had safety and efficacy for patients with hcc, especially in cases with advanced hcc.164 other systemic therapies and tace in a clinical trial of 26 patients, the combination of bevacizumab and tace was evaluated. in this study, all of the cases had bclc stage b or c hcc. in this study, median survival was 10.8 months. the result of this study demonstrated that this combination was an effective therapy for unresectable hcc.165 in a retrospective analysis of 103 patients, combination of sunitinib and tace was assessed. based on the results, this combination therapy elongates the survival period and postpones tumour progression and can be an option for advanced hcc.166 sorafenib and rfa in a retrospective study the effect of sorafenib and rfa was compared with rfa alone. data were collected from 128 patients who had hcc within bclc stage 0 to b1. patients were equally divided in two groups. sorafenib was administered after the rfa procedure. the recurrence rate was lower in the combination group. median os for rfa-sorafenib group and rfa group were 161.8 and 118.6 weeks, respectively. this combination reduced the recurrence rate. it may be an efficient therapeutic option for inoperable hcc.167 in rct the effect of sorafenib and percutaneous radiofrequency ablation was examined. in another study, 62 patients were enrolled, and it was demonstrated that the effect of combination therapy was more efficient for medium-size hcc than rfa alone. this study showed the same results about recurrence rate.168 systemic therapy and radiotherapy radiotherapy and sorafenib. nakazawa, et al. conducted a study to evaluate sorafenib and rt used in the treatment of unresectable hcc with major pvtt. os was compared between cases with pvtt. there is no significant difference in the median survival (4.3 vs. 5.9 months; p = 0.115). after the propensity score matching (n = 28 per group), better survival was observed in the rt group than in the sorafenib group (median survival, 10.9 vs. 4.8 months; p = 0.025).169 in another study yang y, et al evaluated the effect of cryotherapy plus sorafenib as a combination therapy for patients with advanced hcc. a total of 296 patients with hbv-related hcc in advance stage were enrolled and follow up for 2 years. the authors believe that the curative mechanism of sorafenib had a synergic effect on the effect of local cryorx. median os was 12.5 months for cases in the combination group while median os for sorafenib-only group was 8.6 months. this combination is safe and effective therapy for advanced hcc.170 sorafenib and radioembolization. radiation causes activation of intracellular signaling pathway. also, it causes the increasing of vegf in the body. in a phase two clinical trial of 29 hcc cases within bclc stage b and c, safety and efficacy of the sorafenib plus radiembolization was examined. for radiormbolization a form of brachytherapy, 90y-microsphere was administered. twenty five percent of cases showed the best overall response and among these cases, 28% showed cr. median os for cases in bclc stage b was 15.2 months and for bclc stage c was 6.5 months. the results of this study confirmed this combination as a safe and efficient option for advanced hcc.171 surgical resection and minimally invasive ablative therapies. for small hcc, mainly there are two different therapeutic options. one of them is rfa and the other one is surgical resection (sr).172,173 in recent meta-analyses, it has shown that for small hcc (≤3 cm), sr is more efficient than rfa.172,173 in small hcc the one-, threeand five-year os rate is higher in patients who had sr as a treatment option.172,173 the rate of local recurrence of hcc in sr group was lower than rfa group.173 also, for single nodular hcc, sr will be a suitable choice but for multi nodular hcc, rfa is a better choice, because of surgical complications.172 performing rfa 248 j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 hepatocellular treatment review ali kabir et al. by laparoscopic approach shows similar results in os of patients as sr but the rate of local recurrence of hcc is still higher.173 conclusion in summary, recent changes in managing of hcc have shown a trend in using minimally invasive and noninvasive options instead of hepatic resection and transplantation. new technologies in ablative devices, radiotherapy and chemotherapy have improved the accuracy and efficacy of these treatments. in addition, using immune system, gene therapy and new targeted therapy agents have shown the high potential of cellular and molecular mechanisms in managing hcc. however, more rcts are needed to evaluate the effect of these therapies. moreover, the combination of different sort of therapy has become popular recently. many researchers all over the world have reported the benefit of the majority of these combinations. however, in these cases more studies are needed to prove the advantage of this kind of therapy over current curative options. conflict of interest no one of the authors have any conflict of interest. n references 1. ferlay j, soerjomataram i, dikshit r, eser s, mathers c, rebelo m, et al. cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012. int j cancer. 2015;136:e359–e386. 2. wallace mc, preen d, jeffrey gp, adams la. the evolving epidemiology of hepatocellular carcinoma: a global perspective. expert rev gastroenterol hepatol. 2015;9:765–779. 3. bruix j, gores gj, mazzaferro v. hepatocellular carcinoma: clinical frontiers and perspectives. gut. 2014;63:844–855. 4. forner a, llovet jm, bruix j. hepatocellular carcinoma. lancet. 2012;379: 1245–1255. 5. aerts m, benteyn d, van vlierberghe h, thielemans k, reynaert h. current status and perspectives of immune-based therapies for hepatocellular carcinoma. world j gastroenterol. 2016;22:253–261. 6. dong j, li w, dong a, mao s, shen l, li s, et al. gene therapy for unresectable hepatocellular carcinoma using recombinant human adenovirus type 5. med oncol. 2014;31. 7. wei z, doria c, liu y. targeted therapies in the treatment of advanced hepatocellular carcinoma. clinical medicine insights oncology. 2013;7:87–102. 8. zhu ax. systemic therapy of advanced hepatocellular carcinoma: how hopeful should we be?. the oncologist. 2006;11:790–800. 9. lai cl, wu pc, chan gc, lok as, lin hj. doxorubicin versus no antitumor therapy in inoperable hepatocellular carcinoma. a prospective randomized trial. cancer. 1988;62:479–483. 10. petrelli f, coinu a, borgonovo k, cabiddu m, ghilardi m, lonati v, et al. oxaliplatin-based chemotherapy: a new option in advanced hepatocellular carcinoma. a systematic review and pooled analysis. clin oncol. 2014;26: 488–496. 11. qin s, bai y, lim hy, thongprasert s, chao y, fan j, et al. randomized, multicenter, open-label study of oxaliplatin plus fluorouracil/leucovorin versus doxorubicin as palliative chemotherapy in patients with advanced hepatocellular carcinoma from asia. j clin oncol. 2013;31:3501–3508. 12. bruix j, sherman m. management of hepatocellular carcinoma. hepatology. 2005;42:1208–1236. 13. paul sb, sharma h. role of transcatheter intra-arterial therapies for hepatocellular carcinoma. j clin exp hepatol. 2014;4:s112–s121. 14. riemsma rp, bala mm, wolff r, kleijnen j. transarterial (chemo)embolisation versus no intervention or placebo intervention for liver metastases. cochrane database syst rev. 2013:cd009498. 15. european association for the study of the liver, european organisation for research and treatment of cancer. easl–eortc clinical practice guidelines: management of hepatocellular carcinoma. j hepatol. 2012;56:908–943. 16. zhao y, cai g, zhou l, liu l, qi x, bai m, et al. transarterial chemoembolization in hepatocellular carcinoma with vascular invasion or extrahepatic metastasis: a systematic review. asia pac j clin oncol. 2013;9:357–364. 17. xie zb, ma l, wang xb, bai t, ye jz, zhong jh, et al. transarterial embolization with or without chemotherapy for advanced hepatocellular carcinoma: a systematic review. tumour biol. 2014;35:8451–8459. 18. ogawa m, takayasu k, hirayama m, miura t, shiozawa k, abe m, et al. efficacy of a microballoon catheter in transarterial chemoembolization of hepatocellular carcinoma using miriplatin, a lipophilic anticancer drug: short-term results. hepatol res. 2016;46:e60–e69. 19. tawada a, chiba t, ooka y, kanogawa n, saito t, motoyama t, et al. transarterial chemoembolization with miriplatin plus epirubicin in patients with hepatocellular carcinoma. anticancer res. 2015;35:549–554. 20. lewis al, gonzalez mv, lloyd aw, hall b, tang y, et al. dc bead: in vitro characterization of a drug-delivery device for transarterial chemoembolization. j vasc interv radiol. 2006;17:335–342. 21. huang k, zhou q, wang r, cheng d, ma y. doxorubicin-eluting beads versus conventional transarterial chemoembolization for the treatment of hepatocellular carcinoma. j gastroenterol hepatol. 2014;29:920–925. 22. lammer j, malagari k, vogl t, pilleul f, denys a, watkinson a, et al. prospective randomized study of doxorubicin-eluting-bead embolization in the treatment of hepatocellular carcinoma: results of the precision v study. cardiovasc intervent radiol. 2010;33:41–52. 23. sacco r, bargellini i, bertini m, bozzi e, romano a, petruzzi p, et al. conventional versus doxorubicin-eluting bead transarterial chemoembolization for hepatocellular carcinoma. j vasc interv radiol. 2011;22:1545–1552. 24. wiggermann p, sieron d, brosche c, brauer t, scheer f, platzek i, et al. transarterial chemoembolization of child-a hepatocellular carcinoma: drug-eluting bead tace (deb tace) vs. tace with cisplatin/lipiodol (ctace). med sci monit. 2011;17:cr189–cr195. 25. song mj, chun hj, kim hy, yoo sh, park ch, bae sh, et al. comparative study between doxorubicin-eluting beads and conventional transarterial chemoembolization for treatment of hepatocellular carcinoma. j hepatol. 2012;57:1244–1250. 26. kloeckner r, weinmann a, prinz f, pinto dos santos d, ruckes c, dueber c, et al. conventional transarterial chemoembolization versus drug-eluting bead transarterial chemoembolization for the treatment of hepatocellular carcinoma. bmc cancer. 2015;15:465. 27. gao s, yang z, zheng z, yao j, deng m, xie h, et al. doxorubicin-eluting bead versus conventional tace for unresectable hepatocellular carcinoma: a meta-analysis. hepato-gastroenterol. 2013;60:813–820. 28. katsumori t, kasahara t. the size of gelatin sponge particles: differences with preparation method. cardiovasc intervent radiol. 2006;29:1077–1083. 29. wu pz, zhou j, zhang yw. gelatin sponge microparticles for the treatment of the spontaneous rupture of hepatocellular carcinoma hemorrhage. exp therap med. 2016;12:2201–2207. 30. de baere t, arai y, lencioni r, geschwind jf, rilling w, salem r, et al. treatment of liver tumors with lipiodol tace: technical recommendations from experts opinion. cardiovasc intervent radiol. 2016;39:334–343. 31. zhang yw, ao j, liu y, qiao mx, yang xl, tang sx, et al. pharmacokinetics of gelatin sponge microparticles in a rabbit vx2 liver tumor model of hepatic arterial chemoembolization. tumor biol. 2014;35:10905–10910. 32. kamran au, liu y, li fe, liu s, wu jl, zhang yw. transcatheter arterial chemoembolization with gelatin sponge microparticles treated for bclc stage b hepatocellular carcinoma: a single center retrospective study. medicine (baltimore). 2015;94:e2154. 33. qi x, wang d, su c, li h, guo x. hepatic resection versus transarterial chemoembolization for the initial treatment of hepatocellular carcinoma: a systematic review and meta-analysis. oncotarget. 2015;6:18715–18733. 34. kapitanov t, neumann up, schmeding m. hepatocellular carcinoma in liver cirrhosis: surgical resection versus transarterial chemoembolization-a metaanalysis. gastroenterol res pract. 2015;2015:696120. 35. he g, zheng c, huo h, zhang h, zhu z, li j, et al. tace combined with dendritic cells and cytokine-induced killer cells in the treatment of hepatocellular carcinoma: a meta-analysis. int immunopharmacol. 2016;40:436–442. 36. mesiano g, todorovic m, gammaitoni l, leuci v, giraudo diego l, carnevale-schianca f, et al. cytokine-induced killer (cik) cells as feasible and effective adoptive immunotherapy for the treatment of solid tumors. exp opin biol ther. 2012;12:673–684. 37. rude mk, crippin js. liver transplantation for hepatocellular carcinoma. curr gastroenterol rep. 2015;17:1–5. ali kabir et al. 249j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 review hepatocellular treatment 38. o’grady j. liver transplantation. medicine (united kingdom). 2015;43:686–688. 39. sheth ra, patel ms, koottappillil b, shah ja, oklu r, mueller p, et al. role of locoregional therapy and predictors for dropout in patients with hepatocellular carcinoma listed for liver transplantation. j vasc interv radiol. 2015;26:1761–1768; quiz 1768. 40. na gh, kim ey, hong th, you yk, kim dg. effects of loco regional treatments before living donor liver transplantation on overall survival and recurrencefree survival in south korean patients with hepatocellular carcinoma. hpb (oxford). 2016;18:98–106. 41. teng f, wang gh, tao yf, guo wy, wang zx, ding gs, et al. criteria-specific long-term survival prediction model for hepatocellular carcinoma patients after liver transplantation. world j gastroenterol. 2014;20:10900–10907. 42. paredes ah, satoskar r. hepatocellular carcinoma: when to transplant outside of milan criteria. curr hepat rep. 2013;12:37–46. 43. kudo m, izumi n, kokudo n, matsui o, sakamoto m, nakashima o, et al. management of hepatocellular carcinoma in japan: consensus-based clinical practice guidelines proposed by the japan society of hepatology (jsh) 2010 updated version. dig dis. 2011;29:339–364. 44. poon rt, fan st, lo cm, liu cl, wong j. long-term survival and pattern of recurrence after resection of small hepatocellular carcinoma in patients with preserved liver function: implications for a strategy of salvage transplantation. ann surg. 2002;235:373–382. 45. felli e, cillo u, pinna ad, de carlis l, ercolani g, santoro r, et al. salvage liver transplantation after laparoscopic resection for hepatocellular carcinoma: a multicenter experience. updates in surgery. 2015;67:215–222. 46. zhu y, dong j, wang wl, li mx, lu y. shortand long-term outcomes after salvage liver transplantation versus primary liver transplantation for hepatocellular carcinoma: a meta-analysis. transplant proc. 2013;45:3329–3342. 47. rao a, rao g, ahmed i. laparoscopic vs. open liver resection for malignant liver disease. a systematic review. surgeon. 2012;10:194–201. 48. bruix j, sherman m. management of hepatocellular carcinoma: an update. hepatology. 2011;53:1020–1022. 49. dahiya d, wu tj, lee cf, chan km, lee wc, chen mf. minor versus major hepatic resection for small hepatocellular carcinoma (hcc) in cirrhotic patients: a 20-year experience. surgery. 2010;147:676–685. 50. arii s, tanaka s, mitsunori y, nakamura n, kudo a, noguchi n, et al. surgical strategies for hepatocellular carcinoma with special reference to anatomical hepatic resection and intraoperative contrast-enhanced ultrasonography. oncology. 2010;78:125–130. 51. zhou y, xu d, wu l, li b. meta-analysis of anatomic resection versus nonanatomic resection for hepatocellular carcinoma. langenbecks arch surg. 2011;396:1109–1117. 52. liu ph, lee yh, hsu cy, hsia cy, huang yh, chiou yy, et al. surgical resection is better than transarterial chemoembolization for hepatocellular carcinoma beyond milan criteria independent of performance status. j gastrointest surg. 2014;18:1623–1631. 53. topal b, fieuws s, aerts r, vandeweyer h, penninckx f. laparoscopic versus open liver resection of hepatic neoplasms: comparative analysis of shortterm results. surg endosc. 2008;22:2208–2213. 54. santambrogio r, bruno s, kluger md, costa m, salceda j, belli a, et al. laparoscopic ablation therapies or hepatic resection in cirrhotic patients with small hepatocellular carcinoma. dig liver dis. 2016;48:189–196. 55. lencioni r. loco‐regional treatment of hepatocellular carcinoma. hepatology. 2010;52:762–773. 56. crocetti l, de baere t, lencioni r. quality improvement guidelines for radiofrequency ablation of liver tumours. cardiovasc intervent radiol. 2010;33:11–17. 57. vouche m, habib a, ward tj, kim e, kulik l, ganger d, et al. unresectable solitary hepatocellular carcinoma not amenable to radiofrequency ablation: multicenter radiology-pathology correlation and survival of radiation segmentectomy. hepatology. 2014;60:192–201. 58. padia sa, kwan sw, roudsari b, monsky wl, coveler a, harris wp. superselective yttrium-90 radioembolization for hepatocellular carcinoma yields high response rates with minimal toxicity. j vasc interv radiol. 2014;25:1067–1073. 59. garin e, lenoir l, edeline j, laffont s, mesbah h, poree p, et al. boosted selective internal radiation therapy with 90y-loaded glass microspheres (b-sirt) for hepatocellular carcinoma patients: a new personalized promising concept. eur j nucl med mol imaging. 2013;40:1057–1068. 60. garin e, lenoir l, rolland y, edeline j, mesbah h, laffont s, et al. dosimetry based on 99mtc-macroaggregated albumin spect/ct accurately predicts tumor response and survival in hepatocellular carcinoma patients treated with 90y-loaded glass microspheres: preliminary results. j nucl med. 2012;53:255–263. 61. garin e, rolland y, edeline j, icard n, lenoir l, laffont s, et al. personalized dosimetry with intensification using 90y-loaded glass microsphere radioembolization induces prolonged overall survival in hepatocellular carcinoma patients with portal vein thrombosis. j nucl med. 2015;56:339–346. 62. imrt therapy collaborative working group. intensity-modulated radiotherapy: current status and issues of interest. international journal of radiation oncology, biology, physics. 2001;51:880–914. 63. mackie tr, holmes t, swerdloff s, reckwerdt p, deasy jo, yang j, et al. tomotherapy: a new concept for the delivery of dynamic conformal radiotherapy. med phys. 1993;20:1709–1719. 64. kim jy, yoo ej, jang jw, kwon jh, kim kj, kay cs. hypofractionated radiotheapy using helical tomotherapy for advanced hepatocellular carcinoma with portal vein tumor thrombosis. radiat oncol. 2013;8. 65. jung j, kong m, hong se. conventional fractionated helical tomotherapy for patients with small to medium hepatocellular carcinomas without portal vein tumor thrombosis. onco targets ther. 2014;7:1769–1775. 66. yoon hi, lee ij, han kh, seong j. improved oncologic outcomes with image-guided intensity-modulated radiation therapy using helical tomotherapy in locally advanced hepatocellular carcinoma. j cancer res clin oncol. 2014;140:1595–1605. 67. hou jz, zeng zc, wang bl, yang p, zhang jy, mo hf. high dose radiotherapy with image-guided hypo-imrt for hepatocellular carcinoma with portal vein and/or inferior vena cava tumor thrombi is more feasible and efficacious than conventional 3d-crt. jpn j clin oncol. 2016;46:357–362. 68. otto k. volumetric modulated arc therapy: imrt in a single gantry arc. med phys 2008;35:310–317. 69. gong gz, yin y, xing lg, guo yj, liu t, chen j, et al. rapidarc combined with the active breathing coordinator provides an effective and accurate approach for the radiotherapy of hepatocellular carcinoma. strahlenther onkol. 2012;188:262–268. 70. gong g, yin y, guo y, liu t, chen j, lu j, et al. dosimetric differences among volumetric modulated arc radiotherapy (rapidarc) plans based on different target volumes in radiotherapy of hepatocellular carcinoma. j radiat res. 2013;54:182–189. 71. potters l, kavanagh b, galvin jm, hevezi jm, janjan na, larson da, mehta mp, et al. american society for therapeutic radiology and oncology (astro) and american college of radiology (acr) practice guideline for the performance of stereotactic body radiation therapy. int j radiat oncol biol phys. 2010;76:326–332. 72. thariat j, marcié s, marcy py, trimaud r, angellier g, mammar h, et al. [cyberknife robotic stereotactic radiotherapy: technical aspects and recent developments]. bull cancer. 2010;97:807–818. 73. oldrini g, taste-george h, renard-oldrini s, baumann as, marchesi v, troufléau p, et al. implantation of fiducial markers in the liver for stereotactic body radiation therapy: feasibility and results. diagn interv imaging. 2015;96:589–592. 74. kim jw, seong j, lee ij, woo jy, han kh. phase i dose escalation study of helical intensity-modulated radiotherapy-based stereotactic body radiotherapy for hepatocellular carcinoma. oncotarget. 2016;7: 40756–40766. 75. bowen sr, saini j, chapman tr, miyaoka rs, kinahan pe, sandison ga, et al. differential hepatic avoidance radiation therapy: proof of concept in hepatocellular carcinoma patients. radiother oncol. 2015;115:203–210. 76. beddar as, kainz k, briere tm, tsunashima y, pan t, prado k, et al. correlation between internal fiducial tumor motion and external marker motion for liver tumors imaged with 4d-ct. int j radiat oncol biol phys. 2007;67:630–638. 77. law al, ng wt, lee mc, chan at, fung kh, li f, et al. treatment of primary liver cancer using highly-conformal radiotherapy with kv-image guidance and respiratory control. radiother oncol. 2012;102:56–61. 78. kitada t, murakami t, kuzushita n, minamitani k, nakajo k, osuga k, et al. effectiveness of real-time virtual sonography-guided radiofrequency ablation treatment for patients with hepatocellular carcinomas. hepatol res. 2008;38:565–571. 79. nakai m, sato m, sahara s, takasaka i, kawai n, minamiguchi h, et al. radiofrequency ablation assisted by real-time virtual sonography and ct for hepatocellular carcinoma undetectable by conventional sonography. cardiovasc intervent radiol. 2009;32:62–69. 80. sandulescu dl, dumitrescu d, rogoveanu i, saftoiu a. hybrid ultrasound imaging techniques (fusion imaging). world j gastroenterol. 2011;17:49–52. 81. lee mw, rhim h, cha di, kim yj, choi d, kim ys, et al. percutaneous radiofrequency ablation of hepatocellular carcinoma: fusion imaging guidance for management of lesions with poor conspicuity at conventional sonography. ajr am j roentgenol. 2012;198:1438–1444. 250 j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 hepatocellular treatment review ali kabir et al. 82. xu zf, xie xy, kuang m, liu gj, chen ld, zheng yl, et al. percutaneous radiofrequency ablation of malignant liver tumors with ultrasound and ct fusion imaging guidance. j clin ultrasound. 2014;42:321–330. 83. goldberg sn, solbiati l, hahn pf, cosman e, conrad je, fogle r, et al. large-volume tissue ablation with radio frequency by using a clustered, internally cooled electrode technique: laboratory and clinical experience in liver metastases. radiology. 1998;209:371–379. 84. kettenbach j, köstler w, rücklinger e, gustorff b, hüpfl m, wolf f, et al. percutaneous saline-enhanced radiofrequency ablation of unresectable hepatic tumors: initial experience in 26 patients. ajr am j roentgenol. 2003;180:1537–1545. 85. kim jh, kim pn, won hj, shin ym. percutaneous radiofrequency ablation using internally cooled wet electrodes for the treatment of hepatocellular carcinoma. ajr am j roentgenol. 2012;198:471–476. 86. kim jh, kim pn, won hj, shin ym. percutaneous radiofrequency ablation with internally cooled versus internally cooled wet electrodes for small subphrenic hepatocellular carcinomas. j vasc interv radiol 2013;24:351–356. 87. osaki y, ikeda k, izumi n, yamashita s, kumada h, hatta s, et al. clinical effectiveness of bipolar radiofrequency ablation for small liver cancers. j gastroenterol. 2013;48:874–883. 88. mazzaferro v, battiston c, perrone s, pulvirenti a, regalia e, romito r, et al. radiofrequency ablation of small hepatocellular carcinoma in cirrhotic patients awaiting liver transplantation: a prospective study. ann surg. 2004;240:900–909. 89. frericks bb, ritz jp, roggan a, wolf kj, albrecht t. multipolar radiofrequency ablation of hepatic tumors: initial experience. radiology. 2005;237:1056–1062. 90. seror o, n’kontchou g, van nhieu jt, rabahi y, nahon p, laurent a. et al. histopathologic comparison of monopolar versus no-touch multipolar radiofrequency ablation to treat hepatocellular carcinoma within milan criteria. j vasc interv radiol. 2014;25:599–607. 91. toyoda h, kumada t, tada t, kaneoka y, maeda a. placement of a sodium hyaluronate solution onto the liver surface as a supportive procedure for radiofrequency ablation of hepatocellular carcinomas located on the liver surface: a preliminary report. j vasc interv radiol. 2012;23:1639–1645.e1. 92. liu fy, yu xl, liang p, cheng zg, han zy, dong bw, et al. microwave ablation assisted by a real-time virtual navigation system for hepatocellular carcinoma undetectable by conventional ultrasonography. eur j radiol. 2012;81:1455–1459. 93. iannitti da, martin rc, simon cj, hope ww, newcomb wl, mcmasters km, et al. hepatic tumor ablation with clustered microwave antennae: the us phase ii trial. hpb (oxford). 2007;9:120–124. 94. ziemlewicz tj, hinshaw jl, lubner mg, brace cl, alexander ml, agarwal p, et al. percutaneous microwave ablation of hepatocellular carcinoma with a gas-cooled system: initial clinical results with 107 tumors. j vasc interv radiol. 2015;26:62–68. 95. llovet jm, vilana r, brú c, bianchi l, salmeron jm, boix l, et al. barcelona clínic liver cancer (bclc) group. increased risk of tumor seeding after percutaneous radiofrequency ablation for single hepatocellular carcinoma. hepatology. 2001;33:1124–1129. 96. patel pa, ingram l, wilson id, breen dj. no-touch wedge ablation technique of microwave ablation for the treatment of subcapsular tumors in the liver. j vasc interv radiol. 2013;24:1257–1262. 97. kim yj, lee mw, park hs. small hepatocellular carcinomas: ultrasonography guided percutaneous radiofrequency ablation. abdom imaging. 2013;38:98–111. 98. kang tw, rhim h, lee mw, kim ys, choi d, lee wj, et al. radiofrequency ablation for hepatocellular carcinoma abutting the diaphragm: comparison of effects of thermal protection and therapeutic efficacy. ajr am j roentgenol. 2011;196:907–913. 99. kang tw, lim hk, lee mw, kim ys, choi d, rhim h. first-line radiofrequency ablation with or without artificial ascites for hepatocellular carcinomas in a subcapsular location: local control rate and risk of peritoneal seeding at long-term follow-up. clin radiol. 2013;68:e641–e651. 100. zhang d, liang p, yu x, cheng z, han z, yu j, et al. the value of artificial pleural effusion for percutaneous microwave ablation of liver tumour in the hepatic dome: a retrospective case-control study. int j hyperthermia. 2013;29:663–670. 101. wuu c, kliauga p, zaider m, amols h. microdosimetric evaluation of relative biological effectiveness for 103pd, 125i, 241am, and 192ir brachytherapy sources. int j radiat oncol biol phys. 1996;36:689–697. 102. ricke j, wust p, stohlmann a, beck a, cho ch, pech m, et al. ct-guided interstitial brachytherapy of liver malignancies alone or in combination with thermal ablation: phase i–ii results of a novel technique. int j radiat oncol biol phys. 2004;58:1496–1505. 103. collettini f, schnapauff d, poellinger a, denecke t, schott e, berg t, et al. hepatocellular carcinoma: computed-tomography-guided high-dose-rate brachytherapy (ct-hdrbt) ablation of large (5–7 cm) and very large (>7 cm) tumours. eur radiol. 2012;22:1101–1109. 104. collettini f, schreiber n, schnapauff d, denecke t, wust p, schott e, et al. ct-guided high-dose-rate brachytherapy of unresectable hepatocellular carcinoma. strahlenther onkol. 2015;191:405–412. 105. wu f, wang zb, chen wz, wang w, gui y, zhang m, et al. extracorporeal high intensity focused ultrasound ablation in the treatment of 1038 patients with solid carcinomas in china: an overview. ultrason sonochem. 2004;11:149–154. 106. fukuda h, numata k, nozaki a, kondo m, morimoto m, maeda s, et al. high-intensity focused ultrasound ablation assisted using color doppler imaging for the treatment of hepatocellular carcinomas. abdom imaging. 2013;38:1263–1268. 107. anzidei m, napoli a, sandolo f, marincola bc, di martino m, berloco p, et al. magnetic resonance-guided focused ultrasound ablation in abdominal moving organs: a feasibility study in selected cases of pancreatic and liver cancer. cardiovasc intervent radiol. 2014;37:1611– 1617. 108. wu mc. traditional chinese medicine in prevention and treatment of liver cancer: function. saepjcim-c zhong xi yi jie he xue bao. 2003;1(3):163–164. 109. sun z, liang st, zhai xf, lang qb, zhou qh, yue xq, et al. a traditional chinese herbal medicine compound preparation versus interventional therapy after resection of small hepatocellular carcinoma: 22-year followup. j tradit chin med. 2012;32:156–163. 110. jeng ks, sheen is, wang yc, gu sl, chu cm, shih sc, et al. prognostic significance of preoperative circulating vascular endothelial growth factor messenger rna expression in resectable hepatocellular carcinoma: a prospective study. world j gastroenterol. 2004;10:643–648. 111. ye sl, chen x, yang j, bie p, zhang s, liu f, et al. safety and efficacy of sorafenib therapy in patients with hepatocellular carcinoma: final outcome from the chinese patient subset of the gideon study. oncotarget. 2016;7:6639–6648. 112. kudo m. adjuvant therapy after curative treatment for hepatocellular carcinoma. oncology. 2011;81:50–55. 113. wang sn, chuang sc, lee kt. efficacy of sorafenib as adjuvant therapy to prevent early recurrence of hepatocellular carcinoma after curative surgery: a pilot study. hepatol res. 2014;44:523–531. 114. abou-alfa gk, johnson p, knox jj, capanu m, davidenko i, lacava j, et al. doxorubicin plus sorafenib vs doxorubicin alone in patients with advanced hepatocellular carcinoma: a randomized trial. jama. 2010;304:2154–2160. 115. mir o, coriat r, boudou-rouquette p, ropert s, durand jp, cessot a, et al. gemcitabine and oxaliplatin as second-line treatment in patients with hepatocellular carcinoma pre-treated with sorafenib. med oncol. 2012;29:2793–2799. 116. yau tc, cheung fy, lee f, choo sp, wong h, toh hc, et al. a multicenter phase ii study of sorafenib, capecitabine, and oxaliplatin (secox) in patients with advanced hepatocellular carcinoma: final results of hong kong-singapore hepatocellular carcinoma research collaborative group study. in: asco annual meeting proceedings. 2013;2013. p. 4117. 117. zhu ax, rosmorduc o, evans tr, ross pj, santoro a, carrilho fj, et al. search: a phase iii, randomized, double-blind, placebo-controlled trial of sorafenib plus erlotinib in patients with advanced hepatocellular carcinoma. j clin oncol. 2015;33:559–566. 118. zhu ax, kudo m, assenat e, cattan s, kang yk, lim hy, et al. effect of everolimus on survival in advanced hepatocellular carcinoma after failure of sorafenib: the evolve-1 randomized clinical trial. jama. 2014;312:57–67. 119. kelley rk, nimeiri hs, munster pn, vergo mt, huang y, li cm, et al. temsirolimus combined with sorafenib in hepatocellular carcinoma: a phase i dose-finding trial with pharmacokinetic and biomarker correlates. ann oncol. 2013;24:1900–1907. 120. chan sl, mo f, hui ep, koh j, chu c, hui j, et al. a phase i study of temsirolimus as novel therapeutic drug for patients with unresectable hepatocellular carcinoma (hcc). in: asco annual meeting proceedings. 2013;2013. p. e15048. 121. sachdev jc, javed ay, weir ab, korn ri, gulla sm, newbold rg, et al. a phase ii study of temsirolimus in previously treated advanced hepatocellular carcinoma (hcc). in: asco annual meeting proceedings; 2014;2014. p. 4098. ali kabir et al. 251j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 review hepatocellular treatment 122. lim hy, heo j, choi hj, lin cy, yoon jh, hsu c, et al. a phase ii study of the efficacy and safety of the combination therapy of the mek inhibitor refametinib (bay 86-9766) plus sorafenib for asian patients with unresectable hepatocellular carcinoma. clin cancer res. 2014;20:5976–5985. 123. bruix j, tak wy, gasbarrini a, santoro a, colombo m, lim hy, et al. regorafenib as second-line therapy for intermediate or advanced hepatocellular carcinoma: multicentre, open-label, phase ii safety study. eur j cancer. 2013;49:3412–3419. 124. huynh h, ngo vc, fargnoli j, ayers m, soo kc, koong hn, et al. brivanib alaninate, a dual inhibitor of vascular endothelial growth factor receptor and fibroblast growth factor receptor tyrosine kinases, induces growth inhibition in mouse models of human hepatocellular carcinoma. clin cancer res. 2008;14:6146–6153. 125. johnson pj, qin s, park jw, poon rt, raoul jl, philip pa, et al. brivanib versus sorafenib as first-line therapy in patients with unresectable, advanced hepatocellular carcinoma: results from the randomized phase iii brisk-fl study. j clin oncol. 2013;31:3517–3524. 126. kang yk, yau t, park jw, lim hy, lee ty, obi s, et al. randomized phase ii study of axitinib versus placebo plus best supportive care in secondline treatment of advanced hepatocellular carcinoma. ann oncol. 2015;26:2457–2463. 127. cheng al, kang yk, lin dy, park jw, kudo m, qin s, et al. sunitinib versus sorafenib in advanced hepatocellular cancer: results of a randomized phase iii trial. j clin oncol. 2013;31:4067–4075. 128. cainap c, qin s, huang wt, chung ij, pan h, cheng y, et al. linifanib versus sorafenib in patients with advanced hepatocellular carcinoma: results of a randomized phase iii trial. j clin oncol. 2015;33:172–179. 129. trojan j, zeuzem s. tivantinib in hepatocellular carcinoma. expert opin investig drugs. 2013;22:141–147. 130. rota caremoli e, labianca r. tivantinib: critical review with a focus on hepatocellular carcinoma. expert opin investig drugs. 2014;23:1563–1574. 131. santoro a, rimassa l, borbath i, daniele b, salvagni s, van laethem jl, et al. tivantinib for second-line treatment of advanced hepatocellular carcinoma: a randomised, placebo-controlled phase 2 study. lancet oncol. 2013;14:55–63. 132. yakes fm, chen j, tan j, yamaguchi k, shi y, yu p, et al. cabozantinib (xl184), a novel met and vegfr2 inhibitor, simultaneously suppresses metastasis, angiogenesis, and tumor growth. mol cancer ther. 2011;10:2298–2308. 133. siegel ab, cohen ei, ocean a, lehrer d, goldenberg a, knox jj, et al. phase ii trial evaluating the clinical and biologic effects of bevacizumab in unresectable hepatocellular carcinoma. j clin oncol. 2008;26:2992–2998. 134. boige v, malka d, bourredjem a, dromain c, baey c, jacques n, et al. efficacy, safety, and biomarkers of single-agent bevacizumab therapy in patients with advanced hepatocellular carcinoma. oncologist. 2012;17:1063–1072. 135. zhu ax, park jo, ryoo by, yen cj, poon r, pastorelli d, et al. reach trial investigators. ramucirumab versus placebo as second-line treatment in patients with advanced hepatocellular carcinoma following first-line therapy with sorafenib (reach): a randomised, double-blind, multicentre, phase 3 trial. lancet oncol. 2015;16:859–870. 136. patrikidou a, sinapi i, regnault h, fayard f, bouattour m, fartoux l, et al. gemcitabine and oxaliplatin chemotherapy for advanced hepatocellular carcinoma after failure of anti-angiogenic therapies. invest new drugs. 2014;32:1028–1035. 137. sangro b, gomez-martin c, de la mata m, iñarrairaegui m, garralda e, barrera p, et al. a clinical trial of ctla-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis c. j hepatol. 2013;59:81–88. 138. heo j, reid t, ruo l, breitbach cj, rose s, bloomston m, et al. randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia jx-594 in liver cancer. nat med. 2013;19:329–336. 139. segler a, tsimberidou am. lenalidomide in solid tumors. cancer chemother pharmacol. 2012;69:1393–1406. 140. faivre sj, santoro a, kelley rk, merle p, gane e, douillard j-y, et al. a phase 2 study of a novel transforming growth factor-beta (tgf-{beta} 1) receptor i kinase inhibitor, ly2157299 monohydrate (ly), in patients with advanced hepatocellular carcinoma (hcc). in: asco annual meeting proceedings. 2014;2014. p. lba173. 141. chan sl, wong am, lee k, wong n, chan ak. personalized therapy for hepatocellular carcinoma: where are we now?. cancer treat rev. 2016;45:77–86. 142. ahmed m, brace cl, lee ft, goldberg sn. principles of and advances in percutaneous ablation. radiology. 2011;258:351–369. 143. chen k, chen g, wang h, li h, xiao j, duan x, et al. increased survival in hepatocellular carcinoma with iodine-125 implantation plus radiofrequency ablation: a prospective randomized controlled trial. j hepatol. 2014;61:1304–1311. 144. ahlers o, hildebrandt b, dieing a, deja m, böhnke t, wust p, et al. stress induced changes in lymphocyte subpopulations and associated cytokines during whole body hyperthermia of 41.8-42.2 degrees c. eur j appl physiol. 2005;95:298–306. 145. cui j, wang n, zhao h, jin h, wang g, niu c, et al. combination of radiofrequency ablation and sequential cellular immunotherapy improves progression-free survival for patients with hepatocellular carcinoma. int j cancer. 2014;134:342–351. 146. wang xp, xu m, gao hf, zhao jf, xu kc. intraperitoneal perfusion of cytokine-induced killer cells with local hyperthermia for advanced hepatocellular carcinoma. world j gastroenterol. 2013;19:2956–2962. 147. dong y, wu g. analysis of short and long term therapeutic effects of radiofrequency hyperthermia combined with conformal radiotherapy in hepatocellular carcinoma. j buon. 2016;21:407–411. 148. li z, zhang k, lin sm, mi dh, cao n, wen zz, et al. radiofrequency ablation combined with percutaneous ethanol injection for hepatocellular carcinoma: a systematic review and meta-analysis. int j hyperthermia. 2016:1–10. 149. iezzi r, pompili m, la torre mf, campanale mc, montagna m, saviano a, et al. hepatocatt study group for the multidisciplinary management of hcc. radiofrequency ablation plus drug-eluting beads transcatheter arterial chemoembolization for the treatment of single large hepatocellular carcinoma. dig liver dis. 2015;47:242–248. 150. hoffmann r, rempp h, syha r, ketelsen d, pereira pl, claussen cd, et al. transarterial chemoembolization using drug eluting beads and subsequent percutaneous mr-guided radiofrequency ablation in the therapy of intermediate sized hepatocellular carcinoma. eur j radiol. 2014;83:1793–1798. 151. huang zm, li w, li s, gao f, zhou qm, wu fm, et al. cytokine-induced killer cells in combination with transcatheter arterial chemoembolization and radiofrequency ablation for hepatocellular carcinoma patients. j immunother. 2013;36:287–293. 152. cui l, liu xx, jiang y, wu xj, liu jj, zhou xr, et al. comparative study on transcatheter arterial chemoembolization, portal vein embolization and high intensity focused ultrasound sequential therapy for patients. asian pac j cancer prev. 2012;13:6257–6261. 153. bush da, smith jc, slater jd, volk ml, reeves me, cheng j, et al. randomized clinical trial comparing proton beam radiation therapy with transarterial chemoembolization for hepatocellular carcinoma: results of an interim analysis. international j radiat oncol biol phys. 2016;95:477–482. 154. lobo l, yakoub d, picado o, ripat c, pendola f, sharma r, et al. unresectable hepatocellular carcinoma: radioembolization versus chemoembolization: a systematic review and meta-analysis. cardiovasc intervent radiol. 2016;39:1580–1588. 155. zhang y, li y, ji h, zhao x, lu h. transarterial y90 radioembolization versus chemoembolization for patients with hepatocellular carcinoma: a metaanalysis. biosci trends. 2015;9:289–298. 156. ma j, wang jh. 131i-labeled-metuximab plus transarterial chemoembolization in combination therapy for unresectable hepatocellular carcinoma: results from a multicenter phase iv clinical study. asian pac j cancer prev. 2015;16:7441–7447. 157. wu l, yang yf, ge nj, shen sq, liang j, wang y, et al. hepatic artery injection of ¹³¹i-labelled metuximab combined with chemoembolization for intermediate hepatocellular carcinoma: a prospective nonrandomized study. eur j nucl med mol imaging. 2012;39:1306–1315. 158. peng s, yang qx, zhang t, lu mj, yang g, liu zy, et al. lobaplatin-tace combined with radioactive 125i seed implantation for treatment of primary hepatocellular carcinoma. asian pac j cancer prev. 2014;15:5155–5160. 159. jacob r, turley f, redden dt, saddekni s, aal ak, keene k, et al. adjuvant stereotactic body radiotherapy following transarterial chemoembolization in patients with non-resectable hepatocellular carcinoma tumours of ≥ 3 cm. hpb (oxford). 2015;17:140–149. 160. ni s, liu l, shu y. sequential transcatheter arterial chemoembolization, three-dimensional conformal radiotherapy, and high-intensity focused ultrasound treatment for unresectable hepatocellular carcinoma patients. j biomed res. 2012;26:260–267. 161. lin xj, li qj, lao xm, yang h, li sp. transarterial injection of recombinant human type-5 adenovirus h101 in combination with transarterial chemoembolization (tace) improves overall and progressive-free survival in unresectable hepatocellular carcinoma (hcc). bmc cancer. 2015;15. 252 j contemp med sci | vol. 3, no. 11, summer 2017: 239–252 hepatocellular treatment review ali kabir et al. 162. song ds, song mj, bae sh, chung wj, jang jy, kim ys, et al. a comparative study between sorafenib and hepatic arterial infusion chemotherapy for advanced hepatocellular carcinoma with portal vein tumor thrombosis. j gastroenterol. 2015;50:445–454. 163. kawaoka t, aikata h, hyogo h, morio r, morio k, hatooka m, et al. comparison of hepatic arterial infusion chemotherapy versus sorafenib monotherapy in patients with advanced hepatocellular carcinoma. j dig dis. 2015;16:505–512. 164. cosgrove dp, reyes dk, pawlik tm, feng al, kamel ir, geschwind jf. open-label single-arm phase ii trial of sorafenib therapy with drug-eluting bead transarterial chemoembolization in patients with unresectable hepatocellular carcinoma: clinical results. radiology. 2015;277:594–603. 165. buijs m, reyes dk, pawlik tm, blackford al, salem r, messersmith wa, et al. phase 2 trial of concurrent bevacizumab and transhepatic arterial chemoembolization in patients with unresectable hepatocellular carcinoma. cancer. 2013;119:1042–1049. 166. chen j, zhou c, long y, yin x. sunitinib combined with transarterial chemoembolization versus transarterial chemoembolization alone for advanced-stage hepatocellular carcinoma: a propensity score matching study. tumour biol. 2015;36:183–191. 167. feng x, xu r, du x, dou k, qin x, xu j, et al. combination therapy with sorafenib and radiofrequency ablation for bclc stage 0-b1 hepatocellular carcinoma: a multicenter retrospective cohort study. am j gastroenterol. 2014;109:1891–1899. 168. kan x, jing y, wan qy, pan jc, han m, yang y, et al. sorafenib combined with percutaneous radiofrequency ablation for the treatment of mediumsized hepatocellular carcinoma. eur rev med pharmacol sci. 2015;19: 247–255. 169. nakazawa t, hidaka h, shibuya a, okuwaki y, tanaka y, takada j, et al. overall survival in response to sorafenib versus radiotherapy in unresectable hepatocellular carcinoma with major portal vein tumor thrombosis: propensity score analysis. bmc gastroenterol. 2014;14. 170. yang y, lu y, wang c, bai w, qu j, chen y, et al. cryotherapy is associated with improved clinical outcomes of sorafenib for the treatment of advanced hepatocellular carcinoma. exp ther med. 2012;3:171–180. 171. chow pk, poon dy, khin mw, singh h, han hs, goh as, et al. asiapacific hepatocellular carcinoma trials group. multicenter phase ii study of sequential radioembolization-sorafenib therapy for inoperable hepatocellular carcinoma. plos one. 2014;9:e90909. 172. feng q, chi y, liu y, zhang l, liu q. efficacy and safety of percutaneous radiofrequency ablation versus surgical resection for small hepatocellular carcinoma: a meta-analysis of 23 studies. j cancer res clin oncol. 2015;141:1–9. 173. yi hm, zhang w, ai x, li ky, deng yb. radiofrequency ablation versus surgical resection for the treatment of hepatocellular carcinoma conforming to the milan criteria: systemic review and meta-analysis. int j clin exp med. 2014;7:3150–3163. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201701 149j contemp med sci | vol. 5, no. 3, may–june 2019: 149–153 original risk factors of breast cancer among iraqi women sarab k.abedalrahman,a*besmah m.ali,bnajim abid issa al-khalidy,aand ali s. al-hashimia awomen health center, al-alwiyaa maternity teaching hospital, baghdad, iraq. bdepartment of public health, ghazi al-hariri hospital for specialized surgery, medical city, baghdad, iraq. *correspondence to sarab k.abedalrahman(email: sarab.abedalrahman@yahoo.com). (submitted: 25 december 2018 – revised version received: 20 january 2019 – accepted: 25 april 2019 – published online: 26 june 2019) introduction cancer is anticipated to rise by 70% in the coming 25 years in the developing countries. breast cancer is known to represent the main part that increase.1breast cancer has ranked the first malignancy among the iraqi population in general and the leading cause of death among women following cardiovascular diseases.2,3 iraqi breast cancer rates were generally constant between 2000 and2009, but newer statistics from the iraqi cancer registry detect increasing rates since 2009 with females over50s causing the main contribution to the increase.4 breast cancer arises from a multifactorial process. recently, attention was focused on genetic predisposition, and on its association with the modern life style,including diet, alcohol consumption.5oral contraceptive pills was proved to increase risk of breast cancer, which decrease after stopping it.6these risks are associated with more recent formulations of oral contraceptives.7 in iraq with a huge variation in life style modes, cultural, geographical, diets and habits, there are considerably limited information’s sources of on cancer risk factors. actually, the factors for differences in breast cancer incidence in women are not fully understood, which are likely to be explained by reproductive and lifestyle factors such as literacy, diet, age at menarche and menopause, age at first delivery, abortion, family history of breast cancer.8–10 the importance of knowledge of breast cancer risk factors is that it helps in detecting risk group women, and then to use it in the building of breast cancer screening programs and cancer prevention programs. as the risk factors is unique for each community, the study of breast cancer risk factors for iraqi women will be an important step in planning screening and prevention programs. this study attempts to find out some of the various risk factors of breast cancer among iraqi women. patients and methods a retrospective case control study done on patients attended the women health center, in al-alwiyaa maternity teaching hospital, during the period from january 1, 2018to october 30,2018. the study sample consists of 147 females proved to have breast cancer by histopathological examination, and 161 patients proved not to have breast cancer by radiological, histopathological, and cytological investigations and considered as a control group. data collected through standardized questionnaire to gain information about the personal, demographic characteristics, risk factors for breast cancer (e.g. reproductive, hormonal and genetic). the diagnosis of cases and controls done by consultant medical staff, using a triple assessment technique (i.e. physical breast examination, ultrasonography, with or without mammography and fine needle aspiration cytology) according to the patient state. the risk factors are asfollows:early age at menarche,late age at menopause, early age at firstpregnancy, and family history, was categorized according to the centers for disease control and prevention.11 the statistical package for social sciences, version 18 was used for data entry and analysis. pearson chi-square (χ2) test was used to compare proportions of different factors among different groups of study sample. odds ratio (or), and binary univariate logistic regression using enter method were used to find the associations of risk factors. p-value of ≤0.05 was regarded as statistically significant. results the age distribution show that most of cancer cases were 40–50 years (45, 30.6%), and 50–60 years (45,30.6%), versus <30 years (78, 48.4%) among control group, at the same period, this relation was statistically significant as shown in table 1. the mean age among cases 50.6±11.3, was significantly higher than controls 34.7±12.3. the breast cancer starts at the age of 28 years. most of the breast cancer cases and controls were from middle socioeconomic state as follows; 104(70.7%) and 119 (73.9%) respectively. estimating the risk using odds ratio objective this study aimed to identify the risk factors of breast cancer among iraqi women. methods a retrospective case control study, done on 147 breast cancer cases compared with 161 non-malignant cases selected randomly from women health center in al-alwiyaa maternity teaching hospital. results increased risk for breast cancer significantly associated with increased age especially ≥60 years, widow or divorced women[odds ratio (or) 3.7, confidence interval (ci1.5–8.5)], menopause [or 6.43, ci(3.58–11.9)],age at menarche <12 years[or 1.99, ci(1.04–3.8)], and use of contraceptive pills for≥1 year [or 1.99, ci(1.01–3.95)]. conclusion positive risk factor for breast cancer was old age≥60 years, widow or divorced women, menopause, age at menarche <12 years, and use of contraceptive pills for≥1 year. family history, seconddegree relative,not associated with breast cancer. there are some discrepancies between our findings and other studies in the literature need further studies. keywords breast cancer, risk factors, iraqi women issn 2413-0516 150 j contemp med sci | vol. 5, no. 3, may–june 2019: 149–153 risk factors of breast cancer among iraqi women original s.k.abedalrahman et al. demonstrate that socioeconomic state, occupation, and education, were non-significantly associated with breast cancer. however, regarding marital status, when comparing the widow/divorced women to married show [or 3.7(1.1.5–8.5)], this relation was statistically significant, as shown in table 2. the odds ratio of the menopause was 6.43, age at menarche 1.99, hormonal contraception use for ≥1 year 1.99, and menstrual irregularity 1.56 was significantly associated with breast cancer, while age at menopause ≥55 year was non-significantly associated with breast cancer as shown in table 3. the odds ratio for family history of breast cancer was non-significantly associated with breast cancer: family history of breast cancer 0.804, firstdegree relative 1.036, firstand seconddegree relative 1.036, only seconddegree relative 0.56, one relative 0.82, two or more relatives 0.73, as shown in table 6. discussion breast cancer was reported among females in 20s, which is the same to what reported in previous literatures in iraq,4,12,13and neighbor countries14but lower than in cameron 35 years.15this may be due to same environmental exposure. about 52.4% of cases aged <50 years this figure goes with what reported previously in iraq,4,12,13 and differ from what is known globally that, 75% of new cases occurs in women aged older than 50 years,16,17 this difference indicate presence of an environmental factors. the early onset of cancer is accompanied by or increment with increasing age, indicating that factors other than age may affect the breast cancer development.15 most of cases and controls were from middle socioeconomic state 104(70.7%), 119 (73.9%), respectively,this is similar to previous iraqi studies,18 as most of the iraqi women were from middle income families and those with high socioeconomic state usually seek the private health care services, that we cannot catch them. women are at higher risk of breast cancer with high socioeconomic status of 2.5-foldthan those from lower socioeconomic level, which is similar to nkondjock etal., and this may be due to the fact that they tend to carry fewer pregnancies or have children at an older age.19 employed womenhad higher risk of breast cancer of 1.7 than housewives, which agree with ghiasvand et al.14 this may be related to exposure to other risk factors, e.g. carcinogens and stress, as well as they were more educated and had higher socioeconomic status, which by itself risk factors for breast cancer. most of the patients were of low educational level, this agree with previous iraqi studies.13,18,20high educational level had higher odds ratiothan low educational level,this goes with ghiasvand et al.14those patients were more health education to seek medical counseling. menopause had 6.43-fold risk for having breast cancer than menstruating women, goes with what was found in india 1.6-fold,21 which was higher than what was found in iran 0.95fold,22 and cameron 0.37-fold.15this may be due to effect of aging as the menopausal women are already old, or to the high postmenopausal blood estrogen levelswhich is established as risk factors.23sahan et al.24 in iraq documented an increased serum estradiol and prolactin among the preand post-menopausal breast cancer women, and recommends emphasizing the necessity of co-operation between the ministry of health and the ministry of commerce and protecting them from dangerous behavior by providing them with sufficient support and guidance to stay away from the hormonic products and focusing on the extension programs in the protection of the community through educating them with the guidance. late menopause increases the risk of breast cancer. risk increases by almost 3% for each year older at menopause, so that a women who has the menopause at 55 years rather than 45 years, has approximately 30% higher risk.25this study revealed that menopause at the age of 55 yearsincreases the risk of breast cancer by twofoldthan those their age of menopause <55 years. this is consistent with studies in morocco(1.36-fold),26(1.7-fold)27and lower than found by al ramahi28 menopause above 50 years had 9.5-fold risk than under 45 years. this discrepancy may be explained by that most women (especially old age) have difficulties in recalling past events such as age at menarche; therefore, recall bias was unavoidable.13 age at menarche of <12 years had twofold risk than at age ≥12 years, this agree with altaha (3.05).18 this result differs from iranian studies,22,29 which revealed 1.5-fold of risk in women with age at menarche of<13 years was fourfold. use of hormonal contraceptivepills for 1 year or more, increase the risk bytwofold than non-hormonal contraceptive drugs user, this was supported by the following studies:in china (1992), norway and sweden (2002) and malaysia (2005).30–32 however, anafrican study show negative association.33 menstrual irregularity was associated with increased risk of breast cancer by 1.6-fold than women with regular menstruation, this finding is consistent with terry34 that irregular menstruation associated with 1.12-fold risk for breast cancer, and other previous studies.35,36 while opposed by most of the studies which found inverse effect on breast cancer risk.37–41this increase may be due to use table 1 age distribution of study groups cases no (%) controls no (%) total no (%) or (95%ci) p <30 years 6 (4.10) 78 (48.40) 84 (27.30) 1 30–40 years 26 (17.70) 27 (16.80) 53 (17.20) 12.519 <0.05 40–50 years 45 (30.60) 40 (24.80) 85 (27.60) 14.625 <0.05 50–60years 45 (30.60) 15 (9.30) 60 (19.50) 39.000 <0.05 >60 years 25 (17.60) 1(0.60) 26 (8.4) 325.000 <0.05 mean 50.6±11.3 34.7±12.3 χ2= 98.7, p<0.05. 151j contemp med sci | vol. 5, no. 3, may–june 2019: 149–153 original risk factors of breast cancer among iraqi womens.k.abedalrahman et al. table 2. the socio demographic variables among breast cancer cases and women without breast cancer cases no (%) controls no (%) total no (%) or (95% ci) p socioeconomic low 37 (25.2) 30(18.60) 67 (21.80) 1 >0.05 middle 104 (70.7) 119(73.90) 223 (72.40) 1.4 (0.8–2.5) >0.05 high 6 (4.1) 12(7.50) 18 (5.80) 2.5 (0.8–7.5) >0.05 marital status married 108 (73.5) 127(78.90) 235 (76.30) 1.00 unmarried 14 (9.50) 26(16.10) 40 (13.00) 0.63 (0.3–1.5) >0.05 widow/divorced 25 (18.8) 8(5.9) 33 (12.3) 3.7 (1.1.5–8.5) <0.05 occupation housewife 114 (77.60) 137(85.10) 251 (81.50) 1.00 employed 33 (22.4) 24(14.9) 57 (18.5) 1.7 (0.9–2.9) >0.05 education illiterate 44 (29.9) 37 (23) 81 (26.30) 1.00 >0.05 primary 49 (33.3) 61(37.9) 110 (35.70) 1.5 (0.8–2.6) >0.05 intermediate 24 (16.3) 30 (18.60) 54 (17.50) 1.5 (0.7–2.9) >0.05 secondary 12 (8.20) 8 (5.00) 20 (6.50) 0.8 (0.3–2.1) >0.05 university 18 (12.2) 25 (15.50) 43 (14.00) 1.7 (0.0.8–3.5) >0.05 total 147 (100) 161 (100) 308 (100) table 3 the hormonal risk factors among breast cancer cases and women without breast cancer hormonal risk factor cases (%) controls (%) total (%) or (95% ci) p menopause yes 61 (41.5) 16 (9.90%) 77 (25.00%) 6.43 (3.58–11.9) <0.05 no 86 (58.50) 145 (90.10%) 231 (75.00%) age at menarche <12 28 (19.00) 17 (10.60) 45 (14.60) 1.99 (1.04–3.8) <0.05 ≥12 119 (81.00) 144 (89.40) 263 (85.40) age of menopause <55 years 42 (68.90) 13 (81.30) 55 (71.40) 1.96 (0.5–7.96) >0.05 ≥55 years 19 (31.10) 3 (18.80) 22 (28.60) contraceptive <1 year 122 (83) 146 (90.7) 268 (87) 1.99 (1.01–3.95) <0.05 ≥1 year 25 (17.00) 15 (9.30) 40 (13.00) regularity irregular 22 (26.20) 27 (18.50) 49 (21.30) 1.56 (0.8–2.96) >0.05 regular 62 (73.80) 119 (81.50) 181 (78.70) table 6. the genetic risk factors among breast cancer cases and women without breast cancer genetic risk factors cases (%) controls (%) total (%) or (95%ci) p family history of breast cancer yes 35 (23.80) 45(28.00) 80(26.00) 0.8(0.48–1.34) >0.05no 112 (76.20) 116 (72.00) 228 (74.00) 1 relative degree no relative 112 (76.20) 116 (72.00) 228 (74.00) 1 first degree relative 17 (11.60) 17 (10.60) 34 (11.00) 1.04 (0.5–2.1) >0.05 second degree relative 12 (8.20) 22 (13.70) 34 (11.00) 0.56 (0.26–1.2) >0.05 first and second degree 6 (4.10) 6 (3.70) 12 (3.90) 1.036 (0.3–3.3) >0.05 no. of relatives 0 112 (76.20) 116 (72.00) 228 (74.00) 1 1 28 (19.00) 35 (21.70) 63 (20.50) 0.83 (0.47–1.5) >0.05 ≥2 7 (4.80) 10 (6.30) 16 (5.20) 0.73 (0.3–1.97) >0.05 152 j contemp med sci | vol. 5, no. 3, may–june 2019: 149–153 risk factors of breast cancer among iraqi women original s.k.abedalrahman et al. of hormonal therapy for regulation as it was found by titus-ernstoff et al.41 that the apparent inverse association with irregular menstrual cycles was stronger among women who did not use hormonal replacement therapy. positive family history was not significantly associated with increase breast cancerrisk (0.8), while many studies reported the strong association between family history and breast cancer,42in moroccan two to threefold increased risk,26and in southern iran9.07.22,14 family history of the firstdegree relative show increased risk by 1.036-fold than women without family history of breast cancer, thisagree with a study in iraq 2.1-fold increase,28and in cameron 1.3.15 patient with one or more relatives with breast cancer was non-significantly associated with increased risk of breast cancer, this oppositionwhat found by brewer et al.43in large data base study, a cohort of over 113,000 women from the general uk population, found that one relative with breast cancer history increase the risk by 1.7 times and two or more increase the risk by 2.5 times. and the effect of family history was more prominent in women aged <45 years (2.47-fold), while among those aged >45 years the risk was 1.63-fold.the majority of multiple-case families that segregate both breast and ovarian cancer in a dominant fashion are due to mutations in the brca1 gene on chromosome 17q.44 this difference may be explained by the presence of other factors that affect both the cases and control groups, in which its effect appear even on the age distribution also, or may be due to having a relative with breast cancer is promoting for seeking health services, which affect our results. from the results of this study, a need for further investigating the environmental factors that may have direct or indirect effect, on breast cancer, particularly among younger women. conclusion and recommendation positive risk factor for breast cancer was ≥60 years of age, widow or divorced women, menopause, age at menarche <12 years, and use of contraceptive pills for≥1 year,family history, seconddegree relative, not associated with breast cancer. there are some discrepancies between our findings and other studies in the literature which necessity need further studies. conflicts of interest none.  references 1. center m, rebecca s, ahmedin j. global cancer facts &figures.2nded.; american cancer society,atlanta,2011,1–52. 2. iraqi cancer board. results of the iraqi cancer registry 2012. iraqi cancer registry center, ministry of health,baghdad, 2015. 3. alwan nas. breast cancer among iraqi women: preliminary findings from a regional comparative breast cancer research project. jgloboncol.2016;2:255–258. 4. al-hashimi mm, wangxj. breast cancer in iraq, incidence trends from 2000-2009. asian pacjcancer prev.2014;15:281–286. 5. mcpherson k, steel cm, dixonj. m.. abc of breast diseases. breast cancer-epidemiology, risk factors, and genetics. bmj 2000;321:624–628. 6. bhupathiraju sn, grodstein f, stampfer mj, willett wc, hu fb, manson je. exogenous hormone use: oral contraceptives, postmenopausal hormone therapy, and health outcomes in the nurses’health study. am j public health. 2016;106:1631–1637. 7. mørch ls, skovlund cw, hannaford pc, iversen l, fielding s, lidegaard ø. contemporary hormonal contraception and the risk of breast cancer. n engl j med. 2017;377:2228–2239. 8. meshram ii, hiwarkar pa, kulkarni pn. reproductive risk factors for breast cancer: a case control study. online j health allied sci. 2009;8:1–4. 9. pakseresht s, ingle gk, bahadur ak, ramteke vk, singh mm, garg s, et al. risk factors with breast cancer among women in delhi. indian j cancer. 2009;46:132–138. 10. lodha sr, nandeshwara s, pal kd. risk of breast cancer in obese women: a case control study. natl j community med. 2010;1:166–167. 11. joseph da, king jb, miller jw, richardson lc; centers for disease control and prevention (cdc). prevalence of colorectal cancer screening among adults--behavioral risk factor surveillance system, united states, 2010. mmwr suppl. 2012;61:51–56. 12. alwan na. breast cancer: demographic characteristics and clinico-pathological presentation of patients in iraq. east mediterr health j. 2010;16:1159–1164. 13. lafta rk, saeed eq, isa sa. risk factors of breast cancer among women (a sample from baghdad). iraqi j commun med. 2013;26:1–6. 14. ghiasvand r, maram es, tahmasebi s, tabatabaee sh.risk factors for breast cancer among young women in southern iran. int. j. cancer. 2011;129:1443–1449. 15. essiben f, foumane p, meka enu, soh ps, sama,jd osogo e, et al. risk factors for breast cancer: a case-control study of 315 women followed in the gynecology and oncology departments of two university teaching hospitals in yaounde, cameroon. open j obstet gynecol. 2016;6:676–688. 16. robbins sl, cotran rs, kumar v.pocket companion: pathologic basis of disease. 2nded.; wb saunders co, philadelphia, usa, 2001. 17. international agency for research on cancer.iarc hand-books of cancer prevention. weight control and physical activity. vol 6, iarc press, france,2002. 18. altaha ma, al-ani. reproductive factors and risk of breast cancer. al-anbar med j. 2013;11:17–26. 19. nkondjock a, ghadirian p. [risk factors and risk reduction of breast cancer]. med sci (paris).2005;21:175–180 (in french). 20. alwan nas. family history among iraqi patients diagnosed with breast cancer. int j sci res. 2017;6:869–873. 21. butt z, shahbaz u,naseem t,ashfaq u,khan ua, khan mr, et al.reproductive risk factors for female breast cancer: a case – control study. ann king edw med univ. 2009;15:206–210. 22. mahouri k, dehghani zahedani m, zare s. breast cancer risk factors in south of islamic republic of iran: a case-control study.east mediterr health j. 2007;13:1265–1273. 23. graham ac, heather jb, rulla mt. breast cancer. in: david s, joseph ff (eds.) cancer epidemiology and prevention, 3rded.; oxford university press inc., new york, 2006, pp. 995–1012. 24. sahan es, majid ay, hassan as. evaluation of estradiol and prolactin serum levels “in premenopausal; and postmenopausal” women with ((breast cancer)) in baghdad city. iraqi natl j nurs spec. 2017;30:111–117. 25. breast cancer and hormone replacement therapy: collaborative reanalysis of data from 51 epidemiological studies of 52,705 women with breast cancer and 108,411 women without breast cancer. collaborative group on hormonal factors in breast cancer. lancet 1997; 350:1047–1059. 26. laamiri fz, bouayad a, hasswane n, ahid s, mrabet m, amina b.risk factors for breast cancer of different age groups: moroccan data? open j obstet gynecol. 2015;5:79–87. 27. khalis m, charbotel b, chajès v, rinaldi s, moskal a, biessy c, et al. menstrual and reproductive factors and risk of breast cancer: a case-control study in the fez region, morocco. plos one 2018;13:e0191333. 28. al-ramahi s.j.j, al-saffar ajaa. breast cancer risk factors among iraqi women from baghdad/iraq 2012. published thesis, the arab board in community medicine,2013, 37. 29. bahir bh, al-naqeeb aa., niazy sm. risk factors for breast cancer in a sample of women. iraqi j. commun. med.2012;25:4–8. 30. wang qs, ross rk, yu mc, ning jp, henderson be, kimm ht. a case-control study of breast cancer in tianjin, china. cancer epidemiol biomarkers prev. 1992;1:435–439. 153j contemp med sci | vol. 5, no. 3, may–june 2019: 149–153 original risk factors of breast cancer among iraqi womens.k.abedalrahman et al. 31. kumle m, weiderpass e, braaten t, persson i, adami ho, lund e. use of oral contraceptives and breast cancer risk: the norwegianswedish women’s lifestyle and health cohort study. cancer epidemiol biomarkers prev. 2002; 11:1375–1381. 32. norsa’adah b, rusli bn, imran ak, naing i, winn t. risk factors of breast cancer in women in kelantan, malaysia. singapore med j. 2005;46:698–705. 33. balekouzou a, yin p, pamatika cm, bekolo ce, nambei sw, djeintote m, et al.reproductive risk factors associated with breast cancer in women in bangui: a case-control study. bmc womens health. 2017;17:14 34. terry kl, willett wc, rich-edwards jw, hunter dj, michels kb. menstrual cycle characteristics and incidence of premenopausal breast cancer. cancer epidemiol biomarkers prev.2005;14:1509–1513. 35. grabrick dm, vierkant ra, anderson ke, cerhan jr, anderson ve, seller ta. association of correlates of endogenous hormonal exposure with breast cancer risk in 426 families (united states). cancer causes control. 2002;13:333–341. 36. gao yt, shu xo, dai q, potter jd, brinton la, wen w, et al. association of menstrual and reproductive factors with breast cancer risk: results from the shanghai breast cancer study. int j cancer. 2000;87:295–300. 37. clavel-chapelon f. cumulative number of menstrual cycles and breast cancer risk: results from the e3n cohort study of french women. cancer causes control. 2002;13:831–838. 38. den tonkelaar i, de waard f. regularity and length of menstrual cycles in women aged 41-46 in relation to breast cancer risk: results from the domproject. breast cancer res treat. 1996;38:253–258. 39. wu ah, ziegler rg, pike mc, nomura am, west dw, kolonel ln, et al. menstrual and reproductive factors and risk of breast cancer in asianamericans. br j cancer. 1996;73:680–686. 40. parazzini f, la vecchia c, negri e, franceschi s, tozzi l. lifelong menstrual patterns and risk of breast cancer. oncology. 1993;1993:222–250. 41. titus-ernstoff l, longnecker mp, newcomb pa, dain b, greenberg er, mittendorf r, et al. menstrual factors in relation to breast cancer risk. cancer epidemiol biomarkers prev. 1998;7:783–789. 42. collaborative group on hormonal factors in breast cancer. familial breast cancer: collaborative reanalysis of data from 52 epidemiological studies of 58209 women with breast cancer and 101986 women without the disease. lancet. 2001;358:1389–1399. 43. brewer hr, jones me, schoemaker mj, ashworth a, swerdlow aj. family history and risk of breast cancer: an analysis accounting for family structure. breast cancer res treat.2017;165:193–200. 44. ford d, easton df, peto j. estimates of the gene frequency of brca1 and its contribution to breast and ovarian cancer incidence.am j hum genet. 1995;57:1457–1462. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201906 18 j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 review epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review rawan s. abou-assy1, magda m. aly1,3*, reda h. amashah1, samyah d. jastaniah1, hany m. al deen2 1department of biology, college of science, king abdulaziz university, jeddah, saudi arabia. 2department of microbiology, king faisal medical complex, taif, makkah, saudi arabia. 3department of botany and microbiology, faculty of science, kafrelsheikh university, kafrelsheikh govvernorate, egypt. *correspondence to: (email: mmmohammad@kau.edu.sa) (submitted: 10 december 2021 – revised version received: 26 december 2021 – accepted: 17 january 2022 – published online: 26 february 2022) abstract the widespread of multidrug-resistant bacteria, particularly carbapenem-resistant enterobacterales (cre) bacteria, constitutes a major public health threat worldwide, owing to the limited therapeutic options. this review will describe and uncover the saudi experience in the challenge against carbapenem resistance (cr). the different carbapenem resistance prevalence and carbapenemase genes detected from various bacterial species were mapped for saudi regional distribution, based on saudi published data during a period extended from 2017 to 2021. however, vim, imp, and kpc enzymes were usually reported with the predominance of oxa and ndm among enterobacterales. although sim and gim carbapenemases were uncommonly detected in our country. collaborative efforts and raising awareness of the threat of carbapenem resistance are required to minimize the spread of multidrug-resistant bacteria. keywords: carbapenem, enterobacteriaceae, saudi arabia, lactamase, resistant, carbapenemase issn 2413-0516 introduction carbapenem resistance (cr) bacteria is a significant and mounting health concern globally,1,2 this problem is aggravated by inadequate infection control in developing countries due to poor hygiene, resource and structural constraints, deficient surveillance data, and lack of awareness regarding nosocomial infections.3,4 it occurs mainly among gram-negative pathogens such as klebsiella pneumoniae, pseudomonas aeruginosa and acinetobacter baumannii, and may be intrinsic or mediated by transferable carbapenemase-encoding genes,5,6 the most effective carbapenemases, in terms of carbapenem hydrolysis and globally spread, are kpc, vim, imp, ndm and oxa-48 genotypes.7 carbapenem class antibiotics have been a mainstay of treatment for serious infections caused by enterobacterales, but efficacy has been compromised by the widespread acquisition of resistance genes to these critical drugs.8 effective antimicrobial options for carbapenem-resistant enterobacterales (cre) are often lacking, and treatment typically requires reliance on drugs with a risk of toxicity or other safety concerns.9 carbapenem-resistant klebsiella pneumoniae (crkp) is a prominent cause of nosocomial infections associated with high rates of morbidity and mortality, particularly in immune-compromised individuals.10,11 carbapenem resistance causes a broad spectrum of diseases including pneumonia, urinary tract infections, bloodstream infections, skin, and soft tissue infections.12 this resistance is facilitated by complex factors, including the presence of mobile genetic elements, the misuse of antimicrobial drugs, poor infection control practices, and increased international travel.13 in healthcare settings, cre is transmitted from person to person, often via the hands of healthcare personnel or through contaminated medical equipment.14 additionally, sink drains and toilets are increasingly recognized as an environmental reservoir and cre transmission source.15 risk factors for cre colonization and infection have been identified as longer length of hospital stay, prior hospitalization, admission to icu, renal dysfunction, neurological disorders, tracheostomy, mechanical ventilation, central venous catheter (cvc) use, urinary catheter use, nasogastric tube use, implementation of dialysis, prior use of any antibiotic, and specific use of carbapenems.16–18 the aim of this review was to shed light on all studies tackling carbapenem resistance in enterobacteriaceae, carbapenem resistance a. baumannii (crab) and carbapenem resistance p. aeruginosa (crpa) in the saudi arabia regions, with an indication for each region, description of studies timeline, the prevalence of carbapenem resistance, and cr encoding genes detected based on saudi data published over last 5 years from 2017 to 2021. this study enabled us to gain deep insight into the cr problem in saudi arabia, in addition to mapping the regional distribution of carbapenemase enzymes and cr prevalence for each region, which is strongly encouraged by epidemiologists to improve surveillance strategies to minimize the spread of cr gram negative bacteria such as previously described. methods literature review pubmed, sciencedirect and international journals online databases were searched to december 2021. the search key words used were carbapenem resistance in saudi arabia, enterobacteriaceae, cre, escherichia coli, crec, klebsiella, crkp, pseudomonas aeruginosa, crpa, acinetobacter baumannii, crab, carbapenem, -lactam, -lactamase, resistant, carbapenemase, mbl, metallo-b-lactamase, vim, ndm, oxa, oxacillinase, imp, kpc to extract articles published only in english in an attempt to include up to date relevant data. study selection criteria only research articles reporting the prevalence or molecular genotyping frequency of carbapenem resistance in clinical pathogens isolated from patients and hospital environment or 19j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 r.s. abou-assy et al. review epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review showed the proportion of carbapenem resistant isolates of all gram-negative isolates, or clinical data including patient demographics, underlying conditions, and antibiotic treatment in all saudi arabia regions. only studies elaborating bacteria study population, pathogens identified, phenotypic and genotypic methods used to detect carbapenem resistance were used. patient populations of all hospital types were included while case reports and review articles were excluded from this systematic review as it has become conventional.19,20 data extraction a database was created in which study location, sample collection period, bacterial species isolated, a number of isolates tested for cr, cr isolates, cr prevalence, esbl & carbapenemase genes, methods used to identify resistant isolates, references, and outcomes were included. results the emergence and rapid spread of carbapenem-resistant enterobacterales in saudi arabia encouraged scientific researchers and epidemiologists to investigate cr hospital associated infection prevalence and the genes involved in resistance to carbapenems. as described in figure 1a the number of published articles per year was clearly increased and indicated by the highest number recorded in 2021. this is closely related to the dissemination of cre throughout the country and the increased awareness of the importance of surveillance and control of multidrug-resistant bacteria in order to improve health quality. a high prevalence of carbapenemase producers in saudi arabia was mainly identified as a. baumannii, followed by pseudomonas aeruginosa, and klebsiella pneumonia.21–26 the majority number of published research papers per carbapenemase type was oxa, from all genotyping studies, oxa-48 variant in enterobacteriaceae and oxa-23 variant in a. baumannii, and p. aeruginosa, followed by ndm-1 (all the collected studies except two.27,28 however, low number of published articles (8 studies) has reported the detection of vim genes.22,24,28–33 detection of vim genes was reported in 5 studies while imp was reported by el-badawy, abdelwahab et al. 2019, shah, yasir et al. 2019, jawhar, alrashed et al. 2020, khater, alfaki et al. 2020, alqahtani, tickler et al. 2021)30,31,28,34,24 and kpc genes was reported in 5 studies21,24,27,34,35 as described in figure 1b. furthermore, until now no studies reported the presence of sim and gim genes in the collected bacterial isolates from saudi arabia. saudi arabia is divided into 13 administrative regions, and geographically these regions are distributed in five major areas of the country (central, eastern, northern, southern and western areas). most of the studies about cre were conducted in the central, and western areas and small data came from the eastern, southern, and northern areas. the results according to the literature search and study selection indicate that a total of 24 studies met the inclusion criteria and were included for final review, of these 3 (12.5%) reported extended studies for more than three regions or shared with other countries, 8 (33.3%) reported from the central area, 7 (29%) reported from the western area, 2 (8.3%) reported from the eastern area, 3 (12.5%) reported from the south area, and one (4.2%) reported from north area (figure 1c). there was no data reported from fig. 1 (a) number of published papers reporting cre bacteria in saudi arabia from 2017 to 2021, (b) number of published papers per carbapenemase genes in saudi arabia from 2017 to 2021, and (c) number of published papers per saudi regions from 2017 to 2021. a b c najran, tabuk and north border regions. also, 18 studies (75%) reported genotype including carbapenemase or β-lactamase genes distribution and 16 studies (66.7%) reported clinical cr surveillance data. two studies reported systematic reviews of cr from saudi arabia13,36 and three studies reported cr cases37–39 (excluded from this study). the regional distribution of the different carbapenemases gene prevalence were mapped in saudi arabia from five years up to date (figure 2), and regional distribution cr prevalence in enterobacterales over several saudi regions were mapped (figure 3). extended studies from all saudi arabia there is a wide modern study to determine the prevalence of carbapenem resistance gram-negative genes in gulf cooperation council hospitals including saudi arabia (table 1).24 the results indicate the rates of carbapenem resistance genes varied across the gcc hospitals and even among hospitals in the same country and this result was confirmed by the study of al-abdely et al. (2021)40 which explained the circulating strain causing outbreaks in this specific region. in 20 j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review review r.s. abou-assy et al. fig. 2 percentage of epidemiology of carbapenem resistance enterobacterales in saudi arabia regions.21,23–26 cre, carbapenem resistance enterobacterales; cse, carbapenem sensitive enterobacterales. fig. 3 molecular classification of carbapenem resistance k. pneumoniae genes distribution over saudi arabia regions.21,24,29,32,40 table 1. participating cities, cr prevalence and molecular classification of cr isolates in (alqahtani, tickler et al. 2021)24 study city number of isolates cr isolates cr prevalence (%) carbapenemase genes detected dammam 266 49 18.4% oxa-48 (14.3%), ndm (4.1%) khamis mushait 250 50 20.0% oxa-48 (14.0%), ndm; oxa-48 (6.0%) riyadh 73 49 67.3% oxa-48 (51.0%), ndm (2.0%) ndm; oxa-48 (6.1%) vim; oxa-48 (2.0%), vim (6.1%) 21j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 r.s. abou-assy et al. review epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review table 2. participating hospitals, carbapenem resistance prevalence and molecular classification of 456 isolates of k. pneumoniae in (al-abdely, alhababi et al. 2021)40 study saudi hospital name crkp prevalence carbapenemase genes detected 1. king salman bin abdulaziz hospital, riyadh 96% ndm-1 (11%), oxa48 (89%) 2. king khalid hospital and prince sultan center for health service, al kharj 53% ndm-1 (33%), oxa48 (67%) 3. king fahd specialist hospital, buraydah 90% ndm-1 (22%), oxa48 (44%), ndm-1 +oxa48 (33%) 4. regional laboratory and blood bank, microbiology department, dammam 100% oxa48 (100%) 5. gurayat general hospital 96% ndm-1 (32%), oxa48 (50%), ndm-1 +oxa48 (18%) 6. hael general hospital 83% oxa48 (100%) 7. arar central hospital 100% ndm-1 (33%), oxa48 (67%) 8. asir hospital 93% ndm-1 (25%), oxa48 (71%), ndm-1 +oxa48 (4%) 9. king fahd hospital, al baha 100% oxa48 (83%), ndm-1 +oxa48 (17%) 10. king faisal medical complex, taif 80% ndm-1 (17%), oxa48 (67%), ndm-1 +oxa48 (17%) 11. king abdulaziz specialist hospital, taif 100% ndm-1 (13%), oxa48 (88%) 12. king fahad hospital, madina munawara 84% ndm-1 (16%), oxa48 (70%), ndm-1 +oxa48 (4%) 13. hera general hospital, jeddah 80% ndm-1 (25%), oxa48 (35%), ndm-1 +oxa48 (50%) table 3. review of carbapenem resistance studies which include more than 3 regions in saudi arabia regions number of isolates cr prevalence (%) carbapenemase genes detected organism methods used refs. gulf cooperation council 529 of rectal swabs 26.1% one gene; oxa-48 (59.4%), ndm (13.8%), vim (6.5%) & imp (0.7%) two genes; oxa-48 & ndm (8.7%), oxa-48 & vim (6.5%), ndm & kpc (1.5%) & oxa-48 & kpc (0.7%) – xpertcarba-r assay alqahtani et al. ( 2021)24 five regions include central, north, east, south & west 519 of enterobacterales 84.7% oxa-48 (71.2%), ndm-1 (20.7%) & ndm+oxa-48 (8%). k. pneumoniae (90%), k. oxytoca (0.5%), e. coli (4%), e. cloacae (2.5%) & others e-test xpertcarba-r assay al-abdely et al. (2021)40 five hospitals in the east, west and center regions 635 of p. aeruginosa 28.2% for imipenem 23.0% for meropenem 18.7% for meropenem & imipenem carbapenemase genes; ges (9%), vim (3.4%), ndm (6%) & oxa48 (0.5%) esbl genes; per (1.1%) & veb (1.1%) carbapenemase and esbl genes; vim + per (1.1%), vim + veb (0.5%) p. aeruginosa vitek-ii pcr al hassinah et al. (2020)22 addition to the other two studies, table 2 illustrated the presence of carbapenem resistance genes in enterobacteriaceae and p. aeruginosa isolates in more than 3 regions in saudi arabia (table 3).22,40 epidemiology of central area in the last five years, there are eight published studies related to clinical cr infection from a central area (table 4), six of them detect the carbapenemase genes distribution among specific hospitals,28,34,35,41–43 other three studies give a prevalence rated to cr dissemination,34,35,44 two studies focus on crkp,34,43 and one study detect crg in p. aeruginosa and a. baumannii.28 epidemiology of western area the western area includes the makkah region that had two largest cities (makkah and jeddah) with the highest population outside the central region. millions of muslims from across the globe arrive annually in makkah to perform pilgrimage and umrah. these mass gatherings could be a good environment for spreading multi-drug resistant organisms around the world.13,45 there were seven studies published in the last five years from the western region of saudi arabia, three of them from makkah city,21,33,46 two studies from jeddah city,27,31 one study from taif 30 and one study from al-madinah.47 five of these studies detect the carbapenemase genes dissemination ratios and four studies give a cr surveillances,47 and one study focus on crkp33 while two studies detect the crg genes in p. aeruginosa crpa. other studies focus on crab30,31 while carbapenem resistance klebsiella quasipneumoniae in jeddah was described by hala et al. (2019).27 epidemiology of eastern area there were two genotypic studies published recently from the western region of saudi arabia.32,48 the first one aimed to assess different genotypic and phenotypic methods to detect carbapenemases; however, each has a limitation, talal (2019)32 study evaluated the performance of modified carbapenem inactivation method (mcim) test in enterobacteriaceae in 22 j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review review r.s. abou-assy et al. ta bl e 4. r ev ie w o f c ar ba pe ne m re si st an ce st ud ie s i n ce nt ra l, w es te rn , e as te rn , n or th er n an d so ut he rn a re as o f s au di a ra bi a ar ea (c ity ) nu m be r o f is ol at es cr p re va le nc e (% ) ca rb ap en em as e ge ne s or ga ni sm m et ho ds u se d re fs . 1. r iy ad h & q as si m 16 2, 66 5 23 .2 % – en te ro ba ct er al es (1 4% ), e. c ol i ( 6. 4% ), k. p ne um on ia e (9 .6 % ), a. b au m an ii (1 9. 5% ) & p. a er ug in os a (1 9. 2% ) vi te kii bd p ho en ix m ic ro sc an p lu s m ut ai r e t a l. (2 02 1) 44 2. r iy ad h 1, 86 4 7. 6% es bl (5 3. 3% ), kp c (1 1. 5% ), o xa -4 8 (1 .3 % ) & n d m (0 .6 7% ) – vi te kii xp er tc ar ba r a ss ay a le id an e t a l. (2 02 1) 35 3. r iy ad h – – p. ae ru gi no sa ; o xa -2 3 (5 5% ), o xa 4 0 (5 % ), o xa 1 0 (7 .5 % ), o xa 4 8 (3 .7 5% ), im p (1 .2 5% ), vi m (4 6% ), o xa -1 (2 2% ) & g im (1 5% ) a. b au m an ni i; o xa -2 3 (8 5. 7% ), o xa 4 0 (1 7% ), vi m (1 1. 4% ), im p (1 7% ), g im (2 .8 % ), o xa -1 (2 .8 % ) & o xa -4 8 (2 .8 % ) p. ae ru gi no sa (8 0 is ol at es ) a. b au m an ni i ( 35 is ol at es ) ete st vi te kii pc r ja w ha r e t a l. (2 02 0) 28 4. a l-q uw ay iy ah 54 1 al l s am pl es 78 o f k. p ne um on ia e 6. 7% fo r a ll sa m pl es 46 .2 % fo r k. p ne um on ia e o xa -4 8 (7 7. 8% ), n d m (1 3. 9% ), kp c (5 .6 % ), im p (2 .8 % ), & o xa -4 8 & n d m (1 3. 9% ) k. p ne um on ia e vi te kii d 70 c m h t pc r kh at er e t a l. (2 02 0) 34 5. r iy ad h – – – k. p ne um on ia (4 7% ), e. c ol i ( 31 % ), en te ro ba ct er sp . ( 5. 3% ), ci tro ba ct er sp . (5 .3 % ), kl uy ve ra as co rb at a (5 .3 % ), & pr ot eu s ( 5. 3% ) – a lz om or e t a l. (2 01 9) 50 6. r iy ad h – – ch ro m os om al ; c tx -m -1 5 (9 0% ), n d m -1 (2 0% ), n d m -5 (4 0% ), & o xa -1 81 (4 0% ) pl as m id ; c tx -m -1 5 (1 00 % ), n d m -1 (2 0% ), n d m -5 (3 0% ), c tx -m -1 7 (1 0% ), te m -1 8 (9 0% ), o xa -1 (4 0% ), o xa -1 81 (4 0% ), & c m v42 (8 0% ) e. c ol i m ic ro br ot h di lu tio n m et ho d pc r w g s a bd e l g ha ny e t a l. (2 01 8) 41 7. r iy ad h – – o xa -4 8 (5 8. 1% ) n d m (4 1. 9% ) c tx -m -1 (7 7. 4% ) c tx -m -9 (9 .6 % ) t em -1 (7 4. 2% ) o xa -1 (5 4. 8% ) s h v1 (4 .4 % ) k. p ne um on ia e (2 1 is ol at es ), & e . c ol i ( 10 is ol at es ) ete st im ip en em + e d ta st rip m h t pc r a l-a ga m y e t a l. (2 01 8) 42 8. r iy ad h – – o xa -4 8 (6 7. 6% ), n d m -1 (1 2. 7% ), c tx -m -1 5 (6 6. 2% ), & c tx -m -1 4 (2 1% ) k. p ne um on ia e ete st vi te kii pc r (u z za m an , a lro da yy an et a l. 20 18 )43 9. m ak ka h – – vi m (3 1% ), & g es (8 .6 % ) ps eu do m on as a er ug in os a pc r m ls t (a l-z ah ra ni , i br ah im et a l. 20 21 )33 10 . m ed in a 15 ,7 08 38 .4 % fo r im ip en em 46 .1 % fo r m er op en em – k. p ne um on ia e vi te kii a l-z al ab an i et a l. (2 02 0) 47 (c on tin ue d) 23j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 r.s. abou-assy et al. review epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review ta bl e 4. r ev ie w o f c ar ba pe ne m re si st an ce st ud ie s i n ce nt ra l, w es te rn , e as te rn , n or th er n an d so ut he rn a re as o f s au di a ra bi a ar ea (c ity ) nu m be r o f is ol at es cr p re va le nc e (% ) ca rb ap en em as e ge ne s or ga ni sm m et ho ds u se d re fs . 11 . j ed da h 28 6 of k le bs ie lla sp p – kp c2 kl eb sie lla sp p. vi te kii br ot h m icr od ilu tio n pc r w g s h al a et a l. (2 01 9) 27 12 . t ai f 45 o f a ci ne to ba ct er sp p. 71 % o xa -5 1 (1 00 % ), im p (8 7. 5% ), n d m (6 2. 5% ), o xa -2 3 (5 9. 4% ), vi m (9 .3 % ), & o xa -4 0 (3 .1 % ) a. b au m an ni i vi te kii d is k di ffu si on er ic -p cr e l-b ad aw y et a l. (2 01 9) 30 13 . j ed da h 13 5 of a. b au m an ni i 55 .6 % o xa -5 1 (1 00 % ), o xa -2 3 (9 2% ), im p (8 4% ), n d m -1 (1 .3 % ), o xa -2 4/ 40 (5 .3 % ), vi m (9 2% ), te m (8 4% ), & s h v (1 0. 7% ) a. b au m an ni i vi te kii pc r sh ah e t a l. (2 01 9) 31 14 . m ak ka h 86 4 a ll sa m pl es 12 0 of e nt er oba ct er ia ce ae 21 .7 % o xa -4 8 (1 00 % ), n d m -1 (8 4. 7% ), & k pc (7 3. 1% ) k. p ne um on ia e (8 0. 7% ) e. c lo ac ae (7 .7 % ) e . c ol i ( 7. 7% ) p. m ira bi lis (3 .8 % ) vi te kii d is k pc r k ha n et a l. (2 01 9) 21 15 . m ak ka h 4, 80 3 of g ra m ne ga tiv e ba ci lli 58 .2 3% a. b au m an ni i ( 99 .1 3% ) p. ae ru gi no sa (6 2. 4% ) k. p ne um on ia (3 8% ) e. c ol i ( 5. 59 % ) vi te kii fa id ah e t a l. (2 01 7) 46 16 . m ak ka h – – vi m (3 1% ), & g es (8 .6 % ) ps eu do m on as a er ug in os a m ul tip le x pc r m ls t a l-z ah ra ni e t a l. (2 02 1) 33 17 . a l q at if 75 3 of p at ie nt s 20 8 of c lin ic al su rf ac es 2. 8% o f p at ie nt s 36 % o f c lin ic al su rf ac es o xa -6 6/ o xa -2 3 /a rm a (3 7% ), o xa -6 9/ o xa -2 3/ g es -1 1 (4 .2 % ), o xa -9 4/ n d m -1 (2 .1 % ), o xa -6 6/ o xa -2 3 (9 .5 % ), & o xa -5 1/ o xa -2 3 (1 % ) a. b au m an ni i bd p ho en ix pc r w g s a l-h am ad e t a l. (2 02 0) 48 18 . d am m am – – o xa -4 8 (d om in an t) , n d m (s ec on d cr e ge ne d is se m in atio n) , & v im (l ow p re va le nc e) – m ci m pc r ta la l ( 20 19 )32 19 . a l-j ou f 61 7 of e nt er oba ct er al es 32 % – k. p ne um on ia e (6 3% ), p. m ira bi lis (2 9% ), & e . c ol i ( 8% ) bd p ho en ix b an dy a nd ta nt ry (2 02 1) 25 20 . j iz an 50 10 % fo r i m ip en em 12 % fo r m er op ene m c tx -m (7 0% ), sh v (1 6% ), te m (1 2% ), & n d m -1 (0 % ). e. c ol i ( 50 % ), k. p ne um on ia e (4 0% ), a. b au m an ii (4 % ), p. ae ru gi no sa (4 % ), & e. c lo ac ae (2 % ) cd t vi te kii pc r s ob ia e t a l. (2 02 1) 26 21 . a bh a 27 6 of k . p ne um on ia e 61 .7 % fo r m er op en em 44 .5 % fo r im ip en em – k. p ne um on ia e vi te kii a l b sh ab sh e e t a l. (2 02 0) 49 22 . a bh a 54 o f k . p ne um on ia e 63 % fo r i m ip en em 57 .4 % fo r m er op ene m o xa -4 8 (8 1. 5% ), n d m (7 .4 % ), & v im (1 .8 % ) k. p ne um on ia e vi te kii ete st pc r a l-z ah ra ni a nd a la si ri (2 01 8) 29 d 70 c, ca rb ap en em as e d et ec tio n se t; m h t, m od ifi ed h od ge te st ; u ti , u rin ar y tr ac t i nf ec tio n; m ls t, m ul ti lo cu s se qu en ci ng t yp es ; w g s, w ho le g en om e se qu en ci ng ; m ci m , m od ifi ed c ar ba pe ne m in ac tiv at io n m et ho d. 24 j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review review r.s. abou-assy et al. reference to molecular methods. their obtained results confirm that mcim test is a simple tool for the reliable confirmation of carbapenemase activity with 97.3% sensitivity in enterobacteriaceae, especially in clinical microbiological laboratories with limited resources.32 the second study isolated environmental and clinical crab to assess the potential environmental contamination by this pathogen from frequenthand-touch surfaces of intensive care unit (icu), medical, and surgical units were randomly sampled for a month-long period, and the crab identified were compared to clinical isolates of the same period.48 epidemiology of northern area data regarding the cr patterns of enterobacterales in the northern regions are scarce, there was one surveillance non-genotypic report from al-jouf. bandy and tantry (2021) analyzed the antibiograms of enterobacterales identified from january 2019 to december 2019.25 in total, 617 enterobacterales were identified. k. pneumoniae exhibited 62.5%, 62%, and 58.3% resistance towards ertapenem, imipenem, and meropenem, respectively. in e. coli and k. pneumoniae, seasonal variation in the antimicrobial resistance rate was observed for imipenem and meropenem and the resistance were significantly higher in winter. furthermore, the k. pneumoniae meropenem resistance rate was significantly higher in samples received from intensive care units than from other units.25 epidemiology of southern area in the last five years, there were just three studies from southern regions, saudi arabia. two of them from abha city.29,49 the earlier study aims to identify the prevalence of crkp and the most common types of cabapenemases between late april and september in 2015.29 the results of this study indicate that increasing age and intensive care unit admission were associated with crkp isolation, the major type of carbapenemases in the southern region was oxa-48 with 81.5% and it seems to reach an endemic level, ndm was the second most frequent carbapenemase by 7.4% of isolates, this study refers oxa-48 and ndm carbapenem resistance strains dissemination in saudi arabia to receives large numbers of visitors and migrant workers from oxa-48 and ndm endemic countries such as turkey, india, and pakistan every year.29 the third studies aimed to examine k. pneumoniae infections in the icu of aseer central hospital and to determine their antimicrobial susceptibility and their relationship to patients’ clinical outcomes from patients with various infections.49 conclusion carbapenem-resistant enterobacterials (cre) is a growing threat and serious health concern spreading in saudi arabia and worldwide. this dilemma has been documented in many parts of the country and may challenge local health authorities. however, data is still scarce in certain local areas as well as in the eastern, northern border, tabuk, and najran regions. oxa-48 β-lactamase and ndm-1 β-lactamase are a prevalent gene responsible in cr strains in saudi arabia. the high rates of resistant enterobacterials in saudi arabia call for comprehensive surveillance programs to understand the origins and extent of the cr problem in depth as a major step to controlling the menace, developing a local antibiogram database coupled with nationwide antimicrobial stewardship and an infection prevention program might help in improving the knowledge of cr patterns. multiple complex risk factors associated with cr infection to the hospital environment, patient comorbidities, duration of hospital admission, icu complexity, intercurrent illness and the usage of antimicrobial agents contribute to the spread of cr infection. the findings of risk factors associated with cr infection may help clinicians and hospital epidemiologists estimate the likelihood of cr infection in different situations, and thereby initiate timely, targeted treatment and prevention measures. colistin, tigecycline, and a combination of carbapenem containing regimens are the mainstay of the current treatment options. however, new antimicrobials such as avibactam, plazomicin, or siderophore cephalosporins are promising and the research efforts should be focused on the molecular basis of cr and discovering new therapies.  references 1. codjoe, f. s. and e. s. donkor (2018). “carbapenem resistance: a review.” medical sciences 6(1): 1. 2. aslam, b., m. rasool, s. muzammil, a. b. siddique, z. nawaz, m. shafique, m. a. zahoor, r. binyamin, m. waseem and m. khurshid (2020). “carbapenem resistance: mechanisms and drivers of global menace.” pathog. bact. 3. pittet, d., b. allegranzi, j. storr, s. b. nejad, g. dziekan, a. leotsakos and l. donaldson (2008). “infection control as a major world health organization priority for developing countries.” journal of hospital infection 68(4): 285–292. 4. islam, t. a. b., s. shamsuzzaman, n. nehar and j. fardows (2016). “prevalence and antibiogram of microbial agents causing nosocomial urinary tract infection in surgical ward of dhaka medical college hospital.” journal of enam medical college 6(2): 75–79. 5. meletis, g. (2016). “carbapenem resistance: overview of the problem and future perspectives.” therapeutic advances in infectious disease 3(1):15–21. 6. aruhomukama, d., c. f. najjuka, h. kajumbula, m. okee, g. mboowa, i. sserwadda, r. mayanja, m. l. joloba and d. p. kateete (2019). “bla vimand bla oxa-mediated carbapenem resistance among acinetobacter baumannii and pseudomonas aeruginosa isolates from the mulago hospital intensive care unit in kampala, uganda.” bmc infectious diseases 19(1):1–8. 7. dortet, l., g. cuzon, v. ponties and p. nordmann (2017). “trends in carbapenemase-producing enterobacteriaceae, france, 2012 to 2014.” eurosurveillance 22(6): 30461. 8. de oliveira, d. m., b. m. forde, t. j. kidd, p. n. harris, m. a. schembri, s. a. beatson, d. l. paterson and m. j. walker (2020). “antimicrobial resistance in eskape pathogens.” clinical microbiology reviews 33(3):e00181-00119. 9. pouch, s. m. and m. j. satlin (2017). “carbapenem-resistant enterobacteriaceae in special populations: solid organ transplant recipients, stem cell transplant recipients, and patients with hematologic malignancies.” virulence 8(4): 391–402. 10. di domenico, e. g., i. cavallo, f. sivori, f. marchesi, g. prignano, f. pimpinelli, i. sperduti, l. pelagalli, f. di salvo and i. celesti (2020). “biofilm production by carbapenem-resistant klebsiella pneumoniae significantly increases the risk of death in oncological patients.” frontiers in cellular and infection microbiology 10. 11. sundaramoorthy, n. s., s. thothathri, m. bhaskaran, a. ganeshprasad and s. nagarajan (2021). “phages from ganges river curtail in vitro biofilms and planktonic growth of drug resistant klebsiella pneumoniae in a zebrafish infection model.” amb express 11(1):1–11. 12. di domenico, e. g., i. cavallo, f. sivori, f. marchesi, g. prignano, f. pimpinelli, i. sperduti, l. pelagalli, f. di salvo and i. celesti (2021). “biofilm production 25j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 r.s. abou-assy et al. review epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review by carbapenem.” enterobacteriaceae antimicrobial agents and resistance: relationship with the therapeutic approach. 13. ibrahim, m. e. (2019). “prevalence of acinetobacter baumannii in saudi arabia: risk factors, antimicrobial resistance patterns and mechanisms of carbapenem resistance.” annals of clinical microbiology and antimicrobials 18(1):1–12. 14. weber, d. j., e. e. sickbert-bennett, h. kanamori and w. a. rutala (2019). “new and emerging infectious diseases (ebola, middle eastern respiratory syndrome coronavirus, carbapenem-resistant enterobacteriaceae, candida auris): focus on environmental survival and germicide susceptibility.” american journal of infection control 47:a29–a38. 15. julia, l., k. vilankar, h. kang, d. e. brown, a. mathers and l. e. barnes (2017). environmental reservoirs of nosocomial infection: imputation methods for linking clinical and environmental microbiological data to understand infection transmission. amia annual symposium proceedings, american medical informatics association. 16. mills, j. p., n. j. talati, k. alby and j. h. han (2016). “the epidemiology of carbapenem-resistant klebsiella pneumoniae colonization and infection among long-term acute care hospital residents.” infection control & hospital epidemiology 37(1):55–60. 17. zhu, w., z. yuan and h.-y. zhou (2020). “risk factors for carbapenemresistant klebsiella pneumoniae infection relative to two types of control patients: a systematic review and meta-analysis.” antimicrobial resistance & infection control 9(1):1–13. 18. barber, k. e., j. l. wagner, r. c. larry and k. r. stover (2021). “frequency of and risk factors for carbapenem-resistant enterobacteriaceae.” journal of medical microbiology 70(2). 19. morgan, d. j., i. n. okeke, r. laxminarayan, e. n. perencevich and s. weisenberg (2011). “non-prescription antimicrobial use worldwide: a systematic review.” the lancet infectious diseases 11(9):692–701. 20. ssekatawa, k., d. k. byarugaba, e. wampande and f. ejobi (2018). “a systematic review: the current status of carbapenem resistance in east africa.” bmc research notes 11(1):1–9. 21. khan, m. a., a. m. mohamed, a. faiz and j. ahmad (2019). “enterobacterial infection in saudi arabia: first record of klebsiella pneumoniae with triple carbapenemase genes resistance.” the journal of infection in developing countries 13(04):334–341. 22. al hassinah, s., m. alzayer, l. okdah, m. doumith, s. al johani, r. alarfaj, e. alrashidi, n. alhuseinan, b. a. alwan and b. bakhashween (2020). “prevalence of molecular mechanisms of carbapenem resistance in pseudomonas aeruginosa clinical isolates from saudi arabia.” journal of infection and public health 13(2):329. 23. al mutair, a., s. alhumaid, z. al alawi, a. r. z. zaidi, a. j. alzahrani, j. a. al-tawfiq, h. al-shammari, a. a. rabaan, o. khojah and a. al-omari (2021). “five-year resistance trends in pathogens causing healthcare-associated infections at a multi-hospital healthcare system in saudi arabia, 2015– 2019.” journal of global antimicrobial resistance 25:142–150. 24. alqahtani, m., i. tickler, z. al deesi, w. alfouzan, a. al jabri, r. al jindan, s. al johani, s. alkahtani, a. al kharusi and e. mokaddas (2021). “molecular detection of carbapenem resistance genes in rectal swabs from patients in gulf cooperation council hospitals.” journal of hospital infection 112:96–103. 25. bandy, a. and b. tantry (2021). “esbl activity, mdr, and carbapenem resistance among predominant enterobacterales isolated in 2019.” antibiotics 10(6):744. 26. sobia, f., s. n. qurashi and k. y. ghailan (2021). “occurrence of blactxmgp1 and blactx-mgp26 in third generation cephalosporin-resistant and carbapenem-resistant bacterial isolates from southwest region of saudi arabia-a preliminary study.” saudi journal of biological sciences 28(9): 5408–5413. 27. hala, s., c. p. antony, m. alshehri, a. o. althaqafi, a. alsaedi, a. mufti, m. kaaki, b. t. alhaj-hussein, h. m. zowawi and a. al-amri (2019). “first report of klebsiella quasipneumoniae harboring bla kpc-2 in saudi arabia.” antimicrobial resistance & infection control 8(1):1–8. 28. jawhar, w. n. b., m. m. alrashed, a. m. somily and a. m. albarrag (2020). “molecular characterization of carbapenem-resistance genes among pseudomonas aeruginosa and acinetobacter baumannii clinical isolates in riyadh.” pharmacophore 11(6). 29. al-zahrani, i. a. and b. a. alasiri (2018). “the emergence of carbapenemresistant klebsiella pneumoniae isolates producing oxa-48 and ndm in the southern (asir) province, saudi arabia.” saudi medical journal 39(1):23. 30. el-badawy, m. f., s. f. abdelwahab, s. a. alghamdi and m. m. shohayeb (2019). “characterization of phenotypic and genotypic traits of carbapenem-resistant acinetobacter baumannii clinical isolates recovered from a tertiary care hospital in taif, saudi arabia.” infection and drug resistance 12:3113. 31. shah, m. w., m. yasir, m. farman, a. a. jiman-fatani, s. b. almasaudi, m. alawi, d. el-hossary and e. i. azhar (2019). “antimicrobial susceptibility and molecular characterization of clinical strains of acinetobacter baumannii in western saudi arabia.” microbial drug resistance 25(9):1297–1305. 32. talal, l. (2019). “phenotypic and genotypic characterization of carbapenemresistant enterobacteriaceae in bahrain and saudi arabia.” journal of infection and public health 12(2):299. 33. al-zahrani, ibrahim, al-ahmadi and bashaer (2021). “dissemination of vimproducing pseudomonas aeruginosa associated with high-risk clone st654 in a tertiary and quaternary hospital in makkah, saudi arabia.” journal of chemotherapy 33(1):12–20. 34. khater, e. s., a. a. alfaki and s. s. abd elmoaty (2020). “carbapenemresistant klebsiella pneumonia isolated from patients admitted in tertiary care hospital in saudi arabia.” journal of advances in microbiology:76–85. 35. aleidan, f. a., h. alkhelaifi, a. alsenaid, h. alromaizan, f. alsalham, a. almutairi, k. alsulaiman and a. g. abdel gadir (2021). “incidence and risk factors of carbapenem-resistant enterobacteriaceae infection in intensive care units: a matched case–control study.” expert review of anti-infective therapy 19(3):393–398. 36. alotaibi, f. (2019). “carbapenem-resistant enterobacteriaceae: an update narrative review from saudi arabia.” journal of infection and public health 12(4):465–471. 37. alotaibi, f. e., e. e. bukhari, m. m. al-mohizea, t. hafiz, e. b. essa and y. i. altokhais (2017). “emergence of carbapenem-resistant enterobacteriaceae isolated from patients in a university hospital in saudi arabia. epidemiology, clinical profiles and outcomes.” journal of infection and public health 10(5):667–673. 38. abdallah, m., r. alhababi, n. alqudah, b. aldyyat and a. alharthy (2018). “first report of carbapenem-resistant providencia stuartii in saudi arabia.” new microbes and new infections 26:107–109. 39. alghoribi, m. f., k. binkhamis, a. a. alswaji, a. alhijji, a. alsharidi, h. h. balkhy, m. doumith and a. somily (2020). “genomic analysis of the first kpc-producing klebsiella pneumoniae isolated from a patient in riyadh: a new public health concern in saudi arabia.” journal of infection and public health 13(4):647–650. 40. al-abdely, h., r. alhababi, h. m. dada, h. roushdy, m. m. alanazi, a. a. alessa, n. m. gad, a. m. alasmari, e. e. radwan and h. al-dughmani (2021). “molecular characterization of carbapenem-resistant enterobacterales in thirteen tertiary care hospitals in saudi arabia.” annals of saudi medicine 41(2): 63–70. 41. abd el ghany, m., h. sharaf, m. h. al-agamy, a. shibl, g. a. hill-cawthorne and p.-y. hong (2018). “genomic characterization of ndm-1 and 5, and oxa-181 carbapenemases in uropathogenic escherichia coli isolates from riyadh, saudi arabia.” plos one 13(8): e0201613. 42. al-agamy, m. h., a. aljallal, h. h. radwan and a. m. shibl (2018). “characterization of carbapenemases, esbls, and plasmid-mediated quinolone determinants in carbapenem-insensitive escherichia coli and klebsiella pneumoniae in riyadh hospitals.” journal of infection and public health 11(1):64–68. 43. uz zaman, t., m. alrodayyan, m. albladi, m. aldrees, m. i. siddique, s. aljohani and h. h. balkhy (2018). “clonal diversity and genetic profiling of antibiotic resistance among multidrug/carbapenem-resistant klebsiella pneumoniae isolates from a tertiary care hospital in saudi arabia.” bmc infectious diseases 18(1):1–11. 44. mutair, a. a., s. alhumaid, z. a. alawi, a. r. z. zaidi, a. j. alzahrani, j. al-tawfiq, h. al-shammari, a. rabaan, o. khojah and a. al-omari (2021). “five-year resistance trends in pathogens causing healthcare-associated infections at a multi-hospital healthcare system in saudi arabia, 2015-2019.” journal of global antimicrobial resistance. 45. leangapichart, t., p. gautret, k. griffiths, k. belhouchat, z. memish, d. raoult and j.-m. rolain (2016). “acquisition of a high diversity of bacteria during the hajj pilgrimage, including acinetobacter baumannii with bla oxa-72 and escherichia coli with bla ndm-5 carbapenemase genes.” antimicrobial agents and chemotherapy 60(10):5942–5948. 46. faidah, h. s., a. m. momenah, h. m. el-said, a. a. barhameen, s. s. ashgar, a. johargy, a. elsawy, w. almalki and s. al qurashi (2017). “trends in the annual incidence of carbapenem resistant among gram negative bacilli in a large teaching hospital in makah city, saudi arabia.” journal of tuberculosis research 5(04):229. 47. al-zalabani, a., o. a. althobyane, a. h. alshehri, a. o. alrehaili, m. o. namankani and o. h. aljafri (2020). “prevalence of klebsiella pneumoniae antibiotic resistance in medina, saudi arabia, 2014-2018.” cureus 12(8). 26 j contemp med sci | vol. 8, no. 1, january-february 2022: 18–26 epidemiology of carbapenem resistance enterobacterales in saudi arabia: a systematic review review r.s. abou-assy et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i1.1168 48. al-hamad, a., t. pal, h. leskafi, h. abbas, h. hejles, f. alsubikhy, d. darwish, a. ghazawi and a. sonnevend (2020). “molecular characterization of clinical and environmental carbapenem resistant acinetobacter baumannii isolates in a hospital of the eastern region of saudi arabia.” journal of infection and public health 13(4):632–636. 49. al bshabshe, a., a. al-hakami, b. alshehri, k. a. al-shahrani, a. a. alshehri, m. b. al shahrani, i. assiry, m. r. joseph, a. alkahtani and m. e. hamid (2020). “rising klebsiella pneumoniae infections and its expanding drug resistance in the intensive care unit of a tertiary healthcare hospital, saudi arabia.” cureus 12(8). 50. alzomor, o. a., t. s. alfawaz, a. abu-shaheen, m. a. alshehri and d. al shahrani (2019). “a matched case-control study to assess the carbapenemresistant enterobacteriaceae infections among hospitalized children at king fahad medical city, riyadh, saudi arabia.” saudi medical journal 40(11):1105. 39j contemp med sci | vol. 4, no. 1, winter 2018: 39–44 research antioxidants as recipes for efavirenz-induced liver damage: a study in albino rats elias adikwu,a bonsome bokolob adepartment of pharmacology, faculty of basic medical sciences, university of port harcourt, rivers state, nigeria bdepartment of pharmacology, faculty of basic medical sciences, niger delta university wilberforce island, bayelsa state, nigeria correspondence to elias adikwu (email: adikwuelias@gmail.com). (submitted: 02 september 2017 – revised version received: 24 september 2017 – accepted: 11 october 2017 – published online: 26 march 2018) introduction the liver plays vital role in the biotransformation of drugs and toxins.1 in view of the function of the liver, in the biotransformation of drugs and toxins, liver cells which include kupffer, hepatic stellate, endothelial and parenchymal cells are major target of oxidative radicals produced by drugs and toxins.2,3 oxidative stress has been shown to be an essential originating factor in the pathogenesis of drug-induced hepatic damage.4,5 the over-production of oxidative radicals is toxic to hepatocytes and initiates reactive oxygen species (ros)-mediated cascade causing hepatocyte death, and leading to acute or chronic hepatic damage.6,7 the use of efavirenz (efv) a member of the nonnucleoside reverse transcriptase inhibitors (nnrtis) has dramatically reduced human immunodeficiency virus (hiv)-related morbidity and mortality in the world.8 however, studies have associated the use of efv with hepatic damage which is a frequent class problem of the non-nnrtis.9,10 the molecular pathogenesis of this effect is poorly understood, but recent reports have highlighted features of mitochondrial dysfunction in hepatic cells exposed to clinically relevant concentrations of efv.11 also, recent studies have demonstrated endoplasmic reticulum stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cell lines.12 n-acetylcysteine (nac) is the acetylated derivative of the amino acid l-cysteine. it is frequently employed as a source of sulfhydryl groups to cells as an acetylated precursor of reduced gsh. it is an antioxidant that interacts directly with reactive oxygen and nitrogen species and up-regulate endogenous gsh status, thereby preventing oxidative stress-induced alterations in biomolecules.13 furthermore, in experimental animal model and clinical studies, nac showed ameliorative effect on drug-induced acute liver injury and has been clinically adopted for the treatment of acetaminophen associated hepatotoxicity.14,15 moreover, animal and human studies of nac suggest that it is a very safe as an effective tool for the treatment of many diseases considered to be mediated by oxidative radical and it has been used therapeutically in several disorders related to oxidative stress.16 vitamin e is the major lipid-soluble and potent chainbreaking antioxidant that inhibits the production of oxidative radicals when fat undergoes oxidation and during the propagation of free radical reactions.17 it is primarily located in cell and organelle membranes where it can exert its protective effect. it acts as the first line of defence against lipid peroxidation, by protecting polyunsaturated fatty acids present in membrane phospholipids and in plasma lipoproteins from free radical attack.18,19 it is the only major lipid-soluble, chain breaking antioxidant found in plasma, red cells and tissues, allowing it to protect the integrity of lipid structures, mainly membranes.20 in addition, previous studies have demonstrated the potential of vitamin e in the amelioration of drug-induced hepatotoxicity in animal model . 21,22 vitamin c is a six-carbon compound structurally related to glucose. it consists of two inter-convertible compounds: l-ascorbic acid, which is a strong reducing agent, and its oxidized derivative, l-dehydroascorbic acid.23 it is a water-soluble antioxidant with diverse biological functions. its actions include acting as a cofactor for the enzymes involved in collagen hydroxylation, biosynthesis of carnitine and norepinephrine, tyrosine metabolism and peptide hormone amidation.24 its antioxidant property includes inhibition of lipid peroxidation, oxidative cell damage and stimulatory effects on other antioxidants especially issn 2413-0516 objective hepatotoxicity is a clinical challenge associated with the use of efavirenz (efv). this study investigated the effects of n-acetylcysteine (nac), vitamins c and e on efv-induced hepatotoxicity in albino rats. methods rats were divided into groups and administered with nac (20 mg/kg), vitamin c (50 mg/kg), vitamin e (50 mg/kg), vitamins c + e and 60 mg/kg of efv, respectively. rats were also divided into groups and pretreated with nac, vitamins c, e, and combined doses of vitamins c + e prior to treatment with efv for 15 days, respectively. after drug administration rats were sacrificed and serum was collected and evaluated for liver function parameters. rats were dissected, liver was collected weighed and, homogenized and evaluated for alkaline phosphatase (alp), alanine aminotransferase (ast), aspartate aminotransferase (alt), γ-glutamyl transferase (ggt), lactate dehydrogenase (ldh), malondialdehyde (mda), super oxide dismutase (sod), catalase (cat), glutathione (gsh), glutathione peroxidase (gpx) levels and pathological damage. results effects were not significant (p > 0.05) on body and liver weights, however, the levels of ast, alt, ast, ggt, ldh, cb, tb and mda were increased significantly (p < 0.05) whereas sod, cat, sod, gsh and gpx were decreased significantly (p < 0.05) in efv-treated rats in comparison to control. the liver of efv-treated rats showed necrosis of hepatocytes hepatocytes necroses. nevertheless, efv-induced alterations in the above parameters were significantly (p < 0.05) ameliorated in antioxidants pretreated rats. the combined doses of vitamins c and e producedbest and significant (p < 0.05) in comparison to their individual doses. conclusion this study shows the showed prospects of antioxidants as candidates for the treatments of efavirenz-induced hepatotoxicity. keywords antioxidants, efavirenz, liver, toxicity, mitigation, rat 40 j contemp med sci | vol. 4, no. 1, winter 2018: 39–44 antioxidants ameliorate efavirenz-induced liver damage research vitamin e.25,26 furthermore, vitamin c has shown ameliorative effect on drug-induced hepatotoxity as shown in in-vivo animal studies.27,28 the present study was therefore designed to evaluate the effects of nac, vitamins c and e on efavirenz-induced hepatotoxicity in albino rats. materials and methods drugs and chemicals the vitamin e used for this study was manufactured by strides pharmaceuticals india while vitamin c was manufactured by emzor pharmaceuticals industries lagos, nigeria. efavirenz was manufactured by miland laboratory, india. the present study used 20 mg/kg/day of nac, 50 mg/kg/day of vitamin c and 50 mg/kg/day of vitamin e29,30 and 60 mg/kg/ day of efv. nac was dissolved normal saline, vitamin c was dissolved in water while arachis oil was used as the vehicle for vitamin e and efv. grouping of rats and drug administration fifty five albino rats were randomized into six groups a-e. groups a, b, d contained five rats each while group c and e contained twenty rats each which were further divided into four sub-groups of five rats each. the rats in group a served as placebo control whereas group b served as the solvent control and were administered with normal saline and arachis oil respectively. the rats in group c were orally administered with nac (20 mg/kg/day), vitamin c (50 mg/kg/day), vitamin e (50 mg/kg/day) and a combination of vitamins c+ e respectively. the rats in group d were orally administered with 60 mg/kg/day of efv. the rats in groups e were orally pretreated with nac, vitamin c, vitamin e, and combined doses of vitamins c+e prior to the administration of efv for 15 days respectively. collection of samples and biochemical analysis after the termination of drug administration on day 15, the rats were fasted overnight and sacrificed under diethyl ether. the blood was collected via cardiac puncture; serum was extracted and evaluated for alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, gamma glutamyl transferase conjugated and total bilirubin using randox diagnostic kits (randox laboratories ltd., crumlin, u.k.). liver was excised, weighed and washed in cold 1.15% kcl solution, then homogenized and centrifuged. the supernatant was decanted and used for the evaluation of oxidative stress indices. glutathione peroxidase (gpx) activity was evaluated as reported by rotruck et al., 197331 sod activity was determined by the method of sun and zigma (1978)32 while cat activity was measured according to sinha et al., 1972.33 reduced glutathione was analyzed according as described by sedlak and lindsay (1968)34 while malondialdehyde (mda) was measured using the method of buege and aust (1978).35 histological examination of the liver the histological examination of the liver was performed in anatomical pathology department of university of port harcourt teaching hospital, choba, rivers state, nigeria. the liver tissue was excised and rinsed in normal saline. liver sections were taken and processed and embedded in paraffin. liver tissues were then processed into sections 4-5 μm thick, stained with hematoxylin and eosin and observed under a light microscope for any morphological changes. statistical analysis results are expressed as mean ± sd. differences among the experimental groups were identified by one-way analysis of variance and tukey’s multiple comparison test and a p < 0.05 was considered significant. results effects on body, liver weights and liver function parameters the administration of nac, vitamins c and e for 15 days did not produce significant (p > 0.05) effects on serum levels of ast, alt, alp, ggt, ldh, tb and cb when compared to control (table 2 and 3).on the other hand, the above parameters were significantly (p < 0.05) increased in rats administered with 60mg/kg/day of efv for 15 days with no significant (p > 0.05) effects on the body and liver weights in comparison to control (table 1-3). however, rats pretreated with individual doses of nac, vitamins c and e showed significant (p < 0.05) reductions in the serum levels of ast, alt, alp, ggt, ldh, cb and tb when compared to efv-treated rats (table 2 and 3). furthermore, a combination of vitamins c and e produced most and significant (p < 0.05) reductions in the serum levels of ast, alt, alp, ggt, ldh, cb and tb when compared to their individual doses, but effects did not differ significantly (p < 0.05) from pretreatment with nac (table 1and 2). furthermore, treatment with nac, vit c and e did not produce significant (p < 0.05) effects on liver levels of ast, alt, alp, ggt, and ldh in comparison to control. on the contrary, these parameters were significantly (p < 0.05) increased in efv-treated rats in comparison to control (table 1 and 2). however, the liver levels of ast, alt, alp, ggt, and ldh were decreased significantly (p < 0.05) in rats pretreated with nac, vitamins c and e prior to treatment with efv. the observed decreases in these parameters were most and significant (p < 0.05) in rats pretreated with combined doses of vitamins c and e when compared to their individual doses. however, decreases observed in these parameters in rats pretreated with combined doses of vitamins c and e did not differ (p > 0.05) when compared to nac pretreated rats (table 2 and 3). effects on oxidative stress indices and liver histology the administration of nac, vitamins c and e did not produce significant (p > 0.05) effects on the liver levels of sod, cat, gsh, gpx and mda when compared to control. in contrast, liver levels of sod, cat, gsh and gpx were decreased significantly (p < 0.05) whereas mda levels were increased significantly (p < 0.05) in rats treated with efv when compared to control (table 4). however, rats pretreated with nac, vitamin c, and e prior to treatment with efv showed significant (p < 0.05) increases in sod, cat, gsh and gpx levels whereas mda levels were decreased significantly (p < 0.05) when compared to efv-treated rats. interestingly, pretreatment with a combination of vitamins c and e produced the most and significant (p < 0.05) effects on sod, cat, elias adikwu and bonsome bokolo 41j contemp med sci | vol. 4, no. 1, winter 2018: 39–44 research antioxidants ameliorate efavirenz-induced liver damage table 3. effects of n-acetylcysteine, vitamins c and e on liver levels of biomarkers of liver function in efavirenz-treated albino rats treatment alt (u/l) ast (u/l) alp (u/l) ggt (u/l) ldh (u/l) control 70.6 ± 8.07a 65.6 ± 6.33a 63.0 ± 4.12a 80.0 ± 7.15a 1.81 ± 4.12a nac 64.3 ± 5.25a 60.2 ± 4.00a 59.4 ± 3.01a 72.9 ± 6.72a 1.72 ± 3.01a vitamin c 69.4 ± 6.21a 63.5 ± 4.20a 61.8 ± 5.15a 79.8 ± 4.33a 1.79 ± 5.15a vitamin e 67.9 ± 7.18a 62.3 ± 6.06a 60.4 ± 4.01a 77.4 ± 5.42a 1.77 ± 4.01a vitamins c + e 65.3 ± 4.20a 60.0 ± 4.00a 58.6 ± 3.01a 72.6 ± 6.11a 1.73 ± 3.01a efv 190.8 ± 9.13b 194.7 ± 9.34b 186.0 ± 7.55b 200.9 ± 9.95b 4.89 ± 7.55b efv + nac 80.5 ± 6.08c 82.7 ± 5.00c 80.7 ± 5.00c 85.7 ± 6.72c 1.75 ± 5.00a efv + vitamin c 130.6 ± 7.14d 130.2 ± 8.21d 127.6 ± 9.13d 130.6 ± 9.68d 2.99 ± 9.13c efv + vitamin e 125.4 ± 8.05d 127.5 ± 9.15d 120.3 ± 9.24d 125.3 ± 8.65d 2. 73 ± 9.24c efv + vitamins c + e 84.2 ± 7.28c 85.6 ± 7.26c 85.5 ± 6.01c 87.9 ± 7.32c 1.70 ± 6.01d table 4. effects of n-acetylcysteine, vitamins c and e on liver oxidative stress indices of efavirenz-treated albino rats treatment mda (µmol/mg protein) cat (u/mg protein) sod (u/mg protein) gsh (µg/mg protein) gpx (µg/mg protein) control 0.38 ± 0.05a 30.1 ± 3.12a 20.2 ± 1.19a 8.47 ± 0.17a 15.2 ± 1.14a nac 0.30 ± 0.06a 34.8 ± 2.05a 23.3 ± 1.05a 8.79 ± 0.05a 16.0 ± 1.32a vitamin c 0.36 ± 0.02a 31.5 ± 3.23a 21.7 ± 1.20a 8.50 ± 0.26a 15.5 ± 1.00a vitamin e 0.35 ± 0.01a 32.2 ± 4.23a 21.3 ± 1.27a 8.55 ± 0.09a 15.8 ± 1.75a vitamins c + e 0.31 ± 0.02a 34.2 ± 2.15a 23.5 ± 0.21a 8.71 ± 0.18a 17.8 ± 1.63a efv 0.98 ± 0.02b 10.7 ± 1.23b 6.77 ± 0.24b 1.56 ± 0.08b 5.32 ± 0.53b efv + nac 0.48 ± 0.09c 25.3 ± 2.23c 15.9 ± 0.23c 8.27 ± 0.23a 14.0 ± 1.03a efv + vitamin c 0.69 ± 0.06d 15.7 ± 1.20d 10.5 ± 0.13d 3.22 ± 0.15c 8.42 ± 1.22c efv + vitamin e 0.61 ± 0.07d 17.0 ± 1.29d 11.8 ± 0.11d 3.59 ± 0.04c 8.99 ± 0.56c efv + vitamins c + e 0.49 ± 0.01c 23.5 ± 1.15c 15.5 ± 0.17c 8.19 ± 0.13a 13.9 ± 1.17a nac, n-acetylcysteine; efv, efavirenz. data expressed as mean ± sd, n = 5. values with different superscripts on the same column differ significantly at p < 0.05 anova. table 1. effects of n-acetylcysteine, vitamin c and e on body and liver weights of efavirenztreated albino rats treatments initial bw (g) final bw (g) bw gain (g) liver w (g) relative liver w(%) control 220 ± 12.0 250 ± 13.3 18.0 ± 2.00 6.95 ± 0.31 2.78 ± 0.18 efv 230 ± 11.2 248 ± 10.6 18.0 ± 2.11 7.00 ± 0.19 2.82 ± 0.47 efv +nac 234 ± 12.7 253 ± 12.9 19.0 ± 1.00 6.70 ± 0.10 2.64 ± 0.84 efv+vitamin c 225 ± 12.0 243 ± 14.2 18.0 ± 3.00 7.05 ± 0.37 2.90 ± 0.51 efv+vitamin e 240 ± 11.7 260 ± 10.5 20.0 ± 3.13 6.90 ± 0.23 2.65 ± 0.24 efv+vitamins c + e 248 ± 9.66 268 ± 9.06 21.0 ± 2.20 7.01 ± 0.42 2.61 ± 0.89 w,weight; bw,body weight; data expresses ad mean ± sd, n = 5. table 2. effects of n-acetylcysteine, vitamins c and e on serum liver function indices of efavirenz-treated albino rats treatments alt (u/l) ast (u/l) alp (u/l) ldh (u/l) ggt (u/l) tb (g/dl) cb (g/dl) control 38.8 ± 2.09a 32.7 ± 4.12a 34.2 ± 3.38a 50.2 ± 5.22a 0.85 ± 0.08a 9.20 ± 1.44a 4.78 ± 0.08a nac 35.3 ± 3.09a 28.3 ± 3.09a 29.3 ± 3.00a 46.9 ± 3.11a 0.77 ± 0.06a 8.79 ± 0.09a 4.00 ± 0.12a vitamin c 36.7 ± 5.06a 30.9 ± 3.00a 32.3 ± 0.23a 49.3 ± 4.20a 0.83 ± 0.08a 8.92 ± 0.31a 4.53 ± 0.06a vitamin e 35.6 ± 3.04a 31.4 ± 3.06a 31.4 ± 2.11a 48.0 ± 5.20a 0.82 ± 0.05a 8.85 ± 0.22a 4.27 ± 0.01a vitamins c + e 33.8 ± 4.20a 28.3 ± 2.00a 30.6 ± 3.01a 46.3 ± 4.35a 0.78 ± 0.01a 8.75 ± 0.05a 4.09 ± 0.05a efv 114.8 ± 7.13b 130.0 ± 9.34b 120.7 ± 8.55b 160.1 ± 9.23b 3.97 ± 0.02b 21.1 ± 0.11b 14.9 ± 1.15b efv + nac 40.3 ± 3.09a 38.3 ± 3.09c 40.3 ± 3.09c 55.6 ± 5.40a 0.90 ± 0.01c 10.3 ± 1.52a 5.31 ± 0.09c efv + vitamin c 69.8 ± 5.14c 70.2 ± 6.21d 75.6 ± 9.13d 110.6 ± 9.07c 1.73 ± 0.06d 15.6 ± 1.21c 10.7 ± 0.13d efv + vitamin e 67.4 ± 4.05c 62.5 ± 5.15d 70.3 ± 7.24d 100.1 ± 8.22c 1.56 ± 0.05d 15.1 ± 1.31c 10.3 ± 0.24d efv + vitamins c + e 41.2 ± 2.08 a 38.6 ± 4.06c 37.5 ± 4.01c 57.7 ± 6.14a 0.87 ± 0.06a 10.7 ± 1.05c 575 ± 0.01c nac, n-acetylcysteine; efv, efavirenz. data expressed as mean ± sd, n = 5. values with different superscripts on the same column differ significantly at p < 0.05 anova. elias adikwu and bonsome bokolo 42 j contemp med sci | vol. 4, no. 1, winter 2018: 39–44 antioxidants ameliorate efavirenz-induced liver damage research gsh, gpx and mda levels when compared to individual doses. however effects observed in rats pretreated with combined doses of vitamins c and e were not significantly (p > 0.05) different when compared to pretreatment with nac (table 4). furthermore, microscopic examination of the h and e stained sections of the liver of control rat and rats administered with nac, vitamins c and e showed normal histology (fig 1a-e). liver of rats treated with efv shows necrosis of hepatocyte (fig 1.f). however, the liver of rats pretreated with nac, vitamins c and e and combined doses of vitamin c and e showed normal histology (fig 1. g-j) discussion oxidative stress, a consequence of free radicals’ generation is a common mechanism underlying hepatotoxicity caused by drugs and toxicants.36 antioxidants are chemical agents that scavenge, neutralize and mop-up free radicals, thereby preventing oxidative stress.37 therefore, the present study evaluated the benefits of nac, vitamins c and e on efv-induced liver toxicity in albino rats. in the current study, treatment with nac, vitamins c and e did not produce significant effects on ast, alt, alp, ggt, ldh, cb, tb, sod, gsh, and cat levels. these observations are in conjunction with previous reports.38,39 this study did not observe significant effects on the body and liver weights in efv-treated rats, however; levels of ast, alt, alp, ggt, ldh, ct, tb and mda were elevated whereas sod, gsh, cat and gpx levels were reduced significantly. in addition, alterations in hepatic morphology were observed in efv-treated rats. these finding are consistent with previous reports.40-42 ast and alt are enzymes involved in the transfer of amino groups of aspartate and alanine to ketoglutaric acid.43 ast, alt and alp are present in the liver but, alt is primarily located in the liver, and thus is a more specific marker of hepatocellular cell injury.44-46 bilirubin is produced by the normal breakdown of pigment-containing proteins, especially haemoglobin from senescent red blood cells and myoglobin from muscle breakdown.47 ast, alt, alp, ggt, ldh and bilirubin are clinically used as fundamental indices for the assemement of the functionality of the liver. elevations in the levels of these parameters typify hepatocellular injury.48 therefore, the observed elevations in the levels of these parameters in efvtreated rats indicate hepatocellular injury. in various forms of liver disease, serum levels of numerous cytosolic, mitochondrial and membrane associated enzymes are increased. the detailed mechanism by which enzymes released from the cytosol and mitochondria of hepatocytes into the blood stream is not completely known. clinical observations and experimental studies have shown that subtle membrane changes are sufficient to allow passage of intracellular enzymes to the intracellular space.49 a very large concentration gradient between the hepatocytes and the sinusoidal space usually exists for enzymes. cell damage increases membrane permeability, causing cytosolic iso-enzymes to spill into the sinusoids and from there into the peripheral blood.50 oxidative stress has been considered as a pathological mechanism that characterises the initiation and progression of drug-induced liver injury. a network of antioxidant defence which include sod, gat and gsh is structured in the liver of mammals for cellular response to down-regulate oxidative stress under physiological condition and to maintain redox homeostasis in the liver.51 however, the stimulatory production of excessive free radicals from oxygen and nitrogen could alter liver redox homeostasis leading to antioxidant depletions.51-53 hence, the observed decreases in the liver levels of sod, cat, gsh and gpx in efv-treated rats are evidence of hepatic oxidative stress. oxidative stress can cause hepatic damage by inducing antioxidants depletion, irretrievable alteration of lipids, proteins and dna contents and more importantly, modulates pathways that control normal hepatic biological functions.54,55 mda has been widely used for many years as a convenient biomarker for lipid peroxidation which is a free-radical-mediated chain of reactions. its level is always elevated in a state of established oxidative stress and lipid peroxidation.6,57 therefore, the observed elevated liver levels of mda in efv-treated rats suggest lipid peroxidation which a fig 1. (a) liver of the control rat showing normal architecture. (b–e) liver of rat administered with 20 mg/kg of n-acetylcysteine, 20 mg/kg of vitamin c, 20 mg/kg vitamin e and combined doses of vitamins c and e, respectively for 15 days showing normal architecture. (e) liver of rat administered with 60 mg/kg/day of efavirenz (efv) showing hepatocyte and centrilobular necrosis. (f–i) liver of rats pretreated with 20 mg/kg of n-acetylcysteine, 20 mg/kg of vitamin c, 20 mg/kg vitamin e and combined doses of vitamins c and e respectively prior to treatment with 60 mg/kg of efv for 15 days showing normal architecture (h and ex200). b gf c h d i e j a elias adikwu and bonsome bokolo 43j contemp med sci | vol. 4, no. 1, winter 2018: 39–44 research antioxidants ameliorate efavirenz-induced liver damage might have resulted in oxidative deterioration of hepatic polyunsaturated lipids and increase membrane permeability. furthermore, in the present study pretreatment with nac, vitamins c and e ameliorate efv-induced hepatic damage. more so, ameliorative effects were most observed in rats pretreated with combined doses of vitamins c and e than their individual doses. the hepatoprotective effects of antioxidants observed in the present study correlate with findings by ebuehi et al (2012)58 who reported the palliative effects of vitamins c and e on lead-induced hepatotoxicity in rats. similarly awodele and co-researchers reported the ameliorative effects of vitamins c and e on nevirapine-induced hepatotoxicity in rats.59 also, naglaa et al., 201560 reported the beneficial effect of nac on acetaminophen-induced hepatotoxicity in rats. in the current study, these antioxidants might have protected the liver by inhibiting the chain reactions of efv-generated free radicals or scavenged the free radicals before they reached their hepatic targets. also, these antioxidants might have inhibited the depletion of endogenous antioxidants or facilitate their regeneration. nac is a direct and indirect antioxidant that scavenges, neutralizes and chelates free radicals, thereby inhibiting oxidative destruction of tissues and organs.61 it can maintain –sh groups of enzymes and membrane proteins in their reduced state.62 nac can reduce extracellular cystine to cysteine, or produce sh required for the synthesis of hepatic gsh. it can prevent drug-induced hepatic gsh depletion as well; up-regulate the activity of hepatic gsh. also, nac can enhance hepatic glutathione-s-transferase, sod, and cat activities, and promote detoxification mechanisms.63 vitamin c is a water soluble antioxidant that scavenges free radicals in extracellular fluid, trapping radicals and protecting biological membrane from oxidative damage.64 it is an important source of electrons and it easily donates electrons to free radicals such as hydroxyl radicals and super oxide radicals, thereby down-regulating their activities.65 also, vitamin c can up-regulate the levels and activities of some endogenous antioxidants.66 vitamin e is an essential lipophilic antioxidant that can inhibit oxidative radical-induced damage to polyunsaturated fatty acids and act as a membrane-stabilizing agent that can prevent damage to phospholipids.67 it is mainly found in the hydrocarbon part of membrane lipid bilayer towards the membrane interface and in close proximity to oxidise enzymes which can initiate the production of free radicals.68,69 studies have shown that it can inhibit peroxidation of membrane lipids by scavenging lipid peroxyl radicals, as a consequence of which it is converted into a tocopheroxyl radical a potent antioxidant.70 also, it can prevent xenobiotic-induced hepatic mitochondria oxidative stress by down-regulating the activities of oxidative radicals.71 in conclusion, the present study gives an insight on the possible use of antioxidants as recipes for efavirenz-induced liver toxicity in rats. acknowledgments the authors appreciate the support of mr eze ihukwumere of the faculty of pharmacy madonna university, elele, rivers state conflict of interest the authors declare no conflict of interest. source of funding the authors declare no sources of funding. n references 1. duncan sa. transcriptional regulation of liver development. dev dyn. 219:131–142. 2. sanchez-valle v, chavez-tapia nc, uribe m, mendez-sanchez n. role of oxidative stress and molecular changes in liver fibrosis: a review. curr med chem. 2012;194850–194860. 3. cichoż-lach h, michalak a. oxidative stress as a crucial factor in liver diseases. world j. gastroenterol. 2014;20:8082–8091. 4. albano e. free radical mechanisms in immune reactions associated with alcoholic liver disease. free radic biol med. 2002;32:110–114. 5. jaeschke h. reactive oxygen and mechanisms of inflammatory liver injury. j gast hep. 15:718–724. 6. kaplowitz n. biochemical and cellular mechanisms of toxic liver injury. semin liver dis. 2002;22:137–144. 7. lee sj, kim sy, min h. effects of vitamin c and e supplementation on oxidative stress and liver toxicity in rats fed a low-fat ethanol diet. nutr res pract. 2013;7:109–114. 8. palella fj, delaney km, moorman ac, loveless mo, fuhrer j, satten ga, et al. declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. hiv outpatient study investigators. n engl j med. 1998;338:853–860. 9. nunez m, lana r, mendoza jl, martin-carbonero l, & soriano v, risk factors for severe hepatic injury after introduction of highly active antiretroviral therapy. j acquir immune defic syndr, 2001;27:426–31. 10. martinez e, blanco jl, arnaiz ja, perez-cuevas jb, mocroft a, cruceta a, et al. hepatotoxicity in hiv-1-infected patients receiving nevirapine-containing antiretroviral therapy. aids. 2001;15:1261–1268. 11. apostolova n, gomez-sucerquia lj, gortat a, blas-garcia a, esplugues jv. autophagy as a rescue mechanism in efavirenz-induced mitochondrial dysfunction: a lesson from hepatic cells. autophagy. 2011;7:1402–1404. 12. polo m, alegre f, funes ha, blas-garcia a, victor vm, esplugues jv, et al. mitochondrial (dys)function a factor underlying the variability of efavirenzinduced hepatotoxicity? br j pharmacol. 2015;172:1713–1727. 13. zafarullah m, li wq, sylvester j, ahmad m. molecular mechanisms of n-acetylcysteine actions. cell mol life sci. 2003;60:6–20. 14. baniasadi s, eftekhari p, tabarsi p, fahimi f, raoufy mr, masjedi mr, et al. protective effect of n-acetylcysteine on antituberculosis drug-induced hepatotoxicity. eur j gast hepatol. 2010;22:1235–1238. 15. singh s, hynan ls, lee wm. acute liver failure study g, improvements in hepatic serological biomarkers are associated with clinical benefit of intravenous n-acetylcysteine in early stage non-acetaminophen acute liver failure. dig dis sci. 2013;58:1397–1402. 16. cotgreave ia. n-acetylcysteine: pharmacological considerations and experimental and clinical applications. adv pharmacol. 1997;38: 205–227. 17. burton gw, joyce a, ingold ku. is vitamin e the only lipid-soluble, chainbreaking antioxidant in human blood plasma and erythrocyte membranes? arch biochem biophys. 1983;221:281–290. 18. howard ac, anna k, mcneil ak, mcneil pl. promotion of plasma membrane repair by vitamin e. nat commun. 2011;20:597. 19. tran k, wong jt, lee e, chan ac, choy pc. vitamin e potentiates arachidonate release and phospholipase a2 activity in rat heart myoblastic cells. biochem. 1996;31:385–391. 20. burton gw, traber mg. vitamin e: antioxidant activity, biokinetics, and bioavailability. annu rev nutr. 1990;10:357–382. 21. fariss mw. cadmium toxicity: unique cytoprotective properties of alpha tocopheryl succinate in hepatocytes. toxicology. 1991;69:63–77. 22. ben amara n, soudani a, troudi h, bouaziz t, zegha n. antioxidant effect of vitamin e and selenium on hepatotoxicity induced by dimethoate in female adult rats. ecotoxicol enviro safe. 2011;74:811–819. elias adikwu and bonsome bokolo 44 j contemp med sci | vol. 4, no. 1, winter 2018: 39–44 antioxidants ameliorate efavirenz-induced liver damage research this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 23. adikwu e, oputiri d. hepatoprotective effect of vitamin c. pharmacol pharm. 2013;4:84–92. 24. padayatty sj, katz a, wang y, eck p, kwon o, lee jh, et al. vitamin c as anantioxidant: evaluation of its role in disease prevention. jour of ameri coll of nutri. 2001;22:18–53. 25. frei bl, england l, ames bn. ascorbate is an outstanding antioxidant in human blood plasma. proceedings of national academy of science usa. 86:6377–6391. 26. xavier sm, barbosa co, barros do, silva rf, oliveria aa, freitas rm. vitamin c antioxidant in hippocampus of adult wistar rats after seizures and status epilepticus induced by pilocarpine. neurosci lett. 2007;420:76–79. 27. mor f, ozmen o. effect of vitamin c in reducing the toxicity of endosulfan in liver in rabbits. exp toxicol pathol. 2010;62:75–80. 28. al-attar am. hepatoprotective influence of vitamin c on thioacetamideinduced liver cirrhosis in wistar male rats. journal of pharmacology and toxicology. 2011;6:218–233. 29. qader gi, aziz rs, ahmed za, abdullah zf, hussain sa. protective effects of quercetin against isoniazid and rifampicin induced hepatotoxicity in rats.” am jour of pharm scien, 2014;2:56–60. 30. adikwu e, onyedenyifa oj & samuel nk beneficial effects of vitamins c and e on tenofovir and nevirapine-inducedhepatorenal toxicity in male albino rats international journal of biomedical research. 2016;7:211–218. 31. rotruck jt, rope al, ganther hf, swason ab. selenium: biochemical role as a component of glutathione peroxidase. science. 1973;179:588–590. 32. sun m, zigma s. an improved spectrophotometer assay of superoxide dismutase based on epinephrine antioxidation. analalytic biochemistry. 1978;90:81–89. 33. sinha ka. colorimetric assay of catalase. anal biochem. 1972;47:389–394. 34. sedlak j, lindsay rh. estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with ellman’s reagent. analytical biochemistry. 1968;25:1192–1205. 35. buege, ja, aust sd. microsomal lipid peroxidation. methods enzymol. 1978;52:302–310 36. deavall gd, elizabeth am, judith mh, ruth r. drug-induced oxidative stress and toxicity. j toxicol. 2012 (2012), article id 645460, 13 page. 37. li s, tan h, wang n, zhang z, lao l, wong c, et al. the role of oxidative stress and antioxidants in liver diseases. int j mol sci. 2015;16:26087–26124 38. shokrzadeh m, shobi s, attar h, shayegan s, sadat s, payam h, et al. effect of vitamins a, e and c on liver enzyme activity in rats exposed to organophosphate pesticide diazinon. pak jour of biol sci. 2012;15:936–941. 39. mahmoud gs, amer as. protective effects of vitamin c against nicotineinduced oxidative damage of rat liver and kidney iosr journal of environmental science.toxicology and food technology. 2014;8:50–63. 40. sulkowski ms, thomas dl, chaisson re, moore rd. hepatotoxicity associated with antiretroviral therapy in adults infected with human immunodeficiency virus and the role of hepatitis c or b virus infection. jama. 2000;283:74–80. 41. adjene oj, avbunudiogba ja, igbigbi ps. oxidative stress induced by chronic administration of efavirenz on the intracranial visual relay centers of adult wistar rats biology and medicine. 2011;3;16–24. 42. sonderup mw, wainwright h, maughan d, setshedi m, spearman cwn. characteristics of efavirenz drug induced liver injury: a cohort analysis. aids. 2016;30:1483–1485. 43. karmen a, wroblewski f, ladue js. transaminase activity in human blood. j clin invest. 1995;34:126–31. 44. dufour dr, lott ja, nolte fs. diagnosis and monitoring of hepatic injury. i. performance characteristics of laboratory tests. clin chem. 200a;46:2027–2049. 45. dufour dr, lott ja, nolte fs. diagnosis and monitoring of hepatic injury. ii. recommendations for use of laboratory tests in screening, diagnosis, and monitoring. clin chem. 200b;46:2050–2068. 46. roberts wm. variations in the phosphatase activity of the blood in disease. br j exp pathol. 1930;11:90–95. 47. blanckaert m. analysis of bilirubin and bilirubin mono-and di-conjugates. biochem j. 1985;115–28. 48. kwo py, cohen sm, lim jk. practice guideline: evaluation of abnormal liver chemistries am j gastroenterol advance online publication. 2016;517. 49. tredger jm, sherwood ra. the liver: new functional, prognostic and diagnostic tests. ann clin biochem. 1979;34:121–141. 50. freidel r, diedrichs f, lindena j. release and extra cellular turnover of cellular enzyme. in: schmidt e, fw schmidt i, transchold s. munich (eds.), clinical enzymology. karger. 1979;70–105. 51. li sha, tan h, wang n, zhang z, lao l, wong c, et al. the role of oxidative stress and antioxidants in liver diseases. int j mol sci. 2015;16:26087–26124 52. karabulut ab, gui m, karabulut e, kiran tr, ocak sg, otlu o. oxidant and antioxidant activity in rabbit livers treated with zoledronic acid. transplant proc. 2010;42:3820–3822. 53. dey a, lakshmanan j. the role of antioxidants and other agents in alleviating hyperglycemia mediated oxidative stress and injury in liver. food funct. 2013;4:1148–1184. 54. singal ak, jampana sc, weinman sa. antioxidants as therapeutic agents for liver disease. liver int. 2011;31:1432–1448. 55. feng, y, wang n, ye x, li h, feng y, cheung f, et al. hepatoprotective effect and its possiblemechanism of coptidis rhizoma aqueous extract on carbon tetrachloride-induced chronic liver hepatotoxicityin rats. j. ethnopharmacol. 2011;138:683–690. 56. esterbauer h, cheeseman kh. determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. methods in enzymology. 1990;186:407–421. 57. esterbauer h, schaur rj, zollner h. chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes,” free radical biology and medicine. 1991;11:81–128. 58. ebuehi oat, ogedegbe ra, ebuehi om. oral administration of vitamin c and vitamin e ameliorates lead-induced hepatotoxicity and oxidative stress in the rat brain. nig qt j hosp med. 2012;22:85–90. 59. awodele o, popoola t, rotimi k, ikumawoyi v, okunowo w. antioxidant modulation of nevirapine induced hepatotoxicity in rats interdiscip toxicol. 2015;8:8–14. 60. naglaa ab, saad he, mohammed mm, & ahmed msh, protective effect of curcumin versus n-acetylcystein on acetaminophen induced hepatotoxicity in adult albino rats. j cytol histol 2015; s3; 018. 1–8. 61. flanagan rj, meredith tj. use of n-acetyl cysteine in clinical toxicology. am j med. 1991;91:131s–139s. 62. wagner pd, mathien-castello o, bebout de, gray at, natterson pd, glennow c. protection against pulmonary o2 toxicity by n-acetyl cysteine. eur respir j. 1989;2:116–126. 63. zhao c, shichi h. prevention of acetaminophen-induced cataract by a combination of diallyl disulfide and n-acetyl cysteine. j ocul pharmacol her. 1998;14:345–55. 64. harapanhalli rs, yaghmai v, giuliani d, howell rw, rao dv. antioxidant effects of vitamin c in mice following xirradiation. res comm mol pathol pharmacol. 1996;94:271–287. 65. bendich antioxidant micro nutrients and immune responses. in “micronutrients and immune functions” (a. bendich and c.e. butterworth, eds.), 87:168–180. new york academy of science, new york. 66. durak d, uzun fg, kalender s, ogutcu a, uzunhisarcikli m, kalender y. malathion-induced oxidative stress in human erythrocytes and the protective effect of vitamins c and e in vitro. environmental toxicology. 2009;24:235–242. 67. brigelius-flohe r. vitamin e: the shrew waiting to be tamed. free radic biol. med. 46;2009;543–554. 68. mcdowell lr. vitamin c, a and e. in: vitamins in animal nutrition comparative aspects to human nutrition, pp. 93-131. academic press, london, uk. 1989. 69. hosseini-mansoub n, chekani-azar s, tehrani aa, lotfi a. influence of dietary vitamin e and zinc on performance, oxidative stability and some blood measures of broiler chickens reared under heat stress (35°c). j agrobiol. 2010;27:103–110. 70. dowd p, zheng zb. on the mechanism of the anticlotting action of vitamin. e quinone proc natl acad sci usa. 1995;92:8171–8175. 71. fariss mw. oxygen toxicity: unique cytoprotective properties of vitamin e succinate in hepatocytes. free radic biol med. 1990;9:333–343. dx.doi.org/10.22317/jcms.03201809 elias adikwu and bonsome bokolo 88 j contemp med sci | vol. 6, no. 3, may–june 2020: 88–94 review issn 2413-0516 introduction asthma, as a type of allergic diseases, is a common chronic inflammatory disease of the airways characterized by variable and recurring symptoms, reversible airflow obstruction, and bronchospasm1 and common symptoms of it include wheezing, coughing, chest tightness, and shortness of breath.2 the prevalence of asthma has increased significantly since the 1970s. as of 2011, 235–300 million people were affected globally, including about 250,000 deaths.3 it is estimated to affect as many as 339 million people worldwide according to a report from the global asthma network published in 2018.4 globally, asthma is ranked 16th among the leading causes of years lived with disability and 28th among the leading causes of burden of disease.4, 5 it is estimated that the number of people with asthma worldwide may be as high as 334 million according to a report from the global asthma network published in 2014.4 pathophysiological mechanisms of asthma contain the changes occurring in the airways that consist of a chronic eosinophilic and lymphocytic inflammation, together with epithelial and structural remodeling and proliferation, and altered matrix proteins, which underlie airway wall narrowing and bronchial hyper responsiveness (bhr). several inflammatory mediators released from inflammatory cells such as histamine and cysteinyl-leukotrienes induce bronchoconstriction, mucus production, plasma exudation, and bhr. increased expression of t-helper 2 (th2)-derived cytokines such as interleukin-4 and 5 (il-4,5) have been observed in the airway mucosa, and these may cause ige production and terminal differentiation of eosinophils. chemoattractant cytokines (chemokines) such as eotaxin may be responsible for the chemoattraction of eosinophils to the airways.6-9 asthma can be divided into two principal types named intrinsic and extrinsic asthma. intrinsic asthma triggered by boggy membranes, congested tissues, and other native causes such as adrenalin stress or exertion and generally develops later in life and virtually nothing is known of its causes. intrinsic bronchial hyperactivity can be triggered by infection, drugs such as aspirin. in contrast, extrinsic asthma triggered by external agents and allergens. most cases of extrinsic asthma have an allergic origin and are caused by an ige-mediated response to an inhaled allergen.6, 7 this is the type of asthma commonly diagnosed in early life. it carries a better prognosis than extrinsic asthma. many patients with extrinsic asthma may respond to immunotherapy such as using of recombinant allergens. these exciting novel therapies provide not only promise of new therapies for asthma but also valuable tools for investigation of asthma mechanisms.6, 10 this review provides an overview of using of recombinant allergens as new therapeutic options for asthma. allergen allergens are antigens capable of stimulating type-i hypersensitivity reaction in atopic individuals through immunoglobulin e (ige) responses.11, 12 in the other words, allergens are mostly innocuous antigens that elicit powerful t helper cell type 2 (th2) responses leading to hyper-ige production and allergy.9 ige antibodies, bound to basophils in circulation and mast cells in tissue, cause these cells to release chemicals when they come into contact with an allergen. these chemicals can cause injury to surrounding tissue – the visible signs of an allergy. the (detrimental) reaction may result after exposure via ingestion, inhalation, injection, or contact with skin. a greater understanding of the molecular features that make proteins allergenic will help define new therapeutic targets aimed at blocking allergen recognition and protease activity.9 a classic food allergy is an ige-mediated reaction manifesting as any combination of respiratory, cutaneous, gastrointestinal, recombinant allergens in immunotherapy of asthma taiebeh mohammadi farsani1,2, reza yaghubi-tabar1, gholamreza mohammadi farsani2,3 1department of medical biotechnology, isfahan (khorasgan) branch, islamic azad university, isfahan, iran. 2minimally invasive surgery research center, iran university of medical sciences, tehran, iran. 3department of clinical nutrition, school of nutritional sciences and dietetics, tehran university of medical sciences, tehran, iran. *correspondence to : gholamreza mohammadi frasani, (mohammadigh53@gmail.com) (submitted: 18 march 2020 – revised version received: 06 april 2020 – accepted: 04 may 2020 – published online: 26 june 2020) asthma is a chronic inflammatory disorder caused by t-cell-mediated inflammation within airways. the prevalence of allergic diseases is rapidly increasing so that knowing allergens (characterization and types) and strategies for asthma management, prevention, and treatment are very important. the most important strategy is production of recombinant allergens. many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome with use of genetically engineered recombinant allergens. various recombinant allergens are now emerging as strong candidates for allergen-specific immunotherapy. extrinsic asthma (a type of asthma) may respond to immunotherapy such as using of recombinant allergens. these exciting novel therapies provide not only promise of new therapies for asthma but also valuable tools for investigation of asthma mechanisms. this review describes strategies for asthma management, prevention, and treatment and especially recombinant allergens and also recent progresses in the molecular biology of recombinant allergens and then advantage and disadvantage of these allergens are explained. there are many methods for producing allergens such as extraction of serum, ro/ss-a anti-ro/ss-a system, using solid-phase immunoadsorption system and finally recombinant technology for producing recombinant allergens. recombinant allergens can be expressed in many systems such as bacteria, yeast, insect cells, animal cells, and transgenic plants. we describe recombinant allergens produced in these systems. the obtained results hold promise that recombinant allergen-based immunotherapy will improve current immunotherapy practice and may open possibilities for new treatment strategies and possibly even for prophylactic vaccination. 89 review recombinant allergens in immunotherapy of asthmataiebeh mohammadi farsani et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 88–94 cardiovascular, and pulmonary symptoms. the most commonly implicated foods include cow’s milk, egg, peanut, tree nut, soy, wheat, shellfish, and fish.13 progress in molecular biology and analytical chemistry in the past two decades have facilitated the identification of food allergens and their sequential ige-binding epitopes. the production of recombinant allergens and the use of three dimensional structural analysis have also contributed to this progress. although there are a number of limitations associated with studies of food allergen components and epitopes, the detection and quantification of serum ige antibodies specific to allergen components and epitope peptides are useful for the diagnosis and prognosis of food allergy. in addition, clarification of the sensitization patterns of allergen components in individual patients might facilitate allergen-specific immunotherapy of food allergy. however, numerous issues remain to be investigated. for example, conformational ige epitopes and t-cell epitopes have not yet been identified for most food allergens, even those for which the three dimensional structure has been determined. moreover, many allergens remain unidentified because of biodiversity, especially in seafood, and the disposition and digestion of food allergens after ingestion have not been clarified.14 classification and characterization of allergens understanding of classification of allergens is important because allergic patients are sensitized by a variety of allergens. some of them are common such as food allergens (the most common are milk , fruit, fish, eggs, and nuts), pollen, mold, house dust (contains mites as well as dander from house pets), venom from insects (such as bees, wasps, and mosquitoes), or scorpions, plant oil (especially poison ivy, oak, or sumac).15 there are many types of allergens such as oligosaccharides and proteins allergens. protein allergens are very important because they are most abundant allergen in our body and environment.11, 15 characterization of allergens allergens are derived from proteins with a variety of biologic functions, including proteases, ligand-binding proteins, structural proteins, pathogenesis-related proteins, lipid transfer proteins, profilins, and calcium-binding proteins. biological function, such as the proteolytic enzyme allergens of dust mites, might directly influence the development of ige responses and might initiate inflammatory responses in the lung that are associated with asthma. intrinsic structural or biological properties might also influence the extent to which allergens persist in indoor and outdoor environments or retain their allergenicity in the digestive tract. analyses of the protein family database suggest that the universe of allergens comprises more than 120 distinct protein families. structural biology and proteomics define recombinant allergen targets for diagnostic and therapeutic purposes and identify motifs, patterns, and structures of immunologic significance.15 peanut allergy is of particular worry given that it leads to fatal reactions more commonly than other foods. in study evaluating fatal food reactions, 62.5% of fatalities were thought to be by reason of peanut. peanut allergy can develop in adulthood or childhood and is not as likely to be outgrown. a recent study found that a lower peanut-specific ige level and a smaller wheal size on peanut spt anticipated a higher rate of resolution of peanut allergy.13 fish allergy can develop in adulthood. clinical cross reactivity between fish is 50%; consequently, all fish should be abstained unless an oral challenge confirms tolerance to specific fish. most patients with fish allergy are able to tolerate shellfish and vice versa.13 nonceliac gluten sensitivity is a clinical entity with gi and extraintestinal features that requires exclusion of cd and wa for proper interpretation because of the overlapping particularity of these disorders .16 food allergenic chitinases are a relatively small group of proteins, but their relevance as allergens cannot be underestimated given their existence in highly consumed fruits and plant derivatives. it is therefore necessary to have a clear representation of their diffusion, allergenic potency, and structural characteristics. we have reported an updated collection of all the allergenic chitinases identified in food, including information on their molecular analysis. the confirmation that the allergenicity of chitinases is not limited to the hevein-like chitin-binding module and the identification of the first allergenic chitinase outside of the plant kingdom. these findings indicate that further efforts are still needed to achieve a robust characterization of allergenic chitinases (and also of all the other allergens).17 classification of allergens structural classification of allergens a structural classification of allergens whose three dimensional structures have been experimentally determined or inferred from sequence similarity showed a restricted distribution similar to the distribution of allergens into sequence-based pfam families (a large collection of protein families). allergens were found in all structural classes, as defined by scop (structure classification of protein).11,15,18 structural classes of all protein families that contain allergens are classified in all α proteins (392 superfamilies), all β proteins (300 superfamilies), α and β proteins (a/b) (221 superfamilies), α and β proteins (a+b) (424 superfamilies), multidomain proteins (48 superfamilies), membrane and cell-surface proteins (90 superfamilies), small proteins (114 superfamilies), and coiled coil proteins(50 superfamilies).18 functional classification of allergens one-sixth of all allergens in allfam (119 allergens) were inferred to possess hydrolase activity. half of them (58 allergens) were proteases, such as trypsin-like and subtilisin-like serine proteases (14 and 13 allergens, respectively), and papain-like cysteine proteases (10 allergens). other hydrolytic enzymes included polygalacturonases (8 allergens), lipases (8 allergens), and ribosome-inactivating proteins (8 allergens).18 many allergens bound metal ions. these included calcium-binding allergens from the ef-hand family (32 allergens), serum albumins (12 allergens), globins (9 allergens), enolases (9 allergens), and fe/mn superoxide dismutases (7 allergens). allergens with lipid-binding activity comprised nonspecific lipid transfer proteins (nsltps) from the prolamin superfamily (28 allergens), serum albumins (12 allergens), and lipocalins (9 allergens). although not annotated in the gene ontology (go) database, lipid-binding activity was shown for 90 review recombinant allergens in immunotherapy of asthma taiebeh mohammadi farsani et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 88–94 allergens from several other families, such as bet v 1-related allergens that bind plant steroids.18 the nonmetabolic biologic process associated with the greatest number of allergens was transport. this group of allergens comprised lipid-binding proteins, such as the nsltps (28 allergens) and lipocalins (21 allergens), as well as general carrier proteins, such as serum albumins (12 allergens) and caseins (12 allergens). many allergens from the cupin and prolamin superfamilies (26 and 22 allergens, respectively) were annotated as nutrient reservoirs.18 of the 203 allergens without go annotations, 112 were not assigned to a protein family, in most cases because their sequences were too short. the remaining 91 sequences were grouped into 17 allfam families, with tropomyosins (34 allergens), group 2 mite allergens (10 allergens), thaumatin-like proteins (9 allergens), ole e 1–related proteins (9 allergens), and pectate lyases (9 allergens) as the prevailing families.18 there are several epitopes on the surface of protein antigens that are flexible and bind with antibodies.19 allergen proteins are a category of antigens that interact with ige. it is thought that fragments unique to allergens, and that are not found in nonallergen proteins are located at the surface of these proteins.20 fragments unique to allergens that have potential flexibility for binding with ige are thus thought to exist.11 allergens and air pollutants cause mitochondrial dysfunction and mtros-mediated systemic inflammation, which is crucial in the pathogenesis of allergy, asthma, and mets, individually. research is increasingly providing evidence that inflammation during any one of these conditions can drive the pathogenesis of another, establishing a bidirectional causeand-effect relationship. with increased persistence of air pollutants and allergies, a common effective therapy for the comorbidities is needed. while mitochondrial antioxidant coenzymes q10 and mito q are an effort in that direction and have entered clinical trials, mitochondrial transfer therapy is potentially promising in skewing the cellular heteroplasmy in favor of healthy mitochondrial numbers and has met with success in many experimental animal models. mitochondrial targeted therapy, therefore, merits greater research focus in mtros-linked disorders.21 lipocalins may, independent of their mammalian other animal, or plant origin, house siderophore ligands, which critically determine their innate immunomodulatory as well as allergenic characteristics. it has been shown that in loaded state human lipocalins induce regulatory t cells, whereas empty lipocalins rather promote th2 responses and inflammation. to this end, the interplay between exogenous lipocalins and human lcns is not resolved.22 strategies for asthma management, prevention, and treatment during the past two decades, many scientific advances have improved our understanding of asthma and our ability to manage and control it effectively. here, we will describe several strategies for asthma management and prevention that were explained in the following. lifestyle modification avoidance of triggers is a key component of improving control and preventing attacks. the most common triggers include allergens, smoke (tobacco and other), air pollution, non-selective beta-blockers, and sulfite-containing foods.1 cigarette smoking and second-hand smoke (passive smoke) may reduce the effectiveness of medications such as corticosteroids.23 dust mite control measures, including air filtration, chemicals to kill mites, vacuuming, mattress covers, and other methods had no effect on asthma symptoms.24 using of drugs these drugs are included corticosteroids,24 beta-adrenergic receptor agonists,25-27 anticholinergic drugs,28 theophylline,29-34 leukotriene modifiers,35 cromolyn,35 and nedocromil.36, 37 allergen-based immunotherapy immunotherapy is a therapy designed to induce changes in a patient’s immune status in order to treat disease.38 immunotherapy for allergic diseases represents an important but largely unmet medical need. conventional immunotherapy suffers from several breakdowns related to the quality of the extracts used, the risk of inducing anaphylactic reactions, and the extremely long treatment time but recently in immunotherapy for allergic diseases, using of immunologic agents to therapically enhance or suppress the immune system are common. agents used in immunotherapy include monoclonal antibodies, vaccines, ige blocker, and recombinant allergens. these agents may also have a direct antitumor effect.39, 40 new therapeutic strategies based on recombinant technology include peptide-based vaccines, engineered hypoallergens with reduced ige-binding properties, nucleotide-conjugated vaccines that promote th1 responses, and the possibility of developing prophylactic allergen vaccines.41 many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome using genetically engineered recombinant allergens.42 methods for supply allergens there are many methods for gaining allergens contained such as the ro/ss-a anti-ro/ss-a system,43-46 using solid-phase immunoadsorption system,47 extraction of serum,48 recombinant technology.12, 42, 49-51 ro/ss-a anti-ro/ss-a system this system has achieved increasing diagnostic and pathogenetic relevance and several groups have attempted to isolate and characterize this cellular antigen. the methods commonly employed in the isolation of ro/ss-a antigen rely on the use of immune affinity columns using naturally occurring specific autoantibodies.43, 44 however, this approach is limited because of low yield and poor standardization. moreover, a partial denaturation of the antigen can occur during the elution procedures. in the present work, we describe a simple method for the biochemical purification of ro/ss-a antigen from human spleen by fast protein liquid chromatography (fplc).45 dong-hai et al (1998) isolated ro/ss-a antigen from human spleen by a two-step procedure. in the first step, most of the nonantigenic material was removed by means of ammonium sulphate precipitation and ion exchange chromatography. the final purification was obtained by passing the ro/ss-acontaming fractions twice through a mono q ion exchange fplc column. the purified antigen showed identical 91 review recombinant allergens in immunotherapy of asthmataiebeh mohammadi farsani et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 88–94 immunoreactivity with crude material on cie and was composed of two polypeptides with a molecular weight of approximately 60,000 and 55,000, respectively, on sds-page, both reacting on western blotting with a panel of anti-ro/ss-a antisera. this system permits milligrams of highly purified antigen to be obtained from grams of human spleen.46 using solid-phase immunoadsorption system koon yan pak et al (1983) extracted circulating gastrointestinal cancer antigen by using of solid-phase immunoadsorption system of monoclonal antibody-coupled membrane. this system is an immunoadsorption system of monoclonal antibody immobilized on a polyolefin alloy fiber. this methodology provides a selective and convenient means of removing any targeted substance by monoclonal antibody from the serum, and thus overcomes many of the shortcomings associated with conventional plasmapheresis.47 extraction from serum terry et al extracted the carbohydrate-defined class of ia antigens from murine spleen cells and serum. the final extract contains mostly glycolipid, with small amounts of contaminating phospholipids and little or no protein.48 recombinant technology for producing recombinant allergens recombinant technologies, as a part of biotechnology, developed in the 1980s for cloning cdna from low-abundance mrna permitted the cloning of allergens.49 recombinant allergens will enable innovative new strategies for allergen immunotherapy to be developed. these include peptide-based vaccines, engineered hypoallergens with reduced reactivity for ige antibodies, nucleotide-conjugated vaccines that promote th1 responses, and the possibility of developing prophylactic allergen vaccines.52 a great variety of recombinant plant, mite, mold, mammal, and insect allergens have been expressed in heterologous hosts (e.g., escherichia coli), their cdna being used as a template.15, 49 many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome using genetically engineered recombinant allergens.52 recombinant allergens have been used for successful in vitro, as well as in vivo, allergy diagnosis, and work is in progress to produce recombinant allergen derivatives with reduced anaphylactic potential to improve current forms of immunotherapy. recombinant allergens have proven to be valuable tools to investigate t-cell and b-cell recognition of allergens as well as to study mechanisms of specific ige regulation. the immunologic equivalence of many relevant recombinant allergens with their natural counterparts has been demonstrated. the number of biologically active recombinant allergens available for experimental, diagnostic, and therapeutic purposes is increasing tremendously.42 recombinant allergens offer the perspective of molecule-based allergy diagnosis and consequently safe and patient-tailored immunotherapy.12 allergens have diverse biological functions (they may be enzymes, enzyme inhibitors, lipocalins, or structural proteins). recombinant allergens show comparable ige antibody binding to natural allergens and show excellent reactivity on skin testing and in in vitro diagnostic tests.52 advantages and disadvantages of recombinant allergens recombinant allergens have many advantages that make them as drugs for treatment of asthma.12, 42 by using recombinant dna technology for producing recombinant allergens, we use molecules with defined amino acid sequence and other advantages that are preparations of consistent pharmaceutical quality, all batches of one allergen derive from the same master cell bank, avoidance of possible contamination and the risk of infectious agents, dosage in mass units in respect of all components: absolute standardization, inclusion of only the relevant proteins, optimization of the dosage of all components of a preparation, possibility to tailor preparations to a patient’s sensitization profile, precise monitoring and investigation of mechanisms underlying treatment, option to create genetically engineered variants (e.g., with reduced ige reactivity).12,42,51 although recombinant allergens have some disadvantages such as each allergen has to be developed by using a specific approach, for those allergens occurring in many isoforms, there is a need to choose the most relevant, it might be necessary to include >1 isoallergen in cases of limited identity, there are high development costs in relation to limited market potential.51 expression systems of recombinant allergens high-level expression systems have been developed to produce recombinant allergens in bacteria,53-62 yeast,63-66 insect cells,67-71 transgenic plant,72-75 animal cells, and transgenic animal.76-79 these are argued in the following sections. bacteria bacterial system has the advantage that it is easy to handle and often results in high expression levels of the recombinant protein. however proteins expressed in e. coli often accumulate in insoluble inclusion bodies, and therefore require chemical refolding procedures to obtain the protein in a native, biological active form.53 furthermore, bacterial expression systems lack the ability to perform protein glycosylation, and therefore allergens that normally occur as glycoproteins are expressed devoid of carbohydrate moieties. in the case of the glycoprotein phl p 1, the group 1 allergen of the grass p pratense, expression without the carbohydrate component appears not to have any appreciable effect on ige antibody binding or t-cell reactivity54, 55 or on the ability to induce allergen-specific igg1 and igg4 responses.56 valenta et al (2010) described that expression of rbet v 1 and rbet v 2 similar to the natural allergen was also readily accomplished in e coli.19.57 in the other study, synthetic genes coding for 2 hybrid proteins consisting of reassembled der p 1 and der p 2 fragments with (recombinant der p 2 [rder p 2]/1c) and without (rder p 2/1s) cysteines were expressed in e. coli and purified to homogeneity by means of affinity chromatography.58 recombinant fel d 1 chains expressed individually in e. coli do not bind ige as well as natural allergen but can be combined and refolded to produce immunoreactive recombinant fel d 1.59-61 hyaluronidase (hya) is one of several allergens in honeybee venom, has been cloned62 and expressed as a recombinant protein in e. coli.53 92 review recombinant allergens in immunotherapy of asthma taiebeh mohammadi farsani et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 88–94 yeast chua et al.63 in 1992 produced a high level of der p i from a cup1 gene cassette from pyelc5-13t in saccharomyces cerevisiae.63 allergens that did not initially fold correctly in e. coli were eventually produced in other hosts. the hornet allergen dol m 5 is produced as a well-folded allergen when expressed in the yeast pichia pastoris.64 cenk suphioglu et al in australia expressed cyn d 1 (from bermuda grass) in the yeast p. pastoris.65 patricia barral reported the expression of the olive pollen allergen ole e 6 in p. pastoris as a soluble and stable protein. purification to homogeneity, molecular, spectroscopic, and immunological characterization is also described.66 insect cells baculovirus vectors allow the expression of large foreign gene products from single proteins under 20 kda to enzymes and multimeric protein assemblies over 1 million daltons,67, 68 furthermore, this system is adapted for simultaneous expression of several foreign gene, using a single recombinant baculovirus.69 dolm5 from the white-faced hornet (dolichovespula maculate) be cloned with recombinant dna technology.70 one of several allergens in honeybee venom named hya expressed as a recombinant protein in baculovirus-infected insect cells.53 acid phosphatase (api m 3) is a major allergen in honeybee (apis mellifera) venom. recombinant api m 3, expressed in trichoplusia ni cells. the availability of recombinant api m 3 provides a tool for both the development of improved diagnostic tests and the design of safer and more effective immunotherapeutic approaches for honeybee venom allergy.71 transgenic plant smart et al72 produced a genetically modified (gm) plant, narrow leaf lupin (lupinus angustifolius l.), expressing a gene for a potential allergen (sunflower seed albumin) (ssa-lupin) and demonstrate that a gm plant-based vaccine can promote a protective immune response and attenuate experimental asthma, suggesting that plant-based vaccines may be potentially therapeutic for the protection against allergic diseases.72 bet v 1 from birch pollen (betula verrucosa) is one of the first allergens that ca be cloned with recombinant dna technology.69, 70 scientist can produce a transgenic rice plants expressing mouse dominant t cell epitope, peptides of cry j i and cry j ii allergens of japanese cedar pollen as a fusion protein with the soybean seed storage protein glycinin. under the control of the rice seed storage protein glutelin glub-1 promoter, the fusion protein was specifically expressed and accumulated in seeds at a level of 0.5% of the total seed protein.75 animal cells der p 1, a major allergen from dermatophagoides pteronyssinus, which plays a prominent role in ige-mediated immediate hypersensitivity reactions including asthma be cloned with recombinant dna technology76 and be expressed as a recombinant precursor form of der p 1, recproder p 1,77 secreted by anchorage-dependent cho cells cultured in cell factories. to increase the proder p 1 expression level and purification yield, a recombinant cho-k1 clone was adapted78 to growth in serumfree suspension culture. in a study, high-level expression of recproder p 1 were obtained in cultures of the adapted chok1 cell line (4846-6) in a controlled stirred tank bioreactor.79 conclusion and perspective of recombinant allergens the prevalence of asthma is rapidly increasing so that using of allergen–based immunotherapy for the treatment and eventually prevention of ige-mediated allergy are developing. the broad applicability of allergen-specific immunotherapy is limited by the poor quality and allergenic activity of natural allergen extracts that are used for the production of current allergy vaccines. many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome with the use of genetically engineered recombinant allergens. recombinant allergens are effectively proteins that can be produced at will, under defined conditions, and purified with use of single-step procedures such as affinity chromatography. the expression of a recombinant allergen molecule could be a suitable strategy for the development of in vitro diagnosis test as well as for specific immunotherapy. this has tremendous advantages in terms of quality control and standardization. although there are some problems, such as low expression and production of misfolded proteins, in process of using of recombinant technology. problems in recombinant allergen expression in a particular vector can be overcome by choosing a different expression system or by engineering the allergen sequences that enable the protein to assume the correct tertiary structure. recombinant technology holds the promises that recombinant allergen-based immunotherapy will improve current immunotherapy practice and may open possibilities for new treatment strategies and possibly even for prophylactic vaccination. conflicts of interest disclosure authors declare no conflict of interest. references 1. national heart l, institute b. national asthma education and prevention program. expert panel report 3: guidelines for the diagnosis and management of asthma: full report 2007. http://www nhlbi nih gov/ guidelines/asthma/asthgdln pdf. 2007. 2. hill at, sullivan al, chalmers jd, de soyza a, elborn js, floto ra, et al. british thoracic society guideline for bronchiectasis in adults. thorax. 2019;74(suppl 1):1–69. 3. polk bi, dinakar c. management of acute loss of asthma control: yellow zone strategies. curr. opin aller clin immunol. 2019;19(2):154–60. 4. network ga. the global asthma report a, new zealand. 2018. 5. dharmage sc, perret j, custovic a. epidemiology of asthma in children and adults. front pediat. 2019;7:246. 6. matricardi pm, dramburg s, potapova e, skevaki c, renz h. molecular diagnosis for allergen immunotherapy. j allerg clin immunol. 2019;143(3):831–43. 7. westman m, asarnoj a, hamsten c, wickman m, van hage m, editors. windows of opportunity for tolerance induction for allergy by studying the evolution of allergic sensitization in birth cohorts. semin immunol; 2017: elsevier. 8. bush a. pathophysiological mechanisms of asthma. front pediat. 2019;7. 9. shakib f, ghaemmaghami am, sewell hf. the molecular basis of allergenicity. trends immunol. 2008;29(12):633–42. 10. frew aj. immunotherapy of allergic disease. clin immunolo: elsevier; 2019. p. 1227-35. e1. 11. asakawa n, sakiyama n, teshima r, mitaku s. characteristic amino acid distribution around segments unique to allergens. j biochem. 2009;147(1):127–33. 12. egger m, hauser m, himly m, wopfner n, wallner m, ferreira f. development of recombinant allergens for diagnosis and therapy. front biosci (elite edition). 2009;1:77–90. 93 review recombinant allergens in immunotherapy of asthmataiebeh mohammadi farsani et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 88–94 13. patel by, volcheck gw, editors. food allergy: common causes, diagnosis, and treatment. mayo clin proc; 2015: elsevier. 14. matsuo h, yokooji t, taogoshi t. common food allergens and their igebinding epitopes. allergol int. 2015;64(4):332–43. 15. chapman m, smith a, vailes l, arruda l. recombinant mite allergens: new technologies for the management of patients with asthma. allergy. 1997;52(4):374–9. 16. fasano a, sapone a, zevallos v, schuppan d. nonceliac gluten sensitivity. gastroenterology. 2015;148(6):1195–204. 17. leoni c, volpicella m, dileo mc, gattulli ba, ceci lr. chitinases as food allergens. molecules. 2019;24(11):2087. 18. radauer c, bublin m, wagner s, mari a, breiteneder h. allergens are distributed into few protein families and possess a restricted number of biochemical functions. j allerg clin immunol. 2008;121(4):847-52. e7. 19. demchenko ap. recognition between flexible protein molecules: induced and assisted folding. j mol recog. 2001;14(1):42–61. 20. zorzet a, gustafsson m, hammerling u. prediction of food protein allergenicity: a bio-informatic learning systems approach. in silico biol. 2002;2(4):525–34. 21. iyer d, mishra n, agrawal a. mitochondrial function in allergic disease. curr allerg asthma rep. 2017;17(5):29. 22. jensen‐jarolim e, pacios l, bianchini r, hofstetter g, roth‐walter f. structural similarities of human and mammalian lipocalins, and their function in innate immunity and allergy. allergy. 2016;71(3):286–94. 23. stapleton m, howard-thompson a, george c, hoover rm, self th. smoking and asthma. j am board family med. 2011;24(3):313–22. 24. martinez f. genes, environments, development and asthma: a reappraisal. eur resp j. 2007;29(1):179–84. 25. palmqvist m, persson g, lazer l, rosenborg j, larsson p, lotvall j. inhaled dry-powder formoterol and salmeterol in asthmatic patients: onset of action, duration of effect and potency. eur resp j. 1997;10(11):2484–9. 26. usmani os, ito k, maneechotesuwan k, ito m, johnson m, barnes pj, et al. glucocorticoid receptor nuclear translocation in airway cells after inhaled combination therapy. am j resp critic care med. 2005;172(6):704–12. 27. andrew mcivor r, pizzichini e, turner mo, hussack p, hargreave fe, sears mr. potential masking effects of salmeterol on airway inflammation in asthma. am j resp critical care med. 1998;158(3):924–30. 28. barash pg. clinical anesthesia: lippincott williams & wilkins; 2009. 29. essayan dm. cyclic nucleotide phosphodiesterases. j aller clin immunol. 2001;108(5):671–80. 30. deree j, martins jo, melbostad h, loomis wh, coimbra r. insights into the regulation of tnf-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition. clinics. 2008;63(3): 321–8. 31. marques lj, zheng l, poulakis n, guzman j, costabel u. pentoxifylline inhibits tnf-α production from human alveolar macrophages. am j resp critical care. 1999;159(2):508–11. 32. peters-golden m, canetti c, mancuso p, coffey mj. leukotrienes: underappreciated mediators of innate immune responses. j immunol. 2005;174(2):589–94. 33. daly j, jacobson k, ukena d. adenosine receptors: development of selective agonists and antagonists. prog clin biol res. 1987;230:41–63. 34. yano y, yoshida m, hoshino s, inoue k, kida h, yanagita m, et al. anti-fibrotic effects of theophylline on lung fibroblasts. biochem biophys res commun. 2006;341(3):684–90. 35. fanta ch. drug therapy: asthma. n engl j med. 2009;360(10):1002–14. 36. mellis c. benefits of budesonide or nedocromil for mild to moderate asthma in children were not sustained after discontinuation. evidence-based med. 2009;14(6):175–. 37. yang y, lu jy-l, wu x, summer s, whoriskey j, saris c, et al. g-proteincoupled receptor 35 is a target of the asthma drugs cromolyn disodium and nedocromil sodium. pharmacology. 2010;86(1):1–5. 38. fitzhugh dj, lockey rf. history of immunotherapy: the first 100 years. immunol aller clin. 2011;31(2):149–57. 39. moingeon p, batard t, fadel r, frati f, sieber j, van overtvelt l. immune mechanisms of allergen specific sublingual immunotherapy. allergy. 2006;61(2):151–65. 40. cox l, li jt, nelson h, lockey r. allergen immunotherapy: a practice parameter second update. j aller clin immunol. 2007;120(3):s25–s85. 41. crameri r. allergy diagnosis, allergen repertoires, and their implications for allergen-specific immunotherapy. immunol aller clin. 2006;26(2):179–89. 42. zhernov y, curin m, khaitov m, karaulov a, valenta r. recombinant allergens for immunotherapy: state of the art. curr opin aller clin immunol. 2019;19(4):402–14. 43. venables p, smith p, maini r. purification and characterization of the sjögren’s syndrome a and b antigens. clin exp immunol. 1983;54(3): 731. 44. yamagata h, harley jb, reichlin m. molecular properties of the ro/ssa antigen and enzyme-linked immunosorbent assay for quantitation of antibody. j clin investig. 1984;74(2):625–33. 45. bilder r. fast protein liquid chromatography: a new method for analysing large biological molecules. int labmate. 1983;8(5). 46. wu d-h, tavoni a, garzelli c, neri r, vitali c, bombardieri s. a simple method for the biochemical purification of ro/ss-a antigen. j immunol methods. 1989;121(2):219–24. 47. pak ky, randerson dh, blaszczyk m, sears hf, steplewski z, koprowski h. extraction of circulating gastrointestinal cancer antigen using solid-phase immunoadsorption system of monoclonal antibody-coupled membrane. j immunol methods. 1984;66(1):51–8. 48. higgins tj, parish cr. extraction of the carbohydrate-defined class of ia antigens from murine spleen cells and serum. mol immunol. 1980;17(8):1065–73. 49. thomas wr. the advent of recombinant allergens and allergen cloning. j aller clin immunol. 2011;127(4):855–9. 50. valenta r, linhart b, swoboda i, niederberger v. recombinant allergens for allergen‐specific immunotherapy: 10 years anniversary of immunotherapy with recombinant allergens. allergy. 2011;66(6):775–83. 51. cromwell o, häfner d, nandy a. recombinant allergens for specific immunotherapy. j allerg clin immunol. 2011;127(4):865–72. 52. chapman md, smith am, vailes ld, pomés a, editors. recombinant allergens for immunotherapy. allergy and asthma proceedings; 2002: oceanside publications. 53. dudler t, chen w-q, wang s, schneider t, annand rr, dempcy ro, et al. high-level expression in escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase a2. biochim biophys acta lipids lipid metab. 1992;1165(2):201–10. 54. suck r, kamionka t, schäffer b, wahl r, nandy a, weber b, et al. bacterially expressed and optimized recombinant phl p 1 is immunobiochemically equivalent to natural phl p 1. biochim biophys acta proteins proteom. 2006;1764(11):1701–9. 55. cromwell o, fiebig h, suck r, kahlert h, nandy a, kettner j, et al. strategies for recombinant allergen vaccines and fruitful results from first clinical studies. immunol allerg clin. 2006;26(2):261–81. 56. jutel m, jaeger l, suck r, meyer h, fiebig h, cromwell o. allergen-specific immunotherapy with recombinant grass pollen allergens. j allerg clin immunol. 2005;116(3):608–13. 57. valenta r, ferreira f, focke-tejkl m, linhart b, niederberger v, swoboda i, et al. from allergen genes to allergy vaccines. annu rev immunol. 2009;28:211–41. 58. chen k-w, blatt k, thomas wr, swoboda i, valent p, valenta r, et al. hypoallergenic der p 1/der p 2 combination vaccines for immunotherapy of house dust mite allergy. j allerg clin immunol. 2012;130(2):435–43. e4. 59. slunt jb, rogers bl, chapman md. ige antibodies to recombinant forms of fel d i: dichotomy between fluid-phase and solid-phase binding studies. j allerg clin immunol. 1995;95(6):1221–8. 60. keating km, segal db, craig sj, nault ak, semensi v, wasserman as, et al. enhanced immunoreactivity and preferential heterodimer formation of reassociated fel d i recombinant chains. mol immunol. 1995;32(4):287–93. 61. van ree r, van leeuwen wa, bulder i, bond j, aalberse rc. purified natural and recombinant fel d 1 and cat albumin in in vitro diagnostics for cat allergy. j allerg clin immunol. 1999;104(6):1223–30. 62. kuchler k, gmachl m, sippl mj, kreil g. analysis of the cdna for phospholipase a2 from honeybee venom glands: the deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes. eur j biochem. 1989;184(1):249–54. 63. chua k-y, kehal pk, thomas wr, vaughan pr, macreadie ig. high-frequency binding of ige to the der p allergen expressed in yeast. j allerg clin immunol. 1992;89(1):95–102. 64. monsalve ri, lu g. expressions of recombinant venom allergen, antigen 5 of yellowjacket (vespula vulgaris) and paper wasp (polistes annularis), in bacteria or yeast. protein expr purific. 1999;16(3):410–6. 65. suphioglu c, smith pm, ong ek, knox rb, singh mb. recombinant expression and epitope mapping of grass pollen allergens. new horiz allerg immunother: springer; 1996. p. 147–55. 66. barral p, tejera mal, treviño má, batanero e, villalba m, bruix m, et al. recombinant expression of ole e 6, a cys-enriched pollen allergen, in pichia pastoris yeast: detection of partial oxidation of methionine by nmr. protein expr purific. 2004;37(2):336–43. 94 review recombinant allergens in immunotherapy of asthma taiebeh mohammadi farsani et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 88–94 67. maiorella b, inlow d, shauger a, harano d. large-scale insect cell-culture for recombinant protein production. bio/technology. 1988;6(12):1406. 68. trowitzsch s, bieniossek c, nie y, garzoni f, berger i. new baculovirus expression tools for recombinant protein complex production. j struct biol. 2010;172(1):45–54. 69. závodzky p, cseh s. production of multidomain complement glycoproteins in insect cells. insect cell culture: fundam appl aspects: springer; 1996. p. 279–88. 70. fang k, vitale m, fehlner p, king tp. cdna cloning and primary structure of a white-face hornet venom allergen, antigen 5. proc natl acad sci. 1988;85(3):895–9. 71. grunwald t, bockisch b, spillner e, ring j, bredehorst r, ollert mw. molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (api m 3). j allerg clin immunol. 2006;117(4):848–54. 72. smart v, foster ps, rothenberg me, higgins t, hogan s. a plant-based allergy vaccine suppresses experimental asthma via an ifn-γ and cd4+ cd45rblow t cell-dependent mechanism. j immunol. 2003;171(4):2116–26. 73. breiteneder h, hassfeld w, pettenburger k, jarolim e, breitenbach m, rumpold h, et al. isolation and characterization of messenger rna from male inflorescences and pollen of the white birch (betula verrucosa). int arch allerg immunol. 1988;87(1):19–24. 74. breiteneder h, pettenburger k, bito a, valenta r, kraft d, rumpold h, et al. the gene coding for the major birch pollen allergen betv1, is highly homologous to a pea disease resistance response gene. embo j. 1989;8(7):1935–8. 75. takagi h, hiroi t, yang l, tada y, yuki y, takamura k, et al. a rice-based edible vaccine expressing multiple t cell epitopes induces oral tolerance for inhibition of th2-mediated ige responses. proc natl acad sci. 2005;102(48):17525–30. 76. chua k, stewart g, thomas w, simpson r, dilworth r, plozza t, et al. sequence analysis of cdna coding for a major house dust mite allergen, der p 1. homology with cysteine proteases. j exp med. 1988;167(1):175–82. 77. massaer m, mazzu p, haumont m, magi m, daminet v, bollen a, et al. high-level expression in mammalian cells of recombinant house dust mite allergen proder p 1 with optimized codon usage. int arch allerg immunol. 2001;125(1):32–43. 78. scharfenberg k, wagner r. a reliable strategy for the achievement of cell lines growing in protein-free medium. animal cell technology: developments towards the 21st century: springer; 1995. p. 619–23. 79. coulon l, touzani obh, magi m, bollen a, hanus r, jacquet a. production of recombinant allergen proder p 1 by cho cells adapted to grow in serumfree suspension. animal cell technology: from target to market: springer; 2001. p. 110–3. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i3.702 20 j contemp med sci | vol. 1, no. 4, autumn 2015: 20–22 research objective psoriasis is a disease characterised by t-cell-mediated hyperproliferation of keratinocytes initiated by antigen-presenting cells on the skin. environmental factors including streptococcus infections and multiple genetic components may be responsible for the pathogenesis of the disease. this study was performed to assess the association between antistreptolysin-o (aso) serum level and chronic plaque psoriasis. methods serum level of aso was measured in 45 doctor-diagnosed psoriatic patients with an age range of 10–50 years attending the dermatology outpatient clinic in al-hussein medical city in kerbala province – iraq during the period from march 2014 through july 2015. psoriatic patients with chronic plaque type were selectively recruited to the study. another 20 ageand gender-matched persons were chosen as healthy control group. serum level of aso was estimated in all patients and control group using enzyme-linked immunosorbent assay (elisa). findings data revealed a significantly increased serum aso level in the chronic plaque-type psoriatic patients compared to the healthy control group (p-value < 0.05). conclusion the present study suggests that serum aso level could be associated with the immunopathogenesis and/or susceptibility to this type of psoriasis. keywords chronic plaque psoriasis, anti-streptolysin-o association between antistreptolysin-o serum level and chronic plaque psoriasis in iraqi patients abeer t al-hassnawi,a ali mansoor al-ameria & huda h al-hassnawib introduction psoriasis is a chronic skin disease characterised by hyperproliferation of keratinocytes with different clinical types. a number of environmental factors are believed to contribute in the disease causation. streptococcus pyogenes infection is thought to have a distinct relationship with the activation of psoriasis.1 this finding was preceded by norrlind in 1955, who pointed out the occurrence of a streptococcal upper respiratory infection 1–2 weeks prior to the appearance of the skin lesions in about two-thirds of patients with psoriasis.2 following this observation, the presence of streptococci in the throat swab cultures of these patients, as well as the seropositivity of antistreptococcal antibodies were shown. one of the suggested immunopathogenesis of psoriasis is the role of th17 lymphocytes and its related cytokine action, which increase tgf-β production and mucosal defence during streptococcal skin infections. streptococcal infections lead to production of interleukin 6, which along with tgf-β, results in a differentiation of th17 at that location. furthermore, the presence of il-6 and tgf-β enhances neutrophils and mononuclear cells migration to the region. with the production of myeloperoxidase and elastase by neutrophils, skin lesion of psoriasis might be mediated.3,4 the present study aims to investigate the association between serum antistreptolysin-o (aso) level in patients with chronic plaque psoriasis by using elisa technique. methods forty-five patients with chronic plaque psoriasis attending the outpatient clinic of dermatology in al-hussein medical city, kerbala province/ iraq, between march 2014 and july 2015, as well as 20 persons as healthy control group were recruited. informed consent was obtained from each participant prior to the study. patients clinically diagnosed with chronic plaque psoriasis were selected as case group. serum level of aso was estimated in all patients and controls by elisa technique using (creative diagnostics-usa) elisa kit according to the instructions of the kit company. statistical analysis statistical analysis was done by using statistical analysis software, graph pad prism version 6. student’s t-test was used to calculate the significance of difference in mean serum levels of aso between psoriatic patients and control group and f-test was used to compare variances of the two groups. in addition, r2-test was used to predict the association between aso level and occurrence of chronic plaque psoriasis. a p-value < 0.05 was considered significant.5 results data from the current study reveal a significant difference in the mean serum aso level between patients with chronic plaque psoriasis and the control group; 21.4 iu/ml, versus 10.63 iu/ml, with (p < 0.05), as shown in table 1. upon analysis of results to predict the association of elevated serum aso level with occurrence of chronic plaque psoriasis, the following data were obtained: overall, there was significant difference in aso titre status between patients and control. discussion it has been stated that latent or chronic tonsillitis and pharyngitis caused by streptococci are the main infections associated with psoriasis. moreover, some streptococcal dermatitis and vulvovaginitis/balanoposthitis infections mostly seen adepartment of microbiology, college of medicine, university of kerbala, holy kerbala, iraq. bdepartment of microbiology, college of medicine, university of babylon, babylon, iraq. correspondence to ali mansoor al-ameri (email: alimansoor699@gmail.com). (submitted: 18 august 2015 – revised version received: 4 september 2015 – accepted: 6 september 2015 – published online: autumn 2015) issn 2413-0516 21j contemp med sci | vol. 1, no. 4, autumn 2015: 4–8 research antistreptolysin-o serum level and chronic plaque psoriasis in iraqi patientsali mansoor al-ameri et al. during childhood can induce psoriasis. therefore, in acute exacerbations of guttate psoriasis, bacterial culture should be obtained from the patients. determination of serum level of antibodies against streptolysin, a streptococcal exotoxin can also be performed. among these, the most commonly used is antistreptolysin.6,7 in our study, chronic plaque psoriasis patients were shown to have significantly higher aso level compared to the control group (p < 0.05). these findings are consistent with many previous studies in this field.8–12 all these studies showed significant differences (p < 0.05) with their respective controls. in a previous study, association between streptococcal infections and psoriasis was suggested in 18(56%) of the 32 patients, 31% had a history of sore throat 1 to 3 weeks before the appearance of rash and 17(85%) of the 20 patients with acute guttate psoriasis (agp).9 increased levels of antibodies to streptolysin o were also found in the serum of patients in response to infection with haemolytic streptococcus groups a, c or g. streptococcal infection usually leads to eruptive guttate psoriasis, and deterioration of other clinical forms of psoriasis.13 another study found aso titre raised in 60% of agp patients versus 6.6% in healthy control group.11 another prospective cohort study described aso serum level increased 10 times compared with their controls in chronic plaque psoriasis.12 in a retrospective cohort, about 30% of patients with chronic psoriasis stated that they had noted worsening of their disease in association with sore throat.14 an exacerbation of chronic plaque psoriasis only if streptococci were isolated 4 days or later after the onset of sore throat was observed.15 in addition, some studies proposed that the psoriasis patients with high aso titres had guttate psoriasis more frequently compared with patients with normal aso titres.10,16 histopathological studies concerning examination of tonsils performed after tonsillectomies confirmed that streptococci could persist in these tissues and could eventually colonise them.17 the microorganisms’ virulence factors like proteins m, sfd1 and f1 are important regarding their role in tissue internalisation of streptococci and in triggering psoriasis.2 furthermore, antigen-specific t-lymphocyte response against group a streptococci increases in patients with guttate and chronic plaque psoriasis and the association between acute infections and guttate psoriasis has been reported in patients with chronic plaque type psoriasis.18–20 similarly, cd4+ and cd8+ cells isolated from psoriatic lesions were shown to be responsive to streptococcal peptidoglycan by proliferating and secreting interferon-γ.21 it was reported that streptococcal isolates from throat cultures of guttate psoriasis patients secreted streptococcus pyogenes exotoxin c which, by acting as superantigen, triggers polyclonal expansion of vβ2-bearing t cells.22 the outcome of all of the abovementioned studies were similar and supportive of the findings obtained from the current study. however, our study’s result is not in accordance with some previous similar works. these studies found that there is no significant difference in serum aso levels in psoriasis patient and control group.19,23,24 conclusion this study confirms an association between the increased serum level of aso with the immunopathogenesis and/or susceptibility to chronic plaque psoriasis. recommendations further prospective cohort studies with larger sample size are needed to investigate the exact role of streptococcal infection and thus aso level on elaborations of chronic plaque psoriasis. acknowledgments we thank god for help us performing this work. great thanks to the staff members of dermatology outpatient clinic in al-hussein medical city in kerbala for their help in patients’ diagnosis and selection. we are grateful for the staff members of hematology investigation unit in al-hussein medical city for assistance in sample collection and processing. we would like to thank dr. jalal al-karbalaee for his great support and kind cooperation to mount this work. competing interests none declared. conflicts of interest there is no conflicts of interest represented by financial or personal relationships with any other people or organizations regarding this work.  table 1. serum level of aso in patients with chronic plaque psoriasis and control group subject number aso mean (iu/ml ± sem) statistical analysis* psoriatic patients 45 (21.4 ± 0.8521) controls 20 (10.63 ± 0.8023) *table analysed unpaired t-test data column b control aso vs. vs. column a test aso unpaired t-test p-value < 0.0001 p-value summary **** significantly different? (p < 0.05) yes oneor two-tailed p-value? two-tailed t, df t = 7.757 df = 63 how big is the difference? mean ± sem of column a 21.40 ± 0.8521, n = 45 mean ± sem of column b 10.63 ± 0.8023, n = 20 difference between means −10.77 ± 1.389 95% confidence interval −13.55 to −7.997 r2 0.4885 f-test to compare variances f,dfn, dfd 2.538, 44, 19 p-value 0.0306 p-value summary * significantly different? (p < 0.05) yes 22 j contemp med sci | vol. 1, no. 4, autumn 2015: 20–22 risk factors in patients with ihd research ali mansoor al-ameri et.al references 1. bos jd, de rie ma, teunissen mb, piskin g. psoriasis: dysregulation of innate immunity. br j dermatol. 2005;152(6):1098–7. doi: 10.1189/jlb.0109046 pmid: 15948970 2. karabudak aö, dogan b. management of guttate psoriasis in patients with associated streptococcal infection. dovepress j psoriasis target ther. 2012:2: 89–94. doi: http://dx.doi.org/10.2147/ptt.s25211 3. aujla sj, dubin pj, kolls jk. th17 cells and mucosal host defense. semin immunol. 2007;19(6):377–82. doi: http://dx.doi.org/10.1016/j. smim.2007.10.009 pmid: 18054248 4. korn t, oukka m, kuchroo v, bettelli e. th17 cells: effector t cells with inflammatory properties. semin immunol. 2007;19(6):362–71. doi: 10.1016/j.smim.2007.10.007 pmid: 18035554 5. walker ga, shostak j. common statistical methods for clinical research with sas examples, 3rd edn., cary, north calorina, usa: sas. institute inc; 2010: 539. 6. ledoux m, chazerain v, saiag p, mahé e. streptococcal perianal dermatitis and guttate psoriasis. ann dermatol venereol. 2009;136(1): 37–41. doi: 10.1016/j.annder.2008.06.013 pmid: 19171228 7. geerts i, de vos n, frans j, mewis a. the clinical-diagnostic role of antistreptolysin o antibodies. acta clin belg. 2011;66(6):410–5. doi: 10.2143/ acb.66.6.2062604 pmid: 22338301 8. hossain ma, ali me, khondker l, khan msi. association between streptococcal throat infection and psoriasis in bangladesh. j pak assoc dermatol. 2013;23(4):388–93. 9. mukherjee sk. streptococcal infection as trigger for psoriasis. contempor pediatr. 2011;2:207–15. 10. saxena vn, dogra j. long-term use of penicillin for the treatment of chronic plaque psoriasis. eur j dermatol. 2005;15(5):359–62. pmid: 16172045 11. naqqash s, uddee, naqqash sh, butt ak. family history of psoriasis and recent infectious disease are risk factor for the first episode of acute guttate psoriasis. j pak assoc dermatol. 2004;14:124–9. 12. gudjonsson je, thorarinsson am, sigurgeirsson b, kristinsson kg, valdimarsson h. streptococcal throat infections and exacerbation of chronic plaque psoriasis: a prospective study. br j dermatol. 2003;149:530–4. pmid: 14510985 13. baros dn, gajanin vs, gajanin rb, zrnic b. comparative analysis of success of psoriasis treatment with standard therapeutic modalities and balneotherapy. med pregl. 2014;lxvii (5–6):154–60. pmid: 25033574 14. gudjonsson je, kárason a, antonsdóttir aa, rúnarsdóttir eh, gulcher jr, stefánsson k, et al. hla-cw6positive and hla-cw6-negative patients with psoriasis vulgaris have distinct clinical features. j invest dermatol. 2002;118:362–5.no doi. pmid: 11841557 15. bucolo s, torre v, romano g, quattrocchi c, farri f, filidoro m et al. effects of tonsillectomy on psoriasis and tonsil histology ultrastructure. edited by hermenio lima, isbn 978-953-51-1065-1, 190 pages, publisher: intech, chapters published april 17, 2013 under cc by 3.0 license; 2013. doi: http:// dx.doi.org/10.5772/55978 16. kim sk, kang hy, kim yc, lee e-s. clinical comparison of psoriasis in korean adults and children: correlation with serum anti-streptolysin o titers. arch dermatol res. 2010;302:295–9. doi: 10.1007/s00403-009-1025-8 pmid: 20063005 17. park hs, francis kp, yu j, cleary pp. membranous cells in nasal-associated lymphoid tissue: a portal of entry for the respiratory mucosal pathogen group a streptococcus. j immunol. 2003;171(5):2532–7. doi: 10.4049/ jimmunol.171.5.2532 pmid: 12928403 18. raza n, usman m, hameed a. chronic plaque psoriasis: streptococcus pyogenes throat carriage rate and therapeutic response to oral antibiotics in comparison with oral methotrexate. j coll physicians surg pak. 2007;17:717–20. doi: 12.2007/jcpsp.717720 pmid: 18182134 19. rasi a, pour-heidari n. association between plaque-type psoriasis and perianal streptococcal cellulitis and review of the literature. arch iran med. 2009;12(6):591–4. pmid: 19877754 20. wu y, lin y, liu h-j, huang c-z, feng a-p, et al. childhood psoriasis: a study of 137 cases from central china. world j pediatr. 2010;6(3):260–4. doi: 10.1007/s12519-010-0213-0 pmid: 20549408 21. baker bs, laman jd, powles a, van der fits l, voerman js, melief mj, fry l. et al. peptidoglycan and peptidoglycan specific th1 cells in psoriatic skin lesions. pathol. 2006;209(2):174–81. doi: 10.1002/path.1954 pmid: 16493599 22. macias es, pereira fa, rietkerk w, safai b. superantigens in dermatology. j am acad dermatol. 2011;64(3):455–72. doi: 10.1016/j.jaad.2010.03.044 pmid: 21315950 23. cassandra m, conte e, cortez b. childhood pustular psoriasis elicited by the streptococcal antigen: a case report and review of the literature. pediatr dermatol. 2003;20(6):506–10. doi: 10.1111/j.1525-1470.2003.20611.x pmid: 14651571 24. ibrahimbaş y, polat m, serin e, parlak ah. cellular immune response in patients with chronic plaque type psoriasis: evaluation of serum neopterin, procalcitonin, anti-streptolysin o and c reactive protein levels. j clin exp dermatol res. 2010;1:107. doi: 10.4172/21559554.1000107 59j contemp med sci | vol. 2, no. 6, spring 2016: 59–62 research objectives the overall goal of this study was to figure out the effect of ovulation induction on the levels of tnf-α and il-1ß, and to investigate the possible interaction between the cytokines and hormones in respect to anovulation. methods the study comprised of 53 women; 33 sub-fertile and 20 fertile. their ages ranged from 17–39 years. the anovulation in the subfertile group were diagnosed based on the standard criteria (clinical and laboratory investigations). all patients were scheduled for the induction of ovulation by treatment with clomiphene citrate. the blood samples have been drawn on the 2nd day of the menstrual cycle. the hormones and cytokines were measured. 2–4 weeks after commencing the treatment with clomiphene citrate (clomiphene, a nonsteroidal compound, structurally similar to oestrogen, blocks oestrogenic hypothalamic receptors, resulting in blinding of the hypothalamus– pituitary axis to endogenous circulating oestrogen), the second sample of blood was drawn again to re-measure the levels of the same cytokines measured before the treatment. by spss software for windows, version 20, ibm, us, 2010, data of all participants have been entered, descriptive data analysis, paired sample t test, one way anova test and person’s correlations have been used as appropriate. results in this study, the levels of tnf-α and il-1ß before treatment were lower than those in after treatment; however, these differences were significant only in case of tnf-α (p = 0.041). these results indicate that clomiphene citrate has a specific effect on tnf-α expression and to a lesser extent on other cytokines. conclusion the results of this study may implicate a direct role of tnf-α in the fertility and, thus, could raise questions about the possibility of using immune modulation in the treatment of sub-fertility. keywords ovulation stimulation, clomiphene citrate, tumour necrosis factor alpha, interleukin-1ß, fertility, anovulation studying the effect of ovulation stimulation by using clomiphene citrate on serum level of tumor necrosis factor alpha and interleukin-1ß in sub-fertile women in holy kerbala province suha f. mohammed al-ma’aroof, mohanad m. ahmed, narjis h. mansor issn 2413-0516 college of medicine, karbala university, karbala, iraq. correspondence to suha f. mohammed (email: adsa_1980@yahoo.com). (submitted: 3 february 2016 – revised version received: 17 march 2016 – accepted: 19 may 2016 – published online: 26 june 2016) introduction the ovulation is the development and release of an ovum from the ovaries. it is the most fertile period of the menstrual cycle. by about 14 days into the reproductive cycle, an oocyte reaches maturity and is released as an ovum.1 the gonadotropins fsh and lh are produced by the anterior pituitary gonadotrophic cells and are responsible for the ovarian follicular stimulation.2 the fsh targets the receptor expressed only on granulosa cells and induces the maturation of ovarian follicle.3 the lh plays a key role in the initiation of the ovulatory process of preovulatory follicles by activating multiple cellular signaling pathways.4 the most common cause of female infertility is ovulatory disorder characterised by anovulation or by infrequent and/or irregular ovulation.5 anovulatory dysfunction is a common problem and is responsible for about 40% of female infertility.6 sometimes women does not ovulate (release an egg from an ovary each month) or ovulate irregularly, this will interfere with the ability to become pregnant. the ovulation induction is the term for the use of medical therapy to treat women who do not ovulate by themselves.7 the first-line medication for the ovulation induction is clomiphene citrate. it is a nonsteroidal compound, structurally similar to oestrogen and it blocks oestrogenic hypothalamic receptors, resulting in blinding of the hypothalamus–pituitary axis to endogenous circulating oestrogen. this in turn triggers the release of fsh from the anterior pituitary following alterations in gnrh pulsatility.6 the gonadotropins and gonadal steroids play the central role in ovarian folliculogenesis. however, the variable fate of follicles within the same ovary suggests the existence of additional intraovarian modulatory systems.8 the cytokines assist granulosa cell growth during the follicular development and thus, they maintain the normal ovarian function. in addition, the immune mediators may play role in ovulation. in this context, it has been shown that an influx of immune cells takes place during the lh peak and this influx is associated with the release of several cytokines.9 il-1β augmented the fsh-stimulated accumulation of 20-dihydroprogesterone. there are studies in the rat ovary indicate that the rat ovarian theca-interstitial cell is a site of il-1β gene expression, the preovulatory acquisition of which is gonadotropin dependent.10 tnf-α engages in the differentiation of a variety of cell types. in the ovary, the tnf-α was found capable of attenuating the differentiation of cultured granulosa cells from immature rats.11 the detection of tnf-α activity in some luteal tissue on day 5, and the scarcity of macrophages at this stage raise the possibility that cells other than macrophages may also produce tnf in the corpus luteum.12 the tnf-α stimulates progesterone synthesis in differentiated ovaries, while in undifferentiated ovarian cells tnf-α inhibits steroidogenesis.13 the il-1 and tnf-α suppress 17β-estradiol (e2) and progesterone release from granulosa and luteal cells in vitro. the tnf-α affects negatively folliculogenesis and ovarian maturation.14 the principal aim of this study is to investigate the possible side effects of ovulation stimulation by studying its effects on the immune system as well as the certain biochemical systems. it is worthy to mention that understanding the side effects of ovulation stimulation may help better intervention with those effects by therapeutic or preventive measures. 60 j contemp med sci | vol. 2, no. 6, spring 2016: 59–62 effect of ovulation induction on tnf-α and il-1ß research suha f. mohammed al-ma’aroof et al. table 2. characteristics of patients according to the type of sub-fertility type of sub-fertility p-valueprimary n = 15 secondary n = 18 mean (±sd) std. error mean (±sd) std. error age (years) 24.4 (±5.4) 1.8 27.1 (±4.3) 1.4 0.014 bmi 25.7 (±2.1) 0.72 31.7 (±3.2) 1 0.00 fsh (miu/ml) 5.59 (±1.97) 0.65 5.99 (±1.4) 0.48 0.812 lh (miu/ml) 6.8 (±3.8) 1.2 10 (±6.7) 2.2 0.655 oestrogen (pg/ml) 99.3 (±211.2) 70.4 34.9 (±22.4) 7.4 0.377 materials and methods the study comprised of 53 women; 33 sub-fertile and 20 fertile (used as the control group for cytokine profile analyses). their ages ranged from 17 to 39 years. the community of our study was the female partner of sub-fertile couple from karbala city attending the karbala fertility unit (kfu) which is found in the karbala maternity hospital. the samples of our study were female, selected from the local community who were complaining of primary or secondary sub-fertility. the anovulation has been diagnosed based on the patient’s history, hormonal level (day 2) and pelvic u/s in the in the unit of x-ray and sonar in karbala maternity hospital. whereas, the control group (fertile women) has been collected from the local community. the sample collection was during the period from april 2015 to august 2015. the laboratory study has been carried out in the research laboratories of biochemistry department and the microbiology department, college of medicine, university of karbala. the patients and control women have been informed about the study and its aims and their agreement have been taken. the patients have been selected by the gynaecologist after taking history and investigations. the patients were not on any form of ovulation induction strategy by either pharmacological or nonpharmacological way. the blood samples have been drawn on day 2 of the cycle. the patients have been examined by the gynaecologist to ensure that there is no any overt inflammation. in addition, c-reactive protein has been measured in each patient and any patient showed high titers has been excluded from the study. the hormones and cytokines were measured by using standard sandwich enzyme-linked immunosorbent assay technology. after obtaining the blood samples, the patients have been given the ovulation induction drugs (clomiphene citrate) for 3 months. after that, another sample of blood has been drawn again to re-measure the levels of the same cytokines measured before the treatment. by using the statistical package for social sciences, spss software for windows, version 20, ibm, us, 2010, the data of all the participants have been entered, the descriptive data analysis, paired sample t test, one way anova test and person’s correlations have been used as appropriate. results a total of 33 patients were suffering from anovulation and 20 fertile subjects were enrolled. the mean age of the patients was 25.09 years old. as shown in table 1, a significant positive correlation was found between age and bmi (r = 0.364, p = 0.038). a highly significant positive correlation was found between bmi and bmi categories (r = 0.854, p = 0.00). significantly, there were no correlations between age and bmi categories, fsh, lh and oestrogen (r = 0.304, p = 0.085); (r = 0.351, p = 0.092); (r = 0.28, p = 0.185); (r = 0.277, p = 0.266); respectively. in table 2, the mean age of secondary type of infertility was higher in comparison with the primary type of infertility (27.1 vs. 24.4) and this difference was statistically significant (p = 0.014). in addition, the mean bmi of the secondary type of infertility was higher than in the primary type of infertility (31.7 vs. 25.7) and this difference was statistically highly significant (p = 0.00). however, no significant differences were found between both the types of infertility in respect to sex hormones. table 1. correlation matrix among the patient’s characteristics bmi bmi categories fsh lh oestrogen age in years pearson correlation 0.364* 0.304 0.351 0.280 0.277 sig. (2-tailed) 0.038 0.085 0.092 0.185 0.266 bmi pearson correlation 0.854** 0.215 0.241 0.173 sig. (2-tailed) 0.000 0.314 0.257 0.493 bmi categories pearson correlation 0.088 0.159 −0.373 sig. (2-tailed) 0.682 0.457 0.128 fsh pearson correlation 0.273 −0.413 sig. (2-tailed) 0.196 0.089 lh pearson correlation 0.179 sig. (2-tailed) 0.478 61j contemp med sci | vol. 2, no. 6, spring 2016: 59–62 research effect of ovulation induction on tnf-α and il-1ßsuha f. mohammed al-ma’aroof et al. the natural behaviour of patients who tend to engage in more secondary life style with increasing age. the increasing secondary life style may be caused by several reasons such as the social influence, lack of outdoor activities, or accompanied by other changes:life events and the kind of responsibilities in addition to the pathological changes. the various environmental and social factors relating to diet and physical activity have been identified that could contribute to obesity.18 the levels of cytokines before treatment were lower than those in after treatment, however these differences were significant only in case of tnf-α (p = 0.041). these results indicate that the clomiphene citrate affects specifically on tnf-α expression and to a lesser extent on other cytokines. the clomiphene citrate treatment blocks the oestrogenic hypothalamic receptors, resulting in blinding of the hypothalamus–pituitary axis to endogenous circulating oestrogen. this in turn triggers the release of fsh from the anterior pituitary following alterations in gnrh pulsatility.6 and because the oestrogens inhibit il-1 and tnf-α production,19–22 we could assume that the clomiphene citrate treatment has the same role on these cytokines. there is evidence that cytokines are involved in both the inhibition and stimulation of follicular responsiveness to gonadotrophins.23 tnf-α activates neutrophils, induces il-1 gene expression, enhances the expression of class i major histocompatibility complex (mhc) antigens and adhesion molecules on the endothelial cells, and is involved in bone marrow resorption and the production of prostaglandin and collagenase from human synovial cells and fibroblasts.24 the mean concentration of tnf-α in the sub-fertile group was <1 fold lower than its mean concentration in the fertile group. the mean concentration of il-1ß in sub-fertile also was less than its mean concentration in fertile group but they were convergent. abnormally, a low concentrations of sex hormones are associated with a higher serum levels of tnf-alpha and which could suggest a suppressive effect of estradiol and progesterone on pro-inflammatory cytokine secretion.25 the in vitro studies by chao et al. have shown that estradiol can lower the tumour necrosis factor production.26 conclusion the very interesting results were emerged when comparisons, were made between the sub-fertile and fertile groups in respect to the cytokine levels. the levels of tnf-α and il-1ß were significantly lower in the sub-fertile group compared to the fertile subjects indicating a possible role in the aetiology of sub-fertility status. they were low in the sub-fertile groups, and their low concentrations might possibly associate with the anovulation status. generally, the results of the current study may implicate a direct role of certain cytokines (tnf-α and il-1ß) in the fertility and, thus, could raise questions about the possibility of using immune modulation in the treatment of sub-fertility.  table 3. cytokine levels before and after treatment with clomiphene citrate for sub-fertile subject cytokines conc. in pg/ml before treatment mean (±sd) n = 20 after treatment mean (±sd) n = 20 p-value tnf-α 58.47 (±26.49) 74.88 (±23.39) 0.041 il-1ß 10.32 (±7.86) 12.24 (±5.37) 0.266 table 4. correlation of cytokine level between sub-fertile and fertile subject cytokines conc. in pg/ml patients mean (±sd) n = 33 control mean (±sd) n = 17 p-value tnf-α 44.9 (±26.7) 78.6 (±18.2) 0.00 il-1ß 11.2 (±6.2) 13.2 (±10.4) 0.396 as shown in table 3, the paired sample t test has been used to compare the means of the cytokine levels between before and after treatment with clomiphene citrate. the levels of cytokines before treatment were lower than those in after treatment; however, these differences were significant only in case of tnf-α (p = 0.041). table 4 shows that the mean concentration of tnf-α in the sub-fertile group (44.9) was <1 fold lower than its mean concentration (78.6) in the fertile group. the mean concentration of il-1ß in sub-fertile (11.2) also was less than its mean concentration (13.2) in fertile group but they were convergent. discussion the immune cells are associated with the regulation of every level of the hypothalamus–pituitary–ovarian axis. the cytokines assist granulosa cell growth during the follicular development and thus they maintain the normal ovarian function. it has been shown that on the influx of immune cells takes place during the lh peak and this influx of immune cells is associated with the release of several cytokines. in addition, the rupture of the ovarian follicles is believed to be an immune-mediated reaction with il-1, the tnf-alpha are the cytokines playing the major role in this process.9 the crosstalk between the endocrine and immune systems regulates a large number of biological processes that affect target tissues, and this crosstalk involves gene expression, cytokine and/or lymphokine release and hormone action.15 the mean age of the patients was 25.09 years old. the age of the female is the single most important determinant with a gradual decline in fertility especially after the age of 35 years.16,17 a significant positive correlation was found between the age and bmi (p = 0.038). this correlation may be caused by references 1. marieb en, hoehn k. human anatomy & physiology. pearson education; 2007 2. vaitukaitis jl, ross gt, braunstein gd, rayford pl. gonadotropins and their subunits: basic and clinical studies. recent prog horm res. 2013;32:289–33. pmid: 785557 3. themmen apn, huhtaniemi it. mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary– gonadal function. endocr rev. 2000;21(5):551–83. pmid: 11041448 4. russell dl, robker rl. molecular mechanisms of ovulation: co-ordination through the cumulus complex. hum reprod update. 2007;13(3):289–312. pmid: 17242016 5. elghblawi e. polycystic ovary syndrome and female reproduction. br j nurs. 2007;16(18):1118–1121. pmid: 18073681 6. homburg r. clomiphene citrate: end of an era? a mini-review. hum reprod. 2005;20(8):2043–51. pmid: 15878925 62 j contemp med sci | vol. 2, no. 6, spring 2016: 59–62 effect of ovulation induction on tnf-α and il-1ß research suha f. mohammed al-ma’aroof et al. 7. mulders ag, laven js, eijkemans mj, hughes eg, fauser bc. patient predictors for outcome of gonadotrophin ovulation induction in women with normogonadotrophic anovulatory infertility: a meta-analysis. hum reprod update. 2003;9(5):429–449. pmid: 14640376 8. guillemin r. on the word: cybernin. in: franchimont p, channing cp (eds). intragonadal regulation of reproduction. london: academic press; 1981. 9. vinatier d, dufour p, tordjeman-rizzi n, prolongeau jf, depret-moser s, monnier jc. immunological aspects of ovarian function: role of the cytokines. eur j obstet gynecol reprod biol. 1995;63(2):155–68. pmid: 8903772 10. hurwitz a, ricciarelli e, botero l, rohan rm, hernandez er, adashi ey. endocrineand autocrine-mediated regulation of rat ovarian (thecainterstitia006c) intkrleukin-β gene expression: gonadotropin-dependent preoviilatory acquisition. endocrinology. 1991;129(6):3427–3429. pmid: 1954917 11. roby k, terranova p. tumor necrosis factor alpha alters follicular steroidogenesis in vitro. endocrinology. 1988;123(6):2952–2954. pmid: 3058464 12. wuttke w, spiess s, knoke i, pitzel l, leonhardt s, jarry h. synergistic effects of prostaglandin f2alpha and tumor necrosis factor to induce luteolysis in the pig. biol reprod. 1998;58(5):1310–1315. pmid: 9603269 13. bornstein s, rutkowski h, vrezas i. cytokines and steroidogenesis. mol cell endocrinol. 2004;215(1):135–141. pmid: 15026186 14. calogero ae, nicoletti f, palumbo ma, burrello n, di mauro m, lunetta m, et al. macrophage-derived cytokines in the follicular fluids of women with infertility due to immunological causes: elevated levels of interleukin 6 and low levels of granulocyte–macrophage colony-stimulating factor. cytokine. 1998;10(10):814–8. pmid: 9811536 15. sen a, kushnir va, barad dh, gleicher n. endocrine autoimmune diseases and female infertility. nat rev endocrinol. 2014;10(1):37–50. doi: 10.1038/ nrendo.2013.212 pmid: 24189508 16. menken j, trussell j, larsen u. age and infertility. science. 1986;233(4771):1389– 94. pmid: 3755843 17. templeton a, morris jk, parslow w. factors that affect outcome of in-vitro fertilisation treatment. lancet. 1996;348(9039):1402–6. pmid: 8937279 18. koplan jp, dietz wh. caloric imbalance and public health policy. jama. 1999;282(16):1579–1581. pmid: 10546699 19. hu sk, mitcho yl, rath nc. effect of estradiol on interleukin 1 synthesis by macrophages. int j immunopharmacol. 1988;10(3):247–52. 20. polan ml, loukides j, nelson p, carding s, diamond m, walsh a, et al. progesterone and estradiol modulate interleukin-1 beta messenger ribonucleic acid levels in cultured human peripheral monocytes. j clin endocrinol metab. 1989;69(6):1200–6. pmid: 2584355 21. pottratz st, bellido t, mocharla h, crabb d, manolagas sc. 17 beta-estradiol inhibits expression of human interleukin-6 promoter-reporter constructs by a receptor-dependent mechanism. j clin invest. 1994;93(3):944–50. pmid: 8132780 22. pacifici r, rifas l, mccracken r, vered i, mcmurtry c, avioli lv, et al. ovarian steroid treatment blocks a postmenopausal increase in blood monocyte interleukin 1 release. proc natl acad sci u s a. 1989;86(7):2398–402. pmid: 2522659 23. gougeon a. intragonadal regulation of human follicular genesis: facts and hypotheses. ann endocrinol (paris). 1994;55(2):63–73. pmid: 7802429 24. rees r. cytokines as biological response modifiers. j clin pathol. 1992;45(2):93. 25. trenova ag, slavov gs, manova mg, kostadinova ii, vasileva tv. female sex hormones and cytokine secretion in women with multiple sclerosis. neurol res, 2013;35(1):95–9. doi: 10.1179/1743132812y.0000000120 pmid: 23317804 26. chao tc, van alten pj, greager ja, walter rj. steroid sex hormones regulate the release of tumor necrosis factor by macrophages. cell immunol. 1995;160(1):43–9. pmid: 7842485 248 j contemp med sci | vol. 5, no. 5, september-october 2019: 248–253 immunohistochemical expression of sirt1 in oral squamous cell carcinoma and its relationship with clinical-pathological factors maryam seyedmajidi,a shima nafarzadeh,b arezoo rayani,c dariush moslemi,d ali bijani,e and majid sharbatdaranf adental materials research center, school of dentistry, babol university of medical sciences, babol, iran. bdepartment of oral and maxillofacial pathology, school of dentistry, babol university of medical sciences, babol, iran. cdepartment of oral and maxillofacial pathology, school of dentistry, mazandaran university of medical sciences, sari, iran. ddepartment of radiation oncology, babol university of medical sciences, babol, iran. enon-communicable pediatrics diseases research center, babol university of medical sciences, babol, iran. fdepartment of pathology, babol university of medical sciences, babol, iran. correspondence to arezoo rayani (email: arezoo.rayyani@gmail.com). (submitted: 02 august 2019 – revised version received: 18 august 2019 – accepted: 15 september 2019 – published online: 26 october 2019) introduction squamous cell carcinoma (scc) is the most common malignancy of the oral cavity. most samples of oral squamous cell carcinoma (oscc) usually have no visible changes in the early stages. therefore, early detection can be crucial in order to improve the lives of afflicted individuals and constitutes 90–95% of all oral malignancies and the fifth most common cancer worldwide.1–3 the mortality rate is about 50% and has not changed in the past 50 years despite extensive research.4,5 the prevalence of this disease is on the rise in youth. the main contributing factor to oscc in people under 40 years of age is uncertain and the main risk factors (alcohol and tobacco) is often absent in this population.6 sirt1, also known as nad-dependent histone deacetylase3 is an orthologous gene of sir2 and has multiple roles.7 this protein prohibits rrna transcription by deacetylating histone of h3k9 leading to the inhibition of apoptosis. also, the expression of sirt1 with deacetylated histones has an oncogenic effect and reduces the activity of tumor suppressors such as ku70, p53, and foxo protein family.8 sirt1 is involved in dna repair and can act as a tumor suppressor.9 therefore, sirt1 plays different roles in different types of cancer. its expression is increased in stomach, breast, and prostate cancers.3 with the help of b-catenin and c-myc, sirt1 plays an important role in tumor progression and metastasis.3 studies show that this protein has an oncogenic role through the inhibition of apoptosis, differentiation, proliferation, and growth.10 tnm is commonly used to predict the clinicopathological status in cancers. however, the predictive value of the staging system is not enough. there are a number of molecular markers that can be effective in improving the ability of staging system.11,12 studies on the use of sirt1 as a therapeutic molecule goal for oscc are scarce. there is no comprehensive study about the relationship between the expression of sirt1 and clinicopathological factors in patients with oscc and most studies have been done in other parts of the body. therefore, we aimed to investigate the expression of sirt1 in oscc and its relationship with clinicopathological factors such as age, sex, location of tumor, smoking, clinical stage, grade of tumor, metastasis to lymph nodes in the neck, and distant metastasis. materials and methods sampling in this cross-sectional study, 30 paraffin blocks of oscc were obtained from the archives of the department of pathology in hospitals of mazandaran province, northern iran. also, 30 paraffin blocks of normal oral mucosa obtained from patients who referred for crown lengthening surgery and had minimal inflammation or change in clinical and histopathological condition were studied. records of patients diagnosed with oscc were extracted non-randomly and after recording the patients’ demographic data, the relevant blocks were extracted. objective oral squamous cell carcinoma (oscc) is the most common oral cancer in the world that threatens public health. there are a number of molecular markers that can be used to improve tnm staging. one of these markers is sirt1. we aimed to investigate the expression of sirt1 in oscc and its relationship with clinicopathological factors such as age, sex, location of tumor, smoking, clinical stage, grade of tumor, metastasis to lymph nodes in the neck and distant metastasis. methods this cross-sectional study was performed on 30 samples of oscc and 30 samples of normal oral mucosa. required clinical data were collected from patients. after immunohistochemistry staining for sirt1 on sections prepared with paraffin blocks, the percentage of stained cells and staining intensity of the cells were evaluated in the terms of core and cytoplasmic aspects. results positive expression of sirt1 was seen in 93.3% and 96.7% of cells in the core and cytoplasm view, respectively, which was significantly higher than normal tissue (p = 0.021 and 0.001, respectively). we found no significant relationship between the intensity and percentage of core and cytoplasmic staining of squamous cells as well as clinicopathological factors. conclusion the overexpression of sirt1 in oscc samples as compared with normal mouth tissue indicates its role in carcinogenesis. however, further studies on sirt1 may shed more light on the treatment options of oscc. keywords squamous cell carcinoma, immunohistochemical staining, sirt1 issn 2413-0516 original 249j contemp med sci | vol. 5, no. 5, september-october 2019: 248–253 m. seyedmajidi et al. immunohistochemical expression of sirt1 in oral squamous cell carcinoma required information including age, sex, location of tumor, smoking, clinical tumor stage, grade of tumor, neck lymph node metastasis, and distant metastasis were obtained from their pathological reports or during contact with patients. two cuts were prepared from the paraffin blocks. the first cut with a thickness of 4 microns was hematoxylin–eosin stained to confirm the initial diagnosis and select appropriate blocks containing enough samples. the second cut was prepared for immunohistochemical study. sirt1 immunohistochemistry about 3 µm thick slice was prepared from paraffin blocks and mounted on sailyn slide. tissue sections were dehydrated and deparaffined. deparaffined samples for antigen retrieval by lam holder (dako, copenhagen, denmark) was placed in buffer citrate 0.01 m with 2.6–6 ph and subjected to microwave and then the slides were exposed to hydrogen peroxide with for 10 min. next, the surface of the tissue was completely covered for 30 min with primary polyclonal antibodies of anti-sirt1 antibody rabbit (art nosc-15404, santa cruz, biotechnology, santa cruz, ca, usa). then, the tissue surface was covered with secondary antibody [goat anti-rabbit igg h & l (hrp)] for 30 min (art no: ab97051-1 mg, abcam co.) and finally placed on the diamino benzene hydrochloride solution containing 0.03% h2o2 as chromogen for peroxidase activity for 10–15 min. it was then placed in harris hematoxylin for 1 min as counterstain. breast cancer tissue was considered for positive control. gingival tissue was considered for negative control. statistical analysis all slides were evaluated with an optical microscope (olympus bx41, olympus, tokyo, japan) with a magnification of 40 times by two independent pathologists who were unaware of the clinical features of the samples. immunohistochemical expression evaluation of sirt-1 was done so that the five randomly selected histology fields were considered and cell percentage of cytoplasm and nucleus condition was determined with a magnification of 400 in each sample obtaining a score1 as follows: score 0: tumors where 0–5% of cancer cells were stained. score 1: tumors where 6–25% of cancer cells were stained. score 2: tumors where 26–50% of cancer cells were stained. score 3: tumors where 51–75% of cancer cells were stained. score 4: tumors where 76–100% of cancer cells were stained. cytoplasm and nucleus staining intensity of epithelial tumor cells stained with sirt-1 was recorded in four different degrees including: no staining (0), weak staining (1), moderate staining (2) and severe staining (3). finally, based on the total staining intensity and percentage of stained cells, the final score was computed as follows: negative (from 0 to 2) and positive (from 3 to 7).1 statistical analysis was performed using spss statistical software. immunohistochemical staining intensity and percentage differences of staining in each group was determined by chi-square test and mann–whitney test was done to examine the relationship between clinical factors of staining intensity and percentage of staining. results about 60 samples, including 30 paraffin blocks of oscc from 11 (36.7%) men and 19 (63.3%) women with a mean ± sd age 68.70 ± 15.55 years and 30 normal tissue samples of patients referred for crown lengthening surgery were included. the mean ± sd age of the patients in the healthy group was 39.20 ± 12.42 years. among the oscc specimens, 15 (50%) had grade i, 12 (40%) grade ii and three (10%) grade iii oscc. moreover, in this group, buccal mucosa, gums, rethro-molorpad, and lower part of the mouth were involved in 12 (40%), 10 (33.3%), six (20%), and two (6.7%) patients in this group. in this study, sirt1 staining both in the nucleus and in the cytoplasm of squamous cells was evaluated. the frequency distribution of the percentage of stained cells in terms of nuclear and cytoplasmic staining intensity in oscc and normal oral mucosa is shown in table 1. sirt1 expression was significantly seen in the cytoplasm and nucleus of tumor cells showing diffuse distribution in island of oscc. in terms of the percentage of stained cells in the core, 25 (83.3%) cases of oscc had score 4 (76–100%) and in the cytoplasm, 28 (93.3%) cases had score 4 (76–100%), respectively. in terms of intensity of nuclear staining in the cells of osccs, 18 (60%) cases showed severe staining cytoplasm and in terms of staining intensity, 22 (73.3%) cases showed weak staining. according to the results, the mean ± sd final scores of core staining in oscc cells and normal oral mucosa were 6.07 ± 1.780 and 3.00 ± 1.597, respectively. 69.9% of normal mucosa in the mouth and 93.4% of oscc cells expressed a positive marker of sirt1 (score 3 and above) and there was a significant difference in scores between the core staining in oscc and oral mucosa (p < 0.001; table 2). we found that the mean ± sd final scores of cytoplasm staining in oscc cells and normal oral mucosa were 5.10 ± 0.845 and 3.03 ± 2.076, respectively. about 53.4% of normal mucosa in the mouth and 96.7% of oscc cells express a positive marker of sirt1 (score 3 and above) and there was a significant difference in final scores between the oscc and normal oral mucosa between the core and the oral mucosa oral squamous cell carcinoma (p < 0.001; fig. 1 and table 3). table 4 shows the immunoreactivity of oscc in the core and cytoplasm. as shown, sirt1 was expressed significantly in the core and cytoplasm of oscc cells. comparing the sirt1 expression in the two groups using mann–whitney test, we found a significant relationship between all the colors of sirt1 in normal mucosa and oral squamous cell carcinoma in the percentage of cells stained in terms of core (p = 0.001), cytoplasm (p = 0.001), nuclear staining intensity (p = 0.001) and cytoplasm staining intensity (p = 0.001). to examine the relationship between sex and age, clinical tumor stage, location of tumor, distant metastasis and metastasis to lymph nodes and smoking and crying, mann–whitney test was used for sirt1 expression.1 we found no significant relationship between the rate and severity of core and cytoplasmic staining of tumor cells in oscc and samples with clinicopathological factors, such as no lymph node metastasis and distant metastasis, smoking, age, sex, tumor location, original 250 j contemp med sci | vol. 5, no. 5, september-october 2019: 248–253 immunohistochemical expression of sirt1 in oral squamous cell carcinoma m. seyedmajidi et al. table 1. frequency (%) of stained cells and nuclear and cytoplasmic staining intensity of cells in oral squamous cell carcinoma and normal oral mucosa expression grading oral squamous cell carcinoma (%) normal oral mucosa (%) p-value percentage of stained cells in terms of nucleus <5 2 (6.7) 6 (20.0) 0.001 6–25 0 (0) 11 (36.7) 26–50 0 (0) 9 (30.0) 51–75 3 (10.0) 3 (10.0) 76–100 25 (83.3) 1 (3.3) nuclear staining intensity no staining 2 (6.7) 3 (10.0) 0.001 weak 1 (3.3) 7 (23.3) moderate 9 (30.0) 19 (63.3) severe 18 (60.0) 1 (3.3) percentage of stained cells in terms of cytoplasm <5 0 (0) 7 (23.3) 0.001 6–25 1(3.3) 7 (23.3) 26–50 1 (3.3) 2 (6.7) 51–75 0 (0) 88 (26.7) 76–100 28 (93.3) 6 (20.0) cytoplasmic staining intensity no staining 0 (0) 7 (23.3) 0.016 weak 22 (73.3) 15 (50.0) moderate 8 (26.7) 8 (26.7) severe 0 (0) 0 (0) table 2. frequency distribution of the final core staining of cells in oral squamous cell carcinoma and normal oral mucosa expression group grading frequency percentage (%) result final nuclear staining scores oral squamous cell carcinoma 0 2 6.7 − 1 0 0 − 2 0 0 − 3 0 0 + 4 0 0 + 5 3 10.0 + 6 8 26.7 + 7 17 56.7 + normal oral mucosa 0 3 10 − 1 2 6.7 − 2 4 13.3 − 3 10 33.3 + 4 7 23.3 + 5 3 10.0 + 6 0 0 + 7 1 3.3 + original 251j contemp med sci | vol. 5, no. 5, september-october 2019: 248–253 m. seyedmajidi et al. immunohistochemical expression of sirt1 in oral squamous cell carcinoma table 3. frequency distribution of the final score of cytoplasmic staining of cells in oral squamous cell carcinoma and normal oral mucosa expression group grading frequency percentage (%) result final cytoplasmic staining scores oral squamous cell carcinoma 0 0 0 − 1 0 0 − 2 1 3.3 − 3 1 3.3 + 4 0 0 + 5 20 66.7 + 6 8 26.7 + 7 0 0 + normal oral mucosa 0 6 20.0 − 1 0 0 − 2 8 26.7 − 3 2 6.7 + 4 6 20.0 + 5 3 10.0 + 6 5 16.7 + 7 0 0 + fig. 1 immunostaining of sirt1 in (a) normal oral mucosa, (b) well-differentiated oral squamous cell carcinoma, (c) moderately differentiated oral squamous cell carcinoma and (d) poorly differentiated oral squamous cell carcinoma (magnification: 400×). a c b d original 252 j contemp med sci | vol. 5, no. 5, september-october 2019: 248–253 immunohistochemical expression of sirt1 in oral squamous cell carcinoma m. seyedmajidi et al. table 4. immunoreactivity of cells (sirt1) in the core and cytoplasm of cells in oral squamous carcinoma cells location immunoactivity abundance (%) p-value core negative 2 (6.7) 0.021 positive 28 (93.3) cytoplasm negative 1 (3.3) 0.001 positive 29 (96.7) tumor size and tumor clinical stage (p > 0.05). however, in the final check, there was a significant relationship between oscc samples and normal tissue in the mouth with respect to the final scores of the cytoplasmic and nuclear core (p < 0.001). discussion the results of this study suggest the higher expression of sirt1 in oscc compared with normal oral mucosa, which confirms the role of sirt1 in carcinogenesis. oral squamous cell carcinoma is related to genetic changes with lack of cell growth and differentiation.1 tumorigenesis is the result of a series of genetic changes leading to abnormal activity of oncogenes and inactivation of tumor suppressors.2 molecular markers can play an indispensable role in predicting tumor aggressiveness. by identifying the biological markers, we can improve clinical staging system’s ability to increase and predict prognosis and progression of oscc. jung et al.13 reported that sirt1 promotes tumor by p53 inhibition. they found that sirt1 expression is a good prognostic factor for colorectal cancer, and in this regard, is intersected with b-catenin more than p53. also sirt1 reduced protein expression of bcl2. moreover, in our study, the sirt1 expression significantly observed in the nucleus and cytoplasm of tumor cells is shown in diffuse distribution at oral squamous cell carcinoma neoplastic island. its expression in oscc was higher than normal oral mucosa that is consistent with studies on colon cancer,14 squamous cell carcinoma,15 and basal cell carcinoma and bowen’s disease16 showing overexpression of sirt1 in cancer tissue compared to normal tissue. michishita et al.16 also observed that the staining sirt1 in normal tissue and dysplastic and cancerous tissue is mainly appeared in the core in a small number of samples, all of which has cytoplasmic expression. in another study, sirt1 had more gene expression in oscc than the rest of the family.17 byles et al.18 also used human tissue and reported that sirt1 is focused mainly in the core of normal cells, but is apparently seen deformed in the cytoplasm of cancer cells, which is consistent with the results of this study. in this study, core and cytoplasmic expression of sirt1 was not significantly associated with any clinicopathological factors. another study showed that sirt1 expression level was not related to depth of invasion, differentiation and tumor size, type of tumor, lymph node metastasis, and the patient’s age.19 another study also showed a significant relationship between the expression of sirt1 and distant metastasis.20 sirt1 participation in other cancers have been reported including breast,20 colorectal,13 lung,21 and skin16 cancers and bowen’s disease.16 we found no significant relationship among the percentage of core and cytoplasmic staining of tumor cells in a sample of oral squamous cell carcinoma with tumor grade. inconsistently, lin and peng21 found that there was a significant relationship between the survival of sirt1 and average and weak of histological grade. kabra et al.22 also showed that in stage i, ii, and iii colorectal adenocarcinoma sirt1 expression significantly increases. however, in our study there is no link between sirt1 expression and stage of the disease. sirt1 expression plays an important role in predicting oral squamous carcinomas,13 whereas in our study sirt 1 had no relationship with prognostic factors. due to the significant difference in the expression of sirt1, we found a significant relationship between oscc and normal oral mucosa with respect to sirt1 expression indicating that it can be a possible prognostic marker. therefore, by further studies in this regard and studying the role of this protein, it could be used as a therapeutic target molecule in the treatment of patients with oscc. conclusion overexpression of sirt1 in oral squamous cell carcinoma samples compared with normal tissue of the mouth indicates its role in carcinogenesis. with further studies of sirt1 in this regard, sirt1 may be used to treat the target molecule. conflicts of interest the authors declare that they have no conflicts of interest. references 1. yu c, chen k, zheng h, guo x, jia w, li m, et al. overexpression of astrocyte elevated gene-1 (aeg-1) is associated with esophageal squamous cell carcinoma (escc) progression and pathogenesis. carcinogenesis. 2009;30:894–901. 2. noch ek, khalili k. the role of aeg-1/mtdh/lyric in the pathogenesis of central nervous system disease. adv cancer res. 2013;120:159–192. 3. noguchi a, li x, kubota a, kikuchi k, kameda y, zheng h, et al. sirt1 expression is associated with good prognosis for head and neck squamous cell carcinoma patients. oral surg oral med oral pathol oral radiol. 2013;115:385–392. 4. seyedmajidi m, shafaee s, siadati s, khorasani m, bijani a, ghasemi n. cyclo-oxygenase-2 expression in oral squamous cell carcinoma. j cancer res ther. 2014;10:1024–1029. 5. shakib pa, ensani f, abdirad a, valizadeh b, seyedmajidi m, sum s. cd44 and cd74: the promising candidates for molecular targeted therapy in oral squamous cell carcinoma. dent res j (isfahan). 2015;12:181–186. 6. emdad l, das sk, dasgupta s, hu b, sarkar d, fisher pb. aeg-1/mtdh/lyric: signaling pathways, downstream genes, interacting proteins, and regulation of tumor angiogenesis. adv cancer res. 2013;120:75–111. 7. longo vd, kennedy bk. sirtuins in aging and age-related disease. cell. 2006;126:257–268. 8. murayama a, ohmori k, fujimura a, minami h, yasuzawa-tanaka k, kuroda t, et al. epigenetic control of rdna loci in response to intracellular energy status. cell. 2008;133:627–639. 9. bosch-presegué l, vaquero a. the dual role of sirtuins in cancer. genes. cancer. 2011;2:648–662. 10. liu t, liu py, marshall gm. the critical role of the class iii histone deacetylase sirt1 in cancer. cancer res. 2009;69:1702–1705. 11. van der schroeff mp, van schie k, langeveld tp, looman c, baatenburg de jong rj. model-assisted predictions on prognosis in hnscc: do we learn? eur arch otorhinolaryngol. 2010;267:1445–1448. 12. emdad l, sarkar d, su zz, lee sg, kang dc, bruce jn, et al. astrocyte elevated gene-1: recent insights into a novel gene involved in tumor progression, metastasis and neurodegeneration. pharmacol ther. 2007;114:155–170. 13. jung w, hong kd, jung wy, lee e, shin bk, kim hk, et al. sirt1 expression is associated with good prognosis in colorectal cancer. korean j pathol. 2013;47:332–339. original 253j contemp med sci | vol. 5, no. 5, september-october 2019: 248–253 m. seyedmajidi et al. immunohistochemical expression of sirt1 in oral squamous cell carcinoma 14. jin q, yan t, ge x, sun c, shi x, zhai q. cytoplasm-localized sirt1 enhances apoptosis. j cell physiol. 2007;213:88–97. 15. hida y, kubo y, murao k, arase s. strong expression of a longevity-related protein, sirt1, in bowen’s disease. arch dermatol res. 2007;299:103–106. 16. michishita e, park jy, burneskis jm, barrett jc, horikawa i. evolutionarily conserved and nonconserved cellular localizations and functions of human sirt proteins. mol biol cell. 2005;16:4623–4635. 17. lai cc, lin pm, lin sf, hsu ch, lin hc, hu ml, et al. altered expression of sirt gene family in head and neck squamous cell carcinoma. tumour biol. 2013;34:1847–1854. 18. byles v, chmilewski lk, wang j, zhu l, forman lw, faller dv, et al. aberrant cytoplasm localization and protein stability of sirt1 is regulated by pi3k/ igf-1r signaling in human cancer cells. int j biol sci. 2010;6:599–612. 19. yu df, jiang sj, pan zp, cheng wd, zhang wj, yao xk, et al. expression and clinical significance of sirt1 in colorectal cancer. oncol lett. 2016;11: 1167–1172. 20. lee h, kim kr, noh sj, park hs, kwon ks, park bh, et al. expression of dbc1 and sirt1 is associated with poor prognosis for breast carcinoma. hum pathol. 2011;42:204–213. 21. lin sy, peng f. association of sirt1 and hmga1 expression in non-small cell lung cancer. oncol lett. 2016;11:782–788. 22. kabra n, li z, chen l, li b, zhang x, wang c, et al. sirt1 is an inhibitor of proliferation and tumor formation in colon cancer. j biol chem. 2009;284:18210–18217. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201903 original 136 j contemp med sci | vol. 5, no. 3, may–june 2019: 136–139 original acromegaly in iraq: brief look on epidemiology, comorbidities and management berq j. hadi al-yasseri,a* abbas m. rahmah,b and nizar s. shawky al-saffarb acollege of medicine, al-nahrain university, baghdad, iraq. bnational diabetes center, mustansiriyah university, baghdad, iraq. *correspondence to berq j. hadi al-yasseri (email: dr_berq@yahoo.com). (submitted: 15 october 2018 – revised version received: 06 november 2018 – accepted: 25 december 2018 – published online: 26 june 2019) introduction acromegaly is a rare chronic condition in adults resulted from continuous over secretion of growth hormone (gh) and hence increased production of insulin-like growth factor-1 (igf-1). when hypersecretion of gh happens in childhood, the resulted disease is referred to be gigantism rather than acromegaly. in vast majority of cases, the underlying cause of acromegaly has been detected to be microor macro-adenoma of pituitary gland.1 acromegalic patients are manifested by unproportioned growth of skeleton, tissues and organs. they may appear with clinical features like skeletal and acral over/abnormal growth, enlargement of soft tissues with frontal bossing, mandibular protrusion, jaw malocclusion, skin thickening, and increase in size of rings and shoes. other characteristics of this disease are: excessive sweating, paresthesia, goiter, arthritis, kyphoscoliosis, headaches, visual field defects, colon polyps, sleep apnea and day time sleepiness, reproductive disorders and cardiovascular (cv) diseases.2 wide spectrum of manifestations is associated with this disease, ranging from mild to severe clinical phenotypes. thus, a high index of suspicion is required.3 the diagnosis of acromegaly is often delayed for 5–10 years after time of approximate symptoms onset.4 therefore, patients frequently present with significant comorbidities such as hypertension, diabetes mellitus and sleep apnea. untreated conditions lead to decrease life expectancy. causes of death were determined to be mainly due to cv diseases (∼60%), respiratory diseases (25%) and malignancies (15%). with advances in treatment of acromegaly, overall mortality rates have been declined. although mortalities in patients with acromegaly remain higher than in general population, the increase in deaths is currently less than twofolds, compared with two to threefolds seen in earlier series.5 the goals of treatment in acromegaly are symptoms alleviation, tumor size control with preserving pituitary function, biochemical normalization of gh/igf-1 and reversing the raise in morbidity and mortality associated with this disorder. the current available modalities of treatment involve: surgery, pharmacotherapy and radiotherapy. the decision of required treatment option and therapeutic intervention is based on multiple factors, which need careful weighing and tailoring for each patient in a multidisciplinary frame.3 no unified registry has been established in iraq for acromegalic patients and few studies were performed to evaluate health situation and treatment response of them. therefore, in order to start such registration, this pilot study has been conducted to evaluate clinico-epidemiological characteristics for a group of iraqi patients with this disease. materials and methods setting this study was conducted on group of acromegalic patients attending national diabetes center (ndc) which represent one of the largest specialized centers in iraq concerned with management of endocrinological diseases, including acromegaly. the case notes and files of randomly selected 60 patients were reviewed by physician during period of october through december 2017. to be included in the study, the objective acromegaly is a rare chronic condition in adults resulted from continuous over secretion of growth hormone (gh) and hence increased production of insulin-like growth factor-1 (igf-1). pituitary adenoma is the underlying cause in vast majority of cases. this study aims to evaluate clinico-epidemiological characteristics for a group of iraqi individuals with this disease. methods case notes and files of 60 acromegalic patients attending iraqi national diabetes center were reviewed during period of october through december 2017. to be included, the diagnosis of acromegaly should be confirmed by appropriate laboratory and imagining techniques. disease control and biochemical response to management was defined by the criteria of normalized igf-1 for age and sex (z-score ≤ 2) and a mean gh value of ≤2 μg/l. data was entered and analyzed using statistical package for social sciences program, version 20. descriptive statistics was used; presented as frequencies and proportions, or means and standard deviations according to variable type. results patients were almost equally distributed among both sexes. their mean age is 50.6 ± 11.7 years. time interval between symptoms’ onset and diagnosis was nearly 10 years. macroadenoma (>1 cm) was found in 47 (78.3%) patients. diabetes mellitus and hypertension affected 42 (70.0%) and 37 (61.7%) individuals, respectively. for disease control, all acromegalics were put on long-acting somatostatin analogue and 21 (35.0%) of them performed hypophysectomy. in 45 (75.0%) patients, growth hormone value was dropped to ≤2 mg/l; and in 41 (68.3%) patients, igf-1 was normalized for age and sex. conclusion the clinco-epidemiological characteristics of acromegaly in iraq are comparable to that reported in other countries, although there is some delay in time of diagnosis. associated comorbidities (diabetes and hypertension) affected large number of patients. good proportion of study sample, nearly two-thirds, showed good metabolic response to treatment. keywords acromegaly, growth hormone, insulin-like growth factor-1, pituitary adenoma issn 2413-0516 137j contemp med sci | vol. 5, no. 3, may–june 2019: 136–139 original acromegaly in iraq: brief look on epidemiology, comorbidities andmanagementb.j.h. al-yasseri et al. diagnosis of acromegaly should be confirmed by appropriate laboratory and imagining techniques. the files of only those who regularly attended the center were involved. ethical approval for conducting the study was obtained from ncd. each record received a unique study number, so that patients’ identity would not be revealed. data collection the patient’s records were reviewed looking for information concerning demographic factors (age and sex), time of diagnosis, approximate time of symptoms onset; and details of associated comorbidities due to disease or treatment (e.g. hypertension, diabetes mellitus and thyroid disorders). data and results of treatment options (surgery, pharmacotherapy, and radiotherapy), biochemical control parameters (gh and igf-1) and reduction in tumor size were also reported. outcome analysis outcome analysis is restricted only to the results of investigations obtained from ncd laboratories. disease control and biochemical response to management was defined by the criteria of normalized igf-1 for age and sex (z-score ≤2) and a mean gh value ≤2 μg/l. active disease was defined as both an igf-1 z-score >2 and a mean gh value >2 μg/l. patients with conflicting values of igf-1 and gh were considered as not controlled.6,7 statistical analysis data in the records was entered and analyzed using the statistical package for social sciences (spss) program, version 20. descriptive statistics was used; presented as frequencies and proportions (%), or mean and standard deviation according to the variable type. results of the statistical analysis of the data are presented in tables and figures with an explanatory paragraph for each, using microsoft word software for windows, version 2013. results as it has been mentioned previously, this study involved 60 patients. they are almost equally distributed among both sexes. their mean current age is 50.6 ± 11.7 years and nearly two-thirds of them lie in fifth and sixth decades of life. by observing age of disease diagnosis and age when patients first notifying symptoms, it can be concluded that time interval between them is nearly 10 years. the radiological imaging revealed that around three quarters of pituitary tumors were macro-adenoma (> 1 cm) and the remaining were classified as micro-adenoma. table 1 displays these findings. the registered associated medical disorders among acromegalic patients have been shown in fig. 1. as it is obvious, diabetes mellitus and hypertension occupy first and second orders respectively. concerning distribution of thyroid abnormalities among participants; six (10.0%) have hypothyroidism, five (8.3%) have hyperthyroidism, and three (5.0%) have clinical goiter without reference to thyroid function. one patient was reported to have aortic incompetence and another was affected by beckwith-wiedemann syndrome. the modalities of treatment and biochemical indicators of disease control are revealed in table 2. all patients in our study were put on monthly injection of depot long-acting somatostatin analogue (sandostatin-lar) to control disease. almost one-third of them also performed hypophysectomy and only one was subjected to adjuvant radiotherapy. in three-quarters of patients, mean gh value was dropped to ≤2 μg/l; and in more than two-thirds, igf-1 was normalized for age and sex. consequently, more than 60% of patients exhibited completed biochemical control as measured by the two biochemical parameters. decrease in tumor size was observed in more than 70% of patients. collectively, 53.3% (n = 32) of patients have improvement in all the above three measures. discussion although acromegaly is a rare disease but its effect on life of affected individuals is dramatic and make most of them in table 1. basic characteristics of study sample character value number 60 current age; n (%) ≤39 years 7 (11.6%) 40–49 years 18 (30.0%) 50–59 years 19 (31.7%) ≥60 years 16 (26.7%) female sex; n (%) 28 (46.7%) age at diagnosis; (mean ± sd) 46.4 ± 10.8 years age of symptoms onset; (mean ± sd) 36.7 ± 10.2 years type of adenoma; n (%) microadenoma 13 (21.7%) macroadenoma 47 (78.3%) fig. 1 associated medical disorders among patients. table 2. treatment modalities and outcome factor value, n (%) treatment modalities; n (%) pharmacotherapy alone 38 (63.3) pharmacotherapy + surgery 21 (35.0) pharmacotherapy + surgery + radiotherapy 1 (1.7) biochemical control; n (%) safe gh 45 (75.0) normal igf-i 41 (68.3) safe gh and normal igf-i 38 (63.3) tumor size reduction 43 (71.7) 138 j contemp med sci | vol. 5, no. 3, may–june 2019: 136–139 acromegaly in iraq: brief look on epidemiology, comorbidities andmanagement original b.j.h. al-yasseri et al. need for continuous medical care and monitoring.2,5 our study seeks to give a glance on characteristics of disease in iraq and points of similarities and differences from that reported in studies from other countries. demography in current study, sex distribution of patients was nearly the same between males and females, which was consistent with that mentioned previously concerning acromegaly affects both sexes equally.8,9 the mean age of patients at diagnosis (46.4 years) is comparable to that in other studies which reported to be earlyto mid-forties.8–12 the delay period of about 10 years between symptoms onset and diagnosis need to be taken into consideration. although it remained within the limit reported in other places which was 5–10 years,4,13 but prompt action must be taken in order to reduce it. increase awareness of primary health care physicians and other related doctors about collection of symptoms and signs of acromegaly can help to facilitate diagnosis and consequently early management to decrease longterm sequels and comorbidities of this disease. type of adenoma the approximate ratio of macroto micro-adenoma of 3:1 was similar to what found in other countries. the report of spanish acromegaly registry estimated the prevalence of macro adenoma among patients to be 77%.14 other report from belgian registry estimated it to be 79%.7 a review article published in the new england medical journal found that at the time of diagnosis, more than 75% of patients with acromegaly have a macro-adenoma, which often expands laterally to the cavernous sinus or dorsally to the suprasellar region.15 this higher prevalence of macro-adenoma may reflect delay in diagnosis with subsequent additional symptoms and suffering to patients due to impact of local mass. comorbidities the prevalence of diabetes mellitus (dm) among acromegalics is known to be much higher than the general population.16 in previous studies, overt dm were found in 22–52% of participants17–19; depending on patient’s characteristics, clinical setting and treatment type. nevertheless, the prevalence of dm in this study (70%) exceeded all that reported before! this result may be attributed to the fact that all our patients were on octreotide hormone drug for a mean period of 4 years since disease diagnosis. somatostatin analogues (ssas) including octreotide have been reported to increase risk of secondary diabetes through different mechanisms. their metabolic effects are the outcome of pros related to reduction in hyperglycemic hormones (growth hormone or adrenocorticotropic hormone) and the cons from decreasing insulin or incretin secretion.20–24 although meta-analysis performed by mazziotti et al.24 concluded that ssas have a marginal clinical impact on glucose homeostasis in acromegaly, but a pilot study conducted by parkinson et al.21 found that glucose homeostasis tended to deteriorate with octreotide treatment. the prevalence of hypertension in acromegalic people was ranging between 18% and 60% in previous clinical series, and it is almost twice the rate recorded in unaffected people.25–27 this increase is thought to be attributed to expansion in plasma volume and sodium retention due to anti-natriuretic action of gh.25 also, recent studies revealed that the increase in sympathetic tone and/or abnormalities in the circadian rhythm of sympathetic system may have essential role in occurrence of raised blood pressure in acromegalic individuals.26 abnormalities in thyroid gland function are also among registered complications in previous literatures.16 in general, there is increase in sensitivity of the thyroid to tsh, modulated by an increase in sympathetic reaction, which leads to normal t4 concentrations despite decreased tsh levels in patients with active acromegaly.28 the prevalence of hyperthyroidism has been estimated to be 3.5–26%.29 those with high serum thyroxine level were found previously to have inappropriate normal or elevated level of serum thyrotropin which may indicate cosecretion of thyrotropin by adenoma.30,31 hypothyroidism due to hypopituitarism has been reported in nearly 21–26% of patients before. this is expected to result from compression of pituitary gland, especially in cases of macro-adenoma, or from surgical treatment of acromegaly.29,32,33 treatment modalities the portion of patients that performed surgical operations (mostly through trans-sphenoidal approach) for tumor was less than that reported in other countries. in spain, data from acromegaly registry revealed that 81% (n = 995) of patients underwent at least one surgery, most commonly by the trans-sphenoidal route.14 in belgian, 68% (n = 286) of patients underwent 321 surgical procedures, mostly by the trans sphenoidal route (>85%).7 no single factor can be set for the low rate of surgeries in our patients. nevertheless, the nature of thinking of iraqi patients, who afraid from performing operations in head region, may provide partial explanation for this finding. additionally, medical therapy has become globally more commonly used as first-line therapy with availability of long acting – somatostatin analogues. on the other hand, the presence of only one person who underwent radiotherapy is expected. data from spanish acromegaly registry (registro espanol de acromegalia, rea) reported decrease radiotherapy use over time. this is again due to increased usage of long acting – ssas.14 treatment outcomes this study evaluated the “real-life” effectiveness of medical treatment for acromegaly in iraq. the outcome of biochemical measures of disease control was comparable (or even better) than that reported in previous studies in developed countries. in acrobel survey, the overall cure/control rate was 49% (n = 418).7 data from rea revealed cure in 35% of 1084 spanish patients.14 in finnish database, gh and igf-1 levels were normal in 55% (n = 314) and 55% (n = 241) of patients, respectively. the combination of both measures for evaluation was not conducted, but probably the overall control rate using both of them was also below 50%.34 conclusion in general, the clinico-epidemiological characteristics of acromegaly in iraq are comparable with that reported in other countries, although there is some delay in time of diagnosis. associated comorbidities like hypertension and diabetes affected large number of study sample. good proportion of patients, nearly two-thirds, showed good metabolic response. the lag period between starting disease and diagnosis should be reduced. this could be done by increase awareness 139j contemp med sci | vol. 5, no. 3, may–june 2019: 136–139 original acromegaly in iraq: brief look on epidemiology, comorbidities andmanagementb.j.h. al-yasseri et al. acknowledgment none. conflicts of interest none.  of doctors about the disease and conduction of active case detection program. special care must be given to the management of associated comorbidities as it can affect treatment response, life-expectancy, and quality-of-life of patients. further studies involving larger sample and multi-center must be done in order to establish unified registry for iraqi acromegalic patients “acro-iraq”. references 1. banerjee a, patel k, wren am. acromegalyclinical manifestations and diagnosis. pharm j. 2003;13:273–278. 2. chanson p, salenave s. acromegaly. orphanet j rare dis. 2008;3:17. 3. dineen r, stewart pm, sherlock m. acromegaly. qjm. 2017;110:411–420. 4. reid tj, post kd, bruce jn, nabi kanibir m, reyes-vidal cm, freda pu. features at diagnosis of 324 patients with acromegaly did not change from 1981 to 2006: acromegaly remains under-recognized and under-diagnosed. clin endocrinol (oxf ). 2010;72:203–208. 5. sherlock m, ayuk j, tomlinson jw, toogood aa, aragon-alonso a, sheppard mc, et al. mortality in patients with pituitary disease. endocr rev. 2010;31:301–342. 6. alexopoulou o, bex m, abs r, t’sjoen g, velkeniers b, maiter d. divergence between growth hormone and insulin-like growth factor-i concentrations in the follow-up of acromegaly. j clin endocrinol metab. 2008;93:1324–1330. 7. bex m, abs r, t’sjoen g, mockel j, velkeniers b, muermans k, et al. acrobelthe belgian registry on acromegaly: a survey of the ‘real-life’ outcome in 418 acromegalic subjects. eur j endocrinol. 2007;157:399–409. 8. dal j, feldt-rasmussen u, andersen m, kristensen lø, laurberg p, pedersen l, et al. acromegaly incidence, prevalence, complications, and long-term prognosis: a nationwide cohort study. eur j endocrinol. 2016;175:181–190. 9. burton t, le nestour e, neary m, ludlam wh. incidence and prevalence of acromegaly in a large us health plan database. pituitary. 2016;19: 262–267. 10. vallette s1, ezzat s, chik c, ur e, imran sa, van uum s, et al. emerging trends in the diagnosis and treatment of acromegaly in canada. clin endocrinol (oxf ). 2013;79:79–85. 11. lavrentaki a, paluzzi a, wass ja, karavitaki n. epidemiology of acromegaly: review of population studies. pituitary. 2017;20:4–9. 12. cannavò s, ferraù f, ragonese m, curtò l, torre ml, magistri m, et al. increased prevalence of acromegaly in a highly polluted area. eur j endocrinol. 2010;163:509–513. 13. zarool-hassan r, conaglen hm, conaglen jv, elston ms. symptoms and signs of acromegaly: an ongoing need to raise awareness among healthcare practitioners. j prim health care. 2016;8:157–163. 14. mestron a, webb sm, astorga r, benito p, catala m, gaztambide s, et al. epidemiology, clinical characteristics, outcome, morbidity and mortality in acromegaly based on the spanish acromegaly registry (registro espanol de acromegalia, rea). eur j endocrinol. 2004;151:439–446. 15. melmed s. medical progress: acromegaly. n engl j med. 2006;355: 2558–2573. 16. abreu a, tovar ap, castellanos r, valenzuela a, giraldo cm, pinedo ac, et al. challenges in the diagnosis and management of acromegaly: a focus on comorbidities. pituitary. 2016;19:448–457. 17. colao a, auriemma rs, savastano s, galdiero m, grasso lf, lombardi g, et al. glucose tolerance and somatostatin analog treatment in acromegaly: a 12-month study. j clin endocrinol metab. 2009;94:2907–2914. 18. alexopoulou o, bex m, kamenicky p, mvoula ab, chanson p, maiter d. prevalence and risk factors of impaired glucose tolerance and diabetes mellitus at diagnosis of acromegaly: a study in 148 patients. pituitary. 2014;17:81–89. 19. dreval av, trigolosova iv, misnikova iv, kovalyova ya, tishenina rs, barsukov ia, et al. prevalence of diabetes mellitus in patients with acromegaly. endocr connect. 2014;3:93–98. 20. badiu c. effect of somatostatin analogues on glucose metabolism in acromegaly: friend of foe? aace clin case rep. 2016;2:e374–e375. 21. parkinson c, drake wm, roberts me, meeran k, besser gm, trainer pj. a comparison of the effects of pegvisomant and octreotide on glucose, insulin, gastrin, cholecystokinin, and pancreatic polypeptide responses to oral glucose and a standard mixed meal. j clin endocrinol metab. 2002;87:1797–1804. 22. ronchi c, epaminonda p, cappiello v, beck-peccoz p, arosio m. effects of two different somatostatin analogs on glucose tolerance in acromegaly. j endocrinol invest. 2002;25:502–507. 23. baldelli r, battista c, leonetti f, ghiggi mr, ribaudo mc, paoloni a, et al. glucose homeostasis in acromegaly: effects of long-acting somatostatin analogues treatment. clin endocrinol (oxf ). 2003;59:492–499. 24. mazziotti g, floriani i, bonadonna s, torri v, chanson p, giustina a. effects of somatostatin analogs on glucose homeostasis: a metaanalysis of acromegaly studies. j clin endocrinol metab. 2009;94:1500–1508. 25. lombardi g, galdiero m, auriemma rs, pivonello r, colao a. acromegaly and the cardiovascular system. neuroendocrinology. 2006;83:211–217. 26. bondanelli m, ambrosio mr, degli uberti ec. pathogenesis and prevalence of hypertension in acromegaly. pituitary. 2001;4:239–249. 27. vitale g, pivonello r, auriemma rs, guerra e, milone f, savastano s, et al. hypertension in acromegaly and in the normal population: prevalence and determinants. clin endocrinol (oxf ). 2005;63:470–476. 28. roelfsema f, biermasz nr, frolich m, keenan dm, veldhuis jd, romijn ja. diminished and irregular thyrotropin secretion with preserved diurnal rhythm in patients with active acromegaly. j clin endocrinol metab. 2009;94:1945–1950. 29. dąbrowska am, tarach js, kurowska m, nowakowski a. thyroid diseases in patients with acromegaly. arch med sci. 2014;10:837–845. 30. beck-peccoz p, persani l, lania a. thyrotropin-secreting pituitary adenomas. in: de groot lj, chrousos g, dungan k, et al., eds. endotext. mdtext.com, inc.; south dartmouth, ma, 2000. available from: https://www.ncbi.nlm.nih. gov/books/nbk278978/ [updated 2015 may 1] [internet]. 31. socin hv, chanson p, delemer b, tabarin a, rohmer v, mockel j, et al. the changing spectrum of tsh-secreting pituitary adenomas: diagnosis and management in 43 patients. eur j endocrinol. 2003;148:433–442. 32. shestakova t, dreval a, ilovaiskaja i, zaharevich e. study thyroid function in patients with acromegaly. endocr abstr. 2012;29:1715. 33. uchoa hb, lima ga, corrêa ll, vidal ap, cavallieri sa, vaisman m, et al. prevalence of thyroid diseases in patients with acromegaly: experience of a brazilian center. arq bras endocrinol metabol. 2013;57:685–690. 34. kauppinen-mäkelin r, sane t, reunanen a, välimäki mj, niskanen l, markkanen h, et al. a nationwide survey of mortality in acromegaly. j clin endocrinol metab. 2005;90:4081–4086. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201903 39j contemp med sci | vol. 1, no. 4, autumn 2015: 39–41 research objectives to describe a case of disseminated strongyloidiasis in an old age lady with non-specific clinical manifestations. methods and results a 70-year-old lady was admitted to merjan teaching hospital on 28th of july 2008 with 2 months history of generalised abdominal pain. presence of numerous s. stercoralis larvae during intestinal mucosal biopsies confirmed the diagnosis of hyperinfection syndrome in this patient. albendazole therapy did not cure the effects; and the patient died due to this severe illness. the most important clue to prevent such fatal consequences is high index of suspicion, early diagnosis and proper management. conclusion disseminated strongyloidiasis needs high index of suspicion especially in immunocompromised patient who presents with vague gastrointestinal manifestations. keywords disseminated strongyloidosis, gastrointestinal biopsy, old age disseminated strongyloidiasis in a 70-year-old lady monem makki alshok introduction s. stercoralis is distributed in tropical areas and other hot, humid regions like our country. globally between 30 and 100 million people are infected¹,² with disseminated strongyloidiasis, particularly in patients with unsuspected infection who are given glucocorticoids. humans acquire strongyloidiasis when filariform larvae in the fecally contaminated soil penetrate the skin or mucous membranes; so sanitation will not prevent infection.3,4 the larvae then travel through the bloodstream to the lungs, where they break into the alveolar spaces, ascend the bronchial tree, and are swallowed, thereby reach the small intestine. there the larvae mature into adult worms that penetrate the mucosa of the proximal small bowel. the larva repeats the migration that establishes ongoing internal reinfection. this autoinfection cycle allows strongyloidiasis to persist for decades.5 strongyloidiasis can thus persist for decades without further exposure of the host to exogenous infective larvae. in immunocompromised hosts, large numbers of invasive strongyloide larvae can disseminate widely and can be fatal. we describe a fatal disseminated intestinal strongyloidiasis in a 70-year-old lady.6,7 case report a 70-year-old lady was admitted to merjan teaching hospital on 28th of july 2008 with 2 months history of generalised abdominal pain described as heaviness sometime vague in nature. the pain does not radiate to other sites but on few occasions she feels backpain which is dull in nature also the pain was associated with mild distension and flatulence. during the last 2 weeks prior to her last admission to hospital, she developed anorexia, nausea and repeated vomiting and daily low grade fever, with generalised weakness and lassitude to extent that she was unable to do her usual daily activities due dizziness and inability to walk alone for a short distance. she also described a right-sided lower chest pain non-pleuritic dull in nature aggravated by movement, and her bowel motion passed only twice during the last 2 weeks. she is not a smoker, with negative past medical and surgical history. no history of allergy to drugs or abuse of medicine were noted. on physical examination she was very ill, tired lady , mild pallor, dry tongue, no jaundice with signs of generalised wasting, without clubbing of the fingers, but there were bilateral mild leg swelling particularly noticed on her feet associated with mild, non-pruritic evanescent erythema. cardiorespiratory examination was normal. there was a mild distension of her abdomen with no rigidity or guarding, but she had some tenderness on deep palpation of her epigastric and upper right side area of her abdomen, bowel sounds could be heard two times per minute. she was conscious and drowsy with no focal neurologic signs, no neck rigidity. temperature was 38°c, pulse rate was 118/min, and her bp was 95/60 mmhg. at the day of her admission, she was given iv fluid glucose saline, cefatoxime 500 mg qds, ranitidine injection, metoclopramide on need, paracetamol and one alpha hydroxyl cholecalciferol per oral. the following were the investigations: rbs 6.3 mmol/l, fbs 5.2 mmol/l, b. urea 7.2 mmol/l, s. creatinine 108 mmol/l, s. bilirubin 10 µmol/l, alt 13 u/l, ast 9u/l, na 124 mmol/l, k 4.2 mmol/l. urine examination showed no proteinuria, rbc 8–10/hpf, wbc 6–8/hpf, blood picture; pcv 34%, wbc 6 × 10 (78% neutrophil, 20% lymphocytes, 1% monocytes, 1% eosinophil), film normochromic normocytic erythrocytes normal leucocytes, platelets 320 × 10. esr 7 mm/hr, spo2 95%, ecg & echo studies were reported as normal. cxr and skull x-ray and u/s, plain x-ray of abdomen showed normal findings, ct scan of brain and abdomen also were normal. the next day she remained ill and was febrile – bp 90/50 mmhg, pr 99/min, temp 37.8°c. the patient was advised to continue iv normal saline, salt free albumin, stop oral fluids and made arrangement for ogd which showed moderate oesophagitis with reflux, pangastritis with signs of ulcerative duodenitis in the first and second part, multiple gastroduodenal biopsies taken on histopathological examination showed marked chronic superficial gastritis, no malignancy, duodenal mucosal tissues, moderate inflammatory cells infiltration with infestation of the mucosa by helminth strongyloides spp. we put the patient on ngt according to the surgeon’s advice and albendazole (400 mg twice daily for 3 days) was given, iv fluids and ppi were added but the patient remained very ill hypotensive and died on 3 august 2008 6 am. fig. 1a–f shows histopathological and some clinical signs. discussion strongyloides is one of the parasitic infections which has the ability to autoinfect the host without soil or intermediate host. issn 2413-0516 college of medicine, university of babylon, iraq. correspondence to monem makki alshok (email: dr_monem_alshok@yahoo.com). (submitted: 9 may 2015 – revised version received: 29 may 2015 – accepted: 5 june 2015 – published online: autumn 2015) 40 j contemp med sci | vol. 1, no. 4, autumn 2015: 39–41 disseminated strongyloidiasis research monem makki alshok it is often complicated by infections caused by gut flora that gain access to intestinal sites, presumably through ulcers induced by the filariform larvae,8,9 as we had noticed in our case report who presented with severe toxic manifestation. strongyloidiasis can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection. immunocompromised patients are at an increased risk of dissemination because impaired cellular and humoral immunity alter parasite proliferation, resulting in increased parasitic burden and possible dissemination to other organs. in this accelerated phase of autoinfection, enormous numbers of larvae are released into the venous circulation and disseminate throughout the body. as we had noticed in our patient not only having aids or being treated with corticosteroids and/or other immunosuppressive drugs, but possibly she is an elderly patient with disturbed immune function. the patient presented in this case report lived in an endemic area with warm climate area in babylon iraq and her infection with s. stercoralis was undiagnosed till her hospitalization. in some cases of hyperinfection and disseminated strongyloidiasis, eosinophilia is the most important laboratory finding in patients infected with s. stercoralis,10,11 but in our patient eosinophilia is not documented and this might be due to disturbed cellular immune function or possible underlying abuse of non-reported corticosteroid abuse. treatment with ivermectin resulted in remarkable clinical improvement and reversion of eosinophil count to normal.12 we treated our patient by using albendazole antihelmintic which is less effective than ivermectin therapy and it was not available at that time. in this reported case, the initial presentation was c/w features of malabsortion and possible septicemia and the ultimate were made after tissue diagnosis and in strongyloidiasis diagnosis it is important as the infection may persist for decades and immunosuppressed patients with chronic strongyloidiasis are at high risk of developing strongyloides hyperinfection syndrome, a fatal life-threatening complication whereby larval proliferation leads to systemic sepsis and multiorgan failure. if strongyloidiasis is diagnosed early, however, it is easily treatable with oral antihelmintic drugs. therefore, as clinical symptoms and endoscopic findings are non-specific, a high level of suspicion is required for diagnosis especially in patients who present with a vague clinical presentation.13–16 in conclusion, we should know that individuals with disturbed immunity, particularly with cell-mediated immunity defect, hematological malignancy, steroid usage, malnutrition, diabetes and organ transplantation, in addition to elderly subjects are predisposed to this infection. early diagnosis and timely therapy in case of hyperinfection syndrome can have a marked impact on the disease outcome. also endoscopic findings of git could be non specific, a high level of suspicion is required for diagnosis. fig. 1 a-f: histopathological and some clinical signs. a b e fdc 41j contemp med sci | vol. 1, no. 4, autumn 2015: 39–41 research disseminated strongyloidiasismonem makki alshok references 1. keiser pb, nutman tb. strongyloides stercoralis in the immunocompromised population. clin microbiol rev. 2004;17(1):208–17. 2 . pullan rl, brooker sj. the global limits and population at risk of soiltransmitted helminth infections in 2010. parasit vectors. 2012 apr 26;5:81. doi: 10.1186/1756-3305-5-81 pmid: 22537799 3. mejia r, nutman tb. screening, prevention, and treatment for hyperinfection syndrome and disseminated infections caused by strongyloides stercoralis. curr opin infect dis. 2012 aug;25(4):458–63. doi: 10.1097/qco. 0b013e3283551dbd pmid: 22691685 4. feldman m, friedman ls, brandt lj. s. stercoralis in sleisenger and fordtran’s gastrointestinal and liver disease: pathophysiology, diagnosis, management, 10th ed. philadelphia, pa: saunders, an imprint of elsevier inc.; 2015. 5. siddiqui aa, genta rm, berk sl. life cycle of strongyloides stercoralis. in: guerrant rl, walker dh, weller pf, editors. infectious diseases principles, practices and pathogens. philadelphia: churchill-livingstone elsevier; 2006. p. 1274. 6. ramanathan r, nutman t. strongyloides stercoralis infection in the immunocompromised host. curr infect dis rep. 2008 may;10(2):105–10. doi: http://dx.doi.org/10.1007/s11908-008-0019-6 pmid: 18462583 7. fauci as, braunwald e, kasper dl, hauser sl, longo dl, jameson jl, loscalzo j. harrison’s principles of internal medicine, 18th ed. mcgraw-hill companies, inc.; 2012. 8. keiser pb, nutman tb. strongyloides stercoralis in the immunocompromised population. clin microbiol rev. 2004 jan;17(1):208–17. doi: http://dx.doi. org/10.1128/cmr.17.1.208-217.2004 pmid: 14726461 9. kassalik m, mönkemüller k. strongyloides stercoralis hyperinfection syndrome and disseminated disease. gastroenterol hepatol (n y). 2011 nov;7(11):766–8. pmid: 22298975 10. sarangarajan r, ranganathan a, belmonte ah, tchertkoff v. strongyloides stercoralis infection in aids. aids patient care stds. 1997 dec;11(6):407–14. doi: http://dx.doi.org/10.1089/apc.1997.11.407 pmid: 11361861 11. vadlamudi rs, chi ds, krishnaswamy g. intestinal strongyloidiasis and hyperinfection syndrome. clin mol allergy 2006 may;4:8. pmid: 16734908 12. fardet l, généreau t, poirot jl, guidet b, kettaneh a, cabane j. severe strongyloidiasis in corticosteroid-treated patients: case series and literature review. j infect. 2007 jan;54(1):18–27. doi: http://dx.doi.org/10.1016/j. jinf.2006.01.016 pmid: 16533536 13. marathe a , date v. strongyloides stercoralis hyperinfection in an immunocompetent patient with extreme eosinophilia. j parasitol. 2008 jun;94(3):759–60. doi: 10.1645/ge-1392.1 pmid: 18605792 14. lemos lb, qu z, laucirica r, fred hl. hyperinfection syndrome in strongyloidiasis: report of two cases. 2003 apr;7(2):87–94. doi: http://dx.doi. org/10.1053/adpa.2003.50019 pmid: 12715333 15. daniel greaves, sian coggle, christopher pollard, sani h. aliyu, elinor m moore. strongyloidosis stercolaris infection. bmj. 2013;347:f4610 16. buonfrate d, requena-mendez a, angheben a, muñoz j, gobbi f, van den ende j, et al. severe strongyloidiasis: a systematic review of case reports. bmc infect dis. 2013 feb 8,13:78. doi: 10.1186/1471-2334-13-78 pmid: 23394259 1j contemp med sci | vol. 6, no. 1, january–february 2020: 1–7 review issn 2413-0516 introduction premature ovarian failure (pof), also known as premature ovarian insufficiency, is used to describe women under 40 years old with amenorrhea, hypergonadotropic hypogonadism, and infertility as a result of cessation of ovarian function.1, 2 it has been estimated that pof affects 1 in 100 women under 40 years, 1 in 1000 women under 30 years, and 1 in 10,000 of women under 20 years.1,3 there is a higher risk of early menopause in women with poor response to ovarian stimulation undergoing assisted reproductive technology (art). the results of a 10-year follow-up of women younger than 40 years of age with poor ovarian response in ivf cycles indicated that 11% of them developed early menopause and 3% of them developed pof.4 in the large majority of cases, ovarian failure is initiated after puberty.5 secondary amenorrhea associated with premature depletion of ovarian follicles or arrest of folliculogenesis occurs in women with post-pubertal pof. the clinical characteristics of patients with pof include flushes, heat intolerance, irritability, night sweats, sleep disturbance, palpitations, fatigue, anxiety, depression, hair coarseness, infertility, vaginal dryness, and decreased libido.3 furthermore, pof and a long-term delay in estrogen replacement therapy may result in early-onset osteoporosis. deficiency of sex hormones is considered a crucial risk factor for metabolic, cardiovascular, or neurological diseases.3 hormonally, pof is characterized by low levels of estrogens and inhibins and high levels of lh and fsh.3 estrogen/progestin preparations are effective to treat hormonal deficiency in patients with pof. it has been reported that almost 20% of women who consult for infertility have signs of premature ovarian ageing.6 although, restoration of fertility cannot presently be done if the diagnosis of pof is made after the full depletion of the follicular pool, early diagnosis by genetic testing may provide the opportunity for fertility preservation. at present, ovum donation is the only option available for treatment of infertility in women with absence of ovarian reserve.3 although in majority of cases, the underlying cause of pof is not identified, the known causes include: genetic aberrations; autoimmune ovarian damage; iatrogenic complications following surgery, radiation therapy, and chemotherapy; and environmental factors such as viral infections and toxins.7 furthermore, several studies have reported an association between mitochondrial diseases and pof in both animals and humans.8–11 oocyte mitochondrial depletion has been observed in patients with poor recovery rates of mature oocytes after ovarian hyperstimulation and in women with ovarian insufficiency.12,13 due to the maternal inheritance of pof along with the dependence of folliculogenesis upon the mitochondrial biogenesis and bioenergetics, it has been suggested that a generalized mitochondrial defect is likely involved in pof.14 the aim of this review was to illustrate the role of mitochondria in pof. a fuller understanding of the mitochondrial role in pof could contribute to the better management of women with pof in the future. mitochondria and ovarian function mitochondria are the site of cellular respiration/oxidative phosphorylation that produce the energy needed for all aspects of cellular function.15 the oxidative phosphorylation system of mitochondria consists of five multi-enzymatic complexes: complexes i–iv of the electron transport chain and complex v (adenosine triphosphate (atp) synthase).6 the role of mitochondria in premature ovarian failure: a review kajal khodamoradia,b, zahra khosravizadehb, zahra rashidic, ali talebid,e, gholamreza hassanzadehb adepartment of urology, university of miami, miller school of medicine, miami, fl, usa. bdepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. cfertility and infertility research center, health technology institute, kermanshah university of medical sciences, kermanshah, iran. dschool of medicine, shahroud university of medical sciences, shahroud, iran. eclinical research development unit, bahar hospital, shahroud university of medical sciences, shahroud, iran. correspondence to: gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir) (submitted: 11 august 2019 – revised version received: 04 october 2019 – accepted: 15 october 2019 – published online: 26 february 2020) abstract premature ovarian failure (pof) is used to describe women under 40 years old with amenorrhea, hypergonadotropic hypogonadism, and infertility as a result of cessation of ovarian function. it has been reported that almost 20% of women who consult for infertility have signs of premature ovarian ageing. the mitochondrial disorder is one of the critical agents in premature menopause and the occurrence of pof. due to the maternal inheritance of pof along with the dependence of folliculogenesis upon the mitochondrial biogenesis and bioenergetics, it has been suggested that a generalized mitochondrial defect is likely involved in pof. a fuller understanding of the mitochondrial role in pof could contribute to the better management of women with pof in the future. the aim of this review was to illustrate the role of mitochondria in pof. the oocyte mitochondrial dna (mtdna) content in women with diminished ovarian reserve is significantly lower than women with normal ovarian reserve. it has been evidenced that mitochondrial genetic disorders and mitochondrial oxidative stress are associated with pof. according to the maternal inheritance of mtdna, genetic testing should be performed to detect mtdna mutations involved in pof before starting treatment strategies. if these mutations are present, it could suggest that healthy mitochondrial transfer during assisted reproductive technology should be used to prevent the transmission of pof caused by mtdna mutation to the female offspring. future strategies aimed at treatment of pof-related infertility should take into account the significance of the oocyte mitochondrial role in the occurrence of this disorder. keywords premature ovarian failure, low ovarian reserve, mitochondria, infertility 2 review the role of mitochondria in premature ovarian failure k. khodamoradi et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 1–7 it has been reported that disruption of mitochondrial oxidative phosphorylation in mouse oocytes leads to abnormalities in the meiotic spindle and the potential reduction of embryonic preimplantation.16 mitochondria functions directly affect several aspects of the reproductive process including oocyte quality, fertilization process, and embryo development.17 the mitochondrial biogenesis and bioenergetics have an important role in maturation of the oocyte and embryonic development.18,19 moreover, the results of studies showed that the oocyte mitochondrial content could affect fertilizability of the oocyte.17 mature oocytes need large number of mitochondria and it has been shown that the mitochondrial dna (mtdna) content of an oocyte is strictly related to the probability of zygote development.17,20 much of the endogenous reactive oxygen species (ros), as a toxic by-product of oxidative phosphorylation, is generated in the mitochondria. mitochondrial dysfunctions lead to inhibition of oxidative phosphorylation and increased ros generation that can induce apoptosis.6 mitochondria are distributed and localized in regions of the ooplasm that require higher atp levels for an energy-consuming event, resulting in cytoplasmic and nuclear maturation of oocytes including germinal vesicle breakdown, and assembly and disassembly of microtubules for the formation of meiotic spindles.19,21,22 it seems that mitochondrial biogenesis plays a key role in determining the initial size of the ovarian follicular pool during embryonic life.23 age-associated deterioration of mitochondria negatively influences ovarian reserve, segregation of chromosomes, and embryo competence.24 it is well-known that female germline aging is accompanied by mitochondrial dysfunction correlated with reduced levels of oxidative phosphorylation and atp.25 ovarian ageing is correlated with reduced mtdna content in oocytes.6 older women or women with diminished ovarian reserve (dor) have significantly lower oocyte mtdna content compared to younger women or women with normal ovarian reserve.12,26–28 according to the findings of study by hamatani et al., the expression patterns of genes related to mitochondrial functions and oxidative stress show an age-associated alteration on mouse oocytes.29 in women with ovarian ageing, the impaired quality of the oocytes associated with insufficient mtdna content can result in premature biogenesis of mitochondria and embryonic development failure.6 research on mice have shown that mitochondria are involved in follicle pool exhaustion with ageing by affecting the initial size of the follicular pool and the rate of follicular atresia.6 in various mammalian species, including human, the apoptotic process is involved in germ cell elimination at all oogenesis stages and in depletion of ovarian reserve.30 this process in the mammalian oocytes involves both the intrinsic mitochondrial pathway as well as the extrinsic death receptor pathway.30,31 it has been reported that mitochondria and b-cell lymphoma 2 (bcl-2) family members are involved in ovarian apoptosis.32 taken together, these findings strongly support the role of mitochondria in pof. mitochondrial genetic disorders and incidence of pof several human diseases have been identified that are associated with mtdna mutations.33 the higher mutation rate of mtdna (approximately 25 times) compared to that of nuclear dna is due to the proximity of mtdna with the respiratory chain, the absence of histones, and efficient dna repair mechanisms.34 it is known that oocytes have the largest mitochondrial genome content of an organism.17 however, the oocyte mtdna content in women with dor is significantly lower than in women with normal ovarian reserve.6 it has been demonstrated that some cases of pof may be due to follicular atresia and eliminating primary oocytes with harmful mtdna mutations.35 the human mitochondrial genome contains genes coding for 13 polypeptides, 2 rrnas, and 22 trnas.36 deficiency in the mitochondrial trna (mt-trna) genes is known to be an important factor in clinical disease.37 ding and his colleagues assessed the association between mt-trna mutations and premature ovarian insufficiency. their results indicated that levels of fsh, lh, and ros production were increased in pof compared to control groups. in addition to estradiol, total testosterone levels and atp content showed a significant reduction in the pof group. moreover, a high incidence of mt-trna mutations was observed in these patients. the a4435g (trnamet), c3303t (trnaleu), g5821a (trnacys), t4363c (trnagln), and a15951g (trnath) mutations were recognized as pathogenic mutations related to premature ovarian insufficiency. however, their outcomes revealed that the mt-trna mutation itself was inadequate to cause the clinical disorders, and other risk factors may also contribute to pof development.38 in each human cell, mtdna are replicated by dna polymerase gamma (pol γ), which is a holoenzyme consisting of a 140-kda catalytic subunit (polg) and a dimeric form of 52-kda accessory subunit (polg2). the polg subunit has three main functions including dna polymerase, 3′-5′ exonuclease, and 5′ drp lyase activities.39, 40 after identifying the first disease associated with polg gene mutation, progressive external ophthalmoplegia (peo), association with other diseases and syndromes such as alpers syndrome, ataxia-neuropathy syndromes, charcot-marie-tooth disease, and idiopathic parkinsonism were discovered.11, 41, 42 the y955c mutation in polg is commonly associated with autosomal dominant peo. due to this mutation, many women with peo, present indicatives of pof.9, 11 luoma et al., in a clinical and molecular genetic study, investigated female patients with early menopause and used peo as a polg mutation marker. they showed that the co-segregation of premature menopause with polg mutations was significant and suggested polg-associated peo could become an important method in understanding the mechanisms of oxidative stress and mitochondrial dysfunction in premature menopause.9 in 2006, a study investigated three-generation pedigree with familial premature menopause associated with peo which had apparent maternal transmission of disease.11 the proband, her mother, and her maternal grandmother all presented with peo and subsequently developed pof at ages of 28, 35, and 32 years, respectively. the researcher found that peo disease can be distinguished via a dominant y955c mutation in the polg gene and concluded that the variation of the polg mutation can affect the age of women’s menopause.11 however, tong et al. screened the genomic dna of patients with pof for the 5 polg mutations and concluded that these polg mutations cannot be prevalent genetic etiologies for pof.43 in a small size study on the etiology of pof in young girls, a pediatric endocrinologist found that these patients had different etiologies compared to those cases seen in adults which included congenital disorder of glycosylation syndrome and mitochondrial 3 review the role of mitochondria in premature ovarian failurek. khodamoradi et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 1–7 diseases. however, in their study only one pof patient was diagnosed with a mitochondrial disorder. the authors suggested a larger size survey should be done for further clinical recommendations among these cases.44 chen et al. identified two different homozygous missense mutations with variants c.404g>a (p.r135q) and c.605g>a (p.r202h) in four young females from two independent families. these mutations were considered a novel genetic cause of pof in adolescents. this study showed that mrps22 deficiency, especially in the somatic cells of the ovary, had no effect on fertility but that its mutation in germ cells results in the absence of germ cells and infertility in a drosophila model. these findings collectively identify that mrps22 is required for reproduction and ovarian development and may contribute to ovarian dysfunction.45 cytochrome c oxidase (cox) is one of the main enzymes in the electron transport chain of mitochondria. a mutation in the gene encoding mitochondrial cox 1 (mt-co1) reduces cox activity and atp production, which is correlated with dysfunction of mitochondria, increase in apoptosis,46 and follicular depletion in early adulthood, and is followed by pof.47 with this hypothesis, zhen et al. screened the mitochondrial genome of patients with pof and healthy females. they observed a significant incidence of mt-co1 missense mutations in pof patients. also, there were significant increases in fsh, lh, and e2 levels, and a significant decrease in ovarian volume and atp levels compared to the control group. they proposed that mt-co1 gene mutations may be one of the causals in pof.48 further molecular studies are required to uncover other genetic mitochondrial disorders involved in pof. mitochondrial genetic mutations known to be involved in pof are shown in table 1. mitochondrial ros production and incidence of pof mitochondria are responsible for the generation of most of a cell’s energy and their dysfunction lead to increased levels of ros and low atp levels.49 accumulation of ros leads to oxidative stress, which can activate apoptosis in the majority of germ cells in the ovary.50–52 furthermore, pathologic accumulation levels of ros in ovaries may result in tissue inflammation including oophoritis, necrosis, and apoptosis.53 on the other hand, increased levels of ros induce lipid peroxidation and cause increased nuclear dna damage,54 and mitochondrial dna nucleotide changes.55 some of the female reproductive disorders such as endometriosis, polycystic ovary syndrome, pof, and infertility are due to oxidative stress.56,57 it has been well-known that pof is correlated with some mitochondrial disorders, and a correlation between idiopathic pof and increased oxidative stress has been described.58,59 propionic acidemia is an autosomal recessively inherited inborn error of propionate metabolism that is caused by a deficiency of the mitochondrial enzyme propionyl-coa table 1. summary of mitochondrial genetic mutations involved in the pof. authors species gene results luoma et al. (2004) human polg polg mutation is involved in the etiology of parkinsonism, peo, and premature menopause. most women with peo experienced early menopause before age 35. pagnamenta et al. (2006) human polg the y955c mutation in polg can result in mtdna depletion. polg mutations can cause pof and parkinsonism. tong et al. (2010) human polg these polg mutations including y955c and r943h (c2767g>a, c2828g>a, c2857c>t, c2864a>g, and c2869g>t) are not a prevalent genetic etiology for spontaneous 46, xx pof. brauner et al. (2015) human nr5a1, bmp15, gdf9, and nobox in two cases, an nr5a1 gene mutation was detected in the pediatric population with pof. zhen et al. (2015) human mt-co1 a high incidence of mt-co1 missense variants (mt-co1 c.790a>g, mt-co1c.802t>c, mtco1 c.1165a>g, and mt-co1 c.667g>t) was identified in pof patients that could lead to reduction of cox activity, decrease in atp level, and mitochondrial dysfunction. mt-co1 gene mutation may be causal in pof. chen et al. (2018) human mrps22 missense mutations in mrps22 [variants c.404g>a (p.r135q) and c.605g>a (p.r202h)] lead to autosomal recessive inheritance of pof. no changes were detected in mrna expression or protein levels of mrps22, in pof patientderived fibroblasts. also, defects in mitochondrial oxidative phosphorylation or rrna levels were not detected, which suggests a non-bioenergetic or tissue-specific mitochondrial defect. mouse heterozygous mrps22 knockout mice revealed no signs of abnormalities and were fertile. embryonic lethality was observed in heterozygous mrps22 knockout mice. drosophila the mrps22 deficiency in ovarian somatic cells had no effect on fertility, whereas mrps22 deficiency in germ cells resulted in absence of gametes and infertility, demonstrating a cellautonomous requirement for mrps22 in development of germ cell. ding et al. (2019) human mt-trna mitochondrial dysfunction, lower level of atp production, and high levels of ros were detected in pof patients carrying mt-trna mutations including the a4435g (trnamet), c3303t (trnaleu), g5821a (trnacys), t4363c (trnagln), and a15951g (trnath). these mt-trna mutations may have active roles in the pathogensis and progression of pof. pof, premature ovarian failure; polg, mitochondrial dna polymerase γ; peo, progressive external ophthalmoplegia; mtdna, mitochondrial dna; mt-co1, mitochondrial cytochrome c oxidase 1 gene; cox; cytochrome c oxidase; mrps22, mitochondrial ribosomal protein s22; mt-trna, mitochondrial trna. 4 review the role of mitochondria in premature ovarian failure k. khodamoradi et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 1–7 carboxylase. this enzyme plays an important role in catabolizing branched-chain amino acids, odd-chain fatty acids, cholesterol, and other metabolites.60 lam et al. reported a 45-year-old patient who had been diagnosed with propionic acidemia and experienced severe renal failure and pof.61 mitochondrial dysfunction and increased oxidative stress have been considered as possible mechanisms for these complications.61 as a study on mice oocytes has demonstrated, there is an age-associated alteration in the expression patterns of genes involved in mitochondrial functions and oxidative stress.29 there have been several studies on the role of mitochondrial ros in pof. some mitochondrial mutations increase the ros level and decrease atp production in germ cells, and consequently lead to disruption of normal oogenesis and accelerated germ cell apoptosis, ultimately leading to pof.62 as oocytes have greater number of mtdna copies than any cell in the body, pathological levels of ros cause mtdna damage, and mitochondria with nucleotide dna changes produce more free radicals.55 the results of several studies have shown that mutations in the atpase 6 gene, which maintains mitochondrial genome stability and integrity, are associated with excessive ros production and several disorders.63,64 venkatesh et al. found that the mutation of the mitochondrial atpase6 gene can lead to a higher ros level in ovarian cells. the high ros level can damage the mtdna and membrane as a result, and may cause premature cessation of ovarian function by interruption of cell growth, expansion of atresia, and eventually germ cell apoptosis.59 moreover, it has been shown that in patients with ovarian ageing and pof, the ros level in granulosa cells is extremely high.65 according to these findings, mitochondrial ros production and oxidative stress may influence normal oogenesis and ovarian reserve through activation of the apoptotic process, increased follicular atresia, induction of ovarian inflammation, and development of mtdna disorders that can lead to pof. approaches to fertility preservation in patients with pof by influencing the function of mitochondria due to the cumulative adverse effects of pof over time, making a timely diagnosis and initiating appropriate strategies are important for managing the symptoms, supporting patients’ emotional needs, and reducing risk.3 hormone replacement therapy is the most popular treatment for pof-related symptoms, but there are some serious side-effects for this treatment, including increased risks for breast and endometrial cancers.66 other treatments for pof involve influencing the function of mitochondria. coenzyme q10 is one of these treatments studied by ben-meir and their colleagues in aged mice. the results of this study showed that coenzyme q10 supplementation could enhance the activity of mitochondria and amending the mitochondrial gene expression. consequently, the coenzyme q10 supplementation can prevent depletion of ovarian reserve and pof.25 antioxidants are another treatment that can postpone pof. melatonin is known as a highly effective antioxidant and potent ros scavenger67 that protects cells against oxidative stress and diminishes the harmful effects of ros.68 recently, it has been reported that melatonin may be beneficial for reducing and preventing pof in patients who receive chemotherapy treatment.69 melatonin improves pof by reducing oxidative stress damage that was mediated by the sirt1 signaling pathway.70 furthermore, it has been demonstrated that melatonin prevents primordial follicle depletion in cisplatin-treated mice through suppression of the phosphorylation of pten/ akt/foxo3a pathway members.71 although, the specific mechanisms underlying the actions of melatonin protection against ovarian follicle death are not yet clearly understood, it is well-known that its antioxidant effect can maintain the follicular morphology and growth, and can affect oxidative stress response by influencing ros and glutathione production, mitochondrial activity, and apoptosis in follicular cells.67,72 studies have shown that the use of herbal medicine is especially important in pof.73–75 cistanches herba is a parasitic plant that is used in traditional chinese medicine (tcm)76 for treatment of different diseases including female infertility by increasing sex hormone levels, though the exact regulating mechanisms are unknown.77 cistanches herba can prevent cisplatin-induced apoptosis which causes pof in mice. cistanches herba upregulated mitofusin-2 (a mitochondria dynamin-like gtpase) expression and altered mitochondrial membrane structure via interaction with bcl-2/bax proteins.78 dendrobium officinal polysaccharides (dop), which are one of the main active components of dendrobium officinal, another tcm, has recently been found to show good efficacy in producing anti-oxidative and anti-inflammatory effects75,79 and may have the potential to the treatment of pof. wu et al. found that dop improves the function of mitochondria, thus increasing the body’s antioxidant capacity and protecting the body from pof effects on mice. their results also have shown that dop could decrease the symptoms of pof, by increasing the body’s antioxidant capacity, balancing inflammation, and improving mitochondrial function. the potential mechanism signaling pathway may work through regulating the nuclear factor-κb and p53/bcl-2.80 zuogui pills (zgp) are a component in tcm used in the treatment of pof, increasing proliferation and decreasing apoptosis in the ovarian cells.81 the therapeutic effects of zgp on the treatment of pof due to chemotherapy were investigated by peng et al. they illustrated that the number of follicles, ovarian ultrastructures, and the estrous cycle notably ameliorated in rats with pof. furthermore, zgp increased the fsh levels and decreased estradiol levels in serum. moreover, it declines bax, cytochrome c (cyt-c) on both gene and protein levels and elevates bcl-2 gene expression and protein levels. they proposed that zpg can mediate pof through the balance of bax/bcl-2 in ovaries and suppression of mitochondria-dependent apoptosis.75 women with early identified ovarian ageing can have good reproductive potential, and spontaneous pregnancy remains a choice for women with adequate ovarian reserve who are ready to have a child.4 there are a number of treatment options available for women who are infertile due to pof which include ovarian cortical tissue cryopreservation, oocyte, or embryo cryopreservation; oocyte, or embryo donation; and adoption. these treatment options can recommended before or during ovarian failure.82 since mitochondria act as a vehicle for mtdna transmission to subsequent generations, evaluation of mitochondrial disorders prior to any protocol of oocyte reconstruction would allow selection of healthy fertilizable oocytes.83 although, kasteren et al. analyzed the incidence of familial cases of pof and concluded that 5 review the role of mitochondria in premature ovarian failurek. khodamoradi et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 1–7 in these families the risk of other females developing pof will depend on the modes of inheritance and transmission, and further demonstrated that mitochondrial inheritance is not one common mode among their studied families.84 however, different research teams found other conditions that can be investigated for finding occurrence of pof related to mitochondrial genetic diseases, and showed a correlation between mitochondrial diseases and pof.9,48 it has been reported that mitochondrial transfer into an oocyte can lead to prevention of apoptosis in this cell and promotion of embryonic development.85,86 mitochondrial supplementation of oocytes is a strategy for overcoming mtdna deficiency and improving developmental competence. furthermore, supplementation of oocytes exhibiting mtdna deficiency with autologous mitochondria can improve the outcome of in vitro fertilization.83 conclusions the mitochondrial disorder is one of the critical agents in premature menopause and the occurrence of pof. mitochondrial ros production and mitochondrial genetic disorders have been reported as causes of pof. due to the cumulative adverse effects of pof over time, making a timely diagnosis and initiating appropriate strategies are important for managing symptoms, supporting patients’ emotional needs , and reducing risk. the early diagnosis of pof can provide the opportunity to make good reproductive decisions such as having kids earlier and freezing oocytes or embryos. given the maternal inheritance of mtdna, genetic testing should be performed to detect mtdna mutations involved in pof before starting treatment strategies. if these mutations are present, healthy mitochondrial transfer during art should be used to prevent the transmission of pof caused by the mtdna mutation to the female offspring. a fuller understanding of the mitochondrial role in pof could contribute to the better management of women with pof in the future. future strategies aimed at treatment of pof-related infertility should take into account the significance of the oocyte mitochondrial role in the occurrence of this disorder. conflict of interest all authors declare that there is no known conflict of interest regarding this publication. references 1. jankowska k. premature ovarian failure. przeglad menopauzalny= menopause rev.. 2017;16(2):51. 2. conway g, hettiarachchi s, murray a, jacobs p. fragile x premutations in familial premature ovarian failure. lancet. 1995;346(8970):309-10. 3. beck-peccoz p, persani l. premature ovarian failure. orphanet j. rare dis. 2006;1(1):9. 4. maclaran k, nikolaou d. early ovarian ageing. obstetr. gynaecol. 2019;21(2):107-16. 5. santoro n. mechanisms of premature ovarian failure. 2003. 6. may-panloup p, boucret l, chao de la barca j-m, desquiret-dumas v, ferrel’hotellier v, moriniere c, et al. ovarian ageing: the role of mitochondria in oocytes and follicles. human reprod. update. 2016;22(6):725-43. 7. goswami d, conway gs. premature ovarian failure. human reprod. update. 2005;11(4):391-410. 8. trifunovic a, wredenberg a, falkenberg m, spelbrink jn, rovio at, bruder ce, et al. premature ageing in mice expressing defective mitochondrial dna polymerase. nature. 2004;429(6990):417. 9. luoma p, melberg a, rinne jo, kaukonen ja, nupponen nn, chalmers rm, et al. parkinsonism, premature menopause, and mitochondrial dna polymerase γ mutations: clinical and molecular genetic study. lancet. 2004;364(9437):875-82. 10. kujoth gc, hiona a, pugh t, someya s, panzer k, wohlgemuth s, et al. mitochondrial dna mutations, oxidative stress, and apoptosis in mammalian aging. science. 2005;309(5733):481-4. 11. pagnamenta at, taanman j-w, wilson cj, anderson ne, marotta r, duncan aj, et al. dominant inheritance of premature ovarian failure associated with mutant mitochondrial dna polymerase gamma. human reprod. 2006;21(10):2467-73. 12. may-panloup p, chretien m, jacques c, vasseur c, malthiery y, reynier p. low oocyte mitochondrial dna content in ovarian insufficiency. human reprod. 2005;20(3):593-7. 13. santos ta, el shourbagy s, john jcs. mitochondrial content reflects oocyte variability and fertilization outcome. fertil. steril. 2006;85(3):584-91. 14. bonomi m, somigliana e, cacciatore c, busnelli m, rossetti r, bonetti s, et al. blood cell mitochondrial dna content and premature ovarian aging. plos one. 2012;7(8):e42423. 15. zheng h, yu w-m, shen j, kang s, hambardzumyan d, li jy, et al. mitochondrial oxidation of the carbohydrate fuel is required for neural precursor/stem cell function and postnatal cerebellar development. sci. adv. 2018;4(10):eaat2681. 16. zhang x, wu xq, lu s, guo yl, ma x. deficit of mitochondria-derived atp during oxidative stress impairs mouse mii oocyte spindles. cell res. 2006;16(10):841. 17. may‐panloup p, chretien mf, malthiery y, reynier p. mitochondrial dna in the oocyte and the developing embryo. curr. topics dev. biol. 2007;77:5183. 18. van blerkom j, davis pw, lee j. fertilization and early embryolgoy: atp content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. human reprod. 1995;10(2):415-24. 19. dumollard r, duchen m, carroll j. the role of mitochondrial function in the oocyte and embryo. curr. topics dev. biol. 2007;77:21-49. 20. wai t, ao a, zhang x, cyr d, dufort d, shoubridge ea. the role of mitochondrial dna copy number in mammalian fertility. biol. reprod. 2010;83(1):52-62. 21. brevini ta, vassena r, francisci c, gandolfi f. role of adenosine triphosphate, active mitochondria, and microtubules in the acquisition of developmental competence of parthenogenetically activated pig oocytes. biol. reprod. 2005;72(5):1218-23. 22. yu y, dumollard r, rossbach a, lai fa, swann k. redistribution of mitochondria leads to bursts of atp production during spontaneous mouse oocyte maturation. j. cell. physiol. 2010;224(3):672-80. 23. aiken ce, tarry-adkins jl, penfold nc, dearden l, ozanne se. decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet. faseb j. 2015;30(4):1548-56. 24. meldrum dr, casper rf, diez-juan a, simon c, domar ad, frydman r. aging and the environment affect gamete and embryo potential: can we intervene? fertil. steril. 2016;105(3):548-59. 25. ben‐meir a, burstein e, borrego‐alvarez a, chong j, wong e, yavorska t, et al. coenzyme q10 restores oocyte mitochondrial function and fertility during reproductive aging. aging cell. 2015;14(5):887-95. 26. chan c, liu v, lau e, yeung w, ng e, ho p. mitochondrial dna content and 4977 bp deletion in unfertilized oocytes. mol. human reprod. 2005;11(12):843-6. 27. duran he, simsek-duran f, oehninger sc, jones jr hw, castora fj. the association of reproductive senescence with mitochondrial quantity, function, and dna integrity in human oocytes at different stages of maturation. fertil. steril. 2011;96(2):384-8. 28. murakoshi y, sueoka k, takahashi k, sato s, sakurai t, tajima h, et al. embryo developmental capability and pregnancy outcome are related to the mitochondrial dna copy number and ooplasmic volume. j. assist. reprod. genet. 2013;30(10):1367-75. 29. hamatani t, falco g, carter mg, akutsu h, stagg ca, sharov aa, et al. ageassociated alteration of gene expression patterns in mouse oocytes. human mol. genet. 2004;13(19):2263-78. 6 review the role of mitochondria in premature ovarian failure k. khodamoradi et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 1–7 30. tiwari m, prasad s, tripathi a, pandey an, ali i, singh ak, et al. apoptosis in mammalian oocytes: a review. apoptosis. 2015;20(8):1019-25. 31. aitken rj, findlay jk, hutt kj, kerr jb. apoptosis in the germ line. reproduction (cambridge, england). 2011;141(2):139-50. 32. hussein mr, haemel ak, wood gs. apoptosis and melanoma: molecular mechanisms. j. pathol. j. pathol. soc. great br. ireland. 2003;199(3):275-88. 33. lu j, sharma lk, bai y. implications of mitochondrial dna mutations and mitochondrial dysfunction in tumorigenesis. cell res. 2009;19(7):802. 34. lynch m, koskella b, schaack s. mutation pressure and the evolution of organelle genomic architecture. science. 2006;311(5768):1727-30. 35. krakauer dc, mira a. mitochondria and germ-cell death. nature. 1999;400(6740):125. 36. ding y, xia b, yu j, leng j, huang j. mitochondrial dna mutations and essential hypertension. int. j. mol. med. 2013;32(4):768-74. 37. yarham jw, elson jl, blakely el, mcfarland r, taylor rw. mitochondrial trna mutations and disease. wiley interdisc. rev. rna. 2010;1(2):304-24. 38. ding y, xia b-h, zhuo g-c, zhang c-j, leng j-h. premature ovarian insufficiency may be associated with the mutations in mitochondrial trna genes. endocr. j. 2019;66(1):81-8. 39. kaguni ls. dna polymerase γ, the mitochondrial replicase. annu. rev. biochem. 2004;73(1):293-320. 40. chan ss, copeland wc. dna polymerase gamma and mitochondrial disease: understanding the consequence of polg mutations. biochim. biophy. acta (bba)-bioenerg. 2009;1787(5):312-9. 41. van goethem g, dermaut b, löfgren a, martin j-j, van broeckhoven c. mutation of polg is associated with progressive external ophthalmoplegia characterized by mtdna deletions. nat. genet. 2001;28(3):211. 42. stumpf jd, saneto rp, copeland wc. clinical and molecular features of polg-related mitochondrial disease. cold spring harb. perspect. biol. 2013;5(4):a011395. 43. tong z-b, sullivan sd, lawless lm, vanderhoof v, zachman k, nelson lm. five mutations of mitochondrial dna polymerase-gamma (polg) are not a prevalent etiology for spontaneous 46, xx primary ovarian insufficiency. fertil. steril. 2010;94(7):2932-4. 44. brauner r, pierrepont s, bignon-topalovic j, mcelreavey k, bashamboo a. etiology of primary ovarian insufficiency in a series young girls presenting at a pediatric endocrinology center. eur. j. pediatr. 2015;174(6):767-73. 45. chen a, tiosano d, guran t, baris hn, bayram y, mory a, et al. mutations in the mitochondrial ribosomal protein mrps22 lead to primary ovarian insufficiency. human mol. genet. 2018;27(11):1913-26. 46. rzheshevsky a. decrease in atp biosynthesis and dysfunction of biological membranes. two possible key mechanisms of phenoptosis. biochemistry (moscow). 2014;79(10):1056-68. 47. thouas ga, trounson ao, wolvetang ej, jones gm. mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro. biol. reprod. 2004;71(6):1936-42. 48. zhen x, wu b, wang j, lu c, gao h, qiao j. increased incidence of mitochondrial cytochrome c oxidase 1 gene mutations in patients with primary ovarian insufficiency. plos one. 2015;10(7):e0132610. 49. wang t, zhang m, jiang z, seli e. mitochondrial dysfunction and ovarian aging. am. j. reprod. immunol. 2017;77(5):e12651. 50. prasad s, tiwari m, pandey an, shrivastav tg, chaube sk. impact of stress on oocyte quality and reproductive outcome. j. biomed. sci. 2016;23(1):36. 51. khodamoradi k, amini-khoei h, khosravizadeh z, hosseini sr, dehpour ar, hassanzadeh g. oxidative stress, inflammatory reactions and apoptosis mediated the negative effect of chronic stress induced by maternal separation on the reproductive system in male mice. reprod. biol. 2019;19(4):340-8. 52. khodamoradi k, amini-khoei h, khosravizadeh z, hosseini sr, dehpour ar, hassanzadeh g. maternal separation can affect the reproductive system by inflammasome activation in female mice. j. contemp. med. sci. 2019;5(3). 53. behrman hr, kodaman ph, preston sl, gao s. oxidative stress and the ovary. j. soc. gynecol. investig. 2001;8(1_suppl):s40-s2. 54. amidi f, rashidi z, khosravizadeh z, khodamoradi k, talebi a, navid s, et al. antioxidant effects of quercetin in freeze-thawing process of mouse spermatogonial stem cells. asian pac. j. reprod. 2019;8(1):7. 55. john jcs, cooke id, barratt cl. mitochondrial mutations and male infertility. nat. med. 1997;3(2):124-5. 56. lu j, wang z, cao j, chen y, dong y. a novel and compact review on the role of oxidative stress in female reproduction. reprod. biol. endocrinol. 2018;16(1):80. 57. bhardwaj jk, mittal m, saraf p, kumari p. pesticides induced oxidative stress and female infertility: a review. toxin rev. 2018:1-13. 58. simpson jl. genetic and phenotypic heterogeneity in ovarian failure: overview of selected candidate genes. ann. ny acad. sci. 2008;1135(1):146-54. 59. venkatesh s, kumar m, sharma a, kriplani a, ammini a, talwar p, et al. oxidative stress and atpase6 mutation is associated with primary ovarian insufficiency. arch. gynecol. obstet. 2010;282(3):313-8. 60. chalmers r, lawson a. disorders of propionate and methylmalonate metabolism. organic acids in man: springer; 1982. p. 296-331. 61. lam c, desviat lr, perez-cerdá c, ugarte m, barshop ba, cederbaum s. 45-year-old female with propionic acidemia, renal failure, and premature ovarian failure; late complications of propionic acidemia? mol. genet. metab. 2011;103(4):338-40. 62. kumar m, pathak d, kriplani a, ammini a, talwar p, dada r. nucleotide variations in mitochondrial dna and supra-physiological ros levels in cytogenetically normal cases of premature ovarian insufficiency. arch. gynecol. obstet. 2010;282(6):695-705. 63. baracca a, sgarbi g, mattiazzi m, casalena g, pagnotta e, valentino ml, et al. biochemical phenotypes associated with the mitochondrial atp6 gene mutations at nt8993. biochim. biophys. acta (bba)-bioenerg. 2007;1767(7):913-9. 64. venkatesh s, deecaraman m, kumar r, shamsi m, dada r. role of reactive oxygen species in the pathogenesis of mitochondrial dna (mtdna) mutations in male infertility. ind. j. med. res. 2009;129(2). 65. ernst eh, lykke-hartmann k. transcripts encoding free radical scavengers in human granulosa cells from primordial and primary ovarian follicles. j. assist. reprod. genet. 2018;35(10):1787-98. 66. la vecchia c. hormone replacement therapy, breast and endometrial cancer. 1996. 67. gao c, han hb, tian xz, tan dx, wang l, zhou gb, et al. melatonin promotes embryonic development and reduces reactive oxygen species in vitrified mouse 2‐cell embryos. j. pineal res. 2012;52(3):305-11. 68. barberino rs, menezes vg, ribeiro ae, palheta jr rc, jiang x, smitz je, et al. melatonin protects against cisplatin-induced ovarian damage in mice via the mt1 receptor and antioxidant activity. biol. reprod. 2017;96(6):1244-55. 69. jang h, hong k, choi y. melatonin and fertoprotective adjuvants: prevention against premature ovarian failure during chemotherapy. int. jo. mol. sci. 2017;18(6):1221. 70. ma m, chen x-y, li b, li x-t. melatonin protects premature ovarian insufficiency induced by tripterygium glycosides: role of sirt1. am. j. transl. res. 2017;9(4):1580. 71. jang h, lee oh, lee y, yoon h, chang em, park m, et al. melatonin prevents cisplatin‐induced primordial follicle loss via suppression of pten/akt/foxo 3a pathway activation in the mouse ovary. j. pineal res. 2016;60(3):336-47. 72. tamura h, takasaki a, taketani t, tanabe m, kizuka f, lee l, et al. the role of melatonin as an antioxidant in the follicle. j. ovar. res. 2012;5(1):5. 73. ding q, shang f-f. meta-analysis of premature ovarian failure treated combined chinese and western medicines. j. trad. chin. med. univ. hunan. 2011;9. 74. kou m-j, ding x-f, chen j-x, liu y, liu y-y. traditional chinese medicine combined with hormone therapy to treat premature ovarian failure: a meta-analysis of randomized controlled trials. afr. j. trad. complement. altern. med. 2016;13(5):160-9. 75. peng h, zeng l, zhu l, luo s, xu l, zeng l, et al. zuogui pills inhibit mitochondria-dependent apoptosis of follicles in a rat model of premature ovarian failure. j. ethnopharmacol. 2019;238:111855. 76. li z, lin h, gu l, gao j, tzeng c-m. herba cistanche (rou cong-rong): one of the best pharmaceutical gifts of traditional chinese medicine. front. pharmacol. 2016;7:41. 77. li x, yang s, lv x, sun h, weng j, liang y, et al. the mechanism of mesna in protection from cisplatin-induced ovarian damage in female rats. j. gynecol. oncol. 2013;24(2):177-85. 78. pan p, wang y, leng x, deng j, wang c. protective effects of cistanches herba aqueous extract on cisplatin-induced premature ovarian failure in mice. afr. j. trad. complement. altern. med. 2017;14(6):90-101. 79. luo d, qu c, lin g, zhang z, xie j, chen h, et al. character and laxative activity of polysaccharides isolated from dendrobium officinale. j. funct. foods. 2017;34:106-17. 80. wu y-y, liang c-y, liu t-t, liang y-m, li s-j, lu y-y, et al. protective roles and mechanisms of polysaccharides from dendrobium officinal on natural aging-induced premature ovarian failure. biomed. pharmacother. 2018;101:953-60. 81. yao z, wan q, lu h, liu x. effects of zuogui pill, yougui pill and relative compositions on differentiation towards germ cells of mouse embryonic stem cell 1b10. zhongguo zhong yao za zhi= zhongguo zhongyao zazhi= china j. chin. mater. med. 2015;40(3):495-500. 7 review the role of mitochondria in premature ovarian failurek. khodamoradi et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 1–7 82. blumenfeld z, hoek, skillern, goswami, krauss, chen, et al. fertility treatment in women with premature ovarian failure. expert rev. obstet. gynecol. 2011;6(3):321-30. 83. el shourbagy sh, spikings ec, freitas m, st john jc. mitochondria directly influence fertilisation outcome in the pig. reproduction. 2006;131(2):23345. 84. van kasteren y, hundscheid r, smits a, cremers f, van zonneveld p, braat d. familial idiopathic premature ovarian failure: an overrated and underestimated genetic disease? human reprod. 1999;14(10):2455-9. 85. perez gi, trbovich am, gosden rg, tilly jl. reproductive biology: mitochondria and the death of oocytes. nature. 2000;403(6769):500. 86. cagnone gl, tsai t-s, makanji y, matthews p, gould j, bonkowski ms, et al. restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial dna deficiency. scient. rep. 2016;6:23229. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.02202001 254 j contemp med sci | vol. 5, no. 5, september-october 2019: 254–257 premalignant changes associated chronic gastritis in karbala province: multi institutional study rasha abd alraouf neama alsafi, mohammed f. alqanbar, and wafaa redha mohammed al-sabbagh department of pathology, college of medicine, karbala university, karbala, iraq. correspondence to rasha abd alraouf neama alsafi (email: rasha.alsafi78@gmail.com). (submitted: 26 april 2019 – revised version received: 18 may 2019 – accepted: 13 july 2019 – published online: 26 october 2019) introduction the incidence rate of gastric cancer has been decreased gradually and continuously over the last years; however it ranks as the fourth most prevalent cancer and a significant leader to cancer related death over the world.1,2 cancer of the stomach from histopathological aspect is of two types, the intestinal and the diffuse types. the occurrence of the intestinal type may be preceded by a series of precancerous pathological lesions and changes such as chronic atrophic gastritis, intestinal metaplasia (im), and dysplasia.3 however, the genesis of the diffuse type is not clearly recognized.4 helicobacter pylori infection is accompanied with a sequential events such as severe gastritis, chronic atrophic gastritis, im and gastric cancer.5,6 intensely atrophied gastric mucosa that associated with intestinal metaplastic changes may be attributed to infection with certain types of high risk h. pylori.7,8 it was noticed that the infection with h. pylori during early childhood resulted in premalignant state of pan-gastritis in adulthood. so, the infection with h. pylori earlier during life is considered as an important risk factor that increase the opportunity for the evolution of gastric cancer.9,10 other risk factors of im are high salt consumption, cigarette smoking, alcohol drinking, and chronic bile reflux.11 the prevalence of h. pylori infection has been recorded from multiple middle eastern countries such as iraq, iran, turkey, libya, egypt, bahrain, oman, saudi arabia, and the united arab emirates. it has been found that the rate of infection with h. pylori are nearly similar in these countries but still there is little variations in that the prevalence of infection reach its peak in iran while it is nearly uncommon and rare in iraq and egypt.12 according to a study conducted at 2009 in iraq stated that the “the mild pathology and antral-predominant gastritis may help to explain the low cancer rate in iraq”.13 the aim of this study was to investigate the prevalence of gastric premalignant changes like gastric atrophy, intestinal metaplasia and dysplastic changes that associated with chronic gastritis and their relation with h. pylori infection in different age groups in patients underwent esophagogastroduodenoscopy in three health institutions in karbala province. materials and methods the data was collected from patients attended the endoscopy units during a period from january to september 2018 in three medical centers in holy karbala, iraq, al hussein teaching hospital, imam zain al abedin hospital and imam al hujjah hospital. it includes 500 patients who were subjected to esophagogastroduodenoscopy (egd) in this period from whom endoscopic mucosal biopsies were taken. each biopsy contained at least two mucosal pieces that submitted to a conventional histological processing and stained with hematoxylin–eosin stain to assess the presence of gastric mucosal changes. these biopsies were stained by giemsa to detect h. pylori organisms. the collected data include many parameters such as age, sex, and histopathological findings (gastritis, h. pylori infection, atrophic changes, intestinal metaplasia and dysplastic changes). informed written consent was taken from each patient before the inclusion. inclusion criteria all patients who attended hospitals with upper git symptoms and underwent egd with gastric mucosal sampling during the period of the study. objectives the aim of this study was to investigate the prevalence of gastric premalignant changes as gastric atrophy, intestinal metaplasia and dysplastic changes in patients underwent esophagogastroduodenoscopy in three health institutions in karbala province. methods the data was collected from patients attended the endoscopy units during a period from january to september 2018 in three medical centers in holy karbala, iraq, al hussein teaching hospital, imam zain al abedin hospital and imam al hujjah hospital. it includes 215 males and 285 females who were subjected to esophagogastroduodenoscopy (egd) from whom mucosal biopsies were taken. these biopsies were stained by giemsa to detect helicobacter pylori organisms. results the mean age of patients was (40.9 ± 15 years sd). helicobacter pylori infection was demonstrated in (71%) patients, (19.4%) were presented with glandular atrophy, (7.4%) out of 500 patients had intestinal metaplasia and only six (1.2%) patients had dysplastic changes. there was a significant statistical relation between gastric glandular atrophy and age (p-value = 0.03). there was a significant statistical relation between glandular atrophy and infection with h. pylori (p-value = 0.001). there was a significant negative relation between h. pylori infection and the existence of intestinal metaplasia (p-value = 0.0001). this study show a significant statistical relationship between the existence of intestinal metaplasia and the occurrence of dysplastic changes (p-value = 0.001). conclusion the current study show a significant relation between aging and the existence of gastric glandular atrophy. there was a significant relation between each of gastric glandular atrophy and intestinal metaplastic changes with the h. pylori infection. there was a significant relation between gastric intestinal metaplasia and dysplastic changes. keywords premalignant changes, age, h. pylori, gastric atrophy, intestinal metaplasia, dysplasia issn 2413-0516 original 255j contemp med sci | vol. 5, no. 5, september-october 2019: 254–257 r.a.a.n. alsafi et al. premalignant changes associated chronic gastritis in karbala province exclusion criteria this include patients diagnosed to have gastric cancer, those patients undergoing gastric surgery, patients with upper gastrointestinal bleeding and patients with lacking data. statistical analysis chi-square test was used to measure the association between different parameters and p-value of <0.05 was considered significant. results five hundred patients were subjected to egd, enrolled in this study during a period from january to september 2018. the mean age of patients was (40.9 ± 15 years standard deviation). there were 215 (43%) males and 285 (57%) females. all patients have gastritis [500 (100%)]. h. pylori infection was demonstrated in 355 (71%) patients. out of 500 patients, 97 (19.4%) were presented with glandular atrophy, 37 (7.4%) out of total number of patients had intestinal metaplasia, while only six (1.2%) patients had dysplastic changes distributed equally between males and females. the results of this study show that the highest frequencies of patients infected with h. pylori were distributed between 20 and 59 years of age; however, this relation was not significant statistically (p-value = 0.06). there was a significant statistical relation between gastric glandular atrophy and age in that the number of cases with atrophied gastric glands was growing with increasing the age from 20 to 60 years forming about 76 (78.3%) cases (p-value = 0.03). concerning intestinal metaplasia, the results revealed a mounting rate of metaplastic changes with the age and the relationship was statistically near the significant level (p-value = 0.052). all cases with dysplasia were six (1.2%) distributed unevenly through age groups from 30 to 69 years, the relationship of these changes with age was statistically insignificant (p-value = 0.7). the distribution and the relationship of the histopathological findings (h. pylori infection, gastric glandular atrophy, intestinal metaplasia and dysplastic changes) with age was demonstrated in table 1. of 355 patients infected with h. pylori, there were 88 (24.8%) cases presented with glandular atrophy in comparison to only 9/145 (6.2%) of non-infected patients who presented with atrophied gastric glands and this figure reach a significant level statistically (p-value = 0.001). additionally there is a statistical significant negative relationship between intestinal metaplastic changes and the existence of h. pylori infection in that 17 patients (4.8%) with h. pylori infection were presented with intestinal metaplasia in comparison to 20 (13.8%) of non-infected patients presented with intestinal metaplasia on examination as shown in table 2. out of total 37 cases which show intestinal metaplasia there were three (8.1%) cases which were diagnosed to have dysplastic changes while three (0.7%) cases out of the remaining negative cases show dysplastic changes. this result give a significant statistical relationship between the existence of intestinal metaplasia and the occurrence of dysplastic changes (p-value = 0.001) as shown in table 3. discussion in the model of gastric carcinogenesis, h. pylori has a substantial effect in the development of chronic active gastritis. table 1. the histopathological findings of 500 patients underwent esophagogastroduodenoscopy and their relations with age histopathological findings (frequency) frequencies (%) age groups (years) p-value 10–19 20–29 30–39 40–49 50–59 60–69 70–79 ≥80 h. pylori positive 355 (71)* 29 75 87 73 45 33 13 0 (0.06) negative 145 (29)* 5 24 35 34 28 11 6 2 glandular atrophy positive 97 (19.4)* 9 15 19 20 22 5 7 0 (0.03) negative 403 (80.6)* 25 84 103 87 51 39 12 2 intestinal-metaplasia positive 37 (7.4)* 0 4 10 7 9 5 1 1 (0.052) negative 463 (92.6)* 34 95 112 100 64 39 18 1 dysplastic changes positive 6 (1.2)* 0 0 2 1 2 1 0 0 (0.7) negative 494 (98.8)* 34 99 120 106 71 43 19 2 *percentage from total sample size (500). table 2. the relation of h. pylori infection with glandular atrophy and intestinal metaplasia h. pylori infection atrophy (p-value = 0.001) intestinal metaplasia (p-value = 0.0001) total positive (%) negative (%) positive (%) negative (%) positive 88 (24.8)* 267 (75.2)* 17 (4.8)* 338 (95.2) 355 negative 9 (6.2)** 136 (93.8)** 20 (13.8)** 125 (86.2) 145 *percentage out of total positive cases infected with h. pylori. **percentage out of total cases not infected with h. pylori. original 256 j contemp med sci | vol. 5, no. 5, september-october 2019: 254–257 premalignant changes associated chronic gastritis in karbala province r.a.a.n. alsafi et al. chronic h. pylori infection may pave the way to pass through sequential stages of atrophic gastritis, im, and dysplasia to end with gastric adenocarcinoma.14 in this study (according to table 1), h. pylori infection was demonstrated in 355 (71%) patients and this come in congruent with previous study which give a similar figures.15 other studies reported in the prevalence in north europe and north america was <40%, while it was over 70% in east asia, africa, and middle east region.16,17 which comes in consistency with the results of this study while it is far from the figure detected in a turkish study which found h. pylori positivity to be 82%.18 the differences in the reported figures may be related to the point that the incidence actually varies according to living environment, occupation, and geographic region in addition to the diversity in the detection methods. in the current study, 97 (19.4%) patients were presented with glandular atrophy; the prevalence is different from another iraqi study done in dohuk, with smaller sample size which record glandular atrophy in just 3% of gastric biopsies13 and may be more similar to a study conducted in al-kuwait, in which 28.3% of patients had atrophic gastritis while the prevalence still high and were 65% and 54% of examined jordanian and egyptian patients respectively.19–21 in a turkish study, the atrophy was found in 75% of the subjects22 the differences in the results may be attributed to the differences in samples size that affect the results or due to subjectivity between pathologists in making the diagnosis of glandular atrophy. in this study, 37 (7.4%) out of total number of patients had intestinal metaplasia, this figure was similar to that detected in two other studies in united states and netherlands in which the prevalence of intestinal metaplasia were 7% and 8% respectively.23,24 almouradi et al.25 reported that, among the 437 patients who had gastric biopsies performed, 66 were found to have gastric im and they observed that the overall prevalence was 15%. additional turkish study reporting a prevalence of intestinal metaplasia of 13.8%.3 the commonness of gastric intestinal metaplasia publically is still hardly to be ascertain because of the symptomless character of this lesion. there is a wide variation in the prevalence of gastric im between these studies may be related to the variability in localization and sampling of focal metaplastic lesions.3 from 500 cases only six (1.2%) patients had dysplastic changes. a compatible result was detected by a turkish study in which dysplastic changes was 2%18 while it differ from that recorded by iranian study in which the dysplastic changes may reach up to 71% and higher.26 according to the results of this study, there is a significant relationship between the age and glandular atrophy but there was no such a relation with other factors like h. pylori infection, intestinal metaplasia and dysplastic changes. this result come in consistency with other study which find that subjects with glandular atrophy were significantly older27 while it differs from other turkish studies which show that intestinal metaplasia and h. pylori infection is detected in elderly subjects.18,28 this could be attributed to environmental and dietary habits that predispose to early h. pylori infection and its consequences, making them unrelated to the age as an influencing factor, while still glandular atrophy considered part of general degenerative process rather than related to h. pylori infection. in this study (according to table 2), there were 24.8% cases which were diagnosed to have h. pylori infection were presented with glandular atrophy. in two different turkish studies, histopathological examination reveal that the mucosal atrophy was found in 43–75% of h. pylori infected subjects,22,29 in iran, in a study, the atrophied gastric mucosal epithelium was seen in 22% of gastric biopsies.26 in al kuwait, 28.3% of h. pylori infected patients were had atrophic gastritis.19 gastric atrophy was observed in 65% and 54% of examined patients in jordan and egypt, respectively.20,21 again this could be attributed to subjective criteria in determination of gastric atrophy as well as difference in the sample size and ethnicity. the current study show that there is a significant negative relationship between h. pylori infection and gastric intestinal metaplasia in that 4.8% of patients presented with h. pylori infection were diagnosed to have intestinal metaplastic changes in comparison to 13.8% of cases without h. pylori infection show intestinal metaplasia on examination. this figure is similar to that recorded by other studies.8,25,30 intestinal metaplasia may cause lower diagnostic accuracy of h. pylori with histologic examination.11 as clearly known, h. pylori selectively lives in gastric mucosa. hence, the impairment of h. pylori colonization in the areas with intestinal metaplasia is predictable.16 at the same time, although the gastric mucosa with intestinal metaplasia looks like the intestinal mucosa, it would still have the features of gastric mucosa according to the grade of metaplasia.18 according to the results of this study (table 3), there was a significant statistical relationship between the existence of intestinal metaplasia and the occurrence of dysplastic changes. this result may be harmonized with many recent studies.31–33 conclusion the current study provides information about the prevalence of h. pylori infection in patients underwent egds in three main institutions in karbalaa province was 71%. the prevalence of intestinal metaplasia is a premalignant changes recording an alarming figure among patients with chronic gastritis. it was noticed that prevalence of gastric glandular atrophy was increased gradually with age with no such relation with intestinal metaplasia and dysplastic changes as premalignant changes occur in younger age groups. the absence of significant relation between age and im could be considered as alarming feature that these changes could be occur earlier in our population. conflicts of interest none.  table 3. the association between intestinal metaplasia and dysplastic changes metaplasia dysplasia total p-value negative (%) positive (%) negative 460 (99.3)* 3 (0.7)* 463 0.001 positive 34 (91.9)** 3 (8.1)** 37 total 494 6 500 *the percentage is out of total negative cases. **the percentage is out of total positive cases. original 257j contemp med sci | vol. 5, no. 5, september-october 2019: 254–257 r.a.a.n. alsafi et al. premalignant changes associated chronic gastritis in karbala province references 1. ferlay j, shin hr, bray f, forman d, mathers c, parkin dm. estimates of worldwide burden of cancer in 2008: globocan 2008. int j cancer. 2010;127:2893–2917. 2. ferro a, peleteiro b, malvezzi m, bosetti c, bertuccio p, levi f, et al. worldwide trends in gastric cancer mortality (1980–2011), with predictions to 2015, and incidence by subtype. eur j cancer. 2014;50:1330–1344. 3. olmez s, aslan m, erten r, sayar s, bayram i. the prevalence of gastric intestinal metaplasia and distribution of helicobacter pylori infection, atrophy, dysplasia, and cancer in its subtypes. gastroenterol res pract. 2015;2015:434039. 4. park dy, srivastava a, kim gh, mino-kenudson m, deshpande v, zukerberg lr, et al. cdx2 expression in the intestinal-type gastric epithelial neoplasia: frequency and significance. mod pathol. 2010;23:54–61. 5. houghton j, wang tc. helicobacter pylori and gastric cancer: a new paradigm for inflammation-associated epithelial cancers. gastroenterology. 2005;128:1567–1578. doi: https://doi.org/10.1053/j.gastro.2005.03.037 6. catalano v, labianca r, beretta gd, gatta g, de braud f, van cutsem e. gastric cancer. crit rev oncol hematol. 2009;71:127–164. 7. uemura n, okamoto s, yamamoto s, matsumura n, yamaguchi s, yamakido m, et al. helicobacter pylori infection and the development of gastric cancer. n engl j med. 2001;345:784–789. 8. lee yc, chen th, chiu hm, shun ct, chiang h, liu ty, et al. the benefit of mass eradication of helicobacter pylori infection: a community-based study of gastric cancer prevention. gut. 2013;62:676–682. 9. blaser mj, nomura a, lee j, stemmerman gn, perez-perez gi. early-life family structure and microbially induced cancer risk. plos med. 2007;4:e7. 10. dinis-ribeiro m, areia m, de vries ac, marcos-pinto r, monteiro-soares m, o’connor a, et al. management of precancerous conditions and lesions in the stomach (maps): guideline from the european society of gastrointestinal endoscopy (esge), european helicobacter study group (ehsg), european society of pathology (esp), and the sociedade portuguesa de endoscopia digestiva (sped). endoscopy. 2012;44:74–94. 11. correa p, piazuelo mb, wilson kt. pathology of gastric intestinal metaplasia: clinical implications. am j gastroenterol. 2010;105:493–498. 12. globocan iarc. cancer map: male stomach cancer, age-standardized incidence rate per 100,000 in 2002. available from: url: http://www-dep. iarc.fr/ (accessed 30 may 2007). 13. hussein nr, napaki sm, atherton jc. a study of helicobacter pyloriassociated gastritis patterns in iraq and their association with strain virulence. saudi j gastroenterol. 2009;15:125–127. 14. fuccio l, zagari rm, minardi me, bazzoli f. systematic review: helicobacter pylori eradication for the prevention of gastric cancer. aliment pharmacol ther. 2007;25:133–141. 15. yasir s, moin f, akhtar sm. frequency of helicobacter pylori infection on histopathology in patients with dyspepsia. am j clin med res. 2014;2: 53–56. 16. ahn hj, lee ds. helicobacter pylori in gastric carcinogenesis. world j gastrointest oncol. 2015;7:455–465. 17. eusebi lh, zagari rm and bazzoli f. epidemiology of helicobacter pylori infection. helicobacter. 2014;19:1–5. 18. ozakay a, kınacı e, bayrak s, pasaoğlu e, sevinc mm, dursun n. helicobacter pylori, intestinal metaplasia, and the accuracy of biopsies in metaplastic gastric mucosa. int j clin exp med. 2017;10:5332–5337. 19. sarkar c, anim jt, ibrahim bh. atrophic gastritis and intestinal metaplasia in helicobacter pylori-associated antral gastritis. med princ pract. 1994;4: 197–203. 20. korstanje a, den hartog g, biemond i, lamers cb. the serological gastric biopsy: a non-endoscopical diagnostic approach in management of the dyspeptic patient significance for primary care based on a survey of the literature. scand j gastroenterol suppl. 2002;37:22–26. 21. matalka ii, al-omari fa, al-jarrah ma, obeidat fn, kanaan fm. image-based discriminating morphological features for gastric atrophy assessment: a step to go further. pathol res pract. 2008;204:235–240. 22. kaklikkaya n, cubukcu k, aydin f, bakir t, erkul s, tosun i, et al. significance of caga status and vaca subtypes of helicobacter pylori in determining gastric histopathology: virulence markers of h. pylori and histopathology. j gastroenterol hepatol. 2006;21:1042–1047. 23. sonnenberg a, lash rh, genta rm. a national study of helicobactor pylori infection in gastric biopsy specimens. gastroenterology. 2010;139:1894–1901.e2; quiz e12. 24. de vries ac, van grieken nc, looman cw, casparie mk, de vries e, meijer ga, et al. gastric cancer risk in patients with premalignant gastric lesions: a nationwide cohort study in the netherlands. gastroenterology. 2008;134:945–952. 25. almouradi t, hiatt t, attar b. gastric intestinal metaplasia in an underserved population in the usa: prevalence, epidemiologic and clinical features. gastroenterol res pract. 2013;2013:856256. 26. malekzadeh r, sotoudeh m, derakhshan mh, mikaeli j, yazdanbod a, merat s, et al. prevalence of gastric precancerous lesions in ardabil, a high incidence province for gastric adenocarcinoma in the northwest of iran. j clin pathol. 2004;57:37–42. 27. den hoed cm, van eijck bc, capelle lg, van dekken h, biermann k, siersema pd, kuipers ej, et al. the prevalence of premalignant gastric lesions in asymptomatic patients: predicting the future incidence of gastric cancer. eur j cancer. 2011;47:1211–1218. 28. çınar a, sadıç m, atılgan hi̇, baskın a, koca g, demirel k, korkmaz m. prevalence of helicobacter pylori infection in school and pre-school aged children with c-14 urea breath test and the association with familial and environmental factors. mol imaging radionucl ther. 2015;24:66–70. 29. fikret d, kaya ö, suna e, vahap o, mustafa a, şebnem a. relationship between atrophic gastritis, intestinal metaplasia, dysplasia and helicobacter pylori infection. turk j gastroenterol. 2001;12:169–170. 30. galiatsatos p, wyse j, szilagyi a. accuracy of biopsies for helicobacter pylori in the presence of intestinal metaplasia of the stomach. turk j gastroenterol. 2014;25:19–23. 31. gomez jm, patrie jt, bleibel w, frye jw, sauer bg, shami vm, et al. gastric intestinal metaplasia is associated with gastric dysplasia but is inversely correlated with esophageal dysplasia. world j gastrointest endosc. 2017;9:61–69. 32. bozorgnia ma, kashfi smh, ariana m, ghalkhani f, iravani s, lashkari mh, et al. prevalence and correlation of chronic atrophic gastritis, intestinal metaplasia and other precancerous lesions of stomach in iran: a historical cohort study. transl gastrointest cancer. 2015;4:413–422. 33. chon i, choi c, shin cm, park ys, kim n, lee dh. effect of helicobacter pylori eradication on subsequent dysplasia development after endoscopic resection of gastric dysplasia. korean j gastroenterol. 2013;61:307–312. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201904 original 39j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 original issn 2413-0516 introduction skin epithelium disruption occurs by disturbing the continuous functions of living tissues due to physical, chemical, thermal, immunological, and microbial factors in the wound.1 wound healing is a complex process which, involves interactions between immunological and biological systems and includes a cascade of precise steps and events.2-4 wound healing is no longer a generic term; instead, it is considered a series of carefully regulated and interrelated processes.5 these coordinated processes include hemorrhage, coagulation, initiation of acute inflammatory reaction induced by primary injury, remodelling, migration, and proliferation of connective tissue and parenchymal cells, as well as the synthesis of extracellular matrix proteins, new parenchymal remodelling and connective tissue and collagen deposition. finally, in a regular procedure, increasing skin strength is performing and results in healing of the injured tissues.2 it has been proven that many herbal extracts accelerate the wound healing process through angiogenesis,6, 7 activation of nf-kb,8 proinflammatory cytokines,9 inos,10 collagen alpha1 type i upregulation,11 the proliferation of fibroblasts,12 and antioxidant activity.9 in recent years, the concentration of researchers on complementary and herbal medicine has increased throughout the world. iranian traditional medicine is one of the strongest branches of complementary medicine that could be used in phytotherapy. in iranian traditional medicine manuscripts, special attention is paid to wounds and wound healing, and some headings assigned to this topic.13 marham-e-ushaq is an ointment that is used in iranian traditional medicine to heal various types of wounds. this ointment consists of ammoniacum gum (ushaq), sesame oil, and beeswax. the principle and perpendicular component of this product is ushaq (ammoniacum gum).13 when the ushaq gum is applied to the wound area, it can eliminate extra discharge from the wound site, remove corrupt tissues, grow healthy tissue to heal the wounds, and prevent scar formation on the injured site.14 the ushaq or ammoniacum gum is obtained from the dorema ammoniacum, which is one of the most important herbs in the apiaceae family. six species of dorema have been shown in the flora of iran, in which two species are d. ammoniacum d. don. and dorema aucheri boiss are endemic. dorema ammoniacum grows in many arid and semi-arid regions of iran including yazd, isfahan, and semnan provinces and is known by the local persian names such as kandal, vasha, or camakandal.15, 16 in the british pharmacopoeia, the ammoniacum gum is known as antispasmodic and expectorant and used for chronic bronchitis and persistent coughing.17,18 in traditional medicine books, ammoniacum gum is widely used in the gastrointestinal, liver, respiratory, musculoskeletal, and skin disorders as a strong anti-inflammatory, absorbent, and drying agent.13 recent studies show that this herb has antioxidant19, anti inflammatory, analgesic20, antimicrobial21, anticonvulsant 22, ache inhibitor 23, and cytotoxic activity.24 determination the effectiveness of a selected formulations of iranian traditional medicine “ushaq poultice” (ammoniacum gum ointment) on wound healing: an experimental study zeinab zaheri abdevand1, amir siahpoosh1, alireza malayeri2, anayatollah salami3, layasadat khorsandi4 1 department of pharmacognosy, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran. 2 department of pharmacology, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran. 3 department of pharmaceutics, faculty of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran 4 department of anatomical sciences, faculty of medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran corresponding author: zeinab zaheri abdevand (e-mail: zeinab.zaheri@yahoo.com) (submitted: 16 january 2020 – revised version received: 10 february 2020 – accepted: 07 march 2020 – published online: 26 april 2020 objective recent studies showed that this plant had antibacterial, antioxidant, and anti-inflammatory activities. the purpose of this study was to evaluate the therapeutic effects of ammonium gum ointment on wound healing in the animal model. methods thirty-two (32) male wistar rats were selected; then in non-infectious condition, 2 full-thickness wounds with 8 mm in diameter were created bilaterally on shaved skin in 1.5 cm from the dorsal midline and randomly divided into 4 groups: untreated group, phenytoin cream treated group, base ointment (sesame oil and wax) treated group and ammoniacum gum ointment (ago) treated group. the animals received these medicines once daily. on days 5, 7, 10, 12, and 14 wound area was measured. on days 7 and 14, blood samples were taken, and serum level of growth factors [epidermal growth factor (egf), platelet-derived growth factor (pdgf), vascular endothelial growth factor (vegf), and transforming growth factor-β (tgf-b)] were measured. skin samples used for hematoxylin-eosin and trichrome staining. skin tensile strength and hydroxyproline content of skin tissues were also measured. results the percentage of wound healing after 14 days of treatment was significantly increased in the group receiving ago (p<0.05). hydroxyproline content of repaired tissue and tensile strength increased considerably in this group (p<0.001). growth factors were significantly increased in animals treated with ago compared to control groups (egf p<0.05; pdgf p<0.001; vegf p<0.001; tgf-b p<0.05). histological data showed that the topical application of ago compared to other groups resulted in positive effects on enhancing neovascularization and granulation, increased wound healing rate, and decreased wound size. conclusion the topical application of an ointment containing ammoniacum gum can help speed-up the process of wound healing. keywords wound healing, dorema ammoniacum gum, herbal medicine, experimental study 40 original determination the effectiveness of a selected formulations of iranian traditional medicine zeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 in this experimental study, we demonstrated the wound healing potential of marham-e-ushaq (ammoniacum gum ointment, ago) on full-thickness skin wound model in wistar rats and compared with control groups. materials and methods materials dorema ammoniacum gum was purchased from a local herb store in tehran, iran. the gum was identified by the pharmacognosy department of jundishapur university of medical sciences, ahvaz, iran. the sesame oil was purchased from barij co. with batch no. 612138055. preparation of topical products sesame oil and beeswax were warmed in a water bath and mixed. the ammoniacum gum powder (ushaq) was soaked in vinegar and stirred to smoothing. this compound was warmed in a water bath and added to warm oil and wax, stirred regularly for homogenizing and cooling.13 animals and experimental protocol 32 male wistar rats weighing between 200 and 220 g were used in this study. animals were obtained from the animal center of ahvaz jundishapur university of medical science. the animals were housed individually in separated metal cages at 22±1°c, humidity (60±5%), and a 12 h light/dark cycle, and they had free access to standard commercial pellet diet and tap water. the experimental protocol was reviewed and approved by the institutional animal ethics committee (ir.ajums. abhc.rec.1397.039). the rats were anesthetized with intraperitoneal administration of ketamine-xylazine (80 mg/kg: 5 mg/kg). after shaving the hair on their back and disinfecting with 70% ethanol, two circular full-thickness skin wound (8 mm in diameter) was made at a distance of 1.5 cm from the midline of the back of each animal. after wounding, the rats were randomly divided (block randomization using the aabb method) into 4 groups of 8 animals and kept in separate cages. all cages were coded. treatment was started as following: group 1: test group treated with ago applied locally on wounds. group 2: control group treated with phenytoin cream 1% applied locally on wounds. group 3: control group treated with ointment base (beeswax and sesame oil, 1:10) applied locally on wounds. group 4: control group without treatment. the wound area measurement after wounding in non-infectious conditions and taking pictures, the medicine was applied to the wound site from day 0 (day of wounding) and this procedure repeated every 24 h until healing was complete. the measuring method of the wound area is as follows: animals were anesthetized and fixed in the standard position on a flat surface, a ruler placed next to the wound, and a photograph was taken by a digital camera, at a distance of 15 cm from the wound (without zooming in). these conditions were the same for all animals during the experiment. then by the digimizer software, the wound area was measured and recorded. the following equation was used to calculate the percentage of wound healing:25 a at a 0 0 100 − × where a0 is wound area on the first day and at is wound area after time interval. histopathological study on the 7th and 14th day after the complete recovery of the wounds, the animals were sacrificed. the hair was completely shaved. the tissues were collected from all animals and encoded in 10% buffered formalin. after the sequence of processes such as dehydration, clearing, tissue infiltration, and tissue embedding in paraffin, five microns thickness were cut using a microtome and stained with masson-trichrome (for detection of collagen fibers) and hematoxylin and eosin (h & e) (for morphological observations). tissues samples were evaluated qualitatively for the following histological criteria: the extent of re-epithelization, inflammation, neovascularization, the degree of granulation tissue formation, and collagen disorganization.  hydroxyproline assay a certain amount of fresh tissues were weighed, and then samples were hydrolyzed with 6 n hydrochloric acid for 2 h at 120°c. the ph of hydrolyzed samples reached to 7 and subjected to oxidation with chloramine t. colored complex with an erelich reagent at 60°c is read at 550 nm with spectrophotometer (spekol 2000). the values of tissue weight are expressed in μg/mg.26,27 tensile strength measurements the tensile strength indicates tissue integrity. to measure the tensile strength, on the 7th and 14th day, a part of the restored skin is striped with a width of 5 mm and its strength was measured with a tensiometer device.28 growth factors assay on the 7th and 14th day, under sterile conditions, blood samples were taken directly from the heart of the animals. the blood samples were centrifuged at 1000 rpm for 15 min and then stored at -80°c until the test was performed. the level of transforming growth factor-β1 (tgfβ1), platelet-derived growth factor (pdgf), epidermal growth factor (egf), and vascular endothelial growth factor (vegf) in serum samples was measured by elisa, based on the manufacturer’s instructions. statistical analysis data are expressed as mean ± standard error of the means (s.e). statistically significant differences were determined using oneway analysis of variance (anova) with the lsd post hoc for multiple comparisons. for all tests, the values of 41 original determination the effectiveness of a selected formulations of iranian traditional medicinezeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 p≤0.05 were considered as statistically significant. statistical analyze were performed using the spss statistical software (version 16.0 for windows, spss inc., chicago, il, usa). results effect of ammoniacum gum on wound healing the process of wound healing in each group is shown in fig. 1. the data show that the topical application of ago promotes wound contraction with a reduction in wound area. the wound area was measured using digimizer at 0, 5, 7, 10, 12, and 14 days after wounding. reduction in wound area was observed more rapidly with the topical application of ago. the wound healing percentage of animals treated with ago was significantly increased compared to control groups at days 5,7,10, and 12 after wounding (at day 5: p<0.01 vs untreated and base ointment group; at day7: p<0.05 vs untreated and p<0.01 vs base ointment group; at day 10: p<0.05 vs untreated and p<0.01 vs phenytoin and base ointment group; at day 12: p<0.05 vs phenytoin and base ointment group) (fig. 2). on day 12, all animals in the ago group were completely healed. there was a significant difference in wound healing between the ago-treated group and base ointment treated at 14 days (p <0.05). serious infection was not observe in all animals. histopathological study on the 7th day after wounding, the ago-treated group showed a significant increase in granulation compared to the untreated and base ointment-treated groups (p<0.05). in the group treated with ago, the thickness of granulation tissue was uniform. there was no significant increase in vascularization in this group compared to the untreated and phenytoin treated groups. inflammation in this group was significantly lower than the control groups (p<0.05) (figs. 3 and 5). at the end of the first week of treatment, epithelialization was not observed in any of the experimental groups. on the 14th day, the wound surface in all groups coated with freshly epidermal tissue, and keratinization observed, and the thickness of the epithelium layer was uniform in the ago treated group. (fig. 5) the ago-treated group showed no significant difference in the amount of granulation and vascularization compared to the phenytoin treated and untreated groups. edema and inflammation decreased in all groups and this reduction in inflammation were significantly higher than untreated (p<0.001) and base-treated groups (p<0.01). (figs. 4 and 5) trichrome staining in all groups indicated the formation of collagen strands and increased organization and reduction of irregularities in collagen fibers. the collagen disorganization significantly decreased in the ago-treated group compared to the untreated group (p<0.05). (fig. 6) tensile strength measurements the results of this study showed a significant increase in tensile strength in the ago-treated group compared to the untreated and phenytoin-treated groups (p<0.001). the results are shown in table 1. hydroxyproline assay the results of this study showed that on the 7th day after wound healing, the amount of hydroxyproline increased significantly in the group received ago compared to the untreated and the fig. 1 representative photographs of the transition of wound closure in the rat model. untreated; wound surgery only, base ointment; wound topically treated with base (beeswax and sesame oil), phenytoin; wound topically treated with phenytoin 1% cream, ago; wound topically treated with ago. 42 original determination the effectiveness of a selected formulations of iranian traditional medicine zeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 fig. 2 the percentage of wound healing measured by digimizer. values are expressed as mean ± se., n = 8 in each group. *: p<0.05 vs untreated, **: p<0.01 vs untreated, #: p<0.05 vs phenytoin, ##: p<0.01 vs phenytoin, †: p<0.05 vs base treated, ††: p<0.01 vs base treated fig. 3 comparison of histological parameter in experimental groups on day 7. data are expressed as mean ± se. n = 8 in each group. * p<0.05: comparison to untreated groups, † p<0.05: comparison to base ointment-treated groups. 43 original determination the effectiveness of a selected formulations of iranian traditional medicinezeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 base-treated groups (p<0.001), and phenytoin cream treated group (p<0.01). on the 14th day, the hydroxyproline content showed a significant increase (p<0.001) in the ago-treated group compared to all control groups. the results of the hydroxyproline measurement showed in table 2. growth factors assay in the 7th and 14th days after wounding, the serum level of growth factors (egf. pdgf, vegf, tgf-b) in animals treated with ago showed a significant increase (p<0.05). the results showed in figs 7–10. discussion wound healing is a dynamic process that involves several continuous, overlapping, and carefully designed steps. interruptions, deviations, or prolongation of this process can lead to delayed wound healing or occur chronic wounds. in adults, wound healing process includes: (1) rapid homeostasis and inflammation; (2) proliferation; and (3) remodeling.29,30 hemostasis begins shortly after the injury, with vasoconstriction and fibrin clots forming. the clot and tissue around the wound release proinflammatory cytokines and growth factors such as tgf-β, pdgf, fibroblast growth factor, and egf. when the bleeding controlled, inflammatory cells migrate to the wound site (chemotaxis) and advance the inflammatory phase, which is characterized by the consecutive infiltration of neutrophils, macrophages, and lymphocytes.30,31 in the proliferation phase, collagen fibers accumulate and neovascularization occurs, the edges of the wound contracts, the surface of the wound reduces, and then the epithelial tissues produced at the wound site. the main feature of this phase is the collagen deposition in a well-organized network. at this stage, the tensile strength of the tissue enhanced by cross-linking between collagen fibers using vitamin c-dependent hydroxylation.32 at this phase, the migration of fibroblasts to the site of the wound increased.31 the regeneration stage begins several days after injury and remains up to 2 years, resulting in the completion of normal epithelium and scar tissue maturation. the accumulation and deposition of collagen and other cellular matrix proteins give strength to the healing tissue. however, the repaired tissue never achieves initial tissue strength. as the scar matures, the level of blood vessels decreases and the scar changes over time from red to pink and gray.33 dorema ammuniacum from family apiaceae is rich in phenolic compounds, and also antioxidant19, anti-inflammatory20, and antimicrobial21,34 effects of this plant have been approved in various studies. the anti-inflammatory activity of this plant is important in the first stage of the wound healing process because increasing the inflammatory phase duration could postpone wound healing. anti-inflammatory activity is essential to decrease the duration of wound healing fig. 4 comparison of histological parameter in experimental groups on day 14. data are expressed as mean ± sem. n = 8 in each group. *: p<0.05 vs untreated, ***: p<0.001 vs untreated, #: p<0.05 vs phenytoin, †: p<0.05 vs base ointment-treated, ††: p<0.01 vs base ointment-treated group. 44 original determination the effectiveness of a selected formulations of iranian traditional medicine zeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 and reduce pain and scar. in the iranian traditional medicine books, the effects of this plant have been discussed in the treatment of various types of wounds. in this study, the therapeutic effects of dorema ammuniacum gum on the wound healing in animals investigated. no study performed on wound healing effects of ammoniacum gum. however, several studies performed on the anti-inflammatory and antibacterial properties of this compound and the therapeutic effects approved.20,34 in 2017, azam bakhtiarian and colleagues have been studied the analgesic and anti-inflammatory effect of the fig. 5 representative micrographs of wound healing on days 7 and 14 (he staining) in ago, phenytoin, base oint and untreated group under ×40 magnifications. granulation (black arrow), neo-vascularization (red arrow), inflammation (blue arrow) and re-epithelialization (yellow arrow). 45 original determination the effectiveness of a selected formulations of iranian traditional medicinezeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 aqueous extract of dorema ammoniacum gum. the results of this study demonstrated that the gum could decrease the inflammation and pain in experimental rats, in which the anti-inflammatory effect was comparable to indomethacin.20 the antibacterial activity of dorema ammoniacum gum demonstrated by rajani et al.34 in this study, the methanol–dichloromethane extract of ammoniacum gum was assessed against 14 microorganisms which included seven gram-positive and four gram-negative bacteria, one yeast, and two fungi. the results showed that the extract of ammoniacum gum inhibited all the seven gram-positive bacteria, one gram-negative bacterium, one yeast, and one fungus.34 fig. 6 representative micrographs of wound healing on day 7 (trichrome staining) in ago, phenytoin, base oint and untreated group. white arrow indicate representative collagen fibers. 46 original determination the effectiveness of a selected formulations of iranian traditional medicine zeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 it has been reported that tgf-b promotes differentiation of fibroblast to myofibroblasts, which is essential for wound contraction. pdgf can induce matrix metalloproteinase secretion from fibroblasts.35 in 2016, joão de masi et al. studied the therapeutic effect of growth factors on wound healing in rats. the data showed that the injection of growth factors in the margin of the wound can accelerate wound healing.36 this study showed that the wound area in the treated group with ago was significantly reduced compared to the phenytoin treated and untreated groups on different days. the results of imaging confirmed that the treatment with ammoniacum ointment accelerated healing and decreased wound area so that on the 12th day after the wounding, all animals which treated with this medicine had completely healed. the data obtained from the histological test indicate that the topical application of ammoniacum gum accelerated the formation of granulation tissue in the early stages of wound healing which accelerates wound healing. the ammoniacum gum probably accelerated wound healing by rapidly increasing granulation and epithelialization due to increased pdgf and egf, respectively. also, ago increased vascularization and production of epithelial tissue as well as the keratinized layer in these animals on the 14th day. the results of tensile strength and hydroxyproline content of repaired skin on the 7th and 14th days after wounding showed that the use of ammoniacum ointment increased the skin strength. also, the results of the histological parameters confirm that in the treated group the amount of oriented collagen fibers at the wound area, which increases the skin firmness, is significantly increased compared to the control groups. conclusion the results of this study showed that ammoniacum gum increased hydroxyproline content, tensile strength, and serum level of growth factors, and accelerated wound closure, increased the rate of granulation and neovascularization in ago-treated animals. since granulation tissue formation and collagen production in the early stages of wound healing is essential for tissue regeneration, accelerated granulation tissue formation and collagen replacement in the first days of wound healing is one of the causes of rapid healing and wound closure after topical application of ago compared to control groups. acknowledgment this research was supported by jundishapur university of medical sciences, ahvaz, iran. the code of ethics ir.ajums. abhc.rec.1397.039. work was also performed at the medicinal plant research centre of jundishapur university of medical sciences, ahvaz, iran. table 1. comparison of the tensile strength of wound tissue sample after treatment. experimental groups tensile strength (g/cm2) day 7 day 14 ammoniacum gum ointment 877.33±33.19 *** †† # 1511.6 ± 114.9 *** ††† ### phenytoin 724.67±11.83 ** 1078.0 ± 65.7 ointment base 668.67±10.17 982.0 ± 12.1 no-treatment 513±53.69 950.8 ± 28.5 significant differences from untreated, phenytoin and base treatment groups: **: p<0.01 vs untreated, ***: p<0.001 vs untreated; #: p<0.05 vs phenytoin, ###: p<0.001 vs phenytoin; ††: p<0.01 vs base treated, †††: p<0.001 vs base treated. table 2. comparison of hydroxyproline content in wound tissue sample after treatment. day 7 day 14 mean±sem mean±sem ago 13.8100±0.33005 ** †† # 28.5340±0.99748 ** †† ## phenytoin 12.0733±0.48340 ** 22.2060±0.40783 * ointment base 7.7100±0.33171 18.7640±0.31706 no-treatment 8.8300±0.18930 19.7340±0.30323 significant differences from untreated, phenytoin and base treatment groups: *: p<0.01 vs untreated, **: p<0.001 vs untreated; #: p<0.01 vs phenytoin, ##: p<0.001 vs phenytoin; ††: p<0.001 vs base treated. 47 original determination the effectiveness of a selected formulations of iranian traditional medicinezeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 fig. 7 comparison of serum levels of egf in experimental groups. data are expressed as mean ± se. n = 8 in each group. † p<0.05 indicate that values are significantly different from base ointment-treated group. * p<0.05 indicate that values are significantly different from untreated group. fig. 8 comparison of serum levels of pdgf in experimental groups. data are expressed as mean ± se. n = 8 in each group. *: p<0.01 vs untreated, **: p<0.001 vs untreated, #: p<0.01 vs phenytoin, ##: p<0.001 vs phenytoin, ††: p<0.001 vs base treated. 48 original determination the effectiveness of a selected formulations of iranian traditional medicine zeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 fig. 9 comparison of serum levels of tgf-b in experimental groups on days 7 and 14 after wounding. data are expressed as mean ± se. n = 8 in each group. * p< 0.05: values significantly different from untreated group; † p<0.05: values significantly different from base ointment-treated group. fig. 10 comparison of serum levels of vegf in experimental groups. data are expressed as mean ± se. n = 8 in each group. **: p<0.001 vs untreated, ##: p<0.001 vs phenytoin, †: p<0.05 vs base treated, ††: p<0.001 vs base treated. 49 original determination the effectiveness of a selected formulations of iranian traditional medicinezeinab zaheri abdevand et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 39–49 conflict of interest the authors declare that there is no known conflict of interest regarding this publication. references 1. lazarus gs, cooper dm, knighton dr, margolis dj, percoraro re, rodeheaver g, et al. definitions and guidelines for assessment of wounds and evaluation of healing. wound repair regen. 1994;2(3):165–70. 2. attinger ce, janis je, steinberg j, schwartz j, al-attar a, couch k. clinical approach to wounds: debridement and wound bed preparation including the use of dressings and wound-healing adjuvants. plastic reconst surg. 2006;117(7s):72s–109s. 3. george broughton i, janis je, attinger ce. wound healing: an overview. plastic reconst surg. 2006;117(7s):1e-s-32e-s. 4. hunt tk. the physiology of wound healing. ann emerg med. 1988;17(12):1265–73. 5. robson mc, steed dl, franz mg. wound healing: biologic features and approaches to maximize healing trajectories. curr probl surg. 2001;2(38): 72–140. 6. mirmalek sa, parsa t, parsa y, yadollah-damavandi s, salimi-tabatabaee sa, jangholi e, et al. the wound healing effect of iris forentina on full thickness excisional skin wounds: a histomorphometrical study. bangl j pharmacol. 2016;11(1):86–90. 7. shen h-m, chen c, jiang j-y, zheng y-l, cai w-f, wang b, et al. the n-butyl alcohol extract from hibiscus rosa-sinensis l. flowers enhances healing potential on rat excisional wounds. j ethnopharmacol. 2017;198:291–301. 8. nicolaus c, junghanns s, hartmann a, murillo r, ganzera m, merfort i. in vitro studies to evaluate the wound healing properties of calendula officinalis extracts. j ethnopharmacol. 2017;196:94–103. 9. joshi a, joshi vk, pandey d, hemalatha s. systematic investigation of ethanolic extract from leea macrophylla: implications in wound healing. j ethnopharmacol. 2016;191:95–106. 10. pereira ldp, mota mr, brizeno la, nogueira fc, ferreira eg, pereira mg, et al. modulator effect of a polysaccharide-rich extract from caesalpinia ferrea stem barks in rat cutaneous wound healing: role of tnf-α, il-1β, no, tgf-β. j ethnopharmacol. 2016;187:213–23. 11. krishnappa p, venkatarangaiah k, rajanna sks, balan rk. wound healing activity of delonix elata stem bark extract and its isolated constituent quercetin-3-rhamnopyranosyl-(1-6) glucopyranoside in rats. j pharm analy. 2016;6(6):389–95. 12. öz be, ilhan m, ozbilgin s, akkol ek, acikara öb, saltan g, et al. effects of alchemilla mollis and alchemilla persica on the wound healing process. bangl j pharmacol. 2016;11(3):577–84. 13. mh aks. qarabadin e kabir (great qarabadin). tehran, iran: tehran university of medical science. 14. mh aks. makhzan-al-advieh (the storehouse medicaments): encyclopedia of traditional iranian foods and drugs (old times). calcutta, india: tehran university of medical sciences; 1844. 1081 p. 15. trease g, evans w. pharmacognosy (13th edn). bailliere tindall, london. 1989:176–80. 16. mozaffarian v, mozaffarian v, mozaffarian v. dictionary of iranian plant names. 1996. 17. langenheim jh. plant resins: chemistry, evolution, ecology, and ethnobotany: oregon, us: timber press; 2003. 18. yousefzadi m, heidari m, akbarpour m, mirjalili mh, zeinali a, parsa m. in vitro cytotoxic activity of the essential oil of dorema ammoniacum d. don. middle-east j sci res. 2011;7(4):511–4. 19. delnavazi m, tavakoli s, rustaie a, batooli h, yassa n. antioxidant and antibacterial activities of the essential oils and extracts of dorema ammoniacum roots and aerial parts. res j pharmacogn. 2014;1(4):11–8. 20. bakhtiarian a, shojaii a, hashemi s, nikoui v. evaluation of analgesic and antiinflammatory activity of dorema ammoniacum gum in animal model. int j pharm sci res. 2017;8(7):3102–6. 21. kumar vp, chauhan ns, padh h, rajani m. search for antibacterial and antifungal agents from selected indian medicinal plants. j ethnopharmacol. 2006;107(2):182–8. 22. motevalian m, mehrzadi s, ahadi s, shojaii a. anticonvulsant activity of dorema ammoniacum gum: evidence for the involvement of benzodiazepines and opioid receptors. res pharm sci. 2017;12(1):53. 23. adhami h-r, lutz j, kählig h, zehl m, krenn l. compounds from gum ammoniacum with acetylcholinesterase inhibitory activity. sci pharm. 2013;81(3):793–806. 24. shahidi gh, moein mr, foroumadi ar, rokhbakhsh zf. cytotoxic activity of medicinal plants used in iranian traditional medicine on two strains of saccharomyces cerevisiae. 2002. 25. cross s, naylor l, coleman r, teo t. an experimental model to investigate the dynamics of wound contraction. br j plastic surg. 1995;48(4):189–97. 26. woessner jr j. the determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. arch biochem biophysics. 1961;93(2):440–7. 27. edwards c, o’brien jr w. modified assay for determination of hydroxyproline in a tissue hydrolyzate. clin chim acta. 1980;104(2):161–7. 28. fried nm, walsh jr jt. laser skin welding: in vivo tensile strength and wound healing results. lasers surg med off j am soc laser med surg. 2000;27(1):55–65. 29. firdous sm, sautya d. medicinal plants with wound healing potential. bangl j pharmacol. 2018;13(1):41–52. 30. gosain a, dipietro la. aging and wound healing. world j surg. 2004;28(3):321–6. 31. george broughton i, janis je, attinger ce. the basic science of wound healing. plastic reconst surg. 2006;117(7s):12s–34s. 32. guo sa, dipietro la. factors affecting wound healing. j dent res. 2010;89(3):219–29. 33. young a, mcnaught c-e. the physiology of wound healing. surgery (oxford). 2011;29(10):475–9. 34. rajani m, saxena n, ravishankara m, desai n, padh h. evaluation of the antimicrobial activity of ammoniacum gum from dorema ammoniacum. pharm biol. 2002;40(7):534–41. 35. barrientos s, stojadinovic o, golinko ms, brem h, tomic‐canic m. growth factors and cytokines in wound healing. wound repair regen. 2008;16(5):585–601. 36. de masi ecdj, campos acl, de masi fdj, ratti mas, ike is, de masi rdj. the influence of growth factors on skin wound healing in rats. braz j otorhinolaryngol. 2016;82(5):512–21. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i2.719 317j contemp med sci | vol. 5, no. 6, november–december 2019: 317–324 original issn 2413-0516 introduction acute kidney injury (aki) is a complex clinical disorder simply meaning a sudden loss of kidney function induced by damage to the kidneys that causes structural and functional injury.1 aki is diagnosed based on significant increase in serum cr, oliguria, and anuria.2 the causes of aki are divided into three categories: pre-renal, post-renal, and intrinsic kidney disease. among these three categories, only intrinsic disorder represents the actual aki state, while the other two are the result of extra renal disorders, leading to a decrease in glomerular filtration rate (gfr). if the conditions of these two classes are durable, it will eventually lead to cellular damage and intrinsic kidney disease.1 the ischemia-reperfusion (i-r) model is one of the animal models used for the fundamental and therapeutic studies in aki3. i-r activates apoptosis and necrosis. the necrosis is characterized by cellular swelling with a membrane surface rupture. necrotic cells stimulate the immune system resulting in filtration of inflammatory cells along with the release of cytokines.4 apoptosis activates caspase’s complex cascade. in vascular dysfunction, increased vascular permeability, endothelial cell inflammation, activation of the complement system, loss of fluid in the interstitial tissue, vasoconstriction, leukocyte activation, and endothelial-leukocyte reaction result in further damage.5 hydralazine is used as an antihypertensive agent which reduces the contraction responses of a number of contractile agents. hydralazine is a direct arteriolar vasodilator that controls the pregnancy’s high blood pressure with a half-life of 1 h.6 hydralazine activates guanylate cyclase; vasodilation effect does not directly, but indirectly releases endogenous nitric oxide (no) from endothelial cells, by controlling the prolyl-hydroxylase domain protein and activating the hypoxia-inducible factor (hif) pathway. hydralazine also helps to restore the balance between no and superoxide in endothelial dysfunction by inhibiting nadph oxidase.7 hif-1 can increase no production by multiple mechanisms, including increasing the expression of inos and cox4-2.8 considering the characteristics of hydralazine in releasing endogenous no9,10; in this study, we aim to investigate the effects of hydralazine post-treatment administration on renal injury caused by i-r. materials and methods this experimental study was performed on 24 wistar rats weighing 250–300 g. rats were housed at 12 h of darkness and 12 h of brightness at room temperature of 23 ± 2°c with free access to the food and water. the experiments were performed on the rats in accordance with the guidelines and regulations and ethical codes approved by the monitoring committee for laboratory animals of arak university of medical sciences. the study groups included: (1) control group: did not receive the drug; (2) sham group: under anesthesia, only the kidneys were exposed without any ischemia; (3) i-r group: bilateral renal artery and vein occlusion for 20 min; then with 24 h reperfusion; (4) hydralazine treatment group: bilateral renal artery and vein occlusion for 20 min and then hydralazine(5 mg/kg, novaplus, usa) + 24 ho immediately after reperfusion initiation.9–11 to induce aki, the animals were weighed first and then sodium thiopental (25 mg/kg; sandoz, gmbh, estonia) was injected intraperitoneally into the animal,12 and after shaving the back hair, 1.5 cm incisions were made on either side of the midline using scissors and forceps. the artery and vein of both kidneys were blocked using a special clamp for 30 min, instantly after the end of the period, the obstruction was removed and the surgical area sutured with silk thread 3-0 and the animals were kept in a separate cage for recovery.11 after recovery, the rats were placed in metabolic cages and the urine was collected for 12 h. urine specific gravity was therapeutic effects of hydralazine on renal ischemia-reperfusion injury in rat fatemeh ahmadi,a saeed hajihashemi,*,a ali rahbari,b fatemeh ghanbaric adepartment of physiology, faculty of medicine, arak university of medical sciences, arak, iran. bdepartment of pathology, arak university of medical sciences, arak, iran. cdepartment of pharmacology, islamic azad university, arak branch, arak, iran. corresponding author: saeed hajihashemi (email: hajihashemi@arakmu.ac.ir) (submitted: 14 july 2019 – revised version received: 23 august 2019 – accepted: 05 september 2019 – published online: 26 december 2019) objective renal ischemia-reperfusion (i-r) induces acute kidney injury (aki). in this study, the effect of hydralazine was investigated on the renal injury induced by the i-r in rats. methods aki was induced with bilateral obstruction of the renal artery and vein for 20 min following 24 h of reperfusion. hydralazine (5mg/ kg) was injected intraperitoneally as post-treatment. results hydralazine significantly increased the levels of renal clearance of creatinine and renal blood flow, while they were decreased by i-r. also, hydralazine significantly improved levels of serum electrolytes (sodium and potassium) that were impaired by i-r. the tissue malondialdehyde levels were significantly suppressed by hydralazine. conclusion according to the results, the post-treatment of hydralazine had a therapeutic effect on renal i-r because of improved ion reabsorption and excretion and increased renal blood flow and glomerular filtration rate and decreased lipid peroxidation. key words hydralazine, renal ischemia-reperfusion injury, rat 318 original hydralazine and renal ischemia-reperfusion in rat fatemeh ahmadi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 317–324 determined. after 24 h of reperfusion, the rats’ systolic blood pressure was measured from the tail by using a power lab instrument (ad instruments, australia) and the mean of systolic blood pressure was calculated for a rat.13,14 the rats were anesthetized and a longitudinal cut was created in the abdomen with a blade. after separation of the artery and vein of the left kidney, after stabilization of amount of blood flow, the renal blood flow (rbf) was recorded for 30 min by using flow meter of power lab instrument (t402, america)15,16 and the mean of blood flow was measured for 30 min. the blood sample was taken from the abdominal aorta by syringe of cold heparin. after plasma extraction, the concentration of creatinine (cr) and blood urea nitrogen (bun) was measured in the serum and urine samples by using the auto analyzer. (selectra-xl, netherlands).17 the concentration of sodium and potassium were measured by a flame photometer (seac-20fp, italy). osmolarity of urine and plasma samples was measured using a osmometer (gonotec osmomat-030, germany).18,19 renal clearance of creatinine (ccr), absolute and relative excretions of potassium and sodium was calculated using the following formula: ccr(μl/min/gkw) = (v°/1000 × ucr)/pcr absolute excretion of sodium: unav°(μmol/min/gkw) = (v° × una)/1000. absolute excretion of potassium: ukv°(μmol/min/gkw) = (v° × uk)/1000. relative excretion of sodium: fena = (una × pcr)/(pna × ucr) × 100 relative excretion of potassium: fek = (uk × pcr)/(pk × ucr) × 100. both kidneys separately were removed and weighed, then they were cut into two halves. for the mda (malondialdehyde) and frap (ferric reducing ability of plasma) experiments, the right kidney was placed in the liquid nitrogen and immediately transferred to the −20°c freezer. the ohkawa method was used for mda experiment, indicating the level of lipid peroxidation by mda. in addition, the benzie & strain method was used for the frap assay.20,21 for histological study, after removing the left kidney capsule, it was placed in 10% formalin buffer and after fixation, stages of dehydration, clarification, paraffin embedding was performed. after section cutting and preparation of 5-micron sections, slices were mounted and stained with hematoxylin and eosin. after preparation the slides, tissue analysis was performed by an expert pathologist.14,16 the changes in glomerular and tubular and vascular structure were analyzed. glomerulus diameter and bowman’s space size, percentage of injuries in tubule and glomerulus, the number of red blood cells (rbcs) in glomeruli, the shedding of tubular cells, formation of cast proteins in the lumen, tubular cells necrosis and the formation of vacuoles within the cells were examined. based on the severity of injuries in the glomerular and renal parenchyma, the quantity of damage was graded as follows: “grade 0”; 1–25% damage, “grade 1”; 25–50% damage, “grade 2”; 50–75% damage, “grade 3”; and 75–100% damage, “grade 4”.22 finally, the data were presented as the mean ± standard error of the mean (s.e.m.). for statistical analysis, spss software version 25 (spss software, chicago, il, usa), one-way anova, tukey test, kruskal–wallis multiple comparison test and dunnett test were employed at p ≤ 0.05 as the significance level for statistical analysis.23 results post-treatment effects of hydralazine on the rbf and systolic blood pressure the results indicated rbf decreased significantly in the i-r groups (6.42 ± 0.3 ml/min, p < 0.001) compared to the control and sham group (8.5 ± 0.2 ml/min, p < 0.01). in the hydralazine-treated rats the rbf significantly increased (9.04 ± 0.4 ml/ min, p < 0.01) compared to the i-r group, while there was no significant difference between the control group and the sham group. the systolic blood pressure did not show a significant difference between the groups (▶ fig. 1). post-treatment effects of hydralazine on renal clearance of creatinine (c cr ), absolute excretion of sodium (u na v°) and potassium (u k v°) and relative excretion of sodium (fe na ) and potassium (fe k ) results showed that renal creatinine clearance was significantly decreased in the i-r groups (0.008 ± 0.005 μl/min gkw; p < 0.001) compared to the control and sham (0.05 ± 0.01 μl/ min gkw). in the hydralazine-treated rats, the renal clearance fig. 1 comparison of (a) renal blood flow and (b) systolic blood pressure among the groups. similar characters represent significant differences between the groups. results are expressed as mean ± the standard deviation (sd) for six rats in each group. 319 original hydralazine and renal ischemia-reperfusion in ratfatemeh ahmadi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 317–324 of creatinine (0.02 ± 0.009 µl/min  gkw; p < 0.05) significantly increased in compare to the i-r group (0.008 ± 0.005 μl/ min gkw; p < 0.001). the relative excretion of sodium (fena) was not increased significantly in the i-r group compared to the control group (0.42 ± 0.2% vs. 0.39 ± 0.3%). the fena in the hydralazine group was significantly lower than that of the i-r group (0.001 ± 0.0004 vs. 0.42 ± 0.2%; p < 0.01) and control group (0.001 ± 0.0004 vs. 0.39 ± 0.3%; p < 0.05). the fena control group was not significantly different from the sham group (▶table 1). in the i-r group the fek showed a significant increase compared to the control group. (47.28 ± 13.3% vs. 28.6 ± 9.8%; p < 0.05). the fek significantly decreased in the hydralazine-treated rats compared to the control group (8.6 ± 3.1% vs. 28.6 ± 9.8%; p < 0.05), sham group (8.6 ± 3.1% vs. 42.74 ± 13.8%; p < 0.001) and i-r group (8.6 ± 3.1% vs. 47.28 ± 13.3%; p < 0.001) (▶table 1). the absolute excretion of sodium (unav°) did not show any significant difference among the groups. the ukv° in the i-r group (0.82 ± 0.2 µmol/min gkw, p<0.001) showed a significant decrease compared to the control and sham groups (2.68 ± 0.3 and 2.16 ± 0.2 µmol/min gkw, respectively), while the hydralazine group (0.64 ± 0.1 μmol/min  gkw) did not show any significant difference with i-r group (▶table 1). post-treatment effects of hydralazine on urinary levels of sodium ([na] u ), potassium ([k] u ), creatinine ([cr] u ), and osmolality (osmol u ) the urine sodium concentration in the i-r group was significantly higher than the control group. (28.72 ± 8.4 μmol/ml vs. 65.73 ± 6.9 μmol/ml; p < 0.001). the urine sodium concentration in the hydralazine group (28.45 ± 3.7 μmol/ml) was not significantly different from sham (32.33 ± 3.9 μmol/ml) and control groups. the urine sodium concentration decreased significantly in hydralazine group compared to the i-r group (p < 0.001). the urine potassium concentration in the i-r group was significantly lower than the control group (112.9 ± 2.7 μmol/ ml vs. 133.63 ± 13.6 μmol/ml; p<0.01). the urine potassium concentration in the hydralazine group was significantly lower than the sham group (45.03 ± 6.3 μmol/ml vs. 120.28 ± 9.13 μmol/ml; p < 0.001) and the i-r groups (45.03 ± 6.3 μmol/ml vs. 112.9 ± 2.7; p < 0.001). the urinary potassium concentration was not significantly different in the i-r group and sham group. the urinary creatinine concentration was not significantly different between the groups. urine osmolality in the i-r group was significantly lower than the control group. (908.33 ± 48.12 mosm/kgh2o vs. 1515 ± 70.5 mosm/kgh2o; p < 0.001). urine osmolality in the hydralazine group was significantly lower than the sham and control groups (1285.33 ± 162.08 mosm/kgh2o vs. 1493.83 ± 80.85 mosm/kgh2o; p < 0.01), whereas significantly increased compared to the i-r group (1285.33 ± 162.08 mosm/kgh2o vs. 908.33 ± 48.12 mosm/kgh2o; p < 0.001) (▶ table 2). post-treatment effects of hydralazine on plasma concentrations of sodium ([na] p ), potassium ([k] p ), creatinine ([cr] p ), urea ([bun] p ), and osmolality (osmol p ) the results showed that plasma creatinine concentration significantly increased with i-r induction in comparison with control group (1.24 ± 0.35 mg/dl vs. 0.55 ± 0.09 mg/dl; p < 0.001) and sham group (1.24 ± 0.35 mg/dl vs. 0.64 ± 0.08 mg/dl; p < 0.01). the plasma creatinine concentration in the hydralazine group exhibited significant reduction as compared to the i-r group (0.71 ± 0.1 vs. 1.24 ± 0.35 mg/dl; p < 0.001). there was no significant difference between the control and sham groups. in i-r group, bun was significantly higher than the control group (47.81 ± 5.9 mg/dl vs.18.76 ± 2.4; p<0.001) and the sham group (47.81 ± 5.9 mg/dl vs. 24.9 ± 3.1 mg/dl; p < 0.01). there was no significant difference between the hydralazine group and the i-r group. plasma sodium concentration was not significantly different between i-r group and control group (149.23 ± 6.2 μmol/ ml vs. 152.1 ± 6.1 μmol/ml; p<0.01). in the hydralazine group, plasma sodium concentration significantly decreased compared to control group (134.35 ± 2.5 μmol/ml vs. 152.1 ± 6.1 μmol/ml; p < 0.001), sham group (134.35 ± 2.5 μmol/ml vs. 144.45 ± 6.7 μmol/ml; p<0.05) and i-r group (134.35 ± 2.5 μmol/ml vs. 149.23±6.2 μmol/ml; p<0.001). plasma osmolality and potassium concentration were not significantly different among the four groups (▶table3). table 1. comparison of creatinine clearance (ccr), absolute (unav°) and relative (fena) excretions of sodium and absolute (u k v°) and relative (fek) excretions of potassium parameters groups fe k (%) fe na (%) u k v° (µmol/min.gkw) u na v° (µmol/min.gkw) c cr (µl/min.gkw) control 28.6 ± 9.8 0.39 ± 0.3 2.68 ± 0.3 0.56 ± 0.1 0.05 ± 0.01 sham 42.7 ± 13.8 0.28 ± 0.05 2.16 ± 0.2 0.58 ± 0.1 0.05 ± 0.01 i-r 42.7 ± 13.3* 0.42 ± 0.2 0.82 ± 0.2 *** +++ 0.47 ± 0.1 0.008 ± 0.005 *** +++ hyd + i-r 8.6 ± 3.1 * +++ ▫▫▫ 0.001 ± 0.0004 * ▫▫   0.64 ± 0.1 *** +++   0.4 ± 0.1 0.02 ± 0.009 * ▫▫▫ + p < 0.001***, p < 0.01**, p < 0.05* compared to the control group p < 0.001+++, p < 0.01++, p < 0.05+ compared to the sham group p < 0.001□□□, p < 0.01□□, p < 0.05□ compared to the i-r group results expressed in mean ± standard deviation (sd) for 6 rats in each group 320 original hydralazine and renal ischemia-reperfusion in rat fatemeh ahmadi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 317–324 post-treatment effects of hydralazine on mda and frap levels in renal tissue the results of this study indicated that mda level per gram of kidney weight (gkw) significantly increased in i-r group (35.68 ± 6.4 μmol/gkw; p < 0.001) compared to control group (15.33 ± 4.21 μmol/gkw) and sham group (20.41 ± 3.3 μmol/ gkw; p < 0.001). there was no significant difference in the mda level in sham group compared to control group. the mda level in hydralazine-treated rats significantly reduce compared to the i-r rats (21.53 ± 4.19 μmol/gkw vs. 35.68 ± 6.4 μmol/gkw; p < 0.001; ▶fig. 2a). there was no significant difference in the mda levels of hydralazine group compared to the control and sham groups. frap level in the kidney tissue of i-r rats declined significantly compared to the control rats (5.69 ± 0.5 mmol/gkw vs. 8.61 ± 1.06 mmol/gkw; p<0.001) and the sham group (7.59 ± 0.3 mmol/gkw; p<0.001). frap levels of the control group was not significantly different from the sham group. frap level in the hydralazine-treated rats (7.006 ± 0.6 mmpl/gkw) increased but was not significant compared to the i-r rats. frap level in the hydralazine group did not differ significantly from that of the control and sham groups (▶fig. 2b). post-treatment effects of hydralazine on histopathological changes (▶fig. 3) the results of this study showed extensive renal damage in the i-r rats that were significantly different compared to the control group (grade 0), necrosis (grade 3), and vacuolation (grade 2) of tubular cells, increased of bowman’s space (grade 1), presence of protein casts within the tubular lumen (grade 2), scatter of cells into the lumen (grade 3), reduce number of rbcs glomerular (grade 1), and glomerular injury (grade 2) were significantly different from the control group (grade 0; p < 0.05). in the hydralazine-treated rats, necrosis of the tubular cells (grade 2) was significantly different in compared to the control and sham groups (grade 0; p < 0.05) and significantly reduce in comparison with the i-r group (grade 3; p < 0.05) (▶fig. 3). hydralazine had no significant effects on increased bowman’s space (grade 1), formation of protein casts (grade 2), vacuolation (grade 2), glomerular injury (grade 2), cell scattering (grade 2), and reduction in the number of glomerular rbcs (grade 1) compared to the i-r group (▶table 4). table 2. comparison of urinary concentrations of sodium ([na] u ), potassium ([k] u ), creatinine ([cr] u ) and osmolality (osmol u ) among the groups parameters groups [na] u (µmol/ml) [k] u (µmol/ml) [cr] u (mg/dl) osmol u (mosm/kgh 2 o) control 28.72 ± 8.4 133.63 ± 13.6 1.38 ± 0.2 1515 ± 70.5 sham 32.33 ± 3.9▫▫▫ 120.28 ± 9.1 1.33 ± 0.3 1493.83 ± 80.8 i-r 65.73 ± 6.9*** 112.9 ± 2.7 ** 1.25 ± 0.2 908.33 ± 48.1 *** +++ hyd + i-r 28.45 ± 3.7▫▫▫ 45.03 ± 6.3 *** +++ ▫▫▫ 1.28 ± 0.2 1258.33 ± 162.08 ** ++ ▫▫▫ p < 0.001***, p < 0.01**, p < 0.05* compared to the control group p < 0.001+++, p < 0.01++, p < 0.05+ compared to the sham group p < 0.001□□□, p < 0.01□□, p < 0.05□ compared to the i-r group results expressed in mean ± standard deviation (sd) for 6 rats in each group table 3. comparison of plasma concentrations of sodium ([na] p ), potassium ([k] p ), creatinine ([cr] p ) and osmolality (osmol p ) among the groups parameters groups [na] p (µmol/ml) [k] p (µmol/ml) [cr] p (mg/dl) [bun] p (mg/dl) osmol p (mosm/kgh 2 o) control 152.1 ± 6.1 3.66 ± 0.4 0.55 ± 0.09 18.76 ± 2.4 309.16 ± 30.2 sham 144.45 ± 6.7 3.79 ± 0.4 0.64 ± 0.08 24.9 ± 3.1 305.66 ± 6.1 i-r 149.23 ± 6.2 4.02 ± 0.5 1.24 ± 0.3 *** +++ 41.11 ± 6.5 *** ++ 318.83 ± 10.06 hyd + i-r 134.35 ± 5.2 *** + ▫▫▫ 4.14 ± 0.2 0.71 ± 0.1▫▫▫ 47.81 ± 5.9 *** + 333.66 ± 19.7 p < 0.001***, p < 0.01**, p < 0.05* compared to the control group p < 0.001+++, p < 0.01++, p < 0.05+ compared to the sham group p < 0.001□□□, p < 0.01□□, p < 0.05□ compared to the i-r group results expressed in mean ± standard deviation (sd) for 6 rats in each group 321 original hydralazine and renal ischemia-reperfusion in ratfatemeh ahmadi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 317–324 discussion the results of present study showed renal i-r caused aki that is associated with increased plasma creatinine, bun and decreased rbf and clearance of creatinine.24 post-treatment with hydralazine significantly increased the renal clearance of creatinine and rbf and also improved sodium reabsorption and urine osmolality. post-treatment with hydralazine had a nephroprotective effects on renal i-r. these results may be due to the fact that renal i-r injury leads to a lack of integrity and polarity of the epithelial cells with brush border destruction of the tubule. these changes promote the epithelial cellular downfall and the appearance of protein casts, lumen obstruction, and increased intratubular pressure. these factors caused a reduction in ion reabsorption, which during the reperfusion can lead to tubular obstruction and decreasing of gfr. injured proximal tubules cannot reabsorbed sodium ions, which activates tubuloglomerular feedback. this feedback significantly caused pre-glomerular arteriole constriction and reduce gfr.25 hydralazine significantly increased the renal clearance of creatinine rate in the i-r rats. previous studies indicated that hydralazine increased the formation of no by the vascular endothelium and elevated intracellular cgmp levels.26, 27 increased intracellular cgmp levels dilated afferent and efferent arterioles vasodilation leading to increase of gfr. in other study, reetu r. singh showed that no increases gfr by increasing in the ultrafiltration coefficient; no can also reduce tgf and cause constriction of pre-glomerular blood vessels.28, 29 plasma electrolytes in the i-r rats were impaired in comparison with the control rats. fena increased in the i-r rats compared to the control rats but was not significant. fek also increased in the i-r group. this increase indicates the injury to the tubular epithelial cells, especially the proximal tubule during i-r.25 proximal tubules (pt, especially s3), thick ascending limb of henle’s loop (tal) and external medulla are susceptible to i-r injury.30 in addition, i-r causes brush border destruction with reduced reabsorption of sodium by proximal tubules, impaired expression of tubular sodium transporters and unsuitable regulation of na+/k+-atpase expression in the basolateral membranes.19, 30 in the i-r injury lead to direct injury of cells responsible for the secretion of potassium in the distal tubules and collecting ducts.31 post-treatment of hydralazine in the i-r rats reduced urinary excretion of sodium compared to the i-r rats. wu et al. showed the direct inhibitory effect of no on renal na+ transport in proximal tubule and also the renal sympathetic nervous system that stimulated na+ transport in proximal tubule. many studies showed the direct inhibitory effect of no on na+ transport in the isolated tubule segments but not in the intact kidney.32 hydralazine is not a first-line drug for treating fig. 2 comparison of (a) mda and (b) frap levels among the groups. similar characters represent significant differences between the groups. results are expressed as mean ± the standard deviation (sd) for six rats in each group. results are expressed on per gram kidney weight (gkw). fig. 3 comparison of renal histological between different groups. (a) control group with glomerular and normal tubular structure (×40); (b) i-r group with tubular cell necrosis, formation of protein casts inside the tubule lumen, cells scattering into the tubule lumen, vacuolation of tubular cells, increased bowman’s space and reduced number of red blood cells in glomerulus (×40); (c) sham group with tubular cell necrosis (×40); (d) hydralazine group with increased bowman’s space, vacuolation of tubular cells and reduced tubular cell necrosis (×40). rbc: red blood cell, n: necrosis, c: intratubular cast, d: downfall, v: vacuolization 322 original hydralazine and renal ischemia-reperfusion in rat fatemeh ahmadi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 317–324 hypertension due to increased activity of the sympathetic nervous system of the juxtamedullary cells, increased plasma renin activity and salt maintenance.33 therefore, the activation of the renin-angiotensin system tends to neutralize the hydralazine vasodilator effect.34 in the cortical collecting duct, no inhibits basolateral membrane potassium conductance.35 urine osmolality was significantly decreased in the i-r rats compared to the control group because of impairment in the ability of the renal urine concentration. inappropriate regulation of the aqps and na+ transporter proteins in renal tubules and decreasing the expression of na+/k+-atpase in the thick descending limb of the external medulla that causing changes in sodium and water regulation under the influence of i-r injury.19 in the hydralazine-treated rats, urinary osmolality was significantly more than that of the i-r rats. previous studies have shown that hydralazine reduces urinary volume due to water and sodium retention.36 in i-r rats, mda levels significantly increased and frap levels significantly decreased in the kidney tissue. previous studies have shown that i-r causes an imbalance between reactive oxygen species (ros) sources and free radicals scavenger (reduced glutathione peroxidase, catalase, and superoxide dismutase).37 during the reperfusion phase, the oxygen free radicals caused lipid peroxidation which indicated aki and necrosis.38 the mda levels in the hydralazine-treated rats decreased compared to the i-r group and frap levels were increased compared to the i-r group. previous studies have shown that treatment with hydralazine reduces the level of lipid peroxidation in myocardium in rats with spontaneous hypertension.39 the antioxidant effects of hydralazine are via the formation of ros scavenger and inhibitor of o2-generation and peroxynitrite.40 in the present study, blood pressure was not significantly different among the groups. regulation of blood pressure is via the shortand medium-term mechanisms, and the kidneys are long-term regulators of blood pressure that regulated blood volume via the renin-angiotensin system and function of aldosterone hormone. in this study, in the i-r rats the rbf rates showed a significant decrease compared to the control and sham groups, which can be due to the fact that increased renal vascular resistance (rvr) can be a vascular response to ischemia. increase of rvr may activate vasoactive factors, ros and inflammatory pathways that can affect renal perfusion. activation of the sympathetic nervous system, the renin-angiotensin system, prostaglandins, and endothelin a are main vasoconstrictor agents reducing rbf under i-r injury.41 in the sham group, the rbf was significantly lower than the control group, which could be due to the effects of anesthesia and surgical stress. mercatello et al. has shown that anesthesia drugs influenced renal function not only directly, but also through changes in function of cardiovascular and endocrine system. many barbiturates tend to reduce rbf.42 in the hydralazine-treated rats, the rbf was significantly increased in compare to the i-r rats. previous studies indicated that hydralazine significantly reduces rvr and increased cardiac output which ultimately increases rbf.43, 44 the cellular mechanism of vasodilatory effect of hydralazine is not clearly determined, but it may be related to its calcium binding ability that is required for smooth muscle contraction of vessels, inhibiting ip3-induced calcium release from sarcoplasmic reticulum, and inhibiting myosin phosphorylation in arterial smooth muscle cells.27 elkayam uri et al. has shown that stimulating the production of endogenous no in the kidney can consider as a therapeutic target for increasing rbf in patients with heart failure.45 in the present study, histopathological studies showed that compared to the control group, the i-r group induced vacuolation of tubular cells, degeneration of renal tubules, tubular cell necrosis, increased bowman’s space, formation of protein casts within the tubular lumen, cellular cell scattering into the tubular lumen, reduced number of rbcs in glomerula, and glomerular injury. in the hydralazine-treated rats, the improvement was not found in pathologic outcomes. the imbalance between expression and activity of enos and inos (decreased enos activity and increased inos expression) is responsible for the pathophysiology of i-r injury.46 previous studies have not shown the improvement effects of hydralazine on the histological changes.47 authors believe that the time of using hydralazine and also duration of treatment exert a very important role in pathological improvement. table 4. comparison of necrosis level, protein casts, cell scattering, vacuolation, the reduced number of red blood cells, increased bowman’s capsular space, and glomerular injury parameters groups cell necrosis vacuolation increased bowman’s capsular space formation of protein casts cell scattering reduced number of red blood cells glomerular injury control 0 0 0 0 0 0 0 sham 1 * ▫ 0 ▫ 1 * 0 ▫ 0 ▫ 0 ▫ 0 ▫ i-r 3 * + 2 * + 1 * 2 * + 2 * + 1 * + 2 * + hyd + i-r 2 * + ▫ 2 * + 1 * 2 * + 2 * + 1 * + 2 * + p < 0.05* compared to the control group p < 0.05+ compared to the sham group p < 0.05□ compared to the i-r group results expressed in mean ± standard deviation (sd) for 6 rats in each group 323 original hydralazine and renal ischemia-reperfusion in ratfatemeh ahmadi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 317–324 conclusion regarding the results of the present study, post-treatment of hydralazine had therapeutic effects on aki caused by i-r and improved ion reabsorption and excretion, hemodynamic parameters, antioxidant defense, and reduced mda. irb ethical approval number the study ethics committee of arak university of medical sciences approved this research under registration number ir.arakmu.rec. 1396.287. informed consent this research was not on humans. all ethical codes developed by the monitoring committee for laboratory animals at arak university of medical sciences were complied with in the experiments conducted in the present study. acknowledgement the financial support of this study was presented by the research deputy of arak university of medical sciences. support sources deputy of research and education, arak university of medical sciences references 1. makris k, spanou l. acute kidney injury: definition, pathophysiology and clinical phenotypes. clin biochem rev. 2016;37(2):85. 2. case j, khan s, khalid r, khan a. epidemiology of acute kidney injury in the intensive care unit. critic care res pract. 2013;2013. 3. le clef n, verhulst a, d’haese pc, vervaet ba. unilateral renal ischemiareperfusion as a robust model for acute to chronic kidney injury in mice. plos one. 2016;11(3):e0152153. 4. padanilam bj. cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. am j physiol-renal physiol. 2003;284(4):f608-f27. 5. salvadori m, rosso g, bertoni e. update on ischemia-reperfusion injury in kidney transplantation: pathogenesis and treatment. world j transpl. 2015;5(2):52. 6. xu b, bobek g, makris a, hennessy a. antihypertensive methyldopa, labetalol, hydralazine, and clonidine reversed tumour necrosis factor‐α inhibited endothelial nitric oxide synthase expression in endothelial‐ trophoblast cellular networks. clin exp pharmacol physiol. 2017;44(3): 421–7. 7. rocchiccioli jp. hydralazine in heart failure: a study of the mechanism of action in human blood vessels: university of glasgow; 2015. 8. hendrickson md, poyton ro. crosstalk between nitric oxide and hypoxiainducible factor signaling pathways: an update. res rep biochem. 2015;5:147–61. 9. packer m, meller j, medina n, gorlin r, herman mv. dose requirements of hydralazine in patients with severe chronic congestive heart failure. am j cardiol. 1980;45(3):655–60. 10. mikaelian i, coluccio d, hirkaler gm, downing jc, rasmussen e, todd j, et al. assessment of the toxicity of hydralazine in the rat using an ultrasensitive flow-based cardiac troponin i immunoassay. toxicol pathol. 2009;37(7): 878–81. 11. hesketh ee, czopek a, clay m, borthwick g, ferenbach d, kluth d, et al. renal ischaemia reperfusion injury: a mouse model of injury and regeneration. journal of visualized experiments: jove. 2014(88). 12. suleyman z, sener e, kurt n, comez m, yapanoglu t. the effect of nimesulide on oxidative damage inflicted by ischemia–reperfusion on the rat renal tissue. renal fail. 2015;37(2):323–31. 13. zou l, wang w, liu s, zhao x, lyv y, du c, et al. spontaneous hypertension occurs with adipose tissue dysfunction in perilipin-1 null mice. biochim biophys acta (bba)-mol basis dis. 2016;1862(2):182–91. 14. hajihashemi s, hamidizad z, rahbari a, ghanbari f, motealeghi za. effects of cobalamin (vitamin b12) on gentamicin induced nephrotoxicity in rat. drug res. 2017;67(12):710–8. 15. mukai k, kuda y, shibamoto t, tanida m, kurata y, yokoyama h. renal response to anaphylaxis in anesthetized rats and isolated perfused rat kidneys: roles of nitric oxide. j physiol sci. 2018;68(5):689–97. 16. hajihashemi s, jafarian t, ahmadi m, rahbari a, ghanbari f. ameliorative effects of zataria multiflora hydro-alcoholic extract on gentamicin induced nephrotoxicity in rats. drug res. 2018;68(07):387–94. 17. cao y, gao x, yang y, ye z, wang e, dong z. changing expression profiles of long non-coding rnas, mrnas and circular rnas in ethylene glycolinduced kidney calculi rats. bmc genom. 2018;19(1):660. 18. antony as, gomathy s, rajmohan t, anoop p, issaic c. pharmacological evaluation of curcumin for its nephroprotective activity in 5/6 nephrectomized rat model. drug invent today. 2018;10(1). 19. kristensen mlv, kierulf‐lassen c, nielsen pm, krag s, birn h, nejsum ln, et al. remote ischemic perconditioning attenuates ischemia/ reperfusion‐induced downregulation of aqp2 in rat kidney. physiol rep. 2016;4(13):e12865. 20. chole rh, patil rn, basak a, palandurkar k, bhowate r. estimation of serum malondialdehyde in oral cancer and precancer and its association with healthy individuals, gender, alcohol, and tobacco abuse. j cancer res therap. 2010;6(4):487. 21. ustundag y, huysal k, kahvecioglu s, demirci h, yavuz s, sambel m, et al. establishing reference values and evaluation of an in-house ferric reducing antioxidant power (frap) colorimetric assay in microplates. eur res j. 2016;2(2):126. 22. ahmadi m, hajihashemi s, rahbari a, ghanbari f. the effects of diclofenac on renal toxicity disorders induced by gentamicin in rats. j babol univ med sci. 2018;20(2):33-41. 23. yu c, qi d, sun j-f, li p, fan h-y. rhein prevents endotoxin-induced acute kidney injury by inhibiting nf-κb activities. scient rep. 2015;5:11822. 24. basile dp, anderson md, sutton ta. pathophysiology of acute kidney injury. comprehen physiol. 2011;2(2):1303–53. 25. chatauret n, badet l, barrou b, hauet t. ischemia-reperfusion: from cell biology to acute kidney injury. prog urol. 2014;24:s4–s12. 26. miller wl, cavero pg, aarhus ll, heublein dm, burnett jr jc. endothelinmediated cardiorenal hemodynamic and neuroendocrine effects are attenuated by nitroglycerin in vivo. am j hypertens. 1993;6(2):156–63. 27. mccomb mn, chao jy, ng tm. direct vasodilators and sympatholytic agents. j cardiovasc pharmacol therap. 2016;21(1):3–19. 28. krishnan s, kraehling j, eitner f, bénardeau a, sandner p. the impact of the nitric oxide (no)/soluble guanylyl cyclase (sgc) signaling cascade on kidney health and disease: a preclinical perspective. int j mol sci. 2018;19(6):1712. 29. singh rr, easton lk, booth lc, schlaich mp, head ga, moritz km, et al. renal nitric oxide deficiency and chronic kidney disease in young sheep born with a solitary functioning kidney. scient rep. 2016;6:26777. 30. vallon v. tubular transport in acute kidney injury: relevance for diagnosis, prognosis and intervention. nephron. 2016;134(3):160–6. 31. palmer bf. potassium homeostasis in chronic kidney disease. nephrol news issues. 2016;30(4):suppl 8–10, 2–3. 32. mount p, power da. nitric oxide in the kidney: functions and regulation of synthesis. acta physiol. 2006;187(4):433–46. 33. li c, salisbury r, ely d. hydralazine reverses stress-induced elevations in blood pressure, angiotensin ii, testosterone, and coronary pathology in a social colony model. isrn pathol. 2011;2011. 34. haynes w, hand m, dockrell m, eadington d, lee m, hussein z, et al. physiological role of nitric oxide in regulation of renal function in humans. am j physiol-renal physiol. 1997;272(3):f364–f71. 35. sharma s. nitric oxide and the kidney. ind j nephrol. 2004;14(3):77–84. 36. grover a, daniels e. calcium and contractility: smooth muscle: springer science & business media; 2012. 37. sabbahy me, vaidya vs. ischemic kidney injury and mechanisms of tissue repair. wiley interdisc rev syst biol med. 2011;3(5):606–18. 38. hutchens mp, dunlap j, hurn pd, jarnberg po. renal ischemia: does sex matter? anesth analg. 2008;107(1):239–49. 39. cabell ks, ma l, johnson p. effects of antihypertensive drugs on rat tissue antioxidant enzyme activities and lipid peroxidation levels. biochem pharmacol. 1997;54(1):133–41. 40. cheng yw, chiou gc. antioxidant effect of hydralazine on retinal pigment epithelial cells and its potential use in the therapy of age-related macular degeneration. int j ophthalmol. 2008;8(6):1059–64. 324 original hydralazine and renal ischemia-reperfusion in rat fatemeh ahmadi et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 317–324 41. p basile d, c yoder m. renal endothelial dysfunction in acute kidney ischemia reperfusion injury. cardiovasc haematol disord-drug targets (formerly curr drug targets-cardiovasc hematol disord.). 2014;14(1):3–14. 42. mercatello a, editor changes in renal function induced by anesthesia. ann franc anesth reanim.; 1990. 43. elkayam u, weber l, campese vm, massry sg, rahimtoola sh. renal hemodynamic effects of vasodilation with nifedipine and hydralazine in patients with heart failure. j am coll cardiol. 1984;4(6):1261–7. 44. zeisberg e, zeisberg m. a rationale for epigenetic repurposing of hydralazine in chronic heart and kidney failure. j clin epigenet. 2016;2(1). 45. elkayam u, cohen g, gogia h, mehra a, johnson jv, chandraratna pan. renal vasodilatory effect of endothelial stimulation in patients with chronic congestive heart failure. j am coll cardiol. 1996;28(1):176–82. 46. badavi m, naseri mkg, pirmoradi l, hosseini f. beta carotene modulates nitric oxide production in the renal ischemia/reperfusion injury in rat. zahedan j res med sci. 2017;19(3). 47. zuckerman r, patel m, costanzo ej, dounis h, haj ra, seyedali s, et al. hydralazine-associated adverse events: a report of two cases of hydralazine-induced anca vasculitis. braz j nephrol. 2018(ahead)324 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201905 82 j contemp med sci | vol. 5, no. 2, march–april 2019: 82–89 original nano-encapsulated tarragon (artemisia dracunculus) essential oil as a sustained release nano-larvicide mahmoud osanloo,a,b mohammad mehdi sedaghat,c hassan sereshti,d and amir amanie,f* adepartment of medical nanotechnology, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. bdepartment of medical nanotechnology, school of advanced technologies in medicine, fasa university of medical sciences, fasa, iran. cdepartment of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. ddepartment of chemistry, faculty of science, university of tehran, tehran, iran. enatural products and medicinal plants research center, north khorasan university of medical sciences, bojnurd, iran. fmedical biomaterials research center, tehran university of medical sciences, tehran, iran. *correspondence to amir amani (email: aamani@sina.tums.ac.ir). (submitted: 03 january 2019 – revised version received: 20 january 2019 – accepted: 11 february 2019 – published online: 26 april 2019) objective in recent years, essential oil-based larvicides have been introduced as alternatives to industrial ones. however, an appreciable formulation of essential oils with prolonged larvicidal activity (la) has not yet been developed. methods in this study, tarragon essential oil (teo) was encapsulated in chitosan nanoparticles using ion gelation technique. physicochemical properties and duration of la of the prepared nanoformulation were investigated. results encapsulation efficiency of the optimum nanoformulations with a particle size of 168 ± 90 nm was calculated as 39.66% using uv–vis analysis. encapsulating teo in chitosan–tripolyphosphate nanocapsules was shown to increase its efficiency in la at ssf test: perfect la (100% mortality) was achieved at lower concentration (i.e. 31 μg/ml instead of 80 μg/ml). also, perfect la continued for 4 and 3 days, compared with 2 days and 1 day for the non-encapsulated form of teo in the lab and sff tests, respectively. besides, the duration of la of nanoformulation was significantly longer than its corresponding microformulation with the same concentration of ingredients. furthermore, the concentration of teo in the solution tests was also monitored and it was found that the nanoformulations provide a sustained release of teo. moreover, there was a logical relationship between la and concentration of teo in different hours. conclusion this prepared nanoformulation could be introduced as an interesting alternative to synthetic larvicides, due to its easy and fast method of preparation and its green constituents. keywords nanoencapsulation, tarragon essential oil, larvicidal activity, anopheles stephensi, chitosan nanocapsules introduction malaria is still regarded as a global public health problem: an estimated death of 429,000 was caused by malaria just in the year 2015, with transmission occurring in 91 countries around the world.1 controlling mosquito larvae, especially in the endemic region, is probably the easiest and the best cost effective way to control mosquito-borne diseases and eliminate disease transmission.2–4 however, occurring resistance in mosquitoes, environmental pollution, and adverse effects on non-target species have been observed due to the continuous use of synthetic larvicides. in case of resistance, a study by peiris et al.5 was one of the first reports on the occurrence of resistance in mosquitoes (i.e. culex quinquefasciatus) against temephos. since then, several reports on the occurrence of resistance in other mosquitoes population have been published e.g. in aedes aegypti,6–8 and anopheles stephensi.9–11 to overcome the mentioned problems, encapsulations of active agents have been proposed. generally, by encapsulation of active components and providing a sustained release, two effects are expected: reduced side effects and prolonged activity.12–14 for example, encapsulation of temephos in polyethylene glycol (peg), and chitosan/alginate/gelatin against culex spp. led to the reduction of side effects on non-target species and environment by controlling the release of the larvicide.15,16 an interesting alternative for synthetic larvicides is the use of plant-derived essential oil/extraction. lethal concentration 50% (lc50) has been reported for many essential oils such as citrus aurantium (lc50: 31.20 μg/ml), citrus paradise (lc50: 35.71 μg/ml) and kelussia odoratissima (lc50: 4.77 μg/ml) against a. stephensi.17,18 besides, lc50 of leaf extracts of acanthospermum hispidum and cleistanthus collinus against a. stephensi were reported as 20.96 and 67.20 μg/ml, respectively.19,20 however, the main disadvantage of plant-derived essential oils is their very short shelf-life due to evaporation. thus, many attempts have been made to encapsulate essential oils for the control of insects both in agriculture (pests) or urban and rural life (vermin and vectors). for instance, peg nanocapsules containing garlic essential oil showed better mortality (80%) compared with the free eo (11%) after 5 months against tribolium castaneum.21 however, no work so far has reported of a long-lasting larvicidal activity (la) in aqueous media when nanoformulations of essential oil have been prepared.22–26 artemisia dracunculus, known as tarragon belongs to asteraceae family; different properties of its essential oil/ extract were confirmed in recent years. for example, adjusting the level of blood glucose19,20 or the inhibition of blood platelet adhesion.27,28 in this study, for the first time, a sustained release nanoformulation of tarragon essential oil (teo) has been introduced and its release profile has been monitored. furthermore, by encapsulating the teo in chitosan nanocapsules, we tried to overcome the volatility of teo and increasing continuity of la. also, la of nanoformulation was compared with microformulation, with the same concentration of ingredients. issn 2413-0516 m. osanloo et al. 83j contemp med sci | vol. 5, no. 2, march–april 2019: 82–89 original nano-encapsulated tarragon (artemisia dracunculus) essential oil materials and methods materials chitosan (mw = 100 kd, dd = 93%) was bought from easter holding group (china), teo was purchased from zardband pharmaceuticals co (iran), stored at 4–6°c, away from sunlight. tween 20 (tw), ethanol (eth), tripolyphosphate (tpp) and acetic acid were supplied from merck chemicals (germany). evaluation of larvicidal properties of teo and selected nanoformulations larvicidal activity of teo in the lab test was performed in our previous research (lc50 and lc90; 11.36 and 17.54 μg/ml, respectively).29 in this study, la of teo was evaluated under simulated semi-field (ssf) condition i.e. 34 ± 6°c with 12:12 light and dark photoperiods and 25 ± 10% relative humidity, comparable with temperature and humidity in a shaded location, during the hot season of tehran (research location). furthermore, the duration of la of each of the selected nanoformulations (i.e. f1, f2, and f3) was compared with similar concentrations of teo dissolved in eth (bulk teo), in both lab and ssf tests in line with who guidelines with some modifications.30 it should be noted that lab investigations were performed under recommended conditions (i.e. 28 ± 1°c with 12:12 light and dark photoperiods and 65 ± 5% relative humidity). for both types of larvicidal bioassays (i.e. lab and ssf), laboratory-reared 3rd and 4th instar larvae of a. stephensi were obtained from the department of medical entomology, tehran university of medical sciences. tests were repeated 12 times in three different replicates. in each replicate, two control groups were considered; having 1 ml of eth and nanoformulation without teo [f(-oil)]. during the tests, containers were isolated from the environment with help of nets, to prevent environment mosquitoes mixing with larvae of the tests. determining a diagnostic dose of teo in ssf test bulk teo stock solution (20 µl/ml) was prepared by dissolving and diluting with eth to prepare working standard solutions. one ml of working standard solutions was added to containers with 199 ml of no chlorine water to obtain bulk teo concentrations of 20, 40, 80 and 100 μg/ml in each container. containers solution was mixed. then, batches of 25 larvae were added to each container. after 24 h of exposure, dead larvae were counted. the lowest concentration of bulk teo that had perfect la (100%) was selected as the diagnostic dose for ssf test. evaluating the continuity of la of selected nanoformulation in lab and ssf tests in summary, batches of 25 larvae were added to containers having 199 ml with no chlorine water. by adding 1 ml from bulk teo or nanoformulations (i.e. f1, f2, and f3), teo concentration in each container was eventually fixed at 18, 50 or 80 μg/ml. after 24 h of exposure and counting the dead larvae, all the larvae (both dead and live) were removed using rubber pipette and other batches of 25 live larvae were added to the containers. the tests were stopped when la of containers equalled that of containers having controls. the tests were discarded when mortality in control groups (containing eth) increased to 5%, to ensure the accuracy of the test. preparation of chitosan nanocapsules containing teo from the larvicidal bioassays on bulk teo, three different concentrations of teo were selected for encapsulating in chitosan nanocapsules. the lowest amount of teo (i.e. 0.36%) was comparable with lc90 of bulk teo in the lab test and highest amount (i.e. 1.60%) was equal to the concentration of bulk teo having perfect la in ssf test. a third concentration was also studied as a value between the two values mentioned above (i.e. 1.00%). to prepare chitosan nanocapsules containing teo, ion gelation technique with some modifications was used.31 briefly, stock solutions of chitosan were prepared by dissolving chitosan (3% w/w) in an aqueous solution of acetic acid (1% w/w). different working aqueous solutions of chitosan (i.e. 0.05, 0.1, 0.2, 0.4, and 0.8%) were prepared by dilution of stock solution with acetic acid (1% w/w). after mixing teo, tw and eth (600 rpm, room temperature, and 15 min), working chitosan solutions were added dropwise, for preparing a homogenized mixture. for the formation of chitosan nanocapsules containing teo, aqueous solutions of tpp (i.e. 0.02, 0.04, 0.07, and 0.1% w/w) were added to each mixture at once. the mixtures were then stirred for 30 min (1800 rpm) at room temperature. it should be noted that, at each concentration of teo, a fixed amount of tw and eth was used to form a homogeneous mixture under the mentioned condition (600 rpm, room temperature, and 15 min). by using teo 0.36%, tw 2.5% and eth 7.14% was used. however, at teo 1.00%: tw and eth 3.00 and 6.00%, were used respectively. moreover, tw 2.80% and eth 5.80% were added using teo 1.6%. characterization of the nanoformulations particle size (ps) and particle size distribution (psd) of the formulations were determined using dynamic light scattering (dls, scatteroscope-i, k-one, korea), and confirmed by transition electron microscopy (tem, leo 906e zeiss, germany). d50, as reported by the dls instrument, was taken as ps. psd was calculated using eq. (1). measuring the size of the nanocapsules samples was repeated at three different times and the mean ± standard deviation was reported as the final size. psd = d d 75 25 (1) in each of the three concentrations of teo, one formulation with the smallest size (named f1, f2, and f3) was selected for further investigation such as characterization and larvicidal bioassays. for determining the encapsulation efficiency of the selected nanoformulations, the samples were centrifuged under defined conditions (i.e. 4°c, 17 700 g and 60 min). by determining the amount of teo in the supernatant using uv–vis spectroscopy (ce 7250 double beam spectrophotometer, cecil, uk) and deducing it from initial amount, using a specific regression equation, encapsulation efficiency was calculated [see eq. (2)]. nanoformulations without teo [f(-oil)] were also prepared and used as blank samples. encapsulation efficiency % = initial amount of teo the a ( ) mmount of teo in supernatant initial amount of teo ´100 (2) 84 j contemp med sci | vol. 5, no. 2, march–april 2019: 82–89 nano-encapsulated tarragon (artemisia dracunculus) essential oil original m. osanloo et al. to determine the weight of residue after centrifugation of formulation, after discarding the supernatant, upside down falcons containing the residue and were kept at 70°c for 30 min to evaporate its moisture. using eq. (3), the loading capacity of selected nanoformulations was calculated. determining the encapsulation efficiency and loading capacity for three selected nanoformulations were repeated at three different times and the mean ± standard deviation was reported as the final data. loading capacity % = encapsulated teo total weight of prec ( ) iipitate ´100 (3) monitoring concentration of teo during larviciding test to investigate the reason for longer durability of la of the optimum nanoformulation (f3) in comparison with its corresponding bulk teo, the concentration of teo was monitored during lab and ssf tests, using uv–vis spectroscopy. it should be noted that f3 has been selected as optimum nanoformulation due to longer durability of la (see section 3.4). evaluating the effect of encapsulation and size of the capsules in the duration of la larvicidal activity of f3 compared with its non-encapsulated form, i.e. no tpp was added [named f3(-tpp)] to assess the effect of encapsulation of teo in the nanoformulations. a second study was also performed to evaluate the effect of capsules size on the duration of la. for this purpose, a formulation with similar components to that of f3, but with larger size [named f3(micro)] was also prepared using a different processing condition (i.e. dropwise addition of tpp instead of one-shot addition). then, its la was compared with f3. statistical analysis to compare the continuity of la of the selected nanoformulations with each other or in comparison with related bulk teo or results of la in lab and ssf tests, independent-samples t-test with a 95% confidence interval (95% ci) was used. to analyze and compare the durability of la of the optimum nanoformulation (f3) with its related microformulation f3(micro) and non-encapsulated form f3(-tpp), one-way anova (95% ci) was used. statistical analyses were done using spss (v22) software. results and discussion diagnostic dose of teo at ssf larvicidal bioassay the lowest concentration of bulk teo showing perfect la in ssf test was 80 μg/ml (fig. 1). this value for the lab test was 20 μg/ml in our previous report.29 a reason for this difference could be extra evaporation of teo in the ssf test due to a higher temperature, the presence of air circulation, and lower humidity compared with the lab test. furthermore, the ingredients of the used batch of teo in this study were reported in our previous research. totally, 48 components of teo were determined by gc–ms analysis. five major components of teo include estragole (67.623%), cis-ocimene (8.691%), beta-ocimene y (7.577%), limonene (4.338%), and 3-methoxy cinnamaldehyde (1.491%).29 evaluating the effect of chi and tpp concentration on the size of the chitosan nanocapsules containing teo the effect of tpp and chitosan concentrations on the final size of the nanoformulation containing fixed amounts of teo, tw, and eth (i.e. 0.36, 2.50, and 7.14%, respectively) are demonstrated in fig. 2a. samples containing high concentrations of tpp (i.e. 0.07% and 0.10%) and low concentrations of chitosan (i.e. 0.10% and 0.05%) started to precipitate. thus, they were excluded from being reported in fig. 2a. the smallest formulation with the size of 384 ± 60 nm (named f1) had 0.10% and 0.04% of chitosan and tpp, respectively. from fig. 3a, psd of this nanoformulation was calculated as 2.2, showing a satisfactory distribution. in fig. 2b, the effects of concentrations of tpp/chitosan on the final size of the formulations are investigated when the amounts of teo, tw, and eth are fixed at 1.00, 3.00 and 6.00%, respectively. as the details show, to prepare smaller particles, a balance between concentrations of the ingredients is necessary. nanoformulation with the smallest particle size (named f2: 116 ± 40 nm) was obtained using 0.2% and 0.04% of chitosan and tpp, respectively. from fig. 3b, psd of this formulation was calculated as 1.3, an improved distribution compared with that of f1. the effect of concentrations of tpp/chitosan on the size of nanoformulation (containing a fixed amount of teo, tw and eth at 1.60, 2.80, and 5.60%, respectively) are illustrated in fig. 2c. the smallest nanoformulation (named f3) with the size of 168 ± 90 nm was obtained at 0.8% and 0.04% of chitosan and tpp, respectively. from fig. 3c: psd of this formulation was 1.34 with good monodispersity. a tem image of f3 depicted in fig. 3d, shows spherical particles with a size of 155 ± 12. our previous studies indicated that decreasing the size of nanoparticles led to an increase in its efficacy. for instance, mortality of larvae, when using nanoemulsion with the size of 11 nm (92.71%), was significantly better than the corresponding emulsion with the size of 9310 nm (81.67%) at 18 μg/ml concentration of teo.29 in another study, the nanoemulsion of essential oil of anethum graveolens with a particle size of ~10 nm, showed la of 81%, compared with the bulk form with la of 73%.4 in another report, neem nanoemulsion with three difference sizes (31, 93, and 251 nm) showed maximum la when fig. 1 larvicidal activity of essential oil at simulated semi-field (ssf) test. m. osanloo et al. 85j contemp med sci | vol. 5, no. 2, march–april 2019: 82–89 original nano-encapsulated tarragon (artemisia dracunculus) essential oil particle size was minimum (i.e. 31 nm).23 thus, in this study, the formulations with the smallest particle size (i.e. f1, f2, and f3) in each concentration of teo were selected for further evaluations including characterization and larvicidal bioassays. determining the encapsulation efficiency and loading capacity of the selected nanoformulations the results of encapsulation efficiency of the selected nanoformulations are illustrated in table 1. maximum encapsulation fig. 2 effect of tpp and chitosan concentrations on the size of the formulations containing three fixed amounts of essential oil: (a) 0.36%, (b) 1.00%, and (c) 1.60%. tpp: tripolyphosphate. a b c a c d b fig. 3 dls analysis of the selected nanoformulations: (a) f1 (384 ± 60 nm), (b) f2 (116 ± 40 nm) and (c) f3 (168 ± 90), respectively. (d) tem image of f3 nanoformulation. dls: dynamic light scattering, tem: transition electron microscopy. 86 j contemp med sci | vol. 5, no. 2, march–april 2019: 82–89 nano-encapsulated tarragon (artemisia dracunculus) essential oil original m. osanloo et al. efficiency (i.e. 39%) was for f3, probably, due to its larger chitosan content to carry teo. calculated regression equations and their calibration curves are demonstrated in fig. 4a–c. besides, one uv–vis spectrum is also presented as an example in fig. 4d. the range of encapsulation efficiency in this study was close to other similar studies i.e. encapsulation of essential oils in chitosan; such as zataria multiflora (45.24%),32 carum copticum (36.2%),33 oregano (24.72%).31 furthermore, loading capacity in this research was also comparable with similar studies [i.e. loading capacity values of 19.5% for c. copticum33 and 9.05% for z. multiflora].32 comparison of the duration of la of the selected nanoformulations and bulk teo in lab and ssf tests figure 5a compares la of f1 with bulk teo having a similar concentration (i.e. 0.36%) in the lab test. formulation without teo [i.e. f1(-oil)], showed no la. also, la of f1 was significantly lower (independent-samples t-test, p < 0.05) than that of bulk teo during the first day. this could be probably due to the encapsulation of teo which has made some of eo out of access to the larvae. however, during the second day, some la was still observed in f1, while bulk teo showed no la during the same period. it is arguable that this is due to complete evaporation of bulk teo after the first day, while the sustained release of the oil made some of it available on the second day. la of f1 did not continue on the third day. ssf test for f1 was not performed since the formulation failed to show a perfect la in the lab test. the results of the comparison of la of f2 with a similar concentration of bulk teo (i.e. 1.00%) in lab and ssf tests are illustrated in fig. 5b and 5c, respectively. in the lab test, f2 and bulk teo showed a perfect la for 2 days and 1 day, respectively. but, observed la for f2 was significantly higher than bulk teo at 48, 72, and 96 h (independent-samples t-test, p < 0.05). after 5 days, la of f2 did not have any significant difference (independent-samples t-test, p > 0.05) in comparison with la the blank sample [i.e. f2(-oil) ~ 15%]. thus, the test was stopped (data not presented). also, fig. 5c shows the ssf results for f2 and its corresponding bulk teo. from the details, perfect la was not observed in either sample. it is worth noting that la of bulk teo did not continue till the second day, while la of f2 was 32% on the second day which was significantly higher than bulk teo (independent-samples t-test, p < 0.05). this is another indication of the role of chitosan in providing a sustained release and preventing the loss of activity of teo. no la was observed on the third day, due to complete evaporation of teo in the test containers. the comparison between la of bulk teo (1.60%) and corresponding encapsulated nanoformulation (i.e. f3) in both lab and ssf was made and the results are given in fig. 5d and 5e. according to fig. 5d, perfect la was observed at 24 and 48 h in f3 and bulk teo, but la of f3 was significantly higher than at 72, 96, and 120 h (independent-samples t-test, p < 0.05) in a lab test. furthermore, full la of f3 in ssf test (see fig. 5e) continued for 72 h, while, bulk teo showed a full activity for only one day (significantly lower than, independent-samples t-test, p < 0.05). according to the mentioned results, the durability of la of f3 in both lab and ssf tests was significantly higher than other nanoformulations (f1 and f2) (independent-samples t-test, p < 0.05). thus, it was selected for further investigations as optimum nanoformulation. reviewing literature, slow release formulations containing essential oil of lippia sidoides have been found. by encapsulating that essential oil in chitosan/cashew gum beads with a size of around 1.5 mm, larvae of a. aegypti controlled up to 3 days.34 it was not clear what was the meaning of control, but this value was lower than that of this research i.e. 5 days. table 1. encapsulation efficiency and loading capacity of the selected nanoformulations formulations encapsulation efficiency (%) loading capacity (%) f1 31.21 ± 0.90 14.88 ± 1.50 f2 25.10 ± 1.00 16.30 ± 1.70 f3 39.66 ± 0.90 22.24 ± 2.00 fig. 4 specific calibration curves and regression equations for selected nanoformulations: (a) f1, (b) f2, and (c) f3. (d) uv– vis a sample spectrum. a b c d m. osanloo et al. 87j contemp med sci | vol. 5, no. 2, march–april 2019: 82–89 original nano-encapsulated tarragon (artemisia dracunculus) essential oil comparison of the concentration of teo during larviciding test the concentration of available teo in the solutions during larvicidal bioassays for the samples, bulk teo and optimum nanoformulation (f3) is shown in fig. 6. in general, the rate of decrease in concentration of oil in bulk teo in lab test (y = −0.6012x + 60.905) is lower than that of ssf test (y = −0.7333x + 55.4). this phenomenon is also observed in f3: related line equation in lab and ssf tests are y = −0.4479x + 61.393 and y = −0.5286x + 58.048, respectively. these are due to harsh conditions such as higher temperature and air circulation which make higher evaporation of teo in ssf. on the other hand, the decreasing rate in teo concentration is slower in f3 compared with bulk teo in both lab and ssf. as the details show, bulk teo shows a higher teo concentration at the beginning, followed by a sharp drop in teo concentration, in both lab and ssf tests. however, chitosan serves as an oil reservoir. thus, it provides a sustained release of teo, which in turn increases teo concentration. this makes a longer duration of action for the nanoformulation compared with bulk teo. interestingly, teo concentrations are comparable with the activity of samples at a lab test (see fig. 5d). for instance, the concentration of bulk teo at 72 h in the lab was 11 μg/ml, close to lc50 of teo in lab test (11.36 μg/ml) and the la observed was 47%. however, in ssf test (fig. 5e), the concentration and la values do not agree. for instance, the concentration of bulk teo at 24 h (ssf) was 19 μg/ml and its la was 100%. while, according to fig. 1, the lowest concentration to have a perfect la should be 80 μg/ml. in fig. 5e, expected la for this concentration (i.e. 19 μg/ml) occurred at 48 h (i.e. 22% in ssf). it is, therefore, arguably that in ssf test, la strongly depends on the concentration of teo at the beginning time of exposure. in other words, the perfect la which was observed at 24 h was due to the concentration of bulk teo at zero time rather than 24 h after the start of the test. furthermore, according to figs. 1, 5e, and 6, la of f3 significantly improved compared with bulk teo at ssf (independent-samples t-test, p < 0.05): lowest concentration of bulk teo with full la at ssf was 80 μg/ml; while f3 at 48 h with 31 μg/ml concentration of teo showed 100% la (see results of 72 h in fig. 5e). it is, therefore, arguable that, by fig. 5 comparison of duration of larvicidal activities of selected nanoformulation vs. bulk teo in the equal concentration of essential oil and also nanoformulations without oil both in lab and ssf tests. the final concentrations of teo in the tests containers are: (a) 18 μg/ml, (b and c) 50 μg/ml, (d and e) 80 μg/ml. teo: tarragon essential oil, ssf: simulated semi-field. a b d c e 88 j contemp med sci | vol. 5, no. 2, march–april 2019: 82–89 nano-encapsulated tarragon (artemisia dracunculus) essential oil original m. osanloo et al. using nanoformulation of teo, not only durability of la increases, but the efficacy rises too. no report has been found indicating that monitoring concentration of any eo during the larvicidal bioassays and evaluating related la was performed simultaneously. evaluating the encapsulation effect and the size of the capsules on the duration of la figure 7 reports the la of f3 in non-encapsulated form i.e. when no tpp was added to encapsulate the teo: f3(-tpp) and when the particle size was in the range of micro [i.e. 850 ± 35 nm, f3(micro)] in both lab and ssf tests. the results show that perfect la of f3(-tpp) and f3(micro) remain for 2 and 3 days in lab tests, respectively, which are significantly lower than that of f3 i.e. 4 days, (one-way anova, p < 0.05). these values decrease to 1 and 2 days in ssf test, respectively which are significantly smaller than that of f3 i.e. 3 days (one-way anova, p < 0.05). interestingly, la profile of f3(-tpp) is similar to that of bulk teo (1.60%) in fig. 5d. considering the fact that ingredient in f3(-tpp) was similar to f3 except for the presence of tpp which is necessary to prepare chitosan nanocapsules, the important role of encapsulation of teo is clear. whereby non-encapsulated teo acts similar to bulk teo. since tpp had no la (data not given), the preparation of chitosan nanocapsules is the reason for the significant changes in la of f3 and f3(-tpp) samples (independent-samples t-test, p < 0.05). furthermore, to evaluate the effect of particle size, la of f3 was compared with its corresponding microformulation preparation [i.e. f3(micro)], with same ingredients concentration, just with a larger particle size (i.e. 850 ± 35 nm). the duration of la of f3(micro) was significantly lower than f3 (independent-samples t-test, p < 0.05), most probably due to higher precipitation of microformulation compared to nanoformulation, as we noted during the tests. in other words, precipitation instead of evaporation removed teo from the solutions, thus, less activity was observed. it was mentioned that larvae of a. stephensi normally breathe on the surface of the water and avoids the bottom of the container that contained precipitated microparticles.35 this reduces the chance of physical interactions between larvae and the preparation containing teo. these results were in line with a report in which essential oil of l. sidoides was loaded in alginate/cashew gum beads in two forms [i.e. floating and non-floating beads (~1.7 mm)]. floating beads showed very good buoyancy up to 6 days and its la after 48 h was around 85% against a. aegypti, while corresponded mortality to non-floating beads was around 30%.14 conclusion encapsulated teo into chitosan-tpp nanocapsules showed a longer duration of action and also higher efficacy in the lab fig. 7 evaluation of the larvicidal activity of non-encapsulated f3 [f3(-tpp)] and microformulation [f3(micro)], both in the lab (left) and simulated semi-field (ssf) (right) test. the final concentration of teo in the tests containers is 80 μg/ml. tpp: tripolyphosphate, teo: tarragon essential oil. fig. 6 comparison of the concentration of bulk teo (1.6% essential oil) and f3 (nanoformulation with 1.6% essential oil) during the larviciding test in the lab and simulated semi-field (ssf) tests. teo: tarragon essential oil. m. osanloo et al. 89j contemp med sci | vol. 5, no. 2, march–april 2019: 82–89 original nano-encapsulated tarragon (artemisia dracunculus) essential oil and simulated semi-field tests compared with bulk teo, non-encapsulated teo, and chitosan microcapsules. the nanocapsules were able to protect the teo from evaporation to provide long-lasting la. as investigated, there is a logical relationship between la and the concentration of teo in different hours. the possibility of scale-up has been realized by using the very fast and facile method to nanoencapsulation of teo. based on the points mentioned, this formulation could be an interesting alternative to industrial larvicides. acknowledgments this research was supported by tehran university of medical sciences & health services grant no. 95-01-87-31860, and also had ethical approval by its ethical committee centre, ir. tums.rec.1395.2480. conflict of interest there is no conflict of interest among the authors.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1. who. world malaria report 2016, 2016. available from: http://www.who. int/malaria/publications/world-malaria-report-2016/report/en/. 2. bellan se. the importance of age dependent mortality and the extrinsic incubation period in models of mosquito-borne disease transmission and control. plos one. 2010;5:e10165. 3. sumitha kv, thoppil je. larvicidal efficacy and chemical constituents of o. gratissimum l. (lamiaceae) essential oil against aedes albopictus skuse (diptera: culicidae). parasitol res. 2016;115:673–680. 4. osanloo m, sereshti h, sedaghat mm, amani a. nanoemulsion of dill essential oil as a green and potent larvicide against anopheles stephensi. environ sci pollut res int. 2018;25:6466–6473. 5. peiris htr, hemingway j. temephos resistance and the associated crossresistance spectrum in a strain of culex quinquefasciatus say (diptera: culicidae) from peliyagoda, sri lanka. bull entomol res. 1990;80:49–55. 6. wirth mc, georghiou gp. selection and characterization of temephos resistance in a population of aedes aegypti from tortola, british virgin islands. j am mosq control assoc. 1999;15:315–320. 7. melo-santos ma, varjal-melo jj, araújo ap, gomes tc, paiva mh, regis ln, et al. resistance to the organophosphate temephos: mechanisms, evolution and reversion in an aedes aegypti laboratory strain from brazil. acta trop. 2010;113:180–189. 8. rodríguez mm, bisset j, ruiz m, soca a. cross-resistance to pyrethroid and organophosphorus insecticides induced by selection with temephos in aedes aegypti (diptera: culicidae) from cuba. j med entomol. 2002;39:882–888. 9. soltani a, vatandoost h, oshaghi ma, ravasan nm, enayati aa, asgarian f. resistance mechanisms of anopheles stephensi (diptera: culicidae) to temephos. j arthropod borne dis 2015;9:71–83. 10. vatandoost h, hanafi-bojd aa. current resistant status of anopheles stephensi liston to different larvicides in hormozgan province, southeastern iran, 2004. pak j biol sci. 2005;8:1568–1570. 11. vatandoost h, mashayekhi m, abaie mr, aflatoonian mr, hanafi-bojd aa, sharifi i. monitoring of insecticides resistance in main malaria vectors in a malarious area of kahnooj district, kerman province, southeastern iran. j vector borne dis. 2005;42:100–108. 12. li j, li y, dong h. controlled release of herbicide acetochlor from clay/carboxylmethylcellulose gel formulations. j agric food chem. 2008;56:1336–1342. 13. maji tk, baruah i, dube s, hussain mr. microencapsulation of zanthoxylum limonella oil (zlo) in glutaraldehyde crosslinked gelatin for mosquito repellent application. bioresour technol. 2007;98:840–844. 14. paula hc, de oliveira ef, abreu fo, de paula rc. alginate/cashew gum floating bead as a matrix for larvicide release. mater sci eng c mater biol appl. 2012;32:1421–1427. 15. badawy mei, taktak nem, awad om, elfiki sa, el-ela nea. larvicidal activity of temephos released from new chitosan/alginate/gelatin capsules against culex pipiens. int j mosq res. 2015;2:45–55. 16. bhan s, mohan l, srivastava cn. relative larvicidal potentiality of nanoencapsulated temephos and imidacloprid against culex quinquefasciatus. j asia pac entomol. 2014;17:787–791. 17. sanei-dehkordi a, sedaghat mm, vatandoost h, abai mr. chemical compositions of the peel essential oil of citrus aurantium and its natural larvicidal activity against the malaria vector anopheles stephensi (diptera: culicidae) in comparison with citrus paradisi. j arthropod borne dis. 2016;10:577–585. 18. osanloo m, amani a, sereshti h, shayeghi m, sedaghat mm. extraction and chemical composition essential oil of kelussia odoratissima and comparison its larvicidal activity with z-ligustilide (major constituent) against anopheles stephensi. j entomol zool stud. 2017;5:611–616. 19. vivekanandhan p, senthil-nathan s, shivakumar ms. larvicidal, pupicidal and adult smoke toxic effects of acanthospermum hispidum (dc) leaf crude extracts against mosquito vectors. physiol mol plant pathol. 2018;101:156–162. 20. jinu u, rajakumaran s, senthil-nathan s, geetha n, venkatachalam p. potential larvicidal activity of silver nanohybrids synthesized using leaf extracts of cleistanthus collinus (roxb.) benth. ex hook. f. and strychnos nux-vomica l. nux-vomica against dengue, chikungunya and zika vectors. physiol mol plant pathol. 2018;101:163–171. 21. yang fl, li xg, zhu f, lei cl. structural characterization of nanoparticles loaded with garlic essential oil and their insecticidal activity against tribolium castaneum (herbst) (coleoptera: tenebrionidae). j agric food chem. 2009;57:10156–10162. 22. duarte jl, amado jrr, oliveira aemfm, cruz ras, ferreira am, souto rnp, et al. evaluation of larvicidal activity of a nanoemulsion of rosmarinus officinalis essential oil. rev bras farmacogn. 2015;25:189–192. 23. sugumar s, clarke sk, nirmala mj, tyagi bk, mukherjee a, chandrasekaran n. nanoemulsion of eucalyptus oil and its larvicidal activity against culex quinquefasciatus. bull entomol res. 2014;104:393–402. 24. rodrigues ecr, ferreira am, vilhena jce, almeida fb, cruz ras, florentino ac, et al. development of a larvicidal nanoemulsion with copaiba (copaifera duckei) oleoresin. rev bras farmacogn. 2014;24:699–705. 25. ghosh v, mukherjee a, chandrasekaran n. formulation and characterization of plant essential oil based nanoemulsion: evaluation of its larvicidal activity against aedes aegypti. asian j chem. 2013;25:s321–s323. 26. anjali ch, sharma y, mukherjee a, chandrasekaran n. neem oil (azadirachta indica) nanoemulsion—a potent larvicidal agent against culex quinquefasciatus. pest manag sci. 2012;68:158–163. 27. shahriyary l, yazdanparast r. inhibition of blood platelet adhesion, aggregation and secretion by artemisia dracunculus leaves extracts. j ethnopharmacol. 2007;114:194–198. 28. sayyah m, nadjafnia l, kamalinejad m. anticonvulsant activity and chemical composition of artemisia dracunculus l. essential oil. j ethnopharmacol. 2004;94:283–287. 29. osanloo m, amani a, sereshti h, abai mr, esmaeili f, sedaghat mm. preparation and optimization nanoemulsion of tarragon (artemisia dracunculus) essential oil as effective herbal larvicides against anopheles stephensi. ind crops prod. 2017;109:214–219. 30. who. guidelines for laboratory and field testing of mosquito larvicides 2005. available from: http://apps.who.int/iris/handle/10665/69101. 31. hosseini sf, zandi m, rezaei m, farahmandghavi f. two-step method for encapsulation of oregano essential oil in chitosan nanoparticles: preparation, characterization and in vitro release study. carbohydr polym. 2013;95:50–56. 32. mohammadi a, hashemi m, hosseini sm. nanoencapsulation of zataria multiflora essential oil preparation and characterization with enhanced antifungal activity for controlling botrytis cinerea, the causal agent of gray mould disease. innov food sci emerg technol. 2015;28:73–80. 33. esmaeili a, asgari a. in vitro release and biological activities of carum copticum essential oil (ceo) loaded chitosan nanoparticles. int j biol macromol. 2015;81:283–290. 34. paula hcb, sombra fm, de freitas cavalcante r, abreu foms, de paula rcm. preparation and characterization of chitosan/cashew gum beads loaded with lippia sidoides essential oil. mater sci eng c. 2011;31:173–178. 35. nation jl sr. insect physiology and biochemistry. crc press; 2008. dx.doi.org/10.22317/jcms.04201904 31j contemp med sci | vol. 1, no. 4, autumn 2015: 31–35 research objectives this study includes the fungi isolated from apple fruits and diagnosis study with ecological factors for aspergillus terreus. methods the samples were collected from apple varieties which included red importer apples, red local apples, golden yellow importer apples, green importer apples. the collected apples had some obvious lesion or spoilage and isolated number of fungi that differences in appearance percent and used two primers seq id no and its for a. terreus diagnosis by using pcr technique; in addition the study includes using three factors effective on fungal growth with temperature 15, 20, 25, 30 and 35 c° with ph 4, 6, 8, 10 and 12 and the third factor culture medium pda, sda, cza and mea. results results revealed the presence of nine types of fungi which includes p. expansum, rhizopus solonifer, alternaria spp, a. flavus, a. niger, a. terreus, p. digitatum, p. italicum and yeast isolated from various apple fruits. number of isolates p. expansum more than other fungi while less number was found in rhizopus stolonifer. the a. terreus identified by amplicon size of that appear 100 bp for six isolates and it appears that four isolates carrying this gene has size of 600 base pairs. this study showed significant differences (p < 0.05) in effect of five temperatures 15, 20, 25, 30 and 35°c in the growth of fungus a. terreus and ph levels on growth on three isolates of genus the types of culture media (pda, sda, cda, mea) significant at p < 0.05 affected the growth of a. terreus and it had different morphological characteristics. conclusion number of fungi was found in apple and identified a. terreus by pcr method and studied different ecology factors on growth fungus. the best degree for the growth of isolates in the sixth day was 30°c. maximum level of growth of the a. terreus was at ph 6 for all isolates in the sixth day and the growth rate for the same day on sda reached 7.13 cm which was considered the best media for growth. keywords a. terreus, apple fruits, pcr technics, environment factors diagnostic and environmental study of aspergillus terreus isolated from various varieties of apples fruits khadega hamza kadhim & ibtihal muiz al-hussaini introduction the fungi is one of the pathogens that cause damage to the fruits and vegetables after harvest1 where the plants get injured during the harvest, marketing, packaging and storage. the infection begins during wounds that you get during handling and harvesting.2 the apples infected by many types of fungi that cause rot in fruit especialy during post-harvest especially by fungi pencillium expansum, monilininia fracticola, mucor piriformis, alternaria ssp., colletrichum denatum botrytis cinerea, the type of fungi as p. expansum, botrytis cinerea, alternaria ssp may it caused a very large economic losses in italy.3. the apples are rich in salts and vitamins and nutrients, where apple medium-sized contain with peel (182 g), according to the us department of agriculture fat 0.31%, 0.47%, carbohydrates, proteins 25.13%, fiber 4.4%, sugars 18.91%, and the fruit of apples contains 4.46% kind of vitamins a, b, e and folic acid.4 among the benefits of the apples to strengthen the brain, heart, gums its seeds are used to kill the abdomen worm and prevent constipation and indigestion. apples have nutritional values and it is a compromise natural catalyst for the growth of microorganism, especially fungi, where apples are exposed during the harvest, storage and transport to scratches which facilitates the entry of fungi to the texture of the mature fruit and then putrefaction and obtain significant economic losses causing rotting of fruits due to fungus a. terreus during storage and marketing.5 on the other hand, the fungus events to rot in fruit, especially apples stimulates some strains of the fungus to secrete carcinogens mycotoxins. the best ways that reduce crop yields and food mycotoxin contamination with mycotoxin is by preventing appropriate conditions for fungal growth and production of toxins as well as the need to examine all the food processed for human consumption to make sure they do not contain the mycotoxins which harmful to human health6, 7. this study included isolates and diagnosed fungi issn 2413-0516 university of babylon, college of science, department of biology, babylon, iraq correspondence to ibtihal muiz al-hussaini (email ebtihalmuiz@yahoo.com). (submitted: 13 october 2015 – revised version received: 24 october 2015 – accepted: 5 november 2015 – published online: autumn 2015) associated with the fruits of infected apples imported to local markets.babylon’s traditional methods and molecular diagnosis of isolates of fungus a. terreus and also the impact of environmental conditions for the fungus are under study. materials and methods this study was done in the laboratory of mycotoxins and bio-technology in the department of biology. sample collection samples of apples were collected from different local markets between october and december of 2015 in the province of babylon. ten kilograms of the samples were collected for each class of varieties under study places. these included red importer apples, red local apples, golden yellow importer apples, green apple importer apples. the collected apples had some obvious lesion or spoilage. each apple was placed in a sterile plastic bag in room temperature (25–30 c) for 6 days or until fungal growth was evident all over the sample. the purpose was to investigate the frequency of the presence of fungus in apples. the types of culture media which was used in this study were (1) potato dextrose agar (pda), (2) czapeck’s agar with sucrose (cda20s), (3) sabourauds agar (sda) and (4) malt extract (mea). isolate and diagnose fungi associated with the apples under study detection of microorganisms was performed using a moist chamber method. the transfer of apple samples to a laboratory to isolate and diagnose fungi then cut each apple into five small pieces and put in petridishes (diameter 9 cm) containing the pda for each class separately and repeated the process three times (replicates) for each class of the apples then incubated all 32 j contemp med sci | vol. 1, no. 4, autumn 2015: 31–35 diagnostic and environmental study of aspergillus terreus research khadega hamza kadhim & ibtihal muiz al-hussaini table 1. programme of pcr technique seq id no. for a. terreus no. steps steps temperature time no. of cycles 1 initial denaturation 94°c 5 min 1 2 denaturation 94°c 30 min 353 annealing 50°c 45 min 4 elongation 72°c 1 min 5 final extension 72°c 5 min 1 table 2. programme of pcr technique its for a. terreus no .steps steps temperature time no. of cycles 1 initial denaturation 95°c 120 min 1 2 denaturation 95°c 5 min 453 annealing 60°c 45 min 4 elongation 72°c 1 min 5 final extension 72°c 5 min 1 dishes under a temperature of 25 ± 2°c for 7 days.8 developed fungi were identified by their morphological features according to the previously described methods6 and all ratios were calculated according to the following equation: study of effects of some ecological factors in the growth of a. terreus temperature: a series of temperature were arranged (15, 20, 25, 30 and 35) for the growth of a. terreus in sterile petri dish which contains 20 ml pda and the plate was inoculated in a disc 10 mm from the edge of the colony and incubated dishes for 6 days under 25°c (three replicates). radial growth (colony diameter) was estimated at 2 days interval till full expansion of the growth on different temperature treatment was reached.9 ph: various ph were arranged (4, 6, 8, 10 and 12) for growth of a. terreus. the pda was divided pon five flasks glass size 500 ml to 250 ml container, the 4 and 6 by adding drops of hydrochloric hcl acid (5 mm) and a ph of 10 and 12 by adding drops of sodium naoh solution (40%). inoculation steps, incubation condition and growth measurement of fungal isolates were conducted as mentioned earlier.10 effect of culture media: to obtain a suitable medium for enhanced growth by a. terreus various media were arranged (pda , sda, cda and mea ) in sterile petri dish. inculcation steps, incubation condition and growth measurement of fungal isolates were conducted as reported above.11 dna extraction and sequence analyses: after 7 days, growth of fungal colonies on pda was counted and recorded in a colony forming unit per millilitre (cfu/ml). dna was extracted from isolates using the genomic dna purification kit (promega, madison, wi, usa). small subunit ribosomal rna (mtssu rrna) and β-tubulin were then amplified by pcr using primer pairs its1/its4 primers (its1/ttc gta ggt gaa cct gcg g) and (its4/tcc tcc gct tat tga tat gc) and (seq id no: 2f/aca tga acc ctg ttc tga aag) and (seq id no: 2r/cca aga gat cca ttg ttg aag) the conditions described12 here are primers for a. terreus diagnosis. for detection of dna, gel electrophoresis method was used by using agarose gel (1% concentration). this method consisted of three steps: preparation of agarose gel, preparation of casting agarose and the addition of samples. pcr amplification condition: this method is used to amplify dna by using the specific primer. these methods are dependent on the volumes that are found in master mix provided from bioneer (tables 1, 2). the time and vol. which is used for electrophoresis methods: to obtain clear band after pcr running , that used different time and volume during electrophoresis. results table 3 showed that there are many types of fungi in apple varieties, pathogenic fungus p. expansum was recorded high in percentage with p. italicum appearance in most apple varieties (table 3). the highest proportion of the emergence of p. expansum amounted to 90% in the red apples imported, and the lowest rate of 30% in the appearance of golden yellow apples imported. a. terreus was in highest percentage imported. p. digitatium was the highest rate imported (20%), but did not appear in green apple imported. p. digitatium was the highest rate in green apple imported (60%) and the lowest rate appearance was in the golden yellow apples imported at a rate of 30%. the percentage of the emergence of altrenaria spp. in red apple domestic and golden yellow apple imported as 30% did not appear in green apples either. a. niger was the highest proportion of its appearance in the golden yellow apple imported and that 60% did not appear in green apple imported. a. flavus was at high percentage in the golden yellow apples imported did not appear in green apple imported. table 3. percentages of appearance of fungi isolated from apple varieties the percentage of appearance (%) yeastrhizopus stoloniferalternaria sppa. flavusa. nigera. terrusp. digitatump . italicump. expansumtypes of apple fruit 20102050305504090red imported apples 40203060205408060red local apples 701030706020305030golden yellow apples imported 1000000607040green apple imported the percentage of appearance (%) = the number of samples thhat have fungus total number of isolates 100× 33j contemp med sci | vol. 1, no. 4, autumn 2015: 31–35 research diagnostic and environmental study of aspergillus terreuskhadega hamza kadhim & ibtihal muiz al-hussaini as for yeasts the highest rate in the emergence of golden yellow apples by 70% and the lowest percentage of appearance in the green apple imported by 10%. identification of a. terreus based on molecular characteristics the results shown in fig. 1 are the isolated fungi and gene coding for seq id amplified by pcr technique. eight isolates of a.terreus whose diagnosis by using specialized primers seq id 2f: aca tga acc ctg ttg tga aag opposite the primer seq id no: cca aga gat cca ttg ttg aaa. the amplicon size of a. terreus appear 100 bp for six isolated. as well as the universal pair primer was successful in amplification of its region for different isolates of a.terreus had been appears that 4 isolates were carrying this gene has size of 600 base pairs in lane (2, 3, 4,5) (fig. 2). effect of some ecological factors in the growth of a. terreus temperature the results of this experiment (table 4) demonstrated that the growth of a. terreus varied according to the level of temperature. this study showed significant differences (p < 0.05) with effect to five temperatures 15, 20, 25, 30 and 35°c. the best degree for the growth of isolate at-1 on the sixth day was 30°c, with the growth rate of 9 cm, followed by 35°c growth rate of 6.66 cm. for isolate at-2 the best temperature was 30°c with a growth rate of 7.16 cm in diameter, while isolate at-3 had the best growth on the sixth day at 30°c with growth rate of 8.6 cm. as the results showed in table 4 the temperature of 25°c all isolates have less growth in the sixth day with 2.83, 3.21 and 3.13 cm, respectively. but at temperatures of 15°c and 20°c caused reduced growth rates in all isolates of a. terreus. ph the results showed that test three isolates of the a. terreus had variation in growth rates through the use of four ph (table 5) and there were significant differences (p < 0.05) in the growth of a. terreus on pda at a temperature of 25 ± 2°c. maximum level of growth of a. terreus was at ph 6 for isolates af-1 and fig. 1 gel electrophoresis of amplified pcr product from different a. terreus isolate, by using sqe primer (in vol. = 100, agarose = 0.4%). fig. 2 gel electrophoresis of amplified pcr product from different a. terreus isolates by using its primer (in vol. = 100, agarose = 1%) table 4. effect of different temperature on the growth of a. terreus isolates on pda after 6 days of incubation at ph 7 isolates days temperatures 15°c 20°c 25°c 30°c 35°c diameters colonies (cm) at-1 2 0 0 2.5 3.26 2.9 4 0 0 2.5 6.66 4.55 6 1.51 0 2.83 8.33 6.66 at-2 2 0 0 2.16 2.9 3 4 0 1.33 2.8 5.5 3.66 6 0 1.5 3.21 7.16 4.33 at-3 2 0 0 1.66 3.33 3 4 0 0.9 2.58 6 4.66 6 0 1.3 3.13 8.6 6.5 lsd value for temperature = 0.569. lsd value for incubation days= 0.445. lsd value for overlap = 1.707. table 5. effect of different ph on the growth of a. terreus isolates on pda after 6 days of incubation at a temperature of 25 ± 2°c isolates days hydrogen function effect 4 6 8 10 12 diameters colonies (cm) at-1 2 0 2.18 0 0.43 0 4 0 4.16 4.71 0.45 0 6 0 6.23 5 0.47 0 at-2 2 0 3.33 2.81 1.6 0 4 0 5.1 4.55 3.13 0 6 0 7.83 6.23 2.41 0 at-3 2 0 2.8 1.83 1.68 0.8 4 0 5 3.51 2.016 0.96 6 0 6.51 4.33 2.65 1.36 af-2 in the sixth day with 6.23 and 7.83 cm, respectively, and radial growth for isolate af-3 increased at ph 6 (6.23), followed by growth at ph 8, which amounted to 5 cm and 6.23 cm and 4.33 cm for isolates af-1 and af-2 and af-1, respectively. but all the isolates will not growth at ph 4 and 12, except af-3 which will have little growth at ph 12. culture media the results demonstrated that the type of culture media (pda, sda, cda, mea) significant (p < 0.05) affected the growth of a. terreus and its different morphological characteristics was evident that most of the isolates were of colonies of brown colour and characterised isolates at pda and sda were light brown while on mea it was reddish brown colour and cda was dark brown (fig. 3). the growth rate in the sixth day of isolate at-1 was 5.75 cm on pda while the growth rate for the same day on sda reached 7.13 cm, and 6.23 cm growth on cda. the results (table 6) showed the percentage growth on mea was 6.36 cm, but for the isolate at-2 growth rate at sda in the same day reached 8.65 cm while it was 7.86 cm on cza in the same day 34 j contemp med sci | vol. 1, no. 4, autumn 2015: 31–35 diagnostic and environmental study of aspergillus terreus research khadega hamza kadhim & ibtihal muiz al-hussaini table 6. effect of different media on the growth of a. terreus isolates on pda after 6 days of incubation at a temperature of 25 ± 2°c and ph 7 isolates days culture media types pda sda cza mea diameters colonies (cm) at-1 2 3.18 2.25 2.1 1.28 4 4.58 3 4.33 5.33 6 5.75 7.13 6.23 6.36 at-2 2 2.85 3.43 2.83 1.5 4 2.51 4.85 3.51 2.05 6 4.7 8.65 7.86 2.76 at-3 2 2.25 2.66 3.83 1.66 4 2.9 4.35 4.51 2.33 6 3.56 7 6 2.8 lsd value for the type of center = 0.593. lsd value for incubation days = 0.513. lsd value for overlap = 1.781. and frequency of the p. expansum. these results were similar to those reached by churki13 in which he referred that p. expansum may record the highest rate displayed on apples which amounted to 100%, while a. terreus had the lowest emergence and fungi and other isolated from apples the lowest percentage of the appearance of 25%, while the current results do not agree with the findings of pitt,14 in which pencillium ssp. comes second place after rhizopus stolinifer in the damage apples. molecular studies become crucial and necessary for identification of pathogenic fungi.15 based on the data reported12 the molecular weight obtained in the current result agreed with them. on the other hand, the internal transcribed spacer (its) region of the fungal ribosomal dna (rdna) had been used as one of techniques for species identification because it is faster, has accurate species determination, specific, and are less feasible to be affected by exterior effects such as temperature changes and chemotherapy.16 in some isolates which are not product gene (its) and does not appear, resulted that the isolates are able to link with dna supplements appropriately to the absence of the relay so did not give a positive result.17 there have been many studies that deal with environmental conditions, the temperature, ph of culture medium and type of culture medium which were the most important ecology factors that affect the growth of microorganisms and reproduction.18 the degree of appropriate temperature for the growth of a. terrues ranging between 35–40°c19 and in the current study founded that the best growth of the fungus was 30°c. while it was less growth in 15c°c. increasing or decreasing of temperature higher or lower than optimum range may lead to breakdown of enzymes such as cellulose and was importance for the growth and mycotoxigenesis.20 all isolates in this study are grown in ph 6. these results agree with the conclusion of abubakar.21 the ph effect on ions change which enter through cell membrane plasma had done at ph 5.5–6 if increased this value may lead to an imbalance that leading to weakness ions across, and the ph role of the exploits of the entry into force of ions across the membrane. ph becomes the cell membrane and adheres to hydrogen ions by blocking the passage of positive ions, either when the high ph, the hydrogen ions act to prevent the passage of negative ions.23 the culture media sda was found to be the most effective for supporting the maximum growth of a. terrues, because this medium contains nitrogen, potassium and phosphours which provided the fungi with necessary growth requirements.24 conclusion number of fungi in apples were found and a. terreus was identified by pcr method and different ecology factors on growth fungus were studied. the best degree for the growth of isolates in the sixth day was 30°c. maximum level of growth of a. terreus was at ph 6 for isolates in the sixth day and the growth rate for the same day on sda reached 7.13 cm and was considered the best media for growth. figure 3 phenotypic characteristics of a. terreus on four media. reached 8.65 cm while it was 7.86 cm on cza. mea that give colony diameter (2.76 cm) for isolate at-3, lower growth was recorded by pda for isolate at-3 (3.56). it was found that the growth on sda was 7 cm followed by culture media cza (6 cm) at the end of incubation period while this ratio was 2.8 cm on mea. discussion there are a lot of researches that isolates of various fungi of the apples and recorded the proportion of the highest appearance references 1. sommer s. rot of controlled environments in suppression of postharvest diseases. can j plant pathol. 1985;7:331–6. doi: http://dx.doi. org/10.1080/07060668509501700 2. sugar d. control of postharvest diseases of pome fruits by field application of bio control agents. phytopathology. 2001;91:s155. 3. spadaro d, vola r, piano s, gullino ml. mechanisms of action and efficacy of four isolates of the yeast metschnikowia pulcherrima active against postharvest pathogens on apples. postharvest biol technol. 2002;24:12– 134. doi: http://dx.doi.org/10.1016/s0925-5214(01)00172-7 4. al-naimi jh. fruit. basra university press, faculty of agriculture; 1983. p. 552. 5. vinas ijv, teixid n, sanchis v. biological control of major post-harvest pathogens on apple with candida sake. int j food microbiol. 1998;40:9–16. pmid: 9600605 6. kong f, tong z, hen x. rapid identification and differentiation of trichophyton species, based on sequence polymorphisms of the ribosomal 35j contemp med sci | vol. 1, no. 4, autumn 2015: 31–35 research diagnostic and environmental study of aspergillus terreuskhadega hamza kadhim & ibtihal muiz al-hussaini internal transcribed spacer region ,by rolling circle amplification. j clin microbiol. 2008;46(4):1192. pmid:18234865 7. ibrahim ik, al-juboury mi. mycotoxin, 2nd ed. apaa agricultural research; 1998. pp. 343. 8. mikhail s, turki th. seed disease. books for printing press. mosul university; 1982. 9. gupta vk, misra ak, gaur rk. growth characteristics of fusarium spp. causing wilt disease in psidium guajava l. in india. plant protection. 2010;50:4. doi: http://dx.doi.org/10.2478/v10045-010-0076-3 10. al-karkhi da kh. effect some of the physical, chemical and biotic factors on the growth of chao pseudomonas fluorescens strain and efficiency of inhibitory rizoctonia soloni (kuhn) that causes the death of tomato seedlings. master thesis, faculty of agriculture, university of basra; 2001. 11. al-mamory ia kh. assess the efficiency of some anti-fungal agents and opportunistic yeasts isolated from some hospitals in the province of babylon. master thesis, faculty of science for girls, university of babylon; 2010. 12. hooper and dennis g. methods and compositions for detecting aspergillus terreus, aspergillus niger, and mycotoxins. u.s. patent no. 8,956,821; 2015. 13. churki mm. the basics of plant fungus and diseases. al hikma for printing press. the university of baghdad and the ministry of higher education and scientific research; 1991. 14. pitt ji, hocking ad. fungi and food spoilage. blackie academic and professional; 1997. p. 593. 15. malinovschi g, kocsube s, galgoczy l, somogyvari f, vagvolgyi c. rapid pcr based identification of two medically important dermatophyte fungi, icrosporum canis and trichophyton tonsurans. acta biol szegediensis. 2009;53(1):51–54. 16. pitt ji, hocking ad. fungi and food spoilage, 3rd ed. london, new york: springer science and business media; 2009. 17. bulajic a, djekic l, lakic n, krstic b. the presence of alternaria spp. on the seed of apiaceae plants and their influence on seed emergence. arch biol sci. 2009;61:871–81. 18. okuda tm, klich a, seifert ka, ando k. media and incubation effects on morphological characteristic of penicillium and aspergillus. in: samson ra, pitt jl, editors. integration of modern taxonomic methods for penicillium and aspergillus classification. amsterdam, netherland: harwood academic pubs; 2000. pp. 83–99. 19. domsch kh, gams w, anderson th. compendium of soil fungi, 2nd ed. london, uk: academic press; 1980. 20. al-saad mr. principle physiology of microbiology. higher education press in baghdad, mosul university; 1990. p. 400. 21. abubakar a, suberu ha, bello im, abdulkadir r, daudu oa, lateef aa. effect of ph on mycelia growth and sporulation of aspergillus parasiticus. j plant sci. 2013;4:64–7. 22. hassan bh. physiological study of penicillium sp. and aspergillus niger isolated from the seeds of pistachio field and the ability of bacteria pseudomonas fluorescence in inhibition of radial growth of fungi. 2012;4:65–78. 23. fan sq, xu y, liu h. bio-control efficacy of antagonist yeasts to grey mold and blue mold on apple and pears in controlled atmospheres. plant dis. 2002;86:848–53. doi: http://dx.doi.org/10.1094/pdis. 2002.86.8.848 24. zain me. influence of growth medium on diagnostic characters of aspergillus and penicillium species. afr j microbial res. 2009;3:280–86. 78 j contemp med sci | vol. 6, no. 2, march–april 2020: 78–8478 original issn 2413-0516 introduction anticancer medications increase the longevity of many cancer patients but possibly will have adverse effects on other body tissues.1 taxol (paclitaxel) is a chemotherapy drug applicable to treat a number types of malignance and mainly metabolized in the liver by p450 cytochrome and is emitted through bile.2 liver is a vital body structure that shows an essential role in detoxification of toxic agents, and chemical drugs as the initially line of body defense.3 firozi-niyaki et al. demonstrated that taxol administration to female mice significantly raised the hepatic enzymes.4 cure with taxol has also been stated to induce the production of lipid ceramide through lipid membranes and de novo pathway as well as the manufacture of reactive oxygen species (ros) from the mitochondrial matrix.5 free radicals are active molecules or atoms that, owing to their atomic layer, have a strong desire to combine with other surrounding molecules such as proteins, cell membrane lipids, dna, lipoproteins, and rna, can cause tissue damage and diseases such as cancer, hepatic disorders, and cardiovascular if their combining activity is not inhibited.6 one of the most important destructive effects of free radicals is lipid peroxidation (lp), which causes cell membrane injury.7 these free radicals can induce the production of lp and cellular damages, particularly in hepatic cells by alkylating the protein groups and other cellular macromolecules and attacking unsaturated fatty acids.8 oxidative stress plays a vital role in the hepatic tissue impairment induced by toxins and drugs.9 karaduman et al. found that taxol induced hepatic tissue necrosis and damage, caused fibrosis, and increased sinusoidal space and inflammation in the hepatic tissue of rats.10 various herbs with antioxidant properties apply to protective effects against chemoprotective drugs. one of these herbs is nigella sativa (ns), which has a therapeutic and religious history.11 ns with white flowers that turn black in contact with air, is native to asia and east europe belongs to the ranunculaceae family.12 thymoquinone is the chief compound of the aqueous extract of ns, constituting about 72.94% of the weight of its oil.13 this herb has been reported to have frequent pharmacological properties such as attenuation of glucose, lipid, and hypertension, protection of kidney, and hepatic tissues, and antimicrobial effects owing to its antioxidant, anti-inflammatory effects.14 daba et al. described the antioxidant effects of ns against liver toxicity induced by cisplatin.15 additionally, administration of ns oil for lead-induced hepatotoxicity has been informed to prevent pathologica disorders in the hepatic tissue.16 given the antioxidant properties of thymoquinone, it seems that this substantial can shield the liver against taxol-induced oxidative impairment. a review of the literature similarly reveals no study has investigated the protective effects of thymoquinone against destructive effects of taxol through reduce apoptogenic and oxidative stress index and improve histomorphometric change on liver tissue in male rats. hence, the present study was conducted to evaluate the protective effects of thymoquinone against taxol on some hepatic parameters. materials and methods animals for experimental treatments, 64 male rats (wistar, 20–250 g, 8 weeks) were prepared from a specialized center of laboratory animals (pasteur, tehran, iran). standard animal keeping conditions were applied carefully including: 12 h light/12 h dark phase, 22 ± 2°c availability of water and food. the administration of thymoquinone offer a protective effect through the apoptogenic and antioxidant pathway in acute liver failure induced by taxol shiva roshankhah1, cyrus jalili2, amir farmandeh1, mohammad reza salahshoor1 1department of anatomical sciences, medical school, kermanshah university of medical sciences, kermanshah, iran. 2medical biology research center, kermanshah university of medical sciences, kermanshah, iran. corresponding author: mohammad reza salahshoor (e-mail: reza.salahshoor@yahoo.com) (submitted: 15 january 2020 – revised version received: 02 february 2020 – accepted: 09 february 2020 – published online: 26 april 2020) objectives due to the increasing use of chemotherapy drugs and herbs in medicine, this study was designed to assess the effects of thymoquinone and taxol on apoptogenic, oxidative, and histomorphometric changes in liver. methods sixty-four (64) male rats were assigned to 8 groups including control groups (normal group and taxol (20 mg/kg group), thymoquinone groups (4.5, 9, 18 mg/kg) and taxol + thymoquinone (tt) treated groups. all experimental groups were treated intraperitoneally daily for 2 weeks. the relative expression level of apoptotic genes and hepatocyte apoptotic index were analyses. also, nitrite oxide (no), lipid peroxidation (lp), total antioxidant capacity (tac), hepatic enzymes and histomorphometric parameters were evaluated. results in the taxol control group (tcg) all parameters investigated in this study significantly increase except tac level, which was decreased in compared to the normal control group (ncg) (p < 0.01). additionally, all evaluated parameters were reduced in thymoquinone and tt groups, except tac level (which was increased) as compared to the tcg (p < 0.01). conclusion our results discovered that thymoquinone successfully moderate liver injury induced by taxol through the activation of antioxidant pathways, reduce the apoptogenic and the regeneration of histopathological alterations. keywords thymoquinone, apoptogenic, antioxidant, liver, taxol 79 original administration of thymoquinone offer a protective effectshiva roshankhah et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 78–84 experimental medications and treatments conformed to the ethical and humane principles of research. it was accepted by the ethics committee of the related institute committee on the use and care of animals, ir.kums.rec.1397.618. experimental protocol, dissection and sampling the animals were randomly divided into 8 groups (n = 8), including: 1: first group, the ncg, which received normal saline (intraperitoneally injection) equivalent to the amount of experimental groups. second group, the tcg, in this group, the rats was given taxol at a dose of 20 mg/kg (single dose) intraperitoneally injected for 2 weeks. the mice in the both tcg and ncg groups were not given any treatment after taxol and normal saline injection until sacrifice. third to fifth groups, the thymoquinone administration groups, in these groups, each animal, respectively, received 4.5, 9, and 18 mg/kg of thymoquinone intraperitoneally for 2 weeks. sixth to eighth groups, tt administration groups, in this group, each animal received single dose (20 mg/kg) of taxol intraperitoneally in order to induce hepatic damage, then (after taxol injection) they respectively received 4.5, 9, and 18 mg/kg of thymoquinone intraperitoneally for 2 weeks. at the end of the treatment period, all rats were deeply anesthetized by intraperitoneally injection of ketamine hcl (100 mg/kg) and xylazine (10 mg/ kg). the sampling included blood from the hearts (at least 1 ml per animal) for evaluating the antioxidant levels. the animals were then sacrificed and the liver was removed.6,10 evaluation of functional-related enzymes the liver homogenate was centrifuged twice (12,000 rpm, 10 min). then, the supernatant was parted for measurement of hepatic enzymes, including aminotransferase (ast), alanine aminotransferase (alt), and alkaline phosphatase (alp). the alt and ast were tested based on reitman and frankel biochemical methods. the alp protocol was also determined according to the technique which was set out in the practical research laboratory.3 histomorphometric analyses to assess histomorphometric alterations, a section of the liver tissue was fixed in 10% formalin. the tissues were then fixed in paraffin, and the thin sections (4 µm) were prepared using a microtome (leica rm 2125, germany). the slices were then stained with hematoxylin and eosin. thereafter, some morphological assessments including full cellular area, hepatocyte outline, maximum and minimum axis, mean axis, and cvh were determined by light microscope at 40× magnification. at least 50 cells from the separate region were measured to eliminate the probable measurement bias. all procedures were applied with an investigation microscope coupled with a dp12 camera (3.34-million pixel resolution) and using the olysia bio-software (olympus optical co. ltd, tokyo, japan).6 measurement of lp levels the level of molecular reaction among thiobarbituric acid and malondialdehyde (mda) following colorimetry process was considered as the measurement of lipid peroxidation levels. the frozen sample of the pancreas tissue was used. first, the tissue was washed with the phosphate-buffered saline at ph of 7. then, an ultrasonic homogenizer in a cold phosphate buffer containing ethylene diamine tetra-acetic acid was hired to apply tissue homogenization. 20 μl of supernatant was mixed within the test tubes. the test tube contained 4 μl of butylated hydroxytoluene, 20 μl of phosphoric acid (1m), and 20 μl of tba solution. the test tube was incubated for 60 min at 70°c, then it was centrifuged (10000 rpm, 4 min). 80 μl of the supernatant was poured into the spectrophotometer tubes. the produced dye in the commercial kit was read at 532 nm, and in conclusion, the mda level was stated in nmol/mg protein.7 estimation of liver tac total antioxidant capacity (tac) of the serum was analyzed by the frap technique based on the ability of the plasma to reinstate the ferric. a blue color was made when the complex of feiii-tptz in acidic ph payback to feii and the absorption at the extreme wavelength of 500 nm. the element defining the speed of the feii-tptz and blue stain was merely the vitalizing power of the sample. tac values are strategized via the standard curve.3 assessment of no the griess method based on colorimetry approaches was employed to measure the no levels. 500 μl of serum was deproteinized with zinc sulfate (10 mg). after centrifugation at 3000 rpm for 10 min, the equal amounts of griess reagent (1% sulfanilamide, 0.1% naphtylethylenediamide in 2.5% phosphoric acid) were also added to the supernatant in 96-well elisa plates and incubated for 10 min at 37°c. the absorbance was stately at 450 nm with the use of a microplate reader. nitrite concentrations were considered by a sodium nitrite standard curve.6 apoptosis assay the tunel test was performed according to the manufacture’s procedure (roche, germany). paraffin-embedded blocks were prepared using an automatic tissue processor. 5 µm histological slices were cut through a microtome (leica, germany), and five slices per rat were selected. the tissues were subjected to deparaffinization. after routine deparaffinization and blocking, the slices were exposed to tdt with dig. dig labeling and counterstaining were then carried out. to quantitate the rate of apoptosis, the number of positive cells within the liver was calculated.7 real-time quantitative pcr the real-time pcr evaluated the liver genes expression of p53 and bax. the tissue was frozen in nitrogen and then stored in at –70 °c freezer. rna was removed using rneasy mini kit (qiagen, tehran, iran) based on the manufacturer’s instructions. dna was treated via dnase set kit (qiagen, tehran, iran). the rna was hired in order to cdna production through cdna synthesis kit. the expression level of the related genes was measured through glyceraldehyde-3-phosphate dehydrogenase primer as endogenous control by sybr green (fermentas, tehran, iran) through comparative ct method. the primer nucleotide sequences are listed in table 1.16 statistical analyses one way analysis of variance (one-way anova) and tukey post hoc test were used to statistical analysis and differences among the groups, respectively. statistical software package of spss (chicago, il) was used for data analysis. the final outcomes were expressed as mean ± standard error, and p < 0.05 was considered significant. 80 original administration of thymoquinone offer a protective effect shiva roshankhah et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 78–84 results effect of taxol and thymoquinone on liver functional-related enzymes taxol led to a significantly increased in enzymes in tcg comparison with the ncg (p < 0.01). the mean enzymes level concentration was not significant in thymoquinone groups compared to the ncg (p > 0.05). also, in thymoquinone and tt groups significantly decreased in the mean of enzymes were detected in comparison with the tcg (p < 0.01) (table 2). morphometric measurements the mean diameter of hepatocytes and central hepatic vein (chv) in experimental groups showed a significant difference between the ncg and tcg groups (p < 0.01). the mean diameter of hepatocytes and chv was not significant in thymoquinone groups compared to the ncg (p > 0.05). further, in thymoquinone and tt groups the mean diameter of hepatocytes and chv significantly reduced in comparison with tcg (p < 0.01) (fig. 1). histopathological changes histological analysis showed normal liver structure in the ncg, tt, and thymoquinone treatment groups. after treatment with taxol in tcg, the hepatic tissue showed evident changes and injury. these anomalies included increased apoptotic cells, increased irregularities, hyperemia, sinusoidal dilatatio,n and enlargement of chv. treatment with tt at all doses reduced the liver damage caused by taxol harmfulness (fig. 2). apoptotic index the apoptotic index (ai) was significantly higher in tcg compared to the ncg (p < 0.01). no significant differences were similarly found in the ai in all thymoquinone groups as compared to the ncg (p > 0.05). furthermore, whole several doses of thymoquinone in thymoquinone and tt groups fig. 1 effect of taxol and thymoquinone on the hepatocyte diameter (a) and diameter of chv (b) diameters. *significant difference to the ncg (p < 0.01). †significant difference to the tcg (p < 0.01). ‡significant difference to the tcg (p < 0.01). chv: central hepatic vein; thym: thymoquinone; tax: taxol, tcg: taxol control group. ncg: normal control group. table 1. primers used in real-time pcr primer sequences primer id gapdh f : 50aagctcatttcctggtatg-30 r: 50ctgccacaagaactagaga-30 p53 f:50-agagaccgccgtacagaaga-30 r:50-gcatgggcatcctttaactc-30 bax f: 50-ccggcgaattggagatgaact-30 r: 50-ccagcccatgatggttctgat-30 table 2. diverse liver enzymes between treatment groups. enzymes (ng/ml) normal control taxol control thymoquinone (mg/kg) thymoquinone + taxol (mg/kg) 4.5 9 18 4.5 9 18 ast 71.25±4.20 132.21±8.25* 75.85±5.47† 71.32±4.36† 69.11±6.71† 99.01±7.2‡ 85.84±8.12‡ 84.65±5.3‡ alt 29.28±3.25 63.98±5.61* 27.45±3.14† 30.47±4.32† 32.11±3.82† 45.01±2.1‡ 37.2±3.47‡ 35.54±3.2‡ alp 3.02±0.20 7.97±1.24* 2.63±0.52† 3.04±0.41† 3.11±0.21† 5.54±0.83‡ 4.02±0.97‡ 4.01±0.82‡ data were presented as mean ± standard deviation. alp: alkaline phosphatase, alt: alanine aminotransferase, ast: aspartate aminotransferase. *p < 0.01 compared to the normal control group. †p < 0.01 compared to taxol control group. ‡p < 0.01 compared to the taxol control group. 81 original administration of thymoquinone offer a protective effectshiva roshankhah et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 78–84 represented a significant decline in the ai as compared to the tcg (p < 0.01) (figs. 3 and 4). apoptotic gene expression levels upregulated changes of p53 and bax genes in the tcg compared to the ncg were significantly detected (p < 0.01). also, a significant downregulation of these genes similarly was distinguished in all doses of thymoquinone and tt groups as compared to the tcg group (fig. 5). levels of tac, lp, and no in the tcg, the tac levels were lower than the ncg significantly (p < 0.01). also, in whole groups of thymoquinone and tt, the tac levels were considerably elevated as compared to the tcg (p < 0.01) due to the thymoquinone administration. taxol, due to its detrimental effects, significantly increased the no and lp levels in tcg in comparison with the ncg (p < 0.01). it is also found that all doses of thymoquinone can significantly reduce the mean levels of no serum and lp in the thymoquinone and tt groups as compared to the tcg (p < 0.01) (table 3). discussion chemotherapy, the chief factor complicated in oxidative stress, can disrupt the construction and performance of hepatic tissue, one of the vital body structures and the primarily protection barrier that shows an essential role in detoxification fig. 2 microscopic images of liver tissue (five-micron thick sections, h&e staining, magnification×100). ncg (a), thymoquinone (18 mg/kg) group (b) and tt group (d): normal tissue structure. tcg (20 mg/kg) (c): abnormal tissue structure. apoptotic cells (red arrows), central hepatic vein (circles) and hyperemia (block arrow). tcg: taxol control group. ncg: normal control group. fig. 3 apoptosis induction in the hepatocytes following thymoquinone and taxol treatments (400× magnifications, tunel staining). ncg (a), thymoquinone group (18 mg/kg)(b), tcg (20 mg/kg) (c) and tt group (d). shiny red nuclei refers to apoptotic cells. tcg: taxol control group. ncg: normal control group. fig. 4 apoptotic index in the hepatocytes subsequent thymoquinone and taxol aminestration. *significant difference to the ncg (p < 0.01). # significant difference to the tcg (p < 0.01). $ significant difference to the tcg (p < 0.01). thym: thymoquinone; tax: taxol. taxol control group. ncg: normal control group. fig. 5 relative gene expression following thymoquinone and taxol administration. *statistically significant (p < 0.01) between ncg and tcg. $ statistically significant (p < 0.01) between tcg and tt groups. # significant modifications in thymoquinone groups as compared to the tcg (p < 0.01). thym: thymoquinone; tax: taxol. tcg: taxol control group. ncg: normal control group. 82 original administration of thymoquinone offer a protective effect shiva roshankhah et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 78–84 of chemical medications.17 consequently, concurrent consumption of potential antioxidant plants and chemotherapy medications has drastically improved in order to safeguard the cells in contrast to the damaging effects of free radicals.20 the outcomes of the present investigation recommended that taxol administration had adversative effects on hepatic histology and function, increase apoptotic cells and gene expression, and antioxidant imbalance as well, and increase in no level. on the other hand, thymoquinone as an herbal, relieve the diverse effects of taxol administration, perceptibly in liver parameter. the current study outcomes moreover revealed that thymoquinone is able to condense lp and increase tac of hepatic tissue, consequently it is dropping oxidative stress. consistent with these outcomes, some of papers has offered antioxidant properties of thymoquinone.6,14,15 thymoquinone appears to inhibit the lp induced by tbh in the liver tissue.6 additional, thymoquinone is a lipophilic molecule that is capable to prevent lp via fenton reaction.19 gani et al. indicated that extract of ns diminished hepatic enzymes and lp, which is in line with the outcomes of the existing study.20 accordingly, it appears that thymoquinone with its antioxidant properties could condense lp and increase tac in the treatment groups via inhibiting the construction of ros. present study similarly showed the recovery effect of thymoquinone on liver tissue as well as lessening the oxidative stress by displaying falling of lp. the injuriousness of taxol administration can lead to blood and biochemical modifications, oxidative stress, and lp. hence, mechanism for toxicity of taxol drug combinations is oxidative stress.21 the findings discovered that, compared to the ncg, the chv and diameters of hepatocytes in the tcg has significantly decreased. in addition, there was a significant reduction in the diameter of hepatocytes and size of the chv in thymoquinone and tt groups were detected compared to the tcg group. it appears that taxol has hepatotoxic effects, due to increased glutathione, and causes the loss of liver cytoplasmic cells.10 changing the size of chv and liver cells can rise the metabolic action of hepatocytes to defecate the toxin from the body throughout detoxification course.3 the taxol metabolism made free radicals then it causes lp, dna, and membrane proteins reaction and subsequently cell impairment through several ways.7 thymoquinone, as the antioxidant herb, has an inhibitory effect on cytochrome p450, inhibits additional taxol metabolism and diminish the construction of free radicals.6 the outcomes of the existing study agree with the consequences of the study of salahshoor et al., in which they displayed that genistein administration as a herb antioxidant improves liver damage, resulted in the decreasing diameter of hepatocytes.3 additional imperative finding was that some modifications in the liver tissue were perceived in the tcg that were in the form of the plethoric state of the sinuses, kupffer cells accretion around the chv and growth in the apoptotic cells, as well as the chv diameter enlargement. it appears that the attack of free radicals to hepatocyte causes apoptosis in parenchymal cells.7 kupffer cells can prompt inflammatory reactions in the hepatic tissue, which leads to tissue impairment.22 apparently, kupffer cells are triggered in reply to tissue damage and release mediators, such as the tnf and no.6 it may appear that the kupffer cells secretion of toxic mediators in the zones, that have not experienced necrosis yet, are complicated in causing hepatic toxicity and necrosis.25 besides, free radicals’ construction and following oxidative stress can be one of the most dangerous and important causes of the hepatocyte death.3 the current outcomes support the concepts of cresteil et al. who display that the liver harm and apoptosis in liver cells can be caused by taxol.24 thymoquinone appears to carry out a protective result contrary to fibrogenesis on the liver tissue through polyphenol capacity and by stopping the stellate cells activity and stating the cell cycle protein.25 stellate cells show a central part in the enhancement of hepatic fibrosis and oxidative stress.6 it seems that thymoquinone has the ability to inhibit macrophage. thymoquinone can exert its anti-inflammatory properties on nf-kβ by reducing h2o2 production.26 kanter et al. shown that thymoquinone prevents the induction of ethanol’s undesirable properties through the inhibition of lipid’s induction, which is matched with the outcomes of the present study.27 the consequences of this study show that there is a significant alteration between tac and hepatic enzyme levels in the tcg and the ncg. correspondingly, there’s a negative association, between tac in the tcg and enzyme levels in the thymoquinone and tt groups. the progression of the liver enzymes in serum indicates a hepatic damage in the present study. besides, the results of nili et al. confirmed the consequences of the present study in that thymoquinone could decline enzymes serum levels.28 liver enzymes can be released into the serum due to the prevalence of cell membrane impairment.6 it may be possible that taxol induce destruction to the hepatocyte membrane through the inhibition of respiratory sequence.29 the consequences are in agreement with harris et al. conclusions, which show that the taxol administration in rats for 1 week induces the growth of hepatic enzymes and equally condense the tac.30 thymoquinone seems to stabilize hepatocyte membranes and stops outflow of enzymes by stopping lp.15 thymoquinone can apply its antioxidant properties through reducing lp and inducing antioxidant enzymes.6 the  current outcomes are consistent with the results of pari et al. who suggested that table 3. effect of thymoquinone and taxol on antioxidant parameters (n = 8 for each group). groups no (mmol/ml) tac (mmol/ml) lp (nmol/mg ) normal control 42.25±4.36 99.74±7.24 0.92±0.06 taxol control 198.69±8.25* 18.22±3.12* 0.42±0.01* thymoquinone 4.5 mg/kg 44.02±3.52† 98.46±8.06† 0.97±0.03† thymoquinone 9 mg/kg 43.65±6.98† 95.97±5.58† 0.92±0.04† thymoquinone 18 mg/kg 41.82±6.36† 99.11±8.82† 0.96±0.01† thymoquinone + taxol 4.5 mg/kg 93.16±5.14¶ 42.71±5.21¶ 0.61±0.02¶ thymoquinone + taxol 9 mg/kg 72.71±6.74¶ 49.34±3.46¶ 0.59±0.01¶ thymoquinone + taxol 18 mg/kg 73.39±3.31¶ 51.96±3.99¶ 0.57±0.05¶ data are presented as mean ± sem. * p < 0.01 compared to the control group. † p < 0.01 compared to taxol control group. ¶ p < 0.01 compared to the taxol control group. no: nitrite oxide, tac: total antioxidant capacity, lp: lipid peroxidation. 83 original administration of thymoquinone offer a protective effectshiva roshankhah et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 78–84 administration of thymoquinone condenses liver enzymes in diabetic rats and inhibits damages to liver cells.31 in this study, the thymoquinone also showed decreased effects of bax and p53 (apoptotic genes) gene expression and apoptotic hepatocyte index. p53 makes the mitochondrial membrane permeable to the influx of cytochrome-c into the intracellular matrix. accordingly, p53 adjusts the function of apoptotic elements like caspase and bax.32 as cell death is seen in hepatocytes, it is stated that the taxol has upregulation effects on apoptotic factors.33 also, thymoquinone directly translocate within the intranuclear space to induce downregulation of related genes.34 de et al. also found that the apoptotic genes are expressed significantly after taxol administration.35 in the current study, thymoquinone reduced apoptotic cell index and apoptotic gene expression to prevent cell death. based on the obtained results, it can be indicated that thymoquinone owns positive antioxidant effects on molecular function and histological construction of liver cells. the consequences of this study showed a significant rise in the amount of no in blood in the tcg compared to the ncg. besides, there was a significant reduction in the tt group in no level compared to tcg. it seems that oxidative stress increases the production of no synthase, and therefore indications to rise in nitrite production and fall in cell endurance.3 mitochondria dysfunction, due to the extraordinary consumption of o2, may possibly increase the creation of free radicals in most body tissues, including no radicals, and due to the nitrosative and oxidative stress and damage to the tissues, particularly the hepatic tissue.7 the results are in contract with of jalili et al. conclusions, which displayed that thymoquinone can condense no in morphine-induced damage to the hepatic tissue.6 the consequences are in concurrence with new studies representative that taxol, as one of the chemotherapy medications, is seemingly able to impairment hepatocytes, increase apoptogenic, liver enzymes, no, lp, and reduce tac, by inducing oxidative stress. in contrast, thymoquinone, a strong antioxidant herb, can condense the destructive effects of this drug. consequently, consuming thymoquinone can be a good approach to lessen free radicals and inhibit injuries to the hepatic tissue of the patients who are exposed to taxol drug for chemotherapy. conclusion the present study showed that hepatoprotective properties of the thymoquinone in contrary to taxol-induced liver destruction in rats. the outcomes of this study revealed that taxol administration would outbreak dangerous impress from the point of both hepatic histology and function. it was found that thymoquinone reduces ros, cell apoptosis, expression of p53, and bax gens, activating antioxidant agents, and detoxifying enzymes. therefore, thymoquinone might be considered to recover the functional and histological of the hepatocyte exposed to taxol drug. this protecting conclusion can be reconciled through the antioxidant properties of thymoquinone. thymoquinone can be considered as a substitute beneficial agent against oxidative damages induced by toxic drug. but, extra studies are necessary to define its exact mechanism of action. acknowledgments the authors thank kermanshah university of medical sciences for its support. conflict of interest the authors declare no conflict of interest. references 1. cao x, hou j, an q, assaraf yg, wang x. towards the overcoming of anticancer drug resistance mediated by p53 mutations. drug resist updat. 2020;49:100671. 2. nazhand a, durazzo a, lucarini m, mobilia ma, omri b, santini a. rewiring cellular metabolism for heterologous biosynthesis of taxol. nat prod res. 2020;34(1):110–21. 3. salahshoor mr, roshankhah s, hosseni p, jalili c. genistein improves liver damage in male mice exposed to morphine. chin med j 2018;131:1598– 604. 4. firozi-niyaki m, barari ar, abbassi-daloii a. the effect of endurance training and taxol consumption on cyclooxygenase-2 and prostaglandin e2 levels in the liver tissue of mice with cervical cancer. j kashan univ med sci. 2018;22:517–24. 5. karaduman d, eren b, keles on. the protective effect of beta-1, 3-d-glucan on taxol-induced hepatotoxicity: a histopathological and stereological study. drug chem toxicol. 2010;33:8–16. 6. salahshoor mr, vahabi a, roshankhah sh, shabanizadeh darehdori a, jalili c. the effects of thymoquinone against morphine-induced damages on male mice liver. int j prev med. 2018;9:8. 7. salahshoor mr, abdolmaleki a, jalili c, roshankhah s, ziapour a. determination of histopathological and biomedical parameters in protective effects of petroselinum crispum on hepatotoxicity induced by dichlorvos in male wistar rats. comp clin path. 2020;2:1–9. 8. ali bm, velavan b, sudhandiran g, sridevi j, nasar as. radical dendrimers: synthesis, anti-tumor activity and enhanced cytoprotective performance of tempo free radical functionalized polyurethane dendrimers. eur polym j. 2020;122:109354. 9. jayakumar t, huang hc, hsia cw, fong th, khamrang t, velusamy m, et al. ruthenium derivatives attenuate lps-induced inflammatory responses and liver injury via suppressing nf-κb signaling and free radical production. bioorg chem. 2020;96:103639. 10. karaduman d, eren b, keles on. the protective effect of beta-1, 3-d-glucan on taxol-induced hepatotoxicity: a histopathological and stereological study. drug chem toxicol. 2010;33:8–16. 11. mazaheri y, torbati m, azadmard-damirchi s, savage gp. effect of roasting and microwave pre-treatments of nigella sativa l. seeds on lipase activity and the quality of the oil. food chem. 2019;274:480–6. 12. mukhtar h, qureshi as, anwar f, mumtaz mw, marcu m. nigella sativa l. seed and seed oil: potential sources of high-value components for development of functional foods and nutraceuticals/pharmaceuticals. essent oil res. 2019;14:1–3. 13. kausar h, mujeeb m, ahad a, moolakkadath t, aqil m, ahmad a, et al. optimization of ethosomes for topical thymoquinone delivery for the treatment of skin acne. j drug deliv sci technol. 2019;49:177–87. 14. geng d, zhang s, lan j. analysis on chemical components of volatile oil and determination of thymoquinone from seed of nigella glandulifera. zhongguo zhong yao za zhi. 2009;34:2887–90. 15. daba mh. hepatoprotective activity of thymoquinone in isolated rat hepatocytes. toxicol lett. 1998;95:23–9. 16. esfandiari e, roshankhah s, mardani m, hashemibeni b, naghsh e, kazemi m, et al. the effect of high frequency electric field on enhancement of chondrogenesis in human adipose-derived stem cells. iran j basic med sci. 2014;17:571–76. 17. bridgewater ja, pugh sa, maishman t, eminton z, mellor j, whitehead a, et al. systemic chemotherapy with or without cetuximab in patients with resectable colorectal liver metastasis (new epoc): long-term results of a multicentre, randomised, controlled, phase 3 trial. lancet oncol. 2020;21:398–411. 18. zhang y, wu z, yu h, wang h, liu g, wang s, et al. chinese herbal medicine wenxia changfu formula reverses cell adhesion-mediated drug resistance via the integrin β1-pi3k-akt pathway in lung cancer. j cancer. 2019;10:293–304. 19. dera a, rajagopalan p. thymoquinone attenuates phosphorylation of akt to inhibit kidney cancer cell proliferation. j food biochem. 2019;e12793. 20. gani m. evalution of hepatoprotective effect of nigella sativa l. j pharm pharm sci. 2013;4:428–30. 21. meshkini a, yazdanparast r. involvement of oxidative stress in taxol-induced apoptosis in chronic myelogenous leukemia k562 cells. exp toxicol pathol. 2012;64:357–65. 84 original administration of thymoquinone offer a protective effect shiva roshankhah et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 78–84 22. raushan hr, seth pk. behavioral neurochemical and neuromorphological effects of deltamethrin in adult rats. j toxicol environ health. 2010;48:515–6. 23. amouoghli tabrizi b md. protective effect of edible turmeric powder on early hepatic injury in diabetic rats. j kashan univ med sci. 2010;14:190–9. 24. cresteil t, monsarrat b, alvinerie p, tréluyer jm, vieira i, wright m. taxol metabolism by human liver microsomes: identification of cytochrome p450 isozymes involved in its biotransformation. cancer res. 1994;54:386–92. 25. bai t, lian lh, wu yl, wan y, nan jx. thymoquinone attenuates liver fibrosis via pi3k and tlr4 signaling pathways in activated hepatic stellate cells. int j immunopharmacol. 2013;15:275–81. 26. nagi mn, almakki ha, sayed-ahmed mm, al-bekairi am. thymoquinone supplementation reverses acetaminophen-induced oxidative stress, nitric oxide production and energy decline in mice liver. food chem toxicol. 2010;48:2361–5. 27. kumar gn, walle uk, walle t. cytochrome p450 3a-mediated human liver microsomal taxol 6 alpha-hydroxylation. j pharmacol exp ther. 1994;268:1160–5. 28. nili-ahmadabadi a, tavakoli f, hasanzadeh gr, rahimi hr, sabzevari o. protective effect of pretreatment with thymoquinone against aflatoxin b1 induced liver toxicity in mice. daru. 2011;19:282. 29. harris jw, rahman a, kim br, guengerich fp, collins jm. metabolism of taxol by human hepatic microsomes and liver slices: participation of cytochrome p450 3a4 and an unknown p450 enzyme. cancer res. 1994;54:4026–35. 30. harris jw, rahman a, kim br, guengerich fp, collins jm. metabolism of taxol by human hepatic microsomes and liver slices: participation of cytochrome p450 3a4 and an unknown p450 enzyme. cancer res. 1994;54:4026–35. 31. pari l, sankaranarayanan c. beneficial effects of thymoquinone on hepatic key enzymes in streptozotocin–nicotinamide induced diabetic rats. life sci. 2009;85:830–4. 32. al humayed s, al-hashem f, haidara ma, el karib ao, kamar ss, amin sn, et al. resveratrol pretreatment ameliorates p53-bax axis and augments the survival biomarker b-cell lymphoma 2 modulated by paracetamol overdose in a rat model of acute liver injury. pharmacol. 2020;105:39–46. 33. minero vg, de stefanis d, costelli p, baccino fm. and in vivo conditional sensitization of hepatocellular carcinoma cells to tnf-induced apoptosis by taxol. cell cycle.2020;14:1090–1102. 34. chae ig, song ny, kim dh, lee my, park jm, chun ks. thymoquinone induces apoptosis of human renal carcinoma caki-1 cells by inhibiting jak2/ stat3 through pro-oxidant effect. food chem toxicol. 2020;9:253–259. 35. de a, de a, sharma r, suo w, sharma m. sensitization of carboplatinum and taxol-resistant high-grade serous ovarian cancer cells carrying p53, brca1/2 mutations by emblica officinalis (amla) via multiple targets. j cancer. 2020;11:1927–39. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i2.732 107j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 country report oral health status and healthcare system in i.r. iran khoshnevisan m.h,a,b ghasemianpour m,a samadzadeh h,c and baez rjd apreventive dentistry research center, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran. bcommunity oral health department, school of dentistry, sbmu, tehran, iran. coral health bureau, ministry of health, tehran, iran. dwho collaborating centre, school of dentistry, university of texas health science centre at san antonio, texas, usa. correspondence to khoshnevisan m.h (email: khoshmh@gmail.com, mh.khoshnevisan@sbmu.ac.ir). (submitted: 28 may 2018 – revised version received: 07 june 2018 – accepted: 02 july 2018 – published online: 26 september 2018) objective oral and dental health has substantial impact on physical, social and mental well-being of all peoples. the aim of this study was to report the oral health status and system in iran. methods the latest who oral health survey methodology and criteria was used to perform the first comprehensive national oral health survey in iran. all suggested 5 age groups of children and adults were recruited from all provinces in iran. the use of such information is mostly needed for planning appropriate interventions necessary to control and improve the oral health of nation. although oral healthcare services are provided by both public and private institutions, the focus of this report is to describe the current public infrastructure that can have a greater impact on the oral health status nationwide. results the national survey revealed that dental caries, periodontal diseases and tooth loss are increasing compared with previous data. caries-free status is sharply declining from 12 to 15 years old (27%) and number of edentulous people is exceeding 50% in 65–74 years old age groups. these data indicate the urgent need for proper interventions in all age groups, especially in children. the declaration of “2015-oral healthcare reform” by the ministry of health and medical education has paved the way for oral health promotion in children under age 14 at the national level. the target is to halt the progression of oral diseases and maximize the promotion of oral health and quality of life by the year 2025 for the target population. conclusion although the trends of oral diseases are still increasing in almost all areas over the past two decades, the necessary policy has been ratified and evidence-based strategies have been implemented to overcome the identified obstacles. although the decline in the rate of dental caries in 12-year-old children is reported recently, caution must be exercised to safeguard quality of care and supervision to observe the expected outcomes by the target deadline. keywords oral health status, children, adult, iran, healthcare system, promotion introduction oral health is closely associated with overall health. any evidence-based intervention for oral health promotion and oral disease prevention is less expensive and more cost effective than treatment of such conditions. on the other hand, oral diseases are among non-communicable diseases (ncds) with great public-health importance. based on the global strategy for prevention and control of ncds, it is highly important to develop a national policy, establish programs, share regional and global experiences and build capacity to address burden of oral as well as other ncds at the local, the national and international level. availability of reliable current information is greatly needed for achieving adequate improvement in oral health status among the general as well as vulnerable and at-risk population groups in iran. likewise, modification of dental infrastructure, augmentation of productivity and skills improvement of the dental workforce as well as increasing the population’s oral health literacy are the other national priorities. conducting the iranian national oral health survey (inohs-2012) as a cornerstone was successfully accomplished by using the world health organization’s recommended guidelines, methodology and criteria for assessing the current oral health status of population groups and future needs for oral interventions.1 the reported results of this survey demonstrates that oral health disparities are rather extensive due to children, adults and elderly having many carious and missing teeth that are still considered as an “aging process norm” by many people among the iranian population. internationally, it has been recognized that oral diseases including dental caries and the curative dental care needed to restore teeth to normal function are considered as significant economic burden for individuals as well as the communities at large. oral health is more important than healthy teeth; it is an integral part of overall health and well-being and the needs of iranian citizens must be addressed accordingly. declaration of the 2015 national oral healthcare reform in iran was another major event that can potentially improve the nation’s oral health status for years to come. the policy is covering all children up to age 14 nationwide. all resources have been mobilized for these preventive dental services for the priority target groups, aiming for them all to become caries-free by the year 2025. country profile geographically, iran with about 80 million populations, spanning in an area over 1,648,000 km2. being the 18th largest country in the world, iran is located in southwest asia, in the middle east region. the country is bordered by turkmenistan, the caspian sea and the republic of azerbaijan to the north, turkey and iraq to the west, the persian gulf and the sea of oman to the south, and pakistan and afghanistan to the east. about 74% of the country is urban and 26% is considered as rural areas. iran is divided into 31 provinces, 336 districts and over 66,000 villages. historically, persia (now iran) has been the conduit of knowledge from china and india in the east to greece and issn 2413-0516 108 j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 oral health status and healthcare system in i.r. iran country report khoshnevisan m.h. et al. rome in the west. the iranian civilization along with several 1000 years and great indigenous capacities of scholars and philosophers, have permanent records of achievements and contributions in developing world in understanding of nature, medicine, mathematics, chemistry, physics and philosophy. among many people, avicenna, biruni, khayyam and rhazes (razi) are internationally well-known scientists and philosophers whose statutes are currently standing in the “persian scholars pavilion” in vienna international center. in a short description, avicenna has substantial writings on different aspects of medicine (800 ad). biruni was an astronomer; he wrote astronomical encyclopedia in 1000 ad and suggested the possibility of earth’s rotation around the sun. razi was the inventor of alcohol and practical physics; he defined the special or net weight of mater in 10th century. his student abu bakr joveini, wrote the first comprehensive medical book in the persian language. many of those physician’s approaches in medieval persia are still accepted in modern medicine.3 the “gundishapur academy” in persian empire was the first known teaching hospital, where students methodologically mentored and supervised to practice medicine on patients.2 based on expert opinion, the credit for whole hospital system has been given to persia.2 khayyam was an extraordinary mathematician and poet with many metaphysical poems. nevertheless, despite numerous political unrests, iran has made significant scientific advances to date.4 public healthcare system the public healthcare system in iran is composed of a healthcare network called “health and treatment network” (htn) covering most of the urban and rural areas in the country. the health facilities providing basic healthcare in the rural areas, are called health houses (hh); and similar facilities in the urban areas are called health posts (hp). there are over 17,000 hh providing preventive care to villagers. at the next or mid-level in the htn, there are over 5000 health centers (hc) providing care by general physicians, dentists as well as other healthcare providers. hospitals are located in the top level of the network. the chain of referral naturally starts from hh to hc and continues to hospitals when necessary.5 the primary healthcare service facilities includes: 17,325 health houses, 1666 hp, 2407 rural health centers, 2186 urban health center, providing close to 100% of primary healthcare coverage in the urban and about 95% coverage in the rural areas. family as a unit of society is the main focus of the healthcare system in iran. since 1972, major health improvements have been achieved through htn. preventive oral healthcare has been integrated into the htn since 1996. many foreign health administrators have shown interest and have taken the opportunity to visit and become familiar with the operational management and effectiveness of the healthcare system in iran. the level of prevention for preventive oral healthcare at each healthcare network component is described in the following section. level i-a: health houses the hh is the most peripheral rural healthcare units of the national public healthcare system that provides health services to villagers. each hh covers a village population of about 1500 individuals. if the population is less, satellite villages may be covered by an hh. “behvarz” is a native young male or female, in charge of the hh. she/he is selected from village applicants. these individuals with high-school degree are trained for 2 years to provide primary healthcare services for village residents before they are given the responsibility. oral health evaluation and oral hygiene instructions is one of the services provided by the behvarz at the hh, villager’s home or local school. level i-b: health posts a similar healthcare services provided at the hhs, are available in the urban healthcare units called hp. therefore, the oral healthcare services provided by urban hps are performed by all health technicians. oral healthcare is provided at all hps as one of the elements of integrated health services. level ii-a: rural health centers the rural hc is an independent medical unit covering multiple villages with the population of about 10000 individuals in remote areas. the oral healthcare personnel, including dentists and other healthcare providers working in the hcs are supervised by a family physician. aside from supervising responsibilities, the physician is monitoring all hh activities, as well as treating the referred patients from hhs or send referral to the nest level, the city hospital, when specialist services are required. level ii-b: urban health centers this center covers one or more urban hps depending on the local population. duties of urban health centers are similar to those of rural health centers. duties and scope of services for dentists working in urban and rural health centers are also the same. the dental workforce (dentists, dental hygienists and oral health technicians) in urban and rural health centers provide preventive oral healthcare services such as oral hygiene instructions, topical fluoride therapy, fissure sealant therapy, prophylaxis and restorative services, for all patients especially the target populations groups (children, pregnant and lactating mothers) in the community according to their job descriptions. in accordance with new policies of the 2015 oral healthcare reform, level i preventive dental services are mainly provided during the morning shifts in order to comply with new regulations and level ii or therapeutic services are provided during the afternoon shifts. dentists can use the public facilities in order to provide dental treatments with no limitation of services in the afternoon shifts, as long as, the dental materials and dental assistants are provided by the dentist. the government established fee schedules are used at these facilities, and from each payment 20% is deducted by the government and the rest is considered provider’s income. for the morning shift contract, there are additional fee incentives based on remoteness of assigned location and deprivation category of the geographical area. using these payment models, the combination of income from morning and afternoon shifts have been well accepted and considered satisfactory by most recruited dentists. these assignments are usually given to new dental graduates who need to fulfil 2 years of duty service for government in return for free dental education (before being able to have a private practice). khoshnevisan m.h. et al. 109j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 country report oral health status and healthcare system in i.r. iran oral healthcare system the integration of oral health in the primary health care (phc) system was implemented about 20 years ago (1996), with the aim of oral health promotion at the community level. however, the integration of oral health was revisited in the “2015-oral healthcare reform” for further improvements. as a result, the integration of oral health is now actively executed in all medical science universities throughout the country. the prevention of common oral diseases is the main evidence-based strategy currently used by the ministry of health and medical education in iran. there are two target groups selected to provide regular preventive services, primarily in the hhs, hps and hcs. the two major priority target groups are: (1) pregnant women and lactating mothers as well as (2) children under age 14. as a result of active integration, all non-dental health personnel are required to provide the following preventive oral health services throughout the national health network: • oral examination, recording oral health status, oral health education, oral hygiene instruction, application of fluoride varnish for target children and referral to dentist if further preventive care or treatment needed. • all children are required to have biannual screenings and receive preventive care. similarly, all women are required a visit before conception and three visits during pregnancy as well as biannual visits over the 2 years after delivery. referral to dentist (at the health center) is made if any preventive care is necessary. • recording all daily services provided to target populations (women and children) in electronic databases. the dentists, dental hygienists and oral health technicians supervise and support all oral health activities and services provided by the behvarz and other healthcare providers such as family health technician staff at hhs and hps, local schools screening program, etc., in designated geographic areas. other duties are: • providing preventive oral healthcare and treatment services for all patients visiting the center, with special priority given to target populations. • creating records, generating reports for all new patients; accepting referrals from hh and hps. • conducting face to face oral health education for all patients. • coordination and supervision of school management for proper implementation of different projects such as oral health screening, supervised tooth brushing and fluoride varnish application at primary schools. • referring complicated cases requiring specialist services. • supervising and controlling sterilization and infection control procedures for dental units and equipment. • active participation in local and regional oral health-related projects and activities. • supporting inter-sector coordination, advocating for cooperation. • using the available electronic information technology system for reporting the daily activities. • conducting training sessions for health personnel, parents, teachers, etc. • preparing appropriate training material and educational packages for each group. • providing finger toothbrushes designed for infant age group. • program evaluation and publication of reports. • close supervision, critical system and outcomes analysis for providing constructive feed-back. workforce development training of dental specialist, dentist, as well as other allied dental health technicians has now been done locally for over six decades. over this time period, the number of dental schools has increased from five schools that were initially established, to 66 dental schools that are currently fully functional. the numbers of local graduates are about 1500 annually. additionally, about 500 foreign graduates begin practicing in iran each year. the workforce development is rather very fast in iran and the total numbers of graduates are expected to increase by 8–10% annually (table 1). the role of the dental hygienists and oral health technician are the keys to success of the national oral healthcare reform. the initiative for training hundreds of such mid-level personnel is currently underway, to provide level-1 preventive services in local communities, focusing on target population groups. national oral healthcare reform this reform is mainly focused on all three levels of preventive care, the primary, secondary and tertiary prevention. the initial target population is the 7 million primary school students at the national level. the plan is to screen students and provide primary preventive care to all school children. the major objective is the prevention and early diagnosis of dental caries, periodontal diseases and provision of early treatment for any existing conditions by referral to dentist. usually, the first oral health screening for children start at age 6, within the school property right before children begin schooling and continues every 6 months afterward. in 2015, through a formal ceremony in one of the primary schools in tehran, a memorandum of understanding (mou) was signed between the minister of health and the minister of education to facilitate the initiation of free preventive oral healthcare in all primary schools nationwide. based on this reform plan, all students will be checked two times a year for receiving intraoral examination, oral health education, oral hygiene instruction, fluoride varnish application, as well as referral to dentist if sealant therapy or other treatments are needed. due to resource limitation, the permanent dentition of the primary school children has been the primary focus of the table 1. dentist-population ratio in i.r.-iran 1997–2017 year dentists population 1 dentist 1997 10,000 60,000,000 6000 2007 20,000 70,000,000 3500 2017 30,000 80,000,000 2667 110 j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 oral health status and healthcare system in i.r. iran country report khoshnevisan m.h. et al. national oral healthcare reform at this time. although it will be expanded later, the screening of children under age 5 for assessment of possible early childhood caries, is conducted mostly in nurseries based on availability of local/provincial resources. general objectives improving the oral health status of all children under age 14. specific objectives • enhancing knowledge and improving oral healthcare habits. • decreasing caries incidence in primary and permanent dentition. • improving oral healthcare service coverage for target populations. strategies • adopt evidence-based oral disease preventive methodologies. • improve access to oral health education and preventive healthcare services. • use electronic information technology system for process and outcome monitoring and evaluation. • adopt necessary adjustments when reorientation was necessary. activities • establishment of mou between the ministry of health and ministry of education. • monitoring and evaluation of the process as well as the outcomes at the national level. • expansion of activities aiming at 100% coverage of the target populations. • providing annual executive report on achievements and impediments for further improvements. manpower the public health dentists, dental hygienists and oral health technicians who are employed in the national health network are responsible to visit the local primary schools and provide preventive oral healthcare. however, in urban areas, the number of dentists, dental hygienists and oral health technicians are not sufficient to cover all the primary school children. therefore, dental students are helping to provide such preventive care to school children. before starting any school oral health activity, all dental students are trained and calibrated for oral examination, oral health education, oral hygiene instruction and fluoride varnish application to assure the quality and standards of care provided nationwide. as the provision of preventive care expands, more manpower is obviously needed. ideally, it would be best if private dental practitioners will be involved. about 90% (27,000) of the dentists in iran are in private practice. through public– private-partnership more primary school children can receive early dental restorations (level i preventive care) if proper policy is in place. there is still very low interest among the private dentists to sign a contract with insurance companies. the main problem for such outlook is the low rate of insurance reimbursement and delay in payments for services provided by dental practitioners. funding although historically dental insurance coverage has been very minimal and mainly limited to tooth extraction and denture construction in the past; for the first time ever the insurance companies agreed to cover the cost of preventive dental care. the services specified in new dental benefit coverage are: (1) oral examination/screening, (2) oral health education, (3) fluoride varnish application, (4) fissure sealants therapy for posterior permanent teeth, (5) scaling and tooth polishing, (6) dental restorations, (7) pa/bw x-rays, (8) extraction of untreatable primary and permanent teeth. these services are also included as a component of a national plan called the “family physician plan” that provides full medical coverage to populations living in the remote and underserved areas of the country. therefore, anyone with rural insurance coverage can obtain all covered dental services free of charge in the rural areas. similarly, urban citizens can benefit all covered services by paying 30% copay. at this time although participation is not remarkably high, few number of “private dentists” in each province are testing the newly developed program by signing contract with insurance companies for providing dental care to insured children. dental records and surveillance system the students’ unique national identification card (id) card number is used for creation of electronic dental records. when using the id number, students’ demographic information is automatically obtained from the national database and therefore, the dental records will be attached to individuals’ health records. the children’s base-line oral health status is updated biannually. likewise, all preventive care and other treatment services provided will be recorded on a daily basis. all these records are accumulated in the provincial database and at the same time a copy is being sent to the database at the ministry of health for monitoring and evaluation purposes. such data is crucial for sustainable quality control and effectiveness evaluations. currently, the children up to age 14 are defined as the target population. however, the future plan is to gradually expand preventive healthcare services to high school students (through age 18). oral disease epidemiology and oral health research in this section, the current oral health status is explained by reviewing dental caries, caries-free status, periodontal diseases, tooth loss and dental prosthesis used, dental fluorosis, dental erosion, dental trauma, treatment needs indices as well as smoking cessation activities in dental office. controlling tobacco as a common risk factor is one of the who recommended priorities for oral health promotion globally. khoshnevisan m.h. et al. 111j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 country report oral health status and healthcare system in i.r. iran dental caries experience in children the dmft index as a measure of oral health can demonstrate the proportion of children who have decay (d), missing (m) due to caries or filled (f ) deciduous teeth (dmft) and/or in their permanent dentition (dmft). the dmft index for 5–6-year-old children was 5.16 at the national level (table 2). the analysis showed that 85.93% of this index was related to d-component, while the mand f-components were 4.86% and 9.19%, respectively. the mean number of decayed, missing and filled teeth in this sample was 4.5, 0.26 and 0.39, respectively. this means that when conducting a random screening of 100 children, it is expected to see 450 decayed teeth, 26 missing and 39 filled teeth in this age group. when considering distribution by sex and place of residence, this index was higher in rural (5.78) than urban (4.94) areas; and it was also higher among girls (5.24) than boys (5.09), although the difference was not statistically significant (p = 0.384). the dmft index in 12-year-old children was 2.09 at the national level (table 2). the analysis showed that 81.83% of this index was related to d-component, while the mand f-components were 2.94% and 15.22%, respectively. the distribution of d-component showed higher score among boys (85.6%) when compared with girls (78.5%) (fig. 1). also, the rate was higher among the rural dwellers (91.8%) when compared with their counterpart children living in the urban areas (77.5%). the dmft index in 12-year-old children by sex and place of residence, showed significantly higher value in rural (2.29) when compared with urban (2.02) areas (p = 0.032); and it was greater among girls (2.24) when compared with boys (1.94) in the same age group (p = 0.015). the average dmft score in 15-year-old children were reported as 3.29 at the national level (table 2). about 75% of this index was related to d-component and the mand f-components were 5.46% and 19.75%, respectively. children who lived in urban and rural areas demonstrated an index of 3.26 and 3.42, respectively. the corresponding index for 15-year-old boys and girls were 3.27 and 3.31, respectively. although the index was not statistically significant by sex (p = 0.864) and place of residence (p = 0.542). this score was less than the national average in 12 provinces and the lowest was 1.86 in the province of khuzestan. the d-component in 15-year-old boys and girls were 76.3% and 73.9%, respectively. the score was higher in children living in rural areas (85.2%) when compared with their urban living counterparts (72%). the extracted teeth were higher in girls (5.8%) compared to boys (5%). likewise, this statistic was higher among rural living children (7.4%) when compared with children living in urban areas (4.9%). additionally, the number of restored teeth was higher among girls (20.3%) when compared with boys (18.8%). this figure was also higher among urban living children (23.1%) when compared with rural living counterparts (7.4%). caries-free status about 12.7% of the 5–6-year-old children had no dental caries, restoration and/or extraction of only their primary dentition. the index by place of residence showed higher caries -free children living in urban areas (14.1%) when compared to rural areas (10.3%). likewise, the ratio was higher among boys (13.1%) when compared with girls (12.3%). about 13 provinces had a score higher than the national average and the best caries-free score was reported from bushehr province (23.4%). the caries-free rate for permanent dentition among 12-year-old children was 35% (fig. 2). the distribution by sex and place of residence showed a higher score in boys (36.3%) compared to girls (33.7%) (p = 0.227); and a table 2. dmft index by age and gender among iranians (inohs-2012) age group dmft urban rural male female d/dmft (%) m/dmft (%) f/dmft (%) cf (%) 5–6 5.16 4.94 5.78 5.09 5.24 85.93 4.86 9.19 12.7 12 2.09 2.02 2.29 1.94 2.24 81.83 2.94 15.22 27.4 15 3.29 3.26 3.42 3.27 3.31 74.79 5.46 19.75 0.4 35–44 13.20 12.99 13.98 13.51 13.07 32.65 39.81 27.52 — 65–74 25.71 25.29 27.25 26.84 24.84 11.72 84.22 4.05 — fig. 1 dmft components in 12-year-old children by sex and place of residence (inohs-2012). 112 j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 oral health status and healthcare system in i.r. iran country report khoshnevisan m.h. et al. significantly higher rate in urban citizens (36.5%) when compared to rural dwellers (32.5%) (p < 0.001). about 13 provinces reported higher rates than the national average and the highest rate was reported from khorasan razavi province (50.5%). the rate of caries-free in 15-year-old children was reported to be 0.4% at the national level only. the highest value was reported for khozestan province by 1.8%. dental caries experience in adults the dmft index for 35–44-year-old adults was 13.2. the index was significantly (p = 0.037) higher among rural (13.98) dwellers when compared with urban residents (12.99). the index for male (13.51) was higher than females of the same age group (13.07) (p = 0.210). in 9 out of 31 provinces the index was less than national average for this age group. the lowest figure with 8.63 was reported from bushehr province. the d-component in 35–44-year-old adults was 32.65%, while the mand f-components were 39.81% and 27.52%, respectively. the distribution of the d-component showed higher scores among the males (35%) when compared with females (31.6%). also, the rate was higher among the rural dwellers (41.6%) when compared with counterpart group living in the urban areas (30.2%). the distribution of the m-component showed 50.4% in rural areas and 37% in urban areas. the dmft index for 65–74 years old age group was 25.71 at the national level (table 1). the index for eight provinces was less than the national average and the lowest record with 18.16 was reported from bushehr province. the analysis showed that only 11.72% of the index was related to the d-component, while the mand f-components were 84.22% and 4.05%, respectively. obviously, this record shows that majority of permanent teeth have been lost in this age group either due to dental caries, periodontal diseases or both. the distribution by sex and place of residence, showed that the index was significantly higher among male (26.84) than female (24.84) (p = 0.008); and greater in rural dwellers (27.25) than urban residents (25.29) (p = 0.001) (fig. 3). the mean number of decayed, missing and filled teeth in this sample was 2.44, 22.56 and 0.71, respectively. this means that when conducting a random screening of 100 adults, it is expected to observe 244 decayed teeth, 2256 missing and 71 filled teeth in this age group. fig. 4 demonstrates the status of dmft index in all age groups with break-down by sex and place of residence.6 root surface caries the dft index for root caries was reported as 0.78% in 35–44-year-old adults at the national level. the distribution by sex and place of residence showed a higher score in male (1.05%) when compared with females (0.67%). likewise, a higher value was reported for rural living adults (1.15%) when compared with urban living (0.68%) counterparts. total of 15 provinces demonstrated higher than the national average. the highest score was reported from sistan and balochistan province with 3.03 mean roots dft. mean number of decayed and filled roots in 65–74 years old age group was reported 0.79. the dft index by sex and sex and place of residence was higher in female (0.83) than male (0.73); and higher in rural (1.22) than urban areas (0.67). about 21 provinces showed lower and 10 provinces demonstrated higher values. the highest score was reported from sistan and baluchestan province (2.68).6 periodontal diseases bleeding score was 9.7% for 5–6-year-old children at the national level. the distribution by sex and place of residence was 9.4% in boys and 10.1% in girls (p = 0.178), while the score was significantly lower in urban dwellers (8.5%) when compared to residents of rural areas (11.8%) (p = 0.002) (table 3). the national value for bleeding score in 12-year-old children was 26.9%. the distribution by sex and place of residence was 26.9% in boys and 27% in girls (p = 0.263), however, the score was significantly lower in urban living fig. 2 percentage of 12 years old caries-free children by sex and place of residence (inohs-2012). khoshnevisan m.h. et al. 113j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 country report oral health status and healthcare system in i.r. iran fig. 3 dmft components in 65–74 years old adults by sex and place of residence (inohs-2010). table 3. periodontal indices by age, sex and place of residence (percentage) (inohs-2012) age group bleeding pocket loa total urban rural male female 4–5 mm 6+ mm 4+ mm 5–6 9.7 8.5 11.8 9.4 10.1 — — — 12 26.9 25.7 28.8 26.9 27.0 — — — 15 33.8 31.8 37.3 35.3 32.6 0.3 4.6 4.0 35–44 55.5 52.0 61.4 57.0 54.7 3.1 25.6 22.9 65–74 60.9 60.0 62.3 61.5 60.3 8.8 34.6 47.6 fig. 4 the dmft/dmft index among the 5 who suggested age groups by sex and place of residence (inohs-2012). children (25.7%) than their counterparts in rural areas (28.8%) (p = 0.028) (table 3). note: values of recorded bleeding among children under age 15 was evident during the examination. no pocket depth was performed in children of these age groups. the national value for bleeding score in 15-year-old children was 33.8%. the distribution by sex and place of 114 j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 oral health status and healthcare system in i.r. iran country report khoshnevisan m.h. et al. residence was 35.3% in boys and 32.6% in girls (p = 0.216). however, the score was significantly lower in urban (31.8%) versus rural areas (37.3%) (p = 0.003) (table 3). although over 90% of the 15-year-old children at the national level had no periodontal pocket, about 7% had 4–5 mm pockets and 0.4% showed 6+ mm pockets at the national level. the loss of attachment (loa) 4–5 mm was 3.6%; loa 6–8 mm was 0.4%; and loa 9+ was 0% in this age group. as demonstrated in table 3, the national score for bleeding was 55.5% in 35–44-year-old adults. the distribution by sex and place of residence was 57% in male and 54.7% in females (p = 0.001), however, the score was significantly lower in urban (52%) versus rural areas (61.4%) (p = 0.001) (fig. 5). although 71.3% of the 35–44-year-old adults had no periodontal pocket, about 25.6% had 4–5 mm pockets and 3.1% showed 6+ mm pockets at the national level. the rate of periodontal pockets 6+ mm was very close to each other in both sex (male = 3.2%; female = 3%) which were close to the national average (3.1%) as well. however, the distribution by place of residence showed a significantly higher score in rural area (5.4%) when compared with adults living in urban areas (1.8%) (p < 0.001). the loss of attachment with 4+ mm was reported as 22.9% in the 35–44-year-old age group at the national level (loa 4–5 mm = 19.7%; 6–8 mm = 2.8%; 9–11 = 0.3%; 12+ mm = 0.1%). the distribution of index by sex and place of residence showed higher score among male (25.5%) than female (21.6%) and significantly higher values in rural dwellers (30.8%) compared with urban residents (18.3%) (p < 0.001). although the sex difference was not significant, a significantly higher difference was detected in rural residents when compared with city dwellers (p < 0.001). the bleeding score was 60.9% in 65–74-year-old adults at the national level (table 3). the distribution by sex and place of residence was 61.5% in male and 60.3% in females (p = 0.668). however, the score was lower in urban (60%) than rural areas (62.3%) (p = 0.302). the highest score was reported from sistan and baluchistan provinces with 80.5% in 65–74 years old age groups. over 56.6% of 65–74-year-old adults did not have any periodontal pockets. however, about 34.6% had 4–5 mm pockets and 8.8% showed 6+ mm pockets at the national level. the distribution of periodontal pocket of 4–5 mm by sex and place of residence showed higher score in male (35.9%), than females (33.3%) (p = 0.686); and 34% in urban compared to 35.5% in rural areas (p = 0.131) (fig. 6). the distribution of periodontal pocket of 6+ mm by sex and place of residence showed close score values among male (8.8%) and female (8.7%); while the score was higher among rural residents (10.9%) when compared to urban counterparts (8.8%).6 fig. 5 bleeding on probing among 35–44-year-old samples by sex and place of residence (inohs-2012). fig. 6 bleeding on probing among 65–74-year-old samples by sex and place of residence (inohs-2012). khoshnevisan m.h. et al. 115j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 country report oral health status and healthcare system in i.r. iran reported from kohkiloyeh and boyr-ahmad with 7.1% and bushehr with 5.4%. the dental enamel fluorosis score in 15-year-old children were 2% at the national level. the distribution by sex showed 2.1% for boys and 1.9% for girls, the difference by place of residence showed 1.3% in children living in rural areas and 3.1% in their counterparts living in the city (table 4). however, the differences in sex (p = 0.070) and place of residence (p = 0.337) were not statistically significant. although the reported rates were over the national average in 19 provinces; five provinces reported no fluorosis in both sex and children living in urban or rural areas. the highest score was reported from kohkiloyeh and boyr-ahmad (8.1%), and bushehr (7.6%) provinces. the fluorosis score in 35–44-year-old adults was 3.3% at the national level. the distribution by sex showed 2.9% for male and 3.5% for females, the difference by place of residence showed 4.1% in adults living in rural areas and 2.8% in their counterparts living in the city (table 4). however, the differences in sex (p = 0.322) and place of residence (p = 0.106) were not statistically significant in this age group. although three provinces reported no fluorosis, 10 provinces reported rates over the national average in both sex and for adults living in either geographical (urban/rural) area. the highest score was reported from bushehr province with 21%. the fluorosis score in 65–74-year-old adults was 3.5% at the national level. the distribution by sex showed 3.3% for male and 3.8% for females, the difference by place of residence showed 4.1% in adults living in rural areas compared with 3.1% in their counterparts living in the city (table 4). however, no statistical significance was detected in sex (p = 0.441) and place of residence (p = 0.708) in this age group. although 10 provinces reported no fluorosis, seven provinces reported rates more than the national average. the highest score was reported from bushehr province with 28.6%.6 the calculated age-specific community index of enamel fluorosis by region demonstrated eight provinces with records above the national average concentration of fluorosis in all age groups. these rates in these provinces may be considered as an alert for a public health problem. geographically, they are located in the north-west to south-west and south-east of the country. in this case potential recommendations are the use of de-fluoridation system, changing the food or water sources at the community level or using individual (maternal and child) level approaches during tooth development periods when appropriate. dental erosion the national score for dental erosion in 5–6-year-old children was 9.5% with mean number of 4.64 affected teeth (table 5). the distribution by sex and place of residence showed 9.2% in girls, 10.3% in boys (p = 0.001); 12.6% in rural and 7.6% in urban areas (p < 0.001). the highest rate of erosion was reported from kohkiloyeh and boyr-ahmad province with 45.5% with a mean number of 5.23 teeth involved. the national score for dental erosion in 12-year-old children was 4.5% with a mean number of 3.55 affected teeth (table 5). although no sex difference was detected (male = female = 4.5%) (p = 0.558), the distribution by place of residence showed 4.8% in children living in rural area and 4.3% in urban living counterparts (p = 0.501). the difference tooth loss and dental prosthesis about 40% of 35–44-year-old adults have reported tooth loss with 4% of them being complete edentulousness at the national level. two provinces reported no edentulous cases, while 18 provinces reported a score less than the national average. the highest score was reported 13.4% from zanjan province. the analysis showed that 6.3% of 35–44-year-old adults had partial dentures and 5.1% had complete dentures in lower jaw; and 4.4% had partial dentures and 4.1% had complete dentures in upper jaws. the highest rate of adults with partial denture in this age group was reported from alborz province with 16% in the upper jaw and fars province with 12% in lower jaw. although the highest rate of adults with maxillary and mandibular complete dentures were reported 14.4% and 13.4% both in zanjan province, respectively. about 84% adults of 65–74 years old had extracted teeth with 52.2% of them being complete edentulous at the national level. there were 13 provinces with less ratio and 18 provinces with the score above the national average. the highest score was reported from zanjan province with 84% complete edentulousness.6 dental enamel fluorosis although fluorosis of dentin can occur, it requires sophisticated equipment and methods; in oral health surveys. the fluorosis lesions recorded are assessed by visual examination, therefore, the recorded values are the status of enamel fluorosis only. although, 91% of all children had no fluorosis, the national score for 5–6 year old children was 0.8%. while there was not much sex difference (male = female = 0.8%), the distribution by place of residence showed 1.2% of children living in rural areas and 0.6% in their counterparts living in the city affected by fluorosis (p = 0.005) (table 4). although reported rates were over the national average in 10 provinces; eight provinces reported no fluorosis in children of both sexes who were living in urban or rural areas. the highest score was reported from kerman, kohkiloyey and boyrahmad provinces both with score of 3.7%. the national score for dental enamel fluorosis in 12-year-old children was 1.6%. while the sex difference was minimal between boys (1.7%) and girls (1.5%) (p = 0.394), the distribution by place of residence showed 2.6% in children living in rural areas and 0.9% in their counterparts living in the city (p < 0.001) (table 4). although five provinces reported no fluorosis, 12 provinces reported rates over the national average. the highest provincial score was table 4. percent age-specific community index of enamel fluorosis (inohs-2012) age group total (national) place of residence gender urban rural male female 5–6 0.8 0.6 1.2 0.8 0.8 12 1.6 0.9 2.6 1.7 1.5 15 2.0 1.3 3.1 2.1 1.9 35–44 3.3 2.8 4.1 2.9 3.5 65–74 3.5 3.1 4.1 3.3 3.8 116 j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 oral health status and healthcare system in i.r. iran country report khoshnevisan m.h. et al. was not statistically significant. the highest rate of erosion reported from kohkiloyeh and boyr-ahmad province with 24.2% and mean number of 2.01 teeth involved. the national score for dental erosion in 15-year-old children was 6.5% with a mean number of three affected teeth (table 5). the erosion score was higher among girls (6.6%) than boys (6.4%) (p = 0.154); while, the distribution by place of residence showed a statistically significant higher rate in children living in rural areas (7.8%) compared with urban (5.8%) living counterparts (p < 0.001). the highest rate of erosion was reported from kohkiloyeh and boyr-ahmad province with 28.4% with a mean number of 2.3 teeth involved. the national score for dental erosion in 35–44-year-old adults was 27.1% with a mean number of 4.8 affected teeth (table 5). the distribution by sex and place of residence showed statistically significant differences between male (29.4%) and females (26%) (p < 0.001); as well as urban (26.2%) and rural (28.7%) dwellers (p < 0.001). the highest rate of erosion reported from kohkiloyeh and boyr-ahmad provinces with 61.7% and a mean number of 6.64 teeth involved. the national score for dental erosion in 65–74-year-old adults was 49.7% with a mean number of 6.25 affected teeth (table 5). the distribution by sex was 49.5% in male, 50.5% in females (p = 0.174). however, the difference between places of residence was significantly lower in urban (48.7%) versus rural (51.3%) populations (p < 0.006). the highest rate of erosion was reported from ghazvin province with 72.9% and a mean number of 6.81 teeth involved.6 dental trauma the national score for dental trauma in 5–6-year-old children was 1.5% with a mean number of 1.6 affected teeth (table 5). the highest rate of trauma was reported from fars province with 3.4% with a mean number of 1.7 teeth involved. the distribution by sex and place of residence showed 1.1% in girls and 1.9% in boys (p = 0.033); and 1.2% in rural and 1.7% in urban areas (p < 0.201). the highest reported mean number of teeth involved in trauma was 7.5 in 1% of 5–6-year-old children from kurdistan province. the national score for dental trauma in 12-year-old children was 4.9% with mean number of 1.29 affected teeth (table 5). the distribution by sex and place of residence showed 3.4% in girls and 6.5% in boys (p < 0.001); and 4.6% in rural versus 5.1% in urban areas (p = 0.428). the highest rate of trauma was reported from kohkiloyeh and boyr-ahmad province with 10.4% and mean number of 1.13 teeth involved. the national score for dental trauma in 15-year-old children was 6% with a mean number of 1.2 affected teeth (table 5). the distribution by sex and place of residence showed 4.8% in girls and 7.5% in boys (p < 0.001); and 5.7% in rural and 6.2% in urban areas (p = 0.350). the highest rate of trauma was reported from kohkiloyeh and boyr-ahmad province with 11.2% and mean number of 1.2 teeth involved. the national score for dental trauma in 35–44-year-old adults was 5.5% with a mean number of 1.64 affected teeth (table 5). the distribution by sex and place of residence showed 5.1% in females and 6.3% in males (p = 0.381); and 5.2% in rural and 5.6% in urban areas (p = 0.026). the highest rate of trauma was reported from tehran province with 11.9% and a mean of 1.61 teeth involved. interestingly, males in the sistan and baluchistan province had the highest number of teeth involved in trauma reported at 3.71 teeth in 8% of 35–44 year age group. the national score for dental trauma in 65–74-year-old adults was 4% with a mean number of 1.29 affected teeth (table 5). the distribution by sex and place of residence showed 3.5% in females and 4.5% in males (p = 0.090); and 3.7% in rural and 4.2% in urban areas (p = 0.366). the highest rate of trauma was reported from kurdistan province with 18.9% and a mean number of 4.94 teeth involved. although khorasan jonubi province reported the highest number of teeth (9.1) involved in 2.9% of the 65–74-year-old adult populations.6 treatment needs only about 15.7% of the 5–6 year old children did not need any treatment. which means that, about 84.3% of 5–6 year old children were reported to be in need of treatment (table 6). the need was almost equal in both sex (male = 84.0%; female = 84.5%), while children living in rural areas (88.2%) had more need compared to children living in urban areas (81.9%) (p < 0.001). the treatment needs in 6 provinces were reported to be more than 90% of children 5–6 years old. of all those who need treatment, 2.5% were in need of scaling and table 5. prevalence of dental erosion and trauma in different age groups (inohs-2012) age group dental erosion dental trauma % of individuals with erosion mean number of teeth with erosion % of individuals with trauma mean number of teeth with trauma 5–6 9.5 4.64 1.5 1.6 12 4.5 3.55 4.9 1.29 15 6.5 3.0 6.0 1.2 35–44 27.1 4.8 5.5 1.64 65–74 49.7 6.25 4.0 1.29 table 6. treatment needs by age, sex and place of residence (inohs-2012) age group total urban rural male female 5–6 84.3 81.9 88.2 84 84.5 12 75.1 72.3 79.6 75.8 74.3 15 76.0 73 81.2 75.9 76.2 35–44 86.1 84.4 88.9 87.0 85.7 65–74 45.9 43.7 49.4 46.8 45.0 khoshnevisan m.h. et al. 117j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 country report oral health status and healthcare system in i.r. iran fig. 7 distribution of treatment needs among 5–6-year-old children by sex and place of residence (inohs-2012). prophylactic treatment (cleaning of teeth); 69% needed immediate restoration for asymptomatic cavities; 12.3% had emergency treatment need for relief of pain and/or infection; and 0.5% needed referral for specialty evaluation (figure 6). about 75% of 12 year old children were in need of treatment (table 6). the treatment need was very close in boys (75.8%) and girls (74.3%) (p = 0.173). while children living in rural areas (79.6%) had significantly more needs compared to children living in urban areas (72.3%) (p < 0.001). the treatment needs in 10 provinces were reported to be more than 80%. of all those who needed treatment, 11% were in need of scaling and prophylactic treatment (cleaning of teeth); 57.7% needed immediate restoration for asymptomatic carious teeth; 5.7% had emergency treatment for relief of pain and/or infection; and 0.7% needed referral for more comprehensive evaluation. about 76% of 15 year old children were in need of treatment (table 6). the treatment need was very close in boys (75.9%) and girls (76.2%). while children living in rural areas (81.2%) had significantly more needs compared to children living in urban areas (73%) (p < 0.024). of all those who need treatment, 12.2% were in need of scaling and prophylactic treatment (cleaning of teeth); 56.3% needed immediate restoration for asymptomatic carious teeth; 7.1% had emergency treatment for relief of pain and/or infection; and 0.6% needed referral for more comprehensive evaluation. about 86% of the 35–44 year old adults were in need of treatment (table 6). the treatment need was very close in male (87%) and females (85.7%) (p = 0.250). while adults living in rural areas (88.9%) had significantly more needs compared to adults living in urban areas (84.4%) (p < 0.001). of all those who need treatment, 9.4% were in need of scaling and cleaning of teeth; 60.8% needed immediate restoration for asymptomatic carious teeth; 13.4% had emergency treatment need for relief of pain or infection; and 2.5% needed referral for more comprehensive evaluation by specialists. about 45.9% of the 65–74 year old adults were in need of treatment (table 6). the treatment need was higher in male (46.8%) compared with females (45%) (p = 0.485). while adults living in rural areas (49.4%) had significantly more needs compared to adults living in urban areas (43.7%) (p < 0.001). of all those who need treatment, 4.5% were in need of scaling and prophylactic treatment; 24% needed immediate restoration for asymptomatic carious teeth; 8.2% had emergency treatment need for relief of pain and/or infection; and 9.2% needed referral for more comprehensive evaluation.5 new initiative on smoking cessation in dental office tobacco use is a common risk factor and is one of the main root cause of many oraland systemic diseases. the harmful effect of tobacco starts in the mouth, it can affect dental, periodontal as well as overall health, well-being and quality of life in smokers and anyone around them. since there are about 30,000 dentists practicing in iran, it is a unique opportunity to provide smoking cessation program by the dental team in dental clinics. based on the local studies conducted in iran,5 large numbers of dentists have expressed interest in providing consultation and cessation services for patients who smoke. these dentists mostly expressed the need for learning the necessary skills for effective patient communication and counseling. so far, more than a dozen of training of the trainer (tot) workshops has been conducted at the national level. as a result, more than 200 volunteer dentists from all 30 provinces have successfully completed the training program and developed effective communication and consultation skills for smoking cessation. these trainers are running the local workshops at the provincial level. when necessary, referral from dentist to cessation specialist for additional consultation and support can be arranged. further expansion of such trainings can be facilitated by collaborative activities of the who collaborating center for training and research in dental public health; and the who collaborating center for tobacco control, both located at shahid beheshti university of medical science and health services, in tehran, iran. at the same time, ngos collaboration (www.dentii.info) has been instrumental over the past decade to accomplish greater achievements in smoking behavioral change. dental and oral health research activities numbers of dental research projects have been substantially increased over the past 30 years. priorities in dental research have been evaluated by three different institutions independently. although government support has been increased substantially but, funding has not reached to the expected level yet. the research projects supported by the ministry of health have been very effective in leading to great discoveries such as the cem-cement an internationally well-known dental material that was developed as a result of local investigation. it has been successfully used for saving the primary as well as permanent dentitions from early tooth loss both in adults and children. using this material in oral health units of 118 j contemp med sci | vol. 4, no. 3, summer 2018: 107–118 oral health status and healthcare system in i.r. iran country report khoshnevisan m.h. et al. the national healthcare network was instrumental in preventing several thousand teeth from early extractions each year. this is an example of knowledge translation into practice, using evidence-based interventions. dental equipment, instruments and materials although many imported goods are still being used in iran, the move for local production of considerable number of dental equipment, instruments and materials have been started decades ago. the numbers of local products are ever increasing. many of these items with the successful local market are being exported to other countries as well. today, many dental professionals prefer to use local dental products due to good quality, pricing and after sales services available nationwide. discussion and conclusion for many years it has been a problem to bring a deeper understanding of oral and dental public health to the attention of decision makers on what could be done about oral disease prevention and oral health promotion and protection in iran. such diseases not only affecting individuals by pain, infection, discomfort, early tooth loss and eventually cause disability and negative systemic health effects, but also can have greater impact on the community and health system through ever-increasing associated costs. the overall treatment cost far exceeds any assumptions, when considering the systemic diseases caused or aggravated by oral conditions. it has been reported that the cost of treatment in such cases are even higher than the cost of cancer, cardiovascular diseases and stroke to name a few. at the same time, it is extremely disturbing to know that these are all happening while oral diseases are preventable through simple cost-effective measures; but it is still being neglected in most parts of the world. lack of supportive oral health promotion policies for allocation of appropriate resources (money and manpower) has been the major reason for the current challenging situation in the country. at the same time, robust data was needed for proving the need for proper policy as well as preventive program development. such data is also needed for evaluation of any intervention and demonstration of preventive program effectiveness. otherwise, the escalating cost of treatment will greatly impact low income individuals and communities. there is strong evidence that the cost of preventive care is much less than the treatment, for both individuals and governments. the declaration of the national oral healthcare reform, as a fully integrated program in phc, has greatly facilitated better public access to preventive oral care. given the availability of strong evidence-based methods and best practice measures for dental caries and periodontal disease prevention; everyone, especially the designated priority target groups in iran are expected to benefit the most, for all the years to come. the current policy with appropriate investment of resources can be instrumental in increasing awareness, enhancing patient education to encourage healthy life style and self-care. all these good practices are necessary for our nation to become a “caries free nation” by 2025 as it is particularly evident in many scandinavian countries. acknowledgement the authors would like to extend special thanks to many experts from the research institute of dental sciences, shahid beheshti university of medical sciences; oral health bureau, ministry of health and medical education; as well as the oral health units of the medical science universities in all (31) local provinces. likewise, special appreciation goes to all (44200) survey participants who made this nationwide project a reality. conflict of interest none n references 1. petersen pe, baez rj. oral health surveys: basic methods. 5th ed.; world health organization, geneva, 2013. 2. elgood c. a medical history of persia and the eastern caliphate: from the earliest times until the year 1932, cambridge university press, 2010. 3. gorji a, khaleghi ghadiri m. history of headache in medieval persian medicine. lancet neurol. 2002;1:510–515. 4. kharabaf s, abdollahi m. science growth in iran over the past 35 years. j res med sci. 2012;17:275–279. 5. shadpour k. the phc experience in iran. 2nd ed.; unicef, tehran, 1994. 6. oral health status in iran-2012 (inohs-2012). oral health bureau, ministry of health and medical education. 2013. 7. ahmady ae, khoshnevisan mh, heidari n, lando ha. dentists’ familiarity with tobacco cessation programs in dental settings in iran. j public health dent. 2011;71:271–277. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201801 25j contemp med sci | vol. 2, no. 5, winter 2016: 25–27 research effect of countermovement and arm swing on vertical stiffness and jump performance azadeh shadmehra*, s. maryam hejazia, gholamreza olyaeia, saeid talebiana introduction during running or jumping, body’s musculoskeletal system, including muscles, tendons and ligaments are acted together, so that the whole system behaves like a spring. as a result, these behaviours can be explained using a spring–mass model, consisting of a lower extremity as spring and a point mass as body mass.1,2 the leg spring compresses and then lengthens during the ground contact phase, as lower limb joints flex and then extends. the stiffness of the leg spring represents the stiffness of the whole system during the ground contact phase. lower extremity stiffness can affect the response of body to the environment’s perturbations. for assessing this parameter, there are different methods and different functions. vertical stiffness (kvert) represents the vertical displacement of center of mass (com) at the middle of the stance phase during hop in place or vertical jump.1–4 cavagna (1975) explained that displacement of com was determined from double integration of the force–time curve in vertical axis derived from force plate’s data.4 jumping is a functional task that frequently has been used in daily living activity or sport activities. till now, hopping and drop jump frequently have been used for assessment of stiffness but there was lack of adequate evidence about investigating maximum vertical jump as a high demand activity for assessing vertical stiffness. only one study has investigated the contribution of stiffness to vertical jump performance.5 the study of laffaye et al. (2005) has shown that enhancement in jump height will result in lower stiffness.5 these results were in contradiction with farley et al. (1991), which reported that during hopping stiffness increases due to increase in hopping frequency or hopping height.1 it seems that there was no cut-off for the amount of stiffness. high stiffness is related to bony injuries and low stiffness is related to soft tissue injuries.6 understanding the effect of jump performance on stiffness would be expected to augment the efficiency as well as reduce the sport injuries. arm swing and countermovement were the strategies used in jumping to improve jump performance. the effects of these mechanisms on jump performance have been studied before.7–10 these techniques are accompanied with increase in ground reaction force (grf) and work output. although the influences of both arm swing and countermovement on jump performance have been examined by many researchers, the contribution of stiffness to perform in countermovement jump (cmj) and with arm swing (cmja) is still unknown. the purpose of this study was to investigate how the combinations of both strategies can affect vertical stiffness and maximum vertical jump performance. methods subjects twenty-five young healthy females with no training experience participated in this study. their mean (sd), age, weight, height and body mass index (bmi) were 22.6 (1.67) years, 55.92 (5.36) kg, 162.48 (3.94) cm and 21.46 (1.83) kg/m2, respectively. the exclusion criteria were lower extremity abnormalities, previous leg injury, fracture, surgery and balance impairment. all the subjects signed the consent form and then entered in the present study and the project was approved by the ethics committee of tehran university of medical sciences. jumping protocol squat jump (sj): subjects were instructed to dip to ~110° knee flexion, with their hands on their iliac crests, maintain that position and the examiner counted out 2 s on the call of examiner, the subject jumped as high as possible. squat jump with arm swing (sja): for using arm swing, subjects started sj with extended arms and swing them at once the jumping motion had been initiated. countermovement jump (cmj): the subjects started the jump while their hands were on their hips, they were instructed to, with call of examiner, they dip to ~110° knee flexion as quickly as possible and then jump as high as possible. issn 2413-0516 adepartment of physical therapy, school of rehabilitation, tehran university of medical sciences, tehran, iran. correspondence to a. shadmehr (email: shadmehr@tums.ac.ir) (submitted: 28 october 2015 – revised version received:17 december 2015 – accepted: 3 february 2016 – published online: 26 march 2016) objectives to determine the effect of using arm swing and countermovement on vertical stiffness and maximum vertical jump performance. participants a total of 25 young healthy females were participated in the study. they stood on the force plate and performed two models of squat jump with (sja) and without arm swing (sj) and two models of countermovement jump with (cmja) and without arm swing (cmj). main outcome measures: vertical leg stiffness, jump height, flight time, contact time and power were compared in sj, sja, cmj and cmja. results in the cmjs, the stiffness and jump height were significantly higher than sja and sj. contact time in jumps with countermovement and/or arm swing was three times lower than sja and sj. conclusion vertical stiffness and performance parameters can be improved by using countermovement and arm swing during vertical jump and due to enhancement in work output and ground reaction force. keywords vertical stiffness, countermovement, arm swing, jump height 26 j contemp med sci | vol. 2, no. 5, winter 2016: 25–27 effect of countermovement and arm swing for vertical stiffness research azadeh shadmehr et al. countermovement jump with arm swing (cmja): for using arm swing, subjects started cmj with extended arms and swing them at once the jumping motion had been initiated. procedures kvert and performance parameters were assessed within one session and to determine reliability for seven subjects, test repetitions were performed in another session that was 24 h later. testing took place at same time of day and same room. before the test, participants performed enough practice jumps to warm up and familiarised with the procedure. a time up to 5 min was given between practices and jump tests. after that, they were asked to perform randomised maximal jumps with 2 min of rest for prevention of fatigue, from a force platform (9090, kistler, usa). the following variables were calculated with this information: mean and peak force; peak power and flight time. from the force platform, the center of pressure (cop) and the vertical components of grf were obtained.4 the displacement of the com of the body at time t was calculated from double integrating of acceleration of com. the jump height was determined by using flight time according to the formula of jump height (cm) = 1/8 gt2, where g = acceleration due to gravity (9.81 ms−1) and t = flight time of the jump(s).11 for this equation, the body position in the moment of take-off and landing must be the same. subjects were need to extend their hip, knee and ankle joints at initial ground contact of landing.12,13 power was measured as rate of force changes during contact time.13 statistics after data collection, means and standard deviations were calculated. the reliability of procedures was calculated utilising four methods. the pearson product moment (ppm), the intraclass correlation coefficient (icc) and its associated 95% confidence interval, the standard error of measurement (sem) and the paired t-test were determined as outcome measures for reliability and reproducibility. a repeated measures analysis of variance was used to examine the effect of countermovement and arm swing. post-hoc contrast (bonferroni) was table 1. pearson product moment (ppm), intraclass correlation coefficient (icc), lower and upper confidence limits, paired t-test and standard error measurement (sem) for different jump types (n = 7) variables jump types ppm (sig) icc confidence limits % paired t-test sem k vert (kn/m) sj 0.96(0.008) 0.94 0.53–0.99 0.78 0.007 sja 0.99(0.004) 0.98 0.77–0.99 0.6 0.001 cmj 0.97(0.000) 0.97 0.69–0.99 0.36 0.004 cmja 0.92(0.016) 0.94 0.39–0.99 0.14 0.011 jh (m) sj 0.98(0.007) 0.97 0.26–0.99 0.31 0.002 sja 0.97(0.13) 0.91 0.65–0.98 0.81 0.009 cmj 0.99(0.000) 0.98 0.63–0.99 0.28 0.000 cmja 0.92(0.004) 0.96 0.15–0.99 0.19 0.003 k vert : vertical stiffness; jh: jump height; sj: squat jump; sja: squat jump with arm swing; cmj: countermovement jump; cmja: countermovement jump with arm swing. table 2. comparison of mean (sd) for vertical stiffness (k vert ), jump height (jh), flight time (ft), contact time (ct) and power (p), among sj, sja, cmj, cmja variables sj sja cmj cmja k vert (kn/m) 9.88 (2.17) 10.33 (2.09) 10.47 (2.34) 11.02 (2.39) jh (m) 0.139 (0.021) 0.141 (0.022) 0.142 (0.021) 0.155 (0.021) ft (s) 0.338 (0.031) 0.342 (0.025) 0.348 (0.022) 0.35 (0.032) ct (s) 0.085(0.019) 0.084 (0.015) 0.084 (0.017) 0.078 (0.016) p (knm/s) 718 (244.03) 725.49 (224.48) 778.5 (225.24) 733.55 (232.30) sj: squat jump; sja: squat jump with arm swing; cmj: countermovement jump; cmja: countermovement jump with arm swing. used to examine differences among the groups. significance of tests was accepted at an alpha level of 0.05. results it can be observed from table 1 that there was very good reliability in all four jump types (ppm > 0.93, icc > 0.91, sem < 0.01). the vertical stiffness response to use of countermovement and arm swing can be observed in table 2. mean vertical stiffness of subjects significantly was increased from 9.88 ± 2.17 to 11.02 ± 2.39 kn/m across four types (table 2). mean jump height of subjects showed significant increase from 0.199 ± 0.025 to 0.245 ± 0.26 m across four types (table 2). besides, for flight time, an enhancement across all four types was seen from 0.336 ± 0.031 to 0.35 ± 0.032 s (table 2). mean contact time of subjects across four jump types significantly was decreased from 0.177 ± 0.039 to 0.157 ± 0.033 s (table 2). mean power of subjects showed no significant differences between the four jump types (table 2). discussion the purpose of this study was to determine the effect of countermovement and arm swing on vertical stiffness and jump performance and thereafter to establish the relationship between them. an enhancement in vertical stiffness was observed across four types of jump (sj < sja < cmj < cmja), see fig. 1. some 27j contemp med sci | vol. 2, no. 5, winter 2016: 25–27 research effect of countermovement and arm swing for vertical stiffnessazadeh shadmehr et al. previous studies have reported that augmentation in work output and grf occurred by using countermovement and arm swing. stiffness directly associates to the force changes, so enhancement in reaction force can augment the amount of that.9,7,14 increase in jump height also can increase the stiffness.3 in our study, jump height increased with using countermovement and arm swing. similar findings have been reported by other studies, during performance assessment.9,14,7 lees et al. (2004) have reported that during cmj, because of greater work output of the hip extensor muscles, the jump height was higher in cmj than sj.7 on the other hand, grf was increased by utilising the arm movements. the higher grf caused an increase in ground reaction impulse which was the reason for the enhanced jump height.9 ziv & lidor (2010) have been reported that augmented jump height in cmj was associated with the stretchshortening cycle (ssc).15 with using cm, the contractile components store and release energy during eccentric and then concentric phases of jump. some studies investigated the effect of arm swing and have seen an enhancement in grf and net impulse.9,15 our results about flight time are similar to other studies which reported the augmentation in this parameter, with increased jump height.16,17 countermovement and arm swing can augment the energy stored and displacement of com, and so the enhancement occurs in time for transformation of energy or flight time.18 arampatzis et al. (2001) have reported that by increasing the rate of force change during jumps the contact time after landing becomes shorter.19 in our study, no significant differences between the powers of four types of jump were found. samozino et al. (2008) have reported that power was related to velocity rather than jump height and arampatzis et al. (2001) reported that power was related to force rather than stiffness.` our study showed that, with higher levels of performance, from sj to cmja, and increment in force production and work output, there was an enhancement in the amount of stiffness. people utilise different mechanisms for performing maximum vertical jump. based on our research, countermovement and arm swing can positively affect the performance and vertical stiffness. it seemed to be with change in level of activity and need to force production, the body adjusts stiffness and led to change in the amount of resistance against reaction forces and maintain efficiency of system. acknowledgement the authors would like to thank all the subjects who participated in the experiment. this study was a part of an msc thesis and sponsored by tehran university of medical science. the authors would like to acknowledge the assistance of the faculty and staff of the school of rehabilitation, tums.  references 1. farley ct, blickhan r, saito j, taylor cr. hopping frequency in humans: a test of how springs set stride frequency in bouncing gaits. j appl physiol. 1991;71:2127–2132. pmid: 1778902 2. brughelli m, cronin j. a review of research on the mechanical stiffness in running and jumping: methodology and implications. scand j med sci sports. 2008;18:417–426. doi: 10.1111/j.1600-0838.2008.00769.x pmid: 18282225 3. farley ct, morgenroth dc. leg stiffness primarily depends on ankle stiffness during human hopping. j biomech. 1999;32:267–273. pmid: 10093026 4. cavagna ga. force platforms as ergometers. j appl physiol. 1975;39(1): 174–179. pmid: 1150585 5. laffaye g, bardy bg, durey a. leg stiffness and expertise in men jumping. med sci sports exerc. 2005;37(4):536–543. pmid: 15809549 6. butler rj, crowell hp 3rd, davis im. lower extremity stiffness: implication for performance and injury. clin biomech. 2003;18:511–517. pmid: 1282890 7. lees a, vanrenterghem j, de clercq d. the maximal and submaximal vertical jump: implications for strength and conditioning. j strength cond res. 2004;18(4):787–791. pmid: 15574084 8. moran ka, wallace es. eccentric loading and range of knee joint motion effects on performance enhancement in vertical jumping. hum mov sci. 2007;26:824–840. pmid: 17928080 9. hara m, shibayama a, takeshita d, hay dc, fukashiro s. a comparison of the mechanical effect of arm swing and countermovement on the lower extremities in vertical jumping. hum mov sci. 2008;27:636–648. doi: 10.1016/j.humov.2008.04.001 pmid: 18674837 10. tauchi k, endo t, ogata m, matsuo a, iso s. the characteristics of jump ability in elite adolescent athletes and healthy males: the development of countermovement and rebound jump ability. int j sport health sci. 2008;6:78–84. 11. young w. a simple method for evaluating strength, qualities of the leg extensor muscles and jumping abilities. strength cond coach. 1995;2(4):5–8. 12. young w, mclean b, ardagna j. relationship between strength qualities and sprinting performance. j sports med phys fitness. 1995;35(1):13–9. 13. samozino p, morin jb, hintzy f, belli a. a simple method for measuring force, velocity, and power output during squat jump. j biomech. 2008;41: 2940–2945. doi: 10.1016/j.jbiomech.2008.07.028 pmid: 18789803 14. cheng k, wang ch, chen hc, wu cd, chiu ht. the mechanisms that enable arm motion to enhance vertical jump performance: a simulation study. j biomech. 2008;41:1847–1854. doi: 10.1016/j.jbiomech.2008.04.004 pmid: 18514208 15. ziv g, lidor r. vertical jump in female and male basketball players: a review of observational and experimental studies. j sci med sport. 2010;13: 332–339. doi: 10.1016/j.jsams.2009.02.009 pmid: 19443269 16. hobara h, inoue k, muraoka t, omuro k, sakamoto m, kanosue k. leg stiffness adjustment for a range of hopping frequency. j biomech. 2010;43:506–511. doi: 10.1016/j.jbiomech.2009.09.040 pmid: 19879582 17. maulder p, cronin j. horizontal and vertical jump assessment: reliability, symmetry, discriminative and predictive ability. phys ther sport. 2005;6:74–82. 18. vanrenterghem j, lees a, lenoir m, aerts p, clercq dd. performing the vertical jump: movement adaptations for submaximal jumping. hum mov sci. 2004;22:713–727. pmid: 15063050 19. arampatzis a, schade f, walsh m, brüggemann gp. influence of leg stiffness and its effect on myodynamic jumping performance. j electromyogr kinesiol. 2001;11:355–364. pmid: 11595555 63j contemp med sci | vol. 2, no. 6, spring 2016: 63–66 research investigation of inhibition efficiency of punica granatum peel extract against bacteria alaa abdul-hussein al-daamya, ula bahaaa, dhuha alaaddina, marwa hassana introduction bacteria are microscopic single cell called microbes; they are also found in ocean soil and inside the human gut.1 it may be beneficial or harmless (pathogenic). pathogenic bacteria not only cause disease to human, animals and plants, but also develop more interest for researchers to study in detail.2 various bacteria cause different types of diseases such as meningitis caused by neisseria meningitidis; ear infection and strep throat caused by streptococcal bacteria, and toxic shock syndrome caused by staphylococcus aureus.3 multiple researches revealed that punica granatum has pathogen-resistant. p. granatum is one of the oldest known fruits found in writings and artifacts of many culture and religions. it has been revered as a symbol of health, fertility and eternal life.4 p. granatum yields powerful antioxidant such as flavonoids, proanthocyanidins and phenolic.5 research shows that various extracts from p. granatum has antifungal activity against listeria monocytogenes, s. aureus, escherichia coli and yersinia enterocolitica.6 the literature also reveals that p. granatum has antifungal activity against dermatophytic fungi and trichophyton mentagrophytes.7 our study aimed to examine the antibacterial activity of extract of p. granatum peel against five types of pathogenic bacteria, four is gram-negative p. aeruginosa, pseudomonas oryzihabita, proteus varaplis and klebsiella pneumonia and one gram-positive s. aureus using acetone, ethanol, methanol, ethyl acetate and water as solvents. materials and methods microorganisms: in this study, we used five pathogenic bacteria isolated from patient in al-zahra teaching hospital in karbala city. these bacteria are p. aeruginosa, p. vulgaris, k. pneumonia and s. aureus. extraction: p. granatum peel was grinded into powder and was uniformly divided in five beakers weighing 20 g each in a beaker. subsequently, 100 ml of solvent (acetone, ethanol, methanol, ethyl acetate, distill water) with 70% concentration to each beaker that contain plant powder and labeled this beaker with the name of solvent and were kept in a shaking incubator over night with 150 cycle/min at 37°c. the next day, the extracts in all five beakers were filtered using cotton and gauze. further, the purified extracts in tubes were centrifuged at 3000 rpm for 5 min. after centrifugation, the supernatant was transferred to glass dishes and allowed them to dry. later, each dish was scrubbed and stored in a clean container and saved as crude extract. the total weight of the crude was also measured.8 determination of antibacterial activity of the best solvent: first, muller hinton agar medium was prepared and sterilized. it was poured in petri dishes and allowed them to solidify. three wells with 5 mm diameter in every dish were made by cork borer. from the pre-collected powder, 0.05 g from each container was taken and put them in five tubes. each tube contains extract for certain type of our five solvents. 2 ml of ethanol 70% was added to each tube and shaked until the powder was dissolved. the final concentration of extract in each tube was equal to 25 mg/ml. in addition to our five tubes, a sixth tube was also added containing 2 ml for control (70% ethanol without any extract). after activation of bacteria in nutrient broth, we cultured it in the prepared dishes. in each petri dish, 100 μl of each activation bacteria was spread by sterilized spreader, then 50 μl plant extract in each well was added. in the negative control, 50 μl ethanol 70% was added, 50 μg/ml gentamycin was used as a positive control. after culturing, all the dishes were incubated in an incubator for 24 hrs under issn 2413-0516 adepartment of clinical laboratory, college of applied medical sciences, university of karbala, karbala, iraq. correspondence to alaa abdul-hussein al-daamy (email: aakm7789@gmail.com). (submitted: 17 march 2016 – revised version received: 5 april 2016 – accepted: 24 may 2016 – published online: 26 june 2016) objectives the present study aims to determine the inhibitory efficiency of punica granatum peel extract and test its effectiveness against pathogenic bacteria. methods acetone, ethanol, methanol, ethyl acetate and distilled water were used as solvent to obtain of crude extract of p. granatum peel, which tested to determine the effectiveness against five types of pathogens, such as pseudomonas aeruginosa, pseudomonas oryzihabitans, proteus vulgaris, klebsiella pneumonia and staphylococcus aureus under a series of concentrations. it was also used to determine the most efficient concentration of solvent optimization, and then was determined minimum inhibitory concentration (mic) of the extract more efficient. results the results of the current study showed that the most efficient extraction solvent is ethyl acetate showed the diameters of inhibition zone are 48.66, 51.5, 41.66, 28.0, and 40.83 mm for the types of bacteria above, respectively. the results showed that the concentration of ethyl acetate was 40% in the optimal inhibition of bacteria, amounting to diameters of inhibition zone at the concentration of 19.83, 27.33, 17.66, 15.16 and 12.33 mm for each of the bacterial species above, respectively. the results also found that mic is 11 mg/ml of p. aeruginosa and 10 mg/ml of p. varaplis and 9 mg/ml of p. oryzihabitans and 3 mg/ml of k. pneumoniae and s. aureus. conclusion the most effective composites against pathogenic bacteria from p. granatum peel are using ethyl acetate solvent with the concentration of 40%. keywords ethyl acetate, punica granatum, antibacterial activity, mic. 64 j contemp med sci | vol. 2, no. 6, spring 2016: 63–66 investigation of inhibition efficiency of punica granatum peel extract against bacteria research alaa abdul-hussein et al. 37°c. in the next day, after incubation we measure the diameter of zone of inhibition was measured against bacteria by solvent.9 the optimal concentration of the best solvent: depending on the results of the previous experiment, ethyl acetate extract was chosen. five different concentrations of ethyl acetate (20, 40, 60, 80, 100%) were taken in each beaker. in each beaker, 20 g of p. granatum extract powder was added, and the same process of extraction method was repeated that described above and recorded the results. in the next step, 0.05 g of dry extract in five separate tubes was taken, then added 2 ml of 70% ethanol to each tube to obtain on the final concentration 25 mg/ml. after that, 30 petri dishes with muller hinton agar were prepared, and followed the three tests to determine the best concentration of ethyl acetate, which has the largest inhibition zone.9 minimum inhibitory concentration (mic) of ethyl acetate: in a flask, 50 ml of nutrient broth was poured, and in the other flask, 25 ml of nutrient broth was added with 0.625 g from ethyl acetate 40% dry extract to obtain on 25 mg/ml concentration in this flask from extraction. 26 glass tubes were taken and labelled them with numbers from 0 to 25. on each tube, the extract from p. granatum with ethyl acetate 40% concentration which is equal to the number of the label of the tube was taken. for example: in tube number 25, 2 ml from flask 2 was added, which represents 25 mg/ml concentration. in tube number 24, 1.92 ml from flask 2 and 0.08 ml from flask 1 were added, which represents 24 mg/ml concentration, so that with other tubes, in the tube number 0 put only 2 ml from flask 1 to obtain of 0 mg/ml in this tube. the distribution between flask 1 and 2 in these tubes is according to the law c1 v1 = c2 v2. after that, micro titter plate with 96 wells and numbered the wells from 0 to 25, twice for each type of bacteria (each type of bacteria have 52 wells twice for each number), then added 150 μl from each tube in two wells of the same number table 1. the percentage of materials extracted from p. granatum peel no. extraction solvent (100 ml) origin weight of plant powder (g) weigh of extract (g) percentage of extract materials (%) 1 acetone 20 0.49 2.45 2 ethanol 20 0.6 3 3 methanol 20 0.5 2.5 4 ethyl acetate 20 0.65 3.25 5 distil water 20 0.457 2.28 table 2. inhibition zone (mm) to extract p. granatum peel against bacteria bacteria extraction solvent (70%) lsd 0.05gentamycin 10 μg/ml acetone ethanol methanol ethyl acetate distil water p. aeruginosa 21.66 ± 0.88 20.5 ± 0.5 0.0 ± 0.0 27.0 ± 2.59 48.66 ± 3.21 12.83 ± 0.44 4.40 p. oryzihabitans 20.33 ± 0.88 46.66 ± 4.63 39.16 ± 0.83 39.33 ± 0.66 51.5 ± 0.76 39.16 ± 3.0 5.91 p. vulgaris 12.5 ± 0.28 33.0 ± 2.51 31.5 ± 3.25 38.83 ± 1.33 41.66 ± 1.66 23.0 ± 0.57 4.81 k. pneumonia 17.66 ± 0.33 20.66 ± 1.20 18.66 ± 0.16 19.66 ± 0.88 28.0 ± 2.46 26.0 ± 3.60 4.76 s. aureus 17 ± 0.57 39.33 ± 1.83 24.16 ± 1.83 31.0 ± 2.56 40.83 ± 4.4 31.16 ± 0.6 5.94 the numbers refer to mean ± standard error. on each type of bacteria, then added 50 μl of each type of bacteria(after dilute it with both media and take from the 3rd dilution comparison with mcfarland solution) in all the 52 wells from 0 to 25 concentrations. later, the plates were covered and incubated in an incubator for 24 hr in 37°c. the plates were examined to see growth or no growth of bacteria to determine the minimal inhibition concentration from extract for each type of bacteria.10 statistical analysis: statistical analysis included random complete design(rcd) with 3 replicates. 0.05 is the level of probability that was used to identify the significant differences. the significant differences between the averages were also tested using the test less significant difference (lsd) at the level of probability of 0.05.11 results the results show the percentage of materials recoverable from p. granatum when we use the solvents: acetone, ethanol, methanol, ethyl acetate and distilled water. the percentages are 2.45, 3, 2.5, 3.25, 2.28%, respectively (table 1). the higher percentage was noted by ethyl acetate 3.25% and low percentage was noted by distilled water 2.28%. the results show that the extract of p. granatum with ethyl acetate give the most large inhibition zone with significance differences (p < 0.05) in all five type of bacteria p. aeruginosa, p. oryzihabita, p. vulgaris, klebsila pneumonia and s. aureus (table 2). the diameters of inhibition zone are 48.66, 51.5, 41.66, 28.0, 40.83 mm, respectively. the results were significantly different (p < 0.05) comparison with gentamycin and comparison with others solvent. the extract of p. granatum with ( 20, 40, 60, 80, 100)% of ethyl acetate was examined (table 3). larger inhibition zone with significant difference (p < 0.05) against gentamycin from hand and in other hand between ethyl acetate 40% and other concentrations was noted. 65j contemp med sci | vol. 2, no. 6, spring 2016: 63–66 research investigation of inhibition efficiency of punica granatum peel extract against bacteriaalaa abdul-hussein et al. the diameter with the concentration of 40% that gives 19.83, 27.33, 17.66, 15.16, 12.33 mm against p. aeruginosa, p. oryzihabitans, p. vulgaris, k. pneumonia and s. aureus, respectively. the less concentration of extract that caused inhibition to bacteria (table 4). it was noted that the concentration 3 mg/ ml of extraction showed inhibition activity against k. pneumonia and s. aureus, and the concentration 9 mg/ml inhibited p. oryzihabitans, and the concentration 10 mg/ml inhibited p. vulgaris and concentration 11 mg/ml inhibit p. aeruginosa. from this result, it can be noted that bacteria k. pneumonia is more sensitive than the others because it gave less mic, and in contrast p. aeruginosa gave large mic. discussion various extracts from p. granatum have antibacterial activity against listeria monocytogen, s. aureus, e. coli and yersinia enterocytogene.6 in other study, p. granatum has antibacterial activity against the dermatophytic fungi trichophyton mentagrophytes.7 table 3. inhibition zone (mm) of extract p. granatum peel against bacteria by using dilutions series of ethyl acetate bacteria ethyl acetate ratio (%) lsd 0.05gentamycin 10 μg/ml 20% 40% 60% 80% 100% p. aeruginosa 21.66 ± 0.88 14.66 ± 4.1 19.83 ± 3.52 18.73 ± 1.13 4.66 ± 0.33 0.0 ± 0.0 4.80 p. oryzihabitans 20.33 ± 0.88 16.83 ± 4.1 27.33 ± 1.30 20.83 ± 1.16 13.5 ± 0.28 0.0 ± 0.0 4.68 p. vulgaris 12.5 ± 0.28 15.0 ± 0.28 18.16 ± 1.74 17.66 ± 1.09 17.66 ± 1.16 9.83 ± 0.33 2.49 k. pneumonia 17.66 ± 0.33 15.83 ± 1.09 17.16 ± 2.74 17.16 ± 1.48 15.16 ± 0.44 13.5 ± 0.28 3.45 s. aureus 17 ± 0.57 18.33 ± 0.6 21.0 ± 0.28 15.0 ± 1.52 12.33 ± 1.83 5.0 ± 0.5 2.66 the numbers refer to mean ± standard error. table 4. minimum inhibitory concentration (mm) of ethyl acetate extract p. granatum peel extract concentration (mg/ml) types of bacteria p. aeruginosa p. oryzihabitans p. vulgaris k. pneumonia s. aureus 1 + + + + + 2 + + + + + 3 + + + − − 4 + + + − − 5 + + + − − 6 + + + − − 7 + + + − − 8 + + + − − 9 + − + − − 10 + − − − − 11 − − − − − 12 − − − − − 13 − − − − − 14 − − − − − 15 − − − − − 16 − − − − − 17 − − − − − 18 − − − − − 19 − − − − − 20 − − − − − 21 − − − − − 22 − − − − − 23 − − − − − 24 − − − − − 25 − − − − − + means growth − means not growth. 66 j contemp med sci | vol. 2, no. 6, spring 2016: 63–66 investigation of inhibition efficiency of punica granatum peel extract against bacteria research alaa abdul-hussein et al. s. aureus and e. coli but it was found that methanol extract had high inhibitory effect. in one study reported that e. coli is more resistant to all p. granatum extract than other gram -ve bacteria.17 in our study, the difference between previous studies may be in use of different solvents or type of bacteria and also the composition of plant help us to gain more inhibitory effect that contain flavonoids, proanthocyanidins and phenolic. conclusion from the results, we concluded that the extract of p. granatum peel by ethyl acetate 40% has high antibacterial activity against the pathogenic bacteria that isolated from patients. recommendations other study on p. granatum peel is proposed to purify the material, which owns the effectiveness of anti-bacterial pathogenesis.  previous studies reveal that p. granatum has antibacterial activity against s. aureus, and its juice also has inhibitory effect to s. epidermidis and k. pneumonia.12,13 methabe et al14 show that methanol, ethanol, acetone and water extract obtained from p. granatum have antibacterial activity against s. aureus, salmonella typhi, e. coli, s. dysenteriae, vibrio cholera, s. soni, shigella boydii and shigella flexneri . other study15 found that the extract from pnica granatum also showed antibacterial activity against e. coli, enterobacter cloacae, p. fluorescens, proteus vulgaris, alcaligenes faecalis, serratia marcescens, enterobacter aerogenes, s. aureus, micrococcus luteus, bacillus cereus, bacillus subtilis, micrococcus roseus, mycobacterium phlei, bacillus coagulans, micrococcus rodochrus and mycobacterium smegmatis with inhibition zone 11–31 mm. ahmed and beg16 show that alcohol extract of p. granatum have antibacterial activity against e. coli, s. aureus and shigella dysenteriae. also al-zoreky6 started to notice that the water extract of p. granatum have no antibacterial activity against references 1. vidyasagar a. introduction to bacteria, what are bacteria? live science contributor. 2015. 2. mcmahon m. pathogenic bacteria. wise greek, 4 april 2015. 3. health grades editorial staff. bacterial disease. health grades, new york, ny . 2013. 4. seeram np, aviram m, zhang y. comparison of antioxidant potency of commonly consumed polyphenol-rich beverages in the united states. j agric food chem. 2008, 56:1415–1422. 5. reddy mk, gupta sk, jacob mr, khan si, ferreira d. antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from punica granatum l. planta med. 2007 may;73(5):461–467. 6. al-zoreky ns. antimicrobial activity of punica granatum fruit peels. int j food microbial. 15 sep.2009 ; 134(3):242–244. doi:10.1016 /jijj. 7. simone rf, celso vn, tania un, diogenes agc, eliana he and benedito pdf. antifungal activity of pomegranate peel extract and isolated compound punicalagin against dermatophytes. ann clin microbiol antimicrob. 2014; 13(32): 1–6. doi: 10.1186/s12941-014-0032-6. 8. al-daamy a a, abd-al ameer h, zuher h, monather h, ahmmed b and kadhim n. antifungal activity of propolis against dermatophytes and candida albicans isolated from human mouth. j contemp med sci. 2015;1(3):4–8. 9. al-daamy aak, al-shibli mk, al-ghanimi aa. purification and characterization of kojic acid produced by two local isolates (aspergillus flavus and aspergillus fumigatus). thesis. college of education, univesity of al-qadesiah, iraq. 2014. 10. stephen j and cavalieri e. manual of antimicrobial susceptibility testing. american society for microbiology. 2005. 11. dequ g and tessema f. biostatistics. university gondar, funded under usaid from the american people, cooperative agreement no. 663-a-0000-0358-00. 2005. 12. aynechi y, salehisrmaghi m h, shirudi m and souri e. screening of iranian plants for antimicrobial activity of some thai flowers and plant. mahido university, j pharm sci. 1982;10:81. 13. lee j., waston rr. pomegranate: a role in health promotion and aids? in nutrition, foods and aids, waston rr, ed, crc press, boca raton, florida,usa .1998. pp:179 . 14. methabe mc, nikolova rv, la11 n, nyazema nz. antibacterial activities of medicinal plants used for the treatment of diarrhea in limpopo province, south africa. j ethnopharmacol. 2005;105:286–293. 15. kulkani ap, aradhya sm. chemical changes and antioxidant activity in pomegranate arils during fruit development .food chem. 2005;93: 319–324. 16. ahmad ii, beg az. antimicrobial and photochemical studies on 45 indian medicinal plants against multi-drug resistant human pathogens. j ethnophamacol. 2001;74:113–123. 17. negi ps, jayaprakasha gk. j food sci. 2003;68:1473–1477. 59j contemp med sci | vol. 6, no. 2, march–april 2020: 59–65 59 original issn 2413-0516 introduction diabetes mellitus is a chronic disease with a rising prevalence globally. the number of diabetic patients in 2011 was estimated to be around 366 million worldwide, and it is predicted that this figure will rise to more than 50% by 2030. a prevalence of 12.5% was estimated among people aged 20–79 years in middle east and north africa region.1 for the iranian population, prevalence has increased from 7.7% in 2005 to 10.3% in 2016.2–4 diabetes elevates risk of a number of macroand microvascular complications.5, 6 a pooled analysis of 8.49 million person-years at risk, from 102 prospective studies, indicated that hazard ratios (hrs) of coronary heart disease, ischemic stroke and hemorrhagic stroke were 2, 2.27 and 1.56 for diabetic compared with non-diabetic peoples, respectively.7 a population-based study in iran found that hrs of having cardiovascular disease (cvd) were 3.30 and 1.90 for women and men with diabetes compared with people without diabetes, respectively.8 the association between diabetes and complications causes both a shorter life expectancy9, 10 and a poorer health-related quality of life (hrqol) among the diabetic patients compared to those without diabetes.11 similarly, diabetic patients with diabetes-related complications have lower hrqol compared to diabetic patients without any complication.12–15 there has been a growing interest in assessing hrqol in patients with diabetes in iran over the recent years. these studies have reported a negative impact of diabetes-related complications on hrqol.16–18 hrqol is a multidimensional concept that includes domains related to physical, mental, emotional, and social functioning. it goes beyond direct measures of population health, life expectancy, and causes of death, and focuses on the impact health status has on quality of life, for example, for diabetic patients.19 while, hrqol is an important outcome in evaluating the effects of different health states and assessing the effectiveness of various interventions, it is patients’ health utilities (hus) which are the main interest in the context of economic evaluation.20 some approaches to economic evaluation in the health sector are: cost of illness studies, cost benefit analysis, cost effective analysis (cea), cost utility analysis. in cost utility analysis, an extension of cea but enables comparisons of different treatments with quite different outcomes. this is especially when interventions cause differences in the quantity (survival) and quality of life. it does this through combining these in the common metric of the quality adjusted life year (qaly). the costs data for achieving a qaly mean that different interventions for the same illness/conditions can be compared as can interventions for other conditions. it can be used to assess whether drugs should be listed on pbs and to guide resource allocation decisions hus are used to calculate qalys, as a common outcome measure in cost–utility analyses.21 hu is a measure of individual’s preferences for different health outcomes. it is a cardinal value, usually between 0 and 1, covering different health states from the worst to perfect health.20 these hu values are generally combined with survival estimates to generate qaly (e.g., 2 years with a hu value of 0.7 and 2 years with a hu value of 0.5 generate a qaly value of 2.9 for these 5years survival).20 there are two main approaches to elicit the patients’ hu: direct method such as standard gamble and time trade-off, and indirect method using preference-based measures such as health-related quality of life and health utility among patients with diabetes in zabol, southeast iran seyed pouria hedayati1, hassan haghparast-bidgoli2, aliasghar a. kiadaliri3, fatemeh mohabati1 1department of health management and economics, school of public health, tehran university of medical sciences, tehran, iran. 2institute for global health, university college london, london, uk 3department of clinical sciences lund, lund university, faculty of medicine, orthopaedics, clinical epidemiology unit, lund, sweden. corresponding author: fatemeh mohabati (e-mail: mohabatif@gmail.com) (submitted: 16 january 2020 – revised version received: 10 february 2020 – accepted: 14 march 2020 – published online: 26 april 2020) objectives the current study aimed to assess the factors associated with health-related quality of life (hrqol) and health utility (hu) among patients with diabetes in zabol, southeast iran. methods among patients referred to zabol city diabetes clinic, a total of 213 consecutive patients 18 years and older consent to participate in the study in 2015. the persian version of euroqol-5d-3l (eq-5d-3l) using the uk preference weights was applied to derive hu. logistic regression and ordinary least squares were used for data analysis. the stata version 13 (statacorp lp, college station, tx, usa) was used for statistical analysis. results the highest and lowest proportions of “some or extreme problems” were seen in pain/discomfort (86.6%) and self-care (27.8%) dimensions of the eq-5d-3l, respectively. about 33% of women and 14% of men rated their health worse than death (p=0.002). the mean eq-5d-3l index score and visual analogue scale were 0.37 (95% ci: 0.31–0.42) and 51.6 (95% ci: 48.7–54.5), respectively. older age at diagnosis, longer duration of diabetes, lower education, and history of macrovascular complications were associated with lower hrqol and hu. conclusion this study highlights the importance of education and diabetes-related complications in hrqol/hu of diabetes people. the findings suggest that urgent interventions are required to improve hrqol/hu of diabetes patients in zabol. moreover, our results provide inputs for future economic evaluation studies among diabetes patients with similar socioeconomic status in iran. keywords diabetes, health-related quality of life, health utility, eq-5d-3l, iran. 60 original health-related quality of life seyed pouria hedayati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 59–65 euroqol-5d (eq-5d)22 and sf-6d.23–25 among generic instruments, eq-5d, whoqol, health utility index, quality of well-being, and sf-36 have been used internationally. we selected eq-5d because, among these instruments, it has the advantage of being able to calculate a single comprehensive scalar unit of values that can be compared among diseases and used for economic evaluation. eq-5d is a preference-based hrql questionnaire that was developed in europe.26 we measured hrql in patients with diabetes using eq-5d, one of the preference-based measures among hrql instruments that enable calculation of the utility value.27 to our knowledge, only one previous study measured hu among patients with type 2 diabetes using the eq-5d in iran (28). they found that while cvd and nephropathy had a negative impact on hu, there was positive association between having retinopathy and hu.28 the primary aim of the current study was to assess demographic, socioeconomic, and clinical correlates of hrqol/ hu among patients with diabetes in a deprived area of iran. in addition, since event-specific effects on hu are more useful in conducting economic evaluation studies, our secondary aim was to evaluate the effect of macrovascular complications including myocardial infarction (mi), coronary heart disease and stroke as separate events, not pooled as cvd, on patients’ hu. methods design and subjects a cross-sectional study was conducted among patients referred to a hospital-based diabetes clinic in zabol in 2015, 213 of 254 consecutive patients (with either type 1 or type 2 diabetes) who visited the diabetes clinic met the inclusion criteria for the current study: (a) age 18 years or older, (b) consent to participate in the study. these were patients with confirmed diabetes by physicians in the clinic. this is the only diabetes clinic in zabol. the study was approved by the zabol university of medical sciences’ ethical committee. explanatory variables a questionnaire was designed by the research team to collect the data on the variables required for the current study. the main variables included year of birth, year of diagnosis of diabetes, gender, resident place, household income, employment status, years of education, weight (kg), height (cm), and history of self-reported doctor’s diagnosed macrovascular diabetes-related complications. patients were asked if they visited a physician for any of the following complications during last year: stroke, mi, and coronary heart disease. a categorical variable using three quintiles of household income was used as the measure of socioeconomic status in this analysis. health-related quality of life and health utility assessment the persian version of eq-5d-3l was used to assess hrqol/ hu in the study. the eq-5d-3l is simple to use and have shown good performance among people with diabetes in previous studies.29,30 the eq-5d-3l is a multiattribute preference-based instrument which constitute of five dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. each dimension has three levels: no problems, some problems, and extreme problems,22 which result in 24335 potential health states. responses to these dimensions are weighted based on the preference elicited from a sample of general population to compute an index score. the eq-5d-3l index score ranges from less than 0 (negative values) for health states worse than death to 1 for full health. the eq-5d-3l questionnaire also contains a visual analog scale (vas) tool which entails respondent rates his/her current health state on a scale from 0, the worst imaginable health state, to 100, the best imaginable health state. in the current study, due to lack of preference weights for the iranian population, the value sets for uk population31 was used. patients responded to the eq-5d-3l questionnaire through a face-to-face interview, conducted by a trained interviewer. this questionnaire has been translated by the euroqol group into various languages, and for this study, after filling out a form about the current study method on the group’s website, the translated and validated version was sent to the participants. the reliability and validity of the eq-5d have been well-documented in different contexts for different diseases.32–34 data analysis the continuous variables are shown as mean and standard deviation and the categorical as percentages. responses to the eq-5d-3l questions were merged for all five dimensions and a binary outcome as “no problem” or “some or extreme problem” was created. then, χ2 and logistic regression were used to assess the associations between the explanatory variables and these binary variables. the stata version 13 (stata corp lp, college station, tx, usa) was used for statistical analysis. the eq-5d-3l index score and vas were analyzed using t-test, analysis of variance (anova) and ordinary least squares (ols). due to skewed nature of the eq-5d-3l scores, several methods have been used in the literature to analyze the scores.35–37 we chose the ols with robust standard errors in the current study for two main reasons: first, only 5% the patients reported no problem in any dimensions of the eq-5d-3l (i.e., an index score = 1.0) and 1% reported a vas score of 100. it is shown that in this situation ols works as well as other methods.36 second, when hu is the main interest of analysis, as in the current study, the ols with robust standard errors is a valid approach.37 as education level is highly associated with employment and income, we excluded income and employment status from multivariate analysis to avoid any mediation bias. years of education was categorized in two level: 8 years and less, and more than 8 years. three patients with missing value on the year of diagnosis were excluded from the analysis. the design variables and residual plots were used to check the linearity of the continuous variables and continuous covariates were treated as mean-centered values. the stata version 13 (stata corp lp, college station, tx, usa) was used for statistical analysis. participants were asked to participate on a voluntary basis. they were informed about the study objectives, procedures, risks, benefits, alternatives, their rights, and data anonymity and confidentiality. this information was included in the informed consent form signed by the participants. results the mean (sd) age at diagnosis and duration of diabetes were 39.86 (±13.36) years and 9.73 (±7.10) years, respectively. sixty three percent of the sample were male and 31% had a bmi ≥ 61 original health-related quality of lifeseyed pouria hedayati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 59–65 30 (table 1). the macrovascular complications were more common among men. “some or extreme problems” in pain/discomfort dimension has the highest prevalence, with 86.6%, followed by anxiety/depression (84.5%). in total, 33.3% of women and 14.1% of men rated their health worse than death (i.e., eq-5d-3l score < 0, p=0.002). the mean (95% ci) eq-5d-3l index score and vas scale were 0.37 (0.31–0.42) and 51.6 (48.7– 54.5), respectively. the spearman rank correlation between the eq-5d index score and vas scale was 0.65 (p < 0.001). the univariate analysis showed that men and people diagnosed at age older than 25 years had lower problems on the eq-5d-3l dimensions (table 2). better socioeconomic status was generally associated with lower frequency of the problems. the patients with a history of macrovascular events generally suffered from more problems, but this was not statistically significant for pain/discomfort and anxiety/ depression dimensions. patients who received combination of insulin and oral hypoglycemic agents (oha) had statistically significantly more difficulties in doing their usual daily activities. the results of logistic regression showed no gender difference in suffering from problems in the eq-5d-3l dimensions (table 3). except for self-care, there was no statistically significant association between age at diagnosis and having problems in any eq-5d-3l dimensions. longer duration of diabetes and history of macrovascular complications were associated with higher odds of having problems. people with higher education had statistically significantly lower odds of having problems in all dimensions. the results of ols regression revealed that older age at diagnosis, longer duration of diabetes, lower education, and history of macrovascular complications were associated with lower eq-5d-3l index scores (table 4). a similar finding was observed when vas scale was used as dependent variable, except for history of stroke which was no longer statistically significant. discussion in order to support conducting economic evaluation of diabetes preventive or curative interventions in iran, we have estimated hu scores for a range of factors, including demographic, socioeconomic, and clinical factors, among patients referred to a clinic in southeast iran. as one may expect, macrovascular complications’ history was associated with lower hrqol/hu among patients with diabetes. among these complications, history of mi had the highest negative impact on eq-5d-3l index score. in addition, higher education was associated with higher hrqol/hu among patients with diabetes similar to previous national and international studies,28,38–40 people with diabetes had more commonly problem on pain/discomfort and anxiety/depression dimensions of the eq-5d-3l and had least problem in self-care. in addition, physician, nurses, and other caregivers should pay more attention on these dimensions. our findings on the association between education, age at diagnosis, and duration of diabetes with hrqol/hu were in line with previous national and international studies.13,30,41–43. these findings have important clinical and policy-making implications since identifying the most affected dimensions of hrqol and its determinant can guide toward a better management of the disease and improving hrqol in these patients. we found no significant association between treatment modality and hrqol/hu. this might be due to poor sensitivity of the eq-5d-3l to treatment modality in diabetes context as has been previously shown.15,28,44 while the mean vas score was closer to the value reported in the national survey of type 2 diabetes (51.5 vs. 56.8), the mean eq-5d index score in the current study was significantly lower than the national survey (0.37 vs. 0.70).28 there are several possible explanations for this disparity: first, our study included the patients who were referred to a clinic who might be potentially sicker than general diabetes population included in the national survey. second, zabol city located in sistan & baluchestan province that is considered as one of the most deprived provinces in the country. this poor socioeconomic status not only can directly affect the patients’ hrqol/hu, but also influences the quality and access to care and treatment for patients in this city. third, we included both type 1 and type 2 diabetes patients while in the national survey only type 2 diabetes patients were included. patients with type 1 diabetes are diagnosed in younger age, therefore have table 1. demographic, socioeconomic and clinical characteristics of the sample, stratified by sex (n = 213) variable men women n 78 135 age at diagnosis, years 39.87 ± 15.35 39.86 ± 12.12 diabetes duration, years 10.70 ± 8.33 9.17 ± 6.24 bmi 26.08 ± 4.50 28.85 ± 4.57 treatment oha (%) 57.69 48.15 insulin ± oha (%) 42.31 51.85 history of mi (%) 7.69 11.11 history of coronary heart disease (%) 15.38 14.81 history of stroke (%) 5.13 12.59 household income low (%) 38.46 47.41 middle (%) 26.92 31.11 high (%) 34.62 21.48 employment unemployed / housekeeper (%) 12.82 90.37 employed (%) 38.46 5.93 retired (%) 48.72 3.70 education no education (%) 14.10 50.37 1–8 years (%) 33.33 34.07 9–12 years (%) 37.18 13.33 >12 year (%) 15.38 2.22 bmi: body mass index, oha: oral hypoglycaemic agents, mi: myocardial infarct. 62 original health-related quality of life seyed pouria hedayati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 59–65 ta bl e 2. a ss oc ia tio n of th e di m en si on s o f e q5d -3 l a nd e xp la na to ry v ar ia bl es in a na ly si s ( n = 2 13 ) va ria bl e m ob ili ty se lfca re us ua l a ct iv iti es pa in /d is co m fo rt an xi et y/ de pr es si on eq -5 d in de x pe rc en t pva lu e pe rc en t pva lu e pe rc en t pva lu e pe rc en t pva lu e pe rc en t pva lu e m ea n (9 5% c i) pva lu e ge nd er w om en 69 .6 3 0. 00 1 34 .0 7 0. 01 2 53 .3 3 0. 02 3 90 .3 7 0. 04 6 86 .6 7 0. 25 2 0. 31 (0 .2 4– 0. 39 ) 0. 01 6 m en 47 .4 4 17 .9 5 37 .1 8 80 .7 7 80 .7 7 0. 46 (0 .3 7– 0. 54 ) ag e at d ia gn os is, ye ar s ≤ 25 37 .0 4 0. 00 3 7. 41 0. 03 2 18 .5 2 0. 00 4 74 .0 7 0. 07 8 62 .9 6 0. 01 7 0. 67 (0 .5 6– 0. 78 ) < 0. 00 1 26 –3 5 53 .1 9 21 .2 8 46 .8 1 82 .9 8 91 .4 9 0. 37 (0 .2 5– 0. 48 ) 36 –4 5 79 .0 3 35 .4 8 62 .9 0 95 .1 6 87 .1 0 0. 26 (0 .1 6– 0. 37 ) 46 –5 5 60 .3 4 31 .0 3 43 .1 0 87 .9 3 84 .4 8 0. 36 (0 .2 5– 0. 47 ) ≥ 56 63 .1 6 42 .1 1 52 .6 3 84 .2 1 89 .4 7 0. 28 (0 .0 3– 0. 53 ) bm i < 25 55 .7 4 0. 07 8 22 .9 5 0. 05 1 42 .6 2 0. 23 5 81 .9 7 0. 40 2 80 .3 3 0. 52 2 0. 43 (0 .3 2– 0. 54 ) 0. 18 5 25 –3 0 56 .9 8 23 .2 6 44 .1 9 88 .3 7 87 .2 1 0. 37 (0 .2 9– 0. 46 ) ≥ 30 72 .7 3 39 .3 9 56 .0 6 89 .3 9 84 .8 5 0. 30 (0 .1 9– 0. 40 ) du ra tio n of d ia be te s, ye ar s ≤ 5 54 .5 5 0. 22 5 24 .6 8 0. 43 4 44 .1 6 0. 61 4 83 .1 2 0. 25 2 80 .5 2 0. 41 1 0. 40 (0 .3 0– 0. 50 ) 0. 51 1 5– 10 66 .1 0 30 .5 1 45 .7 6 93 .2 2 84 .7 5 0. 32 (0 .2 1– 0. 43 ) 10 –1 5 58 .7 0 23 .9 1 47 .8 3 82 .6 1 84 .7 8 0. 41 (0 .2 9– 0. 54 ) > 15 74 .1 9 38 .7 1 58 .0 6 90 .3 2 93 .5 5 0. 30 (0 .1 7– 0. 43 ) ho us eh ol d in co m e lo w es t q ui nt ile 64 .8 9 0. 22 0 31 .9 1 0. 50 3 50 .0 0 0. 05 0 90 .4 3 0. 20 9 87 .2 3 0. 61 9 0. 32 (0 .2 4– 0. 40 ) 0. 18 9 m id dl e qu in til e 65 .0 8 26 .9 8 55 .5 6 87 .3 0 82 .5 4 0. 36 (0 .2 5– 0. 47 ) h ig he st q ui nt ile 51 .7 9 23 .2 1 33 .9 3 80 .3 6 82 .1 4 0. 45 (0 .3 4– 0. 57 ) em pl oy m en t u ne m pl oy ed 68 .9 4 0. 00 4 33 .3 3 0. 07 7 54 .5 5 0. 15 0 91 .6 7 0. 02 4 87 .8 8 0. 18 2 0. 30 (0 .2 3– 0. 38 ) 0. 01 3 em pl oy ed 39 .4 7 15 .7 9 28 .9 5 76 .3 2 76 .3 2 0. 51 (0 .3 9– 0. 63 ) re tir ed 58 .1 4 23 .2 6 41 .8 6 81 .4 0 81 .4 0 0. 43 (0 .3 0– 0. 56 ) ed uc at io n n o ed uc at io n 81 .0 1 < 0. 00 1 45 .5 7 < 0. 00 1 63 .2 9 < 0. 00 1 94 .9 4 0. 00 1 91 .1 4 < 0. 00 1 0. 18 (0 .0 9– 0. 27 ) < 0. 00 1 1– 8 ye ar s 62 .5 0 26 .3 9 55 .5 6 90 .2 8 88 .8 9 0. 34 (0 .2 5– 0. 43 ) 9– 12 y ea rs 38 .3 0 6. 38 17 .0 2 72 .3 4 63 .8 3 0. 64 (0 .5 4– 0. 73 ) > 12 y ea rs 26 .6 7 13 .3 3 20 .0 0 73 .3 3 93 .3 3 0. 62 (0 .4 4– 0. 80 ) hi st or y o f m i n o 59 .3 8 0. 05 4 24 .4 8 < 0. 00 1 44 .7 9 0. 02 0 85 .9 4 0. 23 1 83 .8 5 0. 42 6 0. 40 (0 .3 4– 0. 46 ) < 0. 00 1 ye s 80 .9 5 61 .9 0 71 .4 3 95 .2 4 90 .4 8 0. 07 (0. 13 –0 .2 8) hi st or y o f c or on ar y h ea rt d ise as e n o 59 .1 2 0. 08 9 22 .1 0 < 0. 00 1 43 .0 9 0. 00 3 86 .1 9 0. 49 3 83 .4 3 0. 29 9 0. 41 (0 .3 5– 0. 47 ) < 0. 00 1 ye s 75 .0 0 62 .5 0 71 .8 8 90 .6 3 90 .6 3 0. 11 (0. 04 –0 .2 6) hi st or y o f s tr ok e n o 58 .3 3 0. 00 4 26 .0 4 0. 03 7 45 .3 1 0. 06 3 85 .9 4 0. 23 1 83 .3 3 0. 15 2 0. 40 (0 .3 4– 0. 46 ) < 0. 00 1 ye s 90 .4 8 47 .6 2 66 .6 7 95 .2 4 95 .2 4 0. 08 (0. 10 –0 .2 5) tr ea tm en t o h a 59 .0 9 0. 45 5 25 .4 5 0. 36 3 40 .9 1 0. 04 9 87 .2 7 0. 85 2 85 .4 5 0. 69 3 0. 39 (0 .3 1– 0. 47 ) 0. 45 0 in su lin ± o h a 64 .0 8 31 .0 7 54 .3 7 86 .4 1 83 .5 0 0. 34 (0 .2 6– 0. 43 ) 63 original health-related quality of lifeseyed pouria hedayati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 59–65 longer duration of diabetes and this might have caused lower hrqol/hu in the current study. in addition, a previous study showed that diabetes-related complications have more negative impact on hrqol among type 1 diabetes patients with younger age. the range of hu decrement due to macrovascular complications was higher in our study than the estimates reported in previous studies.45 this may reflect either poor access to secondary health care or low quality of care for patients with diabetes in such deprived area. in addition, such differences imply that the cost–utility analyses using estimates from previous national study might not be generalizable to diabetes patients in this deprived area of the country. the results of the current study should be interpreted in light of a number of limitations. first, the sampling method was non-random, and this negatively affects representativeness of the patients and limits generalizability of the results presented here. second, the data were self-reported, with risk of potential recall bias and measurement errors that might bias the results. third, we used the preference weights from the uk population to calculate the eq-5d-3l index score. due to intercultural differences in health state preferences,46–48 this might be problematic. fourth, both type 1 and type 2 diabetes were included in the study, which may limit transparency and comparability of the results. fifth, as this is a cross-sectional study, any causal inference from the results should be avoided. table 3. the impact of demographic, socioeconomic, and clinical factors on eq-5d-3l dimensions (n = 213) variable mobility self-care usual activities pain/discomfort anxiety/ depression or p-value or p-value or p-value or p-value or p-value men 0.58 0.147 0.57 0.217 0.96 0.913 0.68 0.398 0.92 0.854 age at diagnosis 1.02 0.183 1.05 0.007 1.02 0.159 1.02 0.396 1.01 0.549 diabetes duration 1.08 0.004 1.10 0.003 1.05 0.058 1.04 0.189 1.05 0.095 body mass index 1.07 0.053 1.07 0.140 1.04 0.233 1.01 0.824 0.99 0.833 education ≤8 years (ref ) 1.00 – 1.00 – 1.00 – 1.00 – 1.00 – >8 years 0.36 0.009 0.23 0.013 0.18 <0.001 0.30 0.013 0.32 0.015 treatment oha (ref ) 1.00 – 1.00 – 1.00 – 1.00 – 1.00 – insulin ± oha 1.14 0.716 1.08 0.846 1.73 0.103 0.85 0.738 0.71 0.458 history of mi 2.84 0.090 6.64 0.003 3.20 0.043 2.77 0.318 1.58 0.549 history of coronary heart disease 1.87 0.193 7.00 <0.001 3.80 0.003 1.25 0.717 1.55 0.526 history of stroke 4.63 0.042 1.35 0.619 1.36 0.589 1.58 0.646 2.50 0.338 or: odds ratio. table 4. the impact of demographic, socioeconomic, and clinical factors on eq-5d-3l index score and visual analog scale (n = 213) variable eq-5d index score visual analog scale coefficient p-value coefficient p-value men 0.005 0.927 1.005 0.756 age at diagnosis –0.004 0.050 –0.279 0.012 diabetes duration –0.007 0.036 –0.567 0.003 body mass index –0.006 0.301 –0.285 0.356 education ≤8 years (ref ) 0.000 – 0.000 – >8 years 0.291 <0.001 11.498 <0.001 treatment oha (ref ) 0.000 – 0.000 – insulin ± oha –0.021 0.682 –3.150 0.282 history of mi –0.295 0.002 –12.099 0.011 history of coronary heart disease –0.250 <0.001 –8.536 0.026 history of stroke –0.188 <0.001 –1.425 0.688 constant 0.376 <0.001 52.035 <0.001 dx.doi.org/10.22317/jcms.v6i2.727 64 original health-related quality of life seyed pouria hedayati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 59–65 conclusion the current study has estimated hu scores for a range of demographic and clinical features of diabetic patients in a deprived area of iran. the findings showed that older age at diagnosis, longer duration of diabetes, lower socioeconomic status, and history of macrovascular complications were associated with lower hrqol/hu. the findings also showed that the mean eq-5d-3l index score in the sample of diabetes patients in the current study was lower than the iranian diabetes general population, implying that specific interventions should be implemented to improve hrqol of patients in this area. using these estimates in conducting cost–utility analyses can assist informed decisions by policy-makers in iran. assessing the effects of microvascular complications on hrqol/hu and evaluating the changes of hrqol/hu over time in a larger sample size are topics for future research. acknowledgments it is acknowledged that the current study was approved by zabol university of medical sciences the current study was not funded. the authors would like to thank the staff of the diabetes center in zabol, iran, for their great assistance in this project. the authors would like to thank the staff of the diabetes center in zabol, iran, for their great assistance in this project. conflicts of interest the authors declare that there is no conflict of interests. references 1. whiting dr, guariguata l, weil c, shaw j. idf diabetes atlas: gglobal estimates of the prevalence of diabetes for 2011 and 2030. diabetes res clin pract. 2011;94(3):311–321. doi:10.1016/j.diabres.2011.10.029 2. esteghamati a, gouya mm, abbasi m, et al. prevalence of diabetes and impaired fasting glucose in the adult population of iran: national survey of risk factors for non-communicable diseases of iran. diab care. 2008;31(1):96–98. doi:10.2337/dc07-0959 3. esteghamati a, meysamie a, khalilzadeh o, et al. third national surveillance of risk factors of non-communicable diseases (surfncd-2007) in iran: methods and results on prevalence of diabetes, hypertension, obesity, central obesity, and dyslipidemia. bmc public health. 2009;9:167. doi:10.1186/1471-2458-9-167 4. world health organization ncd country profiles (2016). available from: http://www.who.int/diabetes/country-profiles/irn_en.pdf 5. mcveigh ge, gibson w, hamilton pk. cardiovascular risk in the young type 1 diabetes population with a low 10-year, but high lifetime risk of cardiovascular disease. diab obes metab. 2013;15(3):198–203. doi:10.1111/ dom.12013 6. goeree r, lim me, hopkins r, et al. excess risk of mortality and complications associated with newly diagnosed cases of diabetes in ontario, canada. can j diab. 2009;33(2):93–104. doi:10.1016/s14992671(09)32006-7 7. emerging risk factors collaboration, sarwar n, gao p, seshasai sr, et al. diabetes mellitus, fasting blood glucose concentration, and risk of vascular disease: a collaborative meta-analysis of 102 prospective studies. lancet. 2010;375(9733):2215–2222. doi:10.1016/s0140-6736(10)60484-9 8. hadaegh f, khalili d, fahimfar n, tohidi m, eskandari f, azizi f. glucose intolerance and risk of cardiovascular disease in iranian men and women: results of the 7.6-year follow-up of the tehran lipid and glucose study (tlgs). j endocrinol invest. 2009;32(9):724–730. doi:10.3275/6399 9. franco oh, steyerberg ew, hu fb, mackenbach j, nusselder w. associations of diabetes mellitus with total life expectancy and life expectancy with and without cardiovascular disease. arch intern med. 2007;167(11):1145–1151. doi:10.1001/archinte.167.11.1145 10. morgan cl, currie cj, peters jr. relationship between diabetes and mortality: a population study using record linkage. diab care. 2000;23(8):1103–1107. 11. koopmanschap m, board c-a. coping with type ii diabetes: the patient’s perspective. diabetologia. 2002;45(7):s18–22. doi:10.1007/s00125-0020861-2 12. fu az, qiu y, radican l, luo n. marginal differences in health-related quality of life of diabetic patients with and without macrovascular comorbid conditions in the united states. qual life res. 2011;20(6):825–832. doi:10.1007/s11136-010-9819-x 13. zhang p, brown mb, bilik d, ackermann rt, li r, herman wh. health utility scores for people with type 2 diabetes in u.s. managed care health plans: results from translating research into action for diabetes (triad). diab care. 2012;35(11):2250–2256. doi:10.2337/dc11-2478 14. o’reilly dj, xie f, pullenayegum e, et al. estimation of the impact of diabetes-related complications on health utilities for patients with type 2 diabetes in ontario, canada. qual life res. 2011;20(6):939–943. doi:10.1007/ s11136-010-9828-9 15. kiadaliri aa, gerdtham ug, eliasson b, gudbjornsdottir s, svensson am, carlsson ks. health utilities of type 2 diabetes-related complications: a cross-sectional study in sweden. int j environ res public health. 2014;11(5):4939–4952. doi:10.3390/ijerph110504939 16. ghanbari a, yekta zp, roushan za, lakeh nm. assessment of factors affecting quality of life in diabetic patients in iran. public health nurs. 2005; 22(4):311–322. doi:10.1111/j.0737-1209.2005.220406.x 17. sanjari m, safari s, shokoohi m, et al. a cross-sectional study in kerman, iran, on the effect of diabetic foot ulcer on health-related quality of life. int j low extrem wounds. 2011;10(4):200–206. doi:10.1177/1534734611428728 18. kiadaliri aa, najafi b, mirmalek-sani m. quality of life in people with diabetes: a systematic review of studies in iran. j diab metab disord. 2013; 12(1):54. doi:10.1186/2251-6581-12-54. 19. ferrans ce. definitions and conceptual models of quality of life. in: lipscomb j, gotay cc, snyder c, (eds.) outcomes assessment in cancer. cambridge, england: cambridge university; 2005. pp. 14–30. 20. torrance gw. utility approach to measuring health-related quality of life. j chronic dis. 1987;40(6):593–603. 21. j. e. sansoni “instruments for economic evaluation: session 5”, managing and measuring health outcomes, menzies school of health research, darwin, 8–11 march 2011. 22. brooks r. euroqol: the current state of play. health policy. 1996;37(1):53–72. 23. brazier j, roberts j, deverill m. the estimation of a preference-based measure of health from the sf-36. j health econ. 2002; 21(2):271–292. 24. hatswell a, pennington b, pericleous l, rowen d, lebmeier m, lee d. patient-reported utilities in advanced or metastatic melanoma, including analysis of utilities by time to death. j health quality of life outcomes 2014; 12:14. 25. cruz l.n, camey s.a, hoffmann j.f, rowen d, brazier j.e. fleck m. p, polanczyk c.a. estimating the sf6d value set for a population-based sample of brazilians. j value in health. 2011;1(4): 108–14. 26. sakamaki h, et al. measurement of hrql using eq-5d in patients with type 2 diabetes mellitus in japan. j int soc pharmaco econ outcomes res (ispor). 2006:9(1):47–53. 27. euroqol group. euroqol—a new facility for the measurement of healthrelated quality of life. health policy 1990;16:199–208. 28. javanbakht m, abolhasani f, mashayekhi a, baradaran hr, jahangiri noudeh y. health related quality of life in patients with type 2 diabetes mellitus in iran: a national survey. plos one. 2012;7(8):e44526. doi:10.1371/journal. pone.0044526 29. glasziou p, alexander j, beller e, clarke p, group ac. which health-related quality of life score? a comparison of alternative utility measures in patients with type 2 diabetes in the advance trial. health qual life outcomes. 2007;5:21. doi:10.1186/1477-7525-5-21 30. janssen mf, lubetkin ei, sekhobo jp, pickard as. the use of the eq5d preference-based health status measure in adults with type 2 diabetes mellitus. diab med. 2011;28(4):395–413. doi:10.1111/j.14645491.2010.03136.x 31. brooks r. euroqol: the current state of play. health policy.1996; 37 (1):53–72. doi:10.1016/0168-8510(96)00822-6. [pubmed: 10158943] 32. brazier j, deverill m, green c. a review of the use of health status measures in economic evaluation. j health serv res policy. 1999;4(3):174–84. [pubmed: 10538884]. 33. longworth l, bryan s. an empirical comparison of eq-5d and sf-6d in liver transplant patients. health econ. 2003;12(12):1061–7. doi:10.1002/hec.787. [pubmed: 14673814]. 65 original health-related quality of lifeseyed pouria hedayati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 59–65 34. dolan p, gudex c, kind p, williams a. the time trade-off method: results from a general population study. health econ.1996;5(2):141–154. doi:10.1002/ (sici)1099-1050(199603)5:2<141::aid-hec189>3.0.co;2-n 35. li l, fu a. some methodological issues with the analysis of preferencebased eq-5d index score. health serv outcomes res methodol. 2009;9(3):162–176. doi:10.1007/s10742-009-0053-3 36. huang ic, frangakis c, atkinson mj, et al. addressing ceiling effects in health status measures: a comparison of techniques applied to measures for people with hiv disease. health serv res. 2008;43 (1 pt 1):327–339. doi:10.1111/j.1475-6773.2007.00745.x 37. pullenayegum em, tarride je, xie f, goeree r, gerstein hc, o’reilly d. analysis of health utility data when some subjects attain the upper bound of 1: are tobit and clad models appropriate? value health. 2010;13(4): 487–494. doi:10.1111/j.1524-4733.2010.00695.x 38. lee wj, song kh, noh jh, choi yj, jo mw. health-related quality of life using the euroqol 5d questionnaire in korean patients with type 2 diabetes. j korean med sci. 2012;27(3):255–260. doi:10.3346/ jkms.2012.27.3.255 39. solli o, stavem k, kristiansen is. health-related quality of life in diabetes: the associations of complications with eq-5d scores. health qual life outcomes. 2010;8:18. doi:10.1186/1477-7525-8-18 40. holmes j, mcgill s, kind p, bottomley j, gillam s, murphy m. health-related quality of life in type 2 diabetes (tardis-2). value health. 2000;3 (suppl 1):47–51. doi:10.1046/j.1524-4733.2000.36028.x 41. bagust a, beale s. modelling euroqol health-related utility values for diabetic complications from code-2 data. health econ. 2005;14(3):217–230. doi:10.1002/hec.910 42. sparring v, nystrom l, wahlstrom r, jonsson pm, ostman j, burstrom k. diabetes duration and health-related quality of life in individuals with onset of diabetes in the age group 15-34 years aa swedish population-based study using eq-5d. bmc public health. 2013;13:377. doi:10.1186/14712458-13-377 43. wexler dj, grant rw, wittenberg e, et al. correlates of health-related quality of life in type 2 diabetes. diabetologia. 2006;49(7):1489–1497. doi:10.1007/ s00125-006-0249-9 44. sakamaki h, ikeda s, ikegami n, et al. measurement of hrql using eq-5d in patients with type 2 diabetes mellitus in japan. value health. 2006;9(1): 47–53. doi:10.1111/j.1524-4733.2006.00080.x 45. beaudet a, clegg j, thuresson po, lloyd a, mcewan p. review of utility values for economic modeling in type 2 diabetes. value health. 2014;17(4):462–470. doi:10.1016/j.jval.2014.03.003 46. greiner w, claes c, busschbach jj, von der schulenburg jm. validating the eq-5d with time trade off for the german population. eur j health econ. 2005;6(2):124–130. 47. badia x, roset m, herdman m, kind p. a comparison of united kingdom and spanish general population time trade-off values for eq-5d health states. med decis making. 2001;21(1):7–16. 48. johnson ja, ohinmaa a, murti b, sintonen h, coons sj. comparison of finnish and u.s.-based visual analog scale valuations of the eq-5d measure. med decision making. 2000;20(3):281–289. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i2.727 93j contemp med sci | vol. 2, no. 7, summer 2016: 93–95 research comparison between manual procedure and automated for determinant of wbcs and pcv in maternity and labor hospital in karbala city karem kdaer karem,a aseel najah sabour,b bara majed kulaifa issn 2413-0516 introduction automated method for the estimation of complete blood count (white blood cells, hematocrit and hemoglobin etc.) is commonly used in routine practice laboratory but many other labs still work on manual procedure for the abnormal automated results as well as health care workers in laboratory can be optimized by doing test on manual microscopic procedure as validation technique for automated method.1 in the recent past, huge progress in automated analysis for hematology examination.2 no automated cell counter can equal the performance of manual differentiation for the presence of old results for leukocyte.3 white blood cells (wbcs), also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders.4 automated systems for white blood cell recognition are currently available in the market. the importance of traceability in the medical diagnostics market is increasing. a technique that can make a determination from a microscope slide (or from a set of images) has the advantage that the data from which a diagnosis is made can be kept on file for future quality assurance needs.5 hematocrit is a test that measures the percentage of blood that is comprised of red blood cell. this is often referred to as packed cell volume (pcv) or erythrocyte volume fraction. it is considered as an integral part of a person’s complete blood count, along with hemoglobin concentration, white blood cell count and platelet counts.6,7 in ethiopia, hematocrit is one of the most common parameters from complete blood count used as a routine examination by physicians in any parts of the country where automated method are unavailable, microhematocrit procedure is used to evaluate hct value of patients.8 materials and methods a comparative cross sectional study was conducted from 28 december 2015 to 28 january 2016 to assess the analytical performance between manual procedure and automated methods for adepartment of environmental health, college of applied medical sciences, university of karbala, karbala, iraq. bdepartment of biology, college of education , university of al-qadisiyah, al-diwaniyah, iraq. correspondence to aseel najah sabour (email: dr_phy@yahoo.com). (submitted: 20 may 2016 – revised version received: 16 june 2016 – accepted: 2 august 2016 – published online: 26 september 2016) objectives this study was designed to determine white blood cells (wbcs) and hematocrit (packed cell volume pcv) by manual procedure and comprised with an automated method in the maternity and labor hospital in karbala city. methods during the period of comparative cross sectional study, 52 cases of blood sample were collected into tri-potassium ethylenediamine tetra-acetic acid (k3edta) from pregnant women admitted to hospital during february to april 2016 for the analysis of pcv and wbcs by sysmex xp-300tm in the heamatological department laboratory performance manual method in the same time. all data analyzed by pearson correlation coefficient as well as using 2 mean & standard deviation for both methods. results for two hematologic parameter test, the correlation coefficient was (r = 0.75) for hematocrit thus improved strong correlation between two procedure manual and automated to evaluate wbcs (r = 0.94), which was significantly different (p < 0.001) for two parameter methods. conclusion from the results of our research, it can be concluded that the automated analyzer (wbcs) and (hb) were well correlated with manual standard method for two hematologic parameter. so health care workers in the laboratory can save performance time for hematologic parameter in automated analyzer. keywords manual procedure, automated method, wbc, pvc hematocrit and white blood cells determination for edta blood sample by sysmex xp-300tm automated hematology analyzer for a clinic sample laboratory or research testing. it provides 17 reportable parameters and three part differential, which include an absolute neutrophil count (anc), the results in histograms for wbc, rbc, and platelet (plt). venous blood was obtained from the patients admitted to maternity and labor hospital in karbala city during study period. the study sample size was 52. about 2–3 ml of blood from the vein of selected patients was collected in a tube containing edta anticoagulant. the percentage of packed cell volume was measured manually by filling a capillary tube (plain) and sealing with modeling clay and centrifuging at 3000 g for 5 minutes, then the result was read using hematocrit reader. also wbc counting was done manually using chamber counter after adding blood (20 µl) to tube containing glacial acetic acid (0.4 ml), and then calculated wbc by equation summation of two parts of chamber and multiplied by 100. the result was obtained from sysmex xp-300tm analyzer. the wbc and pcv values for both method registered were analysed by spss version 23 as a pearson correlation co-efficient. precision was determined using coefficient of variation and the significance of value was decided based on the p-value [0.05] at 95% confidence. results the results in figure 1 show a strong correlation between wbc value in automated sysmex xp-300tm and manual results. at the same time, the relationship between the two methods is extrusive, the value of correlation is +0.95. in the scatter plot chart in figure 2 shows the correlation of result packed cell volume in both methods manual and automated procedure. this value is +0.95. a comparison of automated and manual method to determine the hematocrit for 52 patients is shown by mean ± sd in table 1. 94 j contemp med sci | vol. 2, no. 7, summer 2016: 93–95 comparison between manual procedure and automated research karem kdaer karem et al. obtained, as the mean difference between both methods is significant (p < 0.001), as observed in other studies. a study undertaken in nigeria, using sysmex kx-21n, revealed statistically significant difference (p < 0.0001) when the mean and se values of the two methods (automation and manual) were compared. in another study conducted on canine and feline, there were significant differences between manual and automated hct (p < 0.05). the results of these studies also indicated that the hct values from the automated method could not be used to substitute for those of the manual method, though the values of the two methods were accurate and precise.10,11 unlike to this study which reported higher value of hct in manual method, another study reported a higher pcv value from coulter automated analyzer, even though there was no significant association observed.12 also there was a correlation coefficient in result of hematocrit with both methods fig. 2. in this study, there was highly correlation by using pearson between the wbc result in manual and automated procedure which was done by sysmex xp-300tm (0.94) with highly significant p value (0.001) among (52) cases in our study. similar to revealed result of highly significant and correlation (0.96), p value (0.0001),13 this indicates that the automated hematology analyzer (sysmex kx-21n) readings correlated well with the manual methods. in my opinion, the standard manual procedure is still an optimal method comparing to the automated assessment procedure although progressing. conclusion the results of the present study confirm that the automated hematology analyzer readings are as reliable as the standard manual method. generally, the study showed the hematocrit value obtained from hematology analyzer (sysmex xp 300tm) is different from that of manual, but it is directly proportional in most cases. the automated method cannot replace the manual for hematocrit determination though the result of both methods are close to each other. recommendation all health workers in laboratories should take manual method together automated method to reach accurate result. n table 1. mean ± standard deviation (sd) of hematocrit result by automated and manual methods parameter manual automated p value hematocrit 41.6 ± 5.1 34.5 ± 4.9 0.001 correlation is significant at the 0.01 level (2-tailed). table 2. mean ± sd of wbc result by automated and manual methods parameter manual automated p value white blood cell 9.0 ± 3.4 9.9 ± 3.5 0.001 correlation is significant at the 0.01 level (2-tailed). fig. 1 scatter plot showing the correlation of wbc automated and manual method (r = 0.95). in table 2, a comparison of automated and manual method to determine the white blood cells for 52 patients is shown by mean ± sd. the correlation coefficients for relationships between the manual and automated (sysmex xp-300tm) was calculated using pearsons correlation coefficient formula, which was r = 0.95. the correlation coefficient (r = 0.95) indicated the strong positive correlation between manual and automated methods to determine the hematocrit and white blood cell. the mean ± sd of hct result by the manual method is 41.6 ± 5.1, whereas the automated method is 34.5 ± 4.9. the mean ± sd of wbc result by manual method is 9.0 ± 3.4, whereas in automated method is 9.9 ± 3.5. this implicated that the manual and automated for both tests wbc and pcv were significantly different (p < 0.001) which is less than 0.05 at 95% of confidence interval. discussion automated peripheral blood, leukocyte counts are widely accepted in routine practice. however, many laboratories still reflexively perform manual cbc solely based on abnormal automated results or instruments “flags”, before any manual triage step, to established manual procedure for quality control.9 this study indicated that manual hct is higher than the automated hct. it shows that the hematocrit values determined by the autohematological analyzer (sysmex xp 300tm) cannot replace the manual (microhematocrit) results fig. 2 scatter plot of pcv manual and automated method (r + 0.95). karem kdaer karem et al. 95j contemp med sci | vol. 2, no. 7, summer 2016: 93–95 research comparison between manual procedure and automated references 1. siekmeier r, bierlich a, jaross w. the white blood cell differential: three methods compared. clin chem lab med. 2001;39:432–445. 2. verso ml. the evolution of blood counting techniques. med hist. 1962;8:149–58. 3. pohland d. evaluation of the automated haematology analyser sysmex m-2000. j clin chem clin biochem. 1989;27:41–47. 4. la fleur-brooks m. exploring medical language: a student-directed approach (7th ed.). st. louis, missouri, us: mosby elsevier. (2008, p. 398). 5. alfred rj. katz, (image analysis and supervised learning in the automated differentiation of white blood cells from microscopic images) thesis, 2009. 6. kathleen k. the clinical laboratory improvement act (clia) and the physician’s office laboratory. continuing medical education. 2007. 7. clsi procedure for determining packed cell volume by the microhematocrit method; approved standard (3rd edn) clsi document h7-a3 [isbn 1-56238-413-9]. clsi, 940 west valley road, suite 1400, wayne, pennsylvania 19087–1898, 2000, usa. 8. birhaneselassie m, birhanu a, gebremedhin a, tsegaye a. how useful are complete blood count and reticulocyte reports to clinicians in addis ababa hospitals, ethiopia? bmc hematol. 2013;13:11. 9. lantis kl, harris rj, davis g, renner n, finn wg. elimination of instrumentdriven reflex manual differential leucocyte counts. optimization of manual blood smear review criteria in a high-volume automated hematology laboratory. am j clin pathol. 2003;119(5):656–662. 10. ike so, nubila t, ukaejiofo eo, nubila in, shu en, et al. comparison of haematological parameters determined by the sysmex kx-2in automated haematology analyzer and the manual counts. bmc clin pathol. 2010;10:3. 11. prihirunkit k, lekcharoensuk c, pisetpaisan k. comparison between manual and automated methods for determination of canine and feline hematocrit and hemoglobin concentration. kasetsart j nat sci. 2008;42: 655–659. 12. kakel sj. the evaluation of traditional and automatic coulter method in estimation of haematological parameters in adult rats. beni-suef univ j basic and appl sci. 2013;2:31–35. 13. ike so, nubila t, ukaejiofo eo, nubila in, shu en, ezema i. comparison of haematological parameters determined by the sysmex kx-2in automated haematology analyzer and the manual counts. bmc clin pathol. 2010;10:3. administrator highlight reference 10 and 13 are same. please clarify. administrator highlight 258 j contemp med sci | vol. 5, no. 5, september-october 2019: 258–263 teratogenic effect of the aqueous extract of the foeniculum vulgare (fennel) on fetal development in mice maryam shayan,a kobra mehrannia,b tayebeh rastegar,b maryam khanehzad,b taha ghantabpour,b and gholamreza hassanzadehb,c astudent research committee, school of medicine, shahid beheshti university of medical sciences, tehran, iran. bdepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. cdepartment of neuroscience, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. correspondence to gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir). (submitted: 12 august 2019 – revised version received: 18 september 2019 – accepted: 24 september 2019 – published online: 26 october 2019) introduction congenital anomalies are described as defects that affect the developing fetus during gestation. these malformations can be identified in different prenatal stages through ultrasonography or may remain unidentified until birth or even later in infancy. birth defects may result in abnormality of appearance, structure, organ functions and development.1 in september 2016 who reported that every year, 303,000 newborns die in the first 4 weeks after birth due to congenital anomalies. a child with congenital disorders has several limitations and is a huge burden on the families and health care systems.2 although all of the congenital anomalies cannot be prevented, the environmental causes can be prevented by raising awareness of the mothers in societies.3 at the present time, the use of medicinal plants is increasing worldwide. as a consequence of expensive reproductive assistance methods and side effects of chemical medications, young couples tend to try medicinal herbs before visiting the specialist.4,5 foeniculum vulgare, commonly known as fennel, is an aromatic herb grown in different mediterranean regions and western europe. it is also cultivated in various parts of the world. f. vulgare is a perennial herb with yellow flowers and feathery leaves that falls within the family of apiaceae.6 f. vulgare is called “raziane” in iranian traditional medicine and has been used in both medicine and food preparation since ancient times. fennel has a sweet smell and it also tastes relatively sweet. fennel seeds contain moisture, energy, sugars, essential oils, saturated fatty acids, essential and non-essential amino acids and different types of vitamins and minerals.7 several medicinal applications of f. vulgare have been reported, e.g. antibacterial, anti-fungal, anti-oxidative, anti-carcinogenic, anti-inflammatory, anti-diabetic, anti hypertensive, anti-stress and anxiety, anti-cough, memory enhancing, soothing indigestion, increasing in breast milk production, stimulation in menstrual flow, enhance in libido and facilitation in birth.7–13 pregnant women may assume that consuming herbal medication is less harmful than chemical medication during pregnancy. availability and inexpensiveness of herbs also play an important role in increasing the use of medicinal herbs. safety and dosage of f. vulgare have not been studied during pregnancy and there is no data available about the teratogenicity of f. vulgare throughout gestation. herbal medicine may interact in harmful ways with fetal development. therefore, this study aims to discover whether the administration of various dosages of f. vulgare can cause a teratogenic effect on fetal development in animal models. materials and methods chemical and agents the f. vulgare used in this study was purchased from the pharmacognosy department, school of pharmacy, tehran university of medical sciences, tehran, iran. f. vulgare seeds were milled with an electrical mill. the aqueous extract was prepared by dissolving 40 g of milled powder of f. vulgare objective the present study evaluated the effect of aqueous extract of foeniculum vulgare on fetal development in pregnant mice models. methods a total number of 24 female balb/c mice with a weight range of 25–30 g was divided into four groups. each group received 0.25 ml of the aqueous extract of f. vulgare with different concentrations (2.5, 12.5, and 25 mg) and distilled water as a control group. the aqueous extract of f. vulgare administered through oral gavage on a daily basis from day 6 to day 15 of pregnancy. on day 16, the fetuses were analyzed in terms of morphological changes, skeletal disorders, and cellular alterations. results the result showed the dose-dependent teratogenic effect of the aqueous extract of f. vulgare. at 12.5 and 25 mg concentration, the teratogenic effect was more severe. oral gavage administration of the aqueous extract of f. vulgare increased the number of dead fetuses and reduced the average weight, height, and crown-rump length. subcutaneous hemorrhage, dorsal lesion, wrinkled skin, and considerably lower than normal fetal weight observed in gross morphological inspection at 25 mg concentration. the skeletal studies revealed fetal anomalies, reduction in ossification and reduction in the number of ribs. internal bleeding around the liver and lungs, pulmonary fibrosis and disruption in the arrangement of hepatocytes was also observed in histological analysis. conclusion administration of the aqueous extract of f. vulgare results in embryotoxicity in mice models in morphological, skeletal and cellular levels. keywords foeniculum vulgare, fennel, teratogenicity, embryotoxicity, fetal development, mice issn 2413-0516 original 259j contemp med sci | vol. 5, no. 5, september-october 2019: 258–263 m. shayan et al. teratogenic effect of the aqueous extract of the foeniculum vulgare seeds in 200 ml distilled water. the mixture was refluxed for 2 h. after reflux, the mixture was filtered with a large filter and then büchner funnel. the filtered solution poured in petri dish with 20 cm diameter and heated under 37°c temperature until dried. the dried extract was weighed and kept at 4°c in refrigerator until use. the extraction result of 40 g of milled powder of f. vulgare seeds was 6.67 g dried extract equivalent to 16.7%. from the final aqueous extract three different dosages were prepared for oral gavage administration. animals a total number of 24 balb/c mice were used in this study (razi institute, tehran, iran). the mice utilized in this study were virgin adult females, weighing in the range of 25–30 g. all the animals were maintained in a controlled environment in terms of temperature (23–25°c) and lighting (lights on from 08:00 am to 08:00 pm) and had free access to food and water. all the operational guidelines in the housing, routine husbandry, handling, and experimental procedures were approved by the committee for animal ethics and experiments at the tehran university of medical sciences. adult female mice mating occurred with fertile male mice in the same cages for the establishment of pregnancy. the mating success was monitored by checking the vaginal plugs. the presence of vaginal plugs was defined as day 0 of pregnancy. experimental design and grouping the pregnant mice were caged separately and were randomly divided into four groups. on day 6 (beginning of gastrulation) of pregnancy, each group received a different dosage of aqueous extract of the f. vulgare through oral gavage administration on a daily basis for 9 days until day 15 of pregnancy (completion of organogenesis). group 1 (r1): animals received 0.25 ml of aqueous extract of the f. vulgare with 2.5 mg concentration. group 2 (r2): animals received 0.25 ml of aqueous extract of the f. vulgare with 12.5 mg concentration. group 3 (r3): animals received 0.25 ml of aqueous extract of the f. vulgare with 25 mg concentration. group 4 (co): animals received 0.25 ml of distilled water as a control group. at the end of the experiments, on the 16th day, mice were sacrificed by chloroform and the fetuses were removed. morphological study the fetuses were observed in terms of the total number of fetuses, number of live and dead fetuses, number of abortions, height, weight and crown-rump length (crl). the fetuses were also investigated in terms of morphological and skeletal anomalies using a stereomicroscope. histological study the fetuses were placed in paraffin after fixation in bouin’s solution. after fixation, the samples underwent tissue staining with hematoxylin and eosin (h&e). then the samples were studied using a light microscope. skeletal study the fetuses were placed in acetone for 24 h, then the skin was removed and fetuses were placed in a staining solution containing alizarin red-s, alcian blue, ethanol, and acetic acid. after that, the fetuses were stained through microwave radiation. for further clarification, the fetuses were placed in the solution containing koh 1%, ethanol 95%, and glycerine for 24 h. after these steps, fetuses were observed in terms of possible morphological and skeletal changes. statistical study data were processed (graphpad prism 5.0 graphing and statistics software) by one-way anova along with dunnett’s test or turkey’s multiple comparisons test. the t-test was performed for some data analysis. in all the experiments, p < 0.05 was considered significant. data are presented as mean ± standard error of the mean (sem). results the effect of the aqueous extract of the f. vulgare in the morphological study shown the following results. the gestational factors differed significantly in r2 and r3 groups from those in the r1 and control groups. in comparison to r1 and control group, decreased number of live fetuses, increased number of dead fetuses, reduction in average weight, height and crl were observed in r2 and r3 groups. subcutaneous hemorrhage, wrinkling of the skin, dorsal lesions and considerably lower than normal fetal weight were observed in the r3 group with significant differences while compared with the co group and other experimental groups. the morphological malformations on gross observation are summarized in figs. 1 and 2. skeletal examination revealed a significant difference in skeletal malformation in the r3 group compared to r1, r2, and co group. fetal anomalies, reduction in ossification, reduction in the number of ribs and shortened ribs were observed in higher dosages of f. vulgare. sternal nonunion, branched ribs, shortened fetal long bones, organ rotation abnormalities, and limb shortening were not observed in the fetuses. figure 3 summarizes the results of the skeletal examination. figure 4 shows the morphological malformations observed in mice fetuses. figure 5 (a & b) shows alizarin red-s and alcian blue staining of skeletal analysis. the histological analysis showed internal bleeding in the liver and lungs in r2 and r3 groups. the bleeding was much higher in the r3 group. disruption in the arrangement of hepatocytes and pulmonary fibrosis was also seen in r2 and r3 groups. figure 5c and 5d shows the hemorrhage in the liver and lungs. to summarize the results, morphological, skeletal and histological analysis revealed the teratogenic effect of the aqueous extract of the f. vulgare in r3 (25 mg) and r2 (12.5 mg) groups. teratogenicity of f. vulgare is dose-dependent and is directly increased as the dose increments. no teratogenic effect was observed in the r1 group. figure 6 demonstrated the experimental design and procedure of the present study. discussion foeniculum vulgare (fennel) is an herbal plant used widely in many countries because of its numerous applications.7 fennel is known as a potent exogenous estrogen that can mimic the effect of estrogen in the body. the phytoestrogen in fennel’s structure original 260 j contemp med sci | vol. 5, no. 5, september-october 2019: 258–263 teratogenic effect of the aqueous extract of the foeniculum vulgare m. shayan et al. fig. 1 (a) number of dead fetuses in experimental groups was compared with the co group. (b) comparison of the weight of the fetuses in experimental groups with the co group. experimental groups are also compared with each other. (c) differences between crownrump length (crl) in experimental groups and the control group. (d) comparison of the effect of foeniculum vulgare on fetal growth in experimental groups versus control group. data are expressed as mean ± sem. as it is shown in figure, *p < 0.05 compared experimental group with the co group and experimental groups to each other. each group consists of six mice. the statistical analysis was carried out by one-way analysis of variance (anova) followed by turkey’s post hoc test. a b c d fig. 2 comparison of the effect of foeniculum vulgare in causing subcutaneous hemorrhage (a) and dorsal lesions (b) during pregnancy in mice models. data are expressed as mean ± sem. as it is shown in figure, *p < 0.05 compared experimental group with the co group and experimental groups to each other. each group consists of six mice. the statistical analysis was carried out by one-way anova followed by turkey’s post hoc test. a b original 261j contemp med sci | vol. 5, no. 5, september-october 2019: 258–263 m. shayan et al. teratogenic effect of the aqueous extract of the foeniculum vulgare fig. 3 (a) comparison of the total number of observed skeletal anomalies in experimental groups with the co group. (b) reduction in the number of ribs in experimental groups versus the co group. (c) reduction in the ossification centers in experimental groups was compared with the co group. data are expressed as mean ± sem. as it is shown in figure, *p < 0.05 compared the experimental group with the co group. each group consists of six mice. the statistical analysis was carried out by one-way anova followed by turkey’s post hoc test. aa b c fig. 4 (a) wrinkled skin and subcutaneous bleeding in mice fetus at a concentration of 12.5 mg. (b) severe subcutaneous hemorrhage and wrinkling of the skin at a concentration of 25 mg. (c) fetal growth restriction and reduction in weight, height, and crl of fetuses at concentrations of 12.5 and 25 mg. (d) dorsal lesions at a concentration of 25 mg. a b c d fig. 5 (a) shortened ribs at a concentration of 25 mg. (b) reduction in the number of ribs at a concentration of 25 mg. the method of skeletal analysis was alizarin red-s and alcian blue staining. (c) pulmonary hemorrhage (arrow) and fibrosis (arrowhead) at concentrations of 12.5 and 25 mg. (d) disarrangement of hepatocytes and severe hepatic hemorrhage (arrow) at concentrations of 12.5 and 25 mg. h&e staining was used in histological analysis. a b c d original 262 j contemp med sci | vol. 5, no. 5, september-october 2019: 258–263 teratogenic effect of the aqueous extract of the foeniculum vulgare m. shayan et al. fig. 6 schematic representation of experimental design and procedure of the present study. has an affinity to α and β estrogen receptors. the number of authors has shown that fennel extract can increase the level of estrogen in serum hence it can be used as a treatment in adult women with polycystic ovary syndrome (pcos), in addition, it can be used as an effective prescription to treat female infertility.14,15 it is also shown that fennel increases the number of graafian, antral and multilaminar follicles in mice ovary due to its estrogenic compound.5 moreover, studies have shown that fennel can significantly decrease the toxicity of cyclophosphamide on the ovaries by increasing the level of estrogen and progesterone in serum.16 fennel also showed a protective effect against misoprostol induced threatened abortion in mice models.17 to the best of our knowledge, this is the first study that evaluates the effect of aqueous extract of f. vulgare without any additional substances in pregnant animal models from day 6 (beginning of gastrulation) to day 15 (completion of organogenesis) of pregnancy. in the present study, oral gavage administration of fennel at 12.5 and 25 mg concentration caused an increased number of dead fetuses, reduction in weight, height, and crl of fetuses. besides severe anomalies in skeletal studies, e.g. reduction in ossification, reduction in the number of ribs, and shortening of the ribs were observed. moreover, histological analysis revealed internal bleeding in the liver and lungs, disruption in the arrangement of hepatocytes and pulmonary fibrosis. subcutaneous hemorrhage, wrinkling of the skin and dorsal lesions were also seen at 25 mg concentration. anti-inflammatory and anti-oxidative effects of fennel are shown in several studies.14,18,19 fennel can regulate the levels of liver enzymes in serum and prevent the intoxication effect of cadmium in hepatic cells at a dose of 150 mg/kg in rats.20 even though our findings showed that fennel extract at 12.5 and 25 mg concentration can cause severe bleeding around the liver, along with disruption in arrangement of hepatocytes in mice fetuses. this result may be due to the reducing effect of fennel on liver enzymes. and also the differences in dose toxicity in mice and rats. additionally, this study was conducted during pregnancy in mice, therefore, even the standard dosages can be teratogen during gestation. animal studies in the field of reproduction and infertility presented that fennel can regularize the level of estrogen, progesterone, and prolactin in serum, hence it can be used as a lowcost alternative therapy for infertility.5,14,15,20 intraperitoneal administration at 100 and 200 mg/kg dosages in mice models resulted in significantly increasing the mentioned steroid hormones.15 another study reported that intragastric administration at a 150 mg/kg dosage showed positive results in rat models of pcos. in the study mentioned earlier, fennel showed both positive effects on renal function and on lowering the blood pressure.14 the estrogenic effect of fennel is due to trans-anethole and flavonoids ingredients that are found in this herbal plant.21,22 although fennel showed a positive impact on improving fertility, this study proved that oral gavage administration of this herbal medication in mice models can cause severe anomalies and morphological disorders in fetuses. therefore, it is best to avoid using this medicinal plant during gestation until complete evaluations in experimental studies. the anethole component of fennel also revealed antithrombotic activity through the anti-platelet aggregation mechanism.23 it seems that the internal bleeding around the liver and lungs observed in this study may be the result of the antiplatelet activity of this herbal medicine. in an animal study, the effect of intraperitoneal fennel injection along with misoprostol injection has been assessed. hydroalcoholic extract of fennel at a 2.5, 12.5, and 25 mg/kg dosages were used in the study. pretreatment with fennel showed a protective effect on misoprostol induced-abortion in day 6–15 of pregnancy in mice.17 even though fennel showed positive outcomes on fetuses that have been threatened to abortion, the effect of fennel alone during gestation has not been original 263j contemp med sci | vol. 5, no. 5, september-october 2019: 258–263 m. shayan et al. teratogenic effect of the aqueous extract of the foeniculum vulgare evaluated in the above-mentioned study. in the presented study, it is shown that oral gavage administration of fennel alone has severe teratogenicity effect in mice embryos while using during day 6–15 of gestation in mice models. since it has been proved that fennel can be used as a treatment in infertility and prevention of miscarriage, usage of this medicinal herb should be under the supervision of an expert in this field to determine the exact dosage and prevent the possible side effects. it is also recommended to avoid usage of fennel during pregnancy, particularly in the first trimester that organogenesis occurring. conclusion the utilization of aqueous extract of f. vulgare at a concentration of 12.5 and 25 mg results in embryotoxicity while orally administered in pregnant mice models. the embryotoxicity of this herbal medicine was observed in morphological, skeletal and cellular levels. acknowledgment this study was part of a research grant supported by tehran university of medical sciences, tehran, iran (grant no: 98-02-30-43597). conflicts of interest none.  references 1. connolly c, o’donoghue k, o’mahony m. pfm.54 a review of congenital anomalies in the cork and kerry region from 1996–2010. arch dis childhood fetal neonatal ed. 2014;99:a100. 2. congenital anomalies [updated 2016]. available from: https://www.who. int/news-room/factsheets/detail/congenital-anomalies. 3. sarmah s, muralidharan p, marrs ja. common congenital anomalies: environmental causes and prevention with folic acid containing multivitamins. birth defects res c embryo today 2016;108:274–286. 4. minas a, najafi g, jalali as, razi m. fennel induces cytotoxic effects against testicular germ cells in mice; evidences for suppressed pre-implantation embryo development. environ toxicol. 2018. 5. khazaei m, montaseri a, khazaei mr, khanahmadi m. study of foeniculum vulgare effect on folliculogenesis in female mice. int j fertil steril. 2011;5:122–127. 6. kooti w, ali-akbari s, sharafi-ahvazi n, asadi-samani m, ashtary-larky d, moradi m. therapeutic and pharmacological potential of foeniculum vulgare mill: a review. j herbmed pharmacol. 2015;4:1–9. 7. badgujar sb, patel vv, bandivdekar ah. foeniculum vulgare mill: a review of its botany, phytochemistry, pharmacology, contemporary application, and toxicology. biomed res int. 2014;2014:842674. 8. mesfin m, asres k, shibeshi w. evaluation of anxiolytic activity of the essential oil of the aerial part of foeniculum vulgare miller in mice. bmc complement altern med. 2014;14:310. 9. kacaniova m, mellen m, vukovic nl, kluz m, puchalski c, haščík p, et al. combined effect of vacuum packaging, fennel and savory essential oil treatment on the quality of chicken thighs. microorganisms. 2019;7. pii: e134. 10. juárez-vázquez mdel c, carranza-álvarez c, alonso-castro aj, gonzálezalcaraz vf, bravo-acevedo e, chamarro-tinajero fj, et al. ethnobotany of medicinal plants used in xalpatlahuac, guerrero, mexico. j ethnopharmacol. 2013;148:521–527. 11. javidnia k, dastgheib l, mohammadi samani s, nasiri a. antihirsutism activity of fennel (fruits of foeniculum vulgare) extract. a double-blind placebo controlled study. phytomedicine. 2003;10:455–458. 12. joshi h, parle m. cholinergic basis of memory-strengthening effect of foeniculum vulgare linn. j med food. 2006;9:413–417. 13. el-soud n, el-laithy n, el-saeed g, wahby ms, khalil m, morsy f, et al. antidiabetic activities of foeniculum vulgare mill. essential oil in streptozotocin-induced diabetic rats. maced j med sci. 2011;4:139–146. 14. sadrefozalayi s, farokhi f. effect of the aqueous extract of foeniculum vulgare (fennel) on the kidney in experimental pcos female rats. avicenna j phytomed. 2014;4:110–117. 15. sadeghpour n, khaki af, najafpour a, dolatkhah h, montaseri a. study of foeniculum vulgare (fennel) seed extract effects on serum level of estrogen, progesterone and prolactin in mouse. crescent j med biol sci. 2015;2:23–27. 16. hassanpour a, yousefian s, askaripour m, sharififar f, ezzatabadipour m. ovarian protection in cyclophosphamide-treated mice by fennel. toxicol rep. 2017;4:160–164. 17. shokrzadeh m, dashti a, aghajanshakeri s, pourabbas b, ghassemi barghi n, ogunkunle atj, et al. prevention effects of foeniculum vulgare (fennel) hydroalcoholic extract for threatened abortion by misoprostol induction in experimental mice. int j trad nat med. 2019;9:1–16. 18. choi em, hwang jk. antiinflammatory, analgesic and antioxidant activities of the fruit of foeniculum vulgare. fitoterapia. 2004;75:557–565. 19. oktay m, gülçin i̇, küfrevioğlu öi̇. determination of in vitro antioxidant activity of fennel (foeniculum vulgare) seed extracts. lwt food sci technol. 2003;36:263–271. 20. abdel-wahab a, hashem abdel-razik ar, abdel aziz rl. rescue effects of aqueous seed extracts of foeniculum vulgare and carum carvi against cadmium-induced hepatic, renal and gonadal damage in female albino rats. asian pac j trop med. 2017;10:1123–1133. 21. price kr, fenwick gr. naturally occurring oestrogens in foods—a review. food addit contam. 1985;2:73–106. 22. zhang s, chen x, devshilt i, yun q, huang c, an l, et al. fennel main constituent, trans-anethole treatment against lps-induced acute lung injury by regulation of th17/treg function. mol med rep. 2018;18: 1369–1376. 23. tognolini m, ballabeni v, bertoni s, bruni r, impicciatore m, barocelli e. protective effect of foeniculum vulgare essential oil and anethole in an experimental model of thrombosis. pharmacol res. 2007;56:254–260. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201905 original 71j contemp med sci | vol. 5, no. 2, march–april 2019: 71–76 original breast cancer decisive parameters for iraqi women via data mining techniques suhad faisal behadili,a* mustafa s. abd,a iyden kamil mohammed,b and maha mohammed al-sayyidc acomputer science department, college of science, university of baghdad, baghdad, iraq. bbiomedical department, al-khawarzmy engineering college, university of baghdad, baghdad, iraq. concology teaching hospital, medical city, baghdad, iraq. *correspondence to suhad faisal behadili (email: suhad.behadili@scbaghdad.edu.iq). (submitted: 12 december 2018 – revised version received: 05 january 2019 – accepted: 29 january 2019 – published online: 26 april 2019) objective this research investigates breast cancer real data for iraqi women, these data are acquired manually from several iraqi hospitals of early detection for breast cancer. data mining techniques are used to discover the hidden knowledge, unexpected patterns, and new rules from the dataset, which implies a large number of attributes. methods data mining techniques manipulate the redundant or simply irrelevant attributes to discover interesting patterns. however, the dataset is processed via the waikato environment for knowledge analysis platform. the oner technique is used as a machine-learning classifier to evaluate the attribute worthy according to the class value. results the evaluation is performed using a training data rather than cross validation. the decision tree algorithm j48 is applied to detect and generate the pattern of attributes, which have the real effect on the class value. furthermore, the experiments are performed with three machine-learning algorithms j48 decision tree, simple logistic, and multilayer perceptron using tenfold cross-validation as a test option, and the percentage of correctly classified instances as a measure to determine the best one from them. as well as, this investigation used the iteration control to check the accuracy gained from the three mentioned above algorithms. hence, it explores whether the error ratio is decreasing after several iterations of algorithm execution or not. conclusion it is noticed that the error ratio of classified instances are decreasing after 5–10 iterations, exactly in the case of multilayer perceptron algorithm rather than simple logistic, and decision tree algorithms. this study realized that the tps_pre is the most common effective attribute among three main classes of examined dataset. this attribute highly indicates the bc inflammation. keywords ca 15-3, cea, breast cancer, saliva, mlp, slr, j48, data mining, oner, iraq introduction breast cancer (bc) is the leading cause of death in women in developing countries, and a second cause in developed countries as per the statistics of national cancer institute. the bc may occur in both male and female. however, it has high occurrence in female throughout the world. in addition, bc is most frequently discovered as an asymptomatic nodule on a mammogram.1,2 bc is the most frequent cancer in women worldwide. it is the most mutual cause of cancer death among women (522,000 deaths in 2012), and the most widespread diagnosed cancer among women in 140 out of 184 countries worldwide, that constitutes 1:4 of all cancers in women. it represents the most commonly occurring cancer in women, and the second most common cancer overall. there were over 2 million new cases in 2018. whereas, it has elevated occurrences in iraqi women (all ages), which became one of the major menace to iraqi female health. however, it is essential for clinical researchers to look at several body fluids to identify biomarkers. this study attempt to investigate multiple biomarkers as shown in table 1, which are detectable in blood and saliva of 181 iraqi women in different sites and hospitals for early detection of bc and gynecology during the period from july 2013 to october 2014.3–5 in accordance with the iraqi cancer registry data during period 2000–2009, the 23,792 total incidents registered bc cases among females aged ≥15 years. they stand for 33.8% of all cancers. the prevalence ratio of all female bc in iraq (all ages) incremented from 26.6/100,000 in 2000 to 31.5/100,000 in 2009, which make it one of the major threats to iraqi female health.3 as well as, bc incidence rates in arab women increased. since, improved life expectancy, urbanization growth, adopt the western lifestyles, and retarded and diminished fertility play a role to this augmentation. the lack of breast health programs is obvious in many middle eastern countries. as well as, a deficiency in service provision, in addition to the conservative culture, may be correlated with the increased rate of advanced stages of bc in arab countries.6 although, bc can be diagnosed by classifying tumors. there are two general types of tumors, the malignant and benign tumors. even the physicians need a reliable diagnosis procedure to differentiate between them. nevertheless, it is difficult to distinguish tumors even by the experts. thereby, computerizing the diagnostic system is required to help in tumors diagnosis. the researches endeavored to apply machinelearning algorithms to detect survivability of cancers in human beings.7 however, the researchers realized that these algorithms are done well in cancer diagnosis.2,4 bc survivability prediction is challenging, also it is a complex research assignment. therefore, the existing approaches incorporate statistical methods or supervised machine learning to predict the survival chances of patients.2,7 the groundwork results gifted to the function of the data mining (dm) methods into the survivability forecast problem in bc. in spite of all challenges, dm algorithms have good results in different methodologies of bc, but more work is have to be done in managing different studies, and reviews of dm technologies.2,7,8 the biomarkers are very important not for reliable disease diagnostic only, but also to have a good choice from multiple issn 2413-0516 72 j contemp med sci | vol. 5, no. 2, march–april 2019: 71–76 breast cancer decisive parameters for iraqi women via data mining techniques original s.f. behadili et al. available therapeutic alternatives, that is likely to benefit the patients.3 whoever, it is crucial for clinical researchers to look at multiple body fluids, and different molecular techniques to identify biomarkers. saliva one of body fluids, which is simply and non-invasively gained, and contains several types of potential protein biomarkers. therefore, finding of ca 15-3 protein for breast cancer in saliva proposed renewed interest in the potential use of saliva as a diagnostic fluid.3 it has been stated that person with bc secretes a different profile of proteins compared with the healthy individual.3 as well as, the levels of vascular endothelial growth factor, epidermal growth factor (egf), and carcinoembryonic antigen (cea) in the saliva are considerably raised in bc patients. the salivary biomarkers appearance reflect precisely the normal and disease states. this make saliva an attractive diagnostic fluid beside the sampling benefits compared with blood sampling.3 women with early-stage bc have excellent survival rates. therefore, it is critical to identify factors that anticipate diagnosis of early-stage bc.2,4,9 then, concluding the proportion of bcs that were identified at an early stage (stage i) in different racial/ethnic groups, and whether the ethnic differences well explained by early detection, or by intrinsic biological differences in tumor aggressiveness.2,9 medical organizations generate, and gather large quantity of data. the medical domain is considered as one of the leading areas for applying dm to recognize some significant properties of data.2,10 in medical field, there are various problems, such as in medical imaging like classification, segmentation, extraction, and selection. medical datasets categorized always by huge amount of disease measurements, and comparatively small amount of patient records. these measurements (feature selection) are irrelevant. this irrelevant and redundancy features are difficult to evaluate. meanwhile, to represent the data set, then a large number of features causes memory storage problem. therefore, different dm techniques can convenient with imprecision and uncertainty in data analysis, and can efficiently remove noisy and redundant information.2,11 through the means of big data analytics and machinelearning techniques of patient’s data of precise clinical manifestations. hence, it became possible to identify precise biomarkers in blood or urine, which can be utilized for early diagnosis of bc upon early diagnosis, administration of precise personalized nanomedicine is quite possible, that can reduce the time and cost of drug regimen. by the next decade because of big data analytics, time, and cost of bench to bedside drug discovery lifecycle can be drastically reduced from 9 to 12 years, and current value of around us$ 1.1 billion.12 convergence technology is a revolution, it is not an interdisciplinary collaboration, however taking science, research, and technology development into next revolution by interdisciplinary integration. thereby, foremost barriers faced can be overpowered. the construction of largest patient database (1 million patients), incorporating genetic, behavioral (societal), and clinical information are under progression nowadays. therefore, using big data analytics and machine-learning techniques on huge database, and their outcomes have to be generalized to researchers in engineering, physical, biological, and clinical science, thus to be explored by them.12 this research applied machine-learning algorithms in detecting bc for iraqi women. in this article, the remaining of this paper is organized as follows. section 2 specifies literature review about b.c analysis via dm techniques. section 3 gives information about dm techniques, and its learning rules. section 4 specifies related works on bc using dm. section 5 implies other machine-learning algorithms and its types, with related work on those algorithms. finally, section 6 concludes the results and perspectives. data mining techniques for breast cancer analysis data mining is an interdisciplinary field and is fast reputation because of exploring database technology, information science, machine learning, and neural networks (nn)4,7,10 along with the statistical techniques. however, dm algorithms not applied on the medical data by common people, but the knowledge obtained can be very useful for them if shared within a comprehensible form.2,8 in addition, the machine learning is a branch of artificial intelligence, it is a scientific discipline interested with the design, and development of algorithms, which evolve the computers behavior with regard to empirical data, from either sensor data or databases.4 priyanga and prakasam13 proposed a cancer prediction system based on dm technology. the user’s genetic and non-genetic factors are collected. thus, that helps to predict the bc at early stage. however, it is cost effective to the user. thereafter, waikato environment for knowledge analysis (weka) system analyzed the medical information. whenever, the attributes are finalized, hence the risk range could be determined via the prediction system. this system applied successfully on bc data sets, it achieves better accuracy level comparing to other existing systems. as well as, it gives earlier stage warning to the users, cost and time benefits to the user.2,8,11 kharya14 proposed several efficient dm techniques to classify the bc. they were the soft computing approaches, and decision tree. they provide best predictor with 93.62% accuracy on benchmark and seer data set.2,7 this predictor was used to design the web page application.11 delen et al.15 used common dm algorithms, such as artificial neural network (ann) and decision tree, and along with logistic regression4 to formulate the prediction model for the bc by investigating large database. then, compared the prediction models, which gives 93.6% accuracy with decision tree, 91.2% accuracy with ann, and worst accuracy 89.2% with logistic regression.11 the algorithms such as decision tree, ann, regression,4 support vector machine (svm), naïve bays,16 and backpropagation2 are frequently considered. they introduced various results based on speed, accuracy, performance, and cost. as well as, the effective classification data aids to obtain the patient treatment.11 dm has become a substantial methodology for computing applications in the medicine domain. the progression of dm applications and its implications are indicated in data management of healthcare administrations, epidemiology, patient care, intensive care systems, significant image analysis to information extraction, and automatic identification of unknown subjects. dm includes multiple techniques such as classification, clustering,2 prediction, association rules, decisions tress, and nns. among the diverse classification algorithms, the well-known algorithms id3 and c4.5 play an essential role in bc analysis. plenty of researchers attempt to use machine-learning algorithms for detecting cancers survivability in human beings.7 s.f. behadili et al. 73j contemp med sci | vol. 5, no. 2, march–april 2019: 71–76 original breast cancer decisive parameters for iraqi women via data mining techniques gad17 implemented diagnosis method of bc on wisconsin diagnosis bc (wdbc) dataset and wisconsin prognosis bc (wpbc) dataset,2 which combined unsupervised learning method k-means with svm supervised learning method. consequently, eliminates the inapplicable attributes using feature selection method chi-square.18 shiv shakti et al.19 reviewed the use of dm in bc, they observed that, many researchers are mainly used the nn and decision approach to create a predictive model, and decision rules from the bc data. most of them performed a comparative study of algorithm to take bc data. then, to conduct an experimental work, and find various if….then rules from decision tree, which represented and used j48 classifier of weka.2,8,11 ravi kumar et al.20 explored a comparison among different dm classifiers on the database of bc wbc,2,8 using classification accuracy to establish an accurate classification model for bc prediction. for full usage of invaluable information in clinical data, particularly that often neglected by most of the existing methods, whenever aimed to predict in high accuracies.8 the dataset was divided into training set with 499, and test set with 200 patients. it compares six classification techniques in weka software. so that, comparison results show that svm has higher prediction accuracy than those methods. in addition, the svm are more suitable in handling the classification problem of bc prediction.8 chaurasia and pal21 suggested a diagnosis system for detecting bc based on reptree, rbf network, and simple logistic. in test stage, tenfold cross validation method was applied to the university medical centre, institute of oncology, ljubljana, yugoslavia database to evaluate the proposed system performance. the classification rate of the system is 74.5%. the simple logistic used for reducing the feature space dimension, and proposed rep tree and rbf network model used to obtain fast automatic diagnostic systems for other diseases. zand2 proposed the classification of bc data could be useful to predict the result of some diseases, or discover the genetic behavior of tumors. hence, presented a comparative study on dm techniques in diagnosis and prediction of bc, and prediction analysis for survivability rate of bc patients. ghosh et al.22 applied different classification techniques namely, multilayer perceptron (mlp) using backpropagation nn,2 and svm on bc wisconsin dataset2 from the uci machine-learning repository to bc detection. the conclusion said that svm classifier has the potential to improve significantly the conventional classification methods, hence to be used in medical or general bioinformatics field.10 as well as, delshi howsalya and indra23 used machinelearning algorithm in for automatic examination of hazardous illness, bc has been considered. it identifies cancer occurrence during its beginning, and its reoccurrence, which has three stages. the first stage enclosing the data to number related entities by applying farthest first clustering algorithm. computation time took less time, due to decrease in the size of dataset. the second stage, deviations from the normality (outliers) are detected from bc dataset (bcd) using outlier detection algorithm. thereafter, final stage, j48 classification algorithm identifies whether the cancer is benign or malignant from the pre-processed data set. wisconsin bcd (wbcd) and wdbc show an accuracy of 99.9%, which serves the doctors to diagnose the bc.2,18 various studies indicated factors regarding bc prognosis based on different factors. these studies recently focused on predicting bc through svm, and on survival since the time of first diagnosis.2,7 it is a challenging task. methodology the key intention to this study is to construct data analytical model. this can provide more comprehension of bc, by forming patients’ cohorts (healthy, benign, and malignant), where the most common attributes extracted from 42 attributes of the mentioned three classes, which share several attributes. moreover, identify the explicitly effectiveness of the attributes on these classes.7 data set attributes breast cancer is a heterogeneous disease with varieties in the biological profile, and subsequent clinical prognosis.24 prognostic information of individual patient based on the biological analysis for markers in the primary tumor, that including estrogen receptor (er), progesterone receptor (pr), human epidermal growth factor receptor (her2) and ki67, family history, it is a powerful risk attribute for women <40 years old. its effect increasing when first-degree relatives with breast or ovarian cancer, and if the relatives are young at diagnosis, previous chest radiotherapy, where women with previous radiotherapy at a young age (10–30 years) have about six times as high a risk to develop bc. brca1/2 mutations are rare, only 0.2% of women are estimated generally to be brca1/2 mutation carriers. breast density is a strong independent risk factor for the development of bc. obesity increases the risk of postmenopausal bc, but it is a protective factor for premenopausal women. alcohol and smoking are not risk factors for bc in women aged,25 together with age, tumor size, histological grade, and lymph node involvement. however, there are 42 attributes examined in this study as appears in table 1. table 1. data set attributes before and after space reduction (features selection) before after no. attributes rank no. attributes rank 1 ca_b_pre 93.889 1 ca_b_pre 93.889 2 ca_s_pre 92.222 2 ca_s_pre 92.222 3 e2_b_pr 91.111 3 e2_b_pr 91.111 4 e2_s_pr 91.111 4 e2_s_pr 91.111 5 tp_s_pre 90.556 5 tp_s_pre 90.556 6 pg_b_ps 89.444 6 pg_b_ps 89.444 7 type 89.444 7 type 89.444 8 er 89.444 8 er 89.444 9 e2_s_ps 89.444 9 e2_s_ps 89.444 10 e2_b_ps 89.444 10 e2_b_ps 89.444 11 grade 89.444 11 grade 89.444 12 pg_s_ps 89.444 12 pg_s_ps 89.444 13 pr 89.444 13 pr 89.444 14 her2 89.444 14 her2 89.444 15 ph_s_pos 89.444 15 ph_s_pos 89.444 16 ca_b_pos 89.444 16 ca_b_pos 89.444 (continued ) 74 j contemp med sci | vol. 5, no. 2, march–april 2019: 71–76 breast cancer decisive parameters for iraqi women via data mining techniques original s.f. behadili et al. table 1. data set attributes before and after space reduction (features selection) —continued before after no. attributes rank no. attributes rank 17 cea_b_ps 89.444 17 cea_b_ps 89.444 18 tp_b_pos 89.444 18 tp_b_pos 89.444 19 ca_s_pos 89.444 19 ca_s_pos 89.444 20 cea_s_ps 89.444 20 cea_s_ps 89.444 21 tp_s_pos 89.444 21 tp_s_pos 89.444 22 cea_b_pre 87.778 22 cea_b_ pre 87.778 23 tp_b_pre 87.222 23 tp_b_pre 87.222 24 ph_s_pre 85.556 24 ph_s_pre 85.556 25 cea_s_pre 85 25 cea_s_ pre 85 26 stage 85 26 stage 85 27 bmi 77.778 28 tbf 75.556 29 pg_b_pr 72.222 30 age 72.222 31 pg_s_pr 70.556 32 w_r 65.556 33 blood_g 61.667 34 rh 61.667 35 lew 61.667 36 family_h 61.667 37 lact 61.667 38 s 61.667 39 mns 61.667 40 kind_of_co 61.667 41 pp_meno 61.667 cea, carcinoembryonic antigen. however, these data in primarily stage are classified into obese cancer, healthy obese, healthy non-obese, benign obese, and benign non-obese. thereafter, in advanced preprocessing, they are classified into three main classes namely healthy, malignant, and benign. data analysis approach the descriptive statistics are identified for 181 women with invasive bc. the diagnosed data are captured during july 2013 to october 2014. this investigation accomplished using oner classifier for attributes reduction to extract the most influential features, and j48 for patterns recognition. finally, j48, simple logistic regression (slr), and mlp algorithms were used for classifiers evaluation, and compared their performance with each other according to the correct classified instances. the predominant objective of this research is to explore the dm techniques to enhance the bc diagnosis. peculiarly, this investigation discusses the use of the classification algorithms j48, slr, and mlp in bc analysis. this examination used weka platform2,8,11 to perform the study experiments. figure 1 presents the proposed dm architecture of this study. the preprocessing phase accomplished on the data set to prepare them for manipulation in dm techniques. the raw data set in this phase are extracted, cleaned up, and transfigured into .arff format, which was accepted by weka platform. thereafter, the resulted data would be classified into three main classes. these classes are healthy (h), malignant (m), and benign (b). later on, the feature selection method performed, which measures the weight of attribute according to the class. applying an attribute selection method is to reduce the feature space, and keep only the ones that have highest affect according to a threshold, which is determined by the data analyst. furthermore, implying oner machine-learning classifier to evaluate the worth of an attribute according to the class value. the attribute rank 85 is determined as a threshold of an accepted attributes, and prune the ones that is less than it. it is issued under data analyst control of this study. then, the evaluation performed using training data rather than cross validation. thus, it extracts 26 attributes plus class attribute instead of the initial 42 attributes with their class. the classified classes based on attributes are m, b, and h to indicate cancer, benign, and healthy instances respectively, as demonstrated in table 1. moreover, the decision tree j48 algorithm applied to detect and generate the attributes patterns. these patterns have the real effect on the class value. therefore, it is founded that, the tp_s_pre attribute has a decisive effect on the appearance of bc when it is >0.28. otherwise, its benign or healthy case. as well as, when ca_s_pre attribute > 1.39, and ph_s_pre > 7, then it is healthy case. otherwise, it is benign case unless cea_b_pre ≤ 1.51, then it will be healthy case. hence, when ca_s_pre ≤ 1.07, and ph_s_pre > 7 then it is healthy case. meanwhile, when ph_s_pre ≤ 7 it is benign case. consequently, the ca_s_pre, ph_s_pre, and cea_b_pre are the major attributes to distinguish between the healthy and benign cases. however, the tree in figure 2 demonstrates all the mentioned results. finally, the previous phases discovered hidden knowledge from a raw data. the major three methods examined in this study are explored in table 2. the examined results are produced using tenfold cross-validation as test option, and the percentage of correctly classified instances as a measure to determine the best one from them. furthermore, the iteration control is used to check the accuracy gained from the three mentioned algorithms in the case of 1, 5, and 10 repetitions, and to realize if the error ratio descends during iterations ascending of algorithm execution. accordingly, the error ratio of the correctly classified cases is descending whenever number of repetitions applied increasing, exactly in the case of mlp algorithm rather than slr. table 2 demonstrates the percentage ratios for the correctly classified instances. finally, the classification accuracy is estimated for the used techniques. hence, stated that the highest efficiency achieved by mlp in detection new supposed instances, either they are malignant, benign, or healthy cases. figures 3–5 represent the accuracy classification for used methods j48, slr, mlp respectively. conclusion this study introduced an approach for addressing bc analysis for iraqi women, it is a comparative data mining examination. the data set analysis aims to uncover the ambiguity of the attributes relationships and their patterns. they include s.f. behadili et al. 75j contemp med sci | vol. 5, no. 2, march–april 2019: 71–76 original breast cancer decisive parameters for iraqi women via data mining techniques fig. 1 architecture of data mining for bc investigation of iraqi women. fig. 3 accuracy classification j48 technique. fig. 4 accuracy of classification slr technique. slr, simple logistic regression. table 2. data mining techniques with 1, 5, and 10 iterations algorithm 1-repetition 5-repetition 10-repetition tenfold trees j-48 93.33 91.33 91.72 slr 98.89 98.33 97.89 mlp 97.78 98.56 98.94 mlp, multilayer perceptron; slr, simple logistic regression. fig. 2 pattern visualization of j48 decision tree. fig. 5 accuracy of classification mlp technique. mlp, multilayer perceptron. 76 j contemp med sci | vol. 5, no. 2, march–april 2019: 71–76 breast cancer decisive parameters for iraqi women via data mining techniques original s.f. behadili et al. 42 attributes, which are manipulated, hence reduced to just 26 significant attributes. the acquired attributes are explored as the great influential on the bc inflammation. this study used the information gain or data-driven method (oner classifier) for variable selection instead of manual variable selection methods. furthermore, used machine-learning methods decision tree (j48), mlp, and slr. they can handle numeric and nominal attributes and map raw patient records into subsets of patient cohorts (classes), which share commonalities in attributes. hence, the proposed method formed multiple patient subgroups with different attributes; it helps in improving the baseline accuracy of mentioned classifiers. as a result, data mining techniques could be considered as a powerful research tool for medical researchers, and it is recommended to identify and exploit the patterns and relationships among large number of diseases attributes. therefore, they are able to predict outcome of a disease using the historical datasets. it can also assist in realizing the most effective biomarkers. furthermore, this investigation results supports the results in salih et al.’s3 study, which considered the tps as an effective biomarkers to bc indication, this is explored using decision tree (j48) classifier. as well as, the gained results accuracy is evaluated for each of the mentioned methods. however, it is appeared that mlp is the most accurate classifier with 98.94% of correctness precision, bypassing multiple iterations of execution. as a future perspective, it is counseled to examine big data of bc for iraqi patients, for the sake of deriving more hidden relations, and examine larger attributes. especially, investigates the historical family records of patients, this could help in predicting approach for bc survivability. however, the great challenge of this kind of studies is the lack of data; huge number of disease attributes, electronic registering rareness in iraq, and the lose communication between the medical domain and informatics. conflicts of interest none.  references 1. anupama yk, amutha s, ramesh babu dr. breast cancer prediction using data mining techniques. int j adv res sci eng. 2018;7:41–44. 2. zand hk. a comparative survey on data mining techniques for breast cancer diagnosis and prediction. ind j fundam appl life sci. 2015;5:4330–4339. 3. salih km, mohemmed ak, al-shaikh m, al-sayyid mm. salivary fraction of ca 15-3 and cea as tumor markers for breast cancer in iraqi women. int j eng technol. 2015;4:5114–5117. 4. estanislao gl, campos ra. breast cancer, primary peritoneal malignant mixed mullerian tumor and fallopian tube carcinoma: incidental concomitant malignancies or evidence for a new genetic cancer predisposition syndrome? conference 2018 of the european society of gynaecological oncology, lyon, france, october 4–6, 2018. 5. al-akeedi jm, mahmod as, ali ma. potential prognostic roles for il-6 and crp in iraqi women with breast cancer. int j adv biol res. 2013;3:530–534. 6. madkhali na, santin o, noble h, reid j. understanding breast health awareness in an arabic culture: qualitative study protocol. j adv nurs. 2016;72:2226–2237. 7. shukla n, hagenbuchner m, win kt, yang j. breast cancer data analysis for survivability studies and prediction. comput methods programs biomed. 2018;155:199–208. 8. yuvarani s, jothi v. breast cancer detection in data mining: a review. int j comput appl. 2015;7:45–48. 9. iqbal j, ginsburg o, rochon pa, sun p, narod sa. differences in breast cancer stage at diagnosis and cancer-specific survival by race and ethnicity in the united states. jama. 2015;313:165–173. 10. barot v, brahmbhatt n. a survey on breast cancer diagnosis using data mining technique. int j innov res sci eng technol. 2017;6:147–150. 11. saranya p, satheeskumar b. a survey on feature selection of cancer disease using data mining techniques. int j comput sci mobile comput. 2016;5:713–719. 12. khargonekar p, sinskey a, miller c, ranganathan b. convergence revolution – piloting the third scientific revolution through start-ups for breast cancer cure. cancer sci res open access. 2017;4:1–6. 13. priyanga a, prakasam s. effectiveness of data mining based cancer prediction system (dmbcps). int j comput appl. 2013;83:11–17. 14. kharya s. using data mining techniques for diagnosis and prognosis of cancer disease. int j comput sci eng inform technol. 2012;2:55–66. 15. delen d, walker g, kadam a. predicting breast cancer survivability: a comparison of three data mining methods. artif intell med. 2005;34:113–127. 16. mustafa tk, abd ms. proposed approach for analysing general hygiene information using various data mining algorithms. iraqi j sci. 2017;58:337–344. 17. gad w. svm-kmeans: support vector machine based on kmeans clustering for breast cancer diagnosis. int j comput inform technol. 2016;5:252–257. 18. anupama yk, amutha s, ramesh babu dr. survey on data mining techniques for diagnosis and prognosis of breast cancer. int j recent innov trends comput commun. 2017;5:252–257. 19. shiv shakti s, sant a, aharwal rp. an overview on data mining approach on breast cancer data. int j adv comput res. 2013;3:256–262. 20. ravi kumar g, ramachandra ga, nagamani k. an efficient prediction of breast cancer data using data mining techniques. int j innov eng technol. 2013;2:139–144. 21. chaurasia v, pal s. data mining techniques: to predict and resolve breast cancer survivability. int j comput sci mobile comput. 2014;3:10–22. 22. ghosh s, mondal s, ghosh b. a comparative study of breast cancer detection based on svm and mlp bpn classifier. 2014 first international conference on automation, control, energy and systems (aces), india, ieee, hooghy, india, 2014. 23. delshi howsalya r, indra m. outlier detection algorithm combined with decision tree classifier for early diagnosis of breast cancer. int j adv eng technol. 2016;vii:93–98. 24. fouda ma, sherif fz, ghannam aa, al-shorbagy sh. prognostic value of breast cancer subtypes based on er/ pr, her2 expression and ki-67 index in women received adjuvant therapy after conservative surgery for early stages breast cancer a retrospective clinical study. jsm clin oncol res. 2017. 25. fredholm h. breast cancer in young women aspects on mortality and local recurrence, ph.d., university hospital solna, 2017. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.04201902 158 j contemp med sci | vol. 4, no. 3, summer 2018: 158–162 original effects of genistein treatment on molecular markers related to fibrosis: addition to histological changes in the lung of ovariectomized diabetic rats mohammad reza alipour,a naser ahmadiasl,b pouran karimi,c alireza alihemmati,d masoumeh majidi zolbine and faeze daghighf adrug applied research center, tabriz university of medical sciences, tabriz, iran. bmolecular biology department, tuberculosis and lung research center, tabriz university of medical sciences, tabriz, iran. cneurosciences research center, tabriz university of medical sciences, tabriz, iran. ddepartment of histology and embryology, faculty of medicine, tabriz university of medical sciences, tabriz, iran. edepartment of anatomy and embryology, faculty of medicine, tehran university of medical science, tehran, iran. f tuberculosis and lung research center, tabriz university of medical sciences, tabriz, iran. correspondence to faeze daghigh (email: f_daghigh@yahoo.com). (submitted: 25 march 2018 – revised version received: 18 april 2018 – accepted: 27 june 2018 – published online: 26 september 2018) objective postmenopausal women experience physiological changes in several organs related to estrogen deficiency. on the other hand, the role of estrogen, as a sex hormone, in the physiological function and structure of respiratory system has been stablished. in the present study, we hypothesized that the administration of genistein could reverse lung fibrosis disturbed by estrogen deprivation. methods forty female sprague–dawley rats were assigned into four groups: rats underwent surgery without ovariectomy (sham), rats that underwent ovariectomies (ovx), ovariectomized rats that were fed with high fat diet (ovx.dia), ovariectomized diabetic rats with genistein administration (ovx.dia.gen). western blotting technique and hematoxylin and eosin were used to assess of protein levels and histological changes in the lung. results transforming growth factor beta-1 and matrix metalloproteinase-2 were found to have differential expression among the groups. ovariectomy alone and with diabetes increased the levels of growth factor beta-1 and matrix metalloproteinase-2 as compared with the sham (p < 0.05). on the other hand, genistein treatment reversed ovariectomy-induced changes in the protein levels as compared with ovx.dia group (p < 0.05). conclusion genistein ameliorated expression levels of fibrotic biomarkers in the lung of ovariectomized diabetic rats. our data suggest that genistein may be beneficial for menopausal women through modulation of pulmonary fibrotic biomarkers expression levels. keywords genistein, menopause, lung, fibrosis, tgf-β1 introduction there are few studies done globally on healthy respiratory and lung function in postmenopausal women. estrogen, as a sex hormone exerts important regulatory role in women’s pulmonary function. several lines of evidence suggest that estrogen deficiency associated with decrease in pulmonary function in elderly women.1 likewise, estrogen replacement in ovariectomized (ovx) mice has been shown to reverse lung architecture.2 more importantly, anti-inflammatory and anti-fibrotic effects of estrogen have been established over the past few decades.3 people with diabetes mellitus are suspiciously affected by pulmonary disorders such as edema, inflammation and fibrosis in the lung.4 thus, histopathological findings in diabetic subjects have revealed thickening of basal lamina and fibrosis leading to restrictive lung function.5 the cytokine transforming growth factor beta-1 (tgf-b 1) is known as a main pro-fibrotic factor in diabetic statues. moreover, transient elevation of blood glucose, even in healthy subjects leads to an increase in tgf-β1 urinary levels.6 hormone replacement therapy (hrt) has been proposed to ameliorate insulin sensitivity in postmenopausal diabetic women.7 however, hrt in menopausal women is accompanied by serious concern as it has been linked with breast cancer and cardiovascular diseases.8,9 in recent years, there has been a growing interest in researching new procedures with estrogenic effects in menopausal women. phytoestrogens have been shown to exert similar effects as hrt without unwanted harmful side effects.10 accordingly, genistein, as a phytoestrogen can exert protective effects against metabolic disorders induced by diabetes mellitus.11 thus, genistein is shown to be as an effective therapeutic agent in pulmonary fibrosis.12 in the light of recent data, the present study was designed to evaluate effects of genistein on the lung injury induced by estrogen deficiency in ovariectomized diabetic rats. materials and methods animals and groups forty female wistar rats were obtained from experimental animal research institute, tabriz medical university, tabriz, iran. rats (180–220 g) were kept in cages and acclimated in facility conditions (22–24°c) with a 12-h light, 12-h dark. the study was approved by the ethics committee of tabriz university of medical science (code number: ir.tbzmed. rec.1396.450). animals were randomly divided into four groups (n = 10 in each group): sham, laparotomy without ovariectomies, ovx, rats that underwent bilateral ovariectomy, ovariectomized rats that were fed with high fat diet (hfd) (ovx.dia), for 4 weeks and injected with a single injection of stz (30 mg/kg, intraperitoneally), ovariectomized diabetic rats with genistein administration (ovx.dia.gen) (sigma-aldrich, usa) (1 mg/kg/day; subcutaneously) for 8 weeks with concurrent hfd feeding. issn 2413-0516 mohammad reza alipour et al. 159j contemp med sci | vol. 4, no. 3, summer 2018: 158–162 original effects of genistein treatment on molecular markers related to fibrosis ovariectomy ovaries were removed under anesthesia with a mixture of 50 mg/kg ketamine chloride and 5 mg/kg xylazine chloride. in brief, after skin and muscle walls dissection, peritoneal cavity retrieved and then ovaries were removed with minimal skin and soft disruption.13 diabetes induction ten days after ovariectomy, diabetic rats continued to feed with hfd (25% protein, 58% fat and 17% carbohydrate) for a 4-week period and injected with a single low-dose of stz (30 mg/kg) dissolved in a 0.1-mol/l citrate buffer (ph 4.5) for induction of type ii diabetes.13 at the end of diabetes induction period, animals were given access to standard diet for 4 weeks. blood samples were collected 72 h after stz injection and at the end of the protocol. rats with blood glucose levels higher than 200 mg/dl were considered to have diabetes.14 immunoblotting analysis snap-frozen lung tissue was homogenized on ice in ripa buffer containing proteinase inhibitor cocktail (leupeptin, chymostatin, pepstatin, antipain and aprotinin). after storing at 4°c for 20 min, the samples were centrifuged at 12,000 rpm for 10 min at 4°c. the supernatants were separated and stored at −80°c. proteins were separated by sds-page and transferred onto polyvinylidene difluoride. membranes were blocked with 5% (w/v) nonfat dry milk in tris-buffered saline (tbs) (ph 7.5) for 2 h and incubated with primary antibodies (antitgf-β1 and anti-mmp2, santa cruz, usa) at dilution containing 1% (w/v) nonfat dry milk in 0.05% (v/v) tbs plus 0.05% (v/v) tween 20 (tbst). samples were washed with tbst three times, then incubated for 1 h in secondary antibody (goat anti-rabbit; santa cruz, usa) in antibody dilution buffer. substrates were detected by chemiluminescence (ecl western blotting detection, pierce, rockford, il, usa).15 anti-b -actin antibody has been considered as a loading control. bands on immunoblots were quantified using imagej densitometry software. histological evaluation the lungs were prepared for histopathological evaluation. briefly, after fixing of lungs in 10% formalin and tissues processing, histological sections (5 μm thickness) were stained with standard hematoxylin–eosin. light photomicroscope (olympus bh-2, tokyo, japan) was used for evaluating of fibrosis in the lung tissues. statistical analysis data were expressed as the mean ± sem. differences between groups were determined using one-way anova with tukey’s multiple comparison test. p-value less than 0.05 was considered as statistically significant. results blood glucose levels in the studied groups to evaluate the genistein treatment on blood glucose levels, we measured the changes of blood sugar levels under fasting conditions. ovx.dia rats exhibited hyperglycemia during an overnight fast as compared with sham group (p < 0.05). however, genistein treatment significantly lowered blood glucose levels in comparison to ovx.dia group (p < 0.05, fig. 1). genistein treatment on the levels of tgf-β1 in the lung the lung levels of tgf-β1 significantly augmented in ovx and ovx.dia groups when compared with the sham (p < 0.05). genistein treatment significantly reduced tgf-β1 levels in comparison to ovx.dia group, suggesting that genistein may exert its anti-fibrotic effects by reducing tgf-β1 in the lung of ovariectomized diabetic rats (p < 0.05, fig. 2a and b). genistein treatment on the levels of mmp2 in the lung ovariectomized with or without diabetes led to a markedly increased in matrix metalloproteinase-2 (mmp2) levels (p < 0.05). genistein treatment decreased mmp2 levels in ovx.dia.gen group as compared with ovx.dia, indicating that decrease of mmp2 might be one of the mechanisms by which genistein exerts its protective effects against ovariectomy induced lung fibrosis (p < 0.05, fig. 3a and b). histological changes in the lung tissue histological examination of lung in the sham group showed healthy lung tissues with thin interalveolar septa (fig. 4a, table 1). section of lung in ovx and ovx.dia groups showed thickened interalveolar septa (black arrow) with morphological changes of alveoli (asterisk). alveolar cells deformities with a denuded alveolar epithelium (green arrow) were also detected in ovx and ovx.dia groups. ovariectomy with diabetes were associated with a significant alveolar hemorrhage (blue arrow) and intravascular coagulation (yellow arrow) (p < 0.05, fig. 4b–d, respectively, table 1). genistein treatment declined hemorrhage and intravascular coagulation in ovx. dia.gen group as compared with ovx.dia group (p < 0.05). also, relative decline in number of alveolar cells deformities (green arrow) and alveolar morphological changes (asterisk) were observed in ovx.dia.gen group (fig. 4e, table 1). fig. 1 effect of genistein treatment on blood levels of glucose in the studied groups. ovx: ovariectomized, ovx.dia: ovariectomized diabetic rats, ovx.dia.gen: ovariectomized diabetic rats with 8 weeks of genistein treatment. data are expressed as mean ± sem. *p < 0.05 vs sham group; #p < 0.05 vs ovx. dia group. 160 j contemp med sci | vol. 4, no. 3, summer 2018: 158–162 effects of genistein treatment on molecular markers related to fibrosis original mohammad reza alipour et al. fig. 2 effect of genistein treatment on tgf-β1 expression levels in the lung of studied groups. immunoblotting of tgf-β1 among different studied groups (a), immunoblotting quantitation of tgf-β1 against expression of βactin in lung (b), ovx: ovariectomized group, ovx.dia: ovariectomized diabetic rats, ovx.dia.gen: ovariectomized diabetic rats with genistein treatment. data are expressed as mean ± sem. *p < 0.05 vs sham group; **p < 0.05 vs ovx.dia groups. fig. 3 effect of genistein treatment on mmp2 expression levels in the lung of studied groups. immunoblotting of mmp2 within different groups (a), immunoblotting quantitation of mmp2 against expression level of βactin in the lungs (b), ovx: ovariectomized group, ovx.dia: ovariectomized diabetic rats, ovx.dia.gen: ovariectomized diabetic rats with genistein treatment. data are expressed as mean ± sem. *p < 0.05 vs sham group; **p < 0.05 vs ovx.dia groups. fig. 4 histological micrograph in the lung of different studied groups. sham (4a): normal lung with thin inter alveolar septa, ovx (4b): hemorrhage (blue arrow), thickened inter alveolar septa (black arrow), morphological change of alveoli (asterisk), and alveolar deformity (green arrow) observed in ovx group, ovx.dia (4c and 4d): severe hemorrhage (blue arrow) in ovx.dia group and thickening of interalveolar septa (black arrows) and intravascular coagulation (yellow arrow). ovx.dia.gen (4e) relative thin inter alveolar septa (black arrow) and decrease of hemorrhage (blue arrow), alveolar denudation (green arrow) and intravascular coagulation (yellow arrow) observed in ovx.dia.gen compared with ovx.dia group. ba ba a b c ed mohammad reza alipour et al. 161j contemp med sci | vol. 4, no. 3, summer 2018: 158–162 original effects of genistein treatment on molecular markers related to fibrosis genistein treatment in ovx.dia.gen group revealed a moderate thickened interalveolar septa as compared with ovx.dia group (p < 0.05, fig. 4e, table 1). discussion decrease in ovarian function with surgical or physiological menopause is associated with an increase in tgf-β1 expression levels in several organs.16 similarly, higher blood glucose concentration has been found to stimulate urinary tgf-β1 levels in healthy individuals.6 there appears to be a strong association between the deregulation of tgf-β signaling and various fibrotic diseases. aberrant activation and proliferation of fibroblasts or myofibroblast contribute to an increased collagen deposition in the fibrotic lung. there is also evidence that tgf-β1 might induce fibroblast to myofibroblast trans differentiation. interestingly, the cytokine tgf-β1 has been identified locally at the sites of fibrosis in fibroblast-to-myofibroblast trans-differentiation.17 previous studies demonstrated a strong induction of coagulation factor xii (fxii) expression by tgf-β1. in particular, tgf-β1 expected to assume a major role in increasing the expression of coagulation fxii in the lung fibroblast cells.18 in keeping with previous reports, we observed fibrosis with markedly increased in tgf-β1 expression levels and a marked activation of blood coagulation detecting in the lung of ovx.dia group as compared with the sham. matrix metalloproteinase exert essential roles in functioning of several tissues during aging. thus, aging female mice were characterized by enhanced mmp2 activity, that it was ameliorated by estrogen therapy in the lung.19 interestingly, in a previous study we found an increase in mmp2 expression level in the lung of ovx rats.20 clinical studies have revealed a close relationship between mmp2 expression and pulmonary fibrosis. it is noted that, mmp2 involved in tgf-β1 induced epithelial mesenchymal transition (emt), indicating that production of mmp2 in the lung alveolar cells may exert a major role in the development of pulmonary fibrosis.21 additionally, excess activation of mmps has been shown to destroy extracellular matrix and induce further inflammation in the lung.22 on the other hand, lung epithelial cells that undergo emt show enhanced expression of fibrogenesis markers like mmp2 protein levels.21 there appears to be a strong link between dysregulation of mmp/timp system and diabetes status. thus, blood levels of mmp2 has been shown to significantly increase in type 2 diabetes.23 yusuf et al.4 have found a dramatic change in lung structure in diabetic rats characterized by infiltration of table 1. histological changes in the lung of sham, ovariectomized, ovariectomized diabetic and genistein treatment groups (h&e) group interstitial hemorrhage interstitial thickening alveolar epithelium deformity intravascular coagulation alveolar architectural alteration sham 0 0.20 ± 0.20 0.20 ± 0.20 0 0.40 ± 0.24 ovx 1.20 ± 0.20* 2.60 ± 0.24* 2.80 ± 0.20* 2.80 ± 0.20* 2.80 ± 0.20* ovx.dia 3.80 ± 0.20* 4 ± 0.24* 3.60 ± 0.24* 3.80 ± 0.20* 3.80 ± 0.20* ovx.dia.gen 3 ± 0.50** 2.80 ± 0.20** 3 ± 0.001 2.80 ± 0.20* 3 ± 0.001 a minimum of five fields for each slide were evaluated for lung tissue changes (n = 10 in each group). data are expressed mean ± sem. *p < 0.05 vs sham group. **p < 0.05 vs ovx.dia group. mononuclear cells, edema, hemorrhage, and fibrosis. similarly, ovariectomized diabetic lung is associated with a marked histological abnormalities.4 in the present study, to the best of our knowledge we demonstrated that mmp2 is significantly increased in the lung of ovariectomized diabetic rats. it is also well known that, fibrotic pulmonary remodeling associated with repetitive minor injuries of alveolar epithelial cells lining with a denuded basal lamina.24 our results confirmed previous studies indicating markedly increased in mmp2 expression with severe fibrosis in the lung. we also found significant morphological change in alveolar cells in ovx and ovx.dia groups reflected by denudation of basal lamina. it has become apparent that, phytoestrogens exert beneficial effects on diabetic complications.11 genistein, as a phytoestrogen plays a protective role in the pulmonary fibrosis. as previously reported, genistein could exert a protective effects against interstitial and perivascular lung fibrosis induced by pulmonary hypertension.12 genistein has also been shown to block tgf-β-mediated activation of mmp2 in prostate epithelial cells.25 activation of the ras/erk mapk pathway has been implicated in emt induced by tgf-β1.26 in other study, we showed that genistein can decrease erk expression levels in the lung of ovariectomized diabetic rats.22 in the current study, tgf-β1, and mmp2 significantly decreased in the genistein treatment group as compared with the ovariectomized diabetic rats. there are few researches regarding the role of phytoestrogens on the blood coagulation activities. it is known that genistein increases some coagulation related genes in ovx rats.27 however, in another study high doses administration of isoflavone (up to 300 mg/day) did not change coagulation parameters in the postmenopausal women.28 in the present study, genistein administration partially decreased intensity of intravascular coagulation, but there was no significant change between genistein treatment and ovariectomized diabetic groups. our results are supported by recent studies that highlighted the important role of genistein supplementation in ovariectomy induced lung injury. although the beneficial effects of genistein on respiratory system is not well known. therefore, our results encourage further studies in ovariectomized diabetic states. conclusion estrogen deficiency alone or with hfd increased fibrotic biomarkers such as tgf-β1 and mmp2 in the lung of ovariectomized diabetic rats. genistein treatment ameliorated 162 j contemp med sci | vol. 4, no. 3, summer 2018: 158–162 effects of genistein treatment on molecular markers related to fibrosis original mohammad reza alipour et al. proteins expression and histological changes in the lung of ovariectomized diabetes rats. acknowledgments the present paper is extracted from the phd thesis of faeze daghigh “combination effect of swimming and genistein on the erk signaling leading to apoptosis and histologic changes in the lung tissue of diabetic ovariectomized rats.” the authors would like to thank drug applied. conflict of interest none. n references 1. real fg, svanes c, omenaas er, antò jm, plana e, jarvis d, et al. lung function, respiratory symptoms, and the menopausal transition. j allergy clin immunol. 2008;121:72–80. e3. 2. massaro d, massaro gd. estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice. am j physiol lung cell mol physiol. 2004;287:l1154–l1159. 3. elfattah lia. a comparative study between the effects of dietary soya and estrogen replacement therapy on the lung of ovariectomized albino rats: histological and immunohistochemical study. egypt j histology. 2012;35:34–42. 4. dogru yz, nacar t, unal d, aksak s, albayrak a, odabasoglu f, et al. effects of diabetes mellitus and postmenopausal period on the lungs of rats. afr j pharm pharmacol. 2012;6:1989–2010. 5. yeh hc, punjabi nm, wang ny, pankow js, duncan bb, cox ce, et al. crosssectional and prospective study of lung function in adults with type 2 diabetes: the atherosclerosis risk in communities (aric) study. diabetes care. 2008;31:741–746. 6. mcgowan ta, dunn sr, falkner b, sharma k. stimulation of urinary tgf-β and isoprostanes in response to hyperglycemia in humans. clin j am soc nephrol. 2006;1:263–268. 7. bitoska i, krstevska b, milenkovic t, subeska-stratrova s, petrovski g, mishevska sj, et al. effects of hormone replacement therapy on insulin resistance in postmenopausal diabetic women. open access maced j med sci. 2016;4:83–88. 8. mosca l, collins p, herrington dm, mendelsohn me, pasternak rc, robertson rm, et al. hormone replacement therapy and cardiovascular disease. circulation. 2001;104:499–503. 9. chen cl, weiss ns, newcomb p, barlow w, white e. hormone replacement therapy in relation to breast cancer. jama. 2002;287:734–741. 10. serock mr, wells ak, khalil ra. modulators of vascular sex hormone receptors and their effects in estrogen-deficiency states associated with menopause. recent pat cardiovasc drug discov. 2008;3:165–186. 11. stephenson tj, setchell kd, kendall cw, jenkins dj, anderson jw, fanti p. effect of soy protein-rich diet on renal function in young adults with insulindependent diabetes mellitus. clin nephrol. 2005;64:1–11. 12. matori h, umar s, nadadur rd, sharma s, partow-navid r, afkhami m, et al. genistein, a soy phytoestrogen, reverses severe pulmonary hypertension and prevents right heart failure in rats. hypertension. 2012;60:425–430. 13. irigoyen mc, paulini j, flores lj, flues k, bertagnolli m, moreira ed, et al. exercise training improves baroreflex sensitivity associated with oxidative stress reduction in ovariectomized rats. hypertension. 2005;46:998–1003. 14. su x, meng x, sun c, liu l, su b. intramuscular injection of soluble receptor for advanced glycation endproducts expression vector prevents the development of streptozotocin‐induced diabetes mellitus in rats on high fat diet. j diabetes. 2011;3:309–316. 15. faramoushi m, amir sasan r, sari sarraf v, karimi p. cardiac fibrosis and down regulation of glut4 in experimental diabetic cardiomyopathy are ameliorated by chronic exposures to intermittent altitude. j cardiovasc thorac res. 2016;8:26–33. 16. leask a, abraham dj. tgf-β signaling and the fibrotic response. faseb j. 2004;18:816–827. 17. chambers rc, leoni p, kaminski n, laurent gj, heller ra. global expression profiling of fibroblast responses to transforming growth factor-β 1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching. am j pathol. 2003;162:533–546. 18. jablonska e, markart p, zakrzewicz d, preissner kt, wygrecka m. transforming growth factor-β1 induces expression of human coagulation factor xii via smad3 and jnk signaling pathways in human lung fibroblasts. j biol chem. 2010;285:11638–11651. 19. glassberg mk, choi r, manzoli v, shahzeidi s, rauschkolb p, voswinckel r, et al. 17β-estradiol replacement reverses age-related lung disease in estrogen-deficient c57bl/6j mice. endocrinology. 2014;155:441–448. 20. daghigh f, alihemmati a, karimi p, habibi p, ahmadiasl n. fibrotic and apoptotic markers alteration in ovariectomised rats: addition of swimming training preserves lung architecture. arch physiol biochem. 2017;7:1–6. 21. kasai h, allen jt, mason rm, kamimura t, zhang z. tgf-β1 induces human alveolar epithelial to mesenchymal cell transition (emt). respir res. 2005;6:56. 22. daghigh f, alihemmati a, karimi p, habibi p, ahmadiasl n. genistein preserves the lungs of ovariectomized diabetic rats: addition to apoptotic and inflammatory markers in the lung. iran j basic med sci. 2017;20:1312–1317. 23. lee sw, song ke, shin ds, ahn sm, ha es, kim dj, et al. alterations in peripheral blood levels of timp-1, mmp-2, and mmp-9 in patients with type-2 diabetes. diabetes res clin pract. 2005;69:175–179. 24. geiser t. idiopathic pulmonary fibrosis–a disorder of alveolar wound repair? swiss med wkly. 2003;133:405–411. 25. huang x, chen s, xu l, liu y, deb dk, platanias lc, et al. genistein inhibits p38 map kinase activation, matrix metalloproteinase type 2, and cell invasion in human prostate epithelial cells. cancer res. 2005;65:3470–3478. 26. grände m, franzen a, karlsson jo, ericson le, heldin ne, nilsson m. transforming growth factor-β and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (emt) through a mekdependent mechanism in primary cultured pig thyrocytes. j cell sci. 2002;115:4227–4236. 27. kelly la, o’leary jj, seidlova-wuttke d, wuttke w, norris la. genistein alters coagulation gene expression in ovariectomised rats treated with phytoestrogens. thromb haemost. 2010;104:1250–1257. 28. cheng wc, lo sc, tsai ks, tu st, wu js, chang ci, et al. effects of high-dose phytoestrogens on circulating cellular microparticles and coagulation function in postmenopausal women. j formos med assoc. 2015;114:710–716. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201809 13j contemp med sci | vol. 6, no. 1, january–february 2020: 13–16 original issn 2413-0516 introduction type 1 diabetes mellitus (t1dm) is one of the most common chronic diseases of childhood.1 its incidence is rising worldwide,2 with reported increases of 2–5 percent per year in the middle east.3 in iraq, the incidence of t1dm in basra city is 5–9.99/100,000 per year and was increasing between 2012 and 2016.4 similarly, in al-nassiryah city the incidence was also increasing in the last 5 years,5 and with the existence of only a few national/regional diabetes registries available to support diabetes research, provide reliable data, and help cope with the widespread threat of this disease, there is a need for establishing a population-based arab diabetes registry.6 most children with t1dm grow normally, however, poor glycemic control can result in poor linear growth, poor weight gain, and/ or delayed skeletal development. conversely, treatment with excessive insulin and/or excessive caloric intake can lead to excessive weight gain. and if obesity develops, this can lead to insulin resistance, which complicates diabetes management.7 purpose of the study to estimate the prevalence of t1dm in primary school children in baghdad city, and to evaluate its effect on growth of those children. material and methods this is a comparative cross-sectional study that was conducted in primary schools in catchment area of 12 primary health care centers (phccs) in al-karkh side of baghdad city, selected by multistage cluster sampling (fig. 1), during the period of feb 15, 2018–may 1, 2018. the study population included all primary school students in the selected phccs (all t1dm students in these schools and an equal number of children from the same class and same gender who were t1dmfree were included). research was done in al-karkh side of baghdad/iraq in 141 school in the territory of the following phccs: name of phccs: number of schools which covered by local phcc: (al khadhraa phcc: 8, al jamieaa phcc: 17, the martyr saif zakie phcc: 16, al gazalia the first phcc: 8, al dakhilia phcc: 17, al mansoor exemplary phcc: 14, martyrs of alchalijia phcc: 4, al yarmouk phcc: 13, al huriya exemplary phcc: 12, al jawadin exemplary phcc: 9, al zahraa exemplary phcc: 7, the new iraq phcc: 16). school children with any other chronic disease or comorbidities were excluded from the anthropometric measures only. data collection tools two different types of questionnaires had been used. the first questionnaire was filled by the researcher through direct interview with the primary school teachers. it included questions to gather information on certain student variables, and the anthropometrics’ measures; the centers for disease control epidemiological profile of type 1 diabetes among primary school children in baghdad, iraq sarah hayder zalzala,a faris h. al-lami,b khalid saeed fahadc aal-karkh directorate of health, ministry of health, baghdad, iraq. bcommunity and family medicine department, college of medicine, baghdad university, baghdad, iraq. cdepartment of internal medicine, college of medicine, al-nahrain university, baghdad, iraq. correspondence to: sarah hayder zalzala (email: sarahhaider1988@gmail.com). (submitted: 23 november 2019 – revised version received: 02 december 2019 – accepted: 12 january 2020 – published online: 26 february 2020) objectives to estimate the prevalence of type 1 diabetes mellitus in primary school children in baghdad city, and to evaluate its effect on growth of those children. methods this comparative cross-sectional study was conducted in a sample of primary schools in baghdad city selected by multistage cluster sampling. all primary school students in the selected schools were included. for every diabetic child, we selected a child from the same class who is free from diabetes. information on disease variables were obtained through sending questionnaire to the children’s parents. the centers for disease control and prevention growth charts were used. results the total number of primary school students in the selected 141 schools was 69,115; 110 of them had t1dm (159/100,000). female to male ratio was 1.3:1. obesity and underweight were significantly lower in diabetics than non-diabetic children (p=0.03). conclusion the prevalence of type 1 diabetes mellitus was 159 per 100,000, which was approximate to the prevalence in saudi arabia, less than that in al-kuwait, but higher than that in turkey. percentage of underweight and obesity were lower in the diabetics while overweight percentage was slightly higher compared to the non-diabetics. keywords type 1 diabetes, primary school children, prevalence, obesity, underweight fig. 1 multistage cluster sampling. 14 original epidemiological profile of type 1 diabetes among primary school children sarah hayder zalzala et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 13–16 and prevention (cdc) growth charts were used.8the second questionnaire is translated to arabic language and was sent to parents of the diabetic group only, to gather information about disease variables: clinical presentation for dm (classic new onset of chronic polydipsia, polyuria, and weight loss); diabetic ketoacidosis; or asymptomatic incidental discovery,9 the duration of dm in year(s), the age at onset of diabetes (early onset diabetes has been variously defined as occurring anywhere from age 4 to age 7 years,10 blood glucose monitoring (less than four, four, or more than four times per day) and whether a glucometer in the house was available or not, family history of t1dm, the no. of follow up visit(s) for phcc, diabetic center or private doctor in the last 6 months (routine follow-up should be performed at least four times a year,11 number of er visits and hospital admission in the last 6 months, and the value of the last hba1c test for each child were all inquired. the statistical software (statistical package for the social science, release 11.0 for windows; spss; chicago, il) was used for data entry and analysis. descriptive statistics were used to summarize subject characteristics and questionnaire results. chi-square test of independence was used to test qualitative and frequency data. p-value of <0.05 was considered significant. a written informed consent was obtained from parents of each enrolled student. all personal information was kept anonymous. administrative approval was granted from research committee in ministry of health, iraq. result the total number of primary school students in the selected 141 schools was 69,115; 110 of them had t1dm. the highest prevalence of diabetes was in al mansoor exemplary phcc (338 per 100,000) while the overall prevalence of diabetes was 159 per 100,000. the proportion of female was slightly more than male with female to male ratio of 1.3:1, diabetics’ age ranged 6–14 years old. the distribution of the diabetics according to age, sex, and disease variables is shown in table 1. the distribution of the study group according to anthropometric measures is shown in table 2. table 1. variables no. (n=105) percentage (%) age (year) 6-8 23 21.9 9-11 56 53.3 ≥12 26 24.8 sex male 46 43.8 female 59 56.2 clinical presentation for dm classic new onset 52 62.7 diabetic ketoacidosis 15 18.1 incidental discovery 16 19.2 duration of dm (years) <3 44 53.0 3-6 30 36.2 ≥7 9 10.8 age at onset of diabetes (years) <7 53 63.9 ≥7 30 36.1 the availability of glucometer in the house yes 73 88.0 no 10 12.0 blood glucose monitoring (no. of times per day) <4 56 67.5 ≥4 27 32.5 table 2. distribution of study group by anthropometric measures. variable diabetics nondiabetics chi square p value bmi percentile no.(n=102) percentage (%) no.(n=105) percentage (%) <5 2 2.0 9 8.6 8.929 0.030 ≥5 and <85 76 74.5 66 62.9 ≥85 and<95 17 16.6 14 13.3 ≥95 7 6.9 16 15.2 weight for age percentile <5 5 4.9 2 1.9 3.243 0.198≥5 and ≤ 95 91 89.2 91 86.7 > 95 6 5.9 12 11.4 height for age (percentile) <5 10 9.8 2 1.9 5.931 0.052≥5 and ≤ 95 89 87.2 100 95.2 > 95 3 3.0 3 2.9 15 original epidemiological profile of type 1 diabetes among primary school childrensarah hayder zalzala et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 13–16 the majority of patients (62.7%) were diagnosed by classic symptoms of diabetes. the mean for duration of dm was 3.9 ±2.4 (sd) years and 63.9% have dm before the age of 7 years. around 10% of children didn’t have follow-up visit at all, 50% of them had at least one hospital admission and frequent attacks of hypoglycemia in the last 6 months. only 1 of them had documented hba1c value which was 13%. among the reminder 90% whom had gone for follow-up visit, 14.5% had 1 visit, 13.3% had 2 visits and 62.6% had >2 visits per 6 months. according to where they went for follow-up, 75.9% visited a private doctor and only 54.2% of children had documented hba1c result. discussion the prevalence of t1dm has been reported to vary greatly among different countries, within countries, and among different ethnic populations.12 recently, a study reported that the prevalence and incidence of t1dm were found to be variable among the arabs.6 this study showed that prevalence of diabetes in primary school children in baghdad was 159 per 100,000, which was higher than basrah where prevalence rate was 87 per 100,000,4 despite the age group sample was much wider than this study (all ≤40 years old), but these findings were based on a retrospective data analysis of electronic archives for patients with t1dm registered in faiha specialized diabetes, endocrine, and metabolism center, which is a tertiary referring center in basrah. the prevalence of t1dm was 269.9 per 100,000 among 6–18-year-old kuwaiti children,13 while it was 109 per 100,000 in saudi arabian children (7–12 years old)14 which is approximate to our results. lower prevalence found in children who lived in the nile delta region of egypt (26.8 per 100,000)15 and school children living in istanbul, turkey, (67 per 100,000).16 these variations might be due to the variation in the incidence of t1dm as it varied based upon geography, age, gender, family history, and ethnicity.7 when people relocate from a region of low to high incidence, their risk of developing t1dm also increases, suggesting a causative role for environmental factor(s). however, wide variations in incidence occur between neighboring areas of similar latitude, suggesting the presence of other contributing risk factors and demonstrating the complexity of the pathogenesis of t1dm.7 a study found that there is considerable variation among arab countries, which could not be explained on genetic or climatic variations alone, other environmental factors particularly nutritional ones including high intake of dairy products and vitamin d deficiency are possibly operating.17 the proportion of female was slightly more than male, which agreed with two studies as prevalence for t1dm slightly favors females in australia,18 and japan,19 but disagree with another study which slightly favors males in the usa,20 and it was near parity in north-west england.21 the lifetime risk of developing t1dm is significantly increased in close relatives of a patient with t1dm.22 in this study, around 30.1% of diabetics had family history of t1dm (aunts and uncles were included), as they found that 10%–20% of newly diagnosed childhood cases of t1dm have an affected first-degree relative,2 and familial history was more than 20% when accounting for the extended family history.23 the higher percentage of underweight in the non-diabetics group might be due to the primary cause of poor weight gain in school-aged children and adolescents which is inadequate dietary intake relative to typical metabolic and growth needs.24 children may ingest too much juice or other non-nutritious liquid, resulting in satiation and decreased appetite for higher caloric density or more nutritious solid foods,25, 26 while these beverages might be more restricted in the diabetics, and these results is approximate to turkish study as they found that rates of overweight and obesity were 21.2% and 14.6% (35.8% combined), using the who growth curve,27 compared to 13.3% and 15.2% (28.5% combined) in this study. about 2% of diabetics were underweight, 74% normal, 16.6% overweight and 6.9% obese, which agreed with a canadian study as they found that children with t1dm are more overweight, but not more obese.28 an australasian study showed that the prevalence of being overweight was 19% and of obesity was 6%,29 and another study where none of the study subjects were underweight, 69% were within the normal weight range.30 the challenges for maintaining healthy weight associated with t1dm include weight gain as the result of supra-physiological insulin doses, and overeating to avoid or treat hypoglycemia.29 another study showed that pre‐ and post‐onset body mass index in children with t1dm were both well above the population mean, were closely correlated with each other and (inversely) with age at onset.31 proper attention to diet is a major factor in minimizing hypoglycemia and weight gain while achieving glycemic control.32 the majority of patients were diagnosed by classic symptoms of diabetes which agreed with another iraqi study.33 although routine follow-up should be performed at least four times a year,11 about 10% of parents didn’t go for follow-up visit at all, 50% of those had at least one hospital admission and frequent attacks of hypoglycemia in the last 6 months and only 1 of them had documented hba1c value which was 13%. cardwell et al found that patients attending less than four diabetes clinics in the last year had a significantly higher hba1c level compared to those attending exactly four clinics level.34 acknowledgments authors would like to express deepest thanks for all the staff in the selected primary health care centers and primary schools for their generous help and support during the time of this project, especially to: dr. zainab, al dakhilia phcc. mrs. shatha, al jawadin exemplary phcc. i would like to apologize gratefully to all those people who helped me to complete my work without being able to mention them by names. conflicts of interest disclosure: none references 1. levitsky ll, misra m, wolfsdorf j, hoppin a. management of type 1 diabetes mellitus in children and adolescents. complications and screening in children and adolescents with type. 2011;1. 2. tuomilehto j. the emerging global epidemic of type 1 diabetes. curr. diab. rep. 2013;13(6):795-804. 16 original epidemiological profile of type 1 diabetes among primary school children sarah hayder zalzala et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 13–16 3. mamoulakis d, galanakis e, bicouvarakis s, paraskakis e, sbyrakis s. epidemiology of childhood type i diabetes in crete, 1990–2001. acta paediatr. 2003;92(6):737-9. 4. almahfoodh d, alabbood m, alali a, mansour a. epidemiology of type 1 diabetes mellitus in basrah, southern iraq: a retrospective study. diab. res. clin. pract. 2017;133:104-8. 5. al-rubaee rj. newly diagnosed type 1 diabetes mellitus in dhi-qar city (iraq) sociodemographic study. 6. zayed h, ouhtit a, el bekay r. an arab registry for type 1 diabetes: global benefits for type 1 diabetes patients. curr. med. res. opin. 2016;32(10):16814. 7. levitsky ll, misra m, wolfsdorf j, hoppin a. complications and screening in children and adolescents with type 1 diabetes mellitus. up to date. 2007;17(1). 8. phillips sm, shulman rj, motil k. measurement of growth in children. uptodate, basow, ds (ed), uptodate, waltham, ma. 2011. 9. levitsky ll, misra m. epidemiology, presentation, and diagnosis of type 1 diabetes mellitus in children and adolescents. uptodate waltham, ma: uptodate. 2007. 10. ack m, miller i, weil wb. intelligence of children with diabetes mellitus. pediatrics. 1961;28(5):764-70. 11. chiang jl, kirkman ms, laffel lm, peters al. type 1 diabetes through the life span: a position statement of the american diabetes association. diab. care. 2014;37(7):2034-54. 12. dabelea d. the accelerating epidemic of childhood diabetes. lancet. 2009;373(9680):1999-2000. 13. moussa ma, alsaeid m, abdella n, refai tm, al-sheikh n, gomez je. prevalence of type 1 diabetes among 6-to 18-year-old kuwaiti children. med. princip. pract. 2005;14(2):87-91. 14. al-herbish as, el-mouzan mi, al-salloum aa, al-qurachi mm, al-omar aa. prevalence of type 1 diabetes mellitus in saudi arabian children and adolescents. saudi med. j. 2008;29(9):1285-8. 15. el-ziny mae-m, salem na-b, el-hawary ak, chalaby nm, elsharkawy aa-e. epidemiology of childhood type 1 diabetes mellitus in nile delta, northern egypt-a retrospective study. j. clin. res. pediatr. endocrinol. 2014;6(1):9. 16. akesen e, turan s, güran t, atay z, save d, bereket a. prevalence of type 1 diabetes mellitus in 6–18‐yr‐old school children living in istanbul, turkey. pediatr. diab. 2011;12(6):567-71. 17. abdullah ma. epidemiology of type i diabetes mellitus among arab children. saudi med. j. 2005;26(6):911-7. 18. speight j, browne jl, holmes-truscott e, hendrieckx c, pouwer f. diabetes miles-australia (management and impact for long-term empowerment and success): methods and sample characteristics of a national survey of the psychological aspects of living with type 1 or type 2 diabetes in australian adults. bmc public health. 2012;12(1):120. 19. kawasaki e, eguchi k. is type 1 diabetes in the japanese population the same as among caucasians? ann. ny acad. sci. 2004;1037(1):96-103. 20. menke a, orchard tj, imperatore g, bullard km, mayer-davis e, cowie cc. the prevalence of type 1 diabetes in the united states. epidemiology (cambridge, mass). 2013;24(5):773. 21. anderson sg, narayanan rp, amlesh j, qureshi mz, heald ah. type 1 diabetes in cheshire: cardiometabolic risk factor trends (2004–2009). prim. care diab. 2012;6(2):123-6. 22. mayer‐davis ej, kahkoska ar, jefferies c, dabelea d, balde n, gong cx, et al. ispad clinical practice consensus guidelines 2018: definition, epidemiology, and classification of diabetes in children and adolescents. pediatr. diab. 2018;19:7-19. 23. parkkola a, härkönen t, ryhänen sj, ilonen j, knip m, register fpd. extended family history of type 1 diabetes and phenotype and genotype of newly diagnosed children. diab. care. 2013;36(2):348-54. 24. gahagan s. failure to thrive: a consequence of. pediatrics in review. 2006;27(1):e1-e11. 25. munoz ka, krebs-smith sm, ballard-barbash r, cleveland le. food intakes of us children and adolescents compared with recommendations. pediatrics. 1997;100(3):323-9. 26. kant ak. reported consumption of low-nutrient-density foods by american children and adolescents: nutritional and health correlates, nhanes iii, 1988 to 1994. arch. pediatr. adolesc. med. 2003;157(8):789-96. 27. yardim ms, ozcebe h, araz om, uner s, li s, unlu h, et al. prevalence of childhood obesity and related parental factors in ankara, turkey. 2018. 28. sandhu ν, witmans mb, lemay j-f, crawford s, jadavji n, pacaud d. prevalence of overweight and obesity in children and adolescents with type 1 diabetes mellitus. j. pediatr. endocrinol. metab. 2008;21(7):631-40. 29. phelan h, clapin h, bruns l, cameron fj, cotterill am, couper jj, et al. the australasian diabetes data network: first national audit of children and adolescents with type 1 diabetes. med. j. austr. 2017;206(3):121-5. 30. daneman d. assessment of intakes of artificial sweeteners in children with type 1 diabetes mellitus. can. j. diab. 2004;28(2):00-. 31. betts p, mulligan j, ward p, smith b, wilkin t. increasing body weight predicts the earlier onset of insulin‐dependant diabetes in childhood: testing the ‘accelerator hypothesis’(2). diab. med. 2005;22(2):144-51. 32. delahanty lm, halford bn. the role of diet behaviors in achieving improved glycemic control in intensively treated patients in the diabetes control and complications trial. diab. care. 1993;16(11):1453-8. 33. abd-alrazak om, ghalib ba, abduljabbar ha. growth indices among children and adolescents with type 1 diabetes-baghdad–iraq, 2013. j. facult. med. 2014;56(3):258-63. 34. cardwell c, patterson c, allen m, carson d. diabetes care provision and glycaemic control in northern ireland: a uk regional audit. arch dis. childhood. 2005;90(5):468-73. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.02202003 234 j contemp med sci | vol. 5, no. 5, september-october 2019: 234–241 review alcohol consumption and types of cancer: a review mohammad afshar,a omid otroshi,b elahe elahian,c shahab papi,d razieh aalipour,e mohammad pourebrahimi,f ladan fattah moghaddam,g morteza kameli morandini,h and hamid salehiniyai,j adepartment of operating room, faculty of nursing and midwifery, kashan university of medical sciences, kashan, iran. bschool of medicine, kashan university of medical sciences, kashan, iran. cdepartment of nursing, borujen faculty of nursing, shahrekord university of medical sciences, shahrekord, iran. ddepartment of health education and promotion, faculty of health, tabriz university of medical sciences, tabriz, iran. edepartment of gerontology, university of social welfare and rehabilitation sciences, tehran, iran. fnursing department, university of social welfare and rehabilitation sciences, tehran, iran. gdepartment of nursing, faculty of nursing and midwifery, tehran medical sciences, islamic azad university, tehran, iran. hdepartment of nursing, faculty of medicine, sari branch, islamic azad university, sari, iran. isocial determinants of health research center, birjand university of medical sciences, birjand, iran. jdepartment of epidemiology and biostatistics, tehran university of medical sciences, tehran, iran. correspondence to hamid salehiniya (email: alesaleh70@yahoo.com). (submitted: 12 april 2019 – revised version received: 28 april 2019 – accepted: 10 may 2019 – published online: 26 october 2019) introduction alcohol is one of the most preventable causes of injury and mortality in the world and is one of the major risk factors for diseases.1,2 across the world, adult alcohol consumption has been around 38% over the past 12 months.3 according to a study published in 2011, alcohol accounts for 4% of cancers in the uk.4 most importantly, alcohol is a major risk factor for cancer. evidence related to the role of alcohol in cancer related to the early 20th century.5 epidemiologic studies and numerous meta-analysis have supported this relationship.5 so, the international agency for research on cancer (iarc) in 1988 categorized alcohol as a group one of carcinogens (the highest level in terms of risk).6 after the digestion and processing of alcohol in the body, it is changed to a kind of chemical called acetaldehyde, which is the group one carcinogen2,8 and prevents dna repair, subsequently increases the risk of cancer.7 several epidemiological studies have examined the relationship between alcohol drinking and the risk of developing cancer. based on findings from epidemiological studies, alcohol consumption elevates the risk of various cancers such as breast and intestine8,9 and is one of the main risk factors for liver malignancies.10 in 2007, the iarc concluded that there is ample evidence that alcohol causes cancer of the mouth, throat, esophagus, larynx, and colorectal.11 in some malignancies, such as prostate cancer, this relationship is dosedependent.12 in some other tumors, even taking a small amount of alcohol can increase the risk of cancer; as an illustration, it is associated with colon cancer for every two units of alcohol per day (each unit is 10 ml or 8 g) that risk of malignancy is expanded by 9%.14 however, the findings of previous studies suggested that alcohol could reduce the risk of non-hodgkin’s lymphoma (nhl), thyroid cancer and renal cell carcinoma, but the findings of the studies were inconsistent.15–17 so far, a large prospective study has examined the relationship between alcohol consumption and cancer risk. however, due to the low number of cases and the existence of contradictory results, in most types of cancers no definitive findings have been reported and the epidemiological evidence that systematically addressed is limited. therefore, this study aimed to investigate the relationship between alcohol consumption and common cancers of various organs of the body, taking into account possible confounding variables for each organ based on existing evidence and review of the literature. materials and methods this study was conducted in english by february 2019 to include epidemiological evidence from all available randomized control trials, case-control and cohort studies reporting alcohol consumption related cancer risks through a search in data bases of the pubmed, scopus and web of science. the search strategy included the keywords: “cancer”, objective the purpose of this review study was to investigate the association between alcohol consumption and common cancers. methods this study was conducted in english by february 2019 to include studies reporting alcohol consumption related cancer risks through a search in data bases of the pubmed, scopus and web of science. the search strategy included the keywords: “cancer”, “alcohol consumption or alcohol drinking or underage drinking”. articles that looked at the relationship between each type of cancer and consumption of alcoholic beverages were entered in to the study and summarized in review. results alcohol consumption is associated with a decreased risk of some types of cancers including: renal cell carcinoma and non-hodgkin lymphoma. also, alcohol is independent risk factor for oral and pharyngeal, laryngeal, esophagus, stomach, colorectal, breast and liver cancer. however, further studies are required to confirm the association between alcohol consumption and pancreas, lung, prostate, endometrium, brain tumor and bladder cancer risks. conclusion given the role of excessive alcohol consumption in the occurrence of various types of cancers, there is a need for a comprehensive plan for alcohol abuse in the community. keywords cancer, alcohol consumption, underage drinking, alcohol drinking issn 2413-0516 235j contemp med sci | vol. 5, no. 5, september-october 2019: 234–241 m. afshar et al. review alcohol consumption and types of cancer: a review “alcohol consumption or alcohol drinking or underage drinking”. in addition, the reference lists of relevant articles were manually searched to find any other potentially eligible articles. articles about alcohol consumption amount and types of alcohol beverage are also included in the present study. we excluded reviews, commentaries, articles from overlapping samples, conference abstracts, and articles printed in languages other than english. articles that looked at the relationship between each type of cancer and alcohol consumption were entered in to the study and summarized in review. results study characteristics in the initial electronic literature search, 2236 articles were obtained from data bases and 43 articles were obtained using manual search. after removing duplicates using endnote x7 (n = 1279), the title and abstract of the remaining 1000 articles were reviewed. after this stage, 166 articles were included in the study and 38 of these articles were removed because of scientific reasons and lack of eligible criteria or unrelated to our aim, in all, 128 full papers were reviewed. the most important cancers related to alcohol consumption are summarized in table 1. types of alcohol-related cancers oral and pharyngeal cancer the findings of a review study between 1988 and 2009 indicated that alcohol consumption is a risk factor for oral and throat cancers, and if combined with tobacco, this effect is multiplied and synergist. on the other hand, the findings revealed that even after controlling for smoking, alcohol consumption was still a risk factor for oral and thymus cancer.18 meta-analysis study findings also showed that the risk of oral and throat cancer in alcoholics is dose-dependent.19 the findings of two meta-analysis studies of more than 30 studies with 14,000 cases showed a significant relationship between alcohol intake and the risk of oral and pharyngeal cancer (opc), depending on the dose (relative risks, rrs, increasing from 1.29 for 10 g ethanol/day to 13.02 for 125 g ethanol/day).19 there is a higher rr for cancer of the throat compared with oral cancer, especially in heavy dose.20 it is estimated that 30% of all opcs worldwide are attributed to alcohol consumption.21 findings from several cohort studies in the united states, europe and asia24–22 reported increased risk of oral and throat cancer in alcohol users. adami et al.25 found in a cohort study in sweden that the risk of oral and throat cancer in alcoholics was four times higher than that of non-alcoholic people (95% ci: 2.9–5.6). tønnesen et al.26 also reported that the risk of oral and thoracic cancers in alcoholic men was 3.5 times higher than that of non-alcoholic men (95% ci: 3.0–4.3) and in alcoholic women was 15 times less than non-alcoholic women (95% ci: 10.8–26.0). in oral and thymus cancer the harmful effects of alcohol in both the intracellular and extracellular levels is observed. in this regard, carcinogens can affect the proliferative activity of the stem cells in basal layer. in patients with oral and thoracic cancer, there is a significant increase in salivary acetaldehyde concentration, which can be related to smoking and poor oral health. acetaldehyde production can also be increased by alcohol intake through bacterial fluoride.27 laryngeal cancer the association between receiving alcoholic drinks and the spread of laryngeal cancer was first reported in the early 1900s through clinical reports and mortality statistics, and then confirmed by ecological studies.28–30 several studies have confirmed the role of alcohol in laryngeal cancer.31,32 the findings of a meta-analysis study in north america, europe, japan, and korea also showed that the risk of laryngeal cancer is significantly elevated if more than one unit of alcohol per day is received.33 this risk also increases in non-smokers.34 drinking alcohol through direct contact or solvent action affects laryngeal cancer.35 it is estimated that between 50% and 70% of all esophageal cancer cases are attributed to alcohol in both sexes.36 ethanol changes the motility and tone of the lower esophagus, the gastric reflux to the esophagus after esophagitis.37 this condition increases the risk of barrett’s esophagus or intestinal metaplasia, dysplasia and adenocarcinoma. chronic inflammation causes more esophagus mucosal damage to nitrosamines and polycyclic aromatic carbohydrates. these ingredients are present in alcoholic drinks.38 the relationship between alcohol consumption and the risk of esophageal cancer in various studies (prospective and case-control) has been reported worldwide.39,40 in the most recent evaluation by iarc, ethanol in alcoholic drinks has been grouped as one of the carcinogens for humans.41 in a second report published by the american cancer research association, alcohol was identified as one of the escalating causes of esophageal cancer.42 therefore, drinking alcohol is known as one of the risk factors for esophageal cancer. the risk of esophageal malignancy is dose dependent.1,43,44 when alcohol is consumed with smoking, the relative risk (rr) of the disease increases significantly. in case of 20 cigarettes per day with one to four units per day, rr increases by 1.5 times. by taking four to eight units per table 1. relationship between alcohol consumption and cancer types cancer types risk factor protective controversial oral and pharyngeal cancer * laryngeal cancer * esophagus * stomach * colorectal * liver * pancreas * lung * prostate * bladder * breast * endometrium * renal cell carcinoma * brain tumor * non-hodgkin lymphoma * 236 j contemp med sci | vol. 5, no. 5, september-october 2019: 234–241 alcohol consumption and types of cancer: a review review m. afshar et al. day, rr increases to 12.3; and if you take more than eight units per day, rr will increase 44.4-fold.45 given that most alcoholics, addicts to tobacco, the synergistic effects of this compound are very important.43 the findings of a meta analysis study showed that alcohol consumption could increase the risk of gastric cancer with odds ratio (or) of 39/1 (95% ci: 1.20–1.61), even in the case of low alcohol consumption.46 findings from several recent studies have reported that drinking alcohol can increase the risk of gastric cancer, and its main mechanism is probably related to primary metabolites and stalled individuals, which has a topical toxic effect and soars the risk of gastric cancer. stomach the findings of a meta-analysis study revealed that alcohol consumption even in the case of low dose could increase the risk of gastric cancer with odds ratio (or) of 39/1 (95% ci: 1.20–1.61).46 findings from several recent studies have reported that drinking alcohol can raise the risk of gastric cancer, and its main mechanism is probably related to primary metabolites and acetaldehydes, which has a topical toxic effect and increases the risk of gastric cancer.47,48 the relationship between drinking alcohol and the risk of gastric cancer is biologically plausible; ethanol is one of the soluble fats and may cause damage to the stomach mucosa. acetaldehyde metabolite may have a topical toxic effect that may be related to gastric cancer.49 ethanol pathogenesis is associated with gastric mucosal injury with impairment in the balance of gastric mucus defense and external invasion.50,51 colorectal several epidemiological studies have shown that increasing alcohol consumption is one of the risk factors for colorectal cancer. the findings of the review and meta-analysis studies8,52–54 and several meta-analysis surveys on case and control cohort studies indicate that alcohol intake is associated with an elevation in colorectal cancer.55,56 the findings of several meta-analysis studies showed a positive relationship between alcohol consumption and colorectal cancer, and this relationship is dose-dependent.43,57,58 several studies also found that with a chronic consumption of about 50 g per day of alcohol, the relative risk of colon cancer was 10–20%.43,59 the findings of dashti et al.60 revealed that alcohol consumption, especially more than 28 g per day of ethanol, is associated with colon cancer. liver several studies in various countries have examined the relationship between alcohol consumption and the risk of developing liver cancer (also called hcc) and have confirmed this relationship.61–63 based on systematic and meta-analytic findings, the relative risk of liver cancer for low levels, hazardous and harmful of alcohol consumption, as compared with those who have never consumed alcohol, is respectively 1.45%, 0.33%, and 3.60% (low consumption = 0–19.90 g for females and 039.99 g for males, pure alcohol per day), hazardous consumption = (females 20–39.99 g, males 40–59.99 g), harmful consumption = (females 40+ g, males 60+ g).64 the main role of gastric cancer following alcohol using is acetaldehyde produced by bacterial flora.43 the findings from a meta-analysis study on 19 cohort studies estimated an increase of 16% in liver cancer among those who consumed at least three units of alcohol daily, compared with non-infected individuals.65 in fact, in alcoholic people, prolonged and severe alcohol consumption leads to alcoholic cirrhosis, which is a pathogenic stage in liver carcinogenesis.66 along with the carcinogenicity of acetaldehyde, which is the first metabolite of alcohol, other biological mechanisms also explain the effect of alcohol consumption on hepatocarcinogenesis. these include chronic inflammation, the consequences of increased oxidative stress, the induction of cytochrome p-450 2e1, lipid peroxidation and dna damage, diminished antioxidant defense and dna repair, reduction of hepatic retinoic acid, excess iron loading, and immune deficiency.67 pancreas findings from a prospective study in the united states revealed that alcohol consumption, in particular consumption liquor, at least three units per day increased pancreatic cancer independent of smoking.68 although the findings of some cohort studies62,69,70 and three meta-analysis studies71–73 showed that there is a positive and significant relationship between alcohol consumption and the risk of pancreatic cancer, other cohort studies72,74,75 did not reveal this relationship. heavy alcohol consumption causes acute and chronic pancreatitis, but has never been associated with liver cancer.68 findings from a meta-analysis study in 2016 demonstrated that low to moderate alcohol intake did not significantly correlate with the risk of pancreatic cancer, while receiving high amounts of alcohol as well as alcohol drinks increase the risk of pancreatic cancer.76 several biological mechanisms have been proposed to explain the increased risk of pancreatic cancer following heavy alcohol use. alcohol can cause to inflammatory responses that would lead to overt chronic pancreatitis or diabetes mellitus by inducing mitogenic stimulation.77,78 alcohol consumption also causes asymptomatic chronic pancreatitis, which eventually results in pancreatic cancer,79–81 although the prevalence of pancreatitis history in the total population of patients with pancreatic cancer is very low. lung an elevated risk of lung cancer in the general population has been reported in numerous cohort studies.43 the findings of the study, korte et al.,82 revealed that for doses greater than five units per day, the relative risk was between 1.53 and 1.88. there is a positive correlation between alcohol consumption and lung cancer in several reported studies.83–85 however, the protective effect of low to moderate alcohol in reducing the incidence of lung cancer has been reported in several studies.85–87 rohrmann et al.86 found that the antiinflammatory, antioxidant, and anti-mutagenic effects of intermediate ethanol produce protective effects. although this protective effect is only specific for lung cancer and is not observed for other neoplasms.88 prostate over the past few decades, a number of overviews and meta-analysis have examined the relationship between prostate cancer and alcohol consumption.89–92 according to the findings of longnecker91 and morton et al.,90 there was no association between alcohol consumption and prostate cancer. breslow and weed93 reviewed 32 studies and reported only six studies that found association between alcohol consumption and prostate cancer. 237j contemp med sci | vol. 5, no. 5, september-october 2019: 234–241 m. afshar et al. review alcohol consumption and types of cancer: a review the findings of dennis’s93 meta-analysis on six cohorts and 27 cases-control did not confirm this relationship. dagnelie et al.89 reviewed nine prostate cancer and alcohol studies, and found that in six studies, such a relationship was not confirmed; in two studies, the risk was increased and decreased in one study. the findings of the meta-analysis88 showed that the small, but significant risk of prostate cancer in men who consumed more than 50 g of alcohol per day was observed at a higher risk for men who consumed more than 100 g per day of alcohol, but there was no significant dose– response relation. this study first considered potential confounders, between the study variables and the moderating effects of tobacco use. findings from middleton fillmore et al.92 revealed that there was a significant relationship between heavy alcohol consumption after controlling the effects of age, study population, design, and study variables. rota et al.95 obtained a significantly higher rr of prostate cancer for each drinking alcohol, low (less than or equal to one drink per day) and moderate (greater than one and less than four drink per day) versus abstaining or occasionally alcohol consumption, but the analysis did not show a meaningful relationship with heavy drinking (greater than or equal to four drinks per day). overall, the findings of recent meta-analysis and review studies showed a positive relationship, but none of the studies sufficiently controlled the effects of confounding variables, including incorrect categorization of former alcoholics and occasional alcoholics. bladder the role of alcoholic drinks in bladder carcinogenesis has been studied in several epidemiologic studies.96,97 according to framingham heart study,98 there was no significant relationship between alcohol consumption and bladder cancer. however, the findings of a meta-analysis study of ureteric cancers showed a slight increase of 1.3% for current alcoholic-drinkers compared with non-alcoholic ones.99 the findings of specific drinks analyze also revealed uncertain results. some studies have depicted a higher risk for liquor drinks98–101 and showed an inverse relationship to beer drink.98,102 the average consumption of alcoholic drinks can prevent from coronary artery disease, diabetes mellitus,103,104 renal cell carcinoma,105,106 prostate cancer,107 and nhl108 may be due to mechanisms such as improves immune response and increases insulin sensitivity. one of the possible mechanisms of the protective effect of alcohol consumption in bladder cancer is the urogenous contact hypothesis.109–111 meta-analysis study findings showed that drinking beer and wine reduces the risk of bladder cancer.112 other mechanisms include anti-inflammatory properties of alcohol96,113 and anticholinergic effects of polyphenols in red wine114 and beer. despite the publication of numerous studies in this regard, the role of definitive alcohol on bladder cancer, the effect of various types of alcoholic drinks, patterns of drinking and the role of other factors such as smoking and the frequency of urination for bladder cancer have not yet been proven. breast about 4–5% of all breast cancers are associated with alcohol consumption, and 60% of alcohol-related tumors in women are breast cancer.118 several epidemiological studies have shown a positive relationship between alcohol consumption and breast cancer.119,120 this risk occurs even with the average consumption of alcohol. for increasing the amount of ethanol consumed daily by 10 g, rr growth by 10%.119,121 kwan et al.122 found that the risk of recurrence of breast cancer significantly increased in women who had been breast cancer before (especially after menstruation), taking three to four times per week. systematic study findings showed that alcohol consumption could extend the risk of breast cancer, especially in postmenopausal women.123 the pathogenic mechanism of the disease was certainly due to oxidative stress induced by acetaldehyde and nutritional changes (folate, vitamin b6 and b12), but the main cause of it is interaction with estrogen. it is known that estrogen is metabolized by adh and therefore acts in competition with ethanol. especially high concentrations of acetaldehyde are associated with high levels of estrogen during the menstrual cycle.43 endometrium a cohort study found that alcohol consumption of two units or more could increase the risk of endometrial cancer in women after menstruation. however, there was no such relationship in people who consume less than one drink or one to two drinks per day.124 the role of unopposed estrogens in endometrial cancer etiology has been confirmed.125 daily use of alcohol is associated with high levels of circulating estrogen in postmenopausal women.126–128 alcohol consumption also raise the estrogen levels in postmenopausal women with estrogen replacement therapy.129,130 therefore, it is likely that women drinking alcoholic drinks are at enhanced risk for endometrial cancer. however, the findings of a prospective study between the 1980s and 2010 on 6807 women who participated in the study found that drinking <5 g daily (about half a day) would reduce the risk of endometrial cancer by 22% (multivariable rr = 0.78; 95% ci: 0.66–0.94). getting more alcohol does not; however, produce a protective effect against endometrial cancer [multivariable rrs for 5–14.9 g (one drink), 15–29.9 g (two drinks), or ≥30 g (two drinks)] versus 0 g per day were 0.88, 0.83, and 0.78 (95% ci: 0.49–1.25), respectively.131 renal colon carcinoma the implications of a meta-analysis study on 15 case-control studies revealed an inverse relationship between alcohol consumption and renal cell carcinoma (or 0.67, 95% ci: 0.62–0.73). the dose–response meta-analysis manifested that an increase in alcohol consumption of 12 g of ethanol per day was associated with a significant reduction of 5% risk of renal cell carcinoma.132 other study findings from 12 prospective studies also indicated an inverse relationship between alcohol consumption and renal cell carcinoma.133 the findings of another meta-analysis study in 2012 showed an inverse relationship between alcohol consumption and renal cell carcinoma in both genders. it was also observed that drinking one drink per day has protective effects, but drinking too much does not have protective effects.134 brain tumor alcohol is able to cross the cerebrospinal fluid and is therefore a risk factor for brain cancer.135,136 ethanol is oxidized to acetaldehyde which is a genotoxic metabolite in the brain.137 alcohol consumption is in relation to the risk of brain cancer 238 j contemp med sci | vol. 5, no. 5, september-october 2019: 234–241 alcohol consumption and types of cancer: a review review m. afshar et al. in adults has been studied in a number of studies from the early 1970s138 and usually inconsistent findings revealed in both general use and for various types of alcoholic drinks. for instance, the findings of a cohort study on glioblastoma, which is a type of brain cancer with a low survival rate, showed that there is a dose risk with increasing alcohol consumption for this cancer.139 this finding was similar for beer and wine. on the other hand, according to a study of 908 cases of brain cancer, there was no correlation between alcohol consumption and brain cancer, and a relative risk of 1.17 reported for taking more than 15 drinks per week compared with drinking occasionally (for example less than two drinks per week).140 systematic and meta-analytical findings from 19 studies involving 4200 cases of brain cancer suggest that there is no significant relationship between alcohol consumption and adult brain cancer, although further studies are needed to investigate the potential effects of high doses of alcohol.141 findings of another meta-analysis study on 19 observational studies showed that there is no significant relationship between alcohol consumption and the risk of glioma.142 non-hodgkin’s lymphoma according to the monograph 96 of the iarc, published in 2007,135 the risk of nhl in alcohol users was lower than those who did not consume alcohol. similar results were obtained by the next iarc.143 in addition, the world cancer research fund (wcrf) reported that there was an inverse relationship between drinking alcohol and nhl.143 the results of a meta-analysis study manifested that the risk of nhl among alcohol users is 15% less than those who do not consumed alcohol.144 conclusion the purpose of this review study was to investigate the association between alcohol consumption and the incidence of common cancers. based on the results of studies, alcohol is associated with a reduction in the risk of renal cell carcinoma and nhl. conversely, alcohol is a risk factor for oral and thoracic cancers, larynx, esophagus, stomach, colorectal, liver and breast. however, further studies are needed to confirm definitively the association between alcohol and the development of pancreatic, lung, prostate, bladder, endometrial and brain tumors. given the role of excessive alcohol consumption in the occurrence of various types of cancers, there is a necessity for planning a comprehensive project for alcohol abuse in the community. conflicts of interest none.  references 1. rehm j, mathers c, popova s, thavorncharoensap m, teerawattananon y, patra j. global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders. lancet. 2009;373:2223–2233. 2. ezzati m, lopez ad, rodgers a, vander hoorn s, murray cj, comparative risk assessment collaborating group. selected major risk factors and global and regional burden of disease. lancet. 2002;360:1347–1360. 3. world health organization, unit whomosa. global status report on alcohol and health 2014. world health organization, geneva, 2014. 4. leitzmann mf, koebnick c, freedman nd, park y, ballard-barbash r, hollenbeck ar, et al. physical activity and head and neck cancer risk. cancer causes control. 2008;19:1391–1399. 5. newsholme a. the possible association of the consumption of alcohol with excessive mortality from cancer. br med j. 1903;2:1529–1531. 6. mcguire s. world cancer report 2014. geneva, switzerland: world health organization, international agency for research on cancer, who press, 2015. adv nutr. 2016;7:418–419. 7. iarc working group on the evaluation of carcinogenic risks to humans. personal habits and indoor combustions. volume100 e. a review of human carcinogens. iarc monogr eval carcinog risks hum. 2012;100:1–538. 8. corrao g, bagnardi v, zambon a, la vecchia c. a meta-analysis of alcohol consumption and the risk of 15 diseases. prev med. 2004;38:613–9. 9. fedirko v, tramacere i, bagnardi v, rota m, scotti l, islami f, et al. alcohol drinking and colorectal cancer risk: an overall and dose-response metaanalysis of published studies. ann oncol. 2011;22:1958–1972. 10. stickel f, schuppan d, hahn e, seitz h. cocarcinogenic effects of alcohol in hepatocarcinogenesis. gut. 2002;51:132–9. 11. jemal a, bray f, center mm, ferlay j, ward e, forman d. global cancer statistics. ca cancer j clin. 2011;61:69–90. 12. zhao j, stockwell t, roemer a, chikritzhs t. is alcohol consumption a risk factor for prostate cancer? a systematic review and meta-analysis. bmc cancer. 2016;16:845. 13. burton r, henn c, lavoie d, o’connor r, perkins c, sweeney k, et al. a rapid evidence review of the effectiveness and cost-effectiveness of alcohol control policies: an english perspective. lancet. 2017;389: 1558–1580. 14. ferrari p, jenab m, norat t, moskal a, slimani n, olsen a, et al. lifetime and baseline alcohol intake and risk of colon and rectal cancers in the european prospective investigation into cancer and nutrition (epic). int j cancer 2007;121:2065–2072. 15. world health organization. global strategy on diet, physical activity and health. world health organization, geneva, 2004. 16. phongsavan p, merom d, marshall a, bauman a. estimating physical activity level: the role of domestic activities. j epidemiol community health 2004;58:466–467. 17. levi f, la vecchia c, negri e, franceschi s. selected physical activities and the risk of endometrial cancer. br j cancer. 1993;67:846–851. 18. goldstein by, chang sc, hashibe m, la vecchia c, zhang zf. alcohol consumption and cancer of the oral cavity and pharynx from 1988 to 2009: an update. eur j cancer prev. 2010;19:431–465. 19. tramacere i, negri e, bagnardi v, garavello w, rota m, scotti l, et al. a metaanalysis of alcohol drinking and oral and pharyngeal cancers. part 1: overall results and dose-risk relation. oral oncol. 2010;46:497–503. 20. turati f, garavello w, tramacere i, bagnardi v, rota m, scotti l, et al. a metaanalysis of alcohol drinking and oral and pharyngeal cancers. part 2: results by subsites. oral oncol. 2010;46:720–726. 21. turati f, garavello w, tramacere i, pelucchi c, galeone c, bagnardi v, et al. a meta-analysis of alcohol drinking and oral and pharyngeal cancers: results from subgroup analyses. alcohol alcohol. 2013;48:107–118. 22. boeing h, epic working group on dietary patterns. alcohol and risk of cancer of the upper gastrointestinal tract: first analysis of the epic data. iarc sci publ. 2002;156:151–154. 23. boffetta p, garfinkel l. alcohol drinking and mortality among men enrolled in an american cancer society prospective study. epidemiology. 1990;1:342–348. 24. murata m, takayama k, choi bc, pak aw. a nested case-control study on alcohol drinking, tobacco smoking, and cancer. cancer detect prev. 1996;20:557–565. 25. adami ho, mclaughlin jk, hsing aw, wolk a, ekbom a, holmberg l, et al. alcoholism and cancer risk: a population-based cohort study. cancer causes control. 1992;3:419–425. 26. tønnesen h, møller h, andersen jr, jensen e, juel k. cancer morbidity in alcohol abusers. br j cancer. 1994;69:327–332. 27. seitz hk, meier p. the role of acetaldehyde in upper digestive tract cancer in alcoholics. transl res. 2007;149:293–297. 28. wynder el. some practical aspects of cancer prevention. n engl j med. 1952;246:492–502. 29. tuyns a. incidence trends of laryngeal cancer in relation to national alcohol and tobacco consumption. in: magnus k, ed., trends in cancer incidence causes and practical implications. washington dc, hemisphere, pp. 199–214. 30. devesa ss, blot wj, fraumeni jf. cohort trends in mortality from oral, esophageal, and laryngeal cancers in the united states. epidemiology. 1990;1:116–121. 239j contemp med sci | vol. 5, no. 5, september-october 2019: 234–241 m. afshar et al. review alcohol consumption and types of cancer: a review 31. iarc working group on the evaluation of carcinogenic risks to humans. alcohol consumption and ethyl carbamate. iarc monogr eval carcinog risks hum. 2010;96:3–1383. 32. pflaum t, hausler t, baumung c, ackermann s, kuballa t, rehm j, et al. carcinogenic compounds in alcoholic beverages: an update. arch toxicol. 2016;90:2349–2367. 33. islami f, tramacere i, rota m, bagnardi v, fedirko v, scotti l, et al. alcohol drinking and laryngeal cancer: overall and dose-risk relation—a systematic review and meta-analysis. oral oncol. 2010;46:802–810. 34. hashibe m, brennan p, benhamou s, castellsague x, chen c, curado mp, et al. alcohol drinking in never users of tobacco, cigarette smoking in never drinkers, and the risk of head and neck cancer: pooled analysis in the international head and neck cancer epidemiology consortium. j natl cancer inst. 2007;99:777–789. 35. altieri a, garavello w, bosetti c, gallus s, la vecchia c. alcohol consumption and risk of laryngeal cancer. oral oncol. 2005;41:956–965. 36. popkin bm, armstrong le, bray gm, caballero b, frei b, willett wc. a new proposed guidance system for beverage consumption in the united states. am j clin nutr. 2006;83:529–542. 37. goldenberg d, golz a, joachims hz. the beverage mate: a risk factor for cancer of the head and neck. head neck. 2003;25:595–601. 38. brooks pj, enoch ma, goldman d, li tk, yokoyama a. the alcohol flushing response: an unrecognized risk factor for esophageal cancer from alcohol consumption. plos med. 2009;6:e50. 39. bagnardi v, blangiardo m, la vecchia c, corrao g. alcohol consumption and the risk of cancer: a meta-analysis. alcohol res health. 2001;25:263–270. 40. oze i, matsuo k, wakai k, nagata c, mizoue t, tanaka k, et al. alcohol drinking and esophageal cancer risk: an evaluation based on a systematic review of epidemiologic evidence among the japanese population. jpn j clin oncol. 2011;41:677–692. 41. baan r, straif k, grosse y, secretan b, el ghissassi f, bouvard v, et al. carcinogenicity of alcoholic beverages. lancet oncol. 2007;8:292–293. 42. world cancer research fund and american institute for cancer research. food, nutrition and the prevention of cancer: a global perspective. world cancer research fund, washington, dc, 1997. 43. testino g. the burden of cancer attributable to alcohol consumption. maedica (buchar) 2011;6:313–320. 44. bouvard v, baan r, straif k, grosse y, secretan b, el ghissassi f, et al. a review of human carcinogens—part b: biological agents. lancet oncol. 2009;10:321–322. 45. franke a, teyssen s, singer mv. alcohol-related diseases of the esophagus and stomach. dig dis. 2005;23:204–213. 46. ma k, baloch z, he tt, xia x. alcohol consumption and gastric cancer risk: a meta-analysis. med sci monit. 2017;23:238–246. 47. brzozowski t, konturek pc, konturek sj, kwiecień s, sliwowski z, pajdo r, et al. implications of reactive oxygen species and cytokines in gastroprotection against stress-induced gastric damage by nitric oxide releasing aspirin. int j colorectal dis. 2003;18:320–329. 48. kwiecień s, brzozowski t, konturek sj. effects of reactive oxygen species action on gastric mucosa in various models of mucosal injury. j physiol pharmacol. 2002;53:39–50. 49. he h, chi j. a case-control study of smoking-alcohol consumption and the occurrence of stomach cancer. chinese j dis control. 2012;8:10–20. 50. wu ah, wan p, bernstein l. a multiethnic population-based study of smoking, alcohol and body size and risk of adenocarcinomas of the stomach and esophagus (united states). cancer causes control. 2001;12:721–732. 51. freedman nd, abnet cc, leitzmann mf, mouw t, subar af, hollenbeck ar, et al. a prospective study of tobacco, alcohol, and the risk of esophageal and gastric cancer subtypes. am j epidemiol. 2007;165:1424–1433. 52. kune ga, vitetta l. alcohol consumption and the etiology of colorectal cancer: a review of the scientific evidence from1957 to 1991. nutr cancer 1992;18:97–111. 53. trock b, lanza e, greenwald p. dietary fiber, vegetables, and colon cancer: critical review and meta-analyses of the epidemiologic evidence. j natl cancer inst. 1990;82:650–661. 54. bagnardi v, rota m, botteri e, tramacere i, islami f, fedirko v, et al. alcohol consumption and site-specific cancer risk: a comprehensive dose-response meta-analysis. br j cancer. 2015;112:580–593. 55. longnecker mp, orza mj, adams me, vioque j, chalmers tc. a meta-analysis of alcoholic beverage consumption in relation to risk of colorectal cancer. cancer causes control. 1990;1:59–68. 56. berlin ja, longnecker mp, greenland s. meta-analysis of epidemiologic dose-response data. epidemiology. 1993;4:218–228. 57. seitz hk, stickel f. molecular mechanisms of alcohol-mediated carcinogenesis. nat rev cancer 2007;7:599–612. 58. wang y, duan h, yang h, lin j. a pooled analysis of alcohol intake and colorectal cancer. int j clin exp med. 2015;8:6878–6889. 59. higginson j, muir cs, muñoz n. human cancer: epidemiology and environmental causes. cambridge university press, new york, ny, 1992. 60. dashti sg, buchanan dd, jayasekara h, ait ouakrim d, clendenning m, rosty c, et al. alcohol consumption and the risk of colorectal cancer for mismatch repair gene mutation carriers. cancer epidemiol biomarkers prev. 2017;26:366–375. 61. yu mc, mack t, hanisch r, peters rl, henderson be, pike mc. hepatitis, alcohol consumption, cigarette smoking, and hepatocellular carcinoma in los angeles. cancer res. 1983;43:6077–6079. 62. austin h, delzell e, grufferman s, levine r, morrison as, stolley pd, et al. a case-control study of hepatocellular carcinoma and the hepatitis b virus, cigarette smoking, and alcohol consumption. cancer res. 1986;46:962–966. 63. wang ly, you sl, lu sn, ho hc, wu mh, sun ca, et al. risk of hepatocellular carcinoma and habits of alcohol drinking, betel quid chewing and cigarette smoking: a cohort of 2416 hbsag-seropositive and 9421 hbsagseronegative male residents in taiwan. cancer causes control. 2003;14: 241–250. 64. gutjahr e, gmel g, rehm j. relation between average alcohol consumption and disease: an overview. eur addict res. 2001;7:117–127. 65. turati f, galeone c, rota m, pelucchi c, negri e, bagnardi v, et al. alcohol and liver cancer: a systematic review and meta-analysis of prospective studies. ann oncol. 2014;25:1526–1535. 66. la vecchia c, negri e, la vecchia c, franceschi s. liver cirrhosis and the risk of primary liver cancer. eur j cancer prev. 1998;7:315–320. 67. seitz hk, stickel f. risk factors and mechanisms of hepatocarcinogenesis with special emphasis on alcohol and oxidative stress. biol chem. 2006;387:349–360. 68. gapstur sm, jacobs ej, deka a, mccullough ml, patel av, thun mj. association of alcohol intake with pancreatic cancer mortality in never smokers. arch intern med. 2011;171:444–451. 69. hirayama t. epidemiology of pancreatic cancer in japan. japanese j clin oncol. 1989;19:208–215. 70. heinen mm, verhage ba, ambergen ta, goldbohm ra, van den brandt pa. alcohol consumption and risk of pancreatic cancer in the netherlands cohort study. am j epidemiol. 2009;169:1233–1242. 71. genkinger jm, spiegelman d, anderson ke, bergkvist l, bernstein l, van den brandt pa, et al. alcohol intake and pancreatic cancer risk: a pooled analysis of fourteen cohort studies. cancer epidemiol biomarkers prev. 2009;18: 765–776. 72. michaud ds, vrieling a, jiao l, mendelsohn jb, steplowski e, lynch sm, et al. alcohol intake and pancreatic cancer: a pooled analysis from the pancreatic cancer cohort consortium (panscan). cancer causes control. 2010;21:1213– 1225. 73. tramacere i, scotti l, jenab m, bagnardi v, bellocco r, rota m, et al. alcohol drinking and pancreatic cancer risk: a meta-analysis of the dose-risk relation. int j cancer. 2010;126:1474–1486. 74. shibata a, mack tm, paganini-hill a, ross rk, henderson be. a prospective study of pancreatic cancer in the elderly. int j cancer. 1994;58:46–49. 75. gaziano jm, gaziano ta, glynn rj, sesso hd, ajani ua, stampfer mj, et al. light-to-moderate alcohol consumption and mortality in the physicians’ health study enrollment cohort. j am coll cardiol. 2000;35:96–105. 76. wang yt, gou yw, jin ww, xiao m, fang hy. association between alcohol intake and the risk of pancreatic cancer: a dose-response meta-analysis of cohort studies. bmc cancer. 2016;16:212. 77. go vl, gukovskaya a, pandol sj. alcohol and pancreatic cancer. alcohol. 2005;35:205–211. 78. welsch t, kleeff j, seitz hk, büchler p, friess h, büchler mw. update on pancreatic cancer and alcohol-associated risk. j gastroenterol hepatol. 2006;21:s69–s75. 79. steer ml, waxman i, freedman s. chronic pancreatitis. n engl j med. 1995;332:1482–1490. 80. karlson bm, ekbom a, josefsson s, mclaughlin jk, fraumeni jf, nyrén o. the risk of pancreatic cancer following pancreatitis: an association due to confounding? gastroenterology. 1997;113:587–592. 81. bansal p, sonnenberg a. pancreatitis is a risk factor for pancreatic cancer. gastroenterology. 1995;109:247–251. 82. korte je, brennan p, henley sj, boffetta p. dose-specific meta-analysis and sensitivity analysis of the relation between alcohol consumption and lung cancer risk. am j epidemiol. 2002;155:496–506. 240 j contemp med sci | vol. 5, no. 5, september-october 2019: 234–241 alcohol consumption and types of cancer: a review review m. afshar et al. 83. bagnardi v, randi g, lubin j, consonni d, lam tk, subar af, et al. alcohol consumption and lung cancer risk in the environment and genetics in lung cancer etiology (eagle) study. am j epidemiol. 2010;171:36–44. 84. dosemeci m, gokmen i, unsal m, hayes rb, blair a. tobacco, alcohol use, and risks of laryngeal and lung cancer by subsite and histologic type in turkey. cancer causes control. 1997;8:729–737. 85. prescott e, grønbæk m, becker u, sørensen ti. alcohol intake and the risk of lung cancer: influence of type of alcoholic beverage. am j epidemiol. 1999;149:463–470. 86. rohrmann s, linseisen j, boshuizen hc, whittaker j, agudo a, vineis p, et al. ethanol intake and risk of lung cancer in the european prospective investigation into cancer and nutrition (epic). am j epidemiol. 2006;164:1103–1114. 87. chao c, slezak jm, caan bj, quinn vp. alcoholic beverage intake and risk of lung cancer: the california men’s health study. cancer epidemiol biomarkers prev. 2008;17:2692–2699. 88. bagnardi v, blangiardo m, la vecchia c, corrao g. a meta-analysis of alcohol drinking and cancer risk. br j cancer. 2001;85:1700–1705. 89. dagnelie p, schuurman a, goldbohm r, van den brandt p. diet, anthropometric measures and prostate cancer risk: a review of prospective cohort and intervention studies. bju int. 2004;93:1139–1150. 90. morton ms, griffiths k, blacklock n. the preventive role of diet in prostatic disease. br j urol. 1996;77:481–493. 91. longnecker mp. alcohol consumption and risk of cancer in humans: an overview. alcohol. 1995;12:87–96. 92. middleton fillmore k, chikritzhs t, stockwell t, bostrom a, pascal r. alcohol use and prostate cancer: a meta-analysis. mol nutr food res. 2009;53: 240–255. 93. breslow ra, weed dl. review of epidemiologic studies of alcohol and prostate cancer: 1971–1996. nutr cancer. 1998;30:1–13. 94. dennis lk. meta-analysis for combining relative risks of alcohol consumption and prostate cancer. prostate. 2000;42:56–66. 95. rota m, scotti l, turati f, tramacere i, islami f, bellocco r, et al. alcohol consumption and prostate cancer risk: a meta-analysis of the dose-risk relation. eur j cancer prev. 2012;21:350–359. 96. jiang x, castelao je, groshen s, cortessis vk, ross rk, conti dv, et al. alcohol consumption and risk of bladder cancer in los angeles county. int j cancer. 2007;121:839–845. 97. iarc alcohol drinking. biological data relevant to the evaluation of carcinogenic risk to humans. iarc monogr eval carcinog risks hum. 1988;44:101–152. 98. djoussé l, schatzkin a, chibnik lb, d’agostino rb, kreger be, ellison rc. alcohol consumption and the risk of bladder cancer in the framingham heart study. j natl cancer inst. 2004;96:1397–1400. 99. zeegers mp, tan fe, verhagen ap, weijenberg mp, van den brandt pa. elevated risk of cancer of the urinary tract for alcohol drinkers: a metaanalysis. cancer causes control. 1999;10:445–451. 100. zeegers mp, volovics a, dorant e, goldbohm ra, van den brandt pa. alcohol consumption and bladder cancer risk: results from the netherlands cohort study. am j epidemiol. 2001;153:38–41. 101. chyou ph, nomura am, stemmermann gn. a prospective study of diet, smoking, and lower urinary tract cancer. ann epidemiol. 1993;3:211–216. 102. pelucchi c, negri e, franceschi s, talamini r, la vecchia c. alcohol drinking and bladder cancer. j clin epidemiol. 2002;55:637–641. 103. gunzerath l, faden v, zakhari s, warren k. national institute on alcohol abuse and alcoholism report on moderate drinking. alcohol clin exp res. 2004;28:829–847. 104. beulens jw, stolk rp, van der schouw yt, grobbee de, hendriks hf, bots ml. alcohol consumption and risk of type2 diabetes among older women. diabetes care 2005;28:2933–2938. 105. asal nr, risser dr, kadamani s, geyer jr, lee et, cherng n. risk factors in renal cell carcinoma: i. methodology, demographics, tobacco, beverage use, and obesity. cancer detect prev. 1988;11:359–377. 106. parker as, cerhan jr, lynch cf, ershow ag, cantor kp. gender, alcohol consumption, and renal cell carcinoma. am j epidemiol. 2002;155: 455–462. 107. baglietto l, severi g, english dr, hopper jl, giles gg. alcohol consumption and prostate cancer risk: results from the melbourne collaborative cohort study. int j cancer 2006;119:1501–1504. 108. morton lm, zheng t, holford tr, holly ea, chiu bc, costantini as, et al. alcohol consumption and risk of non-hodgkin lymphoma: a pooled analysis. lancet oncol. 2005;6:469–476. 109. negri e, la vecchia c. epidemiology and prevention of bladder cancer. in: bassi p, pagano f, eds. invasive bladder cancer. springer, london, 2007. pp. 1–14. 110. cohen sm, johansson sl. epidemiology and etiology of bladder cancer. urol clin north am. 1992;19:421–428. 111. michaud ds, spiegelman d, clinton sk, rimm eb, curhan gc, willett wc, et al. fluid intake and the risk of bladder cancer in men. n engl j med. 1999;340:1390–1397. 112. mao q, lin y, zheng x, qin j, yang k, xie l. a meta-analysis of alcohol intake and risk of bladder cancer. cancer causes control. 2010;21: 1843–1850. 113. sierksma a, van der gaag m, kluft c, hendriks h. moderate alcohol consumption reduces plasma c-reactive protein and fibrinogen levels; a randomized, diet-controlled intervention study. eur j clin nutr. 2002;56:1130–1136. 114. luceri c, caderni g, sanna a, dolara p. red wine and black tea polyphenols modulate the expression of cycloxygenase-2, inducible nitric oxide synthase and glutathione-related enzymes in azoxymethane-induced f344 rat colon tumors. j nutr. 2002;132:1376–1379. 115. stevens jf, page je. xanthohumol and related prenylflavonoids from hops and beer: to your good health! phytochemistry. 2004;65:1317–1330. 116. gerhauser c, alt a, heiss e, gamal-eldeen a, klimo k, knauft j, et al. cancer chemopreventive activity of xanthohumol, a natural product derived from hop. mol cancer ther. 2002;1:959–969.8 117. zhao f, nozawa h, daikonnya a, kondo k, kitanaka s. inhibitors of nitric oxide production from hops (humulus lupulus l.). biol pharm bull. 2003;26:61–65. 118. chen wy, rosner b, hankinson se, colditz ga, willett wc. moderate alcohol consumption during adult life, drinking patterns, and breast cancer risk. jama. 2011;306:1884–1890. 119. cancer cgohfib. alcohol, tobacco and breast cancer – collaborative reanalysis of individual data from 53 epidemiological studies, including 58 ,515 women with breast cancer and 95, 067 women without the disease. br j cancer. 2002; 87:1234–1245. 120. smith-warner sa, spiegelman d, yaun ss, van den brandt pa, folsom ar, goldbohm ra, et al. alcohol and breast cancer in women: a pooled analysis of cohort studies. jama 1998;279:535–540. 121. willett wc, stampfer mj, colditz ga, rosner ba, hennekens ch, speizer fe. moderate alcohol consumption and the risk of breast cancer. n engl j med. 1987;316:1174–1180. 122. kwan ml, kushi lh, weltzien e, tam ek, castillo a, sweeney c, et al. alcohol consumption and breast cancer recurrence and survival among women with early-stage breast cancer: the life after cancer epidemiology study. j clin oncol. 2010;28:4410–4416. 123. simapivapan p, boltong a, hodge a. to what extent is alcohol consumption associated with breast cancer recurrence and second primary breast cancer?: a systematic review. cancer treat rev. 2016;50:155–167. 124. setiawan vw, monroe kr, goodman mt, kolonel ln, pike mc, henderson be. alcohol consumption and endometrial cancer risk: the multiethnic cohort. int j cancer. 2008;122:634–638. 125. akhmedkhanov a, zeleniuch-jacquotte a, toniolo p. role of exogenous and endogenous hormones in endometrial cancer: review of the evidence and research perspectives. ann n y acad sci. 2001;943:296–315. 126. gavaler js, van thiel dh. the association between moderate alcoholic beverage consumption and serum estradiol and testosterone levels in normal postmenopausal women: relationship to the literature. alcohol clin exp res. 1992;16:87–92. doi: 10.1111/j.1530-0277.1992.tb00642.x 127. hankinson se, willett wc, manson je, hunter dj, colditz ga, stampfer mj, et al. alcohol, height, and adiposity in relation to estrogen and prolactin levels in postmenopausal women. j natl cancer inst. 1995;87: 1297–1302. 128. onland-moret nc, peeters ph, van der schouw yt, grobbee de, van gils ch. alcohol and endogenous sex steroid levels in postmenopausal women: a cross-sectional study. j clin endocrinol metab. 2005;90: 1414–1419. 129. purohit v. moderate alcohol consumption and estrogen levels in postmenopausal women: a review. alcohol clin exp res. 1998;22:994–997. 130. ginsburg es. estrogen, alcohol and breast cancer risk. j steroid biochem mol biol. 1999;69:299–306. 131. je y, de vivo i, giovannucci e. long-term alcohol intake and risk of endometrial cancer in the nurses’ health study, 1980–2010. br j cancer. 2014;111:186–194. 132. cheng g, xie l. alcohol intake and risk of renal cell carcinoma: a metaanalysis of published case-control studies. arch med sci. 2011;7:648–657. 133. lee je, hunter dj, spiegelman d, adami ho, albanes d, bernstein l, et al. alcohol intake and renal cell cancer in a pooled analysis of 12 prospective studies. j natl cancer inst. 2007;99:801–810 241j contemp med sci | vol. 5, no. 5, september-october 2019: 234–241 m. afshar et al. review alcohol consumption and types of cancer: a review 134. song dy, song s, song y, lee je. alcohol intake and renal cell cancer risk: a meta-analysis. br j cancer 2012;106:1881–1890. 135. international agency for research on cancer. alcohol consumption and ethyl carbamate, vol. 96. international agency for research on cancer, lyon, france, 2010. 136. wrensch m, bondy ml, wiencke j, yost m. environmental risk factors for primary malignant brain tumors: a review. j neurooncol. 1993;17:47–64. 137. deitrich r, zimatkin s, pronko s. oxidation of ethanol in the brain and its consequences. alcohol res health. 2006;29:266–273. 138. choi nw, schuman lm, gullen wh. epidemiology of primary central nervous system neoplasms. ii. case-control study. am j epidemiol. 1970;91:467–485. 139. baglietto l, giles gg, english dr, karahalios a, hopper jl, severi g. alcohol consumption and risk of glioblastoma; evidence from the melbourne collaborative cohort study. int j cancer 2011;128:1929–1934. 140. allen ne, beral v, casabonne d, kan sw, reeves gk, brown a, et al. moderate alcohol intake and cancer incidence in women. j natl cancer inst. 2009;101:296–305. 141. galeone c, malerba s, rota m, bagnardi v, negri e, scotti l, et al. a metaanalysis of alcohol consumption and the risk of brain tumours. ann oncol. 2013;24:514–523. 142. qi zy, shao c, yang c, wang z, hui gz. alcohol consumption and risk of glioma: a meta-analysis of 19 observational studies. nutrients 2014;6: 504–516. 143. secretan b, straif k, baan r, grosse y, el ghissassi f, bouvard v, et al. a review of human carcinogens—part e: tobacco, areca nut, alcohol, coal smoke, and salted fish. lancet oncol. 2009;10:1033–1034. 144. tramacere i, pelucchi c, bonifazi m, bagnardi v, rota m, bellocco r, et al. alcohol drinking and non-hodgkin lymphoma risk: a systematic review and a meta-analysis. ann oncol. 2012;23:2791–2798. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201901 50 j contemp med sci | vol. 6, no. 2, march–april 2020: 50–53 original issn 2413-0516 introduction as with any surgical procedure, the evolution of rhinoplasty is based on improved surgical techniques that survive the test of time and increasing patient expectations.1 aesthetic rhinoplasty is one of the most requested and most demanding facial aesthetic surgical operations nowadays. it is a complex medical procedure, as it is not simply a surgical treatment of a disease but instead involves the alteration of the appearance and characteristics of the nose and the face in general.2 rhinoplasty is arguably one of the most delicate and complex skeletofacial esthetic procedures. its evaluation is also complex and should include the patients’ esthetic goals, nasal function and anatomy and proportion within the face.3 the ventilatory function of the nose must be considered when performing nasal surgery.4 outcomes of any surgical procedure can be measured by quantitative and/or qualitative terms. however, the lack of standardized qualitative assessment makes it difficult to compare objectively the success of different techniques and individual surgeons. nowadays, one of the effective ways to assess the medical procedures is the self-reporting outcomes making it an important factor in clinical trials that’s why, evaluation of quality of life and self-image can be a very good questionnaire to assess the success of any facial aesthetic surgery. because they systemize the gathered information and allow the objective comparison of procedures by measuring pros and cons after the surgery.5 patient satisfaction depends on subjective factors such as patient perception of pre operative appearance, patient expectations, social relationship capacities.6 compared with primary rhinoplasty, revision rhinoplasty is a more challenging surgery because it deals with correction of various deformity of previously operated nose, the anatomy of an operated nose differs completely from its normal counterpart. for this reason, revision surgery requires special attention to anatomy, proper diagnosis, and treatment planning are key to successful revision rhinoplasty and are usually based on psychological evaluation, aesthetic analysis, and functional examination.7 because nasal anatomy can vary significantly between the individuals and artistic quality vary from surgeon to surgeon , beauty comes in many forms, therefore reliable evaluation of face cannot be undertaken by merely taking measurements.8 in 2000, alsarraf et al. created a questionnaire that offered reliability, internal consistency, and validity for several plastic surgeries, including rhinoplasty.9 this questionnaire, the rhinoplasty outcomes evaluation (roe), allowed measure of qualitative aspects such as social, emotional, and psychological. the aim of this study was to use roe preand post-operatively to evaluate the satisfaction of patients who underwent rhinoplasty and to determine the relation with patient characteristics and surgery details. methods this study was approved by kurdistan board for medical speciality prior to initiation. we performed prospective study of all adults who underwent open rhinoplasty between september 2018 and august 2019 in both public and private hospitals (sulaimani surgical teaching hospital and faruk medical city). all patients requested doing rhinoplasty, in addition to septoplasty or turbinoplasty were included in the study. patients younger than 18 years and with congenital or neoplastic nasal deformities were excluded. we included 100 patients who underwent a pre-operative consultation with an oral and maxillofacial and plastic surgeon and answered evaluation esthetic and functional outcomes after rhinoplasty falah abdulla hussein hawrami1, zanyar m. amin1,2, muhammad mahmood3, rebwar a. hama4 1oral and maxillofacial surgery department, sulaimani surgical teaching hospital, sulaymaniyah, kurdistan region, iraq. 2head of oral and maxillofacial surgery department, school of dentistry, university of sulaimani, sulaymaniyah, kurdistan region, iraq. 3faruk medical city, sulaymaniyah, kurdistan region, iraq. 4kurdistan board for medical specialty, department of oral and maxillofacial surgery final stage sulaimani centre, sulaymaniyah, kurdistan region, iraq. corresponding author: rebwar a. hama (email: rebwar.ahmed24@yahoo.com) (submitted: 06 january 2020 – revised version received: 11 february 2020 – accepted: 17 march 2020 – published online: 26 april 2020) objective the aim of this study was to determine patient satisfaction in regard to nose appearance and function with the use of a validated questionnaire, before and after rhinoplasty surgery. methods a prospective study was conducted among all adult patients who underwent open rhinoplasty including other nasal procedures like septoplasty or turbinoplasty between september 2018 and august 2019 in both public and private hospitals (sulaimani surgical teaching hospital and faruk medical city). the rhinoplasty outcome evaluation (roe) questionnaire was used to study the patients’ view. results 100 patients participated in this study by completing the questionnaires and the follow-up period. the main reasons for rhinoplasty in our patients were: aesthetic 54% (n=54), functional 2 %( n=2), and a combination of both in 44% (n=44) patients the mean roe score of all patients pre-operation was 51.8 (males: 49.04, females 54.74) and the mean score post-operation was 75.22 (males 75.64, females 74.81) at 6 months with no statistically significant gender differences p-value=0.79 however, both genders showed a statistically significant improvement between the preand post-operative scores (mean difference = 23.42, p<0.017). in the pre-operative stage, patients recorded worse score for anxious and insecure (p<0.05). there were no difference for gender, age, cause or literacy level in the mean post-operative scores (p>0.05). conclusions we found that patients who consider themselves anxious before surgery were less satisfied with the result of the procedure. additionally, rhinoplasty surgery significantly improved patient quality of life regarding nose shape and function. keywords patient satisfaction, rhinoplasty, questionnaire, surgery 51 original evaluation esthetic and functional outcomes after rhinoplastyfalah abdulla hussein hawrami et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 50–53 the rhinoplasty outcomes evaluation questionnaire (roe) kurdish version. in addition, the questionnaires asked patient’s demographic data such as age, sex, education level, psychological aspects, and reason for doing rhinoplasty. post-operative satisfaction was evaluated by a phone call at 3 and 6 months after surgery, by the same maxillofacial surgeon. the validated kurdish version of the roe questionnaire was used and it is composed of six questions (five about nose shape and one about nasal breathing). each questions scored by the patient on a scale from 0 to 4, where 0 is the most negative answer and 4 the most positive one (fig. 1). a higher score indicates more satisfaction. a positive difference between postand pre operative scores means improvement after intervention. it was also asked if the surgeon explained for the patient what be corrected during rhinoplasty. results after inclusion and exclusion criteria were met, 100 patients participated in this study. the sample was composed of 87 female and 13 male patients. the population was divided into three age groups: 18–29 years old, 30–49 years old and ≥50 years old. the characteristics of the patients are mentioned in detail in table 1. the reasons for undergoing rhinoplasty were aesthetic in 54% (n=54) of patients, functional in 2% (n=2) and a combination of aesthetic and functional in 44% (n=44). it was also asked if the surgeon explained what would be corrected in the nose surgery and 0% answered not at all, fig. 1 kurdish version of rhinoplasty outcomes evaluation questionnaire. table 1. patient’s demographic data. frequency percent gender male 13 13.0 female 87 87.0 total 100 100.0 age 18-29 41 41.0 30-49 58 58.0 50 and above 1 1.0 total 100 100.0 degree 12 grade and less 34 34.0 institution 30 30.0 college degree 36 36.0 total 100 100.0 cause functional 2 2.0 aesthetic 54 54.0 both 44 44.0 total 100 100.0 52 original evaluation esthetic and functional outcomes after rhinoplasty falah abdulla hussein hawrami et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 50–53 2% (n=2) answered somewhat, 21% (n=21) answered moderately, 31% (n=31) answered very much, and 46%(n=46) answered completely. regarding psychological aspects, 56% (n=56) of the patients considered themselves anxious and 44% (n=44) calm pre-operatively and secure patients were 23% and 35% for both calm and anxious patients after 3 months 6 months post-operatively, respectively (table 2). roe score of all patients pre-operation was 51.8 (males 49.04, females 54.74.) and the mean score post-operation was 71.13 (males 71.47, females70.79) at 3 months after rhinoplasty and 75.22 (males 75.64, females 74.81) at 6 months after rhinolasty with no statistical differences between males and females p-value=0.79. however both genders showed a statistically significant improvement between the preand post-operative scores (mean difference = 23.42, p<0.017). also, there was a difference between 3 and 6 months’ post-operative scores after 6 months period most of the patient were satisfied with the results. the correlation between psychological aspects and satisfaction is presented in table 3, showing that anxious patients were significantly less satisfied than calm patients in the pre-operative period. there were no gender, no age, no cause, and no literacy level differences in mean post-operative scores (p>0.05). primary rhinoplasty was performed in 96% and revision rhinoplasty in 4% patients. there was no significant difference in post-operative roe scores between the two groups. discussion this prospective study involved 100 patients who were evaluated by kurdish version roe questionnaire before surgery, at 3 and 6 months post-operatively, to gain more precise results about satisfaction. results of the present study showed statistically significant improvement in roe scores after rhinoplasty surgery, demonstrating a high satisfaction level in this patient population. since rhinoplasty has a huge psychological impact,10 the result of rhinoplasty was found to have a great impact on the psychological status of the patient while present study observed no significant differences in roe scores for sex, age, literacy level, reason for doing rhinoplasty, or primary versus revision. in our study, the mean pre-operative roe score was 51.8 and the mean post-operative score after 6 months was 75.22, indicating an improvement of 23.42 after surgery. these numbers are not in line with those reported by alsarraf et al., which found a mean pre-operative score of 38.8 and a mean improvement of 44.5.11 the reason for that discrepancy is that most of the patients in the present study had already acceptable nose shape because when they were asked about limitation of social activity due the appearance of the nose only one patient (1%) table 2. psychological status of the patients. pre-operative psychology psychology after 3 months psychology after 6 months frequency percent frequency percent frequency percent calm 44 44.0 67 67.0 79 79.0 anxious 56 56.0 33 33.0 21 21.0 total 100 100.0 100 100.0 100 100.0 table 3. mean preand post-operative scores and correlation with psychological aspects. pre-operatively post-operatively 3 months post-operatively 6 months calm mean 54.07 72.99 76.93 sd 15.13 9.64 8.15 anxious mean 53.94 62.89 70.83 sd 14.75 10.77 12.88 p-value 0.97 0.001 0.017 table 4. rhinoplsaty outcomes evaluation questionnaire (roe) pre-operation. roe pre-op most negative answer somewhat moderately very much most positive answer how well do you like the appearance of your nose 44 28 23 5 0 how well are you will able to breath through your nose 2 29 15 20 34 how much friends like your nose 7 23 37 29 4 current nasal appearance limits social activities? 1 10 8 14 67 is nasal appearance the best that can be? 0 2 20 58 20 would you like surgically alter the appearance and function of your nose? 59 9 6 5 21 53 original evaluation esthetic and functional outcomes after rhinoplastyfalah abdulla hussein hawrami et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 50–53 answered that nose shape had always limited social activities and 67 patients (67%) answered never. although a significant improvement was found in roe scores in our population, this study focused on a patient population drawn from both public and private hospital, where rhinoplasty is performed in association with other nasal procedures. in our sample, there were 87 women and 13 men which shows that women are more concerned than men about their physical appearance. regarding age and education level, the majority of patients were between 30 and 49 years old and had college degree and above, respectively. prospective studies are really important, since they improve selection of good candidates for surgery and objective assessment of surgery results. this study was conducted in a maxillofacial department of sulaimani teaching hospital comprising senior specialists, residents, and plastic surgery department in faruk medical city. hence, one limitation of the study is that the rhinoplasty was performed by different surgeons with different levels of experience in the aesthetic area. therefore, expectations in terms of both the pre-operative consultation and the post operative satisfaction may have been affected by this factor. conclusions we found that patients who consider themselves anxious before surgery were less satisfied with the result of the procedure. additionally, rhinoplasty surgery significantly improved patient quality of life regarding nose shape and function. references 1. shahrokh c, bachery r, bryan b, husain ak, current trends in rhinoplasty. sharokh c. bagheri, husain ak. current therapy in oral and maxillofacial surgery. united states, 2012:89. 2. dimitrios k, panagiotis m, stylianos k, nikolaos d, thomas n, assessment of aesthetic results of 100 patients who underwent rhinoplasty— rhinoplasty outcome evaluation, prs global open doi: 10.1097/ gox.0000000000001404; published online 15 september 2017. 3. peter ab, henning s, ge ghali, luke cascarini, basic rhinoplasty, yaria o, faisal aq, maxillofacial surgery, 3rd ed. london, churchill livingstone. 2016:1257. 4. howard bk, rohrich rj understanding the nasal airway: principles and practice. plast reconstr surg 2002;109:1128–46. 5. ching s, thoma a, mccabe re, antony mm. measuring outcomes in aesthetic surgery: comprehensive review of the literature. plast reconstr surg. 2003;111:469–80. 6. meyer l, jacobsson s. the predictive validity of psychosocial factors for patient acceptance of rhinoplasty. ann plast surg. 1986;17:513–20. 7. shahrokh c, bachery r, bryan b, husain ak, revision rhinoplasty. behnam b, sharokh cb current therapy in oral and maxillofacial surgery. united states, 2012:901. 8. alsarraf r, larrabee wf jr, anderson s, et al. measuring cosmetic facial plastic surgery outcomes: a pilot study. arch facial plast surg. 2000;3: 198–201. 9. patnaik u, nilakantan a, bajpai r, addya k comprehensive assessment in cosmetic rhinoplasty: the use of the derriford appearance scale for evaluation of patients. med j armed forces india. 2019;75:184–9. 10. alsarraf r, larrabee wf jr, anderson s, murakami cs, johnson cm jr. measuring cosmetic facial plastic surgery outcomes: a pilot study. arch facial plast surg. 2001;3:198–201. table 5. rhinoplsaty outcomes evaluation questionnaire (roe) 3 months post-operation. roe after 3 months most negative answer somewhat moderately very much most positive answer nose appearance 5 9 19 47 20 nasal breathing 1 6 20 50 23 how much friends like your nose 2 4 16 58 20 current nasal appearance limits social activities? 1 4 0 8 87 is nasal appearance the best that can be? 20 28 22 23 7 would you like surgically alter the appearance and function of your nose? 5 9 8 27 51 table 6. rhinoplasty outcomes evaluation questionnaire (roe) 6 months post-operation. roe after 6 months most negative answer somewhat moderately very much most positive answer nose appearance 1 6 11 43 39 nasal breathing 1 4 14 43 38 how much friends like your nose 2 3 13 49 33 current nasal appearance limits social activities? 1 1 0 3 95 is nasal appearance the best that can be? 33 24 19 17 7 would you like surgically alter the appearance and function of your nose? 6 4 6 19 65 dx.doi.org/10.22317/jcms.v6i2.743 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 20 j contemp med sci | vol. 5, no. 1, january–february 2019: 20–23 original article evaluation the effect of royal jelly on the growth of two members of gut microbiota; bacteroides fragillis and bacteroides thetaiotaomicron vida kazemi,a mojtaba mojtahedzadeh,b seyed davar siadat,c abbas hadjiakhondi,a abdulghani ameri,d sara ahmadi badi,c azadeh manayi,a* and mahdi bagherib amedicinal plants research center, faculty of pharmacy, tehran university of medical sciences, tehran, iran. bdepartment of pharmacotherapy, faculty of pharmacy, tehran university of medical sciences, tehran, iran. cmicrobiology research center (mrc), pasteur institute of iran, tehran, iran. ddepartment of food and drug control, ahvaz jundishapur university of medical sciences, ahvaz, iran. *correspondence to azadeh manayi (email: manayi@sina.tums.ac.ir). (submitted: 05 september 2018 – revised version received: 18 september 2018 – accepted: 10 october 2018 – published online: 26 february 2019) objective in this study the effect of royal jelly on the growth of two important members of bacteroides spp.; bacteroides fragilis and bacteroides thetaiotaomicron, was evaluated. also the physicochemical properties and cytotoxicity effects of royal jelly on caco-2 cell line as gastrointestinal epithelial cell model, was assessed. methods bacteria, b. fragilis and b. thetaiotaomicron were grown on brain heart infusion (bhi) broth medium supplemented with royal jelly in three different concentrations (2.5%, 5% and 10% v/v), both of the bacteria (1.5 × 108 cfu/ml) were inoculated to bhi broth contained royal jelly in anaerobic condition. to calculate the bacterial optical density (od), the absorbance was measured at 600 nm after an overnight. also caco-2 cells, was used to study the effects of royal jelly on epithelial cell viability, and the physicochemical properties consist of total proteins, polysaccharides, phenolic compounds, total lipids, ash and moisture by uv–vis spectrophotometric and gravimetric methods were evaluated. results the growth of b. fragillis and b. thetaiotaomicron were increased by royal jelly (2.5%, 5% and 10% v/v concentrations) and the results indicated that royal jelly increased the growth of bacteria in a dose dependent manner (p < 0.001). in addition mtt assay showed more than 95% viability of caco-2 cells treated with royal jelly. the iranian royal jelly sample contains 59.01% water, 11.57% proteins, 12% lipids, 12.63% polysaccharide, and 5% mineral. conclusion this study showed that royal jelly has a potential effect in the preserving gut microbiota and it is suggested that royal jelly as a complementary and alternative medicine can be used to treatment diseases are associated with gut microbiota–host interactions and immune regulating. although we need to expand our knowledge by designing clinical trials to confirm the therapeutic effects of royal jelly on gut microbiota modulation as a barrier function. keywords bacteroides fragile, bacteroides thetaiotaomicron, gastrointestinal tract microbiota, gut microbiota, royal jelly, traditional medicine introduction royal jelly (rj) as a natural product and healing compound has been widely used since ancient times in the treatment of diseases and health promotion in many countries. this substance is a complex mixture of water, proteins, fats, carbohydrates, amino acids, mineral salts, vitamins, enzymes, hormones, trace elements and antibiotics. rj has been used as a supplement due to its rich carbohydrate, protein, and mineral ingredients.1–3 according to the recent pharmacological studies, rj has antioxidant, antitumor, anti-inflammatory, antiallergic, antiaging, and antihypertensive activities. it has been indicated that oral ingestion of rj ameliorated lipoprotein metabolism and decreased serum total cholesterol and low-density lipoprotein levels. this product may play an important role in the maintenance of the gastrointestinal function due to its components and antioxidant activity. the bacteria are the major part of the adult gut microbiota with an essential metabolic function to degrade polysaccharides of plants. further, formation of the intestinal mucosal barrier and stimulation of angiogenesis within a gut are other beneficial properties of the bacteria for human.1–3 the microbial community of the digestive tract is known as microbiota which is a combination of various micro-organisms and thousands of bacterial species.4,5 gut microbiota are involved in maintaining the gastrointestinal tract function. they also modulate the synthesis of vitamins, metabolism of substances, inhibition of pathogenic bacteria growth, stimulation of mucosal iga production, modulation of immune system, and maintenance of human hemostasis.6–9 bacteroides fragillis and bacteroides thetaiotaomicron belong to the phylum of bacteroidetes. bacteroides spp. are abundance in gut microbiota and have significant roles in complex community. bacteroides spp. mostly colonizes in the distal of gi where fermentation of indigestible carbohydrates occurs.10,11 bacteroides spp. have significant role on the host metabolism and immunity through degradation of proteins and carbohydrate complex and activating the regulatory t cells. furthermore, gut microbiota especially bacteroides spp. have an important role in regulation of immune system and maintenance of homeostasis.5,11,12 the patterns in the growth of bacterial in the gastrointestinal tract can be affected by various diseases, drugs, and diet. recent studies have indicated that the effect of enteral nutrition on the reduction of infectious morbidity in critically ill patients was significantly higher than parenteral nutrition.14 therefore, the modification of gut microbiota–host interactions by royal jelly (viable benefited bacteria) is controversial. however, it is suggested that rj could modulate gut microbiota, the balance between species of living bacteria in human gastrointestinal tract and effect on homeostasis and host functions. issn 2413-0516 v. kazemi et al. 21j contemp med sci | vol. 5, no. 1, january–february 2019: 20–23 original article evaluation the effect of royal jelly on the growth of two members of gut microbiota in this study the physicochemical properties of iranian royal jelly and its effect on the growth of b. fragillis and b. thetaiotaomicron, as important parts of gastrointestinal microbiota and also the cytotoxicity effect of royal jelly on caco-2 cell line as gastrointestinal epithelial cell model evaluated. materials and methods evaluation the physiochemical properties of royal jelly royal jelly preparation freshly harvested royal jelly applied from beekeepers of damavand reign, tehran province (35.7013° n, 52.0586° e) was used. royal jelly composition the composition of rj is depended on seasonal and regional conditions; 100 mg of rj sample were put on small amount of water and to dissolve using ultrasonic homogenizer, subsequently the volume reached to 100 ml with deionized water in a volumetric flask to obtain concentration of 1 mg/ml. total phenol was measured using uv–vis spectrometer (optizen, korea) according to the method of folin–ciocalteau.15 the total polysaccharide was analyzed using uv–vis spectrometer (optizen, korea) according to the previous study conducted by vazirian et al.16 bradford method was applied to determine total protein content of the sample spectrophotometrically.17 total lipid was measured based on the described method by müller et al. (2000).18 moisture and ash content of the sample was measured according to the previous study conducted by horwitz and latimer (2000).19 evaluation the cytotoxicity effect of royal jelly cell culture the human epithelial cell line ibrc c10094 caco-2 was purchased from iranian biological resource center. the cells were grown in dulbecco’s modified eagle medium (high glucose; gibco, usa), supplemented with 10% fetal bovine serum (gibco, usa), 1% penicillin/streptomycin (gibco, usa) and incubated at 37°c in a 5% co2 atmosphere. cells were routinely subcultured every 3 or 4 days by trypsinization until they reached approximately 80% confluence.20 mtt assay caco-2 cells were cultured at the density of 2 × 104 cell/well in a 96-well culture plate and incubated overnight, before rj treatment. cells were treated with rj and incubated for 24 h. then, the cell culture medium was changed. cells were incubated with 100 µl mtt contained medium for 4 h, the medium was removed and then 100 µl dmso was added. the absorbance was measured at 550 nm, using a microplate spectrophotometer (epoch, usa).20 evaluation the effect of royal jelly on bacterial growth bacterial strains and growth condition bacteroides fragillis atcc 23745 and b. thetaiotaomicron atcc 10774 were grown on blood agar plates containing brain heart infusion (bhi) broth supplemented with hemin (5 µg/ml) and menadione (1 µg/ml). incubation performed at 37°c under anaerobic conditions provided 80% n2, 10% co2, and 10% h2 atmosphere. 21 treatment bacteria with medium contained royal jelly samples of rj solution were prepared by deionized distilled water. sterilization of rj solution was performed using a 0.22-µm pore size polyvinylidene difluoride filter (millipore, billerica, ma, usa). this sterile solution was aseptically added to bhi broth at different concentrations (2.5%, 5% and 10% v/v). after preparation of 1.5 × 108 cfu/ml bacterial suspension, 100 µl of b. fragillis and b. thetaiotaomicron suspension were inoculated to bhi broth and rj contained bhi broth. incubation was performed overnight in anaerobic condition. finally, the bacterial optical density was calculated by measuring the absorbance at 600 nm after an overnight. ethics statement this work was part of the research that has been performed in accordance with the ethical rules of the helsinki declaration and all the experiments and procedures used in this research were approved by the ethics committee of tehran university of medical sciences (no.ir.tums. rec.1395.2643). statistical analysis significant differences between groups were determined by one-way anova followed by tukey’s post-hoc test for multiple comparisons. the level of statistical significance was p < 0.05. results the obtained data showed that the total amounts of phenol and polysaccharide in iranian royal jelly sample were 21.99 ± 0.41 µg/mg gae and 12.63%, respectively. the contents of lipid and protein were 12% and 11.57%. the moisture of royal jelly was 59.01% and the amount of ash was 0.1% (table 1). the results demonstrated that the growth of both the bacteria were increased by rj supplementation (table 2). the growth of b. fragillis and b. thetaiotaomicron exposed to all concentration of rj increased in comparison with control group. this product induced growth of b. fragillis higher than b. thetaiotaomicron in all of the tested concentrations. rj with concentration of 2.5% versus 5% of rj significantly increased the growth of b. fragillis (od; 0.330 ± 0.045, 0.610 ± 0.098, respectively) and b. thetaiotaomicron (od; 0.299 ± 0.060, 0.518 ± 0.100, respectively). the results also indicated that there was a significant difference between the growth of b. fragillis (od; 1.020 ± 0.360) and b. thetaiotaomicron (od; 0.751 ± 0.250) exposed to rj at a concentration of 10% v/v in comparison with the growth of them exposed to rj at concentration of 5% v/v (p < 0.001, <0.01, respectively). the mtt assay indicated that rj had no cytotoxicity effect on caco-2 cells after 24 h. the cell growth inhibition values for rj against caco-2 cells were 5% after 24 h. result of this study showed that this substance did not reduce cell viability. table 1. the chemical composition of iranian royal jelly phenol polysaccharide protein lipid moisture ash 21.99 ± 0.41a 12.63 ± 0b 11.57 ± 0b 12.00 ± 0b 59.01 ± 0b 0.1 ± 0b amicrogram per milligram gallic acid equivalent. bpercentage (%). 22 j contemp med sci | vol. 5, no. 1, january–february 2019: 20–23 evaluation the effect of royal jelly on the growth of two members of gut microbiota original article v. kazemi et al. discussion the present findings indicated that rj could increase the bacterial growth with no cytotoxicity effect on caco-2 cells. intestinal epithelial cells are the interface between gut microbiota and host interactions. therefore, the effect of royal jelly on caco-2 cell viability (a human intestinal epithelial cell model) was evaluated. according to our findings, iranian rj contains 59% water, 11.57% proteins, 12% lipids, and up to 12.63% polysaccharide. similar to our study, johansson (1995) indicated that the moisture of rj was near to 60%. it was also indicated that rj contained 12% proteins, 7.6% lipids, and up to 18% polysaccharide.11 compared with johansson’s (1995) study, the amount of protein and polysaccharide in our study was low. while, higher lipid contents in the rj samples was observed in this study. another study indicated that rj from different ecosystems in south america to europe has the wider standard ranges of components (60–70% water, 8–18% proteins, 9–18% total polysaccharide, 3–11% lipids and 0.3–0.5% ash). the findings of this study also were comparable with those examined samples of different origins. the amount of ash in the sample of our study was lower (0.1%) than those reported previously.12,13 the amount of phenolic compounds in our sample was near to the findings of nagai and inoue (21.2 μg/mg) and nabas et al. (23.3 μg/mg).11,13 several factors including the plants species, health of the plant, season, and environmental factors can affect the amount of phenolic compounds in rj.14 the main biological and pharmacological activities (antioxidant, immunomodulation and anti-inflammatory effects) of natural antioxidants may be related to their phenolic constituents. it has been suggested that consuming foods rich in phenolic compounds can increase the population of bacteroides in the gastrointestinal tract (git). therefore, iranian rj could be effective for modulation of several functional organs such as gastrointestinal tract. the git contains many important beneficial microbes. for example, bacteroides spp. are one of the micro-organisms present primarily for the sustenance of a healthy gi system.19 the biological activity and development of this bacterium is further enhanced in the presence of phenolic compounds.19 some in vitro and in vivo experimental studies on polyphenolic compounds have reported that these compounds hastened the growth of lactobacillus and bacteroides and catalyze their probiotic potency in the git.20 under in vitro conditions, polyphenolic compounds promoted the increase in the numbers of bacteroides by 10–100-folds, which was beneficial for the intestinal microbiota. increasing in bacteroides leads to a decrease in the formation of ammonia, skatoles and harmful amine procarcinogens in the large intestine, and reduce acid production that raises fecal ph.21 since microbiota growth was relatively unaffected by most of the aromatic compounds tested, probiotic colonization in the intestine should continue in the presence of phenolic compounds so as to improve the intestinal microbial balance and inhibit pathogen growth. previous research has demonstrated that consumption of other plants rich in polyphenolic compounds such as green tea selectively promotes the growth of bacteroides in the gut wall.18 this data demonstrated that rj contains a high percent of phenolic compounds. however, phenolic compounds increase the growth of intestinal bacteria; antimicrobial activities of polyphenols have been demonstrated.21 the level of inhibition varies depending on the bacterial species, the chemical structure of the compound and phenolic concentration. findings of this study showed that rj could change the bacterial growth. this effect may be related to the rj concentrations and the bacterial species. it has been known that, gastrointestinal microbiota composition has significant roles in the determination of health and disease states.13 as well as scrutiny gut microbiota pattern in conduct an investigation and preclinical studies can be helped in understanding the processes of maintaining gut microbiota by supplementation and prescription of some natural products such as royal jelly in preserving gut microbiota, especially in ill patients undergoing intensive care and receiving medication and variety of drugs with a microbiota weakening effect. this study was designed based on research to recognizing ways to reinstate gut microbiota composition and results were showed that rj has a potential effect in the preserving and energy conversation of gut microbiota to improve human health as a defense barrier, and also royal jelly can be used to formulating preparations for treatment diseases and maintaining health situation. although this assumption needs to expand our knowledge about the mechanism of effects caused by royal jelly on the common microbial community includes pathogens and friendly micro-organisms of healthy voluntaries and patients, by designing clinical trials to confirm the preliminary obtained results on the therapeutic effects of royal jelly on gut microbiota modulation as a barrier function. conclusion according to the importance of bacteroides spp. in gut microbiota–host interactions, it is suggested that gastrointestinal microbiota pattern could be changed by royal jelly supplementation in patients receiving variety of drugs with a gut microbiota weakening side effects. finding of this work indicated that rj has a potential effect in the growth of gut microbiota table 2. the effect of different concentrations (od) of royal jelly on the growth of b. fragillis and b. thetaiotaomicron versus control (non-exposed bacteria to rj) bacterial strain control royal jelly concentration (%v/v) 2.5 5 10 b. fragillis 0.102 ± 0.020 0.330 ± 0.045* 0.610 ± 0.098***,† 1.020 ± 0.360***,†††, §§§ b. thetaiotaomicron 0.116 ± 0.020 0.299 ± 0.060* 0.518 ± 0.100***,†† 0.751 ± 0.250***,††,§§ *significant difference between the control versus others. †significant difference between bacteria exposed to rj 2.5% versus bacteria exposed to other concentrations. §significant difference between bacteria exposed to rj 5% versus bacteria exposed to rj 10%. v. kazemi et al. 23j contemp med sci | vol. 5, no. 1, january–february 2019: 20–23 original article evaluation the effect of royal jelly on the growth of two members of gut microbiota which could improve human health and treatment diseases. however, several clinical trials should be done to cover a large number and wide scope of subjects and confirm the beneficial effects of rj on gut microbiota. in conclusion, royal jelly seems to be a very effective approach to treat and, all results of low sample size and short-term studies have to be taken into account. in this purpose, multi-center study to evaluate the comprehensive application of royal jelly with established treatment modalities in a randomized, controlled trial has been started. 12. hooper lv, littman dr, macpherson aj. interactions between the microbiota and the immune system. science. 2012;336:1268–1273. 13. lin l, zhang j. role of intestinal microbiota and metabolites on gut homeostasis and human diseases. bmc immunol. 2017;18:2. 14. ahmadi badi s, siadat sd, khatami sh, irani sh. induction effect of bacteroides fragilis derived outer membrane vesicles on toll like receptors gene expression and cytokine concentrations in human intestinal epithelial cell. cell j. 2019;21:57–61. 15. elhenawy w, debelyy mo, feldman mf. preferential packing of acidic glycosidases and proteases into bacteroides outer membrane vesicles. mbio. 2014;5:e00909–e00914. 16. ahmed wm, khalaf aa, moselhy wa, safwat gm. royal jelly attenuates azathioprine induced toxicity in rats. environ toxicol pharmacol. 2014;37:431–437. 17. özan f, çörekçi b, toptaş o, halicioğlu k, irgin c, yilmaz f, et al. effect of royal jelly on new bone formation in rapid maxillary expansion in rats. med oral patol oral cir bucal. 2015;20:e651–e656. 18. metwally ibrahim sel, kosba aa. royal jelly supplementation reduces skeletal muscle lipotoxicity and insulin resistance in aged obese rats. pathophysiology. 2018;25:307–315. 19. boger mcl, lammerts van bueren a, dijkhuizen l.cross-feeding amongst probiotic bacterial strains on prebiotic inulin involving the extracellular exo-inulinase of lactobacillus paracasei strain w20. appl environ microbiol. 2018;84:e01539-18. 20. melok al, lee lh, mohamed yussof sa, chu t. green tea polyphenol epigallocatechin-3-gallate-stearate inhibits the growth of streptococcus mutans: a promising new approach in caries prevention. dent j (basel). 2018;6:e38. 21. mayta-apaza ac, pottgen e, de bodt j, papp n, marasini d, howard l, et al. impact of tart cherries polyphenols on the human gut microbiota and phenolic metabolites in vitro and in vivo. j nutr biochem. 2018;59:160–172. acknowledgments this research was supported by the medicinal plants research center, tehran university of medical sciences and the authors would like to thank microbiology research center (mrc), pasteur institute of iran. conflict of interest the authors declare that there is no conflict of interest. n references 1. alvarez-suarez jm, tulipani s, romandini s, bertoli e, battino m. contribution of honey in nutrition and human health: a review. mediterranean j nutr metab. 2010;3:15–23. 2. pasupuleti vr, sammugam l, ramesh n, gan sh. honey, propolis, and royal jelly: a comprehensive review of their biological actions and health benefits. oxid med cell longev. 2017;2017:1259510. 3. rao pv, krishnan kt, salleh n, gan sh. biological and therapeutic effects of honey produced by honey bees and stingless bees: a comparative review. rev bras farmacogn. 2016;26:657–664. 4. benson ak, kelly sa, legge r, ma f, low sj, kim j, et al. individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. proc natl acad sci. 2010;107:18933–18938. 5. clemente jc, ursell lk, parfrey lw, knight r. the impact of the gut microbiota on human health: an integrative view. cell. 2012;148: 1258–1270. 6. lozupone ca, stombaugh ji, gordon ji, jansson jk, knight r. diversity, stability and resilience of the human gut microbiota. nature. 2012;489: 220–230. 7. sekirov i, russell sl, antunes cm, finlay bb. gut microbiota in health and disease. physiol rev. 2010;90:859–904. 8. nicholson jk, holmes e, kinross j, burcelin r, gibson g, jia w, et al. host–gut microbiota metabolic interactions. science. 2012;336:1262–1267. 9. prakash s, tomaro-duchesneau c, saha s, cantor a. the gut microbiota and human health with an emphasis on the use of microencapsulated bacterial cells. j biomed biotechnol. 2011;2011:981214. 10. den besten g, van eunen k, groen ak, venema k, reijngoud dj, bakker bm. the role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. j lipid res. 2013;54:2325–2340. 11. tremaroli v, bäckhed f. functional interactions between the gut microbiota and host metabolism. nature. 2012;489:242–249. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 20 j contemp med sci | vol. 2, no. 5, winter 2016: 20–24 research objectives this study was designed to determine the levels of hormones insulin, testosterone and some female hormones in some male and female hormones in patients with polycystic ovary syndrome (pcos) and find the relationship between the male testosterone hormone and female hormones in patients with pcos and to know the reasons for these changes in view of the prevalence of pcos and its importance as a major problem affecting the health of women and her fertility. methods the hormones included lh, fsh, prl, estradiol (e2), progesterone (p4) and lh/fsh ratio in patients with pcos compared with control group, and to find the relationship between male testosterone hormone and female hormones under the study in patients with pcos. fifty-five female patients with pcos in reproductive age with body mass index (bmi) (25.01 ± 1.70 kg/m2), aged 15–46 years old and who recalculated fertility centre of teaching al-sadr hospital in al-najaf al-ashraf governorate from 1/9/2013 to 1/10/2014, compared with 20 non-patients women with pcos with bmi (23.87 ± 2.66 kg/m2) and age 17–47 years old as control group. results the results showed that there was a significant increasing (p < 0.05) in insulin, testosterone, lh, prl and lh/fsh ratio in patients with pcos compared with control group, while there was a significant decreasing (p < 0.05) in fsh, e2 and p4 in patients with pcos compared with non-patients women. conclusion as the results have shown that there was a positive significant relationship between testosterone and lh, prl and lh/fsh ratio while the relationship was negative significant between testosterone and fsh, e2 and p4 in patients with pcos. keywords polycystic ovary syndrome (pcos), testosterone, lh, fsh, progesterone (p4) the relationship between male testosterone hormone and some female hormones in women with polycystic ovary syndrome (pcos) dalal abdul hussain kadium,a ghsoon ghanem kaem,b zaineb mehdi al saeq,c zaineb assim al safard issn 2413-0516 a,c,dcollege of girls education, kufa university, kufa, iraq. bcollege of applied medical sciences, karbala university, karbala, iraq. correspondence to ghsoon ghanem kaem (email: rose2005rr44@ymail.com). (submitted: 17 november 2015 – revised version received: 21 december 2015 – accepted: 20 january 2016 – published online: 26 march 2016) introduction polycystic ovary syndrome (pcos) was first described in 1935 by stein and leventhal therefore is known stein-leventhal syndrome,1 pcos is one of gynaecological very common in reproductive age and it occurs in about 5–10% of women reproductive age in the world.2,3 pcos is a disease that infected the ovaries which may be associated with a number of clinical symptoms and hormonal disorders; therefore, pcos is not one disease, but a group of related diseases together, which may lead, in the end to infertility resulting from chronic anovulation.4–6 in some cases of pcos did not have any pathogenic symptoms except there are several small cysts in size in the ovaries, which can be observed when vaginal examination by ultrasound,7,8 but in most cases, this syndrome is accompanied by several clinical symptoms, which can be diagnosed in the pcos such as menstrual cycle disorders (amenorrhea or oligomenorrhoea), anovulation, infertility, hirsuitism, acne and obesity9–11 and these symptoms often are produced by hyperandrogenism that caused by insulin resistance and hyperinsulinemia,12,13 also pcos can be diagnosed through some laboratory tests such as measuring hormones levels especially insulin, lh, fsh and male hormones (androgens) especially testosterone hormone14–16 and these symptoms may be shown individually or jointly, but did not require their presence together to diagnose pcos.17,18 the studies also showed that insulin resistance and hyperinsulinaemia in pcos may also cause hyperlipidemia and diabetes mellitus type 2 (niddm),19–21 early arteriosclerosis22 and thyroid dysfunction,23 in addition to a number of complications on the long extent such as chronic cardiovascular diseases,24 hypertension,25,26 cervical cancer, nerve system diseases and stroke.27,28 researches indicated that infection in the pregnant women with pcos may cause abortion, early birth, hypertension and diabetes associated with pregnancy.29,30 the real reasons of pcos are unknown yet, several theories were put to explain mechanisms of pcos, but the recent studies suggested that there are new evidences pointed to transmit this syndrome genetically by gene of prevailing type has not been detected to present time,31–33 the treatment of pcos focuses on treating the different symptoms and disorders accompanying of pcos.34,35 the aim of present study is to examine changes in some male and female hormones in patients with pcos and find the relationship between the male testosterone hormone and female hormones in patients with pcos and to know the reasons for these changes in view of the prevalence of pcos and its importance as a major problem affecting the health of women and her fertility. materials and methods subjects this study was conducted on 75 women of reproductive age. among them 55 women with pcos with body mass index (bmi; 25.01 ± 1.70), aged 15–46 years who recalculated fertility center of teaching al-sadr hospital in al-najaf al-ashraf governorate from 1/9/2013 to 1/10/2014 and diagnosed in brief by the doctor of the fertility center through clinical and biochemical signs of hyperandrogenism as hirsutism, acne and obesity or oligo and/or anovulation that is menstrual disturbance or appearance of the polycystic ovarian on ultrasound, while 20 healthy women with bmi (23.87 ± 2.66) and mailto:rose2005rr44@ymail.com 21j contemp med sci | vol. 2, no. 5, winter 2016: 20–24 research relationship between male testosterone and female pcos hormonesdalal abdul hussain kadium et al. table 1. comparison of hormone concentrations between patients with pcos and control group hormonal parameters patients with pcos n = 55 non-patients with pcos (control group) n = 20 p < 0.05 mean ± sd mean ± sd fasting insulin, iu/ml 14.84 ± 7.80 12.50 ± 5.33 p < 0.05 testosterone, ng/ml 0.88 ± 1.56 0.47 ± 0.31 p < 0.05 lh, iu/ml 9.87 ± 1.79 6.88 ± 1.94 p < 0.05 fsh, iu/ml 3.86 ± 1.39 8.34 ± 1.51 p < 0.05 lh/fsh 2.82 ± 1.57 0.88 ± 0.34 p < 0.05 p r l, ng/ml 26.59 ± 7.75 15.60 ± 3.17 p < 0.05 estradiol (e2), pg/ml 58.14 ± 8.56 75.35 ± 5.9 p < 0.05 progesterone (p4), ng/ml 1.85 ± 0.34 0.87 ± 0.6 p < 0.05 age 17–47 years as control group, their fertility have been confirmed and they have no other diseases like artery diseases, thyroid gland diseases, diabetes mellitus and blood pressure. count bmi weight and height of the body were measured, bmi was calculated by dividing by the weight square of the height (kg/m2).64 hormone assays venous blood samples of pcos patients and non-patients were collected in third and fourth day of menstrual cycle (follicular phase) after 12 h overnight fasting for the estimation of the hormones, serum levels of lh, fsh, testosterone, prl and estradiol (e2) were measured by specific electro chemiluminescence immunoassay (elecsys 2010 cobas, roche diagnostics, mannheim, germany), insulin hormone was measured using chemiluminescence (siemens medical solutions diagnostics, ca, usa), and progesterone (p4) levels were determined using chemiluminescence (advia centaur, siemens healthcare diagnostics, uk). statistical analysis results were entered to spss version 17, mean and standard deviation (mean ± sd) were also calculated, statistical analysis was done using the student’s t test of confirmation of the normal distributed. the correlation among the hormones was performed by pearson correlation test and p value p < 0.05 was considered statistically significant. results comparison of the hormone concentrations between patients with pcos and control group the results (table 1) show a significant increase (p < 0.05) in concentrations of hormones insulin (14.84 ± 7.80), lh (9.87 ± 1.94) testosterone (0.88 ± 1.56) and prl (26.59 ± 7.75) and lh/fsh ratio (2.8 ± 1.57) in patients women with pcos compared with control group (12.50 ± 5.33), (6.88 ± 1.94), (0.47 ± 0.31), (15.60 ± 3.17) and (0.88 ± 0.34) respectively, while there was a significant decreasing (p < 0.05) in hormones levels fsh, estradiol (e2) and progesterone (p4) in women patients with pcos (3.86 ± 1.39, 58.14 ± 8.56, 1.85 ± 0.34) when compared with non-patients women with pcos (8.34 ± 1.51, 75.35 ± 5.9, 0.87 ± 0.6) respectively (table 1). correlation between male testosterone hormone and female hormones in patients women with pcos a significant positive correlation was found between the testosterone and lh (p < 0.05, r = 0.93) and lh/fsh ratio (p < 0.05, r = 0.87) and prl (p < 0.05, r = 0. 79) in patients with pcos (figs. 2–4), while there was a significant negative correlation between testosterone and fsh, e2 and p4 (p < 0.05, r = −0.98, r = −0.89, r = −0.96) respectively in patients with pcos (figs. 1, 5, 6). discussion pcos is a group of disorders in the reproductive and metabolism function of the body, and the mechanism of pcos is unknown so far, but may cause in it one or more of the fig. 2 relationship between testosterone and lh. fig. 1 relationship between testosterone and fsh. fig. 3 relationship between testosterone and lh/ fsh ratio. 22 j contemp med sci | vol. 2, no. 5, winter 2016: 20–24 relationship between male testosterone and female pcos hormones research dalal abdul hussain kadium et al. patients with pcos compared with healthy controls, which was its relationship significantly negative with the testosterone hormone in patients with pcos, the studies have pointed that hyperinsulinemia may affect the system hypothalamic-pituitary system, causing an increase in lh through either the increased frequency pulsating of gnrh47 or by increasing sensitivity of the pituitary gland to gnrh hormone, or increase stimulation of the gland because of disorder feed back mechanism between the pituitary gland and ovary steroids48,16 and hyperandrogenism caused abnormal secretion of the gonadotrophins with relatively high on levels of lh to fsh, which lead to stop growth development of the follicles and production the androgen and the excess of it turns into excess of terminal tissue to estrogen causing abnormal production of the steroids of the ovaries which leads to the continuation of abnormal secretion of gonadotrophins.49,50 also studies have demonstrated that any disorder in the level of lh leads to disorder in the level of fsh because of the pituitary gland disorder or not to respond to gnrh hormone, as each increased the lh may decrease fsh and this leads to height rate of lh/fsh and this rate increases with secretion increased the androgens in women with pcos51,52 and this is indicated by many studies.53–55 and the results of our study also showed a significant increase in the level of prl in patients with pcos compared with non-patients, and the relationship was significant positive between prl and the testosterone hormone, and the reason may be due to hyperinsulinemia, which causes hyperandrogens in the body that affects prolactin-releasing hormone (rph) which is generated by the hypothalamus causing increased hormone,56 or may be due to the disorders in the body of patients with pcos, especially high level of androgens which may lead to a number of neuroendocrine changes such as decreasing level of the dopamine hormone, which is the prolactin inhibiting factor (pif) causing a high level of prolactin hormone57,58 thus, hyperandrogenism in the body increases the level of prolactin hormone, and this result was consistent with the results of many studies.59–61 the study showed that there was a significant decrease in levels of e2 and p4 hormones in patients with pcos compared with control group, and there was a significant negative relationship between e2 and p4 and testosterone hormone in women with pcos, and result of this study agreed with results of many researches, which demonstrated the inability of the ovary to form ovarian hormones (estradiol and progesterone) by granulosa cells62,63 and this would lead to a reduction in levels of these hormones in blood serum against an increase in the levels of androgenic hormones that produce from the ovary which increase secretion of lh which inhibited aromatase enzyme and this reduces transformation the androgen to estradiol.64,65 a few of the fsh leads to low level of estradiol because fsh, the main hormone in the stage of follicular phase of the menstrual cycle, operates on the composition and maturity of ova and thus estrogen hormone secretion of mature ovum66 and the high level lh causes damage to the premature follicles and anovulation and thus, non formation of yellow body in the second half of menstrual cycle, leading to lack of progesterone that works with the estradiol on metamorphosis of the endometrium last stages of the menstrual cycle in recent menstrual cycle of the cession endometriosis in the normal case.67 but in patients with pcos, the secretion of estradiol genetic defects but not the point that starts from and causes rest disorders36,37 is not known. however, recent studies indicate that the resistance of insulin which leads to hyperinsulinemia due to a malfunction in the effectiveness of the insulin hormone receptors on the cell membrane, leading to the inability of insulin to carry glucose particles from blood to the cells and this situation gives a prompt to pancreas to secret increasing of insulin to compensate the lack of effectiveness, causing higher insulin20,38 and this is indicated by our current study which agreed with other results of many studies.39,40 as the results showed that there is a significant increase in the concentration of testosterone in patients with pcos compared with control group, and this due to either an increase in insulin and growth factor-like insulin i (gfi) from the ovary in women with pcos, which causes damage non-mature follicles and anovulation and appearance acne and hirsutism41,42 or due to initial enzymatic defect in adrenal glands43,44 and this result has agreed with results of many studies.45,46 and the results also indicated a significant increase in lh and lh/fsh ratio, and a significant positive relationship between lh, lh/fsh ratio and testosterone hormone, while fsh has been decreased significantly in fig. 4 relationship between testosterone and prl. fig. 5 relationship between testosterone and estradiol (e2). fig. 6 relationship between testosterone and progesterone (p4). 23j contemp med sci | vol. 2, no. 5, winter 2016: 20–24 research relationship between male testosterone and female pcos hormonesdalal abdul hussain kadium et al. from the ovary stimulates growth of the endometrium to become thick and with no enough progesterone leads to severe bleeding or intermittently for a long time and this may cause carcinoma of uterus.68,69 therefore, high secretion of lh increases the secretion of androgens whenever there is a decreased secretion of progesterone and estradiol, this result agreed with the findings of many studies.70,71 23. cakir e, sahin m, topaloglu o, colak nb, karbek b, gungunes a, et al. the relationship between lh and thyroid volume in patients with pcos. j ovarian res. 2012;5:43. doi: 10.1186/1757-2215-5-43 pmid: 23231775 24. orio fj, palomba s, spinelli l, cascella t, tauchmanovà l, zullo f, et al. the cardiovascular risk of young women with polycystic ovary syndrome: an observational analytical, prospective case: control study. j clin endocrinol metab. 2004;89:3696–3701. 25. chen mj, yang ws, yang jh, chen cl, ho hn, yang ys. relationship between androgen levels and blood pressure in young women with polycystic ovary syndrome. hypertension. 2007;49:1442–1447. pmid: 17389259 26. joham ae, boyle ja, zoungas s, teede hj. hypertension in reproductiveaged women with polycystic ovary syndrome and association with obesity. am j hypertens. 2015;28:847–51. doi: 10.1093/ajh/hpu251 pmid: 25542625 27. rosenfield rl. current concepts of polycystic ovary syndrome. baillieres clin obstet gynecol. 1997;11(2):307–333. 28. wild s, pierpoint t, mckeigue p, jacobs h. cardiovascular disease in women with polycystic ovary syndrome at long-term follow-up: retrospective cohort study. clin endocrinol (oxf). 2000;52(5):595–600. pmid: 10792339 29. jakubowicz dj, iuomo mj, jakubowicz s, roberts ka, nestler je. effects of metfomin on early pregnancy loss in the polycystic ovary syndrome. j clin endocrionol metab. 2002;87:524–529. 30. milosavljevic m, stefanovic m, kutlesic r, vukomanovic p, andric a. pregnancy outcomes among infertile patients with polycystic ovary syndrome treated with metformin. med biol. 2006;13(3):172–176. 31. legro rs. the genetics of polycystic ovary syndrome. am j med. 1995;98(1a):9s–16s. 32. urbanek m. the genetics of the polycystic ovary syndrome. nat clin pract endocrinol metab. 2007;3:103–111. 33. koracs gt, norman r. polycystic ovary syndrome. 2nd. cambridge university; 2007. 34. badawy a, elnashar a. treatment options for polycystic ovary syndrome. int j womens health. 2011;3:25–35. 35. asimi zv. evaluation of endocrine changes in women with the polycystic ovary syndrome during metformin treatment. bosn basic med sci. 2013;13(3):180–185. 36. perricone r, pasetto n, dencarolis e, vaquero e, noccioli g, panerai a, fontana l. cystic ovaries in women affected with hereditary angioedema. clin ex immunol. 1992;90(3):401–404. 37. dunaif a, segal kr, futterweit w, dobrjansky a. prefound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. diabetes. 1989;38:1165–1174. 38. pasquali r, gambineri a, pagotto u. the impact of obesity on reproduction in women with polycystic ovary syndrome. bjog. 2006;13:1148–1159. 39. poretski l, piper b. insulin resistance, hypersecretion of lh and a dual defect hypothesis for the pathogenesis of pcos. obstet gynecol. 1994; 84:613–621. 40. pagotto u, gambineri a, vicennati v, heiman ml, tschöp m, pasquali r. plasma ghrelin, obesity and polycystic ovary syndrome: correlation with insulin resistance and androgen levels. j clin endocrinol metab. 2002;87:5625–5629. pmid: 12466363 41. pasquali r, gambineri a. insulin-sensitizing agents in polycystic ovary syndrome. eur j endocrinol. 2006;154:763–775. 42. duleba aj, spaczynski rz, olive dl. insulin and insulin-like growth factor i stimulate the proliferation of human ovarian theca-interstitial cells. fertil steril. 1998;69:335–340. 43. barbieri rl, makris a, randall rw, daniels g, kistner rw, ryan kj. insulin stimulates androgen accumulation in incubations of ovarian stroma obtained from women with hyperandrogenism. j clin endocrinol metab. 1986;62:904–910. 44. yildiz bo, azziz r. the adrenal and polycystic ovary syndrome. rev endocrinol metab disord. 2007;8:331–342. conclusion as the results have shown that there was a positive significant relationship between testosterone and lh, prl and lh/fsh ratio while the relationship was negative significant between testosterone and fsh, estradiol (e2) and progesterone (p4) in patients with pcos.  references 1. stein if, leventhal ml. amenorrhea associated with bilateral polycystic ovaries. am j obstet gynecol. 1935;29:181. 2. franks s. polycystic ovary syndrome. endocrinol metab clin north am. 1995;28(2):379–408. 3. dahiya k, sachdeva a, singh v, dahiya p, singh r, dhankhar r, et al. reproductive hormone and thyroid hormone profile in polycystic ovarian syndrome. endocrinol. 2012;3(6):41–43. 4. speroff l, glass rh, kase ng. anovulation and polycystic ovary. clinical gynaecologic. endocrinology and infertility. in: speroff lg, kase ng, (eds). baltimore: lippincott williams & wilkins; 1999. pp. 487–521. 5. ehrmann d. polycysticovarysyndrome. n eng j med. 2005;352:1223–1236. 6. vause td, cheung ap, sierra s, claman p, graham j, et al. ovulation induction in polycystic ovary syndrome. j obstet gynaecol can. 2010;32: 495–502. 7. parisi l, framonti m, casciano s, zurli a, gazzarrini o. the role of ultrasound in the study of polycystic ovarian disease. j clin ultrasound. 1982;10(14): 167–172. 8. balen ah, laven js, tan sl, dewailly d. ultrasound assessment of the polycystic ovary international consensus definitions. hum reprod update. 2003;9:505–514. 9. theresa l, marx md, mehta md. polycystic ovary syndrome: pathogenesis and treatment over the short and long term. cleveland clin j med. 2003;70(1):31–41. 10. alhalby a, abdel-salam oa, ahmed zm. weight reduction aids antiepileptic therapy to restore ovarian functions of epileptic polycystic ovary women. j neurol psychol. 2014;2(1):1–6. 11. shah d, rasool s. pcos and metabolic syndrome: the worrisome twosome. endocrinol metab syn. 2015;4:2. 12. burghen ca, givens jr, kitabchi ae. correlation of hyperandrogenism with hyperinsulinemia in polycystic ovarian disease. j clin endocrinol metab. 1980;50:113–116. 13. schuring an, schulte n, sonntag b, kiesel l. androgens and insulin: two key players in polycystic ovary syndrome. gynecol geburtshilfliche rundsch. 2008;48:9–15. 14. lewandowski kg, cajdler-luba a, salata i, bienkiewicz m, lewinski a. the utility of the gonadorophin releasing hormone (gnrh) test in the diagnosis of polycystic ovary syndrome (pcos). endocrinol pol. 2011;62(2):120–128. 15. kanagavalli p, muraliswaran p, sathisha tg, thirunaaukarasu d, lakshmi k. a study to assess hormonal profile of polycystic ovarian syndrome in a tertiary care hospital in puducherry. res j pharm bio chem sci. 2013;4(2):1223–1228. 16. fulghesu am, cucinelli f, pavone v, murgia f, guido m, caruso a, et al. changes in luteinizing hormone and insulin secretion in polycystic ovarian syndrome. hum reprod. 1999;14(3):611–617. 17. melissa h, hunter md, james j, sterrett j, pharm d. polycystic ovary syndrome: it´s not just infertility. american academy of family physicians. 2000. 18. marcondes ja, hayashida sa, barcellos cr, rocha mp, maciel ga, baracat ec. metabolic syndrome in women with polycystic ovary syndrome: prevalence, characteristics and predictors. arq bras endocrinol metab. 2007;51(6):972–979. 19. holte j, bergh t, berne c, lithell h. serum lipoprotein lipid profile in women with the polycystic ovary syndrome: relation to anthropometric, endocrine and metabolic variables. clin endocrinol (oxf). 1994;41:463–471. 20. dunaif a. insulin resistance and the polycystic ovary syndrome: mechanisms and implications for pathogenesis. endocrinol rev. 1997;18: 774–800. 21. azziz r, wood ks, reyna r, key tj, knochenhauer es, yildiz bo. the prevalence and features of the polycystic ovary syndrome in an unselected population. j clin endocrinol metab. 2004;89:2745–2749. 22. kelly c, speirs a, gould gw, petrie jr, lyall h, connell jm. altered vascular function in young women with polycystic ovary syndrome. j clin endocrinol metab. 2002;87:742–746. http://www.ncbi.nlm.nih.gov/pubmed/?term=colak nb%5bauthor%5d&cauthor=true&cauthor_uid=23231775 http://www.ncbi.nlm.nih.gov/pubmed/?term=karbek b%5bauthor%5d&cauthor=true&cauthor_uid=23231775 http://www.ncbi.nlm.nih.gov/pubmed/?term=gungunes a%5bauthor%5d&cauthor=true&cauthor_uid=23231775 http://www.ncbi.nlm.nih.gov/pubmed/?term=tsch%c3%b6p m%5bauthor%5d&cauthor=true&cauthor_uid=12466363 http://www.ncbi.nlm.nih.gov/pubmed/?term=pasquali r%5bauthor%5d&cauthor=true&cauthor_uid=12466363 http://www.ncbi.nlm.nih.gov/pubmed/?term=pasquali r%5bauthor%5d&cauthor=true&cauthor_uid=12466363 hari highlight year is inserted as per pubmed. kindly check. 24 j contemp med sci | vol. 2, no. 5, winter 2016: 20–24 relationship between male testosterone and female pcos hormones research dalal abdul hussain kadium et al. 45. gil junior ab, rezende ap, carmo av, duarte ei, de medeiros mm, de medeiros sf. adrenal androgen participation in the polycystic ovary syndrome. rev bars ginecol obstet. 2010;32(11):541–548. pmid: 21271165 46. feuser cs, barbosa js, da silva eb, de medeiros f. current insights into gonadotropic pituitary function in the polycystic ovary syndrome. asian pacific j reprod. 2014;3(1):64–70. 47. mccartney cr, eagleson ca, marshall jc. regulation of gonadotropin secretion: implications for polycystic ovary syndrome. semin reprod med. 2002;20(4):317–326. 48. adashi e, hsueh a, yen ss. insulin enhancement of lh and fsh levels by cultured pituitary cells. endocrinol. 1981;108:1441–1449. 49. ropelato mg, rudaz mg, escobar me, bengolea sv, calcagno ml, veldhuis jd, et al. acute effects of testosterone infusion on the serum luteinizing hormone profile in eumenorrheic and polycystic ovary syndrome adolescents. j clin endocrinnol metab. 2009;94(9):3602–3610. 50. dunaif a. do androgens directly regulate gonadotropin secretion in the polycystic ovary syndrome? j clin endocrinol metab. 1986;63(1):215–221. 51. banaszewska b, spaczynski rz, pelesz m, pawelczyk l. incidence of elevated lh/fsh ratio in polycystic ovary syndrome women with normoand hyperinsulinemia. rocz akad med bialymst. 2003;48:131–4. 52. cho lw, jayagopal v, kilpatrick es, holding s, atkin sl. the lh/fsh ratio has little use in diagnosing polycystic ovarian syndrome. ann clin biochem. 2006;43(3):217–219. 53. adams j, frank s, polson dw. multifollicular ovaries: clinical and endocrine feature and response to pulsatile gonadotrophin releasing hormone. lancet. 1985;1375–1378. 54. kazer rr, kassel b, yen ss. circulating latinizing hormone pulse frequency in women with polycystic ovary syndrome. j clin endocrinol metab. 1987;65:233–236. 55. taylor ae, mccourt b, martin ka, anderson ej, adams jm, schoenfeld d, et al. determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. j clin endocrinol metab. 1997;82:2248–2256. pmid: 9215302 56. chaudhuri s, maiti br. effect of gonadotropin and prolactine on ovarian activity of a wide ovarian species, the tree pie dendrocitta vagabura. indian j exp biol. 1998;36(8):790–795. pmid: 9838881 57. zumoff b, freeman r, coupey s, saenger p, markowitz m, kream ja. a chronobiologic abnormality in luteinizing hormone secretion in teenage girls with the polycystic ovary syndrome. n engl j med. 1983;309:1206–1209. 58. liu jh, park kh. gonadotrophin and prolaction secretion increases during sleep during the puerperium in nonlactating women. j clin endocrinol metab. 1988;66:839–845. 59. falsetti l, eleftheirou g. hyperinsulinemia in the polycystic ovary syndrome: a clinical endocrine and echographic study in 240 patients. gynaecol endocrinol. 1996;10:319–326. 60. scott mg, ladson jh, green ed, gast mj. hormonal evaluation of female infertility and reproductive disorders. clin chem. 1989;35:620–629. pmid: 2522836 61. michelmore kf, balen ah, dunger db, vessey mp. polycystic ovaries and associated clinical and biochemical features in young women. clin endocrinol (oxf ). 1999;51:779–86. 62. mason hd, willis ds, beard rw, winston rm, margara r, franks s. estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and to concentrations of sex steroids in follicular fluid. j clin endocrinol metab. 1994;79:1355–1360. pmid: 7962330 63. wickenheisser jk, quinn pg, nelson vl, legro rs, strauss jf, mcallister lm. differential activity of the cytochrome p450 17α-hydroxylase and steroidogenic acute regulatory protein gene promoters in normal and polycystic ovary syndrome theca cells. j clin endocrinol metab. 2000;85:2304–2311. 64. urban rj, veldhuis jd, dufau ml. estrogen regulates the gonadotrophin releasing hormone-stimulated secretion of biologically active luteinizing hormone. j clin endocrinol metab. 1991;72:660–668. 65. greespan g, gardner d. basic and clinical endocrinology. j endocrinol inverst. 2001;24:491–498. 66. vale w, wiater e, gray p, harrison c, bilezikjian l, choe s. actives and inhibins and their signaling. ann acad sci. 2004;1038:142–147. pmid: 15838109 67. batista mc, cartledge tp, zellmer aw, nieman lk, merriam gr, loriaux dl. evidence for a critical role of progesterone in the regulation of the midcycle gonadotrophin surge and ovulation. j clin endocrin metab. 1992;74(3): 565–570. 68. lasley bl, wang cf, yen ss. the effects of estrogen and progesterone on the functional capacity of the gonadotrophs. j clin endocrinol metab. 1975;41:820–826. 69. conway g. the polycystic ovary syndrome having identifiable infirmity. department of endocrinology. the middle sex hospital mortimer street london. w1n8aa; 2000. 70. pastor cl, griffin-korf ml, aloi ja, evans ws, marshall jg. polycystic ovary syndrome: evidence for reduced sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone. j clin endocrinol metab. 1998;83(2):582–590. 71. de medeiros sf, gil-junior ab, barbosa js, isaias ed, yamamoto mm. new insights into steroidogenesis in normoand hyperandrogenic polycystic ovary syndrome patients. arq bras endocrinol metab. 2013;57:437–444. http://www.ncbi.nlm.nih.gov/pubmed/14737959 http://www.ncbi.nlm.nih.gov/pubmed/?term=anderson ej%5bauthor%5d&cauthor=true&cauthor_uid=9215302 http://www.ncbi.nlm.nih.gov/pubmed/?term=adams jm%5bauthor%5d&cauthor=true&cauthor_uid=9215302 http://www.ncbi.nlm.nih.gov/pubmed/?term=schoenfeld d%5bauthor%5d&cauthor=true&cauthor_uid=9215302 http://www.ncbi.nlm.nih.gov/pubmed/?term=schoenfeld d%5bauthor%5d&cauthor=true&cauthor_uid=9215302 http://www.ncbi.nlm.nih.gov/pubmed/?term=green ed%5bauthor%5d&cauthor=true&cauthor_uid=2522836 http://www.ncbi.nlm.nih.gov/pubmed/?term=gast mj%5bauthor%5d&cauthor=true&cauthor_uid=2522836 http://www.ncbi.nlm.nih.gov/pubmed/?term=winston rm%5bauthor%5d&cauthor=true&cauthor_uid=7962330 http://www.ncbi.nlm.nih.gov/pubmed/?term=margara r%5bauthor%5d&cauthor=true&cauthor_uid=7962330 http://www.ncbi.nlm.nih.gov/pubmed/?term=franks s%5bauthor%5d&cauthor=true&cauthor_uid=7962330 160 j contemp med sci | vol. 5, no. 3, may–june 2019: 160–164 original comparison between gingival pyogenic granuloma and peripheral giant cell granuloma by immunohistochemical detection of cd34 and alpha smooth muscle actin aween auda ablahad,a ameera kamal khaleel,*b and jasim almahanab aministry of health, erbil, kurdistan region, iraq. bdepartment of dentistry, al-hussein university college, karbala, iraq. *correspondence to ameera kamal khaleel (email: amera1kam@yahoo.com). (submitted: 27 november 2018 – revised version received: 16 december 2018 – accepted: 12 march 2019 – published online: 26 june 2019) introduction oral mucosa is constantly exposed to external and internal stimuli and therefore manifests as a spectrum of diseases ranging from developmental, reactive, and inflammatory to neoplastic.1 localized gingival reactive hyperplastic lesions are classified into four sub-types like pyogenic granuloma (pg), peripheral giant cell granuloma (pgcg), focal fibrous hyperplasia, and peripheral ossifying fibroma.2 reactive lesions are non-neoplastic clinically and histologically and nearly they are clinically similar but possess distinct histopathological features.3 in general, pg is a common localized hyperplastic benign vascular lesion of the oral cavity, manifested as exophytic, sessile, erythematous, and painful nodule that is prone to bleeding and ulceration.4 three quarters of all oral pgs occur on the gingiva, and most of them are in response to gingival inflammation and chronic gingival irritants.5–7 peripheral giant cell granuloma is a painless, soft, reddish-bluish tumor-like reactive lesion, clinically bears resemblance to pg,8 however, it has significantly higher rate of recurrence than other reactive lesions and thus has to be treated with caution with complete excision and clearing of the lesion.9 hematopoietic progenitor cell antigen cd34 also known as cd34 antigen is a protein that in humans is encoded by the cd34 gene. the cd34 protein is a member of a family of single-pass transmembrane sialomucin proteins that show expression on early hematopoietic and vascular-associated tissue.10 cd34 is a surface glycophosphoprotein expressed on developmentally early hematopoietic stem and progenitor cells, small-vessel endothelia and embryonic fibroblasts.10 myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing alpha smooth muscle actin (α-sma), and their activation plays a key role in development of the fibrotic response.11,12 they are involved in morphogenesis, inflammation, fibrosis and oncogenesis in many tissues and organs. myofibroblasts help in extracellular matrix reorganization by the production of numerous inflammatory mediators, growth factors and proteins of the extracellular matrix.13 transdifferentiation of fibroblasts into myofibroblasts is an early event in tumorigenesis and it is mediated by cytokines and growth factors which are expressed by tumor cells.14 the pathogenesis of pg and pgcg remains to be not fully understood. in such conditions, immunohistochemistry may provide some practical help and shed a light on the underlying pathogenesis of these lesions. the aim of this research was to study the immunohistochemical expressions of cd34 and α-sma for gingival pyogenic granuloma in comparison with peripheral giant cell granuloma. in such conditions, the immunohistochemistry may provide some practical help and shed a light on the underlying pathogenesis of these lesions. materials and methods the materials used in this study were consisting of 48 formalin fixed, paraffin-embedded biopsy specimens of gingival pg and 39 formalin fixed, paraffin-embedded biopsy specimens of pgcg. they were retrieved from the archives of rizgary teaching hospital, erbil (ministry of health, kurdistan region of iraq) in the period between january, 2013 and objectives the aim of this research was to study the clinical and the immunohistochemical expressions of cd34 and alpha smooth muscle actin (α-sma) for gingival pyogenic granuloma in comparison with peripheral giant cell granuloma. methods formalin fixed, paraffin-embedded biopsy specimens of 48 gingival pyogenic granuloma and 39 peripheral giant cell granuloma were used in the study. immunohistochemical analysis for cd34 and α-sma were studied in pyogenic granuloma (pg), peripheral giant cell granuloma (pgcg). results the mean numbers of the cd34 positive microvessels in pgs and pgcgs were 32.58 ± 17.778 and 22.4 ± 11.208, respectively. statistical analysis showed a highly significant difference present between them (p < 0.01). the mean numbers of blood vessels with vascular surrounding cells non-reactive to α-sma in pgs and pgcgs were 3.81 ± 2.228 and 10.53 ± 3.432, respectively. statistical analysis showed a highly significant differences present between them (p < 0.01). conclusion the mean number of cd34 positive microvessels in pgs was significantly more than that of pgcg, but the mean number of vascular surrounding cells non-reactive to α-sma was significantly less. this can add insight to the clinical behavior and might reflect the differences in pathogenesis of these lesions. keywords pyogenic granuloma, peripheral giant cell granuloma, cd34, α-sma issn 2413-0516 161j contemp med sci | vol. 5, no. 3, may–june 2019: 160–164 original comparison between gingival pyogenic granuloma and peripheral giant cell granulomaa.a. ablahad et al. august, 2016. ten control gingival samples were obtained from clinically healthy patients undergoing orthodontic extractions in erbil specialized dental center. the written consent to carry out biopsies which were required for the study was obtained from healthy volunteers, after the necessary instructions. demographic data and clinical aspects were registered in a special form, and only patients with biopsy proven gingival pg or pgcg were included. the pregnant and edentulous patients (epulis fissuratum) and patients with known systemic disorders such as diabetes and bleeding disorders were excluded from the study. sample collection was authorized by rizgary teaching hospital, ministry of health. the research project was approved by the research ethics committee at college of dentistry, hawler medical university under protocol. novolink™ polymer detection system codes re7140-k from leica microsystems (uk) which includes monoclonal mouse anti-human cd34 (clone qbend/10, dilution 1:100), and an anti-α-sma monoclonal antibody (clone 1a4, diluted 1:100; dako corporation, carpinteria, usa) were used. the staining procedure and the instructions included with each detection system were followed. after necessary data had been collected, the results were given as mean ± standard deviation. the potential difference among groups for histopathological data was evaluated using anova test. all statistical calculations were done using computer programs statistical package for the social science (spss inc., version 19). statistical significance of differences between the groups was tested with the mann–whitney u test. p-value ≤0.05 was considered statistically significant. results the samples used in this study consist of 48 cases of histologically proven pgs and 39 cases of histologically proven pgcgs. the age and sex distribution for both cases are seen in fig. 1. the maxilla was mostly affected by pg (66.7%), followed by the mandible (33.3%), 39.6% cases showed a maximum diameter <1.5 cm, 52.1% showed a maximum diameter 1.5–3 cm, and only 8.3% showed a maximum diameter >3 cm. the results also showed that the mandible was mostly affected by pgcg (72.4%), followed by the maxilla (27.6%). about 39.9% cases showed a maximum diameter <1.5 cm, 48.7% showed a maximum diameter of 1.5–3 cm, and only 15.4% showed a maximum diameter >3 cm. histopathological pictures hematoxylin and eosin results the pyogenic granuloma revealed hyperplastic keratinized stratified squamous epithelium with some areas of epithelial atrophy or ulcerations. the most important features are the occurrence of large numbers of endothelium-lined vascular spaces and the extreme proliferation of fibroblasts cells and inflammatory cells (fig. 2). microscopic examination of the sections of pgcg showed the presence of hyperplastic keratinized stratified squamous epithelium, and the overlying mucosal surface was ulcerated in some areas. histopathologically, fibroblasts in the stroma form a basic element of the lesion and are plump oval to spindle-shaped. multi-nucleated giant cells of variable shapes and sizes and containing multiple nuclei were seen scattered throughout the connective tissue stroma (fig. 3). the connective tissue stroma also reveals some vascularity with different types of inflammatory cell infiltration, and foci of hemorrhage were also observed. immunohistochemical results the number of cd34 positive microvessels was considered as a mvd. any brown staining of endothelial cells or cluster of endothelial cells with or without a lumen that is clearly separate from adjacent microvessels and other connective tissue elements is considered as a single vessel. branching structure fig. 1 age and sex distribution of (a) pyogenic granuloma and (b) peripheral giant cell granuloma. fig. 2 photomicrograph ofpyogenic granuloma with hyperplastic epithelium that overlies a connective tissue that contains numerous inflammatory cells and blood vessels (a1: h&e 100×). fibrodvascular connective tissue consisting of numerous endothelium-lined vascular spaces engorged with red blood cells (arrows), and numerous fibroblasts infiltrated with inflammatory cells (a2: h&e 400×). a b 162 j contemp med sci | vol. 5, no. 3, may–june 2019: 160–164 comparison between gingival pyogenic granuloma and peripheral giant cell granuloma original a.a. ablahad et al. was counted as a single vessel unless there was a break in the continuity of the structure. all samples used in the study demonstrated positive reaction for cd34 (figs. 4 and 5). the mvd for pg and pgcg was ranging (7.7–63.9) and (6.9– 46.4) respectively. statistical analysis of the mvd for pg showed no significant relation with the gender, site and the maximum diameter of the lesions. statistical analysis of mvd for pgcg showed significant relation (p < 0.05) with the gender, but a non-significant relations present with the site and the maximum diameter of the lesions (p > 0.05) as seen in table 1. the mean number of the cd34 positive microvessels in pgs, pgcgs, and normal gingiva was 32.58 ± ,17.778 11.208± 22.4, and 1.197 ± 8.21, respectively. statistical analysis showed a highly significant difference present between pg and normal gingiva, pg and pgcg, pgcg and normal gingiva regarding the mvd (p < 0.01) as seen in table 2. in this study, α-sma positive stromal cells and vascular surrounding spindle cells that showing brown cytoplasmic immunostaining, were considered to be myofibroblasts. vascular surrounding cells and other stromal mesenchymal cells in all samples of normal gingiva revealed immune negativity to α-sma. some of the vascular surrounding cells in pgs and pgcgs studied revealed immune positivity to α-sma especially in pg, but the other stromal mesenchymal cells were negative in both pg and pgcg. the number of blood vessels with vascular surrounding cells non-reactive to α-sma was counted. in pg, the number of non-reactive blood vessels was ranging 1.3–9.4. while in pgcg the number of non-reactive blood vessels was ranging 4.8–18.2. statistical analysis for pg and pgcg showed no significant relation with the gender, site and the diameter of the lesions (p > 0.05) as seen in table 3. the mean number of blood vessels with vascular surrounding cells non-reactive to α-sma in pg was 3.81 ± 2.228, but in pgcg, it was 10.53 ± 3.432. statistical analysis showed a highly significant differences present between pg and pgcg regarding the number of blood vessels with vascular surrounding cells non-reactive to α-sma (p < 0.01) as seen in table 4. discussion pyogenic granuloma and peripheral giant cell granuloma are common lesions of oral cavity. the pathogenesis remains to be fig. 3 photomicrograph of peripheral giant cell granuloma, revealed hyperplastic epithelium that overlies vascular connective tissue which contain numerous multinucleated giant cells (a1: h&e 100×). abundant multinuclenated giant cells (upper arrow) together with inflammatory cell infiltration and areas of hemorrhage (lower arrow) are also seen (a2: h&e 400×). a b fig. 4 photomicrographs revealed the microvessels expressed by cd34 marker in the pyogenic granuloma (arrows) (a1: immunohistochemistry 100×; a2: immunohistochemistry 400×). a b fig. 5 photomicrographs revealed the microvessels expressed by cd34 marker in the peripheral giant cell granuloma (arrows) (a1: immunohistochemistry 100×; a2: immunohistochemistry 400×). a b 163j contemp med sci | vol. 5, no. 3, may–june 2019: 160–164 original comparison between gingival pyogenic granuloma and peripheral giant cell granulomaa.a. ablahad et al. table 3. distribution of the mean and standard deviations of the number of blood vessels with vascular surrounding cells non-reactive to α-sma in relation to the gender, location, and the maximum diameter of pyogenic granuloma and peripheral giant cell granuloma. clinical parameter pg pgcg x ± sd p-value x ± sd p-value gender male 3.90 ± 1.973 0.548 11.43±3.614 0.241 female 4.73 ± 2.458 >0.05 9.91 ± 3.232 >0.05 ns ns site maxilla 3.94 ± 2.208 0.892 10 ± 3.338 0.558 mandible 3.77 ± 2.538 >0.05 10.85 ± 3.26 >0.05 ns ns table 4. the relations between the pyogenic granuloma and peripheral giant cell granuloma for α-sma. α-sma no. (negative) no. (positive) x ± sd p-value pg 0 48 3.81 ± 2.228 0.0001 pgcg 0 39 10.53 ± 3.432 *<0.0001 hs *comparison between pg and pgcg. x: mean, sd: standard deviation, no: number, hs: highly significant. table 1. distribution of the mean and standard deviations of microvessel density in relation to the gender, location, and the maximum diameter of pyogenic granuloma and peripheral giant cell granuloma clinical parameter pg pgcg x ± sd p-value x ± sd p-value gender male 26.79 ± 15.97 0.091 18.56 ± 12.047 0.03 female 37.06 ± 18.154 >0.05 25.06 ± 9.994 <0.05 ns s site maxilla 30.52 ± 16.964 0.852 25.5 ± 12.074 0.379 mandible 31.27 ± 19.216 >0.05 22.01 ± 11.806 >0.05 ns ns maximum diameter (cm) <1.5 35.44 ± 18.363 0.591 18.23 ± 9.807 0.131 1.5–3 30.82 ± 17.15 >0.05 24.22 ± 11.727 >0.05 >3 27.3 ± 21.463 ns 26.35 ± 11.39 ns pg: pyogenic granuloma, pgcg: peripheral giant cell granuloma, x: mean, sd: standard deviation, cm: centimeter, ns: non-significant, s: significant. table 2. the relations between the pyogenic granuloma, peripheral giant cell granuloma and normal gingiva for microvessel density cd34 (mvd) no. (negative) no. (positive) x ± sd p-value pg 0 48 32.58 ± 17.778 0.0001 *<0.01 hs 0.0001 pgcg 0 39 22.4 ± 11.208 **<0.01 hs 0.0001 normal gingiva 0 10 8.21 ± 1.197 ***<0.01 hs *comparison between pg and normal gingiva. **comparison between pg and pgcg. ***comparison between pgcg and normal gingiva. x: mean, sd: standard deviation, no: number, hs: highly significant. continued maximum diameter (cm) <1.5 3.73 ± 1.995 0.119 10.92 ± 3.709 0.143 1.5–3 4.4 ± 2.415 >0.05 11.05 ± 2.837 >0.05 >3 2 ± 0.816 ns 8 ± 3.949 ns pg: pyogenic granuloma, pgcg: peripheral giant cell granuloma, x: mean, sd: standard deviation, cm: centimeter, ns: non-significant. not fully understood. in such conditions, immunohistochemistry may provide some practical help and shed a light on the underlying pathogenesis of these lesions. cd34 immunostaining distribution in this study, the mean value of mvd of normal gingiva specimens was 8.21 ± 1.197. our results were nearly similar to seyedmajidi et al.’s15 result; they found the mvd of all samples of healthy gingiva was 7.95 ± 5.56. the mean value of mvd of samples of pgs was 32.58 ± 17.778. our results were less than vasconcelos et al.’s16 result which was 48.09 ± 30.031, but more than that of seyedmajidi et al.’s15 which was 20.01 ± 11.88. the increased expression of cd34 can be attributed to the increase in the number of blood vessels in pyogenic granuloma and appears to be involved in pathogenesis of oral pyogenic granuloma.15 the mean number of mvd of samples of pgcg was 22.4 ± 11.208. our results were less than hallikeri et al.’s17 result which was 30.05 ± 8.006. the higher expression of cd34 biomarkers in pg as a vessel-rich lesion compared with normal gingiva and pgcg, illustrates the role of these molecules as angiogenesis related markers. the variation of mean mvd among the groups suggests that angiogenesis may be one of the mechanisms possibly contributing to the different biological behavior, architecture or pattern of growth, and may be an important step for the study of new therapy.15 alpha-smooth muscle actin immunostaining distribution the most frequently used myofibroblast marker is α-sma.18 the number of blood vessels with vascular surrounding cells non-reactive to α-sma in pg was significantly less than that of pgcg. most of the small, large blood vessels and abnormal blood vessels and spaces in pg had two or more outer layers of mesenchymal cells (myofibroblast) positive for α-sma. the pattern of the distribution of these mononuclear myofibroblastic cells suggested that these cells might play a role in generating of newly formed blood vessels and spaces, since pg has more blood vessels in comparison with pgcg. epivatianos19 and kawachi20 also found that most perivascular spindle cells in pgs studied were strongly stained to α-sma. 164 j contemp med sci | vol. 5, no. 3, may–june 2019: 160–164 comparison between gingival pyogenic granuloma and peripheral giant cell granuloma original a.a. ablahad et al. the stromal mesenchymal cells between the blood vessels showed no reactivity to α-sma. despite the similarity of pg to granulation tissue, we did not detect any stromal myofibroblasts in all samples of pg. our results come in agreement with damasceno et al.,12 they did not noticed any stromal myofibroblast in pg. regarding the pgcg, this study showed that stromal spindle cells which morphologically resemble myofibroblasts were negative for α-sma, this result agree with that of damasceno et al.’s12 study, but disagree with filioreanu et al.21 and kujan22 studies, they found that smooth muscle specific actin was strongly stained in the spindle mononuclear cells (myofibroblasts) distributed through the lesion. the sub-classification of blood vessels into immature, intermediate and mature is required as this important using the combination of immunohistochemistry of cd34 and α-sma stain to demonstrate pericyte.17 this is required to differentiate the intermediate blood vessels from the mature blood vessels. since immature and intermediate blood vessels are considered as an indicator of the degree of angiogenic activity, and are the main target of anti-angiogenic therapy and not the mature blood vessels, quantification of immature blood vessels may be helpful in estimation of prognosis especially for agents that do selectively target angiogenic endothelial cells, information may provide additional evidence of therapeutic anti-vascular effect for the control and prevention of the growth by inhibition of angiogenesis by anti angiogenic therapy could be a potent therapeutic strategy. conclusion pyogenic granuloma showed more microvessel density and lesser number of blood vessels with vascular surrounding cells non-reactive to α-sma were seen. this can add insight to the clinical behavior and might reflect the differences in pathogenesis of these lesions. conflicts of interest none.  references 1. neville bw, damm dd, allen cm, bouquot je. oral and maxillofacial pathology. 2nd ed., 2005, pp. 371–372. 2. reddy v, saxena s, saxena s, reddy m. reactive hyperplastic lesions of the oral cavity: a ten year observational study on north indian population. j clin exp dent. 2012;4:e136–e140. 3. kamal r, dahiya p, puri a. oral pyogenic granuloma: various concepts of etiopathogenesis. j oral maxillofac pathol. 2012;16:79–81. 4. saghafi s, amoueian s, montazer m, bostan r. assessment of vegf, cd-31 and ki-67 immunohistochemical markers in oral pyogenic granuloma: a comparison with hemangioma and inflammatory gingivitis. iran j basic med sci. 2011;14:185–189. 5. jafarzadeh h, sanatkhani m, mohtasham n. oral pyogenic granuloma: a review. j oral sci. 2006;48:167–175. 6. gomes sr, shakir qj, thaker pv, tavadia jk. pyogenic granuloma of the gingival: a misnomer? a case report and review of literature. j indian soc periodontol. 2013;17: 514–519. 7. sharma a, mathur vp, sardana d. effective management of a pregnancy tumour using a soft tissue diode laser: a case report. laser ther. 2014;23:279–282. 8. ramu s, rodrigues ch. reactive hyperplastic lesions of the gingiva: a retrospective study of 260 cases. world j dent. 2012;3:126–130. 9. motamedi mh, eshghyar n, jafari sm, lassemi e, navi f, abbas fm, et al. peripheral and central giant cell granulomas of the jaws: a demographic study. oral surg oral med oral pathol oral radiol endod. 2007;103:e39–e43. 10. gold m, blanchet m-r, samayawardhena la, bennett j, maltby s, pallen cj, et al. cd34 function in intracellular signaling and mucosal inflammatory disease development. allergy asthma clin immunol. 2010;6:p15. 11. cherng sh, young j, ma h. alpha-smooth muscle actin (α-sma). j am sci. 2008;4:7–9. 12. damasceno ls, gonçalves fda s, costa e silva e, zenóbio eg, souza pe, horta mc. stromal myofibroblasts in focal reactive overgrowths of the gingiva. braz oral res. 2012;26:373–377. 13. rao k b, malathi n, narashiman s, trajan sh. evaluation of myofibroblasts by expression of alpha smooth muscle actin: a marker in fibrosis, dysplasia and carcinoma. j clin diagn res. 2014;8:zc14–zc17. 14. amatangelo md, bassi de, klein-szanto aj, cukierman e. stromaderived three-dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts. am j pathol. 2005;167:475–488. 15. seyedmajidi m, shafaee s, hashemipour g, bijani a, ehsani h. immunohistochemical evaluation of angiogenesis related markers in pyogenic granuloma of gingiva. asian pac j cancer prev. 2015;16: 7513–7516. 16. vasconcelos mg, alves pm, vasconcelos rg, da silveira éjd, medeiros amc, de queiroz lmg. expression of cd34 and cd105 as markers for angiogenesis in oral vascular malformations and pyogenic granulomas. eur arch otorhinolaryngol. 2011;268:1213–1217. 17. hallikeri k, acharya s, koneru a, trivedi d. evaluation of microvessel density in central and peripheral giant cell granulomas. j adv clin res insights. 2015;2:20–25. 18. hinz b. formation and function of the myofibroblast during tissue repair. j invest dermatol. 2007;127:526–537. 19. epivatianos a, antoniades d, zaraboukas th, zairi e, poulopoulos a, kiziridou a, et al. pyogenic granuloma of the oral cavity: comparative study of its clinicopathological and immunohistochemical features. pathol int. 2005;55:391–397. 20. kawachi n. a comparative histological and immunohistochemically study of capillary hemangioma, pyogenic granuloma and cavernous hemangioma in oral region with special reference to vascular proliferation factors. int j oral med sci. 2011;9:241–251. 21. filioreanu am, popescu e, cotrutz c, cotrut ce. immunohistochemical and transmission electron microscopy study regarding myofibroblasts in fibroinflammatory epulis and giant cell peripheral granuloma. rom j morphol embryol. 2009;50:363–368. 22. kujan o, al-shawaf az, azzeghaiby s, almanadille a, aziz k, raheel sa. immunohistochemical comparison of p53, ki-67, cd68, vimentin, α-smooth muscle actin and alpha-1-antichymotrypsin in oral peripheral and central giant cell granuloma. j contemp dent pract. 2015;16:20–24. dx.doi.org/10.22317/jcms.06201908 103j contemp med sci | vol. 2, no. 7, summer 2016: 103–106 research study of antibacterial activity of lawsonia inermis leaf extract alaa abdul hussein kareem al-daamy,a ali abdul hassan,a ali mahmooda issn 2413-0516 introduction bacteria are microorganisms that cause disease and some of which cause fatal diseases in humans. every year millions of people die because of these microorganisms. the increasing of bacteria inside the human body takes advantage of any weakness found in body organs.1 in the past decade, interest on the topic of antimicrobial plant extract has been growing, and the use of herbal medicines in asia represents a long history of human interaction with the environment. the plant used for traditional medicine contains a wide range of substance that can be used to treat chronic as well as infectious disease. a knowledge of how to use the plant against the different illness may be expected to have accumulated in areas where the use of plant is still of great importance the medicinal value of plant lies in some chemical substance that produce a definite physiological action on the human body. the most important of these bioactive compounds of plant are alkaloids, flavonoids, tannins and phenolic.2 the extract of leaves of lawsonia inermis was applied on woollen yarn to investigate the dying characteristics and antimicrobial efficacy against common human pathogens such e. coli, staphylococcus aureus, candida albicans.3 the active component of lawsonia inermis is lawsone (2-hydroxy1,4 naphthoquinone, cas 83-72-7), which is also the principal dye ingredient. current research suggests that lawsone is non-problematic for external use because of it slow toxicity and genotoxicity.4 the bioactive of lawsonia inermis compound with ampicillin (antibacterial) and flucnecoul (antifungal) where found considerably active against test microorganism.3 materials and methods microorganisms five pathogenic bacteria due to disease caused by it are obtained from patient from al-hussein hospital, and given adepartment of clinical laboratories, college of applied medical sciences, university of karbala, karbala, iraq. correspondence to alaa abdul-hussein al-daamy (email: aakm7789@gmail.com). (submitted:3 may 2016 – revised version received: 5 july 2016 – accepted: 28 july 2016 – published online: 26 september 2016) objectives the present study aims to determine antibacterial activity for lawsonia inermis leaf extract against some pathogenic bacteria. methods five solvents used are acetone, ethanol, methanol, ethyl acetate and distilled water to obtain of crude extract of lawsonia inermis leaves, which tested the effectiveness on five types of bacteria are pseudomonas eroginosa, pseudomonas oryzihabita, proteus varaplis, klebsila pneumonia and staphylococcus aureus to determine the most efficient solvent extraction of them, and then was use of a series of concentrations of the solvent is more efficient 0, 20, 40, 60, 80 and 100% to determine the most efficient concentration of solvent optimization, and then was determined the efficiency of minimum inhibitory concentration (mic) of the extract. results the results of the current study showed that the most efficient in the extraction solvent is acetone and the diameters of inhibition zone are 18, 19.83, 17.16, 16.33, 16.5 mm for the types of bacteria above, respectively. the results showed that the concentration of acetone 100% is the best concentration in extraction, amounting to the diameters of inhibition zone at this concentration of 17.33, 20, 19.33, 17.66, 21.66 mm for each of the bacterial species above, respectively. the results also found that mic is 6 mg/ml of pseudomonas eroginosa and 7 mg/ml of pseudomonas oryzihabita and 11 mg/ml of each proteus varaplis, klebsila pneumonia and staphylococcus aureus. conclusion the most effective composites against pathogenic bacteria from lawsonia inermis leaves are using acetone solvent with concentration of 100%. keywords acetone, lawsonia inermis, antibacterial activity, mic numbers 1, 2, 3, 4, and 5. these bacteria are pseudomonas eroginosa, pseudomonas oryzihabita, proteus varaplis, klebsila pneumonia, and staphylococcus aureus. the extraction process we take the dry leaves of lawsonia inermis that obtained from al-fawo farms in basra city, iraq. they were blended into powder by electric blender. then, we take 100 g of this powder and distributed to five beakers each beaker containing 20 g of powder, then it was put in 100 ml of solvent 70% (the ratio of extraction is 1 g: 5 ml) as was the use of solvents are five: acetone, ethanol, methanol, ethyl acetate and distilled water. each beaker was labeled with its solvent name, and incubating it in shaking incubator overnight with 150 cycle/minute at 37°c. this method used for extraction were described by al-daamy et al.5 after 24 hr of shaking, the extract purified by cotton and gauze in other beaker (the filtration must be quietly with pressing the gauze to obtain all the substance) then pour the pre-collected substance of each beaker in tubes and centrifuge them in 3000 rpm for 5 minutes. then pour the substance in the tubes in glass petri dishes carefully without moving the precipitated material, and label each petri dish with the name of the solvent. let the dishes to be dry (these take some time if the weather is cold and the temperature is less than 37°c). after drying of all dishes, we scrabbled each dish to obtain the extract powder. after that, we weighed each extract powder and recorded it to calculate the percentage of extraction and what is the best solvent used (we saved it in separated containers labeled with solvent name). determination of the best solvent for extraction from the pre-collected powder, we weighed 0.05 g from each container and put it in separate five tubes, and each tube contains extract of certain type of our five solvents. add on each 104 j contemp med sci | vol. 2, no. 7, summer 2016: 103–106 study of antibacterial activity of lawsonia inermis leaf extract research alaa abdul hussein kareem al-daamy et al. after that, take a micro titter plate with 96 wells and numbered the wells from 0 to 25 μl, twice for each type of bacteria (each type of bacteria has 52 wells twice for each number), then added 150 μl from each tube in two wells of the same number on each type of bacteria, then added 50 μl of each type of bacteria (after dilute it with both media and take from the 3rd dilution comparison with mcfarland solution) in all the 52 wells from 0 to 25 μl concentrations. after that, cover the plates and incubate them in the incubator for 24 hr in 37°c. then examine the plates to see growth or no growth of bacteria and determine the minimal inhibitory concentration from extract for each type of bacteria.7 statistical analysis statistical analysis included random complete design (rcd) with three replicates. 0.05 is the level of probability that used to identify a significant difference. the significant differences between the averages were also tested by using the test less significant difference (lsd) at the level of probability of 0.05.8 results the results in table 1 show that the percentage of substances using extraction are 2.35, 2.0, 1.7, 2.5, 1.75% by using each of solvents acetone, ethanol, methanol, ethyl acetate, distilled water; respectively. in these results, unclear (show) that the maximum percentage of extraction is 2.35% by using ethyl acetate, and the minimal percentage of extraction is 1.7% using methanol. after determining acetone as the best solvent in the extraction, the results in table 2 shows the extract of, lowsonia inermis leaves with acetone have a larger diameter inhibition zone on all types of bacteria 18, 19.83, 17.16, 16.33, 16.5 mm for each types of bacteria pseudomonas eroginosa, pseudomonas oryzihabita, proteus varaplis, klepsila pneumonia, staphylococcus aureus, respectively, with significant differences (p < 0.05) against the control gentamycin and also against other solvents using. tube 2 ml of ethanol 70% and shaking the tube until the powder melting, the final concentration of extract in each tube equal 25 mg/ml. in addition to our five tubes, we added a sixth tube containing 2 ml for control (70% ethanol without any extract). we prepare 30 petri dishes of muller hinton agar and pour 20 ml in each dish and drill three wells in the media with a diameter of 5 mm to each well. after activation of bacteria in nutrient broth, we culture it in the prepared dishes. in each petri dish, 100 μl of each activation bacteria was added and spread it by sterilized spreader, then added 50 μl plant extract in each well. in the negative control, we added just the 50 μl ethanol 70%, also we used 50 μg/ml gentamycin as a positive control. after culturing, we incubated all the dishes in an incubator for 24 hr in 37°c. then, examine the inhibition zone of each well in each dish. the results were recorded to determine the best solvent that has a high activity against examined bacteria.6 determine the optimal concentration of the best solvent after determining the best solvent which is acetone, in five beakers with different concentrations of acetone 20, 40, 60, 80, 100%, 20 g of lawsonia inermis extract powder was added, and repeated the same process of extraction method which described above and recorded the results. in the next step, in five tubes put 0.05 g of dry extract in each tube, then add 2 ml of 70% ethanol to each tube to obtain on the final concentration 25 mg/ml. after that, we prepared 30 petri dishes with muller hinton agar and follow the three tests method to determine the best concentration of acetone that has a largest inhibition zone.6 determine the minimal inhibitory concentration (mic) prepare two flasks: • first flask has 50 ml nutrient broth media. • second flask have 25 ml nutrient broth and added to it 0.625 g from acetone 100% dry extract to obtain on 25 mg/ml concentration in this flask from extraction. then 26 glass tubes were labelled with numbers from 0 to 25. on each tube put the extract from lawsonia inermis with acetone 100% concentration equivalent to the number of the label of the tube. for example: in tube number 25 put only 2 ml from flask 2 which represents 25 mg/ml concentration. in tube number 24 put 1.92 ml from flask 2 and 0.08 ml from flask 1 which represents 24 mg/ml concentration, so that with other tubes, in the tube number 0 put only 2 ml from flask 1 to obtain of 0 mg/ml in this tube. the distribution between flask 1 and 2 in these tubes according to the law c1 v1 = c2 v2. table 1. the percentage of materials extracted from lawsonia inermis leaves percentage of extract materials (%) weigh of extract (g) origin weight of plant powder (g) extraction solvent (100 ml) no. 2.350.4720acetone1 2.00.4020ethanol2 1.70.3420methanol3 2.50.520ethyl acetate4 1.750.3520distil water5 table 2. inhibition zone (mm) to extract lawsonia inermis leaves against bacteria bacteria extraction solvent (70%) lsd 0.05gentamycin 10 μg/ml acetone ethanol methanol ethyl acetate distil water pseudomonas eroginosa 21.66 ± 0.88 18 ± 2.08 0 ± 0 0 ± 0 0 ± 0 0 ± 0 2.32 pseudomonas oryzihabita 20.33 ± 0.88 19.83 ± 1.36 10 ± 0.2 15.33 ± 1.45 0 ± 0 0 ± 0 2.24 proteus varaplis 12.5 ± 0.28 17.16 ± 1.16 15.6 ± 2.42 0 ± 0 0 ± 0 0 ± 0 2.78 klebsila pneumonia 17.66 ± 0.33 16.33 ± 0.44 10.5 ± 0.86 14.66 ± 0.6 15.3 ± 0.61 0 ± 0 1.37 staphylococcus areus 17 ± 0.57 16.5 ± 1.8 14.5 ± 1.25 0 ± 0 13.83 ± 0.6 0 ± 0 2.41 alaa abdul hussein kareem al-daamy et al. 105j contemp med sci | vol. 2, no. 7, summer 2016: 103–106 research study of antibacterial activity of lawsonia inermis leaf extract table 4. minimum inhibitory concentration (mic) of acetonic extract lawsonia inermis leaves types of bacteria staphylococcus aureus klebsila pneumonia proteus varaplis pseudomonas oryzihabita pseudomonas eroginosa extract concentration (mg/ml) +++++ 1 +++++ 2 +++++ 3 +++++ 4 +++++ 5 ++++− 6 +++−− 7 +++−− 8 +++−− 9 +++−−10 −−−−−11 −−−−−12 −−−−−13 −−−−−14 −−−−−15 −−−−−16 −−−−−17 −−−−−18 −−−−−19 −−−−−20 −−−−−21 −−−−−22 −−−−−23 −−−−−24 −−−−−25 +, means growth; −, means not growth. is 6 mg/ml, and for pseudomonas oryzihabita is 7 mg/ml, while for bacteria proteus varaplis, klebsila pneumonia, staphylococcus aureus is 11 mg/ml. discussion lawsonia inermis leaves have antibacterial activity against many types of bacteria including staphylococcus aureus, klebsila pneumonia, where methanol was used as solvent.4 in comparison with the present study, lawsonia inermis leaves which consist of alkaloids, flavonoids, tannins, and phenolic,2 using acetone 100%. in our study, we found in the results that lawsonia inermis leaves have minimum inhibitory concentration for staphylococcus aureus and klebsila pneumonia is 11 mg/ml, but in other study the minimum inhibition concentration for these bacteria is 25 mg/ml,4 that mean our extract has more activity than that test, and this contrasts may be due to the differences in the solvent and the difference in concentration in the solvent. also the other research showed that lawsonia inermis leaves have antibacterial activity against pseudomonas aeruginosa and escherichia coli, using methanol as solvent.9 comparing this results with our results using lawsonia inermis leaves with acetone 100% in our study and methanolic extract in the other research,9 we see that our extract has antibacterial activity against pseudomonas aeruginosa in mueller hintone agar well diffusion test and the diameter of inhibition zone is 17.33 mm, but in other test,9 the diameter of inhibition zone of lawsonia inermis leaves with methanol against pseudomonas aeruginosa is 15 mm, that mean our extract is more active than methanolic extract, and this is either due to the less activity of their solvent than our solvent or due to the concentration of methanol that they have been used which is less activity. other research showed that lawsonia inermis leaves have antibacterial activity against staphylococcus aureus, pseudomonas aeruginosa, and klebsila pneumonia using methanolic extract.10 when compare our research with this research, we find that in this research with considering the concentration of lawsonia inermis leaves with methanol is 1000 μg/ml, where the diameter of inhibition zone of staphylococcus aureus is 24 mm, and of pseudomonas aeruginosa is 17 mm, and of klebsila pneumonia is 18 mm. but in our test with considering we use lawsonia inermis leaves with acetone 100%, where the diameter of inhibition zone of staphylococcus aureus is 21.66 mm, and of pseudomonas aeruginosa is 17.33 mm, and of klebsila pneumonia is 17.66 mm, that mean in that test their extract more active than our extract against staphylococcus aureus, and few more than our extract against klebsila pneumonia, but our extract the results in table 3 shows the extract of plant with acetone 100% have a larger diameter inhibition zone on all types of bacteria 17.33, 20, 19.33, 17.66, 21.66 mm for bacteria pseudomonas eroginosa, pseudomonas oryzihabita, proteus varaplis, klebsila pneumonia, staphylococcus aureus, respectively, with significant differences (p < 0.05) against gentamycin, also with significant differences (p < 0.05) against other solvents. the results in table 4 shows that the minimal concentration for inhibition (mic) for bacteria pseudomonas eroginosa table 3. inhibition zone (mm) of extract lawsonia inermis leaves against bacteria by using dilutions series of acetone bacteria acetone ratio (%) lsd 0.05gentamycin 10 μg/ml 20% 40% 60% 80% 100% pseudomonas eroginosa 21.66 ± 0.88 0 ± 0 5.5 ± 0.28 7.33 ± 0.88 11.16 ± 0.72 17.33 ± 0.72 1.68 pseudomonas oryzihabita 20.33 ± 0.88 0 ± 0 14.66 ± 0.6 17.5 ± 1.5 19 ± 0.57 20 ± 1.66 1.98 proteus varaplis 12.5 ± 0.28 0 ± 0 12.5 ± 0.28 14.16 ± 0.6 15.33 ± 0.16 19.33 ± 0.62 0.98 klebsila pneumonia 17.66 ± 0.33 9 ± 0.28 12.83 ± 0.72 14 ± 1.2 16.5 ± 1.32 17.66 ± 0.33 1.94 staphylococcus aureus 17 ± 0.57 11.83 ± 0.16 13.33 ± 0.83 13.66 ± 0.16 14.33 ± 0.16 21.66 ± 0.88 1.41 106 j contemp med sci | vol. 2, no. 7, summer 2016: 103–106 study of antibacterial activity of lawsonia inermis leaf extract research alaa abdul hussein kareem al-daamy et al. has more activity than their extract against pseudomonas aeruginosa. this contrast is due to type of solvent and concentration of solvent. conclusion from the results of the present study, we concluded that the extract of lawsonia inermis leaves by acetone 100% has high antibacterial activity against the pathogenic bacteria that isolated from patients. recommendations other study on lawsonia inermis leaves is to purify the compound, which owns the effectiveness of antibacterial pathogenesis. n references 1. beukel dvd. traditional mehndi designs a treasury of henna body art. boston: shambhala, 2000. isbn 978-1570625589. 2. nadia tb, hind ho, amal ma, aiman ah, zainab aa. cytotoxicity effect of aqueous extract of brassica rapa roots on cancer cell lines in vitro. iraqi j sci. 2010;51(4):550–560. 3. mohd y, aijaz a, mohammed s, mohd ik, shafat ak, nikhat a. assessment of colorimetric, antibacterial and antifungal properties of woollen yarn dyed with the extract of the leaves of henna (lawsonia inermis). j clean product. 2015;7: 42–50. doi: 10.1016/j. jclepro.2012.01.005. 4. al-daamy aa, abd-al ameer h, zuher h, monather h, ahmmed b, kadhim n. antifungal activity of propolis against dermatophytes and candida albicans isolated from human mouth. j contemp med sci 2015;1(3):4–8. 5. al-daamy aak, al-shibli mk, al-ghanimi aa. purification and characterization of kojic acid produced by two local isolates (aspergillus flavus and aspergillus fumigatus). thesis. college of education, university of al-qadesiah, iraq. 2014. 6. stephen j, cavalieri e. manual of antimicrobial susceptibility testing. am soc microbiol. 2005. 7. dequ g, tessema f. biostatistics. university gondar, funded under usaid from the american people, cooperative agreement no. 663-a-00-00-0358-00. 2005. 8. abdelraouf ae, amany aa, nedaa aag. antibacterial antifungal and synergistic effect of lawsonia inermis, punica granatum and hibiscus sabdariffa. ann alquds med. 2011;7:33–41. 9. arun p, purushotham kg, johnsy jj, vasantha k. in vitro antibacterial activity and flavonoid contents of lawsonia inermis (henna). int j pharm tech res. 2010;2(2):1178–1181. 10. ali aa, bassam yk, nawres nj. antibacterial activity of lawsonia inermis l. leave extracts on staphylococcus aureus isolates. bas j vet res. 2013; 12(2):256–266. 109j contemp med sci | vol. 6, no. 3, may–june 2020: 109–113 original issn 2413-0516 introduction smoking as a common risk factor has been known to be one of the most important health problems in the current century worldwide. native americans first started using tobacco in ritual ceremonies. in addition, they found some medical benefits for controlling pain when chewing tobacco (e.g. reducing toothache)1. however, it is now well-recognized that smoking is one of the main causes of preventable death especially in developing countries2. about 29% of the total world population are tobacco users3, and smoking-related diseases kill more than five million people globally4 and 75000 people in iran every year5. tobacco use is also considered as the main reason for aggravating deadly non-communicable diseases such as cardiovascular diseases, cancer, asthma, and etc. moreover, there is the high potential for a contagious disease like tuberculosis to be activated and exacerbated by firstor second-hand smoking2. according to the us centres for disease control and prevention (cdc), smoking can directly increase the risk of cardiovascular diseases, brain stroke, lung cancer, chronic obstructive pulmonary disease, peripheral vascular disease, infertility, premature birth and low birth weight6. in addition to the direct impacts of smoking, exposure to second-hand smoke can cause significant health risks for non-smokers. based on the 2010 cdc report, cigarette smoke contains over 7000 harmful chemicals with hundreds being toxic and about 70 of them being carcinogenic. likewise, available evidence shows that cigarette smoke is one of the causing factors for coronary artery disease and lung cancer in adults as well as middle ear infection, respiratory disease, sudden infant death syndrome (sids), low birth weight, brain tumours, leukaemia and asthma in children7. death caused by passive smoking is mainly due to ischemic heart disease in adults and lower respiratory infections in children8. smoking has been reported to be related to different oral diseases like oral and pharyngeal cancers, advanced periodontitis, disorders in oral cavity wound healing, increasing the risk of root decay and soft tissue changes e.g. nicotine induced stomatitis as well as oral premalignant lesions9,10. health professionals are expected to play an active role in controlling tobacco and cigarette consumption at the individual and community levels11. both direct and indirect interventions by health professionals can greatly reduce exposure to environmental tobacco smoke (ets)12. dentists are in a unique position to control smoking for many reasons such as 1) they are preventive oriented, 2) can provide age-specific preventive care and education, 3) meet patients regularly in several sessions and keep good communication with them in a short and long term period to influence on their smoking habit13. beside health professionals, special attention must be paid to health occupations students because they will be the key players of health promotion in society14. before improving the role of our future health professionals in controlling tobacco use, we need to have information about their smoking behaviour. the purpose of this study was to evaluate the smoking behaviour and exposure to ets among the students of shahid beheshti university of medical sciences (sbums). smoking and exposure to environmental tobacco smoke among health occupations students in iran mohammad hossein khoshnevisan1,2, marzie deghatipour2, arezoo ebnahmady3, hooman keshavarz4 1preventive dentistry research centre, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran 2community oral health department, school of dentistry, shahid beheshti university of medical sciences, tehran, iran 3dental public health, faculty of dentistry, university of toronto, toronto, canada 4department of community oral health, school of dentistry, mashhad university of medical sciences, mashhad, iran corresponding author: hooman keshavarz (e-mail: keshavarzh@mums.ac.ir) (submitted: 10 april 2020 – revised version received: 26 april 2020 – accepted: 14 may 2020 – published online: 26 june 2020) objective the purpose of this study was to evaluate the smoking behaviour and exposure to environmental tobacco smoke (ets) among the students of shahid beheshti university of medical sciences (sbums). methods this survey was conducted using a self-administered anonymous questionnaire mainly based on the persian version of the global health professions student survey (ghpss) questionnaire. the students of eight disciplines (dentistry, environmental health engineering, medicine, nursing, nutrition, pharmacy, physiotherapy, and public health) of sbums were invited to participate in this study. chi-square test, mann-whitney u test, and logistic regression served for statistical analyses. the p-value of ≤ 0.05 was considered statistically significant. results a total of 290 students (151 male) participated in the study. about 1.4% of the participants were 15-18 years old, 85.5% were 19-24, 8.6% were 25-29, 3.4% were 30 or older, and 1.0% of them did not report their age. among all the disciplines, dental students reported the highest prevalence (52.0%) for current cigarette smoking while public health students reported the lowest (0.0%). regarding the prevalence of exposure to ets, nutrition students reported the highest (96.4%), and nursing students reported the lowest (57.1%). current cigarette smoking was reported by 34.4% of the participants. this rate was significantly higher in men when compared to women (p<0.001). among participants, 78.6% had been exposed to ets. the frequency distribution of exposure to ets was statistically significantly different between males and females (p<0.001). conclusion this study showed that the prevalence of current cigarette smoking and/or the prevalence of exposure to ets among the students of sbums were alarming. these results have further highlighted the importance and necessity of planning to reduce tobacco use and increase awareness of the harmful effects of tobacco products on overall health among the students of sbums. keywords smoking, environmental tobacco smoke, students, iran 110 original smoking and exposure to ets among students in iran mohammad hossein khoshnevisan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 109–113 materials & methods the present study was done as a questionnaire survey using a self-administered anonymous questionnaire. all the fourthyear medical and dental students of sbums as well as all the third-year students of six other disciplines (environmental health engineering, nursing, nutrition, pharmacy, physiotherapy, and public health) were invited to participate in the study. similar to previous studies done on dental students15-17, in contrast to the other disciplines fourth-year medical and dental students were evaluated because the duration of medical, and dental education is longer in iran compared to most other countries. the difference applied in the case selection, in the authors’ opinion, makes comparison of the results with the results of other studies more valid. the questionnaires were distributed among the students in ordinary classroom settings after explaining study objectives and assuring them of the voluntary participation and confidentiality of collected data. the questionnaire was mainly based on the persian version of the global health professions student survey (ghpss) questionnaire used in previous studies15-17, but according to opinions of an expert panel some questions were added. the final questionnaire was composed of six parts. part 1 with 16 questions assessed tobacco use prevalence among the study participants. part 2 with four questions evaluated exposure to ets. part 3 with 13 questions collected data on attitudes. part 4 with six questions asked participants about their smoking habits or quit status. in part 5, seven questions were used for assessment of available educational programs; and in the last part four questions were used for obtaining demographic information. using a test-retest method with a two-week interval, a pilot study in the school of dentistry, shahed university showed the acceptable reliability of the questionnaire. the collected data were analysed with spss for windows, version 16 (spss inc., chicago, il, usa). the chi-square test was applied to compare the proportion of having experience of smoking (smoking at least once during lifetime), and current smoking (smoking at least once during the previous 30 days) between males and females. the chi-square test was also applied to assess the association between current cigarette smoking and being exposed to ets (exposure to ets at least in one of the previous seven days). mann-whitney u test was used to assess the difference in the distribution of ets exposure frequency between males and females, and also to compare the total scores (range from zero to 13) of smoking control attitudes between male and female subjects, and between current smokers and non-smokers. multivariable logistic regression analysis was applied to evaluate the association between current cigarette smoking, and independent variables including gender, exposure to ets, the total score of smoking control attitudes, and marital status. the p-value of ≤ 0.05 was considered statistically significant. this study was approved by the ethics committee of the shahid beheshti research institute of dental sciences. because the questionnaire used was anonymous and the invited students were completely free to decline participation in the study, the need for consent was waived by the ethics committee. results a total of 290 students participated in this study. table 1 shows the response rates, overall and by disciplines. among all the participants who reported their gender, 151 (52.8%) were male. about 4 (1.4%) of the participants were 15-18 years old, 248 (85.5%) were 19-24, 25 (8.6%) were 25-29, 10 (3.4%) were 30 or older, and 3 (1.0%) of them did not report their age. among all the participants who answered the question on marital status, 234 (82.4%) were single and 50 (17.6%) were married. among all the disciplines, dental students reported the highest prevalence (52.0%) for current cigarette smoking while public health students reported the lowest (0.0%). (table 1) regarding the prevalence of exposure to ets, nutrition students reported the highest (96.4%), and nursing students reported the lowest (57.1%). (table 1) among all, 149 (51.4%) reported having experience of smoking cigarettes, and in this regard, there was a statistically significant difference between the two genders (p<0.001). (table 2) current cigarette smoking was reported by 99 (34.4%) of the participants. this rate was again significantly higher in men when compared to women (p<0.001). (table 2) among all participants, 221 (78.6%) had been exposed to ets. out of which, 11 students (5.0%) had been exposed only at home, 56 (25.3%) only in other places, and 151 (68.3%) both at home and in other places. the frequency distribution table 1. prevalence of current cigarette smoking and exposure to environmental tobacco smoke (ets) among all the participating students. disciplines prevalence of current cigarette smoking (%) prevalence of exposure to ets (%) response rate (%) dentistry (n=25) 52.0 79.2 65.8 environmental health engineering (n=26) 46.2 88.0 47.3 medicine (n=54) 41.5 75.9 42.2 nursing (n=44) 15.9 57.1 72.1 nutrition (n=31) 25.8 96.4 60.8 pharmacy (n=28) 32.1 88.9 48.3 physiotherapy (n=64) 43.8 82.8 65.3 public health (n=18) 0.0 64.7 26.5 total (n=290) 34.4 78.6 52.1 table 2. comparison of having experience of smoking cigarettes, and current cigarette smoking between the two genders. male n(%) female n(%) p-value† experience of smoking cigarettes yes 106(70.2) 42(31.1) p<0.001* no 45(29.8) 93(68.9) current cigarette smoking yes 80(53.0) 18(13.5) p<0.001* no 71(47.0) 115(86.5) †p-values derived from χ2 test. (*=statistically significant) 111 original smoking and exposure to ets among students in iranmohammad hossein khoshnevisan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 109–113 of exposure to ets during the previous seven days was statistically significantly different between males and females. men were more frequently exposed to ets at home and in other places than women. (table 3) out of the current cigarette smokers, 95 students (96.0%) were exposed to ets while 125 (69.4%) of non-smokers reported such exposure (p<0.001). the mean total score (±sd) for smoking control attitudes was 10.6(±3.0). the mean total score (±sd) was 9.9 (±3.3) and 11.4 (±2.4) for men and women, respectively (p<0.001). female students presented statistically significantly more positive approach toward smoking control, when compared to their male counterparts. the mean total score (±sd) for smoking control attitudes was also significantly higher (p<0.001) among non-smokers (11.7±1.9) in comparison with current cigarette smokers (8.8±3.6). the binary logistic regression model showed that current cigarette smoking was significantly associated with gender, exposure to ets, and the total score of smoking control attitudes. (table 4) the majority of students (253 students, 87.2%) recognized that health professionals are role models for their patients and the public, and also believed that health professionals should receive special training on smoking cessation counselling. approximately, 78% of the students expressed their agreement on the fact that it would be less likely for health professionals to advise patients on refraining from smoking if they were cigarette smokers themselves. discussion this study evaluated the smoking behaviour among the students of eight disciplines of sbums. the results showed that the highest prevalence of current cigarette smoking with 52.0% was reported by dental students while the lowest rate (0.0%) was reported by public health students. in line with our results, warren et al. also showed that current cigarette smoking among iranian dental students (10.3%) was more common than among their iranian medical (5.6%) and nursing (4.4%) peers18-20. the present study reported that the prevalence of exposure to ets among nutrition students (96.4%) was considerably more than the prevalence among medical and dental students. warren et al. reported that dental students’ exposure to ets at home was higher (41%) than the exposure among medical (31%) and nursing (37%) counterparts18-20. health professionals, including oral health professionals in a team-work, should work hand in hand in controlling any form of tobacco use. everyone, including lay people and specially health professionals, should also have the required knowledge about the harmful effects of tobacco smoke on health, and should never ignore inhaling the secondhand smoke unintentionally. in this study, having experience of smoking cigarettes was reported by about half of the participants with a remarkable gender difference. keshavarz et al. reported that about onethird of iranian dental students had ever smoked cigarettes. similar to our study, however, they found a significant gender difference for ever use of tobacco products15. our results also showed that more than one-third of the participating students were current cigarette smokers, and the rate among male students was significantly higher than that among their table 3. the frequency distribution of exposure to environmental tobacco smoke (ets) during the previous seven days, by gender. exposure to ets number of days during the previous seven days men n(%) women n(%) total n(%) p-value† home 0 41(28.3%) 74(56.9%) 115(41.8%) <0.001* 1-2 19(13.1%) 14(10.8%) 33(12.0%) 3-4 20(13.8%) 19(14.6%) 39(14.2%) 5-6 18(12.4%) 3(2.3%) 21(7.6%) 7 47(32.4%) 20(15.4%) 67(24.4%) other places 0 21(14.4%) 52(39.4%) 73(26.3%) <0.001* 1-2 29(19.9%) 26(19.7%) 55(19.8%) 3-4 31(21.2%) 26(19.7%) 57(20.5%) 5-6 21(14.4%) 10(7.6%) 31(11.2%) 7 44(30.1%) 18(13.6%) 62(22.3%) †p-values derived from mann-whitney u test. (*=statistically significant) table 4. association of current cigarette smoking with gender, marital status, exposure to environmental tobacco smoke (ets), and total score of smoking control attitudes in a binary logistic regression model. independent variables ß or 95% ci for or p-value gender (0: female, 1: male) 1.6 4.8 (2.3-10.2) <0.001* marital status (0: single, 1: married) 0.4 1.5 (0.6-3.9) 0.439 exposure to ets (0: no exposure to ets, 1: exposed) 2.2 8.8 (2.2-35.2) 0.002* total score of smoking control attitudes -0.4 0.7 (0.6-0.8) <0.001* reference category is indicated by zero. (*=statistically significant) 112 original smoking and exposure to ets among students in iran mohammad hossein khoshnevisan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 109–113 female counterparts. in this regard, our findings are compatible with the results of the study conducted by meysamie et al21. meanwhile, it is common to observe such a difference between male and female students in other countries as well22,23. focusing on dental students, the status of current cigarette smoking seems much more alarming at sbums (52.0%) compared to the status reported at the national level (10.8%) by keshavarz et al15. these results may be extrapolated as a sign of failure or lack of effective smoking control programs among all and especially the male students. our findings on dental students’ exposure to ets are similar to the findings of keshavarz et al.16; both studies showed that about three-fourths of the participating dental students were exposed to ets at home or in other places, and that regarding exposure to ets there is a considerable gender difference. despite the interval between the two studies, at least one can say that there has been no significant improvement in the status of exposure to ets among dental students. it should be noted that more than 18 years before the implementation of this study, the law on banning smoking in public places was adopted in iran. however, it seems that the rule has not been well enforced so far. perhaps the collective efforts of people and professionals are needed to make the world a better place to live. regarding attitudes towards tobacco control policies, our results reflect the fact that most of the participating students were in favour of the policies. these findings are in line with those of many previous studies conducted on health occupations students16-20, 24-27. similar to many previous studies17,24,26, as an example, more than two-thirds of the students in the present study believed that health professionals serve as role models for their patients and the public. as another example, more than two-thirds of the students in this study, in line with many previous studies18-20, believed that health professionals should receive specific training on smoking cessation counselling. in interpreting the statistically significant relationship between gender and the total score for smoking control attitudes, as one of the findings of this study, it should be noted that this relationship was not significant in some previous studies16. this study was a questionnaire cross-sectional study. it was therefore not free from the inherent limitations of such studies. one of the limitations is the relatively high likelihood of a low response rate, which has shown itself in spite of all efforts, especially in some disciplines, in this study. in order to interpret the results of this study, in addition to those who did not participate in the study, the likelihood that some of the participants would not reflect their actual attitudes and behaviours should also be considered. the importance of this point becomes clearer when the social unacceptability of smoking in iranian society, especially for women, is taken into consideration. in this study, we used a questionnaire, as a data collection tool, that has been used in many previous studies. using this questionnaire gives more validity to the comparison between our results and the results of previous similar studies. as another strength of this study, it should be noted that this study has been conducted at one of the largest medical sciences universities in iran and, unlike most previous studies, has evaluated the students of several disciplines of medical sciences. conclusion this study presented that the students’ attitudes towards smoking control policies were generally positive. however, the prevalence of current cigarette smoking and/or the prevalence of exposure to ets among the students were alarming and in some disciplines even much higher than the prevalence reported at the national level for their counterparts. these results have further highlighted the importance and necessity of planning to reduce tobacco use and increase awareness of the harmful effects of tobacco products on health among the students of sbums. acknowledgement this study was funded by the research council at the school of dentistry, shahid beheshti university of medical sciences, tehran, iran. conflicts of interest disclosure the authors declare that there are no conflicts of interest. references 1. axton wf. tobacco and kentucky: university press of kentucky; 1975. 2. world health organization. who global report: mortality attributable to tobacco. geneva, switzerland: world health organization; 2012. 3. world health organization. the world health report: shaping the future. geneva: world health organization, 2003. 4. mathers cd, loncar d. projections of global mortality and burden of disease from 2002 to 2030.plos med. 2006; 3(11):e442. https://doi.org/10.1371/ journal.pmed.0030442 5. world health organization. who report on the global tobacco epidemic, 2008: the mpower package. geneva: world health organization, 2008. 6. centres for disease control and prevention. health effects of cigarette smoking. https://www.cdc.gov/tobacco/data_statistics/fact_sheets/ health_effects/effects_cig_smoking/index.htm. accessed 1 nov 2019. 7. eriksen m, mackay j, ross h. the tobacco atlas. american cancer society; 2013. 8. öberg m, woodward a, jaakkola ms, peruga a, prüss-üstün a. global estimate of the burden of disease from second-hand smoke. geneva, switzerland: world health organization; 2010. 9. u.s. department of health and human services. the health consequences of smoking: a report of the surgeon general. atlanta, ga: us department of health and human services, central for disease control and prevention, national centre for chronic disease prevention and health promotion, office on smoking and health, 2004. 10. mecklenburg re, greenspan d, kleinman dv. tobacco effects in the mouth. nih publication no. 92-3330. bethesda, md: u.s. department of health and human services, public health service, national institutes of health, 1992. 11. roemer r, taylor a, lariviere j. origins of the who framework convention on tobacco control. am j public health. 2005; 95(6):936-8. https://doi. org/10.2105/ajph.2003.025908 12. world health organization. framework convention on tobacco control. geneva: world health organization; 2003. 13. petersen pe. the world oral health report 2003: continuous improvement of oral health in the 21st century– the approach of the who global oral health programme. geneva: world health organization; 2003. 14. beaglehole rh, benzian hm. tobacco or oral health: an advocacy guide for oral health professionals. fdi, world dental press; 2005. 15. keshavarz h, khami mr, jafari a, virtanen ji. tobacco use among iranian dental students: a national survey. east mediterr health j. 2013; 19(8): 704–10. 16. keshavarz h, jafari a, khami mr, virtanen ji. passive smoking and attitudes towards tobacco control programs among iranian dental students. asian pac j cancer prev. 2013; 14(6):3635-9. https://doi.org/10.7314/ apjcp.2013.14.6.3635 dx.doi.org/10.22317/jcms.v6i3.766 113 original smoking and exposure to ets among students in iranmohammad hossein khoshnevisan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 109–113 17. keshavarz h, jafari a, khami mr, virtanen ji. health professionals’ role in helping patients quit tobacco use: attitudes among iranian dental students. isrn public health. 2013; article id: 706451, 5 pages. http://dx.doi. org/10.1155/2013/706451 18. warren cw, sinha dn, lee j, lea v, jones n, asma s. tobacco use, exposure to secondhand smoke, and cessation counselling training of dental students around the world. j dent educ. 2011; 75(3):385-405. 19. warren cw, sinha dn, lee j, lea v, jones nr. tobacco use, exposure to secondhand smoke, and cessation counselling among medical students: cross-country data from the global health professions student survey (ghpss), 2005-2008. bmc public health. 2011; 11(1):72. https://doi. org/10.1186/1471-2458-11-72 20. warren cw, sinha dn, lee j, lea v, jones nr. tobacco use, exposure to secondhand smoke, and training on cessation counselling among nursing students: cross-country data from the global health professions student survey (ghpss), 2005–2009. int j environ res public health. 2009; 6(10):2534-49. https://doi.org/10.3390/ijerph6102534 21. meysamie a, ghaletaki r, haghazali m, asgari f, rashidi a, khalilzadeh o, et al. pattern of tobacco use among the iranian adult population: results of the national survey of risk factors of non-communicable diseases (surfncd-2007). tob control. 2010; 19(2):125-8. https://doi.org/10.1136/ tc.2009.030759 22. sreeramareddy ct, ramakrishnareddy n, rahman m, mir ia. prevalence of tobacco use and perceptions of student health professionals about cessation training: results from global health professions students survey. bmj open. 2018; 8(5):e017477. https://doi.org/10.1136/ bmjopen-2017-017477 23. ng m, freeman mk, fleming td, robinson m, dwyer-lindgren l, thomson b, et al. smoking prevalence and cigarette consumption in 187 countries, 1980-2012. jama. 2014; 311(2):183-92. https://doi.org/10.1001/ jama.2013.284692 24. la torre g, kirch w, bes-rastrollo m, ramos rm, czaplicki m, gualano mr, et al. tobacco use among medical students in europe: results of a multicentre study using the global health professions student survey. public health. 2012; 126(2):159-64. https://doi.org/10.1016/j.puhe.2011.10.009 25. mehrotra r, chaudhary ak, pandya s, mehrotra ka, singh m. tobacco use by indian medical students and the need for comprehensive intervention strategies. asian pac j cancer prev. 2010; 11(2):349-52. 26. khan fm, husain sj, laeeq a, awais a, hussain sf, khan ja. smoking prevalence, knowledge and attitudes among medical students in karachi, pakistan. east mediterr health j. 2005; 11(5-6):952-8. 27. smith m, umenai t. knowledge, attitude and practice of smoking among university students of allied health sciences in japan. asia pac j public health. 2000; 12(1):17-21. https://doi.org/10.1177/101053950001200104 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 51j contemp med sci | vol. 5, no. 1, january–february 2019: 51–58 original article effect of artemisia fruit extract on tnfα and il-6 levels in streptozotocin-induced diabetic mice ekhlass n. ali,a emad mahmoud eltyeaf,b ashwaq a. kadhem,a and jamela joudaa* adepartment of biology, college of science, mustansiriyah university, baghdad, iraq. bdepartment of chemical, college of science, mustansiriyah university, baghdad, iraq. *correspondence to jamela jouda (email: jamela.jouda@uomustansiriyah.edu.iq). (submitted: 14 september 2018 – revised version received: 22 october 2018 – accepted: 27 november 2018 – published online: 26 february 2019) objective this study was conducted to investigate the effects of artemisia extract on some immunological parameters in streptozotocininduced diabetic mice. methods after preparation of artemisia extract, many chemical tests were used to identify the type of element and compounds presented in this plant using many chemical techniques. thirty five streptozocin (stz)-induced diabetes mice were divided into five groups; the first group provided only with water, the other four groups were consumed orally ingested the plant extract in four different concentration (2000, 1000, 500, 250) mg/kg of body weight. another 10 mice did not injected with stz were divided into two groups; one consumed artemisia (art group), and the second consumed only normal saline (cont. group). after 14 days of diabetes induction and artemisia extract treatment, the mice were sacrificed. blood and tissue (brain, spleen and kidney) were collected. fasting blood sugar and insulin levels were determined in the serum. furthermore tumor necrosis factor-alpha (tnf-α) and interleukin-6 (il-6) levels were determined in the serum and aliquots of homogenize tissues. results results declared that the extract of artemisia fruit contains high levels of active compounds especially antioxidant compounds. il-6 and tnf-α levels were decreased while insulin and glucose levels were increased in the stz-induced mice group. artemisia extract effects differently on glucose and insulin levels depending on its concentration. interestingly, il-6 and tnf-α levels increase in serum, brain, and spleen of the stz-induced mice group consumed different concentration of artemisia but it normalized in the stzinduced mice group consumed 250 mg/kg artemisia, as well as insulin and glucose levels for the same group, while there was no difference in kidney. conclusion artemisia can control diabetes in 1000 and 500 mg/kg through controlling insulin level, and in the other hand, using the plant extract in 250 mg/kg, acts as an immune modulator for anti-inflammatory agents like il-6 and tnf-α. keywords artemisia, tumor necrosis factor-alpha, interleukin-6 introduction diabetes mellitus (dm) is the most common metabolic and endocrine disorder worldwide, which is related to metabolism disorder of carbohydrate, fat, and protein.1 diabetes mellitus becomes globally important because of its escalate unabated occurrences. hundred millions of people are suffering from diabetes around the world, and this number increase every year which will reach about 400–500 millions in 2030. diabetes produces many complications which may cause the death.2 in 2011, who reported that more than 80% of diabetes dies in lowand middle-income countries every year.3 one of these complications is the inflammation, which occurs more often in diabetic patients than those without. in this patient, the inflammation course is very complex. immunological disorders are considered as one of the possible causes of the elevated rate of inflammations accompanying to diabetes mellitus.4 the chronic insulitis, inflammation of pancreatic islets caused by destruction pancreatic cells mediated by auto-reactive t cells, play an important role in the diabetes mellitus type-1 development5 while the triggering alteration in the diabetes mellitus type-2 is primary insulin resistance. insulin resistance disrupts the balance of fat, cytokines and the production of adipocins, leading to increased systemic inflammation. thus, inflammatory markers levels increase in the diabetes such as c-reactive protein, interleukin-6 (il-6), tumor necrosis factor-alpha (tnf-α).6–10 many studies explain the role of t-cells and tfn-α producing cells in the glucose homeostasis disorder of both type-1 and 2 dm.11–14 recently, modern medicine include traditional medicine practices15 such as vegetables, culinary herbs, and medicinal plants which are used in the management of diabetes.15,16 who documented that the herbal medicine was used by 80% of world population.17 artemisia is classified as follows: kingdom plant, subkingdom tracheobointa, superdivision spermatophyta, division magnoliophyta, class magnoliopsida, subclass asteridae, order asterales, family asteraceae, subfamily asteroideae, genus artemisia l., which is an important species used as medicinal plant species which is thoroughly studied.18 this plant grows in the limestone wadis of the desert, red sea regions and sinai oases.19 artemisinin is the major active constituent of artemisia. the other compounds include arteether, artemether, artemotil, artenimol, artesunate, dihydroartemisinin, deoxyartemisinin, artemisinic acid, arteannuin-b, stigmasterol, friedelin, friedelan-3 beta-ol, artemetin, and quercetagetin-tetramethyl ether.17,18,20,21 it is widely used to treat drug-resistant and non-drug resistant malaria, for gastrointestinal disorders, and urinary tract disorder as antispasmotic.22 artemisia has many effects such as anti-inflammatory,23 antioxidant,24 antihypertensive, and anti-hyperlipidemia,19 and antitumor.25 it was found that there is no acute or subchronic toxicity of ethanolic extracts of artemisia in mice26 therefore, can open the aspects to use this herb in controlling diabetes. artemisia extract have been used to treat diabetic states in middle east since a long time ago.27 issn 2413-0516 52 j contemp med sci | vol. 5, no. 1, january–february 2019: 51–58 effect of artemisia fruit extract on tnfα and il-6 levels in streptozotocin-induced diabetic mice original article e.n. ali et al. the aim of this work is to study the effect of artemisia extract on the physiological and immunological parameters in streptozotocin-induced diabetic balb/c mice type. materials and methods chemical part the plant tree is collected, cut and dried in the shade at room temperature for 1 week. then, the fruit are taken and grind by an electric mill and kept in glass cans away from light, heat and humidity until use. three grams of artemisia powder was placed in a glass flask. about 8 ml of nitric acid and 2 ml of 60% hydrochloric acid were added and left until the next day after covering with a watch bottle. it was placed in a sandy bath of 80°c for approximately 6 h. the volume is adjusted to 50 ml with distilled ion-free water. finally, the elements (zn, ni, cu, cd, cl, pb) were estimated by atomic absorption device without flame, according to the method referred to in kotani et al.28 about 10 ml of organic solvents mixture composed of hexane and ethyl acetate were added to 0.1 g of the artemisia powder in 1:1 ration. the solution was filtered. the resulting filtrate was used to measure the ftir spectra using ftir spectrum analyzer from shimadzu ftir-8400s and gas chromatography-mass (gc-mass) using gas chromatography–mass spectrometry from shimadzu gcms-qp2010 ultra.29 about 50 g of powdered artemisia fruit were dissolved in each of two volumetric flasks containing 1 l and 500 ml distilled water. the first flask was left in cold water solution. the second was placed in the shaker incubator at 50°c for 48 h as hot solution. both extracts are filtered by the filter paper (whatman 1). the filtrate was centrifuged for 6 min (4000 cycles/min) to completely separate the solution from the remaining minutes of the previous filtration process and take the last solution to make the required measurements. about 1.037 mg of fruit extract and 1.025 mg of the standard material for calibration, using the european30 instrument of the italian company (eurovector s.p.a.) from ea 3000 have been used to analyze the presence of carbon, hydrogen, nitrogen, and sulfur elements in the fruit extract. biology part forty five balb/c mice were used in this study, from prevention research center, baghdad, iraq. these mice aged about 12 week and weighted about 25 g. thirty five mice were injected inter peritoneum (ip) with 50 mg/kg streptozocin (stz) to induce diabetic disease. after 5 days, the fasting blood sugar (fbs) levels were determined. the stz-induced mice with high blood sugar level (<150 mg/dl) were selected as diabetic models. the stz-induced mice were divided into five groups (five mice in each group); the first group was provided only with water and served as control (stz group). the other groups consumed artemisia in different concentrations: 250, 500, 1000, 2000 g artemisia/kg of mouse body weight. another 10 mice did not inject with stz divided into two control groups (five mice in each group); (art group) which consumed 2000 mg/kg artemisia, and (cont. group) which consumed only normal saline. the fbs was measured, using accu-chek active from roche, through the next 5 days after stz-injection to select diabetic mice, and to determine fbs in zero time, in 7 and 14 days after treatment with artemisia. the mice were scarified by cervical distraction after 14 days. spleen, kidney, and brain were homogenized with d.w. by crucible. homogenized samples were centrifuged at 20 000 rpm at 4°c for 10 min, and supernatants were collected and used to determine the il-6 and tnf-α concentrations in tissues. the blood was also collected and centrifuged for 10 min at 10,000 rpm. the serum was collected and was used to determine the insulin, il-6, and tnf-α. serum samples were used to determine insulin level by insulin-products-diesse from diagnostica senese, spain. the serum samples and the eloquent of homogenized tissue were used to determine il-6 by mouse il-6 elisa kit and tnf by mouse tnf-a elisa kit from koma biotech inc. statistical analysis results were expressed as mean ± standard error. data were analyzed by one-way analysis of variance followed by fisher’s test for multiple comparisons, using statview version 5.0. differences were considered significant when p < 0.05. results chemical part the results in table 1 shows that the extract of the fruit of the artemisia contains the elements of carbon, hydrogen, and nitrogen in different percentages. carbon% was the highest element while the lowest element was nitrogen%. the sulfur and oxygen component was not mentioned in table 1 because it was very low. many heavy materials were found in the extract of artemisia fruit in different concentration. the highest was cu while ni was the lowest (table 2). through the analysis of spectroscopy using the technique of gas chromatography–mass spectrometry, there are more than 21 compounds in the organic extract of the artemisia fruit. it was found that it contains antioxidant groups and other compounds. lilac aldehyde a was the highest occurrence in the extract while 2-(triethoxsilyl)propylamine was the lowest occurrence in this extract (table 3 and fig. 1). table 1. chns analysis results of artemisia fruit extract type name n% c% h% s% o% weight (mg) bypass bypass blank blank stander acetanilide 10.363 71.089 6.711 1.025 sample artemisia 1.921 48.015 6.989 1.037 table 2. the elements in the extract of artemisia fruit powder mineral elements amount of concentration (mg/l) pb 0.3 cl 0.35 cu 0.82 ni 0.1 cd 0.6 zn 0.4 e.n. ali et al. 53j contemp med sci | vol. 5, no. 1, january–february 2019: 51–58 original article effect of artemisia fruit extract on tnfα and il-6 levels in streptozotocin-induced diabetic mice ftir spectrum analysis was used to detect the nature of the compounds and the active groups of these compounds present in the extract of artemisia. as a result of vibration of electrons between the bonds, and by the active groups appear peaks in different places within the areas of measurement of the spectrum (fig. 2). biology parts fasting blood sugar of induced diabetic disease and controls groups was determined in zero time and after 7 and 14 days from treatment. in all groups of induced diabetes, the fbs reduced after artemisia treatment compared with the controls groups, significant differences were detected between 14 days and zero time in mice consumed artemisia in 250, 500, and 1000 g/kg concentrations and between 7 days and zero time in mice consumed artemisia in 250 and 500 g/kg concentrations (table 4). in fig. 3, significant decrease in the insulin levels in the induced diabetic disease group consumed only water (stz) compared with other control groups while there was no table 3. gc-mass results of fruit extract of artemisia name a/h height (%) height area (%) area r. time peak# ethoxycarbonyl isothiocyanate 3.04 5.08 46287 4.06 140905 3.705 1 acetic acid 4.15 16.32 148674 17.75 616411 5.886 2 dimethyl sulfoxide 4.15 1.85 16868 2.01 69925 8.130 3 5,5-dimethyl-2(5h)-furanone 3.63 5.73 52220 5.46 189582 8.877 4 lilac aldehyde b 2.73 2.84 25889 1.76 61241 9.768 5 2(3h)-furanone, 5-ethenyldihydro-5-methyl 3.45 5.83 53089 5.28 183279 9.968 6 spiro(tetrahydrofuryl)2.1΄(decalin)5΄,5΄,8΄a-trimethyl 3.00 6.68 60903 5.26 182532 10.84 7 2-(triethoxsilyl)propylamine 1.69 0.53 4795 0.23 8087 11.975 8 7-oxabicyclo[4.1.0]heptane, 2-methyl 2.21 1.01 9218 0.59 20355 12.233 9 2(1h)-naphthlenone, octahydro-4a-methyl-, cis1.60 0.68 6198 029 9909 13.086 10 butylated hydroxytoluene 2.73 2.81 25651 1.75 60786 13.686 11 2-cyclopenten-1-one, 3-methyl-2-(2-pentenyl)-, (z)2.79 2.10 19094 1.54 53331 14.050 12 2-(4-hydroxybutyl)cyclohexanol 2.73 0.87 7914 0.62 21594 14.849 13 nonadecane, 1-chloro 1.85 1.16 10556 0.56 19534 15.003 14 lilac alcohol b 2.91 3.73 34011 2.85 98804 15.233 15 heneicosane 2.37 2.12 19351 1.32 45789 16.265 16 docosane 3.13 5.21 47486 4.29 148838 17.703 17 spiro[2.4]heptane-5-methanol, 5-hydroxy3.33 2.74 24966 2.39 83046 18.434 18 tricosane 3.70 8.05 73467 7.82 271365 19.445 19 lilac aldehyde a 5.37 17.17 156481 24.19 839843 20.922 20 triacontane 5.08 7.49 68252 9.99 347046 21.657 21 total 100.00 911270 100.00 3472211 fig. 1 gc-mass results of fruit extract of artemisia. 54 j contemp med sci | vol. 5, no. 1, january–february 2019: 51–58 effect of artemisia fruit extract on tnfα and il-6 levels in streptozotocin-induced diabetic mice original article e.n. ali et al. significant difference between control groups consumed water (cont) and control groups consumed artemisia (art). insulin level in all stz-induced diabetic mice groups treated with artemisia (250, 500, 1000 and 2000) was significantly high compared with stz control group. it can be observed that insulin level was significantly higher in the stz-induced diabetic mice groups consumed 500 and 1000 g/kg than those consumed 250 and 2000 g/kg artemisia (fig. 3). the differences in serum tnf-α and il-6 levels after 14 days from treatment among all studied groups are shown in fig. 4. in the controls groups, while there was no significant difference between (cont) group and (art) group, significant lower tnf-α and il-6 levels in stz group compared with other controls groups were detected. tnf-α and il-6 levels in all stz-induced diabetic mice groups treated with artemisia (250, 500, 1000 and 2000 g/kg) was significantly elevated as compared with stz group. an extraordinary increasing concentration of tnf-α and il-6 levels were shown in mice group consumed 2000 mg/kg artemisia and significantly lowest in the stz-induced diabetic mice groups consumed 250 mg/kg artemisia extract. the differences in tnf-α and il-6 levels after 14 days from treatment with artemisia were determined in tissues, brain as central nervous system (cns), spleen as lymphoid tissue, and kidney as non-lymphoid tissue. the results are shown in figs. 5 and 6. in kidney, there were no differences in tnf-α level seen, but significant differences were shown in brain and spleen as compared with stz-control group as follows. in the control groups, significant decrease in tnf-α level in the induced diabetic disease group consumed only water (stz) compared with other control groups while there was no significant difference between control groups consumed water (cont) and control groups consumed artemisia (art). tnf-α level in all stz-induced diabetic mice groups treated with artemisia (250, 500, 1000 and 2000 g/kg) was significantly higher compared with stz control. diabetic animals treated with 1000 mg/kg of artemisia showed the highest level of tnf-α in the examined tissue (fig. 5). fig. 2 ftir spectroscopy results in the fruit extract of artemisia. table 4. the fbs level in induced diabetic disease and controls groups in zero time and after 7 and 14 days from treatment groups fbs (mg/dl) zero time 7 days 14 days cont 111.7 ± 3.7 112.0 ± 6.4 107.7 ± 1.5 art 108.0 ± 6.6 112.3 ± 3.3 117.0 ± 0.6 stz 166.4 ± 18.4 152.4 ± 13.2 167.5 ± 15.5 250 157.2 ± 5.0 139.5 ± 7.2* 138.5 ± 3.2§ 500 179.8 ± 17.4 119.3 ± 15.7* 102.0 ± 21.0§ 1000 162.0 ± 6.6 148.4 ± 6.8 130.3 ± 6.8§ 2000 150.9 ± 8.9 150.3 ± 5.7 137.0 ± 4.5 *significant differences in zero time vs. 7 days. §significant differences in zero time vs. 14 days. fig. 3 insulin level in stz-induced diabetic mice and controls groups after 14 days from treatment with artemisia. *significant difference in stz vs. other groups, §significant difference in 2000 vs. other groups, and #significant difference in 250 vs. other groups e.n. ali et al. 55j contemp med sci | vol. 5, no. 1, january–february 2019: 51–58 original article effect of artemisia fruit extract on tnfα and il-6 levels in streptozotocin-induced diabetic mice another significant difference in il-6 level into brain and spleen were shown, while no differences in the kidney were detected, in addition there was no significant difference between control groups consumed water (cont) and control groups consumed artemisia (art) group, but significant low il-6 level in stz control group as compared with other controls groups were detected. il-6 level in all stz-induced diabetic mice groups treated with artemisia (250, 500, 1000 and fig. 4 tnf-α (a) and il-6 (b) levels in stz-induced diabetic mice and controls groups after 14 days from treatment with artemisia. *significant difference in stz vs. other groups, §significant difference in 2000 vs. other groups, and #significant difference in 250 vs. other groups. fig. 5 the tnf-α level into brain, spleen and kidney of stz-induced diabetic mice and controls groups after 14 days from treatment with artemisia. *significant difference in stz vs. other groups, §significant difference in 1000 vs. other groups. 56 j contemp med sci | vol. 5, no. 1, january–february 2019: 51–58 effect of artemisia fruit extract on tnfα and il-6 levels in streptozotocin-induced diabetic mice original article e.n. ali et al. 2000 g/kg) was significantly higher compared with stz into brain, but into spleen only the stz-induced diabetic mice group consumed 1000 g/kg artemisia was significantly higher compared with stz group and other stz-induced diabetic mice groups consumed artemisia (fig. 6). discussion results obtained in this research, using gc-mass technique to elucidate the nature of phytochemical compounds in terms of molecular weight and partial formula, show that the extract of artemisia fruit contains high levels of active compounds especially antioxidant compounds group representing the bulk of the fruit and its ability to link with other active groups. these results agree with many other researches.31,32 as well as artemisia extract found to have certain mineral elements that trigger the activation of enzymes in the body28,33 and the presence of flavonoids, which capture free radicals,34,35 and leprosy. they activate the enzymes and transporter proteins in the membrane of the cell.28 since artemisia has better phytochemical compounds36 which may have role in much of activities.36,37 moreover, swamy et al. in 2016 reported that artemisia enter into various fields such as medical, pharmaceutical, fragrance, and flavor manufacturing. therefore, it became one of the medical plants which form a large part of natural plants.38 the usage of stz to induce diabetes, in congenitally albino mice, was reported in many sources to induce diabetes because stz, a glucosamine-nitrosuria which derived from streptomyces achromogenes, which is used as a chemotherapeutic agent in carcinoma of pancreatic β cell. these cells are damaged by stz leading to hypoinsulinemia and hyperglycemia and induce adiabatic state. stz has chemical structure similar to glucose which allow it to link with the receptor of glut2 glucose transporter by which allow stz to enter and accumulate in β cells.39–41 thus, stz induce immune-mediated insulin deficient diabetes and depression of immune reactivity.42 these evidences can explain the decrease of il-6 and tnf-α, and the increase of insulin and glucose levels in the mice group treated with stz compared with control. it is well established that artemisia extract is very useful in diabetes. antidiabetic ability of various extracts of artemisia due to the presence of any secondary metabolites (flavonoids, alkaloids, phenols, glycosides and terpenes) at various concentrations in artemisia. it has been reported that the secondary metabolites have differently antidiabetic potential, which is the proposed cause of variation in the activities of these extracts.43,44 these evidences can explain the different effects of artemisia on glucose and insulin levels, depending on its concentration, as shown in this study which comes in agreements with chang et al.45 and yagi et al.46 fig. 6 the il-6 levels brain, spleen, and kidney of stz-induced diabetic mice and controls groups after 14 days from treatment with artemisia. *significant difference in stz vs. other groups, §significant difference in 1000 vs. other groups. e.n. ali et al. 57j contemp med sci | vol. 5, no. 1, january–february 2019: 51–58 original article effect of artemisia fruit extract on tnfα and il-6 levels in streptozotocin-induced diabetic mice it seems that artemisia might boost the innate immunity47 which led to use it as antifungal agent in many fungal infections.48 since il-6 and tnf-α soluble mediators are secreted by different innate immune cells,49 decreased by stz effect; artemisia try to boost it.50 in our results, the levels of both il-6 and tnf-α were increased in the stz-induced mice group treated with different concentrations of artemisia but it is normalized in the stz-induced mice group treated with 250 mg/kg artemisia, in which the insulin and glucose levels also normalized. it is at present well established that immune cells produce soluble mediator that can influence the cns and modify its activity in different ways.51,52 on the other hand, cns exerts numerous immunomodulatory functions in lymphoid organs.53 the immunomodulatory effect of cns mediators is mainly exerted by stimulation of β2-adrenoreceptors, which are expressed by b lymphocytes, cd4+, and cd8+ t cells, innate immunity cells, and th1 helper cells.54 in our results, the brain, cns, spleen, and lymphoid organ, were affected by stz and artemisia, in which the il-6 and tnf-α were decreased in stz group compared with control these levels increased in the stz-induced mice group treated with artemisia and normalized in the stz-induced mice group treated with 250 mg artemisia. interestingly, these results were different in the kidney, non-lymphoid organs, in which, there was no differences in il-6 and tnf-α levels among all groups. conclusion from the results in this work, it can be conclude that the stz caused diabetic by increase insulin and glucose levels in the blood resulting from damage in the pancreatic β cell; and depression of immune activity. artemisia can be a good treatment for this case because of the effectiveness of its chemical components by correcting the imbalance in the β cells and raising the insulin and glucose levels in the blood. on the other hand, it boost the innate immunity which secreted il-6 and tnf-α and normalized the levels of il-6 and tnf-α in the blood, cns, and lymphoid organs which connected in different ways, while kidney were not affected by stz or artemisia. acknowledgments ekhlass n. ali, emad mahmoud eltyeaf, ashwaq a. kadhem, jamela jouda would like to thank mustansiriyah university (www.uomustansiriyah.edu.iq), baghdad, iraq for its support in this work. conflict of interest none. n references 1. moore h, summerbell c, hooper l, cruickshank k, vyas a, johnstone p, et al. dietary advice for treatment of type 2 diabetes mellitus in adults. cochrane database syst rev. 2004:cd004097. 2. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetes care. 2004;27:1047–1053. 3. organization wh. fact sheet no 3122011. 4. geerlings se, hoepelman ai. immune dysfunction in patients with diabetes mellitus (dm). fems immunol med microbiol. 1999;26:259–265. 5. atkinson ma, eisenbarth gs. type 1 diabetes: new perspectives on disease pathogenesis and treatment. lancet. 2001;358:221–229. 6. meigs jb, hu fb, rifai n, manson je. biomarkers of endothelial dysfunction and risk of type 2 diabetes mellitus. jama. 2004;291:1978–1986. 7. kroder g, bossenmaier b, kellerer m, capp e, stoyanov b, muhlhofer a, et al. tumor necrosis factor-alphaand hyperglycemia-induced insulin resistance. evidence for different mechanisms and different effects on insulin signaling. j clin invest. 1996;97:1471–1477. 8. freeman dj, norrie j, caslake mj, gaw a, ford i, lowe gd, et al. c-reactive protein is an independent predictor of risk for the development of diabetes in the west of scotland coronary prevention study. diabetes. 2002;51: 1596–1600. 9. esposito k, nappo f, marfella r, giugliano g, giugliano f, ciotola m, et al. inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress. circulation. 2002;106: 2067–2072. 10. emerging risk factors collaboration, kaptoge s, di angelantonio e, pennells l, wood am, white ir, et al. c-reactive protein, fibrinogen, and cardiovascular disease prediction. n engl j med. 2012;367:1310–1320. 11. zeng c, shi x, zhang b, liu h, zhang l, ding w, et al. the imbalance of th17/ th1/tregs in patients with type 2 diabetes: relationship with metabolic factors and complications. j mol med (berl). 2012;90:175–186. 12. jagannathan-bogdan m, mcdonnell me, shin h, rehman q, hasturk h, apovian cm, et al. elevated proinflammatory cytokine production by a skewed t cell compartment requires monocytes and promotes inflammation in type 2 diabetes. j immunol. 2011;186:1162–1172. 13. winer s, chan y, paltser g, truong d, tsui h, bahrami j, et al. normalization of obesity-associated insulin resistance through immunotherapy. nat med. 2009;15:921–929. 14. zhang h, potter bj, cao jm, zhang c. interferon-gamma induced adipose tissue inflammation is linked to endothelial dysfunction in type 2 diabetic mice. basic res cardiol. 2011;106:1135–1145. 15. wazaify m, afifi fu, el-khateeb m, ajlouni k. complementary and alternative medicine use among jordanian patients with diabetes. complement ther clin pract. 2011;17:71–75. 16. lev e, amar z. ethnopharmacological survey of traditional drugs sold in israel at the end of the 20th century. j ethnopharmacol. 2000;72:191–205. 17. ghasemi dn. pharmacopia of iranian plants. 1st ed. isfahan: ministry of health and medical education, deputy of food and medicine, 2002. 18. woerdenbag hj, bos r, salomons mc, hendriks h, pras n, malingre tm. volatile constituents of artemisia annua l. (asteraceae). flavour fragr j. 1993;8:131–137. 19. bhakuni rs, jain dc, sharma rp, kumar s. secondary metabolites of artemisia annua and their biological activity. curr sci. 2001;80:35–48. 20. zheng gq. cytotoxic terpenoids and flavonoids from artemisia annua. planta med. 1994;60:54–57. 21. iqbal s, younas u, chan kw, zia-ul-haq m, ismail m. chemical composition of artemisia annua l. leaves and antioxidant potential of extracts as a function of extraction solvents. molecules. 2012;17:6020–6032. 22. heide l. artemisinin in traditional tea preparations of artemisia annua. trans r soc trop med hyg. 2006;100:802. 23. abad mj, bedoya lm, apaza l, bermejo p. the artemisia l. genus: a review of bioactive essential oils. molecules. 2012;17:2542–2566. 24. al-mustafa ah, al-thunibat oy. antioxidant capacity of some jordanian medicinal plants used traditionally for the treatment of diabetes. pak j biol sci. 2008;1:351–358. 25. ferreira jf, luthria dl. drying affects artemisinin, dihydroartemisinic acid, artemisinic acid, and the antioxidant capacity of artemisia annua l. leaves. j agric food chem. 2010;58:1691–1698. 26. jiang l, liu h, wang j, tan f, zhao k, wu x, et al. characterization and comparison of three transgenic artemisia annua varieties and wild-type variety in environmental release trial. j med plants res. 2010;4:2719–2728. 27. helal ege, abou-aouf n, khattab am, zoair ma. anti-diabetic effect of artemisia annua (kaysom) in alloxan-induced diabetic rats. egypt j hosp med. 2014;57:422–430. 28. kotani m, matsumoto m, fujita a. persimmon leaf extract and astragalin inhibit development of dermatitis and ige elevation in nc/nga mice. j allergy clim immunol. 2000;106:159–166. 29. obistioiu d, cristina rt, schmerold i, chizzola r, stolze k, nichita i, et al. chemical characterization by gc–ms and in vitro activity against candida albicans of volatile fractions prepared from artemisia dracunculus, artemisia abrotanum, artemisia absinthium and artemisia vulgaris. chem cent j. 2014;8:6. 58 j contemp med sci | vol. 5, no. 1, january–february 2019: 51–58 effect of artemisia fruit extract on tnfα and il-6 levels in streptozotocin-induced diabetic mice original article e.n. ali et al. 30. goda a, ohgi s, kinpara k, shigemori k, fukuda k, schneider eb. changes in serum bdnf levels associated with moderate-intensity exercise in healthy young japanese men. springerplus. 2013;2:678. 31. alabdallat ng. in vitro antisickling activity of artemisia herba-alba asso (chih) methanolic extract on sickle cell disease. asian j pharm clin res 2016;1(suppl 1):109–112. 32. yao k, chen h, liu k, langfald a, yang g, zhang y, et al. kaempferol targets rsk2 and msk1 to suppress uv radiation-induced skin cancer. cancer prev res (phila). 2014;7:958–967. 33. duarte j, jimenez r, o’valle f, galisteo m, perez-palencia r, vargas f, et al. protective effects of the flavonoid quercetin in chronic nitric oxide deficient rats. j hypertens. 2002;20:1843–1854. 34. andriantsitohaina r. regulation of vascular tone by plant polyphenols: role of nitric oxide. gen physiol biophys. 1999;18:3–5. 35. baumann j, von bruchhausen f, wurm g. flavonoids and related compounds as inhibition of arachidonic acid peroxidation. prostaglandins. 1980;20:627–639. 36. jeong sh, kim j, min h. in vitro anti-inflammatory activity of the artemisia montana leaf ethanol extract in macrophage raw. food agric immunol. 2018;29:688–698. http://www.tandfonline.com/loi/cfai20:1-11. 37. vijeesh v, usha pn, manju mk. phytochemical analysis and in vitro antifungal studies of medicinal plants elephantopus scaber, cyclea peltata and artemisia japonica. int j pharm phyto res. 2017;9:319–322. 38. swamy mk, akhtar ms, sinniah ur. antimicrobial properties of plant essential oils against human pathogens and their mode of action: an updated review. evid based complement alternat med. 2016;2016:3012462. 39. graham ml, janecek jl, kittredge ja, hering bj, schuurman hj. the streptozotocin-induced diabetic nude mouse model: differences between animals from different sources. comp med. 2011;61:356–360. 40. deeds mc, anderson jm, armstrong as, gastineau da, hiddinga hj, jahangir a, et al. single dose streptozotocin-induced diabetes: considerations for study design in islet transplantation models. lab anim. 2011;45:131–140. 41. furman bl. streptozotocin-induced diabetic models in mice and rats. curr protoc pharmacol. 2015;70:5.47.1-20. 42. kantwerk-funke g, burkart v, kolb h. low dose streptozotocin causes stimulation of the immune system and of anti-islet cytotoxicity in mice. clin exp immunol. 1991;86:266–270. 43. khan i, ahed w, karim n, ahmed m, khan m, ariq s, et al. antidiabetic activity and histopathological analysis of carnosol isolated from artemisia indica linn in streptozotocin-induced diabetic rats. med chem res. 2017;26:335–343. 44. abdallah h, abdelrahman r, jaleel ga, abd el-kader ham, el-marasy s, zaki er, et al. pharmaceutical effects of ethanol extract of artimisia herba alba in streptozotocin-nduced type 1 diabetes mellitus in rats. biochem pharmacol (los angel). 2015;4:196. 45. chang xw, qin y, jin z, xi tf, yang x, lu zh, et al. interleukin-6 (il-6) mediated the increased contraction of distal colon in streptozotocininduced diabetes in rats via il-6 receptor pathway. int j clin exp pathol. 2015;8:4514–4524. 46. yagi m, takabe w, matssumi s, shimode a, maruyama t, yonei y. screening and selection of anti-glycative materials: kuromoji (lindera umbellata). glycative stress res. 2017;4:317–328. 47. bokaeian m, nakhaee a, moodi b, farhangi a, akbarzadeh a. effects of garlic extract treatment in normal and streptozotocin diabetic rats infected with candida albicans. indian j clin biochem. 2010;25:182–187. 48. obolskiy d, pischel i, feistel b, glotov n, heinrich m. artemisia dracunculus l. (tarragon): a critical review of its traditional use, chemical composition, pharmacology, and safety. j agric food chem. 2011;59:11367–11384. 49. lacy p, stow jl. cytokine release from innate immune cells: association with diverse membrane trafficking pathways. blood. 2011;118:9–18. 50. zarasvand ma, madani m, modaresi m. the effect of hydroalcoholic extract of artemisia dracunculus l. (tarragon) on candida albicans infection in mice. jundishapur j nat pharm prod. 2016;11:e29911. 51. kin nw, sanders vm. it takes nerve to tell t and b cells what to do. j leukoc biol. 2006;79:1093–1104. 52. ziv y, ron n, butovsky o, landa g, sudai e, greenberg n, et al. immune cells contribute to the maintenance of neurogenesis and spatial learning abilities in adulthood. nat neurosci. 2006;9:268–275. 53. elenkov ij, wilder rl, chrousos gp, vizi es. the sympathetic nerve--an integrative interface between two supersystems: the brain and the immune system. pharmacol rev. 2000;52:595–638. 54. nance dm, sanders vm. autonomic innervation and regulation of the immune system (1987–2007). brain behav immun. 2007;21:736–745. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 181j contemp med sci | vol. 6, no. 4, july-august 2020: 181–186 original issn 2413-0516 introduction in women aged over 40 years, mammography is the first preferred imaging method for breast cancer screening and has been the only one shown to reduce cancer mortality.1 breast density is associated with collagen, epithelial, and stromal tissues. the interstitium and breast gland tissue are less radiolucent than adipose tissue. mammographic breast parenchymal density is the radiological composition of radiodense fibroglandular tissue and radiolucent adipose tissue. advanced age, high body mass index (bmi), number of births, breastfeeding time, and antiestrogens in breast tissue such as tamoxifen cause a decrease in breast density, while hormone replacement therapy and use of oral contraceptives cause an increase.2 recently, the american college of radiology has recommended the assessment of breast density in the birads (breast imaging reporting and data system) system for the evaluation of lesions in terms of malignancy risk.3 dense breasts restrict the imaging of lesions detected by mammography and reduce the sensitivity of mammography. mammography has been reported to have a sensitivity of 87% and a specificity of 97% in adipose breast tissue (type 1 or type a) and a sensitivity of 63% and a specificity of 89% in high density (type 4 or type d) breast.4 mbd is a dynamic process that differs with age and reproductive changes. therefore, the correlation between mbd and tumor biology and prognosis has been the subject of our study.5–7 in this study, we investigated correlation between breast parenchymal density and prognosis, tumor biology, reoperation rates, and prognostic factors. with the changing life habits and nutrition habits and the new diagnosis and treatment modalities, it is crucial for this correlation to be examined now. materials and methods patients who were operated for breast cancer between january 2017 and december 2019 were included. patients’ surgery and pathology reports, hormone receptor status, preoperative hematological and biochemical parameters, demographic characteristics, and overall survival rates were analyzed retrospectively on an electronic environment. approval was obtained from the ethics committee of our hospital. 143 patients were included. patients who did not receive neoadjuvant therapy, who had no history of hormone replacement therapy or oral contraceptive use, and who were evaluated for breast parenchymal density in their preoperative mammography were included. inoperable breast cancer patients were excluded. the demographic data of the groups (age, bmi, etc.), their postoperative pathological data (tm characteristics) and other radiological evaluations (usg mri. is there any data on this?) were recorded. hormone receptor status was recorded according to the pathology report for human epidermal growth factor receptor 2 (her-2) expression. histological grade was recorded according to the scarff-bloom-richardson grading system. lymphovascular invasion status was recorded according to lymphatic or vascular status indicated in the pathology reports. evaluation of density the craniocaudal and mediolateral images obtained during conventional mammograms were collected in a database. the appearance of radiologically dense and lucent areas on mammography was taken as the mammographic percentage the effect of breast parenchymal density on breast cancer subtypes and prognostic factors cemil yüksel1, serdar çulcu1, batuhan bakırarar2, lütfi doğan1 1ankara abdurrahman yurtaslan oncology training and research hospital, university of health science, ankara, turkey 2department of biostatistics, faculty of medicine, ankara university, ankara, turkey corresponding author: cemil yüksel (e-mail: cemil8537@hotmail.com) abstract objective: mammographic breast parenchymal density is the radiological composition of radiodense fibroglandular tissue and radiolucent adipose tissue. dense breasts restrict the imaging of lesions detected by mammography and reduce the sensitivity of mammography. in this study, we investigated correlation between breast parenchymal density and prognosis, tumor biology, reoperation rates, and prognostic factors. methods: patients who were operated for breast cancer between january 2017 and december 2019 were included. the craniocaudal and mediolateral images obtained during conventional mammograms were collected in a database. the appearance of radiologically dense and lucent areas on mammography was taken as the mammographic percentage consistent with the current literature results: according to breast parenchymal density, there were 21 patients (14.7%) with type 1 breast density, 29 (20.3%) with type 2, 47 (32.9%) with type 3, and 46 (32.1%) with type 4. mean survival time due to breast cancer was 27.45±17.85 months (1–63), and 139 patients (97.2%) lived, while 4 (2.8%) were exitus. conclusion: increased breast parenchymal density is a crucial risk factor for breast cancer. the effects of density on tumor biology and prognosis are still controversial. although increased breast density seems to have an effect on indicators of poor prognosis. additional examinations should be taken into consideration in order not to cause delay or skipping in diagnosis due to the decreased sensitivity of mammography in dense breasts keywords: breast cancer, parenchymal density, prognosis, mammography 182 original efficacy of parenchymal density on breast cancer cemil yüksel j contemp med sci | vol. 6, no. 4, july-august 2020: 181–186 consistent with the current literature.8 in the evaluation of density, the average density of both breasts was taken. the percentage of the area filled by radiologically dense breast tissue was analyzed and then divided into six different percentile categories according to the boyd cutoff value (<5%, 5%–10%, 10%–25%, 25%–50%, 5%–75%, or >75%).24 defined by boyd et al., this categorization includes four types to categorize the mammographic density percentages9 (type i—<25%; type ii—25%–50%; type iii—51%–75%; type iv—>75%). we used this categorization in the current study. the patients were divided into four groups according to their breast parenchymal densities. statistical analysis the spss 11.5 software was used in the analysis of the data. in descriptive statistics, quantitative variables are given as mean ± standard deviation and median (minimum–maximum), and qualitative variables are given as number of patients (percentage). whether there was a difference between more than two categories of a qualitative variable according to a quantitative variable was analyzed using the oneway anova test if normal distribution assumptions were provided and the kruskal–wallis-h test if not. the chi-squared test and fisher’s exact test were used to compare two qualitative variables. p<0.05 was considered as the statistical significance level. results a total of 143 patients were included. the mean ± standard deviation and median values for patients’ ages were found to be 61.36±11.54 and 61.00 (33.00–89.00), respectively. according to breast parenchymal density, there were 21 patients (14.7%) with type 1 breast density, 29 (20.3%) with type 2, 47 (32.9%) with type 3, and 46 (32.1%) with type 4. 58 were grade 0–1 (40.6%), 64 were grade 2 (44.7%), and 21 were grade 3 (14.7%). similarly, 97 patients were node-negative, while 46 were node-positive. 47 (32.8%) of the patients received breast-conserving surgery (bcs), 25 received simple mastectomy (17.4%), 44 (30.7%) received modified radical mastectomy (mrm), 8 (5.6%) received mastectomy upon having positive surgical margin after bcs and axillary dissection (ad), 15 (10.5%) received resection after bcs, 3 (2.1%) received bcs+ad, and 1 (0.9%) received bilateral mastectomy. there was no statistically significant correlation between breast parenchymal density and survival (p=0.105). there was no statistically significant correlation between operation type and survival (p>0.005). mean survival time due to breast cancer was 27.45±17.85 months (1–63), and 139 patients (97.2%) lived, while 4 (2.8%) were exitus. descriptive variables are given in table 1. regarding the correlation between patients’ demographic, histopathological, and clinicopathological characteristics and their breast parenchymal density, the correlations between breast parenchymal density and lymph node status, grade, her-2 expression, surgical margin positivity, and survival were not statistically significant. the p values were 0.129– 0.152–0.569–0.876–0.105, respectively. estrogen and progesterone receptor positivity decrease with increased breast density, which was statistically significant (p=0.001/p=0.039). comparing lymphovascular invasion and breast parenchymal density, we observed that the rate of lymphovascular invasion increased with increased density, which was statistically significant (p=0.002). 38 patients were luminal a, 26 were her-2 positive luminal b, 53 were luminal b, 17 were triple negative and 9 were her-2 positive, and there was a statistically significant correlation between breast parenchymal density and cancer subtype (p=0.011). 14 patients were grade 1, 58 were grade 2 and 71 were grade 3, and the grade increased with increased breast parenchymal density, which was statistically significant (p<0.001). it was observed that there was an increase in in the examinations performed, particularly in mri, with increased breast parenchymal density, which was statistically significant (p=0.09). (table 2) comparing the quantitative variables and breast parenchymal density, we found no statistically significant correlation with survival time (p=0.917). although the number of positive lymph nodes was found to be the highest in patients with type 4 breast parenchyma, the difference was not statistically significant (p=0.132). the decrease in age with the increase in breast parenchymal density was statistically significant (p<0.001). tumor size was statistically significantly the largest in patients with type 4 breast parenchyma at a mean of 3.38 cm (p=0.012). mean ki67 expression was 32.8%, and the highest ki67 expression was found in type 4 breast parenchyma patients at 46.24%, which was statistically significant (p<0.001). (table 3) discussion density is revealed on mammography by measuring the content of fibroglandular tissue. recent research tries to explain the correlation between breast cancer and breast density, but this is made difficult by the lack of standardization for the measurement of breast density. the histopathological type and grade of a tumor determines its prognosis and aggression.10 thus, we investigated the correlation between breast density and prognosis, age, tumor size, stage, hormone receptors, her-2 status, grade, lymphovascular invasion, surgical margin status, reoperation, and additional examinations. increased breast density is a very important risk factor for breast cancer in premenopausal and young women. increased breast density also reduces the sensitivity of mg.11–13 previous research has reported that mmg sensitivity ranges from 100% to 45% between fatty breasts and dense breast.14 this may result in delayed diagnosis and increase the need for additional examination. in fact, parenchymal density had no effect on stage in our study (p=0.152), but it caused a statistically significant increase in required additional examinations (p=0.009). we believe that mri in addition to mammography can be very helpful in diagnosis. there have been numerous studies on breast parenchymal density and its effects on risk of developing breast cancer and prognosis. wolfe et al. (1976) divided mammographic breast parenchymal density findings into 4 groups for the first time and reported that breast density was a risk factor for the development of breast cancer.15, 16 in a meta-analysis including 42 studies, mccormack et al. demonstrated a strong correlation between risk of breast cancer and breast parenchymal density.17 breast density is associated with collagen, epithelial, and 183 original efficacy of parenchymal density on breast cancercemil yüksel j contemp med sci | vol. 6, no. 4, july-august 2020: 181–186 table 1. descriptive. variables age mean±sd 61.36±11.54 median (min.-max.) 61.00 (33.00-89.00) breast parenchyma, n (%) 1 21 (14.7) 2 29 (20.3) 3 47 (32.9) 4 46 (32.1) lymph node, n (%) negative 97 (67.8) positive 46 (32.2) grade, n (%) 0-1 58 (40.6) 2 64 (44.7) 3 21 (14.7) er, n (%) positive 117 (81.8) negative 26 (18.2) pr, n (%) positive 109 (76.2) negative 34 (23.8) cerb2 status, n (%) positive 35 (24.5) negative 108 (75.5) type, n (%) luminal a 38 (26.6) luminal b her-2 26 (18.2) triple negative 17 (11.9) luminal b 53 (37.0) her-2 positive 9 (6.3) lvi, n (%) yes 35 (24.5) no 108 (75.5) grade, n (%) 1 14 (9.8) 2 58 (40.6) 3 71 (49.6) survival, n (%) ex 4 (2.8) alive 139 (97.2) survival time mean±sd 27.45±17.85 median (min.-max.) 24.00 (1.00-63.00) ki67 mean±sd 32.83±26.45 median (min.-max.) 22.00 (2.00-95.00) stromal tissues. increased epithelial and fibroblast activity can contribute to increased breast density and breast cancer development.18, 19 a meta-analysis by petterson et al. (2014) including three studies showed that breast parenchymal density increased the risk of developing breast cancer regardless of the menopausal status.20 research shows that the risk of developing cancer is 4–6 times higher compared in dense breasts compared to fatty breasts.21, 22 preand postmenopausal estrogen receptor positive patients whose breast density showed a higher decrease with tamoxifen have been shown to have better prognosis.7, 23 studies on breast density and breast cancer prognosis are yet to reach a consensus on the matter. there is research suggesting fatty breast density to indicate poor prognosis,5, 6 while there is also research suggesting the opposite.24, 25 in addition, there are also studies arguing that breast density has no correlation to prognosis.11, 26 we found that increased 184 original efficacy of parenchymal density on breast cancer cemil yüksel j contemp med sci | vol. 6, no. 4, july-august 2020: 181–186 breast parenchymal density had no effect on prognosis, but our short follow-up period was an important factor in the lack of effective evaluation of prognosis. of course, these findings are not homogeneous, as there is still no standardization for breast density. we think that more consistent findings can be obtained once standardization increases. similar to the effect of parenchymal density on prognosis, studies on breast parenchymal density and tumor biology have not yet reached a consensus. there is research that found no correlation between breast parenchymal density and hormone receptor positivity,27, 28 while two recent studies have shown that patients with higher breast parenchymal density had higher er positivity.29, 30 yaghjyan et al. found a strong correlation between breast parenchymal density and er positivity and concluded that density had no effect on her-2 status.31 they also demonstrated that parenchymal density had no effect on lymph node positivity. in our study, estrogen and progesterone receptor positivity were found to be increased, particularly in fatty breasts (p=0.129), and her-2 receptor positivity was observed to be more frequent in dense breasts, which was statistically significant (p=0.569). triple negative breast cancers have a pattern of gene expression that shows a course of overexpression of genes responsible for cell proliferation and dna replication pathways regulated by cyclin-dependent kinase inhibitor 1a (cdkn1a). her-2 positive breast cancers show an overexpression of genes table 2. correlation between breast parenchyma and qualitative variables. variables breast parenchyma 1 2 3 4 number % number % number % number % p value lymph node negative 11 52.4 24 82.8 30 63.8 32 69.6 0.129a positive 10 47.6 5 17.2 17 36.2 14 30.4 grade 0-1 6 28.6 18 62.1 19 40.4 15 32.6 0.152b 2 11 52.4 10 34.5 20 42.6 23 50.0 3 4 19.0 1 3.4 8 17.0 8 17.4 er positive 21 100.0 27 93.1 39 83.0 30 65.2 0.001a negative 0 0.0 2 6.9 8 17.0 16 34.8 pr positive 19 90.5 25 86.2 36 76.6 29 63.0 0.039a negative 2 9.5 4 13.8 11 23.4 17 37.0 cerb2 status positive 3 14.3 6 20.7 13 27.7 13 28.3 0.569a negative 18 85.7 23 79.3 34 72.3 33 71.7 type luminal a 10 47.6 12 41.5 11 23.4 5 10.8 0.011b luminal b her-2 3 14.3 5 17.2 9 19.1 9 19.6 triple negative 0 0.0 1 3.4 4 8.5 12 26.1 luminal b 8 38.1 10 34.5 19 40.5 16 34.8 her-2 positive 0 0.0 1 3.4 4 8.5 4 8.7 lvi yes 2 9.5 3 10.3 10 21.3 20 43.5 0.002a no 19 90.5 26 89.7 37 78.7 26 56.5 grade 1 2 9.5 3 10.3 5 10.6 4 8.7 <0.001b 2 13 61.9 18 62.1 19 40.5 8 17.4 3 6 28.6 8 27.6 23 48.9 34 73.9 surgical margin suvival 17 81.0 24 82.8 41 87.2 38 82.6 0.876b positive 4 19.0 5 17.2 6 12.8 8 17.4 additional examination yes 4 19.0 6 20.7 14 29.8 24 52.2 0.009a no 17 81.0 23 79.3 33 70.2 22 47.8 survival ex 2 9.5 1 3.4 0 0.0 1 2.2 0.105b alive 19 90.5 28 96.6 47 100.0 45 97.8 a:chi-squared test, b:fisher’s exact test 185 original efficacy of parenchymal density on breast cancercemil yüksel j contemp med sci | vol. 6, no. 4, july-august 2020: 181–186 responsible for her-2 pathways. epidemiological studies have shown that risk factors for reproduction such as birth information or age of menarche and body mass index are associated with er or pr negative tumors rather than with er or pr positive tumors. er negative breast cancers, especially triple negative breast cancers, are diagnosed at an earlier age.32–34 mema et al. showed that patients with low breast density had a 2.53 times higher risk of developing triple negative breast cancer.35 in our study, patients with dense breasts showed higher triple negativity, which was statistically significant (p=0.011). since density may cause a skipping of possible lesions in breast screening mammograms, some studies have shown increased breast parenchymal density to be associated with larger tumor size, higher number of lymph nodes, and advanced stage.36, 37 in our study, increased breast density was found to be associated with increased tumor size (p=0.012) but not with pathological stage. another study found patients with fatty breasts to be associated with lower stage independently of hormonotherapy and that patients with dense breast parenchyma were at an advanced stage.38 this may be associated with difficulty in diagnosis in dense breasts and with low mammographic sensitivity. lymphovascular invasion is when cancer cells infiltrate the blood vessels or lymphatic vessels inside or around the tumor. lvi is thought to mediate cancer dissemination,39, 40 and is accompanied by poor prognosis and pathological characteristics.41, 42 in our study, lymphovascular invasion positivity was found to be increased with increased density (p=0.002). due to our short follow-up period, we could not evaluate the effect of lymphovascular invasion positivity on prognosis. the limitations of this study were its retrospective nature, the low number of patients, the lack of evaluation of factors affecting prognosis (e.g., additional disease, etc.), short follow-up period, and the lack of evaluation of adjuvant treatment conditions. conclusion increased breast parenchymal density is a crucial risk factor for breast cancer. the effects of density on tumor biology and prognosis are still controversial. although increased breast density seems to have an effect on indicators of poor prognosis, such as lvi and tumor grade, its effect on prognosis could not be shown. in addition, additional examinations should be taken into consideration in order not to cause delay or skipping in diagnosis due to the decreased sensitivity of mammography in dense breasts. disclosure statement: there is no conflict of interest in writing of this article. no financial support or funding has been received. declarations ethics approval and consent to participate: this retrospective study was reviewed and approved by the ethics committee of health science of university ankara abdurrahman yurtaslan oncology training and research hospital. availability of data and materials: the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. funding: there is no funding for this study. table 3. the correlation between breast parenchyma and quantitative variables variables breast parenchyma 1 2 3 4 mean±sd median (min.-max.) mean±sd median (min.-max.) mean±sd median (min.-max.) mean±sd median (min.-max.) pvalue age 66.33±10.80 67.00(40.00-85.00) 60.72±8.59 62.00 (41.00-75.00) 57.06±10.25 56.00 (36.00-80.00) 43.20±9.17 41.50 (26.00-69.00) <0.001 a tumor size 3.15±1.91 2.50(0.50-8.00) 1.93±1.10 1.80 (0.20-4.50) 2.65±1.48 2.10 (0.30-7.50) 3.38±2.20 2.70 (0.80-11.00) 0.012 b number of positive lymph nodes 1.48±2.58 0.00(0.00-9.00) 1.10±5.19 0.00 (0.00-28.00) 1.36±2.92 0.00 (0.00-13.00) 1.54±4.25 0.00 (0.00-24.00) 0.132 b ki67 status 20.57±21.44 15.00 (3.00-80.00) 20.86±21.24 15.00 (2.00-90.00) 32.55±24.00 25.00 (2.00-80.00) 46.24±28.05 50.00 (5.00-95.00) <0.001 b survival time 28.62±16.48 27.00 (7.00-63.00) 27.14±19.16 22.00 (7.00-63.00) 27.38±17.47 24.00 (8.00-63.00) 27.17±18.53 24.00 (1.00-63.00) 0.917 b a:oneway anova, b:kruskal–wallis-h test 186 original efficacy of parenchymal density on breast cancer cemil yüksel j contemp med sci | vol. 6, no. 4, july-august 2020: 181–186 references 1. smith ra, duffy sw, gabe r, tabar l, yen am, chen th. the randomized trials of breast cancer screening: what have we learned? radiol clinics. 2004;42(5):793-806. 2. huo c, chew g, britt k, ingman w, henderson m, hopper j, et al. mammographic density—a review on the current understanding of its association with breast cancer. breast cancer res treatment. 2014;144(3):479-502. 3. sickles ea, d’orsi cj, bassett lw, appleton cm, berg wa, burnside es. acr bi-rads® atlas, breast imaging reporting and data system. reston, va: american college of radiology. 2013:39-48. 4. carney pa, miglioretti dl, yankaskas bc, kerlikowske k, rosenberg r, rutter cm, et al. individual and combined effects of age, breast density, and hormone replacement therapy use on the accuracy of screening mammography. ann intern med. 2003;138(3):168-75. 5. masarwah a, auvinen p, sudah m, dabravolskaite v, arponen o, sutela a, et al. prognostic contribution of mammographic breast density and her2 overexpression to the nottingham prognostic index in patients with invasive breast cancer. bmc cancer. 2016;16(1):833. 6. elsamany s, alzahrani a, elkhalik sa, elemam o, rawah e, farooq mu, et al. prognostic value of mammographic breast density in patients with metastatic breast cancer. med oncol. 2014;31(8):96. 7. masarwah a, auvinen p, sudah m, rautiainen s, sutela a, pelkonen o, et al. very low mammographic breast density predicts poorer outcome in patients with invasive breast cancer. eur radiol. 2015;25(7):1875-82. 8. boyd nf, martin lj, yaffe mj, minkin s. mammographic density and breast cancer risk: current understanding and future prospects. breast cancer res. 2011;13(6):223. 9. boyd n, o’sullivan b, campbell j, fishell e, simor i, cooke g, et al. mammographic signs as risk factors for breast cancer. br j cancer. 1982;45(2):185-93. 10. song t, wang y, du w, cao s, tian y, liang y. the method for breast cancer grade prediction and pathway analysis based on improved multiple kernel learning. j bioinform computat biol. 2017;15(01):1650037. 11. ginsburg o, martin l, boyd n. mammographic density, lobular involution, and risk of breast cancer. br j cancer. 2008;99(9):1369-74. 12. sprague bl, gangnon re, burt v, trentham-dietz a, hampton jm, wellman rd, et al. prevalence of mammographically dense breasts in the united states. jnci j natl cancer inst. 2014;106(10). 13. kolb tm, lichy j, newhouse jh. comparison of the performance of screening mammography, physical examination, and breast us and evaluation of factors that influence them: an analysis of 27,825 patient evaluations. radiology. 2002;225(1):165-75. 14. berg wa, gutierrez l, nessaiver ms, carter wb, bhargavan m, lewis rs, et al. diagnostic accuracy of mammography, clinical examination, us, and mr imaging in preoperative assessment of breast cancer. radiology. 2004;233(3):830-49. 15. wolfe jn. breast patterns as an index of risk for developing breast cancer. am j roentgenol. 1976;126(6):1130-7. 16. wolfe jn. risk for breast cancer development determined by mammographic parenchymal pattern. cancer. 1976;37(5):2486-92. 17. mccormack va, dos santos silva i. breast density and parenchymal patterns as markers of breast cancer risk: a meta-analysis. cancer epidemiol prev biomarkers. 2006;15(6):1159-69. 18. guo y-p, martin lj, hanna w, banerjee d, miller n, fishell e, et al. growth factors and stromal matrix proteins associated with mammographic densities. cancer epidemiol prev biomarkers. 2001;10(3):243-8. 19. li t, sun l, miller n, nicklee t, woo j, hulse-smith l, et al. the association of measured breast tissue characteristics with mammographic density and other risk factors for breast cancer. cancer epidemiol prev biomarkers. 2005;14(2):343-9. 20. pettersson a, graff re, ursin g, dos santos silva i, mccormack v, baglietto l, et al. mammographic density phenotypes and risk of breast cancer: a metaanalysis. j natl cancer inst. 2014;106(5):dju078. 21. boyd nf, guo h, martin lj, sun l, stone j, fishell e, et al. mammographic density and the risk and detection of breast cancer. new engl j med. 2007;356(3):227-36. 22. nyante sj, sherman me, pfeiffer rm, berrington de gonzalez a, brinton la, aiello bowles ej, et al. prognostic significance of mammographic density change after initiation of tamoxifen for er-positive breast cancer. jnci j natl cancer inst. 2015;107(3). 23. li j, humphreys k, eriksson l, edgren g, czene k, hall p. mammographic density reduction is a prognostic marker of response to adjuvant tamoxifen therapy in postmenopausal patients with breast cancer. j clin oncol. 2013;31(18):2249. 24. chiu sy-h, duffy s, yen am-f, tabár l, smith ra, chen h-h. effect of baseline breast density on breast cancer incidence, stage, mortality, and screening parameters: 25-year follow-up of a swedish mammographic screening. cancer epidemiol prev biomarkers. 2010;19(5):1219-28. 25. gierach gl, ichikawa l, kerlikowske k, brinton la, farhat gn, vacek pm, et al. relationship between mammographic density and breast cancer death in the breast cancer surveillance consortium. j natl cancer inst. 2012;104(16):1218-27. 26. maskarinec g, pagano is, little ma, conroy sm, park s-y, kolonel ln. mammographic density as a predictor of breast cancer survival: the multiethnic cohort. breast cancer res. 2013;15(1):r7. 27. chen j-h, hsu f-t, shih h-n, hsu c-c, chang d, nie k, et al. does breast density show difference in patients with estrogen receptor-positive and estrogen receptor-negative breast cancer measured on mri? ann oncol. 2009;20(8):1447-9. 28. yang w-t, dryden m, broglio k, gilcrease m, dawood s, dempsey pj, et al. mammographic features of triple receptor-negative primary breast cancers in young premenopausal women. breast cancer res treatment. 2008;111(3):405-10. 29. conroy sm, pagano i, kolonel ln, maskarinec g. mammographic density and hormone receptor expression in breast cancer: the multiethnic cohort study. cancer epidemiol. 2011;35(5):448-52. 30. j.ding j wr, girling a, thompson d, easton d. . mammographic density, estrogen receptor status and other breast cancer tumor characteristics. breast j. 2010;16(3):279-89. 31. k.ghosh k bk, sellers t, et al. . association of mammographic density with the pathology of subsequent breast cancer among postmenopausal women. cancer epidemiol biomarkers prev. 2008;17(4):872-9. 32. t. fs. molecular portraits of breast cancer: tumour subtypes as distinct disease entities. eur j cancer. 2004;40(18):2667-75. 33. ma h wy, sullivan-halley j, et al. use of four biomarkers to evaluate the risk of breast cancer subtypes in the women’s contraceptive and reproductive experiences study. cancer res. 2010;70(2):575-87. 34. yang x c-cj, goode e, et al. 2011;103(3): 250–263. . associations of breast cancer risk factors with tumor subtypes: a pooled analysis from the breast cancer association consortium studies. j natl cancer inst. 2011;103(3):250-63. 35. mema e sf, chun j, kaplowitz e, price a, goodgal j, moy l. the relationship of breast density in mammography and magnetic resonance imaging in women with triple negative breast cancer. eur j radiol. 2020;124. 36. porter g ea, cornford e, et al.. 2007;188(3):676–683. . influence of mammographic parenchymal pattern in screening-detected and interval invasive breast cancers on pathologic features, mammographic features, and patient survival. ajr am j roentgenol.188(3):676-83. 37. m.sala e sl, warren r, et al. . size, node status and grade of breast tumours: association with mammographic parenchymal patterns. eur radiol. 2000;10(1):157-61. 38. kerlikowske k, cook aj, buist ds, cummings sr, vachon c, vacek p, et al. breast cancer risk by breast density, menopause, and postmenopausal hormone therapy use. j clin oncol. 2010;28(24):3830. 39. ma q, dieterich lc, detmar m. multiple roles of lymphatic vessels in tumor progression. curr opin immunol. 2018;53:7-12. 40. zhang s zd, gong m, wen l, liao c, zou l high lymphatic vessel density and presence of lymphovascular invasion both predict poor prognosis in breast cancer. bmc cancer 2017;17(1):335. 41. rakha ea ms, lee ah, morgan d, pharoah pd, hodi z, macmillan d, ellis io. the prognostic significance of lymphovascular invasion in invasive breast carcinoma. cancer. 2012:3670-80. 42. zhang zq hy, nian q, chen g, cui sq, wang xy tumor invasiveness, not lymphangiogenesis, is correlated with lymph node metastasis and unfavorable prognosis in young breast cancer patients. plos one. 2015. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i4.787 309j contemp med sci | vol. 5, no. 6, november–december 2019: 309–312 original issn 2413-0516 introduction the delivery of quality dental care is a key to the long-term success in oral health promotion. improving the quality of health care services has been a key priority for successful governments.1 clinical governance has so far become an important aspect of quality assurance (qa) and it was first introduced in 1997 with the publication of the “new labors first white paper on health” as a labor’s new approach in public health in the 21st century. the concerns were quality care being delivered to the right patient at right time in a right manner.2 in relation to qa, there are several models available in public health. all models including: international standardization organization (9000, 9001-9004), dental excellence quality model of european federation for quality management, total quality management, european practice assessment model, as well as the clinical governance3 are aiming at improving the quality of health care services. clinical governance would build on (not replace) the existing patterns of qa.2 by definition, clinical governance is “a framework through which dental practitioners are held responsible to improve quality of their services and establish high standards of care by creating an environment in which excellence in clinical care will flourished.” this definition has been produced and applied by nhs clinical governance body, published by the department of health in 1997, for general dental practitioners.4 the clinical governance approach in uk was originally based on seven pillars (n.h.s approach) including: (1) clinical effectiveness, (2) clinical audit, (3) risk management (rm), (4) patient safety, (5) patient and public involvement, (6) use of information, and (7) education and training.5 the clinical governance therefore, covers all activities that help maintaining and promoting patient care standards, and never negates other quality management systems.6 background compliant dental schools can train fully compliant graduates who can use principals and procedures of clinical governance in their daily clinical practice. the university of texas health science center, dental branch at houston began credentialing clinical faculty in 1997 as part of its qa and rm program. credentialing is the process of obtaining, verifying, and assessing the qualifications of a health care practitioner who provides patient services in a health care organization.7 hugh bennet et al, developed a framework for evaluating clinical governance in dental field. this framework included cleanliness and infection control, safety and safeguarding, information and involvement, training and development, quality and improvements, and rm as main domains by focusing on reducing inequities in oral health.6 holden and moore developed a 14 component model to define structure and process control in order to assure the expected outcome in overall clinical performance. they believe it is a robust, flexible, effective, and systematic way of improvements in qa.8 the “medical practitioners and dentists board” in kenya developed a national training and qa standards for dental schools and teaching hospitals including: governance and management, academic program, physical infrastructure, human resources, student affairs, program monitoring, and evaluation, as well developing an innovative clinical governance assessment framework for dental schools in iran dehghanian d,a heydarpoor p,b namdari m,a khoshnevisan mhc acommunity oral health department, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. bschool of management and medical education sciences, shahid beheshti university of medical sciences, tehran, iran. cpreventive dentistry research center, research institute of dental sciences, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. correspondence to khoshnevisan mh (mh.khoshnevisan@sbmu.ac.ir ) (submitted: 07 july 2019 – revised version received: 18 august 2019 – accepted: 26 august 2019 – published online: 26 december 2019) objectives the aim of this study was to develop and validate a dental clinical governance (dcg) assessment framework for use in local dental schools in iran. methods a mixed method (qualitative and quantitative) was used in the present investigation. the study was performed in three steps, including: (a) thorough literature review, (b) focused group discussion, and (c) application of validated instrument. the content validity index (cvi) and content validity ratio (cvr) were calculated for each question. the cronbach’s alpha coefficient was calculated to evaluate the internal consistency and reliability for this questionnaire. the smart pls software was used for calculation of goodness-of-fit (gof) for confirmatory factorial analysis to determine construct validity of this questionnaire. results initially, 140 items covering 7 dcg domains were identified after comprehensive literature review. ten specialists participated in the expert panel, rating independently on the necessity, relevancy, simplicity, and clarity of each question. expert’s ratings were used to calculate the validity for each question. the questions with cvi lower than 0.79 and the cvr less than 0.62 were excluded. reliability analysis was conducted by calculating intraclass coefficient and cronbach’s alpha coefficient which were 0.88 and 0.92, respectively. this shows good reliability and internal consistency of the questionnaire. construct validity determined by computing gof index. the result was 0.622, which indicates a good level of construct validity. after validation process, 124 out of 140 questions left to cover the 7 domains of dcg in our newly developed and validated framework. conclusions the newly developed and standardized dcg framework can be used for assessment of compliance level among iranian dental schools at the national level. key words dental clinical governance, dental schools, validation, questionnaire, framework 310 original developing an innovative clinical governance assessment framework dehghanian et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 309–312 as research and innovation.9 fredekind et al performed a survey to obtain information on whether dental schools integrated qa and rm and what mechanisms have been most effective in measuring accomplishments in those institutions. all 65 dental schools were sent a 29-item survey, of which 46 (71%) responded. sixty-six percent of dental schools (n≈43) had a written qa program supervised by a qa committee.10 kakudate et al evaluated the use of japanese clinical guidelines among 148 dentists in a cross-sectional study. they concluded using clinical guidelines and evidence-based practice is still inadequate in japanese dentistry, but use of the clinical practice guidelines was significantly related to better clinical experience.11 in current study, the aim was to develop and validate a dental clinical governance (dcg) assessment framework for use in local dental schools in iran. methods using descriptive-analytic study design, the investigation was conducted in three steps, including: (a) thorough literature review, (b) semi-structured interview (focused group discussion), and (c) application of validated instrument in selected public dental schools. please note that for the purpose of this publication steps 1 and 2 are presented and the rest will be reported separately. step 1: comprehensive review of literature a comprehensive review of literature was conducted by search in english electronic databases including: pubmed, emerald, science direct, google scholar, and also other published books and documents related to clinical governance to obtain different patterns and frameworks related to clinical governance in dental practice in order to identify the main domains of clinical governance in dentistry. step 2: focused group discussion the outcome of the first step was formatted into a checklist containing 140 questions regarding clinical governance in dental practice prepared. the checklist was categorized in seven main areas. this checklist was provided to focus group participants 2 weeks prior to their meeting. a well-recognized expert (the head of the clinical governance committee at the ministry of health and medical education) was invited as a moderator to manage the focused group discussion. this professional committee was formed with 10 specialists whose qualifications are demonstrated in table 1. the moderator’s goal was to generate the maximum amount of discussion and opinions provided by participants within a given time period.12 first, the moderator explained the importance of clinical governance in quality of health care services and described seven main components of clinical governance based on nhs system that has been applied for evaluating iranian medical hospitals since 2009. discussion on each question was continued and all opinions and comments were written in a board for further explorations and decisions. this cession was recorded and reviewed to make sure nothing was missed. finally, specialists were asked to provide comments independently on the necessity, relevancy, clarity, and simplicity of each question in order to calculate the content validity index (cvi) and content validity ratio (cvr) as well as other analytical procedures for standardization of the questionnaire. results reliability two methods were used for internal consistency and reliability of the newly developed instrument: a) cronbach’s alpha coefficient method was used to assess the internal consistency of a scale which relates to its homogeneity. the calculated value was 0.92 that shows a good internal consistency between questionnaire items.14 b) intraclass correlation coefficient (icc) was calculated to determine the reliability of the scale using test–retest method. the test–retest reliability method was used to assess the stability over time, when applying the same test to the same subjects at different points of time.14 the estimate of icc coefficient was calculated to determine the reliability of the scale using test–retest method. the icc score was 0.88 that demonstrate good reproducibility of questionnaire. content, face and construct validity a) the expert panel’s rating on the necessity, relevancy, simplicity, and clarity of each question was used to calculate the validity for each question. according to lawshe table, an acceptable cvr value for 10 expert panels was 0.62 and acceptable cvi was 0.79.13 therefore, the questions with cvi lower than 0.79 and the cvr less than 0.62 were excluded. b) the face validity of the questionnaire was assessed by using the impact score (frequency × importance). the items related with an impact score of equal to or greater than 1.7 was considered appropriate. the mean score for cvi, cvr, and impact score were calculated for each domain and also for the whole questionnaire. c) construct validity was assessed by confirmatory factorial analysis and computing convergent validity, discriminative validity, and goodness-of-fit (gof) index by computing, r2, t-value, and communality indices. gof index was 0.622, which indicates a good level of construct validity or good relationship between questionnaire items. the convergent validity was determined for each domain by computing average variance extracted (ave). for each domain, table 1. qualifications of focus group participants # specialty/department degree academic rank total 1 social medicine phd assistant professor 1 2 endodontics msc professor 2 3 oral and maxillofacial surgery msc assistant professor 1 4 operative dentistry msc associated professor 1 5 periodontics msc professor 1 6 orthodontics msc professor 1 7 dental public health phd associated professor 2 8 biostatistics and epidemiology phd assistant professor 1 total 10 311 original developing an innovative clinical governance assessment frameworkdehghanian et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 309–312 ave was higher than 0.5 which shows good convergent validity. also, discriminative validity was computed for each question. the results showed that questions in each domain have more correlation with domain topic, compared to other domains. the components of validity and reliability analysis is demonstrated in tables 2 and 3. after validation process, 124 out of 140 questions left to cover the 7 domains of dcg in our newly developed and validated framework. a total of seven questions on leadership and management domain, four questions in clinical effectiveness and audit (prevention) domain, four questions in clinical effectiveness and audit (treatment) domain, and one question in rm domain were excluded because their scores were less than the acceptable thresholds. fig. 1 shows the validation process. components of final validated framework and number of questions left in each domain are shown in table 4. it is important to note that the “clinical effectiveness and audit” domain is originally designated for “treatment.” however, given the importance of “prevention” in dentistry, a separate domain was assigned for its robust independent assessment (numbers 3 and 4 in table 4). discussion dental schools are under increased pressure to demonstrate qa efforts in their clinical practice. qa in any health care institution should meet the acceptable standards of patient safety with highest priority.10 however, the main goal of dental education is to: (a) fulfil the above requirement, (b) train competent dentists who are fully compliant with dcg, and (c) achieve oral health promotion in the public, without forgetting table 2. components of validity analysis domains construct validity (confirmatory factorial analysis) content validity face validity r2 t-value communality ave cvr cvi impact score management and leadership 0.520 2.466 0.693 0.711 0.80 0.82 3.75 clinical audit and effectiveness (prevention) 0.671 25.410 0.756 0.776 0.72 0.95 3.78 clinical audit and effectiveness (treatment) 0.503 10.938 0.739 0.762 0.82 0.97 3.33 patient and public involvement 0.696 28.248 0.775 0.799 0.75 0.91 3.59 risk management 0.403 9.490 0.603 0.624 0.84 0.95 4.15 using information 0.417 9.152 0.821 0.850 0.91 0.87 3.34 staff education 0.552 16.873 0.683 0.706 0.75 0.92 3.64 mean 0.536 14.65 0.724 0.746 0.80 0.92 3.61 g.o.f gof= r communalityy ∗ = ∗ =0 536 0 724 0 622. . . table 3. components of reliability analysis domains reliability composite reliability cronbach’s alpha management and leadership 0.961 0.956 clinical audit and effectiveness (prevention) 0.980 0.978 clinical audit and effectiveness (treatment) 0.957 0.947 patient and public involvement 0.983 0.981 risk management 0.981 0.980 using information 0.992 0.992 staff education 0.981 0.980 mean 0.976 0.973 table 4. framework components and number of questions in each domain # clinical governance domains number of questions 1 management and leadership 10 2 clinical effectiveness and audit (prevention) 15 3 clinical effectiveness and audit (treatment) 8 4 patient and public involvement 14 5 risk management 32 6 using information 23 7 staff education 22 total 124 fig. 1 framework validation process. 312 original developing an innovative clinical governance assessment framework dehghanian et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 309–312 the healthy individuals. despite all scientific innovations and advancements, oral diseases are still highly prevalent based on epidemiological reports, as well as the ever-increasing socioeconomic and demographic changes that occur in most communities. the prevalence of dental caries among adults population is very high (over 95%) in most countries. generally, oral diseases are considered as one of the major public health problems in all regions of the world. they involve individuals and communities and diminishing the quality of life by causing pain, infection, and sufferings that leads to impairment of function as well. based on reported global burden of oral diseases, the condition is mostly concentrated in disadvantaged and poor communities. the current pattern of oral diseases are related to lifestyle and environmental factors, and lack of access to preventive oral health programs.15. the results of national survey regarding the oral health status in iran (2018) showed that periodontal diseases and tooth loss are increasing compared with previous data. caries-free status is sharply declining from 12 to 15 years olds (by 27%) and number of edentulous people is exceeding 50% in our elderly (65–74 years) population. these data indicate the urgent need for effective interventions in all age groups. lack of supportive oral health promotion policies for allocating appropriate resources was considered as the main reason for the current situation in iran.16 therefore, development of new training standards of care within the dcg framework is crucial to fulfil the population’s oral health promotion needs. on the other hand, future accreditation of dental school curriculum and the competencies of their graduates will receive reciprocal recognition at the local, regional, and global levels, if uniform standards of care is provided in harmony with dcg. dcg is dynamic and changing with advancement of science. therefore, innovative dental schools will incorporate dcg framework in order to be qualified for certificate of compliance. this would also help to reduce high current demands for dental services by incorporating preventive principals, while providing the high quality dental care to incoming patients. the newly developed framework has some similarities with hugh bennet’s framework that assessed fewer domains on patient’s satisfaction and patient’s complaints as an independent domain (patient and public involvement) can be considered as an advantage for this framework as well.6 fredekind et al evaluated dental schools based on qa and rm domains10 while, the current validated questionnaire focused on all seven domains of clinical governance for evaluating dental schools. the national training and qa standards for dental schools and teaching hospitals developed by the “medical practitioners and dentists board” in kenya was rather considered as a comprehensive program with measurable standards that can be comparable in some aspects with the newly developed framework, covering both qa and educational needs of students in dental schools and teaching hospitals in kenya.9 kakudate et al evaluated the use of japanese clinical guidelines which is related to clinical effectiveness domain11; while the current validated framework can assess all seven main domains of clinical governance in dental schools. the aim of this study was to develop an innovative framework for assessment of local dental schools in relation to dcg compliance level. the availability of such standardized framework can help all local dental schools to conduct voluntary internal evaluation on their progress in meeting these standards. the ministry of health and medical education can also set the expected national standards of dcg and use this instrument for external evaluation of dental schools in order to ensure that those academic institutions meet the minimum requirements. to recognize such an important achievement, a compliance certificate may be issued by the ministry of health for qualified institutions. this document can play an important role in accreditation of institution as well. regular internal and external evaluations can safeguard qa in dental services delivery for all individuals, healthy, or otherwise. conclusion the current study was conducted to develop a dcg framework for use in local iranian dental schools. the newly developed and validated framework can be used for assessment of compliance level among iranian dental schools at the national level. references 1. mills i, batchelor p. quality indicators: the rationale behind their use in nhs dentistry. br dent j 2011;312:11–15 2. ball g. clinical governance in dental primary care. national dental advisory committee. 2001;1–2 3. yamalik n. quality systems in dentistry part 2.quality assurance and improvement (qa/i) tools that have implications for dentistry. int. dent j 2007;57:459–67. 4. maidment yg. clinical governance. what is it and how can it be delivered in dental practices? prim dent care. 2004 apr; 11(2):57–61. 5. rob mc, paddy p. clinical governance and national health service. john t, editor. clinical governance a guide to implementation for health care professionals, 3rd ed. new delhi: willey-blackwell; 2011. p.2. 6. hugh b, anup k. a model dental governance framework for general dental services provided by the local health boards 2013;1–69. 7. valenza ja, george la, o’neill pn. a model for clinical credentialing of dental school faculty. j dent educ. 2005; 69: 870–8. 8. holden lc, moore rs. the development of a model and implementation process for clinical governance in primary dental care. br dent j 2004;196:21–4. 9. kenya medical practitioners and dentists boards national training and quality assurance standards for dental schools and teaching hospitals in kenya september 2015;1–63. 10. fredekind re, cuny ej, nadershahi na.quality assurance and risk management: a survey of dental schools and recommendations for integrated program management. j dent educ. 2002;66:556–63. 11. kakudate n, yokoyama y, sumida f, matsumoto y, gordan vv, gilbert gh, use of clinical practice guidelines by dentists: findings from the japanese dental practice-based research network. j eval clin pract. 2017 feb;23(1):96–101. 12. tobias o. nyumba, kerrie wilson, christina j. derrick, nibedita mukherjee. the use of focus group discussion methodology: insights from two decades of application in conservation. meth ecol evol.2018; 21–32. 13. lawshe ch. a quantitative approach to content validity. pers psychol. 1975;28:563–575. 14. cronbach lj. coefficient alpha and the internal structure of tests. psychometrika. 1951;16:297–334. 15. poul e, denis b, hiroshi ogawa, saskia e, charlotte n. the global burden of oral diseases and risks to oral health. bull who. 2005;83:661–669. 16. khoshnevisan m.h, ghasemianpour m, samadzadeh h, baez rj. oral health status and healthcare system in i.r. iran. j contemp.med sci 2018;4:107–118. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201903 111j contemp med sci | vol. 2, no. 7, summer 2016: 111–114 research histological study of granulocytic series in the bone marrow of adult goat (caprus hircus) yahia dahash hamdi,a alzubaidi ka,b siraj m al-kafagyb issn 2413-0516 introduction the granulocytic sequence in the active bone marrow is confined to the selected areas of bones, such as the flat bones of the head, pelvis, ribs, vertebral bodies, and the ends shift of the long bones. indeed, the blood cells also produced in the spleen and liver of the fetus.1,2 in adult, the normal marrow consists of hematopoietic precursors of erythrocytes, granulocyte, some lymphocytes, plasma cells, and few scattered precursor of platelets.3,4 generally, hematopoiesis is a complex process in which pluripotent stem cells differentiate and evolves into committed progenitor cells. they in turn proliferate into mature functional blood cells such as erythrocyte, megakaryocytes, monocytes, neutrophils, esinophils, basophiles, tas well as blymphocytes. thus, hematopoiesis takes place within the bone marrow stromal microenvironment.5,6 materials and methods five adult goats were used in this research. these animals were obtained from the animal’s field in the college of veterinary medicine, university of al-anbar. the adult goat weight (23–28) kg. these animals are supplied by ruminates diet supplement with vitamin c. the goats are maintained in a pathogen free environment and femurs are chosen for bone marrow aspiration and biopsy. the great advantage is that serial examinations showing distinct qualitative differentiation of marrow cells may be obtained from femoral bone. each goat was anesthetized with a mixture of xylazine hydrochloride 0.1 mg/kg b.w. and ketamine hydrochloride 4 mg/kg b.w. intramuscularly.7 the hind limb was shaved and prepared aseptically with providone iodine. a small incision is made through the skin to facilitate the entering of the needle. the tip of the needle was pushed through the head of the femur until it reaches the shaft adepartment of anatomy, histology and embryology, college of veterinary medicine, university of al-fallujah, al-anbar, iraq. bdepartment of anatomy, histology and embryology, college of veterinary medicine, university of al-qasim green, babylon, iraq. correspondence to yahia dahash hamdi (email: yahia83mwr@yahoo.com). (submitted: 7 june 2016 – revised version received: 10 july 2016 – accepted: 14 july 2016 – published online: 26 september 2016) objectives the aim of this research is to identify and evaluate the cellular granulocytic series of bone marrow in goat that aids to diagnose bone marrow from a variety of clinical disorders in the field of medicine. methods five adult goats were used in this research. these animals are obtained from the animal’s field in the college of veterinary medicine, university of al-fallujah. results the first indication of the granulocytic series begins from myeloblast, which possesses eccentric, round or oval nucleus that surrounded by a rim of cytoplasm. the smaller nucleus leads to identify the promyelocyte. the cytoplasm of promyelocyte is very light. definitive granulations appear within the cytoplasm of myelocyte, which has round to oval nucleus. the elongation of the nucleus indicates the metamyelocyte. this cell shows the neutrophilic, eosinophilic and basophilic metamyelocyte. a large segment of a circle or twisted nucleus represent the band cell. the last stage of granulocytic series is the segmented cell, which is illustrated by segmented neutrophil, eosinophils, and basophils. conclusion the developmental process of granulocytopioesis series of bone marrow in goat encompasses a lineage of successive morphological alterations involves nuclear and cytoplasmic changes as well as granule transformation resulting in the production of granulocytes. keywords developmental process, granulocytic series, bone marrow, adult goat caprus hircus of femur. special syringe is applied to obtain bone marrow aspiration. syringe is then withdrawn and the marrow expelled onto glass slides. marrow then spreads between the slide and the resultant smear are rapidly air dried.8 all smears were stained with giemsa stain.9 results and discussions the granulocytic events or myeloid series begin with the formation of myeloblast (fig. 1) which is the first precursor cells formed by uni-potent stem cell that has the capacity to give rise to all the granulocytic cell types. myoblast is an oval cell; the cytoplasm is pale, light blue green in color and form a complete collar around the nucleus. the round or oval nucleus in which chromatin is evenly distributed and may form a velvety texture to the nucleus. the promylocyte or progranulocyte possess light cytoplasm and had a slightly smaller nucleus (fig. 2). small nucleoli may be present and its chromatin forms a meshwork. the myelocyte can be recognized as neutrophilic, esinophilic, and basophilic myelocyte (fig. 3–5). the myelocyte has a round to oval nucleus. at this stage, the neutrophilic and esinophilic myelocytes have all the granules or very distinct, but the basophilic myelocyte has only a few dark purple specific granules. regardless of cell type, the granule tends to accumulate near one side of the nucleus. the metamyelocytes are smaller cells; the nuclei tend to be elongated and show a definitive indentation with the absence of nucleoli. the arrangement of the nuclear chromatin appears coarse, with definite clumping. th e cytoplasm of metamyelocytes shows a few granules (fig. 6). band cells or stab cells are characterized by neutral cytoplasm 112 j contemp med sci | vol. 2, no. 7, summer 2016: 111–114 histological study of granulocytic series in the bone marrow of adult goat (caprus hircus) research yahia dahash hamdi et al. fig. 1 myeloblast (arrow). ×100 giemsa stain. fig. 2 promylocyte. ×100 giemsa stain. fig. 3 neutrophilic myelocyte (arrow). ×100 giemsa stain. fig. 4 eosinophilic myelocyte. ×100 giemsa stain. fig. 5 basophilic myelocyte (arrow). ×100 giemsa stain. fig. 6 metamyelocytes. ×100 giemsa stain. containing many granules and appear in the form of neutrophilic, basophilic, and eosinophilic band cells. the cytoplasmic granules course and clumped. their nuclei appear rod shape, kidney-bean shape or in the form of “c” shape of band cell neutrophils (fig. 7). band cell can be identified by their own segmented nuclei (fig. 8). these results were in fig. 7 neutrophilic band cell (arrow). ×100 giemsa stain. yahia dahash hamdi et al. 113j contemp med sci | vol. 2, no. 7, summer 2016: 111–114 research histological study of granulocytic series in the bone marrow of adult goat (caprus hircus) agreement with the previous result.10 eosinophilia granules are found in the cytoplasm of eosinophilic band cells (fig. 9). their nuclei have two segments or in the form of horseshoes. this result coincides with published report.11 the segmented cell showed the form of the three granulocytic series, and it fig. 9 esinophilic band cell (arrow). ×100 giemsa stain. fig. 10 neutrophil (arrow). ×100 giemsa stain. fig. 11 eosinophil (arrow). ×100 giemsa stain. fig. 12 basophil (arrow). ×100 giemsa stain. fig. 8 band cell basiophilic. ×100 giemsa stain. represented the last stage of myeloid cell maturation the neutrophil has a segmented nucleus, possess three to five nuclear lobes joined by short chromatin filaments (fig. 10). the multilobulated nucleus shows a densely packed chromatin. the eosinophilic segmented cells possess two nuclear lobes (fig. 11). the nucleus of basophilicsegmented cell may appear as s-shape or consists of multiple lobes (fig. 12). these marked results in this research were compatible with12 and with.13 medical researcher14 registered that the granulocyte undergo many stages in the granulocytic event but they did not mention the segmented cells in their events. conclusion the developmental process of granulocytopioesis series of bone marrow in goat encompasses lineage of successive morphological alterations involves nuclear and cytoplasmic changes as well as granule transformation resulting in the production of granulocytes. the earliest recognizable progenitor of this granulocytic series were the myeloblast. the daughter cells of myeloblasts were the promylocytes, followed by myelocytes, metamyelocytes, band cells and segmented cells. n references 1. moore mc. clinical implication of positive and negative hematopoietic stem cell regulators. blood j. 1991;78:7–23. 2. williams da. in research of the self-renewing hematopoietic stem cell. blood j. 1991;17:269–299. 3. tavassol m, yoffey j. blood marrow barrier. in bone marrow structure and function, ar liss press, new york, u.s.a. 1983;85–102. 4. lund je. toxicologic effects on blood and bone marrow. in schalms veterinary hematology, 5th ed. lippincott press, philadelphia, u.s.a. 2000; 44–50. 5. morrison sj, hemmat hd, wandyez am, weissman il. the purification and characterization of fetal liver hematopoietic stem cells. proc nat acad sci. 1995;92:10302–10306. 6. gasper pw. the hemapoietic system. in: feldman bf, zinkl jg, jain nc, eds. schalm’s veterinary hematology, 4th ed. philadelphia. u.s.a. lippincott, williams & wilkins. 2000:63–69. 7. riebold tw. ruminant anesthesia. in: greene sa, ed. veterinary anesthesia and pain management secrets. philadelphia, pa: hanley & belfus. 2002; 253–262. 114 j contemp med sci | vol. 2, no. 7, summer 2016: 111–114 histological study of granulocytic series in the bone marrow of adult goat (caprus hircus) research yahia dahash hamdi et al. 8. john wh. atlas of veterinary hematology blood and bone marrow of domestic animals. elsevier press limited, philadelphia, usa. 2001. 9. galger ae, kazolof en. essential of practical microtechnique. lea and febiger. philadiphia, usa. 1964. 10. borregaard n, lollike k, kjeldesen l. human neutrophil granules and secretory vesicles. eur j heamatol. 1993;15:187–198. 11. bruyn pp. structural substrates of bone marrow function. hematol. 1981;18:173–179. 12. panopoulous ad, watowich ss. granulocyte colony-stimulating factor; molecular mechanisms of action during steady state and emergency hematopoiesis. cytokines. 2008;42:277–288. 13. karasuyama h, kaori m, kazushige o, yusuk t, yohei k, yoshiyuki m. roles of basophils in immediate and delayed – onset allergic reactions. allergy j. 2010;3:73–80. 14. porter rl, calui lm. communication between bone cells and hematopoietic stem cells. arch biochem biophys. 2008;473:193–200. 176 j contemp med sci | vol. 6, no. 4, july-august 2020: 176–180 original issn 2413-0516 introduction the total energy consumed by human body consists of basal or resting metabolic rate (bmr), thermic effect of food (tef), and activity thermogenesis (at). bmr is the minimum amount of expended energy that is compatible with life and it is needed to sustain the metabolic activities of cells and tissues as well as to maintain the processes of circulatory, respiratory, gastrointestinal, and renal systems.1, 2 bmr comprises approximately 60–70% of the total energy expenditure which is affected by several factors such as age, sex, body composition, size, heredity and molecular genetics, hormones, and environmental conditions.3-6 some hormonal factors including the autonomous nervous system and thyroid hormones affect bmr. as one of the most important factors affecting bmr, sympathetic–parasympathetic nervous system is influenced by various factors such as age, sex, stress, psychological, and emotional status, etc. moreover, the thyroid function modulates the bmr and following conditions such as cold and heat tolerance, fatigue, euphoria, heart rate, movement speed, etc.7 according to the preceding issues, differences in bmr can be a result of diversities of factors affecting the the evaluation of basic and neurohormonal parameters in hot or cold temperament person proposed in iranian traditional medicine: an observational study gholamreza mohammadi farsani,a mohsen naseri, saeed hosseini,b ali akbar saboor-yaraghi,c mohammad kamalinejad,d taiebeh mohammadi farsani,e ahmadreza dorosti motlagh,f mina movahhedg 1department of clinical nutrition, school of nutritional sciences and dietetics, tehran university of medical sciences, tehran, iran. minimally invasive surgery research center, iran university of medical sciences, tehran, ir iran. mohammadigh53@gmail.com 2iranian traditional medicine clinical trial research center, shahed university, tehran, iran. naserishahed@yahoo.com 3department of clinical nutrition, school of nutritional sciences and dietetics, tehran university of medical sciences, tehran, iran. saeedhmdphd@hotmail.com 4department of immunology, school of public health, tehran university of medical sciences, tehran, iran. asaboor@tums.ac.ir 5department of pharmacognosy, school of pharmacy, shahid beheshti university of medical sciences, tehran, iran. mkamalinejad@yahoo.com 6department of medical biotechnology , isfahan (khorasgan) branch islamic azad university, isfahan, iran, minimally invasive surgery research center, iran university of medical sciences, tehran, ir iran. tmfarsani@gmail.com 7department of clinical nutrition, school of nutritional sciences and dietetics, tehran university of medical sciences, tehran, iran. a_dorosty@yahoo.com 8traditional medicine and materia medica center, shahid beheshti university of medical sciences, tehran, iran. movehhedm54@gmail.com *corresponding authors mina movahhed: traditional medicine and materia medica center, shahid beheshti university of medical sciences, no.8 shams alley, vali-e-asr street, tehran, iran, p.o.box 1516745811, tel/fax: 98-21-8877 6027, mobile: +98-9120617507 email: movahhedm54@gmail.com ahmadreza dorosti motlagh: address: department of clinical nutrition, school of nutritional sciences and dietetics, tehran university of medical sciences, tehran, iran. p.o box 6446 tehran 14155, i.r. iran, tel: +98 (21) 88992710, cell: +98 9129727934 email: a_dorosty@yahoo.com author contributions: gholamreza mohammadi farsani (gmf): study design, statistical analysis, drafted the manuscript. mohsen naseri (mn): study design saeed hosseini (sh): study design masoud yunesian (my): study design ali akbar saboor-yaraghi (aas): study design mohammad kamalinejad (mkn): study design, statistical analysis. taiebeh mohammadi farsani (tmf): assisted to draft the manuscript. mina movahhed (mm): study design, assisted to draft the manuscript ahmadreza dorosti motlagh (adm): conceived the study, study design, statistical analysis. abstract objective: much of energy for life sustainment is consumed as the basal metabolic rate (bmr). temperament is one of the core concepts in iranian traditional medicine. the aim of this study is to assess the bmr and activity of the sympathetic–parasympathetic nervous system and thyroid function in hot and cold temperament people. our study is a repeated cross-sectional study which was implemented in 2 stages on 45 healthy volunteers. methods: bmr was evaluated by indirect calorimetry. thyroid function, sympathetic–parasympathetic nervous system activity, body composition, and nutrition status were also evaluated. we used independent t-test for data analysis by spss ver. 16. results: overall, 45 patients aged 18–40 participated in this study. our results showed that the mean of bmrs were respectively 1664.09 and 1909.48 kcal in cold and hot temperament individuals (p<0.3). systolic and diastolic blood pressure, and heart rate peripheral temperature of individuals with hot temperament were significantly higher (p<0.05), while no statistically significant difference was seen in norepinephrine to epinephrine ratio, norepinephrine to cortisol ratio and core temperature. t3 and tsh levels were respectively, 1.99 and 1.26 in cold temperament individuals and 1.38 and 1.40 in hot temperament individuals (p<0.05). conclusion: it shows signs that there is a relationship between the bmr and neurohormonal system and body temperament meaning cold temperament people have lower bmr, sympathetic nervous system activity and thyroid function in comparison to others. body health is negatively affected by bmr fluctuations. with regarding to this correspondence, the arrangement of a pattern for better bmr regulation is crucial. keywords: temperament; iranian traditional medicine; basal metabolic rate; sympathetic – parasympathetic nervous system; thyroid function abbreviations: basal metabolic rate (bmr), iranian traditional medicine (itm), thermic effect of food (tef), activity thermogenesis (at), bioelectrical impedance analysis (bia), enzyme-linked immunosorbent assay (elisa). 177 original hot and cold temperaments in iranian traditional medicinegholamreza mohammadi farsani j contemp med sci | vol. 6, no. 4, july-august 2020: 176–180 sympathetic–parasympathetic nervous system and thyroid function.8-17 iranian traditional medicine (itm, persian medicine) is one of the oldest schools of traditional medicine.18-22 from itm point of view, temperament is one of the basic concepts of health and disease. temperament is variable among individuals and is affected by several factors such as age, sex, environment, and body composition.23-26 recent investigations have shown that hot (warm) and cold temperaments can be associated with bmr and similarly with sympathetic–parasympathetic nervous system activity.27, 28 some of these relationships are shown in table 1. according to the foregoing discussion, it seems that hot temperament people have a higher bmr in comparison to cold temperament people. a new window is hoped to be created in nutrition science and public health by development of bmr concept in traditional medicine viewpoint. in order to investigate this relationship, comparison between the bmr of both cold and hot temperament individuals was carried out. materials and methods this is a descriptive–analytic study. since the under investigation variables can be affected by different factors, all the variables were investigated in 2 stages with a 2–4 weeks interval. in order to select individuals undergoing the study, a notification was published and distributed among the students residing in tehran university of medical sciences dormitories. inclusion criteria age from 18 to 40 years, healthy individuals (according to history and physical examination), normal body mass index, informed consent. exclusion criteria use of any specific dietary pattern such as vegetarianism, smoking, alcohol imbibing, or regular consumption of coffee, taking any medications or supplements in the past month, abnormal biochemical lab test after evaluation, not having tendency to participate in the study. in this study, demographic characteristics such as height, weight, vital signs, blood group, rh, bmr, body composition, and biochemical assays (measurement of serum levels of adrenaline, noradrenalin, cortisol, t3, t4, and tsh) were evaluated. nutrient intake was assessed using a food frequency questionnaire and three 24-h dietary recalls. temperaments of participants were determined by a validated questionnaire29 and the expert diagnosis (traditional medicine specialist). based on the foresaid temperament determining questionnaire, if the total score of the individual was 19 or greater, he was hot temperament; and if it was 14 or less, the temperament was cold. if the score’s range was from 15 to 18, the individual’s temperament was considered mild that was excluded from the study. the number of gender-divided hot and cold temperament participants was based on required sample size. bmr was measured by the use of indirect calorimetry (metalyzer 1b). in order to control the factors affecting metabolic rate, avoidance of any food especially coffee and cigarette 12 hours before the test was recommended to the participants. they should be also inhibited from moderate and severe aerobic or anaerobic activity 2 and 14 hours before the test, respectively. before performing indirect calorimetry, the participants should rest for 10–20 min. during the test, the room temperature was maintained 20–25°c.30participants’ body composition, including weight, fat, water and muscle percentage, and fat-free mass were measured by bioelectrical impedance analysis (bia) (company inbody model 770). serum thyroid function tests were measured using enzyme-linked immunosorbent assay (elisa) and “pishtaztebcompany kits”, epinephrine and norepinephrine using 2-cat elisa kit manufactured by immunobiological laboratories company, cortisol by dcmo23-4 kit by diametra company, and blood group and rh by cinnaclone ii kit. calculations were performed using 80% power, a 5% significance level, and a 25% dropout rate. the required sample size was approximately 40 participants for each group allowing a 25% withdrawal rate. table 1. factors affecting bmr & temperament. variable basal metabolic rate temperament age *high in the first and second year of life *reduces 2–3% in each decade of life1, 2 *hot temperament is more common in children and the young4 *temperament heat starts to decrease after the age of 35–405 sex in the same weight and height, bmr in women, is 5–10% less than men1, 2 female temperament is colder than the male5 body composition *muscle mass is the most significant determinant of bmr. *it increases bmr1, 2 *high muscle mass indicate shot temperament *high fat mass indicates cold temperament4 body surface those who have more body surface, have higher bmr1 *high muscle mass indicate shot temperament *high fat mass indicates cold temperament4 the sympatheticparasympathetic nervous system stimulating the sympathetic nervous system (e.g. stress) raises the bmr1 some emotional conditions can increase body’s hot temperament6 thyroid function *hypothyroidism reduces the bmr *hyperthyroidism increases the bmr1, 3 *individuals with cold temperament feel cold more than others & tolerate heat better than the cold in normal conditions. *in hot temperament individuals, it is reversed4, 6 178 original hot and cold temperaments in iranian traditional medicine gholamreza mohammadi farsani j contemp med sci | vol. 6, no. 4, july-august 2020: 176–180 we used spss (statistical package for social sciences, ver 16) program for statistical analysis. mean and standard deviation were calculated for quantitative variables and frequency (percent) describes qualitative variables. independent t-test was used to compare bmr, thyroid function and body composition, nutrient intake, clinical characteristics, and biochemical tests between two groups. less than p< 0.05 was assumed as significance. this study was approved by tehran university of medical sciences ethics committee bearing code number 90-04-2715366-55307. all subjects were informed through a written consent form. results totally, 45 patients aged 18–40 years participated in this study. two people were excluded because of not being able to complete the study. finally, 43 patients terminated the study, 20 of whom were male (46.5%) and 23 were females (53.5%). ten men had hot temperament and 10 had cold temperament. among women, 11 had hot temperament and 12 had cold temperament. the average age of the participants was 27.47±4.65 years that this average was more in cold temperament people, but no statistically significant difference existed between them (p value = 0.152). hematocrit and hemoglobin levels were in the normal range in all participating subjects and no statistically significant differences existed among the two hot and cold temperament people (hot and cold groups) (p value>0.05), but the amounts of hemoglobin and hematocrit were significantly higher in men (p value <0.05). the results of the anthropometric indices showed that weight, height, and body mass index had no significant diversity among the two groups (p value> 0.05). as shown in table 2, body fat mass and percentage were significantly greater in the cold groups (p value <0.05) and the percentage of fat free mass and skeletal muscle mass was significantly higher in the hot group (p value <0.05). although the amount and percentage of body water was more in cold group, this difference was not statistically significant (p value> 0.05). nutrient intake results based on ffq, intake of energy (2572.2 vs 1900.61 kcal), carbohydrate (361.40 vs 253.24), fat (89.61 vs 70.08) and protein (94.33 vs 76.24 g) was significantly higher in the hot group (p value <0.05). based on the 24-h recall questionnaire, the individuals’ food intake temperament was assessed relatively due to the expert idea in both days before intervention. based on the results, no significant relationship existed among the two groups in any days before intervention (p value>0.05). biochemical and metabolic findings as shown in table 3, the hot group has a significantly higher bmr (p value = 0.03) even after adjusting bmr for weight (p value = .006). t3 amounts were significantly higher in hot group and tsh was higher in cold group (p value< 0.05). although t4 rate was higher in hot group, it can’t be considered meaningful (p value= 0.457). as shown in fig 1, heart rate, systolic and diastolic blood pressure, and peripheral temperature of the hot group was significantly higher (p value <0.05). but core temperature, the norepinephrine to epinephrine and norepinephrine to cortisol ratio were not significantly diverse among them (p value>0.05). discussion the results indicate that body composition of people with hot temperament significantly differs from people with cold temperament; so that fat tissue is more in cold group and the percentage of fat-free mass and skeletal muscle mass is more in hot group. these findings confirm the traditional medicine viewpoint about the temperament. according to this view, the temperament of fat tissue is cold and muscle tissue is hot. accordingly, the amount of fat is higher in the body of cold group, while the amount of muscle is higher in the body of hot group. in conclusion, body composition can be used as a marker to identify individuals’ temperaments. based on the findings of this study, no significant relationship was seen between the blood group or rh and temperament which may be due to small sample size. for exact assessment of this relation and further studies, greater sample sizes are needed in this regard. our results indicate that bmr varies in hot and cold group. indeed, the mean bmr is higher in hot group. even after weight adjusting (the metabolic rate per kilogram of body weight), the similar results were seen. basal metabolism maintenance and preventing it from slowing down or rising up would affect human health. although traditional concept of temperament is not adverted in modern medicine, investigation of factors influencing temperament indicates that it’s possible fig. 1 the clinical characteristics and biochemical tests to measure the performance of the sympathetic– parasympathetic nervous system. fpo 179 original hot and cold temperaments in iranian traditional medicinegholamreza mohammadi farsani j contemp med sci | vol. 6, no. 4, july-august 2020: 176–180 to consider these effective factors for bmr regulation. in itm, many health protecting orders and diagnostic and therapeutic procedures are determined based on the temperament of individuals. temperament of each individual is based on several physical and psychological characteristics which are completely introduced in itm sources temperament detecting templates have diagnostic roles in determining the temperament. based on itm view, different reactions to pathogenic conditions can be explained by temperament consideration so new abilities may be built to forecast diseases related to temperament susceptibility and to improve treatments on the basis of food and drug temperament. regarding the differences between nutrient intake of hot and cold group, according to the results of our study, it seems that hot group by virtue of having a higher bmr and more muscle tissue, has a greater need for energy and as a result, total calorie and following different macronutrients intake is higher in them. in order to prove the relation between the sympathetic– parasympathetic nervous system and temperament, the clinical indices and vital signs were also measured in addition to plasma epinephrine, norepinephrine, and cortisol level which were assessed in shahabi et al. study. epinephrine and norepinephrine are indices for assessing the balance between environmental sympathetic nervous system and adrenal sympathetic while norepinephrine to cortisol ratio is an index of balance between environmental sympathetic and parasympathetic activity. clinical indices include heart rate, systolic blood pressure, diastolic blood pressure, core temperature, and the ambient temperature. based on the results of this study, the norepinephrine to epinephrine ratio and norepinephrine to cortisol ratio is not significantly different between hot and cold groups. the differences between results of this study and the study of shahabi et al may be due to the difference in the time of measurement. our sampling was carried out early in the morning and in the fasting state while in the study of shahabi et al, the sampling was implemented at 3–5 pm. as so many factors are effective in the balance of the sympathetic–parasympathetic system, more appropriate indicators may be needed to measure this amount. the clinical parameters and vital signs of the individuals were meaningfully different in two groups. the results of this study showed that heart rate, systolic blood pressure, diastolic blood pressure, and ambient temperature were significantly higher in hot group verifying more sympathetic activity in the hot temperament and more parasympathetic activity in cold temperament. similarly symptoms of hypothyroidism in cold temperament and hyperthyroidism signs in hot temperament suggest their relationship with each other in the mind. the results of our study confirm this hypothesis and show that there is a direct correlation between temperament and the level of thyroid function. table 2. body composition of participants based on temperament. variable temperament of individuals significance statistical test*p value cold temperament n=22 hot temperament n=21 mean standard deviation mean standard deviation body fat mass(kg) 13.41 2.15 18.35 4.15 .001> body fat percentage 21.73 5.47 27.98 7.37 .003 skeletal muscle mass (kg) 30.99 7.15 27.03 5.48 .047 fate free mass percentage 78.49 5.72 72.02 7.37 .003 body water (lt) 36.14 8.34 37.41 6.47 .579 body water percentage 55.70 6.23 56.11 462 .807 independent t-test* table 3. bmr and t3, t4, tsh levels based on temperament. variable temperament of individuals significance statistical test*p value cold temperament n=22 hot temperament n=21 mean sd mean sd basal metabolic rate/kgbw(kcal) 25.74 2.96 28.76 3.88 .006 basal metabolic rate(kcal) 1664.09 252.04 1909.48 442.40 .030 t3 1.26 .13 1.40 .18 .008 t4 7.19 .48 7.29 .34 .457 tsh 1.99 1.24 1.38 .63 .049 180 original hot and cold temperaments in iranian traditional medicine gholamreza mohammadi farsani j contemp med sci | vol. 6, no. 4, july-august 2020: 176–180 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. conclusion it seems that there is a relationship between the bmr and neurohormonal system and body temperament meaning hot temperament people have higher bmr, sympathetic nervous system activity, and thyroid function in comparison to others. human health is negatively affected by bmr fluctuations. regarding this correspondence, arrangement of a pattern for better bmr regulation is crucial. obviously, more studies are needed in order to make the two schools of medicine closer to each other in their path stepping forward health maintenance and disease control. furthermore, coldness of temperament may cause fat accumulation in body. this may probably imply that body fat mass is decreased by temperament heating. acknowledgments this study was supported by the school of nutritional sciences and dietetics, tehran university of medical sciences. the school of pharmacy, shahid beheshti university of medical sciences, the school of traditional medicine, shahed university of medical sciences and noorafshar rehabilitation and sports medicine hospital provided this study with cooperation. the authors reported no conflicts of interest. conflict of interest disclosure: the authors reported no conflicts of interest. funding: this study was funded by tehran university of medical sciences, tehran, iran. references: 1. mcmurray rg, soares j, caspersen cj, mccurdy t. examining variations of resting metabolic rate of adults: a public health perspective. med sci sports exercise. 2014;46(7):1352. 2. manini tm. energy expenditure and aging. ageing research reviews. 2010;9(1):1-11. 3. carol d, rachel k, mahan l. escott stump s. krause’s food & nutrition therapy 12e. canada: saunders ei-sevier; 2008. 4. white cr, kearney mr. determinants of inter-specific variation in basal metabolic rate. j comp physiol b. 2013;183(1):1-26. 5. swanson dl, mckechnie ae, vézina f. how low can you go? an adaptive energetic framework for interpreting basal metabolic rate variation in endotherms. j comp physiol b. 2017:1-18. 6. feng p, zhao h, lu x. evolution of mitochondrial dna and its relation to basal metabolic rate. mitochond dna. 2015;26(4):566-71. 7. mccrory p, strauss b, wahlqvist ml. energy balance, food intake and obesity. exercise and obesity london: smith gordon. 1994:115-30. 8. sl. j. alaghraz altebieh and mabahes allaeieh. tehran university of medical sciences, iran2005. 9. khorasani sa. kholasatol hekmah. medicine 1st vol qom: ismaielian. 2006:52-146. 10. sina ai. al-qanun fi al-tibb2005. 11. zhou x, mao d, luo j, wu j, zhuo q, li y. study of basal metabolic rate of 81 young adults aged 20-29 years old in changsha. zhonghua yu fang yi xue za zhi [chin j prev med]. 2017;51(7):642-6. 12. ali n, mahmood s, manirujjaman m, perveen r, nahid a, ahmed s, et al. hypertension prevalence and influence of basal metabolic rate on blood pressure among adult students in bangladesh. bmc public health. 2018;18(1):58. 13. boratyński js, jefimow m, wojciechowski ms. individual differences in the phenotypic flexibility of basal metabolic rate in siberian hamsters are consistent on short-and long-term timescales. physiol biochem zool. 2017;90(2):139-52. 14. lee hj, yang sj. aging-related correlation between serum sirtuin 1 activities and basal metabolic rate in women, but not in men. clin nutr res. 2017;6(1):18-26. 15. anthanont p, jensen md. does basal metabolic rate predict weight gain? am j clin nutr. 2016;104(4):959-63. 16. maximov al, belkin vs, kalichman l, kobyliansky ed. adaptive changes in basal metabolic rate in humans in different eco-geographical areas. colleg antropol. 2015;39(4):887-92. 17. nilsson jf, nilsson jå. fluctuating selection on basal metabolic rate. ecol evol. 2016;6(4):1197-202. 18. rezadoost h, karimi m, jafari m. proteomics of hot-wet and cold-dry temperaments proposed in iranian traditional medicine: a network-based study. scientific reports. 2016;6:30133. 19. parvinroo s, kamalinejad m, sabetkasaei m. pharmacological concepts of temperament in iranian traditional medicine. iran j public health. 2014;43(10):1463-5. 20. emami m, nazarinia ma, rezaeizadeh h, zarshenas mm. standpoints of traditional persian physicians on geriatric nutrition. j evid-based complem altern med. 2014;19(4):287-91. 21. parvinroo s, naghibi f, zahediasl s, kamalinejad m, sabetkasaei m. the effects of seeds with hot and cold temperaments on serum thyroid hormones, corticosterone and urine vanillylmandelic acid concentrations of healthy rats. j ethnopharmacol. 2014;156:216-21. 22. rezaeizadeh h, alizadeh m, naseri m, ardakani ms. the traditional iranian medicine point of view on health. iran j publ health. 2009;38(1):169-72. 23. alizadeh m, khadem e, aliasl j. diagnosis protocol of stomach distemperament for clinical practice in iranian traditional medicine: a narrative review. iran j public health. 2017;46(7):877. 24. zeinalian m, eshaghi m, hadian m, naji h, marandi smm, asgary s. eight essential foods in iranian traditional medicine and their role in health promotion and well-being. int j prev med. 2017;8. 25. miraj s, alesaeidi s, kiani s. a systematic review of the relationship between dystemprament (sue mizaj) and treatments and management of diseases (ilaj and eslah-e-mizaj). electr phys. 2016;8(12):3378. 26. kopaei r, khajegir a, kiani s. the association between dystemperament and prevention of diseases: a systematic review. j clin diag res: jcdr. 2016;10(9):ye01. 27. farsani gm, movahhed m, motlagh ad, hosseini s, yunesian m, farsani tm, et al. is the iranian traditional medicine warm and cold temperament related to basal metabolic rate and activity of the sympatheticparasympathetic system? study protocol. j diab metab disord. 2014;13(1):74. 28. shahabi s, hassan zm, mahdavi m, dezfouli m, rahvar mt, naseri m, et al. hot and cold natures and some parameters of neuroendocrine and immune systems in traditional iranian medicine: a preliminary study. j altern complement med. 2008;14(2):147-56. 29. mojahedi m, naseri m, majdzadeh r, keshavarz m, ebadini m, nazem e, et al. reliability and validity assessment of mizaj questionnaire: a novel self-report scale in iranian traditional medicine. iranian red crescent med j. 2014;16(3). 30. compher c, frankenfield d, keim n, roth-yousey l, group eaw. best practice methods to apply to measurement of resting metabolic rate in adults: a systematic review. j am diet assoc. 2006;106(6):881-903. https://doi.org/10.22317/jcms.v6i4.709 14 j contemp med sci | vol. 5, no. 1, january–february 2019: 14–19 original article demographic and clinical profiles of female patients diagnosed with breast cancer in iraq nada a.s. alwan,* furat n. tawfeeq, and nawar a.g. mallah national cancer research center, university of baghdad, baghdad, iraq. *correspondence to nada a.s. alwan (email:nadalwan@yahoo.com). (submitted: 28 november 2018 – revised version received: 13 december 2018 – accepted: 18 january 2019 – published online: 26 february 2019) objective to highlight the main demographic characteristics and clinical profiles of female patients registered with breast cancer in iraq; focusing on the impact of age. methods this retrospective study enrolled 1172 female patients who were diagnosed with breast cancer at the main center for early detection of breast cancer/medical city teaching hospital in baghdad. data were extracted from an established information system, developed by the principal author under supervision of who, that was based on valid clinical records of iraqi patients affected by breast cancer. the recorded information regarding clinical examination comprised positive palpable lumps, bloody nipple discharge, skin changes, bilateral breast involvement, tumor size, lymph node status, and the stage of the disease. results the mean age at the presentation was 51 years; patients under the age of 50 constituted 46.8%. overall 9.8% were not married, 22.4% were illiterate whereas 19.2% graduated from universities. about 72% of the patients had more than two children, merely 7.5% delivered their first child after the age of 35 years and only 11% were nulliparous. history of lactation and hormonal therapy was recorded in 57.6% and 19.4% respectively. family history of cancer was positive in 28.8% and breast cancer specifically in 18.7%. clinically, the most common presenting symptom was breast lumps (95%) followed by skin changes/ulcerations (6.7%) and bloody nipple discharge (4.3%). bilateral breast involvement was encountered in 4.7%. more than two-thirds of the patients (68.2%) had palpable axillary lymph nodes; classifying 40.5% into advanced stages iii and iv. in general stages i–iv comprised 12%, 47.5%, 31.9%, and 8.6% respectively. upon stratifying the studied sample with respect to age at diagnosis, it was observed that the frequency of unmarried patients was significantly higher among younger women under the age of 50 years, whereas illiteracy and nulliparity features were statistically lower (p < 0.05). conclusion a considerable proportion of breast cancer patients in iraq still present with locally advanced disease at the time of diagnosis. that justifies the necessity to promote public awareness educational campaigns to strengthen our national early detection program. excluding the marital status, level of education and number of parity, there was no statistical difference regarding the impact of age on the demographic and clinical profiles of breast cancer among premenopausal versus postmenopausal iraqi patients. keywords demographic, clinical, profiles, breast, cancer, iraq introduction the latest who estimates reveal that breast cancer is the most prevalent malignancy worldwide in 154 out of 185 countries and is the leading cause of cancer related mortality in more than 100 countries. globally, it remains the most common cancer among women accounting for 25% of the registered female cancers; with approximately 2.1 million newly diagnosed cases in 2018.1 the variations in the incidence rates are often attributable to higher prevalence of risk factors specifically among transitioning regions in south america, africa, and asia.2 apart from genetic predisposition, many other factors could have an impact on developing breast cancer among women including demographic characteristics, clinical, reproductive, and environmental features. increased risk has been associated with advanced age, positive family history, socioeconomic status, diet, endogenous or exogenous hormones, atypical breast diseases, benign tumors, oncogenic viruses, and carcinogenic exposures.3 in iraq, breast cancer ranks the first among the top ten malignant neoplasms affecting the community; comprising 19.5% of total (4996 cases) and 34.3% of female cancers (4922 cases). during 2016, 897 women died from that disease which is the registered as the first cause of cancer related mortality among iraqi females (23.6%) and the second overall among males and females (12.1%) after bronchogenic cancer.4,5 previous cross-sectional studies revealed a considerable lack of knowledge regarding the risk factors for breast cancer in iraq even among the educated strata of the society.6 who introduced guidelines for establishing national strategies to control cancer in the eastern mediterranean region (emr).7 focusing on breast cancer, information on the putative risk factors for the disease and the clinical characteristics of the affected patients are of utmost importance to plan for its early detection and control. within that context, an iraqi national breast cancer research program was established in 2010; through which a comprehensive information system database was developed for patients diagnosed with the disease in collaboration with the international agency for research on cancer (iarc)/who.8 based on that, several studies have documented that iraqi females often present with breast cancer at younger ages, advanced stages and with more aggressive behavior than their western counterparts.9–13 in a recent comparative survey, it was demonstrated that the significant differences in the clinical and tumor characteristics between iraqi and british patients persisted even after adjusting for age among patients younger than 50 years.14 the aim of this study was to highlight the main demographic and clinical profiles of breast cancer among a series of iraqi female patients who were registered with that disease at a main specialized center; focusing on the impact of age. materials and methods this retrospective study enrolled 1172 female patients who were diagnosed with breast cancer at the referral training issn 2413-0516 n.a.s. alwan et al. 15j contemp med sci | vol. 5, no. 1, january–february 2019: 14–19 original article demographic and clinical profiles of female patients diagnosed with breast cancer in iraq center for early detection of breast tumors, medical city teaching hospital in baghdad over a 4-year period from 2014 to 2017. all related data was introduced through an established information system database, developed by the principal author under supervision of iarc, at the national cancer research center of baghdad university. that was part of a designed ongoing national breast cancer research project to document the demographic, clinical and pathological characteristics of iraqi breast cancer patients.12 the analyzed data was based on the information recorded in the file sheet questionnaires and clinical records of the referred patients. the ethical approval was initially obtained by the ethical research committee of the national cancer research center according to the ethical standards laid down by the declaration of helsinki. the study protocol is within the framework of a regional comparative breast cancer research project, approved by the iarc ethics committee, who in 2016. the studied demographic and clinical data included age, marital status, educational background, number of parity, age at first child birth, history of lactation, hormonal therapy, and family history of breast cancer or any other malignancy. only complete valid followed up cases comprising all requested data were included in the clinical presentation part of the study. the recorded variables were positive palpable lumps, bloody nipple discharge, skin changes or ulcerations, bilateral breast involvement, tumor size, lymph node involvement, and the stage of the disease. the clinical stage of breast cancer was defined according to uicc tnm classification system15 and the american joint committee on cancer staging.16 statistical analysis data entry was performed using the statistical program (spss), version no. 23. the findings were statistically analyzed and the digital frequencies and percentages were calculated accordingly. results were considered statistically significant when p-value was ≤0.05. results table 1 illustrates the demographic characteristics and clinical history of 1172 female breast cancer patients. the peak frequency occurred among the age groups (35–49) years and (60–64) years. only 4.4% and 11% were diagnosed before the age of 35 and after 64 years respectively. about 10% were single, 22.4% were illiterate, 10.8% were nulliparous whereas >71% had more than two children. only 7.5% had their first child birth after the age of 35 years. history of lactation was recorded in 57.6%, hormonal intake in 19.4%, and cancer in 28.8%. on the other hand, family history of breast cancer specifically was displayed in 18.7%. clinically, 95% presented with palpable lumps, 4.3% complained of bloody nipple discharge while 6.7% had overlying skin changes and ulcerations. bilateral malignant involvement of the breast was noted in 4.7%. approximately 59% had a tumor size measuring 2–5 cm (t2), more than 68% had lymph node involvement at the time of initial presentation; thus classifying 40.5% of patients into stages iii and iv (table 2). in tables 3 and 4, the patients were stratified according to age into those <50 years versus those aged ≥50 years. a statistical table 1. demographic characteristics and clinical history of the examined iraqi patients diagnosed with breast cancer variable patients age range (years)* 18–90 mean (sd) 51 (10.68) age category n (%) 20–34 52 (4.4) 35–49 497 (42.4) 50–64 494 (42.2) ≥65 129 (11.0) marital status* single 115 (9.8) married 1032 (88) divorced 12 (1.0) widow 13 (1.1) educational status* illiterate 262 (22.4) primary school 355 (30.3) secondary school 271 (23.1) university and over 225 (19.2) unknown 59 (5.0) parity** nulliparous 115 (10.8) 1–2 180 (17.0) 3–5 496 (47.0) ≥6 266 (25.1) age at first child*** <20 257 (27.3) 20–29 466 (49.5) 30–35 124 (13.1) >35 71 (7.5) unknown 24 (2.5) lactation*** yes 543 (57.6) no 399 (42.4) hormonal therapy* yes 227 (19.4) no 945 (80.6) family history (any cancer)* yes 337 (28.8) no 835 (71.2) family history (breast cancer)* yes 219 (18.7) no 953 (81.3) *number of patients = 1172. **number of patients = 1057 (unmarried are excluded). ***number of patients = 942 (unmarried and nulliparous are excluded). difference was noted regarding marital status, level of education, and parity. the frequency of unmarried patients was significantly higher among younger women under the age of 50 years, whereas illiteracy and nulliparity features were statistically lower (p < 0.05). on the other hand, no statistical difference was observed with respect to the clinical presentation of the examined patients including palpable lumps, bloody nipple discharge, skin changes, tumor size, lymph node status and the stage of the disease (table 4). 16 j contemp med sci | vol. 5, no. 1, january–february 2019: 14–19 demographic and clinical profiles of female patients diagnosed with breast cancer in iraq original article n.a.s. alwan et al. the mean age at diagnosis in this study was 51 years; 4.4% were younger than 35 years and 11% were over 64 years. earlier research studies conducted on iraqi women diagnosed with breast cancer have highlighted the relatively young age at presentation of the affected patients.8–12,14,24 similar observations were reported by other researchers from the region who proposed that the differences in the age at presentation of breast cancer between arab and western populations might be attributable to socioeconomic, demographic, and population factors.19,22,25 upon monitoring health for sustainable development goals, who statistics confirmed that countries within the emr have statistically younger population structures compared with the western societies26; overall the age standardized incidence rates for breast cancer among arab women are significantly lower.1 focusing on iraq a previous study, performed a decade table 2. clinical presentation of the examined breast cancer patients variable patients n (%) palpable lump* yes 543 (95) no 28 (5) bloody nipple discharge* yes 25 (4.3) no 546 (95.7) ulceration/skin changes* yes 38 (6.7) no 533 (93.3) bilaterality* yes 27 (4.7) no 544 (95.3) tumor size** ts 15 (2.5) t1 105 (17.7) t2 351 (59.3) t3 94 (15.9) t4 27 (4.6) unknown 43 nodal status** n0 184 (31.7) n1 185 (31.8) n2 126 (21.7) n3 86 (14.8) unknown 54 stage** i 63 (12) ii 250 (47.5) iii 168 (31.9) iv 45 (8.6) unknown 109 *number of patients = 571. **number of patients = 635. table 3. demographic characteristics and clinical history of the study population verified according to age (<50 years versus ≥50 years) variable age <50 age ≥50 chi-square n (%) n (%) p-value marital status* 0.048 significant single 67 (58.2) 48 (41.7) married 473 (45.8) 559 (54.2) divorced 5 (41.6) 7 (58.4) widow 4 (30.8) 9 (69.2) educational status* <0.00001 illiterate 93 (35.5) 169 (64.5) significant primary school 197 (55.5) 158 (44.5) secondary school 140 (51.7) 131 (48.3) university and over 101 (44.9) 124 (55.1) unknown 18 (30.5) 41 (69.5) parity** <0.00001 nulliparous 48 (41.7) 67 (58.3) significant 1–2 102 (56.7) 78 (43.3) 3–5 251 (50.6) 245 (49.4) ≥6 80 (30.1) 186 (69.9) age at first child*** <20 107 (41.6) 150 (58.4) 0.138 20–29 217 (46.6) 249 (53.4) not significant 30–35 67 (54) 57 (46) >35 35 (49.3) 36 (50.7) unknown 10 (41.7) 14 (58.3) lactation*** yes 252 (46.4) 291 (53.6) 0.809 no 182 (45.6) 217 (54.4) not significant hormonal therapy* yes 101 (44.5) 126 (55.5) 0.429 no 448 (47.4) 497 (52.6) not significant family history (any cancer)* yes 166 (49.3) 171 (50.7) 0.293 no 383 (45.9) 452 (54.1) not significant family history (breast cancer)* yes 112 (51.1) 107 (48.9) 0.157 no 437 (45.9) 516 (54.1) not significant *number of patients = 1172. **number of patients = 1057 (unmarried are excluded). ***number of patients = 942 (unmarried and nulliparous are excluded). discussion the “westernization” of the developing world has been claimed to be the main cause of the global rise in the prevalence of breast cancer.17 the increasing incidence of that disease among developing countries could be related to the higher predisposition to risk factors including early menarche, late menopause, nulliparity, late age at delivering the first child, exogenous hormone intake, postmenopausal obesity, and alcohol.1,2,18,19 the demographic and socioeconomic transitions witnessed by most countries in the emr have increased the burden of cancer.20 it is believed that the difference in the impact of sociodemographic characteristics among the arab countries could reflect economic variations in the implementation of the relevant health policies for cancer control.21 in general, the priority cancers in the region could be controlled provisionally though preventive and screening strategies; thus recommending effective interventions to tackle lifestyle risk factors.7,22 in general, regarding breast cancer it has been well documented that screening practices are suboptimum in the arab world.23 n.a.s. alwan et al. 17j contemp med sci | vol. 5, no. 1, january–february 2019: 14–19 original article demographic and clinical profiles of female patients diagnosed with breast cancer in iraq table 4. clinical presentation of iraqi patients diagnosed with breast cancer verified according to age variable age >50 years age ≥50 years chi-square n (%) n (%) p-value palpable lump* yes 274 (50.4) 269 (49.5) 0.289 no 17 (60.7) 11 (39.3) not significant bloody nipple discharge* yes 15 (60) 10 (40) 0.355 no 276 (50.4) 270 (49.5) not significant ulceration/skin changes* yes 20 (52.6) 18 (47.4) 0.831 no 271 (50.8) 262 (49.2) not significant bilaterality* yes 16 (59.2) 11 (40.7) 0.382 no 275 (50.6) 269 (49.4) not significant tumor size** ts 6 (40) 9 (60) 0.224 t1 56 (53.3) 49 (46.7) not significant t2 174 (49.6) 177 (50.4) t3 58 (61.7) 36 (38.3) t4 25 (92.6) 2 (7.4) unknown 19 24 nodal status** n0 90 (48.9) 94 (51.1) 0.584 n1 93 (50.3) 92 (49.7) not significant n2 72 (57.1) 54 (42.9) n3 48 (55.8) 38 (44.2) unknown 27 27 stage** i 33 (52.3) 30 (47.6) 0.354 ii 118 (47.2) 132 (52.8) not significant iii 95 (56.5) 73 (43.4) iv 23 (51.1) 22 (48.9) unknown 61 48 *number of patients = 571. **number of patients = 635. earlier on 721 iraqi female patients diagnosed with breast cancer, displayed that 45.9% presented after the age of 50 years and 75% were married9; corresponding to 53.2% and 88% respectively in this study. that points out to a shift in the presentation of the disease toward postmenopausal age reflecting socioeconomic transitions. on the other hand, a study from oman showed that out of 1230 examined breast cancer patients, 53.5% are still detected under the age of 50 years.27 this study illustrated that 19.2% of the studied cohort graduated from universities and only 10.8% were nulliparous whereas 7.5% had their first child born after the age of 35 years. while these figures were comparable to those recorded in earlier surveys on breast cancer among iraqi women,9,12 the history of hormonal intake in the presented work (19.4%) was significantly lower than that displayed by the iraqi patients in 2010 (29%) probably reflecting better orientation to the draw backs of such medications.9 several studies have recorded a significant association between estrogen therapy and breast cancer among women with a family history.28 longitudinal follow-up in a women’s health initiative randomized trial revealed that family history and hormonal replacement therapy had independent and non-interacting effects on the risk of invasive breast carcinoma.29 in iraq, a former comparative retrospective study involving 204 female patients diagnosed with breast cancer reported very close figures for positive history of cancer and breast cancer (30% and 18.5% respectively) to those observed in this report; however, the demographic and clinical characteristics of patients with positive family history in that study did not reveal any distinct marker for their identification.30 clinically, the vast majority (95%) of iraqi patients in this study presented with palpable lumps, one-third had axillary lymph node involvement; thus classifying 40.5% into advanced stages (31.9% and 8.6% for stages iii and iv respectively). nevertheless, the rate for stage iv breast cancer was significantly lower than that was illustrated in a study conducted on patients visiting the same center in baghdad 10 years ago9; obviously reflecting one of the promising outcomes of establishing the national program for early detection of breast cancer in iraq in 2001.10,12,24 a similar retrospective study performed on 1070 egyptian breast cancer patients displayed that 18.7% were diagnosed before reaching 40 years, 79.5% were married, history of breast feeding, oral contraceptive pills, and breast cancer was noted in 73%, 39.5%, and 7.5% respectively while 62% presented in stages iii and iv.31 in india, the median age at presentation in a survey including 1528 breast cancer patients was 49 years, 69.6% were postmenopausal, family history of breast cancer was positive in 4.2%, the most common symptom was palpable lumps (96%) and 57% were diagnosed in stages iii and iv disease.32 on the other hand, in croatia, europe, where breast cancer is the most common type of female malignancy, analysis of the demographic characteristics and reproductive findings belonging to 870 female patients showed that the mean age was 69 years; <2% were younger than 35 years.33 these findings were consistent with the results reported in other european studies.34 in the same croatian survey, 8% were exposed to oral contraceptives, 15% were nulliparous with a mean number of children not exceeding 1.6, and only 5% delivered their first baby after the age of 35. family history for breast and/or ovarian cancer was reported in 19% and breast lump was the only presenting symptom in 95%. in a comparative study on the clinical and pathological presentation of breast cancer among iraqi and british women, the authors displayed that the patients were significantly younger in iraq and presented with advanced stages; reflecting heterogeneity in the tumor biology and the dilemma of delayed diagnosis.14 several other studies documented the existing gaps in the knowledge and practices toward breast cancer among the iraqi society; emphasizing the necessity to elevate the level of awareness through strengthening public education campaigns and establishing national protocol guidelines for early detection, diagnosis and treatment of the disease.6,8,12,14,35–39 when correlating the impact of age, as an important risk factor,40 with the studied demographic and clinical features of breast cancer in this work no significant association was observed with the clinical stage at presentation. on the other hand, patients diagnosed with breast cancer at the age of 50 years and older were more likely to be married, illiterate and nulliparous compared to younger premenopausal patients (p < 0.05). meta-analytic surveys focusing on breast cancer and reproductive variables in population-based studies 18 j contemp med sci | vol. 5, no. 1, january–february 2019: 14–19 demographic and clinical profiles of female patients diagnosed with breast cancer in iraq original article n.a.s. alwan et al. confirmed that nulliparity, lactation and late age at first birth are significant independent determinants of breast cancer risk.41,42 on the other hand, other studies from developing countries failed to emphasize that the age factor specifically at first child birth was significant.43 among african-american patients, it was documented that age had a dual effect on parity as a risk factor; increasing the risk in women younger than 45 years and decreasing it among those 45 years and older; thus highlighting some of the factors responsible for the observed variations in the incidence of breast cancer in these societies.44 conclusion a considerable proportion of breast cancer patients in iraq is still present with locally advanced disease at the time of diagnosis. that justifies the necessity to promote public awareness educational campaigns to strengthen our national early detection program. excluding the marital status, level of education and number of parity, there was no statistical difference regarding the impact of age on the demographic and clinical profiles of breast cancer among premenopausal versus postmenopausal iraqi patients. acknowledgement the authors thank the working staff at the main referral center for early detection of breast tumors, oncology teaching hospital and the national cancer research center, university of baghdad for their assistance in providing the requested information. author contribution the principal corresponding author designed the study, analyzed the results, wrote the manuscript and presented the final version. the other authors supported in providing relevant information, data entry, and statistical analysis. conflict of interest the authors declare that they have no conflict of interest that competes with any of the contents of the manuscript. funding none.  references 1. bray f, ferlay j, soerjomataram i, siegel rl, torre la, jemal a. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. cancer j clin. 2018;68:394–424. 2. bray f, mccarron p, parkin dm. the changing global patterns of female breast cancer incidence and mortality. breast cancer res. 2004;6: 229–239. 3. brinton la, gaudet mm, gierach gl. breast cancer. in: thun mj, linet ms, cerhan jr, haiman ca, schottenfeld d, eds. cancer epidemiology and prevention. 4th ed. new york, ny: oxford university press, 2018; pp. 861–888. 4. annual statistical report 2016. planning directorate, ministry of health/ environment, republic of iraq, 2017. available from: https://moh.gov.iq/ upload/upfile/ar/513.pdf. 5. annual report. iraqi cancer registry 2016. iraqi cancer board, ministry of health and environment, republic of iraq, 2018. 6. alwan n, al-attar w, eliessa r, al-midfaei z, nidhal f. knowledge, attitude and practice regarding breast cancer and breast self-examination among a sample of the educated population in iraq. east mediterr health j. 2012;18:337–345. 7. world health organization. strategy for cancer prevention and control in the eastern mediterranean region 2009–2013, world health organization. regional office for the eastern mediterranean, 2010. 8. alwan n. iraqi initiative of a regional comparative breast cancer research project in the middle east. j cancer biol res. 2014;2:1016–1020. 9. alwan na. breast cancer: demographic characteristics and clinicopathological presentation of patients in iraq. east mediterr health j. 2010;16:1059–1164. 10. alwan nas, tawfeeq fn, maallah mh, sattar sa. the stage of breast cancer at the time of diagnosis: correlation with the clinicopathological findings among iraqi patients. j neoplasm. 2017;2:1–10. 11. alwan nas, mualla f, al naqash m, kathum s, tawfiq fn, nadhir s. clinical and pathological characteristics of triple positive breast cancer among iraqi patients. gulf j oncolog. 2017;1:51–60. 12. alwan nas. breast cancer among iraqi women: preliminary findings from a regional comparative breast cancer research project. j glob oncol. 2016;2:255–258. 13. alwan nas. tumor characteristics of female breast cancer: pathological review of mastectomy specimens belonging to iraqi patients. world j breast cancer res. 2018;1:1–3. 14. alwan nas, kerr d, al-okati d, pezella f, tawfeeq f. comparative study on the clinicopathological profiles of breast cancer among iraqi and british patients. open public health j. 2018;11:177–191. 15. nccn guidelines. nccn clinical practice guidelines in oncology: breast cancer. v 1. 2016. national comprehensive cancer network. available from: http://www.nccn.org/professionals/physician_gls/pdf/breast.pdf. 16. edge sb, byrd dr, compton cc, fritz ag, greene fl, trotti a, eds. ajcc (american joint committee on cancer) cancer staging manual. 7th ed. new york, ny: springer-verlag, 2010; pp. 347–377. 17. porter p. “westernizing” women’s risks? breast cancer in lower-income countries. n engl j med. 2008;358:213–216. 18. tfayli a, temraz s, abou mrad r, shamseddine a. breast cancer in lowand middle-income countries: an emerging and challenging epidemic. j oncology. 2010;2010:490631. 19. mehdi i, monem ea, al bahrani bj, al kharusi s, nada am, al lawati j, et al. age at diagnosis of female breast cancer in oman: issues and implications. south asian j cancer. 2014;3:101–106. 20. ravichandran k, al-zahrani as. association of reproductive factors with the incidence of breast cancer in gulf cooperation council countries. east mediterr health j. 2009;15:612–621. 21. sweileh wm, zyoud sh, al-jabi sw, sawalha af. contribution of arab countries to breast cancer research: comparison with non-arab middle eastern countries. bmc womens health. 2015;15:25. 22. kulhanova i, bray f, fadhil i, al zahrani a, el basmy a, anwar w, et al. profile of cancer in the eastern mediterranean region: the need for action. cancer epidemiol. 2017;47:125–132. 23. donnelly tt, khater ah, al-bader sb, al kuwari mg, al-meer n, malik m, et al. arab women’s breast cancer screening practices: a literature review. asian pac j cancer prev. 2013;14:4519–4528. 24. alwan n, kerr d. cancer control in war torn iraq. lancet oncol. 2018;19:291– 292. 25. najjar h, easson a. age at diagnosis of breast cancer in arab nations. int j surg. 2010;8:448–452. 26. world health organization. world health statistics 2017: monitoring health for the sdgs, sustainable development goals, 2017. isbn 978-924-156548-6. 27. mehdi i, monem ea, bahrani bj, al kharusi s, nada am, al lawati j, et al. age at diagnosis of female breast cancer in oman: issues and implications. south asian j cancer. 2014;3:101–106. 28. dinger jc, heinemann la, mohner s, thai do m, assmann a. breast cancer risk associated with different hrt formulations: a register-based casecontrol study. bmc womens health. 2006;6:13. 29. gramling r, eaton cb, rothman kj, cabral h, silliman ra, lash tl. hormone replacement therapy, family history, and breast cancer risk among postmenopausal women. epidemiology. 2009;20:752–756. n.a.s. alwan et al. 19j contemp med sci | vol. 5, no. 1, january–february 2019: 14–19 original article demographic and clinical profiles of female patients diagnosed with breast cancer in iraq this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 30. alwan nas. clinical and pathological characteristics of familial breast cancer in iraq. chron j cancer sci. 2017;1:002. 31. gabr a1, razek k, atta h, elsabah t, tamam s. demographic characteristics and clinico-pathological presentation of breast cancer female patients in south egypt cancer institute (2005–2012). seci oncol. 2016;1–6. 32. velappan a, shumugam d. analysis of demographic characteristics and treatment outcome of breast cancer patients in a tertiary cancer centre. iosr j dent med sci. 2017;16:25–28. 33. zitnjak d, soldić z, kust d, bolanča a, kusić a. demographic and clinicopathologic features of patients with primary breast cancer treated between 1997 and 2010: a single institution experience. acta clin croat. 2015;54:295–302. 34. oecd. health at a glance: europe 2012. oecd publishing, 2013;24–26. 35. al alwan n. establishing guidelines for early detection of breast cancer in iraq. int j adv res. 2015;3:539–555. 36. sankaranarayanan r, alwan n, denny l. how can we improve survival from breast cancer in developing countries? breast cancer manage. 2013;2:179– 183. 37. alwan nas, al-attar wm. evaluating the effect of an educational teaching model on the knowledge about breast cancer among female university students in iraq. jj cancer sci res. 2016;2:026. 38. alwan nas, al-attar wm, mallah n, hassoun t. baseline needs assessment for breast cancer awareness and management among paramedical health care providers in iraq. int j sci res. 2017;6:1515–1519. 39. alwan nas, al-attar wm, al mallah. baseline needs assessment for breast cancer awareness among patients in iraq. int j sci res. 2017;6:2088– 2093. 40. chen hl, zhou mq, tian w, meng kx, he hf. effect of age on breast cancer patient prognoses: a population-based study using the seer 18 database. plos one. 2016;11:e0165409. 41. ewertz m, duffy sw, adami ho, kvale g, lund e, meirik o, et al. age at first birth, parity and risk of breast cancer: a meta-analysis of 8 studies from the nordic countries. int j cancer. 1990;46:597–603. 42. babalou a. the association of parity and breastfeeding with breast cancer: a review. health sci j. 2017;11:1. 43. huo d, adebamowo ca, ogundiran to, akang ee, campbell o, adenipekun a, et al. parity and breastfeeding are protective against breast cancer in nigerian women. br j cancer. 2008;98:992–996. 44. palmer jr, wise la, horton nj, adams-campbell ll, rosenberg l. dual effect of parity on breast cancer risk in african-american women. j natl cancer inst. 2003;95:478–483. 187j contemp med sci | vol. 6, no. 4, july-august 2020: 187–190 case report issn 2413-0516 introduction diabetes mellitus (dm) is a metabolic disorder characterized by chronic hyperglycemia which is due to alteration in insulin production and its action.1 dm affects multiple systems in our body leading to complications such as retinopathy, nephropathy, neuropathy, cardiovascular diseases, and other complications such as infections and cognitive impairment.2 diabetic foot ulcer (dfu) is one of the major complications following diabetes in an individual that leads to substantial morbidity. prevalence of dfu in uk is around 7% and usa is 8% and lifetime risk is 15%. in india, around 15% of patients with diabetes are affected by dfu during their lifespan. out of this proportion, 50% is infected that requires hospitalization and 20% need amputation in india. dfu alone contributes 80% of non-traumatic amputation and have huge burden in terms of expenses and morbidity.3,4 there are several factors which contribute in the development of diabetic ulcer; however, neuropathy and ischemia due to peripheral vascular diseases are the major contributing factors. in diabetic patient, production of nerve cell myoinositol that regulates normal neural conduction is hampered. this affects all the components of neural functions including motor, sensory and autonomic functions. deformities such as claw-toe and high arched foot are the result of motor nerve involvement in diabetic patients that results in uneven distribution of force in foot, and thus formation of callus and skin breakdown. dryness of skin and peripheral edema is a result of autonomic dysfunction in diabetic that leads to development of fissured skin. decreased perception of pain and sensation leads to inability to detect any injury in subjects with diabetes due to which wound goes unnoticed for several duration until it gets worsened. endothelial dysfunction associated with diabetes results in atherosclerosis which contributes in decreased blood supply.5-7 wound healing is a complex process that follows a well-designed pattern of various biological and molecular events. certain intrinsic factors and extrinsic factors such as neuropathy, vasculopathy, immunity, wound infection, wound cell abnormalities contributes in delayed wound healing in diabetic population. early detection and intervention is needed in order to enhance healing process in dfu which can also predict the long-term progression.8, 9 identification of associated risk factors and its management can prevent dfu.10 several guidelines have been published that provides information regarding management of diabetic foot ulcer which can be broadly divided into surgical and conservative management. surgical management includes debridement and amputation. conservative management includes prescription of protective footwear, pharmacological management of infection and restoration of perfusion, wound care through regular inspection and debridement, controlling of optimal glucose level, and educating patients regarding self care and management.11-13 adjunctive therapies such as negative pressure wound therapy, hyperbaric oxygen therapy, and electrotherapy along with other physical agents such as ultrasound therapy, ultraviolet ray therapy, low laser therapy have been used to enhance healing of wound.14,15 matrix rhythm therapy is a therapeutic method developed and improved by dr. ulrich randall that is based on a fact effect of matrix rhythm therapy in diabetic foot ulcer healing: a case report varun naik1, chandra bahadur khatri2*, ganesh br1 1department of cardiovascular and pulmonary physiotherapy, kaher’s institute of physiotherapy, belagavi, karnataka, india 2 kaher institute of physiotherapy, nehrunagar, belagavi, karnataka, india corresponding author: chandra bahadur khatri (e-mail: cbkbharat@gmail.com) abstract introduction: diabetic foot ulcer is one of the major complications following diabetes. in india, 15% of diabetic patient develop dfu once in their lifetime. neuropathy associated with diabetes is a major factor in development of foot ulcer in such patients. infection, peripheral arterial diseases, altered functions of white blood cells, stem cells and regenerating tissues, and co-morbidities lead to delayed wound healing. matrix rhythm therapy is a recent advancement in the field of physiotherapy. it provides external oscillation that activates normal rhythm of cell that is between 8 and 12 hz which improves cellular function within cell and extracellular matrix, enhancing tissue healing. a 70-year-old female presented with infected diabetic foot ulcer on her right foot in out-patient department of kle hospital. she had nonhealing ulcer since one and half months and undergone conservative treatment of wound care along with strategies of diabetic foot care. clinical findings: subject complained of pain with score of 2 at rest and 6 while walking which was recorded on the basis of visual analogue scale. wound appeared ischemic and non-healing. on wound assessment, size of wound was 12 mm in diameter and 6 mm depth. it was identified as type d grade i on the basis of classification of wound provided by university of texas health science center. intervention: matrix rhythm therapy was applied on posterior aspect of leg region extending around wound area once a week for 5 weeks with treatment duration of 1 h. result: pain of the subject after 5 weeks of intervention was 0 at rest and 2 while walking on the basis of vas. size of wound decreased with dimension of 6 mm in diameter and 2 mm depth and categorized as type a grade 1. conclusion: matrix rhythm therapy could be considered as adjunct in wound healing of diabetic foot ulcer. key words: diabetic foot ulcer, matrix rhythm therapy 188 case report effect of matrix rhythm therapy in diabetic foot ulcer healing: a case report varun naik j contemp med sci | vol. 6, no. 4, july-august 2020: 187–190 that all cells of biological system follow a rhythmic pattern of vibrations when they are living. this conclusion was drawn from a research conducted at erlangen university in germany. matrix rhythm therapy delivers the vibrations ranging from 8 to 12 hz that mimics normal oscillation that brings changes in cellular level thereby enhancing the function at cellular level (within cell and extracellular matrix).16 patient information a 70-year-old female presented with open wound on the plantar aspect of her right foot which was evident since one-andhalf months. she complained pain at the site of wound which is aggravated on walking or weight bearing on same side. she gave the history of trauma due to uneven foot placement on a pebble. she noticed a wound on foot after a week of injury and applied local wound ointment for a month. wound was increasing in size along with pain during weight bearing. she consulted physician and was referred to physiotherapy department for further management. subject gave the history of diabetes and hypertension since 20 years and 30 years, respectively, and under medication. she takes 16 units and 10 units of insulin in the morning and night for the management of diabetes and diuretics and ace inhibitors for hypertension management. she had undergone coronary artery bypass graft 16 years back. clinical findings pain assessment: visual analogue scale was used to assess pain. it was 2/10 at rest and 6/10 while walking. wound examination: size of wound was 12 mm diameter with 6 mm of depth which was located lateral aspect of posterior one-third on plantar surface of right foot. wound appeared dry with calcified vascular tissues. granulation tissues were minimal in appearance. wound bed was filled with pus suggestive of infection. diagnostic assessment: physical examination was carried out along with an x-ray. x-ray findings were normal to the age of the subject. wound was classified on the basis of diabetic wound classification system provided by the university of texas health science center. wound was identified as type d grade i. pain was assessed on the basis of visual analogue scale which was recorded as 20 mm on rest and 70 mm on weight-bearing activities. therapeutic intervention matrix rhythm therapy was performed on the affected limb. it was applied starting from posterior aspect of knee joint extending around the wound. treatment was given for 5 sessions lasting up to 1 h in each session. frequency of treatment was 1 session per week. follow-up and outcomes outcome measures were recorded in terms of size of the wound, grading of wound and vas for pain. subject has followed up till 1 month after cessation of treatment. figure 1 shows appearance of wound before and after intervention of matrix rhythm therapy. first follow-up: • vas: 2 at rest and 6 on walking • shape of wound: it changed to rectangular shape from circular • size of wound: length=10 mm, breadth: 6 mm and depth: 5 mm • grading: type c, grade i • appearance: ischemic, with partial vasularization of surrounding tissues second follow-up: • vas: 0 at rest and 5 on walking • shape of wound: rectangular • size: length=8 mm, breadth= 8 mm, depth: 5 mm • grading: type c, grade i third follow-up: • vas: 0 at rest and 3 while walking • shape of wound: oval • size: greatest length=8 mm, greatest breadth=5 mm, depth=4 mm • grading: type b, grade i fourth follow up: • vas: 0 at rest and 2 while walking • shape of wound: irregular • size: greatest length: 6 mm, greatest breadth=4 mm, depth=2 mm • grading: type a, grade i no any adverse effects or intolerance was observed or experienced by subject throughout the treatment sessions and follow-ups. at the end of session, wound changed from non-healing ulcer to healing ulcer with vascularization of surrounding tissues. discussion our study aimed to determine the effects of matrix rhythm therapy on diabetic foot ulcer. application of matrix rhythm therapy for five sessions, one session a week showed accelerated wound-healing process in non-healing diabetic foot ulcer along with decreased pain intensity. this study is novel of its kind which is applied for wound healing, and till now no published literature is available. dfu is of the major complications that lead in disability and decreased quality of life in diabetic population. multiple approaches have been applied for the prevention and management of dfu but effective approach that enhances healing of ulcer is not established. several physical modalities such as low level laser, ultraviolet radiation, electrical stimulation have been used in management of wound but most of the studies lack quality of evidence and physiological basis for wound healing.15 most of the studies related to the physiotherapy approaches in wound healing have used multiple therapeutic methods which lead overlapping of physiological effects and results cannot be specified to a particular method.17, 18 in our study, we have used only matrix rhythm therapy and its effect on wound can be specified. 189 case report effect of matrix rhythm therapy in diabetic foot ulcer healing: a case reportvarun naik j contemp med sci | vol. 6, no. 4, july-august 2020: 187–190 use of vibrations as interventions for therapeutic benefit has history since 19th century. vibrations has been used for management of neurological conditions, increasing power and flexibility in athletes, maintenance of bone density in astronauts, and recently it has been used to enhance wound-healing process. a study was done in regarding the effect of vibration in healing process in diabetic mice. it has been found that vibrations produces stimulus that helps in release of growth factors, stem cell proliferation, and cell differentiation when applied to bone. in wound, it increases angiogenesis, formation of granulation tissues, and promotes the accumulation of macrophages, fastens the wound closure, and re-epithelialization. they also found that there is decrease in number of neutrophils cells in wound that decreases the inflammatory conditions within wound. similarly, there was decrease in blood glucose levels and increase in microcirculation after application of vibrations.20,21 in these, studies the frequency of vibrations applied was 35–45 hz compared to physiological oscillations/vibrations in our study. though one of study on effects of vibrations in blood flow in healthy inactive subjects showed that vibration is responsible for increase in blood supply, there are very limited studies related to role of vibrations in wound healing in diabetic subjects.22 in our study, wound healing in dfu is accelerated that can be possibly due to cellular effect of vibrations produced by the application of matrix rhythm therapy. matrix rhythm therapy produces oscillations of 8–12 hz in cells that mimics the normal rhythmic oscillations when cells are alive. in diseased condition, normal rhythmic oscillations are affected thereby impairing functions within cells and its surrounding environment, i.e., extracellular matrix. following application of this therapeutic approach normal oscillations can be restored leading to normal function of cell and its environment.19 similarly, application of matrix rhythm therapy leads to increased circulation which was observed in a study done in athletes. in their study, they compared the effects on circulation by using conventional massage with matrix rhythm therapy. they concluded that, matrix rhythm therapy leads to 190 case report effect of matrix rhythm therapy in diabetic foot ulcer healing: a case report varun naik j contemp med sci | vol. 6, no. 4, july-august 2020: 187–190 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i4.774 increase in circulation through compression effect and soft tissue mobilization along with contraction of muscles produced by stimulation of tonic vibration reflex.23 there are certain limitations of the study despite being first of its kind to intervene matrix rhythm therapy. more emphasis would have been given in detailed objective examination of diabetic foot ulcer and intervention frequency was less which could have been increased. in future, more number of patients can be involved as to develop a case series and can be compared with other physical modalities. in conclusion, matrix rhythm therapy may be used as an adjunctive therapy for wound healing in diabetic foot ulcer. it could be beneficial in not only enhancing healing of wound but also overall improvement on functions of cell and its environment thereby creating optimal atmosphere. informed consent: was duly obtained from subject regarding recoding and publication of data source of funding: nil conflict of interest: nil references 1. world health organization. definition, diagnosis and classification of diabetes mellitus and its complications: report of a who consultation. part 1, diagnosis and classification of diabetes mellitus. geneva: world health organization; 1999. 2. nathan dm. long-term complications of diabetes mellitus. new engl j med. 1993 jun 10;328(23):1676-85. 3. jeffcoate wj, harding kg. diabetic foot ulcers. lancet. 2003 may 3;361(9368):1545-51. 4. ghosh p, valia r. burden of diabetic foot ulcers in india: evidence landscape from published literature. value health. 2017 oct 1;20(9):a485. 5. clayton w, elasy ta. a review of the pathophysiology, classification, and treatment of foot ulcers in diabetic patients. clin diab. 2009 apr 1;27(2):52-8. 6. bowering ck. diabetic foot ulcers. pathophysiology, assessment, and therapy. can family phys. 2001 may 1;47(5):1007-16. 7. noor s, zubair m, ahmad j. diabetic foot ulcer—a review on pathophysiology, classification and microbial etiology. diab metab syndr clin res rev. 2015 jul 1;9(3):192-9. 8. falanga v. wound healing and its impairment in the diabetic foot. lancet. 2005 nov 12;366(9498):1736-43. 9. apelqvist j, larsson j, agardh cd. long‐term prognosis for diabetic patients with foot ulcers. j intern med. 1993 jun;233(6):485-91. 10. singh n, armstrong dg, lipsky ba. preventing foot ulcers in patients with diabetes. jama. 2005 jan 12;293(2):217-28. 11. piaggesi a, schipani e, campi f, romanelli m, baccetti f, arvia c, navalesi r. conservative surgical approach versus non‐surgical management for diabetic neuropathic foot ulcers: a randomized trial. diab med. 1998 may;15(5):412-7. 12. lipsky ba, berendt ar, deery hg, embil jm, joseph ws, karchmer aw, lefrock jl, lew dp, mader jt, norden c, tan js. diagnosis and treatment of diabetic foot infections. clin infect dis. 2004 oct 1:885-910. 13. bakker k, apelqvist j, schaper nc, international working group on the diabetic foot editorial board. practical guidelines on the management and prevention of the diabetic foot 2011. diab metab res rev. 2012 feb;28:225-31. 14. stansby g, avital l, jones k, marsden g. prevention and management of pressure ulcers in primary and secondary care: summary of nice guidance. bmj. 2014 apr 23;348:g2592. 15. zhou k, krug k, brogan ms. physical therapy in wound care: a costeffectiveness analysis. medicine. 2015 dec;94(49). 16. the matrix concept in practice – dr. randoll institut · gemeinnützige gesellschaft für matrix-forschung und -lehre mbh [internet]. [20.04.2019]. available from: https://www.dr-randoll-institut.de/en/matrix-konzept-inder-praxis. 17. ketan bhatikar. effect of matrix rhythm therapy on chronic vein dysfunction deep foot ulcer: a case report. j yoga physio. 2018;6(5):555696. doi: 10.19080/jyp.2018.06.555696. 18. sari z, polat mg, özgül b, aydoğdu o, camcıoğlu b, acar ah, yurdalan su. the application of matrix rhythm therapy as a new clinical modality in burn physiotherapy programmes. burns. 2014 aug 1;40(5):909-14. 19. randoll ug, hennig ff. coherent rhythms (timing frequencies) in biological systems as a basis for the matrix-rhythm. therapy. 2003. 20. weinheimer-haus em, judex s, ennis wj, koh tj. low-intensity vibration improves angiogenesis and wound healing in diabetic mice. plos one. 2014 mar 11;9(3):e91355. 21. yu co, leung ks, jiang jl, wang tb, chow sk, cheung wh. low-magnitude high-frequency vibration accelerated the foot wound healing of n5streptozotocin-induced diabetic rats by enhancing glucose transporter 4 and blood microcirculation. sci rep. 2017 sep 14;7(1):11631. 22. gailiūnienė l, krutulytė g, šiaučiūnaitė v, savickas r, venslauskas m. the effect of low frequency 2-10 hz vibrations on blood circulation in lower extremities. j vibroeng. 2017 sep 30;19(6):4694-701. 23. taspinar f, aslan ub, sabir n, cavlak u. implementation of matrix rhythm therapy and conventional massage in young females and comparison of their acute effects on circulation. j altern complem med. 2013 oct 1;19(10):826-32. 296 j contemp med sci | vol. 6, no. 6, november–december 2020: 296–299 original issn 2413-0516 introduction bronchoscopy is a diagnostic method to treat and observe the airways. a bronchoscope is a device that enters through the nose or mouth and gives the doctor the opportunity to examine abnormalities including foreign bodies, bleeding, tumors, inflammation, and to sample if necessary. the bronchoscope consists of metal tubes with multiple lamps and a camera that displays airway images on the monitor.1 most patients undergoing bronchoscopy, even if local anesthesia and appropriate analgesia are provided during the procedure, see it as a traumatic event. bronchoscope stimulate the mucosa and imminent suffocation, especially when co-administered with hypoxia, may cause fear and anxiety, and its effect on the cardiovascular system may cause complications such as arrhythmia and myocardial infarction, as well as psychological complications including increased patient anxiety.2-4 since bronchoscopy provides valuable diagnostic information and intervention therapies to specialists, it is also a non-invasive procedure. the necessity to do so is unimaginable, and given that most patients are afraid of the procedure and are uncomfortable or painful to perform, a reliable sedation can relieve anxiety, reduce discomfort, increase satisfaction, and create forgetfulness.5 for this reason, the use of sedatives as an inexpensive, safe, and easy method has been considered. these include dexmedetomidine, midazolam, and fentanyl. dexmedetomidine is a selective alpha-2 adrenoceptor agonist with high specificity.6 this drug has been used as an adjuvant in general anesthesia with a central sympatholytic effect, has helped to stabilize the patient’s hemodynamic status and has a potent anesthetic and analgesia effect,7 which reduces the need for opioids and their complications8 and reduces stress response and improves recovery quality.9 midazolam is a sedative and belong to the benzodiazepines. it has the fastest onset among benzodiazepines (about 30–60 s) and is shorter in duration.10 midazolam has various hypnotic, anticonvulsant, and anxiolytic effects as well as a dose-dependent protective effect against cerebral hypoxia. this drug causes a greater drop in blood pressure and a higher respiratory depression than diazepam and lorazepam.11 fentanyl is a fast-starting anesthetic and has a short duration of action. it is an opioid group with a potential of 50–100 times more morphine. it is considered a safe anesthetic drug because of its extensive therapeutic index.12 the aim of this study was to compare the sedative and satisfaction effects of bronchoscopy candidiasis with the combination of dexmedetomidine and fentanyl with midazolam and fentanyl. material and methods this study is a single-blinded randomized clinical trial in which patients undergoing bronchoscopy referred to amir comparison the sedation effect and satisfaction of two combinations, dexmedetomidine and fentanyl with midazolam and fentanyl, in patients undergoing bronchoscopy alireza kamali1, sepideh sarkhosh2, hosein kazemizadeh3 1department of anesthesiology and critical care, arak university of medical sciences, arak, iran. 2medical sciences research committee, arak university of medical sciences, arak, iran. 3department of internal medicine, arak university of medical sciences, arak, iran. corresponding author: hosein kazemizadeh (e-mail: dr.hoseinkazemizadeh@gmail.com ) (submitted: 02 september 2020 – revised version received: 19 september 2020 – accepted: 06` october 2020 – published online: 26 december 2020) abstract objectives: the aim of this study was to compare sedative effects of dexmedetomidine and fentanyl with midazolam and fentanyl in patients undergoing bronchoscopy. methods: this study was a double-blind randomized clinical trial that was performed on 92 patients who referred to amir al momenin hospital in arak for bronchoscopy and underwent asa 1 or 2 underlying grading procedure. patients were randomly divided into two groups of dexmedetomidine and fentanyl (d) midazolam and fentanyl (m). primary vital signs including hypertension and arterial oxygen saturation were monitored and recorded. then, all patients were injected with 2 μg/kg fentanyl as a painkiller and after 3 min, 30 μg dexmedetomidine in syringe with code a and midazolam 3 mg in syringe with code b were injected to patients by an anesthesiologist. then, the two groups were compared in terms of pain at injection, conscious relaxation, satisfaction of operation, recovery time, hypotension, and arterial oxygen saturation and drug side effects and data were analyzed by using statistical tests. results: there was no significant difference between the two groups in terms of mean age and sex distribution. according to the results of this study, there was no significant difference between the two groups in mean blood pressure (p-value = 0.6) and mean heart rate (p-value = 0.4) at the time of bronchoscopy, but at 5 and 10 min after bronchoscopy, there was a significant difference, mean blood pressure and heart rate were significantly lower in dexmedetomidine group. conclusion: both dexmedetomidine and midazolam drug groups contributed to the development of stable and sedative hemodynamics and satisfaction in patients undergoing bronchoscopy, however, the dexmedetomidine and fentanyl group showed a significant decrease in blood pressure and heart rate compared to midazolam and fentanyl and a weaker decrease in arterial oxygen saturation, and patients with bronchoscopy were more satisfied in the dexmedetomidine group. keywords: bronchoscopy, sedation, fentanyl, dexmedetomidine, midazolam. 297 original comparison the sedation effect and satisfaction of two combinations alireza kamali j contemp med sci | vol. 6, no. 6, november–december 2020: 296–299 al momenin hospital of arak – iran were randomly divided into two equal groups, dexmedetomidine, fentanyl (d) and midazolam, fentanyl (m). all patients entered the study after obtaining informed consent and having inclusion criteria. inclusion criteria: 1 age over 18 and under 70 years. 2 bmi below 30 kg/m2 3 no sedative or anxiolytic use or history of substance abuse. 4 pregnancy. 5kidney failure. 6 neurological disorders. 7 no sensitivity to midazolam or soy. 8 no history of heart disease or drug use. exclusion criteria: 1 relaxation level rating 5. 2 ability to drink liquids. 3 sitting alone beside the bed. before doing the research, patients were described with visual analogue scale (vas) and a demographic questionnaire with primary vital signs was recorded. cubital venipuncture was performed and 3 min before bronchoscopy, 2 mg/kg fentanyl was given by anesthesiologist and 30 mg dexmedetomidine to the patient 1 min before surgery or midazolam was injected at a dose of 3 mg at random. after 5 and 15 min from the onset of bronchoscopy and recovery of the patient in terms of blood pressure, heart rate, arterial oxygen saturation with pulse oximeter, and respiratory rate per minute were recorded by the researcher and recorded in a questionnaire. sample size: x z z = + æ è çççç ö ø ÷÷÷÷÷ +( ) -( ) 1 2 1 2 1 2 2 1 2 2 α β δ δ µ µ z z x 1 2 1 1 1 2 1 1 96 10 29 19 11 1 28 92 = = = = = = þ = α β δ µ µ δ . . the number of samples in each group was 46 and 92 in total. data analysis spss software (version 11) was used for data analysis. mean and standard deviation were used for quantitative data and anova was used for data analysis and if the data were significant, bonferroni correction was used to find the exact scattering region. u-test was used to evaluate qualitative data and fisher’s exact test was used to compare the symptoms. ethical considerations a written letter was received from respected university officials to introduce them to the research centers. written letter was obtained from respected authorities of selected research centers. the purpose of the study was described for all research units and their written consent was obtained. all patients’ information is kept confidential by the plan’s administrator. in  all stages of research, all ethics statements in helsinki research and research committees in arak university of medical sciences were considered. this research has a code of ethics ir.arakmu.rec. 1396.241. results 92 patients undergoing bronchoscopy were randomly divided into two equal groups. in chart 1, patients undergoing bronchoscopy in two groups of dexmedetomidine, fentanyl and midazolam, fentanyl were evaluated for age and sex and no significant difference was observed between the two groups in terms of mean age. the mean age of the patients was similar in both groups (p-value = 0.6), and there was no significant difference between the two groups in terms of sex distribution and the frequency of males in both groups was 65%. table 1 compares the changes in mean blood pressure of patients undergoing bronchoscopy at baseline, 5 and 10 min after initiation in the two groups. according to the results, there was no significant difference between the two groups regarding mean blood pressure at the time of initiation bronchoscopy (p-value = 0.6), but at 5 and 10 min after bronchoscopy, there was a significant difference and mean blood pressure was significantly lower in dexmedetomidine group (p-value = 0.04, p-value = 0.03). changes in mean heart rate of patients undergoing bronchoscopy at baseline, 5 and 10 min after initiation were assessed in the two groups; dexmedetomidine + fentanyl and midazolam + fentanyl. mean pr at baseline in the two groups were 93.5±5.3 and 95.4±4.9, mean pr at 5 min after start in 0 10 20 30 40 50 60 70 average age sex frequency distribution (male) sex frequency distribution (female) 53.6 65.4 34.6 52.8 67.6 33.3 group d group m chart 1 comparison of age and sex of patients undergoing bronchoscopy. table 1. comparison of mean blood pressure changes in patients undergoing bronchoscopy. average / group dexmedetomidine + fentanyl (group d) midazolam + fentanyl (group m) p-value average map at base time6.9± 107.85.8± 109.9p= 0.6 average map after 5 minutes4.9±95.87.9 ± 107.7p= 0.04 average map after 10 minutes6.7± 99.79.7± 111.1p= 0.03 298 original comparison the sedation effect and satisfaction of two combinations alireza kamali j contemp med sci | vol. 6, no. 6, november–december 2020: 296–299 the two groups were 89.3±4.8 and 96.5±3.8, respectively, and mean pr of the two groups after 10 min of initiation was 90.4±5.9 and 105.4±8.6, respectively. according to the results, no significant difference was observed between the two groups in terms of mean heart rate at the initiation of bronchoscopy (before the procedure) (p-value = 0.4) but there was a significant difference at 5 and 10 min after the start of bronchoscopy. the mean heart rate was significantly lower in the dexmedetomidine group (p-value = 0.04, p-value = 0.03). comparison of mean arterial oxygen saturation changes in patients undergoing bronchoscopy at baseline, 5 and 10 min after starting in the two groups is discussed in table 2. according to the results between the two groups in terms of mean arterial oxygen saturation of patients at different times onset of bronchoscopy (before surgery) and 5 and 10 min after bronchoscopy, no significant difference was observed (p-value = 0.4, p-value = 0.2). fig. 2 compares sedation score of patients undergoing bronchoscopy at 5, 10, 15 and 20 min after initiation of bronchoscopy in two groups of dexmedetomidine + fentanyl and midazolam + fentanyl. according to the results, there was no significant difference between the two groups in terms of sedation score at 5, 10, 15 and 20 min after the initiation of bronchoscopy (p-value = 0.4, p-value = 0.6). comparison of satisfaction score and ramsay score in recovery of bronchoscopy candidates in two groups in table 3 was evaluated. according to the results, there was a significant difference between the two groups in terms of patient satisfaction score in recovery, there was a significant difference in dexmedetomidine patients’ satisfaction (p-value = 0.04), but there was no significant difference between the two groups ramsay score in the recovery and were almost the same (p-value = 0.6). discussion bronchoscopy is a safe procedure and is often performed to evaluate, diagnose, and treat patients with respiratory diseases.13 bronchoscopy is used as a diagnostic and therapeutic method for the examination of pulmonary diseases, tumors, chronic cough, and respiratory tract infections. this requires a short-term sedation that allows the patient to tolerate an invasive procedure while maintaining cardiorespiratory function.14 the aim of this study was to compare the sedative and satisfaction effects of dexmedetomidine + fentanyl and midazolam + fentanyl in patients undergoing bronchoscopy. no significant differences were observed between the two groups in terms of mean age and sex distribution and mean age of the patients was same in both groups. according to the results of this study, there was no significant difference between the two groups regarding mean blood pressure (p-value = 0.6) and mean heart rate (p-value = 0.4) at the time of bronchoscopy, but at 5 and 10 min after bronchoscopy, there was a significant difference, mean blood pressure and heart rate were significantly lower in dexmedetomidine group. in this study, no significant difference was observed in the mean arterial oxygen saturation of patients at different times of onset bronchoscopy and 5 and 10 min after bronchoscopy. there was no significant difference between the two groups in the score of sedation at 5, 10, 15 and 20 min after the initiation of bronchoscopy. according to the results, there was a significant difference between the two groups in terms of patients satisfaction score in recovery and there was a significant difference in patients satisfaction score in dexmedetomidine group (p-value = 0.04), but ramsay score showed no significant difference between the two groups in recovery and was almost the same (p-value = 0.6). a number of similar studies have been undertaken, including derek’s 2010 study at the department of anesthesiology at haydarpasa teaching hospital, turkey, with the aim of comparing dexmedetomidine and midazolam for sleep, pain, and control of hemodynamic symptoms during colonoscopy under sedation performed on 40 patients (asa i or ii, aged from 20 to 80 years). dexmedetomidine create more stable hemodynamics, higher ramsay score, greater patient satisfaction, and lower numeric rating scale in colonoscopy patients. as a result of this study, dexmedetomidine can be used as a safe sedative in colonoscopy.15 the results of this study were consistent with our study. in our study, dexmedetomidine resulted in more stable hemodynamics and higher ramsay score and more satisfaction. fig. 2 comparison of sedation score. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 average sedation score after 5 minutes average sedation score after 10 minutes average sedation score after 15 minutes average sedation score after 20 minutes 4.9 4.3 4.1 4.44.5 4.01 3.8 4.1 group d group m table 2. comparison of changes in mean arterial blood oxygen saturation. average / group dexmedetomidine + fentanyl (group d) midazolam + fentanyl (group m) p-value average spo2 at base time5.2 ± 94.43.7 ± 93.4p= 0.4 average spo2 after 5 minutes2.8 ± 93.94.9± 92.7p= 0.4 average spo2 after 10 minutes4.8± 93.13.6± 91.8p= 0.2 table 3. comparison of patients’ satisfaction and ramsay score. average / group dexmedetomidine + fentanyl (group d) midazolam + fentanyl (group m) p-value average satisfaction score in recovery 0.98 ± 2.80.76 ± 1.66p= 0.04 average ramsay score in recovery1.1 ± 4.850.98 ± 4.7p= 0.6 299 original comparison the sedation effect and satisfaction of two combinations alireza kamali j contemp med sci | vol. 6, no. 6, november–december 2020: 296–299 another study by demirany in 2007 at the abant izzet baysal university anesthesiology department in turkey, comparing dexmedetomidine and midazolam in sedation of patients undergoing upper endoscopy, it was concluded that dexmedetomidine is a safe drug for sedation in patients with upper gastroscopy and is better than midazolam in terms of side effects and endoscopic satisfaction. as a result, dexmedetomidine is a better drug than midazolam.16 the results of this study were consistent with our study that dexmedetomidine was a safe drug for sedation in patients undergoing bronchoscopy and was superior to midazolam in adverse effects and satisfaction. finally, in a similar study by cheung in 2007, patients were compared with dexmedetomidine and midazolam in the surgical relaxation of molar lesions. there was no significant difference between satisfaction and pain quality, and midazolam caused more amnesia. patients and surgeons satisfaction was similar in the two groups so that patients were satisfied with the same procedure in the future, but heart rate and blood pressure were lower in the dexmedetomidine group during surgery.17 the results of our study were consistent with this study, but this part of the study contradicted our study with significant differences in patient satisfaction and pain quality. but eventually more satisfaction was found in the dexmedetomidine group, which was similar to our study. conclusion according to the results of this study, it can be concluded that in both groups, dexmedetomidine and midazolam were involved in the development of stable and sedative hemodynamics and satisfaction of patients with bronchoscopy. but the dexmedetomidine and fentanyl group had higher drop blood pressure and heart rate and lower drop arterial oxygen saturation compared to midazolam and fentanyl. and patients with bronchoscopy were more satisfied in the dexmedetomidine group. finally, it is suggested that similar studies with larger sample sizes as well as comparisons of other sedatives be conducted in different processes. conflict of interest none references 1. kubba h, cooke j, heartly b. can we develop a protocol for the safe decannulation of tracheostomies in children less than 18 months old? int j pediatr otorhinolaryngol. 2004;6(8):935–937. 2. mitchell rb, hussey hm, setzen g, jacobs in, nussenbaum b, dawson c, brown ca, brandt c, deakins k, hartnick c, et al. clinical consensus statement: tracheostomy care. otolaryngol head neck surg. 2013;14(8):6-9. 3. cristea i, baker cd. ventilator weaning and tracheostomy decannulation in children: more than one way. pediatric pulmonol. 2016;5(1):773–774. 4. cabrini l, gioia l, gemma m, melloni g, carretta a, ciriaco p, et al. acupuncture for diagnostic fiber optic bronchoscopy: a prospective, randomized, placebo-controlled study. am j chin med. 2006;34(3):409-15. 5. simma b, spehler d, burger r, uehlinger j, ghelfi d, dangel p, hof e, fanconi s. tracheostomy in children. eur j pediatr.1996;15(3):291–296. 6. savola jm, ruskoaho h, puurunen j, salonen js, karki nt. evidence for medetomidine as a selective and potent agonist at alpha 2-adrenoreceptors. j auton pharmacol. 1986;6(3):275–84. 7. gurbet a, basagan-mogol e, turker g, ugun f, kaya fn, ozcan b. intraoperative infusion of dexmedetomidine reduces perioperative analgesic requirements. can j anaesth. 2006;53(5):646–52. 8. blaudszun g, lysakowski c, elia n, tramer mr. effect of perioperative systemic alpha2 agonists on postoperative morphine consumption and pain intensity: systematic review and meta-analysis of randomized controlled trials. anesthesiology. 2012;116(4):1312–22. 9. bekker a, haile m, kline r, didehvar s, babu r, martiniuk f, urban m. the effect of intraoperative infusion of dexmedetomidine on the quality of recovery after major spinal surgery. j neurosurg anesthesiol. 2013;25(1):16-24. 10. anand kj, barton ba, mcintosh n. analgesia and sedation in preterm neonates who require ventilatory support: results from the nopain trial. neonatal outcome and prolonged analgesia in neonates. arch pediatr adolesc med. 1999;153(4):331–338. 11. anand kj, hall rw, desai n. effects of morphine analgesia in ventilated preterm neonates: primary outcomes from the neopain randomised trial. lancet. 2004;363(9422):1673–1682. 12. qu w, rippe ra, ma j, scarborough p, biagini c, fiedorek ft, et al. nutritional status modulates rat liver cytochrome p450 arachidonic acid metabolism. mol pharmacol. 1998;54(3):504–513. 13. du rand i, blaikley j, booton r, chaudhuri n, gupta v, khalid s, et al. british thoracic society guideline for diagnostic flexible bronchoscopy in adults. thorax. 2013;6(8):1-4. 14. kremer b, botos-kremer ai, eckel he, schlondorff g. indications, complications and surgical techniques for pediatric tracheostomies an update. j pediatr surg. 2002;3(7):1556-1562. 15. dere k, sucullu i, budak et, yeyen s, filiz ai, ozkan s, dagli g. a comparison of dexmedetomidine versus midazolam for sedation, pain and hemodynamic control, during colonoscopy under conscious sedation. eur j anaesthesiol. 2010;27(7):648-52. 16. demiraran y, korkut e, tamer a, yorulmaz i, kocaman b, sezen g, akcan y. the comparison of dexmedetomidine and midazolam used for sedation of patients during upper endoscopy: a prospective, randomized study. can j gastroenterol. 2007;21(1):25-9. 17. cheung cw, ying cla, chiu wk, wong gtc. a comparison of dexmedetomidine and midazolam for sedation in third molar surgery. 2007;6(2):1132–1138. https://doi.org/10.22317/jcms.v6i6.886 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 96 j contemp med sci | vol. 5, no. 2, march–april 2019: 96–100 original the efficacy of traditional formulation on quality of life and fatigue in multiple sclerosis patients: a randomized double-blind placebo-control clinical trial f. namjooyan,a r. ghanavati,b n. majdinasab,c and h. rezaei zadehd apharmacognosy department, school of pharmacy, jundishapur university of medical sciences, ahvaz, iran. btraditional pharmacy, school of pharmacy, jundishapur university of medical sciences, ahvaz, iran. cneurology department, school of medicine, jundishapur university of medical sciences, ahvaz, iran. dtraditional medicine, school of traditional medicine, tehran university of medical sciences, tehran, iran. *correspondence to rahil ghanavati (email: rahil_ghanavati@yahoo.com). (submitted: 04 december 2018 – revised version received: 26 january 2019 – accepted: 23 february 2019 – published online: 26 april 2019) objective according to effects that cinnamon, ajwain and iranian borago shows in involving mechanisms in ms and ms animal model, we decided to survey the effect of traditional formulation on ms patient by clinical trial. methods in a double blind randomized clinical trial study, 60 patients with ms observed. they take formulation 15 cc per day, fill the msqol-54, and fatigue questionnaires every month. the data were analyzed with 18th version of spss, independent t-test and repeated measure tests. the p-value for tests was 0.05. results the mean quality-of-life and fatigue of patient with ms were significantly changed during 3 months. conclusion most patients with ms had better quality-of-life during 3 month and get less tired. in some patient tremor and pain reduce. because of good result and revenue of formulation, it seems to be useful for ms patient. keywords quality of life, fatigue, multiple sclerosis introduction multiple sclerosis (ms) is a chronic inflammatory autoimmune demyelinated disease of central nervous system (cns) that could be disabling and fatal. ms attack to myelinated axons in cns and destroy it. this disease generally occurs at the age of 20–40 years. the key of pathologic mechanism of ms shows a breakdown in immunologic tolerance or a peripheral infection and following a demineralizing inflammation attack to cns. start of this pathologic process represents the complex interference of genetics and environment that is not understand completely. in ms, neurologic symptoms (visual, sensory and motor) occur because of neurodegenerative process. in cns there are lesions with penetrated specific inflammatory cells and demyelinated. the cause of neurodegenerative process is unknown. one possibility is a chronic infection in cns. various kinds of neurotropic micro-organisms in ms are known although there is no definitive evidence for the reasons. the grade of ms is wildly different and unpredictable. in several patients ms initially diagnosis with reversible neurologic disorder episodes that is often followed by progressive neurologic attack. in 2013 almost 2.5 million patients in the world affected with ms that 50% of them, need help for walking after 15 years of disease onset. women twice the men affected with ms, and northern europe people are in higher risk for ms.1–3 using complementary and alternative medicine (cam), for chronic disease such as ms is an important issue for patients. cam’s methods use in addition or instead of conventional medicine. in summary, cam is wildly described as a positive cure without major defeat and without or with low adverse effect by patients. cam is part of varied medical system which is not considered as a conventional medicine and include mind–body practice and manipulative, traditional medicine and modern medical system. unani medicine is a part of traditional medicine that is according to harrison’s book” unani medicine is a western indian medical system that is derived from iranian medicine and initially use in muslim country and also called hikmet”.4–6 moreover traditional persian medicine (tpm), is an ancient temperament medicine with thousands years history. this kind of medicine is thought in traditional and complementary medicine and traditional pharmacy faculty in iran. recently use of herbal medicine as cam in cure disease is increased.7,8 in tpm one categories of disorders generally called khaddar, that similar point of them is sensory disorders. khaddar is any kind of sensory deficiency or invalidity, which may be associated with motor symptoms. this sensory disorder is painful, like creeping sensation, having a prickly sensation or sense of ant walking on the skin. according to these definitions, it seems that khaddar is similar to hypesthesia and paresthesias.9–13 ajwain or trachyspermum ammi belong to apiaceae that is wildly grown in iran, egypt, pakistan, afghanistan, india and some part of europe. this plant is known as zenyan or nankhah in medicine and pharmaceutical references of tpm. ajwain’s seeds are wildly prescribed by traditional iranian physician’s foe several disorders. because of its various chemical constituents, this seeds have numerous pharmacologic effects. ajwin’s seed has stimulant, carminative, diuretics, analgesic, antimicrobial, antiviral, antiulcer, antihypertensive, and antitussive, and bronchodilator, antiplatelet and hepatoprotective properties.14,15 iranian borago or echium amoenum is belonging to boraginaceae family that is a 2 year or perennial herb. this plant is endemic of north of iran and caucasus. iranian borago is one of the important medicinal herbs in tpm. e. amoenum has different effect such as pain relief, anti-inflammatory, anti oxidant, and analgesic, antianxiety, sedative and anticonvulsant. this plant commercially cultivated for the seed’ oil which issn 2413-0516 f. namjooyan et al. 97j contemp med sci | vol. 5, no. 2, march–april 2019: 96–100 original the efficacy of traditional formulation on quality of life and fatigue in multiple sclerosis patients is prepared from seed. the leaves and flower are also used as drug. they are used for fever, cough and depression. seed’s oil is used for skin disorder such as eczema, seborrhea dermatitis and neuro dermatitis. also it is used for rheumatoid arthritis, alcoholism, obsessive compulsive disorder, pain and swelling and preventing cardiovascular disease.16 cinnamon is prepared from inner bark of an ever green tree which is endemic of sri lanka and south of india. its scientific name is cinnamomum verum or cinnamomum zeylanicum. cinnamon bark is wildly used as a spice and flavoring agent.17,18 in addition to home uses of cinnamon, in ayurveda, cinnamon is used for respiratory digestion and women disease. almost all part of cinnamon tree such as bark, leaves, flower, fruits and root have home and medicinal application. the essential oil which is prepared from roots bark, bark and leaves is significantly different in chemical constituents. so it is suggested that they have different pharmacologic effect. in vitro, in vivo and clinical studies all over the world demonstrated numerous beneficial effect for cinnamon, such as anti-inflammatory, antibacterial, reduce cardiovascular disease, increase cognitive function and reduce risk of colon cancer.18 the aim of this study is evaluate the efficacy of a traditional formulation on quality-of-life and fatigue of ms patients. materials and methods two part of ajwain and one part of iranian borago were soaked in water for 24 h and one part of cinnamon was soaked in water for 72 h then were distilled to yield the drug. drug and placebo were gave to patients 15 cc per day for 3 months. this study was a 3-month, double-blind study of parallel group of patients with multiple sclerosis and was taken in khuzestan ms association in iran from july 2018 to november 2018. sixty adult patient (20–50 years old), who were member of ms association were eligible to participate. patient were required proved ms disease according to clinical examination and disease history, expanded disability status scale score 2–5.5,19 had a regular drug therapy and no change in drugs for last 4 weeks. a neurologist examined all patients. patients with any of the following conditions were not qualified for the study: history of drug or alcohol abuse, pregnancy or lactation during the last 12 months, renal or liver failure, and steroid therapy during last 2 months, another neurologic disease except ms, cardiovascular disease, or infection and sensitivity to cinnamon. this trial is in accordance with the helsinki declaration of 1975. the ethics committee of ahvaz jundishapur university of medical sciences (ethics no. ir.ajums.rec.1397.063) approved the protocol. the patients authorized the testimonial and were informed that they could withdraw from the trial any time they want. a written consent was obtained from all participants. the measurements that include quality-of-life score, fatigue score, and cognition were monitored by filling standardized and validate questionnaire msql-54, fatigue severity score, and california verbal learning test score respectively. all measures were done at baseline and monthly after the treatment started. we used block method for randomization. the investigator provided with a randomization code for each available medication. all randomization codes were opened at the end of the study. patients were randomized to receive drug or placebo in 1:1 ratio. drug and placebo were not visually identified. all participants were supposed to take four capsules per day (every 6 h). results of total 60 patients, who enrolled into the trial, 30 patients were assigned to either drug or placebo group. during follow-up, nine patients were dropped out. three for disease progression, one for moving to another city, one for drug sensitivity, and five for lack of compliance in follow-up. finally, 51 patients completed full 3 months of study period. basic demographic data are presented in table 1. there were no significant differences between the two group’s participants. after 3 months follow-up of all participants, our study, demonstrate that for patient’s fatigue score, the difference between two groups was not significant (p-value: 0.353, f: 1.0464), but interaction difference between drug and placebo group in four levels was significant (p-value <0.001, f: 126.393). mean ± sd score of drug and placebo group and 95% confidence interval were shown in table 2. results showed that fatigue in the drug group was decreased from baseline 15.74%, 23.49% and 40.65% at the end of 1st, 2nd and 3rd month respectively. while in placebo group, fatigue was increased from baseline 15.91%, 32.15% and 43.92% respectively (fig. 1). quality-of-life questionnaire is divided into two parts, physical and mental quality-of-life. for physical quality-of-life score in the cinnamon group increased more than placebo group. according to mauchly’s test, due to lack of sphericity, for comparison between levels and their interactions with drugs used greenhouse-geisser was used. the difference table 2. fatigue score mean ± sd 95% confidence interval down bound upper bound drug baseline 3.729 ± 0.027 3.355 4.103 1st month 3.142 ± 0.033 2.684 3.600 2nd month 2.853 ± 0.032 2.684 3.307 3rd month 2.213 ± 0.32 1.765 2.661 placebo baseline 3.520 ± 0.028 3.121 3.919 1st month 4.186 ± 0.035 3.658 4.635 2nd month 4.652 ± 0.035 4.168 6.135 3rd month 5.066 ± 0.034 4.588 5.543 table 1. demographic data for patients participated in this study drug placebo p-value gender (m/f ) 10/16 9/16 ns age (mean ± sd) years 46 ± 1.52 48 ± 1.78 ns level of education under diploma 6 8 ns diploma 12 10 ns higher diploma 8 7 ns 98 j contemp med sci | vol. 5, no. 2, march–april 2019: 96–100 the efficacy of traditional formulation on quality of life and fatigue in multiple sclerosis patients original f. namjooyan et al. between two groups was significant (greenhouse-geisser, p-value <0.001, f: 11630), and interaction difference between drug and placebo group in four levels was significant (p-value <0.001, f: 41.988). mean ± sd score of drug and placebo group and 95% confidence interval were shown in table 3. results showed that physical quality-of-life in the drug group was increased from baseline 5.29%, 10.69% and 14.63% at the end of 1st, 2nd and 3rd month respectively. while in placebo group, physical quality-of-life was decreased from baseline 0.38%, 2.66% and 12.83% respectively (fig. 2). for mental quality-of-life score in the cinnamon group increased more than placebo group. according to mauchly’s test, due to lack of sphericity, for comparison between levels and their interactions with drugs used greenhouse-geisser was used. the difference between two groups was significant (greenhouse-geisser, p-value <0.001, f: 14.805), and interaction difference between drug and placebo group in four levels was significant (p-value <0.001, f: 75.600). mean ± sd score of drug and placebo group and 95% confidence interval were shown in table 4. results showed that mental quality-of-life in the drug group was increased from baseline 14.37%, 31.31% and 48.88% at the end of 1st, 2nd and 3rd month respectively. while in placebo group, mental quality-of-life was decreased from baseline 2.15%, 8.77% and 16.97% respectively (fig. 3). for overall quality-of-life score in the cinnamon group increased more than placebo group. according to mauchly’s test, due to lack of sphericity, for comparison between levels and their interactions with drugs used greenhouse–geisser was used. the difference between two groups was significant (greenhouse–geisser, p-value <0.001, f: 20.622), and interaction difference between drug and placebo group in four levels was significant (p-value <0.001, f: 93.881). mean ± sd score of drug and placebo group and 95% confidence interval were shown in table 5. results showed that quality-of-life in the drug group was increased from fig. 1 fatigue score curve. table 3. physical quality-of-life score mean ± sd 95% confidence interval down bound upper bound drug baseline 79.280 ± 0.171 76.917 81.643 1st month 83.480 ± 0.162 81.241 85.719 2nd month 87.760 ± 0.161 85.563 89.984 3rd month 90.880 ± 0.171 88.518 93.242 placebo baseline 81.818 ± 0.182 79.299 84.337 1st month 81.500 ± 0.172 79.114 83.886 2nd month 79.636 ± 0.171 77.266 82.007 3rd month 78.318 ± 0.182 75.800 80.836 fig. 2 physical quality-of-life score curve. table 4. mental quality-of-life score mean ± sd 95% confidence interval down bound upper bound drug baseline 50.080 ± 0.312 45.751 54.409 1st month 57.280 ± 0.257 53.721 60.839 2nd month 65.760 ± 0.287 61.790 69.730 3rd month 74.560 ± 0.281 70.675 78.445 placebo baseline 54.909 ± 0.334 50.249 59.524 1st month 53.727 ± 0.275 49.933 57.522 2nd month 50.091 ± 0.306 45.859 54.323 3rd month 45.591 ± 0.300 41.450 49.732 fig. 3 mental quality-of-life score curve. table 5. overall quality-of-life score mean ± sd 95% confidence interval down bound upper bound drug baseline 141.76 ± 2.95 135.81 147.712 1st month 152.68 ± 2.37 147.90 157.46 2nd month 162.56 ± 2.45 157.63 167.48 3rd month 173.52 ± 2.44 168.60 178.44 placebo baseline 159.04 ± 3.15 152.70 165.39 1st month 152.40 ± 2.53 147.31 147.50 2nd month 145.81 ± 2.60 140.56 151.06 3rd month 139.36 ± 2.60 134.122 144.60 f. namjooyan et al. 99j contemp med sci | vol. 5, no. 2, march–april 2019: 96–100 original the efficacy of traditional formulation on quality of life and fatigue in multiple sclerosis patients baseline 8.81%, 18.67% and 27.89 at the end of 1st, 2nd and 3rd month respectively. while in placebo group, overall quality-of-life was decreased from baseline 1.09%, 5.12% and 9.37% respectively (fig. 4). discussion although the etiology of ms is poorly understood, it is becoming clear that widespread inflammation, loss of regulatory t cells (tregs), hyperactivity of autoimmune th1 and th17 cells, breakdown of blood–brain barrier and blood– spinal cord barrier, and loss of neuroprotective molecules in the cns are critical for the manifestation of demyelinating pathology in ms.20 sodium benzoate (nab) is one the direct metabolites of cinamic acid which is find in cinnamon. human body could metabolize cinnamon to nab. studies showed that nab effectively inhibited infiltration of monoulcer and demyelinated cells into spinal cord of experimental autoimmune encephalomyelitis (eae), the animal model of ms, mice. following nab suppress the expression of pro-inflammatory molecules and normalized the expression of myelin in cns. in addition, nab showed that it could change differentiation of myelin basic protein-primed t cells from th1 into th2 mode. nab increase the number of regulatory t cells and reduce the expression of various contact molecules. thus, altogether this evidence showed that nab in multiple steps modulate encephalitogenic t cells so it could be an important therapeutic agent in ms.21 on the other hand, studies showed that cinnamon and its metabolite cinnamon increase neutropic factors (nf) in cns. nab is a fda-approved drug against urea cycle disorder in human and increase the level of brain derived nf (bdnf) and neurotrophin-3 (nt-3) in cns. oral use of cinnamon increases the level of nab and after that level of nf in cns of mice. nab induce activation of protein kinase a (pka), so camp response elements binding (creb) will be activated. thus with oral use of cinnamon pka activate and the level of phospho-creb in mice cns will be increased. this result shows that cinnamon and nab has neurotropic property.22,23 increase and maintenance of regulatory t cells (tregs) during inflammation process could have therapeutic effect in autoimmune disease. nab increase tregs and protect mice against eae.24,25 fig. 4 overall quality-of-life score curve. ajwain is evaluated for its anti-inflammatory effect. both ethanolic and aqueous total extract of ajwain in animal models significantly show anti-inflammatory effect.26 antioxidant effect of ajwain is evaluated with hexachlorocyclohexane extract and showed that in animal model oral extract of ajwain reduce hepatotoxicity induce free radicals stress.26,27 it is well defined that nitric oxide has an important role in inflammatory process and chronic disease such as ms. nitric oxide toxicity increase when it react with superoxide radicals. in vitro studies show that aqueous and ethanolic extract of ajwain inhibited nitric oxide.26,28 in addition ajwain seed oil is effective in neurologic pain.27 clinical trial shows that using ajwain product in curing neuropathy pain in ms patients was useful.29 in summary, we have demonstrated that this drug could improve quality-of-life and fatigue of ms patients through suggested mechanisms. thus this drug may have therapeutic value in ms and other demyelinating conditions. acknowledgment this study was supported by ahvaz jundishapur university of medical sciences grant. conflicts of interest none.  references 1. tullman mj. overview of the epidemiology, diagnosis, and disease progression associated with multiple sclerosis. am j manag care. 2013;19:s15–s20. 2. goldenberg mm. multiple sclerosis review. p t. 2012;37:175–184. 3. siddiqui mk, khurana is, budhia s, hettle r, harty g, wong sl. systematic literature review and network meta-analysis of cladribine tablets versus alternative disease-modifying treatments for relapsing-remitting multiple sclerosis. curr med res opin. 2018;34:1361–1371. 4. honda k, jacobson js. use of complementary and alternative medicine among united states adults: the influences of personality, coping strategies, and social support. prev med. 2005;40:46–53. 5. olsen sa. a review of complementary and alternative medicine (cam) by people with multiple sclerosis. occup ther int. 2009;16:57–70. 6. schwarz s, knorr c, geiger h, flachenecker p. complementary and alternative medicine for multiple sclerosis. mult scler. 2008;14:1113–1119. 7. hamedi a, zarshenas mm, sohrabpour m, zargaran a. herbal medicinal oils in traditional persian medicine. pharm biol. 2013;51:1208–1218. 8. nimrouzi m, zare m. principles of nutrition in islamic and traditional persian medicine. j evid based complementary altern med. 2014;19:267–270. 9. arzani m. tib akbari. jalal-al-din, qom, iran, 2008. 10. jorjani s. medical objectives and excellent researches (al-aghraz al-tibbieh va al-mabahes al-alayieh). 1st ed.; tehran university press, tehran, 2006. 11. kermani ni. explaining the causes and signs (sharh-ol-asbab va alamat). 1st ed.; jalal-al-din; research institute for islamic and complementary medicine (ricm), qom, 2008 (in persian). 12. nazem jahan mohammad ak. eksire azam. dehli: nami monshi nolkshur; book facsimile edition.; 1315:38–70. 13. sina aa. canon of medicine. soroosh press, tehran, 1988. 14. singh g, maurya s, catalan c, de lampasona mp. chemical constituents, antifungal and antioxidative effects of ajwain essential oil and its acetone extract. j agric food chem. 2004;52:3292–3296. 15. zarshenas mm, moein m, samani sm, petramfar p. an overview on ajwain (trachyspermum ammi) pharmacological effects; modern and traditional. j natural remedies. 2013;14:98–105. 16. miraj s, kiani s. a review study of therapeutic effects of iranian borage (echium amoenum fisch). der pharmacia lettre. 2016;8:102–109. 17. dugoua jj, seely d, perri d, cooley k, forelli t, mills e, et al. from type 2 diabetes to antioxidant activity: a systematic review of the safety and efficacy of common and cassia cinnamon bark. can j physiol pharmacol. 2007;85:837–847. 100 j contemp med sci | vol. 5, no. 2, march–april 2019: 96–100 the efficacy of traditional formulation on quality of life and fatigue in multiple sclerosis patients original f. namjooyan et al. 18. ranasinghe p, pigera s, premakumara ga, galappaththy p, constantine gr, katulanda p. medicinal properties of ’true‘ cinnamon (cinnamomum zeylanicum): a systematic review. bmc complement altern med. 2013;13:275. 19. kurtzke jf. rating neurologic impairment in multiple sclerosis an expanded disability status scale (edss). neurology. 1983;33:1444–1452. 20. pahan k. prospects of cinnamon in multiple sclerosis. j mult scler (foster city). 2015;2:1000149. 21. brahmachari s, pahan k. sodium benzoate, a food additive and a metabolite of cinnamon, modifies t cells at multiple steps and inhibits adoptive transfer of experimental allergic encephalomyelitis. j immunol. 2007;179:275–283. 22. jana a, modi kk, roy a, anderson ja, van breemen rb, pahan k. upregulation of neurotrophic factors by cinnamon and its metabolite sodium benzoate: therapeutic implications for neurodegenerative disorders. j neuroimmune pharmacol. 2013;8:739–755. 23. modi kk, jana m, mondal s, pahan k. sodium benzoate, a metabolite of cinnamon and a food additive, upregulates ciliary neurotrophic factor in astrocytes and oligodendrocytes. neurochem res. 2015;40:2333–2347. 24. kundu m, mondal s, roy a, martinson jl, pahan k. sodium benzoate, a food additive and a metabolite of cinnamon, enriches regulatory t cells via stat6-mediated upregulation of tgf-β. j immunol. 2016;197: 3099–3110. 25. mondal s, pahan k. cinnamon ameliorates experimental allergic encephalomyelitis in mice via regulatory t cells: implications for multiple sclerosis therapy. plos one. 2015;10:e0116566. 26. ashok kumar bs, lakshman k, nandeesh r, saran gs. evaluation of antioxidant and anti-amylase activities of sukhasarak churna, an ayurvedic formulation. sci technol arts res j. 2015;4:207–210. 27. kokab s, ahmad s. developing herbal pharmaceutics in pakistan–ii: distinctiveness of selected medicinal herbs and uses. pakistan agriculture research council, islamabad, pakistan. 2011;3:7–8. 28. bajpai vk, agrawal p. studies on phytochemicals, antioxidant, free radical scavenging and lipid peroxidation inhibitory effects of trachyspermum ammi seeds. indian j pharm educ res. 2015;49:58–65. 29. petramfar p, moein m, samani sm, tabatabaei sh, zarshenas mm. trachyspermum ammi 10% topical cream versus placebo on neuropathic pain, a randomized, double-blind, placebo-controlled trial. neurol sci. 2016;37:1449–1455. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.04201906 107j contemp med sci | vol. 2, no. 7, summer 2016: 107–110 research maple syrup urine disease (msud) detected in neurologic disorders iraqi children adel a. kareem,a asaad muhsen abood,b abdul mahdi a. hasanc issn 2413-0516 introduction maple syrup urine disease (msud) is caused by deficiency of the branched-chain alpha-keto acid dehydrogenase enzyme complex characterized by elevated plasma levels of branchedchain amino acids (aas) and urinary excretion of branchedchain keto acids.1-4 msud is inherited in an autosomal recessive mode, with an incidence of 1 in 185,000.5 it is more prevalent in populations with a high frequency of consanguinity. the disease was first described in 1954 by menkes, hurst and craig who observed an unusual odor like that of maple syrup in the urine of four infants who died of a progressive neurological disease in the first weeks of life.6 neonatal screening by tandem mass spectrometry (ms/ ms), also known as expanded newborn screening, enables diagnosis of msud while the patient is still asymptomatic, as well as early treatment onset, two essential factors in improving the clinical course.7 before the introduction of expanded newborn screening, the severe form (classical msud) was believed to account for 75-80% of cases,8 but recent data suggest the milder forms of msud can account for up to 50% of diagnosed cases.9 the iraqi newborn screening program was implemented in 2010 and does not include screening for msud, furthermore, the laboratory tests required for diagnosis of this condition are also not provided through the governmental lab, and are only available at a few selected private medical laboratories. high rates of consanguineous marriages in iraq play an important role in increasing expression of autosomal recessive disorders. in iraq, there are limited data explaining the incidence and clinical presentation of iem. this study reports our local experience in the field of iem over the last 2 years. aconsultant pediatric neurologist, welfare teaching hospital, medical city campus, baghdad, iraq. bpediatrician cabp, al-elwyia pediatric teaching hospital, baghdad, iraq. cpediatric & mental health nursing, college of nursing, babylon university, hilla city, iraq. correspondence to abdul mahdi a. (email: abd_mahdi2003@yahoo.com). (submitted: 18 june 2016 – revised version received: 19 august 2016 – accepted: 21 august 2016 – published online: 26 september 2016) background maple syrup urine disease (msud) is a rare inborn error of metabolism, caused by a deficiency in the activity of the branched chain alpha-keto acid dehydrogenase impairing the degradation of the branched chain amino acids (leucine, isoleucine and valine). objective to examine the demographic and neurological profile in a case of msud. patients and methods a descriptive cross sectional study from 1 february 2014 to 1 february 2016, at neurological ward and clinic of the children welfare teaching hospital, in baghdad, iraq. plasma specimens of 600 patients, with clinical suspicion of inborn error of metabolism (iem) because of unexplained neurological deficits, unexplained developmental delay, recurrent coma and/or neuro-degeneration, msud were confirmed in 29 patients then clinical data of patients were reported and analyzed statistically. results out of 600 patients visiting the neurological outpatient and ward, clinical and neurological findings were recorded as well as the family history and/or other symptoms suggestive of aminoacidopathy, 35 patients were confirmed their diagnosis as msud, 6 patients were excluded because they lost the follow up, therefore only 29 patients were enrolled, most of them (28 patients) were affected by classical msud, where only one patient had intermittent type. considerable delay in diagnosis was noticed, which led to significant neurological abnormalities in most patients and the psychomotor delay was the main clinical presentation. conclusion in the absence of newborn screening, msud is not uncommon in neurologically disorder patients where msud was still diagnosed clinically, but delayed. the importance of clinical awareness and accurate biochemical analysis were the key tools for diagnosis and the necessity for a comprehensive national newborn screening program. keywords maple syrup urine disease, branched-chain amino acids the objective of this study was to outline the profile of iraqi patients with msud from those attend pediatric neurology and neurodisability clinic so as to contribute to the consolidation of specific public policies for msud in the country. patients, materials and methods setting this is a single center descriptive cross sectional study conducted at the pediatric neurology department and clinic at the welfare teaching hospital, medical city, baghdad, for the period from 1 february 2014 to 1 february 2016. a total of 600 patients, who referred to fluorescent high performance liquid chromatography (hplc) analysis for suspected iem, were relying mainly on a high index of clinical suspicion warranting confirmatory testing by laboratory analysis of aa, in addition to those already suspected to being msud that suggested by tandem mass screening. most of patients are well known to the neurological service by their psychomotor delay & behavioral abnormalities who were undiagnosed. the inclusion criteria included the ages of all patients enrolled in the study were between 2 months and 15 years with the following criteria: 1. patients who already well known to neurological clinic with intractable seizures (unclassified with syndrome), unexplained neurological deficits, unexplained developmental delay, recurrent coma, 108 j contemp med sci | vol. 2, no. 7, summer 2016: 107–110 maple syrup urine disease (msud) detected in neurologic disorders iraqi children research adel a. kareem et al. first cousins are the children of two siblings. first cousins have grandparents in common distant cousins: second cousin; the children of first cousins (share the great grandparent) while third cousins: grandchildren of two first cousins (share the great great grandparent). equipment the hplc system consisted of degasser yl 9101, the pump was yl 9110 quaternary pump, and the injection valve rheodyne was equipped with 20 μl sample loading loop, detector model 1100 agilent fluorescence collection of samples venous blood was collected in heparin-containing tubes. after centrifugation (2000 for 10 min). processing of samples add 20 μl from sulfosalicylic acid to deproteinize 200 μl of plasma; mixed on a vortex, incubate at 4°c for 30 min, centrifuged at 3000 g for 15 min remove precipitated protein. the clear supernatants were transferred to polypropylene vials and stored in a refrigerated at −20°c. derivatization to 30 μl of standard solution or plasma add 30 μl from ortho phthaladehyde (opa) reagent and inject 20 μl on to the column. statistical analysis statistical analysis and reporting of obtained data were carried out by descriptive pattern. data were reported and presented as tables to show the frequency distribution (number and percent (n, %) of variables. results out of 600 patients with neurological symptoms and /or sign who referred to quantitative aas by fluorescent hplc, 29 patients were detected to have msud; 28(96.5%) classical type and only one (3.5%) was an intermittent type enrolled in the study that suggested by the significant increase branched chain aa (valin, leucine and isoleucine) and increase leucine to alanine ratio. age of the patients at diagnosis are given in table 1. the main age at diagnosis of msud (table 1) was 2nd year of age. msud were more common in male 72% and the male/ female ratio was 2.6 (table 2). family history was negative in 58.6% (table 3). neurodegeneration, positive family history, unusual urine odour, altered mental status out of proportion of other systemic disorder unusual body or urine odor and hiccup. 2. a significant increase in blood branched chain amino acid (bcaa) levels, on more than one measurement, as determined by a gold-standard method (florescent hplc-based) bcaa quantitation. the exclusion criteria included: 1. age: below 2 months and above 15 years. 2. loss of follow up. out of 600 patients, 35 patients were found to have an msud, and 6 patients are excluded because of loss of follow up. clinical data and study participants msud patients were thoroughly interviewed, clinically examined and investigated according to the approved standard medical and laboratory work up. consanguinity enrolled in the study where it considers a real challenge in our developing country with a high consanguinity rate. basic diagnostic investigations upon clinical suspicion of an aa disorder, basic metabolic investigations were performed using routine methods for the measurement of serum glucose, electrolyte liver function test (aspartate aminotransferase, alanine aminotransferase), arterial blood gases, ammonia, and lactate. ethical approval all patients and their families were informed about the aim and suspected benefit of the study before obtaining their agreements for participation according to the medical research and ethical regulations, thus an oral consent was taken from all enrolled participants and their families. all the medical research ethics rules and instructions regarding patient’s privacy, humanity and security; as well as the medical research, laboratory data and investigation results were strictly considered throughout all the steps of study. definitions psychomotor retardation or developmental delay; refers to the slow progress in the attainment of developmental milestones this may be caused by either static or progressive encephalopathies. intellectual disability; the term intellectual disability is replacing mental retardation, defines as a disability characterized by a significant limitation both intellectual functioning and in adaptive behavior as expressed in conceptual, social, practical and adaptive skills. this disability originates before the age of 18 and manifest with severe problems in the individual’s capacity to perform (i.e. impairment), ability to perform (i.e. activity limitation), and opportunity to function (participation restrictions)12 and in order to get a proper determination of degree of intellectual disability, referred to the psychiatrist’s opinion on the 10th floor of baghdad teaching hospital-medical city and some time to psychology center for research in baghdad university in aljadria. a cousin is a relative with whom a person shares one or more common ancestor, in a general sense, cousins are two or more generations away from any common ancestor table 1. month no. % 0 0 0 3–6 2 6.9 7–12 6 20.6 13–24 7 24.1 25–36 3 10.3 37–48 4 13.8 49–60 4 13.8 >60 3 10.3 total 29 100% adel a. kareem et al. 109j contemp med sci | vol. 2, no. 7, summer 2016: 107–110 research maple syrup urine disease (msud) detected in neurologic disorders iraqi children table 5. no. % closely related 24 83 not closely related 2 7 unrelated 3 10 cases and it is consider significantly higher in our study and it is coincident with study done in libya16 where consanguinity was found in about 87%, in contrast to study from the malaysia14 that parental consanguinity was found in 1/3 of cases, as majority of metabolic diseases are autosomal recessive inherited traits occurring frequently in countries with high consanguinity rates,17 which may suggest possibility of high rate of occurrence of iem in arabian world like iraq although comprehensive published report are not available. delayed diagnosis is common in our case series most of them diagnosed at second year. we believe that the delayed diagnosis is contributed by the lack of newborn screening (nbs) which is considered as effective and important preventive measure, in addition to lack of awareness of iem among physicians. the problem of delayed diagnosis is compounded by the lack of diagnostic facilities. the awareness and understanding of physicians about msud should be heightened through continuous medical education and publicity. therefore, identification and diagnosis of such disorder remain a real challenge and this agree with study done in other developing countries like lebanon18 and malaysia,14 whereas it is inconstant with study done in thailand,19 this discrepancy related to establishment of neonatal screening, which had been established in thailand, but it does not in our country. the psychomotor delay was the main presenting symptom in about 96% from them 65% with seizures while one case (3.5%) experience recurrent encephalopathy, who diagnosed as intermittent msud & this in contrast with thailand study where diagnosed at early infancy (1st 5 mo.) period during work up for neonatal encephalopathy work up,19 and it might be attributed to attended patients in the present study who survive the initial metabolic crisis typically have neuro developmental delay and learning deficit.12 about two third of msud patients in present study experienced a seizure (gtcs) while the seizure present in only ¼ of patients in lebanon study,18 this difference might be related to dietory regimen adapted by different societies. very few parents are able to identify the special burnt sugar smell associated with msud, although this was present in almost all patients in our case series, but when repeat the question or smell the urine, we find it is present in most of cases and this is because some of parents, they don’t experienced maple syrup odour. there for the smell of patient urine may be considered as part of clinical assessment in suspected patient with metabolic diseases. conclusions 1. this study provides useful information for health policy and planning services for future metabolic newborn screening programs, which should include screening for msud. 2. consanguinity had a definite role in the increased frequency of metabolic disease in our population as most of them are autosomal recessive mendelian inheritance. 3. late diagnosis, of aa disorders like msud was still considered challenging issue. recommendations 1. expanding and extending of tandem mass liquid chromatography-metabolic screen mass spectrometry (lc-ms/ms) table 2. gender no. % male 21 79 female 8 21 table 3. no. % negative 17 59 positive 12 41 table 4. no. % psychomotor delay without seizure 9 31 psychomotor delay with seizure 19 66 recurrent encephalopathy 1 3 clinical presentation of msud patients overall, participants show that psychomotor delay represents the main presenting sign (96.5%), with 30% with a seizure while one patient presented with recurrent encephalopathy (3.5%) (table 4). the consanguinity among families of our patients shows about 82% as first cousin and 7% as second cousin (table 5). discussion this is the first study highlighting the clinical and biochemical diagnostic approaches to msud in iraq. although neonatal screening by ms/ms may enhance early detection of msud, hplc remains the key diagnostic tool for confirmation of all suspected cases, whether clinically symptomatic or initially positive by neonatal screening program. however, in a developing country like iraq, resources are carefully allocated and testing is targeted based on clinical awareness. definitive laboratory diagnosis is crucial in confirming the clinical suspicion of msud since its clinical presentation is non-specific and can mimic common conditions such as infections and other iems. with the availability of quantitative, plasma aa analysis by fluorescent hplc, we have started to see an increasing number of children diagnosed msud who their parent concerned about their psychomotor delay with or without seizure. in our case series, msud is found to occur predominantly in male gender (72.5%). most cases are classical type except one case was intermittent type, comparable to a reports from the malaysia14 and the philippines15 were also the majority is in the classical group. another noteworthy finding was that parental consanguinity was found in about 90% of 110 j contemp med sci | vol. 2, no. 7, summer 2016: 107–110 maple syrup urine disease (msud) detected in neurologic disorders iraqi children research adel a. kareem et al. to involve all iraq with involvement other aa disorder particularly msud. 2. establishment of specific metabolic centers in various universities and research institutes and provided them with advanced metabolic diagnostic equipment. 3. establishment of health education program about early detection of metabolic disorder in term of psychomotor delay, urine and sweat odor, positive family history of aa disorders and education about the consanguinity and its role in metabolic disease. competing interests the authors declare that they have no competing interests. authors contributions a. k. did the literature research, analysed the data and drafted the manuscript. a. a. were involved in discussion and evaluation of the data and critically revised the manuscript and also participated in the study coordination and helped to draft the manuscript in addition to contributed clinical data. all authors read and approved the final manuscript. acknowledgment thank for patients, their families. we also thank noor alsebah’s lab administration represented by marwan sami and farqed farhan for their collaboration. n references 1. rezvani i, rosenblatt ds. valine, leucine, isoleucine and related organic acidemias. in: kliegman rm, behrman re, jenson hb, stanton bf, editors. nelson textbook of pediatrics. 18th ed. philadelphia: saunders elsevier; 2007:540–9. 2. nyhan wl, ozand pt. maple syrup urine disease (branched-chain oxoaciduria). in: nyhan wl, ozand pt, editors. atlas of metabolic diseases. london: chapman & hall medical. 1998:138–46. 3. ogier de baulny h, saudubray jm. branched chain organic acidurias. in: fernandes j, saudubray jm, van den berghe g, editors. inborn metabolic diseases: diagnosis and treatment. 3rd ed. berlin: springe-verlag. 2000:196–212. 4. surarit r, srisomsap c, wasant p, svasti j, suthatvoravut u, chokchaichamnankit d, et al. plasma amino acid analyses in two cases of maple syrup urine disease. southeast asian j trop med public health. 1999;30(suppl 2):138–139. 5. chuang dt, shih ve. disorders of branched chain amino acid and keto acid metabolism. in: scriver cr, beaudet al, sly ws, valle d, editors. the metabolic and molecular bases of inherited disease. 9th ed. new york: mcgraw-hill. 2005:1239–1277. 6. menkes jh, hurst pl, craig jm. a new syndrome: progressive familial infantile cerebral dysfunction associated with an unusual urinary substance. pediatrics. 1954;14:462–467. 7. simon e, fingerhut r, baumkötter j, konstantopoulou v, ratschmann r, wendel u. maple syrup urine disease: favourable effect of early diagnosis by newborn screening on the neonatal course of the disease. j inherit metab dis. 2006;29:532–537. 8. morton dh, strauss ka, robinson dl, puffenberger eg, kelley ri. diagnosis and treatment of maple syrup disease: a study of 36 patients. pediatrics. 2002;109:999–1008. 9. fingerhut r, simon e, maier em, hennermann jb, wende lu. maple syrup urine disease: newborn screening fails to discriminate between classic and variant forms. clin chem. 2008;54:1739–41. 10. goodman si, greene cl. metabolic disorders of the newborn. pediatr rev. 1994;15:359–365. 11. eric piña-garza j. fenichel’s, a signs and symptoms approch, clinical pediatric neurology, 7th edition, saunders, tennessee usa. 2014; 122–123:47,113. 12. swaiman k, ashwal s, ferriero d, schor n. aminoacidemia and organic acidemias. swaiman’s pediatric neurology, principles and practice, 2-volume set. 5th ed. elsevier saunders, minneapolis. 2012;1;337– 339,554. 13. fizhugh isbn 0-7136-4859-7. 21, terrick v h (1988). the dictionary of genealogy (5th ed.) london: a & c black. p. 81 14. yunus zm, kamaludin da, mamat m, choy ys, ngu lh. clinical and biochemical profiles of maple syrup urine disease in malaysian children. jimd rep. 2012;5:99–107. 15. lee jy, chiong ma, estrada sc, cutiongco-de la paz em, silao cl, padilla cd. maple syrup urine disease (msud)—clinical profile of 47 filipino patients. j inherit metab dis. 2008;31 suppl 2:s281-s285. 16. alobaidy h. patterns of inborn errors of metabolism: a 12 year singlecenter hospital-based study in libya. qatar med j. 2013;2:57–65. 17. tadmouri go, nair p, obeid t, al ali mt, al khaja n, hamamy ha. consanguinity and reproductive health among arabs. reprod health. 2009;8:6–17. 18. karam pe, habbal mz, mikati ma, zaatari ge, cortas nk, daher rt. diagnostic challenges of aminoacidopathies and organic acidemias in a developing country: a twelve-year experience. clin biochem. 2013;46:1787–1792. 19. vatanavicharn n, ratanarak p, liammongkolkul s, sathienkijkanchai a, wasant p. amino acid disorders detected by quantitative amino acid hplc analysis in thailand: an eight-year experience. clinical chemica acta. 2012;413(13-14):1141–1144. 140 j contemp med sci | vol. 5, no. 3, may–june 2019: 140–144 original diclofenac-induced acute kidney injury is linked with oxidative stress and pro-inflammatory changes in sprague-dawley rats hayder m. alkuraishy, ali i. al-gareeb, and nawar raad hussien* department of clinical pharmacology, medicine and therapeutic, college of medicine, al-mustansiriya university, baghdad, iraq. *correspondence to nawar raad hussien (email: ph.nawar@gmail.com). (submitted: 12 september 2018 – revised version received: 19 november 2018 – accepted: 14 december 2018 – published online: 26 june 2019) introduction diclofenac is a non-steroidal anti-inflammatory drug (nsaid); belong to the acetic acid group. diclofenac is used as analgesic, anti-inflammatory and antipyretic agent. diclofenac sodium acts by inhibiting cyclo-oxygenase-1 (cox-1) and cyclo oxygenase-2 (cox-2) enzymes, which are responsible for prostaglandin synthesis from arachidonic acid.1 diclofenac inhibits cox-1 about 10-fold than cox-2 on contrast to the aspirin which is mainly block cox-1. indeed, it also inhibits lipoxygenase, phospholipase a2 and other pro-inflammatory autacoids.2 diclofenac-induced nephrotoxicity is due to the toxic effect of diclofenac on renal glomeruli and tubules. diclofenac induces formation of glomerular lesions, tubular dilatation and loss of brush border.3 long-term treatments with diclofenac causes dose-dependent reduction in renal pg lead to disturbances in the glomerular function and reduction of glomerular filtration rate (gfr). in addition, diclofenac induces autolysis that increases renal intracellular osmolarity which is responsible for proximal renal tubules dilatations.4 diclofenac is metabolized in the liver to 4-hydroxydiclofenac, after sulfation and conjugation, which are excreted by the urine (65%) and bile (35%). diclofen ac sodium modifies renal function through inhibition of renal prostaglandins leading to reversible renal ischemia.5 although, nsaids related hypertension, salt and water retention, edema and hyperkalemia are highly linked with acute renal failure.6 it has been hypothesized that diclofenac has dose-dependent effects on plasma coagulation parameters, oxidative stress, renal damage and adenosine deaminase activity in diclofenac-induced aki. the toxicity induced by diclofenac sodium result in depletion of atp, lipid peroxidase, mitochondrial dysfunction and change in renal cells calcium concentration.7 prostaglandin play an important role in gfr since; renal plasma current is maintained by balanced between the vasoconstrictor impact of the renin–angiotensin system and the vasodilatory effect of prostaglandin. in fluid depleted state, prostacyclin (pgi2) affect renal homeostatic mechanisms, (pgi2) and (pgd2) cause dilatation of renal vascular bed along with the lowering of renal vascular resistance.8 thus, prostaglandins enhances renal perfusion with redistribution of blood flow from the renal cortex to nephrons in the juxta-medullary region. renal prostaglandins have crucial local function in different parts of renal tissues such as glomeruli and convoluted tubules, thus prostaglandin inhibitors have various deleterious effects.9 diclofenac-induced aki may be related to the ischemia caused by inhibition of prostaglandins synthesis in renal arterioles.10 diclofenac induces oxidative stress in the body and became the main cause of nephrotoxicity and aki leading to the acceleration of lipid peroxidation and reduction of endogenous antioxidant capacity.11 on contrary, a previous study illustrated that inhibition of cox may not be the main mechanism of diclofenac-induced aki, which observed that redox signalling and oxidative stress as the main contributing factors in diclofenac-induced aki.12 normally, reactive oxygen (hydroxyl radical, nitric oxide, peroxynitrite, hydrogen peroxide, superoxide anion and single oxygen) produced in the body are deactivated by non-enzymatic (vitamin c, vitamin d and glutathione) and enzymatic (catalase, glutathione peroxidase and superoxide dismutase) agents.13,14 in general, antioxidants and oxidants are in balance in the body, but oxidative stress happens when the reactive oxygen radicals are produced extremely and/or antioxidants are insufficient, as a result, oxidative stress will be initiated.15 furthermore, the toxic dose of diclofenac provokes renal mitochondrial dysfunction through inhibition of mitochondrial objectives diclofenac induces oxidative stress in the body and became the main cause of nephrotoxicity and acute kidney injury (aki). the traditional markers of aki are blood urea and serum creatinine which are regarded as low sensitive and low specific in detection the early renal damage. therefore, the aim of this study is to evaluate oxidative stress and pro-inflammatory biomarkers in diclofenac-induced aki in rats. methods twenty sprague-dawley male rats were used and randomly divided into two groups. group 1 (n = 10): rats treated with distilled water plus normal saline for 12 days. group 2 (n = 10): rats treated with distilled water plus diclofenac 15 mg/kg for 12 days. rat body weight, body mass index and estimated glomerular filtration rate (egfr) were evaluated in both groups. blood urea, serum creatinine, serum malondialdehyde, superoxide dismutase, glutathione reductase, neutrophil gelatinase associated lipocalin, kidney injury molecules (kim-1) and cystatin-c were estimated. results diclofenac 15 mg/kg led to significant aki through elevation of blood urea and serum creatinine with significant reduction of egfr. kim-1 serum level was significantly elevated with high sensitivity and specificity compared with the other tested biomarkers. conclusion kim-1 serum level is more sensitive and specific with high accuracy compared with the other renal biomarkers in diclofenacinduced aki. estimation of kim-1 serum levels should be regarded as a cornerstone for early detection of aki in high risk patients. keywords diclofenac, estimated glomerular filtration rate, acute kidney injury, nephrotoxicity issn 2413-0516 141j contemp med sci | vol. 5, no. 3, may–june 2019: 140–144 original diclofenac-induced acute kidney injury is linked with oxidative stressh.m. alkuraishy et al. complex causing reduction in atp generation and apoptosis. thus, anti-oxidants play a potential role in prevention of diclofenac-induced aki via attenuation of free radical and oxidative stress effects.16 likewise, diclofenac-induced aki may be related to the induction of pro-inflammatory cytokines including interleukin (il)-6, il-1, il-33 and tumor necrosis factor-α lead to renal nuclear fragmentation and condensation.17 biomarkers of acute kidney injury the traditional markers of acute kidney injury (aki) are blood urea and serum creatinine which are regarded as low sensitive and low specific in detection the former renal damage. thus, detection of the initial renal injure required a new biomarkers which are more sensitive and highly specific that give an insight about the site of underlying renal damage.18 a urinary protein is regarded as a potential marker of acute and chronic renal damage that are induced by nephrotoxic drugs. normally, the glomeruli restrict the transport and migration of high molecular weight proteins from blood into the lumen of nephron, but during pathological conditions high molecular proteins can be identified and detected in the urine due to nephron dysfunction.19 high molecular proteins like albumin, transferrin and immunoglobulin g are more sensitive proteins in early detection of glomerular filtration dysfunction and structural glomerular damage.20 on the other hand, low molecular weight proteins are mainly reabsorbed at renal proximal tubules, when there is an excess in the amount of low molecular weight protein concentrations a nephron overload occurred that exceeding the proximal renal tubules reabsorbing capacity causing proteinuria due to failure of the re-absorption capacity.21 low molecular weight proteins such as α1-microglobuin, β2-microglobin, cystatin-c, retinol binding protein and kidney injury molecule-1 (kim-1) are obviously recorded as the main proteins that reflect the underlying renal glomerular and/or tubular damage during nephrotoxicity.22,23 nephrotoxic agents such as cisplatin chemotherapy, nsaids and aminoglycoside lead to up-regulation of kim-1 due to ischemic reperfusion injury thus; serum level of kim-1 is linked and correlated with the immune-response of renal proximal tubules damage during aki.24 additionally, mrna of kim-1 is vastly expressed in the injured kidney which is exposed and released into the lumen which finally excreted in the urine. also, kim-1 is detected in the blood since; it stable and can be straightforwardly identified.25 besides, neutrophil gelatinase associated lipocalin (ngal) which is a 25 kda protein that attach the granulocytes, it correlated with drug induced aki since; it is responsible for the renal inflammation during ischemia thus; ngal serum level be a sign of acute renal injury.26 additionally, different cytokines such as interleukin, interferon, and colony stimulating factors play an important and integral role in the renal tubular damage and repair so; they are considered as biomarkers of kidney injury during drug induced aki.27 cystatin-c is a low molecular weight protein excreted by glomerular filtration and high level of cystatin-c is linked with reduction of gfr and glomerular filtration. it has been shown that cystatin-c serum levels predict the stage and progression of renal diseases. cystatin-c serum levels also increased in cigarette smoking, cancer, neurological and atherosclerosis.28,29 therefore, novel and new biomarkers may help the determination the site of renal damage and give an important picture about the disease progression and the role of nephro protecting agents.30 therefore, the aim of this study was to evaluate oxidative stress and pro-inflammatory biomarkers in diclofenac induced aki in rats. materials and methods about 20 sprague-dawley male rats were used in this study. these animals were gained from the national center for drug control and research. rats’ age ranged from 3 to 4 months and their body weight ranged from 200 to 400 g. the animals were isolated as three rats in each sterilized cage and placed with suitable temperature (22–25°c) with artificial 12/12 light cycle. they left for 1 week for adaptation without any intervention with free access to normal chow pellets and water. human care for animals was according to the guide to the care and use of laboratory animal. after acclimatization period, weights of rats were taken and rat with wound infection were excluded. then rats were randomly divided into two groups, 10 rats in each group. the study protocol and method for induction of aki was according to singh et al.’s,31 method. control group (n = 10): rats treated with distilled water (5 ml/kg, p.o.) for 12 days, on days 6–12 received an intra peritoneal injection of normal saline (5 ml/kg) daily. aki group (n = 10): rats treated with distilled water (5 ml/kg, p.o.) for 12 days and on days 6–12 received diclofenac 15 mg/kg, i.p. anthropometric measurements length was measured by graduated tape measure from nose to the anus (naso-anal length in cm). rat body weight was done by specific digital balance in gram. body mass index (bmi) equal body weight in grams over the square of length in cm, bmi = bw (g)/length (cm)2. estimated glomerular filtration rate (egfr) was measured according to schwartz formula, egfr = k × height (cm)/serum creatinine (mg/dl), k = 0.55.32 sample collection on day 12, chloroform was used to anesthetize the rats and sharp scissors were used to exudate the rats. the blood sample was allowed to drain in sterile gel tube, and then centrifuged for 10 min at 5000 rpm at room temperature, after that the formed supernatant layer was isolated as serum sample and kept in freezer at −20°c to be assessed later. assessment of biochemical variables blood urea and serum creatinine were estimated by using an auto-analyzer (ilab-300-biomerieux diagnostic, milano, italy) they are expressed as mg/dl. serum malondialdehyde (mda), superoxide dismutase (sod), glutathione reductase (gsh), ngal, kim-1 and cystatin-c were measured by elisa kit methods according to the instruction of the kit manufacture (myo-bio source, usa). statistical analysis statistical package for the social sciences software (spss, inc., chicago, il, usa) was used for data analysis. data of the this study presented as mean ± sd. unpaired student t-test between 142 j contemp med sci | vol. 5, no. 3, may–june 2019: 140–144 diclofenac-induced acute kidney injury is linked with oxidative stress original h.m. alkuraishy et al. control and treated groups was used for detecting the significance of differences. the levels of significance was regarded when p < 0.05. results during diclofenac-induced aki, rat body weight was increased to 286.87 ± 27.24 g compared with the control group 268.00 ± 25.01 g but; not reached to the significant level (p = 0.20). the bmi was increased significantly in aki group compared with the control group (p = 0.01). blood urea was raised significantly in aki group up to 70.5 ± 12.53 mg/dl compared with the control group (41.83 ± 7.46 mg/dl, p = 0.0003). besides, serum creatinine in aki group was increased significantly (1.52 ± 0.49 mg/dl) compared with the control group (0.7 ± 0.14 mg/dl, p = 0.0019). the estimated gfr reduced significantly in aki group (7.59 ± 1.7 ml/min/1.37) compared with the control group (16.89 ± 4.21 ml/min/1.37, p = 0.0001). regarding the oxidative stress and endogenous anti-oxidant capacity, there were insignificant increase in the mda serum levels in aki group (427.65 ± 210.39 ng/ml) compared with the control group (289.85 ± 44.18 ng/ml, p = 0.14) while; both sod and gsh serum level were reduced in aki group compared with the control group (p = 0.2 and 0.4929 respectively). kim-1 was significantly raised in aki group (269.03 ± 29.61pg/ml) compared with the control group (73.78 ± 16.29, p = 0.0001) (table 1). indeed, cystatin-c serum level was insignificantly increased during induction of aki by diclofenac from 0.024 ± 0.0005 ng/ml in the control group to 0.0277 ± 0.009 ng/ml in the aki group (p = 0.33) (fig. 1). blood urea was significantly correlated with serum creatinine (r = 0.99, p = 0.01), mda (r = 0.99, p = 0.008), kim-1 (r = 0.89, p = 0.02) and cys-c (r = 0.98, p = 0.01) but negatively correlated with sod (r = −0.99, p = 0.001) in aki group (table 2). kidney injury molecule-serum level was highly sensitive and specific biomarker in diclofenac-induced aki while; gsh was the least sensitive biomarker (table 3). table 1. effect of diclofenac on the anthropometric variables, biochemical and inflammatory biomarkers in diclofenac induced aki biomarkers control (n = 10) aki (n = 10) p weight (g) 268.00 ± 25.01 286.87 ± 27.24 0.20 height (cm) 21.50 ± 0.83 21.00 ± 0.53 0.19 bmi (g/cm2) 1.02 ± 0.02 1.64 ± 0.03 0.01* blood urea (mg/dl) 41.83 ± 7.46 70.50 ± 12.53 0.0003** serum creatinine (mg/dl) 0.70 ± 0.14 1.52 ± 0.49 0.0019** estimated gfr (ml/min/1.37) 16.89 ± 4.21 7.59 ± 1.7 0.0001** mda (ng/ml) 289.85 ± 44.18 427.65 ± 210.39 0.14 sod (pg/ml) 48.12 ± 32.92 30.62 ± 15.54 0.20 gsh (μg/ml) 15.94 ± 2.39 14.88 ± 3.02 0.4 kim-1 (pg/ml) 73.78 ± 16.29 269.03 ± 29.61 0.0001** ngal (pg/ml) 15.78 ± 3.07 18.76 ± 4.13 0.16 *p < 0.05. **p < 0.01 unpaired t-test. bmi: body mass index; gfr: glomerular filtration rate; mda: malondialdehyde; sod: superoxide dismutase; gsh: glutathione reductase; il-18: interleukin-18; kim-1: kidney injury molecule-1; ngal: neutrophil gelatinase associated lipocalin. fig. 1 cystatin-c serum levels in diclofenac-induced aki. table 2. correlation of blood urea with biochemical parameters and renal biomarkers in diclofenac-induced aki group compared with the control group variables group і group іі r p r p serum creatinine (mg/dl) 0.62 0.33 0.99 0.01** gfr (ml/min/1.37) 0.33 0.50 −0.91 0.08 mda (ng/ml) 0.57 0.23 0.99 0.008* sod (pg/ml) 0.72 0.09 −0.99 0.001* gsh (μg/ml) 0.45 0.43 −0.89 0.09 kim-1 (pg/ml) 0.86 0.06 0.89 0.02** cys-c (ng/ml) 0.66 0.15 0.98 0.01** ngal (pg/ml) 0.50 0.29 0.87 0.05 *p < 0.01. **p < 0.05 unpaired t-test. gfr: glomerular filtration rate; mda: malondialdehyde; sod: superoxide dismutase; gsh: glutathione reductase; kim-1: kidney injury molecule-1; cys-c: cystatin-c; ngal: neutrophil gelatinase associated lipocalin. table 3. sensitivity and specificity of biomarkers in diclofenac-induced aki biomarkers sensitivity specificity accuracy plr nlr blood urea (mg/dl) 70 58.33 63.64 1.6 0.5 serum creatinine (mg/dl) 72.73 63.64 68.18 2.0 0.4 mda (ng/ml) 60.00 33.33 50.00 0.9 1.2 sod (pg/ml) 55.56 40.00 50.00 0.93 1.1 gsh (μg/ml) 50.00 40.00 46.67 0.83 1.25 kim-1 (pg/ml) 90.00 90.00 90.00 9.00 0.11 ngal (pg/ml) 57.14 57.14 57.14 1.33 0.75 cys-c (ng/ml) 81.82 50.00 70.59 1.64 0.36 mda: malondialdehyde; sod: superoxide dismutase; gsh: glutathione reductase; kim-1: kidney injury molecule-1; cys-c: cystatin-c; ngal: neutrophil gelatinase associated lipocalin, plr: positive likelihood ratio; nlr: negative likelihood ratio. discussion this study illustrated significant diclofenac-induced aki which revealed through elevation of blood urea and serum creatinine compared with the control group in the experimental rats as supported by alabi et al.’s,33 study that illustrated diclofenac leads to significant renal tubular necrosis and 143j contemp med sci | vol. 5, no. 3, may–june 2019: 140–144 original diclofenac-induced acute kidney injury is linked with oxidative stressh.m. alkuraishy et al. raising in the renal biomarkers. the induced aki in this study as well showed significant reduction in the estimated gfr which was due to the development of acute tubular necrosis and glomerular damage since; tomohiro et al.,34 exposed that short-term administration of diclofenac leads to proximal renal tubular damage and significant glomerular damage due to potential inhibition of renal prostaglandins which per se reduce renal blood flow and causing renal ischemia. bmi of the experimental rats was higher compared with the control group this might due to daily body weight gain or fluid retention which caused by renal damage. moreover, reduced gfr also take part in raised bmi via reduction of urine output and development of peripheral oedema as a number of rats developed ascites. observational study suggested that fluid retention is the main element of clinical syndrome of kidney disease since; there are similarities in the fluid retention and component in the patients with either heart failure or advanced renal disease.35 oxidative stress, free radical generations, lipid peroxidations and reduction of endogenous anti-oxidant capacity are the main mechanisms of diclofenac-induced aki as described by hickey et al.,36 study that demonstrated a high dose of diclofenac leads to significant lipid peroxidation through elevation of mda serum levels and reduction of sod (marker of anti-oxidant). but in this there were insignificant elevation in mda serum level and insignificant reduction in the anti-oxidant capacity (sod, gsh) respectively which might due to insufficient diclofenac dose, small sample size or short duration of the experimental study. indeed, many cytokines are elevated during diclofenac-induced aki that reflects the inflammatory process in the renal tissues. bunel et al.’s,37 study showed that il-18 and ngal sera levels were significantly increased within short period of renal injury induction (within hours) and return to the normal levels within few days due to partial regeneration of damaged renal tubules. this may explain the insignificant elevation of these biomarkers in this study. in additional, renal biomarkers are subjected to different variability including dynamic of their excretion, diurnal variations and baseline assessment as supported by harrill et al.,38 study that confirmed the variable results of this study. likewise, diclofenac in this study failed to increase serum levels of ngal significantly. furthermore, kim-1 was significantly elevated in this study compared with the control. lan et al.’s,39 study confirmed significant elevation of kim-1 in cadmium-induced nephrotoxicity. kim-1 serum levels are elevated during initiation of acute nephrotoxicity as it increased in both serum and urine since; it shed from renal proximal tubules. kim-1 serum is significantly correlated with different grades of renal damage in acute nephrotoxicity and renal injury because; it is highly sensitive and specific for renal tubular toxicity40 as revealed in our study. on the other hand, luo et al.’s,41 in vitro and in vivo study showed that repeated and consecutive administration of nephrotoxic agents in rats for 7 days result in time dependent increase in both kim-1 and ngal serum levels before the initiation of renal proximal tubules damage suggesting that both kim-1 and ngal are insensitive in detection of early renal damage and these biomarkers may not be appropriate in evaluation and detection of nephrotoxic effect of certain agents. cystatin-c is a sensitive biomarker for detection of acute renal damage seeing as; it more sensitive than blood urea and serum creatinine.42 harman et al.’s,43 study confirmed that cystatin-c is a surrogate biomarker for acute renal injury and should be incorporated in the estimation of gfr as it is more sensitive than creatinine but; cystatin-c serum levels in diclofenac-induced aki of this study were elevated insignificantly compared with the control which might be due to small sample size, biomarker variability and short duration of the study, these factors may contribute to the insignificant elevation of cystatin-c in diclofenac-induced aki in rats model of the current study. conclusion kidney injury molecule-1 serum level is more sensitive and specific with high accuracy compared with the other renal biomarkers in diclofenac-induced aki. therefore, estimation of kim-1 serum levels should be regarded as a cornerstone for early detection of aki in high-risk patients. acknowledgment the authors would like to thank the research committee in college of medicine, al-mustansiriyia university for the great supports. conflicts of interest none.  references 1. verhoeven f, totoson p, marie c, prigent-tessier a, wendling d, tourniernappey m, et al. diclofenac but not celecoxib improves endothelial function in rheumatoid arthritis: a study in adjuvant-induced arthritis. atherosclerosis 2017;266:136–144. 2. inoue t, anai s, onishi s, miyake m, tanaka n, hirayama a, et al. inhibition of cox-2 expression by topical diclofenac enhanced radiation sensitivity via enhancement of trail in human prostate adenocarcinoma xenograft model. bmc urol. 2013;13:1. 3. bickel m, khaykin p, stephan c, schmidt k, buettner m, amann k, et al. acute kidney injury caused by tenofovir disoproxil fumarate and diclofenac co-administration. hiv med. 2013;14:633–638. 4. yasmeen t, qureshi gs, perveen s. adverse effects of diclofenac sodium on renal parenchyma of adult albino rats. j pak med assoc. 2007;57:349–351. 5. yarlagadda sg, perazella ma. drug-induced crystal nephropathy: an update. expert opin drug saf. 2008;7:147–158. 6. schrama yc, van dam t, fijnheer r, hené rj, de groot p, rabelink tj. cyclosporine is associated with endothelial dysfunction but not with platelet activation in renaltransplantation. neth j med. 2001;59:6–15. 7. pendergraft wf 3rd, herlitz lc, thornley-brown d, rosner m, niles jl. nephrotoxic effects of common and emerging drugs of abuse. clin j am soc nephrol. 2014;9:1996–2005. 8. yu y, stubbe j, ibrahim s, song wl, smyth em, funk cd, et al. cyclooxygenase-2-dependent prostacyclin formation and blood pressure homeostasis: targeted exchange of cyclooxygenase isoforms in mice. circ res. 2010;106:337–345. 9. hur j, liu z, tong w, laaksonen r, bai jp. drug-induced rhabdomyolysis: from systems pharmacology analysis to biochemical flux. chem res toxicol. 2014;27:421–432. 10. abu hamad r, berman s, hachmo y, stark m, hasan f, doenyas-barak k, et al. response of renal podocytes to excessive hydrostatic pressure: a pathophysiologic cascade in a malignant hypertension model. kidney blood press res. 2017;42:1104–1118. 11. issa n, kukla a, ibrahim hn. calcineurin inhibitor nephrotoxicity: a review and perspective of the evidence. am j nephrol. 2013;37:602–612. 12. gómez-oliván lm, galar-martínez m, garcía-medina s, valdés-alanís a, islas-flores h, neri-cruz n. genotoxic response and oxidative stress induced 144 j contemp med sci | vol. 5, no. 3, may–june 2019: 140–144 diclofenac-induced acute kidney injury is linked with oxidative stress original h.m. alkuraishy et al. by diclofenac, ibuprofen and naproxen in daphnia magna. drug chem toxicol. 2014;37:391–399. 13. al-kuraishy hm, al-gareeb ai, al-maiahy tj. concept and connotation of oxidative stress in preeclampsia. j lab physicians 2018;10:276–282. 14. al-kuraishy hm, al-gareeb ai. eustress and malondialdehyde (mda): role of panax ginseng: randomized placebo controlled study. iran j psychiatry 2017;12:194–200. 15. al-kuraishy hm, al-gareeb ai. potential effects of pomegranate on lipid peroxidation and pro-inflammatory changes in daunorubicin-induced cardiotoxicity in rats. int j prev med. 2016;7:85. 16. santos-alves e, marques-aleixo i, coxito p, balça mm, rizo-roca d, rocharodrigues s, et al. exercise mitigates diclofenac-induced liver mitochondrial dysfunction. eur j clin invest. 2014;44:668–677. 17. barcelos rp, bresciani g, cuevas mj, martínez-flórez s, soares faa, gonzález-gallego j. diclofenac pretreatment modulates exercise-induced inflammation in skeletal muscle of rats through the tlr4/nf-κb pathway. appl physiol nutr metab. 2017; 42:757–764. 18. li y, deng h, ju l, zhang x, zhang z, yang z, et al. screening and validation for plasma biomarkers of nephrotoxicity based on metabolomics in male rats. toxicol res (camb). 2015;5:259–267. 19. hosohata k, yoshioka d, tanaka a, ando h, fujimura a. early urinary biomarkers for renal tubular damage in spontaneously hypertensive rats on a high salt intake. hypertens res. 2016;39:19–26. 20. bazzi c, rizza v, olivieri g, casellato d, d’amico g. tubular reabsorption of high, middle and low molecular weight proteins according to the tubulo-interstitial damage marker n-acetyl-β-d-glucosaminidase in glomerulonephritis. j nephrol. 2015;28:541–548. 21. park ch, yokozawa t, noh js. oligonol, a low-molecular-weight polyphenol derived from lychee fruit, attenuates diabetes-induced renal damage through the advanced glycation end product-related pathway in db/db mice. j nutr. 2014;144:1150–1157. 22. liu sj, zhai yp, yu yp, liu hn, li f, song p, et al. [significance of low molecular weight urinary protein for assessment of early renal damage in patients with multiple myeloma]. zhongguo shi yan xue ye xue za zhi 2013;21: 410–414 [article in chinese]. 23. zhang j, zeng h, wang n, tian x, dou w, shi p. beneficial effects of creatine phosphate sodium for the treatment of henoch-schönlein purpura in patients with early renal damage detected using urinary kidney injury molecule-1 levels. eur j pediatr. 2016;175:49–55. 24. collier jb, schnellmann rg. extracellular signal-regulated kinase 1/2 regulates mouse kidney injury molecule-1 expression physiologically and following ischemic and septic renal injury. j pharmacol exp ther. 2017;363:419–427. 25. gauer s, urbschat a, gretz n, hoffmann sc, kränzlin b, geiger h, et al. kidney injury molecule-1 is specifically expressed in cystically-transformed proximal tubules of the pkd/mhm (cy/+) rat model of polycystic kidney disease. int j mol sci. 2016;17:e802. 26. kunutsor sk, flores-guerrero jl, kieneker lm, nilsen t, hidden c, sundrehagen e, et al. plasma neutrophil gelatinase-associated lipocalin and risk of cardiovascular disease: findings from the prevend prospective cohort study. clin chim acta. 2018;486:66–75. 27. eren z, günal my, arı e, çoban j, çakalağaoğlu f, çağlayan b, et al. pleiotropic and renoprotective effects of erythropoietin beta on experimental diabetic nephropathy model. nephron. 2016;132:292–300. 28. knight el, verhave jc, spiegelman d, hillege hl, de zeeuw d, curhan gc, et al. factors influencing serum cystatin c levels other than renal function and the impact on renal function measurement. kidney int. 2004;65: 1416–1421. 29. wei l, ye x, pei x, wu j, zhao w. reference intervals for serum cystatin c and factors influencing cystatin c levels other than renal function in the elderly. plos one 2014;9:e86066. 30. zhou x, ma b, lin z, qu z, huo y, wang j, et al. evaluation of the usefulness of novel biomarkers for drug-induced acute kidney injury in beagle dogs. toxicol appl pharmacol. 2014;280:30–35. 31. singh ap, junemann a, muthuraman a, jaggi as, singh n, grover k, et al. animal models of acute renal failure. pharmacol rep. 2012;64:31–44. 32. gacka e, życzkowski m, bogacki r, paradysz a, hyla-klekot l. the usefulness of determining neutrophil gelatinase associated lipocalin concentration excreted in the urine in the evaluation of cyclosporine a nephrotoxicity in children with nephrotic syndrome. dis markers 2016;2016:6872149. 33. alabi qk, akomolafe ro, adefisayo ma, olukiran os, nafiu ao, fasanya mk, et al. kolaviron attenuates diclofenac-induced nephrotoxicity in male wistar rats. appl physiol nutr metab. 2018;43:956–968. 34. mizuno t, ito k, miyagawa y, ishikawa k, suzuki y, mizuno m, et al. short-term administration of diclofenac sodium affects renal function after laparoscopic radical nephrectomy in elderly patients. jpn j clin oncol. 2012;42:1073–1078. 35. hung sc, kuo kl, peng ch, wu ch, wang yc, tarng dc. association of fluid retention with anemia and clinical outcomes among patients with chronic kidney disease. j am heart assoc. 2015;4:e001480. 36. hickey ej, raje rr, reid ve, gross sm, ray sd. diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic dna fragmentation and apoptotic cell death. free radic biol med. 2001;31:139–152. 37. bunel v, tournay y, baudoux t, de prez e, marchand m, mekinda z, et al. early detection of acute cisplatin nephrotoxicity: interest of urinary monitoring of proximal tubular biomarkers. clin kidney j. 2017;10:639–647. 38. harrill ah, lin h, tobacyk j, seely jc. mouse population-based evaluation of urinary protein and mirna biomarker performance associated with cisplatin renal injury. exp biol med (maywood). 2018;243(3):237–247. 39. lan z, bi ks, chen xh. ligustrazine attenuates elevated levels of indoxyl sulfate, kidney injury molecule-1 and clusterin in rats exposed to cadmium. food chem toxicol. 2014;63:62–68. 40. vaidya vs, ozer js, dieterle f, collings fb, ramirez v, troth s, et al. kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies. nat biotechnol. 2010;28: 478–485. 41. luo qh, chen ml, chen zl, huang c, cheng ac, fang j, et al. evaluation of kim-1 and ngal as early indicators for assessment of gentamycininduced nephrotoxicity in vivo and in vitro. kidney blood press res. 2016;41:911–918. 42. dönmez o, korkmaz ha, yıldız n, ediz b. comparison of serum cystatin c and creatinine levels in determining glomerular filtration rate in children with stage i to iii chronic renal disease. ren fail. 2015;37:784–790. 43. harman g, akbari a, hiremath s, white ca, ramsay t, kokolo mb, et al. accuracy of cystatin c-based estimates of glomerular filtration rate in kidney transplant recipients: a systematic review. nephrol dial transplant. 2013;28:741–757. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201904 9j contemp med sci | vol. 2, no. 5, winter 2016: 9–14 research background infertility is the inability of a person to reproduce by natural means. infertility may describe a woman who is unable to get conceived as well as being unable to carry a pregnancy to full term. there are many biological and other causes of infertility, including some of the medical interventions can treat. infertility rates have increased by 4% since the 1980s, mostly from problems with fecundity due to an increase in age. about 40% of the issues involved with infertility are due to the man, another 40% due to the woman, and 20% results from complications with both the partners. in vitro fertilisation and embryo transfer (ivf–et) was first successfully used in humans over 25 years ago; since then, more than one million children have been conceived using this technology. ivf is a procedure designed to enhance the likelihood of conception in couples for whom other fertility therapies have been unsuccessful or are not possible. it is a complex process and involves multiple steps resulting in the insemination and fertilisation of oocytes (eggs) in a laboratory. the embryos created in this process are then placed into the uterus for potential implantation. each stage of the procedure is associated with specific risks; ivf may provide a couple who has been otherwise unable to conceive with a chance to establish a pregnancy2. objective 1. to assess women’s commitment to implementation of ivf 2. to identify short protocol implementation 3. to identify long protocol implementation methodology non-probability (purposive sample), the study sample consists of (60) infertile women who were selected from kamal al-samaraee hospital. the study group consist of (30) infertile women was exposed to follow-up and (30) women is control group the criteria of this sample was infertile women in reproductive age, with different educational levels in the public department were involved in ivf program. results in the study group, 20 women were using short protocol and 10 were using long protocol. the total number of study group was 30, 9 of them become pregnant, 5 of them were using short protocol and 4 of them were using long protocol. in the control group, there were 20 women using short protocol and 10 using long protocol. the total number of study group was 30; 2 of them become pregnant and were using short protocol. conclusions results shows that with respect to study sample, observed significant relationships should be informative and significant level was not achieved. long protocol are much better than short protocol. in addition to that, results shows that with respect to control sample, no significant relationships are accounted, as well as two types of protocol either long or short gives the same responding. results shows that significant relationships are accounted and that the study sample recorded six times better than control. recommendations the study recommended that all the infertile women should be exposed to the implementation of the follow-up and call the patients by phone and through the interview with patients and instruct them about their protocols. keywords infertility, infertile women, ivf, short protocol, long protocol effectiveness of infertile women’s commitment to implement in vitro fertilization (long/short) protocols hawraa hussein ghafela, rabe’a mohsen alia, alaa hazeem al-rahawib issn 2413-0516 adepartment of maternal and neonatal nursing, college of nursing, university of baghdad, baghdad, iraq. bdepartment of obstetrics and gynecology, kamal al-samaraee hospital, ministry of health, baghdad, iraq. correspondence to hawraa hussein (email: hawraa_2004@yahoo.com). (submitted: 16 november 2015 – revised version received: 27 december 2015 – accepted: 23 january 2016 – published online: 26 march 2016) introduction infertility, defined as the inability to become pregnant after 1 year of unprotected sex, is a problem faced by nearly 6.1 million americans that is nearly 10% of men and women of reproductive age. because, this problem is so prevalent, fertility treatments abound. assisted reproductive technology (art) is a group of fertility treatments that involve both the sperm and the egg. in vitro fertilisation (ivf) is the most common type of art. in ivf, the sperm fertilises the egg outside the body, and doctors implant it into the woman’s uterus with hopes of a successful pregnancy. other forms of art include intracytoplasmic sperm injection (icsi), gamete intrafallopian transfer (gift) and zygote intrafallopian transfer (zift). the history of ivf is relatively short. louise brown of england was the first baby born via ivf, in 1978. the next ivf baby was born later that same year in india. soon, people started calling these infants as ‘test-tube babies’ which means fertilisation outside of the body. in 1981, the first american test-tube baby was born, and the number has continued to increase each year3. ivf is the most effective art. it is often used when a woman’s fallopian tubes are blocked or when a man produces very few sperm. doctors treat the woman with a drug that causes the ovaries to produce multiple eggs. once matured, the eggs are removed from the woman. they are put in a dish in the lab along with the man’s sperm for fertilisation. after 3–5 days, healthy embryos are implanted into the woman’s uterus (american college of obstetricians and gynecologists4. the best known of these methods is ivf, which was responsible for the birth of many ‘test-tube babies’. the eggs obtained through the vaginal canal using ultrasound guidance are fertilised with the partner’s sperm outside the woman’s body, not in a test tube, but in a culture dish. a few days later, the eggs, now called zygotes, are placed in the woman’s uterus. mailto:hawraa_2004@yahoo.com 10 j contemp med sci | vol. 2, no. 5, winter 2016: 9–14 effectiveness of infertile women’s commitment to implement ivf protocols research hawraa hussein ghafel et al. if one or more of the zygotes implants successfully, pregnancy results. one study reported that 17% of women who undergone this procedure once, go on to deliver a baby5, in order to maximise success rates with ivf. there are several ovarian stimulation medication protocols that are used to stimulate the ovaries to make enough follicles and eggs. without stimulating medications, the ovaries make and release only one matured egg per menstrual cycle in a month6. ivf is a complex series of procedures used to treat fertility7. ivf is a procedure, used to overcome a range of fertility issues, by which an egg and sperm are joined together outside the body8. in a natural menstrual cycle, the pituitary gland’s luteinising hormone (lh) and follicle-stimulating hormone (fsh) causes the growth of an egg in a fluid-filled follicle within the ovary9. the drugs used for ovarian stimulation have mild side effects in some women, including mild bruising and soreness at the injection site, headaches, an upset stomach and mood swings10. ivf increases the risk of multiple births if more than one embryo is implanted in the uterus11. a pap smear for every 2 years, as well as regular gynaecological and breast examinations are currently the best methods to prevent or detect women’s cancers. use of injectable fertility drugs, such as human chorionic gonadotropin (hcg), to induce ovulation can cause ovarian hyperstimulation syndrome12. the age of the woman has a significant effect on her fertility and the live birth rate decreases significantly from the age 35 years when the woman is using her own eggs13. female body mass index should ideally be in the range of 19–30 before commencing ivf treatment. female body mass index outside this range is likely to reduce the success of assisted reproduction procedures14. approximately 15 years ago, a different type of gnrh analog was developed, called gnrh ‘antagonists’, these medications work differently than the gnrh ‘agonists’ like lupron15. methodology the follow-up is made through the phone and interview with patients when they come to hospital. the instructions given to the women during the follow-up includes: information about ivf procedure, teaching the women about the importance of the commitment in the time of taking their medications such as injections, some of the injection are taken in the morning and the other in the evening, in certain time according to the doctor’s instruction, teaching the women about the side effects of medications, teaching the women about the correct site of injection, because some of the injection should be injected in intramuscular and other is injected subcutaneously. the best way to save the drugs without causing damage during transport from the hospital, because these medications consist of hormone that gets damaged in the hot and in the cold. the woman should keep it in the door of a refrigerator (information about the importance of commitment in a time of injection). information about the complications of ivf may occur. rather than slowly suppress the pituitary over 4–5 days like lupron does, these newer medications, cetrotide and ganirelix acetate—rapidly suppress the pituitary in a matter of hours. gnrh antagonist protocols have several advantages over gnrh agonists protocols. the number of daily injections is fewer (4–5 days) of antagonists versus (3–4 weeks) of agonists and the length of time to stimulate the follicles to maturity is 1 or 2 days shorter, so you may need less total gonadotropins. in addition, your chance of developing ovarian hyperstimulation syndrome is less with gnrh antagonist protocols. multiple research studies have compared ivf agonist and antagonist protocols which mostly showing similar pregnancy rates. results and findings part 1: distribution of socio-demographical characteristics variables table 1 shows distribution of studied groups (with and without) follow-up of ivf (long/short) protocol, in light of ‘socio-demographical characteristics’ variables (sdcv.), as well as comparisons significant are obtained to be sure that two independent groups are thrown from the same population concerning of that variables. results shows that no significant differences at p > 0.05 are accounted between studied groups, which indicating that two independent groups are thrown from the same population in light of sdcv. and that are more reliable for this study, since any meaningful differences may be registered among the studied outcomes, should be interpreted by effectiveness of applying (long/short) ivf protocols in light of follow-up or not. part 2: distributions of reproductive status table 2 shows distribution of observed frequencies and percentages of reproductive status, as well as relationships among studied groups with comparisons significant, which shows that two independent groups are thrown from the same population in light of (reproductive status), and that are more reliable for this study, since any meaningful differences may be registered among final outcomes, should be interpreted by effectiveness of applying (long/short) ivf protocols in light of follow-up or not. part 3: distributions of effectiveness (long/short) ivf protocols in light of follow-up or not: relationship among studied groups (with and without follow-up) protocol and final results of program either success or failure program and contingency coefficients are constructed in table 3 within comparisons significant, as well as an odds ratio and cohort of failure results among (long /short) protocol. results shows that concerning with follow-up group, observed significant relationships should be informative rather than simply stating that statistical significant level was not achieved. in addition to that, long protocol with follow-up stating had four times of success outcomes better than short protocol, as well as cohort to failure outcomes are accounted half effectiveness concerning with follow-up protocol, compared without follow-up group. in addition to that, results shows that concerning without follow-up group, no significant relationships are accounted at p > 0.05, as well as two types of protocol either long or short gives the same responding statistically. figure 1 represents graphically the distribution of studied groups (with and without) ivf protocol. discussion of the results table 1 shows observed frequencies and percentages of the studied sdcv. which are distributed according to studied 11j contemp med sci | vol. 2, no. 5, winter 2016: 9–14 research effectiveness of infertile women’s commitment to implement ivf protocolshawraa hussein ghafel et al. table 1. distribution studied groups according to socio-demographical characteristics variables (sdcv.) sdcv. classes (with follow-up) (without follow-up) c.s. (*) [p-value]no. % no. % age of wife (years) <20 1 3.3 0 0 c.c. = 0.294 p = 0.339 (ns) 20− 5 16.7 7 23.3 25− 7 23.3 9 30.0 30− 8 26.7 8 26.7 35− 5 16.7 6 20.0 40–50 4 13.3 0 0.0 mean ± sd 30.5 ± 6.61 28.83 ± 5.82 age of husband (years) 20− 0 0 2 6.7 c.c. = 0.261 p = 0.497 (ns) 25− 5 16.7 5 16.7 30− 6 20 8 26.7 35− 6 20 8 26.7 40− 11 36.7 6 20 45–50 2 6.7 1 3.3 mean ± sd 36.33 ± 6.37 34.2 ± 6.35 rh : wife pos. 28 93.3 29 96.7 c.c. = 0.076 p = 0.554 (ns)neg. 2 6.7 1 3.3 rh : husband pos. 30 100 29 96.7 c.c. = 0.129 p = 0.313 (ns)pos. 0 0 1 3.3 consanguinity relative 16 53.3 19 63.3 c.c. = 0.101 p = 0.432 (ns)not relative 14 46.7 11 36.7 education: wife illiterate 1 3.3 3 10.0 c.c. = 0.179 p = 0.575 (ns) graduate of primary 11 36.7 11 36.7 graduate of secondary 12 40.0 9 30.0 higher education 6 20.0 7 23.3 education: husband illiterate 1 3.3 1 3.3 c.c. = 0.156 p = 0.681 (ns) graduate of primary 8 26.7 11 36.7 graduate of secondary 9 30 11 36.7 higher education 12 40 7 23.3 occup. wife employer 24 80 26 87.7 c.c. = 0.089 p = 0.488 (ns)house wife 6 20 4 12.3 occup. husband employer 15 50 18 60 c.c. = 0.100 p = 0.436 (ns)free job 15 50 12 40 marriage-wife married before 2 6.7 1 3.3 c.c. = 0.201 p = 0.284 (ns)first wife 26 86.7 23 76.7 second wife 2 6.7 6 20.0 marriage husband married before 2 6.7 3 10 c.c. = 0.193 p = 0.313 (ns)not married before 27 90 23 76.7 polygamous 1 3.3 4 13.3 (*)ns: non sig. at p ≥ 0.05; c.c.: contingency coefficient. samples (with and without follow-up), as well as comparisons significant for relationships. results shows that no significant differences at p > 0.05 are accounted between the two samples, and that are more reliable for this study, since any meaningful deviation may registered between the studied samples should be interpreted for effectiveness of applying studied follow-up relative to subject’s ‘age groups’; majority of the studied samples are reported at the age ranged (25–29) years for wife, and (40–44) years for husband, then followed with subject’s ‘rh’, results indicated that most of the studied individuals had a positive results, and they are accounted in light of with and without follow-up 18 (93.3%) and 29 (96.7%) for wife, as well as 30 (100%) and 29 (96.7%) for husband, then followed with subjects of ‘consanguinity status’, results indicated that sample 12 j contemp med sci | vol. 2, no. 5, winter 2016: 9–14 effectiveness of infertile women’s commitment to implement ivf protocols research hawraa hussein ghafel et al. table 2. distributions of reproductive status at the studied samples with comparisons significant reproductive status resp. (with follow-up) (without follow-up) c.s. (*) [p-value]no. % no. % previous pregnancy yes 12 40 8 26.7 c.c. = 0.140 p = 0.273 (ns)no 18 60 22 73.3 previous ectopic pregnancy yes 2 6.7 2 6.7 c.c. = 0.000 p = 1.000 (ns)no 28 93.3 28 93.3 previous abortion yes 5 16.7 6 20 c.c. = 0.043 p = 0.739 (ns)no 25 83.3 24 80 previous birth of deformed baby yes 0 0 0 0 c.c. = 0.000 p = 1.000 (ns)no 30 100 30 100 previous delivery yes 8 26.7 4 13.3 c.c. = 0.164 p = 0.197 (ns)no 22 73.3 26 86.7 puerperal fever ( in a secondary infertility) yes 0 0 0 0 c.c. = 0.000 p = 1.000 (ns)no 30 100 30 100 fallopian tube obstruction one tub 2 6.7 2 6.7 c.c. = 0.133 p = 0.584 (ns)both of them 1 3.3 3 10 opened tubes 27 90 25 83.3 pituitary gland disorders yes 5 16.7 3 10 c.c. = 0.098 p = 0.448 (ns)no 25 83.3 27 90 elevated of prolactine hormone yes 11 36.7 13 43.3 c.c. = 0.068 p = 0.598 (ns)no 10 63.3 17 56.7 duration of infertility (years) <5 years 5 16.7 5 16.7 c.c. = 0.181 p = 0.566 (ns) 5–9 15 50 13 43.3 10–14 7 23.3 11 36.7 15–19 3 10 1 3.3 type of infertility primary 23 76.7 27 90 c.c. = 0.176 p = 0.166 (ns)secondary 7 23.3 3 10 the causes of infertility related to your husband yes 21 70 23 76.7 c.c. = 0.075 p = 0.559 (ns)no 9 30 7 23.3 (*)ns: non sig. at p > 0.05; c.c.: contingency coefficient. of with follow-up are accounted 16 (53.3%), while without follow-up sample are accounted 19 (63.3%), then followed with subject’s ‘level of education’, results shows that more of 50% of studied sample of ‘wife’ had graduated secondary school and higher educated and they are accounted for 18 (60.0%) and 17 (53.3%), as well as sample of ‘husband’ are accounted 21 (70.0%) and 18 (60.0%), then followed with subject’s ‘occupation’, results shows that most of the studied samples in light of ‘wife’ had recorded employed, and they are accounted 24 (80%) and 26 (87.7%), as well as sample of ‘husband’ had recorded employed, and accounted in light of with and without follow-up 15 (50.0%) and 18 (60.0%) respectively, and the leftover had free job. marriage status for wife had recorded mostly first wife, and accounted in light of with and without follow-up 26 (86.7%) and 23 (76.7%) respectively, then finally followed with subject’s ‘marriage status’ for husband had recorded mostly not married before, and accounted in light of with and without follow-up 27 (90%) and 23 (76.7%) respectively. table 2 shows distribution of the observed frequencies and percentages of reproductive status as well as relationships among studied samples with comparisons significant, and as follows: a. regarding to subjects ‘previous pregnancy’, results indicated that there has been no significant different at p > 0.05 between studied samples, with 8 (26.7%) at the control sample, while 12 (40%) individuals are accounted at the study sample. b. regarding to subjects ‘previous ectopic pregnancy’, results indicated that there has been no significant different at p > 0.05 accounted between studied samples, with 2 (6.7%) individuals are accounted at the control and study samples. c. regarding to subjects ‘previous abortion’, results indicated that there has been no significant different at p > 0.05 accounted between studied samples, with 6 (20%) at the control sample, while 5 (16.7%) individuals are accounted at the study sample. d. regarding to subjects ‘previous birth of deformed baby’, results indicated that there has been no individuals are accounted at the study and control samples. d id y ou h av e or s uff er in g fro m : 13j contemp med sci | vol. 2, no. 5, winter 2016: 9–14 research effectiveness of infertile women’s commitment to implement ivf protocolshawraa hussein ghafel et al. table 3. distribution of final outcomes results of program and protocol types among (with and without follow-up) of ivf protocol groups with comparisons significant groups protocol types no. and percent the final results of program total c.s. (*) [p-value]success failure (w ith fo llo w -u p) long protocol no. 6 5 11 c.c. = 0.295 p = 0.091 (ns) odds ratio (1 : 4.0) cohort: (failure) (1 : 0.54) % type of protocol 54.5% 45.5% 100% % type of result 66.7% 23.8% 36.7% short protocol no. 3 16 19 % type of protocol 15.8% 84.2% 100% % type of result 33.3% 76.2% 63.3% total no. 9 21 30 % type of protocol 30.0% 70.0% 100% % type of result 100% 100% 100% (w ith ou t f ol lo w -u p) long protocol no. 0 10 10 c.c. = 0.186 p = 0.301 (ns) cohort: (failure) (1 : 1.11) % type of protocol 0.0% 100% 100% % type of result 0.0% 35.7% 33.3% short protocol no. 2 18 20 % type of protocol 10% 90% 100% % type of result 100% 64.3% 66.7% total no. 2 28 30 % type of protocol 6.7% 93.3% 100% % type of result 100% 100% 100% (*)ns: non sig. at p > 0.05; c.c.: contingency coefficient. fig. 1 cluster bar charts of final results of program and protocol types at each sample (with and without follow-up) of ivf protocol groups e. regarding to subjects ‘previous delivery’, results indicated that there has been no significant different at p > 0.05 accounted between studied samples, with 4 (13.3%) at the control sample, while 8 (26.7%) individuals are accounted at the study sample. relationship among studied samples (with and without follow-up) and final results of program either success of program (pregnancy occur) or failure, contingency coefficients are constructed in table 3 within comparisons significant, as well as an odds ratio. results shows that concerning with study sample observed, significant relationships should be informative rather than simply significant level, was not achieved as well as long protocol four times concerning study sample are better than short protocol. in addition to that, results shows that concerning with control sample, no significant relationships are accounted at p > 0.05, as well as two types of protocol either long or short gives the same responding. how p-value to be reported: ‘if p-value is found as 0.07, it is more informative for that result to be reported, rather than simply stating that statistical significant was not achieved’. 14 j contemp med sci | vol. 2, no. 5, winter 2016: 9–14 effectiveness of infertile women’s commitment to implement ivf protocols research hawraa hussein ghafel et al. recommendations 1. the follow-up is very important to implement the commitment of ivf protocols through phone and interview with the patient when they come to hospital. 2. the follow-up is to be very effective and guide some of wrong practices of the patients to save the medications by references 1. wikipedia: in vitro fertilization, 2015. available at https://en.wikipedia.org/ wiki/in_vitro_fertilisation 2. georgia reproductive specialists: overview for ivf patients. atlanta; 2007. available at http://www.ivf.com/overview.html 3. jeffries m. in vitro fertilization overview. howstuffworks, 1998–2015. available at http://health.howstuffworks.com/pregnancy-and-parenting/ pregnancy/fertility/in-vitro-fertilization.htm 4. acog (american congress of obstetricians and gynecologists): transferring single prescreened embryo in ivf offers excellent delivery rates. new orleans; 2013. available at http://www.acog.org/about-acog/news-room/ news-releases/2013/transferring-single-prescreened-embryo-in-ivfoffers-excellent-delivery-rates 5. harvard health publications: art, 2010. available at http://www.health. harvard.edu/ 6. advanced fertility center of chicago: ovarian stimulation ivf protocols medications and drugs for in vitro fertilization, advanced fertility center of chicago, s.c; 1996–2015. available at http://www.advancedfertility.com/ ivfstim.htm 7. mayo clinic: in vitro fertilization (ivf), usa; 1998–2015. available at http://www.mayoclinic.org/tests-procedures/in-vitro-fertilization/basics/ definition/prc-20018905 keeping it in the freezer rather than keeping outside the refrigerator which makes the medications exposure to the heat and causes damages. 3. the nurse should instruct the patients about protocols that are used for her. 8. ivf australia, ivf treatment: virtus health, australia; 2015. available at http://ivf.com.au/fertility-treatment/ivf-treatment 9. city fertility center: ivf services, ivf: step-by-step guide, australia; 2013. available at http://www.cityfertility.com.au/fertility-services/ivftreatment/ 10. winfertility: risks of ivf: six rare complications of ivf treatment, usa; 2015. available at https://www.winfertility.com/risks-ivf-six-rare-complicationsivf-treatment/ 11. mayo clinic: in vitro fertilization (ivf). mayo foundation for medical education and research, usa; 1998–2015. available at http://www.mayoclinic.org/testsprocedures/in-vitro-fertilization/basics/definition/prc-20018905 12. coastal ivf, level 4, esplanade and second avenue, maroochydore: sunshine coast, qld, australia; 2015. available at http://www.coastalivf. com.au/risks-21.htm 13. ivf-infertility.com: ivf success, by dr. marcus, 2014. pp. 1–3. available at http://www.ivf-infertility.com/ivf/standard/factors/couples.php 14. ivf-infertility.com: ivf success, by dr. marcus, 2014. pp. 5–7. available at http://www.ivf-infertility.com/ivf/standard/factors/couples.php 15. texas fertility center: ivf stimulation protocols, 2015 texas fertility center. available at http://txfertility.com/fertility-treatments/ivf-stimulationprotocols/ http://www.ivf.com/overview.html http://health.howstuffworks.com/pregnancy-and-parenting/pregnancy/fertility/in-vitro-fertilization.htm http://health.howstuffworks.com/pregnancy-and-parenting/pregnancy/fertility/in-vitro-fertilization.htm http://www.acog.org/about-acog/news-room/news-releases/2013/transferring-single-prescreened-embryo-in-ivf-offers-excellent-delivery-rates http://www.acog.org/about-acog/news-room/news-releases/2013/transferring-single-prescreened-embryo-in-ivf-offers-excellent-delivery-rates http://www.acog.org/about-acog/news-room/news-releases/2013/transferring-single-prescreened-embryo-in-ivf-offers-excellent-delivery-rates http://www.advancedfertility.com/ivfstim.htm http://www.advancedfertility.com/ivfstim.htm http://www.mayoclinic.org/tests-procedures/in-vitro-fertilization/basics/definition/prc-20018905 http://www.mayoclinic.org/tests-procedures/in-vitro-fertilization/basics/definition/prc-20018905 http://ivf.com.au/fertility-treatment/ivf-treatment http://www.cityfertility.com.au/fertility-services/ivf-treatment/ http://www.cityfertility.com.au/fertility-services/ivf-treatment/ https://www.winfertility.com/risks-ivf-six-rare-complications-ivf-treatment/ https://www.winfertility.com/risks-ivf-six-rare-complications-ivf-treatment/ 227j contemp med sci | vol. 4, no. 4, july-august 2019: 227–233 effects of the systemic administration of omega-3 polyunsaturated fatty acid on experimental periodontitis aveen ageel jalal,1 zewar a. al-qassab,1 and rafel a. al-rawi2 1college of dentistry, hawler medical university, erbil, kurdistan region, iraq. 2college of pharmacology, hawler medical university, erbil, kurdistan region, iraq. correspondence to aveen ageel jalal (email: aveenyawar445@gmail.com). (submitted: 02 april 2019 – revised version received: 18 april 2019 – accepted: 19 may 2019 – published online: 26 august 2019) objective the present study was aimed to investigate and evaluate the effects of eicosapentaenoic acid (epa) on ligature induced periodontitis in rats through the biochemical analysis for rat’s serum level of crp, alkaline phosphatase (alp) and mda. methods periodontitis was induced for the studied animals by ligation around the upper central incisor. twenty five animals were used as a control and gavaged by water only, and 100 animals with the induced periodontitis were divided into four equal groups according to the treatment used which was started at time of ligation removal: water (p/w), scaling/root planing (p/srp), 60 mg/kg epa (p/epa), and srp together with epa (p/srp + epa). blood was taken by cardiac puncture, after 3, 24 h, 3 days, 1 week and 2 weeks for biochemical analysis. results the p/srp + epa treatment group showed significant decrease (p < 0.05) in serum crp, significant increase in serum alp, significant decrease in serum mda after 1 week, 3 days, and 3 h respectively in comparison with the p/w treatment group. conclusion the treatment by srp with 60 mg/kg epa is better than each one alone and can be used for treatment of periodontitis. keywords periodontitis, eicosapentaenoic acid, alveolar bone resorption, omega-3 introduction chronic periodontitis is an inflammatory disease affecting the supporting tissues of teeth, and its expression results from the interaction of host defense mechanisms, microbial agents, environmental, and genetic factors.1 a biomarker is a substance used to indicate a biologic state and is an objective measure to evaluate the disease activity and considered as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.2 several biomarkers are associated with periodontal disease like the systemic inflammatory marker such as crp, bone markers such as alkaline phosphatase (alp), and oxidative stress markers such as mda.3–5 ide et al.6 found that crp has been associated with the presence of various bacterial infections, including periodontitis. perumal et al.5 found that there was a decrease in serum bone-specific alp level in severe periodontitis patients as compared with moderate chronic periodontitis group level. other study found that patients with periodontitis had higher values of mda whereas healthy patients had lower levels of mda which is commonly known as a marker of oxidative stress.7 polyunsaturated fatty acids are fatty acids with more than one carbon–carbon double bond. omega-3 are the bioactive lipids which consist of three major omega-3 fatty acids, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) from marine sources, and alpha-linolenic acid (ala) which is from plant sources.8 periodontal disease affects almost 90% of the population,9 and the incidence increase with the growing ageing population.10,11 the most common periodontal treatment is still mechanical removal of plaque and calculus deposit and local antibiotic application. thus, a therapy of natural origin, if effective, might be safer method for treatment of periodontitis. for this reason, the present study was aimed to investigate and evaluate the effects of 60 mg/kg epa12 on ligature-induced periodontitis in rats through the biochemical analysis for serum crp, alp, and mda. materials and methods rats and housing all the wister-albino rats used in the study was aged about 8–10 weeks, weighing 200–300 g and cared in the animal house of college of medicine, hawler medical university, erbil, iraq. they were allowed to adapt to the housing conditions for 1 week prior to the commencement of the study. five rats were housed in each wire cage and maintained on a 12-h light/dark cycle at 20 ± °5c and 20–30% humidity. the animals were kept in standard room conditions and fed with a standard rat chow and allowed to drink water ad libitum. the research project was approved by the research ethics committee at college of dentistry, hawler medical university under protocol. induction of experimental periodontitis the upper incisor was chosen because the induced periodontal disease occurs more rapidly in that location due to the porosity of the spongy bone in the maxilla. the rats were anesthetized by intraperitoneal administration of ketamine (0.5 ml/kg b.w.) and the animals were placed on a proper operating table, which allowed open-mouth maintenance of the rats to facilitate access to the teeth. after that a 3.0 sterile black braided silk threads was placed around the cervix of maxillary right incisor for each animal and kept for 2 weeks. the ligatures are knotted on the labial side of the tooth, resulting in subgingival positioning on the palatal side and supragingival position on the labial side. daily we perform ligatures control and checking, and if any had been lost or become loose, it was replaced. this ligature acts as a gingival irritant for 14 days and promoted the accumulation of plaque and subsequently development of periodontal disease.13 experimental design for biochemical analysis one hundred and twenty five animals were used in the study. twenty five animals (normal control), and 100 animals with the induced periodontitis were randomly assigned into four issn 2413-0516 original 228 j contemp med sci | vol. 4, no. 4, july-august 2019: 227–233 effects of the systemic administration of omega-3 polyunsaturated a.a. jalal et al. experimental groups (25 animals each) according to the treatment used. nc/w normal control + distilled water treatment group. p/w ligature-induced periodontitis + distilled water treatment group. p/srp ligature-induced periodontitis + scaling and root planing treatment group. the right incisors were subjected to srp with manual #1–2 mini-five curettes through 10 distal–mesial traction movements in the labial and palatal aspects. the interproximal areas were scaled with the same curettes using cervico-incisal traction movements.14 p/epa ligature-induced periodontitis + 60 mg /kg epa treatment group. p/srp+epa ligature-induced periodontitis + srp + epa treatment group. the ligatures were removed at day 14 (day 0) and the different types of treatment was started directly. the treatment by intragastric gavage of distilled water or epa were done daily once for 2 weeks. blood sampling for biochemical assays blood was taken by cardiac puncture, 3 h after ligation removal (day 0), 24 h, 3 days, 1 week and 2 weeks for immunological analysis. first by anesthetizing the rat, placed on its back, the left index finger was placed at the level of the lowest ribs without applying any pressure, and the heart is located 1 cm above this point, slightly to the right.15 the 5 ml syringe was hold at a 45° angle and the needle was inserted between two ribs, the plunger was pulled on slowly to fill the syringe. then the samples were placed in non-heparinized tubes, allowed to clot for 2 h at room temperature and centrifuged at 3000 rpm for 10 min at 4°c, and then the supernatant was collected. the serum was frozen at −20°c and used within 1 month to avoid loss of bioactivity and contamination. chemiluminescence immunoassay detection kit was used for determination of high sensitivity crp (23961, diagnostic automation inc., calabasas, ca, usa), colorimetric determination was used of the alp (02160, biolabo, france), and enzyme-linked immunosorbent assay kit was used for mda determination (mbs764641, usa). statistical analysis data were analyzed using spss software version 23, and were summarized using means and standard deviations. statistical analysis with one-way analysis of variance was performed to compare the differences in the means among groups, and when it revealed that there was a statistically significant difference; mann–whitney u-test was performed to assess individual pair of groups for statistically significant finding. p-value ≤0.05 was considered statistically significant. results serum crp the result showed a significant increase (p < 0.05) in serum crp in p/w, p/srp, p/epa, and p/srp + epa treatment groups in the first 3 days in comparison with the nc/w treatment group. the p/epa and p/srp + epa treatment groups showed a non-significant difference in serum crp level from that of the nc/w after 7 days treatment, but not lower level to that of the healthy control group. the p/srp treatment group showed a non-significant difference from that of nc/w treatment group after 2 weeks (table 1). statistical analysis showed non-significant differences (p > 0.05) in serum crp present between all treatment groups in the first 3 days. after 1 week the p/srp + epa treatment group showed significant decrease (p < 0.05) in serum crp in comparison with the p/w, p/srp, and p/epa treatment groups. after 2 weeks a non-significant differences (p > 0.05) were seen between p/srp and p/epa, p/srp and p/srp + epa, p/ epa and p/srp + epa treatment groups (table 2). serum alp the p/w treatment group showed a significant decrease (p < 0.05) in serum alp in comparison with the nc/w. statistical analysis showed a non-significant differences (p > 0.05) present between p/epa and p/srp + epa treatment groups in relation with the nc/w treatment groups only after 1 week treatment, but it was still less than the nc/w treatment group. a significant difference (p < 0.05) was seen between all groups in all other durations studied (table 3). table 1. serum crp (mean ± standard deviation) in normal control/water treatment group in relation with all studied groups groups crp (ng/ml) 3 h p-value 24 h p-value 3 days p-value 1 week p-value 2 weeks p-value nc/w 11.81 ± 1.186 0.012 11.61 ± 1.328 0.012 11.41 ± 1.104 0.012 11.61 ± 1.843 0.012 11.81 ± 1.177 0.012 p/w 14.61 ± 0.354 14.81 ± 1.18 14.56 ± 1.876 13.954 ± 1.45 13.093 ± 0.963 nc/w 11.81 ± 1.186 0.012 11.61 ± 1.32 0.012 11.41 ± 1.104 0.012 11.61 ± 1.843 0.012 11.81 ± 1.177 0.731 p/srp 15.22 ± 0.357 15.32 ± 0.451 15.06 ± 1.96 13.569 ± 1.540 12.03 ± 0.75 nc/w 11.81 ± 1.186 0.012 11.61 ± 1.328 0.012 11.41 ± 1.104 0.012 11.61 ± 1.843 0.144 11.81 ± 1.177 0.756 p/epa 14.45 ± 0.344 14.075 ± 0.256 13.99 ± 1.83 12.94 ± 1.275 12.312 ± 0.785 nc/w 11.81 ± 1.186 0.012 11.61 ± 1.328 0.012 11.41 ± 1.104 0.012 11.61 ± 1.843 0.173 11.81 ± 1.177 0.347 p/srp + epa 14.057 ± 1.467 14.99 ± 1.985 13.89 ± 0.34 12.01 ± 0.834 12.01 ± 1.157 original a.a. jalal et al. 229j contemp med sci | vol. 4, no. 4, july-august 2019: 227–233 effects of the systemic administration of omega-3 polyunsaturated table 2 the relation of serum crp (mean ± standard deviation) between different studied treatments groups groups crp (ng/ml) 3 h p-value 24 h p-value 3 days p-value 1 week p-value 2 weeks p-value p/w 14.61 ± 0.354 0.92 14.81 ± 1.1815 0.731 14.56 ± 1.876 0.920 13.954 ± 1.45 0.833 13.093 ± 0.963 0.012 p/srp 15.22 ± 0.357 15.32 ± 0.451 15.06 ± 1.96 13.569 ± 1.540 12.03 ± 0.75 p/w 14.61 ± 0.354 1.00 14.81 ± 1.18 0.400 14.56 ± 1.876 0.828 13.954 ± 1.45 0.250 13.093 ± 0.9631 0.012 p/epa 14.45 ± 0.344 14.075 ± 0.256 13.99 ± 1.83 12.94 ± 1.275 12.312 ± 0.785 p/w 14.61 ± 0.354 1.00 14.81 ± 1.18 0.417 14.56 ± 1.876 0.528 13.954 ± 1.45 0.012 13.093 ± 0.963 0.012 p/srp + epa 14.057 ± 1.467 14.99 ± 1.985 13.89 ± 0.34 12.01 ± 0.834 12.01 ± 1.157 p/srp 15.22 ± 0.35711 0.828 15.32 ± 0.451 0.920 15.06 ± 1.96 0.674 13.569 ± 1.540 0.144 12.03 ± 0.75 0.920 p/epa 14.45 ± 0.34 14.075 ± 0.256 13.99 ± 1.83 12.94 ± 1.275 12.312 ± 0.785 p/srp 15.22 ± 0.357 0.400 15.32 ± 0.451 1.00 15.06 ± 1.96 0.313 13.569 ± 1.540 0.012 12.03 ± 0.75 1.00 p/srp + epa 14.057 ± 1.467 14.99 ± 1.985 13.89 ± 0.34 12.01 ± 0.834 12.01 ± 1.157 p/epa 14.45 ± 0.344 1.00 14.075 ± 0.256 0.920 13.99 ± 1.83 1.00 12.94 ± 1.275 0.012 12.312 ± 0.785 1.00 p/sra + epa 14.057 ± 1.467 14.99 ± 1.985 13.89 ± 0.34 12.01 ± 0.834 12.01 ± 1.157 table 3. serum alp (mean ± standard deviation) in normal control/water treatment group in relation with all studied groups groups alp (u/l) 3 h p-value 24 h p-value 3 days p-value 1 week p-value 2 weeks p-value nc/w 131.07 ± 2.595 0.012 131.27 ± 2.72 0.012 129.87 ± 3.476 0.012 131.47 ± 4.193 0.012 132.27 ± 4.543 0.021 p/w 114 ± 4.149 114.87 ± 3.827 116.076 ± 4.027 116.9 ± 0.761 117.5 ± 1.014 nc/w 131.07 ± 2.595 0.012 131.27 ± 2.72 0.008 129.87 ± 3.476 0.012 131.47 ± 4.193 0.012 132.27 ± 4.543 0.012 p/srp 114.47 ± 4.009 113.73 ± 1.896 115.476 ± 3.26 114.4 ± 2.80 118.276 ± 4.246 nc/w 131.07 ± 2.595 0.012 131.27 ± 2.72 0.012 129.87 ± 3.476 0.012 131.47 ± 4.193 0.060 132.27 ± 4.543 0.094 p/epa 115.876 ± 4.19 116.45 ± 2.34 120.7 ± 1.256 123.9 ± 2.546 124.5 ± 1.526 nc/w 131.07 ± 2.595 0.012 131.27 ± 2.72 0.021 129.87 ± 3.476 0.021 131.47 ± 4.193 0.060 132.27 ± 4.543 0.060 p/srp + epa 118.5 ± 4.402 120.3 ± 3.389 122.7 ± 1.526 124.1 ± 1.797 124.9 ± 1.968 the p/epa and p/srp + epa treatment groups showed significant increase (p < 0.05) in serum alp after 3 days in comparison with the p/w treatment group. the p/epa and p/ srp + epa treatment groups showed significant increase (p < 0.05) in serum alp after 3 days treatment in comparison with the p/srp treatment group. the p/srp + epa treatment group showed a significant difference from p/epa treatment group after 3 and 24 h only (table 4). serum mda statistical analysis showed that all types of treatment for periodontitis (water, srp, epa, or by srp + epa) showed significant increase (p < 0.05) in serum mda in relation with the nc/w treatment group (table 5). statistical analysis showed that the treatment by srp, epa, srp + epa caused significant decrease (p < 0.05) in serum mda in comparison with the p/w treatment group in all durations studied. no significant differences (p > 0.05) were seen between p/srp and p/epa, p/srp and p/srp + epa, or p/epa and p/srp + epa treatment groups in all durations studied (table 6). discussion serum crp crp is an acute phase protein, synthesized by the liver and circulates in the blood and rise in response to inflammation and used as an inflammatory marker.6 the present study showed that p/w treatment group showed significant increase in level of serum crp. ekuni et al.16 found that the serum crp in normal rats was 1.22 mg/ml, while in the periodontitis group showed a significantly 12% increase compared with that of the control group at time of suture removal. buhlin et al.,17 craig et al.,18 thakare et al.,19 gupta et al.,20 masi et al.,21 aziz et al.,22 and monisha and savitha7 also found that the levels of crp were significantly higher in periodontitis patients than healthy control. the balance between ros and antioxidant defense mechanism is important in the pathogenesis of periodontal disease, and those subjects who were infected with periodontal pathogens had clearly higher levels of crp than those who were not harboring the putative bacteria in their subgingival plaque. these results disagree with that of buduneli et al.23 result, they induced experimental periodontitis in rats by original 230 j contemp med sci | vol. 4, no. 4, july-august 2019: 227–233 effects of the systemic administration of omega-3 polyunsaturated a.a. jalal et al. table 4. the relation of serum alp (mean ± standard deviation) between different studied treatments groups groups alp (u/l) 3 h p-value 24 h p-value 3 days p-value 1 week p-value 2 weeks p-value p/w 114 ± 4.149 0.920 114.87 ± 3.827 0.857 116.076 ± 4.027 0.833 116.9 ± 0.761 0.173 117.5 ± 1.014 0.674 p/srp 114.47 ± 4.009 113.73 ± 1.896 115.476 ± 3.260 114.4 ± 2.80 118.276 ± 4.246 p/w 114 ± 4.149 0.920 114.87 ± 3.827 0.603 116.076 ± 4.027 0.0463 116.9 ± 0.761 0.012 117.5 ± 1.014 0.012 p/epa 115.876 ± 4.19 116.45 ± 2.34 120.7 ± 1.256 123.9 ± 2.546 124.5 ± 1.526 p/w 114 ± 4.149 0.144 114.87 ± 3.827 0.116 116.076 ± 4.027 0.0164 116.9 ± 0.761 0.012 117.5 ± 1.014 0.012 p/srp + epa 118.5 ± 4.402 120.3 ± 3.389 122.7 ± 1.526 124.1 ± 1.797 124.9 ± 1.968 p/srp 114.47 ± 4.009 0.756 113.73 ± 1.896 0.412 115.476 ± 3.260 0.036 114.4 ± 2.80 0.012 118.276 ± 4.246 0.021 p/epa 115.876 ± 4.19 116.45 ± 2.34 120.7 ± 1.256 123.9 ± 2.546 124.5 ± 1.526 p/srp 114.47 ± 4.009 0.144 113.73 ± 1.896 0.083 115.476 ± 3.260 0.012 114.4 ± 2.80 0.012 118.276 ± 4.246 0.036 p/srp + epa 118.5 ± 4.402 120.3 ± 3.389 122.7 ± 1.526 124.1 ± 1.797 124.9 ± 1.968 p/epa 115.876 ± 4.19 0.014 116.45 ± 2.34 0.034 120.7 ± 1.256 0.057 123.9 ± 2.546 0.527 124.5 ± 1.526\ 1.00 p/srp + epa 118.5 ± 4.402 120.3 ± 3.389 122.7 ± 1.526 124.1 ± 1.797 124.9 ± 1.968 table 5. serum mda (mean ± standard deviation) in normal control/water treatment group in relation with all studied groups groups mda (ng/ml) 3 h p-value 24 h p-value 3 days p-value one week p-value two weeks p-value nc/w 22.12 ± 0.920 0.012 21.9 ± 0.663 0.012 22.3 ± 1.204 0.012 22.5 ± 0.707 0.012 22.4 ± 0.894 0.012 p/w 32.8 ± 1.303 32.4 ± 1.516 31.6 ± 1.14 31.2 ± 1.303 30.6 ± 0.894 nc/w 22.12 ± 0.920 0.012 21.9 ± 0.663 0.012 22.3 ± 1.204 0.012 22.5 ± 0.707 0.012 22.4 ± 0.894 0.012 p/srp 27.54 ± 1.143 26.34 ± 1.275 26.94 ± 1.190 26.74 ± 1.26 26.34 ± 1.768 nc/w 22.12 ± 0.920 0.012 21.9 ± 0.663 0.012 22.3 ± 1.204 0.012 22.5 ± 0.707 0.012 22.4 ± 0.894 0.028 p/epa 26.44 ± 1.43 26.54 ± 1.28 26.34 ± 1.275 26.14 ± 1.41 25.74 ± 0.859 nc/w 22.12 ± 0.920 0.012 21.9 ± 0.663 0.012 22.3 ± 1.204 0.012 22.5 ± 0.707 0.028 22.4 ± 0.894 0.028 p/srp+epa 26.5 ± 1.903 25.94 ± 1.663 25.64 ± 1.273 25.34 ± 1.08 25.14 ± 1.431 table 6. the relation of serum mda (mean ± standard deviation) between different studied treatments groups groups mda (ng/ml) 3 h p-value 24 h p-value 3 days p-value 1 week p-value 2 weeks p-value p/w 32.8 ± 1.303 0.012 32.4 ± 1.516 0.012 31.6 ± 1.14 0.012 31.2 ± 1.303 0.012 30.6 ± 0.894 0.012 p/srp 27.54 ± 1.143 26.34 ± 1.275 26.94 ± 1.190 26.74 ± 1.26 26.34 ± 1.768 p/w 32.8 ± 1.303 0.012 32.4 ± 1.516 0.012 31.6 ± 1.14 0.012 31.2 ± 1.303 0.012 30.6 ± 0.894 0.012 p/epa 26.44 ± 1.43 26.54 ± 1.28 26.34 ± 1.275 26.14 ± 1.41 25.74 ± 0.859 p/w 32.8 ± 1.303 0.012 32.4 ± 1.516 0.012 31.6 ± 1.14 0.012 31.2 ± 1.303 0.012 30.6 ± 0.894 0.012 p/srp + epa 26.5 ± 1.903 25.94 ± 1.663 26.34 ± 1.275 25.34 ± 1.08 25.14 ± 1.431 p/srp 27.54 ± 1.143 0.920 26.34 ± 1.275 0.920 26.94 ± 1.190 0.920 26.74 ± 1.26 0.920 26.34 ± 1.768 0.347 p/epa 26.44 ± 1.43 26.54 ± 1.28 26.34 ± 1.275 26.14 ± 1.41 25.74 ± 0.859 p/srp 27.54 ± 1.143 0.920 26.34 ± 1.275 0.674 26.94 ± 1.190 0.920 26.74 ± 1.26 0.920 26.34 ± 1.768 0.250 p/srp + epa 26.5 ± 1.903 25.94 ± 1.663 26.34 ± 1.275 25.34 ± 1.08 25.14 ± 1.431 p/epa 26.44 ± 1.43 0.400 26.54 ± 1.28 0.603 26.34 ± 1.275 0.920 26.14 ± 1.41 0.920 25.54 ± 0.841 1.00 p/srp + epa 26.50 ± 1.903 25.94 ± 1.663 26.34 ± 1.275 25.34 ± 1.08 25.74 ± 0.859 original a.a. jalal et al. 231j contemp med sci | vol. 4, no. 4, july-august 2019: 227–233 effects of the systemic administration of omega-3 polyunsaturated repeated injection of purified lipopolysaccharide (lps) derived from escherichia coli endotoxin and found no significant differences present between any of the studied groups in serum crp. the present study also showed significant increase in serum crp in p/srp treatment group in comparison with the nc/w, but this increase was not significant in relation with the and p/w treatment group. the present result agrees with that of ide et al.’s6 study, they found no statistically significant changes in levels of any of crp after srp. tonetti et al.24 was found that within 24 h after periodontal treatment, there were significant increase in crp indicating an acute systemic inflammatory response, and by day 7, the periodontal treatment group has equal or lower circulating inflammatory biomarkers than the control group. the increase within 24 h following periodontal therapy may be a result of intense transient bacteremia and soft tissue damage resulting from periodontal instrumentation. these two results disagree with that of mattila et al25; they reported a reduction of crp concentrations on 30 patients with chronic periodontitis after non-surgical periodontal treatment. miyajima et al.26 found that the serum crp concentrations were not different between the control and the periodontitis rats (control; 10.9 6 0.6 ng/ml, periodontitis; 11.0 6 0.3 ng/ml). the present study also showed that after 1 week the p/srp + epa treatment group showed significant decrease (p < 0.05) in serum crp in comparison with the p/w, p/srp, and p/epa treatment groups. this means that the treatment by epa with srp is better than each one alone. phillips et al.27 also found that omega-3 supplementation reduces inflammation as measured by decreased levels of crp as a marker of inflammation. naqvi et al.28 found that epa was associated with lower crp in linear secondary analyses. this may be due to its anti-inflammatory effects.29 serhan and savill30 have been found that omega-3 in animal models of periodontitis can be a substrates for neutrophil production of resolvins and protectins, which appear central to the resolution of inflammation. other animal studies have suggested omega-3 may have a protective effect on periodontitis by decreasing the host inflammatory responses to common microbial pathogens such as porphyromonas gingivalis and this result in less tissue breakdown.31 vardar-şengül32 study disagree with the present study, they found that omega-3 cannot be used for treatment of experimental periodontitis induced by repeated injections of e. coli lps in rats, and omega-3 fatty acid administration did not seem to influence the circulating levels of crp. this may be due to different methods used. serum alp alkaline phosphatase is a metalloenzyme anchored to the cell membrane, and it is distributed particularly in the liver, bowel, placenta and bone and its activity and function can be modulated by environmental conditions.33 the present study showed that the ligature-induced periodontitis caused a significant decrease in alp serum levels. kose et al.34 found that the normal serum alp in rats was 124.86 ± 12.40 u/l, while in periodontitis group was 98.86 ± 9.40 u/l after 28 days from ligation removal. cetinkaya et al.35 found that the serum alkaline phosphatase levels were 903.90 ± 6.76 u/l in normal control group and 811.0 ± 6.56 u/l in experimental periodontitis group after 2 months ligation around the mandibular first molar. arabacı et al.36 also found the same results. the significant reduction in alp levels indicates that osteoblastic activity decreased in the periodontal area. contrary to these results, the measurements of periodontal destruction (probing depth, gingival bleeding, and suppuration) are related to higher levels of alp in saliva.37 the present study also showed that the p/srp showed no significant difference from that of the p/w treatment group regarding the alp serum levels. singh et al.38 also found that the changes in the level of alp in serum after srp was not statistically significant in the study groups with gingivitis, aggressive periodontitis and chronic periodontitis patients. this may be probably due to the fact that the local changes in periodontium may not have a direct effect on the levels of this enzyme in serum. the changing concentration in serum depends on function of other organ systems such as bone, kidney, liver etc.39 the p/epa and p/srp + epa treatment groups showed a significant increase (p < 0.05) in serum alp after 3 days in comparison with the p/w treatment group. the statistically significant elevation in alp levels provided by epa treatment in rats with periodontitis suggests that this therapeutic agent have a significant effect on osteoblasts in periodontitis tissues. appleton et al.40 found that omega-3 can inhibit the production of inflammatory cytokines such as il-1, il-6, and tnf-α, which provide an important stimulus for osteoclastic bone resorption, and thus may inhibit bone resorption and prevent bone loss. other study found that supplementation of the diets of growing rats with omega-3 fatty acids results in greater bone formation in rats, and also they found an increase in alp activity in osteoblastic cells in culture.41 al-hashemi et al.42 found that the total alkaline phosphatase in rat’s serum was significantly elevated after treatment with 400 and 600 mg/kg of omega-3 compared with other studied group. attia et al.43 found that rats fed with 20 mg/kg b.w. showed significant increase in serum alp. abdou et al.44 also found the same increase in rat’s serum alp. serum mda periodontal disease is one of the most common chronic inflammatory diseases, and high oxidative stress might have important roles in the etiopathogenesis of periodontitis.45 the present results showed significantly higher levels of serum mda in rats in the p/w treatment group compared with the nc/w treatment group. celec et al.,46 panjamurthy et al.,47 aziz et al.,22 trivedi et al.48 and ahmadi-motamaye et al.49 also found that serum mda level was significantly higher in the periodontitis group. based on the results of these studies, periodontitis can induce systemic oxidative stresses and alter serum mda levels, and synthesis of mda might be due to a decrease in antioxidant in destroyed in periodontal tissues. the present study also showed that treatment by srp can significantly decrease serum mda in comparison with the p/w treatment group. several studies found that successful periodontal therapy increased total antioxidant capacity levels50 and decreased mda levels,50 therefore, periodontal therapy can be very useful for the patient. conclusion the treatment of periodontitis by srp with the epa is better than each one alone. conflict of interest none.  original 232 j contemp med sci | vol. 4, no. 4, july-august 2019: 227–233 effects of the systemic administration of omega-3 polyunsaturated a.a. jalal et al. references 1. offenbacher s, barros sp, singer re, moss k, williams rc, beck jd. periodontal disease at the biofilm-gingival interface. j periodontol. 2007;78:1911–1925. 2. strimbu k, tavel ja. what are biomarkers? curr opin hiv aids 2010;5:463– 466. 3. jayaprakash d, aghanashini s, chatterjee a, bharwani a, vijayendra rr, rosh rm. effect of periodontal therapy on c-reactive protein levels in gingival crevicular fluid of patients with gingivitis and chronic periodontitis: a clinical and biochemical study. j indian soc periodontol. 2014;18: 456–460. 4. liu z, liu y, song y, zhang x, wang s, wang z. systemic oxidative stress biomarkers in chronic periodontitis: a meta-analysis. dis markers. 2014;2014:931083. 5. perumal gcl, mythili r, kumar s, suyambukesan s. serum total alkaline phosphatase enzyme level and severity of chronic periodontitis. int j curr res rev. 2014;6:41–44. 6. ide m, mcpartlin d, coward py, crook m, lumb p, wilson rf. effect of treatment of chronic periodontitis on levels of serum markers of acute-phase inflammatory and vascular responses. j clin periodontol. 2003;30:334–340. 7. monisha k, savitha g. assessment of oxidative stress in periodontitis patients. j pharm sci res. 2016;8:620–622. 8. harris ws. fish oil supplementation: evidence for health benefits. cleve clin j med. 2004;71:208–210. 9. pihlstrom bl, tabak l. the national institute of dental and craniofacial research: research for the practicing dentist. j am dent assoc. 2005;136:728–737. 10. jepsen s, blanco j, buchalla w, carvalho jc, dietrich t, dörfer c, et al. prevention and control of dental caries and periodontal diseases at individual and population level: consensus report of group 3 of joint efp/ orca workshop on the boundaries between caries and periodontal diseases. j clin periodontol. 2017;44:s85–s93. 11. tonetti ms, bottenberg p, conrads g, eickholz p, heasman p, huysmans mc, et al. dental caries and periodontal diseases in the ageing population: call to action to protect and enhance oral health and well-being as an essential component of healthy ageing consensus report of group 4 of the joint efp/orca workshop on the boundaries between caries and periodontal diseases. j clin periodontol. 2017;44:s135–s144. 12. araghizadeh n, paknejad m, alaeddini m, minaii b, abdollahi m, khorasanie r. the efficacy and prophylactic characteristics of omega-3 fatty acids in experimental gingivitis in rats. iran j basic med sci. 2014;17:87–92. 13. ionel a, lucaciu o, moga m, buhatel d, ilea a, flaviu t. periodontal disease induced in wistar rats experimental study. hum vet med. 2015;7:90–95. 14. garcia vg, knoll lr, longo m, novaes vc, assem nz, ervolino e, et al. effect of the probiotic saccharomyces cerevisiae on ligature-induced periodontitis in rats. j periodont res. 2016;51:26–37. 15. parasuraman s, raveendran r, kesavan r. blood sample collection in small laboratory animals. j pharmacol pharmacother. 2010;1:87–93. 16. ekuni d, tomofuji t, irie k, kasuyama k , umakoshi m, azuma t, et al. effects of periodontitis on aortic insulin resistance in an obese rat model. lab invest. 2010;90:348–359. 17. buhlin k, gustafsson a, pockley ag, frostegård j, klinge b. risk factors for cardiovascular disease in patients with periodontitis. eur heart j. 2003;24:2099–2107. 18. craig rg, yip jk, so mk, boylan rj, socransky ss, haffajee ad. relationship of destructive periodontal disease to the acute-phase response. j periodontol. 2003;74:1007–1016. 19. thakare ks, deo v, bhongade ml. evaluation of crp serum levels in periodontitis patients with or without atherosclerosis. indian j dent res. 2010;21:326–329. 20. gupta sc, jindal v, ranbika t. crp and its role in periodontitis. indian j dent sci. 2011;3:31–32. 21. masi s, salpea kd, li k, parkar m, nibali l, donos n, et al. oxidative stress, chronic inflammation and telomere length in patients with periodontitis. free radic biol med. 2011;50:730–735. 22. aziz as, kalekar mg, benjamin t, suryakar an, milsee mol jp. chronic periodontitis and oxidative stress – a biochemical study. indian j dent sci. 2012;4:22–26. 23. buduneli n, baylas h, buduneli e, türkoğlu o, köse t, dahlen g. periodontal infections and pre-term low birth weight: a case-control study. j clin periodontol. 2005;32:174–181. 24. tonetti ms, d’aiuto f, nibali l, donald a, storry c, parkar m, et al. treatment of periodontitis and endothelial function. n engl j med. 2007;356:911–920. 25. mattila k, vesanen m, valtonen v, nieminen m, palosuo t, rasi v, et al. effect of treating periodontitis on c-reactive protein levels: a pilot study. bmc infect dis. 2002;2:30. 26. miyajima s, naruse k, kobayashi y, nakamura n, nishikawa t, adachi k et al . periodontitis-activated monocytes/macrophages cause aortic inflammation. sci rep. 2014;4:5171. 27. phillips t, childs ac, dreon dm, phinney s, leeuwenburgh c. a dietary supplement attenuates il-6 and crp after eccentric exercise in untrained males. med sci sports exerc 2003;35:2032–2037. 28. naqvi az, buettner c, phillips rs, davis rb, mukamal kj. omega 3 fatty acids and periodontitis in u.s. adults. j am diet assoc 2010;110:1669–1675. 29. marion-letellier r, butler m, déchelotte p, playford rj, ghosh s. comparison of cytokine modulation by natural peroxisome proliferator-activated receptor gamma ligands with synthetic ligands in intestinal-like caco-2 cells and human dendritic cells--potential for dietary modulation of peroxisome proliferator-activated receptor gamma in intestinal inflammation. am j clin nutr. 2008;87:939–948. 30. serhan cn, savill j. resolution of inflammation: the beginning programs the end. nat immunol. 2005;6:1191–1197. 31. hasturk h, kantarci a, goguet-surmenian e, blackwood a, andry c, serhan cn et al. resolvin e1 regulates inflammation at the cellular and tissue level and restores tissue homeostasis in vivo. j immunol. 2007;179:7021–7029. 32. vardarşengül s, buduneli n, buduneli e, kardeşler l, baylas h, atilla g, et al. dietary supplementation of omega-3 fatty acid and circulating levels of interleukin-1beta, osteocalcin, and c-reactive protein in rats. j periodontol. 2006;77:814–820. 33. whyte mp. physiological role of alkaline phosphatase explored in hypophosphatasia. ann n y acad sci. 2010;1192:190–200. 34. kose o, arabaci t, yemenoglu h, kara a, ozkanlar s, kayis s, et al. influences of fucoxanthin on alveolar bone resorption in induced periodontitis in rat molars. mar drugs. 2016;14:1–11. 35. cetinkaya bo, acikcoz g, keles gc, ayas b, korkmaz a. the effect of cyclosporin a on alveolar bone in rats subjected to experimental periodontal disease. toxicol pathol. 2006;34:716–722. 36. arabac t, kermen e,özkanlar s, köse o, kara a, kızıldağ a, et al. therapeutic effects of melatonin on alveolar bone resorption after experimental periodontitis in rats: a biochemical and immunohistochemical study. j periodontol. 2015;86:874–881. 37. kinney js, ramseier ca, giannobile wv. oral fluid-based biomarkers of alveolar bone loss in periodontitis. ann n y acad sci. 2007;1098:230–251. 38. singh n, chandel s, singh h, agrawal a, savitha an. effect of scaling and root planing on the activity of alp in gcf & serum of patients with gingivitis, chronic and aggressive periodontitis: a comparative study. j oral biol craniofac res. 2017;7:123–126. 39. weiss mj, rosy k, henthorn ps. structure of the human liver/bone/kidney alkaline phosphatase activity. j periodontal res. 1996;31:66–72. 40. appleto km, fraser wd, rogers pj, ness ar, tobias jh. supplementation with a low-moderate dose of n-3 long-chain pufa has no short-term effect on bone resorptionin human adults. br j nutr. 2011;105; 1145–1149. 41. watkins ba, li y, lippman he, feng s. modulatory effect of omega-3 polyunsaturated fatty acids on osteoblast functions and bone metabolism. prostaglandins leukot essent fatty acids 2003;68:387–398. 42. al-hashemi hm, al-khashab em, hamdoon aa. effect of high doses of omega-3 fatty acids on the metabolism of bones in adult females rats. raf j sci. 2013;24:17–26. 43. attia am, el-banna sg, nomeir fr, abd el-basser mi. lindane-induced biochemical perturbations in rat serum and attenuation by omega-3 and nigella sativa seed oil. indian j biochem biophys 2011;48:184–190. 44. abdou hm, hassan ma. protective role of omega-3 polyunsaturated fatty acid against lead acetate-induced toxicity in liver and kidney of female rats. med res int. 2014;2014:435857. 45. tóthová l, kamodyová n, červenka t, celec p. salivary markers of oxidative stress in oral diseases. front cell infect microbiol. 2015;5:73–79. 46. celec p, hodosy j, celecová v, vodrázka j, cervenka t, halcák l et al.. salivary thiobarbituric acid reacting substances and malondialdehyde--their relationship to reported smoking and to parodontal status described by the papillary bleeding index. dis markers, 2005;21:133–137. original a.a. jalal et al. 233j contemp med sci | vol. 4, no. 4, july-august 2019: 227–233 effects of the systemic administration of omega-3 polyunsaturated 47. panjamurthy k, manoharan s, ramachandran cr. lipid peroxidation and antioxidant status in patients with periodontitis. cell mol biol lett. 2005;10:255–264. 48. trivedi s, lal n, mahdi aa, singh b, pandeys. association of salivary lipid peroxidation levels, antioxidant enzymes, and chronic periodontitis. int j periodontics restorative dent. 2015;35:e14–e19. 49. ahmadi-motamaye f, goodarzi mt, jamshidi z, kebriaei r. evaluation of salivary and serum antioxidant and oxidative stress statuses in patients with chronic periodontitis: a case-control study. front physiol. 2017;8:189. 50. guentsch a, preshaw pm, bremer-streck s, klinger g, glockmann e, sigusch, bw. lipid peroxidation and antioxidant activity in saliva of periodontitis patients: effect of smoking and periodontal treatment. clin oral investig. 2008;12:345–352. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.08201909 original 125j contemp med sci | vol. 5, no. 3, may–june 2019: 125–130 original maternal separation can affect the reproductive system by inflammasome activation in female mice kajal khodamoradi,a,b hossein amini-khoei,c zahra khosravizadeh,a seyed reza hosseini,d ahmad reza dehpour,e and gholamreza hassanzadeha* adepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. bmiller school of medicine, university of miami, miami, fl, usa. cmedical plants research center, basic health sciences institute, shahrekord university of medical sciences, shahrekord, iran. ddepartent of urology, school of medicine, tehran university of medical sciences, tehran, iran. eexperimental medicine research center, tehran university of medical sciences, tehran, iran. *correspondence to gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir). (submitted: 04 december 2018– revised version received: 16 january 2019 – accepted: 23 april 2019 – published online: 26 june 2019) introduction the early childhood is a sensitive age period in which development of brain can be affected harmfully by stressful events. the maternal separation model, as a model mimicking early-life stress, has been broadly studied to evaluate the effect of early-life stress on brain development and associated biological networks. exposure to this stressful event can affect children not only in their early years of life but also in their adult life. it seems that the repeated activation of the hypothalamic pituitary adrenal (hpa) axis and disruptions in pathways of the immune response and the neurotrophin production could play a critical role in the long-lasting effects of early-life stress.1,2 stress can trigger the activity of the adrenergic system that change the function of the endocrine and immune systems.3 cooperation between the immune and endocrine systems is necessary for normal development and function of the female reproductive system.4 according to psychoneuroimmunology studies, the physiological response to stressors affects function of the innate immune system even in the absence of pathogens that is known as sterile inflammatory response.5,6 although it remains unknown how innate immune function is stimulated by stress, the role of different factors including glucocorticoids, danger signals and catecholamines are evidenced by several studies.7–10 the transcription of inflammatory mediators such as cytokines and chemokines are activated during the inflammatory process.11 inflammasomes are intracellular multi-protein complexes which include a nod-like receptor/an aim-like receptor, the adapter molecule apoptosis-associated speck-like protein containing a card (asc), and caspase-1.12–14 nlrp3 along with the adaptor protein asc, active caspase-1 through assembly of the inflammasome, although the exact mechanism for this process is unclear. the nlrp3, the most described inflammasome, can be triggered by a wide range of exogenous and endogenous stress signals. caspase-1 activation leads to the cleavage of pro-interleukin (il)-1beta, pro-il-18 into active forms of il-1β, il-18, which play potential role in modulating innate immune function.15–17 moreover, caspase-1 plays a role in cell apoptosis via stimulation of caspase-3 and -7.18 on the other hand, psychosocial stressors via stimulation of inflammatory cytokines such as tnf-α, il1-β and il-6 can lead to the suppression of gonadotropin-releasing hormone (gnrh) secretion.19 since the main target of gnrh is the gonadotropes of anterior pituitary gland, stress-induced gnrh suppression can affect the secretion of gonadotropic hormones and consequently reproductive functions. mitochondria are responsible for the reactive oxygen species (ros) production, atp generation and apoptotic control.20 owing to the high levels of antioxidant enzymes, the mitochondria can regulate the balance between ros production and degradation.21 the findings of studies have confirmed that chronic stress can lead to disruption of mitochondrial function and free radicals production.22 it is apparent that mitochondria play a significant role in female reproductive processes including oocyte maturation, fertilization and early embryogenesis.23 the evidence for the involvement of oxidative injury in the objective the aim of this study is to investigate effect of maternal separation stress on the ovarian function in adult female mice. methods in this study, maternal separation in pups was performed during post-natal days 2–14. the histological alterations in ovarian tissue, reactive oxygen species (ros) production (using 2′,7′-dichlorofluorescin diacetate assay), gene expression of nlrp3, apoptosisassociated speck-like protein containing a card (asc), caspase-1, tlr4, bax, bcl-2 and tnf-α (using rt-pcr), protein levels of atp, gpx, interleukin (il)-1β and il-18 (using enzyme-linked immunosorbent assay). also, protein expression of caspase-3 and nlrp3 (using immunocytochemistry) were evaluated. results this showed that maternal separation decreased percentage of primordial follicles while increased percentage of secondary and graafian follicles. in addition, maternal separation increased ros production and decreased atp and gpx concentrations. furthermore, maternal separation significantly affected expression of cytokines and genes involved in inflammation and apoptosis including nlrp3, asc, caspase-1, tlr4, tnf-α, il-1β, il-18, bax and bcl2. findings also showed that stress-induced maternal separation significantly increased percentage of caspase-3 and nlrp3 positive cells. we concluded that maternal separation stress has harmful effects on ovarian tissue. conclusion it seems that these harmful effects probably occur through increase of ros production and impact on mitochondrial function, inflammatory process and apoptosis pathways. keywords maternal separation stress, inflammasome, inflammation, oxidative stress, apoptosis, reproductive system issn 2413-0516 126 j contemp med sci | vol. 5, no. 3, may–june 2019: 125–130 maternal separation affects reproductive system in female mice original k. khodamoradi et al. pathogenesis of infertility in females has been accumulating since long.24 since the adverse effects of early-life stress on the neuroendocrine and innate immune system, also adverse effects of oxidative stress on fertility status have been well described in previous studies, stress-induced maternal separation during early life may have an adverse effect on the female reproductive system. therefore, this study was designed to investigate the effect of maternal separation stress on ovarian function in adult female mice. materials and methods experimental animals pregnant naval medical research institute mice were obtained from the pasteur institute of iran. the animals were housed under a cycle of 12 h:12 h light/dark at controlled temperature (22–25°c) and humidity (55–65%). all animal procedures were carried out according to guidelines approved by the ethics committee of tehran university of medical sciences (ir.tums.medicine.rec.1395.2507). to produce maternal separation model, pups were separated from their mothers and placed in a clean cage for 3 h every day (9 am to 12 pm each day) from post-natal days (pnd) 2 to 14. the birthday of pups was considered as pnd 0.25–27 in this study, female pups were randomly divided into two groups: control and maternal separation group. the pups of the control group were left untouched. mice at pnd 70 were sacrificed by deep anesthesia and the ovaries were removed. one ovary from each mouse was used for histological assessment and the second ovary was used for molecular assessments. histological assessment the ovaries for histological assessment were immediately transferred to bouin’s fixative solution. the fixed ovaries were dehydrated in ascending graded series of ethanol (merck, darmstadt, germany) and then emerged in paraffin wax. the serial sections (5 μm thick) were prepared with a rotary microtome (microm, walldorf, germany) and rehydrated in descending graded series of ethanol. the sections were cleared in xylene, stained with hematoxylin and eosin (h&e) and mounted with dpx. for histological assessment, transverse sections from nine different regions of the ovaries were examined. the number of primordial, primary, secondary and graafian follicles were counted by light microscopy and imagej software (imagej u. s. national institutes of health, bethesda, md, usa). ros assay the level concentration of ros production in ovarian tissues was detected with flow cytometry using 2',7' dichlorofluorescin diacetate (dcfh-da; sigma-aldrich, st. louis, mo, usa) after enzymatic digestion of minced tissue.28 the ovarian tissues were mechanically homogenized in ham’s f-10 medium (life technology, carlsbad, ca, usa). the homogenates of ovarian tissue were centrifuged at 10,000g for 5 min and washed with pbs. then, the homogenates were incubated with 20 µm dcfh-da in dark at 37°c for 45 min. the homogenates of ovarian tissue were washed with pbs, and dcf fluorescence (green) was measured in the fl-1 channel using a bd facscan flow cytometer (becton dickinson, san jose, ca, usa).29 real-time reverse transcription polymerase chain reaction analysis the expression level of nlrp3, asc, caspase-1, tlr4, bax, bcl2 and tnfα genes was analyzed by real-time reverse transcription polymerase chain reaction (rt-qpcr). the total rna extraction from ovarian tissues was performed with trizol reagent according to the manufacturer’s instructions (invitrogen, carlsbad, ca, usa). the complementary dna was produced via reverse transcription reaction using a primescript rt reagent kit (takara, south korea) according to the manufacturer’s protocol. the rt-qpcr was carried out with gene specific primers and the hot firepol evagreen qpcr mix plus (solis biodyne, tartu, estonia) by an abi7500 (applied biosystems, foster city, ca, usa). the reference gene glyceraldehydes-3-phosphate dehydrogenase messenger rna (mrna) expression are used as the internal control to normalize mrna expression levels. the level of target genes expression was calculated using 2−δct. list of primer sequences used for rt-qpcr analysis are presented in table 1. enzyme-linked immunosorbent assay the ovarian tissues were homogenized in pbs on ice, then centrifuged at 800 × g for 5 min. the collected supernatants were used in the enzyme-linked immunosorbent (elisa) assay. the level concentrations of atp and gpx were measured with specific elisa kits (r&d systems, minneapolis, mn, usa) and the level concentrations of il-1β and il-18 in ovarian tissue were measured using elisa kits (koma biotech, seoul, korea) according to the manufacturer’s protocols. immunocytochemical analysis to determine caspase-3 and nlrp3 immunoreactivity, the fixed ovarian samples were dehydrated in graded ethanol and embedded in paraffin. after removing paraffin, samples were rehydrated through a graded series of ethanol and permeabilized with 10 mm sodium citrate and 0.05% tween 20. the samples were blocked in a blocking solution including 1% (w/v) bovine serum albumin (sigma-aldrich, st. louis, mo, usa) in pbs. then, the ovarian samples were incubated overnight at 4°c with primary antibodies against caspase-3 (1:1000 dilution, abcam, cambridge, ma, usa) and nlrp3 table 1. primer sequences genes forward primers reverse primers nlrp3 5′-ggacccacagtg-taacttgcaga-3′ 5′-aggctgcagttgtctaattccag-3′ asc 5′-cacaaatcagtctc-caacacc-3′ 5′-taaccattaccttgttccca-3′ caspase-1 5′-cactcgta-cacctcttgccctc-3′ 5′-ctttcacctctttcaccatctcca-3′ tlr4 5′-tgagtggtcagtgt-gattgtggt-3′ 5′-tgtagtgaaggcagaggtgaaag-3′ bax 5′-gcaaactggtgctcaa-gg-3′ 5′-cagccacaaagatggtca-3′ bcl2 5′-acttttaggcgtggct-gatg-3′ 5′-gtgctgctcactgtattttatttt-3′ tnf-α 5′-tgtctcagcctcttct-cattcctg-3′ 5′-aggccatttgggaacttctcatcc-3′ gadph 5′-tgacatcaagaaggtg-gtgaag-3′ 5′-cgaaggtggaagagtgggag-3′ 127j contemp med sci | vol. 5, no. 3, may–june 2019: 125–130 original maternal separation affects reproductive system in female micek. khodamoradi et al. (1:1000 dilution, abcam, cambridge, ma, usa). the samples were incubated with secondary antibody (1:500 dilution, abcam, cambridge, ma, usa) for 2 h at 37°c and then the cells’ nuclei were stained with pi (1:1000, sigma-aldrich, st. louis, mo, usa). the cell counting and merging of the pictures were performed by image j software (imagej u. s. national institutes of health, bethesda, ma, usa). statistical analysis the collected data were analyzed using spss version 20.0 software. the values were tested for normality using the kolmogorov–smirnov test. the statistical significance of the results was determined using the independent samples t-test and mann–whitney u test. the results were presented as the mean ± standard deviation (sd) and p ≤ 0.05 was considered as statistically significant. results histological assessment histological analysis of ovarian follicles was assessed with h&e staining (fig. 1). the results showed that percentage of primordial follicles in the maternal separation group (17.226 ± 9.096) was significantly lower compared with the control group (53.188 ± 4.485) (p < 0.001). there was no significant difference between percentage of primary follicles in the maternal separation group (5.584 ± 3.229) and percentage of primary follicles in the control group (5.839 ± 2.387) (p > 0.05). percentage of secondary follicles in the maternal separation group (28.247 ± 5.621) was significantly higher compared with the control group (16.899 ± 5.639, p < 0.01). in addition, percentage of graafian follicles in the maternal separation group (48.941 ± 7.826) was significantly higher comspared with the control group (24.072 ± 1.908, p < 0.001, fig. 2). ros evaluation reactive oxygen species production in ovarian tissue was detected using the dcfh-da assay. the level of ros production in ovarian tissue of the maternal separation group (614.4183 ± 28.6545) was significantly higher compared with the control group (363.9886 ± 48.3404, p < 0.01, fig. 3). rt-qpcr analysis the results of rt-qpcr analysis showed that expression of the following significantly increased in the maternal separation group compared with the control group: tnf-α (p < 0.001), nlrp3, asc, caspase-1, tlr4 and bax (p < 0.01). in contrast, the expression of bcl2 gene significantly decreased in the maternal separation group compared with the control group (p < 0.01) (fig. 4). enzyme-linked immunosorbent assa enzyme-linked immunosorbent assay results showed that the level concentrations of atp and gpx were significantly lower in the maternal separation group compared with the control group (2.4992 ± 0.03 vs. 3.03497 ± 0.02, p < 0.001 and 147.46 ± 11.58 vs. 399.3414 ± 23.39, p < 0.001 respectively). the il-1β and il-18 concentrations in the maternal separation group were higher than the control group (2.4773 ± 0.01 vs. 1.8753 ± fig. 1 the histopathological features provided from h&e-stained ovarian sections in the control (a) and the maternal separation (b) groups. scale bars are 20 µm. samples were analyzed in triplicate. a b fig. 2 effect of maternal separation on mice ovarian follicles. values are reported as mean ± sd. **p < 0.01, ***p < 0.001. samples were analyzed in triplicate. fig. 3 the level concentrations of ros production in the ovary. values are reported as mean ± sd. **p < 0.01. samples were analyzed in triplicate. ros, reactive oxygen species; sd, standard deviation. 0.02 and 3.3494 ± 0.04 vs. 1.9481 ± 0.08 respectively, p < 0.001 for both) (fig. 5). immunocytochemical analysis caspase-3 and nlrp3 markers were used to label the ovarian cells in the control and maternal separation groups. the nuclei were stained with pi (fig. 6). immunocytochemical analysis showed that the mean percentage of caspase-3 positive cells in the maternal separation group was significantly higher compared with the control group (50.6666 ± 1.76% vs. 24.00 ± 0.58%, p < 0.001). also, the mean percentage of nlrp3 positive cells was significantly higher in the maternal separation group 128 j contemp med sci | vol. 5, no. 3, may–june 2019: 125–130 maternal separation affects reproductive system in female mice original k. khodamoradi et al. inflammation and apoptosis including nlrp3, asc, caspase-1, tlr4, tnf-α, il-1β, il-18, bax and bcl2 were significantly affected in the maternal separation group. our results also showed that maternal separation stress significantly increased percentage of caspase-3 and nlrp3 positive cells. according to a large body of evidence, maternal separation has long-lasting effects on neurodevelopmental and behavioral health that can increase susceptibility to psychopathology in adulthood.30 it is known that the function of hpa axis, an essential hormonal response system to manage stress, adversely affected by maternal separation stress.31 considering the regulatory role of hpa axis in many homeostatic systems, these adverse modifications in hpa axis activity can result in functional alterations in the biological systems such as neuroendocrine, immune and reproductive systems.3,5,32,33 furthermore, stress can affect the function of the adrenergic system that change the activities of the endocrine and immune systems.3 cross-talk between the endocrine and immune systems is necessary for normal development and function of the female reproductive system.4 although it remains unknown how innate immune function is stimulated by stress, the role of different factors including glucocorticoids, danger signals and catecholamines are evidenced by several studies.7–10 previous studies have indicated that nlrs play a regulatory role in the reproductive and innate immune systems in mammals.34 the nlrp3 inflammasome, as a well-known nlr, is a cytoplasmic complex which is activated by danger signals derived from pathogens and metabolic dysregulation. activation of nlrp3 inflammasome lead to regulating the secretion of the pro-inflammatory cytokines il-1β and il-18.35 our findings showed that stress induced by maternal separation significantly affect the nlrp3 inflammasome components and inflammatory molecules including nlrp3, asc, caspase-1, tlr4, tnf-α, il1-β, il18and caspase-3. furthermore, maternal separation stress can significantly affect mitochondrial activation and resulted in increase of ros production and decrease of atp level. in addition, this chronic stress can affect the mrna expression of bcl2 and bax genes which are involved in apoptosis pathways. it is well known that increased ros production is associated with mitochondrial dysfunction and apoptosis. recently, it has been identified that mitochondria play a key role in activation of nlrp3 inflammasome.36 mitochondrial damage can trigger signals of apoptosis and induce nlrp3 inflammasome fig. 4 the gene expression of nlrp3, asc, caspase-1, tlr4, bax, bcl2 and tnf-α using rt-qpcr. values are reported as mean ± sd. **p < 0.01, ***p < 0.001. samples were analyzed in triplicate. sd, standard deviation; rt-qpcr, real-time reverse transcription polymerase chain reaction; asc, apoptosis-associated speck-like protein containing a card. fig. 5 the level concentrations of atp, gpx, il-1β and il-18 measured using elisa. data were analyzed using the mann–whitney u test. values are reported as mean ± sd. ***p < 0.001. samples were analyzed in triplicate. elisa, enzyme-linked immunosorbent assay. fig. 6 immunocytochemical analysis of ovarian cells for caspase-3 and nlrp3 markers. (a) control group; (b) maternal separation group; upper panel: pi stained pictures; lower panel: merged pictures of pi and secondary antibody stained cells. scale bars are 10 µm. samples were analyzed in triplicate. a b in compared with the control group (44.00 ± 1.53% vs. 20.6666 ± 1.45%, p < 0.001) (fig. 7). discussion the findings of this study showed that maternal separation stress led to significant histological alterations in the ovarian tissue, including decreased percentage of primordial follicles and increased percentage of secondary and graafian follicles. in addition, maternal separation stress during early life increased ros production and decreased atp and gpx concentrations. furthermore, expression of cytokines and genes involved in fig. 7 comparison of the mean percentage of positive cells for caspase-3 and nlrp3 markers by immunocytochemical assessment. values are reported as mean ± sd. ***p < 0.001. samples were analyzed in triplicate. 129j contemp med sci | vol. 5, no. 3, may–june 2019: 125–130 original maternal separation affects reproductive system in female micek. khodamoradi et al. activation through release of oxidized mitochondrial dna into cytosol.36,37 furthermore, the findings of studies indicated that mitochondrial ros and dna play an important role in nlrp3 inflammasome activation.38,39 in addition to its role in apoptosis, it has been suggested that bcl2 might play a role in the inhibition of nlrp3 inflammasome activation. shimada et al. have described that increased expression of bcl2 lead to decrease in il-1β levels. from this, it can be concluded that apoptosis plays a role in nlrp3 inflammasome activation.36 in addition, recently some studies have described that tnf, as a proinflammatory cytokine, can regulate the nlrp3 inflammasome activation.40,41 given these data and our findings, plus the known effects of stress on the neuroendocrine system, it seems that maternal separation stress can affect the nlrp3 inflammasome components and inflammatory molecules with alteration in level of ros, bcl2 and tnf-α and subsequently lead to increase of il1-β and il18production. our findings showed that maternal separation resulted in histological alterations in the ovarian tissue, including decreased percentage of primordial follicles and increased percentage of secondary and graafian follicles. in addition to reducing the number of primary follicles, stress likely accelerates the process of evolution of primary follicles to secondary and graafian follicles. it seems that maternal separation stress lead to fast down the folliculogenesis process that may reduce the ovarian follicular reserve and induce a shorter reproductive lifespan. in our study, maternal separation stress cause to increase of ros production and decrease of gpx concentration. cells have defensive strategies to prevent oxidative injury caused by excessive production of ros, such as anti-oxidant enzymes like catalase (cat), superoxide dismutase and gpx.42 although ros play physiological roles during folliculogenesis process, maturation of oocyte, and fertilization,43 increased ros production and oxidative stress can suppress the antioxidant defense and influences the fertilization capacity.44 the findings of studies indicated that oxidative stress lead to follicular atresia and ovarian follicle aging.43,45,46 furthermore, excessive production of ros and oxidative stress can lead to pathologic events in the ovary including inflammation and apoptosis.47 findings of studies described that il-1 is expressed in the mammalian ovary and play a physiological role in ovary. although inflammation is very important in reproductive processes such as ovulation, menstruation and implantation, uncontrolled inflammation has adverse effects on normal function of ovary.48,49 uri-belapolsky et al.50 have reported that il-1 may enhance the inflammatory genes expression and promote apoptotic signaling pathways that leads to exhaust the ovarian reserve. on the other hand, it is well known that follicular atresia can occur via mitochondrial pathway or binding of death receptors to their ligands such as tnf-α and fas.51 according to the above-mentioned data and the results of this study, we can conclude that maternal separation stress via activation of inflammatory and apoptosis pathways may have adverse effects on folliculogenesis process and the number of ovarian follicles. conclusion this study provides a glimpse of the effects of maternal separation stress on ovarian tissue in female mice as adults. based on this study, maternal separation stress had detrimental effects on ovarian tissue, in addition to the known effects of stress on the hpa axis and neuroendocrine system, probably through increase of ros production and impact on mitochondrial function, inflammatory process and apoptosis pathways. an improved insight into the mechanisms by which maternal separation stress affects the reproductive system may provide necessary information to keep the reproductive system healthy. acknowledgments this research has been funded by tehran university of medical sciences (tums); grant no. 32229. the authors thank tehran university of medical sciences for its support. conflict of interest the authors declare that there is no known conflict of interest regarding this publication.  references 1. tractenberg sg, levandowski ml, de azeredo la, orso r, roithmann lg, hoffmann es, et al. an overview of maternal separation effects on behavioural outcomes in mice: evidence from a four-stage methodological systematic review. neurosci biobehav rev. 2016;68:489–503. 2. harrison el, baune bt. modulation of early stress-induced neurobiological changes: a review of behavioural and pharmacological interventions in animal models. transl psychiatry. 2014;4:e390. 3. jóźków p, mędraś m. psychological stress and the function of male gonads. endokrynol pol. 2012;63:44–49. 4. ostanin aa, aizikovich bi, aizikovich iv, kozhin ay, chernykh er. role of cytokines in the regulation of reproductive function. bull exp biol med. 2007;143:75–79. 5. fleshner m. stress-evoked sterile inflammation, danger associated molecular patterns (damps), microbial associated molecular patterns (mamps) and the inflammasome. brain behav immun. 2013;27:1–7. 6. rock kl, latz e, ontiveros f, kono h. the sterile inflammatory response. annu rev immunol. 2009;28:321–342. 7. johnson jd, campisi j, sharkey cm, kennedy sl, nickerson m, greenwood bn, et al. catecholamines mediate stress-induced increases in peripheral and central inflammatory cytokines. neuroscience. 2005;35:1295–1307. 8. mazzeo rs, donovan d, fleshner m, butterfield ge, zamudio s, wolfel ee, et al. interleukin-6 response to exercise and high-altitude exposure: influence of α-adrenergic blockade. j appl physiol. 2001;91:2143–2149. 9. frank mg, thompson bm, watkins lr, maier sf. glucocorticoids mediate stress-induced priming of microglial pro-inflammatory responses. brain behav immun. 2012;26:337–345. 10. campisi j, sharkey c, johnson jd, asea a, maslanik t, bernstein-hanley i, et al. stress-induced facilitation of host response to bacterial challenge in f344 rats is dependent on extracellular heat shock protein 72 and independent of alpha beta t cells. stress. 2012;15:637–646. 11. gómez lópez m, domínguez lópez a, abarca rojano e, rojas hernández s, martínez godínez mde l, miliar garcía a, et al. 17β-estradiol transcriptionally modulates nlrp1 and nlrp3 inflammasomes in gonadectomized rats with inflammation. immunopharmacol immunotoxicol. 2015;37:343–350. 12. bazrafkan m, nikmehr b, shahverdi a, hosseini, hassani f, poorhassan m, et al. lipid peroxidation and its role in the expression of nlrp1a and nlrp3 genes in testicular tissue of male rats: a model of spinal cord injury. iran biomed j. 2018;22:151–159. 13. nikmehr b, bazrafkan m, hassanzadeh g, shahverdi a, sadighi gilani ma, kiani s, et al. the correlation of gene expression of inflammasome indicators and impaired fertility in rat model of spinal cord injury: a time course study. urol j. 2017;14:5057–5063. 14. mohamadi y, noori moghahi smh, mousavi m, borhani-haghighi m, abolhassani f, kashani ir, et al. intrathecal transplantation of wharton’s jelly mesenchymal stem cells suppresses the nlrp1 inflammasome in the rat model of spinal cord injury. j chem neuroanat. 2019;97:1–8. 130 j contemp med sci | vol. 5, no. 3, may–june 2019: 125–130 maternal separation affects reproductive system in female mice original k. khodamoradi et al. 15. yang ca, chiang bl. inflammasomes and human autoimmunity: a comprehensive review. j autoimmun. 2015;61:1–8. 16. jin c, flavell ra. molecular mechanism of nlrp3 inflammasome activation. j clin immunol. 2010;30:628–631. 17. ghaffari, n., hassanzadeh, g., nowrouzi, a., gholaminejhad, m., mokhtari, t., seifali, r., ... & akbari, m. (2018). antioxidative and anti-inflammatory effects of cichorium intybus l. seed extract in ischemia/reperfusion injury model of rat spinal cord. journal of contemporary medical sciences, 4(4). 18. shalini s, dorstyn l, dawar s, kumar s. old, new and emerging functions of caspases. cell death differ. 2015;22:526–539. 19. wagenmaker er, breen km, oakley ae, tilbrook aj, karsch fj. psychosocial stress inhibits amplitude of gonadotropin-releasing hormone pulses independent of cortisol action on the type ii glucocorticoid receptor. endocrinology. 2009;150:762–769. 20. keating dj. mitochondrial dysfunction, oxidative stress, regulation of exocytosis and their relevance to neurodegenerative diseases. j neurochem. 2008;104:298–305. 21. mailloux rj. mitochondrial antioxidants and the maintenance of cellular hydrogen peroxide levels. oxid med cell longev. 2018;2018:7857251. 22. madrigal jl, olivenza r, moro ma, lizasoain i, lorenzo p, rodrigo j, et al. glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain. neuropsychopharmacology 2001;24:420–429. 23. cecchino gn, seli e, alves da motta el, garcía-velasco ja. the role of mitochondrial activity in female fertility and assisted reproductive technologies: overview and current insights. reprod biomed online. 2018;36:686–697. 24. agarwal a, aponte-mellado a, premkumar bj, shaman a, gupta s. the effects of oxidative stress on female reproduction: a review. reprod biol endocrinol. 2012;10:49. 25. amini-khoei h, amiri s, shirzadian a, haj-mirzaian a, alijanpour s, rahimi-balaei m, et al. experiencing neonatal maternal separation increased the seizure threshold in adult male mice: involvement of the opioid system. epilepsy behav. 2015;52:37–41. 26. amini-khoei h, amiri s, mohammadi-asl a, alijanpour s, poursaman s, haj-mirzaian a, et al. experiencing neonatal maternal separation increased pain sensitivity in adult male mice: involvement of oxytocinergic system. neuropeptides. 2017;61:77–85. 27. amini-khoei h, haghani-samani e, beigi m, soltani a, mobini gr, balali-dehkordi s, et al. on the role of corticosterone in behavioral disorders, microbiota composition alteration and neuroimmune response in adult male mice subjected to maternal separation stress. int immunopharmacol. 2019;66:242–50. 28. dym m, jia mc, dirami g, price jm, rabin sj, mocchetti i, ravindranath n. expression of c-kit receptor and its autophosphorylation in immature rat type a spermatogonia. biol reprod. 1995;52:8–19. 29. fatemi n, sanati mh, shamsara m, moayer f, zavarehei mj, pouya a, et al. tbhp-induced oxidative stress alters micrornas expression in mouse testis. j assist reprod genet. 2014;31:1287–1293. 30. slotten ha, kalinichev m, hagan jj, marsden ca, fone kc. long-lasting changes in behavioural and neuroendocrine indices in the rat following neonatal maternal separation: gender-dependent effects. brain res. 2006;1097:123–132. 31. clarke as. social rearing effects on hpa axis activity over early development and in response to stress in rhesus monkeys. dev psychobiol. 1993;26:433–446. 32. kuhn cm, schanberg sm. responses to maternal separation: mechanisms and mediators. int j dev neurosci. 1998;16:261–270. 33. g m. central nervous regulation of the hypothalamic-pituitary-adrenal axis and its impact on fertility, immunity, metabolism and animal welfare-a review. arch anim breed. 2002;45:575–595. 34. peng h, zhang w, xiao t, zhang y. nlrp4g is an oocyte-specific gene but is not required for oocyte maturation in the mouse. reprod fertil dev. 2014;26:758–768. 35. tschopp j, schroder k. nlrp3 inflammasome activation: the convergence of multiple signalling pathways on ros production? nat rev immunol. 2010;10:210–215. 36. shimada k, crother tr, karlin j, dagvadorj j, chiba n, chen s, et al. oxidized mitochondrial dna activates the nlrp3 inflammasome during apoptosis. immunity. 2012;36:401–414. 37. shimada k, crother tr, karlin j, chen s, chiba n, ramanujan vk, vergnes l, ojcius dm, arditi m. caspase-1 dependent il-1β secretion is critical for host defense in a mouse model of chlamydia pneumoniae lung infection. plos one. 2011;6:e21477. 38. zhou r, yazdi as, menu p, tschopp j. a role for mitochondria in nlrp3 inflammasome activation. nature. 2011;469:221–225. 39. nakahira k, haspel ja, rathinam va, lee sj, dolinay t, lam hc, et al. autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial dna mediated by the nalp3 inflammasome. nat immunol. 2011;12:222–230. 40. wree a, mcgeough md, inzaugarat me, eguchi a, schuster s, johnson cd, et al. nlrp3 inflammasome driven liver injury and fibrosis: roles of il‐17 and tnf in mice. hepatology. 2018;67:736–749. 41. mcgeough md, wree a, inzaugarat me, haimovich a, johnson cd, peña ca, et al. tnf regulates transcription of nlrp3 inflammasome components and inflammatory molecules in cryopyrinopathies. j clin invest. 2017;127: 4488–4497. 42. ighodaro om, akinloye oa. first line defence antioxidants-superoxide dismutase (sod), catalase (cat) and glutathione peroxidase (gpx): their fundamental role in the entire antioxidant defence grid. alexandria j med. 2018;54:287–293. 43. agarwal a, gupta s, sekhon l, shah r. redox considerations in female reproductive function and assisted reproduction: from molecular mechanisms to health implications. antioxid redox signal. 2008;10: 1375–1403. 44. agarwal a, virk g, ong c, du plessis ss. effect of oxidative stress on male reproduction. world j mens health. 2014;32:1–17. 45. lim j, luderer u. oxidative damage increases and antioxidant gene expression decreases with aging in the mouse ovary. biol reprod. 2011;84:775–782. 46. tatone c, amicarelli f, carbone mc, monteleone p, caserta d, marci r, et al. cellular and molecular aspects of ovarian follicle ageing. hum reprod update. 2008;14:131–142. 47. behrman hr, kodaman ph, preston sl, gao s. oxidative stress and the ovary. j soc gynecol investig. 2001;8:s40–s42. 48. jabbour hn, sales kj, catalano rd, norman je. inflammatory pathways in female reproductive health and disease. reproduction. 2009;138:903–919. 49. herath s, williams ej, lilly st, gilbert ro, dobson h, bryant ce, et al. ovarian follicular cells have innate immune capabilities that modulate their endocrine function. reproduction. 2007;134:683–693. 50. uri-belapolsky s, shaish a, eliyahu e, grossman h, levi m, chuderland d, et al. interleukin-1 deficiency prolongs ovarian lifespan in mice. proc natl acad sci u s a. 2014;111:12492–12497. 51. hussein mr. apoptosis in the ovary: molecular mechanisms. hum reprod update. 2005;11:162–77. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.06201901 45j contemp med sci | vol. 2, no. 6, spring 2016: 45–48 research objectives oral cancer includes a wide range of malignant neoplasms and is one of the ten main causes of morbidity and mortality worldwide. this study aimed to assess the level of knowledge of adults about the symptoms and risk factors of oral cancer in zanjan city. methods this descriptive, cross-sectional study was conducted on 345 adults presenting to a teaching hospital in zanjan in 2014. the data were collected using a questionnaire, which comprised of four main sections regarding symptoms and risk factors of cancer. the data were analysed using independent t-test and anova. results the mean score of knowledge of adults was 4.88 about the risk factors and 2.86 about the symptoms of cancer out of 12. no significant differences were noted in this regard between males and females or different age groups (p > 0.05). level of knowledge was significantly correlated with the level of education (p < 0.05). conclusion the score of knowledge of adults in zanjan about the symptoms and risk factors of cancer was lower than the average required value in the community. it is absolutely necessary to enhance the public knowledge about oral cancer via the media. keywords oral cancer, symptoms, risk factors, adults, knowledge knowledge of adults about the symptoms and risk factors of oral cancer in zanjan city neda gholamia, sara mehrabib, elham zajkanic, golnaz bashiriniad introduction oral cancer is among the most common cancers and one of the ten main causes of morbidity and mortality worldwide.1 it comprises of 2–3% of all cancers and is the 6th most common cancer in males and 12th most common cancer in females with a male to female ratio of 3:1.2,3 in some cases, the oral cancer occurs following the appearance of precancerous oral mucosal lesions. leukoplakia and erythroplakia are among the most important precancerous lesions.4 major risk factors for oral cancer include tobacco use, alcohol consumption, sunlight exposure, nutritional factors and human papilloma virus.5 oral cancer clinically manifests as a chronic wound, white plaque or red patch, which does not respond to anti-inflammatory treatments.6 studies have reported variable levels of knowledge of adults about oral cancer risk factors. level of knowledge has reported to be 63.3% by monteiro et al. (2012),7 70% by devadiga et al. (2010),8 27.6% by tomar et al. (2005)9 and 6.04 out of 15 by kakoei et al. (2009).10 oral cancer is often detected and diagnosed in advanced stages, which may be due to the lack of public knowledge about its risk factors and symptoms.11 considering the high prevalence of oral cancer in iran and lack of information regarding the level of public knowledge about its symptoms and risk factors in zanjan city, this study aimed to assess the level of knowledge of adults about the symptoms and risk factors of oral cancer in zanjan city. materials and methods this descriptive cross-sectional analytical study was conducted on 345 adults, presenting to a teaching hospital in zanjan. the subjects were randomly selected. first, a pilot study was carried out on 30 randomly selected subjects. the sequence method was used to calculate the sample size. according to the pilot study, the percentage of positive responses (p) was estimated and final sample size was calculated using the formula. a questionnaire with four main parts was used for data collection.12–14 the first part asked for the demographic information of subjects (age, sex and level of education). the second part included 12 questions regarding the risk factors. the third part included ten questions about the symptoms and the fourth part included two questions about the sources of information and the need for further education and information in this regard. the reliability and validity of the questionnaire were assessed using cronbach’s alpha, which was found to be 0.85 for knowledge about risk factors and 0.87 for knowledge about symptoms. in terms of scoring, each correct answer was allocated one positive score, and zero score was given to incorrect or no answers. statistical analysis the data were coded and entered into spss version 18 software (microsoft, il, usa). the frequency percentages were presented in tables and diagrams. independent t-test was used for pairwise comparisons and anova was applied for multiple comparisons. if the anova yielded significant differences, tukey’s post hoc test was applied. the level of significance was set at p = 0.05. results a total of 345 adults presenting to a teaching hospital in zanjan city participated in this study; out of which, 4 were excluded due to incomplete information. of 341 participants, 237 (69.1%) were males and 104 (30.3%) were females. in terms of level of education, 42.6% had a level of education below high school diploma, 53.1% had university education and 3.8% had issn 2413-0516 adepartment of oral and maxillofacial medicine, zanjan school of dentistry, zanjan university of medical science, zanjan, iran. bdepartment of oral and maxillofacial pathology, zanjan school of dentistry, zanjan university of medical science, zanjan, iran. cdepartment of operative dentistry, zanjan school of dentistry, zanjan university of medical science, zanjan, iran. dzanjan school of dentistry, zanjan university of medical science, zanjan, iran. correspondence to elham zajkani (email: elham.zajkanident@gmail.com). (submitted: 28 january 2016 – revised version received: 3 march 2016 – accepted: 15 april 2016 – published online: 26 june 2016) 46 j contemp med sci | vol. 2, no. 6, spring 2016: 45–48 symptoms and risk factors of oral cancer research elham zajkani et al. doctorate or higher degrees. the mean age of participants was 37.7 ± 13.2 years (range of 18–87 years). the frequency of correct answers to questions about the risk factors and symptoms of oral cancer is shown in tables 1 and 2. the highest level of knowledge of individuals was about tobacco use (79%) and alcohol consumption (69%) as risk factors of oral cancer. no significant difference was found in the mean knowledge score of different age groups (p = 0.615). no significant difference was noted in this regard between males and females either (p = 0.668). a significant difference was found in the mean knowledge score of individuals with different levels of education, and the score of knowledge about the risk factors increased with higher level of education (p = 0.000). level of knowledge of adults in zanjan city about oral cancer symptoms is shown in tables 3 and 4. the results showed that the level of knowledge of males in this regard was significantly higher than that of females (p = 0.000). also, a significant difference was found in the mean score of knowledge of individuals based on their level of education (p = 0.000). no significant difference was detected among different age groups (p = 0.307). the most commonly reported sources of acquiring information were the media (radio and television) (38.1%) followed by the internet (34%) and books (12%). discussion oral squamous cell carcinoma (scc) is among the most common oral cancers and is one of the ten most prevalent cancers worldwide.15 scc accounts for 94% of all oral malignancies. the major risk factors for oral cancer include tobacco use, alcohol consumption and exposure to uv radiation (sunlight).16 oral cancer clinically manifests in the form of a chronic wound, prominent lesion, white plaque or red patch. early diagnosis significantly increases the survival rate of patients. however, due to inadequate knowledge of individuals about the symptoms and risk factors of oral cancer, it is commonly detected in advanced stages.17 in our study, the mean knowledge score of participants about oral cancer was 4.88 out of 12, which indicates very low knowledge. kakoei et al. (2009) reported a mean knowledge score of 6 out of 15.10 tomar et al. (2005) reported a low level of knowledge of individuals in florida about cancer.9 devadiga et al. (2010) assessed the level of knowledge of subjects presenting to hospitals in india about oral cancer and found that 70% of the people considered tobacco to be a risk factor for oral cancer.8 the evidence shows that public knowledge about oral cancer is low and attempts must be made to enhance the public knowledge in this regard and encourage the people to quit their unhealthy behaviours and risky habits.18 in our study, the mean knowledge score of individuals about the risk factors of oral cancer was 2.86 out of 12. ariyawardana et al. (2005) stated that <50% of people were aware of the symptoms of cancer.19 horowitz et al. (2000) announced that 44% of the individuals could not even name one of the symptoms of oral cancer.20 tomar et al. (2005) found that only 27.6% of individuals had adequate knowledge about the symptoms of cancer.9 another study showed that people did not have adequate knowledge about the symptoms of oral cancer.21 table 2. the mean, standard deviation, minimum and maximum score of knowledge about the risk factors of oral cancer number mean standard deviation minimum maximum value degree of freedom p value knowledge 341 4.88 1.807 0.00 10.00 11.38 340 0.000 one sample t-test; mean = 6. table 1. the frequency of correct answers of adults in zanjan to knowledge questions about the risk factors of oral cancer number questions correct answers frequency percentage 1 long-term use of antibiotics 111 32.4 2 tobacco use 272 79.3 3 allergy to foods and drugs 115 33.5 4 alcohol consumption 206 60.1 5 old age 185 53.9 6 male gender 71 20.7 7 mouthwashes 191 55.7 8 dentures 149 43.4 9 continuous exposure to sunlight 78 22.7 10 viral infections 165 48.1 11 low fruit and vegetable intake 125 36.4 12 history of cancer in a family member 124 36.2 table 3. the frequency of correct answers of adults in zanjan to knowledge questions about the symptoms of oral cancer number questions correct answers frequency percentage 1 white or red patch on the floor of the mouth or tongue 86 25.1 2 white spot on the cheeks that fades with pulling 125 36.4 3 presence of bilateral bony swellings in the palate 75 21.9 4 paresthesia of the tongue or other parts of the oral cavity 77 22.4 5 chronic earache 68 19.8 6 chronic wound with an indurated margin 120 35 7 any wart-like lesion 82 23.9 8 mobility of the teeth 96 28 9 problem in deglutition 98 28.6 10 feeling of a mass in the neck 107 31.2 47j contemp med sci | vol. 2, no. 6, spring 2016: 45–48 research symptoms and risk factors of oral cancerelham zajkani et al. having no knowledge about the first signs and symptoms of disease may result in negligence, not seeking medical care and subsequent serious consequences. information about the risk factors and early signs and the symptoms of oral cancer may not result in behavioural change (for instance quitting smoking) but can help patients make an informed decision.22 in our study, the participants with higher level of education had greater knowledge about the risk factors of cancer. the mean knowledge score about the risk factors of cancer was not significantly different between males and females or among different age groups. powe et al. (2004) found no association between the level of knowledge and age group or gender of subjects.23 kakoei et al. (2009) found no significant association between knowledge and age group of subjects either.10 a direct correlation between the level of knowledge and level of education was mentioned by kakoei et al. (2009),10 devadiga et al. (2010),8 croucher et al. (2011)24 and powe et al. (2004).23 in this study, 79 and 69% of participants mentioned tobacco use and consumption of alcoholic beverages, respectively as the risk factors of cancer. in a study by ariyawardana et al. (2005), 80.7% of the individuals were not aware of the correlation of tobacco chewing and oral cancer.19 in a study by ashe et al. (2005), the participants believed that cigarette smoking and alcohol consumption were the two important risk factors for the occurrence of pharyngeal carcinoma.25 in a study by huang et al. (2003), only 13% of subjects were aware of the fact that consumption of alcohol increases the risk of oral carcinoma.26 in our study among the risk factors, male gender and exposure to sunlight had the lowest frequency of correct answers. our findings regarding sunlight are in line with the results of powe et al. (2004),23 kakoei et al. (2009)10 and horowitz et al. (2000).20 in general, this study showed that the level of knowledge of adults in zanjan was low about the symptoms and risk factors of oral cancer. also, males had a significantly higher level of knowledge about the symptoms of oral cancer than females. this difference between males and females and higher familiarity of males with the symptoms of oral cancer may be due to the higher prevalence of oral cancer among males.27 in this study, the most commonly reported sources of acquiring information were the media (radio and television) followed by the internet. this finding indicates the role of the media in enhancing the public knowledge about the risk factors and symptoms of oral carcinomas.16 croucher et al. (2011) showed that distribution of brochures significantly promoted the level of knowledge of individuals.24 conclusion the level of knowledge of adults in zanjan about the risk factors and symptoms of oral cancer was lower than the average required value in general population. the knowledge of individuals about the role of cigarette smoking and tobacco consumption in occurrence of oral cancer was greater than about other factors. moreover, the participants expressed the need for broadcasting of informative programs regarding oral and dental health in the media.  table 4. the mean, standard deviation, minimum and maximum score of knowledge about the symptoms of oral cancer number mean standard deviation minimum maximum value degree of freedom p value knowledge 341 2.8622 1.176 0 8 18.139 340 0.000 one sample t-test; mean = 5 references 1. epstein j, van der waal i. oral cancer, 11 th ed., in: greenberg ms, glick m, (eds): burket’s oral medicine, diagnosis and treatment. hamilton: bc decker; 2008. pp.153–189. 2. wen cp, tsai sp, cheng ty, chen cj, levy dt, yang hj, et al. uncovering the relation between betel quid chewing and cigarette smoking in taiwan. tob control. 2005;14(1):16–22. pmid: 15923442 3. nicotera g, gnisci f, biabnco a, angelillo if. dental hygienists and oral cancer prevention: knowledge, attitudes and behaviors in italy. oral oncol. 2004;40:638–644. pmid: 15063393 4. cannick gf, horowitz am, drury tf, reed sg, day ta. assessing oral cancer knowledge among dental students in south carolina. j am dent assoc. 2005;136:373–378. pmid: 15819353 6. casto bc, sharma s, fisher jl, knobloch tj, agrawal a, weghorst cm: oral cancer in appalachia. j heath care pool underserved. 2009;20:274–85. doi: 10.1353/hpu.0.0097 pmid: 19202262 7. monteiro ls, salazar f, pacheco j, warnakalasuriya s. oral cancer awareness and knowledge in the city of valongo, portugal. int j dent. 2012;2012:1–8. 8. devadiga a, prasad kv. knowledge about oral cancer in adults attending a dental hospital in india. asian pac j cancer prev. 2010;11:1609–1613. pmid: 21338205 9. tomar sl, logan hl. florida adult’s oral cancer knowledge and examination experience. j public health dent. 2005;65(4):221–30. pmid: 16468464 10. kakoei s, rad m, mahmoudvand n, mohammadalizadeh s. a survey of the kerman adults’ knowledge about the signs and risk factor of oral carcinoma. shiraz univ dent j. 2009;10(3):234–240. 11. peterson pe. oral cancer prevention and control: the approach of the world health organization. oral oncol. 2009;45:454–60. doi: 10.1016/j. oraloncology.2008.05.023 pmid: 18804412 12. rodriguez t, altieri a, chatenoud l, gallus s, bosetti c, negri e, et al. risk factor for oral and pharyngeal cancer in young adults. oral oncol. 2004;40:207–13. pmid: 14693246 13. pelucchi c, tamani r, negri e, levi f, conti e, franceschi s, la vecchia c. folate intake and risk of oral and pharyngeal cancer. ann oncol. 2003;14:1677–81. pmid: 14581278 14. lambert r, sauvaget c, de camatgo cancela m, sankaranarayanan r. epidemiology of cancer from the oral cavity and oropharynx. eur j gastroenterol hepatol. 2011;23:233–41. doi: 10.1097/ meg.0b013e3283484795 pmid: 21654320 15. saman dm. a review of the epidemiology of oral and pharyngeal carcinoma. head neck oncol. 2012;4:1. doi: 10.1186/1758-3284-4-1 pmid: 22244087 16. loyha k, vatanasapt p, promthet s, parkin dm. risk factor for oral cancer in northeast thailand. asian pac j cancer prev. 2012;13(10):5087–5090. pmid: 23244115 17. soler m, bosetti c, franceschi s, negri e, zambon p, talamini r, et al. fiber intake and the risk of oral, pharyngeal and esophageal cancer. int j cancer. 2001;91:283–7. pmid: 11169948 18. wright jm. oral precancerous lesions and conditions. semin dermatol. 1994;13:125–131. pmid: 8060824 19. ariyawardana a, vithanaarachchi n. awareness of oral cancer and precancer among patients attending a hospital in srilanka. asian pacific j cancer prev. 2005;6:58–61. pmid: 15780034 48 j contemp med sci | vol. 2, no. 6, spring 2016: 45–48 symptoms and risk factors of oral cancer research elham zajkani et al. 20. horowitz am, nourjah p, gift hc. u.s. adult knowledge of risk factors and signs of oral cancers: 1990. j am dent assoc. 2000;126:39–45. pmid: 7822644 21. epstein jb, feldman r, dolor rj, porter sr. the utility of tolonium chloride rinse in the diagnosis of recurrent or second primary cancers in patients with prior upper aerodigestive tract cancer. head neck. 2003;15:911–921. pmid: 14603451 22. gandolfo s, pentenero m, broccoletti r, pagano m, carrozzo m, scully c. toluidine blue uptake in potentially malignant oral lesion in vivo: clinical and histological assessment. oral oncol. 2006;42:89–95. pmid: 16256415 23. powe bd, finnie r. knowledge of oral cancer risk factors among african americans: do nurses have a role? oncol nurs forum. 2004;31:785–791. pmid: 15252432 24. croucher r, islam ss, nunn h. campaign awareness and oral cancer knowledge in uk resident adult bangladeshi: a cross-sectional study. br j cancer. 2011;105:925–930. doi: 10.1038/bjc.2011.317 25. ashe te, elter jr, southerland jh, strauss rp, patton ll. north carolina dental hygienists’ assessment of patients’ tobacco and alcohol use. j dent hyg. 2005;79:9. pmid: 16197766 26. huang wy, winn dm, brown lm, gridley g, bravo-otero e, diehl sr, et al. alcohol concentration and risk of oral cancer in puerto rico. am j epidemiol. 2003;157:881–887. pmid: 12746240 27. mousavi m, gouya m, ramazani r, davanlou m, hajsadeghi n, seddighi z. cancer incidence and mortality in iran. ann oncol. 2009;20(3):556–63. 146 j contemp med sci | vol. 6, no. 4, july-august 2020: 146–149 original issn 2413-0516 introduction the covid-19 has considered an international emergency problem threatening public health on the 30 of january 2020.1 coronaviruses are important human and animal pathogens which can cause diseases such as the middle east respiratory syndrome (mers) and the severe acute respiratory syndrome (sars).2,3 a novel coronavirus was identified as the cause of a cluster of pneumonia cases in wuhan, a city in the hubei province of china.1 in december 2019, 27 cases of pneumonia were recorded in that city.4 wuhan is a very crowded city in china, the first recorded 27 patients were having clinical symptoms like dry cough, dyspnea, fever, and bilateral lung infiltrates on imaging.4 the center of this infection problem is linked to wuhan’s huanan seafood wholesale market, dealing with fish and many other animals including poultry, bats, marmots, and snakes. the local medical center makes the required investigation for the patient and approves that the 27 patients have a severe acute respiratory syndrome coronavirus 2 (sars-cov-2).4 the disease was named covid-19 by the world health organization (who).5 despite that, most of the infected cases have spontaneous recovery but the other developed fatal symptoms include organ failure, septic shock, pulmonary edema, severe pneumonia, and acute respiratory distress syndrom (ards).6 most of the cases which were suffering from fetal symptoms were from the old age group and the people who have other medical issues including cardiovascular, cerebrovascular, endocrine, digestive, and respiratory disease.7 then, it rapidly spread over the adjacent cities, resulting in an epidemic throughout china, followed by an increasing number of cases in other countries around the world. the who declared the chinese outbreak of covid19 to be a public health emergency of international concern posing a high risk to countries with vulnerable health systems. the emergency committee in the who announced that following strict instructions related to isolation, provide the social distance, quarantine for the children, elder peoples, and for those who have symptoms of pneumonia and fever have a role to prevent the spread of covid-19 infection.6 according to the who report,8 a €10,000,000 research fund to contribute to more efficient clinical management of patients infected with the virus, as well as public health preparedness and response. the united kingdom government offers £20,000,000 to develop a vaccine for covid-19.8,9 despite the preventive recommendations to prevent the transition of virus infection in the countries, the governments in most countries around the world have suspended all entry of immigrants and non-immigrants having traveled to high-risk zones.10 hong kong has also suspended several public transport services across the border and many hospital workers and civil servants are currently on strike. strikers are demanding that the border to mainland china be closed completely to prevent further covid-19 transmission.11 according to the last announcement for the who, the general director of the organization announced that until the 9 june, 7 million cases have been reported of covid-19 and 4000 deaths. until the 9 june, new covid-19 has been recorded from different countries including america and south asia. most countries awareness and response to coronavirus disease (covid-19) epidemic among the arab population in the eastern mediterranean region and arab peninsula mhammad saleh1, marwan o m saleh2, mohammed nabil zahid3 1 department of prosthodontics, faculty of dentistry, near east university, nicosia, turkish republic of northern cyprus 2 faculty of medicine, near east university, nicosia, turkish republic of northern cyprus 3 department of preventive dentistry, prince sattam bin abdalaziz university, kingdom saudi arabia. corresponding author: mhammad saleh (e-mail: dr.m.salehh@gmail.com) abstract objectives: the coronavirus (covid-19) causes fatal symptoms especially in areas with poor medical care. this online survey planned to analyze the knowledge and apprehension about coronavirus among the arab populations.  methods: a cross-sectional questionnaire-based survey was conducted from 15 may 2020 to 27 may 2020. the survey included a total of 443 arab participants. divided into four groups according to the age; under 20 years old (28 participants), 20–40 (359), 40–60 (49), and 7 participants were over 60. according to gender; male (318 participants) and female (125). based on education level, participants were categorized as a secondary school (5 participants), high school (28), university graduated (327), and postgraduate (83).  results: most of the participants showed a good adaptation for the precautions concerning isolation and quarantine. 299 participants stayed at home during the covid-19 outbreak by taking a break from jobs or performing their jobs from home. 144 participants performed partial or full-time jobs from 20 to 60 groups of age. most of the population were dependent on social media to receive the update about the virus. 141 participants said that they are not up-to-date enough about the covid-19 related to the language barrier. conclusion: the majority of the participants had heard about covid-19 and were aware of the infection control measures. most of the participants strictly adapted to quarantine during the outbreak. further steps need to be taken to enhance the social media accounts and internet websites in the arabic language which concern medical and educational content.  keywords: covid-19, emr, respiratory infection, aerosol infection. 147 original awareness and response to coronavirus disease (covid-19) epidemic among the arabmhammad saleh j contemp med sci | vol. 6, no. 4, july-august 2020: 146–149 in africa express increasing cases, eastern europe and central asia record some increase in the last days. until the day of writing this paper (11 june 2020) related to the report of the world health organization in the eastern mediterranean region the total report cases were 687,464 and death cases were 15,405 and the daily notification of confirmed covid-19 in emr was stably increasing until the day of writing this articles. the route of the transition of this infection depends on inhaling the airborne droplets or touching surfaces contaminated with the virus and then touching the mucosal membrane in the mouth, nose, or eye. for this reason, the preventive methods depending on isolation and avoiding the crowding area and following the general hygiene instruction are very important to be followed.12 these instructions were very simple to be applied but they need cooperation and a full understanding of people to stop spreading this infection. the risk of contamination will be more in areas where people speak different languages, the language barrier will prevent the ability for these people to be up-to-date with the everyday news and regulations which are important to be followed. with all the restrictions, which have been applied by the governments to apply home isolation to prevent viral infection spread of covid-19, the people will stay in need to get out for their needs. all hospital and dental clinics continue to accept emergency cases during the break, and in these situations, the cooperation of the people and following the preventive instruction is very important to prevent the transition of infection. the dental clinics considered as the most high-risk area to spread infection of covid-19, according to the who;6 moreover, the dental clinic could be a riskier environment for spreading the virus because of the close contact with patients and the nature of the dental treatment.7 although patients diagnosed with covid-19 are not supposed to receive dental treatments, dental emergencies can occur, and close contact would be unavoidable. furthermore, both the relatively prolonged incubation period of the disease (the median incubation period was estimated to be 14 days before any symptoms could even be detected), which could increase the transmission of the disease during these lay periods.13 in this study, a survey has released to evaluate the level of knowledge of the arab people who are living in different countries and to evaluate the response of these people when they are being in a high-risk area like dental clinics as an example, to be able to evaluate the adaptability of these people with preventive instructions of the governments and health organizations. material and method a cross-sectional questionnaire-based survey was conducted from 15 may 2020 to 27 may 2020. ethical clearance was obtained from the institutional review board of prince sattam bin abdulaziz university (psau2020018). the online questionnaire planned to be shared over the social media platforms (facebook, instagram, and whatsapp) to reach the maximum number of participants who belong to arabic ethnic origin and speak arabic as a mother language. the questions in the survey written in the arabic language to be sure that all the participants are from the arab population. the questionnaire consisted of 16 self-prepared questions and had 4 parts. the first part gathered personal information (age, gender, education, and career). the second part is designed to evaluate the ability of the population to understand the new regulations related to covid-19 and see the level of the response to these regulations. in the third part, we ask the population questions about the sources they depend on to be up to date to the regulations related to covid-19 and to evaluate the type of sources which the arabic native speaker is able to follow. according to the report of the world health organization, the dental clinic and dentistry as a career classified as one of the highest risks to spread the covid-19. for this reason, we used dental clinics as an example in the fourth part of the survey. this part of the questionnaire designed to evaluate the response of the population and evaluate their knowledge about the new regulations related to visiting dental clinics during the outbreak. descriptive static analysis performed for participants’ characteristics (gender, age, and level of education). the univariate linear model analysis performed to see the effect of age and level of education as an independent variable on the awareness about the cocid-19 as an outcome (dependent variable). statistical analysis was performed using version 23 of spss statistics (ibm spss statistics, new york, united states). result in the present study, we included a total of 443 arab participants living in different countries of emr. according to age, participants were categorized in 4 groups as under 20 years old (28 participants), between 20 and 40 years old (359 participants), between 40 and 60 years old (49 participants), and over 60 of age (7 participants). 81% of the participants were from one group (20-40). based on gender, participants were categorized into two groups; male (318 participants/71.8%) and female (125 participants/28.2%). based on education level, participants were categorized as a secondary school (5 participants), high school (28 participants), university graduated (327 participants), and postgraduate (83 participants). in the sixth question, the survey estimated information about how this population performed their jobs during the break period, and the answers were as following; 126 participants (24.4% of the participants) get a break from work during the quarantine period, 173 (39.1% of the participants) perform their jobs from home, whereas 144 of the participants (32.5% of the population) were performed partial or full-time jobs. most of the participants who performed the partial or full-time job were between 20 and 60 of age, no one of over 60 of age in our study performs any outdoor job during the quarantine. in the second part of the questionnaire, the goal from the questions was to evaluate the level of arab population knowledge related to the covid-19 and all the recommendations which are very important to be understood and followed to prevent the spread of infection with covid-19. concerning the who’s regulations and instructions, 121 of the participants (37.3%) don’t have enough knowledge related to the risk of the airdrops, and how to avoid this infection. 6 participants out of 443 (1.4%) answered the question as they don’t have any idea related to it. 6 participants who replied negatively were in groups of age between 20 and 40 years old, this group who was performing a part-time or full-time during the covid-19 break. the age and educational level considered a significant factor in determining the level of awareness related to the disease. concerning wearing gloves, 17 participants (3.8%) don’t know any information about the world 148 original awareness and response to coronavirus disease (covid-19) epidemic among the arab mhammad saleh j contemp med sci | vol. 6, no. 4, july-august 2020: 146–149 health organization related to gloves wearing. 422 participants (95.3%) were believed about the importance of the social distance, and they apply it when they are out of their homes. when we asked the population if they are updated to all the information related to the covid-19, 302 of the participation (68.2%) approved that they are updated. 31 of the participants (7%) answered that they are not up-to-date. 110 participants (24.8%) of the population are not sure if they are updated enough. most of this population depends on the internet to get updates and news related to the covid-19 more than television, newspaper, and printed paper. 225 participants (57.6%) obtained the required information related to the covid19 from the websites of the states and the world health organization. 137 participants (30.9%) depend mostly on social media (facebook, instagram, and whatsapp). most of the population (268 participants/ 60.5%) found the medical source related to the covid-19 in the arabic language sources is enough. 134 of the participants (30.2%) didn’t believe that the arabic language sources are enough. 41 of the participants (9.3 %) depend only on un-arabic sources because they believe that it is more trustworthy. when we ask the population if they agree with the decision taken to suspend the work in dental clinics except for emergency cases, 312 of the population (70.4%) answered that this is important and they agree with it. 66 of the population (14.9%) say that they don’t have information about it. 65 of the population (14.7%) did not believe in the importance of this procedure. most of the participants in this study (375/84.7%) complied with the instructions of health organizations and declined to visit dental clinics. 41 of them (9.3%) visited the clinic for emergency cases, and 21 patients (4.7%) visited the clinic to complete the dental treatment which started before the break, 35 patients of the population did not comply to the instruction and continue to visit the dental clinics for esthetic and non-emergency procedures. discussion the center of the covid-19 changed from china where the number of the positively tested patients has reduced sharply, and now cases increase in the eastern mediterranean region (emr). according to the who emr, 19 of the 22 countries/ territories have been affected, mostly in the gulf states, turkey, and the islamic republic of iran.14 the challenges facing some countries in the emr, including complex emergencies, compounded by fragile health systems, weak disease surveillance, poor response capacity, and a suboptimal level of public health preparedness, the ministries of health of 16 of the 22 countries/territories have developed a national action plan against covid-19 with the support of the who regional office for the emr and other united nations agencies.14 the religious mass crowding events which are expected to take place in july 2020 in some countries of the eastern mediterranean region will pose a risk to public health.14 as such, this mass gathering brings the possibility of the propagation of respiratory infections.15 especially, that many of the countries of the eastern mediterranean region start to release the outbreak and life starts to be normal partially or fully gradually. in these circumstances, the public should be responsible to continue the preventive procedures like the social distance and general hygiene to be able to avoid a second wave of the covid-19 infection spread. in addition, laboratory capacities are limited, with 20 of the 22 countries in the emr having a functioning reference laboratory with the ability to detect and confirm mers-cov and other high-threat pathogens. when it comes to the detection of a newly emerging pathogen, a lot of training and guidance with the provision of diagnostic kits and laboratory supplies will be required.14 this survey provides an insight into the level of awareness, perception, and attitude of the arab population who are living in different countries on infection control with a special emphasis on covid-19 at the time of the outbreak in 2020. this study included a random sample of candidates at different groups of age and different educational backgrounds. most of the participants were from the age group between 20 and 40 of age, the reason related to the ability of this group of age to be active on websites and social media more than other groups of age. this could be considered as a limitation of this study because over 60 of age is the group under risk of complication and fetal deterioration related to cover-19. and the elder group of age is the group that is not expected to be up to date for the health precautions. but the only way to reach the participants during the outbreak was the internet and social media. the author expected different results if he had the chance to make a real survey to reach the required groups (elderly people). most of the participants were from the university and postgraduate groups regarding the level of education because this group of people was more reachable over social media to be included in the study. most of the participants show a good adaptation for the precautions concerning isolation and quarantine. 299 participants stayed at home during the covid19 outbreak by taking a break from jobs or performing their jobs from home. 32.5% of participants who perform partial or full-time jobs were from 20 to 60 groups of age which consider the less risky age group related to the complications and fetal deteriorations compensate with covid-19 infection.13 besides that, according to the results of this study, this group of age (20– 60) shows a high level of awareness about transmission methods of the virus and the protected methods to avoid infections. the estimated incubation period of covid-19 is up to 14 days.13 people in this study varied in their knowledge about the incubation period of the disease, but it is essential to know the right incubation period because of its role in determining the safe period to be out of the quarantine especially when the people will come to normal life partially or completely.13 the route of the transition of this disease is expressed by the snowball theory, according to research more than two new cases expected with every new case of covid-19.16 the difficulty of preventing the transition of the infection between the individuals is the probability of transition before the onset of symptoms or very early symptoms.15, 16 for this reason, the present control method which depends on temperature screening is not a trustful method, and there is a critical need for accurate contact tracing starting from the day before the onset of symptoms as well as strict quarantine measures and monitoring before more chains of contagion are established.17 a recent study showed that many countries in africa including some countries that are part of the emr have the variable capacity to respond to outbreaks and high vulnerability.18 besides the protracted conflicts in many countries in the region, lack of infrastructure, limited resources, inadequate prevention control practices, poor preparedness capacity, and inadequate 149 original awareness and response to coronavirus disease (covid-19) epidemic among the arabmhammad saleh laboratory infrastructures and resources in many countries in the emr are among the main barriers to adequately detect and respond to covid-19.17 nsour et al in this study highlights the contribution of emr’s fetps to the preparedness capacities in countries in the emr to respond to the current covid19 threat.17 the purpose of the field epidemiology training program (fetp) is to train the population capacity to detect and respond to health threats and establish local experts in the area of epidemiology.19 these programs establish a real-time and real place to practice and focus on building knowledge trainers to contribute to their country’s health system to detect, notify, report, and respond to events that threaten the national and international health. the programs include many of the smr’s countries like egypt, iraq, jordan, morocco, pakistan, saudi arabia, sudan, tunisia, and yemen.17 the fetps in the emr has a plan for activities to strengthen countries’ preparedness against covid-19. the fetps participated actively in airport surveillance; implemented temperature screening at ports of entry; developed communication materials and guidelines; and shared information to health professionals and the public, often with a 24-hour dedicated hotline. however, some countries remain ill-equipped, have the poor diagnostic capacity, and are in need of further capacity development in response to public health threats. it is essential that fetps continue building the capacity to respond to covid-19 and intensify support for preparedness and response to public health emergencies. despite the findings introduced in this study, it is important to stress that this survey had limitations, including the relatively low response rate, which resulted in a smaller than expected sample size. this could have been caused by a short period of data collection and the limitation in the online methods for reaching the participants over the social media platforms. however, this is considered a moderate sample size. conclusion the majority of the participants had heard about covid19 and were aware of the infection control measures. most of the participants strictly adapted to quarantine during the outbreak, and the young group of age who was in needs to perform the job during the outbreak have a good knowledge related to covid-19 and the method of protection. further steps need to be taken to enhance the social media accounts and internet websites in the arabic language which concern medical and educational content. there is currently no standard therapy for covid-19 and the proposal of digital tools for the contact tracing ready to be applied related to security and privacy issues. so, self-awareness and following health precautions are very important. abbreviations emr: eastern mediterranean region fetp: field epidemiology training program mers: middle east respiratory syndrome mers-cov: middle east respiratory syndrome coronavirus sars: severe acute respiratory syndrome who: world health organization references 1. meng l, hua f, bian z. coronavirus disease 2019 (covid-19): emerging and future challenges for dental and oral medicine. j dent res. 2020;99:481– 487. 2. de wit e, van doremalen n, falzarano d, munster vj. sars and mers: recent insights into emerging coronaviruses. nat rev microbiol. 2016;14:523–534. 3. yin y, wunderink rg. mers, sars and other coronaviruses as causes of pneumonia. respirology. 2018;23:130–137. 4. lu h, stratton cw, tang y. outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle. j med virol. 2020. pp. 401–402. doi:10.1002/jmv.25678 5. world health organization. who global report on traditional and complementary medicine 2019. world health organization; 2019. 6. chen n, zhou m, dong x, qu j, gong f, han y, et al. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet. 2020. pp. 507–513. doi:10.1016/s0140-6736(20)30211-7 7. wang d, hu b, hu c, zhu f, liu x, zhang j, et al. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in wuhan, china. jama. 2020. p. 1061. doi:10.1001/jama.2020.1585 8. sohrabi c, alsafi z, o’neill n, khan m, kerwan a, al-jabir a, et al. world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19). int j surg. 2020;76:71–76. 9. research cm, case medical research. xiyanping injection for the treatment of new coronavirus infected pneumonia. case medical res. 2020. doi:10.31525/ct1-nct04275388 10. el-tholotha m, bau hh, song j. a single and two-stage, closed-tube, molecular test for the 2019 novel coronavirus (covid-19) at home, clinic, and points of entry. doi:10.26434/chemrxiv.11860137.v1 11. parry j. china coronavirus: hong kong health staff strike to demand border closure as city records first death. bmj. 2020. p. m454. doi:10.1136/bmj. m454 12. world health organization. who guidelines on hand hygiene in health care: first global patient safety challenge: clean care is safer care. world health organization; 2009. 13. abbott s, hellewell j, munday j, funk s, cmmid ncov working group. the transmissibility of novel coronavirus in the early stages of the 2019-20 outbreak in wuhan: exploring initial point-source exposure sizes and durations using scenario analysis. wellcome open research. 2020. p. 17. doi:10.12688/wellcomeopenres.15718.1 14. al-tawfiq ja, memish za. covid-19 in the eastern mediterranean region and saudi arabia: prevention and therapeutic strategies. int j antimicrob agents. 2020;55:105968. 15. benkouiten s, al-tawfiq ja, memish za, albarrak a, gautret p. clinical respiratory infections and pneumonia during the hajj pilgrimage: a systematic review. travel med infect dis. 2019;28:15–26. 16. wu jt, leung k, leung gm. nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study. the lancet. 2020. pp. 689–697. doi:10.1016/s0140-6736(20)30260-9 17. nsour ma, al nsour m, bashier h, al serouri a, malik e, khader y, et al. the role of the global health development/eastern mediterranean public health network and the eastern mediterranean field epidemiology training programs in preparedness for covid-19. jmir public health surveill. 2020. p. e18503. doi:10.2196/18503 18. gilbert m, pullano g, pinotti f, valdano e, poletto c, boëlle p-y, et al. preparedness and vulnerability of african countries against importations of covid-19: a modelling study. lancet. 2020;395:871–877. 19. white me, mcdonnell sm, werker dh, cardenas vm, thacker sb. partnerships in international applied epidemiology training and service, 1975-2001. am j epidemiol. 2001;154:993–999. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i4.809 83j contemp med sci | vol. 2, no. 7, summer 2016: 83–87 research survey of congenital malformation concerned to multiple factors in alzahraa hospital childbed and brats in al-najaf al-ashraf al-zubaidi ka,a methak adul razzaq. shemki,b widad h. yahyab issn 2413-0516 adepartment of anatomy, histology and embryology, college of veterinary medicine, university of al-qassim green, babylon, iraq. bdepartment of biology, college of education for girls, university of kufa, najaf, iraq. correspondence to al-zubaidi k.a. (e-mail: kalzubaidi@yahoo.com). (submitted 11 june 2016 – revised version received 30 june 2016 – accepted 2 july 2016 – published online: 26 september 2016) introduction congenital anomalies are also known as birth defect congenital disorders or congenital malformation. congenital anomalies can be defined as structural or functional anomalies can including metallic disorders which are present at the time of the birth approximately 50% of all congenital anomalies cannot be linked to specific causes there are some known causes or risk factors. socioeconomic factors it may be indirect determinant congenital anomalies are more frequent among resource constrained families and countries it is estimated that about 94% of sever birth defect occur in middle and low resource countries where the mothers are more susceptible to macronutrient and micro nutrient malnutrition and may have increased exposure to agents and factors that induce or increase the incidence of abnormal parental development particularly infection and alcohol advanced maternal age also increase the risk of some chromosomal abnormalities including down syndrome. genetic factor consanguinity (relationship by blood) increases the prevalence of rare genetic congenital animosities and nearly doubles the risk for neonatal and childhood death intellectual disability and series birth anomalies in the first cousin unions. some ethnic communities. e.g. ashkenazi jews or finns have comparatively high prevalence of rare genetic mutations leading to a risk to congenital anomalies infection; maternal infections such as syphilis and rubella are significant cause of birth defect in low and middle-income countries. maternal nutritional status; iodine deficiency folate insufficiency; obesity or diabetes mellitus are linked to some congenital anomalies for example folate insufficiency increases the risk of having a baby with neural tube defects environmental factors maternal exposure to pesticides medications alcohol; tobacco and other psychoactive substance certain chemicals high doses of vitamin a during the early pregnancy high doses of radiation increase the risk of having a fetus or infant affected by congenital anomalies working or living near or in waste site, smelters, or mines may also be a risk factor congenital malformation causing deformities have been described since early times primitives man interest in these phenomena has found expression in drawing. carvings and sculptures throughout the world including australians the south pacific islands and the america. written of congenital malformations have come down from najaf in the form of clay tables from the royal library of nineveh which was assembled by the assyrian kink ashurbanipal (c.700bc) these tables include a list of sixty two human malformation with their associated prophetic implication.1,2 these malformation include a cases of a sacral spin bifida in tarxien phase material other abnormalities include absence of the sagittal suture in a skull from hale saflieni hypogeum. the earliest depicted congenital malformations in malta data from the neolithic era and include cases of abnormalities of the hand one with three digits and one with six digits these representation where discovered in an incised decoration frogzibbu tombs at zebbug (malta) and a statuette from hagar qim. polydactyl also evident in a hand print described from the hal saflieni hypogeum,3,4 other skeleton remains showing congenital anomalies were excavated from roman tombs. these included a non-pathological anatomical variation of the sacrum. here the transverse process of the first sacral vertebra was not fused with the rest of the bone. in addition two adult skulls from st. agatha catacombs showed features of non-union of the frontal bone.5,6 early modern congenital anomalies from excavation of burials in maltese churches. these skeletal anomalies included diverse sacral anomalies including a case of spina bifida and other minor anatomical variations.7,8 a case of severe malformation was described in 1788 by dr. saverio fenech. the report records the birth of a monster born to a weman at nadur. the objectives this study was aimed to survey the number of congenital malformation in najaf province throughout all data which be collected from hospital (age; sex; blood group; kind of delivery; weight neonates). methods the present study was carried out on (120) congenial malformation in newborns cases in najaf obstetric hospital; from period extend from august (2014) to march (2015). the age of their mother range from 15to 45-years old. results the higher percentage (49.7%) in the mothers at age was ranged (25–35) years exposed to congenital malformation. the gender percent in the males (n = 72), 59% more than females (n = 49), 40.3% the blood group of neonate was more infected with malformation which involved o+ blood group (n = 43), 36.1% and b+ blood group (n = 36), 30.3% the most common neonate congenital malformation were septicemia (n = 38), 31.9%, cardiovascular and respiratory system malformation (n = 29), 24.4%.the normal delivery was (n = 74), 62% and number of mothers was exposed to surgical operations (cesarean section) estimated (n = 45), 37.8% from the total number of the delivery mothers were suffered from congenital malformation in their neonates. conclusion the male percentage was more risk than female in risk, also the blood group o+ and b+ more risk for anomalies cases. the ages of pregnant range (25–35) high ratio be ours babies to be anomalies. keywords congenital malformation, childbed, brats 84 j contemp med sci | vol. 2, no. 7, summer 2016: 83–87 survey of congenital malformation concerned to multiple factors in alzahraa hospital childbed and brats research al-zubaidi ka et al. table 2. the percentage of gender in the neonates with congenital malformation in najaf obstetric hospital neonates gender frequency percent valid percent cumulative percent valid male female total 72 48 119 59.7 40.3 100.0 59.7 40.3 100.0 59.7 100.0 child had a head and ears which resembled those of a cat. the upper limbs were human like but without articulation the hand being similar. to those of a cat the genital part were also similar to those of female cat the attention given to the description to gather with the added comment that the from requiring attending priest considered these characters as conforming more to human from requiring christian burial suggest that the religious concepts regarding malformations were still prevalent this was not surprising since these concepts remain prevalent until the early decades of the twentieth century.9 there was evidence of three-legged conjoined twin who lived in malta for some time in the early twentieth century. photographs taken in malta of a seven to nine year old child with this anomaly have been found among the belongings of professor ruggiero busuttil. the origins of this child remain unknown and it has been suggested that the child may have been the same three-legged francisco lentini who was born in sicily in 1889 and who later emigrated to america where he joined a number of circuses. this study was aimed to survey the number of congenital malformation in najaf province throughout all data which be collected from hospital (age; sex; blood group; kind of delivery; weight neonates). materials and methods the study was conducted on the delivery pregnant women in the najaf obstetric hospital the study carried out in period august, september, october, november, december of 2014 and january, march of 2015. number of cases study was reached to (290) case with congenital malformations involved (n = 148) male and (n = 144) female. the age of delivery pregnant women was range from 15 to 45-years-old. the study was involved weight of neonates, gender, type of malformation, blood groups kinds of neonate as well as mother and the kind of delivery (normal or caesarean) birth the result will be done by: biostatical analysis used (spss) system and t-test as well as analysis difference test to determine the relationship between: 1. mother age and congenital malformations during period of the study. 2. the relationship among month of years study and congenital malformations. 3. biostatical comparative study between the sex of congenital malformations and gender. results this study that the number of cases (120) of deformity in newborn at najaf obstetric hospital; at period extend from august 2014 to february 2015 the age of delivery mothers (1545) years the high percentage was appeared in (25–35) age was (49.6)% in table 1. this investigated show the gender neonate male (n = 72) at percentage of 59.7% more than neonate females (n = 49) at percentage of 40.3%. this appear in table 2. our observation of the present study was displays relationship between blood group of newborn with their delivery table 3. the relationship between blood groups and neonates congenital defects in najaf obstetric hospital blood group frequency percent valid percent cumulative percent valid ab a b o total 15 26 36 43 119 12.6 21.0 30.3 36.1 100.0 12.6 21.0 30.3 36.1 100.0 12.6 33.6 63.9 100.0 table 1. explain the age of mothers concerned with congenital malformations in babylon obstetric hospital mothers age frequency percent valid percent cumulative percent valid <25 25–35 >35 total 47 59 14 120 39.5 49.6 10.9 100.0 39.5 49.6 10.9 100.0 39.5 89.1 100.0 mothers; which involved the (o+) blood group was no. (43) at percentage (36.1)% and blood group (b+) no. (36) at percentage (30)% from the neonate congenital malformation as show in table 3. the kinds of congenital defect in neonate in their delivery mothers were septicemia (n = 38) and percentage was 31.9%; cardiovascular system (n = 27) at percentage of 22.7% and respiratory defects (n = 29) at percentage of 24.4%. table 4 displays the type of congenital defects with blood group. our results pointed out (o+) blood group and (b+) blood group with high percentage when concerned with congenital malformation. the relationship between congenital defects and the type of delivery note in mothers exposed to normal delivery was (n = 74, at percentage 62% and mothers were exposed to surgical operation (cesarean section) reached (n = 45) at percentage 37% from total number of deliveries mothers as shown in table 5. discussion the result of this study reveals that the number of the cases of deformity during the years of study (2014, 2015) amounted to 120 out of the total 260 live birth in the maternity and children in the province of najaf. the comparison between the previous in the number of cases of deformity during the years (2005– 2008) that amounted to 37810 of deformity that is the highest than the number of deformity in 2014 and 2015) that amounted to 119 cases. the highest number of cases of deformity frequently is the year 2006 from the remaining years of the study, may return this rise to thereturn to the many etiological factors that occurred on the people of wars and subjected to explosions, chemicals pollution, environmental and others10 and also the cases of deformity in 2006 in previous study are higher than the cases of deformity in the present study in the years 2014 and 2015 that return to rhesus group incompatibility, alcohol, 85j contemp med sci | vol. 2, no. 7, summer 2016: 83–87 research survey of congenital malformation concerned to multiple factors in alzahraa hospital childbed and bratsal-zubaidi ka et al. table 4. relation between the types of congenital malformation with blood group at najaf obstetric hospital types of defects blood groups χ2 p-value a b o ab total malformation of the brain and anencephaly count expected count % within defects % within blood group % total 1 1.0 12.5% 6.7% 0.8% 1 1.7 12.5% 4.0% 0.8% 3 2.4 37.5% 8.3% 2.5% 3 2.9 37.5% 7.0% 2.5% 8 8.0 100.0% 6.7% 6.7% 16.120 0.47 prematurity + dyspnea and warp respiratory count expected count % within type of defects % within blood group % of total 4 3.7 13.8% 26.7% 3.4% 4 6.1 13.8% 16.0% 3.4% 9 8.8 31.0% 25.0% 7.6% 12 10.5 41.4% 27.9% 10.1% 29 29.0 100.0% 24.4% 24.4% multiple congenital abnormalities count expected count % within type of defects % within blood group % of total 4 2.1 23.5% 26.7% 3.4% 3 3.6 17.6% 12.0% 2.5% 4 5.1 23.5% 11.1% 3.4% 6 6.1 35.3% 14.0% 5.0% 17 17.0 100.0% 14.3% 14.3% deformation cardiovascular count expected count % within type of defects % within blood group % of total 1 3.4 3.7% 6.7% 0.8% 5 5.7 18.5% 20.0% 4.2% 12 8.2 44.4% 33.3% 10.1% 9 9.8 33.3% 20.9% 7.6% 27 27.0 100.0% 22.7% 22.7% bacterial septicemia count expected count % within type of defects % within blood group % of total 5 4.8 13.2% 33.3% 4.2% 12 8.0 31.6% 48.0% 10.1% 8 11.5 21.1% 22.2% 6.7% 13 13.7 34.2% 30.2% 10.9% 38 38.0 100.0% 31.9% 31.9% total count expected count % within type of defects % within blood group % of total 15 15.0 12.6% 100.0% 12.6% 25 25.0 21.0% 100.0% 21.0% 36 36.0 30.3% 100.0% 30.3% 43 43.0 36.1% 100.0% 36.1% 119 119.0 100.0% 100.0% 100.0% tobacco, and other psychoactive substance, down syndrome, metabolic and hormonal disorders sickle cell disorders and congenital hypothyroidism,11 and this results may correspond to the results of previous study that suggested that the high percent of malformation of pregnant women may be due to hormonal imbalance especially steroid hormones (progesterone, estrogen, or androgen) and gonadotrophic hormones as well (fsh, lg) as other causes of congenital malformations involved the malnutrition, chemical pollutions, and microbial agent, these suggestion which also accordance with the previous studies12,13 and the finding was also identical with the previous studies14–16 that they mentioned the effect of sex hormones on fetus have been documented but this previous studies are based mainly on the exposure of fetus to female sex hormones during the initial period of pregnant from another hand the drug intake during pregnancy include oral contraceptive pills, progesterone analogues to confirm pregnancy medications for medical ailments and sex selection drugs to bear male offspring and also when compared with previous studies in bahrain the congenital malformation caused by joint action of genetic liability and environmental factors17 and there are many environment factors that at one time have been suspected of playing a role in the causation congenital malformation18. the results of study shows that the rate of malformation in male births is more than female by 59.7% when compared with the previous study in (2006). the rate of malformations in the births female is by 55.7%10. the results of this study show that the malformations is higher in the age group of birth mothers (25–35 years) by the ratio of 49.6% when compared with the previous study fined that in 2006 among the age group of 40–44 years by 50% while in (2005), the most age group was 20–24 years by the ratio of 31.6% which in 2007 resembled 2006 that the age group of mothers took place was 40–44 years by 50%10. the results of this study show that the abnormalities by bacterial septicemia is the most frequent and the least frequent is malformation of brain and anencephaly in comparison with the previous study that showed the abnormalities in brain and spinal cord were the most frequent in the years 2005 and 2006–2008) and least frequent was the clefts lip and palate clefts10. the previous study shows that neural malformation (spinal bifida) represented the highest percentage of malformations in bahrain in (1995)17 but in kashan, islamic republic of iran, the most common malformations were genitourinary (32.1%), musculo skeleton (22%) and cardiovascular (14.7%)19. male infants are at greater risk for birth malformations and the highest incidence of birth. malformations is bacterial septicemia presented in this study while in previous studies male infants were greater risk for birth malformations and the highest incidence of birth malformations was facial clefts with cleft palate10,20. in this study, bacterial is the commonest anomaly associated (31.9%) followed by premature and dyspnea and deformity of respiratory system (24.4%) cardiovascular malformation are found in (22.7%) of cases, when you make comparison on the basis of age group of births mothers, we find that the most age group is (25–35) years by the ratio (49.6%) in the present years while in the previous year’s studies show that in (2005) was the most age group (2024) by the ratio (31.6%) the year (2006) was the highest age was (40–44) years by (50%) while in (2007) was resembled that in (2006) that the age group of mothers were (40–44) years by (50%) in (2008) was the common age group (25–29) years by the ratio of (35.5%). 86 j contemp med sci | vol. 2, no. 7, summer 2016: 83–87 survey of congenital malformation concerned to multiple factors in alzahraa hospital childbed and brats research al-zubaidi ka et al. table 5. the relationship between the kinds of delivery and congenital malformation at najaf obstetric hospital types of defects type of delivery total χ2 p-value normal surgical malformation of the brain and anencephaly count 2 6 8 13.2878 .010 expected count 5.0 3.0 8.0 % within type of defect 25.0% 75.0% 100.0% % within type of delivery 2.7% 13.3% 6.7% % of total 1.7% 5.0% 6.7% prematurity + dyspnea and warp respiratory count 23 6 29 expected count 18.0 11.0 290 % within type of defect 79.3% 20.7% 100.0% % within type of delivery 31.1% 13.3% 24.4% % of total 19.3% 5.0% 24.4% multiple congenital abnormalities count 7 10 17 expected count 10.6 6.4 17.0 % within type of defect 41.2% 58.8% 100.0% % within type of delivery 9.5% 22.2% 14.3% % of total 5.9% 8.4% 14.3% deformation cardiovascular count 15 12 27 expected count 16.8 10.2 27.0 % within type of defect 55.6% 44.4% 100.0% % within type of delivery 20.3% 26.7% 22.7% % of total 12.6% 10.1% 22.7% bacterial septicemia count 27 11 38 expected count 23.6 14.4 38.0 % within type of defect 71.1% 28.9% 100.0% % within type of delivery 36.5% 24.4% 31.9% % of total 22.7% 9.2% 31.9% total count 74 45 119 expected count 74.0 45.0 119.0 % within type of defect 62.2% 37.8% 100.0% % within type of delivery 100.0% 100.0% 100.0% % of total 62.2% 37.8% 100.0% conclusions there are more type of neonate anomalies but the high percent is septicemia and cardiovascular defect and respiratory system malformation.the blood group (o+ and b+) more risk to get congenital malformation. the male percentage was more than female in risk for anomalies cases. the ages of pregnant women range (25-35) high ratio be ours babies to be anomalies. n references 1. brodsky i. congenital abnormalities, tetratology and embryology: some evidence of primitive man’s knowledge as expressed in art and lore in oceania. med j australia. 1943;1:417. 2. warkany j. congenital malformations in the past. j chron dis. 1959;10:84–96. 3. savona-ventura c, mifsud a. prehistoric medicine in malta. proprint: malta, 1999; p. 104. 4. zammit t, singer c. neolithic representations of the human form from the islands of malta and gozo. j royal anthrpol lnstitute of great britain and ireland. 1924;54:67–100 5. casser p. medical history of malta. landon; welcome hist med libr;1964;8–9. 6. savona-ventura c, mifsud a, camilleri v. the anthropomorphology of classical skulls from malta. the oracle: journal of the grupp arkeologiku malti. 2000;1:3–7 7. pace jl, ramaswamy s. the finds: skeletal remains. in: blagg tfc, bonanno a, lutrell at, ed. excavations at hal millieri, malta: a report of the 1977 campaign conducted on behalf of the national museum of malta and the university of malta. malta;university press. 1990;84–95. 8. ramaswamy s, pace jl. the medieval skeletal remains from st. gregory’s church at zejtun (malta) part 1. paleopathological studies. arch ital anat embriol. 1979;84(1):43–53. 9. cassar p. change of sex sanctioned by a maltese law court in the eighteenth century. br med j. 1954;2:1413. 10. abdulhadi s, atheer ki, and abdul razzaq ya. survey of malformations at birth in al-najaf al-ashraf province. al-qadisiah medical j. 2011:7;12;99–105. 87j contemp med sci | vol. 2, no. 7, summer 2016: 83–87 research survey of congenital malformation concerned to multiple factors in alzahraa hospital childbed and bratsal-zubaidi ka et al. 11. world health organization. birth defects: sixty third world health assembly a63/10, provisional agenda item 11.7, 1 april. geneva, 2010 12. bandyopadhyay s, singh aj. sex selection through traditional drugs in rural north india. indian j community med. 2007;32(1):32–34. 13. michel fl, helen dh. registries of congenital anomalies: eurocat. enviro health perspectives supplements. 1993;101:153–157. 14. miller e, hare jw, cloherty jp, dunn pj, gleason re, soeldner js, et al. elevated maternal hemoglobin a1c in early pregnancy and major congenital anomalies in infants of diabetic mothers. n engl j med. 1981;304(22):1331–1334. 15. neogi sb. sex selectionan indian perspective online comment in the lancet. available at: www. lancet. com 2006. 16. abdul hs, leena am, methak aa, atheer ki. the relationship among mother ages, months of years, gender of neonate and congenital malformation in al-najaf city al-qadisiah med j. 2010;6(9):27–33. 17. al-arrayed ss. epidemiological of congenital abnormalities in bahrain. eastern mediterranean health j. 1995:2(2);248–252. 18. michel f, helen d. registries of congenital anomalies department of epidemiological and preventive medicine. 1991. denmark. 19. mosayebi z, movahedian ah. pattern of congenital malformation in consaguineous versus nonconsanguineous marriages in kashan university of medical sciences. 2007;13(4). 20. aqrabawi he. facial cleft and associated anomalies: incidence among infant at a jordanian medical center. neonatal unit, king hussein medical center, amman, jordan 2008;14(2). 2 j contemp med sci | vol. 2, no. 5, winter 2016: 2–8 research objectives the findings of the study have proved that there is a high significant positive relationship between the parents’ knowledge and their demographic variables (age, educational level, occupation and residential area). in general, knowledge of parents related to measles was low however, the parents applied preventive practices towards their children with measles. a cross-sectional study was conducted in al-elwyia paediatric teaching hospital in baghdad from the middle of june till the end of september 2014, in order to identify parents’ knowledge of their children with measles. aim to identify the effectiveness of traditional knowledge of parents’ whose children suffer from measles. methodology purposive sample of 100 parents who accompanied their children with measles have been selected. the reliability of the instrument was determined through a test and validating through a panel of experts. the data was analysed through the application of descriptive statistical analysis that include frequency, mean, mean of scores, standard deviation and percentage and the application of inferential statistical analysis that include pearson correlation coefficient, chi-square, and analysis of variance for the differences test of the study group. results the results of the study indicated a positive effect on the knowledge of parents. recommendations the researchers recommended preparing and implementing the knowledge for parents with measles, and for medical and nursing staff to give them knowledge about the condition. keywords measles, children, tradition, parents parents understanding on measles: a study in al-elwyia paediatric hospital in baghdad province, iraq abdul mahdi a. hasana & tareef fadhil rahamb issn 2413-0516 apaediatric & mental health nursing, college of nursing, babylon university, babylon, iraq. bcabp, dch, consultant at al-elwyia paediatric teaching hospital in baghdad city. correspondence to abdul mahdi a. hasan (email: abd_mahdi2003@yahoo.com). (submitted: 3 november 2015 – revised version received: 18 december 2015 – accepted: 12 january 2016 – published online: 26 march 2016) introduction many traditions regarding measles were recognised in many countries. these traditions include traditions regarding red dressings,1 spiritual, supernatural role, herbs and diets.2 these traditions might include amulets or jewellery on it, supernatural believes like diviners and marabouts, sea water.3 in general, ethno pharmacological investigations emphasise the importance of medicinal plants in developing countries, species used regularly with diet are under-investigation and potentially make greater contributions to health.2 measles is one of these diseases which is acute and infectious mostly occurring in children, which is marked by fever, cough, nasal stuffiness and discharge, lacrimation, small, bright-red spots on the buccal mucosa and skin rashes. recently many researchers were done to clarify the role of medical plants in viral diseases including measles,2 furthermore studies regarding plant vaccines e.g., lettuce measles vaccine.4,5 plant-made oral vaccines have the potential to overcome many of the limitations of traditional vaccines.4 one promising approach is the inhalation of aerosolised vaccine, a study was undertaken to try to immunise very young infants using easily accessible vaccine.6 lettuce (lactuca sativa) had been used to treat many diseases.7 it has very low calorie content and is composed primarily of water, about 90–95%. some plant foods and medicinal herbs such as lettuce and garlic contain flavonoids. an antiviral action of some flavonoids has been observed in a number of test tube experiments.8–11 quercetin is found in green tea, onionskins, kale, red cabbage, green beans, tomatoes, potatoes, lettuce, strawberries, cherries and grapes. it is found in especially high amounts in broccoli, red onions and garlic. one study found that quercetin produced this effect against herpes simplex, polio virus and various respiratory viruses, including influenza.12–14 in addition, lettuce has 7% of vit. a. vitamin a supplementation reduced deaths from measles respiratory infection by 70%.15 also lettuce contains zinc (zn; 0.16 mg per 100 g). zinc is another mineral antioxidant nutrient that the immune system requires. zinc deficiency results in lowered immune defenses, and zinc supplementation increases immune activity in people with certain illnesses.16 as with vitamin a, zinc levels have been observed to fall during the early stages of measles infection and to return to normal several days later.17 there is evidence that zinc supplements are helpful in specific viral infections,18–20 but there are no data on the effect of zinc on childhood exanthemas infections. selenium (se) content in lettuce is 0.5 (µ per 100g): selenium is a mineral known to have antioxidant properties and to be involved in healthy immune system activity. recent animal and human research suggests that selenium deficiency increases the risk of viral infection and that supplementation prevents viral infection.21–24 in a controlled trial, children with a specific viral infection (respiratory syncytial virus) who received a single supplement of 1 mg (1,000 mcg) of sodium selenite (a form of selenium) recovered more quickly than children who did not receive selenium.25 vitamin c (ascorbic acid) content in lettuce is 3.9 mg per 100 g: vitamin c has been demonstrated in test tube, animal and human studies to have immune-enhancing and direct antiviral properties.26 preliminary observations made on the effect of vitamin c on viral infections have involved both measles and chicken pox.27,28 vitamin e (alpha-tocopherol) content in lettuce is 0.03 (µg per 100g): healthy immune function also requires adequate http://www.answers.com/topic/kale http://healthlibrary.epnet.com/getcontent.aspx?token=e0498803-7f62-4563-8d47-5fe33da65dd4&chunkiid=21570 http://healthlibrary.epnet.com/getcontent.aspx?token=e0498803-7f62-4563-8d47-5fe33da65dd4&chunkiid=21573 http://www.naturesbounty.com/vf/healthnotes/hn77/hn77_english/supp/zinc.htm http://www.naturesbounty.com/vf/healthnotes/hn77/hn77_english/supp/selenium.htm http://www.naturesbounty.com/vf/healthnotes/hn77/hn77_english/supp/selenium.htm http://www.naturesbounty.com/vf/healthnotes/hn77/hn77_english/supp/vitamin_c.htm 3j contemp med sci | vol. 2, no. 5, winter 2016: 2–8 research parents understanding on measlesabdul mahdi a. hasan et al. amounts of vitamin e. vitamin e deficiency is associated with increased severity of viral infections in mice.29–31 supplementation with vitamin e during viral infections has been shown to increase immune cell activity32 and reduce virus activity33 in mice. research into the effects of vitamin e supplementation on childhood exanthemas has not been done.34 the aim of this study is to know social believes about measles and to know which plants applied in its treatment in local traditional medicine. material and methods the study is cross-sectional and information were taken from 100 mothers whose children were admitted to al-elwyia paediatric hospital in baghdad from june to september during an epidemic in 2009. information includes age, gender, vaccination status, residence, educational status and social habits and believes regarding measles treatment. all cases were diagnosed on clinical and lab bases these children who were admitted to hospital because they had one or more of the following: pneumonia, diarrhea or encephalitis. all cases are confirmed by anti-measles igm antibodies by elisa [dad behringer] non-confirmed cases or equivocal results are excluded from the study. residence of patients in this study was classified according to health sectors of alrusafa health directorate. statistical methods used in analysing and assessing results include: 1. descriptive statistics inform of: a statistical tables included observed frequencies and percentages. b contingency coefficient for the cross tabs (causes correlation ship of the contingency tables). 2. inferential statistics: in order to accept or reject statistical hypotheses they include: a: fissure exact probability (fep) test for testing the interaction (in depen dency among two factors in the 2 × 2 ranks of the contingency tables. b: χ2-test for testing the interaction otherwise in the contingency tables. c: testing the correlation of contingency coefficient. results are considered as highly significant at p < 0.01, significant results at p < 0.05 and non-significant results at p > 0.05. results table 1 shows that 1–4-year-old children constitute 52% of the sample and those bellow 1 year 32% of patient sample. age distribution of mothers is also shown: highest group is 20–29 years age group which compromise 33% of mothers. males constitute 52% of the patient sample. regarding education level of mothers 25% completed college. table 1 also shows that 69% of mothers believe on vaccination while 54% of children were vaccinated and that 53% of mothers believe bad belief treatment. table 1. frequencies and percentages with comparison significant of the studied parameters toward measles’s mother’s believes of treatment variables groups frequency percent cumulative percent c.s. p-value age of child <1 yr 32 32 32 (chi-square) 57.12 p = 0.000 hs 1–4 52 52 84 5–9 14 14 98 10> 2 2 100 age of mother <20 yrs 7 7 7 (chi-square) 24.2 p = 0.000 hs 20–29 33 33 40 30–39 28 28 68 40–49 21 21 89 50> 11 11 100 gender male 58 58 58 (binomial) p = 0.134 nsfemale 42 42 100 education level of mother house wife 21 21 21 (chi-square) 21.8 p = 0.001 hs illiterate 4 4 25 primary school 10 10 35 intermediate school 13 13 48 secondary school 10 10 58 institute 17 17 75 college 25 25 100 mother knowledge for vaccination profit profit 69 69 69 (binomial) p = 0.000 hsnon-profit 31 31 100 vaccination status vaccinated 46 46 46 (binomial) p = 0.484 nsnon-vaccinated 54 54 100 mother’s believes on bad belief treatment yes 53 53 53 (binomial) p = 0.617 nsno 47 47 100 http://www.naturesbounty.com/vf/healthnotes/hn77/hn77_english/supp/vitamin_e.htm 4 j contemp med sci | vol. 2, no. 5, winter 2016: 2–8 parents understanding on measles research abdul mahdi a. hasan et al. table 2 shows that there is significant association between age and gender of children and mothers belief, while meaningful association at confidence 93.9% observed at mothers age group >50 years of age and constitute 90.9% within positive mother’s belief while college level of education observed to be meaningful at confidence 92.7% and were 28.3% for this level. both vaccination status of the child and mothers knowledge of effectiveness of vaccination were insignificantly associated with mother’s belief on traditional treatment and believes. table 3 shows the sample, wearing red clothes was 35% and lettuce soup drink 8%. table 2. significant association between age and gender of children and mothers believes studied parameters groups count & percentages mother’s belief on bad belief treatment total c.s. p-value yes no age of child <1 yr count 15 17 32 χ2 = 3.293 p = 0.349 c.c. = 0.179 p = 0.349 ns % within age of child 46.9% 53.1% 100.0% % within mother’s belief 28.3% 36.2% 32.0% % of total 15.0% 17.0% 32.0% 1–4 count 30 22 52 % within age of child 57.7% 42.3% 100.0% % within mother’s belief 56.6% 46.8% 52.0% % of total 30.0% 22.0% 52.0% 5–9 count 8 6 14 % within age of child 57.1% 42.9% 100.0% % within mother’s belief 15.1% 12.8% 14.0% % of total 8.0% 6.0% 14.0% 10> count 2 2 % within age of child 100.0% 100.0% % within mother’s belief 4.3% 2.0% % of total 2.0% 2.0% gender male count 30 28 58 f.e.p.t. p = 0.461 c.c. = 0.030 p = 0.764 ns % within gender 51.7% 48.3% 100.0% % within mother’s belief 56.6% 59.6% 58.0% % of total 30.0% 28.0% 58.0% female count 23 19 42 % within gender 54.8% 45.2% 100.0% % within mother’s belief 43.4% 40.4% 42.0% % of total 23.0% 19.0% 42.0% age of mother <20 yrs count 2 5 7 χ2 = 9.023 p = 0.061 c.c. = 0.288 p = 0.061 ns confidence 93.9% meaningful % within age of mother 28.6% 71.4% 100.0% % within mother’s belief 3.8% 10.6% 7.0% % of total 2.0% 5.0% 7.0% 20–29 count 15 18 33 % within age of mother 45.5% 54.5% 100.0% % within mother’s belief 28.3% 38.3% 33.0% % of total 15.0% 18.0% 33.0% 30–39 count 14 14 28 % within age of mother 50.0% 50.0% 100.0% % within mother’s believes 26.4% 29.8% 28.0% % of total 14.0% 14.0% 28.0% 40–49 count 12 9 21 % within age of mother 57.1% 42.9% 100.0% 5j contemp med sci | vol. 2, no. 5, winter 2016: 2–8 research parents understanding on measlesabdul mahdi a. hasan et al. table 2. continued studied parameters groups count & percentages mother’s belief on bad belief treatment total c.s. p-value yes no 50> % within mother’s belief 22.6% 19.1% 21.0% % of total 12.0% 9.0% 21.0% count 10 1 11 % within age of mother 90.9% 9.1% 100.0% % within mother’s believes 18.9% 2.1% 11.0% % of total 10.0% 1.0% 11.0% education level of mother house wife illiterate count 13 8 21 χ2 = 11.524 p = 0.073 c.c. = 0.321 p = 0.073 ns confidence 92.7% meaningful % within education level 61.9% 38.1% 100.0% % within mother’s believes 24.5% 17.0% 21.0% % of total 13.0% 8.0% 21.0% primary school count 7 3 10 % within education 70.0% 30.0% 100.0% % within mother’s believes 13.2% 6.4% 10.0% % of total 7.0% 3.0% 10.0% intermediate school count 4 9 13 % within education level 30.8% 69.2% 100.0% % within mother’s believes 7.5% 19.1% 13.0% % of total 4.0% 9.0% 13.0% secondary school count 3 7 10 % within education level 30.0% 70.0% 100.0% % within mother’s believes 5.7% 14.9% 10.0% % of total 3.0% 7.0% 10.0% institute count 7 10 17 % within education level 41.2% 58.8% 100.0% % within mother’s believes 13.2% 21.3% 17.0% % of total 7.0% 10.0% 17.0% college count 15 10 25 % within education level 60.0% 40.0% 100.0% % within mother’s believes 28.3% 21.3% 25.0% % of total 15.0% 10.0% 25.0% vaccination status vaccinated count 25 21 46 f.e.p.t. p = 0.481 c.c. = 0.025 p = 0.803 ns % within vaccination status 54.3% 45.7% 100.0% % within mother’s believes 47.2% 44.7% 46.0% % of total 25.0% 21.0% 46.0% non vaccinated count 28 26 54 % within vaccination status 51.9% 48.1% 100.0% % within mother’s believes 52.8% 55.3% 54.0% % of total 28.0% 26.0% 54.0% mother knowledge for vaccination profit profit count 35 34 69 f.e.p.t. p = 0.322 c.c. = 0.068 p = 0.496 ns continued 6 j contemp med sci | vol. 2, no. 5, winter 2016: 2–8 parents understanding on measles research abdul mahdi a. hasan et al. table 3. social and traditional belief on treatment % within 53 patients frequency within the 53 patients who believes in traditional treatment believe 6635wearing red cloth 13.27gold wearing 7.54red lipstick application 5.33feather under head or in shoulder 15.18lettuce soup drinking 3.82lettuce soup/garlic under head 11.46others % within mother knowledge 50.7% 49.3% 100.0% % within mother’s believes 66.0% 72.3% 69.0% % of total 35.0% 34.0% 69.0% non profit count 18 13 31 % within mother knowledge 58.1% 41.9% 100.0% % within mother’s believes 34.0% 27.7% 31.0% % of total 18.0% 13.0% 31.0% total count 53 47 100 % within mother knowledge 53.0% 47.0% 100.0% % within mother’s believes 100.0% 100.0% 100.0% % of total 53.0% 47.0% 100.0% table 2. continued studied parameters groups count & percentages mother’s belief on bad belief treatment total c.s. p-value yes no discussion like other studies this study shows that infants constitute a remarkable proportion of affected children. in this study affected infants were 32% of the sample; 16.3% of cases in another cross-sectional study done in this hospital in 2008 were below 9 months of age.35 according to muhammad siddiq’s study 6–9 months of age constitute 12.14% of reported measles cases to district head-quarter hospital, bahawalnagar in pakistan.36 there reported 186 (23%) cases in infants aged <1 year in measles outbreak in the republic of the marshall islands, 2003.37 males in this study constitute 58% of affected children with measles. confirmed cases in males are more than females in previous tareef ’s study35 (62%) which contradict muhammad siddiq’s study district head-quarter hospital, bahawalnagar in pakistan who report female cases more than male cases.36 this study shows that 46% of the sample had been vaccinated compared to 37 % in previous study done in 200835 in the same hospital. in muhammad siddiq’s study only 60 cases (42.86%) were immunised against measles36 compared to (46%) buenos aires, argentina, 1997 and 1998 outbreak38 were not vaccinated. while 37% of confirmed measles cases are vaccinated in tariff ’s study.35 measles is an old disease and is managed according to certain traditions and believes including certain herbs and certain diets. in kasak, the magical properties of the shirt colour were attributed to the fact that its red patches protected from measles, yellow ones from jaundice, and deep blue from whooping-cough.1 in chinese medicine treatment of late measles include strawberries, deep red, thick yellow dry coat , according to this study red colour (dressing, gold wearing and lip stick) constitute 35%, 7%, and 4% of total social beliefs, respectively. symbolic function of red colour might be most important of all explanation of widespread among many societies. the most interesting in this study is that 15.1% of mothers believes that lettuce soup drinking is of value in treatment of measles. this might be one good example of accumulative experience of society regarding medical plants and its rule in management of this disease other recognised observation in this study is the odour of lettuce and garlic which constitute 3.8% in frequency in this regard, preliminary studies shows that plant-based inhalational using lettuce is effective for further studies regarding the efficacy of inhalation therapy in measles management (fig. 1). conclusions •   accounting to the review of literature and the finding of the  study, measles is prevalent among children 6–15 years of age. parents should not become overwhelmed with feelings of frustration or failure due to their child’s measles. •   although it is prevalent, measles is seldom a topic of conversation among parents due to the private nature of the topic and perhaps to avoid harming the feeling of the child. •   this research is an attempt to inform the reader of measles  and lists treatment methods and parent practices; it should by no means be a substitute for professional medical care. •   a thorough examination of the child by the family physician  should be done to rule out any medical issues and if necessary professional counseling should be sought to help the child overcome their measles behaviour. http://www.pakmedinet.com/author/muhammad+siddiq http://www.pakmedinet.com/author/muhammad+siddiq http://www.pakmedinet.com/author/muhammad+siddiq http://www.pakmedinet.com/author/muhammad+siddiq 7j contemp med sci | vol. 2, no. 5, winter 2016: 2–8 research parents understanding on measlesabdul mahdi a. hasan et al. fig. 1 pie chart of the studied parameters toward bad beliefs of measles’s treatment. •   based on the present results and the discussion the study has  concluded that parents having minimum level of experiences about measles need a specific education programme and training sessions. recommendations keep the sample group large. in doing so, the researcher will have a good representation of the population. a specific education programme can be designed and presented to parent’s who have minimum level of knowledge in order to improve their level of knowledge. •   the educational programme of the present can be used  as means for knowledge improvement for parents who have children with measles. •   in service, continuous educational programme should  be presented to parents of on a regular base to maintain their level of knowledge. 8 j contemp med sci | vol. 2, no. 5, winter 2016: 2–8 parents understanding on measles research abdul mahdi a. hasan et al. references 1. шаханова н. миртрадиционнойкультурыказахов. алматы, 1998 p 54 2. parker me, chabot s, ward bj, johns t. traditional dietary additives of the maasai are antiviral against the measles virus. j ethnopharmacol. 2007;114(2):146–52. pmid: 17870263 3. general authority for fish resources development. available at: http:// gafrd.kenanaonline.com/topics/57646/posts/89315gafrd} accessed in 1 april 2011 4. webster de, smith sd, pickering rj, strugnell ra, dry ib, wesselingh sl. measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination. vaccine. 2006;24:3538–44. pmid: 16519973 5. huang z, dry i, webster d, strugnell r, wesselingh s. plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice. vaccine. 2001;19:2163–71. pmid: 11228389 6. ekunwe eo. immunization by inhalation of aerosolized measles vaccine. ann trop paediatr. 1990;10(2):145–9. pmid: 1699477 7. alrishahri m. encyclopedia of medical hadiths p609 (1426h) [in arabic]. 8. vrijsen r, everaert l, boeye a. antiviral activity of flavones and potentiation by ascorbate. j gen virol. 1988;69:1749–51. pmid: 2839607 9. debiaggi m, tateo f, pagani l, luini m, romero e. effects of propolis flavonoids on virus infectivity and reaplication. microbiologica. 1990;13:207–13. pmid: 2125682 10. fesen mr, kohn kw, leteurtre f, pommier y. inhibitors of human immunodeficiency virus integrase. proc natl acad sci usa. 1993;90:2399– 403. pmid: 8460151 11. amoros m, simoes cm, girre l, sauvager f, cormier m. synergistic effect of flavones and flavonols against herpes simplex virus type 1 in cell culture: comparison with the antiviral activity of propolis. j nat prod. 1992;55:1732– 40. pmid: 1338212 12. kaul tn, middleton e jr, ogra pl. antiviral effect of flavonoids on human viruses. j med virol. 1985;15:71–79. pmid: 2981979 13. musci i, pragai bm. inhibition of virus multiplication and alteration of cyclic amp level in cell cultures by flavonoids. experientia. 1985;41:930–1. pmid: 2989000 14. ohnishi e, bannai h. quercetin potentiates tnf-induced antiviral activity. antiviral res. 1993;22:327–31. pmid: 8279819 15. glasziou pp, mackerras de. vitamin a supplementation in infectious diseases: a meta-analysis. bmj. 1993;306:366–70. pmid: 8461682 16. fraker pj, king le, laakko t, vollmer tl. the dynamic link between the integrity of the immune system and zinc status. j nutr. 2000; 130:1399s–406s. pmid: 10801951 17. coutsoudis a, coovadia hm, broughton m, salisbury rt, elson i. micronutrient utilisation during measles treated with vitamin a or placebo. int j vitam nutr res. 2001;61:199–204. pmid: 1794947 18. mocchegiani e, muzzioli m. therapeutic application of zinc in human immunodeficiency virus against opportunistic infections. j nutr. 2000;130:1424s–31s. pmid: 10801955 19. novick sg, godfrey jc, pollack rl, wilder hr. zinc-induced suppression of inflammation in the respiratory tract, caused by infection with human rhinovirus and other irritants. med hypotheses. 1997;49:347–57. pmid: 9352505 20. kumel g, schrader s, zentgraf h, brendel m. therapy of banal hsv lesions: molecular mechanisms of the antiviral activity of zinc sulfate. hautarzt. 1991;42:439–45 [review; in german]. pmid: 1657829 21. levander oa, beck ma. selenium and viral virulence. br med bull. 1999;55:528–33. pmid: 10746343 22. beck ma, levander oa. host nutritional status and its effect on a viral pathogen. j infect dis. 2000;182:s93–s96. pmid: 10944489 23. beck ma. nutritionally induced oxidative stress: effect on viral disease. am j clin nutr 2000;71:1676s–81s [review]. pmid: 10837315 24. beck ma. selenium and host defence towards viruses. proc nutr soc. 1999;58:707–11. pmid: 10604206 25. liu x, yin s, li g. effects of selenium supplement on acute lower respiratory tract infection caused by respiratory syncytial virus. zhonghua yu fang yi xue za zhi. 1997;31:358–61 [in chinese]. pmid: 9863072 26. dwyer j, nicholson lm, shircore a, et al. vitamin c intake and progression of carotid atherosclerosis. the los angeles atherosclerosis study. american heart association annual meeting. march 2, 2010 [abstract]. 27. piesse jw. nutritional factors in calcium-containing kidney stones with particular emphasis on vitamin c. int clin nutr rev. 1985;5:110–29. 28. ringsdorf wm, cheraskin wm. medical complications from ascorbic acid: a review and interpretation (part one). j holistic med. 2011;6:49–63. 29. wandzilak tr, d’andre sd, davis pa, williams he. effect of high dose vitamin c on urinary oxalate levels. j urol. 1994;151:834–7. pwid: 8126804 30. levine m. vitamin c and optimal health. presented at the february 25, 1999 60th annual biology colloquium, oregon state university, corvallis, oregon. 31. levine m, conry-cantilena c, wang y, welch rw, washko pw, dhariwal kr, et al. vitamin c pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. proc natl acad sci usa. 1996;93:3704–9. pmid: 8623000 32. han sn, wu d, ha wk, beharka a, smith de, bender bs, et al. vitamin e supplementation increases t helper 1 cytokine production in old mice infected with influenza virus. immunology. 2000;100:487–93. pmid: 10929076 33. hayek mg, taylor sf, bender bs, han sn, meydani m, smith de, et al. vitamin e supplementation decreases lung virus titers in mice infected with influenza. j infect dis. 1997;176:273–6. pmid: 9207381 34. http://www.calorie-counter.net/calories-vegetables/iceberg-lettuce.htm 35. tareef fr. case fatality rate and epidemiological features of measles reported in elwyia pediatric hospital from june to september 2008. risafa med digest. 2009;2(3):55–60. 36. siddiq m, saeed m. a study of measles cases reported to district headquarter hospital, bahawalnagar. pak paed j. 2006;30(1):16–22. 37. hyde tb, dayan gh, langidrik jr, nandy r, edwards r, briand k, et al. measles outbreak in the republic of the marshall islands, 2003. int j epidemiol. 2006;35(2):299–306. pmid: 16299123 38. bilkis md, barrero pr, mistchenko as. measles resurgence in argentina: 1997–8 outbreak. epidemiol infect. 2000;124:289–93. pmid: 10813155 http://www.sciencedirect.com/science/journal/03788741 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235084%232007%23998859997%23670722%23fla%23&_cdi=5084&_pubtype=j&view=c&_auth=y&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=158be8ba41cc2f131d7efe040a5a999e http://gafrd.kenanaonline.com/topics/57646/posts/89315 http://gafrd.kenanaonline.com/topics/57646/posts/89315 http://www.ncbi.nlm.nih.gov/pubmed/?term=strugnell ra%5bauthor%5d&cauthor=true&cauthor_uid=16519973 http://www.ncbi.nlm.nih.gov/pubmed/?term=dry ib%5bauthor%5d&cauthor=true&cauthor_uid=16519973 http://www.ncbi.nlm.nih.gov/pubmed/?term=wesselingh sl%5bauthor%5d&cauthor=true&cauthor_uid=16519973 http://www.ncbi.nlm.nih.gov/pubmed?term=%22ekunwe eo%22%5bauthor%5d file://192.168.1.244/etype/journal%20of%20contemporary%20medical%20sciences/03_edited%20mss/04-04-2016/new%20folder/javascript:al_get(this, 'jour', 'ann trop paediatr.'); http://www.ncbi.nlm.nih.gov/pubmed/?term=luini m%5bauthor%5d&cauthor=true&cauthor_uid=2125682 http://www.ncbi.nlm.nih.gov/pubmed/?term=romero e%5bauthor%5d&cauthor=true&cauthor_uid=2125682 http://www.ncbi.nlm.nih.gov/pubmed/?term=sauvager f%5bauthor%5d&cauthor=true&cauthor_uid=1338212 http://www.ncbi.nlm.nih.gov/pubmed/?term=cormier m%5bauthor%5d&cauthor=true&cauthor_uid=1338212 http://www.ncbi.nlm.nih.gov/pubmed/?term=salisbury rt%5bauthor%5d&cauthor=true&cauthor_uid=1794947 http://www.ncbi.nlm.nih.gov/pubmed/?term=elson i%5bauthor%5d&cauthor=true&cauthor_uid=1794947 http://www.ncbi.nlm.nih.gov/pubmed/?term=effects+of+selenium+supplement+on+acute+lower+respiratory+tract+infection+caused+by+respiratory+syncytial+virus http://www.ncbi.nlm.nih.gov/pubmed/?term=effects+of+selenium+supplement+on+acute+lower+respiratory+tract+infection+caused+by+respiratory+syncytial+virus http://www.ncbi.nlm.nih.gov/pubmed/?term=welch rw%5bauthor%5d&cauthor=true&cauthor_uid=8623000 http://www.ncbi.nlm.nih.gov/pubmed/?term=washko pw%5bauthor%5d&cauthor=true&cauthor_uid=8623000 http://www.ncbi.nlm.nih.gov/pubmed/?term=dhariwal kr%5bauthor%5d&cauthor=true&cauthor_uid=8623000 http://www.ncbi.nlm.nih.gov/pubmed/?term=dhariwal kr%5bauthor%5d&cauthor=true&cauthor_uid=8623000 http://www.ncbi.nlm.nih.gov/pubmed/?term=beharka a%5bauthor%5d&cauthor=true&cauthor_uid=10929076 http://www.ncbi.nlm.nih.gov/pubmed/?term=smith de%5bauthor%5d&cauthor=true&cauthor_uid=10929076 http://www.ncbi.nlm.nih.gov/pubmed/?term=bender bs%5bauthor%5d&cauthor=true&cauthor_uid=10929076 http://www.ncbi.nlm.nih.gov/pubmed/?term=han sn%5bauthor%5d&cauthor=true&cauthor_uid=9207381 http://www.ncbi.nlm.nih.gov/pubmed/?term=meydani m%5bauthor%5d&cauthor=true&cauthor_uid=9207381 http://www.ncbi.nlm.nih.gov/pubmed/?term=smith de%5bauthor%5d&cauthor=true&cauthor_uid=9207381 http://www.pakmedinet.com/author/muhammad+siddiq http://www.pakmedinet.com/author/muddassir+saeed http://www.pakmedinet.com/ppj http://www.ncbi.nlm.nih.gov/pubmed/?term=nandy r%5bauthor%5d&cauthor=true&cauthor_uid=16299123 http://www.ncbi.nlm.nih.gov/pubmed/?term=edwards r%5bauthor%5d&cauthor=true&cauthor_uid=16299123 http://www.ncbi.nlm.nih.gov/pubmed/?term=briand k%5bauthor%5d&cauthor=true&cauthor_uid=16299123 232 j contemp med sci | vol. 2, no. 5, winter 2016: 2–7 applying espghan and wgo guidelines of celiac disease on iraqi patients alaa s. alattabia and israa h. aljerraha* adepartment of microbiology, college of medicine, university of karbala, karbala, iraq. *correspondence to israa h. aljerrah (email: israa4019@gmail.com). (submitted: 03 august 2018 – revised version received: 07 september 2018 – accepted: 18 october 2018 – published online: 26 december 2018) objective the goal of this study is to check the degree to which the physicians and pediatricians can apply european society for paediatric gastroenterology, hepatology and nutrition (espghan) and world gastroenterology organization (wgo) criteria of celiac disease (cd) on their patients in karbala city in iraq. methods this is a cross-sectional study conducted in karbala city hospitals (al-hussein medical city hospital, the pediatric teaching hospital) for the period from august 2017 to february 2018. a random selection of 108 cd patients was performed and the clinical data including the biopsy results were collected directly from patients or their family members through a questionnaire sheets. statistically, the quantitative variables were analyzed using non-parametric t-test and the qualitative variables were analyzed using chi-square test. results the study shows that of the total 108 patients, only n = 43 (39.8%) of candidates who did biopsy even with tissue tg (ttg) iga >10fold upper limit of normal (uln) (200 ru/ml and more) due to the shortage in endomysial antibody (ema)/hla tests recommended by aspghan to omit duodenal biopsy, while n = 51 (47.2%) accomplished only one of the triple tests (only ttg iga >10-fold uln) suggested by aspghan and then applied challenge test and n = 14 (12.9%) achieved none of the triple tests (ttg iga antibody titer >5-fold uln, but <10-fold uln). on the other hand, the wgo guideline is more suitable if properly applied as it considers ttg (iga) a convenient substituent of ema test where high ttg (iga) serum level being diagnostic and to be confirmed by the available dgp (iga and igg) antibodies. conclusion the wgo guideline for symptomatic and asymptomatic patients is more applicable for the areas with limited facilities, while espghan guideline for pediatrics can be applied to a less degree. generally in iraq there is a delay in disease diagnosis due to many reasons to be evaluated. keywords celiac disease, espghan criteria, wgo guideline, ttg (iga) antibodies, challenge test, biopsy findings introduction celiac disease (cd) is a chronic systemic immune-mediated disorder associated with variable small-intestinal mucosal lesion caused by gluten consumption in genetically susceptible individuals.1,2 cd prevalence is about 1% of the general population.3 the continuous ‘westernization’ of the diet4,5 may account for cd being also common in many developing countries, in particular the middle east.6 nowadays, in particular resource-rich countries, more heterogeneous clinical manifestations of cd may be reported, including typical gastroenterological symptoms as well as a myriad of extra-intestinal disorders.7 the gi symptoms (diarrhea and malabsorption syndrome) are predominating in young children, while failure to thrive, short stature, and other growth problems occur in all pediatric age groups.8 generally extra-intestinal (atypical) clinical features occur in children more than 5-year-old and so in adults.9 diagnostic approach in cd includes: clinical manifestations, serological tests, genetic study (hla-dq2/dq8 typing) and small intestinal biopsy histological findings (husby et al., 2013). serum tissue tg (ttg) (iga) measurement is the premier testing recommended for individuals due to its excellent standardization and being a high sensitive and specific test.10,11 the guidelines of the european society for paediatric gastroenterology, hepatology and nutrition (espghan) authorize for cd diagnosis without biopsies in children present a distinctive intestinal clinical features and levels of tg2-iga antibodies 10-fold or more than the upper normal level (unl), detection of anti-endomysial antibody in another test plus genetic study (hla-dq2/dq8 typing) for confirmation [triple test espghan criteria], while proposes shifting to endoscopy and biopsy if endomysial antibody (ema) test and hla-typing are not available.1 recent guidelines from the world gastroenterology organization (wgo) for diagnosis of cd take in consideration the resources available in each country. according to wgo, the experts propose that the primary test for cd patients with or without symptoms is the measurement of anti-ttg (iga) and total iga serum levels with recommendation for adding another test either to confirm the first test positivity or to support the low (borderline) anti-ttg (iga). these tests include ema or dgp antibodies where in complaining patients, the guideline showed that the dgp (iga and igg) have similar performance to ttg (iga) antibodies. the combined tests of dgp (igg) and ttg (iga) are of particular benefit as beside detection of iga-deficient cd patients, igg-dgp can also detect small percent of patients who are normal for iga but are negative for iga-tg2 tests. another point is that some data demonstrate that using these two tests directing toward different antigens to be more useful than using tests act on the same autoantigen like ttg (iga) and ema (iga).12 a recent study showed that a simultaneous testing of ttg (iga) and dgp (igg) antibodies constitutes a potentially accurate diagnostic tool for cd.13 however, a ttg (iga) positivity and clinical response on adherence to a gfd might be enough to consider cd diagnosis in countries with limited facilities.12,14 at the time, wgo necessitates small intestinal biopsy in certain conditions when patients not present the malabsorptive manifestations or when serological tests either low or negative, the wgo also propose omission of intestinal biopsy in other conditions where positive and negative points of cd can be issn 2413-0516 research a.s. alattabi and i.h. aljerrah 233j contemp med sci | vol. 4, no. 4, autumn 2018: 232–236 applying espghan and wgo guidelines of celiac disease on iraqi patients discussed with well-experienced specialists. this is convenient in areas where healthcare facilities are restricted.12 gluten challenge is the mode by which a clinically suspected patient, but uncertain cd where formerly managed by gluten restriction returns to a usual, gluten-containing diet, along medical monitoring, enabling the disease diagnosis.15,16 in the past this test was routinely used to diagnose cd, however a less frequent use nowadays due to the high accuracy of serological tests. the study aims to verify the degree to which we can apply these guidelines on cd diagnosis at the level of karbala city. materials and methods ethically, the selection of the study groups and data collection were accomplished after we have been taken the approval of karbala health director to work in its hospitals and a practical supervisor has been set in each hospital to follow the workflow. a cross-sectional study was carried out for the period from august 2017 to february 2018 in karbala province hospitals. a total of 108 patients (old and new cases, pediatric and adult age groups) who were attending endocrine centers in al-hussein medical city hospital and the pediatric teaching hospital were randomly selected and enrolled in the study according to inclusion and exclusion criteria. the age groups ranging between 1 and 58 years with 68 of them are below 18 years. the patient’s sex was 75 female vs 33 male. clinical data includes: age, sex, residence, complaints, the period of clinical presentation, duration of disease (old cases), family history, skin manifestations, associated autoimmune disorders, serum antibody titer, indications for endoscopy and biopsy result, challenge test, treatment, complications, and more others collected directly from patients or their family members through a questionnaire sheets. the serological markers which currently used as a first step in the screening for cd (ttg iga, igg and dgp iga, igg) are measured by antibody sandwich enzyme-linked immunosorbant assay (elisa) technique which was carried out by the laboratory staff of al-hussein medical city hospital and the pediatric teaching hospital where both antibodies are available in al-hussein medical city lab and only ttg (iga, igg) is available in pediatric teaching hospital. the euroimmun medizinische labordiagnostika ag assay provides a semi-quantitative or quantitative in vitro assay for human antibodies in serum or plasma. data analysis was performed using the spss version 18 software package (statistical package for social science; inc., chicago, il, usa). quantitative data were expressed as mean ± standard deviation (sd), and analyzed using non-parametric t-test. on the other hand, qualitative data were expressed as numbers (n) and percentages, and analyzed using chi-square test. pearson correlation analysis was used to reveal the association between two or more of the related quantitative variables. *a p-values <0.05 were considered as statistically significant. *permission of patients to publish the study. results socio-demographic characteristics of celiac patients children account to about two third (n = 68; 63%) of the celiac patients, while females in this study group are more than males (69.4% vs 30.6%) with the female to male ratio being 2.27:1 table 1. socio-demographic characteristics of celiac patients. the values are in percentage and numbers age (years) number percentage (%) <18 68 63 18 and above 40 37 total 108 100 mean ± sd 18.39 ± 14.08 range 1–58 gender number percentage (%) male 33 30.6 female 75 69.4 total 108 100 residence number percentage (%) urban 80 74.1 rural 28 25.9 total 108 100 table 2. the clinical presentations of celiac patients. the values are in percentage and numbers clinical presentation number percentage (%) intestinal 88 83.8 extra-intestinal 17 16.2 total 105 100 period of presentation number percentage (%) months 48 45.7 years 57 54.3 total 105 100 fig. 1 percentage of ttg iga titer of cd patients. and about three quarters of patients are of urban residence (n = 80; 74.1% vs n = 28; 25.9%). most of patients revealed typical or intestinal clinical features, while atypical or extra-intestinal presentation account for small percentage (83.8% vs 16.2%). another point is that the majority of patients were complaining for at least few months to years before being diagnosed to have cd. research 234 j contemp med sci | vol. 4, no. 4, autumn 2018: 232–236 applying espghan and wgo guidelines of celiac disease on iraqi patients a.s. alattabi and i.h. aljerrah serological markers of cd the candidates also revealed that the percentage of the 10-folds the upper limit of normal (uln) range of the antibody ttg iga (cut-off ), i.e. 200 ru/ml and more, was positive in about three quarters of the patients. notably, fig. 2 shows that there was no significant association of (difference in) ttg iga titer between age groups (p = 0.6). small intestine biopsy results a small intestine endoscopy and biopsy had been done for all adult age group (40) and only three children and the results according to modified marsh17–oberhuber18 classification demonstrates that m2 accounts for about half of cases as shown in fig. 3. the triple tests suggested by aspghan to omit biopsy in symptomatic patients include tg2-iga antibodies table 5. correlation of ttg (iga) antibody titers with biopsy findings biopsy findings no. mean sd p-value* ttg (iga) m2 22 252.56 131.07 0.07 m3 (a–c) 19 337.47 153.87 *pearson correlation was used. correlation is significant at p-value of 0.05. table 4. percentage of aspghan triple test and wgo guide application aspghan triple test application number percentage (%) al-hussein medical city pediatric teaching hospital number percentage (%) number percentage (%) only ttg iga >10-fold uln (1/3) 51 47.3 0 0 51 75 ttg iga >10-fold uln + ema (2/3) 0 0 0 0 0 0 ttg iga >10-fold uln + hla typing (2/3) 0 0 0 0 0 0 ttg iga >10-fold uln + ema + hla typing (3/3) 0 0 0 0 0 0 biopsy if ema/hla tests not available or ttg iga <10-fold uln 43 39.8 40 100 3 4.4 none of the triple test 14 12.9 0 0 14 20.6 total 108 100 40 100 68 100 wgo guideline application number percentage (%) al-hussein medical city pediatric teaching hospital number number high ttg iga titer 108 100 40 68 dgp (iga/igg) for confirmation 0 0 0 0 total 108 100 40 68 fig. 2 association of ttg iga titer with age groups. 10-fold or more than the unl, detection of anti-endomysial antibody in another test plus genetic study (hla-dq2/ dq8 typing) for confirmation.1 table 4 shows that of the total 108 patients, only n = 43 (39.8%) of candidates who did biopsy even with ttg iga >10-fold uln (200 ru/ml and more) due to the shortage in ema/hla tests, while n = 51 (47.2%) accomplished one of the triple tests (only ttg iga >10-fold uln) suggested by aspghan and then applied challenge test, while n = 14 (12.9%) who have ttg iga >5-fold uln, but <10-fold uln and also passed to challenge test. nevertheless, the wgo guideline can be applied in all the patients (n = 108; 100%) as high ttg iga titer is revealed in about three quarter of the patients and the already available dgp (iga, igg) antibodies to confirm positivity. correlation of ttg (iga) level with small intestine biopsy results regarding the biopsy results, patients who had undergone small intestine endoscopy and biopsy were divided into two categories: those with m3 (a–c) grade of modified marsh– oberhuber classification show mean autoantibody level more than that those with m2 grade, however, this difference not reached significance (p-value 0.07). discussion according to the clinical data collected and analyzed, the study demonstrated that children account to about two third (n = 68; 63%) of the celiac patients, a result consistent with the results of other previous studies which showed that 64% of patients with cd in karbala were among age group of 2–10 years.19 the females are more affected than males (n = 75; 69.4% vs n = 33; 30.6%) with the female to male ratio being fig. 3 small intestine biopsy results according to modified marsh–oberhuber classification. research a.s. alattabi and i.h. aljerrah 235j contemp med sci | vol. 4, no. 4, autumn 2018: 232–236 applying espghan and wgo guidelines of celiac disease on iraqi patients 2.27:1, a results which usually observed as there is a higher rate of autoimmune disease among females.20 most of the patients (adults 90% and children 80%) revealed typical or intestinal clinical features, while atypical or extra-intestinal presentation account for small percentage (n = 88; 83.8% vs n = 17; 16.2%) and only three patients of total had silent presentation and diagnosed during follow-up tests for dm type 1. although atypical (non-classical) manifestations are more in western patients,12 however, the predominance of intestinal (typical) manifestations in both age groups of candidates may not reflect the real situation as many undiagnosed cd patients missed among clinics of different specialties searching for symptomatic management of their complaints where low awareness about the extra-intestinal (atypical) manifestations by some clinicians may also contribute to this result. given the increasing rate in cd diagnosis all over the world, many areas including iraq do not always have the sophisticated techniques required for disease diagnosis like anti-ema and genetic study testing. when reviewing the guidelines of the espghan which allow cd diagnosis without biopsies in children with obvious symptoms and levels of (tga-iga) antibodies 10-fold or more the uln, with positive ema in a different blood sample as a confirmatory test and positive hla-dq2/dq8 typing [triple test espghan criteria].1 to verify the study candidates with espghan approach for diagnosis, and although about three quarters of the patients have high serum ttg iga antibody titer [10-folds the uln range] of this antibody cut-off (i.e. 200 ru/ml and more), we have to explain some points: 1. there is no genetic study (hla-dq2/dq8 typing) at least at the level of kabala province to document the positivity of hla-typing. 2. anti-ema assay is also not available in kabala province. nevertheless these points may not represent the stumbling block for the diagnosis as many recent data revealed the possibility to overcome these points, of which werkstetter et al.21 in a prospective study to validate espghan approach on children who complaining of malabsorption symptoms instead of any symptom show that the inclusion of hla analyses did not increase accuracy of diagnosis. also mubarak et al.22 concluded that pediatric patients with a ttga level ≥100 u/ml in whom symptoms improve upon consuming a gfd may not need a small intestinal biopsy to confirm cd. mubarak et al.23 in another study based on prospective data confirms omitting of a small intestinal biopsy for the diagnosis of cd in symptomatic patients with ttg iga serum level ≥100 u/ml. the gastroenterologists in the endocrine center of the ai-hussein medical city hospital when serology result reveals high ttg (iga) antibodies, they consider a small intestine biopsy is a must for diagnosis of cd in adults [biopsy done for n = 43 (40 adults and three children); 39.8%]. on the other hand, pediatricians shift to challenge test (not biopsy) which is regarded as a confirmatory test for children with clinical manifestations of cd and a highly positive ttg (iga) level and their parents refuse a small intestine endoscopy and biopsy as invasive procedure for diagnosis. of note all the pediatric patients (n = 65; 60.2%) were positive for challenge test. hence very high levels of serum anti-ttg iga might be enough on the basis of suggestive intestinal clinical manifestations demonstrated in most of the patients, a point supported by the wgo diagnostic protocol, especially in regions with limited resources (shortage of sophisticated techniques), considered ttg as an acceptable proxy of the ema antibodies (“the ema test needs expert observers, and detection of ttg antibodies by elisa tests should therefore be recommended in settings with low expertise”) [11]. the wgo also recommended the evaluation of the local resources available to develop a diagnostic protocol.24 and his team in a population-based study found that serum level of ttg (iga) can identify celiac disease patients with about 90% ppvs. another supporting point for wgo guideline is the availability of antibodies to dgp (iga and igg), which are in complaining patients having similar performance to ttg (iga) antibodies. however an important fact should be mentioned that dgp (iga and igg) antibodies in the main labs of related hospitals are completely neglected by many gastroenterologists and pediatricians and they think that it is the old low-accurate anti-gliadin antibody test, so they sent only for ttg (iga) antibodies, while others believe that ttg (iga) antibodies are enough for cd serology. this implies that inspite of the specialists ability to apply wgo guideline in most of the patients, but improper application of this guide may occur when some clinicians neglect this test in the absence of ema test. measurement of antibodies serum levels was carried out using quantitative data for caliculation, implying more accuracy for diagnosis in contrast to some laboratories that produce semi-quantitative data, making the assessment uncertain.25 when correlating ttg (iga) level with small intestine biopsy results, we found that patients with m3 (a–c) grade of modified marsh–oberhuber classification have mean autoanti body levels (337.47 ru/ml) more than that those with m2 grade (mean ttg (iga) level 252.56 ru/ml), however, this difference not reached significance (p-value 0.07), an information that meriting further evaluation, where small sample size might be a confounding factor. generally these findings goes with hawamdeh et al.26 findings in 2016 who concluded a significant association between the degree of duodenal damage and anti-ttg titers, where anti-ttg titer more than 10-folds the uln (≥180 u/ml) was significantly associated with marsh iii enteropathy; this strong association of high anti-ttg titer and severity of intestinal damage might provide a remarkable evidence for diagnosis of cd when endoscopy is not applicable and fortified by positive ema test or hla typing. another points deserves discussion are: athe prolonged period of presentation noticed in the majority of patients before diagnosis which may be attributed to many causes including: 1. limited resources of sophisticated techniques important to confirm diagnosis. 2. lacking of screening tests in the primary health centers which are essential to detect many complainers as most people attend these centers for the first time. of note, only the endocrine center in the pediatric teaching hospital carries out screening tests for children with positive family history for cd or children with other autoimmune diseases mostly type 1 dm. 3. celiac disease is the last in the list of differential diagnosis made for patients by most specialists, where irritable bowel, inflammatory bowel disease, giardiasis, and helicobacter pylori infection are in the top of the list. 4. patients jumping from one specialist to another seeking for response while others refuse biopsy at the research 236 j contemp med sci | vol. 4, no. 4, autumn 2018: 232–236 applying espghan and wgo guidelines of celiac disease on iraqi patients a.s. alattabi and i.h. aljerrah this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. beginning of disease and so wasting more time before diagnosis being confirmed. 5. lacking of education about the disease, environmental factors and motivation toward life-long restriction of dietary gluten as most patients think that cd is transient rather than permanent disorder. ball candidates had started a gfd, but unfortunately inadequate adherence noticed in most of the patients, however, some clinical and serological responses had been shown in many of them. all these points are to be considered in the future which may help in applying espghan and wgo guidelines to uncover much of the underdiagnosed cases in iraq. conclusion the wgo guideline is more suitable if properly applied for the iraqi patients due to shortage of sophisticated techniques, while espghan guideline for pediatrics can be applied to a less degree. generally here in iraq there is an obvious delay in disease diagnosis due to many reasons to be evaluated. conflict of interest none.  references 1. husby s, koletzko s, korponay-szabo ir et al. european society for pediatric gastroenterology, hepatology, and nutrition guidelines for the diagnosis of coeliac disease. j pediatr gastroenterol nutr. 2012;54:136–160. 2. ludvigsson jf, leffler da, bai jc, biagi f, fasano a, green ph, et al. the oslo definitions for coeliac disease and related terms. gut. 2013;62:43–52. 3. rubio-tapia a, hill id, kelly cp, calderwood ah, murray ja; american college of gastroenterology. acg clinical guidelines: diagnosis and management of celiac disease. am j gastroenterol. 2013;108:656–676. 4. fasano a, catassi c. celiac disease. n engl j med. 2012;367:2419–2426. 5. sapone a, bai jc, ciacci c, dolinsek j, green ph, hadjivassiliou m, et al. spectrum of gluten-related disorders: consensus on new nomenclature and classification. bmc med. 2012;10:13. 6. dalgic b1, sari s, basturk b, ensari a, egritas o, bukulmez a, et al. prevalence of celiac disease in healthy turkish school children. am j gastroenterol. 2011;106:1512–1517. 7. jeon mk, klaus c, kaemmerer e, gassler n. intestinal barrier: molecular pathways and modifiers. world j gastrointest pathophysiol. 2013;4:94–99. 8. reilly nr, aguilar k, hassid bg, cheng j, defelice ar, kazlow p, et al. celiac disease in normal-weight and overweight children: clinical features and growth outcomes following a gluten-free diet. j pediatr gastroenterol nutr. 2011;53:528–31. 9. akirov a, pinhas-hamiel o. co-occurrence of type 1 diabetes mellitus and celiac disease. world j diabetes. 2015;6:707–714. 10. zintzaras e, germenis ae. performance of antibodies against tissue transglutaminase for the diagnosis of celiac disease: meta-analysis. clin vaccine immunol. 2006;3:187–192. 11. lewis nr, scott bb. meta-analysis: deamidated gliadin peptide antibody and tissue transglutaminase antibody compared as screening tests for coeliac disease. aliment pharmacol ther. 2010;31:73–81. 12. bai jc, ciacci c, corraza gr, fried m, olano c, rostami-nejad m, et al. world gastroenterology organisation practice guidelines: celiac disease. world gastroenterology organisation, milwaukee, wi, usa, vol. 48, 2013, pp. 1–18. . 13. hong yy. deaminated gliadin peptide antibody in the diagnosis of celiac disease. int j celiac dis. 2015;3:56–57. 14. kelly cp, bai jc, liu e, leffler da. advances in diagnosis and management of celiac disease. gastroenterology. 2015;148:1175–1186. 15. lahdeaho ml, mäki m, laurila k, huhtala h, kaukinen k. small-bowel mucosal changes and antibody responses after low-and moderate dose gluten challenge in celiac disease. bmc gastroenterol. 2011; 11:129. 16. leffler, d. schuppan, d. pallav, k. najarian, r. goldsmith, jd, hansen, j, et al. kinetics of the histological, serological and symptomatic responses to gluten challenge in adults with coeliac disease. gut. 2012;62:996–1004. 17. marsh mn. mucosal pathology in gluten sensitivity. in: marsh mn, ed. coeliac disease, blackwell science ltd., oxford, uk, 1992;136–191. 18. oberhuber g. histopathology of celiac disease. biomed pharmacother. 2000;54:368–372. 19. hameed ws, abdul-mehdi rj, tarish hr, alsherees haa. prevalence of dq2, dq8 and dr4 alleles in iraqi celiac patients. int arch biomed clin res. 2016;2:118–121. 20. dixit r, lebwohl b, ludvigsson jf, lewis sk, rizkalla-reilly n, green ph. celiac disease is diagnosed less frequently in young adult males. dig dis sci. 2014;59:1509–1512. 21. werkstetter kj, korponay-szabó ir, popp a, villanacci v, salemme m, heilig g, et al. accuracy in diagnosis of celiac disease without biopsies in clinical practice. gastroenterology. 2017;153:924–935. 22. mubarak a, wolters vm, gerritsen sa, gmelig-meyling fh, ten kate fj, houwen rh. a biopsy is not always necessary to diagnose celiac disease. j pediatr gastroenterol nutr. 2011;52:554–557. 23. mubarak a, wolters vm, gmelig-meyling fh, ten kate fj, houwen rh. tissue transglutaminase levels above 100 u/ml and celiac disease: a prospective study. world j gastroenterol. 2012;18:4399–4403. 24. ermarth a, bryce m, woodward s, stoddard g, book l, jensen mk. identification of pediatric patients with celiac disease based on serology and a classification and regression tree analysis. clin gastroenterol hepatol. 2017;15:396–402.e2. 25. tucci f, astarita l, abkari a, abu-zekry m, attard t, ben hariz m, et al. celiac disease in the mediterranean area. bmc gastroenterol. 2014;14:24. 26. hawamdeh h, al-zoubi b, al sharqi y, qasrawi a, abdelaziz y, barbar m. association of tissue transglutaminase antibody titer with duodenal histological changes in children with celiac disease. gastroenterol res pract. 2016;2016:6718590. research 22 j contemp med sci | vol. 6, no. 1, january–february 2020: 22–25 original issn 2413-0516 introduction uterovaginal prolapse is a common gynecological condition particularly in the grandmultipara.1–3 it is of importance to gynecologist in the developing and low resource countries as women in these environments are predisposed to genital prolapse due to repeated child birth, low-skilled attendant at delivery, and low contraceptive usage.1–4 it has a prevalence of 41–50% in women over the age of 40 years, with a lifetime risk of 7%.5 however, the prevalence is difficult to determine in low resource environment as most of the women do not seek medical attention unless symptoms are pronounced and disturbing.6 the female genital organs are maintained in their normal anatomical position by a number of fascial condensations (endopelvic fascia) such as the transverse cervical (cardinal) and uterosacral ligaments.6 genital prolapse occurs as a result of weakness of these supportive structures. risk factors for genital prolapse include repeated deliveries, difficult vaginal deliveries, increase intra-abdominal pressure, and estrogen withdrawal as in post-menopausal women.7–9 three degrees of uterovaginal prolapse are described and the level of the cervix (the lowest and dependent part) is assessed while the patient is straining. first-degree prolapse is when the descent is still within the vagina; second degree when it has reached the introitus and third degree when it has gone beyond the introitus. the third degree, termed procidentia, is usually accompanied by cystourethrocoele and rectocoele.2 previous surveys have studied the prevalence, risk factors, and management of uterovaginal prolapse; however, there is paucity of information on utilization of reproductive health services and health-seeking behavior of patients with genital prolapse.1–4,10,11 this information is important, especially in low resource environment, as it gives insight on underlying factors that may predispose patients to this condition and behavior that promotes it. this will form basis for formulation of preventive strategies. therefore, this study was designed to determine the prevalence, sociodemographic characteristics, utilization of reproductive health services, and health-seeking attitude of patients with uterovaginal prolapse in university of calabar teaching hospital, south-south, nigeria. methods and materials this was a retrospective descriptive study of women who presented with genital prolapse in university of calabar teaching hospital over a 10-year period from may 1, 2009 to june 1, 2019. data of patient diagnosed with genital prolapse within this period were collected from registers in the gynecological clinic, gynecological ward, gynecological theatre, and from patients’ case records in the medical record department. data obtained included age, parity, menopausal status, occupation, marital status, grade of prolapse, duration of prolapse before presentation, history of antenatal care, history of skilled attendant at previous deliveries, and history of contraceptive use. spss statistics (ibm corp. version 22) program was used for data analysis. results during the period studied, of the 15,543 new gynecological clinic attendees 45 patients were diagnosed with uterovaginal prolapse: the sociodemographic profile and reproductive health service uptake in a low resource setting, calabar, nigeria charles obinna njoku,a,b amarachukwu nnaemezie njoku,b efiok eyo efioka,b adepartment of obstetrics and gynecology, university of calabar, calabar, nigeria. bdepartment of obstetrics and gynecology, university of calabar teaching hospital, calabar, nigeria. correspondence to: njoku charles (email: charlesnjokuobinna@gmail.com). (submitted: 09 september 2019 – revised version received: 04 october 2019 – accepted: 12 october 2019 – published online: 26 february 2020) objective to determine the prevalence, sociodemographic profiles, utilization of reproductive health services, and delay in seeking medical care of patient with uterovaginal prolapse in calabar, nigeria. methods this was a retrospective study of women who presented with uterovaginal prolapse at university of calabar teaching hospital, calabar, nigeria between may 1, 2009 and june 1, 2019. patients’ case records were retrieved and analyzed. statistical analysis was done using spss version 22. results the prevalence of genital prolapse was 0.3%. the mean age and parity were 60.19 ± 8.71 years and 6.31 ± 2.80, respectively. the mean duration of symptoms before presentation was 3.19 ± 2.16 years. genital prolapse was commonest among age group 60–79 years (52.8%), parity 5–9 (66.7%), post-menopausal (97.2%), primary education (55.6%), and farmers (47.2%). grade 3 uterovaginal prolapse was the commonest grade (58.3%). most patients (86.1%) had symptoms of genital prolapse for less than 5 years before seeking medical treatment. the majority of patients had no antenatal care during their pregnancies (80.6%), no skilled attendant at deliveries (86.1%), and no contraceptive use during their reproductive years (77.8%). participants with lower parity (1–4) (p = 0.03), higher educational level (p < 0.001), and teachers/civil servants (p = 0.043) presented earlier (<1 year) to the hospital. conclusion there is poor utilization of reproductive health services among women who develop uterovaginal prolapse in our environment. women with higher social status sought for help earlier. increasing awareness of this condition and providing antenatal care, skilled birth attendants, and contraceptive services will reduce the burden of this condition. keywords uterovaginal prolapse, reproductive health services, delayed presentation. 23 original uterovaginal prolapsec. obinna njoku et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 22–25 genital prolapse, giving a prevalence of 0.3%. however, 39 case records were retrieved of which 36 had adequate information for analysis. the mean age and parity were 60.19 ± 8.71 years and 6.31 ± 2.80, respectively. the mean duration of symptoms before presentation was 3.19 ± 2.16 years. sociodemographic features of patients are shown in table 1. genital prolapse was commonest among age group 60–79 years (52.8%), followed by 40–59 years (44.4%). the modal parity was 5–9 (66.7%). majority (97.2%) of the patients were post-menopausal, 55.6% had primary education, 47.2% were farmers, and 94.4% were married. grade 3 uterovaginal prolapse was the commonest grade (58.3%) and grade 2 was the second commonest (38.9%) while grade 1 was the least (2.8%) as shown in fig. 1. table 2 shows the duration of symptoms before seeking care and usage of reproductive health services of patients. the commonest duration of symptoms before seeking medical treatment was 2 years and below (44.4%), followed by 3–4 years (36.1%), and the least was 7 years and above (8.3%). the majority of patient had no antenatal care during their pregnancies (80.6%), no skilled attendants at deliveries (86.1%), and did not use contraceptive during their reproductive years (77.8%). the relationship between delay in seeking medical care and sociodemographic characteristics of patients is shown in table 3. higher proportion (66.7%) of participants with lower parity (1–4) presented earlier (<1 year) compared to participants with higher parity and the difference is statistically significant (p = 0.03). participants with tertiary (66.7%) and secondary education (100%) presented earlier (<1 year) than those with lower educational level (p < 0.001). teachers (100%) and civil servants (66.7%) sought help earlier (<1 year) than farmers, traders, and housewives (p = 0.043). table 1. sociodemographic features of participants. variables frequency percentage (%) age (years)   40–59 16 44.4 60–79 19 52.8 >80 1 2.8 parity  1–4 9 25 5–9 24 66.7 >10 3 8.3 menopausal status post-menopause 35 97.2 pre-menopause 1 2.8 education level no formal education 9 25 primary 20 55.6 secondary 4 11.1 tertiary 3 8.3 occupation  farmer 17 47.2 trader 11 30.6 housewife 4 11.1 teacher 3 8.3 civil servant 1 2.8 marital status   married 34 94.4 single 2 5.6 fig. 1 grade of genital prolapse among participants. table 2. reproductive health seeking behavior of participants variables frequency percentage (%) duration of symptom before presentation (years)   ≤2     3–4 16 44.4 5–6 13 36.1 ≥7 4 11.1   3 8.3 antenatal care in previous deliveries  yes     no 7 19.4   29 80.6 skilled attendant at deliveries  yes 5 13.9 no 31 86.1 history of contraceptive use  yes 8 22.2 no 28 77.8 24 original uterovaginal prolapse c. obinna njoku et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 22–25 discussion the prevalence of uterovaginal prolapse among gynecological clinic attendees in this study was 0.3%. this was comparable to 0.8% of gynecological clinic attendees south-south nigeria, but lower than 3.9% of gynecological admissions in south-east nigeria, and 1.4% of gynecological admissions in northern nigeria.1,2,4 the prevalence of genital prolapse in this study may not be the true representation of the burden of the condition as this is a hospital-based study. moreover, social and economic barriers that may preclude hospital presentation are highly prevalent in our environment. there is need for community-based study in order to ascertain the actual burden of genital prolapse in this environment. this will help to plan effective preventive strategies. the mean age for genital prolapse in this study was 60.19 ± 8.71 years, with the modal age being 60–79 years. similar findings were reported from previous studies.1.2,3 the mean parity was 6.31 ± 2.80, with uterovaginal prolapse being commonest among grandmultiparous women. similarly, in other studies, genital prolapse was commonest among grandmultiparous women.1,2,10 the present study agrees with previous surveys that uterovaginal prolapse is most prevalent among post-menopausal women.1,2,11 majority of women with genital prolapse in this study were farmers. this agrees with findings from other african studies.1,12 grandmultiparity, post-menopausal status and farming can be explained as risk factors for uterovaginal prolapse as repeated childbirth leads to disruption of the myofascial fibers that support the pelvic organs, estrogen withdrawal weakens the integrity of the pelvic support and the physical activities involved in farming increases the intra-abdominal pressure, leading to genital prolapse. third-degree uterovaginal prolapse was the commonest type in this study. this was similar to findings reported by oraekwe et al. in south-east nigeria.1 studies in enugu, southeast nigeria, port-harcourt, and south-south nigeria showed contrasting findings, with second-degree uterovaginal prolapse being the most prevalent type.10,11 the disparity may be explained by difference in level of awareness and care-seeking attitude of patients in the different study settings. in the present study, majority of women with genital prolapse did not have antenatal care during their pregnancies, there were no skilled attendants during their labors and deliveries, and they did not receive contraceptive services during their reproductive years. these findings underscore the importance of reproductive health services in preventing development of genital prolapse. antenatal care services and presence of skilled personnel during labor and delivery can prevent conditions such as prolonged labor, obstructed labor, traumatic deliveries and perineal lacerations. these obstetric conditions are known to predispose to genital prolapse.5,9 the use of contraceptives enable women to space pregnancies and limit pregnancies to desired number. this prevents high parity which is a known predisposing factor of genital prolapse.1,2,10 majority of women in the present study with lower parity, higher educational level and skilled occupation such as teachers and civil servants presented earlier (<1 year) to the hospital. these findings suggest that women with higher socioeconomic status may be better informed of this condition and were able to overcome socioeconomic barriers against accessing medical treatment. these challenges include depending on husband or other relatives for permission and finance to seek medical treatment, and cultural and religious beliefs against orthodox medical treatments.13 there is a need for increased awareness, especially among women of lower socioeconomic status in order to improve health-seeking behavior of women with genital prolapse. there should also be a scale-up education on the importance of reproductive health services such as antenatal care services, skilled personnel, services in labor and delivery, and family planning services, as preventive tools for genital prolapse. these reproductive health services should be made available and accessible to women to reduce genital prolapse and its antecedent distressful morbidities. conflict of interest none table 3. relationship delay in seeking medical treatment and socio-demographic characteristics of participants. variables total duration of symptom p-value1 year and below (%) above 1 year (%) age (years) 40–59 16 7(43.8) 9(56.2) x2 = 5.456 60–79 years 19 2(10.5) 7(89.5) df = 2 >80 years 1 0(0.0) 1(100.0) p value = 0.065 parity 1–4 9 6(66.7) 3(33.3) x2 = 11.333 5–9 24 3(12.5) 21(87.5) df = 2 >10 3 0(0.0) 3(100.0) p value = 0.03* education level     no formal education 9 0(0.0) 9(100.0)   primary 20 3(15.0) 17(85.0) x2 = 18.844 secondary 4 4(100.0) 0(0.0) df = 3 tertiary 3 2(66.7) 1(33.3) p value < 0.001* occupation     farmer 17 1(5.9) 16(94.1)   trader 11 4(36.4) 7(63.6) x2 = 9.849 housewife 4 1(25.0) 3(75.0) df = 4 teacher 3 2(66.7) 1(33.3) p value = 0.043* civil servant 1 1(100.0) 0(0.0)   25 original uterovaginal prolapsec. obinna njoku et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 22–25 references 1. oraekwe oi, udensi ma, nwachukwu kc, okali uk. genital prolapse: a 5-year review at federal medical centre umuahia, southeastern nigeria. niger. med. j. 2016;57(5):286-289. 2. oyiji ec, dike ei, anolue fc, nzewuihe ace, ejikem cc. uterovaginal prolapse at a university teaching hospital in south-east nigeria. orient j. med.2013;25(3–4):107-112. 3. balogun or. genital prolapse in ilorin: a seven-year review. niger. j. med. 1997;6:77-82. 4. yakubu a, abubakar panti aa, ladan aa, burodo at, hassan ma, nasir s. pelvic organ prolapse managed at usmanu danfodiyo university teaching hospital, sokoto: a 10-year review. sahel med. j. 2017;20:26-9. 5. monga a, dobbs s, editors. gynaecology by ten teachers. 19th ed. london: book power; 2011. p. 154-62. 6. agboola a, editor. urogenital prolapse and the displacement of the uterus. in: textbook of obstetrics and gynaecology for medical students. 2nd ed. ibadan: heinemann educational books plc; 2006. p. 33-88. 7. ogunbode o, aimakhu ve. uterine proplapse during pregnancy in ibadan. am. j. obstet. gynaecol. 1973;16:622-625. 8. cespedes ro, cross ca, meguire ej. pelvic prolapse: diagnosing and treating uterine and vaginal vault proplapse. medscape women’s health 1998;3:3-4. 9. molton pjd. uterovaginal prolapse. in: studd j (ed). progress in obstetrics and gynaecology, vol. 7, edinburgh, churchill livingstone, 1989:319-330. 10. ugboma ha, okpani ao, anya se. genital prolapse in port harcourt, nigeria. niger. j. med. 2004;13:124–9. 11. okeke tc, ani vc, ezenyeaku cc, ikeako lc, enwereji jo, ekwuazi k. an audit of uterovaginal prolapse in enugu, southeast nigeria. am. j. clin. med. res. 2013;1:23–5. 12. akmel m, segni h. pelvic organ prolapse in jimma university specialized hospital, southwest ethiopia. ethiop. j. health sci. 2012;22:85–92. 13. njoku co, njoku an. obstetric fistula: the agony of unsafe motherhood. a review of nigerian experience. jammr 2018; 28(12): 1-7. article no. jammr 46890. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.02202005 274 j contemp med sci | vol. 5, no. 5, september-october 2019: 274–278 accuracy of linear measurements made on cone beam computed tomography scans at different magnifications aisan ghaznavi,a dara ghaznavi,b solmaz valizadeh,c zahra vasegh,c and muna al-shuhayebd adepartment of oral and maxillofacial radiology, school of dentistry, urmia university of medical sciences, urmia, iran. bdepartment of periodontics, faculty of dentistry, tabriz university of medical sciences, tabriz, iran. cdepartment of oral and maxillofacial radiology, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. ddepartment of periodontics, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. correspondence to asian ghaznavi (email: aisanghaznavi@yahoo.com). (submitted: 12 june 2019 – revised version received: 28 july 2019 – accepted: 04 august 2019 – published online: 26 october 2019) introduction before the introduction of cross-sectional imaging, intraoral periapical and panoramic radiography were the most commonly used imaging techniques for implant treatment planning. these radiographic methods could only provide two-dimensional images of three-dimensional structures. since 1980s, multidetector computed tomography (mdct) has been the most accurate imaging technique for evaluation of implant site. however, mdct has certain limitations such as high radiation dose, reduced image quality due to metal artifacts and high cost. furthermore, non-isotropic voxels could lead to lower resolution of the reconstructed images compared with original axial scans. in the1990s, tomography systems such as cone beam ct (cbct) were introduced for the head and neck imaging to overcome the limitations of ct.1,2 in the recent years, cbct has been increasingly used in different fields of dentistry3,6 with several applications in implant treatment.7 one of the advantages of cbct software for dentists is the possibility to make linear measurements between anatomical locations. normally, linear measurements reused to determine the thickness and height of the alveolar ridge for the assessment of implant site before surgery, as well as to measure the distances between anatomical landmarks in orthodontics and calculate the size of pathological lesions of the jaws. since linear measurements are required for implant therapy, orthodontic treatment and surgical management of pathological lesions, clinicians need to be aware of the accuracy of linear measurements made on cbct scans, and the effects of different enhancement features on these measurements.8–9 similar to other digital imaging software programs, cbct offers various enhancement features to modify image quality based on the preferences of the user. primary features used to enhance image quality are magnification (zoom, window/ level) and annotation. moreover, cursor-driven algorithms enable accurate real time measurements.10,11 several studies have evaluated the effects of parameters such as voxel size, thickness and reconstruction angle on the accuracy of cbct linear measurements. the effects of magnification on the diagnostic accuracy of two-dimensional digital radiography have also been the subject of several studies.12–19 according to the literature; no study has assessed the accuracy of linear measurements made on cbct scans with different image magnifications. therefore, this study aimed to assess the effects of image magnification on the accuracy of linear measurements made by new tom vgi cbct device (new tom vgi, quantitative radiology, verona, italy). materials and methods in this study, 42 titanium pins with equal dimensions were inserted into seven dry sheep mandibles. before the placement of implants in the mandibles, pin lengths and diameters were measured using a digital caliper with 0.01 mm accuracy (mitutoyo corp., kawasaki, japan) (figs. 1 and 2). pin lengths were measured from the top to the bottom. since pin diameters were not equal along the pin, square-shaped pin heads were considered as the main indicator of the diameter. objective this study aimed to evaluate the accuracy of linear measurements made on cone beam computed tomography (cbct) (new tom vgi) scans with different image magnifications. methods forty-two titanium pins were inserted into seven dry sheep mandibles. lengths of the pins were measured using a digital caliper with 0.01 mm readability, and the mandibles were radiographed using a cbct unit. after reconstruction of cbct images, three experienced radiologists measured the length and diameter of titanium pins at 100%, 200% and 400% magnifications. accuracy of measurements was analyzed using descriptive statistics and one-way analysis of variance. p < 0.05 was considered statistically significant. interobserver reliability was calculated using the intraclass correlation coefficient test. results the mean differences of linear measurements from the actual lengths of pins were 0.1960, 0.2143 and 0.2047 mm at 100%, 200% and 400% magnifications, respectively (p > 0.05). the mean differences of linear measurements from the actual diameters of pins were 0.2206, 0.2063 and 0.1984 mm at 100%, 200% and 400% magnifications, respectively (p > 0.05). interobserver reliability of pin length measurements was estimated to be 0.285, 0.707 and 0.479 at 100%, 200% and 400% magnifications, respectively. interobserver reliability of pin diameter measurements was 0.078, 0.469 and 0.587 at 100%, 200% and 400% magnifications, respectively. conclusion based on the results, image magnification does not affect the accuracy of linear measurements made on cbct scans. interobserver reliability of pin length measurements was good at 200% magnification, while it was below the acceptable range at other magnifications. for measurement of pin diameters, this index was below the acceptable range at all magnifications. keywords cone beam computed tomography, image magnification, linear measurement issn 2413-0516 original 275j contemp med sci | vol. 5, no. 5, september-october 2019: 274–278 a. ghaznavi et al. accuracy of linear measurements made on cone beam computed tomography scans at different magnifications to radiographically measure the lengths, overall length of the pins was measured in the cross-sectional plane. in addition, square heads of the pins were measured in the axial plane to estimate the pin diameters (figs. 5 and 6). accuracy of linear measurements of the lengths and diameters of the pins in the axial and cross-sectional planes was fig. 1 pin length measurement by digital caliper. fig. 2 pin diameter measurement by digital caliper. to facilitate measurement in the axial plane, diameter of the pin heads was considered as the diameter of the pins. in order to insert the pins into the bone, first, parallel holes were drilled in the edentulous alveolar ridge crest. afterward, the holes were widened to the diameters of the pins using high-speed hand piece and a diamond fissure bur, and the pins were finally inserted into the mandibles (figs. 3 and 4). mandibles were placed in a container filled with water to simulate soft tissues. afterwards, they were placed in the holder of the cbct device (new tom vgi, quantitative radiology, verona, italy). to standardize the position of the mandibles in the cbct device, midsagittal and occlusal planes were adjusted to the vertical and horizontal laser markers, respectively. the mandibles were radiographed using 8 × 12 cm field of view at 110 kvp. after image reconstruction by nnt viewer software, linear measurements were made by three oral and maxillofacial radiologists at 100%, 200% and 400% magnifications. the observers were allowed to change the contrast, sharpness and brightness of the images, which were displayed on a monitor with 1024 × 1280 pixels resolution and 32-bit color depth. fig. 3 parallel drilling of implant holes. fig. 4 pins inserted into dry sheep mandibles. fig. 5 linear measurement of pin lengths at 100%, 200% and 400% magnifications. original 276 j contemp med sci | vol. 5, no. 5, september-october 2019: 274–278 accuracy of linear measurements made on cone beam computed tomography scans at different magnifications a. ghaznavi et al. analyzed using repeated measures analysis of variance (anova). additionally, interobserver reliability was calculated for all magnifications using intraclass correlation coefficient (icc). results in this study, 50% of the observers underestimated the pin lengths by 0.1–0.5 mm compared with the actual size, whereas 7.7% of them overestimated the pin lengths by 0.1–0.5 mm compared with the actual size. errors greater than +0.5 mm did not occur in any of the measurements. error ranges for pin length measurement sat all magnifications are presented in table 1. on the other hand, 65% of the observers overestimated the pin diameters by 0.1–0.5 mm compared with the actual size, whereas 1.6% of them underestimated the pin diameters by 0.1–0.5 mm compared with the actual size. errors greater than +5.0 mm or smaller than −5.0 mm did not occur in any of the measurements. error ranges for pin diameter measurements at all magnifications are shown in table 2. the mean absolute values of differences between the radiographic measurements of lengths and the actual lengths of the pins at 100%, 200% and 400% magnifications were 0.2206, 0.2063, and 0.1984 mm, respectively. according to the results of repeated measures anova, there was no significant difference in the accuracy of pin length measurements at different magnifications (p > 0.05). minimum, maximum, mean and standard deviation of differences of pin length measurements at all magnifications are presented in table 3. table 3. minimum, maximum, mean and sd of pin length measurement differences at all magnifications magnification minimum difference maximum difference mean sd 100 0.00 0.60 0.2206 0.14986 200 0.00 0.50 0.2063 0.12583 400 0.00 0.50 0.1984 0.12266 fig. 6 linear measurement of pin diameters at 100%, 200% and 400% magnifications. table 1. error ranges of pin length measurements at all magnifications by the three observers error range first observer second observer third observer total +0.5 to +1 0 0 0 0 +0.1 to +0.5 7.9 15.1 0 7.7 ±0.1 49.2 53.2 20.6 41 −0.1 to −0.5 41.3 30.1 78.6 50 −0.5 to −1 1.6 1.6 0.8 1.3 table 2. error ranges of pin diameter measurements at all magnifications by the three observers error range first observer second observer third observer total +0.5 to +1 0 0 0 0 +0.1 to +0.5 55.6 57.9 81.7 65 ±0.1 39.7 42.1 18.3 33.4 −0.1 to −0.5 4.7 0 0 1.6 −0.5 to −1 0 0 0 0 table 4. the icc results forth measurement of pin lengths and pin diameters at different magnifications magnification variable 100% 200% 400% length 0.285 0.707 0.479 diameter 0.078 0.469 0.587 the mean absolute values of differences between the radiographic measurements of pin diameters and actual diameters of the pins were 0.1960, 0.2143, and 0.2047 mm at 100%, 200%, and 400% magnifications, respectively. according to the results of repeated measures anova, there was no significant difference in the accuracy of pin diameter measurements using different magnifications (p > 0.05). to calculate interobserver reliability for pin length measurements, we used the icc test. at all magnifications, interobserver reliability was estimated to be <0.6. for measurement of pin diameters, interobserver agreement was 0.7 at 200% magnification and it was <0.6 at other magnifications. the icc test results regarding the measurement of pin lengths and diameters at all magnifications are presented in table 4. discussion the cbct enhancement software programs facilitate and improve the diagnostic accuracy of cbct measurements for oral and maxillofacial radiologists. three-dimensional image reconstruction in different planes and enabling linear and angular measurements are some of the features of cbct.20 this study aimed to evaluate the accuracy of cbct linear measurements using new tom vgi device at different image magnifications. according to the obtained results, the magnification level had no effect on the accuracy of linear measurements. reliability of pin diameter measurements at all magnifications was below the acceptable range, whereas it was good at 200% magnification for pin length measurements. original 277j contemp med sci | vol. 5, no. 5, september-october 2019: 274–278 a. ghaznavi et al. accuracy of linear measurements made on cone beam computed tomography scans at different magnifications the low interobserver reliability was not clinically significant due to low variation among the measurements and the acceptable accuracy of measurements at all magnifications. according to the findings of ganguly et al., cbct imaging is required to evaluate the quality and quantity of the available bone for implant placement, and to determine the exact location of anatomical structures. they also concluded that <1 mm error in radiographic measurements for implant placement was clinically acceptable.21 accordingly, errors in all measurements in our study were within the clinically acceptable range. in the current study, pin lengths were mostly underestimated by 0.1–0.5 mm compared with the actual size, while pin diameters were mostly overestimated by 0.1–0.5 mm compared with the actual size. in a study conducted by fatemitabar et al.22, the accuracy of linear measurements made by cbct (planmeca) was compared with that of ct 64-channel, and the mean difference of measurements from the actual size was estimated to be +0.37 to +0.58 mm using cbct, and +0.37 to +0.72 mm using ct. image quality and accuracy of linear measurements made by cbct are affected by different factors, such as the material of the pin (in pin length measurements), thickness of sections, reconstruction angle and device rotation during imaging.12,16 the findings of the current study indicated that magnification level had no effect on the accuracy of linear measurements. moreover, it was observed that reliability of pin diameter measurements at all magnifications was below the acceptable range, whereas it was good at 200% magnification for pin length measurements. low rate of inter observer agreement was probably due to low variation among the measurements. sherrard et al.,12 and moshfeghi et al.13 reported that size of voxels had no significant effect on the accuracy of cbct linear measurements or interobserver agreement. although the aforementioned studies evaluated the effects of factors other than magnification on the accuracy of linear measurements, similar to our findings, they reported no significant association between the studied parameters and the accuracy of linear measurements. however, the interobserver agreement in the above-mentioned studies was much higher than the value in the current study; the icc was reported to be >0.99 in both of the aforementioned studies. lower interobserver agreement in our study was due to the low variation among the measurements. in the current study, there was no significant difference in the accuracy of linear measurements made at different image magnifications. in a study conducted by hashem et al., there was no significant difference between the radiographic measurements obtained at two different device rotations and direct measurements. similar to our study, hashem et al.14 reported no significant correlation between the accuracy of cbct linear measurements and the studied variable; however, inter observer and intraobserver agreements were reported to be good. according to the results obtained by nikneshan et al., changing the reconstruction angle from −12° to +12° decreased the accuracy of cbct linear measurements. they reported that error rate was below 0.5 mm at all reconstruction angles, which was clinically acceptable.15 similar to our study, titanium pins were inserted into sheep mandibles in the study conducted by nikneshan et al.15 it is noteworthy that titanium pins were used due to low atomic number of the metal,16 which, in turn, decreases the metal artifacts in ct. another similarity between their study and ours was that in both of the studies the mandibles were immersed in water to simulate soft tissues. however, unlike our study, researchers only measured the length of pins for evaluating the accuracy of measurements in the study conducted by nikneshan et al.15 to date, no study has evaluated the effect of image magnification on the accuracy of linear measurements. however, several studies have investigated the effects of image magnification on radiographic diagnostic accuracy. according to the results of the current study, image magnification has no effect on the accuracy of linear measurements. in one research, kositbowornchai et al. reported similar results regarding the diagnostic accuracy of root fractures at different image magnifications (50%, 100% and 200%). consistent with our findings, they observed no significant difference in the criteria for diagnostic accuracy namely positive and negative predictive values, sensitivity and specificity among three different magnifications.17 effects of magnification on interobserver reliability were not assessed in the aforementioned study. in another study, kositbowornchai et al. evaluated the effects of changes in sharpness, magnification and pseudo color on the diagnostic accuracy of digital two-dimensional radiographs for occlusal caries. according to their results, there was no significant relationship between these variables and the diagnostic accuracy of radiography, which is consistent with our findings. the rate of interobserver agreement in the study by kositbowornchai et al.17,18 was reported to be good according to cohen’s kappa coefficient. therefore, the rate of this parameter was higher in the study by kositbowornchai et al. compared with the rate in our study. in another study, morais et al. evaluated the effect of image magnification (100%, 200%, and 400%) in twodimensional digital radiography on the accuracy of diagnosis of periodontal bone lesions in vitro. similar to our study, there was no significant difference in the diagnostic accuracy at different image magnifications.19 in addition, rate of intraobserver agreement for each observer was reported to be moderate at 100%, 200% and 400% magnifications. conclusion in conclusion, it could be stated that the accuracy of linear measurements made on cbct images is not affected by image magnification. moreover, interobserver reliability of the measurement of pin diameter was good at 200%, while it was below the acceptable range at other magnifications. interobserver reliability was below the acceptable range for the measurement of pin lengths at all magnifications. the low interobserver reliability was not of clinical significance because of low variation among the measurements and acceptable accuracy of measurements at all magnifications. conflicts of interest none.  original 278 j contemp med sci | vol. 5, no. 5, september-october 2019: 274–278 accuracy of linear measurements made on cone beam computed tomography scans at different magnifications a. ghaznavi et al. references 1. benavides e, rios hf, ganz sd, an ch, resnik r, reardon gt, et al. use of cone beam computed tomography in implant dentistry: the international congress of oral implantologists consensus report. implant dent. 2012;21:78–86. 2. al-ekrish aa, ekram m. a comparative study of the accuracy and reliability of multidetector computed tomography and cone beam computed tomography in the assessment of dental implant site dimensions. dentomaxillofac radiol. 2011;40:67–75. 3. sheikhi m, ghorbanizadeh s, abdinian m, goroohi h, badrian h. accuracy of linear measurements of galileos cone beam computed tomography in normal and different head positions. int j dent. 2012;2012:214954. 4. ziegler cm, woertche r, brief j, hassfeld s. clinical indications for digital volume tomography in oral and maxillofacial surgery. dentomaxillofac radiol. 2002;31:126–130. 5. guerrero me, jacobs r, loubele m, schutyser f, suetens p, van steenberghe d. state-of-the-art on cone beam ct imaging for preoperative planning of implant placement. clin oral investig. 2006;10:1–7. 6. hellak a, schmidt n, schauseil m, stein s, drechsler t, korbmachersteiner hm. influence of invisalign treatment with interproximal enamel reduction (ier) on bone volume for adult crowding: a retrospective threedimensional cone beam computed tomography study. bmc oral health 2016;16:83. 7. yamamoto k, ueno k, seo k, shinohara d. development of dentomaxillofacial cone beam x-ray computed tomography system. orthod craniofac res. 2003;6:160–162. 8. yepes jf, al-sabbagh m. use of cone-beam computed tomography in early detection of implant failure. dent clin north am. 2015;59:41–56. 9. cavalcanti mg, rocha ss, vannier mw. craniofacial measurements based on 3d-ct volume rendering: implications for clinical applications. dentomaxillofac radiol. 2004;33:170–176. 10. john gp, joy te, mathew j, kumar vr. fundamentals of cone beam computed tomography for a prosthodontist. j indian prosthodont soc. 2015;15:8–13. 11. tyndall da, rathore s. cone-beam ct diagnostic applications: caries, periodontal bone assessment, and endodontic applications. dent clin north am. 2008;52:825–841, vii. 12. sherrard jf, rossouw pe, benson bw, carrillo r, buschang ph. accuracy and reliability of tooth and root lengths measured on cone-beam computed tomographs. am j orthod dentofacial orthop. 2010;137:s100–s108. 13. moshfeghi m, tavakoli ma, hosseini et, hosseini at, hosseini it. analysis of linear measurement accuracy obtained by cone beam computed tomography (cbct-newtom vg). dent res j (isfahan). 2012;9:s57–s62. 14. hashem d, brown je, patel s, mannocci f, donaldson an, watson tf, et al. an in vitro comparison of the accuracy of measurements obtained from highand low-resolution cone-beam computed tomography scans. j endod. 2013;39:394–397. 15. nikneshan s, aval sh, bakhshalian s, shahab s, mohammadpour m, sarikhani s. accuracy of linear measurement using cone-beam computed tomography at different reconstruction angles. imaging sci dent. 2014;44:257–262. 16. boas fe, fleischmann d. evaluation of two iterative techniques for reducing metal artifacts in computed tomography. radiology. 2011;259:894–902. 17. kositbowornchai s, sikram s, nuansakul r, thinkhamrop b. root fracture detection on digital images: effect of the zoom function. dent traumatol. 2003;19:154–159. 18. kositbowornchai s, basiw m, promwang y, moragorn h, sooksuntisakoonchai n. accuracy of diagnosing occlusal caries using enhanced digital images. dentomaxillofac radiol. 2004;33:236–240. 19. de morais j, sakakura ce, lofferdo lc, scaf g. accuracy of zoomed digital image in the detection of periodontal bone defect: in vitro study. dentomaxillofac radiol. 2006;35:139–142. 20. rangel fa, maal tj, bronkhorst em, breuning kh, schols jg, bergé sj, et al. accuracy and reliability of a novel method for fusion of digital dental casts and cone beam computed tomography scans. plos one. 2013;8:e59130. 21. ganguly r, ruprecht a, vincent s, hellstein j, timmons s, qian f. accuracy of linear measurement in the galileos cone beam computed tomography under simulated clinical conditions. dentomaxillofac radiol. 2011;40:299–305. 22. fatemitabar sa, nikgoo a. multichannel computed tomography versus cone-beam computed tomography: linear accuracy of in vitro measurements of the maxilla for implant placement. int j oral maxillofacial implants. 2010;25:499–505. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201907 original 17j contemp med sci | vol. 6, no. 1, january–february 2020: 17–21 original issn 2413-0516 introduction cancers of head and neck area including lips, mouth, tongue, tonsils, pharynx, larynx, hypopharynx, and salivary glands are typically challenging to handle because, unlike malignancies of other organs, they cannot be easily delimited.1 head and neck cancers are the sixth most common cancer in the world. about 40% of these cancers occur in the mouth,2 15% in the hypopharynx, and 25% in the larynx.3 head and neck area is the position of organs that control primary functions such as respiration, swallowing, talking, and hearing and basically any organ that is associated with social interaction.4 in the case of oral cancer, research has shown an increase in the incidence of this cancer worldwide,5 and although this could be seen as a global health problem, two-thirds of the deaths due to this cancer occur in developing countries.6 oral cancer and other head and neck malignancies have major effects such as pain, mucositis, dry mouth, and loss of taste, smell, appetite, and weight, which can severely affect the quality of life (qol) of patients.7 head and neck cancers are known to have significantly high morbidity rates. in addition, the side effects of surgery and radiotherapy can also undermine the qol of patients.8 the effects on appearance and functions often change the patients’ understanding and feelings and their ability to socially interact with others. often, these patients are unable to hide the outward and functional effects of the illness, and this negatively affects their self-esteem and confidence.9 given the major challenges that these patients must face in their lives after treatment and their need to maintain functionality despite their condition, their qol is an important issue worthy of deep consideration.10 the qol of patients with head and neck cancer can be improved through different therapeutic approaches. the efforts in this area have led to the development of several questionnaires and also qol measurements becoming one of the components of treatment protocols. according to the definition of the world health organization, qol is “an individual’s perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards, and concerns.”11 in a study by aquarwal et al. on the qol of patients with tongue cancer, they reported significant improvements in pain, general functions, and psychological state of these patients 12 months after treatment.12 in a research carried out by boyapati et al., an acceptable level of qol outcome was reported for local reconstructive treatment of early tongue/floor of mouth cancer. these researchers stated that local reconstructive surgery could be highly effective in managing early tongue/floor of mouth cancers. the instrument used in this research was the university of washington quality of life (uw-qol) questionnaire.11 in an assessment of qol of head and neck cancer patients undergoing modern radiotherapy, chen showed that 80% of these patients had a good overall qol.13 in another research, head and neck cancer patients who had undergone common surgical procedures with or without complementary therapy earned higher scores in most dimensions of qol.14 there exist strong evidence suggesting a positive relationship between the survival of people with head and neck cancer and their physical activity before treatment, and also between their survival and the change in qol from before treatment to 6 months after.15 it has been shown that patients with advanced cancer have lower qol and higher scores in pain, fatigue, and loss of appetite dimensions.2 a study by villaret et al., which evaluated the qol of treated oral cancer patients with the uw-qol questionnaire, showed that 77% of these patients had maintained their normal or near-normal functions 12 months after treatment. this study concluded that reconstructive treatment can be very effective in maintaining the assessment of quality of life among head and neck cancer patients forouzan rafie,a molook torabi,b ali taheri,c zahra mellata aneuroscience research centre, kerman university of medical sciences, kerman, iran. bdental and oral diseases research center, school of dentistry, kerman university of medical sciences, kerman, iran. cdepartment of oral pathology, school of dentistry, kerman university of medical sciences, kerman, iran. correspondence to: molook torabi (email: torabimolok773@gmail.com). (submitted: 09 september 2019 – revised version received: 04 october 2019 – accepted: 22 october 2019 – published online: 26 february 2020) objective this study evaluates the quality of life of the patients undergoing treatment at the oncology center in yazd, iran. methods this cross-sectional study was conducted on 29 patients with oral and head and neck cancer who were referred to the oncology center of shahid sadoughi hospital in yazd between may 2015 and february 2016. data were collected using the university of washington quality of life questionnaire and a demographic questionnaire. the illness-related information was obtained from the patients’ medical records. questionnaires were administered before the treatment and 6 months after its completion. data analysis was performed in spss 21 using chi-square and anova tests. p-values of less than 0.05 were considered statistically significant. results the sample consisted of 17 (58.6%) women and 12 (41.4%) men with a mean age of 40.00±14.30 years. the most frequent cancer location was the oral cavity and the most frequent treatment method was surgery. the mean score of quality of life before and after treatment was 12.60±2.81 and 11.39±2.63, respectively. the most important issues of the patients before and after treatment were pain and saliva, respectively. stage 3 and 4 patients had a significantly lower quality of life than stage 1 and 2 patients. conclusion the study found that undergoing treatment affects some dimensions of quality of life. hence, choosing the best treatment method with due attention to side effects and follow-up sessions is recommended. keywords cancer, head and neck, uw-qol, quality of life 18 original assessment of quality of life among head and neck cancer patients f. rafie et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 17–21 qol of oral cancer patients.16 for head and neck cancer, qol assessments can play a central role in the patient’s perception and understanding of treatment and provide a preview of the outcome of interventions.11 the present study aimed to assess the qol of head and neck cancer patients who were referred to shahid sadoughi hospital of yazd1 (the only cancer treatment center of yazd province). this assessment was performed using the uw-qol questionnaire, which is a simple and practical instrument containing special questions for patients with head and neck cancer.17 methods this cross-sectional (descriptive–analytic) study was conducted on patients with head and neck cancer in yazd province who were referred to shahid sadoughi hospital from may 2015 to february 2016. the data collection instrument was a qol evaluation questionnaire and a demographic questionnaire querying age, education level, and occupation. illness-related information, including cancer type and location, clinical stage, and treatment type were collected from the patients’ medical files. qol was evaluated using the uw-qol questionnaire, which is a widely accepted instrument for measuring the qol outcomes of head and neck cancers. this questionnaire consists of two parts. the first part has seven questions covering pain, appearance, swallowing, chewing, taste, and saliva. the second part has three global questions about how patients feel relative to before cancer, about their health-related qol, and about their overall qol. of the seven questions contained in the first part, six are four-choice questions with scores between 0 and 3, and one is a three-choice question with a score between 0 and 2. the total score of the questionnaire ranges from 0 (worst) to 20 (best). patients under the age of 18, those with secondary cancers, and those who could not understand the questions due to mental or cognitive impairments were excluded. for illiterate patients and those who could not read or respond for any reason, the researcher read the questions and answers and recorded the responses. all patients were informed that their names and personal information will remain completely confidential and that there is no obligation in responding to questions. subjects were selected by convenience sampling from eligible patients encountered during the study period. data analysis was performed in spss 21 using chi-square and anova tests. in all statistical tests, the significance level was considered to be 0.05. all participants completed written informed consent. the study was approved by the research ethics committee of kerman medical university code number. results a total of 42 patients entered the study. of these, 29 patients responded to all the questions both before and after the treatment. of these 29 patients, 12 were male (41.4%) and 17 were female (58.6%). these patients had a mean age of 40.00±14.30 years and an age range of between 18 and 70 years. among these patients, the most frequent cancer location was oral cavity (48.3%), the most frequent treatment method was surgery 1 the city of yazd is the capital of yazd province in iran (75.9%), and the most frequent education level was grade school (41.4%). demographic characteristics of the patients are provided in table 1. the mean and standard deviation of qol scores in pain, appearance, swallowing, chewing, speech, taste, and saliva dimensions (during the past 7 days) at the onset of treatment and after its end are presented in table 2. the mean qol score in this study was 12.60±2.81 before treatment and changed to 11.39±2.63 after treatment. table 3 shows the overall qol score of the patients before and after treatment and their relations with demographic and clinical variables. as can be seen, a significant difference was table 1. demographic features of the patients. percentnumbervariable 41.412man gender 58.617woman 44.813elementary educational level 27.58high school 17.25diploma 10.33license 31.09employee job 41.412worker 27.58jobless 48.314oral cavity tumor location 24.17larynx 10.33oropharynx 17.25salivary gland 65.519stage 1,2 tumor stage 34.410stage 3,4 75.922surgery type of treatment 6.92chemotherapy 10.33radiotherapy 6.92combination treatment table 2. mean and standard deviation of qol domains before and after treatment. after treatmentbefore treatment quality of life sdmeansdmean 0.272.140.231.15pain 0.311.930.182.18appearance 0.481.230.561.36swallowing 0.211.180.321.27chewing 0.382.230.632.28speech 0.162.140.142.25taste 0.761.750.752.11saliva 19 original assessment of quality of life among head and neck cancer patientsf. rafie et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 17–21 observed between cancer stages in terms of qol before and after treatment, in the sense that persons with stage 3 and 4 cancers had worse qol outcomes. the overall qol after treatment was significantly lower than before treatment. the most frequent answers to the question “which issues have been the most important to you during the past 7 days?” before and after treatment were pain (7 patients or 24.1%) and saliva (8 patients or 27.6%), respectively (fig. 1). answers to “health-related qol during the past 7 days” before and after treatment are illustrated in fig. 2. as can be seen, most people rated their qol as “good” before treatment and “fair” after treatment, and the percentage of people who described their qol as “poor” decreased after treatment. discussion assessment of qol of head and neck cancer patients can contribute to our understanding of the effects of these malignancies and related treatments on their daily activities and to the undergoing efforts to improve the existing support measures.4 the need for effective interventions to improve the health-related qol of cancer patients is widely recognized.18 the mean age of the patients in this study was 40.00±14.30, which is lower than the age range reported in other works.2, 19 this difference can be attributed to the type of study and the population studied.2 in the present study, the most common cancer location was the oral cavity, which is consistent with the observations of oliveira et al.2 in the present study, surgery was the most common method of treatment. the studies carried out by oliveira et al.2 and kumar et al.20 also reported the same finding. however, this is inconsistent with the reports of østhus et al.21 and chiou et al.22 in the present study, pain was the most prevalent problem before treatment but was considerably alleviated after treatment. agarwal et al. also reported a decrease in pain in patients with tongue cancer after treatment.12 the study conducted by gandhi et al. also found that pain was one of the four most important symptoms in patients with head and neck cancer.23 it has been reported that in head and neck cancer cases, pain typically affects oral function. research has also shown that pain is a common complaint in 58% of head and neck cancer patients waiting for treatment and 30% of patients after treatment.24–26 in this study, the most frequent post-treatment complaint was saliva problem. in the study of chen et al., the lowest score 5 years after the treatment was related to salivary dysfunction.13 problems with swallowing, local pain, and mouth dryness are very common complaints among head and neck cancer patients.27 salivary problems of the patients can be attributed to the methods of treatment commonly employed for head and neck cancers, including radiotherapy. in the present study, the patients who had undergone combination therapy had worse qol outcomes. this finding is in agreement with the findings of kessler et al.28 in this study, no statistically table 3. comparison of total score of qol before and after treatment according to demographic and clinical variables. p value after treatmentbefore treatment variable sdmeansdmean 0.05< 2.7411.253.2812.23man gender 2.5211.532.3411.89woman 0.05< 2.1010.273.0412.21elementary educational level 3.1512.524.1213.85high school 2.1011.792.3112.94diploma 3.1710.981.7711.53license 0.05< 3.1210.893.0112.52<40 age group (years) 2.4311.192.6412.5140–59 2.3412.093.7812.85≥60 0.05< 3.2113.124.0112.92oral cavity tumor location 2.5011.842.8013.38larynx 1.5410.422.3611.51oropharynx 3.2712.182.0712.63salivary gland 0.05> 2.0912.093.2914.07stage i, ii tumor stage 3.1710.692.1511.15stage iii, iv 0.05< 2.2812.733.3713.17surgery type of treatment 3.1412.453.2512.56chemotherapy 2.1510.272.1512.26radiotherapy 2.9510.112.5410.41combination treatment 0.05>2.6311.392.8112.60total score 20 original assessment of quality of life among head and neck cancer patients f. rafie et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 17–21 significant relationship was observed between the treatment method and the cancer-related qol relative to before treatment. crombie et al. also did not find a significant relationship between the treatment method and the qol of patients.14 in the present study, overall qol after treatment was lower than before treatment, although some improvements were also observed, most notably in pain. this study found that people with oropharynx and larynx cancer had better post-treatment qol outcomes than those with cancer in salivary gland and oral cavity. this is consistent with the results reported by bjordal et al.29 in the present study, the clinical stage of cancer had a significant relationship with the qol score, as people with stage 3 and 4 cancers had worse qol outcomes. this is not consistent with the findings of other studies such as gandhi et al., lee et al., yucel et al.,30,31,23 and torabi et al., which have found no statistically significant relationship between the clinical stage and oral health-related qol. this inconsistency could be due to the type of the study and the questionnaire used here.32 in this study, the mean total qol score after treatment was significantly lower than before treatment, which may be because 34.4% of the patients were in stage 3 or 4. hammerlid et al. have also reported a higher frequency of dental problems, viscous saliva, loss of taste, dysphagia, and feeling of sickness among stage 3 and 4 patients.33 after treatment, in response to the question “how would you rate your health-related quality of life?” no patient rated their qol as very poor or very well. the most important limitation of this study was reliance on self-reports and lack of clinical examinations to assess pain, dry mouth, swallowing, and other parameters enquired in the questionnaire. conclusion this study found that stage 3 and 4 head and neck cancer patients had significantly lower qol than stage 1 and 2 patients both before and after treatment. before treatment, patients reported that their most important problem during the preceding 7 days was pain, but after treatment, saliva was their biggest problem. patients generally gave a better rating to their qol after treatment. references 1. maccarthy d, nunn j, healy cm, stassen lf, gorman t, et al. outcomes from the first mouth cancer awareness and clinical check-up day in the dublin dental university hospital. j. ir. dent. assoc. 2012;58(2):101–108. 2. oliveira kg, von zeidler sv, jose rv podestá j rv, sena a, evandro d souza ed, jeferson lenzi j, bissoli n s, gouvea sa. influence of pain severity on the quality of life in patients with head and neck cancer before antineoplastic therapy. bmc cancer. 2014 jan 24;14:39 3. zevallos jp. international head and neck cancer epidemiology consortium: update no. 21. head neck. 2016;38:1447–1448. 4. mantia il, rossitto f, andaloro c. quality of life in head and neck cancer: patients’ and family caregivers ‘perceptions. egypt. j. ear nose throat allied scen. 2017;18(3):247–250. 5. awojobi o, scott e s, newton t. patients’ perceptions of oral cancer screening in dental practice: a cross-sectional study. bmc oral health 2012;12:55–63. 6. elango k j, anandkrishnan n, suresh a, iyer s k, ramaiyer sk, kuriakose m a. mouth self-examination to improve oral cancer awareness and early detection in a high-risk population. oral oncol. 2011;47:620–624. 7. ehrsson y t, langius-eklöf a, laurell g. nutritional surveillance and weight loss in head and neck cancer patients. support care cancer 2012;20:757– 765. 8. meyer f, fortin a, gélinas m, nabid a, brochet f, têtu b, bairati i. healthrelated quality of life as a survival predictor for patients with localized head and neck cancer treated with radiation therapy. j. clin. oncol. 2009 jun 20;27(18):2970–6 9. quality of life in patients treated for cancer of the oral cavity requiring reconstruction: a prospective study. acta otorhinolaryngol. ital. 2008;28:120–125. 10. zwahlen ra, dannemann c, grätz kw, studer g, zwahlen d, moergeli h, et al. quality of life and psychiatric morbidity in patients successfully treated for oral carity sguamous cell cancer and their wives. j. oral maxillofac. surg. 2008;66(6):1125–32. 11. boyapati rp, shah kc, flood v, stassen lf. quality of life outcome measures using uw-qol questionnaire v4 in early oral cancer/squamous cell cancer resections of the tongue and floor of mouth with reconstruction solely using local methods. br. j. oral maxillofac. surg. 2013 sep;51(6):502–7. 12. agarwal sk, munjal m, koul r, agarwal r. prospective evaluation of the quality of life of oral tongue cancer patients before and after the treatment. ann. palliat. med. 2014 oct;3(4):238–43. 13. chen am, daly me, farwell dg, vazquez e, courquin j lau dh purdy ja. quality of life among long-term survivors of head and neck cancer treated by intensity-modulated radiotherapy. jama otolaryngol. head neck surg. 2014 feb;140(2):129–33. 14. crombie ak, farah cs, batstone md. health-related quality of life of patients treated with primary chemoradiotherapy for oral cavity squamous cell carcinoma: a comparison with surgery. br. j. oral maxillofac. surg. 2014 feb;52(2):111–7. 15. van nieuwenhuizen aj, buffart lm, brug j, leemans cr, verdonck-de leeuw im. the association between health related quality of life and survival in patients with head and neck cancer: a systematic review. oral oncol. 2015 jan;51(1):1–11. 16. villaret ab, cappiello j, piazza c, pedruzzi b, nicolai p. quality of life in patients treated for cancer of the oral cavity requiring reconstruction: a prospective study. acta otorhinolaryngol. ital. 2008;28:120–125. 17. hassan sj, weymuller ea. assessment of quality of life in head and neck cancer patients. head neck 1993;15:485–496. 18. van der meulen ic, may am, de leeuw jrj, koole r, oosterom m, et al. long-term effect of a nurse-led psychosocial intervention on healthrelated quality of life in patients with head and neck cancer: a randomized controlled trial. br. j. cancer. 2014;110:593–601. 19. parkin dm, bray fi, devesa ss: cancer burden in the year 2000. the global picture. eur. j cancer 2001;37(8):4–66. 20. kumar p, alvi ha, rao j, singh bp, jurel sk, kumar l, aggarwal h.assessment of the quality of life in maxillectomy patients: a longitudinal study. j. adv. prosthodont. 2013 feb;5(1):29–35. 21. østhus aa, aarstad ak, olofsson j, aarstad hj. prediction of survival by pretreatment health-related quality-of-life scores in a prospective cohort of patients with head and neck squamous cell carcinoma. jama otolaryngol. head neck surg. 2013;139(1):14–20. 22. chiou wy, lee ms, ho hc, hung sk, lin hy, su yc, et al. prognostic fators and the relationship of depression and quality of life in head and neck cancer. ind. j. cancer. 2013;50(1):14–20. 23. gandhi ak, roy s, thakar a, sharma a, mohanti bk. symptom burden and quality of life in advanced head and neck cancer patients: aiims study of 100 patients. ind. j. palliat. care. 2014 sep;20(3):189–93. 24. connelly st, schmidt bl: evaluation of pain in patients with oral squamous cell carcinoma. j. pain 2004;5:505–510. 25. cuffari l, de tesseroli sjt, nemr k, rapaport a: pain complaint as the first symptom of oral cancer: a descriptive study. oral. surg. oral med. oral. pathol. oral radiol. endod. 2006;102:56–61. 26. epstein jb, emerton s, kolbinson da, le nd, phillips n, stevenson-moore p, osoba d: quality of life and oral function following radiotherapy for head and neck cancer. head neck 1999;21:1–11. 27. wan leung s, lee tf ,chien c, chao pj, tsai wl fang fm. health-related quality of life in 640 head and neck cancer survivors after radiotherapy using eortc qlq-c30 and qlq-h&n35 questionnaires. bmc cancer 2011;11:128–135. 21 original assessment of quality of life among head and neck cancer patientsf. rafie et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 17–21 28. kessler pa, bloch-birkholz a, leher a, neukam fw, wiltfang j. evaluation of quality of life of patients with oral squamous cell carcinoma. comparison of two treatment protocols in a prospective study. radiother. oncol. 2004;70(3):275–282. 29. bjordal k, ahlner-elmqvist m, hammerlid e, boysen m, evensen jf, biörklund a, jannert m, westin t, kaasa s. a prospective study of quality of life in head and neck cancer patients. part ii: longitudinal data. laryngoscope. 2001 aug;111(8):1440–52. 30. lee y-h, lai y-h, yueh b, chu p-y, chen y-j, chen s-c, et al. validation of the university of washington quality of life chinese version (uwqol-c) for head and neck cancer patients in taiwan. j. formos. med. assoc. 2017;116(4):249–256. 31. yucel b, akkaş ea, okur y, eren aa, eren mf, karapınar h, et al. the impact of radiotherapy on quality of life for cancer patients: a longitudinal study. support. care cancer. 2014;22(9):2479–2487. 32. torabi m, larizadeh m h, safizadeh h, karimi afshar m, modares ahmadi n. quality of life and ohrqol in head and neck cancer patients in kerman, iran. j. oral health oral epidemiol. 2012;1(2):78–82. 33. hammerlid e, bjordal k, ahlner m. prospectivestudy. quality of life in head and neck patients.laryngoscope 2001;111:669–680. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.02202004 168 j contemp med sci | vol. 6, no. 4, july-august 2020: 168–175 original issn 2413-0516 introduction polycystic ovary syndrome (pcos) is also called hyperandrogenic anovulation. it is an endocrine-metabolic disturbance which has features of multiple hormonal imbalances that produce short and long term consequences on women health.1 polycystic ovary syndrome or stein and leventhal syndrome was first diagnosed in 1935. they found a series of seven cases in women suffer from amenorrhea, hirsutism, and bilateral polycystic ovaries, a condition known later as polycystic ovary syndrome (pcos). pcos patients clearly present a higher risk of cardiovascular diseases spatially obese pcos patient.2 it is now categorized as the most common pathology of the endocrine system in females at reproductive age with principle features of menstrual irregularity (amenorrhea, oligomenorrhea), elevated androgens (acne, hirsuitism), and polycystic ovaries by ultrasound (futterweit and ryan, 2006). in pcos, the number of antral follicles and primary follicle pool are much higher than in healthy women and show significant correlation with serum anti-müllerian hormone (amh), which increase by 2–3 folds, and this in combination with higher androgen level give rise to the major characteristic feature of pcos with arrested multiple follicles of less than 10 mm in diameter.3 amh is considered to be a potentially important biomarker of reproductive potential of cattle. it is a growth factor, produced from the granulosa cells of ovary and sertoli cells of testes, and was first discovered to play an important role in sex differentiation in the fetal life.4 hypersecretion of luteinizing hormone (lh) as the result of increased plasticity of the gnrh, is a quite common feature of pcos, particularly in lean women with oligoamenorrhea.5 while many women with pcos have lh and follicle stimulating hormone (fsh) still within the 5–20 miu/ml range, their lh level is often two or three times than that of the fsh level. this situation is called an elevated lh/fsh ratio or a ratio of 3:1. an elevated lh/fsh ratio and increased lh responses to gnrh.6 increased lh secretion with relatively fixed low or normal fsh secretion in women with pcos has been first reported previously. therefore, excess testosterone is produced primarily in the ovaries and is caused by increased lh stimulation from the pituitary and the effect of hyperinsulinemia at the ovary.7 however, increased frequency of pulsatile gnrh release that selectively increases lh secretion.8 pcos is a diagnosis of exclusion, and prolactin levels are routinely measured in newly referred patients to exclude prolactinomas.9 high prolactin may be a marker of low dopaminergic tonus in the central nervous system. prolactin secretion is influenced by several hormonal parameters, age, and smoking status. pcos is associated with hyperandrogenemia and relatively high estrogen levels, which could stimulate prolactin secretion,10 also, depression and a decreased quality of life in pcos could increase dopamine secretion and decrease prolactin levels.11 polycystic ovarian syndrome (pcos) is characterized as a hyperandrogenic state, the lipolytic effect of catecholamine is decreased in subcutaneous adipocytes due to low content of β2-adrenoceptors and hormone sensitive lipase. it is possible that the increased testosterone levels are responsible for these abnormalities in catecholamine signal transduction in subcutaneous fat cells of pcos women. however, in visceral fat cells of pcos women catecholamine-induced lipolysis is enhanced which cannot be explained by testosterone. high levels of insulin in pcos patients leads to excessive production of androgen hormone, which cause some of the unfertilized eggs to disintegrate, leading to inconsistent or nonovulation.12 the early recognition of an ‘insulin resistant phenotype’ is important to prevent cardiovascular involvement in a subset association between anti-müllerian hormone and other biomarkers with ovarian function in polycystic ovarian syndrome of iraqi women huda basim al-lami1*, fadhil jawad al-tu’ma1, and wasan ghazi al-safi2 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. e. mail: fadhil.jawad@uokerbala.edu.iq; f_altoma_56@yahoo.com 2department of gynecology and obstetrics, college of medicine, university of kerbala, kerbala, iraq. *corresponding author: huda b. al-lami, department of chemistry and biochemistry, college of medicine, university of kerbala / kerbala–iraq. e. mail: hudailami1993@ gmail.com abstract: objectives: the current study aimed to investigate the role of of anti-müllerian hormone (amh) and other hormonal biomarkers and insulin resistance in pathogenesis of polycystic ovarian syndrome (pcos) of iraqi women with various ages and body mass index (bmi). method: the sublects include 50 cases of pcos obtained from gynecological and obstetric teaching hospital, kerbala health directorate/ kerbala – iraq and another 50 apparently healthy women as a control group. with age ranged between 18and 37 years during the period from dec. 2019 to june, 2020 which was divided into two groups depending upon their age; first group with age (18–27) years and the second with age (28–37) years. also, they were divided into three subgroups depending upon the bmi, normal weight (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obesity (30–34.9 kg/m2) and measurement of various hormonal levels were performed including luteinizing hormone (lh), follicle stimulating hormone (fsh), insulin, insulin resistance, total testosterone, and amh. results: a significant elevated levels of each of amh, insulin, lh, total testosterone, prolactin, fasting blood glucose and insulin resistance while, a non-significant differences was found in fsh levels in women with pcos as compared with control group. conclusion: a significant high level of the all hormonal parameters including amh, lh, prolactin and total testosterone was found in women with pcos as compared with the control, except the fsh values. keyword: anti-müllerian hormone, luteinizing hormone (lh), follicle stimulating hormone (fsh) and lh/fsh ratio. 169 original association between anti-müllerian hormone and other biomarkers with ovarian functionhuda basim al-lami j contemp med sci | vol. 6, no. 4, july-august 2020: 168–175 of young and susceptible pcos patients without other signs of ir, glycemic hyperinsulinemic clamping is currently the gold-standard for measuring ir. however, it is not suitable for clinical practice since it is complex, time-consuming and not feasible in large populations.13 the association between insulin resistance and hyperandrogenism in pcos is based mainly on two essential concepts: that insulin resistance is an important regulatory factor of ovarian synthesis of the androgens and, in the presence of hyperinsulinism, acts as a true gonadotropic hormone.14 that a condition of hyperinsulinism, occurring as a form of compensation for a state of insulin resistance, may be responsible both for an increased androgenic production and for greater values of free androgens(testosterone), by means of the reduction of hepatic synthesis of the sex hormone binding globulin shbg.13 the presented work aimed to investigate the role of of anti-müllerian in the pathogenesis of pcos of iraqi women and its association with various body mass index (bmi), ages, insulin resistance, and other hormonal biomarkers. materials and methods: this study carried out at research center, department of biochemistry, college of medicine, university of kerbala and the pcos and apparently control group individuals were obtained from gynecological and obstetric teaching hospital/kerbala health directorate/kerbala – iraq from the period dec. 2019 to jul. 2020. subjects of study involved 50 women of pcos and another 50 healthy women with matched age range between 18 and 37 years. women with pcos have been checked medically by ultrasound waves to confirm that they have pcos by radiologist. in the other hand, some women were excluded because they suffer from hypertension, pregnancy, diabetes mellitus, ischemic heart diseases, thyroid disease, pituitary tumors, women taking oral contraceptives and other hormonal drugs. women with pcos were diagnosed by using the rotterdam eshre/asrm criteria from 2003, that including at least two of the following: polycystic ovaries on ultrasound, oligoor a ovulation, biochemical and clinical signs of hyperandrogenism.15 blood samples are collected through a vein puncture during the follicular phase of the menstrual cycle at 9 am, and the blood sample is drawn for both patients and controls. the samples were placed in gel tubes and left for 15 min to clot at room temperature, then the serum was centrifuged at 3000 rpm for 15 min. the serum was divided into eppendorf tubes and the serial number was given in the name for each sample of samples. it was stored in the freezer at -4 °c until it was used to measure serological tests that included measuring the level of the amh, lh, fsh, testosterone (tt), concentration prolactin hormone (prl) and insulin hormone concentration (in). each of these hormones is measured using the elisa technique test for two monoclonal antibodies,16 based on the quantitative sandwich principle according to the manufacturer’s instructions (mannheim, germany). the blood sugar level measured was determined by enzymatic colorimetric (godpap) method, using kit supplied by spinreact, spain. data were analyzed by t-test using spss version 23 statistic program .the comparisons between means were made using least significant differences (lsd) using genstat3statistic program. the difference were considered to be significant at p<0.05 using multivariate model in spss. the data are presented as mean ± s.d (standard division). the correlation coefficient pearson was calculated to examine association among parameters.17 results: the current study was conducted with the aim of studying the measurement of sexual hormones and some biochemical variables in women with pcos in karbala province:iraq. amh levels in patients with pocs of normal weight, overweight and obesity groups were 8.05 ± 1.11 ng/ml, 8.06 ± 1.17 ng/ ml, and 7.78 ± 1.17 ng/ml, respectively, which were increased significantly (p<0.05) when compared with control group of normal weight, over weight, and obesity (2.19 ± 0.67 ng/ml), (2.38 ± 0.62 ng/ml) and (2.46 ± 0.37 ng/ml) groups, respectively, as shown in figure 1 and table 1. the concentration of lh was increased significantly (p <0.05) in pcos patients of first age group (14.96 ± 2.86 m.lu/ ml) and second age group (15.35 ± 4.76 m.lu/ml) as compared with control group of first age group (3.89 ± 1.19 m.lu/ ml) and second age group (4.48 ± 1.29 m.lu/ml), respectively (figure 2). the fsh concentration of women patients with pocs in first (4.87 ± 0.82 m.lu/ml) and second (4.44 ± 1.22 m.lu/ ml) age group was non-significantly different (p<0.05) as compared with control group of first age (4.86 ± 0.94 m.lu/ ml) and second age group (5.09 ± 0.48 m.lu/ml), respectively (figure 3). table 1. the mean ± s.d values of lh, fsh and lh/fsh ratio in control and patients with pocs (according to age) by using t-test. groups parameters control patients p-value first group (18–27) years lh, mlu/ml n = 4 84% n = 39 78% 0.00 3.89 ± 1.19 14.96 ± 2.86* fsh mlu/ml 4.86 ± 0.94 4.87 ± 0.82 0.92 lh/fsh ratio 0.78 ± 0.23 3.11 ± 0.54* 0.00 second group (28–37) years lh mlu/ml n = 8 16% n =11 22% 0.00 4.48 ± 1.29 15.35 ± 4.76* fsh mlu/ml 5.09 ± 0.48 4.44 ± 1.22 0.13 lh/fsh ratio 0.88 ± 0.28 3.51 ± 0.77* 0.00 170 original association between anti-müllerian hormone and other biomarkers with ovarian function huda basim al-lami j contemp med sci | vol. 6, no. 4, july-august 2020: 168–175 fig. 1 the concentration of amh in control and pcos patients. *significant between control and patients at the (p<0.05) fig. 2 the lh concentration in control and pcos patients. *significant between control and patients at the (p<0.05) fig. 3 mean ± sd of serum concentration of fsh in control and pcos patients. 171 original association between anti-müllerian hormone and other biomarkers with ovarian functionhuda basim al-lami j contemp med sci | vol. 6, no. 4, july-august 2020: 168–175 the values of prl was increased significantly (p<0.05) in patients women with pocs of first age group (24.86 ± 4.92 ng/ ml) and second age group (20.50 ± 4.22 ng/ml) as compared with control of first age group (17.51 ± 5.95 ng/ml) and second age group (14.17 ± 7.90 ng/ml), respectively, see table 2 and figure 4. the values of tt observed was increased significantly (p<0.05) in patients women with pocs of first age group (0.73 ± 0.49 ng ng/ml) and second age group (0.55 ± 0.45 ng/ml) as compared with control of first age group (0.22 ± 0.12 ng/ml) and second age group (0.30 ± 0.19 ng/ml), respectively (figure 5). the values of in was increased significantly (p<0.05) in patients women with pocs of first age group (17.97 ± 0.78 μiu/ml) and second age group (17.61 ± 0.95 μiu/ml) compared with control of first age group (11.56 ± 0.78 μiu/ml) and second age group (12.02 ± 0.46 μiu/ml), respectively (figure 6). insulin resistance or homeostatic model assessment (homa-ir) is a technique that calculate insulin resistance and β-cell function. software program was used to solve the equations, so that the estimation of insulin resistance and β-cell function by using fasting glucose and insulin concentration as indicated in the following equation:18 homa ir [ glucose (mg / dl) insulin (u / ml)] / 405= × the homa-ir observed was increased significantly (p<0.05) in patients women with pocs of first age group (4.22 ± 0.36 μiu/ ml) and second age group (4.08 ± 0.40 μiu/ml) as compared with control of first age group (2.38 ± 0.22 μiu/ml) and second age group (2.43 ± 0.06 μiu/ml), respectively (figure 7). the vales of fasting blood glucose (fbg) in women patients with pcos in normal weight (92.07 ± 7.67 mg/di), over weight (95.92 ± 4.60 mg/di), and obesity (95.58 ± 4.79 mg/di) groups increased significantly (p<0.05) when compared with control group of normal weight (81.66 ± 2.38 mg/ di), over weight (84.25 ± 4.04 mg/di) and obesity (80.81 ± 2.48 mg/di) groups, respectively (figure 8). table 2. the mean ± sd values of prl, tt and amh in control and patients with pocs (according to age) by using t-test. groups parameters control patients p-value first group (18–27)years prl, ng/ml n = 42 84% n = 39 78% 0.00 17.51 ± 5.95 24.86 ± 4.92* tt, ng/ml 0.22 ± 0.12 0.55 ± 0.45* 0.00 amh, ng/ml 2.38 ± 0.61 8.18 ± 1.10* 0.00 second group (28–37)years prl, ng/ml n = 8 16% n = 11 22% 0.03 14.17 ± 7.90 20.50 ± 4.22* tt, ng/ml 0.30 ± 0.19 0.73 ± 0.49* 0.02 amh, ng/ml 2.21 ± 0.38 7.29 ± 1.04* 0.00 fig. 4 the concentration of prl in control and pcos patients. *significant between control and patients at the (p<0.05) 172 original association between anti-müllerian hormone and other biomarkers with ovarian function huda basim al-lami j contemp med sci | vol. 6, no. 4, july-august 2020: 168–175 fig. 5 the concentration of tt in control and pcos patients. *significant between control and patients at the (p<0.05) fig. 6 the concentration of in in control and pcos patients. *significant between control and patients at the (p<0.05) fig. 7 the values of homa ir in control and pcos patients. *significant between control and patients at the (p<0.05) 173 original association between anti-müllerian hormone and other biomarkers with ovarian functionhuda basim al-lami j contemp med sci | vol. 6, no. 4, july-august 2020: 168–175 discussion: pcos is one of the most common endocrine disorders in women of reproductive age, characterized by the growth of many follicles instead of one follicle, but these follicles fail to mature. various biomarkers including hormonal and genetic polymorphisms have been performed in pcos patients of iraqi women as reported by al-tu’ma et al.19 the data obtained from the present work showed that pcos was increased in first age group with age 18–27 years, while decreased in second age group with age 28–37 years. this results due to the menstrual cycle tended for normalization with age because of decrease in the follicle population. our result was in agreement with other study which reported the prevalence of pcos seems to decrease with age.20 several other studies, have reported that menstrual cycle a tended for normalization with age, mainly in those women older than 30 years (teede et al., 2018). thus, the ovulatory function seems to improve in pcos patients with advancing age as a consequence of the decrease in the follicle population. the results concerning fbg showed a significantly increase levels in women with pocs groups as compared with control. this result due to insulin resistance causing raised levels of blood glucose, which, sent to liver, than, the glucose is converted into fat and stored in the body, leading to weight gain and finally, obesity that is a key factor in creating pcos. these results was agreement with al-auqbi,21 which found increased fbg in pcos women and about 20–28% of pcos women were prediabetic or diabetic due to insulin resistance and pcos women are at higher risk to develop type 2 diabetes mellitus, gestational diabetes at the reproductive age and even after age of 40 and post menopause. in pcos, women initially glucose metabolism are normal, the rate of conversion to abnormal glucose metabolism can be 25% over just 3 years. women with pcos were commonly obese or overweight impaired glucose tolerance (igt) and increased risk of type ii diabetes compared to obese women.22 the lh levels was increased significantly in pcos women as compared with control group, while no significant difference was observed regarding the fsh levels when compare between pcos and control groups. these results was agreed with another study performed by al-hashimy et al,23 which use pcos women in reproductive age and they found an increased in lh and lh/fsh ratio values in pcos women while no significant difference was found in fsh values. al-mahdawi et al.24 reported that 60–70% of patients with pcos had an increase in their lh level due to increase of pulse frequency, or episodic secretion of lh. the high level of lh causes increased lh/fsh ratio in pcos women groups as compared with control which was agreed with another study found an increased in lh/fsh ratio in women with pcos as compared with control which may be due to primary central disorders involving gnrh secretion or secondary pituitary sensitization to gnrh by an abnormal feedback signals from ovaries.25 in another study on women with pcos in reproductive age found that the ratio of lh/fsh is elevated in women with pcos.26 there was no significant difference in fsh hormone levels reported in this study between pcos and control. this result was agreed with another study done by salehpour et al27 and disagreed with other study performed by dewailly et al,28 which showed that serum lh and lh/fsh ratios were higher in women with pcos than controls. the level of lh and lh/ fsh ratio increased significantly in pcos women in normal weight, overweight, and obesity groups, while no significant different in fsh level in overweight and obesity but increase in pcos of normal weight. results of current study was also agreed with kamran et al,29 which observed that circulating lh and lh/fsh ratio increased significantly in normal weight pcos women. the occurrence of the disorder in the lh and fsh hormones is the result of hypothalamus pituitary gland, which causes the difference in the level of these hormones in the affected women. al-hashimy et al.23 found that 20% of women with pcos did not change with fsh concentration. prl levels was increased significantly in pcos women groups of normal weight and overweight groups which may be due to the pcos pathogenesis related to deficient hypothalamic dopaminergic activity which is also responsible for elevated lh/ fsh ratio. these data were agreed with another studies and may refer to pituitary gland disorders.30 these data may be due to psychological and neurological disorder in women associated with this syndrome, for example, the anxious (anxiety), and this anxiety may be a main factor of prl elevation that explains the fig. 8 the concentration of fbg in control and pcos patients. *significant between control and patients at the (p<0.05) 174 original association between anti-müllerian hormone and other biomarkers with ovarian function huda basim al-lami j contemp med sci | vol. 6, no. 4, july-august 2020: 168–175 progressive of increasing level of this hormone of the pcos women’s beside the other factors. hyperprolactinemia was associated luteal phase dysfunction and may reflect the suppressive effect on elevated prolactin, due to high level of prl blocks binding of fsh to fsh receptor on granulosa cell leading to the suppression of progesterone production.31 tt levels was increased significantly in pcos women as compared with the control. testosterone is one of the most common androgen used as biomarker of hyperandrogenemia in women that cause pcos produced from the ovaries and adrenal glands in women.32 increased lh in pcos leads to an increase in testosterone production by the theca cells within the ovary. women with pcos exhibited a significantly higher tt level, than women without pcos, which is similar to the current study.33 these results were also agreed with previous study done by jumaa and saood,34 which shows that women with pcos raised serum total testosterone. the results of the present study revealed that the amh level is higher in pcos in comparing with control group, which were due to excessive amount of small antral follicles in the ovaries.35 amh is mainly secreted exclusively by the granulosa cells of ovarian early developing follicles from preantral and small antral follicles indicating amh role in folliculogenesis results may be due to increased production per granulosa cell, suggesting an intrinsic granulosa cell dysregulation in pcos or because of impaired access of fsh to follicles. elevated levels of the amh in pcos women groups were associated and related to increased number of follicles in women with pcos agreement with namik et al,36 which found increase levels of the amh in pcos women in their reproductive ages. amh is a member of the transforming growth factor beta superfamily.37 in the women, it is solely produced by the granulosa cells of growing preantral and small antral ovarian follicles. serum amh levels may be used as a marker of ovarian reserve, representing the quantity and quality of the ovarian follicle pool.38 women with pcos are known to have elevated baseline amh levels when compared with agematched normo-ovulatory women.39 the results of the present study revealed significant negative correlation between amh and age. this result indicates a reduction in amh level with increasing age due to a decrease in the number of follicles as women aging and these data agrees with kevenaar et al,40 which reported that circulating levels of amh decline with age, may reflect the age-associated depletion of ovarian follicles. the overweight and obesity was found as a common feature among pcos women studies which was agreed with others.21 high bmi can influence both clinical and pathophysiology manifestations of pcos.41,42 their report indicate that pcos women with bmi >25 kg/m² have a marked increase level of insulin, glucose, lh, tt, and increased insulin resistance. bmi has an opposite relationship with androgen levels in men, whereas it seems to have a synergistic impact in women. the increase in fat tissue and body weight is related to the change in sex steroid equilibrium in pre-and postmenopausal women. increased bmi may be major factor of endocrine and metabolism disorder in pcos women and the obese and overweight pcos women who had increased levels of tt and lh had a high prevalence of overweight/obesity in pcos women. results of current study showed significant difference in serum insulin and insulin resistance (homa-ir), which is higher in pcos patients as compared with control and the results were similar to studies done by others.43 insulin resistance with hyperinsulinemia, is one causes of the pathogenesis of pcos and it lead to development of complications related to pcos and increased ovarian androgen production in pcos stimulates hyperinsulinemia due to ir.8 the proposed mechanisms for insulin-reproductive abnormalities include abnormalities of ovarian steroidogenesis, excessive lh secretion and abnormalities in glucose uptake. there are three possible mechanisms have been proposed for androgen hypersecretion include: an intrinsic functional theca cell defect, hyperinsulinemia following insulin resistance and hypersecretion of pituitary lh resulting in extreme theca stimulation.44 spritzer45 reported that hyperinsulinemia and ir determination of hyperandrogenemia by stimulate ovarian theca cells to secrete androgens and increase the effect of lh on the production of ovarian androgens. insulin and homa-ir in present study was increased significantly in pcos women of normal weight, overweight and obesity groups. insulin resistance is a common feature of pcos and is more marked in obese women, suggesting that pcos and obesity have a synergistic effect on the magnitude of the insulin disorders.46 our results agrees with others which reported that pcos women demonstrate greater variation in insulin parameters compared to controls, independent of weight.47 ir and hyperinsulinemia can be caused by obesity with visceral fat accumulation; hyperinsulinemia affects granulosa cells in small follicles and theca cells and this condition induces early response to lh on granulosa cells of small follicles and causes premature differentiation of these cells, which eventually results in anovulation.48 other stuy found that obesity increases insulin resistance, and the presence of polycystic ovaries increases insulin resistance. the presence of polycystic ovaries appears to have a stronger influence than obesity on insulin resistance.49 the results obtained agree with other study found that weight gain occurs as a result of increased insulin levels and stimulates the ovaries to produce testosterone, so weight gain increase the testosterone hormone. insulin has an inhibitory effect on hepatic production of shbg resulting in increase of free testosterone in addition to its stimulatory effect to the ovarian androgen production augmenting the state of hyperandrogenism.50 conclusion: this study revealed there are significant positive correlation in lh/fsh ratio with bmi and age of pcos women. this study revealed there are significant negative correlation in prl and age of pcos women. a significant rise in the levels of the amh, in, lh, tt, prl, fbg and homa ir was found significantly in women with pcos significantly compared to control, while no significant difference was found in fsh values between a group of infected women and a control group. references: 1. kollmann, m, martins, wp, and raine-fenning, n. terms and thresholds for the ultrasound evaluation of the ovaries in women with hyperandrogenic anovulation. human reprod update, 2014;20(3):463-464. 2. al-tu’ma, fj, ahmed, nn, and al-safi, wg. total antioxidant capacity and homocysteine levels in obese women with polycystic ovary syndrome. int j pharm pharm res, 2017;8:78-86. 3. laven js, mulders ag, visser ja, themmen ap, de jong fh, and fauser bc. anti-mullerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. j clin endocrinol metab, 2004; 89(1):318-23. 175 original association between anti-müllerian hormone and other biomarkers with ovarian functionhuda basim al-lami 4. lee, mm, noto, a, schoenfeld, dt, maclaughlin, g, mol, nc, and grant et. substance in humans. normal 2008;81:2–7. 5. hendriks, ml, brouwer, j, hompes, pg, homburg, r, and lambalk, cb. lh as a diagnostic criterion for polycystic ovary syndrome in patients with who ii oligo / amenorrhoea. reprod biomed online, 2008;16(6):765-771. 6. azziz, r. diagnosis of polycystic ovarian syndrome: the rotterdam criteria are premature. j clin endocrinol metab, 2006;91(3):781-785. 7. sheehan, mt. polycystic ovarian syndrome: diagnosis and management. clin med res, 2004;2(1):13-27. 8. diamanti-kandarakis, e, and dunaif, a. insulin resistance and the polycystic ovary syndrome revisited: an update on mechanisms and implications. endocr rev, 2012;33(6):981-1030. 9. glintborg, d, and andersen, m. an update on the pathogenesis, inflammation, and metabolism in hirsutism and polycystic ovary syndrome. gynecol endocrinol. 2010;26(4):281-296. 10. glintborg, d, altinok, m, mumm, h, buch, k, ravn, p, and andersen, m. prolactin is associated with metabolic risk and cortisol in 1007 women with polycystic ovary syndrome. human reprod., 2014;29(8):1773-79. 11. altinok, ml, glintborg, d, depont christensen, r, hallas, j, and andersen, m. prescription of antidepressants is increased in danish patients with polycystic ovary syndrome and is associated with hyperandrogenism. a population‐based cohort study. clin endocrinol 2014;80(6):884-889. 12. arner, p. effects of testosterone on fat cell lipolysis. species differences and possible role in polycystic ovarian syndrome. biochimie. 2005;87(1):39-43. 13. gonzález, f. inflammation in polycystic ovary syndrome: underpinning of insulin resistance and ovarian dysfunction. steroids 2012;77(4):300-305. 14. denson, tf, mehta, ph, and tan, d.h. endogenous testosterone and cortisol jointly influence reactive aggression in women. psychoneuroendocrinology. 2016;38(3):416-424. 15. bremer, aa, and miller, wl. the serine phosphorylation hypothesis of polycystic ovary syndrome: a unifying mechanism for hyperandrogenemia and insulin resistance. fertil steril. 2008;89(5):1039-1048. 16. nelson, sm, iliodromiti, s, fleming, r, anderson, r, mcconnachie, a, and messow, c. m. reference range for the antimüllerian hormone generation ii assay: a population study of 10,984 women, with comparison to the established diagnostics systems laboratory nomogram. fertil steril. 2014;101(2):523-529. 17. spss. statistical package for social science, users guide for statistics. 2015. 18. kanauchi m. a new index of insulin sensitivity obtained from the oral glucose tolerance test applicable to advanced type 2 diabetes. diabet care. 2002 oct 1;25(10):1891-2. 19. al-tu’ma, fj., farhan nh, and al-safi, wg. association between fat mass and obesity gene (re9939609) polymorphism with pcos women in iraqi poipulation. int j pharm pharm res, 2015;5(1):62–72. 20. lauritsen, mp, bentzen, jg, pinborg, a, loft, a, forman, jl, thuesen, ll, and nyboe andersen, a. the prevalence of polycystic ovary syndrome in a normal population according to the rotterdam criteria versus revised criteria including anti-müllerian hormone. human reprod. 2014;29(4):791-801. 21. al-auqbi, tf obesity, glycemic and hormonal criteria of polycystic ovary syndrome. al-kindy coll med j, 2009; 5(1):33-39. 22. kensara, oa. prevalence of hypovitaminosis d, and its association with hypoadiponectinemia and hyperfollistatinemia, in saudi women with naïve polycystic ovary syndrome. j clin transl endocrinol. 2018;12:20-25. 23. al-hashimy, dh, al-rikaby, hr, and al-khayaat, es. study of some hormonal disorders associated with polycystic ovarian syndrome in women in thi qar governorate. j educ pure sci univ thi-qar, 2019;9(2):25-31. 24. al-mahdawi, ma, khadhem, hk, and al-jebori, sr effect of physical activity on sex hormones in polycystic ovary syndrome iraqi women. iraqi j biotechnol, 2018;17(1):98-107. 25. hassan, bf. measuring the concentration of some hormones in patients sera of polycystic ovaries. baghdad sci j. 2010;7(4):1384-1388. 26. marbut, mm, awwad, ny, yousif, mn, and ahmed, ms. hormonal assessment in women with polycystic ovary syndrome in tikrit city. j madenat alelem univ coll. 2019;11(1):1-9. 27. salehpour, s, tohidi, m, akhound, mr, and amirzargar, n. n acetyl cysteine, a novel remedy for poly cystic ovarian syndrome. j fertil steril. 2009;2:66-73. 28. dewailly, d, catteau-jonard, s, reyss, ac, leroy, m, and pigny, p. oligo anovulation with polycystic ovaries but not overt hyperandrogenism. j clin endocrinol metab. 2006;91(10):3922-27. 29. kamran u, tanzilur r, dan-dan w, xian-hua l, ye l, xiao-yan g, peter ckl, run-ju z, he-feng h and jian-zhong s. concentrations are increased in association with luteinizing hormone and nesfatin-1 concentrations in women with polycystic ovary syndrome, clin chim acta. 2017;471:243-247. 30. hassan, mf. original research the frequency of elevated prolactin level in polycystic ovary syndrome women (pcos) and its’ effect on pregnancy rate. global j public health med. 2020;2(1):109-117. 31. porter, mb, brumsted, jr, and sites, ck, effect of prolactin on folliclestimulating hormone receptor binding and progesterone production in cultured porcine granulosa cells. fertil steril. 2000;73(1):99-105. 32. münzker, j, hofer, d, trummer, c, ulbing, m, harger, a, pieber, t, and obermayer-pietsch, b. testosterone to dihydrotestosterone ratio as a new biomarker for an adverse metabolic phenotype in the polycystic ovary syndrome. j clin endocrinol metab. 2015;100(2):653-660. 33. sung, ya, oh, jy, chung, h, and lee, h. hyperandrogenemia is implicated in both the metabolic and reproductive morbidities of polycystic ovary syndrome. fertil steril. 2014;101(3):840-845. 34. jumaa, mn and saood, na. study of hormonal levels changes in women’s serum with polycystic ovaries syndrome (pcos). j univ anbar pure sci. 2011;5(1):17-23. 35. weenen c, laven js, von bergh ar, cranfield m, groome np, and visser ja. anti-mullerian hormone expression pattern in the human ovary: potential implication for initial and cyclic follicle recruitment. mol hum reprod. 2004;10:77–83. 36. namik, jj, alalaf, sk, and al-tawil, n. g. anti-mullerian hormone and antral follicle count in polycystic ovary syndrome and non-polycystic ovary syndrome women. zanco j med sci. 2018;22(3):292-299. 37. van rooij ia, broekmans fj, te velde er, fauser bc, bancsi lf, de jong fh, et al. serum anti-mullerian hormone levels: a novel measure of ovarian reserve. hum reprod 2002;17:3065-3071 38. la marca a, sighinolfi g, radi d, argento c, baraldi e, and carducci artenisio. anti-mullerian hormone (amh) as a predictive marker in assisted reproductive technology (art). hum reprod update. 2010;16:113-130. 39. piltonen t, morin-papunen l, koivunen r, perheentupa a, ruokonen a, and tapanainen js. serum anti-mullerian hormone levels remain high until late reproductive age and decrease during metformin therapy in women with polycystic ovary syndrome. hum reprod. 2005;20:1820-1826. 40. kevenaar, me, meerasahib, mf, kramer, p, van de lang-born, bm, de jong, fh, groome, np, and visser, ja. serum anti-mullerian hormone levels reflect the size of the primordial follicle pool in mice. endocrinology. 2006;147(7):3228-3234. 41. wang, f, dai, w, yang, xh, guo, yh, and sun, yp. analyses of optimal body mass index for infertile patients with either polycystic or non-polycystic ovary syndrome during assisted reproductive treatment in china. sci rep. 2016;6(1):1-9. 42. al–shammaa, h, el–yassin, hd, and shamam, kh. serum resistin levels, and other hormonal and biochemical parameters in patients with polycystic ovary syndrome (pcos). j facul med. 2009;51(2):200-203. 43. mohammed, dq, hawaa, ad, and husein, sm. correlation between homocysteine and insulin resistance in women with polycystic ovarian syndrome referring to al-yarmook teaching hospital. iraqi j embryo infertil res. 2014;4(2):32-39. 44. cadagan, d, khan, r, and amer, s. thecal cell sensitivity to luteinizing hormone and insulin in polycystic ovarian syndrome. reprod biol. 2016;16(1):53-60. 45. spritzer, pm. polycystic ovary syndrome: reviewing diagnosis and management of metabolic disturbances. arq brasil endocrinol metab. 2014;58(2):182-187. 46. nogueiras, r, gualillo, o, caminos, je, casanueva, f, and diéguez, c. regulation of resistin by gonadal, thyroid hormone, and nutritional status. obesity res. 2003;11(3):408-414. 47. jayagopal, v, kilpatrick, e. s, holding, s, jennings, p. e. and atkin, s. l. the biological variation of insulin resistance in polycystic ovarian syndrome. j clin endocrinol metab, 2002;87(4):1560-1562. 48. sakumoto, t, tokunaga, y, tanaka, h, nohara, m, motegi, e, shinkawa, t, and higashi, m. insulin resistance/hyperinsulinemia and reproductive disorders in infertile women. reprod med biol. 2010;9(4):185-190. 49. cresswell, j, fraser, rb, bruce, c, egger, p, phillips, d, and barker, d. j. relationship between polycystic ovaries, body mass index and insulin resistance. acta obstet gynecol scand. 2003;82(1):61-64. 50. temel, i̇, çelik, ö, hasçalik, ş, çelik, n, şahin, i̇, and aydin, s. serum nonesterified fatty acids, ghrelin, and homocysteine levels in women with polycystic ovary syndrome. turk j med sci. 2010;40(2):221-228. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i4.824 208 j contemp med sci | vol. 6, no. 5, september-october 2020: 208–212 original issn 2413-0516 introduction patients admitted to the hospital are exposed to various invasive procedures during their hospitalization, so one of the biggest challenges in nursing is ensuring patient comfort and pain relief, and this has been always acknowledged in the activities and policies of the health-care system.1 one of the most common invasive procedures in hospitalized patients is direct perforation of the vein to collect blood samples for diagnostic or therapeutic purposes,2 so that almost all hospitalized patients at least once during their hospital stay and approximately one-quarter of them have been exposed to this procedure more than three times.3 this can cause pain and suffering for the patient, which can be a challenge for care and is still a common problem associated with blood sampling in clinical settings.4 studies have shown high levels of acute pain, anxiety, stress, and irritability in patients of different ages following repeated vein punctures in one day to collect blood samples.5, 6 in addition, the chance of vascular injury, bleeding, and nosocomial infection increases and makes future vascular use difficult7, 8 and in rare cases causes peripheral nerve damage.9 it puts nurses at risk of unintended needle entry (needle stick).10 in some patients, such as those with chronic diseases, patients who have had multiple intravenous (iv) injections, iv drug abusers, patients with skin problems at the venous lines, patients taking anticoagulants, the elderly patients with fragile veins, and children, finding a suitable vein may require multiple vein punctures.11, 12iv catheters are commonly used in most hospitalized patients for medical purposes such as injections of fluids, medications, and blood.11, 14 these catheters make it unnecessary to use a needle and make it easier for patients with difficult venous blood sampling.13 this protects patients from repeated injuries by venous puncture sampling and nurses from needle puncture, providing comfort and time saving for patients and nurses, especially those in intensive and acute care, and suffer from collapsed veins, speeding up the results of tests and increasing the speed of medical and nursing care, reducing the risk of infections and other complications of blood sampling.11 sampling for diagnostic tests using arterial and venous catheters has been controversial since half a century ago.15 the results of some studies have shown that by inserting and maintaining an iv catheter and accessing the patient’s bloodstream, the blood sample needed for diagnostic tests can be obtained to reduce the complications of direct blood sampling.4, 5, 10 on the other hand, some studies have concluded that laboratory values obtained from iv catheter blood samples are invalid and recommend further studies in this area.5, 7, 13 on the other hand, the results of some studies suggest that the infusion intake through the iv catheter may affect the results of tests performed on iv catheter blood samples.7, 15. the present study aimed to compare the results of sodium, potassium, creatinine, and urea nitrogen levels in venous blood samples taken by direct blood sampling and peripheral venous catheter. materials and methods this analytical cross-sectional study was performed in 1396 (2017) from poor-sina medical center in rasht. sample size had 95% confidence interval and 90% test power according to the biochemical parameters in venous blood sampling by direct venipuncture and peripheral intravenous catheter fatemeh jaafaraghaei1, monireh aghajany-nasab2*, mahsa aghaei1, nazila javadi pashaki3, ehsan kazemnejhad leili1 1 nursing and midwifery faculty, guilan university of medical sciences, rasht, iran 2 department of biochemistry, molecule and cell research center, school of medicine, guilan university of medical sciences, rasht, iran 3 social determinants of health research center, nursing and midwifery faculty, guilan university of medical sciences, rasht, iran corresponding author: aghajany-nasab monireh, (e-mail: maghajany@gums.ac.ir ) (submitted: 14 july 2020 – revised version received: 21 july 2020 – accepted: 08 august 2020 – published online: 30 october 2020) abstract objectives: the aim of this study was to compare the results of some biochemical values in venous blood samples obtained by direct venipuncture (dv) and peripheral venous catheter (pvc). methods: in this cross-sectional study, 78 hospitalized patients from different wards of poor-sina medical and educational center of rasht in 2017 were divided into 3 equal groups, including patients who were treated with normal saline and dextrose saline and those who did not receive this solution. two separate blood samples were obtained from each patient, one from pvc and the other from dv. the levels of sodium, potassium, creatinine and blood urea nitrogen (bun) were measured and compared. results: difference of pvc with dv in bun was generally 0.32±3.18 mg/dl; creatinine, sodium, and potassium were 0.02±0.12 mg/dl, 1.00±2.49 meq/l and 0.15±0.48 meq/l respectively. there were no statistically significant differences between the two methods in relation to bun and creatinine (p = 0.377 and p = 0.149, respectively), but significant differences in levels of sodium and potassium were observed between the two blood sampling methods (p = 0.001 and p = 0.008, respectively). conclusion: according to the results of this study and other studies, pvc can be used to measure bun and creatinine, not so for sodium and potassium. for other parameters, further investigation is needed. keywords: biochemical values, peripheral catheterization, blood specimen collection 209 original the biochemical parameters in venous blood sampling by direct venipuncturefatemeh jaafaraghaei j contemp med sci | vol. 6, no. 5, september-october 2020: 208–212 the following sampling formula and based on similar studies 26 individuals were included in each group.16 n z z sd d = − + −       ( )2 1 2 1 2 2 2 � � �� α β inclusion criteria included patients over 18 years of age with complete consciousness, with no coagulation problems, available peripheral veins, and peripheral venous with gauge20 catheter for up to 72 hours (receiving normal saline or dextrose saline or no infusion), who were prescribed by a physician who required blood sampling to check for blood sodium, potassium, creatinine, and urea nitrogen levels. exclusion criteria included: patients with anemia and those with an iv arterial fistula and patients who had inoperable peripheral catheter or prolonged blood sampling more than 20 seconds for any reason and hemolyzed blood samples and actually. patients were divided into three groups (26 in each) based on the type of infusion that they received; group 1: normal saline, group 2: dextrose saline, and group 3 did not receive any infusion and the catheter was heparin-locked. two separate blood samples were collected from each patient by one nurse, first sample from a peripheral vein catheter and the other from opposite hand direct vein blood sampling.16 in the first and second groups, serum therapy was stopped for 15 seconds.5 then, in all three groups before blood sampling, the standard tourniquet was closed for 30 seconds, 5 cm above the peripheral venous catheter site.11, 15 the volume of the catheter’s dead space according to the size was set to 0.1 cc, and to ensure that, approximately 0.5 cc of blood (2.5 times and more) was removed and discarded13 and by another syringe, 3 ml was obtained for the test by the same route. immediately after catheter blood sampling in all three groups, another 3 cc blood samples were obtained from the opposite arm via direct blood sampling. after the sampling was completed, the iv fluid flow resumed as before. the test tubes were designated a code by researcher. the laboratory staff and statistical analyst were unaware of the coding and study groups. samples were sent to one laboratory simultaneously to avoid possible variation between laboratories. measurement of blood urea nitrogen (bun) performed by using a diagnostic urea bionic kit (photometric urea and alpha ketoglutarate dehydrogenase method at a wavelength of 340 nm) and creatinine was measured by jaffe’s method. creatinine forms an amber yellow complex with alkaline picrate which was measured photometrically (pars azmoon co., tehran, iran). sodium and potassium were determined by ion selective electrodes. a database sheet was prepared which included the demographic information including age, gender, underlying diseases including diabetes, hypertension, and dyslipidemia, catheter fluid infusion rate, and type of received infusion, along with the results of laboratory tests. data analysis the data were analyzed using spss v18 software. quantitative variables were expressed as mean ± standard deviation and qualitative variables were expressed as frequency and percentage. paired t-test was used to evaluate the differences between the two blood sampling methods and anova or chi-square test was used to assess the differences between the three groups. p <0.05 was considered significant. ethical considerations the present study was conducted under the auspices of the research ethics committee of guilan university of medical sciences (approved code: ir.gums.rec.1395 / 240) and the researchers adhered to all the principles of protocols and guidelines recommended by the helsinki convention on ethics in research. prior to entering the study, informed consent was obtained from all participants. study participants were assured that all patient information would be kept confidential and the outcome of the design would be generally published without mentioning the patient’s name and any specifications. patient participation in this study was completely voluntarily and no changes were made in the treatment or care of their patients due to their participation or not. results in general, the youngest patient was 21 years old and the oldest was 90, with a mean age of 51.28 years. analysis of the results among the three groups showed that there was no statistically significant difference in age between the three groups (p=0.689). frequency of male gender was 61.5% (n=16) in normal saline group, 65.4% (n=17) in dextrose saline group, and 17 cases (65.4%) in non-serum receiving group (p=0.946). based on the history of underlying disease, it was found that overall 10 patients (12.8%) with diabetes, 20 patients (26.6%) with hypertension, and 10 patients (12.8%) with dyslipidemia were included in this study. although the frequency of diabetics was higher in the normal saline group (p=0.014), there was no statistically significant difference between the groups about the hypertension and dyslipidemia (p=0.625). there was no statistically differences between groups with respect to peripheral infusion rate (p=0.22) (table 1). discussion the aim of the present study was to compare the results of sodium, potassium, creatinine, and urea nitrogen levels in venous blood samples obtained by direct blood sampling and peripheral venous catheter pathway, which were performed in order to achieve the primary and basic goals and to improve the quality of nursing care. the main aim is the reduction of pain and complications of direct blood sampling which patients may encounter during hospitalization. the results of the present study showed that there was no statistically significant difference between the two methods of measuring bun and serum creatinine between the study groups. according to the study of hambleton et al in 201410 and zlotowski et al in 200117 aiming comparison of laboratory tests through two methods of direct blood sampling and peripheral catheter and changes in laboratory results following iv normal saline injection indicated that the blood sampling method had no relationship with bun and serum creatinine levels. also, the results of bun and serum creatinine following the two methods of blood sampling in patients receiving dextrose saline in the study of ortels et al in 20143 were not significantly different. in addition, the results of yazdankhah fard et al.’s study in 201411 showed that there is no significant 210 original the biochemical parameters in venous blood sampling by direct venipuncture fatemeh jaafaraghaei j contemp med sci | vol. 6, no. 5, september-october 2020: 208–212 table 1. background variables among different study groups. p valuetotalnl groupds groupns group age 52/31±20/52#52/88±18/7748/65±17/9351/28±18/950/689*age sex 0/946# (%61/5)16(%65/4)17(%65/4)17(%64/1)50male (%38/5)10(%34/6)9(%34/6)9(%35/9)28female diabetes hx 0/014# (%26/9)7(%0)0(%11/5)3(%12/8)10yes (%73/1)19(%100)26(%×8/5)23(%87/2)68no htn hx 0/625# (%,30/8)8(%26/9)7(%19/2)5(%25/6)20yes (%69/2)18(%73/1)19(%80/8)21(%74/4)58no dlp hx 0/892# (%11/5)3(%11/5)3(%15/4)4(%12/8)10yes (%88/5)23(%88/5)23(%84/6)22(%87/2)68no venous infusion rate 0/217# (%,34/6)9(%53/9)14-(%44/2)2310 gtt/min (%42/3)11(%19/2)5-(%30/8)1615 gtt/min (%3/9)1(%11/5)3-(%7/7)420 gtt/min (%19/2)5(%15/4)4-(%17/3)9)25 gtt/min * anova test. # chi-square test. ns: normal saline group, ds: dextrose saline group, nl: no infusion group. table 2. biochemical results using direct and peripheral catheter sampling in groups. variables groups direct sampling catheter sampling sampling methods difference significance level* bun (mg/dl) ns 17/31±7/81 16/81±11/12 -0/50±5/00 0/614 ds 15/15±7/72 14/58±7/20 -0/58±1/50 0/061 ni 14/35±4/38 14/46±3/93 0/12±1/93 0/762 cr (mg/dl) ns 1/10±0/59 1/07±0/61 -0/03±0/14 0/337 ds 1/05±0/36 1/02±0/28 -0/03±0/12 0/153 ni 1/02±0/17 1/02±0/22 0/00±0/11 0/999 na (meq/l) ns 136/19±2/06 137/81±2/42 1/62±2/47 0/003* ds 135/54±2/56 136/50±3/19 0/96±2/86 0/099 ni 136/96±2/46 137/38±2/82 0/42±2/02 0/297 k (meq/l) ns 3/89±0/33 3/96±0/44 0/07±0/40 0/386 ds 3/73±0/52 3/82±0/42 0/08±0/39 0/279 ni 4/00±0/37 4/29±0/63 0/29±0/59 0/020* * paired-t test, significant level p<o.o5. ns: normal saline group, ds: dextrose saline group, ni: no infusion group. 211 original the biochemical parameters in venous blood sampling by direct venipuncturefatemeh jaafaraghaei difference in the results of these two parameters with blood sampling methods in patients receiving normal saline and dextrose saline, which is similar to the results of the present study. according to the results of the present study and the above-mentioned studies, it seems that the two methods of direct blood sampling and peripheral venous catheter path do not cause any differences in bun and serum creatinine. the results of present study showed that sodium levels in the blood samples obtained from peripheral venous catheter was significantly higher than direct blood sampling in the group receiving normal saline. blood potassium levels were higher after taking blood from peripheral venous catheter. in a study aimed comparing the results of biochemical and hematological tests out of two methods, routine blood sampling and peripheral venous catheter conducted by gholipour et al. in 2016,7 it was reported that there was a statistically significant difference between the results of patients’ sodium samples. this finding is consistent with the results of the present study. although another study in 2016, ali asgharpour et al.,4 and rezaei et al.18 showed that the difference in sodium and potassium levels between the two groups is not statistically significant. this is different from the results of our study. in these mentioned studies, the differences in sodium levels was not examined separately for types of infusion, while in the present study the type of infusion considered and higher sodium levels were achieved only in normal saline group. also, in the study of hamilton et al. in 2014,10 hamilton et al. reported that there is no significant difference in terms of these two electrolytes in patients receiving normal saline. in the present study, although results are similar to the mentioned study, there was no significant difference in potassium levels in patients receiving normal saline, but in general, sodium and potassium levels were significantly higher in the peripheral catheter blood sampling method. however, in the hambleton study, normal saline injection was stopped for 2 minutes and 2 cc of blood was discarded, while in the present study, the time of discontinuation of infusion, sampling and discarded blood volume was less, which could be the reason for differences in the results of these two studies.19the effect of injectable drugs on test results was uncontrollable due to receiving through a peripheral catheter, which was one of the limitations of the present study. in addition, the time interval between two samples taken by the two methods may also affect the test results. so, a minimum time interval of about 1 minute was considered in this study. on the other hand, the present study has examined only four laboratory variables and its results cannot be generalized to all biochemical tests, so more studies in this field are needed for other laboratory tests. however, evaluation of the results of two blood sampling methods separately from the infusion received by patients is the strength of this study compared to similar studies in this field. the findings of this study can provide a basis for further studies on the effect of serum therapy on other tests of blood samples of the peripheral venous catheter in hospitalized patients aiming reduction of the complications of direct blood sampling. conclusion the results of the present study showed that there was no statistically significant difference between the two methods of measuring bun and creatinine between the study groups. however, blood sodium levels from peripheral venous catheter were significantly higher than direct blood sampling specifically in the normal saline group and also all cases. blood potassium was higher in patients receiving saline dextrose as well as in total patients following peripheral venous catheter sampling. according to the results of this study and other studies, peripheral venous catheter blood sampling can be used to measure bun and creatinine not for sodium and potassium regarding to the types of infusion. acknowledgments the authors thank all people who helped us make this study and all colleagues in guilan university of medical sciences for their invaluable suggestions during experimental design. conflict of interests: the authors of this manuscript have no conflicts of interest to declare. references 1. bernhofer ei, st marie b, bena jf. a new clinical pain knowledge test for nurses: development and psychometric evaluation. pain manag nurs. 2017 aug; 8(4):224-233. 2. yilmaz kurt f, aytekin ozdemir a, atay s. the effects of two methods on venipuncture pain in children: procedural restraint and cognitive-behavioral intervention package. pain manag nurs. 2019 oct 15. pii: s15249042(18)30213-3. 3. cadacio c, nachamkin i. a novel needle-free blood draw device for sample collection from short peripheral catheters. j infus nurs. 2017;40(3):156–162. 4. aliasgharpour m, jafari r, jalalinia s f, jalal madani s, tabari f, kazemnegad lili a. comparing blood values sampled from venipuncture and continuous infusion catheter. crit care nurs j. 2016;9(3):1–5. 5. ortells-abuye n, busquets-puigdevall t, díaz-bergara m, paguina-marcos m, sánchez-pérez i. a cross-sectional study to compare two blood collection methods: direct venous puncture and peripheral venous catheter. bmj open. 2014;4(2):e004250. 6. kennedy rm, luhmann j, zempsky wt. clinical implications of unmanaged needle-insertion pain and distress in children. pediatrics. 2008 nov;122(suppl 3):s130-3. 7. gholipour baradari a, zargar n, aarabi m, koohsari e, emami zeydi a. comparison of hematologic and biochemical test results in blood samples obtained by venipuncture and peripheral intravenous catheter. j mazandaran univ med sci. 2016;26(139):66–72 (persian). 8. amini m, vaseie m, ansari i. the evaluation of nosocomial urinary tract infections and antimicrobial resistance in icu patients, tehran, iran, 20122016. acta med mediter, 2017;33:945–52 9. asheghan m, khatibi a, holisaz mt. paresthesia and forearm pain after phlebotomy due to medial antebrachial cutaneous nerve injury. j brachial plex peripher nerve inj. 2011;6:5. 10. hambleton vl, gómez ia, andreu fa. venipuncture versus peripheral catheter: do infusions alter laboratory results? j emerg nurs. 2014 jan; 40(1):20-6. 11. yazdankhahfard m, taghizadeganzadeh m, farzaneh m, mirzaei k. comparison of biochemical laboratory values obtained by means of routine method of venipuncture versus peripheral intravenous infusion line after administration of fluids. armaghane danesh. 2015;19(12):1021-1028 (persian). 12. braniff h, decarlo a, haskamp ac, broome me. pediatric blood sample collection from a pre-existing peripheral intravenous (piv) catheter. j pediatr nurs. 2014 sep-oct;29(5):451-6. 13. mofrad zp, shafiee s, mahmoodi n, jahantigh m, samadzadeh h. comparison of the effect of direct venipuncture and venous catheter blood sampling methods on the biochemical results of patients hospitalized in ccu. med surg nurs j. 2016;5(1):11–5. 212 original the biochemical parameters in venous blood sampling by direct venipuncture fatemeh jaafaraghaei j contemp med sci | vol. 6, no. 5, september-october 2020: 208–212 14. cicolini g, manzoli l, simonetti v, flacco me, comparcini d, capasso l, et al. phlebitis risk varies by peripheral venous catheter site and increases after 96 hours: a large multicentre prospective study. j adv nurs. 2014 nov;70(11):2539–49. 15. aghaei-hashjin m, javadi-pashaki n, kazemnezhad-leyli e, aghajaninasab m. comparison of the results of blood sugar between direct blood sampling and peripheral catheter in hospitalized patients. j biochem tech. 2018;special issue (2):189–195. 16. watson kr, o’kell rt, joyce jt. data regarding blood drawing sites in patients receiving intravenous fluids. am j clin pathol. 1983 jan;79(1):119–21. 17. zlotowski sj, kupas df, wood gc. comparison of laboratory values obtained by means of routine venipuncture versus peripheral intravenous catheter after a normal saline solution bolus. ann emerg med. 2001 nov;38(5):497– 504. 18. rezaei k, kohestani h, zand s. comparison of biochemistry values obtained by venipuncture and saline lock after intermittent administration of fluids and drugs. j arak uni med sci. 2009;12(1):49–56 (persian). 19. baker rb, summer ss, lawrence m, shova a, mcgraw ca, khoury j. determining optimal waste volume from an intravenous catheter. j infus nurs. 2013;36(2):92–96. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.829 64 j contemp med sci | vol. 5, no. 2, march–april 2019: 64–70 review effect of xylitol on salivary streptococcus mutans: a systematic review and meta-analysis reza yazdani,a* ammar n.h. albujeer,b,c ebrahim rahnama,d and mohammad javad kharazifarde adepartment of community oral health, faculty of dentistry, tehran university of medical sciences, international campus, tehran, iran. bpublic health unit, nab’a al-hayat foundation for medical sciences and health care, najaf, iraq. cschool of dentistry, tehran university of medical sciences, tehran, iran. dprivate dentist, tehran, iran edepartment of community oral health, faculty of dentistry, tehran university of medical sciences, tehran, iran. *correspondence to reza yazdani (email: ryazdani@tums.ac.ir). (submitted: 18 december 2018 – revised version received: 19 january 2019 – accepted: 25 january 2019 – published online: 26 april 2019) objectives this study aimed to systematically review the available randomized clinical trials (rcts) on the effect of xylitol on the number of streptococcus mutans (s. mutans) colonies. methods an electronic search was carried out in medline and scopus databases for the rcts published during 2002–2014. the inclusion criteria were evaluation of xylitol gums, having at least one control group and counting s. mutans colonies. the articles were divided into three groups based on the subjects’ age group namely 0–6, 6–18 and above 18 years old. to assess the quality of rcts, the retrieved articles were independently reviewed by two reviewers in terms of randomization and to prevent the effect of blinding on the results. review manager (revman) software, heterogeneity test and i2 coefficient as the quantitative scale of heterogeneity were used for statistical analysis. results primary search of the literature using keywords related to sugar alcohols were carried out. after applying the inclusion criteria, 46 articles were found in pubmed and 356 in scopus. heterogeneity was not found in the two age groups of 6–18 and above 18 years old and the i2 coefficient in these two groups was 0%. this rate for the 0–6 years old was 51% (p = 0.15); which indicates moderate heterogeneity. the p-value was 0.25, 0.34 and 0.04 for the 6–18, 0–6 and above 18 years old, respectively. this value was only significant for the age group of above 18 years old. data for all groups were analyzed irrespective of age, which revealed significant differences (p = 0.01). conclusion the available literatures show xylitol as an alternative sweetener, could help to prevent dental caries by reducing the count of s. mutans in the saliva. keywords dental caries, artificial sweetener, adults, child introduction dental caries is a multifactorial disease occurring as the result of an imbalance between the mineral structure of the tooth and oral biofilm. cariogenic bacteria produce acids by metabolizing fermentable carbohydrates. acid causes demineralization by dissolving calcium and phosphate in the structure of enamel and dentin.1 dental caries is a controllable disease. however, it is considered a public health dilemma and affects majority of children and adults.2,3 the main etiology of dental caries is the nutritional habits of people, and consumption of sugar (carbohydrates) is believed to be the main cause. at present, attention is directed toward decreasing the consumption of sugar by the implementation of preventive strategies.4,5 researchers are attempting to find an alternative for the currently used sugars and have come up with nonfermentable sugar alcohols for the purpose of caries prevention. several studies have assessed the efficacy of sugar alcohols, especially xylitol, for prevention of dental caries, and their favorable efficacy in this regard has been previously confirmed.6–8 some previous studies showed that daily consumption of xylitol chewing gums and products were associated with a reduction in prevalence of caries.9–12 however, some others did not report any significant effect related to the consumption of xylitol and refuted its cariostatic effects. xylitol has been introduced as a safe alternative to fermentable sugars for adolescents and the youth and reported to be a non-carcinogenic and even an anti-carcinogenic agent by the american food and drug administration and the european food safety organizations.7 it has been confirmed that xylitol inhibits the growth and proliferation of streptococcus mutans, which is the main microorganism responsible for the occurrence of dental caries.13 fontana et al.,14 evaluated the available review articles and guidelines related to the use of sugar alcohols and stated that extensive use of xylitol and other polyol sugars for preventive or therapeutic purposes requires further assessments with special attention to their efficacy and dosage for use in high-risk communities and their synergy with other preventive modalities. considering the controversial results of previous clinical and review studies, this study sought to assess the effect of xylitol on the number of s. mutans colonies by performing a systematic review. materials and methods this systematic review was designed and carried out according to the cochrane’s handbook for systematic reviews of interventions (version 5.1.0).15 an electronic search was carried out in pubmed and scopus databases for english randomized clinical trials (rcts) or quasi-rcts comparing the efficacy of sugar alcohols with the placebo for prevention of caries. studies published during 2002–2014 with available full texts were searched. non-rcts, historical studies, single group studies with before/after designs, interrupted time series analyses, observational and retrospective studies and controlled before/after prospective cohort trials were excluded. issn 2413-0516 r. yazdani et al. 65j contemp med sci | vol. 5, no. 2, march–april 2019: 64–70 review effect of xylitol on salivary streptococcus mutans: a systematic review and meta-analysis the medline and scopus were searched using keywords in mesh format as well as free words including “sugar alcohols” (mesh) or “sweetening agent” or “sweetener” or “artificial sweetener” or “sugar substitute” and “dental caries” (mesh). after searching the databases, to eliminate the duplicates and also for the purpose of easy citation, the articles were entered into endnote x7 (bld 7072) software. studies on all age groups were searched. articles on a population with specific health conditions (oral or systemic) such as mental retardation were excluded. active intervention groups were those using sugar alcohols in the form of chewing gums, and studies on sugar alcohols consumed in other forms such as pills, drops, lozenges and sugar-saturated wipes were excluded. considering the small number of articles on different types of sugar alcohols, only those including an active intervention group using xylitol and a minimum of one control group not receiving xylitol or receiving placebo or any other form of preventive treatment (such as sealants, fluoridated toothpastes or specific hygienic instructions) were included. with regard to the outcomes, only studies with a methodology based on counting s. mutans colony forming units (cfus) under in vitro conditions were included. based on the variability in dosage of xylitol used in interventional studies and different measurement time points, we tried our best to select studies assessing similar time points, and those reporting data at time points or dosages very different from others were excluded. considering the fact that dental caries is a multifactorial disease and the confirmed effect of age on its occurrence, studies were divided into three groups based on the age range of subjects namely 0–6 years old (which also included studies on infants and pregnant mothers), 6–18 and above 18 years old. data extraction was performed blindly and the reviewers were not aware of the authors’ names, institution, university or journal of the articles. in specific cases, the authors were contacted via email to obtain the raw data. studies collected in a systematic review are widely variable. such diversity among articles is referred to as heterogeneity. determining the type of heterogeneity is valuable. differences among the study subjects (participants), type of intervention performed and the measurement scales used are known as clinical diversity or clinical heterogeneity. diversity in the study design (methodology) and risk of bias of articles is referred to as methodological diversity or methodological heterogeneity. diversity in the effects of interventions measured in different studies is referred to as statistical heterogeneity, which is the outcome of clinical or methodological diversity or both and is more important than other types of heterogeneities. the latter was used in our study and is simply referred to as heterogeneity.15 considering the fact that method of reporting the data was variable in different studies and it was not always possible to contact the corresponding authors, we had to compare studies reporting the logarithm (log10) of the number of bacteria in one group; those reporting the colony counts as frequency values were compared with each other in another group. analyses were carried out using the revman computer program version 5.3.16 after data extraction, heterogeneity tests were used for analysis of data. the final result was obtained by weighing the results of each study based on data dispersion and the assumption of one second colony count in the two groups was calculated using weighted confidence interval. to assess the quality of rcts, the retrieved articles were independently reviewed by two reviewers in terms of randomization and to prevent the effect of blinding on the results. the reviewers were not aware of the name of authors, institution, university or the journal. results a total of 262,944 articles were found in medline and 345,675 in scopus. after applying the above-mentioned inclusion and exclusion criteria regarding the publication year, study design, english language and scope of the journal, 46 articles were found in pubmed and 356 in scopus. the articles were entered into endnote to eliminate duplicates. a total of 404 articles were separately and independently reviewed by the reviewers in terms of their methodology. finally, 22 out of 46 articles found in pubmed and 30 out of 356 found in scopus were included. a total of 52 articles were evaluated in terms of age group of participants, number of study groups, statistical population, sugars compared, time points of assessment, duration of follow-up and measurement scales. with regard to the groups compared, 34 articles compared xylitol with at least one control group; 10 articles compared xylitol with sorbitol; four articles compared sorbitol with one control group; three articles compared xylitol, sorbitol and erythritol; two articles compared erythritol and a control group and five articles compared xylitol and erythritol. among all, those comparing xylitol and a control group were selected and the remainders were excluded. the above-mentioned 52 articles were also compared in terms of outcome; out of which, two had used dmft, and three had used dmfs and 24 counted s. mutans colonies to assess the anti-cariogenic effects of sugars. as stated earlier, counting of s. mutans colonies was an inclusion criterion for our study. the remaining 15 articles were divided into four groups based on the age group of their target population: four articles had been conducted on subjects aged 0–6 years, five articles had been conducted on 6–18 years old and two articles had been conducted on above 18 years old. four articles had evaluated infants and pregnant mothers (fig. 1). among five articles on 6–18 years old, only two by holgerson et al.17 and campus et al.18 had reported the mean and standard deviation of colony counts; the remaining articles had reported frequency values or were not suitable for assessment. the heterogeneity measured for the above-mentioned two articles is depicted in forest diagram. no heterogeneity was found in this respect (χ = 0.05, df = 1, p = 0.83) (fig. 2). the weight of results based on the dispersion of data for the holgerson’s study was 0.8%; the weight of results for the study by campus was 99.2%. the results show the difference in log10 cfus/ml to be 0.08; which was not significant (based on the pand z-values). among articles campus et al.19 and milgrom et al.20 on above 18 years old, the effect of xylitol was found to be significant (p = 0.04 and z = 2.10) (fig. 3); however, it was not significant in the age group of 0–6 years21,22 (p = 0.34) (fig. 4). 66 j contemp med sci | vol. 5, no. 2, march–april 2019: 64–70 effect of xylitol on salivary streptococcus mutans: a systematic review and meta-analysis review r. yazdani et al. fig. 1 search diagram of articles. fig. 2 forest plot for comparing xylitol with control group among 6–18 years old. fig. 3 forest plot for comparing xylitol with control group among above 18 years old. fig. 4 forest plot for comparing xylitol with control group among 0–6 years old. r. yazdani et al. 67j contemp med sci | vol. 5, no. 2, march–april 2019: 64–70 review effect of xylitol on salivary streptococcus mutans: a systematic review and meta-analysis to compare all age groups and assess the effect of age on the results, the three groups were entered into the analysis irrespective of the age of subjects. the results showed complete homogeneity (i2 = 0%, p = 0.01) (fig. 5). since the i2 value is calculated to convert heterogeneity to a quantitative value, this index in the analysis of the two groups was calculated to be 0%; the heterogeneity was calculated to be 51% only in the age group of 0–6 years; based on the definition of i2 values, this group had moderate heterogeneity. 0–40%: might not be important. 30–60%: may represent moderate heterogeneity. 50–90%: may represent substantial heterogeneity. 75–100%: considerable heterogeneity. studies, which were subjected to statistical analyses are presented in table 1. discussion based on the results, in articles on the age groups of 6–18 and 0–6 years old, consumption of xylitol and reduction in s. mutans colony counts were not significantly correlated; but in the age group of above 18 years old, a significant association existed between the consumption of xylitol and reduction in s. mutans colony count (p = 0.04). also, analysis of all articles irrespective of the age of subjects showed a significant correlation in this regard (p = 0.01). however, it should be kept in mind that xylitol has recently found popularity for use in food products due to its anti-cariogenic activity; thus, number of studies, particularly rcts on xylitol is small compared with those on other cariostatic agents such as fluoride. also, use of different methodologies with regard to factors such as dosage of consumption by the participants, time and frequency of consumption, time of sampling, simultaneous use of other preventive measures such as fluoridated toothpastes and duration of follow-up made accurate comparison of articles difficult, if not impossible. also, it should be noted that some previous studies ignored the anti-caries effect of chewing gum and thus, did not provide their control groups with gums to chew to simulate the saliva stimulation effect of xylitol gums in experimental groups. table 1. studies subjected to statistical analyses author methods participants intervention outcome campus et al.,18 italy 204 subjects (acceptance rate 88.3%). inclusion criteria were: >1 and <4 carious lesions, and a salivary s. mutans concentration >105 cfus/ml. xylitol versus control s. mutans cfus/ml in saliva total daily intake of xylitol was 11.6 g. the chewing times were 8.30 a.m. and 1.00, 3.00, 6.00 and 9.00 p.m. plaque ph holgerson et al.,17 sweden double-blind randomized controlled trial with two parallel arms. 128 children (mean age = 12.7 years) consented to participate. the children were stratified as having caries experience (dmfs/ dmfs ≥1) or not. xylitol versus control (sorbitol and maltilol) visible plaque index, salivary mutans streptococci counts and salivary lactic acid production. two pellets, three times daily for 4 weeks random allocation total dose = 6.18 g/day. samples were collected at baseline and immediately after the test period. milgrom et al.,20 usa randomized allocation 132 participants had a mean age of 35 years (range 18–73). controls (g1) (sorbitol/maltitol), or combinations giving xylitol 3.44 g/ day (g2), 6.88 g/day (g3), or 10.32 g/day (g4). s. mutans in saliva and plaque.blinded controls (g1) (sorbitol/ maltitol), or combinations giving xylitol 3.44 g/day (g2), 6.88 g/day (g3), or 10.32 g/ day (g4). groups chewed three pellets/four times/day. groups chewed three pellets/four times/day. samples were taken at baseline, 5 weeks, and 6 months. (continued ) fig. 5 forest plot for comparing xylitol with control group among all ages. 68 j contemp med sci | vol. 5, no. 2, march–april 2019: 64–70 effect of xylitol on salivary streptococcus mutans: a systematic review and meta-analysis review r. yazdani et al. we did not have access to all databases, and non-english manuscripts were not evaluated in our study either. moreover, there were several studies, which met the required criteria to enter into the analysis; however, due to their specific method of reporting data, which was different from that of other articles (not reporting the mean and standard deviation values), they were not included in this study. contacting the corresponding authors was not helpful either. despite all the above-mentioned limitations, we precisely conducted this study using the most recent guideline provided by the cochrane website free of charge.15,16 for more accurate analysis of articles that met our inclusion criteria, only those with similar dosages and sampling time points were entered into the final analysis; which is strength of this systematic review. a review study by mickenautsch and yengopal23 evaluated eight articles on the efficacy of xylitol and sorbitol for caries control and approved the efficacy of xylitol as an alternative anti-caries sugar however, in contrast to our study, they set no age limitation in their inclusion/exclusion criteria; also, their outcome was rate of caries and use of clinical caries indexes such as dmfs. some other review studies have also been performed in this regard in the recent years, with the same results as ours. for instance, mickenautsch et al.10 confirmed the anti cariogenic effect of immediate use of xylitol after eating by reviewing nine studies. also, deshpande and jadad24 and rethman et al.25 reviewed 19 and 15 articles, respectively. antonio et al.26 reviewed three articles on the anti-caries effects of xylitol and confirmed its anti-caries efficacy for areas other than the interproximal surfaces. all the above mentioned studies used xylitol-containing products such as candies and lozenges in addition to chewing gums; these products may have stronger anti-caries effects by further stimulating the secretion of saliva and subsequent increase in the level of ph. however, bader et al.,27 in a review study stated that the available evidence is not sufficient to confirm the anticaries effects of xylitol. similarly, lingström et al.9 compared nine articles and concluded the same result. based on our findings and similar results reported in most previous studies, insignificant efficacy of xylitol in studies on 0–6 and 6–18 years old may be due to several factors. although s. mutans is an important cause of development of caries, our obtained result is exclusively related to this outcome while dental caries is a multifactorial disease. moreover, subjects in these two age groups have less information and control over their personal oral hygiene compared with those above 18 years of age. also, older subjects have better cooperation with the researchers. the results of each study alone confirm the efficacy of xylitol for decreasing s. mutans colony counts. however, the magnitude of this effect remains questionable. further rcts on larger sample sizes and with similar dosages, methods of measurements and equal outcomes are required. one major concern regarding the use of xylitol is its proper daily dosage. fontana and gonzález-cabezas14 evaluated several systematic reviews and reported a suitable mean value of 6 g/day based on a range of 2.9–10.67 g/day, used in most studies. but, they also stated that there were two exceptions to this rule. table 1. studies subjected to statistical analyses—continued author methods participants intervention outcome campus et al.,19 italy double-blinded 346 healthy subjects (age range 18–30 years) subjects who presented more than one carious lesion, but less than four, a salivary s. mutans concentration 6 × 105 cfus/ml and bleeding on probing 25%. subjects with a history of systemic diseases were excluded. magnolia chewing gums contained 30% xylitol, 0.17% mbe (magnolol 0.10% and honokiol 0.07%, respectively), 26% sorbitol, 11% mannitol and 1% maltitol syrup. xylitol chewing gums contained the same percentages of the polyols mentioned, with no other active ingredients. the control chewing gum was sugar-free and contained 28% isomalt, 31% sorbitol, 9% mannitol and 1% maltitol syrup. salivary s. mutans (cfus/ml)randomized allocation three groups: magnolia, xylitol and control. bleeding on probing at baseline, after 7 days, after 30 days of gum use and 7 days after the end of gum use. plaque ph. the total daily intake of magnolol and honokiol was 11.9 mg/day, and for xylitol it was 2.2 g/day. anttonen et al.,21 finland double-blinded 157 children (mean age 5.0 ± 1.4 years) consumed xylitol chewing gum and 149 children (mean age 4.9 ± 1.5 years) sucrose chewing gum. no extra preventive dental measures were taken by the municipal health center for any of the children after the trial. xylitol versus control (sucrose) salivary mutans streptococci (cfus/ml).randomized allocation total amount of 8.4 g sucrose or xylitol in chewing gum was given daily in five doses for 2 months.children used sucrose or xylitol chewing gum regularly for 2 months sample at baseline and after intervention. mäkinen et al.,22 finland double-blinded 123 children with a mean age of 5.03 (0.53) xylitol versus d-glucitol plaque s. mutans and plaque index. daily consumption of xylitol and d-glucitol was 4.5–5 g per subject. average dmfs of children was 11.6 (12.7). randomized allocation interproximal dental plaque was sampled at baseline and after 6 months. r. yazdani et al. 69j contemp med sci | vol. 5, no. 2, march–april 2019: 64–70 review effect of xylitol on salivary streptococcus mutans: a systematic review and meta-analysis first, twice daily use of fluoridated toothpaste with only 10% xylitol (equals approximately 0.02 g/day) caused a significant reduction in the rate of caries.28 the second exception was delay in formation of s. mutans colonies in infants whose mothers used >5 g and in some cases <2 g/day xylitol in their first years of life.29–32 this was also true for one of the articles, which was entered into our analysis. in the study by milgrom et al.,20 three different doses of xylitol namely 3.44, 6.88 and 10.32 g were used; the two groups of 6.88 and 10.32 g showed significant efficacy while 3.44 g xylitol had no significant effect on s. mutans colony count. campus et al.19 assessed the efficacy of consumption of 2.2 g xylitol/day by the age group of above 18 years old and reported significant reduction in the number of s. mutans colonies and a subsequent reduction in the rate of caries. the mean dose of daily consumption of xylitol in 0–6, 6–18 and above 18 years old age groups was 8.34, 5.19 and 2.82, respectively. another reason explaining the significant effect of xylitol reported in previous studies is its significant effect on those above 18 years old and insignificant effects on the other two age groups. the significant effect of xylitol irrespective of age may be due to the difference in its daily dosage. for instance, in the age group of 6–18 years old in the study by campus et al.,18 11.6 g xylitol was used per day; whereas, in the study by holgerson et al.,17 almost half of this dosage (6.18 g) was used daily. also, studies by anttonen et al.,21 and mäkinen et al.,22 reported the use of 8.4 and 4.55 g xylitol/day, which are widely different. this difference can probably result in different outcomes. thus, an effective dose of xylitol for caries reduction is still a matter of debate and further studies are required to find the most effective dosage of xylitol to achieve the highest cariostatic effects. conclusion based on the results, the available literatures show xylitol as an alternative sweetener, which is capable of preventing dental caries by reducing the count of s. mutans in the saliva. the daily dosage of xylitol as an anti-caries agent is still controversial and calls for further investigations. acknowledgments the authors declare no potential conflicts of interest with respect to authorship and/or publication of this article. this manuscript is based on a thesis (thesis number: 44) submitted to faculty of dentistry, tehran university of medical sciences, international campus, tehran, iran, in partial fulfillment of the requirements for the d.d.s degree. conflicts of interest none.  references 1. fejerskov o. changing paradigms in concepts on dental caries: consequences for oral health care. caries res. 2004;38:182–191. 2. marthaler tm. changes in dental caries 1953-2003. caries res. 2004;38: 173–181. 3. petersen pe, bourgeois d, ogawa h, estupinan-day s, ndiaye c. the global burden of oral diseases and risks to oral health. bull world health organ. 2005;83:661–669. 4. gonçalves af, maia lc, vianna r, quintanilha lelp. preventive strategies in oral health promotion. cien saude colet. 2005;10:279–286. 5. burt ba. the use of sorbitoland xylitol-sweetened chewing gum in caries control. j am dent assoc. 2006;137:190–196. 6. isogangas p, mäkinen kk, tiekso j, alanen p. long-term effect of xylitol chewing gum in the prevention of dental caries: a follow-up 5 years after termination of a prevention program. caries res. 1993;27:495–498. 7. mäkinen kk, bennett ca, hujoel pp, isokangas pj, isotupa kp. pape hr, et al. xylitol chewing gums and caries rates: a 40-month cohort study. j dent res. 1995;74:1904–1913. 8. scheinin a, pienihäkkinen k, tiekso j, bánóczy j, szöke j, esztári i, et al. collaborative who xylitol field studies in hungary. vii. two-year caries incidence in 976 institutionalized children. acta odontol scand. 1985;43:381–387. 9. lingström p, holm ak, mejàre i, twetman s, söder b, norlund a, et al. dietary factors in the prevention of dental caries: a systematic review. acta odontol scand. 2003;61:331–340. 10. mickenautsch s, leal sc, yengopal v, bezerra ac, cruvinel v. sugar-free chewing gum and dental caries: a systematic review. j appl oral sci. 2007;15:83–88. 11. riley p, moore d, ahmed f, sharif mo, worthington hv. xylitol-containing products for preventing dental caries in children and adults. cochrane database syst rev. 2015;3:cd010743. 12. lin hk, fang ce, huang ms, cheng hc, huang tw, chang ht, et al. effect of maternal use of chewing gums containing xylitol on transmission of mutans streptococci in children: a meta-analysis of randomized controlled trials. int j paediatr dent. 2016;26:35–44. 13. trahan l. xylitol: a review of its action on mutans streptococci and dental plaque—its clinical significance. int dent j. 1995;45:77–92. 14. fontana m, gonzález-cabezas c. are we ready for definitive clinical guidelines on xylitol/polyol use? adv dent res. 2012;24:123–128. 15. higgins jpt, green s (editors). cochrane handbook for systematic reviews of interventions. version 5.1.0. the cochrane collaboration, 2011. http:// handbook.cochrane.org/2011 [accessed march 2011]. 16. the nordic cochrane centre, the cochrane collaboration. review manager (revman) [computer program]. version 5.3. the nordic cochrane centre, the cochrane collaboration, copenhagen, 2014 [accessed]. 17. holgerson pl, sjöström i, stecksén-blicks c, twetman s. dental plaque formation and salivary mutans streptococci in schoolchildren after use of xylitol-containing chewing gum. int j paediatr dent. 2007;17:79–85. 18. campus g, cagetti mg, sacco g, solinas g, mastroberardino s, et al. six months of daily high-dose xylitol in high-risk schoolchildren: a randomized clinical trial on plaque ph and salivary mutans streptococci. caries res. 2009;43:455–461. 19. campus g, cagetti mg, cocco f, sale s, sacco g, strohmenger l, et al. effect of a sugar-free chewing gum containing magnolia bark extract on different variables related to caries and gingivitis: a randomized controlled intervention trial. caries res. 2011;45:393–399. 20. milgrom p, ly ka, roberts mc, rothen m, mueller g, yamaguchi dk. mutans streptococci dose response to xylitol chewing gum. j dent res. 2006;85:177–181. 21. anttonen v, halunen i, päkkilä j, larmas m, tjäderhane l. a practice-based study on the effect of a short sucrose/xylitol exposure on survival of primary teeth caries free. int j paediatr dent. 2012;22:356–362. 22. mäkinen kk, isotupa kp, mäkinen pl, söderling e, song kb, nam sh, et al. six-month polyol chewing-gum programme in kindergarten-age children: a feasibility study focusing on mutans streptococci and dental plaque. int dent j. 2005;55:81–88. 23. mickenautsch s, yengopal v. effect of xylitol versus sorbitol: a quantitative systematic review of clinical trials. int dent j. 2012;62:175–188. 24. deshpande a, jadad ar. the impact of polyol-containing chewing gums on dental caries: a systematic review of original randomized controlled trials and observational studies. j am dent assoc. 2008;139:1602–1614. 25. rethman mp, beltrán-aguilar ed, billings rj, hujoel pp, katz bp, milgrom p, et al. nonfluoride caries-preventive agents: executive summary of evidencebased clinical recommendations. j am dent assoc. 2011;142:1065–1071. 26. antonio ag, pierro vs, maia lc. caries preventive effects of xylitol-based candies and lozenges: a systematic review. j public health dent. 2011;71:117–124. 27. bader jd, shugars da, bonito aj. systematic reviews of selected dental caries diagnostic and management methods. j dent educ. 2001;65:960–968. 28. sintes jl, elias-boneta a, stewart b, volpe ar, lovett j. anticaries efficacy of a sodium monofluorophosphate dentifrice containing xylitol in a dicalcium phosphate dihydrate base. a 30-month caries clinical study in costa rica. am j dent. 2002;15:215–219. 70 j contemp med sci | vol. 5, no. 2, march–april 2019: 64–70 effect of xylitol on salivary streptococcus mutans: a systematic review and meta-analysis review r. yazdani et al. 29. alamoudi nm, hanno ag, sabbagh hj, masoud mi, almushayt as, el derwi da. impact of maternal xylitol consumption on mutans streptococci, plaque and caries levels in children. j clin pediatr dent. 2012;37:163–166. 30. isokangas p, söderling e, pienihäkkinen k, alanen p. occurrence of dental decay in children after maternal consumption of xylitol chewing gum, a follow-up from 0 to 5 years of age. j dent res. 2000;79:1885–1889. 31. söderling e, isokangas p, pienihäkkinen k, tenovuo j. influence of maternal xylitol consumption on acquisition of mutans streptococci by infants. j dent res. 2000;79:882–887. 32. thorild i, lindau b, twetman s. caries in 4-year-old children after maternal chewing of gums containing combinations of xylitol, sorbitol, chlorhexidine and fluoride. eur arch paediatr dent. 2006;7:241–245. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.04201901 66 j contemp med sci | vol. 6, no. 2, march–april 2020: 66–7266 original issn 2413-0516 introduction the skin is an organ that covers the external surface of the body and protects it from exterior hazards. this organ includes the skin and its derivatives such as skin glands (oil glands and sweat glands), hair, nails, and breast. anatomically, from exterior to interior the skin includes three layers which are called: epidermis, dermis, and hypodermis (or the subcutaneous oil tissues).1 the wound is defined as tissue rupture and/or local destruction of epidermis and dermis which eventually results in the remaining scar. healing includes two steps: (1) contraction and (2) replacement of the destroyed tissue which occurs via cell transfer and division of alongside cells.2, 3 first, a temporary arteriole contraction occurs and afterward the vessels dilate. neutrophils initiate adhesion to blood vessel walls and vessel permeability increases. leukocytes and few lymphocytes appear and glycoprotein networks surround them during the first 3 h.4–6 the number of neutrophils in wound increases during the first 48 h. few monocytes appear in the first 24 h, and then increase in the following days. macrophages increase gradually along with neutrophil number reduction. macrophages produce fibroblast growth factors as well as factors responsible for new vessel production. blood coagulation is the first healing signal.7 fibrinopeptide and thrombin attract the macrophages to the damaged tissue and the activated platelets secrete pdgf, igf-1, and tgfβ which all prepare the target cells for proliferation. subsequently, fibrin stimulates the macrophages to secrete more healing signals.8 when damaged endothelial cells secrete cytokines, integrin and its receptor appear on the surface of passing leukocytes and inflammation begins. newcomer inflammatory cells increase the metabolic need.9 since the local microvascular construction is damaged, energy and o2 reduction, and co2 and lactate accumulation occur at the damaged site. these happenings initiate the healing process and reassure its maintenance.10 in wounds that are initially sewed, the branched vessels quickly join the branched vessel from the front and the blood circulation of the wound is established.12 in remaining or not fully closed wounds, the new vessels only join the neighbor vessels on the same side of the wound and the granulation tissue is created instead.13 angiogenesis is inducible by the addition of chemotactic substances to the endothelial cells in the tissue body of the wound. it seems that wound angiogenesis is a response to local energy reduction and its general mechanism is very similar to the mechanism of collagen sediment regulation.11 in unsuitable conditions (hypoxia or lactate increasing), macrophages secrete a chemotactic peptide for endothelial cells which leads to angiogenesis.14 nad+ reserve protection inhibits this process drastically. therefore, the unanswered metabolic needs cause an anatomical response with means of a series of growth factors that probably have high importance.15 this step starts approximately from day 4 or 5. during healing, fibroplasia (fibroblast proliferation) is stimulated from various mechanisms that start with secretion of pdfg, igf-1, and tgfβ from platelets and resumes with the secretion of cytokines from the macrophage.16 fibroblasts secrete igf-1. epidermal growth and gf1 are also transferred to the damage site via blood circulation. healing fibroblasts are mostly seen near the wound edges.17 at this place, an appropriate growth environment and oxygen pressure close to 40 mmhg are available for them in normal wounds.18 investigating the healing effect of the hydroalcoholic extract of pomegranate seed (punica granatum) on the full thickness wound in rabbit ali asghar hemmati1, iran rashidi2, sedigheh dahanzadeh1, mahmoud moeini1 1departmant of pharmacology, school of pharmacy, ahvaz jundishapur university of medical science, ahvaz, iran. 2department of pathology, school of medicine, ahvaz jundishapur university of medical science, ahvaz, iran. corresponding author: sedigheh dahanzadeh (e-mail:sedigheh.dahanzaadeh@yahoo.com) (submitted: 04 january 2020 – revised version received: 12 february 2020 – accepted: 17 february 2020 – published online: 26 april 2020) objective this research designed to investigate the wound healing process with pomegranate hydroalcoholic seed extract (punica granatum) in comparison with no-treatment, betamethasone, phenytoin, and eucerin in rabbits. methods the positive group including groups that received phenytoin cream (1%) and topical eucerin, respectively, twice a day to complete wound healing. negative control group did not obtain any treatment. treatment groups were received cream of pomegranate seed extract (pse) (2, 5,7,10 w/w) in eucerin and 75% w/w as purified extract twice daily. in order to measure the percentage of wound healing, the zone of the wound was evaluated daily. histological studies were done on the 7th and 15th days of treatment. next, hydroxyproline content of wounds healed and tensile strength of wound tissue samples were measured. results the results demonstrated between pse treatment groups and eucerin animals were statistically significant different (p<0.05) in most of the days reviewed. treatment of rabbits with 10% pse had the best results (complete wound recovery in 12 days). also, this treatment showed higher hydroxyproline content and higher tissue strength. conclusion this research reveals that the extract of 10% pse administrated topically has the proper potential to induce wound recovery in the wound model of rabbits. in addition, 10% pse accelerates the healing of the wound. further study needs to clarify the results of this research. keywords pomegranate (punica granatum), hydroxyproline, rabbit, tensile strength, wound healing 67 original extract of pomegranate seed (punica granatum)ali asghar hemmati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 66–72 this oxygen pressure is optimal for fibroblast proliferation in cell culture. from the newly proliferated fibroblasts, collagen and proteoglycan are secreted which brings close the wound edges.19 both substances are in polymeric form with large molecules which bases the physical strength of the wound. collagen synthesis is not specifically limited to fibroblasts but is regulated gravely via these cells.20 some growth factors (igf-1, tgfβ) cause transcription of the collagen gene. evidence shows the increase of collagen mrna results in the increase of procollagen peptide but this is not enough to raise the amount of collagen sediment, since procollagen peptide cannot transfer to the extracellular environment without the hydroxylation of some of its proline agents.21 epithelial cells respond to many fibroblasts and endothelial stimuli. in the healing process, epithelium mitosis happens a few cells farther from the edge of the wound. new cells travel from the edge of the wound to the unhealed area and probably adhere to the first non-epithelized area by a growth factor or cytokine and form the new edge of the wound.22 oxygen pressure is probably low in the cellular joint. this low o2 pressure stimulates tgfβ production from the squamous epithelial, inhibits the final dissociation, and increases mitosis. this process continues until the wound closes.23 squamous epithelization and dissociation continues at maximum as long as o2 pressure reaches 70 mmhg and the wound surface remains moist. opposed to the classical theory, even short periods of wound drying can disturb this process.24 the exodus of acute, non-infectious, and surface wound also contains growth factor and lactate, therefore, accommodates the growth environment within. the second to the third level of the healing process progresses gradually. the extracellular matrix turnover is a complex process. first fibroblasts replace the initial fibrin matrix with collagen monomers.25 extracellular enzymes (which some of them depend on oxygen pressure) polymerize these monomers quickly.26 this polymerization has a more disordered nature compared to normal situation, and therefore new wounds are weak and fragile. when this initial matrix is replaced by a more mature kind which includes bigger, stronger, more ordered, and more persistent fibers, the wounded delicacy is hindered.27 turnover and reorganization of the new matrix are very important in the healing process. fibroblasts and leukocytes lyse the matrix with the secretion of collagenase. replacement of the old matrix with a new matrix takes place first quickly, and then gradually.28 pomegranate is a small family including a genus, containing two species with characteristics similar to the myrtaceae family. pomegranate has a rugged stem with hardwood and covered with a somewhat green color. its multiple branches have irregular shapes and special colors and usually have sharp thorn-like ends.29, 30 simple dark and usually reciprocal leaves and male– female flowers are among its characteristics. their calyx contains 4–8 flesh-like pieces and is joined to the pod. pomegranate fruit is a sphere as big as an orange and sometimes larger, with relatively thick and red, soft and uneven skin, soft, and uneven.31 some pomegranate breeds have yellowish-white skin. the seeds inside the fruit are surrounded in pink flesh-like covers and its flower is scentless. this plant is dispersed mostly in europe, north africa, asia, and iran. in traditional medicine of iran, all parts of the pomegranate plant are used.32 different parts of pomegranate especially the root skin and stem have 22% tannic acid and picotannic acid, pyrogallic acid, gratanotannic acid, resin, and mucilage. it also contains alkaloids such as pelletierine, iso pelletierine, methyl pelletierine, and pseudo-pelletierine. in addition, many others exist in pomegranate fruit such as vitamins b1, b2, b6, c, and minerals such as potassium, phosphorus, iron, and sodium.33 materials and methods plant material pomegranate fruits were collected in december from the surrounding gardens of isfahan and then seeds were manually separated and dried in the shade for 1 week. to prepare the pomegranate extract, soaking method with ethanol (70%) solvent was used. 500 g of powdered seeds were poured into the beaker and 5 ml of solvent per gram of powder was added. soaking was performed for 72 h. every 12 h, the mixture was stirred while soaking. after 72 h, the mixture was filtered with a clean cloth, and then through filter paper and a buchner funnel. afterwards, extract concentration was performed with the rotary machine at a temperature of 65°c for 70 h. extract obtained per 50 g of powdered pomegranate seeds was 9.4 g and the amount of resulting extract was calculated as 15.8%. animals experiment animals, male and female iranian rabbits were prepared from the animal care center with weight range of 1.2–1.8. they were kept in the school of pharmacy animal room in individual aluminum cages with conditions of 12 h light, 12 h dark, and temperatures of 2 ± 22°c. compact food, carrots, lettuce, and water were available for the animals without limitation. wound creation the place of the wound is on the side of the animal near the spine. for wounding, according to hemmati & mohammadian method, first the hair of wound area was removed using shaving machine and blade and thoroughly cleaned. then local anesthesia was created using lidocaine. animal was placed in standard crouching position. the moving skin was kept with fingers and using a shablon and pen, a 20×20 mm square was drawn on the skin. thereafter, layers of the epidermis, dermis, and hypodermis of animal skin were fully removed by no. 15 scalpel, forceps, and surgical scissors. after wounding, the wound was rinsed with saline, the wound surface was measured and animals were returned to their separate cages. after 24 h, daily measuring of wounds began. to measure, the animal was placed in the standard crouching position, then using a transparent the sides of the wound (wound area) were plotted and its area was measured using graph paper, because this study is basically to compare the level of residual wound. to minimize errors of observation, the animal was placed in standard position and each wound surface was plotted three times. the coagulation on the wound surface should never be physically moved, because it certainly causes an impact to the wound and the wound bed, thus prevents the favorable report of the wound. 68 original extract of pomegranate seed (punica granatum) ali asghar hemmati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 66–72 groups studied 1 – wound group with no treatment. (control group) 2 – wound group receiving1% phenytoin on the wound. (positive control, because this drug is a standard wound healer) 3 – wound group with eucerin ointment base rubbed on their wounds. (as negative control) 4 – wound group receiving 2% extract in eucerin base on the wound 5 – wound group receiving 5% extract in eucerin base on the wound 6 – wound group receiving 10% extract in eucerin base on the wound 7 – wound group receiving the purified extract (75%) in order to observe the effect of purified extract in wound healing and the amount of purified extract was equivalent to other groups. 8 – wound group receiving betamethasone 0.1% ointment on the wound. pathological study a skin sample was prepared on the 7th day from each group for pathological and histological studies. using forceps, scalpel, and surgical scissors, the wound area and some of the healthy skin surrounding it was removed and then immediately transferred to the container with formalin buffer 10%. sections with thickness of 5 μm were prepared and stained with hematoxylin and eosin, and then were examined by light microscopy. determination of hydroxyproline and collagen in skin samples the amount of hydroxyproline in skin samples is measured using obrien et al (1965) method by spectrophotometer. the total amount of collagen is calculated presuming 12.5% of it comprises hydroxyproline. hydroxyproline is separated from collagen by acid hydrolysis and is oxidized to pyrole using chloramine-t solution and produced a red color using paradimethyl amino benzaldehyde (erlich reagent). measuring the elasticity of the skin samples after treatment period, skin strips with dimensions of 20×5 mm were isolated from the wound and placed under tension. two ends of tissue were attached to special and then a gradual stretching was applied to the tissue until it was torn. the amount of resistance in wounded tissues in different groups and healthy skin were compared. ethical considerations due to work on animals using standard methods with consideration of ethics and anesthesia induction when wounding and using sterile pharmaceutical products, moral principles related to the work on experiment animals were precisely observed. data analysis data obtained during the treatment consisted of the amount of hydroxyproline and quantitative tissue resistance. using anova different groups were statistically compared. the results of wound healing comparison of the wound healing in untreatment, eucerinand phenytoin-treated groups in eucerin-treated rabbits group, treatment took 21 days and the percentage of wound healing from macroscopic view reached 100%. statistical tests revealed it doesn’t have a significant difference compared to 2% group (p>0/05). compared to 5%, it is significantly different except on days 6, 11, 12, 13, and 16. compared to 10%, it has significant difference on all days except the first. compared to 75%, it is significantly different on all days except 6, 11, 12, 15, and 16. compared to phenytoin-treated group, it was significantly different on all days except 2, 3, 4, and 7 (p<0/05; fig. 1). comparison of average hydroxyproline amount in different groups on the last day of treatment the aim of study was measuring the amount of hydroxyproline in each treatment groups and comparing the hydroxyproline levels in different groups. the results show that phenytoin-treated group and the group treated with 10% pomegranate seed extract have the highest concentration of hydroxyproline (micrograms per gram), where no treatment group and eucerin-treated group had the lowest amount and hydroxyproline in treatment groups with 2%, 5%, and 75% of pomegranate seed extract and betamethasone ranged between the minimum and maximum concentrations. the results of statistical tests to compare the hydroxyproline amount in different groups shows that the 10% group and the phenytoin-treated group have significant difference with each other and with all other groups (p<0/05). 2%, 5%, and 75 % and betamethasone groups have no significant difference with each other (p<0/05) fig. 1 comparison of the wound healing in untreatment, eucerinand phenytoin-treated groups. data are expressed as (mean±sem). values significantly different from eucerin-treated or no-treatment are indicated as *(p<0·01). 69 original extract of pomegranate seed (punica granatum)ali asghar hemmati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 66–72 but are significantly different compared with other groups. no treatment group and eucerin-treated group also do not have a significant difference with each other but compared to other groups are significantly different (p<0/05; fig. 2). comparison of average tissue resistance against stretching in all treatment groups with no treatment group and betamethasone on the last day of treatment the aim of this study was to measure and compare the average resistance of healed tissue to stretch. the results showed that the group treated with the 10% extract and phenytoin (10%) had the highest resistance to stretching compared with other groups which was significantly different (p<0/05). betamethasone group had the least resistance. eucerin, no treatment, 2%, 5%, and 75% groups had the resistance value ranging between the lowest and highest amount and were significantly different with these groups (p<0/05: fig. 3). discussion given that the wound healing process has many complications, therefore, this seems necessary to know topical drugs for wounds that have the greatest effect on wound healing. in this research, the most aim is the comparison of average wound healing in different groups treated with various concentrations fig. 2 comparative graph of hydroxyproline (µg/g) in treated and untreated groups on the last day of treatment. data are expressed as (mean±sem). values significantly different from eucerintreated or no-treatment are indicated as *(p<0•01). fig. 3 comparison of mean tissue resistance to stretch in all treated groups plus untreated and betamethasone on the last day of treatment. data are expressed as (mean±sem). values significantly different from eucerin-treated or no-treatment are indicated as *(p<0.01). photomicrograph of tissue specimens of rabbits treated with 2% pomegranate seed extract at the end of treatment, formed (a) thin epidermis and granulation in the dermis (b), few inflammatory cells photomicrograph of rabbit tissue specimen treated with 5% pomegranate seed extract at the end of treatment at epidermis: (a) with relatively good thickness and in dermis, (b) granulation tissue with collagen deposited repaired, (c) wound scab has been seen. 70 original extract of pomegranate seed (punica granatum) ali asghar hemmati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 66–72 of the pomegranate (punica granatum) seed extract (pse) in comparison to phenytoin and betamethasone. in this study, phenytoin 1% as control positive and 10% pse can create the optimum result of formation hydroxyproline, whereas eucerin and no treatment groups have a low effect on hydroxyproline constitution in wound healing. phenytoin cream, while being effective, causes some systemic side effects that contain neutropenia, erythema multiform, crystalluria, and hydroxyproline production.34 in the other hand, prolonged and excessive hydroxyproline formation process causes the risk of delayed wound healing.35 a healing tissue synthesizes collagen, which is a constituent of the growing cells. the amount of hydroxyproline is a scale of concentration of collagen. collagen synthesis is a complex construction coordinator of intracellular and extracellular cell growth and repair.36 in addition to the synthesis of the polypeptide chains more than modifications of the molecule occur; most of these are enzymatic and specific for collagen. hydroxyproline as measuring factor of collagen is an essential component of the skin repair and utilized as an adjunctive wound healing therapy stimulates and recruits immune cells and fibroblasts, photomicrograph of rabbit tissue section treated with 10% pomegranate seed extract on the end of epidermal treatment day: (a) thickly formed, (b) fibroblasts stretched, collagen density high, not edema, and inflammatory cells. in this image, fibroblasts have been found to be elongated (400 h&e*) photomicrograph of rabbit tissue section treated with 75% pomegranate seed extract on the end of epidermal treatment day: (a) tinny epidermis formed not fully fibroblasts stretched, collagen density high, low inflammatory cells and (b) wound scab has been seen. (400 h&e*) photomicrograph of rabbit tissue section treated with eucerin on the end of epidermal treatment day in epidermis area. (a) inflammatory cell aggregation is seen, germ cell migration is clear, (b) wound scab has been seen (400 h&e*). photomicrograph of rabbit tissue section treated with phenytoin on the end of epidermal treatment day. (a) tinny epidermis and granulation tissue have formed in dermis, (b) wound scab has been seen (100 h&e*). 71 original extract of pomegranate seed (punica granatum)ali asghar hemmati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 66–72 thereby preserving native ecm structure and promoting healing.37 fibrocytes are one of the most important cells producing cytokines such as (tgf-β1 and tnf-α) involved in the synthesis of collagen during both the inflammatory and the repair phase of the wound healing response.38 commonly, pomegranate has various active substances that can act as an antibacterial, anti-inflammatory, and strong antioxidants. pomegranate including tannin, flavonoid, punicic acid, and phytoestrogen.39 little research has been done in the field extract of pomegranate effect on wound healing the evidence has shown pomegranates protect and strengthen skin cells on the surface and regenerate cells in the deeper layers of skin.40 many substances such as ellagic acid and standardization in pomegranate can strengthen the pharmacologic effect of this fruit as a topical drug.41 chinese and indian complementary medicine use natural herbal materials because herbal medicine has advantages over modern medicine.42 the various active substances of herbal drugs can expand the pharmaceutical spectrum and increased positive results of treatment. these important roles are mainly linked to the mechanism of the body involved in organs and cells in the accountability to repair any disturbance in the body.43 the perturbation creates a lack of balance in the body. all the organs and cells of the body are involved in returning to balance. it is noted that various active components of the herbal material can improve the injuries of organs and tissue and cells which single drugs cannot. in the latest study, pomegranate can be used as antibacterial agents.44 in the inflammation phase, many chemical mediators cause the expression of cyclooxygenase-2 which induced prostaglandins synthesis.45 also, pomegranate has antioxidant properties that can prevent the oxidative stress via sustaining expression level of malondialdehyde, glutathione, glutathione peroxidase, and catalase.46 in this research, the result of skin collagen formation can be related to an optimal healing stage of rat wound. 10% extract of pomegranate seed similar phenytoin shows significant results of collagen formation in wound. from these findings, it is understood that using 10% extract of pomegranate likely promotes a synergic mechanism to provide an optimal process of rat wound healing. the current research shows that topical application of 10% extract of pomegranate represents a proper approach to improve dermal wound. it is noted, according to our previous researches on herbal extracts, we survey 2, 5, 10, 75 extract concentration on wound healing that result shows 10% extract had the most effective wound healing in terms of skin hydroyprolin and tissue resistance against stretching in comparison to other concentration. also, topical 10% extract similar phenytoin shows markedly promotion and improve wound in reducing period of healing from 20 days (for no-treatment or eucerin-treated) to 12 days. the time period results of various concentration of extract were reproducible because they tested in a group of at least six rats. the macroscopic research of 10% extract group was consistent with results from hydroxyproline measure or tensile strength values. 10% extract was almost equal potent with phenytoin. funding information ahvaz jundishapur university of medical science, grand/ award numbers:prc-80. conflict of intrest the authors declare no conflicts of interest. acknowledgment the present study was supported by ahvaz jundishapur university of medical sciences (project grant: prc-80), ahvaz, iran. references 1. rawlings av. ethnic skin types: are there differences in skin structure and function? int j cosmetic sci. 2006;28(2):79–93. 2. rodero mp, khosrotehrani k. skin wound healing modulation by macrophages. int j clin exp pathol. 2010;3(7):643. 3. dorsett‐martin wa. rat models of skin wound healing: a review. wound repair regen. 2004;12(6):591–9. 4. wilgus ta. immune cells in the healing skin wound: influential players at each stage of repair. pharmacol res. 2008;58(2):112–6. 5. martin c, muir i. the role of lymphocytes in wound healing. br j plast surg. 1990;43(6):655–62. 6. peterson jm, barbul a, breslin rj, wasserkrug hl, efron g. significance of t-lymphocytes in wound healing. surgery. 1987;102(2):300–5. 7. koh tj, dipietro la. inflammation and wound healing: the role of the macrophage. expert rev mol med. 2011;13. 8. wolberg as. thrombin generation and fibrin clot structure. blood rev. 2007;21(3):131-42. 9. hertle md, kubler m-d, leigh im, watt f. aberrant integrin expression during epidermal wound healing and in psoriatic epidermis. j clin investig. 1992;89(6):1892–901. 10. waris th, kaarela oi, raatikainen tk, teerikangas he, heikkinen es. microvascular flaps from the lateral arm and radial forearm for the repair of defects of the achilles tendon region. scand j plast reconst surg hand surg. 1991;25(1):87–9. 11. lee pc, salyapongse an, bragdon ga, shears ll, watkins sc, edington hd, et al. impaired wound healing and angiogenesis in enos-deficient mice. am j physiol-heart circul physiol. 1999;277(4):h1600–h8. 12. dietze g, wicklmayr m. pharmaceutical composition and method of stimulating blood circulation and wound healing. google patents; 1979. 13. schindl a, schindl m, schön h, knobler r, havelec l, schindl l. lowintensity laser irradiation improves skin circulation in patients with diabetic microangiopathy. diab care. 1998;21(4):580–4. 14. modarressi a, pietramaggiori g, godbout c, vigato e, pittet b, hinz b. hypoxia impairs skin myofibroblast differentiation and function. j investig dermatol. 2010;130(12):2818–27. 15. hawrylyshyn km. nicotinamide riboside delivery generates nad+ reserves to protect vascular cells against oxidative damage. 2015. 16. clark ra. regulation of fibroplasia in cutaneous wound repair. am j med sci. 1993;306(1):42–8. 17. lewis da, travers jb, somani a-k, spandau df. the igf-1/igf-1r signaling axis in the skin: a new role for the dermis in aging-associated skin cancer. oncogene. 2010;29(10):1475–85. 18. moulin v. growth factors in skin wound healing. eur j cell biol. 1995;68(1):1–7. 19. hehenberger k, brismar k, lind f, kratz g. dose‐dependent hyperbaric oxygen stimulation of human fibroblast proliferation. wound repair regen. 1997;5(2):147–50. 20. jimenez s, freundlich b, rosenbloom j. selective inhibition of human diploid fibroblast collagen synthesis by interferons. j clin investig. 1984;74(3):1112–6. 21. hung cf, rohani mg, lee s-s, chen p, schnapp lm. role of igf-1 pathway in lung fibroblast activation. respir res. 2013;14(1):102. 22. delavary bm, van der veer wm, van egmond m, niessen fb, beelen rh. macrophages in skin injury and repair. immunobiology. 2011;216(7):753–62. 72 original extract of pomegranate seed (punica granatum) ali asghar hemmati et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 66–72 23. scheid a, wenger r, christina h, camenisch i, ferenc a, stauffer u, et al. hypoxia-regulated gene expression in fetal wound regeneration and adult wound repair. pediatr surg int. 2000;16(4):232–6. 24. mortazavi r, chitte s, philip a. 154 regulation of tgf‐β and its signaling components by oxygen tension and steroids in skin cells. wound repair regen. 2005;13(2):a28–a48. 25. toy l. matrix metalloproteinases: their function in tissue repair. j wound care. 2005;14(1):20–2. 26. rousselle p, montmasson m, garnier c. extracellular matrix contribution to skin wound re-epithelialization. matrix biol. 2019;75:12–26. 27. kähäri vm, saarialho‐kere u. matrix metalloproteinases in skin. exp dermatol. 1997;6(5):199–213. 28. rittié l. cellular mechanisms of skin repair in humans and other mammals. j cell commun signal. 2016;10(2):103–20. 29. huan y, peng k-j, wang q-l, gu z-y, lu y-q, jun z, et al. effect of pomegranate peel polyphenol gel on cutaneous wound healing in alloxaninduced diabetic rats. chin med j. 2013;126(9):1700–6. 30. mo j, panichayupakaranant p, kaewnopparat n, nitiruangjaras a, reanmongkol w. wound healing activities of standardized pomegranate rind extract and its major antioxidant ellagic acid in rat dermal wounds. j nat med. 2014;68(2):377–86. 31. holland d, hatib k, bar-ya’akov i. pomegranate: botany, horticulture, breeding. horticult rev. 2009;35(2):127–91. 32. chandra r, babu kd, jadhav vt, jaime a, silva t. origin, history and domestication of pomegranate. fruit vegetab cereal sci biotechnol. 2010;2:1–6. 33. bedigian d. pomegranates. ancient roots to modern medicine. medicinal and aromatic plants—industrial profiles 43. econ bot. 2007;61(1):107–8. 34. bhatia a, prakash s. topical phenytoin for wound healing. dermatol online j. 2004;10(1). 35. park je, abrams mj, efron pa, barbul a. excessive nitric oxide impairs wound collagen accumulation. j surg res. 2013;183(1):487–92. 36. portera ca, love ej, memore l, zhang l, mueller a, browder w, et al. effect of macrophage stimulation on collagen biosynthesis in the healing wound. am surg. 1997;63(2):125–31. 37. ågren m, jorgensen i, andersen m, viljanto j, gottrup p. matrix metalloproteinase 9 level predicts optimal collagen deposition during early wound repair in humans. br j surg. 1998;85(1):68–71. 38. spelman k, burns j, nichols d, winters n, ottersberg s, tenborg m. modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. altern med rev. 2006;11(2):128. 39. de jesus nzt, falcão hds, gomes if, leite tjda, lima grdm, barbosa-filho jm, et al. tannins, peptic ulcers and related mechanisms. int j mol sci. 2012;13(3):3203–28. 40. jurenka j. therapeutic applications of pomegranate (punica granatum l.): a review. altern med rev. 2008;13(2). 41. yoshimura m, watanabe y, kasai k, yamakoshi j, koga t. inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation. biosci biotechnol biochem. 2005;69(12):2368–73. 42. patwardhan b. bridging ayurveda with evidence-based scientific approaches in medicine. epma j. 2014;5(1):19. 43. karimi a, majlesi m, rafieian-kopaei m. herbal versus synthetic drugs; beliefs and facts. j nephropharmacol. 2015;4(1):27–30. 44. kim hg, kim jm, han jm, lee js, choi mk, lee ds, et al. chunggan extract, a traditional herbal formula, ameliorated alcohol-induced hepatic injury in rat model. world j gastroenterol. 2014;20(42):15703–14. 45. afaq f, saleem m, krueger cg, reed jd, mukhtar h. anthocyaninand hydrolyzable tannin-rich pomegranate fruit extract modulates mapk and nf-kappab pathways and inhibits skin tumorigenesis in cd-1 mice. int j cancer. 2005;113(3):423–33. 46. wu y, zhu cp, zhang y, li y, sun jr. immunomodulatory and antioxidant effects of pomegranate peel polysaccharides on immunosuppressed mice. int j biol macromol. 2019;137:504-11. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i2.715 234 j contemp med sci | vol. 6, no. 5, september-october 2020: 234–241 original issn 2413-0516 introduction cancer breast is the utmost frequently identified cancer worldwide and is the principal cause of cancer-linked death in women.1 the american cancer society estimates that 268,600 americans will be detected with invasive breast cancer and 41,760 will die in 2019.2 about 15%–25% of breast cancers show her-2 amplification or overexpression (human epidermal growth factor receptor-2).3 every year, at least 276,000 women worldwide receive a recent diagnosis of breast cancer positive for her-2 (1.38 million × 20%), a number that is estimated to increase to 540,000 by 2030 (2.7 million × 20%).4 although most cancer breast patients can be cured of their disease, up to 20% will develop metastatic breast cancer (mbc) and it has consistently been considered an incurable disease.5 her2-positive patients with breast cancer previously associated with an unfavorable prognosis, since it is a more aggressive disease and significantly reduces disease-free survival in localized diseases and reduces overall survival in metastatic diseases compared to their counterparts without overexpression.6 conversely, the outcome of patients with her2-positive breast cancer has significantly improved through the use of therapeutic agents targeting her2, such as trastuzumab (herceptin), pertuzumab, ado-trastuzumab emtansine (t-dm1), and lapatinib altered disease outcomes in this subgroup of cancer.7 an in-depth analysis of the epidemiological strategy and medical economics (esme) database showed a median overall survival of 51.1 months in 2012, compared to 38.7 months in 2008.8 the use of trastuzumab has meaningfully altered the ordinary history of her2-positive mbc, translating it from a historically aggressive disease subtype to a subtype with better prognosis and prolonged survival.9 trastuzumab (herceptin); f. hoffmann-la roche ltd, basel, switzerland), a humanized monoclonal antibody against her2, revolutionized the treatment of her2-positive breast cancer that was prime approved in 1998 in the america and 2000 in europe, for the treatment of mbc with her2-positive. since then, trastuzumab has been documented in more than 125 nations around the world, leading to a new standard of care for her2-positive disease.10 adjuvant treatment with trastuzumab can reduce the relative risk of recurrence by 46%–52% at 2–3.5 years, and trastuzumab + pertuzumab with combinations of chemotherapy have been displayed to have impressive overall survival in metastatic disease.11 although a large percentage of positive her2 mbc patients treated with trastuzumab recurrence after 12–24 months, few patients have prolonged remission.12 treatment guidelines for metastatic her2-positive patients suggest that her2 target agents must be involved in therapeutic regimens of multiple therapy lines, as this has been associated with better patient outcomes.13 trastuzumab treatment has been connected with longterm survival, particularly in patients who have remained progression-free for >2 years of trastuzumab treatment.14 most studies support maintenance treatment with trastuzumab until progression of the disease or at the request of the long-term continuous trastuzumab use for her2-positive advanced breast cancer haider y. shukur najaf cancer clinic (ncc), departments of oncology, faculty of medicine,, ibn hayyan medical university, najaf, iraq. corresponding author: haider y. shukur (e-mail: shukurhaider@yahoo.com) (submitted: 12 april 2020 – revised version received: 28 may 2020 – accepted: 11 june 2020 – published online: 30 october 2020) abstract objectives: trastuzumab is the standard of care for locally advanced/metastatic her2-positive breast cancer. however, most of these patients will progress within 12 months of trastuzumab therapy. in contrast, there is a paucity of data available on the long-term treatment of patients with trastuzumab. our study was conducted to report efficacy and safety data for patients with locally recurrent/metastatic her2-positive breast cancer who received long-term therapy with trastuzumab (≥5 years). methods: this study was a prospective single-arm study of continuous trastuzumab in patients who were histologically her2-positive and radiologically confirmed inoperable locally recurrent or metastatic breast cancer after complete 1 year of trastuzumab plus chemotherapy (in hormone negative/hormone resistance) treatment then continuous trastuzumab alone , or with hormone therapy (in sensitive hormone positive) without progression [complete or partial response or stable disease]. a total of 50 inoperable local recurrent and metastatic breast cancer patients were treated with continuous intravenous trastuzumab therapy administered according to the standard trastuzumab every 3-weeks (8 mg/kg loading dose followed by 3-weekly 6 mg/kg maintenance doses starting 3 weeks after the loading dose) schedule, from january 2014 to january 2019 at the najaf cancer clinic. results: all 50 patients were evaluated with cr occur only in 20% (10/50) with an oar of 50% (25/50). the cardiac status of these patients remained stable over time for the majority of patients with no marked changes in left ventricular ejection fraction%. no treatment-related death was observed. the median os and median pfs is 61 months and 20 months, respectively. conclusion: in her2-positive recurrent and metastatic breast cancer patients, who initially respond to palliative treatment with trastuzumab, continuous trastuzumab can achieve a long-term tumor remission of several years and had significantly improved survival with tolerated and acceptable adverse events. keyword: trastuzumab, breast neoplasms, her2-positive, iraq 235 original long-term continuous trastuzumab use for her2-positive advanced breast cancerhaider y. shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 234–241 patient, and this remains the standard after response to firstline chemotherapy and an anti-her2 agent that in some cases may last many years.15, 16 the ideal trastuzumab duration for patients who obtain a complete response (cr) is still under debate. cardiac toxicity is the most significant limiting factor for the use of trastuzumab, although cardiac events are rare and mostly asymptomatic in trastuzumab studies beyond progression.17 although there is a lack of evidence regarding treatment discontinuation in response cases, some small clinical case series characterized patients with her2-positive breast cancer who attained crs with chemotherapy with trastuzumab-containing regimens, and the patients persisted disease-free without her2 therapy for years.18 patients and methods patient eligibility this was a prospective study involving patients with her2-positive histology and radiologically confirmed as locally inoperable recurrent or mbc after accomplished 1 year of treatment with trastuzumab plus chemotherapy (hormone negative/hormonal resistance) and then continued trastuzumab, or with hormone therapy (in sensitive hormone positive) without progression [complete or partial response (pr) or stable disease] to continuous trastuzumab received from january 2014 to january 2019 at the najaf cancer clinic (ncc) affiliated clinic, jaber ibn hayyan medical university was eligible for inclusion. in all cases, locally inoperable recurrent or metastatic multidisciplinary breast cancer team was diagnosed according to the definitions of the national comprehensive cancer network .her2-positive status was defined in the primary tumor or in metastatic biopsies as immunohistochemistry (ihc) 3+ staining or 2+ ihc and positive in situ hybridization (sish) or fluorescence in situ hybridization (fish, her2 / cep17 ratio> 2.2) according to asco/cap guidelines.19 inclusion criteria 1) who-performance state (who-ps) of 0–2. 2) age from 20 to 75 years. 3) patients with signs of chronic cardiac failure might be included after an investigator risk–benefit assessment. 4) patient with solitary kidney was included. 5) written informed consent has been requested. exclusion criteria 1) over 75 years old. 2) pregnant or lactating women, female of childbearing time unless they use effective contraceptive measures. 3) severe dyspnea requiring supplemental oxygen therapy and uncontrolled severe systemic illness. 4) patients with a simultaneous diagnosis of another none breast cancer. 5) those who have not received first-line trastuzumab. 6) who-ps ≥ 3. 7) abnormal biochemistry (i.e., bilirubin 1.3-uln, alkaline phosphatase 5-uln, ast/alt 5-uln). 8) inadequate bone marrow. pre-treatment evaluation included all patients were assessed at baseline and then every four doses of trastuzumab (every 3 months) using chest x-ray and high-resolution chest abdomen and pelvic computed tomography (hrct) scan, abdominal and pelvic ultrasound. serum cancer antigen (ca15-3) & carcinoembryonic antigen (cea) levels were analyzed for pretreatment as a basis and at interval when evaluating tumor response every 3 months. physical examination and laboratory tests (cbc & biochemistry) were performed each time before treatment. the tumor response was classified into cr, pr, stable disease (sd) and progressive disease (pd) according to the criteria to estimate the response in solid tumors (version 1.1) mentioned by eisenhauer et al.12 adverse events were classified according to the common terminological criteria of the nci for serious adverse events (version 4.0). overall survival (os) was distinct as the time concerning to the start of trastuzumab maintenance & death date. progression-free survival (pfs) was recognized as the time between the start of trastuzumab maintenance and the first progression and/or death. patients censored are those without an event on the last follow-up date (february 1, 2020). for patients with continuous trastuzumab, information was collected at the time of hospitalization. patients withdrawn from the study in case of intolerable toxic effects or evidence of disease progression, or at their request, move to another line or best supportive care accordingly. for those patients with sd or pr, the duration of trastuzumab continuous until study end. treatment schedule patients formerly treated with trastuzumab therapy sustained to receive intravenous trastuzumab therapy ordered according to the trastuzumab standard program every 21 days (8 mg/kg loading dose then continuous by every 21 days 6 mg/kg maintenance doses). trastuzumab therapy was continued until progression of the disease, intolerable toxicity, patient choice, patient death, or study end. administration of trastuzumab may be delayed in the case of cardiac toxicity by following an algorithm constructed on evaluation of the left ventricular ejection fraction (lvef), but no dose adjustments have been allowed. if delayed, trastuzumab is restarted according to the reload guidelines. for patients whose dose was delayed by ≤14 days, a maintenance dose was ordered as soon as possible and consequent maintenance doses were sustained according to primary program. for those whose dose was postponed >14 days, a loading dose of 8 mg/kg was administered, and then maintenance doses were reinitialized and administered continuous by every 21 days. the cbc & other necessary blood tests were performed regularly every 3 weeks before each dose. treatment evaluations during treatment, assessment, including toxicity assessment and response: treatment evaluations for disease progression were performed as clinically indicated and as previously described & the date of disease progression was recorded. toxicity assessments: cardiac status was assessed by echocardiogram every 4 doses (every 3 months) and as clinically directed. heart failure (new york heart association [nyha] class ii iv) was reported as a serious adverse event (sae) and classified according to the criteria of nci-ctcae version 3 or nci-ctc version 2 and the nyha classification. saes have been reported in accordance with the international council 236 original long-term continuous trastuzumab use for her2-positive advanced breast cancer haider y. shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 234–241 for harmonization (ich) guidelines for the management of clinical safety data, definitions and standards for expedited reporting. hematology and biochemistry laboratory evaluations were performed according to standard medical practice. the main objective of the present study was to evaluate the overall survival (os) of patients with locally non-operable recurrent and mbcs her2-positive who had a long-term response with continuous trastuzumab. secondary goals were progression-free survival and safety. statistical analysis the analyzing of the statistics by ibm spss statistics v22.0. numerical information were expressed as median and range as appropriate. qualitative facts had been expressed as a percentage. the survival curves have been estimated the usage of the kaplan–meier technique. results between january 2014 and january 2019, 50 patients were enrolled in our clinic (ncc) and received trastuzumab treatment with a median follow-up of 60 months (range 4–73 months). the basic patient characteristics listed in table 1. the age group was between 20 and 75 years old, with an average age of 52 years, while 7 (14%) were 65 years & older. forty-four (88%) of our patients were women, while only six (12%) were men. fourteen (29.7%) of our patients were still pre-menopaused, while 33 (70.3%) were post-menopausal. overall, the majority of 45 (90%) of our patients had a good who-ps of 0-1 at the start of trastuzumab therapy, while 5 (10%) patients had a who-ps of 2. one-fifth (20%) of our patients were underweight with a bmi (body mass index) ≤18, while more than half (56%) of our patients had a normal bmi and the remaining 12 (24%) had overweight with bmi >25. most of the patients (86%) had ductal histology and 4 (8%) of the lobular type while remaining 3 patients (6%) of other histology. the tumors were generally aggressive, with 30 (60%) patients with grade iii tumors and 37 (74%) with high-level ki-67% staining (>30%). thirty (60%) of our patients were positive hormones, while 20 (40%) were double negative hormones. at the time of disease recurrence or onset of metastatic disease, the minority of 8 (16%) of our patients suffered from inoperable recurrent locoregional disease, while the majority of 42 (84%) were metastasis. mostly bone metastases found in 17 (34%), liver 13 (26%), and lung 9 (18%). brain metastases were present in only three patients (6%). almost one-fifth of patient (20%) had relevant comorbidities, which were mainly hypertension (16% of all patients) or cardiac arrhythmia (4% of all patients). detailed patient characteristics are listed in table 1. baseline tumor marker results including ca15-3, cea are also listed in table 2. forty-one patients (82%) were found with elevated ca15-3 levels before start trastuzumab, but only nine patients (18%) had normal ca15-3. on the other hand, only 11 patients (22%) found with normal baseline cea, but the remaining 39 patients (78%) had elevated cea. the detailed patient characteristics are listed in table 2. treatment delivery in the majority of 30 (60%) patients, trastuzumab was started with continuation of treatment in combination with endocrine therapy, and for the remaining 40% of patients, trastuzumab table 1. patient clinicopathological characteristics. characteristic no. of patients n=50 age -mean (range ) age ≥ 65 no. (%) 52 (20-75) 7 (14) sex no.(%) female male 44 (88) 6 (12) menopausal status-no.(%) pre post 14 (29.7) 33 (70.3) ecog-ps-no.(%) 0 1 2 25 (50) 20 (40) 5 (10) bmi (kg/m2)no.(%) ≤18 18–25 >25 10 (20) 28 (56) 12 (24) histological type idc ilc other 43 (86) 4 (8) 3 (6) grading g1 g2 g3 2 (4) 18 (36) 30 (60) ki-67 status-no. (%) low (5%–15%) intermediate (16%–30%) high (>30%) 4 (8) 9 (18) 37 (74) hormone statusno.(%) er+/pr+ (double positive) er+/prer-/pr+ er-/pr(double negative) 20 (40) 7 (14) 3 (6) 20 (40) her2 statusno.(%) ihc+3 ihc+2 and positive sish ihc+2 and positive fish 38 (76) 9 (18) 3 (6) primary tumour size (t) -no.(%) t0-1 t2 t3 t4 8 (16) 12 (24) 13 (26) 17 (34) primary nodal status (n) -no. (%) n0 n1 n2 n3 10 (20) 7 (14) 15 (30) 18 (36) disease status before initiation of trastuzumab treatment –no. (%) in operable local recurrence 1. chest wall 2. breast 3. axillary ln 4. supraclavicular ln distant metastasis 1. bone 2. liver 3. lung 4. brain 8 (16) 3 (6) 1 (2) 1 (2) 3 (6) 42 (84) 17 (34) 13 (26) 9 (18) 3 (6) relevant comorbidities hypertension cardiac arrhythmia 10 (20%) 8 (16%) 2 (4%) 237 original long-term continuous trastuzumab use for her2-positive advanced breast cancerhaider y. shukur was continued alone. in the case of endocrine therapy in combination with trastuzumab, the majority of patients 23 (46%) received an aromatase inhibitor and 7 (14%) received tamoxifin with and without zoladex. the median duration of trastuzumab treatment in patients with breast cancer was 3.1 years with an interval from 3 months to 6.0 years. the number of trastuzumab cycles ranged from 4 to 97 cycles with a median of 50 cycles. postponement of treatment was present in 17 (34%) patients. the duration of the treatment delay varied from 3 to 10 days with a median of 5 days. only five (10%) patients on delayed treatment require hospitalization and the duration varies from 2 to 7 days with a median of 4 days. at the end of this study, all patients included in this analysis have withdrawn from study treatment. reasons for withdrawal included the occurrence of serious adverse events (n = 5; 10%); disease progression (n = 13; 26%); and treatment refused (n = 2; 4%). other reasons for discontinuation were reported for 28 patients (56%), study closure, and only 2 (4%) patients agreement to discontinue treatment until further disease progression. (table 3). safety lvef% measurements were available for all patients (100%) included in these analyzes. the cardiac status of these patients remained stable over time for 33 (66%) patients without marked changes in lvef% (range 50–71%). twelve (24%) patients showed a reduction in lvef% to less than 50% (range 47–50%), but the remaining five (10%) showed a marked decrease in lvef% <45% and developed cardiac toxicity that was not corrected with treatment support, and/or delayed in trastuzumab, therefore trastuzumab cancelled permanently. nine (18%) of the 50 patients reported skeletal adverse events, but none of them were related to trastuzumab treatment rather than to hormone therapy and menopause. the reported skeletal side-effects were pain, septic arthritis, fibula fracture, rib fracture, and femur fracture, all of which occurred in one patient (2%) each. there were no patients who died during study treatment. efficacy a clinical response to trastuzumab treatment was evaluated every 3 months and was observed in the majority (74%) of patients with 10 (20%) with complete remission, 15 (30%) partial remission, and 12 (24%) stable disease as the greatest response. a remission (complete or partial) was documented after an average period of 6 months from the continuous start of treatment. unfortunately, until the end of the study, disease progression occurred in 13 (26%) patients during continuous trastuzumab treatment with an estimated mean duration of treatment 3.1 years (95%ci: 0.25–6.0 years). pfs was estimated to be 20 months (95% ci: 17.7–22.2 months) and os 61 months (95% ci: 51.4–70.5 months). it has been estimated that 82% of patients remained in remission for 24 months, 50% for 48 months, and 20% for up to 70 months. (table 5, figs 1 and 2). table 2. patient clinicopathological characteristics ,tumor markers ca15-3 & cea , no.=50. more than 1000 u/ml501-1000 u/ml36-500 u/ml0-35u/mltumor marker 8 (16 %)11 (22 %)24 (48 %)7 (14 %)ca15-3[pts. no.(%)] more than 500 ng/ml101-500 ng/ml4-100 ng/ml0-3 ng/mlcea [pts. no.(%)] 5 (10 %) 16 (32 %)23 (46 %)6 (12 %) table 3. reasons for study withdrawal. reason for study withdrawal, n (%) trastuzumab continuous treatment n = 50 serious adverse events 5 (10%) disease progression 13 (26 %) refusal of treatment 2 (4 %) treatment discontinued until further disease progression 2 (4%) study closure 28 (56%) table 4. over all treatment period, no. of cycles, duration of treatment delay and duration of hospitalization. patients no.= 50variable 3.1 years (0.25-6.0)median duration of trastuzumab treatment (years)-range 50 (4-97)median no. of trastuzumab cycles (range) 5 (3-10)median duration of treatment delay in days (range) 6 (12 %)patient with treatment delay no. (%) 5/17 (30 %)patient with delayed treatment and need hospitalization no. (%) 4 (2-7)median duration of hospitalization in days (range) table 5. objective response rate and survival outcome, no. = 50. response no. of patients (%) complete remission(cr) 10 (20 %) partial response (pr ) 15 (30 %) stable disease (sd) 12 (24 %) progressive disease (pd) 13 (26 %) overall response rate (orr) 25 (50 %) clinical benefit rate (cbr) 37 (74 %) median pfs in months (ci) 20 (17.7-22.2) median os in months(ci) 61 (51.4-70.5) 238 original long-term continuous trastuzumab use for her2-positive advanced breast cancer haider y. shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 234–241 factors associated with better orr and cbr pre-menopausal patients at the start of continuous treatment and of normal bmi with a low or intermediate ki-67% index and for the double or one negative hormone with n0–n2 of grade i–ii disease and metastatic single site disease and without brain metastases (table 6). discussion recurrent and mbc have been consistently considered an incurable disease. however, treatment directed against human epidermal growth factor receptor-2 (her-2) significantly improves survival in those patients, and some patients may respond with prolonged complete remission. a consolidated set of clinical trials has led to worldwide approval of trastuzumab as a standard of care for the treatment of patients with locally advanced/metastatic her2-positive breast.21 discontinuation of anti-her2 drugs in recurrent or metastatic settings remains the main unsolved problem, as studies involving 20–25% of patients with long-term response reported discordant data for patients with prolonged survival who had a break from her2-positive therapies.22 in 108 patients with recurrent and metastatic her2-positive breast cancer who received trastuzumab for more than 2 years as first-line treatment, disease progression occurred in 4 of the 27 patients in those stopped trastuzumab.23 the esmo and asco guidelines do not currently recommend discontinuing anti-her2 treatments until after several years of sustained complete remission.24,25 maintenance treatment with trastuzumab until disease progression remains the standard after response to first-line chemotherapy and an anti-her2 agent. the optimal duration of trastuzumab for patients who obtain a complete response is still under debate. cardiac toxicity is the most significant limiting factor for the use of trastuzumab, although cardiac events are rare and mostly asymptomatic in trastuzumab studies beyond progression, heart attack are and often asymptomatic.26 the tolerability of trastuzumab therapy in these patients has also been well established. in september 2016, more than 2.3 million patients were treated with trastuzumab therapy worldwide. however, despite this abundance of clinical experience, there is a paucity of reported data on long-term treatment with trastuzumab. therefore, the results of our study in patients on long-term therapy with trastuzumab are of considerable interest to the medical oncology community, as many physicians have ongoing questions about the long-term treatment of patients with her2-positive breast cancer locally recurrent/metastatic who are being treated with trastuzumab. the results of our study suggest that long-term trastuzumab therapy from clinical practice had an acceptable safety profile and was well tolerated. lvef% was stable in these patients and in total only 17 serious adverse events were reported, 5 of which were heart-related or led to discontinuation of the treatment. the risk of serious cardiac adverse events is a concern with trastuzumab treatment. previous studies have shown a higher incidence of cardiac dysfunction in patients treated with trastuzumab, mainly when trastuzumab treatment was co-administered with chemotherapy drugs and, in particular, in association with anthracycline chemotherapy, in patients with mbc positive for her2.27 other publications investigating long-term treatment with trastuzumab in locally advanced, relapsing or metastatic her2-positive breast cancer have also reported a low risk of serious cardiac adverse events and a continuous response; however, the need remains for additional information on patients with long-term trastuzumab treatment as reported by our study.28 in our study, five (10%) patients had serious adverse cardiac events (marked reduction in lvef % <45% and developed cardiac toxicity) that were not corrected with supportive treatment and/or postponed treatment, therefore, treatment is permanently suspended. however, 33 (66%) patients without marked changes in lvef percentage (range 50–71%). although 12 (24%) patients showed a reduction in lvef% to less than 50% (range 47–50%) received supportive care and hospitalization encouraged them to complete their treatment, these results suggest that long-term therapy, trastuzumab is feasible and without any additional risk to cardiac safety, particularly with continuous patient care and monitoring in accordance to standard practice. long-term tumor remission over several years was achieved in our group of patients who responded to at least 6 months of trastuzumab treatment for local inoperable recurrent or metastatic breast cancer. pfs was 20 months and os 61 months. fig 1. kaplan–meier curve of progression free survival. fig. 2. kaplan–meier curve of overall survival. 239 original long-term continuous trastuzumab use for her2-positive advanced breast cancerhaider y. shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 234–241 we were able to identify patients who were pre-menopausal at the start of continuous treatment and a normal bmi with a low or intermediate ki-67% index and double or one negative hormone with grade i–ii n0–n2 disease and single site metastatic disease without brain metastases associated with better orr and cbr. trastuzumab therapy has increased response rates and survival times in recurrent and metastatic settings.29one of the problems of our study is that it is a small sample study of a single arm without a comparative arm, so we have not been able to draw unequivocal conclusions, but we were only able to describe the parameters that influence long-term remission within a highly selected sample. another limitation of these analyzes is that recruited patients had previously received trastuzumab treatment and therefore may have had a reduced initial risk of cardiac events and other adverse events in this study. table 6. the response rate according to the different variables. variables cr no. (%) pr no.(%) sd no.(%) pd no.(%) menopuasal status and sex premenapuase patients postmenapuase male 3 (6%) 6 (12%) 1 (2%) 9 ( 18 %) 5 (10%) 1 (2%) 7 ( 14 %) 3 (6%) 2 (4%) 3 ( 6 %) 8 (16%) 2 (4%) body mass index (bmi) under weight [low bmi (<18)] normal weight [bm1 (18–< 25)] over weight [high bmi (≥25)] 1 (2%) 7 (14%) 2 (4%) 2 (4%) 11 (22%) 2 (4%) 1 (2%) 5 (10%) 6 (12%) 5 (10%) 4 (8%) 4 (8%) ki67 % index status low (5%–15%) intermediate (16%–30%) high (>30 %) 5 (10 %) 4 (8 %) 1 (2 %) 8 (16 %) 6 (12 %) 2 (4 %) 2 (4 %) 4 (8 %) 7 (14 %) 2 (4 %) 7 (14 %) 4 (8 %) hormonal status er+/pr+ er+/prer-/pr+ er-/pr0 (0%) 2 (4%) 2 (4%) 5 (10%) 3 (6%) 5 (10%) 3 (6%) 4 (8%) 2 (4%) 3 (6%) 4 (8%) 3 (6%) 5 (10%) 4 (8%) 3 (6%) 1 (2%) disease grade grade i (well differentiated) grade ii (moderate differentiated) grade iii (poor differentiated) 5 (10%) 3 (6%) 2 (4%) 3 (6%) 9 (18%) 3 (6%) 5 (10%) 6 (12%) 2 (4%) 3 (6%) 4 (8%) 6 (12%) tumor size t0-1 t2 t3 t4 2 (4%) 2 (4%) 3 (6%) 3 (6%) 2 (4%) 5 (10%) 3 (6%) 6 (12%) 3 (6%) 4 (8%) 5 (10%) 1 (2%) 3 (6%) 4 (8%) 2 (4%) 4 (8%) nodal state n0 n1 n2 n3 5 (10%) 3 (6%) 2 (4%) 0 (0%) 5 (10%) 4 (8%) 4 (8%) 2 (4%) 0 (0%) 3 (6%) 4 (8%) 5 (10%) 1 (2%) 2 (4%) 4 (8%) 6 (12%) local recurrance supraclavicular ln axillary ln breast chest wall 0 (0%) 1 (2%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (2%) 2 (4%) 2 (4%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) 0 (0%) 0 (0%) 0 (0%) metastatic disease bone only liver only lung only brain only bone with viscera multiple viscera 3 (6%) 4 (8%) 2 (4%) 0 (0%) 2 (4%) 0 (0%) 1 (2%) 4 (8%) 3 (6%) 1 (2%) 2 (4%) 1 (2%) 2 (4%) 1 (2%) 1 (2%) 0 (0%) 3 (6%) 2 (4%) 1 (2%) 0 (0%) 1 (2%) 0 (0%) 4 (8%) 9 (18%) decrease ef% to 47-50 % 5 (10%) 4 (8%) 3(6%) 5 (10%) 240 original long-term continuous trastuzumab use for her2-positive advanced breast cancer haider y. shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 234–241 this selection means that this study population may not be representative of all trastuzumab-treated breast cancer patients. therefore, the safety risks reported here may be underestimated compared to the general population, who may need to discontinue trastuzumab treatment early due to serious adverse events or disease worsening. underestimating safety risks may also be the result of underestimation of serious adverse events or failure to measure lvef%, although significant or serious cardiac adverse events or significant decreases in lvef% are unlikely to be lost in our study through long-term use with trastuzumab, as patients were regularly monitored. finally, there is a paucity of additional information, such as serum and tissue biomarkers, on patients who have performed well with trastuzumab therapy. some studies have identified predictive biomarkers that could provide a clue to the choice of patients who will benefit most from long-term maintenance of trastuzumab after a complete response to firstline combination therapy.30 on the other hand, some other studies have not been able to identify any useful parameter to predict long-term results.31 a randomized clinical trial comparing trastuzumab maintenance with only follow-up after a complete response to first-line treatment may be necessary, although the number of patients would be small and the option of discontinuing trastuzumab may not be attractive to at least some patient. conclusion the results of our study suggest that in patients receiving long-term therapy with trastuzumab for her2-positive disease (≥5 years) for patients with inoperable local recurrent/ metastatic breast cancer, treatment with trastuzumab is well tolerated with an acceptable safety that is well tolerated and of good efficacy. hence, i strongly recommended it. it would be useful to do more research and reports on long-term therapy with trastuzumab. although the percentage of patients with recurrent and mbc with long-term tumor remission is low, we provide evidence indicate that positive her-2 patients who initially respond to trastuzumab treatment possible to achieve long-term tumor remission. abbreviation: asco/cap guidelines: american society of clinical oncology /college of american pathologists ast/alt: aspartate transaminase/alanine transaminase cbc : complete blood count cep17: centromere enumeration probe for chromosome 17 esmo: the european society for medical oncology her2: human epidermal growth factor receptor 2 lvef: left ventricular ejection fraction mbc: metastatic breast cancer who-ps: world health. organization performance status acknowledgments i would like to thank my colleague assistant professor thaer wally for his assistance with information collection and statistical calculations also thanks for other sub-staff for considerable aid and patient support and finally thanks for our patients accepted to treatment and remained in close follow-up. conflict of interest there is absolutely no potential conflict of interest, nor any beneficial relation with drug company. ethical clearance taken from our scientific university committee (jaber bin hayyan medical university) references 1. mohammeed h. forouzanfar kjf, allyne m. delossantos, rafael lozano, alan d. lopez, christopher j. l. murray, mohsen naghavi. breast and cervical cancer in 187 countries between 1980 and 2010: a systemic analysis. the lancet 2011;6736:61351-61352. 2. u.s. breast cancer statistics. [available online at:https://www.breastcancer. org/symptoms/understand_bc/statistics.final.pdf,cited 25 december 2019]. 3. mastro ld, lambertini m, bighin c, et al. trastuzumab as first-line therapy in her2-positive metastatic breast cancer patients. expert rev anticancer ther 2012;12:1391–405. 4. balasubramaniam t. proposal for the inclusion of trastuzumab in the who model list of essential medicines for the treatment of her2-positive breast cancer. geneva, switzerland: knowledge ecology international; 2013. [available online at: http:// www.who.int/selection_medicines/ committees/expert/19/applications/trastuzumab2_8_2_a_ad_final.pdf; cited 4 march 2015]. 5. early breast cancer trialists’ collaborative group (ebctcg) (2005) effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. lancet 365(9472):1687–1717. 6. slamon dj, godolphin w, jones la, et al. studies of the her-2/neu protooncogene in human breast and ovarian cancer. science 1989;244:707–12. 7. baselga j, cortés j, kim sb, et al. pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. n engl j med 2012;366:109–19. 8. gobbini e, ezzalfani m, dieras v, delaloge s et al. time trends of overall survival among metastatic breast cancer patients in the real-life esme cohort. eur j cancer.2018;96:17–24. 9. dawood s, broglio k, buzdar au, hortobagyi gn, giordano sh. prognosis of women with metastatic breast cancer by her2 status and trastuzumab treatment: an institutionalbased review. j clin oncol 2010;28:92–8. 10. national comprehensive cancer network: nccn clinical practice guidelines in oncology (nccn guidelines®): breast cancer, v1. 2017. available at: http://www. nccn.org/professionals/physician_gls/pdf/breast. pdf. accessed 04 dec 2017. 11. swain sm, baselga j, kim sb, et al. pertuzumab, trastuzumab, and docetaxel in her2-positive metastatic breast cancer. n engl j med 2015;372:724–34. 12. witzel i, muller v, abenhardt w, et al. long-term tumor remission under trastuzumab treatment for her2 positive metastatic breast cancer—results from the her-os patient registry. bmc cancer 2014;14:806. 13. von minckwitz g, du bois a, schmidt m, et al. trastuzumab beyond progression in human epidermal growth factor receptor 2-positive advanced breast cancer: a german breast group 26/breast international group 03-05 study. j clin oncol 2009;27:1999–2006. 14. witzel i, müller v, abenhardt w, et al. long-term tumor remission under trastuzumab treatment for her2 positive metastatic breast cancer—results from the her-os patient registry. bmc cancer 2014;14:806. 15. cardoso f. 4th eso–esmo international consensus guidelines for advanced breast cancer (abc 4). ann oncol. 2018;29(8):1634–1657. 16. giordano sh, temin s, chandarlapaty s, crews jr, esteva fj, kirshner jj, krop ie, levinson j, lin nu, modi s, patt da, perlmutter j, ramakrishna n, winer ep, davidson ne. systemic therapy for patients with advanced human epidermal growth factor receptor 2-positive breast cancer: asco clinical practice guideline update. j clin oncol 2018;36(26):2736–2740. 17. tripathy d, slamon dj, cobleigh m, et al. safety of treatment of metastatic breast cancer with trastuzumab beyond disease progression. j clin oncol 2004;22:1063–70. 18. viel e, arbion f, barbe c, et al. prolonged complete response after treatment withdrawal in her2-overexpressed, hormone receptor negative breast cancer with liver metastases: the prospect of disappearance of an incurable disease. bmc cancer 2014;14:690. 19. wolff c, hammond me et al. recommendations for human epidermal growth factor receptor 2 testing in breast cancer: american society of 241 original long-term continuous trastuzumab use for her2-positive advanced breast cancerhaider y. shukur clinical oncology/college of american pathologists clinical practice guideline update. j clin oncol. 2013;31(31):3997–4013. 20. eisenhauer ea, therasse p, bogaerts j, schwartz lh, sargent d, ford r, dancey j, arbuck s, gwyther s, mooney m, rubinstein l, shankar l, dodd l, kaplan r, lacombe d, verweij j. new response evaluation criteria in solid tumours: revised recist guideline (version 1.1). eur j cancer. 2009 jan 1;45(2):228-47. 21. national comprehensive cancer network: nccn clinical practice guidelines in oncology (nccn guidelines®): breast cancer, v1. 2017. available at: http://www. nccn.org/professionals/physician_gls/pdf/breast.pdf. accessed 04 dec 2017. 22. daniels b, kiely be, lord sj, houssami n, lu cy, ward rl, pearson sa. longterm survival in trastuzumab-treated patients with her2-positive metastatic breast cancer: realworld outcomes and treatment: patterns in a wholeof-population australian cohort (2001–2016). breast cancer res treat. 2018;171(1):151–159. 23. niikura n, shimomura a, fukatsu y et a. durable complete response in her2-positive breast cancer: a multicenter retrospective analysis. breast cancer res treat. 2018;167:81–87. 24. cardoso f. 4th eso–esmo international consensus guidelines for advanced breast cancer (abc 4). ann oncol. 2018; 29(8):1634–1657. 25. giordano sh, temin s, chandarlapaty s, crews jr, esteva fj, kirshner jj, krop ie, levinson j, lin nu, modi s, patt da, perlmutter j, ramakrishna n, winer ep, davidson ne. systemic therapy for patients with advanced human epidermal growth factor receptor 2-positive breast cancer: asco clinical practice guideline update. j clin oncol. 2018;36(26):2736–2740. 26. tripathy d, slamon dj, cobleigh m, et al. safety of treatment of metastatic breast cancer with trastuzumab beyond disease progression. j clin oncol. 2004;22:1063–70. 27. seidman a, hudis c, pierri mk, shak s, paton v, ashby m, et al. cardiac dysfunction in the trastuzumab clinical trials experience. j clin oncol. 2002;20:1215–21. 28. smichkoska s, lazarova e. long term trastuzumab in metastatic setting of the patients with her2 positive breast cancer. breast can curr res.2016;1:103. 29. kiely be, soon yy, tattersall mh, stockler mr: how long have i got? estimating typical, best-case, and worst-case scenarios for patients starting first-line chemotherapy for metastatic breast cancer: a systematic review of recent randomized trials. j clin oncol 2010;29(4):456–463. 30. montemurro f, prat a, rossi v, et al. potential biomarkers of long-term benefit from single-agent trastuzumab or lapatinib in her2-positive metastatic breast cancer. mol oncol. 2014;8:20–6. 31. yeo b, kotsori k, mohammed k, walsh g, smith ie. long-term outcome of her2 positive metastatic breast cancer patients treated with first-line trastuzumab. breast 2015;24:751–7. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.862 249j contemp med sci | vol. 6, no. 5, september-october 2020: 249–254 original issn 2413-0516 introduction there are two main groups of cells in the mammalian central nervous system, including neurons and glial (neuroepithelial) cells. glial cells cover 90% of the cells of the central nervous system and play a role in protecting and supporting neurons. in addition, these cells are responsible for providing nutrients, oxygen to the nerves and myelin production, and also play a role in maintaining the homeostasis of the nervous system by destroying pathogens and eliminating dead nerve tissue. glial cells include astrocytes, oligodendrocytes, microglia, and ependymas, which are morphologically distinct from each other.1 primary brain tumors are named based on the tissue from which they originate. pathologically, tumors of glial origin are the most common primary neoplasms of the brain. these brain tumors constitute 45% of intracranial tumors.2 according to the who classification, brain tumors are given low-grade (grade 1 and grade 2) or high grade (grade 3 and 4) depending on the cellular activity and the degree of invasiveness of the tumor. people with glioma eventually have surgery. after surgery, other treatments such as radiation and chemotherapy may be appropriate, depending on the degree of the tumor and other aspects. the standard treatment for grade 4 tumors is radiation therapy and anti-cancer drugs (temozolomide). in patients with slow-growing tumors, magnetic resonance imaging (mri) scans are usually done at regular intervals to check tumor growth before treatment.3,4 technological advances in mri imaging have led to the availability of new techniques for characterizing cns tumors. for example, diffusion weight imaging (dwi), perfusion weight imaging (pwi) and mr spectroscopy (mrs) are relatively new methods that are capable of combining morphological and structural information with physiological and biochemical data. these techniques have recently been used to evaluate glial neoplasms, especially in determining their histological grade.5-8 with the current developments, the metabolic information of the study area can be obtained by brief changes in the method of frequency domain versus time domain analysis by mrs.9 evaluation of diagnostic accuracy of the approved tumor mapping protocol in grading of glial tumors nahid sadighi1, sima fallah arzpeyma2*, mohsen izanlou2, mohamad ali oghabian3, mostafa izanlou4, fateme noori5, saeid sadeghi joni2 1advanced diagnostic and interventional radiology research center (adir), tehran university of medical sciences, tehran, iran. 2department of radiology, razi hospital, guilan university of medical sciences, rasht, iran. 3department of medical physics, imam khomeini hospital, tehran university of medical sciences, tehran, iran. 4imam reza hospital, mashhad university of medical sciences, mashhad, iran. 5department of obstetrics and gynecology, alzahra hospital, guilan university of medical sciences, rasht, iran. *corresponding author: sima fallah arzpeyma (email: simaarzpeyma@gmail.com) (submitted: 07 august 2020 – revised version received: 23 august 2020 – accepted: 10 september 2020 – published online: 30 october 2020) abstract objective: current methods of grading glioma have inherent limitations. the current grading reference standard can be inaccurate when the biopsy specimen is not taken from the most malignant area of the tumor or when the tumor is not completely removed. this is a specific problem in glioma due to the spread of tumor penetration. therefore, the aim of this study was to evaluate the diagnostic accuracy of the approved tumor mapping protocol in the grading of glial tumors. methods: this descriptive cross-sectional study was performed on patients aged 2–82 years with glial tumor. patients were referred to the hospital for tumor mapping and underwent imaging with simultaneous methods of mrs & magnetic resonance (mr) perfusion and conventional mri under the supervision of niag group. then, the results of the second evaluation, including the ratios of the desired metabolites and the amount of blood flow, permeability of the target area were compared with the results of pathology. the results were analyzed by spss software version 24. results: in this study, 30 patients were included. sensitivity, specificity, positive and negative predictive value for the determination of highgrade glioma with peripheral/internal relative cerebral blood volume (rcbv) were 100/100%, 100/93%, 93/100% and 100.100%, respectively. sensitivity, specificity, positive and negative predictive value for the diagnosis of glioma by using peripheral/internal rcbv and thresholds of 2.65 and 1.06 were 100/100%, 93/100%, 93/100% and 100/100%, respectively. sensitivity, specificity, positive predictive value and negative predictive value were determined for diagnosis of high-grade glioma tumor using ch + cr / naa cho / cr and cho / naa ratios with detection threshold of 2.97 (93.3%), 3.5 (78.9%,100%, 100%, and 73.3%), and 2.1 (100%). threshold values of 3.5, 2.1 and 2.97 were obtained using cho / cr, ch + cr / naa and cho / naa, respectively, for the detection of high-grade gliomas. the combination of rcbv, cho / cr, ch + cr / naa and cho / naa had sensitivity, specificity, positive and negative predictive value of 67.7%, 80%, 77% and 70.5%, respectively. significant differences in rcbv and cho / cr, cho / naa and naa / cr ratios were observed between lowand highgrade gliomas (p <0.0001). conclusion: pre-operative grading of glioma based on routine mr imaging is often unreliable. as a result, measuring rcbv and cho / cr and cho / naa ratios independently and somewhat together can significantly improve the sensitivity and predictive values of pre-operative glioma grading. keyword: brain tumor, glial tumor, tumor mapping protocol, tumor grading. 250 original evaluation of diagnostic accuracy of the approved tumor mapping protocol nahid sadighi j contemp med sci | vol. 6, no. 5, september-october 2020: 249–254 dynamic susceptibility contrast, and mri are the most commonly used techniques for measuring tumor perfusion, that typically measured by the relative cerebral blood volume (rcbv). the lack of recurrent blood flow and the deposition of contrast material can be evaluated by measuring rcbv, which is violated in high-grade tumors with leakage pattern.10,11 in clinical studies, 95%–100% sensitivity of differentiating high glioma from low glioma grade has been determined by the threshold of 5.1 rcbv or 75.1. although in similar studies, the specificity was relatively low (5.75%–69%),12 part of which could be due to spatial tumor heterogeneity or potential histological sampling bias.13 in addition, a significant increase in rcbv was found in low-grade glioma (transforming) up to 12 months before the new enhancement, suggesting that rcbv could predict malignancy. perfusion quantitative parameters can also be obtained without the use of corrective methods, including percentage signal recovery (psr) and peak height (ph) that are known as independent parameters in differentiating glioma from metastasis and recurrent metastatic disease from radiation necrosis.14 therefore, the aim of this study was to evaluate the diagnostic accuracy of tumor mapping protocol in grading of glial tumors. materials and methods this descriptive cross-sectional study was performed on patients with brain glioma tumors who were referred to imam khomeini hospital in tehran-iran. data were collected using a checklist in which information such as brain metabolites, ratios, perfusion parameters, and histopathological response were recorded. inclusion criteria were: 1. age between 2 and 82 years 2. approval by neurologists and neurosurgeons. exclusion criteria were: 1. having a history of any brain surgery the sample size was calculated according to the sensitivity and specificity of cho / cr and rcbv parameters. to do this, a formula appropriate to the diagnostic accuracy in a community has been used. considering the specificity of 96%, error 0.15 and prevalence 3.0, the minimum sample size was calculated as 24. sample size (n) basedon specificity z l a s sp p= − × × −( ) × 1 2 1 2 2 / / (11−prevalence) za2 3/84 sp 0.96 1-sp 0.04 prev 0.3 1-prev 0.7 l2 0.02074 n 24 procedure in this study, 30 patients were selected in the initial evaluation by neurological colleagues and diagnosed as glial tumors among patients with brain tumors. patients were referred to imam khomeini hospital for tumor mapping and underwent conventional imaging and simultaneous (mr perfusion techniques and mr spectroscopy (mrs) under the supervision of niag in addition to conventional. mrs imaging was performed by svs and mrsi methods with short and medium tes (20–35 milliseconds and 135–144 milliseconds) from at least three points from the active site of the tumor, peripheral parts and surrounding edema. then, integration of cho, cr, mi, lactate, lipid, naa metabolites was performed from the bottom of the diagram. cho / cr, ch / naa ratios of naa / cr, ll / cr, mi / cr and cho + cr / naa (in tumors with severe heterogeneity) were extracted by data analysis software. patients were also subjected to perfusion imaging (pw) with dynamic susceptibility contrast (dsc) method obtained from t2 * w images according to the protocol. after performing technical correction methods, detailed analysis of the obtained data was performed to extract rcbv, psr, ph parameters. preliminary data were collected in the database so that patients were divided into four subgroups (1, 2, 3, 4) from low-grade astrocytoma to glioblastoma, respectively, after surgery based on the results of resected tissue pathology and who criteria. then the patients were divided into two main groups: low grade and high grade equally (15 low-grade tumors and 15 high-grade tumors). data analysis frequency and percentage were used to describe qualitative data and mean and standard deviation were used for quantitative data. roc curve was used to calculate the sensitivity and specificity. to compare the methods, the area under the roc curve (auc) was examined using medcalc software. spss software version 24 was used for data analysis and the significance level in all tests was 0.05. ethical considerations after obtaining the consent of the university ethics committee, all information was collected and kept confidential. the participants in the project adhered to the declaration of helsinki. a complete description of the research objectives and working methods was provided to the officials of the centers and all research units in written and oral form. written consent was obtained from patients to perform this plan. results in this study, 30 patients with inclusion criteria were included in the study. in the high-grade glioma group, 15 patients included 9 patients (30%) with glioblastoma multiforme (gbm), 4 patients (13.3%) with anaplastic astrocytoma and 2 patients (6.6%) with anaplastic oligodendroglioma. there were 15 patients in the low-grade glioma group, including 10 patients (33%) with astrocytoma and 5 patients (17%) with oligoastrocytoma. table 1 shows the metabolic levels of mrs, and pwi (psr and rcbv) in both highand low-grade glioma groups. among patients, peripheral rcbv levels, internal rcbc, cho 251 original evaluation of diagnostic accuracy of the approved tumor mapping protocolnahid sadighi j contemp med sci | vol. 6, no. 5, september-october 2020: 249–254 / cr, ch / naa and cho + cr / naa, ll / cr was significantly higher in the group with high-grade glioma than in the lowgrade glioma (p < 0.05). also, psr, mi / cr levels were significantly lower in the group with high-grade glioma than in the low-grade glioma (p < 0.05). in the current study, the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) for the diagnosis of high-grade glioma tumor using peripheral rcbv with a diagnostic threshold of 1.06 were 100, 93.3, 93.8 and 100%, respectively. sensitivity, specificity, ppv and npv for the diagnosis of high-grade glioma tumor using internal rcbv with a detection threshold of 2.65 were also determined as 100%. additionally, sensitivity, specificity, ppv and npv for the diagnosis of high-grade glioma tumors using the ch / cr ratio with a detection threshold of 3.5 were 73.3, 100, 100, and 78.9%, respectively. our results revealed that sensitivity, specificity, ppv and npv for the diagnosis of high-grade glioma tumors using the ch / naa ratio with a detection threshold of 2.1 were 100%. furthermore, sensitivity, specificity, ppv and npv for the diagnosis of high-grade glioma tumors using the ch + cr / naa ratio with a detection threshold of 2.97 were 93.3%. sensitivity, specificity, ppv and npv for the diagnosis of high-grade glioma tumors using mi / cr ratio with a detection threshold of 0.5 were also found to be 6.7%. sensitivity, specificity, ppv and npv for the diagnosis of high-grade glioma tumors using ll / cr ratio with a detection threshold of 1.5 were 100, 88.2, 88.2, and 100%, respectively. the diagnostic accuracy of psr in differentiating high-grade tumor from low glioma was found to be 0 and lack of sensitivity, specificity, ppv and npv value was also revealed. the diagnostic thresholds of the combination of metabolic level and rcbv for differentiating high-grade from low-grade glioma had sensitivity, specificity, ppv, and npv of 67, 80, 77, and 70.5%, respectively. table 1. metabolic levels of mrs, pwi (psr and rcbv) in two groups of highand low-grade glioma. variable group n mean std. deviation p-value psr low grade 15 93.700 2.3513 <0.001 high grade 15 85.033 2.9488 <0.001 rcbv periphral low grade 15 0.7253 0.20832 <0.001 high grade 15 1.4780 0.28138 <0.001 rcbv internal low grade 15 1.8300 0.30131 <0.001 high grade 15 3.4093 0.27408 <0.001 ch/cr low grade 15 2.5547 0.49129 <0.001 high grade 15 3.5213 0.81321 <0.001 ch/naa low grade 15 1.5513 0.32443 <0.001 high grade 15 2.8060 0.33749 <0.001 ch+cr/naa low grade 15 2.3853 0.38338 <0.001 high grade 15 3.8600 0.44029 <0.001 mi/cr low grade 15 0.6760 0.16084 <0.001 high grade 15 0.2873 0.11585 <0.001 ll/cr low grade 15 0.7167 0.32778 <0.001 high grade 15 2.6093 0.78733 <0.001 252 original evaluation of diagnostic accuracy of the approved tumor mapping protocol nahid sadighi j contemp med sci | vol. 6, no. 5, september-october 2020: 249–254 fig. 1. roc curve for the sensitivity and specificity of choline to creatine ratio in the diagnosis of high-grade glioma. fig. 3. roc curve for the sensitivity and specificity of the ch + cr / naa ratio in the diagnosis of high-grade glioma. fig. 4. roc curve for the sensitivity and specificity of the mi / cr ratio in the diagnosis of high-grade glioma. fig. 5. roc curve for the sensitivity and specificity of the ll / cr ratio in the diagnosis of high-grade glioma. fig. 6. roc curve for sensitivity and specificity of rcbv peripheral ratio in the diagnosis of high-grade glioma. fig. 2. roc curve for the sensitivity and specificity of the ch / naa ratio in the diagnosis of high-grade glioma. 253 original evaluation of diagnostic accuracy of the approved tumor mapping protocolnahid sadighi j contemp med sci | vol. 6, no. 5, september-october 2020: 249–254 discussion current methods of grading glioma have inherent limitations. the current grading reference standard can be inaccurate when the biopsy specimen is not taken from the most malignant area of the tumor or when the tumor is not completely removed. this particular problem in glioma is due to the spread of tumor infiltration, so the aim of this study was to evaluate the diagnostic accuracy of the approved tumor mapping protocol in the grading of glial tumors.15 to date, few systematic attempts have been made to compare the sensitivity, specificity, ppv and npv of mr perfusion and mr spectroscopy with conventional mr imaging in glioma grading. in a study conducted in 2013, 56 patients with a primary diagnosis of glioma underwent mrp, dwi and mrs, where cbv individually showed a sensitivity and specificity of 100% and 88%, respectively, according to a threshold value of 3.34., in the conclusion of this study, rcbv measurement had the most diagnostic performance in grading glioma tumors, but combining all of the imaging parameters play a more accurate role inclassification of glioma tumors.11 in line with mentioned study, rcbv was able to detect high-grade glioma tumors with a diagnostic threshold of 1.06 where sensitivity, specificity, ppv, and npv were determined to be 100, 93.3, 93.8, 100, respectively internal rcbv showed high sensitivity, specificity, ppv and npv for the diagnosis of high-grade glioma tumor with a detection threshold of 2.65 (100%). another study was performed in 2010 on 40 patients with an initial diagnosis of glioma. after obtaining the parameters of two modalities of perfusion mri and spectroscope, they came to the conclusion that the combination of rcbv, cho / cr, cho / naa and mi / cr was associated with specificity of 68%, sensitivity of 95%, pvp of 76% and nvp of 86.7% for grading of glioma tumors that was in line with our study.9 in a 2003 study by zidan on 160 patients with an initial diagnosis of glioma, rcbv was obtained from the region with the highest perfusion and mrs parameters with moderate and short te. their results showed that the combination of cho / naa, cho / cr, rcbv had sensitivity, specificity, npv and ppv of 3.93%, 60%, 87% and 75% in glioma tumor grading, respectively. contrary to our study, this means that the combination of rcbv with cerebral metabolic parameters is more valuable than conventional mri and each of the above methods alone. furthermore, rcbv is of greater diagnostic value in tumor grading among the parameters measured in this study that was in agreement with our findings.16 a meta-analysis of 83 articles in the ncbj database between 2000 and 2013 showed that lgg had clearly less cho / naa, cho / cr, rcbv than mts and hgg, but no significant difference was observed in adc where the best differentiation and grading between hgg and lgg are formed by combining the parameters of cho / naa, cho / cr, and rcbv, but these criteria cannot accurately differentiate between hgg and metastasis.10 in our study, levels of peripheral rcbv, internal rcbc, cho / cr, ch / naa, and ll / cr were significantly higher in the group with high-grade glioma than in those with lowgrade glioma, and psr, mi / cr levels were significantly lower in the high-grade glioma than low-grade glioma. perfusion parameters and spectroscopic concentration ratios in 55 consecutive patients with histopathologically proved lymphomas, glioblastomas, and metastases have been previously evaluated by vallee et al.17. by obtaining rcbv and psr (percentage signal intensity recovery) parameters and brain metabolites and the corresponding ratios of both modalities, it was concluded that psr, cho / cr, cho / naa have 95% sensitivity and 97% specificity in the diagnosis of glioblastoma from metastasis and lymphoma.17 in line with mentioned study, the ch / cr ratio showed a sensitivity, specificity, ppv, and npvs of 78.9, 100,100, and 73.3, respectively, for the diagnosis of high-grade glioma tumors with a detection threshold of 3.5. ch / naa ratio showed sensitivity, specificity, ppv and bpv of 100% for the diagnosis of high-grade glioma tumors with a detection threshold of 2.1. in contrast, the psr lacked diagnostic accuracy. finally, in addition to vascular proliferation, cellularity, mitotic activity, nuclear pleomorphism, and necrosis are important criteria in grading glioma tissue. ki-67 labeling index is used in histological examination as a marker for cell proliferation. higher levels of ki-67-positive cells correspond to more malignancies in gliomas. metabolite ratios, especially fig. 7. roc curve for the sensitivity and specificity of the rcbv internal ratio in the diagnosis of high-grade glioma. fig. 8. roc curve for sensitivity and specificity of psr ratio in the diagnosis of high-grade glioma. 254 original evaluation of diagnostic accuracy of the approved tumor mapping protocol nahid sadighi j contemp med sci | vol. 6, no. 5, september-october 2020: 249–254 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.844 cho levels, are correlated with ki-67 levels in gliomas,18 so mr spectroscopy of cho / cr and cho / naa ratios is useful in grading gliomas. conclusion pre-operative grading of gliomas based on routine mr imaging is often unreliable. as a result, measuring rcbv and cho / cr and cho / naa ratios independently and to some extent together can significantly improve the sensitivity and predictive values of pre-operative glioma grading. references 1. hill c, nixon c, ruehmeier j, wolf l. brain tumors. phys therapy. 2002;82(5):496-502. 2. amirkhah r, naderi-meshkin h, mirahmadi m, allahyari a, sharifi h. cancer statistics in iran: towards finding priority for prevention and treatment. cancer press. 2017;3(2):27-38. 3. porter kr, mccarthy bj, freels s, kim y, davis fg. prevalence estimates for primary brain tumors in the united states by age, gender, behavior, and histology. neuro oncol. 2010;12(6):520-7. 4. inskip pd, hoover rn, devesa ss. brain cancer incidence trends in relation to cellular telephone use in the united states. neuro oncol. 2010;12(11):1147-51. 5. kaminogo m, ishimaru h, morikawa m, ochi m, ushijima r, tani m, et al. diagnostic potential of short echo time mr spectroscopy of gliomas with single-voxel and point-resolved spatially localised proton spectroscopy of brain. neuroradiology. 2001;43(5):353-63. 6. knopp ea, cha s, johnson g, mazumdar a, golfinos jg, zagzag d, et al. glial neoplasms: dynamic contrast-enhanced t2*-weighted mr imaging. radiology. 1999 211(3) 791-8. 7. moller-hartmann w, herminghaus s, krings t, marquardt g, lanfermann h, pilatus u, et al. clinical application of proton magnetic resonance spectroscopy in the diagnosis of intracranial mass lesions. neuroradiology. 2002;44(5):371-81. 8. sugahara t, korogi y, kochi m, ikushima i, shigematu y, hirai t, et al. usefulness of diffusion-weighted mri with echo-planar technique in the evaluation of cellularity in gliomas. j magnetic resonan imaging jmri. 1999;9(1):53-60. 9. hlaihel c, guilloton l, guyotat j, streichenberger n, honnorat j, cotton f. predictive value of multimodality mri using conventional, perfusion, and spectroscopy mr in anaplastic transformation of low-grade oligodendrogliomas. j neuro-oncol. 2010;97(1):73-80. 10. law m, yang s, wang h, babb js, johnson g, cha s, et al. glioma grading: sensitivity, specificity, and predictive values of perfusion mr imaging and proton mr spectroscopic imaging compared with conventional mr imaging. am j neuroradiol. 2003;24(10):1989-98. 11. roy b, gupta rk, maudsley aa, awasthi r, sheriff s, gu m, et al. utility of multiparametric 3-t mri for glioma characterization. neuroradiology. 2013;55(5):603-13. 12. barajas rf, jr., cha s. benefits of dynamic susceptibility-weighted contrastenhanced perfusion mri for glioma diagnosis and therapy. cns oncol. 2014;3(6):407-19. 13. ohgaki h, kleihues p. epidemiology and etiology of gliomas. acta neuropathol. 2005;109(1):93-108. 14. lev mh, ozsunar y, henson jw, rasheed aa, barest gd, harsh gr, et al. glial tumor grading and outcome prediction using dynamic spin-echo mr susceptibility mapping compared with conventional contrast-enhanced mr: confounding effect of elevated rcbv of oligodendrogliomas [corrected]. ajnr am j neuroradiol. 2004; 25(2): 214-21. 15. cha s, knopp ea, johnson g, wetzel sg, litt aw, zagzag d. intracranial mass lesions: dynamic contrast-enhanced susceptibility-weighted echo-planar perfusion mr imaging. radiology. 2002; 223(1):11-29. 16. zidan s, tantawy hi, makia ma. high grade gliomas: the role of dynamic contrast-enhanced susceptibility-weighted perfusion mri and proton mr spectroscopic imaging in differentiating grade iii from grade iv. egypt j radiol nucl med. 2016;47(4):1565-73. 17. vallee a, guillevin c, wager m, delwail v, guillevin r, vallée j-n. added value of spectroscopy to perfusion mri in the differential diagnostic performance of common malignant brain tumors. am j neuroradiol. 2018;39(8):1423-31. 18. shimizu h, kumabe t, shirane r, yoshimoto t. correlation between choline level measured by proton mr spectroscopy and ki-67 labeling index in gliomas. ajnr am j neuroradiol. 2000;21(4):659-65. 85j contemp med sci | vol. 6, no. 2, march–april 2020: 85–87 letter to editor issn 2413-0516 dear editor-in-chief the coronavirus (covid-19) was one of the unpredictable challenges for all countries. the crisis began in china in late 2019 and other countries around the world were immediately affected by the virus.1 one of the negative consequences of crises is the production high volume of accurate and incorrect information to the extent that it becomes difficult for both people and statesmen to control. rothkopf, in 2003 in the washington post newspaper on the sars topic, noted the issue of infodemy, a combination of information and epidemics. in the years that followed, he used this term regarding to the ebola virus and avian and he considered the spread of false news as a threat to the world.2 it seems that what is threatening, apart from the amount of inaccurate news and information, is the consumption and sharing of this news largely and unmanageable. during covid-19 crisis, this concept was strongly expressed; because the specific features of the virus, such as the speed of its transmission and dissemination and also the presence of new technologies, especially social media, have intensified the issue of producing and publishing false news. besides that, in crises such as ebola, the role of modern media, such as social media, was very weak, but today it has become increasingly important because of social media penetration. in addition to infodemy, a new phenomenon has emerged in the covid-19 crisis, which can be called tumor of misinformation consumption and sharing (tmcs). “although, tumor of consumption has previously been linked to phenomena such as fashion and cultural as well as economic pattern”.3 tmcs is introduced by this articles’ authors according to the conditions governing the production and dissemination of information in the coronavirus crisis. tmcs is a situation in which a person willfully and unwittingly engages in the search and dissemination of inaccurate news and information, and even other private information; so that the person has no control over his or her situation and inflicts serious material and non material damages on him and other people. in addition, this misinformation sharing behavior can also be attributed to covid-19 related misinformation sharing (crms). this article first describes the characteristics of these individuals and then discusses the role of different professions including medical librarians in solving this problem. characteristics of people involved in tmcs people with tmcs choose and share inaccurate news and information. this will be a worrying process if neglected. according to the authors of this article, these individuals have certain characteristics that need to be addressed by health professionals and researchers: 1. spending too much time for consuming and sharing inaccurate information; 2. select fake news quickly and maximize its sharing; 3. superficial handling of incoming messages; 4. inattention to the adverse effects of that information on themselves and society; 5. affected by phenomena such as information obesity and information addiction; 6. the diversity and multiplicity of used media to consume and share information; 7. injecting stress and anxiety into society; 8. cyberchondria experience (uncontrolled and excessive health anxiety); 9. suspicion on other people; 10. critical thinking at a relatively low level; 11. being infected by nomophobia (fear of losing or keeping away from cell phones); 12. low-level emotional intelligence due to lack of self-management and self-control; 13. ignoring people’s privacy by sharing their private information; 14. increasing aggression and isolation among them; 15. interest in being seen by others (people or power holders). treatment of people involved in tmcs it sounds that people involved in tmcs have taken this path, voluntarily and involuntarily. therefore, in order to treat this phenomenon, education is needed in the first stage. some people do not have complete perception of this kind of their behavior. for example they do not know that incorrect information can sometimes lead to human death. in the covid-19 crisis, some people turned to alcohol consume, and this was due to inaccurate news and information being exchanged between people about the benefits of alcohol in prevention and treatment. in fact, the misconception that alcohol can tumor of misinformation consumption and sharing among people in coronavirus (covid-19) crisis; a commentary hasan ashrafi-rizi1, zahra kazempour2 1medical library and information science department, health information technology research center, isfahan university of medical sciences, isfahan, iran. 2library and information science department, faculty of media, payame noor university, tehran, iran. corresponding author: hasan ashrafi-rizi (e-mail: hassanashrafi@mng.mui.ac.ir) (submitted: 04 february 2020 – revised version received: 07 february 2020 – accepted: 19 february 2020 – published online: 26 april 2020) keywords tumor of misinformation consumption and sharing (tmcs), coronavirus (covid-19), misinformation, health media literacy, health information literacy, health literacy 86 letter to editor tumor of misinformation consumption and sharing among people in coronavirus hasan ashrafi-rizi and zahra kazempour j contemp med sci | vol. 6, no. 2, march–april 2020: 85–87 save you from covid-19 or cure you. therefore, these people should receive targeted education. it is the task of the mass media to reduce these misleading and misconceptions ideas in society by inviting health experts. in addition, educating people about health media literacy can also be very important. people should be taught how to distinguish between fake news and true health news. media specialists, cultural management professionals, and medical librarians can handle this task. since medical librarians equipped with the necessary knowledge and awareness about the infrastructures of production and dissemination of health information as well as their familiarity with the typology of information can play an essential role in teaching proper health information seeking behavior when facing with the crises. educating people by health information literacy, health literacy, media health literacy, and training appropriate behavior during the crisis are other actions that medical librarians should consider about the prevention. it should also be said that educating people to identify valid health information from invalid information will be the most important action of medical librarians in the face of crises. besides that, they should be able to identify valid health messages through reliable health-related organizations such as the world health organization and share them through new technologies and appropriate to user characteristics (gender, age, culture,). experiencing this crisis should enhance medical librarians’ intention to be more active and effective in future disasters. in addition, media ethics need to be re-educated to both the public and media stakeholders. “the mass media must know that their mission is to enlighten public opinion and raise people’s knowledge,”4 and violation of that law includes social (pressure and reaction from public opinion) and legal prosecution. people also need to know that their rights in areas such as preserving chastity and public morality, fairness, and impartiality observation in the media, the right to maintaining the persons respect and the prohibition of defamation and insult, mental health observation (avoid violence), the right to media responsiveness, the right to respect religious beliefs, the right to sue the media for wrongdoing and litigation, the right to access correct information and news, and prohibition of false news should be preserved by medias.3 these media can be both mass media and social media. another thing to note is social media. in fact, part of dissemination of inaccurate information is through social media. since the production and sharing of information on social media is less controlled, it should be monitored more by the individual user. therefore, self-care education should take place in different dimension of social, cultural, and social media contexts. “people must ask themselves a few basic questions before using the media message, especially social media: (1) who is the producer and distributor of this information? (knowing the author of the message), (2); what creative technique has been used to attract the attention of the audience? (find structure) (3); to what extent different people may have different perceptions of this message? (audience review); (4) why is this message sent? (ultimate goal of this message); (5) what values, lifestyles, and perspectives will be represented or eliminated in this message? (content review).”5 in addition, apart from educating people in order to promote correct behavior patterns in society in various dimensions, it is needed to make strict rules on some antisocial behaviors, including fake news. in fact, the main pillars of an advanced society are education and culture, and the second is the adoption of rational and ethical laws and adherence to them. conclusion covid-19 will not be the first crisis and the last one in the world. what is important is how people and statesman deal with this crisis. the position of society in terms of hardware (facilities and equipment) and software (knowledge and awareness, policy, ethical, and logical rules), determines the extent of crisis management. the covid-19 crisis in the information and communications dimension created a new phenomenon called “tmcs” or “crms” and it also expanded sharply. some reasons for this phenomenon, apart from the features of covid-19, is people’s staying at home and having no schedule. symptoms of an individual affected by this phenomenon, is spending a lot of time on social media, and the sharing of false news extremely which is very destructive to the mental health of both herself and others. however, disseminating inaccurate news also reduces the proper performance of governments in doing duties. what is harmful in this crisis and similar ones besides the crisis itself, is false news which frustrates people and even make distrust against the government. diagnosing, treating, and rehabilitating people with this immoral behavior depends on training the health information literacy, health literacy, and media health literacy, which medical librarians can fulfill this critical task as before. in addition, identifying valid health messages and sharing them with the general public is another task of medical librarians. likewise, the establishment of rational and ethical laws in that community about the production and sharing of information would be another solution to this problem. according to the authors’ opinion of this article, these individuals have certain characteristics that need to be scientifically investigated by health professionals and researchers, while paying special attention to them in codification or health affair. medical librarians should also conduct effective research and practice appropriate to their discipline. acknowledgment authors wish to thank the staffs of health information technology research center, university of medical sciences, isfahan, iran. authors’ contribution h. a. conceived the original idea, designed the scenarios and collected the data. h. a. and h. a., z. k carried out the analysis of data, approved the final version that was submitted, revised it. h. a. and z. k drafted the manuscript. h. a. and z. k. met the criteria of authorship based on the recommendations of the international committee of medical journal editors. conflict of interest the authors have declared that no competing interests exist. funding and support this research resulted from an independent research without financial support. 87 letter to editor tumor of misinformation consumption and sharing among people in coronavirushasan ashrafi-rizi and zahra kazempour j contemp med sci | vol. 6, no. 2, march–april 2020: 85–87 references 1. ashrafi-rizi h, kazempour z. information typology in coronavirus (covid-19) crisis; a commentary. arch acad emerg med. 2020;8(1):19. 2. tervahauta m. comment: when the world gets infected with the virus, a dangerous infodem also spreads in: juonala j, editor. news conference in helsinki 2020. 3. daneshvar m, moazen a, hoassaini b, amirvaghefi m, haghpanah h. thought, median literacy. tehran: ministry of education; 2016. 171 p. 4. esmaeili m. the press law. tehran: soroush press; 2006. 112 p. 5. toloei a. media literacy: introduction on learning and assesment. tehran: ministry of culture and islamic guidance; 2013. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i2.748 90 j contemp med sci | vol. 5, no. 2, march–april 2019: 90–95 original histological impact of nutritional style alteration in mice khalid m. salih,a jamela jouda,a* shatha salah asad,b yusur falah faraj,c iyden kamil mohammed,d and ali sameer abudlghani altaeea adepartment of biology, college of science, mustansiriyah university, baghdad, iraq. bal-kindy college of medicine, baghdad university, baghdad, iraq. cnational center of hematology, mustansiriyah university, baghdad, iraq. ddepartment of biomedical, college of al-khawarzmy engineering, baghdad university, baghdad, iraq. *correspondence to jamela jouda (email: jamela.jouda@uomustansiriyah.edu.iq). (submitted: 08 november 2018 – revised version received: 19 december 2018 – accepted: 22 january 2019 – published online: 26 april 2019) objectives it is well established that diet and lifestyle are important in the maintenance of health. transition from a plant-based diet mostly to a high-calorie diet of animal products might raise the chronic diseases which is called “degenerative”. this work aimed to study the histopathological effect of transition from complete plant-based diet to 10% animal products (sheep’s brain) on various body organs of mice. methods eight-week-old balb/c male mice were divided into two groups (n = 8); the first is restricted group in which mice were fed on restricted diet containing 10% of sheep’s brain homogenate, while the second is the control group in which fed on ad libitum on the diet for 7 days. during the duration of experiment, body weight and the amount of food intake were recorded daily, then at the end of experiment, all mice were sacrificed and various organs were obtained and processed for histopathological study. results the results showed that food intake by each mouse of restricted group are significantly lower than in control group. although the mean of body weight in both groups revealed non-significant difference, the relative weight of various organs showed significant differences. on the other hand, sever histological changes were detected in all studied organs sections of restricted group. conclusion it can be concluded that changing in nutritional style rather than conventional diet play a crucial role in modifying the architectural aspects of different organs at tissue level. therefore, these findings need further investigation at cellular, physiological, and molecular levels. keywords nutritional style, conventional diet, vegetarian diets introduction it is well established that the diet and lifestyle are important in the maintenance of health.1 a good nutritional status maintaining has important implications for health and well-being, leading to delay and reduced risk of disease, maintaining functional autonomy and thus promoting the continuation of independent living.2 improper nutrition may play a role in the progressive degradation of many body functions3 and caused diseases. diabetes causes by nutrient sparse foods which contained concentrated sugars or refined flour products that contribute to impaired glucose metabolism4 while cancer risk causes by the low fiber intake,5,6 consumption of red meat,7,8 and imbalance of omega-3 and omega-6 fats.9 colorectal cancer development, a major global health concern, is affected by various lifestyle factors and red meat consumption. the processed meat has been associated with increased risk for this cancer.10–12 during human evaluation, the changes in availability of food and composition of dietary have created strong selective pressures on multiple biological processes such as identifying the genetic loci and biological pathways for common diseases.13 vegetarian diets for cats and dogs have health benefits range,14 while the transition from a plant-based diet mostly to a high-calorie diet of animal products; which is high in saturated fats, salt, and sugar, and low in fiber; has been identified as a notable contributor to the rise in chronic diseases which is called “degenerative” diseases such as cardiovascular disease, cancer, obesity and diabetes.15,16 this study aimed to study the effect of transition from complete plant-based diet to 10% animal products (sheep’s brain), contain high fat, on various peripheral body organs of mice. materials and methods eight-week old balb/c male mice were obtained from preventive research center, baghdad, iraq. after 1 week for adaptation on local condition, they are divided into two groups. one group is restricted group (r) which consumed 10% sheep brain, and second group consumed the normal diet ad libitum and served as control group (c). every group contains eight mice. the weight of consumed feed and the weight of body mice were measured. the mice were scarified by cervical dislocation after 7 days. spleen, kidney, liver, heart, small intestine, testes were obtained extracted. after weight recording, these organs were put in 10% formalin solution and processed by standard procedures. sections of paraffin-embedded tissues were stained with hematoxylin and eosin and examined by light microscopy.17 all experiments are carried out in department of biology, college of science, mustansiriyah university. results are expressed as mean ± standard deviation (m ± sd) or mean ± standard error (m ± se). data were anaslyzed by one-way analysis of variance8 followed by fisher’s test for multiple comparisons, using statview version 5.0. differences considered significant at p-value <0.05. issn 2413-0516 k.m salih et al. 91j contemp med sci | vol. 5, no. 2, march–april 2019: 90–95 original histological impact of nutritional style alteration in mice results the food intake by each mouse in restricted group (r) decreased from 2.8 g in the first day down to 0.8 g at the seventh day and with an average of (1.4 ± 0.4 g/day). this result is significantly different from control group 2.9, 3.8 g, 3.0 ± 0.6 g/day respectively as shown in fig. 1. the body and organs weight of mice in both groups showed no significant differences between them except the weight of heart in restricted group (0.250 ± 0.03 g) was significantly higher compared with that in control group (0.206 ± 0.01 g) as shown in table 1. however, calculation of the relative change% in the organ weight of restricted mice in respect to their corresponding organs in control group showed significant decreasing in (spleen, liver, and testis) and increasing in (kidney and heart) as shown in fig. 2. concerning with the histological study of the internal organs in both groups, the results demonstrated several pathological changes in the liver (fig. 3), kidney (fig. 4), spleen (fig. 5), intestine (fig. 6), testis (fig. 7), and heart (fig. 8) of restricted mice. discussion it is well established that most biological membranes have higher proteins/lipids ratio while the membrane of neurons in cns and pns is characterized by a high proportion of lipid and low of protein.18 jennifer et al.19 reported that the myelin had about 78–41% lipid containing of dry weight, white matter had about 49–66% and gray matter had about 36–40%. many studies proved that the amount of fatty in tissues and biofluid are highly valuable in detecting disorderly regulation in biochemical pathways. the circulating fat is derived from both diet and internal metabolism. these fats are interactive biological molecules have highly dynamic. these form most cellular components and signal molecules, dictate energy and control the intake of food.20 eating fatty foods may lead to indigestion and cause gastrointestinal distress because the rate of inhibition of gastric emptying by the inhibitory receptors is most likely found in the duodenum and proximal jejunum. studies conducted by hunt and stubbs (1975)19 indicated that dietary density (kilocalories per milliliter) determines the rate of stomach emptying. this inhibitory activity is mediated by the neural and hormonal mechanisms and their interactions.21 all these evidences can explain the lower food intake in treated group but without significant difference in body weight of this study. this reduction in food intake may be due to disruption in digestion or absorption processes, or negative neural reflexes, or even immunological response in the gut because our results demonstrated infiltration of inflammatory cells in lamina propria of villi with high mucin secretion in the lumen of the intestine. the relationship between liver disease and specific foods is not well understood.22 some researchers suggest that the insulin resistance and an increase in fatty acids after ingestion high fat and sugar-diet contribute to livers inflammation and irreversible scarring.23–25 the various pathological changes in the liver of restricted mice demonstrated in this study agree with several studies that have reported that over-consumption of carbohydrates especially refined carbohydrates, fats (particularly saturated fats), and the protein of meat can cause liver disease.26–28 in compatible with our result concerning with testicular changes, several studies confirmed that environmental factors, diseases and various nutrients may affect the composition of sperm directly or indirectly, thus consuming of high-fat diet by male mice alters global methylation and microrna content in their mature sperm, and transcriptional profiles in their testes.29 another study demonstrated decreasing in the testis/ body weight, seminiferous tubule diameter, spermatogenetic cells and interstitial cells.30 furthermore, the seminiferous tubule diameter also decreased in high-fat diet group which could be related to a disturbance of spermatogenesis.31 there is growing evidence suggest that rich-fat-diet intake affects the heart failure development and discontinuation. table 1. body and organs weight in control and restricted groups groups the absolute weight (in g) (m ± sd) body spleen kidney liver heart testis control 28.34 ± 2.1 0.286 ± 0.03 0.246 ± 0.02 1.83 ± 0.16 0.206 ± 0.01 0.105 ± 0.0008 restricted 29.95 ± 2.8 0.229 ± 0.04 0.251 ± 0.03 1.50 ± 0.23 0.25 ± 0.03* 0.082 ± 0.01 *significant difference at p < 0.05. fig. 1 food intake in control and restricted groups. fig. 2 relative change% of organs in restricted from control group 92 j contemp med sci | vol. 5, no. 2, march–april 2019: 90–95 histological impact of nutritional style alteration in mice original k.m salih et al. fig. 5 pathohistological section in spleen of control group (a), and restricted group (b and c). (a) normal spleen shows no clear lesions. (b) this shows moderate depletion of white pulp. (c) this shows severe congested of red pulp. a b c fig. 3 pathohistological section in liver of control (a) and restricted (b–d) group. (a) histopathological section in the liver of normal animal shows no clear lesions. (b) inflammatory cells particularly neutrophils and mononuclear cells aggregate in liver parenchyma « with megakaryocytes  in the sinusoids. (c) inflammatory cells particularly neutrophils and mononuclear cells aggregate around congested blood vessels with vacuolar degeneration of hepatocytes. (d) this shows dilatation of sinusoids, necrosis, and vacuolar degeneration in the hepatocytes. b dc a fig. 4 pathohistological section in kidney of control group (a), and restricted group (b and c). (a) histopathological section in the kidney of control group shows no clear lesions. (b) histopathological section in the kidney of treated group shows acute cellular degeneration characterized by vacuolation of epithelial cells. ba k.m salih et al. 93j contemp med sci | vol. 5, no. 2, march–april 2019: 90–95 original histological impact of nutritional style alteration in mice fig. 8 pathohistological section in heart of control group (a), and restricted group (b and c). (a) histopathological section in the heart of control group shows no clear lesions. (b) this shows mononuclear cells infiltration in the pericardium. (c) this shows fatty changes in cardiac muscle with edema between cardiac muscle. a b c fig. 7 pathohistological section in testis of control group (a), and restricted group (b and c). (a) histopathological section in the testis of control group shows normal structure. (b) this shows complete disappearance of epithelial layer and only single cell lining of basement membrane of seminiferous tubules. (c) this shows incomplete spermatogenesis. a b c fig. 6 pathohistological section in intestine of control group (a), and restricted group (b and c). (a) histopathological section in the intestine of normal animal shows normal structure. (b) this shows mild mononuclear cells infiltration in lamina propria with mucin secretion. (c) this shows inflammatory cells between villi and lamina propria with mucin in their lumin. a b c 94 j contemp med sci | vol. 5, no. 2, march–april 2019: 90–95 histological impact of nutritional style alteration in mice original k.m salih et al. studies in rodents show that in the absence of obesity, the fat replacement of refined carbohydrates can weaken or inhibit ventricular expansion and contractile dysfunction in response to high blood pressure, myocardial infarction or myocardial genetic.32 moreover, sahraoui et al.33 showed that a relative short period of high fat diet results in sever alterations of cardiac structure. in our results, many cardiac alterations detected such as mononuclear cells infiltration in the pericardium and fatty changes in cardiac muscle with edema between cardiac muscle, so we can suggest that edema is responsible for the increase in the weight of heart. on the other hand, the result of our study demonstrated an increase in the weight of kidney that is compatible with those obtained by other studies. for instances, altunkaynak et al.34 found that fatty diet-consuming animals may lead to increase in the volume of the kidneys and renal deformities due to edema resulted from mononuclear cell infiltrations among the renal tubules.34 in concern with spleen, our results found moderate depletion in white pulp in agreement with other studies. many researchers found that rodents fed with high fat-diet (fish oil) caused many immunological and histological effects in the white pulp of spleen.35–37 a diet rich in saturated fatty acids is thought to stimulate low-grade chronic inflammation, and may contribute to increased serum inflammatory mediators levels.38 the exact mechanism of negative effect of fat diet on white pulp is still not clear.39,40 however, sahraoui et al.33 suggested that the sort period of high fat diet resulting activation not only of inflammatory but also apoptotic processes.33 all these evidences may explain the inflammatory responses demonstrated in this study after feeding mice 10% brain sheep saturated with lipid. conflicts of interest none.  references 1. leslie w, hankey c. aging, nutritional status and health. healthcare (basel). 2015;3:648–658. 2. united nations. world population aging report. 2013. accessed on 27 july, 2015. 3. ahmed fe. effect of nutrition on the health of the elderly. j am diet assoc. 1992;92:1102–1108. 4. foster-powell k, holt sh, brand-miller jc. international table of glycemic index and glycemic load values: 2002. am j clin nutr. 2002;76:5–56. 5. holmes md, liu s, hankinson se, colditz ga, hunter dj, willett wc. dietary carbohydrates, fiber, and breast cancer risk. am j epidemiol. 2004;159: 732–739. 6. slattery ml, curtin kp, edwards sl, schaffer dm. plant foods, fiber, and rectal cancer. am j clin nutr. 2004;79:274–281. 7. slattery ml, boucher km, caan bj, potter jd, ma kn. eating patterns and risk of colon cancer. am j epidemiol. 1998;148:4–16. 8. norat t, lukanova a, ferrari p, riboli e. meat consumption and colorectal cancer risk: dose-response meta-analysis of epidemiological studies. int j cancer. 2002;98:241–256. 9. london sj, sacks fm, stampfer mj, henderson ic, maclure m, tomita a, et al. fatty acid composition of the subcutaneous adipose tissue and risk of proliferative benign breast disease and breast cancer. j natl cancer inst. 1993;85:785–793. 10. norat t, bingham s, ferrari p, slimani n, jenab m, mazuir m, et al. meat, fish, and colorectal cancer risk: the european prospective investigation into cancer and nutrition. j natl cancer inst. 2005;97:906–916. 11. chan ds, lau r, aune d, vieira r, greenwood dc, kampman e, et al. red and processed meat and colorectal cancer incidence: meta-analysis of prospective studies. plos one. 2011;6:e20456. 12. cross aj, ferrucci lm, risch a, graubard bi, ward mh, park y, et al. a large prospective study of meat consumption and colorectal cancer risk: an investigation of potential mechanisms underlying this association. cancer res. 2010;70:2406–2414. 13. luca f, perry gh, di rienzo a. evolutionary adaptations to dietary changes. annu rev nutr. 2010;30:291–314. 14. knight a, leitsberger m. vegetarian versus meat-based diets for companion animals. animals (basel). 2016;6. pii: e57. 15. popkin bm, du s. dynamics of the nutrition transition toward the animal foods sector in china and its implications: a worried perspective. j nutr. 2003;133:3898s–3906s. 16. walker p, rhubart-berg p, mckenzie s, kelling k, lawrence rs. public health implications of meat production and consumption. public health nutr. 2005;8:348–356. 17. drury rav, wallington ea, cameron r. carleton’s histological technique. 4th ed.; oxford university press: new york, 1967. 18. morell p, quarles rh. characteristic composition of myelin. in: siegel gj, agranoff bw, albers rw, editors. basic neurochemistry: molecular, cellular and medical aspects. lippincott-raven: philadelphia, 1999. 19. jennifer t, smilowitz j, german b, zivkovic am. food intake and obesity: the case of fat. in: montmayeur jp, le coutre j, editors. fat detection: taste, texture, and post ingestive effects. boca raton crc press/taylor & francis, 2010. 20. hunt jn, stubbs df. the volume and energy content of meals as determinants of gastric emptying. the journal of physiology. 1975 feb 1;245:209-25. 21. walker hk, hall wd, hurs-t jw. clinical methods: the history, physical, and laboratory examinations. 3rd ed.; butterworths: boston, 1990. 22. dasarathy s. nutrition and alcoholic liver disease: effects of alcoholism on nutrition, effects of nutrition on alcoholic liver disease, and nutritional therapies for alcoholic liver disease. clin liver dis. 2016;20:535–550. 23. ahmed m. non-alcoholic fatty liver disease in 2015. world j hepatol. 2015;7:1450–1459. 24. neuman mg, cohen lb, nanau rm. biomarkers in nonalcoholic fatty liver disease. can j gastroenterol hepatol. 2014;28:607–618. 25. bashiardes s, shapiro h, rozin s, shibolet o, elinav e. non-alcoholic fatty liver and the gut microbiota. mol metab. 2016;5:782–794. 26. zelber-sagi s, nitzan-kaluski d, goldsmith r, webb m, blendis l, halpern z, et al. long term nutritional intake and the risk for non-alcoholic fatty liver disease (nafld): a population based study. j hepatol. 2007;47:711–717. 27. agius l. high-carbohydrate diets induce hepatic insulin resistance to protect the liver from substrate overload. biochem pharmacol. 2013;85: 306–312. 28. colak y, tuncer i, senates e, ozturk o, doganay l, yilmaz y. nonalcoholic fatty liver disease: a nutritional approach. metab syndr relat disord. 2012;10:161–166. 29. soubry a, hoyo c, jirtle rl, murphy sk. a paternal environmental legacy: evidence for epigenetic inheritance through the male germ line. bioessays. 2014;36:359–371. 30. mu y, yan wj, yin tl, yang j. curcumin ameliorates high-fat diet-induced spermatogenesis dysfunction. mol med rep. 2016;14:3588–3594. 31. sohrabi m, hosseini m, inan s, alizadeh z, vahabian m, vahidinia aa, et al. effect of antioxidants on testicular inos and enos after high-fat diet in rat. pak j biol sci. 2017;20:289–297. 32. stanley wc, dabkowski er, ribeiro rf, jr., o‘connell ka. dietary fat and heart failure: moving from lipotoxicity to lipoprotection. circ res. 2012;110: 764–776. 33. sahraoui a, dewachter c, de medina g, naeije r, aouichat bouguerra s, dewachter l. myocardial structural and biological anomalies induced by high fat diet in psammomys obesus gerbils. plos one. 2016;11:e0148117. 34. altunkaynak me, ozbek e, altunkaynak bz, can i, unal d, unal b. the effects of high-fat diet on the renal structure and morphometric parametric of kidneys in rats. j anat. 2008;212:845–852. 35. fan yy, mcmurray dn, ly lh, chapkin rs. dietary (n-3) polyunsaturated fatty acids remodel mouse t-cell lipid rafts. j nutr. 2003;133:1913–1920. k.m salih et al. 95j contemp med sci | vol. 5, no. 2, march–april 2019: 90–95 original histological impact of nutritional style alteration in mice 36. svahn sl, varemo l, gabrielsson bg, peris e, nookaew i, grahnemo l, et al. six tissue transcriptomics reveals specific immune suppression in spleen by dietary polyunsaturated fatty acids. plos one. 2016;11:e0155099. 37. teague h, fhaner cj, harris m, duriancik dm, reid ge, shaikh sr. n-3 pufas enhance the frequency of murine b-cell subsets and restore the impairment of antibody production to a t-independent antigen in obesity. j lipid res. 2013;54:3130–3138. 38. lee jy, sohn kh, rhee sh, hwang d. saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through toll-like receptor 4. j biol chem. 2001;276:16683–16689. 39. cesta mf. normal structure, function, and histology of the spleen. toxicol pathol. 2006;34:455–465. 40. federico a, d‘aiuto e, borriello f, barra g, gravina ag, romano m, et al. fat: a matter of disturbance for the immune system. world j gastroenterol. 2010;16:4762–4772. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.04201905 26 j contemp med sci | vol. 6, no. 1, january–february 2020: 26–31 original issn 2413-0516 introduction cystatin-c (cy/c) is an innovative biomarker of renal function, which seems to share even strong correlation with mortality and vascular risk over serum creatinine or estimated-glomerular filtration rate (egfr) in aging and admitted acute coronary syndrome (acs) subjects. whether a high risk of coronary–vascular diseases related to elevated cyc is due to amplified ischemic load, raised risk of embolism, or another pathway is uncertain.1–3 cy/c is formed and released by cardiac cells, where its production is raised up during myocardial necrosis.1 three clinical subtypes of acs, involving unstable angina, and st and non-st segment elevation myocardial infarction (mi), are primary causes of mortality and debility universally.2 the incremental role of cy/c beyond conventional renal and cardiac markers leftovers moderately discovered.4 cy/c is not influenced by age, sex, race, and lean body mass and may be more suitable for sensing mild–moderate changes in egfr.5 a closer look at the recent data indicates that higher values of cy/c in patients with acs are associated with raised risk for cardiovascular events and death independent of renal function.6 this study is designed to inspect the circulating levels of cy/c in patients with acs and to evaluate the association of cy/c levels with ejection fraction and an angiographic number of stenosed coronaries. materials and methods this is a single-center study, patients were recruited from shahid al-mihrab center for interventional cardiology; all had prearranged admission for diagnostic or therapeutic catheterization. study participants patients were diagnosed as pure acs (n = 136) only without other cardiac pathologies by a cardiologist and referred for further interventional catheterization. sex and age wellmatched healthy controls (n = 94) were selected as being free of any cardiological disabilities or any renal impairment. body mass index (bmi), smoking habits, and history of diabetes mellitus and hypertension were also included in the study. description of acs entire acs patients met the diagnostic standards established by the 2014 american heart association (aha)/american college of cardiology (acc) guidelines for managing non-stsegment elevation acss and the 2013 acc foundation/aha guideline for the management of st-segment elevation mi.1 quartiles of cystatin-c in study subjects we classified acs patients into quartiles based on their blood cy/c levels; <0.9, 0.9–1.49, 1.5–1.99, and >2.0 mg/l.1 the cystatin-c in patients with acute coronary syndrome: correlation with ventricular dysfunction, and affected coronary vessels dhulfiqar ali abed,a raad jasim,b,c hayder abdul-amir al-hindy,d ammar waheeb obaide adepartment of medicinal chemistry, college of pharmacy, university of babylon, babylon, iraq. bpharmacology and therapeutics, monash institute of pharmaceutical sciences, faculty of pharmacy and pharmaceutical sciences, monash university, clayton, australia. cdepartment pharmacology and therapeutics, college of pharmacy, university of babylon, babylon, iraq. dpharmacology & toxicology department, college of pharmacy, university of babylon, babylon, iraq. ef.i.m.s (path.) correspondence to: hayder abdul-amir makki al-hindy (email: makihayder68@gmail.com). https://orcid.org/0000-0001-6232-8501, (submitted: 03 november 2019 – revised version received: 14 december 2019 – accepted: 22 january 2020 – published online: 26 february 2020) objective this study is designed to inspect the circulating levels of cystatin c (cy/c) in patients with acute coronary syndrome (acs) and to evaluate the association of cy/c levels with ejection fraction and angiographic number of stenosed coronaries. methods 136 participants with acs were enrolled in this case–control survey and compared with 94 control. hematological assessments were done for all the participants and included cy/c, creatinine, and uric acid were measured using conventional techniques. creatinine clearance rate was calculated and estimated glomerular-filtration-rate (egfr) were measured. echocardiographic assessment of ejection fraction was done with a cut-off point <40 left ventricular ejection fraction% designated for lv systolic dysfunction. angiographic evaluation involved in this study depending on the severity of stenosed coronaries by calculating number of arteries display not less than 1 critical stenosis (designed as >70% decrease in caliber). results ejection fraction was significantly lower in in acs patients than control group (p < 0.05). serum cy/c was significantly higher in patients’ group. the mean plasma uric acids values were significantly higher in patients’ group (p-0.001) but with no significant differences in creatinine and creatinine clearance (p < 0.05) observed between groups. the egfr were significantly lower in acs people compared to healthy persons. correlation studies of cy/c showed positive correlation with serum creatinine and serum uric acid; as well as nonsignificant negative correlation with egfr (p > 0.05). conclusion besides the role of cy/c in early detection of renal dysfunction, it could have a role in diagnosis of acs and evaluation of lv systolic dysfunction. nevertheless, this study showed cy/c was not related to the number of coronary arteries affected. keywords cystatin c, acute coronary syndrome, left ventricular ejection fraction, coronary angiography. 27 original cystatin-c in patients with acute coronary syndromed. ali abed et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 26–31 study population was stratified according to their levels of serum uric acid (sua) at their first 6–12 h of admission, into two groups: first (<6.3 mg/dl), second (≥6.3 mg/dl). measurements of other biochemical assays all hematological assays were performed on the day of presentation on fasting blood which was drawn and stored at minus 70°c. cy/c was measured by using elabscience® human cy/c elisa kit. while both creatinine and ua were measured using a conventional techniques. the creatinine clearance rate calculated using the cockcroft–gault formula in ml/min from plasma creatinine measured by mol/l as follows:7 creatinine clearance = {(140 age) x wt (kg) x f} / (serum creatinine * 0.8136) where f = 1 for men, and 0.85 for women egfr was measured by means of a new evaluation method established by adjusting of “modification-of-diet-in-renaldisease study” formula, depending on information from “chinese ckd” subjects:8 egfr (ml/min per 1.73 m2) = 175 × scr-1.234 × age-0.179; [scr -mg/dl…...and if female × 0.742 25 subjects who had renal impairment defined as an egfr<60, were omitted. echocardiographic study standard echocardiographic examination was done at rest by means of a vivid 9 apparatus (ge healthcare®) with 2.5/3.5mhz probe. a single qualified physician performed echocardiography. the left ventricular ejection fraction (lvef%) measured by using “modified simpson’s technique”. a cut-off point <40 lvef% designated for expressing lv systolic dysfunction. henceforth, acs patients divided into two-subgroups: ef <40% vis ≥40%.9 coronary angiography at least, two-skillful angiographers inspected angiography images, whereas severity of stenosed coronaries evaluated by calculating number of arteries displays not <1 critical stenosis (designed as >70% decrease in caliber). biostatistics assay all statistics finalized with spss software, version 25. the acs patients were clustered into quartiles based on their serum cy/c levels. anova was used for continuous covariates and chi-square for dichotomous variables. comparisons of continuous data (given as means) were performed by means of t-tests for independent samples. receiver operating characteristic (roc) curve studies were performed to establish specificity/ sensitivity. univariate logistic regression analysis was done wherever required. ethical consideration a conversant consent at the commencement obtained from each participant (or family associate) for acs subjects and controls individually, and our study final approval from the local committee for research ethics at university of babylon. arranged authorization for blood sampling, besides hospital approval by ethical committee of the entire work. results mean age of our acs patients was 59.3 ± 13.1 years, which is not unlike the age of the control group. similarly, bmi did not significantly differ between the two study groups. the males’ number was 92 (67.7%), which were less significant than the control 83 (88.29%). the mean lvef% was incomparable between the groups being lower in the acs group, even though, the mean ef% of both study groups still were within the normal limits. the mean levels of creatinine and calculated creatinine clearance for acs and control subjects were parallels mutually. the mean plasma ua values were significantly higher in the patients (p-0.001). correspondingly, the estimated gfr was significantly lower in acs people compared to healthy persons (109.6–127 ml/min per 1.73 m2), sequentially. a higher incidence of risk-factors in terms of concomitant hypertension and diabetes in acs patients were significant (p-0.05). serum cy/c levels were significantly higher in cardiac patients (table 1). table 1. baseline characteristics of the two groups involved in the study. characteristics group number mean std. deviation p-value age 1 136 59.3 13.1 >0.05 2 94 51.9 12.1 male gender no (%) 1 138 92 (67.7) 0.05 2 94 83 (88.29) bmi (kg/m2) 1 136 27.2 4.6 >0.05 2 94 26.8 4.5 left ventricular ef% 1 129 50.5 10.7 0.05 2 94 57 6.9 uric acid 1 136 5.9 1.8 0.001 2 94 4.4 1.3 creatinine 1 136 0.8 0.4 >0.05 2 94 .87 .11 creatinine clearance 1 129 73.7 60.5 >0.05 2 94 73.9 15.8 egfr (ml/ min/1.73 m2) 1 129 106.7 32.1 0.05 2 76 127.9 27.8 cystatin-c (mg/l) 1 102 1.4 1.0 0.05 2 94 0.50 .26 hypertension no (%) 1 136 74 (54.4) 0.05 2 94 diabetes no (%) 1 136 60 (66.9) 0.05 2 94 16 (16.8) egfr, estimated glomerular filtration rate; ef, ejection fraction. 28 original cystatin-c in patients with acute coronary syndrome d. ali abed et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 26–31 the issue of classification of the subjects’ levels of cy/c into quartiles is well-exposed in fig. 1, which showed that two-thirds of the patients had lower cy/c values than those with higher values. the distribution of the study variables according to cy/c quartiles is demonstrated well in table 2. there was no significant variation in the distribution of following variables according to cy/c quartiles: creatinine, bmi, creatinine clearance, ua, egfr, history of diabetes, hypertension, ef, and angiographic findings regarding the number of stenosed coronaries. meanwhile, significant difference was observed concerning age, gender, and smoking practice. linear regression analysis revealed positive weak significant association of cy/c to creatinine, negative and no significant association of cy/c to egfr and positive significant association of cy/c to sua in acs (figs. 2a–c). roc study verified cy/c 0.955 as a cut-off level to predict incidence of acs. the sensitivity, specificity, and accuracy for cy/c were 84, 86, and 84% sequentially, while the sensitivity, specificity, and accuracy of sua were 58, 72, and 73% sequentially (fig. 3). patients were divided to 2 subgroups: high cy/c group (≥0.95 mg/l) (n = 61) and the low cy/c (<0.95 mg/l) (n = 75) was the second group. discussion as a serious disorder, acs remains a cause of high morbidity and mortality even with considerable therapeutic advances take into consideration risk assessment by using risk factors and risk biomarkers.10 on top of out-of-date risk factors, some novel risk issues found to be associated with acs.11,12 both13,14 table 2. baseline characteristics divided into quartiles of cystatin-c (mg/l) characteristics cystatin-c quartiles (mg/l) total p-valuei (n = 49) <0.9 ii (n = 40) 0.9-1.49 iii (n = 21) 1.5-1.99 iv (n = 26) >2.0 age/years 54.9 57.4 62.8 62.6 <0.05 body mass index(kg/m2) 27.2 28 26.2 26.8 >0.05 gender m(n/total) 29/49 19/40 16/21 24/26 88/136 <0.05 f(n/total) 20/49 21/40 5/21 2/26 48/136 creatinine (mg/ml) .79 .79 .97 .99 >0.05 creatinine clearance 94.7 59.3 76.8 63.5 >0.05 uric acid (mg/dl) <6.3 19 15 11 8 53 >0.05 ≥6.3 11 10 2 8 31 egfr(ml/ min/1.73m2) <60 8/49 8/40 2/21 4/26 22/136 >0.05 >60 41/49 32/40 19/21 22/26 114/136 smoking not smoker 25/49 14/40 5/21 7/26 51/136 <0.05current-smoker 16/49 16/40 6/21 10/26 48/136 ex-smoker 8/49 10/40 10/21 9/26 37/136 history of diabetes 20/49 18/40 11/21 10/26 59/139 >0.05 history of hypertension 29/49 27/40 15/21 7/26 78/136 >0.05 left ventricular ejection fraction ≥40% 8 2 4 4 18 >0.05 <40% 21 21 9 11 62 angiography (number of stenosedcoronaries) 1-vessel 11/49 12/40 9/21 5/26 37/136 >0.05 2-vessels 28/49 12/40 1/9 10/26 51/136 3-vessels 6/49 8/40 9/21 7/26 30/136 4-vessels 4/49 8/40 2/21 4/26 18/136 fig. 1 frequency of cystatin-c (mg/l) quartiles among acs patients. 29 original cystatin-c in patients with acute coronary syndromed. ali abed et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 26–31 found that cy/c is less affected by factors like age, sex, and muscular mass over plasma creatinine and therefore might be a good biomarker of cardiovascular risk and cardiac impairment specially during aging. their findings did not match our study findings of significant variances observed in cy/c quartiles regarding age, sex, and smoking practice. parikh et al. exposed independent association of high levels of cy/c with risk issues like age, female sex, bmi, and smoking, even in people without chronic kidney diseases.15 in this study, cy/c levels in patients and control groups were close to the references of other studies.16,17 cy/c was significantly higher in acs group finding matches with other researches like ge et al.18 this high level of cy/c could reflect the atherotic changes in acs subjects as cy/c is expressed in all nucleating cells, controls action of “cysteine proteases.” cy/c and its fragments may influence the phagochemotactic ability of neutrophil, shares as well as regulates inflammatory responses. inflammation contributes a vital role in the progression of atherosis. thus, cy/c might reflect amplified inflammation which subsidizes to vulnerable coronary-sclerotic plaque.19 our result of a high level of cy/c in acs patients can document the role of cy/c as a diagnostic biomarker, consistent to other studies like eriksson et al.,20 who concluded that cy/c may reflect surely the existence or absence of acs. there is overwhelming evidence corroborating the notion that higher cy/c values is correlated to a higher incidence of coronary–vascular events in symptomless elder peoples, and in cardiac failure. yet, the reasons for such associations are indefinite.21,22 tayeh et al.23 assessed prognostic value of cy/c to predict “major adverse cardiac events” in acs. therefore, cy/c might be a valuable biomarker to diagnose coronary sclerosis. other surveys proposed increased cy/c levels might indicate elevated risk for heart disease/failure, stroke, in addition to mortality.24 meanwhile, close association of cy/c and acute ischemic stroke was also published in another survey.25 the positive correlation of cy/c with serum ua and creatinine observed in this work; are consistent with other analyses.12,26 harada et al.,26 found that cy/c had significant relations with both creatinine and sua mutually in japanian undergraduates and this concomitant “high cy/c and sua values’’ was related with subclinical lipids dysregulation and proposed the cardiometabolic risks. the negative correlation of cy/c and egfr in our study is in agreement with shlipak et al,21 who reported higher cy/c levels which reflect early gfr problems. further evidences supporting a graded association between high blood cy/c levels and increased incidence of cvd in people with gfr ≥60 ml/min/1.73 m2.4,27 fig. 2 scatter blot diagram of cystatin-c to creatinine, estimated glomerular filtration rate, and uric acid in acs patients. fig. 3 roc curve for cystatin-c in acute coronary syndrome. 30 original cystatin-c in patients with acute coronary syndrome d. ali abed et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 26–31 the higher incidence of associated risk factors in term of concomitant hypertension and diabetes mellitus in acs patients was significant, could be related to the finding of muntner et al.28 who displayed the incidence of risk-factors like tobacco-use, hypertension, besides others was higher in individuals with elevated cy/c concentrations. lodh et al., used a cut-off cy/c level of 1 mg/l and gained sensitivity of 77.8 for distinguishing acs patients, nevertheless, the specificity was not much 62 and the area under the curve was 0.831 (29). in this study, the cut-off value used was 0.955, at a higher cut-off we obtained a lesser specificity. the sensitivity was 86, specificity was 84 and 86%, which indicates the predictive value of cy/c as a biomarker for acs and this agreed with qing et al.30 findings, who reported a modest predictive rate of cy/c as a single biomarker for acs. our study showed the association of higher cy/c concentration (at the four quartiles) in acs patients with lower systolic function of the left ventricle reflected by lower ef and this is inconsistent to other analysis like shuaila et al., who found that higher cy/c values associated with lower ef.31 same outcomes were reported by moran et al.,32 who revealed that cy/c can be also a biomarker for cardiac failure. tayeh et al also showed the association between higher cy/c level and the lower ef. from this finding, we can conclude that cy/c can detect the systolic dysfunction of the lv.23 our data provide a convincing evidence that cy/c level not related to the number of stenosed coronaries. our finding was in contrast to other studies that showed that the cy/c level have a tendency to increase with increment of diseased coronaries,30,33 as well as tayeh et al,23 who found that patients with higher level of cy/c (cut-off value 1.141 mg/l) had a significant high number of stenosed vessels in comparison to patients with lower cy/c levels. the small sample size might explain these discrepancies. in acs, the cy/c was not related to the number of coronary arteries affected and this could be due to small size sample. conclusion cy/c is not only a good marker for early detection of renal impairment, but also could be a valuable biomarker for diagnosing coronary artery diseases, and cy/c in patients with acs is a powerful predictor of lv systolic dysfunction, assessing severity of the condition, and predict development of heart failure. it seems important to put factors like age, gender, and smoking habits into consideration during evaluation cy/c level as our study showed it is affected by these factors. acknowledgments we thank the department of pharmacology and toxicology/ college of pharmacy/university of babylon. we thank babylon health directorate/babel hospital for maternity and childhood, for permission to access the data and the community of babel thalassemia center for their official support to the study. abbreviations cy/c – cystatin c acs – acute coronary syndrome bmi – body mass index egfr – estimated glomerular filtration lvef – left ventricular ejection fraction ami – acute myocardial infarction sua – serum uric acid conflicts of interest disclosure there are no conflicts of interest. competing interests the authors declare that they have no competing interests. source of funding self-funded project. ethics approval and consent to participate the institutional review board of the university of babylon approved this study. author contributions all authors confirmed they have contributed to the intellectual content of this paper and have met the following three requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content, and (c) final approval of the published article. ammar had an advisory role and a great cooperative effort for sample collection. references 1. deo r, shlipak mg, ix jh, ali s, schiller nb, whooley ma. association of cystatin c with ischemia in patients with coronary heart disease. clin. cardiol. 2009;32(11):e18–e22. 2. huang q, shen w, li j, luo x, shi h, yan p, et al. association of serum cystatin c levels with acute coronary syndrome in patients of advanced age. j. int. med. res. 2019;47(5):1987–97. 3. mustafa begenc tascanov oy. may cystatin-c be associated with postmyocardial infarction complications? j. clin. anal. med. 2019;10:7. 4. correa s, morrow da, braunwald e, davies ry, goodrich el, murphy sa, et al. cystatin c for risk stratification in patients after an acute coronary syndrome. j. am. heart assoc. 2018;7(20):e009077. 5. wei l, ye x, pei x, wu j, zhao w. reference intervals for serum cystatin c and factors influencing cystatin c levels other than renal function in the elderly. plos one. 2014;9(1):e86066-e. 6. martucheli kfc, domingueti cp. clinical usefulness of cystatin c to assess the prognosis of acute coronary syndromes: a systematic review and metaanalysis. int. j. cardiovasc. sci. 2018;31:290–307. 7. shahbaz h gmuisiisi. creatinine clearance. 2019 2019 jul 5 [cited jan 7 2020]. statpearls publishing, [cited jan 7 2020]. 8. levey as, bosch jp, lewis jb, greene t, rogers n, roth d. a more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. modification of diet in renal disease study group. ann. intern. med. 1999;130(6):461–70. 31 original cystatin-c in patients with acute coronary syndromed. ali abed et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 26–31 9. mentzer g, hsich em. heart failure with reduced ejection fraction in women: epidemiology, outcomes, and treatment. heart failure clin. 2019;15(1):19– 27. 10. amir s al-mumim abmz, hayder aa al-hindy. decayed missed filled scores in iraqi patients with acute myocardial infarction. al-kufa j. biol. sci. 2016;8 (3):5. 11. anderson jl, adams cd, antman em, bridges cr, califf rm, casey de, jr., et al. acc/aha 2007 guidelines for the management of patients with unstable angina/non-st-elevation myocardial infarction: a report of the american college of cardiology/american heart association task force on practice guidelines (writing committee to revise the 2002 guidelines for the management of patients with unstable angina/nonst-elevation myocardial infarction) developed in collaboration with the american college of emergency physicians, the society for cardiovascular angiography and interventions, and the society of thoracic surgeons endorsed by the american association of cardiovascular and pulmonary rehabilitation and the society for academic emergency medicine. j. am. coll. cardiol. 2007;50(7):e1–e157. 12. lodh m, goswami b, parida a, patra s, saxena a. assessment of serum leptin, pregnancy-associated plasma protein a and crp levels as indicators of plaque vulnerability in patients with acute coronary syndrome. cardiovasc. j. afr. 2012;23(6):330-5. 13. nagesh cm, roy a. role of biomarkers in risk stratification of acute coronary syndrome. ind. j. med. res. 2010;132:627–33. 14. sarnak mj, katz r, stehman-breen co, fried lf, jenny ns, psaty bm, et al. cystatin c concentration as a risk factor for heart failure in older adults. ann. intern. med. 2005;142(7):497–505. 15. parikh ni, hwang sj, yang q, larson mg, guo cy, robins sj, et al. clinical correlates and heritability of cystatin c (from the framingham offspring study). am. j. cardiol. 2008;102(9):1194–8. 16. kyhse-andersen j, schmidt c, nordin g, andersson b, nilsson-ehle p, lindstrom v, et al. serum cystatin c, determined by a rapid, automated particle-enhanced turbidimetric method, is a better marker than serum creatinine for glomerular filtration rate. clin. chem. 1994;40(10):1921–6. 17. norlund l, fex g, lanke j, von schenck h, nilsson je, leksell h, et al. reference intervals for the glomerular filtration rate and cell-proliferation markers: serum cystatin c and serum beta 2-microglobulin/cystatin c-ratio. scandinav. j. clin. lab. investig. 1997;57(6):463–70. 18. ge c, ren f, lu s, ji f, chen x, wu x. clinical prognostic significance of plasma cystatin c levels among patients with acute coronary syndrome. clin. cardiol. 2009;32(11):644–8. 19. bharat lochan kbr. cystatin-c as a potential risk factor for acute myocardial infarction with normal renal function. int. arch. integr. med. 2019;6(1):7. 20. eriksson p, jones kg, brown lc, greenhalgh rm, hamsten a, powell jt. genetic approach to the role of cysteine proteases in the expansion of abdominal aortic aneurysms. br. j. surg. 2004;91(1):86–9. 21. shlipak mg, katz r, sarnak mj, fried lf, newman ab, stehman-breen c, et al. cystatin c and prognosis for cardiovascular and kidney outcomes in elderly persons without chronic kidney disease. ann. intern. med. 2006;145(4):237– 46. 22. keller t, messow cm, lubos e, nicaud v, wild ps, rupprecht hj, et al. cystatin c and cardiovascular mortality in patients with coronary artery disease and normal or mildly reduced kidney function: results from the atherogene study. eur. heart j. 2009;30(3):314–20. 23. tayeh o, rizk a, mowafy a, salah s, gabr k. cystatin-c as a predictor for major adverse cardiac events in patients with acute coronary syndrome. egypt. heart j. 2012;64(3):87–95. 24. shlipak mg, matsushita k, arnlov j, inker la, katz r, polkinghorne kr, et al. cystatin c versus creatinine in determining risk based on kidney function. new engl. j. med. 2013;369(10):932–43. 25. huang gx, ji xm, ding yc, huang hy. association between serum cystatin c levels and the severity or potential risk factors of acute ischemic stroke. neurol. res. 2016;38(6):518–23. 26. harada m, izawa a, hidaka h, nakanishi k, terasawa f, motoki h, et al. importance of cystatin c and uric acid levels in the association of cardiometabolic risk factors in japanese junior high school students. j. cardiol. 2017;69(1):222–7. 27. latta f, de filippi c. role for cystatin c-based risk stratification for patients after acute coronary syndrome in the era of high sensitivity cardiac troponin assays. j. am. heart assoc. 2018;7(20):e010589-e. 28. muntner p, mann d, winston j, bansilal s, farkouh me. serum cystatin c and increased coronary heart disease prevalence in us adults without chronic kidney disease. am. j. cardiol. 2008;102(1):54–7. 29. lodh m, parida a, sanyal j, ganguly a. cystatin c in acute coronary syndrome. ejifcc. 2013;24(2):61–7. 30. qing x, furong w, yunxia l, jian z, xuping w, ling g. cystatin c and asymptomatic coronary artery disease in patients with metabolic syndrome and normal glomerular filtration rate. cardiovasc. diabetol. 2012;11:108. 31. shuaila m, abdulamir, hassanain, alshok, monem. prognostic value of cystatin c in acute coronary syndrome. j. contemp. med. sci. 2016;2(4):4. 32. moran a, katz r, smith nl, fried lf, sarnak mj, seliger sl, et al. cystatin c concentration as a predictor of systolic and diastolic heart failure. j. cardiac failure. 2008;14(1):19–26. 33. sekizuka h, akashi yj, kawasaki k, yamauchi m, musha h. cystatin c: a better marker to detect coronary artery sclerosis. j. cardiol. 2009;54(3):359– 67. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.02202006 112 j contemp med sci | vol. 5, no. 2, march–april 2019: 112–116 original estimation of stature from facial indices among iranian medical students soheila madadi, fatemeh tahmasebi, maryam khanehzad, shokofeh kazemzadeh, and gholamreza hassanzadeh* department of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. *correspondence to gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir). (submitted: 26 october 2018 – revised version received: 17 november 2018 – accepted: 27 january 2019 – published online: 26 april 2019) objective anthropometric data, particularly nasofacial dimensions, is important in sex determination, forensic medicine and plastic surgery. the aim of this study is to assess the facial indices, to classify the type of face and to determine the relation between stature and facial height and facial width among iranian medical students. methods this study was done on 200 medical students (100 males and 100 females), in the age range of 18–30 years. stature, facial height and facial width were measured by using a standard spreading caliper and facial index calculated for determination of facial type. the linear regression used for examining the relationship between stature and facial length and facial width. results the result of study showed that the mean and standard deviation of the facial index of males and female students were 87.53 ± 8.81 and 89.25 ± 10.09, respectively. the facial index did not have statistically significant difference between two groups (p = 0.202). according to the results, the dominant type of facial shape in male students was mesoprosopic and in female students were europrosopic. there was a correlation between stature and facial height (r = 0.27, p = 0.000) and facial width (r = 0.54, p = 0.000). conclusion the data of this study can be beneficial in face reconstruction, clinical diagnosis and forensics applications. keywords facial height, facial width, facial index, stature introduction craniofacial measurement is an important part of anthropology and medicine that determine the morphological features of the face and head.1 shape of face is affected by many agents, for instance, gender, race and ethnicity, nutritional, climate, socio-economic, and genetic factors.1,2 the determination of facial parameters is useful in plastic surgery, forensics applications, head and face reconstruction, oral and maxillofacial surgery, orthodontics, clinical diagnosis and treatment planning.3–5 according to craniofacial measurements, shape of faces are classified into five anatomical types that include: hypereuryprosopic, euryprosopic, mesoprosopic, leptoprosopic and hyperleptoprosopic.6 by using facial measurements for example facial height, facial width and facial index is beneficial to identify the genetically and acquired defects. facial type can be an effective element to increase capability of obstr uctive sleep apnea for instance a person with euryproscopic facial type prefers the nasal breathing mode.7,8 comparing the facial index differences among parents, offspring and sibling may be a sign for genetic transition.9 in addition, different studies showed a relation between the stature with body parts measurement, but there is few studies in this field among iranian population.10–12 these kinds of studies might be beneficial for stature estimation in growth defects and skeletal dysplasia cases.13 the aim of this study is to determine the facial indices and to classify the facial type in iranian medical students. and also this study assessed the relationship between facial width and facial height with the stature in iranian medical students. materials and methods this study was carried out on 200 medical students of tehran university of medical sciences, tehran, iran (100 males and 100 females), with age range of 18–30 years with normal craniofacial configurations. the students were sitting on the chair, and their head were in the anatomical position. facial height and facial width were measured by using a standard spreading caliper with scale. the measurement of facial height was taken the distance between nasion (n) and gnathion (gn). and the measurement of face width was taken the distance between zygion (zy) of right and left side. to calculate facial index the following formula was used: facial index = facial length facial width ´100 the anatomical landmarks used in measuring of the parameters were defined as follows: nasion (n): the point on the nasal root in the midpoint of the nasofrontal suture. gnathion (gn): the lowest point on the lower border of the mandible in the midline. zygion (zy): it is the most lateral point on the zygomatic arch. according to facial index, face shapes were classified to five categories that include (banister’s classification) (14): 1. hypereuryprosopic (very broad, short face): facial index <79.9. 2. euryprosopic (broad, short face): facial index 80–84.9. 3. mesoprosopic (average, round face): facial index 85–89.9. 4. leptoprosopic (long, narrow face): facial index 90–94.9. 5. hyperleptoprosopic (very long, narrow face): facial index >95. the stature (standing height) of subjects was measured as distance between the vertex and the heel in centimeters. individuals stood barefoot on a flat surface and their head was stated in the frankfort horizontal plane.15 the data were analyzed by spss 16 software and expressed as mean ± sem. independent-samples t-test was used for comparison of the means of the anthropometric measurements. the relationship issn 2413-0516 113j contemp med sci | vol. 5, no. 2, march–april 2019: 112–116 original estimation of stature from facial indices among iranian medical studentss. madadi et al. between quantitative data was assessed by pearson’s correlation coefficient and linear regression was used to determine the relation between stature with facial length and facial width. statistical significance was considered to be p ≤ 0.05. results in this study, the facial height was assessed and the results revealed that the mean and standard deviation facial height of male and female students were 12.32 ± 1.06 and 11.57 ± 1.12 cm, respectively. there was a significant difference in the facial height of students between two sex groups (p = 0.0001, table 1). according to table 1, mean and standard deviation facial width of male and female students were 14.11 ± 0.76 and 13.00 ± 0.64 cm, respectively. there was a significant difference in the facial width of cases between two sex groups (p = 0.0001). the obtained results showed that mean and standard deviation of the facial index of male and female students were 87.53 ± 8.81 and 89.25 ± 10.09 respectively. also facial index was not statistically significant difference between male and female students (p = 0.202, table 1). according to the results, the common face shape for male and female students were mesoprosopic (round face) and europrosopic (broad face), which was 33% and 36% for male and female students, respectively. the second most dominant face shape for males was europrosopic that was 25% but for females it was mesoprosopic and leptoprosopic (long face) that each of them was 20%. the rare face shape was hypereuryprosopic (very broad face) for both groups, which was 11% and 8% for males and females, respectively (table 2, figs. 1 and 2). mean and standard deviation stature of male and female students were 177.202 ± 6.04 and 161.94 ± 6.17 cm, respectively. there was a significant difference in the stature of students between two groups (p = 0.000, table 3). also, there was a correlation between stature with facial height and facial width that was statistically significant. pearson correlation (r) for stature with facial height and facial width was 0.27 and 0.54, respectively (p = 0.000, table 4). the correlation between stature with facial height and width are shown in scatter graphs (fig. 3). linear regression analysis showed a relation between stature with facial height and facial width in all cases. according to the results, the facial width can predict the stature better than facial height (table 5). discussion in this study the facial index of students of tehran university of medical sciences in the range of 18–30 years old evaluated. facial index calculated by ratio of facial height to facial width and multiplied by 100. the result of study showed that the mean table 1. comparison of facial height, facial width and facial index in male and female students parameters n mean ± sd male female male female facial height 100 100 12.32 ± 1.06 11.57 ± 1.12 facial width 100 100 14.11 ± 0.76 13.00 ± 0.64 facial index 100 100 87.53 ± 8.81 89.25 ± 10.09 sd: standard deviation; n: number of samples. table 2. distribution of facial type in male and female students face shape range of facial index male female (1) hypereuryprosopic (very broad face) <79.9 11 8 (2) europrosopic (broad face) 80–84.9 25 36 (3) mesoprosopic (round face) 85–89.9 33 20 (4) leptoprosopic (long face) 90–94.9 17 20 (5) hyperleptoprosopic (very long face) >95 14 16 table 3. comparison of stature in male and female students groups n mean ± s.d male 100 177.202 ± 6.04 female 100 161.94 ± 6.17 sd: standard deviation; n: number of samples. fig. 1 morphological variation of facial index in male students. fig. 2 morphological variation of facial index in female students. 114 j contemp med sci | vol. 5, no. 2, march–april 2019: 112–116 estimation of stature from facial indices among iranian medical students original s. madadi et al. race, gender and nutritional condition were effective factors on the facial indices and stature.16 our findings were similar to shetti et al.’s study. they showed that mean facial index for males was 87.19 and mean facial index for females was 86.75 in indian students with a statistically significant difference between two groups (p = 0.018).17 kumar et al. reported the mean facial index among adult haryanvi banias was 86.09 in males and 84.84 in females and the dominant face type in males and females were mesoprosopic and europrosopic, respectively. these findings are similar to our research.18 according to wai et al.’s study, the mean facial index in male and female malay young adults were 87.04 and 90.59, respectively, and the face form of males were mesoprosopic and of females were leptoprosopic, respectively. in chinese young adults, the facial index was 85.90 in male cases and 85.40 in female cases with a mesoprosopic face type for both genders. the mean facial index in male and female indian young adults was 92.14 and 92.99, respectively, with leptoprosopic face type for both genders.19 sharma et al.’s study revealed the mean facial index of nepalese students for males and females as 87.20 and 86.81, respectively. the difference between two groups was not statistically significant (p = 0.553). the most common face shape between male nepalese students was euryprosopic and females was mesoprosopic, which is completely contrary to our study, that the dominant type of facial shape for males students was mesoprosopic and females students was europrosopic.20 also, our findings differed from the study of kurnia et al.7 that reported the dominant face type was leptoprosopic type in male and mesoprosopic type in female among chinese in indonesia. azizi et al.21 showed the most common facial type was hyperleptoprosopic type among males and females in qazvin, iran. the study of jahanshahi et al. showed that the face form of the native fars and turkman males were mesoprosopic. and the face form of the native fars and turkman females were euryprosopic, which is similar to the results of this study. the least common facial shape in the native fars males was hyperleptoprosopic and hypereuryprosopic and in females were hyperleptoprosopic. the least common facial shape in the turkman males and females were hypereuryprosopic and leptoprosopic, respectively. in this study, the rare type of face form was hypereuryprosopic for both sexes, which in male students is similar to the findings of jahanshahi et al.’s study in fars and turkman males. also, the mean facial index of fars males and females were 88.22 and 84.48, respectively. the mean facial index of turkman males and females were 87.25 and 81.48, respectively that is closely similar to the results of this study.22 the results of this study resembled with salve et al.23 study that showed the dominant face type in andhra region males and females were mesoprosopic and euryproscopic, respectively. according to gained results the dominant face shape in females are euryprosopic, while in males are mesoprosopic. it indicates that female students have a rather broad face than male students. this study that had been carried out on 200 students, presented the anthropometrical differences between students of tehran university of medical sciences.18 generally, types of face shape are affected by geographical and ethnical factors and show broad, round or long faces in every population.22 the facial indices assessment can be used for sex determination, forensics medicine, and facial plastic surgeries. and more researches are required in a larger sample between iranian population to apply in the medical and surgical purposes.19 table 4. pearson correlation (r) between stature with facial height and facial width variable pearson correlation (r) p facial height 0.27 0 facial width 0.54 0 table 5. linear regression for estimation of stature from facial height and facial width regression equation see r² p fw fh s = 141. 33 + 2.36 (fh) 0.58 0.07 0 s = 89.85 + 5.87 (fw) 0.65 0.29 0 s = 77.01 + 1.51 (fh) + 5.49 (fw) 0.508 0.65 0.32 0 s: stature, fh: facial height, fw: facial width, see: standard error of the estimate, r²: coefficient of determination. fig. 3 correlation between stature (cm) and facial height (cm) and facial width (cm). facial index of male and female cases were 87.53 and 89.25, respectively. it was not statistically significant difference between two gender (p = 0.202). the involved students came from various ethnic and geography. and biological, geographical, 115j contemp med sci | vol. 5, no. 2, march–april 2019: 112–116 original estimation of stature from facial indices among iranian medical studentss. madadi et al. in this study, stature was compared with facial height and facial width of the students and the results of study showed a correlation between them. the correlation coefficient (r) between stature and facial height and facial width was 0.27 and 0.54, respectively. the linear regression equation showed that stature is estimated by facial height and facial width. linear regression analysis showed a poor relation between stature and facial height (s = 141. 33 + 2.36 × facial height (cm), r² = 0.07, see = 0.58) and a moderate relation between stature and facial width (s = 89.85 + 5.87 × facial width (cm), r² = 0.29, see = 0.65). in several studies, the stature estimated from body parts including cephalofacial, upper and lower limb measurements such as forearm length, and foot length.24–28 krishan et al. showed the correlation between stature with facial height (r = 0.34) and facial width (r = 0.46) in north indian population. the result of their study is similar to this study which shows facial width is more reliable for stature estimation than facial height.29 sahni et al. used facial height for prediction of stature in northwest indians and its correlation coefficient (r) was 0.21 in males and 0.18 in females.30 agnihotri et al.’s study showed a correlation between stature with facial height (r = 0.32 in males and r = 0.16 in females) and facial width (r = 0.17 in males and r = 0.27 in females). their result showed that facial height is a more suitable parameter in males than facial width for estimation of stature while in females it is vice versa.31 wankhede et al. reported a relation between stature with facial height in males (r = 0.19) and females (r = 0.14) of central indian population that it was lower than result of this study. this variance could be affected by climatic, geographic or ethnic difference.32 in addition, this study showed that the prediction of facial width for stature estimation is more reliable than facial height among iranian medical students, whereas the anthropometric features are affected by elements such as different races, nutrition, genetic and geographical situation,33 the regression models of the stature from other parts of body can be different in the various regions. also the correlation between facial indices and stature can be useful in identifying nasofacial dysmorphology, maxillofacial and facial reconstruction surgeries, stature estimation, and the application of forensic. hence, further studies in different races are required to estimate the stature. acknowledgment we would like to thank the medical students who participated in this study. conflicts of interest the authors declare no conflicts of interest.  references 1. oladipo g, didia bc, okoh pd, hart js. sexual dimorphism in facial, nasal, maxillary, mandibular and oro-facial heights of adult ijaws. j exp clin anat. 2008;7:10–18. 2. gabriel o, fawehinmi hb, peter o. canthal indices of urhobo and itsekiri ethnic groups. aust j basic appl sci. 2009;3:3093–3096. 3. golalipour mj, hosseinpour kr. estimation of the cranial capacity and brain weight of iranian female newborns. eur j anat. 2006;10:49–52. 4. sakakibara h, tong m, matsushita k, hirata m, konishi y, suetsugu s. cephalometric abnormalities in non-obese and obese patients with obstructive sleep apnoea. eur respir j. 1999;13:403–410. 5. will mj, ester ms, ramirez sg, tiner bd, mcanear jt, epstein l. comparison of cephalometric analysis with ethnicity in obstructive sleep apnea syndrome. sleep 1995;18:873–875. 6. martin r, saller k. lehrbuch der anthropologie, gustav fisher verkag. stuttgart i, 1957, p. 429–547. 7. kurnia c, susiana s, husin w. facial indices in chinese ethnic students aged 20-22. j dent indonesia 2013;19:1–4. 8. bolzan gde p, souza ja, boton lde m, silva am, corrêa ec. facial type and head posture of nasal and mouth-breathing children. j soc bras fonoaudiol. 2011;23:315–320. 9. doni pkr, janaki cs, vijayaraghavan v, delhi raj u. a study on measurement and correlation of cephalic and facial indices in males of south indian population. int j med res health sci. 2013;2:439–446. 10. mohanty bb, agrawal d, mishra k, samantsinghar p, chinara pk. estimation of height of an individual from forearm length on the population of eastern india. j med allied sci. 2013;3:72–75. 11. singh s, nair sk, anjankar v, bankwar v, satpathy dk, malik y. regression equation for estimation of femur length in central indians from inter-trochanteric crest. j indian acad forensic med. 2013;35:223–226. 12. gocha tp, vercellotti g, mccormick le, van deest tl. formulae for estimating skeletal height in modern south-east asians. j forensic sci. 2013;58:1279–1283. 13. hepper ng, black lf, fowler ws. relationships of lung volume to height and arm span in normal subjects and in patients with spinal deformity. am rev respir dis. 1965;91:356–362. 14. saoemes r, williams p, bannister l, berry m, collins p, dyson m, et al. gray’s anatomy. in skeletal system. churchill livingstone, edinburgh, london, 1999, p. 555–560. 15. jit i, singh s. estimation of stature from clavicles. indian j med res. 1956;44:137–155. 16. shah s, koirala s, khanal l. variations in craniofacial anthropometry in 17-25 year-old adult population of nepal. eur j forensic sci. 2014;1:5–8. 17. shetti vr, pai sr, sneha g, gupta c, chethan p. study of prosopic (facial) index of indian and malaysian students. int. j. morphol. 2011;29:1018–1021. 18. kumar m, muzzafar lone m. the study of facial index among haryanvi adults. int j sci res. 2013;2:51–53. 19. wai mm, thwin ss, yesmin t, ahmad a, adnan as, hassan aa, et al. nasofacial anthropometric study among university students of three races in malaysia. adv anat. 2015;2015:5. 20. sharma k, khanal k, mansur di. variations in total facial index among students of kathmandu university school of medical sciences. nepal med coll j. 2014;16:173–176. 21. azizi m, hassanzadeh g, barbarestani m, sadr m, dehbashipour a, alaghbandha n, et al. comparative anthropometric analysis of facial dimensions and types in qazvin, iran and deraghazi khan, pakistan. anat sci j. 2014;11:119–126. 22. jahanshahi m, golalipour mj, heidari k. the effect of ethnicity on facial anthropometry in northern iran. singapore med j. 2008;49:940–943. 23. salve vm, thota nr, naralasetty a. study of facial (prosopic) index of andhra region (india) students. novel sci int j med sci. 2012;1:248–252. 24. krishan k. estimation of stature from cephalo-facial anthropometry in north indian population. forensic sci int. 2008;181:52.e1–52.e6. 25. balvir tk, deshpande jv, badwaik p, rahule as, kasote ap, bashir msm, et al. estimation of stature from the lenth of clavicle in vidarbha region of maharashtra. int j biol med res. 2012;3:2254–2256. 26. moshkdanian g, mahaki zadeh s, moghani ghoroghi f, mokhtari t, hassanzadeh g. estimation of stature from the anthropometric measurement of lower limb in iranian adults. anat sci j. 2014;11:149–154. 27. poorhassan m, mokhtari t, navid s, rezaei m, sheikhazadi a, mojaverrostami s. stature estimation from forearm length: an anthropological study in iranian medical students. j contemp med sci. 2017;3:270–272. 28. mahakizadeh s, moghani-ghoroghi f, moshkdanian g, mokhtari t, hassanzadeh g. the determination of correlation between stature and upper limb and hand measurements in iranian adults. forensic sci int. 2016;260:27–30. 29. krishan k, kumar r. determination of stature from cephalo-facial dimensions in a north indian population. leg med (tokyo). 2007;9:128–133. 116 j contemp med sci | vol. 5, no. 2, march–april 2019: 112–116 estimation of stature from facial indices among iranian medical students original s. madadi et al. 30. sahni d, sharma p, kaur g, aggar wal a. estimation of stature from facial measurements in northwest indians. legal medicine. 2010;12:23-7. 31. agnihotri ak, kachhwaha s, googoolye k, allock a. estimation of stature from cephalo-facial dimensions by regression analysis in indo-mauritian population. j forensic leg med. 2011;18:167–172. 32. wankhede kp, kamdi ny, parchand mp, anjankar vp, bardale rv. estimation of stature from maxillo-facial anthropometry in a central indian population. j forensic dent sci. 2012;4:34–37. 33. akhlaghi m, hajibeygi m, zamani n, moradi b. estimation of stature from upper limb anthropometry in iranian population. j forensic leg med. 2012;19:280–284. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.04201909 285j contemp med sci | vol. 6, no. 6, november–december 2020: 285–290 original issn 2413-0516 introduction breast cancer is a genetic disease caused by a multiple series of mutations in oncogenes, tumor suppressor genes, and stability genes. about 5–10% of all breast carcinomas are caused by germ-line mutations.1 breast cancer occurs in both men and women, although male breast cancer is rare.3 most masses detected on a mammogram and most lumps of the breast grow out to be benign.4 most carcinoma types of cells have an allowing control, while others function actively and are likely metastatic.5 the sign and symptoms of breast cancer differ, sometimes it can be scientifically difficult to differentiate between malignant and benign tumors. a triple test must be carried out: psychiatric assessment, breast screening, and a biopsy examination.13 the concordance of all three modalities is a standard control to prevent false detects. the risk of developing breast cancer is impacted by multiple genetic, behavioral, and environmental factors.4 the involvement of breast cancer in every first-degree female parent almost doubles the risk for a genotype, and the risk grows continuously with the number of relatives affected.18 study shows that breast cancer risk correlates with the acquired dose of radiation. risks became higher in patients under 50 years old than where the individual was diagnosed before 50.6 lung cancer is the most common disease diagnosed, and the major cause of death from cancer. it is closely accompanied by female breast cancer (11.6%), prostate cancer (7.1%), and colorectal cancer (6.1%).7 breast cancer is a malignant tumor dart which begins in breast cells. there are several variables that will increase the chance of developing breast cancer, among most cancers. the immune system typically watches out for and kills cancer cells and cells with weakened dna.8 the failure of such a successful immune response and surveillance could cause breast cancer. some people inherit dna mutations and genes such as brca1, brca2, and p53. anyone with a family history of ovarian or breast cancer was at an elevated risk of breast cancer.9 the pathologist classifies cancer by degree. an often-fatal feature of malignant tumors is the ability of cancer cells to infiltrate other tissues and propagate to distant organs. proteolytic enzymes play a vital role in the development of cancer. in the human genome, over 500 genes encoding proteases or protease-like proteins are present.10 the matrix metalloproteinase-2 (mmp-2) make up a large family of zinc-dependent endopeptidases multidomain spread in all kingdoms except protozoa. each mmp has a signal peptide to guide trafficking.11 mmp-2 is a well-known cancer metastasis mediator but is also believed to be active in many cancer development factors including cell growth and inflammation. that works with the degradation of type iv collagen. the mmp-2 gene is 16q13 in the chromosome, it is 17 kb long with 13 (thirteen) 110–901 bp exons and 175–4350 bp 12 introns.16 genetic variation that modulates the expression of mmp-2 may lead to the individual cancer susceptibility differences. in the mmp-2 promoter, two association between matrix metalloproteinase-2 gene variants and pathogenesis of breast cancer in sera of iraqi women yammamah jawad abbas1, fadhil jawad al-tu’ma1, alaa fraq al-hemerri2 1department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq.2department of chemistry, college of science, university of kerbala, kerbala, iraq. corresponding author: fadhil jawad al-tu’ma (e-mail: f_altoma_56@yahoo.com) (submitted: 04 october 2020 – revised version received: 17 october 2020 – accepted: 25 october 2020 – published online: 30 december 2020) abstract objective: the current study aims to investigate the role of matrix metalloproteinase-2 (mmp-2) in breast cancer pathogenesis in iraqi women. methods: forty-one (41) women with breast cancer and 45 control women were included in this case–control study. body mass index, age, smoking; married status, tumor size, degree, subtype, lymph node status, preand post-menopause were included the phenotypic results. the polymerase chain reaction (pcr)-allele specific restriction was used to observe the rs243865 polymorphism. genomic dna was extracted from whole blood and genotyping with specific prefixes for amplification of the mmp-2 gene was accomplished as enzymerestricted pcr products were digested, followed by electrophoresis on 1.5% agarose gel. in order to interpret the researchers’ results, numerous statistical analyses were applied. results: the amplicon size of mmp-2 gene was 304 bp, and following its amplification reactions by allelic specific pcr. the amplification product for mmp-2 gene amplification snp rs243865 gene polymorphism results exhibited one band of 304 bp, two bands of 304 bp, and one band 304 bp for individuals have genotype as wild type (cc), homozygous (tt), and heterozygous (ct), respectively. genotype frequencies of rs243865 polymorphism were found to be consistent with hardy–weinberg equilibrium. allele frequencies of c allele was 0.57, and the t allele was only 0.43 in cases of breast cancer women patient, while the frequencies of cc, ct, and tt genotypes of the rs243865 snp were statistically significant as 31.7%, 51.2%, 17.1%, respectively. allele frequencies of c and t were 0.78 and 0.22 for the control group, respectively, the heterozygous genotype (ct) was significantly increased the risk of breast cancer women (or=0.3, 95% ci; 0.12–0.8, p≤0.05) with respect to those of the cc wild type. the tt genotype significantly raised the risk of breast cancer women by (or = 0.2, 95% ci; 0.04–0.9, p≤0.05). conclusion: in women with breast cancer, mmp-2 expression’s high association were observed with positive lymph node, histological classification of breast cancer (ll) was higher than other classes and advanced clinical process (ll). keywords: mmp-2, breast cancer, allele, polymerase chain reaction, genotype, snp 286 original association betweenmatrix metalloproteinase-2 gene variants and pathogenesis of breast cancer yammamah jawad abbas j contemp med sci | vol. 6, no. 6, november–december 2020: 285–290 single nucleotide polymorphisms (snps) have been shown to affect in vitro expression; c to t transformations at −1306 (rs243865).2 as it is a part of an extracellular matrix (ecm), this results in the lack of cellular structural support, and thus in the destabilization of the basal membrane, an important step for cancer spread. altered mmp-2 activity arising from the involvement of unique mmp-2, this included variants in the degrade of ecm and disturbance of the membrane barrier in the basement.14 mmp-2 protein has been regulated by immune histochemical approaches in breast carcinoma cells. in processing, growth factors, including egf-like growth factor (hb-egf) and tgfβ are thought to play.12 several studies have shown that genetic polymorphism regulates the expression of mmp-2 in its promoter region and that sustained elevated levels of mmp-2 could make carries more vulnerable and aggressive to tumorogenesis. in this context, we explored the relationship between susceptibility to breast cancer and the existence of variants of mmp2 (promoter): rs243865 (1306 c/t), as possible biomarkers of breast cancer risk. materials and methods forty-one (41) women with breast cancer with age ranged between 16 and 82 years, and another 45 apparently normal women as a control group with age ranged between 16 and 55 years were included in a case–control study during nov. 2019 to sep. 2020. cases and controls were recruited from the imam hussein oncology center, al-hussein teaching hospital, al-hussein medical city/kerbala health directorate – iraq. women in control were selected from the patient’s relatives in a hospital from the general population women-only individuals who did not show signs and symptoms of any chronic diseases such as high blood pressure, kidney disease, heart disease, or others were selected to take part in this study, were free of breast cancer personal or family background, and were comparable in self-declared ethnic origin to patients. the breast cancer diagnosis was consistent with the american cancer society’s recommendations (www. cancer.org). this included mammography and breast biopsy tests to confirm breast cancer; four of these were performed in all patients. unrelated comorbidities are observed in one subject (cases and controls). for patients from medical reports and interviews, the demographic profile and clinical bio-data were gathered using a standardized questionnaire by doctors or senior residents. this included age at research entrance, age at first breast cancer diagnosis, body mass index (bmi), menopause status pre-and post-menopause, tumor size, level of disease at the appearance, marital status, and smoking history. the histological review covered disease stage and nuclear grade status, estrogen receptor (er), and progesterone receptor (pr) status prior to treatment (chemotherapy, surgery, and radiation), and matrix metalloprotease genotypes-2. the study was done and approval was obtained from the research and ethics committee at the college of the medicine / university of kerbala all patients and control subjects provided written informed consent. regarding the mmp-2 gene, one snp, with a minor frequency of alleles. the national center for biotechnology information (ncbi) gene snp gene view was used to identify iraqi women and clinical significance (www.ncbi. nlm.nih.gov / projects / snp/). these included rs243865 (context sequence gagacctgaagctaaagggtg (c / t) aagacataatcg tgacctccaaatg). mmp-2 genotyping was performed by the allelic discrimination method, using dna extraction samples whole blood samples from the patient and healthy control group were collected in edta heat tube cycles (t professional, biometra, germany). dna template supplement, thermus aquatics dna polymerase (taq polymerase), deoxynucleotide triphosphates (dntps) and buffer solution, applied polymerase chain reaction (pcr) performed with a volume of 5 ml primer volumes, 5 ml dna volumes, pcr temperature: 58) 25 ml total volume by gel electrophoresis separates dna fragments by size in an agarose gel. the reproducibility of genotyping was verified by the average rate of successful genotyping per sample and snp. statistical analysis was done on program past version 3.09. mean±sd and percent total were used in presenting continuous and categorical data, respectively. mean differences and intergroup significances were evaluated using student’s t-test and pearson x2 test, respectively. genetic power calculator was employed for calculating the study power, considering the number of study subjects (41 patients and 45 controls), bmi of the included variants, breast cancer prevalence in kerbala/iraq (estimated), and relative risk for heterozygous (1/2) and minor allele homozygous (2/2) genotypes. hardy–weinberg equilibrium (hwe) law was used for genetic calculation which states that allele and genotype frequencies in a population will remain constant from one generation to the next generation in the absence of disturbing factors. hwe can be used to calculate the expected common homozygotes, heterozygotes, expected rare homozygotes, and the frequency range of the 2 (p and q) alleles from the observed genotypes. calculation of odds ratios (ors) and 95% confidence intervals (95% cis) associated with the risk of breast cancer was determined using logistic regression analysis; statistical significance was set at p value≤0.05. results results showed significant differences between breast cancer patients and control women were noted in (age ; p≤0.01) bmi (p≤0.01), number of smokers (p=0.3), and number of married (p≤0.01) were noted in patients than in control women. these covariates were selected as the main covariates that were controlled for later analysis as showed demographic of study participants are listed in table 1. table 1. the demographic characteristics of bc and non-bc. demographical characteristic patients n = 41 control n = 45 statistical analysis age (y) 47.36 ±14.29 32.6 ± 7.02 ≤ 0.01 bmi 29.6 ± 5.5 24.6 ± 3.2 ≤ 0.01 smoking yes 6 11 p value = 0.3 χ2 = 1.3no 35 34 marriage yes 36 23 p value ≤ 0.01 =13.4 χ2no 5 22 bmi: body mass index, age (y) student’s t-test (continuous variables) and pearson’s x2 (categorical variables). mean ± sd. , number of subjects (percent total). 287 original association betweenmatrix metalloproteinase-2 gene variants and pathogenesis of breast canceryammamah jawad abbas j contemp med sci | vol. 6, no. 6, november–december 2020: 285–290 in table 1, patients were stratified to <50 years by age versus those aged <50 years the mean ± sd value of the age of patients breast cancer (47.36±14.29 years) was significantly higher than control (32.6±7.02 years). when analysis was performed on individual stratification age, incidences of breast cancer were significantly different p<0.01. the diagnosis records of female patients with breast cancer were enrolled in the study. most of the patients (56.09%) were diagnosed with breast cancer before the age of 55. however, only 43% of the cases were diagnosed above the age of 55. patients were reported to include a higher prevalence of women with prolonged menstruation (p≤0.01), past oral contraceptive users (p≤0.01), and a lower frequency of women breastfeeding (p≤0.01). a statistical disparity in marital status, educational level, smoking number (p=0.3, χ2=1.3), and married status was observed (p≤0.01=13.4 χ2). the prevalence of married patients among younger women under 50 years of age was substantially higher; while the characteristics of illiteracy and nulliparity were statistically lower (p≤0.05). including palpable lumps, bloody nipple discharge, skin changes, tumor size, lymph node status, and stage of the disease, observational variations were observed with respect to the clinical appearance of the evaluated patients. a statistically significant increase in the frequency of polymorphic genotypes (ct) and (cc) was observed in the breast cancer group compared to the control group with p≤0.05. as regards allelic frequency of mmp-2 gene, c allele was statistically significantly higher in the breast cancer group the c allele significantly higher in the control group with (p value ≤0.05). by calculating the odds ratios for ct and tt, the results is 0.3 and 0.2, respectively (table 2). genotyping frequencies of mmp-2 gene were analyzed with hwe. the results were showed in table 3 for the mmp2 (-1306 c> t) (rs243865) genotype snps the breast cancer patients group (χ2 = 0.09, c allele frequencies =0.57 and the t allele frequency =0.43, p≤0.05), and control (χ2=0.45, c allele frequencies=0.78 and for allele t is 0.22, p≤0.05). the analysis of results showed that the (rs243865) of mmp-2 gene (c→t) genotype frequencies of wild genotype (cc), heterozygous genotype (ct), and homozygous genotype (tt) were 31.7%, 51.2 %, and 17.1%, respectively in breast cancer patients and 62.2%, 31.1%, and 6.7% in the control group. the results of the table 3 showed chi-square of examined snps in the smoking patients and control cc allele is low compared with no smoking in-patient and control, while the chisquare (x2) = 5.07 is significant at p = 0.7. in the ct allele, we show the chi-square examine of snps in smoking patients and table 3. hardy-weinberg equilibrium for mmp2 (1306 c> t) (rs243865). mmp-2 (-1306 c> t) (rs243865) genotype groups patients n= 41 control n= 45 h-w observed frequency % h-w expected frequency % h-w observed frequency % h-w expected frequency % cc 13 31.7 13.47 32.85 28 62.2 27.2 60.4 ct 21 51.2 20.06 48.93 14 31.1 15.6 34.7 tt 7 17.1 7.47 18.22 3 6.7 2.2 4.9 total 41 100 41 100 45 100 45 100 x2 0.09 0.45 p value hwe ≥ 0.05 ≥ 0.05 allele frequency c 0.57 0.78 t 0.43 0.22 mmp-2: matrix metalloproteinase-2; snp: single-nucleotide polymorphism; hwe: hardy–weinberg equilibrium; or: odds ratio. snp genotyping was done by allelic exclusion method, major allele.minor allele. table 2. genotype of mmp-2 gene (-1306 c/t) polymorphism. mmp-2 (1306 c> t) snp (rs243865) genotype patient n = 41 control n = 45 odds ratio ci 95% p value cc (ref ) 13 (31.7%) 28 (62.2%) ct 21 (51.2%) 14 (31.1%) 0.3 0.12 – 0.8 ≤ 0.05 tt 7 (17.1%) 3 (6.7%) 0.2 0.04 – 0.9 ≤ 0.05 mmp-2: matrix metalloproteinase-2; snp: single-nucleotide polymorphism; or: odds ratio. snp genotyping rs243865was done by allelic exclusion method primers. (major allele.minor allele,minor allele c> t number (frequency). crude (unadjusted) or. 288 original association betweenmatrix metalloproteinase-2 gene variants and pathogenesis of breast cancer yammamah jawad abbas j contemp med sci | vol. 6, no. 6, november–december 2020: 285–290 control compared with no smoking number in-patient and control, while the chi-square (x2) value 0.3 at p = 0.6. and the last allele tt, the result of smoking patient and control compared with no smoking patient and control the chi-square (x2) value 2.7 at p = 0.098. the results showed that the chi-square (chi-square) of the snps that were examined in the married patient and control group with a high cc allele compared with the unmarried, the chi-square value (x2) 5.07 at p ≤ 0.05. for a ct allele in a married patient, the chi-square of the snps examined, control compared with his unmarried patient, married, and control, the chi (x2) value is 4.4 at p ≤ 0.05. the result is the last allele tt is the chi-square of the snps examined in a married patient, control compared with unmarried, the chi (x2) value of 0.47 at p = 0.49. most were married with an average of 2–4 children (95% preand 97% post-menopausal), 2% have a close relative of cancer of breast cancer. relationship between bmi and the occurrence of genotypes of mmp-2, we divided the group of patients with breast cancer into two simple categories based on mean ± sd: the first includes patients with a bmi of the cc allele (31.2 ± 6.4) and the second group with a mass index is 24.3 ± 2.3 with a p ≤ 0.01. the second allele is ct included in the patient (28.6 ± 5.5) and control is (24.7 ± 3.98) with p = 0.05. finally, the tt allele is 30 ± 3.7 and the control (26.3 ± 6.4) with a p = 0.3. when comparing the tt and ct genotype versus the wild (cc) genotype, a significant difference was observed between the control group and the patient group with a p ≤ 0.01. table 4. demographic with mmp-2. demographic with mmp-2 (1306 c> t) (rs243865) genotype patients n =41 control n = 45 chi square test smoking cc yes 2 6 p value = 0.7 x2 = 0.2no 11 22 ct yes 3 3 p value = 0.6 x2 = 0.3no 18 11 tt yes 1 2 p value = 0.098 x2 = 2.7no 6 1 demographic with mmp2 (1306 c> t) (rs243865) genotype patients n = 41 control n = 45 chi square test marriage cc yes 12 16 p value ≤ 0.05 x2 = 5.07no 1 12 ct yes 15 5 p value ≤ 0.05 x2 = 4.4no 6 9 tt yes 6 2 p value = 0.49 x2 = 0.47no 1 1 bmi cc 31.2 ± 6.4 24.3 ± 2.3 ≤ 0.01 ct 28.6 ± 5.5 24.7 ± 3.98 ≤ 0.05 tt 30 ± 3.7 26.3 ± 6.4 0.3 discussion breast cancer is the primary cause of cancer-related deaths worldwide. several studies have shown enhanced expression of mmp-2 in breast cancer, and genetic functional variants in the mmp-2 gene were correlated with altered breast cancer susceptibility.19,22 our analysis found that the correlation of rs243865 and mmp-2 snp with the occurrence and aggressiveness of breast cancer was correlated with breast cancer in multiple populations. the mmp-2 gene contains many polymorphisms on chromosome 16, of which the promoter variant rs243865 is related to lower promoter activity.22 the decreased expression is predicted to be associated with a decreased risk of cancer in the development of diseases, including breast cancer. second, several genetically engineered animal studies have linked a low degree of constitutive expression of mmp-2 with a decreased risk of tumor development. it was observed that mice missing the mmp-2 gene developed fewer tumors than wild-type mice when caused by a carcinogenic stimulus.31 it was observed that cancer cells inserted into a vein were less likely to colonize the lungs of mmp-2 knockout mice than those of wild-type mice.32 although some familial risk may be due to shared environmental factors, there may be other common, low penetrance genetic variants affecting susceptibility to breast cancer. because the mmp-2 system has a significant impact on the development and progression of cancer, including breast cancer, and because genetic polymorphisms in the 289 original association betweenmatrix metalloproteinase-2 gene variants and pathogenesis of breast canceryammamah jawad abbas j contemp med sci | vol. 6, no. 6, november–december 2020: 285–290 promoters of mmp-2 are correlated with decreased enzyme activity, we sought to determine whether these polymorphisms may be associated with varying risk of breast cancer. based on an analysis of 41 patients with breast cancer and 45 controls, we found that the mmp-2 polymorphisms influenced the risk of developing the tnm stage and metastasis of breast cancer. subjects carrying the variant genotypes of mmp-2 were at a moderately reduced risk of cancer. in addition, it appeared that the polymorphisms in the two genes had some additive effect regarding reducing breast cancer risk and the protective effects were more pronounced in younger subjects (s55 years old), which is in line with the conception that genetic susceptibility is often associated with an early age of disease onset. our findings were consistent with other research,21,23 which also reported a negative correlation between rs243865 and breast cancer risk. our results were in strong disagreement with other studies,24,25 which showed no link between rs243865 and breast cancer risk. the rs243865 minor (t) allele was functionally associated with disruption of the binding of sp1 binding elements, resulting in decreased activity of the promoter. as in a report, data were kept for the diagnosis of women with breast cancer. by the age of 55, most patients (78.0%) were diagnosed with breast cancer. however, only cases above the age of 55 were diagnosed (21.95%).28 demographic and therapeutic profiles of research subjects. highly substantial bmi (p ≤ 0.01) and percentage of smoke (p ≤ 0.01) between breast cancer patients and control women were found to have relevant differences. a greater proportion of people with extended menstruation (p ≤ 0.01), former oral contraceptive users (p ≤ 0.01), and a lower incidence of women breastfeeding (p ≤ 0.01) compared to control persons have been identified in patients. such covariates were selected, as these covariates were chosen as the primary control covariates.14 as calculated by the bmi, core obesity tends to have a greater effect on the prevalence of breast cancer in african–american females than general adiposity. the pre-menopausal women have 21% an elevated value of bmi and 29% of post-menopausal women with an abdomen diameter of 90 cm are also more likely to develop breast cancer.29 the hwp deviation in the controls is determined by measuring the difference between the observed genotype frequencies and the expected frequencies. it is used in genotyping to identify errors. using only controls for hwp assessment is reasonable when assuming an unusual condition in the study. in the present research, it is clear that the c allele for mmp-2 (-1306 c>t) was linked with breast cancer and may be considered a risk factor for disease progression.27 results from these studies may have implications for possible strategies for cancer prevention, especially for communication and therapy of person-level risk. in this respect, it is reassuring that even for people with the highest decile of risk for those with a low bmi, who did not smoke or drink and who did not use mht, the risks for non-modifiable variables were equal to those of the average woman in the general population.26 despite contradictory effects, local overexpression of mmp-2 is commonly considered facilitating and prevents cancer invasion and metastasis by timp-2. a number of studies have shown that genetic polymorphisms in mmp-1 (1g/2g) or mmp-3 (5a/6a) promoters that modify gene transcription activity may affect the invasiveness or metastasis of certain types of cancer, such as melanoma,34 colorectal cancer,35 and breast cancer.33 however, neither genotype of mmp-2 was substantially associated with the tumor stage or metastatic status of breast cancer at the time of diagnosis in the present study. these observations show that the polymorphisms studied in mmp-2 do not play a major role in suppressing or inducing local mmp-2 expression as a relevant genetic factor, and may therefore not serve as a sole risk marker for metastatic disease. however, there are certain drawbacks to our results on the metastasis of breast cancer since they were collected at the time of diagnosis. it may be appropriate to further analyze wider case series with prospectively followed-up clinical results, especially the survival rate. conclusion the observed data indicated that in women with breast cancer, mmp-2 expression is highly associated with positive lymph node, histological classification of breast cancer (ll) which was higher than other classes, and advanced clinical process (ll). conflict of interest none references 1. hussain sm. detection of estrogen receptor alpha and beta gene mutations in iraqi women with breast cancer. 2016;(june 2016). 2. beeghly-fadiel a, lu w, long j, shu x, zheng y, cai q, et al. matrix metalloproteinase-2 polymorphisms and breast cancer susceptibility. 2009;18(june):1770–7. 3. wolff ac, hammond me, schwartz jn, et al american society of clinical oncology/college of american pathologists: american society of clinical oncology/college of american pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. arch pathol lab med. 2007;131:18-43. 4. tamimi rm, byrne c, colditz ga, hankinson se. endogenous hormone levels, mammographic density, and subsequent risk of breast cancer in postmenopausal women. j natl cancer inst. 2007;99: 1178-1187 5. talvensaari-mattila a, paakko p, blanco-sequeiros g, turpeenniemi-hujanen t. matrix mettaloproteinase-2 (mmp-2) is associated with the risk for a relapse in postmenopausal patients with node-positive breast carcinoma treated with antiestrogen therapy. breast cancer res treat 2001;65:55–6. 6. singh m, agrawal a. assessment of risk factors of breast cancer among women attending tertiary care hospital of chattisgarh: a case control study. ind j surg 2020:1–5. 7. bray f, ferlay j, soerjomataram i. global cancer statistics 2018 : globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 2018;394–424. 8. shah r, rosso k, nathanson, sd. pathogenesis, prevention, diagnosis and treatment of breast cancer. world j clin oncol. 2014;5(3):28. 9. mandal ba. breast cancer pathophysiology. breast cancer pathophysiol. 2019;1–3. 10. das k, prasad r, ahmed s, roy a, mukherjee a. biomedicine & pharmacotherapy matrix metalloproteinase-2 : a key regulator in coagulation proteases mediated human breast cancer progression through autocrine signaling. biomed pharmacother [internet]. 2018;105(may):395–406. 11. radisky es, radisky, dc. matrix metalloproteinases as breast cancer drivers and therapeutic targets. front biosci (landmark edition). 2015;20:1144. 12. saarto t, vehmanen l, blomqvist c, elomaa i. a high serum matrix metalloproteinase-2 level is associated with an adverse prognosis in nodepositive breast carcinoma. 2004;10:1057–63. 13. zgajnar j. ‘clinical presentation , diagnosis and staging of breast cancer’, 2018:159–176. 14. habel af et al. ‘common matrix metalloproteinase-2 gene variants and altered susceptibility to breast cancer and associated features in tunisian women’, 2019;(april):1–8. 15. chen y et al. ‘the impact of matrix metalloproteinase 2 on prognosis and clinicopathology of breast cancer patients : a systematic meta-analysis’, 2015:1–16. 16. ranogajec i, matrix metalloproteinases in breast carcinoma. in proteases in human diseases. springer, singapore. 2017:3-20. 290 original association betweenmatrix metalloproteinase-2 gene variants and pathogenesis of breast cancer yammamah jawad abbas j contemp med sci | vol. 6, no. 6, november–december 2020: 285–290 17. iliyasu y, atanda at, molecular subtyping of carcinoma of the female breast in a tertiary teaching hospital in northern nigeria. ann trop pathol. 2019;10(1):20. 18. kleibl z, kristensen vn ‘women at high risk of breast cancer : molecular characteristics , clinical presentation and management’, the breast. elsevier ltd, 2016;28:136–144. 19. beeghly-fadiel a, lu w, long jr, shu xo, zheng y, cai q, gao yt, zheng w. matrix metalloproteinase-2 polymorphisms and breast cancer susceptibility. cancer epidemiol prev biomarkers. 2009;18(6):1770-1776. 20. habel af, ghali rm, bouaziz h, daldoul a, hadj-ahmed m, mokrani a, zaied s, hechiche m, rahal k, yacoubi-loueslati b, almawi wy. common matrix metalloproteinase-2 gene variants and altered susceptibility to breast cancer and associated features in tunisian women. tumor biol. 2019;41(4):1010428319845749. 21. zhou y, yu c, miao x, tan w, liang g, xiong p, sun t, lin d. substantial reduction in risk of breast cancer associated with genetic polymorphisms in the promoters of the matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 genes. carcinogenesis, 2004;25(3):399-404. 22. néjima db, zarkouna yb, gammoudi a, manai m, boussen h. prognostic impact of polymorphism of matrix metalloproteinase-2 and metalloproteinase tissue inhibitor-2 promoters in breast cancer in tunisia: case-control study. tumor biol. 2014;36:3815-3822. 23. kawal p, chandra a, dhole tn, et al. correlations of polymorphisms in matrix metalloproteinase-1, -2, and -7 promoters to susceptibility to malignant gliomas. asian j neurosurg 2016;11(2):160–166. 24. lei h, hemminki k, altieri a, et al. promoter polymorphisms in matrix metalloproteinases and their inhibitors: few associations with breast cancer susceptibility and progression. breast cancer res treat 2007;103(1):61–69. 25. roehe av, frazzon ap, agnes g, et al. detection of polymorphisms in the promoters of matrix metalloproteinases 2 and 9 genes in breast cancer in south brazil: preliminary results. breast cancer res treat 2007;102(1):123–124. 26. maas p. et al. ‘breast cancer risk from modifiable and nonmodifiable risk factors among white women in the united states’. 2016;21205(10):1295–1302. 27. zhou y. et al. ‘substantial reduction in risk of breast cancer associated with genetic polymorphisms in the promoters of the matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 genes’, 2004;25(3):399–404. 28. nouri mm. ‘breast cancer molecular subtypes in relation to age , stage and grade among sudanese women patients in khartoum oncology hospital (2013–2017)’, 2019;2(august 2017):1–9. 29. desantis c, ma j, bryan l, jemal a. breast cancer statistics, 2013. ca cancer j clin. 2014;64(1):52-62. 30. talvensaari-mattila a, pa¨a¨kko¨ p, turpeenniemi hujanen t. matrix metalloproteinase-2 (mmp-2) is associated with survival in breast carcinoma. br j cancer 2003;89:1270–1275.31. bergers g, brekken r, mcmahon g, vu th, itoh t, tamaki k, tanzawa k, thorp p, itohara s, werb z, hanahan d. matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. nature cell biol. 2000;2:737-744.32. itoh t, tanioka m, yoshida h, yoshioka t, nishimoto h, itohara s. reduced angiogenesis and tumor progression in gelatinase a-deficient mice. cancer res. 1998;58:1048-1051.33. ghilardi g, biondi ml, caputo m, leviti s, demonti m, guagnellini e, scorza r. a single nucleotide polymorphism in the matrix metalloproteinase-3 promoter enhances breast cancer susceptibility. clin. cancer res. 2002;8:3820-3823.34. ye s, dhillon s, turner sj, bateman ac, theaker jm, pickering rm, day i, howell wm invasiveness of cutaneous malignant melanoma is influenced by matrix metalloproteinase 1 gene polymorphism. cancer res. 2001;61:1296-1298. 35. ghilardi g, biondi ml, mangoni j, leviti s, demonti m, guagnellini e, scorza r. matrix metalloproteinase-1 promoter polymorphism 1g/2g is correlated with colorectal cancer invasiveness. clin. cancer res. 2001;7:2344-2346. https://doi.org/10.22317/jcms.v6i6.887 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 32 j contemp med sci | vol. 6, no. 1, january–february 2020: 32–38 original issn 2413-0516 introduction alzheimer’s disease (ad) is the most common progressive disorder in central nervous system (cns) with effects on awareness contents and cognitive abilities.1,2 alois alzheimer introduced ad for the first time by dissecting the brain of a middle-aged woman who had suffered from memory deficits and lack of progressive abilities.1,3 three obvious symptoms of ad are: (1) atrophy of brain in which its weight drops, gyri narrow, sulci widen and ventricles dilate. (2) acceleration of extracellular beta-amyloid plaques (aβ, also called neuritic or senile plaques) formed by the amyloid precursor protein (app) assembling. (3) neurofibrillary tangles attributed neuronal death: abnormalities in the cellular skeleton upset the axonal transport due to disorganization of microtubules involved there-in.4 in terms of prevalence, 1/8th or, in other words, about 10% of people over the age of 65 are afflicted with ad.5 ad is divided into two main types: (1) familial alzheimer’s disease (fad) or the inherited type that occurs prematurely and (2) sporadic alzheimer disease (sad) that occurs individually without genetic mutations, often in the 80’s.5 in this study, intracerebroventricular (icv) injection of streptozotocin (stz), as a diabetogenic factor, was used to suppress glucose metabolism. the stz disrupts the regulating role of insulin and its receptor resulting in destroyed glucose metabolism in brain, lack of energy, lack of cholinergic system, and nerve growth along with learning, memory, general, and local anomalies.6 all of these disorders are attributed to stz that mediates glucose metabolism by means of insulin signaling cascade.4,7 to induce progressive deterioration, like the one seen in sad type, stz is injected which causes a significant reduction of energy in hippocampal and cerebral cortical tissue and therefore, a disruption in the energy associated processes including eating, sorting, and packing of proteins in the golgi organelles. the transmission of neurotransmitters, learning behaviors, memory, and energy-rich compounds are progressively affected.8 the progressive decline in shortand longterm memory in the post-administration phase of intravenous injection and the resultant disorders in behavior and glucose metabolism make stz suitable for sad model. morris water maze (mvm) and passive avoidance memory behavioral tests have confirmed that stz produces ad.9 it was believed for many years that the cns has no neuroregenerative ability at adulthood. nearly a century ago, as the technology progressed, cells that can replicate and differentiate into neurons and glia were identified in some areas of the brain and spinal cord. and until now, active neuronal activity in the subventricular zone (svz) in lateral wall of lateral ventricles, subgranular zone of dentate gyrus, and hypothalamic subependymal zone has been documented.10–12 in the early part of the century, allen identified mitotic cells in the svz of lateral ventricles in mature rats. ventricular region forms during embryonic development near the triiodothyronine improves morphology and upregulates seladin-1 of neurospheres extracted from subventricular zone in streptozotocininduced rat model of alzheimer’s disease tahmineh mokhtari,a,b simin mahahakizadeh,c hadi aligholi,d sahar ijaz,e,f leila noori,e gholamreza hassanzadehg,h,e acas key laboratory of mental health, institute of psychology, beijing, china. bdepartment of psychology, university of chinese academy of sciences, beijing, china. cdepartment of anatomy, school of medicine, alborz university of medical sciences, karaj, iran. ddepartment of neuroscience, school of advanced medical sciences and technologies, shiraz university of medical sciences, shiraz, iran. edepartment of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. fdepartment of anatomy & histology, faculty of biosciences, university of veterinary & animal sciences, lahore, pakistan. gdepartment of neuroscience and addiction studies, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. hlegal medicine research center, legal medicine organization, tehran, iran. correspondence to: gholamreza hassanzadeh (email: hassanzadeh@tums.ac.ir). (submitted: 21 november 2019 – revised version received: 14 december 2019 – accepted: 25 december 2020 – published online: 26 february 2020) objectives in this study, the effects of triiodothyronine (t3) on neurospheres isolated from subventricular zone (svz) of alzheimer’s disease (ad) induced rats were examined. methods eighteen male wistar rats were classified into two groups: sham (sh) and stz (streptozotocin injected, 1.5 mg/kg in each lateral ventricle on days 1 and 3 after recovery). on day 21, the svz was extracted and neurospheres were cultured. t3 (50 nm) was added to the culture medium (stz+t3 group) and then, the morphology and seladin-1 gene expression of neurospheres were evaluated. results the diameter and the number of neurospheres along with the gene expression of seladin-1 were significantly decreased in the stz group compared to sh group (p < 0.05) while the administration of t3 significantly (p < 0.05) increased all these parameters in the stz group. conclusion stz decreases the proliferation of stem cells extracted from svz and administration of t3 to the culture media improves the morphology and upregulates the gene expression of seladin-1 of neurospheres. keywords alzheimer’s disease, subventricular zone, neurospheres, seladin-1. 33 original t3 improved the neurospheres from svz in model of alzheimer’s diseaset. mokhtari et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 32–38 ventricular area and is very noticeable in the ganglionprotuberance region.13 it is composed of a number of cells, possibly, b1 cells, which are the same as astrocytes. they are stem cells in the ventricular region, and these cells divide and make type c-cells that are capable of rapid proliferation. c-type cells, in turn, increase and convert to type a cells that are migratory neuroblasts. a-cells are surrounded by cells of type b1 and c.14,15 preconditioning of stem cells with growth factors can improve their ability of proliferation and development and also promotes neuronal regeneration and survival.16 among these factors, thyroid hormones (ths) such as triiodothyronine (t3) are critical for brain development.17 although, t3 is less than thyroxine [t4], it is the most active form of ths.18 furthermore, the neuroprotective impacts of ths are related to enhancement of neurotrophic factors (nfs) and their anti-apoptotic and anti-inflammatory mechanisms have been proven.19 in this study, we decided to evaluate the effects of this hormone on the level of seladin-1 expression in the neurospheres isolated from svz in ad-induced rats. methods and materials study design and animals a total number of 18 male wistar albino rats (100–150  g) were purchased from shefa neuroscience research center, khatam alanbia hospital, tehran, iran. all animals were maintained in a clean and hygienic environment, on a 12-h light and dark cycle and 23  ±  2  °c temperature, and had access to food and water ad libitum. all procedures were carried out in accordance with the guidelines of the iranian council for use and care of animals and approved by ethical committee of tehran university of medical sciences. this study was granted by iran national science foundation (grant no. 93009595). subjects were randomly divided into two different groups: sham (sh) group which received 5 ml normal saline in each lateral ventricle, stz group which received 1.5 mg/kg stz in 5 ml normal saline in each lateral ventricle on days 1 and 3. all subjects were housed in keeping cages in the resting time. stereotaxic surgery rats were deeply anesthetized with ketamine (100 mg/kg) and xylazine (25  mg/kg) (both razi co., iran) intraperitoneally. for chronic implantation of cannula (27-gauge) into lateral cerebral ventricles (icv) under stereotactic guidance, appropriate narcosis was verified by reflex testing such as the lack of ocular reflex and the absence of a pedal withdrawal response to a hard pinch. therefore, animals were fixed in a stereotactic apparatus, and a midline incision was made on the cranial skin. then, a small hole was induced in the cranial region and the guide cannula was implanted and fixed. coordinates were ap-0.8 mm, l ± 1.5 mm (midline) and 3.6 mm deep from the dura mater. animals were allowed 1 day to recover before stz injection. stz and its vehicle were administered on days 1 and 3 after recovery with a 10 µl hamilton syringe (5 µl in each ventricle at a rate of 1  µl/min) connected via a teflon tube to an injector that exceeded by 2 mm the length of the guide cannula.4,7 morris water maze at the end of the experiment, to confirm the defects in learning and memory of animals, their behaviors were assessed by mwm test based on the previously described methods.20 it consisted of a circular water tank (160 cm diameter, 60 cm height) filled with water (25  ±  1  °c) to a depth of 25  cm. a non-toxic water dispersible emulsion was used to render the water opaque. four equally spaced locations around the edge of the pool (north, south, east, and west) were used as start points which divided the pool into four quadrants. an escape platform (10  cm in diameter) was placed in the pool 2  cm below the surface of water. the escape platform was placed in the middle of one of the randomly selected quadrants of the pool and kept in the same position throughout the entire experiment (north-east for this study). animals received a training session consisting of four trials per session (once from each starting point) for 4 days (days 16, 17, 18, and 19), each trial having a ceiling time of 90  s and a trial interval of approximately 30 s. after climbing onto the hidden platform, animals remained there for 30  s before commencement of the next trial. if the rat failed to locate the hidden platform within the maximum time of 90 s, it was gently placed on the platform and allowed to remain there for the same interval of time. the time taken to locate the hidden platform (latency in s) was measured. twenty-four hours after the acquisition phase, a probe test (day 20) was conducted by removing the platform. rats were allowed to swim freely in the pool for 90 s and the time spent in target quadrant, which had previously contained the hidden platform, was recorded. the time and distance spent in the target quadrant indicated the degree of memory consolidation which had taken place after learning. cell culture on day 21, animals were sacrificed from both the groups (sh and stz) and the svz was isolated. the tissue samples were digested after the mechanical digestion with 250  μl trypsin 0.05% for 4  min and centrifuged by 810  rpm for 5  min after adding trypsin inhibitor. cell deposition in neurosphere cell culture, that was consist of dmem  +  f12 (gibco) glutamine 1% (gibco), b27 supplement 1% (gibco) and epidermal growth factor 20  ng/ml (sigma), was cultivated in incubator with 37  °c temperature, 95% humidity, and 5% co2. within 15 days, nerve/protozoal stem cells were proliferated as a series of spherical cells called neurospheres. by increasing the volume of the neurospheres, the cell passage by collecting the neurons in the falcon tube and centrifuging for 5 min at 810 rpm was performed. to test the effects of t3 on neurospheres, t3 (50 nm) was added to the medium (stz + t3 group). medium was renewed every 2–3 days. morphometric studies in order to evaluate cell proliferation, both the numbers and diameters of neurospheres were considered. on the seventh day of cultivation, five microscopic images were taken randomly from each container. cell counting was performed by using trypan blue and neobar lam and after the second passage, the diameter and number of neurons were measured. number of neurospheres was counted by infinity software. the neurospheres diameter were measured by using the infinity software 34 original t3 improved the neurospheres from svz in model of alzheimer’s disease t. mokhtari et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 32–38 and the results were described as the mean of two perpendicular diameters to the diameter of the neurospheres. the number and diameter of the neurospheres and cells were compared. real-time pcr expression of seladin-1 in neurospheres was measured by real-time pcr. total rna was isolated from hemispheres, then mrna (1  μg) was converted to cdna via reverse transcription using first strand cdna synthesis kit (thermo scientific, usa). specific primers along with cdna and pcr reagents (polymerase, dntp, magnesium and buffer; 5× hot firepol® evagreen® qpcr mix plus [rox] 1  ml, solis bio dyne, estonia) were placed on the three-color real-time pcr machine (applied biosystems step one, usa) for further analysis. at first, incubation of samples was performed at 95 °c for 15 min for initial polymerase activation. then, samples underwent the three subsequent phases as follows: (1) denaturation (at 95  °c for 15  s); (2) annealing (at 60  °c for 20  s); and (3) elongation (at 72 °c for 20 s). finally, quantification of data, its normalization to gapdh and fold change comparison to the control were performed.21 in table 1, the nucleotide sequences of primers are shown. table 1. primer sequence seladin-1 forward 5′-gggtgtttgtgtgcctcttcc-3′ reverse 5′-gctccttccactcccgtacc-3′ b-actin forward ′-ctgtctggcggcaccaccat-3′ reverse 5′-gcaactaagtcatagtccgc-3′ statistical analysis all data were analyzed through one-way analysis of variance (anova) and the post-hoc tukey’s, and two-way anova and the bonferroni’s multiple comparison tests were applied for behavioral data belonging to learning and memory skills using graph-pad prism version 6 software (san diego, california, usa). results were represented as mean  ±  sem and p < 0.05 was considered significant. results effects of icv injection of stz on learning and memory in the rat model of ad injection of stz induced defect in spatial learning and memory of stz group. according to fig. 1, the mean latency time to reach the hidden platform (p < 0.05, fig. 1a) and distance traveled in the mvm (p  <  0.05, fig. 1b) were significantly increased in stz group compared to sh group. moreover, stz-treated groups spent significantly less time (p < 0.05, fig. 1c) and distances in the platform quadrant (p < 0.05, fig. 1d) compared to sh group. effects of t3 on the number of neurospheres extracted from svz in the rat model of ad the number of neurospheres decreased in the ad model of rat compared with sh group (p  <  0.05, fig. 1). culturing with medium contained t3 increased the number of neurospheres in the stz + t3 neurospheres compared to stz group (p < 0.05, fig. 1). effects of t3 on diameter of neurospheres extracted from svz in the rat model of ad based on the results, the diameter of neurospheres decreased in the ad model group (stz group) compared with sh group (p < 0.05, fig. 2). the diameter of neurospheres in the stz + t3 increased compared to stz group (p < 0.05, fig. 1). effects of t3 on seladin-1 gene expression of neurospheres extracted from svz in the rat model of ad based on the results, the seladin-1 gene expression decreased in the ad model group (stz group) compared to sh group (p < 0.05, fig. 2). the seladin-1 gene expression in t3 group increased compared to stz group (p < 0.05, fig. 1). discussion in this study, the effects of t3 on morphology and seladin-1 gene expression of neurospheres extracted from svz were investigated in rat model of ad. in this study, stz was used to induce ad in animals. ad is the most common cause of dementia diagnosed after the age of 60, and it is characterized by neuronal loss as a consequence of neurofibrillary tangles and senile plaques. there are different theories which explain the accumulation of beta amyloid plaques, but abnormal levels of oxidative stress have been reported in both brain and blood stream in ad.22,23 an enhanced oxidative stress induced modified proteins and mitochondrial dysfunction in ad brain have been reported,24,25 and it has also been suggested that oxidative stress is a key for the progression of ad.26 the mechanism of ad development in the brain is unknown. however, different studies have proved that chronic icv injections of stz can reduce uptake of cerebral glucose and induce multiple other effects that resemble behavioral, molecular, pathological characteristics of ad.27 the results of present study revealed that the diameter and the number of neurospheres were significantly decreased in the stz treatment group compared to sh group. ad, the widespread cause of dementia, is characterized by progressive neurodegeneration and the appearance of specific histopathological markers represented by focal extracellular deposits of fibrillar aβ in the brain parenchyma and in the wall of blood vessels, and by the intraneuronal accumulation of neurofibrillary tangles formed as a result of the abnormal hyperphosphorylation of cytoskeletal tau filaments.28 the initial neurodegenerative events of ad appear in the transentorhinal cortex, and subsequently spread to the entorhinal cortex and to the hippocampus.29,30 svz, as a neurogenic niche, contains multipotent neural stem cells (nscs).31,32 the nscs that exhibit slow self-renewal produce neural progenitor cells with a faster dividing cell cycle, which ultimately differentiate into neurons or neuroglia; the differentiation process is regulated by numerous trophic factors.33,34 the majority of studies performed on transgenic animals expressing the mutant app demonstrated decreased neurogenesis either in the dentate gyrus (dg) or in both the dg and the svz.35–38 in a previous similar study, mozhdeh et al. demonstrated that the icv 35 original t3 improved the neurospheres from svz in model of alzheimer’s diseaset. mokhtari et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 32–38 injection of stz impaired the proliferation feature of svz extracted neurospheres in a rat model of ad9 which confirmed the results of present study. the results of present study showed that t3 could increase the seladin-1 gene expression in the stem cells extracted from svz in the rat model of ad. the mechanisms of th levels alteration as a factor to increase the mobility towards ad in older people is unclear. numerous researches tried to find a relation between ad and th levels. in a study by ceresini et al.,39 1171 participants from italy were analyzed and it was reported that dysfunction of thyroid gland tends to be more frequent in older people than in youngsters, with subclinical hyperthyroidism being the most prevalent condition. their findings demonstrated an independent association between cognitive impairment and subclinical hyperthyroidism. their findings were similar to those obtained from kalmijn et al. investigation.40 some studies have demonstrated a very high prevalence of autoimmune thyroid disease in familial ad.41,42 similarly, ganguli et al. reported that the subclinical hypothyroidism state correlates with cognitive disorders in patients aged ≥65.43 an alternative mechanism of th to improve ad has been described. the increase of the seladin-1 gene and protein expression of seladin-1 (selective ad indicator 1) gene cause decrease neuronal death,31,44 promotes cholesterol synthesis inside the neuron,45 and reduce aβ accumulation by inhibition fig. 1 effects of icv injection of stz on spatial memory and learning in the rat model of ad. (a) latency to platform (s), (b) sistance traveled to platform, (c) time, and (d) distance spent in the platform quarter. p <  0.05 compared to sh group. sh: sham group with icv injection of normal saline; stz: animals with icv injection of stz 36 original t3 improved the neurospheres from svz in model of alzheimer’s disease t. mokhtari et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 32–38 fig. 2 effects of t3 on the number of neurospheres extracted from svz in the rat model of ad. (a) p <  0.05 compared to sh group, (b) p <  0.05 compared to stz group. sh: neurospheres isolated from rats received icv injection of normal saline; stz: neurospheres isolated from rats received icv injection of stz, stz + t3: neurospheres isolated from rats received icv injection of stz and cultured with t3. fig. 3 effects of t3 on seladin-1 gene expression of neurospheres extracted from svz in the rat model of ad. the gene expression of seladin-1 was investigated using real-time pcr. (a) p <  0.05 compared to sh group, (b) p <  0.05 compared to stz group. sh: neurospheres isolated from rats received icv injection of normal saline; stz: neurospheres isolated from rats received icv injection of stz; stz + t3: neurospheres isolated from rats received icv injection of stz and cultured with t3. 37 original t3 improved the neurospheres from svz in model of alzheimer’s diseaset. mokhtari et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 32–38 of co-localization of aβ precursor protein.46 th exerts an essential effect on the maintenance and development of cns.47 the effectiveness of th on seladin-1 gene expression was investigated. ishida et al. reported that th upregulated the expression of seladin-1, a gene related to ad in whole cell extract from mouse forebrain.44 benvenuti et al. reported that th increased tr-α1, tr-β1, and tr-β2 and seladin-1 gene expression in an in vitro model of human neuronal precursor cell lines.48 likewise, the beneficial effects of t3 on neurodegenerative diseases have been presented in a study by mokhtari et al., it was proven that injection of t3 improved memory and learning by upregulation of brain-derived neurotropic factor and glial cell-derived neurotropic factor in ca1 hippocampal region in rat model of ischemic brain stroke.49 conclusion the results obtained from this study showed that the icv injection of stz decreases the number and diameter of neurospheres and seladin-1 gene expression in rats (model of ad). additionally, adding t3 to the culture medium of these neurospheres improves their morphological characteristics and increases seladin-1 gene expression. acknowledgement this work was funded by the iran national science foundation (grant 93009595). references 1. katzman r. the prevalence and malignancy of alzheimer disease: a major killer. arch. neurol. 1976;33(4):217–8. 2. hassanzadeh g, hosseini a, pasbakhsh p, akbari m, ghaffarpour m, takzare n, et al. trimetazidine prevents oxidative changes induced in a rat model of sporadic type of alzheimer’s disease. acta med. iran. 2015;53(1):17–24. 3. kumar a, singh a. a review on alzheimer’s disease pathophysiology and its management: an update. pharmacol. rep. 2015;67(2):195–203. 4. hassanzadeh g, fallahi z, khanmohammadi m, elmizadeh h, sharifzadeh m, nouri k, et al. effect of magnetic tacrine-loaded chitosan nanoparticles on spatial learning, memory, amyloid precursor protein and seladin-1 expression in the hippocampus of streptozotocin-exposed rats. int. clin. neurosci. j. 2016;3(1):25–31. 5. peri a, serio m. neuroprotective effects of the alzheimer’s disease-related gene seladin-1. j. mol. endocrinol. 2008;41(5):251–61. 6. condello c, lemmin t, stöhr j, nick m, wu y, maxwell am, et al. structural heterogeneity and intersubject variability of aβ in familial and sporadic alzheimer’s disease. proc. natl acad. sci. 2018:201714966. 7. biasibetti r, dos santos jpa, rodrigues l, wartchow km, suardi lz, nardin p, et al. hippocampal changes in stz-model of alzheimer’s disease are dependent on sex. behav. brain res. 2017;316:205–14. 8. guigon cj, zhao l, lu c, willingham mc, cheng s-y. regulation of β-catenin by a novel nongenomic action of thyroid hormone β receptor. mol. cell. biol. 2008;28(14):4598–608. 9. mozhdeh hp, zeynali b, aligholi h, radgerdi ik, negah ss, hassanzadeh g. the effect of intracerebroventricular administration of streptozocin on cell proliferation in subventricular zone stem cells in a rat model of alzheimer’s disease. neurosci. j. shefaye khatam. 2015;3:80–6. 10. andreotti jp, lousado l, magno lav, birbrair a. hypothalamic neurons take center stage in the neural stem cell niche. cell stem cell. 2017;21(3):293–4. 11. shan x, tomlinson l, yang q, colognato h. distinct requirements for extracellular and intracellular mmp12 in the development of the adult v-svz neural stem cell niche. stem cell rep. 2018;10(3):984–99. 12. corsini ns, knoblich ja. tracing stem cell division in adult neurogenesis. cell stem cell. 2018;22(2):143–5. 13. aligholi h, hassanzadeh g, azari h, rezayat sm, mehr se, akbari m, et al. a new and safe method for stereotactically harvesting neural stem/ progenitor cells from the adult rat subventricular zone. j. neurosci. methods. 2014;225:81–9. 14. doetsch f, caille i, lim da, garcía-verdugo jm, alvarez-buylla a. subventricular zone astrocytes are neural stem cells in the adult mammalian brain. cell. 1999;97(6):703–16. 15. fuentealba lc, obernier k, alvarez-buylla a. adult neural stem cells bridge their niche. cell stem cell. 2012;10(6):698–708. 16. xiong l-l, chen z-w, wang t-h. nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews. neural regen. res. 2016;11(4):591–6. 17. de escobar gm, obregón mj, del rey fe. role of thyroid hormone during early brain development. eur. j. endocrinol. 2004;151(suppl 3):u25–u37. 18. yen pm. physiological and molecular basis of thyroid hormone action. physiol. rev. 2001;81(3):1097–142. 19. genovese t, impellizzeri d, ahmad a, cornelius c, campolo m, cuzzocrea s, et al. post-ischaemic thyroid hormone treatment in a rat model of acute stroke. brain res. 2013;1513:92–102. 20. mahakizadeh s, akbari m, sharifzadeh m, rastegar t, abolhassani f, hassanzadeh g. wharton’jelly mesenchymal stem cells and insulin effect on bdnf expression in ca1 and ca3 regions of rats’ hippocampus after chronic hypoxia. j. contemp. med. sci. 2018;4(2):63–9. 21. sabbaghziarani f, mortezaee k, akbari m, soleimani m, moini a, ataeinejad n, et al. retinoic acid-pretreated wharton’s jelly mesenchymal stem cells in combination with triiodothyronine improve expression of neurotrophic factors in the subventricular zone of the rat ischemic brain injury. metab. brain dis. 2017;32(1):185–93. 22. sutherland gt, chami b, youssef p, witting pk. oxidative stress in alzheimer’s disease: primary villain or physiological by-product? redox rep. 2013;18(4):134–41. 23. schrag m, mueller c, zabel m, crofton a, kirsch w, ghribi o, et al. oxidative stress in blood in alzheimer’s disease and mild cognitive impairment: a meta-analysis. neurobiol. dis. 2013;59:100–10. 24. butterfield da, reed t, newman sf, sultana r. roles of amyloid β-peptideassociated oxidative stress and brain protein modifications in the pathogenesis of alzheimer’s disease and mild cognitive impairment. free radic. biol. med. 2007;43(5):658–77. s h s t z s t z + t 3 0 1 2 3 r el at iv e ge ne e xp re ss io n of s el ad in -1 /b -a ct in a a,b groups fig. 3 effects of t3 on the diameter of neurospheres extracted from svz in the rat model of ad. (a) p <  0.05 compared to sh group, (b) p <  0.05 compared to stz group. sh: neurospheres isolated from rats received icv injection of normal saline; stz: neurospheres isolated from rats received icv injection of stz; stz +t3: neurospheres isolated from rats received icv injection of stz and cultured with t3. 38 original t3 improved the neurospheres from svz in model of alzheimer’s disease t. mokhtari et al. j contemp med sci | vol. 6, no. 1, january–february 2020: 32–38 25. beal mf. mitochondrial dysfunction and oxidative damage in alzheimer’s and parkinson’s diseases and coenzyme q 10 as a potential treatment. j. bioenerg. biomembr. 2004;36(4):381–6. 26. sultana r, butterfield da. role of oxidative stress in the progression of alzheimer’s disease. j. alzheimer’s dis. 2010;19(1):341–53. 27. grieb p. intracerebroventricular streptozotocin injections as a model of alzheimer’s disease: in search of a relevant mechanism. mol. neurobiol. 2016;53(3):1741–52. 28. selkoe dj. alzheimer’s disease: genes, proteins, and therapy. physiol. rev. 2001;81(2):741–66. 29. thompson pm, hayashi km, dutton ra, chiang mc, leow ad, sowell er, et al. tracking alzheimer’s disease. ann. ny acad. sci. 2007;1097(1):183–214. 30. thompson pm, hayashi km, de zubicaray g, janke al, rose se, semple j, et al. dynamics of gray matter loss in alzheimer’s disease. j. neurosci. 2003;23(3):994–1005. 31. al-mahmood s, colin s, schneider c. composition comprising an antisense sequence implicated in the regulation of angiogenesis. google patents; 2012. 32. taupin p, gage fh. adult neurogenesis and neural stem cells of the central nervous system in mammals. j. neurosci. res. 2002;69(6):745–9. 33. seaberg rm, van der kooy d. adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. j. neurosci. 2002;22(5):1784–93. 34. abrous dn, koehl m, le moal m. adult neurogenesis: from precursors to network and physiology. physiol. rev. 2005;85(2):523–69. 35. feng r, rampon c, tang y-p, shrom d, jin j, kyin m, et al. deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. neuron. 2001;32(5):911–26. 36. haughey nj, nath a, chan sl, borchard a, rao ms, mattson mp. disruption of neurogenesis by amyloid β-peptide, and perturbed neural progenitor cell homeostasis, in models of alzheimer’s disease. j. neurochem. 2002;83(6):1509–24. 37. wang r, dineley k, sweatt j, zheng h. presenilin 1 familial alzheimer’s disease mutation leads to defective associative learning and impaired adult neurogenesis. neuroscience. 2004;126(2):305–12. 38. zhang c, mcneil e, dressler l, siman r. long-lasting impairment in hippocampal neurogenesis associated with amyloid deposition in a knock-in mouse model of familial alzheimer’s disease. exp. neurol. 2007;204(1):77–87. 39. ceresini g, lauretani f, maggio m, ceda gp, morganti s, usberti e, et al. thyroid function abnormalities and cognitive impairment in elderly people: results of the invecchiare in chianti study. j. am. geriat. soc. 2009;57(1):89– 93. 40. kalmijn s, mehta km, pols ha, hofman a, drexhage ha, breteler mm. subclinical hyperthyroidism and the risk of dementia. the rotterdam study. clin. endocrinol. 2000;53(6):733–7. 41. lopez ol, rabin bs, huff fj, rezek d, reinmuth om. serum autoantibodies in patients with alzheimer’s disease and vascular dementia and in nondemented control subjects. stroke. 1992;23(8):1078–83. 42. ewins dl, rossor mn, butler j, rogues pk, mullen mj, mcgregor am. association between autoimmune thyroid disease and familial alzheimers disease. clin. endocrinol. 1991;35(1):93–6. 43. ganguli m, burmeister la, seaberg ec, belle s, dekosky st. association between dementia and elevated tsh: a community-based study. biol. psychiatry. 1996;40(8):714–25. 44. ishida e, hashimoto k, okada s, satoh t, yamada m, mori m. crosstalk between thyroid hormone receptor and liver x receptor in the regulation of selective alzheimer’s disease indicator-1 gene expression. plos one. 2013;8(1):e54901. 45. waterham hr, koster j, romeijn gj, hennekam rc, vreken p, andersson hc, et al. mutations in the 3beta-hydroxysterol delta24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis. am. j. human genet. 2001;69(4):685–94. 46. greeve i, hermans-borgmeyer i, brellinger c, kasper d, gomez-isla t, behl c, et al. the human diminuto/dwarf1 homolog seladin-1 confers resistance to alzheimer’s disease-associated neurodegeneration and oxidative stress. j. neurosci. 2000;20(19):7345–52. 47. martin m, dotti cg, ledesma md. brain cholesterol in normal and pathological aging. biochim. biophys. acta (bba)-mol. cell biol. of lipids. 2010;1801(8):934–44. 48. benvenuti s, luciani p, cellai i, deledda c, baglioni s, saccardi r, et al. thyroid hormones promote cell differentiation and up-regulate the expression of the seladin-1 gene in in vitro models of human neuronal precursors. j. endocrinol. 2008;197(2):437–46. 49. mokhtari t, akbari m, malek f, kashani ir, rastegar t, noorbakhsh f, et al. improvement of memory and learning by intracerebroventricular microinjection of t3 in rat model of ischemic brain stroke mediated by upregulation of bdnf and gdnf in ca1 hippocampal region. daru j. pharm. sci. 2017;25(1):4. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.02202007 126 j contemp med sci | vol. 4, no. 3, summer 2018: 126–133 review overview of effective herbal and antioxidant compounds on diabetes mohammad rasool khazaei, fatemeh makalani, elham ghanbari, maryam fayzemahdavi and mozafar khazaei fertility and infertility research center, kermanshah university of medical sciences, kermanshah, iran. correspondence to mozafar khazaei (email: mkhazaei1345@yahoo.com). (submitted: 19 may 2018 – revised version received: 11 june 2018 – accepted: 24 june 2018 – published online: 26 september 2018) objectives the aim of the present study was to review the effect of herbal medicine and antioxidant compounds on diabetes. methods we searched pubmed, science direct, scopus, and medline for studies published from 2000 to 2017 with using keywords of diabetes, herbal medicine, and antioxidant compounds. results various herbal medicines have been introduced to cure the diabetes. the most common types of effective compounds include flavonoids, terpenoid, carotenoids and alkaloids which are able to reduce the levels of blood sugar through increase insulin levels or reduction of intestinal uptake of glucose and regenerate pancreatic tissue through different mechanisms. conclusion herbal medicines with antidiabetic effects can give raise a field to discover drugs with herbal origin for treating the diabetes. different herbal remedies have been known and proved that mechanism of action. keywords diabetes, herbal medicine, herbal antioxidant compounds introduction diabetes is the most common form of endocrine disorders with increasing prevalence within human population.1 according to who reports, diabetic patients account to 366 million up to 2030.2 diabetes is accompanied with loss of quality of life and appearance of risk factors related to mortality. diabetes outbreak seems to be due to disturbance of carbohydrates, fats and proteins metabolism.3 following the relative insulin decrease, acute metabolic consequences are detected including ketoacidosis, hyperosmolar coma and different chronic disorders such as retinopathy, nephropathy, neuropathy and cardiovascular ailment.4 long periods of hyperglycemia in diabetic patient may produce free radicals, especially reactive oxygen species (ros). increased generation of ros may be due to glucose oxidation and protein glycosylation, and inappropriate conditions of tissue could disturb the balance between ros production and defense mechanisms of cells. this imbalance leads to change of function, cell destruction and finally tissue damage, especially in pancreatic tissue damage.5 evidences suggested that free radicals play important role to make changes at molecular level, in turn leads to wide range of human diseases including atherosclerosis, alzheimer’s disease, parkinson’s disease, cancer, arthritis, asthma, immune system deficiency and diabetes.6 three main type of diabetes type 1 diabetes this type of diabetes is due to immune-destruction of beta cells within pancreatic islands, associates with apparent loss of insulin production. type 1 diabetes (insulin-dependent) develops among young adults and is known as juvenile diabetes or insulin-dependent diabetes. in addition to auto immune disorders, diseases caused by insulin production result in insulin-dependent diabetes. also, viral infections may involve in disruption of beta cells. in some cases, inheritance serves as a factor of beta cells destruction.7 decreased level of blood insulin as well as increased level of blood glucose of diabetic patients makes it equisetic to receive exogenous issn 2413-0516 insulin source. recent studies demonstrate that immune system is the key factor of type 1 diabetes pathogenesis, so that immune-suppressor medications like cyclosporine decrease the rate of beta cells destruction during early development of clinical manifestations.8 type 2 diabetes type 2 diabetes (insulin independent) is the most common type of diabetes accounting in 90–95% of patients. this type of diabetes is observed among adults with age more than 40 years due to reduced/inadequate production of insulin or low sensitivity to it (insulin resistance) in which cells fail to use insulin properly.9 also, insulin resistance is associated with gradual dysfunction of beta cells which leads to metabolism disturbance.10 it is necessary to note that endogenous insulin is produced but in lower quantities or because of tissue insensitivity of insulin, its level remains insufficient.11 given that majority of patients are obese, it is seeming that obesity is the key factor of reduced insulin sensitivity; therefore, obese patients suffer from insulin resistance syndrome related metabolic disorders. appropriate diet regimen, weight loss before prescription and life style are important factors to treat type 2 diabetes.12 gestational diabetes mellitus gestational diabetes mellitus is defined as glucose intolerance during second or third trimesters of pregnancy and it occurs in 4% of all pregnancies. about 30–50% of patients face to suffer from diabetes mellitus, especially type 2 ones.13 other types of diabetes additional factors including genetic disorders in insulin secretion of pancreas beta cells as well as different types of exocrine glands diseases such as chronic pancreatitis, cystic fibrosis and even high levels of glucocorticoids strongly damaging pancreas, could dispose one to diabetes.14 in 80% of cases, women suffering from polycystic ovary syndrome, have face to insulin resistance, so that long-term consequences will progress to diabetes mellitus.15 mohammad rasool khazaei et al. 127j contemp med sci | vol. 4, no. 3, summer 2018: 126–133 review overview of effective herbal and antioxidant compounds on diabetes diabetes treatment in traditional medicine herbal therapy is an old field with historical origin and serves as a major base of medicine within ancient civilization including egypt, india, china, greek, iran and islamic medicine. medicinal herbs are so important that pharmacist researchers who explore 21st century medications into traditional medicine believe that medicinal herbs solve issues related to drug discovery in future.16 plants serve as bioactive source of traditional medicine. current century, wide investigations are conducted on medicinal herbs and their effective components which open new way to researchers so that nowadays, plant-derived medications comprise third of those.17 many well-known plants in the world play an important role in treating diabetes. new research suggests that re-attention to old drugs and natural treatments have triggered a new wave of research into the traditional ways diabetes has been treated. traditional anti-diabetic herbs can be a useful source of oral glucose-reducing compounds that can be used as medicines or dietary supplements.18 however, adverse and side effects of chemically-based medications, medicinal herbs with natural components have confirmed harmless or negligible side effects make researchers insist to expand their investigations.19 natural antioxidant agents antioxidant agents play important role in human life. use of antioxidant components is accompanied with reduced risk of table 1. medicinal plants with anti-diabetes activity used in traditional medicine scientific plant family name references active constituent effect zingiber officinale roscoe zingiberaceae 48 phenolic compounds such as ginjervals, shogwaves, paradoxes and zainjorns. terpinaid reduced glucose levels, increased serum levels of insulin, inhibition of glucosidase and amylase enzymes in the intestines trigonella foenumgraecum laguminosae 51–55 alkaloids, saponins, flavonoids and mucilages reduced glucose and cholesterol thymus vulgaris lamiaceae 56–58 tannin, saponin, flavonoids, a phenol compound (thiomol), linalol, cineol, terpenoid, glycoside, caffeic acid, and rhizmarnin acid reduced blood lipids and inhibit ldl oxidation hypoglycemic tribulus terrestris 59–62 saponin gluconeogenstic acid inhibition and hypoglycemic and hypolipidemic serum glucose berberis vulgaris berberidaceae 63–65 berberine alkaloids, oxycetin, berbamine, palmatine, braltlin, berbromine, columbemin, jaterurine increased insulin sensitivity, decreased blood glucose, hypolipidemia, activation of protein kinase b, decreased serum mda and hba1c levels juglans regia juglandaceae 66–70 phenolic compounds, flavonoids quercetin decreased plasma blood glucose camellia sinensis theaceae 71–75 catechin polyphenols, caffeine, teinin, epigallocatechin inhibition of glucose intestinal absorption, increased insulin levels, decreased glucose. triglyceride and fatty acids amygdalus lycioides rosaceae 76–78 amygdalin increased production and release of insulin punica granatum punicaceae 79–84 polyphenols. alkaloids anthocyanin, catechin, quercetin, rutin, alajic acid decreased glucose levels, triglycerides, total cholesterol in the bloodstream olea europaea l oleaceae 85–87 tannin, saponins, glycolic acid, olropin, hydroxy thirozole, ultropeosis enhancing insulin release and increasing glucose uptake urtica dioica urticaceae 88–90 flavonoids, lectins, polysaccharides, salicylic acid, histamine, serotonin, acetylcholine, formic acid and leukotrienes stimulation of beta cells and increased serum insulin, increased glucose uptake by muscle fibroblast cells, inhibition of alpha-amylase activity in serum fbs, cholesterol, tg, ldl reduction rhus coriaria anacardiaceae 91–94 flavonoids quercetin, tannin, quercetin, miristin and anthocyanins inhibition of intake of glucose in the intestine decreases in serum cholesterol levels cannabis sativa cannabaceae 95–101 delta 9 tetrahydrocannabinol, cannabinoid reduced glucose levels panax ginseng araliaceae 102–104 a steroid glycoside called panacillin, a saponin called panoxoside increased insulin production, reduced degradation of beta-pancreatic cells aloe vera liliaceae 105–107 barbaloin, isobarboline, aloenin antioxidant and antidiabetic effects 128 j contemp med sci | vol. 4, no. 3, summer 2018: 126–133 overview of effective herbal and antioxidant compounds on diabetes review mohammad rasool khazaei et al. cancer, diabetes and cardiovascular diseases. plants are the potential sources of antioxidants and many attempts have been performed to replace synthetic antioxidants by natural products. the most common antioxidant compounds of fruits, vegetables and medicinal herbs include flavonoids, alkaloids, terpenoids, carotenoids and vitamins.20 chemical compounds found in plants (also called phytochemicals) have different preventive as well as protective properties,21 which well-known types are phenols, alkaloids and terpenes. also triterpenoids, glycopeptides, various amino acids (hypoglycin, arginin, leucin, isoleucine, lysine, phenylalanine, tryptophan), flavonoids, phenols, coumarins (e.g. scopoletin), guanidines (e.g. galegine), vitamins (e, b, c), polysaccharides (e.g. saccharin), peptides (p peptide with insulin-like properties), anemaran a, aconitan a, galactomannan and sulfur derivatives in onion and garlic were found to have antidiabetic properties.22 flavonoids flavonoids serve as an important compound found in fruits, vegetables and medicinal herbs. major types of flavonoids include flavones, flavanone, flavonols, and anthocyanin23 with antimicrobial, antiviral, antidiabetic, antioxidant and anti-inflammatory effects.24 flavonoids are able to reduce the levels of blood glucose while insulin secretion and sensitivity increases.25 examples of important flavonoids include quercetin, rutin also called rutoside, lycopene, catechin, cinnamic acid, luteolin.26 in general, flavonoids suppress production of ros through inhibition of related enzymes, removing ros, regulation and protection of antioxidant defense system.27 chitin and chitosan after cellulose, chitin is the natural polysaccharide and second biopolymer which is found within crustacean shell such as crab, shrimp, and insect cuticles as well as fungi cell wall.28 chitosan is the deacetylated derivative of chitin and solves into hydro-acidic solutions. chitin and chitosan have low toxicity, antimicrobial and antiallergy characteristics which gain more attention in industrial, medical and pharmaceutical applications.29 chitin and chitosan are novel antioxidant compounds to control diabetes. also, chito-oligosaccharides obtaining from chemical hydrolysis of chitosan30 have several biological effects such as antimicrobial, antioxidant, antidiabetic and immune system improving activities.31 various concentrations of chito oligosaccharides could increase total antioxidant capacity as well as superoxide dismutase activity. also, they can prevent pancreatic island cells to apoptosis, leading to decrease the levels of malondialdehyde, definite product of lipid peroxidation which leads to diabetic keto-acidosis.32 chitosan, chito-oligosaccharides, and their derivatives apply their protective and preventive effects thorough the decrease of oxidative stress and low-density lipoprotein, decrease the inflammation as well as increase of muscular stiffness in age-dependent diseases such as diabetes, atherosclerosis, cancer, etc.33 chito-oligosaccharides have long-term antidiabetic effects in streptozotocin-induced diabetic rats through improve of glucose metabolism and increase of secretory capacity of pancreatic cells.34 chito-oligosaccharides have protective effect for type 2 diabetes which was demonstrated by improving insulin resistance and increasing insulin secretion.35 royal jelly royal jelly (rj) is a slimy, creamy compound that slightly has a spicy taste was secreted from maxillary and subpharyngeal glands of apis mellifera bees. rj has several biological activities on cells and tissues of animal models such as antioxidant, neurotrophic, anti-inflammatory, antitumor, antimicrobial, vasoconstriction, immune-modulatory and regulatory properties, and decrease the levels of blood glucose, cholesterol and hypertension.36 rj applies its antioxidant activity through decrease of lipid peroxidation of rats.37 administration of rj seems to decrease the blood glucose in patients and in contrast, increases serum concentration of insulin38 which may be due to insulin-like activities of rj peptides and presumably, decrease of insulin resistance.38 identification of antioxidant compounds with pharmacologic view has raised; those which not only use in medicine and food industry, but also have minimal side effects to treat diabetes.39 medicinal herbs contain natural products with fewer side effects; therefore, their antioxidant content could improve damages caused by oxidative agents or diseases.40 before the invention of insulin and other antidiabetic medications, patients were treated using traditional medicine. so far, more than 1200 medicinal herbs have been introduced to decrease the blood glucose or its complications.14 in recent years, various experimental and clinical investigations have been conducted based on the medicinal herbs among which significant decrease in patients’ blood glucose has been observed. overall functional mechanisms of antidiabetic herbs different antidiabetic mechanisms of herbs are included into one of following groups: 1. blocking calcium channels of pancreas beta cells. 2. stimulation of camp and inhibition of renal glucose uptake. 3. exciting insulin secretion and inhibiting mechanisms involved into decreased insulin secretion. 4. decreasing insulin resistance. 5. providing essential elements to beta cells including calcium, zinc, magnesium and copper. 6. improving regeneration of beta cells. 7. increasing number and size of cells within pancreas islands. 8. stimulating glycogenesis and hepatic glycolysis. 9. inhibiting activity of αand β-galactosidase. 10. cortisol reducing activities. 11. inhibiting α-amylase activity. 12. prevention of oxidative stress.41–44 to date, wide range of medicinal herbs has been studied to treat diabetes, some of which are discussed as follows. ginger ginger (zingiber officinale) from zingiberaceae family, is one of the most applicable medicinal herb used in iran, china and mohammad rasool khazaei et al. 129j contemp med sci | vol. 4, no. 3, summer 2018: 126–133 review overview of effective herbal and antioxidant compounds on diabetes greek traditional medicine to treat several diseases such as catch cold, rheumatism, neural disorders, gingivitis, asthma, constipation, diabetes, as well as in food industry as flavoring.45 effective compounds of ginger root differ depending upon implant site, and whether the root is dry, or wet. the odor of ginger is due to its volatile oil component. more than 50 effective compound including monoterpenes and sesquiterpene and beta-sesquiphellandrene exists in volatile oil.46 ginger has different pharmaceutical effects, for example, dry root extract of ginger may inhibit the increase of lipid levels, increase of body weight, and fructose-induced hyperglycemia.47 hypo-glycemic, hypo-cholesterolemic, and hypo lipidemic effects of ginger, as well as its antagonist effect on proteinuria and weight loss due to streptozotocin-induced diabetes in rats was demonstrated, suggesting the effectiveness of ginger in diabetic patients. ginger reduces blood sugar with antagonistic activity against serotonin-receptors and blocking them, it also probably inhibits the activity of glucosidase and amylase enzymes in the intestine and thereby reduces glucose uptake in the body.48 fenugreek trigonella foenum-graecum of fabaceae, is one of the most effective antidiabetic herbs used in traditional medicine.49 trigonella has shown to decrease blood glucose levels as a dose-dependent state, in both healthy and diabetic animal models.50 also, in vitro studies demonstrated that 4-hydroxyl lysine, a component of trigonella leads to increase in insulin secretion of pancreatic island cells in response to glucose, both in human and mice. as an interesting result, number of insulin receptors was increased, while alpha-amylase and sucrase (intestinal enzymes involving carbohydrate metabolism) activities were inhibited.51 the compounds in the fenugreek seeds include volatile alkaloids, saponins, flavonoids and mucilages.52 the therapeutic effect of fenugreek seed on diabetes is due to the direct stimulation of an amino acid called 4-hydroxyisoleucine on insulin secretion from beta cells.53 thyme thyme (thymus vulgaris) of lamiaceae family is one of the oldest medicinal herbs containing several compounds such as tannin, flavonoids, saponin, thymol, linalool, cineol, terpenoid, glycosides, caffeic acid, and rosmarinic acid.54 this herb has a wide range of pharmaceutical properties including tonic, digestive, anti-spasmodic, carminative, antifungal, antibacterial, antiseptic, and anti-rheumatoid and antioxidant properties, also the effect of thyme extract on the pancreatic beta cells is shown in experimental model of induced diabetes mellitus.55 both chronic and acute hyperglycemias lead to induction of oxidative stress and increase of lipid peroxidation. thyme extract seems to be effective in oxidative stress prevention.56 tribulus terrestris tribulus terrestris is a plant that contains various types of compounds such as flavonoids, alkaloids, vitamins, tannin and saponin. antioxidant properties of tribulus terrestris seem to be effective to treat cardiovascular diseases, diabetes, and tumors; also it is effective to decrease the inflammation of urinary tract and lower the hypertension.57,58 animal studies show that saponin content could significantly decrease blood glucose and inhibit gluconeogenesis. oral administration of saponin seems to inhibit intestinal alpha-glucosidase, delaying glucose uptake and decrease blood glucose following the feeding.59 antioxidant properties of tribulus terrestris could improve disorders associating with significantly increase in oxidative stress levels, for example diabetes.60 barberry barberry (berberis vulgaris) grows as 1 m spiny shrubs with fragile branches from 0.5 to 3 m height. root of barberry shrub contains several alkaloids such as berberine, berbamine, palmatine, oxycanthine, berberomine, beroulicine, clombamine, jatrorrhizine. in addition to alkaloids, inorganic acids, citric acid, malic acid, resin, tannin, mucilage and pectin exist within the root.61 berberine has several biologic effects such as anti-inflammatory, antioxidant, decreasing the blood glucose and blood pressure.62 since type 1 diabetes is due to the destruction of beta cells by t lymphocytes, therefore it is suggested that barberry may improve type 1 diabetes through its immune-modulatory properties.63 walnut walnut (juglans regia) from juglandaceae family is widely used in traditional medicine.64 walnut leaves are administered to treat diabetes, fever, rheumatic pains, dermal diseases, and its flowers are used to treat malaria and rheumatic pains.65 also, it has been demonstrated that brewed leaf of walnut may be effective to decrease blood glucose of diabetic patients. in iranian traditional medicine, walnut leaves and the pulp of its unripe fruit are used to decrease blood glucose.66 also, walnut leaves extract riches in different antioxidants such as phenolic compounds,67 especially phenolic acids and flavonoids. its main flavonoid is quercetin. studies suggest that flavonoids decrease the blood plasma glucose.68 green tea green tea (camellia sinensis) from theaceae family, contains polyphenol compounds such as catechin, caffeine, theanine, and epigallocatechin.69 green tea serves as an anti-inflammatory, antioxidant and anticancer agent70 and it is rich of poly-phenols, justifying its role to treat diabetes-induced retinopathy.71 administration of green tea powder into animal models of hyperglycemia improves insulin resistance.72 also, human studies have shown that daily administration of 1.5 g of green tea powder increases the glucose tolerance and metabolism of patients.73 peanuts peanut (amygdalus lycioides) is a plant from rosaceae family. it differs from almond because of the presence of amygdalin. also, amygdalin is found within seeds of apricot, cherry, and plum. amygdalin is a cyanogens glycoside with anticancer activity which hydrolyzes into glucose, benzaldehyde, and hydrosyanic acid. amygdalin accelerates pancreatic enzymes activity and enables the pancreas to increase the production and release of insulin. insulin prevents the glycogen breakdown, blocking the increase of blood glucose, as well as facilitates the entrance of glucose into cells. chemical structure of amygdalin consists of two molecules of glucose, one hydrocyanic acid and benzaldehyde with anticancer and anesthesia properties, respectively. oral administration of peanut extract into animal models decreases the blood glucose. amygdalin is used to relief pain caused by cancer, decrease the hypertension, asthma and emphysema.74–76 130 j contemp med sci | vol. 4, no. 3, summer 2018: 126–133 overview of effective herbal and antioxidant compounds on diabetes review mohammad rasool khazaei et al. pomegranate pomegranate (punica granatum) is a plant from punicaceae family. different parts of pomegranate contain poly-phenols, alkaloids, b1, b2, and folic acid vitamins.77 some of the components of pomegranate juice include anthocyanins, glucose, vitamin c, ellagic acid, gallic acid, caffeic acid, catechin, quercetin, rutin also called rutoside, organic compounds of phosphorus, magnesium, potassium, and iron.78 flavonoids of pomegranate prevents both hypertension and cancer cell growth.79 pomegranate juice and its flower extracts are effective to control diabetes, so they decrease the blood glucose, triglyceride, and total cholesterol in animal models of diabetes.80 human studies showed that the pomegranate juice decreases the blood fatty acids, hypertension and increase antioxidant effects, rather than blood glucose; therefore, pomegranate may decrease the diabetes consequences.81,82 olive olive (olea europaea) is a species of small tree in the family oleaceae with constantly green leaves, which can live more than 1000 years under favorable conditions. different compounds have been identified within the olea leaves such as sugar and resin, wax, chlorophyll, tannin, saponins, gallic acid, oleuropein, oleuropeoside, and hydroxytyrosol.83 the most effective part of olea to treat diabetes is its leaves. the experimental studies of diabetes have shown that olea leaves not only decrease the levels of blood glucose and fatty acids,84 but also prevent auto-immune dependent type 1 diabetes to progress.85 nettle nettle (urtica dioica) from utricaceae family, encompasses plants which are generally perennial herb, and most of its aerial parts are covered with hookor cone-shaped piles. in iran, nettle has been introduced as an adjuvant agent to treat diabetes.86 also, nettle is used as anti-inflammatory, lowering the blood glucose, diuretic, analgesic, local anesthetic, and prostatic inflammation.87 its compounds include flavonoids, hydrophilic compounds such as lectins and polysaccharides, substances such as histamine, formic acid, acetylcholine, acetic acid, butyric acid, leukotriene and 5-hydroxy-tryptamine.88 sumac sumac (rhus coriaria) from anacardiaceae family is a shrub plant with long history in traditional medicine. sumac is regarded to prevent cardiovascular diseases, also used as spices.89 phytochemical analyses propose the sumac as rich source of phenolic compounds such as tannin, quercetin, myricetin and anthocyanins.90 tannin has both preventive and anti-cancer properties.91 hypoglycemic activity of quercetin is applied using inhibition of intestinal glucose uptake; therefore, sumac hypoglycemic activity may be attributed to the presence of quercetin.92 cannabis cannabis sativa belongs to cannabaceae family. its seeds contain 3% saturated fatty acids, 28% unsaturated fatty acids and 25% proteins.93 main components of cannabis seeds are tetrahydrocannabinol, canabidiols, and cannabinoids.94 cannabis extract has several anti-tumors, anti-diabetic, anti-bacterial and antioxidant effects.95 chronic inflammation is suggested as a key factor of insulin resistance and type 2 diabetes which is thought to have anti-inflammatory effects of cannabinoid may help to decrease the inflammation and improvement of metabolic state of body. also, effect of hydro-alcoholic extract of cannabis on diabetic animal models, and patients was studied.96 one of the most common complications of diabetes is diabetic neuropathy with main sensory disorders of lower extremity. neurons are unable to regenerate themselves; therefore, destruction of central nervous system is accompanied with irreparable damages. use of alcoholic extract of cannabis seed as a neural protecting compound prevents hyperglycemia induced neural damages as well as following damages.97–99 ginseng ginseng (panax ginseng) an herb from araliaceae family, regarded as less in medicine. the history of its application as medication for several disorders like diabetes reaches to more than 4000 years.100 chemical compounds found in ginseng rhizome include steroid glycoside panakilon, saponin compound named panaxoside and vitamins of b family.101 intraperitoneal injection of ginseng in animal models and its reducing effect on blood glucose and hepatic glycogen to treat the hyper-lipidemia was assessed. hypoglycemic activity of ginseng seems to be through increased insulin production, and decreased destruction of pancreas beta cells.102 aloe vera aloe vera belongs to liliaceae family. the leaves of aloe vera have been used for a long time as medication and contain a clear gel in a central tissue. some of its properties involve in wound healing, anti-inflammatory, anti-cancer, anti-oxidant and anti-diabetic effects.103 this herb is rich in oxidase and catalase enzymes and supports the resistance of vitamins e and c against free radicals.104 barbaloin, isobarbaloin, and aloenin are isolated effective compounds of aloe vera. barbaloin protects the beta cells of pancreatic islets from free radical’s damages. animal model studies have used aloe vera for diabetes treatment, wound healing, tumors and intestine inflammatory diseases, in both injection and oral route.105 discussion diabetes mellitus is known as a chronic and metabolic disorder of endocrine system with increasing incidence. as clinical and experimental studies show, oxidative stress plays pivotal role in pathogenesis and complications of diabetes. also, regarding to side effects of chemical medications, researchers pay more attention to medicinal herbs. herbal medications are considered in diabetes treatment because of less side effects, antioxidant properties and insulin secretion regulatory effects. mechanisms of herbal compounds to reduce blood glucose level include activation of glucose catabolism, increase insulin secretion, inhibition or inactivation of gluconeogenesis, increase antioxidant capacity, leads glucose into the cell and absorption of free glucose and prevents it to bind proteins. animal and human studies have shown beneficial effects of herbs in reducing blood glucose and controlling diabetes. however, more studies are needed to accurately understand the effects of these plants and especially their possible complications in humans. major mechanisms of medicinal plants include: increased insulin secretion, activation of the pathway of glucose catabolism, inhibition or inactivation of the gluconeogenesis pathway, mohammad rasool khazaei et al. 131j contemp med sci | vol. 4, no. 3, summer 2018: 126–133 review overview of effective herbal and antioxidant compounds on diabetes directing of glucose into the cell, absorption of free glucose and prevention of its binding to proteins, increasing the antioxidant capacity, and preventing the harmful effects of oxidants produced in various pathways, which may be due to glucose uptake and the production of ultimate glycemic or other metabolic pathways, which ultimately prevents glucose from absorbing the intestine. conclusion based on the above studies, it can be said that the use of effective herbs in the treatment of diabetes has less side effects and their antioxidant effects regulatesinsulin secretion in the treatment of this disease. although it is unlikely that oral herbs will replace insulin, these natural resources are effective in treating diabetes through stimulation of biosynthesis and secretion of insulin as well as enhancing insulin function. however, it seems unlikely those insulin replacement edible plants, but these natural resources by stimulating endogenous insulin biosynthesis and secretion of insulin as well as strengthening performance, are effective in the treatment of diabetes. conflict of interst none n references 1. tripathi bk, srivastava ak. diabetes mellitus: complications and therapeutics. med sci monit. 2006;12:130–147. 2. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetes care. 2004;27:1047–1053. 3. öztürk e, arslan akk, yerer mb, bishayee a. resveratrol and diabetes: a critical review of clinical studies. biomed pharmacother. 2017;95:230–234. 4. asmat u, abad k, ismail k. diabetes mellitus and oxidative stress—a concise review. saudi pharm j. 2016;24:547–553. 5. sen s, chakraborty r, sridhar c, reddy ysr, de b. free radicals, antioxidants, diseases and phytomedicines: current status and future prospect. int j pharm sci rev res. 2010;3:91–100. 6. reddy ss. health outcomes in type 2 diabetes. int j clin pract suppl. 2000:46–53. 7. chakrabarti r, rajagopalan r. diabetes and insulin resistance associated disorders: disease and the therapy. curr sci. 2002;83:1533–1538. 8. deshpande ad, harris-hayes m, schootman m. epidemiology of diabetes and diabetes-related complications. phys ther. 2008;88:1254–1264. 9. barroso i. genetics of type 2 diabetes. diabet med. 2005;22:517–535. 10. chimen m, kennedy a, nirantharakumar k, pang tt, andrews r, narendran p. what are the health benefits of physical activity in type 1 diabetes mellitus? a literature review. diabetologia. 2012;55:542–551. 11. england lj, levine rj, qian c, soule lm, schisterman ef, yu kf, et al. glucose tolerance and risk of gestational diabetes mellitus in nulliparous women who smoke during pregnancy. am j epidemiol. 2004;160:1205–1213. 12. rani v, deep g, singh rk, palle k, yadav uc. oxidative stress and metabolic disorders: pathogenesis and therapeutic strategies. life sci. 2016;148:183–193. 13. diamanti-kandarakis e, dunaif a. insulin resistance and the polycystic ovary syndrome revisited: an update on mechanisms and implications. endocr rev. 2012;33:981–1030. 14. marles rj, farnsworth nr. antidiabetic plants and their active constituents. phytomedicine. 1995;2:137–189. 15. mccune lm, johns t. antioxidant activity in medicinal plants associated with the symptoms of diabetes mellitus used by the indigenous peoples of the north american boreal forest. j ethnopharmacol. 2002;82:197–205. 16. shapiro k, gong wc. natural products used for diabetes. j am pharm assoc (wash). 2002;42:217–226. 17. aslantürk ös, çelik ta. antioxidant, cytotoxic and apoptotic activities of extracts from medicinal plant euphorbia platyphyllos l. j med plants res. 2013;7:1293–1304. 18. makheswari um, sudarsanam d. phytomedicine for diabetes mellitus: an overview. res. pharm. 2011;1:28–37. 19. grover jk, yadav s, vats v. medicinal plants of india with anti-diabetic potential. j ethnopharmacol. 2002;81:81–100. 20. miura t, itoh c, iwamoto n, kato m, kawai m, park sr, et al. hypoglycemic activity of the fruit of the momordica charantia in type 2 diabetic mice. j nutr sci vitaminol (tokyo). 2001;47:340–344. 21. jarald e, joshi sb, jain dc. diabetes vs herbal medicines. iran j pharm ther. 2008;7:97–80. 22. hanhineva k, törrönen r, bondia-pons i, pekkinen j, kolehmainen m, mykkänen h, et al. impact of dietary polyphenols on carbohydrate metabolism. int j mol sci. 2010;11:1365–1402. 23. atanasov ag, waltenberger b, pferschy-wenzig em, linder t, wawrosch c, uhrin p, et al. discovery and resupply of pharmacologically active plantderived natural products: a review. biotechnol adv. 2015;33:1582–1614. 24. adaramoye oa, adeyemi eo. hypoglycaemic and hypolipidaemic effects of fractions from kolaviron, a biflavonoid complex from garcinia kola in streptozotocin‐induced diabetes mellitus rats. j pharm pharmacol. 2006;58:121–128. 25. rauter ap, martins a, borges c, mota‐filipe h, pinto r, sepodes b, et al. antihyperglycaemic and protective effects of flavonoids on streptozotocin– induced diabetic rats. phytother res. 2010;24:133–138. 26. pietta pg. flavonoids as antioxidants. j nat prod. 2000;63:1035–1042. 27. ngo dn, kim mm, kim sk. chitin oligosaccharides inhibit oxidative stress in live cells. carbohydr polym. 2008;74:228–234. 28. wang x, xing b. importance of structural makeup of biopolymers for organic contaminant sorption. environ sci technol. 2007;41:3559–3565. 29. jayakumar r, nwe n, tokura s, tamura h. sulfated chitin and chitosan as novel biomaterials. int j biol macromol. 2007;40:175–181. 30. rezakhani l, rashidi z, mirzapur p, khazaei m. antiproliferatory effects of crab shell extract on breast cancer cell line (mcf7). j breast cancer. 2014;17:219–225. 31. yuan wp, liu b, liu ch, wang xj, zhang ms, meng xm, et al. antioxidant activity of chito-oligosaccharides on pancreatic islet cells in streptozotocininduced diabetes in rats. world j gastroenterol 2009;15:1339–1345. 32. kerch g. the potential of chitosan and its derivatives in prevention and treatment of age-related diseases. mar drugs. 2015;13:2158–2182. 33. kim jn, chang iy, kim hi, yoon sp. long-term effects of chitosan oligosaccharide in streptozotocin-induced diabetic rats. islets. 2009;1: 111–116. 34. ju c, yue w, yang z, zhang q, yang x, liu z, et al. antidiabetic effect and mechanism of chitooligosaccharides. biol pharm bull. 2010;33:1511–1516. 35. hashimoto m, kanda m, ikeno k, hayashi y, nakamura t, ogawa y, et al. oral administration of royal jelly facilitates mrna expression of glial cell line-derived neurotrophic factor and neurofilament h in the hippocampus of the adult mouse brain. biosci biotechnol biochem. 2005;69:800–805. 36. khazaei m, ansarian a, ghanbari e. new findings on biological actions and clinical applications of royal jelly: a review. j diet suppl. 2018;15:757–775. 37. ghanbari e, nejati v, najafi g, khazaei m, babaei m. study on the effect of royal jelly on reproductive parameters in streptozotocin-induced diabetic rats. int j fertil steril. 2015;9:113–120. 38. guo h, kouzuma y, yonekura m. structures and properties of antioxidative peptides derived from royal jelly protein. food chem. 2009;113:238–245. 39. ahamad j, amin s, mir sr. momordica charantia linn. (cucurbitaceae): review on phytochemistry and pharmacology. res j phytochem. 2017;11:53–65. 40. çoruh n, celep as, özgökçe f. antioxidant properties of prangos ferulacea (l.) lindl., chaerophyllum macropodum boiss. and heracleum persicum desf. from apiaceae family used as food in eastern anatolia and their inhibitory effects on glutathione-s-transferase. food chem. 2007;100:1237–1242. 41. modak m, dixit p, londhe j, ghaskadbi s, devasagayam tp. recent advances in indian herbal drug research guest editor: thomas paul asir devasagayam indian herbs and herbal drugs used for the treatment of diabetes. j clin biochem nutr. 2007;40:163–173. 42. kaneto h, matsuoka ta, nakatani y, kawamori d, matsuhisa m, yamasaki y. oxidative stress and the jnk pathway in diabetes. curr diabetes rev. 2005;1:65–72. 43. marles rj, kaminski j, arnason jt, pazos-sanou l, heptinstall s, fischer nh, et al. a bioassay for inhibition of serotonin release from bovine platelets. j nat prod. 1992;55:1044–1056. 132 j contemp med sci | vol. 4, no. 3, summer 2018: 126–133 overview of effective herbal and antioxidant compounds on diabetes review mohammad rasool khazaei et al. 44. afzal μ, al-hadidi d, menon m, pesek j, dhami ms. ginger: an ethnomedical, chemical and pharmacological review. drug metabol drug interact. 2001;18:159–190. 45. al-amin zm, thomson m, al-qattan kk, peltonen-shalaby r, ali m. antidiabetic and hypolipidaemic properties of ginger (zingiber officinale) in streptozotocin-induced diabetic rats. br j nutr. 2006;96:660–666. 46. mozaffari-khosravi h, talaei b, jalali ba, najarzadeh a, mozayan mr. the effect of ginger powder supplementation on insulin resistance and glycemic indices in patients with type 2 diabetes: a randomized, double-blind, placebo-controlled trial. complement ther med. 2014;22:9–16. 47. shanmugam kr, mallikarjuna k, kesireddy n, reddy ks. neuroprotective effect of ginger on anti-oxidant enzymes in streptozotocin-induced diabetic rats. food chem toxicol. 2011;49:893–897. 48. li y, tran vh, duke cc, roufogalis bd. preventive and protective properties of zingiber officinale (ginger) in diabetes mellitus, diabetic complications, and associated lipid and other metabolic disorders: a brief review. evid based complement alternat med. 2012:516870. 49. abdel-barry ja, abdel-hassan ia, al-hakiem mh. hypoglycaemic and antihyperglycaemic effects of trigonella foenum-graecum leaf in normal and alloxan induced diabetic rats. j ethnopharmacol. 1997;58:149–155. 50. abraham bk, adithan c. review of endocrine pharmacology. indian j pharmacol. 2000;32:s67–s80. 51. kamboj ss, chopra k, sandhir r. hyperglycemia-induced alterations in synaptosomal membrane fluidity and activity of membrane bound enzymes: beneficial effect of n-acetylcysteine supplementation. neuroscience. 2009;162:349–358. 52. basch e, ulbricht c, kuo g, szapary p, smith m. therapeutic applications of fenugreek. altern med rev. 2003;8:20–27. 53. blumenthal m. german federal institute for drugs and medical devices. commission e. herbal medicine: expanded commission e monographs. newton, mass. integrative medicine communications. 2000, pp. 160–169. 54. letchamo w, xu hl, gosselin a. variations in photosynthesis and essential oil in thyme. j plant physiol. 1995;147:29–37. 55. rana p, soni g. antioxidant potential of thyme extract: alleviation of n-nitrosodiethylamine-induced oxidative stress. hum exp toxicol. 2008;27:215–221. 56. lee sj, umano k, shibamoto t, lee kg. identification of volatile components in basil (ocimum basilicum l.) and thyme leaves (thymus vulgaris l.) and their antioxidant properties. food chem. 2005;91:131–137. 57. amin a, lotfy m, shafiullah m, adeghate e. the protective effect of tribulus terrestris in diabetes. ann n y acad sci. 2006;1084:391–401. 58. li m, qu w, chu s, wang h, tian c, tu m. effect of the decoction of tribulus terrestris on mice gluconeogenesis. zhong yao cai. 2001;24:586–588. 59. sharifi am, darabi r, akbarloo n. study of antihypertensive mechanism of tribulus terrestris in 2k1c hypertensive rats: role of tissue ace activity. life sci. 2003;73:2963–2971. 60. zhang sj, qu wj, zhong sy. inhibitory effects of saponins from tribulus terrestris on alpha-glucosidase in small intestines of rats. zhongguo zhong yao za zhi. 2006; 31:910–913. 61. posadzki p, watson lk, ernst e. adverse effects of herbal medicines: an overview of systematic reviews. clin med (lond). 2013;13:7–12. 62. chander v, aswal js, dobhal r, uniyal dp. a review on pharmacological potential of berberine; an active component of himalayan berberis aristata. j phytopharmacol. 2017;6:53–58. 63. fatehi m, saleh tm, fatehi-hassanabad z, farrokhfal k, jafarzadeh m, davodi s. a pharmacological study on berberis vulgaris fruit extract. j of ethnopharmacol. 2005;102:46–52. 64. fallah huseini h, fakhrzadeh h, larijani b, shikh samani ah. review of anti-diabetic medicinal plant used in traditional medicine. j med plants. 2006;1(17):1–8. 65. erdemoglu n, küpeli e, yeşilada e. anti-inflammatory and antinociceptive activity assessment of plants used as remedy in turkish folk medicine. j ethnopharmacol. 2003;89:123–129. 66. cheng cw, wu tx, shang hc, li yp, altman dg, moher d, et al. consort extension for chinese herbal medicine formulas 2017: recommendations, explanation, and elaboration. ann intern med. 2017;167:112–121. 67. fukuda t, ito h, yoshida t. effect of the walnut polyphenol fraction on oxidative stress in type 2 diabetes mice. biofactors. 2004;21:251–253. 68. li wl, zheng hc, bukuru j, de kimpe n. natural medicines used in the traditional chinese medical system for therapy of diabetes mellitus. j ethnopharmacol. 2004;92:1–21. 69. benelli r, venè r, bisacchi d, garbisa s, albini a. anti-invasive effects of green tea polyphenol epigallocatechin-3-gallate (egcg), a natural inhibitor of metallo and serine proteases. biol chem. 2002;383:101–105. 70. weisburger jh, chung fl. mechanisms of chronic disease causation by nutritional factors and tobacco products and their prevention by tea polyphenols. food chem toxicol. 2002;40:1145–1154. 71. tsuneki h, ishizuka m, terasawa m, wu jb, sasaoka t, kimura i. effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. bmc pharmacol. 2004;4:18. 72. ryu oh, lee j, lee kw, kim hy, seo ja, kim sg, et al. effects of green tea consumption on inflammation, insulin resistance and pulse wave velocity in type 2 diabetes patients. diabetes res clin pract. 2006;71:356–358. 73. li y, wang c, huai q, guo f, liu l, feng r, et al. effects of tea or tea extract on metabolic profiles in patients with type 2 diabetes mellitus: a metaanalysis of ten randomized controlled trials. diabetes metab res rev. 2016;32:2–10. 74. moezi l, arshadi ss, motazedian t, seradj sh, dehghani f. anti-diabetic effects of amygdalus lycioides spach in streptozocin-induced diabetic rats. iran j pharm res. 2018;17:353–364. 75. babaei h, sadeghpour o, nahar l, delazar a, nazemiyeh h, mansouri mr, et al. antioxidant and vasorelaxant activities of flavonoids from amygdalus lycioides var. horrida. turk j biol. 2008;32:203–208. 76. sohrab g, ebrahimof s, sotoudeh g, neyestani tr, angoorani p, hedayati m, et al. effects of pomegranate juice consumption on oxidative stress in patients with type 2 diabetes: a single-blind, randomized clinical trial. int j food sci nutr. 2017;68:249–255. 77. jurenka js. therapeutic applications of pomegranate (punica granatum l.): a review. altern med rev. 2008;13:128–144. 78. stowe cb. the effects of pomegranate juice consumption on blood pressure and cardiovascular health. complement ther clin pract. 2011;17:113–115. 79. huang th, peng g, kota bp, li gq, yamahara j, roufogalis bd, et al. antidiabetic action of punica granatum flower extract: activation of ppar-γ and identification of an active component. toxicol appl pharmacol. 2005;207:160–169. 80. li y, wen s, kota bp, peng g, li gq, yamahara j, et al. punica granatum flower extract, a potent α-glucosidase inhibitor, improves postprandial hyperglycemia in zucker diabetic fatty rats. j ethnopharmacol. 2005;99: 239–244. 81. jafri ma, aslam m, javed k, singh s. effect of punica granatum linn. (flowers) on blood glucose level in normal and alloxan-induced diabetic rats. j ethnopharmacol. 2000;70:309–314. 82. sohrab g, sotoodeh g, siasi f, neiestani t, rahimi a, chamari m. effect of pomegranate juice consumption on blood pressure in type 2 diabetic patients. iran j endocrinol metab. 2008;9:399–405. 83. afify amr, el-beltagi hs, fayed sa, el-ansary ae. in vivo correlation of olive leaves extract on some oxidative stress markers in streptozotocin-induced diabetes mellitus in rats. grasas y aceites. 2018;69:e243. 84. eidi a, eidi m, darzi r. antidiabetic effect of olea europaea l. in normal and diabetic rats. phytother res. 2009;23:347–350. 85. jemai h, el feki a, sayadi s. antidiabetic and antioxidant effects of hydroxytyrosol and oleuropein from olive leaves in alloxan-diabetic rats. j agric food chem. 2009;57:8798–8804. 86. cvjetićanin t, miljković d, stojanović i, dekanski d, stošić-grujičić s. dried leaf extract of olea europaea ameliorates islet-directed autoimmunity in mice. br j nutr. 2010;103:1413–1424. 87. patel ss, ray rs, sharma a, mehta v, katyal a, udayabanu m. antidepressant and anxiolytic like effects of urtica dioica leaves in streptozotocin induced diabetic mice. metab brain dis. 2018:33;1281–1292. 88. mehri a, hasani-ranjbar s, larijani b, abdollahi m. a systematic review of efficacy and safety of urtica dioica in the treatment of diabetes. 2011;7: 161–170. 89. zargham h, zargham r. tannin extracted from sumac inhibits vascular smooth muscle cell migration. mcgill j med. 2008;11:119–123. 90. mavlyanov sm, islambekov sy, karimdzhanov ak, ismaikov ai. anthocyans and organic acids of the fruits of some species of sumac. chem nat comp. 1997;33:279–280. 91. cai y, zhang j, chen ng, shi z, qiu j, he c, et al. recent advances in anticancer activities and drug delivery systems of tannins. med res rev. 2017;37:665–701. 92. coskun o, kanter m, korkmaz a, oter s. quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and β-cell damage in rat pancreas. pharmacol res. 2005; 51:117–123. 93. katchan v, david p, shoenfeld y. cannabinoids and autoimmune diseases: a systematic review. autoimmun rev. 2016;15:513–528. 94. baron ep. comprehensive review of medicinal marijuana, cannabinoids, and therapeutic implications in medicine and headache: what a long strange trip it’s been…. headache. 2015;55:885–916. mohammad rasool khazaei et al. 133j contemp med sci | vol. 4, no. 3, summer 2018: 126–133 review overview of effective herbal and antioxidant compounds on diabetes 95. beaulieu p, ware m. reassessment of the role of cannabinoids in the management of pain. curr opin anaesthesiol. 2007;20:473–477. 96. dagon y, avraham y, link g, zolotarev o, mechoulam r, berry em. the synthetic cannabinoid hu-210 attenuates neural damage in diabetic mice and hyperglycemic pheochromocytoma pc12 cells. neurobiol dis. 2007;27:174–181. 97. lotfi n, khazaei m, ali shariatzadeh sm, mehranjani ms, piri f, ansarian a. reproductive parameters in diabetic male rat after exposure to cannabis sativa hydroalcoholic extract. j rep pharma sci. 2014;3:193–200. 98. croxford jl, yamamura t. cannabinoids and the immune system: potential for the treatment of inflammatory diseases? j neuroimmunol. 2005;166: 3–18. 99. tehranipour m, mahdavi shahri n, ekrami koushki a, javad mousavi bz. neuroprotective effects of cannabis sativa alcoholic extract against spinal alpha motoneurons degeneration in male type ii diabetic rats. horizon med sci. 2012;18:141–147. 100. chong sk, oberholzer vg. ginseng–is there a use in clinical medicine? postgrad med j. 1988;64:841–846. 101. ranjbar sh, larijani b, abdollahi m. recent update on animal and human evidences of promising anti-diabetic medicinal plants: a mini-review of targeting new drugs. asian j anim vet adv 2011;6:1271–1275. 102. tripathi ak, bhoyar pk, baheti jr, biyani dm, khalique m, kothmire ms, et al. herbal antidiabetics: a review. int j res pharm sci. 2011;2:30–37. 103. rajasekaran s, sivagnanam k, subramanian s. antioxidant effect of aloe vera gel extract in streptozotocin-induced diabetes in rats. pharmacol rep. 2005;57:90–96. 104. chahardoli m, mahmoodi m, hajizadeh mr, khoramdel azad h, khoshdel ar, mirzaei mr. effect of aloe vera hydroalcoholic extract on blood glucose, serum insulin and the key enzymes in metabolic pathways of glycolysis and gluconeogenesis in hepatocytes of type 1 diabetic rats. j rafsanjan univ med sci. 2015;13:669–682. 105. beppu h, shimpo k, chihara t, kaneko t, tamai i, yamaji s, et al. antidiabetic effects of dietary administration of aloe arborescent miller components on multiple low-dose streptozotocin-induced diabetes in mice: investigation on hypoglycemic action and systemic absorption dynamics of aloe components. j ethnopharmacol. 2006;103:468–477. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.09201803 114 j contemp med sci | vol. 6, no. 3, may–june 2020: 114–119 original issn 2413-0516 introduction surgical site infections (ssis) remain one of the most confounding issues of the surgeons, despite the numerous revolutions in the field of surgical technique, asepsis and antisepsis in the historical process and current technologies.1 although the development of effective antibiotics which are easily applied in clinical practice with the concept of antimicrobial prophylaxis (amp), the incidence of ssi is reported to reach 20% depending on many patients and disease-related factors.2 this situation is no different for stomach cancer surgery, which is one of the most major intaabdominal surgeries, and the incidence of ssi was found to be between 5% and 20%.3 amp is widely applied before many surgical procedures, and studies have shown that it effectively prevents ssi depending on the distribution of patients, the effectiveness of the antibiotic used, and the surgical procedure performed.4,5 although it has been clarified which antibiotic will be used in which surgical procedure and the effective doses of the antibiotics used, the evidence on the duration of amp is limited. although it has been concluded that single dose amp is sufficient in colorectal and hepatobiliary surgery, a clear consensus has not been achieved, except for a few randomized studies that advocate the same idea with limited evidence in gastric cancer surgery.6-10 while single-dose amp prior to gastric cancer surgery was the common concept in the west, it was shown in a survey on surgeons in the far east that more than 60% of surgeons continued prophylaxis in the postoperative period.11 based on the limited number of studies and clinical experience, there are no clear data on the duration of antibiotic use, although the single-dose amp is considered to be more advantageous than the extended-dose amp because of the potential complications of extended-dose amp such as drug side effects and bacterial resistance development. therefore, in this study, we aimed to compare the effectiveness of single-dose and extended-dose amp in preventing the development of ssis in patients undergoing curative gastrectomy with the diagnosis of gastric cancer. material and methods patient selection and data collection a total of 210 patients who underwent curative gastrectomy for gastric cancer between january 2015 and january 2019 were included in the study. patients whose results could not be reached, who underwent palliative procedures, who had metastatic disease at the time of diagnosis, who underwent emergency surgery due to bleeding or perforation, who received neoadjuvant chemotherapy, who received antibiotic treatment preoperatively for various reasons, and who had any immune system disease were excluded from the study. demographic informations, operational results, and pathology reports of patients were analyzed retrospectively using the hospital database. prophylactic antibiotic regimens applied by different surgical teams in our clinic were screened retrospectively via electronic file system. patients who received a single dose of 1 g cefazolin 30 min before the surgical incision were determined as the first group (single-dose amp ) and who received 1 g cefazolin one time 30 min before the surgical incision and every 12 h until the first postoperative day were determined as the second group (extended-dose amp). pathological stages were determined according to the tumor node metastasis (tnm) classification of the 7th edition of the international cancer control.12 deaths occurred comparison of single-dose and extended-dose antimicrobial prophylaxis for preventing surgical site infections after curative surgery for gastric adenocarcinoma ümit mercan*, ogün erşen, ali ekrem ünal surgical oncology, general surgery department, faculty of medicine, ankara university, ankara, turkey. * correspondence to : ümit mercan (umit.mercan@yahoo.com.tr ) (submitted: 03 march 2020 – revised version received: 24 march 2020 – accepted: 10 april 2020 – published online: 26 june 2020) objectives there is no consensus on the duration of antimicrobial prophylaxis (amp) for prevention of surgical site infections (ssi) after curative gastrectomy for gastric adenocarcinoma and the data are limited. in this study, we aimed to compare the effect of single-dose and extended-dose amp on ssi prevention. methods a total of 210 patients who underwent curative gastrectomy for gastric cancer included in the study. patients who received a single dose of 1 g cefazolin 30 min before the surgical incision were determined as single-dose group and the patients who received 1 g cefazolin one time 30 min before the surgical incision and every 12 h until the first postoperative day were determined as extended-dose group. demographic characteristics, postoperative outcomes, and the types and incidence of ssi were compared in two groups and risk factors for ssi development were analyzed. results there was no significant difference in the demographic characteristics and postoperative outcomes in both amp groups in the general patient population and in the subgroups formed based on different clinicopathological and operative factors. there was no significant difference in the incidence of ssi between the two groups (23,4% and 26,7% p=0,346). age, diabetes mellitus, chronic obstructive pulmonary disease, operation time, and operative approach were found to be independent risk factors for ssi development. conclusion in patients undergoing curative gastrectomy for gastric adenocarcinoma, it has been found that the use of extended-dose amp has no effect on reducing the incidence of ssi and single-dose amp is sufficient and safe. keywords antibiotic prophylaxis, stomach neoplasms, surgical wound infection 115 original comparison of single-dose and extended-dose antimicrobial prophylaxisümit mercan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 114–119 within 30 days postoperatively has been determided as in-hospital mortality. complications related to the surgical site were accepted as local complications and the others as systemic complications. the type and severity of postoperative complications were graded and categorized based on the modified clavien dindo classification.13 infections developed within 30 days postoperatively were evaluated as ssi and were determined based on national nosocomial infections surveillance system data.2 ssis were categorized as superficial incisional, deep incisional, and organ/space infections. demographic characteristics, postoperative results, and surgical site infections were compared between two groups. this study was approved by the ethics committee. operation and perioperative care all operations in our clinic were performed in strict accordance with the rules of routine asepsis and antisepsis. preoperative mechanical bowel cleansing was performed. preoperative fasting was achieved for 8 h. intraoperative normothermia was provided by the anesthesia team. laparoscopic gastrectomy was performed in patients with early stage disease and technically appropriate. laparotomy was performed in patients with locally advanced disease and not eligible for laparoscopic surgery. two of 10 mm and three of 5 mm ports were used in patients undergoing laparoscopic surgery. after dissection, the specimens were removed by expanding the umbilical camera port via a spesimen bag. a standard midline incision extending from the xiphoid bone to the umbilicus was performed in laparotomies. depending on the tumor locations and clinical conditions, patients underwent appropriate lymph node dissection with distal or total gastrectomy. silk and polyglactin suture materials were used in the abdomen. the intraabdominal and subcutaneous cavities were washed with sterile saline solutions before closing. while surgical incisions were closing, double-layer polydioxanon (pds) was used in the abdominal fascia and polypropylene suture was used in the skin. two jackson pratt drains and nasogastric tubes were placed in each patient. after anastomotic leakage control using oral methylene blue on the third postoperative day, the nasogastric tubes of the patients were withdrawn and the patients have started oral feeding. patients who had adequate oral intake and who had not required inpatient treatment were discharged with the suggestion of outpatient control. statistical analysis data were presented as numbers with mean± standard deviation or percentage. the suitability of data for normal distribution was determined by kolmogorov–smirnov test and histogram graphs. student’s t-test, mann–whitney u test, χ2 test, or fisher’s exact test were used for comparisons between numerical or categorical data. the binary logistic regression model was used to analyze the risk factors for surgical site infections. all statistical analyzes were performed using ibm spss statistics 23. for all tests, values less than p<0.05 were significant. results of the 210 patients included in the study group, 152 (72,4%) were male and the mean age was 60.89±8.56 years. there were 124 (59%) patients in the single-dose group and 86 (41%) patients in the extended-dose group. the demographic characteristics of the groups are summarized in table 1. there was table 1. comparison of patient characteristics between two groups. variables total (n=210) duration of amp p value single-dose group (n=124) extended-dose group (n=86) age 60,89 ± 8,56 60.71 ±8.42 61,14 ± 8.8 0,722 gender (male) 152(72,4) 90(72,6) 62(72,1) 0,530 bmi (kg/m2) 21,14 ± 3,24 21 ± 3.09 21.21 ± 3.45 0,686 tnm stage* stage 1 stage 2 stage 3 38(18,1) 67(31,9) 105(50) 23(18,5) 31(25) 70(56,5) 15(17,4) 36(41,9) 35(40,7) 0,029 smoking comorbid diseases diabetes mellitus hypertension copd 69(32,9) 40(19) 100(47,6) 28(13,3) 44(35,5) 25(20,2) 60(48,4) 17(13,7) 25(29,1) 15(17,4) 40(46,5) 11(12,8) 0,372 0,379 0,450 0,509 operative approach laparotomy laparoscopy 100(47,6) 110(52,4) 62(50) 62(50) 38(44,2) 48(55,8) 0,246 lnd d1/d1+ d2/d2+ 38(18,1) 172(81,9) 23(18,5) 101(81,5) 15(17,4) 71(82,6) 0,494 gastric resection dg tg 91(43,3) 119(56,7) 50(40,3) 74(59,7) 41(47,7) 45(52,3) 0,180 values are presented as mean±standard deviation or number (%). amp: antimicrobial prophylaxis, bmi: body mass index, tnm: tumor node metastasis, copd: chronic obstructive pulmonary disease, lnd: lymph node dissection, dg: distal gastrectomy, tg: total gastrectomy. *tnm stage was based on the 7th edition of the american joint committee on cancer staging system.12 116 original comparison of single-dose and extended-dose antimicrobial prophylaxis ümit mercan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 114–119 no significant difference between two groups in terms of age, gender, bmi, comorbid diseases, smoking, operative approach, type of operation, and lymph node dissection. the single-dose group had significantly more tnm stage 3 patients than the extended-dose group (p=0,029). the comparison of postoperative outcomes between the two groups are summarized in table 2. the mean length of hospital stay was 8.58±4.06 days and there were 9 (4,3%) patients who developed mortality within 30 days. ssi developed in 52 (24,8%) patients, of which 23 (11%) had wound infection, 12 (5,7%) had intraabdominal abscess, 15 (7,1%) had anastomosis leakage, and 9 (4,3%) due to pancreatic fistula. there was no significant difference between the groups in the incidence of ssi (23,4% and 26,7%, p=0,346). when ssi were categorized according to their types, superficial incisional, deep incisional, and organ/space infections were observed in 5,2%, 3,8%, and 15,7%, respectively. there was no significant difference between the groups in terms of length of hospital stay, mortality rates, postoperative complication severity, ssis, and ssi types. the relation between the incidence of ssi and amp duration in subgroups have been determined according to different clinicopathological and operative factors are summarized in table 3. the incidence of ssi did not differ significantly between the single and extended-dose group in the subgroups formed by age, gender, bmi, duration and type of operation, operative approach, lymph node dissection, or tnm stage. risk factors for ssi determined by univariate and multivariate analyzes in total patient population are summarized in table 4. in univariate analysis, age, bmi, smoking, diabetes mellitus, chronic obstructive pulmonary disease (copd), operative approach, operation time, gastric resection, tnm stage, lymph node dissection, and amp administration time were compared with ssi incidence. age over 65, bmi over 25 kg/m2, long operation time (>180 min), diabetes mellitus and copd, operative approach and gastric resection were statistically significant among these factors. in multivariate analysis of these factors, age over 65 (or: 2,36. 95% ci: 0,74 ~ 4,02. p=0,014), long operation time (or: 2,56. 95% ci: 1,12 ~ 4,48. p=0,001>), presence of diabetes mellitus (or: 5,81. 95% ci: 2,64 ~ 8,28. p=0,003) presence of copd (or: 3,43. 95% ci: 1,20 ~ 6,37. p=0,007) and laparotomy (or: 8,45. 95% ci: 4,48 ~ 16,24. p=0,001>) have been found independent risk factors for the development of ssi after curative gastrectomy for gastric cancer. there was no statistically significant relationship between the duration of amp and the development of ssi (or 1,19. 95% ci: 0,63 ~ 2,25. p=0,349). discusion gastric cancer surgery is one of the major intra-abdominal surgeries, and despite developing surgical methods and experience gained, wound and wound-related infectious complications still remain one of the biggest problems facing surgeons.1 studies have shown that the incidence of ssi reaches 20% despite absolute compliance with asepsis and antisepsis rules.3 in addition to the factors associated with the surgeon and surgical procedures, it is known that many factors related to the patient and the disease play a role in the development of ssi. since the introduction of the concept of amp into clinical practice, there has been a significant decrease in the incidence of ssi that has developed after many surgical procedures.4 with the introduction of more effective antibiotics, the never-ending war between surgeons and microbial agents begins to turn in favor of surgeons and clinicians, but the incidence of ssi still remains high, suggesting that information on this issue is still insufficient. while there is almost a consensus on which antibiotic to be used in which dose and in which surgical procedure, the evidence for amp duration is insufficient and there are different practices in different clinics. there are many clinical studies evaluating amp duration in gastrointestinal malignancies.11,14-17 although the efficacy of single-dose amp has been accepted with limited evidence in colorectal and hepatobiliary surgery, most studies also included liver and pancreatic cancer patients, whose postoperative outcomes were more complex than gastric cancer, table 2. comparison of postoperative outcomes between two groups. duration of amp variables total (n=210) single-dose group (n=124) extended-dose group (n=86) p-value operating time hospital stay mortality 161,46±21,64 8,58 ±4,06 9(4,3) 160,24 ±22 8,48 ± 4,07 6(4,8) 163,22 ± 20,9 8,71 ± 4,06 3(3,5) 0,328 0,786 0,457 clavien-dindo classification grade 3 and above complications 24(11,4) 13(10,5) 11(12,8) 0,380 ssi wound infection inra-abdominal abscess anastomotic leakage pancreatic fistula 52(24,8) 23(11) 12(5,7) 15(7,1) 9(4,3) 29(23,4) 15(12,1) 7(5,6) 9(7,3) 4(3,2) 23(26,7) 8(9,3) 5(5,8) 6(7) 5(5,8) 0,346 0,344 0,592 0,582 0,283 ssi types superficial i̇ncisional deep i̇ncisional organ/space 11(5,2) 8(3,8) 33(15,7) 7(5,6) 5(4) 17(13,7) 4(4,7) 3(3,5) 16(18,6) 0,805 values are presented as mean±standard deviation or number (%). amp: antimicrobial prophilaxis, ssi: surgical site infection. 117 original comparison of single-dose and extended-dose antimicrobial prophylaxisümit mercan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 114–119 making it difficult to apply study data to patients with gastric cancer. there is limited information about the duration of amp in the prevention of ssi development only in gastric cancer patients. although the opinion that a single dose of antibiotic use is sufficient as a result of a few randomized studies, it is seen that long-term use is a common clinical approach in studies from the far east and there are differences in terms of study data and prophylaxis between the eastern and western countries.11,14 a large survey study from japan showed that more than 60% of surgeons continue amp after surgery.11 this may be due to heterogeneity between east and west countries in gastric cancer surgery. the large number of cases in the east causes the number of patients developing ssi to be higher and as a result, it is possible that surgeons may be directed towards more aggressive use of amp. several prospective randomized studies have shown that single dose amp is sufficient in gastric cancer surgery. in a prospective study involving 423 patients by ohashi et al, there table 4. univariate and multivariate analysis of risk factors for ssi. variable univariate analysis multivariate analysis or (%95 ci) p value adjusted or (%95ci) p value age(>65) 3,16(1,65~6,04) 0,014 2,36(0,74~4,02) 0,014 operatingtime(>180) 4,42(2,43~8,42) 0,001> 2,56 (1,12~4,48) 0,001> diabetes mellitus 4,31(2,07~8,94) 0,001> 5,81(2,64~8,28) 0,003 copd 3,49(1,79~5,08) 0,001> 3,43(1,20~6,37) 0,007 operating approach (laparotomy) 6,80(3,98~17,30) 0,003 8,45(4,48~16,24) 0,001> bmi(>25) 3,18(1,26~8,00) 0,014 gastric resection 2,03(1,04~3,96) 0.025 amp duration 0,349 or: odds ratio, copd: chronic obstructive pulmonary disease, bmi: body mass index, amp: antimicrobial prophylaxis. table 3. relation between ssi incidance and amp duration in subgroups. variables duration of amp p-value single-dose group extended-dose group total ssi total ssi age <65 ≥65 81 43 12(14,8) 17(39,5 55 31 11(20) 12(38,7) 0,432 0,944 gender male female 90 43 21(23,3) 8(18,6) 62 24 17(27,4) 6(25) 0,570 0,900 bmi (kg/m2) <25 ≥25 114 10 25(21,9) 4(40) 75 11 17(22,6) 6(54,5) 0,906 0,529 operating time (min) <180 ≥180 102 22 10(9,8) 19(86,3) 69 17 9(13) 14(82,3) 0,511 0,739 tnm stage* stage 1 stage 2-3 23 101 3(13) 26(27,6) 15 71 6(40) 17(23,9) 0,058 0,790 operative approach laparotomy laparoscopy 62 62 21(33,8) 8(12,9) 38 48 13(34,2) 10(20,8) 0,973 0,269 lnd d1/d1+ d2/d2+ 23 101 3(13) 26(25,7) 15 71 6(40) 17(23,9) 0,058 0,790 gastric resection dg tg 50 74 6(12) 23(31) 41 45 10(24,3) 13(28,8) 0,125 0,803 amp: antimicrobial prophylaxis, ssi: surgical site infection, bmi: body mass index, lnd: lymph node dissection, dg: distal gastrectomy, tg: total gastrectomy. *tnm stage was based on the 7th edition of the american joint committee on cancer staging system.12 118 original comparison of single-dose and extended-dose antimicrobial prophylaxis ümit mercan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 114–119 was no significant difference in the incidence of ssi between patients receiving single dose and extended dose amp (10,4% vs 8,1%; or 0,764; 95% ci: 0,395 -1,480; p=0,528).18 in a phase ii study conducted by i̇mamura et al. to investigate the effectiveness of single dose prophylaxis after gastric cancer surgery, the incidence of ssi was similar in the single dose amp group and the control group.19 in a phase iii study conducted with this study and comparing single dose and extended dose amp, it was concluded that single-dose amp is effective and sufficient in preventing the development of ssi.20 similarly in our study, no significant difference was found between the two groups in terms of ssi development. the relationship between the width of the surgical procedure and the duration of amp is still controversial. in our study, although laparotomy and longer operation time were determined as independent risk factors on ssi development, it was found that extended dose amp administration did not have a significant effect on the prevention of ssi development in this subgroup. these results suggest that single-dose amp is sufficient and can be used safely in patients with more invasive procedures such as large lymph node dissection, laparotomy, long surgery time, and even multiorgan resections. it was also found in our study that extended-dose amp use had no effect on the prevention of ssi in the entire spectrum from superficial incisional infections to deep organ infections. determining risk factors for ssis is important in identifying patients at risk, taking necessary precautions and shaping the surgical approach. in our study, age, diabetes mellitus, copd, operative approach, and operation time were determined as independent risk factors for ssi. the previously published study results by hirao et al and utsumi et al determining ssi risk factors appear to be consistent with our results.21,22 in order to prevent ssi development in patients with these risk factors, asepsis and antisepsis rules and intraoperative normotermi precautions should be strictly followed. the main limitations of our study are that it has low generalization rates due to possible selection bias because of being a retrospective study conducted from a single center and it has a relatively low sample size. in addition, although the rates of surgical complications and ssi in our clinic are similar to the literature, the high rates in our study may be due to the selected sample. our results need to be supported by randomized controlled trials with a larger patient population. in conclusion, there is no significant difference between single-dose and extended-dose amp in the prevention of ssi development in patients who underwent curative gastrectomy for gastric cancer. this study proves the effectiveness of single dose preoperative amp in gastric cancer surgery. our results support previous studies demonstrating the effectiveness of single dose prophylaxis and emphasize that the use of extended-dose amp, which is common in eastern countries, is not evidence-based. acknowledgment i would like to thank all members of the turkish surgical oncology association supported writing of this article. mercan u: writing and statistical analysis erşen o: data collection unal ae: critical review disclosure statement there is no conflict of interest in writing of this article. no financial support or funding has been received. references 1. kolasinski w. surgical site infections review of current knowledge, methods of prevention. pol przegl chir. 2018 nov 6;91(4):41-47. 2. burke jp. infection control a problem for patient safety. n engl j med 2003;348:651-656. 3. national nosocomial infections surveillance system. national nosocomial infections surveillance (nnis) system report, data summary from january 1992 through june 2004, issued october 2004. am j infect control 2004;32:470-485. 4. migita k, takayama t, matsumoto s, wakatsuki k, enomoto k, tanaka t et al. risk factors for surgical site infections after elective gastrectomy. j gastrointest surg 2012;16: 1107-1115. 5. ozalp n, zülfikaroğlu b, göçmen e, acar a, ekiz i, koç m et al. risk factors for surgical site infection after gastrectomy with d2 lymphadenectomy. surg today 2009; 39:1013-1015. 6. gilbert dn, moellering rc, sande ma.. the sanford guide to antimicrobial therapy. 33rd ed. hyde park, vt: antimicrobial therapy, 2003:123-124. 7. mangram aj, horan tc, pearson ml, silver lc, jarvis wr. guideline for prevention of surgical site infection, 1999. hospital infection control practices advisory committee. infect control hosp epidemiol 1999;20: 250-278. 8. bratzler dw, houck pm, richards c, steele l, dellinger ep, fry de et al. use of antimicrobial prophylaxis for major surgery: baseline results from the national surgical infection prevention project. arch surg, 2005;140(2), 174-182. 9. kobayashi m, takesue y, kitagawa y, kusunoki m, sumiyama y. antimicrobial prophylaxis and colon preparation for colorectal surgery: results of a questionnaire survey of 721 certified institutions in japan. surg today, 2011;41(10), 1363. 10. mohri y, tonouchi h, kobayashi m, nakai k, kusunoki m. mie surgical infection research group. randomized clinical trial of single-versus multiple-dose antimicrobial prophylaxis in gastric cancer surgery. br j surg 2007;94:683-688. 11. sumiyama y, takesue y. current status of prophylactic antibiotic therapy for prevention of postoperative infections after gastrointestinal surgery: a questionnaire covering 3,823 surgeons. jpn j chemother 2004;52: 474-485. 12. sobin l, gospodarowicz m, wittekind c.. tnm classification of malignant tumours. 7th ed. international union against cancer (uicc). new york: wiley, 2009. 13. dindo d, demartines n, clavien pa. classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. ann surg 2004;240:205–213. 14. ahn hs, yook jh, park ch, park yk, yu w, lee ms et al. general perioperative management of gastric cancer patients at high-volume centers. gastric cancer 2011;14:178-182. 15. dellinger ep, gross pa, barrett tl, krause pj, martone wj, mcgowan je jr et al. quality standard for antimicrobial prophylaxis in surgical procedures. infectious diseases society of america. clin infect dis 1994;18:422-427. 16. takayama t, aramaki o, shibata t, oka m, itamoto t, shimada m et al. antimicrobial prophylaxis for 1 day versus 3 days in liver cancer surgery: a randomized controlled non-inferiority trial. surg today 2019;49(10), 859-869. 17. hentzen jekr, smit ma, bruins mj, rupert cgbm, schreinemakers j, ruijs gjhm et al. efficacy of pre-operative antimicrobial prophylaxis in patients undergoing pancreatoduodenectomy: a multi-center retrospective analysis. surg infect (larchmt). 2018 aug/sep;19(6): 608-613. 18. ohashi m, saka m, katayama h, okinaka k, morita s, fukagawa t et al. a prospective cohort study to evaluate the feasibility of intraoperative antimicrobial prophylaxis in open gastrectomy for gastric cancer. surg infect, 2015;16(6):833-839. 19. imamura h, furukawa h, iijima s, sugihara s, tsujinaka t, tsukuma h,et al. multicenter phase ii study of antimicrobial prophylaxis in low-risk patients undergoing distal gastrectomy for gastric cancer. gastric cancer 2006;9: 32-35. 119 original comparison of single-dose and extended-dose antimicrobial prophylaxisümit mercan et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 114–119 20. imamura h, kurokawa y, tsujinaka t, inoue k, kimura y, iijima s et al. intraoperative versus extended antimicrobial prophylaxis after gastric cancer surgery: a phase 3, open-label, randomised controlled, noninferiority trial. lancet infect dis 2012;12:381-387. 21. hirao m, tsujinaka t, imamura h, kurokawa y, inoue k, kimura y et al; osaka gastrointestinal cancer chemotherapy study group (ogsg). overweight is a risk factor for surgical site infection following distal gastrectomy for gastric cancer. gastric cancer 2013;16:239-244. 22. utsumi m, shimizu j, miyamoto a, umeshita k, kobayashi t, monden m et al. age as an independent risk factor for surgical site infections in a large gastrointestinal surgery cohort in japan. j hosp infect 2010;75: 183-187. dx.doi.org/10.22317/jcms.v6i3.750 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 300 j contemp med sci | vol. 6, no. 6, november–december 2020: 300–305 original issn 2413-0516 introduction dietary supplements are considered as food products that contain dietary ingredients intended to supplement nutritional value to a diet. it encompasses specific vitamins, multivitamins, minerals, enzymes, amino acids, and herbs.1,2 as a balanced diet is essential for development and maintenance of the human body, dietary supplement usage has become commonplace to improve overall health and well-being, and to reduce the risk of certain diseases. dietary supplements use has become very common in the us, europe, and gcc countries.3 a previous study indicated that almost half of saudi participants consumed dietary supplements.4 another study reported 93.3% of saudi athletes consumed supplements,5 while 76.6% of female saudi college students reported consumption of dietary supplements.3 furthermore, dietary supplements were reported to be used by ~36% and ~24% of students in saudi health and non-health colleges, respectively, while 62.3% of saudi adults were reported to be supplement users or had used supplements at least once before.4 such widespread usage has been attributed to increased availability of dietary supplements, and the belief that dietary supplements are more natural and safer to use compared to prescription medication.6 this rise in supplement use, however, is also associated with an increase in the adverse effects caused by supplement mis-/overuse, with studies reporting health complications (and fatalities in severe cases) due to overdose, drug–drug, and drug–disease interactions caused by supplement misuse.7, 8. for example, excessive use of cholecalciferol and vitamin d may adversely affect soft tissues and kidney functions,9 while the medical management of cardiovascular diseases can be affected due to interactions between the supplement and various drugs used in the management of diseases.10 this study aimed to explore the use of dietary supplements among physically active adults and the general population in saudi arabia, and their awareness of positive/adverse effects of dietary supplements. methods study design this is a cross-sectional study using an online self-administered questionnaire. questions included covered sociodemographic and lifestyle characteristics, perceived health status, dietary supplement use, and general awareness regarding adverse effects. study population our target population was adults living in saudi arabia. participants <18 and >65 years of age were excluded. convenience sampling was utilized: assuming the estimated population of saudi arabia of 33.7 million, 95% confidence interval, we calculated a target sample size of 384. after excluding respondents under 18 and over 65 years of age, we analyzed a total of 574 responses. frequency of dietary supplements usage in the saudi arabian public and gym users muhammad raihan sajid1, lama nasser quraiba2, rahaf iyad khames2, noara alhusseini3, haifa alsheikh2, nabel rajab basha2, saad mansoor4, aamir omair5 1department of pathology, alfaisal university, riyadh, saudi arabia. 2alfaisal university, riyadh, saudi arabia. 3alfaisal university, riyadh, saudi arabia. 4department of medical education, alfaisal university, riyadh, saudi arabia. 5research unit, department of medical education, college of medicine, king saud bin abdulaziz university for health sciences, riyadh, saudi arabia. corresponding author: noara alhusseini (e-mail: nalhusseini@alfaisal.edu ) (submitted: 25 august 2020 – revised version received: 22 september 2020 – accepted: 25 october 2020 – published online: 26 december 2020) abstract objectives: the aim of the study is to explore the use of dietary supplements and to assess awareness of its positive/adverse effects among physically active adults and the general population in saudi arabia. methods: we conducted a cross-sectional online survey-based study and received 574 responses showing frequencies and types of dietary supplements. results: the results show that 44.6% of respondents use dietary supplements. the most used dietary supplements were vitamin c and multivitamins, consumed by 44% of the respondents, vitamin d (44%), iron (41%), and calcium (39%). protein sports bars and powder were consumed mainly by gym members, particularly those practicing weight-training exercises. approximately, 4% of respondents reported side-effects secondary to the usage of the dietary supplements. we conclude that the dietary supplement usage is widespread in saudi arabia among both gym members and the general public. conclusion: it is highly recommended to increase awareness measures from official health entities by educating the general population and physically active adults about dietary supplement use and adverse effects to ensure safe practices. keywords: 301 original frequency of dietary supplements usage in the saudi arabian public and gym users muhammad raihan sajid j contemp med sci | vol. 6, no. 6, november–december 2020: 300–305 survey tool (questionnaire) questions included covered sociodemographic and lifestyle characteristics, perceived health status, dietary supplement use, and general awareness regarding adverse effects. we modified and designed our questionnaire from the previously used and validated surveys.11, 12 content validity was performed by an expert in the field. the survey was also translated and made available in arabic as well as english. face validity was performed by an arabic language translation expert. the survey included questions aimed at assessing the knowledge, experience, and awareness of participants regarding dietary supplements in addition to demographic factors (such as age, gender, nationality, and level of education). we also queried the use, purpose, reasons, and duration of taking dietary supplements, as well as short-/long-term side effects, exercising, frequency of going to the gym, and type of supplement used among participants. ethical considerations ethical approval was obtained from alfaisal university institutional review board (approval number irb20026). researchers abided by the rules and regulations of the saudi national committee of bioethics and the research policies & procedures of alfaisal university. no identifying data were collected to ensure anonymity and confidentiality. access to the surveys’ responses was limited to the investigators. data collection and analysis the questionnaire was shared via electronic-social media platforms (instagram, twitter, and whatsapp and work/personal emails). data were extracted and analyzed using the spss statistics 21 data analysis package. the descriptive statistics of mean, standard deviation, percentages, and frequencies were calculated. the chi-square test was employed on categorical variables to examine associations. the significance was determined if the p-value was <0.05. results a total of 636 respondents completed the questionnaire from different regions in saudi arabia, of which 574 respondents fit the inclusion criteria. the sociodemographic characteristics of the participants are summarized in table 1 and their lifestyle choices are presented in table 2. the majority of the respondents were females (67%), from the younger age group of 18–25 years (68%), most had a bachelor’s degree or higher (68%), and almost two-thirds were saudi nationals (66%). the body mass index (bmi) grouping showed that 6% were underweight, while almost half were in the normal weight category (49%); 28% overweight, and 15% obese among the respondents as shown in table 1. table 2 shows that 163 (28%) did not exercise, while most reported exercising 3–5 days per week (37%) and another 14% exercised 6–7 days per week. there were 199 (35%) who were using some specific diet, while 264 (46%) of the participants used dietary supplements in the last month. there were 106 (18%) of the respondents who were members of a gym (table 3). table 1. sociodemographic characteristics of the study participants (n=574). variable n % gender females 383 66.5 males 191 33.2 age (years) 18-25 392 68.1 26-45 156 27.1 >45 28 4.9 educational level high school / diploma / intermediate 183 31.7 bachelor’s degree / graduate 391 67.8 nationality saudi 381 66.1 non-saudi 195 33.8 bmi (kg/m2) <18.5 33 5.7 18.5-24.9 284 49.3 25-29.9 162 28.1 30+ 85 14.8 missing 12 2.1 table 2. lifestyle choices of respondents (n=576). n % exercise no exercise 163 28.3 1-2 days/week 122 21.2 3-5 days/week 212 36.8 6-7 days/week 79 13.7 diet any specific diet 199 34.7 no specific diet 375 65.3 use of dietary supplements yes 264 44.6 no 317 55 302 original frequency of dietary supplements usage in the saudi arabian public and gym users muhammad raihan sajid j contemp med sci | vol. 6, no. 6, november–december 2020: 300–305 length of usage of dietary supplements (n=264) <1 month 40 6.9 1-3 months 73 12.7 >3 months 151 26.2 use of dietary supplements in the last month (n=264) daily 157 27.3 weekly 82 14.2 once a month 25 4.3 gym members yes 106 18.4 no 470 81.3 there were 264 respondents who were using dietary supplements; the most common supplements were vitamin c, vitamin d, and multivitamins consumed by 44% of these 264 respondents. other commonly used supplements included iron (41%) and calcium (39%). protein sports bars and powder were consumed mainly by gym members, particularly those practicing weight-training exercises. a few respondents reported using other specific dietary supplements that included probiotics, zinc, magnesium, and folic acid. there were 39% of the 264 respondents using dietary supplements who reported shortor long-term adverse effects directly resulting from supplement-use, while 4% reported side-effects secondary to the usage of the dietary supplements, including acne, constipation, bloating sensation, nausea, low appetite, change in urine color due to beta-carotene, dizziness, dependency, hair loss, erectile dysfunction, hypotension, changes in bowel movements, increased blood pressure, liver damage, anxiety, tremors, hypervitaminosis, and agitation. the major sources of information regarding dietary supplements were identified as the internet (31%), doctors/pharmacists (18%), and friends/family (8%). the most common reason for supplement use was to complete dietary/nutrient needs (34%), while 16% of the participants used it for muscle building; another (15%) reported use for medical recommendation/ prescription from a doctor. other reported reasons included boosting immunity, enhancing performance (stamina), and improving memory. of those who exercised, 60% reported exercising 1–2 h/session, while 38% reported exercising for <1 h. the most commonly reported type of workout was cardio (37%) and weight training (34%) exercises.table 3 shows the association between the demographic variables and use of dietary supplements. there was a significantly higher proportion of females (48.8%) than males (36.8%) who reported using dietary supplements (p=0004). respondents aged more than 45 years of age were more likely (70.3%) to use dietary supplements (p=0.002) as compared to the younger respondents aged 18–25 years (41.1%) and 26–45 years (49.3%). also, those with bmi greater than 30 kg/m2 (48.2%) and 25–29.9 kg/m2 (50.3%) were more likely to use dietary supplements as compared to those with bmi <18.5 (36.3%) and 18.5–24.9 kg/m2 (41.9%) (p=0.002). the use of dietary supplements was also higher in those who exercised (49.2%) as compared to those who did not (33.7%) (p=0.001). educational level was not significantly related to the use of dietary supplements (p=0.12). there was also no significant difference (p=0.13) in the use between saudis and non-saudis (table 3). table 3. association of demographic variables with use of dietary supplements. n use dietary supplement n (%) do not use dietary supplement n (%) p-value gender females 383 187 (48.8%) 196 (51.1%) 0.004 males 190 70 (36.8%) 120 (63.2%) educational level bachelor / graduate 391 168 (42.9%) 223 (57.1%) 0.12 high school / diploma 183 89 (48.6%) 94 (51.3%) age groups (yrs) 18-25 391 161 (41.1%) 230 (58.9%) 0.002 26-45 156 77 (49.3%) 79 (50.7%) >45 27 19 (70.3%) 8 (29.7%) bmi groups (kg/m2) <18.5 33 12 (36.3%) 21 (63.7%) 0.002 18.5-24.9 284 119 (41.9%) 165 (58.1%) 25-29.9 161 81 (50.3%) 80 (49.7%) >30 85 41 (48.2%) 44 (51.8%) nationality saudi 382 178 (46.6%) 204 (53.4%) 0.13 non-saudi 192 79 (41.1%) 113 (58.9%) exercise yes 410 202 (49.2%) 208 (50.7%) 0.001 no 163 55 (33.7%) 108 (66.2%) 303 original frequency of dietary supplements usage in the saudi arabian public and gym users muhammad raihan sajid j contemp med sci | vol. 6, no. 6, november–december 2020: 300–305 table 4 presents the association between demographic variables with being on a specific diet. there was no difference with regards to gender (p=0.09), educational level (p=0.40), age group (p=0.62), and nationality (p=0.07). it was found that respondents who were overweight or obese (bmi 25 kg/m2 and above) were more likely to be on a specific diet (42%) as compared to those with bmi <18.5 kg/m2 (6%) and those with normal bmi of 18.5 to <25 kg/m2 (31%) (p<0.001). table 5 also shows that there was no difference for exercising or not by gender (p=0.08), education level (p=0.32), and age group (p=0.69). respondents who had bmi <18.5 kg/m2 (48.5%) or bmi >30 kg/m2 were less likely to exercise as compared to those with normal bmi (76%) or bmi between 25 and <30 kg/m2 (74%) (p=0.001). also, saudi nationals were more likely (76%) to exercise as compared to non-saudis (63%) (p=0.001). table 5 shows no difference by gender in those who exercised (p=0.08). also, no statistically significant difference was seen in respondents who exercised based on their education levels (p=0.31). respondents with a higher bmi (>30 kg/m2) were more likely to exercise as compared to those having a normal bmi (p=0.001). saudis were more likely to exercise as compared to the non-saudi respondents (p=0.001). table 4. association of demographic variables with being on a specific diet. n on specific diet n (%) no specific diet n (%) p-value gender females 383 142 (37.1%) 241 (62.9%) 0.09 males 190 57 (30%) 133 (70%) educational level bachelor / graduate 391 140 (35.8%) 251 (64.2%) 0.40 high school / diploma 183 59 (32.2%) 124 (67.8%) age groups (yrs) 18-25 391 126 (32.2%) 265 (67.8%) 0.62 26-45 156 70 (44.9%) 86 (55.1%) >45 27 3 (11.1%) 24 (88.9%) bmi groups (kg/m2) <18.5 33 2 (6.1%) 31 (93.9%) <0.001 18.5-24.9 284 87 (30.6%) 197 (69.4%) 25-29.9 161 68 (42.2%) 93 (57.8%) >30 85 36 (42.4%) 49 (57.6%) nationality saudi 382 141 (36.9%) 241 (63.1%) 0.07 non-saudi 192 58 (30.2%) 134 (69.8%) table 5. association of demographic variables with exercise. n exercise n (%) do not exercise n (%) p-value gender females 383 265 (69.2%) 118 (30.8%) 0.08 males 189 144 (76.2%) 45 (23.8%) educational level bachelor / graduate 390 274 (70.3%) 116 (29.7%) 0.32 high school / diploma 183 136 (74.3%) 47 (25.7%) age groups (yrs) 18-25 391 275 (70.3%) 116 (29.7%) 0.69 26-45 155 118 (76.1%) 37 (23.9%) >45 27 17 (63%) 10 (37%) bmi groups (kg/m2) <18.5 33 16 (48.5%) 17 (51.5%) 0.001 18.5-24.9 284 216 (76.1%) 68 (23.9%) 25-29.9 161 119 (73.9%) 42 (26.1%) >30 84 51 (60.7%) 33 (39.3%) nationality saudi 381 289 (75.9%) 92 (24.1%) 0.001 non-saudi 192 121 (63%) 71 (37%) 304 original frequency of dietary supplements usage in the saudi arabian public and gym users muhammad raihan sajid j contemp med sci | vol. 6, no. 6, november–december 2020: 300–305 discussion the findings of this study demonstrated a high prevalence of dietary supplement use in saudi arabia (45.8%) and showed an association with certain demographics and lifestyle factors. other studies have suggested that dietary supplement use has been very common on a global level.13 the prevalence of dietary supplement use varies globally, however, the findings indicate that it is relatively high in saudi arabia compared to other counties. a cross-sectional study used nationally representative data from the national health and nutrition examination survey in the united states indicated that over half of americans used at least one type of dietary supplements during the previous month of the study.14 in addition, the prevalence of dietary supplement use among pakistanis was 48.2%,15 and 35.6% among college students in the united arab emirates,16 which confirm the findings of the high prevalence of dietary supplements use in general. another cross-sectional study among saudi population revealed that more than half of its participants used dietary supplements, which is similar to our findings.17 moreover, a local study at princess nourah bint abdulrahman university in riyadh also highlighted the finding of the high prevalence of dietary supplement use in saudi as all its participants have used it at some point in time and 32.3% were using it at the time of the study.18 alowais et al have suggested that increased awareness levels about health promotion and disease prevention contributed to the increased use of dietary supplements in many countries,17 which may justify the high prevalence of dietary supplement use in saudi arabia. however, a chinese study revealed that only 24.2% of its participants consumed dietary supplements during the past year.19 the frequency of dietary supplement use in saudi arabia seems to be higher than other countries. a comparison of educational levels (bachelors and graduates vs high school and diploma holders) did not show a statistically significant difference regarding the use of dietary supplements. these findings match another study conducted in saudi arabia that found no association between dietary supplement use and the level of education.1 however, another study in saudi arabia found that dietary supplement use was more prevalent (84.5%) among those with high educational levels.17 also, another study indicated higher dietary supplement use among chinese medical students (58.9%) compared to non-medical students.19 the difference between these results and other studies can be due to the nature of this study, since the survey was distributed electronically and most participants (67.8%) had higher education. since dietary supplements have been very common, sources of information about it vary. the major sources of information regarding dietary supplements identified by the participants were internet, doctors, or pharmacists, and friends, family, or relatives which is in concordance with findings of other studies.17 however, rogza et al. found that physicians were the most frequent source of information for dietary supplement use, which is safer for consumers unlike unreliable sources like internet.20 though only 15% of the participants used dietary supplements due to a medical recommendation/prescription from a doctor, it is recommended to increase awareness measures from official health entities by educating the general population and physically active adults about dietary supplement use and adverse effects to ensure safe practices. the findings show that the most used dietary supplements were vitamin c and multivitamins or both, unlike another saudi-based study among females that showed that cod liver oil, omega 3, multivitamins, ginseng, and vitamin a to be most commonly used supplements. the difference could be a result of limiting the study to college females only as their reason for dietary supplement use was to maintain healthy hair, unlike this study that included both males and females up to 65 years old.3 similarly, a study conducted among saudi females suggested that the primary reason for using dietary supplements was for aesthetic reasons.18 other commonly used supplements in this study were: vitamin d, iron, and calcium. protein sports bars and protein powder were consumed mainly by gym members, particularly those practicing weight-training exercises. a few participants reported using other specific dietary supplements that include probiotics, zinc, magnesium, and finally folic acid. however, jawadi et al found that most commonly used supplements among active adults who go to the gym in saudi were whey protein, amino acids, multivitamins, creatinine, and omega 3 to improve body shape,21 which is slightly similar to the findings in this study as 16% of participants used it for muscle building. other reported reasons included boosting immunity, enhancing performance (stamina), and improving memory. athletes use dietary supplements for various reasons such as maintenance of good health and improving micronutrient deficiencies. maintaining a balanced meal can be a challenge, therefore, dietary supplements replace inadequate energy and macronutrient intake.13 this study mirrors the findings as the most common reason for using dietary supplements reported by the participants was completing dietary/nutrient needs. the results indicate that almost 40% of the responses replied in affirmative regarding dietary supplements having shortor long-term adverse effects. significantly just 4% of respondents reported side-effects secondary to the usage of the dietary supplements. another study conducted among saudis also revealed that dietary supplement consumers (75%) were aware of its adverse effects.4 in contrast, another study in india showed that only 16.9% of dietary supplement users felt that regular use results in side-effects.22 some of the adverse effects reported included acne, constipation, bloating sensation, nausea, low appetite, change in urine color due to beta carotene, dizziness, dependency, hair loss, erectile dysfunction, hypotension, changes in bowel movements, increased blood pressure, liver damage, anxiety, tremors, hypervitaminosis, and agitation. some of the adverse effects including diarrhea, constipation, stomachache, headache, nausea, and vomiting have been reported among japanese adults from using dietary supplements. however, only just under 10% of them have reported the symptoms due to the inability to link the cause and effect relationship of these adverse effects.23 the results showed that respondents who exercised were more likely to use dietary supplements. a cross-sectional study among saudi females mirrors our findings as it showed that more physical activity was significantly positively associated with dietary supplements use.3 while another study in saudi arabia showed that dietary supplement users believed that its use is most needed among pregnant women and during recovery from diseases.17 since this study sample included physically active participants, this difference can be justified by the type of dietary supplements and the purpose of its use. 305 original frequency of dietary supplements usage in the saudi arabian public and gym users muhammad raihan sajid j contemp med sci | vol. 6, no. 6, november–december 2020: 300–305 study limitations since our study was conducted during coronavirus covid-19 pandemic, all sports activities including gyms were closed due to social distancing and quarantine regulations, which might have impacted physical activity levels and dietary supplement use. therefore, this factor can affect the generalizability of our study. in addition, due to the nature of the cross-sectional studies, causal inferences cannot be achieved. moreover, recall bias in the type and frequency of supplement use may be present. selection bias is another limitation given that only people with internet access were included in our study. conclusion the dietary supplement usage is widespread in saudi arabia among both gym members and general public. the incidence of reported adverse effects is low as compared to other published studies. since there are many dietary supplements available, future studies should be conducted to assess different types of dietary supplements and its adverse effects in saudi arabia. in addition, it is highly recommended to increase awareness measures from official health entities by educating the general population and physically active adults about dietary supplement use and adverse effects to ensure safe practices. conflicts of interests authors report no conflict of interests. references 1. alfawaz ha, krishnaswamy s, al-faifi l, atta ha bin, al-shayaa m, alghanim sa, et al. awareness and attitude toward use of dietary supplements and the perceived outcomes among saudi adult male members of fitness centers in saudi arabia. int j sport nutr exerc metab. 2018. 2. muwonge h, zavuga r, kabenge pa, makubuya t. nutritional supplement practices of professional ugandan athletes: a cross-sectional study. j int soc sports nutr. 2017. 3. alfawaz h, khan n, alfaifi a, shahrani fm, al tameem hm, al otaibi sf, et al. prevalence of dietary supplement use and associated factors among female college students in saudi arabia. bmc womens health. 2017. 4. algaeed h, aljaber m, alwehaibi a, aljaber l, arafah a, aloyayri m, et al. general public knowledge and use of dietary supplements in riyadh, saudi arabia. j fam med prim care. 2019. 5. aljaloud so, ibrahim sa. use of dietary supplements among professional athletes in saudi arabia. j nutr metab. 2013. 6. dennehy ce, tsourounis c, horn aj. dietary supplement-related adverse events reported to the california poison control system. am j heal pharm. 2005. 7. deuster pa. dietary supplements and adverse events. mil med. 2020. 8. timbo bb, ross mp, mccarthy p v., lin ctj. dietary supplements in a national survey: prevalence of use and reports of adverse events. j am diet assoc. 2006. 9. drüeke tb, massy za. role of vitamin d in vascular calcification: bad guy or good guy? nephrology, dialysis, transplantation : official publication of the european dialysis and transplant association european renal association. 2012. 10. dwyer jt, coates pm, smith mj. dietary supplements: regulatory challenges and research resources. nutrients. 2018. 11. lieberman hr, stavinoha tb, mcgraw sm, white a, hadden ls, marriott bp. use of dietary supplements among active-duty us army soldiers. am j clin nutr. 2010. 12. lieberman hr, marriott bp, williams c, judelson da, glickman el, geiselman pj, et al. patterns of dietary supplement use among college students. clin nutr. 2015. 13. maughan rj, burke lm, dvorak j, larson-meyer de, peeling p, phillips sm, et al. ioc consensus statement: dietary supplements and the highperformance athlete. br j sports med. 2018. 14. kantor ed, rehm cd, du m, white e, giovannucci el. trends in dietary supplement use among us adults from 1999-2012. jama j am med assoc. 2016. 15. naqvi aa, ahmad r, zehra f, yousuf r, kachela b, nehal nadir m. dietary supplement use among students of pharmacy colleges in the city of karachi, pakistan: prevalence, opinions, and attitudes. j diet suppl. 2019. 16. radwan h, hasan ha, ghanem l, alnajjar g, shabir a, alshamsi a, et al. prevalence of dietary supplement use and associated factors among college students in the united arab emirates. j community health. 2019. 17. alowais m, selim me-h. knowledge, attitude, and practices regarding dietary supplements in saudi arabia. j fam med prim care. 2019. 18. altamimi jz. awareness of the consumption of dietary supplements among students in a university in saudi arabia. j nutr metab. 2019. 19. liu h, yang y, xu d, xia h, pan d, wang s, et al. investigation and comparison of nutritional supplement use, knowledge, and attitudes in medical and non-medical students in china. nutrients. 2018. 20. tindle ha, wolsko p, davis rb, eisenberg dm, phillips rs, mccarthy ep. factors associated with the use of mind body therapies among united states adults with musculoskeletal pain. complement ther med. 2005. 21. jawadi ah, addar am, alazzam as, alrabieah fo, al alsheikh as, amer rr, et al. prevalence of dietary supplements use among gymnasium users. j nutr metab. 2017. 22. joseph n, kumar a, singh h, shaheen m, das k, shrivastava a. nutritional supplement and functional food use among medical students in india. j diet suppl. 2018. 23. chiba t, sato y, kobayashi e, ide k, yamada h, umegaki k. behaviors of consumers, physicians and pharmacists in response to adverse events associated with dietary supplement use. nutr j. 2017. https://doi.org/10.22317/jcms.v6i6.891 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 36 j contemp med sci | vol. 2, no. 6, spring 2016: 36–41 research assessment of psychosocial aspects of patients with rectal cancer under chemotherapy in baghdad city kareem r. sagta, wissam j. qassimb, rajaa ibrahim abedc introduction colorectal cancer (also known as colon cancer, rectal cancer or bowel cancer) is the development of cancer in the colon or rectum (parts of the large intestine).1 it is due to the abnormal growth of cells that have the ability to invade or spread to other parts of the body.2 signs and symptoms may include blood in the stool, a change in bowel movements, weight loss and feeling tired all the time.3 risk factors for colorectal cancer include lifestyle, older age and inherited genetic disorders that only occur in a small fraction of the population.4,5 other risk factors include diet, smoking, alcohol, lack of physical activity, family history of colon cancer and colon polyps, presence of colon polyps, race, exposure to radiation and even other diseases such as diabetes and obesity. a diet, high in red, processed meat, while low in fiber, increases the risk of colorectal cancer. other diseases such as inflammatory bowel disease, which includes crohn’s disease and ulcerative colitis, can also increase the risk of colorectal cancer. some of the inherited conditions that can cause colorectal cancer include: familial adenomatous polyposis and hereditary non-polyposis colon cancer; however, these represent <5% of cases. it typically starts as a benign tumour, often in the form of a polyp, which over time becomes cancerous.4,5 bowel cancer may be diagnosed by obtaining a sample of the colon during a sigmoidoscopy or colonoscopy. this is then followed by medical imaging to determine whether the disease has spread. screening is effective for early detection to reduce colorectal cancer death, and consistent screening is recommended starting from the age of 50–75.6 during colonoscopy, small polyps may be removed if found. if a large polyp or tumour is found, a biopsy may be performed to check whether it is cancerous. aspirin and other non-steroidal anti-inflammatory drugs decrease the risk. their general use is not recommended for this purpose due to side effects.7,8 treatments used for colorectal cancer may include some combination of surgery, radiation therapy, chemotherapy and targeted therapy. cancers that are confined within the wall of the colon may be curable with surgery while cancer that has spread widely is usually not curable, with management focusing on improving quality of life and symptoms. five-year survival rates in the united states are around 65%. this, however, depends on how advanced the cancer is, whether or not all the cancer can be removed with surgery, and the person’s overall health. globally, colorectal cancer is the third most common type of cancer making up about 10% of all cases. in 2012, there were 1.4 million new cases and 694,000 deaths from the disease. it is more common in developed countries, where more than 65% of cases are found. it is less common in women than men.9,10 materials and methods objectives of the study the study objectives are to determine the effect of chemotherapy upon psychological and social aspects of the quality of issn 2413-0516 adepartment of psychiatric mental health, college of nursing, university of baghdad, iraq. bdepartment of community health nursing, college of nursing, university of baghdad, iraq. cdepartment of fundamentals of nursing, college of nursing, university of baghdad, iraq. correspondence to wissam j. qassim (email: drwissamjk@yahoo.com). (submitted: 17 february 2016 – revised version received: 12 march 2016 – accepted: 20 april 2016 – published online: 26 june 2016) objectives the study objectives are to determine and identify the association between the effect of chemotherapy upon psychological and social aspects of the quality of life for patients with rectal cancer treated by chemotherapy and demographic characteristics including age, gender, housing, marital status, educational level, occupation and income. methods quantitative design (a descriptive study): the study was conducted at the medical city/baghdad teaching hospital, al-kadhimiya teaching hospital and radiation and nuclear medicine hospital in baghdad, starting from december 30, 2014 to february 15, 2015. to achieve the objectives of the study, according to special criteria, a non-probability (purposive) samples of (50) patient who reviewed the hospitals above to receive chemotherapy drugs to treatment disease. the data were collected by self-reporting of patients with rectal cancer. instrument validity was determined through content validity by a panel of experts. reliability of the instrument was determined through the use of pearson correlation coefficient for the test–retest approach, which was 0.88. the analysis of data was performed through the application of descriptive statistics (frequency, percentage and mean of score) and inferential statistics (chi-square [χ2] test). results the results of the study indicated most of patients with rectal cancer have side effects of chemotherapy related to psychological and social aspects through the increase significant of items related to side effects during assessment of these items, and there is no significant association between the effect of chemotherapy upon quality of life for patients and gender, housing, occupation and income. while, there is significant relationship between the effect of chemotherapy upon the quality of life for patients and age, marital status and educational level. conclusion the researcher can conclude most of patients with rectal cancer have side effects of chemotherapy related to the psychological and social aspects. keywords rectal cancer, chemotherapy, psychosocial 37j contemp med sci | vol. 2, no. 6, spring 2016: 36–41 research effect of chemotherapy on rectal cancer patientswissam j. qassim et al. life for patients with rectal cancer. and to identify the association between the effect of chemotherapy upon psychological and social aspects of the quality of life for patients and their demographic characteristics including age, gender, housing, marital status, educational level, occupation and income. design of the study quantitative design (a descriptive study) was carried out to determine the effect of chemotherapy upon psychological and social aspects of the quality of life for patients with rectal cancer. setting of the study the study was conducted at the medical city/baghdad teaching hospital, al-kadhimiyia teaching hospital and radiation and nuclear medicine hospital in baghdad. sample of the study a non-probability (purposive) sample of (50) patient who reviewed the hospitals above to receive chemotherapy drugs to treat the disease. instrument construction after extensive review of relevant literature which includes the effect of chemotherapy upon psychological and social aspects of the quality of life for patients with rectal cancer. the questionnaire was constructed for the purpose of the study consisted of 40 items which include two parts: part i: patients’ demographic characteristics the first part concerned with determination of the demographic characteristics of these patients through designated sheet which include ten items: age, gender, housing, marital status, level of education, occupation, income, method of taking the treatment, duration of the disease and body max index. part ii: questionnaire to side effect of treatment this part is concerned with data to side effects of chemotherapy drugs upon patients with rectal cancer which include: 1. effects related to the psychological situation consisted of 18 items. 2. side effects related to the personal and social consisted of 12 items. the questionnaire to side effect of treatment were ordinal according to the three level scale which were scored as (never = 1, sometimes = 2, always = 3) for each level respectively so the cutoff point was 2. content validity was determined through the use of panel of experts. data collection the data were collected by self-reporting by patients with lung cancer for the period from 15th january to 14th february 2015. statistical data analysis appropriate statistical approach is used that includes descriptive statistics (frequency, percentage and mean of score) and inferential statistics (chi-square [χ2] test). results table 1 reveals that the majority (56%) of patients were 60 years old and more. 90% of patients were male, 62% of patients table 1. distribution of the samples according to demographic characteristics no. variables 1. age* (years) f % 1.1. less than 20 2 4 1.2. 20–29 2 4 1.3. 30–39 4 8 1.4. 40–49 5 10 1.5. 50–59 9 18 1.6. 60 and more 28 56 total 50 100 2. gender f % 2.1. male 45 90 2.2. female 5 10 total 50 100 3. housing f % 3.1. urban 19 38 3.2. rural 31 62 total 50 100 4. marital status f % 4.1. single 5 10 4.2. married 45 90 total 50 100 5. level of education f % 5.1. illiteracy 23 46 5.2. able to read and write 4 8 5.3. primary school graduate 10 20 5.4. intermediate school graduate 7 14 5.5. high school graduate 2 4 5.6. institute and college graduate 4 8 total 50 100 6. occupation f % 6.1. student 1 2 6.2. government employee 3 6 6.3. retired 5 10 6.4. self-employee 8 16 6.5. other 33 66 total 50 100 7. income f % 7.1. adequate 17 34 7.2. not adequate 33 66 total 50 100 8. body mass index f % 8.1. less than 20 26 52 8.2. 20–25 10 20 8.3. 26–30 12 24 8.4. 31–35 2 4 total 50 100 f: frequency; %: percent. 38 j contemp med sci | vol. 2, no. 6, spring 2016: 36–41 effect of chemotherapy on rectal cancer patients research wissam j. qassim et al. live in rural house and 90% of patients were married. concerning the level of education, 46% was illiteracy and 66% of patients had other occupation. in relation to income, 66% of patients were not adequate, 52% of patients had the body mass index of less than 20. table 2 shows that the significant side effect of treatment related to the psychological aspect was non-significant on items (3, 6, 8, 11 and 12) and significant on items (1, 2, 4, 5, 7, 9, 10, 13, 14, 15, 16, 17 and 18). table 3 reveals that the significant side effect of treatment related to social aspect was non-significant on item (6) and significant on items (1, 2, 3, 4, 5, 7, 8, 9, 10, 11 and 12). table 4 shows that there is high significant association between the level of education of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores. and indicates that there is significant association between ages and marital status of sample and the effect of chemotherapy upon psychological and social aspects of the table 2. the mean of scores a. side effects related to the psychological aspect no. items never sometime always ms s. f % f % f % 1. bank is very concerned for the future of your family 32 64 9 18 9 18 1.54 s 2. concerned about the length of treatment 25 50 20 40 5 10 1.6 s 3. disturbed for no reason 31 62 14 28 5 10 1.48 ns 4. feel pessimistic about the future 26 52 16 32 8 16 1.64 s 5. feel the loss your important when the other 13 26 25 50 12 24 1.98 s 6. feel a desire to cry 37 74 10 20 3 6 1.32 ns 7. feel that life is difficult 25 50 21 42 4 8 1.58 s 8. feel uncomfortable 45 90 2 4 3 6 1.16 ns 9. feel you are useless to your family 17 34 23 46 10 20 1.86 s 10. feel remorse for your actions the previous 22 44 19 38 9 18 1.74 s 11. feel sorry for yourself 32 64 14 28 4 8 1.44 ns 12. feel the fear of disease 34 68 11 22 5 10 1.42 ns 13. having difficulty adapting to the disease 27 54 18 36 5 10 1.56 s 14. is terrified of the disease 26 52 12 24 12 24 1.72 s 15. is fragmented and confused 22 44 20 40 8 16 1.72 s 16. suffer from sleep disturbances 23 46 25 50 2 4 1.58 s 17. suffers from disturbing dreams 11 22 22 44 17 34 2.12 s 18. you feel you have become a secret passion 14 28 28 56 8 16 1.88 s ms: mean of scores; s: significant; ns: non-significant. table 3. the mean of scores and significant side effect of treatment for section: b. side effects related to social aspect no. items never sometime always ms s. f % f % f % 1. attention to yourself experiencing difficulty 18 36 21 42 11 22 1.86 s 2. changing family relationships suffer 17 34 18 36 15 30 1.96 s 3. experience to rely on family members 27 54 17 34 6 12 1.58 s 4. experiencing lack of social activities 19 38 23 46 8 16 1.78 s 5. experiencing unity 19 38 21 42 10 20 1.82 s 6. experiencing fear of the future 34 68 10 20 6 12 1.44 ns 7. stop experiencing the fear of family support 18 36 14 28 18 36 2 s 8. suffer change of family responsibilities 17 34 19 38 14 28 1.94 s 9. suffer from the impact of the disease on your work or study 29 57.9 6 12 15 30 1.72 s 10. suffer the loss of financial security 25 50 9 18 16 32 1.82 s 11. suffers from the difficulty of social integration 12 24 25 50 13 26 2.02 s 12. treatment of people suffering a difference to you about other 18 36 21 42 11 22 1.86 s ms: mean of scores; s: significant; ns: non-significant. 39j contemp med sci | vol. 2, no. 6, spring 2016: 36–41 research effect of chemotherapy on rectal cancer patientswissam j. qassim et al. continued table 4. association between age, gender, marital status, housing, level of education, occupation, income and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores scores age (years) good fair poor total χ² obs. sig. f f f f less than 20 2 0 0 2 19.316 s 20–29 0 2 0 2 30–39 3 1 0 4 40–49 1 2 2 5 50–59 1 7 1 9 60 and more 8 19 1 28 total 15 31 4 50 p ≤ 0.05; df: 10; χ² crit.: 18.31. scores gender good fair poor total χ² obs. sig. f f f f male 17 15 13 45 5.858 nsfemale 1 2 2 5 total 18 17 15 50 p ≤ 0.05; df: 2; χ² crit.: 5.99. scores housing good fair poor total χ² obs. sig. f f f f urban 10 4 5 19 0.573 nsrural 5 24 2 31 total 15 28 7 50 p ≤ 0.05; df: 2; χ² crit.: 5.99. scores marital status good fair poor total χ² obs. sig. f f f f single 2 3 0 5 6.523 smarried 15 25 5 45 total 17 28 5 50 p ≤ 0.05; df: 2; χ² crit.: 5.99. scores level of education good fair poor total χ² obs. sig. f f f f illiteracy 7 16 0 23 22.988 hs able to read and write 0 4 0 4 primary school graduate 3 5 2 10 intermediate school graduate 3 2 2 7 high school graduate 2 0 0 2 institute and college graduate 3 1 0 4 total 18 28 4 50 p ≤ 0.05; df: 10; χ² crit.: 18.31. scores occupation good fair poor total χ² obs. sig. f f f f student 0 1 0 1 11.425 ns government employee 3 0 0 3 retired 0 5 0 5 self-employee 1 5 2 8 other 12 17 4 33 total 16 28 6 50 p ≤ 0.05; df: 8; χ² crit.: 15.51. 40 j contemp med sci | vol. 2, no. 6, spring 2016: 36–41 effect of chemotherapy on rectal cancer patients research wissam j. qassim et al. quality of life of patient’s scores. also reveals that there is no significant association between income, occupation and housing of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores. discussion through the data analysis, distribution of demographic variables in table 1 reports that, most of patients with rectal cancer are 60 years old and more and this account for 28 (56%). this result is similar to the results obtained from the study done by fu et al. (2007). these results indicate that the majority of aged patients with rectal cancer are diagnosed at the age of 55–64 years old.8 regarding gender of patients with rectal cancer, 45 (90%) of patients were male. this finding is similar to the results obtained from the study done by centers for disease control and prevention (2009).9 concerning the housing, most of patients with rectal cancer are living in rural house (31 [62%]). these findings are supported by world cancer report (2014). these results indicate that the majority of patients with rectal cancer are living in urban house.10 with regard to the marital status of patients with rectal cancer, it is demonstrated that most of the patients were married, 45 (90%). this finding is similar to the results obtained from the study which indicates that most of patients with rectal cancer are married.11 forty two (84%) patients have children, while 90% of patients do not have children after the disease. this result is consistent with the study which indicates that the majority of patients do not have children after the disease (90%).12 in relation to level of education, the majority of patients with rectal cancer, 23 (46%) was illiterate. this result is inconsistent with the study which indicates that the majority of patients with rectal cancer are high school graduate (70%).13 the majority of patients in this study, 33 (66%), have other occupations. this result is disagreement with the study that the majority of patients in this study are working in industrial occupations (60%).14 the researcher refers that the occupations related to industrial materials have great effect for injuries with rectal cancer. the monthly income was not adequate for 33 (66%) patients. this result is in agreement with a study which indicates that the monthly income of majority of patients is not adequate (65%).15 twenty four (48%) of patients use intravenous for the administration of drugs. 47 (94%) of patients are suffering from disease for about 1–4 months. this result is in agreement with a study, which indicates that the majority of patients taking drugs of chemotherapy by intravenous administration and increase the side effects of chemotherapy.16 in relation to the body mass index of patients with rectal cancer, most of the patients are <20 which account 26 (52%). this result is inconsistent with the study which indicates that the majority of patients are above 25 (78%).17 table 2 shows that the significant to side effect of treatment related to the psychological aspect was non-significant on items (feel a desire to cry, feel sorry for yourself, feel the fear of disease, disturbed for no reason and feel uncomfortable). significant on items (feel pessimistic about the future, feel that life is difficult, feel remorse for your actions than the previous, feel you have become a secret passion, bank is very concerned about the future of your family, feel the loss your important when the other, feel you are useless to your family, suffers from disturbing dreams, concerned about the length of treatment, fragmented and confused, having difficulty adapting to the disease, suffer from sleep disturbances, and terrified of the disease). the result of this study disagrees with the study that indicates to increase the side effect of chemotherapy for patients with pulmonary cancer, especially these related to psychological aspects to feel pessimistic about the future, feel that life is difficult, feel remorse for your actions than the previous, feel you have become a secret passion, bank is very concerned about the future of your family.19 table 3 reveals that the significant side effect of treatment related to social aspect was non-significant on item (experiencing fear of the future). significant on items (attention to yourself experiencing difficulty, suffer from the impact of the disease on your work or study, suffer the loss of financial security, stop experiencing the fear of family support, suffer change of family responsibilities, changing family relationships to suffer, experiencing lack of social activities, experience to rely on family members, treatment of people suffering a difference to you about other, experiencing unity and suffers from the difficulty of social integration). this result disagrees with the study, which showed the high significant of score for all items of personal and social.20 table 4 indicates that there is significant association between ages of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores. this result agrees with the study which indicated there were no significant differences between the age of patients and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores.21 the study shows that there is no significant association between gender of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores. this results contability with the result obtained from study which indicated that there is no relationship between scores income good fair poor total χ² obs. sig. f f f f adequate 10 7 0 17 4.071 nsnot adequate 12 15 6 33 total 22 22 6 50 p ≤ 0.05; df: 2; χ² crit.: 5.99. sig.: level of significance; hs: highly significant; ns: non-significant; p: probability value; χ2: chi-squared test; df: degree of freedom. table 4. continued 41j contemp med sci | vol. 2, no. 6, spring 2016: 36–41 research effect of chemotherapy on rectal cancer patientswissam j. qassim et al. gender and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores.22 the analysis of the result of the study shows that there is no significant association between housing of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores. this result agrees with the study which showed that there is no significant relationship between housing of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores.23 there is significant association between marital status of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores. these finding agree with the results obtained from a study done by möslein et al. 2003, which indicated that there is significant association between marital status of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores.24 regarding the relationship between the level of education and the effect of chemotherapy upon quality of life of patient’s scores, this study indicates that there is high significant association between level of education of sample and the effect of chemotherapy upon quality of life of patient’s scores. this result is in agreement with the study which indicates significant association between level of education of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores.25 the analysis of the result of the study shows that there is no significant association between occupation of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores. this result agrees with the study which showed that there is no significant relationship between occupation of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores.25 there is no significant association between income of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores. these findings agree with the results obtained from a study which indicated that there is no significant association between income of sample and the effect of chemotherapy upon psychological and social aspects of the quality of life of patient’s scores.26 conclusion the study concluded that most of patients with rectal cancer have side effects of chemotherapy related to the psychological and social aspects. recommendations 1. making quality information more available to assist patients in making informed decisions about change quality of life after treatment with chemotherapy. 2. increased health education by focusing on the side effect of chemotherapy upon quality of life and how to prevent these effects through t.v. programs, radio, newspaper and medical magazines…etc.  references 1. colon cancer treatment (pdq®). nci. 2014-05-12. retrieved on 29 june 2014. 2. defining cancer. national cancer institute. retrieved on 10 june 2014. 3. general information about colon cancer. nci. 2014-05-12. retrieved on 29 june 2014. 4. world cancer report 2014. world health organization. 2014. pp. chapter 5.5. 5. colorectal cancer prevention (pdq®). national cancer institute. 2014-02-27. retrieved on 29 june 2014. 6. screening for colorectal cancer. u.s. preventive services task force. october 2008. retrieved on 29 june 2014. 7. thorat ma, cuzick j. role of aspirin in cancer prevention. curr oncol rep. 2013;15(6):533–40. doi: 10.1007/s11912-013-0351-3 pmid: 24114189 8. rostom a, lewin g, dubé c, code c. routine aspirin or nonsteroidal anti-inflammatory drugs for the primary prevention of colorectal cancer: recommendation statement. am fam phys. 2007;76(1):109–13. 9. seer stat fact sheets: colon and rectum cancer. nci. retrieved 18 june 2014. 10. world cancer report 2014. world health organization. 2014. pp. chapter 1.1. 11. yamada t. principles of clinical gastroenterology. in: alpers dh (ed). chichester: wiley-blackwell; 2008. p. 381. 12. astin m, griffin, t, neal, rd, rose, p, hamilton, w. the diagnostic value of symptoms for colorectal cancer in primary care: a systematic review. br j gen pract. 2011;61(586):231–43. doi: 10.3399/bjgp11x572427 pmid: 21619747 13. adelstein ba, macaskill, p, chan, sf, katelaris, ph, irwig, l. most bowel cancer symptoms do not indicate colorectal cancer and polyps: a systematic review. bmc gastroenterol. 2011;11:65. doi: 10.1186/1471-230x-11-65 pmid: 21624112 14. watson aj, collins, pd. colon cancer: a civilization disorder. dig dis. 2011;29(2):222–8. doi: 10.1159/000323926 pmid: 21734388 15. cunningham d, atkin w, lenz hj, lynch ht, minsky b, nordlinger b, et al. colorectal cancer. lancet. 2010;375(9719):1030–47. doi: 10.1016/s01406736(10)60353-4 pmid: 20304247 16. lee im, shiroma ej, lobelo f, puska p, blair sn, katzmarzyk pt. effect of physical inactivity on major non-communicable diseases worldwide: an analysis of burden of disease and life expectancy. lancet. 2012;380(9838):219–29. doi: 10.1016/s0140-6736(12)61031-9 pmid: 22818936 17. fedirko v, tramacere i, bagnardi v, rota m, scotti l, islami f, et al. alcohol drinking and colorectal cancer risk: an overall and dose-response metaanalysis of published studies. ann oncol. 2011;22(9):1958–72. doi: 10.1093/ annonc/mdq653 pmid: 21307158 18. valtin h. “drink at least eight glasses of water a day.” really? is there scientific evidence for “8 × 8”? am j physiol regul integr comp physiol. 2002;283(5):r993–r1004. pmid: 12376390 19. jawad n, direkze n, leedham sj. “inflammatory bowel disease and colon cancer”. recent results cancer res. 2011;185:99–115. doi: 10.1007/978-3642-03503-6_6 pmid: 21822822 20. xie j, itzkowitz sh. cancer in inflammatory bowel disease. world j gastroenterol. 2008;14(3):378–89. pmid: 18200660 21. triantafillidis jk, nasioulas g, kosmidis pa. colorectal cancer and inflammatory bowel disease: epidemiology, risk factors, mechanisms of carcinogenesis and prevention strategies. anticancer res. 2009;29(7): 2727–37. pmid: 19596953 22. juhn e, khachemoune a. gardner syndrome: skin manifestations, differential diagnosis and management. am j clin dermatol. 2010;11(2):117–22. doi: 10.2165/11311180-000000000-00000 pmid: 20141232 23. half e, bercovich d, rozen p. familial adenomatous polyposis. orphanet j rare dis. 2009;4:22. doi: 10.1186/1750-1172-4-22 24. möslein g, pistorius s, saeger hd, schackert hk. preventive surgery for colon cancer in familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer syndrome. langenbecks arch surg. 2003;388(1):9–16. pmid: 12690475 25. stein u, walther w, arlt f, schwabe h, smith j, fichtner i, et al. macc1, a newly identified key regulator of hgf-met signaling, predicts colon cancer metastasis. nature med. 2008;15(1):59–67. doi: 10.1038/nm.1889 pmid: 19098908 26. stein u. macc1: a novel target for solid cancers. expert opin ther targets. 2013;17(9):1039–52. doi: 10.1517/14728222.2013.815727 pmid: 23815185 126 j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 original issn 2413-0516 introduction it is well-known that better health for a greater portion of population leads to higher productivity and more equitable economic development. in the health sector, the benefits of implementing policies derived from sound evidence-based research may often yield desirable outcomes. focusing on essential research leads to better use of available resources for health and ultimately to more health gains per dollar spent. in fact, one of the basic tenets of economics is that resources are always limited and there can never be enough of it to meet all the perceived needs. there will never be enough resources to address all the peoples’ health needs, especially in developing countries. in resource rich settings, a high proportion of available research funds go to investigator-driven initiatives, but in limited resource settings, there is an expectation that research must respond more directly to community health needs, and therefore be conducted according to recognized priorities. the outcomes and benefits cannot always be evaluated in economic terms. hence, prioritizing domains for research has to be evaluated on several factors including burden of illness, impact on specific population groups, especially vulnerable sections of the society, health promotion, disease prevention, rehabilitation, societal impact etc. priority setting is increasingly recognized as essential to deliver widespread population health changes that respond to critical needs and contribute to sustainable developmental outcomes. numerous world health assembly resolutions have stressed the need for action on prioritizing the health research activities for the following reasons: – health research prioritization is regarded as a key part of efforts needed to strengthen national health research systems. – most importantly, priority setting can provide valuable direction for the allocation of public and private research funds into areas of strategic importance.1 – setting priorities for health research is essential to maximize the impact, which is especially relevant in resourcescarce environments. – setting priorities in research can serve to act as a catalyst for public debate, for bringing together different stakeholders and creating networks. these networks would ideally comprise researchers in the public and private sectors, decision-makers in governments, and civil society. – prioritization mechanisms are necessary to facilitate the current demand for increased harmonization of health research at a global level particularly in combination with analyses of financial flows for health research and burden of disease studies. due to the immense importance of health research in improving the health system and health service, the decision to prioritize health research was the first step in the direction to enhance health research, and thereby enhance health service in oman by the centre of studies & research. this concept paper aims to portray the steps and strategies followed in setting the health research priorities in sultanate of oman in 2019. health research priority setting in oman: towards better utilization of the available resources adhra al-mawali*1,2 ,ayaman al-harrasi**1, sathish kumar jayapal 1, , hilal al-kharusi 1 , mariam al-rashdi 1, , avinash daniel pinto1 1centre of studies & research, ministry of health, sultanate of oman 2the research council. sultanate of oman *first author: adhra al-mawali **first author: ayaman al harrasi correspondence to adhra al-mawali (adhra.almawali@moh.gov.om) (submitted: 03 may 2020 – revised version received: 14 may 2020 – accepted: 28 may2020 – published online: 26 june 2020) objective this concept paper aims to portray the steps and strategies followed in setting the health research priorities in sultanate of oman. methods this exercise was developed based on council on health research for development management process to priority setting for planning a high-quality health research priority setting exercise at national and subnational levels. ranking for the diseases and risk factors in oman was based on the estimates obtained from the global burden of disease 2016 study by the institute for health metrics and evaluation. the most important topics were generated from a panel of experts to tackle the above-ranked problems which was supported with a feasibility score to plan for conducting research. results a list of top 30 diseases and 10 risk factors based on disability-adjusted life years estimates was ranked and listed. subresearch topics were listed under each category. feasibility scores were obtained from all the subnational levels based on available human resources, infrastructure, financing, technology, legality, and ethical consideration. a plan for the next 15 years was developed for each subnational level based on the feasibility score. conclusion prioritization mechanisms are necessary to facilitate the current demand for increased harmonization of health research to meet health services needs, particularly in combination with translating the results into actions that improve overall population health. due to the immense importance of health research in improving the health system and health services, the decision to prioritize health research was the first step in the direction to enhance health research and thereby enhance health service in oman by the centre of studies & research. this intervention will lead the health system to achieve better planning for effective utilization of available resources. keywords oman, health research, priorities, health research priorities, health research priority setting, cohred 127 original health research priority setting in omanadhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 why second edition? the first edition of health research priorities in oman was published in april 2014. research domains were identified based on delphi method, considering the priorities of research emanating from the eighth 5-year plan for the development of health, regional research priorities, research priorities recommended by international organizations and research priorities that serve the health vision for 2050. as we move towards 2050, these domains have to be reviewed and updated periodically in the light of the changing spectrum of health problems. in oman between 1970 and 1990, communicable diseases were a major health problem. through active intervention under wise leadership, considerable progress has been achieved and most preventable communicable diseases have been almost eliminated. by the turn of this century, the burden of disease has transitioned to noncommunicable diseases and a few of the newer communicable diseases.2 it is easy to see why this dynamic process of periodic recasting of the priority in research has to be practiced, so that the focus remains on the important issues that can produce the most beneficial effects in health care and health delivery. what will be the health issues that require research by 2050? it will be difficult to predict now, but through a dynamic process of prioritizing health research it can be on track at all times and in changing situations. materials and methods the question of how priority setting processes work remains topical, contentious, and political in every health system across the globe. it is particularly acute in the context of developing countries because of the mismatch between needs and resources, which is often compounded by an underdeveloped capacity for researchers and weak institutional infrastructures for research.2 yet, there is limited research into how the process of setting and implementing health research priorities works in developing countries. in 1990, the commission on health research and development drew attention to the need for essential national health research for developing countries. the commission on health research for development advocated the use of a systematized approach to priority setting within each country’s essential national health research (enhr) strategy. the council on health research for development (cohred) was established to assist developing countries with the implementation of this strategy.4 cohred emphasized the following principles: • there is a need for solid evidence to underpin an inclusive health research agenda. • there is a need to involve all stakeholders in the prioritization process. • and there is a need to link research results to policy and action. based on cohred’s experience, there is no one best method for priority setting. they strongly suggest those responsible for priority setting to weigh complexity of methods against what is to be achieved and what resources are available. what is the process of research priority setting followed in oman? for health research priority setting exercises to effectively target research with the greatest public health benefit, it is important that they are of high quality. there are various approaches available to guide priority setting for health research which differ on important aspects of the process. therefore, taking the heterogeneous nature of research priority setting exercises and the different contexts for which priorities can be set, the optimal approach varies per exercise.5 consensus on a gold-standard or best practice for health research prioritization, thus seems difficult to achieve and is, more importantly, not an appropriate response. but there are nine common themes of good practice for health research prioritization processes which have been used by the centre of studies and research in oman based on cohred recommendation to provide assistance for planning a high-quality health research priority setting exercise at national and subnational levels. there are nine common themes of areas to focus during the exercise which are:6 1. assessing the contextual factors 2. choosing the approach 3. assess the inclusiveness 4. identify the information 5. choosing the criteria 6. adapt methods 7. evaluation 8. transparency 9. planning for implementation. assessing the contextual factors there are several contextual factors that underpin the process of research priority setting, namely practical considerations about available resources, the focus of the exercise, the values that stakeholders adhere to, and the health, research, economic, and political environment in a country. these factors influence the prioritization process and the eventual research priorities and should therefore be discussed explicitly from the beginning of the exercise. careful planning of the prioritization exercise is important to establish an exercise that meets the initial expectations. it is necessary to identify available financial, human, and time resource. the contextual elements were determined as following: ◦ the information resources which were available for the exercise were: • institute for health metrics and evaluation’s burden of diseases 2016 study • annual health reports • health research priorities 2014 (1st version) • health vision 2050 • 5 years plan for health research (csr) • experts’ opinion • review of literature ◦ the focus of the exercise: • to identify the research priorities in diseases and their risk factors and health system. • to ensure that the priorities are used by practicing health workers, students of health sciences, academicians or researchers from other institutions with common interest, and by stakeholders and decision-makers. ◦ health, research, and the political environment in which the process will take place is detailed in the “health vision 2050”and was revised thoroughly.7 here are the main aspects related to the research prioritizing exercise at the time of health research priority setting (2016): 128 original health research priority setting in oman adhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 • health status (2016):8 ◦ the health system in oman is characterized by its universal health coverage for both citizens and nonnationals. health care is directly provided in facilities mainly owned and operated by the government. the government provides about 79% of hospitals and about 91% of hospital beds. public health services are run by 78% of doctors, 84% of nurses, and 78% of other paramedics. about 66% of dentists and 71% of pharmacists work in the private sector. ◦ the ministry of health (moh) is the main healthcare provider and is responsible for ensuring the availability of health policies and plans and monitoring their implementations. other health-care providers in the country include: armed forces medical services, royal oman police medical services, sultan qaboos university hospital, diwan medical services, petroleum development oman medical services, and the private sector. ◦ the omani health system is a free-medical care health system, chiefly financed through government revenues. the government is committed to providing health care and services to all citizens free of charge and has considered equity in financing health services across different health governorates with the aim of ensuring financial protection for all.9 nonomani residents receive their medical care mainly in private health-care facilities ◦ moh provides health care mainly to omani citizens through 49 hospitals and 205 health centers scattered across the country. moh distributed hospitals across all governorates, such that each governorate is served by a governorate hospital (gh) (10) aided by a wilayat hospital (wh) (5) in some populated governorates to provide secondary care to their inhabitants (secondary health care is also provided by two extended health centers and one local hospital). ◦ these secondary care hospitals are apexed by four national referral hospitals (nrh) (located in muscat governorate) that provide tertiary care to citizens of oman. moh provides primary health care through health centers (182 hcs), extended health centers (21 ehcs) and local hospitals (29 lh) distributed across all governorates. ◦ a total of 6,393 physicians, 14,675 nurses, 358 dentists, 554 pharmacists, and 6,234 paramedical staff in addition to 12,050 medical orderlies and support staff run health services in moh health-care facilities (2016). • political and economic status (2016): ◦ it is important to scan the political situation in oman to show the geopolitical stability supporting different aspects of development including health. oman was one of the least known countries and remained largely isolated from the rest of the world until 1970 when his majesty sultan qaboos bin said came to power. his majesty’s reign signaled the renaissance or the beginning of a bright new era that renewed oman’s historic past and opened a new chapter of development, prosperity and social and economic progress. ◦ oman is currently described as a high-income country. it is a relatively large country with an area of about 309,500 km. it has difficult terrain and an intricate topography, with high and rugged mountains and barren valleys. its small population of 4.2 million, of which 43.6% are expatriates or nonnationals, is scattered over large areas of sparsely populate settlements. ◦ the sultanate of oman evolved to become a modern country with state-of-the-art services under the rule of sultan qaboos, which began in 1970. in 2015, its gross domestic product (gdp) at current prices has grown to omani rials (omr) 26,850.3 million (us69,922.7 million). the graph below showing the expenditure on health in oman from 1995 to 2015. ◦ administratively, the country is divided into 11 governorates with 61 wilayats (districts). each governorate is considered a health region. these governorates are: muscat, dhofar, musandam, al-buraimi, ad dakhiliyah, north batinah, south batinah, south sharqiyah, north sharqiyah, al-dhahirah, and al-wusta. ◦ leadership and governance in the omani health system has responsibly and wisely managed resources and revenues to the benefit of the health of the people of oman and has responded to their needs during its different stages of development. sound policies, strategies, and development plans have been adopted. the government of oman, through the ministry of health, has a health policy that is based upon several basic principles: provision of comprehensive public and personal health services to its population through a health system with primary health care as its cornerstone. equity in the distribution of health services and fairness of financial contribution among different population groups according to health needs. community involvement in planning and implementation of its health care aimed at developing community self-reliance for sustainable health development, responsiveness to health and nonhealth needs of the community and intersectoral cooperation with other health-related sectors to ensure positive impact on community health.10 ◦ the consultative council: in november 1991, sultan qaboos replaced the 10-year-old state consultative council with the consultative assembly (majlis al-shura) to systematize and broaden public participation in government. the assembly has 84 elected members and exercise some legislative powers. representatives were chosen in the following manner: local caucuses in each of the 59 districts sent forward the names of 3 nominees, whose credentials were reviewed by a cabinet committee. these names were then forwarded to the sultan, who made the final selection. the consultative assembly serves as an information channel between the people and the government ministries. it is empowered to review drafts of economic and social legislation prepared by service ministries, such as communications and housing, and to provide recommendations. service ministers also may be summoned before the majlis 129 original health research priority setting in omanadhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 to respond to representatives’ questions. it has no authority in the areas of foreign affairs, defence, security, and finances. the council of state (majlis al-dawla) has 83 appointed members including 14 women. • research status ◦ the centre of studies & research (csr) is the official body and focal point within the ministry of health which is responsible for implementing the policies of the ministry of health for promoting research culture and governing research activities in the health sector in oman. it has been providing evidence-based information to decision makers and other researchers/academicians.11 ◦ the research council oman (trc) was established on june 22, 2005, pursuant to the royal decree number 54/2005. it carries tasks related to research and endeavors to promote and support research using all the material and moral means possible. the research council in its capacity is the main national authority in this area, acts as a focal point for research and innovation and liaise with various institutions concerned with research. the research council is both a policy-making institution and a funding agency. it is offering funding called block funding program (bfp) which is an institutionaland performance-based funding. this program allocates small-to-medium size research grants to support shortand mid-term research projects in areas defined by researchers from academic and research institutions in oman and serve the national research priority areas as well as trc priority themes. the main goal of this program is to sustain and develop further excellent in research and to create a competitive economy through advanced and evidence –based research (fig. 2). ◦ however, research in oman is still considered of limited benefits. the scimago journal and country rank portal assessed countries for the number and citations of publications during 1996–2010. it showed that oman had only 1,522 publications with 7,357 citations. this was not considered favorable and oman was ranked ninth among 15 countries in the middle east and north africa region. a study published in 2011 showed that more than one-quarter of biomedical publications by omani researchers during the period 2005–2009 were published in journals with no impact factor (if) and more than half were in journals having if of less than 1. the study concluded that the quality of research originating from oman is of limited usefulness. ◦ almost all of the health-care facilities run by moh and other public health-care providers are fully computerized. patients “records are managed electronically, and a wealth of patient information is available. the situation is not the same in the private sector. in spite of the fact that moh hospitals and primary health-care units are fully computerized, data are not directly extracted from health institutions” databases by the nhsis. coordination between nhsis and it is a challenge for sustaining the flow of health data and information. the absence of a countrywide electronic connectivity of all health-care facilities is another challenge for enhanced health service delivery and for extracting health data. fig. 1 expenditure on health in oman from 1995 to 2015. fig. 2 health research bodies in oman and the types of health research they covered. 130 original health research priority setting in oman adhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 ◦ there are a number of challenges from enhancing and developing health research; these would include: • the lack of sufficient and allocated funds for health research in ministry of health. • non-adherence of the academia and national programs to the identified research priorities of the moh. • weak co-ordination of research activities between the moh and academia-supported researchers within and outside the country and poor communication of the research results. • a limited number of identified research topics in the health development plans are implemented. • lack of close monitoring of research activities and their outcome. • a “research culture” has not permeated sufficiently among health care professionals. • although collaboration with reputed institutions in other countries exists in the field of health-care services and academia, collaborative research is minimal. • health service in oman is highly reliant on medical products originating in and based on research conducted in other countries. • private donor agencies are motivated to grant donations for health care but not for research due to lack of sufficient awareness of the relevance of health research. • infrastructure for conducting innovative research, particularly in the area of medical products and technology, is lacking. • lack of sufficient experience for research in congenital anomalies and genetic disorders is an important challenge to the health system in oman. • poor access to the benefits and products of research, despite dramatic advances in knowledge and technology. choosing the approach used to health research priorities setting there are number of comprehensive approaches to health research priority setting. these approaches are comprehensive because they provide structured, detailed, step-by-step guidance for the entire priority setting process, covering many of the points on this checklist. they assist in the preparatory work of an exercise, in deciding on priorities, and in what to do after priorities have been set. use of these approaches is therefore in general advantageous and their use should be at least considered. the four commonly used comprehensive approaches are: 1. 3d combined approach matrix (cam).14 2. essential national health research (enhr) approach. 3. the child health and nutrition research initiative (chnri) approach. 4. the cohred management process to priority setting. the one adapted in oman’s exercise of priority setting is cohred (commission on health research for development) as it focuses on the management process for national-level exercises. this high-level approach delineates important steps of a priority setting process for national-level exercises and discusses a wide range of options for tools and approaches to use in the process. over the past 15 years, cohred has supported countries in setting national priorities for health research. based on this experience, cohred has developed an integrative approach that countries can use to manage their priority setting process. the present approach has been structured as a comprehensive guide that will help the users in designing the most appropriate priority setting process for their countries. to facilitate action practical ideas, management tools, existing priority setting methods and techniques, reference documents and country examples are proposed. the approach reveals priority setting as a cyclic management process where six key practical steps are identified: 1. assessing the situation. 2. setting the scene. 3. choosing the best method. 4. planning priority setting. 5. setting priorities. 6. making priorities work. cohred is an international nongovernmental organization whose primary objective is to strengthen research for health and innovation systems, with a focus on lowand middle-income countries. cohred supports countries to use research for health and innovation to: improve health and reduce health inequities. this guide of cohred is meant for any country, region or institution that wants to make a difference in health, equity and development through research.15 they recommend the following: • research priorities should be credibly set and regularly updated: set a date for an update already at the start. • ensure the process is inclusive. this is as important as the methodology used to define priorities. • cohred suggest not to allocate resources to the defined priorities at once. allow some financial flexibility for innovation, blue sky research or unexpected health challenges and opportunities assess the inclusiveness of health research priorities research setting process in principle, broad stakeholder involvement (multisectoral and multidisciplinary) is beneficial for the outcomes of a research priority setting exercise for several reasons. firstly, it minimizes the chances of research options being overlooked. different groups of stakeholders tend to prioritize research differently. secondly, participation in the exercise fosters ownership of the established priorities among those involved, thus increasing the chances of implementation of the priorities. thirdly, broad participation makes priorities correspond to the needs of those that will implement and those that will benefit from the research priorities. as such, the prioritized research will be a better response to societal and policy needs, increasing the overall credibility of the exercise and the potential impact on health and health equity. finally, broad stakeholder involvement may prevent unnecessary duplication of prioritization efforts and hence wasting of resources. lastly, appropriate leadership of the priority setting process needs to be identified. this can for example be in the form of an executive committee or an advisory group that provides overall guidance on the prioritization process, while a 131 original health research priority setting in omanadhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 larger core working group or decision-making group actually decides on priorities. good leadership can be pivotal in creating and sustaining a high-quality priority setting process. in second edition of health research priority setting, there was appropriate representation of expertise and balanced gender and regional participation. all elements of health sectors have been included. identify the information needed to be gathered for health research priorities setting there are many ways to make the priority setting process better informed and choices should be made on which types of information are necessary. these can include the collection of technical data that are often needed to inform discussion on research priorities (see criteria), such as burden of disease, cost-effectiveness of interventions, current resource flows towards particular research areas, or determinants of disease. furthermore, in order to be able to prioritize research, one must first know where the gaps in knowledge are; a literature review to identify those gaps is often necessary.16 also, an initial survey of broader stakeholder views on priorities or opinions on matters related to the research area, or a review or impact analysis of previously established priorities can serve as preparation before the actual exercise. furthermore, research priority setting is needed at different geographical levels: national, local within governorates, and within wilayats. for most health research topics, priorities will be the same on all levels. for most however, priorities will reflect the context they are seeking to address. the information gathered to conduct the exercise were: 1. literature reviews on how to conduct the exercise of priority setting. 2. collection of technical data (e.g., burden of disease, mortality, morbidity). 3. assessment of broader stakeholder views. 4. expert opinions. 5. reviews or impact analyses of previous priority setting exercises and exercises from other geographical levels. choosing the criteria for health research priorities setting commonly, criteria can be categorized into one of three dimensions: public health benefit (should we do it?), feasibility (can we do it?) and cost. participants in the priority setting exercise should decide by consensus on appropriate criteria at the beginning of the exercise. the following questions were answered as following to reach consensus on the criteria used by the centre of studies and research to set the health research priorities: 1 (should we do it?) public health benefit in order to answer this question, the focus of the second edition of priority setting was to answer the following three questions: magnitude of a health problem: in order to align health systems with the populations they serve, policymakers first need to understand the true nature of their country’s health challenges – and how those challenges are shifting over time. that means more than just estimating disease prevalence, such as the number of people with depression or diabetes in a population. global burden of disease (gbd) data incorporate both the prevalence of a given disease or risk factor and the relative harm it causes. the tools allow decision-makers to compare the effects of different diseases, such as malaria versus cancer, and then use that information at home. the disability-adjusted life year (daly) is a measure of overall disease burden. the daly metric is composed of years of life lost (yll) due to disease causing mortality and years lived with disability (yld). this provides high-quality epidemiological data on health status that are independent of interest groups. the health research priorities setting (2018) by csr adapted the dalys estimates from the global burden of disease (gbd) 2016 study by the institute for health metrics and evaluation (ihme).15,16 to make these estimates more accessible and useful, ihme has distilled large amounts of complicated information into a suite of interactive data visualizations that allow people to make sense of the over 1 billion data points generated. collected and analyzed by a consortium of more than 3,000 researchers in more than 130 countries, the data capture premature death and disability from more than 300 diseases and injuries in 195 countries, by age and sex, from 1990 to the present, allowing comparisons over time, across age groups, and among populations. the flexible design of the gbd machinery allows for regular updates as new data and epidemiological studies are made available. in that way, the tools can be used at the global, national, and local levels to understand health trends over time, just like gross domestic product data are used to monitor a country’s economic activity. policymakers in brazil, china, india, indonesia, mexico, the united kingdom, and other countries worldwide are collaborating with gbd researchers to adopt this approach for measuring their population’s health and how it varies by different regions, socioeconomic status, or ethnic groups in their country. the global burden of disease (gbd) 2016 study is a systematic assessment of the disability and mortality of major diseases and risk factors worldwide. it is a collaborative effort of scientists and researchers from the world health organization (who), world bank, institute for health metrics and evaluation (ihme), harvard school of public health, and university of auckland school of population health. gbd 2016 estimates the burden of 291 diseases and injuries in 187 countries from 1990 to 2015. thus, list of 30 diseases and 10 risk factors where ranked based on dalys estimates from the above-mentioned study. thus, the ranking is independent of interest group. what about research to strengthen health systems this is particularly true when a systems perspective is used, i.e., by considering all the positive and negative effects of a particular system-level intervention, this research can provide a robust and accurate understanding of health systems challenges and their potential solutions, thereby improving the utility of the findings in other settings. this systems approach, in combination with stakeholder engagement, also informs the definition of priority research questions to 132 original health research priority setting in oman adhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 address health systems challenges. health systems research by necessity is highly multidisciplinary, with a strong emphasis on social sciences, economics, and anthropological investigations. much ongoing research consists of descriptive, comparative, and evaluation studies and secondary analytical research. although experimental studies are less common, partly because of operational and ethical challenges in experimenting at the health system level, they can be very informative and provide convincing evidence on the benefit of innovations in health system efficiency and health impact. the research in this domain falls under the general definition by the alliance for health policy and systems research (hpsr) as: “the production of new knowledge to improve how societies organize themselves to achieve health goals.” the alliance for hpsr further clarifies that the prime focus of health policy and systems research is not a specific disease or service, but rather the health system as a whole. however, health systems research sometimes adopts a disease or service specific. more specifically, it can address any or all of the six building blocks of health systems identified by the who: service delivery, information and evidence, medical products and technologies, health workforce, health financing, and leadership and governance. in doing so, it should explicitly acknowledge the importance of the continuous interactions between the different building blocks of the health systems and the different sectors (including nonhealth sectors) involved, as well as all the other characteristics of complex health systems. another definition refers to health systems research as “research that enhances the efficiency and effectiveness of the health system.” research on health systems addresses a huge research area that has only been marginally covered to date. because of the multitude of system challenges and their complex multidimensional environment, research prioritization is essential, and some recent priority-setting initiatives are being timely. due to the relative scarcity of research capacity to undertake this type of research, efforts to improve the design, robustness, and applicability of the evidence generated in one setting to another would be highly desirable. systems thinking methods and approaches can offer tremendous help and guidance on this. by using a systematic, comprehensive way of examining the design and evaluation of potential health systems interventions, and ensuring involvement and ownership of all stakeholders involved, the utility and pay back from the evidence generated from this research greatly increases. the centre of studies & research with collaboration with health policy and system department in ministry of health had categorized the research in health systems into two main categories (fig. 6) 1. research related to functions of health system. (input + process). 2. research related to outcome of health system. (output). likelihood of reducing disease burden the list of diseases and risk factors and health system issues ranked by dalys were assessed for the likelihood of reducing the burden. they all are having moderate to high likelihood for reducing their burden according to the experts. present level of knowledge the lists were assessed for the present level of knowledge. it has been found that there is a need for more and accurate information in order to tackle them effectively. thus, list of recommended topics where integrated with the above list of diseases and risk factors as can be seen in table 1. feasibility (can we do it?) instead of assessing the feasibility of the main research list of health problems and risk factors, topics or research questions of each health problem are assessed from the following six feasibility perspectives: • infrastructure (is). • human resources/workforce (hr). • technology (t). • finance (fin). • ethical aspects (eth). • legality (l). each perspective will be scored from 0 to 2 denoting: 0: not available / not currently legal / not currently approved 1: available but not sufficient /legal /ethically approved 2: sufficient the maximum score is 10; and the higher the score, the higher feasibility the topic will have. the feasibility score can be used to categorize the specific research questions into short, intermediate and long-term list of specific research topics as following: • short-term priorities (to be done with 5 yrs.): feasibility score = (7–10) • intermediate-term priorities (to be done with 10 yrs.): feasibility score = (4–6) • long-term priorities (to be done with 5 yrs.): feasibility score = (0–3). the above feasibility analysis has been integrating into the list of the diseases and risk factors as in table 1. cost effectiveness (is it cost effective?) a) there is big gap of knowledge in this area which should be filled through building capacity. b) can be used in the next edition of research priorities exercise as a tool to rank health research priorities. adapt methods for health research priorities setting as cohred is the approach adapted by the centre of studies & research, its recommendation is to use methods suited to local context and needs. as recommended by cohred, the exercise of priorities setting should consider the use of more than one method to optimise the usefulness of results, and adapting methods to specific setting, available data and resources, and to local needs.1,22 in general, there are two main categories of methods for identifying the health issues: • first category: using and compiling existing data (compound approaches) for example: ▪ essential national health research ▪ burden of disease ▪ 3d combined approach matrix23 ▪ child health priorities 133 original health research priority setting in omanadhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 ta bl e 1. e xa m pl e fo r o ne o f t he to p 30 d is ea se s( ne ck a nd b ac k pa in ) w ith in te gr at ed fe as ib ili ty a na ly si s a nd p la nn in g pr oc es s. e ac h di se as es a nd ri sk fa ct or s i s p re se nt ed w ith a ta bl e in cl ud in g al l re co m m en de d to pi cs w hi ch a re cl as si fie d ac co rd in g to h ea lth ca re co m po ne nt s a nd h ea lth sy st em b ui ld in g bl oc ks .f ea si bi lit y an al ys is a re d on e by e ac h go ve rn or at e ba se d on th ei r f ea si bi lit y st at us to co nd uc t t he re se ar ch a nd a cc or di ng to th e re su lts o f t he fe as ib ili ty sc or e , a p la n is to b e bu ilt , f ol lo w ed a nd e va lu at ed b y th e ce nt re o f s tu di es a nd r es ea rc h . sp ec if ic r es ea rc h to pi cs co m po ne nt o f he al th c ar e he al th sy st em fe as ib il it y co st ef fe cti ve ne ss sh or t t er m (w ith in 5 ye ar s) in te rm ed ia te (w ith in 1 0 ye ar s) lo ng te rm (w ith in 1 5 ye ar s) hr (0 -2 ) is (0 -2 ) fi n (0 -2 ) t (0 -2 ) l (0 -1 ) et h (0 -1 ) to ta l (0 -1 0) (7 -1 0) (4 -6 ) (0 -3 ) st ud y to a ss es s th e ep id em io lo gi ca l ch ar ac te ris tic s of b ac k an d ne ck p ai n si tu at io n an al ys is                   st ud y to a ss es s th e m ec ha ni sm o f ba ck a nd n ec k pa in si tu at io n an al ys is                   st ud ie s to id en tif y th e un de rly in g ris k fa ct or s of g et tin g ba ck a nd n ec k pa in in o m an . pr & po                   st ud y to a ss es s th e bu rd en o f b ac k an d ne ck p ai n pr & po                   st ud y to e va lu at e th e eff ec tiv en es s of th e av ai la bl e m an ag em en t s tr at eg ie s fo r b ac k an d ne ck p ai n tx                   st ud y to a ss es s th e eff ec tiv en es s of cu rr en t p re ve nt iv e m ea su re s of b ac k an d ne ck p ai n tx                   st ud y to a ss es s th e eff ec tiv en es s of p ha rm ac ol og ic a nd n on ph ar m ac ol og ic m ea su re s in tr ea tm en t o f b ac k an d ne ck p ai n tx                   st ud y to a ss es s th e eff ec tiv en es s of ph ys io th er ap y tr ea tm en t i n ba ck a nd ne ck p ai n tx                   st ud y to a ss es s th e ch al le ng es o f in di vi du al a nd h ea lth c ar e sy st em in tr ea tm en t                   st ud y to a ss es s th e qu al ity o f l ife o f pa tie nt s w ith b ac k an d ne ck p ai n                   (c on tin ue d) 134 original health research priority setting in oman adhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 ta bl e 1. c on t’d sp ec if ic r es ea rc h to pi cs co m po ne nt o f he al th c ar e he al th sy st em fe as ib il it y co st ef fe cti ve ne ss sh or t t er m (w ith in 5 ye ar s) in te rm ed ia te (w ith in 1 0 ye ar s) lo ng te rm (w ith in 1 5 ye ar s) hr (0 -2 ) is (0 -2 ) fi n (0 -2 ) t (0 -2 ) l (0 -1 ) et h (0 -1 ) to ta l (0 -1 0) (7 -1 0) (4 -6 ) (0 -3 ) st ud y to e va lu at e th e pa in -r el ie vi ng m ea su re s in tr ea tm en t o f b ac k an d ne ck p ai n tx                   st ud y to e xp lo re th e ps yc ho lo gi ca l, so ci al a nd c ul tu ra l f ac to rs in de ve lo pm en t a nd tr ea tm en t o f b ac k an d ne ck p ai n tx                   ec on om ic e va lu at io n of tr ea tm en t o f ba ck a nd n ec k pa in h b: f in                   st ud y to a ss es s th e av er ag e w ai tin g tim e fo r b ac k an d ne ck p ai n h p: q                   ph ar m ac oe co no m ic s of a na lg es ic s in vo lv ed in th e tr ea tm en t o f b ac k an d ne ck p ai n h b: f in                   st ud y to a ss es s th e pa tie nt sa tis fa ct io n w ith th e he al th c ar e sy st em h p: q                   he al th ca re co m po ne nt s: he al th s ys te m b lo ck s: he al th s ys te m p er fo rm an ce : fe as ib ili ty a sp ec ts : si tu at io n a na ly si s (s a ) h ea lth w or kf or ce (h w ) sa fe ty (s a f) h um an r es ou rc e (h r ) pr ev en tiv e & p ro m ot iv e ca re (p r & p o ) se rv ic e d el iv er y (s d ) q ua lit y (q ) in fra st ru ct ur e (i s) d ia gn os tic (d x) h ea lth in fo rm at io n (h i) eq ui ty (e ) fi na nc in g (f in ) cu ra tiv e (t x) m ed ic al p ro du ct s, va cc in es a nd te ch no lo gy (m v t ) te ch no lo gy (t ) pr og no st ic (p ro g ) fi na nc in g (f in ) le ga lit y (l ) re ha bi lit at iv e ca re (r eh ab ) g ov er na nc e (g o v ) et hi ca l c on si de ra tio ns (e th ) 135 original health research priority setting in omanadhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 • second category: providing insight in future health priorities (foresight techniques): ▪ visioning ▪ scenario creation ▪ delphi ▪ roadmaps what is best for oman in setting health research priorities? in oman, both methods are applicable, as we have reliable mortality and morbidity data for substantial parts of the population with ihme estimates of gbd (2016) as well as having” health vision 2050” which is considered to be the roadmap for the planning in the future.23,24,25 so, combining both methods for setting health research priority in oman will give the most reliable list of priorities.26 evaluation the identification of health research priorities should be seen in the broader context of health research coordination and inform funding and policymaking for health research in a sustainable manner.27 hence, previously set priorities should be periodically reviewed to ensure that priorities are up to date. besides updating research priorities, other forms of evaluation can be considered. evaluation of the process used to set priorities can increase the quality and acceptability of that process. furthermore, to make research prioritization legitimate and fair, an appeals mechanism for the established priorities can be considered, providing opportunity for feedback.28 finally, performing an impact analysis, for example in the form of a review of research performed and/or funding allocated based on previously established priorities, can be valuable. not only can this provide insight into priorities that have remained devoid of attention, but it can also enforce discussion on implementation issues. the evaluation of the established priorities and the priority setting process will take place in periodic manner. health research priority setting will not be a one-time exercise.29 the achievement of each governorate will be evaluated yearly and graded by star grading system: • 1 star: achieving 20% of 5 year (short term) specific research questions list of the governorate. • 2 stars: achieving 40% of 5 year (short term) specific research questions list of the governorate. • 3 stars: achieving 60% of 5 year (short term) specific research questions list of the governorate. • 4 stars: achieving 80% of 5 year (short term) specific research questions list of the governorate. • 5 stars: achieving 100% of 5 year (short term) specific research questions list of the governorate. transparency documenting all steps followed in building the second edition of health research priorities by publishing a report, to be as transparent as possible is crucial. potential implementers of health research priorities are unlikely to adopt or use priorities unless they are fully informed of all aspects of the priority setting process; transparency increases the credibility and thus the acceptability of the final result.30 therefore, the report should not be limited to stating a list of priorities, but should also explain how those priorities were established, and by who.31 this entails providing details on which choices were made for points one through eight on this checklist, and why those choices were made.32 the documented was distributed through the official channels of ministry of health and workshops was documented at each governorate to ensure full understanding of the objectives of the prioritizing process. planning for implementation the plan for implementation is considered to be as follows:33 • all research regions will follow the national health research priorities for diseases, risk factors and the health system. • feasibility assessment of the specific research questions for each health issue or problem will be done by each research region to generate short-, intermediateand long-term lists of specific health research questions or topics. • the list of priorities to be announced in a ceremony in each research region with the presence of all community sectors (including private parties) • research groups for the short-term topic to be established within each research region. • recruitments of researchers for the research groups with the needed qualifications to be announced in the media channels including the webpage of each research region in the csr website. • research methodology training programs to be linked with the research groups activities: ▪ level one: introduction + basic concepts ▪ level two: advanced research methodology skills ▪ level three: writing the proposal • research activities to be followed by a team from research section in each governorate. • a report of achievements to be submitted to csr at the end of each year. • achievements to be graded by star grading system. • grade of each research region to be announced in the webpage of each governorate in the csr website. • all research regions will compete every year to win the top best achievement in the 5 years short-term health research priorities plan. results following the above-mentioned methodology, a list of 30 diseases and 10 risk factors where ranked based on dalys estimates from the global burden of diseases study. top risk factors list in general, the risk factors can be categorized into metabolic, behavioral and environmental/occupational risk factors. globally, behavioral risks are causing the most disease burden, but in the oman, metabolic risks are the most causative risks of the disease burden17 (fig. 3). in 2016, the risk factors that drive the most disease burden (dalys) in oman are as following: 1. high body-mass index (bmi) 2. high fasting plasma glucose 3. dietary risks 4. high blood pressure 5. high total cholesterol 6. occupational risks 7. tobacco 136 original health research priority setting in oman adhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 each risk factors were discussed separately with listing of the most urgent specific research questions. example of a table constructed for each risk factor can be seen in table (1). top diseases list list of 30 diseases where ranked based on dalys estimates as follow in figs 4 and 5: each disease were discussed separately with listing of the most urgent specific research questions. example of a table constructed for each disease can be seen in table 1. health system research for the purposes of this exercise, which has been undertaken with limited time and resources, we have categorized the research agenda in hsr into:18 1-functions: we have identified what appear to be the key characteristics and core indicators of a well performed health system as recommended by who in each building block.19 list can be seen in appendix (1) 2-outcomes: a number of principles can inform priority setting in hsr in relation to the outcome. firstly, there needs to be clear evidence that the problem related to health systems is preventing attainment of the sdgs and that this problem is potentially tractable if addressed by new knowledge from research. thus, we need to distinguish between the need for better research evidence and solutions that do not require new knowledge, e.g.. an absolute lack of resources that prevents delivery of basic services. research can however contribute to problem-solving in resource poor environments by, for example, leading to more appropriate policies for financing of health systems, for prioritizing the use of resources and developing an appropriate workforce.20 secondly, it is important to understand the degree to which methodological development is necessary in order to tackle a given research problem and finally to understand how new research can bridge gaps in the existing research portfolio. resources used in this exercise of prioritizing health research agenda in hsr: to accomplish this exercise, we have built on work undertaken by the who commission on macroeconomics and health that developed a taxonomy of constraints to achieving the sdgs. we have also considered the health policy and systems research key characteristics of health system building blocks derived from who publications. we have also taken into account priorities suggested by researchers. finally, we have taken into account experience from high income countries of research on service delivery fig. 3 the risk factors that drive the most disease burden (dalys) in oman. fig. 4 list of top 10 diseases where ranked based on dalys estimates. 137 original health research priority setting in omanadhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 and organization. we have taken a broad view of hsr and have included within it, issues of management, organization and delivery of health services and research relevant to the development and implementation of policy related to health systems. list of the topics related to the outcome can be found in appendix 2. feasibility analysis taking into consideration the subnational differences in each perspective of the feasibility assessment, the feasibility assessment done for each governorate separately. so, there will be common national health research priorities but different fig. 5 list of top 30 diseases by daly, yld, yll. the red highlighted topics are removed from the main list due to lack of specifications and wide range of diseases included in each category with each single disease might contribute less to overall daly. fig. 6 the complex integration between the inputs, process and output within the health system. 138 original health research priority setting in oman adhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 specific research questions for each health problem or issue. table 1 is showing how the diseases or risk factor was integrated with the research questions and feasibility perspectives. discussion there are number of comprehensive processes for health research priority setting. consensus on a gold-standard or best practice for health research prioritization thus seems difficult to achieve and is, more importantly, not an appropriate response. however, a nine common themes of good practice for health research prioritization processes have been followed by the centre of studies and research in oman based on cohred recommendation to provide assistance for planning a high-quality health research priority setting exercise at national and sub national levels which are ;assessing the contextual factors, choosing the approach, assess the inclusiveness, identify the information, choosing the criteria, adapt methods, evaluation, transparency and planning for implementation . few countries in the world followed the above-mentioned comprehensive process of prioritization as it is a challenging process to follow.6 based on the available resources in oman, all themes were taken into account and the results were shaped into a 15 years plan. dissemination of the results were satisfactory at all subnational levels. focus groups to continually work on the proposed topics from each subnational level will be managed through a website that also will help in bringing junior researchers and seniors in one channel and for united purposes. limitations in each project are inevitable but bringing the maximum quality of the output was the ultimate aim of the exercise of health research priority setting in oman .resources were always not enough but getting experts opinions was the only way to fill some gaps in the knowledge needed to continue the exercise. adherence to the plan will be a challenge that we are planning to compete by regular follow-up and updating. conclusion in resource rich settings, a high proportion of available research resources go to investigator-driven initiatives, but in limited resource settings, there is an expectation that research must respond more directly to community health needs, and therefore be conducted according to recognized priorities. prioritization mechanisms are necessary to facilitate the current demand for increased harmonization of health research to meet health services needs particularly in combination with translating the results into actions that improve overall population health. due to the immense importance of health research in improving the health system and health services, the decision to prioritize health research was the first step in the direction to enhance health research and thereby enhance health service in oman by the centre of studies & research. this intervention will lead the health system to achieve better planning for effective utilization of available resources. competing interests the authors declare that they have no competing interests. references 1. mcgregor s, henderson kj, kaldor jm. how are health research priorities set in lowand middle-income countries? a systematic review of published reports. plos one, 2014;9:e108787. 2. al-mawali a. non-communicable diseases: shining a light on cardiovascular disease, oman’s biggest killer. oman med j. 2015 jul;30(4):227-8. doi: 10.5001/omj.2015.47. pmid: 26366254; pmcid: pmc4561645 3. angelis a, kanavos p, montibeller g. resource allocation and priority setting in health care: a multi‐ criteria decision analysis problem of value? global policy, 2017;8:76-83. 4. council on health research for development (cohred). essential national health research and priority setting: lessons learned. 1997 5. dolan p, tsuchiya a. health priorities and public preferences: the relative importance of past health experience and future health prospects. j health econ, 2005;24:703-714. 6. viergever rf, olifson s, ghaffar a, terry rf. a checklist for health research priority setting: nine common themes of good practice. health res policy syst, 2010;8:36. 7. ministry of health oman. ninth five year-plan for health development. 2015 8. ministry of health oman. annual health report 2016. 9. fryatt r, mills a, nordstrom a. financing of health systems to achieve the health millennium development goals in low-income countries. lancet, 2010;375:419-426. 10. deng g, weber w, sood a, kemper kj. research on integrative healthcare: context and priorities. explore: j sci heal, 2010;6: 143-158. 11. al-mawali, a, ia john, dp avinash. fraud and misconduct in clinical research: a step to improve ethical practice in research. j contemp med sci, 2010;4(3 sep. 2018). issn 2413-0516. available at: <http://www.jocms.org/ index.php/jcms/article/view/449>. date accessed: 29 june 2020 12. ghaffar a, collins t, matlin s, olifson s. the 3d combined approach matrix: an improved tool for setting priorities in research for health. geneva: global forum for health research. 2009. 13. okello d, chongtrakul p. a manual for research priority setting using the enhr strategy. cohred. 2000 14. el lawindi mi, galal ys, khairy wa. health research and millennium development goals: identifying the gap from public health perspective. global j health sci, 2016;8:1. 15. institute for health metrics and evaluation (ihme). global burden of disease study 2016 (gbd 2016). 2016. 16. world health organization. the global burden of disease concept. (available from http://www.who.int/quantifying_ehimpacts/publications/ en/9241546204chap3.pdf ). accessed on july 1, 2018. 17. mitchell s, shaw d. the worldwide epidemic of female obesity. best pract res clin obstet gynaecol, 2015;29:289-99. 18. world health organization. framework for developing a health systems research agenda. (available from http://www.who.int/rpc/meetings/ framework_for_developing_a_health_systems_research_agenda.pdf ). accessed on july 3, 2018. 19. viergever rf, terry r, matsoso m. health research prioritization at who: an overview of methodology and high-level analysis of who led health research priority setting exercises. geneva: world health organization. 2010 20. kutzin j. a descriptive framework for country-level analysis of health care financing arrangements. health policy, 2001;56:171-204. 21. montorzi g, de haan s, ijsselmuiden c. priority setting for research for health: a management process for countries. council on health research for development (cohred). 2010 22. whear r, thompson‐ coon j, boddy k, et al. establishing local priorities for a health research agenda. health expect, 2015;18:8-21. 23. national association of county and city health officials (naccho). tip sheet: prioritizing issues in a community health improvement process. community health assessments and community health improvement plans for accreditation preparation demonstration project. 2010. 24. organization wh. a research policy agenda for science and technology to support global health development: a synopsis. geneva: world health organization. 1997. 25. al mawali ahn, al qasmi am, al sabahi sms, idikula j, elaty maa, morsi m, al hinai at. oman vision 2050 for health research: a strategic plan for the future based on the past and present experience. oman med j. 2017 mar;32(2):86-96. doi: 10.5001/omj.2017.18. pmid: 28439378; pmcid: pmc5397084. 139 original health research priority setting in omanadhra al-mawali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 126–139 26. oman health vision 2050, published by undersecretary of planning, ministry of health, sultanate of oman. 2015 27. ranson mk, bennett sc. priority setting and health policy and systems research. health res. policy syst, 2009;7: 27. 28. garcia ab, cassiani shdb, reveiz l. a systematic review of nursing research priorities on health system and services in the americas. rev. panam. de salud públ., 2015;37:162-171. 29. cooke j, ariss s, smith c, read j. on-going collaborative priority-setting for research activity: a method of capacity building to reduce the researchpractice translational gap. health res policy syst, 2015;13:25 30. madon t, hofman kj, kupfer l, glass ri. implementation science. american association for the advancement of science. 2007. 31. world health organization. a prioritized research agenda for prevention and control of noncommunicable diseases. 2011. 32. mirzoev t, kane s. what is health systems responsiveness? review of existing knowledge and proposed conceptual framework. bmj global health, 2017;2:e000486. 33. remme jh, adam t, becerra-posada f, et al. defining research to improve health systems. plos med, 2010;7:e1001000. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i3.791 46 original issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 46 – 52 introduction kidney transplantation is the treatment of choice for patients with end-stage renal disease (esrd), but because of the limited availability of dialysis in developing countries, it is the only option for the long-term survival of the great majority of the world’s esrd patient.1–3 over the past decades, studies repeatedly documented kidney biopsy as the gold-standard for the diagnosis of allograft dysfunction.4 kidney allograft biopsy diagnoses are primarily based upon histological findings and immunofluorescence staining for c4d, and treatment is directed toward the pathologist diagnosis.5 but this histological assessment is restricted by the subjective interpretation of the lesions, making the histopathological diagnosis of limited reproducibility.6 since 2009, many groups have investigated gene expression profiling by microarray or pcr in an attempt to improve the diagnostic accuracy of biopsy sample analysis. sis et al.7 and reeve et al.8 reported that biopsies with rejection (acute and chronic) showed three molecular profiles: endothelial transcript, natural killer (nk) cell-associated transcript, and ifn-γ. the collaboration of edmonton study group with european countries led to the intercomex study, in which they used microarrays and algorithms to measure mrna levels in 519 transplant biopsies from 10 north america and european centers, to assess t cellsand antibody-mediated rejection, the diagnoses assigned based on molecular microscopic diagnostic system (mmdx). this study was able to differentiate rejection from non-rejection injury as kidney injury or inflammatory glomerular disease.9 despite that molecular studies have contributed to our understanding of the mechanisms of renal graft pathology, the logistical issues and high cost of these sophisticated technics make it not practical to be used in daily diagnostic practice and management of graft recipients, especially in a developing country like iraq.4 this study aims to evaluate whether reasonably inexpensive testing methods, as immunohistochemistry (ihc) and molecular expression of von willebrand factor (vwf) and interferon (ifn), might help discriminate acute rejection from other transplant disorder. material and methods patients and sample collection this study included a total of 49 for-cause biopsies obtained between january 1, 2019 and december 31, 2019. a satisfactory biopsy was defined as having at least five glomeruli and one arterial cross-section. the biopsies were performed for poor function, deterioration in function, or proteinuria. basic demographic and clinical parameters were retrieved from medical records. histopathology and diagnostic criteria all biopsies were processed in the histopathology laboratory of shorsh hospital/sulaymaniyah governorate and studied by light microscopy in 18 serial sections using hematoxylin and eosin, periodic acid–shiff, masson trichrome stains. jones periodic acid-methenamine silver stains were performed on expression of vwf and interferon γ in renal allograft biopsies and correlation with inflammatory cells. single center experience. alaa abbas ali1*, kais h al-taee2, zana sidiq m saleem2 1 department of pathology, university of sulaimani college of medicine and shoresh teaching hospital, sulaimani, iraq. 2 department of nephrology, university of dohuk college of medicine, dohuk, iraq. *correspondence to: alaa abbas ali (e-mail: dr.alaaabbas@yahoo.com) (submitted: 11 november 2020 – revised version received: 25 november 2020 – accepted: 17 december 2020 – published online: 26 february 2021) abstract objective gene expression profiling by microarrays or rt pcr has been studied in certain western centers to enhance the diagnostic accuracy of allograft biopsy, however, such sophisticated tests are difficult to apply in developing countries. this study was conducted to evaluate the expression of von willebrand factor (vwf) and interferon-gamma (ifnγ) as an end pcr product in renal allograft biopsy with different pathological categories. methods forty-nine (49) indicated renal allograft biopsies were analyzed histologically by the banff 2017 classification, inflammatory cell infiltration was analyzed by immunohistochemistry study for cd4, cd8, cd16, and cd68 markers, and a fresh tissue used for molecular study. results the biopsy findings were acute t-cell mediated rejection (a-tcmr) 30.6%, interstitial fibrosis and tubular atrophy 22.4%, c4dtransplant glomerulopathy 12.2%, calcineurin inhibitor toxicity 10.2%, c4d+antibody mediated rejection 10.2% and normal histology 14.3%. the only significant difference in vwf expression was between acute tcmr and normal, p=0.01, spearman’s correlation also showed a significant relationship between vwf and acute tcmr (r=0.53, p=0.01), and vwf was found to correlate with the numbers of interstitial cd4+ (r=0.29, p=0.03) and cd68+ (r=0.37, p=0.007) cells. ifnγ expression was significant in acute tcmr versus normal, p=0.009. spearman’s pair-wise testing showed that infγ correlated with cd8 in both the glomerular (r=0.38, p=0.006) and interstitial (r=0.30, p=0.04) compartments and with cd16+ interstitial cells (r=0.36, p=0.01). conclusions molecular and immunohistochemistry data of this study distinguish acute tcmr from other forms of transplant pathology and mildly dysfunctional kidneys with normal histology. keywords renal allograft biopsy; vwf; ifnγ; pcr; kidney transplantation 47 original vwf and ifnγ in renal allograftalaa abbas ali, kais h al-taee, zana sidiq m saleem j contemp med sci | vol. 7, no. 1, january-february 2021: 46 – 52 selected cases to better see basement membrane changes. none of the biopsies were studied by electron microscopy. direct immunofluorescence was performed on frozen sections using fluorescein-conjugated anti-human igg, igm, iga, c3, c1q (dako, santa clara, ca). c4d staining was performed using a monoclonal anti-c4d antibody (bio-rad, inc) by indirect immunofluorescence. the histological findings were categorized based on the banff 2017 classification5,10 into normal or no specific changes, acute tcmr, c4d+ amr, c4d-tg, if/ta-nos, and cni toxicity. transplant glomerulopathy (tg) is diagnosed by the presence of double contours of the glomerular basement membrane (gbm) graded at cg1b and above after excluding other causes of double contour gbm such as thrombotic microangiopathy and glomerulonephritis.10 immunohistochemistry positively charged slides were used for ihc staining with the following markers cd4 (clone: rbt; cd4, bio bs, usa), cd8 (clone: ep334, bio sb, usa), cd16 (clone; ep364, bio sb, usa), and cd68 (dako, glostrup, denmark). these markers represent the cells involved in the major non-antibody pathways of innate and adaptive immunity. ihc stain scoring in all of the biopsies, we used ihc stain to assess the number of cd4+ cells, cd8+ cells, cd16+ cells, and cd68+ cells in two tissue compartments: intraglomerular and interstitium. the counting was performed manually at 400x. staining for cd4, cd8, cd16, and cd68 was quantified by averaging the number of cells stained in five glomeruli or five 400x microscopic fields of the interstitial areas. molecular study rna extraction. messenger rna was extracted from rna later preserved kidney allograft specimens using total rna purification kit (norgen biotek, on, canada) according to the manufacturer’s instructions. the rna was eluted in depc-treated water and stored at -80°c. rna quality and concentration was assessed using biophotometer (eppendorf, hamburg, germany) at wavelengths 260/280. complementary dna (cdna) was constructed by goscript reverse transcriptase pcr (promega, madison, wi, usa) according to the manufacturer’s procedure. primer design and polymerase chain reaction. the primers sequences used in this study were taken from online articles (bonthron et al.11 and kim et al.12). the primers were purchased from genewiz (nj, usa). we used conventional polymerase chain reaction (pcr) (applied biosystemsveriti 96-well thermal cycler, usa), to detect the expression of vwf nucleotide sequences were as follows: sense primer: 5΄ agggacagctcctggatga 3΄, anti-sense primer 5΄ actggcagatcccactgaag 3΄, interferon-gamma (ifn-γ) nucleotide sequences were as follows: sense primer: 5΄ tgaatgtccacccgaaagca 3΄, anti-sense primer 5΄ cgacctcgaaacagcatctga 3΄, human β-actin was employed as a housekeeping gene verify the integrity of target gene mrna with nucleotide sequences were as follows: sense primer: 5΄ caccaactgggacgacat 3΄, anti-sense primer 5΄acagcctggatagcaacg 3΄.11,12 the reaction mix and thermal cycling. 25 µl go taq g2 green master mix2x (promega corp., madison, wi, usa) reaction volume was prepared by adding the following: 2.5  µl for each forward and reversed primer, 12.5 µl go tag g2 green mix, 5.5 µl nuclease-free water, and 2.0 µl dna template (c dna). the mixture was placed in a pcr tube, then centrifuged for 10 sec, and placed into applied biosystem thermal cycler. the pcr reaction was performed as follows: initial denaturation at 95°c of 2 min for 1 cycle, denaturation at 95°c for 30 sec. for 35 cycles, annealing at 55°c for 1 min. for 35 cycles and extension at 72°c for 1 min. the resulting pcr products were analyzed by 2% agarose gel electrophoresis stained with ethidium bromide. the gel was run at 100 volts for 45 min, and the cdna fragments were visualized by uv-light as shown in fig. 1. statistical procedures the data were analyzed with ibm spss (statistical package for social science-version 26.0) c4d, vwf, ifn-γ expression was recorded as binary positive or negative variables (0 or 1) and banff scores as ordinal variables (scores 0, 1, 2, 3). the number of inflammatory cells in the glomerular or interstitial compartments were expressed as mean ± sd. the continuous variables of age, time post-transplant (in days), and serum creatinine were not normally distributed and were expressed as the median and interquartile range (iqr). for ihc and molecular marker expression, each marker was analyzed by one-way analysis of variance (anova) with post-hoc tukey’s test to determine differences between diagnostic groups. paired comparisons between two diagnostic groups used mann-whitney u-tests. spearman correlation was used to evaluate the paired relationships between molecular and ihc findings and diagnoses. differences in categorical variables between-groups were evaluated by chi-square or fisher exact tests. differences between two groups were considered significant at p<0.05. fig. 1 pcr bands of vwf and ifn γ on gel electrophoresis. abbreviation: vwf, von willebrand factor; ifn interferon, b-actin, beta actin. 48 original vwf and ifnγ in renal allograft alaa abbas ali, kais h al-taee, zana sidiq m saleem j contemp med sci | vol. 7, no. 1, january-february 2021: 46 – 52 results patient characteristics the study included 49 kidney transplant recipients’ patients who underwent a kidney biopsy. the transplant units do not perform protocol biopsies and all biopsies were for clinical indications. median patient age was 37 years with an iqr of 24-48 years old. recipients were 77.8% male. median serum creatinine (s.cr) was 2.0 mg/dl with an iqr of 1.7–2.8 mg/dl. all 54 patients were found to have a median time of 365 days (iqr 29.5–1095). the patient characteristics are summarized in table 1. abbreviations: continuous variables of age and serum creatinine and time of biopsy are expressed as median and interquartile range. iqr, interquartile range, mmf: mycophenolate mofetil. histopathological results the histological diagnosis and number of specimens consisted of acute tcmr, n=15; c4d positive antibody-mediated rejection, n=5 (including two cases of mixed acute t-cmr and acute c4d+amr); cni toxicity, n=5; c4d-transplant glomerulopathy n=6; interstitial fibrosis and tubular atrophy, n=11 and normal histology, n=7. the normal histology was associated with mild dysfunction post-transplantation and would represent mild aki without identifiable pathological findings and serve as a non-rejection control. ihc evaluation of cd4, cd8, cd16, and cd68 positive cells the expression of different cellular markers in five major histological categories is shown in table 2 with significant p value in relation to normal histology. as shown in table 2, cd16+ (p=0.02) expression in the glomerular compartment was higher in c4d+amr (fig. 2), while cd68 (p=0.04) was significantly expressed in the c4d-tg group (fig. 3). table 1. clinical characteristic of recipients, n(%). data number (%) total number of recipients 49(100) median age of recipients (yr) 36 (iqr: 24-47) gender(male) 37(75.5) living/deceased donor 49/0 (100) related/unrelated donor 6/43 (12.2/87.8) median serum creatinine at biopsy (mg/dl) 2 (iqr:1.7-2.8) previous transplant 4 (8.2) mediate time of biopsy (days) 365(iqr:43-1095) maintenance immunosuppressive regimens at biopsy mmf, tacrolimus, steroid 34(69.4) mmf, cyclosporine, steroid 15(30.6) indication for biopsy: primary non function 5(10.2) rapid deterioration of graft function 18(36.7) slow deterioration of graft function 20(40.8) proteinurea 2(4.1) follow up biopsy 4(8.1) abbreviations: continuous variables of age and serum creatinine and time of biopsy are expressed as median and interquartile range. iqr, interquartile range, mmf: mycophenolate mofetil. table 2. one way anova comparing ihc inflammatory cell marker expression in the major diagnostic categories of transplant pathology. ihc compartment normal (n=7) a-tcmr (n=15) c4d+amr (n=5) c4d-tg (n=6) ifta (n=11) cni toxicity (n=5) cd4 glomerular 0.2±0.31.000 1.1±2.8 0.808 0.2±0.3 1.000 0.5±0.7 0.945 0.0±0.0 0.737 0.2±3.1 1.000 interstitial 6.3±3.11.000 22.6±11.7 0.001 24.7±19.9 0.447 16.2±13.4 0.556 11.2±6.4 0.321 15.9±10.8 0.490 cd8 glomerular 0.2±0.31.000 2.1±3.1 0.229 0.1±0.2 1.000 1.5±1.8 0.556 0.7±1.1 0.698 0.8±0.6 0.400 interstitial 6.3±51.000 24.7±16.7 0.012 21.4±7 0.037 17.2±10.2 0.279 16±10 0.144 20.9±9.0 0.115 cd16 glomerular 0.7±0.81.000 4.7±4.7 0.060 4.1±1.4 0.023 4.9±4.9 0.433 0.8±1.8 1.000 2.3±1.0 0.181 interstitial 7.1±7.51.000 19.6±11.1 0.062 18.2±11.8 0.501 8.9±3.6 0.845 11±4.7 0.818 14.3±9.6 0.736 cd68 glomerular 1.5±1.61.000 2.9±4.1 0.917 5.4±1.7 0.430 7.6±7.2 p=0.044 1.1±1.4 0.994 1.2±1.1 0.999 interstitial 7±51.000 19.8±8.6 0.004 24±11.0 0.084 19.1±10.1 0.204 12.2±4.6 0.317 12.6±6.0 0.880 note: in each cell upper numbers are mean ± standard deviation and lower numbers are p values. significant differences are bolded with the reference having a p value of 1.000. abbreviation: a-tcmr; acute t cell-mediated rejection, amr; antibody mediated rejection; tg, transplant glomerulopathy; ifta; interstitial fibrosis and tubular atrophy; cni; calcineurin inhibitor toxicity. 49 original vwf and ifnγ in renal allograftalaa abbas ali, kais h al-taee, zana sidiq m saleem j contemp med sci | vol. 7, no. 1, january-february 2021: 46 – 52 fig. 2 compartment-specific cd16+cell expression. a and c, glomerular. b and d, interstitial in renal allograft biopsies in different diagnostic categories. # p<0.05 vs. normal histology. *p<0.05 vs. ifta. fig. 3 compartment-specific cd68+cell expression. a and c, glomerular. b and d, interstitial in renal allograft biopsies in different diagnostic categories. # p<0.05 vs. normal histology.* *p<0.05 vs. ifta. 50 original vwf and ifnγ in renal allograft alaa abbas ali, kais h al-taee, zana sidiq m saleem j contemp med sci | vol. 7, no. 1, january-february 2021: 46 – 52 cd4+, cd8+, and cd68+ cells staining were pronounced in the interstitial compartment of acute tcmr. molecular result of von willebrand factor (vwf) and interferon-gamma (ifnγ) expression in different diagnostic groups molecular result for vwf. vwf was found in 20% of acute tcmr, 8% of if/ta, 6% of c4d-amr, 2% of cni toxicity, 2% of tin, 2% of aki, and 2% of normal biopsies. when these proportions were compared using a fisher exact test, the only significant difference in vwf expression was between acute tcmr and normal, p=0.01. fig. 4. spearman’s correlation also showed a significant relationship between vwf and acute tcmr (r=0.53, p=0.01), and vwf was found to correlate with the numbers of interstitial cd4+ (r=0.29, p=0.03) and cd68+ (r=0.37, p=0.007) cells (table 3). molecular results for ifnγ. ifnγ was expressed in acute tcmr (16%), if/ta (8%), c4d-amr (2%), tin (2%), atn (4%), and cni toxicity (4%). there was no expression of ifnγ in c4d-amr nor normal histology. a fisher exact test was used to compare the expression of ifnγ in different diagnostic groups and found that the only significant difference was between acute tcmr and normal, p=0.01 fig. 5. spearman’s pair-wise testing showed that infγ correlated with cd8 in both the glomerular (r=0.38, p=0.006) and interstitial (r=0.30, p=0.04) compartments and with cd16+ interstitial cells (r=0.36, p=0.01) (table 3). discussion in this study, we applied molecular testing and ihc to a subset of 2019 biopsies was to evaluate whether reasonably inexpensive testing methods might help discriminate between acute and the several chronic transplant disorders, and whether this might have any application in an under-resourced medical transplant setting. we selected primers for two cdna transcripts: 1. vwf was used as endothelial activation markers. 2. ifnγ was used as a general inflammatory marker.13, 14 our results showed intra-glomerular cd16+cells predominate in c4d+amr while cd68+cells were increased in the glomerular compartment of c4dtg as compared to normal histology and to if/ta (p=0.04 &0.01). glomerular cd68+cells correlate with c4dtg, and c4d+amr, while cd16+cells correlate with c4d+amr only. these findings are similar to studies performed by others using ihc study dos santos et al.15 and divella et al.16 or by using macrophages associated transcript hayde et al.17 and lefaucheur et al.18 we obtained good results for vwf and ifnγ, vwf and ifnγ are the end-products of activation cascades and are expressed constitutively at fairly high levels in an inflammatory reaction. in expression arrays, the great majority of the markers are stimulatory molecules that upregulate the expression of constitutive end-products, but these molecules are often transiently expressed at much lower levels than the end-products and can be difficult to detect without the proper systems.13 we were able to demonstrate that ifnγ levels were significantly associated with increased numbers of tissue infiltrating cd8+ t cells and cd16+ cells. the results also showed that an increased expression of vwf was significantly associated with interstitial infiltrates of cd68+ macrophages, which agrees with batal et al19 study, which stated that vwf expression by ihc associated with peritubular capillaritis. the numbers of tissue infiltrating cd4+ t cells, cd8+ t cells, cd16+ cells, and cd68+ macrophages were significantly associated with each other, and with the diagnosis of acute tcmr, these findings were similar to dos santos et al. study.15 this produced a high degree of discrimination between acute tcmr and kidneys with mild dysfunction and normal histology (i.e.  non rejection). these inflammatory markers were just as elevated in c4d+ abmr as they were for acute tcmr, but the small number of biopsies in the c4d+ abmr group did not allow fig. 4 vwf expression in renal allograft biopsies showing acute t cell-mediated rejection (tcmr) versus normal histology. #p<0.05. 51 original vwf and ifnγ in renal allograftalaa abbas ali, kais h al-taee, zana sidiq m saleem j contemp med sci | vol. 7, no. 1, january-february 2021: 46 – 52 table 3. correlation of inflammatory cells, vwf and inf-γ in biopsies of different diagnostic categories. compartments atcmr c4d-tg c4d+amr if/ta vwf ifn-γ glomerular statistic cd4 r * 0.25 0.15 0.05 -0.44 0.10 0.13 p ns ns ns 0.06 ns ns ns cd8 r * 0.49 0.41 -0.27 0.15 0.05 0.38 p 0.02 ns ns ns ns 0.006 cd16 r * 0.37 0.45 0.86 -0.20 0.06 0.13 p ns ns <0.001 ns ns ns cd68 r * 0.08 0.60 0.81 -0.12 0.15 0.17 p ns 0.03 0.001 ns ns ns interstitial cd4 r * 0.68 0.53 0.61 0.38 0.29 0.18 p 0.0004 0.05 ns 0.03 ns 0.03 ns cd8 r * 0.63 0.57 0.80 0.58 0.05 0.30 p 0.002 0.03 0.001 0.01 ns 0.04 cd16 r * 0.59 0.41 0.61 0.42 0.27 0.36 p 0.004 ns 0.03 ns ns 0.01 cd68 r * 0.66 0.71 0.85 0.50 0.37 0.22 p 0.001 0.008 0.001 0.03 0.007 ns molecular vwf r * 0.53 0.47 0.35 0.26 p 0.01 ns ns ns ifn-γ r * 0.55 0.35 not detected 0.46 p 0.009 ns ns ns abbreviation: a tcmr, acute t-cell mediated rejection; amr, antibody mediated rejection; tg; transplant glomerulopathy; if/ta, interstitial fibrosis and tubular atrophy; ns, not significant; r *, spearman’s correlation coefficient. p<0.05 was bolded. fig. 5 ifn-γ expression in acute t cell-mediated rejection (tcmr) versus normal histology. #p<0.05. 52 original vwf and ifnγ in renal allograft alaa abbas ali, kais h al-taee, zana sidiq m saleem j contemp med sci | vol. 7, no. 1, january-february 2021: 46 – 52 for the differences between most markers to reach significance. similarly, the values of the markers were generally low to intermediate for tg and if/ta suggesting that our testing tended to identify these disorders as inflammatory poor processes, which they are by routine light microscopy. in summary, our molecular and ihc data distinguish acute tcmr from other forms of transplant pathology and mildly dysfunctional kidneys with normal histology. this is an indication that some form of basic molecular testing could be designed for our transplant services. if a true quantitative system could be developed and appropriate controls established, this might be of clinical importance for distinguishing between post-transplant dysfunction, borderline rejection, and acute tcmr. among our patients, these categories comprise over 50% of our biopsies. such testing has been performed in the united states by whole-genome expression profiling on peripheral blood samples, with the results being as discriminative between acute tcmr, normal histology, and acute dysfunction with non-rejection as the results reported from the intercomex trials.19 conclusion based on our findings, we can suggest that cd16 and cd68 markers can express themselves in different compartments depending on the underlying pathology. our result also showed that some form of basic molecular testing could be designed for our transplant services to differentiate acute tcmr from other forms of transplant pathology and mildly dysfunctional kidneys. declarations acknowledgments the authors would like to acknowledge the kurdistan institution for strategic studies and scientific research, the university of sulaimani, and histopathological laboratory of shorsh hospital for their facilities, services, and support. competing interests none to declare. ethical approval this study was approved by the ethical committee of the faculty of medical sciences /university of sulaimani in the kurdistan region, iraq under protocol number (#11n/29). it carried out on indicated graft biopsies and it involved no additional patient intervention that did not require informed consent. references 1. jha v, chugh k. dialysis in developing countries: priorities and obstacles. nephrology. 1996;2(2):65-71. 2. zhou j, d’agati v, laszik z, nadasdy t. silva’s diagnostic renal pathology. cambridge: cambridge university; 2016. 3. parajuli s, aziz f, garg n, panzer s, joachim e, muth b et al. histopathological characteristics and causes of kidney graft failure in the current era of immunosuppression. world j transplant. 2019;9(6):123-1 4. williams w, taheri d, tolkoff-rubin n, colvin r. clinical role of the renal transplant biopsy. nat rev nephrol. 2012;8(2):110-121. 5. haas m, loupy a, lefaucheur c, roufosse c, glotz d, seron d et al. the banff 2017 kidney meeting report: revised diagnostic criteria for chronic active t cell–mediated rejection, antibody‐mediated rejection, and prospects for integrative endpoints for next‐generation clinical trials. am j transplant. 2018;18(2):293-3 6. mengel m, sis b, halloran p. swot analysis of banff: strengths, weaknesses, opportunities and threats of the international banff consensus process and classification system for renal allograft pathology. am j transplant. 2007;7(10):2221-2226. 7. sis b, jhangri g, bunnag s, allanach k, kaplan b, halloran p. endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of c4d staining. am j transplant. 2009;9(10):2312-2323. 8. reeve j, einecke g, mengel m, sis b, kayser n, kaplan b et al. diagnosing rejection in renal transplants: a comparison of molecularand histopathology-based approaches. am j transplant. 2009;9(8):1802-1810. 9. halloran p, reeve j, akalin e, aubert o, bohmig g, brennan d et al. real time central assessment of kidney transplant indication biopsies by microarrays: the intercomex study. am j transplant. 2017;17(11):2851-2862. 10. roufosse c, simmonds n, clahsen-van groningen m, haas m, henriksen k, horsfield c et al. a 2018 reference guide to the banff classification of renal allograft pathology. transplantation. 2018;102(11):1795-1814. 11. bonthron d, handin r, kaufman r, wasley l, orr e, mitsock l et al. structure of pre-pro-von willebrand factor and its expression in heterologous cells. nature. 1986;324(6094):270-273. 12. kim s, kim yk, lee h, cho je, kim hy et al. interferon gamma mrna quantitative real-time polymerase chain reaction for the diagnosis of latent tuberculosis: a novel interferon gamma release assay. diagn microbiol infect dis. 2013, 75, 68-72. 13. halloran p, venner j, madill-thomsen k, einecke g, parkes m, hidalgo l et al. review: the transcripts associated with organ allograft rejection. am j transplant. 2017;18(4):785-795. 14. adam b, afzali b, dominy k, chapman e, gill r, hidalgo l et al. multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue. clin transplant. 2016;30(3):295-305. 15. dos santos d, campos e, saraiva câmara n, david d, malheiros d. compartment-specific expression of natural killer cell markers in renal transplantation: immune profile in acute rejection. transplant int. 2015;29(4):443-452. 16. divella c, rossini m, loverre a, schena a, maiorano a, gesualdo v et al. immunohistochemical characterization of glomerular and tubulointerstitial infiltrates in renal transplant patients with chronic allograft dysfunction. nephrol dial transplant. 2010;25(12):4071-4077. 17. hayde n, bao y, pullman j, ye b, calder r, chung m et al. the clinical and genomic significance of donor-specific antibody–positive/c4dnegative and donor-specific antibody–negative/c4d-negative transplant glomerulopathy. clin j am soc nephrol. 2013;8(12):2141-2148. 18. lefaucheur c, viglietti d, hidalgo l, ratner l, bagnasco s, batal i et al. complement-activating anti-hla antibodies in kidney transplantation: allograft gene expression profiling and response to treatment. j am soc nephrol. 2017;29(2):620-635. 19. batal i, azzi j, el-haddad n, riella l, lunz j, zeevi a et al. immunohistochemical markers of tissue injury in biopsies with transplant glomerulitis. human pathol. 2012;43(1):69-80. 20. kurian s, williams a, gelbart t, campbell d, mondala t, head s et al. molecular classifiers for acute kidney transplant rejection in peripheral blood by whole genome gene expression profiling. am j transplant. 2014;14(5):1164-1172. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.912 313j contemp med sci | vol. 5, no. 6, november–december 2019: 313–316 original issn 2413-0516 introduction the pelvimetry refers to the evaluation of the diameters of the bony pelvis to assess the pelvic cavity for the passage of a fetus.1 the maternal pelvis morphological feature is important predictive factor of vaginal delivery. the female pelvis was classified by caldwell-moloy based on shape in 1933. this classification contain four main pelvic types; gynaecoid (50%), android (25%), anthropoid (20%), and platy pelloid (0.5%) (fig. 1).2 the prevalence of pelvic varieties is different according to sex and races.3 for successful vaginal delivery proper shape and size of the pelvis is necessary. the contracted pelvis leads to disturbances like premature rupture of the membrane, ineffective contractions, abnormal conditions of the fetus, labor dystocia, and finally increasing in rate of cesarean section.3 the elective cesarean in these cases could improve the result of delivery. although cesarean delivery rate is increased in last decades.5–8 the result of recent studies showed that rate of cesarean is raised.9–12 in normal female pelvis, a longer diameter of the inlet (the transverse diameter) and a longer diameter of the midpelvis (the anteroposterior diameter) are placed perpendicularly. therefore, a fetal head rotates from a transverse position in the pelvic inlet to a sagittal position in the midpelvis. narrowing of the pelvic cavity in the midpelvis prevent this rotation. klemt et al. demonstrated that narrow midpelvis and inadequate proportion of the pelvic inlet causes the emergency cesarean section.13 the pelvic inlet and midpelvis are important factors to the anthropological analysis of the female pelvic typology.14 nowadays evaluation of pelvic types based on clinical examination and imaging techniques including ct, mri, radiography and us are available.14,15 the previous studies have shown that pelvimetry with ct is convenient and accurate technique.16 the assessment of pelvic types in addition to another anatomy parameters has clinical importance in predicting the procedures of pregnancy and childbirth in evaluation of the link between pelvimetry based on computed tomography and predicting status’ delivery shahla mirgalobayat,a laya ghahari,b leila allahqoli,a seyed reza saadat mostafavi,c katayoun safari,d masih rikhtehgar,c ali ramezanghorbani,c mohmoodreza madadiane aendometriosis research center, rasoul-e-akramhospital, iran university of medical science (iums), tehran, iran bdepartment of anatomy, school of medicine, aja university of medical sciences, tehran, iran cdepartment of radiology, rasoul-e-akram hospital, iran university of medical sciences, tehran, iran ddepartment of small animal internal medicine,islamic azad university, tehran, iran efaculty of pharmacy, islamic azad university, tehran, iran corresponding author: shahla mirgalobayat (email: mirgaloybayat.sh@iums.ac.ir) (submitted: 14 august 2019 – revised version received: 02 september 2019 – accepted: 12 september 2019 – published online: 26 december 2019) objectives the aim of this study is an anthropometric study in non-pregnant reproductive-aged women to predicting kind of delivery. methods this study was based on the archived information of hazrat-e rasool university hospital on 157 generative age women from 16 to 60 years old who underwent ct between march 2015 and march 2018. results the obtained results showed that the three conjugates on sagittal plane, transverse diameter, anteroposterior sagittal diameter, interspinous diameter, and intertuberous diameter on the coronal plane. the obstetrical conjugate was 123.3 mm in normal vaginal delivery and 113.9 mm in cesarean section, which was slightly longer than delivery group. the mean interspinous and intertuberous diameters measured by ct scans were 105.0 and 107.4 mm. conclusion the results of our study showed that because of the reduction in diameter of the inlet and middle pelvis, the rate of cesarean section was higher in females under 35 years ages . keywords pelvimetry, computed tomography, non-pregnant women fig. 1 different types of pelvic inlets according to classification of caldwell-moloy.4 314 original evaluation of the link between pelvimetry based on computed tomography shahla mirgalobayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 313–316 reproductive-aged women. in the present work, results of pelvimetry of 157 non-pregnant reproductive-aged women admitted hazrat-e rasool university hospital in tehran evaluated. materials and methods study design and participants this research was a retrospective study. the clinical records and reports of abdominal ct scans of women referred to radiology department of hazrat-e rasool akram university hospital, iran university of medical sciences, tehran, iran during march 2015 to march 2018 were recruited. subjects according to inclusion and exclusion criteria were eligible. one hundred and fifty-five non-pregnant reproductive-aged women were included and pelvic fractures, lumbosacral spondylolisthesis, pelvic bone tumor, or anomalies and diseases or trauma affected the bony pelvic structure were excluded. in this work, ct examination was conducted using ct scanner (siemens medical systems inc., madison, wi, usa).17 the obstetrical importance of anteroposterior diameter (diagonal conjugate) is from the tip of the sacral promontory to the lower border of the symphysis pubis (fig. 2). the true conjugate was determined by measuring the distance from the tip of the sacral promontory to the upper border of the symphysis pubis. the obstetric conjugate is the shortest distance between the tip of the sacral promontory and the most bulging point on the back of symphysis pubis that was measured on the sagittal radiograph. the transverse diameter of the pelvic inlet is the distance between the farthest two points on the iliopectineal lines that were measured on the anteroposterior radiograph. the interspinous diameter of the midpelvis is the distance between the ischial spines that were measured on the axial radiograph. the anteroposterior diameter of the pelvic outlet is determined by measuring the distance from the lower border of the symphysis pubis to the sacrococcygeal joint that was measured on the sagittal radiograph (fig. 2). the intertuberous diameter of the outlet is the distance between the inner borders of ischial tuberosities that is measured on the anteroposterior radiograph.18 the pelvimetry measurements were assessed by the radiographs and using electronic calipers with an internal scale. ethics statement this study was performed in accordance with the ethical rules of helsinki declaration. all the procedures used in this research was approved by the ethics committee of iran university of medical sciences (no.ir.iums.rec.1397.234). statistical analysis the obtained data and analytical measurements were recorded and compared. analysis of collected maternal data including age, gravidity, parity, weight, and height were used as covariates to understand the relationship between pelvic types and kind of delivery. in this study. categorical variables were presented as number (percent) and continuous variables as mean ± sd with min–max or percentage as appropriate. statistical analyses were performed using commercially available software (ibm spss statistics version 22; ibm, chicago, il, usa). comparison and correlation of variables were conducted with and correlation tests and a p<0.05 was considered statistically significant. results pelvic anatomical measurements of 157 subjects were analyzed in the present study. among the studied subjects, age ranged from 16 to 60 years, height ranged from 150 to 180 cm, and population weight ranged from 43 to 98 kg. out of these, 102 (65.0%) had at least one normal vaginal delivery (nvd group), and 55 individuals (35.0%) had undergone at least one cesarean section (c-sec group). the indication of cesarean delivery was cephalopelvic disproportion. the demographic and anatomical characteristics of the investigated women are summarized in table 1. subjects with cesarean section had a lower height than those with nvd (mann–whitney u-test, p = 0.035). besides, the c-section group of mothers had shorter obstetrical conjugate and interspinous diameter than those in the nvd group (p < 0.05 for both parameters) (table 1). no significant difference was observed between two arms of the study in terms of age, weight, parity, true conjugate, diagonal conjugate, transverse diameter, posterior sagittal diameter, intertuberous diameter, and anteroposterior diameter (table 1). the frequency distribution of different types of pelvis has been presented in table 2. the chi square findings unveiled that the relative frequency of android pelvis were statistically higher in c-sec group (p = 0.036). the percentages of other types of pelvis were comparable between the two groups of females (table 2). the obtained results demonstrated that scatter plots of pelvic anatomical measurements was stratified according to fig. 2 pelvic inlet, outlet and diagonal conjugate according to snell clinical anatomy.4 315 original evaluation of the link between pelvimetry based on computed tomographyshahla mirgalobayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 313–316 the median age of the population. statistical analysis revealed that the median values of true conjugate (fig. 3a), obstetrical conjugate (fig. 3b), diagonal conjugate (fig. 3c), transverse diameter (fig. 3d), posterior sagittal diameter (fig. 3e), and interspinous diameter (fig. 3f) were significantly lower in women aged <35 years than women aged >35 years in c-sec group (p < 0.05). although no significant differences were shown between women more or less than 35 years of age who had undergone spontaneous vaginal delivery (nvd group) (fig. 3a–h). discussion in this study, all 157 subjects had a true conjugate (122.0 ± 9.0  mm) and a diagonal conjugate (123.9 ± 9.5 mm). the obstetrical conjugate was 123.3 mm in nvd and 113.9 mm in c-sec. it was slightly longer than the delivery group. this difference in diameter of conjugates is assumed to originate from the measurements made in younger women who have a cesarean section and they have not experienced vaginal delivery. the cesarean-section is also called cesarean delivery. however, the c-section has many risks such as wound infection, blood loss, injury to an organ just like the gastrointestinal tract or bladder, adverse reactions, although the cesarean-section has been grown in iran.19 regarding to evidences based on the anatomy and anthropology sciences, the pelvimetry technique has an important role in determination of the bony pelvis diameters and estimating the pelvic diameters.1 in this study, results of pelvimetry using ct in study population and its association with increased cesarean section rate were evaluated. in this study, the height of mothers did not match with type of delivery and pelvis dimensions. furthermore, no relationship was found between pelvis type and body mass index. previous studies have shown that maternal height is related to the risk of cesarean section. cesarean section rises with shortness of maternal height.20 the data of our study showed that the gynaecoid pelvis (58%), platypelloid (24.2%), anthropoid (8.9%), and android (8.9%). the size and shape of the pelvic canal vary in women, and the gynecoid pelvis is usually the most appropriate type of pelvis for vaginal delivery. however, we know that android and platypelloid are not suitable for childbirth. a previous study showed that mothers with gynecoid pelvis had better outcomes (71.6%–47.8%).21 in this research, we evaluated the pelvic diameter in women ≤35and ≥35 years old. our result proved that the inlet and midpelvis diameters of <35 years old group are significantly decreased in compared to the >35 years old group. lin et al indicated that there was a link between increasing maternal age and tendency to cesarean section.22 but in the present study, the rate of the cesarean section has been more in <35 years old. table 1. sensitivity and specificity of diagnostic values of ck-18 and fli for detection of nafld demographic/anatomic characteristic total (n = 157) nvd (n = 102) c-sec (n = 55) p-value* age [yr], median (iqr) 35 (30–39) 35 (29–37) 35 (31–40) 0.725 height [cm], median (iqr) 165 (160–169) 167 (164–170) 161 (157–164) 0.035 weight [kg], median (iqr) 70 (63–78) 71 (64–78) 70 (62–77) 0.913 no. of delivery, median (range) 1 (1–8) 1 (1–8) 1 (1–3) 0.739 true conjugate [mm], median (iqr) 122.0 (114.5–130.0) 123.7 (113.0–132.2) 120.1 (111.9–131.5) 0.335 obstetrical conjugate [mm], median (iqr) 118.2 (113.4–125.4) 123.3 (115.7–127.1) 113.9 (108.0–120.1) 0.013 diagonal conjugate [mm], median (iqr) 123.9 (114.1–129.5) 126.5 (117.3–134.3) 125.5 (112.1–131.3) 0.136 transverse diameter [mm], median (iqr) 220.3 (210.3–230.7) 223.4 (212.1–233.1) 219.1 (210.0–228.2) 0.259 posterior sagittal diameter [mm], median (iqr) 35.2 (28.0–39.5) 36.9 (29.1–41.2) 35.8 (28.9–38.1) 0.417 interspinous diameter [mm], median (iqr) 105.0 (99.3–110.5) 109.4 (104.2–114.1) 101.0 (95.4–107.0) 0.029 intertuberous diameter [mm], median (iqr) 107.4 (101.3–112.5) 109.8 (104.4–115.3) 107.2 (101.5–111.1) 0.281 anteroposterior diameter [mm], median (iqr) 113.3 (106.5–117.4) 113.9 (105.8–119.0) 111.8 (105.5–118.2) 0.363 *computed by mann–whitney u-test abbreviation: c-sec, cesarean section, iqr, interquartile range (25th–75th percentile); nvd, normal vaginal delivery table 2. frequency of different types of pelvis in the study population type of pelvis total (n = 157) nvd (n = 102) c-sec (n = 55) p-value* gynaecoid pelvis (round), n (%) 91 (58.0) 62 (60.8) 29 (52.7) 0.716 android pelvis (transverse oval), n (%) 14 (8.9) 5 (4.9) 9 (16.4) 0.036 anthropoid pelvis (long oval), n (%) 14 (8.9) 8 (7.8) 6 (10.9) 0.564 platypelloid pelvis (flat), n (%) 38 (24.2) 27 (26.5) 11 (20.0) 0.437 *computed by fisher’s exact test 316 original evaluation of the link between pelvimetry based on computed tomography shahla mirgalobayat et al. j contemp med sci | vol. 5, no. 6, november–december 2019: 313–316 conclusion regarding the results of this study, shortening of diameter of the inlet and midpelvis causes the increase in rate of cesarean section and it has been more in females <35 years old. this study need to consider more variables such as economic status and psychological conditions. acknowledgements this research was funded by iran university of medical sciences, fund grant ir.iums.rec.1397.234. the authors would like to thank our colleagues at hazrat-e rasool university hospital. conflict of interest the authors declare no conflict of interest. references 1. aubry s, padoin p, petegnief y, vidal c, riethmuller d, delabrousse e. can three-dimensional pelvimetry using low-dose stereoradiography replace low-dose ct pelvimetry? diagn intervent imaging. 2018;99(9):569–76. 2. caldwell w, moloy hc. anatomical variations in the female pelvis and their effect in labor with a suggested classification. am j obstet gynecol. 1933;26(4):479–505. 3. williams obstetrics. 23th edition.translation: valadan m rs, fathollahi a. tehran: arjmand medical publisher; 2010. volume 2. p. 141–142. 4. s.snell r. clinical_anatomy_by_regions2012. 5. bregar at. indications for caesarean delivery between 1955 and 2005 indicationen fuer kaiserschnitt geburth zwieschen 1955 und 2005. 6. eskew jp, saywell jr, zollinger t, erner b, oser t. trends in the frequency of cesarean delivery. a 21-year experience, 1970–1990. j reprod med. 1994;39(10):809–17. 7. krychowska a, kosińska k, karwan-płońska a. comparison of indications for cesarean section in 1985–86 and 2000–01. analysis of changes. ginekologia polska. 2004;75(12):926–31. 8. tampakoudis p, assimakopoulos e, grimbizis g, zafrakas m, tampakoudis g, mantalenakis s, et al. cesarean section rates and indications in greece: data from a 24-year period in a teaching hospital. clin exp obstet gynecol. 2004;31(4):289–92. 9. shareferad g, fathean z, terane m, mahake b. the survey of pregnant women views about delivery and cesarean according behavioral intention model. ilam uni med sci j. 2007;15:19–23. 10. bani s, seied ra, shamsi gt, ghojazadeh m, hasanpoor s. delivery agents preferences regarding mode of delivery for themslves and pergnant women (obstetrics, gynecologists, midwives). 2010. 11. rafiei m, saei ghare m, akbari m, kiani f, sayehmiri f, sayehmiri k, et al. prevalence, causes, and complications of cesarean delivery in iran: a systematic review and meta-analysis. int j reprod biomed (yazd, iran). 2018;16(4):221–34. 12. azami-aghdash s, ghojazadeh m, dehdilani n, mohammadi m, asl amin abad r. prevalence and causes of cesarean section in iran: systematic review and meta-analysis. iran j public health. 2014;43(5):545–55. 13. klemt a-s, schulze s, brüggmann d, louwen f. mri-based pelvimetric measurements as predictors for a successful vaginal breech delivery in the frankfurt breech at term cohort (frabat). eur jof obstet gynecol reprod biol. 2019;232:10–7. 14. perlman s, raviv-zilka l, levinsky d, gidron a, achiron r, gilboa y, et al. the birth canal: correlation between the pubic arch angle, the interspinous diameter, and the obstetrical conjugate: a computed tomography biometric study in reproductive age women. j matern-fetal neonat med. 2018:1–11. 15. zaretsky mv aj, menintire dd, hatab mr, leveno kj. magnetic resonance imagining pelvimetry and the prediction of labor dystocia. obstet gynecol. 2005;106(5):919–926. 16. kolesova o, kolesovs a, vetra j. age-related trends of lesser pelvic architecture in females and males: a computed tomography pelvimetry study. anat cell biol. 2017;50(4):265–74. 17. federle mp, cohen ha, rosenwein mf, brant-zawadzki mn, cann ce. pelvimetry by digital radiography: a low-dose examination. radiology. 1982;143(3):733–5. 18. morris cw, heggie jc, acton cm. computed tomography pelvimetry: accuracy and radiation dose compared with conventional pelvimetry. austr radiol. 1993;37(2):186–91. 19. ham sj, koops hs, veth rp, van horn jr, eisma wh, hoekstra hj. external and internal hemipelvectomy for sarcomas of the pelvic girdle: consequences of limb-salvage treatment. eur j surg oncol (ejso). 1997;23(6):540–6. 20. liselele hb, boulvain m, tshibangu kc, meuris s. maternal height and external pelvimetry to predict cephalopelvic disproportion in nulliparous african women: a cohort study. bjog int j obstet gynaecol. 2000;107(8):947–52. 21. salk i, cetin m, salk s, cetin a. determining the incidence of gynecoid pelvis using three-dimensional computed tomography in nonpregnant multiparous women. med princip pract. 2016;25(1):40–8. 22. lin hc, sheen tc, tang ch, kao s. association between maternal age and the likelihood of a cesarean section: a population‐based multivariate logistic regression analysis. acta obstet gynecol scand. 2004;83(12):1178–83. fig. 3 scatter plot of (a) true conjugate, (b) obstetrical conjugate, (c) diagonal conjugate, (d) transverse diameter, (e) posterior sagittal diameter, (f) interspinous diameter, (g) intertuberous diameter, and (h) anteroposterior diameter in subjects with ≥35 and <35 years of age who had undergone either nvd (normal vaginal delivery) or c-sec (cesarean section). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.12201904 208 j contemp med sci | vol. 4, no. 4, july-august 2019: 208–213 valproic acid enhances the paclitaxel activity in respiratory tract cancer cells ahmed salim kadhim al-khafaji,1,2 ghaliah alnefaie,2 and ahmed majeed al-shammari3 1department of biology, college of science, university of baghdad, baghdad, iraq. 2department of molecular & clinical cancer medicine, institute of translational medicine university of liverpool, liverpool, uk. 3experimental therapy department, iraqi center for cancer and medical, genetic research, mustansiriyah university, baghdad, iraq. *correspondence to ahmed salim kadhim al-khafaji (email: khafaji@scbaghdad.edu.iq). (submitted: 12 april 2019 – revised version received: 26 april 2019 – accepted: 21 may 2019 – published online: 26 august 2019) objective epigenetic therapies have already been introduced into clinical cancer management. the main objective of this study is to explore the potential of modulating paclitaxel efficiency using two epigenetic modifiers; valproic acid and decitabin. methods the potential sensitisation of lung and oral cancer cells to paclitaxel was examined by two well-known epigenetic modifiers; the dna methyltransferase inhibitor decidabine and histone deacetylase class i inhibitor valproic acid (vpa). the effect epigenetic modifiers were tested using qpcr and pyrosequencing techniques utilising respiratory tract cancerous tissues and cell lines. results the results exhibited that vpa was an effective epigenetic sensitizer for treating lung and head and neck cancerous cells (a549, sklu1 and bhy). about 48 h prior to paclitaxel addition, a significant increase (p < 0.01) of the paclitaxel toxicity was observed when the cancer cells pre-treated with vpa for 48 h and subsequently with paxlitaxel for 72 h. interestingly, mrna expression of aurka was reduced by vpa treatment. the result also demonstrated that p53 status was involved in vpa-mediated paclitaxel sensitisation of hbec cell lines to paclitaxel. vpa seems to potentiate p53 wild type cells (hbec-3kt) to paclitaxel, while p53 hbec knockouts showed less cytotoxic effect of paclitaxel after exposure to 0.5 mm vpa. on the other hand, decitabin was not efficient to sensitise any of the cell lines to paclitaxel when used in either a synchronous or a preceding manner. in addition, the pyrosequencing analysis of the methylation status of the different gene promoters in the lung tumour and normal tissues showed that all the promoters were unmethylated. conclusion it can be concluded that the epigenetic modifier vpa can alter the response of cancer cells to paclitaxel treatment. further investigation is needed to explore the epigenetic mechanism of sensitising cancerous cells to paclitaxel. keywords valproic acid, paclitaxel, lung cancer, head and neck cancer, epigenetic introduction epigenetic therapies have recently been introduced into clinical cancer management.1,2 previous evidence suggested that valproic acid (vpa) enhances paclitaxel cytotoxic effects in cancerous cells3 due to hdac6 deactivation, which results in tubulin hyperacetylation4 and sensitises lung cancer cells to apoptosis.5 the combinatory effect of vpa-paclitaxel was also tested in hnscc tumours.6 aurora kinases also play a transcriptional regulatory role in hdac inhibitors-mediated cytotoxicity in lung cancer cells.7 recent reported data showed that enhancing p53 acetylation due to hdac inhibition leads to enhance paclitaxel-induced apoptosis.8 in addition, a combination of vpa and decitabin has been introduced in clinical trials to treat nsclc patients.1,2 epigenetic therapies and epigenetic sensitization of cancer cells to common chemotherapeutics have come to focus in the last decade.9,10 as sensitization to taxanes was a major objective of this study, the potential of modulating paclitaxel efficiency was examined using two epigenetic modifiers; valproic acid to induce histone acetylation and decitabin to induce global dna hypomethylation. both epigenetic drugs is used as a combination treating patients with lung and head and neck in phase i clinical studies1,2 which demonstrated that decreased dna methylation and induction of histone acetylation were associated with prolonged stable disease for 6 months as a median (4–12 months). materials and methods cell lines nsclc cell lines (a549 and sk-lu-1), hnscc cell line (bhy) and hbec cell lines (hbec-3kt and hbec-3kt-53). paclitaxel exposure cells were seeded in 48-well plates in six biological replicates, cultured in 500 μl of medium and exposed to increasing concentrations of paclitaxel (1–35 nm) for 72 h. growth was measured using the mtt assay. primary lung tumours one hundred and thirty three primary lung tumours [57 adenocarcinomas, 76 squamous cell carcinomas (sqccl)], and 44 adjacent normal tissues (from 20 adenocarcinoma and 24 sqccl patients), have been utilised in this study. the mean age of those patients were 67 (45–82). fifty-six patients were females and 77 males. rna extraction total rna was extracted from cell lines and primary lung tumours using mirneasy mini kit (qiagen), and then quantified by thermo scientific nanodrop 2000 spectrophotometer. reverse transcription was undertaken using high capacity cdna reverse transcription kits (life technologies). issn 2413-0516 original a.s.k. al-khafaji et al. 209j contemp med sci | vol. 4, no. 4, july-august 2019: 208–213 vpa potentiates the ptx effect in cancer cells qpcr predesigned taqman expression assays (life technologies) were employed using vic-labeled actb as endogenous control. real-time pcr assays were performed in triplicate. the genes tested were: aurka, aurkb, aurkc, ckap5, tpx2, ttk, kif11, dlgap5, tubb and tubb3. dna methylation analysis dna extraction dna extraction from cell lines and primary lung tumours were performed using dneasy® blood and tissue kit (qiagen) using dneasy 96 protocol for purification of total dna from tissues and spin-column protocol for purification of total dna from cell lines. dna methylation in order to generate positive control for methylation-specific pcr or bisulfate sequencing, reaction of four units sssi per µg of unmethylated dna for 2 h was prepared according the modified manufacturing protocol. bisulphite treatment of dna to investigate the methylation status of the different gene promoters tested in this study, 500 ng dna from primary tissues was bisulphite treated utilising the ez-96 dna methylation-gold™ kit (zymo research) following the manufacturer’s protocol. pyrosequencing methylation analysis pyrosequencing (psq) is a method that can identify the sequence from small dnas efficiently and with high fidelity. the samples were prepared for psq. the targeted dna sequence was amplified by pcr using forward biontinylated (fb), reverse (r) and sequencing (s) primers. the primers were designed by the pyromark assay design 2.0 software. decitabine efficiency this was achieved by pyrosequencing-methylation analysis of the line-1.2 (genebank accession no m80343) retrotransposon (daskalos 2009). forward primer: bio-tagggagtgttagatagtgg, reverse primer: aactccctaaccccttac, sequencing primer: caaataaaa caatacctc. pcr amplification was performed using qiagen hotstartaq plus master mix kit. statistical analysis the kolmogorov–smirnov test, mann–whitney test and wilcoxon test were employed for statistical analysis using spss 20. bonferroni correction was used to adjust for multiple comparisons. the ic50 values were calculated using graphpad prism 5. kaplan–meier curves were constructed for survival analysis and the log-rank test was used to examine the differences between groups. results based on aforementioned reported data, the epigenetic role of the histone acetylator (vpa) and dna methylator (decitabin) in sensitising rtc cells to paclitaxel was investigated. the cellular response to vpa was next examined alone in order to select the concentrations below ic50 for further investigation of vpa ability to sensitise cancerous cells to taxanes. mtt analysis of vpa exposure of lung cell lines (a549 and sklu1) and the second most paclitaxel resistant oral cancer cell line (bhy) demonstrated that these cells are resistant to very high vpa micro-molar concentrations (fig. 1) with ic50s of 6.63 mm of a549, 20 mm of sklu1 and 2.1 mm of bhy) (table 1) at 95% ci. in order to examine the ability of valproate to potentiate the anti-tumour efficacy of paclitaxel in controlling cellular viability, two different doses of valproate 0.5 and 1 mm that are below ic50s of all these three cell lines were used. a fixed dose of paclitaxel (10 nm) was utilised to test our hypothesis. this was also under the ic50s of the examined cell lines; 13.6 nm of a549, 16.7 nm of sklu1 and 14 nm of bhy. the growth inhibitory effects of 1 mm vpa and 10 nm paclitaxel were determined as optimal doses utilised either in combination or as successive treatments of the cell lines, a549, sklu1 and bhy. the synchronous treatment of vpa and paclitaxel produced only a minor additive effect (data not shown). in contrast, when vpa used to treat the cell for 48 h prior to paclitaxel addition, a significant increase of the paclitaxel toxicity was observed in the subsequent 72 h (fig. 2). interestingly, mrna expression of aurka in bhy cell line was significantly reduced to around 65% after treatment with 1 mm vpa for 48 h (fig. 3). fig. 1 mtt line graph showing the cellular survival rates of lung (a549 and sklu1) and hnscc (bhy ) cell lines to vpa. error bars represent 95% confidence intervals. table 1 the data demonstrating the ic50 values of vpa in a549, sklu1 and bhy cell lines and their respective 95% ci. the results demonstrate that sklu1 cell line is the most resistant to vpa with ic50 value 20 mm vpa, while bhy is the most sensitive one ic50 value 2.1 mm vpa cell line vpa ic 50 (nm) 95% ci a549 6.63 5.77–7.63 sklu1 20.00 11.80–33.92 bhy 2.10 19.40–2.27 original 210 j contemp med sci | vol. 4, no. 4, july-august 2019: 208–213 vpa potentiates the ptx effect in cancer cells a.s.k. al-khafaji et al. fig. 2 bar charts of cytotoxic effects of 48-hour treatment with 1 mm vpa flowed by 72 h of 10 nm paclitaxel on a549, bhy and sklu1 cell lines. interestingly, sklu1 cell line exhibited more response to paclitaxel after 48 h exposure to vpa. bhy showed different trend of response to treatment of vpa and paclitaxel separately compared to a549 and sklu1 cell lines. error bars represent 95% confidence fig. 3 aurka mrna expression in vpa-treated bhy cells with 1 mm concentration compared with non-treated in comparison with hbec-3kt control. vpa treatment seems to reduce aurka expression to around 65%. error bars were represented 95% confidence then it was examined how the status of tumour suppressor gene p53 could affect the paclitaxel sensitisation of hbec cell lines to paclitaxel. the results demonstrated that the vpa exhibited more efficiency in sensitising p53 wild type hbec cells to paclitaxel than that exhibited in sensitising p53 knockouts and thus p53 expression seems to increase the cytotoxic effect of paclitaxel after course exposure to 0.5 mm vpa although pre-treatment of hbecs with 1 mm vpa shows different trend (fig. 4). the efficiency of decitabine treatment of a549 was determined at different concentrations (50, 100 and 200 µm) by measuring the global methylation levels (line-1 element) (fig. 5). this agent showed a dose-dependent efficiency to demethylate a549 cellular dna (fig. 6). although decitabine was efficient in reducing global line methylation, it did not sensitise any of the cell lines to paclitaxel when used either in a synchronous (fig. 7) or in a preceding manner (data not shown). fig. 4 bar charts of cytotoxic effects of 48 h treatment with 1 and 0.5 mm vpa followed by 72 h of 10 nm paclitaxel on hbec-3kt (a) and hbec-3kt-p53 (b) cell lines. p53 wild type hbec cells showed more response to 10 nm paclitaxel after 48 h exposure to 0.5 mm vpa compared with p53 knockouts. however, increasing vpa concentration to 1 mm no more effect in hbec-3kt as opposed to more effect in hbec-3kt-p53. error bars represent 95% confidence intervals. original a.s.k. al-khafaji et al. 211j contemp med sci | vol. 4, no. 4, july-august 2019: 208–213 vpa potentiates the ptx effect in cancer cells fig. 5 pyrograms of line-1 global methylation analysis demonstrating the cellular dna methylation status of a549 cell line in the absence (a) and presence (b) of decitabine at 200 µm. fig. 6 the bar chart demonstrating the change in the methylation status of line-1 following treatment of a549 with the demethylating agent decitabine at different concentration (0, 50, 100 and 200 µm). the results showed that reduction of the methylation level correlated with increasing decitabine dose. in order to provide insight into the inability of decitabine to sensitise cell lines to paclitaxel, the methylation status of the different gene promoters tested in this study was investigated. the pyrosequencing analysis demonstrated that none of the gene promoters examined in this study demonstrated altered methylation status; in fact all promoters were unmethylated in all tumour and normal tissues tested (fig. 8). discussion the data obtained demonstrated that pre-treatment of three different rtc cell lines with vpa sensitised these cells to paclitaxel, while decitabin has no such sensitising effect. the maximum concentration that has been used in this study was corresponded to levels in the plasma of patient treated for epilepsy that ranged from 30 to 111 mg/l as opposed to 0.2–0.8 mm and exhibited low risk side effects,11 while resulting in histone acetylation.12 these findings are consistent with chen et al.,13 who established that vpa enhanced paclitaxel response in resistant human lung adenocarcinoma cells but in dose-dependent manner, but in contrast to erlich et al.,6 who could original 212 j contemp med sci | vol. 4, no. 4, july-august 2019: 208–213 vpa potentiates the ptx effect in cancer cells a.s.k. al-khafaji et al. fig. 7 mtt line graphs showing the sensitivity of a549 (a), sklu1 (b) and (c) skmes1 cell lines to paclitaxel in the presence of differing concentrations of decitabine (0, 50 and 100 µm). the data showed no significant difference in cellular response to treatment with paclitaxel alone or in combination with decitabine. error bars were represented 95% confidence intervals. fig. 8 representative pyrograms showing dna demethylation status of aurka gene promoter in (a) tumour and (b) normal lung samples. the figures show that the gene promoters were unmethylated in both malignant and normal tissues of the lung. original a.s.k. al-khafaji et al. 213j contemp med sci | vol. 4, no. 4, july-august 2019: 208–213 vpa potentiates the ptx effect in cancer cells not deduce that vpa can potentiate the cytotoxic effect to paclitaxel in hnscc cells. this inconsistency probably exists because the researchers did not try to pre-treat the cells with vpa prior paclitaxel treatment rather they examined only the vpa-paclitaxel combination. however, the findings demonstrated a minor effect of vpa and paclitaxel in rtc cells. this epigenetic sensitisation of cancer cells to paclitaxel might result through induction of apoptosis due to enhancement of tubulin acetylation.4 although paclitaxel-induced apoptosis in nsclc is well documented and p53-independent,14–17 following the finding that vpa pre-treatment potentiates paclitaxel cytotoxic effect in rtc cell lines. the association of vpa-mediated paclitaxel cytotoxicity with p53 status was also investigated. the results indicated that p53 status was a determinant of epigenetic sensitisation of hbec cells to paclitaxel cytotoxicity. it was evident that 0.5 mm vpa enhanced paclitaxel activity in p53 wild type hbec cells but to a lesser extent in the p53-knockout derivatives. however, increased vpa dose to 1 mm showed similar paclitaxel sensitising effect in both p53 wild type and p53 null cells. further investigation is required to provide compelling evidence on the exact mechanism of p53 involvement on vpa-based sensitization of paclitaxel. the present study also demonstrated that vpa exposure of bhy cells led to the reduction of aurka mrna expression. this suggests that aurka transcription is under epigenetic control.18 while the mechanism behind vpa-mediated sensitisation to paclitaxel is still unclear, the reduction of aurka expression may be one of the mediators due to the fact that higher levels of aurka transcripts are associated with poor prognosis of nsclc.19 conclusion in conclusion, the results indicate that hdac inhibitors could be beneficial in sensitising rtc cells to paclitaxel, which is a very common and inexpensive chemotherapeutic agent. such sensitisation could lead to lowering the effective dose of paclitaxel and subsequently reducing the adverse effects of this drug to the patient. additional preclinical and clinical evidence is required to provide further support to our observation. the great advantage of vpa is that it is in routine clinical use for many years demonstrating minor side effects. further research is required to establish the exact molecular mechanisms modulating this epigenetic sensitisation of cancer cells to paclitaxel. acknowledgment this project is co-funded by the university of baghdad and ministry of higher education and scientific research (mohesr)/iraq alongside the roy castle lung cancer foundation, uk. the authors would like to acknowledge triantafillos liloglou for critically reading the manuscript. conflicts of interest none. ■ references 1. braiteh f, soriano ao, garcia-manero g, hong d, johnson mm, silva lde p, et al. phase i study of epigenetic modulation with 5-azacytidine and valproic acid in patients with advanced cancers. clin cancer res. 2008;14:6296–6301. 2. chu bf, karpenko mj, liu z, aimiuwu j, villalona-calero ma, chan kk, et al. phase i study of 5-aza-2’-deoxycytidine in combination with valproic acid in non-small-cell lung cancer. cancer chemother pharmacol. 2013;71:115–121. 3. roy choudhury s, karmakar s, banik nl, ray sk. valproic acid induced differentiation and potentiated efficacy of taxol and nanotaxol for controlling growth of human glioblastoma ln18 and t98g cells. neurochem res. 2011;36:2292–2305. 4. catalano mg, poli r, pugliese m, fortunati n, boccuzzi g. valproic acid enhances tubulin acetylation and apoptotic activity of paclitaxel on anaplastic thyroid cancer cell lines. endocr relat cancer. 2007;14:839–845. 5. tesei a, brigliadori g, carloni s, fabbri f, ulivi p, arienti c, et al. organosulfur derivatives of the hdac inhibitor valproic acid sensitize human lung cancer cell lines to apoptosis and to cisplatin cytotoxicity. j cell physiol. 2012;227:3389–3396. 6. erlich rb, rickwood d, coman wb, saunders na, guminski a. valproic acid as a therapeutic agent for head and neck squamous cell carcinomas. cancer chemother pharmacol. 2009;63:381–389. 7. bailey vj, easwaran h, zhang y, griffiths e, belinsky sa, herman jg, et al. ms-qfret: a quantum dot-based method for analysis of dna methylation. genome res. 2009;19:1455–1461. 8. kim ch. druggable targets of squamous cell lung cancer. tuberc respir dis (seoul). 2013;75:231–235. 9. timofeeva mn, hung rj, rafnar t, christiani dc, field jk, bickeböller h, et al. influence of common genetic variation on lung cancer risk: meta-analysis of 14 900 cases and 29 485 controls. hum mol genet. 2012;21:4980–4995. 10. jones se, erban j, overmoyer b, budd gt, hutchins l, lower e, et al. randomized phase iii study of docetaxel compared with paclitaxel in metastatic breast cancer. j clin oncol. 2005;23:5542–5551. 11. zighetti ml, fontana g, lussana f, chiesa v, vignoli a, canevini mp, et al. effects of chronic administration of valproic acid to epileptic patients on coagulation tests and primary hemostasis1. epilepsia. 2015;56: e49–e52. 12. tremolizzo l, difrancesco jc, rodriguez-menendez v, riva c, conti e, galimberti g, et al. valproate induces epigenetic modifications in lymphomonocytes from epileptic patients. prog neuropsychopharmacol biol psychiatry. 2012;39:47–51. 13. chen j, liu j. spatial-temporal model for silencing of the mitotic spindle assembly checkpoint. nat commun. 2014;5:4795. 14. king tc, akerley w, fan ac, moore t, mangray s, hsiu chen m, et al. p53 mutations do not predict response to paclitaxel in metastatic nonsmall cell lung carcinoma. cancer 2000;89:769–773. 15. vogt u, zaczek a, klinke f, granetzny a, bielawski k, falkiewicz b. p53 gene status in relation to ex vivo chemosensitivity of non-small cell lung cancer. j cancer res clin oncol. 2002;128:141–147. 16. das gc, holiday d, gallardo r, haas c. taxol-induced cell cycle arrest and apoptosis: dose-response relationship in lung cancer cells of different wildtype p53 status and under isogenic condition. cancer lett. 2001;165:147– 153. 17. duarte ml, de moraes e, pontes e, schluckebier l, de moraes jl, hainaut p, et al. role of p53 in the induction of cyclooxygenase-2 by cisplatin or paclitaxel in non-small cell lung cancer cell lines. cancer lett. 2009;279:57– 64. 18. zhang xh, rao m, loprieato ja, hong ja, zhao m, chen gz, et al. aurora a, aurora b and survivin are novel targets of transcriptional regulation by histone deacetylase inhibitors in non-small cell lung cancer. cancer biol ther. 2008;7:1388–1397. 19. al-khafaji ask, marcus mw, davies mpa, risk jm, shaw rj, field jk, et al. aurka mrna expression is an independent predictor of poor prognosis in patients with non-small cell lung cancer. oncol lett. 2017;13: 4463–4468. dx.doi.org/10.22317/jcms.08201905 original 42 j contemp med sci | vol. 2, no. 6, spring 2016: 42–44 research objectives this study aimed to assess the relationship between serum inflammatory marker of high-sensitivity c-reactive protein (hs-crp) with the presence and severity of angiographically evaluated coronary artery disease (cad). methods this cross-sectional study was conducted in cardiac catheterisation unit at al-hussein medical city, al-hussein teaching hospital/ karbala from november 2014 to september 2015. it included 76 patients (49 males and 27 females) who presented with the signs and symptoms of cad and has undergone angiography. serum levels of hs-crp were measured in patients with cad before entering into the catheterisation room. after coronary angiography was done for all patients, the extent and severity of cad was correlated with serum levels of hs-crp. the extent of cad was determined by angiography according to the number of coronary arteries involved and degree of narrowing in coronary artery diameter. the serum level of hs-crp was measured by elisa technique. results the mean ± sd age of patients was 57.76 ± 9.69 (ranged 35–79) years, 64.4% males and 35.6% females. regarding the angiographic finding in patient group, it was normal in 22 patients (28.9%), single vessel involvement in 18 patients (23.7%), two vessels disease in 14 patients (18.4%) and three vessels disease in 16 patients (21.1%). the left main stem disease (lmd) was found in 6 patients (7.9%). the extent of cad as obtained from angiographic finding had significant correlation with the serum level of hs-crp (r = 0.736), (p = 0.000000001). conclusion there is a significant strong positive correlation between the extent of cad and the serum level hs-crp. keywords high sensitive c-reactive protein (hs-crp), coronary arteries disease (cad), body mass index (bmi) association between hs-crp levels and the severity of coronary atherosclerosis fadhil jawad al-tu’maa, zainab abdul-hussein abd-yasera, karim obais al-naffib introduction coronary heart disease is the most prevalent chronic disease and the main leading cause of death in the world, with more than half a million newly diagnosed coronary artery disease (cad) patients each year.1,2 cardiac catheterisation and coronary angiography are often important for definitive evaluation of coronary artery anatomy, the presence of evaluation of cads and the presence of interventional therapy.3 the use of biomarkers as a tool for the diagnosis of plaque rupture has generated great interest in clinicians due to their promise to either ‘rule-in’ or ‘rule-out’ acute coronary syndromes (acs).4 there is strong evidence that cardiovascular conditions are linked to inflammation. likewise, there is a role of inflammation in the pathogenesis of atherosclerosis.5 crp is a sensitive, but non-specific acute-phase reactant, which (when elevated to ≥3 mg/l) is a predictor of cardiovascular events in otherwise asymptomatic individuals.6 increases in crp levels detected by assays with expanded sensitivity to very low levels of crp, so-called high-sensitivity c-reactive protein (hs-crp), showed a strong correlation as an independent risk factor for future cardiac events. importantly, crp elevation is not only related directly to plaque burden, but also rather to plaque inflammation and instability. though crp can be elevated in other conditions, it possesses several traits that make it an attractive biomarker: (a) it is highly stable in plasma with a limited coefficient of variation, (b) it has been demonstrated to have predictive capacity in multiple ethnic groups, (c) it predicts both short and long-term outcomes and (d) it provides independent predictive value in asymptomatic individuals, high-risk patients and also in disease states such as stroke, peripheral arterial disease and sudden death.7 blood measurements of hs-crp are often performed to assess the risk of future heart disease. it has also been suggested that hs-crp can be used to target therapy and tailor-risk modification to prevent cardiovascular disease (cvd).8 in most of the studies reported, the association of hs-crp with cardiovascular risk has been found to be highly significant in global risk-assessment programmes.9 this study aimed to assess the relationship of serum inflammatory marker hs-crp, with the presence and severity of angiographically evaluated cad. materials and methods this study was conducted at the departments of medicine (angiographic department) in al-hussein teaching hospital/ holy karbala, iraq and in the department of biochemistry, college of medicine, university of karbala from november 2014 to september 2015. in this cross sectional study, 76 patients (49 males and 27 females) were studied who had undergone angiography and were found to have cad. all the selected cases met the inclusion criteria of the study. inclusion criteria consisted of adult patients of both sexes with ischemic heart disease, who had attacks of angina or myocardial infarction and had undergone coronary angiography. exclusion criteria included stable angina, unstable angina, acute mi, acute or chronic renal diseases, thyroid disorders, recent stroke, diabetic ketoacidosis, non-ketotic hyperosmolar diabetes, any recent surgery in the last 2 months, tumours and autoimmune disease. other parameters measured include blood sugar, blood pressure, age, gender and smoking state to compare the effect of these factors on the level of hs-crp levels. the classification of atherosclerotic patient depends on the extent of cad. there are four main coronary arteries: left issn 2413-0516 adepartment of biochemistry, college of medicine, university of kerbala, holy kerbala, iraq. bdepartment of internal medicine, college of medicine, university of kerbala, holy kerbala, iraq. correspondence to fadhil jawad al-tu’ma (email: f_altoma_56@yahoo.com). (submitted: 9 january 2016 – revised version received: 27 february 2016 – accepted: 7 april 2016 – published online: 26 june 2016) 43j contemp med sci | vol. 2, no. 6, spring 2016: 42–44 research association of hs-crp levels with cadfadhil jawad al-tu’ma et al. main coronary artery, left anterior descending artery (lad), left circumflex (lcx) and right coronary arteries (rca) were assessed. all patients underwent coronary angiography, and the results were collected from the catheterisation laboratory according to the patient’s name and file number. the angiographic results concern the presence of significant (lesions more than or equal to 70% diameter stenosis for coronary arteries and more than or equal to 50% diameter stenosis for left main coronary artery by visual estimation)10 coronary artery lesion and the numbers of arteries involved by a significant lesion and classified as: • normal (no significant lesion) • left main stem (lms) disease • one vessel involvement • two vessels involvement • three vessels involvement. after explaining the aims of the study and obtaining the patients’ approval for participation, 1 ml blood samples were taken from the patients and were transferred to the laboratory after clotting, hs-crp was measured using elisa assay (hs-crp elisa cat. no. de740011). coronary angiography is performed under local anaesthesia. the procedure is sterile, and all potential access sites must be disinfected, shaved and sterilised. at the beginning of the procedure, the patient lies down in supine position on the cardioangiograph table, and is prepared for the procedure in sterile conditions. coronary angiography is performed with the patient in the fasting state. results the mean ± sd of patient age was 57.76 ± 9.69 (ranged 35–79) years, in which 64.4% of males and 35.6% of females. regarding the angiographic finding in patient group, there was no significant finding in 22 patients (28.9%), single vessel involvement in 18 patients (23.7%), two vessels disease in 14 patients (18.4%) and three vessels disease in 16 patients (21.1%). the lmd was found in 6 patients (7.9%), while the remaining 70 patients (92.1%) were free of lmd. these findings are shown in table 1. the atherosclerotic patients presented with higher hs-crp that mean significant correlation of the extent of coronary atherosclerosis disease with hs-crp. there was no significant difference in hs-crp titer between three vessel disease and lmd (p = 0.903). while, there is a significant difference of that titer between any two of the other angiographic findings (p = 0.000000001). there is strong positive association between the extent of cad and hs-crp titer (r = 0.736) as shown in table 2. table 2. the difference of hs-crp titer between angiographic findings angiographic findings (hs-crp mean ± sd) significance of difference (p value) no significant finding (1.01 ± 1.07) one vessel disease (2.64 ± 2.14) 0.006 no significant finding (1.01 ± 1.07) two vessel disease (4.33 ± 2.01) 0.000 no significant finding (1.01 ± 1.07) three vessel disease (6.01 ± 2.14) 0.000 no significant finding (1.01 ± 1.07) left main stem disease (6.12 ± 1.68) 0.000 one vessel disease (2.64 ± 2.14) two vessel disease (4.33 ± 2.01) 0.012 one vessel disease (2.64 ± 2.14) three vessel disease (6.01 ± 2.14) 0.000 one vessel disease (2.64 ± 2.14) left main stem disease (6.12 ± 1.68) 0.000 two vessel disease (4.33 ± 2.01) three vessel disease (6.01 ± 2.14) 0.014 two vessel disease (4.33 ± 2.01) left main stem disease (6.12 ± 1.68) 0.049 three vessel disease (6.01 ± 2.14) left main stem disease (6.12 ± 1.68) 0.903 table 3. correlation between hs-crp titer and extent of cad angiographic finding no. hs-crp titer p value (between groups) pearson r (correlation)mean std. deviation non 22 1.01 1.07 0.000000001 0.736 one vessel disease 18 2.64 2.14 two vessel disease 14 4.33 2.01 three vessel disease 16 6.01 2.14 left main stem disease 6 6.12 1.68 total 76 3.46 2.68 table 1. angiographic findings of the studied group (n = 76) angiographic findings no. % non (normal or <70% stenosis) 22 28.9 one vessel disease 18 23.7 two vessel disease 14 18.4 three vessel disease 16 21.1 left main stem disease (>50% stenosis) 6 7.9 total 76 there was a significant positive correlation between the extent of cad and serum level of hs-crp (r = 0.736), (p = 0.000000001) which means that the higher levels of hs-crp are found in patients with more extensive cad as shown in table 3. discussion the results obtained indicated that hs-crp level is a good marker of the presence and extent of coronary atherosclerosis disease. this study was done to non-invasively assess the extent and severity of coronary atherosclerosis disease patients by measure serum level of hs-cpr. numerous studies have provided the evidence that inflammation has important role in the occurrence and the development of coronary vessel disease.11–13 the statical correlation between extent of cad and level of hs-crp are shown in fig. 1. accordingly, there are several mediators of the inflammatory response, including acute-phase 44 j contemp med sci | vol. 2, no. 6, spring 2016: 42–44 association of hs-crp levels with cad research fadhil jawad al-tu’ma et al. proteins, cytokines and cellular adhesion molecules have been evaluated as potential indicators of the risk of a first acute atherothrombotic event, as well as of recurrent complications after initial presentation.14 as the prototypical acute-phase reactant, hs-crp has been the focus of much of the clinical investigation.15 this can be explained as that, hs-crp is an acute-phase protein marker that can demonstrate the subclinical inflammatory states detecting lower serum levels of crp. there are several advantages in hs-crp measurements related to coronary vessel disease. the first advantage is that, it is a stable compound, and it can be measured at any time of the day without special relevance to biological clock of the day.16 while, the other markers such as lipids and il-6 exhibit circadian rhythm and are related to meals also. thus, we can perform hs-crp testing in clinical settings at any time in a day.17 cushman et al. have revaluated the prevalence and correlates between increased hs-crp and reported a significant effect of hs-crp measurement on coronary heart disease risk reclassification. they observed that with the inclusion of hs-crp in their testing data, the reynolds risk score classified the population differently compared to the new framingham risk scores.18 this observation is in agreement to our study regarding hs-crp and its significant correlation with the presence and the severity of coronary vessel disease.19 the publication of that article as this study was being performed indicates the novelty of the subject. in another study performed on 140 patients with cad in india, the researchers found a positive correlation between the serum levels of hs-crp and the severity of coronary atherosclerosis in the patients without diabetes mellitus and since only non-diabetic patients who had coronary atherosclerosis were studied, the findings could not be generalised.20 to the best of our knowledge, no such study has been done in iraq till now. the novelty of our study, its controversy and its strength concerning the effect of regional factors with the regard to life conditions make the study distinguished. the possible limitations of our study are limited the number of subjects and cross-sectional design. the prospective studies on large scale are needed to explore the true pathogenic role of hs-crp in assessing cardiovascular risk as not indicator for atherosclerotic extent. additionally, the study was carried out in a tertiary center, so the findings could not be generalised to the whole population. conclusion we conclude that the coronary atherosclerosis patient has high level of hs-crp on comparing with healthy people. also the level of the increase in hs-crp indicates the level of cad extent.  fig. 1 correlation between extent of cad and level of hs-crp. references 1. roger vl, go as, lloyd-jones dm, benjamin ej, berry jd, borden wb, et al. heart disease and stroke statistics: 2012 update: a report from the american heart association. circulation. 2012;125:e2–e220. doi: 10.1161/ cir.0b013e31823ac046 pmid: 22179539 2. xu jq, kochanek kd, murphy sl, tejada-vera b. deaths: final data for 2007. natl vital stat rep. 2010;58(19);1–6. 3. kolansky dm. acute coronary syndromes: morbidity, mortality, and pharmacoeconomic burden. am j manag care. 2009;15(2):s36–s41. pmid: 19355807 4. gotto am. role of c-reactive protein in coronary risk reduction: focus on primary prevention. am j cardiol. 2007;99:718–25. pmid: 17317380 5. osman r, l’allier pl, elgharib n, tardif jc. critical appraisal of c-reactive protein throughout the spectrum of cardiovascular disease. vasc health risk manag. 2006;2(3):221–237. pmid: 17326329 6. ridker pm, cook nr. biomarkers for prediction of cardiovascular events. n engl j med. 2007;356:1472–1475. doi: 10.1056/nejmc070079 7. hein tw, singh u, vasquez-vivar j, devaraj s, kuo l, jialal i. human c-reactive protein induces endothelial dysfunction and uncoupling of enos in vivo. atherosclerosis. 2009;206:61–8. doi: 10.1016/j. atherosclerosis.2009.02.002 pmid: 19268941 8. heeschen c, hamm cw, bruemmer j, simoons ml. predictive value of c-reactive protein and troponin t in patients with unstable angina: a comparative analysis. the chimeric c7e3 antiplatelet therapy in unstable angina refractory to standard treatment (capture) investigators. j am coll cardiol. 2000;35:1535–1542. pmid: 10807457 9. danesh j, whincup p, walker m, lennon l, thomson a, appleby p, et al. low grade inflammation and coronary heart disease: prospective study and updated meta-analyses. bmj. 2000;321:199–204. pmid: 10903648 10. walldius g, jungner i. rationale for using apolipoprotein b and apolipoprotein a-i as indicators of cardiac risk and as targets for lipidlowering therapy. eur heart j. 2005;26:210–2. pmid: 15618031 11. hak ae, stehouwer cd, bots ml, polderman kh, schalkwijk cg, westendorp ic, et al. associations of c-reactive protein with measures of obesity, insulin resistance, and subclinical atherosclerosis in healthy, middle-aged women. arterioscler thromb vasc biol. 1999;19:1986–1991. pmid: 10446082 12. ridker pm, rifai n, pfeffer ma, sacks fm, moye la, goldman s, et al. inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels: cholesterol and recurrent events (care) investigators. circulation. 1998;98:839–844. pmid: 9738637 13. lagrand wk, visser ca, hermens wt, niessen hw, verheugt fw, wolbink gj, et al. c-reactive protein as a cardiovascular risk factor: more than an epiphenomenon? circulation. 1999;100:96–102. pmid: 10393687 14. blake gj, ridker pm. c-reactive protein and other inflammatory risk markers in acute coronary syndromes. j am coll cardiol. 2003;41:37s–42s. pmid: 12644339 15. liuzzo g, biasucci lm, gallimore jr, grillo rl, rebuzzi ag, pepys mb, et al. the prognostic value of c-reactive protein and serum amyloid a protein in severe unstable angina. n engl j med. 1994;331:417–424. pmid: 7880233 16. ockene is, matthews ce, rifai n, ridker pm, reed g, stanek e. variability and classification accuracy of serial high-sensitivity c-reactive protein measurements in healthy adults. clin chem. 2001;47:444–450. pmid: 11238295 17. meier-ewert hk, ridker pm, rifai n, price n, dinges df, mullington jm. absence of diurnal variation of c-reactive protein levels in healthy human subjects. clin chem. 2001;47:426–430. pmid: 11238292 18. cushman m, mcclure la, howard vj, jenny ns, lakoski sg, howard g. implications of increased c-reactive protein for cardiovascular risk stratification in black and white men and women in the us. clin chem. 2009;55(9):1627–1636. doi: 10.1373/clinchem.2008.122093 pmid: 19643839 19. habib ss, abdel-gader am, kurdi mi, al-aseri z, soliman mm. lipoproteina(a) is a feature of the presence, diffuseness, and severity of coronary artery disease in saudi population. saudi med j. 2009;30:346–352. pmid: 19271061 20. mahajan n, malik n, bahl a, sharma y, dhawan v. correlation among soluble markers and severity of disease in non-diabetic subjects with pre-mature coronary artery disease. mol cell biochem. 2009;330(1–2):201–9. doi: 10.1007/s11010-009-0134-1 pmid: 19412573 262 j contemp med sci | vol. 6, no. 6, november–december 2020: 262–266 original issn 2413-0516 introduction glucose-6-phosphate dehydrogenase (g6pd) deficiency is known as a disorder transmitted by an x-linked enzymatic mutation in the g6pd gene. it has been known that g6pd is the most common human enzymatic defect, represented in around 400 million humans around the world. the enzyme catalyzes pentose phosphate pathway’s first reaction, offering antioxidant protection to the cells counteracting any oxidative stress. hence, individuals with deficiency of this enzyme are unable to protect red blood cells (rbc) against myriad of oxidative stresses.1 the most recent worldwide assessment of the disease prevalence had shown that the middle eastern region stands second in prevalence (7.2%).2 g6pd deficiency is common among iraqis; with an estimated prevalence around 6% in the middle part (baghdad),3 and 10.9% in northern iraq.4 the disease is manifested silently, unless an affected individual eats fava beans or takes some hemolysis-inducing medicines.5 the presentation of this disease depends mainly on the degree of deficiency and the severity of hemolytic attack.1 it presents usually with a sudden episode of acute hemolysis including fatigue, pallor, and dark colored urine.6g6pd deficiency represents a major health problem in iraq; particularly during the harvest of fava beans, which peaks during spring season. moreover, the diagnosis of g6pd deficiency continues to be a challenge for the health department, especially in regions where diagnostic facilities are not available and growing fava beans are widespread. this study was conducted to study the profile of g6pd and to examine the presence of any demographic, clinical, or biochemical factors that may be associated with the severity of hemolysis in patients treated at emergency room of children welfare teaching hospital in medical city/baghdad. patients and methods patients and design this cross-sectional study was conducted at the emergency room of children welfare teaching hospital in medical city, baghdad, iraq. this hospital is a tertiary center and one of the largest hospitals in baghdad with over 250 beds for patients. a purposive sampling method was used to recruit 57 patients who were visiting the pediatric emergency department from march to may 2017 with a presentation of acute hemolytic episode of g6pd. this study included patients who are younger than 14 years, were previously diagnosed with g6pd deficiency via g6pd enzymatic assay, and had a history of ingestion of fava beans or with the first presentation of a hemolytic hemolysis in children with glucose-6-phosphate dehydrogenase deficiency after ingestion of fava beans; facts predicting severity hasanein h. ghali1, doaa alem al mamoori2 1department of pediatrics, college of medicine, university of baghdad, children welfare teaching hospital, baghdad medical city, baghdad, iraq. 2department of emergency medicine, baghdad medical city, baghdad, iraq. corresponding author: hasanein h. ghali (e-mail: hasaneinghali@comed.uobaghdad.edu.iq) (submitted: 02 september 2020 – revised version received: 24 september 2020 – accepted: 26 october 2020 – published online: 26 december 2020) abstract objectives: this study aimed to assess the demographic, clinical, and biochemical characteristics as predictors of hemolysis after ingesting fava beans. methods: a cross-sectional study was undertaken. a total of 57 patients with glucose-6-phosphate dehydrogenase (g6pd) deficiency were recruited from the emergency department of children welfare teaching hospital, medical city. data were collected using a specially designed form. a purposive sampling method was used to recruit 57 patients (49 males and 8 females) who were visiting the pediatric emergency department from march to may 2017 with a presentation of acute hemolytic episode of g6pd. patients were classified into mild or severe hemolysis groups based on their hemoglobin level at the time of admission. results: younger age group patients tend to present with the severe form of hemolysis (3.59 years with a p value of 0.001). no significant gender susceptibility between both types of hemolysis. the urban area-based living individuals tend to present with mild hemolysis while those from rural areas tends to present with more severe episodes of hemolysis (p value 0.001). there was a significant correlation between the type of fava bean ingestion (fresh or dried) and the severity of hemolysis, those who presented with more severe hemolysis usually had a history of ingestion of fresh type of fava bean. eight individuals of severe type recorded previous episodes of hemolysis while three individuals of the mild type recorded previous episodes (p value 0.001). family history of g6pd was significant in 88.2% of individuals with the severe form (p value 0.005). conclusion: this study is aimed to report several factors that might predict the severity of hemolysis among patients with g6pd deficiency. younger age, residence in a rural region, ingestion of fresh fava beans, and history of frequent hemolysis incidents are predictors of developing severe hemolysis among children admitted to the emergency room of children welfare teaching hospital in medical city/ baghdad. keywords: favism, g6pd deficiency, severe haemolysis, iraqi children 263 original hemolysis in children with glucose-6-phosphate dehydrogenase deficiency after ingestion of fava beanshasanein h. ghali j contemp med sci | vol. 6, no. 6, november–december 2020: 262–266 episode of the disease. the medical diagnosis of g6pd deficiency was confirmed according to the medical history, clinical presentation at the time of admission and/or laboratory investigations. patients presented with sudden onset of pallor, jaundice, dark colored urine, with features of hemolysis (increased bilirubin, low hemoglobin, increased reticulocyte count if available, and increased urinary urobilinogen if available) were further investigated to exclude other hereditary hemolytic disorders and/or acquired autoimmune hemolytic anemia. those patients with suspicious blood film results and negative coombs test results were asked to do a g6pd enzymatic assay after hemolysis resolves. because the g6pd enzyme can be falsely normal during a hemolysis episode, the enzymatic activity is usually assessed 1–3 months after a crisis for those with negative history of diagnosis. information about recent infection or ingestion of drugs that may induce hemolysis was also elicited. patients who had ingested these drugs were excluded. the legal guardians of eligible patients were approached and invited to participate in this study. in accordance with the helsinki declaration of ethical principles,7 informed verbal consent was obtained from the legal guardians before enrolment in this study. data collection and quantification relevant demographic information about the patients were elicited, including age, gender, and residence (urban, or rural). clinical data involved medical history and physical examination. data about the form of fava bean ingested (fresh or dried), duration of exposure to fava beans before admission (in days), duration of onset of symptoms before admission (in days), presence of vomiting during the attack, history of previous episodes of hemolysis, history of neonatal jaundice, and personal or family history of g6pd deficiency were recorded. information from the physical examination included fever (defined as temperature ≥38°c axillary corrected), pallor, jaundice, and organomegaly. data about hematological and biochemical investigations that were performed at admission included blood group (a, b, ab or o), rh (negative or positive), hemoglobin level, white blood cell (wbc) count, platelets, total serum bilirubin (tsb), reticulocyte count, alanine aminotransferase (alt), and aspartate aminotransferase (ast). data were quantified and expressed as either frequencies (for categorical variables) or means and standard deviation (for continuous variables). the severity of hemolysis was assessed based on hemoglobin level at admission. cases with a hemoglobin level ≤5 g/dl were considered severe, while patients with a hemoglobin level of more than 5 g/dl were classed as having mild-moderate hemolysis. statistics descriptive analysis was performed to describe the study sample. the differences between the mild and severe hemolysis groups were assessed by bivariate analyses. these included independent samples t-test for continuous data and chi square analysis for categorical data. because the outcome variable is dichotomous (mild vs severe hemolysis). all analyses were performed using ibm spss (spss, chicago, il, usa) statistics for mac. results a total of 57 were recruited in this study. of 57 children with g6pd deficiency, 17 (29.8%) were diagnosed as severe cases depending on the level of hemoglobin at time of presentation to the emergency room in children welfare teaching hospital. the total number is 57 patients. the mean age is 4.35 years, males were constituting 85.9%, 32 patients were living in urban area mostly from baghdad, two-thirds of the patients have recorded ingestion of fresh type of fava beans (64.9%), while one-third ingested dried type (35%). ten patients were already diagnosed with g6pd prior to the presentation; six of them were diagnosed based on previous episodes of hemolysis, and four depending on routine assessment for a suspected family member. analysis of the demographic and clinical characteristics of the 57 patients according to the severity of hemolysis (mild vs severe) and the results of the independent samples t-test (for continuous variables) and chi square (for categorical variables) are represented in table 1. younger age group patients tend to present with the severe form of hemolysis (3.59 years with a p value of 0.001). no significant gender susceptibility was present between both types of hemolysis. the urban areabased living individuals tend to present with mild hemolysis while those from rural areas tend to present with more severe episodes of hemolysis (p value 0.001). moreover, there was a significant correlation between the type of fava bean ingestion and the severity of hemolysis, those who presented with more severe hemolysis usually had a history of ingestion of fresh type of fava bean. the mean duration of exposure was longer in those with mild hemolysis (2.87 days), and the mean onset of symptoms was also longer in severe hemolysis (2.20 days) with a significant p value of 0.005. eight individuals of severe type recorded previous episodes of hemolysis while three individuals of the mild type recorded previous episodes (p value 0.001). eightytwo percent of the severe cases recorded history of neonatal jaundice early in life treated with phototherapy, or exchange transfusion while 35% of the mild type recorded previous neonatal episode of jaundice (p value 0.001). family history of g6pd was significant in 88.2% of individuals with the severe form (p value 0.005). no significant correlation was seen between the type of hemolysis and the presence of vomiting, fever, jaundice, organomegaly, or pallor in those patients. pallor and jaundice detection in this study depend mainly on the clinical findings explored by the physician who was in charge in the er. baseline laboratory characteristics of 57 children with the diagnosis of g6pd had shown the following results: blood group was distributed as following: group o was detected in 43.8% of patients, group b was detected in 26.3%, this is followed by group a in 22.8%, and ab in 7%. rh positive was seen in 50 patients. the cbc profile showed the mean hemoglobin level detected was 6.1 g/dl, ranging from 2.1 to 8.5 g/dl, the mean reticulocyte count was 8% ranging from 3.5 to 25%. liver function tests (tsb, alt, and ast) were carried out to all patients where the mean serum bilirubin detected is 3.88 mg/dl, ranging from 1.9 to 9.5 mg/dl.table 2 shows the analysis of the laboratory characteristics of the 57 patients according to the severity of hemolysis (mild vs severe) and the results 264 original hemolysis in children with glucose-6-phosphate dehydrogenase deficiency after ingestion of fava beans hasanein h. ghali j contemp med sci | vol. 6, no. 6, november–december 2020: 262–266 of the independent samples t-test (for continuous variables) and chi square (for categorical variables). no significant correlation was detected between both types of hemolysis and blood grouping or rh type. significant correlation between hemoglobin level and the severity of hemolysis is expected to be detected as the classification of hemolysis was mainly based on the hemoglobin level. additionally, tsb level and reticulocyte count were found to be significantly higher in the severe form type (tsb mean of 5.07 mg/dl and reticulocytes count of 11.9% with p values of 0.02 and <0.001, respectively). there was no statistical difference in the level of platelets and wbcs between both types of hemolysis. discussion this report discusses the relationship between demographic profile, clinical, hematological and biochemical characteristics, and the severity of hemolysis among favic patients. the results of this study showed that patients with severe hemolysis were younger as compared to mild cases. this is consistent with a previous study conducted in jordan, which reported similar results.8 although the mechanism for this relationship is not clear, patients with more severe deficiency in the g6pd enzyme tend to present at earlier age in the course of the disease. because acute hemolysis in favic patients is triggered by ingestion of fava beans, younger children may not be intellectually able to realize the impact of ingesting fava beans, especially if unsupervised by their parents. but this is not the case here, as those who already knew that they are g6pd-deficient are three patients only in the severe form. despite the fact that the results of the present study revealed that the majority (85.9%) of patients are male, the gender difference between mild and severe hemolysis groups was not significant. male predominance in patients with g6pd deficiency was widely reported.9-11 because g6pd deficiency follows an x-linked pattern of inheritance, it is fully expressed in heterozygous males and homozygous females.12 therefore, heterozygous females are usually asymptomatic. the results also revealed that in comparison to city living, favic patients living in rural areas were more likely to develop severe hemolysis. several factors may render people living in rural areas more susceptible to the severe form of hemolysis. first, the health beliefs in rural areas are still primitive, especially when it comes to disease and illness.13 second, there is a lack of health awareness and education in the regional area regarding the nature of the disease and the factors that may table 1. baseline demographic, history and clinical characteristics according to severity of hemolysis. item mild hemolysis (n = 40) severe hemolysis (n = 17) p value age (years)(sd) 5.57 (2.11) 3.59 (1.98) 0.001 gender male 34 (85.0%) 15 (88.20%) female 6 (15.0%) 2 (11.76%) 1 residence urban 28 (70.0%) 4 (23.5%) rural 12 (30.0%) 13 (76.4%) 0.001 form of fava beans fresh 15 (37.5%) 12 (70.5%) dried 25 (62.5%) 5 (29.4%) 0.04 history mean duration of exposure(sd) 2.87 (1.69) 2.50 (1.45) 0.1 mean onset of symptoms(sd) 1.42 (0.51) 2.20 (1.01) 0.005 previous episodes 3 (7.5%) 8 (41.1%) 0.001 history of neonatal jaundice 14 (35.0%) 14 (82.3%) 0.001 g6pd cases 7 (17.5%) 2 (17.6%) 0.9 family history of g6pd 15 (37.5%) 15 (88.2%) 0.005 clinical characteristics presence of vomiting 15 (37.5%) 5 (29.4%) 0.1 presence of fever 17 (42.5%) 6 (35.2%) 0.7 presence of pallor 36 (90.0%) 17 (100.0%) 0.3 presence of clinical jaundice 37 (92.5%) 17 (100.0%) 0.5 presence of organomegaly 1 (2.5%) 1 (5.8%) 0.5 265 original hemolysis in children with glucose-6-phosphate dehydrogenase deficiency after ingestion of fava beanshasanein h. ghali j contemp med sci | vol. 6, no. 6, november–december 2020: 262–266 contribute to inducing hemolysis.13 parents may not be aware about the role of fava beans in inducing hemolysis among their children, even if they are not diagnosed with g6pd, and even if there is family history of g6pd, people from urban areas tend to avoid ingesting fava bean. third, regional areas in iraq are well-known for growing fava beans, unlike the urban region. the higher exposure to fava beans in rural areas, especially the fresh form during the harvest season, may explain the higher probability of developing severe hemolysis. this was also evident in this study as patients who had consumed fresh beans had more severe hemolysis as compared to those who had consumed dried beans. this is consistent with several reports from other middle-eastern countries, which showed that seasonal peaks of favism are corresponding to harvesting fresh fava beans.14,15the frequency of previous episodes of hemolysis could predict the severity of hemolysis in this study. patients with a history of more frequent hemolysis incidents had more severe hemolysis. the same was found for those who had a history of neonatal jaundice. the frequency of previous incidents may reflect the level of g6pd enzyme activity. according to the who classification of g6pd deficiency, the severity of g6pd enzyme deficiency corresponds to the chronicity of hemolysis.16excessive heme metabolism resulting from severe intravascular hemolysis leads to significant increases in bilirubin, which overwhelms the hepatic conjugation process resulting in unconjugated hyperbilirubinemia state.17 biochemically, our results showed that the more increase in tsb, and reticulocyte count could predict the severity of hemolysis. owing to intravascular hemolysis and destruction of rbcs, increased breakdown of hemoglobin results in increased tsb.18 as the bone marrow attempts to compensate for the continuous rbc destruction, reticulocytes release into peripheral blood drastically increases, reflecting the increasing amount of erythropoiesis.19this study has several noteworthy limitations. first, part of the data were collected based on patients’ and parents’ recall. therefore, recall bias might have been present. second, study participants were recruited using non-probability sampling, which has a limited generalizability.20 therefore, these findings should be interpreted with caution, as it does not represent the entire iraqi population. in addition, the clinical assessment of patients was carried out by physicians, which can be subjective. therefore, measurement bias might have been present as well. references 1. cappellini md, fiorelli g. glucose-6-phosphate dehydrogenase deficiency. lancet (london, england), 2008;371(9606):64-74. 2. nkhoma et, poole c, vannappagari v, hall sa, beutler e. the global prevalence of glucose-6-phosphate dehydrogenase deficiency: a systematic review and meta-analysis. blood cells mol dis, 2009;42(3):267-278. 3. al-musawi bm, al-allawi n, abdul-majeed ba, eissa aa, jubrael jm, hamamy h. molecular characterization of glucose-6-phosphate dehydrogenase deficient variants in baghdad city iraq. bmc blood disord. 2012;12:4. 4. al-musawi, b.m., al-allawi n., abdul-majeed, b.a. et al. molecular characterization of glucose-6-phosphate dehydrogenase deficient variants in baghdad city iraq. bmc hematol, 2012;12:4. 5. merdin a, avci f, guzelay n,glucose-6-phosphate dehydrogenase deficiency presented with convulsion: a rare case. hematol rep, 2014;6(1):5266. table 2. baseline laboratory characteristics according to severity of hemolysis. item mild hemolysis (n = 40) severe hemolysis (n = 17) p value blood group o 17 (42.5%) 8 (47.0%) 0.6 b 10 (25.0%) 5 (29.4%) a 9 (22.5%) 4 (23.5%) ab 4 (10.0%) 0 (0.0%) rh positive 35 (87.5%) 15 (88.2%) 1negative 5 (12.5%) 2 (11.7%) hematological parameters haemoglobin g/dl(sd) 7.05 (1.22) 4.46(0.58) 0.001 wbc x 109/l(sd) 4.90 (3.51) 5.78(4.40) 0.4 platelets x 109/l(sd) 165.45 (81.16) 184.86(132.63) 0.5 tsb, mg/dl(sd) 3.09 (1.80) 5.07(0.95) 0.02 reticulocytes, %(sd) 6.92(4.88) 11.91 (5.56) 0.001 biochemistry tsb, mg/dl(sd) 3.09 (1.80) 5.07(0.95) 0.02 alt, iu/l(sd) 39.81(17.91) 47.19 (11.51) 0.4 ast, iu/l(sd) 23.39(22.41) 25.65 (19.81) 0.19 alt: alanine aminotransferase; ast: aspartate aminotransferase; tsb: total serum bilirubin; wbc: white blood cells. all baseline and laboratory values are expressed as mean ± standard deviation (sd) except as indicated otherwise. 266 original hemolysis in children with glucose-6-phosphate dehydrogenase deficiency after ingestion of fava beans hasanein h. ghali j contemp med sci | vol. 6, no. 6, november–december 2020: 262–266 6. frank je. diagnosis and management of g6pd deficiency. am family physician, 2005;72(7):1277-1282. 7. world medical association declaration of helsinki. ethical principles for medical research involving human subjects, jama, 2013;310(20):2191-2194 8. al-sweedan sa, jdaitawi h, khriesat wm, khader yy, al-rimawi hs. predictors of severe hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency following exposure to oxidant stresses. hematol/ oncol stem cell ther. 2009;2(2):354-357. 9. yao lq, zou tb, wang xt, quan x, chen q, yang fb, hu ls, fan lm, wang m, feng xy, liu jt, zhao zm [g6pd deficiency among children under 7 years old from yunnan with unique ethnic minority origin]. chin j med genet. 2013;30 (2):189-194. 10. von fricken me, weppelmann ta, eaton wt, alam mt, carter te, schick l, masse r, romain jr, okech ba ,prevalence of glucose-6-phosphate dehydrogenase (g6pd) deficiency in the ouest and sud-est departments of haiti. actatropica. 2014;135:62-66. 11. ntaios g, chatzinikolaou a, tomos c, manolopoulos c, karalazou p, nikolaidou a, alexiou-daniel s, prevalence of glucose-6-phosphate dehydrogenase deficiency in northern greece. intern med j. 2008;38(3):204-206.guindo a, fairhurst rm, doumbo ok, wellems te, diallo da ,x-linked g6pd deficiency protects hemizygous males but not heterozygous females against severe malaria. plos med. 2007;4(3):66. 12. sultan as, medicine in the 21st century: the situation in a rural iraqi community. patient educ counsel. 2007;68(1):66-69. 13. belsey ma, the epidemiology of favism. bull world health organ 1973;48(1):1-13. 14. kattamis ca, kyriazakou m, chaidas s. favism: clinical and biochemical data. j med genet. 1969;6(1):34-41. 15. glucose-6-phosphate dehydrogenase deficiency. who working group. bull world health organ. 1989;67(6):601-611. 16. roche sp, kobos r, jaundice in the adult patient. am. family phys. 2004;69(2):299-304. 17. tabbara ia hemolytic anemias. diagnosis and management. med clin north am. 1992;76(3):649-668. 18. gilmer pr, jr., koepke ja. the reticulocyte. an approach to definition. am j clin pathol. 1976;66(1):262-267. 19. catania ja, dolcini mm, orellana r, narayanan v, nonprobability and probability-based sampling strategies in sexual science. j sex res. 2015;52 (4):396-411. https://doi.org/10.22317/jcms.v6i6.851 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 140 j contemp med sci | vol. 6, no. 3, may–june 2020: 140–145 original issn 2413-0516 introduction novel coronavirus disease 2019 (covid-19) is a serious contagious disease result from severe acute respiratory syndrome coronavirus 2 (sars-cov-2), initially diagnosed in wuhan, hubei, china in december 2019, then a rapid epidemic of coronavirus 2019 infection occurs. on march 11, 2020, the world health organization (who) declared it a pandemic. it’s almost the most serious pandemic in recent history. whereas far as writing this article, it affects more than 4 million people all over the world with more than 250,000 deaths.1-3 the rt-pcr test is the reference standard for the definitive diagnosis of covid-19 infection. despite the indicators of its lower sensitivity and specificity at levels 30-60%.4,5 further, the rt-pcr test is not easily available and need a relatively time consuming for processing. meanwhile, the early diagnosis of covid-19 infection is a cornerstone in the management, by which the complications and the numbers of contacts can be reduced. the ct scan suitable alternative where it is available and can be done in a relatively short time as it was in previous sars epidemics accordingly.6,7 however, the ct can be normal especially in the early period of the disease as reported.8-10 most covid-19 patients are presented with signs and symptoms of upper and/or lower respiratory tract infection. severe respiratory distress may occur, fever reportedly being the most common presentation. the reference standard for its diagnosis was the real-time reverse transcription-polymerase chain reaction (rt-pcr). chest computed tomography (ct) reported having higher sensitivity than pcr as high as 98% in the early detection of covid-19 patients.10,11 main ct findings were peripheral ground-glass opacities (ggos), consolidation opacities, and crazy paving appearance,9,12 gradually ct became an important part of the official diagnostic criterion of covid-19, ct provides information about early diagnosis, differential diagnosis, clinical classification, and can assess disease progression. furthermore, it detects pulmonary complication and follows up after discharge, the most important findings were: • the ggo: which is an area of increased attenuation in the lung parenchyma, with the preservation of bronchial and vascular margins.  • consolidation: which is an area of increased attenuation in the lung parenchyma, which obscures the pulmonary vessels and bronchiolar walls. and • crazy-paving alternating areas of ggo with thickened interlobular septa and intralobular lines in between.10,13,14 in iraq, the first confirmed case was reported in al-najaf city on february 24, 2020, and by april 30, more than 2000 cases were confirmed including more than 90 deaths.11,15 the first case of covid-19 reported in karbala was on march 3. and karbala is well-known as a religious city that is located in the middle of iraq with a population of nearly 1.5 million peoples. the holy city is attended by tens of million visitors each year, and several big mass gathering visits including the arbaeen visit, which represents the biggest annual mass gathering in the world occur in karbala.16 this study aims to describe ct scans’ findings of covid19 patients, particularly ggos, and to estimate the association between these opacities and the time of clinical presentation. patients and methods a cross-sectional study conducted during the period march 1 to april 20, 2020, at the diagnostic imaging department in al-imam al-hussein medical city in karbala\iraq, which is the biggest hospital in the governorate with more than 500 beds and represent the referral site for other hospitals and the one that receives all covid-19 cases. spectrum of ground-glass opacities in chest ct of covid-19 patients in karbala dhia mahdey alghazali1*, maytham a. maamera 2, haider fadel alkazraji1, ali abdulridha abutiheen3 1 department of diagnostic radiology, al-imam al-hussein medical city, karbala, iraq. 2 department of pulmonology, respiratory care unit, al-imam al-hussein medical city, karbala, iraq. 3 department of family & community medicine, college of medicine, university of kerbala, karbala, iraq. correspondence to : dhia mahdey alghazali (dhia2603mff@yahoo.com ) (submitted: 04 may 2020 – revised version received: 17 may 2020 – accepted: 24 may2020 – published online: 26 june 2020) objective to describe the ground-glass opacities (ggo) seen in chest ct scans of covid-19 patients and to estimate the association between these opacities and the time of clinical presentation. methods a cross-sectional study involving 81 covid-19 confirmed patients in imam al-hussein medical city in karbala-iraq during the period from march 1 to april 20, 2020. chest ct scan findings were evaluated by two radiologists and categorized accordingly. chi-square test was used for statistical analysis and a p-value of less than 0.05 was considered statistically significant. results the mean age ± standard deviation of patients was 53.5 ± 17.1 years, with male predominance as 63 (77.8%) of cases were males. nearly half of the patients were presented within the second week of starting the sign and symptoms. ggo was present in 79 scans (97.5%), followed by consolidation opacity in 29 patients (35.8%). four types of ggo were described. bilateral multiple subpleural ggo was the most prevalent type. there was a significant association between late time of patient presentation and more extensive ggo type. conclusion chest ct scan is valuable in diagnosis and management of covid-19 cases. the presence of ggo in ct scan of a patient that previously had no chest illness is highly suggestive of covid-19 disease, different types of ggo were seen. bilateral confluent type of ggo is associated with more serious and delayed status and warns the need for intensive care unit admission. keywords covid-19, chest ct, corona covid-19, ground-glass opacity, rt-pcr, consolidation. 141 original spectrum of ground-glass opacities in chest ct of covid-19 patients in karbaladhia mahdey alghazali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 140–145 patients with positive ct scan findings and positive rt-pcr were included in the study. those patients were referred from the respiratory clinic, for suspicion with covid19 infection. they had signs and symptoms of upper and/or lower respiratory tract infection. an unenhanced ct scan was done for all patients routinely following the guidelines of karbala health directorate for management of covid-19. where every suspected case or symptomatic person who had contact with covid-19 patient or travel to an endemic area should manage by complete blood count lab test, rt-pcr, and ct scan. so, 81patients with a positive ct scan and a positive pcr test were included out of total 86 diagnosed cases including 6 deaths, 5 patients were excluded, 2 with heart failure, 2 with pulmonary tuberculosis, and 1 was a known case of churg-strauss syndrome. chest ct examinations were performed by using a multidetector-row scanner (somatom definition edge siemens medical system, germany). the examination was carried out without a patient preparation, in supine, hands up position, from the upper neck to the upper renal poles depending on the scanogram, in a single breath-hold cluster, with 5-mm collimation, reconstruction at 1 mm interval and energy level of 120 kv and 200–250 ma. the ct scan results were evaluated by two independent radiologists to decrease interobserver bias. ethical approval was obtained from the research ethics committee in karbala health directorate. further, the patient’s names and personal information were kept confidential. demographic information of patients and ct findings were entered and analyzed using statistical package for social sciences (spss. version 21). data were expressed as frequencies and percentages while age was expressed as mean and standard deviation (sd). chi-square test was used for statistical analysis and a probability (p) value of less than 0.05 was considered statistically significant. results a total of 81 covid-19 confirmed patients with positive ct scans were included in the study. their age ranged from 12 to 83 years with a mean ± sd of 53.5 ± 17.1 years. the male to female ratio was 3.9:1 where 63 (77.8%) of the patients were men. all patients had symptoms at the onset of the disease, including fever, cough, and shortness of breath. complete blood count tests were performed for all patients. 23 (28.4%) patients had leukopenia, 66 (81.5%) had lymphocytopenia, while c-reactive protein (crp) was elevated in 56 (69.1%) patients. nearly half of patients, 41 (50.6%), were presented within the first week of symptoms’ appearance and half of them 40 (49.4%) were presented late in the second week as shown in fig. 1. ggos were present in 79 scans (97.5%). according to the distribution and severity of ggo, four configurations were described as shown in figs 2,3 and 4 as follows: type 1: unilateral single ggo in 17(21.52%) patients. type 2: bilateral multiple discrete ggo in 21(26.58%) patients. type3: bilateral multiple predominantly subpleural ggo in 25(31.65%) patients, and type 4: bilateral diffuse confluent patches of ggo in 16(20.25%) patients. there was a significant association (p-value <0.001) between the type of ggo and the time of clinical presentation, most patients with unilateral single ggo (type 1) and bilateral subpleural ggo (type 3) presented in the first week. while patients presented in second week exhibiting bilateral multiple discrete ggo (type 3) and bilateral confluent ggo (type 4), as shown in table 1. the second most common finding was consolidation opacities (as seen in fig 5a,b) were seen in 29 (35.8%) patients. the crazy-paving appearance was seen only in two ct scans represent 2.4%, also there was a significant association between the presence of consolidation and time of presentation (p-value < 0.001) as shown in table 1. pleural effusion was seen only in two (2.4%) very ill patients, who sooner died. discussion chest ct is a mainstay in the diagnosis and management protocol of covid-19 cases in iraq. as the rt-pcr is limited to certain centers and with a limited number of tests per day. especially in the beginning of the spread of disease in iraq, where ct is highly sensitive and almost all cases with positive fig. 1 pie chart of patient’s presentation time within first and second weeks. fig. 2 categories of ggo finding among 79 patients. 142 original spectrum of ground-glass opacities in chest ct of covid-19 patients in karbala dhia mahdey alghazali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 140–145 rt-pcr had positive findings, and as ct is more sensitive than rt-pcr.10,12 perhaps, some of those with positive ct findings who test negative for pcr might have false-negative rt-pcr. anyhow, cases with negative rt-pcr were excluded from the study. the higher male to female ratio of nearly fourfold increase in the current study could be attributed to the social customs in our region, where men can move freely and spend more time outside doors, unlike women. this male predominance is higher than the 60% male involvement reported by a meta-analysis and the 68% reported by chen et al.17, 18 further, males had shown higher severity and fatality with covid-19 infection.19 this was related to social causes including higher cigarette smoking rates among males as well as genetic or hormonal causes.17-19 regardless of how the respiratory system is affected, the pathological process involves alveolar edema, proteinaceous exudate, focal reactive hyperplasia of pneumocytes with patchy inflammatory cellular infiltration, and multinucleated giant cells.20,21 these changes are still not sufficient to fill the smallest air space (alveoli). in ct, this process is seen as a mild increase attenuation of lung parenchyma what is called ggo.22,23 according to the severity and distribution of these opacities, four patterns of ggo were identified and highlighted by many researchers.10,24-26 the unilateral ggo pattern seen in first week seems to be increased in number and becoming multiple discrete ggo by second week, while the bilateral subpleural patches of ggo seen in first week seems to change to diffuse confluent patches of ggo as most of these seen in second week, this agreed by han et al.27 the most extensive diffuse confluent patches of ggo which is associated with consolidation representing a late disease, and usually resulting in a bad outcome where six of the cases with type 4 had been fig. 3 axial ct scans lung window showing different types of ggo: (a, b) left lower lobe unilateral ground-glass pulmonary opacity (type 1). (c, d) shows bilateral discrete foci of ground-glass opacities (type 2). a b dc 143 original spectrum of ground-glass opacities in chest ct of covid-19 patients in karbaladhia mahdey alghazali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 140–145 intubated and then lost. these results are in agreement with what other researchers who had highlighted that the single ggo on the initial ct scan can indicate early-phase of disease while advance-phase disease reflected by ct signs of aggravation perhaps within the second week.28-30 so, on the era of covid-19 pandemic, patients with no previous medical history of chest illness, diagnosed having any type of ggo in chest ct should carry the possibility of covid-19 until prove otherwise. the number of cases in kerbala and iraq, in general, is probably higher than what was reported. as a significant percentage of the people are afraid of consequences of diagnosis with covid-19 including being quarantined with their families and relative. as well as, the stigmatization effect of being infected with covid-19 is present to a certain level. further, this could explain why nearly half of patients being diagnosed lately and many being presented at a late stage and denying any history of travel to endemic countries or contact with covid19 patients. a related issue noticed that patients presented earlier within first week were more identified to have a phobia of being sick with covid-19. while others presented late within the second week were complaining from serious respiratory symptoms. consolidation was the second most common ct finding, that been seen in nearly third of patients, of those (93.1%) were presented within the second week. so, consolidation might be considered as a sign of severe disease, which probably will be in need to be admitted to intensive care, and this agrees with das et al.31 while pulmonary cavitation, and significant lymphadenopathy that were characteristic of previous sars fig. 4 axial ct scans lung window showing different types of ggo; (a, b) show bilateral subpleural patchy ground-glass pulmonary opacity (type 3), (c, d) show bilateral diffuse confluent ground-glass opacities (type 4).  a b dc 144 original spectrum of ground-glass opacities in chest ct of covid-19 patients in karbala dhia mahdey alghazali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 140–145 disease,32 had not been recognized with patients with covid19 in kerbala and this agrees with bernheim et al.8 conclusion chest ct scan is valuable in diagnosis and management of patients with covid-19 infection. the presence of ggo in ct scan of patient that previously had no chest illness is highly suggestive of covid-19 disease. different types of ggo are seen; bilateral confluent type of ggo is associated with more serious and delayed status and warns the need for intensive care unit management. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. table 1. association between time of patient’s presentation and both consolidations and the type of ggo. presentation total p valuein 1st week in 2nd week consolidation no consolidation 39 13 52 < 0.001 75.0% 25.0% 100.0% consolidation 2 27 29 6.9% 93.1% 100.0% total 41 40 81 (50.6%) (49.4%) 100.0% type of ggo* unilateral single patch 17 0 17 < 0.001 100.0% 0.0% 100.0% bilateral multiple discrete patches 2 19 21 9.5% 90.5% 100.0% bilateral multiple subpleural 21 4 25 84.0% 16.0% 100.0% bilateral diffuse confluent patches 1 15 16 6.3% 93.7% 100.0% total 41 38 79* 51.9% 48.3% 100.0% *ggo was present among 79 patients fig. 5 axial thin-section unenhanced ct scan images (a) shows right lower lobe consolidation pulmonary opacity. (b) shows left lower and upper lobes consolidation pulmonary opacities. a b 145 original spectrum of ground-glass opacities in chest ct of covid-19 patients in karbaladhia mahdey alghazali et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 140–145 conflict of interest no potential conflict of interest relevant to this article was reported. reference 1. zhonghua l, x bing, xz zhi. novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china [in chinese]. 2020 feb 17;41(2):145-51. 2. world health organization, “situation report 80.”, 9 apr. 2020. https:// www.who.int/docs/default-source/coronaviruse/situationreports/20200409-sitrep-80-covid-19.pdf?sfvrsn=1b685d64_2 accessed 11 apr 2020. 3. world health organization “situation report 51, 11 mar. 2020, https:// www.who.int/docs/default-source/coronaviruse/situation reports/20200311-sitrep-51-covid-19. pdf?sfvrsn=1ba62e57_10 accessed 11 apr 2020. 4. general office of national health committee. office of state administration of traditional chinese medicine. notice on the issuance of a program for the diagnosis and treatment of novel coronavirus (2019-ncov) infected pneumonia (trial fifth edition) (2020-02-26). 5. yang y, yang m, shen c, wang f, yuan j, li j, zhang m, wang z, xing l, wei j, peng l. evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections. medrxiv 2020:2020.02. 11.20021493. 6. ajlan am, ahyad ra, jamjoom lg, alharthy a, madani ta. middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings. ajr am j roentgenol 2014 oct;203(4):782-787. 7. paul ns, roberts h, butany j, chung t, gold w, mehta s, konen e, rao a, provost y, hong hh, zelovitsky l, weisbrod gl. radiologic pattern of disease in patients with severe acute respiratory syndrome: the toronto experience. radiographics 2004;24(2):553–563.55 8. bernheim a, mei x, huang m, yang y, fayad za, zhang n, diao k, lin b, zhu x, li k, li s. chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection. radiology. 2020 feb 20:200463. 9. fang y, zhang h, xie j, lin m, ying l, pang p, ji w. sensitivity of chest ct for covid-19: comparison to rt-pcr. radiology, 2020:200432. doi: 10.1148/ radiol.2020200432.  10. ai t, yang z, hou h et al. correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases [published online ahead of print, 2020 feb 26]. radiology. 2020;200642.  11. world health organization “situation report 36”, 25 feb. 2020. https:// www.who.int/docs/default-source/coronaviruse/situationreports/20200225-sitrep-36-covid-19.pdf?sfvrsn=2791b4e0_2 accessed 11 apr 2020. 12. wang w, tang j, wei f. updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan, china. j med virol. 2020 apr;92(4):441-7. 13. ye z, zhang y, wang y, huang z, song b. chest ct manifestations of new coronavirus disease 2019 (covid-19): a pictorial review. eur radiol. 2020 mar 19:1-9. 14. wormanns d, hamer ow. glossary of terms for thoracic imaging--german version of the fleischner society recommendations. rofo: fortschritte auf dem gebiete der rontgenstrahlen und der nuklearmedizin. 2015 aug;187(8):638-61. 15. world health organization “situation report 45.”  5 mar. 2020 https://www. who.int/docs/default-source/coronaviruse/situation-reports/20200305sitrep-45-covid-19.pdf?sfvrsn=ed2ba78b_4 accessed 11 apr 2020. 16. abutiheen aa. clients’ satisfaction with referral system in karbala. am j appl sci. 2014;11(2):216-22. 17. li lq, huang t, wang yq, wang zp, liang y, huang tb, zhang hy, sun w, wang y. covid-19 patients’ clinical characteristics, discharge rate, and fatality rate of meta-analysis. j med virol. 2020 jun;92(6):577-83. 18. chen n, zhou m, dong x, qu j, gong f, han y, qiu y, wang j, liu y, wei y, yu t. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. lancet. 2020 feb 15;395(10223):507-13. 19. jin jm, bai p, he w, wu f, liu xf, han dm, liu s, yang jk. gender differences in patients with covid-19: focus on severity and mortality. front public health. 2020 apr 29;8:152. 20. tian s, hu w, niu l, liu h, xu h, xiao sy. pulmonary pathology of early phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer. j thoracic oncol. 2020 feb 28. 21. yoshikawa a, bychkov a. coronavirus disease 2019 (covid-19). pathologyoutlines.com website. http://www.pathologyoutlines.com/topic/ lungnontumorcovid.html. accessed may 5th, 2020. 22. park cm, goo jm, lee hj et-al. nodular ground-glass opacity at thinsection ct: histologic correlation and evaluation of change at follow-up. radiographics. 2007;27(2):391-408. doi:10.1148/rg.272065061. 23. chang m, jin m, hyun j, chang h, eun j, jung-gi i. nodular ground-glass opacity at thin-section ct: histologic correlation and evaluation of change at follow-up. published online:mar 1 2007radiographicsvol. 27, no. 2 https://doi.org/10.1148/rg.272065061 24. kanne jp. chest ct findings in 2019 novel coronavirus (2019-ncov) infections from wuhan, china: key points for the radiologist. radiology. 2020 feb 4;295(1):16-7. 25. chung m, bernheim a, mei x, zhang n, huang m, zeng x, cui j, xu w, yang y, fayad za, jacobi a. ct imaging features of 2019 novel coronavirus (2019ncov). radiology. 2020 feb 4;295(1):202-7. 26. ng my, lee ey, yang j, yang f, li x, wang h, lui mm, lo cs, leung b, khong pl, hui ck. imaging profile of the covid-19 infection: radiologic findings and literature review. radiology: cardiothoracic imaging. 2020 feb 13;2(1):e200034.  27. han r, huang l, jiang h, dong j, peng h, zhang d. early clinical and ct manifestations of coronavirus disease 2019 (covid-19) pneumonia. am j roentgenol. 2020 mar 17:1-6. 28. zhou s, wang y, zhu t, xia l. ct features of coronavirus disease 2019 (covid-19) pneumonia in 62 patients in wuhan, china. ajr am j roentgenol. 2020 mar 5:1-8. doi: 10.2214/ajr.20.22975. 29. pan f, ye t, sun p, gui s, liang b, li l, zheng d, wang j, hesketh rl, yang l, zheng c. time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia. radiology. 2020 feb 13:200370. doi: 10.1148/radiol.2020200370. 30. wang y, dong c, hu y, li c, ren q, zhang x, shi h, zhou m. temporal changes of ct findings in 90 patients with covid-19 pneumonia: a longitudinal study. radiology. 2020 mar 19:200843. 31. das km, lee ey, enani ma, aljawder se, singh r, bashir s, al-nakshbandi n, aldossari k, larsson sg. ct correlation with outcomes in 15 patients with acute middle east respiratory syndrome coronavirus. am j roentgenol. 2015 apr;204(4):736-42. 32. ooi g, khong p, müller n, yiu w, zhou l, ho j, lam b, nicolaou s, tsang k. severe acute respiratory syndrome: temporal lung changes at thin-section ct in 30 patients. radiology 2004;230(3):836–844. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i3.769 53j contemp med sci | vol. 2, no. 6, spring 2016: 53–55 case report the calcifying epithelial odontogenic tumour (ceot), also known as pindborg tumour, is an uncommon lesion that included <1% of all odontogenic tumours. this is a rare benign, but locally aggressive odontogenic tumour, usually seen in the posterior area of the mandible and is mostly found in patients between 30 and 50 years of age, without sex predilection. the tumour has a recurrence rate of 10–15% and rare malignant transformation. keywords odontogenic tumour, pindborg tumour, mandible a case report of calcifying epithelial odontogenic (pindborg) tumour in the mandible shima nafarzadeha, reza imanib, mansooreh mohammadic, sina haghanifard issn 2413-0516 aassistant professor, oral and maxillofacial pathology department, dentistry school, babol university of medical sciences, babol, iran. bpost graduate student, oral and maxillofacial pathology department, dentistry school, babol university of medical sciences, babol, iran. cassistant professor, oral and maxillofacial surgery department, dentistry school, babol university of medical sciences, babol, iran. dassociate professor, oral and maxillofacial radiology department, dentistry school, babol university of medical sciences, babol, iran. correspondence to reza imani (email: rezaimani83@yahoo.com). (submitted: 10 february 2016 – revised version received: 24 march 2016 – accepted: 26 may 2016 – published online: 26 june 2016) introduction calcifying epithelial odontogenic tumour (ceot), also known as pindborg tumour, is an uncommon tumour, including <1% of all odontogenic tumours.1–3 the tumour was first described in 1956 by the late dr. jens j pindborg.1 it is usually intraosseous and mostly occurs in the jaw bones. the mandible is affected twice more than the maxilla. the most involved site is the mandibular posterior area.4 the tumour shows a variable radiographic view based on its development; mixed radiolucent–radiopaque feature is the most prevalent, seen in 65% of cases.5,6 the mean occurrence age is 40 years with a range between 2nd and 6th decades.4 here, we present a rare case of huge pindborg tumour in the mandibular body of a 48-year-old man. case report a 48-year-old male patient was referred to babol dentistry school, babol, iran, for fixed prosthetic treatment. during examinations, an asymmetry with a swelling at posterior area of the left side of his mandible was observed (fig. 1). the patient was aware of the lesion from 15 years ago, but, as there were no sign or symptom, he did not seek treatment. his past medical and habitual history was otherwise clear. on extra oral examination, we found a well-defined bony hard swelling in the mandibular molar area extending to the angle. the overlying skin was intact with no tenderness. on intra-oral examination, the mass was palpated with intact mucosal coverage (fig. 2). in his panoramic view, the multilocular lesion measuring was 7 × 3 cm, with coarse trabeculae and radiopaque foci and impacted molar tooth was detected in the left mandibular body. the inferior border of mandible and alveolar crest in the lesion area were expanded and the third molar tooth was displaced to the border of the mandible (fig. 3). an incisional biopsy under local anaesthesia was performed (fig. 4) and the sample was sent to the pathology laboratory for histopathological evaluations. the microscopic views showed an odontogenic tumour composed of nests and islands of epithelioid cells with oeosinophilic cytoplasms. the production of amyloid-like material was evident, so pindborg tumour was considered as a diagnosis (figs. 5, 6). the patient was scheduled for a surgical excision and reconstruction as treatment plan. discussion the ceot, which also known as pindborg tumour, is an uncommon lesion that included <1% of all odontogenic tumours. approximately about 200 cases have been reported to date.7 pindborg tumour was previously described in the literature as adenoid adamantoblastoma, ameloblastoma of unusual type with calcification.8 thoma and goldman described the tumour as a neoplasm arising from the odontogenic epithelium; subsequently, the german pathologist jorgen pindborg recognised it as a separate entity in 1955, and in honour of him, this lesion was termed as the pindborg tumour.4 in 1967, abrams and howell reported the first case of ceot consist of clear cells.9 the term ‘ceot’ has been generally accepted by the who in the first edition of ‘histological typing of odontogenic tumours, jaw cysts and allied lesions’, where it was recognised as a distinct entity. for more than 30 years, the ceot has been known widely as ‘pindborg tumour’.10 ceot is a rare benign, but locally aggressive odontogenic tumour.11 it is a slow-growing neoplasm, which has a recurrence rate of 10–15% and rare malignant transformation.12 this neoplasm is mainly intraosseous with a strong tendency to the mandible.13 peripheral tumours usually arise in the anterior gingiva and account for <5% of cases. tumour histogenesis is not exactly clear, but it is believed to arise from remnants of dental lamina and stratum intermedium.13,14 odontogenic tissue is able to produce dentin and enamel because of the interactions between odontogenic mesenchyme and epithelium. thus, when odontogenic tissue undergoes tumoural changes, it can produce abnormal calcifications resembling enameloid, dentinoid and cementum in histologic features.15 clinically, pindborg 54 j contemp med sci | vol. 2, no. 6, spring 2016: 53–55 pindborg tumour case report shima nafarzadeh et al. fig. 2 exophitic growth on the left side of mandibular ridge. fig. 3 a multilocular mixed radiolucent–radiopaque lesion. fig. 4 surgical view. fig. 5 histopatholoic view showing tumoural nests (×100). fig. 1 clinical view showing facial asymmetry. tumour usually is seen in the posterior area of mandible and is mostly found in patients between 30 and 50 years of age, without sex predilection.16 this case was also seen in mandibular premolar molar area of a 48-year-old male patient. the most common clinical features of ceot, when detectable, are a localised swelling of the involved jaw. pain or paresthesia may exist which is depended to the size of the tumour, the growth pattern or its location, and proximity to the neurovascular structures.17 our case had no pain or paresthesia, despite the noticeable size of the tumour and its long duration. radiographically, ceot is characterised as a unilocular or multilocular radiolucent lesion that often exhibits a mixed radiopaque–radiolucent pattern. the mixed pattern shows areas of scattered flecks of calcification (driven snow pattern).5,6 however, calcifications sometimes, may not be observed on radiographs.17 our case also revealed a radiolucent– radiopaque mass 55j contemp med sci | vol. 2, no. 6, spring 2016: 53–55 case report pindborg tumourshima nafarzadeh et al. references 1. rajendiran r, sivapathasundaram b. shafer’s textbook of oral pathology, 6th ed. india: elsevier; 2009. pp. 279–81. 2. reichart p, philpsen hp. odontogenic tumors and allied lesions, 1st ed. usa: quintessence publishing co; 2004. pp. 93–103. 3. neville bw, damm dd, allen cm, bouquat je. oral & maxillofacial pathology, 3rd ed. india: saunders; 2009. pp. 716–8. 4. rani v, masthan mk, aravindha b, leena s. aggressive calcifying epithelial odontogenic tumor of the maxillary sinus with extraosseous oral mucosal involvement: a case report. iran j med sci. 2016;41(2):145–149. 5. ching as, pak mw, kew j, metreweli c. ct and mr imaging appearances of an extraosseous calcifying epithelial odontogenic tumor (pindborg tumor). am j neuroradiol. 2000;21:343–5. pmid: 10696021 6. uchiyama y, murakami s, kishino m, furukawa s. ct and mr imaging features of a case of calcifying epithelial odontogenic tumor. jbr-btr. 2012; 95:315–9. doi: 10.5334/jbr-btr.676 pmid: 23198374 7. nevill bw, damm dd, allen cm, chi ac. oral and maxillofaciall pathology, 4th ed. philadelphia: saunders; 2016. p. 674 8. shetty d, jayade bv, jayade g, gopalkrishnan k. peripheral calcifying epithelial odontogenic tumor: case report. j oral biol craniofac res. 2014;4(2):147–150. doi: 10.1016/j.jobcr.2014.03.002 9. turatti e, brasil j, de andrade ba, romañach mj, de almeida op. clear cell variant of calcifying epithelial odontogenic tumor: case report with immunohistochemical findings. j clin exp dent. 2015;7(1):e163–e166. doi: 10.4317/jced.51995 pmid: 25810830 10. vinayakrishna k, soumithran cs, sobhana cr, biradard v. peripheral and central aggressive form of pindborg tumor of mandible: a rare case report. j oral biol craniofac res. 2013;3(3):154–158. doi: 10.1016/j.jobcr.2013.07.001 pmid: 25737906 11. rydin k, sjöström m, warfvinge g. clear cell variant of intraosseous calcifying epithelial odontogenic tumor: a case report and review of the literature. oral surg oral med oral pathol oral radiol. 2016. pii: s2212– s4403. doi: 10.1016/j.oooo.2016.01.001 pmid: 26953043 12. more cb, vijayvargiya r. intraosseous calcifying epithelial odontogenic (pindborg) tumor: a rare entity. 2015;19(2):269. doi: 10.4103/0973029x.164561 pmid: 26604515 13. regezi ja, sciubba jj, jordan rck. oral pathology clinical pathologic correlations, 6th ed. st. louis: wb saunders; 2016. p. 277. 14. vigneswaran t, naveena r. treatment of calcifying epithelial odontogenic tumor/pindborg tumor by a conservative surgical method. j pharm bioallied sci. 2015;7(1):s291–s295. doi: 10.4103/0975-7406.155961 pmid: 26015736 15. lee sk, kim ys. current concepts and occurrence of epithelial odontogenic tumors: ii: calcifying epithelial odontogenic tumor versus ghost cell odontogenic tumors derived from calcifying odontogenic cyst. korean j pathol. 2014;48(3):175–187. doi: 10.4132/koreanjpathol.2014.48.3.175 pmid: 25013415 16. chen cy, wu cw, wang wc, lin lm, chen yk. clear-cell variant of calcifying epithelial odontogenic tumor (pindborg tumor) in the mandible. int j oral sci. 2013;5(2):115–119. doi: 10.1038/ijos.2013.29 pmid: 23703711 17. sahni p, nayak mt, singhvi a, sharma j. clear cell calcifying epithelial odontogenic (pindborg) tumor involving the maxillary sinus: a case report and review of literature. j oral maxillofac pathol. 2012;16(3):454–459. doi: 10.4103/0973-029x.102520 pmid: 23248488 18. gnepp dr. diagnostic surgical pathology of the head and neck, 2nd ed. philadelphia: saunders; 2009. p. 810. 19. philipsen hp, reichart pr. calcifying epithelial odontogenic tumour: biological profile based on 181 cases from the literature. oral oncol. 2000;36:17–26. pmid: 10889914 20. ai-ru l, zhen l, jian s. calcifying epithelial odontogenic tumors: a clinicopathologic study of nine cases. j oral pathol. 1982;11(5):399–406. pmid: 6815318 fig. 6 histopathologic view showing amyloid-like material production (×400). without driven snow appearance. association with impact teeth are often seen,13 but was not seen in our case. ceot is microscopically characterised by cords and nests of round to polygonal oeosinophilic epithelial cells with nuclear pleomorphism and conspicuous intercellular bridges in a fibrous stroma that typically contains variable amounts of the congo red positive amyloid-like material and calcified structures.18–20 epithelial island, pleomorphism and amyloid-like material were seen in our case, and amyloid presence was confirmed by congo red staining. in the mandible, the treatment of choice is marginal resection with a rim of normal tissue. wide resection seems to be unnecessary in typical cases. in the maxilla, more aggressive treatment should be done because of the rapid-growth potential of the neoplasms.18 our treatment plan was an excisional surgery with reconstruction, and the patient was asked to attend follow-up sessions to monitor the recurrence.  264 j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy ameer z. wohaib, nidhal k. maraie, and gaith a. jassim department of pharmaceutics, college of pharmacy, mustansiriyah university, baghdad, iraq. department of pharmacology, college of pharmacy, mustansiriyah university, baghdad, iraq. correspondence to ameer z. wohaib (email: azw_alw@yahoo.com). (submitted: 23 august 2019 – revised version received: 08 september 2019 – accepted: 22 september 2019 – published online: 26 october 2019) introduction controlled release dosage form is a dosage forms that release the drug continuously in predetermined pattern for a fixed period of time, either systemically or locally to specified target organ.1 it occurs when the active constituents and polymer both combined in such manner that the release from the bulk mate­ rials was pre­designed; its role is to alter the pharmacokinetics and or pharmacodynamics of pharmacologically active moie ­ ties by using novel drug delivery system or by modification of molecular structure or its physiological parameters.2 emboliza­ tion technique is a technique in which an occlusive drug is delivered through a catheter to block flow within a targeted blood vessel. it is performed for many medical conditions such as discontinue bleeding from a hemorrhagic ulcer or to tumor by blocking its blood supply.3 ion exchange resin microspheres can be used to induce emboli after loading with the suitable drug and in order to obtain the suitable particle size to block the blood supply microencapsulation technique can be applied.4 the polymer used as a coated material is poly(lactic­co­glycolic acid) (plga) which is a family of fda­approved biodegradable polymers that are physically strong and highly biocompatible and have been extensively studied as sustained delivery vehicles for drugs.5 coating (microencapsulation) of the microspheres loaded drug like drug–resin complex (drc) provided better control on the release of the drug by utilizing rate­controlling membrane that can modify the action of the therapeutic emboli for a longer time.6 sulfasalazine is tasteless, odorless, brownish­yellow powder with 542°c melting point. it is indicated in the treatment of mild to moderate ulcerative colitis, it is also indicated in rheuma­ toid arthritis, juvenile rheumatoid arthritis, crohn’s disease and psoriatic arthritis.7 sulfasalazine has a potent inhibitory property on system xc -( ) since it markedly reduced cystine uptake, glu­ tathione levels, growth and viability of human cancer cells.8 the aim of this work is to prepare and evaluation (in vitro/ in vivo) microcapsules containing sulfasalazine­ion exchange resin complex (resinate) to induce emboli for treatment of solid cancer through cutting off blood supply to area and slowly release the drug for a long period enough to treat the disease, to be used as alternative to surgery, chemotherapy and radiotherapy that had serious side effects. materials and methods materials sulfasalazine purchased hyperchem, china, deae­sephadex a25 from pharmacia pharmaceutical company, sweden, plga [lactic acid:glycolic acid (85:15)] from advanced biomedical technology, taiwan. methods preparation of drug–resin complex the drc had been prepared in our laboratory but it required further optimization therefore specific amount of resin (deae sephadex a­25) was used to prepare a drug:resin ratio (1:8) by suspending the resin in sulfasalazine aqueous solution with at 400 rpm for 120 min at 50°c using hot plate magnetic stirrer then the obtained residue allowed to dry in a hot air oven at 40°c overnight.9 the drug entrapment efficiency was objective this work involves preparation and evaluation (in vitro/in vivo) of microcapsules containing sulfasalazine-ion exchange resin complex (resinate) to induce emboli for treatment of solid cancer. methods the drug–resin complex (resinate) had been optimized by using drug:resin ratio (1:8), by suspending the resin in sulfasalazine aqueous solution with at 400 rpm for 120 min at 50°c. for controlling the release of drug, microencapsulation for the resinate was applied where 21 formulas with different resinate:poly(lactic-co-glycolic acid) ratios 2:1, 1:1 and 1:2 were prepared by solvent evaporation method to study the effect of different variables including resinate:polymer ratio, stirring speed, effect of temperature and aqueous phase volume on microencapsulation efficiency and percent of yield. results the in vitro release study for the prepared resinate, which had 72% entrapment efficiency, showed 80.992% of drug released within 15 min and the release continued until 99.83% within 75 min formula was found to be f19 had 76.70% encapsulation efficiency and 89.40% yield. the in vitro release study for selected formula showed that 32% of drug released within 1 h and 78% of drug released within 20 days and the release continued up to 96% within 45 days indicating a controlled release manner with spherical microcapsule of 595 µm. the preliminary in vivo work using rabbits showed instant occlusion of the central auricular artery of the rabbit ear leading to ischemia within 3 days that continued to the end of study period. conclusion this work show the suitability of microcapsules size to prevent blood flow and forming embolization with controlling release of the drug that may treat the solid tumor. keywords embolization therapy, sulfasalazine, deae sephadex a-25 ion exchange resin, plga coated microcapsules issn 2413-0516 original a a b a b 265j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 ameer z. wohaib et al. application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy determined by analyzing the supernatant obtained for the free drug by measuring its uv absorbance at 359 nm, the amount of sulfasalazine entrapped was determined according to this equation.10 entrapment efficiency ee entrapped drug content initial ( ) = drug content é ë ê ù û ú in vitro dissolution study for the prepared drug–resin complex the release of the drug from the prepared drc (resinate; 1:8) was studied using dissolution apparatus ii and 900 ml phos­ phate buffer (ph 7.4) as dissolution medium fixed at 50 rpm stirring speed and 37°c. samples (5 ml) were withdrawn at predetermined time, filtered and analyzed using uv spectro­ photometer at 359 nm,11 the withdrawn samples were replaced by the same of fresh phosphate buffer.12 microencapsulation of the prepared drug–resin complex microcapsules were prepared by solvent evaporation method, as follows: (a) aqueous phase: specific amount of the selected resinate (ratio 1:8) was suspended in 300 ml aqueous media con­ taining 50 ml of glycerol in polyvinyl alcohol solution (0.15% w/v). (b) organic phase: different amounts of plga had been dis­ solved in 20 ml dichloromethane (dcm). then organic phase had been added to the aqueous phase with continuous shaking at 35°c for 45 min (until complete evapo­ ration of dcm). the mixture was filtered and the microcap­ sules obtained had been washed with sufficient quantity of deionized distilled water and normal hexane to remove any residual organic solvent, and then washed with formaldehyde (5%) to solidify the coated layer. the prepared microcapsules were dried overnight in a hot air oven at 40°c. twenty­one formulas containing different resinate:plga ratio 2:1, 1:1 and 1:2 were prepared using different volumes of aqueous phase at different stirring speed and stirring temperature in order to optimize the preparation,13 as shown in table 1. encapsulation efficiency and percentage of product yield were the main parameters used to evaluate microencapsula­ tion methods and conditions and they were calculated as the follows:14 encapsulation efficiency total drug free drug total drug = ´ 1100% percent yield actual yield theoretical yield = ´100% variables affecting percent of yield and encapsulation efficiency of the prepared microcapsules effect of resinate:plga ratio the effect of using different ratios of resinate:plga (1:2, 1:1 and 2:1) on the encapsulation efficiency and percent of yield of the table 1. the prepared formulas of sulfasalazine microcapsules formula no. amount of resinate (mg) organic phase aqueous phase (ml) stirring temperature (°c) stirring speed (rpm) resinate:plga ratioplga (mg) dcm (ml) f1 30 60 20 300 35 600 1:2 f2 30 60 20 400 35 600 1:2 f3 30 60 20 500 35 600 1:2 f4 30 30 20 300 35 600 1:1 f5 30 30 20 400 35 600 1:1 f6 30 30 20 500 35 600 1:1 f7 30 15 20 300 35 600 2:1 f8 30 15 20 400 35 600 2:1 f9 30 15 20 500 35 600 2:1 f10 30 60 20 500 45 600 1:2 f11 30 60 20 500 55 600 1:2 f12 30 30 20 500 45 600 1:1 f13 30 30 20 500 55 600 1:1 f14 30 15 20 500 45 600 2:1 f15 30 15 20 500 55 600 2:1 f16 30 60 20 500 55 700 1:2 f17 30 60 20 500 55 800 1:2 f18 30 30 20 500 55 700 1:1 f19 30 30 20 500 55 800 1:1 f20 30 15 20 500 55 700 2:1 f21 30 15 20 500 55 800 2:1 original 266 j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy ameer z. wohaib et al. prepared microcapsules was studied using f1, f4 and f7 using 300 ml aqueous medium, 600 rpm stirring speed at 35°c.15 effect of aqueous phase volume the effect of volume of aqueous phase (300, 400 and 500 ml) on the encapsulation efficiency and percent of yield of the pre­ pared microcapsules from each resinate:plga ratios (2:1, 1:1 and 1:2) was studied using formulas f1–f9.15 effect of temperature the effect of temperature (35, 45 and 55°c) on encapsulation efficiency and percent of yield of the prepared microcapsules from each resinate:polymer ratios (2:1, 1:1 and 1:2) was studied using formulas f3, f6, f9–f15, keeping 500 ml aqueous phase and 600 rpm stirring speed.16 effect of stirring speed the effect of different stirring speeds (600, 700 and 800 rpm) on the encapsulation efficiency and percent of yield of the pre­ pared microcapsules from each resinate:plga ratios (2:1, 1:1 and 1:2) was studied using formulas f11, f13, f15–f21 with 500 ml aqueous phase and temperature 55°c.17 in vitro dissolution studies for the selected prepared microcapsules for each resinate:plga ratio, the best formula of microcapsules was selected depending on entrapment efficiency and percent of yield and they were f17, f19 and f21. the release of drug from selected formulas were measured by modified dissolution method using hot plate magnetic stirrer at 100 rpm and 40 ml phosphate buffer (ph 7.4) dissolution medium volume at 37°c. certain amount from each formula (equivalent to 3.2 mg of sul­ fasalazine) were placed in dialysis membrane (3000 da) sealed and introduced in 40 ml of phosphate buffer (ph 7.4). samples (1 ml) were withdrawn at predetermine time and diluted as need by fresh phosphate buffer (ph 7.4), filtered and analyzed using a uv spectrophotometer at 359 nm,18 the withdrawn samples were replaced by the same volume of fresh phosphate buffer.12 particle size analysis the ion exchange resin, selected resinates and selected micro­ capsules formula (f19) each one separately have been exam­ ined using laser diffraction particle size analyzer. the particles was suspended in distilled water and characterized by volume and number distribution using laser diffraction and polariza­ tion intensity differential scattering.19 morphological characterization the morphological characterization of the selected microcap­ sules formula (f19) was carried out using scanning electron microscopy (sem) together with deae sephadex a­25 micro­ spheres and the selected resinate.20 in vitro degradation study the in vitro degradability of plga coat of the selected micro­ capsules formula (f17) was assessed using fresh human serum. three samples (20 mg) of (f17) were suspended separately in 5 ml of human serum and incubated at 37°c for 10, 20 and 30 days. sample at each interval has been centrifuged at 1200 rpm for 5 min and the sediment was washed by deionized distilled water then dried at hot air oven overnight at 40°c and exam ­ ined under sem.21 preliminary in vivo test for embolization property two rabbits were used to evaluate the embolization property for selected microcapsules formula (f19), the rabbits ears had been prepared for injection by wiping with sterile tissue then (24g) cannula had been inserted in ear central artery through which 3 ml suspension containing (30 mg) of sterile microcap­ sules (f19) was injected slowly.22,23 results and discussion preparation of drug–resin complex sulfasalazine is a weak acid for which a weak base anion exchange resin is helpful for preparation of drc. deae sephadex a­25 is one type of the anion exchange resins, which is a weak base in nature and it is biocompatible so it is suitable for formation of complex with sulfasalazine.24 drc was pre­ pared previously in our laboratory but it needs further optimi­ zation, therefore in this study drc 1:8 was prepared and its entrapment efficiency was 72% which is higher than lower drug:resin ratio since the increase in the amount of resin lead to increase in the number of chemical equivalent that available for ionic exchange per unit weight or volume of resin.25 in vitro dissolution study for the prepared drug–resin complex figure 1 shows the cumulative percent of drug release for the prepared drug:resin ratio (1:8). the results showed that the percentage of sulfasalazine released within 15 min at phos­ phate buffer (ph 7.4) was 80.992%, and the release continued up to 99.83% within 75 min, this may be due to the fact that the complex will release the drug only when it is replaced by the ions which have the same charge after hydration of drc by the medium. the ionic exchange may reach equilibrium, and this depend on the ionic constitution and the fluid volume, also the drug must diffuse from the resin through the internal exchange sites and it mainly depends on the efficient complex formed between the drug and the resin.26 microencapsulation (coating) of selected drug–resin complex (resinate) microencapsulation of the drc (resinate) is adapted to con­ trol release of the drug. different formulas (21 formulas) of the coated (microencapsulated) resinate had been prepared with different resinate:plga ratios, aqueous phase volume, stirring speed and temperature. the encapsulation efficiency and per­ cent of yield were calculated to estimate the best formula. fig. 1 cumulative drug release of drug:resin ratio (1:8) with best entrapment efficiency. original 267j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 ameer z. wohaib et al. application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy variables affecting percent of yield and encapsulation efficiency of the prepared microcapsules effect of resinate:plga ratio the results showed that as plga amount was increased (from 2:1 to 1:1) the encapsulation efficiency and percent of yield increased that may be as a result of high viscosity of the con­ tinuous phase that may delay the drug diffusion from the res­ inate and enhance precipitation of plga polymer on the surface of the resinate.15 but the result also showed decrease in encapsulation efficiency with no effect on percent of yield as a plga ratio increased from 1:1 to 1:2 because the observation indicated that an increase in the polymer concentration increases the entrapment of the drug inside the microsystems but on a high polymer amount it can form a loose network in the matrix which decreased the encapsulation efficiency as the network allows the drug particles to leach out during the microcapsules production27 as shown in fig. 2. effect of aqueous phase volume the results showed a significant increase in encapsulation effi­ ciency and percent of yield by increasing the volume of aqueous phase that may extract the solvent instantly which lead to fast precipitation of the coating polymer (plga) in large volume of continuous phase,28 as shown in fig. 3. therefore, 500 ml aqueous phase volume was selected for further study. fig. 2 effect of resinate:plga ratio on percent of yield and encapsulation efficiency for three prepared formula f1 (ratio 1:2), f4 (ratio 1:1) and f7 (2:1). fig. 3 effect of aqueous phase volume on drug encapsulation efficiency and percent of yield for the prepared formula for different resinate:plga ratio (a) 2:1, (b) 1:1 and (c) 1:2. a b c original 268 j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy ameer z. wohaib et al. effect of temperature it was found that there was a significant increase in the encap­ sulation efficiency and percent of yield upon increasing the temperature since the high temperature facilitated the evapo­ ration of dcm leading to precipitation of plga on the res­ inate in the dispersed phase,15,17 as shown in fig. 4, therefore, the temperature selected for further study was kept at 55°c (using 500 ml aqueous phase volume). effect of stirring speed the results showed as stirring speed increased the percent of yield was significantly increased since the stirring process may enhance the emulsification process.29 the results also showed that as the stirring speed increased the encapsulation effi­ ciency decreased (not significantly) that may be due to vortex formation at high agitation rates which resulted in leaching of drug from the resinate.30 therefore, the best stirring speed was 800 rpm (keeping the temperature at 55°c and 500 ml aqueous phase volume). accordingly, for each resinate:plga ratio one best formula was selected which had best encapsulation effi­ ciency and percent of yield (f17, f19 and f21), as shown in fig. 5. in vitro dissolution studies for the selected prepared microcapsules the results showed that the percentage of sulfasalazine released from formula (f21) within 1 h at phosphate buffer (ph 7.4) was 28%, and the release after 20 day was 81% which con­ tinued up to 98% within 45 days. on another hand the results showed that the percentage of drug released from formula (f17) within 1 h was 29%, and the release after 20 days was 70% which continued up to 93% within 45 days. while the percentage of drug released from f19 within 1 h was 32%, and the release after 20 days was 78% which continued up to 96% within 45 days. the initial burst effect may be due to the pres­ ence of some drug particles on the surface of the microspheres, which can be considered as a desired effect to ensure the initial therapeutic action of drug.31 it was also observed that the drug release from f17 (containing resinate:plga ratio 1:2) showed significantly lower drug release than f21 (containing resin­ ate:plga ratio 2:1) since the drug release rates decreases as the amounts of polymer increases which may create thicker coat around the particles in the microcapsules.32,33 while f19 (containing resinate:plga ratio 1:1) showed no significant decrease in drug release in comparison to f21, but f19 showed fig. 4 effect of temperature on drug encapsulation efficiency and percent of yield for the prepared formula for different resinate:plga ratio (a) 2:1, (b) 1:1 and (c) 1:2. a b c original 269j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 ameer z. wohaib et al. application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy fig. 5 effect of stirring speed on drug encapsulation efficiency and percent of yield for the prepared formula for different resinate:plga ratio (a) 2:1, (b) 1:1 and (c) 1:2. higher encapsulation efficiency and higher percent of yield than f21, therefore, it was selected for further study as shown in fig. 6. particle size analysis the results showed an increase in particle size of the selected resinate (588 µm) and selected prepared microcapsules (f19) (595 µm) in comparison to resin microsphere (306 µm). this was due to adsorption of the drug particles on the resin micro­ sphere during drc process and coating the resinate particles by plga polymer in the microencapsulation process.34 the result also showed that 90% of the selected prepared micro­ capsules (f19) was in 595 µm (particle size) which is more suitable for embolization therapy.35 fig. 6 cumulative percent of drug released of the selected prepared microcapsules f17, f19 and f21. a b c original 270 j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy ameer z. wohaib et al. differential scanning calorimetry figure 7 shows the thermogram of sulfasalazine, deae sephadex a­25, selected resinate, selected prepared microcap­ sules (f19). sulfasalazine exhibited a sharp exothermic peak at 246.5°c indicating the temperature of drug melting. the resin had no detected peaks at the range of temperature used. the drc showed that the drug exothermic peak was absent indi­ cating the formation of drc and no drug crystals available.36 the result also showed the absence of the drug exothermic peak in the prepared microcapsules indicating that microen­ capsulation process had no effect on drc. determination of morphological characterization the results showed the spherical shape of resin microspheres and the resinate showed similar appearance to resin micro­ sphere. the feature of sulfasalazine crystals was not detected indicating formation of drug resin mixture and the drug was dispersed molecularly in the resonates.36,37 the results also showed the prepared microcapsules had spherical shape with slightly irregular surface indicating the efficient deposition of plga on the surface of resinate with complete coating and no pores referring to the efficient conditions used for optimiza­ tion of the solvent evaporation method applied38 as shown in fig. 8. in vitro degradation study the results showed the gradual biodegradation of plga coat of selected formula in human serum where pores formed upon incubation and became more clear with time causing gradual controlled drug release with time accordingly. similar results obtained with sorafenib loaded imageable microspheres for embolization where plga polymer degradation gave lactic acid and glycolic acid in the biological fluid39,40 as shown in fig. 9. fig. 7 differential scanning calorimetry thermogram of (a) sulfasalazine, (b) deae sephadex a-25, (c) selected resinate, (d) selected prepared microcapsules (f19). a b c d original 271j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 ameer z. wohaib et al. application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy fig. 8 scanning electron microscopy (sem) of (a) deae sephadex a-25 microspheres (b) selected resinate (c) selected microcapsules formula (f19). a b c fig. 9 incubated samples of selected formula (f19) in human serum at 37°c for (a) 10, (b) 20 and (c) 30 days. a b c original 272 j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy ameer z. wohaib et al. preliminary in vivo test for embolization property figure 10 showed the rabbits ears images at 1, 3, 9, 15, 20 days after selected microcapsules formula (f19) had been injected in the ear central artery to evaluate the embolization property . the results showed the instant occlusion of the central auricular artery near the site of injection causing ischemia in the region within 3 days that continued to the end of the study period. this indicated that the prepared microcapsules having diameter (595 µm) aggregated enough to prevent arterial blood flow to the injected area forming embolization.41 there was no sign of tissue necrosis upon embolization during the study period. conclusion this work succeeded in preparation of microcapsules con­ taining sulfasalazine­ion exchange resin and optimized to get spherical microcapsules with 595 µm size which was suitable to induce embolization upon in vivo test using rabbits to pre­ vent blood supply to the region as well as controlling drug release for 45 days that may treat the solid tumor to be used as alternative to chemotherapy, radiotherapy and surgery. acknowledgment the authors would like to thank mustansiriyah university (www.uomustansiriyh.edu.iq) baghdad­iraq for its support in the present work. conflicts of interest none.  references 1. gahlyan m, jain s. oral controlled release drug delivery systema review. pharmatutor. 2014;2:170–178. 2. nidhi p, anamika c, twinkle s, bhakta mehul s, hitesh j, umesh u. controlled drug delivery system: a review. indo am j pharm sci. 2016;3:227–233. 3. sheth ra, sabir s, krishnamurthy s, avery rk, zhang ys, khademhosseini a, et al. endovascular embolization by transcatheter delivery of particles: past, present, and future. j funct biomater. 2017;8:1–12. 4. inal o, boz d. ion exchange resins and applications in pharmaceutical technology. adv j pharm life sci res. 2015;3:16–27. 5. makadia hk, siegel sj. poly lactic-co-glycolic acid (plga) as biodegradable controlled drug delivery carrier. polymers (basel). 2011;3:1377–1397. 6. chai f, sun l, he x, li j, liu y, xiong f, et al. doxorubicin-loaded poly (lactic-co-glycolic acid) nanoparticles coated with chitosan/alginate by layer by layer technology for antitumor applications. int j nanomedicine. 2017;12:1791–1802. 7. azad khan ak, howes dt, piris j, truelove sc. optimum dose of sulphasalazine for maintenance treatment in ulcerative colitis. gut. 1980;21:232–240. 8. lo m, ling v, low c, wang yz, gout pw. potential use of the antiinflammatory drug, sulfasalazine, for targeted therapy of pancreatic cancer. curr oncol. 2010;17:9–16. 9. singh i, kumar p, nagpal m, arora s. ion-exchange resin complexation: masking the bitter taste of cefuroxime axetil. rev cubana de farm. 2011;45:171–180. 10. mensah-biney r, reid kj, hepworth mt. the loading capacity of selected cation exchange resins and activated carbons for gold-thiourea complex. miner eng. 1995;8:125–146. 11. anand v, kandarapu r, garg s. ion-exchange resins: carrying drug delivery forward. drug discov today. 2001;6:905–914. 12. kaushik d. development of taste masked levofloxacin oral suspension using ion exchange resinates. j chem pharm res. 2016;8:385–394. 13. obeidat wm, price jc. evaluation of enteric matrix microspheres prepared by emulsion–solvent evaporation using scanning electron microscopy. j microencapsul. 2004;21:47–57. 14. yadav av, shete as, dabke ap, shinde vr. formulation and in-vitro evaluation of aceclofenac microcapsules. int j pharmtech res. 2009;1: 135–138. 15. jyothi nv, prasanna pm, sakarkar sn, prabha ks, ramaiah ps, srawan gy. microencapsulation techniques, factors influencing encapsulation efficiency. j microencapsul. 2010;27:187–197. 16. jafari sm, assadpoor e, he y, bhandari b. encapsulation efficiency of food flavours and oils during spray drying. drying technol. 2008;26:816–835. 17. dhakar rc, maurya sd, sagar bps, bhagat s, prajapati sk, jain cp. variables influencing the drug entrapment efficiency of microspheres: a pharmaceutical review. pharm lett. 2010;2:102–116. 18. yan j, wang f, chen j, liu t, zhang t. preparation and characterization of irinotecan loaded cross-linked bovine serum albumin beads for liver cancer chemoembolization therapy. int j polym sci. 2016;1:1–8. 19. kippax p. appraisal of the laser diffraction particle-sizing technique. pharm technol. 2005;29:88–96. 20. prakash k, raju pn, shanta kk, lakshmi mn. preparation and characterization of lamivudine microcapsules using various cellulose polymers. trop j pharm res. 2007;6:841–847. 21. lee sy, choi jw, lee jy, kim dd, kim hc, cho hj. hyaluronic acid/ doxorubicin nanoassembly-releasing microspheres for the transarterial chemoembolization of a liver tumor. drug deliv. 2018;25:1472–1483. 22. sharma kv, bascal z, kilpatrick h, ashrafi k, willis sl, dreher mr, et al. longterm biocompatibility, imaging appearance and tissue effects associated with delivery of a novel radiopaque embolization bead for image-guided therapy. biomaterials. 2016;103:293–304. 23. zhou x, kong m, cheng xj, feng c, li j, li jj, et al. in vitro and in vivo evaluation of chitosan microspheres with different deacetylation degree as potential embolic agent. carbohydr polym. 2014;113:304–313. fig. 10 rabbits ears which injected with selected prepared microcapsules (f24) after (a) control ears, (b) 1 day, (c) 3, (d) 9, (e) 15 and (f ) 20 days. a b c d e f original 273j contemp med sci | vol. 5, no. 5, september-october 2019: 264–273 ameer z. wohaib et al. application of plga-ion exchange resin microcapsules of sulfasalazine for embolization therapy 24. wikströms. sephadex® ion exchange media. edition aa data file ion exchange chromatography 2007:1-8. 25. irwin wj, belaid ka, alphar ho. drug delivery by ion exchange resin part iiiinteraction of ester prodrugs of propranolol with cation exchange resin. drug dev ind pharm. 1987;13:2047–2066. 26. srikanth mv, sunil sa, rao ns, uhumwangho mu, ramana murthy kv. ionexchange resins as controlled drug delivery carriers. j sci res. 2010;2: 597–611. 27. banerjee s, siddiqui l, bhattacharya ss, kaity s, ghosh a, chattopadhyay p, et al. interpenetrating polymer network (ipn) hydrogel microspheres for oral controlled release application. int j biol macromol. 2012;50:198–206. 28. othman m, ariff ab, kapri mr, rios-solis l, halim m. growth enhancement of probiotic pediococcus acidilactici by extractive fermentation of lactic acid exploiting anion-exchange resin. front microbiol. 2018;9:2554. 29. alexandridou s, kiparissides c. production of oil-containing polyterephthalamide microcapsules by interfacial polymerization. an experimental investigation of the effect of process variables on the microcapsule size distribution. j microencapsul 1994;11:603–614. 30. mukkala bvp, tegk m. studies on influence of process variables on performance of gliclazide mucoadhesive microcapsules. asian j pharm clin res. 2011;5:167–174. 31. chella n, yada kk, vempati r. preparation and evaluation of ethyl cellulose microspheres containing diclofenac sodium by novel w/o/o emulsion method. j pharm sci res. 2010;2:884–888. 32. jelvehgari m, hassanzadeh d, kiafar f, loveym bd, amin s. preparation and determination of drug-polymer interaction and in-vitro release of mefenamic acid microspheres made of cellulose acetate phthalate and/or ethylcellulose polymers. iran j pharm res. 2011;10:457–467. 33. jelvehgari m, nokhodchi a, rezapour m, valizadeh h. effect of formulation and processing variables on the characteristics of tolmetin microspheres prepared by double emulsion solvent diffusion method. indian j pharm sci. 2010;72:72–78. 34. nepal pr, chun mk, choi hk. preparation of floating microspheres for fish farming. int j pharm sci. 2007;341:85–90. 35. swaine t, tang y, garcia p, john j, waters lj, lewis al. evaluation of ion exchange processes in drug-eluting embolization beads by use of an improved flow-through elution method. eur j pharm sci. 2016;93: 351–359. 36. aman rm, meshali mm, abdelghani gm. ion-exchange complex of famotidine: sustained release and taste masking approach of stable liquid dosage form. drug discov ther. 2014;8:268–275. 37. samprasit w, akkaramongkolporn p, ngawhirunpat t, rojanarata t, opanasopit p. meloxicam taste-masked oral disintegrating tablet with dissolution enhanced by ion exchange resins and cyclodextrin. aaps pharmscitech. 2013;14:1118–1128. 38. akbarzadeh a, mikaeili h, zarghami n, mohammad r, barkhordari a, davaran s.preparation and in vitro evaluation of doxorubicin-loaded fe₃o₄ magnetic nanoparticles modified with biocompatible copolymers. int j nanomed. 2012;7:511–526. 39. kapoor dn, bhatia a, kaur r, sharma r, kaur g, dhawan s. plga: a unique polymer for drug delivery. ther deliv. 2015;6:41–58. 40. choi jw, park jh, cho hr, chung jw, kim dd, kim hc, et al. sorafenib and 2,3,5-triiodobenzoic acid-loaded imageable microspheres for transarterial embolization of a liver tumor. sci rep. 2017;7:554. 41. saralidze k, koole lh, knetsch mlw. polymeric microspheres for medical applications. materials (basel). 2010;3:3537–3564. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201906 original 218 j contemp med sci | vol. 4, no. 4, july-august 2019: 218–223 identification of strategies for collaboration of medical librarians with family physicians in advancing the goals of the family physician program based on experts’ viewpoints hasan ashrafi-rizi,1 mahdi amraei,1 hossein kargar,1 zahra ghazavi,2 and zahra kazempour3 1medical library and information science, health information technology research center, isfahan university of medical sciences, isfahan, iran. 2medical library and information science, iran university of medical sciences, tehran, iran. 3department of knowledge and information science, payame noor university, tehran, iran. correspondence to hasan ashrafi-rizi (email: hassanashrafi@mng.mui.ac.ir). (submitted: 08 may 2019 – revised version received: 21 may 2019 – accepted: 16 june 2019 – published online: 26 august 2019) objective the present study was carried out with the aim of identifying ways of collaboration of medical librarians with family physicians in advancing the goals of the family physician program based on the views of experts. methods this research was qualitative content analysis. the statistical population included experts in the fields of family physicians. sampling method was targeted and in order to identify qualified people, snowball sampling was used as well. the data collection tool was semi-structured interview, and the researchers exploited thematic analysis in the data analysis. to determine the validity and reliability, the lincoln and guba method was used based on four criteria of credibility, transferability, conformability and dependability. results regarding the common areas of medical librarians and family physicians, four concepts were extracted including helping physicians in scientific and professional mastering, upgrading research methodology, upgrading information literacy, and the content of information resources for patient training. moreover, four concepts were identified: appropriate policy-making, education suitability, participation in clinical research, and promotion of the quality of patient training in addition to six components of interaction strategies. conclusion strategies for the interaction of medical librarians with family physicians can become practical in areas such as proper policymaking, suitability of education, participation in clinical research, and the improvement of the quality of patient training. keywords collaboration strategies, family physicians, family physician program, medical librarians introduction the national family physician program was launched with the aim of establishing social justice and promoting family health as well as reducing the cost of treatment in iran. this program plays a pivotal role in all health system programs, and its proper implementation and achievement of its goals can ensure the achievement of high-level health indicators.1 the family physician places the patients in the referral system and supervises them from the time they are admitted to the system until the end of treatment and post-treatment care.2 the service package in the family physician program includes prevention, health promotion, initial treatment and emergency management, referral, and health management.1 in summary, the family physician program is performed to control the costs of repetitive services, increase accountability against patients, track services provided to patients, establish an electronic health record for the community individuals, increase the ability to control the quality of services and establish financial discipline, and direct the patients in the three levels of the service delivery system in a fully guided manner centered by the family physician. family physicians need to be trained to provide high-quality services. the results of a study by asadi showed that the knowledge and skills of family physicians are not sufficient to meet the expectations of the health system, and it is necessary to enhance the level of information and skills of physicians by holding workshops. in this study, the most important family physician weaknesses were the information gap between providers and recipients of information, lack of specialized training for the health team, especially physicians, and the inadequacy of culture-building and informing the public.1 in the study by movahedi et al.,3 lack of sufficient time to meet the specialized information by the physicians was the main reason for their need for the up-to-date information seeker, i.e. the medical librarians. in the study by habibi et al.,4 the main reasons for failure to use electronic resources by general practitioners were lack of familiarity with the internet, electronic resources, and medical databases, and general practitioners need to learn the internet, tools, and methods of searching the web and medical databases. in the study by rahimianfar et al.,5 it was shown that nurses understood the need for up-to-date information, but barriers such as lack of time and lack of familiarity with resources responding to their requirements caused them not to be able to seek information. the results of the investigation by papi et al. indicated that the information needs of physicians included conducting research, preparing articles, obtaining new and up-to-date information, managing patient care, teaching, and performing everyday tasks. however, physicians’ lack of comprehensive knowledge of electronic information resources has led to less use of such resources.6 findings by ciarlo et al. revealed that general practitioners prefer textbooks, and oncologists prefer magazines and the internet to meet their information requirements. moreover, based on the results, the causes of uncertainty during the patient counseling session were lack of time, knowledge and information. in fact, oncologists choose different ways to access information.7 findings in the study by traver et al.8 showed that physicians need a set of capabilities, organizational resources, and knowledge to acquire accurate digital literacy skills to help patients. the findings by bryant revealed that family physicians had to seek information to address their issn 2413-0516 original h. ashrafi-rizi et al. 219j contemp med sci | vol. 4, no. 4, july-august 2019: 218–223 identification of strategies for collaboration of medical librarians specialized needs including information on patient care, and use medical libraries less. nevertheless, they believed that using the potential of medical librarians is very effective in the work process. therefore, it can be claimed that medical librarians can play an effective role in meeting the information needs of family physicians.9 besides, the results of some studies indicate that over 40% of patients did not receive best-evidence-based care, and more than 20% of them received inadequate or inappropriate care.10 today, the health system should be safe, patient-centered, based on the best evidence and according to the patient’s values, and in the meantime, the doctor is responsible for maintaining the patient’s health and should complete the different stages of his patients’ treatment based on the latest scientific evidence. however, there is a gap between what the physicians do and what they should do in their daily practice,11 and despite the development of medical knowledge over the past years,12 there are still reports released about medical errors, endangering the lives of patients, and hence increased health care costs. given the investigations, it seems that the health sector has a special focus on information in all its dimensions including education, research, and treatment, and at all levels of individuals including policy-makers and health care providers and receivers. in this way, immediate access to valid and up-to-date information is indisputable for making correct decisions.13 in short, lack of physicians’ access to appropriate information causes a reduction in the quality of health services, an increase in health care costs for the patient and the health system, endangering the lives of patients, and the reduction of patient confidence in the health system. therefore, using the knowledge and skills of medical librarians by physicians will be very effective in dealing with various occupational issues. medical librarians, in addition to communicating with academic communities, have close links with patients and information seekers. in fact, medical librarians have a great mission in the health system by providing access to information, and help all institutions, organizations, and individuals whose main goals are specifically to protect and improve the health of individuals and society with one of these groups in the health system being family physicians. moreover, the health system is one of the most important parts constantly evolving under the influence of technological advances. in this area, the need for the dissemination and use of high quality health information in the field of education, research, and health care, on the one hand, and the change in the storage and retrieval of information, on the other hand, has made the presence of medical librarians essential in this field. medical librarians play an active role in the continuous education and modernity of information of the treatment team through presence in various sectors of the health system,14 and the family physician program is no exception. now, given the above issues, the basic question is raised: how can medical librarians play their role by utilizing their ability to meet the educational and information needs of the family physician program? and what are these roles? the family physician program will be successful when the basis for decisions in the education, research, treatment, and even management of the program is valid and up-to-date health information. family physicians need to use the potential and capabilities of medical librarians due to lack of adequate time, and sometimes the inability to seek and evaluate the information they need. so far, there has been no study in iran regarding the cooperation between medical librarians and family physicians. therefore, in this study, in addition to identifying these roles, it was decided to provide the necessary ground for using the capabilities of the medical librarians in the family physician program. therefore, the purpose of this study was to identify strategies for collaboration between medical librarians with family physicians in advancing the goals of the family physician program. materials and methods this research was qualitative content analysis. the statistical population included experts in the fields of family physicians, general practitioners in clinics and health centers, and medical librarians who were well informed about the family physician or who owned scientific works in this field in addition executive directors working in the field of family physician in iran. sampling method was purposeful and in order to identify qualified people, snowball sampling was used as well (10 participants remained after data saturation). the data collection tool was semi-structured interview. after identifying the samples, the time and place of the interview were coordinated and, before any interview, informed consent was received regarding participation in the interview. data saturation occurred through interviewing with 10 people. during the interview, the interviewer also took notes, all of which were used in the data analysis process. researchers used thematic analysis in the data analysis. in this way, each topic was read several times and a code was assigned to each topic in the end. similar codes formed a component on a more abstract level, which were more general than the original code, and eventually the same concepts were combined and, with more abstraction, the main concepts were formed. the data analysis method was manually executed. in order to determine the validity and reliability, the lincoln and guba method was used based on four criteria of credibility, transferability, conformability, and dependability.15 for data credibility, the researcher used the peer debriefing and providing the results for the participants. for data transferability, the researchers described the phenomenon in question thoroughly, and explained the research conditions and details of the procedure. in addition, for conformability, they accurately and thoroughly noted and reported the interview process, data collection, decision-making, and interpretations. for data dependability, the researchers’ notes were provided to peers outside the study (referees) during the interviews. the work process was also provided to the peers, and they were asked to express their views on the results. at the same time, the participants in the study entered the interview with satisfaction and could easily leave the interview at any time. results the present study was carried out with the aim of identifying the strategies of collaboration of medical librarians and family physicians in advancing the goals of the family physician program. the results of the analysis led to the creation of eight concepts and 15 components. findings regarding the common areas between medical librarians and family physicians to advance the goals of the family physician program resulted in the following four concepts. original 220 j contemp med sci | vol. 4, no. 4, july-august 2019: 218–223 identification of strategies for collaboration of medical librarians h. ashrafi-rizi et al. scientific and professional mastering the knowledge and skills of family physicians in achieving the goals of the family physician program are called the scientific and professional mastering. in fact, access to and use of clinical information is one of the main requirements of this concept. family physicians should be able to use the latest scientific findings to upgrade their specialized knowledge and skills. participants stated that family physicians should be informed of the patients’ lifestyles, the use of medications, the recognition of the basic principles of family health, environmental harmful factors, the epidemiological study of diseases, the recognition of various diseases, knowledge of common diseases, patient screening, etc. (table 1). upgrading research methodology the research methodology is a set of factors used by researchers to solve the research problem in a precise and systematic way. family physicians should themselves perform research for better prevention, diagnosis, treatment, and rehabilitation of patients, and performing high quality research requires exploiting various specialized fields including medical librarians. in this regard, participants pointed out some of their needs including more familiarity with the research process, conducting research in specific fields, conducting systematic research, understanding the information needs, the need for research colleague, recognition of modern technologies in conducting research, unwillingness to perform research, and the need for individual education, and stated that the presence of medical librarians can help them in the research (table 1). improving information literacy the ability to identify information needs, find information, and evaluate and effectively use information is called the information literacy. participants declared that they needed help from medical librarians in searching for information resources, using new technologies in accessing information, evaluating information resources, searching for specific topics, searching for work tasks, correct information, and information updating practices (table 1). content of the patient training information resources high-quality information to improve the health of the general public is one of the main tools used by family physicians. however, in the preparation and dissemination of this content, two main components should be considered: (a) information resources characteristics, (b) methods of providing information resource content. regarding the characteristics of the information resources, the participants stated that the content should be easy to understand, and specific attention should be paid to the characteristics such as the level of literacy, cultural issues, being rural or urban, simplicity and quality of content for patients. they also stated that the methods of receiving information should be different according to the patients’ needs. for example, printed brochures are needed to be used for a person, and films and other social media facilities may be used for others. anyway, both the type of content and the tool and method of accessing this information are affected by various variables, and family physicians and medical librarians table 1. common areas between medical librarians and family physicians in advancing the goals of the family physician program concept component codes scientific and professional mastering upgrading the professional knowledge of family physicians information associated with life style (p1), manner of medication use (p1), ability to understand the results of tests (p1), understanding the basic principles of family health (p2), identification and control of mental and physical health (p2), harmful factors in environment (p2), interpretation of premarital routine tests (p2), epidemiological study of diseases (p3), recognition of various diseases (p3), recognition of different cultures (p4), knowledge on common diseases (p5), recognition of communication skills (p5), how to work in groups (p5), information about specialized medical groups (p5), disease tracking (p7), patient screening (p10), understanding of diseases and patients (p10), information associated with the use of medication in families (p10), updating of clinical information of physicians (p10) upgrading research methodology understanding the research process familiarization with research process (p2), conducting research in specific fields (p3), contribution to research (p5), performing systematic research (p5), family physician research associate (p5) research prerequisites need for individual education (p10), recognition of information needs (p5), recognition of different databases (p5), recognition of new technology in conducting research (p8), unwillingness to conduct research (p10) improving information literacy correct information seeking help to search databases (p2), understanding how to use modern technology to access information (p5), finding information sources (p6), evaluation of information resources (p7), ability to search in specific topics (p10), searching in the field of work tasks (p10), correct information (p3) search-related factors searching for resources by others (p10), unwillingness to search resources (p10), lack of knowledge about the benefits of searching (p10), methods of updating information by physicians themselves (p3) education information resource content information resources characteristics comprehensibility of patient training brochures (p1), impact of residence location in the provision of information resources for patients (p1), patient training through images (p1), literacy and knowledge of people in the dissemination of information (p7), simplification of information for patients (p7), updating information resources for patients (p7), quality of information resources for patient training (p9), providing useful information to patients (p7) methods of providing information resource content variation of patient training information resources (p1), patient training through virtual social networking (p1), providing patients with suitable information (p4), provision of information resources for patients in various formats (p7), prioritization of some information resources in patient training (p8), information of the public of health issues (p9), appropriate information transfer to patients (p10) original h. ashrafi-rizi et al. 221j contemp med sci | vol. 4, no. 4, july-august 2019: 218–223 identification of strategies for collaboration of medical librarians should pay attention to these issues in performing their mission (table 1). findings about strategies of interaction of medical librarians with family physicians to advance the goals of the family physician program resulted in the following four concepts. appropriate policy-making systematic establishment of interaction between medical librarians and family physicians for advancing the goals of the family physician program is called the appropriate policy-making. in this regard, first, the position of medical librarians in the family physician program should be considered, and then the type and extent of cooperation and efforts should be developed and implemented for their mutual understanding. in the meantime, medical librarians should increase their level of knowledge and ability including improving communication skills, mastering the medical terminology, and recognizing all kinds of diseases (table 2). suitability of training training is essential for improving the patient’s health and increasing the quality of the services provided by physicians. the participants stated that in order to achieve this goal, medical librarians should help family physicians in two components. diversification of training today, education is very diverse, and physicians as well as patients should be able to benefit from these trainings according to their own circumstances. the participants argued that information resources should be provided in various forms to physicians and they should encourage them to use modern technologies. in addition, the cultural and social issues should be regarded in the provision of information resources for patients. training is needed to be provided sometimes in groups, sometimes virtually, and sometimes even individually. these conditions can be true for patients as well, and they must benefit from diversity in education depending on their conditions and facilities. educational content the content of information resources available to patients and physicians should be of high quality. information resources for patient training should be useful and valid. at the same time, these resources should be up-to-date and accessible to patients easily. family physicians should also use the latest scientific findings in the prevention, diagnosis, treatment, and rehabilitation process. medical librarians provide the necessary context for this important issue by providing valid information resources and facilitating access to information (table 2). contribution to clinical research targeted and systematic activities of medical librarians to help family physicians in conducting clinical research are called contribution to clinical research. this concept is explained by two components. table 2. strategies of interaction of medical librarians with family physicians to advance the goals of the family physician program concept component codes appropriate policy-making establishment of formal interaction lack of common understanding (p6), identification of the type of cooperation (p6), preparation of joint meetings (p7), establishment of formal relations in cooperation (p5), improvement of relationships (p10), lack of a legal framework for interaction (p6) effective interaction professionalization of medical librarians (p9), lack of confidence in medical librarians (p9), more familiarity with medical terms (p9), enhancing communication skills of medical librarians (p6) suitability of education diversification of education providing information resources in various formats (p7), using modern technologies in teaching physicians (p9), paying attention to cultural and social issues in the provision of patient training information resources (p1), group training of physicians (p1), virtual training of physicians (p9), singletraining of physicians (p10) education content proper response to the physician’s information needs (p3), provision of suitable materials for physicians (p5), identification of information needs of physicians (p9), comprehensibility of patient training brochures (p1), impact of residential area in the provision of information resources for patients (p1), training patients through images (p1), considering people’s literacy and knowledge in the dissemination of information (p7), simplifying information for patients (p7), updating information resources for patients (p7), quality of information resources for patient training (p9) contributing to clinical research information acquisition methods assistance in searching in the field of job tasks (p10), helping to search in specialized databases (p2), helping to conduct the research process (p2), contribution in family physician research (p5), evaluating information resources (p7), training virtual search methods (p10), providing access to databases (p10), training using different databases (p5) clinical research principles research process training (p2), contributing to research in specific fields (p3), research assistance (p5), collaboration in conducting systematic research (p5), helping physicians recognize knowledge gaps (p5), training new technologies in conducting research (p8), suitability of research methodology tailored to the needs of physicians (p10) upgrading the quality of patient training principles of information dissemination participation in patient information provision (p7), ability to work with physicians on patient training (p9), adequate knowledge of the proper dissemination of information among the general public (p10), assistance in updating information of physicians related to patient training (p10), identification of patient habits (p10) information dissemination tools providing appropriate tools for disseminating information for patients (p10), using modern information dissemination technologies (p8), teaching new technologies to patients (p7) original 222 j contemp med sci | vol. 4, no. 4, july-august 2019: 218–223 identification of strategies for collaboration of medical librarians h. ashrafi-rizi et al. information acquisition methods understanding the proper ways of information acquisition is one of the basic concepts in conducting clinical research by family physicians. the participants stated that they needed medical librarians to find information resources such as libraries and databases. moreover, the family physicians expected medical librarians to provide access to databases for them. principles of clinical research in the process of promoting community health, family physicists not only need the scientific findings of others, but they must also perform research themselves. learning and performing research require special expertise and exploitation of the ability of the expertise such as medical librarians. the participants stated that they had problems in the process of conducting research, research in specific fields, conducting systematic research, identifying knowledge gaps, and benefiting from new technologies in conducting clinical research, and that medical librarians could help them remarkably in this process (table 2). improving the quality of patient training patient training is subject to certain principles and rules and, if not followed, all the efforts of family physicians and health professionals will be eliminated. in patient training, one first needs to know the patients’ information needs and then use the appropriate tools for disseminating information among them. the content of these resources, while being up-to-date, should follow the principles of reading materials of health in simple language. medical librarians should also have the ability to work with physicians in patient training, which is because of the sensitivity of providing patients with services. furthermore, medical librarians should provide appropriate information in order to better train patients and update the knowledge and skills of family physicians (table 2). discussion since access to and use of clinical information is one of the essential requirements of the scientific and professional mastering of family physicians, they must be able to use the latest scientific findings on the types of diseases, screening of patients, recognition of the basic principles of family health, epidemiological investigation of diseases, and mastering in common diseases. some studies show that physicians need to be equipped with the latest scientific evidence regarding the scientific and professional mastering and to make accurate clinical decisions.15–17 also, the participants stated that the presence of medical librarians could help them in various research stages. in some studies, emphasis has been placed on performing research by physicians in addition to pointing out the role of medical librarians in the process of health research.18,19 family physicians need help from medical librarians to seek information resources, use new technologies for access to information, evaluate information resources, search for specific topics, search for work tasks, correct information, and information updating practices. physicians must gain the necessary information literacy skills in order to obtain the best evidence for treatment.20 according to different studies, information literacy of physicians is an inevitable necessity.21–23 in addition, helping physicians in conducting research and efforts to reduce the level of research anxiety of faculty members, especially clinical faculty members, have been predicted to be among the duties of medical librarians.24 high-quality information to improve the health of the general public is one of the main tasks of family physicians. therefore, the provision and dissemination of this content should focus on two main components: the characteristics of information sources and the ways of provision of the information resources content. several studies have emphasized the readability, simplicity, and intelligibility of information resources for patients.20,25–27 furthermore, in studies such as ashrafi-rizi et al.,28 developing credible information content for patient training has been considered one of the common responsibilities of medical librarians and healthcare professionals. experts believe that the position of medical librarians must first be identified in the family physician program, and then the type and extent of cooperation should be determined, and efforts should be made to mutually understand each other. at the same time, medical librarians need to increase their level of knowledge and ability including upgrading communication skills, more mastering the medical terminology, and recognizing all kinds of diseases. there were enough scientific works to explain, but the study by mcgowan29 focused on the economic analysis of responding of the medical librarians to the physicians’ questions, the results of which and other works can establish the context for interactions between medical librarians and family physicians. in some studies, the variety of training was emphasized, especially with regard to patients’ specific needs for information.30,31 at the same time, physicians themselves also need specific training given during their working conditions, lack of time, ability level, etc. and medical librarians should provide this context for them. in patient training, one first needs to know the patients’ information needs and then use the appropriate tools for disseminating information among them. medical librarians should also have the ability to work with physicians in patient training, which is because of the sensitivity of providing patients with services. furthermore, medical librarians should provide appropriate information in order to better train patients and update the knowledge and skills of family physicians. in some studies, the interaction of medical librarians and healthcare professionals regarding patient training has been emphasized.30–32 conclusion in this study, the platform for cooperation between the two professions was identified in four areas including helping physicians in scientific and professional mastering, promoting research methodology, improving information literacy, and the content of information resources for patient training. moreover, strategies for interaction of medical librarians with family physicians were presented in the frameworks of appropriate original h. ashrafi-rizi et al. 223j contemp med sci | vol. 4, no. 4, july-august 2019: 218–223 identification of strategies for collaboration of medical librarians policy-making, suitability of training, participation in clinical search, and upgrading quality of patient training. funding this paper has been sponsored by health information technology research center, the deputy of research and technology at isfahan university of medical sciences, isfahan, iran (ethics no: ir. mui.research.rec.1397.216). competing interests the authors have declared that no competing interests exist. conflicts of interest none.  references 1. asadi s. family physician assessment implementation based on the model (swot) in iran. j med educ dev. 2014;6:72–77. 2. shiri m, asgari h, talebi m, karamalian h, rohani m, narimani s. educational needs assessment of family (general) physicians working in rural health centers of esfahan districts in five domains. iran j med educ. 2011;10:726– 734 (article in persian). 3. movahedi f, ashrafi rizi h, sharif moghadam h. physicians’ perception about the role of clinical librarianship at alzahra medical center. j health adm. 2014;16:71–81. 4. habibi s, farzi j, lotfollahzadeh r. gps’ information seeking behavior in ardabil and their approach towards electronic sources. j ardabil univ med sci. 2008;8:136–141. 5. rahimianfar aa, hakimian r, salimi t. assessment of information seeking behavior of nurses in academic hospitals of yazd. health inf manage. 2014;10:706. 6. papi a, ghazavi r, moradi s. the awareness and use of electronic information resources by physicians in educational hospitals. health inf manage. 2015;11:712–727. 7. ciarlo g, liebl p, zell j, fessler j, koester m, ruetters d, et al. information needs of oncologists, general practitioners and other professionals caring for patients with cancer. eur j cancer care (engl). 2016;25:1015–23. 8. traver m, basagoiti i, martinez-millana a, fernandez-llatas c, traver v. experiences of a general practitioner in the daily practice about digital health literacy. the real needs. conf proc ieee eng med biol soc. 2016;2016:5644–5647. 9. bryant sl. the information needs and information seeking behaviour of family doctors. health info libr j. 2004;21:84–93. 10. grol r, grimshaw j. from best evidence to best practice: effective implementation of change in patients’ care. lancet 2003;362:1225–1230. 11. manavi s, olyaee manesh a, yazdani s, shams l, nasiri t, shirvani a, et al. model for implementing evidence based health care system in iran. iran j public health. 2013;42:758–766. 12. dawes m. critically appraised topics and evidence-based medicine journals. singapore med j. 2005;46:442–448; quiz 449. 13. shahrzadi l, ashrafi-rizi h. health information consulting: key role of medical librarians. j med educ dev. 2016;9:1–4. 14. hodhodinezhad n, ashrafi-rizi h, shahrzadi l, soleymani m. effect of using social marketing techniques on the knowledge and attitudes of students and faculty members of isfahan university of medical sciences to the medical librarianship services. j health adm. 2016;19:31–42. 15. hariri n. principles and methods of qualitative research. kerman: islamic azad university, science and research branch of tehran; 2016. 16. sahapong s, manmart l, ayuvat d, potisat s. a systematic review of the roles and competencies of medical information professionals(mips) in evidence-based medicine in thailand; 2006. p. 426–435. 17. schuers m, griffon n, kerdelhue g, foubert q, mercier a, darmoni sj. behavior and attitudes of residents and general practitioners in searching for health information: from intention to practice. int j med inform. 2016;89:9–14. 18. ashrafi-rizi h, fateme z, khorasgani zg, kazempour z, imani st. barriers to research activities from the perspective of the students of isfahan university of medical sciences. acta inform med. 2015;23:155–159. 19. ashrafi-rizi h, najafi ns, kazempour z, taheri b. research self-efficacy among students of isfahan university of medical sciences. j educ health promot. 2015;4:26. 20. cedars be, cohen aj, fergus kb, baradaran n, ndoye m, kamal p, et al. qualitative analysis of the content found in online discussion boards for urethral stricture disease and urethroplasty. urology 2019;130:155–161. 21. farokhzadian j, ahmadian l, khajouei r, mangolian shahrbabaki p. information literacy and training needs of nursing managers for evidencebased practice. iran j nurs. 2016;29:43–56. 22. kalavani a, kazerani m, shekofteh m. evaluation of sbmu residents’ knowledge and use of evidence-based medical databases and concepts. j payavard salamat 2018;12:34–43. 23. ross j. information literacy for evidence-based practice in perianesthesia nurses: readiness for evidence-based practice. j perianesth nurs. 2010;25:64–70. 24. ashrafi-rizi h, zarmehr f, bahrami s, ghazavi-khorasgani z, kazempour z, shahrzadi l. study on research anxiety among faculty members of isfahan university of medical sciences. mater sociomed. 2014;26:356–359. 25. abu-heija aa, shatta m, ajam m, abu-heija u, imran n, levine d. quantitative readability assessment of the internal medicine online patient information on annals.org. cureus 2019;11:e4184. 26. alfonso ar, demitchell-rodriguez em, ramly ep, noel dy, levy-lambert d, wang mm, et al. assessment of american cleft palate-craniofacial association-approved teams’ websites for patient-oriented content and readability. cleft palate craniofac j. 2019;56:1213–1219. 27. yiu a, ng kk, lee vw, bajorek bv. evaluating the understandability and actionability of web-based education materials for patients taking non-vitamin k oral anticoagulants. ther innov regul sci. 2019:2168479019849878. 28. ashrafi-rizi h, shahrzadi l, dehghani-champiri z. identification of patients’ rights to benefit from consumer health information services: a delphi study. j educ health promot. 2019;8:102. 29. mcgowan j, hogg w, zhong j, zhao x. a cost-consequences analysis of a primary care librarian question and answering service. plos one 2012;7:e33837. 30. truccolo i. providing patient information and education in practice: the role of the health librarian. health info libr j. 2016;33:161–166. 31. zhu y, ghajar m, mitre e. share: spreading health awareness with resources and education -librarians’ role in patient education, a case study. j hosp librariansh. 2016;16:319–327. 32. sollenberger jf, holloway rg jr. the evolving role and value of libraries and librarians in health care. jama 2013;310:1231–1232. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.08201907 original 279j contemp med sci | vol. 6, no. 6, november–december 2020: 279–281 original issn 2413-0516 introduction liposuction remains one of the most widely cosmetic surgical procedures and is increasingly widespread every year.1 however, since its introduction, legitimate fears over patient welfare have contributed to restrictions on the amount of fat that can be sucked.2-4 these limits are primarily determined by hemodynamic variations and blood loss that may arise during liposuction.4 previously, it has been shown that tranexamic acid, as an anti-fibrinolytic agent, could prevent the conversion of plasminogen to plasmin in a competitive manner, and prevent the binding and degradation of fibrin and maintaining the basis of its matrix structure.5 studies in various medical areas, such as orthopedic surgery, cardiology, and gynecology, have demonstrated that it can minimize blood loss and transfusion needs.6-8 in recent years, the surgical use of tranexamic acid to prevent blood loss has been resurrected and the use of tranexamic acid has been co-opted by cosmetic surgeons to limit intraoperative bleeding.9 this has been found to be especially successful in burns and craniomaxillofacial and cosmetic procedures. although several publications have cited its use in liposuction, its effectiveness in reducing pre-operative blood loss during liposuction has not yet been studied.10 on the other hand, epinephrine is the most widely used blood vessel constrictor and blood coagulation accelerator, particularly on the skin or mucous membranes to control bleeding at the site of the operation.6, 7 it can reduce the absorption of local anesthetics into the bloodstream, resulting in decreased systemic toxic side effects, longer therapeutic duration of action, and decreased blood loss.11 the effect of epinephrine, with its non-selective adrenergic properties on the skin and subcutaneous tissues, in a local anesthetic solution is exerted by the constriction of local vasculatures and a decrease in local blood flow.8, 9 epinephrine, however, has serious side effects and there are some drawbacks owing to possible dose-related heart effects.7 in addition, the change in its concentration tends to have various effects on the severity of bleeding in the surgical cut.12 the optimum dosage of epinephrine for the prevention of bleeding has still not been specifically established and is controversial in dermatological surgery.9-12 this research was conducted to assess the influence of dose-dependent epinephrine supplementation in local anesthesia on intraoperative bleeding regulation, and also to assess its effects on hemodynamic properties during dermatological surgery. based on the above-mentioned information, we aimed to figure out the effectiveness of using epinephrine in tumescent solution during liposuctions surgery. material and methods in this study, we present a prospective, double-blind, nonrandomized study evaluating the effects of adding epinephrine to tumescent solution intraoperative in patients undergoing liposuction. thirty-six patients including 6 males and 29  females undergoing liposuction were divided into two groups (n=18 in each group). in this study, inclusion criteria were as follows: patients who were referred for abdominal liposuction surgery, have a complete pre-operative examination with a history and examination without any problem or liposuction contraindication. comparative evaluation about the efficacy of the use of variable concentrations of epinephrine in tumescent solution for control of bleeding in patients with abdominal liposuction mahtab farhadi, abdolreza khorshidifar, abdolazim ghalambor, alireza bakhshaeekia department of plastic and reconstructive surgery, faculty of medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran corresponding author: abdolreza khorshidifar (email: abdolrezakhorshidifar@yahoo.com) (submitted: 12 september 2020 – revised version received: 29 september 2020 – accepted: 23 october 2020 – published online: 26 december 2020) abstract objective: the use of epinephrine for controlling the blood loss has gained out in many dermatological surgeries; however, its use in liposuction has not been studied. in this regard, we aimed to figure out the effectiveness of using epinephrine in tumescent solution during liposuctions surgery. methods: in this study, we present a prospective, double-blind, non-randomized study evaluating the effects of adding epinephrine to tumescent solution intraoperative in patients undergoing liposuction. thirty-six patients including 6 males and 29 females undergoing liposuction were divided into two groups. in case group, we use 1–1.4 mg/l epinephrine (based on the location of surgery) in tumescent solution; and control group did not receive epinephrine. lab data such as hemoglobin and hematocrit as well as clinical data including blood pressure and heart rate were recorded before, after 1 h, and 6 h of liposuction. results: in this study, we observed that both case and control group faced a significant drop in their hemoglobin and hematocrit levels; however, the decrement was significantly lower in case group. in addition, both groups had a stable hemostasis after 1 h and 6 h of surgery. in this regard, we did not observe any significant difference between heart rate and blood pressure of two groups. conclusion: the results of this study suggest that using epinephrine as vasoconstriction agent in tumescent solution might decrease the rate of bleeding and increase the chance of stable hemostasis both during and after abdominal liposuction. keywords: liposuction, epinephrine, blood loss, bleeding control, hemostasis. 280 original comparative evaluation about the efficacy of the use of variable concentrations mahtab farhadi j contemp med sci | vol. 6, no. 6, november–december 2020: 279–281 in this study, liposuction surgery of the abdomen and flanks liposuction are conducted without any other surgery. all cases in both control and experimental groups are performed by the same expert cosmetic surgeon and team in one surgical center with the same lipomatic device. the body mass indexes were between 30 and 40 and the volume of transfused tumescent liquid was equal to the volume of lipid which has been aspirated from patient. due to the lower density of fat tissue in lower abdominal and lower risk of bleeding in this region, we used 1 mg/l of epinephrine in these cases and we add epinephrine at the dose of 1.4 and 1.2 mg/l in cases with liposuction of right/ left flanks and upper abdomen, respectively. in addition, we monitor the blood pressure and pulse rate during the surgery and every 6 h after surgery. also, we routinely check anemia and hypovolemia by evaluating the hemoglobin and hematocrit before and 24 h after surgery to possible blood transfusion after surgery. the data were described by frequency (percentage (%)) or mean ± sd, and the comparison between the two groups was tested by k2 test. all statistical analysis was implemented in spss 25.0, p-values <0.05 were considered statistically significant. results of the 36 cases enrolled in the current study, 18 were enrolled in the control group and 18 in the control group, with a total of 7 men (19.4%) and 29 women (80.6%). there were 3 men (16.7%) and 15 women (83.3%) in the control group. in the control group, there were 4 men (22.2%) and 14 women (77.8%). the overall mean age of case group was 40.69±8.75 years with a maximum age of 56 years and a minimum of 26 years. in the control group, the mean was 43±9.09 with a maximum age of 56 and a minimum of 28 years. the mean age in case group was 38.3±7.9 years with a maximum age of 56 and a minimum of 26 years. all details have been mentioned in the table 1. our results showed no significant difference alongside with these two groups regarding to age and genders (case and control, p>0.05). an average of 3.5 l of fat has been sucked during the liposuction procedure, with a maximum of 7 l and a minimum of 3.5 l. in addition, the results failed to show any significant difference between tow control and case groups (p>0.05). the hemoglobin of case group was 13.2 before liposuction became 12.5 after 24 h; and the control group had the mean hemoglobin of 12.9 before and 11.35 after surgery. the results showed a significant higher rate of hemoglobin in case group in comparison to control group after surgery but not before (p<0.05). hematocrit also increased from 39.2% in the control group to 37.78 after the intervention, but in the control group hematocrit dropped from 38.68 to 34.65 after the intervention, which was also a significant difference between the two groups after intervention (p<0.05). on the other hand, the systolic blood pressure in the control group increased from 124.7 to 127.5 in the first hour and dropped into 124.72 in the next 6 h; and in the control group, the systolic blood pressure decreased from 123.33 to 120.0 in the first hour to 116.38 in the next 6 h. the difference was not significantly different between two groups before and after surgery (p>0.05). in addition, the diastolic blood pressure was not statistically significant between the two groups (p>0.05). in terms of heart rate, in the control group, the mean heart rate before the intervention was 83.66 bpm and 1 h after the intervention was 88.61, and 6 h after the intervention was 83.72; in the control group heart rate before the surgery was 79.61 and 1 h after was 78.61, and 6 h later, it was 77.5. there was no significant difference between two groups (p>0.05). table 1. the statistical reports in two case and control groups. item total no. patients mean±sd p value age 36 40.69±8.75 0.328 case 18 38.3±7.9 control 18 43±9.09 gender (m:f) 36 0.873 case 18 4:14 control 18 3:15 hemoglobin before 36 0.491 case 18 13.2±1.3 control 18 12.9+1.1 hemoglobin after 36 0.039 case 18 12.5±1.2 control 18 11.35±0.92 hematocrit before 36 0.383 case 18 39.2±3.9 control 18 38.68±3.2 hematocrit after 36 0.048 case 18 37.78±3.3 control 18 34.65±3.5 blood pressure before 36 0.771 case 18 124.7±10.9 control 18 123.33±11.3 blood pressure 1 h later 36 0.569 case 18 127.5±10.5 control 18 120.0±9.4 blood pressure 6 h later 36 0.388 case 18 124.7±9.7 control 18 116.38±8.8 pulse rate before 36 0.719 case 18 83.6±5.0 control 18 79.61±5.8 pulse rate 1 h later 36 0.823 case 18 88.6±5.6 control 18 78.61±5.1 pulse rate 6 h later 36 case 18 83.7±5.2 0.533 control 18 77.5±4.1 281 original comparative evaluation about the efficacy of the use of variable concentrationsmahtab farhadi j contemp med sci | vol. 6, no. 6, november–december 2020: 279–281 discussion according to the current study, it seems that increasing the dose of epinephrine is effective in reducing bleeding due to abdominal suction; and increasing the dose of epinephrine significantly reduced the both hemoglobin and hematocrit, but this issue was seen in control as well. in addition, there was no statistically significant change in blood pressure and heart rate of the cases because these parameters might be affected by other conditions in the subject, such as pain and anxiety due to surgery. several randomized experiments were justified in order to offer definitive proof as to the impact of epinephrine on hemorrhage during various forms of operations.13-15 epinephrine is a sympathomimetic amine with beta-adrenergic agonist receptor effects. various arterioles, particularly in the skin and mucosa, exhibit vasoconstriction due to the prevailing stimulation of the α-receptor.4 the minimum dosage of epinephrine to achieve sufficient and minimal toxic effect on hemostasis is yet to be specifically defined in dermatological surgery. few experiments remain in the literature to determine the optimum dosage of epinephrine in human subjects. in this regard, previous studies have shown that local anesthetics with 1:100,000 epinephrine, have been suggested to be consistent of sufficient vasoconstriction.16 regulation of the dosage of epinephrine as a vasoconstrictor not only decreases the magnitude of unintended results, but can also decrease intraoperative bleeding.17 epinephrine in combination with local anesthetic solution has been found to minimize perioperative bleeding from surgical wound sites in a variety of surgical environments.18-20 it has been shown that the 1:50,000 dose of epinephrine can have the highest hemostasis when used as an infiltration injection.21 however, owing to its rebounding activity and systemic cardiovascular side effects such as tachycardia, it can be used sparingly.22 in our research, hemodynamic changes such as heart rate and systolic blood pressure were not observed following surgery. also, in the current study, no side effects were observed due to increasing the dose of epinephrine, and also considering the cheapness and availability of this drug in terms of cost-effectiveness, it seems reasonable to use a higher dose of this drug depending on the surgeon. major hemodynamic changes, especially hypotension, have been reported in a lidocaine-receiving community with 1:200,000 epinephrine. however, these improvements were found to have lasted no longer than 4 min.23 in another analysis of 3rdto 6th-min heart rate, systolic arterial pressure from 3rdto 5th-min and diastolic arterial pressure from 2ndto 6th minute following local injection were both higher in patients with higher epinephrine concentrations.24 overall, this study showed that using epinephrine is safe in liposuction surgery when used with tumescent solution. this issue could decrease the rate of blood transfusion to reduce the viral and bacterial infection, and increase the economically and psychologically imposed on the patient. conclusion the results of this study suggest that using epinephrine as vasoconstriction agent in tumescent solution might decrease the rate of bleeding and increase the chance of stable hemostasis both during and after abdominal liposuction. references 1. folwaczny c, heldwein w, obermaier g, schindlbeck n. influence of prophylactic lo-cal administration of epinephrine on bleeding complications after polypectomy. endoscopy 1997;29:31-3. 2. koay j, orengo i. application of local anesthetics in dermatologic surgery. dermatol surg 2002;28:143-8. 3. fink br, aasheim gm, levy ba. neural pharmacokinetics of epinephrine. anesthesiology 1978;48:263-6. 4. dunlevy tm, o’malley tp, postma gn. optimal concentration of epinephrine for vasoconstriction in neck surgery. laryngoscope 1996;106:1412-4. 5. pallasch tj. vasoconstrictors and the heart. j calif dent assoc 1998;26:668-76. 6. brown rs, rhodus nl. epinephrine and local anesthesia revisited. oral surg oral med oral pathol oral radiol endod 2005;100:401-8. 7. milam sb, giovannitti ja jr. local anesthetics in dental practice. dent clin north am 1984;28:493-508. 8. o’malley tp, postma gn, holtel m, girod da. effect of local epinephrine on cutaneous blood flow in the human neck. laryngoscope 1995;105:140-3. 9. santos cf, modena kc, giglio fp, sakai vt, calvo am, colombini bl, et al. epinephrine concentration (1:100,000 or 1:200,000) does not affect the clinical efficacy of 4% articaine for lower third molar removal: a doubleblind, randomized, crossover study. j oral maxillofac surg 2007;65:2445-52. 10. sisk al. comparison of etidocaine and lidocaine for control of intraand post-operative bleeding and pain. j oral maxillofac surg 1986;44:16-20. 11. agresti a. categorical data analysis. 2nd ed. boston: john wiley & sons, 2002. 12. guinard jp, carpenter rl, morell rc. effect of local anesthetic concentration on capillary blood flow in human skin. reg anesth 1992;17:317-21. 13. cansanção al, cansanção aj, cansanção bp, vidigal ra. lipoabdominoplasty in obese patients: is it safe? has good results? plast reconstr surg. 2015;136(suppl):93–94. 14. rajesparan k, biant lc, ahmad m, field re. the effect of an intravenous bolus of tranexamic acid on blood loss in total hip replacement. j bone joint surg br. 2009;91:776–783. 15. amer km, rehman s, amer k, haydel c. efficacy and safety of tranexamic acid in orthopaedic fracture surgery: a meta-analysis and systematic literature review. j orthop trauma 2017;31:520–525. 16. horrow jc, hlavacek j, strong md, et al. prophylactic tranexamic acid decreases bleeding after cardiac operations. j thorac cardiovasc surg. 1990;99:70–74. 17. gai my, wu lf, su qf, tatsumoto k. clinical observation of blood loss reduced by tranexamic acid during and after caesarian section: a multi-center, randomized trial. eur j obstet gynecol reprod biol. 2004;112:154–157. 18. tang ym, chapman tw, brooks p. use of tranexamic acid to reduce bleeding in burns surgery. j plast reconstr aesthet surg. 2012;65:684–686. 19. murphy gr, glass ge, jain a. the efficacy and safety of tranexamic acid in cranio-maxillofacial and plastic surgery. j craniofac surg. 2016;27:374–379. 20. nair as, sriprakash k, nirale am, et al. large volume lipo-suction: perioperative considerations. int j sci res publications 2013;3:1–4. 21. nair as, verma s. use of tranexamic acid in megaliposuction. int j pharm pharm sci. 2015;7:8. 22. ors s, ozkose m. late postoperative massive bleeding in septorhinoplasty: a prospective study. plast surg (oakv.) 2016;24:96–98. 23. butz dr, geldner pd. the use of tranexamic acid in rhytidectomy patients. plast reconstr surg glob open 2016;4:e716. 24. ausen k, fossmark r, spigset o, pleym h. randomized clinical trial of topical tranexamic acid after reduction mammoplasty. br j surg. 2015;102:1348–1353. https://doi.org/10.22317/jcms.v6i6.857 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 197j contemp med sci | vol. 5, no. 4, july-august 2019: 197–201 1department of pediatrics, college of medicine, kerbalaa university, kerbalaa, iraq. 2department of pathology, college of medicine, kerbalaa university, kerbalaa, iraq. 3department of pediatrics, children teaching hospital, kerbalaa, iraq. 4college of medicine, kerbalaa university, kerbalaa, iraq. correspondence to usama al-jumaily (email: drusama2004@yahoo.com). (submitted: 22 april 2019 – revised version received: 28 april 2019 – accepted: 17 may 2019 – published online: 26 august 2019) clinico-pathological spectrum of childhood central nervous system tumors in iraq: a single-institutional study usama al-jumaily,1 rasha al-safi,2 sabah al-mosawi,3 homam al-obaidy,4 and mohammed fawzi2 introduction tumors of the nervous system in childhood occupy the second most common tumor after leukemia.1–4 they comprise approximately one-third of all childhood malignancies. in the setting of intricacy of treatment, physical and neuropsychological deficits, and neuroendocrine sequelae, they are considered one of the main causes of morbidity and mortality in children.5,6 childhood central nervous system (cns) tumors differ from adult brain tumors with respect to the primary site of involvement, clinical behavior, early metastases, histological and biologic characteristics. recently, with the advent of biological investigations and the evolution of molecular and genetic studies, therapeutic approaches direct toward each specific tumor type.6–9 this mandates a thorough knowledge of the incidence and distribution of the various cns neoplasms. although data from the western countries regarding the epidemiology of cns tumors are well documented, such reliable studies are scant and nascent in developing nations. specifically, till date, there is no documented data pertinent to the profile of pediatric cns tumors in iraq, which is actually very fastidious because of disparity in the management of such tumors. the present study therefore attempts to profile the clinico-pathological features of pediatric cns tumors (according to the who classification) at a single tertiary center in iraq. the data has been compared with published statistics from population and hospital-based series. materials and methods data of primary cns tumors of the brain and spinal cord in the pediatric age group (<18 years of age) was collected from the medical records of single tertiary hospital in iraq, imam hussein cancer center at kerbalaa governorate that was established in january, 2014. because of complexity of management, the necessity for multidisciplinary approach, and practical inexperience of treating children with primary cns tumors, this center was the first in iraq managing those children with primary cns tumors in addition to other malignancies. children with various neurological symptoms with a recent diagnosis of primary cns tumors, from different parts of the country visiting or referred to the above mentioned cancer center were properly managed according to internationally adopted protocols by surgery, radiotherapy, and chemotherapy. the conducted time period was from january 2014 to december 2017 (i.e., 4 year period study). patients with primary cns tumors who were younger than 18 years at the time of diagnosis was conducted. all cases with secondary cns involvement (i.e., the primary tumor is elsewhere in the body with secondary metastases to the cns) were excluded from the study. data were retrieved from medical records and pathology databases. patients included in this study were diagnosed depending on the characteristic site of the primary tumor and histological characteristics categorized according to the world health organization classification 2007.3,10 we reviewed patients’ characteristics [demographics, tumor location, pathology, living site], treatment plan (chemotherapy, surgery, and radiotherapy), and outcome. the collected data were analyzed and compared with available published data in tumor registries and hospital-based studies. results patients’ characteristics demographics from january 2014 to december 2017, 54 patients <18 years old with primary cns tumors were enrolled in this study. objectives the present retrospective study analyzed the spectrum of pediatric central nervous system (cns) tumors in a single tertiary hospital in iraq. methods data regarding frequencies of various primary cns tumors (diagnosed according to the world health organization classification), in pediatric patients (<18 years of age), were collected from a single tertiary care hospital in iraq for a period of 2014–2017. results fifty-four children were diagnosed with primary cns tumors. the most common primary pediatric cns tumors were medulloblastoma (37%), followed by low grade gliomas (lggs) (29.6%), high grade gliomas (0.011%), cns germinoma (0.09%), supratentorial primitive neuro-ectodermal tumors and ependymomas (0.037% for each). rare tumors encountered were oligodendrogliomas, choroid plexus carcinoma, and pineoblastoma (0.18% for each). the most common lggs tumor was pilocytic astrocytoma. conclusion this is the first study reporting the spectrum of cns tumors in children in iraq. except for a higher frequency of cns germinoma, the profile of other pediatric cns tumors in iraq is relatively similar to that reported in other countries. keywords astrocytoma, epidemiology, medulloblastoma, pediatric brain tumor, world health organization classification issn 2413-0516 original 198 j contemp med sci | vol. 5, no. 4, july-august 2019: 197–201 childhood central nervous system tumors u. al-jumaily et al. there were 31 males (57.4%) and 23 females (42.6%) with a median age of 6 years (range 1–18 years). regarding the age at the time of diagnosis, 23 patients were 5 years and less, 21 patients aged between 5 and 10 years, and 10 patients were above 10 years of age. tumor sites of the 54 patients, 27 (50%) patients had a tumor at the posterior fossa/cerebellum site; the next common sites of involvement in descending order were cerebrum (eleven patients; i.e., 20.4%), suprasellar/hypothalamic (five patients; i.e., 9.35%), brain stem (four patients, i.e., 7.4%), spinal cord (two patients; i.e., 3.7%), optic nerve (one patient; i.e., 1.9%), and pineal gland (one patient; i.e., 1.9%) (table 1). pathological diagnosis of the 54 patients, 23 (42.59%) patients had embryonal tumors histology (20 patients, medulloblastoma; two patients, supratentorial primitive neuroectodermal tumor pnet, and one patient, pineoblastoma); the second most common histology was low grade glioma (lgg) comprising 31.48% (16, pilocytic astrocytoma; one, oligodendroglioma) of all cohort group. high grade gliomas (hggs) were the third most common tumors (six patients, 11.1%) followed by cns germinoma histology (five patients, 9.25%), ependymoma (two patients, 3.7%), and choroid plexus carcinoma (one patient, 1.85%) (table 2). table 3 showed the distribution of various histological subtypes according to the original site of involvement. living place thirty-four patients (i.e., 63%) reside in urban cities; the rest of the cohort group (i.e., 20 patients, 37%) lives in rural regions. modalities of treatment surgery. forty-six patients were submitted to surgery including either biopsy/pr (partial resection), subtotal resection (str), or gross total resection (gtr). for the remaining eight patients who did not undergo any kind of surgery, the site of tumor was considered inaccessible (brain stem gliomas) or detrimental (optic nerve gliomas), and the diagnosis was made depending on the radiological findings. another one patient with choroid plexus carcinoma was not subjected to surgery because of bad clinical status and the diagnosis was also established based on the consistent radiological appearance (table 4). chemotherapy. forty-six patients received chemotherapy adopted according to the primary histology of the tumor. well-known international chemotherapy protocols were followed (cog and siop cns tumors protocols). only eight patients were not received chemotherapy; three of them had brain stem diffuse pontine gliomas, two were having ependymoma, and three patients with pilocytic astrocytoma who underwent complete surgical resection of their primary tumors (table 4). radiotherapy. radiotherapy (either as craniospinal irradiation or focal radiotherapy) was given to 33 patients according to recommended protocol based on the histological type and site of the tumor. the remaining 21 patients did not receive radiotherapy because of the following: 13 patients with lggs who underwent surgery other than gtr followed by chemotherapy; another three patients with lggs who underwent complete surgical resection and were only followed clinically and radiologically; three patients with medulloblastoma who were under 3 years of age and received only chemotherapy after surgery; one patient with choroid plexus carcinoma who received only one cycle of chemotherapy and died because of tumor progression; and another one patient with germinoma who received chemotherapy and parents refused further treatment (table 4). outcomes. out of 54 cases diagnosed at our institute, there were 14 deaths (25.9%) in our study; the highest mortality were in embryonal tumors (seven patients) and hgg (five patients) due to disease progression; one patient with choroid plexus carcinoma also died because of advanced tumor disease, and another one patient with lgg who died because of intracranial hemorrhage related to thrombocytopenia as a sequel of chemotherapy effects. nine patients (16.7%) abandoned treatment or lost follow-up. moreover, 26 patients (48.1%) have completed treatment and are on regular follow-up, and four (7.4%) patients are currently on treatment. one patient (1.9%) with ependymoma is still alive with disease recurrence until the date of writing this study (table ). discussion pediatric cns tumors accounted on an average 14.8% of total intracranial tumors (ranging from 10% to 21%). in our study, there is a slight male preponderance (the overall male to female ratio was 1.34:1); this ratio is variable in other reported series worldwide.2,5,7,11–13 table 1 frequency and percentage of pediatric cns tumors according to the primary site of involvement site frequency percentage cerebellum 27 50.0 cerebrum 11 20.4 suprasellar 5 9.3 brain stem 4 7.4 spinal cord 3 5.6 optic nerve 3 5.6 pineal gland 1 1.9 total 54 100.0 table 2 frequency and percentage of pediatric cns tumors according to histological type histology frequency percentage medulloblastoma 20 37.0 pilocytic astrocytoma 16 29.6 hgg 6 11.1 cns germinoma 5 9.3 supratentorial pnet 2 3.7 ependymoma 2 3.7 pineoblastoma 1 1.9 oligodendroglioma 1 1.9 choroid plexus carcinoma 1 1.9 total 54 100.0 pnet, primitive neuroectodermal tumors; hgg, high grade gliomas; cns, central nervous system original 199j contemp med sci | vol. 5, no. 4, july-august 2019: 197–201 childhood central nervous system tumorsu. al-jumaily et al. table 3 distribution (with its percentage) of various histological types according to primary site of involvement bs cerebellum spinal cord suprasellar cerebrum pineal optic nerve mb 0 20 (100) 0 0 0 0 0 pilocytic astrocytoma 1 (6.3) 5 (31.3) 3 (18.8) 0 4 (25) 0 3 (18.8) cns germinoma 0 0 0 5 (100) 0 0 0 hgg 3 (50) 1 (16.7) 0 0 2 (33.3) 0 0 pineoblastoma 0 0 0 0 0 1 (100) 0 supratentorial pnet 0 0 0 0 2 (100) 0 0 ependymoma 0 1 (50) 0 0 1 (50) 0 0 oligodendroglioma 0 0 0 0 1 (100) 0 0 choroid plexus carcinoma 0 0 0 0 1 (100) 0 0 total 4 27 3 5 11 1 3 bs, brain stem gliomas; mb, medulloblastoma; hgg, high grade gliomas; pnet, primitive neuroectodermal tumor. table 4 modalities of treatment according to various histological subtypes subtype of tumor-modalities of treatment frequency percentage mb—surgery, radiotherapy, chemotherapy 18 33.3 pilocytic astrocytoma—surgery, chemotherapy 9 16.7 pilocytic astrocytoma—chemotherapy 4 7.4 hgg—radiotherapy 3 5.6 hgg—surgery, radiotherapy, chemotherapy 3 5.6 pilocytic astrocytoma—surgery 3 5.6 mb—surgery, chemotherapy 2 3.7 cns germinoma—surgery, radiotherapy, chemotherapy 2 3.7 cns germinoma—surgery, chemotherapy 2 3.7 supratentorial pnet—surgery, radiotherapy, chemotherapy 2 3.7 ependymoma—surgery, radiotherapy 2 3.7 cns germinoma—radiotherapy, chemotherapy 1 1.9 pineoblastoma—surgery, radiotherapy, chemotherapy 1 1.9 oligodendroglioma—surgery, radiotherapy, chemotherapy 1 1.9 choroid plexus carcinoma—chemotherapy 1 1.9 total 54 100.0 mb, medulloblastoma; hgg, high grade gliomas; pnet, primitive neuroectodermal tumor; cns, central nervous system. table 5 frequency of different types of pediatric cns tumors reported in different countries (in percentage) tumor brazil2 korea13 germany18 canada17 beijing21 sweden19 morocco20 japan22 india6 iraq (current study lgg 33.4 30.4 42.8 41.1 36.7 51 38.8 35.7 35.8 31.5 mb and pnet 13.9 19.8 25.7 15.4 14.6 17 28.9 10 22.4 40.7 ependymoma 7.4 8.1 10.4 7 5.6 8 12 4.8 9.8 3.7 gct 3.6 8.1 na 3.1 7.9 1.5 0.9 14.3 2 9.3 pineal tumor na na 1.3 0.5 0.6 2.7 0.7 0 1.3 1.9 cpc 3 2.2 na 2.3 1.8 1.9 na 0 1.8 1.9 neuronal and mixed neuronal glial 7.6 6.2 3.2 <2 3.1 0 1.3 0 2.4 0 craniopharyngioma 11 9.2 4.4 6.8 18.4 4.6 6.6 10.5 10.2 0 hgg na na na na na na na na na 11.1 meningeal 3 2.6 1.2 <2 3.1 1.6 2.2 1.9 3.2 0 nerve sheath na 0.4 na 3.1 2.8 1.1 na 0 3.6 0 lgg, low grade gliomas; mb, medulloblastomas; pnet, primitive neuroectodermal tumors; gct, germ cell tumors; cpc, choroid plexus carcinomas; hgg, high grade gliomas; na, no available data. original 200 j contemp med sci | vol. 5, no. 4, july-august 2019: 197–201 childhood central nervous system tumors u. al-jumaily et al. about two-third of children with cns tumors in this study were living in urban areas, which anticipate higher incidence of such tumors in civilized rather than rural regions; this may suggest an environmental impact in the etiology of such tumors.14,15 in this study, the most common brain tumors in the pediatric age group in descending order are embryonal tumors (medulloblastoma and supratentorial pnet, and pineoblastoma), lgg (pilocytic astrocytoma and oligodendroglioma), hgg, cns germinoma, ependymoma, and choroid plexus carcinoma. compared with other previous reported series nationwide, it was seen that there is a higher incidence of embryonal tumors in our current study.1,2,5–7,11–13 low grade gliomas occupy the second most common encountered tumor (i.e., 31.5%); in other reported studies, lgg were the most common cns tumors in pediatric age group with a percentage ranging from 30.4% to 51%.6,7,11–13 high grade gliomas were the third most common cns tumors in our study, while data were lacking from most of published studies, the incidence of hgg is variable in other studies.1,2 germ cell tumors (gct) are the fourth most common type comprising 9.3%. there was a higher frequency of gct from studies of south-east asia including korea, china, and japan which may suggest a genetic and environmental influence.2,16 although ependymomas are the third most common tumors from the international-based data, they only occupy the fifth place in this current study comprising only 3.9% of the total histological types.1,6,7,13 the incidence of craniopharyngioma reported from the various published studies is variable, ranging from 4.4% to 18.4%. it was the third most common cns tumor from data reported from korea, brazil, and india, while figures from canada, germany, sweden, and morocco showed that it was the fourth most common pediatric cns tumor.2,6,13,17–20 data from beijing and japan, however, showed craniopharyngiomas to be the second and third commonest tumor, respectively.21,22 our current study, and as there is no incidence of craniopharyngioma, may not reflect the accurate burden of such tumor as most of the patients may be circulated between neurosurgeons and radiation oncologists by passing pediatric oncologists; another factor is the lack of complete registration of newly diagnosed cases with local cancer registries, leading to underestimation of such diseases. regarding the outcome, our results is relatively similar to those reported in developing countries based on the rate of death and frequency of abandonment, and lower than that in developed countries.5,23–25 our major obstacles that lead to low outcome were late referral to pediatric oncologist, lack of multidisciplinary teamwork, misconception of our community that cancer will eventually lead to death, poverty and financial problems that hinder the family consulting pediatric oncologist. we emphasize on hospital-based prevalence data to form the basis for estimating the disease incidence. this data is essential to determine the required healthcare basis in the management of these diseases, and for evaluating geographical variance in their molecular and genetic outlines. as the incidence of cns tumors is increasing in developing countries, and because of higher morbidity and mortality comparing with other pediatric malignancies, it necessitates accurate hospital-based estimation of such tumors by collecting data from all oncology centers in iraq in an attempt to represent the profile of the entire country. as our center is the first tertiary center managing pediatric cns tumors in iraq, our study may not reflect the real distribution of patients with primary cns tumors in children. another limitation of our study was that not all children with cns tumors were managed by a pediatric oncologist; some of the children are managed by adult oncologists, neurosurgeons, and radiation oncologists who they focused on surgery followed by radiotherapy without awareness of detrimental effects of radiotherapy in growing brains of children. this highlights the necessity of encouraging physicians to refer every child with cns tumor to a tertiary center under the care of pediatric oncologists; subsequently, this will facilitate and accurately estimate the incidence of cancer in developing countries like iraq; moreover, the value of epidemiological-based data collection by hospitals will be improved. despite of these limitations, this study may represent an initiative to launch for further collaborative studies between cancer centers in different areas in iraq for more precise estimation of primary cns tumors in iraqi children, as an endeavor to assess the whole patient group. to conclude, there is a rising global trend in the incidence of pediatric cns tumors. the present study may form the basis for a more wide study to provide the first profile of pediatric cns tumors in iraq. conflicts of interest none. ■ table 6 outcome of patients with cns tumors treatment status frequency percentage complete treatment with no evidence of tumor recurrence 26 48.1 death 14 25.9 abandoned treatment 9 16.7 on treatment 4 7.4 alive with disease 1 1.9 total 54 100.0 pineal gland 1 1.9 total 54 100.0 references 1. lacour b, guyot-goubin a, guissou s, bellec s, désandes e, clavel j. incidence of childhood cancer in france: national children cancer registries, 2000-2004. eur j cancer prev. 2010;19:173–181. epub 2010/04/03. 2. rosemberg s, fujiwara d. epidemiology of pediatric tumors of the nervous system according to the who 2000 classification: a report of 1,195 cases from a single institution. childs nerv syst. 2005;21:940–944. 3. louis dn, ohgaki h, wiestler od, cavenee wk, burger pc, jouvet a, et al. the 2007 who classification of tumours of the central nervous system. acta neuropathol. 2007;114:97–109. 4. ishihara h, ohno y, fujii m, hara j, soda m. epidemiological analysis of childhood cancer in japan based on population-based cancer registries, 1993-2009. jpn j clin onco. 2017;47:660–663. 5. suresh sg, srinivasan a, scott jx, rao sm, chidambaram b, chandrasekar s. profile and outcome of pediatric brain tumors experience from a tertiary original 201j contemp med sci | vol. 5, no. 4, july-august 2019: 197–201 childhood central nervous system tumorsu. al-jumaily et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. care pediatric oncology unit in south india. j pediatr neurosci. 2017;12:237– 244. 6. jain a, sharma mc, suri v, kale ss, mahapatra ak, tatke m, et al. spectrum of pediatric brain tumors in india: a multi-institutional study. neurol india 2011;59:208–211. 7. rickert ch, paulus w. epidemiology of central nervous system tumors in childhood and adolescence based on the new who classification. childs nerv syst. 2001;17:503–511. 8. diamandis p, aldape k. world health organization 2016 classification of central nervous system tumors. neurol clin. 2018;36:439–447. 9. desouza rm, jones br, lowis sp, kurian km. pediatric medulloblastoma update on molecular classification driving targeted therapies. front oncol. 2014;4:176. 10. fuller gn, scheithauer bw. the 2007 revised world health organization (who) classification of tumours of the central nervous system: newly codified entities. brain pathol 2007;17:304–307. 11. asirvatham jr, deepti an, chyne r, prasad ms, chacko ag, rajshekhar v, et al. pediatric tumors of the central nervous system: a retrospective study of 1,043 cases from a tertiary care center in south india. childs nerv syst. 2011;27:1257–1263. 12. madhavan r, kannabiran bp, nithya am, kani j, balasubramaniam p, shanmugakumar s. pediatric brain tumors: an analysis of 5 years of data from a tertiary cancer care center, india. indian j cancer 2016;53:562–565. 13. suh yl, koo h, kim ts, chi jg, park sh, khang sk, et al. tumors of the central nervous system in korea: a multicenter study of 3221 cases. j neurooncol. 2002;56:251–259. 14. gilli io, joaquim af, tedeschi h, dos santos aguiar s, morcillo am, ghizoni e. factors affecting diagnosis of primary pediatric central nervous system neoplasias in a developing country. childs nerv syst. 2019;35:91–96. 15. johnson kj, cullen j, barnholtz-sloan js, ostrom qt, langer ce, turner mc, et al. childhood brain tumor epidemiology: a brain tumor epidemiology consortium review. cancer epidemiol biomarkers prev. 2014;23:2716–2736. 16. kang jm, ha j, hong ek, ju hy, park bk, shin sh, et al. a nationwide, population-based epidemiologic study of childhood brain tumors in korea, 2005-2014: a comparison with united states data. cancer epidemiol biomarkers prev. 2019;28:409–416. 17. kaderali z, lamberti-pasculli m, rutka jt. the changing epidemiology of paediatric brain tumours: a review from the hospital for sick children. childs nerv syst. 2009;25:787–793. 18. kaatsch p, rickert ch, kühl j, schüz j, michaelis j. population-based epidemiologic data on brain tumors in german children. cancer 2001;92:3155–3164. 19. hjalmars u, kulldorff m, wahlqvist y, lannering b. increased incidence rates but no space-time clustering of childhood astrocytoma in sweden, 1973-1992: a population-based study of pediatric brain tumors. cancer 1999;85:2077–2090. 20. karkouri m, zafad s, khattab m, benjaafar n, el kacemi h, sefiani s, et al. epidemiologic profile of pediatric brain tumors in morocco. childs nerv syst. 2010;26:1021–1027. 21. zhou d, zhang y, liu h, luo s, luo l, dai k. epidemiology of nervous system tumors in children: a survey of 1,485 cases in beijing tiantan hospital from 2001 to 2005. pediatr neurosurg. 2008;44:97–103. 22. makino k, nakamura h, yano s, kuratsu j. population-based epidemiological study of primary intracranial tumors in childhood. childs nerv syst. 2010;26:1029–1034. 23. wagner hp, antic v. the problem of pediatric malignancies in the developing world. ann n y acad sci. 1997;824:193–204. 24. gajjar a, sanford ra, heideman r, jenkins jj, walter a, li y, et al. lowgrade astrocytoma: a decade of experience at st. jude children’s research hospital. j clin oncol. 1997;15:2792–2799. 25. rutkowski s, bode u, deinlein f, ottensmeier h, warmuth-metz m, soerensen n, et al. treatment of early childhood medulloblastoma by postoperative chemotherapy alone. n engl j med. 2005;352:978–986. dx.doi.org/10.22317/jcms.08201903 original 272 j contemp med sci | vol. 7, no. 5, september–october 2021: 272–275 original single center study of vaccination breakthrough infection with sars-cov-2 among erbil population in august 2021 zakarea abdullah yaseen al-khayat1*, sharmeen qadr faqi2, dlshad abdullah hasan2, dalia yaqoub falo2 1department of microbiology, college of medicine, hawler medical university, erbil, iraq 2molecular diagnostic department, central public health laboratory (cphl), erbil, iraq. *correspondence to: zakarea abdullah yaseen al-khayat (e-mail: zakarea.abdullah@hmu.edu.krd) (submitted: 05 september 2021 – revised version received: 18 september 2021 – accepted: 09 october 2021 – published online: 26 october 2021) abstract objectives: this is a first study to determine the incidence of covid-19 infection in the post-vaccinated in erbil city population. methods: this prospective study was conducted in the central health laboratory in erbil city over a period from 1st to 31st of august 2021. all vaccinated & non vaccinated persons (18 years of age and above) who had symptoms suggestive of covid-19 were engaged in the study. nasopharyngeal swabs were collected and examined for sars-cov-2 by real-time rt-pcr implemented to all attendance according to world health organization guidelines. results: a total of 2934 persons had attended the central laboratory for checking during the study period. from this total number, 551 (18.8%) were vaccinated while 2383 were nonvaccinated (81.2%). sixty one persons (61, 11.1%) of the vaccinated group showed a positive pcr. the highest incidence of pcr positivity according to age range, gender and vaccine dose, were as follows: male (42; 68.9%), 33–47 years (36; 59%), second dose (36; 59%) respectively. statistically, the differences of distribution of the pcr positivity concerning the these factors were: non-significant. the higher incidence of pcr positivity was among the nonvaccinated group (449, 18.8% ) and statistically, the differences of distribution of the pcr positivity between both groups of vaccinated and the nonvaccinated was significant (p ≤ 0.05). conclusion: primarily the present study conclude that sars-cov-2 incidence among vaccinated persons was 11.1%. overall the present data provides further assurances of the effectiveness of the vaccines even when the vaccine was not able to prevent completely the infection. further studies are needed to explore this topic. keywords: sars-cov-2, vaccines, polymerase chain reaction, post-vaccination, breakthrough, erbil issn 2413-0516 introduction covid-19 is a novel infectious malady caused by a sars-cov-2 virus, which is demonstrated mostly as an acute respiratory disease with an interstitial alveolar pneumonia. yet, it can involve different organs encompassing the heart, kidneys, nervous system, blood and the digestive tract1. covid-19 vaccines are efficient and pivotal weapons to boost the control of this pandemic2. vaccination against covid-19 has been considered as the most influential mean to halt the dispersal of the virus, beside the protective measures utilized by each individual3. the covid-19 mrna vaccines (from biontech/pfizer and moderna) have been authorized in different countries (including the usa and the eu) after successful clinical trials and are already in prevalent use4. the two products contain a nucleoside-modified mrna, encoding the sequence of the full-length s protein with two stabilizing proline mutations in s2 to preserve the native prefusion conformation. both vaccines use lipid nanoparticles for delivery5. in the real-world settings, the pfizer-bnt162b2 vaccine is approximately 90% effective in preventing sars-cov-2 infection and 94–100% effective in preventing severe or fatal disease6. meanwhile, several vaccines based on different non replicating adenovirus vectors and the full-length s protein are used for vaccination campaigns in many countries after approval by national and international authorities3. these vaccines include products of the gamaleya institute in moscow (sputnik v), university of oxford/astrazeneca (astrazeneca, cambridge, uk) (chadox1-s/azd1222), the beijing institute of biotechnology/cansino (cansino biologics, tianjin, china) and janssen pharmaceutica (janssen pharmaceutica, beerse, belgium)7. in these vaccines different adenoviruses are used as vectors but the basic principle of production platforms and mechanism of action is the same8. the gene for the sars-cov-2 s protein is synthesized as a dna and engineered into the dna genome of adenoviruses, replacing an adenovirus gene (e1) that is essential for virus replication9. through this manipulation, the adenovirus can no longer replicate and cannot give rise to a full infectious cycle (it is therefore referred to as non-replicating viral vector), but it can still enter cells and express the inserted foreign gene to produce the coronavirus s protein10,11. however, early studies propose that covid-19 vaccines shelter against severe illness; however, postvaccination sars-cov-2 infections (i.e., breakthrough infections) can occur because covid-19 vaccines do not offer 100% protection12. more recently, postvaccination infections in fully vaccinated persons have been mentioned though little is known about the risk factors, clinical presentation and outcomes for breakthrough infections compared with demographically and clinically similar controls13. thus, the objectives of this study were to report any postvaccination infections among fully or partially vaccinated persons, and to determine some factors like: age, gender, and vaccine dose which may have an association with infections in the vaccinated persons. our aim was not to determine specific vaccine efficacy or effectiveness. materials and methods study protocol this prospective study was carried out in the molecular diagnostic department at the central public health laboratory 273j contemp med sci | vol. 7, no. 5, september–october 2021: 272–275 z. abdullah yaseen al-khayat et al. original vaccination breakthrough infection with sars-cov-2 (cphl) in erbil city through a period from august 1 2021 to august 31 2021. this study was achieved with the cooperation of department of microbiology, college of medicine, hawler medical university, erbil, iraq. moral considerations this study was confirmed by the: ethics committee of hawler medical university, erbil and approved by erbil director health for collecting samples and data from referred patients at cphl-erbil. acquainted endorsement was possessed from each patient. the patients were aware of study’s goals and they could regress thereof if they wished so to do. study population the study population included all suspected cases of covid-19 attending cphl for pcr screening of sars-cov 2 infection during study period. inclusion criteria were: 18 year old and above, and inhabitants of erbil governorate. all the attendance who conferred agreement for sars-2 pcr testing and to share in this study were involved in the study. relevant clinical information, including patients’ vaccination status (type and dose number) age, gender, date of birth, was collected upon recruitment. non-consenting or unwilling patients were excluded from this study. sars-cov-2 testing by real-time rt-pcr nasopharyngeal and/or throat swab samples were collected in viral transport medium from the enrolled patients by a trained laboratory technician and examined for sars-cov-2 by real-time rt-pcr following world health organization guidelines. viral rna was extracted using sphaeramag dna/rna isolation kit on automate phoenix-pure96 system (procomcure com.) following manufacture’s instruction. real-time rt-pcr was done using diagnovital®sars-cov-2 realtime pcr kit (rta laboratories biological products pharmaceutical and machinery industry) on rotor-gene q (qiagen) real-time pcr detection system. diagnovital®sars-cov-2 detects the presence of 2 different and highly specific gene sequences of sars-cov-2: e gene and rdrp gene. all 2 assays must be tested positive to confirm the sample as sars-cov-2-positive. statistical analysis the data analysis was performed using descriptive statistics, including frequency, and frequency percentage. comparisons were made using chi2 test by using standard equations. the results were announced with p ≤ 0.05 or p ≤ 0.01 as the acceptable level of significance. results a total of 2934 persons had attended the central laboratory for checking during the study period. of this total number, 551 (18.8%) were vaccinated while 2383 were nonvaccinated (81.2%). table 1 delineates the incidence of pcr positivity among vaccinated persons according to age, gender and number of dose. it is noted that sixty one persons (11.1%) of the vaccinated group showed a positive pcr. the highest incidence of pcr positivity according to age range, gender and dose, were as follows: male (42; 68.9%), 33–47 years (36; 59%), second dose (36; 59%) respectively. statistically, the differences of distribution of the pcr positivity concerning the abovementioned factors were: non-significant. table 2 clarified the incidence of pcr positivity among vaccinated persons in comparison to the nonvaccinated persons (control). the higher incidence of pcr positivity was among the nonvaccinated group (449, 18.8%) and statistically, the differences of distribution of the pcr positivity between both groups of vaccinated and the nonvaccinated was significant (p ≤ 0.05). discussion to the best of our knowledge, no published data are available on the occurrence of sars-cov-2 infections among vaccinated citizens in erbil governorate, hence this study can be considered as the first study of such quality to deal with and investigate the incidence, correlated factors among these persons. the incidence of positive pcr in the post-vaccinated group was 11.1%, which was differed significantly from the incidence in the nonvaccinated group (18.8%). it seems that from the results of the present study, factors like age, gender and dose had no impact on the high incidence of pcr positivity among vaccinated group. table 1. incidence of pcr positivity among vaccinated persons in relation to age, gender, number of dose parameter n pcr (+) % pcr (–) % age 18–32 171 18 29.5 153 31.2 33–47 301 36 59 265 54.1 48–62 54 3 4.9 51 10.4 63–82 25 4 6.6 21 4.3 total 551 61 100 490 100 df = 3 ns gender male 421 42 68.9 379 77.3 female 130 19 31.4 111 22.7 total 551 61 100 490 100 df = 1 ns dose first 179 25 41 154 31.4 second 372 36 59 336 68.6 total 551 61 100 490 100 df = 1 ns table 2. incidence of pcr positivity among vaccinated and nonvaccinated persons parameter vaccinated % nonvaccinated % total pcr(+) 61 11.1 449 18.8 510 pcr (–) 490 88.9 1934 81.2 2424 total 551 100 2383 100 2934 χ2 = 18.8187 df = 1 significant (p ≤ 0.05) 274 j contemp med sci | vol. 7, no. 5, september–october 2021: 272–275 vaccination breakthrough infection with sars-cov-2 original z. abdullah yaseen al-khayat et al. post-vaccinations infections are a matter of interest but sufficient data concerning these infections are not available in real world setting14. vaccines have effectiveness in decreasing risk of getting covid-19 infections by 70–90%, and also shield from severe infection13. it is possible, therefore, some people who are fully vaccinated against covid-19 may get covid-19 infection4. a recent study by amit et al.15 showed that the ratio of vaccinated people infected with covid-19 after receiving the first dose vaccine was 0.5%, while a study achieved by hatmal mm et al. in jordan16 declared that the incidence was 7.02% after the first dose only. this variation in ratios could be due to the fact that amit et al.’s study involved only healthcare workers; this class of the population is assumed to be well educated and has a furthermore strict commitment in following prevention regulations to avoid covid-19 infection. in amit’s study, individuals infected with covid-19 were also tested in the early post-vaccination period (1–10 days), while there was no time limit in the present study. anecdotal report (unpublished) from india & declared records from other part of world (refer website of center for disease control, usa) deduce that these infections are existing. in addition, it seems that these breakthrough infections are either asymptomatic or mild in nature14. despite the fact that the preventive efficacy of covid-19 vaccines is argued in clinical trials, the knowledge about what happens following vaccination in the real world is still modest, especially among the general population16. thus, knowing what to expect after vaccination will help with public education, dispelling myths, and lowering the apprehension about covid-19 vaccines15. fear and suspicion, as well as a lack of information about clinical trials, have all been identified as factors that may lead to hesitancy in receiving the covid-19 vaccines10. the present study found a relatively high sars-cov-2 following receipt of the second dose. polack et al.,4 and butt aa et al.13 concluded that while the vaccine’s protective effect may not be obvious during the first two weeks after vaccination so recipients may wrongly realize themselves to be at a reduced risk of sarscov-2 infection and become less firm to nonpharmacological preventive measures such as social distancing and face covering. careful education and counselling during the vaccination process could help efforts to minimize such risk-compensation behavior17. the concept of vaccination is dependent on triggering an adaptive immune response, b and t cells, from which memory cells will evolve and furnish a long-lasting immunity. although an immune response can take place as early as within the first week, long-lasting immunity can take up to 4 weeks to develop7. the bnt162b2 vaccine was shown to elicit an effective humoral (antibody-mediated) and cellular (t-cell-mediated) responses a week after the booster dose. however, the response between the first and second doses was negligible18. vaccine efficacy does not always portend vaccine effectiveness, i.e., the protection advantage related to a vaccine when administered non-randomly under field conditions. equally, randomized controlled trials were done in a nominated age group, or geographical setting might not predict effectiveness if the vaccine is more widely prevailed. alternative vaccine platforms or the addition of adjuvants may be required for adequate immunogenicity, as for influenza vaccines19,20. it is noted from the results of the present studies, old ages persons had a lower incidence of pcr positivity while the highest incidence was in the age range of 33–47 years (59%). a study by butt aa et al.21 in qatar announced that increasing age was independently associated with a higher risk of severe disease or death in persons with breakthrough infection. in addition, encumbrance of comorbidities was not linked with the higher risk of severe disease or death21, 22. it is worthy to mention that central laboratory in erbil offers its services for public and military personals so it is expected that most attendance will be males and of age ranges mostly detected in the present study. the strengths of our study include: a national population, appropriately matched control group, it is the first step to elucidate such an important and newly arising topic in this vaccination campaign. limitations of the present study include small sample size, short duration, bias and shortages in collecting data regarding demography and risk factors like on obesity, smoking and co-morbid diseases which are important determinants of severity of covid-19. further studies are recommended to study and investigate this vital topic taking into consideration type of the vaccine, interval between the emergence of symptoms and the last dose. in addition follow up such persons are mandatory to explore any complications associated with vaccination. acknowledgments the authors are grateful to the management of health directorate of erbil for their allowing us to conduct the study. a words of thank are directed to the staff of the central laboratory for their assistance. our thanks and regards to the people who share in the study and we appreciate their patience during the study. financial support and sponsorship nil. conflicts of interest there are no conflicts of interest.  references 1. dawood h, hwayyiz a, ibrahim i, rahman ia. the clinical features of covid-19 in a group of iraqi patients: a record review. journal of the faculty of medicine baghdad. 2021 may 11;63(1):8-12. 2. baden lr, el sahly hm, essink b, et al. efficacy and safety of the mrna-1273 sars-cov-2 vaccine. n engl j med 2021; 384(5):403-16. 3. voysey m, clemens sac, madhi sa, et al. safety and efficacy of the chadox1 ncov-19 vaccine (azd1222) against sars-cov-2: an interim analysis of four randomised controlled trials in brazil, south africa, and the uk. the lancet 2021;397(10269): 99-111. 4. polack fp, thomas sj, kitchin n, absalon j, gurtman a, lockharts, et al. safety and efficacy of the bnt162b2mrna covid-19 vaccine. n engl j med. 2020;383(27):2603–15. 5. dagan n, barda n, kepten e, et al. bnt162b2 mrna covid-19 vaccine in a nationwide mass vaccination setting. n engl j med 2021;384:1412–23. 275j contemp med sci | vol. 7, no. 5, september–october 2021: 272–275 z. abdullah yaseen al-khayat et al. original vaccination breakthrough infection with sars-cov-2 6. hacisuleyman e, hale c, saito y, et al. vaccine breakthrough infections with sars–cov-2 variants. n engl j med 2021;384:2212–18. 7. heinz fx, stiasny k. profiles of current covid-19 vaccines. wiener klinische wochenschrift. 2021 apr;133(7):271-83. 8. zhu f-c, guan x-h, liy-h, huang j-y, jiang t, hou l-h, et al. immunogenicity and safety of a recombinant adenovirus type-5-vectored covid-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo controlled, phase 2 trial. lancet. 2020;396(10249): 479–88. 9. logunov dy, dolzhikova iv, shcheblyakov dv, tukhvatulin ai, zubkova ov, dzharullaeva as, kovyrshina av, lubenets nl, grousova dm, erokhova as, botikov ag. safety and efficacy of an rad26 and rad5 vector-based heterologous prime-boost covid-19 vaccine: an interim analysis of a randomised controlled phase 3 trial in russia. the lancet. 2021 feb 20;397(10275):671-81. 10. voysey m, clemens sac, madhi sa, weckx ly, folegatti pm, aley pk, et al. safety and efficacy of the chadox1 ncov19 vaccine (azd1222) against sars-cov-2: an interim analysis of four randomised controlled trials in brazil, south africa, and the uk. lancet. 2021;397(10269):99–111. 11. sadoff j, le gars m, shukarev g, heerwegh d, truyers c, de groot am, stoop j, tete s, van damme w, leroux-roels i, berghmans pj. interim results of a phase 1–2a trial of ad26. cov2. s covid-19 vaccine. new england journal of medicine. 2021 may 13;384(19):1824-35. 12. bleier bs, ramanathan jr m, lane ap. covid-19 vaccines may not prevent nasal sars-cov-2 infection and asymptomatic transmission. otolaryngology–head and neck surgery. 2021 feb;164(2):305-7. 13. butt aa, khan t, yan p, shaikh os, omer sb, mayr f. rate and risk factors for breakthrough sars-cov-2 infection after vaccination. j infect 2021;83:237–79. 14. tyagi k, ghosh a, nair d, dutta k, bhandari ps, ansari ia, misra a. breakthrough covid19 infections after vaccinations in healthcare and other workers in a chronic care medical facility in new delhi, india. diabetes & metabolic syndrome: clinical research & reviews. 2021 may 1; 15(3):1007-8. 15. amit, s.; regev-yochay, g.; afek, a.; kreiss, y.; leshem, e. early rate reductions of sars-cov-2 infection and covid-19 in bnt162b2 vaccine recipients. lancet 2021, 397, 875–877. 16. hatmal mm, al-hatamleh ma, olaimat an, hatmal m, alhaj-qasem dm, olaimat tm, mohamud r. side effects and perceptions following covid-19 vaccination in jordan: a randomized, cross-sectional study implementing machine learning for predicting severity of side effects. vaccines. 2021 jun;9(6):556. 17. zaqout a, daghfal j, alaqad i, hussein sa, aldushain a, almaslamani ma, abukhattab m, omrani as. the initial impact of a national bnt162b2 mrna covid-19 vaccine rollout. international journal of infectious diseases. 2021 jul 1;108:116-8. 18. ni l, ye f, cheng m-l, feng y, deng y-q, zhao h, et al. detection of sarscov-2-specific humoral and cellular immunity in covid-19 convalescent individuals. immunity 2020;52. 19. hodgson sh, mansatta k, mallett g, harris v, emary krw, pollard aj. what defines an efficacious covid-19 vaccine? a review of the challenges assessing the clinical efficacy of vaccines against sars-cov-2. lancet infect dis 2021;21:e26–e35. 20. allam aa, sayed aa. active covid-19 infection and transmission after the first dose of the bnt162b2 mrna vaccination in saudi arabia: a case report. journal of infection and public health. 2021 aug 1;14(8):1123-5. 21. butt aa, nafady-hego h, chemaitelly h, abou-samra ab, al khal a, coyle pv, al kanaani z, kaleeckal ah, latif an, al masalmani y, bertollini r. outcomes among patients with breakthrough sars-cov-2 infection after vaccination: breakthrough sars-cov-2 infection .international journal of infectious diseases 110 (2021) 353–358. 22. butt aa, yan p, shaikh os, mayr fb. outcomes among patients with breakthrough sars-cov-2 infection after vaccination in a high-risk national population. e clinical medicine. 2021, 1;40:101117. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i5.951 54 j contemp med sci | vol. 6, no. 2, march–april 2020: 54–58 original issn 2413-0516 introduction modern humans exhibit great diversity of physical differences because of some factors such as sexual dimorphism, topography, climate, geographical location, and diet.1, 2 these physical differences account for different races, tribes, and ethnicities.3ÿ it also has very important influence on the health and identification of an individual; therefore, becomes very necessary to establish baseline for health and physical identification of humans for each population.4, 5 physical anthropology relies mainly on external measurements (anthropometry) and descriptions (morphology) of the human body. such measurements are useful in the analysis and classification of fossil remains as well as study of living population. anthropometry comes from a greek word “anthropos” which means human and “metron” which means measure. it is a non-invasive technique to measure the dimensions of the human body and skeleton.1, 6 anthropometry has become essential tool of biological anthropology that involves a series of standardized measuring techniques that express quantitatively, the dimensions of human body. cephalic and facial indexes are very important anthropometric index used in describing head and facial dimensions and morphology, respectively.7-9 cephalic index (ci) is very useful in racial, ethnic, and gender identification of individuals. it is defined as the ratio of maximum cephalic width to maximum cephalic length multiplied by hundred percent. based on the value of ci, there are six different types of heads.10 facial index (prosopic index), on the other hand, is used to describe the facial proportion, prognosis of orthodontic treatments as well as indicating the direction of growth of craniofacial complex. the facial index is also defined as the ratio of morphological facial height to the ratio of facial width (bizygomatic width) multiplied by hundred percent. there are five different types of face based on facial index.10, 11 the facial and cephalic indices of an individual are determined by growth and developmental pattern. according to virchow’s law of parallel and perpendicular bone expansion, patency of the metopic and sagittal sutures are responsible for the adequate growth of cranial vault in the anterior–posterior while that of coronal and lambdoid sutures account for the growth in the transverse directions.12 the fusion of metopic suture occurs between 3 and 9 months while the others fuse between 22 and 39 months.13 premature fusion of the sagittal suture may result an increase the entire length of the head in the anterior–posterior direction but in early fusion of metopic suture, only the anterior length is reduced.13 although a high amount of report on cephalic and facial indices exist among various tribes and ethnic groups but there are little or no published data of such kind exist for the akan people in ghana. this study therefore aims at establishing for the first time, the baseline of cephalic and facial indices among the akan people of ghana. materials and methods a total of 100 (50 males and 50 females) akan volunteer adults between the age of 20 and 58 years (mean age of 29.7 years for males and 30.7 years for females) were recruited for the study. these volunteers were randomly selected from teachers and students of obiriyeboah shs and college of distant education, ucc (obiriyeboah shs center) as well as inhabitants of assinfosu and the neighboring towns within the cephalofacial characteristics of the akan people in the assin districts in central region of ghana: anthropometric studies gordon kyei1,2, ghazaleh moshkdanian3, parichehr pasbakhsh1, farid abolhassani1, tayebeh rastegar1, gholamreza hassanzadeh1,4,* 1international campus, tehran university of medical science, tehran, iran. 2department of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. 3anatomical sciences research center, kashan university of medical sciences, kashan, iran. 4legal medicine research center, legal medicine organization, tehran, iran. *corresponding author: gholamreza hassanzadeh (e-mail: hassanzadeh@tums.ac.ir ) (submitted: 23 january 2020 – revised version received: 06 february 2020 – accepted: 03 march 2020 – published online: 26 april 2020) objective cephalic and facial indices are very important in the classification and identification of populations. the present study aims at determining the cephalic and facial indices among the akan ethnic group living the assin districts in the central region of ghana. methods a total of 100 (50 males and 50 females) akan volunteer adults between the age of 20 and 58 years were recruited for the study. cephalic length (cl), cephalic width (cw), facial width (fw) and total facial height (tfh) were measured using a spreading caliper. cephalic index (ci) [(cw/cl) × 100] and facial index (fi) or prosopic index (pi) [(tfh/fw) ×100] as well statistical analysis such as z-test and pearson correlation were performed. the p-value of less than 0.05 was considered statistically significant. results our results are comparable with other authors with mean (for both male and females) ci and fi of 78.6± 4.66 and 96.51 ± 12.55, respectively. there was a correlation between cw and fw as well as some other cephlaofacial parameters. there was no sexual dimorphism of both ci and fi. the predominant head type among the study populations was mesocephalic and brachycephalic while hyperleptoprosopic was the face type. conclusion this study provides baseline data for the akan people of the assin districts of the central region of ghana, which will be a valuable in cephalometric anthropometry and in forensic science. keywords cephalic index; facial index; anthropometry; akan people, ghana. 55 original cephalofacial characteristics in central region of ghanagordon kyei et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 54–58 assin districts. subjects with history of craniofacial injury or trauma and obstructive hairstyle were excluded from the study. verbal informed consent was obtained from each participant. the hrdlicka’s method was used in assessing the cephalofacial parameters.14 the cephalic width (cw) was also measured as the maximum transverse diameter between two fixed points of the head (euryon), above the mastoid prominence (fig. 1a). the cephalic length (cl) was measured with the help of spreading calipers, from glabella to mid-point of external occipital protuberance (fig. 1b). then the ci was calculated and the head type determined for each subject. table 1 illustrates the types of heads and their designated values of cephalic indices. ci was calculated using the following formula (no. 1) 15: ci cw cb = ×100 (1) as well, total facial height (tfh) was measured from the nasion to mental tubercle (fig 1. c). and facial (bizygomatic) width (fw) was measured as a distance between the right and left zygion (fig. 1d). additionally, facial index (fi) was calculated and face type was determined for each case. table 2 illustrates the types of faces and their designated values of cephalic indices. fi was calculated using the following formula (no. 2) 16: fi fl fw = ×100 (2) all the measurements were taken with the subject sitting in relaxed condition and head in anatomical position. to reduce the technical error of the measurements, each dimension was taken twice and the average recorded. all measurements were taken to the nearest 1.0 cm. the following give descriptions of the measured parameters: • cephalic length (cl): glabella to inion • cephalic width (cw): distance between parietal eminences (zygion) • total facial length (tfl): nasion to gnathion • facial width (cw): distance between zygomatic arches. the data obtained were statistically analyzed using ibmspss version 22 and microsoft excel 2016 for windows. basic descriptive statistics and independent sample t-test were performed at 95% confident interval. the p-value of less than 0.05 was considered statistically significant. results tables 3–7 show the results of present study. basic statistics of cl, cw, ci, tfh, fw, and fi are shown in table 3. the mean cl and cw of males were 18.26 ± 0.85 and 14.20 ± 0.76, while those in females were 17.52 ± 0.91 and 13.88 ±0.82, respectively. as the data show, there were significant differences (p<0.05) in both mean cl and cw between male and female subjects. the means of ci were also 77.92 ± 5.37 and 79.28 ± 5.08 for males and females, respectively, but there were no significant differences between the two genders (p=0.052) (table 4). the mean ci of both males and females was 78.60 ± 4.66, which falls within mesocephalic )a head of medium proportions (. this type together with brachycephalic (a moderately fig. 1 a. the cephalic width (cw) was also measured as the maximum transverse diameter between two fixed points of the head (euryon), above the mastoid prominence. b. the cephalic length(cl) was measured with the help of spreading calipers, from glabella to mid-point of external occipital protuberance. c. total facial height (tfh) was measured from the nasion to mental tubercle. d. facial (bizygomatic) width (fw) was measured as a distance between the right and left zygion. table 1. classification of head type based on cephalic index. type of head cephalic index hyperdolicocephalic 65.5–69.9 dolichocephalic 70.0–74.9 mesocephalic 75.0–79.9 brachycephalic 80.0–84.9 hyperbrachycephalic 85.0–89.9 ultrabrachycephalic ≥90 table 2. classification of face type based on facial index. type of face facial index hypereuryprosopic <79.9 europrosopic 80.0–84.9 mesoprosopic 85.0–89.9 leptoprosopic 90.0–94.9 hyperleptoprosopic ≥95.0 56 original cephalofacial characteristics in central region of ghana gordon kyei et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 54–58 broad and short head) were the predominant types of head (tables 1, 3 and 5). the basic statistics of tfh and fw are shown in table 3. the mean of tfh and fw were 11.58 ± 0.97 and 12.02 ± 1.12, while those in females were 11.52 ± 0.95 and 12.14 ± 1.23, respectively. there was no significant difference in both mean tfh and fw between male and female subjects (p>0.05, table 4). table 4 shows the mean facial index 97.21 ± 12.70 and 95.82 ± 12.49for males and females respectively, and the difference was not statistically significant (p = 0.291). the mean fi of the studied population was 96.51 ± 12.55 (table 3), which table 3. basic descriptive statistic of cephalofacial anthropometric characteristics among the akan population in assin districts. parameters n mean ± sd(cm) sem range 95% ci cv % cl 100 17.89 ± 0.95 0.095 16.00–20.00 17.70–18.08 5.31 cw 100 14.04 ± 0.80 0.080 12.00–16.00 13.88–14.20 5.70 ci 100 78.60 ±4.66 0.466 66.67–88.88 77.68–79.53 5.93 tfh 100 11.55±0.96 0.096 10–14 11.36–11.74 8.31 fw 100 12.08±1.17 0.117 10.00–15.00 11.85–12.31 9.67 fi 100 96.51 ±12.55 1.255 71.43–130 94.03–99 13.00 cephalic length (cl)/ cephalic width (cw)/ cephalic index (ci)/ total facial height (tfh)/ facial (bizygomatic) width (fw)/ facial index (fi). table 4. comparing the cephalofacial characteristics between males and females among the akan population in assin districts. parameters gender mean ± sd (cm) sem range 95% ci cv % z /p-value cl male 18.26 ± 0.85 0.121 16–20 18.26–18.50 4.65 z = 4.199 p< 0.001 female 17.52 ± 0.91 0.129 16–20 17.26–17.78 5.19 cw male 14.20 ± 0.76 0.107 13–16 13.99–14.41 5.35 z = 2.023 p = 0.021 female 13.88 ± 0.82 0.119 12–16 13.65–14.11 5.91 ci male 77.92 ± 5.37 0.759 68.42–88.89 76.39–79.45 6.89 z = -1.628 p=0.052 female 79.28 ± 5.08 0.532 66.67–83.33 78.21–80.35 6.41 tfh male 11.58 ± 0.97 0.137 10.00–14.00 11.30–11.86 8.38 z = 0.331 p=0.370 female 11.52 ±0.95 0.135 10.00–14.00 11.25–11.79 8.25 fw male 12.02 ± 1.12 0.158 10.00–14.00 11.70–12.34 9.31 z = -0.511 p=0.304 female 12.14±1.23 0.174 10.00–15.00 11.79–12.49 10.13 fi male 97.21 ±12.70 1.796 76.92–127.2 93.6–100.82 13.06 z = 0.552 p= 0.290 female 95.82 ±12.49 1.766 71.43–130 92.27–99.37 13.03 table 5. frequency (percentage) of head types among the akan population in assin districts. nose type male n (%) female n (%) all n (%) hyperdolicocephalic 3 (6) 1 (2) 4 (4) dolichocephalic 14 (28) 5 (10) 19 (19) mesocephalic 18 (36) 18 (36) 36 (36) brachycephalic 10 (20) 26 (52) 36 (36) hyperbrachycephalic 5 (10) – 5 (5) ultrabrachycephalic – – – table 6. frequency (percentage) of face types among the akan population in assin districts. nose type male n (%) female n (%) all n (%) hypereuryprosopic 3 (6) 3 (6) 6 (6) europrosopic 9 (18) 9 (18) 18 (18) mesoprosopic 1 (2) 2 (4) 3 (3) leptoprosopic 14 (28) 16 (32) 30 (30) hyperleptoprosopic 23 (46) 20 (40) 43 (43) table 7. simple pearson correlation of the cephalofacial variables (r-value /p-value). variables cl cw tfl fw cl 0.415/0.0001 0.244/0.007 0.099/0.164 cw 0.142/0.080 0.179/0.037 tfl 0.023/0.408 fw cephalic length (cl)/ cephalic width (cw)/ total facial height (tfh)/ facial (bizygomatic) width (fw). 57 original cephalofacial characteristics in central region of ghanagordon kyei et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 54–58 falls within hyperleptoprosopic (a very long narrow face) type of face (table 2). according to our data, hyperleptoprosopic is also the predominant type of face as depicted in table 6. furthermore, the correlation between the head width and facial width has been shown in table 7. discussion in the present study, cephalofacial anthropometric characteristics of the akan people in the assin districts (central region of ghana) were investigated. this study has revealed that the ci of the akan people in the assin districts was 78.60 ± 4.66. this value is in agreement with the results obtained by many authors such as basu17 and bhargav and kher.29 however, in a study conducted by madadi et al18 among iranian medical students, the ci of 83.51 was recorded.18 anupama et al. (2009) also obtained a ci of 85.53 among punjabi students.19 these results do not correlate with the result of the current study, however it confirms the fact that there are differences in the ci among different populations. according to the results, no significant difference was observed between males and females ci. our findings corroborated with the results of oladipo and paul19 as well as shema et al.20 in contrast, the results of babatunde21 and anupama et al22 were not in accordance with our findings. the reasons for the absence of difference in ci between male and female are not clear but may be due to the fact that the effect and interplay amongst growth, thyroid, and sex hormones seem to have the similar effect in both sexes. the dominant types of head in our study are mesocephalic and brachycephalic. the existence of more than one dominant head type with an equal percentage of distribution within the same study population is uncommon in the published literatures. however, both head types observed in the present study are among the most common head type in the african population.19, 23 the facial index of the studied population was 96.51 ± 12.55. by comparing with the results in other populations, our value showed a degree of variations. among fars and turkmen of iran, males and females showed a prosopic index of 74.3 and 81.6, respectively.24 bini tribe of nigeria recorded a prosopic index of 86.93,25 while in malaysia, males and females respectively had a prosopic index of 85.72 and 87.91.26 however, our results were comparable with ukwuani indigenes in nigeria who had a prosopic index of 99.15 ± 6.11 and 94.54 ± 9.89 among males and females, respectively.27 as well, jaberi et al recorded 92 ± 6 for an iranian population.7 indigenes of maiduguri also had prosopic indexes of 99.59 and 97.54 for males and females, respectively.28 among the qazvin people of iran, azizi et al recorded a prosopic index of 102.88 in males and 96.69 in females, respectively.11 in this study, similar to the ci, there was no significant sexual dimorphism between males and females in the prosopic index as well. the reasons for this observation are not clear but can be the same with ci reasons. the dominant type of face in the study population was hyperleptoprosopic in both males and females. this is in the direct agreement of the face type amongst bini people of nigeria. finally, our results indicate that there are a significant positive correlation between cephalic length and cephalic width, cephalic length and total facial length as well as cephalic width and facial width. these findings agree with that of umar et al30 among hausa and yoruba ethnic groups of nigeria. obaje et al30 also came to the same conclusion in their study of cephalic indices among benue ethnic groups of nigeria. conclusion this study has revealed that the ci and facial index among the akan people of the assindistricts in the central region of ghana are, respectively, 78.60 and 96.51. this population has both mesocephalic and brachycephalic as dominant types of head. the dominant face type among them is hyperleptoprosopic. there was a positive correlation between head width and facial width among them. data obtained from this study will serve as a baseline for forensic scientist and anthropologists, and for comparisons between the akan people of assin districts and other ethnic groups within or outside ghana. acknowledgment we appreciate the effort of all those who helped to enrich this project specially miss pricilla afuwa, mr. emmanuel adjei, mr. yussifalhassan and third year psychology students (2018/ 2019 academic year) of code, ucc (oyess centre). special gratitude also goes to all the 100 volunteered akan people of assin districts who participated in this study. conflict of interest there was no conflict of interest. references 1. navid s, mokhtari t, alizamir t, arabkheradmand a, hassanzadeh g. determination of stature from upper arm length in medical students. anat sci j. 2014;11(3):135–40. 2. akhlaghi m, bakhtavar k, bakhshandeh h, mokhtari t, farahani mv, parsa va, et al. sex determination based on radiographic examination of metatarsal bones in iranian population. int j med toxicol foren med. 2017;7(4 (autumn)):203–8. 3. moshkdanian g, mahaki zadeh s, moghani ghoroghi f, mokhtari t, hassanzadeh g. estimation of stature from the anthropometric measurement of lower limb in iranian adults. anat sci j. 2014;11(3): 149–54. 4. mojaverrostami s, mokhtari t, malekzadeh m, noori l, kazemzadeh sh is. stature estimation based on fingers anthropometry in iranian population. anat sci. 2017;14(4):163–8. 5. eftekhar vaghefi sh, sheikhbahaei f, mokhtari t, khademi f, bahari h, ghorbani r. a model for individual height estimation from forearm length in natives of kerman, iran. anat sci j. 2014;11(3):141–4. 6. asharani s, hiremarali lt, rajendra r, surendra m. study of nansal index among students of tertiary medical care institute in southern india. 2015. 7. jaberi kr, kavakebian f, mojaverrostami s, najibi a, safari m, hassanzadeh g, et al. nasofacial anthropometric study among students of shiraz university of medical sciences, iran: a population based study. ind j otolaryngol head neck surg. 2019;71(2):206–11. 8. mohammed i, mokhtari t, ijaz s, ngaski aa, milanifard m, hassanzadeh g. anthropometric study of nasal index in hausa ethnic population of northwestern nigeria. j contemp med sci. 2018;4(1). 9. hassanzadeh g, sadr m, alaghbandha n, dehbashipour a, abbas ma, heydar zeidi o. anthropometric characteristics of craniums in residents of qazvin, iran and dera ghazi khan, pakistan: a comparative study. anat sci j. 2013;10(1):43–9. 10. kumari kl, babu pv, kumari pk, nagamani m. a study of cephalic index and facial index in visakhapatnam, andhra pradesh, india. int j res med sci. 2015;10:2320–6012. 11. azizi m, hassanzadeh g, barbarestani m, sadr m, dehbashipour a, alaghbandha n, et al. comparative anthropometric analysis of facial 58 original cephalofacial characteristics in central region of ghana gordon kyei et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 54–58 dimensions and types in qazvin, iran and deraghazi khan, pakistan. anat sci j. 2014;11(3):119–26. 12. shah g, jadhav h. the study of cephalic index in students of gujarat. j anat soc india. 2004;53(1):25–6. 13. cohen jm, kreiborg s. cranial size and configuration in the apert syndrome. j craniofac genet dev biol. 1994;14(3):153-62. 14. hrdlička a. practical anthropometry. wistar institute of anatomy and biology; 1952. 15. yagain vk, pai sr, kalthur sg, chethan p, hemalatha i. study of cephalic index in indian students. int j morphol. 2012;30:125–9. 16. wai mm, thwin ss, yesmin t, ahmad a, adnan as, hassan aa, et al. nasofacial anthropometric study among university students of three races in malaysia. adv anat. 2015;2015. 17. basu a. anthropometry of the kayasthas of bengal. j anat soc india. 1963;3:20–5. 18. madadi s, khanehzad m, tahmasebi f, gordon k, hassanzadeh g. correlation of horizontal cephalic index and cranial parameters in iranian medical students. acta med iran. 2018;56(9):577–84. 19. oladipo g, olotu j, suleiman y. anthropometric studies of cephalic indices of the ogonis in nigeria. asian j med sci. 2009;1(2):15–7. 20. nair sk, anjankar vp, singh s, bindra m, satpathy d. the study of cephalic index of medical students of central india. asian j biomed pharm sci. 2014;4(28):48. 21. akinbami bo. measurement of cephalic indices in older children and adolescents of a nigerian population. biomed res int. 2014;2014. 22. mahajan a, khurana bs, batra aps. the study of cephalic index in punjabi students. j punjab acad foren med toxicol. 2009;9(2):66-70. 23. oladipo gs, paul cw. anthropometric comparison of cephalic indices between the urhobo and itsekiri ethnic groups of nigeria. global j pure appl sci. 2009;15(1). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i2.723 40 original issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 40 – 45 introduction due to the current covid-19 pandemic, considering isolation as the solution, many countries have gone through total lockdown. in april, approximately 3 billion people, in more than 130 countries were locked down, and nearly 90% students were stopped from going to school. this was the fact due to which numerous children and their parents were compelled to use electronic devices more frequently to connect themselves with the outside world.1 unfortunately, this frequent use of electronic devices lead them towards computer vision syndrome (cvs). american optometric association recognized cvs as a complex of eye and vision problems associated with near work experienced during computer use.2 globally, around 60 million computer workers experience discomfort from cvs.3 approximately, 45 million workers used electronic devices by staring at the screen for hours daily.4 american optometric association found that 14.25% people who visited the optometry clinics have complained primarily of the symptoms related to computer use.5 cvs occurs due to an increased visual demand to the extent that it exceeds the person’s visual ability.6 it could also be explained by reduced blinking reflex, while staring at the screen, resulting in exaggeratedly dry eyes.7 dry eyes were found as a significant determinant factor in cvs.8 in early 2020, the world health organization (who) declared the disease covid-19 as a global pandemic. the people of saudi arabia experienced many changes due to the pandemic including disruption of the daily life routine and prolonged use of electronic devices. thereby, the study aimed to determine the prevalence of cvs, its associated risk factors, and common related symptoms associated with the prolonged use of electronic devices during the period of covid-19 curfew in jeddah city. subjects and methods it was a non-interventional cross-sectional study conducted among the participants from jeddah, saudi arabia over a period of covid-19 curfew from april to june 2020. participants aged >20 years and using electronic devices were included in the study. the electronic questionnaire used in the current study was previously used in a recent study by abudawood et al.9 the questionnaire consisted of four parts. the first part included the demographic characteristics; age, gender, occupation, permission to go out during lockdown, any ocular problems, wearing eyeglasses/contact lens for vision correction during the use of electronics, any chronic illnesses, previous eye surgeries, and current medications. the second part included the participant’s habits like using the computer; the third part included the frequency and intensity of eye symptoms, and the final part included habits to reduce symptoms of cvs. descriptive data were analyzed using spss version 21 program. quantitative data were shown as mean, standard deviation (sd), and qualitative data. numerical values expressed in n = numbers and shown as mean, sd, percentage (%), while non-numerical values were expressed as frequency and percentage. a comparison of variables between patients from different ethnicities were analyzed by independent t-test for continuous variables and the chi-square (χ2) test for categorical variables. p-value <0.05 was considered statistically significant. results one thousand two hundred twenty-seven (1227) participants from jeddah city were included in the present study. the participants were between 20 and 60 years old. the majority of them digital eye strain during covid-19 lockdown in jeddah, saudi arabia nawaf almarzouki1*, konooz faisal2, arwa nassief2, noura najem2, rayana eid2, renad albakri2, dana alhibshi2, shehana alwethinani2, heba ashi3 1 department of ophthalmology, king abdulaziz university hospital, jeddah, saudi arabia. 2 king abdulaziz university, faculty of medicine, jeddah, saudi arabia. 3 department of dental public health, faculty of dentistry, king abdulaziz university, jeddah, saudi arabia. *correspondence to: nawaf almarzouki (e-mail: nawaf.almarzouki@gmail.com) (submitted: 22 november 2020 – revised version received: 05 december 2020 – accepted: 27 december 2020 – published online: 26 february 2021) abstract objective this study aimed to determine the prevalence of computer vision syndrome (cvs) and associated risk factors of prolonged use of electronics. methods this was an online non-interventional cross-sectional study conducted over the period of covid curfew in jeddah city from april to june 2020, through a questionnaire. participants were between 20 and 60 years of age and used electronic devices. results totally, 1227 participants were recruited between 20 and 60 years of age. the majority of them were females (69.9%). almost 1048 participants used smartphones or laptops. more than half (54.5%) of them used electronics for more than 4 h daily. a high symptom severity score was found in 44% of the respondents. taking breaks during electronics use <30 minutes (p=0.018), viewing computers at a distance less than arm length (p=0.001), and the use of screen protectors (p=0.014) were significant factors related to the symptom’s severity score. conclusions cvs was prevalent among the participants who used electronics for more than 4 h daily. taking breaks during electronics use, viewing computers at an appropriate distance, and the use of screen protectors were effective practices to relieve the eye symptoms severity score. keywords computer, eyestrain, lockdown, symptoms, dry eye, vision, saudi arabia. 41 original digital eye strain during covid-19 lockdownnawaf almarzouki et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 40 – 45 were females (69.9%). most of them had eye diseases (63.6%), myopia (41.2%), astigmatism (20.9%), dry eye (15.5%), and other eye diseases. almost, 43.8% participants were wearing glasses, and 8.9% were wearing contact lenses. out of 1227, 1048 participants were using smartphones or laptops. more than half (54.5%) of them were using electronic devices for more than 4 h daily. majority of the participants (87.3%) were taking breaks during the use of electronic devices and lifting distance from the screen was less than the arm’s length (76.3%). a high symptom severity score was found in 44% individuals in the present study (table 1). the common symptoms associated with the prolonged use of electronic devices during the period of covid-19 curfew was assessed. burning sensation in the eyes, itchy eyes, increased sensitivity to light, headache, and pain in the neck or back were the most common symptoms associated with prolonged use of electronics during the period of covid-19 curfew (tables 2–4). table 1. description of different study variables (n=1227).   frequency (%) age 20-30 411 (33.5) 31-40 271 (22.1) 41-50 239 (19.5) 51-60 227 (18.5) >60 79 (6.4) gender male 369 (30.1) female 858 (69.9) occupation medical 171 (13.9) non-medical 490 (39.9) housewife 190 (15.5) not working 34 (2.8) retired 96 (7.8) student 246 (20) permission to break lockdown 381 (31.1) eye diseases 780 (63.6) myopia* 506 (41.2) astigmatism* 256 (20.9) dry eye* 190 (15.5) hypermetropia* 148 (12.1) cataract* 35 (2.9) glaucoma* 7 (0.6) keratoconus* 7 (0.6) wearing glasses 537 (43.8) wearing contact lenses 109 (8.9) diabetes mellitus 121 (9.9) (continued) table 1. continued hypertension 160 (13) previous eye operations 183 (14.9) use of medications affecting vision 57 (4.6) use of laptop or tablets 1048 (85.4) frequency of use of smartphones or laptops (n=1048) <1 hr 54 (5.2) 1-2 hrs 152 (14.5) 3-4 hrs 271 (25.9) >4 hrs 571 (54.5) taking breaks during use (n=1048) 915 (87.3) frequency of breaks (n=1048) no breaks 133 (12.7) < 30 minutes 343 (32.7) 30-60 minutes 318 (30.3) > 60 minutes 254 (24.2) distance to screen (n=1048) less than arm length 800 (76.3) more than arm length 248 (23.7) position during use (n=1048) sitting 453 (43.2) supine 30 (2.9) both 565 (53.9) level during use (n=1048) below eye level 514 (49) same eye level 521 (49.7) above eye level 13 (1.2) light source (n=1048) * ceiling / wall 849 (81) windows 334 (31.9) lampshades 228 (21.8) light source (n=1048) single 765 (73) multiple 283 (27) screen brightness (n=1048) faint 377 (36) pale 23 (2.2) bright 584 (55.7) very bright 64 (6.1) use of screen protector (n=1048) 138 (13.2) symptoms severity score (n=1048) $ high 461 (44) low 587 (56) *more than one answer allowed, $ developed from the following questionnaire. 42 original digital eye strain during covid-19 lockdown nawaf almarzouki et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 40 – 45 table 2. presence of symptoms questionnaire (n=1048).   never sometimes often always burning sensation in the eyes 371 (35.4) 479 (45.7) 156 (14.9) 42 (4) itchy eyes 368 (35.1) 508 (48.5) 127 (12.1) 45 (4.3) a sense of dust inside the eye 674 (64.3) 272 (26) 72 (6.9) 30 (2.9) flooding of tears 639 (61) 304 (29) 70 (6.7) 35 (3.3) too much blinking 624 (59.5) 322 (30.7) 79 (7.5) 23 (2.2) redness of the eye 538 (51.3) 395 (37.7) 86 (8.2) 29 (2.8) pain in the eye 518 (49.4) 404 (38.5) 95 (9.1) 31 (3) eyelid heaviness 714 (68.1) 266 (25.4) 51 (4.9) 17 (1.6) blurred vision 389 (37.1) 486 (46.4) 132 (12.6) 41 (3.9) double vision 756 (72.1) 220 (21) 58 (5.5) 14 (1.3) impaired near vision 622 (59.4) 240 (22.9) 111 (10.6) 75 (7.2) increased sensitivity to light 485 (46.3) 367 (35) 143 (13.6) 53 (5.1) seeing rainbows or halos around lights 745 (71.1) 222 (21.2) 52 (5) 29 (2.8) vision deterioration 802 (76.5) 189 (18) 42 (4) 15 (1.4) a headache 284 (27.1) 418 (39.9) 237 (22.6) 109 (10.4) pain in the neck or back 161 (15.4) 433 (41.3) 277 (26.4) 177 (16.9) numbness in the fingers of the hands 463 (44.2) 336 (32.1) 164 (15.6) 85 (8.1) table 3. severity of symptoms questionnaire (n=1048).   never intermediate severe burning sensation in the eyes 561 (53.5) 453 (43.2) 34 (3.2) itchy eyes 578 (55.2) 434 (41.4) 36 (3.4) a sense of dust inside the eye 771 (73.6) 259 (24.7) 18 (1.7) flooding of tears 778 (74.2) 238 (22.7) 32 (3.1) too much blinking 778 (74.2) 248 (23.7) 22 (2.1) redness of the eye 669 (63.8) 347 (33.1) 32 (3.1) pain in the eye 685 (65.4) 330 (31.5) 33 (3.1) eyelid heaviness 828 (79) 206 (19.7) 14 (1.3) blurred vision 600 (57.3) 405 (38.6) 43 (4.1) double vision 858 (81.9) 178 (17) 12 (1.1) impaired near vision 704 (67.2) 299 (28.5) 45 (4.3) increased sensitivity to light 636 (60.7) 372 (35.5) 40 (3.8) seeing rainbows or halos around lights 850 (81.1) 180 (17.2) 18 (1.7) vision deterioration 860 (82.1) 167 (15.9) 21 (2) a headache 388 (37) 543 (51.8) 117 (11.2) pain in the neck or back 327 (31.2) 538 (51.3) 183 (17.5) numbness in the fingers of the hands 616 (58.8) 359 (34.3) 73 (7) 43 original digital eye strain during covid-19 lockdownnawaf almarzouki et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 40 – 45 the determinant factors associated with the symptom’s severity score were also assessed. age (40–50 or >60 years), male gender, retired people, and having permission to break lockdown were statistically significant determinant factors related to the symptom’s severity score (p <0.05). additionally, the symptoms severity score was significantly associated with having eye diseases (p=0.000), wearing glasses (p=0.000), or contact lenses (p=0.001), and spending more than 4 h on electronic devices (p=0.048). moreover, taking breaks during electronic devices use <30 min (p=0.018), viewing computers at a distance less than an arm’s length (p=0.001), and the use of screen protectors (p=0.014) were also significant factors related to the symptom’s severity score in the current study (table 5). table 4. practices to relieve symptoms (n=1048).   never rare sometimes often always you position the screen 50-71cm from the eye (more than arm’s length) and below eye level 182 (17.4) 307 (29.3) 366 (34.9) 146 (13.9) 47 (4.5) an anti-glare screen is used to reduce the amount of light reflected from the screen 416 (39.7) 373 (35.6) 135 (12.9) 70 (6.7) 54 (5.2) an overhead lighting opens from the ceiling plus a desk lamp or light hits your eyes 260 (24.8) 250 (23.9) 267 (25.5) 182 (17.4) 89 (8.5) take short breaks every 20 minutes for 20 seconds and look at objects at least 20 feet away (the 20-20-20 rule) 186 (17.7) 277 (26.4) 316 (30.2) 176 (16.8) 93 (8.9) you do eye exercises moving them with frequent blinking 298 (28.4) 316 (30.2) 251 (24) 124 (11.8) 59 (5.6) avoid sitting in a place where air hits your eyes directly 208 (19.8) 242 (23.1) 214 (20.4) 183 (17.5) 201 (19.2) table 5. comparison regarding symptoms severity score (n=1048).   symptoms severity score p value#   high (n=461) low (n=587) age 20–30 172 (37.3) 218 (37.1) 0.954 31–40 95 (20.6) 139 (23.7) 0.236 41–50 99 (21.5) 96 (16.4) 0.034$ 51–60 83 (18) 86 (14.7) 0.143 >60 12 (2.6) 48 (8.2) 0.000$ gender male 102 (22.1) 213 (36.3) 0.000$ female 359 (77.9) 374 (63.7) occupation medical 65 (14.1) 90 (15.3) 0.577 non-medical 199 (43.2) 237 (40.4) 0.363 housewife 58 (12.6) 69 (11.8) 0.684 not working 15 (3.3) 11 (1.9) 0.154 retired 17 (3.7) 48 (8.2) 0.003$ student 107 (23.2) 132 (22.5) 0.782 (continued) table 5. continued permission to break lockdown 129 (28) 201 (34.2) 0.030 $ eye diseases 358 (77.7) 305 (52) 0.000$ myopia* 234 (50.8) 208 (35.4) 0.000$ astigmatism* 136 (29.5) 100 (17) 0.000$ dry eye* 119 (25.8) 48 (8.2) 0.000$ hypermetropia* 56 (12.1) 52 (8.9) 0.082 cataract* 19 (4.1) 8 (1.4) 0.005$ glaucoma* 3 (0.7) 1 (0.2) 0.326 keratoconus* 4 (0.9) 1 (0.2) 0.175 wearing glasses 237 (51.4) 207 (35.3) 0.000$ wearing contact lenses 61 (13.2) 42 (7.2) 0.001 $ diabetes mellitus 44 (9.5) 47 (8) 0.380 hypertension 65 (14.1) 60 (10.2) 0.055 previous eye operations 73 (15.8) 83 (14.1) 0.444 use of medications affecting vision 30 (6.5) 25 (4.3) 0.105 frequency of use of smartphones or laptops (n=1048) <1 hour 25 (5.4) 29 (4.9) 0.726 1-2 hours 61 (13.2) 91 (15.5) 0.300 3-4 hours 108 (23.4) 163 (27.8) 0.111 >4 hours 267 (57.9) 304 (51.8) 0.048$ taking breaks during use (n=1048) 403 (87.4) 512 (87.2) 0.925 frequency of breaks (n=1048) no breaks 58 (12.6) 75 (12.8) 0.925 <30 min 133 (28.9) 210 (35.8) 0.018$ 30–60 min 145 (31.5) 173 (29.5) 0.489 (continued) 44 original digital eye strain during covid-19 lockdown nawaf almarzouki et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 40 – 45 discussion the aim of the present study was to determine the prevalence of cvs, its associated risk factors, and common symptoms associated with prolonged use of electronic devices during the period of covid-19 curfew in jeddah city. out of 1227 participants recruited in the current study, 1048 participants used smartphones or laptops, and more than half (54.5%) of them used electronic devices for more than 4 h daily. a high symptom severity score was found in 44% participants in the present study. burning sensation in the eyes, itchy eyes, increased sensitivity to light, headache, and pain in the neck or back were the most common symptoms associated with prolonged use of electronic devices, during the period of covid-19 curfew. similarly, these symptoms were found in a recent study carried out by abudawood et al, who assessed the prevalence of cvs among medical students.9 in the current study, age particularly between 40 and 50 or >60 years old, was a determinant factor associated with cvs. in agreement with this result, a significant high prevalence of cvs was found among patients aged more than 40 years as compared to patients aged less than 20 years.3 in another study, significantly higher tear film evaporating rate was found in patients older than 45 years as compared to younger ones, which showed exaggerated symptoms of dryness related to cvs.10 in the current study, the symptoms severity score was significantly associated with eye diseases and astigmatism (p=0.000). similarly, a significant increase in cvs was found in people with uncorrected residual astigmatism.11, 12 wearing glasses (p=0.000) or contact lenses (p=0.001) were significant factors associated with the symptom’s severity score among the current study participants. similar results were found by ranasinghe et al. that showed a significant high eye symptoms severity score among contact lens wearers; moreover, contact lens wearers were more prone to dryness, which increased their cvs symptoms.3, 13 in another study, it was found, people who wore glasses had a significant effect on cvs symptoms.14 another study carried out in chennai found a significantly higher risk of having a headache and blurred vision among individuals wearing glasses or contact lenses.15 in the present study, spending more than 4 h on electronic devices (p=0.048) was a statistically significant risk factor associated with cvs. similar results were found by abudawood et al, who reported that the duration of studying using computers was the most significant risk factor correlated with cvs.9 additionally, students who use computers for more than 4 h daily had significantly high risk of cvs.14, 16 the current study results revealed that, taking breaks during computer use of <30 min (p=0.018) was significantly associated with cvs. in consistence, hassan et al found that taking short breaks decreased visual discomfort.17, 18 students who were not taking breaks during using computers were associated significantly with visual symptoms and neck and shoulder pain. in previous studies, cvs found significantly among the computer users who did not take frequent breaks.14, 19 the american optometric association recommended viewing computer at a distance 20–28 inches.20 the current results revealed that viewing a computer at a distance less than arm length (p=0.001) had a significant effect on eye symptoms severity score. in agreement with this result, recently it was found that most students who used computers at a distance of less than arm length significantly had high cvs symptoms.21 additionally, it was reported that the frequency of headache was decreased in computer users who watched the screen from a long-distance.22 further, the use of a screen protector (p=0.014) was also significant factor related to the eye symptoms severity score in the current study. this could be explained as using screen filters might help reduce glare and reflections from the screen.22 ranasinghe et al found significantly high cvs among patients who do not use screen filters.3 moreover, it was reported that the risk of eye diseases was increased among students who do not use screen filters.23 table 5. continued >60 min 125 (27.1) 129 (22) 0.054 distance to screen (n=1048) less than arm length 375 (81.3) 425 (72.4) 0.001 $ more than arm length 86 (18.7) 162 (27.6) position during use (n=1048) sitting 185 (40.1) 268 (45.7) 0.073 supine 13 (2.8) 17 (2.9) 0.942 both 263 (57) 302 (51.4) 0.071 level during use (n=1048) below eye level 212 (46) 302 (51.4) 0.079 same eye level 239 (51.8) 282 (48) 0.222 above eye level 10 (2.2) 3 (0.5) 0.016$ light source (n=1048) * ceiling/wall 385 (83.5) 464 (79) 0.067 windows 152 (33) 182 (31) 0.498 lampshades 99 (21.5) 129 (22) 0.845 light source (n=1048) single 324 (70.3) 441 (75.1) 0.079 multiple 137 (29.7) 146 (24.9) screen brightness (n=1048) faint 161 (34.9) 216 (36.8) 0.531 pale 12 (2.6) 11 (1.9) 0.424 bright 257 (55.7) 327 (55.7) 0.989 very bright 31 (6.7) 33 (5.6) 0.459 use of screen protector (n=1048) 74 (16.1) 64 (10.9) 0.014 $ *more than one answer allowed, #chi square test, $statistically significant. 45 original digital eye strain during covid-19 lockdownnawaf almarzouki et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 40 – 45 conclusion cvs was prevalent among individuals who used electronic devices for more than 4 h daily. taking breaks during electronics use, viewing computers at appropriate distance, and the use of screen protectors were significant practices to relieve the eye symptoms severity score. acknowledgment the authors like to acknowledge dr.nawaf almarzouki, an ophthalmologist who examined the questionnaire. abbreviations computer vision syndrome (cvs) world health organization (who) standard deviation (sd) conflict of interests the authors declare that there is no conflict of interest regarding the publication of this article. funding none consent to participate informed consent was obtained from all participants. ethical approval ethical approval was obtained from the research ethics committee rec at king abdulaziz university. reference no 387-20. references 1. al zobbi m, alsinglawi b, mubin o, alnajjar f. measurement method for evaluating the lockdown policies during the covid-19 pandemic. int j environ res public health. 2020;17(15):5574. https://doi.org/10.3390/ ijerph17155574 2. zainuddin h, isa mm. effect of human and technology interaction: computer vision syndrome among administrative staff in a public university. int j busin humanit technol. 2014;4(3):38-44. 3. ranasinghe p, wathurapatha ws, perera ys, lamabadusuriya da, kulatunga s, jayawardana n, et al. computer vision syndrome among computer office workers in a developing country: an evaluation of prevalence and risk factors. bmc res notes. 2016;9(1):150. https://doi.org/10.1186/s13104-0161962-1 4. noreen k, batool z, fatima t, zamir t. prevalence of computer vision syndrome and its associated risk factors among under graduate medical students of urban karachi. pak j ophthalmol. 2016;32(3). 5. sheedy je. vision problems at video display terminals: a survey of optometrists. j am optomet assoc. 1992;63(10):687. 6. rosenfield m. computer vision syndrome: a review of ocular causes and potential treatments. ophthal physiol optics. 2011;31(5):502-15. https://doi. org/10.1111/j.1475-1313.2011.00834.x 7. girard bc, lévy p. dry eye syndrome in benign essential blepharospasm. j franç ophtalmol. 2019;42(10):1062-7. https://doi.org/10.1016/j. jfo.2019.06.007 8. yan z, hu l, chen h, lu f. computer vision syndrome: a widely spreading but largely unknown epidemic among computer users. comput human behav. 2008;24(5):2026-42. https://doi.org/10.1016/j.chb.2007.09.004 9. abudawood ga, ashi hm, almarzouki nk. computer vision syndrome among undergraduate medical students in king abdulaziz university, jeddah, saudi arabia. j ophthalmol. 2020;2020. https://doi. org/10.1155/2020/2789376 10. guillon m, maïssa c. tear film evaporation-effect of age and gender. contact lens anterior eye. 2010;33(4):171-5. https://doi.org/10.1016/j. clae.2010.03.002 11. boyles m. the changing face of computer vision; new studies show that cvs is a growing concern for patients, providers and even employers. rev optomet. 2004;141(2):43-7. 12. bali j, neeraj n, bali rt. computer vision syndrome: a review. j clin ophthalmol res. 2014;2(1):61. https://doi.org/10.4103/2320-3897.122661 13. tauste a, ronda e, molina mj, seguí m. effect of contact lens use on computer vision syndrome. ophthal physiol optics. 2016;36(2):112-9. https://doi.org/10.1111/opo.12275 14. reddy sc, low ck, lim yp, low ll, mardina f, nursaleha mp. computer vision syndrome: a study of knowledge and practices in university students. nepal j ophthalmol. 2013;5(2):161-8. https://doi.org/10.3126/nepjoph. v5i2.8707 15. logaraj m, madhupriya v, hegde sk. computer vision syndrome and associated factors among medical and engineering students in chennai. ann med health sci res. 2014;4(2):179-85. https://doi.org/10.4103/21419248.129028 16. mashalla yj. impact of computer technology on health: computer vision syndrome (cvs). med pract rev. 2014;5(3):20-30. 17. fenety a, walker jm. short-term effects of workstation exercises on musculoskeletal discomfort and postural changes in seated video display unit workers. phys ther. 2002;82(6):578-89. https://doi.org/10.1093/ ptj/82.6.578 18. hassan a, mmk b. prevalence of computer vision syndrome (cvs) amongst the students of khyber medical university, peshawar. in islamabad congress of ophthalmology. 2017;15(2):59. 19. mowatt l, gordon c, santosh ab, jones t. computer vision syndrome and ergonomic practices among undergraduate university students. int j clin pract. 2018;72(1):e13035. https://doi.org/10.1111/ijcp.13035 20. loh ky, redd sc. understanding and preventing computer vision syndrome. malay family phys off j acad family phys malay. 2008;3(3):128. 21. al tawil l, aldokhayel s, zeitouni l, qadoumi t, hussein s, ahamed ss. prevalence of self-reported computer vision syndrome symptoms and its associated factors among university students. eur j ophthalmol. 2020;30(1):189-95. https://doi.org/10.1177/1120672118815110 22. blehm c, vishnu s, khattak a, mitra s, yee rw. computer vision syndrome: a review. surv ophthalmol. 2005;50(3):253-62. https://doi.org/10.1016/j. survophthal.2005.02.008 23. shantakumari n, eldeeb r, sreedharan j, gopal k. computer use and vision related problems among university students in ajman, united arab emirate. ann med health sci res. 2014;4(2):258-63. https://doi.org/10.4103/21419248.129058 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.937 https://doi.org/10.4103/2141-9248.129058 https://doi.org/10.4103/2141-9248.129058 53 original issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 53 – 60 introduction the world health organization (who) states that oral health is considered a part of the general well-being and is an essential element for good quality of life.1 good oral health helps us speak, smile, taste, chew, swallow, and express ourselves confidently.2,3 besides, it affects appearance, by allowing people to perform their daily activities without psychological or social limitations (shah and elhaddad, 2015)not many people are aware of the relationship of smoking with potential oral diseases. therefore, the aims of this study were to analyze oral hygiene behavior, smoking habits, and perceived oral health problems among a sample of university students in al-kharj, saudi arabia. materials and methods: a self-administered questionnaire about oral hygiene behavior, smoking, and perceived oral health problems was developed. the questionnaires were mainly distributed in medical, dental, and pharmacy colleges of the university. questionnaires completed at other colleges were included under the term “other colleges.” results: overall, 380 questionnaires were returned. majority of the students (92.4%. this depends on several factors, such as personal attitudes, behaviors, and knowledge. in the literature, the focus was mainly on the cure for oral diseases rather than prevention.4 several behavioral models, such as the health belief model and reasoned action theory, established the significant role of knowledge in behavioral changes explanation.5 parents’ knowledge and attitudes play an essential role in raising the awareness in their children and encouraging them to have a healthy life.6 additionally, parents’ involvement in health promotion programs can improve children’s health, and further, the mothers’ higher education elevated their children’s oral health.7 moreover, the lack of the mothers’ knowledge about oral hygiene may be one of the contributing factors for the dental caries’ high prevalence among children.8 therefore, poor parenting may be one of the etiological factors for dental caries.9 it was reported that children of mothers with positive oral attitudes were more likely to brush their teeth twice daily (z, virtanen, ghofranipour, & murtomaa, 2008). so far, no studies have been conducted in saudi arabia to assess the effect of mothers’ education on the oral hygiene of children. the study aimed to evaluate the association between gender and the mothers’ education, and the oral hygiene of their children. methods this comparative cross-sectional study included 531 children in jeddah, saudi arabia. an electronic database search was performed using google scholar and pubmed to gather background information and data related to the research question and to determine the knowledge gap. arabic was chosen for this survey to avoid possibilities of language barriers, followed by data analysis and segregation from the survey. students were stratified according to their gender and their mother’s education. oral health of children in association with gender and mothers’ education: a comparative cross-sectional study heba ashi department of dental public health, faculty of dentistry, king abdulaziz university, jeddah, saudi arabia correspondence to: heba ashi (e-mail: hmashi@kau.edu.sa) (submitted: 12 december 2020 – revised version received: 28 december 2020 – accepted: 19 january 2020 – published online: 26 february 2021) abstract objective to evaluate the association between gender and mothers’ education, and the oral hygiene of their children. methods this comparative cross-sectional study included 531 children in jeddah, saudi arabia. arabic was chosen for this survey to avoid possibilities of language barriers, followed by data analysis and segregation from the survey. students were stratified according to gender and their mother’s education. data were analyzed using the statistical package for the social sciences (spss), version 25. results brushing twice daily, using the dental floss, using fluoridated toothpaste, using mouthwash regularly, and bruxism were higher among females than males (58% vs. 28%, 28.2% vs. 10.9%, 71% vs. 30%,55% vs. 35% and 16.5% vs. 9.8%, respectively) with a significant difference (p<0.001, <0.001, <0.001, <0.001 and <0.001, respectively). meanwhile, using toothpick, clenching, and biting on hard objects were higher among males than females (49.3% vs. 34.9%, 21.7% vs. 7.1% and 46.7% vs. 34.9%, respectively) with a significant difference (<0.001 and <0.001, respectively). going to dentists during the last 3 months was significantly higher among females than males (47% vs. 33%), with a significant difference (p<0.001). on the other hand, going to dentists every 6 months was notably higher among males than females (22% vs.. 17%) (p<0.001). regular check-ups were higher among females (27.8% vs. 16.7%), with a significant difference (p=0.007). in accordance with the mothers’ education, brushing more than twice daily, manual toothbrush, electrical toothbrush, dental floss, and using toothpaste were significantly higher among those who received a university and post-graduate education (p<0.001, <0.001, <0.001, and 0.049, respectively). going to dentists during the previous 3 and 6 months was higher among those who received a university and post-graduate education (p=0.001). conclusions girls and children whose mothers had a university and post-graduate education had better attitudes toward oral hygiene. therefore, we highly recommend more oral hygiene health education programs, especially for mothers with less education. keywords oral health, oral hygiene, mothers education 54 original oral health of children in association with gender and mothers’ education heba ashi j contemp med sci | vol. 7, no. 1, january-february 2021: 53 – 60 data were analyzed using the statistical package for the social sciences (spss), version 25. categorical variables are presented as numbers and percentages. results were compared using the chi-square test and the monto carlo test. all the tests were 2-tailed, and a p-value <0.05 was considered statistically significant. ethical considerations: the study was approved by the research ethics committee of the faculty of dentistry (rec-fd), and consent was approved by the parents before any data collection. results out of the 531 students, the mothers of 250 (47.1%) students had a university and post-graduate education, whereas the number of students with fathers having a university or post-graduate education were 353 (66.5%) (table 1). according to oral hygiene habits, when comparing both genders, brushing twice daily, using the dental floss, fluoridated toothpaste, and mouthwash regularly, and bruxism were higher among females than males (58% vs. 28% , 28.2% vs. 10.9%, 71% vs. 30%, 55% vs. 35% and 16.5% vs. 9.8%, respectively) with a significant difference (p<.001, <.001, <.001, <.001, and <.001, respectively). however, using toothpick, clenching, and biting on hard objects were higher among males than females (49.3% vs. 34.9%, 21.7% vs. 7.1%, and 46.7% vs. 34.9% respectively) with a significant difference (<.001 and <.001, respectively) (table 2, figs 1, 2, 3, 4, and 5). when comparing dental history between both sexes, going to dentists during the last 3 months was higher among females than males (47% vs. 33%), with a significant difference (p<.001). contrarily, going to dentists every 6 months was significantly higher among males than females (22% vs. 17%) (p<.001). additionally, regular check-ups were higher among females (27.8% vs. 16.7%), with a significant difference (p=.007) (table 3, figs 6 and 7). while evaluating the oral hygiene habits with regard to the mothers’ education, brushing more than twice daily, using a manual/electrical toothbrush, using dental floss, and using toothpaste were significantly higher among those with a university and post-graduate education (p<.001, <.001, <.001, and <.01, respectively) (table 4, figs 8 and 9). when comparing dental history according to the mothers’ education, going to the dentist during the previous 3 and 6 months was higher among those with a university and post-graduate education (p=.001) (table 5). discussion this comparative cross-sectional study was conducted at king abdul-aziz university and included 531 children. it assessed the association between mothers’ education and gender and oral health practices among their children. the outline suggests that females and children of mothers with a university and post-graduate education showed better attitudes toward oral hygiene habits and dentist visits. in the gender-based oral health comparison in our sample, brushing twice daily, using the dental floss, fluoridated toothpaste, and mouthwash regularly, and bruxism was higher among females than males. however, using toothpick, clenching, biting on hard objects was higher among males than females. therefore, we can conclude that females are more committed to oral health and hygiene. this could be due to their aesthetic concerns and the presentation of their smile. this finding is similar to a study by singh et al10, who found that the male gender was significantly related to dental caries. additionally, a study by kumar et al11 found that females had better knowledge and oral health practices than males.11 similarly, a study by jaber et al found that males had good knowledge but poor practice toward oral health.12 when comparing the dental history and commitment to visits between both genders, females showed more commitment than males, which is similar to a study by mcdonald,13 who also reported similar results. regarding the oral hygiene habits, in association with the mothers’ education, brushing twice daily, using the manual toothbrush and electrical toothbrush, was higher among those with a university and post-graduate education. this is on par with a study by hallas et al8, who found that lack of knowledge of mothers regarding oral hygiene might be one of the contributing factors for the high dental caries prevalence in children. additionally, a study by z, virtanen, ghofranipour, and murtomaa (2008) found that children of mothers with positive attitudes were more likely to brush their teeth twice daily. this study showed the difference in oral health and attitudes among girls and boys, children of mothers with a university or post-graduate education, and with secondary or primary education. thus, we highly recommend more oral hygiene health education programs, especially for mothers with less education. data were collected via an online survey that had some restrictions, such as the likelihood of inaccurate information, as the survey is in the form of multiple-choice questions. respondents may not feel comfortable providing answers that present themselves in an unfavorable manner. conclusion females had a better attitude toward oral hygiene than males. moreover, children whose mothers had a university and post-graduate education also had better attitudes toward oral hygiene habits and dentist visits. thus, we highly recommend more oral hygiene health education programs, especially for mothers with less education. the literature review suggests that no studies have been conducted in saudi arabia to assess the effects of the mothers’ education on oral hygiene of children. further research is needed to determine oral health problems based on clinical examination and comprehensive table 1. family education of the sample (n=531). n percent 1. what education does your mother have? university and postgraduate 250 47.1 secondary and preparatory 75 14.1 others 206 38.8 2. what education does your father have? university and postgraduate 353 66.5 secondary and preparatory 31 5.8 others 147 27.7 note. all variables are summarized as percentage 55 original oral health of children in association with gender and mothers’ educationheba ashi j contemp med sci | vol. 7, no. 1, january-february 2021: 53 – 60 table 2. oral hygiene habits according to gender. boys girls p-value n % n % 1. how often do you brush your teeth? never 2 1% 0 0% <0.001* once or a few times a week 43 16% 4 2% once a day 119 43% 30 12% twice a day 78 28% 149 58% more than twice a day 23 8% 61 24% other 11 4% 11 4% 2. what do you use to brush your teeth? manual toothbrush 233 84.4% 217 85.1% 0.003* manual and electrical toothbrush 6 2.2% 2 0.8% manual toothbrush and miswak 12 4.3% 2 0.8% electrical toothbrush 20 7.2% 26 10.2% miswak 0 0.0% 5 2.0% none 2 0.7% 3 1.2% other 3 1.1% 0 0.0% 3. which of the following product do you use to clean between your teeth? dental floss 30 10.9% 72 28.2% <0.001* dental floss and interdental brush 5 1.8% 7 2.7% dental floss and toothpick 5 1.8% 2 0.8% interdental brush 26 9.4% 21 8.2% interdental brush and toothpick 6 2.2% 0 0.0% toothpick 136 49.3% 89 34.9% none 65 23.6% 60 23.5% other 3 1.1% 4 1.6% 4. do you use any toothpaste while brushing? yes 269 97% 236 93% 0.009** no 7 3% 19 7% 5. what kind of toothpaste do you use? fluoridated 82 30% 182 71% <0.001* non-fluoridated 8 3% 0 0% do not know 184 67% 73 29% 6.do you use any mouthwash regularly? yes 96 35% 141 55% <0.001** no 178 65% 114 45% 7. do you have any of the following oral habits? bruxism 27 9.8% 42 16.5% <0.001** clenching 60 21.7% 18 7.1% biting on a hard object 129 46.7% 89 34.9% other 60 21.7% 106 41.6% note. all variables are summarized as percentage. the test of significance was carried out at 0.05 level. *monto carlo test was used. **chi-square test was used. significant results are in bold. 56 original oral health of children in association with gender and mothers’ education heba ashi j contemp med sci | vol. 7, no. 1, january-february 2021: 53 – 60 fig. 3 toothpaste type according to gender. fig. 4 mouthwash use according to gender.fig. 1 toothbrushing according to gender.2017 (n=8665). fig. 2 cleaning product use according to gender. fig. 5 oral habits according to gender. fig. 6 dentist last visit according to gender. 57 original oral health of children in association with gender and mothers’ educationheba ashi j contemp med sci | vol. 7, no. 1, january-february 2021: 53 – 60 table 3. dental history according to gender. boys girls p-value n % n % 1. have you ever been to a dentist? yes 266 96% 250 98% 0.248** no 10 4% 5 2% 2. if yes, how often do you visit a dentist? every 6 months 61 22% 43 17% 0.001* every year 5 2% 16 6% irregularly 61 22% 39 15% only when in pain 119 43% 109 43% other 30 11% 45 18% 3. when was the last visit? previous 1–3 months 90 33% 121 47% <0.001** previous 4–6 months 39 14% 44 17% previous 7–12 months 41 15% 19 7% >1 year ago 33 12% 38 15% >2 years ago 31 11% 23 9% >5 years ago 42 15% 10 4% 4. what was the purpose of the visit? regular check-up (dental examination) 46 16.7% 71 27.8% 0.007** cleaning the teeth 40 14.5% 35 13.7% filling 68 24.6% 46 18.0% extraction 33 12.0% 18 7.1% orthodontic treatment 63 22.8% 51 20.0% other 26 9.4% 34 13.3% note. all variables are summarized as percentage. the test of significance was carried out at 0.05 level. *monto carlo test was used. **chisquare test was used. significant results are in bold. fig. 7 purpose of the visit according to gender. fig. 8 toothbrushing according to mothers’ education. 58 original oral health of children in association with gender and mothers’ education heba ashi j contemp med sci | vol. 7, no. 1, january-february 2021: 53 – 60 table 4. oral hygiene habits according to mother’s education. university and postgraduate secondary and preparatory others p-value n % n % n % 1. how often do you brush your teeth? never 0 0% 2 3% 0 0% <0.001* once or a few times a week 11 4% 11 15% 25 12% once a day 65 26% 9 12% 75 36% twice a day 97 39% 41 55% 89 43% more than twice a day 60 24% 10 13% 14 7% other 17 7% 2 3% 3 1% 2. what do you use to brush your teeth? manual toothbrush 207 82.8% 66 88.0% 177 85.9% <0.001* manual and electrical toothbrush 0 0.0% 0 0.0% 8 3.9% manual toothbrush and miswak 2 0.8% 0 0.0% 12 5.8% electrical toothbrush 33 13.2% 7 9.3% 6 2.9% miswak 5 2.0% 0 0.0% 0 0.0% none 0 0.0% 2 2.7% 3 1.5% other 3 1.2% 0 0.0% 0 0.0% 3. which of the following product do you use to clean between your teeth? dental floss 69 27.6% 15 20.0% 18 8.7% <0.001* dental floss and interdental brush 9 3.6% 0 0.0% 3 1.5% dental floss and toothpick 2 0.8% 2 2.7% 3 1.5% interdental brush 28 11.2% 4 5.3% 15 7.3% interdental brush and toothpick 2 0.8% 0 0.0% 4 1.9% toothpick 89 35.6% 38 50.7% 98 47.6% none 49 19.6% 14 18.7% 62 30.1% other 2 0.8% 2 2.7% 3 1.5% 4. do you use any toothpaste while brushing? yes 236 94% 68 91% 201 98% 0.049* no 14 6% 7 9% 5 2% 5. what kind of toothpaste do you use? fluoridated 144 58% 45 60% 75 37% <0.001* non-fluoridated 5 2% 0 0% 3 1% do not know 101 40% 30 40% 126 62% 6. do you use any mouthwash regularly? yes 121 49% 29 39% 87 42% 0.193** no 127 51% 46 61% 119 58% 7. do you have any of the following oral habits? bruxism 42 16.8% 9 12.0% 18 8.7% 0.004** clenching 21 8.4% 15 20.0% 42 20.4% biting on a hard object 103 41.2% 29 38.7% 86 41.7% other 84 33.6% 22 29.3% 60 29.1% note. all variables are summarized as percentage. the test of significance was carried out at 0.05 level. *monto carlo test was used. **chisquare test was used. significant results are in bold. 59 original oral health of children in association with gender and mothers’ educationheba ashi j contemp med sci | vol. 7, no. 1, january-february 2021: 53 – 60 fig. 9 toothbrushing way according to mothers’ education. table 5. dental history according to mother education. university and postgraduate secondary and preparatory others p-value n % n % n % 1. have you ever been to a dentist? yes 246 98% 72 96% 198 96% 0.275** no 4 2% 3 4% 8 4% 2. if yes, how often do you visit a dentist? every 6 months 45 18% 19 25% 40 19% 0.001* every year 7 3% 7 9% 7 3% irregularly 57 23% 5 7% 38 18% only when in pain 107 43% 40 53% 81 39% other 34 14% 4 5% 37 18% 3. when was the last visit? previous 1–3 months 116 46% 34 45% 61 30% 0.001* previous 4–6 months 43 17% 11 15% 29 14% previous 7–12 months 20 8% 14 19% 26 13% >1 year ago 28 11% 7 9% 36 17% >2 years ago 24 10% 6 8% 24 12% >5 years ago 19 8% 3 4% 30 15% 4. what was the purpose of the visit? regular check-up (dental examination) 57 22.8% 19 25.3% 41 19.9% 0.634** cleaning the teeth 36 14.4% 10 13.3% 29 14.1% filling 48 19.2% 20 26.7% 46 22.3% extraction 23 9.2% 3 4.0% 25 12.1% orthodontic treatment 56 22.4% 13 17.3% 45 21.8% other 30 12.0% 10 13.3% 20 9.7% note. all variables are summarized as percentage. the test of significance was carried out at 0.05 level. *monto carlo test was used. **chisquare test was used. significant results are in bold. 60 original oral health of children in association with gender and mothers’ education heba ashi j contemp med sci | vol. 7, no. 1, january-february 2021: 53 – 60 detailed interviews to overcome internal validity errors that might occur in an electronics-based survey. conflict of interest none references 1. leader g, petersen pe. chief, oral health programme, world health organization, department for chronic disease and health promotion. commun dent health. 2005;22. 2. conway di, mcmahon ad, robertson d, macpherson lmd. epidemiology of dental diseases. in: handbook of epidemiology: second edition. new york: springer; 2014:2321–53. doi:10.1007/978-0-387-09834-0_51. 3. halawany hs. a review on miswak (salvadora persica) and its effect on various aspects of oral health. saudi dental j 2012;24(2):63–69. elsevier. doi:10.1016/j.sdentj.2011.12.004. 4. levin l, shenkman a. the relationship between dental caries status and oral health attitudes and behavior in young israeli adults. j dent educ. 2004;68(11):1185–91. doi:10.1002/j.0022-0337.2004.68.11.tb03864.x. 5. noar sm. a health educator’s guide to theories of health behavior. int q commun health educ. 2005-2006;24:75–92. doi:10.2190/dalp-3f95gct3-m922. 6. christensen p. the health-promoting family: a conceptual framework for future research. soc sci med. 2004;59(2):377–87. doi:10.1016/j. socscimed.2003.10.021. 7. nourijelyani k, yekaninejad ms, eshraghian mr, mohammad k, foroushani ar, pakpour ah. the influence of mothers’ lifestyle and health behavior on their children: an exploration for oral health. iran red crescent med j. 2014;16(2). retrieved 12 5:2020. https://ncbi.nlm.nih.gov/pmc/articles/ pmc3965884. 8. hallas d, fernandez jb, lim lj, catapano p, dickson sk, blouin kr, et al. ohep: an oral health education program for mothers of newborns. j pediatr health care. 2015;29(2):181-90. retrieved 12 5, 2020. https://sciencedirect. com/science/article/abs/pii/s0891524514003563. 9. stillman-lowe c. oral health — educating mothers with young children. br dent j. 2000;188(4):199. retrieved 12 6. https://nature.com/ articles/4800429. 10. singh a, purohit b, sequeira ps, acharya s. oral health status of 5-year-old aborigine children compared with similar aged marginalised group in south western india. int dent j. 2011;61(3):157-62. retrieved 12 6, 2020. https:// onlinelibrary.wiley.com/doi/10.1111/j.1875-595x.2011.00033.x. 11. kumar h, behura ss, ramachandra s, nishat r, dash kc, mohiddin g. oral health knowledge, attitude, and practices among dental and medical students in eastern india a comparative study. j int soc prev commun dent. 2017;7(1):58–63. doi:10.4103/jispcd.jispcd_30_17. 12. jaber mf, khan a, elmosaad y, mustafa mm, suliman n, jamaan a. oral health knowledge, attitude and practices among male qassim university students. int j commun med public health. 2017;4(8):2729. doi:10.18203/2394-6040.ijcmph20173316. 13. mcdonald b. marital status as a predictor of dental service utilization. retrieved 12 6. http://ncurproceedings.org/ojs/index.php/ncur2013/article/ download/419/308, 2020; 2013. further reading 14. ghofranipour ji, f, murtomaa h, murtomaa hinfluence of mothers’ oral health knowledge and attitudes on their children’s dental health. eur arch paediatr dent. 2008;9(2):79-83. retrieved 12 6. https://link.springer.com/ article/10.1007/bf03262614. 15. sayegh a, dini el, holt rd, bedi r. caries prevalence and patterns and their relationship to social class, infant feeding and oral hygiene in 4-5-year-old children in amman, jordan. commun dent health. 2002;19(3):144-51. retrieved 12 6, 2020. https://ncbi.nlm.nih.gov/pubmed/12269460. 16. shah ah, elhaddad sa. oral hygiene behavior, smoking, and perceived oral health problems among university students. j int soc prev commun dent. 2015;5(4):327–33. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.944 https://doi.org/10.1007/978-0-387-09834-0_51 https://doi.org/10.1016/j.sdentj.2011.12.004 https://doi.org/10.1002/j.0022-0337.2004.68.11.tb03864.x https://doi.org/10.2190/dalp-3f95-gct3-m922 https://doi.org/10.2190/dalp-3f95-gct3-m922 https://doi.org/10.1016/j.socscimed.2003.10.021 https://doi.org/10.1016/j.socscimed.2003.10.021 https://doi.org/10.4103/jispcd.jispcd_30_17 https://doi.org/10.18203/2394-6040.ijcmph20173316 223j contemp med sci | vol. 6, no. 5, september-october 2020: 223–228 original issn 2413-0516 introduction one of the common causes of refractive errors globally is myopia, and myopic astigmatism.1-3 this refractive error can be permanently corrected by the well-known laser refractive surgeries such as laser-assisted in situ keratomileusis (lasik) or small incision lenticule extraction (smile).4-7. in 2008, smile was introduced for the first time to be a novel refractive surgical approach.8 a paradigm shift has been marked by refractive lenticule extraction in the refractive surgery field from the well-known and practiced flap-based corneal ablative techniques to flapless extraction method of femtosecond laser-produced intrastromal lenticules.9 the extraction of an intrastromal lenticule has been described by sekundo et al following lifting a flap in 2008, and the procedure has been developed to the smile, a procedure in which a small 2–5-mm side cut incision is made through which the refractive lenticule is extracted.8, 10, 11 as of the time this paper is being prepared, femtosecond lasik and smile are considered to be the most widely accepted procedures by both refractive surgeons and myopic patients for the correction of refractive errors.12 the procedure of smile involves the production of an intrastromal lenticule using femtosecond laser, and then this lenticule is going to be extracted through a small peripheral corneal incision, thereby, considering it as a minimally invasive refractive corneal surgery.9, 13 although, pervious reports have shown comparable efficacy, with respect to long-term visual acuity results, between femto-lasik and smile, the later operation is superior for having a number of advantages including greater corneal sensitivity, superior biomechanics, less induction of higher-order aberrations and fewer dry eye symptoms.14-17 probably, the major disadvantage of smile is the delayed optimum recovery of visual acuity following surgery in comparison with lasik.11, 18-20 smile offers precise visual quality and acuity and because of the enhanced patient satisfaction, the technique is increasingly being preferred for the correction of refractive errors associated with myopia and myopic astigmatism.9 nevertheless, smile has a steep learning curve and this is a challenge for beginners.21, 22 in order to familiarize the beginner surgeon to delicate procedures of smlie, a stepwise approach is needed and this involves observation, wetlab training, followed by flap-based refractive lenticule extraction (pseudo-smile).8, 21, 23 during early learning phase, a high rate of intraoperative complications may be seen due to difficult lenticule dissection and extraction.21, 24, 25 in order to ease the process of lenticule extraction easier and to minimize complications, various modifications of the surgical procedure have been developed as smile is gaining global acceptance among occular surgeons.9 the scanning trajectory of the femtosecond laser is being modified in order to improve visual outcome following smile. a number of approaches have been tried in order to improve short-term post-operative visual acuity results following smile including lowering laser energy levels, changing cap thickness, and intraoperative cap repositioning.26-29 on the other hand, the effect of the difference between diameters of the cap and lenticule on early visual and refractive outcome has been investigated by some authors.30 in the current study, the aim was to evaluate the effect difference in lenticule thickness (≤55 µm versus >55 µm) on the visual outcome in a cohort of 61 patients with low myopia. patients and methods the current prospective cohort study included 61 patients undergoing smile during the period extending from november 14, 2019 to june 14, 2020, a total of 99 eyes. the the effect of thin lenticule versus thick lenticule (a cut-off value of 55 micrometer) on the outcome of smile in low myopia zaid yousif hameed shukur college of medicine, university of kufa, najaf, iraq. corresponding author: zaid yousif hameed shukur (e-mail: zaid.shukur@uokufa.edu.iq) (submitted: 19 july 2020 – revised version received: 29 july 2020 – accepted: 17 august 2020 – published online: 30 october 2020) abstract objectives: in the current study, the aim was to evaluate the effect difference in lenticule thickness small incision lenticule extraction (smile) cases (≤55 µm versus >55 µm) on the visual outcome in a cohort of 61 patients with low myopia (≤3.0 diopters) methods: the current prospective cohort study included 61 patients undergoing smile refractive procedure during the period extending from november, 14 2019 to june 14, 2020, a total of 99 eyes. the study was conducted in ibsar ophthalmology surgery centre «private medical centre» in the wholly najaf city, mid-euphrates region of iraq. the cases enrolled in the study were all operated by the same ophthalmologist. according to lenticule thickness, eyes were classified into two groups, (≤55 µm versus >55 µm). the main outcome variable was visual acuity postoperative within two short period of time, 1 week and 3 months. results: comparison of visual acuity between patients with lenticule thickness of ≤55 µm and patients with lenticule thickness of >55 µm are shown in table 6. there was no significant difference in ucva (p = 1.000), significant difference in bcva (better with lenticule thickness of ≤55 µm) (p = 0.037), no significant difference in vaas (p = 0.065), and no significant difference in va3m (p = 0.599). conclusion: thin lenticule of less than or equal 55 micrometer to as low as 41 micrometer can be chosen for smile with efficacy and safety comparable to that of thicker lenticule provided that the operation is done by well trained and skilled refractive surgeon. key words: thin lenticule, smile, low myopia 224 original the effect of thin lenticule versus thick lenticule zaid yousif hameed shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 223–228 study was conducted in ibsar ophthalmology surgery centre «private medical centre» in the wholly najaf city, mid-euphrates region of iraq. the cases enrolled the study were all operated on by the same ophthalmologist. the main inclusion criterion was patients with low myopia (≤-3.0 diopter). other inclusion criteria were stable refraction in the last 2 years, and normal corneal topography. exclusion criteria were eyes with severe dryness, cataract, uveitis, or progressive corneal degeneration. according to lenticule thickness, eyes were classified into two groups, (≤55 µm versus >55 µm). the main outcome variable was visual acuity post-operative within two short period of time, 1 week and 3 months. the study was approved by the institutional ethical approval committee associated with faculty of medicine and health directorate, in addition, a verbal consent was obtained from every participant. regular pattern of corneal topography was confirmed, before the smile procedure, by oculus pentacam® hr from oculus inc, usa. in all enrolled patients, the mesopic (4 lux) pupil diameter was ≤6.5 mm and the residual thickness of the stromal bed was >250 μm. performance of smile was done using the visumax femtosecond laser (carl zeiss meditec, jena, germany) with a 500 khz. in order to produce the four cleavage planes (lenticule horizontal cut, lenticule vertical cut, the cap cut, and the peripheral external opening cut), the femtosecond laser with an energy of 130 nj was utilized. all participants were subjected to a complete ophthalmic examination pre-operatively and at day 1, week 1, and month 3, post-operatively. visual acuity was assessed using a snellen chart at 6 m in a well-illuminated room. examination also included slit-lamp biomicroscopy, dilated fundoscopy examination, and corneal topography. data were analyzed using spss for windows (statistical package for social sciences, ver. 23.0, spss inc., chicago, illinois, usa) software. quantitative data were expressed as mean, range, and standard deviation, while, categorical data were expressed as number and percentage. independent samples t-test was used to study difference in means between the two groups, while, yates correction and fischer exact tests were used to evaluate association between categorical variables. the level of significance was considered at p ≤ 0.05, and high significance at p ≤ 0.01. results the current study included 61 patients with low myopia (≤3 diopter) with a mean age of 27.67 ±7.75 years and an age range of 18–56 years. the study enrolled 23 (37.7%) males and 38 (62.3%) females. total number of eyes included in this study was 99, 52 (52.5%) right and 47 (47.5%) left, as shown in table 1. according to lenticule thickness, eyes were categorized into two groups, (≤55 µm versus >55 µm. baseline uncorrected visual acuity of patients enrolled in the current study categorized according to lenticule thickness into (≤55 versus >55 µm) was shown in table 2. the mean uncorrected visual acuity of eyes with thinner lenticule (≤55 µm thickness) was 6/ (34.95 ±22.70) and that of eyes with thicker lenticule (>55 µm thickness) was 6/ (15.81 ±5.50); the difference was highly significant, as shown in table 2. these visual acuity results assessed retrospectively. best corrected visual acuity of patients enrolled in the current study categorized according to lenticule thickness into (≤55 versus >55 µm) was shown in table 3. the mean visual acuity of eyes with ≤55 µm thickness was 6/ (6.76 ± 1.17) and that of eyes with >55 µm thickness was 6/ (8.59 ± 4.38); the difference was not significant (p = 0.075), as shown in table 3. visual acuity 1 week after surgery of patients enrolled in the current study categorized according to lenticule thickness into (≤55 versus >55 µm) are shown in table 4. the mean uncorrected visual acuity of eyes with ≤55 µm thickness was 6/ (6.74 ±0.99) and that of eyes with >55 µm thickness was 6/ (8.16 ±3.24); the difference was not significant (p = 0.063), as shown in table 4. uncorrected visual acuity 3 months after surgery of patients enrolled in the current study categorized according to lenticule thickness into (≤55 versus >55 µm) are shown in table 5. the mean uncorrected visual acuity of eyes with ≤55 µm thickness was 6/ (7.08 ±1.66) and that of eyes with >55 µm thickness was 6/ (7.34 ±2.15); the difference was not significant (p = 0.625), as shown in table 5. comparison of visual acuity between patients with lenticule thickness of ≤55 µm and patients with lenticule thickness of >55 µm are shown in table 6. there was no significant difference in ucva (uncorrected visual acuity) (p = 1.000), significant difference in bcva (better with lenticule thickness of ≤ 55 µm) (p = 0.037), no significant difference in vaas (visual acuity after surgery) (p = 0.065) and no significant difference in va3m (visual acuity 3 months after surgery) (p = 0.599). in order to produce the four cleavage planes (lenticule horizontal cut, lenticule vertical cut, the cap cut, and the peripheral external opening cut), the femtosecond laser with an energy of 130 nj was utilized (fig 1). the beginning of the femtosecond laser from outside toward inside to create the lenticule lower surface which is called the lenticule interphase (green arrow). the peripheral cut ring represents the surrounding vertical cut of the lenticule periphery to be then separated from the rest of the stroma. the upper surface femtosecond laser cut of the lenticlue beginning from the center towards the far periphery which is called the cap interphase (blue arrow), the last femtosecond laser cut which is the surface incision cut at the periphery of the cap interphase (red arrow) table 1. characteristics of patients enrolled in this study. characteristic value number of cases 61 age (years) mean ± sd 27.67 ±7.75 range (minimum–maximum) 38 (18-56) gender male, n (%) 23 (37.7 %) female, n (%) 38 (62.3 %) eye total 99 od, n (%) 52 (52.5 %) os, n (%) 47 (47.5 %) sd: standard deviation; n: number of cases; od: right eye; os: left eye. 225 original the effect of thin lenticule versus thick lenticulezaid yousif hameed shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 223–228 table 2. baseline uncorrected visual acuity of eyes enrolled in the current study categorized according to lenticule thickness into (≤55 versus >55 µm). ucva total n = 99 thickness group ≤55 n = 19 >55 n = 80 6/ 9.5 12 (12.1 %) 2 (10.5 %) 10 (12.5 %) 6/ 12 27 (27.3 %) 3 (15.8 %) 24 (30.0 %) 6/ 15 17 (17.2 %) 1 (5.3 %) 16 (20.0 %) 6/ 18 20 (20.2 %) 3 (15.8 %) 17 (21.3 %) 6/ 24 12 (12.1 %) 1 (5.3 %) 11 (13.8 %) 6/ 36 3 (3.0 %) 1 (5.3 %) 2 (2.5 %) 6/ 60 8 (8.1 %) 8 (42.1 %) 0 (0.0 %) mean ±sd 6/ (19.48 ±13.28) 6/ (34.95 ±22.70) 6/ (15.81 ±5.50) range 6/ 9.5 -6/ 60 6/ 9.5 –6/ 60 6/ 9.5 -6/ 36 p < 0.001 †hs n: number of cases; sd: standard deviation; ucva: uncorrected visual acuity; †: independent samples t-test; hs: highly significant at p ≤ 0.01 table 3. best corrected visual acuity of patients enrolled in the current study categorized according to lenticule thickness into (≤55 versus >55 µm). bcva total n = 99 thickness group ≤55 n = 19 >55 n = 80 6/6 52 (52.5 %) 12 (63.2 %) 40 (50.0 %) 6/7.5 21 (21.2 %) 5 (26.3 %) 16 (20.0 %) 6/9.5 9 (9.1 %) 2 (10.5 %) 7 (8.8 %) 6/12 10 (10.1 %) 0 (0.0 %) 10 (12.5 %) 6/15 2 (2.0 %) 0 (0.0 %) 2 (2.5 %) 6/19 3 (3.0 %) 0 (0.0 %) 3 (3.8 %) 6/24 1 (1.0 %) 0 (0.0 %) 1 (1.3 %) 6/30 1 (1.0 %) 0 (0.0 %) 1 (1.3 %) mean ±sd 6/ (8.24 ±4.03) 6/ (6.76 ±1.17) 6/ (8.59 ±4.38) range 6/ (6 -30) 6/ (6 -9.5) 6/ (6 -30) p 0.075 †ns n: number of cases; sd: standard deviation; bcva: best visual acuity; †: independent samples t-test; ns: not significant at p> 0.05. table 4. visual acuity one week after surgery of patients enrolled in the current study categorized according to lenticule thickness into (≤ 55 versus > 55 µm). thickness group vaas total n = 99 ≤55 n = 19 >55 n = 80 6/5 1 (1.0 %) 0 (0.0 %) 1 (1.3 %) 6/6 44 (44.4 %) 11 (57.9 %) 33 (41.3 %) 6/7.5 32 (32.3 %) 7 (36.8 %) 25 (31.3 %) 6/9 1 (1.0 %) 0 (0.0 %) 1 (1.3 %) 6/9.5 7 (7.1 %) 1 (5.3 %) 6 (7.5 %) 6/12 9 (9.1 %) 0 (0.0 %) 9 (11.3 %) 6/15 3 (3.0 %) 0 (0.0 %) 3 (3.8 %) 6/19 1 (1.0 %) 0 (0.0 %) 1 (1.3 %) 6/24 1 (1.0 %) 0 (0.0 %) 1 (1.3 %) mean ±sd 6/ (7.88 ±2.99) 6/ (6.74 ±0.99) 6/ (8.16 ±3.24) range (5 -24) (6 -9.5) (5 -24) p 0.063 †ns n: number of cases; sd: standard deviation; vaas: visual acuity one week after surgery; †: independent samples t-test; ns: not significant at p> 0.05. table 5. visual acuity 3 months after surgery of patients enrolled in the current study categorized according to lenticule thickness into (≤55 versus >55 µm). va3m total n = 99 thickness group ≤55 n = 19 >55 n = 80 6/5 4 (4.0 %) 0 (0.0 %) 4 (5.0 %) 6/6 51 (51.5 %) 11 (57.9 %) 40 (50.0 %) 6/7.5 24 (24.2 %) 5 (26.3 %) 19 (23.8 %) 6/9 1 (1.0 %) 0 (0.0 %) 1 (1.3 %) 6/9.5 11 (11.1 %) 2 (10.5 %) 9 (11.3 %) 6/12 6 (6.1 %) 1 (5.3 %) 5 (6.3 %) 6/15 2 (2.0 %) 0 (0.0 %) 2 (2.5 %) mean ±sd 6/ (7.29 ±2.06) 6/ (7.08 ±1.66) 6/ (7.34 ±2.15) range 6/ (5 -15) 6/ (6 -12) 6/ (5 -15) p 0.625 †ns n: number of cases; sd: standard deviation; va3m: visual acuity 3 months after surgery; †: independent samples t-test; ns: not significant at p> 0.05 226 original the effect of thin lenticule versus thick lenticule zaid yousif hameed shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 223–228 discussion laser refractive surgery field has been revolutionized following the introduction of femtosecond laser. this laser type has been widely used in lasik and it resulted in the development of newer refractive surgery technique which uses single platform rather than the known two platforms for lasik and this technique was named femtosecond lenticule extraction (flex).7, 31 the later technique has also the advantage of avoiding corneal stromal ablation which is a necessary step in flex; however, it was similar to lasik in that it needs formation of a corneal flap.31 the newly introduced laser refractive technique, smile, has the advantages of formation of corneal stromal pocket rather than corneal flap of old techniques, since it utilizes a small peripheral corneal incision in order to extract the lenticule.32 because of minimal disruption of the peripheral collagen networks in the anterior stroma, the corneal stability will be preserved following smile much better than following oldest operations, lasik and flex, in addition to avoidance of possible injury to the subbasal nerve plexus.33, 34 nowadays, smile is regarded as the standard of care for surgical correction of refraction errors by most ocular surgeons.8 acar b. t. and acar, in 201730, compared the results of smile procedure between two groups of patients depending on the cap lenticule diameter difference (cldd) and found that “in smile, 0.4 mm cldd is associated with better visual outcome than 1.0 mm and that narrow cldd should be considered in smile to increase the visual acuity particularly in the early post-operative period”. table 6. comparison of visual acuity between patients with lenticule thickness of ≤55 µm and patients with lenticule thickness of >55 µm. visual acuity thickness group p≤55 n = 19 >55 n = 80 ucva ≥ 6/ 9.5 2 (10.5 %) 10 (12.5 %) 1.000 y ns< 6/ 9.5 17 (89.5 %) 70 (87.5 %) bcva ≥ 6/ 9.5 19 (100.0 %) 63 (78.8 %) 0.037 f s< 6/ 9.5 0 (0.0 %) 17 (21.3 %) vaas ≥ 6/ 9.5 19 (100.0 %) 66 (82.5 %) 0.065 f ns< 6/ 9.5 0 (0.0 %) 14 (17.5 %) va3m ≥ 6/ 9.5 18 (94.7 %) 73 (91.3 %) 0.599 y ns< 6/ 9.5 1 (5.3 %) 7 (8.8 %) n: number of cases; ucva: uncorrected visual acuity; bcva: best corrected visual acuity; vaas: visual acuity after surgery; va3m: visual acuity 3 months after surgery; y: yates correction; f: fischer exact test; ns: not significant at p> 0.05; s: significant at p ≤ 0.05. fig. 1. this figure shows the different stages of the femtosecond laser applied during docking procedure for the correction of myopia or myopic astigmatism. fig. 2. through the peripheral opening the surgeon is accessible to introduce the dissector to separate the lenticule on both upper and lower surfaces and then removing it out of the pocket. 227 original the effect of thin lenticule versus thick lenticulezaid yousif hameed shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 223–228 a study published in 2018, carried out by siedlecki et al.35 was aiming at reporting the effect of increasing minimum lenticule thickness on the efficacy and safety and smile in patients with low myopia (up to -3.50 diopters). in the later study, thick lenticule group was compared in terms of visual and refractory outcome to standard thickness group and the results obtained pointed out to that “increasing minimum lenticule border thickness seems to improve the safety and efficacy of smile in low myopia”. in the current study, we were able to show that there seems to be no significant difference in the efficacy of smile with thin lenticule, in the contrary to the belief of siedlecki et al.35, since it was noticed in the present study that the visual acuity of patients with thin lenticule was similar, and sometimes even better, than that of patients with thicker lenticule. it seems that experienced hand can do smile with results that are of sufficient efficacy and safety when lenticule is as thin as <55 µm and this will increase the scope of selected myopic patients in need to do refractive surgery with thinner corneas as the minimum lenticule thickness we chose to preserve much residual stromal thickness to the acceptable limits. conclusion thin lenticule of less than or equal 55 µm to as low as 41 µm can be chosen for smile with efficacy and safety removal in one piece comparable to that of thicker lenticule provided that the operation is done by well-trained and skilled ocular surgeon hand to avoid tear of the lenticule and extract it as one piece (fig. 2), and we can increase the scope of myopic patients in need to do smile procedure by using thinner smile lenticule and preserve the stromal tissue to the acceptable residual stromal thickness. acknowledgment the author have no conflicts of interest the author have no supported funding, the author have no financial disclosure the author of the current study would like to express deep thanks and appreciation to all patients participating in the study. references 1. n warner, “update on myopia,” curr opin ophthalmol. 2016;27:402–406. 2. wu pc, huang hm, yu hj, fang pc, chen ct. epidemiology of myopia. asia pac j ophthalmol (phila). 2016;5(6):386-393. 3. foster pj, jiang y. epidemiology of myopia. eye (lond). 2014;28(2):202-208. 4. gl ehlke and rr krueger, “laser vision correction in treating myopia,” asiapac j ophthalmol. 2016;5:434–437. 5. a agca, a demirok, y yıldırım et al., “refractive lenticule extraction (relex) through a small incision (smile) for correction of myopia and myopic astigmatism: current perspectives,” clin ophthalmol. 2016;10:1905–1912. 6. kuryan j, cheema a, chuck rs. laser-assisted subepithelial keratectomy (lasek) versus laser-assisted in-situ keratomileusis (lasik) for correcting myopia. cochrane database syst rev. 2017;2(2):cd011080. 7. ganesh s, brar s, arra rr. refractive lenticule extraction small incision lenticule extraction: a new refractive surgery paradigm. ind j ophthalmol. 2018;66(1):10-19. 8. sekundo w, kunert k, russmann c, et al. first efficacy and safety study of femtosecond lenticule extraction for the correction of myopia: six-month results. j cataract refract surg. 2008;34(9):1513–1520. 9. titiyal js, kaur m, shaikh f, gagrani m, brar as, rathi a. small incision lenticule extraction (smile) techniques: patient selection and perspectives. clin ophthalmol. 2018;12:1685-1699. 10. sekundo w, kunert ks, blum m. small incision corneal refractive surgery using the small incision lenticule extraction (smile) procedure for the correction of myopia and myopic astigmatism: results of a 6 month prospective study. br j ophthalmol. 2011;95(3):335–339. 11. shah r, shah s, sengupta s. results of small incision lenticule extraction: all-in-one femtosecond laser refractive surgery. j cataract refract surg. 2011;37(1):127–137. 12. m liu, y chen, d wang et al., “clinical outcomes after smile and femtosecond laser-assisted lasik for myopia and myopic astigmatism: a prospective randomized comparative study,” cornea. 2016;35:210–216. 13. e chansue, m tanehsakdi, s swasdibutra, and c mcalinden, “efficacy, predictability and safety of small incision lenticule extraction (smile),” eye vision. 2015:2:14. 14. dz reinstein, tj archer, and m gobbe, “small incision lenticule extraction (smile) history, fundamentals of a new refractive surgery technique and clinical outcomes,” eye vision. 2014:1:3. 15. y zhang, q shen, y jia, d zhou, and j zhou, “clinical outcomes of smile and fs-lasik used to treat myopia: a meta-analysis,” j refract surg. 2016;32:256–265. 16. mj ye, cy liu, rf liao, g. zy, by zhao, and y liao, “smile and wavefrontguided lasik out-compete other refractive surgeries in ameliorating the induction of high-order aberrations in anterior corneal surface,” j ophthalmol. 2016;2016:article id 8702162, 7 pages. 17. wt cai, qy liu, cd ren et al., “dry eye and corneal sensitivity after small incision lenticule extraction and femtosecond laser-assisted in situ keratomileusis: a meta-analysis,” int j ophthalmol. 2017;10:632–638. 18. r shah and s shah, “effect of scanning patterns on the results of femtosecond laser lenticule extraction refractive surgery,” j cataract refractive surg. 2011;37:1636–1647. 19. a vestergaard, a ivarsen, s asp, and jq hqjortdal, “femtosecond (fs) laser vision correction procedure for moderate to high myopia: a prospective study of relex® flex and comparison with a retrospective study of fs-laser in situ keratomileusis,” acta ophthalmol. 2013;91:335–362. 20. k kamiya, k shimizu, a igarashi, and h kobashi, “effect of femtosecond laser setting on visual performance after small incision lenticule extraction for myopia,” br j ophthalmol. 2015;99:1381–1387. 21. titiyal js, kaur m, rathi a, et al. learning curve of small incision lenticule extraction: challenges and complications. cornea. 2017;36(11):1377–1382. 22. pradhan k, reinstein d, carp g, et al. learning curve for small incision lenticule extraction (smile) of a novice corneal laser refractive surgeon evaluated by procedure time for consecutive cases. european society of cataract & refractive surgeons. 2013. escrs website. available from: http://escrs.org/amsterdam2013/programme/free-papers-details. asp?id=15825&day=0. 23. ang m, mehta js, chan c, et al. refractive lenticule extraction: transition and comparison of 3 surgical techniques. j cataract refract surg. 2014;40(9):1415–1424. 24. ivarsen a, asp s, hjortdal j. safety and complications of more than 1500 small-incision lenticule extraction procedures. ophthalmology. 2014;121(4):822–828. 25. ramirez-miranda a, ramirez-luquin t, navas a, graue-hernandez eo. refractive lenticule extraction complications. cornea. 2015;34(suppl 10):s65–s67. 26. r shetty, r shroff, l kaweri, c jayadev, mk kummelil, and a sinha roy, “intraoperative cap repositioning in small incision lenticule extraction (smile) for enhanced visual recovery,” curr eye res. 2016;41:1532–1538. 27. m he, w wang, h ding, and x zhong, “comparison of two cap thickness in small incision lenticule extraction: 100 μm versus 160 μm,” plos one. 2016;11, article e0163259. 28. yw. ji, m kim, ds y. kang et al., “lower laser energy levels lead to better visual recovery after small-incision lenticule extraction: prospective, randomized clinical trial,” am j ophthalmol. 2017;179:159–170. 29. d donate and r thaëron, “lower energy levels improve visual recovery in small incision lenticule extraction (smile),” j refractive surg. 2016;32:636– 642. 30. acar bt and acar s. effect of cap-lenticule diameter difference on the visual outcome and higher-order aberrations in smile: 0.4 mm versus 1.0 mm. j ophthalmol. 2017;2017:8259546. 31. moshirfar m, mccaughey mv, reinstein dz, shah r, santiago-caban l, fenzl cr. small-incision lenticule extraction. j cataract refract surg. 2015 mar;41(3):652-65.  228 original the effect of thin lenticule versus thick lenticule zaid yousif hameed shukur j contemp med sci | vol. 6, no. 5, september-october 2020: 223–228 32. miruna n, andrei f, vasile fm, rotaru e. smile--the next generation of laser vision correction. rom j ophthalmol. 2016;60(1):6-8. 33. moshirfar m, albarracin jc, desautels jd, birdsong oc, linn sh, hoopes pc. ectasia following small-incision lenticule extraction (smile): a review of the literature. clin ophthalmol. 2017;11:1683-1688. 34. moshirfar m, bruner cd, skanchy df, shah t. hyperopic small-incision lenticule extraction. curr opin ophthalmol. 2019 jul;30(4):229-235. 35. siedlecki j, luft n, keidel l, et al. variation of lenticule thickness for smile in low myopia. j refract surg. 2018;34(7):453-459. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.860 261j contemp med sci | vol. 7, no. 4, july–august 2021: 261–263 case report the supply of cellular energy by changing in diet and life-style a possible treatment for uterine myoma and cyst: a case report hassan akbari1,2, khadije heydarolad3* 1school of persian medicine, tehran university of medical sciences, tehran, iran. 2department of pathology, shahid beheshti university of medical science, tehran, iran. 3iranian traditional medicine doctor, university of traditional medicine of armenia, utma. *correspondence to: khadije heydarolad (e-mail: hassanakbari23@yahoo.com) (submitted: 17 april 2021 – revised version received: 29 april 2021 – accepted: 23 may 2021 – published online: 26 august 2021) introduction the uterine myoma or fibroids with or without cyst is the most common benign tumor of the female reproductive system causing significant morbidity and impairing their quality of life. it is frequently found in fertile women aged 16 to 50 years old. the etiology of the disease is not clearly known. gene abnormality and hormonal effects are associated with the disease.1 while many cases remain asymptomatic, myomas may presents with abnormal uterine bleeding, pelvic pain, sciatica, urinary frequency, dysuria, constipation, infertility, and, in extreme cases, ureteral obstruction and death according to the site, type, and size of the lesion.2 todays, surgery (hysterectomy) is the most attempted treatment in cases with large tumor size or severe symptoms. myomas are the underlying cause of half all performed hysterectomies. removal of the uterus is inacceptable to many women whether they are willing to bearing a child later or simply because of psychological burden. besides, surgical interventions carry lots of possible complications, including bleeding, injury to bladder, intestine or ureters, subsequent adhesion formation and obstruction, complications of anesthesia and of hospitalization in general. also, surgeries impose many costs to the patients and health systems.3 all of these consequences lead the current treatment approaches to non-surgical and conservative ones and over the years conservative approaches have been introduced. changing in cellular energy can cause numerous diseases including myomas and cysts. supplying cellular energy by changing in nutrition and life style could be a possible treatment for myomas and cysts. in this case report we treated a 48-year-old woman who was diagnosed with uterine myoma and cervical cyst by changing pattern of nutrition and lifestyle and using some traditional persian medicine approaches from september 2020 to december 2020. she was candidate for hysterectomy. however, she was not willing to receive any surgical intervention or hormone therapy. case description patient characteristics and medical history a 48-year-old woman without any past medical history referred to baqiatallah hospital, tehran, iran in september 2020 with the chief complaint of excessive bleeding during menstruation from one year ago. other complaints were 1) feeling a pressure in hypo-gastric area which begins 2–3 days before the menstruation and lasts for 1–2 days after menstruation 2) dysmenorrhea, from one year ago menstrual pain worsened and the volume of bleeding increased so she took painkillers on the first day of menstruation. she had not any complaint of dysuria, frequency in urination and leucorrhea. her menstrual cycle was 25–28 days long and regular and her menstruation duration was 7 days. she had a obstetric history of three full-term births, pre-term birth 0, abortion 0 and live offspring 3 (gpal: 3–0–0–3). physical examinations of the patient were as follows: 1) general appearance: pale, 2) pulse: weak, thin and symmetric, 3) sleep: normal, 4) defecation: normal, 5) urination: normal, 6) abdomen: a mass was palpable in the hypo gastric area without tenderness, 7) vaginal examination with speculum: no abnormal secretion or bleeding was observed, a 2 cm cyst was observed on the cervix, 8) anthropometrics: 160 cm, 61.7 kg, bmi: 24.1. after examination and taking history the patient temperament was diagnosed as a cold-wet type. the gynecologist requested ultrasound of uterus and ovaries and appendages. a 22 mm myoma was observed in the anterior septum of the uterine and along with a 15 mm cyst in abstract objectives uterine myoma and cervical cyst are common benign tumors. if the size is too large or the symptoms are severe or get worse, the patient will receive surgery or hormone therapy. a 68 years old woman presented to a hospital in iran with abnormal uterine bleeding (aub). after performing sonography and suitable imaging, the patient was diagnosed with cervical cyst and uterine myoma. the patient was candidate for hysterectomy. the patient was not willing to do surgery. we tried a traditional medicine approach for this case. we recommended the lifestyle change by following the health triangle instruction that is based on six essential principles. these principles are as following: eating and drinking according to temperament, proper sleeping and awaking, correct motion and motionless, retention and vomiting, mental disorders. she received traditional medicine treatment including dietary and life-style change, using vaginal honey and olive oil, frasion sodden, aftimoni honey vinegar, docein, horsehair sodden and phlebotomy, wet cupping and dry cupping on specific regions. the treatment course was 4 months long. the complete cure of cervical cyst and reduction in size of myoma and bleeding was resulted. and the patient had no problem. keywords uterine myoma, cervical cyst, traditional medicine, health triangle issn 2413-0516 262 j contemp med sci | vol. 7, no. 4, july–august 2021: 261–263 the supply of cellular energy by changing in diet and life-style case report h. akbari and k. heydarolad the cervical area. the patient was candidate for hysterectomy by the gynecologist physician. the patient was not willing to do the surgery because of psychological burden. she visited the traditional medicine clinic seeking the non-surgical treatment. treatment and progress of symptoms therapeutic map we recommended the patient to change the lifestyle by following the health triangle instruction that is based on four essential principles, including eating and drinking a wellbalanced diet and according to temperament, proper sleeping and awaking (getting at least 8 hours of sleep every night) and physical activity. dietary intervention the recommended dietary changes according to temperament were as follows: 1) replacing the current consumable oils with consumed sesame, olive and animal oil, 2) replacing the iodized salt with natural organic salt, 3) not using the white sugar, cub sugar, tomatoes and tomatoes sauce. pharmaceutical intervention the ordered pharmaceutical interventions were as follows: 1) using vaginal honey and olive oil one other night, for 7 session a week, to healing only vaginal honey and olive oil, 2) daily drinking of glass of frasion sodden with honey, for four months, 3) drinking one glass of aftimoni honey vinegar daily (one third of the glass should be honey vinegar, and the rest should be water) for two month, and then typical honey vinegar for 40 days, 4) consuming docein every 12 hours one tablespoon up to 2 months, 5) using horsehair sodden to stop bleeding, 2 glass daily, 6) consuming bark pomegranate pollen a tablespoon every 8 hours. manual practices for the perfect result we also carry out right hand phlebotomy, then after a month we performed wet cupping between the two shoulders, and after two weeks we performed sacral cupping, waist and hypogastria warm and dry cupping for 21 sessions by chamomile oil. progress of the symptoms the main symptoms of the patient were resolved after the treatments and the hysterectomy was not accomplished. the excessive bleeding which the patient was complaining about was reduced to the normal amount. and the patient did not report any side effects during the course of treatment. in the follow up solography it was specified that the 15 mm cervical cyst was completely vanished and the myoma size reduced to 17 mm. the following months the patient had not any problem. informed consent and ethics for using of personal medical information for publication the informed consent was obtained from the patient. discussion in this case study, traditional persian medicine was found to be effective for women with uterine myoma and cervical cyst who did not want surgical interventions or hormone therapy. patients may refuse surgical interventions because of fear of surgery, financial and psychological burdens or individual situation. these patients could be alternatively treated with conservative and traditional medicine instead of invasive surgery. for this purpose, we should provide enough information on the advantages and disadvantages of various treatments. uterine myoma (fibroids) can form in reproductive organs of women especially in ages of 16 to 50. the genetic and environmental factors have both associated with this disease. the environmental factors that affect health could be perfect targets to treat and prevent myomas. one of these factors is undoubtedly life-style and diet. studies support the idea that regular intake of fruits and cultured milk food, availability of quality preferences in diet, regular exercises, frequent foot walks at clean air could prevent the formation of uterine myoma.4 besides, cellular energy deprivation could lead to many diseases and lesions including uterine cysts and myomas. if the energy is not sufficient, the immune system and mechanism that destroy the pathologies in the body are the firsts that damage.5 by improving the cellular energy and supplying this energy deficiency for immune system cells, smooth muscle cells of uterine myomas and epithelial cells of cysts, the body as a complex system can automatically eliminate the pathologic cells. the underlying mechanism includes pathologic cells apoptosis with the aim of macrophages and other immune system cells.6 in addition, some specific temperament are associated with gynecological diseases.7 these results highlight the importance of balanced diet which is in accordance with temperament on preventing and improving the uterine myoma. the main component of our therapeutic approach was honey. there are several studies on the anti-tumor effect of honey.8 the possible mechanisms for anti-tumor activity of honey and its ingredients are antioxidant, anti-inflammatory, ant proliferative, apoptotic, tumor necrosis factor inhibiting, immune modulatory and estrogenic effects.9 honey along with bark pomegranate pollen have benefits that we found is suitable for preventing and improving uterine myoma, including anti-oxidant and anti-tumor compounds in pomegranate and honey. the pharmaceutical therapy has also the effect of relieving blood clots and improving dysmenorrhea by promoting blood circulation.10, 11 along with pharmaceutical therapies and life-style change traditional medicine manual practices have also been found to be effective in numerous diseases.12 a case report of the effectiveness of dry cupping on uterine fibroid has been recently published.13 according to the traditional persian medicine, cupping therapy has a favorable effect on uterus and ovaries functions improvement and is recommended for their related diseases.14 the possible therapeutic effects of cupping therapy have been studied previously. cupping therapy increases opioid production in body which can induce comfort, muscle relaxation and pain reduction. it has also increases blood flow to the muscles and skin, stimulates the peripheral and autonomic nervous systems, and modulates the immune system and hormonal balance.15 it is shown that dry cupping can decrease pain intensity and menstrual bleeding. cupping can also divert blood flow away from the pelvic area and reduce the congestion in female reproductive system and finally reducing pain and tumor size.16 263j contemp med sci | vol. 7, no. 4, july–august 2021: 261–263 h. akbari and k. heydarolad case report the supply of cellular energy by changing in diet and life-style conclusion in conclusion, supplying the cellular energy deprivation of the immune system cells and smooth muscle cells of myomas and epithelial cells of cysts by changing in nutrition and lifestyle could be an alternative therapy for uterine myomas and cysts for patients who are unwilling to receive hormonal therapy and perform surgeries.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1. sparic r, mirkovic l, malvasi a, tinelli a. epidemiology of uterine myomas: a review. int j fertil steril. 2016;9(4):424-35. 2. el-balat a, dewilde rl, schmeil i, tahmasbi-rad m, bogdanyova s, fathi a, et al. modern myoma treatment in the last 20 years: a review of the literature. biomed research international. 2018;2018:4593875. 3. okolo s. incidence, aetiology and epidemiology of uterine fibroids. best practice & research clinical obstetrics & gynaecology. 2008;22(4):571-88. 4. feofilova ma, pavlov og, geimerling ve. [the effect of life-style and occupational hazards on development of hysteromyoma]. probl sotsialnoi gig zdravookhranenniiai istor med. 2018;26(6):406-10. 5. delmastro-greenwood mm, piganelli jd. changing the energy of an immune response. am j clin exp immunol. 2013;2(1):30-54. 6. gerkau nj, rakers c, petzold gc, rose cr. differential effects of energy deprivation on intracellular sodium homeostasis in neurons and astrocytes. j neurosci res. 2017;95(11):2275-85. 7. sahebi l. the association between temperament and gynecological disease from persian medicine point of view. 2020. 8. cianciosi d, forbes-hernández ty, afrin s, gasparrini m, reboredorodriguez p, manna pp, et al. phenolic compounds in honey and their associated health benefits: a review. molecules. 2018;23(9). 9. waheed m, hussain mb, javed a, mushtaq z, hassan s, shariati ma, et al. honey and cancer: a mechanistic review. clin nutr. 2019;38(6):2499-503. 10. pyo sj, kang dg, jung c, sohn hy. anti-thrombotic, anti-oxidant and haemolysis activities of six edible insect species. foods. 2020;9(4). 11. kandylis p, kokkinomagoulos e. food applications and potential health benefits of pomegranate and its derivatives. foods. 2020;9(2). 12. cao h, li x, liu j. an updated review of the efficacy of cupping therapy. plos one. 2012;7(2):e31793. 13. majid d, mohamad hossein a, sara r, elham a. effectiveness of dry cupping therapy in the management of uterine fibroid: a case report. traditional and integrative medicine. 2020;5(2). 14. mokaberinejad r, rampisheh z, aliasl j, akhtari e. the comparison of fennel infusion plus dry cupping versus metformin in management of oligomenorrhoea in patients with polycystic ovary syndrome: a randomised clinical trial. j obstet gynaecol. 2019;39(5):652-8. 15. al-bedah amn, elsubai is, qureshi na, aboushanab ts, ali gim, el-olemy at, et al. the medical perspective of cupping therapy: effects and mechanisms of action. j tradit complement med. 2018;9(2):90-7. 16. inanmdar w, sultana a, mubeen u, rahman k. clinical efficacy of trigonella foenum graecum (fenugreek) and dry cupping therapy on intensity of pain in patients with primary dysmenorrhea. chin j integr med. 2016. https://doi.org/10.22317/jcms.v7i4.986 122 original issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 122 – 127 introduction metabolic syndrome is associated with an increased risk of type 2 diabetes mellitus (dm) and cardiovascular disease (cvd).1-4 metabolic syndrome is the coincidence in a person of interrelated risk factors for cvd and diabetes. these factors include hyperglycemia, raised blood pressure, elevated triglyceride level, reduced hdl-c, and obesity (particularly central adiposity).5 various reports indicate that the individual of the metabolic syndrome is associated with increased risk for both cvd and type 2 diabetes.6,7 higher blood pressure, the shorter life expectancy, people with high blood pressure run a higher risk of having a stroke (which damages the brain) or a heart attack. if left untreated for a long time, high blood pressure can lead to kidney failure and even damage the eye. it can also make the heart abnormally large and less efficient (a condition called ‘left ventricular hypertrophy’). this can lead to heart failure, which is when the pumping action of the heart becomes less effective. it also known as hypertension – rarely makes people feel ill. it is sometimes called a ‘silent threat’ because there are usually no symptoms, and it very often goes undiagnosed.8 blood pressure is regulated by renin–angiotensin system (ras). dm is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, activation, or action.9 the effects of dm include long-term damage, dysfunction, and failure of various organs. dm may present with characteristic symptoms such as thirst, polyuria, blurring of vision, and weight loss. most cases of dm fall into three general broad categories: type 1 dm (t1dm), type 2 dm (t2dm), and gestational dm (gdm) and another type is a specific type of diabetes.10 blood pressure is the force of blood against the walls of arteries. blood pressure rises and falls throughout the day. when blood pressure stays elevated over time, it’s called high blood pressure.11 the myosin heavy chain 9 (myh9) gene, which is located on chromosome 22 q12.3-13.2, has 40 exons that encode a protein of approximately 224,000 kda. the protein is a non-muscle myosin heavy chain that dimerizes to form the motor domain of non-muscle myosin iia, an important motor protein found in most cells of the body.12 the myh9 gene is expressed in fibroblasts, erythroblasts, and kidney cells.13 abnormal myh9 expression, positioning, or function change leads to cytoskeleton damage, causing proteinuria, hematuria, or renal failure in some cases.14 myh9 has also been shown to be a major susceptibility gene for nephropathy, and hypertension. this association has also been demonstrated in various populations, ranging from african-americans and hispanic americans to europeans.15–18 freedman et al. found that mutations in myh9 association between microalbuminuria and myh9 gene polymorphisms in hypertensive iraqi patients with metabolic syndrome alaa fahem jasim1, fadhil jawad al-tu’ma1*, dhafir abdul-mahdi faisal2 1 department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 2 college of pharmacy, university of kufa, najaf, iraq. *correspondence to: fadhil jawad al-tu’ma (e-mail: fadhil.jawad@uokerbala.edu.iq) (submitted: 21 december 2020 – revised version received: 09 january 2021 – accepted: 19 february 2021 – published online: 26 april 2021) abstract objective the presented work aimed to study the correlation between microalbuminuria and myh9 (t>c) (rs3752462) gene polymorphisms in metabolic syndrome of iraqi patients. methods this study was a cross-sectional study. sample size was 140 persons of both gender selected randomly between april 1, 2019 and aug. 15, 2020. they are divided into two groups, 80 with metabolic syndrome patients and 60 with control groups subdivided into another two groups: 30 persons with obese and 30 persons without obese. the distribution of sample according to the gender, the number of males had metabolic syndrome were 47 and female 33, but in obese healthy control the number of males were 19 and female 11, while in non-obese healthy control the number of males were 15 and female 15. the age groups were ranged between 40 and 60 years. typing of myh9 was performed by refractory mutation system-polymerase chain reaction (arms-pcr). the current study was performed to investigate the snps that affected myh9 gene which was (t>c) (rs3752462). results there was a significant difference between blood sugar, hba1c, total cholesterol, tg, vldl-c, ldl-c, blood urea, microalbuminuria, bmi, and homa-ir in metabolic syndrome patients as compared with healthy control groups (p ≤ 0.01). the significant result (p ≤ 0.01) were appeared between tt for myh9 genotype patients and obese control with biochemical parameters (blood glucose, hba1c, tc, tg, vldl-c, and ldl-c). the correlation between tt snp for myh9 genotype patients and non-obese control with biochemical parameters is significant result (p ≤ 0.01 and p ≤ 0.05) (blood glucose, hba1c, insulin, tc, tg, vldl-c, ldl-c, urea, and microalbuminuria). the significant result (p ≤ 0.01 and p ≤ 0.05) appeared between ct for myh9 genotype patients and obese control with biochemical parameters (blood glucose, hba1c, tc, tg, and vldl-c) respectively. conclusions there is a significant difference between blood glucose, hba1c, total cholesterol, tg, vldl-c, ldl-c, blood urea, microalbuminuria, bmi, and homa-ir in metabolic syndrome patients and healthy control groups (p ≤ 0.01). also, a significant result (p ≤ 0.01) obtained between tt for myh9 genotype patients and obese and non-obese control with some biochemical parameters. (p ≤ 0.01 and p ≤ 0.05). the significant results (p ≤ 0.01 and p ≤ 0.05) appear between ct for myh9 genotype patients and obese control with some biochemical parameters. keywords metabolic syndrome, albumins, myh9, genotype, arms-pcr http://en.wikipedia.org/wiki/diabetes_mellitus_type_1 http://en.wikipedia.org/wiki/diabetes_mellitus_type_2 123 original association between microalbuminuria and myh9 gene polymorphismsfadhil jawad al-tu’ma et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 122 – 127 associated with african-american proteinuria caused by high blood pressure were associated with end-stage renal disease (esrd).19 other investigator show that the rs3752462 was associated with chronic kidney disease (ckd).20 the present study was aimed to investigate the correlation between microalbuminuria and myh9 (t>c) (rs3752462) gene polymorphisms in metabolic syndrome of iraqi patients and its correlation with various biochemical markers. materials and methods this work was designed as a cross-sectional study, 140 subjects were recruited, about 80 of them with metabolic syndrome and the other 60 of apparently healthy individuals randomly with age ranged between 40 and 50 years between april 1, 2019 and aug. 15, 2020. the patients were diagnosed by a specialist physician at al-hassan medical center for endocrinology, in the al-hussein medical city / kerbala health directorate iraq. it should be noted that kerbala is a holy city and one of the biggest cities in iraq. the study protocol was approved by the medical ethics committee of college of medicine/ university of kerbala and the kerbala health directorate. the glycated hemoglobin (hba1c) level was assessed, using the cobas hba1c kit (roche, germany), by cobas integra® 400 (roche, germany); the turbidity measurement was made with a spectrophotometer to determine the light absorbance. the amount of light blocked by particle suspension depends not only on concentration, but also on the particle size, because particles tend to aggregate and settle out of suspension, consequently sample handling becomes critical. blood samples were collected from t2dm patients and the control group in edta tubes. total genomic dna was extracted from the peripheral blood sample (1 ml), using the kit reliaprep™ blood gdna miniprep system obtained from (promega usa). the protocol was followed according to the manufacturers’ recommendations. dna concentration and purity were measured, using a biodrop (uk). genotyping of myh9 (t>c) (rs3752462) polymorphism was performed by tetra-primer amplification refractory mutation system-polymerase chain reaction technique (t-armspcr) with the use of thermocycler (cleaver/uk). outer forward: 5ʹ-agctgcagcccagagcatctcctctaat-3ʹ; outer reverse: 5ʹ-tcgtttgagcagctgtgcatcaattaca-3ʹ; inner forward: 5ʹ-caggtgtg aggtcaaagcaagccttgt-3ʹ. inner reverse: 5ʹ-atcgacc tcattgaga agccagtgaggag-3ʹ. the amplification was performed in a total volume of 25 μl consisted of 12.5 μl of taq green master mix (promega, usa) 1, 1, 1 and 1 μl of outer forward, reverse forward, inner forward and inner reverse primers, respectively and 5 μl genomic dna as a template. the pcr reaction program protocol was 94 °c for 5 min followed by 35 cycles of 94 °c for 35 s, 55 °c for 45 s, 72 °c for 55 s and a final cycle 72 °c for 5 min. the amplification products were 381 bp for control band. the product of amplification was analyzed by 2% agarose gel electrophoresis. results in the present study that is shown in table 1, there was a significant difference between blood glucose, hba1c, total cholesterol, tg, vldl-c, ldl-c, blood urea, microalbuminuria, bmi, and homa-ir in metabolic syndrome patients and table 1. correlation between biochemical parameters with metabolic syndrome and compared with healthy control. p value mean ± sd parameters control groups patients non-obeseobese ≤ 0.01110.87 ± 17.31112.33 ± 27.22244.31 ± 98.28blood glucose, mg/dl ≤ 0.015.64 ± 0.486.08 ± 0.739.30 ± 2.15hba1c% 0.1313.13 ± 2.3014.37 ± 3.2314.8 ± 4.48insulin, µu/ml ≤ 0.01120.00 ± 22.57124.07 ± 23.71178.28 ± 47.32tc, mg/dl ≤ 0.01107.50 ± 20.89114.30 ± 25.09254.29 ± 101.50tg, mg/dl 0.6835.47 ± 6.7833.60 ± 5.7434.95 ± 9.96hdl-c, mg/dl ≤ 0.0120.40 ± 4.2322.83 ± 4.5650.81 ± 20.76vldl-c, mg/dl ≤ 0.0161.97 ± 27.2463.93 ± 25.59100.70 ± 43.28ldl-c, mg/dl ≤ 0.0126.40 ± 7.0829.93 ± 6.7635.09 ± 12.97urea, mg/dl 0.500.87 ± 0.180.90 ± 0.220.95 ± 0.426creatinine, mg/dl ≤ 0.0113.40 ± 5.3313.83 ± 20.8550.08 ± 77.95microalbuminuria ≤ 0.0123.78 ± 1.0037.01 ± 1.8237.48 ± 1.98bmi, kg/m2 ≤ 0.013.66 ± 1.14.05 ± 1.58.4 ± 3.02homa-ir (hba1c) glycated hemoglobin a1c, (tg) triglycerides, (tc) total cholesterol, (hdl-c) high-density lipoproteincholesterol, (vldl-c) very low density lipoprotein-cholesterol, (ldl-c) low density lipoprotein-cholesterol, (bmi) body mass index, (homa-ir) homeostatic model assessment of insulin resistance. https://www.mayoclinic.org/diseases-conditions/chronic-kidney-disease/symptoms-causes/syc-20354521 http://www.webmd.com/cholesterol-management/default.htm 124 original association between microalbuminuria and myh9 gene polymorphisms fadhil jawad al-tu’ma et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 122 – 127 healthy control groups (p ≤ 0.01). while a non-significant differences between metabolic syndrome patients and healthy control groups regarding insulin (p = 0.13), hdl-c (p = 0.68), and serum creatinine (p = 0.50). the amplification of myh9 (t>c) gene polymorphism was observed in 381 bp as shown in fig. 1. lane 1: represented dna marker 100 1000 bp. lane 4, 8 and 9: represented heterozygote (ct) and internal control of (t>c) snp of myh9 gene were showed in (381 bp, 234 bp and 201 bp). lane 2 and 6: represented mutant type (cc) and internal control of (t>c) snp of myh9 gene were showed in (381 bp and 201 bp). lane 3 : represented wild type (tt) and internal control of (t>c) snp of myh9 gene were showed in (381 bp and 234 bp). lane 5 and 7: represented internal control of (cc) of myh9 gene were showed in (382 bp). fig. 1 amplification of snp of myh9 (t>c) was showed in this figure table 2. correlation between snp (t>c) for myh9 genotype and biochemical parameters in patients with mets. myh9 (t>c)patients group mean of parameters cccttt 54233no. of patients 185.25256.67239.78blood glucose, mg/dl 9.429.3959.236hba1c% 1614.315.42insulin, µu/ml 172.75173.97182.09tc, mg/dl 146.75268.82245.57tg, mg/dl 4032.8537.12hdl-c, mg/dl 27.2553.4550.03vldl-c, mg/dl 113.2595.42103.57ldl-c, mg/dl 36.534.9535.06urea, mg/dl 0.880.940.97creatinine, mg/dl 1755.8750.30microalbuminuria 36.5737.6337.28bmi, kg/m2 (hba1c) glycated hemoglobin a1c, (tg) triglycerides, (tc) total cholesterol, (hdl-c) high-density lipoprotein-cholesterol, (vldl-c) very low density lipoprotein-cholesterol, (ldl-c) low density lipoprotein-cholesterol, (bmi) body mass index. table 3. correlation between control group had snp (t>c) for myh9 genotype and biochemical parameters. myh9 (t>c) control groups mean of parameters non obese controlobese control ccctttcccttt 00301821no. of subjects //110.8691117.12111.52blood glucose, mg/dl //5.635.76.236.03hba1c, % //13.131113.514.85insulin, µu/ml //120123130.75121.57tc, mg/dl //107.5119116.37113.28tg, mg/dl //35.463135.2533.09hdl-c, mg/dl //20.4212322.85vldl-c, mg/dl //61.966870.8761.09ldl-c, mg/dl //26.42029.2530.66urea, mg/dl //0.870.80.950.88creatinine, mg/dl //13.4611.515.09microalbuminuria //23.7836.337.6536.80bmi, kg/m2 (hba1c) glycated hemoglobin a1c, (tg) triglycerides, (tc) total cholesterol, (hdl-c) high-density lipoproteincholesterol, (vldl-c) very low density lipoprotein-cholesterol, (ldl-c) low density lipoprotein-cholesterol, (bmi) body mass index. http://www.webmd.com/cholesterol-management/default.htm http://www.webmd.com/cholesterol-management/default.htm 125 original association between microalbuminuria and myh9 gene polymorphismsfadhil jawad al-tu’ma et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 122 – 127 discussion metabolic syndrome and hypertension verified the convincing association with microalbuminuria in both females or = 3.34; 95% (ci 2.454.55) and males or = 2.51; 95% (ci 1.633.86)21(table 3). the most common component of metabolic syndrome in our sample was shown to be central obesity and dm followed by hypertriglyceridemia and hypertension; this disagrees with the indian study, which showed that the main component was hypertension (98.37%), followed by dyslipidemia (77.05%), hyperglycemia (75.41%), and obesity (59.02%).22 most of the obese who showed metabolic syndrome were not exercising, but no significant association was found between metabolic syndrome and practicing exercises. the heritage family study in the united states showed that exercise training resulted in improvement in the metabolic profile of the participants; the prevalence of metabolic syndrome was decreased from 16.9% before training to 11.8% after training.23 the higher prevalence of high tg in our country relative to most of the studies elsewhere might be due to higher intake simple carbohydrates and higher ratio of simple to complex carbohydrate consumption by the persian people.24 we observed higher prevalence of hypertriglyceridemia in iranian males similar to findings in most other countries.25 older adults are highly susceptible to overweight and obesity due to reduced mobility.26 kuchak and colleagues, as well as simoes and colleagues, reported that a lack of physical activity program and movement in older adults can intensify the growth of body mass and obesity.27,28 dyslipidemia is one of the major risk factors for coronary heart disease besides age, family history, cigarette smoking, hyperglycemia, and hypertension. the prevalence of dyslipidemia varies widely according to the ethnic, socioeconomic, and cultural characteristics of distinct population groups.29 hypertriglyceridemia was independently associated with dm. a similar association between hypertriglyceridemia and hba1c was reported in a russian study.30 the present study showed the independent risk factors of the different types of dyslipidemia by multivariate analysis. dm and hypertension were independent risk factors for hypercholesterolemia. a study from saudi arabia also reported same association between dm, hypertension, and hypercholesterolemia.31 the mean value of homa-ir was significantly higher in adult male with essential hypertension when compared with that of controls. systolic blood pressure and diastolic blood pressure show positive correlation with homa-ir but was significant only with systolic blood pressure. this finding is consistent with the findings of some other investigators of other countries.32,33 pa sarafidis and an lasaridis was designed to determine the validity and the reproducibility of homa-ir, 1/homa-ir, quicki and mcauley’s index in a population of patients that have developed hypertension before type ii diabetes.34 an elevated serum creatinine level is also a late sign of renal damage in essential hypertension with frankly elevated serum creatinine values predict a poor prognosis in patients with hypertension.35 a study by wachtell et al in 2002 showed an association with raised microalbuminuria and increased risk of heart attacks and stroke. among the patients with hypertension.36 the prevalence of microalbuminuria was also higher among those with hypertensive retinopathy in our study and it was statistically significant. microalbuminuria is more prevalent in essential hypertensive with target organ dysfunction.36 table 4. the correlation between snp (t>c) for myh9 genotype and biochemical parameters myh9 (t>c) cccttt patients vs. non-obese control patients vs. obese control patients vs. non-obese control patients vs. obese control patients vs. non-obese control patients vs. obese control biochemical parameters -0.3-≤ 0.01≤ 0.01≤ 0.01blood sugar, mg/dl -0.097-≤ 0.01≤ 0.01≤ 0.01hba1c% -0.6-0.7≤ 0.010.6insulin, µu/ml -0.6-≤ 0.01≤ 0.01≤ 0.01tc, mg/dl -0.5-≤ 0.01≤ 0.01≤ 0.01tg, mg/dl -0.2-0.40.50.2hdl-c, mg/dl -0.4-≤ 0.01≤ 0.01≤ 0.01vldl-c, mg/dl -0.7-0.1≤ 0.01≤ 0.01ldl-c, mg/dl -0.6-0.16≤ 0.010.24urea, mg/dl -0.7-0.90.30.5creatinine, mg/dl 0.6-0.13≤ 0.050.07microalbuminuria (hba1c) glycated hemoglobin a1c, (tg) triglycerides, (tc) total cholesterol, (hdl-c) high-density lipoprotein-cholesterol, (vldl-c) very low density lipoproteincholesterol, (ldl-c) low density lipoprotein-cholesterol. http://www.webmd.com/cholesterol-management/default.htm 126 original association between microalbuminuria and myh9 gene polymorphisms fadhil jawad al-tu’ma et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 122 – 127 the amplification of snp of myh9 (t>c) was showed in 381 bp as in fig 3. association between myh9 (t>c) snp and biochemical parameters the results in current study are shown in tables 2-4. the correlation between metabolic syndrome patients and control groups have myh9 (t>c) snp with biochemical parameters (blood glucose, hba1c, insulin, tc, tg, hdl-c, vldl-c, ldl-c, urea, creatinine, microalbuminuria, and bmi). the significant results (p ≤ 0.01) appear between tt for myh9 genotype patients and obese control with biochemical parameters (blood glucose, hba1c, tc, tg, vldl-c, and ldl-c). the correlation between tt snp for myh9 genotype patients and non-obese control with biochemical parameters is significant result (p value ≤ 0.01 and p ≤ 0.05) (blood glucose, hba1c, insulin, tc, tg, vldl-c, ldl-c, bl. urea, and microalbuminuria). the significant results (p ≤ 0.01 and p ≤ 0.05) appear between ct for myh9 genotype patients and obese control with biochemical parameters (blood glucose, hba1c, tc, tg, and vldl-c). the non-significant results (p-value > 0.01) are appear between cc for myh9 genotype patients and obese control with biochemical parameters (blood glucose, hba1c, insulin, tc, tg, hdl-c, vldl-c, ldl-c, urea, creatinine, and microalbuminuria). the correlation between tt snp for myh9 genotype patients and obese control with biochemical parameters (insulin, hdl-c, urea, creatinine and microalbuminuria) is non-significant (p > 0.01). the non-significant results (p > 0.01) showed between tt snp for myh9 genotype patients and non-obese control with biochemical parameters (hdl-c and creatinine). the correlation between ct snp for myh9 genotype patients and obese control with biochemical parameters (insulin, hdl-c, ldl-c, urea, creatinine, and microalbuminuria) is non-significant (p > 0.01). the results in current study that are shown in the same tables that the correlation between metabolic syndrome patients and control groups have myh9 (t>c) snp with biochemical parameters (blood glucose, hba1c, insulin, tc, tg, hdl-c, vldl-c, ldl-c, urea, creatinine, microalbuminuria, and bmi). there is a difference in the distribution of the cc, ct, and tt genotypes at rs3752462 in patients with ckd with sbp ≥ 140 mmhg and sbp < 140 mmhg. the multivariate analysis showed that the cc genotype at rs3752462 was associated with a lower risk of concurrent higher sbp than the tt genotype in ckd patients. this result provides evidence that there is a genetic link between myh9 and hypertension in patients with ckd in china.37 it is found that myh9 snp rs3752462 is involved in the susceptibility of ckd patients to hypertension. multivariate logistic regression analysis indicated that the cc genotype was a protective factor against higher sbp in patients with ckd. it also indicated that the mutation of a c allele to a t allele can lead to an sbp increase.38,39 the minor allele of snp rs3752462 is associated with an increased risk of dkd. the results suggest that myh9 rs3752462 might play an important role in the risk of dkd in the chinese han population.40 myh9 rs3752462 was associated with cerebrovascular blood flow (cbf) in patients with type 2 diabetes.41 albuminuria is highly associated with indian blood quantum in this population suggesting a strong genetic susceptibility to ckd.42 conclusion 1. there is a significant difference between blood glucose, hba1c, total cholesterol, tg, vldl-c, ldl-c, blood urea, microalbuminuria, bmi, and homa-ir in metabolic syndrome patients and healthy control groups (p ≤ 0.01). 2. most of metabolic syndrome patients are found in age group of 40–49 years. 3. the significant result (p ≤ 0.01) appear between tt for myh9 genotype patients and obese control with biochemical parameters (blood glucose, hba1c, tc, tg, vldl-c, and ldl-c). 4. the correlation between tt snp for myh9 genotype patients and non-obese control with biochemical parameters is significant result (p ≤ 0.01 and p-value ≤ 0.05) (blood glucose, hba1c, insulin, tc, tg, vldl-c, ldlc, urea, and microalbuminuria). 5. the significant result (p ≤ 0.01 and p ≤ 0.05) appear between ct for myh9 genotype patients and obese control with biochemical parameters (blood sugar, hba1c, tc, tg, and vldl-c). conflict of interest none references 1. grundy, sm. metabolic syndrome: connecting and reconciling cardiovascular and diabetes worlds. j am coll cardiol, 2006;47.6:1093-1100. 2. grundy sm. does a diagnosis of metabolic syndrome have value in clinical practice? june 2006;83(6). 3. grundy, sm. drug therapy of the metabolic syndrome: minimizing the emerging crisis in polypharmacy. nat rev drug discov, 2006;5.4:295-309. 4. 4. moller, de, kaufman, k d. metabolic syndrome: a clinical and molecular perspective. annu rev med, 2005;56:45-62. 5. eckel, rh., et al. the metabolic syndrome. the lancet, 2010;375.9710: 181-183. 6. lakka, h-m, et al. the metabolic syndrome and total and cardiovascular disease mortality in middle-aged men. jama, 2002;288.21:2709-2716. 7. hunt, kelly, et al. ncep versus who metabolic syndrome in relation to all cause and cardiovascular mortality in the san antonio heart study (sahs). diabetes, 2003;52. 8. hansen, ml, gunn, pw, kaelber, dc. underdiagnosis of hypertension in children and adolescents. jama, 2007;298.8:874-879. 9. american diabetes association, et al. standards of medical care in diabetes—2010. diabetes care, 2010;33(1):s11-s61. 10. baynes, hw. classification, pathophysiology, diagnosis and management of diabetes mellitus. j diabe metab, 2015;6.5:1-9. 11. chobanian, av. national heart, lung, and blood institute joint national committee on prevention, detection, evaluation, and treatment of high blood pressure; national high blood pressure education program 127 original association between microalbuminuria and myh9 gene polymorphismsfadhil jawad al-tu’ma et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 122 – 127 coordinating committee: the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure: the jnc 7 report. jama, 2003;289:2560-2572. 12. arrondel, christelle, et al. expression of the nonmuscle myosin heavy chain iia in the human kidney and screening for myh9 mutations in epstein and fechtner syndromes. j am soc nephrol, 2002;13.1:65-74. 13. marini mbm, pecci a, romagnoli r, musante l, candiano g, ghiggeri gm, balduini c, seri m, marini m, bruschi m, pecci a, romagnoli r, musante l, candiano g, ghiggeri gm, balduini c, seri m,marini m, bruschi m, pecci a, romagnoli r, musante lc, non-muscle myosin heavy chain iia and iib interact and co-localize in living cells: relevance for myh9-related disease. 2016;41(6). 14. cheng, wenrong, et al. polymorphisms in the nonmuscle myosin heavy chain 9 gene (myh9) are associated with the progression of iga nephropathy in chinese. nephrol dial transplant, 2011;26.8:2544-2549. 15. behar, dm., et al. african ancestry allelic variation at the myh9 gene contributes to increased susceptibility to non-diabetic end-stage kidney disease in hispanic americans. human mol genet, 2010;19.9:1816-1827. 16. pattaro, cristian, et al. genome-wide linkage analysis of serum creatinine in three isolated european populations. kidney int, 2009;76.3:297-306. 17. kopp, jeffrey b., et al. myh9 is a major-effect risk gene for focal segmental glomerulosclerosis. nat genet, 2008;40.10:1175-1184. 18. liu, liping, et al. association of myh9 polymorphisms with hypertension in patients with chronic kidney disease in china. kidney blood press res, 2016;41.6:956-965. 19. freedman, bi., et al. the non-muscle myosin heavy chain 9 gene (myh9) is not associated with lupus nephritis in african americans. am j nephrol, 2010;32.1:66-72. 20. lipkowitz, ms, et al. association analysis of the non-muscle myosin heavy chain 9 gene (myh9) in hypertensive nephropathy: african american study of kidney disease and hypertension (aask). j am soc nephrol, 2009;20:56a. 21. palaniappan, l, carnethon, m, fortmann, sp. association between microalbuminuria and the metabolic syndrome: nhanes iii. am j hypertension, 2003;16.11:952-958. 22. kaur, j. assessment and screening of the risk factors in metabolic syndrome. med sci, 2014;2.3:140-152. 23. katzmarzyk, pt., et al. targeting the metabolic syndrome with exercise: evidence from the heritage family study. med sci sports exercise, 2003;35.10:1703-1709. 24. bahreinian, maryam, and ahmad esmaillzadeh. “opinion: quantity and quality of carbohydrate intake in iran: a target for nutritional intervention.” (2012): 648-649. 25. tabatabaei-malazy o, qorbani m, samavat t, sharifi f, larijani b, fakhrzadeh h. prevalence of dyslipidemia in iran: a systematic review and meta-analysis study. int j prev med. 2014;5(4):373. 26. nelson me, rejeski wj, blair sn, duncan pw, judge jo, king ac, et al. physical activity and public health in older adults: recommendation from the american college of sports medicine and the american heart association. med sci sports exercise. 2007;39(8):1435-45. 27. koochek a, johansson s, kocturk t, sundquist j, sundquist k. physical activity and body mass index in elderly iranians in sweden: a populationbased study. eur j clin nutr. 2008;62(11):1326-32. 28. simoes ej, kobau r, kapp j, waterman b, mokdad a, anderson l. associations of physical activity and body mass index with activities of daily living in older adults. j commun health. 2006;31(6):453-67. 29. bayram f, kocer d, gundogan k, kaya a, demir o, coskun r, et al. prevalence of dyslipidemia and associated risk factors in turkish adults. j clin lipidol. 2014;8(2):206-16. 30. karpov y, khomitskaya y. prometheus: an observational, cross-sectional, retrospective study of hypertriglyceridemia in russia. cardiovasc diabetol. 2015;14(1):115. 31. abujbara m, batieha a, khader y, jaddou h, el-khateeb m, ajlouni k. the prevalence of dyslipidemia among jordanians. j lipids. 2018;2018. 32. penesova a, cizmarova e, belan v, blazicek p, imrich r, vlcek m, et al. insulin resistance in young, lean male subjects with essential hypertension. j human hypertension. 2011;25(6):391-400. 33. sinha s, qazi sa, banik s, islam mz. correlation study of insulin resistance and essential hypertension among bangladeshi male volunteers. j young pharm. 2015;7(3):200. 34. sarafidis p, lasaridis a, nilsson p, pikilidou m, stafilas p, kanaki a, et al. validity and reproducibility of homa-ir, 1/homa-ir, quicki and mcauley’s indices in patients with hypertension and type ii diabetes. j human hypertension. 2007;21(9):709-16. 35. schillaci g, reboldi g, verdecchia p. high-normal serum creatinine concentration is a predictor of cardiovascular risk in essential hypertension. arch intern med. 2001;161(6):886-91. 36. kumar a, rekha n, raghav ed. a study of microalbuminuria in patients with essential hypertension. int j contemp med res. 2016. 37. liu l, wang c, mi y, liu d, li l, fan j, et al. association of myh9 polymorphisms with hypertension in patients with chronic kidney disease in china. kidney blood pressure res. 2016;41(6):956-65. 38. masconi k. the occurance of genetic variations in the myh9 gene and their association with ckd in a mixed south african population: stellenbosch: stellenbosch university; 2012. 39. bondzie pa. new insights into the molecular regulation of kidney disease: contributions of apol1 and myh9: boston university; 2014. 40. zhao h, ma l, yan m, wang y, zhao t, zhang h, et al. association between myh9 and apol1 gene polymorphisms and the risk of diabetic kidney disease in patients with type 2 diabetes in a chinese han population. j diab res. 2018;2018. 41. ling c, cai c, chang b, shi w, wei f, yu p, et al. myh9 gene polymorphisms may be associated with cerebrovascular blood flow in patients with type 2 diabetes. genet mol res. 2015;14(1):1008-16. 42. franceschini n, voruganti vs, haack k, almasy l, laston s, göring hh, et al. the association of the myh9 gene and kidney outcomes in american indians: the strong heart family study. human genet. 2010;127(3):295-301. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.963 70 j contemp med sci | vol. 2, no. 7, summer 2016: 70–73 research pharmacy college, al-qadisiyah university, al-qadisiyah, iraq. correspondence to hams hussain al-fattli (email: hamshussainhashim@gmail.com). (submitted: 15 may 2016 – revised version received: 29 june 2016 – accepted: 1 july 2016 – published online: 26 september 2016) objectives this study was conducted to disclose the specific antibodies against m. bovis of bovine tuberculosis (btb) in blood and milk serum samples, with detection of the most prevalent clinical signs in positive cows. methods in some rural areas of two iraqi provinces (wasit and dhi-qar), 119 lactating cows were submitted to the clinical examination with obtaining of blood and milk to tested by using the idexx elisa test. results the overall seroprevalence in blood and milk was (20.16%) and (15.12%), respectively. in wasit, the prevalence was (22.85%) and (15.71%), while in dhi-qar, the prevalence was (16.32%) and (14.28%) in blood and milk, respectively. as well as, marked significant differences in seroprevalence were observed between and within the two study’s provinces and samples. according to clinical examination, a significant rising (p > 0.05) was revealed in respiratory disorders, decreasing in milk production, emaciation, rough hair coat and repetitive reproductive problems, whilst a significant decreasing (p < 0.05) in persistent feces abnormalities, mastitis, lymph nodes enlargement and loss of appetite. conclusion the study demonstrated, for the first time in iraq, the efficient of idexx elisa, as a screening test in the detection of btb in lactating cows by using blood and milk serum samples, and the competence of milk, as sample, in exhibition of infection. also, the study exposed the high infection rate of btb in cows of rural areas of wasit and dhi-qar provinces. keywords mycobacterium bovis, lactating cows, idexx elisa, clinical and serological, wasit and dhi-qar, iraq the clinical and serological diagnosis of mycobacterium bovis in blood and milk serums of lactating cows by idexx elisa test in wasit and dhi-qar provinces/iraq hams hussain hashim al-fattli issn 2413-0516 introduction bovine tuberculosis (btb) is a chronic debilitating infectious disease of domestic and wild animals as well as humans, which is generated by very slow growing bacilli, mycobacterium bovis.1,2 btb has no geographical boundaries and existing in most developing countries with an inadequate or unavailable surveillance control activities.3 although, btb manifested, essentially, by a respiratory symptoms, the disease may be found in lymph nodes, intestine, liver, kidneys, bones and central nervous system; resulting in a different clinical signs depending on the site of lesions.4,5 cattle are, extremely, liable to become infected with m. bovis in first live by long subclinical phase without or with short interrupted shedding, and terminated in an advance disease with symptoms in a slight rate of diseased cattle.6,7 so, the diseased cows are the main source for infection to spread it by inhalation, ingestion and drinking of contaminated water or infected milk.1 the precise routes of transmission and genealogical significance of infective paths have not been determined because the disease diagnosis had, broadly, been focused on diseased animals that have a scandalous apparent damage.8 in cattle, the damage, firstly, evidenced in respiratory system as granulomatous lesions, then, it may spread to localize in other parts, involved udder to aggregate and calcified causing in mastitis.9 in industrialized countries, btb monitoring with removal schedules, and milk pasteurization had drastically decreasing in extent of infection in both cattle and human.10 iraq is one of these countries that many of the epidemiological and public health aspects of the disease remaining, in general, undetermined.11,12 btb is intractable by scarcity of the feasible techniques and presence of reservoirs; and distribution of it in most areas.13 the accurate diagnosis for btb is essential; for con trol, eradication, treatment of affected individu als and prevents transmission of infection to humans.14 the control strategy that used in most countries through using a single or comparative tuberculin skin test, which depended mainly on the basis of delayed hypersensitivity reactions, is not effective in the diagnosis of infection in first and in terminal stages.15 antibody responding to bacteria showed an invariably related to the mycobacterial emerged pathology and antigen burden.16 thus, the development of serological techniques was increasing a point of diagnosis of the diseased cattle and act as a complementary to tuberculin test.17 in addition, multiple tests, if used, might increase the total diagnostic power by detecting the subclinical cases of infected animals that missed by skin test.18 in addition to easiness of sample obtaining with technique procedures, the technique can apply in several purposes to supply an extra experiment chances didn’t provided by other tests.19 idexx m. bovis antibody kit is a new serological and commercial elisa test that manufactured by idexx laboratories to detect the infection in blood, or milk of cows.20 it’s validated and certified by oie in 2012 with approval number of 20120107.21 this test is easy to use, cost effective for surveillance, and the need for a little time.19,22 the aims were: 1. estimating the idexx elisa, under field conditions, in diagnosing of infected lactating cows by using the milk and blood serum samples, for the first time in iraq. 2. the comparison, in accuracy, between blood and milk serum samples in detection of btb. 3. providing the more practical and actual morbidity rate for btb in cows of wasit and dhi-qar provinces. 4. identification the relationships between clinical signs and positive cases. hams hussain hashim al-fattli 71j contemp med sci | vol. 2, no. 7, summer 2016: 70–73 research the clinical and serological diagnosis of mycobacterium bovis in blood and milk serums of lactating cows materials and methods samples and data collection a total of 119 lactating cows were submitted to this study, 70 cows from different rural areas of wasit province and 49 cows from different rural areas of dhi-qar province/ iraq, between august 2015 and march 2016. the data of case history were recorded in depending on clinical examination and owner’s information. under aseptic condition, from each cow, 10 ml blood sample from jugular vein was collected by using a vacutainer syringe, and 50 ml milk sample was collected by manual milking into plastic tubes. the blood and milk samples, transported to the laboratory for obtaining sera, centrifuged at 3700 rpm for 15 minutes, and a portion of skim milk was pipetted off from below the cream layer. every serum sample saved in 1ml, labeled and numbered, eppendorf tubes and frozen under −20°c.23,24 idexx elisa “the blood and milk serum samples of lactating cows were examined by indirect idexx elisa kit for btb (mycobacterium bovis) according to the manufacturer’s instructions (idexx laboratories). the serum samples and kit controls were diluted 1:50 in sample diluents that provided with the kit as a first step, then, 100 μl was added into the wells and incubated at room temperature (37°c) for 1 hour. this step was followed by removal of the contents of the wells by washing the plates with pbs tween buffer solution, 4 times after which 100 μl of a monoclonal anti-bovine igg hrp conjugate was added into each well and incubated at room temperature for 30 minutes. again, the plates were washed 4 times by a pbs tween buffer solution that followed by the addition of 100 μl of tetramethylbenzidine substrate (tmb) into each well, and incubated for 15 minutes at room temperature. in direct, the further reactions were stopped by addition of 50 μl h2so4 and the optical density (od) value was read by using an elisa microplate reader (biotek, usa) at a wavelength of 450 nm. the idexx elisa results were recorded as positives or negatives based on a sample to positive (s/p) ratio and the result of each sample are submitted to the following formula: (15.12%) were positives with milk serum samples by idexx elisa test. depending on the results of blood serums samples (table 2), 16/70 (22.85%) were positives with btb in wasit province and 8/49 (16.32%) positive cows in dhi-qar province. while in (table 3) the results of milk serum samples were 11/70 (15.71%) and 7/49 (14.28%) in wasit and dhi-qar provinces, respectively. in table 4, the results of data collection and clinical case history examination in24 positive cows with blood testing s/p ratio = sample result at (450) mean of negative controlsmmean of positive controls mean of negative controlsmanufacturer’s recommended cut-off is an s/p ratio of 0.3. the (s/p) ratio ≥ 0.3 is considered that the test was positive.” statistical analyses all data were arranged and labeled with a computerized program (word and excel v. 2013), then transferred to the ibm spss (v. 23) to analysed by chi-square test. statistically, the significant differences were used at (p > 0.05) to compare between the results of blood and milk serum samples, and to detect the associations between infection and the clinical signs (25). results during 8 months, the blood and milk serum samples of 119 lactating cows were tested by idexx elisa test in 2 provinces, wasit and dhi-qar, to detect the positive cases with m. bovis infections. according to (table 1), 24/119 (20.16%) were positives with blood serum samples testing while 18/119 table 2. total infected cases by blood serum groups according to provinces results total tested number total infected cases wasit / 70 cows dhi-qar / 49 cows no. % no. % 119 16 22.85a 8 16.32b horizontally, the different small letters refers to a significant difference at level p < 0.05. table 3. total infected cases by milk serum according to provinces results total tested number total infected cases wasit / 70 cows dhi-qar / 49 cows no. % no. % 119 11 15.71a 7 14.28b horizontally, the different small letters refers to a significant difference at level p < 0.05. table 1. total infected cases according to blood and milk serum results total tested number total infected cases in blood serum in milk serum no. % no. % 119 24 20.16a 18 15.12b horizontally, the different small letters refers to a significant difference at level p < 0.05. table 4. clinical signs of positives cows with blood and milk serum samples clinical signs with blood samples (24) with milk samples (18) no. % no. % 1 respiratory disorders 5 20.83a 3 16.66b 2 emaciation 9 37.5b 7 38.88b 3 lymph nodes enlargement 2 8.33 b 2 11.11a 4 rough hair coat 5 20.83b 4 22.22b 5 loss of appetite 2 8.33b 1 5.55a 6 decreasing of milk production 7 29.16 a 4 22.22b 7 reproductive problems 3 12.5 b 2 11.11b 8 persistent feces abnormalities 1 4.16 b 1 5.55b 9 mastitis 4 16.66b 3 16.66b horizontally, the different small letters refers to a significant difference at level p < 0.05. 72 j contemp med sci | vol. 2, no. 7, summer 2016: 70–73 the clinical and serological diagnosis of mycobacterium bovis in blood and milk serums of lactating cows research hams hussain hashim al-fattli group and18 positive cows with milk testing group were as follow, respectively; the respiratory disorders (cough and/or dyspnea) were found in 5/24 (20.83%) and 3/18 (16.66%), continual decreasing in milk production in 7/24 (29.16%) and 4/18 (22.22%); emaciation in 9/24 (37.5%) and 7/18 (38.88%), rough hair coat in 5/24 (20.83%) and 4/18 (22.22%), repetitive reproductive problems (abortions, uterine discharges, infertility) in 3/24 (12.5%) and 2/18 (11.11%), persistent feces abnormalities (diarrhea or constipation) in 1/24 (4.165) and 1/18 (5.55%), mastitis in 4/24 (16.66%) and 3/18 (16.66%), lymph nodes enlargement (locally or systemically) in 2/24 (8.33%) and 2/18 (11.11%), and loss of appetite in 2/24 (8.33%) and 1/18 (5.55%). discussion in countries that do not commence routine screening tests for their herds, their herds are expected to contain animals at different stages of tuberculosis. therefore, for assaying their herds, the better use of serological test, idexx elisa test would be useful in early detection and advance cases which otherwise cannot be detected by tuberculin skin test.26,19 in this study, btb had an overall prevalence 24/119 (20.16%) with blood testing group and 18/119 (15.12%) with milk testing group. thirteen cows were positives with both tests, while 11 and 5 cows, respectively, were positive to test in blood and milk groups only. thus, idexx elisa had a sensibility to detect the antibodies in blood more than in milk, and several causes may be concluded in the interpretation of this difference such as the individual differences in animal’s antibody responses, lactation stage, time of milk collection, herd size, proportion of milk dilution, cutoff level changing, and the producing of nonspecific antibody responses to test antigens.20 however, the test sensitivity is increased markedly with severity of the disease.27 also, the high relationship among an antibody’s reactions in milk and blood had been reported, at same animal, in btb and john’s disease.28,29 as well as, milk might become, widely, extra-suitable after collection of it during the routine examination of herds.30 milk can be contributed in identifying the diseased cattle, although about 50 percentage of technique’s sensitivity would decrease.31 also, it would be missed; apparently, their benefit through an examination of a few diseased cattle that persist at large groups.20 the results of tables 2 and 3 revealed that in wasit province, the morbidity rate was more than this reported in dhi-qar province. although, the role of local spread of btb in some areas is not well understood, btb testing had a significant impact on the expansion and long distance spread of disease, especially on transmission to areas with relatively low incidence.32 a variable incidence of btb may effect through several modes like the geographical position features; agro ecological system; public health condition for humans and animals; herd size, farm management and grazing practice; age, breed, gender and body condition score of animals; compelled organizing schemes of the veterinary departments; concurrent diseases, host genetic variation, immune suppression, cattle behavior, physiological status, cows scheme form, feeding system, treatments with control program, environments or weather, pathological variations.33–35 whilst the respiratory disorders, emaciation, rough hair coat, decrease in milk production and presence of reproductive failures shows the most prevalent signs of positive cows in blood and milk groups; the enlargement of lymph nodes, loss of appetites and feces abnormalities manifested the lowers, with presence of some differences between both serum groups. these resultants were approval to studies revealed by various researchers.36–39 historically, btb is troublesome and tired to detect in depending, solely, on obvious symptoms particularly in advanced countries that had a low proportion of diseased cows with acutely intense infection, and the diagnosis is employ, mostly, through the tuberculin skin test or discovered after slaughter.40,41 in early stages of infection, the clinical signs are not visible, but with advance stages, the signs begin to appear in depending on species of animal, point of entry, sites of localization, and the afflicted organs.42 many animals may be infected sub-clinically and remained asymptomatic until the development of disseminated lesions, or infected again with m. bovis; submitted to bad feeding, progress of age, and if undergo from more one infection by other pathogens.43–45 referred that the most btb infections, infrequently, shown to be diseased clinically and seems apparently healthy. the acuteness of infection in cattle is depending, generally, on several factors such as the infective dose, point of entry, troubled immunity, age, stress, and the genetic variation.24,46 also, the high cattle density may provide a chance for incorporate throughout the unleash feeding on pasture or during persistence of healthy and diseased cattle with each other under commercial schemes with low nutrition that make these cattle very liable for diseases.47 a recent literature review concluded that the role of positive btb cows, with minimal or no observed signs, is far from clear.48 “despite the successes of the idexx elisa in detection of infection with m. bovis, but the reliability of it is depending on several factors including the efficiency of testing procedure, mode of interpretation of result as well as the immunological responsiveness of the animal at the time of test. furthermore, the negative results to test doesn’t mean that the animal is not infected with m. bovis, while on the other hand, the positive results represented an immunological response that might be due to the current infections or a previous exposure to m. bovis but, may less commonly, due to the infection or exposure to other bacteria that share an antigens similar to those of m. bovis.”49,50 acknowledgment i grant this work to my family for great supporting, contributing in completion of this work, and to everyone help me during collection samples, data, and providing me with required references. n references 1. radostits om, gay cc, hinchcliff kw, constable pd. veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs and goats. 10th ed. saunders elsevier company ltd, london. part 2, diseases associated with bacteria – iv. 2007; pp. 1007–1017. 2. abubakar u, ameh j, abdulkadir i, salisu i, okaiyeto s, kudi ac. bovine tuberculosis in nigeria: a review. vet res. 2011;4(1):24–27. 3. gumi b, schelling e, firdessa r, aseffa a, tschopp r, yamuah l, et al. prevalence of bovine tuberculosis in pastoral cattle herds in the oromia region, southern ethiopia. trop anim health prod. 2011;43(6):1081–1087. 4. smith bp. large animal internal medicine. elsevier health sciences. chapter 31, diseases of respiratory systems. 2014; pp. 576. hams hussain hashim al-fattli 73j contemp med sci | vol. 2, no. 7, summer 2016: 70–73 research the clinical and serological diagnosis of mycobacterium bovis in blood and milk serums of lactating cows 5. terefe d. gross pathological lesions of bovine tuberculosis and efficiency of meat inspection procedure to detect-infected cattle in adama municipal abattoir. j vet med anim health. 2014;6(2):48–53. 6. sweeney rw. pathogenesis of paratuberculosis. vet clin food anim prac. 2011;27(3):537–546. 7. brooks-pollock e, roberts go, keeling mja. dynamic model of bovine tuberculosis spread and control in great britain. nature. 2014;511 (7508):228–231. 8. murphy d, gormley e, costello e, o’meara d, corner lal. the prevalence and distribution of mycobacterium bovis infection in european badgers (meles meles) as determined by enhanced post mortem examination and bacteriological culture. res vet sci. 2010;88(1):1–5. 9. michel al, de klerk lm, pittius ncg, warren rm, van helden pd. bovine tuberculosis in african buffaloes: observations regarding mycobacterium bovis shedding into water and exposure to environmental mycobacteria. bmc vet res. 2007;3(1):1. 10. alvarez j, perez am, bezos j, casal c, romero b, rodriguez-campos s, et al. eradication of bovine tuberculosis at a herd-level in madrid, spain: study of within-herd transmission dynamics over a 12 year period. bmc vet res. 2012;8(1):1. 11. hasso sa. confirmed pathogens of cows and buffaloes in iraq. iraqi journal of veterinary sciences. 2004;18(1):1–14. 12. mudhar asa. application of tuberculin screening tests for determination the prevalence of bovine tuberculosis in basra governorate/iraq. mirror res vet sci anim. 2015;4(3):1–8. 13. andrews ah, blowey rw, boyd h, eddy rg. bovine medicine: diseases and husbandry of cattle. john wiley and sons. part 2, diseases, growing cattle, respiratory diseases. 2008;ch.49, pp. 862–864. 14. claridge j, diggle p, mccann cm, mulcahy g, flynn r, mcnair j, et al. fasciola hepatica is associated with the failure to detect bovine tuberculosis in dairy cattle. nat commun. 2012;3:853. 15. shitaye je, tsegaye w, pavlik i. bovine tuberculosis infection in animal and human populations in ethiopia: a review. veterinari medicinapraha. 2007;52(8):317. 16. waters wr, whelan ao, lyashchenko kp, greenwald r, palmer mv, harris bn, et al. immune responses in cattle inoculated with mycobacterium bovis, mycobacterium tuberculosis, or mycobacterium kansasii. clin vac immunol. 2010;17(2):247–252. 17. whelan c, shuralev e, kwok hf, kenny k, duignan a, good m, et al. use of a multiplex enzyme linked immunosorbent assay to detect a subpopulation of mycobacterium bovis – infected animals deemed negative or inconclusive by the single intradermal comparative tuberculin skin test. j vet diagnos invest. 2011;23(3):499–503. 18. ameni g, erkihun a. bovine tuberculosis on small-scale dairy farms in adama town, central ethiopia, and farmer awareness of the disease. revue scientifique et technique-office international des epizooties. 2007;26(3):711–720. 19. hirpa e, ameni g, lawrence jc, tafess k, worku a, sori t, et al. performance evaluation of mycobacterium bovis antibody test for the diagnosis of bovine tuberculosis in ethiopia. 2014;5:9. 20. buddle bm, wilson t, luo d, voges h, linscott r, martel e, et al. evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis from milk samples from dairy cows. clin vac immunol. 2013;20(12):1812–1816. 21. oie, procedure for registration of diagnostic kits abstract sheet idexx m.bovis antibody test kit, idexx laboratories, www.oie.int.accessed. (2012). 22. mukundan h, chambers m, waters r, larsen m. tuberculosis, leprosy and mycobacterial diseases of man and animals: the many hosts of mycobacteria. cabi. 2015; pp. 221–224. 23. belić b, cincović mr, stojanović d, kovačević z, medić s, simić v. hematology parameters and physical response to heat stress in dairy cows. savremena poljoprivreda. 2010;59(1-2):161–166. 24. mahmud maa, belal smsh, shoshe, nz. prevalence of bovine tuberculosis in cattle in the selected upazila of sirajganj district in bangladesh. bangladesh j vet med. 2014;12(2):141–145. 25. petrie a, watson p. statistics for veterinary and animal science, second edition. ames: blackwell publishing. (2006); pp. 312. 26. who/iuatld. anti-tuberculosis drug resistance in the world. fourth global report. who/htm/tb/.394. geneva, switzerland: world health organization. (2008). 27. mosaad aa, abdel-hamed as, fathalla si, ghazy aa, elballal s, elbagory a, et al. sensitive and specific diagnostic assay for detection of tuberculosis in cattle. global veterinaria. 2012;8(6):555–564. 28. lombard je, byrem tm, wagner ba, mccluskey bj. comparison of milk and serum enzyme-linked immunosorbent assays for diagnosis of mycobacterium avium subsp. paratuberculosis infection in dairy cattle. j vet diagn invest. 2006;18:448–458. 29. jeon by, kim sc, je s, kwak j, cho je, woo jt, et al. evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in korea. res vet sci. 2010;88:390–393. 30. waters wr, buddle bm, vordermeier hm, gormley e, palmer mv, thacker tc, et al. development and evaluation of an enzyme-linked immunosorbent assay for use in the detection of bovine tuberculosis in cattle. clin vac immunol. 2011;18(11):1882–1888. 31. marassi cd, medeiros l, figueiredo e, fonseca ls, duarte r, paschoalin v, et al. a multidisciplinary approach to diagnose naturally occurring bovine tuberculosis in brazil. pesquisa veterinária brasileira. 2013;33(1):15–20. 32. green dm, kiss iz, mitchell ap, kao rr. estimates for local and movementbased transmission of bovine tuberculosis in british cattle. proc r soc lond [biol]. 2008;275(1638):1001–1005. 33. moiane i, machado a, santos n, nhambir a, inlamea o, hattendorf j, et al. prevalence of bovine tuberculosis and risk factor assessment in cattle in rural livestock areas of govuro district in the southeast of mozambique. plos one. 2014;9(3):e91527. 34. more sj, good m. understanding and managing btb risk: perspectives from ireland. vet microbiol. 2015;176(3):209–218. 35. cezar rd, lucena-silva n, filho af, borges jde m, de oliveira pr, lúcio éc, et al. molecular detection of mycobacterium bovis in cattle herds of the state of pernambuco, brazil. bmc vet res. 2016;12(1):1. 36. collins ch. the bovine tubercle bacillus. br j biomed sci. 2000;57(3):234. 37. cassidy jp. the pathogenesis and pathology of bovine tuberculosis with insights from studies of tuberculosis in humans and laboratory animal models. vet microbiol. 2006;112(2):151–161. 38. renwick ar, white pc, bengis rg. bovine tuberculosis in southern african wildlife: a multi-species host–pathogen system. epidemiol infect. 2007;135(04):529–540. 39. skuce ra, allen ar, mcdowell sw, branch b. bovine tuberculosis (tb): a review of cattle-to-cattle transmission, risk factors and susceptibility. agri food biosci ins, belfast. 2011;1(3):32–37. 40. fentahun t, luke g. diagnostic techniques of bovine tuberculosis: a review. african j basic appl sci. 2012;4(6):192–199. 41. ramos df, silva pe, dellagostin oa. diagnosis of bovine tuberculosis: review of main techniques. brazilian j biol, (ahead). 2015; http://dx.doi.org/ 10.1590/1519-6984.23613. 42. green le, medley g. cattle to cattle transmission of bovine tuberculosis: risk factors and dynamics. cattle practice. 2008;16(2):116–121. 43. cosivi o, meslin fx, daborn cj, grange jm. epidemiology of mycobacterium bovis infection in animals and humans, with particular reference to africa. revue scientifique et technique (international office of epizootics). 1995;14(3):733–746. 44. donnelly c, cox d. bovine tb in cattle and badgers. significance. 2007;4(4):164–167. 45. collins jd. tuberculosis in cattle: strategic planning for the future. vet microbiol. 2006;112(2):369–381. 46. bojkovski j, renata re. the case of bovine tuberculosis at the slaughter house. bulletin of university of agricultural sciences and veterinary medicine cluj-napoca. vet med. 2011;2:68. 47. adesokan hk, ijagbone if, oputa he, cadmus sib, stack ja. serological survey of brucellosis in livestock animals and workers in ibadan, southwestern, nigeria. african j biomed res. 2006;9:163–168. 48. sosnik a, carcaboso ám, glisoni rj, moretton ma, chiappetta da. new old challenges in tuberculosis: potentially effective nanotechnologies in drug delivery. advanced drug deliv rev. 2010;62(4):547–559. 49. bermúdez hr, rentería et, medina bg, hori-oshima s, de la mora valle a, lópez vg. evaluation of a lateral flow assay for the diagnosis of mycobacterium bovis infection in dairy cattle. j immunoassay and immunochem. 2012;33(1):59–65. 50. bhanurekha v, gunaseelan l, pawar g, nassiri r, bharathy s. molecular detection of mycobacterium tuberculosis from bovine milk samples. j advan vet anim res. 2014;2(1):80–83. 255 letter to editor issn 2413-0516 dear editor-in-chief: as the coronavirus pandemic exacerbates, its disproportionate impacts affects all aspects of the community and life. the dental schools and dentistry profession were in the front line of this changes and bear a large portion of this crisis.1 this burdens added to the dental education in iraq in addition to its current challenges.2,3 until october 28, 2020, iraq registered 463,951 cases, and 10,770 persons died from covid-19, of which 2.3% from the infected cases and 6% of the death cases were dentists.4 upon on the who recommendations along with this high mortality rate made the policy makers in iraqi ministry of health (moh) and ministry of higher education and scientific research (mohesr) take the decisions for national-wide lockdown and closure of dental schools in iraq and minimize the dentists presence in public hospitals and dental centers to 25%, and just for emergency patients need since march 1, 2020. this decision affected the 5th stage students which had already graduate in september 2020 from taking the enough clinical training and patient management, while the successful e-learning coverage the theoretical educational sessions well. as its impossible to certain the end of this pandemic and this will be a real threaten for the new graduated dentists skills and knowledge to manage patients, the policy makers must change the dental education program in iraq and to add the (exit interview) for all graduated students which would make a need assessment for the real need, and design an intensive course for them upon on their needs that would help dental schools and educators to ensure the dental education quality assurance. this intensive course could be like the general practice residency (gpr) which is accredited in usa.5 this model could be considered in other effected countries which has the same lockdown and closed policy like iraq. keywords: covid-19, dental education, dentistry, iraq references: 1. machado, ra, bonan, prf, perez, dedc and martelli júnior, h. covid-19 pandemic and the impact on dental education: discussing current and future perspectives. braz oral res. 2020;34. 2. khoshnevisan, mh, albujeer, an, taher, aa and almahafdha, a. dental education in iraq: issues, challenges and future. j contemp med sci, 2017;3(11):260-263. 3. albujeer, an, khami, mr and almahafdha, a. private dental schools in iraq: a real threat to the dental profession. iran j public health. 2020;49(1):201-202. 4. statistics of covid-19, 2020; ministry of health, iraq. 5. lau, a, dodson, tb, sonis, st and kaban, lb. an outcomes study of 40 years of graduates of a general practice dental residency. j dental educ. 2015;79(8):888-896. covid-19 impact on dental education in iraq; challenges and future implications ammar n.h. albujeer1,2,3, 1 nab’a al-hayat foundation for medical sciences and health care, najaf, iraq. 2 college of dentistry, al-farahidi university, baghdad, iraq. 3 school of dentistry, tehran university of medical sciences, tehran, iran. corresponding author: ammar n.h. albujeer (e-mail: ammar.dent@yahoo.com) (submitted: 20 october 2020 – revised version received: 28 october 2020 – accepted: 29october 2020 – published online: 30 october 2020) this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.863 66 review issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 66 – 72 introduction supervision term in the cambridge vocabulary is defined as the act of looking at the person or activity and making sure that everything is done properly and safely. supervision in two terms of overlapping circles can be focused on the development of performance, or both.1 kilminster (2007) noted that supervision “the provision of guidance and feedback on substances of personal, professional, and educational development in the background of a trainee’s experience of providing safe and appropriate patient care”.2 undergraduate medical education (ugm) includes theoretical and clinical knowledge designed to provide efficient human resources to meet a community’s needs.3 the terms of clinical education refer to the acquisition of supervised professional skills.4 the purposes of clinical supervision are ensuring patient safety/care, educating the trainee, promoting high standards, identifying trainee problems, monitoring trainee progress, and supporting the trainee.2 in the systematic review, a study conducted from 2002 to 2014, results show that the competencies of general physicians in assessed skills were not desirable.5 problems in ugm display the differences in the education level between universities and weaknesses in providing teaching by the residents.6 also, several clinical supervisors do not have an efficient performance in teaching hospitals which needs to be more evaluated and improved.7 according to the literature, the availability of supervision for medical trainees and specializing physicians is inadequate.8 new curriculums strive to move from time-based model to the current trend towards competency-based medical education (cbme).9 some trainees have a concern about job future,6, 10 tests time condition,11 on-call and rotation schedules in the hospital and preparation for residency,12 fear of lack of knowledge and lack of skill, and criticism of trainee activities by clinical educator,13 that has made them psychical and spiritual problems. they use many mechanisms to deal with stress, but the using of many negative coping strategies. high levels of stress and anxiety during the clinical course can affect the professional effectiveness of trainees and reducing their attention and concentration, impair their decision-making skills, and reduce the trainee’s ability to communicate effectively with patients.11 stress has negative effects on trainees learning and clinical success, and their proper functioning of overshadowing.13 the availability and emotional support of the supervisor provided a positive relationship with learning progress and increased trainee satisfaction.14 therefore, trainees require pastoral, and spiritual care, especially when trainees experience a crisis in their personal or professional life.1 the pattern of clinical supervision of medical trainees in iran includes a range of internships to attending physicians. the attending physicians or faculty members and senior residents provide supervision for junior residents and internship in a hierarchical supervisory structure. the supervision level is reduced by attending physicians for ugm. current supervision doesn’t meet the needs of medical trainees. there is an agreement in the general literature that supervision has three functions managerial or administrative, educative, and supportive.2 the idea of three functions or roles of supervision is reflected across professions; for example, nursing,15,16 social work,17,18 and recommended for the use in medicine.2,8 the managerial function is responsible for the trainee’s casework and ensuring that the supervisee complies with the rules, and norms of organizations, in which the work is carried out.19 the educational function is developing skills and enhancing understanding.20 the supportive function acknowledges the emotional effects on an individual of work, and in particular of work with people in distress.19 each of the functions may come to the fore or remain in the background in clinical supervision and circumstances.21 there is needing to expand clinical supervision of medical trainees, and students must be considered about a general and holistic view, but only through providing and teaching several core components of three functions of clinical supervision in undergraduate medical education during a clinical course soleiman ahmady1* id , masoumeh seidi2 1 shahid beheshti university of medical sciences, tehran, iran; research affiliated faculty at department of lime, karolinska institutet, sweden. 2 virtual school of medical education and management, shahid beheshti university of medical sciences, tehran, iran. *correspondence to: soleiman ahmady (e-mail: soleiman.ahmady@gmail.com) (submitted: 12 november 2020 – revised version received: 26 january 2021 – accepted: 04 february 2021 – published online: 26 april 2021) abstract objective attending physicians must holistically consider medical trainees. the quality control aspects of clinical practices and skills development as well as increase in trainees’ knowledge and alleviated stress. current clinical supervision does not meet the needs of trainees. this study aimed to identify the components of functions of clinical supervision in undergraduate medical education. the process of the literature review was implemented in five phases following the method developed by carnwell and daly. the scope of the review was limited to clinical supervision of medical trainees and the functions of clinical supervision models. methods using the keywords in combination and separately, they were searched in the databases of pubmed, eric, scopus, and wos from 1970 to 2019. results a total number of 49 documents (policy /guideline/ standard) and 19 articles (functions of clinical supervision) were obtained. conclusions to make efforts to empower attending physicians in the clinical supervision functions and these functions be simultaneously applied for trainees supervision. keywords clinical supervision, medical trainee, model, policy, guideline, standard. https://orcid.org/0000-0003-0551-6068 67 review core components of three functions of clinical supervision in undergraduate medical educationsoleiman ahmady, masoumeh seidi j contemp med sci | vol. 7, no. 2, march-april 2021: 66 – 72 skills and competencies. moreover, supervisors must not expect students to abide by several laws and regulations, and other aspects as individuals should not be overlooked. there is no similar study on the application functions of clinical supervision to medical students in the clinical setting. the purpose of this study was a response to the question being investigated. what are the components, and the characteristics of three functions associated with the clinical supervision of medical trainees in the ugm? methods a critical review was utilized as the research design. to note, the main purpose of this literature review was to appraise and synthesize the current state of knowledge related to the topic under investigation, as a means of identifying gaps of knowledge addressed by this new study.22 in this critical review, the researcher did not claim to cite all relevant articles in the desired field, but rather morally committed to presenting different views in a fair manner.23 in this study, the process of conducting the literature review was implemented according to carnwell and daly.22 the search strategy was comprised of two steps. at the first step, the search was done by focusing on the following keywords, separately and in combination: clinical supervision (and) (“medical student*” or trainee* or clerkship) (and) (policies or guidelines or standards). besides, manual library searches along with free internet ones were performed. all the available sources were thus identified and each database was respected by the instructions specified for it. the search in the databases of web of science (wos), scopus, pubmed, google scholar, and the education resources information center (eric) on clinical supervision of ugm was accordingly done on articles published from 1970 to 2019. after excluding the irrelevant studies, a total number of 49 documents (namely, 42 policies, 4 guidelines, and 3 standards) were selected to review, as shown in the prisma flow diagram (fig. 1). forty-nine (49) documents were reviewed by the researchers in their full-texts. two criteria were also considered for the final selection of the policies, guidelines, and standards. being updated over the last 10 years and considering aspects such as supervisor, trainee, context, supervisor–supervisee relationship, and tasks.24, 25 the number of policies, guidelines, and standards at medical schools were correspondingly written very briefly and if they had pointed out a new component, that component was extracted. after assessing the given documents with the above-mentioned criteria, 30 documents remained. at the second step, studies using the models of clinical supervision functions were searched. the searches correspondingly revealed the models of clinical supervision functions employed in numerous studies and resources associated with nursing, social work, and other fields. from the obtained articles, 19 studies were selected (tuck 2017, basa 2017, gillieatt 2014, beddoe 2012, turner 2011, hughes 2010, gardner 2010, brunero 2008, clark 2006, scaife 2003, sloan, watson 2002, morrison 2001, morton-cooper 2000, bowels 1999, cutcliffe 2010, butterworth 1997, butterworth 1996, jones 1996, erera 1994). then, 19 studies were scrutinized in relation to the functions of clinical supervision. the definition, characteristics, and components of each function of clinical supervision were extracted separately from the studies. three functions of clinical supervision were defined separately. results the main components of documents: clinical supervision for medical trainees in medical schools the u.s. department of education identifies the liaison committee on medical education (lcme) as an accrediting agency for medical education programs leading to the md degree.26 the committee on the accreditation of canadian medical schools (cacms) standards provide for the quality of medical education programs leading to the m. d.27 in standard no. 9-3 two, committees emphasizing on clinical supervision for medical students, and medical schools have attempted to prepare this standard 9-3 clinical supervision in the form of policy/guideline/standard. most medical schools wrote in the form of policy. a researcher designed a checklist of 30 documents and obtained clinical supervision for medical students. the document of each medical school was compared with the checklist. to avoid the lengthy text of the documents and prevent readers’ fatigue, a sample of 10 medical schools (including university of mc gill faculty of medicine,28 university of 22 fig. 1. prisma flowchart. records excluded: (n=205) 28 duplicates and irrelevant texts 50 university & school supervision, postgraduate research supervision 37 physician, preceptor, faculty member & surveys 44 masters or doctoral students supervision 29 medical residency 17 non-full -text records identified through database searching (n=273) full-text reviewed: (n=68) 19 articles 49 documents (42 policies, 4 guidelines, and 3 standards) excluded after reviewing the full-text documents 11 not addressing aspects such as supervisor, trainee, context, supervisorsupervisee relationship 8 the date the was not updated 19 articles 30 (policies/ guidelines/ standards) 49 included in review id en tif ic at io n s cr ee ni ng e lig ib ili ty in cl ud ed fig. 1 prisma flowchart. 68 review core components of three functions of clinical supervision in undergraduate medical education soleiman ahmady, masoumeh seidi j contemp med sci | vol. 7, no. 2, march-april 2021: 66 – 72 alberta,29 university of british columbia,30, 31 university of boston school of medicine (busm),32 university of nebraska college of medicine,33 university of florida atlantic charles e. schmidt college of medicine (fau com),34 university of washington school of medicine (uwsom),35 university of north carolina school of medicine (unc som),36 university of minnesota medical school (umms),37 and university of pennsylvania38 are listed in table 1. while reviewing the general medical council (gmc) standards,39 the generic standards for training by the postgraduate medical education and training board (pmetb),40 and a guideline of the royal australian college of  general practitioners (racgp),41 15 components were identified. these components were then added to the ones obtained from the aforementioned 30 medical schools (table 2). accordingly, a total number of 25 components were achieved. characteristics of three function of clinical supervision the models for functions of clinical supervision have been explained in a theoretical and conceptual manner. models that divided the function of clinical supervision into three functions,42 including bridget proctor (1986): formative, restorative, and normative,43 and emphasis on supervisee. alfred kadushin (1976): educational, supportive, and managerial/ administrative,44 and emphasis on supervisor.45 hawkins and shohet (2006): developmental, resourcing, and qualitative,46 emphases on both supervisor and supervisee.47 proctor’s model (1986) provides a good framework and is one of the most cited models in the uk nursing literature.48 proctor’s model has been criticized for lack of detail in its application. however, its positive that allows space for the necessary creativity that developing clinical practice requires.49 gillieatt (2014) suggests that adapting proctor’s model for student clinical supervision is relevant across a broad range of health disciplines and clinical areas.50 managerial/normative/qualitative this function relates to the accountability of supervisees’ clinical performance and clinical outcomes to ensure ethical quality services.42 maintaining standards of practice, care, and professionalism providing honest feedback and constructive criticism,46 assessment,50-52 quality control aspects of clinical practice,48 organizational responsibility,53 ensure the identification and self-assessment of professional and ethical competence.20 educational/formative/developmental this function focuses on learning, training, and teaching.42 evaluating observed performance,54 responsibility for the development of the student,43,54 and the ongoing monitoring and evaluating of the student at certain times at end of the course or point of promotion,43 skills development, and increasing the trainee’s knowledge,19,44,48,50,52,53 learning through reflection and sharing good practice, opportunities for teaching and skill acquisition,46 enabling personal and professional development.52 table 1. the components of clinical supervision policy/guideline/standard in 10 medical schools. school of medicine m c g ill al be rt a br iti sh c ol um bi a bo st on ne br as ka ch ar le s e . c hi de w as hi ng to n no rt h ca ro lin a m in ne so ta pe nn sy lv an ia components duties or expectations or responsibilities of clinical trainees 1 1 1 1 1 1 1 1 1 1 setting purpose 1 1 1 0 1 1 1 1 1 1 levels of clinical supervision 1 0 1 1 1 1 1 1 1 1 defining clinical supervisor 1 0 1 1 1 1 1 1 0 1 recommendations for the safety of patients and trainees 1 1 1 1 1 0 0 1 1 1 assessment of clinical trainees 1 1 1 0 1 0 1 1 1 1 expectations or roles or responsibilities of the supervisor 1 1 1 0 1 0 0 1 1 1 delegate responsibility to trainees 0 1 1 1 0 1 1 1 1 0 report clinical trainees concerns of the supervision process 1 0 0 1 1 0 1 1 0 1 consider professional and ethical standards 1 0 1 1 0 0 0 1 0 1 monitoring of supervision program 1 0 1 1 1 0 0 0 0 1 determinant factors to require the amount of supervision 1 0 1 0 0 1 0 0 1 1 provide feedback and constructive criticism on performance 0 0 1 0 1 0 1 1 0 0 how requests help trainees in clinical situations 1 1 0 0 0 1 1 0 0 0 delineate work procedures of trainees 0 0 1 1 0 0 0 0 1 0 the position of clinical trainees in the treatment team 1 0 0 0 0 0 0 0 0 1 69 review core components of three functions of clinical supervision in undergraduate medical educationsoleiman ahmady, masoumeh seidi j contemp med sci | vol. 7, no. 2, march-april 2021: 66 – 72 supportive/restorative/resourcing this function provides support to supervisee’s emotional responses,42 adequately refreshed and recreative,52 to alleviate the stress,54,55 well-being,50,53 self-care,50 acknowledges the emotional effects on the individual work, and in particular work with people in distress,19 real-time supervision, positive achievements, coping for adverse incidents.56 space for trainees to vent their feelings in a listening environment.46 student support is complemented by the form of peers and peer groups, and the existence of personal tutors or educational advisers,52 facilitates the discharging of emotions and recharging of energies.20 discussion this study aimed to identify the characteristics and the components of clinical supervision functions of medical trainees during a clinical course. it is of note that supervision in the education system is regarded as an umbrella term,1, 21, 57 covering all one-on-one professional encounters.21 there are also some terminologies and words, which contain dimensions of supervision as their core concepts,57 such as clinical supervision, educational supervision, remedial supervision, mentoring, preceptorship, and coaching. in this sense, each term can have different effects on educational goals and specific characteristics.1, 21 in this respect, educational supervision refers to regular supervision taking place in the context of a recognized training to establish learning needs and to review progress. it is also the most common one among senior and junior postgraduate residents.1, 58 as well, remedial supervision is a subset of educational supervision aimed at retraining.21 clinical supervision is also regarded as an interchange between practicing professionals to assist the development of professional skills.21 mentoring is likewise rooted in socialization theories in psychology. the focus of these theories is on how individuals learn new behaviors and social roles.59 a preceptorship represents a time-defined relationship with externally defined objectives, and has the teaching of a novice in the proficiencies of a new role as its goal.60 coaching is also a process that can guide students table 2. main components of clinical supervision functions of supervised medical trainees. function components managerial supervision determine the theory or conceptual framework define ethical, professional and legal standards in the clinical environment existence of a contract/agreement between the supervisor and trainee eligible manpower to be the supervisor determine the knowledge, skills, and competencies of a clinical supervisor expectations, duties and responsibilities of the clinical supervisor expectations, responsibilities and tasks of clerkship/internship delineate work procedures for clerkship/internship assessment or evaluation of clerkship/internship monitoring of education programs educational supervision setting educational goals supervisor and students as teacher (intern, resident, and fellow) training determining to achieve the minimum competencies expected in the clerkship/internship course determinant factors of the amount of supervision required trainees presentation feedback and constructive criticism determine progressive levels of clerkship/internship provide reflection opportunities during and after the action supportive supervision the type of relationship between the supervisor and trainee safety of trainees in all clinical settings design flowchart for request help trainees in clinical situations guidelines for report trainees concerns about the clinical, administrative, vocational and educational affairs and supervision process planning for real-time supervision (during activity) considering personal needs and balance between work and life trainees existence activities to increase the confidence, well-being, and creativity of trainees define the position of clerkship/internship in the treatment team 70 review core components of three functions of clinical supervision in undergraduate medical education soleiman ahmady, masoumeh seidi j contemp med sci | vol. 7, no. 2, march-april 2021: 66 – 72 towards performance improvement.61 it is noteworthy that preceptorship,21,62 and coaching are a subset of mentoring.63,64 in the literature review, the types of supervision had been cited in these articles (e.g. clark, 2006; launer, 2014; scaife, 2003; morton-cooper, 1997; jarvis, 2004; rousseau 2008). the researchers in the present study also designed a form for different types of supervision in medical education (fig. 2). studies show that clinical and educational supervision are effective as they apply mentoring principles and characteristics.65, 66 the mentoring during supervision of a fortuitous relationship can also foster the development of medical students.58 it seems that supervision and mentoring are far from being mutually exclusive and they are potentially complementary in many respects. although the mentoring role signals a concerted emphasis on support, encouragement, advocacy, and collegial connection, supervision may encompass a distinct mandate for evaluation and gatekeeping.66, 67 the effectiveness of supervision can be accordingly improved during formal meetings, augmenting quality of feedback and ensuring that occurrence of regular mentoring dialogues would be highly valuable.58 with regard to the review of 30 documents (i.e., policies, guidelines, standards) on clinical supervision of medical students, it seems that all medical schools have attempted to compose and adhere to standard no. 9.3 of clinical supervision of medical students, introduced in the lcme and the cacms, in a brief manner or in detail. according to the review of the given documents, it seems that many have been merely designed in response to agencies for the accreditation of medical education programs. the focus of the components are also laid more on enforcing student regulations and discipline and concentrated on duties, responsibilities, and expectations of supervisors, to ensure the supervision process has been presented. these tasks dominate and focus supervision on compliance with organizational and legislative requirements. within these situations, the underlying power relations are arranged to privilege managerial functions at the expense of educational and supportive functions, which result in trainees’ displeasure and possibly compromised supervision outcomes.68, 69 therefore, it is imperative that all three supervision functions are simultaneously applied. besides, two committees (namely, lcme and cacms) have thus far reviewed the requirements of supervision of the gmc for the school of medicine.39 in this document, the responsibility of students are achieving all outcomes as well as ensuring patient safety by working within the limits of their competence.70 the elements in the supervision of medical trainees have been thus announced by the pmetb and gmc as guaranteeing trainee safety, ensuring an appropriate level and amount of clinical duties, as well as monitoring progress, feedback on performance, and provision of career advice.71,72 the racgp has also guided supervision and support trainees to maintain general practitioners’ teaching standards.41 accordingly, supervisors’ roles, mentioned for trainees’ supervision and support, take account of assisting students to understand the requirements of the education course and providing direct observation sessions, which should be at an appropriate level considering the students’ knowledge and experience, ensuring that students are introduced to all members of staff. moreover, the given roles are also related to taking direct and principal responsibility of patients and being physically present at the workplace at all times, whilst students are providing clinical care as the ultimate management of patients.41 iranian doctor of medicine (m.d.) program differs from that in the united states and canada. in these countries, students complete an undergraduate university degree before enrolling in medical schools. the curriculum is also characterized by 2+2 structures for basic sciences and clinical years.73 in iran, students enter in medical schools from high school. the curriculum is additionally characterized by 3+3.3 for basic sciences and clinical years. in addition, clinical courses have two phases, clerkship (minimum of 21 months) and internship (18 months). these two phases in some of the supervision components must be separately defined on basic educational goals as experiences, expectations, and needs of trainees. in many little policies, guidelines, and standards, there are difference between clerkship and internship. for example, roles and work procedures for medical students,34 at charles e. schmidt college of medicine or orders in hospital medical records, teaching and learning, observation, assessment, and feedback at columbia faculty of medicine are divided into two groups: medical students in 1 and 2 and 3 and 4 years.30 accordingly, the components that should be provided for the clerkship and internship courses were separately identified (table 2). on the other hand, clinical supervision function models demonstrated that each function had different characteristics. each function is also part of the supervisory activities for medical students because each function has its own definition and includes specific tasks and the students must benefit from all these components. management supervision blind spots as well as poor practices are correspondingly intended to ensure corporate ethics and client safety standards.74 educational supervision can thus provide knowledge and skills needed to perform effective services and is associated with cognitive aspects and learning in trainees,44,75 training and development, as well as performance evaluation observed.54 besides, supportive supervision indirectly enhances effective emotional states and promotes resilience to stress.76 so, in supervisory support, supervisors provide the space for trainees to reflect on the impact of work on their personal and professional well-being.74 twenty-five (25) components were broken down into managerial supervision, educational supervision, and supportive supervision. it should be noted that these three functions were challenging as they were not all mutually exclusive and a degree of overlap was inevitable (table 2). fig. 1 relationship between types of supervision in medical education. 71 review core components of three functions of clinical supervision in undergraduate medical educationsoleiman ahmady, masoumeh seidi j contemp med sci | vol. 7, no. 2, march-april 2021: 66 – 72 attending physicians should be thus given necessary training for the supervisory roles, knowledge of supervision models, trainees’ developmental levels, together with adult learning principles. clinical supervision training can accordingly facilitate improved patient outcomes, supervisor competence, and trainee satisfaction. in this line, curtin university course provide supervision training to allied health, nursing, and medical staff. this training course is comprised of five 1-day introductory and three 4-day advanced workshops. the 1-day training provides an overview of an expanded version of the proctor’s model with its own roots in the kadushin’s work on social work supervision.50 medical students are thus expected to benefit more from clinical supervision, to achieve a sense of personal support and well-being, to increase knowledge and awareness of possible solutions to clinical problems, to boost confidence, to reduce emotional stress and burnout, and to enhance participation in reflection practices.54 however, these models address three central supervision functions, but suggest the need to take account of other factors. this means, expanding the model so that supervisors become empowered to match the supervision tasks to the developmental stage of trainees, critically reflecting on human diversity such as culture, ethnicity, disability, socioeconomic status, religion, and life experiences. conclusion the perception of clinical supervision is yet a monitoring tool. it is apparent that managerial/administrative role is much more extensive and it mostly includes assuming responsibilities for trainees’ work and ensuring that they have conformed to rules and norms of organizations. it seems that two educational and supportive roles have faded or disappeared due to managerial roles. there are also efforts to define functions for clinical supervision in other field of medical education in which trainees can spend part of their education at patient bedside. the researchers reviewed documents to achieve objective and tangible characteristics for functions associated with clinical supervision of medical trainees. the results of the present study on clinical supervision function models demonstrated that each function had different characteristics, and three supervisions were separately defined. the main purpose was to help graduate a person being without any stress and concerns, as a qualified doctor, a respectable citizen, and a competent manager, etc. it is thus necessary that the three clinical supervision functions be simultaneously applied by supervisors. it is recommended to make efforts to empower attending physicians in clinical supervision functions and developing tools for student evaluation based on these three functions. the results of this study should be operationally implemented for medical students to detect challenges and inconsistencies disclosure the author declares no conflict of interest. references 1. launer j. supervision, mentoring and coaching. understanding medical education evidence, theory and practice oxford: wiley blackwell/asme. , 2014:111-22. 2. kilminster s, cottrell d, grant j, jolly b. amee guide no. 27: effective educational and clinical supervision. med teacher. 2007;29(1):2-19. 3. bass eb, vi ahf, morrison g, wills s, mumford lm, goroll ah. national survey of clerkship directors in internal medicine on the competencies that should be addressed in the medicine core clerkship. am j med. 1997;102(6): 564-71. 4. zeind cs, zdanowicz m, macdonald k, parkhurst c, king c, wizwer p. developing a sustainable faculty mentoring program. am j pharm educ. 2005;69(5):100. 5. changiz t, fakhari m, jamshidian s, zare s, asgari f. systematic review of studies in the field of competencies of new or soon to-be-graduate general physicians in iran. sdmej. 2015;12(2). 6. amiresmaili m, nekoei moghadam m, moosazadeh m, pahlavan e. challenges of general practice education in iran: a qualitative study. strides dev med educ. 2013;9(2):118-31. 7. razmjou s, baradaran hr, kouhpayehzadeh j, soltani-arabshahi k. comparison of quality of clinical supervision as perceived by attending physicians and residents in university teaching hospitals in tehran. med j islamic repub of iran. 2015;29:248. 8. kilminster s, jolly bc. effective supervision in clinical practice settings: a literature review. med educ. 2000;34(10):827-40. 9. wimmers pf, mentkowski m. assessing competence in professional performance across disciplines and professions. evaluating the paradigm shift from time-based toward competency-based medical education (chapter 19): springer; 2016. 10. nasri k, kahbazi m, nourouzi a, nasri s. the medical education problems and possible solutions in stagers and intern’s view points of arak university of medical sciences. 2010:2006-07. 11. shapiro sl, shapiro de, schwartz ge. stress management in medical education a review of the literature. acad med. 2000;75(7):748-59. 12. ministry. ministry of health and medical education in iran,doctor of medicine curriculum. approved sixty-seventh meeting of the supreme council for medical planning 2018. 13. yazdankhafard m, poladi sea. the stressors of clinical education from students’ point of view in bushehr university of medical sciences. iran j med educ. 2008;8:341-50. 14. caspi j, reid wj. educational supervision in social work: a task-centered model for field instruction and staff development: columbia university press; 2002. 15. brunero s, stein-parbury j. the effectiveness of clinical supervision in nursing: an evidenced based literature review. aust j adv nurs. 2008. 16. bowles n, young c. an evaluative study of clinical supervision based on proctor’s three function interactive model. j adv nurs. 1999;30(4):958-64. 17. kadushin, editor supervision in social work1976: new york and london. columbia university press. 18. erera ip, lazar a. operationalizing kadushin’s model of social work supervision. j social service res. 1994;18(3-4):109-22. 19. inskipp f, proctor b. the art, craft and tasks of counselling supervision, part 1. making the most of supervisors twickenham, uk: cascade publications. 1993. 20. morton-cooper a, palmer a. mentoring, preceptorship, and clinical supervision: a guide to professional roles in clinical practice: university of warwick.blackwell science; 2000. 21. clark p, jamieson a, launer j, trompetas a, whiteman j, williamson d. intending to be a supervisor, mentor or coach? which, what for and why? educ primary care. 2006;17(2):109-16. 22. carnwell r, daly w. strategies for the construction of a critical review of the literature. nurse educ pract. 2001;1(2):57-63. 23. norman g, eva kw. quantitative research methods in medical education. understanding medical education oxford, uk: wiley-blackwell. 2010:301-22. 24. sterkenburg a, barach p, kalkman c, gielen m, ten cate o. when do supervising physicians decide to entrust residents with unsupervised tasks? acad med. 2010;85(9):1408-17. 72 review core components of three functions of clinical supervision in undergraduate medical education soleiman ahmady, masoumeh seidi j contemp med sci | vol. 7, no. 2, march-april 2021: 66 – 72 25. hauer ke, ten cate o, boscardin c, irby dm, iobst w, o’sullivan ps. understanding trust as an essential element of trainee supervision and learning in the workplace. adv health sci educ. 2014;19(3):435-56. 26. lcme. functions and structure of a medical school .standards for accreditation of medical education programs leading to the md degree. liaison committee on medical education. 2014;28. 27. cacms. committee on accreditation of canadian medical schools (cacms) standards and elements effective 2018. 28. mcgill. supervision policy for trainees in the clinical team . the university of mc gill faculty of medicine , 2016 [available from: https://mcgill.ca/ ugme/files/ugme/supervision_policy_for_trainees_in_clinical_team_ v1.1.pdf. 29. alberta. supervision of medical students on clinical rotations policy. university of alberta 2014 [available from: https://www.ualberta.ca/ medicine/-/media/medicine/ume/policy/clinicalsupervision.pdf. 30. columbia. expectations of clinical supervisors and preceptors of students in clinical settings. the university of british columbia, 2018 [available from: https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20 guidelines/supervision%20of%20students%20in%20%20required%20 clinical%20learning%20experiences%20(031).pdf. 31. columbia. expectations of medical students in supervised clinical settings. the university of british columbia 2017 [available from: https:// mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20 guidelines/supervision%20of%20students%20in%20%20required%20 clinical%20learning%20experiences%20(031).pdf. 32. boston. clinical supervision policy. boston university school of medicine, 2018 [available from: https://www.bumc.bu.edu/busm/files/2018/11/ clinical-supervision-policy.pdf. 33. nebraska. clinical supervision of medical students. university of nebraska college of medicine (uncom), 2017 [available from: https://www. unmc.edu/com/education/com-student-policies/com-policies/clinicalsupervision.htmluniversity 34. florida. medical student roles and supervision policy.medical student handbook.florida atlantic university charles e. schmidt college of medicine (fau com), 2019. 35. washington. policy on supervision of medical students in clinical settings. the university of washington school of medicine (uwsom) 2017 [available from: https://medicine.wsu.edu/documents/2017/08/clinical-supervisionof-medical-students.pdf/. 36. carolina. clinical supervision of medical students. university of north carolina school of medicine (unc som) 2019 [available from: https://www. med.unc.edu/md/policies/files/2019/01/clinical-supervision-of-medicalstudents-lcme-9.3.pdf. 37. minnesota. student supervision during clinical activities. the university of minnesota medical school (umms), 2019 [available from: https://med.umn. edu/sites/med.umn.edu/files/ocu.0119.006.1_student_supervision.pdf. 38. pennsylvania. supervision of medical student clinical activity. (upenn) 2019 [available from: https://www.med.upenn.edu/flpd/assets/usercontent/documents/9.3%20medical%20student%20supervision%20policy. docx. 39. gmc. developing teachers and trainers in undergraduate medical education. advice suppl tomorrow’s doctors 2009:12. 40. pmetb. generic standards for training: postgraduate medical education and training board; 2008. 41. racgp. guidelines for the supervision of medical students in general practice. the royal australian college of general practitioners (racgp). 2007. 42. basa v. models of supervision in therapy, brief defining features. eur j counsel theory res pract. 2017;1(4):1-5. 43. proctor b. supervision: a cooperative exercise in accountability. in m marken and mpayne (eds) enabling and ensuring leicester: leicester national youth bureau and council 44. for education and training in youth and community work. 1988b. 45. kadushin a, harkness d. supervision in social work: columbia university press; 2002. 46. morrison t. staff supervision in social care: an action learning approach: pavilion publishing; 2001. 47. hawkins p, shohet r. supervision in the helping professions: mcgraw-hill education (uk); 2012. 48. hughes jm. the role of supervision in social work: a critical analysis. critic soc think policy pract. 2010;2:59-77. 49. turner j, hill a. implementing clinical supervision (part 1): a review of the literature. mental health nurs (online). 2011;31(3):8. 50. sloan g, watson h. clinical supervision models for nursing: structure, research and limitations. nursing stand (through 2013). 2002;17(4):41. 51. gillieatt s, martin r, marchant t, fielding a, duncanson k. evaluation of an inter-professional training program for student clinical supervision in australia. human resour health. 2014;12(60):1-9. 52. butterworth tc, j. ; white, e.;. it is good to talk an evaluation of clinical supervision and mentorship in england and scotland. university of manchester. gb_ 1997:12245; gb; 1997. 53. morton-cooper a, palmer a. mentoring, preceptorship and clinical supervision. nurse educ today. 2000;20:418-21. 54. butterworth t, bishop v, carson j. first steps towards evaluating clinical supervision in nursing and health visiting. i. theory, policy and practice development. a review. j clin nurs. 1996;5(2):127-32. 55. cutcliffe jr. an alternative training approach in clinical supervision. routledge handbook of clinical supervision: routledge; 2010. p. 63-78. 56. jones a. clinical supervision: a framework for practice. int j psychiatric nurs res. 1996;3:290-307. 57. tuck ja. a new approach to team clinical supervision on an acute admissions unit. mental health pract. 2017;20(5). 58. scaife j. supervision in the mental health professions: a practitioner’s guide: routledge; 2003. 59. patel p. an evaluation of the current patterns and practices of educational supervision in postgraduate medical education in the uk. perspect med educ. 2016;5(4):205-14. 60. chang s, baldwin cd, cameron c. mentoring mentors in scientific communication for trainees. acad med j assoc am med coll. 2015;90(3):265-. 61. barker er, pittman o. becoming a super preceptor: a practical guide to preceptorship in today’s clinical climate. j am acad nurse pract. 2010;22(3):144-9. 62. landreville j, cheung w, frank j, richardson d. a definition for coaching in medical education. can med educ j. 2019;10(4):e109. 63. morton-cooper a. preceptorship via action research: a reflective account: university of warwick; 1997 [available from: https://ethos.bl.uk/ orderdetails.do?uin=uk.bl.ethos.263611/. 64. rousseau m. structured mentoring for sure success: pennwell corp; 2008. 65. jarvis j. coaching and buying coaching services: a guide: chartered institute of personnel and development; 2004. 66. passi v. the importance of mentoring during educational supervision. perspect med educ. 2016;5(4):195-6. 67. johnson wb, skinner cj, kaslow nj. relational mentoring in clinical supervision: the transformational supervisor. j clin psychol. 2014;70(11):1073-81. 68. johnson wb. transformational supervision: when supervisors mentor. prof psychol res pract. 2007;38(3):259. 69. gardner a, mccutcheon h, fedoruk m. superficial supervision: are we placing clinicians and clients at risk? contemp nurse. 2010;34(2):258-66. 70. beddoe l. external supervision in social work: power, space, risk, and the search for safety. austr social work. 2012;65(2):197-213. 71. council gm. tomorrow’s doctors: outcomes and standards for undergraduate medical education. manchester, uk: general medical council. 2009. 72. pmetb. operational guide for the pmetb quality framework 2009 [available from: http://www.pmetb.org.uk/fileadmin/user/qa/qf/qf_ operational_guide.pdf 73. gmc. general medical council.the early years. london: gmc1999. 74. kennedy tj, regehr g, baker gr, lingard l. preserving professional credibility: grounded theory study of medical trainees’ requests for clinical support. bmj. 2009;338:b128. 75. farmer s tn. trainor nev .supervision chapter four : effective interventions with offenders 2016 [107]. available from: http://www.hma.co.nz/wpcontent/uploads/2016/01/chapter-4-supervision.pdf. 76. basa v. supervisor-supervisee relationship and alliance. eur j counsel theory res pract. 2017;1:1-5. 77. lakey b, tardiff ta, drew jb. negative social interactions: assessment and relations to social support, cognition, and psychological distress. j soc clin psychol. 1994;13(1):42-62. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.875 https://mcgill.ca/ugme/files/ugme/supervision_policy_for_trainees_in_clinical_team_v1.1.pdf https://mcgill.ca/ugme/files/ugme/supervision_policy_for_trainees_in_clinical_team_v1.1.pdf https://mcgill.ca/ugme/files/ugme/supervision_policy_for_trainees_in_clinical_team_v1.1.pdf https://www.ualberta.ca/medicine/-/media/medicine/ume/policy/clinicalsupervision.pdf https://www.ualberta.ca/medicine/-/media/medicine/ume/policy/clinicalsupervision.pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://mednet.med.ubc.ca/aboutus/policiesandguidelines/policies%20guidelines/supervision%20of%20students%20in%20%20required%20clinical%20learning%20experiences%20(031).pdf https://www.bumc.bu.edu/busm/files/2018/11/clinical-supervision-policy.pdf https://www.bumc.bu.edu/busm/files/2018/11/clinical-supervision-policy.pdf https://www.unmc.edu/com/education/com-student-policies/com-policies/clinical-supervision.htmluniversity https://www.unmc.edu/com/education/com-student-policies/com-policies/clinical-supervision.htmluniversity https://www.unmc.edu/com/education/com-student-policies/com-policies/clinical-supervision.htmluniversity https://medicine.wsu.edu/documents/2017/08/clinical-supervision-of-medical-students.pdf/ https://medicine.wsu.edu/documents/2017/08/clinical-supervision-of-medical-students.pdf/ https://www.med.unc.edu/md/policies/files/2019/01/clinical-supervision-of-medical-students-lcme-9.3.pdf https://www.med.unc.edu/md/policies/files/2019/01/clinical-supervision-of-medical-students-lcme-9.3.pdf https://www.med.unc.edu/md/policies/files/2019/01/clinical-supervision-of-medical-students-lcme-9.3.pdf https://med.umn.edu/sites/med.umn.edu/files/ocu.0119.006.1_student_supervision.pdf https://med.umn.edu/sites/med.umn.edu/files/ocu.0119.006.1_student_supervision.pdf https://www.med.upenn.edu/flpd/assets/user-content/documents/9.3%20medical%20student%20supervision%20policy.docx https://www.med.upenn.edu/flpd/assets/user-content/documents/9.3%20medical%20student%20supervision%20policy.docx https://www.med.upenn.edu/flpd/assets/user-content/documents/9.3%20medical%20student%20supervision%20policy.docx https://ethos.bl.uk/orderdetails.do?uin=uk.bl.ethos.263611/ https://ethos.bl.uk/orderdetails.do?uin=uk.bl.ethos.263611/ http://www.pmetb.org.uk/fileadmin/user/qa/qf/qf_operational_guide.pdf http://www.pmetb.org.uk/fileadmin/user/qa/qf/qf_operational_guide.pdf http://www.hma.co.nz/wp-content/uploads/2016/01/chapter-4-supervision.pdf http://www.hma.co.nz/wp-content/uploads/2016/01/chapter-4-supervision.pdf 275j contemp med sci | vol. 6, no. 6, november–december 2020: 275–278 original issn 2413-0516 introduction pressure ulcer or bedsore is an area of necrosis caused by external pressure on bony prominences and areas without adequate blood flow.1, 2 in recent decades, some researches have shown a 10–22% prevalence of pressure ulcer.3 even in most developed countries, despite new technologies and providing the best nursing care, the rate of death is high because of pressure ulcer. the mortality rate of pressure ulcer is approximately 6000 people each year in the united states.4 the cost of bedsore treatment is a large burden on patients and healthcare facilities. an estimated $500–$40,000 is needed for the treatment of each bedsore in the united states.5 bedsore is classified into four types: (1) erythema or redness without pallor, (2) slight decrease in skin thickness, (3) severe decrease in skin thickness, and (4) bone, tendon, and muscle damage.3 most pressure ulcers tend to become chronic, for example, it takes 10–12 weeks for a grade 4 ulcer to heal.4 intensive care can help to improve a bedsore. the use of preventive devices can also inhibit the worsening of sores. hydrocolloid dressings can also promote healing.4 the most important preventative step is to find high-risk patients and treatment method depends on the pressure ulcer grade. prevention of pressure ulcer can have important health consequences and can be more effective than treatment.6 although not all pressure ulcers can be avoided, most of them can be prevented by appropriate care. the initial assessment in preventative strategies should identify risk factors.3 wound healing may not be rapid; however, factors such as pressure distribution, adequate nutrition, and optimal management can help accelerate this process.3 one of the main points, which reflect the quality of care of patients at risk, is to detect developing marks at the onset of pressure ulcer. moreover, the feeling of responsibility in caregivers can be effective in the quality of care and preventing of sore worsening.7 clinical audit is a quality enhancement process that aims to improve patient care and their outcomes through intervention and the change with the use of clear criteria.3 the aim of this study was to evaluate patients at risk, promote prevention of pressure ulcer, increase the quality of patient care, and ultimately reduce the number of patients with pressure ulcer through clinical audit. materials and methods this prospective study was performed on patients at risk for ulcers hospitalized in ahvaz golestan hospital’s general, intensive, and emergency units from september 2013 to march 2014. a total of 208 patients at the risk of pressure ulcer were enrolled in the study according to hospitalization date. children under 12 years of age were excluded from the study. the ethics committee of ahvaz jundishapur university of medical sciences approved this research project. hospital supervisors using a questionnaire and achieved the initial evaluation. three professors of dermatology verified a standard clinical audit questionnaire. in this study, variables were assessed and measured (table 1). after measurement, a report of clinical audit for prevention and improvement of bedsore in southwest iran nader pazyar1, nasrin fatemi2, nosrat mirzai3, mohammad jahangir4, farahnaz rasooli5, shahrzad attarzadeh5 1emam khomeini university hospital, dermatology department, ahvaz jundishapur university of medical science, ahvaz, iran. 2golestan university hospital, department of clinical audit, ahvaz jundishapur university of medical science, ahvaz, iran. 3golestan university hospital, neurosurgery intensive care unit, ahvaz, ahvaz jundishapur university of medical science, ahvaz, iran 4ahvaz jundishapour university of medical science, ahvaz, iran 5golestan university hospital, clinical nurse manager, ahvaz, iran corresponding author: nasrin fatemi (email: nasrin.fatemi@yahoo.com) (submitted: 05 august 2020 – revised version received: 12 september 2020 – accepted: 04 october 2020 – published online: 26 december 2020) abstract objective: pressure ulcer is areas of necrosis caused by external pressure on bony prominences with a prevalence of 10–22%. methods: this study was conducted to improve prevention and care of bedsore by clinical audit at ahvaz golestan hospital. we included hospitalized patients at the risk of bedsore in general, special, and emergency units at ahvaz golestan hospital during a 6-month period. in accordance to the clinical audit cycle, the current situation was assessed by observation and consultation. after finding weakness points, proper interventions were implemented based on nice guidelines for bedsore. subsequently, another audit was performed to assess the effectiveness of intervention. results: comparison of results before and after intervention showed an increase in all studied variables. the changes in studied variables are listed as follows: bedsore reduction (p=0.001), patients’ assessment during the first 6 hours of hospitalization (p=0.008), assessment of external pressure (p=0.001), change position (p=0.001), care standards (p=0.170), and skin friction (p=0.001). the highest increase was seen in change position (p=0.001) and the lowest increase was seen in maintaining adequate skin hygiene (p=0.360). conclusions: clinical audit led to improvement of prevention and treatment quality of bedsore, and also formulation and implementation of standards of care. keywords: clinical audit; nice guideline; pressure ulcer; prevention 276 original a report of clinical audit for prevention and improvement of bedsore in southwest iran nader pazyar j contemp med sci | vol. 6, no. 6, november–december 2020: 275–278 the variables were compared to nice guidelines in order to estimate the difference between our care and the standards of care and treatment (table 2). three months after the initial surveillance, a supervisor used questionnaires, observations, and interviews to review the patients’ status. data were analyzed using spss16, and statistical tests of chi-square and fisher’s mcnemar. p value of less than 0.05 was considered significant. results the access of patients to the preventive devices was 31% before the study which escalated to 60.3% after the intervention (p=0.001) (fig. 1). evaluation of patients in the first 6 hours of hospitalization was 43.6% prior to intervention in comparison with 55.8% after the intervention (p=0.008). inspection of vulnerable and high-risk areas of the body was 61.5% which reached to 69% after the intervention (p=0.27). the modification of external pressure was 51.3% before the study which rose to 80.6% (p=0.001) (fig. 2). patients position changing by nurses went from 0% to 44.9% (p=0.001). implementation of standards of care changed from 76.7% to 84.6% (p=0.17), which did not show a significant difference. the patients’ skin friction increased from 75.6% during start the study to 92.3% after the intervention (p=0.001) (fig. 3). fig. 1 access of patients to preventive devices before and after implementation of audit. fig. 2 assessment of external pressure points before and after audit. fig. 3 consideration of skin friction before and after audit. table 1. influential bedsore variables before and after intervention. variables 1 access of patients to preventive devices 2 evaluation of patients in the first six hours of hospitalization 3 inspection of vulnerable and high-risk areas of the body 4 the assessment of external pressure in patients 5 patients position changing by nurses 6 implementation of standards of care for patients 7 understanding of the patients’ skin friction 8 the use of pressure distributors in patients during surgery 9 participation of patients in the implementation of care process 10 recording of the results of assessment and interventions for patients with pressure ulcer 11 patients’ participation in the implementation of care process 12 effective monitoring of the implementation of the treatment and care protocol for patients with pressure ulcer table 2. interventions used in clinical audit to standardize bedsore care. intervention design of colored cards to indicate patients at risk for pressure ulcer 1 use of intermittent inflation mattress2 use of cushions for position changing3 placement of evaluation form at patients’ bedside4 training of all caregivers according to guidelines5 intervening in the choice of material used for clothing and bedding and changing non-absorbable fabric to high quality cotton types 6 finding appropriate placements as soon as possible for intubated patients in the internal emergency ward 7 increasing medical documenting personnel in attempt to decrease the time used by nurses for documentation 8 necessitating nutrition consultation for icu patients9 necessitating all clinical wards to report all grade ii or higher pressure ulcer to the hospitals safety unit 10 277 original a report of clinical audit for prevention and improvement of bedsore in southwest irannader pazyar j contemp med sci | vol. 6, no. 6, november–december 2020: 275–278 the use of pressure distributors in patients during surgery went from 0% to 41.6% (p=0.001) (fig. 4). participation of patients in the care process increased from 76% to 93.1% (p=0.001). recording of the results of assessment and interventions for patients increased from 46.2% to 65.9% post-intervention (p=0.005). effective monitoring of the treatment and care protocol changed from 46.2% to 54.3% after the intervention which did not show a significant difference (p=0.25). discussion the skin is the largest organ of the body that acts as a deterrent against bacteria, chemicals, and physical actions and maintains body homeostasis. damage to the epidermis and dermis can lead to systemic infections, increased morbidity, and cost of care, and has negative psychosocial consequences.8 out of 16 variables examined in our study, 8 were increased considerably after intervention. they were including the access of patients to bedsore preventive devices, assessment of external pressure, patients position changing, skin friction, use of pressure distributors, patients’ participation in care process, recording of the assessment, and interventions. our interventions lead to improvement of following items: patients’ assessment at the first 6 hours, inspection of vulnerable and high-risk areas of the body, the implementation of standards of care, effective monitoring of the treatment, and care protocol (fig. 5). 1. the preparation of guidelines for prevention and treatment of pressure ulcer in hospitalized patients. 2. preparation of training manuals and pamphlets for the prevention and treatment of pressure ulcer. 3. participation of hospital managers in improvement of the patients and standards of care. 4. increasing the nurses’ knowledge in the prevention and treatment of pressure ulcer. other achievements were the stabilization of the audit team to assess consistently high quality patient care and increase the willingness of staff to report patients suffering from pressure ulcer. guidelines were updated and attention was increased to the care of the wound bed following a multilevel analysis study of hospitals and nursing homes for the elderly performed in berlin, germany between the years 1991 and 2003 in which 522 nurses answered questionnaires in attempt to balance bedsore treatment. the study also showed that nursing knowledge was slightly better in 2003 than in 1991, nurses were more cautious and aware, more knowledgeable of preventive factors, and that the implementation of audit led to higher knowledge.9 in a systematic review, nursing training for early detection and recognition of pressure ulcer caused a decrease in the prevalence of this complication from 78.13% to 15.5%, which showed the importance of nurses’ knowledge of bedsore prevention.9 a survival analysis study by pusan and colleagues in south korea conducted on patients in intensive care wards emphasized the importance of regular position changing. the average time needed to cause a bedsore in patients with moderate, high, and very high risk was 5, 3.5, and 3 hours, respectively.10 in a clinical trial by defloor and colleagues on 838 patients in nursing homes, position changing was studied during 4 weeks. it was concluded that position changing every 2 hours with a conventional mattresses compared to position changing every 4 hours with a conventional mattresses significantly reduced the incidence of bedsore.11 a study in a houston hospital (2009) showed that upgrading the quality of evidence-based nursing practice through training, ongoing monitoring of patients’ skin, nurse participation, and sharing of knowledge are advanced monitoring tools for reducing pressure ulcer in rehabilitation wards. not only will skin monitoring and staff training lead to an increase in the accountability of nurses and documentation development, but it will also improve patients’ skin status.12 all of the above studies emphasize on the proper care, and early identification of high-risk patients. the most important factors in the prevention of pressure ulcer include reducing pressure with changing positions, training of nursing staff, removing moisture and external pressure to the skin, and assessing the nutritional status of patients. the weaknesses of our study include incomplete data in primary patients’ evaluation forms because of admission nurses errors, a shortage of personnel in clinical units, and the misuse of nursing care protocols by some nurses. conclusion the interventions implemented in our study improved care, prevention, and treatment of patients with or at risk for pressure ulcer. it seems that the clinical audit process can lead to improved patient care standards.fig. 5 radar chart of studied criteria. fig. 4 the rate of use of pressure distributors during surgery before and after audit. 278 original a report of clinical audit for prevention and improvement of bedsore in southwest iran nader pazyar j contemp med sci | vol. 6, no. 6, november–december 2020: 275–278 acknowledgments we would like to thank ahvaz golestan hospital’s chief (dr.  arti), manager (dr. safarinia), treatment representative (mr. cheraghi), nursing administrator (ms. attarzadeh), and all head nurses and nurses who helped us in this study. this paper was derived from a research project approved by the university of ahvaz is therefore we would also like to thank ahvaz jundishapour clinical research center and ahvaz golestan clinical research development unit. references 1. national guideline clearing house (ngc). www.guidline.gov/index. asp. national institute for clinical excellence (nice). [accessed on 2013 sep 28]. 2. the management of pressure ulcers in primary and secondary care. a clinical practice guideline. available from: http://www.nice.org.uk/ nicemedia/pdf/cg029fullguideline.pdf [accessed on 2013 sep 28]. 3. dealey c. best practice guide pressure ulcers. british geriat soc. 20 jun 2012. http://www.org.uk; http://www.bgs.org.uk/index.php/topresources/ publicationfind/goodpractice/2078-bpgpressureulcers 4. shahin es, dassen t, halfens rj. incidence, prevention and treatment of pressure ulcers in intensive care patients: a longitudinal study. int j nurs stud. 2009;46:413–21. 5. courtney h, lyder nd. prevention and management pressure ulcer. jama 2003;289(2):223–226. 6. chou r, dana t, bougatsos c, blazina i, starmer aj, reitel k, buckley di. pressure ulcer risk assessment and prevention: a systematic comparative effectiveness review. ann intern med. 2013;159(1):28–38. 7. irvine a, black c. pressure sore practices. nurs times. 1990;86:74–8. 8. schindler ca, mikhailov ta, kuhn em, christopher j, conway p, ridling d, scott am, simpson vs. protecting fragile skin: nursing interventions to decrease development of pressure ulcers in pediatric intensive care. am j crit care. 2011;20(1):26–34;quiz 35. 9. wilborn d, grittner u, dassen t, kottner j. the national expert standard pressure ulcer prevention in nursing and pressure ulcer prevalence in german health care facilities: a multilevel analysis. j clin nurs. 2010;19:3364-7. 10. kim hj, jeong is. optimal time interval for position change for icu patients using foam mattress against pressure ulcer risk. j korean acad nurs. 2012;42:730-7. 11. defloor t, de bacquer d, grypdonck mh. the effect of various combinations of turning and pressure reducing devices on the incidence of pressure ulcers. int j nurs stud. 2005;42:37–46. 12. anderson jj, mokracek m, lindy cn. a nursing quality program driven by evidence-based practice. nurs clin north am. 2009;44:83–91. https://doi.org/10.22317/jcms.v6i6.826 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 34 original issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 34 – 39 introduction diabetes mellitus (dm) and chronic kidney disease (ckd) (as a part of non-communicable diseases) have replaced infection as a main cause of death in last century. dm is the seventh leading cause of death and responsible for 14.5% of global all-cause mortality. dm is an out of control pandemic, each year 10 million new cases are added globally. in iraq, it affects 517,080 adults and expected to be 20,09,000 by year 2030, 46.5% of them are unaware of their status.1–9 dm may be subclinical for years; 25–50% of patients have complications at the time of diagnosis like diabetic nephropathy which is the leading cause of renal failure and increases death rate 20–40 times, rise in dm morbidity, mortality, and cost; is parallel to that of ckd.4,10–15 ckd is defined as drop in glomerular filtration rate (gfr) below 60 ml/min/1.73 m2 for 3 months or more. initial assessment should include gfr and not by serum creatinine (scr) alone.16–18. staging based on gfr is a good way to assess ckd severity; stage 1 (gfr ≥ 90), stage 2 (60–89), stage 3 (30–59), stage 4 (15–29), and stage 5 (<15).17,19 most of the diabetics with ckd will die from cardiovascular complication; death is two times more at stage 3 and three times at stage 4 than individuals with normal kidney function.4,20,21 many gfr estimating formulae are available like; cockcroft gault (cg), and ckd–epidemiology collaboration (ckd-epi) (the best for diverse populations).22,23 in primary health care (phc), most patients with ckd stages 1, 2 are undetected as ckd is silent and reversible. gfr would be half normal before scr level would be abnormal. kidney disease improving global outcome (kdigo) guidelines consider annual ckd initial screening by scr-based estimated-gfr (egfr) in diabetics as cost-effective; starting 5 years after diagnosis in type 1 dm, and at time of diagnosis in type 2 dm. early detection, slow progression, decrease complications, and need for dialysis. primary care providers are well positioned to manage ckd patients as early referral to nephrologist improve prognosis but they are only detecting 10.8% of them at stage 3.4,11,16,17,19,24–26 this study aims; to assess diabetic adults attending diabetes centers; socio-demographic profile, ckd prevalence, associated risk factors; and to increase family physicians awareness that gfr estimated by ckd-epi equation is better than scr alone to assess renal function and detect ckd among diabetic adults in phc. methods an outpatient-based cross-sectional study with some analytic elements conducted on registered diabetic adults attending diabetes and endocrine-related diseases tertiary centers in baghdad (rusafa & karkh) during the period from december 15, 2019 to august 15, 2020. study project was reviewed and approved by scientific and ethics committees of; training & human development national center/ imoh, and baghdad/ rusafa and karkh health directorates, and informed consents were obtained from them. after satisfied study explanation, a verbal consent was obtained from all registered diabetic adults who attended study place during study period, met eligibility, and inclusion criteria, who consciously agreed to participate in study. exclusion criteria were (age <18 or >70 years, diabetes for <5 years, body mass index (bmi) >30, (cimetidine, trimethoprim, cephalosporin, aspirin) medications in last 5 days, high meat meal last night, urinary tract structural anomaly, sickle cell anemia, spinal cord injury, or pregnancy). a convenience non-selective sampling method was used; researcher made regular study place visits for data collection in system of 5 h a day/1 day a week for 9 months. based on eligibility criteria, 800 diabetic adults were recruited in study and data were collected from them. for each visit, 20–25 detecting chronic kidney disease in diabetic adults by estimating glomerular filtration rate and serum creatinine ahmed kh. mohammed almaawi* senior specialist physician, studies and research department, commission of medical tertiary centers, ministry of health, baghdad, iraq. *correspondence to: ahmed kh. mohammed almaawi (e-mail: dr.ahmedaswad@gmail.com) (submitted: 28 november 2020 – revised version received: 25 december 2020 – accepted: 15 january 2021 – published online: 26 february 2021) abstract objective to assess chronic kidney disease (ckd) prevalence and risk factors including socio-demography among diabetics by estimating glomerular filtration rate (gfr) rather than serum creatinine (scr). methods a cross-sectional study was conducted in december 15, 2019 through august 15, 2020, among 800 diabetics attending tertiary diabetes centers, baghdad. data were collected by self-administered questionnaire. spss was used for data analysis by (mean, standard deviation and t-test) for quantitative variables and (frequency, percentage, chi-square test and kappa index) for qualitative variables. p-value less than 0.05 was considered significant. results 800 diabetics for last 5–40 years, 95.6% with type 2. aged 52.1±13.2 years, with male:female ratio of 1.03:1, 63.6% were with no income, scr level was 0.86±0.3 mg/dl, and egfr by cockcroft gault (cg) and ckd-epi equations was 100.4 ± 36.5 & 92.2 ± 25.5 ml/min/1.73 m2, respectively. ckd prevalence based on scr, and egfr assessed by above equations was 13.3%, 20% and 15.9%, respectively (p<0.001). those with ckd were hypertensive, females, and living in peripheries. conclusions diabetic patients, mainly those with risk factors are more likely to develop ckd. it is better to detect ckd initially by estimating the gfr, rather than scr level alone. furthermore, using ckd-epi equation might be better than the cg formula to estimate the gfr. keywords diabetes, kidney, creatinine 35 original detecting chronic kidney disease in diabetic adultsahmed kh. mohammed almaawi j contemp med sci | vol. 7, no. 1, january-february 2021: 34 – 39 patients were privately interviewed with their medical records checking. for data collection, a self-administered structured questionnaire-form paper was developed, and a pilot study had been done on a randomly selected 10 patients to figure out unclear questions and assess time needed to fill questionnaire. according to pilot study questionnaire, adjustment was done to be more acceptable, and patients who were subjected to pilot study weren’t included in study. questionnaire-form collect data; (age, gender, residence, occupation, income, marital status, smoking, diabetes type and duration, hypertension, heart disease, weight, height, bmi (weight(kg)/height(m2)), exclusion criteria, egfr, and scr), scr and egfr values were used to detect ckd; scr ≥ 1.2 mg/dl for female, and ≥1.4 mg/ dl for male and egfr (below 60 ml/min/1.73 m2 estimated by standard cg and ckd-epi equations) were considered as ckd. in order to compare results from both equations; values estimated by original cg equation ((140-age)*weight/ scr*72(*0.85 if female)) in (ml/min), were normalized to 1.73 m2 body surface area by multiplying gfr by (1.73/(weight0.4 25*height0.725)*0.007184) according to du bois equation.22,23,27 for data entry, storage, and analysis, computerized software spss 20 ibm (statistical package for social science version 20) was used. data were stratified according to age, egfr, and ckd. mean, standard deviation, and t-test were used to present continuous variables and to check association significance. frequency and percentage, chi-square test, and kappa index cross-tabulation were used to present discrete variables and to check association significance. probability (p-values) of less 0.05 was considered as statistically significant. results study included 800 adults with dm for the last 11.7±6.9 (5–40) years (yr), majority 95.6% (765) with type 2 dm, and only 4.4% (35) with type 1, aged 52.1±13.2 (18–70) yr; (53.75%(430) aged 41–60 yr, 28.38%(227) aged 61–70 yr and 17.87% (143) aged 18–40 yr). study included 405 (50.6%) males and 395 (49.4%) females, with male:female ratio was 1.03:1. 88.4% (707) of patients live in nearby places. table 1 shows that 90.5% (724) of patients are married while 9.5% (76) were either single, divorced, or widow. 63.6%(509) with no income, and only 31.9%(255) had fixed monthly income salary (from a job 16.5%(132), retirement 13.3%(106), or governmental allowance 2.1%(17)), and 4.5%(36) had no fixed income. 90.9% (727) had secondary school educational level or less and only 9.1% (73) had a college level or above. 14.8% (128) of them were smokers. the mean bmi of study group was (25.6±2.6 (16.730) kg/m2). table 2 shows that mean scr level was 0.86±0.3 mg/dl, mean egfr detected by cg and ckd-epi equations was 100.4±36.5 & 92.2±25.5 ml/min/1.73m2, respectively (p<0.001). ckd prevalence detected by abnormal scr was 13.3% (106/800) (57/800 (7.13%) females and 49/800 (6.13%) males, p=0.045). this is significantly different from ckd prevalence by using cg equation 20% (160/800) (p<0.001), and using ckd-epi equation 15.9%(127/800), results by cg was higher than that by ckd-epi equation (p<0.001). according to ckd-epi, 61.4%(78/127) of those with ckd aged 61–70 yr. ckd prevalence among males aged between 61 and 70 yr was higher by using cg than ckd-epi equation (p<0.001). table 1. socio-demographic profile of study sample in different age groups (n: total sample size = 800). age (yr) gender married n. % income n. % education n. % bmi kg/m2 mean ±sd (range) fixed income not fixed no incom e job retired allow ance salary 2ndary college 18-40 male no.=66 39 59.1% 23 34.8% 0 0% 1 1.5 % 11 16.7% 31 47% 54 81.8% 12 18.2% 24.4±2.8 (17.3-29.8) female no.=77 50 64.9% 7 9.1% 0 0% 1 1.3% 2 2.6% 67 87% 65 84.4% 12 15.6% 25.2±2.3 (19.5-30) 41-60 male no=209 208 99.5% 65 31.1% 28 13.4% 8 3.8% 18 8.6% 90 43.1% 188 90% 21 10% 25.4±2.6 (19.2-29.98) female no=221 209 94.6% 18 8.1% 6 2.7% 0 0% 1 0.5% 196 88.7% 209 94.6% 12 5.4% 26.1±2.6 (18.3-30) 61-70 male no=130 127 97.7% 16 12.3% 65 50% 4 3.1% 4 3.1% 41 31.5% 115 88.5% 15 11.5% 25.2±2.5 (16.7-30) female no.=97 91 93.8% 3 3.1% 7 7.2% 3 3.1% 0 0% 84 86.6% 96 99% 1 1% 26.3±2.8 (19.5-30) 18-70 male no=405 374 92.3% 104 25.7% 93 23% 13 3.2% 33 8.1% 162 40% 357 88.1% 48 11.9% 25.2±2.6 (16.7-29.98) female no=395 350 88.6% 28 7.1% 13 3.3% 4 1% 3 0.8% 347 87.8% 370 93.7% 25 6.3% 26±2.6 (18.3-30) total n=800 724 90.5% 132 16.5% 106 13.3% 17 2.1% 36 4.5% 509 63.6% 727 90.9% 73 9.1% 25.6±2.6 (16.7-30) n.: number of subjects with specific characteristic; %: percentage= (n. /no); yr: year; sd: standard deviation; bmi: body mass index. 36 original detecting chronic kidney disease in diabetic adults ahmed kh. mohammed almaawi j contemp med sci | vol. 7, no. 1, january-february 2021: 34 – 39 to study different associated risk factors, sample was divided into two groups; with or without ckd (defined by egfr level of less than 60 ml/min/1.73 m2 detected by ckd-epi). as shown in table 3, 265/800 (33.1%) patients were hypertensive for the last 1–32 years, and 48/800 (6%) gave a history of heart disease for the last 1–25 years. those with ckd were more likely to be hypertensive (p<0.001), female (p=0.029), and lived in periphery (p<0.001), and more likely to be older, less educated with heart disease and no income. to compare cg and ckd-epi equations, sample was classified into five stages by egfr level estimated by using two equations as shown in table 4; comparing result from each stage showed significant difference (p<0.001). according to ckd-epi, 122/127 (96.1%) of patients with ckd were in stage 3. ckd (stage 3) prevalence was higher by using cg while stage 4 prevalence was higher by ckd-epi. table 5 shows that cross-tabulating results of both equations showed a strong agreement; 684 (85.5%) and by using kappa index (kappa value: 0.73, 95% confidence interval (ci): 0.68–0.77, p < 0.001). ckd-epi: chronic kidney disease epidemiology equation; cg: cockcroft gault equation; egfr: estimated glomerular filtration rate; n: number of subjects with specific character; %: percent= (n /n). discussion this study aims to assess ckd prevalence in diabetic adults and to check whether estimating egfr using ckd-epi formula can detect ckd better than cg formula or scr level. ckd screening is cost-effective as earlier intervention can slow renal damage progression and serves oriented family physician to use modest phc resources judgmentally where scr is feasible, albuminuria is not; but ckd is underestimated whether by phc providers or by international classification of diseases-9(icd-9).4,24,28,29 to achieve this aim, 800 patients’ data were analyzed regarding socio-demography, risk factors, and ckd prevalence in different age groups and stages. the study diabetic population was with 1.03:1 male:female ratio and was middle aged, overweighed, modestly educated, with no job or fixed income, and lived in nearby city areas. this in agreement with narenpitak et al where 760 dm patients aged 58.7 ± 9.8 yr. with bmi 25.6 ± 4.2 kg/m2.30 both genders are somewhat equally affected by dm, but obese people are 80 times more likely to be affected. in iraq, genetics, socio-demographic changes, urbanization, increasing sedentary lifestyle, and overweight had led to emerging dm epidemics mainly in age above 40.4,6,8,10,12 ckd prevalence in this study was significantly higher by using cg equation (20%) than with ckd-epi (15.9%), or abnormal scr level (13.3%). this is in agreement with zaman et al; ckd prevalence by using cg (31.4%), by ckd-epi (21.9%) and by scr (18.6%) though prevalence was higher as the last is a hospital-based and ours is outpatient-based study.31 and in agreement with bouzid et al; prevalence using cg: 19.8%32 and fiseha et al; prevalence using cg: 23.8%.33 all this resembles a worldwide over/underestimation of ckd with a wide variation depending on the used approach, obesity, high age, economic problems, and dm prevalence.15,19,34 in phc scr isn’t enough for renal function table 2. serum creatinine, egfr and ckd prevalence of study sample in different age groups (n: total sample size = 800). age (yr) gender serum creatinine ckd-epi equation cg equation p values.cr mean±sd range ckd prevalence n. % egfr mean±sd range ckd prevalence n. % egfr mean±sd range ckd prevalence n. % 18-40 male no.=66 0.81±0.28 0.5-2.15 5 7.6% 119.2±24.3 41.6-161 1 1.5% 137.4±37.6 54-237.8 1 1.5% 0.076 female no.=77 0.71±0.26 0.3-1.42 10 13% 111.3±27 47.6-165.4 5 6.5% 137.1±45.9 59.7-323.1 2 2.6% 0.015 41-60 male no=209 0.9±0.3 0.5-3.9 15 7.2% 96.4±18.3 15.9-128.2 14 6.7% 100.9±25 21.04-167.4 19 9.1% <0.001 female no=221 0.74±0.23 0.34-1.6 23 10.4% 92.6±20 37.3-124.1 29 13.1% 104.6±29.3 40.8-189.7 29 13.1% <0.001 61-70 male no=130 1.07±0.43 0.54-4.-8 29 22.3% 76.8±21.5 14.1-111.9 38 29.2% 73.9±22.5 14.8-134.6 57 43.8% <0.001 female no.=97 0.94±0.34 0.47-2.33 24 24.7% 69.1±21.9 20.6-105.5 40 41.2% 71.2±23.9 26.2-129.6 52 53.6% <0.001 18-70 male no=405 0.94±0.36 0.5-4.08 49 12.1% 93.8±24.8 14.1-161 53 13.1% 98.2±34 14.8-237.8 77 19% <0.001 female no=395 0.78±0.28 0.3-2.33 57 14.4% 90.5±26.1 20.6-165.4 74 18.7% 102.8±38.8 26.2-323.1 83 21% <0.001 total n=800 0.86±0.3 0.3-4.08 106 13.3% 92.2±25.5 14.1-165.4 127 15.9% 100.4±36.5 14.8-323.1 160 20% <0.001 n.: number of subjects with specific characteristic; %: percent = (n. /no); sd: standard deviation; s.cr: serum creatinine; yr: year; egfr: estimated glomerular filtration rate in (ml/min/1.73 m2) units; ckd-epi: chronic kidney disease epidemiology equation; cg: cockcroft gault equation; abnormal s.cr: ≥1.4mg/dl in male: ≥1.2 mg/dl in female; abnormal egfr: less than 60 ml/min/1.73 m2. 37 original detecting chronic kidney disease in diabetic adultsahmed kh. mohammed almaawi j contemp med sci | vol. 7, no. 1, january-february 2021: 34 – 39 assessment as its level will keep normal till renal function drop by 50%,19,25 compared to measured gfr; estimating egfr have systematic bias but it is minimal (10–20%) in cg and ckd-epi. cg formula is affected by weight while ckd-epi is the most accurate one for diverse population.18,23 majority (61.4%) of ckd (by ckd-epi) were in patients aged 61–70 yr which is in agreement with nakata.35 this is expected as there is continuous renal function drop after age of 30 yr, reaching a ckd prevalence of 25% by age of 70.16,17 in order to study ckd provoking factors, sample was divided into two groups; with and without ckd, hypertension prevalence was higher in those with ckd, in agreement with bradshaw et al, narenpitak et al, hooi et al, and jolly et al; dm and hypertension are independent risk factors and responsible for 15% of ckd.4,30,36–39 table 3. risk factors prevalence of study sample in association with ckd (n: total sample size = 800). associated risk factor presence of ckd p. value with ckd without ckd male no.=53 female no.=74 total no.=127 male no.=352 female no.=321 total no.=673 hypertension n. % 29 45.7% 39 52.7% 68 53.5% 87 24.7% 110 34.3% 197 29.3% <0.001 heart disease n. % 4 7.5% 6 8.1% 10 7.9% 19 5.4% 19 5.9% 38 5.6% 0.332 female n. % 74 100% 74 58.3% 321 100% 673 47.7% 0.029 periphery living n. % 4 7.5% 24 32.4% 28 22% 28 8% 37 11.5% 65 9.7% <0.001 2ndary school or less n. % 40 75.5% 71 95.9% 111 87.4% 317 90.1% 299 93.1% 616 91.5% 0.138 smoking n. % 15 28.3% 3 4.1% 18 14.2% 98 27.8% 2 0.6% 100 14.9% 0.842 no income n. % 19 35.8% 63 85.1% 82 64.6% 143 40.6% 284 88.5% 472 63.4% 0.81 n.: number of subjects with specific characteristic; %: percentage= (n. /no); dm: diabetes mellitus, yr: year; sd: standard deviation; ckd: chronic kidney disease; egfr less than 60 ml/min/1.73 m2 detected by ckd-epi equation. table 4. ckd classification into different stages according to egfr level detected by ckd-epi & cg equations (n: total sample size = 800). ckd stage egfr (ml/min/1.73m2) gender ckd prevalence by ckd-epi equation n= 800 ckd prevalence by cg equation n= 800 p. value 1 >90 male n. (%) 284 (35.5%) 234 (29.3%) <0.001 female n. (%) 252 (31.5%) 246 (30.8%) 2 60-89 male n. (%) 68 (8.5%) 94 (11.8%) <0.001 female n. (%) 69 (8.6%) 66 (8.3%) 3 30-59 male n. (%) 51 (6.4%) 75 (9.4%) <0.001 female n. (%) 71 (8.9%) 81 (10.1%) 4 15-29 male n. (%) 1 (0.1%) 1 (0.1%) <0.001 female n. (%) 3 (0.4%) 2 (0.3%) 5 <15 male n. (%) 1 (0.1%) 1 (0.1%) female n. (%) 0 (0%) 0 (0%) 3-5 <60 male n. (%) 53 (6.6%) 77 (9.6%) 0.001 female n. (%) 74 (9.3%) 83 (10.4%) ckd-epi: chronic kidney disease epidemiology equation; cg: cockcroft gault equation; egfr: estimated glomerular filtration rate; n.: number of subjects with characteristic; %: percent= (n. /n). 38 original detecting chronic kidney disease in diabetic adults ahmed kh. mohammed almaawi j contemp med sci | vol. 7, no. 1, january-february 2021: 34 – 39 heart disease prevalence was higher in those with ckd in agreement with bradshaw et al and (jolly et al; cardiovascular risk increase as egfr drop, death related to heart disease was 2–3 times in those with stage 3 and 4 ckd, respectively, than that with normal kidney function.4,21,36,38 those with ckd are more likely to be unemployed with no income which is in agreement with bradshaw et al. females’ percent was higher in those with ckd which is in agreement with hooi et al37; as age-related diabetic kidney disease progression differ between sexes.40 those living in periphery are more likely to have ckd; in low income countries where unaffordable costly health service, unawareness,41 unemployment and dm epidemic in iraq, making ckd a leading cause of death and lately referred ckd.36,42–44 study sample was categorized to five stages according to egfr level19 using two equations; standard ckd-epi (the most accepted index one23) and cg, comparing between above equations showed strong agreement: 85.5%, kappa: 0.73 (95% ci: 0.68–0.77, p < 0.001), 96.1% of ckd patients were with stage 3 that was higher by depending on cg which is in agreement with zaman et al that found strong agreement: 70.9%, kappa: 0.56 (95% ci: 0.44–0.67, p < 0.001) and considered high cg prevalence as overestimation compared to ckd-epi,31 and in agreement with kitiyakara et al 2012;45 that is related to continuously increasing ckd (stage 3) prevalence among adult in correlation with increasing dm, advanced age, and obesity.19 cg doesn’t take diverse people in consideration and requires weight and height which isn’t available in laboratories while ckd-epi does not; ckd-epi is better to assess ckd prevalence in diabetics despite strong agreement between them. conclusion diabetic adults especially those with risk factors are likely to develop ckd and it is better to detect ckd initially in phc and in tertiary centers by estimating gfr rather than depending on scr alone. ckd-epi formula may be better than cg to achieve that goal. recommendations – family physicians, general practitioners, and phc providers should be aware for ckd among risky patients including diabetics attending phc centers. – initial ckd screening for risky patients should be done by egfr estimation using a creatinine-based formula rather than scr alone. – family physicians are in the best position for ckd detection, initial management, and follow up in coordination with nephrologist. references 1. omran a. the epidemiologic transition: a theory of the epidemiology of population change. milbank q 2005:83(4);731–757. available at: https:// www.ncbi.nlm.nih.gov/pmc/articles/pmc2690264/#. 2. stenvinkel p. chronic kidney disease: a public health priority and harbinger of premature cardiovascular disease. j int med 2010;268:456–467. available at: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2796.2010.02269. 3. alwan a. chronic non-communicable diseases. in: health in iraq: the current situation, our vision for the future and areas of work, 2nd ed. bgd (iraq): al-adib press; 2004;31-40. available online at: https://www.who.int/ hac/crises/irq/background/iraq_health_in_iraq_second_edition.pdf?ua=1. 4. bakris g, ritz e. the message for world kidney day 2009: hypertension and kidney disease— a marriage that should be prevented. j hum hypertens 2009;23:222–225. available at: https://www.nature.com/articles/ jhh2008169. 5. pearson er, mccrimmon rj. diabetes mellitus. in: walker br, colledge nr, ralston sh et al. davidson’s principles &practice of medicine, 22nd ed. edinburgh: elsevier; 2014:797-836. 6. powers ac, niswender kd, evans-molina c. diabetes mellitus: diagnosis, classification and pathology. in: jameson jl, fauci as, kasper dl et al, harrison’s principles of internal medicine, 20thed. new york: mcgrow-hill education; 2018:2850-2859. 7. world health organization. country and regional data on diabetes: who eastern mediterranean region [internet] .geneva, switzerland; 2020 [cited 2020 aug 25]. available at: https://www.who.int/diabetes/facts/world_ figures/en/index2.html. 8. al-obaidi s, abdulrazaq m, muhammed s et al. diagnosis of diabetes mellitus in patients with tuberculosis. in: iraqi moh national tuberculosis program. tuberculosis in patients with diabetes mellitus: a guide of management, 1st ed. iraq: moh; 2019:2-3: 7-8. available at: http://phd.iq/lionimages/ pdfstore/tuberculosis%20in%20patients%20with%20dm%20final.pdf 9. powers ac, stafford jm, rickels mr. diabetes mellitus: complications. in: jameson jl, fauci as, kasper dl et al, harrison’s principles of internal medicine, 20th ed. new york: mcgrow-hill education; 2018:2875-2883. 10. crandall j, shamoon h. diabetes mellitus. in: goldman l, shafer ai, goldman-cesil medicine, 25th ed. philadelphia: elsevier; 2016:1527-1548. 11. bargman jm, skorecki kl. chronic kidney disease. in: jameson jl, fauci as, kasper dl, et al, harrison’s principles of internal medicine, 20th ed. new york: mcgrow-hill education; 2018:2111-2121. table 5. ckd staging by using ckd-epi compared to cg equations (n= 800) (kappa value: 0.73, 95% confidence interval (ci): 0.68 0.77, p < 0.001). ckd staging by ckd-epi n (%) total 1 2 3 4 5 ckd staging by cg n (%) 1 472 (59%) 8 (1%) 0 (0%) 0 (0%) 0 (0%) 480 (60%) 2 64 (8%) 91 (11.4%) 5 (0.6%) 0 (0%) 0 (0%) 160 (20%) 3 0 (0%) 38 (4.8%) 117 (14.6%) 1 (0.1%) 0 (0%) 156 (19.5%) 4 0 (0%) 0 (0%) 0 (0%) 3 (0.4%) 0 (0%) 3 (0.4%) 5 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (0.1%) 1 (0.1%) total 536 (67%) 137 (17.1%) 122 (15.2%) 4 (0.5%) 1 (0.1%) 800 (100%) ckd-epi: chronic kidney disease epidemiology equation; cg: cockcroft gault equation; egfr: estimated glomerular filtration rate; n: number of subjects with specific character; %: percent= (n /n). https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2690264/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2690264/ https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2796.2010.02269 https://www.who.int/hac/crises/irq/background/iraq_health_in_iraq_second_edition.pdf?ua=1 https://www.who.int/hac/crises/irq/background/iraq_health_in_iraq_second_edition.pdf?ua=1 https://www.nature.com/articles/jhh2008169 https://www.nature.com/articles/jhh2008169 39 original detecting chronic kidney disease in diabetic adultsahmed kh. mohammed almaawi j contemp med sci | vol. 7, no. 1, january-february 2021: 34 – 39 12. gale e, anderson j. diabetes mellitus. in: kumar p, clark m, kumar & clark’s clinical medicine, 9th ed. edinburgh: elsevier; 2017:1241-1276. 13. alicic r, rooney m, tuttle k. diabetic kidney disease: challenges, progress, and possibilities. clin j am soc nephrol 2017;12:2032-2045. available at: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5718284/. 14. karalliedde j, viberti g. microalbuminuria and cardiovascular risk. ajh 2004;17:986–993. available at: https://academic.oup.com/ajh/ article/17/10/986/107224. 15. alaini a, malhotra d, rondon-berrios h, et al. establishing the presence or absence of chronic kidney disease: uses and limitations of formulas estimating the glomerular filtration rate. world j methodol 2017;7(3):73-92. available at: http://dx.doi.org/10.5662/wjm.v7.i3.73. 16. grill a, brimble s. approach to the detection and management of chronic kidney disease: what primary care providers need to know. can fam physician 2018;64(10):728-735. available at: https://www.ncbi.nlm.nih.gov/ pmc/articles/pmc6184972/. 17. upadhyay a, inker l, levey a. chronic kidney disease: definition, classification and approach to management. in: turner n, lameire n, goldsmith d, et al. oxford textbook of clinical nephrology, 4th ed. glasgow: bell &bain; 2016:743-755. 18. levey a. becker c, inker, l. glomerular filtration rate and albuminuria for detection and staging of acute and chronic kidney disease in adults: a systematic review. jama 2015;313(8):837–846. available at: https://www. ncbi.nlm.nih.gov/pmc/articles/pmc4410363/. 19. mitch w. chronic kidney disease. in: goldman l, shafer a, goldman-cesil medicine, 25th ed. philadelphia: elsevier; 2016:833-841. 20. berl t, henrich, w. kidney-heart interactions: epidemiology, pathogenesis, and treatment. clin j am society nephrol 2006;1:8–18. available at: https:// cjasn.asnjournals.org/content/1/1/8. 21. goldsmith d. cardiovascular disease and chronic kidney disease in the developed world. in: turner n, lameire n, goldsmith d, et al. oxford textbook of clinical nephrology, 4th ed. glasgow: bell & bain; 2016:777-780. 22. yaqoob m, ashman n. kidney and urinary tract disease. in: kumar p, clark m, kumar & clark’s clinical medicine, 9th ed. edinburgh: elsevier; 2017:723-794. 23. levey a, stevens l, schmid c et al. a new equation to estimate glomerular filtration rate. ann intern med 2009;150(9):604-612. available at: https:// www.ncbi.nlm.nih.gov/pmc/articles/pmc2763564/. 24. ravera, m, noberasco g, weiss u, et al. ckd awareness and blood pressure control in the primary care hypertensive population. am j kidney dis 2010;57(1):71–77. available at: https://www.ajkd.org/article/s02726386(10)01395-1/fulltext. 25. mcfarlane p, cherney d, gilbert r, et al. chronic kidney disease in diabetes. cjd 2018;42:201–209. available at: https://www.canadianjournalofdiabetes. com/article/s1499-2671(17)30985-1/fulltext. 26. komenda p, ferguson t, macdonald k, et al. cost-effectiveness of primary screening for ckd: a systematic review. am j kidney dis 2014;63(5):789-797. available at: https://www.ajkd.org/article/s0272-6386(14)00024-9/fulltext. 27. du bois d, du bois e. a formula to estimate approximate surface area if height and weight be known. arch int. med 1917;17:863-871. available at: https://www.scirp.org/(s(i43dyn45teexjx455qlt3d2q))/reference/ referencespapers.aspx?referenceid=1332732. 28. levey as, atkins r, coresh j, et al. chronic kidney disease as a global public health problem: approaches and initiatives – a position statement from kidney disease improving global outcomes. kidney int 2007;72(3):247–259. available at: https://www.kidney-international.org/article/s00852538(15)52649-5/fulltext. 29. stevens l, fares g, fleming j, et al. low rates of testing and diagnostic codes usage in a commercial clinical laboratory: evidence for lack of physician awareness of chronic kidney disease. jasn 2005;16(8):2439–2448. available at: https://jasn.asnjournals.org/content/16/8/2439. 30. narenpitak s, narenpitak a. prevalence of chronic kidney disease in type 2 diabetes in primary health care unit of udon thani province, thailand. j med assoc thai 2008;91(10):1505-1513. available at: http://citeseerx.ist.psu.edu/ viewdoc/download?doi=10.1.1.463.7206&rep=rep1&type=pdf. 31. zaman s. detection of chronic kidney disease by using different equations of glomerular filtration rate in patients with type 2 diabetes mellitus: a cross-sectional analysis. cureus 2017;9(6):e1352. available at: https://www. ncbi.nlm.nih.gov/pmc/articles/pmc5510968/. 32. bouzid c, smida h, kacem a, et al. renal failure in tunisian patients with type 2 diabetes: frequency and related factors. la tunisie med 2011;89(1):10–15. available at: https://www.latunisiemedicale.com/articlemedicale-tunisie_1502_en. 33. fiseha t, kassim m, yemane t. chronic kidney disease and under diagnosis of renal insufficiency among diabetic patients attending a hospital in southern ethiopia. bmc nephrol 2014;15:198. available at: https:// bmcnephrol.biomedcentral.com/track/pdf/10.1186/1471-2369-15-198. 34. de boer i, rue t, hall y, et al. temporal trends in the prevalence of diabetic kidney disease in the united states. jama 2011;305(24):2532–2539. available at: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3731378/. 35. nakata j, ohsawa i, onda k. et al. risk of overestimation of kidney function using gfr‐estimating equations in patients with low insulin clearance. j clin lab anal 2012;26(4):248-253. available at: https://www.ncbi.nlm.nih.gov/ pmc/articles/pmc6807624/. 36. bradshaw c, kondal d, montez-rath m, et al. early detection of chronic kidney disease in low-income and middle-income countries: development and validation of a point-of-care screening strategy for india. bmjgh 2019;4(5):e001644. available at: https://gh.bmj.com/content/4/5/e001644. long. 37. hooi ls, ong lm, ahmad g., et al. a population-based study measuring the prevalence of chronic kidney disease among adults in west malaysia. kidney int 2013;84(5):1034–1040. available at: https://www.kidney-international. org/article/s0085-2538(15)56071-7/fulltext. 38. jolly s, navaneethan s, schold j, et al. chronic kidney disease in electronic health record problem list: quality of care, esrd, and mortality. am j nephrology 2014;39:288–296. available at: https://www.karger.com/ article/fulltext/360306 39. kazancioglu r. risk factors for chronic kidney disease: an update. kidney int suppl 2013;3(4):368–371. available at: https://www.ncbi.nlm.nih.gov/pmc/ articles/pmc4089662/. 40. hemmelgarn b, zhang j, manns b, et al. progression of kidney dysfunction in community-dwelling elderly. kidney int. 2006;69(12):2155–2161. available at: https://www.kidney-international.org/article/s00852538(15)51433-6/fulltext. 41. ene-iordache b, perico n, bikbov b, et al. chronic kidney disease and cardiovascular risk in six regions of the world (isn-kddc): a cross-sectional study. lancet glob health 2016;4(5):307–319. available at: https://www. thelancet.com/journals/langlo/article/piis2214-109x(16)00071-1/fulltext 42. parameswaran s, geda s, rathi m, et al. referral pattern of patients with end-stage renal disease at a public sector hospital and its impact on outcome. natl med j india 2011;24(4):208–213. available at: https:// pubmed.ncbi.nlm.nih.gov/22208139/. 43. george c, mogueo a, okpechi i, et al. chronic kidney disease in low-income to middle-income countries: the case for increased screening. bmj glob health 2017;2:e000256. available at: https://gh.bmj.com/content/2/2/ e000256. 44. mansour a, douri f. diabetes in iraq: facing the epidemic. a systematic review. wulfenia 2015;22(3):258-273. available at:https://www.researchgate. net/publication/280084146_diabetes_in_iraq_facing_the_epidemic_a_ systematic_review. 45. kitiyakara c, yamwong s, vathesatogkit p, et al. the impact of different gfr estimating equations on the prevalence of ckd and risk groups in a southeast asian cohort using the new kdigo guidelines. bmc nephrol 2012;13:1. [cited 2020 aug 25]. available at: https://bmcnephrol. biomedcentral.com/articles/10.1186/1471-2369-13-1. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.926 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5718284/ https://academic.oup.com http://dx.doi.org/10.5662/wjm.v7.i3.73 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6184972/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6184972/ https://cjasn.asnjournals.org/content/1/1/8 https://cjasn.asnjournals.org/content/1/1/8 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2763564/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2763564/ https://www.ajkd.org/article/s0272-6386(10)01395-1/fulltext https://www.ajkd.org/article/s0272-6386(10)01395-1/fulltext https://jasn.asnjournals.org/content/16/8/2439 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5510968/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5510968/ https://www.latunisiemedicale.com/article-medicale-tunisie_1502_en https://www.latunisiemedicale.com/article-medicale-tunisie_1502_en https://bmcnephrol.biomedcentral.com/track/pdf/10.1186/1471-2369-15-198 https://bmcnephrol.biomedcentral.com/track/pdf/10.1186/1471-2369-15-198 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6807624/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6807624/ https://www.kidney-international.org/article/s0085-2538(15)56071-7/fulltext https://www.kidney-international.org/article/s0085-2538(15)56071-7/fulltext https://www.karger.com/article/fulltext/360306 https://www.karger.com/article/fulltext/360306 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4089662/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4089662/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4089662/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4089662/ https://www.kidney-international.org/article/s0085-2538(15)51433-6/fulltext https://www.kidney-international.org/article/s0085-2538(15)51433-6/fulltext https://www.thelancet.com/journals/langlo/article/piis2214-109x(16)00071-1/fulltext https://www.thelancet.com/journals/langlo/article/piis2214-109x(16)00071-1/fulltext https://pubmed.ncbi.nlm.nih.gov/22208139/ https://pubmed.ncbi.nlm.nih.gov/22208139/ https://gh.bmj https://bmcnephrol.biomedcentral.com/articles/10.1186/1471-2369-13-1 https://bmcnephrol.biomedcentral.com/articles/10.1186/1471-2369-13-1 140 j contemp med sci | vol. 7, no. 3, may-june 2021: 140–144 original eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds armin s. hariri1, sevda shayesteh2, parina asgharian3, mohsen chamanara4,5, maryam-sadat sadrzadeh-afshar6* 1oral and maxillofacial medicine department, faculty of dentistry, aja university of medical sciences, tehran, iran 2 department of pharmacology and toxicology, faculty of pharmacy, alborz university of medical sciences, karaj, iran 3department of pharmacognosy, faculty of pharmacy, tabriz university of medical sciences, tabriz, iran 4department of pharmacology, faculty of medicine, aja university of medical sciences, tehran, iran 5 toxicology research center, aja university of medical sciences, tehran, iran 6oral and maxillofacial medicine department, faculty of dentistry, aja university of medical sciences, tehran, iran *correspondence to: maryam-sadat sadrzadeh-afshar, m_sadrzade@alumnus.tums.ac.ir (submitted: 07 april 2021 – revised version received: 18 april 2021 – accepted: 27 april 2021 – published online: 26 june 2021) introduction gingiva is the first tissue destructed in periodontal diseases and gingivitis leads to oral cavity complications if remained untreated. the healing process includes 4 main phases hemostasis, inflammation, proliferation and remodeling; and proliferation and migration of fibroblasts, play crucial role in healing process.1 despite a rapid healing process, the presence of bacteria in oral cavity accounts for the complicated wounds accompanied with inflammation.2 chronic increased levels of pro-inflammatory factors such as interleukin (il-1β) and tumor necrosis factor (tnf-a) activates apoptotic pathways in fibroblasts as well as inducing the production of il-6 which leads to a positive inflammatory cycle.3,4 in addition, inflammation diminishes the migration and proliferation of the cells, resulting in delayed wound healing.5 moreover, enhanced inflammatory response leads to loss of connective tissue attachments and alveolar bone destruction.6 on the other hand, the administration of anti-inflammatory drugs such as corticosteroids, decelerate the healing process by reducing the proliferation rate of the fibroblasts as well as decreasing collagen and glycosaminoglycan synthesis.7 therefore, investigating anti-inflammatory agents not impairing the healing process is of great importance for gingiva wounds treatment. eremostachys binalodensis is an iranian endemic specie of eremostachys genus from lamiaceae family. it is mostly grown in middle-east and west asia, having thick roots.8 several bioactive compounds have been isolated from the eremostachys species including: terpenoids, monoand sesqui tepenes, linear and branched hydrocarbons and derivatives.9 this plant was topically used for wound healing of snake bites and rheumatism joint pains.10 the rhizomes, rich in iridoid glycosides, are known for analgesic effects.11 in addition, the anti-inflammatory effects of eremostachys rhizomes have been reported in allergies and auto-immune diseases.12 it has been suggested that, attenuating the prostaglandins formation is responsible for eremostachys anti-inflammatory effects.13 besides mentioned properties, the anti-bacterial effects of eremostachys on escherichia coli and staphylococcus aureus species suggests it for oral wounds and infections which have not been studied yet.14 taking this information into consideration, the purpose of this study was to evaluate the effectiveness of e. binalodensis on gingival wound healing through altering fibroblast proliferation and secretion of three important inflammatory cytokines. materials and methods e. binalodensis extract preparation the rhizomes of e. binalodensis were collected on july from binalud mountains, mashhad iran [(3 36° 17’ 60.00” n) latitude (58° 32’ 60.00” e) longitude and altitude 1950 m above sea level]. a voucher specimen has been deposited in the herbarium of the faculty of pharmacy, tabriz university of medical sciences, iran under the accession code tbz-fph 4033. air-dried rhizomes of e. binalodensis (100 g each) were extracted with methanol. all obtained extracts were separately concentrated using a rotary evaporator at a maximum temperature of 45°c. cell culture human gingival fibroblasts (hgfs) (hgf1-pi 1) were obtained from pasteur institute, tehran, iran. cells were cultured in abstract objectives in this study we aimed to evaluate the effect of e. binalodensis on gingival inflammatory wounds. materials and methods in-vitro wound was induced by scratching the surface layer of human gingival fibroblasts (hgfs). cells were retreated with 1,10,100,1000 µg/ml of e. binalodensis methanol extract prior to 1 µg/ml lps stimulation. hgfs proliferation was assessed by mtt test. also levels of critical inflammatory cytokines such as il-1b, il-6 and tnf-a were determined by enzyme-linked immunosorbent assay (elisa). results wound induction was associated with secretion of il-1β, il-6 and tnf-a from hgfs. e. binalodensis enhanced the hgfs proliferation besides reducing the level of il-1β, il-6 and tnf-a in lps-scratch-stimulated hgfs. conclusions regarding anti-inflammatory and proliferative effects of e. binalodensis on hgfs, availability and safety of it, it is suggested for enhancing the wound healing process in gingival inflammatory wounds. key words eremostachys, gingivitis, fibroblast, inflammation, wound issn 2413-0516 141j contemp med sci | vol. 7, no. 3, may-june 2021: 140–144 a. s. hariri et al. original eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds dulbecco’s modified eagle medium (sigma-aldrich, germany) including 10% fetal bovine serum, 2 mm l-glutamine, 100 mm l-ascorbate-2-phosphate, 50 u/ml streptomycin, 50 u/ml penicillin and 1 mm sodium pyruvate.15 cells were seeded (1 ´ 105 cells/well) in 24-well plates and incubated for 24 hours in 37 ˚c, 5% co2 and 95% humidity. in-vitro wound and lps induction in-vitro wound was induced by scrapping the 2-3 mm of surface layer of hgfs creating a scratch to the cells followed by mentioned incubation method.16 in order to induce inflammation, 1 µg/ml bacterial endotoxin (lipopolysaccharides (lps)) was added 1 hour after e. binalodensis extract application on cells. experimental groups first, the effective concentration of e. binalodensis on hgfs’ proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (mtt) test. 1, 10, 100 and 1000 µg/ml of the methanolic e. binalodensis extracts in 1% dimethyl sulfoxide (dmso) were selected for mtt test. regarding the results of mtt test, the work was performed in five groups: 1. control, 2. scratch, 3. scratch + extract, 4. scratch + lps, 5. scratch + lps + extract. groups 1, 2, 4 were pretreated with the same amount of dmso. extract was performed 1 hour prior to lps and scratch and cells were incubated for 48 hours. mtt assay the effect of e. binalodensis on hgfs proliferation was assessed by mtt test. mtt assay is based on the reduction of mtt to formazan crystals by mitochondria of viable cells.17 first, cells which were 48 hours incubated with 1, 10, 100 and 1000 µg/ml of the methanolic extracts of e. binalodensis, were seeded at a density of 1 × 104 cells/well in a 96-well plate. second, the medium was removed and 10 µl mtt was added to the wells and incubated for 4 hours. after observing formazan crystals, 200 µl dmso was added to dissolve the crystals. finally, the absorbance of the plate was read at 450 nm by spectrophotometer (specord 250, analytik jena), cell viability was expressed relative to the control group which was regarded as 100%. the test was repeated three times. elisa assay of inflammatory markers in order to evaluate the main inflammatory markers involved in periodontal diseases, the protein levels of il-1β, il-6 and tnf-a in culture supernatants were measured by enzymelinked immunosorbent assay (elisa). human il-1β elisa kit (ab100562), human tnf alpha elisa kit (ab181421), human il-6 elisa kit (ab46027) were purchased. for this purpose, 50 µl of cells were added to a 96-well plate. 50 µl of the antibody cocktail, regarding the type of measured marker, was added to each well and incubated for 2 hours on a plate shaker with 400 rpm. after washing the plate regarding the kit instruction, 100 µl 3,3',5,5'-tetramethylbenzidine (tmb) development solution was added and incubated. finally, after washing and adding the stop solution, the plate absorbance was read at 450 nm by spectrophotometer. the levels of il-1b, l-6 and tnf-a in cell culture supernatants, expressed as pg/ml, were quantified based on each corresponding standard curve. each experiment was repeated three times. statistical analysis the data was analyzed by graphpad prism 6 through one-way analysis of variance with post-hoc tukey tests. in this study, p < 0.05 was considered a significant difference. results effect of e. binalodensis on hgfs proliferation effects of e. binalodensis on hgfs proliferation was evaluated by mtt assay. as shown in figure 1, the cell viability did not change by 1 and 10 µg/ml of extract; however, 100 and 1000 µg/ml e. binalodensis extract enhanced the hgfs proliferation significantly (p < 0.001, p < 0.0001). the results illustrated the proliferative effect of e. binalodensis on hgfs. considering mtt test results, 100 µg/ml of e.binalodensis, the lowest proliferative concentration in this research, was selected for further elisa tests. effect of e. binalodensis on production of il-1β in lps-stimulated hgfs as presented in figure 2, scratch induction raised the il-1β significantly compared to control group (p < 0.01). also, lps application (scratch + lps group) induced 1l-1β significantly in comparison with scratch group (p < 0.0001). however, e. binalodensis administration significantly reduced the il-1β level in comparison with scratch + lps group (p < 0.0001). effect of e. binalodensis on production of il-6 in lps-stimulated hgfs regarding figure 3, il-6 elevated in scratch group compared to control group (p < 0.05). in addition, e. binalodensis extract application reduced the il-6 level in comparison with scratch fig. 1 the effect of e. binalodensis on hgfs proliferation. e: eremostachys binalodensis. (*** p < 0.001, **** p < 0.0001 compared to control group). data are presented as mean ± sd. fig. 2 effect of e. binalodensis on production of il-1β in lps stimulated hgfs. data are presented as mean ± sem. (**p < 0.01 compared to control group, ####p < 0.0001 compared to scratch group, &&&&p < 0.0001 compared to scratch + lps group). 142 j contemp med sci | vol. 7, no. 3, may-june 2021: 140–144 eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds original a. s. hariri et al. group (p < 0.05). lps induction (scratch + lps group), raised the il-6 level significantly compared to scratch group (p < 0.0001). in contrast, e. binalodensis extract administration significantly decreased the il-6 level compared to scratch + lps group (p < 0.0001). effect of e. binalodensis on production of tnf-a in lps-stimulated hgfs as shown in figure 4, tnf-a significantly increased in scratch-applied group in comparison with control group (p < 0.05, p < 0.0001). in group receiving e. binalodensis extract, tnf-a level declined significantly in comparison with scratch group (p < 0.05). similarly, lps administration (scratch + lps group) induced tnf-a compared to scratch group (p < 0.0001). contrarily, e. binalodensis extract administration reduced the tnf-a level when compared to scratch + lps group (p < 0.0001). discussion in the present study, we evaluated the possible effect of e. binalodensis on gingival inflammatory wounds. gingival wounds, leading in gingivitis, are the first stage of periodontal diseases. in comparison with dermal or mucosal wounds, gingival wounds are thought to be more complicated due to presence of microbial plaques, low accessibility of mouthwashes and antimicrobial agents to the bacteria and ph variations.18 the untreated gingivitis can lead to periodontal disease accompanied with sever medical conditions such as cardiovascular disease, diabetes, and adverse pregnancy outcomes.19 inflammation is regarded as the first stage of healing process of gingival wounds followed by fibroblast-mediated tissue formation and remodeling.20 it starts with infiltration of leukocytes to the wound site, resulting in pathogen combating, tissue degradation and regeneration.21 however, excessive inflammatory response diminishes mitogenic cell activity and tissue remodeling.22 in this study, it was observed that il-1β was elevated in scratched fibroblasts, confirming fibroblasts as one of the major sources of il-1β. it has been reported that, besides fibroblasts the accumulated neutrophils post-injury, produce pro-inflammatory cytokines such as il-1β and tnf-a.23 lps or pathogen-activated toll-like receptors induce nuclear factor kappa b (nf-kb) expression which results in pro il-1β production.24 il-1β increases neutrophil infiltration and triggers inflammatory cascades in wound area. also, elevated levels of il-1β induces prostaglandin e2 formation, together inducing collagenase which leads to periodontal attachment loss.25 moreover, il-1β stimulated matrix metalloproteinases, contribute in extracellular matrix degradation and tissue destruction.26 in contrast, our results showed that e. binalodensis markedly decreases the scratch + lps induced il-1β level. it has been reported that there is a strong relationship between increasing gingival cervical fluid levels of il-1β and severity of gingivitis and blocking il-1β activity or reducing it by anti-inflammatory agents, have improving effects on gingivitis.27,28 similarly, in this research tnf-a was elevated postscratch in fibroblasts. tnf-a is involved in late steps of wound healing, shifting the cells to tissue remodeling phase; however, increased levels of it results in tissue damaging by impairing fibroblast activity and stimulating osteoclastogenesis.29 also tnf-a induces fibroblast apoptosis through foxo signaling pathway.30 although increase of tnf-a level was observed following scratch + lps application, pretreatment with e.binalodensis attenuated the tnf-a rise in fibroblasts. regarding the synergistic effects of il-1β and tnf-a, it has been reported that administration of antagonists of both cytokines has more beneficial effects on gingivitis.27 moreover, the level of il-6, one of the predictors of periodontal disease initiation, was evaluated in this study. the results showed that, both scratch and lps induction increases the il-6 level. it is suggested that, enhanced levels of il-1β and tnf-a are involved in stimulating fibroblast-secretion of il-6 through mitogen-activated protein kinase (mapk) pathway, resulting in positive inflammatory feedback.31 il-6 is highly associated with the pocket depth and severity of gingivitis.32 also the differentiation of cd4 to t cells is impaired by high levels of il-6, resulting in reduced bacterial inhibition in oral cavity.33 in addition, il-6, known as stimulator of osteoclasts, is responsible for bone resorption followed by gingivitis.34 our results showed that e.binalodensis reduced the il-6 level post scratch as well as post lps application. similarly, it has been shown that, agents reducing il-6, decrease bone loss in inflamed gingival mucosa. also tocilizumab (il-6 receptor inhibitor) administration have been reported to improve gingivitis and reduce bleeding sites.35 therefore, e. binalodensis inhibits inflammation-induced complications by reducing the secretion of three critical pro-inflammatory cytokines. the potential anti-bacterial effects of eremostachys genius, have been reported in previous studies.14 besides inhibiting inflammation, anti-bacterial effects of e. binalodensis may enhance the gingival wound healing process. fig. 3 effect of e. binalodensis on production of il-6 in lps stimulated hgfs. data are presented as mean ± sem. (*p < 0.05 compared to control group, #p < 0.05 and ####p < 0.0001 compared to scratch group, &&&&p < 0.0001 compared to scratch + lps group). fig. 4 effect of e. binalodensis on production of tnf-a in lps stimulated hgfs. data are presented as mean ± sem. (**p < 0.01 compared to control group, ##p < 0.05 and ####p < 0.0001 compared to scratch group, &&&&p < 0.0001 compared to scratch + lps group). 143j contemp med sci | vol. 7, no. 3, may-june 2021: 140–144 a. s. hariri et al. original eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds the second stage in reformation of periodontal tissue centrally involves fibroblasts, which generate and organize the collagen fibers that attach the alveolar bone and gingiva to the cementum covering the tooth root.36 in this study, it was observed that e.binalodensis extract, not only reduces the inflammatory cytokines, but also enhances the fibroblast proliferation. this is the remarkable feature of proposing e. binalodensis for gingival wounds. the increased rate of gingival fibroblast proliferation, results in enhanced wound healing process. our results are similar to liao et al. study indicating that, traditional medicine inhibiting il-6 and inducing fibroblast proliferation are suggested for gingival wound healing.37 the treatment choices for gingivitis include anti-inflammatory drugs which usually impair fibroblast activation.7 regarding the crucial role of fibroblasts in gingival wound healing, it is of a great importance to administer agents that prevent inflammation without attenuating fibroblast proliferation and activity. regarding the previous studies, it is suggested that the anti-inflammatory effects of e. binalodensis is related to the presence of iridoid glycosides in this plant.13 taken together, it can be concluded that, the methanol extract of e. binalodensis has improving effects on inflammatory gingival wounds. also availability of this plant and previously confirmed safety of it, reduces the cost of treatment besides enhancing the healing process. further in-vivo investigations can be applied for evaluating the effects of e. binalodensis in details. funding sources this study was supported by aja university of medical sciences (grant:97001404). acknowledgment the authors would like to thank sara laboratory (tabriz, iran) and pharmacognosy department of tabriz university of medical sciences for their contribution in this study. conflicts of interest the authors have no conflicts of interest to disclose.  references 1. smith pc, cáceres m, martínez c, oyarzún a, martínez j. gingival wound healing: an essential response disturbed by aging? journal of dental research. 2015;94:395–402. 2. bhattacharya r, xu f, dong g, li s, tian c, ponugoti b, et al. effect of bacteria on the wound healing behavior of oral epithelial cells. plos one. 2014;9:e89475. 3. palmqvist p, lundberg p, lundgren i, hänström l, lerner u. il-1β and tnf-a regulate il-6-type cytokines in gingival fibroblasts. journal of dental research. 2008;87:558–63. 4. shirasugi m, nishioka k, yamamoto t, nakaya t, kanamura n. normal human gingival fibroblasts undergo cytostasis and apoptosis after longterm exposure to butyric acid. biochemical and biophysical research communications. 2017;482:1122–8. 5. basso fg, soares d, pansani t, turrioni a, scheffel d, de souza costa c, et al. effect of lps treatment on the viability and chemokine synthesis by epithelial cells and gingival fibroblasts. archives of oral biology. 2015;60:1117–21. 6. taylor jj. protein biomarkers of periodontitis in saliva. international scholarly research notices. 2014;2014. 7. roques c, téot l. the use of corticosteroids to treat keloids: a review. the international journal of lower extremity wounds. 2008;7:137–45. 8. mozaffarian v. a dictionary of iranian plant names. tehran: farhang moaser. 1996;396. 9. asgharian p, delazar a, asnaashari s. chemical constituents of eremostachys macrophylla montbr. & auch. aerial parts. pharmaceutical sciences. 2020;26:203–8. 10. mosaddegh m, naghibi f, moazzeni h, pirani a, esmaeili s. ethnobotanical survey of herbal remedies traditionally used in kohghiluyeh va boyer ahmad province of iran. journal of ethnopharmacology. 2012;141:80–95. 11. khan s, nisar m, simjee su, rehman w, khan r, jan i, et al. evaluation of micronutrients level and antinociceptive property of eremostachys laciniata (l) bunge. african journal of biotechnology. 2010;9. 12. delazar a, sarker sd, nahar l, jalali sb, modaresi m, hamedeyazdan s, et al. rhizomes of eremostachys laciniata: isolation and structure elucidation of chemical constituents and a clinical trial on inflammatory diseases. advanced pharmaceutical bulletin. 2013;3:385. 13. khan s, nisar m, rehman w, khan r, nasir f. anti-inflammatory study on crude methanol extract and different fractions of eremostachys laciniata. pharmaceutical biology. 2010;48:1115–8. 14. modaressi m, delazar a, nazemiyeh h, fathi-azad f, smith e, rahman mm, et al. antibacterial iridoid glucosides from eremostachys laciniata. phytotherapy research: an international journal devoted to pharmacological and toxicological evaluation of natural product derivatives. 2009;23:99–103. 15. xiong g, ji w, wang f, zhang f, xue p, cheng m, et al. quercetin inhibits inflammatory response induced by lps from porphyromonas gingivalis in human gingival fibroblasts via suppressing nf-κb signaling pathway. biomed research international. 2019;2019. 16. weinreb m, nemcovsky ce. in vitro models for evaluation of periodontal wound healing/regeneration. periodontology 2000. 2015;68:41–54. 17. xiong g, ji w, wang f, zhang f, xue p, cheng m, et al. quercetin inhibits inflammatory response induced by lps from porphyromonas gingivalis in human gingival fibroblasts via suppressing nf-κb signaling pathway. biomed research international. 2019. 18. politis c, schoenaers j, jacobs r, agbaje jo. wound healing problems in the mouth. frontiers in physiology. 2016;7:507. 19. nazir ma. prevalence of periodontal disease, its association with systemic diseases and prevention. international journal of health sciences. 2017;11:72. 20. ahangar p, mills sj, smith le, gronthos s, cowin aj. human gingival fibroblast secretome accelerates wound healing through anti-inflammatory and proangiogenic mechanisms. npj regenerative medicine. 2020;5:1–10. 21. polimeni g, xiropaidis av, wikesjö um. biology and principles of periodontal wound healing/regeneration. periodontology 2000. 2006;4:30–47. 22. chiquet m, katsaros c, kletsas d. multiple functions of gingival and mucoperiosteal fibroblasts in oral wound healing and repair. periodontology 2000. 2015;68:21–40. 23. schett g, dayer j-m, manger b. interleukin-1 function and role in rheumatic disease. nature reviews rheumatology. 2016;12:14. 24. cheng r, wu z, li m, shao m, hu t. interleukin-1β is a potential therapeutic target for periodontitis: a narrative review. international journal of oral science. 2020;12:1–9. 25. reinhardt ra, masada mp, kaldahl wb, dubois lm, kornman ks, choi ji, et al. gingival fluid il-1 and il-6 levels in refractory periodontitis. journal of clinical periodontology. 1993;20:225–31. 26. kobayashi m, squires gr, mousa a, tanzer m, zukor dj, antoniou j, et al. role of interleukin-1 and tumor necrosis factor a in matrix degradation of human osteoarthritic cartilage. arthritis & rheumatism: official journal of the american college of rheumatology. 2005;52:128–35. 27. zhang x, kohli m, zhou q, graves dt, amar s. short-and long-term effects of il-1 and tnf antagonists on periodontal wound healing. the journal of immunology. 2004;173:3514–23. 28. oh h, hirano j, takai h, ogata y. effects of initial periodontal therapy on interleukin-1β level in gingival crevicular fluid and clinical periodontal parameters. journal of oral science. 2015;57:67–71. 29. nathan c. points of control in inflammation. nature. 2002;420:846–52. 30. declercq w, denecker g, fiers w, vandenabeele p. cooperation of both tnf receptors in inducing apoptosis: involvement of the tnf receptorassociated factor binding domain of the tnf receptor 75. the journal of immunology. 1998;161:390–9. 31. xiao w, hodge dr, wang l, yang x, zhang x, farrar wl. nf-kappab activates il-6 expression through cooperation with c-jun and il6-ap1 144 j contemp med sci | vol. 7, no. 3, may-june 2021: 140–144 eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds original a. s. hariri et al. site, but is independent of its il6-nfkappab regulatory site in autocrine human multiple myeloma cells. cancer biology & therapy. 2004;3:1007–17. 32. johannsen a, rydmark i, söder b, åsberg m. gingival inflammation, increased periodontal pocket depth and elevated interleukin-6 in gingival crevicular fluid of depressed women on long-term sick leave. journal of periodontal research. 2007;42:546–52. 33. lin s-j, chen y-l, kuo my-b, li c-l, lu h-k. measurement of gp130 cytokines–oncostatin m and il-6 in gingival crevicular fluid of patients with chronic periodontitis. cytokine. 2005;30:160–7. 34. choi dh, moon is, choi bk, paik jw, kim ys, choi sh, et al. effects of sub-antimicrobial dose doxycycline therapy on crevicular fluid mmp-8, and gingival tissue mmp-9, timp-1 and il-6 levels in chronic periodontitis. journal of periodontal research. 2004;39:20–6. 35. ancuța c, chirieac r, ancuța e, țănculescu o, solomon sm, fătu am, et al. exploring the role of interleukin-6 receptor inhibitor tocilizumab in patients with active rheumatoid arthritis and periodontal disease. journal of clinical medicine. 2021;10:878. 36. smith pc, martínez c, martínez j, mcculloch ca. role of fibroblast populations in periodontal wound healing and tissue remodeling. frontiers in physiology. 2019;10:270. 37. liao j, azelmat j, zhao l, yoshioka m, hinode d, grenier d. the kampo medicine rokumigan possesses antibiofilm, anti-inflammatory, and wound healing properties. biomed research international. 2014;2014. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. doi: https://doi.org/10.22317/jcms.v7i3.976 306 j contemp med sci | vol. 6, no. 6, november–december 2020: 306–312 original issn 2413-0516 introduction liver cancer is the sixth most common cancer in the worldwide.1 the mechanisms of hepatocarcinogenesis are not fully understood; however, the theory of cancer stem cells (cscs) has recently gained traction as a potential contributor to hepatic cancer. cscs display great plasticity and self-renewal potential and play a decisive role in tumor formation and growth.2 these cells are highly drug-resistant and metastatic, which may underlie the recurrence and drug resistance of liver cancer.3, 4 therefore, the identification of liver cscs markers and therapeutic targets associated with them is necessary for improving treatment outcomes.5, 6 biomarker is defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” biomarkers play an important role in the early diagnosis of diseases.7, 8 microarray technology has revolutionized gene expression analysis and has been used widely for the identification of cancer biomarkers. the use of high-throughput techniques has resulted in an exponential growth in the amount of information available in biomedical databases, which then can be exploited to integrate gene expression data for applications such as biomarker discovery, disease classification, or phenotype comparisons, among others.9 however, these types of data are characterized by high dimensionality as the number of genes is far bigger than the number of samples. one of the biggest challenges of high-dimensional data are the curse of dimensionality, which describes the exponential increase in volume associated with adding extra dimensions in the euclidean space. it is responsible for the breakdown of the optimal statistical model fitting.10 to address the problem, researchers have applied various machine learning methods to reduce both cardinality and redundancy of gene expression data during the classification process, and most of these methods were developed to facilitate the analysis of microarray data to identify the best discriminative genes or biomarkers.11 extreme gradient boosting (xgboost) is a machine learning algorithm that assigns an importance score to each feature in the training phase, and these scores can then be used as the basis for identification of importance features. since it uses multiple subsets of features to predict outcomes in the area of dimension cursed problem ensemble models may have better performance. moreover, xgboost is an optimized distributed gradient boosting that achieves state-of-the-art prediction performances.12 feature importance ranking using the common tree ensemble models such as xgboost and gbm r packages may provide inconsistent results. these methods only consider the effect of splits along the decision path. therefore, when the model relies more on a given feature, the importance assigned to that feature changes incorrectly.13 regarding model interpretation, which is especially important when using machine learning models that are often difficult to interpret, several studies have used shapley additive explanations (shap).14-16 proposed by lundberg and lee, 17 shap is based on game theory18 and local explanations19 that offers a means to estimate the contribution of each feature. shap provides a consistent importance value, which is an alternative to permutation feature importance.20 in the present study, we applied machine learning to gene expression data from previous studies on liver cscs to identify putative biomarkers for identification and characterization of these cells. potential biomarker detection for liver cancer stem cell by machine learning approach ali farzane1, maryam akbarzadeh2, reza ferdousi3, mohammadreza rashidi4, reza safdari1 1department of health information management, school of allied medical sciences, tehran university of medical sciences, tehran, iran. 2department of biochemistry, erasmus university medical center, rotterdam, netherlands. 3department of health information technology, school of management and medical informatics, tabriz university of medical sciences, daneshgah st, tabriz, iran. 4stem cell and regenerative medicine institute, tabriz university of medical sciences, tabriz, iran. corresponding author: reza safdari (e-mail: rsafdari@tums.ac.ir ) (submitted: 05 october 2020 – revised version received: 19 october 2020 – accepted: 27 october 2020 – published online: 30 december 2020) abstract objectives: in this study, we aimed to identify putative biomarkers for identification and characterization of these cells in liver cancer. methods: we employed a supervised machine learning method, xgboost, to data from 13 geo data series to classify samples using gene expression data. results: across the 376 samples [129 cancer stem cell, cscs and 247 non-cscs cases], xgboost displayed high performance in the classification of data. xgboost feature importance scores and shap (shapley additive explanation) values were used for the interpretation of results and analysis of individual gene importance. we confirmed that expression levels of a 10-gene set (ptger3, aurkb, c15orf40, idi2, or8d1, naca2, serpinb6, l1cam, smc1a, and rasgrf1) were predictive. the results showed that these 10 genes can detect cscs robustly with accuracy, sensitivity, and specificity of 97%, 100%, and 95%, respectively. conclusions. we suggest that the 10-gene set may be used as a biomarker set for detecting and characterizing cscs using gene expression data. key words: liver cancer, cancer stem cell, machine learning, biomarker, gene expression 307 original potential biomarker detection for liver cancer stem cell by machine learning approachali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–312 materials & methods data preparation the study was approved by the ethics committee at tehran university of medical sciences, tehran, iran (ethics code: ir.tums.rec.1394.1589). since we used non-identifiable information from a publicly available data set, no specific consent was required. datasets gene expression data for samples of liver csc and non-csc were downloaded from geo through accession numbers gse56771, gse59713, gse66529, gse84223, gse68778, gse126121, gse112788, gse66515, gse42318, gse131680, gse103866, and gse62905, and a data series including only non-csc samples were downloaded via accession number gse112790. the first nine of the above data series were expression profiling by array, while the remaining four were high-throughput sequencing (hiseq). we excluded samples that had undergone intervention. finally, a total of 129 csc and 247 non-csc samples were included in the study. preprocessing r language was used for all processing steps, including preprocessing, modeling, and pathway analysis. the dropout effect was eliminated in the hiseq series. we performed log transformation on data series, followed by quartile normalization where it was needed. next, the ensembl database (https:// www.ensembl.org/index.html) was used to convert ids from different data series to ensembl ids. data series were integrated using the merge function, and then the combat function from the sva r package was applied to remove batch effects. finally, we had 8409 common transcript genes across all data series. xgboost model for biomarker signature identification data classification was performed using xgboost, which is an efficient implementation of the gradient boosting framework proposed by chen and guestrin.21 gradient boosted decision tree is an ensemble learning method based on sequential decision trees whereby each decision tree learns from the previous tree to improve the model and build a strong learner. model tuning in xgboost, several parameters need to be selected to maximize model performance. however, the multiplicity of parameters may result in a model learning noises and random fluctuations and considering them meaningful, a phenomenon referred to as overfitting. overfitting is a modeling error that occurs when a function is too closely fit to a limited set of data points.22 parameter tuning is an essential step in avoiding overfitting or undue complexity. the hyperparameters adopted in this study were “nrounds,” “eta,” “min_child_weight,” “max_ depth,” “gamma,” “colsample_bytree,” “subsample,” “lambda,” and “alpha.”20 the parameter “nrounds” is the number of trees that are fitted in the model. the “eta” parameter refers to the learning rate, which is used to make the model more robust. the “min_ child_weight” is the minimum sum of instance weight needed in a child. if the tree partition step results in a leaf node with the sum of instance weight less than “min_child_weight,” the building process will stop further partitioning.21 the “max_depth” parameter defines the maximum number of partitions, with greater maximum depth increasing the risk of overfitting. the minimum loss reduction required to make a further partition on a leaf node of the tree is defined as “gamma,” with a larger “gamma” resulting in a more conservative algorithm. the “subsample” parameter refers to the fraction of observations randomly selected for the training instances, which is inversely related to overfitting. another parameter useful in avoiding overfitting is “colsample_bytree.” finally, the parameters “lambda” and “alpha” are l2 and l1 regularization terms, respectively, that keep the weights small, thus preventing overfitting. the random search method was used for model tuning. random search means that hyperparameters are randomly picked from the predefined searching domain uniformly and the searching does not depend on the previous boosting result. it has been shown to be efficient for problems with high dimensions in some studies.23 model evaluation in this study, accuracy, sensitivity, and specificity were assessed to evaluate model performance. they are defined in equations (1)–(3). the goal is to develop a model with high accuracy, sensitivity, and specificity. 307 original potential biomarker detection for liver cancer stem cell by machine learning approachali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–313 materials & methods data preparation the study was approved by the ethics committee at tehran university of medical sciences, tehran, iran (ethics code: ir.tums.rec.1394.1589). since we used non-identifiable information from a publicly available data set, no specific consent was required. datasets gene expression data for samples of liver csc and non-csc were downloaded from geo through accession numbers gse56771, gse59713, gse66529, gse84223, gse68778, gse126121, gse112788, gse66515, gse42318, gse131680, gse103866, and gse62905, and a data series including only non-csc samples were downloaded via accession number gse112790. the first nine of the above data series were expression profiling by array, while the remaining four were high-throughput sequencing (hiseq). we excluded samples that had undergone intervention. finally, a total of 129 csc and 247 non-csc samples were included in the study. preprocessing r language was used for all processing steps, including preprocessing, modeling, and pathway analysis. the dropout effect was eliminated in the hiseq series. we performed log transformation on data series, followed by quartile normalization where it was needed. next, the ensembl database (https:// www.ensembl.org/index.html) was used to convert ids from different data series to ensembl ids. data series were integrated using the merge function, and then the combat function from the sva r package was applied to remove batch effects. finally, we had 8409 common transcript genes across all data series. xgboost model for biomarker signature identification data classification was performed using xgboost, which is an efficient implementation of the gradient boosting framework proposed by chen and guestrin.21 gradient boosted decision tree is an ensemble learning method based on sequential decision trees whereby each decision tree learns from the previous tree to improve the model and build a strong learner. model tuning in xgboost, several parameters need to be selected to maximize model performance. however, the multiplicity of parameters may result in a model learning noises and random fluctuations and considering them meaningful, a phenomenon referred to as overfitting. overfitting is a modeling error that occurs when a function is too closely fit to a limited set of data points.22 parameter tuning is an essential step in avoiding overfitting or undue complexity. the hyperparameters adopted in this study were “nrounds,” “eta,” “min_child_weight,” “max_ depth,” “gamma,” “colsample_bytree,” “subsample,” “lambda,” and “alpha.”20 the parameter “nrounds” is the number of trees that are fitted in the model. the “eta” parameter refers to the learning rate, which is used to make the model more robust. the “min_ child_weight” is the minimum sum of instance weight needed in a child. if the tree partition step results in a leaf node with the sum of instance weight less than “min_child_weight,” the building process will stop further partitioning.21 the “max_depth” parameter defines the maximum number of partitions, with greater maximum depth increasing the risk of overfitting. the minimum loss reduction required to make a further partition on a leaf node of the tree is defined as “gamma,” with a larger “gamma” resulting in a more conservative algorithm. the “subsample” parameter refers to the fraction of observations randomly selected for the training instances, which is inversely related to overfitting. another parameter useful in avoiding overfitting is “colsample_bytree.” finally, the parameters “lambda” and “alpha” are l2 and l1 regularization terms, respectively, that keep the weights small, thus preventing overfitting. the random search method was used for model tuning. random search means that hyperparameters are randomly picked from the predefined searching domain uniformly and the searching does not depend on the previous boosting result. it has been shown to be efficient for problems with high dimensions in some studies.23 model evaluation in this study, accuracy, sensitivity, and specificity were assessed to evaluate model performance. they are defined in equations (1)–(3). the goal is to develop a model with high accuracy, sensitivity, and specificity. accuracy true positive true negative total sample = +∑ ∑ ∑ ( )1 sensitivity true positive predicted positive = ( )∑ ∑ 2 specificity true negative predictednegative = ∑ ∑ ( )3 gene selection two gene importance ranking lists obtained from xgboost and shap values were considered for the selection of candidate genes. we selected the 10 highest-ranking genes common to both lists as the marker genes. in xgboost, feature importance is measured using three metrics, namely, gain, cover, and frequency. gain is the contribution of a feature to the accuracy of the branches on them it is located. cover measures the relative quantity of observations concerned by a feature. frequency is a simpler way to measure the gain. it just counts the number of times a feature is used in all generated trees. we used the gain score to create a ranked list of genes.24 the second ranking list was created with shap, which explains the prediction of an instance x by computing the contribution of each feature to the prediction. the shapley value for a feature j is the feature’s contribution to the prediction, weighted and summed over all possible feature value combinations that determined through formula 4: gene selection two gene importance ranking lists obtained from xgboost and shap values were considered for the selection of candidate genes. we selected the 10 highest-ranking genes common to both lists as the marker genes. in xgboost, feature importance is measured using three metrics, namely, gain, cover, and frequency. gain is the contribution of a feature to the accuracy of the branches on them it is located. cover measures the relative quantity of observations concerned by a feature. frequency is a simpler way to measure the gain. it just counts the number of times a feature is used in all generated trees. we used the gain score to create a ranked list of genes.24 the second ranking list was created with shap, which explains the prediction of an instance x by computing the contribution of each feature to the prediction. the shapley value for a feature j is the feature’s contribution to the prediction, weighted and summed over all possible feature value combinations that determined through formula 4: 308 original potential biomarker detection for liver cancer stem cell by machine learning approach ali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–312308 original potential biomarker detection for liver cancer stem cell by machine learning approach ali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–313 ∅ = − −( ) { }( )− ( )( ) ⊆ … ∑ ∪j s x x x i p i s p s p v s x v s { , , }\{ } ! ! ! ( ) 1 1 4 shap values can be used as feature importance scores: features with large absolute shapley values are important. since we wanted global importance, we averaged the absolute shapley values per feature across the data (formula 5): i j i n j i = ∅ = ( )∑ 1 5| | ( ) shap feature importance is an alternative to permutation feature importance. however, while permutation feature importance is based on the decrease in model performance, shap is based on the magnitude of feature attributions.25 enrichment analysis pathway analysis for the top 10 genes was carried out using reactome, an open-source, open access, manually curated, and peer-reviewed pathway database. reactome provides intuitive bioinformatics tools for the visualization, interpretation, and analysis of pathway knowledge to support basic and clinical research, genome analysis, modeling, systems biology, and education. gene ontology analysis was performed with enrichr web-based tools and services. results data preparation datasets. in this study, our aim was to screen and mine for specific biomarkers for liver cscs using the online available data. at the first stage, we obtained the gene expression profile of liver cscs cell line and tissue including 376 samples (129 liver cscs and 247 non-cscs) from published data in the geo database. preprocessing. the id for each data series, which was captured from its own platforms annotation file, was mapped to an ensembl id via the biomart package in r to unify the datasets. then, the data series were merged, followed by a batch correction accomplished using the combat function from the sva package. the distributions of expression values before and after the batch effect correction for the combined datasets are shown in fig. 1a–f. the difference in gene expression distribution between cscs (fig. 1a) and non-csc samples was also magnified after batch effect correction (fig. 1b). the quantile–quantile (q–q) plots (fig. 1c–d) revealed a decrease in the distance between dots and the normal distribution line after removal of the batch effect. finally, the pca plots display the batch effect due to the integration of data from various studies (fig. 1e), which has been resolved after applying the batch effect correction (fig. 1f ). therefore, the gene expression data sets would be reliable for the subsequent analysis after batch effect correction. xgboost model for a biomarker signature model tuning. the hyperparameters adopted in this study were “eta,” “nrounds,” “max_depth,” “gamma,” “lambda,” “alpha,” “min_child_weight,” “subsample,” and “colsample_ bytree.” table 1 presents the search domains and optimal values for the hyperparameters. gene selection and models evaluation. we randomly selected 65% of the data to train xgboost and used the remaining 35% to test the model. then, we employed a sevenfold cross-validation process to assess model performance stability. fig. 2a displays the results of cross-validation for the three performance measures, i.e., accuracy, sensitivity, and specificity. the obtained accuracy was 88.68–94.45, sensitivity 86.68–94.11, and specificity 87.89–94.87. overall, these indicators are significantly high and suggest that xgboost can be used to model cell classification. xgboost was finally retrained on the 75% of the training set and tested on the 25% of the testing set. the final performance indicators achieved were as follows: accuracy: 90%, sensitivity: 94%, and specificity: 89%, which is again indicative of significantly high performance. for gene ranking, we created 1000 models using the same hyperparameters, with each model assigning an importance score to each gene. then, the median of the 1000 scores for each gene was computed to obtain the average score for the gene. we re-ranked these genes based on shap values and the gain scores. the 10 top-ranking genes remained the same in both rankings. we select these genes as a potential biomarker set. the biomarker genes were ptger3, aurkb, c15orf40, idi2, or8d1, naca2, serpinb6, l1cam, smc1a, and rasgrf1. to see if the selected marker genes could serve as “universal” markers for cell classification using gene expression data, we trained the xgboost model using only the selected marker genes. interestingly, the selected genes did reasonably better than all-gene modeling. the performance indicators achieved were as follows: accuracy: 97%, sensitivity: 100%, and specificity: 95% (fig. 2b). we also calculated the shap value for each marker gene (fig. 3). enrichment analysis we further tested the top 10 genes for enriched gene ontology terms by the enrichr web service and analysis tools of the reactome website for pathways (fig. 4). results a total of 57 were recruited in this study. of 57 children with g6pd deficiency, 17 (29.8%) were diagnosed as severe cases depending on the level of hemoglobin at time of presentation to the emergency room in children welfare teaching hospital. the total number is 57 patients. the mean age is 4.35 years, males were constituting 85.9%, 32 patients were living in urban area mostly from baghdad, two-thirds of the patients have recorded ingestion of fresh type of fava beans (64.9%), while one-third ingested dried type (35%). ten patients were already diagnosed with g6pd prior to the presentation; six of them were diagnosed based on previous episodes of hemolysis, and four depending on routine assessment for a suspected family member. analysis of the demographic and clinical characteristics of the 57 patients according to the severity of hemolysis (mild vs severe) and the results of the independent samples t-test (for continuous variables) and chi square (for categorical variables) are represented in table 1. younger age group patients tend to present with the severe form of hemolysis (3.59 years with a p value of 0.001). no significant gender susceptibility shap values can be used as feature importance scores: features with large absolute shapley values are important. since we wanted global importance, we averaged the absolute shapley values per feature across the data (formula 5): 308 original potential biomarker detection for liver cancer stem cell by machine learning approach ali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–313 ∅ = − −( ) { }( )− ( )( ) ⊆ … ∑ ∪j s x x x i p i s p s p v s x v s { , , }\{ } ! ! ! ( ) 1 1 4 shap values can be used as feature importance scores: features with large absolute shapley values are important. since we wanted global importance, we averaged the absolute shapley values per feature across the data (formula 5): i j i n j i = ∅ = ( )∑ 1 5| | ( ) shap feature importance is an alternative to permutation feature importance. however, while permutation feature importance is based on the decrease in model performance, shap is based on the magnitude of feature attributions.25 enrichment analysis pathway analysis for the top 10 genes was carried out using reactome, an open-source, open access, manually curated, and peer-reviewed pathway database. reactome provides intuitive bioinformatics tools for the visualization, interpretation, and analysis of pathway knowledge to support basic and clinical research, genome analysis, modeling, systems biology, and education. gene ontology analysis was performed with enrichr web-based tools and services. results data preparation datasets. in this study, our aim was to screen and mine for specific biomarkers for liver cscs using the online available data. at the first stage, we obtained the gene expression profile of liver cscs cell line and tissue including 376 samples (129 liver cscs and 247 non-cscs) from published data in the geo database. preprocessing. the id for each data series, which was captured from its own platforms annotation file, was mapped to an ensembl id via the biomart package in r to unify the datasets. then, the data series were merged, followed by a batch correction accomplished using the combat function from the sva package. the distributions of expression values before and after the batch effect correction for the combined datasets are shown in fig. 1a–f. the difference in gene expression distribution between cscs (fig. 1a) and non-csc samples was also magnified after batch effect correction (fig. 1b). the quantile–quantile (q–q) plots (fig. 1c–d) revealed a decrease in the distance between dots and the normal distribution line after removal of the batch effect. finally, the pca plots display the batch effect due to the integration of data from various studies (fig. 1e), which has been resolved after applying the batch effect correction (fig. 1f ). therefore, the gene expression data sets would be reliable for the subsequent analysis after batch effect correction. xgboost model for a biomarker signature model tuning. the hyperparameters adopted in this study were “eta,” “nrounds,” “max_depth,” “gamma,” “lambda,” “alpha,” “min_child_weight,” “subsample,” and “colsample_ bytree.” table 1 presents the search domains and optimal values for the hyperparameters. gene selection and models evaluation. we randomly selected 65% of the data to train xgboost and used the remaining 35% to test the model. then, we employed a sevenfold cross-validation process to assess model performance stability. fig. 2a displays the results of cross-validation for the three performance measures, i.e., accuracy, sensitivity, and specificity. the obtained accuracy was 88.68–94.45, sensitivity 86.68–94.11, and specificity 87.89–94.87. overall, these indicators are significantly high and suggest that xgboost can be used to model cell classification. xgboost was finally retrained on the 75% of the training set and tested on the 25% of the testing set. the final performance indicators achieved were as follows: accuracy: 90%, sensitivity: 94%, and specificity: 89%, which is again indicative of significantly high performance. for gene ranking, we created 1000 models using the same hyperparameters, with each model assigning an importance score to each gene. then, the median of the 1000 scores for each gene was computed to obtain the average score for the gene. we re-ranked these genes based on shap values and the gain scores. the 10 top-ranking genes remained the same in both rankings. we select these genes as a potential biomarker set. the biomarker genes were ptger3, aurkb, c15orf40, idi2, or8d1, naca2, serpinb6, l1cam, smc1a, and rasgrf1. to see if the selected marker genes could serve as “universal” markers for cell classification using gene expression data, we trained the xgboost model using only the selected marker genes. interestingly, the selected genes did reasonably better than all-gene modeling. the performance indicators achieved were as follows: accuracy: 97%, sensitivity: 100%, and specificity: 95% (fig. 2b). we also calculated the shap value for each marker gene (fig. 3). enrichment analysis we further tested the top 10 genes for enriched gene ontology terms by the enrichr web service and analysis tools of the reactome website for pathways (fig. 4). results a total of 57 were recruited in this study. of 57 children with g6pd deficiency, 17 (29.8%) were diagnosed as severe cases depending on the level of hemoglobin at time of presentation to the emergency room in children welfare teaching hospital. the total number is 57 patients. the mean age is 4.35 years, males were constituting 85.9%, 32 patients were living in urban area mostly from baghdad, two-thirds of the patients have recorded ingestion of fresh type of fava beans (64.9%), while one-third ingested dried type (35%). ten patients were already diagnosed with g6pd prior to the presentation; six of them were diagnosed based on previous episodes of hemolysis, and four depending on routine assessment for a suspected family member. analysis of the demographic and clinical characteristics of the 57 patients according to the severity of hemolysis (mild vs severe) and the results of the independent samples t-test (for continuous variables) and chi square (for categorical variables) are represented in table 1. younger age group patients tend to present with the severe form of hemolysis (3.59 years with a p value of 0.001). no significant gender susceptibility shap feature importance is an alternative to permutation feature importance. however, while permutation feature importance is based on the decrease in model performance, shap is based on the magnitude of feature attributions.25 enrichment analysis pathway analysis for the top 10 genes was carried out using reactome, an open-source, open access, manually curated, and peer-reviewed pathway database. reactome provides intuitive bioinformatics tools for the visualization, interpretation, and analysis of pathway knowledge to support basic and clinical research, genome analysis, modeling, systems biology, and education. gene ontology analysis was performed with enrichr web-based tools and services. results data preparation datasets. in this study, our aim was to screen and mine for specific biomarkers for liver cscs using the online available data. at the first stage, we obtained the gene expression profile of liver cscs cell line and tissue including 376 samples (129 liver cscs and 247 non-cscs) from published data in the geo database. preprocessing. the id for each data series, which was captured from its own platforms annotation file, was mapped to an ensembl id via the biomart package in r to unify the datasets. then, the data series were merged, followed by a batch correction accomplished using the combat function from the sva package. the distributions of expression values before and after the batch effect correction for the combined datasets are shown in fig. 1a–f. the difference in gene expression distribution between cscs (fig. 1a) and non-csc samples was also magnified after batch effect correction (fig. 1b). the quantile–quantile (q–q) plots (fig. 1c–d) revealed a decrease in the distance between dots and the normal distribution line after removal of the batch effect. finally, the pca plots display the batch effect due to the integration of data from various studies (fig. 1e), which has been resolved after applying the batch effect correction (fig. 1f ). therefore, the gene expression data sets would be reliable for the subsequent analysis after batch effect correction. xgboost model for a biomarker signature model tuning. the hyperparameters adopted in this study were “eta,” “nrounds,” “max_depth,” “gamma,” “lambda,” “alpha,” “min_child_weight,” “subsample,” and “colsample_ bytree.” table 1 presents the search domains and optimal values for the hyperparameters. gene selection and models evaluation. we randomly selected 65% of the data to train xgboost and used the remaining 35% to test the model. then, we employed a sevenfold cross-validation process to assess model performance stability. fig. 2a displays the results of cross-validation for the three performance measures, i.e., accuracy, sensitivity, and specificity. the obtained accuracy was 88.68–94.45, sensitivity 86.68–94.11, and specificity 87.89–94.87. overall, these indicators are significantly high and suggest that xgboost can be used to model cell classification. xgboost was finally retrained on the 75% of the training set and tested on the 25% of the testing set. the final performance indicators achieved were as follows: accuracy: 90%, sensitivity: 94%, and specificity: 89%, which is again indicative of significantly high performance. for gene ranking, we created 1000 models using the same hyperparameters, with each model assigning an importance score to each gene. then, the median of the 1000 scores for each gene was computed to obtain the average score for the gene. we re-ranked these genes based on shap values and the gain scores. the 10 top-ranking genes remained the same in both rankings. we select these genes as a potential biomarker set. the biomarker genes were ptger3, aurkb, c15orf40, idi2, or8d1, naca2, serpinb6, l1cam, smc1a, and rasgrf1. to see if the selected marker genes could serve as “universal” markers for cell classification using gene expression data, we trained the xgboost model using only the selected marker genes. interestingly, the selected genes did reasonably better than all-gene modeling. the performance indicators achieved were as follows: accuracy: 97%, sensitivity: 100%, and specificity: 95% (fig. 2b). we also calculated the shap value for each marker gene (fig. 3). enrichment analysis we further tested the top 10 genes for enriched gene ontology terms by the enrichr web service and analysis tools of the reactome website for pathways (fig. 4). discussion liver cancer is a leading global health issue associated with high morbidity and mortality rate.26 in recent years, cscs have been reported to make important contributions to tumor recurrence, progression, and therapeutic resistance. therefore, therapeutic targeting of liver stem cells is necessary.5 in this study, we integrated data series from geo to identify potential biomarkers for liver cscs via machine learning classification-based gene selection. one application of integrated gene expression is biomarker discovery. the integration of data from multiple studies increases the sample size by incorporating samples from different cohorts, increasing the statistical power and the robustness of the results. however, it should be mentioned that increasing the sample size reduces the gene count in the integrated data set, resulting in information loss.9, 27, 28 thirteen (13) data series from geo were integrated producing a total of 385 samples in two groups (cscs and noncscs). batch correction was conducted with combat of the sva r package, which is frequently used in this area.10, 29-32 we used xgboost for cell type prediction, as it offers high prediction accuracy and has stronger interpretability owing to its state-of-the-art algorithms. because of these advantages, 309 original potential biomarker detection for liver cancer stem cell by machine learning approachali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–312 researchers are increasingly using xgboost in biomarker discovery.12, 33, 34 to improve the prediction performance, we tuned hyperparameters using random search, which is more efficient than either a traditional manual or grid search and evaluates more of the search space, especially when the search space has more than three dimensions.35 as we did not have an external data set to evaluate our model, we performed a sevenfold cross-validation with accuracy as the overall metric and sensitivity and specificity as the class-specific metrics. in our model, the values for the three metrics indicate high performance at both training and testing stages, suggesting that xgboost can effectively distinguish the two classes.12, 20 table 1. tuned hyperparameter and searching domain in xgboost. name domain transformation function optimal hyperparameter value eta [0.01 , 0.10] 0.088 nrounds [100 , 1000] 475 max_depth [4 , 10] 5 gamma [-1 , 0] f(x) = 10x 0.62 lambda [-1 , 1] f(x) = 10x 1.59 alpha [-1 , 1] f(x) = 10x 1.08 min_child_weight [1 , 12] 3.28 subsample [0.5 , 1] 0.61 colsample_bytree [0.5 , 1] 0.85 fig 1. the density, q–q, and pca plots for evaluating the effect of the batch removal method on overall data. (a) the density plot before batch effect removal. (b) the density plot after batch effect removal (c) the q–q plot before batch effect removal. (d) the q–q plot after batch effect removal. (e) the pca plot before batch effect removal. (f ) the pca plot after batch effect removal. the dashed line in the density plot represents cscs samples, and the continuous line represents non-cscs samples. n, the gene number in the combined data set. 309 original potential biomarker detection for liver cancer stem cell by machine learning approachali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–313 table 1. tuned hyperparameter and searching domain in xgboost. name domain transformation function optimal hyperparameter value eta [0.01 , 0.10] 0.088 nrounds [100 , 1000] 475 max_depth [4 , 10] 5 gamma [-1 , 0] f(x) = 10x 0.62 lambda [-1 , 1] f(x) = 10x 1.59 alpha [-1 , 1] f(x) = 10x 1.08 min_child_weight [1 , 12] 3.28 subsample [0.5 , 1] 0.61 colsample_bytree [0.5 , 1] 0.85 fig 1. the density, q–q, and pca plots for evaluating the effect of the batch removal method on overall data. (a) the density plot before batch effect removal. (b) the density plot after batch effect removal (c) the q–q plot before batch effect removal. (d) the q–q plot after batch effect removal. (e) the pca plot before batch effect removal. (f ) the pca plot after batch effect removal. the dashed line in the density plot represents cscs samples, and the continuous line represents non-cscs samples. n, the gene number in the combined data set. 310 original potential biomarker detection for liver cancer stem cell by machine learning approach ali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–312 fig. 2. models performance presentation. (a) the xgboost model’s sevenfold cross-validation plot. (b) selected gene xgboost model roc generates an auc value greater than that achieved using all-gene xgboost. fig. 3. shap summary plot. contribution of each gene to model (xgboost with top 10 genes) output. 311 original potential biomarker detection for liver cancer stem cell by machine learning approachali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–312 xgboost assigns an importance score for each feature, which can be used for feature selection. shap values also provide a means of ranking features as well as providing a measure of prediction consistency. therefore, for more certainty, we used both xgboost importance and shap values.13, 20 the shap values and gain scores for genes were imputed, as described by riberio et al and chen et al, respectively.19, 21 we created 1000 xgboost models by the same tuning parameters and then obtained feature importance score (gain) and shap values for each of the 1000 models. finally, the median of 1000 scores was calculated for each gene. these scores are reliable and can be used as gene ranks.12 genes were ordered separately by gain score and shap value. the top 10 genes from the shap list were selected, 9 of which were also among the top 10 genes in the gain score list and the remaining 1 corresponded to the 11th gene in this list. so, the 10 highest-ranking genes from the shap list were selected as the biomarker set, including ptger3, aurkb, c15orf40, idi2, or8d1, naca2, serpinb6, l1cam, smc1a, and rasgrf1. to ensure that this gene set can be used as a biomarker set, we trained our model using only these 10 genes, which offered better prediction performance compared with all-gene models. many studies have shown that cscs have one or more abnormalities in signaling pathways that regulate cell cycle and self-renewal. the cellular pathways in which the key genes are most involved are pathways associated with cell cycle regulation.36 for example, the aurora kinase b (aurkb)protein phosphatase 1 (pp1) axis has been shown to mediate the resetting of oct4 during the cell cycle in embryonic stem cells. aurkb-pp1 axis also plays a critical role in cell cycle-dependent changes in kinetochore assembly by regulating the balance between phosphorylation and dephosphorylation of kinetochore substrates.37 smc1a has also a key role in tumor metastasis and resistance to radiation therapy. this gene is associated with cscs, epithelial-to-mesenchymal transition, and dna–damage response pathways. yadav et al demonstrated that suppression of smc1a expression reduces the self-renewal capacity of prostate cancer cells.38 ptger3 induces tumorigenesis and drug resistance in ovarian cancer.36 lmbr1 is a regulator of nuclear stemness marker bmi1 in gastrointestinal stromal tumors.39 another study has reported pfkl to play a vital role in the maintenance of csc-like phenotype in hepatocellular carcinoma.40 l1cam has been implicated in maintaining the growth and survival of cd133+ glioma cells both in vitro and in vivo and has been suggested to be a csc-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors.41 fig. 4. pathway and gene ontology analysis of selected genes. (a) selected genes-involved pathways. (b) overall view of cell cycle pathways in which the selected genes were involved. (c) selected genes involved in biological processes. (d) selected genes involved in molecular functions. (e) selected genes involved in cellular component. 312 original potential biomarker detection for liver cancer stem cell by machine learning approach ali farzane j contemp med sci | vol. 6, no. 6, november–december 2020: 306–312 conclusions these 10 key genes were found to play important roles in liver csc maintenance. it seems that aurkb is more important for controlling the stemness and may help in the treatment of liver cancer. this gene may be a therapeutic target for inhibiting liver cancer stemness characteristics. however, this conclusion is based on retrospective data, and validation of these findings warrants further biological studies. acknowledgments this work supported by the department of health information management, school of allied medical science, tehran university of medical sciences. conflict of interest the authors of this paper declare that they do not have any conflict of interest. references 1. dasgupta p, henshaw c, youlden dr, et al. global trends in incidence rates of primary adult liver cancers: a systematic review and meta-analysis. front oncol 2020;10:171. 2. bai kh, he sy, shu ll, et al. identification of cancer stem cell characteristics in liver hepatocellular carcinoma by wgcna analysis of transcriptome stemness index. cancer med 2020. 3. prasad s, ramachandran s, gupta n, et al. cancer cells stemness: a doorstep to targeted therapy. biochim biophys acta (bba)-mol basis dis 2020;1866:165424. 4. shibata m and hoque mo. targeting cancer stem cells: a strategy for effective eradication of cancer. cancers 2019; 11: 732. 5. xiang y, yang t, pang b-y, et al. the progress and prospects of putative biomarkers for liver cancer stem cells in hepatocellular carcinoma. stem cells int 2016;2016. 6. najafi m, farhood b and mortezaee k. cancer stem cells (cscs) in cancer progression and therapy. j cell physiol 2019;234:8381-8395. 7. chang n-w, dai h-j, shih y-y, et al. biomarker identification of hepatocellular carcinoma using a methodical literature mining strategy. database 2017;2017. 8. shahrjooihaghighi a, frigui h, zhang x, et al. an ensemble feature selection method for biomarker discovery. in: 2017 ieee international symposium on signal processing and information technology (isspit) 2017, pp. 416-421. ieee. 9. toro-domínguez d, martorell-marugán j, lópez-domínguez r, et al. imageo: integrative gene expression meta-analysis from geo database. bioinformatics 2019;35:880-882. 10. diegues i, vinga s and lopes mb. identification of common gene signatures in microarray and rna-sequencing data using network-based regularization. in: international work-conference on bioinformatics and biomedical engineering 2020, pp.15–26. springer. 11. shukla ak, singh p and vardhan m. a two-stage gene selection method for biomarker discovery from microarray data for cancer classification. chemomet intell lab syst 2018;183:47-58. 12. li y, umbach dm, bingham a, et al. putative biomarkers for predicting tumor sample purity based on gene expression data. bmc genom 2019;20:1-12. 13. lundberg sm and lee s-i. consistent feature attribution for tree ensembles. arxiv preprint arxiv:170606060 2017. 14. guthrie nl, carpenter j, edwards kl, et al. emergence of digital biomarkers to predict and modify treatment efficacy: machine learning study. bmj open 2019;9:e030710. 15. caly h, rabiei h, coste-mazeau p, et al. pregnancy data enable identification of relevant biomarkers and a partial prognosis of autism at birth. biorxiv 2020. 16. hathaway qa, roth sm, pinti mv, et al. machine-learning to stratify diabetic patients using novel cardiac biomarkers and integrative genomics. cardiovasc diabetol 2019;18:78. 17. lundberg sm and lee s-i. a unified approach to interpreting model predictions. in: advances in neural information processing systems 2017, pp.4765-4774. 18. štrumbelj e and kononenko i. explaining prediction models and individual predictions with feature contributions. knowl inform syst 2014;41:647-665. 19. ribeiro mt, singh s and guestrin c. “ why should i trust you?” explaining the predictions of any classifier. in: proceedings of the 22nd acm sigkdd international conference on knowledge discovery and data mining 2016, pp.1135-1144. 20. parsa ab, movahedi a, taghipour h, et al. toward safer highways, application of xgboost and shap for real-time accident detection and feature analysis. accident anal prev 2020;136:105405. 21. chen t and guestrin c. xgboost: a scalable tree boosting system. in: proceedings of the 22nd acm sigkdd international conference on knowledge discovery and data mining 2016, pp.785-794. 22. werpachowski r, györgy a and szepesvári c. detecting overfitting via adversarial examples. in: advances in neural information processing systems 2019, pp.7858-7868. 23. wang y and ni xs. a xgboost risk model via feature selection and bayesian hyper-parameter optimization. arxiv preprint arxiv:190108433 2019. 24. developers x. understand your dataset with xgboost r-project. https:// xgboost.readthedocs.io/en/latest/r-package/discoveryourdata.html (2020). 25. molnar c. interpretable machine learning. lulu com 2019. 26. wang w, smits r, hao h, et al. wnt/β-catenin signaling in liver cancers. cancers 2019;11:926. 27. toro-domínguez d, villatoro-garcía ja, martorell-marugán j, et al. a survey of gene expression meta-analysis: methods and applications. brief bioinform 2020. 28. ghosheh n, küppers-munther b, asplund a, et al. human pluripotent stem cell-derived hepatocytes show higher transcriptional correlation with adult liver tissue than with fetal liver tissue. acs omega 2020;5:4816-4827. 29. bai j, zhang x, kang x, et al. screening of core genes and pathways in breast cancer development via comprehensive analysis of multi gene expression datasets. oncol lett 2019;18:5821-5830. 30. xia l, su x, shen j, et al. anln functions as a key candidate gene in cervical cancer as determined by integrated bioinformatic analysis. cancer manage res 2018;10:663. 31. kuang y, wang y, zhai w, et al. genome-wide analysis of methylation-driven genes and identification of an eight-gene panel for prognosis prediction in breast cancer. front genet 2020;11:301. 32. guo t, ma h and zhou y. bioinformatics analysis of microarray data to identify the candidate biomarkers of lung adenocarcinoma. peerj 2019;7:e7313. 33. zhang x, li t, wang j, et al. identification of cancer-related long non-coding rnas using xgboost with high accuracy. front genet 2019;10:735. 34. ding w, chen g and shi t. integrative analysis identifies potential dna methylation biomarkers for pan-cancer diagnosis and prognosis. epigenetics 2019;14:67-80. 35. si m, xiong y, du s, et al. evaluation and calibration of a low-cost particle sensor in ambient conditions using machine-learning methods. atmos measure tech 2020;13. 36. rodriguez-aguayo c, bayraktar e, ivan c, et al. ptger3 induces ovary tumorigenesis and confers resistance to cisplatin therapy through upregulation ras-mapk/erk-ets1-elk1/cftr1 axis. ebiomedicine 2019;40:290304. 37. shin j, kim tw, kim h, et al. aurkb/pp1-mediated resetting of oct4 during the cell cycle determines the identity of embryonic stem cells. elife 2016;5:e10877. 38. yadav s, kowolik cm, lin m, et al. smc1a is associated with radioresistance in prostate cancer and acts by regulating epithelial-mesenchymal transition and cancer stem-like properties. mol carcinogen 2019;58:113-125. 39. bai c, liu x, xu j, et al. expression profiles of stemness genes in gastrointestinal stromal tumor. human pathol 2018;76:76-84. 40. lin s-h, liu t, ming x, et al. regulatory role of hexosamine biosynthetic pathway on hepatic cancer stem cell marker cd133 under low glucose conditions. scient rep 2016;6:1-10. 41. bao s, wu q, li z, et al. targeting cancer stem cells through l1cam suppresses glioma growth. cancer res 2008;68:6043-6048. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i6.898 56 j contemp med sci | vol. 2, no. 6, spring 2016: 56–58 research objectives this study was designed to investigate the correlation between serum lipid profile and lipid peroxidation as malondialdehyde (mda) in psoriatic patients. methods this case–control study was performed on 70 psoriatic patients and 30 healthy individuals as control, matched for age and sex. the blood samples were collected after 14 h fasting. the serum lipid profile was assayed using the standard kit and mda was assayed using the elisa kit. results the certain parameters, including serum triglyceride, cholesterol, low-density lipoprotein-cholesterol (ldl-c) and very low-density lipoprotein (vldl), were significantly higher in the case group compared to the controls (p < 0.05), while high-density lipoproteincholesterol (hdl-c) remained within normal limit in the patients group compared to the control, while there was a significantly higher levels in mda in case group as compared to that found in the controls (p < 0.05). conclusion these results have revealed the higher plasma level of lipid profiles in psoriatic patients. this may elevate the risk of atherosclerosis, particularly cardiovascular disorders. therefore, from the epidemiological point of view, on screening the psoriatic patients, particularly those with severe psoriasis, is recommended. keywords psoriasis, lipid profile, malondialdehyde correlation between malondialdehyde and dyslipidemia in psoriatic patients fadhil jawad al-tu′ma,a ali tariq abd al-hassan,b eman musa abed al-da,amya issn 2413-0516 adepartment of biochemistry, college of medicine, university of karbala, holy karbala, iraq. bcollege of dentistry, university of karbala, holy karbala, iraq. correspondence to fadhil jawad al-tu′ma (email: f_altoma_56@yahoo.com). (submitted: 21 january 2016 – revised version received: 3 march 2016 – accepted: 13 may 2016 – published online: 26 june 2016) introduction psoriasis is a chronic inflammatory and autoimmune disorder with unknown etiology.1 the prevalence rate of psoriasis ranges from 1 to 4.8%. although it may involve in all age groups, the mean age of its incidence is 17.8 years.1 the disease is characterised by an increased keratinocyte proliferation and alteration in the dermal and epidermal t-cells, monocytes macrophages and neutrophils.2 the increased antigen presentation by dendrites cells and their presentation to tlymphocytes lead to the following changes: t-cell activation and secretion of type1 (th1) cytokines like interferon, interleukin-2 and tumour necrosis factor alpha (tnf-α). these cytokines induce inflammatory changes in the epidermis, yielding thick, scaly plaques.3 recently, the role of tlymphocytes in pathogenesis of psoriasis and atherosclerosis has been clarified. the psoriasis has been associated with an abnormal plasma lipid metabolism and diabetes, probability related to alterations in insulin secretion and sensitivity.4 furthermore, there is an increased oxidative stress which is accompanied by a high frequency of cardiovascular disease.5 the high rate of cardiovascular events is related to the severity of the disease which occurs more frequently in patients with large areas of the body affected by psoriasis lesions.6 although hyperlipidemia is one of the cardiovascular risk factors, the findings of various studies are not consistent, and some researches even disagree with the role of hyperlipidemia in psoriasis (or the role of cardiovascular disorders in psoriasis).7–11 this study was designed to investigate the serum lipid profile of patients with various grades of psoriasis. materials and methods there is a cross-sectional study conducted over a period from march 2015 through march 2016. the samples were collected from the dermatology clinic in al-hussein teaching hospital in karbala city. the practical side of the study was performed at the laboratory of clinical chemistry department and the laboratory of the immunology department in al-hussein teaching hospital. this study included 70 patients, attending a dermatology clinic in al-hussein teaching hospital (50 males and 20 females) with an age range of 7–70 years. they were diagnosed by a dermatology specialist as having psoriasis. on the other hand, 30 apparently healthy persons (15 males and 15 females) with an age range of 9–60 years were chosen as a control group. a questionnaire was designed to obtain the information from psoriasis patients and control group. it contained the name, age, weight, height, duration of disease and site of lesion. the exclusion criteria for both the groups were: diabetes, hypertension, cardiovascular disease, smoking, history of alcohol intake, liver obstructive disease, kidney problems, connective tissue diseases, hypothyroidism, family history of hyperlipidemia and using lipid-lowering drugs, cyclosporine, corticosteroids, β-blockers, thiazide, retinoid and methotrexate. the subjects who had high-fat foods during dinner were excluded. after explaining the purpose of the study and obtaining the consent letter, the data were recorded on questionnaires for each patient. after a 14 h fasting period, 5 ml venous blood was taken in a sterile syringe in the morning from all cases and submitted to the laboratory. the serum levels of total cholesterol, triglyceride, ldl-c, hdl-c and vldl-c were measured by an enzymatic method with the standard kits made by biolabo, sa, france. in addition, the serum levels of malondialdehyde (mda) were measured by elisa method with the standard kits made by elabscience, china. the severity of psoriasis was evaluated based on the standard criteria of psoriasis. the clinical severity of the disease was determined according 57j contemp med sci | vol. 2, no. 6, spring 2016: 56–58 research correlation between mda and dyslipidemia in psoriatic patients fadhil jawad al-tu′ma et al. to the pasi score. by estimating the extent of the body surface in evolvement, the scaling in percentage and scoring the erythematic, thickening of the affected as (scalp, trunk, the lower limb and upper limb), the severity of the disease was determined. the collected data were analysed with student’s t-test to assess the difference between the two groups. a logistic regression was used for correlation, and the multi-varieties egression was used to investigate the effect of serum lipid level on the severity of psoriasis. the p values of <0.05 were considered statistically significant. results in this case–control study, 70 psoriatic patients and 30 normal individuals as the control group were enrolled for investigation. as in table 1, the total cholesterol, triglycerides, vldl-c, hdl-c and ldl-c were significantly altered (p < 0.05). table 2 depicts the levels of mda (p < 0.05) which were significantly elevated in psoriatic patients as compared to normal healthy controls. in the patient group, the serum total cholesterol, triglyceride, vldl-c, and ldl-c were significantly higher than the controls while the hdl-c level was no change (p < 0.05). in the patient group, the serum mda level was significantly higher than the controls (p < 0.05). discussion the coronary atherosclerosis is common and the prevalence is increasing. the disorders are mediated by the t-helper cytokines, such as psoriasis, are associated with an increased risk of atherosclerosis and cardiovascular events.12 it seems that the prevalence of cardiovascular events is associated with the severity of the disease and the body surface area involvement.13 one of the causes of cardiovascular diseases in psoriatic patients may be the elevation of plasma lipid and other inflammatory mediators.14 there is a contraindicating reports about the association between serum triglyceride, cholesterol, ldl-c, vldl-c and hdl-c with psoriasis; the discrepancy goes farthest, some studies indicate normal11,15 higher13,16,17 or even lower serum triglyceride levels in psoriatic patients.16 in this study, the serum triglyceride level was significantly in patients with psoriasis compared to the controls (p < 0.05). there have been controversial results on serum cholesterol level in psoriatic patients; different studies report higher,18 lower17 or even normal levels.9,19 our results indicate significantly higher serum cholesterol levels in psoriatic patients compared to controls (p < 0.05). in a numerous studies, the serum ldl-c levels in psoriatic patients are reported normal7 or higher.9 in our investigation, the serum ldl-c in the case group was higher than the control group. also in our study, the vldl-c level was higher which contrasts the other data that indicate normal range.17,19 also, the hdl-c level was nonsignificant in psoriasis patients compared with control group (p > 0.05) and inconsistent with other studies.13,17,19 the differences in the results of various studies might reflect genetic factors, lifestyle, severity of disease, daily activity and diet in each region. the causes of dyslipidemia (abnormal amount of lipids) in psoriasis may be multiple; the immune mechanisms involving il-6 and tumour necrosis factor, c-reactive protein and cellular oxidative stress may be responsible for the altered lipid metabolism.5 the increased leukocyte count is linked to cell activation, as the activated neutrophils are the important sources of oxygen metabolites, which may trigger oxidative modifications in plasma constituents and in cell membranes, which probably accounted for the significant rise in plasma lipid peroxidation along with a significantly reduced antioxidants, clearly reveals the development of an oxidative stress conditions in psoriasis.20–23 the results of this study show significantly increased plasma mda levels in psoriatic patients as compared to controls and the levels were positively correlated to disease severity, a result which supports the involvement of oxidative damage in the etiopathogenesis of psoriasis. these results are in agreement with those of other investigators.24,25 these studies suggested that the increased production of ros/rns in patients with psoriasis results in an increased lipid peroxidation with a subsequent formation of high levels of mda. however, some other investigators26 did not detect any difference in serum mda levels in psoriatic patients as compared to controls in spite of the significantly increased levels in tissues. also found nonsignificant values of plasma mda in patients with psoriasis in relation to controls, in spite of high significant values in erythrocytes.27 agreeing with our results, a higher platelet, erythrocyte, tissue, serum and plasma levels of mda and a correlation with disease severity have been reported in previous studies.28 although we did not find a correlation between levels of mda and severity of psoriasis, some previous studies of attwa et al. (2011)29 and jyothi et al. (2011)30 had reported a positive correlation between the levels of mda and the disease severity. they suggested that their results might support the proposal that serum mda level could be helpful in predicting the prognosis of psoriasis and add further support for the involvement of oxidative stress in the pathogenesis of psoriasis.  table 1. lipid profile of psoriatic and control parameters group mean ± sd range p-value tc (mg/dl) patient 212.5 ± 52.46 105.0–311.0 <0.05 control 127.3 ± 18.87 165.0–101.0 tg (mg/dl) patient 170.8 ± 62.71 403.0–80.0 <0.05 control 108.2 ± 11.56 140.0–87.0 hdl-c (mg/dl) patient 39.59 ± 7.58 68.0–25.0 >0.05 control 40.70 ± 4.10 51.0–35.0 ldl-c (mg/dl) patient 137.6 ± 47.71 235.6–34.20 <0.05 control 66.55 ± 19.52 103.2–39.0 vldl-c (mg/dl) patient 34.33 ± 12.58 16.00–80.60 <0.05 control 21.63 ± 2.313 28.0–17.40 table 2. malondialdehyde of psoriatic and control parameters group mean ± sd range p-value mda ng/ml patient 354.9 ± 112.2 31.74–497.0 <0.05 control 232.0 ± 70.35 111.0–369.0 58 j contemp med sci | vol. 2, no. 6, spring 2016: 56–58 correlation between mda and dyslipidemia in psoriatic patients research fadhil jawad al-tu′ma et al. references 1. bijlmakers mj, kanneganti sk, barker jn, trembath rc, capon f. functional analysis of the rnf114 psoriasis susceptibility gene implicates innate immune responses to double-stranded rna in disease pathogenesis. hum mol genet. 2011;15:3129–3137. doi: 10.1093/hmg/ddr215 pmid: 21571784 2. ortonne jp. recent developments in the understanding of the pathogenesis of psoriasis. br j dermatol. 1999;140:1–7. pmid: 10731127 3. kraeger jg, bowcock a. psoriasis pathophysiology: current concepts of pathogenesis. ann rheum dis. 2005;64(suppl 2):ii30–ii36. pmid: 15708932 4. shapiro j, chohen ad, david m, hotak e, chodik g, viner a, et al. the association between psoriasis, diabetes mellitus and atherosclerosis in israel: a case control study. j am acadnermatol. 2007;56:629–634. pmid: 17157411 5. gupta m, charis s, borkar m, chandankhede m. dyslipidemia and oxidative stress in patients of psoriasis. biomed res. 2011;22(2):221–224. 6. gelfand jm, neiman al, shin db, wang x, margollis dj, tormel ab. risk of myocardial infarction in patients with psoriasis. jama. 2006;296:1733–1741. pmid: 17032986 7. seckin d, tokgozoglu l. akkoya s. are lipoprotein profile and lipoprotein (a) levels altered in men with psoriasis? j am acad dermatol. 1994;31:445–449. pmid: 8077470 8. pietrzak a, lecewicz-torun b. activity of serum lipase [ec300]. and the diversity of serum lipid profile in psoriasis. j mol cat b enzym. 2006;40: 144–154. pmid: 11782673 9. piskin s, gurkok f, ekuklu g, senol m. serum lipid levels in psoriasis. yousei med j. 2003;44:24–26. pmid: 12619171 10. mallbris l, granath f, hamsten a, stahle m. psoriasis is associated with lipid abnormalities at the onset of skin disease. j am acad dermatol. 2006;54:614–621. pmid: 16546581 11. vyanik bs, ari z, onur e, gunduz k, tanulka s, durkan k. serum lipids and apolipoproteins in patients with psoriasis. clin chem lab med. 2002;40: 65–68. pmid: 11916273 12. frostegard j, ulfgren ak, nyberg p, hedin u, swedenborg j, andersson u, et al. cytokine expression in advance human atherosclerotic plaques: dominance of pro-inflammatory (th1) and macrophage-stimulating cytokines. atherosclerosis. 1999;145:33–43. pmid: 10428293 13. akhyani m, ehsani ah, robati rm, robati am. the lipid profile in psoriasis: a controlled study. j eur acad dermatol venercol. 2007;21:1330–1332. 14. javidi z, tayyebimeibodi n, nahidi y. serum lipids abnormalities and psoriasis. indian j dermatol. 2007;52:89–92. pmid: 17958837 15. reynoso-von drateln c, martínez-abundis e, balcázar-muñoz br, bustossaldaña r, gonzález-ortiz m. lipid profile, insulin secretion, and insulin sensitivity in psoriasis. j am acad dermatol. 2003;48:882–885. pmid: 12789179 16. uyanik bs, ari z, onur e, gündüz k, tanülkü s, durkan k. serum lipids and apolipoproteins in patients with psoriasis. clin chem lab med. 2002; 40:65–68. pmid: 11916273 17. bajaj dr, mahesar sm, devrajani br, iqbal mp. lipid profile in patients with psoriasis presenting at liaquat university hospital hyderabad. j pak med assoc. 2009;59:512–515. pmid: 19757693 18. fortinskaia es, torkhovskaia ti, sharapova gia, loginova tk, kliuchnikova zhi, khalilov em. features of distribution of free and esterified cholesterol in the epidermis, biological membranes and plasma lipoproteins in psoriasis. klin lab diagn. 1996;38–43. pmid: 8963558 19. farshchian m, zamanian a, farshchian m, monsef ar, mahjub h. serum lipid level in iranian patients with psoriasis. j eur acad dermatol venereol. 2007;21:802–805. pmid: 17567311 20. gornicki a, gutsze a. erythrocyte membrane fluidity changes in psoriasis: an epr study. dermatol set. 2001;27:27–30. pmid: 11457641 21. therond b, gerbaud p, dimon s. antioxidant enzymes in psoriatic fibroblasts and erythrocytes. j invest dermatol. 1996;106:1325–8. pmid: 8752678 22. santos-silva a, rebelo i, castro emb. erythrocyte damage and leukocyte activation in ischemic stroke. clin chim acta. 2002;320:29–35. pmid: 11983197 23. weiss sj. neutrophil-mediated met hemoglobin formation in the erythrocyte: the role of superoxide and hydrogen peroxide. bio chem. 1982;257:2947–53. pmid: 6277918 24. baz k, cimen my, kokturk a, yazici ac, eskandari g, ikizoglu g, et al. oxidant/ antioxidant status in patients with psoriasis. yonsei med j. 2003;44:987–990. pmid: 14703605 25. wozniak a, drewa g, krzyzynska-malinowska e, czajkowski r, protas-drozd f, mila-kierzenkowska c, et al. oxidant–antioxidant balance in patients with psoriasis. med sci monit. 2007;13:cr30–cr33. pmid: 17179907 26. yildirim m, inaloz hs, baysal v, delibas n. the role of oxidants and antioxidants in psoriasis. j eur acad dermatol venereol. 2003;17:34–36. pmid: 12602965 27. kökçam i, naziroğlu m. antioxidants and lipid peroxidation status in the blood of patients with psoriasis. clin chim acta. 1999;289:23–31. pmid: 10556650 28. bariaa aj. thesis; babylon university, college of medicine. oxidant and antioxidant status in psoriasis. 2010;26–28. 29. attwa e, swelam e. relationship between smoking-induced oxidative stress and the clinical severity of psoriasis. j eur acad dermatol venereol. 2011;25:782–787. doi: 10.1111/j.1468-3083.2010.03860.x pmid: 21039915 30. jyothi rs, govindswamy ks, gurupadappa k. psoriasis: an oxidative stress condition. j clin diagn res. 2011;5:120–121. 150 j contemp med sci | vol. 6, no. 4, july-august 2020: 150–155 original issn 2413-0516 introduction coronavirus disease of 2019 (covid-19) is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the outbreak was first identified in  wuhan, hubei,  china, in december 2019, and was recognized as a pandemic by the  world health organization  (who) on 11 march 2020.   iraq  reported its first confirmed cases on 24 february 2020, while the first case appeared in nineveh was on 22 march 2020. nineveh is iraq’s third largest and second most populated governorate, with its capital named mosul city. in iraq, until 3 july 2020, the number of cases had reached 53,708, with 2,160 deaths, and 27,912 recoveries.1-3 during the conflict between the so-called islamic state in iraq and syria (isis) group and the iraqi forces, 9 out of 13 public hospitals were damaged in nineveh, slashing healthcare capacity and the number of hospital beds by 70 percent. the reconstruction of health facilities has been extremely slow and there are still less than 1,000 hospital beds for a population of more than 3.5 million people. this is half of the internationally recognized minimum standard for health service delivery in a humanitarian context.4 looking ahead, the fear is that the pandemic will exacerbate growing inequality in nineveh society “without a doubt, the economic impact of covid-19 will plunge the most vulnerable families in nineveh deeper into poverty,” therefore, special attention should be paid on infection prevention and control (ipc) measures and people awareness of its importance.5 ipc refers to policies and procedures used to minimize the risk of spreading infections, particularly in health facilities. the purposes of ipc are to protect ourselves, our patients, and our family and community.6-7 according to current evidence, the main routes of covid-19 virus transmission are through respiratory droplets and contact.8-13 hence, in order to prevent and control infection, the centers for disease control and prevention (cdc) recommend the following: [14] 1. hand washing: hand washing is recommended to prevent the spread of the disease. the cdc recommends that people wash hands often with soap and water for at least 20 s. it further recommended using an alcohol-based hand sanitizer with at least 60% alcohol.15 2. respiratory hygiene: health organizations recommended that people cover their mouth and nose with a bent elbow or a tissue when coughing or sneezing (the tissue should then be disposed of immediately).16 3. physical distancing: (also commonly referred to as social distancing). methods include quarantines; travel restrictions; and the closing of schools, workplaces, stadiums, theatres, or shopping centers.17 in iraq, limited previous studies to document compliance of iraqi medical staff in general, and those in nineveh governorate in particular, with regards to ipc measure to any of the infectious diseases. it is speculated that due to shortage of time, lack of resources, and reduced number of staff/patients ratio, there is lack of compliance to such important measures. moreover, despite the fact that number of covid-19 cases are not too many in mosul city due to city lockdown and other measures, asymptomatic infections have been well-documented as well as the fact that incubation period is thought to infection prevention and control measures for covid-19 among medical staff in nineveh governorate, iraq zakria a kassim1, sharef w al-mulaabed2*, saif w younis3, ali a abutiheen4 1 department of medicine, mosul general hospital, mosul, iraq. 2 department of pediatrics, presbyterian medical group, new mexico, united states. 3 department of public health, mosul, iraq. 4 department of family & community medicine, college of medicine, university of kerbala, karbala, iraq. *corresponding author: sharef w al-mulaabed (e-mail: drsharefw@gmail.com) abstract: objectives: to assess the adherence to infection prevention and control (ipc) measures among medical staff working in nineveh governorate during covid-19 pandemic. methods: a cross-sectional study, using an online survey sent to different medical staff in nineveh. the questionnaire was composed of two parts, the first included demographic information, and the second covered ipc measures. survey was completed by medical staff witnessed covid-19 pandemic in nineveh governorate. results: the total sample was 412, of whom, 316 (77%) were males and 142 (35%) were physicians. overall, the percentage of staff following different ipc measures was ranging from 31% in wearing head cover, to 97% in keeping clean hands constantly. main missed points were found in respiratory hygiene, physical distancing, and self-isolation. females were more likely to apply bandages to wounds and wearing gloves before examining patients. compared to other medical staff, physicians were less compliant to washing hands, putting waste in designated places, and wearing protective cloths. those who work in hospitals were better compliant with sterilizing hands before entering home. conclusion: the adherence to ipc measures was ranging from less than one-half in wearing sterile head cover, to nearly all respondents in keeping clean hands. there is a great need to provide support as well as training in regards to ipc in nineveh governorate city. keywords: infection prevention and control, covid-19, nineveh, mosul, iraq. 151 original infection prevention and control measures for covid-19 among medical staff in ninevehzakria a kassim j contemp med sci | vol. 6, no. 4, july-august 2020: 150–155 be within 14 days following exposure.9, 18 hence, ipc measures should be taken into consideration with all patients as much as possible. this study aims to assess the attitude and adherence to ipc measures among medical staff working in nineveh governorate during covid-19 pandemic. materials and methods a cross-sectional study conducted at nineveh governorate, in which there are about 3,500,000 inhabitants. using computer application called google forms, the survey was sent online via (facebook messenger application) directed to members of different facebook groups of medical staff in nineveh. all those witnessed covid-19 pandemics in nineveh were invited to participate in the study. data collection was done during 1-week period from 6 april 2020 to 13 april 2020. the survey was especially designed to provide information on the infection prevention and control for covid-19 after thorough review of the literatures.6, 7, 14-17, 19 the questionnaire was anonymous and composed of two parts: the first covered demographic information of participants including gender, age, marital status, and educational level, site of work, and medical specialty. the second part represented the specific ipc questions consisting of 14 questions on general ipc measures, attitude about the most infected areas to work within, any suffering from lack of personal protective equipment, source of covid-19 obtaining information, and finally if the responders gain benefit from the information of survey. for the ipc questionnaire, answer options were given according to 4-point likert-type scale (1= strongly agree, 2= agree, 3= disagree, and 4= strongly disagree). for the purpose of data analysis, answers “1” and “2” were grouped as a positive answer (yes) and expressed in tables, while responses “3” and “4” were considered as (no) and analyzed accordingly. ethical approval was obtained from nineveh health directorate. the questionnaire was anonymous and did not include any data on other personal identifiers. furthermore, the participation was voluntary with assuring the confidentially of responses. data were analyzed using spss (statistical package for social sciences) software, version 20 (ibm, chicago, illinois, usa). number and percentage (%) of respondents to each ipc measure were calculated in overall sample and then compared between different groups. comparison was done using chi–squared (χ2) test. a p-value of < 0.05 was considered as statistically significant. results: the total studied sample was 412 health personnel. out of them, 316 (77%) were males, 221 (54%) were of age group <35 years of age. while 142 (35%) of them were physicians, 270 (65%) were other health-care professionals such as dentists, pharmacists, nurses, and medical ancillary staff. demographic characteristics of those responded to the survey are shown in table 1. table 1. demographic characteristics of all respondents (n=412) characteristic number percentage gender male female 316 96 77% 23% age/ years < 25 25-34 35-44 ≥ 45 27 194 132 59 7% 47% 32% 14% marital status married single divorced \ widow 308 99 5 75% 24% 1% specialty medicine surgery obgyn pediatric radiology / laboratory isolation centers er other – multiple specialties or administration 39 58 19 31 67 23 39 125 9% 14% 5% 8% 16% 6% 9% 30 position physician nurse or nurse technician pharmacist or pharmacy tech dentists other healthcare providers (lab techs, radiographers) administrative (drivers, security, others) 142 98 28 7 113 24 35% 24% 7% 2% 27% 6% location of work nineveh health department administration doh hospitals inside mosul doh hospitals outside mosul primary health care sectors other * 20 235 53 48 51 5% 57% 13% 12% 12 source of information health authority instructions media both 55 54 300 13% 13% 73% * mosul university, humanitarian organization, or medical detachment at entrances to the city. 152 original infection prevention and control measures for covid-19 among medical staff in nineveh zakria a kassim j contemp med sci | vol. 6, no. 4, july-august 2020: 150–155 the percentage of staff following different ipc measures was widely ranging from as low as 40% in wearing a head cover specialized for health personnel, to as high as 97% in keeping clean hands and trimming fingernails constantly (table 2). main defects were found in respiratory hygiene, physical distancing, and self-isolation. comparison between responses to different ipc measures among males and females is illustrated in table 2. females were more likely to sterilize and apply bandages to wounds/scratches as well as trend to wear gloves before examining patients. concerning adherence to ipc measures in physicians compared to other health-care staff, this is summarized in table 3. physicians were less likely to wash hands from elbows and below for 20 s, put medical and general waste in designated places, wear a protective cover; however, they were more likely to cover mouth and nose completely on cough or sneeze, and wear masks while dealing with patients. on the other hand, physicians were less likely to report deficiency in health protection supplies. compared to health-care staff working outside hospitals, those who work in hospitals were better compliant with placing medical and general waste designated areas, as well as sterilizing hands before entering home (table 4). respondents from surgical specialties were found more likely to wash hands from the elbows and below for 20 s, but less likely to cover mouth/nose completely when cough or sneeze as shown in table 5. of note, 332 (81%) of respondents reported benefit from this questionnaire, which was not significantly different between male and female, or those work in hospital and outside hospitals, while more benefit was reported in non-physician respondents compared to physicians. discussion health workers are at the front line of the covid-19 outbreak response, and as such, they are exposed to hazards that put them at risk of infection. between 10 and 20 percent of united states’ coronavirus cases are health-care worker.20 even in iraq, many covid-19 cases are being reported daily among health workers, therefore, following proper ipc is very important. overall, there are wide ranges of many issues; medical staff is following regarding ipc measures. this can make a sense on how to do proper ipc and how to improve the defective points in future, regarding each entity. the lowest rate was for wearing head were nearly 2/5 indicating its use. however, this might be explained by the fact that head cover use is not indicated in those who had regular work. moreover, many health workers do not work in places that have direct contact with covid-19 patients. other than table 2. percentage of answers (as strongly agree or somewhat agree) to different questions among all respondents as well as comparison between male and female (total n=412) question all (n=412) male (n=316) female (n=96) p value q1. keeping at least 1 meter away from others. 296 (72%) 230 (73%) 66 (70%) 0.471 q2. removing ring, keys, watch or metal bracelets while at work. 291 (71%) 221 (70%) 70 (74%) 0.534 q3. keeping clean and trimmed fingernails. 398 (97%) 306 (98%) 92 (97%) 0.751 q4. sterilizing and applying bandages to scratches/wounds. 327 (79%) 243 (77%) 84 (88%) 0.019* q5. washing hands up to elbows x 20 seconds, or use medical sanitizers before & after examining patients. 357 (87%) 276 (89%) 81 (85%) 0.362 q6. covering mouth and nose on cough or sneeze. 390 (95%) 300 (96%) 90 (95%) 0.744 q7. wearing a mask while dealing with infected patients. 382 (93%) 293 (94%) 89 (94%) 0.979 q8. wearing rubber gloves before examination. 382 (93%) 289 (92%) 93 (98%) 0.052 q9. ensuring to keep hands away from the face. 372 (90%) 288 (91%) 84 (88%) 0.376 q10. disposing medical waste in designated places. 392 (95%) 298 (95%) 94 (99%) 0.100 q11. wearing a protective cover specialized for health personnel at work. 273 (66%) 215 (69%) 58 (62%) 0.192 q12. wearing a head cover (for workers in extremely dangerous areas).** 163 (40%) 130 (58%) 33 (51%) 0.316 q13. cleaning hands or other sterilizing measures before entering home. 376 (91%) 288 (95%) 88 (94%) 0.774 other q1. did you get benefit from this questionnaire? 330 (80%) 255 (82%) 75 (80%) 0.672 other q2. did you suffer from deficiency in health protection supplies during the outbreak? 321 (78%) 243 (77%) 76 (81%) 0.476 *statistically significant **number of answers in each group is different in this q: all, n=290; male, n=225; female n=65. 153 original infection prevention and control measures for covid-19 among medical staff in ninevehzakria a kassim j contemp med sci | vol. 6, no. 4, july-august 2020: 150–155 table 3. comparison of percentage answered as yes (extremely, or to some extent) to the following qs between physicians and all other healthcare staff (total n=412) question physicians (n=142) all others (n=270) p value q1. keeping at least 1 meter away from others. 102 (%71) 196 (73%) 0.696 q2. removing ring, keys, watch or metal bracelets while at work. 105 (73%) 186 (69%) 0.393 q3. keeping clean and trimmed fingernails. 139 (%99) 260 (97%) 0.251 q4. sterilizing and applying bandages to scratches/wounds. 108 (76%) 221 (82%) 0.141 q5. washing hands up to elbows x 20 seconds, or use medical sanitizers before & after examining patients. 116 (82%) 243 (91%) 0.004* q6. covering mouth and nose on cough or sneeze. 140 (98%) 252 (94%) 0.075 q7. wearing a mask while dealing with infected patients. 139 (97%) 245 (92%) 0.031* q8. wearing rubber gloves before examination. 133 (93%) 251 (94%) 0.692 q9. ensuring to keep hands away from the face. 132 (92%) 241 (90%) 0.370 q10. disposing medical waste in designated places. 134 (94%) 260 (98%) 0.067 q11. wearing a protective cover specialized for health personnel at work. 87 (61%) 187 (70%) 0.064 q12. wearing a head cover (for workers in extremely dangerous areas).** 48 (45%) 119 (61%) 0.044* q13. cleaning hands or other sterilizing measures before entering home. 130 (94%) 247 (95%) 0.650 other q1. did you get benefit from this questionnaire? 106 (74%) 226 (85%) 0.006* other q2. did you suffer from deficiency in health protection supplies during the outbreak? 126 (89%) 195 (73%) <0.001* *statistically significant **number of answers in each group is different in this q: doctors, n=94; all others, n=197 table 4. comparison of percentage answered as yes (extremely, or to some extent) to the following qs between those working in hospital versus those outside hospitals (total n=412) question work in hospital (n=288) work outside hospital (n=124) p value q1. keeping at least 1 meter away from others. 205 (71%) 93 (75%) 0.457 q2. removing ring, keys, watch or metal bracelets while at work. 208 (72%) 83 (68%) 0.333 q3. keeping clean and trimmed fingernails. 281 (98%) 118 (96%) 0.257 q4. sterilizing and applying bandages to scratches/wounds. 226 (79%) 103 (83%) 0.315 q5. washing hands up to elbows x 20 seconds, or use medical sanitizers before & after examining patients. 248 (%87) 111 (90%) 0.531 q6. covering mouth and nose on cough or sneeze. 275 (96%) 117 (94%) 0.516 q7. wearing a mask while dealing with infected patients. 271 (95%) 113 (91%) 0.166 q8. wearing rubber gloves before examination. 270 (94%) 114 (92%) 0.346 q9. ensuring to keep hands away from the face. 261 (91%) 112 (90%) 0.923 q10. disposing medical waste in designated places. 279 (98%) 115 (93%) 0.021* q11. wearing a protective cover specialized for health personnel at work. 198 (69%) 76 (63%) 0.288 q12. wearing a head cover (for workers in extremely dangerous areas). ** 120 (57%) 44 (55%) 0.774 q13. cleaning hands or other sterilizing measures before entering home. 266 (96%) 111 (90%) 0.022* other q1. did you get benefit from this questionnaire? 236 (83%) 96 (78%) 0.257 other q2. did you suffer from deficiency in health protection supplies during the outbreak? 227 (79%) 94 (76%) 0.548 *statistically significant. **number of answers in each group is different in this q: work in hospital, n=211; work outside hospital, n=80. 154 original infection prevention and control measures for covid-19 among medical staff in nineveh zakria a kassim j contemp med sci | vol. 6, no. 4, july-august 2020: 150–155 this, nearly 70% of the sample does not remove their rings and watches while performing hand washing. in addition, a similar percentage does not follow social distancing and keeping 1 m or more distance than other people or patient. this might be a core step in ipc but not well-known by the medical staff due to poor ipc training courses or may be due to increasing number of patients in very small health facilities. no significant difference was identified between male female practices, except for sterilizing and applying bandages to scratches/wounds, where females are more adhere compared to male. there is a similar finding from another study done in usa.21 with regards to physicians compared to other medical staff, physicians are less adherent to hand sanitations, wearing head cover while they are in their shift, but they are more careful about wearing facemasks during their shifts than others. this equipment was recommended by us food and drug administration society as personal protective measures.22 compared to medical staff who work outside the hospital, those who work inside the hospital are more adherent about throwing medical waste in their special places, and they are more careful to avoid spreading the infection to their houses than those who are working outside the hospital. this may due to more serious types of infections and invasive techniques faced by medical staff working inside the hospital.23 at another comparison, surgical staff is more careful for hand sanitation but they are less serious in covering their mouth during coughing and sneezing. this finding might be due to the special work environment facing each specialty. most germs spread airborne which means that while sneezing or coughing millions of germs into the air are sending and other people might breathe them in and get infected.24 in general, 22% of respondents indicated shortage in personal protective equipment (ppe). ppe shortages had been recognized in many areas of the world with covid-19 pandemic; however data collection was just at beginning of the pandemic in nineveh and few cases had been registered by that time. this study has a number of limitation and challenges. firstly, there are no national data existing on the ipc control for iraqi medical staff related to covid-19 or other infectious diseases, for comparison. additionally, due to the shortage of time and the situation related to covid-19 lockdown, we could collect 412 respondents, which represent major part of the medical staff but not all of them. conclusion ipc is a crucial element when medical staff handles or treat any infectious disease. hand washing, respiratory hygiene, physical distancing, and self-isolation were the main issues to control infection. staff from all medical specialties had some defects in one of the areas relevant to adherence to infection prevention and control. this might be due to lack of medical educational materials, poor working environment, or lack of seriousness when dealing with infectious diseases in general, or the current pandemic in specific. so, training on how to do proper ipc is important for all of them. regardless of covid-19 pandemic, the study was done in order to improve the concept of infection control. table 5. comparison of percentage answered as yes (extremely, or to some extent) to the following qs between surgical specialties and medical specialties (total n=412) question surgical specialties (n=77) medical specialties (n=70) p value q1. keeping at least 1 meter away from others. 49 (64%) 52 (74%) 0.164 q2. removing ring, keys, watch or metal bracelets while at work. 60 (78%) 48 (69%) 0.200 q3. keeping clean and trimmed fingernails. 75 (98%) 66 (97%) 0.900 q4. sterilizing and applying bandages to scratches/wounds. 62 (81%) 52 (75%) 0.452 q5. washing hands up to elbows x 20 seconds, or use medical sanitizers before & after examining patients. 67 (87%) 52 (75%) 0.070 q6. covering mouth and nose on cough or sneeze. 70 (91%) 70 (100%) 0.010* q7. wearing a mask while dealing with infected patients. 68 (88%) 67 (96%) 0.102 q8. wearing rubber gloves before examination. 69 (90%) 65 (93%) 0.489 q9. ensuring to keep hands away from the face. 67 (87%) 66 (94%) 0.134 q10. disposing medical waste in designated places. 75 (96%) 65 (93%) 0.196 q11. wearing a protective cover specialized for health personnel at work. 49 (64%) 43 (62%) 0.869 q12. wearing a head cover (for workers in extremely dangerous areas). ** 25 (50%) 26 (52%) 0.841 q13. cleaning hands or other sterilizing measures before entering home. 71 (96%) 61 (92%) 0.370 other q1. did you get benefit from this questionnaire? 57 (75%) 55 (79%) 0.610 other q2. did you suffer from deficiency in health protection supplies during the outbreak? 62 (82%) 58 (83%) 0.840 *statistically significant. **number of answers in each group is different in this q: surgical specialties, n=50; surgical specialties, n=50. 155 original infection prevention and control measures for covid-19 among medical staff in ninevehzakria a kassim as this study provided assessment of the situation and adherence to ipc measures in nineveh governorate, it can build a basic stone for conducting further studies in future and collaborate with other cities to compare the medical staff attitude as well as follow-up after education or other measures to improve this poor adherence. the medical staff still needs more governmental support for protective measures; however, there are certain measures that all staff can take on their own to protect themselves as well as others whom they encounter. in addition, we urge non-governmental organizations to improve currently available services and provide more protective supplies as much as possible. conflicts of interest disclosure all authors indicate that there is no actual or potential conflicts of interest with regard to this manuscript. references: 1. world health organization, “coronavirus disease 2019”.  url available at: https://www.who.int/emergencies/diseases/novel-coronavirus 2019?gclid =cj0kcqjwoub3brc6arisabghnyzd1crd-zueutqbtpir7_9utkeepmaq0nvz uyywtqz6tqvnpcjcogeaalucealw_wcb. 2. world health organization, “who director-general’s opening remarks at the media briefing on covid-19 – 11 march 2020”.  url available at: https:// www.who.int/dg/speeches/detail/who-director-general-s-openingremarks-at-the-media-briefing-on-covid-19---11-march 2020 3. worldometer, “covid-19 coronavirus pandemic”. accessed on 3 july 2020. available at: https://www.worldometers.info/coronavirus/country/iraq. 4. medecins sans frontiers, “a year on from battle, mosul’s healthcare system is still in ruins” 9 july 2018. accessed on 11 may 2020. url available at: https:// www.msf.org/year-battle-mosul%e2%80%99s-healthcare-system-still-ruins 5. unicef, “fighting covid-19 deepens iraq’s humanitarian crisis” 21 april 2020. accessed on 11 may 2020. url available at: https://blogs.unicef.org/ blog/fighting-covid-19-deepens-iraqs-humanitarian-crisis/ 6. center for disease control and prevention, “guidelines for environmental infection control in health-care facilities” 2003 updated: july 2019. assessed at 11 may. available at: http://www.faqs.org/health/topics/74/infectioncontrol.html. 7. medicine for humanity, “covid-19 global pandemic”. url available at: https://medicinesforhumanity.org/wp-content/uploads/2020/04/covid19-curriculum_mfh-final version_4.14.20.pdf 8. liu j, liao x, qian s, yuan j, wang f, liu y, et al. community transmission of severe acute respiratory syndrome coronavirus 2, shenzhen, china, 2020. emerg infect dis. 2020 jun 17;26(6). 9. chan jf, yuan s, kok kh, to kk, chu h, yang j, et al. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster. the lancet. 2020 feb 15;395(10223):514-23. 10. li q, guan x, wu p, wang x, zhou l, tong y, et al. early transmission dynamics in wuhan, china, of novel coronavirus–infected pneumonia. n engl j med. 2020 jan 29. 11. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. the lancet. 2020 feb 15;395(10223):497-506. 12. burke rm, midgley cm, dratch a, fenstersheib m, haupt t, holshue m, et al. active monitoring of persons exposed to patients with confirmed covid19-united states, january-february 2020. mmwr. morbid mortal weekly rep. 2020 mar 6;69(9):245-6. 13. world health organization. report of the “who-china joint mission on coronavirus disease 2019 (covid-19)” 16-24 february 2020. url available at: https://www.who.int/docs/default-source/coronaviruse/who-chinajoint-mission-on-covid-19-final-report.pdf 14. center for disease control and prevention, “coronavirus disease 2019-using ppe” last accessed on june 9, 2020. url available at: https://www.cdc.gov/ coronavirus/2019-ncov/hcp/using-ppe.html 15. center for disease control and prevention, “prevention & treatment”. 15 february 2020. archived from the original on 15 december 2019. url available at: https://www.cdc.gov/coronavirus/mers/about/prevention. html. 16. world health organization, “coronavirus disease (covid-19) advice for the public”. url available at: www.who.int/emergency/diseases/novelcoronavirus-2019/advice-for-public. 17. adigun a, johnson j. physical distancing, masks can dramatically reduce covid-19 transmission. abcnews. url available at: https://abcnews. go.com/health/optimal-physical-distancing-facial-protection-reducecovid-19/story?id=71019436 . 18. hersh e, goodwin m. how long is the incubation period for the coronavirus?. healthline. march 13, 2020 url available at: https://www. healthline.com/health/coronavirus-incubation-period. 19. world health organization, “coronavirus disease (covid-19) advice for public”. url available at: https://www.who.int/emergencies/diseases/novelcoronavirus-2019/advice-for-public. 20. nicholson l. “thousands of health care workers infected with coronavirus, cdc report finds”. reuters news. april 15, 2020. url available at: https:// www.nbcnews.com/health/health-news/thousands-health-care-workersinfected-coronavirus-cdc-report-finds-n1183886. 21. hamblin j. “evidence of the superiority of female doctors” the atlantic. health. december 2016. url available at: https://www.theatlantic.com/ health/archive/2016/12/female-doctorssuperiority/511034/. 22. us food and drug administration “isolation gown” last update 11-3-2020. url available at: https://www.fda.gov/medical-devices/general-hospitaldevices-and-supplies/personal-protective-equipment-infection-control. 23. better health channel, “preventing healthcare associated infection”. feb 2019. url available at: https://www.betterhealth.vic.gov.au/health/ conditionsandtreatments/infections-in-hospital-reduce-the-risk. 24. center for disease control and prevention, “water, sanitation and environmentally-related hygienecoughing and sneezing” updated on april 22, 2020. url available at: https://www.cdc.gov/healthywaterlhygiene/ etiquettelcoughing_sneezing.html. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i4.820 77j contemp med sci | vol. 2, no. 7, summer 2016: 77–82 research estrogen-degrading bacteria in women with premature ovarian failure juman khaleel al-sabbagh, mohammad sabri a. razzaq, milal m. abd-al-rudha issn 2413-0516 department of microbiology, college of medicine, babylon university, babylon province, iraq. correspondence to juman khaleel al-sabbagh (email: jkphoenix05@yahoo.com). (submitted: 29 may 2016 – revised version received: 7 june 2016 – accepted: 10 june 2016 – published online: 26 september 2016) introduction premature ovarian failure (pof) is an early ovarian malfunction different from normal men opause, which disturbs the production of follicles, re sulting in amenorrhea under the age of 40 in 1–3% of reproductive age women.1 affected women show menstrual problems followed by an elevated level of gonadotropins, such as follicle stimulating hormone (fsh) ≥ 40 iu/l and hypoes trogenism for an average four months, measur ing serum fsh is a routine diagnosis procedure for the disease.2,3 intestinal microbial richness and functions, influence levels of estrogens via enterohepatic circulation; thus, the gut microbial community likely affects the risk for estrogen-related conditions.4 aerobic and anaerobic estrogen-degrading microorganisms are phylogenetically diverse; they are mainly isolated from different environment; estrogens can be degraded via growthlinked and non-growth-linked reactions, as well as through abiotic degradation in the presence of selective microorganisms.5 sex steroid hormones play an important physiological role in reproductive and non-reproductive tissues including the immune cells, these hormones exhibit their functions by binding to their specific intracellular receptors, that act as either ligand dependent transcription factors, or membrane receptors that stimulate several signal transduction pathways.6 development, reproduction, cell proliferation, inflammation, metabolism, differentiation, apoptosis, homeostasis, and brain function a number of physiological roles that these hormones played.7 steroid hormones consisting of estrogens, progestagens, mineralocorticoids, and androgens glucocorticoids; these are powerful signal molecules that regulate a host of organismal functions; among them, estrogens are responsible for the development of female secondary sex characteristics.8 interestingly, these steroid hormones also participate in the communication between microorganisms and their mammalian hosts; this type of communication is commonly called “interkingdom signaling”, and can be used by microbial pathogens to activate their virulence factors and, control the course and outcome of infection.9 natural estrogens are a group of steroid components named for their importance in the estrous cycle of humans and animals. three major naturally occurring estrogens are called estrone (e1), 17estradiol (e2) and estriol (e3), which are synthesized from pregnenolone, these are excreted from the humans and cattle in the urine.10 and some of these natural estrogens are discharged into environments without processing, it is estimated that, estrogen discharge is increasing in the urban areas.11 estrogens are often used as contraceptives, especially synthetic estrogens. thus, large amounts of estrogens are constantly excreted into the environment from humans; these hormones are chemically very stable in the environment and that is because of the aromatic ring in its structure.8 also12 found that, the major one of estrogens for environmental contamination is e2, which is considered about 50 times more potent than e1 and 6 times than e3, and they show that, estrogen excreted from women at a reproductive age (15–59 years old) differs from 5–31 μg per day for e1 and 3–19 μg per day for e2; while during these periods, a woman estrogen excretion is similar to that of a man, with levels about 4–12 μg per day for e1 and 1.5–7 μg per day for e2. some organisms have the pathways to make use of estrogen compounds as growth substrates and the bacteria, which can degrade estrogens were mainly isolated from different origins.8 under aerobic conditions, steroids can be degraded by different bacterial species of different genera; although bacteria are generally able of growing on estradiol or estrone as a sole source of carbon and energy, only a few degradation objectives estimation of the level of follicle stimulation hormone (fsh), luteinizing hormone (lh), and estrogen hormone, isolation of bacteria from premature ovarian failure (pof) women, detection of the ability of these bacteria to degrade the estrogen hormone using hplc in aerobic and anaerobic condition. methods in this study, 60 women who suffered from pof; and have menopause at least 8 months after the last period, whose ages range between 20 and 39 years, venous blood samples and high vaginal swabs were collected from these patients who are admitted to the children and maternity hospital and al-hilla teaching hospital in hilla city/iraq from the period of february to july 2015. also, 40 women with no history of menopause with the age range approximately matched to that of patients as control group. these women were subjected to investigate fsh, lh, and estrogen hormone. different types of bacteria isolated from the patients vagina were examined for their ability to lyse estrogen in vitro by using hplc technique. results fsh and lh hormones showed a significant increase (p < 0.05) in the concentration of these hormones in blood of patients when compared to healthy control women. while there is a significant decreasing in the concentration of est hormone in these women as compared to healthy control. out of 60 women patients with pof only 42 patients showed positive bacterial growth, while 18 patients show no growth. the bacterial isolates show a ability to degrade estradiol in aerobic and anaerobic condition. and the results demonstrating the ability of vaginal bacteria to exhaust the estrogen efficiently and that may affect the systemic estrogen levels in these women. conclusion fsh and lh used as diagnostic tools for detection of premature ovarian failure. different bacterial types isolated from patients vagina show an efficient degradation of estrogen. keywords pof, fsh, lh, est. estdegrading bacteria 78 j contemp med sci | vol. 2, no. 7, summer 2016: 77–82 estrogen-degrading bacteria in women with premature research mohammad sabri a. razzaq et al. mechanisms have been suggested.11,13 the oxygen-dependent pathway with its manner of cleavage of the steroid nucleus looks to be a general degradation pathway of steroids and cholesterol in aerobic bacteria.14 another important feature is that, the genes for steroid degradation in some bacterial species are not constitutively expressed, but are induced by their respective steroid substances; moreover, studies of the mechanisms regulating the steroidinducible gene expression exposed that regulator proteins, binding proteins or intermediate compounds produced in the course of steroid degradation are probable involved in microbial steroid metabolism.15 anoxic environments improve where organic matter is degraded through microbial activity, and oxygen has only limited access, for example, during the denitrification step in wastewater treatment or in sediments of rivers and lakes; steroids that arise in such environments have in common with aliphatic hydrocarbons and monoaromatic compounds a chemical inertness that makes them recalcitrant substrates for bacteria.14 materials and methods clinical samples: this case control study involved women diagnosed with premature ovarian failure, they were referred to babylon province and al-hilla teaching hospital from the period february to july 2015, all subjects underwent a standard diagnostic work-up to rule out any verifiable cause of pof prior to inclusion into the study. a. patients: sixty patients participating in this case control study were related to women who suffered from pof; these women have menopause at least 8 months after the last period, whose ages range between (20– 39) years, venous blood samples and high vaginal swabs were collected from these patients under supervision of specialist gynecologist. b. control: venous blood samples and high vaginal swabs from 40 women as a control that had a normal menstrual cycle, with no history of menopause with age range approximately matched to that of patients. c. ethical approval: the study was done and the samples were collected after getting the agreement of the patients and also gynecologist. collection of vaginal swabs: high vaginal swabs were taken from case control women by using cotton tipped swab. those specimens were collected under the help of advisory to avoid any possible contamination. swab for culturing should be placed in tubes containing normal saline to maintain the swab moist until taken to laboratory or placed in transport media and transformed to the laboratory for culturing. these samples were inoculated on macconkey agar, nutrient agar, blood agar and chocolate agar plates and incubated aerobically and anaerobically at 37°c for 24–48 hr. estimation of fsh, lh and estrogen hormone: all cases of patients and control were subjected to estimate the fsh, lh and estrogen hormone serum level. this assay was achieved according to the method described by the manufacturing company vidas/germany. microscopic examination and colonial morphology: accor ding to the diagnostic procedures recommended macfaddin,16 a single colony was taken from each primary positive culture and its identification depended on the morphological properties (colony size, shape, color, nature of pigments, translucency, edge, elevation and texture). bacterial smear stained with gram stain was used to check the morphological properties of bacterial cells. minimal salt medium (msm): 3.5 g of k2hpo4, 1.5 g of kh2po4, 0.5 g of nacl, 0.5 g of (nh4)2so4 and 0.15 g of mgso.7h2o and trace element were added and they contained, 2 g of nahco3.10 h2o, 0.3 g mnso4.4h2o, 0.2 g znso4.7h2o, 0.02 g (nh4)mo7o2.4h2o, 0.1 g cuso4.5h2o, 0.5 g cocl2.6h2o, 0.05 cacl2.2h2o and 0.5 g feso4.7h2o dissolved in 1000 ml of distilled water, and then sterilized in an autoclave at 121°c for 15 minutes. after cooling the mixture to 50°c, estradiol was added to the mixture as the only carbon source in the media in concentration between 20–30 µg/ml. this media used to detection of estrogen-degrading activity.11 estimation of estrogen degradation ability of bacterial isolates by using hplc: 17 β-estradiol (c18h24o2) was added to the medium as the only carbon source in the media in concentrations between 20–30 μg/ml. each sample was inoculated directly into the medium. the samples were incubated aerobically and anaerobically at 37°c for 48 hours. and the concentration of estradiol was measured in the containing media using hplc on a c18 column with gradient elution with acetontrile at elution rate 1 ml per minute at absorption 210 nm.17 this analytical technique grounded on the separation of molecules due to the differences in their composition and/or structure; these involve moving of the sample through the system over the stationary phase, the molecules in the sample will have dissimilar interactions and affinities with the stationary phase, that leading to separation of molecules.18 the sample components that exhibit stronger interactions with the stationary phase will move more slowly through the column, than components with weaker interactions; so different compounds can be separated from each other when they move through the column.19 the schematic of an hplc instrument typically involves a sampler, pumps and a detector; the sampler transports the sample mixture into the mobile phase stream which conveys it into the column; the pumps supply the desired flow and composition of the mobile phase through the column; while the detector creates a signal proportional to the amount of sample component emerging from the column, therefore allowing for quantitative analysis of the sample components.20 statistical analysis: this study used statistical analysis that included the calculation of mean values and percentage. the statistical package for the social sciences version 18 (spss inc., chicago, usa) was used for statistical analysis by calculation of p-value with 95% confidence interval (95% ci). t-test was used to compare means of (fsh, lh and estrogen hormone) serum level between two groups (women with pof and controls). a p-value ≤ 0.05 was considered as a significant at p-value = 0.001. results pof diagnosed women: from february to july 2015, only 60 patients with pof were included in this study who admitted to the-children and maternity hospital and al-hilla teaching hospital for surgery. these women have menopause at least 8 months after the last period, whose ages ranging from (20– 39) years. besides 40 healthy women, that had a normal menstrual cycle, with no history of menopause were also included 79j contemp med sci | vol. 2, no. 7, summer 2016: 77–82 research estrogen-degrading bacteria in women with premature mohammad sabri a. razzaq et al. as a control group. according to the clinical findings of the gynecologist and according to the hormones levels (fsh, lh and e2) the results of pof patients were scored. the results showed that, most of women with pof have a high levels of fsh and lh and low level of e2. and the levels of fsh and lh are highly a significant at (p value < 0.05), figs 1 and 2, respectively. moreover, the levels of e2 decreased significantly in women with pof as shown in fig. 3. high vaginal swabs were collected from the patients and control groups to detect the presence of bacterial infection in these women. only 42 patients showed positive bacterial growth, while 18 patients show negative results for the bacterial growth table 1. as compared with a control group who show no growth. among the bacteria which isolated from the patients̓ vagina are klebsiella pneumonia (10), enterococcus fecalis (3), enterobacter coloaca (2), acinetobacter baumannii (2), escherichia coli (8), proteus vulgaris (4), streptococcus agalactia (4), staphylococcus aureus (3), pseudomonas aeroginosa (2) and streptococcus mutans (4), as shown in table 2. and these isolates were examined to show their ability to degrade estrogen in vitro by using high performance liquid chromatography (hplc), where the peak of standard appears at 1.63 min. the results are scored according to the ability of these bacteria to consume estrogen as the sole source of fig. 1 the mean and standard deviation (sd) of fsh iu/ml for patients and control groups. *significant at p-value < (0.05). sd for patients ± 30.06; sd for control ± 2.81. fig. 2 the mean and standard deviation (sd) of lh iu/ml for patients and control groups. *significant at p-value < (0.05). sd for patients ± 15.09; sd for control ± 2.47. fig. 3 the mean and standard deviation (sd) of e2 pg/ml for patients and control groups. * significant at p-value < (0.05). sd for patients ± 3.99; sd for control ± 86.90. table 2. the types and the number of bacteria isolated from women with pof types of bacteria number of isolates enterococcus fecalis 3 enterobacter coloaca 2 klebsiella pneumoniae 10 acinetobacter baumannii 2 escherichia coli 8 proteus vulgaris 4 streptococcus agalactia 4 staphylococcus aureus 3 pseudomonas aeroginosa 2 streptococcus mutans 4 table 1. number of bacterial isolates in case and control women groups positive growth no growth patients (n = 60) 42 18 control (n = 40) no pathogenic bacteria carbon. the results showed that the consumption didn’t depend on bacterial species. however, consumption of estradiol at any extent facilitate the growth of bacteria in vitro and also the results revealed that the consumption of estradiol increased anaerobically when compared to the aerobic condition as seen in table 3, and the bacterial isolates show a different peaks and different areas of estradiol derivatives according to the hplc results. discussion hormone investigation (fsh, lh and e2) is necessary and fsh above 40 iu/l and estradiol under 50 pmol/l in women aged below 40 years approve the diagnosis.21 along with, both primary and secondary forms of ovarian failure, are biochemically described by low levels of gonadal hormones (like, estrogens) and extraordinary gonadotropins (lh and fsh) (hypergonadotropic amenorrhea); the elevation of fsh is 80 j contemp med sci | vol. 2, no. 7, summer 2016: 77–82 estrogen-degrading bacteria in women with premature research mohammad sabri a. razzaq et al. frequently more marked than that of lh and, an fsh value >30 iu/l is indicative of ovarian failure.22 fsh stimulates follicle growth in the ovary and is normally negatively regulated by inhibin and estrogen released from the maturing follicle; in ovarian failure, however, the nonexistence of a maturing follicle results in a lack of negative feedback and raised fsh levels; additionally, they also have low estrogen levels and elevated lh levels characteristic of post-menopausal women.23 however, there are several reasons for ovarian failure as a result of endocrine dysfunction and these comprise: deficiencies in enzymes essential for steroid hormone synthesis as a result of either genetic abnormalities or autoimmune attack.24 mutations in steroid hormone or hormone receptor genes, and targeted damage of endocrine organs including the pituitary, hypothalamus or ovaries by disease processes autoimmune sources, or iatrogenic assault.25 it is advocated that a decrease in e2 bioactivity could affect ovarian follicle reserve in a number of means; it could supply a less potent negative feedback on the pituitary, leading to increased fsh levels, ovarian stimulation and abnormal folliculogenesis.26 the ordinary ovarian activity requires accurate endocrine regulation and hormone signaling, and lh is needed to stimulate ovulation and therefore indirectly offers negative feedback on fsh production, a failure of response could therefore cause infertility and elevated fsh.25 several studies show that, there were a decline of fsh levels in pof patients having follicular growth or ovulatory cycles, however, these fsh levels continued higher than the normal fsh levels.27 similarly, among premenopausal patients with ovulatory cycles, variability in fsh levels exceeding the normal range has been described, and fsh levels are reliant on underlying follicular activity.28 the etiology of bacterial vaginitis is poorly understood and remains a question for discussion, bacterial vaginitis can arise and remit spontaneously or progress into a chronic or recurrent disease.29 bacterial vaginitis may sometimes affect women after menopause; the reduction in estrogen levels in perimenopausal and postmenopausal women has been related to an abnormal vaginal flora of 35% and 70%, respectively when compared to the normal flora.30 vaginal lactobacilli have a crucial role in maintaining an environment that restricts the growth of pathogenic microorganisms in the vagina.31 it has been proposed that, estrogen and lactobacillus are required to achieve an optimal vaginal ph of 4.0 to 4.5.32 after puberty, under the effect of estrogen, glycogen is deposited in the vaginal epithelial cells, which is metabolized by vaginal epithelial cells to glucose, lactobacilli produce lactic acid from glucose, keeping the vagina at an acidic ph.33 however the enterobacteriaceae family is one of efficacious bacterial isolate was capable to grow in the presence of β-estradiol 17. and the intestinal contents can hydrolyze numerous estrogens and estrogen metabolites (em), these reactions have been attributed to gut luminal bacteria, which greatly effect on systemic estrogen and em levels.4 furthermore34 found that, escherichia spp. are highly proficient in degradation of estradiol. since most intestinal bacteria are the main source of bacterial vaginitis so, this will give evidence on the role of intestinal bacteria in degradation of estrogen. so35 published that, pseudomonas strains display estrogen-metabolizing activity; the ability of the isolated strains to metabolize a mixture of the different estrogens types was observed, and demonstrated that the transformations were involved the oxidation of βe2 into e1. the fecal microbiome (number and species) was very directly and strongly associated with systemic estrogens level; these associations were robust to different classifications of microbiome diversity, and they held for estradiol, estrone and em; and the diversity analysis advises that estrogen levels are not associated with any particular cluster or class of that microbiome.36,37 moreover, the bacteria can improve many mechanisms to exhaust or to eliminate sex hormones in their benefit, by using them as carbon and energy sources, in principal through their modification or chemical degradation.6 degradation pathways of the natural estrogens in addition to e2 are still questionable; and all these pathways are only valid under aerobic circumstances and the breakup of c-c bonds and the oxidation of quaternary carbon atoms are dependent on oxygen; besides, it is not yet clear how the degradation pathway resembles if oxygen is absent although it was displayed that, steroids can be degraded under anaerobic conditions.38 so while another bacteria were reported by39 to be able to completely degrade 20, 000 μg of e2 within 18 hours, klebsiella sp. exhibited a good estrogen degrading activity when it was inoculated in estradiol anaerobically.17 the multiplicity of the gut microbiota, could influence systemic estrogen levels through enzymatic and other pathways.4,40 besides41 finding that, intestinal microbial richness and specific taxa may contribute to systemic estrogen levels and associated diseases. utmost of pof patients women do not have any symptoms; however, in untreated pof, typical symptoms of estrogen withdrawal may be present, they include irritability, nervousness, restlessness, hot flushes, insomnia, depression, loss of concentration, loss of libido, etc; physical examination may reveal painful bones, thinness of the skin, stiffness or weight gain.42 young women with pof are at an enlarged risk of coronary heart disease, osteoporosis, cardiovascular accidents and depression, and exogenous estrogens have been shown to have advantageous effects on cardiovascular status and bone density, they also have increasing levels of cardioprotective high density lipoproteins and reduction total cholesterol and low density lipoprotein level.43 reproductive organs which are highly modified by estrogen, such as ovary, uterus, vagina and table 3. the ability of bacterial isolates to consume β-estradiol as the sole source of carbon by hplc bacterial types and no. mean of conc. of est ± sd µg/ml in aerobic condition mean of conc. of est ± sd µg/ml in anaerobic condition k. pneumonia (10) 12.9978 ± 8.17 8.4022 ± 1.08 e. fecalis (2) 15.396 ± 0.035 9.2565 ± 0.39 e. coloaca (2) 12.659 ± 0.78 10.7205 ± 0.08 a. baumannii (2) 11.8457 ± 0.37 8.6575 ± 0.50 e. coli (8) 7.7126 ± 1.10 7.0985 ± 0.90 p. vulgaris (4) 8.3155 ± 0.38 7.9272 ± 0.22 s. agalactia (4) 7.2937 ± 0.33 6.8747 ± 0.56 s. aureus (2) 7.2545 ± 0.34 6.1325 ± 0.08 p. aerogenosa (2) 7.167 ± 0.06 6.570 ± 0.04 s. mutans (4) 10.004 ± 2.29 8.5497 ± 0.41 81j contemp med sci | vol. 2, no. 7, summer 2016: 77–82 research estrogen-degrading bacteria in women with premature mohammad sabri a. razzaq et al. cervix; these changes are consistent with increased estrogen levels in treated females.44 nevertheless, the capability and the time intervals in which the tested miscellaneous bacteria which degraded estradiol is more or less acceptable compared to other bacterial isolates previously testified as potential βestradiol-degrading bacteria; such as rhodococcus zopfii, which completely degrade 100 mg/l of β-estradiol plus ethinyl estradiol estrone, and estriol within 24 hours, and they found that, these substance had no estrogenic activity at all and, estrogens are decomposed entirely.11 the longer the duration of estrogen deficiency, the more severe are the consequences; hence starting treatment early affords longer-term benefits to the health of women; hormone replacement therapy (hrt) should be used in younger women with pof, except contraindicated, until the age of menopause and then reviewed.45 as well46 found that, hormone replacement therapy use before the age of 60 years results in a 24% decrease in coronary heart disease and 30% reduction in total mortality. from the systemic adverse effects of estrogen insufficiency were minimum estrogenise the vaginal epithelium and loss of ovarian activity, that lead to reduce androgen production by 50%, which can have profound effects on general and sexual wellbeing.45 loss of ovarian function at an early age disturbs bone architecture at the very time when bone buildup is at its maximum due to the estrogen deficiency.47 even though early loss of ovarian function has been allied as a risk factor for cv mortality, there are no enough data indicating that these patients are at an increased risk of cv adverse effects from hormonal therapy.48 so estrogen replacement therapy is the mainstay for treatment of women with pof.49 the first line therapy is a trial with estradiol replacement with close observing of ovulation; exogenous estrogens could act by sensitizing the granulosa cells to the effect of fsh leading to ovulation; estrogens may act similarly by down regulation of the lh and fsh receptors; they can be counseled until the age of 55 years.21 n references 1. pouresmaeili f, fazeli z. premature ovarian failure: a critical condition in the reproductive potential with various genetic causes. int j fertil steril. 2014;8(1):1–12. 2. welt ck. primary ovarian insufficiency: a more accurate term for premature ovarian failure. clin endocrinol. 2009;68:449–509. 3. wieacker p. genetic aspects of premature ovarian failure. j reprod med endocrinol. 2009;6(1):17–18. 4. flores r, shi j, fuhrman b, xu x, veenstra td, gail mh, et al. fecal microbial determinants of fecal and systemic estrogens and estrogen metabolites: a cross-sectional study. j trans med. 2012a;10:253–264. 5. yu cp, deeb ra, chu kh. microbial degradation of steroidal estrogens. chemosphere. 2013;91(9):1225–1235. 6. garcía-gómez e, gonzález-pedrajo b, camacho-arroyo i. role of sex steroid hormones in bacterial-host interactions. bio med res intl. 2013;13:1–10. 7. edwards dp. regulation of signal transduction pathways by estrogen and progesterone. ann rev physiol. 2005;67:335–376. 8. zhang t. identification of a new marine steroid-degrading bacterium s19 1 and isolation of estradiol-inducible genes and a novel promoter from this bacterium. thesis of doctorate, christian-albrechts-university/china. 2012. 9. hughes dt, sperandio v. inter-kingdom signaling: communication between bacteria and their hosts. nat rev microbiol. 2008;6(2):111–120. 10. berg jm, tymoczko jl, stryer l. biochemistry, 6th edn. (2006), w. h. freeman and company, new york. 11. yoshimoto t, nagai f, fujimoto j, watanabe k, mizukoshi h, makino t, et al. degradation of estrogens by rhodococcus zopfii and rhodococcus equi isolates from activated sludge in wastewater treatment plants. appl environ microbiol. 2004;70(9):5283–5289. 12. hernandez-raquet scg. occurrence, fate and biodegradation of estrogens in sewage and manure. appl microbiol biotechnol. 2010,86:1671–1692. 13. fujii k, satomi m, morita n, motomura t, tanaka t, kikuchi s. novosphingobium tardaugens sp. nov., an oestradiol-degrading bacterium isolated from activated sludge of a sewage treatment plant in tokyo. int j syst environ microbiol. 2003;53:47–52. 14. fahrbach m, kuever j, meinke r, kämpfer p, hollender j. denitratisoma oestradiolicum gen. nov. sp. nov., a 17β-oestradiol-degrading, denitrifying beta proteobacterium. int j syst evol microbiol. 2006;56:1547–1552. 15. pruneda-paz jl, linares m, cabrera je, genti-raimondi s. teir, a luxr-type transcription factor required for testosterone degradation in comamonas testosteroni. j bacteriol. 2004;186:1430–1437. 16. macfaddin jf. biochemical tests for the identification of medical bacteria. 3rd ed., (2000). the williams and wilkins-baltimor, usa. 17. elnwishy n, hanora a, afifi r, omranf h, matiasson b. a potential 17β estradiol degrader bacterium isolated from sewage water. egypt acad j biolog sci. 2012;4(1):27–34. 18. kupiec t. quality-control analytical methods: high-performance liquid chromatography. int j pharm compound. 2004;8(3):223–227. 19. gerber f, krummen m, potgeter h, roth a, siffrin c, spoendlin c. practical aspects of fast reversed-phase high-performance liquid chromatography using 3 μm particle packed columns and monolithic columns in pharmaceutical development and production working under current good manufacturing practice. j chromatography a. 2004;1036(2):127–133. 20. xiang y, liu y, lee ml. ultrahigh pressure liquid chromatography using elevated temperature. j chromatography a. 2006;1104(1–2):198–202. 21. vujović s, ivović m, tančić-gajić m, marina l, barać m, arizanović z, et al. premature ovarian failure. srp arh celok lek. 2012;140(11–12):806–811. 22. beck-peccoz p, persani l. premature ovarian failure. orphanet j rare dis. 2006;6:1–9. 23. nippita ta, baber rj. premature ovarian failure: a review. climacteric. 2007;10:11–22. 24. chen b, suo p, wang b, wang j, yang l, zhou s, et al. mutation analysis of the wnt4 gene in han chinese women with premature ovarian failure. reprod biol endocrinol. 2011;9:75–78. 25. bretherick kl. genetic factors in premature ovarian failure. doctorate thesis in the university of british columbia (2008). 26. gowri v, al shukri m, al-farsi fa, al-busaidi na, dennison d, al kindi s, et al. aetiological profile of women presenting with premature ovarian failure to a single tertiary care center in oman. post reprod health. 2015;21(2):1–6. 27. bidet m, bachelot a, bissauge e, golmard jl, gricourt s, jerome d, et al. resumption of ovarian function and pregnancies in 358 patients with premature ovarian failure. j clin endocrinol metab. 2011;96:3864–3872. 28. broekmans fj, soules mr, fauser bc. ovarian aging: mechanisms and clinical consequences. endocr rev. 2009;30(5):465–493. 29. donders g. diagnosis and management of bacterial vaginosis and other types of abnormal vaginal bacterial flora: a review. obstet gynecol surv. 2010;65(7):462–473. 30. wilson jd, lee ra, balen ah, rutherford aj. bacterial vaginal flora in relation to changing oestrogen levels. int. j. std aids. 2007;18:208–311. 31. mania-pramanik j, kerkar sc, salvi vs. bacterial vaginosis: a cause of infertility. int. j. std aids. 2009;20:778–781. 32. melvin l, glasier a, elton r, cameron st. ph-balanced tampons: do they effectively control vaginal ph? bjog. 2008;115:639–645. 33. suresh a, rajesh a, bhat rm, rai y. cytolytic vaginosis: a review. indian j sex transm dis aids. 2009;30(1):48–50. 34. yi t, harper wf. the link between nitrification and biotransformation of 17 alphaethinylestradiol. environ sci technol. 2007;41(12):4311–4316. 35. isabelle m, villemur r, juteau p, lépine f. isolation of estrogen-degrading bacteria from an activated sludge bioreactor treating swine waste, including a strain that converts estrone to β-estradiol. can j microbiol. 2011;57:559–568. 36. falk rt, xu x, keefer l, veenstra td, ziegler rg. a liquid chromatography spectrometry method for the simultaneous measurement of 15 urinary estrogens and estrogen metabolites: assay reproducibility and 82 j contemp med sci | vol. 2, no. 7, summer 2016: 77–82 estrogen-degrading bacteria in women with premature research mohammad sabri a. razzaq et al. interindividual variability. cancer epidemiol biomarkers prev. 2008;17: 3411–3418. 37. bjornerem a, emaus n, berntsen gk, joakimsen rm, fonnebo v, wilsgaard t, et al. circulating sex steroids, sex hormone-binding globulin, and longitudinal changes in forearm bone mineral density in postmenopausal women and men: the tromso study. calcif tissue int. 2007;81:65–72. 38. moschet c, hollender j. microbial degradation of steroid hormones in the environment and technical systems. swiss fed ins aquat sci technol. 2009;13:133–153. 39. lee h, liu d. degradation of 17β-estradiol and its metabolites by sewage bacteria. water, air, and soil pollution. 2002;134:353–368. 40. plottel cs, blaser mj. microbiome and malignancy. cell host microbe. 2011; 10:324–335. 41. flores r, shi j, gail mh, gajer p, ravel j, goedert jj. assessment of the human faecal microbiota: ii. reproducibility and associations of 16s rrna pyrosequences. eur j clin invest. 2012b;42:855–863. 42. vujovic s, stojanovic m, penezic z, ivovic m, ivanisevic m, barac b et al. endocrine and metabolic characteristics of women with premature ovarian failure. cic international, roma. 2005;757–760. 43. khastgir g, studd jw, fox sw, jones j, alaghband-zadeh j, chow jw. a longitudinal study of the effect of subcutaneous estrogen replacement on bone in young women with turner’s syndrome. j bone miner res. 2003;18:925–932. 44. ghadami m, el-demerdash e, salama sa, binhazim aa, archibong a, chen ex, et al. toward gene therapy of premature ovarian failure: intraovarian injection of adenovirus expressing human fsh receptor restores folliculogenesis in fshr(−/−) forko mice. mol hum reprod. 2010;16(4):241–250. 45. arora p, polson dw. diagnosis and management of premature ovarian failure. obstet gynaecol. 2011;13(2):67–72. 46. rossouw je, prentice rl, manson je, wu l, barad d, barnabei vm, et al. postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. jama. 2007;297:1465–1477. 47. nelson lm. clinical practice. primary ovarian insufficiency. n engl j med. 2009;5:360(6):606–614. 48. rebar rw. premature ovarian failure. obstet gynecol. 2009;113:1355–1363. 49. vujovic s. aetiology of premature ovarian failure. menopause intl. 2009;15:72–75. 67j contemp med sci | vol. 2, no. 6, spring 2016: 67–69 research objectives the present study was conducted to investigate the effect of nigella sativa on hormone reproduction and thyroid function in female rats. methods twelve females rats were used and divided randomly into two groups (6 animals for each) first group served as treatment and gave nigella sativa oil (1ml /kg bw/day ) orally according to body weight for a period of 30 consecutive days. while other group drenched with normal saline at same dose that above mentioned and served as control. results the result revealed significant increase in levels of lh, estrogen t3 and t4 and significant decrease in level of tsh. histological sections of thyroid gland revealed presence vaculation in colloid of thyroid follicle. conclusion in conclusion, the nigella sativa oil caused the elevation of thyroid hormones as well as lh and estrogen but still within normal value. keywords nigella sativa, rat, lh, t3, t4, tsh study the effect of nigella sativa on thyroid function and reproductive hormone of female rat wafaa kadhim jasima, mayada sahib hassana, ghsoon ghanem keamb issn 2413-0516 adepartment of veterinary physiology and pharmacology, college of veterinary medicine, university of karbala, karbala, iraq. bdepartment of clinical laboratories, college of applied medical sciences, university of karbala, karbala, iraq. correspondence to wafaa kadhim jasim (email: wafaa.gasom@uokerbala.edu.iq ). (submitted:12 march 2016 – revised version received: 8 april 2016 – accepted: 30 april 2016 – published online: 26 june 2016) introduction a herbaceous plant, called nigella sativa (ns) has different names throughout the world such as black seed, black caraway and black cumin. the black seed oil is reported to be beneficial due to its composition of many components such as aromatic oils trace elements and vitamins.1 since nigella sativa oil possesses high ratio of unsaturated fatty acids such as linoloic acid which in turn play an important positive role in function of reproductive system.2 a high percentage of medicine plants have pharmacological principles, so they are useful as curatives for many ailments. according to the reports of world health organization (who), traditional medicine won the trust of 70–80% of people in primary health care.3 the plants and their derivatives have a key role in world health. they have long been known to possess biological activity. and from which it draws about 30% of all modern medicines.4 in addition, the plants used for a long time in the folklore medicine to improve fertility by enhancing fertility recipes and aphrodisiacal qualities.5,6 nigella sativa l. plant belonging to the family called ranunculaceae,7 which is also called black seed and often their seeds used in folk medicine in some asian countries and in the middle east to promote the health and well used in the treatment of various diseases.8,9 several reports confirmed the usefulness of black seed oil because of having more than one hundred of components as volatile oil, vitamins and trace elements.1 recently, clinical and animal studies revealed that the black seed extract has many therapeutic effects. n. sativa seeds have also been used as a natural remedy to promote female menstruation, galactagogue, carminative, laxative and gastroprotective in traditional medicine.12 anti-tumor,13,14 anti-anxiety,15 anti-microbial,16,17 anti parasitic properties,10,11 anti-inflammatory18 and anti-oxidant,19 diuretic and hypotensive, genoprotective, hepato-protective and antidiabetic11 as well as bronchodilator activity and estrogenic activity.20 despite the progress in n. sativa seeds research in the last decade, the biological and physiological effects of n. sativa seeds still controversial and needs more investigation. so the present study aimed to evaluate n. sativa seeds oil as a supportive traditional medicine on hormones involved in female reproduction and on thyroid function. material and methods animal and experimental design: the present study was conducted on the female rats (rattus norvegicus) to evaluate the effect of nigella sativa oil on some hormonal indices of healthy female rats. twelve female rats were divided randomly into two groups (6 animals for each) first group served as treatment and gavaged nigella sativa oil (1ml/kg bw/day) orally according to body weight for a period of 30 consecutive days. while other group drenched with normal saline at same dose that above mentioned and served as control group. at the end of the experiment, blood samples were collected under chloroform anaesthesia via cardiac puncture using sterile disposable syringes at baseline(pre-treatment), day 30 after treatment. the blood samples were then centrifuged at 3000 rpm for 10 minutes to separate the serum. the serum was stored at −80°c until assays were carried out. all tests were performed according to the manufacturer’s instructions.21 hormonal assay was performed. estrogen and lh, t3, t4 and tsh levels were measured by using elisa kit (bio merieux). all rats were sacrificed by cervical dislocation under anesthesised and then midline laparotomy was performed. resected thyroid gland specimens of each rat in all groups were fixed in 10% buffered formaldehyde for 24 hours and histological procedure was performed according to ref. (22 ). statistical analysis: two sample independent t test was used, and the data were expressed as means ± standard deviation. the data were analyzed using spss windows program version 15.23 results and discussion the hormonal activities of lh and estrogen showed significant increase in the treated groups (table 1) while stayed the same in the control group. 68 j contemp med sci | vol. 2, no. 6, spring 2016: 67–69 study the effect of nigella sativa on thyroid function and reproductive hormone of female rat research wafaa kadhim jasim et al. 6. mcdonald a. a botanical perspective on the identity of soma (nelumbo nucifera gaertn) 2004. based on scriptural and iconographic records. new york: botanical garden press. 7. d’cruz sc, vaithinathan s, jubendradass r, mathur pp. effects of plants and plant products on the testis. asian j androl. 2010;12:468–479. 8. mozaffarian va. dictionary of iranian plants names. tehran: farhang moaser publishers; 1998. p. 365. 9. el-kadi a, kandil o. the black seed (n sativa) and immunityits effects on human t-cell subset. fed proc. 1987;46:122–126. 10. swamy smk, tan bkh. extraction, isolation and characterization of antitumor principle, alpha-hederin from the seeds of nigella sativa. plant med. 2001;67:29–32. references 1. ali bh, blunden g. pharmacological and toxicological properties of nigella sativa. phytotherapy res. 2003;17:299–305. 2. feller sj, sauer fd, kramer jg. steady-state rates of linoleic acid biohydrogenation by ruminal bacteria in continuous culture. j dairy sci. 1995;78:1815. 3. bashandy aes. effect of fixed oil of nigella sativa on male fertility in normal and hyperlipidemic rats. int j pharmacol. 2007;3: 27–33. 4. world health organization. traditional medicine strategy 2002–2005. geneva: 2002. 5. burns mm. alternative medicine: herbal preparation. clin ped emerg med. 2000;1:186–190. in present experiments, the ns oil treatment led to significant increase in lh levels which may be due to the direct effect of oil on hypothalamus which in turn increases gonadotropic releasing hormone (gnrh), furthermore fatty acids can stimulate gnrh-dependent pathways that initiate changes in gonads function.24 the positive increased effect of estrogen concentration in treated groups is may be attributed to the contents of the ns oil especially thymoquinone that enter in building of cholesterol which is important source of cholesterol esters that may have a role in estrogen synthesis.25 regarding thyroid function, the results of t3, t4 and tsh serum level of control and treatment groups are shown in table 1, the plasma concentration of t3 and t4 are significantly (p ≤ 0.005) increased and tsh serum level significantly (p < 0.005) decreased in treatment groups compared with control groups. the treatment with oral administration of ns increased t4 levels in rabbits.26 these results indicated that oral administration of ns lead to hyperthyroidism in mice; likewise, other study reports that the oral administration of ns not only increased serum t3 and decreased tsh but also has an anti-oxidant effect.27 the oral administration with ns could raise t3 level without changing t4 and tsh serum concentration levels.28 further work is suggested for evaluating the effect of ns on the serum concentration of thyroid hormones to clarify the possible mechanism of action. histological result of thyroid gland revealed normal appearance of thyroid tissue and intact follicular structure in control rats, thyroid follicles lined with cuboidal epithelial lining filled with colloid (fig. 1). thyroid gland of female rats treated with ns showed no histological changes when compare with the control except that some of the follicles were enlarged and containing vacuolated colloid as appear from figure 2. this might be attributed to increased endocytotic activity of many follicular cells reflecting a compensatory mechanism to the augmented release of the stored hormones in the follicular lumen.29 conclusion in conclusion, the nigella sativa oil caused elevation of thyroid hormones as well as lh and estrogen but still within normal value and histological sections of thyroid gland revealed presence vaculation in colloid of thyroid follicle.  table 1. shows effect of nigella sativa oil on some hormones in female rats (mean ± se) parameter groups lh (μiu/ml) estrogen (pg/ml) total t3 (ng/dl) total t4 (μg/dl) tsh (μlu/ml) control 0.42 ± 0.01 29.08 ± 0.75 0.13 ± 0.02 72.04 ± 1.29 0.05 ± 0.006 treatment 0.31 ± 0.12* 41.27 ± 1.42* 1.17 ± 0.07* 93.64 ± 1.27* 0.02 ± 0.01* *represent significant difference at (p ≤0.05). fig. 1 light micrograph for thyroid gland of control rat, shows normal architecture, thyroid follicles, filled with colloid (star) and lined by cuboidal thyrocytes (thin arrow) (h&e) 100x. fig. 2 light micrograph of histological changes in thyroid gland of female rat treated with nigella sativa shows thyroid follicles vary in size (arrow), some of follicles with vacuolated colloid (stars). h&e, 100x. 69j contemp med sci | vol. 2, no. 6, spring 2016: 67–69 research study the effect of nigella sativa on thyroid function and reproductive hormone of female ratwafaa kadhim jasim et al. 11. semsam shariat h. medicinal plants. 2nd ed. esfahan: chaharbagh; 2007. p. 32. 12. zargari a. medicinal plants. 6th ed. tehran: university press; 1990. pp. 43–44. 13. kanter m, demir h, karakaya c, ozbek h. gastroprotective activity of nigella sativa l oil and its constituent, thymoquinone against acute alcohol-induced gastric mucosal injury in rats. world j gastroenterol. 2005;11:6662–6666. 14. david rw, omar ag, peter ac. the in vitro antitumor activity of some crude and purified components of block seed (nigella sativa) anticancer res. 1998;18:1527–1532. 15. salomi mj, nair sc, panikkar kr. inhibitory effects of nigella sativa and saffron (crocus sativus) on chemical carcinogenesis in mice. nutr cancer. 1991;16:67–72. [pubmed]. 16. perveen t, haider s, kanwal s, haleem dj. repeated administration of nigella sativa decrease 5-ht turnover and produces anxiolytic effects in rats. pak j pharm sci. 2009; 22:139–144. 17. hanafy ms, hatem me. studies on the antimicrobial activity of nigella sativa seed (black cumin) j ethnopharmacol. 1991;34:275–278. 18. salem ml, hossain ms. protective effect of black seed oil from nigella sativa against murine cytomegalovirus infection. int j immunopharmacol. 2000;22:729–740. 19. houghton pj, zarka r, de las heras b, hoult jr. fixed oil of nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. planta med. 1995;61:33–36. 20. burits m, bucar f. antioxidant activity of nigella sativa essential oil. phytother res. 2000;14:323–328. 21. bamosa ao, ali ba, sawayan sa. effect of oral ingestion of nigella sativa seed on some blood parameters. saudi pharm j. 1997;5:126–129. 22. mescher al. junqueira s. basic histology text and atlas.12th ed. 2010. p. 1. 23. spss statistical packages for the social sciences. (2001). statistical software for windows version 13.0 micrisoft. spss, chicago, il, usa. 24. boukhliq r, martin gb, white cl, blackberry ma, murray pj. role of glucose, fatty acids and protein in regulating of testicular growth and secretion of gonadotrophin, prolactin, somatotrophin and insulin in the mature ram. j reprod fertil. 1997;9:515–524. 25. wainwarin wlp. the androgen in: austin cr and short rv (eds). reproduction in mammals. cambrige uni press london; 1979. 26. sharif sh, elmahdi bm, ali mohammed am, mohammed ah. the effects of nigella sativa l. ethanolic extract on thyroid function in normal and alloxaninduced diabetic rats. thyroid res pract. 2012;9:48–54. 27. khalawi aa, al-robai aa, khoja sm, shaker as. can nigella sativa oil (nso) reverse hypothyroid status induced by ptu in rat? biochemical and histological studies. life sci j. 2013;10:802–811. 28. meral i, yener z, ozbek h, ustun r. effects of nigella sativa l. on serum concentrations of thyroid hormones, thyroid stimulating hormone and glucose in alloxan-induced diabetic rabbits. j vet med. a physiology, pathology, clinical medicine. 2001;48:593–599. 29. saikia uk, saikia m. drug-induced thyroid disorders. j indian med assoc. 2006;104(10):585–587. 242 j contemp med sci | vol. 5, no. 5, september-october 2019: 242–247 umbilical cord length and cord abnormalities in term singleton pregnancy: a review of pregnancy outcome in a tertiary health institution in nigeria charles njoku,a,b patience odusolu,a,b emechebe cajetan,a,b emmanuel ekanem,b and amarachukwu njokub adepartment of obstetrics and gynaecology, university of calabar, calabar, nigeria. bdepartment of obstetrics and gynaecology, university of calabar teaching hospital, calabar, nigeria. correspondence to charles njoku (email: charlesnjokuobinna@gmail.com). (submitted: 22 july 2019 – revised version received: 08 august 2019 – accepted: 14 august 2019 – published online: 26 october 2019) introduction the umbilical cord (also called the naval string, birth cord or funiculus umbilicus) is a conduit between the fetus and the placenta and indeed the lifeline of the fetus.1 it develops from the remnants of the yolk sac and allantois.1 the umbilical cord serves very important functions which includes delivering oxygen rich blood through the umbilical vein to the fetus. the umbilical cord also serves as a source of nutrient, including calories, proteins, fats, as well as vitamins to the fetus.2 the umbilical cord also transfers waste products and deoxygenated blood away from the fetus to the maternal circulation, where it can be processed and excreted. the umbilical cord length at term has appreciable variation with extremes ranging from no cord to lengths up to 300 cm.3,4 at birth, the mature normal umbilical cord is between 50 and 60 cm in length and 37.7 ± 7.73 mm in diameter.1,3 the umbilical cord can be long or short and there may be as many as 40 spiral twists in the cord as well as false and true knots.3 short cords, defined as cords <32 cm long, are seen in 0.4–0.9% of pregnancies and some studies revealed an incidence of up to 3–10% of all umbilical cords.1,5–7 long cords, defined as cords length longer than 80 cm according to some studies or 100 cm according to others, are seen in 3.7–4% and 0.5% of all umbilical cords during pregnancies respectively.1,6 umbilical cord length is one of the factors documented as a definite risk for poor fetal outcome.8 there is an association of abnormal cord length with neurological abnormalities and low iq values.6,9 excessively long or short cord may be the cause of hematoma and thrombosis of cord vessels and the placental surface, thus causing fetal death and or thrombocytopenia.10 causes of differences in cord length are unknown; however, the length of the umbilical cord is thought to reflect the sufficient space in the amniotic cavity for movement and the tensile strength applied to the umbilical cord during fetal movement.11 a short cord may be due to reduced fetal activity (such as with twinning; monoamniotic and conjoined), as a primary failure of elongation, and in association with sirenomelia (lack of adequate fetal blood pressure), schisis, anencephaly (lack of hypothalamic hormones), acardia (cardiac output) and adhesions (early amniotic rupture sequence).6 it may also be due to oligohydramnios, amnion rupture, uterine structural anomalies as well as substances such as alcohol and beta blockers.5 short cords can interfere with the mechanics of labor and delivery while exhibiting changes in fetal heart rate patterns.6,12 this restriction of decent (which is relative to the placental position and insertion) leads to an increase in the incidence of caesarean section, forceps and vacuum extractions. other complications of excessively short cords include delay in second stage of labor, retained placenta, placental abruption, rupture of umbilical cord, inversion of uterus, birth asphyxia, and cord herniation.3 on the other hand, long umbilical cord is directly associated with poor fetal outcome and umbilical cord accidents especially; fetal entanglement and true knots.6 placental changes are associated with long cords suggesting blood flow disruption or objective to determine the association of feto-maternal outcomes of term pregnancies with fetal umbilical cord lengths and cord abnormalities in calabar. methods a cross-sectional study of 600 women with singleton pregnancies who delivered either virginally or caesarean section between 37 and 42 completed weeks. examination of cord was done at delivery for loop round neck, cord length, knots and cord abnormalities. outcomes recorded include fetal presentation, sex, birth weight, length of newborn and apgar scores at 1st and 5th min. also, mode of delivery, labor duration and maternal complications were noted. cords with abnormalities were sent for histological examination. data was analyzed using spss version 20. level of significance was set at p-value <0.05. results the mean cord length was 61.07 ± 14.931 cm, short cords were 23 (7.7%), long cords were 37 (12.3%) and 480 (80.0%) were normal length. male fetuses had longer cords, mean cord length in the vertex presentations were significantly longer than in breech presentation and increased with increase in birth weight. abruptio placentae was higher among fetuses with short umbilical cord (17.4% vs. 0.4%) (p-value = 0.000). there was positive correlation between fetal weight and umbilical cord length. cord coiling index showed a negative correlation with cord length (r = −0.261; p-value = 0.000). conclusion abnormal umbilical cord lengths significantly predispose to obstetric complications but cord abnormalities are rare and does not affect pregnancy outcome. high index of suspicion and careful evaluation of cord may inform and reduce untoward feto-maternal outcome. keywords umbilical cord, length, complications, obstetric outcome issn 2413-0516 original 243j contemp med sci | vol. 5, no. 5, september-october 2019: 242–247 c. njoku et al. umbilical cord length and cord abnormalities in term singleton pregnancy increased resistance.12 male cords are longer than female cords and term vertex fetuses may have longer lengths than term breech fetuses.6 multigravida cord length may be longer than primagravida cord length. other complications of excessively long umbilical cords include cord prolapse, torsion, and delivery complications. there are also more cases of fetal distress, fetal anomalies, and respiratory distress.3 a multivariate analysis of a small sample of pregnancy reported an association with long cords and intrauterine growth restriction.6 the length of the umbilical cord and its position within the amniotic fluid are of great clinical relevance and is associated with a wide range of unfavorable obstetric outcome and this study aims to determine the relationship between length and abnormalities of the umbilical cord and obstetric outcome. though the pathogenesis of variability of umbilical cord length remains largely unclear, fetal demise or compromise due to cord complication could be a source of depression to the obstetrician, medical community and the mother. in our locality, there is scarcity of data on umbilical cord length and cord abnormalities with relation to feto-maternal outcome. this study would provide information about the length of umbilical cord and cord abnormalities, its association with adverse obstetric outcome in our locality. this will increase awareness, guide obstetricians to pay more attention to, and carefully evaluate umbilical cord length and possible abnormalities to avoid untoward perinatal complications. materials and methods this study is a prospective cross-sectional study carried out at the labor ward and theatre of obstetrics and gynaecology department of university of calabar teaching hospital, calabar. it is a tertiary health facility located in calabar, south of nigeria. ethical approval was obtained from the research and ethics committee of the hospital. the study was carried out over a 5-months period between february 15 and july 14 2016. simple random sampling was used to select participants for the study. the inclusion criteria were primigravida or multigravida with singleton pregnancies who had either vaginal delivery or caesarean section between 37 and 42 completed weeks. pregnancies complicated by intrauterine fetal death, multiple gestations, congenital malformations of fetus, diabetes, preeclampsia, chorioamnionitis, preterm labor and intrauterine growth restriction were excluded from the study. the socio demographic characteristics were obtained using pretested questionnaire and this includes initials of clients, hospital number, age, marital status, parity, last menstrual period and gestational age in weeks. before delivery, fetal presentation, lie and position were determined by abdominal palpation. the mode of delivery was also noted. at delivery the umbilical cord was examined for the following: the presence of any loop around neck, trunk, cord knots (true or false) and any cord abnormalities such as cyst, hematoma, and velamentous insertion. after the delivery of fetus, the umbilical cord was clamped at two places and cut in between. the length of the umbilical cord was measured from the cut end up to fetal umbilicus and from the other cut end to the placental attachment and the two measurements added to obtain the umbilical cord length. it was measured with flexible tape in centimeters. other abnormalities of the umbilical cord after delivery were noted clinically. lengths of cords were measured and labelled as long (≥80 cm), short (≤32 cm) or normal (between 32 and 80 cm). thereafter umbilical cords with abnormalities were sent for histological analysis. type of insertion on the placenta was also noted. the placenta was examined under running water to remove blood clots and the placental weight measured using a weighing scale and recorded in grams. fetal parameters that were recorded after the time of delivery include apgar scores at 1 and 5 min, sex of the newborn, weight of the newborn and length of newborn (by measuring the distance between the crown and the heel). maternal complications of labor and delivery which includes poor decent of presenting part, prolonged second stage of labor and placental abruption were also noted. data obtained were analyzed using the statistical package for social sciences version 20. level of significance was set at p-value <0.05. results during the study period, there were 1433 deliveries, 600 of the parturient were recruited for the study. umbilical cord length varied from 20 to 110 cm with mean length of 61.07 ± 14.931 cm. short umbilical cords were 46 (7.7%), normal cord were 480 (80.0%) while long cords were 74 (12.3%). vaginal delivery was 412 (68.7%) while caesarean section was 188 (31.3%). the mean umbilical cord length was 61.07 ± 14.93 cm, mean gestational age 38.94 ±1.326 weeks and mean parity 2.21 ± 1.239. table 1 outlines the socio-demographics characteristics of the parturient. majority of the parturient were between 30 and 34 years of age (38.0%), married (97.7%) and women in their first and second pregnancy (para 1 and 2) (66.0%). mean umbilical cord length was longer among the age group of 20–24 (63.13 ± 15.69 cm), married women (61.30 ± 14.86 cm) and women in their third and fourth pregnancy (para 3 and 4) (63.27 ± 14.82 cm). the male fetuses generally had statistically significant longer umbilical cord length than female fetuses (63.74 ± 15.66 cm vs. 57.97 ± 13.44 cm) as shown in table 2. the mean cord length in the vertex presentation group was significantly longer than the breech group (61.23 ± 14.95 cm vs. 54.29 ± 13.25 cm). the mean umbilical cord length increased with increase in birth weight and there was statistically significant difference between umbilical cord lengths and birth weight. longer umbilical cords were significantly hypocoiled while shorter umbilical cords were hypercoiled. placental weight group of 0.9–1.0 kg had the longest mean umbilical cord length and there was statistically significant difference between placenta weight and umbilical cord length (p-value = 0.018). there was no statistically significant difference between umbilical cord lengths and umbilical cord abnormalities (p-value = 0.925). figure 1 shows the distribution of umbilical cord complications. most parturient had no umbilical cord complications (82.3%) and the most common complication was nuchal cord (12.0%). true knot occurred in 1%, cord round body in 4% while cord prolapse was 0.7%. table 3 shows the correlation between umbilical cord length and feto-maternal outcome. when the umbilical cord length was compared with placental weight using the pearson’s correlation, there was significant positive correlation between placental weight and umbilical cord length (r = 0.212; p-value = 0.000). there was also significant positive correlation between umbilical cord length and fetal weight. umbilical original 244 j contemp med sci | vol. 5, no. 5, september-october 2019: 242–247 umbilical cord length and cord abnormalities in term singleton pregnancy c. njoku et al. table 1. the feto-placental characteristics in relation to mean cord length variables frequency (%) cord length (cm) test statistics mean ± sd sex male 161 63.74 ± 15.66 4.8428 <0.001 female 139 57.97 ± 13.44 lie/presentation vertex 293 61.23 ± 14.95 6.0171 <0.001 breech 7 54.29 ± 13.25 fetal weight <2.5 kg 9 60.52 ± 15.05 1.813 0.046 2.5 ≤ 4.0 kg 269 64.33 ± 14.57 4.0 kg and above 22 66.41 ± 12.85 length of the neonate below normal <45 4 64.75 ± 18.998 normal 45–55 289 60.98 ± 15.012 0.216 0.806 greater normal >55 7 63.43 ± 9.846 cord coiling index hypocoiled <0.17 129 (43.0) 65.32 ± 12.61 10.904 0.000 normal 0.17–0.37 165 (55.0) 58.18 ± 15.46 hypercoiled >0.37 6 (2.0) 49.17 ± 22.61 types of umbilical cord insertion centric 151 (50.4) 61.21 ± 14.775 eccentric 103 (34.3) 61.58 ± 15.288 0.241 0.869 marginal 45 (15.0) 59.38 ± 15.01 vilamentous 1 (0.15) 63.00 placental weight (kg) 0.1–0.2 1 (0.3) 64.00 0.3–0.4 24 (8.0) 57.25 ± 15.098 0.5–0.6 121 (40.4) 58.4 ± 15.837 2.777 0.018 0.7–0.8 129 (43.0) 62.96 ± 13.832 0.9–1.0 22 (7.3) 68.82 ± 12.764 >1.0 3 (1.0) 59.67 ± 14.154 cord abnormalities none 297 (99.0) 61.10 ± 14.973 single umbilical artery 2 (0.7) 57.50 ± 16.263 0.078 0.925 umbilical cord cyst 1 (0.3) 58.00 table 2. correlation between length of umbilical cord with maternal characteristics and obstetric outcome obstetrics outcome umbilical cord length p-value correlation (r) birth weight 0.179 0.037* gestational age 0.089 0.124 5th minute apgar score −0.022 0.703 length of neonate 0.066 0.255 maternal age 0.002 0.975 parity 0.065 0.260 duration of labor 0.100 0.085 umbilical cord coiling index −0.261 0.000* placental weight 0.212 0.000* *correlation is significant at <0.05 levels (two-tailed). fig. 1 umbilical cord complications. table 3. the relationship between umbilical cord length and feto-maternal outcomes variables feto-maternal outcome chi-square p-value 1st minute apgar <7 ≥7 short cord 2 (8.6) 21 (91.3) 0.60 0.752 long 8 (21.6) 29 (78.4) 1.21 0.271 normal 35 (14.6) 205 (85.4) referenced 5th minute apgar <7 ≥7 short cord 2 (8.6) 21 (91.3) 0.30 0.639 long 3 (8.1) 34 (91.9) 0.29 0.483 normal 14 (5.8) 226 (94.2) referenced delayed 2nd stage yes no (continued) original 245j contemp med sci | vol. 5, no. 5, september-october 2019: 242–247 c. njoku et al. umbilical cord length and cord abnormalities in term singleton pregnancy table 3. the relationship between umbilical cord length and feto-maternal outcomes—continued variables feto-maternal outcome chi-square p-value short cord 2 (8.7) 21 (91.3) 3.54 0.117 long 1 (2.7) 36 (97.3) 0.06 0.581 normal 5 (2.1) 235 (97.9) referenced abruptio placentae yes no short cord 4 (17.4) 19 (82.6) 32.42 0.000 long 1 (2.7) 36 (97.3) 2.34 0.249 normal 1 (0.4) 239 (99.6) referenced low birth weight (<2.5 cm) yes no short cord 2 (8.7) 21 (91.3) 2.12 0.181 long 1 (2.7) 36 (97.3) 0.01 1.000 normal 7 (2.9) 233 (97.1) referenced macrosomia (>4 kg) yes no short cord 1 (4.3) 22 (95.7) 0.04 1.000 long 3 (8.1) 34 (91.9) 0.08 0.968 normal 19 (7.9) 221 (92.1) referenced cord round neck yes no short cord 0 (0.00) 23 (100) 2.88 0.088 long 9 (24.3) 28 (75.7) 4.85 0.028 normal 27 (11.3) 213 (88.7) referenced breech presentation yes no short cord 2 (8.7) 21 (91.3) 4.65 0.031 long 1 (2.7) 36 (97.3) 0.19 0.514 normal 4 (1.7) 236 (98.3) referenced cord coiling index showed a significant negative correlation with umbilical cord length (r = −0.261; p-value = 0.000). abruptio placentae was significantly higher among fetuses with short umbilical cord than normal umbilical cord length (17.4% vs. 0.4%) and this difference was statistically significant (p-value = 0.000). cord round neck was significantly higher among long umbilical cord than the normal umbilical cord length control (p-value = 0.036). discussion several studies concerning umbilical cord length at term showed that umbilical cord length varies from no cord to lengths up to 300 cm and mature umbilical cord is between 50 and 60 cm in length.1,2,4 in this study the mean umbilical cord length was 61.07 cm and ranged from 20 to 110 cm. this study is comparable with the findings in sudan where the mean cord length was 60.5 cm13 and agwu et al.14 in abakaliki, nigeria where the mean cord length was 57 cm and ranged from 22 to 124 cm. however, this was higher than 52.7 cm reported in ilorin by adesina et al.15 this showed that human neonates exhibit wider variations in terms of the length of their umbilical cord as earlier documented by jaya et al.16 and stefos et al.17 respectively. the reason for the difference in the mean values of umbilical cord length obtained in these studies is not very clear but may be because umbilical cord length is influenced by environmental and genetic factors.16 although it is not fully understood what controls cord length, various authors correlate cord length with fetal activity and movement.18 it is suggested that sufficient space in the amniotic cavity for movement and the tensile force applied to the umbilical cord during fetal movements are two main factors that determine cord length. however, more recent studies using animal models have argued against the “stretch hypothesis,” stating that the umbilical cord continues to grow throughout pregnancy in an almost linear fashion.5,15 the incidence of short cords in this study was 7.7% of all deliveries is similar to the incidence of 5.9% in the study by balkawade and shinde3 and 7.2% by adesina et al. 15 this finding is also similar to other reported incidence of short cords which ranged from 2% to 10%.13,14,19 the incidence of 7.7% in this study is however higher than 0.7% reported in abakaliki, nigeria.14 the marked difference in the reported low prevalence of short cord in that study may have been influenced by the low cut off of <32 cm used in the definition of short umbilical cord. in this present study, the incidence of long umbilical cord was 12.3% which is higher than 7% by agwu et al.14 and 9.3% by adesina et al.15 the significance of long umbilical cords resides in the fact that they may be directly associated with poor fetal outcome and umbilical cord accidents such as fetal entanglement, knot formation (multiple) and torsion.9 this study showed that the male fetuses had statistically significant longer umbilical cord length than the female fetuses and there was a significant positive correlation between the sex of neonate and umbilical cord length. this may be explained by the fact that male fetuses normally weigh more than their female counterparts and also by the fact that length of umbilical correlates significantly with birth weight. this is consistent with the findings of krakowiak et al.5 who demonstrated that female infants had shorter cords than male infants. in this present study, the mean umbilical cord length increased with increase in birth weight and umbilical cord table 4. the maternal characteristics in relation to mean cord length of the study population variables frequency (%) cord length (cm) mean ± sd age (years) 14–19 11 (3.7) 58.64 ± 23.61 20–24 30 (10.0) 63.13 ± 15.69 25–29 97 (32.3) 60.47 ± 14.57 30–34 114 (38.0) 61.24 ± 14.93 35 and above 48 (16.0) 61.13 ± 13.17 marital status married 293 (97.7) 61.30 ± 14.86 single 7 (2.3) 51.14 ± 15.65 parity 1–2 198 (66.0) 60.20 ± 15.09 3–4 86 (28.7) 63.27 ± 14.82 5 and above 16 (5.3) 59.94 ± 13.03 original 246 j contemp med sci | vol. 5, no. 5, september-october 2019: 242–247 umbilical cord length and cord abnormalities in term singleton pregnancy c. njoku et al. length was positively correlated with birth weight. fetuses with longer umbilical cord were likely to have normal birth weight or to be macrosomic. this could be explained by the fact that as the fetus grows the umbilical cord also increases in length. agboola20 also reported a significant positive correlation between cord length and fetal weight. wu et al.21 in their series using stepwise logistic regression for multivariate analysis of the relationship between umbilical cord length and obstetric outcome found a statistically significant relationship between umbilical cord length and birth weight. concerning umbilical cord index, 43% were hypocoiled, 55% normocoiled and 2% hypercoiled. umbilical cord coiling index showed a significant negative correlation with umbilical cord length which means that with increase in umbilical cord length, the coiling index decreases. abnormal coiling (hypo-coiling and hyper-coiling) is known to have chronic (growth retardation) and acute (fetal intolerance to labor and fetal demise) effects on fetal wellbeing.22,23 the vessels of the umbilical cord are prone to torsion, compression, tension, and subsequent interruption of the blood flow. this risk is minimized by their helical disposition. it is possible that the coiled umbilical cord has elastic properties that enable it to resist external forces that might compromise the umbilical vascular flow. it may be that, the coiled umbilical cord acts like a semi-erectile organ that is more resistant to snarling torsion, stretch, and compression than the non-coiled one. hypercoiled cords often have more thrombi in placental surface veins because the flow is more sluggish, and when coiling becomes excessive, the fetus can strangle due to decreased circulation in the cord vessels.24,25 ercal et al.23 when comparing neonates with hypocoiled and normocoiled found a higher incidence of meconium staining, interventional delivery, apgar scores, fetal blood ph and intrapartum fetal heart rate disturbance in neonates with hypocoiled cords and concluded that umbilical cord index has a strong relationship with perinatal outcome. thus, detection of an abnormal coiling index can lead to identification of fetuses at risk which need special care to improve perinatal outcome. there was statistically significant difference between umbilical cord length and placenta weight in this study. the placental weight showed significant positive correlation with umbilical cord length which means that umbilical cord length increases with increase in placental weight. it then implies that, factors which directly affect the weight of the placenta will indirectly affect the length of the umbilical cord. such factors include nutrition, maternal anemia, altitude, hypertension, maternal diabetes mellitus and other chronic medical illness. an abnormal placental cord insertion site has been associated with a number of complications of pregnancy that may result from compression or rupture of poorly supported umbilical vessels.1 intrapartum hemorrhage, fetal bradycardia, stillbirth, intrauterine growth restriction and preterm labor have all been linked to velamentous and to a lesser extent, marginal cord insertions.1,26 in this present study, there was only a single reported case of velamentous umbilical cord insertion and there was no adverse maternal, fetal or perinatal outcome associated with it. in a study conducted by adesina et al.,15 velamentous cord insertions occurred in 1.1% and 0.9% respectively of singleton pregnancies. several articles concerning umbilical cord length and feto-maternal outcome have been published in the past with conflicting reports.1,6,14,21 various reports have demonstrated that a short umbilical cord was associated with birth asphyxia, abruptio placentae, breech presentation and prolonged second stage of labor. krakowiak et al.5 in their study reported an increase in hypoxic–ischemic encephalopathy, fetal distress, and infant death and low birth weight and a twofold increase in risk of death among term infants born with short cords. on the contrary, a long umbilical cord was frequently associated with cord accidents and fetal distress. in this study, only abruptio placentae and breech presentation were significantly higher among fetuses with short umbilical cord than normal umbilical cord length control. also, only cord round neck was significantly higher among long umbilical cord than the normal umbilical cord length control. low birth weight, macrosomia and delayed second stage of labor were not significantly different among long and short umbilical cord compared with normal cord length control. this study also did not found an increase in intrapartum fetal distress and birth asphyxia with length of umbilical cord. this finding is similar to the study in taiwan where multivariate analysis of the relationship between umbilical cord length and obstetric outcome found no association between umbilical cord length and antepartum and intrapartum fetal wellbeing.21 however, this is in contrast to the study where agwu et al.14 found an increase intrapartum fetal distress and birth asphyxia in the long cord arm group. the difference with study may be related to the difference in patients’ selection for the study, intrapartum monitoring and caesarean sections rates in different studies. umbilical cord abnormality was 1% of total deliveries. there was no significant association between varying umbilical cord lengths and umbilical cord abnormalities. this may be due to rarity of these abnormalities as it occurs in 1% of live birth from other studies also.14,15,27 another study also reported an incidence of 0.2%. adesina et al.15 noted that single umbilical artery is associated with low birth weight babies and preterm deliveries. neonates with single artery have increased risks of congenital and chromosomal abnormalities as well as adverse perinatal outcome.27 the two neonates with single umbilical cord had moderate birth asphyxia but made good recovery in the perinatal period and there were no obvious congenital anomalies among them. this study did not utilize the intrapartum evaluation with doppler ultrasound to possibly determine cord length and abnormalities as to reduce feto-maternal complications during delivery. further research is therefore needed to determine the umbilical cord characteristics using high definition doppler ultrasound and relationship with obstetric outcome. conclusion the study showed that the length of umbilical cord varied from 20 to 110 cm with mean of 61.07 ± 14.931 cm. abruptio placentae and breech presentation were significantly higher among fetuses with short umbilical cord than normal and cord round neck was significantly higher among long umbilical cord than the normal cord length. cord abnormalities were very rare and there were no significant association between umbilical cord lengths and umbilical cord abnormalities from this study. appropriate examination and documentation of umbilical cord length and abnormalities is necessary. this will original 247j contemp med sci | vol. 5, no. 5, september-october 2019: 242–247 c. njoku et al. umbilical cord length and cord abnormalities in term singleton pregnancy provide more information on fetal well-being, neonatal outcome and basis for further studies. ante partum or intrapartum obstetric ultrasound scan for possible abnormal cord lengths and cord abnormalities may reduce untoward pregnancy outcome. conflicts of interest none.  references 1. kulkarni ml, matadh ps, ashok c, pradeep n, avinash t, kulkarni am. absence of wharton’s jelly around the umbilical arteries. indian j pediatr 2007;74:787–789. 2. pathak s, hook e, hackett g, murdoch e, sebire nj, jessop f, et al. cord coiling, umbilical cord insertion and placental shape in an unselected cohort delivering at term: relationship with common obstetric outcomes. placenta. 2010;31:963–968. 3. balkawade nu, shinde ma. study of length of umbilical cord and fetal outcome : a study of 1,000 deliveries. j obstet gynecol india. 2012;62: 520–525. 4. yadav b, kurdukar, dv, darade rv, mahadar na. correlation of umbilical cord length and foetal outcome. j evol meical dent sci. 2013;2:412–417. 5. krakowiak p, smith en, de bruyn g, lydon-rochelle mt. risk factors and outcomes associated with a short umbilical cord. obstet gynecol. 2004;103:119–127. 6. collins jh. umbilical cord accidents. bmc pregnancy childbirth. 2012;12:a7. 7. lamonica ge, wilson ml, fullilove am, rayburn we. minimum cord length that allows spontaneous vaginal delivery. j reprod med. 2008;53: 217–219. 8. hershkovitz r, silberstein t, sheiner e, shoham-vardi i, holcberg g, katz m, et al. risk factors associated with true knots of the umbilical cord. eur j obstet gynecol reprod biol. 2001;98:36–39. 9. sørnes t. umbilical cord knots. acta obstet gynecol scand. 2000;79: 157–159. 10. sangwan v, nanda s, sangwan m, malik r, yadav m. cord complications : associated risk factors and perinatal outcome. open j obstet gynecol. 2011;1:174–177. 11. beall mh, ross mg. umbilical cord complications. medscape. 2014. 12. collins cl, collins cl, collins cc. umbilical cord accidents. pregnancy inst. 2010:1–84. 13. ahmed m, nuggedalla a. a preliminary study on the morphological variations in the umbilical cord sundanese. time j med sci rep res. 2013;1:10–15. 14. agwu um, umeora ouj, ogbonnaya lu, iyare fe, obuna ja, umahi ve, et al. fetal umbilical cord length and associated intrapatum complications in a tertiary institution, southeast nigeria. ebonyi med j. 2010;9:112–119. 15. adesina kt, ogunlaja oo, aboyeji ap, olarinoye oa, adeniran as, fawole aa, et al. umbilical cord parameters in ilorin: correlates and foetal outcome. east afr med j. 2014;19:274–280. 16. jaya ds, kumar ns, bai ls. anthropometric indices, cord length and placental weight in newborns. indian pediatr. 1995;32:1183–1188. 17. stefos t, sotiriadis a, vasilios d, tsirkas p, korkontzelos i, avgoustatos f, et al. umbilical cord length and parity—the greek experience. eur j obstet gynecol reprod biol. 2003;107:41–44. 18. bimpong s. qualitative evaluation of umbilical cord and placental indices and pregnancy outcome (master’s thesis). kwame nkrumah university of science and technology, kumasi, 2012. 19. georgiadis l, keski-nisula l, georgiadis s, räisänen s, harju m, heinonen s. short umbilical cord – a risk factor for placental abruption? placenta. 2014;35:275–280. 20. agboola a. correlates of human umbilical cord length. int j gynaecol obstet. 1978-1979;16:238–239. 21. wu jf, chang sy, hsu ty, hsieh ch, kung ft, hwang fr, et al. multivariate analysis of the relationship between umbilical cord length and outcome. chang gung med j. 1996;19:247–252. 22. chitra t, sushanth ys, raghavan s. umbilical coiling index as a marker of perinatal outcome: an analytical study. obstet gynecol int. 2012;2012:213689. 23. ercal t, lacin s, altunyurt s, saygili u, cinar o, mumcu a. umbilical coiling index: is it a marker for the foetus at risk? br j clin pract. 1996;50:254–256. 24. kashanian m, akbarian a, kouhpayehzadeh j. the umbilical coiling index and adverse perinatal outcome. int j gynaecol obstet. 2006;95:8–13. 25. de laat mw, franx a, van alderen ed, nikkels pg, visser gh. the umbilical coiling index, a review of the literature. j matern fetal neonatal med. 2005;17:93–100. 26. appia pk. relationship between the morphology of the placenta, umbilical cord and perinatal outcome (master’s thesis). kwame nkrumah university of science and technology, 2009. 27. martínez-frías ml, bermejo-sánchez e, rodríguez-pinilla e, prieto-merino d, grupo de periférico del ecemc. [characteristics of neonates with and without a single umbilical artery. analysis of two consecutive series of neonates with and without congenital defects]. ann pediatr (barc). 2006;65:541–550. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201902 original 145j contemp med sci | vol. 7, no. 3, may-june 2021: 145–151 original design, synthesis, and biological evaluation of camptothecin loaded biotinylated cellulose nanowhiskers as anticancer agents osamah n. wennas1*, mohammed h. mohammed1, raid m. al abood2, and dhulfiqar ali abed3,4 1 department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. 2 department of pharmacy, al safwa university college, karbala, iraq. 3 department of pharmaceutical chemistry, college of pharmacy, university of babylon, babylon, iraq. 4 department of medicinal chemistry, ernest mario school of pharmacy, rutgers, the state university of new jersey, 160 frelinghuysen road, piscataway, nj 08854, united states). *correspondence to: correspondence to: osamah n. wennas (e-mail: osamawennas@gmail.com) (submitted: 13 february 2021 – revised version received: 28 march 2021 – accepted: 12 april 2021 – published online: 26 june 2021) introduction cancer is a broad group of diseases that involves uncontrolled proliferation of abnormal cells that are capable of de-differentiation, invasion, and metastasis through the bloodstream or the lymphatic system.1–5 camptothecins, like camptothecin (cpt, 1, figure 1) are regarded as one of the most promising anticancer drugs of the 21st century.6 however, camptothecins have many challenges that need to be overcome. the problems include fast and ph-dependent hydrolysis of the lactone structure to give inactive carboxylate form, poor water solubility, and low and variable oral bioavailability.7 different approaches are being investigated to modulate camptothecins’ systemic delivery, such as the development of prodrugs, or semi-synthetic analogs such as topotecan (2) and irinotecan (3), which are used clinically for the treatment of colon and ovarian cancers, respectively. another approach is the polymer conjugates such as amphiphilic peg−cpt conjugates (4),8,10 which improve the biological distribution, increase the body’s retention times, reduce systemic toxicity, and improve the therapeutic efficacy.8,11 cellulose molecules with at least one dimension within the nanoscale (1–100 nm) are considered as a nanocellulose.12 nanocellulose can be divided into two main categories, cellulose nanowhiskers (cnws), also known as cellulose nanocrystals (cncs), and cellulose nanofibrils.13,14 among them, cnws drew a lot of attention due to their properties like a high degree of crystallinity, smaller nanoparticle sizes, and better dispersion.15 cnws are considered interesting nanosize carriers for drug delivery because of their numerous advantages, like being biocompatible and not triggering an immune response.16 cnws have high hydrophilicity that impedes opsonin proteins’ adsorption.17 they are also suitable for different types of chemical linkages because the hydroxyl groups on the surface of cnws can be easily modified to other chemical groups.18 in the current study, reduction-sensitive nanoparticles were designed from biotndecorated cnwscpt with disulfide linkage. the presence of disulfide between the cnws and cpt was suggested to release the active drug in response to the elevated intracellular gsh. biotin has been used as targeting moiety with many anticancers to prevent the non-specific normal cells attack and to increase the uptake by the target cells. the in vitro drug release was studied at different gsh concentrations, and the cytotoxic effects of the nano conjugate on mcf7, hpeg2, and cho cell lines were studied and compared with cpt prodrug without the nanocarrier. materials and methods materials camptothecin, biotin, and 3,3'-dithiodipropionic acid were purchased from beijing yibai biotechnology co., ltd. cnws were purchased from the process development centerthe university of maine u.s.a. other chemicals were purchased abstract objective using biotinylated cellulose nanowhiskers (cnws), we designed and synthesized a glutathione (gsh) sensitivecamptothecin (cpt) prodrug for selective cpt delivery (compound 12). methods cpt-biotin (compound 9), was synthesized by direct conjugation of cpt to the biotin via gsh sensitive linkage to evaluate the role of cnws in compound 12. the chemical structures of the synthesized prodrugs were confirmed by ft-ir, 1h nmr, 13c nmr, and esi-ms, while the nanoparticles were characterized by dls and tem. results the in-vitro drug release assay demonstrated that only 18.6% of cpt was released from the nano conjugate under gsh stimulation at micromolar level (100 μm), while 83.1% accumulative release rate was achieved under gsh stimulation at millimolar level (10 mm). the in-vitro cytotoxicity assay (mtt assay) demonstrated that compound 9 showed higher inhibition ratios on biotin positive cells, mcf-7, and hepg2, and lower cytotoxicity on biotin negative, cho. compound 12 showed good activity against mcf-7, hepg2, and much lower cytotoxicity on cho. conclusion this work demonstrates cpt-biotinylated cellulose nanowhiskers for selective chemotherapy and may have the potential to be used for cancer targeting. keywords anticancer, camptothecin, cellulose nanowhiskers, biotin, glutathione. issn 2413-0516 146 j contemp med sci | vol. 7, no. 3, may-june 2021: 145–151 design, synthesis, and biological evaluation of camptothecin loaded biotinylated cellulose nanowhiskers original o. n. wennas et al. from sigma–aldrich. all chemicals are of analytical grade, and they were used as received without further purification. characterization of compounds 5-9 and compound 11 melting points, fourier transform infrared spectroscopy: ftir, nmr: 1h nmr and 13c nmr spectra, and electrospray ionization mass spectrometry: esi-ms were performed for compound characterization. chemical synthesis the target compounds were synthesized by multi-step reactions, as shown in schemes 1&2. synthesis of biotin methyl ester (compound 5) 0.75 g (3.075 mmol) of biotin was suspended in 75 ml methanol, 0.5 g of the catalyst (amberlite ir-120 resin) was added, and the suspension was stirred at room temperature for 24 h. after filtration and solvent’s evaporation under reduced pressure, the title compound was obtained as a white powder, mp. 165-166oc, yield 88%. 1h nmr (500 mhz, dmso-d6) 6.46 (1 h, s), 6.38 (1 h, s), 4.35 – 4.29 (1 h, m), 4.14 (1h, m), 3.59 (3 h, s), 3.12 (1 h, dd, j 4.9, 3.2), 2.83 (1 h, dd, j 12.4, 5.1), 2.59 (1 h, d, j 12.5), 2.31 (2 h, t, j 7.5), 1.68 – 1.42 (4 h, m), 1.35 (2 h, tt, j 14.3, 6.6). 13c nmr (126 mhz, dmso-d6) 173.78, 163.22, 61.52, 59.68, 55.81, 51.66, 40.33, 33.57, 28.48, 28.44, 24.95. esi (ms) calcd for c11h19n2o3s (m+h) + 259.11, found 259.3. synthesis of biotin hydrazide (compound 6) 258 mg (1 mol, 1.00 equiv) of compound 5 was dispersed in 4 ml of methanol, 0.5 ml, (10 mmol, 10.0 equiv) of hydrazine was added. the suspension was stirred for 24 h at room temperature. the solvent was removed under reduced pressure, and the title compound was obtained as a white powder after washing with chloroform, mp. 245-247oc, yield 90%. 1h nmr (500 mhz, dmso-d6) 8.93 (1 h, s), 6.44 (1 h, s), 6.37 (1 h, s), 4.32 (1 h, dd), 4.15 (3 h, m), 3.10 (1 h, m), 2.83 (1 h, dd, j 12.4, 5.0), 2.59 (1 h, d, j 12.4), 2.02 (2 h, t, j 7.4), 1.67 – 1.41 (4 h, m), 1.31 (2 h, tt, j 14.3, 6.5). 13c nmr (126 mhz, dmso-d6) 172.02, 163.21, 61.52, 59.68, 55.89, 33.71, 28.70, 28.49, 25.70. ms (esi) calcd for c10h19n4o2s (m+h) + 259.12, found 259.3. synthesis of 3,3'-dithiodipropionic anhydride (compound 7) 3,3'-dithiodipropionic acid 2 g was dissolved in 5 ml of acetyl chloride in a round bottom flask and refluxed at 65ºc for 4 h until the solution was clear. after most of the solvent is removed under reduced pressure at 50ºc, iced ethyl ether was added to precipitate dithiodipropionic anhydride. after filtration and washing with petroleum ether, the title compound was obtained as a white powder, mp. 65-68oc, yield 38%. synthesis of camptothecin -3,3'-dithiodipropionic acid cpt-ss-cooh. (compound 8) compound 7 (1.923 g, 10 mmol) and compound 1 (0.348 g, 1 mmol) were dissolved in pyridine (30 ml), a solution of dmap (0.61 g, 5 mmol) in 10 ml of pyridine was added dropwise at 0 °c under nitrogen atmosphere. the reaction mixture was heated to 70 °c, and the reaction proceeded for 48 h. then, the reaction solution was precipitated by using an excess of methanol, washed with dilute hcl, and dried to give the title compound as a yellow powder mp. 228-230oc, yield 42%. 1h nmr (500 mhz, dmso-d6) 8.66 (1 h, s), 8.15 (1 h, d, j 8.5), 8.10 (1 h, d, j 8.2), 7.89 – 7.82 (1 h, m), 7.70 (1 h, t, j 7.5), 7.18 (1 h, s), 5.51 (2 h, s), 5.26 (2 h, s), 3.09 – 2.89 (6 h, m), 2.59 (2 h, t, j 7.0), 2.18 (2 h, qd, j 7.2, 3.8), 0.92 (3 h, t, j 7.4). 13c nmr (126 mhz, dmso-d6) 173.43, 170.84, 167.60, 157.00, 152.76, 148.36, 146.41, 145.71, 131.97, 130.87, 130.19, 129.42, 128.95, 128.41, 128.15, 119.30, 95.71, 76.69, 66.76, 50.68, 34.37, 33.72, 33.62, 32.90, 30.69, 8.06. ms (esi) calcd for c10h19n4o2s (2m+h) + 1081.21, found 1081.00. synthesis of camptothecin-ss-biotin hydrazide (compound 9) to a stirred solution of compound 8 (540 mg, 1 mmol) in 10 ml of dmf, edc (288 mg, 1.5 mmol) and nhs (172 mg, 1.5 mmol) were added. the solution was stirred at room temperature for 1 h to form nhs ester of compound 8. compound scheme. 1 synthesis schematics of compound 9. reagents and conditions: (a) acetyl chloride, 65ºc, 4 h, (38%); (b) dmap, pyridine, 70°c, 48 h, (42%); (c) amberlite ir 120 resin, methanol, rt, 24 h, 88%; (d) hydrazine, methanol, rt, 24 h, 90%; (e) edc/ nhs, dmso, rt, 48h, 72%. fig. 1 chemical structures of cpt and -some cpt analogs and prodrugs. 147j contemp med sci | vol. 7, no. 3, may-june 2021: 145–151 o. n. wennas et al. original design, synthesis, and biological evaluation of camptothecin loaded biotinylated cellulose nanowhiskers 6 (258 mg, 1 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 48 h. the product was precipitated by the addition of water (100 ml). the product was purified by washing with dilute hcl, and then dried to give the title compound as a white powder, mp. 234-236, yield 72%. 1h nmr (500 mhz, dmso-d6) 9.85 (1 h, d, j 2.1), 9.74 (1 h, d, j 2.1), 8.68 (1 h, s), 8.17 (1 h, d, j 8.5), 8.13 (1 h, d, j 8.2), 7.91 – 7.84 (1 h, m), 7.72 (1 h, t, j 7.6), 7.18 (1 h, s), 6.41 (1 h, s), 6.36 (1 h, s), 5.51 (2 h, s), 5.29 (2 h, s), 4.31 (1 h, m), 4.12 (1 h, m), 3.13 – 3.06 (1 h, m), 3.01-2.92 (6 h, m), 2.82 (1 h, dd, j 12.5, 5.1), 2.61 – 2.51 (3 h, m), 2.18 (2 h, qd, j 7.2, 3.8), 2.12 (2 h, t, j 7.4), 1.56 – 1.43 (4 h, m), 1.35 (2 h, tt, j 14.3, 6.5), 0.94 (3 h, t, j 7.4). 13c nmr (126 mhz, dmso-d6) 171.35, 170.86, 169.41, 167.62, 163.17, 157.02, 152.80, 148.37, 146.43, 145.69, 132.02, 130.92, 130.25, 129.45, 128.99, 128.45, 128.19, 119.33, 95.70, 76.67, 66.78, 61.50, 59.66, 55.87, 50.71, 33.93, 33.65, 33.42, 33.39, 32.82, 30.72, 28.55, 28.48, 25.51, 8.06. ms (esi) calcd for c10h19n4o2s (m+h) + 781.21, found 781.00. synthesis of carboxylated cnws (cnw-cooh, compound 10) 1.02 g of cnws were suspended in 100 ml of distilled water and sonicated for 5 min. 29.5 mg, 0.188 mmol of tempo, and 324 mg, 3.15 mmol nabr were added to the suspension. then, 3.15 mmol of naocl was added slowly to the cellulose suspension. the ph of the mixture was kept at 10-11 by using 0.5 m naoh while stirring the suspension. after about 45 min, the oxidation was terminated by adding 2 ml of methanol. 0.5 m hcl was used to adjust the ph 7. the oxidized cnws were dialyzed against deionized water for 48 h. the concentration of the suspension was determined gravimetrically. synthesis of camptothecin -3,3'-dithiodipropionic hydrazide (compound 11) compound 8 (540 mg, 1 mmol) was dissolved in 15 ml of dmf at room temperature. hobt (162 mg, 1.2 mmol) and edc (230 mg, 1.2 mmol) were added. the mixture was stirred at room temperature; the progress of the reaction was monitored by tlc. after 2 h when all the acid was converted to activated ester intermediates. the activated intermediate was then slowly added (by inverse addition) to a solution of hydrazine (2 mmol) and cyclohexene (0.05 ml) in dmf (10 ml) while the temperature was kept at 0-10 °c. the reaction was complete upon the completion of the addition. the product was precipitated by the addition 100 ml of cold distilled water. after filtration, the product was washed with distilled water and dried to get the title compound as a yellow powder, mp. 215218oc, yield 61%. 1h nmr (500 mhz, dmso-d6) 9.06 (1 h, s), 8.62 (1 h, s), 8.13 (1 h, d, j 8.6), 8.07 (1 h, d, j 8.3), 7.89 – 7.81 (1 h, m), 7.67 (1 h, t, j 7.5), 7.17 (1 h, s), 5.50 (2 h, s), 5.22 (2 h, s), 4.36 (2 h, s), 3.02 – 2.89 (6 h, m), 2.51 (2 h, t, j 7.1), 2.17 (2 h, qd, j 7.2, 3.8), 0.93 (3 h, t, j 7.6). 13c nmr (126 mhz, dmso-d6) 170.88, 169.91, 167.62, 156.97, 152.71, 148.32, 146.38, 145.71, 131.94, 130.86, 130.11, 129.40, 128.91, 128.36, 128.12, 119.31, 95.69, 76.67, 66.78, 50.63, 34.30, 33.65, 32.78, 30.72, 8.07. ms (esi) calcd for c10h19n4o2s (m+h) + 555.13, found 555.10. synthesis of cpt-cnw-biotin (compound 12) 100 ml of compound 10 (0.74 w/v%) was flocculated by adding 6 ml of saturated nacl, separated by centrifugation and rinsed (by redispersion, followed by centrifugation at 3000 rpm for 5 min) three times with 50 ml of acetone, then two times with 50 ml of dmf. the pellets were dispersed in 100 ml of dmf and subjected to 5 min of sonication. the concentration of cnwcooh was determined gravimetrically (0.65% containing 0.75 mmol of cooh). 72 mg of edc (0.37 mmol) and 43 mg of nhs (0.37 mmol) were added to activate half of the cnwcooh. the suspension was stirred for 1 h at rt. 129 mg (0.5 mmol) of compound 6 was added, and the suspension was stirred for 24 h, and then the suspension was transferred to a dialysis tube and dialyzed against dmf for 24 h (3 times). the suspension was transferred to a round bottom flask. 115 mg of edc (0.6 mmol) and 69 mg of nhs (0.6 mmol) were added to activate the remaining cnw-cooh. the suspension was stirred for 1 h at rt. 333 mg (0.6 mol) of compound 11 was added, and the suspension was stirred for 24 h and then the suspension was transferred to a dialysis tube and dialyzed against dmf for 24 h (3 times) and then against di water for 48 h. characterization of nanoparticles conductometric titration the carboxyl content of compound 10 was determined using conductometric titration,19 as shown in the titration curve (figure 2). determination of biotin and cpt content elemental analysis was used to determine biotin content in compound 12 depending on the n%; leco elemental analyzer was used. the amount of cpt was determined by uv-visible spectrophotometer at 372 nm using carry 100 device. particle size and zeta potential horiba sz100 instrument was used to determine the particle size and zeta potential. transmission electron microscopes (tem) after sonication in a water bath, very diluted suspensions of the nanoparticles (0.001 wt.%) were dropped on carbon film-covered copper grids. 2% uranyl acetate solution was scheme. 2 synthesis schematics of compound 12. reagents and conditions; (a) tempo, nabr, naclo, rt, 45 min; (b) hobt, edc, dmf, rt, 2 h; (c) inverse addition of hobt ester to hydrazine, dmf, cyclohexene 0-10oc, 78%; (d) edc, nhs, dmf, rt, 24 h. 148 j contemp med sci | vol. 7, no. 3, may-june 2021: 145–151 design, synthesis, and biological evaluation of camptothecin loaded biotinylated cellulose nanowhiskers original o. n. wennas et al. table 1: characterization data of cnws and cnw conjugates. sample particle size pdi zeta potential biotin % cpt % cnws 68.1±4.19 0.383±0.124 -34.33±1.59 compound 10 81.23±0.3.53 0.462±0.052 -65.2±2.40 compound 12 87.26±3.496 0.369±0.035 -45.16±1.20 8.67 5.6 used for staining the samples on the grid, and the drying was done at ambient conditions. then, the samples were examined by tem (philips model: cm120). in vitro drug release study the cpt release experiments were carried out at 37oc. using two different media, acetate buffer (ph 5.8) with 10 mm gsh and phosphate buffer saline, ph 7.4 (fbs) with 100 μm gsh to simulate the different microenvironments in cancer cells and blood vessels, respectively. in vitro cytotoxicity assay the in vitro cytotoxicity of compounds 9 and 12 was evaluated by mtt assay on biotin-positive cell lines, human breast cancer cell (mcf-7) and human hepatic carcinoma (hepg2), and biotin negative cell line, noncancerous chinese hamster ovarian (cho). mtt was performed to determine the cytotoxic effect of the samples at various concentrations. the results were given as the mean of three independent experiments and the ic50 values were then calculated. results chemical synthesis of (5-9) and 11 compound 5 was prepared by the esterification of biotin with methanol in the presence of high excess of methanol according to fischer esterification using amberlite ir120 resign as acid catalysis.20 ft-ir showed the disappearance of carboxylic acid carbonyl stretching band (1700 cm-1) and the appearance of new band at 1743 cm-1 due to ester carbonyl stretching. 1h nmr of the compound 5 was characterized by the disappearance of cooh proton signal at 12.00 p.p.m. and the appearance of a new signal as an indication of biotin’s successful esterification, the signal was related to cooch3 protons at 3.59 p.p.m. as singlet. compound 6 was obtained by the hydrazinolysis of biotin methyl ester in methanol using an excess of hydrazine hydrate.21 ft-ir showed the disappearance of ester carbonyl stretching band (1743 cm-1) and the appearance of a new band at 1685 cm-1 due to hydrazide carbonyl stretching. 1h nmr of compound 6 was characterized by the disappearance of cooch3 protons at 3.59 p.p.m. and the appearance of new signals to indicate the success of hydrazinolysis of compound 5, the signals were related to conhnh2 protons at 8.93 p.p.m. and 4.15 p.p.m. respectively both of them as a singlet. compound 7, the ft-ir showed the new bands at 1793 cm-1 and 1739 cm-1 due to asymmetric and symmetric stretching vibration of anhydride carbonyl. compound 8 was obtained by the esterification of the hydroxyl of compound 1 with the anhydride (compound 7) using dmap as an acylating catalyst.8 ft-ir showed a new band at 1720 cm-1 due to the carboxylic acid carbonyl stretching. 1h nmr of compound 8 was characterized by the disappearance of alcoholic proton signal at 6.53 p.p.m. and the appearance of new signals to indicate cpt esterification’s success; the signals related to aliphatic protons at 3.00-2.59 p.p.m. compound 9 was obtained by the conjugation of compound 8 and compound 6 (biotin hydrazide) using edc as a coupling agent.22 ft-ir showed the disappearance of the carboxylic acid band at 1720 cm-1 due to amide formation. 1h nmr of compound 9 was characterized by the disappearance of conhnh2 signal at 4.15 p.p.m. and the appearance of new signals that were related to conhnhco protons at 9.85 p.p.m. and 9.74 p.p.m. both of them as a singlet. compound 11 was obtained by the reaction of compound 8 with hydrazine using hobt and edc as coupling agents.23 ft-ir showed the disappearance of the carboxylic acid band at 1720 cm-1 due to azide formation. 1h nmr of compound 11 was characterized by the appearance of new signals to indicate the success of the reaction, the signals were related to conhnh2 protons at 9.06 p.p.m. and 4.36 p.p.m., respectively, both of them as a singlet. characterization of nanoparticles conductometric titration the determination of carboxyl content of compound 10 was done using a method derived for the titration of cellulosic fibers by applying the following equation. [cooh] = ((v*c naoh) / m cnws) [oso3h] in mmol kg-1 where v is the volume of naoh (14.63 ml), cnaoh is the naoh concentration (0.01 mol/l), m cnws is the weight of the sample (0.00015 kg), and [oso3h] was determined by elemental analysis and it was 175 mmole. by applying the equation, the [cooh] was calculated, and it was found to be 1150 mmole/kg. fig. 2 conductometric titration of compound 10. 149j contemp med sci | vol. 7, no. 3, may-june 2021: 145–151 o. n. wennas et al. original design, synthesis, and biological evaluation of camptothecin loaded biotinylated cellulose nanowhiskers determination of biotin and cpt content the biotin and cpt contents of compound 12 were shown in table 1. particle size and zeta potential the studies via dls and zeta potential analysis revealed that the nanoparticles (cnws, compounds 10, and 12) have a hydrodynamic radius between 68 and 87 nm and an overall negative surface charge, as shown in table 1. transmission electron microscopes figure 3 shows the tem images of cnws, and compound 12. no significant changes in the morphology or the degree of agglomeration were observed. in vitro release study the disulfide linkage between the cnws and cpt can be cleaved readily in the presence of a reducing agent such as gsh,8 as shown in scheme 3.24 the accumulative release of cpt from compound 12 is only 18.6% in pbs with 100 μm gsh after 48 h, while the cumulative release in the acetate buffer medium ph 5.8 with 10 mm gsh is 83.1%, as shown in figure 4. in vitro cytotoxicity assay the anticancer activity of compound 9, and compound 12 were examined in three cell lines with different expression levels of biotin receptor. mcf-7 and hepg2 cells overexpress biotin receptors on the surface, while biotin receptors were rarely expressed on cho cells.25 figure 5a, b, and c show the viability of the cells after 72 h incubation. the half-maximal inhibitory concentration was determined to show the effectiveness of the tested compounds on growth inhibition of the three cell lines (figure 5d). fig. 3 tem images of (a) cnws (b) compound 12. scheme. 3 cpt release mechanism from compound 12. fig. 4 accumulative release of cpt from compound 12 at different conditions. 150 j contemp med sci | vol. 7, no. 3, may-june 2021: 145–151 design, synthesis, and biological evaluation of camptothecin loaded biotinylated cellulose nanowhiskers original o. n. wennas et al. fig. 5 cytotoxicity of free cpt, compound 9, and compound 12 against (a) mcf-7, (b) hepg2, and (c) cho cells after incubation for 72 h (n = 3) (d) ic50s of free 6-mp, compound 9, and compound 12 in mcf-7, hepg2, and cho cells. discussion chemical structure of the intermediates and the final compounds was confirmed using ft-ir, 1h nmr, 13c nmr, and esi-ms. characterization of nanoparticles was done using dls and tem. in figure 4. in vitro-release study results suggest that the release of cpt is accelerated in the cellular environment (especially tumors) with elevated gsh levels. in contrast, the release is minimum in the extracellular environment with a low gsh level. regarding the in vitro cytotoxicity, the ic50 of compound 9 is less than half or third of that of cpt in biotin positive cell lines, mcf-7 and hepg2, respectively, and about seven-folds higher than cpt for the biotin negative cho cell line. for compound 12, it showed comparable activity to cpt against mcf-7 and higher activity against hepg2 with ic50 about half that of cpt, and much lower activity against cho with ic50 is about eleven times higher than that of cpt. the relatively small molecule compound 9 has a cellular uptake by passive diffusion beside the cell endocytosis; conversely, compound 12 as a nanoparticle, mainly internalized only by cells endocytosis. these results suggest that the uptake and the cytotoxicity of compounds 9 and compound 12 were higher for cell lines with biotin overexpression than for cells with low biotin expression, and they can potentially reduce side effects of cpt by selectively killing biotin-positive tumor cells conclusions in the recent decade, there have been several cpt-macromolecule prodrugs to address cpt limitations. however, there is still an unmet need to develop different types of macromolecule prodrugs to reduce systemic toxicity and improve the therapeutic efficacy. in the present study, we synthesized two cpt prodrugs using a biotin receptor and a gsh sensitive disulfide linker for intracellular delivery of cpt. the chemical structures of the synthesized prodrugs were confirmed by ft-ir, 1h nmr, 13c nmr, and esi-ms, while the nanoparticles were characterized by dls and tem. the release study showed that gsh could promote the drug release at the intracellular millimolar level compared with gsh at a micromolar level. mtt assay demonstrated that this system 151j contemp med sci | vol. 7, no. 3, may-june 2021: 145–151 o. n. wennas et al. original design, synthesis, and biological evaluation of camptothecin loaded biotinylated cellulose nanowhiskers had a higher inhibition ratio on biotin positive cells, mcf-7, and hepg2, and lower cytotoxicity on negative biotin cells, cho. therefore, these biotin-guided, gsh dependent prodrugs had the potential to be used for cancer targeting. acknowledgments we thank the department of pharmaceutical chemistry/college of pharmacy/university of baghdad for their official support in this study. conflicts of interest disclosure there are no conflicts of interest. competing interests the authors declare that they have no competing interests. source of funding self-funded project.  this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. references 1. ghorab, m.m.; el-gazzar, m.g.; alsaid, m.s. synthesis and anti-breast cancer evaluation of novel n-(guanidinyl)benzenesulfonamides. int. j. mol. sci. 2014, 15, 5582–5595, doi:10.3390/ijms15045582. 2. eggen, m.; georg, g.i. the cryptophycins: their synthesis and anticancer activity. med. res. rev. 2002, 22, 85–101, doi:10.1002/med.10002. 3. al-amily, d.h.; hassan mohammed, m. design, synthesis, and docking study of acyl thiourea derivatives as possible histone deacetylase inhibitors with a novel zinc binding group. sci. pharm. 2019, 87, 28, doi:10.3390/ scipharm87040028. 4. al-darraji, a.s.; mohamed, m.h. synthesis and preliminary anticancer evaluation of 6-mercaptopurine-methotrexate conjugate as possible mutual prodrug. iraqi j. pharm. sci. 2019, 28, 114–124, doi:10.31351/ vol28iss1pp114-124. 5. mohammed, m.h.; taher, m.a. synthesis of new two derivatives of 6-mercaptopurine (6mp) 6-[5-pyridine-4-yl1, 2, 3, 4-oxazolezole-2-yl) dithiol]-9h-purine (38) and 9h-purine-6-yl-benyldithiocarbamate (45) with cytotoxicity results from the national cancer institute’s anticancer drug. int j pharm sci res 2012, 3, 2613–2622. 6. sriram, d.; yogeeswari, p.; thirumurugan, r.; ratan bal, t. camptothecin and its analogues: a review on their chemotherapeutic potential. nat. prod. res. 2005, 19, 393–412, doi:10.1080/14786410412331299005. 7. gupta, e.; vyas, v.; ahmed, f.; sinko, p.; cook, t.; rubin, e. pharmacokinetics of orally administered camptothecins. ann. n. y. acad. sci. 2000, 922, 195–204, doi:10.1111/j.1749-6632.2000.tb07038.x. 8. liu, c.; yuan, j.; luo, x.; chen, m.; chen, z.; zhao, y.; li, x. folate-decorated and reduction-sensitive micelles assembled from amphiphilic polymercamptothecin conjugates for intracellular drug delivery. mol. pharm. 2014, 11, 4258–4269, doi:10.1021/mp500468d. 9. mi, z.; burke, t.g. differential interactions of camptothecin lactone and carboxylate forms with human blood components. biochemistry 1994, 33, 10325–10336, doi:10.1021/bi00200a013. 10. wang, j.; cooper, r.c.; he, h.; li, b.; yang, h. polyamidoamine dendrimer microgels: hierarchical arrangement of dendrimers into micrometer domains with expanded structural features for programmable drug delivery and release. macromolecules 2018, 51, 6111–6118, doi:10.1021/acs. macromol.8b01006. 11. paranjpe, p. v.; chen, y.; kholodovych, v.; welsh, w.; stein, s.; sinko, p.j. tumortargeted bioconjugate based delivery of camptothecin: design, synthesis and in vitro evaluation. j. control. release 2004, 100, 275–292, doi:10.1016/j. jconrel.2004.08.030. 12. ioelovich, m. cellulose as a nanostructured polymer: a short review. bioresources 2008, 3, 1403–1418, doi:10.15376/biores.3.4.1403-1418. 13. isogai, a. wood nanocelluloses: fundamentals and applications as new biobased nanomaterials. j. wood sci. 2013, 59, 449–459, doi:10.1007/s10086013-1365-z. 14. abitbol, t.; rivkin, a.; cao, y.; nevo, y.; abraham, e.; ben-shalom, t.; lapidot, s.; shoseyov, o. nanocellulose, a tiny fiber with huge applications. curr. opin. biotechnol. 2016, 39, 76–88. 15. xu, x.; liu, f.; jiang, l.; zhu, j.y.; haagenson, d.; wiesenborn, d.p. cellulose nanocrystals vs. cellulose nanofibrils: a comparative study on their microstructures and effects as polymer reinforcing agents. acs appl. mater. interfaces 2013, 5, 2999–3009, doi:10.1021/am302624t. 16. klemm, d.; schumann, d.; udhardt, u.; marsch, s. bacterial synthesized cellulose artificial blood vessels for microsurgery. prog. polym. sci. 2001, 26, 1561–1603. 17. lemarchand, c.; gref, r.; couvreur, p. polysaccharide-decorated nanoparticles. eur. j. pharm. biopharm. 2004, 58, 327–341, doi:10.1016/j. ejpb.2004.02.016. 18. eyley, s.; thielemans, w. surface modification of cellulose nanocrystals. nanoscale 2014, 6, 7764–7779, doi:10.1039/c4nr01756k. 19. fraschini, c.; chauve, g.; bouchard, j. tempo-mediated surface oxidation of cellulose nanocrystals (cncs). cellulose 2017, 24, 2775–2790, doi:10.1007/ s10570-017-1319-5. 20. zör, k.i̇.; amberl, o. deactivation of amberlite ir-120 used in the esterification of acetic acid with isobutanol. 2006. 21. murar, c.e.; thuaud, f.; bode, j.w. kaha ligations that form aspartyl aldehyde residues as synthetic handles for protein modification and purification. j. am. chem. soc. 2014, 136, 18140–18148, doi:10.1021/ja511231f. 22. gong, x.y.; yin, y.h.; huang, z.j.; lu, b.; xu, p.h.; zheng, h.; xiong, f.l.; xu, h.x.; xiong, x.; gu, x.b. preparation, characterization and in vitro release study of a glutathione-dependent polymeric prodrug cis-3-(9h-purin-6-ylthio)acrylic acid-graft-carboxymethyl chitosan. int. j. pharm. 2012, 436, 240–247, doi:10.1016/j.ijpharm.2012.06.043. 23. zhang, x.; breslav, m.; grimm, j.; guan, k.; huang, a.; liu, f.; maryanoff, c.a.; palmer, d.; patel, m.; qian, y.; et al. a new procedure for preparation of carboxylic acid hydrazides. j. org. chem. 2002, 67, 9471–9474, doi:10.1021/ jo026288n. 24. meng, x.; gao, m.; deng, j.; lu, d.; fan, a.; ding, d.; kong, d.; wang, z.; zhao, y. self-immolative micellar drug delivery: the linker matters. nano res. 2018, 11, 6177–6189, doi:10.1007/s12274-018-2134-5. 25. ren, w.x.; han, j.; uhm, s.; jang, y.j.; kang, c.; kim, j.h.; kim, j.s. recent development of biotin conjugation in biological imaging, sensing, and target delivery. chem. commun. 2015, 51, 10403–10418, doi:10.1039/ c5cc03075g. https://doi.org/10.22317/jcms.v7i3.960 74 j contemp med sci | vol. 2, no. 7, summer 2016: 74–76 research objectives according to previous studies, the aim of the presented work is to investigate the possible association between the codon 72 polymorphism (pro72arg, rs1042522) of the tumor suppressor gene (p53) with acute myocardial infarction (ami) in iraqi patients of karbala province. methods a total of 58 smokers and non-smokers patients with ami diagnosed by clinical history, electrocardiography and troponin i level and another 35 healthy controls were included in this study. the (pro72arg) polymorphism of the p53 gene was evaluated by molecular techniques. results the genotype distribution for the pro72arg variant of the p53 gene in ami non-smokers patients (pp: n = 9, 47.4%; rp: n = 2, 10.5%; rr: n = 8, 42.1%) and controls (pp: n = 9, 50.0%; rp: n = 9, 50.0%; rr: n = 0, 0.0%) was significantly different (p = 0.002). and the genotype distribution for the pro72arg variant of the p53 gene in ami smokers patients (pp: n = 20, 51.3%; rp: n = 1, 2.6%; rr: n = 18, 46.2%) and controls (pp: n = 14, 82.4%; rp: n = 2, 11.8%; rr: n = 1, 5.9%) was significantly different (p = 0.009). conclusion these findings suggest that the pro72arg polymorphism of tumor suppressor p53 gene is associated with ami patient studies. keywords ami, pcr, rflp, polymorphism polymorphism of tumor suppressor gene (p53) codon 72 in iraqi patients with acute myocardial infarction fadhil jawad al-tu’maa, riyadh d. al-zubaidib, abdul-mutalib badr al-khaleelic, and hassan m. abo-almaalid issn 2413-0516 adepartment of biochemistry, college of medicine, university of karbala, karbala, iraq. bdepartment of internal medicine, college of medicine, university of karbala, karbala, iraq. cdepartment of chemistry, pure science education college, university of karbala, karbala, iraq. ddepartment of clinical laboratory sciences, college of pharmacy, university of karbala, karbala, iraq. correspondence to abdul-mutalib badr al-khaleeli (email: abd742007@yahoo.com). (submitted: 25 january 2016 – revised version received: 7 may 2016 – accepted: 23 april 2016 – published online: 26 september 2016) introduction different genes including the tumor suppressor (p53) gene have been implicated in the etiology of coronary artery diseases.1 by regulating gene expression and other indirect means p53 participates in the regulation of glucose, fatty acid, amino acid and purine metabolism, in addition it influences mitochondrial integrity and oxidative phosphorylation, insulin sensitivity, antioxidant response.2 furthermore p53 gene plays an important role in regulating vascular smooth muscle cell growth and may mediate and abnormal occurrence of apoptosis in atherosclerotic lesions by attenuating or accelerating the apoptotic death process.3 on the other hand p53 gene could regulate cell division and apoptosis within atherosclerotic plaque depending on the level of p53 gene expression induced by dna damage and cell type. affected mutations in p53 gene can induce dysfunction of p53 and inhibit apoptosis and loss of gene activity could which play a relevant role in the pathogenesis of atherosclerosis.4 a common p53 tumor suppressor gene polymorphisms occur at codon 72 of exon 4, with two alleles encoding either arginine (cgc) or proline (ccc). the distribution of the three genotypes (r/r, r/p and p/p) depends largely on the ethnic composition of the studied population.5 the pro or arg of codon 72 has variants reported to differ in functional activity because this polymorphism is located in the proline rich domain of p53, which is necessary for the p53 protein to fully induce apoptosis.6 the aim of the presented work is to investigate the possible association between the codon 72 polymorphism (pro72arg, rs1042522) of the tumor suppressor gene (p53) with (smoker and non-smoker) acute myocardial infarction in iraqi patients of karbala province. materials and methods subjects the study was conducted during the period from nov., 2014 till sep., 2015. fifty eight patients presented with typical chest pain to the cardiac care unit (ccu) in al-hussein teaching hospital, al-hussein medical city/ kerbela health directorate and department of biochemistrycollege of medicine/university of karbala. thirty five persons age – matched healthy volunteers were selected as a control group. both groups were divided into smokers and nonsmokers (39 ami smokers patient and 17 smoker control) and (19 nonsmokers ami patient and 18 nonsmoker control). the diagnosis was based on the clinical history, presentation confirmed by ecg and various investigations of cardiac biomarker. dna extraction about 10 ml of venous blood sample was drawn from each patient and control groups. two ml of blood sample collected in edta tube for genomic dna extraction used for molecular analysis. the dna extraction kit was purchased from bioneer, south korea. the remaining sample was transferred into another tube at room temperature and stand for 20 min for clotting and then centrifuged at 3000 rpm for serum collection used for various cardiac biomarkers determinations. tumor suppress gene p53 polymorphism four primers (table 1) were used in a single pcr reaction, p1 and p2 to amplify a 281 bp band (control band) which make sure the success of the amplification, p3 specific to amplify a 193 bp band which indicate the present of proline allele and p4 75j contemp med sci | vol. 2, no. 7, summer 2016: 74–76 research polymorphism of tumor suppressor gene (p53) codon 72 in iraqi patients with acute myocardial infarction abdul-mutalib badr al-khaleeli et al. specific to amplify a 131 bp band which indicate the present of arginine allele as shown in (fig. 1).7 analysis for different subjects (m = molecular weight marker) pcr premix™ kit was used to amplify. it contained 5 µl of extracted dna, (0.5 µl from each p1 and p2 and 1µl from each p3 and p4 ) of 10 pmol/µl primers were mix in total volume of 20 µl. then the mixture was added to lyophilized pcr premix formula. the mixture was heated for 5 min at 94°c and underwent 40 cycles of amplification: annealing (65°c for 30 s), extension (72°c for 30 s) and denaturation (94°c for 30 s). the pcr product was analyzed on 1.5% agarose gel stained with ethidium bromide.7 result the results showed a significant difference in distribution of p53 (codon 72) allelic polymorphism (p < 0.01) as shown in table 1. higher percentage in rr(arginine/arginine) allele (42.1%) in non-smoker ami patient in compared (0%) with non-smoker normal control groups, while low percentage in rp (arginine/proline) allele (10.5%) in non-smoker ami patient in compared (50%) with non-smoker normal control groups (table 2). significant difference in distribution of p53 (codon 72) allelic polymorphism (p < 0.01) as shown in table 3. higher percentage in rr (arginine/arginine) allele (46.2%) in smoker ami patient in compared to smoker normal control groups (5.9%), while low percentage in pp (proline/proline) allele (51.3%) in smoker ami patient in compared to smoker normal control groups (82.4%) (table 3). discussion the results showed a significant difference in distribution of p53 (codon 72) allelic polymorphism, high percentage in rr allele in smoker and non-smoker ami patient in comparing to smoker and non-smoker normal control group. these findings were agreeing to chilean study by (josé caamaño et al. 2011). this study showed increasing in rr allele in patients and explained that apoptosis in endothelial cells have related to plaque instability and thrombus formation. the disturbance in the apoptotic response may lead to accumulation of intimal cells through a therogenesis and functional consequence of the pro72arg polymorphism has related to inhibition of p73 function, a member of the p53 family of nuclear transcription factors, implicated in tumor suppression, arg polymorphic allele is more efficient in binding to p73, blocking its action and facilitating the proliferation of vascular smooth muscle cells. by this mechanism arg variant of the p53 pro72arg polymorphism is more susceptible to suffer deregulation of apoptosis during atherosclerosis progression.8 also another brazilian study by (disciplina de genética et al 2007) suggested that arg72 allele is associated with cardiovascular disease.9 in additional type 2 diabetes mellitus t2dm which is risk factors for ami and the fact that the carriers of genotypes containing arg72 allele previously associated with susceptibility to type 2 diabetes mellitus t2dm than pro72 genotype carriers.10 conclusion in this study, we ascertain that a significant association between the codon 72 polymorphism (pro72arg, rs1042522) of the tumor suppressor gene (p53) with acute myocardial infarction (ami). n table 3. comparison between smoker myocardial infarction patients and smoker control groups in the distribution of p53 (codon 72) allelic polymorphism codon 72 allelic polymorphism pvalue rr (n =), % pp (n =), % rp (n =), % smoker patient (18) 46.2% (20) 51.3% (1) 2.6% p < 0.01 control (1) 5.9% (14) 82.4% (2) 11.8% *rr: arginine/arginine, pp: proline/proline, rp: arginine/proline. table 1. sequence of primers used for pcr of p53 gene polymorphism primer sequence p1 5’-gccgtcccaagcaatggatgatt-3’ p2 5’-ggcaactgaccgtgcaagtcacag-3’ p3 5’-agaatgccagaggctgctccacc-3’ p4 5’-cttctggtgcaggggccaagc-3’ fig. 1 agarose gel electrophoretogram of the p53 arg72pro polymorphism. table 2. comparison between non-smoker myocardial infarction patients and non-smoker control groups in the distribution of p53 (codon 72) allelic polymorphism codon 72 allelic polymorphism pvalue rr (n =), % pp (n =), % rp (n =), % non smoker patient (8) 42.1% (9) 47.4% (2) 10.5% p < 0.01 control (0) 0.0% (9) 50.0% (9) 50.0% *rr: arginine/arginine, pp: proline/proline, rp: arginine/proline. 76 j contemp med sci | vol. 2, no. 7, summer 2016: 74–76 polymorphism of tumor suppressor gene (p53) codon 72 in iraqi patients with acute myocardial infarction research abdul-mutalib badr al-khaleeli et al. references 1. geng yj. molecular signal transduction in vascular cell apoptosis. cell res. 2001;11:253–264. 2. maddocks od, vousden kh. metabolic regulation by p53. j mol med (berl). 2011;89:237–245. 3. geng yj. biologic effect and molecular regulation of vascular apoptosis in atherosclerosis. curr atheroscler rep. 2001;3:234–242. 4. mercer j, mahmoudi m, bennett m. dna damage, p53, apoptosis and vascular disease. mutat res. 2007;621:75–86. 5. omori s, yoshida s, kennedy s. polymorphism at codon 72 of the p53 gene is not associated with endometriosis in a japanese population. gynecol investing. 2004;11:232–236. 6. baptiste n, friedlander p, chen x, prives c. the proline-rich domain of p53 is required for cooperation with anti-neoplastic agents to promote apoptosis of tumor cells. oncogene. 2002;21:9–21. 7. bassam l, amal a. detection of arg72pro polymorphism of tumor suppressors gen tp53 by a rapid one-step amplification refractory mutation system-pcr. am j biochem mol biol. 2011;2: 231–236. 8. caamaño j, saavedra n, jaramillo pc, lanas c, lanas f, salazar la. tp53codon 72 polymorphism is associated with coronary artery disease in chilean subjects. med princ pract. 2011;20:171–176. 9. smith ma, silva md, cendoroglo ms, ramos lr, araujo lm, labio rw, et al. tp53codon 72 polymorphism as a risk factor for cardiovascular disease in a brazilian population. braz j med biol res. 2007;40:1465– 1472. 10. katarina k, lukas p, veronika d, katerina k. association of the arg72pro polymorphism in p53 with progression of diabetic nephropathy in t2dm subjects. j nephrol ther. 2014;4:153. 49j contemp med sci | vol. 2, no. 6, spring 2016: 49–52 research objectives this study aims to estimate the rate of acute stress disorder (asd) among burn patients. the sample consists of 100 patients admitted to burn unit in al-kindy teaching hospital in baghdad city. there is a significant relationship between asd and burn patients; it is the mean majority of patients with burn developed asd. methods a total of 100 patients are attended to burn unit in al-kindy teaching hospital in baghdad city. dsm-iv criteria were applied. ghq and socio demographic data were used for diagnosis. all the patients who do not meet the criteria were excluded from the sample by using asd symptom questionnaire; proper consent was already taken from all patients. results a total of 100 patients with asd were identified in the burn unit, academic psychiatrists made diagnosis to evaluate the positive symptoms (positive symptoms scale). asd represent 80% of all admissions and was more common among females than males, more than half of the cases were between 15 and 24 years of age with the mean age of 30 years. the majority of cases were married, and the mortality rate of burned patients was 69.4, and the median total body surface area (tbsa) burnt was 60%. 18.6% of patients had previous attempt for self-inflicted burn. conclusion both the disease process and treatment of burned patients may affect the mental state of patients producing a variety of symptoms. asd is the most common psychiatric disorder in burned patients. there is a significant relationship between asd and burned patients. keywords burned patients, stress-related disorder, acute stress disorder, dissociation, early identification psychosocial and medical patterns of acute stress disorder in burn unit in baghdad, iraq abbas saeed al-eessaa, abdulmahdi a. hasanb issn 2413-0516 aspecialist, psychiatrist in marjan teaching hospital, hilla city, iraq. bdepartment of paediatric and psychiatric mental health nursing, college of nursing, university of babylon, hilla city, iraq. correspondence to abbas saeed al-eessa (email: drabbasaleessa@yahoo.com). (submitted: 23 december 2015 – revised version received: 27 february 2016 – accepted: 1 april 2016 – published online: 26 june 2016) introduction acute stress disorder (asd) refers to an anxiety and disturbed behaviour that can occur in the time after an extreme trauma. the symptoms usually start during or soon after the trauma, but if the symptoms continue longer than a month, then the condition called ptsd.1–3 the signs and symptoms related to asd includes sense of impending doom, nervousness, instability tachycardia, hyperventilation, paraesthesia, diaphoresis, flushing, headache, and there are many causes and methods and behaviours of burn contribute to asd. the cause of burn is variable and difficult, and the psychiatric morbidity between burned patients and asd is believed to represent either primary manifestations of the disease caused by skin involvement or psychological reaction to living with a disability and chronic illness.5–7 materials and methods a total of 100 patients were attended to burn unit in al-kindy teaching hospital in baghdad city, dsm-iv criteria were applied for the diagnosis of persons with asd, ghq and sociodemographic data were used and applied for each person, and the aim was to know the targeting patients; all the patients who do not meet the criteria were excluded from the sample and the study was conducted by using asd scale. subjects and sample a total of 100 patients with asd were identified in the burn unit, academic psychiatrists made diagnosis to evaluate the positive symptoms (positive symptoms scale) was used. statistical analysis all the statistical analysis were performed using p-value and chi-square test (χ2). the relations among the categorical variables were investigated by χ2. p-value more than or equal to 0–0.5 were considered statistically significant. all the data were analysed by χ2 at a confidence level of 95%. the data were analysed using χ2 test for the differences between the groups. the odds ratio was computed for estimating the strength of association of the risk factors and the occurrence of the disease. if odds ratio equal to 1 or more means that there is a positive association. if odds ratio less than one means, there is a protection against the occurrence of the disorder. after studying this table and the statistical data, we found there is a strong correlation between asd and age groups, 35–54 more than other group. table 7 shows that every patient in this group is free from asd. sociodemographic characteristics, gender and age of groups (p < 0.05) are shown in tables 1–8, further no significant relationship was found among sociodemographic characteristics, and the prevalence of asd was higher in female than in male in asd of 89.8% (45), 65.8% (35), respectively. table 8 shows that by analysing this table, we found the correlation is positive between the asd and the severity of burn. discussion in this study, the ratio of female to male patients is 3:2, which is slightly differing from the ratio recorded by other authors. there is a significant relation between female and male genders; in our 50 j contemp med sci | vol. 2, no. 6, spring 2016: 49–52 psychosocial and medical patterns of asd research abbas saeed al-eessa et al. fig. 1 the ratio of female to male patients. fig. 2 significant relation between female sex married divorce, widow. fig. 3 significant relation between female sex married divorce, widow. fig. 5 close association between severity of burn injury and asd. table 1. distribution of patients according to the burning agent burning agent no. of patients and % kerosene 46 gas 40 benzene 8 hot fluid 3 electricity 1 other 2 (one is by hot iron bar and wielding oxygen the other case is unknown) total 100 table 2. distribution of patients according to the cause of burn the cause no. of patients and % accidental 82 induced attempted suicide definite 10 suspected 8 criminal induced 0 study, we found that asd is more common in females, and this difference could be due to the impact of life and bodily constitution, and the male could be more denial than females, many studies found that mood disorders are common in burn unit,4,8,9,12 and the depression is the commonest that vary from 25 to 50% in our study; the commonest psychiatric morbidity in burn unit is asd that was predominated in the patients with burn, and a close association between the severity of burn injuries and asd was found. a significant relation between adult and middle age, female sex and widowed, married and divorced, unemployed. significantly, a large number of the sample table 3. distribution of acute stress disorder cases in burn patients no. of patients and % male female total cases 27 53 80 non cases 14 6 20 total 41 59 100 fig. 4 close association between severity of burn injury and asd. 51j contemp med sci | vol. 2, no. 6, spring 2016: 49–52 research psychosocial and medical patterns of asdabbas saeed al-eessa et al. presented from rural area. also there is a low level of education and poverty which are the risk factor for accidents as burn and for asd. also, we have found a considerable number of patients in burn unit with attempted suicide either definite or suspected self-immolation among young muslim women in parts of the middle east and central asia, which is increasingly becoming a cause of death and disability, and very little is known about this phenomenon.13–16 male victims generally predominate in western countries and females in the middle east and the indian sub-continent.15–17 in jordan, burning by kerosene is a common traditional and dramatic way of attempting suicide by females. sati was described as a custom in india, in which the widow was burnt to ashes on her dead husband. in view of the selected variables of our study, we have found high number of patients with asd in the age between 15 and 24 years, this may be explained by difficult economic and social situations and burden.11,12 the females out-numbered males. this may be explained by the tendencies in females to have mental disturbances more than males.14,15 the high number of patients with asd are in the group of married, divorced and widowed, which is because of each group has their own stress in their difficult socio-economic state and being responsible for many family demands. asd was high in unemployed. this may be explained due their financial problems and lack of economic resources and social insecurity and their psychological effect of being without work.9 by studying the result of severity of burn, we have found that asd increases with the severity of burn, in moderate and severe burn, while we have less or no asd in minor burns. recommendations the patient with burn needs psychological assessment from beginning of disease and at regular intervals thereafter to deal with psychiatric morbidity. liaison work between plastic surgeon and psychiatrist is required for the management of the burned patients. abbreviations asd: acute stress disorder; ghq: general health questionnaire; dsm-iv diagnostic statistical of mental health disorder-fourth version; tsab: total surface area burnt; ptsd: post traumatic stress disorder.  table 4. distribution of patients according to age groups age group patients no. cases no. % of the cases p-value χ2 odd’s ratio 15–24 45 35 77.7 0.0631 0.78 25–34 25 21 84 0.12 1.42 35–44 16 14 87.5 1.01 1.9 45–54 8 8 100 3.063 e* 55 & above 6 2 33.3 7.32 0.102 total 100 80 e* means that every patient burned in this age group is having acute stress disorder. table 5. distribution of patients according to sex difference sex patients cases no. % of the cases p-value χ2 odd’s ratio male 41 27 65.8 8.593 0.218 female 59 53 89.8 total 100 80 table 6. acute stress disorder according to marital status marital status patients no. cases no. % of the cases p-value χ2 odd’s ratio single 33 24 27.7 1.558 0.523 married 59 49 83 0.705 1.58 widowed 4 4 100 2.166 e* divorced 4 3 75 0.291 0.74 total 100 80 e* means every patient in this group is having asd. table 7. distribution of patients with acute stress disorder according to an employment employment patients no. cases no. % of the cases p-value χ2 odd’s ratio housewife 57 48 84.2 1.479 1.833 self employed 26 20 76.9 0.213 0.778 unemployed 11 10 90.9 1.435 2.714 government employed 2 2 100 2.041 e* pupils 4 0 0 . . . . . . total 100 80 e* means every patient in this group is having asd; . . . means every patient in this group of pupils is free from asd. table 8. distribution of patients with acute stress disorder according to the severity of burn burn severity patients no. cases no. % of the cases p-value χ2 odd’s ratio minor 2 0 0 . . . . . . moderate 33 19 57.5 9.596 0.355 severe 65 61 93.8 21.875 12.842 total 100 80 . . . means every patient in this group of pupils is free from asd. references 1. johnstone ev, owens dgc, lawrie sm, sharpe m, freeman cpl. companion to psychiatric studies, 7th ed. churchill livingstone: elsevier; 2013. 2. michel k. suicide risk factors: a comparison of suicide attempters with suicide completers. br j psychiatr. 1987;150:78–82. pmid: 3651731 3. bancroft jh, skrimshire am, reynolds f, simkin s, smith j. self-poisoning and self-injury in the oxford area: epidemiological aspects. br j prev soc med. 1975;29:170–177. 4. haider is, haider i. deliberate self harm. pak j med sci. 2001;17(3):151–155. 5. warr p. unemployment and mental health. oxford: oxford university press; 2008. 52 j contemp med sci | vol. 2, no. 6, spring 2016: 49–52 psychosocial and medical patterns of asd research abbas saeed al-eessa et al. 6. roy a. depression, attempted suicide and suicide in patients with chronic schizophrenia. psychiatr clin north am. 2005;9:193–206. 7. van der does aj, hinderink em, vloemans af, spinhoven p. burn injuries, psychiatric disorders and length of hospitalization. j psychosom res. 1997;43(4):431–5. pmid: 9330243 8. ashton jr, donnan s. suicide by burning as an epidemic phenomenon: an analysis of 82 deaths and inquests in england and wales in 1978–9. psychol med. 1981;11(4):735–9. pmid: 7323229 9. laloë v, ganesan m. self-immolation a common suicidal behaviour in eastern sri lanka. burns. 2002;28(5):475–80. pmid: 12163288 10. dsm-iv. diagnostic and statistical manual of mental disorder, 4th ed. in: american psychiatric association. washington dc: apa; 2010. 11. abu ragheb s, qaryoute s, el-muhtaseb h. mortality of burn injuries in jordan. burns incl therm inj. 2000;10(6):439–43. pmid: 6478292 12. geller-jl. self-incineration: a review of the psychopathology of setting oneself afire. int j law psychiatr. 1997;20:355–72. pmid: 9347397 13. gelder m, gath d, mayou r. concise oxford textbook of psychiatry. great britain: oxford university press; 2003. p. 85. 14. davidson ti, brown lc. self-inflicted burns: a five-year retrospective study. burns. 1985;11:157–60. 15. campbell ea, guiao iz. muslim culture and female self-immolation: implications for global women, health research and practice. health care women int. 2004;25(9):782–93. pmid: 15513806 16. suokas j, lönnqvist j. outcomes of attempted suicide and psychiatric consultation, risk factors and suicide mortality during a five year follow-up. acta psychiatr scand. 1991;84:545–9. pmid: 1792928 17. horner bm, ahmadi h, mulholland r, myers sr, catalan j. case-controlled study of patients with self-inflicted burns. burns. 2005;31(4):471–5. pmid: 15896510 291j contemp med sci | vol. 6, no. 6, november–december 2020: 291–295 original issn 2413-0516 introduction childhood acute lymphoblastic leukemia (all) is the most common cancer diagnosed among pediatrics, consisting nearly 25% of malignant disease in children.it is more common among males, with a peak incidence between 1 and 4 years of age.1 symptom of all are generally non-specific and numerous and include prolonged fever, petechia, bone pain, and palpable liver or spleen detected during physical examination.2 the typical duration of chemotherapy treatment is usually 2–3 years, and consists of induction, consolidation, and maintenance therapy.3 most of the drugs used for treating all in children have several adverse effects, which can be mild or even fatal, so risk-directed stratification contributes to suppress relapse risk and avoid excess complication.4 granulocyte colony-stimulating factor (g-csf) has been used to ameliorate drug induced myelosuppression.5 guidelines indicate the use of g-csf whenever the chance of febrile neutropenia is up to 20% or more.6 several adverse effects including bone pain, arthralgia, myalgia, thrombocytopenia, epistaxis, cough, dyspnea, fever, nausea, back pain, acute respiratory distress syndrome, alveolar hemorrhage and hemoptysis, severe sickle cell crises in patients with sickle cell disorders, cutaneous vasculitis, sweet’s syndrome, and osteoporosis have been reported following g-csf administration.7-11 royal jelly (rj) is a bee product secreted from the hypopharyngeal and mandibular glands of worker bees. there are many reports on pharmacological activities of rj in experimental animals, but there are few about its antioxidative properties connected to aging. in addition, preliminary studies have showed that use of rj as an immunomodulatory supplement can have numerous positive in vivo and in vitro effects.12-13 nowadays, researchers have been studying rj benefits on healing different types of disease including peptic ulcer, malignancies, and diabetes. the mixture of both workers jelly and rj is called n-chromosome rj, which have elucidated to have a significant healing feature to treat peptic ulcers.14 the present study was undertaken for the first time in the world to investigate the effect of fresh oral prescribed n-chromosome rj, a new product of rj on immune cell counts of patients with childhood all who were chemotherapeutically treated and were in complete remission and maintenance state. various blood chemical analyses were conducted to count peripheral wbc, absolute neutrophil (anc) and lymphocyte count (alc) to analyze concurrent change in blood immune cells during the trial. materials and methods this single-center, randomized study was carried out on 26 patients who were previously diagnosed with all and were in complete remission and maintenance state of their chemotherapy treatment. patients were visited in the pediatric oncology clinic of ali asghar hospital in tehran (iran) between april 2020 and june 2020, after obtaining approval from our local ethics board and after receiving registry codes from the iranian registry of clinical trials (registration code: irct) and after taking ethical consent and explaining different aspects of study, patients were enrolled in the trial. evaluation of the effectiveness and safety of n-chromosome royal jelly on the number of peripheral white blood cells in patients with acute lymphoblastic leukemia gholamreza bahoush, rozhin pahlevani department of pediatric hematology and oncology, ali-asghar children`s hospital, faculty of medicine, iran university of medical sciences, tehran, iran. corresponding author: rozhin pahlevani (email: rozhinpahlevani64@hotmail.com) (submitted: 02 september 2020 – revised version received: 19 september 2020 – accepted: 06 october 2020 – published online: 26 december 2020) abstract objectives: this study aims to investigate the immune response blood cells following n-chromosome royal jelly (rj) administration in patients with childhood acute lymphoblastic leukemia (all). methods: this single center before and after 12-weeks clinical trial was done in ali-asghar children hospital on 26 patients diagnosed with all during maintenance phase of chemotherapy. after collecting their demographics, patients received a starter dose of 2-g processed new natural n-chromosome rj before breakfast and were followed up every 2 weeks, counting their peripheral white blood cells (wbcs), absolute neutrophil count (anc), absolute lymphocyte count (alc) by a blood cell count and differential analysis. results: a total of 26 patients were enrolled in this study (16 males and 10 females). mean peripheral wbc count of total patients were raised significantly after administering n-rj. being 2510±1192 cells per cubic millimeter at the beginning, and then raised to 4549±1500 cells per cubic millimeter at the end of trial. (p<0.005). none of patients suffered from any adverse reaction during the trial. there was a positive statistical relationship between total peripheral wbc, anc, and alc count and n-chromosome rj increased dosage. being more prominent at the beginning of trial (p<0.001) and the last 2 weeks of follow-ups. (p<0.0005) conclusion: this study has successfully demonstrated that n-chromosome rj can be a promising immune-enhancing supplement in patients diagnosed with all in their complete remission and maintenance therapy time. in addition, it is a natural alternative for drugs like granulocyte colony-stimulating factor, but without those long-term adverse effects, we see in g-csf drugs. keywords: acute lymphoblastic leukemia, n-chromosome royal jelly, white blood cell 292 original evaluation of the effectiveness and safety of n-chromosome royal jelly on the number of peripheral white blood cells gholamreza bahoush j contemp med sci | vol. 6, no. 6, november–december 2020: 291–295 inclusion criteria were as follows: 1. age less than 18-year-old. 2. having the confirmed diagnosis of all. 3. all enrolled patients have been treated by ic-bfm2009,1 in complete remission and in the maintenance phase of chemotherapy. 4. approvement of clinical trial taken by ethical consent. 5. not having a previous history of honey-related compound allergic reaction. patients who received g-csf or other hormonal immune-targeted therapy were excluded from the trial. in addition, children with underlying disorders (such as down syndrome) were excluded from the study. demographic data including age, sex, type of malignancy and its stage, duration of treatment, and type of immunophenotyping were recorded for each patient. also, a cardiac monitoring was done by a simple echocardiography by an expert pediatric cardiologist to rule out any cardiac background disease. processed new natural n-chromosome rj is a homogenous component of honey used by queen bee due to its rich nutritional and immunological characteristics. it is yellowish in color with brown tinges and semi-soluble in water. n-chromosome rj refers to the rj produced by nurse bees for queen cells that have drone larvae grafted onto them. study participants received a starting dose of 2 g processed new natural n-chromosome rj before breakfast during their maintenance phase of chemotherapy and dosage were doubled according to their immune-cell counts and response. each patient was followed up by taking a total cbc and cell differential every 2 weeks and assessment was done till the end of 12 weeks of treatment. data analysis patients’ information was entered into spss v23.0. descriptive data were analyzed by descriptive tests. parametric and non-parametric tests was employed and p-value less than 0.05 was considered as significant. ethical considerations patient information was only available to the executor and the name of the patient remained confidential. research team members were aware of the details of helsinki statement about ethic principles in medical research and were strictly committed to follow them in this research study. this project was approved at the ethics committee of the university of medical sciences. results this study is a single center pilot clinical trial. all patients were evaluated before treatment, and every 2 weeks lasting for 12 weeks after intervention. during each visit, a complete cbc and differential cell count analysis were done and n-chromosome rj dosages were titrated according to immune-cell response in each patient. this study included 26 patients diagnosed with all (16 males and 10 females) who were during maintenance phase of 1 the berlin-frankfurt-münster (bfm) 2009 protocol for all chemotherapy. their chemotherapy protocol. the mean age of total patients was 5.78 years. cellular immunophenotyping showed that 24 patients (92.3%) had b-cell all and the others (7.7%) were treated as t-cell all (table 1). none of patients had an allergic reaction, hypotension, or respiratory adverse effect after taking n-chromosome rj and mean wbc count of total patients at the beginning of trial was 2510 ±1192 cells per cubic millimeter, while it raised to 4549±1500 cells per cubic millimeter at the end of trial. there was significant statistical relationship between total peripheral wbc, anc, and alc count and n-chromosome rj increased dosage. being more prominent at the beginning of trial (p<0.001) and the last two weeks of follow-ups (p<0.0005) (table 2). there was a positive correlation among peripheral wbc, anc, and tlc count during every 2 weeks, especially when the n-chromosome rj was started (before trial and during first 2 weeks) showing an increase in total number of cells (p=0.0001) (figs. 1–3). discussion during recent decades, the prognosis of childhood all has improved dramatically, nowadays, reaching a cure rate of almost 90%.15 chemotherapy is the mainstay treatment in patients diagnosed with all and it consisted of three different phase including induction, consolidation, and total remission and maintenance phase.16 chemotherapy-induced neutropenia is one of major dose limiting effects of chemotherapy, usually known as the most serious hematologic toxicity of chemotherapy,17-18 thus clinicians have been using different type of drugs, trying to ameliorate neutrophil counts in patients who have undergone chemotherapy. for example, in our prior study, we have evaluated g-csf positive effects on patients with all. however, administering g-csf can cause various adverse effects in patients.17,19 since previous studies have successfully indicated that rj can have anti-oxidative and immunomodulation effects.12-13 in a study published by kaftanoglu published in 1997, he and his colleagues have successfully indicated that using rj in patients suffering from leukemia can increase the average peripheral wbc, neutrophil and lymphocyte.12 in addition, shirzad and colleagues in a study published in 2013 have concluded that use of rj in syngeneic mice suffering from fibrosarcoma can significantly reduce tumor size (p<0.05).20 our purpose in this study was to evaluate n-chromosome rj effects on blood cell counts, especially lymphocyte and neutrophil absolute count and to see its potentials in patients diagnosed with all in complete remission and maintenance phase. none of patients who were included experienced an allergic reaction to administered n-chromosome rj. additionally, this study indicated that administration of n-chromosomal rj can significantly increase peripheral wbc, anc, and tlc in patients suffering from neutropenia (p<0.0005). more recently, the lipophilic fraction of rj was reported to have extraordinary anti-proliferative activities in a neuroblastoma cell line (sh-sy5y) compared with hydrophilic fraction.21 in addition, this study also found that the biological activities in neuroblastoma cells were stronger than immortalized murine myoblasts and prostate cancer cells. thus, we also agree with their opinion that the search for more effective and disease-specific fractions of rj may be critical for improvements in the anti-cancer effects. 293 original evaluation of the effectiveness and safety of n-chromosome royal jelly on the number of peripheral white blood cells gholamreza bahoush j contemp med sci | vol. 6, no. 6, november–december 2020: 291–295 table 1. hematological markers according to royal jelly dosage per day in patients with childhood all. he m at ol og ic m ar ke r ti m e of tr ia l rj d os ag e m ea n (s d) he m at ol og ic m ar ke r ti m e of tr ia l rj d os ag e m ea n (s d) he m at ol og ic m ar ke r ti m e of tr ia l rj d os ag e m ea n (s d) w bc c ou nt be fo re tr ia l qd 2553(1537) a n c co un t be fo re tr ia l qd 1097(647) a lc c ou nt be fo re tr ia l qd 670(363) bid 2590(797) bid 1069(355) bid 758(388) tid 2638(909) tid 1104(380) tid 650(182) w ee k 1 qd 3533(936) w ee k 1 qd 1638(389) w ee k 1 qd 1099(409) bid 3383(739) bid 1550(353) bid 949(335) tid 3604(1151) tid 1611(563) tid 897(298) w ee k 2 qd 3241(339) w ee k 2 qd 1547(274) w ee k 2 qd 862(286) bid 4240(1493) bid 2095(766) bid 1228(572) tid 4171(1248) tid 1990(624) tid 1051(307) w ee k 3 qd 3637(646) w ee k 3 qd 1766(316) w ee k 3 qd 991(298) bid 4640(1706) bid 2458(961) bid 1360(604) tid 4312(822) tid 1996(537) tid 1260(335) w ee k 4 qd 4205(593) w ee k 4 qd 2368(267) w ee k 4 qd 1281(437) bid 5520(1972) bid 3142(1197) bid 1627(717) tid 4225(1023) tid 2343(471) tid 1326(442) w ee k 5 qd 4322(742) w ee k 5 qd 2660(362) w ee k 5 qd 1268(477) bid 6557(2871) bid 4150(1874) bid 2097(921) tid 6188(762) tid 3751(423) tid 1970(279) w ee k 6 qd 4903(778) w ee k 6 qd 3025(528) w ee k 6 qd 1524(361) bid 7221(3048) bid 4969(2130) bid 2309(995) tid 8654(464) tid 5719(213) tid 2838(167) qd: once daily; bid: twice daily; tid: three times a day. table 2. clinical and demographic profile of children diagnosed with all. demographics number (%) age (mo.) 5.78 sex 1. male 2. female 16(61.5%) 10(38.5%) all type 1) b-cell 2) t-cell 24(92.3%) 2(7.7%) hypotension none cardiac disease none respiratory disease none allergic reaction none fig. 1 whole blood cell count before and after administering n-chromosome royal jelly. 294 original evaluation of the effectiveness and safety of n-chromosome royal jelly on the number of peripheral white blood cells gholamreza bahoush j contemp med sci | vol. 6, no. 6, november–december 2020: 291–295 consistent with previous studies, we showed that administration of rj can successfully increase immunological cell counts and it is an effective supplement which can be effectively administered without any adverse effect in comparison with other drugs such as g-csf. since this study was done in a private oncologic clinic, one limitation of our study could be a small sample size. secondly, we couldn’t evaluate dose–response effect of n-chromosomal rj effect in patients. future investigation is warranted to further characterize these findings and their relevance to prognosis in patients with all. conclusion to our knowledge, this is the first pilot study to suggest that use of n-chromosomal rj supplement can significantly increase immune cells count in patients that were in total remission and maintenance phase during chemotherapy–chromosome rj should be considered as an effective alternative supplement in comparison with g-csf. clinical trial registration the study was registered in the iranian clinical trial registry center (http://www.irct.ir) with the registration code: irct20190106042260n1 acknowledgments the authors would like to thank the caspian apiaries company for providing n-chromosome royal jelly, helping us to conduct this study. funding the authors received no financial support for the research, authorship, and/or publication of this article. conflict of interest there is no contradiction in the article. references 1. horibe k, saito am, takimoto t, tsuchida m, manabe a, shima m, et al. incidence and survival rates of hematological malignancies in japanese children and adolescents (2006–2010): based on registry data from the japanese society of pediatric hematology. int j hematol. 2013;98(1):74-88. 2. inaba h, greaves m, mullighan cg. acute lymphoblastic leukaemia. the lancet. 2013;381(9881):1943-55. 3. schmiegelow k, attarbaschi a, barzilai s, escherich g, frandsen tl, halsey c, et al. consensus definitions of 14 severe acute toxic effects for childhood lymphoblastic leukaemia treatment: a delphi consensus. lancet oncol. 2016;17(6):e231-e9. 4. kato m. pediatric acute lymphoblastic leukemia: springer; 2020. 5. lieschke gj, burgess aw. granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. new engl j med. 1992;327(1):28-35. 6. wang y, chen l, liu f, zhao n, xu l, fu b, et al. efficacy and tolerability of granulocyte colony-stimulating factors in cancer patients after chemotherapy: a systematic review and bayesian network meta-analysis. scient rep. 2019;9(1):1-12. 7. pfeil am, allcott k, pettengell r, von minckwitz g, schwenkglenks m, szabo z. efficacy, effectiveness and safety of long-acting granulocyte colonystimulating factors for prophylaxis of chemotherapy-induced neutropenia in patients with cancer: a systematic review. supportive care cancer. 2015;23(2):525-45. 8. bronchud mh‚ scarffe jh‚ thatcher n‚ et al. phase i/ii study of recombinant human granulocyte colony-stimulating factor in patients receiving intensive chemotherapy for small cell lung cancer. br j cancer. 1987;56:809-813. 9. heil g, hoelzer d, sanz ma, et al. a randomized, double-blind, placebocontrolled, phase iii study of filgrastim in remission induction and consolidation therapy for adults with de novo acute myeloid leukemia. blood. 1997;90:4710-4718. 10. dale dc‚ bonilla ma‚ davis mw‚ et al. a randomized controlled phase iii trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia. blood. 1993;81:2496-2502. 11. highlights of prescribing information: these highlights do not include all the information needed to use neupogen safely and effectively; initial fda approval: 1991. 12. kaftanoglu o, tanyeli a. the use of royal jelly during treatment of childhood malignancies. bee products: springer; 1997:179-83. 13. gasic s, vucevic d, vasilijic s, antunovic m, chinou i, colic m. evaluation of the immunomodulatory activities of royal jelly components in vitro. immunopharmacol immunotoxicol. 2007;29(3-4):521-36. 14. mirabzadeh a. the use of n-chromosome royal jelly to treat h. inpilory ulcers. abstract-xxxxiii international apicultural congress (vol. 29, pp. 76-77). fig. 2 absolute lymphocyte cell count before and after administering n-chromosome royal jelly. fig. 3 absolute neutrophil cell count before and after administering n-chromosome royal jelly. 295 original evaluation of the effectiveness and safety of n-chromosome royal jelly on the number of peripheral white blood cells gholamreza bahoush j contemp med sci | vol. 6, no. 6, november–december 2020: 291–295 15. smith ma, seibel nl, altekruse sf, ries la, melbert dl, o’leary m, et al. outcomes for children and adolescents with cancer: challenges for the twenty-first century. j clin oncol. 2010;28(15):2625. 16. kliegman rm sb, geme jst, schor nf. nelson, ed. t. textbook of pediatrics (chapter 495): philadelphia: elsevier; ; 2016. 17. badr m, hassan t, sakr h, karam n, rahman da, shahbah d, et al. chemotherapy-induced neutropenia among pediatric cancer patients in egypt: risks and consequences. mol clin oncol. 2016;5(3):300-6. 18. ozer h, armitage jo, bennett cl, crawford j, demetri gd, pizzo pa, et al. 2000 update of recommendations for the use of hematopoietic colonystimulating factors: evidence-based, clinical practice guidelines. american society of clinical oncology growth factors expert panel. j clin oncol off j am soc clin oncol. 2000;18(20):3558. 19. zahran am, elsayh ki, saad k, eloseily em, osman ns, alblihed ma, et al. effects of royal jelly supplementation on regulatory t cells in children with sle. food nutr res. 2016;60:32963. 20. shirzad m, kordyazdi r, shahinfard n, nikokar m. does royal jelly affect tumor cells? j herbmed pharmacol. 2013;2(2). 21. gismondi a, trionfera e, canuti l, di marco g, canini a. royal jelly lipophilic fraction induces antiproliferative effects on sh-sy5y human neuroblastoma cells. oncol rep. 2017;38(3):1833-44. https://doi.org/10.22317/jcms.v6i6.856 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 88 j contemp med sci | vol. 2, no. 7, summer 2016: 88–92 research the role of bacteria bacillus subtilis in improving rooting response of mung bean (vigna ratiata) cuttings khalid ali hussein,a najeh hashem kadhum,a yass kudhir yassera issn 2413-0516 introduction bacteria that colonize plant roots in many cases are useful for the growth and development and plant productivity,1 and the synthesis of plant hormone growth regulators such as auxin, gibberellins and cytokines) by microorganisms, believed as one of the main forms of interaction between the plant and the microorganisms. the ability of production has been found in different types of bacteria (pathogens and commensally as well as free is common with the plant, khalid (2004)2 revealed that many bacteria rhizophere are able to synthesis iaa including streptomyces, bacillussp, azotobatersp. in addition to bacteria, fungi and algae have shown to stimulate plant growth by the synthesis of iaa. the bacteria synthesize and release auxins as secondary metabolites.3 indole-3-acetic acid (iaa) is the main member of the auxin family that controls many important physiological processes including cell enlargement and division, tissue differentiation, and responses to light and gravity.4 the auxin, iaa has been identified as a rooting hormone. exogenous application of iaa induces rooting in several stem cuttings, formation of adventitious roots can be induced by the treatment with auxins.5 iaa produced by the bacteria works in conjunction with the internal auxin plant to stimulate root proliferation and division of cells and nutrient uptake from the culture media.6 the main precursor for the synthesis of iaa in plants and bacteria is tryptophan. high production of iaa results in adding tryptophan to the growth medium.7 dobbelare et al., (2003)8 reported the bacteria plant growth promoting rhizobacteria (pgpr) led to an increase in the number of root hairs and lateral roots and decrease of the lengths of the roots. these changes attributed to the effect of auxin produced by the bacteria root colonies of strains of bacteria, b. subtilis, which enhanced rooting in plants. roots are the most the sensitivity of to changes iaa levels, by elongation primary roots and the formation adventitious roots and lateral roots.9 the hormone produced by the bacteria can be used as a relationship between bacteria and adepartment of biology, college of science, university of karbala, karbala, iraq. correspondence to khalid ali hussein (email: kalbio1970@yahoo.com). (submitted: 21 june 2016 – revised version received: 17 july 2016 – accepted: 10 august 2016 – published online: 26 september 2016) objectives this study includes the investigative ability of bacillus subtilis isolated from soil to produce indole acetic acid (iaa) and effect on rooting response of mung bean cuttings. methods the some of the optimal conditions for iaa production have been indicated. the effect of indole acetic acid production in bacillus subtilis filtrate on the rooting response of mung bean cuttings. the effect of iaa production in bacillus subtilis filtrate in carbohydrate, protein, iaa contents and activity of iaao in cutting hypocotyls. results results revealed that, maximum iaa production was obtained by inoculating with an inoculum size of 125 µl/10 ml production culture and incubation at 40°c for 72 hrs. iaa produced by bacteria was significantly enhanced the average of root number/cutting to its levels in control treatment and caused an increase in iaa level and a decrease iaa oxidase activity in hypocotyls, which were significantly different from the control. whereas, carbohydrate and protein contents in hypocotyls were increased significantly to its levels in control. the highest level values were found in synthetic auxin treatment. conclusion the bacterial auxin can be used to improve rooting response of mung bean cuttings and this method very easy and not costly compared with synthetic hormones. keywords mung bean, indole acetic acid, rooting response and bacillus subtilis plants.10 overall, industrial rooting hormones are widely used in vegetative propagation of stem cuttings which show variation in rooting ratios. so the goal of this study is to use b. subtilis bacteria in the production of iaa in growth medium as well as with the study some of the conditions that affect the production of bacteria that isolated from soil and to find out the role of the bacteria iaa in rooting response of mung bean cuttings and improvement the ability of the mung bean cuttings rooting by using relatively easy and inexpensive way. materials and methods bacillus subtilis, bacteria was used that was obtained from the department of biology sciences / college of education / university of karbala isolated from soil and dried to form a dry powder and loaded with a substance calcium carbonate and working bacterial suspension in normal saline solution 0.85% by dissolving 50 mg of bacteria in 4.5 ml of physiological salt solution. bacteria active in nutrient broth by taking 1 ml of suspension bacteria was added to 9 ml of nutrient broth and incubated for 24 hours for the parapets incubated10 to identify the optimal conditions for indole acetic acid production by bacteria bacillus subtilis a number of factors were studied affecting indole acetic acid production of by bacillus subtilis in nutrient broth media. after sterilization, it was supported by amino acid tryptophan in the concentration of 1 mg/ml. some of the optimal culture conditions of production were studied.10 effect of incubation period on iaa production 100 µl of 24 h bacterial broth was inoculated in 9.9 ml of nutrient broth medium in 25 ml flask containing 0.1 mg ml−1 l-tryptophan. the flask was incubated at 35°c temperature for (24, 48, 96, 120 and 196 hr), to determine the optimum period khalid ali hussein et al. 89j contemp med sci | vol. 2, no. 7, summer 2016: 88–92 research the role of bacteria bacillus subtilis in improving rooting response for the production. the concentration of indole acetic acid was estimated according to bent et al., (2001)11 in production media (nutrient broth support by tryptophan), the bacteria was removed from the culture by use of the centrifuge (10000 r/ min), then the amount of auxin estimated in the bacterial filtrate using salkowski reagent (fecl31 ml, hclo3 35 ml) absorbance was noted at 530 nm. the amount of iaa produced was calculated based on standard graph prepared from iaa solution (0–100 μg ml−1). effect of incubation temperature on iaa production the bacteria bacillus subtilis was incubated for 48 hours at various temperatures (30, 35, 40, 45 and 50°c) to determine the optimum temperature for the production of auxin. effect of inoculum size on iaa production production medium of indole acetic acid incubate by inoculum size (25, 50, 75, 100, 125 and 150) µl/ 10 ml production culture and incubation at 40°c for 72 hrs to statement effect of the inoculum size iaa production from bacteria. the effect of indole acetic acid production in bacillus subtilis filtrate on the rooting response of mung bean cuttings after determining the optimal conditions for iaa production, temperature, period incubation and inoculum size, its effect on rooting mung bean cuttings were being tested. seeds of mung bean (vigna ratiata l. wilezek) were germinated in sterile sawdust supplied with water after soaking overnight. in addition to seedling growth were carried out in growth cabinet at 25 ± 1°c, under continuous illumination supplied by warm white fluorescent tubes (1500–1800 lux) and relative humidity of 60–70% (binder gmbh, germany). stem cuttings were prepared according to12 from 10-day-old light grown seedlings (fig. 1). the cutting had apical bud, pair of fully expanded primary leaves, epicotyls and 3 cm of hypocotyl under cotyledonary nodes, after removal of root system (fig. 2). boric acid was prepared at concentrations 5 µg/ml (as rooting medium) auxin solution (iaa) was prepared at concentration 10−4 m. basal part of the hypocotyl of fresh cuttings were treated for 24 h with tested solutions (distilled water, bacterial filtrate and synthetic auxin) thereafter, cuttings were transferred to boric acid (5 µg/ml) for 6 days due to its necessity in the formation of root primordia and its subsequent growth and development to visible roots.13 twelve cuttings per treatment for rooting test were placed 4 per glass vial containing 15 ml (3 cm depth) of the appropriate solution under the same conditions of growing seedlings, then calculate the number root/cuttings. the effect of iaa production in bacillus subtilis filtrate in carbohydrate, protein, iaa contents and activity of iaao the carbohydrate contents of hypocotyle according to dubois et al. (1956)14 and proteins according to bishop et al., (1985)15 and according to the method unyayar et al. (1996)16 iaa was estimated, the activity of iaao estimated according to sequeria and nineo, 1966)17, iaao activity is represented by the amount of iaa degraded (µg) starting from 1 mg. all experiments were designed completely randomized, data were subjected to analysis of variance (anova) and the means were compared using l.s.d test (p ≤ 0.05) by sas software. results and discussion the iaa production by bacteria in a growth medium was watched for five days, and then the model was to draw every 24 hours to measurement iaa produced by bacteria. figure 3 shows that the iaa production begins after 24 hours of incubation. iaa production reached to (55.34) mg/ml and then rose gradually to a maximum in the third day, when they reached to (106.38) mg/ml, then began to decline, reaching below the fifth day with a concentration of (71.62) mg/ml. the highest value of auxin production was obtained at 72-hours (the third day) which is not significantly different compared to 48-hours. mutluru and konada, (2007)18 observed that the combination of bacteria with plant roots produces iaa to the liquid medium, and the amount of auxin product depends on the growth conditions, phase growth of the bacteria and the abundance of material basis. bharucha et al., (2013)19 showed fig. 1 mung bean seedling in culture media (left) and mung bean seedling (right). fig. 2 mung bean cuttings were prepared from 10-day-old light grown seedlings after removal of root system. 90 j contemp med sci | vol. 2, no. 7, summer 2016: 88–92 the role of bacteria bacillus subtilis in improving rooting response research khalid ali hussein et al. 4. the effect of iaa produced by bacteria in rooting response of mung bean cuttings figure 6 shows a significant increase in rooting response of mung bean cuttings terms number of adventitious roots per cutting (8.5, 21.8, 38.4) root/cutting each of the control, synthetic auxin, respectively. data show that the number of roots increased three-fold compared with the roots of cuttings treatment with distilled water and an increase percentage 156.47% when using bacterial filtrate attributed the cause to increase rooting percentage in bacterial filtrate to the bacteria ability to produce auxin, which is impact effective in rooting response. many of microorganisms can interact with the plant and produced hormones similar to those produced by plant such as regulated growth auxins and gibberellins and cytokines,26 among these hormones and most important in initiation and development roots is auxin.27 results of the study showed that bacillus subtilis bacteria produces growth regulator (iaa), that impact on rooting response and thus success of rooting. this is agreeing with bae (2007)28 which found that bacillus subtilis stimulate the emergence and differentiating adventitious roots in cucumber cuttings. tavkelova et al., (2005)29 showed that kidney bean cuttings treated with liquid growth media of bacillus subtilis enhanced adventitious root formation from 4.7 to 4.13 times more than control samples. srinivasan et al., (1996)30 showed that the treated phaseolus vulgaris plant with bacillus (non pathogenic) enhanced adventitious root formation. also erturk et al., (2010)31 found that bacillus rc23 and bacillus subtilis and bacillus rc03 and bacillus simplex rc19 stimulates adventitious root formation in kiwifruit cuttings. 5. effect of bacterial and synthetic iaa in hypocotyl content of iaa figure 7 shows the effect of each bacterial and synthetic filtrate auxin in hypocotyl cutting content of iaa, the iaa content in fig. 3 the effect of incubation periods on iaa production from bacillus subtilis. fig. 4 the effect of temperature on iaa production from bacillus subtilis. fig. 5 the effect of inoculum size on iaa production from bacillus subtilis. fig. 6 effect of iaa produced by bacteria in rooting response of mung bean cuttings. that the higher productivity of iaa at 96 hours by bacteria pseudomonas putida. swain et al., (2007)20 indicated that the iaa production by the bacillus subtilis increased linearly from 2 to 8 days, and then fell with a decreased bacteria in a culture which was supported with amino acid tryptophan. decline in iaa production may be arising from the release of analysis enzymes for auxin such as peroxidase and iaa oxidase.3 2. temperature figure 4 indicates that the highest value of iaa production was obtained at a temperature of 40°c, and gave (133.27) mg/ ml, and then iaa production decrease significantly on level of 0.05. so this degree was adopted in all experiments. varied thermal grades used in the production of indole acetic acid of microbiology, shahab et al. (2009)21 found that the 30°c is the optimal for auxin production from bacilllus thuringiensis. while the use of 36 ± 2 temperature in the auxin production from azotobacter, rhizobium, bacillus and pseudomonas.22 on the other hand, while sudha et al., (2012)23 showed that 37°c is the optimum temperature for iaa production by bacillus ssp. any change in the optimum temperature affected on the biosynthesis enzymes of iaa and thus reflected on the auxin production.24 3. inoculum size results in fig. 5 show that the iaa production increases the inoculum size until it reached its maximum by using 125 μl, where the auxin production reached (108.52 μg/ml) thereafter iaa declined, so the inoculum size, period of incubation for 2 days and at a temperature of 40°c were adapted in practical experience on mung bean cuttings. volumes of the inoculum size used varied in iaa production from microorganisms, swain et al., (2007)20 had used the inoculum size 2% in iaa production from b. subtilis. huu da et al., (2015)25 indicated that the percentage of inoculum size 1.65% gave the highest iaa productivity from b. subtilis tib6 isolated from root rhizosphere of the pepper plant. khalid ali hussein et al. 91j contemp med sci | vol. 2, no. 7, summer 2016: 88–92 research the role of bacteria bacillus subtilis in improving rooting response fig. 10 effect of bacterial and synthetic iaa in hypocotyl content of protein. hypocotyl as the primary content papers in control treatment (distilled water for 24 hours) is 0.875 μg/g fresh weight, this content rise to 1.041 μg/g fresh weight of hypocotyl cutting in treated with bacterial filtrate and increase percentage 19%. however in the synthetic auxin treatment, iaa content has increased significantly for both treatment control and bacterial filtrate and increased by 145.91% compared to the treatment of bacterial filtrate as 100%, increase percentage 334.36% compared to the control treatment as 100%. internal auxin produced in the vegetative and transport basipetally to hypocotyl may be complementary to the exodogenous iaa levels, which improved the rooting percentage,32 the increase in iaa content may be due to the decline in iaao activity fig. 8, and this was confirmed by33 auxin also known as the key signal initiation and development adventitious roots formation,34 lower iaao activity in the cuttings might be among the critical factors that improve rooting and this is reflected in rooting response. 6. effect of bacterial and synthetic iaa in iaa oxidase activity in hypocotyl the activity of iaao in hypocotyl cutting is 0.337 μg iaa oxidase/1 h. g. fw in cuttings that treated with distilled water for 24 hours. this activity lowered to 0.114 μg iaa oxides/1 h. g. fw in cuttings treatment of bacterial filtrate, reduce percentage 66.17%, lowers activity is 0.051 μg iaa oxides/1 h.g. fw. it was recorded in the treatment in synthetic iaa fig. 8. this reflects increase iaa in hypocotyl (fig. 5), which revealed the highest number of adventitious roots (fig. 6). yan et al., (2014)33 found reduced iaao activity in hemarthria compressa plant when treated with auxin. 7. effect of bacterial and synthetic iaa in hypocotyl content of carbohydrate the effect of bacterial filtrate in hypocotyl content of carbohydrate illustrated in fig. 9. the results showed that the bacterial filtrate raise carbohydrate content from 3.10 mg/g f.w in control sample to 4.26 when exposed to bacterial filtrate for 24 hours, which increased significantly whereas carbohydrate content in hypocotyl cuttings treated by synthetic iaa hypocotyl raise to 6.89 mg/g. f.w. adventitious root formation seems that depends on a sufficient supply of carbohydrates to adventitious roots formation zone where the roots processed energy and carbon necessary to initiation and development adventitious roots formation.35 the relationship between auxin and carbohydrate metabolism by creating spin-off roots have studied the outward processing of auxin and control carbohydrate levels and the transfer of carbon and stimulate the enzymes used to metabolize sugar in the rooting zone and the increase observed in the base of the stem in carbohydrates mind alqrnql levels after outward processing of auxin and suggested that auxin stimulates the new sink of carbohydrates, which directly contributes to the formation of the root primordial,36 druge et al., (2009)37 found that the accumulation of auxin in the rooting zone contributes to the establishment of a new sink of carbohydrates in the rooting zone by stimulating the activity of enzymes invertase cell wall and vascular.38 8. effect of bacterial and synthetic iaa in hypocotyl content of protein figure 10 shows the effect of the bacterial filtrate synthetic auxin in protein content of hypocotyl mung bean cuttings increased fig. 7 effect of bacterial and synthetic iaa in hypocotyl content of iaa. fig. 8 effect of bacterial and synthetic iaa in iaa oxidase activity in hypocotyl. fig. 9 effect of bacterial and synthetic iaa in hypocotyl content of carbohydrate. 92 j contemp med sci | vol. 2, no. 7, summer 2016: 88–92 the role of bacteria bacillus subtilis in improving rooting response research khalid ali hussein et al. from 0.109 in the control treatment to 0.114 when treated with bacterial auxin. an increase percentage 4.58%. in case supplying cuttings with a solution of synthetic iaa in concentration 5 × 10−5 for 24 hours, protein content in hypocotyl was increa sed by 82.23% compared to treatment with distilled water. bacterial filtrate is succeeded in increasing the protein content, but did not reach the level of a protein hypocotyl cuttings-treatment of synthetic auxin. these ratios were significant at the 5% the bacillus subtilis. the ability to produce iaa quantities improved when tryptophan percent increases in growth media,25 the bacterial filtrate containing iaa form the bacteria isolated from the soil efficiently improving rooting mung bean response by increasing rooting requirements such as auxin carbohydrates, protein and reduced activity of iaao analysis for auxin in basal part cutting. thus we can recommend their use in raising rooting rates in stem cuttings as an alternative to synthetic auxin in the plant propagation which is resulting environmental problems and may increase the cost of propagation. n references 1. arshad m, frankenberger wt. plant growth regulating substances in the rhizosphere: microbial production and functions. adv agron. 1998;62: 146–151. 2. khalid a, tahir s, arshad m, zahirza. relative efficiency of rhizobacteria for auxin biosynthesis in rhizosphere and non-rhizosphere soils. aus j soil res. 2004;42:921–926. 3. khamna s, yokota a, peberdy jf, lumyong s. indole-3-acetic acid production by streptomyces sp. isolated from some thai medicinal plant rhizosphere soils. eurasia j bio sci. 2010;4:23–32. 4. teale wd, paponov ia, palme k. auxin in action: signaling, transport and the control of plant growth and development. nat rev mol cell biol. 7:847–859. 5. srivastava hs. plant physiology and biochemistry. rastogi publications. india. 2012. 6. leveau jhj, lindow se. utilization of the plant hormone indole-3-acetic acid for growth by pseudomonas putida strain 1290. appl environ microbiol. 2005;71:2365–2371. 7. rifat h, safdar a, ummay a, rabia k, iftikhar a. soil beneficial bacteria and their role in plant growth promotion: a review. ann microbiol. 2010;60: 579–598. 8. dobbelaere s, vanderleyden j, okon y. plant growthpromoting effects of diazotrophs in the rhizosphere. crc crit rev plant sci. 2003;22:107–149. 9. davies jr, davis ft, kester de. commercial importance of adventitious rooting to horticulture. in: davis td, haissig be, eds. biology of adventitious root formation. new york: plenum press, 1994;53–59. 10. spaepen s, vanderleyden j, remans r. indole-3-acetic acid in microbial and microorganism-plant signaling. fems microbiol. 2007;31:4. 11. bent e, tuzun s, chanway cp, enebak s. alterations in plant growth and in root hormone levels of lodge pole pines inoculated with rhizobacteria. can j microbiol. 2001;47:793–800. 12. hess ce. the mung bean bioassay for detection of root promoting substances. plant physiol. 1961;36(i):suppl. 21. 13. middleton w, jarvis bc, booth a. the effects of ethanol on rooting and carbohydrate metabolism in stem cutting of phaseolus aureus roxb. new phytol. 1978;81:279–285. 14. dubois m, gilles ka, hamilton jk, rebers pa, smith f. colorimetric method for determination of sugars and related substances. anal. chern. 1956;28:350–356. 15. bishop mc, dben-von laufer jl, fody ep, thirty tcu. clinical chemistry principles, procedures and correlations. 1985;181–182. 16. ünyayar s, topcuoglu sf, ünyayar a. a modified method for extraction and identification of indole-3-acetic acid (iaa), gibberellic acid (ga 3 ), abscisic acid (aba) and zeatin produced by phanerochate chrysosporium me 446. bulg j plant physiol. 1996;22(3–4);105–110. 17. sequeria l, mineo l. partial purification and kinetics of indole acetic acid oxidase from tobacco roots. plant physiol. 1966;41:1200–1208. 18. sridevi m, mallaiah kv. bio production of indole acetic acid by rhizobium strains isolated from root nodules of green manure crop, sesbania sesban (l.) merr. iranian j biotechnol. 2007;5(3):178–182. 19. bharucha u, patel k, trivedi u. optimization of indole acetic acid production by pseudomonas putida ub1 and its effect as plant growth-promoting rhizobacteria on mustard (brassicanigra). natl acad agri sci, 2013. 20. swain mr, naskar sk, ray rc. indole -3-acetic acid production and effect on sprouting of yam (dioscorea rotundata l.) minisetts by bacillus subtilis isolated from culturable cowdung microflora. polish j micrbiol. 2007;56(2):103–110. 21. shahab s, ahmed n, khan ns. indole acetic acid production and enhanced plant growth promotion byindigenous psbs. afr j agri res. 2009;4(11):1312–1316. 22. joseph b, patra rr, lawrence r. characterization of plant growth promoting rhizobacteria associated with chickpea (cicerarietinum l.). int j plant prod, 2007;2:141–152. 23. sudha m, shyamala gr, prbhavati p, astapritya p, yamuna dy, saranya a. production and optimization of indole acetic acid by indigenous microflora using agro waste as substrate. pak j biol sci. 2012;15:39–43. 24. bilkay is, karakoc s, aksoz n. indole-3-acetic acid and gibberellic acid production in aspergillusniger. turk j biol, 2010;34:313–318. 25. huu dat tt, kim cuc nt, cuong pv. optimization of indole-3-acetic acid production by bacillus subtilis tib6 using responses surface methodology. int j develop res. 2015;5(4):4036–4042. 26. melo is. rizobacteriaspromotoras de crescimento de plantas: descriçao e potencial de agricultura. in: melo, i.s.: azevedo, j.l. (eds). ecologiamicrobiana. embrapa meioambiente, jaguariuna, 1998;86–116. 27. gaudin v, vrain t, jouanin l. bacterial genes modifying hormonal balances in plants. plant physiol biochem. 1994;32:11–28. 28. bae ys, park k, lee yg, choi oh. a simple and rapid method for functional analysis of plant growth promoting rhizobacteria using the development of cucumber adventitious root system. plant pathol j. 2007;23(3): 223–225. 29. tsavkelova e, cherdyntseva t, netrusov a. auxin production by bacteria associated with orchid roots. mikrobiologiya. 2005;74:46–53. 30. srinivasan m, petersen dj, holl fb. influence of indolaceticacid-producing bacillus isolates on the nodulation of phaseolus vulgaris by rhizobium etli under gnotobiotic conditions. can j microbiol. 1996;42:1006–1014. 31. erturk y, ercisli s, haznedar a, cakmakci r. effects of plant growth promoting rhizobacteria (pgpr) on rooting and root growth of kiwifruit (actinidiadeliciosa) stem cuttings. biol res. 2010;43:91–98. 32. pop ti, pamfil d, bellini c. auxin control in the formation of adventitious rooting. not bot hort agrobot cluj. 2011;39:307–316. 33. yan yh, li jl, zhang xq, yang wy, wan y. effect of naphthalene acetic acid on adventitious root development and associated physiological changes in stem cutting of hemarthriacompressa. plos one. 2014;9(3):34–55. 34. laskowski m, biller s, stanley k, kajstura t, prusty r. expression profiling of auxin-treated arabidopsis roots: toward a molecular analysis of lateral root emergence. plant cell physiol. 2006;47:788–792. 35. corrêa lr, paim dc, schwambach j, fett-neto ag. carbohydrates as regulatory factors on the rooting of eucalyptus saligna smith and eucalyptus globuluslabill. plant growth regulation. 2005;45:63–73. 36. agulló-antón ma, sánchez-bravo j, acosta m, druege u. auxins or sugars: what makes the difference in the adventitious rooting of stored carnation cuttings? j plant growth regul. 2011;30:100–113. 37. druege u. involvement of carbohydrates in survival and adventitious root formation of cuttings within the scope of global horticulture. in: niemi k, scagel c (eds) adventitious root formation of forest trees and horticultural plants from genes to applications. research signpost, kerala, 12009;87–208. 38. ahkami ah, melzer m, ghaffari mr, pollmann s, ghorbanijavid m. shahinnia f. distribution of indole-3-acetic acid in petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation. planta. 2013;238(3):499–517. 23 original issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 23 – 27 introduction chronic kidney disease (ckd) is a systemic condition that adversely affects the patient’s quality of life. if left untreated, it can progress to advanced stages, which are associated with high morbidity and mortality. the american society of nephrology defined ckd and classified it into five stages based on the glomerular filtration rate (gfr).1-3 given the increasing prevalence of ckd, there are ongoing attempts to identify and control the associated risk factors such as obesity, malnutrition, hypertension, diabetes mellitus, as well as periodontal diseases. such attempts are imperative to decrease the burden of disease on ckd affected patients.4-7 it has been suggested that periodontitis was a potential independent risk factor for ckd.8 by definition, periodontitis is a chronic inflammatory condition that causes periodontal tissue destruction. it often occurs due to poor oral hygiene, microbial plaque, and calculus accumulation around the tooth surface causing subsequent gingival inflammation, periodontal pocket formation, and loss of tooth supporting structures.9 in this case, the local inflammatory factors and c-reactive protein (crp) pass through the affected gingiva into the peripheral blood circulation and exert systemic effects.4,10 continuation of this process in ckd patients can aggravate their disease condition and even it may lead to renal failure if left untreated. based on a cross-sectional study, a mutual correlation was reported between periodontal diseases and ckd.11 likewise, such correlations have been assumed between periodontal diseases and a number of other systemic conditions such as cardiovascular diseases, diabetes mellitus, osteoporosis, respiratory diseases, rheumatoid arthritis, low birth weight, and immature birth.4,12-19 in general, periodontitis is a multifactorial disease, and therefore, several factors are affecting the susceptibility and development of periodontal diseases.20,21 the prevalence of periodontal disease has been reported 57% in ckd patients.22 thus, considering the role of periodontitis on systemic conditions, the purpose of this study was to assess the correlation between periodontal diseases and ckd duration. materials and methods this descriptive cross-sectional study was approved by the regional research ethics committee with reference number “ir.sbmu.ribs.rec.1394.170” and performed in complete accordance with the declaration of helsinki. participation in this study was voluntary and written informed consent was obtained from all subjects. after explanation of the study objectives, potential questions were answered by the lead author. likewise, oral examination was conducted by a single calibrated examiner (lead author) prior to their hospital appointments. a convenient sampling technique was used to recruit ckd adult patients who attended the “labbafinejad hospital” in tehran city during 2017. for sample size calculation, we used the reported prevalence (57%) with 95% confidence level, and 7% margin of error. based on this calculation, at least 192 cases were needed for conducting this study. two instruments were used for data collection in this project. the first questionnaire assessed the oral health behavior of ckd patients and it was a self-reported questionnaire. the second questionnaire was a standard who instrument which was used for clinical evaluation of the oral health status of ckd patients. likewise, who recommended methodology was used for oral examination in order to determine the decayed, missing, and filled teeth (dmft) in our sample population. for gingival health status, we used the clinical periodontal and chronic kidney diseases: a modifiable association foujan jabbarzadehkhoei1, soheila bakhshandeh2, mahshid namdari2, mina pakkhesal3, mohammad hossein khoshnevisan2,4* 1 school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 2 community oral health department, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 3 community oral health department, school of dentistry, golestan university of medical sciences, gorgan, iran. 4 dental research center, research institute of dental sciences, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. *correspondence to: mohammad hossein khoshnevisan (e-mail: khoshmh@gmail.com) (submitted: 12 november 2020 – revised version received: 29 november 2020 – accepted: 07 december 2020 – published online: 26 february 2021) abstract objective the purpose of this study was to assess the relationship between periodontal diseases and chronic kidney disease (ckd) duration. methods this descriptive cross-sectional study was conducted on referral ckd patients to a teaching hospital in 2017. two instruments were used for data collection. the first one was a self-reported questionnaire regarding oral health status and patients’ behaviors. the second questionnaire was used for the clinical assessment of oral health status. results out of 192 patients, 46.9% were male and 53.1% female with a mean (sd) age of 51.9 (±15.1) years. the mean duration of ckd was 7.70 (±7.34) years. about 67.7% of patients experienced toothache in the past year. also, 67.7% had gingival bleeding (bop), 34.4% had clinical attachment loss (cal) > 4 mm, and over 50% of patients had a pocket depth (pd) > 4 mm. by controlling the patient’s age, a direct correlation was detected between the duration of ckd and decayed, missing, and filled teeth index (r=0.64, p<0.001). moreover, the prolongation of the disease period was detected in patients with cal>4 mm (p=0.02). likewise, a direct correlation was detected between the duration of ckd and the periodontal index (r=0.48, p<0.001). conclusions given the direct correlation between the periodontal conditions and duration of ckd, regular biannual dental visits are essential for ckd patients. all physicians are encouraged to include regular oral health checkups in the treatment protocol for ckd patients. keywords periodontal disease; chronic kidney disease; oral health. 24 original periodontal & chronic kidney diseases association mohammad hossein khoshnevisan et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 23 – 27 attachment loss (cal), periodontal index (pi), community periodontal index (cpi), and oral hygiene index (ohi). the cal was used for measuring the position of the soft tissue in relation to the cemento-enamel junction (cej) which is a fixed point. two measurements were used to calculate the cal index: the probing depth and the distance from the gingival margin to the cej. the pi was used by evaluating and scoring all teeth using five well-distinguished categories (0, 1, 2, 6, and 8), representing incremental degrees of disease severity. the cpi was based on three features of bleeding, dental calculus, and depth of gingival sulcus. a special “whoprobe” was used for periodontal examinations. the ohi was reported as the sum of two indices; the amounts of debris, and calculus present on the tooth surface. thus, the ohi may ranges from 0, meaning no debris or calculus to as high as 6. the debris index consists of the following scores: 0, no debris on the tooth surface; 1, less than onethird of tooth surface covered with soft debris; 2, more than one-third and less than two-thirds of tooth covered with soft debris; 3, over two-thirds of the tooth surface covered with soft debris. likewise, the calculus index, composed of the following scores: 0, no calculus on the tooth surface; 1, less than onethird of the tooth surface covered with supra-gingival calculus; 2, over one-third and less than two-thirds of the tooth surface covered with supra-gingival calculus; 3, over two-thirds of the tooth surface covered with supra-gingival calculus.12 for oral health behaviors evaluation, several questions were used to assess daily tooth brushing frequency, use of fluoridated toothpaste, flossing, consuming sugary snacks between main meals, frequency of dental visits per year, the time of last dental check-up, and cigarette smoking habit. data were collected anonymously and analyzed using spss version 22. the frequency of qualitative variables, and the mean and standard deviation of quantitative variables were reported for the sample population. likewise, demographic variables, oral health-related behaviors and oral health-related indices were reported for ckd patients. the independent t-test, mann–whitney test, and pearson correlation coefficient were used for statistical analysis. the p-value less than 0.05 was considered statistically significant. results out of 192 participants, 90 (46.9%) patients were male and 102 (53.1%) were female. the mean age of patients was 51.9 (±15.1) years, ranging from 18 to 79 years. the mean duration of ckd in patients was 7.70 (±7.34) years. additional patients’ demographic information was presented in table 1. the frequency distribution of patients’ past dental experiences were provided in table 2. out of 67.7% of patients who experienced toothache in the past year, only 52% of them had a dental visit. as reported in table 3, a high percent of patients had gingival bleeding (67.7%), more than half of the them had periodontal pocket higher than 4mm, and about 34% had cal>4mm as reported in table 4. the mean dmft index was 11.61 (±6.23) (table 4). gender differences in mean dmft and periodontal index was minimal (p=0.99). however, advance state of periodontal diseases as demonstrated by cal index, was slightly higher among female when compared with male ckd patients (p=0.87). although, no significant association was detected between dmft and pi with patients’ education (p>0.05), but the prevalence of cal>4mm and pi was higher among patients with lower education (p=0.12, p=0.92). as demonstrated in table 5, a direct correlation was detected between duration of ckd and dmft index (r=0.64, p<0.001) as well as the pi (r=0.48, p<0.001). also, a longer disease duration was detected in patients with cal>4 mm (p=0.02). discussion several studies have reported on the correlation between periodontal diseases and ckd, emphasizing on the regular oral and dental care as one of the most important strategies to decrease the burden of ckd.23,24 in other words, entry of local inflammatory factors from affected gingiva into patient’s blood stream can aggravate systemic inflammation process and exacerbate the ckd. obviously, oral health is not only about optimal esthetics, mastication, phonetic, and speech; there is a direct and often mutual correlation between oral health and general health. not paying enough attention to this critical table 1. demographic information in ckd patients. variable category number percentage gender male 90 46.9 female 102 53.1 age groups (yrs.) 18-40 46 23.9 41-60 92 47.9 61-80 54 28.2 place of residence tehran 138 71.9 other provinces 54 28.1 educational level illiterate 22 11.5 below high school diploma 74 38.5 high school diploma 61 31.8 over high school diploma 35 18.2 disease duration <5 years 85 44.3 5-10 years 49 25.5 ≥10 years 57 29.7 underlying disease/ health conditions hypertension 71 36.9 anemia 40 20.8 diabetes mellitus 30 15.6 cardiac disease 30 15.6 liver disease 30 2.1 cancer 1 0.5 none 29 15.1 25 original periodontal & chronic kidney diseases associationmohammad hossein khoshnevisan et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 23 – 27 point, it can impose a significant financial burden on patients and the health-care system. financial resources are important for financing public health plans for overall health promotion. however, most developing countries have excluded oral health from the list of their priorities due to budgetary constraints. this can cause several problems and exerts additional burden on patients and the health-care system. thus, this study aimed to assess the correlation between periodontal disease and ckd duration in order to provide further information that may be useful for medical professionals and authorities, policy makers in the health-care systems in developing countries. in 2016, ausavarungnirun et al assessed the correlation between periodontal disease and different stages of ckd in table 2. dental experiences in ckd patients. variable score/response number frequency oral complaints bad taste in the mouth 126 65.6 halitosis 115 59.9 xerostomia 114 59.4 oral ulcers 55 28.6 oral and dental self-assessment good 66 34.4 moderate 65 33.8 poor 53 27.6 i do not know 8 4.2 gingival selfassessment good 84 42.7 moderate 54 28.1 poor 49 25.5 i do not know 7 3.6 tooth cleaning tooth brushing 185 96.4 dental flossing 52 27.1 wooden toothpick 21 19.9 plastic toothpick 19 9.9 toothbrush stick 4 2.1 frequency of daily tooth brushing twice or more 51 26.6 once 79 41.1 less than one a day 62 2.1 frequency of dental visits per year twice or more 17 8.8 once 60 31.3 never 113 58.9 i do not know 2 1 time of last dental visit < 1 year ago 79 40.1 between 1-5 years ago 87 45.6 ≥5 years ago 24 12.6 never 2 1 table 3. frequency distribution of ckd patients based on dental and gingival indices. variable score number percentage dmft <10 102 53.1 10-20 72 37.5 >20 18 9.4 gingival bleeding no bleeding 60 31.3 bleeding 130 67.7 edentulous 2 1 periodontal pocket no pocket 81 42.3 4-6 mm pocket 109 56.7 edentulous 2 1 clinical attachment loss 0-3 mm 126 65.6 4-5 mm 61 31.8 6-8 mm 5 2.6 debris index 0 48 25 >1/3 111 57.8 1/3-2/3 25 13 >2/3 8 4.2 calculus index 0 37 19.3 >1/3 95 49.5 1/3-2/3 48 25 >2/3 12 6.3 dmft: decayed, missing, and filled teeth table 4. comparison of dmft, pi, cal indices by gender and education level dmft p-value pi p-value cal<4mm cal>4mm p-value mean (sd) mean (sd) n (%) n (%) gender male 11.63 (6.17) 0.99 1.43 (0.92) 0.99 58 (65.2%) 31 (34.8%) 0.87 female 11.64 (6.31) 1.43 (0.88) 67 (66.3%) 34 (33.7%) education level under diploma 11.57 (6.34) 0.75 1.43 (0.91) 0.92 98 (63.2%) 57 (36.8%) 0.12 over diploma 11.94 (5.84) 1.42 (0.87) 27 (77.1%) 8 (22.9%) dmft (decayed, missing, and filled teeth); pi (periodontal index); cal (clinical attachment loss); p-value (probability value). level of significance: p<0.05 significant. 26 original periodontal & chronic kidney diseases association mohammad hossein khoshnevisan et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 23 – 27 thai patients. they found that advanced periodontitis was more common in higher stages of ckd than early stages of disease.25 also, grubbs et al in a retrospective cohort study in 2016 evaluated the correlation between periodontal disease and kidney function and found that, the incidence of ckd was doubled in patients with advanced periodontitis.26 grubbs et al in 2015 evaluated the correlation of periodontal disease (in different severities) with the occurrence of ckd in africanamericans and reported that patients with advanced periodontitis were four times more susceptible to ckd (p=0.002). they showed that periodontal diseases were more common in patients at high risk of ckd, and found a significant correlation with clinical impairment of renal function.8 in their study, 86.3% of patients had gingival bleeding and 29.8% had pocket depth > 4 mm. also, cal > 4 mm was noted in 33.8% of patients.8 in present study, 67.7% of patients had gingival bleeding and 34.4% had cal > 4 mm. also, 56.7% of patients had pocket depth > 4 mm. thus, the majority of our findings are in agreement with the results of grubbs et al.8 in the same study, smoking had a significantly higher frequency among patients with advanced periodontitis.8 thus, they concluded that smoking was an aggravating factor for periodontitis. in present study, the majority of patients were non-smokers; this probably explains why some periodontal indices such as pi in our patients were not as high as those reported in other studies. a review study by ismail et al in 2013 reported higher prevalence of moderate to severe periodontal diseases in ckd patients compared with healthy controls. likewise, they reported that periodontitis was correlated with higher level of systemic pro-inflammatory markers.27 in another review study by ioannidou et al in 2013, the prevalence of periodontitis was assessed in ckd patients. in line with findings of other studies, this report also indicated higher prevalence of periodontitis in patients with ckd compared with healthy individuals. moreover, increased prevalence of periodontitis was associated with decreased renal function in patients with ckd.28 joseph et al in 2009 evaluated the prevalence of periodontal disease in ckd patients in comparison with healthy controls. they compared the oral hygiene, gingival inflammation, periodontal pocket, and cal in 77 ckd patients with different etiologies and 77 healthy controls. the patients were divided into four groups of no periodontitis, mild, moderate, and severe periodontitis. they reported significantly higher periodontal indices in the ckd group compared with the control group. also, the prevalence and severity of periodontal diseases were significantly higher in the ckd group (p<0.001).29 in present study, when comparing periodontal parameters measured in ckd patients with the national data, it was revealed that, most indices in ckd patients were considerably different from the national mean values reported by the latest national oral health survey conducted in 2012.30 based on who recommendations, in this survey, three children and two adult age groups were considered. therefore, our data were compared with national average in two adult age groups of 35–44 and 65–74 years old. the majority of our study participants fall within these two age categories (18–40=24%, 41–60=50%, 61+=28%). although, patients’ age in our study population was slightly younger than the mean age of patients in the national survey, the measured indices were found to be higher than the national average. one limitation of this study can be improved by using multicenter design and make further assessments in order to determine if prevention and treatment of periodontal diseases can prevent gfr decline in ckd patients. conclusion given the direct correlation between the periodontal conditions and duration of ckd, regular biannual dental visits are essential for ckd patients. all physicians are encouraged to include regular oral health check-ups in the treatment protocol for ckd patients. thus, the active cooperation between medical and dental teams can be helpful in minimizing the ckd progression and severity. financial support and sponsorship nil. conflicts of interest there are no conflicts of interest. a list of abbreviations ckd: chronic kidney disease cal: clinical attachment loss pd: pocket depth pi: periodontal index dmft: decayed, missing, and filled teeth gfr: glomerular filtration rate cej: cementoenamel junction cpi: community periodontal index who: world health organization references 1. levey as, coresh j, bolton k, culleton b, harvey ks, ikizler ta, et al. k/ doqi clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. national kidney foundation. am j kidney dis 2002;39:1-266. 2. rosansky, sj. renal function trajectory is more important than chronic kidney disease stage for managing patients with chronic kidney disease. am j nephrol 2012;36:1–10. 3. khajehdehi p, malekmakan l, pakfetrat m, roozbeh j, sayadi m. prevalence of chronic kidney disease and its contributing risk factors in southern table 5. correlation of dmft, pi, and cal indices by ckd duration (years). dmft pi cal ckd duration (years) correlation coefficient* 0.64 0.48 0.87 p-value <0.001** <0.001** 0.02** dmft (decayed, missing, and filled teeth); pi (periodontal index); cal (clinical attachment loss); ckd (chronic kidney disease); p-value (probability value). * calculated by pearson correlation coefficient. ** level of significance: p<0.05 significant. 27 original periodontal & chronic kidney diseases associationmohammad hossein khoshnevisan et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 23 – 27 iran: a cross-sectional adult population-based study. iran j kidney dis 2014;8:109-15. 4. han ss, shin n, lee sm, lee h, kim dk, kim ys. correlation between periodontitis and chronic kidney disease in korean adults. kidney res clin pract 2013;32:164-70. 5. hakemi ms. chronic kidney disease epidemiology. iran j kidney dis 2014;8:261. 6. kshirsagar av, moss kl, elter jr, beck jd, offenbacher s, falk rj. periodontal disease is associated with renal insufficiency in the atherosclerosis risk; in communities (aric) study. am j kidney dis 2005;45:650-57. 7. fisher ma, taylor gw, shelton bj, jamerson ka, rahman m, ojo ao, et al. periodontal disease and other nontraditional risk factors for ckd. am j kidney dis 2008;51:45-52. 8. grubbs v, vittinghoff e, beck jd, kshirsagar av, wang w, griswold me, et al. association between periodontal disease and kidney function decline in african americans: the jackson heart study. j periodontol 2015;86:1126-32. 9. armitage gc. development of a classification system for periodontal diseases and conditions. ann periodontol 1999;4:1-6. 10. arigbede ao, babatope bo, bamidele mk. periodontitis and systemic diseases: a literature review. j ind soc periodontol 2012;16:487-91. 11. ricardo ac, athavale a, chen j, hampole h, garside d, marucha p, et al. periodontal disease, chronic kidney disease and mortality: results from the third national health and nutrition examination survey. bmc nephrol 2015;16:97. 12. butchibabu kalakonda d, koppolu p, kusai baroudi d, mishra a. periodontal systemic connections-novel associations-a review of the evidence with implications for medical practitioners. int j health sci 2016;10:293-307. 13. joshy g, arora m, korda rj, chalmers j, banks e. is poor oral health a risk marker for incident cardiovascular disease hospitalisation and all-cause mortality? findings from 172 630 participants from the prospective 45 and up study. bmj open 2016;6:e012386. 14. broder hl, tormeti d, kurtz al, baah-odoom d, hill rm, hirsch sm, et al. type ii diabetes and oral health: perceptions among adults with diabetes and oral/health care providers in ghana. community dent health 2014;31:158-62. 15. jeffcoat m, parry s, sammel m, clothier b, catlin a, macones g. periodontal infection and preterm birth: successful periodontal therapy reduces the risk of preterm birth. bjog int j obstet gynaecol 2011;118:250-6. 16. haerian-ardakani a, eslami z, rashidi-meibodi f, haerian a, dallalnejad p, shekari m, et al. relationship between maternal periodontal disease and low birth weight babies. iran j reprod med 2013;11:625–30. 17. esfahanian v, sadighi shamami mz, sadighi shamami mh. relationship between osteoporosis and periodontal disease: review of the literature. j dent (tehran) 2012;9:256–64. 18. venkataraman a, almas k. rheumatoid arthritis and periodontal disease. an update. n y state dent j 2015;81:30-6. 19. kim hd, sim sj, moon jy, hong yc, han dh. association between periodontitis and hemorrhagic stroke among koreans: a case-control study. j periodontol 2010;81:658-65. 20. ruospo m, palmer sc, craig jc, gentile g, johnson dw, ford pj, et al. prevalence and severity of oral disease in adults with chronic kidney disease: a systematic review of observational studies. nephrol dial transplant 2014;29:364-75. 21. kawar n, gajendrareddy pk, hart tc, nouneh r, maniar n, alrayyes s. periodontal disease for the primary care physician. dis mon 2011;57:174-83. 22. brito f, almeida s, figueredo cm, bregman r, suassuna jh, fischer rg. extent and severity of chronic periodontitis in chronic kidney disease patients. j periodontal res 2012;47:426-30. 23. craig rg. interactions between chronic renal disease and periodontal disease. oral dis 2008;14:1-7. 24. wahid a, chaudhry s, ehsan a, butt s, ali khan a. bidirectional relationship between chronic kidney disease & periodontal disease. pak j med sci 2013;29:211-5. 25. ausavarungnirun r, wisetsin s, rongkiettechakorn n, chaichalermsak s, udompol u, rattanasompattikul m. association of dental and periodontal disease with chronic kidney disease in patients of a single, tertiary care centre in thailand. bmj open 2016;6:e011836. 26. grubbs v, vittinghoff e, taylor g, kritz-silverstein d, powe n, bibbinsdomingo k, et al. the association of periodontal disease with kidney function decline: a longitudinal retrospective analysis of the mros dental study. nephrol dial transplant 2016;31:466-72. 27. ismail g, dumitriu ht, dumitriu as, ismail fb. periodontal disease: a covert source of inflammation in chronic kidney disease patients. int j nephrol 2013;2013:1-6. 28. ioannidou e, hall y, swede h, himmelfarb j. periodontitis associated with chronic kidney disease among mexican americans. j public health dent 2013;73:112-9. 29. joseph r, krishnan r, narayan v. higher prevalence of periodontal disease among patients with predialytic renal disease. braz j oral sci 2009;8:14-8. 30. khoshnevisan m.h, ghasemianpour m, samadzadeh h, baez rj. oral health status and healthcare system in i.r. iran. j contemp med sci 2018;4:107-18. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.919 73j contemp med sci | vol. 6, no. 2, march–april 2020: 73–77 73 original issn 2413-0516 introduction although pancreatic adenocarcinoma constitutes a low percentage of gastrointestinal malignancies, it is still a type of cancer with a high mortality and 5-year survival rates that cannot exceed 1%, since it is usually diagnosed at an advanced stage and few of the diagnosed patients have a chance of curative surgery.1,2 despite numerous successes in medical treatments in the historical process, surgical resection is the basic step of pancreatic cancer treatment.3 although surgical treatment can cure patients, despite the increased experience, the postoperative serious complication and morbidity rates are quite high.4 recognizing the postoperative complications and ensuring that patients are delivered to adjuvant treatment as soon as possible by applying the necessary treatments are perhaps as important as the success of surgical resection.5,6 moreover, these complications should not only be recognized and treated appropriately, but also various methods should be developed to predict the complications. thus, the surgeon will be able to better identify the patient at high risk for postoperative complications.7,8 in recent studies, it has been found that sarcopenia – considered as a syndrome characterized by progressive loss of skeletal muscle mass – is one of many factors that increase the risk of serious postoperative complications especially in elderly patients.9–12 in addition to anthropometric measurements, the diagnosis of sarcopenia can be made by measurement of the cross-sectional images of psoas muscle in abdominal computed tomography (ct).13 in addition to the effect of sarcopenia on postoperative results, it has been shown in many studies that it affects oncologic outcomes in other gastrointestinal cancers such as colorectal cancer and gastric cancer.14–16 however, there are limited studies on the relationship between postoperative outcomes and sarcopenia in patients with pancreatic cancer. therefore, in this study, we aimed to investigate the effect of sarcopenia on severe postoperative complications in patients undergoing curative resection for pancreatic adenocancer. material and methods patient selection and data collection between january 2014 and january 2020, 120 patients who underwent curative resection for pancreatic cancer at ankara university surgical oncology clinic were evaluated retrospectively. demographic data, laboratory results, preoperative ct images, surgical and pathology reports, medical follow-up records were obtained through the hospital database. patients with distant metastasis, who were evaluated as intraoperative unresectable, who underwent palliative operations, whose data could not be accessed and whose ct scans could not be measured appropriately or ct images were not found excluded from the study. pancreaticoduodenectomy (pd), distal pancreatectomy (dp) or total pancreatectomy (tp) were performed when appropriate and the patients were divided into two groups as pd and non-pd. pathological stages of patients were determined according to japan pancreas society (2009) general rules for the study of pancreatic cancer, 6th edn.17 postoperative complications were categorized and graded acoording to the modified clavien dindo classification.18 pancreatic fistulas were categorized using the international pancreatic fistula study definitions.19. effect of sarcopenia on postoperative complications after curative pancreatectomy for adenocarcinoma ümit mercan, ogün erşen, ali ekrem ünal* surgical oncology, general surgery department, faculty of medicine, ankara university, ankara, turkey. *corresponding author: ümit mercan (e-mail: umit.mercan@yahoo.com.tr) (submitted: 25 february 2020 – revised version received: 09 march 2020 – accepted: 26 march 2020 – published online: 26 april 2020) objective despite the increased experience in pancreatic cancer surgery, morbidity and mortality rates remain very high. many factors play a role in the development of postoperative complications and in studies it has been shown that sarcopenia, which is defined as progressive muscle mass loss is also an important factor. despite the proven effect of sarcopenia on postoperative complications and oncological outcomes in many types of gastrointestinal cancer, there are very few studies on pancreatic cancer. therefore, in this study, we aimed to investigate the effect of sarcopenia on the development of serious postoperative complications in patients who underwent curative pancreatectomy for pancreatic cancer. methods total psoas index (tpi) was calculated for sarcopenia diagnosis by measures of psoas muscle area on the level of l3 spine from preoperative staging computed tomograhpy (ct) images in pancreatic cancer patients undergoing curative resection. patient demographics and postoperative outcomes were analyzed in sarcopenic and non-sarcopenic group. results: it has been found to be statistically significant relation between severe postoperative complication and sarcopenia (p=0,001>). sarcopenia was more associated with cardiac and pulmonary complications among others (p=0.007, 0.003 respectively). in multivariate analysis, age [odds ratio (or): 1,08. 95% confidence interval (ci): 1,01~1,15, p=0,013], asa score (or: 2,84. 95% ci 1,62 ~ 4,97. p=0,043) and tpi (or: 3,61. 95% ci: 1,58 ~ 5,74. p=0,001>) has been found independent risk factors for severe postoperative complications. conclusion our results suggest that sarcopenia determined by using tpi, which can be easily obtained by examining the preoperative ct imaging, is an independent risk factor of severe postoperative complications. determining the degree of sarcopenia can affect patient selection, predictability of possible serious complications, elective operation preparation process with a combination of nutrition and exercise therapy in a particular patient group, and decisions regarding adjuvant or neoadjuvant therapy. key words pancreatectomy, postoperative complication, sarcopenia 74 original effect of sarcopenia on postoperative complications ümit mercan et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 73–77 computed tomography scanning measurements preoperative contrast-enhanced ct scans were analyzed to create a sarcopenia profile for each patient. the mean values of all measurements were taken as a basis. cross-sectional area of both psoas muscles were obtained by manually drawing the outer edges of the psoas muscles on the lumbar (l) 3 vertebra image where both spinous proceses were observed. sarcopenia quantification patients in the lowest 25% of the values obtained as a result of the total posoas index (tpi) determined specifically for gender were evaluated sarcopenically. measurement of the psoas muscle area at the l3 lumbar vertebra level and the calculation of the patient’s height and tpi have been described in other studies.9 if formulated, tpi is calculated by dividing the right and left psoas muscle by the total area of the individual in square meters of height [(right psoas muscle area + left psoas muscle area) / height (m2)]. statistical analysis numerical data are given as mean ± standard deviation. student’s t-test, mann–withney u test, chi-square test or fisher exact test were used when appropriate for numerical and categorical variables. binary logistic regression analysis was applied for possible factors affecting postoperative complications. p-values below 0,05 were considered statistically significant. ibm statistics version 23.0 was used for statistical analysis. results there were 120 patients who met the study and analysis criteria. the relationship between clinicopathological variables and sarcopenia have been summarized in table 1. the mean age of the patients was 58,03 ± 7,61, of which 76 (63,3%) were male. patients in the lowest 25% of the values obtained as a result of the tpi determined specifically for gender were evaluated sarcopically. with this method, 30 (25%) patients were considered sarcopenic. the lower quarter for men was 4,23 ± 1,52 cm2/m2, while for women it was 2,73 ± 1,08 cm2/ m2. thirty-four (28,3%) patients were obese and 3 (8,4%) of them were evaluated as sarcopenic obese. pd was performed in 97 (80,8) patients, while the remaining 23 (19,2%) patients underwent dp or tp. seven (5,8%) patients were operated after neoadjuvant therapy. stage 2 and 3 patients made up 90,9% of the study population. 72 (60%) patients were reported as negative node. no significant difference was found between sarcopenic and non-sarcopenic groups in terms of age, gender, operation, asa score, tnm stage, and lymph node status. the relationship between postoperative results and sarcopenia has been summarized in table 2. the average operation time was 212,16 ± 35,25 min. 45 (37,5%) patients had table 1. the relationship between clinicopathological variables and sarcopenia variables total sarcopenic (n=30) non-sarcopenic (n=90) p-value age 58,03±7,61 59,03±6,74 57,7±7,88 0,408 gender (male) 76(63,3) 19(63,3) 57(63,3) 0,590 asa score 0,670 1 12(10) 1(3,3) 11(12,2) 2 49(40,8) 9(30) 40(44,4) 3 59(49,2) 20(66,7) 39(43,4) bmi (kg/m2) 27,49±4,41 25,43±4,77 28,17±4,08 0,003 weight loss % 6,27±8,60 8,65±9,57 5,48±8,16 0,102 operation 0,034 pd 97(80,8) 28(93,3) 69(76,7) non-pd 23(19,2) 2(6,7) 21(23,3) tnm stage 0.754 1 11(9,1) 2(6,7) 9(10) 2 59(49,2) 14(46,7) 45(50) 3 50(41,7) 14(46,7) 36(40) lymph node status 0,258 n0 72(60) 16(53,3) 56(62,2) n1 48(40) 14(46,7) 34(37,8) neoadjuvant treatment 7(5,8) 3(10) 4(4,4) 0,239 tpi (cm2/m2) 4,68±1,61 3,06±0,87 5,22±1,43 0,001> numerical data were given as mean ± standard deviation. asa: american society of anesthesiology score, bmi: body mass index, pd: pancreaticoduodenectomy, tnm: tumor-node-metastasis, tpi: total psoas index. 75 original effect of sarcopenia on postoperative complicationsümit mercan et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 73–77 clavien-dindo grade 3 and above complications. cardiac complications developed in 20 (16,7%) patients, pulmonary in 56 (46,7%) patients, infective in 29 (24,2%) patients, and wound-related complications in 26 (21,7%) patients. delayed gastric emptying occurred in 22 (18,3%) patients and nasogastric decompression was performed. based on the clinical condition of the patients and the postoperative drain and blood amylase levels, grade a pancreatic fistula was observed in 9 (7,5%) patients, grade b fistula in 12 (10%) patients but grade c fistula was not observed. the average length of hospital stay was 11,42 ± 7,02 days, and the patients needed an average of 3,38 ± 4,40 days of intensive care. there was a significant difference between the two groups in terms of length of hospital stay, more in the sarcopenic group (p = 0,003). there were 8 (6,7%) patients who developed mortality within 1 month after the operation or during hospitalization. the incidence of cardiac and pulmonary complications was significantly different between the groups (p = 0,007, p = 0,003). when all complications were evaluated, a strong relationship was found between clavien-dindo grade 3 and above complications and sarcopenia (p = 0,001>). univariate and multivariate analysis of risk factors effective on postoperative serious complications have been summerized in table 3. when all variables that may affect postoperative serious complications are analyzed, age (or: 1,08. 95% ci: 1,01 ~ 1,15. p = 0,013), asa score (or: 2,84. 95% ci 1,62 ~ 4,97. p = 0,043) and tpi (or: 3,61. 95% ci: 1,58 ~ 5,74. p = 0,001>) have been identified as independent risk factors. discussion many factors affect postoperative results in pancreatic cancer, which has a very low survival rate even after curative resection. serious morbidities may occur after postoperative complications and disease prognosis is affected. while improved surgical technique, increased surgical experience, better anesthesia and intensive care process and improvements in parenteral nutrition make pd a safer procedure, surgery still carries a high risk of postoperative morbidity and mortality.20,21 in addition to knowing the treatment and table 2. the relationship between postoperative outcomes and sarcopenia. variables total sarcopenic (n=30) non-sarcopenic (n=90) p-value operation time (min.) 212,16±35,25 220±31,48 209±36,20 0,161 blood loss 0,045 >1000ml 12(10) 6(20) 6(6,7) 1000ml> 108(90) 24(80) 84(93,3) clavien dindo 3 and above complications 45(37,5) 20(66,7) 25(27,8) 0,001>* pancreatic fistula grade a 0,552 grade b 9(7,5) 5(16,7) 4(4,4) grade c 12(10) 4(13,3) 8(8,9) 0(0) 0(0) 0(0) cardiac complications 20(16,7) 10(33,8) 10(11,1) 0,007* pulmonary complications 56(46,7) 21(70) 35(38,9) 0,003* infective complications 29(24,2) 4(13,3) 25(27,8) 0,084 wound complications 26(21,7) 4(13,3) 22(24,4) 0,153 delayed gastric emptying 22(18,3) 5(16,7) 17(18,9) 0,512 hospital stay (day) 11,42±7,02 14,23±7,41 10,48±6,67 0,003* intensive care stay (day) 3,38±4,40 4,23±3,71 3,09±4,59 0,219 30 day/in-hospital mortality 8(6,7) 2(6,7) 6(6,7) 0,681 reoperation 8(6,7) 1(3,3) 7(7,8) 0,359 numerical data were given as mean ± standard deviation. table 3. univariate and multivariate analysis of risk factors effective on postoperative serious complications. variable univariate analysis multivariate analysis or (95% ci) p-value adjusted or (95% ci) p-value age 3.37(0,07~0,28) 0,001 1,08(1,01~1,15) 0,013* asa score 4,37(1.96~9.72) 0,001> 2,84(1,62~4,97) 0,043* tpi 4,81( 2,42~8,70) 0,001> 3,61(1,58~5,74) 0,001> * or: odds-ratio, ci: confidence interval, asa: american society of anesthesiologist,tpi: total psoas index. 76 original effect of sarcopenia on postoperative complications ümit mercan et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 73–77 management of postoperative complications, it is necessary to predict these complications. this is important in determining the necessary clinical treatment options and appropriate timing in the application of adjuvant therapy.5,6 in addition, this prediction may improve patient selection prior to surgical resection.7,8 in recent years, sarcopenia has been defined as a syndrome characterized by progressive loss of skeletal muscle mass and can cause consequences such as physical disability, low quality of life, and even death.10–12 it also reflects the patient’s nutritional status and condition. like many other factors, sarcopenia has been proven by several randomized studies that affect postoperative results. some studies have shown that sarcopenia is a negative prognostic factor for esophageal, gastric,22 and colorectal cancer.23 several methods for the diagnosis of sarcopenia have been described in the literature. the diagnostic accuracy and sensitivity of the methods based on measurement of muscle mass with radiological imaging compared to anthropometric methods were found to be higher. based on the fat density values, hounsfield unit average calculation is one of the methods used in the diagnosis of sarcopenia. tpi is a special measurement based on the ratio of psoas muscle area to the patient’s height and reflects the patient’s sarcopenia status. according to the international cancer cachexia consensus, tpi value was defined as <55 cm2/m2 for men and <39 cm2/ m2 for women as sarcopenia.24 in our study, patients in the lowest 25% of the tpi value that was evaluated specifically for gender were evaluated as sarcopenic. according to the results obtained in our study, it is seen that tpi value has a significant relationship with postoperative results in pancreatic cancer patients. tpi values can be easily determined by the patient’s preoperative staging ct images and are not a time-consuming procedure. considering that ct is applied to each patient for preoperative staging, there is no additional radiation exposure and no cost burden. there are also data supporting the use of magnetic resonance imaging (mri) in staging and operative planning in pancreatic cancer patients.25 mri has previously been used to measure sarcopenia, and can therefore be used preoperatively to perform this function, but ct is considered to be more appropriate for use due to additional cost and possible contraindications of mri, and therefore we used ct data in our study. while the focus of our study was the correlation between sarcopenia and surgical outcomes, its relationship with survival has been previously studied. as a result of the analysis of the data, sarcopenia was found as an independent predictor in severe postoperative complications. the most important reason for this may be postoperative limited mobilization due to low muscle strength and the inability to tolerate the negative catabolic state after surgery due to the current cachexia. also in our study, cardiac and pulmonary complications were more common in the sarcopenic group. the most common extra-abdominal complications in pancreatic surgery are of lung origin and the detection and treatment of sarcopenia in elderly patients with underlying chronic lung disease can contribute to reducing the risk of mortality. in our study, the length of hospital stay was found to be significantly longer in the sarcopenic group. effective preoperative treatment of sarcopenia is also important in terms of preventing secondary complications originating from hospital and reducing treatment costs. in the light of these data, the potential of reducing the patient’s degree of sarcopenia by interfering with medical and nutritional support can direct the selection of the right patient for the operation and clinical decision-making, especially in the elderly population. patients who are in the high-risk group in terms of complications may be directed to neoadjuvant chemotherapy during this period by applying concurrent treatments such as nutrition and exercise treatments to reduce the degree of sarcopenia before surgical resection. this approach can lead patients to a better outcome that can ultimately alter patients’ prognosis by reducing the postoperative complication rates as well as potential benefits of neodjuvant therapy such as increased surgical curability by reducing tumor size and predicting adjuvant therapy response. the most important limitation of our study is being a retrospective study performed from a single center and a study with a limited number of samples. it is also possible that performing the operations by different surgeons and possible selection bias would have affected postoperative results. in addition, there is no universally accepted definition of ct-based assessment for sarcopenia in the literature due to differences in physical condition among people. despite all these limitations, we believe our results are valuable to help understanding the effect of sarcopenia on postoperative outcomes in patients with pancreatic cancer. as a result, sarcopenia is an important indicator in predicting serious complications after resection in pancreatic cancer. tpi is a patient-specific value that can be easily obtained from preoperative imaging, and given its ease of measurement and its relationship to postoperative complications, it can be widely used to help predict patient morbidity, potentially alter the clinical course of patients by identifying sarcopenic patients, and reduce postoperative complication rates. determining the degree of sarcopenia can affect patient selection, predictability of possible serious complications, elective operation preparation process with a combination of nutrition and exercise therapy in a particular patient group and decisions regarding adjuvant or neoadjuvant therapy. acknowledgment i would like to thank all members of the turkish surgical oncology association supported writing of this article. author contribution mercan u: data collection and writing erşen o: statistical analysis unal ae: critical review disclosure statement there is no conflict of interest in writing of this article. no financial support or funding has been received. 77 original effect of sarcopenia on postoperative complicationsümit mercan et al. j contemp med sci | vol. 6, no. 2, march–april 2020: 73–77 references 1. siegel r, naishadham d, jemal a. cancer statistics, 2012. ca cancer j clin 2012;62(1):10–29. 2. bilimoria ky, bentrem dj, ko cy, ritchey j, stewart ak, winchester dp et al. validation of the 6th. edition ajcc pancreatic cancer staging system: report from the national cancer database. cancer 2007;110(4):738–44. 3. winter jm, brennan mf, tang lh, d’angelica mi, dematteo rp, fong y et al. survival after resection of pancreatic adenocarcinoma: results from a single institution over three decades. ann surg oncol. 2012;19(1):169– 75. 4. sun rc, button am, smith bj, leblond rf, howe jr, mezhir jj a comprehensive assessment of transfusion in elective pancreatectomy: risk factors and complications. j gastroint surg. 2013;17(4),:627–35. 5. van der gaag na, harmsen k, eshuis wj, busch orc, van gulik tm, gouma dj. pancreatoduodenectomy associated complications influence cancer recurrence and time interval to death. eur j surg oncol. 2014;40(5):551–8. 6. valle jw, palmer d, jackson r, cox t, neoptolemos jp, ghaneh p et al. optimal duration and timing of adjuvant chemotherapy after definitive surgery for ductal adenocarcinoma of the pancreas: ongoing lessons from the espac-3 study. j clin oncol. 2014;32(6):504–12. 7. vollmer cm, sanchez n, gondek s, mcauliffe j, kent ts, christein jd et al. pancreatic surgery mortality study group. a root-cause analysis of mortality following major pancreatectomy. j gastroint surg. 2012;16(1): 89–103. 8. kneuertz pj, pitt ha, bilimoria ky, smiley jp, cohen me, ko cy et al. risk of morbidity and mortality following hepato-pancreato-biliary surgery. j gastroint surg. 2012;16(9):1727–35. 9. peng p, hyder o, firoozmand a, kneuertz p, schulick rd, huang d et al. impact of sarcopenia on outcomes following resection of pancreatic adenocarcinoma. j gastroint surg, 2012;16(8):1478–86. 10. cruz-jentoft aj, baeyens jp, bauer jm, boirie y, cederholm t, landi f et al. sarcopenia: european consensus on definition and diagnosis: report of the european working group on sarcopenia in older people. age ageing. 2010;39(4):412–23. 11. delmonico mj, harris tb, lee js, visser m, nevitt m, kritchevsky sb et al. alternative definitions of sarcopenia, lower extremity performance and functional impairment with aging in older men and women. j am geriatr soc. 2007; 55(5):769–74. 12. goodpaster bh, park sw, harris tb, kritchevsky sb, nevitt m, schwartz av et al. the loss of skeletal muscle strength, mass, and quality in older adults: the health, aging and body composition study. j gerontol a biol sci med sci. 2006;61(10):1059–64. 13. kim tn, choi km. sarcopenia. definition, epidemiology, and pathophysiology. j bone metab. 2013; 21–30. 14. reisinger kw, van vugt jl, tegels jj, snijders c, hulsewé kw, hoofwijk ag et al. functional compromise reflected by sarcopenia, frailty, and nutritional depletion predicts adverse postoperative outcome after colorectal cancer surgery. ann surg, 2015;261(2):345–52. 15. ongaro e, buoro v, cinausero m, caccialanza r, turri a, fanotto v et al. sarcopenia in gastric cancer: when the loss costs too much. gastric cancer. 2017;20(4):563–72. 16. shen y, hao q, zhou j, dong b. the impact of frailty and sarcopenia on postoperative outcomes in older patients undergoing gastrectomy surgery: a systematic review and meta-analysis. bmc geriatr., 2017;17(1):188. 17. japan pancreas society (2009) general rules for the study of pancreatic cancer, 6th. edn. kanehara, tokyo. 18. dindo d, demartines n, clavien pa classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. ann surg, 2004;240:205–13. 19. dusch n, lietzmann a, barthels f, niedergethmann m, rückert f, wilhelm tj. international study group of pancreatic surgery definitions for postpancreatectomy complications: applicability at a high-volume center. scand j surg. 2017;106(3):216–23. 20. uzunoglu fg, reeh m, vettorazzi e, ruschke t, hannah p, nentwich mf et al. preoperative pancreatic resection (prepare) score: a prospective multicenter-based morbidity risk score. ann surg. 2014;260:857–63. 21. winter jm, cameron jl, campbell ka, arnold ma, chang dc, coleman j et al. 1423 pancreaticoduodenectomies for pancreatic cancer: a singleinstitution experience. j gastrointest surg. 2006;10:1199–210. 22. fukuda y, yamamoto k, hirao m, nishikawa k, nagatsuma y, nakayama t et al. sarcopenia is associated with severe postoperative complications in elderly gastric cancer patients undergoing gastrectomy. gastric cancer. 2016;19(3):986–93. 23. miyamoto y, baba y, sakamoto y, ohuchi m, tokunaga r, kurashige j et al. sarcopenia is a negative prognostic factor after curative resection of colorectal cancer. ann surg oncol. 2015;22(8):2663–68. 24. fearon k, strasser f, anker sd, bosaeus i, bruera e, fainsinger rl et al. definition and classification of cancer cachexia: an international consensus. the lancet oncol., 2011;12(5):489–95. 25. grimm a, meyer h, nickel md, nittka m, raithel e, chaudry o et al. repeatability of dixon magnetic resonance imaging and magnetic resonance spectroscopy for quantitative muscle fat assessments in the thigh. j cachexia sarcopenia muscle., 2018;9(6):1093–100. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i2.738 218 j contemp med sci | vol. 6, no. 5, september-october 2020: 218–222 original issn 2413-0516 introduction cervical cancer is ranked as the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women worldwide. cervical cancer continues to be a major public health problem affecting middle-aged women in 42 low-resource countries.1 in iraq, cervical cancer is 12th cancer among women, and the 10th among those aged between 15 and 44 years old.2 virtually, the cervical precancerous changes occur mainly due to the infection with human papillomavirus (hpv), which has many oncogenic subtypes, including: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 which is classified as group 1 carcinogens by the iarc monographs.3 in spite of the majority of hpv infections are curable spontaneously and don’t lead to precancerous cell changes, but the persistent infection with carcinogenic hpv types is the main cause in triggering the development of cervical cancer.4 other important cofactors include immunosuppression (particularly human immunodeficiency virus), smoking, parity (a higher number of full-term pregnancies increases risk), and oral contraceptive might also contribute to precancerous changes.5 prevention, early detection, control, and treatment of the pre-invasive uterine cervical lesions are essential methods to limit cervical cancers and are imperative ways in decreasing early death throughout the productive period in a woman’s life. early detection occurs through screening test, which include cytology, hpv testing, and via. screening through cytology has undoubtedly led to a major decline in cervical cancer burden in several resource-rich countries, however, the method might have reached its limits, with reports from several countries with longstanding high-quality cytology-based programmers indicating that trends have either stabilized or began to rise.6 meta-analyses and pooled analyses of randomized trials have shown that screening with hpv tests protects better against future cervical precancerous lesions and invasive cancers than screening by cytology7-9 and, therefore, virological screening programmes are becoming increasingly recommended.10, 11 routine cervical screening of women 30 years and older using papanicolaou (pap) cytology and hpv together (co-testing) was first approved by the us food and drug administration in 2003.12 currently, there are two different major guidelines for hr-hpv testing. in the usa, combined hr-hpv and cytology testing every 5 years is an alternative to cytology testing every 3 years.13 in europe, co-testing is discouraged at any age, and european guidelines recommend hr-hpv as a primary screening test for women above 30 years of age, owing to convincing evidence for more efficacious screening.14 early detection in our institute till may 2019 was done by primary cytology test, visual acetate acid inspection (iva) inspection method. after that, hr hpv co testing was established as a screening test and as hr-hpv testing is not offered in the public health sector in iraq, and there are no data available on hpv prevalence in the general, asymptomatic female population. so, we aimed to estimate the prevalence of abnormal cytology and positive hr-hpv test results in a screened woman and to evaluate the feasibility of integrating the hr-hpv co-testing as a primary test in the national cervical cancer screening program. hpv co-testing as cervical screening test. experience of alweiya early detection cervical clinic: one-year analysis zainab j al-jobawi1, besmah m. ali2, asan a al-niyazee3, mustafa h ibraheem3 1 cancer screening fellowship, babil health directorate, ministry of health, iraq. 2 gazi al hariri hospital, ministry of health, baghdad, iraq. 3 al-alwiyaa maternity teaching hospital, ministry of health, baghdad, iraq. correspondence: dr.zainab j. al-jobawi (e-mail: zainabjalil5@gmail.com) (submitted: 18 july 2020 – revised version received: 26 july 2020 – accepted: 19 august 2020 – published online: 30 october 2020) abstract objectives: the high-risk human papilloma virus test with conventional cytology (hpvco-test) was firstly introduced in our institute in may 2019. so, we aimed to estimate the prevalence of abnormal cytology and positive high-risk human papillomavirus test results in a screening woman and to assess the accuracy between cytology and human papilloma virus testing to evaluate the feasibility of integrating the latter as a primary test in the national cervical cancer screening program. methods: a prospective study for women attending to early detection cervical clinic, during the period from may 2019 to may 2020. patients who were sexually active were included in the study. samples for conventional cytology and hpv by using real-time polymerase chain reaction technique for high risk types were taken concurrently. the prevalence of positive screening results and the difference in accuracy between two testing were estimated by mcnemar’s χ2 test. result: a total of 388 women were included in the study. the prevalence of positive test for hr-hpv was 2.1% (8) while the prevalence of abnormal cytology test was 19.1% (74). concerning discordant pairs, 0.8% (3) of women had normal cytology with a positive hr-hpv test result and 17.8% (69) of women had abnormal cytology with a negative hr-hpv test result. a total of 311 (80.1%) women had normal cytology and negative hr-hpv test results. the proportion of women with abnormal cytology and positive hr-hpv test results was 1.3% (5 women). the difference in accuracy between the two results was statistically significant (<0.0001). the prevalence of positive hr-hpv test decreased with increasing age, whereas the prevalence of abnormal cytology showed a bimodal age pattern. conclusion: the prevalence of abnormal cytology was high to that of hr-hpv testing, which could not allow for the implementation of hrhpv as a primary test in the national screening program in iraq. keywords: conventional cytology, hr-hpv, hpv co-test, abnormal cytology, cervical cancer. 219 original hpv co-testing as cervical screening test. experience of alweiya early detectionzainab j al-jobawi j contemp med sci | vol. 6, no. 5, september-october 2020: 218–222 patients and methods a prospective study for women attending to alweiya early detection cervical clinic, which belongs to alrosafa health directorate, baghdad, iraq, during the period from may 2019 to may 2020. these women either suffered from genital health problems or just attended the aforementioned center for screening. all patients were aware of cytological examination and its purpose. patients who were sexually active were included in the study. samples for conventional cytology and hr-hpv testing were taken concurrently. samples were obtained by rotating a spl cyto pap brush (spl life sciences, pocheon, south korea) five full turns in the cervical region. cytology was performed at the reference laboratory of the al-alwiyaa maternity teaching hospital. samples were analyzed by one cytopathologist. cytological results were classified based on the 2001 bethesda system into,15 where the smears that interpreted negative for intraepithelial lesion, were considered normal cytology result and the smears that interpreted as atypical squamous cells of undetermined significance (asc-us, possibly nonneoplastic); atypical squamous cells (asc-h, cannot exclude hsil); atypical glandular cells (agc); low-grade squamous intraepithelial lesion (lsil); high-grade squamous intraepithelial lesion (hsil); invasive squamous cell carcinoma (scc); adenocarcinoma in situ (ais); invasive adenocarcinoma and invasive endometrial adenocarcinoma, were considered as abnormal cytology result. hr-hpv testing was done at the central public health laboratory, molecular biology unit, to detect hpv dna in cervical swabs by using real-time polymerase chain reaction (pcr) technique for high risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59). all samples were blinded to cytology results. the data obtained were tabulated in a microsoft excel spreadsheet and subsequently exported to the spss statistics software package, version 23. descriptive data on the study population are presented in absolute and relative frequencies. continuous variables are presented as mean ± sd. the overall prevalence of positive cytology and hr-hpv test result and by age was reported. differences in positive hr-hpv test result by type of abnormal cytology result were tested with mcnemar’s χ2 test. p values less than 0.05 were considered statistically significant. result a total of 388 women were enrolled in this study, the mean age (sd) of women was 40.4 (10.1) years, age range was between 23 and 65 years. from all women, only 40 (10.3%) women was came for screening, other women were referral cases (table 1). the prevalence of positive test for hr-hpv was 2.1% (8), while the prevalence of abnormal cytology test was 19.1% (74), concerning discordant pairs, 0.8%(3) of women had normal cytology with a positive hr-hpv test result and 17.8%(69) of women had abnormal cytology with a negative hr-hpv test result. a total of 311 (80.1%) women had normal cytology and negative hr-hpv test results. the proportion of women with abnormal cytology and positive hr-hpv test results was 1.3% (5 women). the difference in accuracy between the two results was statistically significant (p=<0.0001) (table 2) the pattern of cytological abnormality shown by cytology was, 33.8% (25) of participants had atypical squamous cell of undetermined significant (ascus), 58.1% (43) of women had low squamous intraepithelial lesion (lsil), 4% (3) of women had high squamous intraepithelial lesion (hsil), 2.7% (2) of women had atypical glandular cell (ags), and only 1 woman had ca. cervix (fig 1). the prevalence of positive hr-hpv test result varied considerably with age, the prevalence decreased from 3.2% in women aged <30 years to 2.5% in women aged 30–39 years, and 1.6% in women aged 40–59 years (fig 2). there was a slight increase in the prevalence of abnormal cytology test among women 30–39 years and 50–59 years compared with women aged <30 years and 40–49 years (fig 3). discussion this is the first study in iraq to assess the prevalence of abnormal cytology and positive hr-hpv test results using hr-hpv co test. we found that among 388 women with acceptable results for both tests, the population prevalence of abnormal cytology results was 19.1% and the prevalence of positive hr-hpv was 2.1% and the difference in accuracy between the two results was statistically significant. we found a higher prevalence of abnormal cytology than found in populations tested on a regular basis such as in romania and turkey (the 12–11.5%. respectively).16, 17 this could be explained by the prevalent and incident cases discovered in our sample, in contrast to only incident cases found in regularly tested women found in that studies and also in our sample, majority of women were symptomatic (89.7%) and the frequency of abnormal cytological result in symptomatic women was higher and this proved by many study.18, 19 the prevalence of abnormal cytology result showed a bimodal age pattern result which not in line to that reported in some other populations, where shown increased in prevalence of abnormal cytology with increasing age.20, 21 other studies showed a reversed result where the prevalence of abnormal cytology decrease by increase in age.22, 23unfortunately, in iraq there table 1. distribution of age and mode of detection among studied women. characteristic number percentage age <30 years 62 16% 30–39 years 121 31.2% 40–49 years 128 33% 50–59 years 62 16% ≥60 years 15 3.9% mode of detection screening 40 10.3% symptomatic 348 89.7% table 2. concordance of cytology and hr hpv test in hpv co testing. result of hpv co test cytology test total p value abnormal normal hr hpv positive 5 3 8(2.1%) <0.0001negative 69 311 380(97.9%) total 74(19.1%) 314(80.9%) 388 mcnemar’s χ2 test, significant ≤0.05. 220 original hpv co-testing as cervical screening test. experience of alweiya early detection zainab j al-jobawi j contemp med sci | vol. 6, no. 5, september-october 2020: 218–222 fig 1. the pattern of cytological abnormality shown by cytology. fig 2. prevalence of positive hr-hpv infection result by 10-year age group. 221 original hpv co-testing as cervical screening test. experience of alweiya early detectionzainab j al-jobawi were no population-based study which investigated the prevalence of hpv in women, in this study, the prevalence of positive hr-hpv was low compared to other population. the most plausibly explanation related to societal factors and sexual behavior, also our country exhibit low rates of other sexually related infections, such as hiv.24 in our study, the peaks in hr-hpv prevalence was in women aged less than 30 years and the prevalence decrease by increase age. this explained that younger age had a high proportion of positive results which reflects infections that will clear spontaneously (transient hr-hpv infections that eliminated by the immune system in 6–18 months) without reflected in cellular atypia and this in line of other studies.23, 25 interestingly, we found that the likelihood of hr-hpv positive testing but cytologic negative to be a less common than the likelihood of hr-hpv negative testing but cytologic positive (0.8% vs. 17.8%), the 0.8% of women with normal cytology and positive hr-hpv results was lower to the percentage obtained in the artistic trial (9%),26 lower than worldwide estimate of 11.7%,27 and substantially lower than previously reported percentages from eastern europe,28, 29 14.0% in southeastern asia to 14.4% in south central asia.30 variations between studies most likely reflect differences in the population studied with respect to risk factors for exposure to hpv and methods of evaluations. the 17.7% of women with negative hr-hpv test results and abnormal cytology, where the cytological abnormality in our study where primarily asc-us cytology and lsil and those abnormality harbor low prevalence of hpv infection by many studies,31, 32 also those abnormality bears a low risk of cin2/3.33 these women require surveillance and follow-up according to guidelines, as they have an elevated risk for cervical lesions over time.34 recognition of 80.2% of women with cytologically normal and hr-hpv-negative women is important because extension of screening intervals from 3 years based on negative cytology alone to 5 years based on hr-hpv co test. the present study has several limitations, including the use of data from a single provider of health-care services and not based on the iraqi population as a whole. also, cytological abnormalities were not compared with histology results and data were based on opportunistic screening as opposed to an organized screening program. conclusion the prevalence of abnormal cytology was high to that of hr-hpv testing, which could not allow the implementation of hr-hpv as a primary test in the national screening program in iraq. our data indicate that combined cytology and hr-hpv testing does not identify the same at-risk women and would entail higher diagnostic work-up, health-care costs. the high prevalence of abnormal cytology was enough to highlight the importance of the early detection of cervical cancers, thus saving lives. further population-based prospective studies are needed to eliminate the drawbacks of our study and to determine nonhospital-based hpv prevalence in iraqi women. acknowledgments we think ateaf hamza, chief lad assistant who took part in this study. we also grateful to all patients involved in this study fig 3. prevalence of abnormal cytology by 10-year age group. 222 original hpv co-testing as cervical screening test. experience of alweiya early detection zainab j al-jobawi j contemp med sci | vol. 6, no. 5, september-october 2020: 218–222 conflict of interest no conflict of interest. references 1. arbyn m, weiderpass e, bruni l, de sanjosé s, saraiya m, ferlay j, et al. estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis. lancet global health. 2020;8(2):e191-e203. 2. board ic. annual report of cancer disease in iraq 2013. in: board c, editor.: ministry of health; 2017. 3. ghittoni r, accardi r, chiocca s, tommasino m. role of human papillomaviruses in carcinogenesis. ecancer med sci. 2015;9. 4. papillomaviruses h. iarc monographs on the evaluation of carcinogenic risks to humans. lyon, france: iarc. 2011. 5. thun m, linet ms, cerhan jr, haiman ca, schottenfeld d. cancer epidemiology and prevention: oxford university press; 2017. 6. de kok im, van der aa ma, van ballegooijen m, siesling s, karim-kos he, van kemenade fj, et al. trends in cervical cancer in the netherlands until 2007: has the bottom been reached? int j cancer. 2011;128(9):2174-81. 7. arbyn m, ronco g, anttila a, meijer cj, poljak m, ogilvie g, et al. evidence regarding human papillomavirus testing in secondary prevention of cervical cancer. vaccine. 2012;30:f88-f99. 8. ronco g, dillner j, elfström km, tunesi s, snijders pj, arbyn m, et al. efficacy of hpv-based screening for prevention of invasive cervical cancer: follow-up of four european randomised controlled trials. lancet. 2014;383(9916):524-32. 9. ogilvie gs, van niekerk d, krajden m, smith lw, cook d, gondara l, et al. effect of screening with primary cervical hpv testing vs cytology testing on high-grade cervical intraepithelial neoplasia at 48 months: the hpv focal randomized clinical trial. jama. 2018;320(1):43-52. 10. smith ra, andrews ks, brooks d, fedewa sa, manassaram-baptiste d, saslow d, et al. cancer screening in the united states, 2019: a review of current american cancer society guidelines and current issues in cancer screening. ca: a cancer j clin. 2019;69(3):184-210. 11. von karsa l, arbyn m, de vuyst h, dillner j, dillner l, franceschi s, et al. european guidelines for quality assurance in cervical cancer screening. summary of the supplements on hpv screening and vaccination. papillomavirus res. 2015;1:22-31. 12. wright jr tc, schiffman m. adding a test for human papillomavirus dna to cervical-cancer screening. new engl j med. 2003;348(6):489-90. 13. curry sj, krist ah, owens dk, barry mj, caughey ab, davidson kw, et al. screening for cervical cancer: us preventive services task force recommendation statement. jama. 2018;320(7):674-86. 14. elfström km, arnheim-dahlström l, von karsa l, dillner j. cervical cancer screening in europe: quality assurance and organisation of programmes. eur j cancer. 2015;51(8):950-68. 15. solomon d, davey d, kurman r, moriarty a, o’connor d, prey m, et al. the 2001 bethesda system: terminology for reporting results of cervical cytology. jama. 2002;287(16):2114-9. 16. suteu o, blaga ml, nygård m, leinonen mk, nicula f, pais r, et al. prevalence of positive screening test results and agreement between cytology and human papillomavirus testing in primary cervical cancer screening in north-western romania. eur j cancer prev. 2020;29(2):141-8. 17. çalişkan e, coşkun sk, öztürk ce, cangür ş, önal b. analysis of hpv genotypes and liquid-based cervical cytology: results from a tertiary academic center in northwestern turkey. jpn j infect dis. 2020:jjid. 2020.072. 18. tahir qa, bukhari mh. evaluation of pre-malignant cervical lesions in females presenting with abnormal pelvic complaints. jpma j pak med assoc. 2020;70(2):272-5. 19. nayak pk, mitra s, agrawal s, hussain n, thakur p, mishra b. role of various screening techniques in detecting preinvasive lesions of the cervix among symptomatic women and women having unhealthy cervix. 2020. 20. gravitt pe, paul p, katki ha, vendantham h, ramakrishna g, sudula m, et al. effectiveness of via, pap, and hpv dna testing in a cervical cancer screening program in a peri-urban community in andhra pradesh, india. plos one. 2010;5(10):e13711. 21. kim m-j, kim jj, kim s. type-specific prevalence of high-risk human papillomavirus by cervical cytology and age: data from the health checkups of 7,014 korean women. obstet gynecol science. 2013;56(2):110-20. 22. castle pe, fetterman b. five-year experience of human papillomavirus dna and papanicolaou test cotesting. obstet gynecol. 2009;113(3):595. 23. wright jr tc, stoler mh, behrens cm, apple r, derion t, wright tl. the athena human papillomavirus study: design, methods, and baseline results. am j obstet gynecol. 2012;206(1):46. e1-. e11. 24. vardell e. global health observatory data repository. med ref serv quart. 2020;39(1):67-74. 25. monsonego j, cox jt, behrens c, sandri m, franco el, yap p-s, et al. prevalence of high-risk human papilloma virus genotypes and associated risk of cervical precancerous lesions in a large us screening population: data from the athena trial. gynecol oncol. 2015;137(1):47-54. 26. kitchener h. hpv primary cervical screening: time for a change. 2015. 27. bruni l, diaz m, castellsagué m, ferrer e, bosch fx, de sanjosé s. cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. j infect dis. 2010;202(12):1789-99. 28. forman d, de martel c, lacey cj, soerjomataram i, lortet-tieulent j, bruni l, et al. global burden of human papillomavirus and related diseases. vaccine. 2012;30:f12-f23. 29. tachezy r, smahelova j, kaspirkova j, salakova m. human papillomavirus type-specific prevalence in the cervical cancer screening population of czech women. plos one. 2013;8(11):e79156. 30. bao y-p, li n, smith j, qiao y-l. human papillomavirus type distribution in women from asia: a meta-analysis. int j gynecol cancer. 2008;18(1). 31. ciotti m, sesti f, paba p, benedetto a, patrizi l, criscuolo a, et al. human papillomavirus (hpv) testing in the management of women with abnormal pap smears. experience of a colposcopy referral clinic. eur j gynaecol oncol. 2004;25(5):577-84. 32. gonzalez-bosquet e, almagro mm, mora i, suñol m, callejo j, lailla j. prevalence of human papilloma virus infection of the uterine cervix in women with abnormal cervical cytology. eur j gynaecol oncol. 2006;27(2):135-8. 33. perkins rb, guido rs, castle pe, chelmow d, einstein mh, garcia f, et al. 2019 asccp risk-based management consensus guidelines for abnormal cervical cancer screening tests and cancer precursors. j lower genital tract dis. 2020;24(2):102. 34. wright jr tc, massad ls, dunton cj, spitzer m, wilkinson ej, solomon d. 2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests. am j obstet gynecol. 2007;197(4):346-55. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.859 103j contemp med sci | vol. 6, no. 3, may–june 2020: 103–108 original issn 2413-0516 introduction stroke is the most prevalent vascular accident of central nervous system among middle aged individuals. the sensory motor dysfunction, cognitive impairments, and declined quality of life are the side effects of stroke in suffered patients.1 unfortunately, no effective pharmaceutical treatment has been introduced, only endovascular approaches or rehabilitation may reduce the severity of symptoms.2 in the field of regenerative medicine, there are numerous preclinical and clinical studies that demonstrated the therapeutic effect of stem cell transplantation in various neurodegenerative diseases such as ischemic stroke.3-5 embryonic stem cells (esc), the pluripotent stem cells, can differentiate to different lineages. so that, under defined protocol, embryonic stem cells has been shown to differentiate to neural progenitor cells (npc).6 after transplantation of embryonic derived neural progenitor cells (es-npcs) in model of middle cerebral artery occlusion (mcao) in the rats, the cells are capable to migrate toward ischemic site, proliferate and differentiate to the neurons and glial cells, and replace the dead cells.7 on the other hand, they induce angiogenesis and neurogenesis, decrease the neuroinflammation, and preserve the integrity of blood–brain barrier through bystander effects.8 following the es-npcs injection, the size of ischemic area reduced and the sensory-motor function improved. the histological investigation also revealed positive findings in favor of neural tissue repair.6, 9, 10 moreover, different types of biomarkers such as micrornas (mirnas) appear in blood and brain tissue following ischemia. the mirnas are small non-coding and single-stranded rnas that regulate many internal processes such as cell proliferation, differentiation, development, cell cycle, apoptosis, etc. in addition to tissues, they are present in serum or plasma in the form of complexes and macrovesicles.11 the previous studies exposed that a wide spectrum of mirnas identified, in the blood and brain tissue after mcao in rats by microarray with both upand downregulated manner. clinical studies also confirmed these findings in patients with ischemic stroke. therefore, the mirnas are considered as a promising biomarker for prognosis of stroke patients.12-16 mirna-210 has been shown to prevent neuronal apoptosis and with neuroprotective role by suppressing the caspase pathway, induce a balance between bcl-2 and bax expression.17 in ischemic condition, mir-210 plays role as a proangiogenic factor and involves in cell-cycle regulation, dna damage reconstruction, and neural tissue restoration.17-19 human embryonic derived neural progenitor cells improves neurological scores following brain ischemia/ reperfusion: modulation of blood and brain tissue microrna-210 leila arab1, aslan fanni2, shiva nemati#2, ehsan arefian3, jafar ai#4, tahmineh mokhtari 5,6, maryam farahmandfar7, nasser aghdami2, gholamreza hassanzadeh1,8,9 1department of neuroscience and addiction studies, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. 2department of stem cells and developmental biology, cell science research center, royan institute for stem cell biology and technology, acecr, tehran, iran. 3department of microbiology, school of biology college of science, university of tehran, tehran, iran. 4department of tissue engineering and applied cell sciences, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. 5cas key laboratory of mental health, institute of psychology, beijing, china; 6department of psychology, university of chinese academy of sciences, beijing, china 7department of neuroscience and addiction studies, school of advanced technologies in medicine, electrophysiology research center, neuroscience institute, tehran university of medical sciences, tehran, iran. 8legal medicine research center, legal medicine organization, tehran, iran. 9department of anatomy, school of medicine, tehran university of medical science, tehran, iran. #2 co-second author #3 co-third author correspondence to: gholamreza hassanzadeh (hassanzadeh@tums.ac.ir ) (submitted: 13 march 2020 – revised version received: 28 march 2020 – accepted: 11 may 2020 – published online: 26 june 2020) objective in this study, we evaluated the effects of human embryonic derived neural progenitor cells on neurological score, histopathological changes, and mirna-210 as biomarkers of regeneration. methods the animals were randomly divided into the four groups: sh (sham), mcao (middle cerebral artery occlusion), mcao+pbs, and mcao+cell. one day after mcao induction, embryonic derived neural progenitor cells (hesc-npcsgfp) or pbs were injected intracerebroventriculary in mcao+cell or mcao+pbs groups. on day 1, 2, 3, and 7 after ischemia induction, the neurological score was tested in each rat. at 48 h, the expression of mirna-210 was evaluated and 7 days after, the pathological assessments were performed by h&e staining. results neurological score showed the promotion of functional recovery in mcao+cell group. based on h&e staining, the percentage of neural death in ischemic region reduced in mcao+cell group. the mirna-210 significantly upregulated in both brain and blood samples. conclusion according to the findings, hesc-npcsgfp injection could upregulate the mirna-210 of tissue and blood to support the neuroprotective and regenerative effect of hesc-npcsgfp in the ischemic lesion and improved the neurological score and reduce the neural death in ischemic region. keywords embryonic stem cell; neural death; micro-rna-210; brain ischemia; rat 104 original hesc-npcsgfp after brain ischemia/ reperfusion leila arab et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 103–108 also, mirna-210 improves the stem cell survival via regulation of apoptosis-related protein (caspase 8 associated protein 2).20, 21 also, overexpression of mir-210 induces angiogenesis and neurogenesis in ischemic tissues to compensate decrease hypoperfusion.22 we designed this study to evaluate the effect of intracerebroventricul injection (icvi) of es-npcs on fold changes of mirna-210 in ischemic brain tissue and compared with mcao and mcao+placebo groups, 24 h, and 48 h after ischemia. method and material animals the male wistar albino rats (260–300 g, 12-week-old) were enrolled into the study, purchased from laboratory animal department of royan institute. they were kept in animal room with temperature of 18-24°c, 40-–70% humidity, 12h light–12h dark cycle and free access to food and water. they were treated according to the guidelines of iranian council for use and care of animals and approved by ethical committee of tehran university of medical sciences. the rats were randomly divided into four groups: 1. sham group (sh): operated rats without any vascular occlusion which underwent stereotaxic intervention and with experience of icvi injection (n=8 rats). 2. mcao: rats only with mcao for 60 min (n= 8 rats). 3. mcao + pbs (phosphate buffered saline) group: rats with mcao for 60 min which followed by icvi injection of pbs (5 µl) (n= 8 rats). 4. mcao + cell group: rats with mcao for 60 min which was followed by icvi injection of cell suspension. (105 cells in 5 µl pbs) (n=8 rats). model induction before mcao induction, each rat was held in the induction chamber with vaporization of 5% isoflurane. then, it was put immediately in supine position on heating pad and heating light with nosecone mask to inhalation of 1–2% isoflurane during surgery. with a middle neck incision and dissection of the neck soft tissues and muscles, we accessed to the common carotid artery (cca). in next step, the proximal of cca and external carotid artery (eca) were ligated, and an intraluminal 4-0 nylon monofilament (doccol co., usa) was inserted into the mca to occlude its origin under monitoring of blood flow by laser doppler flowmeter (moor instruments). after 60 min, the filament was removed and the neck incision was sutured.23 the body temperature was monitored with rectal temperature probe to remain at 37°c.24 cell preparation and generating of hesc-npcsgfp the hesc (rh6) was received from royan cell bank, then, their differentiation procedure toward hnpcs has been done according to a standard procedure.25 the characteristics of hesc-npcsgfp were evaluated using immunofluorescence staining. immunofluorescence staining to perform immunofluorescence staining, hnpc-gfp were fixed using 4% paraformaldehyde (mallinckrodt, phillipsburg, nj), and permeabilized with 0.1% triton x-100 (sigma) for 15 min at ambient temperature. the cells were incubated with primary antibody for 1 h at room temperature (rt), washed, and incubated with fluorescein isothiocyanate-conjugated secondary antibodies, antimouse immunoglobulin m (igm) (1:100), antimouse igg (1:200), and antirat igm (1:200), as appropriate, for 1 h at rt. primary antibodies were nestin (1:100), sox1(1:100), gfap (1:400) to confirm the undifferentiated stage. the cells were analyzed with a fluorescent microscope (olympus). icv injection to perform the icv injection, after 24 h the rats were anesthetized with isoflurane (5% for induction and 2% for maintenance), then fixed in stereotaxic frame. the es-npcs (1×105 cells in 5 µl pbs) or pbs was injected with using hamilton syringe into the right cerebral ventricle at: bregma: ap=-0.12 mm, ml=1.6 mm, and dv=4.3 mm. modified neurological severity score (mnss) to assess the sensory, motor, reflexes, and balance of rats after mcao, we use mnss test,26 while the worst score is 0 and the best one is 18. the test was performed for each rat on day 1, 2, 3, and 7 after ischemia induction. mirna real-time quantitative pcr the mirna expression was measured in the ischemic area and blood samples 48 h after mcao. the rats were anesthetized with isoflurane 5%. the cardiac blood samples were taken and the ischemic area of the right hemisphere was isolated and stored in -80°c freezer. total rna (plus mirna) was extracted from brain samples. single-strand cdna was synthesized using universal cdna synthesis kit (exiqon, vedbaek, denmark). quantification of mirna-210 was performed with stem-loop real-time pcr. qpcr was performed in triplicate in three separate experiments on an applied biosystems step one plus real-time pcr machine. the relative expression of mirna was normalized to the endogenous control u6 expression using the comparative cycle threshold (ct) method. hematoxylin and eosin staining for light microscopy study, the rats were anesthetized with ketamine/xylazine (razico, iran), and perfused by 0.9% saline and 4% paraformaldehyde (pfa, sigma), respectively. the brains were dissected and cut into the sections with 3–5 mm thickness. then, the sections were post-fixed in 10% formalin 72 h at 4°c. in order to light microscopy analysis, the samples were embedded in paraffin and 5 μm coronal sections were prepared by using a rotary microtome (leica biosystems, milan, italy). one section from each five section was selected and the tissue sections stained with hematoxylin and eosin (h&e). afterward, graded alcohols (70, 80, 90, and 100% [2 times]) was used to dehydrate the sections. finally, they were mounted in canada balsam and prepared for analysis. study and survey of sections was performed by using a light field microscope (olympus, cx31, tokyo, japan). in cortex field, the intact and ischemic cells considered as dark neurons, were counted in the ×400 images by using a connected camera to the microscope.27 statistical analysis data analysis was performed with standard statistical software graphpad prism, version 6 (graphpad, la jolla, ca). one-way 105 original hesc-npcsgfp after brain ischemia/ reperfusionleila arab et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 103–108 anova followed by bonferroni’s post-hoc comparisons tests were performed in all statistical analyses. to test the feasibility, we built statistical model by regression analysis. correlations were estimated by pearson correlation test. differences were considered significant at p < 0.05. result characterization of hesc-npcsgfp generated hnpc (fig. 1) was evaluated for expression of neural progenitor markers by immunofluorescence staining. the phase contrast microscopy photograph of normal hnpc was shown in fig. 1a. the hnpc population had highly expressed nestin (fig. 1b), sox1 (fig. 1d) with the lower expression of gfap (fig. 1c) at their progenitor stage. effects of icv injection of hesc-npcsgfp on the modified neurological severity score (mnss) following i/r injury the results of behavioral functional test (mnss) showed that the neurological function outcomes significantly improved in mcao+cell during a week after injection (3.71±0.76/18, p-value<0.001) compared with mcao (8.29±1.11, p-value<0.059) and mcao+pbs (7.71±1.11, p-value: 0.230) groups on day 7 (table 1). effects of icv injection of hesc-npcsgfp on the neural cells death of ischemic area following i/r injury evaluation of apoptosis by h&e staining showed that the count of neural cell death (shrunken cells) in mcao+cell group is much lesser than (~50%) mcao+pbs group (80%) and mcao group (~80%) (fig. 2). effects of icv injection of hesc-npcsgfp on mirna-210 profile (rt-pcr) of blood and brain samples of ischemic area following i/r injury brain and blood samples from rats in different groups were screened for a total of 72 rattus norvegicus. the mirna-210 was found to be present in both the blood and brain 48 h after reperfusion. then, the correlation between mcao-blood/ tissue-48h was examined (fig. 3). fig. 1 characterization of hesc-npcsgfp. (a) phase contrast microscopy of normal hnpc after generation of rh6. (b–d) immunofluorescence staining for neural progenitor markers (nestin, sox1, and gfap). 106 original hesc-npcsgfp after brain ischemia/ reperfusion leila arab et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 103–108 discussion in this study, we evaluated the effects of h-esc-npc on brain impairments induced by ischemia reperfusion. for this purpose, the neurological score, histopathological changes, and the mirnas-120 level of ischemic area and peripheral blood were investigated in each group. the mcao was applied by a similar method in all rats and the formation of ischemic tissue was confirmed by h&e staining. the mcao+cell group significantly had better sensory-motor function. as the previous studies showed, transplantation of esc–npcs in brain ischemic lesion could promotes functional recovery after ischemia reperfusion via migration proliferation, and differentiation in the ischemic region. human esc-npcs proliferate and differentiate to neurons and glia cells in one step. administration of esc-npcs was approved to reduce the volume of ischemic region via induction of neurogenesis and angiogenesis.8 on day 7, after cell injection, the rats were perfused and their brains were extracted for more studies. the h&e staining results confirmed the reduced percentage of neural death in ischemic region. the migration, proliferation and differentiation of h-esc-npcs in the ischemic lesion has been confirmed in several studies.28-31 on the other hand, this amount of transplanted cells cannot be differentiated to replace the damaged tissue in the ischemic region. so, they might apply their alterations via other mechanisms. the transplanted cells secrete different trophic factors including cytokines, chemokines, and extracellular proteins to the surrounding environments which act as antiapoptotic factor, immunomodulators, angiogenesis factors, and antioxidant molecules. these progenitor cells are capable to endogenous neurotropic factors such as brain-derived neurotrophic factor (bdnf), stromal cell-derived factor 1 (sdf1), vascular endothelial growth factor (vegf), nerve growth factor (ngf), etc. all these factors play important and beneficial role to repair the ischemic impairments following stem cell transplantation. the analysis of neural stem cells’ secretome showed that they secrete different growth factors, fig. 2 effects of hesc-npcsgfp on percentage of neural death of ischemic region in rat. (a) h&e staining in different groups (100×). (b) comparing the percentage of neural death in different groups ****p < 0.0001 compared to sh group; sh: sham operated group; mcao: ischemia induction group, mcao+pbs: ischemia induction group with icv injection of pbs; mcao+cell: ischemia induction group with icv injection of hesc-npcsgfp. table 1. effects of icv injection of hesc-npcsgfp on the modified neurological severity score (mnss) following i/r injury. time (day) group day 1 day 2 day 3 day 7 p-value pairwise comparisons mcao (m) 10.14 (1.86) 9.43 (1.27) 9.29 (0.95) 8.29 (1.11) 0.059 mcao+ pbc (p) 9.43 (1.81) 8.57 (2.23) 7.29 (3.09) 7.71 (1.11) 0.230 mcao+ cell (c) 10.14 (2.27) 7.57 (1.40) 6.29 (1.38) 3.71 (0.76) <0.001 1>3; 1>7; 2>7 p-value 0.744 0.164 0.044 <0.001 pairwise comparisons c<m c<m; c<p pbs: phosphate buffer saline. values are given as mean (sd). 107 original hesc-npcsgfp after brain ischemia/ reperfusionleila arab et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 103–108 fig. 3 real-time pcr quantification of target gene expression in the rats’ blood of all groups at 48 hours after ischemia. (a) mirna-210 expression in brain samples, (b) mirna-210 expression in blood samples. the figure shows that mirna-210 upregulated in blood and tissue in mcao+cell group compared to mcao+pbs group 48h after ischemia. ****p<0.0001. data are expressed as mean±sem. all reactions were performed in triplicate. (c) correlation between rats’ blood/tissue-48h after cell injection. e.g., vegf, glial cell-derived neurotrophic factor (gdnf), triiodothyronine, ngf, and fibroblast growth factor 8 (fgf8), etc.28, 32-36 in our previous studies, the regulation of upstream genes (nlrp1, nlrp3, asc, and caspase-1) and downstream genes (tnf-α, il-1β, il-18, and il-6) of inflammasome complex has been investigated. these studies demonstrated that transplanted stem cells from different sources from human can reduce the inflammation by inhibit the formation and also action of inflammasome complexes and consequently prevent the secondary injury in nervous system.37-39 brain and blood mirnas helped the transplanted cells to progress the regeneration process. mirnas keep the neural cells survived in hypoxic environment by regulating different pathway that activates in this critical condition. mirnas has been shown to significantly enhance the survival and neuroprotective efficacy of mesenchymal stem cells for treatment of intracerebral hemorrhage model.40 the previous studies have shown that in the hypoxic events, upor downregulation of mirnas, induce the expression of different proteins that control the inflammation and ionic hemostasis41, 42 and also has been shown that inflammation may disturb the neural regeneration.43 therefore, to realize the effect of transplanted cells on fold change of mirnas following ischemia, we assessed the changes of mirna-210. the studies showed that these mirnas might be upregulated following ischemic attack to survive affected cells.44 our findings showed that mirna-21 upregulated in blood and brain 48 h after mcao. the most upregulation of all mirnas was observed in mcao+cell group compared with mcao+pbs, mcao, and sham groups. upregulation of mirnas was intensively seen in the blood after 48 h of ischemia.15, 44, 45 we demonstrated that upregulation of these mirnas is strengthened by stem cell injection, in other words, stem cells collaborate with mirna to promote regeneration of neural cells in the ischemic region. as a detectable biomarker in blood, this factor can be used as a predictor of regeneration and prognosis of disease. we can conclude that mirnas are a part of regenerative process in coordination with stem cells. hence, measuring the mirnas in blood sample of patients with stroke can show the prognosis of patient as a biomarker of regeneration. conclusion icv injection of h-esc-npcs in mcao model could improve the sensory-motor condition by decreasing the neural death and promotion of mirna-210 in ischemic region and blood samples. acknowledgment this study was registered in department of research, school of advanced technologies in medicine, tehran university of medical science with register number of 95-02-87-32255. 108 original hesc-npcsgfp after brain ischemia/ reperfusion leila arab et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 103–108 references 1. romano jg, sacco rl. decade in review-stroke: progress in acute ischaemic stroke treatment and prevention. nat rev neurol. 2015;11(11):619–21. 2. bagheri a, talei s, hassanzadeh n, mokhtari t, akbari m, malek f, et al. the neuroprotective effects of flaxseed oil supplementation on functional motor recovery in a model of ischemic brain stroke: upregulation of bdnf and gdnf. acta med iran. 2017:785–92. 3. cheng y, zhang j, deng l, johnson nr, yu x, zhang n, et al. intravenously delivered neural stem cells migrate into ischemic brain, differentiate and improve functional recovery after transient ischemic stroke in adult rats. int j clin exp pathol. 2015;8(3):2928–36. 4. alizamir t, akbari m, mokhtari t, hassanzadeh g. associated functional motor recovery induced by intracerebroventricular (icv) microinjection of wharton’s jelly mesenchymal stem cells following brain ischemia/ reperfusion injury in rat: decreased dark neurons and bax gene expression in the cerebral corte. j contemp med sci. 2017;3(12). 5. mehrannia k, mokhtari t, mogehi smhn, akbari m, bazzaz jt, mahakizeh s, et al. intracerebroventricular injection of wharton’s jelly mesenchymal stem cells attenuates brain damage in rat model of hypoxia: optimization of vascular endothelial growth factor and downregulation of inflammatory factors. j contemp med sci. 2018;4(3). 6. hao l, zou z, tian h, zhang y, zhou h, liu l. stem cell-based therapies for ischemic stroke. biomed res int. 2014;2014:468748. 7. takagi y, nishimura m, morizane a, takahashi j, nozaki k, hayashi j, et al. survival and differentiation of neural progenitor cells derived from embryonic stem cells and transplanted into ischemic brain. j neurosurg. 2005;103(2):304–10. 8. baker ew, kinder ha, west fd. neural stem cell therapy for stroke: a multimechanistic approach to restoring neurological function. brain behav. 2019;9(3):e01214. 9. daadi mm, maag al, steinberg gk. adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model. plos one. 2008;3(2):e1644. 10. chang dj, oh sh, lee n, choi c, jeon i, kim hs, et al. contralaterally transplanted human embryonic stem cell-derived neural precursor cells (enstem-a) migrate and improve brain functions in stroke-damaged rats. exp mol med. 2013;45:e53. 11. yang zb, li tb, zhang z, ren kd, zheng zf, peng j, et al. the diagnostic value of circulating brain-specific micrornas for ischemic stroke. intern med. 2016;55(10):1279–86. 12. li m, zhang j. circulating micrornas: potential and emerging biomarkers for diagnosis of cardiovascular and cerebrovascular diseases. biomed res int. 2015;2015:730535. 13. bhalala og, srikanth m, kessler ja. the emerging roles of micrornas in cns injuries. nat rev neurol. 2013;9(6):328–39. 14. wang c, ji b, cheng b, chen j, bai b. neuroprotection of microrna in neurological disorders (review). biomed rep. 2014;2(5):611–9. 15. wang y, wang y, yang gy. micrornas in cerebral ischemia. stroke res treat. 2013;2013:276540. 16. martinez b, peplow pv. blood micrornas as potential diagnostic and prognostic markers in cerebral ischemic injury. neural regen res. 2016;11(9):1375–8. 17. qiu j, zhou xy, zhou xg, cheng r, liu hy, li y. neuroprotective effects of microrna-210 on hypoxic-ischemic encephalopathy. biomed res int. 2013;2013:350419. 18. huang x, le qt, giaccia aj. mir-210--micromanager of the hypoxia pathway. trends mol med. 2010;16(5):230–7. 19. meng zy, kang hl, duan w, zheng j, li qn, zhou zj. microrna-210 promotes accumulation of neural precursor cells around ischemic foci after cerebral ischemia by regulating the socs1-stat3-vegf-c pathway. j am heart assoc. 2018;7(5). 20. chan yc, banerjee j, choi sy, sen ck. mir-210: the master hypoxamir. microcirculation. 2012;19(3):215–23. 21. kim hw, haider hk, jiang s, ashraf m. ischemic preconditioning augments survival of stem cells via mir-210 expression by targeting caspase-8associated protein 2. j biol chem. 2009;284(48):33161–8. 22. zeng l, he x, wang y, tang y, zheng c, cai h, et al. microrna-210 overexpression induces angiogenesis and neurogenesis in the normal adult mouse brain. gene ther. 2014;21(1):37–43. 23. zendedel a, habib p, dang j, lammerding l, hoffmann s, beyer c, et al. omega-3 polyunsaturated fatty acids ameliorate neuroinflammation and mitigate ischemic stroke damage through interactions with astrocytes and microglia. j neuroimmunol. 2015;278:200–11. 24. mokhtari t, akbari m, malek f, kashani ir, rastegar t, noorbakhsh f, et al. improvement of memory and learning by intracerebroventricular microinjection of t3 in rat model of ischemic brain stroke mediated by upregulation of bdnf and gdnf in ca1 hippocampal region. daru. 2017;25(1):4. 25. nemati s, hatami m, kiani s, hemmesi k, gourabi h, masoudi n, et al. long-term self-renewable feeder-free human induced pluripotent stem cell-derived neural progenitors. stem cells dev. 2011;20(3):503–14. 26. chen j, hu r, liao h, zhang y, lei r, zhang z, et al. a non-ionotropic activity of nmda receptors contributes to glycine-induced neuroprotection in cerebral ischemia-reperfusion injury. sci rep. 2017;7(1):3575. 27. atlasi ma, naderian h, noureddini m, fakharian e, azami a. morphology of rat hippocampal ca1 neurons following modified two and four-vessels global ischemia models. arch trauma res. 2013;2(3):124. 28. takahashi k, yasuhara t, shingo t, muraoka k, kameda m, takeuchi a, et al. embryonic neural stem cells transplanted in middle cerebral artery occlusion model of rats demonstrated potent therapeutic effects, compared to adult neural stem cells. brain res. 2008;1234:172–82. 29. daadi mm, hu s, klausner j, li z, sofilos m, sun g, et al. imaging neural stem cell graft-induced structural repair in stroke. cell transplant. 2013;22(5): 881–92. 30. beyer f, samper agrelo i, kury p. do neural stem cells have a choice? heterogenic outcome of cell fate acquisition in different injury models. int j mol sci. 2019;20(2). 31. ouyang q, li f, xie y, han j, zhang z, feng z, et al. meta-analysis of the safety and efficacy of stem cell therapies for ischemic stroke in preclinical and clinical studies. stem cells dev. 2019;28(8):497–514. 32. barzegar m, kaur g, gavins fne, wang y, boyer cj, alexander js. potential therapeutic roles of stem cells in ischemia-reperfusion injury. stem cell res. 2019;37:101421. 33. bliss t, guzman r, daadi m, steinberg gk. cell transplantation therapy for stroke. stroke. 2007;38(2 suppl):817–26. 34. liao ly, lau bw, sanchez-vidana di, gao q. exogenous neural stem cell transplantation for cerebral ischemia. neural regen res. 2019;14(7): 1129–37. 35. willis cm, nicaise am, peruzzotti-jametti l, pluchino s. the neural stem cell secretome and its role in brain repair. brain res. 2020;1729:146615. 36. baraniak pr, mcdevitt tc. stem cell paracrine actions and tissue regeneration. regen med. 2010;5(1):121–43. 37. mousavi m, hedayatpour a, mortezaee k, mohamadi y, abolhassani f, hassanzadeh g. schwann cell transplantation exerts neuroprotective roles in rat model of spinal cord injury by combating inflammasome activation and improving motor recovery and remyelination. metab brain dis. 2019;34(4):1117–30. 38. mahdavipour m, hassanzadeh g, seifali e, mortezaee k, aligholi h, shekari f, et al. effects of neural stem cell-derived extracellular vesicles on neuronal protection and functional recovery in the rat model of middle cerebral artery occlusion. cell biochem funct. 2019. 39. ijaz s, mohammed i, gholaminejhad m, mokhtari t, akbari m, hassanzadeh g. modulating pro-inflammatory cytokines, tissue damage magnitude, and motor deficit in spinal cord injury with subventricular zone-derived extracellular vesicles. j mol neurosci. 2020;70(3):458–66. 40. zhang h, wang y, lv q, gao j, hu l, he z. microrna-21 overexpression promotes the neuroprotective efficacy of mesenchymal stem cells for treatment of intracerebral hemorrhage. front neurol. 2018;9:931–. 41. slota ja, booth sa. micrornas in neuroinflammation: implications in disease pathogenesis, biomarker discovery and therapeutic applications. noncoding rna. 2019;5(2). 42. roitbak t. micrornas and regeneration in animal models of cns disorders. neurochem res. 2019. 43. obora k, onodera y, takehara t, frampton j, hasei j, ozaki t, et al. inflammation-induced mirna-155 inhibits self-renewal of neural stem cells via suppression of ccaat/enhancer binding protein β (c/ebpβ) expression. scientific reports. 2017;7:43604. 44. xu w, gao l, zheng j, li t, shao a, reis c, et al. the roles of micrornas in stroke: possible therapeutic targets. cell transplant. 2018;27(12):1778–88. 45. jeyaseelan k, lim ky, armugam a. microrna expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. stroke. 2008;39(3):959–66. dx.doi.org/10.22317/jcms.v6i3.781 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 95j contemp med sci | vol. 6, no. 3, may–june 2020: 95–102 review issn 2413-0516 introduction concerning the history of human life, for a long time, “infer­ tility” has been a topic of interest for scientists that lead to the discovery of the causes of this disease. however, 15–25% of infertility cases are still called idiopathic.1 who describes infertility as below: infertility is a genital disorder and includes failure to get pregnant after 12 months or more of regular and unpro­ tected sexual intercourse (without any other causes such as breastfeeding or postpartum amenorrhea). primary infertility including infertility in couples who have any children, second­ ary infertility is an inability to get pregnant again. infertility has a high prevalence and occurs in 10–15% of couples. in 45% of cases, infertility is related to female dis­ orders such as ovarian diseases (polycystic ovary), disorders of fallopian tubes, uterine abnormalities (endometriosis). 30% of infertility is related to men and 25% of cases, have no clear cause.1 in all iranian traditional manuscripts, infertility and uter­ ine diseases have been investigated and their causes and symp­ toms described in detail and categorized. in iranian traditional medicine (itm), sterility and infertility are called “oghr” and “osr ol­habal” which means “infertility” and “difficult to get­ ting pregnant”.2­4 the use of complementary medicine in the treatment of infertility is increasing. acupuncture, homeopathy, herbal remedies, massage therapy, etc. are the methods that are now promoted by their proponents.5­7 in traditional medicine, various reasons have been expressed for infertility in women, including disorders in female semen, diseases that occur in the uterus such as types of uterine abnormalities, uterine hemorrhoids, cervical inclina­ tion, and flatulence in uterine and severe swelling in cervix.2,4,8 one of the reasons for infertility that is mentioned in traditional manuscripts is uterus flatulence. in this study, the etiology and treatment of this disease have been investigated from the perspective of hakim ali ibn abbas ahvazi in the kamel al­sina’ah al­tebiyah. method this is a review article in which one of the authentic books in itm called kamel al­sina’ah al­tebiyah, written by hakim ali ibn abbas ahvazi, was investigated. related content to nafkhat­ol­rahem disease, causes and symptoms were col­ lected and the role of this disease in infertility was investigated. the treatment method and the measures needed to improve the disease were studied. some of the compounds that are used to treat this disease were collected along with their profiles. also, some of the compound medications used in the treat­ ment of this disease in the kamel al­sina’ah were also collected in separate tables. to investigate and compare this disease in current med­ icine, the pubmed, science direct, and google scholar data­ bases were searched using the keywords vaginal flatus, vaginal flatulence, and pelvic floor weakness. based on the results, the cause of uterine bloating and vaginal flatus was investigated and some of its causes were identified. also, treatment meth­ ods of this disorder in current medicine was adapted to the treatment and remedies described in itm. evaluation of flatulence of uterus (nafkhat-ol-rahem) as one of the causes of infertility in women from the viewpoint of ali ibn abbas ahvazi and modern medicine zeinab zaheri1, mojgan tansaz2, fereshteh golfakhabadi1* 1medicinal plant research center, department of pharmacognosy, school of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran 2school of traditional iranian medicine, shahid beheshti university of medical sciences, tehran, iran *correspondence to: fereshteh golfakhabadi (golfakhrabadi@yahoo.com) (submitted: 03 march 2020 – revised version received: 23 march 2020 – accepted: 11 april 2020 – published online: 26 june 2020) objective today, infertility and its complications are one of the largest problems because it would impose economic burden and psychological pressure on the people. one of the infertility reasons in most iranian traditional medicine (itm) manuscripts are focused on nafkhat-ol-rahem. methods in this study, the ibn abbas theories in kamel al-sina’ah about nafkhat-ol-rahem, causes, reasons, and treatment were investigated. to achieve recent studies, searches were performed in the databases with the keywords including vaginal flatus, vaginal flatulence, and pelvic floor weakness. results this disease is caused by bad cold temper, hit, and obstruction of cervix, etc. traditional medicine considered the bloating of uterus (nafkhat-ol-rahem), the cause of some miscarriages and infertility, because dense gas that accumulated in the uterus, prevents implantation and establishment of the embryo. in itm, treatment involves the use of warm and mohallel riyah (gas dissolvent) herbal medicines in the form of oral, poultice (topical ointment), forzajeh and homol (vaginal suppository). conclusion nafkhat-ol-rahem is a highly prevalent disease in women, but because of the shame, the patient does not speak with her doctor. bloating of uterus and vaginal flatulence are seen in the pelvic floor muscle relaxation, which can be harmless or associated with major complications such as rectovaginal fistula and infection of the uterus. keywords nafkhat-ol-rahem, infertility, ali ibn abbas, iranian traditional medicine 96 review nafkhat-ol-rahem and infertility in women zeinab zaheri et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 95–102 results special attention has been paid to infertility in itm, so part of the great manuscripts of itm dedicated to the causes and treatment of infertility. infertility is called oghr in traditional medicine.2,4,8­11 the structure of the uterus from the perspective of ali ibn abbas ali ibn abbas ahvazi has been described as the structure of the uterus. the uterus is similar to bladder in the shape and has two branches on both sides, where the semen is entering in. during pregnancy and development of the fetus, the uterus can be enlarged and drawn in all directions without getting damage. the uterus is composed of a layer, and this layer con­ sists of a series of woven warps that are in various directions (longitudinal, diagonal, and transvers). uterine has two spaces on the right and left side of the ends to a single depth that is named cervix (neck of the uterus). the uterus is located on the rectum and under the bladder, and thus is protected; it is also connected by ligaments to the surrounding limbs, so it can be drawn in all directions during pregnancy.12 causes of infertility from the perspective of ali ibn abbas iranian medicine, is a humoral system. according to the principles of itm, the human body consists of four humors: dam (blood), balgham (phlegm), safra (yellow bile), and soda (melancholy). these humors have specific qualitative and quantitative characteristics. the imbalance in temperament (mizaj) and humors (akhlat) is the cause of disease, therefore treatment is based on restoring and maintaining this balance. infertility in a couple is due to the presence of diseases in men and women. infertility in women has several causes that ibn abbas categorized them in two groups: diseases related to su’mizaj and structural disorders.11,12 su’mizaj su’mizaj occurs when an organ’s temperament goes out of range. diseases related to su’mizaj are in two categories: simple su’mizaj and material su’mizaj.9,11,12 simple su’mizaj occurs when one of the four qualities (warmth, coldness, dryness, or wet) overcome an organ. material su’mizaj is caused by humor overcoming and caused corruption in the organ and altering the quality of that organ.9 diseases due to uterus structure the most common cause of infertility is related to the uterus. pregnancy is only caused if the uterus is healthy and any cause that disrupts its structure can lead to fertility disorder. some of these structural disruptions include: • cervical obstruction due to factors such as severe swelling, excess tissue, and coldness. • retroverted uterus, or coming down and ejection • uterus wounds and hemorrhage that causes bleeding. • blockage in the blood vessels and seminal tubs in the uterus, which prevents semen from reaching the uterus. • uterus swellings • bloating in the uterus and wind accumulation in it that causing swelling under the umbilical region.11,13 effect of uterus flatulence on infertility the cold su’mizaj of the uterus causes contraction of the vas­ cular and, along with a thick wind (rih) that penetrates the tissue of the uterus, in the early stages of pregnancy, prevents the implantation of the fetus in the uterus. the probability of abortion in the first 3 months of pregnancy was higher. in traditional medicine, one of the causes of abortion is uterus flatulence,14 because the thick winds (riyah) in the uterus can separate the embryo sheath (amniotic sac) and the naghr in the uterus (the vascular openings that the blood flows from them into the uterus), which the choroid is attached to, and causes abortion.15 uterus flatulence from the perspective of ali ibn abbas ibn abbas in kamel al­ sina’ah al­tebiyah says “nafkhat­ol­ rahem is accumulation of thick winds (riyah) in the uterus space for various reasons”.11 some causes of uterine flatulence include: • cold su’mizaj is the main cause of nafkhat­ol­rahem. cold su’mizaj causes weakness and suppress the uterus instinc­ tive heat and produce riyah and bloating in it. this riyah can accumulate in the uterus, or penetrate into the depths of the uterus and its tissue.11 • abortion and hard delivery (osr al­veladat) can cause pain and weakness in the uterus, and as a result, the instinct heat of the uterus reduced.11 • cervical closure due to the presence of blood clots in the cervix which prevents the outflow of riyah in the uterus.11 if the riyah and bloating are in the uterus, it is easier to treat, but the riyah that penetrates into the depth of the uterus, is not easily treatable. if the patient does not follow all the instructions, she may endure with the disease forever.11 signs and symptoms of this disease include:2,9,11 • bloating, pain and stretch in the pubis and under the umbilical region. • due to the presence of bloating, when hit underneath the navel, its sound like a drum. • rigidity (stiffness) and abdominal sturdiness. • swelling of the pubis area • stretching the pain up to the thigh and thigh lymph nodes. due to the accumulation of lymph material in this area, pain is spread to the legs. • the stretching of the pain to the stomach and the diaphragm, due to the involvement and attachment of the ligaments of the uterus to the proximity of the stomach and the diaphragm. • pain can be relocated from place to place and from the womb to other organs. • disposal of gas from the uterus during intercourse. • eating flatulent foods will make the patient get worse. uterus flatulence in recent medicine in the review of the articles, there was no case in which uter­ ine flatulence and its complications were considered as a dis­ ease. the uterine bloating is described as an outflow of air or odorless gas from the vagina during walking, physical activity, intercourse, or vaginal examination. normally, this is a natural phenomenon that occurs when the air enters the vagina and 97 review nafkhat-ol-rahem and infertility in womenzeinab zaheri et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 95–102 then driven out by the normal contraction of the uterus and abdomen muscles. although this phenomenon is usually safe, it is very uncomfortable for many women and makes them feel embarrassed.16,17 studies have shown that, gas comes out of the vagina is considered as one of the symptoms of pelvic floor muscle dys­ function (pfmd). pfmd is a disorder that causes many social problems and affects family and social life, but its prevalence is not clear. many women do not report their own problems because they either feel shame about it or they see it as a nat­ ural consequence of pregnancy and childbirth or because of their age. for this reason, they prefer to limit their social con­ nections so that they are less likely to be distressed. common problems with pfmd include prolapse of the pelvic organs (cystourethrocele, rectocele, uterine or vagina prolapse), and urinary incontinence. vaginal flatulence (vaginal wind) is another symptom of this disorder.17,18 many women do not know about pelvic floor muscles and their function, and they are unaware of the problems that can be caused by inappropriate functioning of these muscles in their bodies after pregnancy and childbirth. most women do not have enough knowledge about the association between reduced pelvic floor muscle function with vaginal gas outflow, constipation, and sexual dysfunction.19 normally, the air comes out of the vagina is odorless, but if it has odor, it is a serious disorder. the disorders associ­ ated with this gas include the presence of rectal and vaginal fistulas, vaginal surgery, and internal injuries. in the event of any of these, treatment should be taken as soon as possible, otherwise it may cause a uterine infection. rectovaginal fistula (rf) sometimes occurs after rectal surgery, in which there is disposal of vaginal gas, fecal leakage, and vaginal discharge, which is very annoying. treatment of these conditions is possible with surgery. in all of these cases, the gas outflow of vagina is not an independent disease, but one of the symptoms associated with these conditions. rf or uterine and vagina prolapse have specific causes and treatment of these disorders is usually done by surgery. in all of these cases, vaginal gas disposal is not an independent disease, but is one of the symp­ toms associated with these diseases. rf or uterine and vagina prolapse have specific causes, and treatment of these disorders is usually done by surgery.20,21 treatment the itm is based on maintaining health and preventing illness. each person should know his temperament and adjust his life­ style accordingly. in order to maintain health, it is essential to observe the six principles of health preservation (proper nutrition and adequate drinking of healthy water, healthy air, proper sleep, adequate exercise, mental conditions control, preserving essentials, and eliminating useless substances from the body). when disease occurs, therapeutic approaches are required to restore the body to its original temperament and cure the disease. 2 in the case of uterus flatulence, the treatment is: • correcting the nutrition: the patient should avoid eating flatulent and cold temperament foods. considering that one of the causes of uterine flatulence is cold su’mizaj of uterine, eating foods with hot temperament that produce delicate humors, can prevent uterine bloating and some­ what modulate the cold su’mizaj of the uterus.4 • suitable sleep: sleeping helps maintain the strength of the various organs and returns health to the body. at the time of sleep, blood goes to the internal organs of the body, which improves their actions and eliminates wastes from them.22 • sufficient exercise: one of the major tools used by the body in treatment is thermal therapy that, by increasing body temperature, mildly breaks down and excretes the sub­ stance. in the canon of medicine, ibn sina has said that nature, by instinctive heat, repels toxins. in the event of cold su’mizaj, the instinctive heat of the limb is decreased, such as that caused by the uterus flatulence, exercise and mobility creates heat that breaks down the riyah of the uterus.9,22 • drug therapy: given that cold su’mizaj is the main cause of this disease, herbal medicine with hot temperament that reduce riyah are considered as the first line of treatment.4 the drugs listed in the kamel al­ sina’ah are prescribed in several ways: (a) oral medicines, such as jovaresh (an oral dosage form) and beverages. 12 (b) topical medications • oils and poultices – liquid or semi­solid dosage form that can be rubbed on the skin.23 • hoghneh (vaginal enema) – boil some herbs in water, filter the liquid and use it vaginally.23 • forzajeh (vaginal suppository) – a piece of wet wool fabric with dry or wet medicines that used vaginally.23 • humol (vaginal suppository) – a piece of cotton cloth containing a drug that is used vaginally.23 (c) performing manual actions such as using a hot cup­ ping under the umbilicus. most of the medicines used to treat this disease have hot and dry temperament, which can be useful in mizaj modulat­ ing and eliminating the cold su’mizaj in the uterus. also, these medications have functions such as dissolvent of thickened winds (mohalel), cleansing (monaghi), cleaner (jaly), cocative (monzej), phlegm repellent, etc., which are used together in the treatment of uterus flatulence.24 specifications and proper­ ties of some of the materials (mofradat) that are used in prepa­ ration of combinational medicines in kamel al­sina’ah (such as the scientific name, traditional and general name, temper­ ament, functions, and properties of these drugs) have been collected from the makhzan al­adviyeh book and presented in table 1. several formulas of combinational medicines from the kamel al­sina’ah book in the treatment of uterus flatulence are listed in tables 2–5. treatment in modern medicine motion and exercise cause warmth and heat in the body, which helps breaking down the moisture in the uterus. with less humidity, fewer winds are produced in the uterus, which helps reduce the uterus flatulence.22 in modern medicine, to increase the function of the pelvic floor muscle and reduce its complications, these muscles should be tonicated by exercising and moving. one of these exercises is kegel movements. this exercise should be done regularly and always. the method of doing sports is that the person is sitting or lying on the ground. the pelvic floor muscle then contracts for 5 s, and then relaxes 98 review nafkhat-ol-rahem and infertility in women zeinab zaheri et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 95–102 table 1. specifications of some of medicinal herbs (mofradat) that used in preparation of medicines for the treatment of uterus flatulence from the kamel al-sina’ah book of ibn abbas’s and their properties in the makhzan al-adviyeh book.11,24 scientific name traditional name temperament (mizaj) properties and functions application in uterine diseases 1 pimpinella anisum anison hot and dry attenuant, wind dissolvent, cleaner, increase the flow of urine, milk, and menstrual blood the decoction of this plant interrupts the bleeding of the uterus. 2 foeniculum vulgare raziyanaj hot and dry thick wind dissolvent, increase the flow of urine and menstrual blood 3 carum cupticum nankhah hot and dry wind dissolvent, increase the flow of urine, milk, and menstrual blood vaginal suppository and vaginal enema (forzajeh and hoghneh) is used to clean the uterus from moisturs and dry it. 4 carum petroselinum fotrasalion hot and dry wind and bloat dissolvent, increase the flow of urine and menstrual blood forzajeh is useful in abortion 5 apium graveolens karafs hot and dry blockage opener, dissolvent, increase the flow of urine and menstrual blood 6 legoecia cuminoides ghordemana hot and dry dissolvent for thick bloating 7 ruta graveolense sodab hot and dry wind and bloat dissolvent, increase the flow of urine and menstrual blood forzajeh and humol is useful in abortion and improve the flow of menstrual blood. 8 anethum graveolens shebet hot and dry dissolvent, cocative, blockage opener and increase the flow of urine and menstrual blood sitting in the decoction of leaves and seeds is useful for the diseases of the uterus. drinking decoction of its leaves and fresh or dry seeds is beneficial to remove and breaking down the wind. 9 opopanax chironium javshir hot and dry wind dissolvent, blockage opener and eliminate uterus bloat the decoction is useful to improve the flow of urine and menstrual blood and modifying the uterus and breaking down its rigidity. its humol is extremely powerful in improve the flow of menstrual bleeding. orally or vaginally, it breaks down winds in the uterus and eliminates the stiffness of the uterus. 10 origanum majorana marzanjosh hot and dry attenuant, dissolvent, blockage opener, cleaner, absorbent rubbing it on the limbs causes disintegration of phlegmatic swelling. its forzajeh is a modulator of the flow of menstrual bleeding. 11 matricaria chamomilla babonaj hot and dry attenuant, dissolvent, blockage opener, increase the flow of urine, milk and menstrual blood, relaxation of fatigue and pain in the uterus and breaking down the accumulated winds in the limbs. oral and topical is beneficial. in many diseases of the uterus, sitting in the decoction and pouring it on the place is useful. 12 artemisia vulgaris berenjasf hot and dry attenuant, blockage opener, increase the flow of urine sitting in the decoction to relieve menstrual blood and urine retention, difficult delivery, cervical closure and swelling are beneficial. pouring the decoction of this plant and rubbing it on the abdomen can help relieve urinary and menstrual blood retention, swelling and pain relief. humol of its extract with myrrh is useful for disposal of waste materials from the uterus. (continued) 99 review nafkhat-ol-rahem and infertility in womenzeinab zaheri et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 95–102 table 1. continued scientific name traditional name temperament (mizaj) properties and functions application in uterine diseases 13 artemisia absinthium afsantin hot and dry blockage opener, attenuant, astringent, laxative for bile and other bitter humors its forzajeh with honey is beneficial to menstrual bleeding. facilitate the exit of urine, menstrual blood, milk and sweat. 14 thymus serpyllum namam hot and dry blockage opener, strengthening it facilitates the removal of urine and menstrual bleeding and relieves the infection. 15 artemisia herba-alba shih hot and dry blockage opener, phlegm deferent , wind dissolvent, laxative for infectious humors 16 carum carvi cummon hot and dry attenuant, deferent, dissolvent, astringent orally used to breakdown the wind and bloat and facilitate urine and menstrual blood output. enema (hoghneh) with other herbal medicine decoction leads to depletion of wind and bloat. 17 peganum harmala harmal hot and dry cleaner, dissolvent for intestinal bloating and thick material, increase the flow of urine, milk and menstrual blood, laxative for melancholy and thick phlegm its enema (hoghneh) is useful in kidney and uterus coldness 18 valeriana celtica nardin hot and dry increase the flow of urine and menstrual blood sitting in its decoction is beneficial in uterus diseases 19 jasminum officinalis yasamin hot and dry blockage opener, increase the flow of urine and menstrual blood cathartic for melancholy, bile and phlegm and it eliminates thick wind. 20 costus sp. ghost hot and dry facilitates menstrual blood and urine output. eating it with honey is used to absorb moisture and phlegm and removing them. vaginal suppositories and sitting in the decoction of this herb is beneficial for menstrual bleeding and relief of uterine pains. 21 melilotus officinalis eklil al-malek hot and dry dissolvent, cocative, astringent drinking decoction of the leaves and branches is useful for facilitate the menstrual blood and urination. 22 citrus medica var. cedrata otroj hot and dry blockage opener 23 ficus carica tin hot and wet dissolvent, attenuant, strong cleaner and mild laxative. vaginal suppository (humol) with honey in wool fabric is used to clean up uterus wounds, absorb infectious moisture and discontinue uterine bleeding. removes the rigidity of the uterine. 24 althea officinalis khatmi cold and dry dissolvent, coactive, relaxant and laxative sitting in the decoction is useful for anal swellings and cervical adhesion. with duck fat and gum (samgh al-botom), are experienced in the uterus swellings and cervical adhesion. 25 commiphora mukul mukul hot and dry attenuante, cleaner, laxative, blockage opener facilitates the exit of urine, menstrual blood, and milk. it is useful in difficult delivery and menstrual retention. vaginal suppository (humol) is useful for absorbing moistures from the uterus and drying them, removing the cervical adhesion and lowering the fetus and abortion. (continued) 100 review nafkhat-ol-rahem and infertility in women zeinab zaheri et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 95–102 table 1. continued scientific name traditional name temperament (mizaj) properties and functions application in uterine diseases its poultice is suitable for rigid swelling, breakdown the thick winds and diseases of the uterus. its smoke is beneficial for bloat of the uterus and its cervical adhesion. 26 seasamum indicum samsem hot and wet blockage opener, laxative, moisturizing correcting the burnt substances and melancholic materials. 27 glycyrrhiza glabra soos hot and dry cocative, moisture cleanser, improve flow of urine and menstrual blood 28 viola odorata banafsaj cold and wet mild cathartic for bile, dissolve soft and rigid swells 29 marrubium vulgare faraseion dissolvent, cleanser, blockage opener in liver and spleen dissolvent for thick wind and viscose phlegm sitting in the decoction of this plant or rubbing it on the site is effective in cleaning the uterus, facilitating the removal of menstrual bleeding 30 trigonella foenumgraecum holbeh laxative, cocative, dissolvent, improve flow of menstrual blood eliminate viscose phlegm sitting in the decoction of this plant is beneficial to facilitate birth, removal of the placenta and cleansing the uterus. vaginal suppository (forzajeh) with goose fat is beneficial to soften the stiffness of the uterus and open the cervix. 31 commiphora myrrha morr hot and dry cleaner, desiccative, astringent, blockage opener, cocative, cathartic, laxative, dissolvent for winds and cold phlegmatic swellings protecting the humors from infection. improve the flow of urine and menstrual bleeding. eating one and a half grams of it with fried egg yolk, stop the bleeding from the uterus. together with water of ruta graveolense or artemisia absinthium, are useful for the diseases of the uterus. enema with fenugreek is helpful for relieving the rigidity of uterus. vaginal suppository (humol) with myrtle leaf water eliminates the bad smell of the uterus. 32 borax boraq hot and dry attenuante, cleaner and thick substance deferent table 2. formulation of an oral combinational medicine used in the treatment of uterine bloating from kamel al-sina’ah.11,24 dosage form scientific name of ingredient preparation method oral pimpinella anisum apium graveolens foeniculum vulgare carum cupticum ruta graveolense carum petroselinum legoecia cuminoides all spices are boiled in water. then mix it with jasmine oil and nervine oil and costus oil and enter it in the uterus. table 3. formulation of a topical medicine used in the treatment of uterine bloating from kamel al-sina’ah.11,24 dosage form scientific name of ingredient preparation method topical ruta graveolense anethum graveolens a mixture of both oil is rubbed below the umbilicus and above the pubic. 101 review nafkhat-ol-rahem and infertility in womenzeinab zaheri et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 95–102 for 10 s. the contraction and release of the muscles should be done 10 times and repeated 3 times a day.25,26 this exercise can be performed in any state (sitting, sleeping, working, etc.). it should be noted that exercising in the abdomen or back after having performed this exercise indicates that exercise has not been properly performed. during the contraction of pelvic floor muscles, other muscles, such as the abdomen, back, and hips, should be relaxed.26 there was no study on the effects of this exercise on the prevention of vaginal flatus. however, a study on the effect of pelvic floor muscle exercise on urinary incontinence has shown that 3 months of exercise in 36 patients resulted in 56% improvement in patients, but did not effect on patients with moderate to severe incontinence.27 discussion in most iranian traditional manuscripts, including the kamel al­sina’ah, the canon of medicine, kholaseh al­hekmah, akbari medicine, etc. the uterus flatulence is an indepen­ dent disease and its causes, complications, and treatments are detailed. however, in modern medicine, they have diagnosed this disease in a lesser amount and do not consider it as a disease. on the other hand, due to the lack of reporting by patients, the exact incidence and complications of this disease have not been determined. treatment of uterus flatulence in itm includes series of measures such as improving nutrition, avoiding cold and flat­ ulent foods, doing exercise, and appropriate sleep. in addition to these measures, the principles of treating the disease in tra­ ditional medicine include cleansing the body, strengthening the uterus, and adjusting the temperament of the uterus. first, the patient’s body should be cleaned of substances causing the disease. cleansing the body and the uterus is done with using appropriate laxatives. one of the most famous laxatives is iyarej fiqera, that is also introduced in the avicenna book (the canon of medicine) for cleaning the body in the uterus flat­ ulence. in order to strengthen and modify the temperament of the uterus, various types of vaginal suppository (forzajeh and humol), and other appropriate dosage forms are used. examples of these medicines are presented in tables 4 and 5. in order to achieve treatment, the patient should observe all these remedies, failure to complete these treatments do not result in complete treatment, and the disease will not be erad­ icated, and will return again. one of the cases that is very effective in the treatment of this condition and is partly taken into account in modern medicine is exercise. exercise causes the body to warm and elevate intrinsic heat, and this heat can help to reduce winds in uterine. in traditional medicine, it is recommended that exer­ cise is carried out in a general way throughout the body so that the heat produced is uniformly dispersed in the body. while in modern medicine, exercises designed to reduce vaginal fla­ tus are localized and only related to the pelvic and perineal muscles, and do not involve the rest of the body. on the other hand, in modern medicine, uterus flatulence is less common and no complete studies have been done on it, there is no clear strategy for treating this disease. given the relatively complete recognition of itm practi­ tioners about this disease, a full description of this disease is available in most itm books. ibn abbas in kamel al­sina’ah said that the uterus bloating is the result of overcoming cold su’mizaj in the uterus, which causes reducing heat of the womb and ultimately leads to the creation of wind in the womb. avicenna also considered the uterus flatulence as a result of overcoming coldness in the uterus and cervix. this coldness causes the closure of the cervix and retention winds in the uterus. this wind does not reside and penetrates into the tis­ sue of the womb. aqili in the kholaseh al­hekmah book said, the dominance of cold su’mizaj over uterus and the production of winds due to heat weakness is one of the causes of infertil­ ity. hakim arzani in akbari’s medicine book introduces the uterus flatulence as a result of weakness in the uterus due to overcoming cold su’mizaj. cold su’mizaj is one of the main causes of infertility in women.2,3,9,12 as a result of this cold, su’mizaj and winds production in the uterus, pain, and stiffness are felt in the pubes area, the abdomen is stiff and pain is felt in the groin and even in the stomach. the area between the umbilicus and the pubes swells, so that when it’s hit with the finger it gives out drum sound. treatment of the disease is initially a cleansing of the body using laxatives, including iyarajat. in the next step, warming up the uterus and breaking down the wind in the uterus using table 4. a formula of combination medicine of vaginal enema (hoghneh) used in the treatment of uterine bloating from kamel al-sina’ah.11,24 dosage form scientific name of ingredient preparation method vaginal eneme (hoghneh) matricaria chamomilla anethum graveolens origanum majorana artemisia vulgaris artemisia absinthium thymus serpyllum artemisia herba-alba ruta graveolense apium graveolens foeniculum vulgare pimpinella anisum carum carvi carum cupticum peganum harmala valeriana celtica jasminum officinalis costus sp. all spices are boiled in water. then mix it with jasmine oil and nardine oil and costus oil and enter it in the uterus. table 5. a formula of combination medicine of vaginal suppository (forzajeh) used in the treatment of uterine bloating from kamel al-sina’ah.11,24 dosage form scientific name of ingredient preparation method vaginal suppository (forzajeh) ficus carica commiphora mukul carum carvi borax milk dried figs are well peeled and mixed with milk. then combine borax and mukul and cumin with it and prepare forzajeh. 102 review nafkhat-ol-rahem and infertility in women zeinab zaheri et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 95–102 a variety of jovaresh and syrups. in preparing these medicines, the herbs with warming and cleansing characteristics are used, such as chamomile, dill, and marjoram.12 conclusion it is important to note that uterine flatulence is an indication of the cold su’mizaj of the uterus, which is the source of many of the problems in women, including infertility. therefore, fur­ ther studies are needed on this disease and its causes, as well as its relation with infertility, and the treatments provided in itm is also should be considered. conflict of interest the authors declare that there is no known conflict of interest regarding this publication. acknowledgment this study was supported by ahvaz jundishapour university of medical science in iran. references 1. danforth dn. danforth’s obstetrics and gynecology: lippincott williams & wilkins; 2008. 2. shah arzani m. akbari medicine (tib-e-akbari). qom: resuscitation institute of natural medicine, jalal al-din publications; 2008. 3. mh ak. kholasat al hekmat. qum: esmaeilian publication; 2006. 4. njm nkc. eksire azam. tehran: iran university of medical sciences, institute of history of medicine, islamic medicine and supplement; 2008. 5. rayner j-a, willis k, burgess r. women’s use of complementary and alternative medicine for fertility enhancement: a review of the literature. j altern complement med. 2011;17(8):685-90. 6. coulson c, jenkins j. complementary and alternative medicine utilisation in nhs and private clinic settings: a united kingdom survey of 400 infertility patients. j exp clin assist reprod. 2005;2(1):5. 7. xue cc, zhang al, lin v, da costa c, story df. complementary and alternative medicine use in australia: a national population-based survey. j altern complement med. 2007;13(6):643-50. 8. jorjani i. zakhire kharazmshahi. qum: natural resuscitation institute; 2012. 9. (avicenna) sa. canon of medicine (al qanun fil tibb). beirut, lebanon: arab heritage revival house; 2005. 10. na s. sharh al-asbab va al-alamat. qum: natural resuscitation institute; 2008. 11. aia ma. kamel al sanaat al tebbiyat (the perfect book of the art of medicine). qum: natural resuscitation institute jalalaldin; 2007. 12. aia ma. kamel al sanaat al tebbiyat (the perfect book of the art of medicine). qum: natural resuscitation institute jalalaldin; 2007. 13. tansaz m, adhami s, mokaberinejad r, namavar jahromi b, atarzadeh f, jaladat am. an overview of the causes and symptoms of male infertility from the perspective of traditional persian medicine. iran j obstet gynecol infertil. 2016;18(182):11-7. 14. sl e. medicine during the the safavieh. tehran: tehran university; 1978. 15. s b. infertility and contraception in iran medicine. tehran: ismaeelian; 2010. 16. allahdin s. flatus vaginalis a distressing symptom. int j colorect dis. 2011;26(11):1493-. 17. krissi h, medina c, stanton sl. vaginal wind–a new pelvic symptom. int urogynecol j. 2003;14(6):399-402. 18. kjølhede p, wahlström j, wingren g. pelvic floor dysfunction after burch colposuspension-a comprehensive study. part i. acta obstet gynecol scand. 2005;84(9):894-901. 19. buurman mbr, lagro‐janssen alm. women’s perception of postpartum pelvic floor dysfunction and their help‐seeking behaviour: a qualitative interview study. scand j car sci. 2013;27(2):406-13. 20. nakagoe t, sawai t, tuji t, nanashima a, yamaguchi h, yasutake t, et al. successful transvaginal repair of a rectovaginal fistula developing after double-stapled anastomosis in low anterior resection: report of four cases. surg today. 1999;29(5):443-5. 21. schloericke e, hoffmann m, zimmermann m, kraus m, bouchard r, roblick uj, et al. transperineal omentum flap for the anatomic reconstruction of the rectovaginal space in the therapy of rectovaginal fistulas. colorect dis. 2012;14(5):604-10. 22. (avicenna) sa. translation and description of avicenna’s healthcare management. tehran: iran university of medical sciences; 2008. 23. s a. iranian traditional pharmacy and pharmaceutical dosage forms. tehran: chogan publication; 2013. 24. mh ak. makhzan ol advieh (the storehouse medicaments). tehran: tehran university of medical sciences; 2008. 25. cammu h, van nylen m, amy j. a 10‐year follow‐up after kegel pelvic floor muscle exercises for genuine stress incontinence. bju int. 2000;85(6):655-8. 26. kegel ah. progressive resistance exercise in the functional restoration of the perineal muscles. am j obstet gynecol. 1948;56(2):238-48. 27. elia g, bergman a. pelvic muscle exercises: when do they work? obstet gynecol. 1993;81(2):283-6. dx.doi.org/10.22317/jcms.v6i3.718 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 189j contemp med sci | vol. 7, no. 3, may-june 2021: 189–191 case report effect of early initiation of combination therapy of anti-inflammatory and anti-fibrotic drugs on post covid-19 interstitial lung disease: a case report rahmah alsilmi division of pulmonology, department of internal medicine, faculty of medicine, king abdulaziz university, jeddah, saudi arabia. correspondence to rahmah alsilmi (email: ralsilmi@kau.edu.sa). (submitted: 12 april 2021 – revised version received: 27 april 2021 – accepted: 01 may 2021 – published online: 26 june 2021) introduction as the covid-19 pandemic hits the world repeatedly, our understanding of this novel virus infection regarding pathophysiology, clinical sequelae, and proper treatments continues to evolve. the current understanding of the potential long-term sequelae of covid-19 is premature and mostly extrapolated from other viral infections and post acute respiratory distress syndrome (ards) interstitial lung abnormalities. during the evolvement of pathological stages of ards, different stages can occur, including diffuse alveolar damage with exudative phase, edema and hyaline membrane formation that can form into more organized material, and then the progression into the fibrotic phase, which seems to occur after the initial two weeks of insult.1 that is being said, the clinical significance and outcome of this proportion of patients with post-ards fibrosis seem to be less significant, especially with the strict use of lung-protective ventilation during ards management.2 although the data on severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) infections initially confirmed a significant number of patients with interstitial lung abnormalities;3,4 the follow-up data from sars infected patients demonstrated that the interstitial changes could substantially resolve with a minority having residual changes after a prolonged follow-up period.5,6 regarding covid-19 related interstitial lung disease (ild), we still lack evidence based data regarding predicting, following and treating this outcome. case history a 64-year-old non-smoker male with no previous known medical conditions tested positive for sars-cov2 virus pcr on nov 4th, 2020. nine days later, he was admitted to our hospital with worsening dyspnea, cough, diarrhea, and highgrade fever. he had severe covid-19 disease characterized by severe pneumonia, acute respiratory failure, and increased inflammatory markers. initially, his oxygen requirement was 5 l via nasal cannula, but his condition progressed, and subsequently, he required non-invasive mechanical ventilation with bipap alternating with high flow nasal cannula. he was monitored in the intensive care unit (icu) with the awake proning position. he received ten days of dexamethasone 6 mg daily, and his clinical status has slightly improved on the subsequent two weeks, but he still was requiring supplemental oxygen with resting hypoxemia and was then shifted to a regular ward. as there was no further improvement in his clinical status, a chest computed tomography (ct), which was done three weeks after his presentation, was done to investigate pulmonary embolism, and it came back negative for this concern. however, it demonstrated marked infiltrate with bilateral consolidation and ground-glass opacities (fig. 1). before discharge, he was started on prednisone 30 mg po daily, azithromycin 250 mg po thrice weekly, and pirfenidone with escalating regimen over two weeks to reach a maximum 801 mg po tid afterward. he was discharged home on 3 l oxygen given the resting hypoxemia. ten days post-hospital discharge, our clinic’s assessment demonstrated significant improvement in the resting oxygen desaturation and the degree of dyspnea. spirometry revealed a moderately severe reduction in forced vital capacity which was 59% predicted, and a six-minute walk test showed exertional desaturation with nadir oxygen saturation of 87% and a walking distance of 78% predicted. given the clinical improvement, a taper prednisone regimen was initiated at 5 mg off every two weeks while continuing azithromycin and pirfenidone without dose modification. a repeat ct chest image was obtained after seven weeks of the initial one, while he was maintained on 15 mg prednisone, azithromycin, and pirfenidone, and it displayed remarkable improvement of the previous parenchymal infiltrates with no evidence of pulmonary fibrosis (fig. 1). abstract the persistence of lung parenchymal changes post covid-19 is increasingly recognized as a vital outcome observed on some patients recovering from sars-cov2 infection with limited evidence-based management so far. here we present a 64-year-old male developed extensive interstitial lung changes post sars-cov2 infection and was successfully managed with oral prednisone, pirfenidone, and azithromycin maintenance regimen with significant improvement in his clinical and radiographic parameters noted within a relatively short time. the role of a combined therapeutic approach with anti-inflammatory and anti-fibrotic drugs might be provocative action in patients with covid-19 related interstitial lung disease, especially those at risk for persistent long-term abnormalities and pulmonary fibrosis development. keywords case report, covid-19, pirfenidone, anti-fibrotic, systemic corticosteroid, macrolide abbreviation ild; interstitial lung disease, covid-19; coronavirus disease of 2019, bipap; bilevel positive airway pressure, ard; acute respiratory distress syndrome, sars; severe acute respiratory syndrome, mers; middle east respiratory syndrome, ct; chest computed tomography issn 2413-0516 190 j contemp med sci | vol. 7, no. 3, may-june 2021: 189–191 effect of early initiation of combination therapy of anti-inflammatory and anti-fibrotic drugs on post covid-19 ild case report r. alsilmi discussion our patient had severe covid-19 illness and a prolonged hospital stay that resulted in significant morbidity. his clinical response to the initial systemic corticosteroid therapy was slow, and he was still left with major respiratory impairment. thus, we initiated combined therapy with anti-inflammatory and anti-fibrotic drugs to alleviate the burden of the ild and try to prevent fibrosis development which would lead to a considerable unfavorable outcome. the available data on the radiographic abnormalities post-acute covid-19 infection were reported to persist in up to two third of the patients on a short follow-up period7 even in non-critical cases.8 nevertheless, the overall lung parenchymal acute changes, particularly the inflammatory ones such as ground-glass opacities and consolidations, seem to improve in a considerable proportion of patients on subsequent follow-up images regardless of the severity of the initial illness.7 the concern of the evolution of some of these radiographic changes, particularly the reticulation into fibrosis, warrants a close follow-up of these patients to early identify those who might progress into fibrosis on a more extended follow-up period.7,9 the rate of pulmonary fibrosis development post-acute covid-19 was reported in up to 46% of the patients on a relatively short follow-up period, with the overall score of fibrosis being mild.10 that is being said, the persistence of the radiographic abnormalities with the development of fibrotic parenchymal changes in the long term is not clear at this time with the availability of the relatively short follow-up data. some predictors of the outcome of lung fibrosis post-acute covid-19 were reported to include older age, length of hospital stay, icu admission, maximum ct score of the parenchymal changes at the initial illness, and elevated inflammatory markers.9,10,11 moreover, the factors that might correlate with persistence of the lung parenchymal changes on subsequent chest images and thus might predict subsequent pulmonary fibrosis were noted to include the severity of the initial illness, elderly age, and comorbidities.7 this observation signifies the importance of close follow-up on the patients with these potential risk factors for early identification, monitoring, and adequately treating this substantial outcome. post-acute covid-19, physiologic lung abnormalities, i.e., restrictive ventilator defect and reduction in diffusion capacity, have been frequently reported. furthermore, it seems that the physiologic lung dysfunction correlates with the extent of lung parenchyma changes and the severity of acute illness.12,13 the persistence of symptoms after the recovery of the acute sars-cov2 infection has been frequently reported in the literature, with respiratory symptoms of dyspnea and cough being encountered more often.7,14 our patient has residual exertional dyspnea though it improved since his first presentation correlating with the overall improvement in his chest images and physiological lung function. understanding the pathophysiology of covid-19 related ild and its long-term sequelae unfolds into two primary processes, i.e., intense inflammation as part of ards and the probability to heal up into a fibrotic process as short and longterm consequences, respectively. concerning the acute covid-19 infection, systemic corticosteroid has shown significant results regarding its beneficial effect on short term outcomes such as progression of acute respiratory failure and mortality when given to patients needing respiratory support.15 however, the continuation of this therapy beyond the acute illness is not yet clear. the current practice of prescribing systemic corticosteroids beyond the acute illness to target the inflammatory component of the lung pathology is extrapolated from other interstitial lung disease management, particularly organizing pneumonia, which is a component of ards seen in covid-19 disease. this practice needs more robust longitudinal studies to evaluate its effectiveness on long-term outcomes. macrolide antibiotics have been addressed with special recognition in the literature regarding their underlying anti-inflammatory and immunomodulatory effect in addition to an antimicrobial and potentially anti fibrotic role.16 the effectiveness of macrolide in organizing pneumonia as a first-line agent for mild diseases, as adjuvant therapy with a systemic corticosteroid and as a steroid-sparing agent have been reported.17,18,19 our knowledge about the longterm behaviour of post covid-19 related ild is premature at this time of the pandemic, and in particular, the rate of complete responsiveness to corticosteroid and the relapse rate of ild during the taper of corticosteroid or upon its discontinuation. the use of macrolide at the start of the covid-19 related ild management would add potential anti-inflammatory effect and would allow a rapid corticosteroid taper regiment, thus minimizing its potential side effects. anti-fibrotic drugs, including pirfenidone and nintedanib were initially approved for the treatment of idiopathic pulmonary fibrosis, given their effect on slowing the fibrotic lung process.20 besides, nintedanib eventually granted regulatory agencies approval for other ild such as ild with progressive fibrosing phenotype and systemic sclerosis-related ild based on robust evidence.21,22 fig. 1 chest computed tomography (lung window) at three weeks of positive sars-cov2 pcr detection (right) and seven weeks later (left) with axial (top), coronal (middle), and sagittal (bottom) views demonstrating significant improvement in the inflammatory opacities within a considerably short period of time with no signs of fibrosis. 191j contemp med sci | vol. 7, no. 3, may-june 2021: 189–191 r. alsilmi case report effect of early initiation of combination therapy of anti-inflammatory and anti-fibrotic drugs on post covid-19 ild this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.10201901 pirfenidone and nintedanib modulate the fibrotic process, in addition to a potential anti-inflammatory effect, by targeting different pathways, including transforming growth factor-beta, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, and fibroblast growth factor receptor23,24 respectively. we do not know at this time the magnitude of postcovid 19 pulmonary fibrosis; hence aiming to prevent its development rather than mitigating its progression after well-established fibrosis ensues would perhaps be a reasonable approach. current large clinical trials are ongoing to address this important clinical outcome. our patient had features of severe infection and potential risk factors to progress into persistent lung parenchymal abnormalities and probably pulmonary fibrosis. he responded quite well to the combination therapy; the further plan is to taper corticosteroid relatively quickly while maintaining azithrmycin and pirfenidone with further taper regimen upon complete resolution of his clinical, physiological and radiological abnormalities. conclusion identifying high-risk patients post covid-19 who might develop persistent lung parenchymal abnormalities and thus might progress into pulmonary fibrosis is crucial. similarly, investigating the proper treatment of the intense lung parenchymal inflammation post-covid-19, with multi-target therapeutics is one of the critical unmet needs given the massive magnitude of this pandemic and its significant burden on patients’ outcome and health-related cost. conflicts of interest none. references 1. thille aw, esteban a, fernández-segoviano p, et al. chronology of histological lesions in acute respiratory distress syndrome with diffuse alveolar damage: a prospective cohort study of clinical autopsies. lancet respir med. 2013;1:395–401. 2. burnham el, janssen wj, riches dwh, et al. the fibroproliferative response in acute respiratory distress syndrome: mechanisms and clinical significance. eur respir j. 2014;43:276–285. 3. ooi gc, khong pl, müller nl, et al. severe acute respiratory syndrome: temporal lung changes at thin-section ct in 30 patients. radiology. 2004;230:836–844. 4. das km, lee ey, singh r, et al. follow-up chest radiographic findings in patients with mers-cov after recovery. indian j radiol imaging. 2017;27: 342–349. 5. zhang p, li j, liu h, et al. long-term bone and lung consequences associated with hospital-acquired severe acute respiratory syndrome: a 15-year follow-up from a prospective cohort study. bone res 2020;8:8. 6. wu x, dong d and ma d. thin-section computed tomography manifestations during convalescence and long-term follow-up of patients with severe acute respiratory syndrome (sars). med sci monit. 2016;22:2793–2799. 7. sonnweber t, sahanic s, pizzini a, et al. cardiopulmonary recovery after covid-19 – an observational prospective multi-center trial. eur respir j 2020; in press 8. yu-miao zhao, yao-min shang, wen-bin song, et al. follow-up study of the pulmonary function and related physiological characteristics of covid-19 survivors three months after recovery. eclinicalmedicine 2020;25:100463. 9. yu m, liu y, xu d, et al. prediction of the development of pulmonary fibrosis using serial thin-section ct and clinical features in patients discharged after treatment for covid-19 pneumonia. korean j radiol. 2020;21:746–755 10. yang zl, chen c, huang l, et al. fibrotic changes depicted by thin-section ct in patients with covid-19 at the early recovery stage: preliminary experience. front med (lausanne). 2020;7:605088. 11. wei j, yang h, lei p, et al. analysis of thin-section ct in patients with coronavirus disease (covid-19) after hospital discharge journal of x-ray science and technology. 2020;28:383–389. 12. mo x, jian w, su z, et al. abnormal pulmonary function in covid-19 patients at time of hospital discharge. eur respir j. 2020; published online may 12. 13. guler sa, ebner l, beigelman c, et al. pulmonary function and radiological features four months after covid-19: first results from the national prospective observational swiss covid-19 lung study. eur respir j 2021; in press. 14. carfı a, bernabei r, landi f. for the gemelli against covid-19 post-acute care study group. persistent symptoms in patients after acute covid-19. j am med assoc. 2020;324:603–605. 15. horby p, lim ws, emberson jr, et al. dexamethasone in hospitalized patients with covid-19. n engl j med. 2021;384:693–704. 16. faverio p, bini f, vaghi a, et al. long-term macrolides in diffuse interstitial lung diseases. eur respir rev. 2017;26:170082. 17. ding ql, lv d, wang bj, et al. macrolide therapy in cryptogenic organizing pneumonia: a case report and literature review. exp ther med. 2015;9:829– 834. 18. chang wj, lee ej, lee sy, et al. successful salvage treatment of steroidrefractory bronchiolar cop with low-dose macrolides. pathol int. 2012;62:144–148. 19. pathak v, kuhn jm, durham c, et al. macrolide use leads to clinical and radiological improvement in patients with cryptogenic organizing pneumonia. ann am thorac soc. 2014;11:87–91. 20. an official ats/ers/jrs/alat clinical practice guideline: treatment of idiopathic pulmonary fibrosis: an update of the 2011 clinical practice guideline. am j respir crit care med 2015;192:e3–e19. available at http://www.atsjournals.org/doi/abs/10.1164/rccm.201506–1063st. 21. flaherty kr, wells a u, cottin v et al. nintedanib in progressive fibrosing interstitial lung diseases. n engl j med. 2019;381:1718–1727. 22. distler o, highland kb, gahlemann m, et al. nintedanib for systemic sclerosis-associated interstitial lung disease. n engl j med. 2019. 23. iyer sn, gurujeyalakshmi g, giri sn. effects of pirfenidone on transforming growth factor-beta gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis. j pharmacol exp ther. 1999;291:367–373. 24. wollin l, wex e, pautsch a, et al. mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis. eur respir j. 2015;45:1434–1445. 267j contemp med sci | vol. 6, no. 6, november–december 2020: 267–274 original issn 2413-0516 introduction multiple sclerosis (ms) is among the most prevalent progressive chronic neurological diseases that leads to neurodegeneration in adults and about two and a half million people worldwide1 and about 60,000 afflicts in iran. the prevalence rate of this disease in iran has been 20 times as high as other countries in the region.2 in the past, iran was known as a country with a low prevalence of ms. yet, recent research has shown that the rate of the disease has been significantly increased, and the maximum prevalence rate of the disease has been reported in isfahan province (80 per 100,000 populations).3 due to its unpredictable nature, this disease can be stressful to all family members. its occurrence during youth and early adulthood, marked by a critical period of seeking employment and marriage, can add to the psychological burden of this disease.4 in the long run, a patient might require primary carer, due to failed daily activities, which is often provided by a family member. most often, these caregivers are neglected.5, 6 caregivers are referred to as hidden patients, who tolerate a great deal of emotional burden in fulfilling their tasks.7 taking care of a patient with a chronic disease creates a lot of tension for the family and caregivers. caregivers are particularly vulnerable to stress because the patient’s biological, social, and psychological needs are often prioritized to their own needs, and they get burned out due to the high loads of pressure and needs.8 taking care of people with mc differs from other chronic diseases, as this disease does not follow a certain trend and is inherently unpredictable. concerning this disease, the caregiver is unable to precisely predict the occurrence, recurrence, and progression of the disease and even the functional ability of patient during a day.9 this instability and unpredictability of needs would add to the caregiving burden of this disease compared to other chronic diseases.10 caregivers suffer from many physical and psychological adverse of the stress involved in caregiving. there is research evidence that the care provided by a spouse or parent at home or in the long run is accompanied by functional limitations and psychological symptoms such as depression in caregivers.11 developments in solving health issues have replaced care-providing institutes with family caregivers.4 as the topic of home care has attracted the attention of health professionals in the present century to reduce costs and increase the quality of care services, on the other hand, a vast majority of research has addressed the patient rather than the patient’s family or caregivers, though they are faced with many problems in taking care of patients, therefore, the present research aimed to explore the factors affecting psychological problems of primary caregivers of individuals with multiple sclerosis (pcims). materials and methods in the present research, conventional content analysis was used to collect and analyze the required data directly based on participants’ experiences and perceptions with no assumptions. an inductive process was followed to extract the codes, categories, and subcategories.12, 13 in this research, which was conducted in isfahan ms association in 2018–19, purposive sampling was used to select the participants. ms patients, caregivers who were usually family members and the medical staff including a neurologist, nurse, and psychologist comprised the participants of this research. the inclusion criteria were willingness to participate. primary caregivers with at least one year experience of factors affecting psychological problems in primary caregivers of people with multiple sclerosis: a qualitative study bita sadeghi1, fatemeh estebsari1, maryam rassouli2, abbas ebadi3 1department of community health nursing, school of nursing and midwifery, shahid beheshti university of medical sciences, tehran, ir iran. 2cancer research center, shahid beheshti university of medical sciences, tehran, ir iran. 3behavioral sciences research center, life style institute, nursing faculty, baqiyatallah university of medical sciences, tehran, ir iran corresponding author: fatemeh estebsari (e-mail: fatemeestebsari@yahoo.com) (submitted: 15 august 2020 – revised version received: 01 september 2020 – accepted: 27 october 2020 – published online: 26 december 2020) abstract objectives: the present research aimed to explore factors affecting psychological problems among primary caregivers of individuals with multiple sclerosis (pcims). methods: the present qualitative research employed a content analysis approach and was conducted in isfahan, iran in 2018–19. the participants were selected through purposive sampling method. semi-structured interviews were conducted to explore psychological problems involved in providing care to ms patients and the factors affecting them with 8 patients, 10 caregivers and 3 healthcare providers. conventional content analysis was used to analyze the data. results: the acquired data were put in four main categories: (i) “isolation and loneliness,” (ii) “caregiver’s concerns,” (iii) “frustration and hopelessness,” and (iv) “disruption in the family foundation,” each with certain subcategories. conclusion: the present results can be helpful to manage psychological disorders and the underlying factors of pcims. thus, healthcare providers and policymakers should consider these areas and make special attempts to improve the performance and conditions of these caregivers to maximize the quality of care provided. keywords: multiple scleroses, primary caregiver, family caregiver, qualitative research, psychological burden. 268 original factors affecting psychological problems in primary caregivers of people with multiple sclerosis bita sadeghi j contemp med sci | vol. 6, no. 6, november–december 2020: 267–274 caregiving for ms patients, healthcare providers with at least one year of work experience in sections related to ms disease, and patients with at least one year of diagnosis. according to the above-mentioned inclusion criteria, interviews were held with 32 participants (16 caregivers of ms patient and 10 ms patients aware of caregivers’ experience, and 6 healthcare providers with experience of working with ms patients and their families). table 1 lists the participants’ demographic characteristics. table 1. demographic characteristics of primary caregivers. years of disease relation with individual with ms educationjobmarital statussexageid code 6motherprimaryhousekeepermarriedfemale49c1 7husbandbsemployeemarriedmale38c2 5husbandprimaryunemployedmarriedmale50c3 15motherprimaryunemployedwidowfemale58c4 4wifediplomaemployeemarriedmale46c5 8wifediplomahousekeepermarriedfemale28c6 8wifediplomahousekeepermarriedfemale38c7 5motherbsemployeewidowfemale61c8 4wifebshousekeepermarriedmale38c9 10daughterprimaryunemployedmarriedfemale33c10 6husbandmsemployeemarriedmale41c11 3daughterbsemployeesinglefemale22c12 5motherbsemployeedivorcedfemale48c13 4wifebsemployeemarriedfemale45c14 17fatherdiplomaretiredmarriedmale52c15 9motherprimaryhousekeepermarriedfemale57c16 5--primaryhousekeepermarriedfemale44p1 6--primaryunemployeddivorcedfemale43p2 4--bsunemployedmarriedmale32p3 5--bshousekeepermarriedfemale40p4 6--bshousekeepermarriedfemale30p5 5--bsunemployeddivorcedfemale40p6 8--diplomaemployeemarriedmale42p7 3--bs studentstudentsinglefemale22p8 5--bs studentstudentsinglefemale25p9 10--msunemployedmarriedmale33p10 ----bsnurse--female38h1 ----bsnurse--female26h2 ----bsnurse--female30h3 ----bsphysiotherapist--male36h4 ----specialistneurologist--male52h5 ----phdpsychologist--male41h6 id code (c: caregiver, p: patient, h: healthcare provider). 269 original factors affecting psychological problems in primary caregivers of people with multiple sclerosisbita sadeghi j contemp med sci | vol. 6, no. 6, november–december 2020: 267–274 data collection the research data were collected via in-depth semi-structured interviews from september 2018 to august 2019. each interview started with open questions regarding the experience of caregiving and gradually followed by more detailed questions about the psychological problems caused by caregiving. the data collection continued with patients and medical staff dealing with these caregivers. at first, they were asked to share their experience of taking care of ms patients. examples of open questions: “please talk about the psychological problems you had with caregiving”, “what factors do you consider to be involved in creating these problems?” when the participants had problems describing their experiences or when the interview needed to be more specialized, follow-up questions were asked for clarification: “can you explain more?,” “what do you think about this?,” “can you provide an example?” all the interviews were held in a calm and private place (in ms association, participants’ home, or workplace). each interview, depending on participants’ physical and psychological conditions, took between 30 and 80 minutes. all interviews were tape-recorded. non-verbal behaviors were noted as field notes by the interviewer. they were also used as complementary data and were used for trustworthiness. the sample size was based on the data saturation. thus, selection of eligible cases continued until adequate data were collected (data saturation phenomenon).14 in this research, data saturation occurred after conducting 21 interviews; then, the list of codes was merged and categories were extracted. data analysis the data collected in this research were analyzed through conventional content analysis as a systematic data coding and categorizing method.15 in this method, the researchers immerse themselves in the data to allow new insights to emerge.16 at the same time of data collection, data analysis began too. the interviews were transcribed in persian and analyzed by the first author. initially, the tape-recorded interviews were transcribed word by word. before the coding process, the interviews were read several times by the researcher to get a general sense of the text. then, the transcribed interviews were reviewed line by line and the main words, phrases, and sentences were identified as meaning units. the main ideas were extracted from them and they took the title of the code. afterwards, the different codes formed their own subcategories based on their similarities and differences. the subcategories were also compared and those who were similar in terms of features and content merged to form the main categories. peer check, member check were used to achieve a consensus in coding. instances of data analysis are included in table 2. rigor according to lincoln and guba, four criteria are essential for trustworthiness. these include: credibility, dependability, transferability, and conformability.17 to increase credibility, prolonged engagement with data and emerging codes and categories was followed (here, engagement with data for more than 11 months) as well as peer checking and member checking. several sessions were held by the researcher with the participants to delve into their experiences in depth. moreover, each participant was asked for feedback on the codes and their interpretation. supplementary comments were collected and essential changes were made. peer checking was one way to make sure of the dependability and confirmability in research. it was held through weekly meetings between the second and third authors, and two other phd candidates of nursing. all research procedures were checked including codes and categories. conformability refers to the objectivity and implies that the data accurately represent the information that the participants provided and the interpretation of those data are not invented by the researcher.18 furthermore, trustworthiness includes the question of transferability, which refers to the extent to which the findings can be transferred to other settings or groups.18 it was attempted in the present research to include rich quotes from the participants that form the main categories, in order to increase conformability. to make sure of the transferability, the data collection and analysis procedure was described comprehensively. ethical considerations in this research, all participants were informed before the interview that their participation is fully voluntary. they could withdraw from the research without any conditions, whenever they wanted. having signed a letter of informed consent, the participants were ensured of the confidentiality of the information they provided. each participant was interviewed only once. the purpose and significance of study and the research protocol were explained to the participants. their voice was recorded as they consented to. this research was part of a phd dissertation approved by the research ethics committee of sbmu (ir.sbmu.phnm.1397.53). results four main categories were extracted and are shown along with their subcategories in table 3. table 2. examples of the analysis process. meaning unit code sub-category category as far as i can stand on my feet, i will take care of my daughter. but i am worried about the upcoming years, when i lose my abilities, if my daughter cripples, who will take care of her? concerns about the patient’s disability physical concerns caregiver’s stresses and concerns when my patient’s symptoms return or a new problem arises, i feel there is nothing i can do to help her. inability to control conditions feeling incompetent frustration and hopelessness 270 original factors affecting psychological problems in primary caregivers of people with multiple sclerosis bita sadeghi j contemp med sci | vol. 6, no. 6, november–december 2020: 267–274 table 3. categories and subcategories extracted from the data. subcategory category unwillingness and lack of motivation sense of pity patient’s low self-confidence isolation and loneliness physical concerns psychological concerns social concerns caregiver’s stresses and concerns feeling incompetent lack of recovery in the disease’s course lack of acceptance of the disease inadequate knowledge of disease’s nature frustration and hopelessness conflict in interactions reduced emotional and sexual relations pessimism toward spouse continue married life as a pity disruption in the family foundation isolation and loneliness caregivers, due to unwillingness and lack of motivation, freedom from other’s pity for the patient and patient’s low self-confidence, began to reduce or stop communication with others. such isolation and loneliness adversely affected the caregiver’s psychology. unwillingness and lack of motivation the unfavorable psychological state of the patient and the caregiver made them reluctant to social interactions and reduced social relations and activities. “i can’t take my son home alone for a moment now,” said one of the participating caregivers. with this respect, a participating caregiver stated: “now i cannot even leave my son alone for a moment at home. if i want to go to my brother’s or my sister’s house and come back, my mind is constantly on him. it’s like my heart is dead and don’t feel like talking to anyone. what good is it if i go somewhere and sit in a quiet corner and think about everything?” sense of pity on the patient the participants believed that when a patient receives excessive attention, she/he feels pity. to get rid of this pity, the caregiver and the patient would use methods such as concealment of the disease and keeping a social distance. concerning this, a participant patient quoted: “people’s excessive attention would convey a bad feeling to me. they keep asking me: how are you? is it better? even when some of them see me, they keep whispering thank god i am not so! we think that if we see less people, we will be less bothered” (p4: female). patient’s low self-confidence according to the present research, there are certain factors that can reduce patient’s self-confidence. to compensate for this defect, the patient and caregiver might reduce social relations or even cease them entirely. a participating caregiver stated: “my wife, due to her walking problems, prefers to stay home and not to go out. at home, she uses a wheelchair easily but she avoids going out with the fear of others staring at her on the wheelchair. when she doesn’t come with me, i also don’t like to go to family parties and ceremonies alone” (c3: male). caregiver’s stresses and concerns this category is made up of the caregiver’s physical concerns, social concerns, and family concerns subcategories, as introduced in the following: caregiver’s physical concerns these concerns deal more with the patient’s possible future physical problems, such as fear of the patient’s disability, and his/her own probable physical problems, which in both cases have a negative psychological impact on the caregiver as an instance, one participating caregiver stated: “as long as i can stand on my feet, i will take care of my daughter. but i am worried about the upcoming years, when i lose my abilities, if my daughter cripples, who will take care of her??” (c1: female). caregiver’s social concerns some caregivers were concerned about the future of their patients. these concerns were more about the patient’s career, income, and marriage which would eventually affect the caregiver’s psychology negatively. with this regard, a participating caregiver mentioned: “before my daughter was sick, she used to work and had a good income. she was busy with her job. now that she is sick, she has to stay home and i keep wondering what’s going to happen to her. not only her job, but also her chances of marriage worry me. can she ever marry?” (c8: female). caregiver’s family concerns the majority of participants in this research believed that this disease affected their mood tremendously and would get on the patient’s nerves in many cases. a concern of the caregivers was the patient’s constant moody behavior toward other family members which could gradually disrupt effective communication with family members and fade away the existing emotions they had for each other. as an example, a participating caregiver maintained: “my daughter has become quick-tempered due to her illness. she gets easily offended and insists that we should not come to her house any longer. she keeps shouting at her child and arguing with her husband. i’m afraid soon her husband’s patience will end and leave her. i’m much worried” (c1: female). frustration and hopelessness as some caregivers stated, while they take care of an ms patient, they gradually feel tired and frustrated and move little by little to disappointment. this category consists of such subcategories as feeling incompetent, lack of recovery in the disease’s course, lack of acceptance of the disease, and inadequate knowledge of disease’s nature. feeling incompetent when the symptoms of disease recurred, the caregivers felt unable to control conditions; thus, their sense of control, competence, and identity was disrupted. 271 original factors affecting psychological problems in primary caregivers of people with multiple sclerosisbita sadeghi j contemp med sci | vol. 6, no. 6, november–december 2020: 267–274 with this regard, a participating caregiver commented: “when my patient’s symptoms return or a new problem arises, i feel there is nothing i can do to help her. i feel i am a different person” (c9: male). lack of recovery in the disease’s course as the data analysis revealed, caregivers and patients experienced many challenges during the disease. while tolerating the disease problems and hardship, in the face of no improvement in patient’s physical and psychological conditions, the patient and the caregiver both find themselves in a submissive state and give in to the disease and its adverse effects. as an instance, a caregiver commented: “when i see the time passes, yet the patient does not get any better, i lose hope and see no sense in seeing different doctors. sometimes, my poor son says let’s stop buying expensive ampoules. it’s too late and i only wish to die calmly and proudly” (c4: female). lack of acceptance of disease as the data analysis showed, patients and caregivers who did not manage to accept their condition experienced more problems with the disease. they had a lower self-confidence. in the face of the disease, they showed less resistance and sooner felt exhausted and frustrated. concerning this, a participating caregiver said: “after so long a time, my husband hasn’t accepted his disease. he tends to hide his disease, as he is somehow proud. he says there is nothing. he doesn’t help to treat himself. even for visiting doctor and taking his medication, i have to insist every time to be satisfied. whenever the symptoms recur, he gets nervous and complains. he always complains why it all happened to him. it makes everything harder for me. it’s really hard for me. i am very upset by this situation” (c7: female). inadequate knowledge of disease’s nature not knowing the reliable and accurate information sources and consequently not knowing the disease’ nature led patients and caregivers to seek information at unreliable databases. or they might easily yield to incorrect medical advice by unreliable sources and act accordingly. this can impose high costs for the act of caregiving. besides, it can add to the symptoms of disease and finally leave the patient frustrated when no sign of improvement is observed. as an example, a patient quoted: “my wife found someone via the net who claimed to be an herbal medicine specialist. he claimed to find new treatments for ms. when we paid him, he sent over some home-made tablets which left me hospitalized for a week” (p3: male). disruption in the family foundation as the data analysis revealed, disruption in the family foundation was another main category extracted from the present research. damages to family caused by decrease in the patient’s/ caregiver’s tolerance threshold and their introspection and the disintegration of coherence, all attested to the vulnerability of family with an ms patient. this class was mostly in cases where the caregiver was the patient’s spouse. the underlying factors as the subcategories leading to such problems were: conflict in interactions, reduced emotional and sexual relations (of couples), pessimism toward the spouse, and continue married life as a pity. these subcategories will be introduced below. conflict in interactions as the research data showed, one reason for the creation of familial and marital problems was conflict in interactions among family members, and it is dramatically affected by sudden changes to patient’s moods and their unreasonable anger. as an instance, a participant described: “unfortunately, many ms patients develop moody behavior and thus misbehave on different conditions which manifests itself mostly in the form of anger and aggression. moreover, many families and caregivers are not aware that this moodiness are due to the disease. so, they react inappropriately and get into conflict in interactions” (h2: female). reduced emotional and sexual relations of couple as the data revealed, this disease disrupts the sexual relationship of couples. occasionally, besides the patient, the caregiver also has sexual problems. spouses who act as the caregiver stated that such sexual problems usually result from the psychological burden of the caregiving and the consequent mental concerns, and there existed no physiological defect. as an instance, a caregiver stated: “my wife is totally frigid. my sexual desire has decreased too. it must be due to too much stress and tension” (c9: male). as the participants commented, this problem would cause emotional coldness in patient–caregiver relations and it was unpleasant to the caregiver. here is part of a patient’s comment: “i really feel i am not able to meet my husband’s sexual desire. we are no longer warm and friendly. we look more like home mates. my husband grumble sometimes. i don’t expect him to understand me. as a man, how long is he going to tolerate it?” (p2: female). pessimism toward the spouse as the data revealed, some patients got pessimistic toward their spouse and did not feel secure in life anymore. thus, they misbehaved toward their caregiver. often, the patient’s spouse reacts inappropriately which would gradually disrupt emotional relations. this disruption can in turn have such consequences as marital and social problems and even divorce. here is a comment made by a participating caregiver: “one factor that turns us away from each other is that my wife thinks now that she has ms, i intend to marry someone else. she distrusts me and makes me very angry, she keeps being sarcastic and keeps checking my telephone. that’s why we have argued so often” (c3: male). continuing married life due to pity in some cases, the patient believed that his spouse continued their married life with no true love and only due to pity. as an instance, a patient commented: “i feel that my wife only continues our life for god’s sake. she pities me. only her religious beliefs make her stay with me. otherwise, she no longer feels happy in this life. i even told her i don’t need her sympathy. i asked her to leave me and continue her own life. i don’t know why she insists that i think everything is as ever before. when i says this, she gets upset. this thought that comes to mind bothers me a lot. actually, our love has turned artificial and forced. it’s as if we are just pretending” (p3: male). 272 original factors affecting psychological problems in primary caregivers of people with multiple sclerosis bita sadeghi j contemp med sci | vol. 6, no. 6, november–december 2020: 267–274 discussions the present research showed that different factors are involved in creating caregivers’ psychological problems in the case of ms patients. according to the participants’ experiences, four categories were involved in creating caregivers’ psychological problems. these included “isolation and loneliness,” “caregiver’s concerns,” “frustration and hopelessness,” and “disruption in the family foundation.” in the qualitative research, shapiro et al. had 4 groups with a focus on 15 ms patients and their caregivers to explore caregivers’ misbehaviors toward the patients. they concluded that a key reason why caregivers misbehaved toward patients was their social isolation.19 others’ sense of pity on ms patients showed to be an effective factor in patient’s social isolation followed by caregiver’s isolation. as the participants stated, patients disliked being pitied. to avoid a sense of pity, they attempted to hide their disease and communicate less with others. to this aim, caregivers also had to cut down on their social relations so as to keep others in the dark about the disease. social isolation has been also discussed in a body of research on ms patients and their caregivers.20-22 such subcategories as “sense of pity on patients” have been in common with the present research.21, 22 similarly, in a qualitative study conducted by bogosin et al., the aim was to explore the experience of 15 caregivers of ms patients. in this research, one reason the caregivers brought for their isolation was that wherever the patients went, they thought they were the center of attention; they were pitied by others, and thought they were incapable of doing anything. they might actually be cognitively mistaken as this might not be as others truly think about them. thus, they try to hide their disease in an attempt to get along with the psychological burden of pity and similar to other related studies,22 they might end in social isolation and the same can go for their caregivers.23 caregiver’s concerns were another main category in the present research and showed to be an effective factor in caregivers’ psychological problems. in this research, the participants maintained that their physical and social concerns created a psychological burden for them. with this regard, mutch in another study mentioned “caregiver’s concerns” as a category, as he found that caregivers of ms patients were concerned about their patient’s prospective life and his/her physical and social conditions.24 in the present research, the participating caregivers also maintained that their family concerns were more about disrupting dynamic relations among family members. with this concern, kouzoupis indicated that ms patients influenced all family members and this influence can differ across members. that is why each member can use a different strategy to cope with the effects of the disease. such strategies might be in conflict, which can in turn cause more stress for family members and disrupt family dynamics.25 caregivers often worry about their own future, their patient, and their family in general. concerns about the aggravation of patient’s conditions prevailed among caregivers, as this could imply further needs and care burden.24, 26, 27 many caregivers worry about the loss of their own health which could restrict their ability to take care of their beloved patient. and, it may eventually force them to leave their patients in care centers.9, 27 frustration and hopelessness was another category in the present research. it was partly induced by no sign of improvement in patient’s conditions. in his research, masoudi found that continuous stress and no sign of improvement in patient’s conditions can frustrate the caregiver and reduce the quality of caregiver’s performance and the quality of the care s/he provides.28 another subcategory of this category was inadequate knowledge of the nature of disease. similar research findings revealed that primary caregivers required the knowledge and awareness of caregiving, so that they could act effectively as a caregiver. this could reduce their distress and burden. the lack of knowledge could negatively affect caregiver’s physical, psychological, and social health.29, 30 in the present research, some participants talked about traditional and cultural beliefs such as medical and nutritional beliefs rooted in their limited awareness of the disease. by implementing these beliefs, they caused more disabilities in the patient and this factor would add to the caregiver’s pressure. some other related studies discussed the medical and nutritional beliefs of ms patients’ caregivers (such as the benefits of consuming linseed oil, aromatherapy or bee therapy). it has also been pinpointed that these cases in combination with pharmacology can have adverse effects and can even reduce the effect of medicines.31 in the qualitative study by bogosian et al.24, some participants stated that they had met ms patients before; this background might have meant inability to control the disease and its symptoms to them.23 and according to this belief, the person has used strategies to avoid and hide the disease to deal with the psychological burden of the disease. such perceptions of the disease can tremendously affect the results, adaptive strategies, and self-management such as accepting treatments and medical priorities.32 disruption in the family foundation was found to be another category in this research. it resulted from contact in interactions, reduced emotional and sexual relations of the couples, pessimism toward the spouse, and continuing life due to pity. a body of related literature on ms showed that this disease can cause many marital problems and can disrupt the relations of the couples.23, 33, 34 in the present research, one reason for the occurrence of marital problems was patient’s sexual problems followed by the caregiver’s unmet emotional needs. in another study, rollero35 drew attention to the theme of “changes in the couple” and admitted that pcims often suffered from sexual problems and their needs were left unmet. participants in this research stated that sex mattered to them but for many reasons including patient’s constant fatigue, this need was left unmet. this had disrupted their marital relations.35 the same was acknowledged by the participants in the qualitative research by courts et al. who complained about problems in sexual relations, and they mentioned it as a factor involved in changing their communication with the spouse.31 in their review study, schulz et al. explored the psychological and physical effects of caregiving on primary caregivers and concluded that sexual problem was a key factor involved in primary caregivers’ stress and depression and low mental health.36 one factor found among reasons for creating contact in family interactions was patient’s distrust in the caregiver and patient’s changing mood and continuous aggression. in some qualitative research, courts et al.31 explored the experiences of those with an ms afflict spouse. some of the participants maintained that ms could cause aggression and marital problems for the couples and could even lead to divorce. yet, in the same study, some of the other participants, contrary to our study, stated that with the diagnosis of ms, their marital relationship has become stronger.31 in this regard, mutch in 273 original factors affecting psychological problems in primary caregivers of people with multiple sclerosisbita sadeghi j contemp med sci | vol. 6, no. 6, november–december 2020: 267–274 its qualitative study (content analysis) (2010) on the spouses of people with ms, about their experience of caring for these patients, according to the participants’ statements concluded that after the disease was diagnosed, their marital relations were further strengthened. such strength was perceived by some participants as a motivation for continuing the caregiving tasks and adapting to the caregiving burden.24 another finding of the present research was pessimism toward the spouse which could disrupt marital relations. the qualitative research by courts et al., conducted on 12 spouses of people with ms, some acknowledged that their spouses thought they would leave them soon. however, the patients did not share these negative thoughts with the spouses. accordingly, a participant in this research mentioned that when he got to know what his wife thought, he began to support her cognitively and ensured her that she was wrong.31 in the content analysis by bogosian et al., some participants maintained that patient’s mood swings and aggression often disrupts the relationship between the couples and it causes them to quarrel.23 other related body of literature also showed that the caregivers should not only care about the physical symptoms of ms disease but also be aware of the psychological aspects often manifested as moodiness.37 this moodiness can impose much distress on the caregiver and can adversely affect her/his quality of life. it can disrupt caregiver’s adaptation to the caregiving role and the relevant problems and can even entirely disrupt the patient–caregiver relations.38 conclusion the present findings revealed the factors affecting psychological problems of pcims. they are all of a great significance and psychological consultation seems to be able to help caregivers in all stages of the disease to solve these problems and be better prepared to act efficiently in taking care of ms patients. as this research is qualitative in type, its findings need to be interpreted carefully. the generalization of results is limited to the scope of the present research and to delve into the different aspects of the problems and challenges these caregivers are faced with, further research is required in different contexts. acknowledgments the present researcher should like to express gratitude to isfahan ms association and the research committee of shahid beheshti university of medical sciences. the gratitude is extended to all participants who shared their experience and information with the researchers and took part in this research. financial disclosure: none to declare. conflict of interest: none to declare. references 1. topcu g, buchanan h, aubeeluck a, garip g. caregiving in multiple sclerosis and quality of life: a meta-synthesis of qualitative research. psychol health. 2016;31(6):693-710. 2. gafari s, khoshknab mf, nourozi k, mohamadi e. informal caregivers’ experiences of caring of multiple sclerosis patients: a qualitative study. iran j nursing midwifery res. 2017;22(3):243. 3. izadi s, nikseresht a, sharifian m, sahraian ma, jahromi ah, aghighi m, et al. significant increase in the prevalence of multiple sclerosis in iran in 2011. iran j med sci. 2014;39(2):152. 4. buhse m. assessment of caregiver burden in families of persons with multiple sclerosis. j neurosci nursing. 2008;40(1):25-31. 5. navab e, negarandeh r, peyrovi h, navab p. stigma among i ranian family caregivers of patients with a lzheimer’s disease: a hermeneutic study. nursing health sci. 2013;15(2):201-6. 6. masoudi r, abedi h, abedi p, mohammadianinejad se. the perspectives of iranian patients with multiple sclerosis on continuity of care: a qualitative study. j nursing res. 2015;23(2):145-52. 7. galushko m, golla h, strupp j, karbach u, kaiser c, ernstmann n, et al. unmet needs of patients feeling severely affected by multiple sclerosis in germany: a qualitative study. j palliative med. 2014;17(3):274-81. 8. boyd ma. psychiatric nursing: contemporary practice: lippincott williams & wilkins; 2008. 9. mckeown l, porter-armstrong a, baxter g. caregivers of people with multiple sclerosis: experiences of support. multiple sclerosis j. 2004;10(2):219-30. 10. figved n, myhr k-m, larsen j-p, aarsland d. caregiver burden in multiple sclerosis: the impact of neuropsychiatric symptoms. j neurol neurosurg psychiatry. 2007;78(10):1097-102. 11. caputo j, pavalko ek, hardy ma. the long‐term effects of caregiving on women’s health and mortality. j marriage family. 2016;78(5):1382-98. 12. corbin j, strauss a. basics of qualitative research: techniques and procedures for developing grounded theory: sage publications; 2014. 13. speziale hs, streubert hj, carpenter dr. qualitative research in nursing: advancing the humanistic imperative: lippincott williams & wilkins; 2011. 14. estebsari f, taghdisi mh, mostafaei d, jamshidi e, latifi m. determining the factors contributing to quality of life of patients at the last stage of life: a qualitative study. iran red crescent med j. 2013;15(12). 15. grbich c. qualitative data analysis: an introduction: sage; 2012. 16. hsieh h-f, shannon se. three approaches to qualitative content analysis. qual health res. 2005;15(9):1277-88. 17. guba eg. criteria for assessing the trustworthiness of naturalistic inquiries. educ technol res dev. 1981;29(2):75-91. 18. estebsari f, taghdisi mh, mostafaei d, rahimi z. elements of healthy death: a thematic analysis. med j islamic republic of iran. 2017;31:24. 19. shapiro j, wiglesworth a, morrison eh. views on disclosing mistreatment: a focus group study of differences between people with ms and their caregivers. multiple sclerosis related disorders. 2013;2(2):96-102. 20. masoudi r, khayeri f, rabiei l, zarea k. a study of stigma among iranian family caregivers of patients with multiple sclerosis: a descriptive explorative qualitative study. appl nursing res. 2017;34:1-6. 21. ghafari s, fallahi-khoshknab m, nourozi k, mohammadi e. patients’ experiences of adapting to multiple sclerosis: a qualitative study. contemp nurse. 2015;50(1):36-49. 22. abolhassani s, yazdannik a, taleghani f, zamani a. social aspects of multiple sclerosis for iranian individuals. disab rehab. 2015;37(4):319-26. 23. bogosian a, moss-morris r, yardley l, dennison l. experiences of partners of people in the early stages of multiple sclerosis. multiple sclerosis j. 2009;15(7):876-84. 24. mutch k. in sickness and in health: experience of caring for a spouse with ms. br j nursing. 2010;19(4):214-9. 25. kouzoupis ab, paparrigopoulos t, soldatos m, papadimitriou gn. the family of the multiple sclerosis patient: a psychosocial perspective. int rev psychiatry. 2010;22(1):83-9. 26. heward k, molineux m, gough b. a grounded theory analysis of the occupational impact of caring for a partner who has multiple sclerosis. j occup sci. 2006;13(2-3):188-97. 27. cheung j, hocking p. the experience of spousal carers of people with multiple sclerosis. qual health res. 2004;14(2):153-66. epub 2004/02/11. 28. masoudi r, abedi ha, abedi p, mohammadianinejad se. iranian family caregivers’ challenges and issues in caring of multiple sclerosis patients: a descriptive explorative qualitative study. iran j nursing midwifery res. 2014;19(4):416. 29. fukui s. information needs and the related variables of japanese family caregivers of terminally ill cancer patients. nursing health sci. 2004;6(1):29-36. 30. given b, sherwood pr, given cw. what knowledge and skills do caregivers need? j social work educ. 2008;44(sup3):115-23. 31. courts nf, newton an, mcneal lj. husbands and wives living with multiple sclerosis. j neurosci nursing. 2005;37(1):20. 32. obiwuru o, joseph s, liu l, palomeque a, tarlow l, langer-gould am, et al. perceptions of multiple sclerosis in hispanic americans: need for targeted messaging. int j ms care. 2017;19(3):131-39. 274 original factors affecting psychological problems in primary caregivers of people with multiple sclerosis bita sadeghi j contemp med sci | vol. 6, no. 6, november–december 2020: 267–274 33. eriksson m, svedlund m. ‘the intruder’: spouses’ narratives about life with a chronically ill partner. j clin nursing. 2006;15(3):324-33. epub 2006/02/10. 34. boeije hr, van doorne-huiskes a. fulfilling a sense of duty: how men and women giving care to spouses with multiple sclerosis interpret this role. community work family. 2003;6(3):223-44. 35. rollero c. the experience of men caring for a partner with multiple sclerosis. j nursing scholarship. 2016;48(5):482-9. 36. schulz r, sherwood pr. physical and mental health effects of family caregiving. j social work educ. 2008;44(sup3):105-13. 37. coleman j, rath l, carey j. multiple sclerosis and the role of the ms nurse consultant. aust nursing j anj, the. 2001;9(3):cu1. 38. sherman t, rapport l, hanks r, ryan k, keenan p, khan o, et al. predictors of well-being among significant others of persons with multiple sclerosis. multiple sclerosis jo. 2007;13(2):238-49. https://doi.org/10.22317/jcms.v6i6.846 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 404 not found 282 j contemp med sci | vol. 6, no. 6, november–december 2020: 282–284 original issn 2413-0516 introduction periodontitis affects the soft and mineralized tissues surrounding the tooth. it is a disease characterized by a decrease in the level of alveolar bone and attachment caused by subgingival biofilm that contains periodontal pathogenic microorganisms.1 the most important part of the treatment of periodontitis is scaling and root planing (srp). srp generally aims to eliminate subgingival microorganisms and remove bacterial endotoxins which penetrated into the cementum surface, thereby controlling the progression of periodontal destruction creating a healthy subgingival enviroment.2 the function of srp is to reduce the bacterial population in the subgingival flora, inflammation, pocket depth, and clinical attachment level.3 however, as the depth of the pocket increases, the effectiveness of these methods may decrease.4 therefore, there is a need for methods to be applied in addition to the srp process performed with ultrasonıc and hand tools. 5 antimicrobial agents have been developed for use with srp to delay recurrence of colonization with existing pathogens, and improved clinical results have been observed in the evaluations made in the treated area.6 one of these antimicrobial agent is chlorhexidine belonging to the biguanide family, and are widely used in dental and medical treatments as an antiseptic agent. it is a broad-spectrum antiseptic effective on a variety of gram positive and gram negative bacteria as well as yeasts and viruses.7 the ability of chlorhexidine (chx) to adhere to the dental pellicle and oral mucosa extends its anti-plaque effect.8 chx mouthwash (0.2%) are widely used before surgical applications, but can also be used as an irrigant in addition to mechanical instrumentation at varying concentrations. besides these effects of chx, there are many side effects. one of the common side effects of chx is brown discoloration of teeth, some restorative materials, and dorsum of the tongue.9 histological studies have shown that chx may lead to delay in tissue healing processes by causing inhibition of gingival fibroblast proliferation depending on the in-vitro dose, and other studies with fibroblast cultures have explained that it can be severely cytotoxic at concentrations between 17 and 100 mm.10,11 ribeiro et al, in their study on rats, showed that the use of chlorhexidine digluconate caused primary dna damage in leukocytes and oral mucosal cells.12 air-polishing devices (apds) are highly effective in removing plaque and extrinsic staining by generating a slurry of pressurized air, water, and abrasive powder. for this purpose, fine-grained (dv90: 63 mm) glycine powder applied directly to the periodontal pocket has been shown to be effective and safe in removing subgingival biofilm from periodontal pockets.13 glycine means sweet in ancient greek. non-essential amino acid and polypeptide are important components of glycine. it is odorless, colorless, non-allergenic, and dissolves quickly in water. also, it has anti-inflammatory, immunomodulatory, and cytoprotective (cell protective) effects.14 in one study, glycine powder air-polishing (gpap) reduced the total viable bacterial counts in periodontal pockets with probing depth (pds) ranging from 3 to 5 mm and to a significantly greater extent than srp using curettes.15 however, no studies air polishing and chx effects in addition to subgingival ultrasonic instrumentation on clinical periodontal parameters: a randomised clinical trial ayse caygur department of periodontolgy, faculty of dentistry, near east university, nicosia, cyprus. corresponding author: ayse caygur (e mail address: ayse.caygur@gmail.com ) (submitted: 04 september 2020 – revised version received: 23 september 2020 – accepted: 11 october 2020 – published online: 26 december 2020) abstract objectives: this study was performed to investigate the efficacy of using glycine powder air-polishing (gpap) or antiseptics (chlorhexidine, chx) adjunctively to scaling and root planing (srp) in the treatment of periodontitis. methods: in this trial, 90 patients (between 28 and 80 years old) who had at least three teeth with 3to 7-mm periodontal pockets were ıncluded from the department of periodontology. the patients were divided into three groups randomly. in the control group, ultrasonic instrumentation was performed with distilled water and hand instrumentation. in the chx group, ultrasonic instrumentation was performed with chx and hand instrumentation. in the gpap group, in addition to srp with ultrasonic performed with distilled water and hand instrumentation. gpap was performed for 10 s per periodontal pocket using a perio-flow device. all treatments were performed in one session. resuls: the scores of plaque index (pi), gingival index (gi), probing depth (pd), bleeding on probing (bop), and clinical attachement level (cal) were decreased in all groups after 1 month, and the results were statistically significant in intergoup comparisions. pi and cal scores were statistically significant in intragroup comparisions in gpap group. altough gi, pd, and bop scores were lower in the gpap group than chx and control group, the differences after 1 month were not statistically significant. conclusion: within the limits of this study, it was seen that srp was effective alone in the treatment of periodontitis. however, using a chx with ultrasonic devices has little beneficial effects on periodontal parameters when compared with control and gpap groups. gpap groups was found more effectively in pi and cal scores. keyword: dental plaque, root planning, chlorhexidine, glycine, periodontal index 283 original air polishing andchx effects in addition to subgingival ultrasonic instrumentation on clinical periodontal parametersayse caygur j contemp med sci | vol. 6, no. 6, november–december 2020: 282–284 have investigated the efficacy of using gpap adjunctively or antiseptics (chx) with srp in the treatment of periodontal parameters. therefore, the aim of this clinical study was to evaluate the effect of using gpap or chx adjunctively with srp on periodontal parameters. materials and methods ninety (90) patients (28–80 years of age) who applied to periodontology department and had periodontal pockets between 3 and 7 mm in at least three teeth were included in this study. patients with known pregnancy, lactation, and systemic disease (diabetes), those who have taken antibiotics in the last 4 months or have used other drugs permanently, and individuals who have undergone any periodontal treatment within 6 months and those with furcha defect were not included. periodontal examination the plaque index (pi),16 gingival index (gi),17 clinical attachment level (cal), pd and bleeding on probing (bop) were measured at baseline and 1 month after treatment by a single calibrated examiner who was not aware of the type of treatment applied. the pi, gi, pd, position of the gingival margin, and bop were evaluated with a periodontal probe at six sites on all teeth. bop was assessed by the percentage of sites that bled after probing. study design this was a single-blinded, computer-randomized, controlled clinical study. written informed consent was obtained from each study participant after all procedures had been fully explained. this study was approved by the ethics committee (2015/33-236). all patients had previously undergone and completed initial periodontal therapy. the patients were divided into three groups. in the control group, ultrasonic instrumentation (piezonmaster 700; electro medical systems, nyon, switzerland) was performed with distilled water and hand instrumentation. in the first group, ultrasonic instrumentation was performed with chx (drogsan, istanbul, turkey, 0.2%) and hand instrumentation. in the second group, in addition to srp with ultrasonic performed with distilled water and hand instrumentation, gpap (air-flow perio powder; electro medical systems) was performed for 10 s per periodontal pocket using a perio-flow device (air-flow master; electro medical systems). all treatments were performed in one session. statistical analysis for all three groups, the mean values of the clinical parameters were calculated. to evaluate the changes over time within each group, one-way repeated anova was used. levene test was used to evaluate the homogeneity of variances of the values of p > 0.05 anova test, if the values of p ≤ 0.05 kruskal–wallis variance analysis was used. a t-test was used for comparison among groups at each time. results all 90 patients were involved in a 1-month study period. the standard deviations and mean of the pi, gi, pd, bop, gr, and cal in the first, second, and control groups are presented in table 1. the scores of pi, gi, pd, bop, and cal were table 1. the means and standard deviations of pi, gi, pd, bop and cal from baseline to the 30th day of treatment in all groups. baseline 1 month pi chx group gpap group control group 0,920,73 1,610,82 0.63 ± 0.74 0,280,40* 0,41 0,52* 0.39 ± 0.58* gi chx group gpap group control group 1.42 ± 0.95 1,56±0,70 1.80 ± 0.78 0.81 ± 0.40* 0.40 ± 0.51* 0.76± 0.71* pd chx group gpap group control group 4.78 ± 0.57 4.72 ± 0.49 4.90 ± 0.68 3.78 ± 0.61* 3.50 ± 0.34* 3.94 ± 0.93* bop chx group gpap group control group 0.57 ± 0.25 0.44 ± 0.11 0.44 ± 0.21 0.13 ± 0.22* 0.04 ±0.08* 0.08 ± 0.19* cal chx group gpap group control group 0.98 ± 0.86 1.62 ± 1.43 0.93 ± 0.71 0.64 ± 0,80* 0,89 ± 1,12* 0.11 ± 0.23* data are presented as mean ± standard deviation. *differences between t0 and t4 were statistically significant in the intergroup comparisons for both groups; p<0.05. decreased in all groups on 1 month and was statistically significant in intergoup comparisions. pi and cal scores were statistically significant in intragroup comparisions in second group. altough gi, pd, and bop scores were lower in the second group than first and control group, the differences on 1 month were not statistically significant. discussion dental plaque in the periodontal pocket and on the root surface was recently shown that cause changes in biological structure. bacterial exotoxins that penetrate the root surface cause changes of antibody complexes, and microbial metabolism.18 the efficacy of periodontal therapy is directly related to the percentage of bacteria in the pocket.19 mechanical debridement is arguably the most important part of treating periodontal disease. studies have shown that the effectiveness of mechanical debridement is superior to measurable indexes. these end points include cal, pd, bop, and alterations in the subgingival microflora.4 due to anatomical limitations, it is not always possible to reach the root surface and perform effective debridement.20 for this reason, new searches have been started. petersilka et al,21 demostrated that, up to 30% of the total surface area of treated roots can be reinfected with pathogenic bacteria, therfore influencing the reoccurence of periodontal disease. yilmaz and bayindir, 22 used essential oils and chx as a cooling agent and found that there were significant reduction in bop, gi, pd, and pi in all groups on 1 month. the difference among the groups were not significant at any point except in bop scores. on the other hand, a similar study by tumer et al,8 there was a significant decrease in all parameters but there were not statistically significant difference between the groups. 284 original air polishing andchx effects in addition to subgingival ultrasonic instrumentation on clinical periodontal parameters ayse caygur j contemp med sci | vol. 6, no. 6, november–december 2020: 282–284 guarnelliet al.,23 demonstrated that the use of chx adjunctive to umi in patients with aggressive periodontitis, indicated no additional efficacy over umi alone. similar with this report, in our study, the reduction of the clinical parameters in the chx group was not statistically significant when compared with the other groups. also, the effectiveness of the antimicrobial agents may be affected due to the contamination of antimicrobial agents with blood and gingival crevicular fluid contents.24 the present study revealed that mechanical instrumentation and gpap had the same effect on pi scores when used in periodontal pockets with moderate pd. similarly, flemmig et al.25 showed that hand instrumentation and gpap had the same effect on pi when used in periodontal pockets with pds of up to approximately 3–5 mm. because the use of low-abrasive powder led to a significantly higher reduction in subgingival bacteria than hand instrumentation, it may be speculated that the clinical outcomes of periodontal maintenance therapy using subgingival air polishing may be equivalent to or even better than the clinical outcomes of conventional modes of debridement.26 in our study, all scores decreased in all groups, but the difference between the groups was not statistically significant exept in pi and cal. pi and cal scores were statistically significant in intragroup comparisions in gpap group. in some studies, it has been shown that it fails to remove subgingival calculus and is effective in bacterial elimination due to its low abrasiveness. although bleeding decreased after both treatments, better results were obtained with glycine.27 on the other hand, it was determined that it caused a decrease in both the removal of the biofilm and the recolonization of bacteria (p. gingivalis) in the applied area. on the contrary, muller et al.28 also found a significant decrease in the pd and bop in their ultrasonic and perio-flow groups when used with srp at 3-month intervals, but no significant difference was found between the two groups. gpap has been shown to be more efficient in debriding deep periodontal sites than srp using curettes. similar to this study, caygur et al.20 found no statistically significant difference between the control group and the gpapl group, although there was a significant decrease in periodontal parameters in the gpap group in their study. conclusion within the limits of this study, it was seen that srp was effective alone in the treatment of periodontitis. however, using a chx with ultrasonic devices has little beneficial effects on periodontal parameters when compared with control and gpap groups. gpap groups was found more effectively in pi and cal scores. acknowledgments none. references 1. american academy of periodontal. glossary of periodontal terms,4th ed. chicago: american academy of periodontology. 2001; 40. 2. nagarakantı s, gunupatı s, chava vk, reddy bvr. effectivess of subgingival irrigation as an adjunt to scaling and root planing in the treatment of chronic periodontitis: a systematic review. jcdr. 2015;9(7):6-8. 3. ioannou i, dimitriadis n, papadimitrious k, et al. hand instrumentation versus ultrasonic debridement in the treatment of chronic periodontitis: a randomized clinical and microbiological trial. j clin periodontol. 2009;36:132-141. 4. cobb cm. clinical significance of non-surgical periodontal therapy: an evidence-based perspective of scaling and root planing. j clin periodontol. 2002;2: 6-16. 5. quirynen m, teughels w, de soete m, et al. topical antiseptics aspects. periodontol 2000. 2002;28:72-92. 6. cosyn, j, sabzevar mm. a systematic review on the effects of subgingival chlorhexidine gel administration in the treatment of chronic periodontitis. j periodontol. 2005;76:1805-13. 7. gunsolley jc. clinical efficacy of antimicrobial mouthrinses. j dent. 2010;38:6-10. 8. tumer h, berberoglu a, caygur a, et al. clinican evaluation of chlorhexidine and essential oils adjunctive effects in subgingival ultrasonıc ınstrumentation on periodontal parameters and halitosis. j essent oils bearing plants. 2019;22(1):169-175. 9. walker cb, karpani k, baehn, p. chemotherapeutics: antibiotics and other antimicrobials. periodontol 2000. 2004;36:146-165. 10. dogan s, gunay h, leyhausen g, et al. effect of low-concentrated chlorhexidine on growth of streptococcus sobrinus and primary human gingival fibroblasts. clin oral investig. 2003;7(4):212-216. 11. boisnic s, ben slama l, branchet-gumila mc, et al wound healing effect of eludril in a model of human gingival mucosa(serbian). rev stomatol chir maxillofac. 2006;107:431-35. 12. ribeiro da, bazo ap, da silva franchis ca, et al. chlorhexidine induces dna damage in rat peripheral leukocytes and oral mucosel cells. j periodontal res. 2004;39(5):358-361. 13. petersilka g, faggon cm, stratmann u, et al. effect of glycine powder airpolishing on the gingiva. j clin periodontol. 2008; 35:324-332. 14. zhong z, wheeler md, li x, et al l-glycine: a novel antiinflammatory, immunomodulatory, and cytoprotective agent. curr opin clin nutr metab care. 2003; 6:229-240. 15. petersılka gj. subgingival air-polishing in the treatment of periodontal biofilm infections. periodontol 2000. 2011;55:124-142. 16. silness j, loe h. periodontal disease in pregnancy ii. correlation between oral hygiene and periodontal condition. acta odontol scand. 1964;22:121-135. 17. loe h, silness j. periodontal disease in pregnancy i. prevalance and severity. acta odontol scand. 1963;21:533-551. 18. khosravi m, baharami zs, atabaki msj. et al. comparative effectiveness of hand and ultrasonic instrumentations in root surface planing in vitro. j clin periodontol. 2004;31:160-165. 19. chaves es, jeffcoat mk, ryerson cc. et al. persistent bacterial colonization of porphymonas gingivalis, prevotella intermedia, and actinobacillus actinomycetemcomitans in periodontitis and its association with alveolar bone loss after 6 months of therapy. j clin periodontol. 2000;27:897-903. 20. caygur a, albaba mr, berberoglu a, et al. efficacy of glycine powder air-polishing combined with scaling and root planing in the treatment of periodontitis and halitosis: a randomised clinical study. 2017;45(3):1168-1174. 21. petersilka gj, ehmke b, flemming tf. antimicrobial effects of mechanical debriment. periodontol 200. 2002;28: 56-71. 22. yilmaz hg, bayındır h. clinical evaluation of chlorhexidine and essential oils for adjuctive effects in ultrasonic instrumentation of furcation involvements: a randomized control trial. j clin periodontol. 2011;38:637-643 23. guarnelli me, franceschetti g, manfrini r. et al. adjuctive effect of chlorhexidine in ultrasonic instrumentation of aggresive periodontitis patients: a pilot study. j clin periodontol. 2008;35:333-341. 24. preus hr, koldsland oc, aass an, et al. the plaque-and-gingivitis-inhibiting capacity of a commercially available essential oil product. a parallel, splitmouth, single blind,randomized, placebo-controlled clinical study. acta odontol scand. 2013;71:1613-19. 25. flemming tf, hetzel m, topoll h, et al. subgingival debridement efficacy of glycine powder air polishing. j periodontol. 2007;78:1002-1010. 26. petersilka gj, tunkel j, barakos k, et al. subgingival plaque removal at interdental sites using a low abrasive air polishing. j periodontol. 2003;74:307-311. 27. moene r, decaillet f, andresan e, et al. subgingival plaque removal using new air-polishing device. j periodontol. 2010; 81:79-88. 28. müller n, moene r, cancela ja, et al. subgingival air polishing with eryhriol during periodontal maitenanace. j clin periodontol. 2014;41:883-889. https://doi.org/10.22317/jcms.v6i6.883 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 191 letter to editor issn 2413-0516 j contemp med sci | vol. 6, no. 4, july-august 2020: 191–192 dear editor in chief the journal of contemporary medical sciences vitamin d, a pre-hormone, regulates and/or affects multiple functions such as immune-, coagulation-, cardiovascular-, central nervous system, inflammation, cancer rates, deep-vein-thrombosis, sleep-apnea, and respiratory tract infections (rtis).1-8 the role of vitamin d in reducing rti is outstanding as reported by a meta-analysis.2 our first reference is a must read document with “over 150 references” regarding the rti reduction in patients with vitamin d level (vdl) >40-ng/ml versus those <20-ng/ml.1 vdl and covid severity has been well-shown in a very recent study.9 the two most important causes of covid-19 severity, needing ventilators and possible death are: 1. cytokine storm and complement dysfunction ending on a death spiral of respirator and drowning in their fluids.2-6 2. hyper-coagulation state ending with multiple peripheral system diseases, such as cardiomyopathy or encephalopathy.7,8 vitamin d plays a major role in both processes.2-9 over 5,000 patients of neuro-ophthalmology department had vdl checked from 2010 to 2020; 66% had deficiency (<20-ng/ml), prevalence changed to 83% if <30-ng/ml was chosen, and 93% when <35-ng/ml was set as deficient. in between 2010 and 2012 after patients stopped vitamin d once reaching normal levels, vdd recurred in all cases after 4 month follow-ups. table 1 shows suggested cut-offs accepted by most endocrine societies. vdd is multifactorial and endemic worldwide. insult to vitamin d may come from artificial coloring and flavoring found in processed foods, soft drinks, lack of sun exposure, any chronic illness such as diabetes, hypertension, chronic kidney disease, obesity, and more. heat (avoiding sun exposure) and color of skin (less uv effect for vitamin d in darker skins) cause african countries to have higher vdd than the scandinavian nations. with inadequate sun exposure and constant poisoning of vitamin d by food additives and chronic illnesses, lifelong vitamin d supplementation (llvds) for urban residents is a must. we have only seen a few good vdl (>40-ng/ml without vitamin d supplement), in patients that live in rural areas who consume their own farms’ food including milk with no soft drink usage. the majority of people who consume processed foods encounter vdd. in the latter subgroup, many clinician stop supplementation when vitamin d reaches normal levels and this will almost always result in vdd recurrence. in the neuro-ophthalmology department, we have been using 70–100-iu of vitamin d3/kg/day for maintenance since 2010. we used 70-iu/kg/day in patients with normal eye exams and 100-iu/kg/day for retinal and optic neuropathy patients. after supplementation all patients had vdl >40ng/ml with some in-between 60 and 89, and none over 90 in the last 9 years. in a subset of over 500 patients on continuous 1–8-year-treatment/ follow-up, we have not seen even one case of toxicity. since covid-19 outbreak, we have had 21 patients, all with vdl >40ng/ml (including 2 health-care workers and several with chronic disease, like diabetes, hypertension and obesity), who were on regular follow-up for their eye disease informed us that, they had covid-19 but the hospitalization period was all under 4 days. this finding prompted us recommending this dosage for all other cases in the hospital. subsequently, we started supplementation of vitamin d as routine care from early june 2020 in all sars-cov-2+ and covid-19 patients (sars-cov-2+ with typical signs and symptoms that needed admission) in the iranian red-crescent hospital in dubai, a dramatic and complete resolution of icu admissions was observed in the last 8 weeks. we cannot overemphasize the role of vitamin d in controlling all infectious diseases especially in covid-19.1 we had no patients with initial vitamin d levels of >40 that required more than 2–3 days of hospitalization, hence no cytokine storm, hypercoagulation, nor complement deregulation occurred. prior to this change, we had several deaths of covid-19 patients on respirators. our vitamin d supplementation (in addition to the required therapies such as chloroquin, remdesivir or others) was: 1. 300,000-iu, im vitamin d3 once, followed by: 2. 100-iu/kg/day. 3. we know that, one bullous injection is not enough and patient must continue with daily dosage for maintenance. this regimen is very safe and very far from toxicity. 4. it is also a known fact that hydroxychloroquin decreases vdl. 5. checked vitamin d on oral vitamin d3 in hospitalized patients but 1 week after im dosage. 6. if patient recovers in shorter period they are discharged on daily dosage and vdl is checked in 3 months but 6 days after last dosage. suggested role of vitamin d supplementation in covid-19 severity parviz afshar1, mohammad ghaffaripour2, hamid sajjadi3 1 hospital director, iranian hospital dubai, dubai, uae2 icu director, iranian hospital dubai, dubai, uae 3 neuro-ophthalmology director, iranian hospital dubai, dubai, uae corresponding author: hamid sajjadi (email: hsajjadi@yahoo.com) table 1. typical endocrine societies guideline. very low <20 ng/ml insufficient 21-29 ng/ml sufficient 30-60 ng/ml ideal 40-60 ng/ml considered safe up to 100 ng/ml toxic >120-150 ng/ml 192 letter to editor suggested role of vitamin d supplementation in covid-19 severity parviz afshar j contemp med sci | vol. 6, no. 4, july-august 2020: 191–192 because vitamin d3 is fat soluble, raising its level is definitely weight-related. that is why in many studies with a constant dose it is difficult to raise vdl in obese patients. this may be the main cause of cytokine storm and death in young healthy obese persons. although a daily dose of 70–100-ng/ml/kg is preferred, since the 50,000-iu capsule of vitamin d3 supplement is widely available, we would recommend that individuals between 50 and 100-kg to take 50,000-iu of vitamin d3 weekly in order to protect against cytokine storm and hypercoagulation syndromes of covid-19 and other respiratory viruses. under 50 and over 100-kg need the daily calculated dose. for other patients/healthy individuals llvds for all ages: <30-ng/ml 1. vitamin d3, 300,000-iu one im injection. 2. vitamin d3, 70–100 iu/kg/day. for example, at least: 10-kg =700-iu-daily, 70-kg =5,000-iu-daily, 100-kg = 7,000-iu-daily. 30-40 ng/ml 1. vitamin d3, 70–100 iu/kg/day. >45-ng/ml: no additional treatment needed, recheck vitamin d in few days for possible error and if in the same range, then recheck every 4 months. the correct time for checking vdl is 6 days after last dose of vitamin d3 to avoid artificial high levels. unfortunately, many colleagues are afraid of an extremely-rare vitamin d toxicity (vdt). vdt is reported in usage over 40,000-iu/day.10 there are 88 diseases including sleep disorder that may resolve or reduce severity with vdl of 60–80-ng/ml.4 we achieved this by using 100-iu/kg/day in our patients and with llvds, without vdt. because of recently available scientific evidence and reported numerous new functions of this pre-hormone, we would like to propose changing the vdl to 40–100-ng/ml as normal and consider below 40 as deficient. references: 1. https://www.mdpi.com/2072-6643/12/4/988 2. https://www.bmj.com/content/356/bmj.i6583 3. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6164284/ #:~:text=multiple%20studies%20have%20shown%20that, for%20cancer%20prevention%20and%20therapy 4. vitamindwiki.com 5. https://europepmc.org/article/ppr/ppr149846 6. https://www.jstage.jst.go.jp/article/jnsv/63/3/63_155/_pdf 7. https://www.mdpi.com/2218-273x/9/11/649/xml 8. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4069050/ 9. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7455115/ 10. https://www.merckmanuals.com/professional/nutritional-disorders/ vitamin-deficiency-dependency-and-toxicity/vitamin-d-toxicity? this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i4.822 113 original issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 113 – 115 introduction placenta accreta is a potentially life-threatening complication of pregnancy characterized by implantation that occurs when placental trophoblasts invade into the uterine wall.1 placenta accreta precise pathogenesis is unknown. a suggested hypothesis involves low deciduous development, excessive invasion of trophoblastic, or a combination of both.2 based on the depth of attachment and invasion into the muscular layers of the uterus, three grades of irregular placental attachment are defined: accreta chorionic villi, rather than being confined within the decidua basalis, are connected to the myometrium, increta: invade chorionic villi into the myometrium, percreta: chorionic villi enter via the perimetrium (uterine serosa). placenta accreta is associated with an elevated risk of excessive bleeding at the time of attempted vaginal delivery due to irregular attachment to the myometrium. there is a constant need for blood supplies to be transfused, and surgical uterine removal (hysterectomy) is often important to control life-threatening bleeding.3,4 previous cesarean delivery, uterine instrumentation, and intrauterine scarring are risk factors for placenta accreta, both of which can be associated with disruption or absence of decidua basalis, as well as placenta previa, obesity, maternal age over 35, grand multiparty, and persistent miscarriage.5 6 methodology a retrospective study among pregnant women diagnosed with placenta accreta who undergo a cesarean section (c/s) at maternity wards in baghdad city’s teaching hospitals. a non-probability purposive sample consists of 410 pregnant women. the data were collected from patient records in the statistical department of the maternity hospitals, for the last 3 years (january/2018 to december/2020). this questionnaire was composed of three parts, part1: sociodemographic characteristics include: age, level of education of the mother, women’s occupation, blood groups, monthly income, smoking, bmi, rh factor, and residency. part 2: obstetrical and past medical history includes: gravidity, parity, abortion, gestational age, mode of the previous delivery, gestational age in weeks (a pregnancy with which placenta accreta occurred, interval between the last c/s and the current pregnancy. anemia, heart disease, hypertension, diabetes, goiter, viral hepatitis, food allergy, drugs allergy, and do not suffer from diseases. part 3: placental complications and risk factors for placenta accreta. results table 1 shows that pregnant women are with age 32±6 years in which 50.7% of them is with age 30–39 years and 30.3% are with age group 20–29 years. the blood group presents that 46.1% of pregnant women are of blood group “o”, 23.1% of them are of “ab” group, 19.3% are of “b” blood group, and only 11.5% are of “a” blood group. the rh factors refers that 76.6% of pregnant women are with positive rh factors while 23.4% are with negative factor. regarding occupation, 71.2% of pregnant women are housewives and only 22.4% are governmental employee. the residency variable shows that 65.4% of pregnant women residents at urban area and 21.5% are resident at suburban area. the smoking status shows that 88.5% of pregnant women are not smoking and only 11.5% of them are smoking. table 2 reveals that pregnant women having history of 5–7 gravidity (43.4%) in which average refers to 6±2 pregnancies. the table shows that 45.4% of pregnant women having history of 1–3 lived children and 38% having 4–6 lived child. all of pregnant women were getting c/s as a mode of delivery (100%), 45.9% of them are having 4–6 c/s and 32.4% are having 1–3. more than half of pregnant women show they have no abortion but 25.6% of them have one abortion previously. the gestational age by ultrasound at birth among pregnant women refer to third semester among most of them (99%). the gestational age by ultrasound at birth refers to 28–32 weeks among most of the pregnant women (99%). the interval between last c/s and current pregnancy refers to 2 years among the highest percentage of pregnant women (37.8%). this table depicts that there is high significant relationship between placenta accreta and history of c/s among correlation between placenta accreta and history of cesarean section among iraqi pregnant women: retrospective study nour ali abdulla*, hawraa hussein ghafel* maternal and neonate nursing department, college of nursing, university of baghdad, baghdad, iraq. *correspondence to: hawraa hussein ghafel (e-mail: hawraah@conursing.uobaghdad.edu.iq) (submitted: 24 january 2021 – revised version received: 09 february 2021 – accepted: 08 march 2021 – published online: 26 april 2021) abstract objective to identify demographic characteristics of pregnant women with placenta accreta and to find out the correlation between placenta accreta and history of cesarean section (c/s). methods a retrospective study was conducted at maternity wards in baghdad city’s teaching hospitals. the study sample consists of 410 pregnant women diagnosed with placenta accreta who undergo a c/s. retrospective study for the last 3 years (january/2018 to december/ 2020). the data were collected from patient records in the statistical department of the maternity hospitals. results the findings of the study shows that a highly significant relationship between placenta accreta and history of c/s among pregnant women as indicated by a strong positive correlation at p-value= 0.001. conclusions there is a correlation between placenta accreta and history of cesarean section among iraqi pregnant women keywords placenta accreta, cesarean section, pregnant women, iraq 114 original correlation between placenta accreta and history of cesarean section among iraqi pregnant women nour ali abdulla, hawraa hussein ghafel j contemp med sci | vol. 7, no. 2, march-april 2021: 113 – 115 pregnant women as indicated by strong positive correlation at p-value = 0.001. discussion the result in the table 1 shows that the highest percentage of pregnant women with placenta accreta is 50.7% among the age group 30–39 years. also, the present study found a high significant relationship among age and incidence of placenta accreta at p-value= 0.001. the results show that the highest percentage of pregnant women (46.1%) of their blood group is “o”, and this result is consistent with that obtained with salman et al.7 who found it was 56% with blood group (o) and this could be due to race and genetic. the majority of the sample (71.2%) were housewives, and 22.4% of them are governmental employees. the women’s occupation have a role in increasing placenta accreta during pregnancy especially exhaustion work and may have an effect on her pregnancy and may lead to antepartum hemorrhage, low birth weight, and preterm labor. regarding residency, the highest percentage (65.4%) of the study sample table 1. distribution of pregnant women according to their general information. list characteristics f % 1 age (m±sd=32±6) ≤19 years 11 2.7 20–29 year 124 30.3 30–39 year 208 50.7 40≤ year 67 16.3 total 410 100 2 blood group a 47 11.5 b 79 19.3 o 189 46.1 ab 95 23.2 total 410 100 3 rh factor positive 96 23.4 negative 314 76.6 total 410 100 4 occupation housewife 292 71.2 employee 92 22.4 retired 2 0.5 student 24 5.9 total 410 100 5 residency urban 268 65.4 rural 54 13.2 suburban 88 21.5 total 410 100 6 smoking no 363 88.5 yes 47 11.5 total 410 100 f: frequency, %: percentage, m: mean, sd: standard deviation. table 2. distribution of pregnant women according to their obstetrical history. list history f % 1 gravid a (m±sd=6±2) 2 – 4 143 34.9 5 – 7 178 43.4 8 – 10 74 18 11 ≤ 15 3.7 total 410 100 2 para (m±sd=4±2) none 3 0.7 1 – 3 186 45.4 4 – 6 156 38 7 – 9 52 12.7 10 ≤ 13 3.2 total 410 100 3 mode of previous delivery normal delivery 0 0 cesarean section 410 100 total 410 100 4 number of previous delivery 1 – 3 133 32.4 4 – 6 188 45.9 7 – 9 67 16.3 10 – 12 22 5.4 total 410 100 5 number of abortion none 223 54.4 1 105 25.6 2 45 11 3 27 6.6 4 + 10 2.4 total 410 100 6 pregnancy trimester (pregnancy with placenta accrete) first semester 0 0 second semester 4 1 third semester 406 99 total 410 410 7 gestational age by ultrasound at birth 1 – 12 week 0 0 13 – 27 week 4 1 28 – 38 week 406 99 total 410 100 8 time between last cesarean and current pregnancy (m±sd=2±1) none 1 0.2 1 years 81 19.8 2 years 155 37.8 3 years 96 23.4 4+ years 77 18.8 total 410 100 f: frequency, %: percentage. 115 original correlation between placenta accreta and history of cesarean section among iraqi pregnant womennour ali abdulla, hawraa hussein ghafel j contemp med sci | vol. 7, no. 2, march-april 2021: 113 – 115 were living in an urban area. this result does not agree with salman et al7 who found the pregnant women with placenta accreta live in rural areas (60%). women who live in rural have low awareness and low commitment about prenatal visits and have poor education about signs and symptoms of accreta. the result shows that the majority of the sample (88.5%) of pregnant women is not smoker but there is a highly significant relationship between smoking and placenta accreta at p-value= 0.001. smoking is a biologically plausible risk factor in contribution to compensatory placenta hypertrophy. table 2 indicates that result the highest percentage (43.4%) of study sample were multigravida5-7 pregnancies, there is a significant relationship between gravidity and placenta acreta at p-value= 0.025. the lack of health education about family planning and the number of gravidity, and refusing use of family planning methods (especially in rural areas) lead to increased number unplanned pregnancy. regarding mode of previous delivery, the highest percentage (100%) of the study sample were had a previous history of c/s. this result agreed with abdelfatah et al8 who found that 100% of pregnant women were getting c/s as a mode of delivery. most of pregnant women undergo cesarean delivery because of their fear of natural childbirth pain, they believe the cesarean delivery is less painful than a normal birth. regarding of the number of previous delivery, 45.9% of them have 4–6 multiparty and 32.4% of them is having 1–3 cesarean delivery. these results show that there is a significant association between the number of c/s and incidence of placenta accreta. the result indicates that 54.4% of the study sample did not have abortion, while 25.6% of them had one previous abortion. sofiah and fung9 who said that a number of previous curettage increase the incidence of placenta accreta. the highest percentage of the study sample (99%) were in 28-40 weeks of gestational age (pregnancy with placenta accreta). this result refers to most of pregnant women with placenta accrete at the third trimester of their pregnancy. mohamed and ahmed10 found that the gestational age more than 30 weeks of women with placenta accreta. the majority of patients with placenta accreta was diagnosed with placenta accreta at the moment of birth in iraqi, there is no previous diagnosis during prenatal visits. regarding the detecting of gestational age by using of ultrasound at among pregnant women refers to most of pregnant women with placenta accreta were diagnosed at third semester (99%). placenta accreta is detected at the moment of birth and not during prenatal visits in iraq because most of women were not committed in prenatal visits. the results indicate the interval between the last c/s and current pregnancy is 2 years, the highest percentage of pregnant women (37.8%) have 2 years interval between one delivery and another. women’s need to increase their awareness about family planning for regulating the intervals between pregnancies to avoid incidence and complications of placenta accreta. table 3 shows that there is a highly significant relationship between placenta accreta and history of c/s among pregnant women as indicated by a strong positive correlation at p-value= 0.001. fitzpatrick et al11 who found a relationship between placenta accreta and history of c/s. performing repeated cesarean deliveries increases the chances of placenta accreta due to a wound in the uterine muscle in every c/s, causing abnormality in the area of placenta implantation and preventing it from attaching normally and causing its implantation into the uterine muscle rather than implant in to the myometrium. conclusion there is a correlation between placenta accreta and the history of c/s among pregnant women. conflict of interest none. references 1. eshkoli, t, weintraub, ay, sergienko, r, sheiner, e. placenta accreta: risk factors, perinatal outcomes, and consequences for subsequent births. am j obstet gynecol, 2013;208(3):219-e1. 2. garmi, g, goldman, s, shalev, e, salim, r. the effects of decidual injury on the invasion potential of trophoblastic cells. obstet gynecol, 2011;117(1):55–59. 3. smith, zl, sehgal, ss, van arsdalen, kn, goldstein, is. placenta percreta with invasion into the urinary bladder. urol case rep, 2014;2(1):31–32. 4. cahill, ag, beigi, r, heine, rp, silver, rm, wax, jr, gynecologists, ac of o. placenta accreta spectrum. am j obst gynecol, 2018;219(6):b2–b16. 5. gielchinsky, y, rojansky, n, fasouliotis, sj, ezra, y. placenta accreta— summary of 10 years: a survey of 310 cases. placenta, 2002;23(2–3): 210–214. 6. practice, c. on o. acog committee opinion. placenta accreta. number 266, january 2002. american college of obstetricians and gynecologists. int j gynaecol obstet off organ int feder gynaecol obstet, 2002:77(1):77. 7. salman, st, mohammed, ah, noaman, ng, ibrahim, hk. prevalence of placenta accreta in diyala governorate during 2010-2012 years. med j tikrit, 2013;19(2):286–295. 8. abdelfatah, ea, awad, eem, abd-eldaym, tm, ali, zh. outcome of patients with placenta accreta at el shatby maternity university hospital. open j obstet gynecol, 2017;7(7):725. 9. sofiah, s, fung, yc. placenta accreta: clinical risk factors, accuracy of antenatal diagnosis and effect on pregnancy outcome. med j malay, 2009;64(4):298–302. 10. mohamed, ah, ahmed, imm. incidence of placenta accreta and its complications in cases of previous cesearean sections with placenta previa anterior at al hussein university hospital. egypt j hosp med, 2018;73(3):6310–6315. 11. fitzpatrick, ke, sellers, s, spark, p, kurinczuk, jj, brocklehurst, p, knight, m. incidence and risk factors for placenta accreta/increta/percreta in the uk: a national case-control study. plos one, 2012;7(12):e52893. table 3. correlation between placenta accreta and history of cesarean section among iraqi pregnant women (n=80). correlation placenta accreta cesarean section placenta accreta pearson correlation 1.00 0.216** p-value -0.001 cesarean section pearson correlation 0.216** 1.00 p-value 0.001 -**correlation is significant at the 0.01 level (2-tailed). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.961 167j contemp med sci | vol. 7, no. 3, may-june 2021: 167–170 original risk factors associated with poor glycemic control in patients with type two diabetes mellitus in zakho city brisik h. rashad1,*, basheer a. abdi1, ibrahim a naqid1, nawfal r. hussein1, zubaida a. ibrahim2, jodi a. rashad2, shangist m. saleem3, avan s. saleh3, alaa a. mustafa3 1department of biomedical sciences, college of medicine, university of zakho, kurdistan region, iraq. 2college of medicine, university of duhok, kurdistan region, iraq. 3college of medicine, university of zakho, kurdistan region, iraq. *correspondence to: brisik h. rashad (email: brisk.rashad@uoz.edu.krd) (submitted: 13 april 2021 – revised version received: 24 april 2021 – accepted: 29 may 2021 – published online: 26 june 2021) introduction dm is a chronic condition caused by relative lack of insulin due to impaired insulin secretion or insulin resistance.1 dm is the ninth major cause of death and with obesity, they represent the biggest epidemics in human history.2, 3 the burden of dm is represented by its enormous breadth as dm has been quadrupled in the past three decades.2 diabetes was seriously underrated as a global public health issue; it was proposed that the total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030.4 however, in 2015, there were already 415 million people with diabetes, far above what was predicted in 2000 for 30 years later according to international diabetes federation.5 a better understanding of the traditional drivers “risk factors” of the diabetes is required in order to combat the disease.3 there are considerable number of risk factors associated with poor glycemic control including age; sex; family history; smoking history; hypertension; chronic diseases such as ischemic heart disease, bronchial asthma and thyroid diseases; raised body mass index and raised waist circumference.6-10 few studies have been conducted in neighboring countries to iraq to assess the risk factors associated with poor glycemic control.11-13 the aim of this study was to investigate the prevalence of poor glycemic control in zakho city in kurdistan region of iraq and to explore the modifiable risk factors that may help controlling the disease. materials and methods study design and setting this retrospective observational study was conducted in zakho city, kurdistan region of iraq. in the city, diabetes center was established in 2010. all patients with diabetes who receive treatment from governmental hospitals were registered in the center. patients in this study, we recruited patients with known history of type 2 diabetes (t2dm) receiving oral anti-diabetic medications. those patients were registered in zakho diabetes center with regular visits. demographic data were collected via face-to-face interview. all patients who were 18 years or older, with type 2 diabetes, registered in the center for at least 12 months, and agreed to participate were included in the study. controlled diabetes was defined as hba1c below 7%.14 anthropometric assessment the measurement of anthropometric indices was conducted by trained personnel. the measurement of participants weight was conducted by a balance while the height of the participants was measured by a wall-fixed tape measure. then, the waist circumference was measured by a plastic tape measure. the waist circumference was considered at the narrowest level between the lowest rib and iliac crest. lab measurements plasma glucose was determined using colorimetric enzymatic method with glucose oxidase. hba1c concentrations were measured in whole blood samples using high performance liquid chromatography. hyperlipidemia was confirmed by history taking. uncontrolled diabetes was defined as hba1c of 7% or more. abstract objectives: the aim of this study was to investigate the prevalence of poor glycemic control in zakho city and to explore the modifiable risk factors that may help controlling the disease. methods: we recruited patients with known history of diabetes receiving oral anti-diabetic medications. those patients were registered in zakho diabetes center with regular visits. the measurement of anthropometric indices was conducted by trained personnel. plasma glucose was determined using colorimetric enzymatic method with glucose oxidase. hba1c concentrations were measured in whole blood samples using high performance liquid chromatography. results: in this study, 520 patients were recruited. the average age of the patients was 56.92 ± 9.62. among those, 190 were male. the blood sugar was controlled in 122 (23.4%) patients. we found a significant association between sex and hba1c level (p = 0.000; or = 0.4796; ci = 0.3175–0.7243). in addition, waist circumference was significantly associated with hba1c levels (p = 0.018; or = 1.02; ci = 1.0031–1.0373). conclusions: the vast majority of the patients had uncontrolled diabetes. we found that sex and waist circumference were risk factors for uncontrolled diabetes. any diabetes controlling program should focus on those two factors. keywords: risk factors, diabetes mellitus, poor glycaemic control, zakho city issn 2413-0516 168 j contemp med sci | vol. 7, no. 3, may-june 2021: 167–170 risk factors associated with type 2 diabetes mellitus original b.h. rashad et al. ethics the study protocol and informed consent were approved by the research and ethics committee of the college of medicine, zakho university, kurdistan region of iraq. informed consent was obtained from all recruited subjects. statistical analysis statistical analysis was conducted using minitab 18. linear regression was used to investigate the relationship between variables and outcomes when the variables were continuous while chi square test was used for categorical data. p value of <0.05 value was considered as significant. results patients in this study, 520 patients with t2dm were recruited. the average age of the patients was 56.92 ± 9.62. among those, 190 were male. the blood sugar was controlled in 122 patients (table 1). amongst our patients, 65.96% had a family history of t2dm. hypertension (ht) was found in 53.07% of our recruited samples. out of 520 patients, 13.46% were smokers. factor affecting sugar level then, we studied the factors affecting blood sugar levels. we found a significant association between sex and hba1c level. our results showed that males were at higher risk of uncontrolled diabetes (p = 0.000; or = 0.4796; ci = 0.3175– 0.7243) (table 2). additionally, waist circumference was significantly associated with hba1c levels (p = 0.018; or = 1.02; ci = 1.0031–1.0373). other variables did not show significant influence on hba1c levels (table 2). other risk factors did not show any significant impact on hba1c levels. discussion to prevent organ damage and other complication of diabetes, glycemic control remains the main therapeutic objective.15 it was previously shown that diabetic patients who control their hba1c below 7% will have less microvascular diabetic complications.14 age, sex, waist circumference, smoking history, family history, body mass index and hypertension were shown to play role in the control of hba1c.6,9,10,16,17 studying risk factors associated with poor glycemic control is crucial for the control of diabetes in any society. therefore; this study was conducted to check the rate of t2dm with good glycemic control and risk factors associated with poor glycemic control. in this study, the mean hba1c was 8.19 ± 1.6 which was comparable to what was found by marilia et al.18 in this study, 50.19%, 23.65% and 26.15% of recruited patients had hba1c < 8.0%, ≥ 8.0 to < 9.0%, and ≥ 9.0%, respectively. again these results were comparable to marilia et al., results.18 additionally, 23.46% of the patients were having their hba1c being controlled which was comparable to a study performed in saudi arabia.19 in studies conducted in turkey, sudan and tanzania, 32.5%, 28% and 30% of the patients showed good glycemic control, respectively.20-22 this discrepancy in hba1c control may be related to cultural and genetic variations. in this study, male to female ratio was 0.57 [male 36.5%, female 63.5%]. this is in contrast to a study that was conducted in the usa and tanzania where the male to female ratios were 1.15, and 1.5, respectively.23 in contrast to a previous study from iraq,13 in our study, males were at a statistically significant increased risk of suboptimal t2dm control in comparison to females with, which was also noticed in a study conducted in the usa,23 while in a study conducted in india, male were predominant and a significantly higher risk of poor table 2. factor affecting hba1c levels factors controlled uncontrolled p value or ci 95% age (average ± std) 55.36 ± 10.52 57.25 ± 9.29 0.057 1.0208 0.9992–1.0428 bmi (average ± std) 30.59 ± 5.49 31.51 ± 5.57 0.102 1.0319 0.9931–1.0723 waist circumference (average ± std) 104.4 ± 12.98 107.42 ± 12.58 0.018 1.0200 1.0031–1.0373 sex (female) 61/122 (50.00%) 269/398 (67.58%) 0.000 0.4796 0.3175–0.7243 ihd 11/122 (9.01%) 58/398 (14.57%) 0.100 1.7214 0.8728–3.3952 asthma 2/122 (1.63%) 9/398 (2.26%) 0.667 1.3882 0.2959–6.5128 thyroid dysfunction 3/122 (2.45%) 11/398 (2.76%) 0.854 1.1275 0.3094–4.1083 hyperlipidemia 39/122 (31.96%) 154/398 (38.69%) 0.175 1.3432 0.8732–2.0662 family history 75/122 (61.47%) 268/398 (67.33%) 0.235 1.2919 0.8484–1.9672 smoking 22/122 (18.03%) 48/398 (12.06%) 0.090 0.6097 0.3482–1.0677 ht 60/122 (49.18%) 216/398 (54.27%) 0.325 1.2264 0.8171–1.8406 table 1. characteristics of patients recruited in the study variable no % sex (female) 330 63.46% ihd 69 13.26% asthma 11 0.021% thyroid 14 0.026% hyperlipidemia 193 37.11% family history 343 65.96% smoking 70 13.46% ht 276 53.07% 169j contemp med sci | vol. 7, no. 3, may-june 2021: 167–170 b.h. rashad et al. original risk factors associated with type 2 diabetes mellitus glycemic control was associated with females.6 these variations may be in part related to genetic variation and discrepancy of the predominant sex in one study upon the other. other risk factor which was at statistically significant association with raised hba1c% was the central obesity represented by waist circumference, while body mass index was statistically not associated with poor glycemic control, this was consistent to a study done in india by rohit et al.,24 to explain the variation between bmi and waist circumference, it has been suggested that age-related increases in total body fat and visceral adiposity compromise the reliability of bmi as a sensitive marker of adiposity in older age groups25. in another study, kayar et al., study20 found that both bmi and waist circumference were statistically associated with poor glycemic control. in contrast to a previous study in iraq where younger age was associated with an increased risk of poor glycemic control,13 in our study there was a trend that age increases the chance of uncontrolled diabetes, though this was not statistically significant. in our study, hypertension, thyroid dysfunction, asthma and ischemic heart disease, hyperlipidemia and family history and smoking history were not statistically associated with poor glycemic control. this in in contrast to other studies where these factors were found to play an important role in controlling diabetes.6, 13, 21, 22 this is difficult to explain and more studies are needed with larger sample size to explore the role of these factors in controlling diabetes. to conclude, out of 520 patients, 377 (72.5%) patients were having hba1c% of equal or more than 7% reflecting uncontrolled diabetes. we found that sex and waist circumference are risk factors for uncontrolled diabetes. any diabetes controlling program should focus on those two factors. conflicts of interest the authors declare that there is no conflicts of interest. acknowledgment we would like to thank the staff of diabetes centre/zakho city for their kind help and support. funding/support the authors received no financial support for the publication of this article. authors’ contribution all authors equally contributed to designing the study, analyzing data, and writing the paper. further, all authors approved the final draft and the authors’ order.  references 1. diagnosis and classification of diabetes mellitus. diabetes care. 2013;36(supplement 1):s67-s74. 2. zheng y, ley sh, hu fb. global aetiology and epidemiology of type 2 diabetes mellitus and its complications. nature reviews endocrinology. 2017;14:88. 3. zimmet pz. diabetes and its drivers: the largest epidemic in human history? clin diabetes endocrinol. 2017;3:1. 4. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes. diabetes care. 2004;27(5):1047. 5. cho nh, shaw je, karuranga s, huang y, da rocha fernandes jd, ohlrogge aw, et al. idf diabetes atlas: global estimates of diabetes prevalence for 2017 and projections for 2045. diabetes research and clinical practice. 2018;138:271-81. 6. haghighatpanah m, nejad asm, haghighatpanah m, thunga g, mallayasamy s. factors that correlate with poor glycemic control in type 2 diabetes mellitus patients with complications. osong public health res perspect. 2018;9(4):167-74. 7. peng k, chen g, liu c, mu y, ye z, shi l, et al. association between smoking and glycemic control in diabetic patients: results from the risk evaluation of cancers in chinese diabetic individuals: a longitudinal (reaction) study. journal of diabetes. 2018;10(5):408-18. 8. zhu h-t, yu m, hu h, he q-f, pan j, hu r-y. factors associated with glycemic control in community-dwelling elderly individuals with type 2 diabetes mellitus in zhejiang, china: a cross-sectional study. bmc endocr disord. 2019;19(1):57. 9. black mh, anderson a, bell ra, dabelea d, pihoker c, saydah s, et al. prevalence of asthma and its association with glycemic control among youth with diabetes. pediatrics. 2011;128(4):e839-e47. 10. ogbonna su, ezeani iu, okafor ci, chinenye s. association between glycemic status and thyroid dysfunction in patients with type 2 diabetes mellitus. diabetes metab syndr obes. 2019;12:1113-22. 11. alzaheb ra, altemani ah. the prevalence and determinants of poor glycemic control among adults with type 2 diabetes mellitus in saudi arabia. diabetes metab syndr obes. 2018;11:15-21. 12. esteghamati a, larijani b, aghajani mh, ghaemi f, kermanchi j, shahrami a, et al. diabetes in iran: prospective analysis from first nationwide diabetes report of national program for prevention and control of diabetes (nppcd-2016). scientific reports. 2017;7(1):13461. 13. mansour aa, alibrahim nty, alidrisi ha, alhamza ah, almomin am, zaboon ia, et al. prevalence and correlation of glycemic control achievement in patients with type 2 diabetes in iraq: a retrospective analysis of a tertiary care database over a 9-year period. diabetes & metabolic syndrome: clinical research & reviews. 2020;14(3):265-72. 14. selvin e, coresh j, golden sh, boland ll, brancati fl, steffes mw. glycemic control, atherosclerosis, and risk factors for cardiovascular disease in individuals with diabetes. diabetes care. 2005;28(8):1965. 15. kakade aa, mohanty ir, sandeep r. assessment of factors associated with poor glycemic control among patients with type ii diabetes mellitus. integr obes diabetes. 2018;4(3). 16. peng k, chen g, liu c, mu y, ye z, shi l, et al. association between smoking and glycemic control in diabetic patients: results from the risk evaluation of cancers in chinese diabetic individuals: a longitudinal (reaction) study. journal of diabetes. 2018;10(5):408-18. 17. zhu h-t, yu m, hu h, he q-f, pan j, hu r-y. factors associated with glycemic control in community-dwelling elderly individuals with type 2 diabetes mellitus in zhejiang, china: a cross-sectional study. bmc endocr disord. 2019;19(1):57. 18. gomes mb, tang f, chen h, cid-ruzafa j, fenici p, khunti k, et al. 1279-p: socioeconomic factors associated with poor glycemic control in people with type 2 diabetes: the discover study. am diabetes assoc; 2019. 19. alramadan mj, magliano dj, almigbal th, batais ma, afroz a, alramadhan hj, et al. glycaemic control for people with type 2 diabetes in saudi arabia an urgent need for a review of management plan. bmc endocr disord. 2018;18(1):62. 20. kayar y, ilhan a, kayar nb, unver n, coban g, ekinci i, et al. relationship between the poor glycemic control and risk factors, life style and complications. 2017. 170 j contemp med sci | vol. 7, no. 3, may-june 2021: 167–170 risk factors associated with type 2 diabetes mellitus original b.h. rashad et al. 21. kamuhabwa ar, charles e. predictors of poor glycemic control in type 2 diabetic patients attending public hospitals in dar es salaam. drug healthc patient saf. 2014;6:155-65. 22. omar sm, musa ir, osman oe, adam i. assessment of glycemic control in type 2 diabetes in the eastern sudan. bmc research notes. 2018;11(1):373. 23. roy s, sherman a, monari-sparks mj, schweiker o, jain n, sims e, et al. association of comorbid and metabolic factors with optimal control of type 2 diabetes mellitus. n am j med sci. 2016;8(1):31-9. 24. rohit s, rahul m. diabetes risk diagnosis using obesity markers and glycemic control in indian population. journal of the association of physicians of india. 2019;67:22. 25. goodpaster bh, krishnaswami s, resnick h, kelley de, haggerty c, harris tb, et al. association between regional adipose tissue distribution and both type 2 diabetes and impaired glucose tolerance in elderly men and women. diabetes care. 2003; 26(2):372-9. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i3.970 119j contemp med sci | vol. 2, no. 8, autumn 2016: 119–122 research prognostic value of cystatin c in acute coronary syndrome mohammed abdulwahid shuaila,a hassanain m saeed abdulamir,b monem makki alshokb ambchb, merjan teaching hospital hilla babylon, ministry of health, babylon, iraq. bdepartment of medicine, college of medicine, university of babylon, iraq. correspondence to professor dr monem alshok (email: dr_monem_alshok@yahoo.com). (submitted: 9 september 2016 revised version received: 2 october 2016 accepted: 7 october 2016 – published online: 26 december 2016) objective coronary artery disease associated with an increment with a variety of markers, one of them cystatin c, in this study we evaluate its role in a prospective study as prognostic marker, and evaluate the correlation between cystatin c plasma level and variety of acute coronary syndrome (acs) complications. methods a total 51 patients who admitted for merjan teaching hospital coronary care unit whom acs was diagnosis was made depend on history, clinical examination and investigation, and then blood sample was taken to measure plasma level and follow up for 6 months of any new events including new ischemia, rehospitalization, electrical and mechanical complication. results patient who admitted with acs with high level of cystatin c associated with more mortality (p value: 0.09) and more electrical complication (p value: 0.035) and more rehospitalization (p value: 0.01), but failed to show a correlation with mechanical complication. conclusion elevated level of cystatin c in patient admitted to hospital with acs associated with an increase in hospital mortality, electrical complication, and rehospitalization and lower ejection fraction than a patient with a normal cystatin c level. keywords cystatin c, prognostic value, acute coronary syndrome introduction cystatin c (cys-c) is a small protein molecule (120 amino acid peptide chain, approximately 13kda) produced by virtually all nucleated cells in the human body. it belongs to the family of papain-like cysteine proteases and its main biological role is the extracellular inhibition of cathepsins. it’s near constant production rate, the fact that it is freely filtered from the glomerular membrane and then completely reabsorbed without being secreted from the proximal tubular cells, made it an almost perfect candidate for estimating renal function. the strong correlation between chronic kidney disease (ckd) and cardiovascular disease (cvd) along with the growing understanding of the role of cysteinyl cathepsins in the pathophysiology of cvd inspired researchers to explore the potential association of cys-c with cvd. a high level of cystatin c in the blood corresponds to a decreased glomerular filtration rate (gfr) and hence to kidney dysfunction, recent studies suggest that increased levels of cystatin c may also indicate an increased risk of heart disease, heart failure, stroke, and mortality.1,2,3,4 there is a close relationship between cystatin c and acute ischemic stroke, independently of conventional risk factors.5 several studies mentioned that increased levels of cystatin c are found in patients with coronary artery disease.6,7,8,9,10 cystatin c recently, it has been studied for its role in predicting new-onset or deteriorating cardiovascular disease. also cystatin c may be an indicator of acute pte (pulmonary thromboembolism) in patients with normal renal function. also elevated serum cystatin c levels may predict venous thrombosis beyond reflecting impaired kidney function. cystatin c is known to modulate the neutrophil chemotactic activity and may inhibit prothrombotic activity of proteolytic substances secreted by activated neutrophils. thus, it may be hypothesized that increased serum levels of cystatin c represent an inadequate counterbalancing mechanism to avoid thrombosis formation.11–17 a recent study conducted by urbonaviciene et al. demonstrated that higher serum cystatin c levels independently predicted 5-year all-cause and cvs mortality in symptomatic peripheral arterial disease patients with normal renal function. in accordance with urbonaviciene et al.18 loew and colleagues19 have reported that only high plasma cystatin c levels(> 1.24 mg/l) were associated with risk of fatal and nonfatal cardiovascular events during the follow-up. furthermore, in a recent meta-analysis, higher cystatin c levels were strongly and independently associated with specific endpoints like stroke, myocardial infarction and heart failure.20,21 the aim of the present study was to evaluate whether the concentration of cys c could predict the severity of coronary artery disease after myocardial infarction in patients with normal renal function estimated from the concentration of serum creatinine, and to determine the prognostic value of cys c in predicting cardiovascular mortality during the follow up of patients with acute coronary syndrome (acs). methods a short-term prospective study was conducted on 51 patients with acs admitted to merjan teaching hospital from the first of march 2015 to first of july 2015. patients were excluded from the study if there is any neoplasia in any organ, pregnancy, thyroid dysfunction, altered mental state, renal impairment on admission, suspected dissected aortic aneurysm, recent mi (6 month ago), patients with egfr less than 60 ml/ kg/min, patient received steroid and any patient refuse participation. the diagnosis of acs was established by symptoms, electrocardiography and cardiac biomarkers. detailed history was taken from the patients, and physical examination was performed on admission. echocardiography was done usually in the next day of admission, but bed-side echocardiography was done if there is a strong indication to assess hemodynamically unstable patients and to rule out mechanical complication in stemi patients. potential risk factors for acs including hypertension (hpt), diabetes (dm), smoking, as well as age of patients, and other information were reviewed by direct questionnaire to the patients or their relatives. risk factors were defined according to the protocol. the patients with a history of hpt or without history but compatible with jnc9 definition of hypertension mm hg were defined hypertensive. issn 2413-0516 120 j contemp med sci | vol. 2, no. 8, autumn 2016: 119–122 prognostic value of cystatin c in acute coronary syndrome research mohammed abdulwahid shuaila et al. the patients who have the history of dm, being on glucose-lowering medication prior to acs onset or without a history but who had fasting blood sugar more than 126 mg/dl or random blood sugar > 200 mg/dl on two occasions were defined diabetics. the patients were regarded as current smokers if they smoked until admission or stopped smoking within the last 3 months. gfr measured using mdrd equation (modification of diet in renal disease) as follows: egfr ml/min. 73m² = 175 × s.(s.cr) ¹ ־¹⁴⁵ × (age)־ º²º³× (0742 if female) × (1.212 if black) we followed the patients daily during the period of hospitalization. clinical assessment and echocardiography was done to the patients who discharged from hospital, on average of 6 to 8 weeks after discharge. the patients were followed up by telephone or outpatient clinic visits for up to 3 months. blood sampling and laboratory methods blood was drawn on admission at the time of inserting an intravenous line. the sample was collected into a plain tube, allowed to clot and then separate the serum by centrifuge using {hettich rotofix22} (a german 2005), centrifuge at a rate of 3000 rpm for 5 minutes. 0.5–1.0 ml of serum is stored at 2–8 celsius for a few days for the measurement of cystatin c level. serum cystatin c is measured by using (nephlometric immunoterbidimetric method). the other laboratory investigation included a complete blood count, and random blood glucose, urea and creatinine. cardiac enzymes, including troponin, if indicated we perform a thyroid function test. statistical analysis statistical analysis was performed using statistical package for social science version 20 “spss 20”. categorical variable such as sex, diabetes, hypertension, smoking, occurrence of death was expressed as a percentage. the correlations between categorical variables were assessed using chi square or fisher exact test as appropriate. continuous variables that were normally distributed such as the age of the patient, serum cystatin c, duration of hospitalization were expressed as mean ± standard deviation. the correlation between continuous variables were assessed using annova test. p value of 0.05 or less considered to be significant. results in this study, 51 patients were enrolled. the age ranged from 45 to 65 (mean ± sd 52.12 ± 8.93), 32 of them were male, 19 were female, 12 patients (23.5%) were diabetes, 15 patients (29.4%) were hypertensive, 13 patients (25.4%) were current smokers. of those patients, 36 patients (71%) were admitted with the diagnosis of stemi, 15 patients (29%) admitted with the diagnosis of non-stemi/ua, among patients with stemi 20(28%) had anterior stemi, 16(22%) had inferior stemi. in the ua/ nstemi group 13(86%) of them have st depression/t inversion, and 2(14%) have normal ecg findings. 33 patients (91%) had successful reperfusion therapy, 25 patients (69%) had pci primary or rescue, and 8(22%) patients had thrombolysis as a sole reperfusion therapy. one patient developed acute ischemic stroke with slurred speech and dysphagia on the second day of hospitalization after primary pci (with normal brain ct scan done immediately after the event) with the resolution of the neurological deficit during the follow up. the duration of hospitalization was 4 ± 1.82 days and mortality was 4 patients (7.5%). table 2 shows the correlation between mortality and the patients’ clinical characteristics. the mean age of patients who died (55 ± 6.272), 3(75%) of them where males and 1(25%) where females, 2 (50%) were hypertensive with (0.22 p value), and 3(75%) were diabetics (0.036 p value), 3 (75%) were smokers (0.046 p value). table 3 shows the correlation between cystatin table 1. baseline characteristics of the patients variables no./percent no. 51 patients age (52.12 ± 8.93) sex 32 males/19 females dm 12 patients (23.5%) hypertension 15 patients (29.4%) smoking 13 patients (25.4%) stemi 36 patients 70.5%) nonstemi\ua 15 patients (28.3%) reperfusion in stemi 33 patients (62.2%) thorombolysis 8 patients (15%) pci 25 patients (47.1%) ecg findings anterior stemi 20(39.2%) inferior stemis 16(30.1%) st depression\t inversion 13(24.5%) normal ecg 2(3.7%) mortality 4 patients (7.5%) s. cystatin duration of hospitalization (4 days ± 1.82) table 2. correlation between mortality and patients’ clinical characteristics variables in hospital mortality p value yes (4) no (49) age 55 ± 6.272 51.87 ± 9.138 ns sex 3 (5.6%) males 1 (1.8%) 0.22 hypertension 2(3.7%) 2(3.7%) 0.22 dm 3(5.6%) 1(1.8%) 0.036 smoking 3(5.6%) 1(1.8%) 0.046 table 3. correlation among cystatin level, mortality electrical complications and mechanical complications variables no. mean cystatin c ± sd p values mortality died 4 0.8538 0.48531 0.009 no 47 1.5250 0.30838 electrical complications yes 4 1.4150 0.61016 0.035 no 47 0.8632 0.47869 rehospitalization yes 9 1.3833 0.40159 0.01 no 42 0.8043 0.46864 mechanical complications yes 1 1.7000 0.1 no 50 0.8906 0.49770 mohammed abdulwahid shuaila et al. 121j contemp med sci | vol. 2, no. 8, autumn 2016: 119–122 research prognostic value of cystatin c in acute coronary syndrome level and mortality, re hospitalization, electrical and mechanical complications. in patients who died during the hospital course were 1.5250 ± 0.30838, while the mean cystatin c level in patients who were discharged alive from the hospital were between 0.8538 and 0.8531. the correlation was statistically significant (p value 0.009). 4 patients (19%) developed electrical complications in hospital. two patients developed primary ventricular fibrillation (vf) were successfully resuscitated, one patient developed atrial fibrillation in which rate controls done with intravenous amiodarone infusion, and one patient developed sustained ventricular tachycardia (vt) was successfully resuscitated with mean cystatin c (1.4150 ± 61016) sd developed electrical (p value 0.035 significant). also our results showed correlation between cystatin c level and ejection fraction (ef) using spearman’s correlation coefficient, which showed a negative correlation coefficient (–154). nine patients (17.6%) readmitted to hospital, of those patient 4 patients admitted with decompensated heart failure, 2 patients admitted due to post mi angina, 2 patients due to acute pulmonary edema, and 1 patient due to vsr. with mean 1.3833 and std. deviation 0.40159. vsr in which 1 patient (1.9%) developed vsr. this patient developed clinical deterioration with hemodynamic instability and developed a new murmur with auscultation, echo approves the presence of acquired vsd and the patient referred for urgent surgical intervention (p value 0.1 non-significant) discussion in this study, we have found that an increase in cystatin-c level is associated with an increase in hospital mortality. this result has agreed with osama tayeh et al.23 their study was a non-randomised controlled trial took place in egypt, prospectively has conducted on 75 patients with (acs) and also an equal number of controls. patients who have included in this study are presented with acs and evaluated the prognostic value of it as a predictor for the major acute coronary event). our concern study has shown that group with a high cystatin c level is associated with significant in hospital mortality (p value 0.025), and also it is going with leila abid et al.,24 which showed (p value 0.01 clinically significant). and another study where cystatin c was used as a prognostic biomarker in stemi and shlipak et al.25 eriksson et al. have asserted that an increase in cholesterol or ldl-cholesterol levels are considered as risk factors of ihd, but cys-c may reflect precisely the presence or absence of cad.26 we found in this study that diabetes and smoking are associated with increasing mortality that agrees with osama tayehetal. and presenting significant correlation with smoking and disagree with leila abidetal. (non-significant). while in a multivariate regression analysis between the prevalence of high cys-c and other common traditional risk factors, including: hypertension, diabetes, and smoking if all risk factors are present, the patient had a high cys-c level. meanwhile hypertensive was clinical regarded as nonsignificant (p value 0.22). the reasonable explanation for our results is that serum cystatin c has regarded as the most sensitive marker of early renal dysfunction, due to that it may be involved in the process of coronary heart disease. despite the uncertainty of the exact mechanisms underlying the predictive role of cystatin c in chd, evidence suggests that elevated serum cystatin c is associated with worse prognosis in patients with chd. a study by zethelius et al. have declared whether a combination of biomarkers, including n-terminal pro-brain natriuretic peptide, cystatin c, troponin, and hs-crp, have improved patient stratification of risk compared with established cardiovascular risk factors. those researchers have discovered that adding cystatin c to the system significantly will improve predictive efficacy.27 also, we found that higher patient with higher level of cystatin c levels have associated with increased incidence in hospital electrical complications (p value 0.035), and this agrees with osama tayah et al. who have confirmed that higher level of cystatin c is associated with a higher chance of electrical complications (p value 0.029). and we also found that high level of cystatin c associated with higher rehospitalization due to various causes (decompensated heart failure, post mi angina, pulmonary edema, etc.) that agreed with ichimoto et al.,28 whom investigated the cystatin level of 71 patients with stemi, had also suggested the prognostic value of cys c, high concentrations of this marker were associated with greater frequency of rehospitalization and acute heart failure episodes. association of cystatin c with greater mortality rate during follow-up. kilic et al.29 have investigated 160 patients hospitalized with acs and demonstrated that the admission of the serum cysc level was significantly associated with future cardiovascular complications and rehospitalization and mortality during 12 months of follow-up. it also agrees with osama tayeh and axel akerblom et al.30 higher level of cystatin c is associated with lower ejection fraction (spearman’s correlation coefficient which showed a negative correlation coefficient (–0.154). this also has agreed with moran et al. who have concluded that cys-c can be a marker of heart failure, and its combination with n-terminal pro-b-type natriuretic peptide (nt– pro-bnp) considered to be as good marker than cys-c alone for predicting cardiovascular mortality, especially in elderly patients with heart failure.31 our study agrees with garcıa acuna et al. who indicated that an elevated serum cys-c level will predict the development of myocardial infarction, heart failure and cardiovascular death in 203 patients hospitalized with high-risk acs, independent of other classical risk factors either in-hospital or during a 6-month follow-up period.32 the results of our study also have agreed with silva et al.33 who have suggested that patients admitted for st elevation myocardial infarction and who presented elevated cys c levels (p 0.84 mg/l) on admission, had greater risk of progression to cardiogenic shock or death during hospitalization. in this same study, only cys c levels p 0.84 mg/l and impaired lvef < 40% were the predictors of the risk of death during the follow-up. and also agreed with ichimoto et al. and osama et al. mechanical complications have shown non-significant results (p value 0.1), which had disagreed with osama tayeh et al. (p value 0.002), and this might be due to small a number of data that we have used in our study. conclusion elevated level of cystatin c in patients admitted to the hospital with acs associated with an increase in hospital mortality, electrical complication, and re hospitalization and lower ejection fraction than a patient with a normal cystatin c level. we recommend measuring the cystain c level with early hours of admission for any patient of acs for risk stratification and early possible intervention. conflict of interest none. n 122 j contemp med sci | vol. 2, no. 8, autumn 2016: 119–122 prognostic value of cystatin c in acute coronary syndrome research mohammed abdulwahid shuaila et al. references 1. angelidis c, deftereos s, giannopoulos g, anatoliotakis n, bouras g, hatzis g, et al. cystatin c: an emerging biomarker in cv disease. curr top med chem athens greece. 2013;13:164–179. 2. shlipak mg, et al. cystatin c versus creatinine in determining risk based on kidney function. n engl j med 2013;369:932–943. online at: http:// www.nejm.org/doi/full/10.1056/nejmoa1214234 through http://www. nejm.org. 3. henry’s clinical diagnosis and management by laboratory methods. 21st ed. mcpherson r, pincus m, eds. philadelphia, pa: saunders elsevier: 2007, 153–154. 4. inker la, schmid ch, tighiouart h, eckfeldt jh, feldman hi, greene t, et al. estimating glomerular filtration rate from serum creatinine and cystatin c. n engl j med. 2012;367:20–29. 5. huang gx, ji xm, ding yc, huang hy. association between serum cystatin c levels and the severity or potential risk factors of acute ischemic stroke. neurol res. 2016;38(6):518–523. 6. dupont m, wu y, hazen sl, tang wh. cystatin c identifies patients with stable chronic heart failure at increased risk for adverse cardiovascular events. circ heart fail. 2012;5:602–609. 7. ix jh, shlipak mg, chertow gm, ali s, schiller nb, whooley ma, cystatin c. left ventricular hypertrophy, and diastolic dysfunction: data from the heart and soul study. j card fail. 2006;12:601–607. 8. ix jh, shlipak mg, liu hh, schiller nb, whooley ma. association between renal insufficiency and inducible ischemia in patients with coronary artery disease: the heart and soul study. j am soc nephrol. 2003;14:3233. 9. ni l, lü j, bo hou l, tao yan j, fan q, hui r, et al. cystatin c, associated with hemorrhagic and ischemic stroke, is a strong predictor of the risk of cardiovascular events and death in chinese. stroke. 2007;38:3287–3288. 10. windhausen f, hirsch a, fischer j, van der zee pm, sanders gt, van straalen jp, et al. cystatin c for enhancement of risk stratification in non-st elevation acute coronary syndrome patients with an increased troponin t. clin chem. 2009;55:1118–1125. 11. ellen e. brodin, sigrid k. brækkan, anders vik, jan brox, john-bjarne hansen haematologica. 2012;97:1008–1013. 12. massberg s, grahl l, von bruehl ml, manukyan d, pfeiler s, goosmann c, et al. reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. nat med. 2010;16:887–896. 13. leung-tack j, tavera c, martinez j, colle a. neutrophil chemotactic activity is modulated by human cystatin c, an inhibitor of cysteine proteases. inflammation. 1990;14:247–258. 14. helmersson-karlqvist j, johan ärnlöv axel c. carlsson, härmä j, larsson a. increased urinary cystatin c indicated higher risk of cardiovascular death in a community cohort. atherosclerosis. 2014;234:108–113. 15. johannes a. cystatin c and the relation to cardiovascular disease: studies on the relative importance of genetic and environmental factors. doctoral theses department of medicine karolinska university etssjukhuset, solna, 2015. 16. dardashti a, nozohoor s, algotsson l, ederoth p, bjursten h. the predictive value of s-cystatin c for mortality after coronary artery bypass surgery. j thorac cardiovasc surg. 2016;152:139–146. 17. dent th. predicting the risk of coronary heart disease. ii. the role of novel molecular biomarkers and genetics in estimating risk, and the future of risk prediction. atherosclerosis. 2010;213:352–362. 18. urbonaviciene g, shi gp, urbonavicius s, henneberg ew, lindholt js. higher cystatin c level predicts long-term mortality in patients with peripheral arterial disease. atherosclerosis. 2011;216:440–445. 19. loew m, hoffmann mm, koenig w, brenner h, rothenbacher d. genotype andplasma concentration of cystatin c in patients with coronary heart disease and risk for secondary cardiovascular events. arterioscler thromb vasc biol. 2005;25:1470–1474. 20. lee m, saver jl, huang wh, chow j, chang kh, ovbiagele b. impact of elevatedcystatin c level on cardiovascular disease risk in predominantly high cardio-vascular risk populations: a meta-analysis. circ cardiovasc qual outcomes. 2010;3:675–683. 21. salgado jv, souza fl, salgado bj. how to understand the association between cystatin c levels and cardiovascular disease: imbalance, counterbalance, or consequence? j cardiol. 2013;62:331–335. 22. servais a, giral p, bernard m, bruckert e, deray g. isnard, bagnis, c. is serum cystatin-c a reliable marker for metabolic syndrome? am j med. 2008;121:426–432. 23. tayeh o, rizk a, mowafy a, salah s, gabr k. cystatin-c as a predictor for major adverse cardiac events in patients with acute coronary syndrome. egyptian heart j. 2012;64:87–95. 24. abid l, charfeddine s, kammoun s, turki m, ayedi f. cystatin c. a prognostic marker after myocardial infarction in patients without chronic kidney disease. j saudi heart assoc. 2016;28:144–151. 25. shlipak mg, katz r, sarnak mj, fried lf, newman ab, stehman-breen c, et al. cystatin c and prognosis for cardiovascular and kidney outcomes in elderly persons without chronic kidney disease. ann intern med. 2006;145:237–246. 26. eriksson p, jones kg, brown lc, greenhalgh rm, hamsten a, powell jt. genetic approach to the role of cysteine proteases in the expansion of abdominal aortic aneurysms. br j surg. 2004;91:86–89. 27. zethelius b, berglund l, sundstro ¨ m j, et al. use of multiple biomarkers to improve the prediction of death from cardiovascular causes. n engl j med. 2008;358:2107–2116. 28. ichimoto e, jo k, kobayashi y, inoue t, nakamura y, kuroda n, et al. prognostic significance of cystatin c in patients with st-elevation myocardial infarction. circ j. 2009;73:1669–1673. 29. teoman k, gokhan o, ertan u, zeki y, ulas s, ulas b, et al. comparison of the long-term prognostic value of cystatin c to other indicators of renal function, markers of inflammation and systolic dysfunction among patients with acute coronary syndrome. j atheroscler. 2009;207: 552–558. 30. ix jh, shlipak mg, chertow gm, whooley ma. association of cystatin c with mortality, cardiovascular events, and incident heart failure among persons with coronary heart disease: data from the heart and soul study. circulation 2007;115:173–179. 31. moran a, katz r, smith nl. cystatin c concentration as a predictor of systolic and diastolic heart failure. j card fail. 2008;14:19–26. 32. jose gam, eva gb, lilian gd, carlos pg, rafael vp, ana ll. prevalence of disease of the large arteries in an elderly belgian population: relationship with some metabolic factors. acta cardiol. 1984;39: 365–372. 33. silva d, cortez-dias n, jorge c, marques js, carrilhoferreira p, magalhães a, et al. cystatin c as prognostic biomarker in st-segment elevation acute myocardial infarction. am j cardiol. 2012;109:1431–1438. 193j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 review issn 2413-0516 introduction to ensure reconstruction of the recipient immune system after grafting from the donor cells, the recipient is subjected to immunosuppressive and myelosuppressive conditioning treatments before transplantation, thereby avoiding the problems of rejection.1 however, the donor’s immunocompetent t-lymphocytes may detect the expressed antigens by the recipient cells as foreign after transplantation, hence provoking an immune reaction according to the intensity of inflammatory responses which may damage the tissues and organs of the recipient. these circumstances known as graft-versus-host disease (gvhd) is a result of the incompatibility between the antigens of human leukocyte antigen (hla) system in recipient and donor.2 allogenic hematopoietic stem cell transplantation (hsct) is a common procedure used for treatment of various benign and malignant hematological diseases. hsct has some complications, most frequent and serious of which is gvhd, regarded as the major cause of late mortality not associated with underlying disease process.3, 4 the risk of gvhd is considered as an important cause of insufficient popularity of hsct.5 gvhd is an alloand autoimmune disorder with a variable clinical course that commonly involves various tissues and organs. it has a negative effect on life expectancy of patients with this disorder. the prevalence of acute gvhd (agvhd) has remained almost constant in the last 10 years, however, cgvhd has increased.6 the reason for this is the use of hematopoietic cells rather than of bone marrow transplants, the lymphocyte infusions especially into decreased intensity allotransplants, using donors who are not wholly hla-compatible with or without blood ties to the recipient, performing more transplants in the elderly, and the number of yearly performed transplants. in allogenic transplant patients, a rate of agvhd between 50% and 70% is observed, while this rate for cgvhd is between 30% and 50%.8 in contrast to previous few years, these two forms of gvhd is usually distinguished from each other by their clinical characteristics rather by timing criteria.1,3 the agvhd usually affects liver, gastrointestinal tract, and skin and is potentially fatal. in the cgvhd, the oral cavity is mainly affected, and sometimes may be the only affected part of the body by the disease.9 the cgvhd is more common in first 3 years post-transplantation, and normally developed by the acute form. the cgvhd has similar clinical characteristics to the other immune-mediated diseases including lupus erythematosus, lichen planus, and systemic sclerosis. the drugs used for agvhd prevention are not effective on preventing cgvhd.6 although there are no specific treatments for gvhd, corticosteroids and immune modulators can be used for this condition.10 this study attempts to provide a modified remedial method for oral cgvhd within these criteria: (i) identifying the most important causes of oral cgvhd development, (ii) the pathogenic role of the t-cells, (iii) the major risk factors for cgvhd, (iv) determining when a biopsy is needed to confirm the oral cgvhd diagnosis, (v) the frequency of the oral cavity affecting by cgvhd, and (vi) determination of the effectiveness rate of oral cgvhd treatment. a review on therapeutic management for oral chronic graft-versus-host disease mina khayamzadeha, hanieh kavianib adepartment of oral and maxillofacial medicine, faculty of dentistry, tehran university of medical sciences, international campus, tehran, iran. bdepartment of oral and maxillofacial radiology, faculty of dentistry, tehran university of medical sciences, international campus, tehran, iran. corresponding author: hanieh kaviani (e-mail: haniehkaviani@yahoo.com ) (submitted: 19 june 2020 – revised version received: 07 july 2020 – accepted: 14 august 2020 – published online: 30 october 2020) abstract chronic graft-versus-host disease (cgvhd) is one of the main complications of transplantation in allogeneic hematopoietic cells which results in reduced quality-of-life. cgvhd mostly affects the oral cavity, producing various symptoms and manifestations. it can lead to both shortand long-term complications such as limited oral intake, mucosal sensitivity, secondary malignancy, and even early death. gvhd is an alloand autoimmune disorder with a variable clinical course that commonly involves various tissues and organs. it can occur in both the acute and chronic forms. in the case of cgvhd, a large number of organs are affected including the oral cavity. however, in some cases of cgvhd only the oral cavity gets affected. the clinical indications of chronic oral gvhd may include sclerosis, hyperkeratotic plaques, lichenoid lesions, and limited oral aperture. the level of oral involvement is commonly mild, but medium to high severity erosive as well as ulcerated lesions may also occur. although diagnosed through clinical examination, its confirmation is usually done by biopsy study. the first treatment option is using local corticosteroids with the potency to treat half of patients. extracorporeal photopheresis and systemic corticosteroids are in the next ranks. patient survival after diagnosis of oral chronic gvhd is within 4.5 years in 50% of the cases, thus it is not regarded as a determinant factor in patient survival. this study attempted to provide a detailed approach for clinical diagnosis and management of patients suffering from oral cgvhd. it particularly considered the factors such as differential diagnosis, symptom control, screening for, and prevention of secondary complications. it tried to provide practical and relevant considerations and recommendations for all clinicians who deal with oral cgvhd patients in order to achieve improved care and outcomes. keywords: chronic graft-versus-host disease (cgvhd), risk factors, oral gvhd, biological pathogens, therapeutic management 194 review a review on therapeutic management for oral chronic graft-versus-host disease mina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 material and methods a systematic review of recent literature on topical agents used for treatment of the inflammatory mucosal lesions found in ogvhd patients was conducted. in order to identify potentially relevant articles with the subject of this study, an extensive research was performed over pubmed, national library of medicine’s medline, and embase electronic databases using keywords “gvhd,” “chronic gvhd,” “graft vs. host,” “diagnosis,” “prevention,” “grading,” and “treatment”. accordingly, 274 articles were found from the mentioned databases which was further categorized into 4 main groups including diagnosis, prevention, grading, and treatment. after elimination the unrelated articles, finally 36 articles were selected as related articles within this comprehensive study. to obtain more detailed studies on the subject of this study, 9 other relevant articles were identified from relevant databases. eventually, 46 publications were included in this review (fig. 1). oral cgvhd epidemiology the oral cavity is usually affected in cgvhd, so that around 80% of patients with cgvhd demonstrate oral cavity involvement. when diagnosis of cgvhd, oral characteristics are commonly present, demonstrating the initial clinical manifestations. oral involvement can be regarded as the most common single-site involved in cgvhd patients. although there are no significant difference in severity, extent, and complications of oral cgvhd among all age groups, the overall occurrence of cgvhd in adults is higher than in pediatric patients.13 there are several factors associated with higher risk of cgvhd incidence after allogeneic hct such as hla mismatch, antecedent acute gvhd, and stem cell source, however, risk factors specific of oral cgvhd have remained unknown. acute graft-versus-host disease gvhd disease can be naturally categorized into agvhd and cgvhd. previously, this categorization was based on inception time of incidence in which agvhd specified as incidence within 100 days of post-transplantation.15 recently, gvhd status is largely defined based on clinical properties. presentation of acute symptoms such as liver dysfunction, upper and lower gastrointestinal involvement, oral mucositis, and maculopapular cutaneous lesions can be classified as classic, recurrent, persistent, or late-inception agvhd. incidence of agvhd within 100 days post-allogeneic hsct is defined as classic agvhd, while incidence later than 100 days after figure 1. flow chart illustrating the article selection steps in this study according with prisma technique. records identified through database searching (n = 274) sc re en in g in cl ud ed el ig ib ili ty id en tif ic at io n additional records identified through other sources (n = 16) removed similar and duplicate articles during screening (n = 142) final screened records (n = 148) excluded unrelated records (n = 94) qualified full-text articles (n = 54) excluded records for other reasons (n=4) high quality selected records (n = 46) treatment (n=18) prevention (n=12) screening (n=6) diagnosis (n=10) extra related records founded from available resources (n=5) fig. 1. the procedure of choosing relevant articles used in the present study in accordance with prisma method. 195 review a review on therapeutic management for oral chronic graft-versus-host diseasemina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 allogeneic hsct indicates recurrent, persistent or late-inception agvhd.16 chronic graft-versus-host disease cgvhd is the most frequent complication of post-allogeneic hsct, which occurs in about 50% of patients who survive more than 1 year after transplantation. manifestations of cgvhd is commonly developed within the first 3 years following hsct. either the involvement of one organ or several areas of the body is possible.16 cgvhd may target the mouth, eyes, skin, liver, lungs, gi tract, genitourinary tract, and joints of patients, producing pain and function disability, thus weakening the quality-of-life. moreover, it is considered as the major cause of death among survivors of transplant, so that it accounts for about 70% of deaths before 5 years of transplantation.70 this high rate of morbidity and mortality can largely be attributed to immune dysregulation and suppression which result in recurrent infections.16 the cgvhd syndrome has clinical and histological similarities to many immunologic disorders such as sjögren’s syndrome, scleroderma, bronchiolitis obliterans, primary biliary cirrhosis, chronic immunodeficiency, and immune cytopenias. as a result, cgvhd is defined as a multisystem autoimmune and alloimmune disorder which is specified by immune deficiency, immune dysregulation, end-organ dysfunction, and reduced survival.16 clinical features and risk factors of oral cgvhd although usually considered as a singular clinical entity, oral cgvhd has its own clinical features in terms of sclerodermatous, mucosal, and salivary gland involvement, each of which can result in significant morbidity and late complications. clear understanding of their distinct clinical features is necessary to appropriate diagnosis and effective management.7 the risk factors described for cgvhd includes hla inconsistency or lack of blood ties between the recipient and donor; a male recipient and female donor; higher ages of the recipient and donor; child-bearing in female donors; donor lymphocyte infusions; the transplantation of mobilized peripheral blood cells; and a history of agvhd.14, 18 the risk factors described for cgvhd includes hla inconsistency or lack of blood ties between the recipient and donor; a male recipient and female donor; higher ages of the recipient and donor; child-bearing in female donors; donor lymphocyte infusions; the transplantation of mobilized peripheral blood cells; and a history of agvhd.14, 18 among these, three factors of a male recipient and female donor, higher ages, and the transplantation of mobilized peripheral blood cells seem to be especially associated to cgvhd. the factors of conditioning treatment intensity before transplantation and whole body irradiation appears to have no effect on cgvhd occurrence. although the mechanism of the disease in terms of immunopathogenic is not completely apparent, the major provoking factor of gvhd is known to be reactivity of donor t-cells against the recipient tissues which occurs as intensified direct or indirect inflammatory responses.3, 19 despite the existence of common risk factors, each type of gvhd has its own risk factors, suggesting the difference between their underlying pathogenesis. activation of the interferon-1 pathway seems to have a role in oral cgvhd.6 some authors have specified the thymus gland destruction by alloreactive t cells as the major provoking factor in cgvhd, arguing that thymopoiesis and t cell renovation phenomena may be observed after autologous hematopoietic cell transplantation, and cgvhd is not common. it has been proposed that cgvhd is distinguished via a humoral immune response mediated by th2 lymphocyte, while agvhd is specified by a cellular immune response mediated by th1 lymphocyte.6 both the b lymphocytes and t-cells are involved in the depletion of general immune tolerance. the capability of b lymphocytes to produce antibodies on one hand, and the good outcomes obtained in using anti-cd20 drugs for autoimmune diseases with similar features to those of gvhd on other hand, caused many recent studies on the role of the b lymphocytes in gvhd pathogenesis. although there is no established autoantibody panel specific to cgvhd, more symptoms are seen in patients with autoantibodies and cgvhd.20 moreover, antigen-presenting cells like dendritic cells as well as cytokines like b-cell activating factor which belongs to the family of tumor necrosis factor appears to play a role.20 tables 1 and 2 demonstrate the criteria for characterization of different gvhd presentation. table 1. diagnostic differentiation between agvhd and cgvhd. derived from maria et al.21 diagnostic differentiation between agvhd and cgvhd. agvhd cgvhd maculopapular rash secretory hepatitis nausea and vomiting anorexia at least one diagnostic or distinctive manifestation of cgvhd persists with no characteristic features of agvhd without time restrictions. presence of both cgvhd and agvhd features with no time restrictions. occurring in 100 days post-transplant, or donor lymphocyte infusion, with no diagnostic or distinctive demonstrations of cgvhd after 100 days posttransplant, or donor lymphocyte infusion, with no diagnostic or distinctive demonstrations of cgvhd classic cgvhd overlap syndrome classic agvhd recurrent or late-inception agvhd 196 review a review on therapeutic management for oral chronic graft-versus-host disease mina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 chronic gvhd immunopathogenesis the basic cgvhd pathogenesis requires alloreactive donor t-cells to detect and invade the host tissues in immunocompromised recipients.22 currently, it is known that there is a nuanced interaction between multiple immune cell types of the recipient and donor. the factors such as inflammation induced by the post-transplant infectious and conditioning regiment results in complexity of such a relationship. cgvhd has several fibrotic and autoimmune characteristics. the cgvhd syndrome has clinical and histological similarities to many classic autoimmune disorders such as sjögren’s syndrome, scleroderma, primary biliary cirrhosis, systemic lupus erythematosus, and lichen planus.16 the procedure consists of a potent pro-inflammatory t-cell component in addition to b cells involvement and regulatory t-cells.23 absence of a preclinical model to simulate the clinical aspects of human cgvhd is the major challenge in comprehending the immunopathogenesis of cgvhd. the focus of animal models are largely on mortality and weight loss, missing the major mhc factors. despite using some minor-mhc mismatch models, none of them sufficiently replicate the temporal and multiorgan features of human cgvhd.24 it is challenging to set up prospective longitudinal studies in patients with cgvhd, since this requires long-term tracking of the patients. the samples used in these studies are often limited to clinically indicated tissue biopsies, clinical laboratory values, and peripheral blood with a variable nature, making it difficult to get a clear conclusion on cgvhd development and pathogenesis. below, the results of both animal models and studies of human cgvhd are provided with more emphasis on human data. also, a discussion on the contribution of various factors and cell types to pathogenesis of cgvhd is presented.25 oral cgvhd oral cgvhd clinical presentation various organs can be affected by cgvhd, of which the skin is most commonly involved organ followed by the oral cavity with the prevalence of 45–83% in cgvhd patients.26 oral cgvhd can manifest as salivary gland dysfunction, restricted mouth opening and mucosal lesions. there are a variety of clinical manifestation of cgvhd in severity and type of tissue changes, and any location in oral cavity can be attacked by cgvhd. clinical examination involved tongue, labial and buccal mucosa, lips, gingiva, floor of mouth, hard and soft palate, as well as mouth movement, and salivary function evaluations.26 subjective oral dryness, oral pain, and sensitivity of the patient, and oral movement restriction or dysfunction can be regarded as important elements in clinical diagnosis of cgvhd, and convince the practitioner of performing more site specific evaluations.27 oral mucosal lesions the affected area of intraoral mucosa and the severity of lesion could vary and should be evaluated when examination of oral mucosal lesions.26 oral mucosal lesions of cgvhd are usually characterized as ulcerative, lichenoid, mucoceles, or erythema (fig. 2).16 erythema, described as the oral mucosa redness with no breakdown in tissue, is typically a symptom of inflammation or infection. it can be associated with edema and/or atrophy of the mucosa (fig. 1a). complaint of oral sensitivity with mucosal erythema is common among patients. lichenoid oral cgvhd, defined as lacey lines, or milky to white reticular streaks on oral mucosa, is similar to the wickham’s striae seen in oral lichen planus (fig. 2b). these can be a sign of table 2. diagnostic clinical signs of cgvhd. derived from maria et al.21 organ or location cgvhd common characteristics observed in both the agvhd and cgvhd skin poikiloderma morphea-like features lichen planus-like features lichen sclerosus-like features erythema pruritus maculopapular rash mouth lichen-type features restriction of mouth opening from sclerosis hyperkeratotic plaques gingivitis erythema mucositis pain genitalia lichen planus-like featuresvaginal scarring or stenosis gastrointestinal tract esophageal web strictures or stenosis in the upper to mid third of the esophagus anorexia diarrhea nausea vomiting weight loss lung bronchiolitis obliterans liver overall bilirrubin, alkaline phosphatase > 2 x upper limit of normal alt or ast > 2 x upper limit of normal muscles fascia, joints fasciitis joint rigidity or contractures secondary to sclerosis 197 review a review on therapeutic management for oral chronic graft-versus-host diseasemina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 hyperkeratotic leukoplakias which is white plaques or thickened, hyperplastic mucosa. the oral mucosal lesions are not painful most of the times and considered as diagnostic signs for oral cgvhd while evaluation of post-hsct.16 oral mucosa breakdown results in ulcerations which are associated with pseudomembranes, because weak healing of wounds developed over the time (fig. 2c). ulcerations may be painful to some extent that result in reduced eating and prevention of oral hygiene.26 infection can easily reach to patient’s bloodstream through oral cavity ulcers which completely breakdown the solidarity of oral mucosal barrier. mucoceles are surface subepithelial secretions of saliva which are transferred from minor salivary glands to the epithelial–connective tissue interface induced by fibrotic occlusions of the glandular duct openings.26 they manifest as dome-shaped lesions filled by fluid which are surrounded and covered by oral mucosa (fig. 2d). they are commonly asymptomatic and seen solely on the lips and palate. salivary ducts may be blocked by salivary gland inflammation, which is deteriorated by reduced secretion and increased viscosity of saliva, results in mucocele formation.16 salivary dysfunction patients with oral cgvhd grow inflammatory damages to salivary gland tissue, experiencing reduced salivary flow and dry mouth. salivary dysfunction induced by cgvhd have similar characteristics with sjögren syndrome manifestations, that is, xerostomia or subjective dry mouth, and hyposalivation or reduction of objective saliva flow.29 a patient with dry mouth experiences problems in swallowing, chewing, speaking, increased dental, and oral mucosal disease such as oral caries and candida infections (fig. 3a-c).30 salivary dysfunction induced by oral cgvhd presents different manifestations and appears to be a distinct entity from the mucosal. there are few correlations between their manifestations. however, evaluation of cgvhd requires a formal salivary function test as an important symptom and indication in diagnosis, staging, and treatment of the disease.29 diagnosis of oral cgvhd oral cgvhd diagnosis is mainly based on the precedent, findings of clinical examination, and beginning time of signs and symptoms, for example, the time after allogeneic hematopoietic cell transplantation (hct), weakening of immunosuppressive regiment, and involvement of other affected regions in cgvhd.13 the national institutes of health consensus development project on criteria for clinical trials in chronic graft-versus-host disease has introduced a standardized criteria regarding the diagnosis of salivary gland and oral mucosal cgvhd, though these criteria have not been validated and were based on the opinion of experts (table 3). diagnostic aspects required to make a clinical decision on oral (mucosal) cgvhd are hyperkeratotic plaques and lichenoid or reticular lesions along with ulcerative and erythematous variations without lichenoid reticulations manifestation. however, these aspects are considered to be “distinctive”, b.lichenoid lesions a.erythema erythema lichenoid lesions c.ulcerations ulcerations d. mucoceles e. sclerosis fig. 2. various clinical manifestations of oral cgvhd. (a) erythema of the tongue and oral mucosa, (b) lichenoid lesions occurred on the lips, buccal mucosa, and other regions of the oral cavity, (c) oral ulcers, (d) mucoceles formed on the hard palate and occasionally below labial mucosa, and (e) peri-oral sclerosis that restricts the opening of mouth. derived from jw et al.28 a b c fig. 3. extreme dry mouth induced by salivary gland dysfunction (a). this paves the way for fungal overgrowth and opportunistic infections (b), and weakens the remineralization of tooth enamel, producing cervical carious lesions (c). derived from jw et al.28 198 review a review on therapeutic management for oral chronic graft-versus-host disease mina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 not diagnostic.31 in the cases that making a clinical diagnosis is difficult, the biopsies of minor salivary gland and/or oral mucosal are helpful, though they are typically not necessary in practice. in overall, the diagnosis of salivary gland cgvhd is more difficult than oral mucosal cgvhd, due to the more obvious clinical characteristics and restricted differential diagnosis of oral mucosal cgvhd.32 the staging score established by the national institutes of health is a simple measure for determining the functional impact of oral cgvhd. it is also useful to evaluate the changes in severity of oral cgvhd over the time (table 4). it is essential that oral cgvhd be differentiated from other common oral conditions that do not require therapy. to distinguish oral cgvhd from other common oral complications is essential, since they do not need therapy. these complications include: lesions induced by cheek biting with a spread and ragged” white appearance; linea alba which is a hyperkeratotic white line, forming along the bite plane; geographic tongue, which appears in the form of erythematous and atrophic patches, enclosed by circular white hyperkeratotic alterations that grow and wane regularly; and leukoedema, which is white faintly reticular that entirely disappears when the tissue be stretched.1 although not routinely performed, tissue biopsy is only laboratory test available for confirmation of oral cgvhd diagnosis, thus, experience and good diagnostic skills are critical in diagnosis of this condition. in the cases with equivocal findings or atypical presentations, consultation with oral medicine specialty can be beneficial, especially when indications are disproportionate with clinical properties.3 oral cgvhd predictive factors it is reported that a history of acute gvhd and using peripheral blood stem cell source are the risk factors for oral cgvhd.34 moreover, the involvement of salivary gland in oral cgvhd has been reported to be a result of pre-transplant conditioning tbi.35 the results of a recent comprehensive multivariate logistical regression analysis showed that oral cgvhd had significant relationships with mouth pain in patients as well as with a number of inflammation laboratory markers such as higher levels of total complement and lower levels of albumin.36 the oral cgvhd can have many health consequences such as reduced oral epithelial integrity and limited repair ability, increased oral infections, oral pain, negative impacts on eating, nutrition, speech, quality-of-life, and increased caries ris.19 oral cgvhd is not related to poor long-term survival, but it has negative impacts on functional capacity, oral health, symptoms, and quality-of-life of patients. oral cgvhd has is directly related to the severity of patients’ self-reported oral pain.37 the risk of extensive cervical decay is very high in oral cgvhd patients during the first 2 years of transplantation. patients with oral cgvhd have been reported to experience taste changes and higher levels of oral pain and dryness than patients without oral cgvhd.38. moreover, the risk of reduced oral cavity-related quality-of-life and lower scores of body mass index is increased in oral cgvhd patients. atrophy or dysfunction of salivary gland in patients results in difficulty swallowing, higher risk of occurring dental carious lesions as the result of impaired remineralization, and repetitious co-infections because of decreased salivary defenses as secretary iga.19 oral cgvhd management systemic corticosteroids alone or in combination with other immunomodulatory agents are required for cgvhd, particularly in the cases with multisystem involvement. this will provide adequate control of oral disease symptoms in many cases. management of cgvhd by systemic immunosuppressive therapy requires 2–3 years averagely. however, the data on the long-run course and management approaches of oral table 3. clinical aspects for salivary gland and oral mucosal cgvhd. derived from nathaniel et al.33 oral mucosal cgvhd signs hyperkeratotic plaques, lichen-type appearance, erythema/atrophy, atrophic glossitis, ulcerations with pseudomembranes, superficial mucoceles symptoms taste changes, sensitivity to brushing/mint-flavored toothpaste, sensitivity to foods/drinks including acidic foods, spicy/seasoned foods, alcoholic beverages and mouth rinses containing alcohol, hard, crunchy or crusty foods, salty foods, warm foods or drinks. salivary gland cgvhd signs saliva deficiency or lack of mouth floor, thickened or sticky saliva, ropey or foamy saliva, oropharyngeal candidiasis, atrophic mucosa, food debris inside the mouth, dental caries at the cervical margins and interproximal, tongue “clicking” during speaking, repeated water sipping, inability to swallow dry foods without fluids symptoms throat constriction and difficulty swallowing, difficulty chewing, difficulty speaking, xerostomia, sensitivity to foods or drinks, nightly waking due to severe dryness, taste alterations sclerotic cgvhd signs leathery skin, mucosal bands, limitation in mouth opening from sclerosis symptoms jaw pain, difficulty eating, tightness table 4. staging system for oral cgvhd in accordance with national institutes of health consensus criteria. derived from nathaniel et al.33 zero i ii iii asymptomatic there are mild symptoms along with disease signs, but oral intake is not significantly limited. there are moderate symptoms along with disease signs. oral intake is partially limited. there are severe symptoms along with disease signs. oral intake is severely restricted. 199 review a review on therapeutic management for oral chronic graft-versus-host diseasemina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 cgvhd therapy are unavailable.39 according to our experience, management of many patients with oral cgvhd require long-term intensive ancillary therapy, even after stoppage of therapies with systemic immunosuppressives. the accessibility of local and topical therapies is beneficial to oral cavity, owing to the fact that continuous oral disease is usually manageable without systemic immunosuppression. similarly, in the cases with limited oral involvement, ancillary measures without systemic therapy may be sufficient to manage cgvhd.1, 3 oral cgvhd therapy aimed to reduce the symptoms, eliminate the painful lesions, prevention, and management of secondary complications. all patients need to be informed on the importance of good oral hygiene, keep daily flossing and tooth brushing, and visit professional dental clinics at least twice annually. there are no proofs for antibiotic prophylaxis in patients who their cgvhd has been treated, however, because of precaution aspects during immune reconstitution, most centers suggest that elective dental visits to be restricted during the first year after transplantation.31 toothpaste contains sodium lauryl sulfate and flavoring agents, resulting severe sensitivity in patients with oral cgvhd. these patients should be recommended to use children’s toothpaste which are free of sodium lauryl sulfate and less intensely flavored. moreover, oral cgvhd patients cannot tolerate mouthwashes that are heavily flavored agents and those containing alcohol, and should be avoided. the tolerability of oral cgvhd patients is largely variable. they may face difficulty to eat outdoor where food options are limited and highly seasoned. patients may need to be avoided from consumption of rough/hard, spicy, acidic, hot, and carbonated drinks/foods. in some patients, nutritional counseling may be required, although it is not routinely indicated.40 tooth decay due to the reduced antifungal and anticariogenic activities in patients with salivary gland cgvhd, they are at higher risk for evolving secondary infectious complications. patients with oral cgvhd often developed accelerated and rampant dental caries due to under-recognized reasons, resulting widespread dental therapy, teeth extraction, and considerable economic and social costs.41 dental caries is more prominent at interproximal surfaces and cervical margins where dental plaque in absence of salivary flow can be accumulated. the difficulty of tooth brushing in patients with oral mucosal cgvhd may result in neglecting oral hygiene, exacerbating the problem of salivary gland changes. apart from the effects on teeth, the risk of recurrent oral candidiasis threatens the patients with salivary gland cgvhd, especially if they are being treated by topical corticosteroid therapy.42 dental caries prevention is an important constituent of salivary gland cgvhd management. we recommend the measures given in table 5 for all patients with clinically significant disease. it is of great importance to continuously notify these patients of maintaining good oral hygiene and non-cariogenic diet. in the case of intense salivary gland hypofunction in which brushing after eating is impossible, patients should be encouraged to water-rinse their mouths after eating.43 there are increasing evidence on using calciumor phosphate-based remineralizing agent (e.g., gc mi paste plus, gc america) just before applying the topical fluoride.44 in order to further protection, dentists can apply fluoride varnish twice a year during patient referrals. oral cgvhd treatment oral pain or soft tissue sensitivity is the major complaint of patients with oral cgvhd. although several formulations of lidobenalox (magic mouthwash) exist, the basic three ingredients are the same which include an antihistamine (benadryl), a local anesthetic (lidocaine), and an aluminum/magnesium hydroxide (maalox®) to coat the oral cavity. two other formulations for this compound rinse are corticosteroid and antifungal ingredients which are beneficial for cgvhd patients. liquid dyclonine (usually 0.5%) is a potent topical anesthetic which can be prescribed for palliation of pain in the cases with table 5. recommendations regarding the screening, prevention, and management of late complications in oral cgvhd patients. derived from nathaniel et al.33 late complication prevention screening management -oral squamous cell carcinoma smoking interruption moderate consumption of alcohol yearly clinical examination atypical/suspicious lesions biopsy visiting a multidisciplinary headand-neck oncology center -rampant dental caries minimal consumption of refined carbohydrates, especially soft drinks containing sugar brushing at least twice a day after eating daily flossing daily applying of fluoride 1.1% gel paint on or in custom trays application of remineralizing agent with fluoride application of professional fluoride varnish the risk is greater in patients with considerable salivary gland cgvhd the risk is greater in patients with orofacial sclerotic cgvhd the risk is greater in in patients which severe mucosal disease who avoiding oral hygiene teeth examination for any evidence of cervical demineralization or decay referral to dental clinics twice a year • examination of soft and hard tissues • annual bitewing radiographs treatment of dental caries soon after diagnosed follow-up carefully for new or recurrent dental caries reinforcement of dietary habits and oral hygiene reinforcement of daily preventive measures -fibrosis unknown preventive measures asking patients on the existance of any tightness /limited opening widespread sclerotic skin disease, particularly if the patient’s neck is involved examination of intraoral buccal fibrotic bands via palpation -physical therapy -intralesional steroid therapy -surgery -systemic therapy for systemic involvement 200 review a review on therapeutic management for oral chronic graft-versus-host disease mina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 severe and intolerable pain. additionally, longor short-acting narcotics can be used before meals to allow for adequate nutrition.10 titration of effective systemic therapy is used to manage oral cgvhd. moreover, topical immunosuppressive treatment as well as ancillary supportive care may be also required for this purpose. oral cgvhd flares can be occurred in the following situations; when a patient stops or tapers a systemic immunosuppression, or when adjustment of drug levels. currently, management of mucosal symptoms of oral cgvhd is largely included the use of topical corticosteroids with high and ultra-high potency (table 6), calcineurin inhibitors and analgesics.44 the topical treatments are not always efficient. they may breakdown in mucosal integrity, increasing the risk of systemic absorption in patients with oral cgvhd. to improve the efficacy, some topical agents may be added to oral adhesive or oral rinse formulation. nevertheless, there are no clinical trials to determine the specific agents and dosing plans.31 oral rinses are largely applied in management of mucosal disease due to the effectiveness and easiness of this delivery mode. a number of oral rinses are usually used which may be titrated by systemic medications. dexamethasone and budesonide elixirs are two types of corticosteroid rinses able to be used for alleviation of oral ulcers symptoms, lichenoid/ hyperkeratotic reactions, and soft tissue sensitivity. topical therapy of the oral cavity by corticosteroid may result in thinning of the oral mucosal and increasing oral sensitivity over the time. moreover, topical corticosteroid therapy in short-term may result in accumulation of oral yeast species.33 generally, it is suggested that patients to be prophylactically treated by an antifungal troche or rinse, in addition to any systemic antifungal treatment, during treatment of patients with oral topical steroid. steroid drugs are lipophilic, with the ability to transverse the oral mucosa. in cases of severe oral cgvhd, the mucosal barrier integrity is also a failure. monitoring of patients for cushingoid features and increased adrenal suppression needs to be performed, and in the case of occurring such symptoms, topical treatments should be modified as accordingly.45 there are oral rinse formulations designed for directed topical therapies that can be used as systemic immunosuppressive medications, most of which were initially designed for oral lichen planus, which have many similar clinical features with oral cgvhd.46 ointments are another mechanism of topical delivery which are extremely efficient for patients with isolated symptoms of oral cgvhd. in these cases, ointments can be applied table 6. topical therapy recommendations for oral cgvhd. derived from jw et al, 2013. agent oral rinse corticosteroid oral ulcers oral ulcers soft tissue sensitivity caries hyperkeratosis/lichenoid candidiasis budesoinde yes yes yes yes chlorhexidine yes clobetasol yes yes yes yes cyclosporine yes yes yes dexamethasone yes yes yes yes dyclonine yes yes yes lidobenalox (magic mouth wash) only when formulated with dexamethasone yes yes yes nystatin yes sodium fluoride rinse yes tacrolimus yes yes yes other topicals azathioprine ointment yes clobetasol ointment yes yes clotrimazole troches yes cyclosporine ointment yes fluoride gel yes thalidomide ointment yes yes 201 review a review on therapeutic management for oral chronic graft-versus-host diseasemina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 as “spot treatment.” currently available topical ointments are azathioprine, tacrolimus, thalidomide, and cyclosporine. as an adjunct to oral treatment, local phototherapy has increasingly gained attention, and resulted in positive outcomes in some cases of dermatological cgvhd. for treatment of oral puva, an oral tablet of 8-methoxypsoralen is first administered, which sensitizes the oral mucosa of patients against uv exposure.33 chlorhexidine oral rinse has potent bactericidal attributes. its alcohol-free version is effective in controlling the oral bacterial flora of patients, especially those having difficulty with oral hygiene (e.g., flossing and brushing) or those with xerostomia who are exposed to dental decay. there are several studies supporting the effectiveness of shortand longterm pilocarpine therapy for salivary agitation in patients with cgvhd. however, using pilocarpine had been associated with increased gastric fluid secretions, making it unsuitable to be used in patients with gi tract gvhd.40 conclusion oral cgvhd is one of the common complications of hsct. the diagnosis of oral cgvhd is mostly done via clinical findings, but sometimes a biopsy is needed to confirm the condition. although is typically mild, the manifestations of this disorder are in the forms of hyperkeratotic plaques and confined oral opening secondary to sclerosis. oral cgvhd is not considered as a determinative factor of patients’ survival and its treatment is usually through locally applied corticosteroids. however, the first option to diagnosis of this disorder is conducting clinical trials. new studies on the pathogenesis of cgvhd are underway, and new therapy strategies are under active investigations such as expanding t-regulatory cells, targeting the processes implicated in fibrosis, and targeting at b-cells which are promising for future advances in cgvhd treatment. establishment of best treatment for cgvhd patients entails well-designed prospective studies. references [01]. dignan fl, amrolia p, clark a, et al. diagnosis and management of chronic graft-versus-host disease. br j haematol. 2012;158:46-61. [02]. kuten-shorrer m, woo sb, treister ns. oral graft-versus-host disease. dent clin north am. 2014;58:351-68. [03]. dignan fl, clark a, amrolia p, et al. diagnosis and management of acute graft-versus-host disease. br j haematol. 2014;158:30-45. [03]. jaglowski sm, devine sm. graft-versus-host disease: why have we not made more progress? curr opin hematol. 2014;21:141-7. [05]. paczesny s. discovery and validation of graft-versus-host disease biomarkers. blood. 2013;121:585-94. [06]. linhares yp, pavletic s, gale rp. chronic gvhd: where are we? where do we want to be? will immunomodulatory drugs help? bone marrow transplant. 2013;48:203-9. [07]. travnik r, beckers m, wolff d, et al. graft-versus-host disease (gvhd) an update: part 1: pathophysiology, clinical features and classifcation of gvhd. hautarzt. 2011;62:139-54. [08]. elad s, zeevi i, resnick ib, et al. valida-tion of the national institutes of health (nih) scale for oral chronic graft-versus-host disease (cgvhd). biol blood marrow transplant. 2010;16:62-9. [09]. michael t. b, bengt h, allan j. h, impact of oral side effects from conditioning therapy before hematopoietic stem cell transplantation: protocol for a multicenter study. jmir res protoc. 2018;7(4): e103. [10]. salmasian h, rohanizadegan m, banihosseini s, et al. corticosteroid regimens for treatment of acute and chronic graft versus host disease (gvhd) after allogenic stem cell transplantation. cochrane database syst rev. 2010;20:cd005565. [11]. arai s, jagasia m, storer b, et al. global and organ-specific chronic graftversus-host disease severity according to the 2005 nih consensus criteria. blood. 2011;118(15):4242-4249. [12]. claudio a, brent r. l, stephanie j. l, peripheral-blood stem cells versus bone marrow from unrelated donors. n engl j med. 2013;367(16): 10.1056/ nejmoa1203517. [13]. jacobsohn da. optimal management of chronic graft-versus-host disease in children. br j haematol. 2010;150(3):278-292. [14]. flowers me, inamoto y, carpenter pa, et al. comparative analysis of risk factors for acute graft-versus-host disease and for chronic graftversus-host disease according to national institutes of health consensus criteria. blood. 2011;117(11):3214-3219. [15]. kelli p. a, geoffrey r. h, bruce r. b. chronic graft-versus-host disease: biological insights from preclinical and clinical studies. blood. 2017;129:13-21. [16]. division of hematopoietic stem cell transplantation, national cancer center hospital. 2018 update on chronic graft-versus-host disease. rinsho ketsueki. 2018;59(10):2300-2306. [17]. arora m, klein jp, weisdorf dj, et al. chronic gvhd risk score: a center for international blood and marrow transplant research analysis. blood. 2011;117:6714–20. [18]. ferrara jl, levine je, reddy p, et al. graft-versus-host disease. lancet. 2009;373:1550-61. [19]. meier jk, wolff d, pavletic s, et al. oral chronic graft-versus-host disease: report from the international consensus conference on clinical practice in cgvhd. clin oral investig. 2011;15:127–39. [20]. mays jw, fassil h, edwards da, et al. oral chronic graft-versus-host disease: current pathogenesis, therapy, and research. oral dis.2013;19:327-46. [21]. maria m, josé v. b, yolanda j, et al. graft-versus-host disease affecting oral cavity. a review. j clin exp dent. 2015;7(1): e138-45. [22]. mahesh k, sujay k. acute graft versus host disease. indian journal of dermatology, venereology and leprology. 2011;77(2):217-9. [23]. blazar br, murphy wj, abedi m. advances in graft-versus-host disease biology and therapy. nat rev immunol. 2012;12:443–58. [24]. robert z, bruce r. b. preclinical models of acute and chronic graft-versushost disease: how predictive are they for a successful clinical translation? blood 2016;127:3117-3126. [25]. schroeder ma, dipersio jf. mouse models of graft-versus-host disease: advances and limitations. dis model mech. 2011;4:318–33. [26]. piccin a, tagnin m, vecchiato c, et al. graft-versus-host disease (gvhd) of the tongue and of the oral cavity: a large retrospective study. int j hematol. 2018;108(6):615-621. [27]. stefanie s, adela rc, keith ms. how i treat refractory chronic graft-versushost disease. blood. 2019;133(11):1191-1200. [28]. jw mays, h fassil, da edwards, et al. oral chronic graft-versus-host disease: current pathogenesis, therapy, and research. oral dis. 2013;19(4): 327–346.’ [29]. imanguli mm, atkinson jc, mitchell sa, et al. salivary gland involvement in chronic graft-versus-host disease: prevalence, clinical significance, and recommendations for evaluation. biol blood marrow transplant. 2010;16:1362–9. [30]. julia s, rosa-maría l. p, josé g. s, oral lesions in sjögren’s syndrome: a systematic review. med oral patol oral cir bucal. 2018 jul 1;23 (4): e391-400. [31]. carpenter p. a, kitko c. l, elad s, et al. national institutes of health consensus development project on criteria for clinical trials in chronic graft-versushost disease: v. the 2014 ancillary therapy and supportive care working group report. biol blood marrow transplant. 2015;21(7):1167-87. [32]. diana mc, claire jd, michael js, et al. use of the national institutes of health consensus guidelines improves the diagnostic sensitivity of gastrointestinal graft-versus-host disease. arch pathol lab med. 2017;142:1098–1105. [33]. nathaniel t, c duncan, c cutler, et al. how we treat oral chronic graftversus-host disease. blood. 2012;120(17). [34]. hull km, kerridge i, schifter m. long-term oral complications of allogeneic haematopoietic sct. bone marrow transplant. 2012;47(2):265–70. [35]. garming-legert k, remberger m, ringden o, hassan m, dahllof g. longterm salivary function after conditioning with busulfan, fractionated or single-dose tbi. oral dis. 2011;17:670–6. [36]. fassil h, bassim cw, mays j, et al. oral chronic graft-vs.-host disease characterization using the nih scale. j dental res. 2012;91:s45–s51. [37]. laura gc, renata lf, carmem b, et al. oral manifestations compatible with chronic graft-versus-host disease in patients with fanconi anemia. 2015;21(2):275-280. [38]. hani m, shahrukh kh, sharon e, et al. chronic graft‐versus‐host disease: current management paradigm and future perspectives. 2018;25(4):931-948. [39]. daniel w, armin g, francis a, et al. consensus conference on clinical practice in chronic graft-versus-host disease (gvhd): first-line and 202 review a review on therapeutic management for oral chronic graft-versus-host disease mina khayamzadeh j contemp med sci | vol. 6, no. 5, september-october 2020: 193–202 topical treatment of chronic gvhd. biol blood marrow transplant. 2010;16(12):1611-1628. [40]. castellarin p, stevenson k, biasotto m, et al. extensive dental caries in patients with oral chronic graft-versus-host disease. biol blood marrow transplant. extensive dental caries in patients with oral chronic graft-versushost disease biol blood marrow transplant. 2012 oct;18(10):1573-9 . doi: 10.1016/j.bbmt.2012.04.009 2012 [41]. hamid r. m, masoud s, ladan j, et al. oral manifestation and dental management in a patient with chronic graft versus host disease with around a 10-year follow-up biomedical research and therapy. 2018;5(6):2370-2377. [42]. bollero p, passarelli pc, d’addona a, et al. oral management of adult patients undergoing hematopoietic stem cell transplantation. eur rev med pharmacol sci. 2018;22(4):876-887. [43]. valéria cc. cochrane fluoride reviews: an overview of the evidence on caries prevention with fluoride treatments. 2014;5(2):78-83. [44]. cochrane nj, cai f, huq nl, et al. new approaches to enhanced remineralization of tooth enamel. j dent res. 2010;89(11):1187-1197. [44]. wolff d, gerbitz a, ayuk f, et al. consensus conference on clinical practice in chronic graftversus-host disease (gvhd): first-line and topical treatment of chronic gvhd. biol blood marrow transplant. 2010;16:1611–28. [45]. joseph p. graft-vs-host disease following allogeneic hematopoietic cell transplantation. blood and marrow transplant program, 2011;18(4):268-276. [46]. elad s, zeevi i, finke j, et al. improvement in oral chronic graft-versushost disease with the administration of effervescent tablets of topical budesonide-an open, randomized, multicenter study. biol blood marrow transplant. 2012;18:134–40. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.852 229j contemp med sci | vol. 6, no. 5, september-october 2020: 229–233 original issn 2413-0516 introduction celiac disease (cd) is an immune-mediated enteropathy.1 it is characterized by an immune-mediated response to gluten present in foods.2 cd-associated symptoms are often triggered by production of autoantibodies and in many cases flattening of the intestinal villi.3 its prevalence is currently estimated at 1:100 of the western countries,4 and is rising in the middle eastern populations to 1.5:100.5-7 cd may occur in adults and children,8 with a female/male ratio of 3:1.9 about 90% of the cd patients carry a major histocompatibility complex class ii molecule, human leukocyte antigen (hla) variant dq2 and most of the remainder of patients carry an hla variant named dq8.10 the clinical presentation of cd ranges from asymptomatic form to a symptomatic which could represent a general malabsorption (typical or classical) or extra intestinal symptoms (atypical or symptomatic).11, 12 the long-term consequences of undiagnosed and/or untreated cd can lead to a form of unresponsiveness (refractory celiac) to gluten-free diet (gfd) and malignancies.11 because of its atypical presentation, a large portion of cd patients remain undetectable. this atypical form include the presentation of patients with extra intestinal symptoms, a silent form which can present with positive serology and histological changes of intestinal mucosa with no symptoms13; a potential cd form which can present with a positive serology with no architectural changes in mucosa histology; and a latent form which can be defined as normal histological mucosa with no serological abnormalities.14, 15 the diagnosis of cd requires a joint clinicopathological approach; the recommended first-line test is serology with immunoglobulin a (iga) tissue transglutaminase, deaminated gliadin iga and/or iga endomysial antibodies. these serological tests show high levels of sensitivity and specificity, but biopsy is the gold-standard to confirm the diagnosis.16 the latter being based on the modified marsh grading system and is the most conclusive current test for cd.17 the modified marsh grading criteria includes an architectural changes of the mucosa (atrophy of villi and crypts hyperplasia) with a rise in the number of intraepithelial lymphocytes (iels).18 the classical histopathology changes of cd are partial or total villous atrophy and increase of iels. however, the pathology classification of cd is changing, with recognition that cd may show minimal pathology (normal architecture and an iel count ≥25⁄100 enterocytes). this entity is also described as lymphocytic duodenitis, and recommendation of follow-up serology testing is paramount in this condition.19, 20 an increase in the number of iel is the hallmark of early stage cd as it is the first and most sensitive index of the effects of gluten on the mucosa.21 yet, the threshold of what is considered normal is still debatable. studies have suggested a count of 20-25/100 ec to be borderline lymphocytosis with a value equal or above 25 as pathologic22 other authors suggests even a lower number to be as an increase in iels.23, 24 the distribution of the iels is also being considered as a morphological feature of cd.21, 25 the iels comprise of a significant lymphocyte population residing in close immediacy to the intestinal lumen between enterocytes in the intestinal epithelium.21, 26 phenotypically, in cd patients iels are t lymphocytes, of which the majority 70% are cd8+ t cells and less than 20% are cd4+ t-cells. b cells are not present.21, 26-28 several studies have suggested cd to be of a th1-mediated immune response.29, 30 the predominant cytokine produced by t-cell isolated from cd patients mucosa which have a significant role in the lesion formation is ifn gamma.31 this view is supported by other studies on evaluation of t-bet immunostaining in the counting of intraepithelial lymphocytes in celiac disease amna e. al-araji1, tuqa z. omran2*, mohanad m. ahmed3, nazar j. metib4 1 department of pathological analysis, university of kafeel, al-najaf, iraq. 2 department of medical sciences, university of al-ameed, kerbala, iraq. 3 department of medical microbiology and immunology, kerbala university, kerbala, iraq. 4 consulting pathologist, al-hussein medical city, kerbala, iraq. *corresponding author: tuqa z. omran ( email: tukhafaji@alameed.edu.iq ) (submitted: 10 july 2020 – revised version received: 19 july 2020 – accepted: 21 august 2020 – published online: 30 october 2020) abstract objective: in this study, we aimed to evaluate cd3, t-bet, and gata3 staining of intraepithelial lymphocytes (iels) in comparison to the routine h&e stains in celiac disease. methods: a total of 50 patients, in whom celiac disease was diagnosed based on a combination of clinicopathological features, were enrolled in the study. duodenal biopsied tissues were processed routinely into formalin fixed paraffin embedded (ffpe) blocks. sections were prepared and stained with h&e and each of cd3, t-bet and gata3. a number of histological criteria were measured to calculate the marsh score. the results were analyzed using the ibm spss analytic software. results: a positive correlation was found between the count of t-bet stained cells and the increase of iels (p-value = 0.001). in addition, low count of gata3 stained cells was seen in almost all cases. the count of gata3 stained cells was not affected by the increase in iels count. conclusions: the majority of increased iels were stained with t-bet. whereas in normal iels count is less than half the iels were stained with t-bet. this would indicate that t-bet immunostaining is a potential alternative to h&e and/or cd3-based counting of iels. key words: t-bet; immunostaining; gluten sensitive enteropathy; distribution pattern; marsh grading; immunohistochemistry. 230 original evaluation of t-bet immunostaining in the counting of intraepithelial lymphocytes amna e. al-araji j contemp med sci | vol. 6, no. 5, september-october 2020: 229–233 the intestinal lamina propria isolated t cells, underlined by the increase of cytokines ifn gamma and il-18.32, 33 t-bet is a specific t box transcription factor which correlates with ifn gamma expression in th1 and natural killer cells.34 reports suggest that the t-bet transcription factor is upregulated in active cd then down regulated to normal levels in treated (on gfd) cd patients.32 however, the significance of counting t-bet expressing cells was not evaluated thoroughly. this study was aimed to investigate the value of the counting and distribution of types of iels in relation to other parameters to improve the diagnosis of cd. materials and methods this is a cross-sectional study of which the samples collected were formalin-fixed paraffin embedded (ffpe) proximal duodenal tissue of 50 consecutive cd patients. they were collected from al-hussainy teaching hospital, al-kafeel super-specialty hospital and al-sajjad medical laboratory in the city of kerbala from august 2019 to february 2020. cd was diagnosed based on a combination of suggestive clinical presentation, positive serology, and/or indicative histology based on the h&e staining results. processing and staining of the ffpe samples was performed. h&e staining of the sections was done and marsh grading was judged according to the modified marsh criteria.35 the marsh grading system was chosen in this research as it provides a wide more detailed view of the histopathological changes (lesions) that appears in the duodenal mucosa of cd patients.36 these changes include the count of iels and the villus architecture, being preserved or atrophied in addition to the degree of atrophy and the hyperplasia of the crypts. immunohistochemistry was preformed, on 3–5 μm sections, using monoclonal antibodies directed against cd3 (clone pc3/188a, mouse monoclonal igg1, dako a/s denmark, gata3 (clone hg3-31, mouse monoclonal antibodies, santa cruz biotechnology, inc.) and t-bet (clone 4b10, mouse monoclonal igg1, santa cruz biotechnology, inc.). prior to incubation with the primary antibodies, the de-waxed 3–5 μm thick sections were subjected to an antigen retrieval procedure consisting of a high-temperature heating of the sections immersed in envision™ flex target retrieval solution, high ph for 30 min. bound antibodies were made visible using a detection kit purchased from leica biosystems newcastle ltd. uk, novolinktm polymer detection system. the resulting image was a nuclear staining for the t-bet and gata3, while the cd3 showed a membranous staining of brown color due to the dab chromogen. data were analyzed using ibm spss analytic software. results of the 50 samples, 38 (76%) were females and 12 (24%) were males, female to male ratio was 3.2:1. their ages range 3–65 years. of those patients, only 34 (68%) of them had a positive serology (table 1). on the h&e stain, 40 (80%) of the samples showed an increase of the iels (30 iels/100 ec) in the duodenal villi while the other 10 samples showed only a borderline increase (20–29 iels/100 ec). on staining with the cd3 monoclonal antibody, of the 40 (80%) samples only 32 (64%) cases showed an increase of iels of 30/100 ec. while the remaining of the 40 (80%) had either a borderline or a normal count of iels, as did the other 10 (20%) cases. the t-bet monoclonal antibodies were used to label and track the th1 lineage of iels in cd. the iels in the 32 (64%) samples counted by cd3, were counted by t-bet, with the t-bet most of the cases had 30 iels/100 ec, the remaining few showed a borderline count 20–29 iels/100ec. in general, the staining with t-bet showed a lower count of iels. this decrease of the iels count could be attributed to the fact that t-bet can only stain the th1 lymphocyte cell, while the cd3 stains all of the cd3 barring t-lymphocytes including th1 and th2, making the t-bet to be a more specific marker for cd than do cd3. on staining with gata3, a markedly low percentages of 5% of the iels in cd samples were stained with gata3. deducing that the th2 has no significant role in the pathogenesis of cd. the weak presence of th2 cells in the duodenal biopsy of cd-diagnosed patients further supports the previous finding, which is that cd is of a th1-mediated immune response. table 1. patient’s characteristics. characteristics frequency percent% gender female 38 76.0 male 12 24.0 age groups 0-10 1 2.3 11-20 12 27.3 21-30 14 31.8 31-40 9 20.5 41-50 7 15.9 >51 1 2.3 serology negative 14 28.0 positive 34 68.0 borderline 2 4.0 marsh grade 0* 17 34.0 1 10 20.0 2 6 12.0 3* 17 34.0 * all grades of marsh (iiia, iiib, iiic) are expressed in this table as grade iii. * grade 0 are patients with positive serology and a borderline increase of iels which is counted as (20-29/100ec) in this study. table 2. means and slandered deviations of the iels count with different stains in celiac disease diagnosed patients. stain iels count (mean sd) p value compared to h&e h&e 41.25±6.2 cd3 34±5.8 0.001 t-bet 30.2±6.8 0.001 gata 3 3.2±3.49 0.001 231 original evaluation of t-bet immunostaining in the counting of intraepithelial lymphocytesamna e. al-araji j contemp med sci | vol. 6, no. 5, september-october 2020: 229–233 a highly significant correlation was found between the number of iels stained by t-bet and the marsh grades (p = 0.001). it was observed that with the increase of iels, counted by h&e and/or by cd3 staining, there was also an increase in the t-bet stained iels (th1 lineage), showing that most of the increased iels were t-bet positive. these results suggest the possibility of using the t-bet as an important tool for the counting of iels and possibly the diagnosis of cd. nineteen cases of the diagnosed cd (positive serology), marsh stages i, ii, iiia, iiib, showed a high staining percentage of the iels with t-bet (70–90%), and cd3 (80–100%), while the gata3 showed a markedly low staining percentage (1–5%) (fig. 1, table 3). in a comparison with the diagnosed celiac cases (which either had a positive serology or an indicative histology), of the negative serology cases which showed an indicative histology (marsh i-iiic), all showed a staining percentage of no less than 70% with t-bet while the gata3 showed less than 5% staining positivity. as for the cases with positive serology, some with borderline increase in iel showed increased t-bet positivity with also no gata3 staining. interestingly, of the positive serology cases, two of them had a normal count of iels (18) yet they had a high staining positivity for the t-bet of no less than 80%. while another case with negative serology and a borderline increase iel count (20–29/100ec) with cd3 showed a high positive staining of the iels with the t-bet monoclonal antibodies. regarding the distribution patterns of the t-bet stained iels in the villi of the duodenum mucosa, table 4 shows the majority 68% of the cases showed regular distribution of the t-bet stained iels throughout the villi body, while only 2% of the cases showed an aggregation of the iels in the base of villi. the relation between marsh stages and the distribution pattern of t-bet stained iels is highly significant. discussion histopathological assessment of biopsied duodenal tissue is of vital role for the diagnosis of cd. however, a subjective grading system that is currently being used tends to misdiagnose table 3. percentage of stained cells in different counts of iels. stain normal iels count increases iels count cd3 <40 80-100 % t-bet 0-40 % 70-90 % gata3 1-5 % 0-7 % * a different expression level of t-bet stained cells were observed when there was a borderline increase of iels. d b c a fig 1. high power view of cd diagnosed patient marsh iiia, the same tissue sample compared in different stains. black arrows: shows the stained intraepithelial lymphocytes. a: h&e, b: cd3 cytoplasmic staining pattern, c: t-bet staining of the th1 type iels (the responsible in cd pathogenesis) p-value < 0.001, d: gata-3 staining of th2 type iels shows minimal count of iels. x40. 232 original evaluation of t-bet immunostaining in the counting of intraepithelial lymphocytes amna e. al-araji j contemp med sci | vol. 6, no. 5, september-october 2020: 229–233 some cases, especially in the absence of an architectural changes.37 counting the iels in h&e stained sections is not a straightforward process. it is not always possible to distinguish the iels nuclei from the enterocytes as they may be present in a multitude of shapes or due to the overlapping and lack of contrast between cells. the usage of immunohistochemistry is very helpful in the counting of iels.38 owing to the fact that it is cell type specific staining, ihc offers the ability to distinguish the type of iels, and the nuclei form the enterocytes. studies have suggested that the ihc staining of iels by cd3 can improve the diagnosis in cd patients of the marsh i lesions,38, 39 as the t-lymphocytes are cd3+ cells. the guidelines of british society of gastroenterology stipulate the that use of ihc staining in borderline cases is vital.37, 40 many other studies used the cd3 mononuclear antibody to improve the count of iels in an attempt to either detect the early stages of cd or improve contrast to count iels in seropositive cases or simply to study the celiac-related iels.19, 41 in the current study, counting of iels based on cd3 was compared to h&e based counting. we found that the count of the cd3 stained cells was lower than that stained with h&e. t-bet staining of duodenal iels in celiac diagnosed cases was investigated in this study. a highly significant correlation was found between the number of iels and the percentage of t-bet staining. cd3 eliminated the issue of overcounting the iels and their overlapping with other cell nucleus that was faced with the h&e. however, it is not specific for cd. recent study proposed that the cd3 was of no more use in the diagnosis of the normal histology cases of cd than is the staining with h&e (20). therefore, the search of a more specific marker for cd would serve as a more reliable method for diagnosing cd, especially in early and potential cases. very few studies examined t-bet expression in limited number of cd patients.42, 43 they found the t-bet expression in iels of duodenal biopsy was increased, therefore a role for t-bet was suggested in cd. however, the use of t-bet expression in the diagnosis of cd was not investigated. the current study aimed to investigate t-bet role in cd. these results shows that most of the increased iels were t-bet positive, suggesting that t-bet expression in iels of cd patient is of significance. it also is supportive of the studies which suggests that cd is a th1-mediated inflammatory reaction. the t-bet staining was found to be superior to the cd3 ihc staining in settings of the contrast, intensity, and background staining probably due to the fact that t-bet selectivity of only th1 cells rather than all the cd3+ cells like with the cd3 monoclonal antibodies. these findings suggests the possibility of using the t-bet monoclonal antibodies for counting iels in cd as a useful diagnostic tool, while the gata3 was found to be of low expression. this was similar to what was found in the literature.43, 44 of the 50 cases, 2 cases with positive serology had a high positivity for t-bet even with a normal iel counts (0–19). this result could be interpreted with a potential or a latent cd of the 2 cases indicating the usefulness of using t-bet in detecting latent or potential cd cases. in 19 cases of serology positive (including anti-ttg iga) and histology indicative of cd, increased iels in cd3 and h&e staining with crypt hyperplasia and some cases showed atrophy in the villi in addition to the clinical indications, the tbet expression in iels was of no less than 70% of the cells. in 10 cases of serology positive (but anti-ttg iga negative) and a negative histology, meaning no increase in iels, no crypt hyperplasia nor villus atrophy, they showed less than 40% expression of t-bet by iels. while in 10 cases of seronegative with an architectural changes including villus atrophy (iiia, iib, iiic), the t-bet staining positivity was also high (70–10%). another criteria that have been suggested in the literature by some authors is to distinguish early stages of cd via the distribution pattern of the iels. a study of over 400 biopsies suggested that iels in cd were not normally distributed.41 dickson et al. suggested that the loss of the normal “decrescendo” pattern of normal iels distribution of duodenal villi of patients is suggestive of cd (dickson et al., 2006). mino et  al. found that an aggregation of iels in the tip of villi observed in cd3 staining is suggestive of cd (mino and lauwers, 2003). goldstein et al. found that a regular distribution pattern along the villi was most sensitive of an association of celiac marsh i lesion (goldstein and underhill, 2001), it was also suggested that the increase of mean iel in the villus tip is associated with cd (goldstein and underhill, 2001). in other words, they suggested that the regular distribution of iels ≥25 in architecturally normal villus is indicative of cd, they also note that a gestalt approach should be applied in assessing the biopsy by the pathologists. these findings are similar to what we found in the duodenal biopsy of cd patient, regular distribution of increased iels in the duodenal villus. these results were highly significant in correlation with the marsh stages. all of the mentioned cases have studied the iels distribution with either the cd3 staining or with the h&e stain. in the current study, the iels distribution was examined with the t-bet monoclonal antibodies which showed 10 cases of marsh iii with an aggregation of cells in the tip. another 6 cases had a regular distribution. on the other hand, 17 cases of grade 0 marsh with seropositive and ≤19 iels had also a regular distribution of iels along the villi, which showed a statistical significance in comparison with marsh grading system (p < table 4. distribution pattern of tbet stained iels in correlation with marsh grades in the duodenal villi of celiac disease diagnosed patients. marsh stages total percent % p value 0 1 2 3* distribution pattern of t-bet stained iels in villi flat villi 0 0 0 3 3 6 % p<0.01 aggregated in the tip 0 2 0 10 12 24 % aggregated in the base 0 0 1 0 1 2 % regular distribution 17 8 3 6 34 68 % marsh grade 3 includes (iiia, iiib, iiic). 233 original evaluation of t-bet immunostaining in the counting of intraepithelial lymphocytesamna e. al-araji 0.001) and (p < 0.017) in comparison with the t-bet stained iels. this could be suggestive that the distribution pattern of iels can be indicative of early stage cd. conclusion the majority of increased iels were stained with t-bet, whereas in normal iels count, there was less than half the iels stained with t-bet. this would indicate that t-bet immunostaining is a potential alternative to h&e and/or cd3-based counting of iels. compliance with ethical standards the authors declare that they have no conflict of interest. the author declare that research involved human participants and consent was obtained. this research was funded by the authors. references 1. sharma a, wang xj, russo pa, wu t-t, nehra v, murray ja. features of adult autoimmune enteropathy compared with refractory celiac disease. clin gastroenterol hepatol. 2018;16(6):877-83. e1. 2. lerner a. serological diagnosis of celiac disease–moving beyond the tip of the iceberg. int j celiac dis. 2014;2:64-6. 3. leon f. flow cytometry of intestinal intraepithelial lymphocytes in celiac disease. j immunol methods. 2011;363(2):177-86. 4. mustalahti k, catassi c, reunanen a, fabiani e, heier m, mcmillan s, et al. the prevalence of celiac disease in europe: results of a centralized, international mass screening project. ann med. 2010;42(8):587-95. 5. barada k, bitar a, mokadem ma-r, hashash jg, green p. celiac disease in middle eastern and north african countries: a new burden? world j gastroenterol. wjg. 2010;16(12):1449-57. 6. barada k, daya ha, rostami k, catassi c. celiac disease in the developing world. gastroint endosc clin. 2012;22(4):773-96. 7. al-hussaini a, troncone r, khormi m, alturaiki m, alkhamis w, alrajhi m, et al. mass screening for celiac disease among school-aged children: toward exploring celiac iceberg in saudi arabia. j f pediat gastroenterol nutr. 2017;65(6):646-51. 8. cappello m, morreale gc, licata a. elderly onset celiac disease: a narrative review. clin med insights gastroentero. 2016;9:cgast.s38454. 9. gonzález da, de armas lg, rodríguez im, almeida aa, garcía mg, gannar f, et al. strategies to improve the efficiency of celiac disease diagnosis in the laboratory. j immunol methods. 2017;449:62-7. 10. reinton n, helgheim a, shegarfi h, moghaddam a. a one-step real-time pcr assay for detection of dqa1*05, dqb1*02 and dqb1*0302 to aid diagnosis of celiac disease. j immunol methods. 2006;316(1):125-32. 11. green ph, cellier c. celiac disease. new engl j med. 2007;357(17):1731-43. 12. agardh d, lee h-s, kurppa k, simell v, aronsson ca, jörneus o, et al. clinical features of celiac disease: a prospective birth cohort. pediatrics. 2015;135(4):627-34. 13. fei bao, peter h.r. green, govind bhagat. an update on celiac disease histopathology and the road ahead. arch pathol lab med. 2012;136(7):73545. 14. volta u, villanacci v. celiac disease: diagnostic criteria in progress. cell mol immunol. 2011;8:96. 15. ludvigsson jf, leffler da, bai jc, biagi f, fasano a, green ph, et al. the oslo definitions for coeliac disease and related terms. gut. 2013;62(1):43-52. 16. walker mm, murray ja. an update in the diagnosis of coeliac disease. histopathology. 2011;59(2):166-79. 17. charlesworth rpg, andronicos nm, scott dr, mcfarlane jr, agnew ll. can the sensitivity of the histopathological diagnosis of coeliac disease be increased and can treatment progression be monitored using mathematical modelling of histological sections? – a pilot study. adv med sci. 2017;62(1):136-42. 18. bai jc, fried m, corazza gr, schuppan d, farthing m, catassi c, et al. world gastroenterology organisation global guidelines on celiac disease. j clin gastroenterol. 2013;47(2):121-6. 19. kurppa k, ashorn m, iltanen s, koskinen lle, saavalainen p, koskinen o, et al. celiac disease without villous atrophy in children: a prospective study. j pediat. 2010;157(3):373-80.e1. 20. hudacko r, kathy zhou x, yantiss rk. immunohistochemical stains for cd3 and cd8 do not improve detection of gluten-sensitive enteropathy in duodenal biopsies. mod pathol off j us can acad pathol, inc. 2013;26(9):1241-5. 21. abadie v, discepolo v, jabri b. intraepithelial lymphocytes in celiac disease immunopathology. semin immunopathol. 2012;34(4):551-66. 22. corazza g, villanacci v. coeliac disease. j clin pathology. 2005;58(6):573-4. 23. pellegrino s, villanacci v, sansotta n, scarfì r, bassotti g, vieni g, et al. redefining the intraepithelial lymphocytes threshold to diagnose gluten sensitivity in patients with architecturally normal duodenal histology. aliment pharmacol therap. 2011;33(6):697-706. 24. siriweera eh, qi z, yong jl. validity of intraepithelial lymphocyte count in the diagnosis of celiac disease: a histopathological study. int j celiac dis. 2015;3:156-8. 25. kabiraj a, gupta j, khaitan t, bhattacharya pt. principle and techniques of immunohistochemistry – a review. 2015. 5204-10 p. 26. cheroutre h, madakamutil l. acquired and natural memory t cells join forces at the mucosal front line. nat rev immunol. 2004;4(4):290. 27. neutra mr, mantis nj, kraehenbuhl j-p. collaboration of epithelial cells with organized mucosal lymphoid tissues. nat immunol. 2001;2(11):1004. 28. chang f, mahadeva u, deere h. pathological and clinical significance of increased intraepithelial lymphocytes (iels) in small bowel mucosa. apmis. 2005;113(6):385-99. 29. mcallister cs, kagnoff mf, editors. the immunopathogenesis of celiac disease reveals possible therapies beyond the gluten-free diet. seminars in immunopathology; 2012: springer. 30. harris km, fasano a, mann dl. monocytes differentiated with il-15 support th17 and th1 responses to wheat gliadin: implications for celiac disease. clin immunol. 2010;135(3):430-9. 31. di sabatino a, corazza gr. coeliac disease. the lancet. 2009;373(9673):1480-93. 32. rajendran a, sivapathasundharam b. shafer’s textbook of oral pathology: elsevier health sciences; 2014. 33. jöhrens k, anagnostopoulos i, stein h. t‐bet expression patterns in coeliac disease, cryptic and overt enteropathy‐type t‐cell lymphoma. histopathology. 2005;47(4):368-74. 34. salvati v, macdonald t, bajaj-elliott m, borrelli m, staiano a, auricchio s, et al. interleukin 18 and associated markers of t helper cell type 1 activity in coeliac disease. gut. 2002;50(2):186-90. 35. stanford medicine. celiac disease 2018. available from: http:// surgpathcriteria.stanford.edu/gi/celiac-disease/marsh.html. 36. villanacci v, ceppa p, tavani e, vindigni c, volta u. coeliac disease: the histology report. digest liver dis. 2011;43:s385-s95. 37. ludvigsson jf, bai jc, biagi f, card tr, ciacci c, ciclitira pj, et al. diagnosis and management of adult coeliac disease: guidelines from the british society of gastroenterology. gut. 2014:gutjnl-2013-306578. 38. mubarak a, wolters vm, houwen rhj, ten kate fjw. immunohistochemical cd3 staining detects additional patients with celiac disease. world j gastroenterol. wjg. 2015;21(24):7553-7. 39. veress b, franzén l, bodin l, borch k. duodenal intraepithelial lymphocyte‐ count revisited. scand j gastroenterol. 2004;39(2):138-44. 40. ensari a. gluten-sensitive enteropathy (celiac disease): controversies in diagnosis and classification. arch pathol lab med. 2010;134(6):826-36. 41. rostami k, marsh mn, johnson mw, mohaghegh h, heal c, holmes g, et al. roc-king onwards: intraepithelial lymphocyte counts, distribution & role in coeliac disease mucosal interpretation. gut. 2017:gutjnl-2017-314297. 42. holtmann mh, neurath mf. t helper cell polarisation in coeliac disease: any (t-)bet ? gut. 2004;53(8):1065-7. 43. jöhrens k, grünbaum m, anagnostopoulos i. differences in the t-bet and gata-3 expression patterns between lymphocytic colitis and coeliac disease. virchows archiv. 2010;457(4):451-6. 44. omran tz, ahmed mm, metib nj. assessment of gata3 expression in duodenal biopsies of celiac disease suspected and diagnosed patients. iraq med j. 2018(3):72-4%v 2. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.861 156 j contemp med sci | vol. 6, no. 4, july-august 2020: 156–160 original issn 2413-0516 introduction: the restorative treatment threshold can be defined as the point at which dentists would begin drilling a carious tooth to make a restoration.1 this process can be continued with other restorations in the earlier lesion or another, which is against the newer concept of caries prevention in dentistry. dental caries can be remineralized in early, non-cavitated stages.2 because the demineralization is an inverse action and can shift to remineralization, it is possible to delay the operative treatment for non-cavitated lesions.3there are variations in dentists’ decisions to do the restorative treatment when the caries lesion limited to enamel or at the beginning of extension to dentin. these variations are mainly related to their knowledge and working experiences.4–6 variations also depend on the opinions of the dentists in extending the restorations, where many of them tend to restore the carious teeth in the early stages. a study showed that some dentists had more propensity to replace the whole affected restorations, while dentists who prefer to delay the starting of operative treatment until the lesion developed to advanced stage, recommended repair instead of replacement.7 the consideration of caries risk level in patients by dentist may differ based on the age and work experience.8numerous epidemiological studies were conducted in iraq concerning dental caries and its related factors; however, most of them concerned with preschool or primary school children. the percentage of tooth decay in iraq for the age group of 12 years is considered low, ranging from 1.2 to 2.5. however, dental treatment need is high, especially in adolescents who will make the future of country.9–11 minimal invasive restorative treatments are in the scope of modern dentistry. to our knowledge, there is no study regarding dentists’ decision on the start point of restorative treatment in iraq and although it is influenced by caries risk level of the patients, most of the iraqi dentists may also tend to restore the carious teeth in the early stages.12 the main aim of the present study was to explore the iraqi dentists’ decision-making regarding the restorative treatment for dental caries. method and materials ethical considerations dentists’ participations were under a voluntary basis, and followed declaration of helsinki ethical principles for medical research involving human subjects. tehran university of medical sciences research ethics committee gave the ethical approval (id number: ir.tums.vcr.rec.1396.1984) study design and population a cross-sectional study was performed among general dental practitioners (gdps) who worked in specialist dental centers and primary health-care centers in baghdad based on a self-administered questionnaire in 2016.13 a convenient sampling method was adopted to select the gdps from the centers. the response rate was 94% (n=150) from 159 questionnaires distributed. questionnaire and variables survey questionnaire was based on a questionnaire that was previously established by ghasemi et al.1, 11 it covered iraqi dentists’ risk-based restorative treatment decisions fatin abd al sattar aledhari1,2, katayoun sargeran3*, mahdia gholami4, ahmad reza shamshiri4 1 department of technical affairs dental division, diwaniyah health office, iraqi ministry of health, diwaniyah, iraq 2 school of dentistry, university of qadisiyah, diwaniyah, iraq 3 research center for caries prevention, dentistry research institute, department of community oral health, school of dentistry, tehran university of medical sciences, tehran, iran 4 research center for caries prevention, dentistry research institute, department of community oral health, school of dentistry, tehran university of medical sciences, tehran, iran corresponding author: katayoun sargeran, (e-mail: k-sargeran@tums.ac.ir ) abstract objective: the aim of the present study was to investigate the decision-making by iraqi dentists concerning the use of restorations in the treatment of dental caries. methods: a cross-sectional study based on a self-administered questionnaire was conducted among 159 dentists who worked in public health sector in baghdad during summer of 2016. a questionnaire which obtained information on age, attitude, knowledge, and barriers, in addition to two scenarios designed to investigate dentists’ restorative treatment threshold, was used. paper patient cases (ppcs) were indicated high-risk (hr) and low-risk (lr) patients were also provided. dentists were asked to point out at when they will start restoration of the teeth according to each ppc. results: after checking for completeness, 90 questionnaires remained for data analysis. for hr scenario: dentists with medium and high barrier scores (74.2%), and those with lowand medium preventive attitude (78.6%) showed the lower scores in restorative treatment threshold. in lr scenario, we found an interaction between age and other predictors. dentists ≤40 years old made better preventive decisions. conclusion: the present study showed that age and good preventive attitude play an important role in making the restorative decision by dentists. continuing education programs must be provided for iraqi dentists to improve their preventive treatment decisions. keywords: knowledge, attitude, dentist, restorative treatment 157 original iraqi dentists’ risk-based restorative treatment decisionsfatin abd al sattar aledhari j contemp med sci | vol. 6, no. 4, july-august 2020: 156–160 information on variables such as age, gender, type of working sector (private, governmental), duration of working as gdp, and history of previous attendance to any continuing education program. other variables were: dentists’ knowledge (5-point likert scale answers, later scored from 0 to 4), attitude (answers in the form of adjective pairs, scored from 7 to 1) and the perceived barriers (5-point likert scale answers, later scored from 0 to 4) about preventive dental care. there were two paper patient cases (ppcs) scenarios for evaluation of dentists’ restorative treatment threshold: high-risk (hr) & low-risk (lr). the dentists were asked to specify for each case, when they would start drilling a carious tooth for making a restoration.1, 14 statistical analysis acceptance missing level was set ≤20% for each question.13 knowledge, attitude, and perceived barrier were categorized into low (0–33.3), medium (33.3–66.6), and high (66.6–100) based on questionnaire scores standardized between 0 and 100. to test the associations among subgroups for evaluating the most potential predictor’s, simple logistic regression was used then followed by multiple logistic regression (stepwise method) for testing the association among the dependent and independent variables, adjusted for confounders. because of interaction between age and other predictors, we performed data analyses in each age group, separately. significant level was set at p-value< 0.05.13 results in the present study, 159 questionnaires were distributed and the response rate was 94%. however, only 90 questionnaires that contain complete data were included in the analyses. of all the participated dentists (n=150), 71% were female. the mean age was 40±9.88, ranged from 27 to 65 years. dentists who were ≤40 years old represent 51.1% from all the participants. high risk (hr) dentists with mediumand high barrier scores and those with lowand medium preventive attitude showed the lower scores in restorative treatment threshold in hr patient as shown in table 1. multiple regression analysis confirmed the above-mentioned results regarding the association between attitude and treatment threshold (or=0.31; 95% ci: 1.08-8.00; p-value= 0.02) and the association between barrier score and treatment threshold (or=3.45; 95% ci: 0.10-0.77; p-value= 0.02), (table 2). low risk (lr) we found an interaction between age and other predictors, consequently the results are presented separately for 2 age categories. in dentists ≤40 years old, those with better preventive attitude showed better restorative treatment threshold. conversely, those who reported they had not been attended an educational course about caries prevention got the lowest scores (table 3). study result shown in table 4. revealed that there was no significant relationship between independent variables and restorative treatment decision regarding low risk patients in dentists >40 years old. however, for those ≤40 years old multiple regression analysis confirmed that those with better preventive attitude (or=5.36; 95%ci: 1.26-22.83; p-value=0.02), and those who had been attended to educational course in caries prevention (or=5.75; 95%ci: 0.998-33.17; p-value=0.05), had better restorative treatment threshold (table 5). discussion dentist’s decision-making regarding the operative treatment is influenced by their attitude towards controlling active lesions and preventing new ones. attitude follows their evidence based knowledge about caries progression and understanding of different stages of caries as well as risk-based caries treatment and prevention modalities. this will empower them in avoiding unnecessary treatment decisions.16 this study showed an interesting point in case of lr patient. younger dentists (≤40) who showed good preventive attitude and attended preventive educational courses, had higher restorative treatment threshold. a good preventive attitude was the same predictor of the decision of iraqi dentists regarding hr patient. this study revealed that one-third of the iraqi dentists would restore carious teeth when reached the outer half of the dentin in hr patients which is in contrast with the opinion of california dentists that most of them tended to restore the carious lesion when reached dentin–enamel junction (dej).14 in lr patient, our results were similar to the swedish dentists’ decisions regarding lr patients. they decided to delay the restorative treatment when there was no dentinal involvement.17another important predictor that affected the iraqi dentists’ decision was their attendance to continuing educational courses regarding preventive dentistry. this is in line with the findings of mai e. khalaf et al. among general dentists in kuwait.18to improve restorative treatment threshold of iraqi dentists, dental schools should put more emphasis on the topics about preventive dentistry in general dental education. continuing education courses will extremely help to update evidence-based knowledge and practice of dentists. this will consequently improve the oral health of the community. to give the dentists a room for preventive action, more research in provision of preventive dental care barriers is necessary. conclusion: the present study showed that age and good preventive attitude play an important role in making the restorative decision by dentists. continuing education programs must be provided for iraqi dentists to improve their preventive treatment decisions. acknowledgments: the authors would like to thank the participants for their sincere cooperation in this study. this research was part of a msc thesis by fatin abd al sattar aledhari in tehran university of medical sciences. conflicts of interest disclosure: there are no conflicts of interest. 158 original iraqi dentists’ risk-based restorative treatment decisions fatin abd al sattar aledhari j contemp med sci | vol. 6, no. 4, july-august 2020: 156–160 table 2. predictors of restorative treatment threshold in case of high-risk patient; results of multiple logistic regression analysis or 95%ci p-value attitude score low and medium ref. high 0.31 0.11-0.81 0.02 barrier score low ref. medium and high 3.45 1.27-9.34 0.02 table 1. distribution (n, %) of dentists belonging to the categories of restorative treatment threshold for high-risk (hr) patient and simple logistic regression results restorative treatment threshold score or 95%ci p-value low and medium high gender female 41 (65.1%) 22 (34.9%) 1.20 0.45-3.219 0.71 male 18 (69.2%) 8 (30.8%) ref. age (years) ≤40 29 (64.4%) 16 (35.6%) 1.18 0.49-2.85 0.71 >40 30 (68.2%) 14) 31.8 %) ref working duration <10 years 19 (57.6%) 14 (42.4%) ref 10-19 years 21 (75.0%) 7 (25.0%) 0.45 0.15-1.35 0.16 ≥20 years 19 (67.9%) 9 (32.1%) 0.64 0.22-1.84 0.41 working in private sector no 35 (70.0%) 15 (30.0%) ref yes 24 (61.5%) 15 (38.5%) 1.45 0.60-3.53 0.40 education course attendance no 14 (66.7%) 7 (33.3%) ref. yes 45 (66.2%) 23 (33.8%) 1.02 0.36-2.88 0.97 attitude score low and medium 33 (78.6%) 9 (21.4%) ref. high 26 (55.3%) 21 (44.7%) 2.96 1.16-7.54 0.02 knowledge score low 17 (68.0%) 8 (32.0%) ref medium 37 (71.2%) 15 (28.8%) 0.86 0.31-2.42 0.78 high 5 (41.7%) 7 (58.3%) 2.97 0.72-12.34 0.13 barrier score low 13 (48.1%) 14 (51.9%) ref medium and high 46 (74.2%) 16 (25.8%) 0.32 0.12-0.83 0.02 159 original iraqi dentists’ risk-based restorative treatment decisionsfatin abd al sattar aledhari j contemp med sci | vol. 6, no. 4, july-august 2020: 156–160 table 3. distributions (n, %) of dentists’ belonging to the categories of restorative treatment threshold for low risk patient and simple logistic regression results restorative treatment threshold patient b (score) lr or 95%ci p-value low and medium high dentists ≤40 years old gender female 6(54.5%) 5 (45.5%) 2.0 0.498.09 0.33 male 24 (70.6%) 10 (29.4%) ref. working in private sector no 19 (73.1%) 7 (26.9%) ref yes 11 (57.9%) 8 (42.1%) 0.51 0.14-1.78 0.29 education course attendance no 13 (86.7%) 2 (13.3%) ref. yes 17 (56.7%) 13(43.3%) 4.97 0.9526.00 0.058 attitude score low and medium 19 (82.6%) 4 (17.4%) ref. high 11 (50.0%) 11 (50.0%) 1.0 0.05-0.82 0.03 knowledge low 13 (76.5%) 4 (23.5%) ref medium 14 (60.9%) 9 (39.1%) 2.09 0.528.46 0.30 high 3 (60.0%) 2 (40.0%) 2.17 0.26-17.89 0.47 barrier score low 10 (71.4%) 4 (28.6%) ref medium and high 20 (64.5%) 11 (35.5%) 1.38 0.35-5.43 0.65 dentists >40 years old gender female 12 (42.9%) 16 (57.1%) 0.86 0.243.02 0.81 male 7 (46.7%) 8 (53.3%) ref. working in private sector no 11 (45.8%) 13 (54.2%) ref yes 8 (42.1%) 11 (57.9%) 0.86 0.262.89 0.81 education course attendance no 3 (50.0%) 3 (50.0%) ref. yes 16 (43.2%) 21 (56.8%) 1.31 0.23-7.38 0.76 attitude score low and medium 8 (42.1%) 11 (57.9 %) ref. high 11 (45.8%) 13 (54.2%) 0.86 0.35-3.92 0.81 knowledge score low 4 (50.0%) 4 (50.0%) ref medium 13 (46.4%) 15 (53.6%) 1.15 0.245.56 0.86 high 2 (28.6%) 5 (71.4%) 2.50 0.2921.40 0.40 barrier score low 8 (61.5%) 5 (38.5%) ref medium and high 11 (36.7%) 19 (63.3%) 2.76 0.72-10.57 0.14 160 original iraqi dentists’ risk-based restorative treatment decisions fatin abd al sattar aledhari j contemp med sci | vol. 6, no. 4, july-august 2020: 156–160 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i4.801 table 4. predictors of restorative treatment threshold of young (≤40 years old) iraqi dentists on low-risk patient; results of multiple logistic regression analysis.   or 95%ci p-value attitude score low and medium ref. high 5.36 1.26-22.83 0.02 education course attendance no ref. yes 5.75 0.998-3.17 0.05 references: 1. ghasemi h. preventive orientation and caries management by iranian dentists. ethesis; university of helsinki, finland. 2008. http://hdl.handle. net/10138/20253. accessed 26 may 2020. 2. fejerskov o, nyvad b, kidd e. dental caries: the disease and its clinical management. oxford, wiley-blackwell, 3rd ed., 2015:160166. 3. rechmann p, doméjean s, rechmann bmt, kinsel r, featherstone jdb. approximal and occlusal carious lesions. restorative treatment decisions by california dentists. j am dent assoc. 2016;147(5):328-338. 4. baraba a, domejean s, juric h, espelid i, tveit ab, anic i. restorative treatment decisions of croatian university teachers. coll antropol. 2012;36(4):1293-1299. 5. tveit ab, espelid i, skodje f. restorative treatment decisions on approximal caries in norway. int dent j. 1999;49(3):165-172. 6. innes npt, schwendicke f. restorative thresholds for carious lesions: systematic review and meta-analysis. j dent res. 2017;96(5):501-508. 7. heaven tj, gordan v v, litaker ms, fellows jl, brad rindal d, firestone ar, et al. agreement among dentists’ restorative treatment planning thresholds for primary occlusal caries, primary proximal caries, and existing restorations: findings from the national dental practice-based research network. j dent. 2013;41(8):718-725. 8. riley jl, gordan v v, ajmo ct, bockman h, jackson mb, gilbert gh, et al. dentists’ use of caries risk assessment and individualized caries prevention for their adult patients: findings from the dental practice-based research network. community dent oral epidemiol. 2011;39(6):564-573. 9. matloob mh. dental caries in iraqi 12-year-olds and background fluoride exposure. community dent health. 2015;32(3):163-169. 10. khoshnevisan mh, albujeer an, taher aa, almahafdha a. dental education in iraq: issues, challenges and future. j contemp med sci. 2017 sep 26;3(11):260-3. 11. albujeer an, taher a. dental education and oral health service in iraq. iran j public health. 2017 may 9;46(5):713-4. 12. kakudate n, sumida f, matsumoto y, manabe k, yokoyama y, gilbert gh, et al. restorative treatment thresholds for proximal caries in dental pbrn. j dent res. 2012;91(12):1202-1208. 13. aledhari faa, sargeran k, gholami m, shamshiri ar. preventive orientation of iraqi dentists in baghdad in 2016. j dent (tehran). 2017;14(5):246–253. 14. ghasemi h, murtomaa h, torabzadeh h, vehkalahti mm. knowledge of and attitudes towards preventive dental care among iranian dentists. eur j dent. 2007;1(4):222-229. 15. szklo m, nieto j. epidemiology: beyond the basics. jones & bartlett learning, 4th ed., 2018:69-75. 16. rechmann p, doméjean s, rechmann bmt, kinsel r, featherstone jdb. approximal and occlusal carious lesions restorative treatment decisions by california dentists. j am dent assoc. 2016;147(5):328-38. 17. sundberg h, mejàre i, espelid i, tveit ab. swedish dentists’ decisions on preparation techniques and restorative materials. acta odontol scand. 2018;58(3):135-41. 18. khalaf me, alomari qd, ngo h, domjean s. restorative treatment thresholds: factors influencing the treatment thresholds and modalities of general dentists in kuwait. med princ pract. 2014;23(4):357-62. 80 review issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 80 – 85 introduction two main forms of beta-thalassemia have been differentiated by their clinical severity, first severe anemia that occur in the initial year of life as thalassemia major (tm) and the second mild anemia as thalassemia intermedia.1-3 the tm consequences occurred in diverse range in developing countries, from lack of public access to blood screening tests to timely iron removal from patients.4,5 the two main causes of death in patients with tm were excess serum ferritin and iron overload followed by complications that occur including liver and endocrine disorders, cardiac disease, anemia, infection, and eventually death.6-8 the survival rate of tm in the last few decades has progressively increased in order that only 2% of tm died before 10 years after diagnosis.9 some factors affect on this increasing picture of tm survival including improving of surveillance in some parts of the world, improved treatment of cardiac complications, better accessibility to blood donor, blood screening for pathogens, treatment of infections, and treatment with deferoxamine.10, 11 in the modell et al. study, decreasing of cardiac disease in tm was reported as the main factor in increasing of survival.12 lower survival rate in some reigns in the world associated with availability of deferoxamine treatment and also related to burdensome iron chelation regimen refusal by tm patients.13 studies about survival in tm patients reported different rates in which these differences may be due to several factors, but some factors such as clinical phenotype of patients in different reign possibly are important.14 to assess the current survival rate in patients with tm worldwide, to appraise the long-term survival (10-, 15-, 20-, and 30-year), and to obtain the important limitations toward long-term survival in thalassemia patients, this systematic review and meta-analysis was conducted. materials and methods this is a systematic review and meta-analysis of patients with beta-thalassemia major. this study was designed in 2020. the methodology of the present study is based on the prisma (preferred reporting items for systematic reviews and metaanalysis) statement.15 search strategy the researchers searched five international databases including web of knowledge, pubmed, embase, scopus, and proquest until march 2020. we also searched the google scholar for detecting gray literature. selected keywords for international databases included: “beta-thalassemia”, survival rate”, “survival analysis” and “kaplan–meier estimate” the initial search was conducted by two researchers (shr and sh). the searched record entered the endnote x7 software, and duplicate articles were removed automatically. inclusion and exclusion criteria all observational studies (cross-sectional, case–control, and  cohort) stated the survival rate of patients with beta thalassemia major, published in english language without time period restriction were included in the study. it should be  noted that studies that did not report the confidence interval  or sample size were not included into the final analysis. quality assessment the newcastle-ottawa quality assessment checklist was used to evaluate the quality of selected papers. this tool has three different parts including selection (4 questions), comparability (1 question) and outcome (3 questions), and based on the final scores divided into three categories: good, fair, and poor.16-19 the global survival rate of patients with beta-thalassemia major: a systematic review and meta-analysis maryam soltani1, soheil hassanipour2, yousef veisani3, mitra darbandi4, shahab rezaiean4* 1 razi clinical research development unit (rcrdu), birjand university of medical sciences (bums), birjand, iran. 2 cardiovascular diseases research center, department of cardiology, heshmat hospital, school of medicine, guilan university of medical sciences, rasht, iran 3 psychosocial injuries research center, ilam university of medical sciences, ilam, iran 4 research center for environmental determinants of health, kermanshah university of medical sciences, kermanshah, iran *correspondence to: shahab rezaiean (e-mail: shahab.rezaian.kums@gmail.com) (submitted: 08 january 2020 – revised version received: 21 january 2021 – accepted: 13 february 2021 – published online: 26 april 2021) abstract objective thalassemia is a public health challenge in the countries entitled belt thalassemia, but there is no pooled estimate of the survival rate on thalassemia major patients. the aim of this meta-analysis was to evaluate the pooled 10-, 15-, 20-, and 30-year survival rates of the patients with beta-thalassemia major around the world. methods a comprehensive literature search of five international databases including medline/pubmed, scopus, embase, web of knowledge, and proquest was conducted to identify studies reporting survival rate of beta-thalassemia major until march 2020. results from 714 retrieved studies, 7 studies with 8777 subjects were included in the meta-analysis. base on random effect model, the 10-, 15-, 20-, and 30-year survival rates were 98.39, 95.07, 90.41, and 82.93 percent, respectively. conclusions this meta-analysis provided acceptable results for estimating survival rate of beta-thalassemia compared to other studies. hence, these results can be effectively used to develop and implement prevention and treatment interventions for policymakers. keywords survival rate; beta-thalassemia major, systematic review, meta-analysis 81 review the global survival rate of patients with beta-thalassemia majorshahab rezaiean et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 80 – 85 screening of studies screening of studies, extraction of results, and evaluation of quality control of articles were performed separately by two authors (shr and sh). if there was no agreement between the two, the supervisor would announce the decision on that article. data extraction form all final articles entered into the study process were provided by a checklist and were arranged to extract the data. this checklist includes the name of the author, the year of publication, the period of the study, the country of origin, the survival rate by year for each survival period. statistical analysis the heterogeneity of the studies was assessed by cochran test (with significance less than 0.1). also, i2 statistics was used for heterogeneity assessment. in the case of heterogeneity, the random effects model was utilized. all analyses were performed by the stata (version 13) software. results study selection after searching all the international databases, 524 articles were selected and after deleting duplicate articles, 431 studies entered the review phase in terms of title and abstract. after reviewing the abstract of articles, 15 articles entered the next stage, at which point the full text was examined and 7 studies entered the final analysis. it should be noted that the referenced articles were also reviewed to add related articles. in the screening stages of studies, some articles were removed for a variety of reasons, which included the unrelated topic (n=406), the unrelated population (n=10), inadequate information (n=6), and the repeated results (n=2). the study selection process is outlined in fig. 1. result of quality assessment based on our results, six studies have good and one study had a fair quality. the result of quality assessment presented in table 1. study characteristics the included studies were published from 1994 to 2018. base on geographical location, five studies conducted in iran9, 20-23, one in italy,24 and one in cyprus.25 characteristics of the included studies presented in table 2. heterogeneity the result of chi-squared test and the i2 index indicated that there was a considerable difference between-study heterogeneity. for 10(i2= 88.1 %, p<0.001), 15(i2= 94.1%, p<0.001), 20(i2= 93.8%, p<0.001), and 30-year survival rate (i2= 98.2%, p<0.001). results of the meta-analysis first, the articles were sorted according to the year of publication and then analyzed by 1-, 10-, 15-, 20-, and 30-year table 1. quality assessment of included studies. author (year) selection comparability outcome total quality telfer, 2006 2 1 2 5 fair kosaryan, 2007 2 2 2 6 good di bartolomeo, 2008 3 1 2 6 good roudbari, 2008 3 2 2 7 good rajaeefard, 2015 3 1 2 6 good zamani, 2015 2 2 2 6 good ansari-moghadam, 2018 3 2 2 7 good table 2. basic information of included studies. author (year) location time period sample size survival rate 10 15 20 30 telfer, 2006 cyprus 1980-2004 539 100 98.5 92.7 kosaryan, 2007 iran 1984-2006 1010 98 88 70 di bartolomeo, 2008 italy 1983-2006 115 89.2 roudbari, 2008 iran 1998-2006 578 97 92.1 rajaeefard, 2015 iran 911 97 92 zamani, 2015 iran 1997-2013 133 98.3 88.4 80.5 ansari-moghadam, 2018 iran 1971-2015 5491 99 97 95 88 82 review the global survival rate of patients with beta-thalassemia major shahab rezaiean et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 80 – 85 survival. it should be noted that the number of papers of 5, 25, and other survival rate was very low. 10-year survival rate based on the random-effect model, the results of the study demonstrated that 10-year survival rate of btmp was 98.39% (95 % ci; 97.51–99.26, i2=88.1%, p<0.001). the highest survival was reported from cyprus (100%, 95% ci; 99.12–100) by telfer et.al. 10-year survival rate of btmp has been shown in fig. 2. 15-year survival rate the results of the study show that 15-year survival rate was 95.07% (95 % ci; 92.50–97.64, i2=94.1%, p<0.001). the highest survival was reported from cyprus (98.5%, 95% ci, 96.74– 99.18) by telfer et.al. 15-year survival rate of btmp has been shown in fig. 3. 20-year survival rate based on the random-effect model, the 20-year survival rate was 90.41% (95 % ci; 85.43–95.40, i2=93.8%, p<0.001). the highest survival was reported from iran (95%, 95% ci, 94.37– 95.54) by ansari-moghadam et.al. 20-year survival rate of btmp has been shown in fig. 4. 30-year survival rate the results of the study show that 30-year survival rate was 82.93% (95 % ci; 74.21–91.66, i2=98.2%, p<0.001). the highest survival was reported from cyprus (92.7%, 95% ci, 90.15– 94.74) by telfer et.al. 30-year survival rate of btmp has been shown in fig. 5. publication bias given that the number of articles entered in each of analyses was less than 10, investigating the publication bias was not rational.26 discussion the present meta-analysis was carried out to determine the survival rate of patients with beta-thalassemia major until 10, 15, 20, and 30 years of age. the 10-, 15-, 20-, and 30-year survival rates were 98.39, 95.07, 90.41, and 82.93 percent, respectively. the findings of this study show that about 82% of the patients with beta-thalassemia major survived until the age of 30 years. in recent decades, several factors have been shown to improve survival rate of beta-thalassemia patients including implementation of thalassemia prevention program, increased id en ti fi ca ti o n sc re en in g el ig ib ili ty in cl u d ed studies identified through international database search (n= 524) studies remained after removal of the duplicates (n= 431) studies remained for qualitative and quantitative analyses (n=7) full-text papers assessed for eligibility (n=15) studies excluded after tittle and abstract screening (n= 416) papers excluded for various reasons (n=8) fig. 1 flowchart of the included eligible studies in systematic review. 83 review the global survival rate of patients with beta-thalassemia majorshahab rezaiean et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 80 – 85 fig. 2 forest plot of 10year survival rate. fig. 3 forest plot of 15year survival rate. fig. 4 forest plot of 20-year survival rate. 84 review the global survival rate of patients with beta-thalassemia major shahab rezaiean et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 80 – 85 quality of healthcare services, provision of appropriate treatment, and essential services for these patients, such as blood transfusions, administration of appropriate drugs, and screening of donated blood before blood transfusion.22, 27-30 telfer and colleagues investigated factors associated with long-term survival of tm, and showed an unexpected increasing trend of survival rate since 2000 compared to before 1999. they also stated that excessive exposure of patients to toxins produced in iron chelation therapy, and subsequently increased rates of cardiac complications, the survival rate of thalassemia patients was significantly lower until 2000.27 the findings of previous studies showed that delayed documentation of tm cases in registration system and delayed diagnosis in these patients are associated with low survival rate for beta-thalassemia major over the past years.31-33 the results of this study show that the highest 10and 15-year survival rates for beta-thalassemia major were reported in cyprus, and the highest 20-year survival rate was reported in iran. efthimiadis and colleagues reported the 15-year survival rate for beta-thalassemia major patients as 58%, and 88% for all.34 bartolomeo and colleagues also revealed that the 20-year survival rates for beta-thalassemia major patients and for all as 89.2%, and 85.7%, respectively.35 rajaeefard and colleagues reported the 20-, 40-, and 60-year survival rates of beta-thalassemia major patients as 85%, 63%, and 54%, respectively.22 wu et al.36 also reported the survival rate of tm patients as higher than 97% due to improvements in treatment and growth of chelation therapy between 2007 and 2011. although, excess iron due to long-term blood transfusion among these patients is the main cause of various complications, especially cardiac complications (as one of the main causes of death in beta-thalassemia major patients), nowadays optimal iron chelation therapy and administration of deferiprone as a chelator in the treatment of iron overload in thalassemia patients, considerably increased survival rate among these individuals.37-39 limitations this meta-analysis had some limitations. the high proportion of retrieved studies were conducted in iran due to high prevalence of beta-thalassemia major in this country. in the two studies, the combined survival rate has been reported for both major and intermedia thalassemia patients, and then these studies were excluded in the analysis. the meta regression was not done because of small sample of retrieved studies. in addition, due to various reports of survival rates in the retrieved studies, we failed to obtain an overall survival rate from all collected survival reports. conclusion in conclusion, this meta-analysis provided acceptable results for estimating survival rate of beta-thalassemia compared to other studies. hence, these results can be effectively used to develop and implement prevention and treatment interventions. conflicts of interest disclosure all authors declare no conflicts of interest. references 1. aessopos a, farmakis d, karagiorga m, voskaridou e, loutradi a, hatziliami a, et al. cardiac involvement in thalassemia intermedia: a multicenter study. blood. 2001;97(11):3411-6. 2. olivieri nf. the beta-thalassemias. n engl j med. 1999;341(2):99-109. 3. wang hc, hsieh ll, liu yc, hsiao hh, lin sk, tsai wc, et al. the epidemiologic transition of thalassemia and associated hemoglobinopathies in southern taiwan. ann hematol. 2017;96(2):183-8. 4. borgna-pignatti c, cappellini md, de stefano p, del vecchio gc, forni gl, gamberini mr, et al. survival and complications in thalassemia. ann ny acad sci. 2005;1054:40-7. 5. zafari m, kosaryan m, gill p, alipour a, shiran m, jalalli h, et al. non-invasive prenatal diagnosis of beta-thalassemia by detection of the cell-free fetal dna in maternal circulation: a systematic review and meta-analysis. ann hematol. 2016;95(8):1341-50. fig. 5 forest plot of 30-year survival rate. 85 review the global survival rate of patients with beta-thalassemia majorshahab rezaiean et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 80 – 85 6. borgna-pignatti c, rugolotto s, de stefano p, zhao h, cappellini md, del vecchio gc, et al. survival and complications in patients with thalassemia major treated with transfusion and deferoxamine. haematologica. 2004;89(10):1187-93. 7. ladis v, chouliaras g, berdousi h, kanavakis e, kattamis c. longitudinal study of survival and causes of death in patients with thalassemia major in greece. ann ny acad sci. 2005;1054:445-50. 8. tripathi p, pati hp, mahapatra m, tyagi s, ahuja a, saxena r. utility of labile plasma iron assay in thalassemia major patients. ind j hematol blood transf off j ind soc hematol blood transf. 2019;35(2):272-7. 9. zamani r, khazaei s, rezaeian s. survival analysis and its associated factors of beta thalassemia major in hamadan province. iran j med sci. 2015;40(3):233-9. 10. wu h-p, lin c-l, chang y-c, wu k-h, lei r-l, peng c-t, et al. survival and complication rates in patients with thalassemia major in taiwan. pediat blood cancer. 2017;64(1):135-8. 11. ansari-moghaddam a, adineh ha, zareban i, mohammadi m, maghsoodlu m. the survival rate of patients with beta-thalassemia major and intermedia and its trends in recent years in iran. epidemiol health. 2018;40(0):e2018048-0. 12. modell b, khan m, darlison m, westwood ma, ingram d, pennell dj. improved survival of thalassaemia major in the uk and relation to t2* cardiovascular magnetic resonance. j cardiovasc magnetic resonance off j soc cardiovasc magnetic resonance. 2008;10(1):42. 13. pakbaz z, fischer r, treadwell m, yamashita r, fung eb, calvelli l, et al. a simple model to assess and improve adherence to iron chelation therapy with deferoxamine in patients with thalassemia. ann ny acad sci. 2005;1054:486-91. 14. porter jb, davis ba. monitoring chelation therapy to achieve optimal outcome in the treatment of thalassaemia. best pract res clin haematol. 2002;15(2):329-68. 15. moher d, shamseer l, clarke m, ghersi d, liberati a, petticrew m, et al. preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015 statement. syst rev. 2015;4(1):1. 16. penson df, krishnaswami s, jules a, seroogy jc, mcpheeters ml. evaluation and treatment of cryptorchidism. 2012. 17. nikbakht ha, hassanipour s, shojaie l, vali m, ghaffari-fam s, ghelichighojogh m, et al. survival rate of colorectal cancer in eastern mediterranean region countries: a systematic review and meta-analysis. cancer control. 2020;27(1):1073274820964146. 18. hassanipour s, vali m, gaffari-fam s, nikbakht ha, abdzadeh e, joukar f, et al. the survival rate of hepatocellular carcinoma in asian countries: a systematic review and meta-analysis. excli j. 2020;19:108-30. 19. hassanipour s, delam h, arab-zozani m, abdzadeh e, hosseini sa, nikbakht ha, et al. survival rate of prostate cancer in asian countries: a systematic review and meta-analysis. ann glob health. 2020;86(1):2. 20. kosaryan m, vahidshahi k, karami h, forootan ma, ahangari m. survival of thalassemic patients referred to the boo ali sina teaching hospital, sari, iran. hemoglobin. 2007;31(4):453-62. 21. roudbari m, soltani-rad m, roudbari s. the survival analysis of beta thalassemia major patients in south east of iran. saudi med j. 2008;29(7):1031-5. 22. rajaeefard a, hajipour m, tabatabaee hr, hassanzadeh j, rezaeian s, moradi z, et al. analysis of survival data in thalassemia patients in shiraz, iran. epidemiol health. 2015;37:e2015031. 23. ansari-moghaddam a, adineh ha, zareban i, mohammadi m, maghsoodlu m. the survival rate of patients with beta-thalassemia major and intermedia and its trends in recent years in iran. epidemiol health. 2018;40:e2018048. 24. di bartolomeo p, santarone s, di bartolomeo e, olioso p, bavaro p, papalinetti g, et al. long-term results of survival in patients with thalassemia major treated with bone marrow transplantation. am j hematol. 2008;83(7): 528-30. 25. telfer p, coen pg, christou s, hadjigavriel m, kolnakou a, pangalou e, et al. survival of medically treated thalassemia patients in cyprus. trends and risk factors over the period 1980-2004. haematologica. 2006;91(9):1187-92. 26. schneck a. examining publication bias-a simulation-based evaluation of statistical tests on publication bias. peerj. 2017;5:e4115. 27. telfer p, coen pg, christou s, hadjigavriel m, kolnakou a, pangalou e, et al. survival of medically treated thalassemia patients in cyprus. trends and risk factors over the period 1980-2004. haematologica. 2006;91(9):1187-92. 28. hassanzadeh j, mirahmadizadeh a, karimi m, veisani y, rezaeian s. trends in 5-, 10-, 20-, and 30-year survival rates of beta-thalassemia patients in southern iran, 1995-2016: a retrospective cohort study. j public health res. 2017;6(3). 29. charafeddine k, isma’eel h, charafeddine m, inati a, koussa s, naja m, et al. survival and complications of beta-thalassemia in lebanon. acta haematol. 2008;120(2):112-6. 30. caocci g, orofino mg, vacca a, piroddi a, piras e, addari mc, et al. longterm survival of beta thalassemia major patients treated with hematopoietic stem cell transplantation compared with survival with conventional treatment. am j hematol. 2017;92(12):1303-10. 31. miri m, tabrizi namini m, hadipour dehshal m, sadeghian varnosfaderani f, ahmadvand a, yousefi darestani s, et al. thalassemia in iran in last twenty years: the carrier rates and the births trend. iran j blood cancer. 2013;6(1):11-7. 32. hassanzadeh j, mirahmadizadeh a, karimi m, rezaeian s. delay in diagnosis of hemoglobulinopathies (thalassemia, sickle cell anemia): a need for management of thalassemia programs. iran j pediat. 2017;27(2). 33. hadipour dehshal m, ahmadvand a, yousefi darestani s, manshadi m, abolghasemi h. secular trends in the national and provincial births of new thalassemia cases in iran from 2001 to 2006. hemoglobin. 2013;37(2): 124-37. 34. efthimiadis gk, hassapopoulou hp, tsikaderis dd, karvounis hi, giannakoulas g, parharidis ge. cardiac determinants of survival in betathalassemia. haematologica. 2006;91(12 suppl):elt11. 35. di bartolomeo p, santarone s, di bartolomeo e, olioso p, bavaro p, papalinetti g, et al. long-term results of survival in patients with thalassemia major treated with bone marrow transplantation. am j hematol. 2008;83(7):52830. 36. wu hp, lin cl, chang yc, wu kh, lei rl, peng ct, et al. survival and complication rates in patients with thalassemia major in taiwan. pediat blood cancer. 2017;64(1):135-8. 37. peng c-t, tsai c-h, wu k-h. effects of chelation therapy on cardiac function improvement in thalassemia patients: literature review and the taiwanese experience. hemoglobin. 2008;32(1-2):49-62. 38. peng c-t, chang j-s, wang l-y, chiou s-s, hsiao c-c, wang s-c, et al. update on thalassemia treatment in taiwan, including bone marrow transplantation, chelation therapy, and cardiomyopathy treatment effects. hemoglobin. 2009;33(5):304-11. 39. huang y-c, chang j-s, wu k-h, peng c-t. regression of myocardial dysfunction after switching from desferrioxamine to deferiprone therapy in β-thalassemia major patients. hemoglobin. 2006;30(2):229-38. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.903 128 j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 original exogenous acylated ghrelin promotes but un-acylated ghrelin prevents hepatic steatosis by modulating peripheral insulin resistance and hepatic oxidative and endoplasmic reticulum stress samah alharbi1*, refaat a. eid2 1department of physiology, faculty of medicine, umm al-qura university, makkah, saudi arabia. 2electron microscope unit, college of medicine and medical science, king khalid university, abha, saudi arabia. *correspondence to: samah alharbi (email: saaharbi@uqu.edu.sa). (submitted: 03 march 2021 – revised version received: 22 march 2021 – accepted: 15 april 2021 – published online: 26 june 2021) introduction non-alcoholic fatty liver disease (nafld) is one of the most common chronic liver disease that is linked with obesity, diabetes mellitus (dm), insulin resistance (ir), and other metabolic disorders.1–3 it develops by over-accumulation of triglyceride (tgs) in the liver and its complications range from simple steatosis to non-alcoholic steatohepatitis (nash).2–4 up-to-date, the underlying mechanisms involved in the pathogenesis of nafld are still not fully understood with oxidative stress being the key player.1–3 currently, the emerging role of the endoplasmic reticulum (er) stress in the development and progression of nafld is well-established.5 the er is an essential cellular organelle that is found in large quantities in the liver of mammals and plays an essential role in the process of protein and lipid synthesis.5 however, the accumulation of unfolded proteins in the er during the high metabolic activity triggers unfolded protein response (upr) and er stress via the activation of different transmembrane signal transducers.5 although the initial activation of the upr is a protective mechanism that suppresses protein synthesis and enhances cell survival,5 the unresolved sustained upr and er stress is associated with hepatic lipid accumulation, ir, steatosis, and hepatocytes apoptosis.6 the precise mechanisms behind this are well-demonstrated in excellent reviews and studies.5, 7–9 indeed, the role of er stress in the development of hepatic steatosis and ir has been demonstrated in several conditions including obesity,7 alcoholism,8 viral hepatitis,9 and nafld.5, 12–17 however, suppressing er by pharmacological approaches or by weight loos reduced hepatic steatosis and improved hepatic insulin signaling.7, 15, 18–20 on the other hand, the currently available data suggest that nafld is no longer considered an exclusively hepatic disorder. in this setting, the gut-liver axis has been recently shown to play a crucial role in the regulation of hepatic lipogenesis and has been identified as a key player in the development/prevention of nafld.3, 21–27 ghrelin is the major gut hormone released from the stomach and the gut, and to a lesser extent from other peripheral tissues.28 in the circulation, ghrelin circulates in two forms, a common dominant form (>90%) named acylated ghrelin (ag, n-octanoylated at ser3) or des-acyl (unacylated) ghrelin (uag) without this post-translational modification.28 in mammals, such acylation is mediated by the activation of ghrelin-ghrelin o-acyltransferase (goat) enzyme, which is widely distributed in the stomach and other peripheral tissues including the adrenal cortex, breast, and colon.3,29 it is currently well-accepted that ag acts peripherally by activating its receptors, secretagogue receptor type 1a (ghs-r1a) where the effects exerted by uag are through other unidentified receptors.3 studies on the effect of the two forms of ghrelin on nafld progression revealed contradictory effects where ag induces but uag inhibits hepatic steatosis. indeed, the direct effect of ag on the liver was stimulating hepatic lipogenesis and inducing hepatic steatosis, ir and apoptosis through different pathways including: 1) activation of fatty acids (fas) synthesis-related transcription factors and genes (i.e. sterol regulatory element-binding protein-1c (srebp-1c) acetyl coa carboxylase 1 (acc-1), fatty acid synthase (fas), 2) activation of p53, mammalian target of rapamycin (mtor), peroxisome proliferator-activated receptor-gamma (pparγ), abstract objectives: this study tested the effects of acylated (ag and un-acylated ghrelin (uag) on hepatic lipid synthesis and insulin resistance (ir) from prospective to their effect on endoplasmic reticulum stress and investigated the possible underlying mechanisms. methods: healthy rats were divided as 4 groups (n = 12/each) as control, control + ag, control + uag, and control + ag + uag (1:1). ga or uag were given subcutaneously (200 ng/kg/each) for 8 weeks. results: ag increased fasting levels of glucose and insulin resistance, increased hepatic glucose production, and impaired glucose and insulin tolerance. besides, it increased serum levels of free fatty acids (ffas), enhanced serum and hepatic levels of triglycerides and cholesterol, and increased lipid deposition in the livers of rats. concomitantly, it stimulated the mrna levels of srebp1/2, fatty acid synthase, and protein levels of all arms of er stress including xbp-1, chop, atf-6, and p-eif2α, thus activating lipid synthesis and er stress. it also reduced protein levels of p-irs (tyr612), p-akt (ser307), and increased levels of ros, tnf-α, il-6, and protein levels of cleaved caspase-12, p-irs (ser307), and p-jnk (the183/tyr186) in rats’ livers. administration of uag alone or in combination with ag produced contradictory effects. however, both ag and uag significantly increased mrna levels of ampk and pparα suggesting fas oxidation. conclusion: ag induces hepatic steatosis and suppresses hepatic insulin signaling mainly by inducing peripheral ir that is associated with hepatic oxidative stress, inflammation, and er stress. however, uag alone or in combination exerts opposite effects. keywords: ghrelin, acylated, insulin resistance, steatosis, liver, endoplasmic reticulum, oxidative stress. issn 2413-0516 129j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 s. alharbi and r. a. eid original exogenous acylated ghrelin promotes and c (pkc) signalling pathways, and 3) inhibition of the mitochondria fatty acids (fas) oxidation by suppressing the activity and levels 5′ amp-activated protein kinase (ampk), carnitine palmitoyltransferase i (cpt-1), and ppraa.25, 30–35 on the other hand, the majority of studies have shown that uag administration is associated with suppressing hepatic lipogenesis mainly due to stimulating fas oxidation.35–37 opposing these data, a single study has shown that exogenous chronic administration of ag in combination with hfd for 8 weeks limited liver triglyceride content, oxidative stress, and inflammation in a rat model of hfd-induced obesity.24 until now, the effect of ag and dag on hepatic lipogenesis from perspective of their effect of upr arms and er stress was never tested yet which was worthy for us. therefore, in this study, we aimed to investigate the effect of chronic individual or combined administration of ag and uga on hepatic steatosis and ir in healthy rats with respect to their effects on upr and markers of er stress. materials and methods animals all procedures of the current study were approved by the animal ethical committee at umm al-qura university (uqu), makkah, saudi arabia (irb # hapo-02-2019-03-307) which their regulations follow the animal ethics and guidelines established by the us national institutes of health (nih publication no. 85-23, revised 1996). adult male sprague-dawley rats (120-140 g, 7-8 weeks) were supplied by the animal house at king khalid university, abha, ksa. during the experimental procedure, all rats were always maintained under the stable condition of a controlled temperature (22 ± 1°c), humidity (40−65%), and light/dark cycle (12 hours/each) and had free access to their drinking water and diet. experimental design rats were divided into 4 groups (n = 12/each) as follows: 1) lfd control group: received a daily dose normal saline, 2) control +ag-treated group: received a daily dose of ag (200 ng/kg) (cat. no. g8903, sigma, aldrich, uk), 3) control + uag-treated group: received a daily dose of uag (200 ng/kg) (cat no. 2951, sigma, aldrich, uk), and 4) control + ag + uag-treated group: received a concomitant daily dose of both ag and uag (200 ng/ml/each). the vehicle, ag and uag were administered to rats subcutaneously (s.c) every day for consecutive 8 weeks. the dose selections of ag and dag were adopted from the study of dallak,27 who showed diabetogenic and lipogenic effects of ag that was resolved by an equal dose of uag, effects that were independent of food intake. oral glucose tolerance test (ogtt) by the end of the experimental procedure, all rats/groups were fasted for 12 h and received an oral dose of glucose (2 g/kg).38 then, blood samples (300 µl) were withdrawn from all rats into k2edta microtubes (bd microtainers, cat. no. 363706, bd, usa) at 0.0, 0.0, 15, 30, 60 and 120 minutes at or post-glucose administration. all blood samples were centrifuged at 3000 xg for 10 minutes to collect plasma. plasma samples were stored at –20°c for later determination of glucose and insulin levels using rat’s special colorimetric and elisa kits purchased from (cat. no. 81693 and 90010, chrystalchem, il, usa respectively). hepatic insulin resistance (ir) was determined for each rats using the homeostasis model assessment of insulin resistance index (homa-iri) ([fpg(mg/dl) × fasting iri(ng/ml)]/405). serum and tissue collection directly after ogtt, rats (n = 6) were fasted for 4 hours and then anesthetized with pentobarbital sodium (60 mg/kg). blood samples (1ml) were withdrawn from the rat’s hearts directly and centrifuges at 3000 xg for 10 min to prepare sera which were stored at –20oc for determination of tgs and chol levels. then, the liver of each rat was rapidly removed on ice, washed with 0.01m ice cold-phosphate buffer (ph = 7.4), and cut into small pieces. some liver samples were snapfrozen in liquid nitrogen and stored at –80°c for further biochemical and molecular analysis. other parts of livers were placed in 10% buffered formalin and processed for histopathological evaluation. besides, gastrocnemius muscle samples were collected from all rats for determination of glycogen content. hepatic lipid extraction hepatic lipid extraction for the measurements of tgs and cholesterol (chol) levels were prepared according to the method of folch et al. (1957). to prepare total cell homogenates, parts of frozen livers (30 mg) were homogenized, individually, in 0.5 ml ice-cold pbs (ph 7.4) containing 10 µl protease inhibitor (cat. no. p8340 sigma-aldrich, mo, usa), centrifuged at 10000 xg for 10 min to collect supernatants. biochemical analysis in the serum and liver homogenates serum and hepatic levels of tgs were measured using a commercially available kit (cat. no. mbs726298 and mbs168179, mybiosource, ca, usa respectively). hepatic and muscle glycogen content was determined using a glycogen assay colorimetric kit (cat. no. ka8061, abnova, usa). hepatic levels of ros were measured using an assay kit (cat. no. sta-347, cell biolabs, inc. san diego, ca). hepatic levels of reduced and oxidized glutathione (gsh) were determined using a rat’s colorimetric assay determination kit (cat. no. ab138881/ abcam, uk). hepatic levels of malondialdehyde (mda) were measured using a colorimetric determination kit (cat. no. ab118970/ abcam, uk). serum and hepatic levels of free fatty acids (ffas) levels were assayed using a colorimetric kit (e1001, applygen technologies inc., beijing, china). hepatic levels of tumour necrosis factor-α tnf-α and interleukin-6 (il-6) were measured using elisa kits (cat. no. mbs267737 and mbs355410 mybiosource, usa, respectively). isolated liver preparations and insulin sensitivity the inhibition of insulin on glucagon-induced glucose output in all experimental groups (6 rats/group) was studied in freshly isolated perfused livers as previously described by others.39 in brief, after stabilization of the liver, bovine glucagon (55.4 pg in 50 ml perfusion buffer) (lt1504; lifetein, llc, nj, usa) was perfused for 20 minutes followed by perfusion of another buffer containing glucagon + insulin (100 µu) for the next 15 minutes. perfusion rate of hormones was carried out at a constant flow rate of 0.17ml/minute to achieve portal concentrations of glucagon and insulin of 100 pg/ml and 100 µu, 130 j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 exogenous acylated ghrelin promotes original s. alharbi and r. a. eid respectively. the perfusate was collected from the suprahepatic inferior vena cava every five minutes and then used for biochemical analysis of glucose levels using the same glucose kit used to measure plasma glucose. the glucose production rate was presented as mg/min/g liver weight for every 10 minutes of hormone perfusion. the integrated insulin responses were expressed as glucose production rate during the last 10 minutes divided by glucose production rate during the first 10 minutes of the hormone perfusion protocol. during the perfusion, the liver was always checked by gross appearance and color. at the end of the experimental procedure, the liver was removed, weighed, and stored at –80oc for hepatic glycogen level. real-time pcr primers used to study mrna levels of srebp-1c, ppraα fatty acid synthase, srebp1/2 are shown in table 1. total rna was extracted from frozen livers (30 mg/sample) using an rneasy mini kit (qiagen, victoria, australia). superscript ii reverse transcriptase and oligo (dt) (thermo fisher, ma, usa) was used to synthesize the single-stranded cdna. qpcr runs were performed in a cfx96 real-time pcr system (bio-rad, ca, usa) using ssofast evergreen supermix (bio-rad, montreal, canada). pcr components were 10 μl ssofast evergreen supermix, 2 μl diluted cdna (500 ng/µl), 0.4 μl of 10 μm forward primer (200 nm/reaction), 0.4 μl of 10 μm reverse primers (200 nm/reaction n), and 6.2 μl of nuclease-free water. all reaction was run as an initial heating step (1 cycle/95°c/30 sec), a denaturation step (95°c/5 sec), annealing/extension (60°c/30 sec) (each of 34 cycles), and a final melting step (1 cycle/95°c/1 sec). levels of mrna of each gene were quantified by the associated software using the δδct method. wells with no template dna served as a negative control. all procedures were conducted according to the manufacturer’s instructions and will be done for 6 samples/groups. western blotting total proteins were extracted from frozen livers (40 mg) after homogenization in radioimmunoprecipitation assay (ripa) buffer (0.5% sodium deoxycholate, 0.1% sds,150 mm nacl, 1.0% np-40, 50 mm tris (ph 8.0) and supplied with a protease inhibitor (sigma-aldrich, st. louis, mo, usa). protein levels in all samples were measured using a pierce bca protein assay kit (cat. no. 23225, thermofisher scientific). equal protein samples (40 µg) were separated on an 8–12% gradient sds-page gel, transferred nitrocellulose membrane, and then blotted with primary antibodies table 2. membranes were then blotted with an hrp-conjugated secondary antibody. all washings and antibodies dilution were done in tbs buffer with 0.1% tween 20 (tbst). each membrane was stripped up to 4 times maximum using restore™ plus stripping buffer (thermo fisher). bands will be visualized and their intensities will be evaluated using chemiluminescence (pierce ecl reagents, thermo fisher, usa, piscataway, nj) and c-di git blot scanner (li-cor, usa). data were analyzed for at least 3 samples/groups. statistical analysis statistical analyses for all measured parameters will be using graph pad prism statistical software package (version 6). differences among the experimental groups were assessed by one-way anova, followed by tukey’s test. results of ogtt and glucose production in the isolated liver preparations were analysed using the same software but with 2-way with repeated measures. data will be presented as means pulse standard deviation (mean ± sd). values will be considered significantly different when p < 0.05. results metabolic parameters and ogtt there were no changes in food intake between all experimental groups of rats. ag-treated rats had higher fasting plasma glucose and insulin levels, as well as homa-iri but had lower hepatic and muscle glycogen content as compared to control rats (fig. 1, a-f). they also showed an increase in the levels of fasting plasma glucose and insulin at all analysis intervals after the ogtt (fig. 2, a-d). however, with stable fasting insulin levels and homa-iri, uag-treated rats showed a significant decrease in fasting plasma glucose levels, and their livers and muscles had higher glycogen content as compared to control rats (fig. 1, a-f). besides, the administration of uag to rats significantly lowered blood glucose and insulin levels between 30-120 minutes of the ogtt (fig. 2, a-d). on the other hand, co-administration of uag to ag-treated rats attenuated all the above-mentioned effects exerted by ag (figs. 1&2). table 1. primers used in the quantitative real-time pcr reaction gene primers genbank accession product length srebp-1c f:5’-ggagccatggattgcacatt-3’ r:5’aggaaggcttccagagagga-3’ af286470.2 191 srebp-2 f:5’-ctgaccacaatgccggtaat-3’ r:5’-cttgtgcatcttggcatctg-3’ nm_001033694.1 204 fas f:5’-aggtgctagaggccctgcta-3’ r:5’-gtgcacagacaccttcccat-3’ x62888.1 281 ppar-α f:5’-tcacacaatgcaatccgttt-3’ r:5’-ggccttgaccttgttcatgt-3’ nm_013196.1 119 ampk-α1 f:5’-tgtgacaagcacatt ttccaa-3’ r:5’-ccgatctctgtggagtag cag -3’ nm_0191 42 131 18srna f:5’-ggatccattggagggcaagt-3’ r:5’-acgagctttttaactgcagcaa-3’ nm_017008.4 150 131j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 s. alharbi and r. a. eid original exogenous acylated ghrelin promotes table 2. characteristics of primary antibodies used in western blotting analysis antibody cat. no. mw (kda) manufacturer xbp-1 sc-8015 29:1:250 santa cruze biotechnology gadd 153 (chop-10) sc-7351 39:1:1000 santa cruze biotechnology atf-6α sc-166659 90:1000 santa cruze biotechnology (p-eif2α) (ser51) 9721 38:1:1000 cell signaling technology pro and cleaved caspase 12 2202α 44/55,1:1000 cell signaling technology jnk sc-7345 46/54:1:1000 santa cruze biotechnology p-jnk (thr183 and tyr185) sc-6254 46/54:1:1000 santa cruze biotechnology akt1 sc-5298 62 ,1:1000 santa cruze biotechnology p-akt (ser473) sc-514032 52.1:500 santa cruze biotechnology p-irs-1/2 (tyr612) sc-17195 170,1:500 cell signaling technology p-irs-1 (ser307) 2381 180,1:500 cell signaling technology irs-1 2382 180,1:1000 cell signaling technology β-actin 4970 45,1:2000 cell signaling technology, fig. 1 metabolic parameters in the all experimental groups. data are presented as mean ± sd in 6 rats/ group. α: vs. control rats, β: vs. ag-treated rats, and γ: vs. uag-treated rats. ag: acylated ghrelin. uag: unacylated ghrelin. a b c d fe 132 j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 exogenous acylated ghrelin promotes original s. alharbi and r. a. eid alterations in glucose production and glycogen content in isolated liver preparation livers isolated from ag-treated rats showed glucose production during the first 20 minutes after being challenged with glucagon alone (fig. 3, a&b). however, glucose levels remained significantly higher than all other groups during the last 15 minutes when they were perfused with a solution containing insulin and glucagon, indicating ir (fig. 3, a&b). consequently, they had the highest integration glucose production rate and had the lowest glycogen content (fig. 3, c&d). on the other hand, glucose production during glucagon perfusion alone or with perfusion of insulin and glucagon, as well as glucose production rate was significantly decreased with a concomitant increase in glycogen content was seen in isolated livers obtained from uag treated rats (fig. 3, a-d). however, normal glucose response, glucose production, and glycogen levels were observed in the isolated livers of ag + uag-treated rats (fig. 3, a-d). hepatic and serum lipids profile and liver histology in all groups of rats significantly higher serum and hepatic levels of tg, chol, and ffas (fig. 4, a-f) with increased lipid vacuoles accumulation and ballooning of the hepatocytes (fig. 5b) were observed in the livers of ag-treated rats as compared to control rats. however, normal architectures with the absence of lipid vacuoles (fig. 5d) and normal levels of tg, chol, and ffas (fig. 4, a-e) were observed in ag + uag-treated group. also, lowered serum and hepatic levels of tg, chol, and ffas (fig. 4, a-e) with almost normal liver architectures (fig. 5c) with were reduced fat vacuoles were observed in the livers of rats administered uag. oxidative stress and inflammation in the livers of all groups levels of gsh and ratio of gsh/gssg were significantly decreased whereas hepatic levels of gsgg, mda, ros, tnf-α, and il-6 were significantly increased in the livers of ag-treated rats as compared to control rats (fig. 6, a-f). however, normal levels of all these parameters were significantly observed in uag or ag +uag-treated rats as compared to control rats (fig. 6, a-f). alterations in mrna levels mrna levels of srebp1c, srebp2, fas, pparα, and ampkα1 were significantly increased in ag-treated rats as compared to control rats (fig. 7, a&b). on the other hand, mrna levels of srebp1c, srebp2, and fas, were significantly decreased but mrna levels of ampkα1 and pparα were significantly increased in the livers of uag-treated rats as compared to control rats (fig 7, a&b). however, normal levels of all these mrnas were detected in the livers of ag + uagtreated rats. protein markers of hepatic insulin signaling and er stress protein levels of all measured parameters were within expected weights. total levels of irs-1, akt, and jnk, were not significantly changed between all groups of rats (fig 8, a&b, fig 9d). protein levels of p-akt (ser473) and p-irs1/2 (tyr612) and their activation ratios were significantly decreased in fig. 2 outputs of the oral glucose tolerant test (ogtt) in all experimental groups. data are presented as mean ± sd in 6 rats/group. α: vs. control rats, β: vs. ag-treated rats, and γ: vs. uag-treated rats. ag: acylated ghrelin. uag: unacylated ghrelin. a b c d 133j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 s. alharbi and r. a. eid original exogenous acylated ghrelin promotes fig. 3 glucose production and hepatic glycogen levels in isolated liver preparations collected from all experimental groups. data are presented as mean ± sd in 6 rats/group. the first 20 minutes represents the period of glucose production during perfusion of glucagon alone. the last 15 minutes represents the period of glucagon + insulin perfusion. in figures a&b; α: vs. control rats, β: vs. ag-treated rats, and γ: vs. uag-treated rats. ag: acylated ghrelin. uag: unacylated ghrelin. fig. 4 serum and hepatic levels of triglycerides (tg) (a and c), total cholesterol (chol) (b and d) and free fatty acids (ffas) (e and f) in all experimental groups. data are presented as mean ± sd in 6 rats/group.α: vs. control rats, β: vs. ag-treated rats, and γ: vs. uag-treated rats. ag: acylated ghrelin. uag: unacylated ghrelin. a b c d a b c d e f 134 j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 exogenous acylated ghrelin promotes original s. alharbi and r. a. eid fig. 5 photomicrographs of liver tissues obtained from sample rats of experimental groups a and c: were taken from control rats and uag-treated rats, respectively, and both showing intact polyhedral hepatocytes radiating from central vein (cv) with rounded euchromatic nuclei and intact blood sinusoids. c: was taken from ag-treated rats showing disrupted hepatocytes in which most of them filled of fat and have balooning structures. d: was taken from rat treated with ag + uag and showing normal architectures like those observed in control rats with absence of fat vacuoles. however, some sinusoids were dilated. fig. 6 hepatic levels of markers of oxidative stress and inflammation all experimental groups. data are presented as mean ± sd in 6 rats/ group. α: vs. control rats, β: vs. ag-treated rats, and γ: vs. uag-treated rats. ag: acylated ghrelin. uag: unacylated ghrelin. ag-treated rats but significantly increased in uag-treated rats. in addition, protein levels of p-irs (ser307) and p-jnk (thr183 and tyr186) were significantly increased in ag-treated rats but were significantly decreased in uag-treated rats (fig. 8, a-d, fig. 9d). besides, protein levels of perk, p-eif2α, atf-6, chop, xbp-1 were increased in ag-treated rats but significantly decreased in uag-treated rats as compared to control rats (fig. 9, a-d). however, protein levels of all these proteins were returned to their baseline levels in ag + uag –treated rats as compare to ag-treated rats (fig. 9, a-d). discussion in this study, we have shown that chronic administration of low dose of ag35 of not only induced fasting hyperglycaemia, hyperinsulinemia, and systemic ir, but also enhanced hepatic fig. 7 mrna levels of some selected genes in the livers of all experimental groups. data are presented as mean ± sd in 6 rats/ group. α: vs. control, β: vs. ag-treated rats, and γ: vs. uag-treated. ag: acylated ghrelin. uag: unacylated ghrelin. a b c d e f 135j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 s. alharbi and r. a. eid original exogenous acylated ghrelin promotes fig. 8 protein levels of markers of hepatic insulin signaling in the livers experimental groups. data are presented as mean ± sd in 6 rats/ group. α: vs. control rats (lane a), β: vs. ag-treated rats (lane b), and γ: vs. uag-treated rats (lane c). lane d was taken from a rat treated with ag + uag. ag: acylated ghrelin. uag: unacylated ghrelin. fig. 9 protein levels of er stress components in the livers experimental groups. data are presented as mean ± sd in 6 rats/group.α: vs. control rats (lane a),β: vs. ag-treated rats (lane b), and γ: vs. uag-treated rats (lane c). lane d was taken from a rat treated with ag + uag. ag: acylated ghrelin. uag: unacylated ghrelin. 136 j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 exogenous acylated ghrelin promotes original s. alharbi and r. a. eid lipogenesis and suppressed hepatic insulin signaling associated with upregulation of srebp1/2 and fas expression, oxidative stress, inflammation, and activation of upr and er stress. on the contrary, chronic administration of uag afforded opposite effects with no effect of fasting insulin levels but concomitant with activation of atf-6 arm of upr and upregulation of ampk and pparα, major inducers of mitochondria fas oxidation. besides, co-administration of uag with ag, in a ratio of 1:1, attenuated systemic and hepatic effects exerted by ag and suppressed hepatic lipogenesis, ir, oxidative stress, inflammation, and er stress. the first interesting observation in this study is that all the biochemical and molecular alterations observed in ag and uag-treated rats of this study seem to be independent of food intake and orexigenic role of ghrelin as all experimental groups of rats consumed a similar amount of diet during the whole period of the study. supporting these findings, the s.c. individual or combination of both ag and uag at the same doses and 14 days used in this study didn’t modify food intake in rats.27 also, a very weak increase in food intake was shown after ag injection and neither the s.c nor the centrally infused ag increased food consumption.40–42 on the other hand, ag-treated rats developed peripheral and hepatic ir. peripheral ir was evident by the observed fasting hyperglycaemia and hyperinsulinemia, lower hepatic and muscle glycogen contents, a higher value of homa-iri, and the increase in plasma glucose and insulin levels at all measured intervals after the ogtt. however, we also confirmed the hepatic ir in ag-treated rats by the increase in glucose production during the infusion of combined glucagon and insulin in the isolated liver preparation that indicates the inability of insulin to suppress glucagon-induced gluconeogenesis. to confirm these results, we have also studied the activity of insulin signaling in the livers of ag-treated rats and found that ag increased the irs (ser307) and p-jnk (thr183 and tyr185) and decrease protein levels of irs (tyr612) and p-akt (ser473). similar to these data, short and long-term administration of ag, and independent of food intake or growth hormones, increased hepatic gluconeogenesis, increased glucose output, and/or impaired hepatic and peripheral insulin sensitivity.27, 32, 43, 44 however, some other studies have shown that ag administration could not affect,45 increases insulin levels,46 or improves peripheral insulin sensitivity.27, 47 such variation in our data and those studies could be attributed to inter-experimental conditions which could reflect different doses, treatment period, administration way (systemic vs. central), and/or species used. on the contrary, the opposite effects of fasting hypoglycaemia but with no alterations in fasting insulin levels were seen in uag-treated rats. besides, co-administration of uag with ag at a ratio of 1:1 reversed ag-induced alteration in all the above-mentioned biochemical parameters and stimulated hepatic insulin signaling. these data suggest that uag can reduce fasting hyperglycemia and improves peripheral and hepatic insulin sensitivity which is also supported by other previous studies.27, 31, 48, 49 what is considered unique is that we have also found that livers obtained from uag have reduced glucose production upon infusion of glucagon alone, thus illustrating uag as an inhibitor of gluconeogenesis. this could be attributed to the improvement in hepatic insulin sensitivity. interestingly, we also found a slight but significant increase in atf-6 protein levels (a transmembrane protein that is released in er stress) in the liver of these rats (as discussed later). glucagon stimulates hepatic gluconeogenesis through the creb regulated transcription coactivator 2 (crtc2).50 atf6 transient activation was shown to inhibit hepatic glucose output by inhibiting crtc2 occupancy at the promoters of genes that are involved in gluconeogenesis.50 this is could be a novel mechanism by which uag inhibits glucose production in the hepatocytes. under ir, the adipose tissue and muscular lipolysis are increased. as a result, the influx of the ffas to the liver increases and stimulated the generation of ros and inflammatory cytokines, lipid peroxidation, and hepatocytes damage due to increased β-oxidation and mitochondria dysfunction and leads to the generation of ros, lipid peroxidation, depletion of antioxidants, inflammation, which in turn damage the hepatocytes.51 ffas can directly induce er stress in the liver cells by stimulating store-operated ca2+ entry and activation of calpain-2.52 nonetheless, ros and inflammatory cytokines activate hepatic er stress.53 moreover, er stress is known to induce hepatic ir through ire1α-mediated activation of c-jun n-terminal kinase (jnk), which in turn, impairs insulin signalling through increasing ser307 phosphorylation of irs-1.54–57 also, oxidative stress can suppress hepatic insulin signaling in different tissues by activating stress pathways involving a family of serine/threonine kinases and oxidation of the mediator proteins.58 in the same line with these studies, we have also found an increase in the serum and hepatic levels of ffas with a concomitant increase in hepatic ros, tnf-α, and il-6 and a reduction in hepatic and muscles glycogen, thus suggesting that ag pro-oxidant effect is mediated by stimulating peripheral lipolysis. this possibly due to stimulating muscle lipolysis but not that of adipose tissue as suggested by other authors.32, 59, 60 similar to our data, the infusion of ag increased plasma levels of nefa plasma non-esterified fas (nefas).42, 61 besides, ag-increased the expression of ire1α and induced activation of jnk and phosphorylation of irs (ser307) which were reduced in the livers of uag-treated rats and attenuated in the livers of ag-treated rats which co-administered uag. also, individual or combined administration of uag with ag reduced the levels of ffas and suppressed the hepatic oxidative stress and inflammatory response. since the liver lacks ag receptors (gshr-1a),62 these data suggest that the hepatic effect of both ag and ug on markers of oxidative stress, inflammation, and insulin signaling are controlled by modulating peripheral insulin sensitivity, ffas influx and possibly through the unidentified peripheral or hepatic receptor. this requires further studies. on the other hand, previous studies have shown that ag-stimulates hepatic fas synthesis related genes and suppresses fas oxidation whereas uag suppresses hepatic lipid accumulation by increasing fas oxidation.25, 27, 30, 32, 36 ampkα1 is a negative inhibitor of srebps and potent inducer of pparα and other β-oxidation genes (dallak, 2018b). on the other hand, insulin is a potent inducer of srebp1/2 in the mammalian liver.51, 53 similar to these studies, we have also found that ag-stimulated hepatic lipogenesis and fat accumulation are associated with increasing the mrna levels of srebp1/2 and fas. on the other hand, individual administration of uag to healthy rats or in combination with ag reduced the hepatic mrna levels of these lipogenic enzymes. however, both ag and uag increased the mrna of ampk and pparα. 137j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 s. alharbi and r. a. eid original exogenous acylated ghrelin promotes therefore, it seems reasonable that the anti-lipogenic effect of uag is mediated by suppressing fas synthesis genes and stimulating fas oxidation by upregulating/activating ampk/ pparα axis. however, it seems logical that the activation of ampk/pparα by ag treatment is a compensatory mechanism to reduce fatty acid synthesis and fat accumulation or due to increased ffas influx. indeed, ffas can activate ampk and pparα in the different tissues including the livers and cardiomyocytes.58, 63, 64 also, and given the higher fasting insulin levels, it could be possible that ag increases the activation of srebp1/2 and fas by the resulted hyperinsulinemia. however, since ffas were significantly decreased and insulin levels were not significantly altering in uag-treated rats, we could suggest that uag suppresses lipogenic genes and stimulates ampk by a direct effect on the liver and possibly by reducing oxidative stress and inflammation. although this provides some possible explanation for the effect of ag and uag on hepatic lipid metabolism, the precise mechanisms by which both ghrelin forms regulate this is largely unknown. on interesting area is the emerging role of er stress in hepatic lipid metabolism.65 within most cells including the hepatocytes, accumulation of unfolded proteins in the er due to the increase in metabolic activity, triggers unfolded protein response (upr) via activation of three transmembrane signal transducers, namely inositol-requiring enzyme-1 (ire-1), activating transcription factor-6 (atf-6), and protein kinase rna-like er kinase (perk), mediated by dissociation of chaperone immunoglobulin heavy-chainbinding protein (bip; also known as grp78.5 these events are associated with phosphorylation and inhibition eukaryotic translation initiation factor 2α (eif2α), activation of transcription factor 4 (atf4), splicing of x-box-binding protein-1 (xbp1), all of which aid to inhibit protein synthesis to enhance cell survival.65 as mentioned before, higher levels of ffas, ros, and inflammatory cytokines can also activate hepatic upr and er stress.52, 53 nonetheless, unresolved upr and er stress activates numerous arms that induce hepatic steatosis.65, 66 indeed, the activation of the perk/eif2α-/atf4 pathway induces hepatic steatosis by upregulating srebps, fas, and stearoyl-coa desaturase-1 (scd1).8, 50, 67, 68 also, phosphorylation of eif2a induces glucose intolerance and exaggerates hepatic lipid synthesis and steatosis in rodent.8 in the same line, upregulation of chop, a downstream target of p-eif2α, increases hepatic gluconeogenesis and lipid deposition and stimulates hepatocytes apoptosis.66, 69 besides, ire1α/xbp-1pathway induced hepatic lipogenesis by the binding of xbp-1 to the promoters of scd1, diacylglycerol acyltransferase-2 (dgat2), and acc-1, thus inducing their activation.9 this could explain the findings of dallak27 who failed to explain why the livers of ag-treated rats have increased expression of dgat-2 which was reduced with the treatment with uag. in this study, chronic administration of ag activated upr and er stress in the liver of rats as shown by the significant increase in hepatic levels of perk, atf6, ire1α, and their downstream targets p-eif2α, atf4, chop, and xbp-1. this could be due to increased hepatic levels of ffas, ros, and inflammatory cytokines due to the peripheral ir as described above. on the contrary, chronic administration of uagsignificantly inhibited the expression of all these biochemical markers, except for atf-6 which showed a slight but significant increase. besides, co-administration of uag with ag reversed all its effects on these markers. based on these data, we concluded that that ag induces hepatic steatosis and ir by inducing er stress where uag prevents these events by suppressing er and activating atf6 possibly due to improving insulin sensitivity. interestingly, atf6 causes an increase in ampk expression and fas oxidation to rescue cells from lipotoxicity.70 this could help us to explain the increased fas oxidation in the livers of uag-treated rats. in conclusion, our data suggest that ag induces hepatic steatosis and ir by increasing oxidative stress, inflammation, and er stress, at least through inducing peripheral ir. however, uag protective effect is mediated by contradictory mechanisms. also, our data suggest that a balanced ratio of ag/uag is crucial to preserve normal glucose, insulin, and lipid metabolism. although our data suggest that the lipogenic effect of ag and anti-lipogenic effects of uag mediated by modulating the peripheral ir, other mechanisms including unidentified hepatic or systemic receptor and modulating growth hormones and parasympathetic flow were not investigated in this study. indeed, several authors have suggested that the hepatic effect of ag is mediated indirectly by altering the levels of growth hormones, the vagus nerve, and unidentified hepatic receptors.28, 71–73 besides, our data remain descriptive and if the effects of ag and uag er stress are mediated by a direct effect or through peripheral insulin-dependent mechanism can’t be definitely judged form these data. therefore, further studies in genetically modified animals and isolated hepatocytes lacking some arms of er are needed to validate our data. besides, further studies using an er inhibitors may help to better understanding these results. acknowledgment the authors would like to express their gratitude to the college of medicine at king khalid university for their help in conducting the animal part of this study. they also would like to thank the technical staff for their help in measurements of some parameters under the supervision of dr. refaat eid for his significant contribution to this study at the department of biology. the authors can extend their thanks to the deanship of scientific research at umm al-qura university for supporting this project by grant code: 10-med-1-03-0004. conflicts of interest the authors declare no conflicts of interest.  references 1. malaguarnera, m., di rosa, m., nicoletti, f. & malaguarnera, l. 2009. molecular mechanisms involved in nafld progression. journal of molecular medicine, 87, 679. 2. cohen, j. c., horton, j. d. & hobbs, h. h. 2011. human fatty liver disease: old questions and new insights. science, 332, 1519 -1523. 3. zhang, s. r. & fan, x. m. 2015. ghrelin-ghrelin o-acyltransferase system in the pathogenesis of nonalcoholic fatty liver disease. world journal of gastroenterology: wjg, 21, 3214. 4. te sligte, k., bourass, i., sels, j., driessen, a., stockbrűgger, r. & koek, g. 2004. non-alcoholic steatohepatitis: review of a growing medical problem. european journal of internal medicine, 15, 10 -21. 138 j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 exogenous acylated ghrelin promotes original s. alharbi and r. a. eid 5. zhang, x. q., xu, c. f., yu, c. h., chen, w. x. & li, y.-m. 2014a. role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. world journal of gastroenterology: wjg, 20, 1768. 6. tabas, i. & ron, d. 2011. integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. nature cell biology, 13, 184 -190. 7. özcan, u., cao, q., yilmaz, e., lee, a.-h., iwakoshi, n. n., özdelen, e., tuncman, g., görgün, c., glimcher, l. h. & hotamisligil, g. s. 2004. endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. science, 306, 457 -461. 8. oyadomari, s., harding, h. p., zhang, y., oyadomari, m. & ron, d. 2008. dephosphorylation of translation initiation factor 2α enhances glucose tolerance and attenuates hepatosteatosis in mice. cell metabolism, 7, 520 -532. 9. lee, a. h., scapa, e. f., cohen, d. e. & glimcher, l. h. 2008. regulation of hepatic lipogenesis by the transcription factor xbp1. science, 320, 1492 -6. 10. ji, c. 2014. new insights into the pathogenesis of alcohol-induced er stress and liver diseases. international journal of hepatology, 2014. 11. chan, s.-w. 2014. unfolded protein response in hepatitis c virus infection. frontiers in microbiology, 5, 233. 12. wang, d., wei, y. & pagliassotti, m. j. 2006. saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis. endocrinology, 147, 943 -951. 13. rahman, s. m., schroeder-gloeckler, j. m., janssen, r. c., jiang, h., qadri, i., maclean, k. n. & friedman, j. e. 2007. ccaat/enhancing binding protein β deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis. hepatology, 45, 1108 -1117. 14. puri, p., mirshahi, f., cheung, o., natarajan, r., maher, j. w., kellum, j. m. & sanyal, a. j. 2008. activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease. gastroenterology, 134, 568 -576. 15. gregor, m. f., yang, l., fabbrini, e., mohammed, b. s., eagon, j. c., hotamisligil, g. s. & klein, s. 2009. endoplasmic reticulum stress is reduced in tissues of obese subjects after weight loss. diabetes, 58, 693 -700. 16. rinella, m. e., siddiqui, m. s., gardikiotes, k., gottstein, j., elias, m. & green, r. m. 2011. dysregulation of the unfolded protein response in db/db mice with diet-induced steatohepatitis. hepatology, 54, 1600 -1609. 17. fang, d.-l., wan, y., shen, w., cao, j., sun, z.-x., yu, h.-h., zhang, q., cheng, w.-h., chen, j. & ning, b. 2013. endoplasmic reticulum stress leads to lipid accumulation through upregulation of srebp-1c in normal hepatic and hepatoma cells. molecular and cellular biochemistry, 381, 127 -137. 18. ozawa, k., miyazaki, m., matsuhisa, m., takano, k., nakatani, y., hatazaki, m., tamatani, t., yamagata, k., miyagawa, j.-i. & kitao, y. 2005. the endoplasmic reticulum chaperone improves insulin resistance in type 2 diabetes. diabetes, 54, 657 -663. 19. nakatani, y., kaneto, h., kawamori, d., yoshiuchi, k., hatazaki, m., matsuoka, t.-a., ozawa, k., ogawa, s., hori, m. & yamasaki, y. 2005. involvement of endoplasmic reticulum stress in insulin resistance and diabetes. journal of biological chemistry, 280, 847 -851. 20. kammoun, h. l., chabanon, h., hainault, i., luquet, s., magnan, c., koike, t., ferré, p. & foufelle, f. 2009. grp78 expression inhibits insulin and er stress induced srebp-1c activation and reduces hepatic steatosis in mice. the journal of clinical investigation, 119, 1201 -1215. 21. gualillo, o., lago, f. & dieguez, c. 2008. introducing goat: a target for obesity and anti-diabetic drugs? trends in pharmacological sciences, 29, 398 -401. 22. soares, j.-b., roncon-albuquerque jr, r. & leite-moreira, a. 2008. ghrelin and ghrelin receptor inhibitors: agents in the treatment of obesity. expert opinion on therapeutic targets, 12, 1177 -1189. 23. estep, m., abawi, m., jarrar, m., wang, l., stepanova, m., elariny, h., moazez, a., goodman, z., chandhoke, v. & baranova, a. 2011. association of obestatin, ghrelin, and inflammatory cytokines in obese patients with nonalcoholic fatty liver disease. obesity surgery, 21, 1750 -1757. 24. li, y., hai, j., li, l., chen, x., peng, h., cao, m. & zhang, q. 2013. administration of ghrelin improves inflammation, oxidative stress, and apoptosis during and after non-alcoholic fatty liver disease development. endocrine, 43, 376 -386. 25. li, z., xu, g., qin, y., zhang, c., tang, h., yin, y., xiang, x., li, y., zhao, j. & mulholland, m. 2014. ghrelin promotes hepatic lipogenesis by activation of mtor-pparγ signaling pathway. proceedings of the national academy of sciences, 111, 13163 -13168. 26. arslan, n., sayin, o. & tokgoz, y. 2014. evaluation of serum xenin and ghrelin levels and their relationship with nonalcoholic fatty liver disease and insulin resistance in obese adolescents. journal of endocrinological investigation, 37, 1091 -1097. 27. dallak, m. a. 2018b. acylated ghrelin induces but deacylated ghrelin prevents hepatic steatosis and insulin resistance in lean rats: effects on dag/pkc/jnk pathway. biomedicine & pharmacotherapy, 105, 299 -311. 28. zhang, y., fang, f., goldstein, j. l., brown, m. s. & zhao, t.-j. 2015. reduced autophagy in livers of fasted, fat-depleted, ghrelin-deficient mice: reversal by growth hormone. proceedings of the national academy of sciences, 112, 1226 -1231. 29. lim, c. t., kola, b., grossman, a. & korbonits, m. 2011. the expression of ghrelin o-acyltransferase (goat) in human tissues. endocrine journal, 58, 707 -710. 30. kola, b., hubina, e., tucci, s. a., kirkham, t. c., garcia, e. a., mitchell, s. e., williams, l. m., hawley, s. a., hardie, d. g. & grossman, a. b. 2005. cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via amp-activated protein kinase. journal of biological chemistry, 280, 25196 -25201. 31. barazzoni, r., zanetti, m., ferreira, c., vinci, p., pirulli, a., mucci, m., dore, f., fonda, m., ciocchi, b. & cattin, l. 2007. relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome. the journal of clinical endocrinology & metabolism, 92, 3935 -3940. 32. barazzoni, r., bosutti, a., stebel, m., cattin, m. r., roder, e., visintin, l., cattin, l., biolo, g., zanetti, m. & guarnieri, g. 2005. ghrelin regulates mitochondrial-lipid metabolism gene expression and tissue fat distribution in liver and skeletal muscle. american journal of physiology-endocrinology and metabolism. 33. rigault, c., le borgne, f., georges, b. & demarquoy, j. 2007. ghrelin reduces hepatic mitochondrial fatty acid β oxidation. journal of endocrinological investigation, 30, rc4-rc8. 34. porteiro, b., díaz-ruíz, a., martínez, g., senra, a., vidal, a., serrano, m., gualillo, o., lópez, m., malagón, m. m. & diéguez, c. j. e. 2013. ghrelin requires p53 to stimulate lipid storage in fat and liver. 154, 3671 -3679. 35. dallak, m. a. 2018a. acylated ghrelin induces but deacylated ghrelin prevents hepatic steatosis and insulin resistance in lean rats: effects on dag/pkc/jnk pathway. 105, 299 -311. 36. delhanty, p. j., sun, y., visser, j. a., van kerkwijk, a., huisman, m., van ijcken, w. f., swagemakers, s., smith, r. g., themmen, a. p. & van der lely, a.-j. 2010. unacylated ghrelin rapidly modulates lipogenic and insulin signaling pathway gene expression in metabolically active tissues of ghsr deleted mice. plos one, 5, e11749. 37. ezquerro, s., méndez-giménez, l., becerril, s., moncada, r., valentí, v., catalán, v., gómez-ambrosi, j., frühbeck, g. & rodríguez, a. 2016. acylated and desacyl ghrelin are associated with hepatic lipogenesis, β-oxidation and autophagy: role in nafld amelioration after sleeve gastrectomy in obese rats. scientific reports, 6, 1 -12. 38. khaleel, e. f., abdel-aleem, g. a. & mostafa, d. g. 2018. resveratrol improves high-fat diet induced fatty liver and insulin resistance by concomitantly inhibiting proteolytic cleavage of sterol regulatory element-binding proteins, free fatty acid oxidation, and intestinal triglyceride absorption. canadian journal of physiology and pharmacology, 96, 145 -157. 39. roden, m., prskavec, m., fürnsinn, c., elmadfa, i., könig, j., schneider, b., wagner, o. & waldhäusl, w. 1995. metabolic effect of sodium selenite: insulin-like inhibition of glucagon-stimulated glycogenolysis in the isolated perfused rat liver. hepatology, 22, 169 -174. 40. wertz-lutz, a., knight, t., pritchard, r., daniel, j., clapper, j., smart, a., trenkle, a. & beitz, d. 2006. circulating ghrelin concentrations fluctuate relative to nutritional status and influence feeding behavior in cattle. journal of animal science, 84, 3285 -3300. 41. iqbal, j., kurose, y., canny, b. & clarke, i. j. 2006. effects of central infusion of ghrelin on food intake and plasma levels of growth hormone, luteinizing hormone, prolactin, and cortisol secretion in sheep. endocrinology, 147, 510 -519. 42. roche, j., sheahan, a., chagas, l., blache, d., berry, d. & kay, j. 2008. longterm infusions of ghrelin and obestatin in early lactation dairy cows. journal of dairy science, 91, 4728 -4740. 43. murata, m., okimura, y., iida, k., matsumoto, m., sowa, h., kaji, h., kojima, m., kangawa, k. & chihara, k. 2002. ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. journal of biological chemistry, 277, 5667 -5674. 44. gauna, c., meyler, f., janssen, j., delhanty, p., abribat, t., van koetsveld, p., hofland, l., broglio, f., ghigo, e. & van der lely, a. j. 2004. administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. the journal of clinical endocrinology & metabolism, 89, 5035 -5042. 45. kamegai, j., tamura, h., shimizu, t., ishii, s., sugihara, h. & wakabayashi, i. 2001. chronic central infusion of ghrelin increases hypothalamic https://doi.org/10.22317/jcms.v7i3.998 139j contemp med sci | vol. 7, no. 3, may-june 2021: 128–139 s. alharbi and r. a. eid original exogenous acylated ghrelin promotes neuropeptide y and agouti-related protein mrna levels and body weight in rats. diabetes, 50, 2438 -2443. 46. stevanović, d., nešić, d., milošević, v., starčević, v. & severs, w. b. 2008. consummatory behavior and metabolic indicators after central ghrelin injections in rats. regulatory peptides, 147, 52 -59. 47. theander-carrillo, c., wiedmer, p., cettour-rose, p., nogueiras, r., perez-tilve, d., pfluger, p., castaneda, t. r., muzzin, p., schürmann, a. & szanto, i. 2006. ghrelin action in the brain controls adipocyte metabolism. the journal of clinical investigation, 116, 1983 -1993. 48. zhang, w., chai, b., li, j. y., wang, h. & mulholland, m. w. 2008. effect of des-acyl ghrelin on adiposity and glucose metabolism. endocrinology, 149, 4710 -4716. 49. cederberg, h., koivisto, v. m., jokelainen, j., surcel, h. m., keinänenkiukaanniemi, s. & rajala, u. 2012. unacylated ghrelin is associated with changes in insulin sensitivity and lipid profile during an exercise intervention. clinical endocrinology, 76, 39 -45. 50. wang, c., huang, z., du, y., cheng, y., chen, s. & guo, f. 2010. atf4 regulates lipid metabolism and thermogenesis. cell research, 20, 174 -184. 51. utzschneider, k. m. & kahn, s. e. 2006. the role of insulin resistance in nonalcoholic fatty liver disease. the journal of clinical endocrinology & metabolism, 91, 4753 -4761. 52. cui, w., ma, j., wang, x., yang, w., zhang, j. & ji, q. 2013. free fatty acid induces endoplasmic reticulum stress and apoptosis of β-cells by ca 2+/ calpain-2 pathways. plos one, 8, e59921. 53. kawasaki, n., asada, r., saito, a., kanemoto, s. & imaizumi, k. 2012. obesityinduced endoplasmic reticulum stress causes chronic inflammation in adipose tissue. scientific reports, 2, 799. 54. du, k., herzig, s., kulkarni, r. n. & montminy, m. 2003. trb3: a tribbles homolog that inhibits akt/pkb activation by insulin in liver. science, 300, 1574 -1577. 55. zhang, x. q., xu, c. f., yu, c. h., chen, w. x. & li, y. m. 2014c. role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. world j gastroenterol, 20, 1768 -76. 56. nakae, j., biggs, w. h., kitamura, t., cavenee, w. k., wright, c. v., arden, k. c. & accili, d. 2002. regulation of insulin action and pancreatic β-cell function by mutated alleles of the gene encoding forkhead transcription factor foxo1. nature genetics, 32, 245 -253. 57. kim, j. j., li, p., huntley, j., chang, j. p., arden, k. c. & olefsky, j. m. 2009. foxo1 haploinsufficiency protects against high-fat diet -induced insulin resistance with enhanced peroxisome proliferator -activated receptor γ activation in adipose tissue. diabetes, 58, 1275 -1282. 58. rains, j. l. & jain, s. k. 2011. oxidative stress, insulin signaling, and diabetes. free radical biology and medicine, 50, 567 -575. 59. grala, t., kay, j., walker, c., sheahan, a., littlejohn, m., lucy, m. & roche, j. 2010. expression analysis of key somatotropic axis and liporegulatory genes in ghrelin-and obestatin-infused dairy cows. domestic animal endocrinology, 39, 76 -83. 60. vestergaard, e. t., buhl, m., gjedsted, j., madsen, m., jessen, n., nielsen, s., gaylinn, b. d., liu, j., thorner, m. o. & moller, n. 2011. acute peripheral metabolic effects of intraarterial ghrelin infusion in healthy young men. the journal of clinical endocrinology & metabolism, 96, 468 -477. 61. thidarmyint, h., yoshida, h., ito, t. & kuwayama, h. 2006. dose-dependent response of plasma ghrelin and growth hormone concentrations to bovine ghrelin in holstein heifers. journal of endocrinology, 189, 655 -664. 62. sun, y., garcia, j. m. & smith, r. g. 2007. ghrelin and growth hormone secretagogue receptor expression in mice during aging. endocrinology, 148, 1323 -1329. 63. suchankova, g., tekle, m., saha, a. k., ruderman, n. b., clarke, s. d. & gettys, t. w. 2005. dietary polyunsaturated fatty acids enhance hepatic amp-activated protein kinase activity in rats. biochemical and biophysical research communications, 326, 851 -858. 64. watt, m. j., steinberg, g. r., chen, z. p., kemp, b. e. & febbraio, m. a. 2006. fatty acids stimulate amp-activated protein kinase and enhance fatty acid oxidation in l6 myotubes. the journal of physiology, 574, 139 -147. 65. zhang, x. q., xu, c. f., yu, c. h., chen, w. x. & li, y. m. j. w. j. o. g. w. 2014b. role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. 20, 1768. 66. basseri, s. & austin, r. c. 2008. er stress and lipogenesis: a slippery slope toward hepatic steatosis. developmental cell, 15, 795 -796. 67. seo, j., fortuno, e. s., suh, j. m., stenesen, d., tang, w., parks, e. j., adams, c. m., townes, t. & graff, j. m. 2009. atf4 regulates obesity, glucose homeostasis, and energy expenditure. diabetes, 58, 2565 -2573. 68. xiao, g., zhang, t., yu, s., lee, s., calabuig-navarro, v., yamauchi, j., ringquist, s. & dong, h. h. 2013. atf4 protein deficiency protects against high fructose-induced hypertriglyceridemia in mice. journal of biological chemistry, 288, 25350 -25361. 69. kim, i., xu, w. & reed, j. c. 2008. cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. nature reviews drug discovery, 7, 1013 -1030. 70. yamamoto, k., takahara, k., oyadomari, s., okada, t., sato, t., harada, a. & mori, k. 2010. induction of liver steatosis and lipid droplet formation in atf6αknockout mice burdened with pharmacological endoplasmic reticulum stress. molecular biology of the cell, 21, 2975 -2986. 71. iwasaki, y., dezaki, k., kumari, p., kakei, m. & yada, t. 2015. ghrelin counteracts insulin-induced activation of vagal afferent neurons via growth hormone secretagogue receptor. neuropeptides, 52, 55 -60. 72. chen, j. a., splenser, a., guillory, b., luo, j., mendiratta, m., belinova, b., halder, t., zhang, g., li, y. p. & garcia, j. m. 2015. ghrelin prevents tumour-and cisplatin-induced muscle wasting: characterization of multiple mechanisms involved. journal of cachexia, sarcopenia and muscle, 6, 132 -143. 73. guillory, b., jawanmardi, n., iakova, p., anderson, b., zang, p., timchenko, n. a. & garcia, j. m. 2018. ghrelin deletion protects against age-associated hepatic steatosis by downregulating the c/ebpα-p300/dgat1 pathway. aging cell, 17, e12688. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 102 original issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 102 – 107 introduction tuberculosis (tb) is a paramount reason of morbidity and is amongst the peak 10 causes of death universally, standing over hiv as a sole infectious cause of death.1 based on speculation from 2017, there were 10 million tb cases and 1.6 million tb-linked deaths worldwide, despite of a 2% incline in incidence proportion per annum in recent years.1 according to the world health organization (who), in 2019, 770,000 persons had passed away from hiv. an estimated third of these deaths were pertained to tb.2 hiv and tb are well known as a syndemic which is clarified as “the concourse of two or more diseases that perform in an interactive pattern to aggrandize the encumbrance of disease”.3 al-salihy4 concluded that iraq had been classified as a low-prevalence hiv epidemic, with a low number of formally reported cases (0.1% of the total population). the first case was announced in 1986 among hemophilic patients who had received contaminated blood products. furthermore, the cumulative number of hiv/aids registered cases from 1986 up to 2011 was 306. among the registered cases, 85% were male and the most common mode of transmission (66%) was via imported blood products, 17% by heterosexual route, and 5% by vertical transmission from infected mother. no cases of transmission due to homosexual or drug addicts were reported. since 2003, the transmission mode shifted towards the heterosexual route, as the government adopted strict measures to ensure blood safety. today, tb continues to be the main infectious reason of death in people with hiv, who are probably 15–22 times more likely to be infected with tb than people without.5 presently, the center for disease control and prevention (cdc) advises that all patients with active or latent tb be checked for hiv because hiv is a quite-recognized stimulator of hidden tb.6 nearly one-quarter of the universal inhabitance is appraised to be latently infected with mycobacterium tuberculosis (mtb) also referred to as latent tuberculosis (ltbi).7 a person with ltbi has an mtb infection, but the bacteria continues to be recumbent and ineffectual within the host’s body with no clinical markers.8 in hiv negative patients, a latent tb infection has a 10% chance of proceeding into an active infection throughout a lifetime. however, this jeopardy is considerably boosted in hiv positive patients and it progressively magnifies as immune function descends.9 individuals with hiv and tb also have the potency to impact the health of hiv negative individuals because the brisk of ltbi makes mtb highly infectious.10 awareness about the predominance of hiv-positive infection in active tb patients is mandatory, as prevalence of hiv among newly diagnosed tuberculosis patients in erbil governorate, iraq zakarea abdullah yaseen al-khayat1 id *, nabaz fisal shakir agha2 id , pshtiwan dhahir majeed3, kawthar ibrahim fatah alharmni4 id , derin nabaz fisal agha5 1 department of microbiology, college of medicine, hawler medical university, erbil, iraq. 2 department of anesthesia, erbil polytechnic university, erbil medical technical institute, iraq. 3 department of nursing, erbil polytechnic university, erbil medical technical institute, iraq. 4 department of anatomy & histology, college of medicine, hawler medical university, erbil, iraq. 5 department of pharmacology, erbil polytechnic university, erbil medical technical institute, iraq. *correspondence to: zakarea abdullah yaseen al-khayat (e-mail: dr_zakarea@yahoo.co.uk) (submitted: 03 january 2021 – revised version received: 22 january 2021 – accepted: 06 february 2021 – published online: 26 april 2021) abstract objective this study was accomplished with a purpose to determine the sociodemographic profile and the prevalence of hiv among tuberculosis (tb) patients. methods this prospective study was carried out in the department of microbiology at the chest and respiratory disease specialized centre in erbil city (in collaboration with the specialist physician) through a period from january 2017 to december 2019. new tb patients were interviewed on a predesigned questionnaire. collected samples were processed in a special laboratory in tb center. the samples were subjected to microscopy with ziehl–neelsen staining and inoculated on solid medium; the third sputum sample was tested directly by genexpert test. hiv testing was done using screening test and if the screening result was positive, the diagnosis was confirmed by western blot. results a total of 397 approved new tb patients underwent hiv testing. among them, 41 cases (10.3%) were found to be positive on elisa screening, and subsequently they were all confirmed by the western blot test. the highest prevalence of hiv positivity according to gender, age range, and occupation, were as follows: male (29; 70.7%), 30–42 years (21; 51.2%), laborers (13; 31.7%) respectively. the male to female ration is 2.7 statistically, the differences of distribution of the hiv positivity concerning the above-mentioned demography were as follows: gender: significant (p ≤ 0.05), age range: not significant, occupation: not significant. the highest prevalence of hiv positivity was among pulmonary tb (25; 61%). rifampicin resistant prevalence was higher among hiv positive in comparison to hiv negative tb case (23; 56.1%) (134; 37.4%), respectively. statistically, the differences of distribution of the hiv positivity in relation to both tb pattern & rifampicin monodrug resistant were significant (p ≤ 0.05). conclusions the prevalence of hiv infection in tb patients in current study was 10.3%. if hiv testing was done for all tb patients, then routine reporting of hiv status for all tb patients would provide even better information which may provide a base to future planning. keywords hiv prevalence, tuberculosis, erbil, sociodemographic determinants, rifampicin resistance. https://orcid.org/0000-0001-8353-4777 https://orcid.org/0000-0001-7212-959x https://orcid.org/0000-0002-9658-4682 103 original prevalence of hiv among newly diagnosed tuberculosis patients in erbil governorate, iraqzakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 102 – 107 it is increasingly being realized that such facts would motivate arranging and may also be pivotal for setting the convenient treatment regime.11 the significance of hiv monitoring in tb patients is increasingly being recognized as the hiv epidemic keeps on to uphold the global tb epidemic.10 in numerous territories, the hiv dispersal in tb patients is a critical signal of the propagation of hiv into the overall inhabitance. information about the hiv prevalence in tb patients is indispensable for supplying overall hiv/aids attention and assistance, encompassing antiretroviral therapy (art) to hiv-positive patients.11,12 thus, the objectives of this study were to explore the feasibility of hiv screening among tb patients registered for treatment in a selected tb treatment center in erbil governorate and to depict the prevalence of hiv amongst tb patients in the research district. in addition, the study also describes various clinical presentations among these co-infected cases. materials and methods study protocol this prospective study was carried out in the department of microbiology at the chest and respiratory disease specialized centre in erbil city (in collaboration with the specialist physician) through a period from january 2017 to december 2019. this study was achieved with the cooperation of prevention health department, erbil medical technical institute, erbil polytechnic university, with departments of: microbiology, anatomy & histology, college of medicine, hawler medical university, erbil, iraq. moral considerations this study was confirmed by: the ethics committee of hawler medical university, erbil; the committee of erbil medical technical institute, erbil polytechnic university, iraq; health directorate of erbil. acquainted endorsement was possessed from each patient. the patients were aware of study’s goals and they could regress thereof if they wished so to do. study population in the current study, all newly approved cases of tb (18 years of age and above) who were inhabitants of erbil governorate were embroiled during the study period. all tb patients who conferred agreement for hiv testing (after pretest counseling) in the inand outpatient wards of the department of medicine were involved in the study. using a structured, pre-tested questionnaire including all personal information, such as age, gender, socioeconomic background, education level, profession, sexual preferences and promiscuity, history of past surgery, or blood transfusion were collected by patient interview. patients receiving or who received anti-tb treatment in the previous month and non-consenting patients were excluded from this study. in addition, cases on art who were attending the hospital for follow-up were also excluded. sample and diagnostic procedures suspected tb patients for collecting specimens were allocated in this study on the basis of presenting symptoms and chest radiography findings. pulmonary specimens were taken from the sputum. patients meeting the clinical eligibility criteria were asked to provide three sputum specimens: two spot samples and one obtained in the morning. extra-pulmonary specimens were taken from pleural fluid, lymph node biopsy, gastrointestinal, cerebrospinal fluid, skin, and genitourinary. afb and culture collected samples were processed in a special laboratory in tb center, then two of the three samples were randomly selected and processed with n-acetylcysteine and sodium hydroxide followed by centrifugation.13 the samples were resuspended in 1.5 ml of sample buffer and underwent microscopy analysis with ziehl–neelsen staining and cultured on solid medium (löwenstein–jensen, biomerieux, france). the third sputum sample was tested directly by genexpert test. solid cultures were considered negative after 42 days of incubation without isolation of any mycobacteria.14 non-respiratory specimens from closed and normally sterile sites were not decontaminated prior to smear preparation and culture but were concentrated by centrifugation at 3,000g for 20 min. processed specimens from non-sterile sites and centrifuged specimens from sterile sites were directly cultured.13,14 genexpert assay the genexpert assay was used as previously used.15 briefly, the provided buffer was added at a 3:1 ratio to clinical samples. the tubes were mixed manually twice in 15-min period at room temperature before 2 ml of the inactivated material was transferred to the test cartridge. the cartridge was then inserted into the test platform, and the hand on work ended. then, the machine automatically filtered, washed, and ultrasonically lyzed to release dna. real-time pcr amplification and detection were performed in an integrated reaction tube. primers used for this assay were forward: (cgtggaggcgatcacaccgcagac) and reverse: (agctccagcccggcacgctcacgt) (applied biosystems). the results were finally read after 1 h 45 min, in which fluorescent signal was measured automatically. negative or positive and defined susceptible or resistant to rifampin depending on the detection of mutations in rpob gene (mtbrif instructions). the patient confirmed to be infected with tb disease when: bacteriologically or microscopically confirmed pulmonary tb (ptb), i.e., positive culture and/or at least one sputum sample with a positive acid-fast bacillus (afb) test; afb positive smear which was a fluorochromestained smear with at least one acid fast bacilli in 40 fields (or xpert mtb/rif positive).7,8 hiv test hiv testing was done using khb (shanghi kehua bioengineering, ltd, shanghai, china) as a screening test. if the screening result was positive, the diagnosis was confirmed by western blot test (hivblot 2.2, genelabs diagnostics, singapore). statistical analysis the data analysis was performed using descriptive statistics, including frequency, and frequency percentage. comparisons were made using chi2 test by using standard equations. the results were announced with p ≤ 0.05 or p ≤ 0.01 as the acceptable level of significance. 104 original prevalence of hiv among newly diagnosed tuberculosis patients in erbil governorate, iraq zakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 102 – 107 results a total of 1436 tb patients were registered for treatment during the study period. of the total tb patients, 397 had met the present study criteria mentioned above. the total 397 approved tb patients underwent hiv testing. among them, 41 cases (10.3%) were found to be positive on elisa screening, and subsequently they were all confirmed by the western blot test. the remaining 356 tb cases were hiv negative. table 1 delineates the prevalence of hiv infection among new tuberculosis patients according to age, gender and occupation. the highest prevalence of hiv positivity according to gender, age range and occupation, were as follows: male (29; 70.7%), 30–42 years (21; 51.2%), laborers (13; 31.7%) respectively. the male to female ration is 2.7. statistically, the differences of distribution of the hiv positivity concerning the above-mentioned demography were as follows: gender: significant (p ≤ 0.05), age range: non-significant, occupation: non-significant table 2 illustrates tb pattern i.e.: pulmonary and extra pulmonary, and the rifampicin drug resistant among hiv seropositive and seronegative cases. the highest prevalence of hiv positivity was among pulmonary tb (25; 61%). rifampicin resistant prevalence was higher among hiv positive in comparison to hiv negative tb case (23; 56.1%) (134; 37.4%) respectively. statistically, the differences of distribution of the hiv positivity in relation to both tb pattern and rifampicin monodrug resistant were significant (p ≤ 0.05). discussion to the best of our knowledge, no published data are available on the prevalence of hiv among newly diagnosed tb patients in erbil governorate, hence this study can be considered the first study of such quality to deal with and investigate the prevalence, correlated risk factors, and impact of such an infection among these patients. according to the cdc recommendation, hiv screening to be accomplished for all tb patients comprising persons with tb disease, latent tb disease, and persons suspected of having tb and contact to tb patients. thus, the current study was achieved to define the prevalence of hiv among diagnosed tb patients in erbil. this study expounds that hiv seroprevalence among tb patients attending the chest and respiratory disease specialized centre in erbil city in erbil in 2017–2019 was 10.3%. prevalence rates of hiv tb patients vary internationally.16 the present prevalence of hiv among tb patients was found to be less than 12%, who’s estimate of hiv among the world tb patients.17 the prevalence of tb/ hiv co-infection in india,18 and ethiopia19 were 18.9% and 28.6%, respectively, and were higher than our rates. in a study carried by van der werf et al. (20), the highest co-infection rates were recorded from latvia (19.5%), malta (17.1%), portugal (14.7%), and estonia (10.1%). gao et al21 performed a systematic review and meta-analysis to determine the prevalence of tb/hiv co-infection by recognizing 47 studies comprising 272,466 persons except china. their prime findings were that, a high prevalence of tb/hiv co-infection and they deduced that tb/hiv co-infection dispersal was higher among tb patients than among hiv/aids patients, but which were not significantly different. studies from sub-saharan africa had enrolled those hiv seroprevalence rates of 50–70% in patients with tuberculosis.16 hospital-based hiv seroprevalence studies amongst tb patients from various territories of india had exhibited table 1. prevalence of hiv infection among new tuberculosis patients in relation to age, gender, occupation. parameters n hiv+ (%) hiv(%) gender male 223 29 (70.7) 194 (54.5) female 174 12 (29.3) 162 (45.5) total 397 41 (100) 356 (100) χχ2 = 3.9373 df=1, significant (p ≤ 0.05) age 18-30 116 11 (26.8) 105 (29.5) 30-42 177 21 (51.2) 156 (43.8) >42 104 9 (22) 95 (26.7) total 397 41 (100) 356 (100) df = 2 ns occupation farmer 71 6 (14.6) 65 (18.3) service 83 7 (17.1) 76 (21.3) (clerk/ tailor) drivers 79 10 (24.4 ) 69 (19.4) housewives 66 5 (12.2) 61 (17.1 ) laborers 98 13 (31.7) 85 (23.9) total 397 41 (100) 356 (100) df = 4 ns table 2. tuberculosis pattern and rifampicin status among hiv seropositive & seronegative patients. type of tb hiv positive (n)% hiv negative (n)% total pulmonary 25 (61) 269 (75.6) 294 extra pulmonary 16 (39) 87 (24.4) 103 total 41 (100) 356 (100) 397 χχ2 = 4.0712 df = 1, significant (p ≤ 0.05) rifampicin sensitive 18 (43.9) 222 (62.4) 240 resistant 23 (56.1) 134 (37.4) 157 total 41 (100) 356 (100) 397 χχ2 = 5.2389 df = 1, significant (p ≤ 0.05) 105 original prevalence of hiv among newly diagnosed tuberculosis patients in erbil governorate, iraqzakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 102 – 107 a considerable divergences in the prevalence which varied from 0.4% to 28.1% have been reported.22 studies performed in various provinces of brazil had manifested that the prevalence of hiv infection in individuals with tuberculosis varies immensely, i.e., from 0.8% to 30%, pertaining to the location studied and the technique applied.23 furthermore, a research by thanh et al24 determined fundamental geographical distinctions in hiv prevalence among tb patients. these enormous diversities in the co-infection rates of tb/ hiv across the planet, as declared, can partially be attributed to: under-recording, diagnostic procedures used, discrepancies in tb diagnosis, epidemiology of tb in different nations, and study methodology utilized.20,23,24 table 2 explicates the prevalence of hiv seropositivity in regarding to gender, age, and occupational status. like other studies in different developing countries,17,18,25 the present study declared more prevalence of hiv co-infection amongst male tb patients. excepting a few nations in africa (sub-saharan africa), the prevalence of co-infection has been proclaimed to be higher among males than females.25–28 the prevalence of hiv is known to be higher among women than men in sub-saharan africa as a consequence of several reactions to sociodemographic risk factors, sexual attitude, and hiv/aids knowledge. also, women are renowned to have a higher predisposition to hiv infection and are usually subjected to sexual behaviors earlier than men at most due to economic circumstances.27,28 sentinel observational study accomplished in delhi by jain et al.29 in 1997–98 proposed a higher jeopardy of hiv co-infection existing among males complaining from tb which is in harmony with the present study. in the current study, hiv/tb patients were found to have a male-to-female ratio of 2.4. others have found that the male/female ratio of hiv-associated tb in africa was 0.83,28 although in other territories such as the western pacific, that ratio was 3.1.32 the ration of the current study is akin to the ratio of 2.7 revealed in 16 who european countries in 2005 and signaled that both infectious diseases are more widespread in the male population.30,31 with regard to the gender structure, it had been presumed that women with tb are not announced owing to a numerous of sociocultural issues, or diversified affairs affined with access in the health-care system and health services.16 therefore, in spite of the biological elucidations, there are sturdy evidences that corroborate such a liaison between female under proclamation rates in the context of specific cultural factors which perform an important role in developing and transitional societies.25 studies have been suggested that higher rate of tb infection in men may be due to the more vulnerability of males to pulmonary tb, difficulty in diagnosing tb in women, women with pulmonary tb have different symptoms from men and may not test positive on microscopic examination of the sputum, or that tb lung lesions might not be as severe in women as in men, resulting in women not being accurately diagnosed.18,22,32 in spite of the non-significant differences, the current study illustrate that hiv sero-positivity was higher among tb patients of age range 30–42 years and in laborer: 21 (51.2%); 13 (31.7%), respectively. studies had deduced that seroprevalence rates were highest among males in the age group between 31 and 40 years.19,22,27 hiv infection among tb patients continue to be astonishing, as it has been declared that more than 1 in 6 tb patients aged 25–44 year infected with hiv.25 the tb/hiv encumbrance was also had been observed to be higher within the age group that is mostly affected by tb.28 a study notified that the greatest leverage of the embarkation in tb extent had been on people between 25 and 45 years of age, since this is the age group mainly affected by hiv.29 this age prevalence of hiv co-infection among tb patients likely point out the age-specific prevalence of hiv in the community. this may be related to patients’ being in a sexually active age group in which both tb and hiv dominate most.29,33 the other possible elucidation for this may be their increased family, organizational, and societal responsibilities as people in this age group involve themselves in various peculiar and distinctive daily activities in order to win the socioeconomic tribulation which increases the frequency of their contact with other patients in their society.28,34 the aforementioned outcomes of this study support this fact as the majority of the tb-hiv co-infected patients were in the productive age group 31–40 years, mostly illiterate and lack of skills. all these factors in linking with unsafe sexual practice were found to be the major cause of hiv transmission.16,22,24 tb is a disease of destitution. it is excessively realized that poorer community, the greater the probability of being infected with tb. insufficiency of basic health services, poor nourishment and inappropriate living circumstances, all participate in the propagation of tb and its influence upon the society.16,24,27 in india, a study had determined that 76% of tb/hiv cases were in the age group of 21–40 years and also the occupational profile of their patients disclosed that a majority of them were farmers and laborers followed by transport drivers.18 channa et al.16 announced that 36.8% of tb/hiv patients were working as manual laborers while thanh et al.24 found majority (55.6%) of patients were working as farmers. manjareeka22 had detected seropositivity proportion was elevated among those who were jobless (40%) followed by the laborers (35%). the percentage of the professions is thus noted to alter in various studies, hugely due to the divergences in the occupational style and the origin from where the patients were picked.22 some studies pointed out a strong interconnection of seropositivity with socioeconomic factor, whilst other studies linked knowledge and educational level.22,24 the current study revealed that the highest prevalence of hiv positivity was among pulmonary tb (25; 61%). this result is consistent with findings reported by chandra et al,35 kamath et al,18 and olowe et al36 where higher seropositivity of hiv were detected in cases of pulmonary tb. in addition, a study by jha et al37 from eastern nepal showed that 71.4% of the tb/hiv co-infected cases were suffering more commonly from pulmonary tb than with extra-pulmonary tb (28.6%). the pulmonary tb (85.7%) was more common than extra-pulmonary tb (14.3%) was also reported by the study done in western nepal.38 contrasting results have been found in studies conducted by dahiya et al,39 mitku et al25, and kavya et al40 with higher incidence of extra-pulmonary tb than pulmonary tb among hiv-tb co-infected patients. globally, the tb-associated hiv mortality in co-infected patients is three times higher than mortality among tb patients.3 there are a number of possible explanations that 106 original prevalence of hiv among newly diagnosed tuberculosis patients in erbil governorate, iraq zakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 102 – 107 have been proposed for the increased mortality among co-infected patients.16,19 the location and the extent of tb are influenced by the degree of immunosuppression, often increasing the difficulty of diagnosis and hence delaying treatment initiation, which may result in higher mortality.9,11 with the scarcity of checking studies for eptb and the deprivation of an appropriate diagnostic equipment, it is strenuous to estimate the actual spread of the disease or precisely evaluate the anatomical distribution of disease.20, 22, 24 enormous retrospective explorations have been incapable to denote an appropriate matching amidst characteristics of eptb because of the fickle appearance of the disease.16 a study in brazil found similar non-specific symptoms in their patients with eptb.23 the differential diagnosis for the existence of these fundamental symptoms is spacious, but stay as a substantial hint for the diagnosis of tb in a high tb/hiv load situation. whilst symptoms and signs are beneficial in guidance; moreover, checking to confirm the location of disease, there is a shortage of data on distinguishing clinical findings that may be utilized to assist the diagnosis of tb over other pathologies at these sites. the clinical presentation of eptb is protean, and establishing the diagnosis presents significant challenges in resource-limited settings.41,42 the deficiency of diagnostic devices take part to a diagnostic predicament, and future evolution in molecular established technology may ameliorate our capability to diagnose the disease adequately. the application of obtainable technologies demand better guide-based direction. due to the reliance on non-microbiological diagnostic tests for eptb, diagnostic certainty remains elusive.22,42 the complex relationship, however, between patient demographic characteristics, behavioral risk factors, clinical characteristics and comorbidities, and eptb remains poorly understood.42 studies revealed that newly diagnosed tb/hiv infected have greater risk of mdr-tb than those negative to hiv.43,44 while most studies in north america showed an association between hiv infection and mdr-tb, not a single study from africa demonstrated such association, and results from other regions were conflicting. individual studies varied widely in study design and sample size, and results were rarely adjusted for potential confounding.44 these restrictions and the noted diversity imped a comprehensive deductions considering the inclusive linking between hiv infection and mdr-tb. when stratified by type of mdr-tb, the analysis suggests that primary, but not acquired, mdr-tb is associated with hiv infection.45 different biological mechanisms relating drug-resistant tb to hiv infection have been proposed. drug malabsorption in hiv-infected patients, particularly rifampin and ethambutol, can produce drug resistance leading to treatment failure.46 the linkage between hiv infection and mdr-tb may be discomposed by risk factors such as injection drug use, imprisonment, socioeconomic status, alcohol use, and hospitalization which are shared between both. hiv-infected patients and mdr-tb patients are more likely to be hospitalized compared to those who are hiv negative.47 hiv-infected patients may thus be more likely to be exposed to patients with drug-resistant isolates, and thus be infected or re-infected with a resistant isolate as the associations between mdrtb and hiv infection observed in many north american studies, which included in part patients involved in institutional outbreaks in new york city, support this possibility.46,47 it is of worthy to mention that albujeer48 had concluded in their study in iraq that preventing the spread of hiv in conservative communities such as middle eastern communities requires a comprehensive strategy that includes effective, continued health education and health promotion programs at both community and health professional levels. conclusions the prevalence of hiv infection among tb patients in this study was 10.3%. greater focus of health intervention should be required on reproductive age group and in area in which high immigration and overcrowding present. even with unavoidable restrictions, we can still determine that the hiv prevalence among tb patients was high. involvement planes aiming sociodemographic and behavioral factors linking with higher risk of tb-hiv co-infection are imperatively needed for. acknowledgments the authors are grateful to the management of health directorate of erbil for their allowing us to conduct the study. we are also thankful to the senior staff and colleagues at the in the department of microbiology at the chest and respiratory disease specialized centre in erbil city for their cooperation and support. financial support and sponsorship nil. conflicts of interest there are no conflicts of interest. references 1. harding e. who global progress report on tuberculosis elimination. the lancet resp med. 2020 jan 1;8(1):19. 2. world health organization. global tuberculosis report 2020: executive summary. 3. sharan r, bucşan an, ganatra s, paiardini m, mohan m, mehra s, khader sa, kaushal d. chronic immune activation in tb/hiv co-infection. trends microbiol. 2020 apr 22. 4. al-salihy sr, enad om. knowledge and attitude of health care workers in baquba teaching hospital toward hiv/aids infection. iraqi j public health. 2017 sep 20;1(2):42-6. 5. wong k, nguyen j, blair l, banjanin m, grewal b, bowman s, boyd h, gerstner g, cho hj, panfilov d, tam ck. pathogenesis of human immunodeficiency virus-mycobacterium tuberculosis co-infection. j clin med. 2020 nov;9(11):3575. 6. melgar m, nichols c, cavanaugh js, kirking hl, surie d, date a, ahmedov s, maloney s, fukunaga r, offices’ tuberculosis cc, advisors hi. tuberculosis preventive treatment scale-up among antiretroviral therapy patients—16 countries supported by the us president’s emergency plan for aids relief, 2017–2019. morbid mortal week rep. 2020 mar 27;69(12):329. 7. sterling tr, njie g, zenner d, cohn dl, reves r, ahmed a, menzies d, horsburgh jr cr, crane cm, burgos m, lobue p. guidelines for the treatment of latent tuberculosis infection: recommendations from the national tuberculosis controllers association and cdc, 2020: 11961206 . 107 original prevalence of hiv among newly diagnosed tuberculosis patients in erbil governorate, iraqzakarea abdullah yaseen al-khayat et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 102 – 107 8. bares sh, swindells s. latent tuberculosis and hiv infection. curr infect dis rep. 2020 jul;22:1-8. 9. letang e, ellis j, naidoo k, casas ec, sánchez p, hassan-moosa r, cresswell f, miró jm, garcía-basteiro al. tuberculosis-hiv co-infection: progress and challenges after two decades of global antiretroviral treatment roll-out. arch bronconeumol. 2020 jan 10. 10. ganatra sr, bucşan an, alvarez x, kumar s, chatterjee a, quezada m, fish a, singh dk, singh b, sharan r, lee th. antiretroviral therapy does not reduce tuberculosis reactivation in a tuberculosis-hiv coinfection model. j clin investig. 2020 aug 24;130(10). 11. canetti d, riccardi n, martini m, villa s, di biagio a, codecasa l, castagna a, barberis i, gazzaniga v, besozzi g. hiv and tuberculosis: the paradox of dual illnesses and the challenges of their fighting in the history. tuberculosis. 2020 may 1;122:101921. 12. maher d, watt cj, williams bg, raviglione m, dye c. tuberculosis deaths in countries with high hiv prevalence: what is their use as an indicator in tuberculosis programme monitoring and epidemiological surveillance?[unresolved issues]. int j tuberculosis lung dis. 2005 feb 1;9(2):123-7. 13. steingart kr, ng v, henry m, hopewell pc, ramsay a, cunningham j, urbanczik r, perkins md, aziz ma, pai m. sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. lancet infect dis. 2006 oct 1;6(10):664-74. 14. steingart kr, henry m, ng v, hopewell pc, ramsay a, cunningham j, urbanczik r, perkins m, aziz ma, pai m. fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. the lancet infect dis. 2006 sep 1;6(9):570-81. 15. world health organization. rapid implementation of the xpert mtb/rif diagnostic test: technical and operational ‘how-to’; practical considerations. world health organization; 2011. 16. channa aa, jameel n, khalil r. prevalence of human immunodeficiency virus infection among the diagnosed tuberculosis patients in karachi, pakistan. int j res med sci. 2016 mar;4(3):789. 17. pourakbari b, mamishi s, banar m, keshtkar aa, mahmoudi s. prevalence of tb/hiv co-infection in iran: a systematic review and meta-analysis. ann ig. 2019 jul 1;31(4):333-48. 18. kamath r, sharma v, pattanshetty s, hegde mb, chandrasekaran v. hiv-tb coinfection: clinico-epidemiological determinants at an antiretroviral therapy center in southern india. lung india off organ ind chest soc. 2013 oct;30(4):302. 19. belay m, bjune g, abebe f. prevalence of tuberculosis, hiv, and tb-hiv co-infection among pulmonary tuberculosis suspects in a predominantly pastoralist area, northeast ethiopia. global health action. 2015 dec 1;8(1):27949. 20. van der werf mj, ködmön c, zucs p, hollo v, amato-gauci aj, pharris a. tuberculosis and hiv coinfection in europe: looking at one reality from two angles. aids (london, england). 2016 nov 28;30(18):2845. 21. gao j, zheng p, fu h. prevalence of tb/hiv co-infection in countries except china: a systematic review and meta-analysis. plos one. 2013 may 31;8(5):e64915. 22. manjareeka m, nanda s. prevalence of hiv infection among tuberculosis patients in eastern india. j infect public health. 2013 oct 1;6(5):358-62. 23. e silva ho, gonçalves ml. prevalence of hiv infection in tuberculosis patients treated at primary health care clinics in the city of fortaleza, brazil. j bras pneumol. 2012;38(3):382-5. 24. thanh dh, sy dn, linh nd, hoan tm, dien ht, thuy tb, hoa np, tung lb, cobelens f. hiv infection among tuberculosis patients in vietnam: prevalence and impact on tuberculosis notification rates. int j tuberculosis lung dis. 2010 aug 1;14(8):986-93. 25. abdallah tm, siddig mf, ali aa. seroprevalence of hiv infection among tuberculosis patients in kassala, eastern sudan. j aids hiv res. 2011 aug 31;3(8):161-3. 26. mitku aa, dessie zg, muluneh ek, workie dl. prevalence and associated factors of tb/hiv co-infection among hiv infected patients in amhara region, ethiopia. afric health sci. 2016 jul 1;16(2):588-95. 27. pennap g, makpa s, ogbu s. sero-prevalence of hiv infection among tuberculosis patients in a rural tuberculosis referral clinic in northern nigeria. pan african med j 2010;5:22. 28. baluku jb, anguzu g, nassozi s, babirye f, namiiro s, buyungo r, sempiira m, wasswa a, mulwana r, ntambi s, worodria w. prevalence of hiv infection and bacteriologically confirmed tuberculosis among individuals found at bars in kampala slums, uganda. scient rep. 2020 aug 10;10(1):1-9. 29. jain sk, aggarwal jk, rajpal s, baveja u. prevalence of hiv infection among tuberculosis patients in delhi — a sentinel surveillance study. ind j tuberculosis. 2000;47:21—6. 30. fiebig l, kollan c, hauer b, gunsenheimer-bartmeyer b, an der heiden m, hamouda o, haas w. hiv-prevalence in tuberculosis patients in germany, 2002–2009: an estimation based on hiv and tuberculosis surveillance data. plos one. 2012 nov 7;7(11):e49111. 31. tavares am, fronteira i, couto i, machado d, viveiros m, abecasis ab, dias s. hiv and tuberculosis co-infection among migrants in europe: a systematic review on the prevalence, incidence and mortality. plos one. 2017 sep 28;12(9):e0185526. 32. hiatt t, nishikiori n. epidemiology and control of tuberculosis in the western pacific region: analysis of 2012 case notification data. western pacific surveil resp j: wpsar. 2014 jan;5(1):25. 33. jaiswal rk, srivastav s and mahajan h. socio demographic profile of tb-hiv coinfected patients in bundelkhand region,uttar-pradesh. nat jn med research 2012; 2(2):149-151. 34. soyam vc, das j, rajeeva tc, boro p, kohli c. prevalence and sociodemographic correlates of hiv among tuberculosis patients of dots centre in delhi. asian j med sci. 2016;7(1):53-8. 35. chandra nm, babu ra, prasad dts, devulapalli m, banu ssk, avanthi b, et al. epidemiological surveillance of tuberculosis among hiv/aids seropositive individuals attending art center at a tertiary care teaching hospital. int j community med public health 2017;4:2816-2824. 36. olowe oa, makanjuola ob, adekami as, adefioye oj. epidemiological characteristics and clinical outcome in a population of tb patients in south– western nigeria. eur j microbiol immunity 2017; 2: 127-132. 37. jha n, khanal b, karki pp, rijal s, deo bk, khadka dk, malla p. tb/hiv coinfection status among the newly diagnosed tb patients: a study from eastern nepal. saarc j tubercul lung dis hiv/aids. 2008;5(2):22-5. 38. ghimire p, dhungana gr, bam ds, rijal bp. tuberculosis and hiv co-infection status in united mission hospital, tansen, western nepal. saarc j tubercul lung dise hiv/aids. 2004;1:32-7. 39. dahiya n, bachani d, das r, rasania sk. socio-demographic and clinical profile of hiv positive patients attending integrated counseling and testing center of a primary health centre in delhi. saarc j tubercul lung dis hiv/ aids 2017;15:22-26 . 40. kavya s, anuradha k, venkatesha d. cd4 count evaluation in hiv-tb co infection before and after anti-tubercular treatment. int j res med sci. 2017;2(3):1031-4. 41. leeds il, magee mj, kurbatova ev, del rio c, blumberg hm, leonard mk, kraft cs. site of extrapulmonary tuberculosis is associated with hiv infection. clin infect dis. 2012 jul 1;55(1):75-81. 42. namme lh, marie-solange d, hugo bertrand mn, elvis t, achu jh, christopher k. extrapulmonary tuberculosis and hiv coinfection in patients treated for tuberculosis at the douala general hospital in cameroon. ann trop med public health 2013;6:100-4. 43. van den hof s, tursynbayeva a, abildaev t, adenov m, pak s, ismailov s. hiv and multidrug-resistant tuberculosis: overlapping risk factors. eur resp j. 2015 feb 1;45(2):567-9. 44. adetunji so, donbraye e, ekong mj. hiv infection among rifampicin resistant tuberculosis patients in ibadan, southwest nigeria. world j. med. sci. 2018;15(4):139-43. 45. singh a, prasad r, balasubramanian v, gupta n. drug-resistant tuberculosis and hiv infection: current perspectives. hiv/aids (auckland, nz). 2020;12:9. 46. saldanha n, runwal k, ghanekar c, gaikwad s, sane s, pujari s. high prevalence of multi drug resistant tuberculosis in people living with hiv in western india. bmc infect dis. 2019 dec;19(1):1-6. 47. baluku jb, mugabe p, mulwana r, nassozi s, katuramu r, worodria w. high prevalence of rifampicin resistance associated with rural residence and very low bacillary load among tb/hiv-coinfected patients at the national tuberculosis treatment center in uganda. biomed res int. 2020 jul 25;2020. 48. albujeer an, shamshiri ar, taher a. hiv/aids awareness among iraqi medical and dental students. j int soc prev commun dentistry. 2015 sep;5(5):372. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.955 61 original issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 61 – 65 introduction nowadays, the human life style cause a lot of stress for individuals which could be ended to depression and anxiety disorders.1 stress affect different body organs and their functions and also increase the level of cortisol in blood. high level of cortisol decreases the muscle mass, increases the blood glucose and body fat, induces insomnia, anorexia, fatigue, and brain atrophy.2, 3 the neurotrophins including brain-derived neurotrophic factor (bdnf) and glial cell line-derived neurotrophic factor (gdnf) have important roles in central nervous system like neuroprotective effects against neuroinflammation and oxidative toxicity that help the neurons to survive, maintain, migration, and differentiation.4, 5 but, as a consequence of stress, these neurotrophic factors (ntfs) decrease and cause mood disorders specially depression.6, 7 ntfs, including bdnf and gdnf, are involved in the different steps of development and function of neurons.8 previous studies demonstrated that the level of bdnf and gdnf was significantly lower than healthy individuals.9, 10 exposure to the chronic stress induced hippocampal atrophy and decrease of neurogenesis in hippocampus and impairments in cognition and memory.11-13 impairment of serotonergic system lead to depression and anxiety disorders. bdnf and gdnf stimulate the growth of 5-ht neurons and also have direct effect on gene expression in serotonergic system.14 various drug treatments are used in anxiety disorders and depression but the harms of side effects for the patients lead to evaluation of herbal drugs as a natural harmless treatment. flaxseed (fs, linum usitatissimum) is a 1-year planet which is a valuable source of phenol complex, antioxidant agents, alpha-linolenic acid (ala), fibers, minerals, and vitamins.15, 16 the ala of fs oil increase the expression of bdnf gene and related mrna in hippocampus. in rats with middle cerebral artery occlusion, nutrition with fs, increase the bdnf and gdnf gene expression in motor cortex and ca1 area of hippocampus.15 according to decrease of bdnf and gdnf in depression,17 we designed this study to evaluate the effect of fs oil on bdnf and gdnf gene expression in ca1 area of hipocampus in mice with immobility stress. method and materials animals in this study, 32 white laboratory male mice (20–25 g, 12-week old) were enrolled. they were kept in a room with temperature of 18–24°c, 40–70% humidity, 12h light–12h dark cycle, and flaxseed oil (linum usitatissimum) attenuates restraint stress-induced depressive-like behavior: upregulation of neurotrophic factors in ca1 region of hippocampus sahand talei1, tahmineh mokhtari2, ilia asadi3, leila arab4, negar hassanzadeh5, emadoddin hosseinjani5, gholamreza hassanzadeh6,7,8* 1 school of medicine, tehran university of medical sciences, tehran, iran. 2 cas key laboratory of mental health, institute of psychology, beijing, china; department of psychology, university of chinese academy of sciences, beijing, china. 3 student research committee, semnan university of medical sciences, semnan, iran. 4 department of neuroscience and addiction studies, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. 5 legal medicine research center, legal medicine organization, tehran, iran. 6 department of neuroscience and addiction studies, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran. 7 department of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. 8 legal medicine research center, legal medicine organization, tehran, iran. *correspondence to: ghomareza hassanzadeh (e-mail: hassanzadeh@tums.ac.ir) (submitted: 10 november 2020 – revised version received: 25 november 2020 – accepted: 14 december 2020 – published online: 26 february 2021) abstract objective depression results from oxidative and inflammatory neural damages caused by life stressors. flaxseed (fs) oil exerts antioxidant activity in various studies. in this study, we aimed to evaluate the antidepressant effects of fs oil on the depressive-like behavior following immobility stress in mice. methods in this study, 32 male mice were divided into 4 groups: control group; fs oil group; model group: the immobility stress (using falcon for 20 day, 6 h a day) + feeding with normal saline by oral gavage for another 20 days); model+fs group: the immobility stress + feeding with 0.2 ml/kg of fs oil by oral gavage for another 20 days. at the end, all mice were evaluated with tail suspension test (tst) and sacrificed through cardiac puncture method. the brain was isolated and prepared for molecular and histopathological studies. results the gene expression and protein level of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor were significantly increased in fs oil treated groups (p<0.05). in microscopic evaluations, the number of dead neurons significantly deceased in fs group (p<0.05). tst results showed a significant decrease in immobility time and depressive-like behaviors in fs oil treated group (p<0.05). conclusions fs oil feeding may be effective in treatment of depressive like disorders through its antioxidative and neurotrophic promoting features. therefore, it can be used as an appropriate alternative choice in daily diet to prevent depression in clinical investigations. keywords depression, immobility stress, flaxseed, neurotrophic factors, antioxidant 62 original flaxseed oil (linum usitatissimum) attenuates restraint stress-induced depressive-like behavior gholamreza hassanzadeh et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 61 – 65 free access to food and water. they were treated according to the guidelines of iranian council for use and care of animals and approved by ethical committee of tehran university of medical science. the mice were randomly divided in 4 groups with 15 mice: group 1: control; the mice were fed with water and routine food for 20 days without any stress or treatment. group 2: flaxseed (fs) oil; mice were fed with water and routine food, and they also received fs oil (0.2 ml/daily by gavage) for 20 days without any stress. group 3: immobility stress model (model); mice with depressive-like behavior (immobility stress for 6 h/day for 20 days) and feeding with normal saline by oral gavage for another 20 days. group 4: model+fs oil; mice with depressive-like behavior (immobility stress for 6 h/day for 20 days) and fs oil (0.2 ml/daily by gavage) for another 20 days. immobility stress induction for model induction, the mice were kept in 50 ml falcon tubes which made them totally immobile for 6 h a day. during this period, other stressors like noise, light, or temperature changes were omitted. at the end of induction period, the mice were returned to their cage. after 20 days, all the mice were sacrificed by cardiac puncture under anesthesia to extract their whole brain for more evaluation. brain tissue sampling fresh samples of hippocampus were extracted immediately after sacrificing and put in freezing tube and kept in -80°c. for histological studies, the brains were prefixed by a transcardial perfusion of normal saline (ns) followed by 4% paraformaldehyde (pfa, sigma in 0.1 m phosphate buffer pre-fixation). the post-fixed was done by using 10% formalin at 4°c for 72 h. hematoxylin & eosin staining after fixation and tissue processing, the 5 um-thick coronal sections were prepared by rotary microtome (leica biosystems, milan, italy). for light microscopy observation, the tissue sections stained with hematoxylin and eosin (h & e) according to the standard protocol and analyzed via a light field microscope (olympus, cx31, tokyo, japan). the photomicrographs were prepared from ca1 region and the number of dead neurons (eosinophilic neurons) were calculated in each photomicrographs. total rna extraction and quantitative real-time pcr gene expression of bdnf and gdnf were evaluated after 24 h of sacrificing. under anesthesia, the brain was extracted and placed in ice-cold saline (0.9%). then, the hippocampal ca1 region was isolated. the expression of bdnf and gdnf genes in ca1 region was assessed by quantitative real-time pcr (abi prism 7500 real-time pcr system, roche diagnostics, germany). the total rna was extracted, and cdna was synthesized from 1 μg total rna using primescript rt reagent kit (takara bio inc., otsu, japan). quantitative real-time pcr was performed in a cycler (light cycler 2.0, roche) by sybr green (takara bio inc., otsu, japan). the primers were designed using generunner software (table 1). the b-actin gene was as the internal control standard. protein concentration the protein concentrations of bdnf and gdnf in hippocampal ca1 region were assessed by abcam elisa kits in accordance with their manufacturer’s guidelines. hippocampus ca1 area was homogenized and after the centrifuge (14,000 rpm, 4°c for 3 min), the supernatant was diluted with sample buffer. then, it was incubated in 96-well flat-bottom plates, coated by antibdnf and anti-gdnf monoclonal antibodies. then, the plates were incubated with polyclonal anti-rabbit antibody for 2 h. for measuring the protein concentration, the color reaction with tetramethyl benzidine was quantified in a plate reader at 450 nm. tail suspension test (tst) the test was performed in a container with posterior, left, and right lateral white walls that a rod (50 cm length) was fixed between left and right lateral walls at a distance of 70 cm from the floor. the mice were hanged in the way that about 1 cm length of tail end was fixed at the rod using an adhesive tape and left dangling for 6 min. immobility time was the seconds that hanged mouse remained inactive and immobile. for 6 min, all the movement of mice was recorded by a video camera and analyzed.18 statistical analysis results were expressed as mean ± s.e.m. the data were analyzed via one way anova with tukey’s post hoc. nonparametric test of kruskal–wallis and dunn’s multiple comparisons for post-test. p < 0.05was considered as the significance level. results effects of fs oil on depressive-like behavior tst was used for evolutions of fs oil effects on depressive like behavior. the mean time of immobility was compared between groups. an increased time of immobility was seen model group (p<0.05, fig. 1). treatment of normal mice with fs oil (fs group) decreased the mean time of immobility compared to control group (p<0.05, fig. 1). also, a decreased mean time of immobility was observed in model +fs group compared to model group (p<0.05, fig. 1). table 1. list of primers. gene forward primer reverse primer bdnf caggattgacagtttgatagctctatc atcgctccagcaactaacaacg gdnf aatgtgtccgtcgtggatctg caacctggtcctcagtgtatc b-actin ctgggactacatggagaagagc gtctcaatcacgatctgggtcatt 63 original flaxseed oil (linum usitatissimum) attenuates restraint stress-induced depressive-like behaviorgholamreza hassanzadeh et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 61 – 65 effects of fs oil on gene expression of ntfs in ca1 region of mice with depressive-like behavior the mean gene expression of ntfs, including ddnf and gdnf was evaluated in ca1 region of hippocampus in each group. according to fig. 2, there was a significant decrease in the gene expression of bdnf and gdnf in model group compared to control and fs groups (p< 0.05, fig. 2a,b). there was a significant increase in the gene expression of bdnf and gdnf in model+fs group compared with control, fs, and model+fs groups (p < 0.05, fig. 2a,b). effects of fs oil on protein concentration of ntfs in ca1 region of mice with depressive-like behavior the mean protein concentration of ntfs, including ddnf and gdnf was evaluated in ca1 region of hippocampus in each group. fig. 3 showed that there was a significant decrease in the protein concentration of bdnf and gdnf in model group compared to control and fs groups (p < 0.05, fig. 3a,b). there was a significant increase in the protein concentration of bdnf and gdnf in model+fs group compared to control, fs, and model+fs groups (p < 0.05, fig. 3a,b). effects of fs oil on histopathological alterations of ca1 region of mice with depressive-like behavior the mean number of dead neurons was evaluated in the ca1 region of hippocampus in each group. fig. 4 showed that there was an increase in the number of dead neurons of ca1 region in model group compared to control and fs groups (p < 0.05, fig. 3a,b). also, a significant decrease in the number of dead neurons of ca1 region of model+fs group was recorded compared to control, fs, and model+fs groups (p < 0.05, fig. 3a,b). discussion in the present study, we evaluated the effects of fs oil on the depressive-like behavior induced by immobility stress. we used immobility stress to induce depressive like behavior in mice. in this group, the immobility time increased in tst which confirmed the depressive behavior of these animals. additionally, the number of dead neurons increased and gene expression and protein concentration of nfs decreased in ca1 region of hippocampus. a group of models have been developed to induce depressive-like behaviors in animals through exerting chronic stress. chronic immobility stress is one of the most convenient models used in preclinical studies in case of time, cost, and energy and its similarity to the available stressors causing depression in human life.19, 20 chronic stress is supposed to disrupt oxidant/antioxidant balance and lead to oxidative stress which damage the neuronal cell components such as dna, plasma membrane and stimulates the inflammatory signaling pathways, results in neuronal death in hippocampal region and depression.21 another studies reported that enhanced inflammation and oxidative stress in brain tissue can lead to depression.22, 23 bdnf and gdnf are well-known neurotrophic factors, being in a continuous interaction with serotonergic system.24 reduced levels of ntfs in hippocampus were seen in several neurological disorders.25 in the preclinical and clinical studies, the important role of bdnf in depression has been proven. exposure of animal models to chronic stress may decrease the levels of bdnf in fig. 1 effects of treatment with fs oil on immobility time (s) in tst in mice with depressive-like behavior. a, p< 0.05 compared to control group, b, p< 0.05 compared to fs group, c, p< 0.05 compared to model group. control: normal mice, fs: flaxseed oil group (0.2 ml/daily for 20 days), model: immobility stress group and received normal saline (20 days), model+fs: immobility stress+flaxseed oil. fig. 2 effects of fs oil on gene expression of a) bdnf and b) gdnf in ca1 region of mice with depressive-like behavior. a, p< 0.05 compared to control group, b, p< 0.05 compared to fs group, c, p< 0.05 compared to model group. control: normal mice, fs: flaxseed oil group (0.2 ml/daily for 20 days), model: immobility stress group and received normal saline (20 days), model+fs: immobility stress+flaxseed oil. 64 original flaxseed oil (linum usitatissimum) attenuates restraint stress-induced depressive-like behavior gholamreza hassanzadeh et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 61 – 65 the hippocampus.8, 26 on the other hand, treatment of patients with major depressive disorder with antidepressants resulted in alterations in serum bdnf and gndf levels after 12 weeks.27 targeting the bdnf and gdnf pathways and developing antidepressive strategies to enhance the levels of these ntfs in serum and brain were considered in several studies.28-30 nowadays, it is possible to extract herbal concentrates with acceptable qualities to be used for pharmaceutical purposes.31-33 among a wide range of herbs with beneficial characteristics, fs has been shown to have antioxidant and anti-inflammatory features.34 as a functional food, this seed or its oil can be consumed in daily diets.35 in the present study, we used oral fs oil for treatment of depressive like disorders following chronic stress. administration of fs oil for 20 days could decrease the immobility time in tst, reduced dead neurons of ca1 region following induction of depressive behavior. this neuroprotective effects were shown to be related to enhanced levels of ntfs in this critical region. the neuroprotective effects of fs has been demonstrated in several studies. gholamineghad et al. confirmed the antioxidative and neuroprotective properties of fs in a rat model of spinal ischemic injury. in this study, disrupted functions of rats due to ischemia were significantly improved after treatment with fs daily diet (10% fs ) for 4  weeks after ischemic surgery.36 fs could significantly decreased the levels of inflammatory meditators.37 furthermore, navaie et al showed the antioxidative, protective, and preventive effects of fs (10% fs for 4 weeks) on hypoxia-induced hippocampal impairment in rat model.38 fs oil compounds, such as ala, are capable to enhance the gene expression of neurotrophic factors like bdnf and gdnf.39, 40 rahmati et al also indicated that fs oil administration (10 mg/kg) for 10 weeks enhanced the levels of bdns in hippocampus and changed the pain feeling in rat model.41 bagheri et al also reported that fs oil administration (0.2 ml daily for 3 weeks) also increased the levels of bdnf and gdnf in motor cortex of brain stroke model of rats.42 taken together, fs oil (0.2 ml daily for 3 weeks) could decrease the depressive-live disorder via decreasing the neural death in hippocampal ca1 region of mice following a chronic immobility stress for 20 days. accordingly, it seems that fs oil enhanced the levels of ntfs (bdnf and gdnf) in ca1 region and protected the pyramidal cells of this region against increased levels of inflammatory mediators and oxidative factors, and survived these neurons. conflict of interest none fig. 3 effects of fs oil on protein concentration of a) bdnf and b) gdnf in ca1 region of mice with depressive-like behavior. a, p< 0.05 compared to control group, b, p< 0.05 compared to fs group, c, p< 0.05 compared to model group. control: normal mice, fs: flaxseed oil group (0.2 ml/daily for 20 days), model: immobility stress group and received normal saline (20 days), model+fs: immobility stress+flaxseed oil. fig. 4 effects of fs oil on histopathological changes of ca1 region of hippocampus of mice with depressive-like behavior. (a) h&e staining (scale bar: 50 µm), (b) comparing the mean number of dead neurons (arrows) of ca1 region in different groups. a, p< 0.05 compared to control group; b, p< 0.05 compared to fs group; c, p< 0.05 compared to model group. control: normal mice, fs: flaxseed oil group (0.2 ml/daily for 20 days), model: immobility stress group and received normal saline (20 days), model+fs: immobility stress+flaxseed oil. 65 original flaxseed oil (linum usitatissimum) attenuates restraint stress-induced depressive-like behaviorgholamreza hassanzadeh et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 61 – 65 references 1. joo y, choi km, lee yh, kim g, lee dh, roh gs, et al. chronic immobilization stress induces anxietyand depression-like behaviors and decreases transthyretin in the mouse cortex. neurosci lett. 2009;461(2):121-5. 2. sapse at. stress, cortisol, interferon and “stress” diseases. i. cortisol as the cause of “stress” diseases. med hypotheses. 1984;13(1):31-44. 3. carpenter wt, jr., gruen ph. cortisol’s effects on human mental functioning. j clin psychopharmacol. 1982;2(2):91-101. 4. martinowich k, manji h, lu b. new insights into bdnf function in depression and anxiety. nat neurosci. 2007;10(9):1089-93. 5. riccio a, ahn s, davenport cm, blendy ja, ginty dd. mediation by a creb family transcription factor of ngf-dependent survival of sympathetic neurons. science. 1999;286(5448):2358-61. 6. emon mpz, das r, nishuty nl, shalahuddin qusar mma, bhuiyan ma, islam mr. reduced serum bdnf levels are associated with the increased risk for developing mdd: a case-control study with or without antidepressant therapy. bmc res notes. 2020;13(1):83. 7. numakawa t, adachi n, richards m, chiba s, kunugi h. brain-derived neurotrophic factor and glucocorticoids: reciprocal influence on the central nervous system. neuroscience. 2013;239:157-72. 8. ducray a, krebs sh, schaller b, seiler rw, meyer m, widmer hr. gdnf family ligands display distinct action profiles on cultured gabaergic and serotonergic neurons of rat ventral mesencephalon. brain res. 2006;1069(1):104-12. 9. sjors dahlman a, blennow k, zetterberg h, glise k, jonsdottir ih. growth factors and neurotrophins in patients with stress-related exhaustion disorder. psychoneuroendocrinology. 2019;109:104415. 10. karege f, perret g, bondolfi g, schwald m, bertschy g, aubry jm. decreased serum brain-derived neurotrophic factor levels in major depressed patients. psychiatry res. 2002;109(2):143-8. 11. liu rj, aghajanian gk. stress blunts serotoninand hypocretin-evoked epscs in prefrontal cortex: role of corticosterone-mediated apical dendritic atrophy. proc natl acad sci usa. 2008;105(1):359-64. 12. popoli m, yan z, mcewen bs, sanacora g. the stressed synapse: the impact of stress and glucocorticoids on glutamate transmission. nat rev neurosci. 2011;13(1):22-37. 13. yaribeygi h, panahi y, sahraei h, johnston tp, sahebkar a. the impact of stress on body function: a review. excli j. 2017;16:1057-72. 14. popova nk, ilchibaeva tv, naumenko vs. neurotrophic factors (bdnf and gdnf) and the serotonergic system of the brain. biochemistry (mosc). 2017;82(3):308-17. 15. bagheri a, talei s, hassanzadeh n, mokhtari t, akbari m, malek f, et al. the neuroprotective effects of flaxseed oil supplementation on functional motor recovery in a model of ischemic brain stroke: upregulation of bdnf and gdnf. acta med iran. 2017;55(12):785-92. 16. connor we. alpha-linolenic acid in health and disease. am j clin nutr. 1999;69(5):827-8. 17. tsybko as, il’chibaeva tv, kondaurova em, bazovkina dv, naumenko vs. the effect of central administration of the neurotrophic factors bdnf and gdnf on the functional activity and expression of the serotonin 5-ht2a receptors in mice genetically predisposed to depressive-like behavior. mol biol (mosk). 2014;48(6):983-9. 18. can a, dao dt, terrillion ce, piantadosi sc, bhat s, gould td. the tail suspension test. j vis exp. 2012(59):e3769. 19. son h, yang jh, kim hj, lee dk. a chronic immobilization stress protocol for inducing depression-like behavior in mice. jove (j of visual exp). 2019(147):e59546. 20. liu l, zhou x, zhang y, liu y, yang l, pu j, et al. the identification of metabolic disturbances in the prefrontal cortex of the chronic restraint stress rat model of depression. behav brain res. 2016;305:148-56. 21. duman rs. neuronal damage and protection in the pathophysiology and treatment of psychiatric illness: stress and depression. dialogues clin neurosci. 2009;11(3):239-55. 22. yanik m, erel o, kati m. the relationship between potency of oxidative stress and severity of depression. acta neuropsychiatrica. 2004;16(4):200-3. 23. lopresti al, maker gl, hood sd, drummond pd. a review of peripheral biomarkers in major depression: the potential of inflammatory and oxidative stress biomarkers. prog neuro-psychopharmacol biol psychiatry. 2014;48:102-11. 24. popova n, ilchibaeva t, naumenko v. neurotrophic factors (bdnf and gdnf) and the serotonergic system of the brain. biochemistry (moscow). 2017;82(3):308-17. 25. mokhtari t, akbari m, malek f, kashani ir, rastegar t, noorbakhsh f, et al. improvement of memory and learning by intracerebroventricular microinjection of t3 in rat model of ischemic brain stroke mediated by upregulation of bdnf and gdnf in ca1 hippocampal region. daru j pharm sci. 2017;25(1):4. 26. tsankova nm, berton o, renthal w, kumar a, neve rl, nestler ej. sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action. nat neurosci. 2006;9(4):519-25. 27. park y-m, lee b-h. alterations in serum bdnf and gdnf levels after 12 weeks of antidepressant treatment in female outpatients with major depressive disorder. psychiatry investig. 2018;15(8):818-23. 28. angelucci f, aloe l, jiménez-vasquez p, mathé aa. lithium treatment alters brain concentrations of nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in a rat model of depression. int j neuropsychopharmacol. 2003;6(3):225-31. 29. tunca z, ozerdem a, ceylan d, yalçın y, can g, resmi h, et al. alterations in bdnf (brain derived neurotrophic factor) and gdnf (glial cell line-derived neurotrophic factor) serum levels in bipolar disorder: the role of lithium. j affect disorders. 2014;166:193-200. 30. varela rb, valvassori ss, lopes-borges j, mariot e, dal-pont gc, amboni rt, et al. sodium butyrate and mood stabilizers block ouabain-induced hyperlocomotion and increase bdnf, ngf and gdnf levels in brain of wistar rats. j psychiatric res. 2015;61:114-21. 31. ashaari z, hadjzadeh m-a-r, hassanzadeh g, alizamir t, yousefi b, keshavarzi z, et al. the flavone luteolin improves central nervous system disorders by different mechanisms: a review. j mol neurosci. 2018;65(4): 491-506. 32. ghaffari n, hassanzadeh g, nowrouzi a, gholaminejhad m, mokhtari t, seifali r, et al. antioxidative and anti-inflammatory effects of cichorium intybus l. seed extract in ischemia/reperfusion injury model of rat spinal cord. j contemp med sci. 2018;4(4). 33. mokhtari t, hussein osman h-e, el-meghawry el-kenawy a, dashti n. ameliorative effect of virgin olive oil against nephrotoxicity following subchronic administration of ethephon in male rats. j tradit complement med. 2020;10(5):487-95. 34. mokhtari t, faghir ghanesefat h, hassanzadeh g, moayeri a, haeri smj, rezaee kanavee a, et al. effects of flaxseed oil supplementation on renal dysfunction due to ischemia/reperfusion in rat. ilam univ med sci. 2017;4(1):22-9. 35. goyal a, sharma v, upadhyay n, gill s, sihag m. flax and flaxseed oil: an ancient medicine & modern functional food. j food sci technol. 2014;51(9):1633-53. 36. gholaminejhad m, arabzadeh s, akbari m, mohamadi y, hassanzadeh g. anti-oxidative and neuroprotective effects of flaxseed on experimental unilateral spinal cord injury in rat. j contemp med sci. 2017;3(10):213-7. 37. gholaminejhad m, hassanzadeh g, akbari m. o2: flaxseed reduces proinflammatory factors il-1β, il-18 and tnf-α in injured spinal cord rat model. shafaye khatam neurosci j 2018;6(2):2. 38. navaie f, hassanzadeh g, mahakizadeh s, mehrannia k, alizamir t, dashti n, et al. anti-oxidative and neuroprotective effects of supplementary flaxseed on oxidative damage in the hippocampus area of a rat model of hypoxia. arch neurosci. 2018;5(4). 39. hadjighassem m, kamalidehghan b, shekarriz n, baseerat a, molavi n, mehrpour m, et al. oral consumption of α-linolenic acid increases serum bdnf levels in healthy adult humans. nutr j. 2015;14(1):20. 40. razi ss, latif mj, li x, afthinos jn, ippagunta n, schwartz g, et al. dietary flaxseed protects against lung ischemia reperfusion injury via inhibition of apoptosis and inflammation in a murine model. j surg res. 2011;171(1):e113-e21. 41. rahmati-ahmadabad s, azarbayjani m, nasehi m. the effects of highintensity interval training with supplementation of flaxseed oil on bdnf mrna expression and pain feeling in male rats. ann appl sport sci. 2017;5(4):1-12. 42. bagheri a, talei s, hassanzadeh n, mokhtari t, akbari m, malek f, et al. the neuroprotective effects of flaxseed oil supplementation on functional motor recovery in a model of ischemic brain stroke: upregulation of bdnf and gdnf. acta med iran. 2017:785-92. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.945 203j contemp med sci | vol. 6, no. 5, september-october: 203–207 original issn 2413-0516 introduction: a novel type of coronavirus has been recognized at the beginning of december 2019.1 the virus was initially named as 2019-cov which was later called severe acute respiratory syndrome coronavirus 2 (sars-cov-2).2 the epidemic was first started in wuhan, china in december 2019. covid-19 was found to be a zoonotic pathogen according to a recent research with pangolins and chinese horseshoe bats, similar to the severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome (mers) coronavirus.3-5 as of 15 june, covid-19 has been recognized in more than 215 countries, with a total of over 24 million laboratory-confirmed cases and more than 441,000 deaths.6 this novel viral infection spreads faster and has higher contagious rate than sars and mers.5 according to studies, the incubation period of covid-19 was estimated between 1 and 14 days.1,7 the transmission route of covid-19 to human was reported to be via air-borne droplets, blood-borne droplets, saliva, body secretions, touching, hand-shaking, and close contact with infected person as well as virus contaminated surfaces. in addition to these transmission routes, fecal–oral route could carry a risk as coronavirus was detected in the stool of patients.8,9 the majority of patients with covid-19 represent mild clinical manifestations with fever, shortness of breath, fatigue, muscle pain, headache, diarrhea, vomiting, and dry cough.9-11 in some severe conditions, development of pneumonia, acute respiratory distress syndrome, shock, and arrhythmia were investigated. patients with severe conditions were kept under consent of intensive-care units.9-13 the most susceptible individuals were found to be immune-compromised individuals with older age and people with existing clinical conditions such as diabetes, hypertension, and cardiovascular diseases. these susceptible individuals were associated with severe clinical manifestations and poor prognosis.13-15 the travel history of patients 14 days prior to symptom onset was regarded as an important parameter in terms of diagnosis of covid-19. apart from the travel history, various laboratory tests [e.g., reverse transcriptase polymerase chain reaction (rt-pcr), chest computed tomography (ct), serological tests] were applied to patients with clinical symptoms in order to diagnose the disease. until now, there is no specific anti-ncov treatment, so majority of the treatment methods applied were supportive treatments.16 all these findings suggested the importance of early diagnosis and self-isolation in terms of covid-19. covid-19 was found to be very contagious to some of the professions, especially the health-care workers. huge numbers of health-care workers were reported to have acquired covid-19 while working with covid-19 infected individuals.17 these findings indicate that dental practitioners were also under high risk of getting covid-19 due to their working environments (close contact with patients, usage of high speed rotary instruments, expose to body fluids such as saliva and blood).5 although covid-19 patients were not allowed to have dental treatments, some asymptomatic cases, or individuals with post infection would carry a huge risk in terms of these dental practitioners.3,18 these unavoidable contacts with patients during this pandemic period made dentists to have an extreme awareness and integrity to deal with disease and manage the control of its spread. awareness and perception of dentistry students studying in cyprus regarding covid-19: a cross-sectional study cenk serhan özverela,b, sevcan kurtulmus-yilmazc adepartment of basic medical sciences, faculty of dentistry, near east university, lefkosa, cyprus, mersin10, turkey bnear east university, desam institute, lefkosa, cyprus, mersin10, turkey cdepartment of prosthodontics, faculty of dentistry, near east university, lefkosa, cyprusmersin10, turkey corresponding author: cenk serhan özverel (e-mail: cenkozverel@gmail.com ) (submitted: 05 august 2020 – revised version received: 11 august 2020 – accepted: 27 august 2020 – published online: 30 october 2020) abstract: objectives: the present study aimed to evaluate the awareness and assessing the knowledge of dental students and dentists on covid-19. methods: the study population consisted of undergraduate students who are currently studying dentistry in cyprus. students were sent an online survey and data were collected on 16 april. survey comprised of questions about the demography, education level of the participants, their infection control knowledge, and conditions that require treatment during pandemic. results: a total of 594 students with equal gender distribution joined the survey. the age group of the participants were mostly between 18 and 25 age (94%) with 63% pre-clinical and 37% clinical students. the majority of participants were aware that dentists were at highrisk group in terms of covid-19 (98.5%). when clinical questions were asked, data indicated strong relationship between participants’ responses with their level of study. upon asking covid-19-specific questions, participants that have taken microbiology course were reported to have higher correct answer rate. conclusions: dental students studying in cyprus demonstrated high awareness of covid-19 and the higher risks of covid-19 exposure that dental professionals face. study demonstrated the importance of microbiology courses, as it significantly affected the answers to the questions on diagnosis methods and immune response to covid-19. keywords: dentistry students, survey, dental education, coronavirus, covid-19, sars-cov-2 204 original awareness of dentistry students regarding covid-19 cenk serhan özverel j contemp med sci | vol. 6, no. 5, september-october: 203–207 the presence of infection prevention guidelines by country’s ministry of health provide minimum requirements that are needed to be applied by health-care workers. following these precautions precisely would help patients’ protection and also the dental professionals. this study aimed to evaluate the awareness and assessing the knowledge of dental students and dentists on covid-19. materials and methods: sample a total of 1370 undergraduate students who are currently studying dentistry in cyprus were invited to take part in an online questionnaire. invitation was posted on student’s class representatives and students’ social media groups. the period of the survey was april 15–16, 2020. materials questions were developed following the current guidelines16 and literature19 regarding covid-19. an online google forms was used to construct a questionnaire comprising the following areas: (1) demographic questions (gender, age, ethnicity), (2) questions related to their education year (pre-clinical or clinical classes, whether they have taken microbiology courses), (3) general questions to determine their covid-19 knowledge, (4) questions related to diagnosis of covid-19, (5) questions to measure their infection control knowledge, and (6) questions related to covid-19 and dental applications. ‘no opinion’ option has also been added to some of the suitable questions asked during the survey. all participants were informed about the questionnaire beforehand. participation of respondents was voluntary and anonymous. ethical approval was obtained from the institutional ethics committee of near east university (ydu/2020/79-1105). data analysis data were collected in an excel file and statistical analysis was done on spss 16.0 (ibm corp, new york, usa). chi-square and spearman correlation tests were performed to assess the relation of the dentistry students’ responses with respect to gender and to their clinical experience (according to their education level) and whether they have taken microbiology courses.  results: demography and education this study covers a total of 594 dentistry students (291 females and 303 males), with a responsive rate of 49.5% of total invited students. the age of participants were ranged from 18 to 40+, with most common group belonging to 18–25 ages (94.5%, 561). the vast majority of survey participants are people who come from international countries such as; turkey (69%, 410), iran (5%, 30), jordan (3%, 18), iraq (4%, 24), syria (4%, 24), united arab emirates (2%, 12), lebanon (2%, 12), and germany, france, canada, nigeria, sudan with <1%. international students were also asked whether they returned to their home countries after the start of covid-19 and 47% (280) of the participants reported that they have returned to their countries. the majority of students (40%) who turned back their countries indicated that they wanted to be with their families during the pandemic (fig. 1). the participants of survey were also asked which classes they went to during the academic year 2019–2020, so that the differences between pre-clinical and clinical classes were aimed to take into account. according to the data, 375 (63%) participants were in pre-clinical classes (1st, 2nd and 3rd years) and 219 (37%) participants were clinical classes (4th and 5th years). participants’ microbiology knowledge was also assessed during the survey and 64% (343) of the participants reported that they had microbiology classes during their dentistry education. awareness of participants the vast majority of respondents said they were first aware of covid-19 pandemic through tv news (43%, 261) and social media (51%, 301). only small population of participants was first aware of covid-19 pandemic through newspapers (2%, 12) and university announcements (4%, 24). participants were asked about the sources of information they are following to be aware of covid-19 status. the main platform participants used was social media (35%, 208), tv news (26%, 154), the ministry of health website (21%, 125), and to a lesser extent, research journals related to covid-19 (12%, 71) were reported. participants’ awareness were also investigated by asking current worldwide status (april 15) of covid-19 pandemic. almost all of the participants knew the country where pandemic originated (99.5%, 591). the majority defined the country with the highest number of death rate as usa (70%, 416) and the country with the highest recovery rate as china (53%, 315). fig. 1 graphical representation of participants demography 205 original awareness of dentistry students regarding covid-19cenk serhan özverel j contemp med sci | vol. 6, no. 5, september-october: 203–207 participants’ general knowledge about coronavirus was also assessed during the survey. vast majority of the participants (90%, 534) were aware of the incubation period of the coronavirus (1–14 days) and knew that 40+ individuals were under high risk when compared to younger individuals (82%, 487). when asked about the symptoms of covid-19, majority of participants reported fever (97%, 576), dry-cough (94%, 558), and shortness of breath (97%, 576) as symptoms. one-third of the participants reported headache (34%, 202) and diarrhea (35%, 208), two-third of the participants reported sore throat (64%, 380) as symptom of the disease. less than 20% of the participants selected runny nose and dizziness as symptoms of covid-19. when asked about the measures for preventing covid-19, majority of the participants reported that cleaning hands (94%, 558), use of gloves (82%, 487), use of face masks (89%, 529), not going out unless needed (98%, 582), avoiding family and neighbor visits (94%, 558), maintaining social distance (98%, 582), disinfecting clothes worn outside (85%, 505) can help to prevent transmission of covid-19. when asked about the transmission of covid-19 via hand contact, 73% (434) of the participant confirmed that. covid-19 transmission type was also asked to the individuals and 75% (446) of the participants knew that the disease is zoonotic. the present study reported no significant relationship between participants’ responses with gender, age, or home country. the study also indicated a significant relationship between participants’ education level (whether they have taken microbiology courses) and responses to questions like transmission routes, whether the disease is zoonotic or not. measurement of covid-19 precautions in the eye of dentists almost all of the participants 98.5% (585) reported that, dentists are at high-risk group in terms of covid-19 and measurement of patients’ temperatures should be mandatory (93%, 551). a total of 89% (527) students indicated that exceeding social distance limit with patient, exposure to body fluids such as blood or saliva (94%, 562) and use of high speed rotary  instruments  (54%, 325) are the causes that increases dentists tendency to the disease. participants reported that it is important to change gloves (97.5%, 580), face masks (96%, 570), wearing face shields (93%, 553). they were aware of importance of disinfecting units after treatment of each patient (90%, 538) and sterilization of instruments used (91%, 546). majority of participants mentioned that during thecovid-19 outbreak, painful tooth requiring root canal treatment (90%, 535), swelling of face (82%, 492), and trauma (87%, 518) were accepted reasons for patient treatments, whereas, only small percentage of participant population which significantly comprised of pre-clinical students selected dental caries (14%, 88), routine control (2.5%, 15), and completion treatment started before outbreak (36%, 207) as acceptable reasons in treatment. the clinical questions reported statistically significant relationship between participants’ responses with their level of study (pre-clinical and clinical students). measurement of perception levels of participants on covid-19 participants were asked about the mortality rates of sarscov-2 in comparison to some other viral diseases (sars, mers, and seasonal influenza). one-fourth (25.9%, 202) of the individuals reported that mortality related to seasonal influenza is lower than sars-cov-2, whereas, 22% (175) and 16.9% (132) of the respondents respectively indicated sars and mers have lower mortality rates than sars-cov-2. onethird (271, 34.7%) of the participants indicated that they have no knowledge about the mortality rates. when information about current covid-19 diagnostic tests such as serological tests and rt-pcr were asked, minor level of participants were aware of the importance of igg (16%, 99) and igm  (20%, 122) antibody measurements, however, nearly half of them were aware of the importance of rt-pcr analysis (45%, 286) in terms of covid-19 diagnosis. participants who had selected ige (8.5%, 51) and iga (12%, 76) measurements were reported as minorities (fig 2). however, 49% (295) of the participants indicated that they had no opinion regarding the question, which were significantly belonging to dental students did not have microbiology course yet (1st year students). moreover, the participants’ knowledge about the speed of the diagnostic tests was also asked. only 27% (162) of the participants were aware of the fact that serological tests (measuring antibody levels) were responsible for quicker diagnosis of the disease, but 30% (182) of the individuals reported rt-pcr analysis is quicker and 43% (250) of them reported that they had no opinion regarding question. general questions regarding the diagnostic tools were also included in the survey. nearly, onethird of the participants (30%, 174) reported that, rt-pcr analysis could possibly give false-negative result when sample is not taken properly and 10% (56) of them were aware of that rt-pcr has relatively more accuracy when compared to the serological tests (fig 2). some other chosen answers were igm fig. 1 graphical representation of participants answers on covid-19 diagnostic methods. 206 original awareness of dentistry students regarding covid-19 cenk serhan özverel j contemp med sci | vol. 6, no. 5, september-october: 203–207 response develops in the late stages of disease (7.2%, 43), iga titer increases upon encountering covid-19 (7.6%,45), igg titer peaks towards late stages of the disease, and individuals who had recovered disease (13.9%, 35), ige response peaks in newly infected individuals (5.89%, 35). two-third (66.6%) of the participants had indicated that they had no opinion about the test principles. the present study reported significant relationship between participants’ responses on covid-19specific questions, serological tests etc. and whether participants received microbiology course or not. discussion: this survey aims to give an insight on the level of knowledge and awareness of dentistry students on covid-19 who are studying in cyprus. the data obtained provide important clues when it is assumed that dentists are at the top of the professions with the highest risk group5 and 37% of the participants who responded to the survey were actively working in the clinic. in addition to these, the importance of microbiology classes, the depth of the curriculum and the level of awareness about covid-19 were determined in line with the responses given by the students of pre-clinical and clinical classes. present study covered a total of 594 pre-clinical and clinical dental students with equally distributed male and female populations. international students were asked whether they returned to their home countries and the reason of it. as cyprus is an island, there are only two ways of transportation, air and sea. upon confirmation of the first case reported in cyprus on 10 march, airports and borders were closed on the 16 march.20 while leaving of the country were maintained with a controlled manner and foreign citizens living/studying in the country were allowed to return to their homes, only 47% of the participants returned to their countries, taking into account the difficulties that might be experienced during returns. this might be the main reason why most of the participants stayed in cyprus. when asked about the reason for leaving cyprus, the majority of the participants mentioned that they preferred to be with their families during the pandemic and some of them specified the economical issues. the vast majority of the participants had known the origin of the pandemic was china, the country with highest mortality was usa, and more than half of the participants had known that the highest recovery rate was in china.6 this showed, the participants were following the current worldwide status of the covid-19 and well aware of the situation. the estimated incubation time for covid-19 was reported to be between 1 and 14 days and older individuals were more susceptible.1,7 study participants knowledge about incubation period of the and susceptibility of the covid-19 were impressive. this knowledge was very important for participants working in dental clinics in terms of the determination safe period for suspected patients. knowledge of the participants about common symptoms of the covid-19 was also investigated in the study. previous literature indicated that dentists’ knowledge on contagiousness of respiratory diseases was limited amongst other health-care professionals.21,22 despite the previous researches, majority of participants were found to be highly informed about the common symptoms of the covid-19 (dry-cough, high fever, and shortness of breath).9-11 the essential actions in disease management were also investigated in the survey. participants were reported to have a good knowledge in general disease management protocols, for instance; importance of washing hands, use of gloves and face masks, not going out unless necessary, maintaining social distance, avoiding of family and neighbor visits, and disinfecting clothes worn outside. some more questions were also added in the survey to measure the knowledge about precautions needed to be taken in the field of dentistry. nearly all of the participants (99%) were aware of the fact that dentists are at the top of the professions with highest risk group due to the normal exceeding social distance with patient and exposure to body fluids of patients, with more than half of the individuals were aware of risk created due to the usage of rotary instruments during treatment.5, 23 the individuals who are aware of the risk of using rotary instruments were significantly found to be the ones with clinical experience. these have a good knowledge in rotary instruments as they are actively using them during clinical practices. after asking about the risks that may cause danger in clinic, participants were asked about necessary measures to minimize these risks. the answers received were quite satisfactory. nearly all of the participants were aware of the importance of using face shields, face masks, gloves, disinfecting units after each treatment, proper sterilization of instruments, measuring patients’ temperatures prior to entry to hospital in terms of protecting both patients and dentists. when asked about which condition should be a reason to apply to dentists during pandemic period, vast majority of them have mentioned that patients with painful tooth requiring root canal treatment, patients with swollen face and trauma patients should only apply to dentists. dental caries and completion of treatment started before pandemic (15% and 35%, respectively) were also selected as conditions should be cared during covid-19 pandemic which can be attributed to their insufficient clinical experiences. studying at clinical classes significantly affected the answer of the students and only students at pre-clinical classes selected these two choices. it was aimed to measure the actual knowledge of the students and evaluate its relationship with their education level. therefore, ‘no opinion’ option was added to the appropriate questions in the survey for the prevention of random selection among choices. participants’ detailed knowledge about the rate of covid-19 infection in comparison to other viral diseases, diagnosis methods and immune response formed during and after infection was also investigated during the survey. the mortality rates of sars-cov-2, seasonal influenza, sarscov, and mers-cov were also asked to the participants. more than on-third of the participants knew that, seasonal influenza has lower mortality rate than sars-cov-2 whereas sars and mers had higher mortality rates.5 however, 45% of the participants, especially those who are first-year students, did not answer this question correctly. the main reason for this may be due to the lack of microbiology classes in the first years of dentistry curriculum. test methods for covid-19 diagnosis were also asked during the survey. nearly half of the participants had knowledge about the importance of rt-pcr tests during disease diagnosis, however only one-fifth of the participants were aware of the importance of antibody titer (igg and igm) measurements by serological tests. nearly half of the participants had no knowledge about the diagnostic tests according to the survey. further detailed questions were also asked about the diagnostic tests. only one-fourth of the participants stated that the serological tests respond faster 207 original awareness of dentistry students regarding covid-19cenk serhan özverel than the rt-pcr tests, while the remaining three-fourth either gave wrong answer or had no opinion. the detailed questions were asked against covid-19 diagnostic tools and about one-third of the participants showed that they knew the importance of sampling while performing rt-pcr tests, and that if the sample was not taken properly, false-negative data might be obtained. one in ten participants stated that rt-pcr had high accuracy as well. in addition to these correct answers,24,25 approximately two-third of the participants stated that they did not know about this question. nearly one-third of the participants are aware that serological tests and rt-pcr tests together provide more accurate results and one-fifth of them indicated that quick-serological tests are advantageous in screening large populations. however, more than half of the students, especially the ones in early years of education had no opinion about these aspects of the covid19 diagnostic tests. these might be attributed to condense immunological and pharmacology classes are held during the second and third years of the curriculum. dental students studying in cyprus showed high awareness of covid-19 and the higher risks of covid-19 exposure that dental professionals face. preventing the spread of corona virus during dental applications and the conditions that require treatment during pandemic was dependent on the clinical experience of participants. having microbiology courses significantly affected the answers to the questions on diagnosis methods and immune response to covid-19. conflicts of interest disclosure: all authors declare no conflicts of interest. references: 1. li q, guan x, wu p, wang x, zhou l, tong y, et al. early transmission dynamics in wuhan, china, of novelcoronavirus-infected pneumonia. n engl j med 2020 mar 26;382(13):1199-1207. doi: 10.1056/nejmoa2001316 2. world healthorganization. who timeline – covid-19 [accessed 15 june 2020]. https://www.who.int/news-room/detail/27-04-2020-who-timeline--covid-19 3. chan jf, yuan s, kok kh, to kk, chu h, yang j, xing f, liu j, yip cc, poon rw, et al. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. lancet. 2020;395(10223):514–523. 4. lu r, zhao x, li j, niu p, yang b, wu h, wang w, song h, huang b, zhu n, et al. genomic characterisation and epidemiology of 2019 novel coronavirus: i̇mplications for virüs origins and receptor binding. lancet 2020;395(10224):565–574. 5. meng l, hua f, bian z. coronavirus disease 2019 (covid-19): emerging and future challenges for dental and oral medicine. j dental res. 2020;99(5):481487. doi:10.1177/0022034520914246 6. worldometer. 2020. covid-19 coronaviruspandemic. [accessed 15 june 2020]. https://www.worldometers.info/coronavirus/ 7. backer ja, klinkenberg d, wallinga j. incubationperiod of 2019 novelcoronavirus (2019-ncov) infections among travellers from wuhan, china, 20–28 january 2020. euro surveill. 2020;25(5). doi:10.2807/15607917. es.2020.2825.2805.2000062. 8. holshue ml, debolt c, lindquist s, lofy kh, wiesman j, bruce h, spitters c, ericson k, wilkerson s, tural a, et al. first case of 2019 novelcoronavirus in the united states. n engl j med 2020. doi:10.1056/nejmoa2001191. 9. hejaz ha, zalloum a, attili r. novel coronavirus disease-2019: epidemiology, diagnosis, therapeutics and guideline protocol for disease management in different countries. iraq med j. 2020 jun 26;4(2). 10. guan w-j, ni z-y, hu y, liang w-h, ou c-q, he j-x, liu l, shan h, lei c-l, hui ds, et al. clinical characteristics of 2019 novel coronavirus infection in china. medrxiv2020. doi:10.1101/2020.1102.1106.20020974 11. zhou f, yu t, du r, fan g, liu y, liu z, et al. clinical course and risk factors for mortality of adult in patients with covid-19 in wuhan, china: a retrospective cohort study. the lancet 2020;395(10229):1054-1062. doi: https://doi.org/10.1016/ s0140-6736(20)30566-3 12. saleh m, saleh m, zahid mn. awareness and response to coronavirus disease (covid-19) epidemic among the arab population in the eastern mediterranean region and arab peninsula. j contemp med sci. 2020 aug 26;6(4). 13. wang d, hu b, hu c, zhu f, liu x, zhang j, wang b, xiang h, cheng z, xiong y, et al. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in wuhan, china. jama 2020. doi:10.1001/ jama.2020.1585. 14. kui l, fang yy, deng y, liu w, wang mf, ma jp, xiao w, wang yn, zhong mh, li ch, et al. clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province. chin med j (engl) 2020. doi:10.1097/ cm1099.0000000000000744 15. yang y, lu q, liu m, wang y, zhang a, jalali n, dean n, longini i, halloran me, xu b, et al. epidemiological and clinical features of the 2019 novel coronavirus outbreak in china. j dental res. 2020. doi:10.1101/2020.1102 .1110.20021675. 16. world health organization. clinical management of severe acutere spiratory infection when novel coronavirus (2019-ncov) infection is suspected: interim guidance [accessed 15 june 2020]. 2020. https://www.who.int/ publications-detail/clinical-management-of-severe-acute-respiratoryinfection-when-novel-coronavirus-(ncov)-infection-is-suspected. 17. secon h. nearly 3,400 chinese healthcare workers have gotten the coronavirus, and 13 have died. business insider 2020 mar 04. 18. rothe c, schunk m, sothmann p, bretzel g, froeschl g, wallrauch c, zimmer t, thiel v, janke c, guggemos w, et al. transmission of 2019-ncov infection from an asymptomatic contact in germany. n engl j med. 2020. doi:10.1056/nejmc2001468 19. spagnuolo g, de vito d, rengo s, tatullo m. covid-19 outbreak: an overview on dentistry. int j environ res public health 2020;17(6):2094. doi: https://doi.org/10.3390/ijerph17062094 20. trnc public information office. is north cyprus set to become first country to eradicate covid-19? [accessed 15 june 2020]. https://pio.mfa.gov.ct.tr/ en/is-north-cyprus-set-to-become-first-country-to-eradicate-covid-19/ 21. baseer m, ansari s, alshamrani s, alakras a, mahrous r, alenazi a. awareness of droplet and airborne isolation precautions among dental health professionals during the outbreak of coronavirus infection in riyadh city, saudi arabia. j clinexpdent 2016 oct;8(4):e379-e387. doi: 10.4317/ jced.52811 22. abolfotouh ma, alqarni aa, al-ghamdi sm, salam m, al-assiri mh, balkhy hh. an assessment of thelevel of concern among hospital-based healthcare workers regarding mers outbreaks in saudi arabia. bmc infectdis 2017 jan 03;17(1):4. doi: 10.1186/s12879-016-2096-8 23. centers for disease control and prevention 2020. cdc recommendation: postponenon-urgent dental procedures, surgeries, andvisits. [accessed 15 june 2020]. https://www.cdc.gov/oralhealth/infectioncontrol/statementcovid.html 24. winter ak, hegde st. the important role of serologyfor covid-19 control. the lancet infectious diseases 2020. doi: https://doi.org/10.1016/s14733099(20)30322-4 25. long q, liu b, deng h, wu g-c, deng k, chen y-k. et al. antibody responses to sars-cov-2 in patients with covid-19. nat med 2020. https://doi. org/10.1038/s41591-020-0897-1 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.828 242 j contemp med sci | vol. 6, no. 5, september-october 2020: 242–248 original issn 2413-0516 introduction sepsis is a common cause of inpatient mortality in both genders and all age groups, particularly in the intensive care units of hospitals. annually, more than 19 million people are affected by sepsis (with a mortality rate as high as 18%) worldwide.1, 2 in sepsis, the cause of death is generally multiorgan dysfunction syndrome (mods). despite the recent decrease in its mortality rate which can be attributed to the advancements in antibiotics and intensive care equipment, the incidence of sepsis is increasing worldwide.3 sepsis-induced shock is associated with multiorgan failure and mortality rate of patients with sepsis. the most frequently affected organs by sepsis are those of the cardiovascular and respiratory systems.4 although the precise mechanisms of sepsis-induced cardiomyopathy are not yet fully elucidated, it suggested that severe inflammation, metabolism insufficiencies, and defective beta-adrenergic response may be involved in this process.3 it is demonstrated that pulmonary complications of sepsis are due the cumulative effect of circulating cellular elements, soluble inflammatory mediators, and cytokines on the pulmonary tissues.5 considering its high fatality rate, sepsis leaves little opportunity for clinical research in the field. therefore, the clp procedure (which is considered as the gold-standard model of human sepsis) provides the opportunity for an investigation into different aspects of this condition. during this procedure, secretion of intestinal polymicrobial flora into the abdomen leads to development of systemic presentations very similar to those caused by sepsis in humans.6 clp-induced damages (and sepsis in humans) stimulate the immune system, activate oxidative stress pathways, increase caspase levels (due to apoptosis), lead to the production of early and late proinflammatory cytokines such as tnf-α and hmgb1 and upregulation of autophagy and hypoxia-related genes.7 reactive oxygen species (ros) are major signaling molecules involved in the oxidative stress response. in sepsis, the excessive accumulation of ros impairs cellular homeostasis, and this leads to oxidative stress and mitochondrial dysfunction. this oxidative stress process, caspases activation, and hyperinflammation also promote autophagy, a defensive cytoprotective process for recycling waste organelles and other intracellular material following cellular damages.8 therefore, indicators of oxidative stress and autophagy processes have been widely used to investigate the intensity of sepsis induced through clp procedure.9 similarly, measurement of the enzyme amp-activated protein kinase (ampk) is extensively applied for assessment of severity of sepsis.10this ros elevation also promotes autophagy, a defensive cytoprotective process for recycling waste organelles and other intracellular material following cellular damages.8 consequently, autophagy proteins such as lc3b are considered most valuable evaluation the cardiopulmonary markers in cecal ligation and punctureinduced sepsis in wistar rats tina didari1, shokoufeh hassani1, maryam baeeri1, vida kazemi2 #, mohammad abdollahi1,3, mojtaba mojtahedzadeh1,4 # 1 pharmaceutical sciences research center (psrc), the institute of pharmaceutical sciences (tips), tehran university of medical sciences, tehran, iran. 2 medicinal plants research center, faculty of pharmacy, tehran university of medical sciences, tehran, iran. 3 department of toxicology and pharmacology, school of pharmacy, tehran university of medical sciences, tehran, iran. 4 department of clinical pharmacy, school of pharmacy, tehran university of medical sciences, tehran, iran. # corresponding authors: mojtaba mojtahedzadeh (email: mojtahed@tums.ac.ir ) dr.vida kazemi (e-mail: kazemi-v@razi.tums.ac.ir ) (submitted: 29 july 2020 – revised version received: 19 august 2020 – accepted: 07 september 2020 – published online: 30 october 2020) abstract: objective: sepsis is a clinical problem caused by host immune disability against pathogens. rodent cecal ligation and puncture (clp) models mimic sepsis in humans. gauges needle size in clp is related to cytokine storm, inflammation, and organ failure. this study focus, for the first time, on precise and inexpensive biochemical markers to evaluate the difference of sepsis severity in the heart and lung tissues, one day after cecal ligation and puncture-induced sepsis with needle gauge 18 (g-18). methods: twelve adult male wistar rats were randomly allocated into two groups of six animals. these groups include; sham operation as the control group and underwent clp procedure with g-18. all rats were sacrificed 24 hours after clp. then lungs and heart samples were collected for biochemical and histological assessment. following the procedure, reactive oxygen species (ros), myeloperoxidase activity (mpo), tumor necrosis factor-alpha (tnf-α), high mobility group box 1 (hmgb1), lactate generation, caspases (-3 and -9), gene expression of autophagy, cellular hypoxia, and pathological assessment of both tissues were measured. results: increased level of ros, mpo, pro-inflammatory cytokines, hyperlactatemia, caspases production, overexpression of hypoxia (prkaa1 gene), and autophagy (map1lc3b gene) in the lungs were higher compared to heart 24 hours after the procedure. moreover, hyperplasia of pneumocyte and inflammatory cells, and myocardial necrosis were found in the pathological assessment. conclusion: the purpose of study was to determine the severity of sepsis by means of cost-effective and precise inflammatory markers. our findings demonstrated that injury-related indicators in lungs meaningfully increased compared to heart 24 hours after clp. keywords: sepsis, cecal ligation and puncture (clp), inflammation, oxidative stress, needle gauge 243 original gauge-18 is related to organ inflammationtina didari j contemp med sci | vol. 6, no. 5, september-october 2020: 242–248 biomarkers for evaluation of cellular impairment.9 similarly, measurement of the enzyme ampk and autophagy proteins are employed for assessment of molecular dysfunction.10 it is demonstrated that the size of the needle used in the clp procedure can be influential in different septic outcomes such as mortality, cytokines concentrations, apoptosis, lactic acidosis, and autophagy. previous studies has showed that g-18 induced mods more efficiently compared to other gauge groups.6, 11, 12 there exist limited experiments on factors affecting mods with g-18. the current study was designed and carried out with different views. firstly, a period of 24 hours was chosen for investigation into the severity of sepsis in the cardiovascular and pulmonary systems. doing this, we managed to minimize the duration of the study and reduce the costs involved in animals’ care following the procedure. moreover, in the current study, for the first time, we employed autophagy gene expression and cellular hemostasis indicators (along with simultaneous assessment of cellular hemostasis and measurement of primary and secondary inflammatory cytokines) for assessment of cytokine storm. moreover, appropriate target tissues (pulmonary and cardiac) were chosen for investigation into sepsis induced through clp procedure. lastly, we employed low-cost, convenient, and precise laboratory tests for assessment of the extent of tissue damage (such as lactate, oxidative stress, and apoptosis). material and methods animals adult male wistar rats 3–4 months old (250–300 g) were procured from the animal breeding house of the faculty of the pharmacy of tums. animals were kept according to the animal standard care facility under controlled temperature (23 ± 10c), 12 h light/dark cycle, 55± 10% humidity, and ad libitum feed. the protocol of the study was approved by the institutional ethical committee under code number ir.tums.vcr. rec.1396.2341. reagents for high mobility group box 1 (hmgb1) and tnf-α, elisa kits were obtained from zellbio gmbh (ulm, germany) and diaclone (france), respectively. the lactate isolation kit was produced from zellbio gmbh (ulm, germany). rnase solution, iscript cdna synthesis kit, and propidium iodide were manufactured by sigma-aldrich gmbh (munich, germany). other chemicals not specifically mentioned were all purchased from sigma-aldrich gmbh ( munich, germany). animal groups all male wistar rats were randomly divided into the following three groups: sham or control group (which underwent the same operation without the clp procedure) and clp group with g-18 (which was operated with two punctures of g-18) the procedure adult male wistar rats first underwent clp as described in detail elsewhere.11 rats were anesthetized with ketamine and xylazine. then, the cecum was ligated with a 3.0 silk suture at its base right below the ileocecal valve, and two punctured using g-18. next, the abdomen was closed using silk suture. following the operation, animals were hydrated through intravenous administration of an isotonic saline solution, returned to a cage in 24 hours.13 according to previous research, sepsis induced 6 hours after clp.11, 12 sample collection twenty-four hours after clp, the animals were sacrificed. heart and lung tissues were harvested, then put into two parts for further histopathological and biochemical assessments. assessment of biochemical variables ros assay tissue samples were all homogenized and centrifuged accordingly. subsequently, they were stored in 75 μl extraction buffer, mixed with 80 μl of assay buffer, and kept for 30 min at 37 °c. production of ros was absorbed by 2’, 7’-dichlorofluorescein diacetate (dcf-da) as a fluorogenic reagent. identification of the absorbance change of dcf-da was identified using elisa fluorometer (biotec, tecan u.s.) with maximum excitation (488 nm) and emission (529 nm) spectra for an hour. myeloperoxidase (mpo) activity briefly, tissue samples were homogenized in 50 mm potassium buffer (ph 6.0) containing 0.5% htab. subsequently, the samples were centrifuged (30,000g, 15 min, 4 °c) and the supernatant was mixed with phosphate buffer (ph 6, 50 mm). change of absorbance was recorded through spectrophotometric analysis at 460 nm 5 minutes later, as described in detail elsewhere.14 lactate levels in tissue samples according to the manufacture’s protocol, lactate assay was performed for analysis of all samples. in short, tissue samples (100 mg) were homogenized in 8% perchloric acid and lactate level evaluated using a standard curve. caspase-3 and -9 activity colorimetric assays were utilized for measurement of the caspases-3 and -9 through specific identification of particular amino acid sequences. briefly, the color changed and detected by spectrophotometry as setup by rahimifard.15 assessment of pro-inflammatory cytokines (tnf-α and hmgb1) the quantity of rat-specific tnf-α and hmgb1 in test samples were examined by a rat-specific elisa kit. all test steps were performed according to the manufacturer’s protocol. gene expression evaluation for real-time reverse transcription-polymerase chain reaction (rt-qpcr), 1 μg of total rna was reverse transcribed to cdna using a primescript rt reagent kit (takara, japan). rt-qpcr was performed using the step one plus abi system (applied biosystems). rt-qpcr was carried out under manufacturer’s instruction.16 the sequences of primers used for rt-qpcr are presented in table 2. histopathological studies the animals were euthanized 24 hours after the procedure, and heart and lung tissues were isolated and fixed in the 10% 244 original gauge-18 is related to organ inflammation tina didari j contemp med sci | vol. 6, no. 5, september-october 2020: 242–248 neutral buffered formalin (nbf, ph 7.26) for 48 hours, and then were embedded in paraffin and stained with hematoxylin and eosin (h&e), then observed using a light microscope (olympus bx51, japan). any suspected changes were recorded. 200 x (scale bar=100μm), 400 x (scale bar=20μm). statistical analysis the results were presented as the mean ± standard error of the mean (sem). one-way analysis of variance (anova) and tukey’s multicomparison tests was applied with the degree of significance set at p< 0.05. statistical analyses were performed using graphpad prism version 5 (graphpad software, la jolla, ca, usa). results ros generation ros levels were raised significantly in heart of clp compared to the sham group (p<0.01). moreover, ros production in the lung was increased significantly in clp compared to the sham group (p<0.001). a significant difference was not found between the lung and heart tissue of the clp group (table 1). myeloperoxidase activity the result of mpo assay as neutrophil infiltration marker showed that a significant elevation in the heart (p<0.01) and lung (p<0.001) samples of the clp group in comparison with related sham groups. moreover, no significant differences were found between the heart and lung tissues of clp groups (table 1). lactate levels lactate production, a reliable marker of tissue hypoperfusion and hypoxia was increased in both heart (p<0.01) and lung (p<0.001) samples of clp group, compared to related sham groups. hyperlactatemia was significantly increased in the lung (p<0.001) compared to the heart sample of the clp group (table 1). caspase 3 and caspase 9 caspases as cysteine proteases family activate during apoptosis. the result showed meaningfully hyperactivation of caspase-3 in the heart (p<0.001) and lung (p<0.001) specimens of clp groups, compared to related sham groups. also, caspase-3 activity was increased in the lung compared to heart tissue in the clp group (p<0.05). moreover, it found that caspase-9 significantly elevated in the heart (p<0.001) and lung (p<0.001) samples of clp groups, compared to related sham groups. a significant increase of caspase-9 activity was observed in the lung compared to the heart tissue of the clp group (p<0.05) (table 1). proinflammatory cytokines tnf-α which is an early modulator of inflammation during sepsis was found to be significantly increased in the heart and lung tissue of the clp group (p<0.001) in comparison with the related sham groups. the levels of tnf-α in lung tissue were increased compared to the heart sample in the clp group (p<0.001). the amount of hmgb1, a late phase inflammatory biomarker, was significantly elevated in the heart and lung tissue of the clp group (p<0.001) in comparison with the related sham groups. the levels of hmgb1in lung tissue were increased compared to the heart sample in the clp group (p<0.001) (fig. 1). real-time reverse transcription-polymerase chain reaction (rt-qpcr) the mrna expression level of the prkaa1 gene (related gene to ampk activity, as the stress protein) in the heart (1.77-fold) and lung sample (3.66-fold) was significantly increased in clp group compared to the sham group. also, the mrna expression level of prkaa1 was increased significantly in lung tissue compared to the heart specimens in the clp group (p<0.001). the mrna expression level of the map1lc3b gene (related gene to activation of lc3iib, as an autophagy indicator) in the heart (4.38-fold) and lung samples (5.86-fold) was significantly elevated in clp group compared to the sham group. the analysis of the mrna expression level of map1lc3b data showed that lung tissue has been significantly increased compared to the heart samples in the clp group (p<0.05) (fig. 2). the abbreviations and primers of proteins used for realtime rt-qpcr listed in table 2. histopathological analysis all the samples were visualized by an independent reviewer. the results of each sample have reported that micrographs of the lung in the clp group showed various degrees of lung table 1. effect of gauge-18 on biochemical markers of lung and heart tissue, 24 hrs. after clp. variables groups sham-heart clp-heart sham-lung clp-lung ros (u/mg protein) 0.66±0.05 1.21±0.08** 0.27±0.03 0.98±0.16*** mpo (unit/gr tissue) 77.83±3.64 112.64±7.44** 75.97±7.50 130.11±7.20*** lactate levels (mmol/l) 2.90±0.07 4.18±0.12** 6.47±0.11 10.06±0.45*** ••• caspase 3 (% of content) 83.04±1.85 117.60±1.45*** 100.12±2.52 138.29±8.97*** • caspase 9 (% of content) 85.17±7.35 114.94±1.43*** 83.18±3.10 132.77±2.35*** • results are expressed as mean ± sem for six animals in each group. ***: significant difference from the related sham group at p<0.001, **: significant difference from the related sham group at p<0.01, •••: significant difference from clpheart group at p<0.001, •: significant difference from clp-heart group at p<0.05. 245 original gauge-18 is related to organ inflammationtina didari sh am -h ea rt cl phe ar t sh am -l un g cl plu ng 0 200 400 600 800 1000 24 hrs. pg /m g pr ot ei n *** *** tnf-� � �� sh am -h ea rt cl phe ar t sh am -l un g cl plu ng 0 10 20 30 40 50 24 hrs. ng /m l *** *** hmgb1 � �� fig. 1. assessment of proinflammatory cytokines level in heart and lung injury, one day after clp with gauge 18. tumor necrosis factor-α (tnf-α), b: high mobility group box 1 (hmgb1). results are expressed as mean ± sem for six animals in each group. ***: significant difference from the related sham group at p<0.001, •••: significant difference from clp-heart group at p<0.001. sh am -h ea rt cl phe ar t sh am -l un g cl plu ng 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 24 hrs. r el at iv e fo ld c ha ng e ampk (prkaa1 gene) * � �� *** fig. 2. the m-rna gene expression level of prkaa1 (ampk related gene) and map1lc3b (lc3iib related gene) using rt-qpcr. results are expressed as mean ± sem for six animals in each group. samples were analyzed in triplicate. rt-qpcr (real-time reverse transcription-polymerase chain reaction); prkaa1 (protein kinase amp-activated catalytic subunit alpha 1); map1lc3b (microtubule associated protein 1 light chain 3 beta). ***: significant difference from the related sham group at p<0.001, *: significant difference from the relatedsham group at p<0.05, •••: significant difference from clp-heart group at p<0.001, •: significant difference from clp-heart group at p<0.05. sh am -h ea rt cl phe ar t sh am -l un g cl plu ng 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 24 hrs. r el at iv e fo ld c ha ng e lc3iib (map1lc3b gene) *** *** � 246 original gauge-18 is related to organ inflammation tina didari j contemp med sci | vol. 6, no. 5, september-october 2020: 242–248 injury such as hyperplasia of pneumocyte-type a, peribronchiolar inflammation, and hyperemia compared to the sham group. the thickness of the alveolar septa significantly increased in comparison to the normal area. however, lung inflammation was verified by the presence of inflammatory cells (lymphocyte) and edema. moreover, the histopathological evaluation of the heart sample in the clp group showed myocardial cell necrosis (fig. 3). discussion we designed the current study to illuminate differences in cardiac and pulmonary tissue damages following the clp g-18 procedure in rats 24 hours after the procedure. to our best knowledge, this is the first study which aimed to investigate the differences between pulmonary and cardiac dysregulation with g-18 clp. this was carried out through assessment of cellular and molecular sepsis mechanisms such as oxidative stress, expression of homeostatic–autophagic related genes, proinflammatory cytokines, apoptosis and tissue perfusion in male wistar rats. the findings of our study demonstrated that 24 hours after the clp procedure, inflammatory markers, tissue lactate levels, proinflammatory cytokines, caspases, and gene expression of cellular homeostasis and autophagy in samples were more pronouncedly increased in the lung tissues. moreover, the increase of the aforementioned factors was more highly elevated in the pulmonary tissue in with the heart specimen of the clp group. this should be taken into consideration for the development and execution of related protocols to increase the accuracy of the results and avoid waste of resources. oxidative stress is a major indicator of sepsis induced by the clp procedure. oxidative stress pathways and ros generation are consisted of several components such as increased lpo levels, alternated metabolic gene expression, and increased mpo levels during the procedure.17, 18 in this regard, many consider mpo as a major indicator of the neutrophil infiltration process.18-23 it is demonstrated that sepsis-induced by g-18 can elevate ros levels in the liver, colon, and kidney 16–24 hours following the procedure.24-27 congruently, the findings of our study demonstrated that 24 hours after the clp procedure, pulmonary tissue was impaired more intensely than cardiac tissue in clp-g18. lactate level is generally considered as a reliable indicator of tissue hypoperfusion, hypoxia, and altered microcirculation table 2. the abbreviations and primers of proteins used for real time rt-qpcr. gene name gene symbol primer sequence (5´-3´) glyceraldehyde3-phosphate dehydrogenase gapdh f: agtctactggcgtcttcacc r: ccacgatgccaaagttgtca amp-activated catalytic subunit alpha 1 (ampk) prkaa1 f : ccgtcttatagttcaaccat r : ttcgttcattattctcctgtt microtubuleassociated protein 1 light chain 3 beta (lc3iib) map1lc3b f : cagtgattatagagcgataca r : gccttctaattatcttgatgag fig. 3. the histopathology of the lung and heart in sham and clp groups. arrowheads: hyperplasia of pneumocyte type a, thick arrow: perivascular inflammation and infiltration of inflammatory cells, thin arrows: myocardial necrosis. 200 x (scale bar=100μm), 400 x (scale bar=20μm). 247 original gauge-18 is related to organ inflammationtina didari j contemp med sci | vol. 6, no. 5, september-october 2020: 242–248 in clp models of sepsis. previous studies have separately reported raised lactate levels following clp-g18 in blood, ileum, and liver, and noted the elevation of the lactate levels in the colon following clp procedure with g22 needles.18, 20, 28-31 in this regard, we observed that tissue lactate levels increased more intensely following clp-g18 procedure in lung cells compared to heart tissue. regarding proinflammatory cytokines, it is demonstrated that the blood levels of these indicators of inflammation, particularly tnf-α and hmgb1, is altered during the clp procedure.32-35 previous reports have demonstrated high levels of tnf-α in different organs of rats up to 72 hours after the clp-g18 procedure.23, 24, 31, 36 in this regard, we observed that in comparison with the cardiopulmonary system, the levels of both aforementioned cytokines were more intensely increased in pulmonary tissue compare to cardiac samples 24 hours after clp-g18. mitochondrial membrane disturbance during ros elevation results in activation caspases 9 and 3 with the natural consequence of cellular apoptosis.31 this programmed cell death process during the clp procedure is observed and reported in different tissues such as the kidney and brain.37, 38 correspondingly, findings of our study revealed that the levels of the aforementioned caspases increased in the lung more significantly in comparision of the heart in clp-g18. a combination of ros elevation and enhanced autophagy is responsible for the elimination of abnormal proteins and the promotion of organ failure-related apoptosis during sepsis.39-43 several studies have demonstrated that the lc3-ii/lc3-i ratio is increased in the liver, kidney, spleen, and mesenteric nodes after the clp procedure.44-47 moreover, hsaio et al showed an increase in lc3-ii levels in the renal tissues of rat’s 3 hours after clp-g18. correspondingly, escobar concluded that 8 hours after the clp-g22 procedure, lc3-ii was increased in parallel with phosphorylated ampk in the kidney and liver.39 in this regard, we observed that the gene expression levels of mrna of lc3-iib and ampk after clpg18 was more pronouncedly increased in the lung of the animals in comparison with the cardiac cells. moreover, histopathological changes we observed confirmed the superiority of pulmonary impairment compared to cardiac cells in clp-g18 in terms of provoking inflammation through demonstration of edematous and thickened lung tissues and myocardial cell necrosis. these observations support the hypothesis that in comparison with lung and heart organs 24 hours after clp, pulmonary samples provoke oxidative stress reactions and cellular damage more pronouncedly compared to cardiac cells. conclusion in conclusion, findings of this study demonstrated that lung is superior to heart in terms of demonstration of the severity of sepsis induced in a rat model with clp-g18, and both the rise of inflammatory markers and the extent of the ensuing organ damage is greater in the pulmonary cells undergone the procedure compared to cardiac cells with clp g-18. it can be concluded that the choice of the tissue can have a great influence on the research outcomes, and this should be considered in the design and implementation of clp studies so that researchers can cause the precise level of sepsis they intend to induce without waste of resources. acknowledgments this study was in part supported by a grant from tums coded 96-01-45-34820. conflict of interest the authors declare that there is no known conflict of interest regarding this publication. references 1. drosatos k, lymperopoulos a, kennel pj, pollak n, schulze pc, goldberg ij. pathophysiology of sepsis-related cardiac dysfunction: driven by inflammation, energy mismanagement, or both? curr heart failure reprts. 2015;12(2):130-40. 2. seymour cw, gesten f, prescott hc, friedrich me, iwashyna tj, phillips gs, et al. time to treatment and mortality during mandated emergency care for sepsis. new engl j med. 2017;376(23):2235-44. 3. sun y, cai y, zang qs. cardiac autophagy in sepsis. cells. 2019;8(2). 4. gonzalez ma, ochoa cd. multiorgan system failure in sepsis. sepsis: springer; 2018. p. 67-71. 5. park i, kim m, choe k, song e, seo h, hwang y, et al. neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury. eur resp j. 2019;53(3). 6. rittirsch d, huber-lang ms, flierl ma, ward pa. immunodesign of experimental sepsis by cecal ligation and puncture. nat protoc. 2008;4(1):31. 7. dejager l, pinheiro i, dejonckheere e, libert c. cecal ligation and puncture: the gold standard model for polymicrobial sepsis? trends microbiol. 2011;19(4):198-208. 8. li l, tan j, miao y, lei p, zhang q. ros and autophagy: interactions and molecular regulatory mechanisms. cell mol neurobiol. 2015;35(5):615-21. 9. aoki h, kondo y, aldape k, yamamoto a, iwado e, yokoyama t, et al. monitoring autophagy in glioblastoma with antibody against isoform b of human microtubule-associated protein 1 light chain 3. autophagy. 2008;4(4):46775. 10. faubert b, boily g, izreig s, griss t, samborska b, dong z, et al. ampk is a negative regulator of the warburg effect and suppresses tumor growth in vivo. cell metab. 2013;17(1):113-24. 11. wichterman ka, baue ae, chaudry ih. sepsis and septic shock--a review of laboratory models and a proposal. j surg res. 1980;29(2):189-201. 12. ebong s, call d, nemzek j, bolgos g, newcomb d, remick d. immunopathologic alterations in murine models of sepsis of increasing severity. infect immun. 1999;67(12):6603-10. 13. zapelini ph, rezin gt, cardoso mr, ritter c, klamt f, moreira jc, et al. antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model. mitochondrion. 2008;8(3):211-8. 14. krawisz je, sharon p, stenson wf. quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. assessment of inflammation in rat and hamster models. gastroenterology. 1984;87(6):1344-50. 15. rahimifard m, navaei-nigjeh m, baeeri m, maqbool f, abdollahi m. multiple protective mechanisms of alpha-lipoic acid in oxidation, apoptosis and inflammation against hydrogen peroxide induced toxicity in human lymphocytes. mol cell biochem. 2015;403(1-2):179-86. 16. baeeri m, momtaz s, navaei-nigjeh m, niaz k, rahimifard m, ghasemi-niri sf, et al. molecular evidence on the protective effect of ellagic acid on phosalone-induced senescence in rat embryonic fibroblast cells. food chem toxicol. 2017;100:8-23. 17. husain-syed f, mccullough pa, birk hw, renker m, brocca a, seeger w, et al. cardio-pulmonary-renal interactions: a multidisciplinary approach. j am coll cardiol. 2015;65(22):2433-48. 18. goto m, samonte v, ravindranath t, sayeed mm, gamelli rl. burn injury exacerbates hemodynamic and metabolic responses in rats with polymicrobial sepsis. j burn care res off publ am burn assoc. 2006;27(1):509. 19. liu h, wu j, yao jy, wang h, li st. the role of oxidative stress in decreased acetylcholinesterase activity at the neuromuscular junction of the diaphragm during sepsis. oxidat med cell longevity. 2017;2017:1-6. 20. sener g, sehirli o, cetinel s, ercan f, yuksel m, gedik n, et al. amelioration of sepsis-induced hepatic and ileal injury in rats by the leukotriene receptor blocker montelukast. prostagl leukotr essent fatty acids. 2005;73(6):453-62. 248 original gauge-18 is related to organ inflammation tina didari j contemp med sci | vol. 6, no. 5, september-october 2020: 242–248 21. sener g, toklu h, ercan f, erkanli g. protective effect of beta-glucan against oxidative organ injury in a rat model of sepsis. int immunopharmacol. 2005;5(9):1387-96. 22. sener g, toklu h, kapucu c, ercan f, erkanli g, kacmaz a, et al. melatonin protects against oxidative organ injury in a rat model of sepsis. surg today. 2005;35(1):52-9. 23. xiao x, yang m, sun d, sun s. curcumin protects against sepsis-induced acute lung injury in rats. j surg res. 2012;176(1):e31-9. 24. chen hh, lin kc, wallace cg, chen yt, yang cc, leu s, et al. additional benefit of combined therapy with melatonin and apoptotic adiposederived mesenchymal stem cell against sepsis-induced kidney injury. j pineal res. 2014;57(1):16-32. 25. zhu w, lu q, wan l, feng j, chen hw. sodium tanshinone ii a sulfonate ameliorates microcirculatory disturbance of small intestine by attenuating the production of reactie oxygen species in rats with sepsis. chin j integr med. 2016;22(10):745-51. 26. gonzalez as, elguero me, finocchietto p, holod s, romorini l, miriuka sg, et al. abnormal mitochondrial fusion-fission balance contributes to the progression of experimental sepsis. free radic res. 2014;48(7):769-83. 27. wang p, huang j, li y, chang r, wu h, lin j, et al. exogenous carbon monoxide decreases sepsis-induced acute kidney injury and inhibits nlrp3 inflammasome activation in rats. int j mol sci. 2015;16(9):20595608. 28. liaw wj, chen th, lai zz, chen sj, chen a, tzao c, et al. effects of a membrane-permeable radical scavenger, tempol, on intraperitoneal sepsisinduced organ injury in rats. shock. 2005;23(1):88-96. 29. tsao cm, jhang jg, chen sj, ka sm, wu tc, liaw wj, et al. adjuvant potential of selegiline in attenuating organ dysfunction in septic rats with peritonitis. plos one. 2014;9(9):1-9. 30. yang s, zhou m, chaudry ih, wang p. novel approach to prevent the transition from the hyperdynamic phase to the hypodynamic phase of sepsis: role of adrenomedullin and adrenomedullin binding protein-1. ann surg. 2002;236(5):625-33. 31. zhai x, yang z, zheng g, yu t, wang p, liu x, et al. lactate as a potential biomarker of sepsis in a rat cecal ligation and puncture model. mediators inflamm. 2018;2018:1-9. 32. abdulmahdi w, patel d, rabadi mm, azar t, jules e, lipphardt m, et al. hmgb1 redox during sepsis. redox biol. 2017;13:600-7. 33. léger t, charrier a, moreau c, hininger-favier i, mourmoura e, rigaudière jp, et al. early sepsis does not stimulate reactive oxygen species production and does not reduce cardiac function despite an increased inflammation status. physiol rep. 2017;5(13):1-12. 34. stevens ne, chapman mj, fraser ck, kuchel tr, hayball jd, diener kr. therapeutic targeting of hmgb1 during experimental sepsis modulates the inflammatory cytokine profile to one associated with improved clinical outcomes. sci rep. 2017;7(1):1-14. 35. lin wj, yeh wc. implication of toll-like receptor and tumor necrosis factor alpha signaling in septic shock. shock. 2005;24(3):206-9. 36. chang cl, leu s, sung hc, zhen yy, cho cl, chen a, et al. impact of apoptotic adipose-derived mesenchymal stem cells on attenuating organ damage and reducing mortality in rat sepsis syndrome induced by cecal puncture and ligation. j transl med. 2012;10:1-14. 37. olguner cg, koca u, altekin e, ergur bu, duru s, girgin p, et al. ischemic preconditioning attenuates lipid peroxidation and apoptosis in the cecal ligation and puncture model of sepsis. exp therap med. 2013;5(6):1581-8. 38. zhou j, chen y, huang gq, li j, wu gm, liu l, et al. hydrogen-rich saline reverses oxidative stress, cognitive impairment, and mortality in rats submitted to sepsis by cecal ligation and puncture. j surg res. 2012;178(1):390-400. 39. escobar da, botero-quintero am, kautza bc, luciano j, loughran p, darwiche s, et al. adenosine monophosphate-activated protein kinase activation protects against sepsis-induced organ injury and inflammation. j surg res. 2015;194(1):262-72. 40. nishida k, kyoi s, yamaguchi o, sadoshima j, otsu k. the role of autophagy in the heart. cell death different. 2009;16(1):31-8. 41. zaha vg, young lh. amp-activated protein kinase regulation and biological actions in the heart. circul res. 2012;111(6):800-14. 42. mizumura k, cloonan s, choi me, hashimoto s, nakahira k, ryter sw, et al. autophagy: friend or foe in lung disease? ann am thoracic soc. 2016;13(supplement 1):s40-s7. 43. mungai pt, waypa gb, jairaman a, prakriya m, dokic d, ball mk, et al. hypoxia triggers ampk activation through reactive oxygen speciesmediated activation of calcium release-activated calcium channels. mol cell biol. 2011;31(17):3531-45. 44. hsieh ch, pai py, hsueh hw, yuan ss, hsieh yc. complete induction of autophagy is essential for cardioprotection in sepsis. ann surg. 2011;253(6):1190-200. 45. lee s, lee sj, coronata aa, fredenburgh le, chung sw, perrella ma, et al. carbon monoxide confers protection in sepsis by enhancing beclin 1-dependent autophagy and phagocytosis. antioxidants redox signal. 2014;20(3):432-42. 46. watanabe e, muenzer jt, hawkins wg, davis cg, dixon dj, mcdunn je, et al. sepsis induces extensive autophagic vacuolization in hepatocytes: a clinical and laboratory-based study. lab investig j techn methods pathol. 2009;89(5):549-61. 47. takahashi w, hatano h, hirasawa h, oda s. protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model. critical care. 2013;17(2):1-13. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.864 120 j contemp med sci | vol. 6, no. 3, may–june 2020: 120–125 original issn 2413-0516 introduction type 2 diabetes is related to considerable morbidity and mortality all over the world. interactions between factors of genetics and environment, which include diet, have an important impact on the development of this condition.1-3 over the last decade, accumulating evidence has indicated that the concentration of 25-hydroxy vitamin d3 ([25(oh)d3]) in the blood is inversely proportional with diabetes mellitus, hypertension and some other diseases.4 most of the required cholecalciferol or vitamin d3 is derived from the biosynthetic pathway under the skin from 7dehydrocholesterol through exposure to sunlight.5 most experts recommend an optimal concentration greater than 30 ng/ml in serum and vitamin d3 deficiency is less than 30 ng/ml. vitamin d3 deficiency is even more prevalent among patients with type 2 diabetes mellitus (t2dm), endothelial dysfunction predicts cardiovascular events and represents an essential event for vascular abnormalities observed in t2dm patients.6 vitamin d3 [25(oh) d3] has numerous biological actions, like calcium homeostasis and metabolism, stimulation of the innate immune response, maintaining of blood glucose level, and insulin secretion stimulation in order to therapeutically use it in diabetics.7 there is a relation between diabetes mellitus and vitamin d3 deficiency (vdd) as well as a relation with cancer, dyslipidemia, endothelial dysfunction, and hypertension.8 it was observed that the estimation of 25(oh)d3 insufficiency and deficiency is experienced by 1 billion people and its impact on abnormal metabolism of glucose as well as in diabetes type 2 was exhibited.9 inadequate concentrations related to the circulating 25(oh)d3 are related to metabolic syndrome, insulin resistance (ir), glucose intolerance, and increased diabetes risk10 due to the fact that there are many indications related to the vdd may possibly have contributing impact on developing type 2 diabetes. initially, the pancreatic beta cells has enzyme of 1 α-hydroxylase and vitamin d receptor (vdr) and the other indication implies that treating with vitamin d improves ir and glucose tolerance.11 also, it has been shown that vitamin d moderates the glycolytic pathway, stimulates the general activation regarding the protein synthesis in pancreatic β-cells, improves ca+2 influx to β-cells in addition to promoting converting pro-insulin to insulin.12 the vdd is considered to be related to decreased release of insulin, ir, and type 2 diabetes, with regard to epidemiological and experimental researches, for example, many studies of animals showed that 1α,25–dihydroxy vitamin d3 (1,25(oh)2d3) promote pancreatic β-cell in secreting insulin. the association between ir and vdd might begin from inflammation, as vdd is related to increased inflammation markers. moreover, genetic polymorphisms of genes that are related to vitamin d could impact impaired glycemic control and diabetes of type 2.13 a direct advantageous influence related to vitamin d on insulin action was approved by an insulin receptors increment after 24 h of treatment through the use of 1,25(oh)2d3 in u-937 cells of human promonocytic.1 in addition, gathering evidence from experimental researches have suggested that activation of vdr could increase the secretion of insulin and improve insulin sensitivity, so vdd was linked to diabetes mellitus onset and progression.14 many observational studies confirmed an association of the deficiency of 25(oh) d3 with diabetes of type 2 and disturbed glucose metabolism. on the other hand, was considerably unsuccessful in showing good [25(oh)d3] impacts on metabolism of glucose and therefore, it is still not clear whether or not 25(oh) d3 is relevant for the pathogenesis of diabetes of type 2, so this study will clarify association between hypovitaminosis d3 and type ii diabetes mellitus of iraqi patients shaymaa m. hadi1, fadhil j. al-tu’ma2, riyadh d. al-zubaidi3 1department of pharmaceutical chemistry, college of pharmacy, university of kerbala, kerbala, iraq. 2department of chemistry and biochemistry, college of medicine, university of kerbala, kerbala, iraq. 3department of internal medicine, college of medicine, university of kerbala, kerbala, iraq. correspondence to: shaymaa m. hadi (shaymaa.hadi88@gmail.com) (submitted: 18 april 2020 – revised version received: 07 may 2020 – accepted: 14 june 2020 – published online: 26 june 2020) objective this research has been carried out to evaluate the levels of 25-hydroxy vitamin d3 ([25(oh)d3]) as well as the correlation between the deficiency levels with the risk of experiencing type 2 diabetic mellitus (t2dm) in sample of iraqi population. materials and methods the levels regarding the [25(oh)d3] and the fasting blood sugar (fbg) have been estimated in group of 58 t2dm patients and 31 control subjects of age between 25 and 65 years. the [25(oh)d3] has been estimated via elisa and fbg was measured spectrophotometrically. results the levels of [25(oh)d3] have been considerably lower in individuals experiencing t2dm (9.465 ± 3.567 ng/ml) than in the control group (14.146 ± 11.045 ng/ml), (p = 0.02), whereas fbg levels were considerably higher in patients experiencing t2dm (218 ± 66 mg/dl) widespread in comparison to control group (89.8 ± 9 mg/dl), (p = 0.009). the levels related to the [25(oh)d3] are inversely related to fbg in both diabetic and control group (p = 0.01, r2 = 0.1), (p < 0.01, r2 = 0.2), respectively. vitamin d3 deficiency (vdd) is considered to be related to the hazard of experiencing t2dm; also it is highly prevalent in subjects of iraqi nationality. conclusion vdd is associated to the risk of experiencing t2dm. hypovitaminosis d is very prevalent among the study participants and its percent was higher in female and have a therapeutic implications as cautious supplementation of vitamin d could enhance glycemic control in t2dm. keywords hypovitaminosis d, 25-hydroxyvitamin d3, 25(oh)d3, type ii diabetes mellitus, t2dm, 25-hydroxy vitamin d3. 121 original association between hypovitaminosis d3 and type ii diabetes mellitus of iraqi patientsshaymaa m. hadi et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 120–125 the existence of an association between t2dm and vdd as well as estimating the levels of 25(oh) d3 among the study participants. materials and methods eighty-nine iraqi individuals (58 t2dm cases and 31 controls) of age ranged between 25 and 65 years (male and female) have taken part in this case control study. patients that have type 2 diabetes mellitus disease have been appointed from al-hussein teaching hospital, al-hussein medical city/ kerbala health directorates between april 2018 and march 2019 and diagnosed according to criteria of american diabetes association (2014) defining diabetes as (fbg ≥126 mg/dl). healthy subjects that didn’t match the standards which are used to select t2dm were assigned as control. the levels related to fbg and 25(oh)d3 have been estimated for all the participants in this research. the levels of [25(oh) d3] have been estimated via enzyme-linked immunosorbent assay (elisa), fbg was measured spectrophotometrically. demographic information, medical history and past family were recorded to every participating subject. those with comorbidities have been excluded from the study, which has been permitted via the clinical research ethics committee of the college of medicine, university of kerbala. the data were presented either by mean ± standard deviation (mean ± sd) or ratio or percent. the t-test of the student has been utilized for comparing the difference between the groups of patients and controls from numerical variables. analysis of regression has been carried out for analyzing the correlation between levels of fbg and 25(oh) d3. a chi-squared test has been carried out for comparing the number of individuals that have insufficiency and vdd in control and patients groups. in all statistical analysis, the significance value was <0.05. results levels of fbg have been higher in patients group having t2dm in comparison to control group, levels of fbg are (218 ± 66 mg/dl) and (89.8 ± 9 mg/dl) in the group of patients and group of controls, respectively (p <0.01). in patients’ group that have t2dm, the levels of 25(oh) d3 have been lower compared to the levels in the group of controls, the levels of 25(oh) d3 were 9.465 ± 3.567 ng/ml and 14.146 ± 11.045 ng/ ml in the group of patients and the group of controls, respectively (p=0.02) as seen in (table 1). general observation to table 2a showed that patient had greater percent of vdd in comparison with control group (100% vs 87%), respectively. while the remaining of control group 9,68% had insufficiency of vitamin d (vdi) and only 3.23% had optimum level of vitamin d (vo). with regard to the patient group, 0% of vi and vo is recorded. generally, 95.5%, of all population whose serum 25(oh) d3 was measured experienced vdd, 3.4% experienced vi and 1.1% experienced vo level. also, it has been shown that female had larger percent of vdd (51.7%, 48.4%) than male (48.3 %, 38.7%) in both patient and control group respectively. because most study subjects experienced vdd, they have been subclassified into mild, moderate, and severe, and it is found that patient group had a greater percent of severe and moderate vdd (13.8%, 44.8%), respectively in comparison with the control group which had only 3.7%, 25.9% in an order. for mild vdd, patients also had large percent (41.38%), but it is not the greater one because the control group had it 70% as seen in table 2b. after performing the analysis in the whole population of the study, patients with t2dm and controls, 25(oh)d3 levels were discovered to have inverse association with fbg levels in the patients’ group that had type 2 diabetes (p = 0.01, r = 0.1, analysis of linear regression) (fig. 1) and in control group (p <0.01, r2 = 0.22) (fig. 2). table 1. biochemical and clinical characteristics related to the research subjects. parameters controls mean ± sd n = 31 t2dm subjects mean ± sd n = 58 p-value no. (m/f) 31 (15/16) 58 (28/30) 0.9 age, year 42.6 ± 1.9 46.13 ±1.3 0.1 fbg (mg/dl) 89.5 ± 1.8 229.7 ± 9.8 < 0.01 1,25(oh)d3 (ng/ml) 14.14 ± 1.1 9.46 ± 0.46 0.02” table 2. comparison of serum vitamin d status among the research subjects. case control total vitamin d status n = 89 male frequency (%) female frequency (%) total frequency (%) male frequency (%) female frequency (%) total frequency (%) frequency % vitamin d deficiency (vdd) 28 (48.3) 30 (51.7) 58 (100) 12 (38.7) 15 (48.4) 27 (87.1) 85 95.5 vitamin d insufficiency (vis) 2 (6.45) 1 (3.225) 3 (9.675) 3 3.4 vitamin d optimum, (vo) 1 (3.225) 1 (3.225) 1 1.1 total 58 31 89 p=0.019 *serum vitamin d status based on us endocrine society classification (22). insufficiency 21-29 ng/ml, deficiency ≤ 20 ng/ml, sufficiency ≥30 ng/ml. 122 original association between hypovitaminosis d3 and type ii diabetes mellitus of iraqi patients shaymaa m. hadi et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 120–125 discussion several studies associated between the deficiency of 25-(oh) d3 and various clinical conditions of iraqi patients have been studied including hyperthyroidism, unstable angina, coronary artery diseases, and t2dm.8,15,16 the main goal of the presented research has been investigating the relation between t2dm and vdd. in this research, lower 1,25(oh)2d3 levels have been noticed in patient that have t2dm compared to a control group and an inverse correlation has been noticed between levels of fbg and 1,25(oh)2d3 in t2dm group, which implies that the 1,25(oh)2d3 levels have an impact on glucose control in t2dm. in addition to that, an inverse correlation has been discovered between fbg and vitamin d levels in the entire study population, patients who have t2dm and controls when they have been examined together. it has appeared that vitamin d could be related with glucose control in t2dm. moreover, from a statistically significant perspective, more subjects experiencing t2dm had vitamin d insufficiency and deficiency in comparison to control population. vitamin d, which is secosteroid synthesized in skin via the action of uv sun irradiation, is associated with bone metabolism. extra skeletal impacts related to vitamin d are presently a topic of interest for researchers.17 the association of immune system and vitamin d is under an intense discussion. it has been proved that vitamin d stimulates the immune tolerance, vdd is related with certain autoimmune diseases, like multiple sclerosis, rheumatoid arthritis, and t1dm.18-21 the correlation between t2dm and vitamin d and metabolic syndrome is recently under a debate. vitamin d has exhibited a relation with glucose metabolism and the progression of t2dm and the metabolic syndrome.22,23 in a cross-sectional study to sample of population from eastern finland, an inverse correlation has been noticed between levels of [25(oh)d3] and fbg, fasting insulin, and 2 h glucose tolerance tests,24 which have implied that low level of serum [25(oh)d3] could have an association with reduced metabolism of glucose. in one of the recent studies, an inverse correlation of ir with the levels of [25(oh)d3] has been noticed that has been mainly found at levels of [25(oh)d3] in the range of 16– 36 ng/ml.25 in one of the nested case–control studies that have been carried out amongst 608 females with recently identified diabetes type 2, higher concentration of plasma [25(oh)d3] was related to a lower risk of diabetes table 2b. subclassification and comparison of vdd amongst study participants. case control total vitamin d deficient, (vdd) n = 85 frequency (%) frequency (%) frequency (%) mild 24 (41.38) 19 (70.38) 43 50.6 moderate 26 (44.82) 7 (25.92) 33 38.8 sever 8 (13.8) 1 (3.7) 9 10.6 total 58 27 85 100 *a deficiency in vitamin was sub classified to mild vdd (10-20 ng/ml), moderate vdd (5-10 ng/ml), severe (<5 ng/ml) (23). fig. 1 inverse correlation between fbg (mg/dl) and 25(oh)d3 (ng/ml) in subjects experiencing t2dm, (p = 0.01, r2 = 0.1, analysis of linear regression). 25(oh)d3; fbg. 123 original association between hypovitaminosis d3 and type ii diabetes mellitus of iraqi patientsshaymaa m. hadi et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 120–125 type 2 in females.26 in a potential observational research with an average follow up of 2.7 years, higher plasma [25(oh)d3] has been related to lower risks of occurrence of diabetes in high-risk subjects.27 in a potential research in high-risk asian individuals, deficiency of [25(oh)d3] has been related with higher risks of developing diabetes mellitus of type 2. in a longitudinal research of ir determinants and metabolic syndrome, a considerable inverse correlation of baseline [25(oh) d3] with fasting glucose at follow-up has been recorded. in a massive group research of older individuals that involved 7791 individuals, that were at first diabetes-free, levels of serum [25(oh)d3] have been inversely related to incident diabetes in females but not in males.28 earlier researches indicated that low vitamin d ingestion might be associated to higher hazard of developing the metabolic syndrome and t2dm.29,30 in one of the earlier studies, vdd has been discovered to be associated with higher hazard for the metabolic syndrome and insulin resistance.31 in a research that involved postmenopausal females, the levels of fasting glucose were observed to be negatively related to serum [25(oh)d3].32 one of the new studies indicated that vitamin d levels could have inverse association to the levels of glycosylated hemoglobin in gestational diabetes mellitus.33 moreover, it was proposed that sufficient vitamin d intake could be related with lower risks of developing gestational diabetes mellitus.34 in this research, lower levels of 1,25(oh)2d3 were recorded in individuals who had t2dm compared to controls, which can be attributed the consideration that the receptors of vitamin d were discovered in pancreatic beta cells, which, in addition were discovered for expressing the enzyme 1-α-hydroxylase.35 vitamin d facilitates insulin secretion from pancreatic β-cells, which is why it appears to control the insulin secretion.36 therefore, vdd could be related with reduced insulin secretion in t2dm in addition to that, as vitamin d promotes insulin receptor expression,37 vdd could be associated with ir.38 according to those results it would be physiologically suitable recommending supplementation of vitamin d for the sake of improving glucose control in patients with t2dm.39 in accordance with that, vitamin d was administered to t2dm patients.40 nevertheless, those researches didn’t show steady results. in some of the researches supplementations of vitamin d were discovered to enhance glucose regulation in t2dm,39 while in others no such effect was observed.41 in a randomized controlled trial, administrating 2000 international units (iu) cholecalciferol each day for a period of 16 weeks has been discovered to enhance the function of beta cells in adults at high diabetes risks.42 moreover, vitamin d was administered to diabetics of type 2 with nephropathy and has been discovered to ameliorate albuminuria.43 at a molecular level, vitamin d seems to have an impact on reducing oxidative stress.44 this research has a number of obstacles. due to the fact that it’s an observational research, it means that conclusion cannot be made whenever any cause and effect association is concerned between t2dm and vdd. moreover, 1,25(oh)2d3 has been selected as a marker for vdd, as presently suggested. nevertheless, vitamin d circulates in the blood in different forms and the active form of this vitamin is 1,25(oh)2d3. therefore, a larger number of researches are required utilizing finer tools for determining the vdd in humans and particularly, in diabetes patients. a larger number of researches are as well required with supplementation of vitamin d and glucose control long-term observation in t2dm. after discussing those results, the hidden correlation between vdd and a variety of diseases is still to be discovered.45 vitamin d could be associated with autoimmunity in addition to metabolic diseases.46 moreover, recent research was set forward, it is concerned with discussing the correlation between vitamin d and adipose tissue. it seems that vitamin d could influence adipogenesis,47,48 thereby it modulates energy consumption in adipose tissue. those innovative discoveries could give an explanation to the reason why vitamin d administration to diabetes mellitus patients and the metabolic syndrome patients seems to have contradictory effects. the results that have been presented in this study are of therapeutic implications. in t2dm patients, normal vitamin d levels in the blood could facilitate regulation of glucose. moreover, in individuals that tend to develop t2dm, the optimum vitamin d levels in the blood could delay the clinical development of t2dm. fig. 2 inverse correlation between fbg (mg/dl) and 25(oh)d3 (ng/ml) in controls (p < 0.01, r2 = 0.229, analysis of linear regression). 25(oh)d3; fbg. 124 original association between hypovitaminosis d3 and type ii diabetes mellitus of iraqi patients shaymaa m. hadi et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 120–125 conclusions vdd is associated to the risk of experiencing t2dm. hypovitaminosis d is very prevalent among the study participants and its percent was higher in female and patients than this in male and controls, respectively. those results could have therapeutic implications as cautious supplementation of vitamin d could enhance glycemic control in t2dm. references 1. hadi, sm, al-tu’ma, fj, and al-zubaidi, rd. genotyping of vitamin d receptor foki polymorphism as a predictor for type 2 diabetes mellitus by a tetra primer-arms-pcr assay. gene rep. (2019):15:100365. 2. zisser h, gong p, kelley cm, seidman js, riddell mc. exercise and diabetes. int j clin pract suppl. 2011;170:71–5.  3. prokopenko i, mccarthy mi, lindgren cm. type 2 diabetes: new genes, new understanding. trends genet. 2008;24:613–21. 4. pilz s, tomaschitz a, ritz e, pieber tr. vitamin d status and arterial hypertension: a systematic review. nat rev cardiol. 2009 oct;6(10): 621–30. 5. wang tj, pencina mj, booth sl, jacques pf, ingelsson e, lanier k, et al. vitamin d deficiency and risk of cardiovascular disease. circulation. 2008 jan 29;117(4):503–11. 6. targher g, bertolini l, padovani r, zenari l, scala l, cigolini m, et al. serum 25-hydroxyvitamin d3 concentrations and carotid artery intima-media thickness among type 2 diabetic patients. clin endocrinol (oxf ). 2006 nov;65(5):593–97. 7. demay mb, kiernan ms, deluca hf, kronenberg hm. sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxy vitamin d3 receptor and mediate transcriptional repression in response to 1,25-dihydroxy vitamin d3. proc natl acad sci usa. 1992;89:8097–101. 8. al-tu’ma, fj, and yosuf, lmz. vitamin d deficiency and hypertension in type 2 diabetic iraqi patients. j contemp med sci., 2015;1(1):17–20. 9. melamed, ml, michos, ed, post, w, and astor, b. 25-hydroxyvitamin d levels and the risk of mortality in the general population. arch intern med, 2008;168(15):1629-37. 10. palomer, x, gonzález‐clemente, jm, blanco‐vaca, f, and mauricio, d. role of vitamin d in the pathogenesis of type 2 diabetes mellitus. diab obesity metab, 2008;10(3):185-197. 11. martin, t, and campbell, rk. 2011. vitamin d and diabetes. diab spect, 2011;24(2):113-118. 12. reis af, hauache om, velho g. vitamin d endocrine system and the genetic susceptibility to diabetes, obesity and vascular disease. a review of evidence. diab metab. 2005;31:318–25.  13. lips, p, eekhoff, m, van schoor, n, oosterwerff, m, de jongh, r, krul-poel, y, and simsek, s. vitamin d and type 2 diabetes. j steroid biochem mol biol, 2016;173:280–285. 14. nakashima, a, yokoyama, k, yokoo, t, and urashima, m. role of vitamin d in diabetes mellitus and chronic kidney disease. world j diab, 2016;7(5): 89–100. 15. al-tu’ma, fj, mohammed, am, and al-saraf, hh. role of vitamin d3 and apo-b/apoa-1 ratio in patients with unstable angina in kerbala province: iraq. j contemp med sci, 2017;3(10):224–28. 16. rahi, saz, al-tu’ma, fj, al-ganabi, am and al-tu’ma, j.f. polymorphism of vitamin d receptor (re2228570) in sera of coronary artery diseases and biochemical parameters. al-qadisiyah med j 2018;14(25):1-8. 17. rosen, c, adams, j, bikle, d, black, d, demay, m, manson, j. et al. the nonskeletal effects of vitamin d: an endocrine society scientific statement. endocr rev, 2012;33:456–492. 18. weiss, s. bacterial components plus vitamin d: the ultimate solution to the asthma (autoimmune disease) epidemic? j allergy clin immunol, 2011;127:1128–1130. 19. weinstock-guttman, b, mehta, b, ramanathan, m, karmon, y, henson, l, halper, j. et al. vitamin d and multiple sclerosis. neurologist, 2012;18: 179–183. 20. haga, h., schmedes, a., naderi, y., moreno, a. and peen, e. severe deficiency of 25-hydroxyvitamin d(3) (25(oh)d3) is associated with high disease activity of rheumatoid arthritis. clin rheumatol, 2013;32: 629–633. 21. hyppönen, e., lärä, e., reunanen, a., järvelin, m. and virtanen, s. intake of vitamin d and risk of type 1 diabetes: a birth-cohort study. lancet, 2001;358:1500–1503. 22. maxwell, c. and wood, r. update on vitamin d and type 2 diabetes. nutr rev, 2011;69:291–295. 23. lim, s., kim, m., choi, s., shin, c., park, k., jang, h. et al. association of vdd with incidence of type 2 diabetes in high-risk asian subjects. am j clin nutr. 2013;97:524–530. 24. hurskainen, a, virtanen, j, tuomainen, t, nurmi, t, and voutilainen, s. association of serum 25-hydroxyvitamin d with type 2 diabetes and markers of insulin resistance in a general older population in finland. diab metab res rev 2012;28:418–423. 25. heaney, r, french, c, nguyen, s, ferreira, m, baggerly, l, brunel, l. et al. a novel approach localizes the association of vitamin d status with insulin resistance to one region of the 25-hydroxyvitamin d continuum. adv nutr, 2013;4:303–310. 26. pittas, a, sun, q, manson, j, dawson-hughes, b. and hu, f. plasma 25-hydroxyvitamin d concentration and risk of incident type 2 diabetes in women. diab care, 2010;33:2021–2023. 27. pittas, a, nelson, j, mitri, j, hillmann, w, garganta, c, nathan, d. et al. plasma 25-hydroxyvitamin d and progression to diabetes in patients at risk for diabetes: an ancillary analysis in the diabetes prevention program. diab care, 2012;35:565–573. 28. schöttker, b, herder, c, rothenbacher, d, perna, l, müller, h, and brenner, h. serum 25-hydroxyvitamin d levels and incident diabetes mellitus type 2: a competing risk analysis in a large population-based cohort of older adults. eur j epidemiol, 2013;28:267–275. 29. liu, s, song, y, ford, e, manson, j, buring, j, and ridker, p. dietary calcium, vitamin d, and the prevalence of metabolic syndrome in middle-aged and older u.s. women. diab care, 2005;28:2926–2932. 30. pittas, a, dawson-hughes, b, li, t, van dam, r, willett, w, manson, j. et al. vitamin d and calcium intake in relation to type 2 diabetes in women. diab care, 2006;29:650–656. 31. chiu, k, chu, a, go, v, and saad, m. hypovitaminosis d is associated with insulin resistance and beta cell dysfunction. am j clin nutr, 2004;79: 820–825. 32. need, a, o’loughlin, p, horowitz, m, and nordin, b. relationship between fasting serum glucose, age, body mass index and serum 25 hydroxyvitamin d in postmenopausal women. clin endocrinol (oxford), 2005;62: 738–741. 33. lau, s, gunton, j, athayde, n, byth, k, and cheung, n. serum 25-hydroxyvitamin d and glycated haemoglobin levels in women with gestational diabetes mellitus. med j aust, 2011;194:334–337. 34. alzaim, m, and wood, r. vitamin d and gestational diabetes mellitus. nutr rev, 2013; 71:158–167. 35. bland, r, markovic, d, hills, c, hughes, s, chan, s, squires, p. et al. expression of 25-hydroxyvitamin d3–1alpha-hydroxylase in pancreatic islets. j steroid biochem mol biol 2004;89–90:121–125. 36. zeitz, u, weber, k, soegiarto, d, wolf, e, balling, r, and erben, r. impaired insulin secretory capacity in mice lacking a functional vitamin d receptor. faseb j 2003;17:509–511. 37. maestro, b, campión, j, dávila, n, and calle, c. stimulation by 1,25-dihydroxyvitamin d3 of insulin receptor expression and insulin responsiveness for glucose transport in u-937 human promonocytic cells. endocr j 2000;47:383–391. 38. talaei, a, mohamadi, m, and adgi, z. the effect of vitamin d on insulin resistance in patients with type 2 diabetes. diabetol metab syndr, 2013; 5:8. 39. osei, k. 25-oh vitamin d: is it the universal panacea for metabolic syndrome and type 2 diabetes? j clin endocrinol metab, 2010;95:4220-22. 40. al-daghri, n.m., alkharfy, k.m., al-othman, a., el-kholie, e., moharram, o., alokail, m.s. et al. vitamin d supplementation as an adjuvant therapy for patients with t2dm: an 18-month prospective interventional study. cardiovasc diabetol, 2012;11:85. 41. heshmat, r, tabatabaei-malazy, o, abbaszadeh-ahranjani, s, shahbazi, s, khooshehchin, g, bandarian, f. et al. effect of vitamin d on insulin resistance and anthropometric parameters in type 2 diabetes: a randomized doubleblind clinical trial. daru 2012;20:10. 42. mitri, j, dawson-hughes, b, hu, f, and pittas, a. effects of vitamin d and calcium supplementation on pancreatic β cell function, insulin sensitivity, and glycemia in adults at high risk of diabetes: the calcium and vitamin d for diabetes mellitus (caddm) randomized controlled trial. am j clin nutr, 2011;94:486–494. 43. huang, y, yu, h, lu, j, guo, k, zhang, l, bao, y. et al. oral supplementation with cholecalciferol 800 iu ameliorates albuminuria in chinese type 2 diabetic patients with nephropathy. plos one, 2012;7:e50510. 44. salum, e, kals, j, kampus, p, salum, t, zilmer, k, aunapuu, m. et al. vitamin d reduces deposition of advanced glycation endproducts in the aortic 125 original association between hypovitaminosis d3 and type ii diabetes mellitus of iraqi patientsshaymaa m. hadi et al. j contemp med sci | vol. 6, no. 3, may–june 2020: 120–125 wall and systemic oxidative stress in diabetic rats. diab res clin pract , 2013;100:243–249. 45. chagas, c, borges, m, martini, l, and rogero, m. focus on vitamin d, inflammation and type 2 diabetes. nutrients 2012;4:52–67. 46. holick, m. evidence-based d-bate on health benefits of vitamin d revisited. dermatoendocrinology, 2012;4:183–190. 47. ochs-balcom, h, chennamaneni, r, millen, a, shields, p, marian, c, trevisan, m. et al. vitamin d receptor gene polymorphisms are associated with adiposity phenotypes. am j clin nutr, 2011;93:5–10. 48. landrier, j, marcotorchino, j, and tourniaire, f. lipophilic micronutrients and adipose tissue biology. nutrients, 2012;4:1622–1649. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.v6i3.790 213j contemp med sci | vol. 6, no. 5, september-october 2020: 213–217 original issn 2413-0516 introduction anthropology is a science study about characteristics of the human body skeleton.1 the central axis of anthropology is estimating race, gender, and the other human features from the body skeleton.2 anthropometric data are collected from the dead or alive samples.3 physical characteristics of the human skeleton depend on the race, age, sex, and lifestyle.4 the forearm is one of the important parts in the upper extremity and the hand portion is the most mobile part, and plays an important role in the movement of the upper limb.5 through measuring the length of some parts, like forearm and hand, estimating the body size and height can be occurred.6 we can use these indicators to determine gender, sex, and environmental conditions.7 moreover, for designing pre-fabricated upper limb prosthesis or designing the ergonomic instrument in different nations, having information about the length of the different parts of the body such as upper limb, can be useful.2therefore, this study was carried out for measurement of forearm and hand length in the students of hamadan university of medical sciences. materials and methods in this analytical–descriptive study, 160 students (age range: 18–22) were included and the samples were randomly selected. exclusion criteria were subjects who had a fracture in the forearm and hand bones. a measurement protocol to guide the project team colleagues were used.2 anatomical position and body landmarks recognition were taught to all members in the measurement team as a training program. through the indirect measurement methods, superficial forearm and hand size of 160 samples were evaluated. measured parameters included: stature, weight, forearm, and hand length (the apparent size of the forearm was measured from the tip of the olecranon to the point between the radius and ulnar styloid). the hand length measurement was done from the midpoint between the radius and ulnar styloid to the tip of the middle finger. plastic meters and scales were used for measurements. statistical analysis spss 22.0 software was used for statistical analysis. the association of parameters was assessed by pearson’s correlation coefficient r. regression equations were computed to examine the relationship between the stature and hand length, in addition to the stature and forearm length. the p values less than 0.05 were considered statistically significant. results in this study, 160 samples were examined. the mean of stature, weight, forearm, and hand length for all samples are mentioned in table 1. table 2 shows the comparison between males and females stature in addition to the other parameters. moreover, it demonstrates the significant differences between two genders in all parameters (p=0.000). statistically, significant correlation was observed among stature and other parameters in all cases (p=0.000) (table 3). our study demonstrated an obvious correlation and significant differences among the stature the stature estimation from students’ forearm and hand length in iran babak ebrahimi1, soheila madadi2, leila noori3,1, shadan navid4, melika darvishi5, tahereh alizamir6* 1. department of anatomy, school of medicine, tehran university of medical sciences, tehran, iran. 2. department of anatomy, faculty of medicine, arak university of medical sciences, arak, iran. 3. department of anatomical sciences, school of medicine, babol university of medical sciences. 4. department of anatomy, gonabad university of medical sciences, gonabad, iran. 5. department of radiology, paramedical faculty, hamadan university of medical sciences, hamadan, iran. 6. department of anatomy, school of medicine, hamadan university of medical sciences, hamadan, iran. corresponding author: tahereh alizamir (email: alizamirt@yahoo.com) (submitted: 08 july 2020 – revised version received: 21 august 2020 – accepted: 17 september 2020 – published online: 30 october 2020) abstract objectives: the purpose of this study was to determine the stature from student’s forearm and hand length in hamadan university of medical sciences. methods: for measurements, the forearm and hand length of 160 students were measured. the range of the sample ages were between 18 and 22 years, selected randomly. in this descriptive and analytical study, the cluster sampling method was used to select the subjects. for anthropometric measurements, we used metal and plastic tape, goniometer, caliper, and scale. the height and length of the forearm and hand were measured separately. results: the mean ± sd of the stature were 164.435±5.072 cm and 180.446±5.569 cm, in females and males, respectively. the mean ± sd of the forearm length were 24.906±1.347 cm and 27.751±1.294 cm, in females and males, respectively. the mean ± sd of hand length were 17.356±2.223 cm and 19.418±0.888 cm, in females and males respectively. besides, there was a correlation between height and forearm length of all cases. also, this correlation was seen for the stature and hand length. conclusion: according to our study, forearm and hand length have correlation with the stature, so they can be used as factors for stature estimation. keywords: anthropology, stature, forearm length, hand length, hamadan 214 original stature estimation from students’ forearm and hand length babak ebrahimi j contemp med sci | vol. 6, no. 5, september-october 2020: 213–217 and all parameters in males and females (p=0.000), except the comparison between the stature and hand length in females, which was not meaningful (p=0.102) (table 4). the analysis of the linear regression with the stature as the dependent variable and weight as independent variable showed for all samples. also, the regression model is meaningful in both males and females (p=0.000, p=0.016, p=0.007, respectively) (table 5). the analysis of the linear regression for study population with stature as the dependent variable and hand length as independent variable demonstrated that the regression model is meaningful in total samples and males (p=0.000) but it is not meaningful in females (p=0.102) (table 6). the analysis of the linear regression for study population with stature as the dependent variable and forearm length as independent variables exhibited that the regression model is meaningful in total samples, separately (p=0.000) (tables 7, 8). correlation between stature (cm) with weight (kg), hand length (cm) and forearm length (cm), in total samples, both in males and females are shown in figs 1, 2, and 3. discussion for the forensic experts, identification of the corpse is an important challenge, and estimation of the stature can be useful for this subject.8 in this study, we examined 160 healthy male and female participants. our findings indicated a higher height for males as compared with females which is in the line with ilayperuma et al. results.9 linear regression is considered as a suitable method for estimating the relationship between stature and body fragments.10 in our study, it was shown that there was a strong positive correlation between forearm length and stature, and a moderate positive correlation between the stature and hand length. (stature=66.268+4.033×forearm length, see= 0.230 and r2= 0.661) and hand length (stature= 122.327+2.725×hand length, see= 320 and r2= 0.314) was found for all cases based on linear regression equation. an anthropological study on the sample of a turkish adult population reported that the correlations between stature and hand length for both genders are statistically significant which is in the same line with our results.11 our statistical analysis showed a significant correlation among the forearm, hand length, and the examinee’s stature. the mean stature were 180.446±5.569 and 164.435±5.072 for males and females, respectively. the stature factor between the two sexes was statistically significant (p=0.000). we observed remarkable differences in forearm and hand length between male and female groups. akhlaghi et al. (2012) in their study reported the correlation between forearm length and stature (r=0.580).12 however, in their study, the correlation coefficient value was lower than our results which can be considered as the differences between distinct iranian ethnic groups.12 gender and stature determination are among the most important issues in the context of forensic science.13 in the case of human remains or incomplete skeleton, parts of the body such as forearm or hand bones can be helpful to find more evidence.14 according to the findings of the present study, it is concluded that forearm and hand length are longer in males compared with females in the iranian population. however, the effective role of nutrition and genetic factors should be considered.14 another study determined, the relationship between stature and long bones through the radiographic images of individuals, whose results are in accordance with our findings.15 mccluskey et al. showed that athletes with longer limb had high speed and in this study, all examined parameters showed a significant difference between the sexes.15 accordingly, an appropriate strategy seems to be necessary for product designing based on natural differences among body dimensions in males and females.15 consequently, anthropometric indexes can provide supportive information for designing of equipment.16in conclusion, our results showed that there is a statistically significant correlation among stature, forearm, and hand length for students of hamadan university of medical sciences. furthermore, the forearm length is a more predictive factor than hand length in stature estimation. table 1. comparison of mean of stature and other parameters for total subjects (n=160). parameters n mean ± sd stature 160 172.440±9.627 weight 160 68.776±11.974 hand length 160 18.387±1.979 forearm length 160 26.328±1.941 foot length 160 25.170±1.864 table 2. comparison of mean of stature and other parameters between males and females (n=80). parameters n mean ± sd p-value significance male female male female stature 80 80 180.446±5.569 164.435±5.072 0.000 sig. weight 80 80 75.413±11.098 62.138±8.729 0.000 sig. hand length 80 80 19.418±0.888 17.356±2.223 0.000 sig. forearm length 80 80 27.751±1.294 24.906±1.347 0.000 sig. foot length 80 80 26.566±1.337 23.775±1.123 0.000 sig. table 3. correlation between stature and other parameters in total sample (n=160). variables pearson correlation (r) p-value significance stature 1 weight 0.593 0.000 sig. hand length 0.560 0.000 sig. forearm length 0.813 0.000 sig. foot length 0.849 0.000 sig. 215 original stature estimation from students’ forearm and hand lengthbabak ebrahimi j contemp med sci | vol. 6, no. 5, september-october 2020: 213–217 table 4. correlation between stature and other parameters in between males and females (n=80). male female variables pearson correlation (r) p-value significance pearson correlation (r) p-value significance stature 1 1 weight 0.269 0.016 sig. 0.301 0.007 sig. hand length 0.546 0.000 sig. 0.184 0.102 ns forearm length 0.619 0.000 sig. 0.449 0.000 sig. foot length 0.576 0.000 sig. 0.657 0.000 sig. table 5. linear regression analysis for study population with stature as dependent variable and weight as independent variable. population r r² adjusted r² se b p-value total (160) 0.593 0.351 0.347 3.596 139.657 0.477 0.000 male (80) 0.269 0.072 0.060 4.172 170.273 0.135 0.016 female (80) 0.301 0.091 0.079 3.937 153.566 0.175 0.007 table 6. linear regression analysis for study population with stature as dependent variable and hand length as independent variable. population r r² adjusted r² se b p-value total (160) 0.560 0.314 0.310 5.926 122.327 2.725 0.000 male (80) 0.546 0.298 0.289 11.563 113.975 3.423 0.000 female (80) 0.184 0.034 0.021 4.442 157.147 0.420 0.102 ns table 7. linear regression analysis for study population with stature as dependent variable and forearm length as independent variable. population r r² adjusted r² se b p-value total (160) 0.813 0.661 0.659 6.060 66.268 4.033 0.000 male (80) 0.619 0.383 0.375 10.632 106.489 2.665 0.000 female (80) 0.449 0.202 0.192 9.501 122.302 1.692 0.000 table 8. linear regression analysis for study population with stature as dependent variable and foot length as independent variable. population r r² adjusted r² se b p-value total (160) 0.849 0.720 0.719 5.483 62.137 4.382 0.000 male (80) 0.576 0.332 0.323 10.250 116.735 2.398 0.000 female (80) 0.657 0.431 0.424 9.172 93.946 9.965 0.000 216 original stature estimation from students’ forearm and hand length babak ebrahimi j contemp med sci | vol. 6, no. 5, september-october 2020: 213–217 fie 1. correlation between stature (cm) with weight (kg), hand length (cm), forearm length (cm) and foot length (cm). fie 2. correlation between stature (cm) with male weight (kg), hand length (cm), forearm length (cm) and foot length (cm). 217 original stature estimation from students’ forearm and hand lengthbabak ebrahimi acknowledgments the authors of the paper sincerely thank their co-participants and hamadan university of medical sciences. references 1. jaberi kr, kavakebian f, mojaverrostami s, najibi a, safari m, hassanzadeh g, et al. nasofacial anthropometric study among students of shiraz university of medical sciences, iran: a population based study. indian journal of otolaryngology and head & neck surgery. 2019;71(2):206-11. 2. navid s, mokhtari t, alizamir t, arabkheradmand a, hassanzadeh g. determination of stature from upper arm length in medical students. anatomical sciences journal. 2014;11(3):135-40. 3. jabalameli m, moradi a, bagherifard a, radi m, mokhtari t. evaluation of distal femoral rotational alignment with spiral ct scan before total knee arthroplasty (a study in iranian population). archives of bone and joint surgery. 2016;4(2):122. 4. poorhassan m, mokhtari t, navid s, rezaei m, sheikhazadi a, mojaverrostami s, et al. stature estimation from forearm length: an anthropological study in iranian medical students. journal of contemporary medical sciences. 2017;3(11):270-2. 5. bridge al, oxenham mf, miszkiewicz jj. estimating stature using human forearm and leg anthropometric data in an australian female sample. australian journal of forensic sciences. 2020;52(1):83-95. 6. atamtürk d, pelin c, duyar i̇. estimation of human physique in forensic anthropological cases. eurasian journal of anthropology. 2019;10(2):56-66. 7. vignesh m, mohanraj kg. determination of sex using forearm–a morphometric study. drug invention today. 2019;11(9). 8. hassanzadeh s, alemohammad zb, mokhtari t, arabalidoosti f, rezaei f. correlation between craniofacial parameters and obstructive sleep apnea syndrome in iranian population. iraq medical journal. 2019;3(2). 9. ilayperuma i, nanayakkara g, palahepitiya n. prediction of personal stature based on the hand length. galle medical journal. 2009;14(1). 10. singh b, krishan k, kaur k, kanchan t. stature estimation from different combinations of foot measurements using linear and multiple regression analysis in a north indian male population. journal of forensic and legal medicine. 2019;62:25-33. 11. sanli sg, kizilkanat ed, boyan n, ozsahin et, bozkir mg, soames r, et al. stature estimation based on hand length and foot length. clinical anatomy: the official journal of the american association of clinical anatomists and the british association of clinical anatomists. 2005;18(8):589-96. 12. akhlaghi m, hajibeygi m, zamani n, moradi b. estimation of stature from upper limb anthropometry in iranian population. journal of forensic and legal medicine. 2012;19(5):280-4. 13. sharma s, peddawad r. estimation of stature and gender from biacromial breadth measurements in adult population of bareilly. indian journal of forensic medicine & toxicology. 2019;13(4):167-71. 14. mohammed i, mokhtari t, ijaz s, ngaski aa, milanifard m, hassanzadeh g. anthropometric study of nasal index in hausa ethnic population of northwestern nigeria. journal of contemporary medical sciences. 2018;4(1). 15. mccluskey l, lynskey s, leung ck, woodhouse d, briffa k, hopper d. throwing velocity and jump height in female water polo players: performance predictors. journal of science and medicine in sport. 2010;13(2):236-40. 16. castellucci h, viviani c, arezes p, molenbroek jf, martínez m, aparici v, et al. applied anthropometry for common industrial settings design: working and ideal manual handling heights. international journal of industrial ergonomics. 2020;78:102963. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i5.830 fie 3. correlation between stature (cm) with female weight (kg), hand length (cm), forearm length (cm) and foot length (cm). 161j contemp med sci | vol. 6, no. 4, july-august 2020: 161–167 original issn 2413-0516 introduction colorectal cancer (crc) is one of the most frequent malignant tumors worldwide, accounting for nearly 19.7% of whole cancers and 8.9% of cancer deaths.1the evolution of cancer is affected by the sequence of events including both epigenetic and inherited epithelial cells changes and is also directed by tumor–host interaction.2 the strong reaction of tumor-infiltrating immune cells in blocking tumor dissemination is a familiar model of this interaction.2 however, certain types of malignancies have the ability to escape immune response through the upregulation of the host immune checkpoints in the tumor microenvironment.3 programmed cell death1 ligand1 (pd-l1, cd274) is a key immune checkpoint transmembrane physiological ligand for programmed-death1 (pd1), expressed by lymphocytes, macrophages, and dendritic cells.2, 4 the pd-1/pd-l1 interaction plays an important role in the inhibition of t cell-mediated immune response leading to the exhaustion of effectors t cells2. it is also expressed in various malignancies, serve as a receptor transferring an anti-apoptotic signal to guard tumor cells against apoptosis and immune escape of tumor cells.2 the pd1-pdl1 pathway, therefore, has been involved in cancer progression.5 this observation leads to the production of pd1–pdl1 pathway inhibitors to counteract tumor cells of evading from host immune responses, and to intensify antitumor immunity, thus providing a promising approach in oncology toward immunotherapy.6 it is noted that expression of pd-l1, by tumor-infiltrating immune cells (tiics), and or neoplastic cells, affects patients’ prognosis in several cancers and correlates with a therapeutic response.7nonetheless, the role concerning pd-l1 tissue expression in crc and its microenvironment in correspondence with clinicopathological characteristics remains elucidated with different studies reporting conflicting results.6, 8, 9 therefore, the aim of the current study is to investigate pd-l1 immunoexpression in crc, and its microenvironment, and to reveal whether there is a correlation between them with variable clinicopathological parameters. materials and methods patients and primary tissue specimens characteristics a retrospective tissue microarrays study carried out on 99 formalin-fixed, paraffin-embedded specimens of colonic and rectal carcinoma, obtained at random from the department of pathology laboratories at azadi teaching hospital and some own clinical laboratories in duhok city, iraq during the period between january 2015 and december 2019. we maintained a routine protocol and proper ethics approval from the medical research ethics committee, college of medicine, university of mosul (approval number uom/com/mrecl2019(8), date 17/2/2020), to access the clinicopathological data included: patient age, sex, the primary site of the tumor, histological differentiation, the number of the involved lymph nodes, angiolymphatic invasion, and tnm stage. the pathologic staging based on the 8th edition of the american joint committee on cancer staging manual.10 correlation between programmed cell death ligand1 (pd-l1) expression and clinical parameters in colorectal carcinoma zainab waleed aziz al-hayali1, asmaa mohammadsheet mahmood1, zahraa osama yahiya1, wahda mohammed taib al –nuaimy2 1 department of pathology, ninevah medical college, ninevah university, mosul, iraq. 2 department of pathology, college of medicine, university of mosul, nineveh province, iraq. corresponding author: zainab waleed aziz al-hayali (email: zainabwaleed90@yahoo.com ) abstract objectives: programmed cell death ligand1 (pd-l1) tissue expression in colorectal cancer (crc) displays conflicting results among various studies. we aimed to identify the rate of pd-l1 positivity in colorectal carcinoma, and its immune infiltrating cells, their relationship with clinicopathological parameters of patients, and to correlate the results with other studies. methods: pd-l1 antibody retrospectively analyzed immunohistochemically in tissue microarray blocks of 99 specimens with colonic and rectal carcinomas operated between january 2015 and december 2017. a comparison performed between pd-l1 expression in tumor cells (tcs) as well as tumor-infiltrating immune cells (tiics) for age, sex, histological differentiation, the primary tumor location, number of involved lymph nodes, angiolymphatic invasion, and tnm stage. results: of the 99 patients, the median age was 54.5 (range: 18–3) years. fourteen samples were pd-l1 positive in tcs, increased to 32% in tiics. a significant expression of pd-l1 in tcs was correlated with medullary histology (p= 0.03), number of the involved lymph nodes (p= 0.02), distant metastasis (p= 0.001), and tnm stage (p= 0.0001). the pd-l1 status in tiics was again connected with adverse clinical and pathological parameters. conclusions: the expression of pd-l1 in tcs and tiics is associated significantly with advanced cancer or lymphatic invasion in patients who underwent surgery after a diagnosis of crc. the research designates the significance of estimation of tcs and tiics in correlation to clinicopathological characteristics of patients a finding that could produce a piece of evidence for precise electing immunotherapy. keywords: programmed cell death ligand1, colorectal carcinoma, tissue microarray study, immunohistochemistry. 162 original pd-l1 in colorectal carcinoma zainab waleed aziz al-hayali j contemp med sci | vol. 6, no. 4, july-august 2020: 161–167 tissue microarray construction in the current study, two independent pathologists retrospectively analyzed h&e stained slides and selected the representative archival paraffin‐embedded blocks for each case. four cases were excluded due to lack of sufficient tissue in the block. tissue microarray (tma) blocks made with a hand-operated tma kit(3dhistech manual tma kit) includes two ingredients (puncher extractor device and moulder).11during the procedure, 2 mm in diameter three tissue cores were selected from regions of concern in paraffin-embedded tissues using a hollow needle, along with those of normal colonic tissue as negative controls. all tissue cores arranged in recipient paraffin blocks (tma blocks). subsequently, sections of 3 µm from this block were cut, mounted then transferred to glass slides. later 2 tma block sections were chosen one was stained with hematoxylin-eosin to confirm the histology and to certify the tumor tissue availability (≥ 50% of the core area) in the slide while another slide submitted to pd-l1 immunohistochemistry test. immunohistochemistry technique immunohistochemical (ihc) staining was performed on tma sections with dako automated autostainer link 48 with zytochem plus hrp polymer kit detection system used in the work of the current study. briefly, 3 μm tma thick sections were baked overnight at 58 °c, deparaffinized in xylene, rehydrated through graded ethanol. a pretreatment procedure was performed later on the tissue sections with heat-induced epitope retrieval (hier), hindered for endogenous peroxidase activity in 3% hydrogen peroxide solution at 37 °c for 10 min, ensued by high-pressure cooking in citrate antigen retrieval solution (ph= 6.0) for about 10 min for pd-l1. tissue sections were incubated at 37 °c for 60 min with rabbit igg monoclonal antibodies against pd-l1 (1:100, cat. no.rbk063-05, zytomed systems, berlin, germany). immunostaining was performed using the zytochem plus hrp polymer (dab) (polhrp-006, zytomed systems, berlin, germany), in which a brown-colored precipitate appeared at the antigen site. finally, counterstaining and blueing with hematoxylin (sigmaaldrich, st louis, mo, usa), and mounting in a non-aqueous mounting medium.8 scoring of pd-l1 immunohistochemical expression pd-l1 positivity designated as cells with membranous and/ or cytoplasmic immunohistochemical staining on ≥5% of all tcs or tiics.12 trivial changes observed in the staining intensity, therefore, was not estimated. pd-l1 immunoexpression on tumor cells (tcs), and tumor-infiltrating immune cells (tiics) was analyzed separately. tiics were designated as lymphocytes, macrophages, and dendritic cells infiltrating nests of neoplastic cells.8 statistical analyses statistical analyses using the spss statistical package for social sciences (version 18.0 for windows, spss, chicago, il, usa) were performed. analysis of the data was made by using the t-test and chi-square test. the microsatellite instability (msi) status and the relapse/survival free relapse were unknown; thus, not assessed. all p-values considered statistically significant when retained less than or equal to 0.05.12 results clinical characteristics of patients the baseline clinicopathological characteristics of 99 primary crc samples are listed within table 1. briefly, the median age was 54.5 years (range 18–83 years old), by the dominance of males (55.5%), with a male to female ratio 1.25:1. the predominant primary tumor locations were the right colon (34.3%), and the rectum (31.3%). histologically, there were 93.9% moderate to poorly differentiated adenocarcinoma, 15.1% of which show medullary type. most of the tumors invade through the muscularis propria into the pericolorectal tissues (t3 and t4% = 84.8%). fifty cases were presented with regional lymph node involvement by metastatic disease. approximately 7.1% of cases were at stage iv. table 1. clinicopathological characteristics of crc patients. variables sample no. age ( years) mean 54.5 gender female male 44 55 tumor location lt colon rectum right colon transverse colon 28 31 34 6 medullary type yes no 15 84 tumor differentiation well moderate poor 6 80 13 primary tumor (pt) t2 t3 t4 15 77 7 no .of involved lymph nodes mean 3.18 angio-lymphatic invasion negative positive 30 69 regional lymph node (pn) n0 n1 n2 49 28 22 distant metastasis (pm) m0 m1 91 8 tnm stage i ii iii iv 9 40 43 7 163 original pd-l1 in colorectal carcinomazainab waleed aziz al-hayali j contemp med sci | vol. 6, no. 4, july-august 2020: 161–167 immunohistochemical expression of pd-l1 the pd-l1 staining was at various frequencies both within tcs and tiics. with a ≥5% cut-off, tumor cells were positive in 14% of specimens, while in 32% of tiics. the normal colonic mucosa did not show any staining. cytoplasmic and/ or membranous staining was observed in positive tcs cells and tiics with the predominance of the former. in tcs, focal pd-l1 positive pattern was prevailing. however, in tiics the diffuse positivity was the most common. the expression of pd-l1 is shown in figures 1 and 2. the correlation between the expression of pd-l1 within tcs and the clinicopathological parameters shown in table 2. on tcs, expression of pd-l1 was significantly linked with medullary histology (p=0.03), number of the involved lymph nodes (p=0.02), distant metastasis (p=0.001), and tnm stage (p=0.0001). on the contrary, other variables such as age, gender, primary tumor location, tumor differentiation, angiolymphatic invasion, and regional lymph node (pn) status were of no significance, although primary tumor (pt) status approached the borderline of significance (p=0.07). a b c figure 1. programmed cell death ligand-1 immunohistochemical expression in crc neoplastic cells (original magnification, ×100, ×400). (a) a lack of pd-l1 expression; (b) negative pd-l1 expression (<5% of stained cells); (arrows); (c) positive pd-l1 expression (≥5% of stained cells). 164 original pd-l1 in colorectal carcinoma zainab waleed aziz al-hayali j contemp med sci | vol. 6, no. 4, july-august 2020: 161–167 the pd-l1 status in tiics was studied and its association with the variable clinical and pathological features of crc patients were analyzed (table 3). the tiics exhibited higher pd-l1 immunoexpression were significantly associated with medullary histology (p=0.01), primary tumor (pt) status (p=0.05), regional lymph node (pn) status (p=0.04), angiolymphatic invasion (p=0.02), distant metastasis (p=0.002), and tnm stage (p=0.002) while other variables provided no significant association. discussion in oncology, the detection of a prognostic and predictive biomarker remains very important to select patients who benefit from a given therapy while sparing others. in this rapidly evolving field, pd-l1 becomes one of the emerging biomarkers that play a major role in immune evasion and distant metastasis.9 however, the role of this biomarker is less clear in colorectal cancer, as to whether its expression indicates a better or worse a b c figure 2. programmed cell death ligand-1 immunohistochemical expression in colorectal carcinoma immune cells (original magnification, ×400). (a) a lack of pd-l1 expression; (b) focal pd-l1 expression (<5% of stained cells); (arrow); (c) positive pd-l1 expression (≥5% of stained cells). 165 original pd-l1 in colorectal carcinomazainab waleed aziz al-hayali j contemp med sci | vol. 6, no. 4, july-august 2020: 161–167 table 2. immunohistochemical tissue expression of pd-l1 in tcs across crc patients. variable pd-l1 expression in tumor cell (tc) p-value(negative) n=85 (85.9%) (positive) n=14 (14.1%) age, mean± sd 53.68±16.85 56.57±8.06 0.3 gender female male 35 (79.5%) 50 (90.9%) 9 (20.5%) 5 (9.1%) 0.09 tumor location lt colon rectum right colon transverse colon 26 (92.9%) 28 (90.3%) 27 (79.4%) 4 (66.7%) 2 (7.1%) 3 (9.7%) 7 (20.6%) 2 (33.3%) 0.2 medullary type no yes 75 (90.2%) 10 (66.7%) 9 (10.8%) 5 (33.3%) 0.03 tumor differentiation well moderate poor 5 (83.3%) 70 (87.5%) 10 (76.9%) 1 (16.7%) 10 (12.5%) 3 (23.1%) 0.5 primary tumor (pt) t2 t3 t4 13 (86.7%) 68 (88.3%) 4 (57.1%) 2 (13.3%) 9 (11.7%) 3 (42.9%) 0.07 no .of involved lymph nodes, mean± sd 2.66±4.53 5.84±6.45 0.02 angio-lymphatic invasion negative positive 28 (93.3%) 57 (82.6%) 2 (6.7%) 12 (17.4%) 0.1 regional lymph node (pn) n0 n1 n2 45 (91.8%) 23 (82.1%) 17 (77.3%) 4 (8.2%) 5 (17.9%) 5 (22.7%) 0.2 distant metastasis (pm) m0 m1 83 (91.2%) 2 (25.0%) 8 (8.8%) 6 (75.0%) 0.001 tnm stage i ii iii iv 9 (100.0%) 37 (92.5%) 37 (86.0%) 2 (28.6%) 3 (7.5%) 6 (14.0%) 5 (71.4%) 0.0001 *p≤ 0.05 s prognosis.13, 14 besides, pd-l1 expression in an association with the clinical and pathological features are not well-defined in such tumor. in this study, pd-l1 positivity recognized in 14% of the patients with colorectal carcinoma. this result is agreed by two prior studies published very comparable issues with pd-l1 immunoexpression in 14.6% and 12% of cases, respectively.12, 15 in contrast, masugi et al. analyzed 823 rectal and colonic cancer cases and noticed pd-l1 tissue expression in 89% of them.16 these discrepancies might be due to the various scoring systems, positivity cut-offs applied, intratumoral staining heterogeneity, and the different antibodies used.12 in terms of histopathological parameters, pd-l1 in tcs was significantly associated with medullary histology, number of the involved lymph nodes, distant metastasis, and advanced stage. interestingly, a nice research by zhu et al17 has also pointed that high pd-l1 expression in tumor cells is correlated significantly with the metastatic progression, advancement of tumor stage, and poor outcomes in crcs. although pd-l1 expression was more common in rightsided tumors with female predominance similar to previous studies,3, 18 they didn’t approach a statistic significance. from our point of view, this attributed to the small sample size of the study. on the contrary, other variables such as age, tumor differentiation, angiolymphatic invasion, and regional lymph node status were of no significance compatible with shan et al.19 according to our study, these findings indicate that pd-l1 expression in tcs is closely linked to aggressive histological features in crc in consistence with other studies that linked pd-l1 expression to unfavorable prognosis.9, 15 however, these studies did not evaluate pd-l1 expression on immune cells, which now implemented in the scoring strategy for numerous 166 original pd-l1 in colorectal carcinoma zainab waleed aziz al-hayali j contemp med sci | vol. 6, no. 4, july-august 2020: 161–167 neoplasms like triple-negative breast cancers.20 moreover, the prognostic value regarding pd-l1 positivity in these immune cells still awaits investigations.21 to address these questions, we separately studied pd-l1 immunoexpression in tiics and correlated the results with the clinicopathological data of the patients. the results identified pd-l1 positivity in 32% of the tumor immune cells. this immunoexpression was significantly associated with medullary histology, angiolymphatic invasion, distant metastasis, and advanced stage. so, again connected with adverse clinical and pathological parameters. lin et al22 and wang et al23 reported similar results but not in masugi et al.16 the metastatic progression and advancement of tumor stage in crc patients with pd-l1 positive tumor infiltrating immune cells could be explained by the fact that such expression might lead to the inhibition of activated t lymphocytes and immune evasion, thus to poor outcomes.24, 25 another possible explanation is that alteration of the intestinal microbes by some abnormal intestinal status may induce a shift in the immunologic function accordingly.26 therefore, intestinal dysfunction as a consequence of crc may modify the intestinal immune system. so, from our point of view, our findings highlight support to the hypothesis that pd-l1 expression correlated with the poor clinical and pathological features in colorectal carcinoma, because we also shed lights on the frequency of pd-l1 positivity in tiics in rectal and colonic carcinomas, rather than tcs alone. it suggested that patients with advanced cancer or lymphatic invasion were more likely to express pd-l1. the current study has some limitations that are somewhat of small sample size (99 cases), its retrospective nature as well table 3. pd-l1 immunoexpression in tiics across crc patients. variable pd-l1 expression in tumor infiltrating immune cells (tiics) p-value (negative) n=67 (67.7%) (positive) n=32 (32.3%) age, mean± sd 15.61±16.63 53.00±14.46 0.6 gender female male 28 (63.6%) 39 (70.9%) 16 (36.4%) 16 (29.%1) 0.2 tumor location lt colon rectum right colon transverse colon 21 (75.0%) 26 (83.9%) 18 (52.9%) 2 (33.3%) 7 (25.0%) 5 (16.1%) 16 (47.1%) 4 (66.7%) 0.1 medullary type no yes 61 (72.6) 6 (40.0%) 23 ( 27.4) 9 (60.0%) 0.1 tumor differentiation well moderate poor 4 (66.7%) 57 (71.3%) 6 (46.2%) 2 (33.3%) 23 (28.8%) 7 (53.8%) 0.2 primary tumor (pt) t2 t3 t4 12 (80.0%) 53 (68.8%) 2 (28.6%) 3 (20.0%) 24 (31.2%) 5 (71.4%) 0.05 no .of involved lymph nodes, mean± sd 2.50±4.61 4.40±5.37 0.07 angio-lymphatic invasion negative positive 25 (83.3%) 42 (60.9%) 5 (16.7%) 27 (39.1%) 0.02 regional lymph node (pn) n0 n1 n2 38 (77.6%) 18 (64.3%) 11 (50.0%) 11 (22.4%) 10 (35.7%) 11 (50.0%) 0.04 distant metastasis (pm) m0 m1 66 (72.5%) 1 (12.5%) 25 (27.5%) 7 (87.5%) 0.002 tnm stage i ii iii iv 9 (100.0%) 30 (75.0%) 27 (62.8%) 1 (14.3%) 10 (25.0%) 16 (37.2%) 6 (85.7%) 0.002 *p≤ 0.05 s 167 original pd-l1 in colorectal carcinomazainab waleed aziz al-hayali as the msi status, and the relapse/survival free relapse not addressed. however, to our knowledge, no similar study has conducted in iraq, despite the relatively high prevalence of colorectal carcinoma. to achieve further strong evidence, sizable well-designed cohort studies and investigations on pd-l1 immunoexpression on tiics, are required. conclusions the immunoexpression of pd-l1 in tcs and its microenvironment is associated significantly with advanced cancer or lymphatic invasion in patients who underwent surgery after a diagnosis of crc. the research designates the significance of estimation of tcs and tiics together in correlation to clinicopathological characteristics of patients, a finding that could produce a piece of evidence for precise electing patients toward immunotherapy, aiming for better outcomes. statement conflict of interest: no conflict of interest. funding statement: no funding. references 1. bray f. ferlay j, soerjomataram i, siegel rl, torre la, jemal a. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin. 2018;68:394-424. 2. schott ds, pizon m, pachmann u, pachmann k. sensitive detection of pd-l1 expression on circulating epithelial tumor cells (cetcs) could be a potential biomarker to select patients for treatment with pd-1/pd-l1 inhibitors in early and metastatic solid tumors. oncotarget. 2017;8(42):72755. 3. valentini am, di pinto f, cariola f, guerra v, giannelli g, caruso ml, et al. pdl1 expression in colorectal cancer defines three subsets of tumor immune microenvironments. oncotarget. 2018;9(9):8584. 4. topalian sl, hodi fs, brahmer jr, gettinger sn, smith dc, mcdermott df, et al. safety, activity, and immune correlates of anti–pd-1 antibody in cancer. new engl j med. 2012;366(26):2443-54. 5. bertucci f, finetti p, mamessier e, pantaleo ma, astolfi a, ostrowski j, et al. pdl1 expression is an independent prognostic factor in localized gist. oncoimmunology. 2015;4(5):e1002729. 6. zhou c, tang j, sun h, zheng x, li z, sun t, et al. pd-l1 expression as poor prognostic factor in patients with non-squamous non-small cell lung cancer. oncotarget. 2017;8(35):58457. 7. gatalica z, snyder c, maney t, ghazalpour a, holterman da, xiao n, et al. programmed cell death 1 (pd-1) and its ligand (pd-l1) in common cancers and their correlation with molecular cancer type. cancer epidemiol prev biomarkers. 2014;23(12):2965-70. 8. li y, liang l, dai w, cai g, xu y, li x, et al. prognostic impact of programed cell death-1 (pd-1) and pd-ligand 1 (pd-l1) expression in cancer cells and tumor infiltrating lymphocytes in colorectal cancer. mol cancer. 2016;15(1):55. 9. song m, chen d, lu b, wang c, zhang j, huang l, et al. pten loss increases pd-l1 protein expression and affects the correlation between pd-l1 expression and clinical parameters in colorectal cancer. plos one. 2013;8(6):e65821. 10. amin mb, edge sb. ajcc cancer staging manual: springer; 2017. 11. shaban zm, al-aubaidy sr, hameedi ad. idh1 mutation in gliomas in baghdad by immunohistochemical study. int j genet genomics. 2018;6(1):1-7. 12. inaguma s, lasota j, wang z, felisiak-golabek a, ikeda h, miettinen m. clinicopathologic profile, immunophenotype, and genotype of cd274 (pdl1)-positive colorectal carcinomas. modern pathol. 2017;30(2):278-85. 13. rosenbaum mw, bledsoe jr, morales-oyarvide v, huynh tg, mino-kenudson m. pd-l1 expression in colorectal cancer is associated with microsatellite instability, braf mutation, medullary morphology and cytotoxic tumorinfiltrating lymphocytes. modern pathol. 2016;29(9):1104-12. 14. droeser ra, hirt c, viehl ct, frey dm, nebiker c, huber x, et al. clinical impact of programmed cell death ligand 1 expression in colorectal cancer. eur j cancer. 2013;49(9):2233-42. 15. lee ks, kim bh, oh hk, kim dw, kang sb, kim h, et al. programmed cell death ligand‐1 protein expression and cd 274/pd‐l1 gene amplification in colorectal cancer: implications for prognosis. cancer sci. 2018;109(9):295769. 16. masugi y, nishihara r, yang j, mima k, da silva a, shi y, et al. tumour cd274 (pd-l1) expression and t cells in colorectal cancer. gut. 2017;66(8):1463-73. 17. zhu h, qin h, huang z, li s, zhu x, he j, et al. clinical significance of programmed death ligand-1 (pd-l1) in colorectal serrated adenocarcinoma. int j clin exp pathol. 2015;8(8):9351-9. pubmed pmid: 26464688. pubmed central pmcid: pmc4583920. epub 2015/10/16. eng. 18. elfishawy m, abd esa, hegazy a, el-yasergy df. immunohistochemical expression of programmed death ligand-1 (pdl-1) in colorectal carcinoma and its correlation with stromal tumor infiltrating lymphocytes. asian pacific j cancer prev : apjcp. 2020 jan 1;21(1):225-32. pubmed pmid: 31983188. pubmed central pmcid: pmc7294013. epub 2020/01/28. eng. 19. shan t, chen s, wu t, yang y, li s, chen x. pd-l1 expression in colon cancer and its relationship with clinical prognosis. int j clin exp pathol. 2019;12(5):1764-9. pubmed pmid: 31933995. pubmed central pmcid: pmc6947132. epub 2020/01/15. eng. 20. kowanetz m, zou w, gettinger sn, koeppen h, kockx m, schmid p, et al. differential regulation of pd-l1 expression by immune and tumor cells in nsclc and the response to treatment with atezolizumab (anti–pd-l1). proc natl acad sci. 2018;115(43):e10119-e26. 21. lee lh, cavalcanti ms, segal nh, hechtman jf, weiser mr, smith jj, et al. patterns and prognostic relevance of pd-1 and pd-l1 expression in colorectal carcinoma. modern pathol. 2016;29(11):1433-42. 22. lin h, wei s, hurt em, green md, zhao l, vatan l, et al. host expression of pd-l1 determines efficacy of pd-l1 pathway blockade–mediated tumor regression. j clin investig. 2018;128(2):805-15. 23. wang l, ren f, wang q, baldridge la, monn mf, fisher kw, et al. significance of programmed death ligand 1 (pd-l1) immunohistochemical expression in colorectal cancer. mol diag ther. 2016;20(2):175-81. 24. park j-j, omiya r, matsumura y, sakoda y, kuramasu a, augustine mm, et al. b7-h1/cd80 interaction is required for the induction and maintenance of peripheral t-cell tolerance. blood. 2010;116(8):1291-8. 25. butte mj, keir me, phamduy tb, sharpe ah, freeman gj. programmed death-1 ligand 1 interacts specifically with the b7-1 costimulatory molecule to inhibit t cell responses. immunity. 2007 jul;27(1):111-22. pubmed pmid: 17629517. pubmed central pmcid: pmc2707944. epub 2007/07/17. eng. 26. pflughoeft kj, versalovic j. human microbiome in health and disease. annu rev pathol mech dis. 2012;7(1):99-122. pubmed pmid: 21910623. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v6i4.810 217j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 original evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells haleh forouhandeh1†, mahtab zarchini2†, elham safarzadeh3, ommoleila molavi4, parina asgharian5,6*, vahideh tarhriz1* 1molecular medicine research center, biomedicine institute, tabriz university of medical sciences, tabriz, iran. 2student research committee, tabriz university of medical sciences, tabriz, iran. 3department of microbiology & immunology, faculty of medicine, ardabil university of medical sciences, ardabil, iran. 4department of pharmaceutical biotechnology, faculty of pharmacy, tabriz university of medical sciences, tabriz, iran. 5department of pharmacognosy, faculty of pharmacy, tabriz university of medical sciences, tabriz, iran. 6drug applied research center, tabriz university of medical sciences, tabriz, iran. *correspondence to: parina asgharian (e-mail: parina.asgharian@gmail.com), vahideh tarhriz (e-mail: t.tarhriz@yahoo.com) †these authors contributed equally to this work. (submitted: 03 march 2021 – revised version received: 28 march 2021 – accepted: 12 april 2021 – published online: 26 august 2021) abstract objective the present study concerns the cytotoxic activity of a. nemorosa different extracts on breast cancer cells (mcf-7) and normal cell lines (hfff). methods different extracts of aerial parts of a. nemorosa were prepared using soxhlet apparatus. the cytotoxicity of samples was assessed by mtt assay on breast cancer cells (mcf-7) and noncancerous cells (hfff) with different concentrations of extracts in 24 and 48 hours. the most potent extract was fractioned and cytotoxic activity of fractions was considered, as well. a flow cytometry (annexin v/pi) assay has been used for detecting the mechanism of cell death in sample treated cell lines. moreover, for clarifying volatile components of n-hexane extract and its 80% and 100% vlc fractions were subjected to gc-ms apparatus. results results indicated that n-hexane extract and its 80% and 100% vlc fractions exhibited a significant (p < 0.001) inhibitory effect on the growth of the mcf-7 cell line compared to the control group. meanwhile, flow cytometry analysis revealed that potent extract caused cell death through necrosis and 80% and 100% fractions showed different mechanisms (such as autophagy). the major compounds, which maybe were in charge of showing cytotoxic activity were non-terpenoids. conclusion this study provides the evidence that in vitro cytotoxic activity of n-hexane extract and 80% and 100% vlc fractions of a. nemorosa inhibited the proliferation of breast cancer cells (mcf7) via a different mechanism. keywords anthriscus nemorosa, cancer, gc-ms, cytotoxic, flow cytometry issn 2413-0516 graphical abstract introduction cancer is a disease that begins with the abnormal proliferation of cells in the body. all cancers have an uncontrolled growth pattern and a tendency to detach from the source and metastasize. cancer mortality is on the rise and has become one of the leading causes of human mortality today. breast cancer is one of the most common malignancies in iranian women.1 various factors such as old age of reproduction and lactation, radiation, smoking, alcohol, and many other factors increase the risk of developing this disease in women.2,3 chemotherapy, surgery, radiotherapy, immunotherapy, gene therapy, hormone therapy, 218 j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells original h. forouhandeh et al. and cell therapy are some of the treatments available for this disease.4 chemical drugs available on the market due to the side effects of the drug are less popular. in recent years, attention has been paid to drugs with natural sources and fewer side effects.5 nature is an amazing source of suitable new drug compounds with great chemical diversity found in millions of plants, animal, marine, and microbial species.6,7 plant compounds that have anti-cancer and anti-tumor properties are in the chemical groups of aldehydes, alkaloids, flavonoids, glycosides, terpenoids, and phenolic compounds.8-10 it is noteworthy that more than 60% of common anticancer drugs are now derived from natural sources, including plants, marine organisms, and microorganisms.11,12 among the well-known medicinal plants, the genus anthriscus from the umbelliferae family has valuable compounds that are used in many cases, and has the following effects: anti-cough, diuretic, antipyretic,13 analgesic,14 antiviral,15 anti-asthma,16 insecticide,17 anti-inflammatory,18 an indirect inhibitor of cutaneous anaphylactic reactions,19 liver protector,20 a competitive inhibitor of cyp2c9 and cyp3a4 enzymes,21 reduction of skin pigmentation caused by uv light in guinea pigs.22 however, most studies show the anti-cancer effects of the compounds of this plant.13,18,23 considering the increasing prevalence of breast cancer due to the importance of medicinal plants in cancer treatment and regarding less investigation on the cytotoxic effects of this plant on breast cancer, the present study aimed to evaluate the cytotoxic effects of the plant extract on the category of breast cancer cells. materials and methods preparation of plant samples anthriscus nemorosa (mb) spreng (a. nemorosa) was collected in july 2016 from ardabil province, jafarabad moghan region and was matched and identified with its herbarium specimen and identified as tbz.fph 1037 in the school herbarium. the sample was maintained in the faculty of pharmacy, tabriz university of medical sciences, tabriz, iran. preparation of extracts the amount of 200 g of a. nemorosa plant powder ensuring weighing and loading was placed in a suitable cartridge paper in a 1-liter soxhlet apparatus and extracted with n-hexane (n-hex), dichloromethane (dcm), and methanol (meoh) solvents, respectively, for 72 hours. the soxhlet was placed on the balloon in the electric oven securing it with a clamp. then, we added the desired solvents from the top of the soxhlet chamber little by little until the solvent reaches half of the balloon. after adding the solvent, it was placed in the refrigerant on the soxhlet and opened the tube water. then was turned on the electric stove and adjusted the temperature until the solvent boiled evenly inside the balloon. after the extraction, we turned off the device and discarded the plant pulp that did not contain the extract, and filtered the liquid extract inside the balloon with a funnel and filtered paper, and kept it in suitable containers. fractionation vlc method was used to fractionate non-polar extracts. in this method, the extracts were first dried completely by rotary evaporator at 45°c and low air pressure. fractionation was then performed using silica gel as the stationary phase and increasing percentages of ethyl acetate in hexane. identification of volatile compounds in non-polar samples gas chromatography-mass spectrometry (gc-ms) was used to identify the compounds in the n-hex extract and its 80% and 100% fractions. samples were injected into the device under the following conditions: injection temperature: 270ºc, column flow rate: 1.2 ml/min, split ratio: 1:10, total flow: 18.4 ml/min, and helium gas were used as carriers. cell lines used in experiments mcf-7 cell: this cell line includes breast cancer cells from a 69-year-old caucasian woman from the pasteur institute of iran. hfff cell: this cell line contains normal fibroblast cells that are from a newborn human and have been prepared by the pasteur institute of iran. cell cultures the culture medium used is roswell park memorial institute (rpmi-1640), purchased from sigma company in powder form. cell lines were cultured in rpmi 1640 liquid culture medium containing 10% fbs and 1% penicillin/streptomycin antibiotic. the cells were incubated in sterile flasks at 37°c, 5% co2, and 95% moisture. confluent cells (upon 70%) are trypsinized and then subcultured at lower numbers in new culture flasks. chemicals all solvents used in this research were purchased from caledon canada. all chemicals were analytical grade. evaluation of cytotoxicity of extracts by mtt method to perform this test, 200 microliters of cell suspension containing the appropriate number of cells (approximately 5000 cells) first was cultured uniformly in each of the plate wells and allowed the cells to adhere to the bottom of the plate and become stable. the plate is then incubated for 24 hours at 37°c, 95% humidity and 5% carbon dioxide until the cells reach the desired density. after this time, the cells are treated with different concentrations of n-hex, dcm, and meoh extracts. these concentrations have been determined through a previous pilot test. then we moved the plates gently on the horizontal surface to homogenize and incubate them for 24 and 48 hours. the supernatant was then discarded and 150 μl of the prepared mtt solution was added to each well, and the plates were transferred to a carbon dioxide incubator at 37°c for 4 hours in sterile aluminum foil. in this case, mtt (dimethylthiazole-2 and 5-diphenyltetrazolium bromide) is converted to insoluble purple formazan crystals by mitochondrial enzymes in living cells due to the rupture of the tetrazolium ring. after 4 hours, the contents of the wells are gently drained and 200 μl of dmso is added to each well to dissolve the purple crystals of formazan. finally, the light absorption of the obtained solution is read by a plate reader with a wavelength of 570 nm. detection by flow cytometry to perform the 2 × 105 cell test, 6 wells were added to each well from the plate. the plates were placed in a cell culture incubator for 24 hours. after this time and reaching the appropriate 219j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 h. forouhandeh et al. original evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells confluency of the cells, the cells were treated with concentrations equal to ic50 of n-hex extract and its fractions. for this purpose, the top culture medium of each well was emptied by pasteur pipette and after washing with pbs, the cells were trypsinized from the bottom of the well and transferred to separate microtubes. the microtubes were then centrifuged and repeated after removing the medium on the cells and washing with pbs. after this step, disperse the cells in 100 μl of diluted annexin v binding buffer and add 5 μl of annexin solution and pi dye to each microtubule. we incubated the contents in the dark for 30 minutes at room temperature. the microtubes are then centrifuged and the cells are dispersed in 200 μl of annexin v binding buffer. finally, the absorbance of each sample at 488 and 617 nm is read by flow cytometry. all steps of this test are performed except incubation in ice. statistical analysis of data the results obtained from uv absorption at a wavelength of 570 nm from the plate reader were entered into graphpad prism 8.0.2 software and analyzed (curve fit) nonlinear regression to calculate ic50 (concentration of sample that inhibits 50% cell growth) was used. anova analysis and tukey post hoc test were used for comparison between groups. in all tests, the minimum level of significance was considered p <0.05. results amounts of extracts the results of weighing the extracts obtained from the extraction of 340 g of plant shoot powder by n-hex, dcm, and meoh solvents are shown in table 1. because n-hex extract showed a significant cell inhibitory effect compared to the other extracts and control group, 2 g of this extract were fractionated by the vlc method for further studies. the results of the weight of these fractions are also given in table 1. cytotoxicity test results evaluation of cytotoxic effects of plant samples by mtt method the ic50 values of the samples (the concentration of the drug that causes 50% of cell death) were calculated by graphpad prism 8.0.2 software. these numbers are based on the nonlinear regression diagram log (inhibitor) vs. normalized responses—variable slope and drug concentration are calculated. cells were treated with different concentrations of dcm, n-hex, and meoh extracts for 24 and 48 hours. n-hex extract with the lowest amount of ic50 was the strongest fractionated extract and the relevant experiments were performed on the fractions. the ic50 values obtained are according to table 2. to evaluate and determine the cytotoxic effects of n-hex, dcm, and meoh extracts, mcf-7 cells were treated with concentrations of 0, 50, 100, 200, 400, 500, 600, and 800 μg/ml of each sample. the results of the mtt test at 24 and 48 hours are shown in figure 1. due to the significant cytotoxicity of the n-hex extract on mcf-7 cells compared to other extracts and the control group, in the next step, the fractions of n-hex extract were tested by mtt. the results are available in figure 2. besides, for evaluation of the effect of cytotoxicity of a. nemorosa extracts and fractions on non-cancerous cells, an mtt assay was performed on a normal hfff cell line. the results of ic50 from 24 and 48 hours treatment are as shown in table 3. table 1. results of weighing n-hex, dichloromethane, and methanol extracts and fractions from 340 g of a. nemorosa aerial powder meoh dcm n-hex extract 18.96 5.34 6.73 weight of plant extract (g/100 g of extract) 100% 80% 60% 40% 20% 10% fractions 7.48 15.25 9.65 13.80 14.43 7.35 weight of n-hexane extract fraction (g/100 g of extract) table 2. results of cytotoxic tests on mcf-7 cancer cell line n-hex, dcm, and meoh extracts of a. nemorosa ic 50 (μg/ml) mcf -7 extracts 24 h 48 h meoh extract 195.9 ± 6.36 259.8 ± 16.26 dcm extract 161.6 ± 5.09 203.8 ± 5.09 n-hex extract 129.2 ± 4.17 75.63 ± 7.94 a. nemorosa fractions of n-hexane extract 10% 83 ± 8.54 24.13 ± 5.06 20% 113.9 ± 7.63 56.9 ± 15.62 40% 69.04 ± 14.95 29.04 ± 4.49 60% 46.16 ± 4.35 22.6 ± 7.04 80% 8.66 ± 1.68 26.82 ± 0.67 100% 8.88 ± 0.48 14.71 ± 3.7 220 j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells original h. forouhandeh et al. fig. 1 viability of mcf-7 cancer cells after exposure to increasing concentrations of n-hex, dcm, and meoh extracts of a. nemorosa after 24 and 48 hours of incubation. fig. 2 viability of mcf-7 cells treated with different concentrations of a. nemorosa n-hex extract fractions using mtt method in 24 and 48 hours of incubation. table 3. cytotoxic effect of n-hex, dcm, and meoh extracts of a. nemorosa on hfff cells ic 50 (μg/ml) hfff extracts 24 h 48 h meoh extract >1000 >1000 dcm extract 401.2 ± 27.93 249.1 ± 25.73 n-hex extract 196.8 ± 28.7 150.1 ± 26.44 a. nemorosa fractions of n-hexane extract 10% 148.1 ± 23.68 474.5 ± 84.9 20% 52.08 ± 0.85 137 ± 27.44 40% 91.55 ± 8.85 25.43 ± 4.31 60% 55.88 ± 13.82 13.71 ± 1.7 80% 69.58 ± 15.57 18.96 ± 0.66 100% 35.67 ± 6 15.85 ± 2.4 significant comparison of the effect of different samples with controls two way anova test and tukey post hoc test were used to compare the cytotoxic effects of different plant samples at 24 and 48 hours. in statistical tests, the significance level is defined as ns (p > 0.05), * (p < 0.05), ** (p, 0.01), *** (p < 0.001). the viability of mcf-7 and hfff cells after treatment with n-hex, dcm, and meoh extracts after 24 and 48 hours of incubation is shown in figures 3 and 4, respectively. statistical analysis of 221j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 h. forouhandeh et al. original evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells fig. 3 statistical comparison of viability of mcf-7 cells treated with n-hex, dcm, and meoh extracts of a. nemorosa compared to the control group, using mtt method. fig. 4 statistical comparison of viability of hfff cells treated with n-hex and dcm extracts of a. nemorosa compared to the control group, using mtt method. fig. 5 statistical comparison of viability of mcf-7 cells treated with n-hex extracts fractions compared to the control group, using mtt method. anova and tukey post hoc test showed that the relevant extracts at both incubation times (24 and 48 hours) were significantly different from the dmso control group. the results of the statistical comparison of n-hex fractions at 24 and 48 hours are also shown in figure 5. according to figure 6, the viability of mcf-7 cells in the presence of dcm and meoh extracts in 24 hours and n-hex and meoh extracts in 48 hours relative to the cell hfff levels are significantly reduced. flow cytometry test results on mcf-7 cells in this test, n-hex extract and 60%, 80%, and 100% fractions were incubated with cancer cell lines for 24 hours at ic50 concentration. flow cytometry was performed to determine the extent of apoptosis. the test histogram view is shown in figure 7. the amount of apoptotic and necrotic cells can be seen in the diagram figure 7. identification of compounds in n-hex extract and its 80% and 100% fractions via gc-ms data on compounds identified in n-hex extract and its 80% and 100% fractions obtained by injection into gc-ms are available in the following tables 4, 5, and 6. according to obtain results the major components are belong to non-terpenoid compounds. discussion global cancer status around the world using globocan 2018 estimates, the incidence and mortality of cancer by the international agency for research on cancer, with a focus on geographical diversity in 20 regions of the world is estimated that in 2018 about 18.1 million new cancers.24 breast cancer has been diagnosed as the most common cancer among women. diagnosis of cancer and the leading cause of death varies from country to country depending on the rate of economic development and lifestyle-related factors.25 in a review, medicinal plants in iran with anti-cancer effects in different cell lines have been studied. in most studies, phenolic compounds and alkaloids have anticancer effects on various cancers. plants and their active compounds have anti-cancer effects by eliminating free radicals and antioxidant effects, stopping the cell cycle, inducing apoptosis, and inhibiting angiogenesis. therefore, extracts and active compounds of medicinal plants can greatly help researchers and pharmacists in developing new anticancer drugs.26 in the present study, the cytotoxic effects of n-hex, dcm, and meoh extracts of a. nemorosa on mcf-7 and hfff cell lines were investigated for the first time. for this purpose, at the beginning of the work, cytotoxicity and ic50 values obtained from the treatment of these cells with different concentrations of extracts were investigated. the results showed 222 j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells original h. forouhandeh et al. fig. 6 mcf-7 cancer cell viability due to exposure to n-hex, dcm, and meoh extracts of a. nemorosa compared to normal hfff cells in 24 and 48 hours of incubation. fig. 7 flow cytometric test of n-hex extract and some of its fractions on mcf-7 cells and comparison of apoptotic and necrotic cells in mcf-7 cell line. that n-hex extract had significantly (p < 0.001) more cytotoxicity on mcf-7 cancer cells in 24 and 48 hours treatment compared to dcm, meoh extracts and dmso control. in addition, according to the findings of this study, the cytotoxic effect of n-hex extract on mcf-7 cells is dose-dependent and time-dependent with a p-value < 0.001. the percentages of apoptosis and necrosis in n-hex extract are 4.57% and 86.8%, respectively. then, n-hex extract was selected as the most potent extract and fractionation was performed by the vlc method. the 80% and 100% fractions of n-hex extract showed the highest effect of cytotoxicity on the tested cell line compared to other fractions. these fractions with a p-value <0.001 showed more cytotoxicity than control (dmso) at both incubation times (24 and 48 hours). the effect of n-hex extract fractions is dose-dependent and all fractions except 80% and 100% fractions with p-value <0.001 are time-dependent. 80% and 100% fractions did not show significant differences in 24 and 48 hours of treatment. the percentages of apoptosis and necrosis in the 60% fraction are 2.97% and 95.9%, in the 80% fraction 4.36% and 1.37%, and in the 100% fraction 0.98% and 21.4%, respectively. the two fractions 80% and 100% act in a way other than apoptosis and necrosis, which can probably be attributed to other methods such as autophagy. of course, it is 223j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 h. forouhandeh et al. original evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells https://doi.org/10.22317/jcms.v7i3.960 table 5. characteristics of volatile compounds identified in the 80% fraction of n-hex extract of a. nemorosa no. molecular formula ki amount (percentage) retention time (minutes) compounds 1 c 13 h 28 o 1456.2 11.26 25.425 1-tridecanol 2 c 7 h 12 o 2 1488.51 2.04 26.3 cyclopropanecarboxylic, 1-methyl-, ethyl ester 3 c 14 h 30 o 1659.26 2.54 30.608 n-tetradecanol 4 c 14 h 30 o 2 1698.51 25.22 31.558 ethanol, 2-(dodecyloxy)5 c 20 h 38 1835.71 1.26 34.675 neophytadiene 6 c 16 h 32 o 2 1937.35 9.98 36.858 n-hexadecanoic acid 7 c 20 h 40 o 2097 1.78 40.142 phytol 8 c 18 h 32 o 2 2109 1.34 40.258 9,12-octadecadienoic acid (z,z)9 c 22 h 24 o 7 3225 4.96 60.392 deoxypodorhizon 10 c 22 h 22 o 7 3267 4.22 61.558 desoxypodophyllotoxin 11 c 29 h 50 o 3351 3.64 67.45 .gamma.-sitosterol 12 c 29 h 48 o – 7.8 65.117 trans-stigmasta-5,22-dien-3.beta.-ol 72.04 identified 51.38 non-terpenoid 22.66 terpenoid table 4. characteristics of volatile compounds identified in the n-hex extract of a. nemorosa no. molecular formula ki amount (percentage) retention time (minutes) compounds 1 c 10 h 16 o 1148.77 1.10 16.450 (s)-cis-verbenol 2 c 12 h 26 1198.74 1.91 18.000 dodecane 3 c 12 h 16 o 2 1247.81 2.24 19.475 chrysanthenyl acetate 4 c 14 h 3 o 1398.74 9.97 23.867 tetradecane 5 c 15 h 24 1515.81 3.17 25.192 (z)-.beta.-farnesene 6 c 12 h 26 o 1538.27 3.57 25.442 1-dodecanol 7 c 5 h 8 o 2 1619.13 0.97 26.342 (e)2-methylcrotonic acid 8 c 14 h 30 o 1659.94 0.98 30.625 1-tetradecanol 9 c 14 h 30 o 2 1698.8 2.77 31.567 ethylene glycol monododecyl ether 10 c 18 h 38 1798.08 1.39 33.850 octadecane 11 c 20 h 40 o 1836.48 2.59 34.692 trans-phytol 12 c 16 h 32 o 2 1940.57 6.65 36.925 palmitic acid 13 c 20 h 42 o 2 1969.03 2.03 37.517 2-octadecoxyethanol 14 c 18 h 36 o 2 1975.04 0.85 37.642 hexadecanoic acid, ethyl ester 15 c 19 h 4 0 1997.5 1.02 38.108 n-nonadecane 16 c 20 h 40 o 2097 1.76 40.158 phytol 17 c 20 h 36 o 2 2155 1.02 40.917 linoleic acid, ethyl ester 18 c 23 h 46 o 2 2502 1.52 47.417 docosanoic acid, methyl ester 19 c 25 h 50 o 2 2712 3.10 50.808 methyl tetracosanoate 20 c 31 h 64 3100 6.02 59.517 n-hentriacontane 21 c 22 h 24 o 7 3225 2.77 60.417 deoxypodorhizon 22 c 22 h 22 o 7 3267 1.99 61.608 desoxypodopyllotoxin 23 c 18 h 34 o – 2.32 40.317 9,12-octadecadien-1-ol 61.71 identified 35.7 non-terpenoid 26.01 terpenoid 224 j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells original h. forouhandeh et al. table 6. characteristics of volatile compounds identified in 100% fraction of a. nemorosa no. molecular formula ki amount (percentage) retention time (minutes) compounds 1 c 13 h 28 o 1456.2 0.67 25.425 1-tridecanol 2 c 5 h 8 o 2 1491.32 3.68 26.367 (e)2-methylcrotonic acid 3 c 12 h 24 o 2 1539.28 0.68 27.617 lauric acid 4 c 14 h 30 o 2 1698.1 1.90 31.550 2-dodecyloxyethanol 5 c 14 h 28 o 2 1736.81 0.77 32.442 myristic acid 6 c 20 h 38 1835.71 0.61 34.675 neophytadiene 7 c 16 h 32 o 2 1940.19 13.22 36.917 palmitic acid 8 c 14 h 30 o 2 1968.6 12 37.508 laureth-1 9 c 19 h 32 o 2 2092 7.35 40.400 linolenic acid, methyl ester 10 c 19 h 40 o 2176 9.58 42.708 2-nonadecanol 11 c 22 h 24 o 7 3225 3.06 60.383 deoxypodorhizon 12 c 22 h 22 o 7 3276 4.45 61.575 desoxypodophyllotoxin 13 c 18 h 34 o – 5.89 40.317 9,12-octadecadien-1-ol 63.86 identified 55.74 non-terpenoid 8.12 terpenoid worth mentioning that the fraction has a necrosis mechanism 100% relative to itself. furthermore, flow cytometry results showed that in the 80% fraction, the mechanism of cytotoxicity was other than necrosis and apoptosis. the results of statistical analysis showed that the effect of cytotoxicity depends on the concentration of extracts and fractions as, with increasing the concentration of plant samples, the viability of cells decreases. the viability of cells after treatment with meoh and dcm extracts does not depend on the exposure time of cells in the mcf-7 cell line. various studies on the anticancer effects of different species of the genus anthriscus have proven the cytotoxic properties of these plants in the treatment of cancer, and several anti-cancer compounds of this genus have been purified. the essential oil derived from plant a. caucalis was tested on hepg2 and mcf-7 cell lines by mtt method and reported ic50 = 67.50 μg/ml and ic50 = 55.83 μg/ml, respectively, which was also significant in terms of dose and time. its pronounced anti-cancer effect can be attributed to the pronounced presence of two beta-compounds (β-bisabolene) and (z, e)-αfarnesene.27 although the mechanism of extracts and fractions cytotoxicity of a. nemorosa which we discussed was necrosis or other than necrosis and apoptosis, in another study an active compound with antitumor activity was isolated from the root of a. sylvestris and structural studies showed that this compound was deoxy podophyllotoxin (dppt) and its biological activity in human cervical cancer cells (hela) was examined. flow cytometric analysis showed that dppt stops the cell cycle in the g2/m phase before apoptosis, and dppt functional mechanisms include inhibition of tubulin polymerization and activation of caspases 3 and 7.28 another study in a. sylvestris showed that deoxypodophyllotoxin, as the most important constituent of this plant, is converted to epipodophyllotoxin, which is the main substance for the semi-synthesis of cytostatic agents of etoposide and teniposide.29 autophagy plays an important role in the development of this type of cancer, although the exact underlying mechanisms are unknown. the data confirmed that anthracin causes apoptosis in 2 breast cancer cell lines, mcf-7 (estrogen receptor-positive) and mda-mb-231 (estrogen receptor, progesterone receptor, and her2 / neu negative receptor). anthracin reduces the phosphorylated levels of akt and mtorc1 and subsequently inhibits cell growth.30 interestingly, prevention of autophagy by a drug inhibitor or genetic deletion of ulk1 and atg13 accelerates the process of anthracin-induced apoptosis, and autophagy has protective effects on the cell.30 overall, our results suggest that anthracin is an inhibitor of mtor and that a combination of an autophagy inhibitor and anthracin may serve as a promising new strategy for the treatment of breast cancer cells. the predominant escape composition of this extract by phytochemistry by gc-ms is non-terpenoid and these substances are probably responsible for the toxic effects of cancer cells. the most common ingredient in this extract is palmitic acid. in a study on marine algal extract, palmitic acid was identified as a selective anti-cancer compound. this compound induces apoptosis in human leukemia cancer cell lines and has shown an anti-cancer effect on mice in vitro.31 the second major component of n-hex extract is n-hentriacontane, which is an antioxidant and cytotoxic compound against cancer cells.32 in the 80% fraction, tri-decanol and hexadecanoic acid are the main compounds. in previous studies, the cytotoxicity of various alkanes has been investigated, which includes a wide range, and tetradecane has a weaker cytotoxic effect than other alkanes.33 fukuzawa et al., (2008), showed the biologically active compounds, which were isolated from ganoderma 225j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 h. forouhandeh et al. original evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells lucidum spores and studied to inhibit the growth of human promyelocytic leukemia cells (hl60) by several different types of long-chain fatty acids, including hexadecanoic acid. they indicated that hexadecanoic acid can inhibit the growth of this cell line with ic50 = 132 ± 25 μg/ml. 34 in the 100% fraction, it is the major non-terpenoid compound and the most common compound is palmitinic acid. desoxypodophyllotoxin is an important compound among the extracts and fractions. podophyllotoxin, which has a significant anti-cancer effect,35 has been obtained from other genera of the same family as this plant. podophyllotoxin is one of the most popular plant lignans that has been used as the main ingredient in the preparation of potent anticancer drugs such as etoposide and teniposide.36 conclusion in general, it can be concluded that the n-hex extract of a. nemorosa and its 60%, 80%, and 100% fractions showed the greatest effects of inhibiting cancer cell growth on mcf-7 cancer cell lines. it is noteworthy that the mechanism of action of n-hex extract and its 60% fraction on mcf-7 is mainly necrosis and 80% and 100% fractions on mcf-7 are in a path other than apoptosis and necrosis. according to the obtained results, the anti-cell growth effects of the shoots of these plants can be attributed to the n-hex extract, which is probably due to the presence of non-terpenoid compounds. extensive and detailed phytochemical studies can determine the exact amount and nature of these substances in different extracts of the plant. declarations ethics approval and consent to participate there is no involvement of human or animal in this study. consent for publication all other authors declare no conflict of interest. availability of data and materials the authors confirm that the data supporting the findings of this study are available within the article. competing interests we declare that we have no significant competing financial, professional, or personal interests that might have influenced the performance or presentation of the work described in this manuscript. funding this work was supported and funded scheme by faculty of pharmacy (grant no: 61103), tabriz university of medical sciences. authors’ contributions mz and hf performed the experiments; mz, es, om and pa analyzed of data; hf, and vt prepared the manuscript; pa and vt wrote and edited the manuscript; pa designed the experiments; pa led and supervised the project. acknowledgements we would like to thank dr. baradaran for their supportive help in performing the anti-proliferative part.  references 1. mousavi sm, montazeri a, mohagheghi ma, jarrahi am, harirchi i, najafi m, et al. breast cancer in iran: an epidemiological review. the breast journal. 2007;13(4):383-91. 2. zeinomar n, knight ja, genkinger jm, phillips k-a, daly mb, milne rl, et al. alcohol consumption, cigarette smoking, and familial breast cancer risk: findings from the prospective family study cohort (prof-sc). breast cancer research. 2019;21(1):1-14. 3. trichopoulos d, macmahon b, cole p. menopause and breast cancer risk. journal of the national cancer institute. 1972;48(3):605-13. 4. taraphdar ak, roy m, bhattacharya r. natural products as inducers of apoptosis: implication for cancer therapy and prevention. current science. 2001:1387-96. 5. harvey al. natural products in drug discovery. drug discovery today. 2008;13(19-20):894-901. 6. seyfi r, kahaki fa, ebrahimi t, montazersaheb s, eyvazi s, babaeipour v, et al. antimicrobial peptides (amps): roles, functions and mechanism of action. international journal of peptide research and therapeutics. 2020;26(3):1451-63. 7. alipour m, khanmohammadi o. antibacterial activity of plant extracts against oral and skin pathogens. african journal of microbiology research. 2011;5(19):2909-11. 8. fulda s, efferth t. selected secondary plant metabolites for cancer therapy. world journal of traditional chinese medicine. 2015;1(1):24. 9. mohammadzadeh f, monirifar h, saba j, valizadeh m, haghighi ar, zanjani bm, et al. genetic variation among iranian alfalfa (medicago sativa l.) populations based on rapd markers. bangladesh journal of plant taxonomy. 2011;18(2):93-104. 10. safarzadeh e, shotorbani ss, baradaran b. herbal medicine as inducers of apoptosis in cancer treatment. advanced pharmaceutical bulletin. 2014;4(suppl 1):421. 11. elujoba aa, odeleye o, ogunyemi c. traditional medicine development for medical and dental primary health care delivery system in africa. african journal of traditional, complementary and alternative medicines. 2005;2(1):46-61. 12. maikhuri r, nautiyal s, rao k, saxena k. role of medicinal plants in the traditional health care system: a case study from nanda devi biosphere reserve. current science. 1998:152-7. 13. jeong g-s, kwon o-k, park b-y, oh s-r, ahn k-s, chang m-j, et al. lignans and coumarins from the roots of anthriscus sylvestris and their increase of caspase-3 activity in hl-60 cells. biological and pharmaceutical bulletin. 2007;30(7):1340-3. 14. lee sh, son mj, ju hk, lin cx, moon tc, choi h-g, et al. dual inhibition of cyclooxygenases-2 and 5-lipoxygenase by deoxypodophyllotoxin in mouse bone marrow-derived mast cells. biological and pharmaceutical bulletin. 2004;27(6):786-8. 15. sudo k, konno k, shigeta s, yokota t. inhibitory effects of podophyllotoxin derivatives on herpes simplex virus replication. antiviral chemistry and chemotherapy. 1998;9(3):263-7. 16. lin cx, lee e, jin mh, yook j, quan z, ha k, et al. deoxypodophyllotoxin (dpt) inhibits eosinophil recruitment into the airway and th2 cytokine expression in an ova-induced lung inflammation. planta medica. 2006;72(09):786-91. 17. kozawa m, baba k, matsuyama y, kido t, sakai m, takemoto t. components of the root of anthriscus sylvestris hoffm. ii. insecticidal activity. chemical and pharmaceutical bulletin. 1982;30(8):2885-8. 18. jin m, moon tc, quan z, lee e, kim yk, yang jh, et al. the naturally occurring flavolignan, deoxypodophyllotoxin, inhibits lipopolysaccharide-induced inos expression through the nf-κb activation in raw264. 7 macrophage cells. biological and pharmaceutical bulletin. 2008;31(7):1312-5. 19. lin cx, son mj, ju hk, moon tc, lee e, kim sh, et al. deoxypodophyllotoxin, a naturally occurring lignan, inhibits the passive cutaneous anaphylaxis reaction. planta medica. 2004;70(05):474-6. 20. kiso y, konno c, hikino h, yagi y, hashimoto i. liver-protective actions of desoxypodophyllotoxin and its analogs. journal of pharmacobio-dynamics. 1982;5(8):638-41. 226 j contemp med sci | vol. 7, no. 4, july-august 2021: 217–226 evaluation of the cytotoxic activity of anthriscus nemorosa on breast cancer cells original h. forouhandeh et al. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 21. lee sk, kim y, jin c, lee sh, kang mj, jeong tc, et al. inhibitory effects of deoxypodophyllotoxin from anthriscus sylvestris on human cyp2c9 and cyp3a4. planta medica. 2010;76(07):701-4. 22. choi h, lee j, shin h-j, lee b-g, chang i, hwang j-s. deoxypodophyllotoxin reduces skin pigmentation of brown guinea pigs. planta medica. 2004; 70(04):378-80. 23. chen h, jiang h-z, li y-c, wei g-q, geng y, ma c-y. antitumor constituents from anthriscus sylvestris (l.) hoffm. asian pacific journal of cancer prevention. 2014;15(6):2803-7. 24. beheshtirouy s, mirzaei f, eyvazi s, tarhriz v. recent advances on therapeutic peptides for breast cancer treatment. current protein & peptide science. 2020. 25. bray f, ferlay j, soerjomataram i, siegel rl, torre la, jemal a. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca: a cancer journal for clinicians. 2018;68(6):394-424. 26. asadi-samani m, kooti w, aslani e, shirzad h. a systematic review of iran’s medicinal plants with anticancer effects. journal of evidence-based complementary & alternative medicine. 2016;21(2):143-53. 27. lai p, rao h, gao y. chemical composition, cytotoxic, antimicrobial and antioxidant activities of essential oil from anthriscus caucalis m. bieb grown in china. records of natural products. 2018;12(3):290-4. 28. yong y, shin sy, lee yh, lim y. antitumor activity of deoxypodophyllotoxin isolated from anthriscus sylvestris: induction of g2/m cell cycle arrest and caspase-dependent apoptosis. bioorganic & medicinal chemistry letters. 2009;19(15):4367-71. 29. olaru ot, niţulescu gm, orțan a, dinu-pîrvu ce. ethnomedicinal, phytochemical and pharmacological profile of anthriscus sylvestris as an alternative source for anticancer lignans. molecules. 2015;20(8):15003-22. 30. jung ch, kim h, ahn j, jung sk, um my, son k-h, et al. anthricin isolated from anthriscus sylvestris (l.) hoffm. inhibits the growth of breast cancer cells by inhibiting akt/mtor signaling, and its apoptotic effects are enhanced by autophagy inhibition. evidence-based complementary and alternative medicine. 2013;2013. 31. harada h, yamashita u, kurihara h, fukushi e, kawabata j, kamei y. antitumor activity of palmitic acid found as a selective cytotoxic substance in a marine red alga. anticancer research. 2002;22(5):2587-90. 32. kim sj, chung ws, kim ss, ko sg, um jy. antiinflammatory effect of oldenlandia diffusa and its constituent, hentriacontane, through suppression of caspase-1 activation in mouse peritoneal macrophages. phytotherapy research. 2011;25(10):1537-46. 33. yang j-h, lee c-h, monteiro-riviere na, riviere je, tsang c-l, chou c-c. toxicity of jet fuel aliphatic and aromatic hydrocarbon mixtures on human epidermal keratinocytes: evaluation based on in vitro cytotoxicity and interleukin-8 release. archives of toxicology. 2006;80(8):508-23. 34. fukuzawa m, yamaguchi r, hide i, chen z, hirai y, sugimoto a, et al. possible involvement of long chain fatty acids in the spores of ganoderma lucidum (reishi houshi) to its anti-tumor activity. biological and pharmaceutical bulletin. 2008;31(10):1933-7. 35. van uden w, bos ja, boeke gm, woerdenbag hj, pras n. the large-scale isolation of deoxypodophyllotoxin from rhizomes of anthriscus sylvestris followed by its bioconversion into 5-methoxypodophyllotoxin β-dglucoside by cell cultures of linum flavum. journal of natural products. 1997;60(4):401-3. 36. lv m, xu h. recent advances in semisynthesis, biosynthesis, biological activities, mode of action, and structure-activity relationship of podophyllotoxins: an update (2008-2010). mini reviews in medicinal chemistry. 2011;11(10):901-9. doi: https://doi.org/10.22317/jcms.v7i2.940 96 original issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 96 – 101 introduction childhood obesity is one of the major public health challenges, raising concerns for developing serious health conditions.1,2 studies in developing countries and the middle east show an increase in childhood obesity and its growing trend.3, 4 comparative patterns have been observed in iranian children, with 7–12% obesity rate, reported in children aged 6–12 years.5 according to a nationwide study in iran, overall prevalence of elevated body mass index (bmi), including obesity and overweight in children and adolescents has increased over the years.6 potential contributors of increased obesity includes genetic factors, increased energy intake, physical inactivity, environmental, social and psychological factors, neurological disorders, and overeating in childhood.4 obesity has a strong genetic basis, and numerous genetic variants have been associated with its phenotypes.7 genomewide association studies (gwas) have identified several genetic variants that are related with bmi and obesity in adulthood.8 in 2007, the first bmi locus was found in the fat mass and obesity-associated (fto) locus, taken after by melanocortin 4 receptor (mc4r) in 2008, and 18 further loci in 2010.9 while the focus of these studies has been on bmi or obesity in adult populations, reports have showed that some of these genetic variations are also significant associations with childhood bmi.9 leptin gene (lep) and leptin receptor gene (lepr) are commonly studied genes in the field of obesity. several common polymorphisms of both lep and lepr genes (located on chromosome 7q31and 1p31, respectively) have been studied in various populations for their potential association with the development of human obesity and its related complications.10,  11 among these single nucleotide polymorphisms (snps), the lep a19g polymorphism (rs2167270) of the untranslated region of exon 1 affects leptin concentration and has been investigated in detail for its effects on obesity related metabolic disease.12 some studies indicate that the a allele is associated with higher leptin levels and lower bmi13 and it could be concluded that the g variant may be considered a disadvantageous factor in the context of obesity-related traits.14 variants of lepr have also been described to influence leptin receptor activity.12 the first to identify was a snp in the coding region of the leptin receptor gene, gln223arg.15 the gln223arg polymorphism, characterized by an adenine (a) to guanine (g) transition at position 668 (codon 223) in exon 6 in the extracellular domain of lepr results in an amino acid substitution (glutamine with an arginine). this substitution of amino acid glutamine (gln) to arginine (arg) occurs in the intracellular domain of the receptor.16 it has been suggested that the change of gln by an arg causes a change of electric charge from neutral to positive within the protein, which can affect the functional characteristics of the receptor. this seems to be associated with an impaired signaling capacity of the leptin receptor and with higher mean circulating level of leptin.17 the g allele has mainly been connected with increased adiposity, bmi, percent fat mass (% fm), as well as higher circulating insulin and leptin levels.14 but, the results obtained so far are ambiguous. some studies suggested no association between g allele and obesity-related parameters, while the others found the association with a lean phenotype.18, 19 on the other hand, the a allele has also been found to be associated with total abdominal fat mass, increased insulin and leptin levels, and higher risk of developing type 2 diabetes.20 not all the studies have demonstrated the association between the polymorphisms and obesity.12 previous studies showed genetics factors can affect body responses to weight loss programs.21, 22 however, few studies evaluated the impact of polymorphisms on metabolic lep and lepr polymorphisms influences anthropometric outcome in response to 8 weeks of combined training in obese boys aliakbar jahandideh1*, hadi rohani1, hamid rajabi1,2, mohammad shariatzadeh1, sahar razmjou3 1sport sciences research institute, tehran, iran. 2department of exercise physiology, faculty of physical education and sports sciences, kharazmi university, tehran, iran. 3clinical epidemiology and chronic diseases programs, the ottawa hospital research institute, ottawa, on, canada. *correspondence to: ali akbar jahandideh (e-mail: aliakbarjahandideh3@gmail.com) (submitted: 13 january 2021 – revised version received: 28 january 2021 – accepted: 16 february 2021 – published online: 26 april 2021) abstract objective the purpose of the present study was to investigate whether lep19 g>a and lepr 668 a>g polymorphisms would influence the effect of an 8-week combined aerobic and resistance training. methods thirty obese boys (bmiz>+2) aged 11–13 (12.66±0.47) years were recruited from three middle schools in quchan. the changes in body composition parameters and metabolic factors in response to 8-weeks combined aerobic and resistance training program were analyzed regarding lep and lepr polymorphism. dna was extracted from cheek cells donated by the 30 participants and genotyping was carried out using pcr. results our results suggest that carriers of rs2167270g and rs1137101a allele were characterized by a greater reduction in body mass and waist-to-hip ratio (whr) (p<0.05). also, a significant decrease was observed in leptin levels in carriers of rs2167270g allele after the training program (p=0.031). moreover, the lep and lepr polymorphisms were associated with changes in lipid profile in response to training. conclusions in response to 8 weeks of regular physical activity, obese boys with g (rs2167270) and a (rs1137101) alleles had the best likelihood of losing weight which was associated with a decrease in body mass, fat mass (%), whr and leptin concentrations. keywords body mass index, health promotion, physical activity, genetic, boy 97 original lep and lepr modify post-training losing weightali akbar jahandideh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 96 – 101 parameters changes during weight loss, especially in children.23 therefore, considering that combined aerobic and resistance training has been proposed as an interesting training strategy for maintaining and/or improving health and seems to be an effective strategy for preventing and managing obesity.24 therefore, the aim of our study is to investigate the effect of snps in lep (rs2167270) and lepr (rs1137101) on metabolic response and weight loss after 8-weeks of combined training in obese children boys. methods study design fifty-eight obese boys aged 11–13 years (mean ± sd 12.66 ± 0.47) were recruited from three middle schools in quchan, iran. inclusion criteria for the present study were the following: (a) age from 11 to 13 years old (b) sds-bmi>2 (c) stage i and ii in the tanner classification. sexual maturation was examined using tanner stages at the beginning of the study by a self-reported questionnaire. written instructions and a depiction of pubertal development stages were used as a guide.25 none of these individuals had engaged in regular activity in the previous 2 months. they had no history of any metabolic, diabetes, and cardiovascular diseases. participants were refrained from taking any medications or supplements known to affect metabolism. fifty-eight samples were assessed for lep and lepr gene variations. among those, 30 participants were selected based on representing all three genotypes in each polymorphism (aa, ag, and gg for lep and aa, ga, and gg for lepr). for each participant (30 subjects), blood samples, height and weight, waist-to-hip ratio (whr) and body mass index for sex/age z-score (bmiz) were measured 48 h before and after training program. written informed consent was obtained from the participants and their parents after they had received a detailed explanation of the study aims and procedures. the study was approved by the sport science research institute of iran research (ir.ssrc.rec.1299.111). physical exercise training protocol physical exercises were supervised by professional kinesiologists. training program was three times per week (each session lasted 70 min) during 8 weeks. training program includes 5 min of warm-up (stretching exercises, light running), 30 min of low-intensity rhythmic aerobic movements, 1 set of 7 resistance exercises (dumbbell squat, dumbbells chest press, medicine ball trunk rotation, dumbbell shoulder press, dumbbell bicep curl, band lat pull-down on the chair and dumbbell lunge). five minutes of static and stretching exercises were performed to cool down. in order to observe the principle of overload, 5–10 percent was added to the number of repetitions or activity time every 2 weeks. blood analyses blood sampling was at two stages: 48 h before and after 8 weeks of exercise training program. blood samples were taken after a 12-h overnight fast at 7 am from the elbow vein. blood samples for biochemical analyses were centrifuged 300 × g for 15 min at room temperature in order to receive blood plasma. blood plasma was used to determine lipid profile levels including: triglycerides (tg), total cholesterol (tc), high-density lipoprotein cholesterol (hdl-c), low-density lipoprotein cholesterol (ldl-c) (using assay kits from pars azmoon company, iran), and leptin concentrations (mecrodia elisa kits, sweden). all samples were measured in duplicate. genetic analyses participants were asked to refrain from eating or drinking 30 min prior to saliva collection. each individual was instructed to rub their tongues around the inside of their mouths for about 30 sec, and then expectorate approximately 2 ml saliva into the tube. dna was extracted from cheek cells of 58 saliva samples using the oragen company kit. the pcr product of 5’-cagttgcgcaagttgtgatcg-3’ (forward) and 5’-gctggttctgcaaggtcctg-3’ (reverse) for rs2167270 and 5’ gaatgtcttgtgcctgtgcca-3’ (forward) and 5’-gaagccactcttaatacccccagt-3’ (reverse) for rs1137101 were used. amplicons of 308 bp were visualized with 1% agarose gel. in this step, pcr reaction of temperature gradient was used for each pairs of primers designed in order to find the best primer annealing temperature. after completing the stopping steps, 3 µl of pcr product to ensure optimal replication on 1% agarose gel was examined. after identifying the appropriate temperature for all three pairs of pcr synthesized primers of all samples, 2 µl of pcr product was brought to 0.1% agarose gel to confirm the correct replication of the desired fragment. restriction fragment length polymorphism method was used for determining the genotype of the lep and lepr genes. enzymatic digestion at 65 °c overnight consisted of 1 µl of the enzyme, 3 µl of pcr product, 2 µl of the specific buffer, and 15 µl of deionized water. after enzymatic digestion, the enzyme digestion product was electrophoresed on 12% polyacrylamide gel to observe the cut fragments, and three samples with different genotypes were sent to sina codun for sequencing. then, the digested dna (lep rs2167270: 330 (252-77) g/a, lepr rs1137101: 274 (189-85) a/g) was visualized using a gel documentation system. statistical analysis the kolmogorov–smirnov test of data was used to evaluate the distribution for normality. allele frequencies were determined by gene counting. to test the influence of the lep a19g (rs2167270) and lepr a66g (rs1137101) polymorphisms on training response, two way mixed analysis of variance (anova) was used with genotype as between subject factor and training program as within subject factor. follow-up verification was made with the bonferroni post hoc test. the hardy–weinberg equilibrium analysis was evaluated using a chi-square test with one degree of freedom. the level of statistical significance was set at p < 0.05. statistical analysis was carried out with ibm spss statistics for windows version 21.0. results lep a19g (rs2167270) and lepr a66g (rs1137101) polymorphisms both polymorphisms conformed to hardy–weinberg expectations (chi-square = 0.5776, p = 0633; chi-square = 0.465, p = 0.55, for the lep rs2167270 and lepr rs1137101, respectively). two-way mixed anova revealed a significant effect of the lep rs2167270 genotype for whr (sig = 0.0.34, f = 3.831); nevertheless, no significant effect of lepr rs1137101 genotype was observed on measurement variables (tables 1, 2). https://www.google.com/search?q=dumbbells+chest+press&spell=1&sa=x&ved=2ahukewjcnzrz7ldpahxp-sokhfp_cmwqkeeckab6bagqecm https://www.google.com/search?q=,+dumbbell+shoulder+press&spell=1&sa=x&ved=2ahukewjtke6n7bdpahwbloskha_ld-kqkeeckab6bagtecm 98 original lep and lepr modify post-training losing weight ali akbar jahandideh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 96 – 101 values are presented as mean ± standard deviation; bmi: body mass index; fm: body fat percentage; whr: waist-tohip ratio; tc: total cholesterol; tg: triglycerides; hdl-c: high-density lipoprotein cholesterol; ldl-c: low-density lipoprotein cholesterol. ns: not significant; p-value significantly different (p<0.05). values are presented as mean ± standard deviation; bmi: body mass index; fm: body fat percentage; whr: waist-tohip ratio; tc: total cholesterol; tg: triglycerides; hdl-c: high-density lipoprotein cholesterol; ldl-c: low-density lipoprotein cholesterol. ns: not significant; p-value significantly different (p<0.05). table 1. the lep rs2167270 genotypes and response to training (two-way mixed anova). parameter gg (n = 13) ag (n = 12) aa (n = 5) two way mixed anova p-value pretraining posttraining pre-training posttraining pretraining posttraining genotype training genotype × training interaction body mass (kg) 77.6 ± 7.58 74.2 ± 7.7 76.4 ± 10.2 75.1 ± 10.2 87.1 ± 15.6 86.4 ± 15.3 0.1 <0.01* 0.032* fm (%) 32.1 ± 4.7 29.3 ± 4.7 33.3 ± 4.07 31.4 ± 4.7 34.3 ± 2.6 33.6 ± 3.1 0.32 <0.01* 0.15 bmiz 2.13 ± 0.12 2.02 ± 0.15 2.1 ± 0.09 2.06 ± 0.15 2.22 ± 0.17 2.21 ± 0.15 0.13 <0.01* 0.008* whr 0.93 ± 0.03 0.91 ± 0.04 0.95 ± 0.02 0.95 ± 0.03 0.98 ± 0.05 0.98 ± 0.04 0.034* <0.01* 0.049* tg (mg/dl) 196.61 ± 42.4 182.84 ± 34.7 192.66 ± 35.7 164.16 ± 32.6 201.4± 19.35 181 ± 18.47 0.61 <0.01* 0.41 hdl-c (mg/dl) 40.46 ± 4.75 45.07 ± 5.54 39.71 ± 4.59 42 ± 4.8 39.2 ± 3.11 42.6 ± 3.57 0.09 0.093 0.059 ldl-c (mg/dl) 105.07 ± 31 97.84 ± 30.3 108.58 ± 33.4 96.75 ± 27.6 110.8 ± 14.6 92.4 ± 9.07 0.99 <0.01* 0.44 tc (mg/dl) 198.07 ± 40.8 179.92 ± 33.3 188.16 ± 38.6 168.66 ± 34.8 190.6 ± 22.1 180.4 ± 23.7 0.72 <0.01* 0.8 leptin (ng/ ml) 13.17 ± 1.42 11.09 ± 1.83 13.62 ± 1.61 11.71 ± 1.57 14.11 ± 1.41 13.53 ± 1.44 0.12 <0.01* 0.03* values are presented as mean ± standard deviation; bmi: body mass index; fm: body fat percentage; whr: waist-to-hip ratio; tc: total cholesterol; tg: triglycerides; hdl-c: high-density lipoprotein cholesterol; ldl-c: low-density lipoprotein cholesterol. ns: not significant; p-value significantly different (p<0.05). table 2. the lepr rs1137101 genotypes and response to training (two-way mixed anova). parameter aa (n = 10) ga (n = 13) gg (n = 7) two way mixed anova p-value pretraining posttraining pretraining posttraining pre-training posttraining genotype training genotype × training interaction body mass (kg) 82.07 ± 11.6 78.43 ± 12.83 77.5 ± 9.03 75.7 ± 9.09 76.4 ± 12.12 75.69 ± 12.1 0.66 <0.01* 0.04* fm (%) 32.96 ± 4.14 30.25 ± 5.42 32.02 ± 4.24 31.4 ± 4.7 34.88 ± 3.84 33.44 ± 3.85 0.28 <0.01* 0.5 bmiz 2.17 ± 0.14 2.06 ± 0.21 2.13 ± 0.12 2.07 ± 0.13 2.09 ± 0.91 2.08 ± 0.14 0.93 <0.01* 0.06 whr 0.95 ± 0.54 0.93 ± 0.06 0.95 ± 0.03 0.94 ± 0.04 0.95 ± 0.02 0.95 ± 0.02 0.95 <0.01* 0.03* tg (mg/dl) 196.1 ± 29.39 172 ± 33.07 192.46 ± 45.32 180.38 ± 31.34 201.7 ± 27.87 169.57 ± 35.65 0.98 <0.01* 0.26 hdl-c (mg/dl) 40.3 ± 3.52 43.6 ± 5.98 42.3 ± 3.72 43.76 ± 4.18 39.65 ± 4.57 40 ± 5.44 0.22 0.07 0.44 ldl-c (mg/dl) 105.4 ± 28.22 92.2 ± 17.05 106.07 ± 29.68 101.9 ± 28.02 112.85 ± 33.92 95.42 ± 34.26 0.84 <0.01* 0.14 tc (mg/dl) 193.7 ± 33.57 175.1 ± 37.65 191.3 ± 41.67 180.84 ± 24.95 194.57 ± 36.52 166.14 ± 38.2 0.93 <0.01* 0.34 leptin (ng/ml) 13.74 ± 1.29 11.74 ± 1.21 12.97 ± 1.58 11.26 ± 2.18 14.17 ± 1.39 12.65 ± 1.74 0.2 <0.01* 0.68 values are presented as mean ± standard deviation; bmi: body mass index; fm: body fat percentage; whr: waist-to-hip ratio; tc: total cholesterol; tg: triglycerides; hdl-c: high-density lipoprotein cholesterol; ldl-c: low-density lipoprotein cholesterol. ns: not significant; p-value significantly different (p<0.05). 99 original lep and lepr modify post-training losing weightali akbar jahandideh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 96 – 101 there was a significant effect of interaction between training × genotypes (rs2167270) on body mass (p = 0.032, f  = 3.934), whr (p = 0.049, f = 3.38) and leptin hormone (p = 0.031, f = 3.95). reduction in the gg allele in response to training was greater than the other two alleles (ag and aa) (fig. 1, table 1). furthermore, there was a significant effect interaction between training × rs1137101 leptin receptor gene on body mass (p = 0.04, f = 3.625) and whr (p  =  0.037, f  =  3.75). as opposed to ag and gg, aa homozygotes demonstrated a training-related greater reduction in body mass and whr (fig. 2, table 2). we observed positive effect of training on the other lipid profile markers in carriers of different alleles, although it was not significant (tables 1, 2). discussion lifestyle interventions, including increased physical activity, are recommended as an effective treatment for weight loss as well as to improve biochemical parameters in obese boys and adolescents.26 our genetic study was designed to test whether variation in the lep and lepr genes can modulate changes in selected body mass, body composition, and leptin hormone following 8 weeks of supervised combined training in obese boys. an important finding of this study was the significant interaction between training and the lep rs2167270 genotype for body mass, bmiz, whr, and leptin level. we also find a significant interaction between training and the lepr rs1137101 genotype for body mass and whr. a training-related decrease in the lep gg and lepr aa homozygotes differed significantly from the change in other alleles. therefore, the results of our study support the assumption that lep and lepr genes variation play an important role in inter-individual changes of body mass, fat mass, lipid profile, and leptin hormone in response to regular physical activity. given the important role of leptin in the regulation of energy metabolism, we suggest that genetic variants of lep and lepr may modulate physiological responses to lifestyle interventions and its influence on health in pre-pubertal obese boys. when analyzed separately, lowest leptin levels were observed in people with g homozygous (rs2167270) at 7 there was a significant effect of interaction between training × genotypes (rs2167270) on body mass (p = 0.032, f = 3.934), whr (p = 0.049, f = 3.38) and leptin hormone (p = 0.031, f = 3.95). reduction in the gg allele in response to training was greater than the other two alleles (ag and aa) (fig. 1, table 1). furthermore, there was a significant effect interaction between training × rs1137101 leptin receptor gene on body mass (p = 0.04, f = 3.625) and whr (p = 0.037, f = 3.75). as opposed to ag and gg, aa homozygotes demonstrated a training-related greater reduction in body mass and whr (fig. 2, table 2). we observed positive effect of training on the other lipid profile markers in carriers of different alleles, although it was not significant (tables 1, 2). fig. 1. training × genotype lep rs2167270 interaction for body mass, bmiz, whr and leptin level (mean±sd). 0.93 0.95 0.98 0.91 0.95 0.98 0.86 0.88 0.9 0.92 0.94 0.96 0.98 1 gg ag aa w h r (c m ) lep rs2167270 pretraining posttraining 77.6 76.4 87.1 74.2 75.1 86.4 65 70 75 80 85 90 gg ag aa bo dy m as s (k g) lep rs2167270 pretraining posttraining 2.13 2.1 2.22 2.02 2.06 2.21 1.9 1.95 2 2.05 2.1 2.15 2.2 2.25 gg ag aa bm iz lep rs2167270 pretraining posttraining 13.17 13.62 14.11 11.09 11.71 13.53 0 2 4 6 8 10 12 14 16 gg ag aa le pt in (n g/ m l) lep rs2167270 pretraining posttraining fig. 1 training × genotype lep rs2167270 interaction for body mass, bmiz, whr and leptin level (mean±sd). 8 fig. 2. training × genotype lepr rs1137101 interaction for body mass and whr (mean±sd). discussion lifestyle interventions, including increased physical activity, are recommended as an effective treatment for weight loss as well as to improve biochemical parameters in obese boys and adolescents.26 our genetic study was designed to test whether variation in the lep and lepr genes can modulate changes in selected body mass, body composition, and leptin hormone following 8 weeks of supervised combined training in obese boys. an important finding of this study was the significant interaction between training and the lep rs2167270 genotype for body mass, bmiz, whr, and leptin level. we also find a significant interaction between training and the lepr rs1137101 genotype for body mass and whr. a training-related decrease in the lep gg and lepr aa homozygotes differed significantly from the change in other alleles. therefore, the results of our study support the assumption that lep and lepr genes variation play an important role in inter-individual changes of body mass, fat mass, lipid profile, and leptin hormone in response to regular physical activity. given the important role of leptin in the regulation of energy metabolism, we suggest that genetic variants of lep and lepr may modulate physiological responses to lifestyle interventions and its influence on health in pre-pubertal obese boys. when analyzed separately, lowest leptin levels were observed in people with g homozygous (rs2167270) at baseline. these results are consistent with previously published studies that reported obese people with gg homozygotes had lower leptin levels compared to those with heterozygous or homozygous for a allele.13,27 a study by jiang et al. predicted that rs2167270 would change the binding site of the transcription factor and it could affect the transcription of the leptin gene.28 consequently, this snp may affect gene expression at the transcription level, leading to impaired leptin production.28 in addition, participants with g homozygous experienced a greater reduction in leptin levels in response to training which is same as previous studies.10 in a study conducted on 242 european participants, walsh et al. showed that individuals with gg homozygous may gain additional health benefits from expending more energy in training due to their genetic predisposition compared to carriers of the a allele.10 in rs1137101 leptin receptor gene, the lowest leptin level was observed in ga heterozygous at baseline. data on the association between polymorphism in lepr gene and serum leptin levels 82.07 77.5 76.4 78.43 75.7 75.69 72 74 76 78 80 82 84 aa ga gg bo dy m as s (k g) lepr rs1137101 pretraining posttraining 0.95 0.95 0.95 0.93 0.94 0.95 0.92 0.925 0.93 0.935 0.94 0.945 0.95 0.955 aa ga gg w h r (c m ) lepr rs1137101 pretraining posttraining fig. 2 training × genotype lepr rs1137101 interaction for body mass and whr (mean±sd). 100 original lep and lepr modify post-training losing weight ali akbar jahandideh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 96 – 101 baseline. these results are consistent with previously published studies that reported obese people with gg homozygotes had lower leptin levels compared to those with heterozygous or homozygous for a allele.13,27 a study by jiang et al. predicted that rs2167270 would change the binding site of the transcription factor and it could affect the transcription of the leptin gene.28 consequently, this snp may affect gene expression at the transcription level, leading to impaired leptin production.28 in addition, participants with g homozygous experienced a greater reduction in leptin levels in response to training which is same as previous studies.10 in a study conducted on 242 european participants, walsh et al. showed that individuals with gg homozygous may gain additional health benefits from expending more energy in training due to their genetic predisposition compared to carriers of the a allele.10 in rs1137101 leptin receptor gene, the lowest leptin level was observed in ga heterozygous at baseline. data on the association between polymorphism in lepr gene and serum leptin levels have been observed in a number of groups; although they are contradictory. while some studies have reported the association between glutamine allele (q) with higher serum leptin levels in plasma,29, 30 others have found that allele arginine (r) is associated with increased leptin levels.31, 32 at the amino acid level, this polymorphism results in a change in the electric charge from neutral to positive in this position, thereby affecting the performance of the receptor and changing its signaling capacity. in our study, both types of alleles (gg and aa) experienced a decrease in leptin in response to training, and the decrease rate was higher in homozygous aa, although these changes were not significant. whether the regular physical activity can affect leptin production through this polymorphism needs further investigation. leptin has been shown to negatively regulate hepatic function of 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase (hmg-coa) and reduce cholesterol biosynthesis.12 moreover, leptin positively regulates cholesterol catabolism and reduce plasma very-low density lipoprotein cholesterol levels.33 the researchers founded that the lepr polymorphism is associated with significant increase of serum tc, tg, ldl-c, and hdl-c levels in obese and non-obese individuals.12 in our study, boys with gg genotypes (rs1137101) had higher tg and ldl-c and lower hdl-c levels than boys with ag and aa genotypes which is consistent with the results of other studies.34-36 also, boys with aa genotypes (rs2167270) had higher tg, tc and ldl-c and lower hdl-c levels than boys with ga and gg genotypes. hence, our results show that the presence of the g (rs1137101) and a (rs2167270) alleles increases the risk of anthropometric and lipid abnormalities in the obese boys. in response to 8 weeks of combined exercise in obese boys, the greatest improvement in lipid profile was observed in people with genotypes aa (rs2167270) and gg (rs1137101). to our knowledge, there is only one study that examines the interaction between leptin and leptin receptor polymorphisms and changes in lipid profile response to a training program. in a recent study by leońska-duniec et al., highest decrease in ldl-c was observed in young women carrying homozygous aa (rs1137101) following 12 weeks of aerobic exercise.12 it is worth noting that the subjects in our study were obese boys, while the subjects studied by leońska-duniec et al. were young women with normal bmi. another factor could be different type of training protocol (aerobic vs combined training). leptin is commonly thought to be an important hormone in reducing obesity, but studies have shown that obese people have higher leptin levels, resulting in ‘leptin resistance’,37 i.e., increased production of leptin from adipose tissue that occurs in obese people leads leptin-sensitive tissues like skeletal muscle and liver to resist its effects.38 leptin resistance is associated with impaired leptin transport from the blood–brain barrier; hence, jak/stat leptin messaging will be reduced and a suppressor of cytokine-3 messaging will be induced.39 weakening of leptin sensitivity in the brain will result in additional accumulation of tg in adipose tissue, muscle, liver, and pancreas, ultimately leading to impaired insulin sensitivity. exercise plays an important role in improving leptin and insulin sensitivity. since premature puberty in children is one of the side effects of increasing leptin levels, reducing leptin levels through reducing adipose tissue is considered as a positive effect of exercise on obese children.40 moreover, it improves insulin sensitivity and increases glucose transporter type 4 (glut4) by activating amp-activated protein kinase and reduces leptin resistance through lowering leptin levels.35 the present study has a number of limitations that should be noted. first, our study consisted of exclusively male samples; therefore, the results should not be generalized to girls. second limitation was small sample size of this study which may reduce the external validity of the research. further studies with larger sample size in other populations are warranted. conclusion our results showed that a variant of rs2167270 and g variant of rs1137101 may be considered as disadvantageous factor in the context of training-induced effects on body mass traits in obese boys. unfortunately, the results of profile lipid are inconsistent. practical implications the present study provides evidence that dna sequence variations in the lep and lepr genes are associated with the magnitude of the effects of regular physical activity on body mass, whr and leptin concentration among obese boys. therefore, the screening of polymorphisms in the lep and lepr as well as other genetic markers may be useful to identify individuals who are expected to respond well or poorly to exercise. as a result, the main challenge in treating obesity in the near future will be discovering the chains behind genetic communications with obesity and regular physical activity. acknowledgments we thank the technicians at the participating institutions for their time and effort along with the subjects for their participation. conflict of interest. the authors declare no conflict of interest 101 original lep and lepr modify post-training losing weightali akbar jahandideh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 96 – 101 references 1. wijtzes ai, bouthoorn sh, jansen w, franco oh, hofman a, jaddoe vw, et al. sedentary behaviors, physical activity behaviors, and body fat in 6-year-old children: the generation r study. int j behav nutr phys activ. 2014;11:96. 2. ng m, fleming t, robinson m, thomson b, graetz n, margono c, et al. global, regional, and national prevalence of overweight and obesity in children and adults during 1980–2013: a systematic analysis for the global burden of disease study 2013. the lancet. 2014;384(9945):766-81. 3. farrag ns, cheskin lj, farag mk. a systematic review of childhood obesity in the middle east and north africa (mena) region: prevalence and risk factors meta-analysis. adv ped res. 2017;4. 4. samaras k, kelly pj, chiano mn, spector td, campbell lv. genetic and environmental influences on total-body and central abdominal fat: the effect of physical activity in female twins. ann intern med. 1999;130(11):873-82. 5. kelishadi r. childhood overweight, obesity, and the metabolic syndrome in developing countries. epidemiol rev. 2007;29(1):62-76. 6. rahmanian m, kelishadi r, qorbani m, motlagh me, shafiee g, aminaee t, et al. dual burden of body weight among iranian children and adolescents in 2003 and 2010: the caspian-iii study. arch med sc: ams. 2014;10(1):96. 7. wang k, li wd, zhang ck, wang z, glessner jt, grant sf, et al. a genomewide association study on obesity and obesity-related traits. plos one. 2011;6(4):e18939. 8. mărginean co, mărginean c, voidăzan s, meliţ l, crauciuc a, duicu c, et al. correlations between leptin gene polymorphisms 223 a/g, 1019 g/a, 492 g/c, 976 c/a, and anthropometrical and biochemical parameters in children with obesity: a prospective case-control study in a romanian population—the nutrichild study. medicine. 2016;95(12). 9. lakshman r, elks ce, ong kk. childhood obesity. circulation. 2012;126(14):1770-9. 10. walsh s, haddad c, kostek m, angelopoulos t, clarkson p, gordon p, et al. leptin and leptin receptor genetic variants associate with habitual physical activity and the arm body composition response to resistance training. gene. 2012;510(1):66-70. 11. lakka ta, rankinen t, weisnagel sj, chagnon yc, lakka h-m, ukkola o, et al. leptin and leptin receptor gene polymorphisms and changes in glucose homeostasis in response to regular exercise in nondiabetic individuals: the heritage family study. diabetes. 2004;53(6):1603-8. 12. leonska-duniec a, jastrzebski z, jazdzewska a, krzysztof f, cieszczyk p. leptin and leptin receptor genes are associated with obesity-related traits changes in response to aerobic training program. j strength condition res. 2018;32(4):1036-44. 13. hager j, clement k, francke s, dina c, raison j, lahlou n, et al. a polymorphism in the 5’ untranslated region of the human ob gene is associated with low leptin levels. int j obesity. 1998;22(3):200-5. 14. enns je, taylor cg, zahradka p. variations in adipokine genes adipoq, lep, and lepr are associated with risk for obesity-related metabolic disease: the modulatory role of gene-nutrient interactions. j obes. 2011;2011. 15. bruce thompson d, ravussin e, bennett ph, bogardus c. structure and sequence variation at the human leptin receptor gene in lean and obese pima indians. human mol genet. 1997;6(5):675-9. 16. paracchini v, pedotti p, taioli e. genetics of leptin and obesity: a huge review. am j epidemiol. 2005;162(2):101-14. 17. chagnon yc, wilmore jh, borecki ib, gagnon j, pérusse l, chagnon m, et al. associations between the leptin receptor gene and adiposity in middleaged caucasian males from the heritage family study. j clin endocrinol metab. 2000;85(1):29-34. 18. ogawa t, hirose h, yamamoto y, nishikai k, miyashita k, nakamura h, et al. relationships between serum soluble leptin receptor level and serum leptin and adiponectin levels, insulin resistance index, lipid profile, and leptin receptor gene polymorphisms in the japanese population. metabolism. 2004;53(7):879-85. 19. portolés o, sorlí jv, francés f, coltell o, gonzález ji, sáiz c, et al. effect of genetic variation in the leptin gene promoter and the leptin receptor gene on obesity risk in a population-based case-control study in spain. eur j epidemiol. 2006;21(8):605-12. 20. guizar-mendoza j, amador-licona n, flores-martinez s, lopez-cardona m, ahuatzin-tremary r, sanchez-corona j. association analysis of the gln223arg polymorphism in the human leptin receptor gene, and traits related to obesity in mexican adolescents. j human hypertension. 2005;19(5):341-6. 21. bouchard c. gene–environment interactions in the etiology of obesity: defining the fundamentals. obesity. 2008;16(s3):s5-s10. 22. ordovas jm, shen j. gene–environment interactions and susceptibility to metabolic syndrome and other chronic diseases. j periodontol. 2008;79:1508-13. 23. gajewska j, kurylowicz a, ambroszkiewicz j, mierzejewska e, chelchowska m, szamotulska k, et al. adipoq− 11377c> g polymorphism increases the risk of adipokine abnormalities and child obesity regardless of dietary intake. j pediat gastroenterol nutr. 2016;62(1):122-9. 24. alberga as, prud’homme d, sigal rj, goldfield gs, hadjiyannakis s, phillips p, et al. effects of aerobic training, resistance training, or both on cardiorespiratory and musculoskeletal fitness in adolescents with obesity: the hearty trial. appl physiol nutr metab = physiologie appliquee, nutrition et metabolisme. 2016;41(3):255-65. 25. marshall wa, tanner jm. variations in the pattern of pubertal changes in boys. arch dis childhood. 1970;45(239):13-23. 26. ho m, garnett sp, baur l, burrows t, stewart l, neve m, et al. effectiveness of lifestyle interventions in child obesity: systematic review with meta-analysis. pediatrics. 2012;130(6):e1647-e71. 27. poitou c, lacorte j-m, coupaye m, bertrais s, bedel j-f, lafon n, et al. relationship between single nucleotide polymorphisms in leptin, il6 and adiponectin genes and their circulating product in morbidly obese subjects before and after gastric banding surgery. obesity surg. 2005;15(1):11-23. 28. jiang y, wilk j, borecki i, williamson s, destefano a, xu g, et al. common variants in the 5’ region of the leptin gene are associated with body mass index in men from the national heart, lung, and blood institute family heart study. am j human genet. 2004;75(2):220-30. 29. furusawa t, naka i, yamauchi t, natsuhara k, kimura r, nakazawa m, et al. the serum leptin level and body mass index in melanesian and micronesian solomon islanders: focus on genetic factors and urbanization. am j human biol. 2011;23(4):435-44. 30. ali sb, kallel a, sediri y, ftouhi b, feki m, slimene h, et al. lepr p. q223r polymorphism influences plasma leptin levels and body mass index in tunisian obese patients. arch med res. 2009;40(3):186-90. 31. suriyaprom k, tungtrongchitr r, thawnasom k. measurement of the levels of leptin, bdnf associated with polymorphisms lep g2548a, lepr gln223arg and bdnf val66met in thai with metabolic syndrome. diab metab synd. 2014;6(1):6. 32. oliveira rd, cerda a, genvigir fdv, sampaio mf, armaganijan d, bernik mms, et al. leptin receptor gene polymorphisms are associated with adiposity and metabolic alterations in brazilian individuals. arq brasil endocrinol metab. 2013;57(9):677-84. 33. vanpatten s, ranginani n, shefer s, nguyen lb, rossetti l, cohen de. impaired biliary lipid secretion in obese zucker rats: leptin promotes hepatic cholesterol clearance. am j physiol gastroint liver physiol. 2001;281(2):g393-g404. 34. takahashi-yasuno a, masuzaki h, miyawaki t, ogawa y, matsuoka n, hayashi t, et al. leptin receptor polymorphism is associated with serum lipid levels and impairment of cholesterol lowering effect by simvastatin in japanese men. diab res clin pract. 2003;62(3):169-75. 35. shabana n, hasnain s. association of the leptin receptor gln223 arg polymorphism with lipid profile in obese pakistani subjects. nutrition. 2015;31(9):1136-40. 36. becer e, mehmetçik g, bareke h, serakıncı n. association of leptin receptor gene q223r polymorphism on lipid profiles in comparison study between obese and non-obese subjects. gene. 2013;529(1):16-20. 37. daghestani m, purohit r, daghestani m, daghistani m, warsy a. molecular dynamic (md) studies on gln233arg (rs1137101) polymorphism of leptin receptor gene and associated variations in the anthropometric and metabolic profiles of saudi women. plos one. 2019;14(2). 38. steinberg gr, smith ac, wormald s, malenfant p, collier c, dyck dj. endurance training partially reverses dietary-induced leptin resistance in rodent skeletal muscle. am j physiol endocrinol metabol. 2004;286(1):e57-e63. 39. steinberg gr, rush jw, dyck dj. ampk expression and phosphorylation are increased in rodent muscle after chronic leptin treatment. am j physiol endocrinol metab. 2003;284(3):e648-e54. 40. de araujo acc, roschel h, picanço ar, do prado dml, villares smf, de sá pinto al, et al. similar health benefits of endurance and high-intensity interval training in obese children. plos one. 2012;7(8). this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.928 123j contemp med sci | vol. 2, no. 8, autumn 2016: 123–125 research inflammatory status as a contributor for anemia in patients with chronic kidney disease in karbala, iraq hasanain salah al-khayat,a ali mansoor al-ameri,b moaid abdulbari abodea aal-hussein medical city, ministry of health, karbala, iraq. bcollege of medicine, university of karbala, kerbala, iraq. correspondence to ali mansoor al-ameri (email: alimansoor699@gmail.com). (submitted: 16 august 2016 – revised version received: 07 october 2016 – accepted: 10 october 2016 – published online: 26 december 2016) objective in chronic kidney disease (ckd), there is primary deficiency of erythropoietin, thereby leading to anemia. in addition, chronic immune activation and the inflammatory process could be involved in the pathophysiology of anemia in patients with (ckd). however, anemia and inflammation are present in earlier stages of renal impairment. the objective of this study was to evaluate the possible association of some of the markers of inflammation with anemia in patients with ckd with and without dialysis treatment. methods the markers of inflammation such as white blood cell (wbc) count, granulocytes % and c-reactive protein (crp) were determined. similarly, serum albumin, platelets count and hemoglobin level were measured in blood samples from 61 patients with ckd attending nephrology unit at al-hussein medical city and 20 healthy controls. the selected patients were stratified into two subgroups; 31 patients without hemodialysis (hd) and other 30 patients on hd. results a significantly lower hemoglobin level, serum albumin and platelets count were observed in patients with ckd on hemodialysis therapy compared to controls, (p value = 0.0001). there was a significant correlation between wbc count and granulocytes % with regard to the low hemoglobin level in patients with ckd especially those on hemodialysis hd, (p value < 0.05). conclusion renal failure anemia was found to be strongly associated with high wbc count and gronulocytes percentage which are known markers of inflammatory status. key words inflammatory markers, anemia, chronic kidney disease introduction anemia is a well-known complication in patients with chronic kidney disease (ckd). in those patients, there is primary deficiency of erythropoietin production, thereby leading to anemia.1 however, it was stated that erythropoetin deficiency isn’t the only cause of renal anemia and that inflammatory process is one of the causes involved in the pathophysiology of this type of anemia especially in patients with end-stage renal failure and dialysis. however, a controversy was developed in this regards, because anemia and inflammation are present in patients with previous stages of renal failure.2 the objective of this study was to evaluate the possible association of inflammatory status with anemia in patients with ckd with and without hemodialysis (hd) treatment. subjects and methods seventy patients (34 women; and 36 men); age range 21–70 years who attended nephrology unit at al-hussein medical city. the selected patients were doctor-diagnosed with ckd for varying time of disease onset. the patients were recruited to participate in this study during the period from october, 2015 through february, 2016. meanwhile, other 20 healthy persons were chosen as control group. the selected patients were stratified into two subgroups; (31 patients) with ckd without hd (mean s. creatinine 2.87 ± 0.71) and other (30 patients) on regular hd therapy (mean s. creatinine 5.65 ± 1.79). blood specimens were collected from 61 patients and 20 healthy unrelated controls after taking informed consent from each participant. nine patients were excluded because they were found to be crp positive. markers of inflammatory status such as white blood cell (wbc) count, granulocytes % and c-reactive protein (crp) were determined. in addition, serum albumin, platelets count and hemoglobin level were measured in patients’ and controls’ blood. statistical analysis the data were analyzed using the statistic software “graph pad, prism”. results are expressed as mean ± sd. differences of the studied markers among the groups were compared using one-way anova test. to predict the association of different inflammatory markers with anemia in patients with ckd, pearson’s correlation coefficient in normally distributed variables was used. the results with p value < 0.05 were regarded statistically significant. results data of the current study revealed a significantly lower blood hemoglobin level, serum albumin and platelets count in patients with ckd on hd therapy compared to those without hd and the healthy controls, p value = 0.0001 tested by one-way anova, (see fig. 1). about 58% of ckd group and 87% of hd group were found to be anemic. furthermore, there is a significant association of wbc count and granulocytes % with hemoglobin level in patients on hd, p value = 0.0213 and 0.0081, respectively. however, no significant association was observed among all the tested markers with hemoglobin level in the other two groups (ckd without hd and healthy control groups), except an association between serum albumin and hemoglobin level in the control group, see (table 1). discussion anemia is a common complication in the case of kidney function impairment, as it was found in the current study. herein, about 61.7% of the ckd group and 87.8% of the hd group were found to have abnormally low level of blood hemoglobin. likewise, obrador et al. stated that among predialysis patients, 68% of issn 2413-0516 124 j contemp med sci | vol. 2, no. 8, autumn 2016: 123–125 inflammatory status as a contributor for anemia research hasanain salah al-khayat et al. table 1. correlation of hemoglobin level with wbc count, platelets count, granulocyte % and serum albumin in patients with ckd (± hd) and control group 1. patients with ckd hb vs. granulocyte % hb vs. wbc count hb vs. platelets hb vs. serum albumin pearson r 0.3501 0.1197 0.05983 –0.3161 95% confidence interval –0.004 to 0.62 –0.245 to 0.454 –0.3009 to 0.405 –0.6 to 0.04 r squared 0.1226 0.0143 0.0035 0.0999 p value(two-tailed) 0.0535 0.5212 0.7492 0.0888 2. patients with hd pearson r –0.6185 –0.4186 0.1432 0.1256 95% confidence interval –0.847 to –0.196 –0.676 to –0.068 –0.228 to 0.478 –0.26 to 0.48 r squared 0.3826 0.1753 0.02052 0.01577 p value (two-tailed) 0.0081* 0.0213* 0.4501 0.5326 3. control group pearson r 0.4387 0.3276 0.1444 0.5603 95% confidence interval –0.004 to 0.737 –0.134 to 0.672 –0.318 to 0.551 0.15 to 0.8 r squared 0.1924 0.1073 0.02087 0.3139 p value (two-tailed) 0.0530 0.1586 0.5435 0.0102* those with advanced ckd who required renal replacement therapy had a hematocrit less than 30 mg/dl; of these, 51% of patients had a hematocrit less than 28 mg/dl.3 furthermore, although anemia is not as common in the earlier stages of ckd, patients with stage iii disease have a prevalence of concurrent anemia of 5.2%, whereas those with stage iv disease have a prevalence of concurrent anemia of 44.1%.4 data of the current study revealed a significant association between renal failure anemia and some markers of inflammation, namely wbc count granulocytes percentage especially in patients on regular hd therapy. previous studies have widely investigated the role of cellular and molecular markers of inflammation in this type of anemia. it was stated that inflammatory condition is not only a cause of anemia, but that it is the most prevalent cause of temporary resistance to erythropoietin therapy in renal anemia.2,5 the data of the current study revealed abnormally increased number of wbcs (especially granulocytosis) in patients with fig 1. a comparison of hemoglobin level (a), wbc count (b), granulocytes % (c), serum albumin (d) and platelets count (e) in patients with ckd, patients on hd and control group. key ckd chronic kidney disease; hd hemodialysis; hb hemoglobin. a b c d e hasanain salah al-khayat et al. 125j contemp med sci | vol. 2, no. 8, autumn 2016: 123–125 research inflammatory status as a contributor for anemia ckd, and it is significantly associated with anemia in these patients. several molecular factors contribute to polymorphonuclear (pmn) leukocytosis by inhibiting apoptosis potential of these inflammatory cells. some of these molecules are free immunoglobulin light chains (iglcs) as pmnl apoptosis inhibiting proteins,6 phenylacetic acid7 and p-hydroxy-hippuric acid,8 an erythrocyte plasma membrane ca2+-atpase inhibitor accumulating in uremic sera,9 attenuate pmnl apoptosis. the complement factor c5a also delays apoptosis of human pmnls.10,11 the role of leukocytosis in renal anemia could be summarized by the increased expression of pro-inflammatory cytokines which interfere with erythropoietin action in varying mechanisms including competitive inhibition due to receptor domain similarity. an increased level of the proinflammatory cytokine, interferon gamma, was detected in renal failure and some sort of competition for common receptors between erythropoietin and interferon gamma was suggested.2,12 similarly, it was found that inflammatory factors contribute to anemia in renal patients in all the stages of renal failure and cytokines like interleukin 1 and 6 were associated with anemia in those patients.13 another proposed role of the inflammatory cytokines, such as interleukins (il-1 and il-6), and tumor necrosis factor (tnf-alpha), is that they are believed to cause the destruction of rbc precursors and decrease the number of erythropoietin receptors on progenitor cells.14 data analysis of this study also express significantly reduced level of serum albumin especially in hd group. this finding is consistent with many previous studies which investigated this state of dialysis-associated hypoalbuminemia. different physiologic mechanisms and suggestions to alleviate this condition were suggested by such studies.15–25 in addition, an association of serum albumin level with hemoglobin level in healthy control group. this finding could be due to confounding results related to inadequately small sample size of the study group. however, a previous finding in patients with ckd was detected, in which the author found an association between serum albumin and responsiveness to erythropoietin in renal failure anemia and the author recommended further investigation regarding this association.2 conclusion owing to the above data, renal failure anemia was found to be strongly associated with high wbc count and gronulocytes percentage which are known markers of inflammatory status. recommendation future studies with larger sample size are recommended to investigate other cellular and molecular markers of inflammatory status namely t-h1 derived cytokines and their role in “renal” anemia. conflict of interest none. n references 1. mcclellan w, aronoff sl, bolton wk, et al. the prevalence of anemia in patients with chronic kidney disease. curr med res opin. 2004;20: 1501–1510. 2. eschbach jw. anemia management in chronic kidney disease: role of factors affecting epoetin responsiveness. j am soc nephrol. 2002;13: 1412–1414. 3. obrador gt, ruthazer r, arora p, kausz at, pereira bj. prevalence of and factors associated with suboptimal care before initiation of dialysis in the united states. j am soc nephrol. 1999;10:1793–800. 4. center for disease control and prevention. prevalence of chronic kidney disease and associated risk factors—united states, 1999–2004. mmwr morb mortal wkly rep. 2007;56:161–165. 5. atanasiu v, manolescu b, stoian i. hepcidin the link between inflammation and anemia in chronic renal failure. rom j intern med. 2006;44:25–33. 6. cohen g, rudnicki m, deicher r, hörl wh. immunoglobulin light chains modulate polymorphonuclear leucocyte apoptosis. eur j clin invest. 2003;33:669–676. 7. cohen g, raupachova j, hörl wh. the uraemic toxin phenylacetic acid contributes to inflammation by priming polymorphonuclear leucocytes. nephrol dial transplant. 2012. 8. cohen g, raupachova j, wimmer t, deicher r, hörl wh. the uraemic retention solute para-hydroxy-hippuric acid attenuates apoptosis of polymorphonuclear leukocytes from healthy subjects but not from haemodialysis patients. nephrol dial transplant. 2008;23:2512–2519. 9. jankowski j, tepel m, stephan, n, van der giet m, breden v, zidek w, et al. characterization of p-hydroxy-hippuric acid as an inhibitor of ca2+-atpase in end-stage renal failure. kidney int. 2001;78:s84–s88. 10. perianayagam mc, balakrishnan vs, king aj, pereira bj, jaber bl. c5a delays apoptosis of human neutrophils by a phosphatidylinositol 3-kinasesignaling pathway. kidney int. 2002;61:456–463. 11. perianayagam mc, balakrishnan vs, pereira bj, jaber bl. c5a delays apoptosis of human neutrophils via an extracellular signal-regulated kinase and bad-mediated signaling pathway. eur j clin invest. 2004;34:50–56. 12. ali mj, jaafer kn, baqur as. a study of erythropoietin\interferon-γ interactions in response to leishmania donovani antigens in chronic renal failure, an msc thesis, 2006. 13. de lima ga, mazzali m, gentil af, plotegher l, grotto hz. anemia in chronic renal disease: evaluation of inflammatory activity on erythropoiesis and iron metabolism in patients not submitted to dialysis treatment. clin lab. 2012;58:695–704. 14. means rt. recent developments in the anemia of chronic disease. curr hematol rep. 2003;2:116–121. 15. eustace ja, coresh j, kutchey c, et al. randomized double blind trial of oral essential amino acids for dialysis-associated hypoalbuminemia. kidney int. 2000;57:2527–2658. 16. bronich l, te t, shetye k, et al. successful treatment of hypoalbuminemic hemodialysis patients with a modified regimen of oral essential amino acids. j ren nutr. 2001;11:194–201. 17. leon jb, majerle ad, soinski ja, et al. can a nutrition intervention improve albumin levels among hemodialysis patients? a pilot study. j ren nutr. 2001;11:9–15. 18. kaysen ga, dubin ja, müller hg, et al. levels of alpha1 acid glycoprotein and ceruloplasmin predict future albumin levels in hemodialysis patients. kidney int. 2001;60:2360–2366. 19. kaysen ga, chertow gm, adhikarla r, et al. inflammation and dietary protein intake exert competing effects on serum albumin and creatinine in hemodialysis patients. kidney int. 2001;60:333–340. 20. kaysen ga, dubin ja, müller hg, et al. relationships among inflammation nutrition and physiologic mechanisms establishing albumin levels in hemodialysis patients. kidney int. 2002;61:2240–2249. 21. pupim lb, kent p, caglar k, et al. improvement in nutritional parameters after initiation of chronic hemodialysis. am j kidney dis. 2002;40:143–151. 22. guarnieri g, antonione r, biolo g. mechanisms of malnutrition in uremia. j ren nutr. 2003;13:153–157. 23. neyra r, chen ky, sun m, et al. increased resting energy expenditure in patients with end-stage renal disease. j parenter enteral nutr. 2003;27:36–42. 24. johansen kl, kaysen ga, young bs, et al. longitudinal study of nutritional status, body composition, and physical function in hemodialysis patients. am j clin nutr. 2003;77:842–846. 25. hasegawa t, bragg-gresham jl, pisoni rl, robinson bm, fukuhara s, akiba t, et al. changes in anemia management and hemoglobin levels following revision of a bundling policy to incorporate recombinant human erythropoietin. kidney int. 2011;79:340–346. 28 original issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 28 – 33 introduction the wrong use of antibiotics in medicine has been considered a principal start leading to the emergence of bacteria resist to many antibiotics (multidrug resistance).1,2 because of the main sources for transmission of extended-spectrum β-lactamase (esbl)-producing bacteria to humans are animals, hence some measures were raised recently to reduce antimicrobial agents use in husbandry of animal in europe.3 this esbls may be transmitted either directly or indirectly by contaminated meat products consumption.4 esbls bacteria can hydrolyze cephalosporin and penicillin antibiotics by production of β-lactamases enzymes. so, gram-negative bacteria in the intestine belonging to enterobacteriaceae becomes resistant to these antibiotic classes when they possess an esbl gene. esbls are prevalent globally with more than 1.5 billion people colonized enterobacteriaceae especially with esbls.5 most of this load falls on the developing countries, but the pervasiveness of organisms producing esbls increased in the developed countries. there are many esbls groups with the same attitude but different in evolution. the largest ones are tem and shv β-lactamases mutants. some critical amino acids were affected by the mutation resulting in enlargement in active sites and enable it to shield the ring of β-lactam by deflecting the oxyimino substitutes.6 ctx-m enzymes is the second largest group. these are divided into five subgroups based on sequence homology. enterobacteriaceae (especially e. coli) that produce the ctx-m enzymes have been identified to cause urinary tract causing infections.7,8 many studies reported the esbls ctx-m-type is the most frequent esbl type worldwide.6 esbls are enzymes leading to increase resistance to aztreonam, cefotaxime, ceftazidime, cephalosporins, and penicillin, while clavulanic acid inhibits them. the ctx-m, tem, and shv are the three major types of esbls. the ctx-m is more prevalent than tem and shv has a distribution among a broad range of clinically important bacteria.7,8 the critical patients are susceptible to infection particularly, and the nature of causative agents and epidemiology can very extremely. the pathogens that are drug-resistant are considered as a source of concern as they carry a higher mortality and morbidity, and are difficult to be routinely identified by laboratory assays. this leads to delay in diagnosis and finding the appropriate therapy by antimicrobial. there is also a rising worry regarding the new antibiotics deficiency,9 especially for esbls gram-negative bacteria. drug-resistant strains of enterobacteriaceae are important10; thus, the aim of the present study was to determine the enterobacteriaceae strains among patients who are suffering from diarrhea, phenotypic, and genotypic characterization of esbl-producing isolates in jeddah, saudi arabia. extended-spectrum β-lactamase enterobacteriaceae from patients in jeddah, saudi arabia: antibiotic susceptibility and molecular approaches wafa a. alshehri1, tarek a. a. moussa2* 1 department of biology, college of science, university of jeddah, jeddah, saudi arabia. 2 botany and microbiology department, faculty of science, cairo university, giza, egypt. *correspondence to: wafa a. alshehri (e-mail: waalshehri@uj.edu.sa) (submitted: 23 november 2020 – revised version received: 05 december 2020 – accepted: 19 december 2020 – published online: 26 february 2021) abstract objective this study aimed to determine the enterobacteriaceae strains among patients who are suffering from diarrhea, phenotypic, and genotypic characterization of extended-spectrum β-lactamase (esbl)-producing isolates in jeddah, saudi arabia. methods stool samples were collected and cultured to determine the different enterobacteriaceae strains. pcr was done for the isolates to detect the different esbls genes and antibiotic susceptibility against different antibiotics. results the total number of patients in this study was 200 (114 males [57%] and 86 females [43%]). the patients were categorized to teenagers (21, 10.5%), adults (92, 46%), middle age (25, 12.5%), and elderlies (25, 12.5%) according to age. five enterobacteriaceae strains were found: enterobacter cloaca (7, 3.5%), escherichia coli (111, 55.5%), klebsiella pneumoniae (75, 37.5%), proteus mirabilis (6, 3.0%), and pseudomonas aeruginosa (1.0, 0.5%). p. aeruginosa was absent in all female patients under investigation. the response of the isolated (e. coli, k. pneumoniae, and e. cloaca) strains to ampicillin, cefotaxime, cefotaxime-clavulanate, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, nalidixic acid, streptomycin, tetracycline, and trimethoprim-sulphamethoxazole was highly resistant, while the response was highly susceptible to ampicillin sulbactam, ceftazidime, ceftazidime-clavulanate, and imipenem. the most frequent gene was bla ctx-m (195) followed by bla tem (149), bla shv (73), and bla oxa (3), while the highest pair of genes in the same organism was bla tem +bla ctx-m (134) followed by bla shv +bla ctx-m (64), bla shv +bla tem (52), and the least pairs were bla tem +bla oxa (3) and bla ctx-m +bla oxa (2). bla shv +bla ctx-m +bla tem was found in 44 organisms and bla ctx-m +bla tem +bla oxa in 2 organisms only. the bla shv +bla oxa , bla shv +bla ctx-m +bla oxa , bla shv +bla tem +bla oxa , and bla ctxm +bla tem +bla oxa +bla shv were not present in any organism under investigation. conclusions in teenager group, there were no organism that contained bla oxa gene, while bla oxa was present in e. cloaca and p. mirabilis only. bla shv gene was absent in e. coli but present in e. cloaca and k. pneumoniae. the most susceptible group to infection with enterobacteriaceae was adults’ group, while teenage was more resist to infection. keywords esbls, antibiotics, molecular, virulence genes, enterobacteriaceae 29 original extended-spectrum β-lactamase enterobacteriaceae from patients in jeddah, saudi arabiawafa a. alshehri, tarek a. a. moussa j contemp med sci | vol. 7, no. 1, january-february 2021: 28 – 33 materials and methods sample collection and isolation stool samples were collected from 200 diarrheal patients at different hospitals in jeddah, saudi arabia. the samples were transferred to the laboratory within 2 h for isolation of bacteria. the samples were streaked on macconkey agar with crystal violet plates (difco, detroit, mi, usa). after incubation at 37°c overnight, the colonies were picked up and transferred to new plates. the purified isolates were identified biochemically by the api 20e (biomerieux, france). antibiotic susceptibility and detection of esbls the susceptibility of isolated bacteria to antibiotics was tested by the kirby-bauer agar disc diffusion method. susceptibility to ampicillin (amp, 10 μg), amoxicillin/clavulanate (amc, 30 μg), ampicillin sulbactam (sam, 20 μg), cefotaxime (ctx, 30 μg), cefotaxime/clavulanate (ctl, 30 μg/10 μg), ceftazidime (caz, 30 μg), ceftazidime/clavulanate (cal, 30/10 μg), ceftriaxone (cro, 30 μg), cephalothin (cef, 30 μg), chloramphenicol (chl, 30 μg), ciprofloxacin (cip, 5 μg), imipenem (ipm, 10 μg), nalidixic acid (nal, 10 μg), streptomycin (str, 10 μg), tetracycline (tet, 30 μg), and trimethoprim-sulphamethoxazole (sxt, 25 μg) was determined according to the criteria of the clinical laboratory standards institute.11 all antibiotics were purchased from oxoid (italy). the double-synergy test was used to screen the esbl activity of the isolated bacteria.12 characterization of esbl producing bacteria genomic dna was extracted from the esbl producing bacteria according to the manufacturer instructions using mericon  dna bacteria (plus) kit (qiagen, valencia, ca, usa). the encoding genes of β-lactamase enzymes was amplified using the primers as follows: blactx-m f-atgtgcagyaccagtaargtkatggc, r-tgggtraartargts accagaaycagcgg (593 bp); blaoxa f-acacaatacatatcaacttcgc, ragtg tgtttagaatggtgatc (813 bp); blashv f-ctttatcggccctcactcaa, r-aggtg ctcatcatgggaaag (327 bp); blatem f c g c c g c a t a c a c t a t t c t c a g a a t g a , r-acgctcaccggctccagatttat (445 bp). pcr was then performed in total volume (50 µl) reaction mixture: dna templet (50 ng), dntps (0.25mm), mgcl2 (1.5 mm), pfu dna polymerase (0.2 u), primers (50 pmol) and complete to 50 µl with distilled h2o. the temperature profile included an initial denaturation step at 95°c for 10 min, followed by 35 cycles of 95°c for 30 s, 55°c for 1 min, and 72°c for 1 min and a final extension step at 72°c for 7 min. results in modern medicine, one of the greatest challenges is antimicrobial resistance.13,14 the most used classes in the infection treatment caused by gram-negative pathogenic bacteria are the combination of β-lactam/β-lactamase, cephalosporins, and fluoroquinolones inhibitor due to their safety, available in both oral forms and parenteral, and efficacy. the resistance to these agents would restrict the empiric treatment efficacy of gram-negative infections and also, limit the options of their treatment. in this study, the total patient’s number was 200 (114 males [57%] and 86 females [43%]) (fig. 1). the patients were categorized to teenagers 13–19 years (21, 10.5%), adults 20–40 years (92, 46%), middle age 41–59 years (25, 12.5%) and elderlies 60 < years (25, 12.5%). it was found that the number of females was greater than males in teenager category only (15 vs 6), while the number of males was greater than females in the rest three categories (table 1, fig. 2). five enterobacteriaceae strains was found in the samples collected where enterobacter cloaca (7, 3.5%), escherichia coli (111, 55.5%), klebsiella pneumoniae (75, 37.5%), proteus mirabilis (6, 3.0%), and pseudomonas aeruginosa (1.0, 0.5%) (table 2). e. coli and k. pneumoniae were the most dominant enterobacteriaceae strains in the patients under investigation. the prevalence of e. coli was noticed among males of adults, middle age, and elderlies categories in comparing to the other four strains. p. aeruginosa was absent in all female patients under investigation, also it is noticed that the high prevalence of e. cloaca in both female and male in middle age category (table 3, fig. 3). fig. 1 percentage of male and female in the collected samples. (n=8665). table 1. age grouping of the patients suffering from diarrhea. category age group total female male number % number % number % teenager 13-19 21 10.5 15 71.4 6.0 28.6 adults 20-40 92 46.0 38 41.3 54 58.7 middle age 41-59 62 31.0 25 40.3 37 59.7 elderlies 60 < 25 12.5 8.0 32.0 17 68.0 total 200 100 86 43.0 114 57.0 30 original extended-spectrum β-lactamase enterobacteriaceae from patients in jeddah, saudi arabia wafa a. alshehri, tarek a. a. moussa j contemp med sci | vol. 7, no. 1, january-february 2021: 28 – 33 fig. 2 percentage of male and female in the different category. male: outer circle and female inner circle. table 3. distribution of esbls organisms among the group categories. category e. coli k. pneumoniae e. cloacae p. aeruginosa p. mirabilis female male female male female male female male female male teenager 8.0 3.0 7.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 adults 19.0 34.0 15.0 16.0 1.0 1.0 0.0 1.0 2.0 2.0 middle age 14.0 18.0 8.0 18.0 2.0 2.0 0.0 0.0 0.0 1.0 elderlies 5.0 10.0 3.0 5.0 0.0 1.0 0.0 0.0 0.0 1.0 total 46.0 65.0 33.0 42.0 3.0 4.0 0.0 1.0 2.0 4.0 total 111.0 75.0 7.0 1.0 6.0 discussion e. coli  (65%),  klebsiella  spp. (25%) pseudomonas (5%), enterobacter spp. (4%), and  acinetobacter  spp. (2%) were detected in three tertiary care hospitals  samples in lahore, pakistan.15 a few enterobacteriaceae species including enterobacter aerogenes, e. cloacae, e. coli, k. pneumoniae, p mirabilis, and serratia marcescens are responsible for most infections produced by this family.16,17 the crucial factor in increasing antimicrobial resistance was the production of β-lactamase in gram-negative bacteria, like esbls ctx-m and shv and k. pneumoniae carbapenemase.14,18 the broad spreading of the esbls has left patients and clinicians with very limited options in the infections treatment caused by multidrug resistant (mdr) enterobacteriaceae.19–21 the antibiotic susceptibility test was performed, and the results presented in table 4. the response of the isolated e. coli strains to ampicillin (amp), cefotaxime (ctx), cefotaxime-clavulanate (ctl), ceftriaxone (cro), cephalothin (cef), chloramphenicol (chl), ciprofloxacin (cip), nalidixic acid (nal), streptomycin (str), tetracycline (tet), and trimethoprim-sulphamethoxazole (sxt) was highly resistant while the response of the isolated e. coli strains to amoxicillin-clavulanic acid (amc) was moderately susceptible, while to ampicillin sulbactam (sam), ceftazidime (caz), ceftazidime-clavulanate (cal), and imipenem (ipm) was highly susceptible. the response of k. pneumoniae strains to amp, ctx, ctl, cro, cef, chl, cip, str, tet and sxt was highly resistant while the response to amc and nal was moderately susceptible, and to sam, caz, cal, and ipm was highly susceptible. the response of e. cloaca was highly resistant to amp, amc, ctx, cro, cef, chl, nal, str, tet and sxt, while the response was moderately susceptible to ctl and cip, and highly susceptible to sam, caz, cal, and ipm (table 4). enterobacteriaceae isolates resistant to ceftazidime-avibactam were evaluated for the presence of esbls encoding genes.22,23 more than 99.9% of enterobacteriaceae was inhibited by ceftazidime-avibactam. only 82.2% of mdr enterobacteriaceae and 64.2% of ceftriaxone-non-susceptible table 2. esbls producing organisms detected in the collected samples. isolate total female male number % number % number % enterobacter cloacae 7.0 3.5 3.0 42.9 4.0 57.1 escherichia coli 111.0 55.5 46.0 41.4 65.0 58.6 klebsiella pneumoniae 75.0 37.5 33.0 44.0 42.0 56.0 proteus mirabilis 6.0 3.0 2.0 33.3 4.0 66.7 pseudomonas aeruginosa 1.0 0.5 0.0 0.0 1.0 100 total 200 100 84 42.0 116 58.0 31 original extended-spectrum β-lactamase enterobacteriaceae from patients in jeddah, saudi arabiawafa a. alshehri, tarek a. a. moussa j contemp med sci | vol. 7, no. 1, january-february 2021: 28 – 33 fig. 3 the number of the five isolated organisms in different categories. table 4. the percentage of antimicrobial resistance esbl producing isolates. antibiotic abb. conc. (µg) e. coli (n = 111) k. pneumoniae (n = 75) e. cloacae (n = 7) n (%) n (%) n (%) ampicillin amp 10 111 (100) 75 (100) 7 (100) amoxicillin-clavulanic acid amc 30 44 (39.6) 32 (42.7) 7 (100) ampicillin sulbactam sam 20 11 (10) 11 (14.7) 0 (0.0) cefotaxime ctx 30 100 (90) 64 (85.3) 7 (100) cefotaxime-clavulanate ctl 30/10 88 (79.2) 53 (70.7) 3 (42.9) ceftazidime caz 30 11 (10) 11 (14.7) 1 (14.3) ceftazidime-clavulanate cal 30/10 9 (8.1) 8 (10.7) 0.0 (0.0) ceftriaxone cro 30 111 (100) 75 (100) 7 (100) cephalothin cef 30 111 (100) 75 (100) 7 (100) chloramphenicol chl 30 111 (100) 74 (98.7) 7 (100) ciprofloxacin cip 5 83 (74.8) 64 (85.3) 4 (57.1) imipenem ipm 10 11 (10) 11 (14.7) 0.0 (0.0) nalidixic acid nal 10 89 (80.1) 26 (34.7) 6 (85.7) streptomycin str 10 94 (84.7) 68 (90.7) 7 (100) tetracycline tet 30 100 (90.1) 66 (88) 7 (100) trimethoprim-sulphamethoxazole sxt 25 106 (95.5) 70 (93.3) 7 (100) 32 original extended-spectrum β-lactamase enterobacteriaceae from patients in jeddah, saudi arabia wafa a. alshehri, tarek a. a. moussa j contemp med sci | vol. 7, no. 1, january-february 2021: 28 – 33 k. pneumoniae isolates were susceptible to meropenem. among e. cloacae strains (99.8%) were susceptible to ceftazidime-avibactam. only 0.06% enterobacteriaceae isolates were non-susceptible to ceftazidime-avibactam.24 a potent activity of ceftazidime-avibactam was shown against p. aeruginosa, where 97.1% isolates was susceptible, and 71.8% was inhibited of non-susceptible isolates to ceftazidime, meropenem, and piperacillin-tazobactam.22,23 most of the  e. coli  (87.5%) and k. pneumoniae (75%) isolates were mdr and showed also mdr phenotypes.17,21–23 the most frequent gene was blactx-m (195) followed by blatem (149), blashv (73), and blaoxa (3). the blactx-m was the most widespread gene among the e. coli strains and in all age categories, while the rarest gene was blaoxa (table 5). only e.  coli and k. pneumoniae were detected in teenagers. the presence of these genes in pairs in the same strain was detected, where the highest pair of genes in the same organism was blatem + blactx-m (134) (e. coli (74), k. pneumoniae (51) and e. cloaca (9)), followed by blashv + blactx-m (64) (k. pneumoniae (63) and e. cloaca (1)), blashv + blatem (52) (k. pneumoniae (51) and e. cloaca (1)) and the least pairs were blatem + blaoxa (3) (e. cloaca (2) and p. mirabilis (1)) and blactx-m + blaoxa (2) in e. cloaca only. the blashv + blactx-m + blatem was found in 44 organisms and blactx-m + blatem + blaoxa in two organisms only. the blashv + blaoxa, blashv + blactx-m + blaoxa, blashv + blatem + blaoxa and blashv + blactx-m + blatem + blaoxa was not found in any organism under investigation (table 6). e. coli strains had 32.4%  blactx-m, 81% blatem, and 16.2%  blashv genes, while in k. pneumoniae strains had 41.1% blactx-m, 64.7% blatem, and 35.2% blashv genes. 24 pcr showed that blactx − m  gene represented by 76% followed by  52% blaoxa,  28% blatem and 21% blashv were most predominant detected by cdst among esbls. the 78% of blaoxa, 65% of blactx − m − 1, and 57% of blatem genes were found on plasmids. amplicon sequencing demonstrated that blactx − m − 15 (75%), blaoxa − 1 (49%) and blatem − 1b (34%) and 21 isolates carried three genes in them.15,25 conclusion in conclusion, in teenager group, there were no organism that contained oxa gene, while oxa present in e. cloaca and p.  mirabilis only. shv gene was absent in e. coli but present in e. cloaca and k. pneumoniae in this study. the most susceptible group to infection with enterobacteriaceae was adults’ group, while teenage group was more resist to infection. conflict of interest none table 5. the frequency occurrence of esbl encoding genes in the isolated bacteria. isolate bla ctx-m bla oxa bla shv bla tem number % number % number % number % e. cloacae 9.0 4.5 2.0 66.7 1.0 0.5 11.0 5.5 e. coli 111.0 57.0 0.0 0.0 0.0 0.0 78.0 39.0 k. pneumoniae 74.0 38.0 0.0 0.0 72.0 36.0 59.0 29.5 p. mirabilis 0.0 0.0 1.0 33.3 0.0 0.0 1.0 0.5 p. aeruginosa 1.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 total 195 3.0 73 149 table 6. the prevalence of the coexistence of esbl encoding genes in the isolated bacteria. genes e. cloaca e. coli k. pneumoniae p. mirabilis p. aeruginosa total bla tem +bla ctx-m 9 74 51 0 0 134 bla shv +bla ctx-m 1 0 63 0 0 64 bla shv +bla tem 1 0 51 0 0 52 bla tem +bla oxa 2 0 0 1 0 3 bla ctx-m +bla oxa 2 0 0 0 0 2 bla shv +bla oxa 0 0 0 0 0 0 bla shv +bla ctx-m +bla tem 1 0 43 0 0 44 bla ctx-m +bla tem +bla oxa 2 0 0 0 0 2 bla shv +bla ctx-m +bla oxa 0 0 0 0 0 0 bla shv +bla tem +bla oxa 0 0 0 0 0 0 bla shv +bla ctx-m +bla tem +bla oxa 0 0 0 0 0 0 33 original extended-spectrum β-lactamase enterobacteriaceae from patients in jeddah, saudi arabiawafa a. alshehri, tarek a. a. moussa j contemp med sci | vol. 7, no. 1, january-february 2021: 28 – 33 references 1. collignon p, aarestrup fm, irwin r, mcewen s. human deaths and thirdgeneration cephalosporin use in poultry, europe. 2013; 2. chantziaras i, boyen f, callens b, dewulf j. correlation between veterinary antimicrobial use and antimicrobial resistance in food-producing animals: a report on seven countries. j antimicrob chemother. 2014;69(3):827–34. 3. murphy d, ricci a, auce z, beechinor jg, bergendahl h, breathnach r, et al. ema and efsa joint scientific opinion on measures to reduce the need to use antimicrobial agents in animal husbandry in the european union, and the resulting impacts on food safety (ronafa). efsa j. 2017;15(1):e04666. 4. lazarus b, paterson dl, mollinger jl, rogers ba. do human extraintestinal escherichia coli infections resistant to expanded-spectrum cephalosporins originate from food-producing animals? a systematic review. clin infect dis. 2015;60(3):439–52. 5. woerther p-l, burdet c, chachaty e, andremont a. trends in human fecal carriage of extended-spectrum β-lactamases in the community: toward the globalization of ctx-m. clin microbiol rev. 2013;26(4):744–58. 6. livermore dm, paterson dl. pocket guide to extended-spectrum β-lactamases in resistance. current medicine group; 2006. 7. pitout jdd, nordmann p, laupland kb, poirel l. emergence of enterobacteriaceae producing extended-spectrum β-lactamases (esbls) in the community. j antimicrob chemother. 2005;56(1):52–9. 8. paterson dl, bonomo ra. extended-spectrum β-lactamases: a clinical update. clin microbiol rev. 2005;18(4):657–86. 9. boucher hw, talbot gh, bradley js, edwards je, gilbert d, rice lb, et al. bad bugs, no drugs: no eskape! an update from the infectious diseases society of america. clin infect dis. 2009;48(1):1–12. 10. mosqueda-gómez jl, montaño-loza a, rolón al, cervantes c, bobadilladel-valle jm, silva-sánchez j, et al. molecular epidemiology and risk factors of bloodstream infections caused by extended-spectrum β-lactamaseproducing klebsiella pneumoniae: a case–control study. int j infect dis. 2008;12(6):653–9. 11. clsi. performance standards for antimicrobial susceptibility testing. twenty first informational supplement, clinical and laboratory standards institute (clsi) document. 2011;m 100-s21. 12. jarlier v, nicolas m-h, fournier g, philippon a. extended broad-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in enterobacteriaceae: hospital prevalence and susceptibility patterns. clin infect dis. 1988;10(4):867–78. 13. deak d, outterson k, powers jh, kesselheim as. progress in the fight against multidrug-resistant bacteria? a review of us food and drug administration–approved antibiotics, 2010–2015. ann intern med. 2016;165(5):363–72. 14. mehrad b, clark nm, zhanel gg, lynch iii jp. antimicrobial resistance in hospital-acquired gram-negative bacterial infections. chest. 2015;147(5):1413–21. 15. abrar s, ain nu, liaqat h, hussain s, rasheed f, riaz s. distribution of bla (ctx m) , bla (tem), bla (shv) and bla (oxa) genes in extended-spectrumβ-lactamase-producing clinical isolates: a three-year multi-center study from lahore, pakistan. antimicrobial resistance and infection control [internet]. 2019 may 22;8:80. available from: https://pubmed.ncbi.nlm.nih. gov/31139363 16. garrity g. bergey’s manual® of systematic bacteriology: volume 2: the proteobacteria, part b: the gammaproteobacteria. vol. 2. springer science & business media; 2007. 17. ahmed sf, ali mmm, mohamed zk, moussa ta, klena jd. fecal carriage of extended-spectrum β-lactamases and ampc-producing escherichia coli in a libyan community. ann clin microbiol antimicrob. 2014;13(1). 18. castanheira m, mendes re, jones rn, sader hs. changes in the frequencies of β-lactamase genes among enterobacteriaceae isolates in us hospitals, 2012 to 2014: activity of ceftazidime-avibactam tested against β-lactamaseproducing isolates. antimicrob agents chemotherapy. 2016;60(8):4770–7. 19. jacob jt, klein e, laxminarayan r, beldavs z, lynfield r, kallen aj, et al. vital signs: carbapenem-resistant enterobacteriaceae. mmwr morb mortal week rep. 2013;62(9):165. 20. gupta n, limbago bm, patel jb, kallen aj. carbapenem-resistant enterobacteriaceae: epidemiology and prevention. clin infect dis. 2011;53(1):60–7. 21. ali mmm, ahmed sf, klena jd, mohamed zk, moussa taa, ghenghesh ks. enteroaggregative escherichia coli in diarrheic children in egypt: molecular characterization and antimicrobial susceptibility. j infect develop count. 2014;8(5):589–96. 22. sader hs, castanheira m, shortridge d, mendes re, flamm rk. antimicrobial activity of ceftazidime-avibactam tested against multidrug-resistant enterobacteriaceae and pseudomonas aeruginosa isolates from us medical centers, 2013 to 2016. antimicrob agents chemother. 2017;61(11). 23. sader hs, castanheira m, shortridge d, mendes re, flamm rk. antimicrobial activity of ceftazidime-avibactam tested against multidrug-resistant enterobacteriaceae and pseudomonas aeruginosa isolates from u.s. medical centers, 2013 to 2016. antimicrob agents chemother [internet]. 2017 oct 24;61(11):e01045-17. available from: https://pubmed.ncbi.nlm.nih. gov/28827415 24. pishtiwan ah, khadija km. prevalence of blatem, blashv, and blactx-m genes among esbl-producing klebsiella pneumoniae and escherichia coli isolated from thalassemia patients in erbil, iraq. mediterr j hematol infect dis. 2019;11(1). 25. ali mmm, mohamed zk, klena jd, ahmed sf, moussa taa, ghenghesh ks. molecular characterization of diarrheagenic escherichia coli from libya. am j trop med hyg. 2012;86(5):866–71. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.920 164 j contemp med sci | vol. 7, no. 3, may-june 2021: 164–166 original detection rate of fetal cns anomalies by first and second trimester ultrasound screening mahyar mohammadifard1, nasrin khorashadizadeh2, maryam zarouni3* 1department of radiology, imam reza educational hospital, birjand university of medical sciences, birjand, iran 2department of radiology, valie-asr hospital, birjand university of medical sciences, birjand, iran. 3department of radiology, birjand university of medical sciences, birjand, iran *corresponding author: maryam zarouni, (e-mail: maryamzarouni10@yahoo.com) (submitted: 23 april 2021 – revised version received: 18 may 2021 – accepted: 27 may 2021 – published online: 26 june 2021) introduction the central nervous system (cns) is the first system to develop in the embryonic period1 and neural tube defects (ntds) are the most common central nervous system defect. these defects are caused by neural tube closure problems between the third and fourth weeks of embryonic development2,3 and are among the causes of stillbirth, neonatal mortality, and severe lifelong disability.4,5 neural tube defects are the most common congenital anomaly after cardiac defects and its incidence is 1.4-2 per 1000 live births.6 although the exact cause of neural tube defects is not known, but factors such as radiation, medication, malnutrition, chemical and genetic factors are involved in the development of the central nervous system and neural tube defects at the time of fertilization.7 cns abnormalities are the most common reason for referral for prenatal diagnosis and concern of many patients. almost all cns abnormalities occur as a result of abnormalities in the period of embryogenesis and its development. although many cns abnormalities can be assessed in the first and second trimesters, others develop or become visible later in pregnancy.8 cns anomalies are also the most important factor in morbidity and mortality during infancy and in children, some of which become symptomatic only in early childhood.9 ultrasound is the selective modality and effective diagnostic method for the diagnosis of congenital cns anomalies. however, ultrasound is currently performed worldwide to detect cns abnormalities because brain structures continue to develop until the second trimester, at 22 to 19 weeks of gestation.10 early detection of anomalies, especially in the first and early second trimesters, helps to plan for intervention and management during pregnancy. early detection of cns abnormalities during pregnancy reduces emotional stress and economic cost.9 attempts have been made to reduce the age of screening for cns anomalies. screening is recommended at approximately 13-13 weeks of gestation at the same time as routine ultrasound is performed for fetal morphogenetic evaluation.11 therefore, the aim of this study was to detect the first and second trimester detection of cns abnormalities in fetuses of pregnant mothers with ultrasonography. materials and methods this cross-sectional study was performed on pregnant women who referred to the radiology department of imam reza and valiasr hospitals in tehran, iran during 2019-2020. in the present study, a checklist was used to collect information. the checklist includes measurement of nuchal translucency (nt), nasal bone, lateral ventricles, ossification of fetal skull and spine in first trimester anomaly scan, and measurement of biparietal diameter, fetal head circumference, cavum septum pellucidum (csp) corpus callosum, cerebellum view, transverse cerebellar diameter, cerebellar vermis, cisternagna, nuchal fold , falx cerebri e, fetal spine. then, women who met the inclusion and exclusion criteria were included in the study. inclusion criteria included: pregnant women, gestational age of 11-13 weeks and 18-20 weeks, and having informed consent. procedure after obtaining informed consent, pregnant women were screened in the first trimester at week 13-13 and in the second abstract objectives the aim of this study was to detect cns abnormalities in first and second trimester by ultrasound. methods this cross-sectional study was performed on pregnant women who referred to the radiology department of imam reza and valiasr hospitals in tehran-iran during 2019-2020. after obtaining informed consent, pregnant women were screened in the first trimester at week 13-13 and then in the second trimester at week 18-20 by a 5-8 mhz ultrasound transducer. each ultrasound included examination of the fetal brain and vertebrae at the axial coronal and sagittal planes in the most important anatomical areas, including the trans thalamic (tt) or the trans-ventricular (tv) plane, transverse cerebellar, and vertebral canal plan. ultrasound of the first and second trimesters of all mothers was performed. information of all pregnant mothers was collected and recorded. data analysis was performed using spss software version 25. results in this study, 2234 pregnant women were included in the study. the total rate of detected anomalies was found to be 1.3%. the rate of abnormalities detected in the first trimester was far less than in the second trimester. the prevalence of cns anomalies in the population under 30 years of age was also found 0.9%, while it was 1.6% in the population over 30 years of age. conclusion second trimester ultrasound is the method of choice in diagnosing central nervous system abnormalities. however, first trimester ultrasound is the diagnostic method for structural abnormalities of the skull. keywords cns anomaly, ultrasound, first trimester, second trimester issn 2413-0516 165j contemp med sci | vol. 7, no. 3, may-june 2021: 164–166 m. mohammadifard et al. original detection rate of fetal cns anomalies by first and second trimester ultrasound screening trimester at week 18-20 by a 5-8 mhz ultrasound transducer. each ultrasound included examination of the fetal brain and vertebrae at the axial, coronal, and sagittal planes in the most important anatomical areas, including the trans-thalamic (tt) or the trans-ventricular (tv) plane, transverse cerebellar, and vertebral canal plan. ultrasound of the first and second trimesters of all mothers was performed. information of all pregnant mothers was collected and recorded. data analysis central and dispersion indices were used to describe the data. frequency was used for quantitative variables and percentage for qualitative variables. chi-square tests were used to investigate the relationship between the variables. kappa statistics were calculated to confirm the prevalence of cns anomalies in the first and second trimesters. data analysis was performed using spss software version 25. a p value of 0.05 was considered to be statistically significant. ethical considerations a written letter of introduction was obtained from the university and the selected researcher centers. the purpose of the study was described for all research units and written consent was obtained from after describing the purpose of the study. the information of all patients remained confidential. ethics declarations of helsinki and ethics research committees of the university of medical sciences were also considered. the code of ethics was taken from the relevant authorities to conduct the research (ir.bums.rec.1399.070). results a total of 2234 pregnant women were included in the study. the overall prevalence of central nervous system abnormalities in the first and second trimesters was assessed. in the first and second trimester 2230 (99.8%), and 2208 patients (98.8%) had no central nervous system abnormalities, respectively. the prevalence of cns anomalies in the first and second trimester scans was 0.2% and 1.3%, respectively. table 1 shows the prevalence of various central nervous system abnormalities in the first and second trimesters. the most common type of anomaly was related to choroid plexus cyst. the prevalence of central nervous system anomalies in the first trimester by age groups is shown in table 2. the prevalence of cns anomalies was 0.1% in the population under 30 years of age, while it was reported to be 0.3% in the population over 30 years of age. however, no significant difference was observed between the two groups (p = 0.617). table 3 shows the prevalence of central nervous system anomalies in the second trimester by age groups. the prevalence of cns anomalies in the population under 30 years of age was 0.9%, while it was 1.6% in the population over 30 years of age. however, no statistically significant difference was observed between the two groups (p = 0.122). the level of agreement for the detection of cns anomalies in the first and second trimester ultrasounds is summarized in table 4. the agreement between the detection of cns anomalies in the first and second trimester sonography was 0.233, indicating a weak agreement between the detection of cns anomalies in the first and second trimester sonography. discussion early detection of anomalies, especially in the first trimester and early second trimester, helps plan for intervention and management during pregnancy, leading to reduction of emotional stress and economic cost.12 therefore, the aim of this table 1. prevalence of various anomalies of the central nervous system in the first and second trimesters variable frequency lumbosacral meningocell 1 chiari type 2 malformation 3 ventriculomegaly 4 choroid plexus cyst 12 hydrocephalus 3 dandy-walker malformation 3 anencephalus 2 occipital encephalitis 1 skull deformity 1 table 2. prevalence of central nervous system anomalies in the first trimester by age groups variable frequency p-value age 30 (year)≥ no 1443 (99.9%) 0.617 yes 2 (0.1%) total 1445 (100%) 30 (year)≤ no 787 (99.7%) yes 2 (0.3%) total 789 (100%) table 3. prevalence of central nervous system anomalies in the second trimester by age groups variable frequency p-value age 30 (year)≥ no 1442 (99.9%) 0.122 yes 13 (0.1%) total 1445 (100%) 30 (year)≤ no 776 (98.4%) yes 13 (1.6%) total 789 (100%) table 4. the level of agreement between of cns anomalies in first and second trimester ultrasound the second quarter statistics of kappa negative number (percent) positive number (percent) first three months negative 2204 (98.7%) 26 (1.2%) 0.233positive 0 (0%) 4 (0.2%) total 2204 (98.7%) 30 (1.3%) 166 j contemp med sci | vol. 7, no. 3, may-june 2021: 164–166 detection rate of fetal cns anomalies by first and second trimester ultrasound screening original m. mohammadifard et al. study was to detect cns anomalies in first and second trimester through sonography. in this study, 2234 were included. the total rate of detected anomalies was 1.3%. the rate of abnormalities detected in the first trimester was far less than in the second trimester. the prevalence of cns anomalies in the population under 30 years of age was 0.9%, while it was found to be 1.6% in the population over 30 years of age. various studies have been performed in this regard. one study by onkara13 a prevalence of 0.31% for cns anomalies, which is less than the present study. the reason for this difference may be the gestational age. another study by neeta natu10 reported that the detection rate of congenital anomalies in low-risk and high-risk pregnancies was 2.6%, which is higher than our study. the reason for this difference could be the study population. in lorie’s study,14 the number of undiagnosed anomalies in the first trimester among the lowrisk and high-risk population was higher than our study, which could be due to the number of pregnant women and the type of population studied. carroll15 reported that 77% of ultrasound results in the diagnosis of brain abnormalities were consistent with fetal autopsy. dulgheroff16 found that the prevalence of structural abnormalities in the first, second and third trimesters of pregnancy was 2.95%, which is higher than the present study; the reason for this difference may be related to gestational age. sefidakht et al.17 compared the sonographic findings with the diagnosis of ventriculomegaly and its correlation with the mri findings. the results of the study showed that mri is superior to ultrasound and clinical examination at birth in the diagnosis of cns anomalies. in the present study, the diagnosis of anomaly by ultrasound in the second trimester was the method of choice, but the use of mri for future studies and its comparison with ultrasound are recommended. in the study by struksnaes et al.,18 the correlation between the results of ultrasound at week 11-33 and autopsy was investigated. they concluded that there was a 96.9% agreement between the findings of ultrasound and autopsy, and ultrasound was recommended as a useful and reliable method for diagnosing cns anomalies. however, in the present study, the second trimester ultrasound was found to be a useful diagnostic method for diagnosing cns anomalies. conclusion first trimester ultrasound is a useful diagnostic method in diagnosing structural abnormalities of the skull. second trimester ultrasound is the method of choice for diagnosing central nervous system abnormalities. references 1. e. v. voss and m. stangel, “nervous system involvement of connective tissue disease: mechanisms and diagnostic approach,” current opinion in neurology. 2012;25:306-315. 2. mclachlan nm, wilson sj. the contribution of brainstem and cerebellar pathways to auditory recognition. front psychol. 2017;8:265. 3. s. sciascia, m. l. bertolaccini, d. roccatello, m. a. khamashta, and g. sanna, “autoantibodies involved in neuropsychiatric manifestations associated with systemic lupus erythematosus: a systematic review,” journal of neurology. 2014;261:1706-1714. 4. d. aletaha, t. neogi, a. j. silman et al., “2010 rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative,” annals of the rheumatic diseases. 2010;69:1580-1588. 5. p. chiewthanakul, k. sawanyawisuth, c. foocharoen, and s. tiamkao, “clinical features and predictive factors in neuropsychiatric lupus,”asian pacific journal of allergy and immunology. 2012;30:55-60. 6. j. zavada, p. nytrov a, k. p. wandinger et al., “seroprevalence ´ and specificity of nmo-igg (anti-aquaporin 4 antibodies) in patients with neuropsychiatric systemic lupus erythematosus,” rheumatology international. 2013;33:259263. 7. b. florica, e. aghdassi, j. su, d. d. gladman, m. b. urowitz, and p. r. fortin, “peripheral neuropathy in patients with systemic lupus erythematosus,” seminars in arthritis and rheumatism. 2011;41:203-211. 8. rumack c. the fetal brain. in diaghnostic ultrasound, 5th edition. elsevier mosby philadephia. 2018;166-1212. 9. iliescu g, comanescu c. cns abnormalities at 11-14 weeks. neonate pediatrmed. 2017;3:2572-4983. 10. natu n, wadnere n, yadav s, kumar r. utility of first trimester anomaly scan in screeining of congenitial abnormalities in low and high risk pregnancies. jk science. 2014;16:151-155. 11. dias t. screening for early fetal structural anomalies. seri lanka journal of obsteterics and gynaecology. 2011;33:183-188. 12. s. hirohata, y. sakuma, t. yanagida, and t. yoshio, “association of cerebrospinal fluid anti-sm antibodies with acute confusional state in systemic lupus erythematosus,” arthritis research & therapy. 2014;16:450. 13. onkara d, onlor p, kajl m. evaluation of fetal central nervous system anomalies by ultrtrasound and its anatomical co-reelation. jcdr. 2014;8:05-07. 14. lorie m, harper md, sheri m. the performance of first trimester anatomy scan: a decision analysis. pmc. 2017;33:957-965. 15. carroll, s. g., h. porter, s. abdel-fattah, p. m. kyle, and p. w. soothill. ‘correlation of prenatal ultrasound diagnosis and pathologic findings in fetal brain abnormalities’, ultrasound obstet gynecol. 2000;16:149-153. 16. dulgheroff f, et al. fetel structural anomalies diagnosed during the first, second and third trimesters of pregnancy using ultrasonography: a retrospective cohort. sao paulo med. 2019;137. 17. sefidbakht s, dehghani s, safari m, vafaei h, kasraeian m. fetal central nervous system anomalies detected by magnetic resonance imaging: a two year experience.iranian journal of pediatrics. 2016;26:4589. 18. struksnaes c, karl blaas h, voget c. autopsy finding of central nervous system anomalies in intact fetuses following termination of pregnancy after prenatal ultrasound diagnosis. pediatr dev pathol. 2019;22:546557. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i3.982 152 j contemp med sci | vol. 7, no. 3, may-june 2021: 152–157 original seroprevalence and risk factors associated with helicobacter pylori infection among children aged less than 18 years old in duhok province, iraq azad mohammed taher al-brefkani1, ibrahim a. naqid2,*, nizar bakir yahya3, nawfal r. hussein2 1department of medical lab technology, shekhan technical college of health, duhok polytechnic university, duhok, kurdistan region, iraq 2department of biomedical sciences, college of medicine, university of zakho, kurdistan region, iraq 3department of paediatric, college of medicine, university of duhok, kurdistan region, iraq *correspondence to: ibrahim a. naqid (email: ibrahim.naqid@uoz.edu.krd) (submitted: 13 march 2021 – revised version received: 28 april 2021 – accepted: 02 may 2021 – published online: 26 june 2021) introduction helicobacter pylori (h. pylori) is a spiral, gram-negative bacterium that is recognized as a worldwide problem. it colonizes the human stomach in approximately half of the world’s population and can cause gastritis and peptic ulceration.1,2 infection with this microorganism plays an essential role in the pathogenesis of gastric adenocarcinoma (ga) and primary gastric lymphoma in adulthood.3 ga is the second most common cause of cancer-related deaths worldwide.4 in a previous study conducted in iraq, the prevalence of h. pylori infection varied according to age group, ranging from 27% in children to 58% in teenagers.5 in the same study, the infection rate was 78%, which was significantly higher than the prevalence in children.5 several studies have described a significant variation in the seroprevalence of h. pylori infection between and within countries due to differences in ethnicity and geographical settings of each population.6 it was previously proposed that the acquisition of infection with h. pylori occurs during early childhood; adults may carry the same bacterial strain for consecutive decades.7,8 it has been previously shown that the prevalence of h. pylori infection increases significantly with age, which is largely due to a birth cohort effect rather than a late acquisition of infection.5 additionally, previous report linked h. pylori infection with low socioeconomic status, crowded or poor living conditions, low education level or quality of drinking water, and inadequate sanitation practices.9 studies of seroprevalence and the determination of potential risk factors associated with h. pylori infection are essential in establishing appropriate health strategies for preventing h. pylori-related diseases in the community. therefore, the present study aimed to evaluate the potential risk factors associated with the seroprevalence rate of h. pylori infection among individuals aged 18 years and younger in duhok province, kurdistan region, iraq. materials and methods study design and population this cross-sectional study was conducted at the heevi and azadi teaching hospital and shekhan hospital, kurdistan region, iraq. over a four-year period (2016–2020), a total of 381 blood samples were collected randomly from children aged four to 18 years and evaluated for specific igg antibodies against h. pylori. information about sociodemographic characteristics including age, gender, hygienic practices, sources of drinking water, education level of parents, accommodation, smoking status, number of family members, family income history of gastrointestinal diseases, and frequency of eating fast food and french fries obtained from each subject after obtaining consent using the questionnaire. all subjects were invited to answer the questionnaire through a face-to-face interview, which was the same as in a previous study.10 abstract objectives this study aimed to detect the prevalence of h. pylori infection and identify potential factors sociated with infection in duhok province, iraq. methods this cross-sectional study was conducted in duhok province, kurdistan region, iraq. over a four-year period from 2016 to 2020, a total of 381 children aged four to 18 years who attended the hospitals and were evaluated for specific igg antibodies against h. pylori using serological tests were included. a questionnaire was completed at the start of the study. h. pylori serology data were analyzed using the chi-square test. results the seroprevalence of h. pylori igg-specific antibodies among the study participants was 31%. the infection rate significantly increased with increasing age of the participants (p < 0.001), from 19.8% among four to five-year-olds to 42.9% among 16 and 18-yearolds. the infection rate was significantly higher in girls (35.9%) than that in boys (26.5%) (p < 0.047). infections were also higher among university students (41%; p < 0.001), children from the country-side population (41%; p < 0.001), children from crowded households (67.3%; p < 0.001), and children with a history of gastrointestinal pain (44.4%; p > 0.001). conclusion the prevalence rate of h. pylori infection among children in duhok province is quite similar to that reported in previous tudies in the kurdistan region, iraq, and increases with age. university ttendance, history of gastrointestinal pain, countryside population, and overcrowding were risk factors associated with h. pylori infection. valuable approaches to improve sanitary purposes and educational and socioeconomic status should be emphasized and promoted to reduce the risk of h. pylori infection among children. keywords h. pylori, seroprevalence, risk factors, children, duhok, iraq issn 2413-0516 153j contemp med sci | vol. 7, no. 3, may-june 2021: 152–157 a.m. taher al-brefkani et al. original prevalence and risk factors for h. pylori infection inclusion and exclusion criteria all patients who attended the heevi and azadi teaching hospital and whose guardians agreed were included in this study. patients who did not agree to participate were excluded. other exclusion criteria included a self-reported history of h. pylori-positivity and other gastric disorders. serological detection of anti-h. pylori igg a 5-ml syringe and needle were used to bleed about 5 ml of blood from each subject under aseptic conditions. the blood samples were then centrifuged at 1500 rpm for 5 min to obtain the serum and kept frozen at –20°c until required. anti h. pylori igg positivity was determined using a commercial kit (bioactiva diagnostica, germany) following the manufacturer’s instructions. the cut-off was determined by an index value obtained from the ratio of sample absorbance to the absorbance of a cut-off calibrator. the result was considered negative if the h. pylori igg index was <0.90, and positive if the h. pylori igg index was >1.00. ethics approval the study was approved by the ethics committee of the college of medicine, university of zakho, kurdistan region of iraq, based on the ethical principles of human research and experimentation. informed consent was obtained from the children’s parents or legal guardians before enrollment in the study. statistical analysis statistical analysis was performed using spss (spss, inc., cary, nc, usa). to investigate the risk factors for h. pylori seropositivity and influential factors, the chi-square test was used. a significance level of p < 0.05 was used for all analyses. results the sociodemographic variables associated with the seroprevalence of h. pylori infection in children aged less than 18 years are presented in table 1. during the study period, 381 (200 girls, 181 boys) were tested for h. pylori-specific igg. the ages of the participants ranged from four to 18 years. the seroprevalence of h. pylori igg-specific antibodies was 118 (31.0%). the seroprevalence rate of h. pylori according to age varied from 19.8% in the age group of four to six years to 42.9% in the age group of 16 to 18 years. there was a positive association between age and seroprevalence; older age had a higher prevalence rate of h. pylori igg antibodies (p < 0.001). the seropositivity of h. pylori was significantly associated between male (26.5%) and female (35.9%) participants (p < 0.047). according to the students’ education level, the highest seropositivity was found among university students (44.4%) and the lowest at the pre-school level (20%), with statistically significant differences (p < 0.001). in addition, infection prevalence was significantly higher in the country-side population (41.0%) than that in the city population (25.2%) (p < 0.001). there was also a significant association between the fathers’ education levels and h. pylori positivity (p < 0.039), while no association was found between the mothers’ education levels and h. pylori positivity (p > 0.31). no significant relationships were found between sources of drinking water (p > 0.52), frequency of table 1. relationship between h. pylori seroprevalence and associated risk factors h. pylori infection (no. [%]) demographic data positive n = 118 (31.0%) negative n = 263 (69.0%) total χ2 p value age ( year) 4-6 26 (19.8%) 105 (80.2%) 131 16.61 0.002 7-9 9 (29.0%) 22 (71.0%) 31 10-12 14 (26.9%) 38 (73.1%) 52 13-15 30 (39.5%) 46 (60.5%) 76 16-18 39 (42.9%) 52 (57.1%) 91 gender boys 53 (26.5%) 147 (73.5%) 200 3.93 0.047 girls 65 (35.9%) 116 (64.1%) 181 education qualification of students pre-school 20 (20.0%) 80 (80.0%) 100 21.63 0.001 primary school 21 (21.0%) 79 (79.0%) 100 secondary school 38 (42.2%) 52 (57.8%) 90 high school 35 (42.7%) 47 (57.3%) 82 university 4 (44.4%) 5 (55.6%) 9 accommodation city 61 (25.2%) 181 (74.8%) 242 10.31 0.001 (continued) 154 j contemp med sci | vol. 7, no. 3, may-june 2021: 152–157 prevalence and risk factors for h. pylori infection original a.m. taher al-brefkani et al. table 1. relationship between h. pylori seroprevalence and associated risk factors h. pylori infection (no. [%]) demographic data positive n = 118 (31.0%) negative n = 263 (69.0%) total χ2 p value country side 57 (41.0%) 82 (95.0%) 139 students father education qualification non-literate 31 (34.8%) 58 (65.2%) 89 10.1 0.039 primary school 28 (34.6%) 53 (65.4%) 81 secondary school 27 (41.5%) 38 (58.5%) 65 high school 11 (21.6%) 40 (78.4%) 51 university 21 (22.1%) 74 (77.9%) 95 students mother education qualification non-literate 33 (34.4%) 63 (65.6%) 96 4.73 0.31 primary school 33 (31.1%) 73 (68.9%) 106 secondary school 30 (36.1%) 53 (63.9%) 83 high school 11 (25.6%) 32 (74.4%) 43 university 11 (20.8%) 42 (79.2%) 53 source of drinking water tape water 82 (32.0%) 174 (68.0%) 256 0.41 0.52 mineral water 36 (28.8%) 89 (71.2%) 125 fast food daily 16 (34.8%) 30 (65.2%) 46 1.75 0.62 weekly 39 (28.1%) 100 (71.9%) 139 monthly 34 (35.1%) 63 (64.9%) 97 occasionally 29 (29.3%) 70 (70.7%) 99 chips and cakes daily 87 (32.3%) 182 (67.7%) 269 3.2 0.36 weekly 22 (26.8%) 60 (73.2%) 82 monthly 7 (41.2%) 10 (58.8%) 17 occasionally 2 (15.4%) 11 (84.6%) 13 history of gastrointestinal pain in last 3 weeks yes 52 (44.4%) 65 (55.6%) 117 14.33 0.001 no 66 (25.0%) 198 (75.0%) 264 habit of hand washing before eating yes 68 (28.5%) 171 (71.5%) 239 1.9 0.16 no 50 (35.2%) 92 (64.8%) 142 washing raw vegetables yes 114 (30.4%) 261 (69.6%) 375 3.63 0.57 no 4 (66.7%) 2 (33.3%) 6 smoking status yes 4 (30.8%) 9 (69.2%) 13 3.86 0.145 no 28 (41.2%) 40 (58.8%) 68 not regularly 7 (70.0%) 3 (30.0%) 10 teeth decay yes 48 (31.4%) 105 (68.6%) 153 0.019 0.89 (continued) —continued 155j contemp med sci | vol. 7, no. 3, may-june 2021: 152–157 a.m. taher al-brefkani et al. original prevalence and risk factors for h. pylori infection table 1. relationship between h. pylori seroprevalence and associated risk factors h. pylori infection (no. [%]) demographic data positive n = 118 (31.0%) negative n = 263 (69.0%) total χ2 p value no 70 (30.7%) 158 (69.3%) 228 family income very bad 6 (35.3%) 11 (64.7%) 17 1.72 0.78 bad 12 (29.3%) 29 (70.7%) 41 middle 73 (32.4%) 152 (67.6%) 225 good 25 (29.1%) 61 (70.9%) 86 very good 2 (16.7%) 10 (83.3%) 12 number of family members 2-4 14 (23.0%) 47 (77.0%) 61 5-7 36 (20.3%) 141 (79.7%) 177 45.84 0.001 8-10 31 (35.2%) 57 (64.8%) 88 >10 37 (67.3%) 18 (32.7%) 55 p values were determined using chi-square test (χ2). p value <0.05 were considered statistically significant. fast food consumption (p > 0.62), frequency of eating chips and cakes (p > 0.36), and h. pylori positivity. the prevalence of h. pylori infection was also significantly higher in patients with a history of gastrointestinal pain than that in patients without (p < 0.001). other potential risk factors such as the habit of handwashing before eating, washing raw vegetables, smoking status, tooth decay, and family income were not statistically associated with h. pylori infection. additionally, a significant association was found between the number of family members with h. pylori positivity (p < 001); larger families had a higher prevalence rate (67.3%). discussion h. pylori infection is one of the most common chronic infections worldwide; it is estimated that approximately half of the world’s population is infected with this pathogen.1,11 h. pylori infection is a risk factor for gastric ulcer disease and plays a significant role in gastric cancer and gastric lymphoma.4 the seroprevalence study and detection of common risk factors for h. pylori infection have the potential to underpin appropriate health strategies for preventing h. pylori-associated diseases in the community. h. pylori infection has been studied extensively in iraq; it has been found to be one of the most common infectious agents causing gastric diseases.2,12–15 in addition, the prevalence of h. pylori infection has been studied in different age groups.5 however, no study has determined the other potential risk factors associated with this infection. therefore, this study examined the seroprevalence of h. pylori infection and its associated risk factors. in this study, the prevalence of h. pylori infection was 31.0% among children aged four to 18 years who attended the heevi and azadi teaching hospital. our findings are comparable to those reported in other community-based studies conducted in sulaimani province, iraq, where the prevalence of infection was 32.3%.16 in another study conducted in iraq, the overall seropositivity of h. pylori was 36% in children and 78% in adults.5 another study also found that 28% of recruited samples were seropositive for h. pylori infection in duhok province, iraq, which is slightly lower than the prevalence reported in our study.17 the prevalence of h. pylori infection ranges from more than 70% in developing countries, such as india and bangladesh, to around 20% in developed countries, such as australia and the netherlands.18 however, the prevalence of infection in our study was much lower than that reported in several studies in iran, where h. pylori infection rates were found to be at 47% to 64%19 and at 72.8%.20 nguyen et al. conducted another study in langson province, vietnam, and found an overall seropositivity rate of 41.4% to 46.8% in children.21 the results of our study were also inconsistent with those of a study conducted in egypt, saudi arabia, and turkey, where the prevalence rates of h. pylori infection were at 60%, 75%, and 77.5%, respectively.22,23 this study noted possible reasons for this discrepancy in the infection rate between our study and previous reports, which could be due to variations in the studied regions and groups and as well as the laboratory techniques used in the studies. the observed low prevalence in our study could also be the result of frequent usage of antibiotics and proton pump inhibitors or the gradual improvement of socioeconomic status and sanitation during the last decades.24 this practice could have led to the increased clearance of h. pylori, thereby resulting in lowered prevalence. different risk factors are associated with h. pylori infection during childhood. our results showed a significant relationship between h. pylori seropositivity and several variables related to socio-demographic characteristics such as age, gender, student and parents’ education, accommodation, history of gastrointestinal pain, and number of family members. according to age group, the rate of h. pylori infection varied —continued 156 j contemp med sci | vol. 7, no. 3, may-june 2021: 152–157 prevalence and risk factors for h. pylori infection original a.m. taher al-brefkani et al. from 19.8% to 42.9%. there was a significant difference between age seroprevalence; older age and those between 16 to 18 years had a higher prevalence rate of h. pylori igg antibodies (p < 0.001). our results are similar to those of a previous study conducted in iraq, which reported that the prevalence of infection varied according to age, ranging from 27% in children to 58% in teenagers.5 another study conducted in vietnam reported an increase in the seroprevalence of h. pylori, from 30.9% in children under three years to 53.1% in those 15 to 18 years of age in a study population of 476 children.21 additionally, the highest infection rate was found in girls (35.9%), with a statistically significant difference (p < 0.05). in contrast to our study, studies from developed countries indicated a higher h. pylori infection rate in boys than that in girls.25 other studies conducted in developing countries found that seropositivity rates in men and women seem to be similar.26 gender-specific h. pylori seropositivity rates often vary according to the developmental index of the country and its geographic location. in this study, the highest infection rate of h. pylori was found among university students (44.4%) and the lowest rate at the pre-school level (20%), with significant differences (p < 0.001). this could be due to university students living in dormitories with other students, which could increase the chance of acquiring the infection among themselves. there is also crowding at the universities that increases the chance of person-to-person transmission. our results are inconsistent with those of a previous study conducted in duhok province, iraq, which showed that there were no significant differences between different levels of education.17 in a study conducted in taiwan, lower education levels of mothers were associated with higher h. pylori infection rates.27 in another study conducted in russia, a strong positive association between the mothers’ education level and h. pylori infection rate was observed.28 in contrast, in the present study, there was a positive association between the fathers’ education level, rather than the mothers’, and h. pylori infection rate (p < 0.039). this could be partly due to the number of samples used in the present study. further studies are required to recruit a larger number of samples to explore this area. additionally, the prevalence rate of h. pylori infection was significantly higher in rural areas than in urban areas (p < 0.001). this could be due to poor personal, environmental, and residential hygiene conditions, differences in socioeconomic conditions, sources of drinking water, low levels of education, crowded families, and poor residential facilities in rural areas. public health education on preventable risk factors associated with h. pylori infection, such as personal hygiene and sanitation, should be emphasized and promoted in such areas. in this study, h. pylori seropositivity was 44.4% among those with a history of gastrointestinal disease, with a significant difference (p < 0.001). our finding of a positive association between h. pylori infection and gastrointestinal disease could be, at least, partly explained by the confounding effects of nutritional habits and several lifestyles accompanying such diseases. the number of children is the main reason for the increase in household size. household size, in turn, increases the possibility of h. pylori infection. our study found a lower infection rate of h. pylori (20.3%) among children from households with one to seven members, compared to 67.3% among children from households with more than 10 members (p < 0.001). this could mean that overcrowding increases the chances of acquiring h. pylori infection among children.29 in large families, children often share their bedrooms or beds with their siblings. sharing beds can significantly increase the risk of h. pylori infection exposure.25 the seropositivity rates of h. pylori infection are also high in adults living in large households.6,30 on the other hand, in the present study, no positive associations were found between the mothers’ education levels, habit of hand washing before eating, washing raw vegetables, smoking status, tooth decay, family income, and h. pylori infection. a study conducted in vietnam found a significant relationship between h. pylori seropositivity and several variables related to the socioeconomic status of their household, such as monthly income, mother’s education, and father’s occupation.31 another study showed that the prevalence of infection was lower in children from families of higher socioeconomic status (p < 0.005).32 the poor socioeconomic status of children in iran was associated with a higher prevalence of infection (p < 0.005).33 furthermore, a study found that the prevalence of h. pylori infection in children without regular hand washing after defecation was significantly higher than in those doing regular hand washing (p = 0.021).31 therefore, our findings disagree with those of previous studies.32,33 these differences could be partially due to the number of samples, geographical locations, and techniques used for the detection of infection. further studies are needed to explore the potential risk factors associated with h. pylori infection. in conclusion, the infection rate of h. pylori was found to be 31% in children aged less than 18 years. this rate is quite similar to other previous studies reported in the kurdistan region, iraq, and in neighboring countries. our data found a low prevalence of h. pylori infection among children at an early age; prevalence increases along with increasing age. the highest infection rate was also observed in girls than in boys. student and parents’ education, accommodation, history of gastrointestinal pain, and overcrowding were the risk factors associated with h. pylori infection in children. no significant association was found between the habit of handwashing before eating, washing raw vegetables, smoking status, tooth decay, family income, and the rate of h. pylori infection. therefore, effective approaches to improve sanitation and as well as educational and socioeconomic status should be emphasized and promoted to reduce and control h. pylori infection. acknowledgment we would like to thank the staff of heevi, azadi teaching hospital and shekhan hospital for their kind help and support. funding/support the authors received no financial support for the publication of this article. conflicts of interest the authors declare that there is no conflict of interests. 157j contemp med sci | vol. 7, no. 3, may-june 2021: 152–157 a.m. taher al-brefkani et al. original prevalence and risk factors for h. pylori infection references 1. hussein nr. helicobacter pylori and gastric cancer in the middle east: a new enigma? world j gastroenterol. 2010;16:3226-34. 2. hussein nr, saleem zs, balatay aa, abd kh, daniel s, taha aa, et al. seroprevalence of helicobacter pylori infection in renal transplant recipient attending duhok kidney disease center. transplantation proceedings. 2016;48(1):92-5. 3. ishaq s, nunn l. helicobacter pylori and gastric cancer: a state of the art review. gastroenterology and hepatology from bed to bench. 2015; 8(suppl 1):s6-s14. 4. atherton jc. the pathogenesis of helicobacter pylori-induced gastroduodenal diseases. annual review of pathology. 2006;1:63-96. 5. hussein nr, robinson k, atherton jc. a study of age-specific helicobacter pylori seropositivity rates in iraq. helicobacter. 2008;13(4):306-7. 6. mitchell h, megraud f. epidemiology and diagnosis of helicobacter pylori infection. helicobacter. 2002;7 suppl 1:8-16. 7. malaty hm, el-kasabany a, graham dy, miller cc, reddy sg, srinivasan sr, et al. age at acquisition of helicobacter pylori infection: a follow-up study from infancy to adulthood. lancet (london, england). 2002;359(9310):931-5. 8. kivi m, johansson al, reilly m, tindberg y. helicobacter pylori status in family members as risk factors for infection in children. epidemiology and infection. 2005;133(4):645-52. 9. al-shamahy ha. seroprevalence of helicobacter pylori among children in sana’a, yemen. annals of saudi medicine. 2005;25(4):299-303. 10. kim jh, kim hy, kim ny, kim sw, kim jg, kim jj, et al. seroepidemiological study of helicobacter pylori infection in asymptomatic people in south korea. journal of gastroenterology and hepatology. 2001;16(9):969-75. 11. go mf. review article: natural history and epidemiology of helicobacter pylori infection. alimentary pharmacology & therapeutics. 2002;16 suppl 1:3-15. 12. hussein nr, mohammadi m, talebkhan y, doraghi m, letley dp, muhammad mk, et al. differences in virulence markers between helicobacter pylori strains from iraq and those from iran: potential importance of regional differences in h. pylori-associated disease. journal of clinical microbiology. 2008;46(5):1774-9. 13. hussein nr, napaki sm, atherton jc. a study of helicobacter pyloriassociated gastritis patterns in iraq and their association with strain virulence. saudi j gastroenterol. 2009;15(2):125-7. 14. salih am, goreal a, hussein nr, abdullah sm, hawrami k, assafi m. the distribution of caga and dupa genes in helicobacter pylori strains in kurdistan region, northern iraq. annals of saudi medicine. 2013;33(3):290-3. 15. hussein nr, mirkhan sa, ramadhan aa, issa rs, naqid ia, yassin b, et al. levofloxacin-based regimen versus bismuth quadruple regimen for helicobacter pylori eradication in kurdistan region, iraq. avicenna j clin microbiol infect. 2020;7(3):81-4. 16. bayati a, al-windi a, ahmad n. seroprevalence of anti-helicobacter pylori antibodies in population of sulaimani governorate/kurdistan region/iraq. journal zankoy sulaimani. 2013;15:175-85. 17. yahya n. helicobacter pylori seropositivity in children in duhok city, iraq. science journal of university of zakho. 2018;6:82-7. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. 18. khalifa mm, sharaf rr, aziz rk. helicobacter pylori: a poor man’s gut pathogen? gut pathogens. 2010;2(1):2. 19. falsafi t, valizadeh n, sepehr s, najafi m. application of a stool antigen test to evaluate the incidence of helicobacter pylori infection in children and adolescents from tehran, iran. clinical and diagnostic laboratory immunology. 2005;12(9):1094-7. 20. salehi m, ghasemian a, shokouhi mostafavi sk, najafi s, rajabi vardanjani h. sero-prevalence of helicobacter pylori infection in neyshabur, iran, during 2010-2015. iran j pathol. 2017;12(2):183-8. 21. viet t, nguyen h, nguyen v, thanh t, phan b, hoang h, et al. epidemiology of helicobacter pylori infection in tay children in vietnam. annals of clinical and laboratory research. 2016;04. 22. frenck rw, jr., fathy hm, sherif m, mohran z, el mohammedy h, francis w, et al. sensitivity and specificity of various tests for the diagnosis of helicobacter pylori in egyptian children. pediatrics. 2006;118(4):e1195-202. 23. salih ba. helicobacter pylori infection in developing countries: the burden for how long? saudi j gastroenterol. 2009;15(3):201-7. 24. egan bj, holmes k, o’connor hj, o’morain ca. helicobacter pylori gastritis, the unifying concept for gastric diseases. helicobacter. 2007;12 suppl 2:39-44. 25. moayyedi p, axon at, feltbower r, duffett s, crocombe w, braunholtz d, et al. relation of adult lifestyle and socioeconomic factors to the prevalence of helicobacter pylori infection. int j epidemiology. 2002;31(3):624-31. 26. yilmaz e, doğan y, gürgöze mk, unal s. seroprevalence of helicobacter pylori infection among children and their parents in eastern turkey. j paediatr child health. 2002;38(2):183-6. 27. wu mc, sung ch, chang yc, ho cl, wu cc, wu kh, et al. seroprevalence of helicobacter pylori and hepatitis a virus among children in rural central taiwan. jpn j infect dis. 2015;68(6):494-503. 28. malaty hm, paykov v, bykova o, ross a, graham dp, anneger jf, et al. helicobacter pylori and socioeconomic factors in russia. helicobacter. 1996;1(2):82-7. 29. ding z, zhao s, gong s, li z, mao m, xu x, et al. prevalence and risk factors of helicobacter pylori infection in asymptomatic chinese children: a prospective, cross-sectional, population-based study. alimentary pharmacology & therapeutics. 2015;42(8):1019-26. 30. fraser ag, scragg r, metcalf p, mccullough s, yeates nj. prevalence of helicobacter pylori infection in different ethnic groups in new zealand children and adults. australian and new zealand journal of medicine. 1996;26(5):646-51. 31. nguyen v, nguyen k, phung c, kremp o, kalach n, dupont c, et al. prevalence of and factors associated with helicobacter pylori infection in children in the north of vietnam. the american journal of tropical medicine and hygiene. 2006;74:536-9. 32. etukudo om, ikpeme ee, ekanem ee. seroepidemiology of helicobacter pylori infection among children seen in a tertiary hospital in uyo, southern nigeria. pan afr med j. 2012;12:39-. 33. alborzi a, soltani j, pourabbas b, oboodi b, haghighat m, hayati m, et al. prevalence of helicobacter pylori infection in children (south of iran). diagnostic microbiology and infectious disease. 2006;54(4):259-61. doi: https://doi.org/10.22317/jcms.v7i3.972 116 original issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 116 – 121 introduction depression is a commonly prevalent mental illness affecting more than 264 million people globally and is one of the major causes of disability.1 people with depression suffer from a constant feeling of sadness and a loss of interest in previously pleasurable activities.2 in contrast, anxiety causes feelings of tension, worry-related cognitions, and physical changes including increased blood pressure.3 university is a point of transition from being a teenager to an adult that includes considerable changes in several life domains, such as finance, housing, emotional, and social aspects. the transition period creates a burden that young adults may experience as stressful.4 university students are considered a high-risk group for developing mental disorders and their mental health is a major public health concern.5,6 moreover, they are often subject to high expectations from their families to be academically successful and have a desire to be more successful than their peers. such demands can trigger mental health problems including depression, anxiety, and stress.7,8 many students become trapped in a vicious cycle as depression and anxiety affect the learning process by impairing memory and reducing concentration, which in turn will only reinforce pre-existing feelings of inadequacy, hopelessness, and low self-esteem.6,9 depression and anxiety symptoms are widely prevalent in university students.5 both are known to lower the quality of life and academic performance.10 more importantly, some students are more prone to developing mental disorders than others. for instance, students who are enrolled in demanding and challenging courses,11 students with financial struggles,11,12 and students with rural backgrounds11 are more vulnerable to mental illness. ibrahim et al conducted a systemic review on depression prevalence in university students, including 24 studies from 12 different countries—2 of them were within the middle-east: egypt and lebanon. the mean age of the students was 15–26. they found that depression was present in nearly one-third of the students with weighted mean prevalence of 30.6%—ranging from 10% to 84.6%. sixteen of the included studies reported gender differences, with females being more prone to depression in a majority of them. importantly, six studies found inverse relationship between year of study and rates of depression.5 in saudi arabia, a study conducted on preclinical medical and dental students in umm al-qura university showed high levels of depression (69.9%), anxiety (66.4%), and stress (70.9%). a more alarming issue is that, based on the depression anxiety stress scales (dass), the prevalence of students with severe to extremely severe symptoms of depression, anxiety, and stress, was 25.4%, 21.8%, and 34.1%, respectively.13 there is still stigma within the saudi community about seeking help when it comes to mental health.14 currently, awareness is much better compared to 10 years ago among developed countries, which helps doctors and teachers to correct erroneous concepts (e.g., mental illnesses are real, and negative emotions and thoughts need to be relieved because, with time, they can lead to physical harm and vice versa).15–17 most research findings outline the need to address mental health problems in young adults, students in particular. there have been no studies in saudi arabia regarding the risk of depression, anxiety, and stress among first-year university students. our study’s goal is to provide necessary data as evidence to raise awareness among college students to improve their mental health and offer early treatment and detection. furthermore, academic institutions need to provide sustainable mental health services to support university students to succeed in their academic years. therefore, this study aims to determine the rates of depression, anxiety, and stress among first-year university students at king abdulaziz university, and to determine the predisposing factors that trigger mental-health problems in students. materials and methods a descriptive cross-sectional study was conducted at king abdulaziz university, jeddah, saudi arabia. it was conducted from october 1, 2019, until january 30, 2020. the sample size rates of depression, anxiety, and stress in king abdulaziz university freshmen, jeddah, saudi arabia: a cross-sectional study sulhi a. alfakeh1, khaled mesfer alghamdi2, rawan mostafa hilal2, marwa mohamed abdelmoaty2 1 department of internal medicine, faculty of medicine, king abdulaziz university, jeddah, saudi arabia. 2 faculty of medicine, king abdulaziz university, jeddah, saudi arabia. *correspondence to: sulhi a. alfakeh (e-mail: salfakeh@kau.edu.sa) (submitted: 16 february 2021 – revised version received: 27 february 2021 – accepted: 09 march 2021 – published online: 26 april 2021) abstract objective this cross-sectional study aimed to determine the rates of depression, anxiety, and stress among first-year university students in saudi arabia. methods we distributed an online survey through to 861 first-year students, aged 17–25 years, at king abdulaziz university. the survey used a validated arabic short version of the depression anxiety and stress scales, and sociodemographic factors. results the prevalence of depression, anxiety, and stress was 80.4%, 71.8%, and 69.3%, respectively. a plurality showed extremely severe depression and anxiety, and severe stress symptoms. the frequency of exercise was inversely related to depression, anxiety, and stress. a total of 8.2% of students had visited a psychiatrist at some point in their lives, and 40.5% of students had suicidal thoughts. conclusions programs to identify students suffering from mental illness and offer adequate care and support are essential. keywords depression, anxiety, stress, saudi arabia, students 117 original rates of depression, anxiety, and stress in king abdulaziz university freshmen, jeddah, saudi arabiasulhi a. alfakeh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 116 – 121 needed for this study was calculated using the raosoft sample size calculator18 as 377 participants of an estimated population of 19,000. the study included 861 first-year (preparation year) king abdulaziz university students, with 457 men (53.1%) and 404 women (46.9%), aged 17-25. this study was approved by the unit of biomedical ethics at king abdulaziz university. all participants signed an online consent form to participate in the study. all of the students’ information ensures collected confidently. the participants completed a self-reported questionnaire using the university blackboard system. the questionnaire was distributed through secured online links sent by the university counseling center. the university identification number was used for verification, and only first-year university students were granted electronic access to the questionnaire. demographic data such as gender, age, family income, smoking, residency, and weekly exercise level were included. the arabic short version of the depression anxiety stress scales (dass-21) was used to measure symptoms of depression, anxiety, and stress during the past 2 weeks, with 21 questions classified into 3 sub-scales, each containing 7 items on a scale from 0 (did not apply to me at all) to 3 (applied to me very much or most of the time). it has been adapted and validated in arabic.19 based on the scoring instructions of the dass scale, the scores of the seven questions for each subscale were summed and multiplied by 2 and then classified based on the severity of symptoms. for the depression subscale, scores between 0 and 9 were normal, 10–13 mild, 14–20 moderate, 21–27 severe, and above 28 extremely severe symptoms. for the anxiety subscale, scores between 0 and 7 were normal, 8–9 mild, 10–14 moderate, 15–19 severe, and above 20 extremely severe symptoms. for the stress subscale, scores between 0 and 14 were normal, 15–18 mild, 19–25 moderate, 26–33 severe, and above 34 extremely severe symptoms.19 fifteen, yes or no, questions were added regarding 12-month and lifelong prevalence of depression diagnosis, anxiety diagnosis, seeing a psychiatrist or a health worker because of mental health struggles, seeing a psychologist because of mental health struggles, prescribed medications for struggles with mental health, suicidal ideation, and suicidal attempts, and if they wanted to visit the academic consultation center at the king abdulaziz university. data were entered and analyzed using the statistical package for the social sciences (spss) version 21. frequencies and percentages were used to describe qualitative variables, where the chi-squared test was used to evaluate the differences between two qualitative variables. a p-value < 0.05 was considered significant. results a total of 871 responses were collected, of which 10 were excluded because of the age criteria and 861 responses were included for the final analysis. the majority 81.1% had never smoked, 92.8% were living with their families, 37.0% had a high family monthly income, and 73.9% exercised once a week only. table 1 shows the characteristics of the sample. the prevalence of depression, anxiety, and stress symptoms among students was 80.4%, 71.8%, and 69.3%, respectively. ranging from mild to extremely severe, a plurality of participants with depression and anxiety showed extremely severe symptoms, specifically, 33.4%, and 35.5%, respectively. for stress, a plurality of participants showed severe symptoms, specifically, 22.2% (table 2). there were no significant gender differences in depressive symptoms. however, female students had higher anxiety symptoms than male students (76.7%, vs. 67.6%, p-value = <0.001, chi-squared value = 27.852, and df = 4) and higher stress symptoms (71.4% vs. 67.1%, p-value = <0.001, chisquared value = 24.011, and df = 4). smokers and ex-smokers had higher symptoms of depression (smoker = 88.3%, and ex-smoker= 84.6%) and anxiety (smoker = 79.3%, and ex-smoker = 84.7%) compared to non-smokers (depression: 78.8%, anxiety: 69.7%, p-value = 0.026, chi-squared value = 17.440, and df = 8), while there were no significant differences in stress. the analysis showed a negative relationship between the frequency of exercise during the week and symptom levels for depression and anxiety. students exercising 2–3 times per week showed significantly lower symptoms (depression = 71.2%, and anxiety = 64.4%), as well as students exercising 4 times and more per week (depression = 72.2%, and anxiety = 58.3%) compared to students exercising once a week only (depression = 83.5%, p-value = .007, chi-squared value = 21.063, and df = 8) (anxiety: 75.3%, p-value = .007, chisquared value = 20.901, and df = 8) (tables 3, 4, and 5). table 1. characteristics of the sample. characteristic n (%) gender male 457 (53.1) female 404 (46.9) family income <10000 sr 304 (35.3) 10000 150000 sr 238 (27.6) >15000 sr 319 (37) smoking status non-smoker 698 (81.1) smoker 111 (12.9) ex-smoker 52 (6) place of residence alone 35 (4.1) with family 799 (92.8) university dorm 27 (3.1) exercise once a week 636 (73.9) two to three times a week 146 (17) four or more times a week 79 (9.2) table 2. symptom severity of depression, anxiety, and stress. depression n (%) anxiety n (%) stress n (%) normal 169 (19.6) 242 (28.1) 264 (30.7) mild 84 (9.8) 56 (6.5) 98 (11.4) moderate 174 (20.2) 151 (17.5) 145 (16.8) severe 146 (17) 106 (12.3) 191 (22.2) extremely severe 288 (33.4) 306 (35.5) 163 (18.9) 118 original rates of depression, anxiety, and stress in king abdulaziz university freshmen, jeddah, saudi arabia sulhi a. alfakeh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 116 – 121 we also asked questions about visits to a psychiatrist, psychologist, or health-care worker because of mental health issues, students who used medication for their mental struggles, and students who had suicidal ideation or attempted suicide, during the last 12 months or at some point in their life, in relation to their dass-21 scores. a large percentage of students who had never before been diagnosed with depression or anxiety showed various symptoms of depression, anxiety, and stress. the majority had never seen a physician, psychiatrist, or a psychologist for mental health difficulties before. moreover, the majority had never taken any medication for mental health difficulties before. additionally, 40.5% of the students have had suicidal thoughts, and a plurality of them had extremely severe symptoms of depression. on the other hand, 17.5% of the students had ever attempted suicide before. finally, 79% wanted to visit the university counselling center. discussion our study showed a high prevalence of depression, anxiety, and stress symptoms, specifically, in 80.4%, 71.8%, and 69.3% of participants, respectively, among first-year king abdulaziz university students in jeddah, saudi arabia. to our knowledge, there have been no local studies done in saudi arabia or in the middle-east region among freshmen to compare with our results. however, compared to our results, research worldwide reported lower rates of depression, anxiety, and stress among university students, such as in okinawa (11.3%, 18.0%, and 5.7% respectively), germany (24.6% ,33.6%, and 26.3% respectively), thailand (35.6%, 43.3%, 19.0% respectively),40 united states (33%, 40%, and 38% respectively),20 and malaysia (37.2%, 63%, and 23.7%, respectively).7 a plurality of the sample showed extremely severe symptoms of depression and anxiety (33.4% and 35.5%, respectively), whereas a plurality showed severe stress symptoms (22.2%). by comparison, a study in egypt found that a plurality of students showed moderately severe symptoms of depression, anxiety, and stress (48.5%, 43.6%, and 30.1% respectively).21 a study in mecca, saudi arabia found that a plurality of participants had severe to extremely severe symptoms of depression, anxiety, and stress (25.4%, 21.8%, and 34.1% respectively).13 there were no significant differences between genders in depression symptoms. however, female students had higher anxiety symptoms than male students (76.7% vs. 67.6%) and higher stress symptoms (71.4% vs. 67.1%). in a study of undergraduate students from 15 universities in china, female students had significantly higher anxiety symptoms than males, which was reported as 40% for males and 45% for females in the first and second years.22 egyptian female medical students were at risk for any lifetime disorder, any 12-month disorder, and the persistence of symptoms.21 males were found to be more prone to depression than females (53.9% vs. 46.1%), whereas females were more vulnerable than males to anxiety (55.2% vs. 44.8%).22 table 3. distribution and significance of sociodemographics and depression scores. depression subgroups normal mild moderate severe extremely severe p-value gender             male 79 (17.3%) 48 (10.5%) 100 (21.9%) 84 (18.4%) 146 (31.9%) 0.168 female 90 (22.3%) 36 (8.9%) 74 (18.3%) 62 (15.3%) 142 (35.1%) smoking status             smoker 13 (11.7%) 8 (7.2%) 18 (16.2%) 19 (17.1%) 53 (47.7%) 0.022non-smoker 148 (21.2%) 72 (10.3%) 148 (21.2%) 117 (16.8%) 213 (30.5%) ex-smoker 8 (15.4%) 4 (7.7%) 8 (15.4%) 10 (19.2%) 22 (42.3%) family monthly income             less than 10 k riyals 53 (17.4%) 39 (12.8%) 59 (19.4%) 53 (17.4%) 100 (32.9%) 0.342between 10 k and 15 k riyals 45 (18.9%) 19 (8.0%) 44 (18.5%) 42 (17.6%) 88 (37.0%) more than 15 k riyals 71 (22.3%) 26 (8.2%) 71 (22.3%) 51 (16.0%) 100 (31.3%) living status             alone 4 (11.4%) 2 (5.7%) 6 (17.1%) 13 (37.1%) 10 (28.6%) 0.124with family 159 (19.9%) 77 (9.6%) 163 (20.4%) 131 (16.4%) 269 (33.7%) dorm/out 6 (22.2%) 5 (18.5%) 5 (18.5%) 2 (7.4%) 9 (33.3%) exercise             once a week 105 (16.5%) 63 (9.9%) 124 (19.5%) 115 (18.1%) 229 (36.0%) 0.008two to three times a week 42 (28.8%) 14 (9.6%) 34 (23.3%) 22 (15.1%) 34 (23.3%) more than four times a week 22 (27.8%) 7 (8.9%) 16 (20.3%) 9 (11.4%) 25 (31.6%) 119 original rates of depression, anxiety, and stress in king abdulaziz university freshmen, jeddah, saudi arabiasulhi a. alfakeh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 116 – 121 we demonstrated a positive relation between smoking and the severity of depression and anxiety. several studies have demonstrated such a correlation and that the risk of psychiatric illnesses including depression and anxiety increases with smoking.23–26 however, a study in syria found that smoking does not cause significant psychiatric symptoms.27 although using the same tool to evaluate psychiatric symptoms (dass21), this difference may be due to an unbalanced sample distribution. results showed that an increase in the frequency of exercise per week is associated with a reduced risk of psychological distress. exercise is known to be an effective intervention to help reduce the symptoms of depression and anxiety,28 which raises the question of whether exercise may be an effective first-line intervention option. a landmark meta-analysis of randomized controlled trials found that current literature is insufficient to answer this question and emphasized the need to conduct adequate quality studies to demonstrate this effect.29 in comparison to the results of our study, multiple surveys published in 2018 done with first-year college students in 19 colleges across 8 countries including australia, germany, northern ireland, spain, south africa, the united states, mexico, and belgium, found that major depressive disorder (mdd) was the most common of the disorders surveyed across all countries combined (21.2% lifetime prevalence; 18.5% 12-month prevalence) followed by generalized anxiety disorder (18.6% lifetime prevalence–16.7% 12-month prevalence).30 recently, the saudi national mental health survey (snmhs) results were published, showing a lifetime prevalence of depression and generalized anxiety disorder (gad) of 6.0% and 1.9%, respectively.31 in our survey, we asked about anxiety disorder diagnoses in general, while the prevalence data in the snmhs were specific to gad. this in addition to the surprising fact provided by snmhs that more educated saudis were more prone to have mental illness, and the increased risk for our sample population might explain the difference between our result and that of the snmhs. around 21% of our participants wanted to visit the academic consular office and 8.2% had visited a psychiatrist regarding their mental issues. in comparison, another study in saudi arabia found that faith healing setting visitors had a high rate of being diagnosed with mental illness and a small portion reported a positive history of psychiatric disorder, while more than 50% of participants had not sought medical help regarding their concerns.32 from 13,984 of first-year university students among different countries, only 24.6% would definitely seek mental therapy.33 similarly, in riyadh, 25.2% were willing to ask for a psychiatric consultation if they experienced a mental illness, and the main reason for not visiting a psychiatrist was feeling ashamed.34 apparently, there are many people with mental disorders who are not seeking help or willing to visit mental health professionals because they feel embarrassed to express their emotions. people who had already sought mental health treatment were in a minority. table 4. distribution and significance of sociodemographics and anxiety scores. anxiety subgroups normal mild moderate severe extremely severe p-value gender             male 148 (32.4%) 34 (7.4%) 92 (20.1%) 56 (12.3%) 127 (27.8%) <0.0001 female 94 (23.3%) 22 (5.4%) 59 (14.6%) 50 (12.4%) 179 (44.3%) smoking status             smoker 23 (20.7%) 8 (7.2%) 23 (20.7%) 10 (9.0%) 47 (42.3%) 0.026non-smoker 211 (30.2%) 44 (6.3%) 112 (16.0%) 92 (13.2%) 239 (34.2%) ex-smoker 8 (15.4%) 4 (7.7%) 16 (30.8%) 4 (7.7%) 20 (38.5%) family monthly income             less than 10 k riyals 83 (27.3%) 12 (3.9%) 52 (17.1%) 34 (11.2%) 123 (40.5%) 0.076between 10 k and 15 k riyals 62 (26.1%) 17 (7.1%) 40 (16.8%) 39 (16.4%) 80 (33.6%) more than 15 k riyals 97 (30.4%) 27 (8.5%) 59 (18.5%) 33 (10.3%) 103 (32.3%) living status             alone 11 (31.4%) 1 (2.9%) 7 (20.0%) 4 (11.4%) 12 (34.3%) 0.417with family 221 (27.7%) 55 (6.9%) 139 (17.4%) 101 (12.6%) 283 (35.4%) dorm/out 10 (37.0%) 0 (0.0%) 5 (18.5%) 1 (3.7%) 11 (40.7%) exercise             once a week 157 (24.7%) 44 (6.9%) 107 (16.8%) 83 (13.1%) 245 (38.5%) 0.007two to three times a week 52 (35.6%) 8 (5.5%) 28 (19.2%) 18 (12.3%) 40 (27.4%) more than four times a week 33 (41.8%) 4 (5.1%) 16 (20.3%) 5 (6.3%) 21 (26.6%) 120 original rates of depression, anxiety, and stress in king abdulaziz university freshmen, jeddah, saudi arabia sulhi a. alfakeh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 116 – 121 there is a widely accepted belief in society that mentally ill people are dangerous and hiring them will lessen working network functionality.35 almost 800,000 people end their life by attempting suicide every year worldwide, among males and females aged between 15 and 29 years. furthermore, suicide was the second leading cause of death in 2016.36 surprisingly, 40.5% of the students had ever thought of suicide and 17.5% had attempted suicide. a study assessing repeated suicidal attempts among youth in saudi arabia illustrated that 26.1% had recurrent suicide attempts between 10 and 24 years old and 51% had family problems.37 although being a muslim is thought to be a protective factor against suicide acceptability, society still suffers from stigmatizing mental illnesses.38 our findings differ from those in first-year canadian university undergraduate students, wherein 14% of students admitted having suicidal thoughts and 1.6% had attempted suicide.39 these results justify proper canadian academic support and improved mental health awareness toward seeking help from professionals when it is needed. we used the dass-21 to measure the prevalence of depression, anxiety, and stress, along with brief sociodemographic factors associated with them in a specific high-risk group. further studies including much larger samples from different places and with different risk levels are needed to detect the exact extent of these issues, and to detect the nature of the relationship between the mental illnesses and sociodemographic factors. furthermore, dass-21 is not a diagnostic tool, thus it might yield an overestimated prevalence rate. also, it is difficult to establish if our sample is representative of the population. our research might have the risk of selection bias as the students who are more likely to experience mental illness could be more likely to fill the questionnaire. conclusion college students are often viewed as a privileged population, but they are not immune from suffering and disability associated with mental illness. youth mental health has a high impact and a significant role in academic performance, especially among the junior years, as they are yet to adapt to the new and challenging environment. in addition, our study highlighted that a significant number of students are at high risk for mental struggles. we suggest establishing programs to identify students who are in distress and offer adequate care and support to help them overcome mental illness. increasing the number of mental health facilities and raising awareness will increase assessment of suicidal risk and would facilitate an effective response to the elevated suicide rates. data availability statement the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. table 5. distribution and significance of sociodemographics and stress scores. stress subgroups normal mild moderate severe extremely severe p-value gender             male 148 (32.4%) 58 (12.7%) 93 (20.4%) 95 (20.8%) 63 (13.8%) <0.0001 female 116 (28.7%) 40 (9.9%) 52 (12.9%) 96 (23.8%) 100 (24.8%) smoking status             smoker 23 (20.7%) 11 (9.9%) 20 (18.0%) 26 (23.4%) 31 (27.9%) 0.177non-smoker 226 (32.4%) 82 (11.7%) 114 (16.3%) 155 (22.2%) 121 (17.3%) ex-smoker 15 (28.8%) 5 (9.6%) 11 (21.2%) 10 (19.2%) 11 (21.2%) family monthly income             less than 10 k riyals 88 (28.9%) 36 (11.8%) 54 (17.8%) 65 (21.4%) 61 (20.1%) 0.741between 10 k and 15 k riyals 65 (27.3%) 30 (12.6%) 40 (16.8%) 56 (23.5%) 47 (19.7%) more than 15 k riyals 111 (34.8%) 32 (10.0%) 51 (16.0%) 70 (21.9%) 55 (17.2%) living status             alone 9 (25.7%) 4 (11.4%) 5 (14.3%) 12 (34.3%) 5 (14.3%) 0.653with family 245 (30.7%) 91 (11.4%) 138 (17.3%) 174 (21.8%) 151 (18.9%) dorm/out 10 (37.0%) 3 (11.1%) 2 (7.4%) 5 (18.5%) 7 (25.9%) exercise             once a week 177 (27.8%) 72 (11.3%) 105 (16.5%) 148 (23.3%) 134 (21.1%) 0.047two to three times a week 56 (38.4%) 15 (10.3%) 29 (19.9%) 28 (19.2%) 18 (12.3%) more than four times a week 31 (39.2%) 11 (13.9%) 11 (13.9%) 15 (19.0%) 11 (13.9%) 121 original rates of depression, anxiety, and stress in king abdulaziz university freshmen, jeddah, saudi arabiasulhi a. alfakeh et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 116 – 121 references 1. gbd 2017 disease and injury incidence and prevalence collaborators. global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the global burden of disease study 2017. lancet, 2018;392(10159):1789–1858. https://doi.org/10.1016/s01406736(18)32279-7 2. american psychiatric association. anxiety. 2019. https://www.apa.org/ topics/anxiety/index 3. american psychiatric association. major depressive disorder and the “bereavement exclusion.” american psychiatric association. 2013. 4. grøtan, k, sund, er, bjerkeset, o. mental health, academic self-efficacy and study progress among college students – the shot study, norway. front psychol, 2019;10:article 45. https://doi.org/10.3389/fpsyg.2019.00045 5. ibrahim, ak, kelly, sj, adams, ce, glazebrook, c. a systematic review of studies of depression prevalence in university students. j psychiat res, 2013;47(3):391–400. https://doi.org/10.1016/j.jpsychires.2012.11.015 6. stallman, hm. prevalence of psychological distress in university students: implications for service delivery. austral family phys, 2008;37(8):673–677. 7. shamsuddin, k, fadzil, f, ismail, wsw, shah, sa, omar, k, muhammad, na, et al. correlates of depression, anxiety and stress among malaysian university students. asian j psychiat, 2013;6(4), 318–323. https://doi.org/10.1016/j. ajp.2013.01.014 8. silverstein, st, kritz-silverstein, d. a longitudinal study of stress in first-year dental students. j dental educ, 2010;74(8):836–848. 9. rice, kg, leever, ba, christopher, j, porter, jd. perfectionism, stress, and social (dis)connection: a short-term study of hopelessness, depression, and academic adjustment among honors students. j counsel psychol, 2006;53(4):524–534. https://doi.org/10.1037/0022-0167.53.4.524 10. kessler, rc, foster, cl, saunders, wb, stang, pe. social consequences of psychiatric disorders i: educational attainment. am j psychiatry, 1995;152(7):1026–1032. https://doi.org/10.1176/ajp.152.7.1026 11. bayram, n, bilgel, n. the prevalence and socio-demographic correlations of depression, anxiety and stress among a group of university students. social psychiat psychiatr epidemiol, 2008;43(8):667–672. https://doi.org/10.1007/ s00127-008-0345-x 12. adewuya, ao, ola, ba, afolabi, oo. validity of the patient health questionnaire (phq-9) as a screening tool for depression amongst nigerian university students. j affect disord, 2006;96(1-2):89–93. https://doi. org/10.1016/j.jad.2006.05.021 13. aboalshamat, k, hou, x-y, strodl, e. psychological well-being status among medical and dental students in makkah, saudi arabia: a cross-sectional study. med teacher, 2015;37(sup1):s75–s81. https://doi.org/10.3109/01421 59x.2015.1006612 14. alhabeeb, aa, alasmari, ss, alduraihem, ra, qureshi, na. mental health awareness phone polling survey: focus on community knowledge, attitude and practice, saudi arabia. int neuropsych dis j, 2019:1–14. https://doi. org/10.9734/indj/2019/v13i230106 15. druss, bg. mental health quality improvement goes global. world psychiat, 2018;17(1):44–45. https://doi.org/10.1002/wps.20487 16. gilbert, p, mcewan, k, irons, c, bhundia, r, christie, r, broomhead, c, et al. self-harm in a mixed clinical population: the roles of self-criticism, shame, and social rank. brit j clin psychol, 2010;49(4):563–576. https://doi. org/10.1348/014466509x479771 17. srivastava, k, chatterjee, k, bhat, ps. mental health awareness: the indian scenario. indust psychiatry j, 2016;25(2), 131–134. https://doi.org/10.4103/ ipj.ipj_45_17 18. raosoft. sample size calculator. raosoft, inc. 2004. http://www.raosoft.com/ samplesize.html. 19. moussa, mt, lovibond, p, laube, r, megahead, ha. psychometric properties of an arabic version of the depression anxiety stress scales (dass). res social work pract, 2017;27(3):375–386. https://doi. org/10.1177/1049731516662916 20. beiter, r, nash, r, mccrady, m, rhoades, d, linscomb, m, clarahan, m, et al. the prevalence and correlates of depression, anxiety, and stress in a sample of college students. j affect disord, 2015;173:90–96. https://doi. org/10.1016/j.jad.2014.10.054 21. abdallah, ar, gabr, hm. depression, anxiety and stress among first year medical students in an egyptian public university. int res j med med sci, 2014;2(1):11–19. 22. gao, w, ping, s, liu, x. gender differences in depression, anxiety, and stress among college students: a longitudinal study from china. j affect disord, 2020;263:292–300. https://doi.org/10.1016/j.jad.2019.11.121 23. byeon, h. association among smoking, depression, and anxiety: findings from a representative sample of korean adolescents. peerj, 2015;3(10):e1288. https://doi.org/10.7717/peerj.1288 24. khaled, sm, bulloch, a, exner, dv, patten, sb. cigarette smoking, stages of change, and major depression in the canadian population. can j psychiatry, 2009;54(3), 204–208. https://doi.org/10.1177/070674370905400309 25. kulsoom, b, afsar, na. stress, anxiety, and depression among medical students in a multiethnic setting. neuropsychiat dis treatment, 2015;11:1713–1722. https://doi.org/10.2147/ndt.s83577 26. martini, s, wagner, fa, anthony, jc. the association of tobacco smoking and depression in adolescence: evidence from the united states. subst use misuse, 2002;37(14):1853–1867. https://doi.org/10.1081/ja-120014087 27. al saadi, t, zaher addeen, s, turk, t, abbas, f, alkhatib, m. psychological distress among medical students in conflicts: a cross-sectional study from syria. bmc med educ, 2017;17(1):173. https://doi.org/10.1186/s12909-0171012-2 28. callaghan, p. exercise: a neglected intervention in mental health care? j psychiat mental health nurs, 2004;11(4):476–483. https://doi.org/10.1111/ j.1365-2850.2004.00751.x 29. lawlor, da, hopker, sw. the effectiveness of exercise as an intervention in the management of depression: systematic review and meta-regression analysis of randomised controlled trials. bmj, 2001;322(7289):763–767. https://doi.org/10.1136/bmj.322.7289.763 30. auerbach, rp, mortier, p, bruffaerts, r, alonso, j, benjet, c, cuijpers, p, et al., who wmh-ics collaborators. who world mental health surveys international college student project: prevalence and distribution of mental disorders. j abnorm psychol, 2018;127(7):623–638. https://doi.org/10.1037/ abn0000362 31. al-subaie, as, al-habeeb, a, altwaijri, ya. overview of the saudi national mental health survey. int j methods psychiat res, 2020;29(3):e1835. https:// doi.org/10.1002/mpr.1835 32. alosaimi, fd, alshehri, y, alfraih, i, alghamdi, a, aldahash, s, alkhuzayem, h, et al. the prevalence of psychiatric disorders among visitors to faith healers in saudi arabia. pak j med sci, 2014;30(5):1077–1082. https://doi. org/10.12669/pjms.305.5434 33. ebert, dd, mortier, p, kaehlke, f, bruffaerts, r, baumeister, h, auerbach, rp, et al. barriers of mental health treatment utilization among first-year college students: first cross-national results from the who world mental health international college student initiative. int j methods psychiatric res, 2019;28(2):1–14. https://doi.org/10.1002/mpr.1782 34. mahmoud, ma. knowledge and awareness regarding mental health and barriers to seeking psychiatric consultation in saudi arabia. asian j pharm res health care, 2019;10(4):109–116. https://doi.org/10.18311/ ajprhc/2018/23359 35. corrigan, pw, druss, bg, perlick, da. the impact of mental illness stigma on seeking and participating in mental health care. psychol sci public interest, 2014;15(2):37–70. https://doi.org/10.1177/1529100614531398 36. world health organization. suicide. 2019. https://www.who.int/newsroom/fact-sheets/detail/suicide 37. ahmed, ae, alaqeel, m, alasmari, na, jradi, h, otaibi, ha, abbas, et al. risk assessment of repeated suicide attempts among youth in saudi arabia. risk manag healthcare policy, 2020;13:1633–1638. https://doi.org/10.2147/ rmhp.s245175 38. ejjennane, fe. religion and psychological well-being: does islam offer protection against suicide? [master’s thesis]. harvard extension school. 2019. https://nrs.harvard.edu/urn-3:hul.instrepos:37365390 39. duffy, a, keown-stoneman, c, goodday, s, horrocks, j, lowe, m, king, et al. predictors of mental health and academic outcomes in first-year university students: identifying prevention and early-intervention targets. bjpsych open, 2020;6(3):e46. https://doi.org/10.1192/bjo.2020.24 40. china, t, ratanasiripong, p, toyama, s, nann, n. the first-year experience: mental health of university students in okinawa, germany, and thailand. 2020 41. kessler, rc, walters, ee, forthofer, ms. the social consequences of psychiatric disorders, iii: probability of marital stability. am j psychiatry, 1998;155(8), 1092–1096. https://doi.org/10.1176/ajp.155.8.1092 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.962 https://www.apa.org/topics/anxiety/index https://www.apa.org/topics/anxiety/index http://www.raosoft.com/samplesize.html http://www.raosoft.com/samplesize.html https://www.who.int/news-room/fact-sheets/detail/suicide https://www.who.int/news-room/fact-sheets/detail/suicide 92 original issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 92 – 95 introduction diagnosis is a fundamental part of every branch of medicine and plays a critical role in choosing the treatment plan and subsequent procedures.1 diagnostic radiographic images are commonly used to identify, diagnose, rating, and screen the diseases that affect the head and neck area or dental system.2,3 in recent years, cone-beam computed tomography (cbct) has become an increasingly popular diagnostic tool in dentistry, as it can provide three-dimensional (3d) images of the maxillofacial region that greatly facilitate diagnosis and treatment in this field, especially implant placement and treatment of pathogenic lesions. with the introduction and advancement of cbct, 3d imaging has turned into a practical, accessible tool for dental practitioners. clinical applications of cbct in the diagnosis and treatment procedures involving oral and maxillofacial areas are increasing by the day. one of the most important treatment methods in modern dentistry is implant placement. however, the success of this treatment largely depends on the quality and quantity of bone in the implant recipient site. research has shown that the implants inserted in bones of poor or low quantity are more likely to fail, which is why it is critical to carefully assess the bone structure before implant placement.1, 2 with the emergence of new imaging techniques such as multidetector computed tomography (mdct), cbct, dual-energy x-ray absorptiometry (dxa), and digital subtraction, it is now easier to measure bone mineral density (bmd) for such purposes.3-5 among these methods, cbct is increasingly used in dentistry for 3d imaging of oral and maxillofacial regions.6-8 the main advantages of cbct include high accessibility, ease of use, and the ability to provide real-size data and cross-sectional, multiplan, and 3d reconstructions of the area of interest.9-11 cbct can provide a clear view of the location of impacted teeth, their relationship with, and effects on other teeth.11 the data obtained from cbct can be reconstructed to illustrate incisions in the axial, coronal, and sagittal planes. cbct also allows the surgeon to reconstruct 3d images of the target area.12 some researchers have argued that cbct voxel numbers can be used to estimate the hounsfield unit (hu) and bmd.13, 14, but many others believe that cbct does not offer a reliable assessment of bmd.15, 16 the hu is one of the main quantitative measures of bone density in mdct. finding a relationship between the hu of identical specimens in cbct and mdct could indicate the accuracy and validity of this unit for cbct. it may open up new avenues for research on this subject. while many new cbct machines offer a built-in tissue density estimation feature, only a few studies have evaluated the accuracy of these estimates. therefore, the authors decided to investigate this issue. for this purpose, a comparison was made between the hu values obtained for a series of specimens with different bone densities from cbct and mdct images. comparison of the hounsfield unit values obtained from cone-beam computed tomography (cbct) and multidetector computed tomography (mdct) images for different bone densities atefeh khavid1 id , mojgan sametzadeh2 id , mostafa godiny3* id , mohammad mehdi moarrefpour4 id 1 department of oral and maxillofacial radiology, school of dentistry, kermanshah university of medical sciences, kermanshah, iran 2 radiology department, faculty of medical science, jundishapour university of medical sciences, ahvaz, iran 3 department of endodontics, school of dentistry, kermanshah university of medical sciences, kermanshah, iran 4 students research committee, school of dentistry, kermanshah university of medical sciences, kermanshah, iran *correspondence to: mostafa godiny (e-mail: mostafa_goodin@yahoo.com) (submitted: 02 february 2021 – revised version received: 17 february 2021 – accepted: 20 march 2021 – published online: 26 april 2021) abstract objective in recent years, cone-beam computed tomography (cbct) has become a key diagnostic tool in dentistry. cbct can provide 3d images of the maxillofacial area to help dental practitioners in diagnosis and treatment, especially implant placement and treatment of pathogenic lesions. this study aimed to compare the hounsfield unit (hu) values obtained from cbct images for bones of different densities with the corresponding hu values from multidetector ct (mdct) images. methods cube-shaped bone blocks of identical size were cut from the middle section of the cow ribs and femur area such that they had a layer of cortical bone in their buccal, lingual, and top surfaces and trabecular bone in the middle. mdct scans were performed using a somatom sensation ct scanner. after determining hu from the results of these scans, nine suitable specimens from different ranges of hu were chosen for comparison. hu of the cbct images was computed by the dedicated software of the cbct machine. finally, hu values obtained from mdct and cbct were compared. data analysis was performed using spss version 25 at the 0.05 significance level. results the results showed a statistically significant difference between the mean hu from mdct images and the mean hu from cbct images (p<0.05). for similar specimens, cbct produced higher mean hu values than mdct. the pearson correlation test detected a significant direct relationship between the hu values of specimens in mdct and cbct (p<0.05). conclusions for the tools and software used in this study, there was no significant difference between the hu values obtained from mdct and cbct, but the mean hu obtained from cbct was higher than that from mdct. keywords hounsfield unit, cone beam computed tomography, multidetector computed tomography https://orcid.org/0000-0001-5505-7571 https://orcid.org/0000-0002-3402-4321 https://orcid.org/0000-0002-8451-0483 https://orcid.org/0000-0002-9179-6029 93 original cbct and mdct images for different bone densitiesmostafa godiny et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 92 – 95 method this study was carried out with the approval of the ethics committee (ir.kums.rec.1399.322) of kermanshah university of medical sciences and the relevant officials of the school of dentistry at this university. the specimens were made from the bone of a slaughtered cow with due attention to measurement accuracy. the pilot study was performed with three dry mandibles. mdct and cbct scanned the specimens, and their hu in the same anterior and posterior regions were determined. it was observed that the hu values obtained from cbct images were very close to those of mdct images (fig. 1a, 1b). to create the specimens, bone blocks of the same size were cut from the middle section of a cow’s ribs and femur area. the blocks were cube-shaped with right angles and identical dimensions and were cut such that they had a layer of cortical bone in their buccal, lingual, and top surfaces and trabecular bone in the middle. according to previous studies,27 cow bone was chosen mainly; a cow’s femur bone has a density of d1, and different parts of its rib bone have a density of d2 to d4. other reasons for choosing cow bone included easier access and the possibility of repeated imaging without significant ethical limitations. at all stages of the study, the specimens were placed in a refrigerator with a humid environment to prevent drying, which would change the tissue density (this is why dry mandibles were not used). also, the delay between specimen preparation and final imaging was minimized to minimize density change during this period. mdct scans were obtained using a somatom sensation ct scanner. after determining the bone density of all specimens fig. 1 (a) an example of cbct images. fig. 1 (b) an example of cbct images. fig. 1 (c) specimens fixed in the wax model of the mandible. 94 original cbct and mdct images for different bone densities mostafa godiny et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 92 – 95 in hu, nine suitable specimens from each range of hu were selected for use in subsequent steps. the selected specimens were numbered and then fixed on the wax model of the mandible in random order (fig. 1c). after taking another mdct scan from the fixed specimens, cbct scans were performed using a newtom giano cbct machine. in all of these scans, tissue density was obtained from the machine’s dedicated software, which an expert operated. in the end, the results obtained from cbct and mdct were compared with each other. a total of 52 specimens were scanned by each imaging method (104 specimens in total). the required data, which included the hu values obtained from cbct images and those from mdct images, were collected with a checklist. data analysis was performed in spss version 25 using both descriptive and inferential statistical methods at the 0.05 significance level. the normality of data distribution was checked with the kolmogorov–smirnov test. descriptive statistics (mean, standard deviation) were used to summarize and describe the quantitative characteristics of the variables. the pearson correlation test and independent t-test were used to test the hypotheses. results in this study, the tissue density of 52 cow bone specimens was determined by mdct and cbct determined the tissue density of another 52 specimens. the mean hu (tissue density) of the specimens in mdct, and cbct were 805.9±591.8 and 1084.5±772.6, respectively (table 1). after establishing the normality of the data, the independent t-test was performed to detect differences between the means of hu values. this test showed a statistically significant difference between the mean hu in mdct images, 805.9, and the mean hu in cbct images, 1084.5 (p<0.05). as can be seen, for similar specimens, cbct provided a higher mean of hu than mdct (table 2). the pearson correlation test found a significant direct relationship with a correlation coefficient of 0.978 (p<0.01) between the hu values of the specimens in mdct and those of the specimens in cbct (fig. 2). as shown in table 3, the chi-square test detected no statistically significant difference between the hu values obtained from mdct and cbct images (p<0.05), suggesting that the two imaging methods offer similar results in terms of hu. discussion bone density assessments are essential for deciding when implant placement is a viable treatment and for the ultimate success of this procedure. therefore, these assessments need to be made based on reasonably reliable imaging tests. thus, there have been many studies on the estimation of bone density with different methods.17 initially, these assessments were being table 1. the mean and standard deviation of hu (tissue density) of bone specimens in mdct and cbct. variable mean standard deviation min max hu of specimens in mdct 805.9 591.8 16 1792 hu of specimens in cbct 1084.5 772.6 134 2346 table 2. comparison of mean hu of the specimens in mdct and cbct images. variable mdct cbct t p-valuemean ± standard deviation mean ± standard deviation hu of specimens 805.9±591.8 1084.5±772.6 -2.064 0.042 table 3. relative and absolute frequency distribution of mu of the specimens from mdct and cbct images. mu / group mdct cbct chisquare p-valuefrequency (percentage) frequency (percentage) d1 < 1250 19(50) 19(50) 6.47 0.091 850-1250 7(50) 7(50) 350-850 6(28.6) 15(71.4) 0-350 20(64.5) 11(35.5) fig. 2 scatter plot of mu of the specimens from mdct and cbct images. 95 original cbct and mdct images for different bone densitiesmostafa godiny et al. j contemp med sci | vol. 7, no. 2, march-april 2021: 92 – 95 performed theoretically, but eventually, the hu was introduced to standardize the relationship between theoretical assessments and bone density. research has shown that high bone density is correlated with the success of implants. also, high density is associated with the good initial stability of the implant. recently, cbct has replaced mdct in many areas of the dental practice and is increasingly used for pre-implant imaging, thanks to its ability to capture mineralized tissues. examining the hu values obtained for identical specimens from two different imaging techniques can shed more light on the accuracy and the differences of the two methods, which can have important implications for the diagnosis and the subsequent treatments. in this study, the findings showed no significant difference between the two imaging methods in terms of the obtained mu values. however, the mean hu in cbct images was higher than the mean hu in mdct. this finding is consistent with the results of a study by sliva et al.18, in which, after examining cbct and mdct images taken from 20 dried mandibles, the mean hu obtained from cbct images was significantly higher. in a study by nackaerts et al., different cbct machines reported different hu values for the same specimens, but this was not an issue in mdct.15 after comparing the hu and gray values obtained with 11 different cbct machines for the same specimens, mah et al.19 also reported differences in the densities estimated by these machines. however, other studies such as nomura et al.14 have obtained similar voxel values from cbct and mdct, indicating that cbct could still be a good imaging method for face assessments. the discrepancies mentioned above could be due to various reasons, including the low accuracy of the software. these differences may also be attributed to the cone angle of the beams, which is the main difference between cbct and mdct imaging techniques, because as the region of interest gets larger and the said angle increases, so does the inaccuracy of measurements. according to nackaerts et al.,15 tissue density estimates obtained from cbct are unreliable because they vary with the machine, imaging parameters, and imaged area. this issue could be extremely important for implant placement because an error in the reported hu may result in the misidentification of the bone type (d1, d2, d3, d4), which may put the operation at risk. the most important limitation of this study was the inability to use live specimens because of high x-ray exposure. conclusion considering the tools and software used in this study, there was no statistically significant difference between the hu values obtained from mdct and cbct images. still, the mean hu obtained from cbct was higher than mdct. it is recommended to design and perform a similar study to investigate the role of the software error in the observed differences. references 1. payahoo s, jabbari g. the ability of cone beam computed tomography to predict osteopenia and osteoporosis via radiographic density derived from cervical vertebrae. int j scient res dental med sci. 2019;1(2):18-22. 10.30485/ijsrdms.2019.89758. 2. resnik rr, kircos lt, misch ce. diagnostic imaging and techniques. contemporary implant dentistry. missouri: mosby. 2007:38-67. 3. isoda k, ayukawa y, tsukiyama y, sogo m, matsushita y, koyano k. relationship between the bone density estimated by cone‐beam computed tomography and the primary stability of dental implants. clini oral implants res. 2012;23(7):832-6. https://doi.org/10.1111/j.16000501.2011.02203.x. 4. gonzález‐garcía r, monje f. the reliability of cone‐beam computed tomography to assess bone density at dental implant recipient sites: a histomorphometric analysis by micro‐ct. clin oral implants res. 2013;24(8):871-9. https://doi.org/10.1111/j.1600-0501.2011.02390.x. 5. fuster-torres má, peñarrocha-diago m, peñarrocha-oltra d, peñarrochadiago m. relationships between bone density values from cone beam computed tomography, maximum insertion torque, and resonance frequency analysis at implant placement: a pilot study. int j oral maxillofac implants. 2011;26(5): 1051-56. 6. yabroudi f, sindet-pedersen s. cone beam tomography (cbct) as a diagnostic tool to assess the relationship between the inferior alveolar nerve and roots of mandibular wisdom teeth. smile dent j. 2012;7(3):12-6. 7. feldkamp la, davis lc, kress jw. practical cone-beam algorithm. josa a. 1984;1(6):612-9. https://doi.org/10.1364/josaa.1.000612. 8. aponte mendez m, kayasöken g, afjeh soleymani b, ravanbakhsh b. evaluation outcome of cone beam computed tomography for treatment plan success and failure: a systematic review. int j scient res dental med sci. 2020;2(2):46-51. 10.30485/ijsrdms.2020.233140.1061. 9. kipp dp, goldstein bh, weiss ww. dysesthesia after mandibular third molar surgery: a retrospective study and analysis of 1,377 surgical procedures. j am dental assoc. 1980;100(2):185-92. https://doi.org/10.14219/jada. archive.1980.0074. 10. libersa p, savignat m, tonnel a. neurosensory disturbances of the inferior alveolar nerve: a retrospective study of complaints in a 10-year period. j oral maxillofac surg. 2007;65(8):1486-9. https://doi.org/10.1016/j. joms.2007.03.023. 11. monaco g, montevecchi m, bonetti ga, gatto mr, checchi l. reliability of panoramic radiography in evaluating the topographic relationship between the mandibular canal and impacted third molars. j am dental assoc. 2004;135(3):312-8. https://doi.org/10.14219/jada.archive.2004.0179. 12. deepak c, saravanan b, kumar sk. cbct-a paradigm shift in the management of dental impactions. ind j multidisc dent. 2011;1(2). 13. naitoh m, hirukawa a, katsumata a, ariji e. evaluation of voxel values in mandibular cancellous bone: relationship between cone‐beam computed tomography and multislice helical computed tomography. clin oral implants res. 2009;20(5):503-6. https://doi.org/10.1111/j.1600-0501.2008.01672.x. 14. nomura y, watanabe h, honda e, kurabayashi t. reliability of voxel values from cone‐beam computed tomography for dental use in evaluating bone mineral density. clin oral implants res. 2010;21(5):558-62. https://doi. org/10.1111/j.1600-0501.2009.01896.x. 15. nackaerts o, maes f, yan h, couto souza p, pauwels r, jacobs r. analysis of intensity variability in multislice and cone beam computed tomography. clin oral implants res. 2011;22(8):873-9. https://doi.org/10.1111/j.16000501.2010.02076.x. 16. hua y, nackaerts o, duyck j, maes f, jacobs r. bone quality assessment based on cone beam computed tomography imaging. clin oral implants res. 2009;20(8):767-71. https://doi.org/10.1111/j.1600-0501.2008.01677.x. 17. arisan v, karabuda zc, avsever h, özdemir t. conventional multi‐slice computed tomography (ct) and cone‐beam ct (cbct) for computer‐ assisted implant placement. part i: relationship of radiographic gray density and implant stability. clin implant dent related res. 2013;15(6):893-906. https://doi.org/10.1111/j.1708-8208.2011.00436.x. 18. silva im, freitas dq, ambrosano gm, bóscolo fn, almeida sm. bone density: comparative evaluation of hounsfield units in multislice and cone-beam computed tomography. braz oral res. 2012;26(6):550-6. https://doi. org/10.1590/s1806-83242012000600011. 19. mah p, reeves te, mcdavid wd. deriving hounsfield units using grey levels in cone beam computed tomography. dentomaxillofac radiol. 2010;39(6):323-35. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.943 86 original issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 86 – 91 advances in knowledge this is the first study to assess the risk factors of dfd in oman. the study revealed high incidents of uncontrolled risk factors in oman. it opens the door for future articles to study this disease and explore more about it. application to patient care this study gives the community chance to identify dfd risk factors and by that it is possible to prevent getting high numbers of new dfd cases by controlling these modifiable risk factors. creating a new management technique to control these risk factors. increased public awareness of dfd risk factors and its substantial impact on the incidence and outcomes of the disease. introduction one of the most difficult challenges that faces modern medicine is chronic diseases,1 and diabetes mellitus (dm) is one of the devastating conditions with many complications, the world health organization (who) estimated that the global prevalence of diabetes among adults (above 18 years) was 8.5% in 2014.2 based on the international diabetes federation (idf), the prevalence of diabetes in the middle east and north africa (mena) was 9.6% in the population with age from 20 to 79 in 2017.3 the idf predict that the prevalence will increase to 12.1% in 2045.3 oman is of no exception in this regard. 71,625 patients of the omani population are suffering from diabetes based on a ministry of health (moh) records in 2011.4 the annual health report of ministry of health (moh) in 2016 showed that the number of diabetic patients in oman was 89,246 for all those who are registered in moh al shifa system.5 the recent steps survey for non-communicable diseases (ncd) 2107 revealed that approximately 14.5% of the omani population are diabetic.6 86.6% were identified by the health system and 13.4% were newly diagnosed during the survey.6 diabetes is a very serious condition if not controlled.2 this disease can lead to various consequences. there are many manifestations of diabetic complications, but the most important ones are cardiovascular diseases, neuropathy, nephropathy, retinopathy, skin conditions, hearing impairment, alzheimer’s disease and diabetic food disease (dfd). dfd is a disastrous complication of diabetes, and it is defined as foot ulcers associated with peripheral arterial disease and neuropathy which can lead to development of gangrene that may eventually require lower limb amputation.7 the prevalence of dfd has a large variation depending on the region and the income of the country.3 the average prevalence of dfd worldwide is 6.4% among diabetic patients.3 in the mena region, the prevalence of dfd in saudi arabia was 4.3%, while it was a bit higher in jordan and algeria with prevalence of 5% and 11.9% respectively.8 dfd is a serious complication and it can impact the patient’s quality of life.7 based on idf, those who have diabetes are at risk of requiring surgical amputation 10–20 times more than those who do not have it.3 almost half of the lower limbs amputation in oman from 2003 to 2013 are diabetes-related and it is anticipated to rise in the upcoming years.9 11 in 2007, one-third of diabetes treatment cost was spent on foot ulcers, risk factors associated with diabetic foot disease in diabetes patients: first national study in oman abdullah rashid al jabri1, adhra al mawali2,3,* 1 college of medicine, sultan qaboos university, muscat, oman. 2 center of studies and research, ministry of health, muscat, oman. 3 strategic research program for non-communicable diseases, the research council, muscat, oman. *correspondence to: adhra al mawali (e-mail: adhra.almawali@gmail.com) (submitted: 16 january 2021 – revised version received: 14 february 2021 – accepted: 25 february 2021 – published online: 26 april 2021) abstract objective the aim of this study is to identify the correlation between multiple risk factors in the development of diabetic foot disease (dfd) in oman. it also aims to identify the effect of having multiple significant risk factors on the disease progression, and to explore which risk factor shows the highest correlation with disease development. methods a retrospective case–control study was conducted with 100 patients and 200 controls. data of the participants were extracted from hospital’s electronic patient record system (alshifa) from 2000 to 2018. chi-square, fisher exact test, odds ratio and multiple regression analysis were used to determine the significance of various risk factors. results having a hba1c > 7, body mass index > 30 kg/m2, and blood pressure over 140/90 mmhg showed a strong correlation with the development of dfd. other risk factors such as age of diabetes, gender, total blood cholesterol, triglyceride levels, ldl, and hdl did not show any significant correlation with dfd. conclusions risk factors for dfd are highly prevalent in our society, controlling these risk factors could minimize the morbidity and the mortality related to this disease as well as reducing the economic impact related to it. proper education for those at a higher risk could play an important role in the control of this disease. keywords diabetic foot, risk factors, body mass index, oman 87 original risk factors associated with diabetic foot diseaseabdullah rashid al jabri, adhra al mawali j contemp med sci | vol. 7, no. 2, march-april 2021: 86 – 91 and this shows that dfd is a true burden from the economic aspect worldwide.3 there are many risk factors that have been shown to have a direct relation in developing dfd. these risk factors are age, gender, smoking, diabetes duration, hypertension, neuropathy, peripheral vascular disease and poor glycemic control.12 al-rubeaan et al in saudi arabia showed that dfd is associated with certain risk factors such as duration of diabetes, hypertension, and poor glucose control.12 there is a paucity in studies related to dfd as there are only two studies conducted in oman since 1991 about dfd; and they did not investigate the different risk factors associated with dfd.13 there was a call from oman medical journal for more research in dfd in april 2017 in this area.13 thus, to the best of our knowledge, this is the first study in oman that focuses mainly on the risk factors associated with development of dfd. this study aims to identify the correlation between multiple risk factors in the development and progression of dfd in oman. methods a retrospective case–control study was conducted from december 2018 to march 2019. this study covered all moh health centers from all governorates of oman. data were collected from the case records of those who were clinically diagnosed to have dfd due to type 2 dm from 2000 to 2018, almost two decades regardless of their onset of diabetes. the population surveyed adults aged 20 years or older, men and women, as well as nationals and non-national residents. those younger than 20 years old, tourists, and persons from labor camps were excluded. patient’s anonymity was maintained to ensure patient confidentiality. true crypt (program used to keep data protected) was used as a safety measure to ensure confidentiality. all retrieved case files were studied thoroughly to find out all the relevant data related to dfd patients including their presence of foot ulcers, date of diagnosis, onset of disease, age, gender, hbac1 (levels of glycosylated hemoglobin), body mass index (bmi), ldl, hdl, triglyceride, and total blood cholesterol before getting dfd. average of three consecutive results was taken for the highest representation of the patients controlling status. other risk factors that were planned to be included are smoking status, social status, and educational status but unfortunately these risk factors were not available for most of the patients. sample size calculation sample size calculation for this study was done using epi info software. a 95% confidence level and a power of 90% was used with a ratio of controls to cases of two. the sample size for the study group (cases) was calculated to be 100 patients. a total of 100 dm case files with dfd and 200 dm controls without dfd were studied from december 2018 to march 2019, using case files extracted from 79 medical centers, representing all governorates of oman. case selection strategy to minimize bias and to ensure generalizability, the patients included in the study were chosen through random sampling by choosing random case number from the records (regardless of date of diagnosis, age, and gender). microsoft excel was used to get random sequence of patients according to their number from each health center. ethical approval prior to its initiation, the ethical approval was obtained from research and ethical review & approve committee (rerac) of moh. inclusion criteria all samples (cases and controls) were type 2 dm, they should be above 20-year-old and they must have a patient record in moh system (alshifa). the selection criteria for cases is that they must have dfd while controls do not have dfd. exclusion criteria patients with type 1 dm and those who are younger than 20 years were excluded. diabetes unrelated ulcers and amputations were excluded. pregnant women who have gestational diabetes were excluded as well. in addition, those who have genetic diseases such as sickle cell disease and rheumatoid arthritis were eliminated because of the impact of these diseases on ulcers formation. these exclusion criteria were applied for cases and controls. the inclusion and exclusion criteria were strictly followed to avoid any confusion that could affect the quality of the results. statistical analysis statistical analysis was calculated using the spss 23.0 software. further analysis using chi-square test was done to assess the correlation of multiple risk factors and dfd. multiple risk factors such as age of diabetes, gender, hypertension, bmi, total blood cholesterol, triglyceride levels, ldl, hdl, and poor glycemic control were studied. odds ratio were also calculated. reference ranges of the international standard of who was used for the risk factors. data were safely stored and encrypted using truecrypt software to ensure confidentiality and integrity of patient records. results three-hundred patients (100 cases and 200 controls) were included in this study from all governorates of oman. mean age of the population was calculated and the gender distribution was demonstrated in fig. 1 and table 1. diabetic males mean age was slightly less (56) and it was very near to diabetic females mean age which was 58 years (table 3). females who are suffering from dfd had mean age of 64 and the number was higher in dfd male patients with a mean age of 66 (table 2). poorly controlled blood glucose (hba1c) among patients of dfd 91% (n=91) had poorly controlled blood sugar while only 9% (n=9) had controlled blood glucose (table 3). table 3 shows that 52% (n=104) of diabetic patients had poorly controlled blood sugar and 48% (n=96) were having a good control. poorly controlled diabetic patients showed a significant association in developing dfd. dm patients with dfd (cases) are 9.3 times more likely to have elevated hba1c than the controls without dfd (univariate analysis p-value <0.001) (multivariate analysis p-value <0.001) (table 4). body mass index the second risk factor studied is bmi, which also showed a big variation between cases and controls. obesity appeared in 40% 88 original risk factors associated with diabetic foot disease abdullah rashid al jabri, adhra al mawali j contemp med sci | vol. 7, no. 2, march-april 2021: 86 – 91 (n=80) of the controls and 60% (n=120) were non-obese. on the other hand, 62% (n=62) of dfd patients were obese and 38% (n=38) were non-obese. our study demonstrated that being an obese is a significant risk factor that is associated with dfd. dm patients with dfd are 3.2 times more likely to be obese than controls without dfd (univariate analysis p-value <0.001) (multivariate analysis p-value = 0.034) (table 4). blood pressure table 3 shows that blood pressure (bp) was high in 78% (n=78) of dfd patients while only 28% (n=28) had controlled bp. percentages were significantly different in diabetic patients, 66.5% (n=135) had controlled bp and 33.5% (n=70) were not. bp was a significant risk factor dm patients with dfd are 5.3 times more likely to have high blood pressure than controls without dfd (univariate analysis p-value <0.001) (multivariate analysis p-value <0.001) (table 4). gender females were slightly more in both cases 52% (n=52) and control 55.5% (n=111) when compared to males in both cases 48% (n=48) and controls 44.5% (n=89). but the difference was not statistically significant (fig. 1) (p-value = 0.56). age at onset of diabetes age at onset of diabetes was divided into three groups (20–44, 45–65, >65). among dfd patients, age at onset of diabetic patients showed the following: 26% (n=26) had diabetes in the age between 20 and 44 years and 64% (n=64) got the disease in the age between 45 and 65 years, while only 10% (n=10) acquired the disease above 65 years old. on the other side, diabetic patient’s age showed the following: 37.5% (n=75) of them had diabetes in the age between 20 and 44 years and table 3. variables achieving independent statistical significance as risk factors for dfd by univariate analysis.   t2dm patients dfd patients p valuea odds ratio total normal abnormal total normal abnormal total number 200 100  hba1c 200 96(48%) 104(52%) 100 9(9%) 91(91%) <0.001 9.3 bmi 200 120(60%) 80(40%) 100 32(32%) 68(68%) <0.001 3.2 blood pressure 200 133(66.5%) 67(33.5%) 100 28(28%) 78(78%) <0.001 5.5 total cholesterol 200 158(79%) 42(21%) 100 70(70%) 30(30%) 0.09 1.6 triglyceride 200 136(68.5%) 64(31.5%) 100 57(57%) 43(43%) 0.06 1.6 hdl 200 196(98%) 4(2%) 100 98(98%)  2(2%)  1 1 ldl 200 126(63%) 74(37%) 100 58(58%) 42(42%) 0.40 1.2 table 4. variables achieving independent statistical significance as risk factors for dfd by multivariate analysis. p value odds ratio 95% c.i. for odds ratio lower upper hba1c .000 8.6 18.9 4.0 blood pressure .000 4.5 8.1 2.5 bmi .034 1.9 3.3 1.1 total cholesterol .510 1.3 3.0 0.6 triglyceride .800 1.1 2.0 0.6 hdl .553 1.4 4.1 0.5 ldl .871 0.9 2.0 0.4 constant .028 4.004 fig. 1 patients demographic characteristics. table 1. age at onset of diabetes among dm patients and dfd patients. dfd patients (cases) age at onset diabetes 20-44 26% (n=26) 45-65 64% (n=64) >65 10% (n=10) diabetic patients (controls) age at onset of diabetes 20-44 37.5% (n=75) 45-65 54.5% (n=109) >65 8% (n=16) table 2. mean age of each sex among dm patients and dfd patients. diabetic patients female male (controls) mean age 58 years 56 years dfd patients female male (cases) mean age 64 years 66 years 89 original risk factors associated with diabetic foot diseaseabdullah rashid al jabri, adhra al mawali j contemp med sci | vol. 7, no. 2, march-april 2021: 86 – 91 54.5% (n=109) got the disease in the age between 45 and 65 while only 8% (n=16) acquired the disease in the age above 65 (p-value = 0.14) (table 1). total cholesterol total blood cholesterol was also studied as a risk factor for dfd and the percentages was as the following: 70% (n=70) of dfd patients had a normal total cholesterol and 30% (n=30) had a high total blood cholesterol. the numbers were slightly better in diabetic patients with 79% (n=158) having normal total cholesterol and 21% (n=42) had a high total blood cholesterol. the difference between cases and controls was not statically significant (odds ratio = 1.6) (univariate analysis p-value =0.09) (multivariate analysis p-value = 0.51) (table 4). triglyceride triglyceride also followed the same trend as total cholesterol. among diabetic patients, 31.5% (n=63) had a high triglyceride and 68.5% (n=137) had a normal triglyceride. the percentages were slightly high in dfd patients 43% (n=43) who have high triglyceride and 57% (n=57) had controlled level. triglyceride showed a trend to significant correlation with dfd. (odds ratio =1.6) (univariate analysis p-value =0.06) (multivariate analysis p-value = 0.80) (table 4). hdl percentages of hdl were the same in both cases and controls with 92% having a normal hdl level in the blood and 8% had low hdl levels. hdl did not show any association with dfd (odds ratio = 1) (univariate analysis p-value =1) (multivariate analysis p-value=0.55) (table 4). ldl ldl did not show any association with the progression of dfd. sixty-three percent (63%) (n=126) of diabetic patient had a normal triglyceride level while 37% (n=74) had a high triglyceride level. on the other side, 42% (n=42) of dfd patients had a high level of triglyceride and 58% (n=58) were within the normal limits. ldl did not show a statistically significant correlation with dfd (odds ratio = 1.2) (univariate analysis p-value=0.40) (multivariate analysis p-value = 0.87) (table 4). multiple risk factors eighty-six percent (86%) of dfd patients had two or more significant risk factors; and only 14 (14%) of them got one or no significant risk factor. on the other hand, 70 (35%) of diabetic patients had two or more significant risk factors and 130 (65%) got one or no significant risk factor. multivariate analysis showed that those who had two or more significant risk has a 10 times higher risk of developing dfd in comparison to those who do not have any of these risk factors (table 5). discussion dfd progression and risk factors have been investigated in many previous studies. this study has shed the light on nine different risk factors and identified their correlation with the presence of dfd. three of the risk factors were found to have a statistically significant correlation with dfd. poorly controlled blood glucose (hba1c) was shown to be a strong contributing risk factor with dfd. this finding is in line with many other studies.14-17 our study found a strong correlation between hba1c and dfd, and this is probably due to the effect of hba1c on the development of neuropathy and microvascular injury.18 bmi is also a common risk factor for dfd but it’s impact is less than that of hba1c. many studies demonstrated an association between bmi and dfd.6,12,14 our study revealed that having a bmi of >30 has a significant correlation with the development of dfd which could be due to external or internal influence of the increased weight on the presence of foot ulcers.19,20 furthermore, our study confirmed that increased blood pressure has a strong association with the development of dfd. this finding was also supported by many other studies.12,15 this could be due to the occlusion of the blood vessels in the limb.21 gender is a very controversial subject in dfd progression, many studies showed that males are likely to develop dfd while others did not confirm this finding.22 in our study, we did not find any correlation between gender and dfd. based on our results, it was obvious that the general control (blood glucose level hba1c, blood pressure and bmi) in females was worse than males and this could play a very important role in developing dfd, but at the same time, males are more prone to foot ulcers due to probably physical activity and less foot care in comparison to females. it was found that males with dfd were slightly more than females but the difference was not statistically significant. larger sample size is required to confirm this finding. age at onset of dm was also studied and it did not show any correlation with the occurrence of dfd. most of the studies showed that longer duration of diabetes increases the chance of getting diabetes complications which means those who got diabetes at a younger age have a higher chance of getting manifestations later in life.12 some studies demonstrated that age of diabetic patients does not play an important role in dfd development.14,.23 this study revealed that lipid profile do not have any correlation with dfd. our finding is similar to many other studies.14,23 lipid profile in general do not appear to play an important role in developing dfd. multivariate analysis of the risk factors showed a strong association with the development of dfd for those who had two or more risk factors. it has been found that patients with two or more significant risk factors have 11 times higher risk of dfd in comparison to those who have less or no risk factors. limitations being of a retrospective nature, this is one of the limitations of this study and therefore limited our ability to analyze some risk factors, which was intended to be studied initially. in addition, direct access to alshifa system is available only for the past 5 years. furthermore, larger sample size is required to confirm these findings. table 5. the effect of multiple risk factors on the development of dfd. dfd yes no p value exposed 86 (86%) 70 (35%) not exposed 14 (14%) 130 (65%) <0.001 *exposed= two or more significant risk factors 90 original risk factors associated with diabetic foot disease abdullah rashid al jabri, adhra al mawali j contemp med sci | vol. 7, no. 2, march-april 2021: 86 – 91 recommendations prevention and control of dm and dfd requires that it be prioritized at the national and governorate levels while strengthening multisectoral, legislative and societal aspects, as it increases risks to health, social, and financial progress. second, encourage large-scale awareness campaigns to control modifiable risk factors for people with diabetes: physical inactivity, unhealthy diet, and alcohol consumption. third, improve the health system to ensure access to basic techniques for testing, diagnosing, valid, and safe drugs as well as continuous monitoring of diabetes in primary health-care sessions. fourth, implement evidence-based practices and decision-making in diabetes management through the use of a simplified information system to ensure the best quality of data. fifth, developing health clinics in all health institutes to promote early detection and examination of diabetes, as well as acting as data sources for prevention and health progress. sixth, improving the implementation of the omani national policy on diet, physical activity, and health, and engaging with the agricultural sector to promote healthy diets and eating habits. seventh, strengthening laws related to the manufacture, packaging, and marketing of food and beverages to reduce consumption of unhealthy foods. eighth, encouraging an active lifestyle and reducing the sedentary lifestyle through implementing the physical activity toolkit in the country. conclusion dfd is a real-life threating complication of diabetes. poorly controlled diabetes (hba1c > 7), obesity (bmi > 30 kg/m2) and high blood pressure (>140/90 mmhg) were significantly correlated with progression to dfd. these risk factors are modifiable, and so it is important to raise the level of awareness among diabetic patients. controlling these risk factors can help in reducing future dfd related morbidity and mortality. there are few studies with low output and impact in the fields of diabetic foot care and dfd in oman which focused on epidemiology, prevention, and management of dfd. more studies are required to further improve our understanding of the scope of dfd, and the impact of the existing diabetes programs on the level of diabetic foot care in oman. continuous support governmental and financial support for diabetic foot research is important. cooperation with major regional and international diabetic foot research organizations, such as the ‘gulf diabetic foot working group’ (gdfwg) and the ‘international working group on the diabetic foot’, is essential (i.e., through sharing intervention, programs, and initiating collaborative multicenter studies). there is a dearth of studies related to dfd in oman, thus more studies are urgently needed, and stronger screening programs are required to obtain the exact number of dfd patients in oman who are at high risk. the investigated results are essential to support the formulation and implementation of initiation and action plans for dm and dfd that enhance patients’ health status. developing this plan can ensure not only a huge financial return on investment in the health budget but more importantly, improving the quality of life for omanis. conflict of interest the authors declare that they have no conflict of interest. references 1. al-mawali a. non-communicable diseases: shining a light on cardiovascular disease, oman’s biggest killer. oman med j [internet]. 2015 jul;30(4):227–8. available from: https://pubmed.ncbi.nlm.nih.gov/26366254 2. world health organization. diabetes mellitus who [internet]. definition, diagnosis and classification of diabetes mellitus and its complications. 1999. p. 49. available from: https://www.staff.ncl.ac.uk/philip.home/ who_dmg.pdf 3. international diabetes federation. idf diabetes atlas [internet]. 2017. 1–150 p. available from: http://www.diabetesatlas.org/resources/2017atlas.html 4. annual health report 2011 moh [internet]. moh. 2011. available from: https://www.moh.gov.om/en/web/statistics/-/201-6 5. annual report moh 2016 [internet]. 2016. p. 81–2. available from: https:// www.moh.gov.om/en/web/statistics/-/20-46 6. almobarak ao, awadalla h, osman m, ahmed mh. prevalence of diabetic foot ulceration and associated risk factors: an old and still major public health problem in khartoum, sudan? ann transl med. 2017 sep;5(17):340. 7. alexiadou k, doupis j. management of diabetic foot ulcers. diabetes ther. 2012;3(1):1–15. 8. alkhier ahmed d, elsharief e, alsharief a. the diabetic foot in the arab world. j diabet foot complicat. 2011;3(3):55–61. 9. annual health report 2003 moh [internet]. 2002. available from: https:// www.moh.gov.om/documents/274609/1595174/2002+التقريرالسنوي+اإلحصايئ/ 7f90306b-27bc-49d6-9c20-9f831a11e78c 10. annual health report 2013 moh [internet]. 2003. available from: https:// www.moh.gov.om/en/web/statistics/annual-reports?p_p_id=122_ instance_trutpmhsllz9&p_p_lifecycle=0&p_p_state=normal&p_p_ mode=view&p_p_col_id=column-1&p_p_col_pos=1&p_p_col_ count=2&p_r_p_564233524_resetcur=true&p_r_p_564233524_ categoryid=274760 11. al-busaidi is, abdulhadi nn, coppell kj. care of patients with diabetic foot disease in oman. vol. 16, sultan qaboos univ med j. 2016:e270–6. 12. al-rubeaan k, al derwish m, ouizi s, youssef am, subhani sn, ibrahim hm, et al. diabetic foot complications and their risk factors from a large retrospective cohort study. plos one [internet]. 2015;10(5). available from: http://dx.doi.org/10.1371/journal.pone.0124446 13. al-busaidi is. diabetic foot disease in oman: a call for more research. oman med j. 2017;32(4):354–5. 14. nyamu pn, otieno cf, amayo eo, mcligeyo so. risk factors and prevalence of diabetic foot ulcers at kenyatta national hospital, nairobi background : diabetic foot ulcers contribute significantly to the morbidity and mortality of patients with diabetes mellitus . the diabetic patients with foot ulce. east africa med j. 2003;80(1):36–43. 15. fawzy ms, alshammari ma, alruwaili aa, alanazi rtr, alharbi jam, almasoud amr, et al. factors associated with diabetic foot among type 2 diabetes in northern area of saudi arabia: a descriptive study. bmc res notes. 2019 jan;12(1):51. 16. dubský m, jirkovská a, bem r, fejfarová v, skibová j, schaper nc, et al. risk factors for recurrence of diabetic foot ulcers: prospective follow-up analysis in the eurodiale subgroup. int wound j [internet]. 2013;10(5):555–61. available from: https://doi.org/10.1111/j.1742-481x.2012.01022.x 17. lavery la, armstrong dg, wunderlich rp, mohler mj, wendel cs, lipsky ba. risk factors for foot infections in individuals with diabetes. diabetes care [internet]. 2006 jun 1;29(6):1288 lp – 1293. available from: http://care. diabetesjournals.org/content/29/6/1288.abstract 18. nabil abd el fatah ea etc. study of risk factors of diabetic foot ulcers. 2014;28–34. available from: www.mmj.eg.net/article.asp?issn=11102098;year=2014;volume=27;issue=1;spage=28;epage=34;aulast=al;aid=m enoufiamedj_2014_27_1_28_132298%0d 19. boyko ej, ahroni jh, stensel v, forsberg rc, davignon dr, smith dg. a prospective study of risk factors for diabetic foot ulcer. the seattle diabetic foot study. diab care [internet]. 1999 jul 1;22(7):1036 lp – 1042. available from: http://care.diabetesjournals.org/content/22/7/1036.abstract 91 original risk factors associated with diabetic foot diseaseabdullah rashid al jabri, adhra al mawali j contemp med sci | vol. 7, no. 2, march-april 2021: 86 – 91 20. mantey i, foster a v, spencer s, edmonds me. why do foot ulcers recur in diabetic patients? diab med. 1999 mar;16(3):245–9. 21. ogbuawa o, williams jt, henry wlj. diabetic gangrene in black patients. south med j. 1982 mar;75(3):285–8. 22. dinh t, veves a. the influence of gender as a risk factor in diabetic foot ulceration. vol. 20, wounds : a compendium of clinical research and practice. 2015. 127–131 p. 23. manda v, sreedharan j, muttappallymyalil j, das r, hisamatsu e. foot ulcers and risk factors among diabetic patients visiting surgery department in a university teaching hospital in ajman, uae. int j med public heal. 2012;2(3):34–8. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.947 108 original issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 108 – 112 introduction uncontrolled pregnancy is one of the most important challenges of the century.1,2 oral contraceptive pills (ocp) continued to be one of the most common methods of contraception used by the majority of women. across all ages, women in their 20s are the most likely to use ocp.3 the ocp is the commonest contraceptive method used among saudi women.4 combined pills and progestin-only pills are the two types of ocp.5 the usage of ocp has been shown to cause oxidative stress by enhanced depletion of antioxidant and increased lipid peroxidation.6 oxidative stress is defined as an imbalance between antioxidants and pro-oxidants in the cells, which are manifested by high levels of free radicals.7 free radicals are deleterious to the human body because they can retrieve electrons from various molecules provoking the formation of oxidized forms so that severe oxidative stress can even trigger cell apoptosis and necrosis.8 antioxidants systems, both non-enzymatic and enzymatic, are produced in the body to block too much free radicals production.9 non-enzymatic antioxidants are known as synthetic antioxidants or dietary supplements, including vitamin c, vitamin e, β-carotene, and so on.10 mounting evidence suggests that oxidative stress could play a pivotal role in the pathogenesis of several diseases including inflammatory, muscular, cardiovascular, and neurodegenerative diseases.11 nitric oxide (no) is a key organizer of the endothelia functional in the cardiovascular system. change of the redox equilibrium in the vascular system intervenes with no product and modifies vascular homeostasis bioavailability, promoting the development of metabolic and cardiovascular diseases.12 estrogen generally has diverse vascular actions via increasing the bioavailability of no on activation of no synthase, and no is inversely associated with oxidative stress.13 lobysheva et al.14 observed a correlation between the use of combined oral contraceptives pills (cocp) and increased oxidative stress and the decreased no in women who used ocp. hassan et al.15 reported that no is significantly reduced in cocp users than nonusers. vitamins also immediately scavenged ros and upregulated the activity of antioxidant enzymes. vitamin e is considered one of the most vital antioxidants; vitamin e prevent peroxyl radicals generated by peroxidation of polyunsaturated fatty as a result of ros, therefore, protecting cells against oxidation of membrane phospholipids.16 hassan et al.15 concluded that serum vitamin e levels are significantly lower among the ocp users’ groups. vitamins b6 and b12 play crucial inter-related roles in dna synthesis throughout the lifecycle, especially during childhood, adolescence, and the reproductive years for women.17 low vitamin b6 status has been associated with an increased risk of cardiovascular disease.18 the use of ocp causes many biochemical changes in; clotting factors, thrombosis, platelet changes, atherosclerosis, and inflammatory profiles.19-21 jamil et al.22 concluded that hemoglobin levels are normal in the ocp users. baker et al. (2016) reported that the wbc count elevates in the woman using ocp due to that the ocp may cause some internal infection in the women. crp is generated via the liver, and crp levels rise within the presence of any inflammatory in the body.23 serum levels of crp are often used to detect inflammatory signs to estimate cardiovascular sickness and stroke.24 ferreira et al. (2017) found a significant increase in crp levels, especially among overweight women who use cocp more than 3 mg/dl (a boundary value related to the evolution of cardiovascular diseases). this study aimed to evaluate the effect of ocp on oxidative stress in saudi women. materials and methods the study was carried out on 55 saudi women were divided into two groups: women taking an ocp (n=30) and women who had never taken the ocp (n=25). the ethical approval was obtained through the local ethics committee at the king effect of oral contraceptive pills on oxidative stress in saudi women nadia n. osman1,2, dalal mhammed al-mutairi1,3* 1 biochemistry department, faculty of science, king abdulaziz university, jeddah, saudi arabia. 2 food irradiation research department, national center for radiation research and technology, atomic energy authority, cairo, egypt. 3 biochemistry department, faculty of science, tabuk university, tabuk, saudi arabia. *correspondence to: dalal mhammed al-mutairi (e-mail: dalal_9911@hotmail.com) (submitted: 05 january 2021 – revised version received: 29 january 2021 – accepted: 21 february 2021 – published online: 26 april 2021) abstract objective the aim of this study was to evaluate the effect of oral contraceptive pills (ocps) on oxidative stress in saudi women. methods a total of 55 saudi women were divided into two groups: users of ocp (n=30) for at least 1 year and non-users (nocp, n=25). the demographic data were obtained through face-to-face interviews performed by the researcher. blood specimen from both groups were drawn after 8 h of fasting to estimate serum total antioxidant (taoc), nitric oxide (no), c-reactive protein (crp), vitamins (e, b6, b12), and some hematological parameters: hemoglobin (hb), red blood cell (rbc), white blood cell (wbc). results the results showed a significant decreased in serum taoc, no, vitamins e, and b6 accompanied with high significant increase in crp level while no significant changes b12, hb, rbc, and wbc were observed in the ocp users as compared to the control group. conclusions the results of this study indicated that the use of ocp resulted in low levels of the total antioxidant, nitric oxide, vitamins e, and b6 with a significant increase in crp. women who used ocp may be more susceptible to oxidative stress by enhanced depletion of antioxidants. 109 original effect of oral contraceptive pills on oxidative stress in saudi womennadia n. osman, dalal mhammed al-mutairi j contemp med sci | vol. 7, no. 2, march-april 2021: 108 – 112 abdul-aziz university hospital. all relevant information was recorded on predesign questionnaires. a blood specimen (6  ml) was collected by venipuncture after 8–12 h of fasting were collected to estimate various parameters: total antioxidants, no, vitamins e, b6, and b12 by colorimetric method using elisa kit. hb, rbc, and wbc were estimated using a hematology analyzer. the crp level was also determined by a commercial kit from simens. statistical package for social science (spss) computer software was used for data analysis. means ± standard deviation (sd) was calculated. independent t-test and spearman’s correlation were used to analyze the differences between bath ocp users and non-user control. the results were considered statistically significant at p ≤ 0.05. results the current results showed that total antioxidant and nitric oxide were significantly decreased (p=0.000) among women using oral contraceptive when compared to control group (table 1). as shown in table 2, there was a highly significant (p=0.000) reduction in the vitamins e and b6 in ocp users compared to the control group. however, there was no significant difference (p=0.32) in vitamin b12 between the cases and control. data demonstrated in table 3 showed that ocp induced a non-significant changes in hb, rbc, and wbc (p=0.06, 0.52, 0.90 respectively), associated with a highly significant increase (p=0.000) in crp, compared to noc. from table 4, it could be observed that there was a significant changes association between duration of ocp use and taoc, no, crp, vitamins e, and b6. moreover, hb was significantly higher started after 5 years, while, vitamin b12 and wbc were significantly different started 3–4 years. no significant differences in the rbc were noticed along with the tested duration (1–7 years) (table 4). discussion oral contraceptive steroids are one of the factors that enhance oxidative stress and lead to the formation of free radicals. oxidative stress constitutes a disturbance caused by an imbalance between the generation of free radicals and the antioxidant system, which causes damage to biomolecules. this, in turn, may lead to the occurrence of many chronic degenerative diseases.25 the free radical-mediated peroxidation of membrane lipids increases permeability and membrane fluidness with loss of its integrity that leads to cell damage.26 antioxidants work cooperatively in biological systems, and it is important to be able to correlate antioxidant measurements with antioxidant defenses and disease prevention. it is therefore recommended to study “total antioxidant capacity”, rather than monitoring individual antioxidant levels, which may be less affected by dietary habits.27 in this study, the total antioxidant level was significantly decreased among women using ocp. this result was in agreement with palan et al. (2010) and adejumo et al.28 the behavior of molecules related to oxidative stress can differ according to types and doses of estrogen, progestogen, or the particular compounds of estrogen and progestogen.29 estrogens display an antioxidant activity by inhibiting the expression and function of the nadp+/nadph oxidase,30 by increasing the expression and level of activation of the endothelial isoform of the nitric oxide synthase (enos)31 and by stimulating the expression and activity of the manganese sod (mnsod) and of the extracellular sod (ecsod).32 these antioxidant activities of estrogens are counteracted by progestins via the activation of the nadph oxidase and the inhibition of the expression and activity of the mnsod and of the ecsod.33 therefore it implies that the counteractive effect of progestin would result in a decrease in the serum total antioxidant status especially, in women taking either cocp.28 in the current study, there is a significant decrease in serum no in the ocp users as compared to the control group. table 1. the effect of ocp on total antioxidant and no. variables group n mean p-value sig taoc noc 25 21.82 0.000 h. sig ocp 30 4.89 no noc 25 0.000 h. sig ocp 30 22.05 noc non-oral contraceptives, ocp oral contraceptives pills, taoc total antioxidant, no nitric oxide. values are as mean±sd, p<0.05 significant as compared to the non-users. table 2. the effect of ocp on vitamins. variables group n mean p-value sig vitamin e noc 25 0.000 h. sig ocp 30 24.379.87 vitamin b6 noc 25 627.65123.38 0.000 h. sig ocp 30 274.1870.24 vitamin b12 noc 25 267.4867.05 0.32 n. sig ocs 30 248.46 noc non-oral contraceptives, ocp oral contraceptives pills. values are as mean±sd, p<0.05 significant as compared to the non-users. table 3. the effects of ocp on some hematological parameters and crp. variables group n mean p-value sig hb noc 25 0.06 n. sig ocp 30 12.16 1.77 rbc noc 25 4.32 0.58 0.52 n. sig ocp 30 4.23 0.42 wbc noc 25 6.99 1.73 0.90 n. sig ocp 30 7.08 3.59 crp noc 25 0.16 0.000 h. sig ocp 30 noc non-oral contraceptives, ocp oral contraceptives pills, hb haemoglobin, rbc red blood cells, wbc white blood cells, crp c-reactive protein. values are as mean±sd, p<0.05 significant as compared to the non-users. 110 original effect of oral contraceptive pills on oxidative stress in saudi women nadia n. osman, dalal mhammed al-mutairi j contemp med sci | vol. 7, no. 2, march-april 2021: 108 – 112 a similar result was reported by lobysheva et al.14 hassan et al.15 observed that taking cocp resulted in a significant decrease in no level. estrogen is generally has diverse vascular actions via increasing the bioavailability of no on activation of no synthase, and no is inversely associated with oxidative stress.13 decreased bioavailability of no may result in abnormal reactions between the vessel wall and platelets and is thus involved in the initiation and progression of atherosclerosis.34 the present study reported a significant decrease in vitamins e, b6 and no significant change in b12 in the ocp users as compared to the control group. a similar result was reported by palan et al. (2010), wilson et al. (2011) and singh et al. (2018). the study by hassan et al.,15 observed a decrease in the serum vitamin e, especially in women taking either cocp. in contrast to our results, mcarthur et al. (2013) and berenson and rahman,35 have reported that the young women using ocp had significantly lower serum vitamin b12 concentrations. vitamin e level was significantly decreased among women using ocp. the mechanism of such an effect is unknown, but it was thought that, at its high level, certain estrogens, in particular diethylstilbestrol, may produce reactive oxygen species that may attack a number of biological substances, including lipids and dna, and could natural antioxidants may be consumed to trap such compounds.36 the ocp may have some relatively specific effect on tryptophan’s metabolism, which is independent of vitamin b6 levels. this effect may be primarily on the activity of tryptophan oxygenase since studies in rats have shown directly as well as adrenal-mediated effects of estrogens on the activity of this enzyme.37 previous studies38,39 indicated that steroids or steroid conjugates could affect the activity of the plp-dependent enzymes of the kynurenine pathway, i.e., kynureninase and kynurenine aminotransferase. thus, it seems most likely that the altered tryptophan metabolism of oral contraceptive users is the summation of hormonal effects on tryptophan oxygenase and kynureninase rather than the production of a general vitamin b6 deficiency.40.41 vitamin b12 level was no significant difference between ocp users and nonusers because that absorption is not affected and that redistribution of b12 throughout the body could be responsible.42 in this study, level hb and wbc were showed a non-significant decrease in non-oral contraceptive users. regarding hematological parameters, different results were observed by several researchers. toryila et al.43 have reported a reduction in hb, rbc, wbc among women using cocp compared to the controls. jamil et al.22 showed hb levels are normal in the ocp. al-zayadi44 and yeasmin et al.45 have reported increases in hb level comparing with control groups. toryila et al.46 observed an increase in wbc count in coc-treated female wistar rats. the difference may be as a result of the use of different coc with different concentrations of estrogen and progesterone and the duration of use. mooij et al. (1992) reported no significant difference on hematological parameters due to ocp use in women, but serum iron status was significantly increased for the users of ocp. in this study a no significant change in rbc among women using ocp. a similar result was reported by al-zayadi.44 due to the confounding of the effects of dose and the type of hormone, and the formulation of the drug, it was not easy to estimate the effect of ocp on rbc. even though the combination of estrogen and progesterone would be considered as advance formal of the ocp, the use of it still strongly associated with vascular disease.47 table 4. effect of duration of ocp use on biochemical parameters. control n = 25 duration of ocs 1-2 years n = 7 3-4 years n = 10 5-7 years n = 13 taoc 21.828 ± 5.674 5.357 ± 1.651p= 0.000* 4.860 ± 1.706 p= 0.000* 4.669 ± 2.200 p= 0.000* no 171.316± 34.8 45.771 ± 21.229p= 0.000* 41.220 ± 25.322 p=0.000* 40.892 ±21.422 p= 0.000* crp 0.160 ± 0.071 1.614 ± 0.467p= 0.000* 1.440 ± 0.384 p=0.000* 1.608 ± 0.528 p=0.000* vit e 96.524±25.026 23.843 ±11.652p= 0.000* 22.460 ± 8.921 p=0.000* 26.139 ± 10.081 p= 0.000* vit b6 627.652±123.389 284.343 ± 69.892p= 0.000* 289.060 ± 92.414 p= 0.000* 257.262 ± 50.212 p= 0.000* vit b12 267.480 ± 67.053 278.00 ± 50.471p= 0.523 224.300 ± 70.539 p=0 .049* 251.154 ± 85.517 p= 0.508 hb 11.204 ± 2.054 11.450 ± 2.441p= 0.600 12.019 ± 1.125 p= 0.547 12.657 ± 1.760 p= 0.047* rbc 4.321 ± 0.588 4.134 ± 0.360p= 0.218 4.234 ± 0.260 p=0.371 4.289 ± 0.555 p= 0.712 wbc 7.156 ± 1.604 6.370 ± 1.382p= 0.210 5.729 ± 0.811 p=0.008* 6.869 ± 1.673 p= 0.470 the values are the mean ± s.d. of parameters measured. significantly different from control value at p<0.05*, 0.01**, 0.001*** 111 original effect of oral contraceptive pills on oxidative stress in saudi womennadia n. osman, dalal mhammed al-mutairi j contemp med sci | vol. 7, no. 2, march-april 2021: 108 – 112 the results indicated a significant increase in crp in the ocp users as compared to the control group; this finding is in agreement with divani et al.48, ferreira et al., (2017), guedes et al.49, and oliveira et al.50 it was indicating that ocp leads to an increased subclinical inflammatory process. divani et al.51 established that progestin increases interleukin 6 (il-6, an inflammatory-mediated stimulation) of crp in combination with conjugated equine estrogen and showed that il-6 change was negatively linked to crp change when subjects were treated only with equine estrogen. the involvement of progestin has been suggested to induce il-6-mediated crp stimulation, whereas other mechanisms are likely to be responsible for the development of crp in women receiving only conjugated equine estrogen.52 this study evaluates the effects of duration of ocp use on oxidative stress, which is represented by a significant decrease in taoc, no, vitamins e, and b6 levels. in contrast, crp has shown a significant increase. egoro et al. (2018) demonstrated that elevated levels of plasma crp in long-term users of ocs containing lower doses of estrogen and progestin composition for ≥2 years as compared to (control group). however, it was observed a significant change in hb, wbc, and b12 after 3 years of using ocp. similarly, toryila et al.43 reported that longterm use of cocp might lead to more complications than shorttime use. however, the long-term users of the ocp on rbc has shown no significant difference (p≥ 0.05) compared to the mean value of non-users of ocs (control group). conclusion the results of this study indicated that the use of ocp resulted in low levels of total antioxidant, no, vitamins e, and b6 with a significant increase in crp. moreover, there is a statistically significant relationship between ocp use and increased oxidative stress. conflict of interest none references 1. minahan, c, o’neill, h, sikkema, n., joyce, s, larsen, b, sabapathy, s. oral contraceptives augment the exercise pressor reflex during isometric handgrip exercise. physiol rep, 2018;6:e13629. 2. meier, tb, drevets, wc, teague, tk, wurfel, be, mueller, sc, bodurka, j, dantzer, r, savitz, j. kynurenic acid is reduced in females and oral contraceptive users: implications for depression. brain behav immun, 2018;67:59-64. 3. al-mass, aa, al-shahrani, bs, al-mweisheer, an, tulbah, sa, syed, s, anwer, r, haque, s. user experience, knowledge and practice of oral contraceptive: a study from riyadh, saudi arabia. ann med health sci res. 2018. 4. alawwad, m, alamri, f, amer, s. prevalence and determinants of contraceptive use among saudi women: a descriptive national study. j preg child health, 2019;6:2. 5. mustafa, r, afreen, u, hashmi, ha. contraceptive knowledge, attitude and practice among rural women. j coll physicians surg pak, 2008;18:542-545. 6. osman, n, jambi, e, bashaikh, s. effect of oral contraceptive pills on oxidative stress in diabetic rats. int j pharmaceut res allied sci, 2017;6. 7. nayak, sb, bhat, vr, mayya, ss. serum copper, ceruloplasmin and thiobarbituric acid reactive substance status in patients with ovarian cancer. ind j physiol pharmacol, 2004;48:486-488. 8. halliwell, b. free radicals and antioxidants: updating a personal view. nutr rev, 2012;70:257-265. 9. chasan-taber, l, willett, wc, manson, je, spiegelman, d, hunter, dj, curhan, g, colditz, ga, stampfer, mj. prospective study of oral contraceptives and hypertension among women in the united states. circulation, 1996;94: 483-9. 10. pierce, jd, cackler, ab, arnett, mg. why should you care about free radicals? because these molecules appear to play a role in the development of heart disease, cancer, diabetes, and other diseases. understanding how free radicals form and what you can do to help keep them in balance is essential to maximizing the care you provide now--and in the future. rn, 2004;67:38-44. 11. lichtenberg, d, pinchuk, i. oxidative stress, the term and the concept. biochem biophys res commun, 2015;461:441-444. 12. tousoulis, d, kampoli, a-m, tentolouris nikolaos papageorgiou, c, stefanadis, c. the role of nitric oxide on endothelial function. curr vasc pharmacol, 2012;10:4-18. 13. andozia, mb, vieira, cs, franceschini, sa, torqueti tolloi, mr, silva de sa, mf, ferriani, ra. ethinylestradiol and estradiol have different effects on oxidative stress and nitric oxide synthesis in human endothelial cell cultures. fertil steril, 2010;94:1578-82. 14. lobysheva, ii, van eeckhoudt, s, dei zotti, f, rifahi, a, pothen, l, beauloye, c, balligand, j-l. heme-nitrosylated hemoglobin and oxidative stress in women consuming combined contraceptives. clinical application of the epr spectroscopy. free radic biol med, 2017;108:524-532. 15. hassan, as, al-salih, rm, al-naser, ah. long term oral contraceptive administration is associated with low serum levels of nitric oxide, vitamin c and vitamin e. ind j public health res dev, 2020;11:1514-1519. 16. morganti, p, bruno, c, guarneri, f, cardillo, a, del ciotto, p, valenzano, f. role of topical and nutritional supplement to modify the oxidative stress. int j cosmetic sci, 2002;24:331-339. 17. mann, j, truswell, as. essentials of human nutrition, oxford university press. 2017. 18. shen, j, lai, c-q, mattei, j, ordovas, jm, tucker, kl. association of vitamin b-6 status with inflammation, oxidative stress, and chronic inflammatory conditions: the boston puerto rican health study. am j clin nutr, 2010;91:337-342. 19. haarala, a, eklund, c, pessi, t, lehtimäki, t, huupponen, r, jula, a, viikari, j, raitakari, o, hurme, m. use of combined oral contraceptives alters metabolic determinants and genetic regulation of c‐reactive protein. the cardiovascular risk in young finns study. scand j clin lab investig, 2009;69:168-174. 20. wiegratz, i, stahlberg, s, manthey, t, sänger, n, mittmann, k, palombo-kinne, e, mellinger, u, lange, e, kuhl, h. effects of an oral contraceptive containing 30 mcg ethinyl estradiol and 2 mg dienogest on lipid metabolism during 1 year of conventional or extended-cycle use. contraception, 2010;81:57-61. 21. sitruk-ware, r, nath, a. characteristics and metabolic effects of estrogen and progestins contained in oral contraceptive pills. best pract res clin endocrinol metab, 2013;27:13-24. 22. jamil, s, khan, ra, dilshad, h, fatima, s. haematologic variations associated with the long term use of contraceptives in young females. int j med res rev, 2014;2:580-586. 23. bastard, j-p, maachi, m, lagathu, c, kim, mj, caron, m, vidal, h, capeau, j, feve, b. recent advances in the relationship between obesity, inflammation, and insulin resistance. eur cytokine network, 2006;17:4-12. 24. zieske, aw, tracy, rp, mcmahan, ca, herderick, ee, homma, s, malcom, gt, mcgill jr, hc, strong, jp. elevated serum c-reactive protein levels and advanced atherosclerosis in youth. arterioscl thrombosis vasc biol, 2005;25:1237-1243 25. aguilar, taf, navarro, bch, perez, jam. endogenous antioxidants: a review of their role in oxidative stress. a master regulator of oxidative stress-the transcription factor nrf2. 2016. 26. abdoljalal, m, gholamreza, v, mohammad, t. serum lipid peroxidtion nd leptin level in ml end female type 2 diabetic patients in gorgan, iran. j chin clin med, 2010;5:26-35. 27. lands, lc, grey, v, grenier, c. total plasma antioxidant capacity in cystic fibrosis. pediat pulmonol, 2000;29:81-87. 28. adejumo, en, adediji, io, akinmulero, ao. effect of hormonal contraceptives on the total antioxidants status of women from isolo, lagos state, nigeria. j biosci med, 2016;4:107. 29. chen, j-t, kotani, k. different effects of oral contraceptive and dydrogesterone treatment on oxidative stress levels in premenopausal women. j clin med res, 2018;10:146. 112 original effect of oral contraceptive pills on oxidative stress in saudi women nadia n. osman, dalal mhammed al-mutairi j contemp med sci | vol. 7, no. 2, march-april 2021: 108 – 112 30. laufs, u, adam, o, strehlow, k, wassmann, s, konkol, c, laufs, k, schmidt, w, böhm, m, nickenig, g. down-regulation of rac-1 gtpase by estrogen. j biol chem, 2003;278:5956-5962. 31. chambliss, kl, shaul, pw. estrogen modulation of endothelial nitric oxide synthase. endocr rev, 2002;23:665-686. 32. strehlow, k, rotter, s, wassmann, s, adam, o, grohe, c, laufs, k, böhm, m, nickenig, g. 2003. modulation of antioxidant enzyme expression and function by estrogen. circul res, 2003;93:170-177. 33. wassmann, k, wassmann, s, nickenig, g. 2005. progesterone antagonizes the vasoprotective effect of estrogen on antioxidant enzyme expression and function. circul res, 2005;97:1046-1054. 34. fallah, s, nouroozi, v, seifi, m, samadikuchaksaraei, a, aghdashi, em. influence of oral contraceptive pills on homocysteine and nitric oxide levels: as risk factors for cardiovascular disease. j clin lab anal, 2012;26:120-3. 35. berenson, ab, rahman, m. effect of hormonal contraceptives on vitamin b12 level and the association of the latter with bone mineral density. contraception, 2012;86:481-487. 36. roy, d, kalyanaraman, b, liehr, jg. xanthine oxidase-catalyzed reduction of estrogen quinones to semiquinones and hydroquinones. biochem pharmacol, 1991;42:1627-1631. 37. braidman, ip, rose, d. effects of sex hormones on three glucocorticoidinducible enzymes concerned with amino acid metabolism in rat liver. endocrinology, 1971;89:1250-1255. 38. mason, m, manning, b. effects of steroid conjugates on availability of pyridoxal phosphate for kynureninase and kynurenine aminotransferase activity. am j clin nutr, 1971;24:786-791. 39. rose, d, brown, r. the influence of sex and estrogens on liver kynureninase and kynurenine aminotransferase in the rat. biochim biophys acta, 1969;184:412-419. 40. lumeng, l, cleary, re, li, t-k. effect of oral contraceptives on the plasma concentration of pyridoxal phosphate. am j clin nutr, 1974;27:326-333. 41. rios-avila, l, coats, b, chi, y-y, midttun, ø, ueland, pm, stacpoole, pw, gregory iii, jf. metabolite profile analysis reveals association of vitamin b-6 with metabolites related to one-carbon metabolism and tryptophan catabolism but not with biomarkers of inflammation in oral contraceptive users and reveals the effects of oral contraceptives on these processes. j nutr, 2015;145:87-95. 42. wertalik, lf, metz, en, lobuglio, af, balcerzak, sp. decreased serum b 12 levels with oral contraceptive use. jama, 1972;221:1371-4. 43. toryila, j, amadi, k, odeh, s, egesie, u, adelaiye, a, achie, l. effects of combined oral contraceptive (duofem) on some physiological parameters in female wistar rats. j afr assoc physiol sci, 2018;6:65-72. 44. al-zayadi, t. contraceptive effects in hematological and biochemical parameters of healthy women at alsamawah city. j chem biol phys sci, 2018;8. 45. yeasmin, t, haque, s, yeasmin, s, amin, m. iron status in women using oral contraceptives. bangl j physiol pharmacol, 2014;26. 46. toryila, j, amadi, k, odeh, s, adelaiye, a, ug, e, na. dynamics of combined oral contraceptive: a study of some haematological parameters in female wistar rats. iosr j pharm (iosrphr), 2014;4:15-19. 47. durand, p, blache, d. enhanced platelet thromboxane synthesis and reduced macrophage-dependent fibrinolytic activity related to oxidative stress in oral contraceptive-treated female rats. atherosclerosis, 1996;121:205-216. 48. divani, aa, luo, x, datta, yh, flaherty, jd, panoskaltsis-mortari, a. effect of oral and vaginal hormonal contraceptives on inflammatory blood biomarkers. mediat inflam, 2015a. 49. guedes, jvm, nunes, nr, ferreira, lg, vilar, tg, pinheiro, mb, domingueti, cp. evaluation of lipid profile, high-sensitivity c-reactive protein and d-dimer in users of oral contraceptives of different types. jornal brasileiro de patologia e medicina laboratorial, 2018;54:14-20. 50. oliveira, sds, petto, j, diogo, dp, santos, acnd, sacramento, mdsd, ladeia, amt. plasma renin in women using and not using combined oral contraceptive. int j cardiovasc sci, 2020;33:208-214. 51. divani, aa, luo, x, datta, yh, flaherty, jd, panoskaltsis-mortari, a. effect of oral and vaginal hormonal contraceptives on inflammatory blood biomarkers. mediat inflam, 2015b;379501-379501. 52. reuben, db, palla, sl, hu, p, reboussin, ba, crandall, c, herrington, dm, barrett-connor, e, greendale, ga. progestins affect mechanism of estrogeninduced c-reactive protein stimulation. am j med, 2006;119:167. e1-167. e8. 53. norouzi, v, seifi, m, fallah, s, korani, m, samadikuchaksaraei, a. effect of oral contraceptive therapy on homocysteine and c-reactive protein levels in women: an observational study. anatolian journal of cardiology/anadolu kardiyoloji dergisi, 2011;11. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.956 1 review issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 1 – 5 introduction the main element of anchorage control is proposed to be resistance to the adverse movement of maxillary mesial molar when the maxillary arch spaces close, which can improve the treatment results.1-3 in a person with full class ii malocclusion, treatment is successful when the extraction spaces are entirely closed from the front with different methods and with maximum anchorage.4-6 extraoral appliances can seem useful in anchorage control, but it depends on the individual’s adaptation and anchorage control. isolated cases of facial injury should also be associated with the patient.7 the effectiveness of intra-oral appliances such as the arch of nance holding, transpalatal bar is still in question and can be answered with prospective studies and treatment planning.8, 9 according to a study in the field, temporal intra-oral skeletal anchorage devices (tisads), which are called mini-implant or miniscrews, have been developed as the little titanium screws embedded in palatal or vestibular mucosa across the bone for creating an individual inflexible anchor unit. furthermore, tisads are capable of connecting to the adjacent tooth for reinforcing anchorage.10 tisads can be a conventional option that, unlike older methods, is non compliant. tisads do not attach directly to the teeth and are considered simple, differing from other methods. according to studies, the survival rate of tisads was between 80 and 94%.10-12 therefore, it can be used as a potential method to require an anchorage reinforcement in the treatment process. the evidence review also shows discrepancies in tisads’ efficiency vs. traditional approaches to anchorage supplementation.13-18 becker et al.’s meta-analysis and systematic review answered this question: “how do the orthodontic miniimplants perform for the quality of anchorage quality in comparison to the traditional devices in cases which require en-masse retractions of the upper front teeth?” and the answer was orthodontic mini-implants can achieve maximum anchorage en-masse retraction and direct anchorage.16 due to the discrepancy in the results and reaching an overall conclusion, anchorage reinforcement is facilitated by our meta-analysis study and systematic review aimed to assess the effects of tisads on conventional comparison anchorage in the maxillary arch. method search strategy pubmed, cochrane library, embase, isi, scopus, web of science, lilacs, bbo, opengrey, and google scholar, were used from the electronic databases until 2020 to perform systematic literature. therefore, for managing electronic titles, a software program (endnote x8) was used. with mesh terms, searches were performed: (“implant-supported”[mesh], dental prosthesis, or “prostheses and implants”[mesh] or “dental implants”[mesh], or “micro-implant” [mesh], or “mini-screw” [mesh], or evaluating the effect of the temporal intra-oral skeletal anchorage device (tisad) for facilitating the anchorage reinforcement: a meta-analysis and systematic review ali amiri1*, setareh khosravi2, shiva torabi3, hadi golshekan4, fan qi1 1 department of orthodontics, college of stomatology, the first affiliated stomatological hospital, xi’an jiaotong university, xi’an 710004, pr china 2 department of orthodontics, school of dentistry, shahed university, tehran, iran 3 department of orthodontics, school of dentistry, shiraz university of medical sciences, shiraz, iran 4 department of oral and maxillofacial surgery, school of dentistry, guilan university of medical sciences, rasht, iran *correspondence to: ali amiri (e-mail: draliamiri2020@gmail.com) (submitted: 04 january 2021 – revised version received: 19 january 2021 – accepted: 28 january 2021 – published online: 26 february 2021) abstract objective in this meta-analysis and systematic review, we aimed to evaluate the effects of the temporal intra-oral skeletal anchorage devices (tisads) to facilitates anchorage reinforcement. methods pubmed, cochrane library, embase, isi, scopus, web of science, lilacs, bbo, opengrey, and google scholar, were used from the electronic databases until 2020 to perform systematic literature. two reviewers extracted data blindly and independently from various abstracts as well as full texts of articles they considered for data extraction. using the cochrane collaboration’s tool, we evaluated the publications’ quality. then, we computed the mean difference of tisads and conventional anchorage groups with a confidence interval (ci) of 95%, restricted maximum likelihood, and random effect model of the mesial movement of molars and their tipping. moreover, we employed stata/mp 16 that has been considered the most rapid version of stata for evaluating meta-analysis. results according to our electronic searches, 134 topics and abstracts with potential relevance were identified according to the research design. finally, five publications matched the required inclusion criteria of the study. in addition, the cochrane collaboration instrument exhibited all studies with low to moderate biases. also, the mean difference of mesial molar movement showed less anchorage loss in the tisads group vs. the controls, and a significant difference between these two groups (md= -1.74 with a ci of 95%, -2.76, -0.71. p = 0.00). conclusions tisads can reduce treatment time, and tisads are more effective in enabling the anchorage than other methods and higher tipping in the tisads. keywords implant-supported, dental implants, molar movement, meta-analysis. 2 review evaluating the effect of the temporal intra-oral skeletal anchorage device (tisad) ali amiri et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 1– 5 “temporary anchorage device” [mesh] or “temporary intraoral skeletal anchorage devices” [mesh] or “miniplates” [mesh] and “orthodontics” [mesh]) and keywords dental prosthesis, prostheses, implants, micro-implant, temporary intra-oral skeletal anchorage devices, anchorage reinforcement, miniplates, implant-supported, orthodontics were used for other databases. with regard to the core criterion of prisma, we carried out our present systematic review and meta-analysis19 and pico strategy. inclusion criteria 1. the randomized-controlled trials (rcts), prospective and retrospective cohort investigations as well as controlled clinical trials. 2. anchorage in the maxillary arch. 3. intervention (tisads) and comparison group (conventional anchorage). 4. change of mesial molar movement and tipping of molars. 5. english language. exclusion criteria 1. case reports and studies, reviews, in-vitro studies, non-control group animals. 2. studies incomplete or inconsistent data for the purpose of the present study. data extraction and quality assessment two reviewers extracted data blindly and independently from abstracts as well as full texts of the publications for data extraction. in case of disagreement, the third referee examined or confirmed the opinions of the two referees. the study’s data, years, study design, number of patients, mean/range of age, force, diameter, and length of tisads, mean of treatment duration were extracted from the study, mean of treatment duration, the diameter of tisads, length of tisads, sample size, mean/ range of age. with the use of cochrane collaboration’s instrument, the quality of the studies included was evaluated.20 finally, the scale score for low and high and unclear risks was 1 and 0, respectively. this score ranges between 0 and 6, and the greater quality results from the greater score. meta-analysis in this step, the mean difference of the tisads and conventional anchorage group with a confidence interval (ci) of 95%, restricted maximum likelihood, and random effect model of the molars’ mesial movement and their tipping were calculated. rct studies were statistically evaluated by analyzing the subgroup and establishing significance by p<0.05, both jointly and separately. to deal with potential heterogeneity, random effects were used, showing i2 heterogeneous cases. ultimately, forest plots and meta-analysis were assessed through software stata16. a p-value below 0.05 is statistically significant (typically 0.05). results in the related searches, 134 topics and abstracts with potential relevance were found. we excluded 95 publications from our study due to incompatibility with our inclusion criteria in the abstract. therefore, in the next stage, the full-text papers from 36 studies have been thoroughly reviewed, so we excluded 31 investigations because of incompatibility with our inclusion criteria. in this way, five investigations matched the required inclusion criteria (fig. 1). in this meta-analysis, table 2 gives each study. study characteristics therefore, we considered five investigations of the rcts. as mentioned earlier, cases in tisads groups and control group were 125 and 149, respectively; a total was 274; the mean age the included studies (n=5) studies excluded (n=95) in-vitro studies, case reports, case studies, reviews, non-control group animal. not meet eligibility criteria in the abstract. full content article excluded (n=31) studies incomplete or inconsistent data for the purpose of the present study. not meet eligibility criteria in full text. studies after copies expelled (n=131) studies screened (n=131) full content article surveyed for eligibility (n=36) id en ti fi ca ti o n sc re en in g el ig ib ili ty in cl u d ed studies identified (n=134) fig. 1 study attrition. 3 review evaluating the effect of the temporal intra-oral skeletal anchorage device (tisad)ali amiri et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 1 – 5 in tisads groups was 21.58 years according to the mean age of studies included (table1). the magnitude of the force in two studies21, 22 was not reported; one study23 used 100g and al-sibaie et al.24 300g, another study25 150g. the diameter of tisads was 1.3, 1.5, and 1.6 mm. also, the length of tisads was 7–10 mm. the mean treatment duration in tisads groups and the control group was 21.27 and 26.52 months, respectively. one study did not report treatment duration25 (table 2). evaluation of bias based on the cochrane collaboration’s instrument, three investigations acquired a 5/6 total score, one study had a 4/6 overall score, and one study had a 2/6 overall score. in all studies, except victor et al. 2014, this finding indicated a low to moderate risk of bias25 (table 2). as a result, the quality of the studies was high. mesial molar movement mean difference was (md = -1.74 with a ci of 95%, –2.76, –0.71. p = 0.00) among three studies (i2 = 0.00%; p = 0.55) and heterogeneity identified. in the group of tisads vs. the control group, anchorage loss was significantly less. we did not observe any significant differences ws found (p=0.55) between the studies (fig. 2). table 1. selected studies for systematic and meta-analysis review. study. year design number of cases mean/range of age (years) force (g) diameter of tisads (mm) length of tisads (mm) mean of treatment duration (months) tisads control tisads controls m f m f tisads control ganzer et al.2019 [21] rct 80 16.4 15.0 nr 1.5 8–10 28.4 21.1 40 40 14 26 14 26 sandler et al. 2014 [23] rct 78 14.15 14.26 100 1.6 8 26.83 28.01 27 51 11 16 30 21 al-sibaie et al. 2014 [24] rct 56 23.02 20.46 300 1.6 7 12.9 16.97 28 28 9 19 12 16 victor et al. 2014 [25] rct 20 nr nr 150 1.3 8 nr nr10 10 nr nr lee et al. 2011 [22] rct 40 24.64 22.16 nr 1.6 8 24.95 28.00 20 20 0 20 0 20 rct: a randomized controlled trial. tisads: temporary intra-oral skeletal anchorage devices. m: male. f: female. nr: not reported. table 2. risk of bias assessment. study random generation of sequences concealment of allocation participants and personnel blinding blinding of outcomes evaluation insufficient data on outcomes selective reporting total score ganzer et al.2019 [21] + + + + + ? 5 sandler et al.2014 [23] + + – + + + 5 al-sibaie et al.2014 [24] + + – + + + 5 victor et al.2014 [25] ? ? – ? + + 2 lee et al.2011 [22] + + – + + ? 4 low (+), unclear (?), high (-). 4 review evaluating the effect of the temporal intra-oral skeletal anchorage device (tisad) ali amiri et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 1– 5 tipping of molars mean difference was (md, -1.71 95% ci -6.61, 3.19. p= 0.48), two studies have found heterogeneity (i2= 96.52%; p=0.00). more tipping in the tisads group was shown in fig. 3, but the difference between the tisads and the control group was not statistically significant. discussion we put tisads in palatal or vestibular mucosae across the bone to create an individual inflexible anchor unit. as mentioned earlier, we evaluated the effects of tisads in the maxillary arch in this meta-analysis and systematic review. our finds indicate that in the tisads group vs. the control group, anchorage loss was significantly less. more tipping was observed in the tisads group. the anchorage must be maintained by eliminating undesired mesial molar movement for a better choice in treatment strategy. before using tisads, the headgear, nance support arch, and transpalatal bar were reinforced in the anchorage, but the anchorage may be destroyed again.26-28 in accordance with the present study results, distal movement of molar and anchorage increase is also observed for tisads.16, 29 these results can be estimated from the friction in the molar between the archwire and the bracket slot; it can also depend on the size of the archwire. generally, the results of this study indicate that tisads are better for anchorage preservation than other conventional methods. on the other hand, according to the outputs of the present study, we did not find any significant differences between tisads and the controls in the molars’ tilting. still, according to fig. 3, it can be seen that distal tipping of the molar in the tisads group compared to other methods, the usual ones were more consistent. also, the duration of treatment was mentioned, which was approximately 4 months less in tisads in comparison to the controls. other studies have confirmed these results,2, 7, 21, 30 which means that using tisads will also reduce the duration of treatment. according to the selected studies in the present study, data were insufficient for meta-analysis to examine changes in the vertical molar position. the limitations of the current study include the limited numbers of clinical trials addressing the application of the tisads and almost more studies focused on typically cephalometric outcomes. as a result of newer studies with high and adequate sample size, the study of all variables and the duration of treatment is needed, also; due to the limited rct studies in the use of tisads; it is better to do more rct studies with high quality and low risk of bias in this area. however, patient-centered outcomes are incorporated and focused on technical outcome measures, incorporating relevant results for both patients and clinicians. we assume these research findings will be of great help for future dental research. fig. 2 mesial molar movement outcomes in tisads group and conventional anchorage. fig. 3 tipping of molars outcomes in tisads group and conventional anchorage. 5 review evaluating the effect of the temporal intra-oral skeletal anchorage device (tisad)ali amiri et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 1 – 5 conclusion tisads could reduce treatment time, more tipping, and more effectively enable the anchorage than conventional methods. recommended in the future, high-quality studies on this aim must be done for a more comprehensive and better conclusion. conflict of interest none references 1. lai eh, yao cc, chang jz, chen i, chen yj. three-dimensional dental model analysis of treatment outcomes for protrusive maxillary dentition: comparison of headgear, miniscrew, and miniplate skeletal anchorage. am j orthodont dentofacial orthoped. 2008;134(5):636-45. https://doi. org/10.1016/j.ajodo.2007.05.017. 2. park co, sa’aed nl, bayome m, park jh, kook ya, park ys, han sh. comparison of treatment effects between the modified c-palatal plate and cervical pull headgear for total arch distalization in adults. korean j orthodont. 2017;47(6):375-83. https://doi.org/10.4041/kjod.2017.47.6.375. 3. melsen b, bosch c. different approaches to anchorage: a survey and an evaluation. angle orthodont. 1997;67(1):23-30. https://doi. org/10.1043/0003-3219(1997)067<0023:dataas>2.3.co;2. 4. wahl n. orthodontics in 3 millennia. chapter 15: skeletal anchorage. am j orthodont dentofacial orthoped. 2008;134(5):707-10. https://doi. org/10.1016/j.ajodo.2008.04.015. 5. alharbi f, almuzian m, bearn d. anchorage effectiveness of orthodontic miniscrews compared to headgear and transpalatal arches: a systematic review and meta-analysis. acta odontol scand. 2019;77(2):88-98. https:// doi.org/10.1080/00016357.2018.1508742. 6. kyung hm. progress of anchorage in lingual orthodontic treatment. temp anchor devices clin orthodont. 2020:489-96. https://doi. org/10.1002/9781119513636.ch47. 7. antoszewska-smith j, sarul m, łyczek j, konopka t, kawala b. effectiveness of orthodontic miniscrew implants in anchorage reinforcement during enmasse retraction: a systematic review and meta-analysis. am j orthodont dentofacial orthoped. 2017;151(3):440-55.https://doi.org/10.1016/j. ajodo.2016.08.029. 8. diar-bakirly s, feres mf, saltaji h, flores-mir c, el-bialy t. effectiveness of the transpalatal arch in controlling orthodontic anchorage in maxillary premolar extraction cases: a systematic review and meta-analysis. angle orthodont. 2017;87(1):147-58. https://doi.org/10.2319/021216-120.1. 9. feldmann i, bondemark l. orthodontic anchorage: a systematic review. the angle orthodontist. 2006 may;76(3):493-501. https://doi.org/10.1043/00033219(2006)076[0493:oa]2.0.co;2. 10. kuroda s, sugawara y, deguchi t, kyung hm, takano-yamamoto t. clinical use of miniscrew implants as orthodontic anchorage: success rates and postoperative discomfort. am j orthodont dentofacial orthoped. 2007;131(1):9-15. https://doi.org/10.1016/j.ajodo.2005.02.032. 11. jing z, wu y, jiang w, zhao l, jing d, zhang n, cao x, xu z, zhao z. factors affecting the clinical success rate of miniscrew implants for orthodontic treatment. int j oral maxillofac implants. 2016;31(4):835-841. 12. antoszewska j, papadopoulos ma, park hs, ludwig b. five-year experience with orthodontic miniscrew implants: a retrospective investigation of factors influencing success rates. am j orthodont dentofacial orthoped. 2009;136(2):158-e1.https://doi.org/10.1016/j.ajodo.2009.03.032. 13. barthélemi s, desoutter a, souaré f, cuisinier f. effectiveness of anchorage with temporary anchorage devices during anterior maxillary tooth retraction: a randomized clinical trial. kor j orthodont. 2019;49(5):279-85. https://doi.org/10.4041/kjod.2019.49.5.279. 14. benson pe, tinsley d, o’dwyer jj, majumdar a, doyle p, sandler pj. midpalatal implants vs headgear for orthodontic anchorage—a randomized clinical trial: cephalometric results. am j orthodont dentofacial orthoped. 2007;132(5):606-15.https://doi.org/10.1016/j.ajodo.2006.01.040. 15. sandler j, benson pe, doyle p, majumder a, o’dwyer j, speight p, thiruvenkatachari b, tinsley d. palatal implants are a good conventional to headgear: a randomized trial. am j orthodont dentofacial orthoped. 2008;133(1):51-7. https://doi.org/10.1016/j.ajodo.2007.04.032. 16. becker k, pliska a, busch c, wilmes b, wolf m, drescher d. efficacy of orthodontic mini implants for en masse retraction in the maxilla: a systematic review and meta-analysis. int j implant dentist. 2018;4(1):35.doi: 10.1186/s40729-018-0144-4. 17. khlef hn, hajeer my, ajaj ma, heshmeh o. evaluation of treatment outcomes of en masse retraction with temporary skeletal anchorage devices in comparison with two-step retraction with conventional anchorage in patients with dentoalveolar protrusion: a systematic review and meta-analysis. contemp clin dentist. 2018;9(4):513-523. doi:10.4103/ ccd.ccd_661_18. 18. upadhyay m, yadav s, patil s. mini-implant anchorage for en-masse retraction of maxillary anterior teeth: a clinical cephalometric study. am j orthodont dentofacial orthoped. 2008;134(6):803-10. https://doi. org/10.1016/j.ajodo.2006.10.025. 19. moher d, liberati a, tetzlaff j, altman dg, altman d, antes g, atkins d, barbour v, barrowman n, berlin ja, clark j. preferred reporting items for systematic reviews and meta-analyses: the prisma statement (chinese edition). j chin integr med. 2009;7(9):889-96. https://doi.org/10.1371/ journal.pmed.1000097 20. higgins jp, altman dg, gøtzsche pc, jüni p, moher d, oxman ad, savović j, schulz kf, weeks l, sterne ja. the cochrane collaboration’s tool for assessing risk of bias in randomised trials. bmj. 2011;343:d5928. 21. ganzer n, feldmann i, petrén s, bondemark l. a cost-effectiveness analysis of anchorage reinforcement with miniscrews and molar blocks in adolescents: a randomized controlled trial. eur j orthodont. 2019;41(2):1807. https://doi.org/10.1093/ejo/cjy041. 22. lee ay, kim yh. comparison of movement of the upper dentition according to anchorage method: orthodontic mini-implant versus conventional anchorage reinforcement in class i malocclusion. isrn dentistry. 2011. https://doi.org/10.5402/2011/321206. 23. sandler j, murray a, thiruvenkatachari b, gutierrez r, speight p, o’brien k. effectiveness of 3 methods of anchorage reinforcement for maximum anchorage in adolescents: a 3-arm multicenter randomized clinical trial. am j orthodont dentofacial orthoped. 2014;146(1):10-20. https://doi. org/10.1016/j.ajodo.2014.03.020. 24. al-sibaie s, hajeer my. assessment of changes following en-masse retraction with mini-implants anchorage compared to two-step retraction with conventional anchorage in patients with class ii division 1 malocclusion: a randomized controlled trial. eur j orthodont. 2014;36(3):275-83. https://doi.org/10.1093/ejo/cjt046. 25. victor d, prabhakar r, karthikeyan mk, saravanan r, vanathi p, vikram nr, reddy pa, sudeepthi m. effectiveness of mini implants in three-dimensional control during retraction-a clinical study. journal of clinical and diagnostic research: jcdr. 2014;8(2):227-232. doi:10.7860/jcdr/2013/7801.4066. 26. geron s, shpack n, kandos s, davidovitch m, vardimon ad. anchorage loss—a multifactorial response. angle orthodont. 2003;73(6):730-737. https://doi.org/10.1043/0003-3219(2003)073<0730:almr>2.0.co;2. 27. dodeja t, bhat sr, talwar a. a comparative study to evaluate the effectiveness of lateral cephalogram and study cast to measure anteroposterior anchorage loss with preadjusted edgewise appliance. ind j orthodont dentofacial res. 2018;4(3):156-60. doi: 10.18231/24556785.2018.0030. 28. dai ff, xu tm, shu g. comparison of achieved and predicted tooth movement of maxillary first molars and central incisors: first premolar extraction treatment with invisalign. angle orthodont. 2019;89(5):679-87. https://doi.org/10.2319/090418-646.1. 29. upadhyay m, yadav s, nagaraj k, patil s. treatment effects of mini-implants for en-masse retraction of anterior teeth in bialveolar dental protrusion patients: a randomized controlled trial. am j orthodont dentofacial orthoped. 2008;134(1):18-29. https://doi.org/10.1016/j.ajodo.2007.03.025. 30. park hs, yoon dy, park cs, jeoung sh. treatment effects and anchorage potential of sliding mechanics with titanium screws compared with the tweed-merrifield technique. am j orthodont dentofacial orthoped. 2008;133(4):593-600. https://doi.org/10.1016/j.ajodo.2006.02.041. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.932 73 review issn 2413-0516 j contemp med sci | vol. 7, no. 2, march-april 2021: 73 – 79 introduction: inflammatory bowel disease (ibd) is a chronic inflammatory condition of unknown etiology that is thought to result from a complex interaction of genetic predisposition (leading to immunological abnormalities), dysbiosis of the gut microbiota, and environmental influences. however, none of the risk factors alone were sufficient for disease development, but the complex interactions between each factor play a major role leading to the development of ibd.1 these risk factors alone or in combination may play a significant role in the development of ibd.2 the incidence of ibd has been increasing in both developing and developed nations and this is hypothesized to be in part related to the change in dietary and lifestyle factors associated with modernization.3 huang et al. (2016) found that abnormal innate immune responses may induce adaptive immunity imbalance, where inflammatory cytokines produced may increase innate immune damages, abate intestinal barrier functions, and aggravate inflammation.4 additionally, an immunological imbalance of the intestinal mucosa, mainly associated with cells of the adaptive immune system, which responds against self-antigens, produces chronic inflammatory conditions in these patients, and it is thought that aberrant and continuing immune response to the microbes in the gut, catalyzed by genetic susceptibility of the individual, maintains ongoing intestinal mucosal inflammation.5 subsequently, this ongoing inflammation damages the intestinal wall, leading to diarrhea, bleeding and abdominal pain.6 fig. 1 shows the complex pathogenesis of ibds.3 ibd has an increasing incidence and prevalence in most countries and becomes a global emerging disease.3 a westernized lifestyle, personal habits, and environmental factors have been found to contribute to the pathogenesis of ibd.7 the relevant risk factors (including smoking, hygiene hypothesis, microorganisms, appendectomy, medication, nutrition, and stress) have all been associated with the pathogenesis of ibd.8 however, the exact specific mechanism underlying the association between environmental factors and ibd is still poorly understood.6 the treatment of ibd in any given patient may have several different goals, such as relief of symptoms: abdominal pain, rectal bleeding, and diarrhea; induction of remission in patients with active disease; prevention of relapse; correction of nutritional deficiencies; healing of fistulas; and avoidance of emergency surgery with the ultimate goal of improving quality of life.9 the current drug therapy including the use of anti-inflammatory agents and immunomodulators and biologic agents may reduce the symptoms associated with ibd and achieve long-term remission, and also therapy may be modified to some extent based on the severity, disease location, extraintestinal manifestations, and associated complications.10 materials and methods a critical review was performed by defining a scope review and collect relevant data sources and review literature, the application of literature to the current study.11 electronic databases such as pubmed, google scholar, and relevant journals nutrition and lifestyle factors associated with inflammatory bowel disease tamara r. qalqili1, yaser m. rayyan2, reema f. tayyem3* 1department of nutrition and food technology, faculty of agriculture, the university of jordan, amman, jordan. 2department of gastroenterology & hepatology, school of medicine, the university of jordan, amman, jordan. 3department of human nutrition, college of health sciences, qatar university, doha, qatar, doha, qatar. *correspondence to: reema f. tayyem (e-mail: reema.tayyem@qu.edu.qa) (submitted: 10 february 2021 – revised version received: 27 february 2021 – accepted: 04 april 2021 – published online: 26 april 2021) abstract objective many dietary and lifestyle factors are found to be associated with the pathogenesis of inflammatory bowel disease (ibd). the purpose of this study is to review the dietary and lifestyle factors associated with ibd. in addition too, this review attempts to investigate the association between dietary patterns and ibd risk and compare lifestyle factors among ibd patients. methods google scholar and pubmed were searched together with relevant journals for english studies from september 2018 to august 2020. the original studies which evaluated the lifestyle factors and dietary patterns as risk factors for inflammatory bowel disease were included. results several studies in ibd were discussed, and highlighted the independent effects of various dietary and lifestyle factors on the risk of ibd. forty-nine (49) articles met the inclusion criteria and indicated that dietary factors tend to play a pivotal role in the disease etiopathogenesis and course. however, research on food and ibd is contradictory. conclusions an excessive intake of sugar and animal fat is considered a risk factor for the development of ibd, whereas a high fiber diet and high intake of fruits and vegetables may play a protective effect. the role of lifestyle factors in ibd is crucial. amply of evidence suggested that smoking is a causative agent in crohn’s disease while it is protective against ulcerative colitis. stress, depression, vitamin d deficiency, and impaired sleep have all been all associated with incident ibd. a diet with a modified carbohydrate composition, a semi-vegetarian diet, a diet low in protein and fat, and a diet low in fermentable oligosaccharides, disaccharides, monosaccharides and polyols should be taken into consideration for ibd patients. keywords inflammatory bowel disease; ulcerative colitis; crohn’s disease; lifestyle factors; nutrition. 74 review nutrition and lifestyle factors associated with inflammatory bowel disease tamara r. qalqili, yaser m. rayyan, reema f. tayyem j contemp med sci | vol. 7, no. 2, march-april 2021: 73 – 79 were systematically searched for english language publications from september 2018 to august 2020. the inclusion criteria were: (1) studies concerning ibd (including crohn’s disease (cd) and ulcerative colitis) diagnosed using any well-established criteria. (2) studies examining quality of life. (3) studies with adult populations; controlled studies, including randomized controlled trials (baseline data only), with prospective, retrospective, or cross-sectional designs. (4) peer-reviewed papers. (5) studies focusing on other psychological variables such as depression, anxiety, distress, coping, or personality, and lifestyle factors like smoking and physical activity. articles with interventional studies (e.g., medication trials); studies in languages other than english; conference abstracts or any short papers with incomplete data presented; case reports, case series, or qualitative research and animal studies were excluded. titles and abstracts were screened by two authors (tq and rt) for possible inclusion in the study following the preferred reporting items for systematic reviews and metaanalyses (prisma) guidelines.12 appropriate studies were assessed with regards to the inclusion and exclusion criteria. full texts of selected studies were carefully shortlisted regarding the eligibility criteria and methodological aspects. original articles investigated the lifestyle factors and dietary patterns as risk factors for ibd were included. after reviewing the full texts, all authors were involved in analysis and reaching consensus on the factors emerging from the data. the data extraction included authors, year of publication, country of origin, design, setting, participant characteristics (ibd subtype, age, sex, disease activity status) and sample size, outcome measures, and results for main outcome measures. results of the 786 studies identified during the database searches, 205 were removed as duplicates. titles and abstracts were screened for the remaining 581 papers, and 346 did not meet the inclusion criteria, leaving 217 included for full review. a total of 49 unique studies were included in the final review by reading a full text. fig. 2 shows the paper selection process based on prisma 2009 flowchart.12 inflammatory bowel disease and body mass index the relationship between body mass index (bmi) and problems associated with ibd, such as morbidity, complications, prognosis, the stage of the disease, and medical therapy, have been found in many studies. one study revealed that in ibd patients, bmi was lower than that in non-ibd controls.13 another study indicated that ulcerative colitis (uc) patients have higher bmi than controls or have a bmi in the normal range.14 other studies showed that bmi was decreased in patients with active uc, but not in patients with cd, compared with healthy controls.15 on the contrary, others found that patients with inactive cd had a lower body weight and bmi value when compared with both uc and healthy controls.16 few studies have shown that medical therapy may decrease lean mass.17,18 another study indicated that patients’ bmi is lower than controls before the use of therapy.19 dong et al. (2015) found that medical therapy, including corticosteroid, azathioprine, mesalamine, and tnf-alpha antagonists, may improve bmi in patients with cd. however, the authors found that without therapy, crohn’s patients’ bmi was significantly lower than non-ibd controls, while with medical treatment, the difference was not statistically significant.14 obesity may contribute to ibd development through several mechanisms. ibd was found to be associated with excess adipocyte hypertrophy generating a pro-inflammatory state through secretion of inflammatory cytokines and chemokines, including interleukin (il)-1β, il-6, il-8, monocyte chemoattractant factor (mcp-1), tumor necrosis factor-α (tnfα), and c-reactive protein; crp.15 these biofactors are closely related to the pathogenesis of ibd.15 epidemiological studies have shown a parallel rise in the prevalence of obesity and immune-mediated conditions, including ibd. this association may produce a host pro-inflammatory response that originates from adipokines produced by visceral and subcutaneous fat.15,20 reduced level of adiponectin plays a permissive role in the release of pro-inflammatory cytokines (tnfα; crp; interleukin-6: il-6; vascular cell adhesion molecule-1: vcam 1; and mcp-1), additionally, insulin resistance is promoted by the interaction of tnfα and insulin receptor through a decrease in tyrosine kinase, and this promotes oxidative stress which can impact intestinal bacteria.21 moreover, alterations in the intestinal microbiome contribute to dysbiosis as well as to altered intestinal permeability which then, in turn, contributes to promoting the pro-inflammatory state like ibds.20 fig. 3 shows the effect of obesity on the development of ibd.20 mendall et al. (2011) documented that there was a significant association between obesity at diagnosis for cd but not with uc in their case–control study for ibd patients attending gastroenterology clinics. their study aimed to determine whether obesity at diagnosis is a risk factor for cd and uc versus community controls and whether there is a u-shaped relationship between bmi at diagnosis and risk of cd versus uc. the authors revealed that obesity at diagnosis was more common in subjects with cd versus uc with an odds ratio of 2.02 (1.18–3.43) p = 0.0096 and also cd versus community controls in the 50–70 year age group (odds ratio 3.22 (1.59– 6.52) p=0.001).22 there was evidence of a ‘dose–response’ fig. 1 the complex pathogenesis of inflammatory bowel diseases (ibd).3 abbreviations: ngs (next generation sequencing), wgs (whole genome sequencing), wes (whole exome sequencing), gwas (genome-wide association studies) and mirnas (micro ribonucleic acids). 75 review nutrition and lifestyle factors associated with inflammatory bowel diseasetamara r. qalqili, yaser m. rayyan, reema f. tayyem j contemp med sci | vol. 7, no. 2, march-april 2021: 73 – 79 phenomenon with increasing degrees of obesity-associated with increased disease risk. low bmi at diagnosis was also associated with the risk of cd versus uc. a u-shaped relationship between bmi and risk of crohn’s was supported by the strong inverse association of bmi at diagnosis (p=0.0001) and positive association of bmi at diagnosis squared (p = 0.0002).22 however, another hypothesis of the relationship between obesity and ibd was reported by chan et al. (2013), they found out that there was no association with the four higher categories of bmi compared with a normal bmi for uc (p trend=0.36) or cd (p trend=0.83). the lack of association was consistent when bmi was analyzed as a continuous or binary variable (bmi 18.5<25.0 vs. ≥ 25 kg/m2), they concluded that obesity, as measured by bmi, is not associated with the development of incident uc or cd.23 inflammatory bowel disease and malnutrition nutrition plays a pivotal role in the clinical care of patients with ibd. in broad terms, nutritional therapy can be considered as supportive or even a primary treatment. supportive therapy aims to correct malnutrition and micronutrient deficiencies and reverse their metabolic pathological consequences, in addition to providing advice on specific dietary regimes.24 patients with ibd often suffer from nutritional deficiencies. malnutrition is characterized by weight loss, growth failure, and micronutrient depletion.24 a study by nguyen et al. (2008) examined the prevalence of clinically diagnosable malnutrition among hospitalized patients with ibd in the united states and whether this malnutrition influenced health outcomes. the authors revealed that the prevalence of malnutrition was greater in cd and uc patients than in non-ibd patients (6.1% and 7.2% vs 1.8%, p<0.0001). the adjusted odds ratio for malnutrition among ibd admissions compared with non-ibd admissions was 5.57 [95% ci: 5.29–5.86]. additionally, there was an increased likelihood of malnutrition among those with fistulizing cd (or 1.65; 95% ci: 1.50–1.82) and those who had undergone bowel resection (or 1.37; 95% ci: 1.27–1.48). malnutrition was associated with increased in-hospital mortality (or 3.49; 95% ci: 2.89–4.23). this study suggests that malnutrition may serve as a clinical marker of poor ibd prognosis in hospitalized patients.25 inflammatory bowel disease and physical activity physical activity may play a role in reducing the risk of diverticulosis, gastrointestinal hemorrhage, and inflammatory fig. 2. the paper selection process based on preferred reporting items for systematic reviews and meta-analyses prisma 2009 flowchart.12 records identified through database searching (n = 786) sc re en in g in cl ud ed e lig ib ili ty id en ti fi ca ti on additional records identified through other sources (n = 0) records after duplicates removed (n = 205) records screened (n = 581) records excluded (n = 346) full-text articles assessed for eligibility (n = 217) full-text articles excluded, with reasons (n =168) studies included in the review (n = 49) fig. 2 the paper selection process based on preferred reporting items for systematic reviews and meta-analyses prisma 2009 flowchart.12 76 review nutrition and lifestyle factors associated with inflammatory bowel disease tamara r. qalqili, yaser m. rayyan, reema f. tayyem j contemp med sci | vol. 7, no. 2, march-april 2021: 73 – 79 bowel disease.26 current research revealed that exercise may be beneficial to reduce stress and symptoms of ibd, and also recommends exercise to help counteract some ibd specific complications by improving bone mineral density, immunological response, psychological health, weight loss, and stress management ability.27 a host of environmental factors may contribute to the onset of ibd. several studies examined the role of physical activity and active lifestyle as potential protective factors in the development of ibd. ng et al. (2015) found a protective effect of exercise and onset of cd (or: 0.58; 95% ci: 0.4–1.0), but none for uc, when comparing 186 cd patients, 256 uc patients and 940 controls matched for age, sex, and geographical location.28 a case–control study in a slovakian population found a statistically significant association between lower weekly rates of sport participation among adolescent and the development of ibds (or: 2.7, 95% ci: 1.5–5.0).29 however, chan et al. (2013) found in a prospective cohort study of 177 uc and 75 cd patients that there was no association between physical activity levels and uc, (p-trend=0.79) or cd (p-trend=0.42) 23. inflammatory bowel disease and lifestyle factors the role of lifestyle factors in ibd is crucial. smoking was first established as a risk factor in the development of ibd more than 30 years ago.30 smoking is one of the most important and well-characterized environmental risk factors for ibd, but its pathogenic mechanism is not clear. evidence from studies suggested that smoking is a causative agent in cd while it is protective against uc.31,32 the association between smoking and uc was first described by harries et al. (1982) who noted a reduced frequency of smokers among patients with uc compared to healthy controls.30 the effect of smoking on the localization and clinical course of 231 patients with cd was studied by lindberg et al. (1992) who found that heavy smokers (greater than 10 cigarettes/d) had small bowel cd more fig. 3 the effect of obesity on developing inflammatory bowel disease (ibd).20 abbreviations: tnfα (tumor necrosis factor-alpha), crp (c reactive protein), vacam-1 (vascular cell adhesion molecule-1), il-6 (interleukin-6) and mcp-1 (monocyte chemoattractant protein-1). 77 review nutrition and lifestyle factors associated with inflammatory bowel diseasetamara r. qalqili, yaser m. rayyan, reema f. tayyem j contemp med sci | vol. 7, no. 2, march-april 2021: 73 – 79 often than patients that smoked (less than 10 cigarettes/d) (p = 0.045).33 van der heide et al. (2009) found that no detrimental effects of active smoking on cd were observed, but passive smokers had higher need of immunosuppressant and infliximab-based therapy more frequently than non-passive smokers. additionally, the authors found that active smoking had beneficial effects on uc, indicated by reduced rates of colectomy, primary sclerosing cholangitis, and backwash-ileitis in active smokers compared to never smokers.34 furthermore, cosnes et al. (1999) found that early smoking significantly increases the risk of cd [or 2.0 (1.65–2.47)], also cd patients who smoke had a worse course of illness and quality of life, and were more likely to develop complications. additionally, they had a higher rate of hospitalizations, showed a worse response to treatments, and had a greater need for surgery.35 the mechanisms of the effect of smoking in ibd are complex, involving different chemicals found in the cigarette, such as nicotine, free radicals, and carbon monoxide, that can act on different targets, including mucus layer, cytokine production, macrophage function, and microvasculature.36 both smoking and nicotine affect the cytokine profile, predominantly by reducing the production of proinflammatory cytokines. smokers with active inflammatory bowel disease have significantly lower concentrations of il-1β and il-8 in patients with uc, and il-8 in patients with cd than in non-smokers.37 other evidence suggested that in patients with cd, when a high concentration of cigarette particulate matter reaches the ileum, it may alter the interaction of the intestinal mucosa with the microbiota through several mechanisms, e.g., affecting bacterial clearance, changing microbiota composition, increasing the permeability of the intestinal barrier, and dysregulating immune responses.32 on the contrary, people who have uc have a thinner mucus layer in the left colon and rectum when compared to healthy people. thus, it has been proposed that nicotine may increase the production of mucus and suppress the immune system which subsequently prevents inflammation in the colon.37 another theory states that nitric oxide, released by nicotine, may reduce muscle activity in the colon.39 many other lifestyle factors are likely to enhance the risk of developing ibd. stress reduces mucus secretion and increases intestinal permeability of colon in a mouse model.40 bitton et al. (2008) documented the association between major life stress factors, anxiety, depression, and ibd risk. the authors showed that participants with higher stress suffered from more relapses of both uc and cd. also, the presence of anxiety or depression has been associated with increased disease activity and an increased risk of surgery in cd patients.41 inflammatory bowel disease and dietary patterns diet was suspected to be an environmental factor involved in the etiology of ibd.42 experimental models showed that diet contributed to gut inflammation through several mechanisms including antigen presentation, alteration of gut permeability, and changes in the composition of the gut microbiota.42-44 the mediterranean diet has long been accepted as an example of a well-balanced diet but a gold-standard “mediterranean diet” is lacking.45 at least 16 countries border the mediterranean sea, variation in mediterranean diet among these countries as well as among regions within the same country is well known.46 however, despite the above variation, a mediterranean diet has the following common characteristics: high consumption of fruits, vegetables, bread, other cereals, potatoes, beans, nuts, and seeds; low to moderate consumption of dairy products, fish, eggs, and poultry; and infrequent consumption of red meat.47 in the mediterranean diet, olive oil is frequently consumed which is an important monounsaturated fat source and wine is consumed in low to moderate amounts as well.48 the combination and range of foods included in this dietary pattern provide plenty of antioxidants such as flavonoids, carotenoids, and antioxidant vitamins, lots of phytochemicals including phytoestrogens, sufficient quantities of fiber, adequate folate, and a favorable fatty acid profile.45 a growing body of scientific evidence indicated that the mediterranean dietary pattern was associated with significant improvements in health status and a decrease in inflammatory markers.47,49 the protective effect of the mediterranean diet was hypothesized to be derived from the balance in the omega-6/ omega-3 ratio of the mediterranean diet pattern (35% total fat: 15% monounsaturated fatty acids (mufa) (mainly from olive oil), 13% saturated fatty acids (sfa), and 6% polyunsaturated fatty acids (pufa).50 additionally, adherence to the mediterranean diet pattern has been shown to beneficially affect the gut microbiome and gut metabolites (metabolome).51 also, there was a large body of evidence showing that mediterranean dietary patterns regulate inflammation in chronic disease.51,52 a case–control study (n = 264 ibd subjects and 203 controls) found that low adherence to the mediterranean dietary pattern was a significant risk factor in the development of pediatric uc (or: 2.3; ci: 12–4.5).51 in addition, other dietary patterns were found to be associated with the risk of ibd, such as western dietary pattern which is high in meat, dairy products, fat, sugary foods, processed meats, pastries, baked goods, confectionery, sweetened drinks, alcohol, and limited amounts of vegetables and fruits.9 ibd has traditionally been thought of as a disease of the western hemisphere, however, there is an increased incidence in japan, hong kong, korea, and eastern europe, although still not as common, an increasing incidence of ibd is identified in south africa, south america, and saudi arabia as well.53 the dramatic rise in the incidence of ibd, particularly in south asia, india, and japan, traditionally low incidence countries, suggested that environmental factors, such as western dietary pattern, may play an important role in disease pathogenesis.28 migration from a low incidence country to a high incidence country increases the risk of developing ibd.54 a western diet that contains pro-inflammatory food cytokines, can modulate intestinal permeability, and alter the intestinal microbiota promoting a low-grade chronic inflammation in the gut which is considered an important risk factor and a prerequisite in the development of uc.54 a case–control study conducted in iran on newly diagnosed uc patients (n = 62 uc patients, 124 controls) showed that subjects that had a higher dietary inflammatory index (pro-inflammatory diet) had an increased risk of developing uc (or: 1.55, 95% ci: 1.04–2.32.55 shivappa et al. (2016) concluded that encouraging intake of more anti-inflammatory dietary factors, such as plant-based foods rich in fiber and phytochemicals, and reducing intake of pro-inflammatory factors, such as fried or processed foods rich in transfatty acids, could be a potential strategy for reducing the risk of uc.55 a meta-analysis showed an inverse association between the intake of vegetables and fruit and cd, with a pooled or for the highest versus lowest 78 review nutrition and lifestyle factors associated with inflammatory bowel disease tamara r. qalqili, yaser m. rayyan, reema f. tayyem j contemp med sci | vol. 7, no. 2, march-april 2021: 73 – 79 consumption of fruit = 0.57(95% ci 0.44–0.74).56 however, patients who consumed a pro-inflammatory diet (e.g., high in animal protein, low in fruit and vegetables) have demonstrated a higher risk of ulcerative colitis.56 a prospective trial conducted in japan in hospitalized subjects with cd (n=22) examined the effect of a semi-vegetarian diet on maintaining remission. the diet was lacto-ovo vegetarian, in which eggs and milk were allowed with small portions, meat offered once every 2 weeks and fish weekly. the remission rate, a period which symptoms of disease were reduced or disappeared, achieved with the semi-vegetarian diet was 100% after 1 year and 92% after 2 years. a semi-vegetarian diet showed significant prevention of relapse (p = 0.003 log-rank test). based on these observations, the semi-vegetarian diet may be a highly effective way to maintain a remission cd.57 furthermore, the rise in the incidence of ibd in countries that had a very low incidence suggested that industrialization and adoption of the westernized diet may be a risk factor in the development of ibd, additionally reduced consumption of fruits and possibly vegetables, resulting in a reduced overall intake of fiber, with higher intake of meats, fast foods, and transfatty acids appeared to be associated with an overall increase in the risk of developing ibd.58 a case–control study carried out by sakamoto et al. (2005) revealed that a higher consumption of sweets was positively associated with uc risk [or for the highest vs lowest quartile, 2.86; 95% ci, 1.24–6.57], whereas the consumption of sugars and sweeteners (or, 2.12; 95% ci, 1.08–4.17), sweets (or, 2.83; 95% ci, 1.38–5.83), fats and oils (or, 2.64; 95% ci, 1.29– 5.39), and fish and shellfish (or, 2.41; 95% ci, 1.18–4.89) were positively associated with cd risk. for nutrients, the intake of vitamin c (or, 0.45; 95% ci, 0.21–0.99) was negatively related to uc risk, while the intake of total fat (or, 2.86; 95% ci, 1.39–5.90), mufas (or, 2.49; 95% ci, 1.23–5.03) and polyunsaturated fatty acids (or, 2.31; 95% ci, 1.12–4.79), vitamin e (or, 3.23; 95% ci, 1.45–7.17), and n-3 fatty acids (or, 3.24; 95% ci, 1.52–6.88) and n-6 fatty acids (or, 2.57; 95% ci, 1.24–5.32) were positively associated with cd risk.59 geerling et al. (2000) found that high intakes of monounsaturated fat (or: 33.9 [95% ci 2.6–443.1]), polyunsaturated fat (or: 5.1 [95% ci 1.0–26.7]), and vitamin b6 (or: 6.9 [95% ci 1.6–30.7] were associated with an increased risk to develop uc.60 in a case–control study conducted in jordan by rayyan et al. (2021), the authors showed that ibd patients showed significant increase in the intake of saturated fat, protein, carbohydrates, sugars, fiber, mufa, trans-fat cholesterol, vitamins a, d, e, b12, c and folate, beta-carotene, retinol, calcium, potassium, iron, omega-3 and omega-6 compared to the control group. however, the control group showed higher intake of vitamin k and caffeine when compared to the ibd patients (p < 0.05).61 conclusion in this review, we found that ibd is chronic relapsing intestinal inflammatory disease characterized by complex interactions of multiple factors including smoking, major life stressors, diet, and lifestyle. various dietary and nutritional factors have been suggested as being significant etiological factors both for cd and uc. however, research on food and ibd is contradictory. an excessive intake of sugar and animal fat is considered a risk factor for the development of ibd, whereas a high fiber diet and high intake of fruits and vegetables may play a protective effect. the role of lifestyle factors in ibd is crucial. amply of evidence suggested that smoking is a causative agent in cd while it is protective against uc. stress, depression, vitamin d deficiency, and impaired sleep have all been all associated with incident ibd. a diet with a modified carbohydrate composition, a semi-vegetarian diet, a diet low in protein and fat, and a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols should be taken into consideration for ibd patients. competing interests the authors declare that they have no competing interest. funding no fund has been requested for this work. references 1. molodecky, na, kaplan, gg (2010), environmental risk factors for inflammatory bowel disease. gastroenterol hepatol 2010;6(5), 339. 2. frolkis, a, dieleman, la, barkema, hw, panaccione, r, ghosh, s, fedorak, rn, alberta ibd consortium, environment and the inflammatory bowel diseases. can j gastroenterol, 2013:27. 3. loddo, i, romano, c, inflammatory bowel disease: genetics, epigenetics, and pathogenesis. front immunol 2015;6:551. 4. huang, y, chen, z, inflammatory bowel disease related innate immunity and adaptive immunity. am j transl res 2016;8(6):2490. 5. bonen, dk, cho, jh, the genetics of inflammatory bowel disease. gastroenterology, 2003;124(2):521-536. 6. o’sullivan, m, o’morain, c, nutrition in inflammatory bowel disease. best pract res clin gastroenterol, 2006;20(3):561-573. 7. manzel, a, muller, dn, hafler, da, erdman, se, linker, ra, kleinewietfeld, m. role of “western diet” in inflammatory autoimmune diseases. curr allergy asthma rep, 2014;14(1):404. 8. ye, y, pang, z, chen, w, ju, s, zhou, c. the epidemiology and risk factors of inflammatory bowel disease. int j clin exp med 2015;8(12), 22529. 9. pithadia, ab, jain, s., treatment of inflammatory bowel disease (ibd). pharmacol rep, 2011;63(3), 629-642. 10. rutgeerts, p, van assche, g, vermeire, s. optimizing anti-tnf treatment in inflammatory bowel disease. gastroenterology, 2004;126(6):1593-1610. 11. carnwell, r, daly, w. strategies for the construction of a critical review of the literature. nurse educ pract,2001;1(2):57-63. 12. moher, d, liberati, a, tetzlaff, j, altman, dg, prisma group. preferred reporting items for systematic reviews and meta-analyses: the prisma statement. plos med,2009;6(7):e1000097. 13. ghosha c, shukla, a. malnutrition in inflammatory bowel disease patients in northern india: frequency and factors influencing its development. 2008. 14. dong, j, chen, y, tang, y, xu, f, yu, c, li, y, dai, n. body mass index is associated with inflammatory bowel disease: a systematic review and metaanalysis. plos one, 2015;10(12):e0144872. 15. flores, a, burstein, e, cipher, dj, feagins, la. obesity in inflammatory bowel disease: a marker of less severe disease. digest dis sci, 2015;60(8):2436-2445. 16. capristo, e, mingrone, g, addolorato, g, greco, av, gasbarrini, g. metabolic features of inflammatory bowel disease in a remission phase of the disease activity. j intern med, 1998;243(5):339-347. 17. geerling, bj, badart-smook, a, stockbrügger, rw, brummer, rj. comprehensive nutritional status in patients with long-standing crohn disease currently in remission. am j clin nutr, 1998;67(5):919-926. 18. mijač, dd, janković, gl, jorga, j, krstić, mn. nutritional status in patients with active inflammatory bowel disease: prevalence of malnutrition and methods for routine nutritional assessment. eur j intern med, 2010;21(4):315-319. 79 review nutrition and lifestyle factors associated with inflammatory bowel diseasetamara r. qalqili, yaser m. rayyan, reema f. tayyem j contemp med sci | vol. 7, no. 2, march-april 2021: 73 – 79 19. cravo, m, guerreiro, cs, dos santos, pm, brito, m, ferreira, p, fidalgo, c, pereira, ad. risk factors for metabolic bone disease in crohn’s disease patients. inflam bowel dis,  2010;16(12):2117-2124. 20. szilagyi, a. relationship (s) between obesity and inflammatory bowel diseases: possible intertwined pathogenic mechanisms. clin j gastroenterol, 2019;1-14. 21. balistreri, cr, caruso, c, candore, g. the role of adipose tissue and adipokines in obesity-related inflammatory diseases. mediat inflam, 2010. 22. mendall, ma, gunasekera, av, john, bj, kumar, d. is obesity a risk factor for crohn’s disease? digest dis sci, 2010;56(3):837-844. 23. chan, ss, luben, r, olsen, a, tjonneland, a, kaaks, r, teucher, b, bergmann, mm. body mass index and the risk for crohn’s disease and ulcerative colitis: data from a european prospective cohort study (theibdin epic study). am j gastroenterol, 2010;108(4):575-582. 24. goh, j, o’morain, ca. nutrition and adult inflammatory bowel disease. aliment pharmacol therap, 2003;17(3):307-320. 25. nguyen, gc, munsell, m, harris, ml. nationwide prevalence and prognostic significance of clinically diagnosable protein-calorie malnutrition in hospitalized inflammatory bowel disease patients. inflam bowel dis, 2008;14(8):1105-1111. 26. strate, ll, liu, yl, aldoori, wh, giovannucci, el. physical activity decreases diverticular complications. am j gastroenterol, 2009;104(5):1221. 27. narula, n, fedorak, rn. exercise and inflammatory bowel disease. can j gastroenterol hepatol, 2008;22(5):497-504. 28. ng, sc, tang, w, leong, rw, chen, m, ko, y, studd, c, kasturiratne, a. environmental risk factors in inflammatory bowel disease: a populationbased case-control study in asia-pacific. gut, 2015;64(7):1063-1071. 29. hlavaty, t, toth, j, koller, t, krajcovicova, a, oravcova, s, zelinkova, z, huorka, m. smoking, breastfeeding, physical inactivity, contact with animals, and size of the family influence the risk of inflammatory bowel disease: a slovak case–control study. united eur gastroenterol j, 2013;1(2):109-119. 30. harries, ad, baird, a, rhodes, j. non-smoking: a feature of ulcerative colitis. br med j, 1982;284(6317):706. 31. legaki, e, gazouli, m. influence of environmental factors in the development of inflammatory bowel diseases. world j gastroint pharmacol therap, 2016;7(1):112. 32. qalqili t, rayyan y, tayyem r. nutrition and lifestyle factors associated with inflammatory bowel disease. j gastroint liver dis jgld, 2021;30(1), in press. 33. lindberg, e, järnerot, g, huitfeldt, b. smoking in crohn’s disease: effect on localisation and clinical course. gut,1992; 33(6):779-782. 34. van der heide, f, dijkstra, a, weersma, rk, albersnagel, fa, van der logt, em, faber, kn, dijkstra, g. effects of active and passive smoking on disease course of crohn’s disease and ulcerative colitis. inflam bowel dis, 2009;15(8):1199-1207. 35. cosnes, j, carbonnel, f, carrat, f, beaugerie, l, cattan, s, gendre, j. effects of current and former cigarette smoking on the clinical course of crohn’s disease. aliment pharmacol therap, 1999;13(11):1403-1411. 36. loftus jr, ev. clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. gastroenterology, 2004;126(6):1504-1517. 37. bhatti, ma, hodgson, hjf. peripheral blood pro-inflammatory cytokine profile in active inflammatory bowel disease (ibd) differ between smokers and non-smokers. gut, 1997;40(3s). 38. salih, a, widbom, l, hultdin, j, karling, p. smoking is associated with risk for developing inflammatory bowel disease including late onset ulcerative colitis: a prospective study. scand j gastroenterol, 2018;53(2):173-178. 39. mahid, ss, minor, ks, soto, re, hornung, ca, galandiuk, s. smoking and inflammatory bowel disease: a meta-analysis. in mayo clinic proceedings (vol. 81, no. 11, pp. 1462-1471). elsevier. 2006. 40. cosnes, j. smoking, physical activity, nutrition and lifestyle: environmental factors and their impact on ibd. digest dis, 2010;28(3):411-417. 41. bitton, a, dobkin, pl, edwardes, md, sewitch, mj, meddings, jb, rawal, s, wild, ge. predicting relapse in crohn’s disease: a biopsychosocial model. gut, 2008;57(10):1386-1392. 42. racine, a, carbonnel, f, chan, ss, hart, ar, bueno-de-mesquita, hb, oldenburg, b, key, t. dietary patterns and risk of inflammatory bowel disease in europe: results from the epic study. inflam bowel dis, 2016;22(2):345-354. 43. chapman-kiddell, ca, davies, ps, gillen, l, radford-smith, gl, role of diet in the development of inflammatory bowel disease. inflam bowel dis, 2010;16(1):137-151. 44. nickerson, kp, mcdonald, c. crohn’s disease-associated adherent-invasive escherichia coli adhesion is enhanced by exposure to the ubiquitous dietary polysaccharide maltodextrin. plos one, 2012;7(12):e52132. 45. demetriou, ca, hadjisavvas, a, loizidou, ma, loucaides, g, neophytou, i, sieri, s, kyriacou, k. the mediterranean dietary pattern and breast cancer risk in greek-cypriot women: a case-control study. bmc cancer, 2012;12(1):113. 46. karimi, z, jessri, m, houshiar-rad, a, mirzaei, hr, rashidkhani, b. dietary patterns and breast cancer risk among women. public health nutr, 20141;7(5):1098-1106. 47. estruch, r. anti-inflammatory effects of the mediterranean diet: the experience of the predimed study. proc nutr soc, 2010;69(3):333-340. 48. castello, a, pollán, m, buijsse, b, ruiz, a, casas, am, baena-cañada, jm, lluch, a. spanish mediterranean diet and other dietary patterns and breast cancer risk: case–control epigeicam study. br j cancer, 2014;111(7):1454-1462. 49. altomare, r, cacciabaudo, f, damiano, g, palumbo, vd, gioviale, mc, bellavia, m, monte, ail. the mediterranean diet: a history of health. iran j public health, 201342(5):449. 50. de filippis, f, pellegrini, n, vannini, l, jeffery, ib, la storia, a, laghi, l, turroni, s. high-level adherence to a mediterranean diet beneficially impacts the gut microbiota and associated metabolome. gut, 2016;65(11):1812-1821. 51. strisciuglio, c, giugliano, f, martinelli, m, cenni, s, greco, l, staiano, a, miele, e. impact of environmental and familial factors in a cohort of pediatric patients with inflammatory bowel disease. j pediat gastroenterol nutr, 2017;64(4):569-574. 52. marlow, g, ellett, s, ferguson, ir, zhu, s, karunasinghe, n, jesuthasan, ac, ferguson, lr. transcriptomics to study the effect of a mediterraneaninspired diet on inflammation in crohn’s disease patients. human genomics, 2013;7(1):24. 53. lovasz, bd, golovics, pa, vegh, z, lakatos, pl. new trends in inflammatory bowel disease epidemiology and disease course in eastern europe. digest liver dis, 2013;45(4):269-276. 54. hart, ar, luben, r, olsen, a, tjonneland, a, linseisen, j, nagel, g, appleby, p. diet in the aetiology of ulcerative colitis: a european prospective cohort study. digestion, 2008;77(1):57-64. 55. shivappa, n, hébert, jr, rashvand, s, rashidkhani, b, hekmatdoost, a. inflammatory potential of diet and risk of ulcerative colitis in a case–control study from iran. nutr cancer, 2016;68(3):404-409. 56. li, f, liu, x, wang, w, zhang, d. consumption of vegetables and fruit and the risk of inflammatory bowel disease: a meta-analysis. eur j gastroenterol hepatol, 2015;27(6):623-630. 57. chiba, m, abe, t, tsuda, h, sugawara, t, tsuda, s, tozawa, h, imai, h. lifestylerelated disease in crohn’s disease: relapse prevention by a semi-vegetarian diet. world j gastroenterol wjg, 2010;16(20):2484. 58. lee, d, albenberg, l, compher, c, baldassano, r, piccoli, d, lewis, jd, wu, gd. diet in the pathogenesis and treatment of inflammatory bowel diseases. gastroenterology, 2015;148(6):1087-1106. 59. sakamoto, n, kono, s, wakai, k, fukuda, y, satomi, m, shimoyama, t, kobashi, g. dietary risk factors for inflammatory bowel disease a multicenter case‐ control study in japan. inflam bowel dis, 2005;11(2):154-163. 60. geerling, bj, dagnelie, pc, badart-smook, a, russel, mg, stockbrügger, rw, brummer, rj. diet as a risk factor for the development of ulcerative colitis. am j gastroenterol, 2000;95(4):1008-1013. 61. qalqili t, rayyan y, abu-sneineh a, tayyem r. nutrients consumed by the inflammatory bowel disease jordanian patients. ann cancer res ther, 2021;29(1):22-29. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.910 https://www.jstage.jst.go.jp/search/global/_search/-char/en?item=8&word=awni+t.+abu-sneineh 170 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 review application of platelet rich fibrin in the regeneration of intra oral defects: a systematic review muna al-shuhayeb,1 mansour meymandi,1 mohammad reza talebi ardakani,1 reza amid,1,2 farzaneh poursafar,3 hossein mohammad rahimi,4 sarah al-maawi,5 shahram ghanaati,5 and anahita moscowchi6 1department of periodontics, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 2dental research center, research institute of dental sciences, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 3department of periodontics, school of dentistry, tehran university of medical sciences, tehran, iran. 4students research office, school of dentistry, shahid beheshti university of medical sciences, tehran, iran. 5department for oral, cranio-maxillofacial and facial plastic surgery, frankfurt orofacial regenerative medicine (form) lab, university hospital frankfurt goethe university, frankfurt, germany. 6 department of community oral health, school of dentistry, tehran university of medical sciences, tehran, iran. correspondence to farzaneh poursafar (email: farzaneh.poursafar70@gmail.com). (submitted: 12 may 2019 – revised version received: 28 may 2019 – accepted: 10 june 2019 – published online: 26 august 2019) introduction the second generation of platelet concentrate, platelet-rich fibrin (prf) was introduced by choukroun et al.1 to accelerate the healing procedures. prf can be easily prepared following the centrifugation of non-coagulated blood without requiring additional anticoagulants such as bovine thrombin.2,3 the obtained product would be an autologous matrix of dense fibrin which is rich in platelets, growth factors and platelet cytokines.4,5 various growth factors and cytokines were detected in prf including platelet-derived growth factor (pdgf), vascular endothelial growth factor, insulin-like growth factors, transforming growth factor-beta 1 (tgf-β), interleukin-1β (il-1β), interleukin-4 (il-4), and interleukin-6 (il-6).6,7 the prf concentrate is highly biocompatible. as no anticoagulants are used in this system, prf forms a fibrin matrix in the later stages of the coagulation. this formed fibrin matrix contains platelets which are gradually releasing mentioned cytokines and growth factors, following the fibrin fabrication.6 as a result of these features, the fibrin network of prf can be used as the source of autologous growth factors especially in damaged tissues.8 the biological characteristics of prf was shown to positively influence the differentiation and proliferation of osteoblasts, fibroblasts, endothelial cells, and chondrocytes9–11 and facilitate the osteointegration process.12 all these procedures may initiate and accelerate the healing and regeneration process.13 over the course of many years, platelets were identified to promote bone repair and the bone healing.14,15 it has also been shown by various studies that growth factors may stimulate the bone regeneration in the intra-oral bone defects.16 prf provide an autologous concentrate of fibrin, platelets, leukocytes and their growth factors. based on the previous data, it can be expected that the application of the platelet concentrates in the intraoral defects may lead to clinical success in less duration of time in addition to reduced postoperative symptoms.17,18 platelet-rich fibrin, as a type of platelet concentrate, has been used widely in the various studies solely or in combination with other approaches in case of maxillofacial defects, such as facial plastic surgery,19 maxillary sinus augmentation in combination with bone substitute materials,20 root coverage with assistant of coronally displaced flap,21 and the treatment of furcation defects.22 these studies suggested that prf can be used as the treatment or supplement in the bone defects of oral region. however, the additional benefit of prf in clinical trials did not present as clinically significant in every situation. the aim of the present systematic review is to investigate the effect of prf on bone regeneration and healing in the intra-oral region including ridge preservation, sinus augmentation, endodontal and periodontal defects. objective to systematically review the randomized clinical trials (rcts) on the effect of platelet rich fibrin (prf) concentrate on the tissue regeneration of intra-oral defects. methods an electronic search was performed in pubmed/medline and cochrane library using relevant keywords until june 2018. rcts which used prf concentrate for treatment of intrabony defects, sinus augmentation, furcation involvement, endo-periodontal lesions, gingival recession, and ridge preservation were included in the present review. results in total, 79 studies that were used prf either solely or mixed in human trials were included and divided based on the defect type. most of the studies used prf to treat intra-bony defects showed that it would improve treatment outcomes. in case of furcation involvement, the application of leukocyte-prf in addition to open flap debridement improved the bone regeneration in grade ii mandibular furcation involvement. in case of ridge preservation and sinus floor augmentation, the results were controversial. conclusion the result of the current systematic review implied that the treatment outcome of prf application for periodontal and soft tissue repair depends on the treatment strategies and type of the defect. it was shown that prf application is a practical approach to accelerate and enhance new bone formation in human studies in intrabony defects and furcation involvements. however, further clinical trials for evaluation of other types of intra-oral defects are required. keywords platelet rich fibrin, bone regeneration, periodontal defects, sinus floor augmentation issn 2413-0516 171j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects materials and methods study design this systematic review was conducted based on the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines.23 the search protocol was considered based on the patient, intervention, comparison, outcome (pico) question of the study (table 1). clinical studies regarding prf application for accelerating tissue regeneration of the selected periodontal defects (intrabony defects, sinus augmentation, furcation involvement, endo-periodontal lesions, root coverage, gingival recession, and ridge preservation) were included. in this review only randomized clinical trials (rcts) with parallel or splitmouth design were included. inclusion criteria was english papers, more than 10 patients, and comparison of the effect of treatment with the control group (conventional treatment). case reports and series, animal studies and review articles were excluded. search strategy a comprehensive electronic search was conducted in pubmed/ medline and cochrane library from january 2009 to june 2018 using the following terms: “platelet rich fibrin”, “bone regeneration”, “bone augmentation”, “periodontal defect”, “apical lesion”, “maxillofacial surgery”, and “gingival recession”. related published papers were found using the various combinations of the related search terms based on the pico question (table 1). study selection and data extraction study selection was performed by two independent reviews. disagreements were resolved by discussion. the initial paper selection was done by assessing the titles and abstracts. the full texts of the potentially suitable articles were obtained for final assessment according to the inclusion and exclusion criteria. to perform the study, the following data were extracted: study design, study groups, the number of patients and samples, variables, data evaluation methods, the follow-up periods, and the treatment outcomes. extracted data from selected articles were summarized in the tables for each defect type and compared in a qualitative manner. due to the variety of defect types and various protocols for using prf meta-analysis could not be performed. results study selection after removing duplicate topics, a total of 370 articles were found in the electronic search. following the initial screening of titles and abstracts, 106 studies were chosen for further evaluations as relevant for the aim of the study. finally, 79 records completely fulfilled the inclusion criteria of the present study. figure 1 demonstrated the details of the search strategy and study selection. selected studies were categorized based on their defect type: intrabony defect, ridge preservation, sinus augmentation, endo-periodontal defect, furcation defect, and gingival recession. because of the various approaches for the prf application and different type of defects, conducting the meta-analysis was not possible. prf application on the treatment of intrabony defects totally, 29 clinical trials used prf concentrates in the treatment of intrabony defects (table 2). prf concentrates were used solely24–37 or in combination with other treatment approaches including demineralized freeze-dried bone allograft (dfdba),38 nanocrystalline hydroxyapatite (ncha),39 metformin,40 bovine porous bone mineral (bpbm),41 demineralized bone matrix (dbm),42 rosuvastatin,43 alendronate,44 etc. (table 2). the follow-up period was varied from 3 to 12 table 1 study question and related keywords based on patient, intervention, comparison, outcome format question of the review search keywords population (p) patients need bone or tissue regeneration for their defects (intrabony defects, sinus augmentation, furcation involvement, endo-periodontal lesions, gingival recession, and ridge preservation) periodontal defects, intrabony defects, sinus augmentation, furcation involvement, endoperiodontal lesions, root coverage, gingival recession, ridge preservation intervention (i) application of platelet concentrates including platelet-rich fibrin (prf), leukocyte-prf (l-prf), and advanced prf (a-prf) alone or beside other conventional approaches as a supplement prf, l-prf, a-prf comparison (c) various kind of intra oral regenerative treatment approaches outcome (o) regeneration of bone and the periodontal tissue tissue regeneration, bone regeneration, new bone formation, healing fig. 1 flow diagram of study selection strategy. 172 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. ta bl e 2. r ev ie w o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on in tr ab on y de fe ct s au th or s ( ye ar ) st ud y de si gn st ud y si ze , de fe ct ty pe pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l pr ad ee p et a l.4 5 rc t 90 , p id 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in pr f + h a + o fd o fd a nd o fd + p rf cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in m ea n pd re du ct io n, m ea n ca l ga in a nd p er ce nt ag e of m ea n bo ne fil l w ith th e ad va nt ag e of o fd + p rf (3 .9 0 ± 1 .0 9, 3 .0 3 ± 1. 16 m m , a nd 5 6. 46 ± 9 .2 6% , r es pe ct iv el y) a nd p rf + h a + o fd g ro up s (4 .2 7 ± 0 .9 8, 3 .6 7 ± 1 .0 3 m m , a nd 6 3. 39 ± 16 .5 2% , r es pe ct iv el y) in c om pa ris on to o fd g ro up (2 .9 7 ± 0. 93 , 2 .6 7 ± 1 .0 9 m m , a nd 1 5. 96 ± 1 3. 91 % , r es pe ct iv el y) . n aq vi e t a l.4 6 sp lit -m ou th rc t 10 p at ie nt s w ith pa ire d pi d 30 00 rp m (4 00 g ) fo r 1 0 m in pr f + b io ac tiv e gl as s pu tt y + o fd bi oa ct iv e gl as s pu tt y + o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed o nl y in m ea n ib d re du ct io n w ith th e ad va nt ag e of te st g ro up (7 .1 ± 1 .3 7 m m ) gr ou ps in c om pa ris on to c on tro l g ro up (5 .7 ± 1 .6 4 m m ). th or at e t a l.3 5 sp lit -m ou th rc t 10 p at ie nt s w ith pa ire d pi d a nd la p 30 00 rp m (4 00 g ) fo r 1 2 m in pr f + m fo m fo (k irk la nd fla p) cl in ic al a nd ra di ogr ap hi c ev al ua tio n 12 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in m ea n pd re du ctio n, m ea n ca l ga in a nd r bf w ith th e ad va nt ag e of te st gr ou ps (w ith m ea n ca l ga in a nd b on e fil l o f 4 .0 ± 0 .6 3 an d 3. 09 m m ) i n co m pa ris on to c on tr ol g ro up . a bo ut 8 0% of th e pr ftr ea te d si te s sh ow ed ≥ 50 % b on e fil l. ya ja m an ya e t a l.3 7 c t 38 p at ie nt s w ith 90 p id s 30 00 rp m fo r 1 0 m in pr f + o fd o fd a nd p er io g la ss + o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in m ea n pd re du ct io n, m ea n ca l ga in a nd p er ce nt ag e of m ea n bo ne fil l w ith th e ad va nt ag e of o fd + p rf (6 .1 1 ± 0 .9 2, 6 .7 4 ± 1. 55 m m , a nd 7 5. 01 ± % 7. 85 ) a nd p er io g la ss + o fd (5 .5 7 ± 1 .1 0, 6 .5 7 ± 1 .4 5 m m , a nd 7 4. 44 ± 8 .5 7% ) g ro up s in co m pa ris on to o fd g ro up (3 .6 8 ± 0 .7 2, 4 .1 4 ± 0 .7 6 m m , an d 69 .2 9 ± 7 .7 3% ). ya sa sw in i e t a l.4 7 sp lit -m ou th rc t 14 p at ie nt s w ith pa ire d pi d 30 00 rp m (4 00 g ) fo r 1 2 m in pr f + p er io g la ss m pp g + p er io g la ss cl in ic al & r ad io gr ap hi c ev al ua tio n 6 an d 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed o nl y in c as e of b on e fil l w ith th e ad va nt ag e of p rf g ro up s in c om pa ris on to m pp g g ro up (7 0. 55 ± 1 5. 99 v s. 55 .3 0 ± 1 1. 87 a t m on th 6 an d 84 .5 5 ± 1 1. 74 v s. 72 .2 ± 9 .9 1 at m on th 9 ). pa te l e t a l.3 0 sp lit -m ou th rc t 13 p at ie nt s w ith pa ire d pi d 30 00 rp m fo r 1 0 m in pr f + o fd o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6, 9 a nd 1 2 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed o nl y in m ea n pd re du ct io n, m ea n ca l ga in a nd p er ce nt ag e of m ea n bo ne fil l w ith th e ad va nt ag e of p rf g ro up (4 .1 ± 0 .3 1, 3 .4 ± 0 .6 9 m m , a nd 4 5. 18 ± 7 .5 7% ) i n co m pa ris on to o fd g ro up (5 .5 ± 0 .5 2, 4 .7 ± 0 .6 7 m m a nd 2 1. 6 ± 9 .3 % ) ba ja j e t a l.2 5 rc t 17 p at ie nt s w ith 44 p id s 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in pr f + o fd o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed o nl y in m ea n pd re du ct io n, m ea n ca l ga in , m ea n ib d re du ct io n an d pe rc en ta ge o f m ea n bo ne fi ll w ith th e ad va nt ag e of p rf gr ou p (3 .1 4 ± 1 .2 6, 2 .6 6 ± 1 .0 7, 2 .2 4 ± 0 .6 6 m m , a nd 4 6. 14 ± 1 1. 39 % ) i n co m pa ris on to o fd g ro up (2 .1 4 ± 1 .2 6, 1 .5 9 ± 1 .0 1, 0 .8 4 ± 0 .9 9 m m , 1 5. 76 ± 1 8. 77 % ). (c on tin ue d) 173j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects ta bl e 2. r ev ie w o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on in tr ab on y de fe ct s— co nt in ue d au th or s ( ye ar ) st ud y de si gn st ud y si ze , de fe ct ty pe pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l g al av e t a l.2 7 rc t 20 , p id 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in pr f + o fd a bg + o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 3, 6 , a nd 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed o nl y in c as e of r bf w ith th e ad va nt ag e of a bg g ro up (3 0. 34 % c om pa re d w ith pr f 20 .2 2% ). ch ad w ic k et a l.2 6 rc t 36 , p id 30 00 rp m fo r 1 0 m in o fd + p rf o fd + d fd ba cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6 m on th s th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n th e gr ou ps (d fd ba : m ea n ca l ga in = 1 .1 6 ± 1 .3 3 m m , m ea n bo ne fi ll = 1 .5 3 ± 1 .6 4 m m , a nd m ea n ra di og ra ph ic b on e fil l = 1 .1 4 ± 0 .8 8 m m ; p rf : m ea n ca l ga in = 1 .0 3 ± 0 .8 6 m m , m ea n cl in ic al b on e fil l = 1 .3 5 ± 1 .6 0 m m , a nd m ea n ra di og ra ph ic bo ne fi ll = 1 .1 0 ± 1 .0 1 m m ). ch an dr ad as e t a l.4 2 rc t 36 , p id 30 00 rp m fo r 1 2 m in o fd + p rf + d bm o fd , o fd + pr f cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s m ea n pd re du ct io n an d ra l ga in o f p rf (4 .2 5 ± 1 .4 8 an d 3. 92 ± 0 .9 0 m m ) a nd p rf + d bm (4 .2 5 ± 1 .4 8 an d 3. 92 ± 0. 90 m m ) g ro up s w as s ig ni fic an tly b et te r t ha n th e co nt ro l gr ou p (3 .0 0 ± 1 .2 1 an d 2. 25 ± 0 .6 2 m m ). lb g a nd p er ce nt ag e of m ea n bo ne fi ll pr f + d bm g ro up (3 .4 7 ± 0 .5 3 m m , an d 61 .5 3 ± 4 .5 4% ) w as s ig ni fic an tly b et te r t ha n ot he r gr ou ps (2 .5 5 ± 0 .6 1 m m , a nd 4 9. 60 ± 1 4. 08 % in p rf g ro up an d 1. 21 ± 0 .8 0 m m , 2 4. 69 ± 1 5. 59 % in c on tr ol g ro up ). pr ad ee p et a l.4 3 rc t 90 , p id 30 00 rp m (4 00 g ) fo r 1 0 m in o fd + p rf + 1. 2% r sv o fd a nd o fd + p rf cl in ic al a nd e va lua tio n 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in m ea n pd re du ctio n, m ea n ca l ga in a nd m ea n de fe ct d ep th re du ct io n w ith th e ad va nt ag e of o fd + p rf + 1 .2 % r sv g ro up (4 .9 0 ± 0 .3 1, 3 .9 3 ± 0 .7 8, a nd 3 .6 3 ± 0 .6 7 m m c om pa re d w ith p rf g ro up 4 .0 3 ± 0 .1 8, 3 .3 0 ± 0 .6 5, a nd 3 .1 7 ± 0 .6 5 m m a nd c on tr ol g ro up 3 .1 0 ± 0 .3 0, 2 .4 7 ± 0 .7 7, a nd 1 .4 3 ± 0 .5 0 m m ). ka no riy a et a l.4 4 rc t 90 , p id 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in ac ce ss th er ap y + p rf + 1 % a ln ac ce ss th er ap y, ac ce ss th er ap y + p rf cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in m ea n pd re du ctio n, m ea n ca l ga in a nd m ea n de fe ct d ep th re du ct io n w ith th e ad va nt ag e of a cc es s th er ap y + p rf + 1 % a ln gr ou p (4 .5 3 ± 0 .8 1, 5 .1 6 ± 0 .4 6, a nd 2 .8 4 ± 0 .2 6 m m co m pa re d w ith p rf g ro up 3 .7 ± 0 .9 1, 4 .2 ± 0 .6 6, a nd 2 .4 2 ± 0 .2 1 m m a nd c on tr ol g ro up 2 .8 6 ± 0 .6 8, 3 .0 3 ± 0 .1 8, a nd 0. 38 ± 0. 26 m m ). m ar ta nd e et a l.4 9 rc t 96 , p id 30 00 rp m fo r 12 –1 4 m in o fd + p rf + 1. 2% a tv o fd a nd o fd + p rf cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in m ea n pd re du ct io n, m ea n ca l ga in a nd p er ce nt ag e of m ea n de fe ct de pt h re du ct io n w ith th e ad va nt ag e of o fd + p rf + 1 .2 % at v gr ou p (4 .0 6 ± 1 .2 2, 3 .6 6 ± 1 .4 2 m m , a nd 5 0. 96 ± 4. 88 % ) i n co m pa ris on to o fd g ro up (2 .7 6 ± 1 .4 3, 2 .5 0 ± 1. 33 m m , 5 .5 4 ± 1 .7 1% ). ay de m ir tu rk al et a l.5 0 sp lit -m ou th rc t 28 p at ie nt s w ith pa ire d pi d 30 00 rp m fo r 1 0 m in em d + p rf + o fd em d + o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6 m on th s th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n th e gr ou ps . 174 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. ta bl e 2. r ev ie w o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on in tr ab on y de fe ct s— co nt in ue d au th or s ( ye ar ) st ud y de si gn st ud y si ze , de fe ct ty pe pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l ag ra w al e t a l.3 8 sp lit -m ou th rc t 30 p at ie nt s w ith pa ire d pi d 40 0 g fo r 1 2 m in lpr f + d fd ba d fd ba cl in ic al a nd ra di ogr ap hi c ev al ua tio n 12 m on th s cl in ic al a nd ra di og ra ph ic o ut co m es o f l -p rf tr ea te d gr ou ps w as s ig ni fic an tly b et te r t ha n th e co nt ro l g ro up (p d : 4. 15 ± 0 .8 4 vs . 3 .6 0 ± 0 .5 1 m m ; c a l: 3 .7 3 ± 0 .7 4 vs . 2 .6 1 ± 0 .6 8 m m ; r ec : 0 .4 7 ± 0 .5 6 vs . 1 .0 0 ± 0 .6 1 m m ; b on e fil l: 3. 50 ± 0 .6 7 vs . 2 .4 9 ± 0 .6 4 m m ; a nd d ef ec t r es ol ut io n: 3 .7 3 ± 0 .6 3 vs . 2 .7 5 ± 0 .5 7 m m ). aj w an i e t a l.2 4 sp lit -m ou th rc t 30 p at ie nt s w ith pa ire d pi d 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in lpr f + o fd o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s o nl y ra di og ra ph ic o ut co m es o f l -p rf tr ea te d gr ou ps w as s ig ni fic an tly b et te r t ha n th e co nt ro l g ro up (c em en t en am el ju nc tio n to b as e of th e de fe ct : 2 .6 0 ± 1 .1 0 vs . 1 .3 0 ± 0 .2 m m ; a nd a lv eo la r c re st to b as e of th e de fe ct : 1 .4 5 ± 0. 49 v s. 0. 80 ± 0 .3 5 m m ). el ge nd y et a l.3 9 sp lit -m ou th rc t 20 p at ie nt s w ith pa ire d pi d 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in lpr f+ n ch a + o fd n ch a + o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6 m on th s bo th c lin ic al a nd ra di og ra ph ic o ut co m es (p pd , c a l, b on e de ns ity ) o f l -p rf tr ea te d gr ou p (3 .4 2 ± 0 .4 9, 3 .5 5 ± 0 .5 1, an d 10 7. 59 ± 9 .4 5 m m ) w er e si gn ifi ca nt ly b et te r t ha n th e co nt ro l g ro up (3 .8 2 ± 0 .5 4, 3 .9 0 ± 0 .4 4, a nd 9 3. 20 ± 5 .7 8 m m ). pr ad ee p et a l.4 0 rc t 12 0, p id 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in 1% m et fo rm in + l -p rf + o fd 1% m et fo rm in + o fd , l -p rf + o fd , o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s pd re du ct io n, r a l ga in o ut co m es , a nd p er ce nt ag e of de fe ct d ep th re du ct io n of s tu dy g ro up (4 .9 0 ± 0 .3 0, 4 .9 0 ± 0 .3 0 m m , 5 2. 65 ± 0 .0 31 % ) w er e si gn ifi ca nt ly b et te r th an th e co nt ro l g ro up s (m f: 3 .9 3 ± 0 .2 5, 3 .9 3 ± 0 .2 5 m m , 48 .6 9 ± 0 .0 26 % , p rf : 4 .0 0 ± 0 .1 8, 4 .0 3 ± 0 .1 8 m m , 4 8 ± 0. 02 9% , a nd o fd a lo ne : 3 .0 0 ± 0 .1 8, 2 .9 6 ± 0 .1 8 m m , 9 .1 4 ± 0 .0 4% ). m at hu r e t a l.2 9 c t 38 , p id 30 00 rp m fo r 1 0 m in o fd + l -p rf o fd + a bg cl in ic al e va lu at io n 6 m on th s th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n th e gr ou ps . (p pd c ha ng e: p rf g ro up : − 2. 67 ± 1 .2 9, a bg g ro up : − 2. 40 ± 1 .0 6, c a l ga in : p rf g ro up : − 2. 53 ± − 1. 06 , a bg g ro up : − 2. 53 ± − 1. 63 ). sh ah e t a l.3 3 sp lit -m ou th rc t 20 p at ie nt s w ith pa ire d pi d 30 00 rp m fo r 1 0 m in o fd + l -p rf o fd + d fd ba cl in ic al e va lu at io n 6 m on th s th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n th e gr ou ps (t he m ea n re du ct io n in p d : p rf g ro up : 3 .6 7 ± 1 .4 8 m m , d fd ba g ro up : 3 .7 0 ± 1 .7 8 m m . g ai n in r a l: p rf g ro up : 2. 97 ± 1 .4 2 m m , d fd ba g ro up : 2 .9 7 ± 1 .5 4 m m , g in gi va l m ar gi n m ig ra te d ap ic al ly : p rf g ro up : 0 .4 3 ± 1 .3 1 m m , d fd ba g ro up : 0 .7 2 ± 2 .3 m m ). g up ta e t a l.2 8 rc t 44 , p id w ith c p 30 00 rp m fo r 1 2 m in lpr f + o fd em d + o fd cl in ic al a nd c bc t 6 m on th s o nl y de fe ct re so lu tio n w as s ig ni fic an tly h ig he r i n em d gr ou p. (4 3. 07 ± 1 2. 21 % v s. 32 .4 1 ± % 14 .6 1) . ba ns al e t a l.5 2 sp lit -m ou th rc t 10 p at ie nt s w ith pa ire d pi d 30 00 rp m fo r 1 0 m in d fd ba + l -p rf + o fd d fd ba + o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6 m on th s pd re du ct io n an d ca l ga in o f t he s tu dy g ro up (4 .0 ± 0 .8 16 an d 3. 4 ± 0 .6 06 m m ) w er e si gn ifi ca nt ly b et te r t ha n th e co nt ro l g ro up (3 .1 ± 0 .7 38 a nd 2 .3 ± 0 .6 99 m m ). (c on tin ue d) 175j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects ta bl e 2. r ev ie w o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on in tr ab on y de fe ct s— co nt in ue d au th or s ( ye ar ) st ud y de si gn st ud y si ze , de fe ct ty pe pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l pr ad ee p et a l.3 1 c t 90 , p id 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in lpr f + o fd pr p + o fd , o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s cl in ic al a nd ra di og ra ph ic o ut co m es o f l -p rf a nd p rp tr ea te d gr ou ps w as s ig ni fic an tly b et te r t ha n th e co nt ro l gr ou p (m ea n pd re du ct io n an d ca l ga in : p rf : 3 .7 7 ± 1 .1 9 an d 3. 17 ± 1 .2 9 m m , p rp : 3 .7 7 ± 1 .0 7 an d 2. 93 ± 1 .0 8 m m , co nt ro l g ro up : 2 .9 7 ± 0 .9 3 an d 2. 83 ± 0 .9 1 m m . p er ce nt ag e of m ea n bo ne fi ll: p rf : 5 5. 41 ± 1 1. 39 % , p rp : 5 6. 85 ± 14 .0 1% , c on tr ol g ro up : 1 .5 6 ± 1 5. 12 % ). le ko vi c et a l.4 1 sp lit -m ou th rc t 17 p at ie nt s w ith pa ire d pi d 10 00 g fo r 1 0 m in lpr f + b pb m + o fd lpr f + o fd (c on tr ol ) cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6 m on th s cl in ic al a nd ra di og ra ph ic o ut co m es o f s tu dy g ro up s w er e si gn ifi ca nt ly b et te r t ha n th e co nt ro l g ro up (r ed uc tio n in po ck et d ep th : p rf -b pb m : 4 .4 7 ± 0 .7 8 m m o n bu cc al a nd 4. 29 ± 0 .8 2 m m o n lin gu al s ite s. pr f: 3 .3 5 ± 0 .6 8 m m o n bu cc al a nd 3 .2 4 ± 0 .7 3 m m o n lin gu al s ite s. ca l ga in : pr fbp bm : 3 .8 2 ± 0 .7 8 m m o n bu cc al a nd 3 .7 1 ± 0 .7 5 m m o n lin gu al s ite s. pr f: 2 .2 4 ± 0 .7 3 m m o n bu cc al a nd 2. 12 ± 0 .6 8 m m o n lin gu al s ite s. d ef ec t fi ll: p rf -b pb m : 4. 06 ± 0 .8 7 m m o n bu cc al a nd 3 .9 4 ± 0 .7 3 m m o n lin ogu al s ite s, pr f: 0 .2 1 ± 0 .6 8 m m o n bu cc al a nd 2 .0 6 ± 0 .6 4 m m o n lin gu al s ite s) . th or at e t a l.3 6 c t 32 , p id 40 0 g fo r 1 2 m in lpr f + o fd o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s m ea n pd re du ct io ns , c a l ga in a nd b on e fil l o f l -p rf gr ou ps (4 .5 6 ± 0 .3 7, 3 .6 9 ± 0 .4 4 m m , a nd 4 6. 92 % ) w as si gn ifi ca nt ly b et te r t ha n th e co nt ro l g ro up (3 .5 6 ± 0 .2 7, 2. 13 ± 0 .4 3 m m , a nd 2 8. 66 % ). sh ar m a et a l.3 4 rc t 56 , p id 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in lpr f + o fd o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s ra di og ra ph ic o ut co m es (b on e fil l) of l -p rf tr ea te d gr ou ps w as s ig ni fic an tly b et te r t ha n th e co nt ro l g ro up (4 8. 26 ± 5. 72 % v s. 1. 80 ± 1 .5 6% ). rc t: ra nd om iz ed c lin ic al tr ia l, c t: c lin ic al tr ia l, pi d : p er io do nt al in tr ab on y de fe ct , l a p: lo ca liz ed a gg re ss iv e pe rio do nt iti s, d fd ba : d em in er al iz ed fr ee ze -d rie d bo ne a llo gr af t, o fd : o pe n fla p de br id em en t, n ch a : n an oc ris ta lin e hy dr ox ya pa tit e, a bg : a ut og en ou s bo ne g ra fti ng , e m d : e na m el m at rix d er iv at iv e, b pb m : b ov in e po ro us b on e m in er al , d bm : d em in er al iz ed b on e m at rix , r sv : r os uv as ta tin , a ln : a le nd ro na te , a tv : a to rv as ta tin , g tr : gu id ed ti ss ue re ge ne ra tio n, h a : h yd ro xy ap at ite , m fo : m od ifi ed fl ap o pe ra tio n, m pp g : m ar gi na l p er io st ea l p ed ic le g ra ft. 176 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. months. all the studies evaluated the treatment outcomes through clinical improvement including periodontal pocket depth (ppd) reductions and clinical attachment loss (cal) gains. most of them also did the radiographic assessment for further evaluations (table 2). most of the studies showed that the application of prf improved treatment outcomes at least in one measured parameter (table 2). only, ajwani et al.24 reported that additional use of l-prf did not lead to improving the treatment outcome in comparison with open flap debridement (ofd) alone. moreover, shah et al.33 and chadwick et al.26 compared the effect of prf in combination with ofd versus dfdba in combination with ofd. in both studies, no advantages were observed by both treatment approaches. also, mathur et al.29 and galav et al.27 showed that prf has no advantage in comparison with autogenous bone graft (abg). only thorat et al.35 selected patients with aggressive periodontitis. they reported that the additional use of prf in comparison to kirkland flap alone improved both clinical and radiographic outcomes. prf application in the ridge preservation a total of 17 clinical trials used prf approach for the ridge preservation (table 3). prf concentrates were used solely53–65 or in combination with plaster of paris66 and dfdba.67 only one study used advanced platelet-rich fibrin (a-prf) in addition to fdba for ridge preservation.68 the follow-up period was varied from 1 week to 6 months. unlike intrabony defects, various methods including scintigraphic evaluation,54,57 serial radio visio-graphic analysis,56 and histomorphometric evaluation65,68 were used to evaluate the treatment outcome other than clinical and radiographic assessment. in all the studies with radiographic evaluation, improvement in the bone filling was observed in the ridge preserved by prf concentrates. in case of the additional application of prf concentrates in the socket when compared with not using any intervention, the results were controversial. in this case, approximately half of the clinical trials shows the advantage of using prf.53,59,62,63,65 thakkar et al.67 showed that using additional prf besides dfdba enhanced ridge preservation process in comparison to dfdba alone. furthermore, the effectiveness of prf when compared with beta-tri-calcium phosphate with collagen was observed in das et al.55 study. in the case of a-prf, clark et al.68 showed the supplemental use of a-prf for the ridge preservation in combination with fdba did not lead to better results. sinus floor augmentation similarly, prf concentrates has been evaluated in seven clinical trials with the aim of the sinus floor augmentation (table 4). six studies utilized supplemental l-prf besides bio-oss20,70–73 or nanobone74 in a two-stage method for sinus augmentation. only kanayama et al.75 used sole l-prf in the crestal approach of sinus floor elevation. the measured outcomes and follow up periods were varied among the selected studies. most of the measured outcomes in the included clinical trials showed the additional application of l-prf has no additional benefit compared with the bone graft alone. tatullo et al.73 reported that supplemental l-prf can reduce the healing time. furthermore, in the bolukbasi et al.’s study,70 less change in the bone length/the implant length ratio was observed in the l-prf treated patients. kanayama et al.75 reported that l-prf only approach led to from 4.00 ± 1.63% to 4.38 ± 1.67% bone gain in the sandblasted acid-etched implants and the hydroxyapatite implants respectively. endo-periodontal defects in the case of endo-periodontal lesions, only two clinical trials included in the present systematic review (table 5). dhiman et al.76 found that the application of l-prf in apicomarginal lesions can improve the clinical outcomes. moreover, the complete bone filling was observed in the l-prf treated patients in the singh et al.’s study.77 management of furcation defects eight clinical trials were aimed at evaluating the bone regeneration in grade ii mandibular furcation involvement (table 6). three studies evaluated the treatment outcomes of l-prf besides ofd compared with ofd alone.22,78,79 in four studies, l-prf was applied mixed with other materials including metformin gel,80 bioactive ceramic composite granules (bccg),81 rosuvastatin,82 hydroxyapatite (ha),82 and alendronate.83 asimuddin et al.84 compared the effect of prf alone with allograft and healiguide collagen membrane. all the studies did clinical and radiographic assessments to evaluate the treatment outcomes. the follow-up period was from 6 to 9 months (table 5). bajaj et al.78 and sharma et al.22 reported that the application of l-prf in addition to ofd improved the bone regeneration defects in grade ii mandibular furcation involvement. siddiqui et al.79 suggested that using β-tricalcium phosphate (β-tcp) lead to better treatment outcomes when compared with prf. it was shown that when prf applied mixed with rosuvastatin and ha,82 alendronate,83 and metformin,80 the treatment outcome improved when compared with prf alone. furthermore, lohi et al.81 reported that supplemental use of prf besides bccg led to significant improvements in all measured parameters. coverage of gingival recessions in case of gingival recessions, 16 articles using prf concentrates were met the requirements for inclusion in the present systematic review. the included clinical trials used prf for root coverage of patients with class i or ii miller gingival recession (table 7). six studies evaluated the clinical outcomes of additional prf to coronally advanced flap (caf),85–88 modified caf (mcaf),21 and lateral sliding bridge flap89 compared with flap sole approach. culhaoglu et al.90 compared the effect of twoand four-layers prf in root coverage procedure. other studies compared the outcomes of using supplemental prf in gingival recession with other supplemental approaches including connective tissue graft (ctg),90–94 subepithelial ctg (sctg),95,96 enamel matrix derivative,97 amniotic membrane,98 and resin-modified glass ionomer cement.99 all studies assessed clinical parameters in their follow-up periods. the follow-up period was varied from 121,87,90 to 24 months.89 except for padma et al.87 when the application of prf was compared with flap alone, no additional benefit by prf was observed in the treatment outcomes.21,85,86,88,89 in two studies conducted by eren et al.,95 and öncü96 the use of prf did not lead to a significant improvement in clinical parameters when compared with sctg. agarwal et al.98 reported a significant advantage of prf compared with the amniotic membrane in case of root coverage percentage. in studies comparing prf to ctg, the outcomes were controversial. only mufti et al.93 177j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects ta bl e 3 re vi ew o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on ri dg e pr es er va tio n au th or s ( ye ar ) st ud y de si gn si te (p at ie nt ) pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt te st c tr cl ar k et a l.6 8 rc t 40 n on -m ol ar te et h 13 00 rp m (2 00 g ) fo r 8 m in a -p rf + fd ba a -p rf , f d ba , bl oo d cl ot (c on tr ol ) m ic ro -c t an d hi st om or ph om et ric an al ys is 15 w ee ks m or e bo ne d en si ty (5 51 ± 5 8 m g/ cm 3 v s. 48 7 ± 6 4 m g/ cm 3 ), m or e vi ta l b on e (4 6 ± 1 8% v s. 29 ± 1 4% a nd le ss lo ss o f r id ge h ei gh t ( 1. 8 ± 2 .1 m m v s. 1. 0 ± 2 .3 m m ) w as pr es en t i n th e a -p rf g ro up c om pa re d w ith th e fd ba gr ou p an d m or e bo ne m in er al d en si ty w as o bs er ve d in th e fd ba g ro up c om pa re d w ith c on tr ol s ig ni fic an tly . zh an g et a l.6 5 c t 28 m ol ar 40 0 g fo r 1 0 m in pr f n o in te rv en tio n ra di og ra ph ic a nd hi st om or ph om et ric ev al ua tio n 7 d ay s, 1 an d 3 m on th s th e hi st om or ph om et ric e va lu at io n (t he o st eo id a re a/ tis su e ar ea ) s ho w s si gn ifi ca nt b on e fo rm at io n (9 .7 62 4 ± 4. 01 21 % ) i n pr f gr ou p in c om pa ris on to c on tr ol (2 .8 05 6 ± 1 .2 09 4% ). g iri sh k um ar e t a l.6 6 rc t 90 s oc ke ts in 4 8 pa tie nt s 30 00 rp m fo r 1 0 m in pr f + p la st er of p ar is n o in te rv en tio n an d pr f cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6 m on th s th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n gr ou ps (a lv eo la r h ei gh t l os s: po p– pr f: 2 .8 ± 0 .4 6, p rf : 3 ± 0 .8 , co nt ro l: 3. 3 ± 0 .6 1; a lv eo la r w id th lo ss : p o p– pr f: 2 .9 ± 0. 8, p rf : 3 ± 0 .6 4, c on tr ol : 3 ± 0 .8 3; b on e fil l: po p– pr f: 10 .1 ± 2 .9 , p rf : 9 .8 3 ± 2 .2 4, c on tr ol : 7 .9 ± 1 .5 ). a lz ah ra ni e t a l.5 3 rc t 24 s oc ke ts 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in pr f n o in te rv en tio n cl in ic al a nd ra di ogr ap hi c ev al ua tio n 1, 4 a nd 8 w ee ks si gn ifi ca nt im pr ov em en t w as o bs er ve d th e te st g ro up co m pa re d w ith th e co nt ro l g ro up in c as e of ri dg e w id th pr op or tio ns (1 1. 33 ± 2 .3 0 m m v s. 12 .0 4 ± 2 .5 0 at w ee k 4 an d 10 .9 7 ± 2 .3 3 m m v s. 11 .5 4 ± 2 .4 2 at w ee k 8) a nd ra di og ra ph ic b on e fil l p er ce nt ag e (7 4. 05 ± 1 .6 6% v s. 68 .8 2 ± 1. 07 % a t w ee k 1, 8 1. 54 ± 3 .3 3% v s. 74 .0 3 ± 2 .6 1 at w ee k 4 an d 88 .8 1 ± 1 .5 3% v s. 80 .3 4 ± 2 .6 1% a t w ee k 8) . th ak ka r e t a l.6 7 rc t 36 s ite s, si ngl ero ot ed te et h 30 00 rp m fo r 1 0 m in pr f + d fd ba d fd ba cl in ic al a nd ra di ogr ap hi c ev al ua tio n 3 an d 6 m on th s th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n gr ou ps [r id ge w id th d iff er en ce (f ro m b as el in e to 1 80 d ay s) : d fd ba : 1 .3 61 1 ± 0 .7 03 05 m m , d fd ba -p rf : 0 .7 5 ± 0 .4 93 m m . r id ge h ei gh t d iff er en ce (f ro m b as el in e to 1 80 d ay s) : d fd ba : − 1. 38 89 ± 0 .5 01 63 m m , d fd ba –p rf : − 1. 08 33 ± 0. 42 87 5 m m ]. te m m er m an e t a l.6 3 sp lit -m ou th rc t 22 s oc ke ts 27 00 rp m fo r 1 0 m in lpr f n o in te rv en tio n cl in ic al a nd ra di og ra ph ic (c bc t) ev al ua tio n 3 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in c as e of a ll ev al ua te d pa ra m et er s w ith th e ad va nt ag e of l -p rf g ro up (m ea n ve rt ic al h ei gh t c ha ng es a t t he b uc ca l w er e − 1. 5 ± 1. 3 m m fo r c on tr ol s ite s vs . 0 .5 ± 2 .3 m m fo r t es t s ite s (p < 0 .0 05 ). at th e bu cc al s id e, c on tr ol s ite s va lu es w er e − 2. 1 ± 2 .5 , − 0. 3 ± 0 .3 (p < 0 .0 05 ) a nd − 0. 1 ± 0 .0 m m , a nd te st s ite s va lu es w er e − 0. 6 ± 2 .2 (p < 0 .0 05 ), − 0. 1 ± 0 .3 , an d 0. 0 ± 0 .1 m m . s ig ni fic an t d iff er en ce s w er e fo un d fo r to ta l w id th re du ct io n be tw ee n te st (− 22 .8 4% ) a nd c on tr ol si te s (− 51 .9 2% ) a t 1 m m b el ow c re st le ve l.) . 178 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. ta bl e 3 re vi ew o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on ri dg e pr es er va tio n— co nt in ue d au th or s ( ye ar ) st ud y de si gn si te (p at ie nt ) pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt te st ct r ku m ar e t a l.5 8 rc t 31 m an di bu la r m ol ar s 30 00 rp m fo r 1 0 m in lpr f n o in te rv en tio n cl in ic al a nd ra di ogr ap hi c ev al ua tio n 1 an d 3 m on th s si gn ifi ca nt ly m ea n po ck et d ep th re du ct io n w as o bs er ve d in b ot h gr ou ps . t he re w er e no s ig ni fic an t d iff er en ce s be tw ee n gr ou ps . ba sl ar li et a l.5 4 sp lit -m ou th c t 40 m an di bu la r m ol ar s 30 00 rp m fo r 1 0 m in lpr f n o in te rv en tio n sc in tig ra ph ic e va lu at io n 1 an d 3 m on th s th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n gr ou ps in c as e of te ch ne tiu m -9 9m m et hy le ne d ip ho sp ho na te up ta ke (l -p rf 4 .1 ± 1 .0 v s. co nt ro l 3 .9 ± 1 .1 ). ye la m al i e t a l.6 4 sp lit m ou th rc t 20 p at ie nt s w ith bi la te ra l i m pa ct ed th ird m ol ar 30 00 rp m fo r 1 0 m in lpr f pr p cl in ic al e va lu at io n 4 m on th s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in c as e of s of t t is su e he al in g w ith th e ad va nt ag e of l -p rf g ro up . m ar en zi e t a l.5 9 sp lit m ou th rc t 26 p at ie nt s w ith ex tr ac te d 27 00 rp m fo r 1 2 m in lpr f n o in te rv en tio n cl in ic al e va lu at io n 7 d ay s si gn ifi ca nt d iff er en ce s w er e ob se rv ed in c as e of h ea lin g an d po st -o pe ra tiv e pa in w ith th e ad va nt ag e of lpr f gr ou p. g iri sh r ao e t a l.5 6 c t 44 t hi rd m an di bul ar m ol ar s 36 0– 40 0 rp m fo r 20 m in lpr f n o in te rv en tio n se ria l r ad io v isi ogr ap hi c an al ys is 1 d ay a nd 1 , 3 a nd 6 m on th s th e m ea n pi xe ls re co rd ed w as n ot s ig ni fic an tly d iff er en t be tw ee n gr ou ps . su tt ap re ya -s ri et al .62 sp lit -m ou th c t 20 p re m ol ar s 30 00 rp m fo r 1 0 m in lpr f n o in te rv en tio n cl in ic al e va lu at io n 1 w ee k h or iz on ta l r es or pt io n bu cc al a sp ec t w as s ig ni fic an tly lo w er in p rf tr ea te d gr ou p (l -p rf g ro up : 1 .9 ± 1 .1 m m v s. 2. 6 ± 0 .7 m m ). pr f ha d fa st er b on e he al in g th an c on tr ol (n ot s ig ni fic an t) . h au se r e t a l.6 9 rc t 23 p re m ol ar s 27 00 rp m fo r 1 2 m in lpr f, lpr f + m uc os al fla p n o in te rv en tio n m ic ro -c t an d hi st olo gi c ev al ua tio n 8 w ee ks a s ig ni fic an t d iff er en ce in c as e of in tr in si c bo ne q ua lit y se en p re se rv at io n of th e al ve ol ar w id th w as s ee n us in g pr f an d. l -p rf + fl ap in c om pa ris on to c on tr ol g ro up (b on e vo lu m e/ to ta l v ol um e fo r p rf : 0 .2 81 ± 0 .0 37 , p rf + fla p: 0 .1 97 ± 0 .0 27 , a nd c on tr ol : 0 .2 49 ± 0 .0 37 ). si ng h et a l.6 1 c t 40 t hi rd m an di bul ar m ol ar s 30 00 rp m fo r 1 0 m in lpr f n o in te rv en tio n cl in ic al a nd ra di ogr ap hi c ev al ua tio n 12 w ee ks th e tra be cu la r b on e fo rm at io n w as se en in a ll of b ot h gr ou ps , th er e w er e no si gn ifi ca nt d iff er en ce s b et w ee n gr ou ps . ( gr ay sc al e va lu e fo r l -p rf : 1 46 .9 a nd fo r c on tro l: 1 23 ). si m on e t a l.5 0 c t si x m ol ar s an d 15 p re m ol ar s 14 50 g fo r 1 5 m in lpr f n o in te rv en tio n cl in ic al a nd ra di ogr ap hi c ev al ua tio n 4 m on th s th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n gr ou ps [m ea n w id th re so rp tio n fo r l -p rf : 0 .3 2 m m (4 .7 1% ) a nd co nt ro l: 0. 57 m m (7 .3 8% )]. g ur bu ze r e t a l.5 7 rc t 14 p at ie nt s w ith bi la te ra lly s of t tis su e im pa ct ed 3r d m an di bu la r m ol ar s 20 30 rp m (4 00 g ) fo r 1 0 m in pr f n o in te rv en tio n sc in tig ra ph ic e va lu at io n 4 w ee ks th er e w er e no s ig ni fic an t d iff er en ce s be tw ee n gr ou ps in c as e of te ch ne tiu m -9 9m m et hy le ne d ip ho sp ho na te up ta ke (l -p rf 4 .5 ± 1 .0 v s. co nt ro l 4 .6 ± 1 .0 ). rc t: ra nd om iz ed c lin ic al tr ia l, c t: c lin ic al tr ia l, d fd ba : d em in er al iz ed fr ee ze -d rie d bo ne a llo gr af t, βtc pcl : b et atr i-c al ci um p ho sp ha te w ith c ol la ge n, f d ba : f re ez edr ie d bo ne a llo gr af t. 179j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects ta bl e 4 re vi ew o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on si nu s a ug m en ta tio n au th or s ( ye ar ) st ud y de si gn si te (p at ie nt ) pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l n iz am e t a l.7 2 rc t sp lit m ou th 13 , 2 6, 5 8 40 0 g fo r 1 2 m in t: b io -o ss ® + lpr f tw ost ag e (la te ra l) c: b io -o ss ® ra di og ra ph ic a nd h a 6 m on th s t: f bh 1 3. 60 ± 1 .0 9, c : f bh 1 3. 53 ± 1 .2 0. t : n bf 2 1. 38 ± 8. 78 % ; r g m 2 5. 95 ± 9 .5 4% ; b on eto -b on e su bs tit ut e co nt ac t 4 7. 33 ± 1 2. 33 % ; c t 52 .6 7 ± 1 2. 53 % , c : n bf 2 1. 25 ± 5 .5 9% ; r g m 3 2. 79 ± 5 .8 9% . b on eto -b on e su bs tit ut e co nt ac t 5 4. 04 ± 8 .3 6% ; c t 45 .9 6 ± 8 .3 6% , i m pl an t s r (1 8 m on th s) : 1 00 % . bo lu kb as i e t a l.7 0 rc t pa ra lle l 25 , 3 2, 6 6 40 0 g fo r 1 2 m in t: b io -o ss ® + lpr f tw ost ag e (la te ra l) c: b io -o ss ® + rc m ra di og ra ph ic a nd h a 6 m on th s t: n bf 3 5. 0 ± 8 .6 0% : r g m 3 3. 05 ± 6 .2 9% ; c t 30 .6 3 ± 7. 53 % . c : n bf 3 2. 97 ± 9 .7 1% ; r g m 3 3. 79 ± 8 .5 7% ; c t 33 .9 4 ± 9 .1 5% . t he d iff er en ce s be tw ee n gr ou ps w er e no t s ta tis tic al ly s ig ni fic an t. t: 1 0 da ys a fte r s in us li fti ng : g sh /o sh 4 .2 6; 1 0 da ys a fte r i m pl an t p la ce m en t: bl /il 1. 43 , g sh /o sh 4 .7 8; 6 m on th s af te r i m pl an t p la ce m en t: bl /il 1 .3 8, g sh /o sh 4 .7 8; 6 m on th s af te r l oa di ng : b l/ il 1 .3 7, g sh /o sh 4 .3 9; 1 2 m on th s af te r l oa di ng : b l/ il 1. 32 , g sh /o sh 4 .3 9; 2 4 m on th s af te r l oa di ng : b l/ il 1 .3 0, g sh /o sh 4 .3 6. c : 1 0 da ys a fte r s in us li fti ng : g sh /o sh 4. 2; 1 0 da ys a fte r i m pl an t p la ce m en t: bl /il 1 .4 6, g sh / o sh 4 .5 5; 6 m on th s af te r i m pl an t p la ce m en t: bl /il 1 .4 3, g sh /o sh 4 .5 2; 6 m on th s af te r l oa di ng : b l/ il 1 .3 7, g sh / o sh 4 .0 9; 1 2 m on th s af te r l oa di ng : b l/ il 1 .2 9, g sh /o sh 3. 81 ; 2 4 m on th s af te r l oa di ng : b l/ il 1 .2 3, g sh /o sh 3 .6 7. t sh ow ed s ta tis tic al ly le ss c ha ng e in th e bl /il ra tio . t he di ffe re nc e of g sh /o sh ra tio w as in si gn ifi ca nt b et w ee n gr ou ps . i m pl an t s r (3 0 m on th s) : 1 00 % . ka na ya m a et a l.7 5 c t 27 , , 3 9 40 0 g fo r 1 0 m in o nl y lpr f on est ag e (o st eot om e) n on e bo ne g ai n (r ad io gr ap hi c) 12 m on th s th e m ea n bo ne g ai ns : i n th e sa nd bl as te d ac id -e tc he d im pl an ts 4 .3 8 ± 1 .6 7 an d in th e hy dr ox ya pa tit e im pl an ts 4. 00 ± 1 .6 3 m m . bo ss ha rd t e t a l.7 4 rc t pa ra lle l 12 , 1 6, 1 6 n ot m en tio ne d t: n an ob on e + lpr f (t w ost ag e) c: n an ob on e + rc m ra di og ra ph ic a nd h a 7– 11 m on th s t: n bf 2 8. 6 ± 6 .9 0% ; r g m 2 5. 7 ± 8 .8 % . c : n bf 2 8. 7 ± 5 .4 ; rg m 2 5. 5 ± 7 .6 . i m pl an t s r (1 2 w ee ks ): 10 0% . g as sl in g et a l.7 1 rc t sp lit m ou th 6, 1 2, 3 2 40 0 g fo r 1 2 m in t: b io -o ss ® + a bg + l -p rf m em br an e tw ost ag e (la te ra l) c: b io -o ss ® + a bg + r cm (b io -g id e® ) ra di og ra ph ic a nd h a 5 m on th s t: n bf 1 7% ; r g m 1 5. 9% . c : n bf 1 7. 2% ; r g m 1 7. 3% . t he di ffe re nc es b et w ee n gr ou ps w er e no t s ta tis tic al ly s ig ni fica nt . i m pl an t s r (1 2 m on th s) : 1 00 % . zh an g et a l.2 0 rc t pa ra lle l 10 , 1 1, 30 0 g fo r 1 0 m in t: b io -o ss ® + lpr f tw ost ag e (la te ra l) c: b io -o ss ® h a 6 m on th s t: n bf 1 8. 35 ± 5 .6 2% (1 .4 ti m es o f t ha t i n co nt ro l); r g m 19 .1 6% ± 6 .8 9% (1 .5 ti m es le ss er th an th at in c on tr ol ); bo ne -t obo ne s ub st itu te c on ta ct 2 1. 45 ± 1 4. 57 % . c : n bf 12 .9 5 ± 5 .3 3% ; r g m 2 8. 54 ± 1 2. 01 % ; b on eto -b on e su bst itu te c on ta ct 1 8. 57 ± 5 .3 9% . t he d iff er en ce s be tw ee n gr ou ps w er e no t s ta tis tic al ly s ig ni fic an t. 180 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. ta bl e 5 re vi ew o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on e nd ope rio do nt al le si on au th or s ( ye ar ) st ud y de si gn si te (p at ie nt ) pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l d hi m an e t a l.7 6 rc t 30 , a pi co m ar gi na l le si on s 30 00 rp m fo r 1 0 m in lpr f cl in ic al a nd ra di og ra ph ic ev al ua tio n 12 m on th s th e st at is tic al ly s ig ni fic an t d iff er en ce in l -p rf g ro up w ith th e co nt ro l gr ou p w as o bs er ve d in c as e of p d r (8 ± 0 .9 2 m m v s. 7. 27 ± 0 .9 6 m m ). in c as e of c a l (7 ± 0. 92 m m v s. 6. 6 ± 1 .1 8 m m ) a nd s pl r (9 3. 41 ± 7 .0 0 vs . 9 4. 57 ± 5 .8 7) n o si gn ifi ca nt d iff er en ce s w er e ob se rv ed . si ng h et a l.7 7 c t 15 , p er i-a pi ca l l es io ns 30 00 rp m fo r 1 0 m in lpr f cl in ic al a nd ra di og ra ph ic ev al ua tio n 6 m on th s co m pl et e bo ne re ge ne ra tio n w as o bs er ve d in th e lpr f tr ea te d pa tie nt s. rc t: ra nd om iz ed c lin ic al tr ia l, c t: c lin ic al tr ia l, ca l: c lin ic al a tt ac hm en t l os s. ta bl e 4 re vi ew o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on si nu s a ug m en ta tio n au th or s ( ye ar ) st ud y de si gn si te (p at ie nt ) pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt ta tu llo e t a l.7 3 rc t pa ra lle l 60 , 7 2, 2 40 30 00 rp m fo r 1 0 m in t: b io -o ss ® + lpr f tw ost ag e (la te ra l) c: b io -o ss ® ra di og ra ph ic a nd h a 10 6, 1 20 , 1 50 d ay s t: a fte r 1 06 d ay s: tra be cu la r b on e 22 .7 9% , o st eo id ti ss ue 7. 01 % , m ed ul la ry sp ac es 7 0. 2% ; a fte r 1 20 d ay s: tra be cu la r bo ne 2 6. 15 % , o st eo id ti ss ue 3 .8 4% , m ed ul la ry sp ac es 70 .1 % ; a fte r 1 50 d ay s: tra be cu la r b on e 37 .0 6% , o st eo id tis su e 3. 53 % , m ed ul la ry sp ac es 6 1. 41 . c : a fte r 1 06 d ay s: tra be cu la r b on e 26 .4 4% , o st eo id ti ss ue 5 .1 2% , m ed ul la ry sp ac es 6 8. 44 % ; a fte r 1 20 d ay s: tra be cu la r b on e 28 .7 % , os te oi d tis su e 3. 12 % , m ed ul la ry sp ac es 6 8. 18 % ; a fte r 1 50 da ys : t ra be cu la r b on e 38 .9 7% , o st eo id ti ss ue 2 .8 8% , m ed ul la ry sp ac es 5 8. 15 % . i sq v al ue s: af te r 1 06 d ay s: 37 .2 ± 4 .2 ; af te r 1 20 d ay s: 36 .8 ± 6 .1 ; a fte r 1 50 d ay s: 39 .1 ± 9 .0 . t he d iffe re nc es b et w ee n gr ou ps w er e no t s ta tis tic al ly si gn ifi ca nt . lpr f re du ce d th e he al in g tim e. im pl an t s r: 1 00 % . rc t: ra nd om iz ed c lin ic al tr ia l, c t: c lin ic al tr ia l, h a : h is to m or ph om et ric a na ly si s, a bg : a ut ol og ou s bo ne g ra ft, c t sc an : c om pu te d to m og ra ph ic s ca n, r cm : r es or ba bl e co lla ge n m em br an e, g sh /o sh : t he g ra fte d an d th e or ig in al s in us h ei gh t r at io , n bf : n ew b on e fo rm at io n, r g m : r es id ua l g ra ft m at er ia l. 181j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects ta bl e 6 re vi ew o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on fu rc at io n de fe ct s au th or s (y ea r) st ud y de si gn sa m pl e si ze , si te pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l sh ar m a et al .80 rc t 30 , f ur ca tio n in vo lv em en t (d eg re e ii) o f m an di bu la r m ol ar s 30 00 rp m fo r 1 0 m in o fd + p rf + 1 % m et fo rm in g el o fd + p rf cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6 m on th s cl in ic al p ar am et er s (p d re du ct io n, r va l an d rh a l ga in ) sh ow ed s ta tis tic al ly s ig ni fic an t i m pr ov em en t a t o fd + p rf + 1 % m et fo rm in g ro up s w he n co m pa re d w ith co nt ro l g ro up . as im ud di n et a l.8 4 rc t 22 , f ur ca tio n in vo lv em en t (d eg re e ii) o f m an di bu la r m ol ar s 30 00 rp m fo r 1 0 m in pr f a llo gr af t + h ea lig ui de c ol la ge n m em br an e cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s th er e w er e no s ig ni fic an t d iff er en ce s in m os t o f t he ev al ua te d pa ra m et er s. th e rv ca l at b as el in e w as 1 2. 03 ± 1. 04 a nd a t n in e m on th s af te r s ur ge ry w as 8 .4 2 ± 0 .9 7 in gr ou p a , t he ra di og ra ph ic li ne ar b on e fil l ( rv g bf ) a t b as elin e w as 1 3. 0 ± 0 .8 9 in g ro up a a nd a t 9 m on th s af te r su rg er y w as 9 .9 1 ± 0 .5 4. in te rg ro up c om pa ris on s ho w ed st at is tic al ly s ig ni fic an t d iff er en ce o f r vc a l an d rv g bf in re la tio n to p rf g ro up (g ro up a ) w he n co m pa re d w ith al lo gr af t + g tr g ro up (g ro up b ), 9 m on th s po st -s ur ge ry . lo hi e t a l.8 1 rc t 20 , f ur ca tio n in vo lv em en t (d eg re e ii) o f m an di bu la r m ol ar s 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in pr f + b io ac tiv e ce ra m ic c om po si te gr an ul es bi oa ct iv e ce ra m ic co m po si te g ra nul es cl in ic al a nd ra di ogr ap hi c ev al ua tio n 6 m on th s si gn ifi ca nt im pr ov em en t w as o bs er ve d in th e te st g ro up co m pa re d w ith th e co nt ro l g ro up in a ll th e m ea su re d pa ra m et er s (t he m ea n pp d re du ct io n of 3 .3 8 ± 1 .0 6 m m an d m ea n cl in ic al a tt ac hm en t g ai n of 3 .0 0 ± 0 .9 3 m m , m ea n re du ct io n in v d f 1. 38 ± 0 .5 2 m m a nd in b ph 2 .0 0 ± 0 .7 6 m m in c om pa re s w ith a m ea n re du ct io n in v d f of 0. 60 ± 0 .7 0 m m a nd b ph o f 1 .1 0 ± 0 .8 8 m m in c on tr ol gr ou p, m ea n pe rc en t h or iz on ta l a nd v er tic al d ef ec t fi ll in th e te st g ro up w as 4 7. 06 % a nd 4 0. 68 % w he n co m pa re d w ith 2 4. 44 % a nd 2 0% in c on tr ol g ro up , m ea n in cr ea se in ra di og ra ph ic b on e de ns ity a fte r 6 m on th s fo llo w -u p w as 20 .0 8 ± 1 9. 53 g ra y le ve ls in te st g ro up a nd in th e co nt ro l gr ou p 5. 26 ± 5 .9 4 gr ay le ve ls ). ka no riy a et al .83 rc t 32 , f ur ca tio n in vo lv em en t (d eg re e ii) o f m an di bu la r m ol ar s 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in ac ce ss th er ap y w ith p rf a nd 1 % a ln (1 ) ac ce ss th er ap y al on e (2 ), ac ce ss th er ap y w ith p rf (3 ) cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s g ro up 3 s ite s sh ow ed a s ig ni fic an tly g re at er p er ce nt ag e of ra di og ra ph ic d ef ec t fi ll (5 6. 01 ± 2 .6 4% ) w he n co m pa re d w ith g ro up 2 (4 9. 43 ± 3 .7 0% ) a nd g ro up 1 (1 0. 25 ± 3. 66 % ) a t 9 m on th s. pr ad ee p et al .82 rc t 10 5, f ur ca tio n in vo lv em en t (d eg re e ii) o f m an di bu la r m ol ar s 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in 1. 2 m g rs v ge l + p rf + h a w ith o fd (1 ) o fd + p la ce bo g el (2 ), pr f + h a w ith o fd (3 ) cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s m ea n pd re du ct io n w as g re at er in g ro up 2 (3 .6 8 ± 1 .0 7 m m ) a nd g ro up 3 (4 .6 2 ± 1 .0 3 m m ) t ha n gr ou p 1 (2 .1 1 ± 1. 25 m m ), an d m ea n rv ca l an d rh ca l ga in w er e gr ea te r in g ro up 2 (3 .3 1 ± 0 .5 2 an d 2. 97 ± 0 .5 6 m m , r es pe ct iv el y) an d gr ou p 3 (4 .1 7 ± 0 .7 0 an d 4. 05 ± 0 .7 6 m m ) c om pa re d w ith g ro up 1 (1 .8 2 ± 0 .7 8 an d 1. 62 ± 0 .6 4 m m ). a s ig ni fic an tly g re at er p er ce nt ag e of m ea n bo ne fi ll w as fo un d in g ro up 2 (5 4. 69 ± 1 .9 3% ) a nd g ro up 3 (6 1. 94 ± 3 .5 4% ) co m pa re d w ith g ro up 1 (1 0. 09 % ± 4 .2 8% ). 182 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. ta bl e 6 re vi ew o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on fu rc at io n de fe ct s— co nt in ue d au th or s (y ea r) st ud y de si gn sa m pl e si ze , si te pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l ba ja j e t a l.7 8 rc t 72 , b uc ca l f ur ca tio n (d eg re e ii) of m an di bu la r m ol ar s a bo ut 4 00 g fo r 10 m in lpr f + o fd pr p + o fd , o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s re la tiv e ve rt ic al c lin ic al a tt ac hm en t l ev el g ai n w as a ls o gr ea te r i n pr f (2 .8 7 ± 0 .8 5 m m ) a nd p rp (2 .7 1 ± 1 .0 4 m m ) s ite s as c om pa re d w ith c on tr ol s ite (1 .3 7 ± 0 .5 8 m m ). sh ar m a et al .22 rc t 36 , b uc ca l f ur ca tio n (d eg re e ii) o f m an di bu la r m ol ar s 30 00 rp m (a bo ut 40 0 g) fo r 1 0 m in lpr f + o fd o fd cl in ic al a nd ra di ogr ap hi c ev al ua tio n 9 m on th s re la tiv e ve rt ic al c lin ic al a tt ac hm en t l ev el g ai n w as g re at er in p rf (2 .8 7 ± 0 .8 5 m m ) a nd p rp (2 .7 1 ± 1 .0 4 m m ) s ite s as c om pa re d w ith c on tr ol s ite (1 .3 7 ± 0 .5 8 m m ). rc t: ra nd om iz ed c lin ic al tr ia l, d fd ba : d em in er al iz ed fr ee ze -d rie d bo ne a llo gr af t, o fd : o pe n fla p de br id em en t, rs v: ro su va st at in , a ln : a le nd ro na te , h a : h yd ro xy ap at ite , β -t cp : b et atr i-c al ci um p ho sp ha te , p d : p ro bi ng de pt h, r va l: re la tiv e ve rt ic al a tt ac hm en t l ev el , r h a l: re la tiv e ho riz on ta l a tt ac hm en t l ev el . ta bl e 7. r ev ie w o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on g in gi va l r ec es si on au th or s (y ea r) st ud y de si gn si te (p at ie nt ) pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt p r f g ro u p s c o n tr o l ra m ire dd y et a l.9 9 rc t 20 p at ie nt s w ith m ill er c la ss i or ii (7 8 de fe ct s) 27 00 rp m fo r 1 2 m in pr f + c a f ca f + r m g ic cl in ic al e va lu at io n 3 an d 6 m on th s bo th th e gr ou ps s ho w ed o pt im al ro ot co ve ra ge . i n ca se o f k tt , p rf g ro up s ho w s si gn ifi ca nt a dv an ta ge s (2 .9 5 ± 0 .1 8 m m v s. 2. 19 ± 0 .1 2 m m ), in c as e of d en tin s en si tiv ity , rm g ic w as s ig ni fic an tly lo w er (8 3% o f s ite s w ith ou t s en si tiv ity v s. 46 % ). cu lh ao gl u et al .90 rc t 63 p at ie nt s w ith m ill er c la ss i 27 00 rp m fo r 1 2 m in tw o la ye rs p rf + ca f (i) a nd fo ur la ye rs p rf + c a f (ii ) co nn ec tiv e tis su e gr af t ( c tg ) + c a f cl in ic al e va lu at io n 1, 3 , a nd 6 m on th s in c as e of k tt , c on tr ol g ro up s ho w s si gn ifi ca nt a dv an ta ge s (2 .3 5 ± 1 .0 2 m m v s. 1. 86 ± 0. 49 m m fo r t es t i a nd 1 .7 8 ± 0 .4 2 m m fo r te st ii ). in c as e of ro ot c ov er ag e sc or es , t es t i gr ou p sc or e w as s ig ni fic an tly lo w er (5 6. 34 ± 14 .5 1 vs . 8 0. 13 ± 1 8. 93 fo r c on tr ol a nd 6 9. 65 ± 1 5. 28 fo r t es t i i). (c on tin ue d) 183j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects ta bl e 7. r ev ie w o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on g in gi va l r ec es si on au th or s (y ea r) st ud y de si gn si te (p at ie nt ) pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l m uf ti et a l.9 3 rc t 32 p at ie nt s w ith m ill er c la ss i 30 00 rp m fo r 1 0 m in ca f + p rf ca f + c tg cl in ic al e va lu at io n 6 m on th s in th e te st g ro up , s ig ni fic an t i m pr ov em en t w as s ee n in a ll pa ra m et er s fro m b as el in e to 6 m on th s (u nl ik e co nt ro l). m or eo ve r, in s om e pa ra m et er s (k tt , c a l) , p rf g ro up s ho w s si gni fic an t a dv an ta ge s. (k tt m ea n ra nk : c a f + pr f: 1 3. 34 , c a f + c tg : 1 9. 66 , c a l m ea n ra nk : ca f + p rf : 1 0. 28 , c a f + c tg : 2 2. 72 ). ag ar w al e t al .98 sp lit m ou th rc t 30 p at ie nt s w ith m ill er c la ss i or ii n ot m en tio ne d ca f + p rf ca f, ca f + a m ni ot ic m em br an e cl in ic al e va lu at io n 3 an d 6 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 5 6% fo r t he c a f + p rf g ro up , 3 6% fo r t he c a f + am ni ot ic m em br an e gr ou p an d 33 % fo r t he ca f gr ou p (p < 0 .0 5) . ke ce li et a l.9 2 sp lit m ou th rc t 40 p at ie nt s w ith m ill er c la ss i or ii n ot m en tio ne d ca f+ c on ne ct iv e tis su e gr af t + p rf ca f + c tg cl in ic al e va lu at io n 3 an d 6 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 7 9. 9% fo r t he c on tr ol g ro up a nd 8 9. 6% fo r t he te st gr ou p (p < 0 .0 5) . tu na lio ta e t al .94 sp lit m ou th rc t 22 p at ie nt s w ith m ill er c la ss i or ii 27 00 rp m fo r 1 2 m in ca f + p rf ca f + c tg cl in ic al e va lu at io n 12 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 7 7. 4% fo r t he c on tr ol g ro up a nd 7 6. 6% fo r t he te st gr ou p (n o si gn ifi ca nt d iff er en ce s) . th am ar ai se lva n et a l.8 8 sp lit m ou th rc t 20 p at ie nt s w ith m ill er c la ss i or ii 30 00 rp m fo r 1 0 m in ca f + p rf ca f cl in ic al e va lu at io n 3 an d 6 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 6 5% fo r t he c on tr ol g ro up a nd 7 4. 2% fo r t he te st gr ou p (n o si gn ifi ca nt d iff er en ce s) . g up ta e t a l.8 6 sp lit m ou th rc t 26 p at ie nt s w ith m ill er c la ss i or ii 27 00 rp m fo r 1 2 m in ca f + p rf ca f cl in ic al e va lu at io n 3 an d 6 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 8 6. 6% fo r t he c on tr ol g ro up a nd 9 1% fo r t he te st gr ou p (n o si gn ifi ca nt d iff er en ce s) . bo zk ur t d oğ an e t al .85 sp lit m ou th rc t 20 p at ie nt s w ith m ill er c la ss i or ii ac ce le ra tio n fo r 3 0 s, 27 00 rp m fo r 2 m in , 2 40 0 rp m fo r 4 m in , 2 70 0 rp m fo r 4 m in , 30 00 rp m fo r 7 m in , a nd de ce le ra tio n fo r 3 6 s ca f + p rf ca f cl in ic al e va lu at io n 6 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 8 2. 1% fo r t he c on tr ol g ro up a nd 8 6. 7% fo r t he te st gr ou p (n o si gn ifi ca nt d iff er en ce s) . ra ja ra m e t al .89 sp lit m ou th rc t 20 p at ie nt s w ith m ill er c la ss ii 27 00 rp m fo r 1 2 m in pr f + la te ra l s lid in g br id ge fl ap la te ra l s lid in g br id ge fl ap cl in ic al e va lu at io n 12 a nd 2 4 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 8 0% fo r t he c on tr ol g ro up a nd 7 8. 8% fo r t he te st gr ou p (n o si gn ifi ca nt d iff er en ce s) . er en e t a l.9 5 sp lit m ou th rc t 22 p at ie nt s w ith m ill er c la ss i or ii 40 0 g fo r 1 2 m in pr f + c a f ca f + s c tg cl in ic al e va lu at io n 6 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 9 4. 2% fo r t he c on tr ol g ro up a nd 9 2. 7% fo r t he te st gr ou p (n o si gn ifi ca nt d iff er en ce s) . pa dm a et al .87 sp lit m ou th rc t 15 p at ie nt s w ith m ill er c la ss i or ii 30 00 rp m fo r 1 0 m in ca f + p rf ca f cl in ic al e va lu at io n 1, 3 , a nd 6 m on th s fu ll ro ot c ov er ag e ob ta in ed in s tu dy g ro up s (1 00 % ). th e ro ot c ov er ag e pe rc en ta ge in th e st ud y gr ou p w as s ig ni fic an tly h ig he r t ha n th e co nt ro l g ro up (6 8/ 4% ) ( p < 0 .0 00 1) . 184 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. ta bl e 7. r ev ie w o f p ub lis he d cl in ic al tr ia ls e va lu at ed th e ap pl ic at io n of p rf co nc en tr at e on g in gi va l r ec es si on — co nt in ue d au th or s (y ea r) st ud y de si gn si te (p at ie nt ) pr f pr ep ar at io n pr ot oc ol in te rv en tio n va ria bl e ou tc om e fo llo w -u p pe rio d re su lt pr f gr ou ps co nt ro l ja nk ov ic e t al .97 sp lit m ou th rc t 20 p at ie nt s w ith m ill er c la ss i or ii 30 00 rp m (a bo ut 4 00 g ) f or 10 m in ca f + p rf ca f + e m d cl in ic al e va lu at io n 12 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 7 0. 5% fo r t he c on tr ol g ro up a nd 7 2. 1% fo r t he te st gr ou p (n o si gn ifi ca nt d iff er en ce s) . a ro ca e t a l.2 1 sp lit m ou th rc t 20 p at ie nt s w ith m ill er c la ss i or ii 30 00 rp m fo r 1 0 m in pr f + m ca f m ca f cl in ic al e va lu at io n 1, 3 , a nd 6 m on th s th e pe rc en ta ge o f r oo t c ov er ag e w as 9 1. 5% fo r t he c on tr ol g ro up a nd 8 0. 7% fo r t he te st gr ou p (p < 0 .0 04 ). rc t: ra nd om iz ed c lin ic al tr ia l, ca f: c or on al ly a dv an ce d fla p, m ca f: m od ifi ed c or on al ly a dv an ce d fla p, s c tg : s ub ep ith el ia l c on ne ct iv e tis su e gr af t, c tg : c on ne ct iv e tis su e gr af t, rm g ic : r es in m od ifi ed g la ss in om er ce m en t, em d : e na m el m at rix d er iv at iv e, k tw : k er at in iz ed ti ss ue w id th , k tt : k er at in iz ed ti ss ue th ic kn es s, ca l: c lin ic al a tt ac hm en t l os s. reported advantages of prf in some measured parameters (root coverage percentage and cal, respectively). discussion choukroun’s6 prf is a second-generation platelet concentrate that contains a vast amount of cytokines and growth factors within an autologous dense fibrin matrix rich in platelets. prf is used as a surgical additive to accelerate healing and regeneration processes.100 due to its advantages, prf have recently gained much attention among researchers as a safe approach in various procedures solely or in combination with other treatment approaches including the regeneration of soft and hard tissue defects of periodontium,21,38,101 maxillary sinus-lift procedures,102,103 wound healing104,105 and facial reconstruction plastic surgery.19 the present systematic review aimed to assess the current evidenced in regards to the clinical benefit of prf concentrates in the regenerative procedures of soft and hard tissue intra-oral defects. application of prf was performed either solely or in combination with other regenerative materials in intra-oral defects. the evaluated clinical trials were categorized according to the types of defects including intrabony defects, sockets preservation, sinus floor augmentation, endo-periodontal defects, furcation defects, gingival recessions. in each included study, the type of used concentrate, study groups, defect location, sample size, follow-up periods, evaluation method, and treatment outcome were reported. as mentioned before, due to the application of various types of prf concentrates on different kinds of defects, conducting the meta-analyze was not possible in the current systematic review. biochemical analysis of the prf concentrate structure showed that it consists of platelets, leukocyte, and cytokines; and circulating hematopoietic precursor cells within a fibrin 3d network polymerized in a tetra molecular structure.106 there are various components and growth factors in prf including fibrin, fibronectin, pdgf, and tgf-β that all are important in the tissue healing and regeneration process.106,107 it is also found that using prf can lead to an increase in leukocyte degranulation and cytokine release of pro-inflammatory mediators including il-1β and il-6 as well as anti-inflammatory cytokines, such as il-4. furthermore, the dense fibrin scaffold of prf leads to a slow release of cytokines and glycoproteins (such as thrombospondin-1) in the first 7 days in the site.5,9 with all these features, it has been shown in the literature that prf can play an important role through its growth factors in modulating the healing and regeneration procedures by inducing the proliferation and recruitment of endothelial cells, gingival fibroblast, chondrocytes, and osteoblasts.108,109 however, recent studies addressed a new aspect in evaluating the effect of prf on different regenerative approaches. in this case, the influence of the applied relative centrifugal force (rcf) during the centrifugation of prf on its composition and bioactivity was investigated. interestingly, different in vitro and in vivo preclinical studies proved that the value of the applied rcf has an enormous influence on prf bioactivity.110–113 therefore, a high rcf was shown to produce a prf matrix with significantly lower number of platelets and leukocytes and a significantly lower concentrations of different growth factors. not only the composition and bioactivity were influenced by the applied rcf, but also its function. recent preclinical studies have shown that prf that was prepared 185j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects with low rcf induced significantly higher rate of vascularization in vitro and in vivo compared with a prf matrix that was prepared using a high rcf.114,115 based on these studies, the low speed centrifugation concept (lscc) was introduced to standardize the centrifugation protocols in the preparation of blood concentrates.110 application of autologous prf is safe and low-cost methods when compared with using growth factors concentrations in the recombinant form.7 in respect to the intrabony defects in consequence of chronic periodontitis, studies showed that using prf lead to favourable outcomes in case of ppd reductions and cal gains when compared with the flap only approach.24,25,30,32,34,36 however, when prf concentrates compared the other novel treatment approaches additional benefits were observed. furthermore, application of prf in combination with dfdba lead to more ppd reductions and cal gains. also, in the case of aggressive periodontitis, only one study was found suggesting that using prf cause better clinical outcomes.35 the present study confirmed the outcomes of miron et al.’s7 systematic review and ghanaati et al.116 that reported the beneficial effect of prf in the treatment of intrabony defects. in case of using prf concentrates on accelerating the ridge preservation and dimensional changes following tooth extraction, the results of clinical trials were controversial. although, it was shown that using prf besides a bone graft material can result in a better clinical outcome in regards to the reduction of ridge width.67 the only included clinical trial that applied a-prf in the present systematic review was clark et al.’s68 study that showed using a-prf besides fdba has no significant advantages when compared with a-prf or fdba alone. despite the limited number of clinical trials concerning sinus floor augmentation, when supplemental l-prf used in addition to bone graft materials, no additional benefits were observed. however, it has been reported that using l-prf may reduce the healing time70 and can cause less change in the bone length/the implant length ratio.75 regarding endo-periodontal defects, both included studies showed that the use of l-prf acts as an ideal material by accelerating the bone filling.76,77 however, since only two studies evaluated the outcomes of prf concentrates on endo-periodontal lesions, further research is required to support the advantage of prf application. platelet-rich fibrin concentrates showed a significant effect on the improvement of clinical and radiographic parameters when used as an adjunctive treatment or sole treatment for furcation involvement of mandibular molars.22,78–84 however, when prf was used as alternative material compared with β-tcp, it showed a significant advantage in ppd reducitions, cal gains and bone filling.79 in case of the root coverage of miller class i and ii defects, significant enhancement of root coverage procedures was not observed when prf concentrates were applied. similar to our study, in a systematic review has been reported that platelet concentrates do not lead to improvement in soft tissue root coverage of gingival recessions.13 however, it was found that prf had similar advantages in the treatment of gingival recessions when compared with ctg. in one study, it has been reported that using prf instead of ctg can lead to significant improvement in higher keratinized tissue and cal gain.93 application of ctg approach showed high patient morbidity and the reaching a desirable treatment outcome is depends on the technical ability of the clinician.92,93,96 for these reasons, prf concentrates may be applied for similar indication as the application of ctg, when autologous transplantation is undesired or too complex to be performed. other than evaluated defects, prf concentrates have been utilized with the aim of bone regeneration in various oral and maxillofacial defects including reconstruction of unilateral alveolar cleft,117 ridge augmentation,118 cystic bone defect.119,120 in addition to the regenerative use of prf, hoaglin et al.121 reported that prf might help wound healing process of extraction sites by the prevention of localized osteitis. antibacterial effect of prf is mostly because of the recruitment of white blood cells and macrophages to the site.7 all together the present systematic review evaluated the recent advances in the clinical application of prf in different defects morphologies. at this point, it is noteworthy to outline that many different centrifugation protocols were used throughout the studies showing different results, that were sometimes controversial. additionally, some studies did not report the specific centrifugation setting they used in their studies, which makes the evaluation of such studies even more difficult. however, this aspect is currently a topic of discussion in the literature. some researchers and clinicians are keen to provide guidelines on the report of clinical studies when using blood concentrate.122 the authors strongly encourage to use these guidelines to at least define the type of prf used in the studies. additionally, attempt to standardize the preparation protocols according to the previously introduced lscc are ongoing. this concept was thoroughly investigated and proved in preclinical studies.113–115 however, randomized controlled clinical studies following this standardization concept are still needed to eventually prove its benefit for the clinical application. conclusion in conclusion, the final treatment outcome utilizing prf concentrates depends on the treatment strategies and the type of the defect. in case of intrabony defects and furcation involvement, the studies clearly showed that the use of prf concentrates alone or as a supplement could be helpful. in case of ridge preservation, sinus floor augmentation, and endo-periodontal lesions due to a limited number of evidence or controversial results, further clinical trials are required. future clinical studies with histological evaluation are needed. additionally, the clinical applciation of prf requires more standardization in the preparation protocols of blood concentrated to provide reproducable clinical sucsess. conflicts of interest the authors report no other conflicts of interest related to this study. ■ 186 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. references 1. dohan dm, choukroun j, diss a, dohan sl, dohan aj, mouhyi j, et al. platelet-rich fibrin (prf): a second-generation platelet concentrate. part i: technological concepts and evolution. oral surg oral med oral pathol oral radiol endod. 2006;101:e37–e44. 2. behnia h, khojasteh a, kiani mt, khoshzaban a, mashhadi abbas f, bashtar m, et al. bone regeneration with a combination of nanocrystalline hydroxyapatite silica gel, platelet-rich growth factor, and mesenchymal stem cells: a histologic study in rabbit calvaria. oral surg oral med oral pathol oral radiol. 2013;115:e7–e15. 3. dohan ehrenfest dm, rasmusson l, albrektsson t. classification of platelet concentrates: from pure platelet-rich plasma (p-prp) to leucocyteand platelet-rich fibrin (l-prf). trends biotechnol. 2009;27:158–167. 4. kumar rv, shubhashini n. platelet rich fibrin: a new paradigm in periodontal regeneration. cell tissue bank. 2013;14:453–463. 5. dohan ehrenfest dm, de peppo gm, doglioli p, sammartino g. slow release of growth factors and thrombospondin-1 in choukroun’s platelet-rich fibrin (prf): a gold standard to achieve for all surgical platelet concentrates technologies. growth factors 2009;27:63–69. 6. dohan dm, choukroun j, diss a, dohan sl, dohan aj, mouhyi j, et al. platelet-rich fibrin (prf): a second-generation platelet concentrate. part ii: platelet-related biologic features. oral surg oral med oral pathol oral radiol endod. 2006;101:e45–e50. 7. miron rj, zucchelli g, pikos ma, salama m, lee s, guillemette v, et al. use of platelet-rich fibrin in regenerative dentistry: a systematic review. clin oral investig. 2017;21:1913–1927. 8. kang yh, jeon sh, park jy, chung jh, choung yh, choung hw, et al. platelet-rich fibrin is a bioscaffold and reservoir of growth factors for tissue regeneration. tissue eng part a. 2011;17:349–359. 9. dohan ehrenfest dm, diss a, odin g, doglioli p, hippolyte mp, charrier jb. in vitro effects of choukroun’s prf (platelet-rich fibrin) on human gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts in primary cultures. oral surg oral med oral pathol oral radiol endod. 2009;108:341–352. 10. he l, lin y, hu x, zhang y, wu h. a comparative study of platelet-rich fibrin (prf) and platelet-rich plasma (prp) on the effect of proliferation and differentiation of rat osteoblasts in vitro. oral surg oral med oral pathol oral radiol endod. 2009;108:707–713. 11. vahabi s, yadegari z, mohammad-rahimi h. comparison of the effect of activated or non-activated prp in various concentrations on osteoblast and fibroblast cell line proliferation. cell tissue bank. 2017;18:347–353. 12. öncü e, alaaddinoğlu ee. the effect of platelet-rich fibrin on implant stability. int j oral maxillofac implants. 2015;30:578–582. 13. del fabbro m, bortolin m, taschieri s, weinstein r. is platelet concentrate advantageous for the surgical treatment of periodontal diseases? a systematic review and meta-analysis. j periodontol. 2011;82:1100–1111. 14. gruber r, karreth f, frommlet f, fischer mb, watzek g. platelets are mitogenic for periosteum-derived cells. j orthop res. 2003;21:941–948. 15. metzler p, von wilmowsky c, zimmermann r, wiltfang j, schlegel ka. the effect of current used bone substitution materials and platelet-rich plasma on periosteal cells by ectopic site implantation: an in-vivo pilot study. j craniomaxillofac surg. 2012;40:409–415. 16. intini g. the use of platelet-rich plasma in bone reconstruction therapy. biomaterials 2009;30:4956–4966. 17. cheung ws, griffin tj. a comparative study of root coverage with connective tissue and platelet concentrate grafts: 8-month results. j periodontol. 2004;75:1678–1687. 18. el-sharkawy h, kantarci a, deady j, hasturk h, liu h, alshahat m, et al. platelet-rich plasma: growth factors and proand anti-inflammatory properties. j periodontol. 2007;78:661–669. 19. charrier jb, monteil jp, albert s, collon s, bobin s, dohan ehrenfest dm. relevance of choukroun’s platelet-rich fibrin (prf) and smas flap in primary reconstruction after superficial or subtotal parotidectomy in patients with focal pleiomorphic adenoma: a new technique. rev laryngol otol rhinol (bord). 2008;129:313–318. 20. zhang y, tangl s, huber cd, lin y, qiu l, rausch-fan x. effects of choukroun’s platelet-rich fibrin on bone regeneration in combination with deproteinized bovine bone mineral in maxillary sinus augmentation: a histological and histomorphometric study. j craniomaxillofac surg. 2012;40:321–328. 21. aroca s, keglevich t, barbieri b, gera i, etienne d. clinical evaluation of a modified coronally advanced flap alone or in combination with a plateletrich fibrin membrane for the treatment of adjacent multiple gingival recessions: a 6-month study. j periodontol. 2009;80:244–252. 22. sharma a, pradeep ar. autologous platelet-rich fibrin in the treatment of mandibular degree ii furcation defects: a randomized clinical trial. j periodontol. 2011;82:1396–1403. 23. moher d, liberati a, tetzlaff j, altman dg, prisma group. preferred reporting items for systematic reviews and meta-analyses: the prisma statement. plos med. 2009;6:e1000097. 24. ajwani h, shetty s, gopalakrishnan d, kathariya r, kulloli a, dolas rs, et al. comparative evaluation of platelet-rich fibrin biomaterial and open flap debridement in the treatment of two and three wall intrabony defects. j int oral health. 2015;7:32–37. 25. bajaj p, agarwal e, rao ns, naik sb, pradeep ar, kalra n, et al. autologous platelet-rich fibrin in the treatment of 3-wall intrabony defects in aggressive periodontitis: a randomized controlled clinical trial. j periodontol. 2017;88:1186–1191. 26. chadwick jk, mills mp, mealey bl. clinical and radiographic evaluation of demineralized freeze-dried bone allograft versus platelet-rich fibrin for the treatment of periodontal intrabony defects in humans. j periodontol. 2016;87:1253–1260. 27. galav s, chandrashekar kt, mishra r, tripathi v, agarwal r, galav a. comparative evaluation of platelet-rich fibrin and autogenous bone graft for the treatment of infrabony defects in chronic periodontitis: clinical, radiological, and surgical reentry. indian j dent res. 2016;27:502–507. 28. gupta sj, jhingran r, gupta v, bains vk, madan r, rizvi i. efficacy of plateletrich fibrin vs. enamel matrix derivative in the treatment of periodontal intrabony defects: a clinical and cone beam computed tomography study. j int acad periodontol. 2014;16:86–96. 29. mathur a, bains vk, gupta v, jhingran r, singh gp. evaluation of intrabony defects treated with platelet-rich fibrin or autogenous bone graft: a comparative analysis. eur j dent. 2015;9:100–108. 30. patel gk, gaekwad ss, gujjari sk, s c vk. platelet-rich fibrin in regeneration of intrabony defects: a randomized controlled trial. j periodontol. 2017;88:1192–1199. 31. pradeep ar, rao ns, agarwal e, bajaj p, kumari m, naik sb. comparative evaluation of autologous platelet-rich fibrin and platelet-rich plasma in the treatment of 3-wall intrabony defects in chronic periodontitis: a randomized controlled clinical trial. j periodontol. 2012;83:1499–1507. 32. rosamma joseph v, raghunath a, sharma n. clinical effectiveness of autologous platelet rich fibrin in the management of infrabony periodontal defects. singapore dent j. 2012;33:5–12. 33. shah m, patel j, dave d, shah s. comparative evaluation of platelet-rich fibrin with demineralized freeze-dried bone allograft in periodontal infrabony defects: a randomized controlled clinical study. j indian soc periodontol. 2015;19:56–60. 34. sharma a, pradeep ar. treatment of 3-wall intrabony defects in patients with chronic periodontitis with autologous platelet-rich fibrin: a randomized controlled clinical trial. j periodontol. 2011;82:1705–1712. 35. thorat m, baghele on, s rp. adjunctive effect of autologous plateletrich fibrin in the treatment of intrabony defects in localized aggressive periodontitis patients: a randomized controlled split-mouth clinical trial. int j periodontics restorative dent. 2017;37:e302–e309. 36. thorat m, pradeep ar, pallavi b. clinical effect of autologous platelet-rich fibrin in the treatment of intra-bony defects: a controlled clinical trial. j clin periodontol. 2011;38:925–932. 37. yajamanya sr, chatterjee a, hussain a, coutinho a, das s, subbaiah s. bioactive glass versus autologous platelet-rich fibrin for treating periodontal intrabony defects: a comparative clinical study. j indian soc periodontol. 2017;21:32–36. 38. agarwal a, gupta nd, jain a. platelet rich fibrin combined with decalcified freeze-dried bone allograft for the treatment of human intrabony periodontal defects: a randomized split mouth clinical trail. acta odontol scand. 2016;74:36–43. 39. elgendy ea, abo shady te. clinical and radiographic evaluation of nanocrystalline hydroxyapatite with or without platelet-rich fibrin membrane in the treatment of periodontal intrabony defects. j indian soc periodontol. 2015;19:61–65. 40. pradeep ar, nagpal k, karvekar s, patnaik k, naik sb, guruprasad cn. platelet-rich fibrin with 1% metformin for the treatment of intrabony defects in chronic periodontitis: a randomized controlled clinical trial. j periodontol. 2015;86:729–737. 41. lekovic v, milinkovic i, aleksic z, jankovic s, stankovic p, kenney eb, et al. platelet-rich fibrin and bovine porous bone mineral vs. platelet-rich fibrin in the treatment of intrabony periodontal defects. j periodontal res. 2012;47:409–417. 187j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 muna al-shuhayeb et al. review application of platelet rich fibrin in the regeneration of intra oral defects 42. chandradas nd, ravindra s, rangaraju vm, jain s, dasappa s. efficacy of platelet rich fibrin in the treatment of human intrabony defects with or without bone graft: a randomized controlled trial. j int soc prev community dent. 2016;6:s153–s159. 43. pradeep ar, garg v, kanoriya d, singhal s. platelet-rich fibrin with 1.2% rosuvastatin for treatment of intrabony defects in chronic periodontitis: a randomized controlled clinical trial. j periodontol. 2016;87:1468–1473. 44. kanoriya d, pradeep ar, singhal s, garg v, guruprasad cn. synergistic approach using platelet-rich fibrin and 1% alendronate for intrabony defect treatment in chronic periodontitis: a randomized clinical trial. j periodontol. 2016;87:1427–1435. 45. pradeep ar, bajaj p, rao ns, agarwal e, naik sb. platelet-rich fibrin combined with a porous hydroxyapatite graft for the treatment of 3-wall intrabony defects in chronic periodontitis: a randomized controlled clinical trial. j periodontol. 2017;88:1288–1296. 46. naqvi a, gopalakrishnan d, bhasin mt, sharma n, haider k, martande s. comparative evaluation of bioactive glass putty and platelet rich fibrin in the treatment of human periodontal intrabony defects: a randomized control trial. j clin diagn res. 2017;11:zc09–zc13. 47. yasaswini ms, prabhakara rao kv, tanuja p, motakatla nr. comparison of marginal periosteal pedicle graft and bioactive glass with plateletrich fibrin and bioactive glass in the treatment of intrabony defects a clinicoradiographic study. j pharm bioallied sci. 2017;9:s96–s102. 48. chatterjee a, pradeep ar, garg v, yajamanya s, ali mm, priya vs. treatment of periodontal intrabony defects using autologous platelet-rich fibrin and titanium platelet-rich fibrin: a randomized, clinical, comparative study. j investig clin dent. 2017;8. 49. martande ss, kumari m, pradeep ar, singh sp, suke dk, guruprasad cn. platelet-rich fibrin combined with 1.2% atorvastatin for treatment of intrabony defects in chronic periodontitis: a randomized controlled clinical trial. j periodontol. 2016;87:1039–1046. 50. aydemir turkal h, demirer s, dolgun a, keceli hg. evaluation of the adjunctive effect of platelet-rich fibrin to enamel matrix derivative in the treatment of intrabony defects. six-month results of a randomized, splitmouth, controlled clinical study. j clin periodontol. 2016;43:955–964. 51. panda s, sankari m, satpathy a, jayakumar d, mozzati m, mortellaro c, et al. adjunctive effect of autologus platelet-rich fibrin to barrier membrane in the treatment of periodontal intrabony defects. the j craniofac surg. 2016;27:691–696. 52. bansal c, bharti v. evaluation of efficacy of autologous platelet-rich fibrin with demineralized-freeze dried bone allograft in the treatment of periodontal intrabony defects. j indian soc periodontol. 2013;17:361–366. 53. alzahrani aa, murriky a, shafik s. influence of platelet rich fibrin on postextraction socket healing: a clinical and radiographic study. saudi dent j. 2017;29:149–155. 54. baslarli o, tumer c, ugur o, vatankulu b. evaluation of osteoblastic activity in extraction sockets treated with platelet-rich fibrin. med oral patol oral cir bucal. 2015;20:e111–e116. 55. das s, jhingran r, bains vk, madan r, srivastava r, rizvi i. socket preservation by beta-tri-calcium phosphate with collagen compared to platelet-rich fibrin: a clinico-radiographic study. eur j dent. 2016;10:264– 276. 56. girish rao s, bhat p, nagesh ks, rao ghr, mirle b, kharbhari l, et al. bone regeneration in extraction sockets with autologous platelet rich fibrin gel. j maxillofac oral surg. 2013;12:11–16. 57. gurbuzer b, pikdoken l, tunali m, urhan m, kucukodaci z, ercan f. scintigraphic evaluation of osteoblastic activity in extraction sockets treated with platelet-rich fibrin. j oral maxillofac surg. 2010;68:980–989. 58. kumar n, prasad k, ramanujam l, k r, dexith j, chauhan a. evaluation of treatment outcome after impacted mandibular third molar surgery with the use of autologous platelet-rich fibrin: a randomized controlled clinical study. j oral maxillofac surg. 2015;73:1042–1049. 59. marenzi g, riccitiello f, tia m, di lauro a, sammartino g. influence of leukocyteand platelet-rich fibrin (l-prf) in the healing of simple postextraction sockets: a split-mouth study. biomed res int. 2015;2015:369273. 60. simon bi, gupta p, tajbakhsh s. quantitative evaluation of extraction socket healing following the use of autologous platelet-rich fibrin matrix in humans. int j periodontics restorative dent. 2011;31:285–295. 61. singh a, kohli m, gupta n. platelet rich fibrin: a novel approach for osseous regeneration. j maxillofac oral surg. 2012;11:430–434. 62. suttapreyasri s, leepong n. influence of platelet-rich fibrin on alveolar ridge preservation. j craniofac surg. 2013;24:1088–1094. 63. temmerman a, vandessel j, castro a, jacobs r, teughels w, pinto n, et al. the use of leucocyte and platelet-rich fibrin in socket management and ridge preservation: a split-mouth, randomized, controlled clinical trial. j clin periodontol. 2016;43:990–999. 64. yelamali t, saikrishna d. role of platelet rich fibrin and platelet rich plasma in wound healing of extracted third molar sockets: a comparative study. j maxillofac oral surg. 2015;14:410–416. 65. zhang y, ruan z, shen m, tan l, huang w, wang l, et al. clinical effect of platelet-rich fibrin on the preservation of the alveolar ridge following tooth extraction. exp ther med. 2018;15:2277–2286. 66. girish kumar n, chaudhary r, kumar i, arora ss, kumar n, singh h. to assess the efficacy of socket plug technique using platelet rich fibrin with or without the use of bone substitute in alveolar ridge preservation: a prospective randomised controlled study. oral maxillofac surg. 2018;22:135–142. 67. thakkar dj, deshpande nc, dave dh, narayankar sd. a comparative evaluation of extraction socket preservation with demineralized freezedried bone allograft alone and along with platelet-rich fibrin: a clinical and radiographic study. contemp clin dent. 2016;7:371–376. 68. clark d, rajendran y, paydar s, ho s, cox d, ryder m, et al. advanced platelet-rich fibrin and freeze-dried bone allograft for ridge preservation: a randomized controlled clinical trial. j periodontol. 2018;89:379–387. 69. hauser f, gaydarov n, badoud i, vazquez l, bernard jp, ammann p. clinical and histological evaluation of postextraction platelet-rich fibrin socket filling: a prospective randomized controlled study. implant dent. 2013;22:295–303. 70. bolukbasi n, ersanlı s, keklikoglu n, basegmez c, ozdemir t. sinus augmentation with platelet-rich fibrin in combination with bovine bone graft versus bovine bone graft in combination with collagen membrane. j oral implantol. 2015;41:586–595. 71. gassling v, purcz n, braesen jh, will m, gierloff m, behrens e, et al. comparison of two different absorbable membranes for the coverage of lateral osteotomy sites in maxillary sinus augmentation: a preliminary study. j craniomaxillofac surg. 2013;41:76–82. 72. nizam n, eren g, akcalı a, donos n. maxillary sinus augmentation with leukocyte and platelet-rich fibrin and deproteinized bovine bone mineral: a split-mouth histological and histomorphometric study. clin oral implants res. 2018;29:67–75. 73. tatullo m, marrelli m, cassetta m, pacifici a, stefanelli lv, scacco s, et al. platelet rich fibrin (p.r.f.) in reconstructive surgery of atrophied maxillary bones: clinical and histological evaluations. int j med sci. 2012;9:872–880. 74. bosshardt dd, bornstein mm, carrel jp, buser d, bernard jp. maxillary sinus grafting with a synthetic, nanocrystalline hydroxyapatite-silica gel in humans: histologic and histomorphometric results. int j periodontics restorative dent. 2014;34:259–267. 75. kanayama t, horii k, senga y, shibuya y. crestal approach to sinus floor elevation for atrophic maxilla using platelet-rich fibrin as the only grafting material: a 1-year prospective study. implant dent. 2016;25:32–38. 76. dhiman m, kumar s, duhan j, sangwan p, tewari s. effect of platelet-rich fibrin on healing of apicomarginal defects: a randomized controlled trial. j endod. 2015;41:985–991. 77. singh s, singh a, singh s, singh r. application of prf in surgical management of periapical lesions. natl j maxillofac surg. 2013;4:94–99. 78. bajaj p, pradeep ar, agarwal e, rao ns, naik sb, priyanka n, et al. comparative evaluation of autologous platelet-rich fibrin and plateletrich plasma in the treatment of mandibular degree ii furcation defects: a randomized controlled clinical trial. j periodontal res. 2013;48:573–581. 79. siddiqui zr, jhingran r, bains vk, srivastava r, madan r, rizvi i. comparative evaluation of platelet-rich fibrin versus beta-tri-calcium phosphate in the treatment of grade ii mandibular furcation defects using cone-beam computed tomography. eur j dent. 2016;10:496–506. 80. sharma p, grover hs, masamatti ss, saksena n. a clinicoradiographic assessment of 1% metformin gel with platelet-rich fibrin in the treatment of mandibular grade ii furcation defects. j indian soc periodontol. 2017;21:303–308. 81. lohi hs, nayak dg, uppoor as. comparative evaluation of the efficacy of bioactive ceramic composite granules alone and in combination with platelet rich fibrin in the treatment of mandibular class ii furcation defects: a clinical and radiographic study. j clin diagn re. 2017;11:zc76–zc80. 82. pradeep ar, karvekar s, nagpal k, patnaik k, raju a, singh p. rosuvastatin 1.2 mg in situ gel combined with 1:1 mixture of autologous platelet-rich fibrin and porous hydroxyapatite bone graft in surgical treatment of mandibular class ii furcation defects: a randomized clinical control trial. j periodontol. 2016;87:5–13. 83. kanoriya d, pradeep ar, garg v, singhal s. mandibular degree ii furcation defects treatment with platelet-rich fibrin and 1% alendronate gel combination: a randomized controlled clinical trial. j periodontol. 2017;88:250–258. 188 j contemp med sci | vol. 5, no. 4, july-august 2019: 170–188 application of platelet rich fibrin in the regeneration of intra oral defects review muna al-shuhayeb et al. 84. asimuddin s, koduganti rr, panthula vnr, jammula sp, dasari r, gireddy h. effect of autologous platelet rich fibrin in human mandibular molar grade ii furcation defectsa randomized clinical trial. j clin diagn res. 2017;11:zc73–zc77. 85. bozkurt doğan ş, öngöz dede f, ballı u, atalay en, durmuşlar mc. concentrated growth factor in the treatment of adjacent multiple gingival recessions: a split-mouth randomized clinical trial. j clin periodontol. 2015;42:868–875. 86. gupta s, banthia r, singh p, banthia p, raje s, aggarwal n. clinical evaluation and comparison of the efficacy of coronally advanced flap alone and in combination with platelet rich fibrin membrane in the treatment of miller class i and ii gingival recessions. contemp clin dent. 2015;6:153–160. 87. padma r, shilpa a, kumar pa, nagasri m, kumar c, sreedhar a. a split mouth randomized controlled study to evaluate the adjunctive effect of plateletrich fibrin to coronally advanced flap in miller’s class-i and ii recession defects. j indian soc periodontol. 2013;17:631–636. 88. thamaraiselvan m, elavarasu s, thangakumaran s, gadagi js, arthie t. comparative clinical evaluation of coronally advanced flap with or without platelet rich fibrin membrane in the treatment of isolated gingival recession. j indian soc periodontol. 2015;19:66–71. 89. rajaram v, thyegarajan r, balachandran a, aari g, kanakamedala a. platelet rich fibrin in double lateral sliding bridge flap procedure for gingival recession coverage: an original study. j indian soc periodontol. 2015;19:665–670. 90. culhaoglu r, taner l, guler b. evaluation of the effect of dose-dependent platelet-rich fibrin membrane on treatment of gingival recession: a randomized, controlled clinical trial. j appl oral sci. 2018;26:e20170278. 91. jankovic s, aleksic z, klokkevold p, lekovic v, dimitrijevic b, kenney eb, et al. use of platelet-rich fibrin membrane following treatment of gingival recession: a randomized clinical trial. int j periodontics restorative dent. 2012;32:e41–e50. 92. keceli hg, kamak g, erdemir eo, evginer ms, dolgun a. the adjunctive effect of platelet-rich fibrin to connective tissue graft in the treatment of buccal recession defects: results of a randomized, parallel-group controlled trial. j periodontol. 2015;86:1221–1230. 93. mufti s, dadawala sm, patel p, shah m, dave dh. comparative evaluation of platelet-rich fibrin with connective tissue grafts in the treatment of miller’s class i gingival recessions. contemp clin dent. 2017;8:531–537. 94. tunaliota m, özdemir h, arabacι t, gürbüzer b, pikdöken l, firatli e. clinical evaluation of autologous platelet-rich fibrin in the treatment of multiple adjacent gingival recession defects: a 12-month study. int j periodontics restorative dent. 2015;35:105–114. 95. eren g, atilla g. platelet-rich fibrin in the treatment of localized gingival recessions: a split-mouth randomized clinical trial. clin oral investig. 2014;18:1941–1948. 96. öncü e. the use of platelet-rich fibrin versus subepithelial connective tissue graft in treatment of multiple gingival recessions: a randomized clinical trial. int j periodontics restorative dent. 2017;37:265–271. 97. jankovic s, aleksic z, milinkovic i, dimitrijevic b. the coronally advanced flap in combination with platelet-rich fibrin (prf) and enamel matrix derivative in the treatment of gingival recession: a comparative study. eur j esthet dent. 2010;5:260–273. 98. agarwal sk, jhingran r, bains vk, srivastava r, madan r, rizvi i. patientcentered evaluation of microsurgical management of gingival recession using coronally advanced flap with platelet-rich fibrin or amnion membrane: a comparative analysis. eur j dent. 2016;10:121–133. 99. ramireddy s, mahendra j, rajaram v, ari g, kanakamedala ak, krishnakumar d. treatment of gingival recession by coronally advanced flap in conjunction with platelet-rich fibrin or resin-modified glass-ionomer restoration: a clinical study. j indian soc periodontol. 2018;22:45–49. 100. bastami f, khojasteh a. use of leukocyte-and platelet-rich fibrin for bone regeneration: a systematic review. regen reconstr restor 2016;1:47–88. 101. marrelli m, tatullo m. influence of prf in the healing of bone and gingival tissues. clinical and histological evaluations. eur rev med pharmacol sci. 2013;17:1958–1962. 102. mazor z, horowitz ra, del corso m, prasad hs, rohrer md, dohan ehrenfest dm. sinus floor augmentation with simultaneous implant placement using choukroun’s platelet-rich fibrin as the sole grafting material: a radiologic and histologic study at 6 months. j periodontol. 2009;80:2056–2064. 103. tajima n, ohba s, sawase t, asahina i. evaluation of sinus floor augmentation with simultaneous implant placement using platelet-rich fibrin as sole grafting material. int j oral maxillofac implants. 2013;28:77–83. 104. bielecki t, dohan ehrenfest dm, everts pa, wiczkowski a. the role of leukocytes from l-prp/l-prf in wound healing and immune defense: new perspectives. curr pharm biotechnol. 2012;13:1153–1162. 105. ozcan m, ucak o, alkaya b, keceli s, seydaoglu g, haytac mc. effects of platelet-rich fibrin on palatal wound healing after free gingival graft harvesting: a comparative randomized controlled clinical trial. int j periodontics restorative dent. 2017;37:e270–e278. 106. choukroun j, diss a, simonpieri a, girard mo, schoeffler c, dohan sl, et al. platelet-rich fibrin (prf): a second-generation platelet concentrate. part iv: clinical effects on tissue healing. oral surg oral med oral pathol oral radiol endod. 2006;101:e56–e60. 107. choukroun j, adda f, schoeffler c, vervelle a. une opportunit?? en paroimplantologie: le prf. implantodontie 2001;42:55–62. 108. chen fm, wu la, zhang m, zhang r, sun hh. homing of endogenous stem/ progenitor cells for in situ tissue regeneration: promises, strategies, and translational perspectives. biomaterials 2011;32:3189–209. 109. roy s, driggs j, elgharably h, biswas s, findley m, khanna s, et al. plateletrich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation. wound repair regen. 2011;19:753–766. 110. choukroun j, ghanaati s. reduction of relative centrifugation force within injectable platelet-rich-fibrin (prf) concentrates advances patients’ own inflammatory cells, platelets and growth factors: the first introduction to the low speed centrifugation concept. eur j trauma emerg sur. 2018;44:87–95. 111. el bagdadi k, kubesch a, yu x, al-maawi s, orlowska a, dias a, et al. reduction of relative centrifugal forces increases growth factor release within solid platelet-rich-fibrin (prf)-based matrices: a proof of concept of lscc (low speed centrifugation concept). eur j trauma emerg surg. 2019;45:467–479. 112. fujioka-kobayashi m, miron rj, hernandez m, kandalam u, zhang y, choukroun j. optimized platelet-rich fibrin with the low-speed concept: growth factor release, biocompatibility, and cellular response. j periodontol. 2017;88:112–121. 113. wend s, kubesch a, orlowska a, al-maawi s, zender n, dias a, et al. reduction of the relative centrifugal force influences cell number and growth factor release within injectable prf-based matrices. j mater sci mater med. 2017;28:188. 114. herrera-vizcaíno c, dohle e, al-maawi s, booms p, sader r, kirkpatrick cj, et al. platelet-rich fibrin secretome induces three dimensional angiogenic activation in vitro. eur cell mater. 2019;37:250–264. 115. kubesch a, barbeck m, al-maawi s, orlowska a, booms pf, sader ra, et al. a low-speed centrifugation concept leads to cell accumulation and vascularization of solid platelet-rich fibrin: an experimental study in vivo. platelets 2019;30:329–340. 116. ghanaati s, herrera-vizcaino c, al-maawi s, lorenz j, miron rj, nelson k, et al. fifteen years of platelet rich fibrin in dentistry and oromaxillofacial surgery: how high is the level of scientific evidence? j oral implantol. 2018;44:471–492. 117. findik y, baykul t. secondary closure of alveolar clefts with mandibular symphyseal bone grafts and with platelet-rich fibrin under local anesthesia: three case reports. j contemp dent pract. 2013;14:751–753. 118. reddy pk, bolla v, koppolu p, srujan p. long palatal connective tissue rolled pedicle graft with demineralized freeze-dried bone allograft plus plateletrich fibrin combination: a novel technique for ridge augmentation three case reports. j indian soc periodontol. 2015;19:227–231. 119. dar m, hakim t, shah a, najar l, yaqoob g, lanker f. use of autologous platelet-rich fibrin in osseous regeneration after cystic enucleation: a clinical study. j oral biol craniofac res. 2016;6:s29–s32. 120. meshram vs, lambade pn, meshram pv, kadu a, tiwari ms. the autologous platelet rich fibrin: a novel approach in osseous regeneration after cystic enucleation: a pilot study. indian j dent res. 2015;26:560–564. 121. hoaglin dr, lines gk. prevention of localized osteitis in mandibular thirdmolar sites using platelet-rich fibrin. int j dent 2013;2013:875380. 122. miron rj, pinto nr, quirynen m, ghanaati s. standardization of relative centrifugal forces in studies related to platelet-rich fibrin. j periodontol. 2019;90:817–820. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. dx.doi.org/10.22317/jcms.08201901 6 review issn 2413-0516 j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 introduction viruses are microscopic particles ranging in size from 30 to 300 nm, lacking typical cellular structures. they are not able to reproduce outside the living host cell and are more like a large chemical compound. for this reason, they are also called compulsive intracellular parasites. viruses can infect hosts ranging from bacteria to animals, plants to humans.1-3 viruses rely on host cell surface receptors to enter the cell and employ host cellular machinery to replicate, assemble, and release new virus particles. each virus has a specific mechanism to infect the host through the attachment of the virus to host cell membrane via binding of molecules of the outer surface of the virion to a receptor molecule on the host cell (protein or carbohydrate); penetration and uncoating of the virus and subsequent release of the virions into host cell; reverse transcription of viral rna into dna (i.e., retroviruses); integration of the viral dna into host cell genome; use of the cellular system for gene duplication and translation (i.e., human immunodeficiency virus (hiv)), or use of their system to produce mrna coding for viral proteins (early genes); synthesis and assembly of nucleocapsids (late genes); release of the naked virions by cell lysis. alternatively, viruses with envelopes can be released by a process known as budding, in which the nucleocapsid is wrapped by the membrane and pinched off4-8 (fig. 1). as of now, viral infections are significant threats to human and animal health, imposing a tremendous economic burden. during the past two decades, the epidemic of ebola virus, chikungunya virus, zika virus, yellow fever, the severe acute respiratory syndrome (sars) virus and the middle east respiratory syndrome coronavirus (mers-cov), influenza, nipah and henipaviral diseases, and lassa fever raised significant challenges for public health authorities all over the world, highlighting the emerge of strategies to predict, prevent, or control the pathogens geographic spread, and the human-tohuman transmission.9 in the 21st century, three members of the β-coronavirus genus caused deadly infections in humans. in 2002, the outbreak of sars-cov began in northern china and extended globally. after that, mers-cov spread out in the middle east in 2012, and lately, sars-cov-2 (covid-19) was reported on december 30, 2019, in wuhan city, hubei province, china. sars is an 80–160 nm single-stranded positive rna virus surrounded by a coat containing the e2 virus binding protein, the e1 matrix protein, and the nucleocapsid n protein. to date, no proven effective therapeutics or vaccines exist for human coronaviruses infections.10 current treatment options for coronavirus infections include medications such as lopinavir/ritonavir, oseltamivir, chloroquine, hydroxychloroquine, remdesivir, favipiravir; nucleoside analogs; neuraminidase (na) inhibitors; peptide (ek1); abidol; rna synthesis inhibitors (such as tdf, 3tc); anti-inflammatory drugs (such as hormones and other molecules); angiotensin-converting enzyme inhibitors or angiotensin receptor blockers; and chinese traditional medicine such shufengjiedu capsules and lianhuaqingwen capsule. a patent review study offered that proteases inhibitors (since proteases are essential for viruses replication), monoclonal antibodies, and interferons (infs) are definite targets for coronavirus infection treatment. this study also concluded that traditional formulations containing natural compounds might help to improve symptoms of infection such as fever, cough, sore throat, and shortness of breath.11,12 a recent a narrative review of herbal preparations against rna viruses eghbal jasemi1¥, saeideh momtaz1,2,4¥*, reza ghaffarzadegan1, amir hossein abdolghaffari3,2,4, mohammad abdollahi2 1 medicinal plants research center, institute of medicinal plants, acecr, tehran, iran. 2 toxicology and diseases group, pharmaceutical sciences research center (psrc), the institute of pharmaceutical sciences (tips), and department of toxicology and pharmacology, school of pharmacy, tehran university of medical sciences, tehran, iran. 3 department of toxicology and pharmacology, school of pharmacy, tehran medical sciences, islamic azad university, tehran, iran. 4 gastrointestinal pharmacology interest group, universal scientific education and research network, tehran, iran. ¥ eghbal jasemi and saeideh momtaz contributed equally in this work. *correspondence to: saeideh momtaz (e-mail: saeideh58_momtaz@yahoo.com) (submitted: 03 january 2021 – revised version received: 28 january 2021 – accepted: 08 february 2021 – published online: 26 february 2021) abstract objective although several therapeutics were designed to control infectious diseases, viral infections are still fatal. currently, evidence extracted from in vivo, in vitro, and in silico studies support the antiviral activity of many herbs scientifically; however, the therapeutic potential of many other herbs is still unknown. plants and their products may potentially control the propagation of viruses in a variety of conditions. methods data were extracted from pubmed, scopus, google scholar, and science direct from 1983 to 2020. we gathered a list of plant extracts, phytochemicals, and herbal formulations that can inhibit rna viral infections, mainly those are originated from the coronaviruses family. we also provided an overview of their inhibitory mechanism of actions. results plant families, including lamiaceae, asteraceae, and myrtaceae, contain the highest number of species with anti-coronaviruses activities, respectively. conclusions it can be suggested that the combination of these antiviral ingredients with each other, any synthetic compound, or already approved drugs or inhibitors can be a novel approach for antiviral therapies. keywords viruses, coronaviruses, herbal extracts, phytochemicals, essential oils https://en.wikipedia.org/wiki/lamiaceae 7 review a narrative review of herbal preparations against rna virusessaeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 study reported that some natural products such as lycorine, homoharringtonine, silvestrol, ouabain, tylophorine, and 7‐ methoxycryptopleurine may possess potential antiviral activities at nanomolecular equivalent concentrations.13 natural isolates or extracts such as scutellarein, silvestrol, tryptanthrin, saikosaponin b2, lectins such as griffithsin, lycorine and polyphenolics including quercetin, myricetin, caffeic acid, psoralidin and isobavachalcone were also shown to be active against human coronaviruses.14 given the significance of natural products, particularly herbal preparations and phytochemicals, on viral infections, we tried to collect a list of plant species/bioactive components or antiviral herbal medicine formulations that are capable of combating viral infections in rna viruses with emphasize on coronaviruses. we also provided a summary of different types of viruses and the family of coronaviruses. methods study design data were extracted from pubmed, scopus, google scholar, and science direct, using the keywords “antiviral” or “coronavirus” or “rna virus” or “herbals” or “plant species” or “herbal preparation” or “herbs” or “herbal formulation” in the title/summary and the keywords “plant, herb, phytochemistry” in the whole text. the research results were included in the study, regardless of the time limitation. the final articles used in the current review were from 1983 to 2020. 32% of the references were from 2015 until 2020. two persons independently evaluated studies and non-english and duplicates were excluded. we avoided studies on dna viruses and focused on rna viruses, specifically the coronavirus family. we also prioritized our work over studies that examined the antiviral mechanisms of plants and their derivatives. data extraction a summary of information including the name of plants, essential oil, phytochemical compounds, and herbal formulation, method of study such as in vitro or in vivo, the model used in the assay, dosage of treatment, and finally the results of the study are presented in two tables. study selection and characteristics out of a total of 930 studies, 440 studies were deleted due to irrelevant title and abstract, 186 studies were excluded in repetitive, and 50 non-english studies were left out. in total, 22 studies remained to review the full text. a further 53 studies were discontinued due to the irrelevance of criteria in this study. types of viruses viruses only reproduce within individual species. therefore, to facilitate the study, they are divided into vertebrate viruses, invertebrate viruses, bacterial viruses (bacteriophage), and plant viruses, depending on the type of host. virus families are classified by the suffix–viridae. today, viruses are classified considering to three major characteristics: nature and structure of the genome, viral symmetry nucleocapsids (icosahedral or helical), and general morphology. nevertheless, other features are also used to viral classification: size, physiochemical properties; mechanisms of gene expression and virus replication; serological relationships; host and tissue susceptibility; and pathology. vertebrate viruses are often classified according to their genomic content (dna or rna) (table 1), single or double-stranded, and linear or annular.15, 16 double-stranded dna (dsdna) viruses there are many viruses with the dsdna genome that infect mammals. they are divided into seven families: hepadnaviridae, polyomaviridae, papillomaviridae, adenoviridae, herpesviridae, poxviridae, and asfarviridae. with the exception of the poxviridae and asfarviridae families, all families have members that can cause persistent infection in humans or animals. hepadenoviruses, polyomaviruses, papillomaviruses, and viral herpes are causally linked to human cancers. this suggests that dsdna viruses have many ways of disrupting and influencing cell division. many details of their reproduction cycle indicate that they evolved from retroviruses.17 genomic content of these viruses enter the host cell nucleus and mimics the genome of the host cell. typically, the viral genome is replicated from the host cell using a dna polymerase, and the viral genome is transcribed from the host cell by rna polymerase. the resulting transcripts are then fig. 1 a schematic model of a coronavirus. 8 review a narrative review of herbal preparations against rna viruses saeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 transported to the cytoplasm and are replicated by the host cell ribosomes. several replicated viral dna molecules are converted to virions. virions contain an entire virus particle, consisting of an outer protein shell called a capsid and an inner core of nucleic acid. virions use the machinery of host cells to complete their life cycle and target other host cells, initiating new infection cycles.18 single-stranded dna (ssdna) viruses this type of virus is often identified by the presence of two genes; a gene for viral nucleocapsid protein and another gene as a dna replication enzyme. once these viruses enter the cells, viral ssdna converts to dsdna using host cell dna polymerase using the 3’ end of viral dna as the base template for transcription, resulting in the production of viral proteins. then replicated viral dna is reconverted into an ssdna genome, which later might form virions. parvoviruses in dogs and cats belong to the ssdna virus family.19-22 (+) single-stranded rna or (+) ssrnas viruses this class is by far the largest group of viruses (i.e., many common cold viruses and the poliovirus) and consequently has considerable variety in terms of size, structure, organization, and observed replication strategy. ssrnas are also known as picornaviruses because they have small rna genomes. the ssrna genome can act as an mrna molecule, thus called “+”. however, there are several common theme in genome organization, particularly unsegmented and single open reading frame (orf) genomes, where the proteolytic lysis of a long “polyprotein” leads to mature gene products. non-segmented genomes with multiple orfs require two rounds of translation or subgenomic mrna to express structural and non-structural proteins. according to this situation, there are multi-part genomes, each component has a single orf. the virus genome in this class range in size from 5 kb (i.e., leviviruses) to 30 kb (i.e., coronaviruses).23 (-) single-stranded rna or (-) ssrnaviruses the negative-strand rna viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, ebola, and hantaviruses. all these viruses are enveloped viruses whose genomes is consisted of either one (in paramyxoviruses, rhabdoviruses, filoviruses, and bornadisease virus) or several (in orthomyxoviruses, bunyaviruses and arenaviruses) rna segments. the virus carries its rnadependent rna polymerase, which is responsible for the transcription and replication of the viral genome in the infected cell.24, 25 double-stranded rna (dsrna) viruses double-stranded rna (dsrna) molecules belong to a limited group of viruses such as reoviruses with 10 different dsrnas in their genomes.26 these viruses also contain the rna replicase enzyme as a part of the virus structure. this enzyme transcribes positive rna strands and helps the virus to complete the steps of its replication cycle alone, such as rotaviruses.27, 28 (+) ssrna retroviruses the retroviruses encompass a large family of infectious agents (retroviridae) unified by a typical virion structure and mode of replication. retroviruses genomes may serve as mrna, consisting of a dimer of identical single-stranded rna molecules, each 7–10 kb in length. viruses with genomes higher than 8 kb are those that have other genes in addition to gag, pol, and env. they use an enzyme called reverse transcriptase, giving them the unique property of transcribing their rna into dna after entering a cell. once the virus enters the cell, instead of the rna (+) strand, the virion rna is used as a template to create a dna copy of the viral genome through a viral enzyme called reverse transcriptase. this viral dna becomes integrated into the host-cell dna. such viral dna is called a “provirus”, identical to the genes of host cells. this integrated provirus is transcribed into rna (+) and is transported to the cytoplasm to be utilized for the synthesis of viral proteins, or as a genome for new viruses. retroviruses are in effect retrograde because the flow of genetic information is reversed compared with the normal pathway of molecular biosynthesis: dna to rna and then to protein. these viruses are referred to as human endogenous retroviruses or hervs. hiv is a retrovirus and a member of the lentivirus.29, 30 approaches to counter virus infection should be adjusted according to the specific viral characteristics. to this end, today’s medicines hit targets that minimize the risk of disease by disrupting the vital functions of the virus. table 2 provides a summary of current medications with their mechanism of action for viral infections. there are several therapeutic targets for these drugs, which are also used to design new drugs. considering the side effects of chemical drugs, the search for new drugs with similar properties and fewer side effects could fix a significant part of the health problems area. coronaviruses; structure, genome, and lifecycle the family coronaviridae includes the genus coronavirus and torovirus. coronaviruses belong to the family coronaviridae, suborder cornidovirineae, order nidovirales, and realm riboviria. coronaviruses are large enveloped positive-sense, ssrna viruses of vertebrates by importance in medical and veterinary diseases.31 the same other rna viruses, table 1. types of viruses, examples and their typical properties. virus type example properties dna viruses poxviruses, herpes, adenoviruses, papillomaviruses contain mostly double-stranded dna, a small number singlestranded dna. dna viruses enter the cell nucleus and direct the generation of new viruses rna viruses influenza, measles, mumps, cold, meningitis, poliomyelitis, retroviruses (aids, t-cell leukemia), arenaviruses, coronaviruses contain largely ssrna. rna viruses do not enter the cell nucleus (except the influenza virus). rna retroviruses use the viral reverse transcriptase to make a dna copy of the viral rna, which is then integrated into the host genome ssrna, single-stranded rna. 9 review a narrative review of herbal preparations against rna virusessaeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 coronaviruses are highly mutant. however, the mutation rate might be somewhat lower than other rna viruses because of its genome-encoded exonuclease, enabling the viruses to become more virulent and to extend more efficiently through different hosts.32 coronaviruses are likely to have a seasonal distribution and may cause asymptomatic as well as lower and upper respiratory tract infections.33 it has been reported that human coronaviruses can infect neurons since viral rna has been detected in the brain of patients with multiple sclerosis.34 interest in this family of viruses has increased in recent years due to the identification of a newly emerging coronavirus as the causative agent of sars and mers.35, 36 until the appearance of sars in 2003, only four human low-pathogenic; hcov-oc43, hcovhku1, hcov-nl63, and hcov-229e, were recognized, of which types 229e and oc43 constitute a significant cause of the common cold and therefore were not high precedence for centralized research.37-39 coronaviruses contain three significant proteins in their structure: the very large (200 k) glycoprotein s (spike) is the major inducer of neutralizing antibody, which forms the large (15–20 nm) peplomers in the virus envelope; an unusual transmembrane protein (m); and an internal phosphorylated nucleocapsid protein (n). moreover, there is a tiny transmembrane protein e, and some coronaviruses contain another coat protein with hemagglutination and esterase (he) functions.40, 41 their 30-kb (+) ssrna is the largest known rna virus genome with g+c contents varying from 32% to 43%.42 the viral genome contains distinctive features, including a unique n-terminal fragment within the spike protein. sars-cov-2 binds to ace2 (the angiotensin 2 converting enzyme) via its s protein and enables the virus to penetrate and infect cells. to complete the virus entrance into the cells, the s protein must be enzymatically prepared by a protease, for example, protease tmprss2 in the case of sars-cov and sarscov-2. tmprss2 facilitates the linkage of the virus receptor (s protein) to its cellular ligand (ace2)43, 44 (fig. 1). since the s glycoprotein is surface-exposed and mediates the virus entry into host cells, also, the ace2 could mediate sars-cov-2 s-mediated entry into cells, they both are the main targets of ongoing vaccine and therapeutic design efforts and for neutralizing polyclonal antibodies upon infection.45 within the host cell, the virus undergoes uncoating, and then the genome table 2. antiviral mechanisms of compounds as therapeutic targets in medical applications. antiviral mechanism target virus (es) compounds approved selected compounds in development for the indicated target virus reference virus adsorption inhibitors hiv, herpesviruses (hsv), cmv, rsv, and other enveloped viruses polysulphates, polysulphonates, polycarboxylates, polyoxometalates, chicoric acid, zintevir, cosalane derivatives, negatively charged albumins 51-55 viral dna polymerase inhibitors hsv-1 & -2,vzv, cmv, ebv, hhv-6, -7, -8 acyclovir, valaciclovir, ganciclovir, valganciclovir, penciclovir, famciclovir, brivudin, foscarnet bicyclic furopyrimidine nucleoside analogues, a5021, cyclohexenylguanine 56-58 reverse transcriptase hiv nrtis: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir nnrtis: nevirapine, delavirdine, efavirenz emtricitabine, amdoxovir emivirine, uc781, dpc083, tmc125 (r165335) 59-63 virus–cell fusion inhibitors hiv, rsv, and other paramyxoviruses hiv: amd3100, tak779 and t20 derivatives 64-66 acyclic nucleoside phosphonates dna viruses (polyoma, papilloma-, herpes-, adenoand poxviruses), hiv, hbv cmv: cidofovir hiv: tenofovir hbv: adefovir 67-69 viral protease inhibitors hiv, herpesviruses, rhinoviruses, hcv hiv: saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir hiv: atazanavir, mozenavir, tipranavir human rhinovirus: ag7088 70-74 inhibitors of processes associated with viral rna synthesis hiv, hcv 75 viral neuraminidase inhibitors influenza a and b virus zanamivir, oseltamivir§ rwj270201 76-79 imp dehydrogenase inhibitors hcv, rsv ribavirin mycophenolic acid, eicar, vx497 80-82 s-adenosylhomocysteine hydrolase (–) rna hemorrhagic fever viruses (for example, ebola) 83, 84 https://www.sciencedirect.com/topics/immunology-and-microbiology/neutralizing-antibody https://www.sciencedirect.com/topics/immunology-and-microbiology/virus-nucleocapsid 10 review a narrative review of herbal preparations against rna viruses saeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 is transcribed and translated. as mentioned earlier, the positive-stranded viral rna serves as mrna for the translation of the two large orfs (orf 1a and 1b), each coding for the rna polymerase units, depending on the rna. upon cleavage, these proteins covert to the active rna polymerase, forming a full-length complementary rna (negative sense). cytoplasmic membranes are the site of coronavirus replication and transcription. these membranes modulate both continuous and discontinuous rna synthesis through a viral replica, a large protein complex encoding the 20 kb replicase gene.46 based on the genome size, numerous genetic recombination can happen between different but related coronavirus genomes, which is a critical mechanism for the genetic diversity of coronaviruses in nature.47 during maturation and assembly, protein s is co-translationally inserted into the rough endoplasmic reticulum (rer) and is glycosylated with n-linked glycans. glycosylation is a crucial stage for protein s function and transportation. protein s forms trimers before being exported from er, and then interacts with proteins m and e to translocate to the site of virus assembly. sars-cov expresses another structural protein which is called 3a. this protein is associated with both the intracellular and plasma membranes, and can induce programmed cell death (apoptosis). protein s is crucial for virus entry, but not for its assembly48-50 (fig. 2). herbal preparations and antiviral mechanisms medicinal herbs, especially those used in folk medicine, have been highly regarded in the treatment of viral diseases because they contain bioactive substances that can be used to produce antiviral drugs with minimal side effects. several secondary plant metabolites such as essential oils, flavonoids, saponins, tannins, alkaloids, lignans, terpenes, and phenolic acids shown significant antiviral activities against various viruses.85, 86 indeed, those elements that interfere with certain stages of viral biosynthesis, for example, the replication cycle, are the best for clinical antiviral approaches. low concentrations and minimum effects on host cell machinery are the main privileges of these drugs, which ultimately leads to cure of infected cells. on the other hand, virucidal drugs denature viral structural proteins or glycoproteins, which results in a total loss of the infectivity of the viral particles. the therapeutic approach, as well as herbal derivatives, could apply different strategies to inhibit virus function. it is known that both protein and lipid–protein of capsids must protect the nucleic acid inside the virus from the harmful substances, and facilitate the surface absorption of the virion into the host cell. the invasion of a cell by a viral particle always depends on its specific and close connection to one of the surface components of the host cell’s plasma membrane.87 some plants use this mechanism to abolish the virus entry into the host cell by blocking their attachments to the cell surface.88 most rna viruses propagate in the cytoplasm because they have all the enzymes needed for in their genome.89 this step may be a therapeutic target for some herbal compounds due to their inhibitory effects.90 the hiv protease enzyme is responsible for the proteolytic cleavage between gag and gag-pol precursor polypeptide, which in turn converts them into functional forms and ensures the viral maturation and infectivity. the hiv polymerase inhibitors show their activity at the end of virus replication and therefore interfere with active virus formation functionally.91, 92 many active plant compounds act as hiv protease inhibitors are shown in in-vitro assays.93 besides, the hiv integrase enzyme performs two main functions: the entry of pre-viral complexes into the nuclear pores and then the integration of the viral dna genome into the host cell chromosome.94 the effectiveness of some herbs in inhibiting this enzyme has reduced the proliferation of the hiv-1 virus.95 some phytochemicals have been studied, and results indicated their efficiency in integrase activity.96 the influenza virus na causes the release of the virus from the host cell surface by catalysis a breakdown in the silicic acid attached to glycoprotein and glycolipid.97, 98 the inhibition of this enzyme can be considered as one of the therapeutic targets in medicinal plant studies.99 it is well evidenced that many plants contain ribosome‐inactivating proteins (rips) with n-glycosidase activity that can affect ribosomal function through depurination of large ribosomal rrnas in infected cell and inhibition of viral protein synthesis. rips fig. 2 a viral cycle of a coronavirus in the host cell. 11 review a narrative review of herbal preparations against rna virusessaeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 can inhibit viral mrna and dna replication. depurination is defined as inactivation of ribosomes by removal of a specific adenine from the sarcin/ricin (s/r) loop of the large rrna, thereby inhibiting translation. to date, rips were shown effective against hiv, hbv, and hsv. for example, trichosanthin (trichosanthes kirilowii), pap (pokeweed americana), gap31 (gelonium multiflorum) and map30 (momordica charantia) have been reported to inhibit hiv-1 replication in vitro.100, 101 in following sections, we prepared a list of herbal preparations, essential oils, and phytochemicals that were able to inhibit the activity of the coronaviruses family. herbal extracts to date, numerous herbal preparations were investigated against various types of viruses. table 3 represents plant species, and those were shown valid on coronaviruses. morus spp. antiviral properties of the leaves and the stem bark of the mulberry tree (morus spp.) were evaluated in human coronavirus (hcov 229e) in l-132 cells. it was shown that morus spp. reduced the viral titer and the cytopathogenic effects. the hydroalcoholic extract of the leaves exhibited the highest antiviral activity. the inhibition percentage of viral infectivity ranged from 34% to 36% for the aqueous stem bark extracts and from 37% to 45% for the hydromethanolic stem bark extracts. in comparison, this inhibition ranged from 67% to 100% for the hydromethanolic extracts of leaves.102 echinacea purpurea antiviral potential of e. purpurea examined against hcov 229e and the highly pathogenic mersand sars-covs in vitro. hcov-229e was irreversibly inactivated when exposed to echinaforce (a commercial standardized extract of  e. purpurea) at an ic50 of 3.2 µg/ml. pre-treatment of cell lines had only a marginal effect on virus propagation at 50 µg/ml.103 uvaria angolensis the methanolic extract of the stem bark of u. angolensis inhibited both the hiv-1 rnase h enzyme and the reverse transcriptase activities with ic50 values of 1.0  ±  0.2 and 0.62 ± 0.15 μg/ml, respectively. rds1643 and efavirenz were considered as control of rnase h inhibitor and reverse transcriptase, respectively. the ic50 values for rds1643 were measured as 2.7 ± 0.2 μg/ml.104 pometia pinnata leaf and bark of p. pinnata have traditionally been used to treat fever and fester. the ethanolic extracts of p. pinnata (sapindaceae) leaves have shown one of the most potent inhibitory activity against hiv-1 integrase in vitro with an ic50 value of 8.8 µg/ml. in this study, proanthocyanidin a2 was isolated as an anti-hiv-1 integrase compound, with an ic50 value of 30.1 µm.95 anthemis hyalina antimicrobial activities of different species in the genus of anthemis have been documented. treating hela ceacam1 cells infected coronavirus mhv-a59 with the ethanoic extract of anthemis hyaline decreased the proliferation and function of this virus. however, this herb has a positive effect on il-8 secretion and expression of transient receptor potential proteins (trp) family genes, and its primary role was reducing the viral load.105 phyllanthus amarus the hydroalcoholic extract of p. amarus leaves inhibited the interaction of hiv-1 envelope gp120 protein with its cd40 cell receptor up to 50%. inhibition of virus envelope protein gp120 binding to the cellular receptor cd4 was the primary mechanism underlying the blocked virus entry. the extract also showed an inhibitory effect on other enzymes of the virus, such as reverse transcriptase, integrase, and protease.106 glycine max or black soybean an aqueous/ethanol extract of black soybean inhibited respiratory tract viruses such as human adenovirus type 1, coxsackie b1, and influenza a in vero, hela, mdck, and fl cell lines in a dose-dependent manner. ethanolic extract from black soybean demonstrated dose-dependent inhibitory activity against human adenovirus type 1 replication. the antiviral index was about 1.5 mg/ml, and significant activity was recorded at 3.5 mg/ml.107 sambucus nigra in a placebo-controlled randomized, double-blind study, oral administration of elderberry (sambucus nigra) extract was shown to be an effective, safe, and cost-saving healing agent in influenza patients. in this study, patients received 15 ml of elderberry syrup for 5 days, and they recorded their symptoms on a visual analog scale. compared with placebo, respiratory influenza symptoms improved 4 days earlier, and their need for medication decreased.108 geranium sanguineum geranium sanguineum belonging to geraniaceae is one of the medicinal plants rich in polyphenols, which reduces infection of various types of influenza viruses (h7n1, h7n7, and h3n2) in chicken embryo fibroblasts, mdck cell lines and icr mice (10 mg/kg in mice). n-butanol/ethanol and the ethanol/acetic acid fractions showed the highest antiviral effects in vitro and in vivo, respectively.109 boehmeria nivea boehmeria nivea root extract reduced hepatitis b virus (hbv) replication in vitro (in hepg2 2.2.15 cell). real-time pcr, southern blot and northern blot techniques were used in this study to assay viral gene expression and replication.110 polygonum cuspidatum different extracts of p. cuspidatum was shown to inhibit various viruses. this effect was associated with the presence of a class of the chemical group called anthraquinones. anthraquinones have been reported to possess antiviral and virucidal activities against various types of viruses. ethanolic extract of p. cuspidatum inhibited the hbv replication in a dose-dependent manner in the stable hepg2 2.2.15 hepatoblastoma cell line.111 guazuma ulmifolia g. ulmifolia, also known as mutamba, has therapeutic effects such as wound healing, antiulcerogenic, hypoglycemic and https://www.sciencedirect.com/topics/medicine-and-dentistry/echinacea-purpurea https://www.sciencedirect.com/topics/medicine-and-dentistry/echinacea-purpurea https://en.wikipedia.org/wiki/geraniaceae 12 review a narrative review of herbal preparations against rna viruses saeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 ta bl e 3. m ed ic in al p la nt s i nv es tig at ed a ga in st co ro na vi ru se s a nd re la te d vi ru se s. sc ie nt ifi c n am e  fa m ily m ec ha ni sm he rb al p ro du ct ty pe o f st ud y eff ec tiv e do sa ge ce ll ty pe /a ni m al m od el vi ru s re fe re nc e m or us sp p. m or ac ea e in hi bi tio n of v ira l i nf ec tiv ity le av es a nd s te m b ar k ex tr ac t in v itr o 5 µg /m l l13 2 co ro na vi ru s (h co v 22 9e ) (1 02 ) ec hi na ce a pu rp ur ea as te ra ce ae vi ru ci da l a ct iv ity h er ba l e xt ra ct in v itr o 3. 2 µg /m l h uh -7 ve ro a 9 m er s a nd s a rs co vs 10 3 a nt he m is h ya lin a as te ra ce ae d ec re as e vi ru s lo ad a nd il -8 de cr ea se t rp g en es e xp re ss io n et ha no lic e xt ra ct in v itr o h el a ce ac am i co ro na vi ru s m h va 59 10 5 sa m bu cu s n ig ra ad ox ac ea e im pr ov e sy m pt om s an d ov er al l w el lb ei ng h er ba l e xt ra ct a c lin ic al tr ia l 15 m l o f s yr up fo ur tim es a d ay , f or 5  d ay s in flu en za 10 8 u va ri a an go le ns is a nn on ac ea e h iv -1 r n as e h e nz ym e an d re ve rs e tr an sc rip ta se a ct iv ity m et ha no l s te m b ar k ex tr ac t in v itr o 1. 0  ±  0 .2 a nd 0. 62  ±  0 .1 5  μg /m l a 54 9 h iv -1 10 4 po m et ia p in na ta sa pi nd ac ea e ac tiv ity a ga in st h iv -1 in te gr as e et ha no l l ea ve s ex tr ac t in v itr o 8. 8 µg /m l h iv -1 95 bo eh m er ia n iv ea u rt ic ac ea e in hi bi t h bv d n a s ec re tio n in to su pe rn at an t ro ot e xt ra ct in v itr o 10 m g/ l h ep g 2 2. 2. 15 hu m an he pa to bl as to m a h bv 11 0 po ly go nu m c us pi da tu m po ly go na ce ae in cr ea se e xp re ss io n of h bs ag de cr ea se h bv d n a in th e su sp en si on et ha no lic e xt ra ct in v itr o 30 μ g/ m l h ep g 2 2. 2. 15 hu m an he pa to bl as to m a h bv 11 1 g ua zu m a ul m ifo lia m al va ce ae in hi bi te d re pl ic at io n bl oc k th e sy nt he si s of vi ra la nt ig en s h er ba l e xt ra ct in v itr o 5 m g/ m l h ep -2 (h um an la ry nx c ar ci no m a) ce lls po lio vi ru s1 & bo vi ne h er pe sv iru s 11 2 o le a eu ro pa ea o le ac ea e d ec re as e vh sv ti te rs a nd v ira l pr ot ei n ac cu m ul at io n in hi bi ts re pl ic at io n m od ul at e ho st c el l g en e ex pr es si on p ro fil e le af e xt ra ct in v itr o 0. 6– 1 m g/ m l ep ith el io m a pa pu lo su m cy pr in id m t2 c el l l in e h em or rh ag ic se pt ic ae m ia v iru s (v h sv ) a nd h iv -1 11 4, 1 40 ph yl la nt hu s a m ar us ph yl la nt ha ce ae bl oc ke d th e in te ra ct io n of h iv -1 gp 12 0 w ith c d 4 in hi bi tio n of v iru s en tr y in hi bi te d ke y en zy m es r t, in a nd p r in hi bi tio n of h iv -1 re pl ic at io n af te r o ra l a pp lic at io n h er ba l e xt ra ct in v itr o & ex v iv o 1 m g/ m l i n vi tro 12 00 m g; s in gl e do se in h um an m t4 c el ls h iv -1 10 6 tr ic hi lia g la br a m el ia ce ae re du ct io n in v sv ti te r le af m et ha no lic e xt ra ct in v itr o 0. 25 m g/ m l ve ro c el ls ve si cu la r st om at iti s vi ru s (v sv ) a nd h sv -1 14 1 g ly ci ne m ax fa ba ce ae in hi bi t v iru s re pl ic at io n h er ba l e xt ra ct in v itr o 0. 15 m g/ m l h el a ce lls fl c el ls m d ck c el ls ve ro c el ls h um an ad en ov iru s ty pe 1, c ox sa ck ie b 1 an d in flu en za a 10 7 a za di ra ch ta in di ca m el ia ce ae in hi bi t v iru s re pl ic at io n in hi bi tio n of v ira l r n a re pl ic at io n le af e xt ra ct in v itr o & in v iv o 1. 89 7 m g/ m l 12 0– 30 m g/ m l c6 /3 6 ce ll lin e m ic e d en gu e vi ru s ty pe -2 (d en -2 ) 10 7 eu ca ly pt us c am al du le ns is m yr ta ce ae vi ru ci da l a ct iv ity es se nt ia l o il le av es e xt ra ct in v itr o 0. 85 ± 0. 15 μ g/ m l 0. 1± 0. 00 8 μg /m l 0. 3± 0. 00 2 μg /m l h ep -2 m a 10 4 bg m ve ro h sv , v ar ic el la zo st er , p ol io vi ru s, ec ho vi ru s, co xs ac ki e b4 , ro ta vi ru s 12 412 6 a rt em is ia p ri nc ep s as te ra ce ae pl aq ue fo rm at io n in hi bi to r es se nt ia l o il in v itr o 0. 1% (v /v ) ( es se nt ia l oi l) ra w 2 64 .7 cr fk ca lic iv iru sf9 a nd m n v1 12 7, 1 28 m os la d ia nt he ra la m ia ce ae re pl ic at io n in hi bi to r d ec re as e of c yt ok in e pr od uc tio ns (i fn -γ an d il -4 ) e nh an ci ng a nt io xi da nt ac tiv iti es es se nt ia l o il in v iv o 90 –3 60 m g/ kg ic r m ic e in flu en za a 12 1 h et er ot ha la m us a lie nu s, bu dd le ja c or do be ns is as te ra ce ae & sc ro ph ul ar ia ce ae vi ru ci da l a ct iv ity es se nt ia l o il in v itr o cc 50 (p pm ): 14 7. 6 ± 5. 7 an d 15 7. 2 ± 3. 5 ve ro h sv -1 , d en v2, an d ju n v 12 9 co ri do th ym us c ap it at us , o ri ga nu m d ic ta m nu s, s al vi a fr ut ic os a la m ia ce ae re pl ic at io n cy cl e an d pr og en y vi ru s pr od uc tio n m ix es e ss en tia l o il in v itr o 15 m l/ l h el a m d ck h 1n 1 in flu en za vi ru s an d h rv 14 rh in ov iru s 12 2 m el al eu ca a lte rn ifo lia m yr ta ce ae vi ru ci da l a ct iv ity es se nt ia l o il in v itr o 0. 00 06 % (v ⁄ v ) m d ck in flu en za a su bt yp e h 1n 1 12 3 la ur us n ob ili s la ur ac ea e ic 50 re pl ic at io n in hi bi to ry es se nt ia l o il (β -o ci m en e, 1, 8ci ne ol e, α -p in en e, an d βpi ne ne ) in v itr o 12 0 m g/ m l ve ro co ro na vi ru s sa rs 12 0 li pp ia ju ne lli an a, l ip pi a tu rb in at e ve rb en ac ea e & as te ra ce ae vi ru ci da l a ct iv ity es se nt ia l o il in v itr o vc 50 = 1 4– 20 p pm ve ro ju n v 13 0 eu pa to ri um p at en s as te ra ce ae vi ru ci da l a ct iv ity es se nt ia l o il in v itr o vc 50 = 1 50 p pm ve ro d en gu e vi ru s ty pe 2 (d en -2 ) 13 0 ci nn am om um z ey la ni cu m , d au cu sc ar ot a, e uc al yp tu s gl ob ul us , r os m ar in us offi ci na lis la ur ac ea e, a pi ac ea e, m yr ta ce ae , la m ia ce ae re du ce d vi ra l u ni ts v iri on en ve lo pe s tr uc tu re s es se nt ia l o il bl en d in v itr o ve ro h 1n 1 su bt yp e of in flu en za a v iru s 13 113 3 fo rt un el la m ar ga ri ta ru ta ce ae vi ru ci da l a ct iv ity fr ui ts a nd le av es es se nt ia l o il (α -t er pi ne ol ) in v itr o 6. 77 µg /m l (fr ui ts ) 38 .8 9 µg /m l (le av es ) m d ck in flu en za v iru s su bt yp e h 5n 1 13 4 m el is sa o ffi ci na lis la m ia ce ae vi ru ci da l a ct iv ity re pl ic at io n in hi bi to ry es se nt ia l o il in v itr o 0. 50. 1 m g/ m l m d ck av ia n in flu en za h 9n 2 su bt yp e 13 5 po go st em on c ab lin la m ia ce ae vi ru ci da l a ct iv ity im pr ov in g th e im m un e re sp on se a tt en ua tin g sy st em ic in fla m m at or y re sp on se s es se nt ia l o il in v iv o 10 -8 0 m g/ kg m ic e h 1n 1 & h 2n 2 in flu en za v iru s 13 613 8 tr ac hy sp er m um a m m i a pi ac ea e re du ct io n vi ru s tit er vi ru ci da l a ct iv ity es se nt ia l o il in v itr o 0. 5m g/ m l ve ro ja pa ne se en ce ph al iti s vi ru s 13 9 a lo e ve ra (l .) as ph od el ac ea e 3c lp ro in hi bi to ry h er ba l e xt ra ct in v itr o 42 8 μg /m l sa rs co v2 11 6 https://en.wikipedia.org/wiki/moraceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/adoxaceae https://en.wikipedia.org/wiki/sapindaceae https://en.wikipedia.org/wiki/urticaceae https://www.google.com/search?sxsrf=alekk019roo1ju0h1i1zncfdtk7d58a5hg:1587287356728&q=polygonaceae&stick=h4siaaaaaaaaaongvulqz9u3mc3jzvjeyhoqn1oznp-xmjyamaoaaotq0bsaaaa&sa=x&ved=2ahukewi2lbeykvtoahvfkuwkhb0qc3yqmxmoatadegqidhad&sxsrf=alekk019roo1ju0h1i1zncfdtk7d58a5hg:1587287356728 https://www.google.com/search?sxsrf=alekk01fdynwjhxp35coe5f2vzsl5w5_ta:1587287454997&q=malvaceae&stick=h4siaaaaaaaaaongvuluz9u3mmwuzclexmrpm5htlpicmpgkadzzvnizaaaa&sa=x&ved=2ahukewi8-qthkvtoahweduwkhqxbadeqmxmoatavegqidhad&sxsrf=alekk01fdynwjhxp35coe5f2vzsl5w5_ta:1587287454997 https://www.google.com/search?biw=1455&bih=717&sxsrf=alekk00byhxkwokf_azrr02pbv38cwoera:1587287489917&q=phyllanthaceae&stick=h4siaaaaaaaaaongvuluz9u3mm0xzjzaxmoxkfgzk5oyv5krmjyamaoarapbwr4aaaa&sa=x&ved=2ahukewivrvjxkvtoahxqzaqkhez2araqmxmoatadegqidhad https://en.wikipedia.org/wiki/fabaceae https://www.google.com/search?sxsrf=alekk02okdel39yyni4b_97umweujd0k2w:1587287568262&q=meliaceae&stick=h4siaaaaaaaaaongvulqz9u3slzmylneyumbmpozmjyamaoa4_jqmbgaaaa&sa=x&ved=2ahukewjxnkb9kvtoahwpm-wkheujci4qmxmoatahegqidrad&sxsrf=alekk02okdel39yyni4b_97umweujd0k2w:1587287568262 https://en.wikipedia.org/wiki/myrtaceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/scrophulariaceae https://en.wikipedia.org/wiki/lamiaceae https://en.wikipedia.org/wiki/myrtaceae https://en.wikipedia.org/wiki/lauraceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/asteraceae https://www.google.com/search?sxsrf=alekk00pwpsa5caipyv80-soo-nhipyaea:1587284962097&q=lauraceae&stick=h4siaaaaaaaaaongvuluz9u3mmxol7fcxmrpk1haljicmpgkab7iw7gzaaaa&sa=x&ved=2ahukewiizcqiiftoahwvh1wkhww5cg8qmxmoataeegqidxad&sxsrf=alekk00pwpsa5caipyv80-soo-nhipyaea:1587284962097 https://en.wikipedia.org/wiki/apiaceae https://en.wikipedia.org/wiki/myrtaceae https://www.google.com/search?sxsrf=alekk01q-cejppx7rnwm3iooswqyvgtfeg:1587285072773&q=lamiaceae&stick=h4siaaaaaaaaaongvulqz9u3mmnkmv_eyumtmjuzmjyamaoa5oowihgaaaa&sa=x&ved=2ahukewjpza3xiftoahxc6aqkhxr5d10qmxmoataeegqidrad&sxsrf=alekk01q-cejppx7rnwm3iooswqyvgtfeg:1587285072773 https://www.google.com/search?sxsrf=alekk03p5r-oh8ibbyz-uwhnxgpa6p7dzg:1587285112214&q=rutaceae&stick=h4siaaaaaaaaaongvulqz9u3yc4omhre6mgt8plhpwepi0lrtl5jnolics7il3fnk8ksqrrs4wkdsqs4ektgmjqypli44dyerawcqaulicmpiakadunxuvsaaaa&sxsrf=alekk03p5r-oh8ibbyz-uwhnxgpa6p7dzg:1587285112214 https://en.wikipedia.org/wiki/lamiaceae https://en.wikipedia.org/wiki/lamiaceae https://en.wikipedia.org/wiki/apiaceae https://www.google.com/search?rlz=1c1gcea_enir901ir901&sxsrf=alekk00lq8id-hai0watxnceujedfn6qdw:1591690208373&q=asphodelaceae&stick=h4siaaaaaaaaaongvuluz9u3mcovt6pyxmrrwfyqkz-smpoynjqycgcdpohhhqaaaa&sa=x&ved=2ahukewiondmlpptpahxtzxuihdkzcksqmxmoatapegqibxad 13 review a narrative review of herbal preparations against rna virusessaeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 ta bl e 3. m ed ic in al p la nt s i nv es tig at ed a ga in st co ro na vi ru se s a nd re la te d vi ru se s. sc ie nt ifi c n am e  fa m ily m ec ha ni sm he rb al p ro du ct ty pe o f st ud y eff ec tiv e do sa ge ce ll ty pe /a ni m al m od el vi ru s re fe re nc e m or us sp p. m or ac ea e in hi bi tio n of v ira l i nf ec tiv ity le av es a nd s te m b ar k ex tr ac t in v itr o 5 µg /m l l13 2 co ro na vi ru s (h co v 22 9e ) (1 02 ) ec hi na ce a pu rp ur ea as te ra ce ae vi ru ci da l a ct iv ity h er ba l e xt ra ct in v itr o 3. 2 µg /m l h uh -7 ve ro a 9 m er s a nd s a rs co vs 10 3 a nt he m is h ya lin a as te ra ce ae d ec re as e vi ru s lo ad a nd il -8 de cr ea se t rp g en es e xp re ss io n et ha no lic e xt ra ct in v itr o h el a ce ac am i co ro na vi ru s m h va 59 10 5 sa m bu cu s n ig ra ad ox ac ea e im pr ov e sy m pt om s an d ov er al l w el lb ei ng h er ba l e xt ra ct a c lin ic al tr ia l 15 m l o f s yr up fo ur tim es a d ay , f or 5  d ay s in flu en za 10 8 u va ri a an go le ns is a nn on ac ea e h iv -1 r n as e h e nz ym e an d re ve rs e tr an sc rip ta se a ct iv ity m et ha no l s te m b ar k ex tr ac t in v itr o 1. 0  ±  0 .2 a nd 0. 62  ±  0 .1 5  μg /m l a 54 9 h iv -1 10 4 po m et ia p in na ta sa pi nd ac ea e ac tiv ity a ga in st h iv -1 in te gr as e et ha no l l ea ve s ex tr ac t in v itr o 8. 8 µg /m l h iv -1 95 bo eh m er ia n iv ea u rt ic ac ea e in hi bi t h bv d n a s ec re tio n in to su pe rn at an t ro ot e xt ra ct in v itr o 10 m g/ l h ep g 2 2. 2. 15 hu m an he pa to bl as to m a h bv 11 0 po ly go nu m c us pi da tu m po ly go na ce ae in cr ea se e xp re ss io n of h bs ag de cr ea se h bv d n a in th e su sp en si on et ha no lic e xt ra ct in v itr o 30 μ g/ m l h ep g 2 2. 2. 15 hu m an he pa to bl as to m a h bv 11 1 g ua zu m a ul m ifo lia m al va ce ae in hi bi te d re pl ic at io n bl oc k th e sy nt he si s of vi ra la nt ig en s h er ba l e xt ra ct in v itr o 5 m g/ m l h ep -2 (h um an la ry nx c ar ci no m a) ce lls po lio vi ru s1 & bo vi ne h er pe sv iru s 11 2 o le a eu ro pa ea o le ac ea e d ec re as e vh sv ti te rs a nd v ira l pr ot ei n ac cu m ul at io n in hi bi ts re pl ic at io n m od ul at e ho st c el l g en e ex pr es si on p ro fil e le af e xt ra ct in v itr o 0. 6– 1 m g/ m l ep ith el io m a pa pu lo su m cy pr in id m t2 c el l l in e h em or rh ag ic se pt ic ae m ia v iru s (v h sv ) a nd h iv -1 11 4, 1 40 ph yl la nt hu s a m ar us ph yl la nt ha ce ae bl oc ke d th e in te ra ct io n of h iv -1 gp 12 0 w ith c d 4 in hi bi tio n of v iru s en tr y in hi bi te d ke y en zy m es r t, in a nd p r in hi bi tio n of h iv -1 re pl ic at io n af te r o ra l a pp lic at io n h er ba l e xt ra ct in v itr o & ex v iv o 1 m g/ m l i n vi tro 12 00 m g; s in gl e do se in h um an m t4 c el ls h iv -1 10 6 tr ic hi lia g la br a m el ia ce ae re du ct io n in v sv ti te r le af m et ha no lic e xt ra ct in v itr o 0. 25 m g/ m l ve ro c el ls ve si cu la r st om at iti s vi ru s (v sv ) a nd h sv -1 14 1 g ly ci ne m ax fa ba ce ae in hi bi t v iru s re pl ic at io n h er ba l e xt ra ct in v itr o 0. 15 m g/ m l h el a ce lls fl c el ls m d ck c el ls ve ro c el ls h um an ad en ov iru s ty pe 1, c ox sa ck ie b 1 an d in flu en za a 10 7 a za di ra ch ta in di ca m el ia ce ae in hi bi t v iru s re pl ic at io n in hi bi tio n of v ira l r n a re pl ic at io n le af e xt ra ct in v itr o & in v iv o 1. 89 7 m g/ m l 12 0– 30 m g/ m l c6 /3 6 ce ll lin e m ic e d en gu e vi ru s ty pe -2 (d en -2 ) 10 7 eu ca ly pt us c am al du le ns is m yr ta ce ae vi ru ci da l a ct iv ity es se nt ia l o il le av es e xt ra ct in v itr o 0. 85 ± 0. 15 μ g/ m l 0. 1± 0. 00 8 μg /m l 0. 3± 0. 00 2 μg /m l h ep -2 m a 10 4 bg m ve ro h sv , v ar ic el la zo st er , p ol io vi ru s, ec ho vi ru s, co xs ac ki e b4 , ro ta vi ru s 12 412 6 a rt em is ia p ri nc ep s as te ra ce ae pl aq ue fo rm at io n in hi bi to r es se nt ia l o il in v itr o 0. 1% (v /v ) ( es se nt ia l oi l) ra w 2 64 .7 cr fk ca lic iv iru sf9 a nd m n v1 12 7, 1 28 m os la d ia nt he ra la m ia ce ae re pl ic at io n in hi bi to r d ec re as e of c yt ok in e pr od uc tio ns (i fn -γ an d il -4 ) e nh an ci ng a nt io xi da nt ac tiv iti es es se nt ia l o il in v iv o 90 –3 60 m g/ kg ic r m ic e in flu en za a 12 1 h et er ot ha la m us a lie nu s, bu dd le ja c or do be ns is as te ra ce ae & sc ro ph ul ar ia ce ae vi ru ci da l a ct iv ity es se nt ia l o il in v itr o cc 50 (p pm ): 14 7. 6 ± 5. 7 an d 15 7. 2 ± 3. 5 ve ro h sv -1 , d en v2, an d ju n v 12 9 co ri do th ym us c ap it at us , o ri ga nu m d ic ta m nu s, s al vi a fr ut ic os a la m ia ce ae re pl ic at io n cy cl e an d pr og en y vi ru s pr od uc tio n m ix es e ss en tia l o il in v itr o 15 m l/ l h el a m d ck h 1n 1 in flu en za vi ru s an d h rv 14 rh in ov iru s 12 2 m el al eu ca a lte rn ifo lia m yr ta ce ae vi ru ci da l a ct iv ity es se nt ia l o il in v itr o 0. 00 06 % (v ⁄ v ) m d ck in flu en za a su bt yp e h 1n 1 12 3 la ur us n ob ili s la ur ac ea e ic 50 re pl ic at io n in hi bi to ry es se nt ia l o il (β -o ci m en e, 1, 8ci ne ol e, α -p in en e, an d βpi ne ne ) in v itr o 12 0 m g/ m l ve ro co ro na vi ru s sa rs 12 0 li pp ia ju ne lli an a, l ip pi a tu rb in at e ve rb en ac ea e & as te ra ce ae vi ru ci da l a ct iv ity es se nt ia l o il in v itr o vc 50 = 1 4– 20 p pm ve ro ju n v 13 0 eu pa to ri um p at en s as te ra ce ae vi ru ci da l a ct iv ity es se nt ia l o il in v itr o vc 50 = 1 50 p pm ve ro d en gu e vi ru s ty pe 2 (d en -2 ) 13 0 ci nn am om um z ey la ni cu m , d au cu sc ar ot a, e uc al yp tu s gl ob ul us , r os m ar in us offi ci na lis la ur ac ea e, a pi ac ea e, m yr ta ce ae , la m ia ce ae re du ce d vi ra l u ni ts v iri on en ve lo pe s tr uc tu re s es se nt ia l o il bl en d in v itr o ve ro h 1n 1 su bt yp e of in flu en za a v iru s 13 113 3 fo rt un el la m ar ga ri ta ru ta ce ae vi ru ci da l a ct iv ity fr ui ts a nd le av es es se nt ia l o il (α -t er pi ne ol ) in v itr o 6. 77 µg /m l (fr ui ts ) 38 .8 9 µg /m l (le av es ) m d ck in flu en za v iru s su bt yp e h 5n 1 13 4 m el is sa o ffi ci na lis la m ia ce ae vi ru ci da l a ct iv ity re pl ic at io n in hi bi to ry es se nt ia l o il in v itr o 0. 50. 1 m g/ m l m d ck av ia n in flu en za h 9n 2 su bt yp e 13 5 po go st em on c ab lin la m ia ce ae vi ru ci da l a ct iv ity im pr ov in g th e im m un e re sp on se a tt en ua tin g sy st em ic in fla m m at or y re sp on se s es se nt ia l o il in v iv o 10 -8 0 m g/ kg m ic e h 1n 1 & h 2n 2 in flu en za v iru s 13 613 8 tr ac hy sp er m um a m m i a pi ac ea e re du ct io n vi ru s tit er vi ru ci da l a ct iv ity es se nt ia l o il in v itr o 0. 5m g/ m l ve ro ja pa ne se en ce ph al iti s vi ru s 13 9 a lo e ve ra (l .) as ph od el ac ea e 3c lp ro in hi bi to ry h er ba l e xt ra ct in v itr o 42 8 μg /m l sa rs co v2 11 6 https://en.wikipedia.org/wiki/moraceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/adoxaceae https://en.wikipedia.org/wiki/sapindaceae https://en.wikipedia.org/wiki/urticaceae https://www.google.com/search?sxsrf=alekk019roo1ju0h1i1zncfdtk7d58a5hg:1587287356728&q=polygonaceae&stick=h4siaaaaaaaaaongvulqz9u3mc3jzvjeyhoqn1oznp-xmjyamaoaaotq0bsaaaa&sa=x&ved=2ahukewi2lbeykvtoahvfkuwkhb0qc3yqmxmoatadegqidhad&sxsrf=alekk019roo1ju0h1i1zncfdtk7d58a5hg:1587287356728 https://www.google.com/search?sxsrf=alekk01fdynwjhxp35coe5f2vzsl5w5_ta:1587287454997&q=malvaceae&stick=h4siaaaaaaaaaongvuluz9u3mmwuzclexmrpm5htlpicmpgkadzzvnizaaaa&sa=x&ved=2ahukewi8-qthkvtoahweduwkhqxbadeqmxmoatavegqidhad&sxsrf=alekk01fdynwjhxp35coe5f2vzsl5w5_ta:1587287454997 https://www.google.com/search?biw=1455&bih=717&sxsrf=alekk00byhxkwokf_azrr02pbv38cwoera:1587287489917&q=phyllanthaceae&stick=h4siaaaaaaaaaongvuluz9u3mm0xzjzaxmoxkfgzk5oyv5krmjyamaoarapbwr4aaaa&sa=x&ved=2ahukewivrvjxkvtoahxqzaqkhez2araqmxmoatadegqidhad https://en.wikipedia.org/wiki/fabaceae https://www.google.com/search?sxsrf=alekk02okdel39yyni4b_97umweujd0k2w:1587287568262&q=meliaceae&stick=h4siaaaaaaaaaongvulqz9u3slzmylneyumbmpozmjyamaoa4_jqmbgaaaa&sa=x&ved=2ahukewjxnkb9kvtoahwpm-wkheujci4qmxmoatahegqidrad&sxsrf=alekk02okdel39yyni4b_97umweujd0k2w:1587287568262 https://en.wikipedia.org/wiki/myrtaceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/scrophulariaceae https://en.wikipedia.org/wiki/lamiaceae https://en.wikipedia.org/wiki/myrtaceae https://en.wikipedia.org/wiki/lauraceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/asteraceae https://www.google.com/search?sxsrf=alekk00pwpsa5caipyv80-soo-nhipyaea:1587284962097&q=lauraceae&stick=h4siaaaaaaaaaongvuluz9u3mmxol7fcxmrpk1haljicmpgkab7iw7gzaaaa&sa=x&ved=2ahukewiizcqiiftoahwvh1wkhww5cg8qmxmoataeegqidxad&sxsrf=alekk00pwpsa5caipyv80-soo-nhipyaea:1587284962097 https://en.wikipedia.org/wiki/apiaceae https://en.wikipedia.org/wiki/myrtaceae https://www.google.com/search?sxsrf=alekk01q-cejppx7rnwm3iooswqyvgtfeg:1587285072773&q=lamiaceae&stick=h4siaaaaaaaaaongvulqz9u3mmnkmv_eyumtmjuzmjyamaoa5oowihgaaaa&sa=x&ved=2ahukewjpza3xiftoahxc6aqkhxr5d10qmxmoataeegqidrad&sxsrf=alekk01q-cejppx7rnwm3iooswqyvgtfeg:1587285072773 https://www.google.com/search?sxsrf=alekk03p5r-oh8ibbyz-uwhnxgpa6p7dzg:1587285112214&q=rutaceae&stick=h4siaaaaaaaaaongvulqz9u3yc4omhre6mgt8plhpwepi0lrtl5jnolics7il3fnk8ksqrrs4wkdsqs4ektgmjqypli44dyerawcqaulicmpiakadunxuvsaaaa&sxsrf=alekk03p5r-oh8ibbyz-uwhnxgpa6p7dzg:1587285112214 https://en.wikipedia.org/wiki/lamiaceae https://en.wikipedia.org/wiki/lamiaceae https://en.wikipedia.org/wiki/apiaceae https://www.google.com/search?rlz=1c1gcea_enir901ir901&sxsrf=alekk00lq8id-hai0watxnceujedfn6qdw:1591690208373&q=asphodelaceae&stick=h4siaaaaaaaaaongvuluz9u3mcovt6pyxmrrwfyqkz-smpoynjqycgcdpohhhqaaaa&sa=x&ved=2ahukewiondmlpptpahxtzxuihdkzcksqmxmoatapegqibxad 14 review a narrative review of herbal preparations against rna viruses saeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 antimicrobial. g. ulmifoli extract inhibited poliovirus-1 replication by 26% in hep-2 cells and restricted the synthesis of viral antigens in the infected cell culture.112 azadirachta indica azadirachta indica (neem) is grown in tropical countries and has been reported to possess anti-inflammatory, antipyretic, and hypoglycemic activities. the aqueous extract of the leaves of a. indica inhibited dengue virus type-2 (den-2) in vitro c6/36 cell line and in vivo (mice).113 olea europaea olive leaf extract inhibited replication of viral hemorrhagic septicaemia virus (vhsv).114 the extract also inhibited acute infection and cell-to-cell transfer of hiv-1 virus in mt2 cell line.115 aloe vera a set of experiments suggested that a. vera has potent antiviral activity. a. vera contains bioactive virucidal compounds such as anthraquinones. as mentioned, anthraquinones like some antiviral drugs (lopinavir, ritonavir), alone or in combination with other medications, can target sarscov-2 protease 3clpro.116 acemannan is the predominant acetylated polysaccharide mannan extracted from a. vera gel and has been approved by the us fda for the treatment of hiv-1 in humans. acemannan inhibits glycosylation of viral proteins and inhibits cell fusion and suppression of virus release.117 essential oils essential oils contain a large number of compounds and may target several purposes. however, in most cases, synergistic mechanisms are involved in their antimicrobial activity. studies have shown essential oils are complex mixtures of lipophilic and volatile secondary metabolites isolated from plants such as monoterpenes (hydrocarbon and oxygenated monoterpens), sesquiterpenes (hydrocarbon and oxygenated sesquiterpens), and/or phenylpropanoids that are responsible for a broad biological functions such as antimicrobial, antioxidant, anti-inflammatory, anticancer, cancer chemoprotective, repellent and insecticidal, allelopathic, cytotoxicity, and antiviral activities.118, 119 laurus nobilis the essential of the leaves of l. nobilis has been shown to have potent antiviral activity against coronavirus sars. the most important compounds in the essence of this herb include β-ocimene, 1,8-cineole, α-pinene, and β-pinene. the ic50 of l. nobilis was measured 120 mg/ml with a selective index of 4.2 (si; tc50/ic50).120 mosla dianthera this herb is used as an aromatic herb in traditional medicine in the treatment of cough, colds, fever, bronchitis, nasal hyperemia, and headache. the effect of the essential oil of this herb on mice infected by influenza a was investigated. it was found that this essence has significant effects, including reducing viral lung titers, inhibiting pneumonia, lowering serum levels of ifn-γ and interleukin 4 (il-4), and enhancing the antioxidant activity in lung tissue of mice infected with influenza a.121 coridothymus capitatus, origanum dictamnus and salvia fruticosa a mixture of aromatic plant essential oils consisted of c. capitatus, o. dictamnus, and s. fruticosa was tested against the h1n1 influenza virus and hrv14 rhinovirus in hela and mdck cells. it was found that this compound caused a defect in the nucleoprotein trafficking of the virus in vitro, thereby, has the potential to be used as an herbal drug against respiratory system viruses such as h1n1 influenza and rhinovirus hrv14.122 melaleuca alternifolia the antiviral effect of this essence on the mdck cell line was investigated using plaque reduction assay. the results of this study confirmed the antiviral activity of the essence of m. alternifolia against influenza a subtype h1n1, which was implicated to the presence of terpinen-4-ol, as the essence active ingredient.123 eucalyptus camaldulensis the essential oil of e. camaldulensis reduced the proliferation of coxsackie b4, rotavirus, and herpes simplex virus (hsv). moreover, the methanolic extract of this plant attenuated the activity of hsv, varicella zoster, poliovirus, and echovirus.124-126 artemisia princeps the essential oil of a. princeps var. orientalis and its bioactive components, including borneol, athujone, and camphor, were studied on two noroviruses; mnv-1 and calicivirus-f9 by time-of-addition plaque assays. the a. princeps essence at concentrations of 0.1 and 0.01 inhibited the growth of calicivirus-f9 and mnv-1 by 48% and 64% (v/v), respectively. borneol and camphor showed no antiviral activity, whereas athujone, the major compound of the essence, strongly and dose-dependently inhibited virus infection.127, 128 heterothalamus alienus and buddleja cordobensis in a study on seven herbs (pectis odorata, gaillardia megapotamica, heterothalamus alienus, aloysia triphylla, artemisia mendozana, jungia polita, buddleja cordobensis) indigenous to the south america, their cellular properties (cytotoxicity) and their inhibitory effects on hsv-1, dengue virus type 2 (denv-2) (causing dengue fever, dengue hemorrhagic fever, and dengue shock syndrome), and junin virus (junv) (the cause of argentine hemorrhagic fever) were evaluated. the strongest association between cytotoxicity and antiviral activities was observed for the essence of hetero-thalamus alienus and b. cordobensis against junv virus.129 lippia junelliana and lippia turbinate the essential oils of south american species named as l. junelliana and l. turbinate could temperatureand time dependently inhibit junv in vero cells in vitro (vc50: 14–20 ppm).130 eupatorium patens the essential oil of the leaves, flowers, and fruits of e. patens (asteraceae), indigenous to south america, potently restrained the den-2 replication (vc50: 150 ppm). the https://www.sciencedirect.com/topics/medicine-and-dentistry/dengue-fever https://www.sciencedirect.com/topics/medicine-and-dentistry/dengue-fever 15 review a narrative review of herbal preparations against rna virusessaeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 main components of the essential oil were d-germacrene; β-caryophyllene; bicyclogermacrene; α-pinene; caryophyllene oxide.130 cinnamomum zeylanicum, daucuscarota, eucalyptus globulus and rosmarinus officinalis the blend of essential oil of these herbs inhibited h1n1 subtype of influenza a virus function, probably through interfering the formation of the virus coat and inhibiting its dna polymerase enzyme. for h1n1, a reduction was greater than 99%.131-133 fortunella margarita the essential oil compound of the fruit and leaves of f. margarita was found effective against avian influenza virus subtype h5n1 in mdck cell line, which was associated with the presence of α-terpineol in its fruit. the fruit essential oil has been shown to be more effective and caused an 80% inhibition of the virus activity.134 melissa officinalis the inhibitory effect of the essential oil of m. officinalis on the avian influenza h9n2 subtype was investigated in the mdck cell line. it has been found that this herb has a synergistic effect in inhibiting the h9n2 virus with noseltamivir. m. officinalis can interact with cell surface proteins (i.e., masking the cell surface), thereby blocking the virus binding to cellular receptors.135 pogostemon cablin the antiviral activity of the essential oil obtained from p. cablin has been demonstrated against h1n1 and h2n2 influenza virus. oral administration of p. cablin essential oil in mice has also been shown to protect against influenza virus infection by enhancing the immune response and decreasing the systemic and pulmonary inflammatory response.136-138 trachyspermum ammi the essential oil of t. mumammi was found efficient against the japanese encephalitis virus (jev) in vitro. jev titration was determined by plaque assay, and the antiviral activity of this plant was measured in vitro using a plaque reduction neutralization test. treatment of vero cell line with this essential oil in both preand post-exposure treatments reduced the virus titers by 80% and 40%, respectively.139 herbal active biochemicals cinnamaldehyde cinnamaldehyde, an abundant aromatic phenylpropanoid in cinnamomi cortex (cinnamomum verum) has been studied for its potential properties against the influenza virus. the results of reverse transcription (rt-pcr) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) analyses showed that cinnamaldehyde inhibited the synthesis of influenza virus proteins at the post-transcriptional level. in mice infected with influenza virus, inhalation (50 mg in the cage daily) and nasal inoculation (250 g for each rat daily) of this compound over 8 days increased the survival rate from 20% to 100% and 70%, respectively. importantly, inhalation of cinnamaldehyde reduced virus titers in the bronchoalveolar lavage on the sixth day after infection142 (table 4). 5,7,3’, 4’-tetrahydroxy-2’ (3,3-dimethylallyl) isoflavone 5,7,3’, 4’-tetrahydroxy-2’ (3,3-dimethylallyl) isoflavone, is extracted from psorothamnus arborescens. the anti leishmanial property of this compound has already been proven in the scientific literature. chymotrypsin virus-like cysteine protease enzyme (3clpro) is an essential factor for the replication cycle of coronavirus, representing a potential target for the treatment of such disease. this compound has a high affinity to the catalytic domain of 3clpro enzyme. it also can bind receptor-binding residues of the 3clpro143, 144 (table 4). 1,2,3,4,6-penta-o-galloyl-β-d-glucoside a compound called 1,2,3,4,6-penta-o-galloyl-β-d-glucoside, extractable from the aerial parts of saxifraga melanocentra, is responsible for the anti-hepatitis c virus (hcv) effect of the herb. the compound was also a potent inhibitor of the hcv serine protease enzyme145 (table 4). myricitrin and scutellarin myricitrin can be obtained from a plant called myrica cerifera. it also can bind to and inhibit the catalytic domain of 3clpro enzyme with high affinity. modeling analysis also showed that scutellarin or myristine from scutellaria baicalensis could directly link the atp/adp binding site of the sars-cov virus helicase enzyme (nsp13), thereby preventing direct binding of atp/adp to it. in particular, myricetin interferes with the atpase activity of the sars-cov protein helicase. it is likely to do so by directly interacting with essential residues of the atpase domain, such as n265, y269, and r443146, 147 (table 4). glycyrrhizin and licorine glycyrrhizin, the bioactive component of licorice and lycorine from red spider lily (lycoris radiata), exhibited a potent anti-sars-cov corona activity. however, the effective concentration of glycyrrhizin (ec50) for inhibiting viral infection was very high (ec50 300 mg/l). glycyrrhizin was most effective when given both during and after the adsorption period148, 149 (table 4). quercetin quercetin is the most abundant dietary plant pigment flavonoid (i.e., in onion and garlic). it was reported that this compound inhibited the activity of 3clpro of sars-cov with a fret method. its shown quercetin has low toxicity to the cells in vitro. their mers‐cov 3clpro inhibitory activity is lower than their corresponding compounds at 40 μm150 (table 4). baicalein scutellaria baicalensis is one of the genus that may use as a source of this flavonoid. ethanolic extract of s. baicalensis inhibited sars-cov-2 3clpro activity in vitro and the replication of sars-cov-2 in vero cells with an ec50 of 0.74 µg/ml. baicalein strongly inhibited sars-cov-2 3clpro activity with an ic50 of 0.39 µm151 (table 4). 16 review a narrative review of herbal preparations against rna viruses saeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 ta bl e 4. a nt iv ira l p hy to ch em ic al s e ffe ct iv e on r na v iru se s. co m po un d so ur ce he rb al fa m ily vi ru ci da l t ar ge t ty pe o f st ud y eff ec tiv e do sa ge ce ll ty pe /a ni m al m od el vi ru s re fe re nc e m yr ic it ri n m yr ic a ce rif er a m yr ic ac ea e 3c lp ro in hi bi to ry (a n ec es sa ry pr ot ea se fo r v ira l g ro w th ) 3d st ru ct ur e si m ul at io n sa rs -c ov -2 14 6 sc ut el la ri n sc ut el la ria b ai ca le ns is la m ia ce ae h el ic as e (n sp 13 ) i nh ib ito ry in v itr o 0. 86 ± 0 .4 8 μm m cf 10 a sa rs -c ov -2 14 7 g ly cy rr hi zi n an d li co ri ne ly co ris ra di at e a m ar yl lid ac ea e in hi bi t c pe (v iru sin du ce d cy to pa th ic e ffe ct ) r ep lic at io n in hi bi to r in v itr o m ts a ss ay 1 5. 07 ± 1. 2 nm 30 0 m g/ l ve ro sa rs -c ov -2 14 8, 1 49 ci nn am al de hy de ci nn am om i c or te x m yr ic ac ea e su rv iv al ra te (i n vi vo ) p ro te in sy nt he si s (in v itr o) in v iv o & in vi tro 1 in ha la tio n (5 0 m g/ ca ge / da y) 2 i no cu la tio n (2 50 μ g/ m ou se /d ay ) 2 0– 20 0 μm ic r fe m al e m ic e m d ck in flu en za a (h 1n 1 su bt yp e) 14 2 5, 7, 3 ‘, 4 ’-t et ra hy dr ox y2’ ( 3, 3di m et hy la lly l) is ofl av on e ps or ot ha m nu s ar bo re sc en s fa ba ce ae vi ru ci da l a ct iv ity in v itr o 7. 1 µm ve ro co ro na vi ru s 14 3, 1 44 q ue rc et in 3c lp ro in hi bi to ry in v itr o 40  μ m e. c ol i b l2 1 sa rs -c ov 15 0 ba ic al en si s sc ut el la ria b ai ca le ns is la m ia ce ae 3c lp ro in hi bi to ry in v itr o 0. 39 µ m ve ro sa rs -c ov -2 15 1 se nn os id e a rh eu m p al m at um l . an d rh eu m o ffi ci na le ba ill . po ly go na ce ae re ve rs e tr an sc rip ta se rn as e h r tas so ci at ed fu nc tio ns in te gr as e in v itr o 2– 5 μm ju rk at h iv -1 15 2 te tr ao -g al lo yl -β -d gl uc os e ch in es e he rb s vi ru ci da l a ct iv ity in v itr o 4. 5 m ve ro sa rs -c ov 15 3 lu te ol in o no po rd um il ly ric um l . as te ra ce ae rn as e h r tas so ci at ed in v itr o 12 .8  μ m ju rk at h iv -1 15 4 tr yp ta nt hr in st ro bi la nt he s cu si a ac an th ac ea e in hi bi ts e ar ly a nd la te s ta ge s of re pl ic at io n, b y bl oc ki ng v ira l r n a sy nt he si s an d pa pa in -li ke p ro te as e 2 ac tiv ity in v itr o 1. 52 µ m ll cm k2 co ro na vi ru s n l6 3 15 5 te lli m ag ra nd in eu ge ni a ca ry op hy lla ta , m yr ta ce ae vi ru sce ll fu si on in v itr o 16 .1 2 ± 1 .9 8 m g/ m l sy nc yt ia h iv -1 15 9 g lo bo id na n a eu ca ly pt us g lo bo id ea m yr ta ce ae h iv -1 in te gr as e ac tiv ity in v itr o 0. 64 μ m h ut 78 t -c el l h iv -1 16 0 g in kg ol ic a ci d ch in es e he rb al m ed ic in es in hi bi te d h iv p ro te as e ac tiv ity in v itr o 31 .2 μ g/ m l ju rk at c el ls h iv -1 93 k ae m pf er ol d er iv at iv es bl oc k th e 3a c ha nn el p ro te in s in v itr o 2. 3 µm xe no pu s oo cy te co ro na vi ru s 15 6 sa ik os ap on in s a bs or pt io n an d pe ne tr at io n of th e vi ru s in v itr o 25 m m ol /l m rc -5 co ro na vi ru s 15 8 1, 2, 3, 4, 6pe nt ao -g al lo yl βd -g lu co si de sa xi fra ga m el an oc en tra sa xi fra ge s in hi bi ts n s3 s er in e pr ot ea se in v itr o 0. 1.0 01 m g/ m l co s7 c el ls h ep at iti s c vi ru s 14 5 a rt es un at e ar te m isi a an nu a as te ra ce ae in hi bi tio n of h iv -1 re pl ic at io n in v itr o 60 0 nm h iv -1 15 7 https://en.wikipedia.org/wiki/myricaceae https://en.wikipedia.org/wiki/lamiaceae https://en.wikipedia.org/wiki/myricaceae https://en.wikipedia.org/wiki/fabaceae https://en.wikipedia.org/wiki/lamiaceae https://en.wikipedia.org/wiki/polygonaceae https://en.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/acanthaceae https://www.google.com/search?rlz=1c1gcea_enir901ir901&sxsrf=alekk02ce63oqrnlmgvz4e_i_hph89tgzw:1591089346063&q=myrtaceae&stick=h4siaaaaaaaaaongvuluz9u3mmxlskxfxmrpw1lukpicmpgkakb0cv8zaaaa&sa=x&ved=2ahukewjbnzza5elpahwnwqykhyx6bhsqmxmoataeegqierad https://en.wikipedia.org/wiki/myrtaceae https://www.google.com/search?sxsrf=alekk03cffsnsjhh-egw6bn2x7a0pfv7bg:1587287430614&q=saxifragaceae&stick=h4siaaaaaaaaaongvuluz9u3mlywni1-xojmlfdyxz1hkatja05eyzth4groyc93zsvjlkkuuunig7jkuhilelo0gks4urbcnkwsvmgjfzlprynpicmpiaka-zhom2maaaa&sxsrf=alekk03cffsnsjhh-egw6bn2x7a0pfv7bg:1587287430614 https://www.google.com/search?rlz=1c1asuc_enir712ir712&sxsrf=alekk01vqo9cs2hasxmrzpj2xtx2xxj-sa:1591684252425&q=asteraceae&stick=h4siaaaaaaaaaongvmlxz9u3ydetwstk5vhcklqumjyamaoace7djhgaaaa&sa=x&ved=2ahukewji8tbzjftpahxby6qkhbnraisqmxmoataeegqidbad 17 review a narrative review of herbal preparations against rna virusessaeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 sennoside a sennoside a from dried roots of rheum palmatum l. and rheum officinale baill is hiv-1 rt inhibitor effective phytochemical on both hiv-1 reverse transcriptase and rnase h rt-associated functions in biochemical assays. besides, sennoside a affected the hiv-1 integrase activity in vitro and hiv-1 replication in jurkat cell line. sennoside a inhibited both hiv-1 rt-associated functions with ic50 values of 2–5 μm range.152 (table 4). tetra-o-galloyl-β-d-glucose this small molecule from chinese herbs exhibited prominent anti-sars-cov activity with a ec50 concentration of 4.5 m in vero e6 cells153 (table 4). luteolin among seven compounds isolated from onopordum illyricum l., luteolin was the most effective on hiv-1 rnase h rt-associated function in a low concentration without cytotoxicity (ic50 of 12.8 μm)154 (table 4). tryptanthrin the antiviral activity of tryptanthrin isolated from strobilanthes cusia was investigated against coronavirus nl63 in llc-mk2 cell line. tryptanthrin effectively inhibited the cytopathic effect and virus yield (ic50 = 1.52 µm) in hcov-nl63-infected cells. this molecule prevented the early and late stages of hcov-nl63 replication, mainly by blocking the viral rna genome synthesis and papain-like protease 2 activity155 (table 4). kaempferol derivatives these phytochemicals have a potency to block the 3a channel proteins of coronaviruses. the yxxφ domain of 3a channel is a protein internalization signal which is involved in clathrin-mediated endocytosis, therefore its involved in virus cell entry. in a research study, using xenopus oocyte for heterologous expression and applied voltage-clamp techniques, the glycoside juglanin (carrying an arabinose residue) was found as the most effective kaempferol derivatives with an ic50 value of 2.3 µm for inhibition of the 3a-mediated current156 (table 4). artesunate bioingredients of artemisia annua can play potential protective roles against infections by viruses, specifically hsv-1, hbv, hcv, bovine viral diarrhea virus, and epstein-barr virus. it was demonstrated that 10 days administration of artesunate (a semi-synthetic derivative of artemisinin used to treat malaria) at 600 nm inhibited hiv-1 replication157 (table 4). saikosaponins among saikosaponins (a, b2, c, and d) tested on coronavirus 229e in human fetal lung fibroblasts, saikosaponin b2 demonstrated a potent anticoronaviral activity at a concentration of 25 mmol/l. although, the mode of action of this compound possibly involved interference in the early stage of viral replication such as absorption and penetration of the virus158 (table 4). tellimagrandin initially, the interaction between the hiv envelope glycoprotein gp120 and the cell membrane protein cd4 results in virus-cell fusion. out of isolated compounds from eugenia caryophyllata, tellimagrandin significantly inhibited the viruscell fusion and syncytia formation in hiv-1 with an ic50 value of 16.12±1.98 mg/ml159 (table 4). ginkgolic acid in jurkat cells, ginkgolic acid (31.2 μg/ml) isolated from ginkgo leaves, inhibited the hiv protease activity by 60% in a dose-dependent manner. moreover, ginkgolic acid treatment (50 and 100 μg/ml) effectively inhibited the hiv infection at day 7 dose-dependently93 (table 4). globoidnan a it was reported that globoidnan a, a lignan obtainable from eucalyptus globoidea, can interfere with hiv-1 integrase activity. this compound was found to inhibit the combined 3’ processing and strand transfer activity of hiv integrase with an ic50 = 0.64 μm160 (table 4). antiviral herbal medicine formulations kangbingdu kangbingdu (kbd) is a chinese traditional medicinal formula in form of a classic oral liquid that has been modified based on the traditional chinese “baihutang” and “qingwenbaiduyin” medicine formulations. kbd is often used to improve the clinical symptoms of viral diseases, especially the influenza virus. kbd is composed of radix isatidis, rhizoma phragmitis, radix rehmanniae, radix curcumae, rhizoma anemarrhenae, rhizoma acori tatarinowii, herba pogostemonis, fructus forsythiae and gypsum fibrosum. kbd significantly reduced the sensitivity of male kunming mice to influenza viruses, which is evidenced by reduced mortality, decreased inflammation, and inhibited viral replication in the pulmonary system. in a549 cells, administration of kbd increased the protein expression of mavs (mitochondrial antiviral signaling protein) and the levels of ifn-β protein and interferon-induced transmembrane-3 protein, resulting in inhibition of viral infection. it was shown that (r, s) -goitrine, mangiferine, forsythine, and forsythoside a were other active ingredients of kbd against the influenza virus. the mitochondrial antiviral signaling pathway was introduced as the primary mechanism of action of kbd.161 maxingshigan–yinqiaosan in a prospective rct, the efficacy and safety of oseltamivir and a traditional chinese mixture named “maxingshiganyinqiaosan” in treatment of uncomplicated influenza h1n1 subtype were compared. maxingshigan–yinqiaosan consisted of 12 herbs: zhimahuang (honey-fried herba ephedrae); zhimu (rhizoma anemarrhenae); qinghao (herba artemisiae annuae); shigao (gypsum fibrosum); yinhua (flos lonicerae japonicae); huangqin (radix scutellariae); chaoxingren (stir-baked semen armeniacae amarum); lianqiao (fructus forsythiae); bohe (fructus forsythiae); zhebeimu (bulbus fritillariae thunbergii); niubangzi (fructus arctii tosum); and gancao (radix et rhizoma glycyrrhizae). clinical interventions and control were given for 5 days with oseltamivir, 75 mg twice daily; maxingshigan–yinqiaosan, 200 ml 4 times daily. oseltamivir and maxingshigan–yinqiaosan, alone or together, reduced time of fever resolution in patients with h1n1 influenza.162 18 review a narrative review of herbal preparations against rna viruses saeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 sheng jiang san sheng jiang san (sjs) is a chinese multi-herbal formulation made of four herbs consist of rhei radix et rhizoma, bombyx batryticatus, cicadae periostracum, and curcumae longae rhizoma. the inhibitory effect of sjs against different strains of influenza virus a/wsn/33 (h1n1) on mdck cells was examined. the ic50 of sjs was lower than 35 μg/ml against h1n1. sjs at 2 mg/ml inhibited the na activity up to 80%. this enzyme cleaves terminal neuraminic acid residues of glycan structures on the surface of the infected cell, thereby facilitating the release and spread of viruses progeny to reach the surrounding uninfected cells. the ic50 of oseltamivir acid was 250 μm against neuraminidase. to evaluate the efficacy of sjs in the influenza virus, infected balb/c mice were employed as in vivo model. oral administration of 1 g/kg/day of sjs for 7 days, exhibited 50% protection of infected mice from h1n1 symptoms. sjs also significantly down-regulated tumor necrosis factor (tnf-α) and up-regulated il-2 of influenza virus induced mice.163 lianhuaqingwen lianhuaqingwen (lh) is another chinese plant medicine composed of 13 herbs with positive impact on sars-cov-2 by inhibiting viral replication. a research study revealed that lh significantly inhibited sars-cov-2 replication in vero e6 cells (600 μg/ml) and markedly reduced mrna transcription of pro-inflammatory cytokines (tnf-α, il-6, ccl-2/mcp-1, and cxcl-10/ip-10).164 in addition, lh in the form of capsule (4 capsules, thrice daily for 14 days) in patients who were suffering covid-19 conferred therapeutic effects by improving the recovery rate of symptoms, shortening recovery time, and improving the recovery of chest radiologic abnormalities.165 san wu huangqin decoction san wu huangqin decoction (swhd) is a chinese compound formulation consists of sophora flavescens, scutellaria baicalensis, and rehmannia glutinosa. this herbal formulation could effectively inhibit the influenza a/pr/8/34 (h1n1) virus at all steps of virus replication in vitro. the rna expression of four h1n1 target viral proteins (hemagglutinin, na, np nucleoprotein, and matrix-2) was significantly down-regulated in mdck cells. in vivo, swhd at 23.40 and 11.70 g/kg significantly attenuated clinical symptoms, reduced mortality, and increased the survival time of infected animals. swhd mediated the pulmonary index, viral titer, pathological changes in lung tissue, and expression of the main ifv proteins.166 kabasura kudineer cresset flare is a software that used for molecular docking studies against the spike protein sars-cov-2. in silico molecular docking assay, examining pharmacokinetics parameters, have shown that six plant species in kabasura kudineer formula (sida acuta, adhatoda vasica, andrographis paniculata, tinospora cordifolia, costus speciosus, plectranthus ambonicus) have potential to directly suppress the sars-cov-2 spike protein.167 poly-herbal gel the anti-hiv activity of an aqueous gel formula containing ethanolic extract of the heartwood of acacia catechu, leaves of lagerstroemia speciosa, and fruits of aegle marmelos, phyllanthus emblica, and terminalia chebula was examined against cxcr4 tropic and ccr5 tropic viruses. the gel inhibited viral activity with ic50 values of 58.17 ± 4.4 and 63.54 ± 6.8 μg/ml for cxcr4 and ccr5, respectively. cxcr4 and ccr5 are two of several chemokine receptors used by the hiv to infect t cd4+ lymphocytes. furthermore, this gel could inhibit three key enzymes of the hiv-1 reverse transcriptase, protease, and integrase enzymes significantly.168 qing fei pai du tang qing fei pai du tang (qfpdt) is a chinese medicinal preparation consisting of 21 plants derivation of 5 conventional formula. some studies have reported the qfpdt inhibitory effect of qfpdt on covid-19. by several mechanisms, qfpdt could prevent the progression of mild covid-19 cases and shorten the average duration of symptoms and hospitalization. necessary scientific studies, supported by network pharmacology, reviewed the possible therapeutic targets of qfpdt and its constituents, including ephedra sinica, bupleurum chinense, pogostemon cablin, cinnamomum cassia, and scutellaria baicalensis. their findings indicated that main herbs of qfpdt have antiviral effects via different mechanisms including direct effect on virus replication and autophagy; regulation of host pathways like toll-like receptors, rig-1-like helicases, ampactivated protein kinase, phosphatidyl inositol-3-kinase/ protein kinase b or extracellular regulated kinase 1/2/mitogen-activated protein kinase signal pathways; elevation of the human defense system via t and b cell functions, and free radical scavenging activities by enhancing antioxidant enzymes. qfpdt also modulated inflammation conditions through suppression of inflammatory-related genes, signal pathways and cytokines.169 chai-ling chai-ling decoction (cld), derived from a modification of two decoctions, include xiao-chai-hu (xch) and wu-lingsan (wls) decoction, has been used to treat early-stage of covid-19. the possible mechanisms of cld in covid-19 were preliminarily investigated, relying on network pharmacology and molecular docking method. cld might reduce the inflammatory response and improve lung damages of covid19 through interleukin 17 signaling, t helper cell 17 differentiation, tumor necrosis factor signaling, and hypoxia-inducible factor-1 signaling. besides, molecular docking assay indicated that beta-sitosterol, kaempferol, and stigmasterol were the primary three components in cld with the highest affinity to sars-cov-2 and ace2.170 san yao san san yao san fang (sysf) was shown effective in patients with covid-19. the synergy of sysf and western interventions improved the recovery rate of covid-19 symptoms such as fever, cough, and fatigue, and other symptoms such as headache, gastrointestinal symptoms, myalgia, dyspnoea, and chest tightness.171 conclusion throughout history, humans have been dependent on plant/ natural sources for the treatment of various infections. over the last few decades, hundreds of plant and herb species were shown to have potential antiviral activities, which 19 review a narrative review of herbal preparations against rna virusessaeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 were attributed to a variety of active phytochemicals including flavonoids, terpenoids, lignans, sulphides, polyphenolics, coumarins, saponins, furyl compounds, alkaloids, polyines, thiophenes, proteins, peptides, and some essential oils. plant extracts or substances derived from plants possess their antiviral effects through inhibition of viral replication and by deterring the activity of viral reproduction or interacting with vital viral proteins that are associated with virulence.172 their ability to act as bioreactors and production of vaccines under plant platforms are another advantage of plant systems to induce mucosal or long-term immunity against viral infection. very recently, it was proposed that highly polar glycosylated compounds and polyphenols are more effective antiviral substances, particularly when being administered orally.13 in our study, we gathered information about plant extracts, essential oils, and phytochemicals that are favorable for life-threatening coronaviruses diseases and can be potential candidates for further development of antivirals. we found those plant families, including lamiaceae, asteraceae, and myrtaceae contain the highest number of species with anti-coronaviruses activities, respectively. it can be suggested that the combination of these antiviral ingredients with each other, any synthetic compound, or already fda-approved drugs or inhibitors can be a novel approach for antiviral therapies. evaluation of efficacy and adverse effects of herbal medicines for the treatment of viral infection is warranted. recent findings exhibited that a combination of herbal therapy with modern medicines significantly improved the total effective rate and syndrome score of cough, fever, dry, and sore throat, and fatigue in covid-19 patients, signifying the advantage of herbal therapy for viral infections.173 conflict of interest none references 1. sinkovics j, horvath j, horak a. the origin and evolution of viruses (a review). acta microbiol immunol hung. 1998;45(3-4):349-90. 2. woolhouse m, scott f, hudson z, howey r, chase-topping mjptotrsbbs. human viruses: discovery and emergence. 2012;367(1604):2864-71. 3. lodish h, berk a, zipursky sl, matsudaira p, baltimore d, darnell j. viruses: structure, function, and uses. molecular cell biology 4th edition: wh freeman; 2000. 4. moss jajrt. hiv/aids review. 2013;84(3):247-67. 5. van bm, debyser zjar. hiv-1 integration: an interplay between hiv-1 integrase, cellular and viral proteins. 2005;7(1):26-43. 6. kilcher s, mercer jjv. dna virus uncoating. 2015;479:578-90. 7. hagan mfjaicp. modeling viral capsid assembly. 2014;155:1. 8. lindenbach bd. virion assembly and release. hepatitis c virus: from molecular virology to antiviral therapy: springer; 2013. p. 199-218. 9. kraemer m, cummings d, funk s, reiner r, faria n, pybus o, et al. reconstruction and prediction of viral disease epidemics. epidemiol infect. 2019;147. 10. walls ac, park y-j, tortorici ma, wall a, mcguire at, veesler d. structure, function, and antigenicity of the sars-cov-2 spike glycoprotein. cell. 2020;181(2):281-92.e6. 11. nascimento junior jac, santos am, quintans-júnior lj, walker cib, borges lp, serafini mr. sars, mers and sars-cov-2 (covid-19) treatment: a patent review. expert opinion on therapeutic patents. 2020(just-accepted). 12. lu h. drug treatment options for the 2019-new coronavirus (2019-ncov). biosci trends. 2020;14(1):69-71. 13. islam mt, sarkar c, el-kersh dm, jamaddar s, uddin sj, shilpi ja, et al. natural products and their derivatives against coronavirus: a review of the non-clinical and pre-clinical data. phytother res. 14. mani js, johnson jb, steel jc, broszczak da, neilsen pm, walsh kb, et al. natural product-derived phytochemicals as potential agents against coronaviruses: a review. virus res. 2020;284:197989. 15. richman dd, whitley rj, hayden fg. clinical virology: john wiley & sons; 2016. 16. fields bn, knipe dm, howley pm, everiss kd, kung h-j. fundamental virology: lippincott-raven philadelphia^ epa pa; 1996. 17. modrow s, falke d, truyen u, schätzl h. molecular virology: springer berlin heidelberg; 2013. 18. tsurumi t, fujita m, kudoh a. latent and lytic epstein‐barr virus replication strategies. rev med virol. 2005;15(1):3-15. 19. kim k-h, bae j-w. amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded dna viruses. appl environ microbiol. 2011;77(21):7663-8. 20. weitzman md, fradet-turcotte a. virus dna replication and the host dna damage response. annu rev virol. 2018;5(1):141-64. 21. charman m, weitzman md. replication compartments of dna viruses in the nucleus: location, location, location. viruses. 2020;12(2):151. 22. tattersall p, cotmore sf. parvoviruses. e ls. 2001. 23. murphy f. virus taxonomy; classification and nomenclature of viruses; 6th report of the international committee on taxonomy of viruses. arch virol suppl. 1995;10:415-33. 24. palese p, zheng h, engelhardt og, pleschka s, garcía-sastre a. negativestrand rna viruses: genetic engineering and applications. proc natl acad sci. 1996;93(21):11354-8. 25. garcía-sastre a. negative-strand rna viruses: applications to biotechnology. trends biotechnol. 1998;16(5):230-5. 26. zarbl h, millward s. the reovirus multiplication cycle. the reoviridae: springer; 1983. p. 107-96. 27. murray pr, rosenthal ks, pfaller ma. medical microbiology: elsevier health sciences; 2015. 28. libonati m, carsana a, furia a. double-stranded rna. mol cell biochem. 1980;31(1):147-64. 29. coffin jm. structure and classification of retroviruses. in: levy ja, editor. the retroviridae. boston, ma: springer us; 1992. p. 19-49. 30. stocking c, kozak c. endogenous retroviruses. cell mol life sci. 2008;65(21):3383-98. 31. gorbalenya ae. severe acute respiratory syndrome-related coronavirus–the species and its viruses, a statement of the coronavirus study group. biorxiv. 2020. 32. wang c, horby pw, hayden fg, gao gf. a novel coronavirus outbreak of global health concern. the lancet. 2020;395(10223):470-3. 33. kristoffersen aw, nordbø sa, rognlien a-gw, christensen a, døllner h. coronavirus causes lower respiratory tract infections less frequently than rsv in hospitalized norwegian children. ped infect dis j. 2011;30(4): 279-83. 34. stewart jn, mounir s, talbot pj. human coronavirus gene expression in the brains of multiple sclerosis patients. virology. 1992;191(1):502-5. 35. lai mm. coronaviridae: the viruses and their replication 2001. 36. wang l-f, shi z, zhang s, field h, daszak p, eaton bt. review of bats and sars. emerg infect dis. 2006;12(12):1834. 37. cui j, li f, shi z-l. origin and evolution of pathogenic coronaviruses. nat rev microbiol. 2019;17(3):181-92. 38. cauchemez s, van kerkhove m, riley s, donnelly c, fraser c, ferguson n. transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart. euro surveill bull eur sur les maladies transmissibles eur commun dis bull. 2013;18(24). 39. andersen kg, rambaut a, lipkin wi, holmes ec, garry rf. the proximal origin of sars-cov-2. nat med. 2020;26(4):450-2. 40. brian d, baric r. coronavirus genome structure and replication. coronavirus replication and reverse genetics: springer; 2005. p. 1-30. 41. holmes kv. coronaviruses (coronaviridae). encyclopedia of virology. 1999:291-8. 42. tan y-j, lim sg, hong w. characterization of viral proteins encoded by the sars-coronavirus genome. antiviral res. 2005;65(2):69-78. https://en.wikipedia.org/wiki/lamiaceae 20 review a narrative review of herbal preparations against rna viruses saeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 43. hoffmann m, kleine-weber h, schroeder s, krüger n, herrler t, erichsen s, et al. sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor. cell. 2020. 44. guo y-r, cao q-d, hong z-s, tan y-y, chen s-d, jin h-j, et al. the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak–an update on the status. military med res. 2020;7(1):1-10. 45. walls ac, park y-j, tortorici ma, wall a, mcguire at, veesler d. structure, function, and antigenicity of the sars-cov-2 spike glycoprotein. cell. 2020. 46. sola i, almazan f, zuniga s, enjuanes l. continuous and discontinuous rna synthesis in coronaviruses. annu rev virol. 2015;2:265-88. 47. wu h-y, guy js, yoo d, vlasak r, urbach e, brian da. common rna replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavius molecules. virology. 2003;315(1):174-83. 48. hussain s, chen y, yang y, xu j, peng y, wu y, et al. identification of novel subgenomic rnas and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus. j virol. 2005;79(9):5288-95. 49. qinfen z, jinming c, xiaojun h, huanying z, jicheng h, ling f, et al. the life cycle of sars coronavirus in vero e6 cells. j med virol. 2004;73(3):332-7. 50. van boheemen s, de graaf m, lauber c, bestebroer tm, raj vs, zaki am, et al. genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mbio. 2012;3(6):e00473-12. 51. baba m, nakajima m, schols d, pauwels r, balzarini j, de clercq ejar. pentosan polysulfate, a sulfated oligosaccharide, is a potent and selective anti-hiv agent in vitro. 1988;9(6):335-43. 52. baba m, snoeck r, pauwels r, de clercq ejaa, chemotherapy. sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus. 1988;32(11):1742-5. 53. pluymers w, neamati n, pannecouque c, fikkert v, marchand c, burke tr, et al. viral entry as the primary target for the anti-hiv activity of chicoric acid and its tetra-acetyl esters. 2000;58(3):641-8. 54. witvrouw m, fikkert v, pluymers w, matthews b, mardel k, schols d, et al. polyanionic (ie, polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle. 2000;58(5):1100-8. 55. baranova e, shastina n, shvets vjrjobc. polyanionic inhibitors of hiv adsorption. 2011;37(5):527. 56. andrei g, de clercq e, snoeck r. viral dna polymerase inhibitors. viral genome replication: springer; 2009. p. 481-526. 57. mcguigan c, barucki h, blewett s, carangio a, erichsen jt, andrei g, et al. highly potent and selective inhibition of varicella-zoster virus by bicyclic furopyrimidine nucleosides bearing an aryl side chain. 2000;43(26):4993-7. 58. wang j, froeyen m, hendrix c, andrei g, snoeck r, de clercq e, et al. the cyclohexene ring system as a furanose mimic: synthesis and antiviral activity of both enantiomers of cyclohexenylguanine. 2000;43(4):736-45. 59. verweij-van wissen c, aarnoutse r, burger djjocb. simultaneous determination of the hiv nucleoside analogue reverse transcriptase inhibitors lamivudine, didanosine, stavudine, zidovudine and abacavir in human plasma by reversed phase high performance liquid chromatography. 2005;816(1-2):121-9. 60. richman ddjat. antiretroviral activity of emtricitabine, a potent nucleoside reverse transcriptase inhibitor. 2001;6(2):83-8. 61. das k, clark ad, lewi pj, heeres j, de jonge mr, koymans lm, et al. roles of conformational and positional adaptability in structure-based design of tmc125-r165335 (etravirine) and related non-nucleoside reverse transcriptase inhibitors that are highly potent and effective against wildtype and drug-resistant hiv-1 variants. 2004;47(10):2550-60. 62. pauwels rjcoip. new non-nucleoside reverse transcriptase inhibitors (nnrtis) in development for the treatment of hiv infections. 2004;4(5): 437-46. 63. de clercq ejc, biodiversity. non‐nucleoside reverse transcriptase inhibitors (nnrtis): past, present, and future. 2004;1(1):44-64. 64. donzella ga, schols d, lin sw, esté ja, nagashima ka, maddon pj, et al. amd3100, a small molecule inhibitor of hiv-1 entry via the cxcr4 coreceptor. 1998;4(1):72-7. 65. ding x, zhang x, chong h, zhu y, wei h, wu x, et al. enfuvirtide (t20)-based lipopeptide is a potent hiv-1 cell fusion inhibitor: implications for viral entry and inhibition. 2017;91(18):e00831-17. 66. eggink d, berkhout b, w sanders rjcpd. inhibition of hiv-1 by fusion inhibitors. 2010;16(33):3716-28. 67. de clercq e, holý ajnrdd. acyclic nucleoside phosphonates: a key class of antiviral drugs. 2005;4(11):928-40. 68. de clercq ejbp. acyclic nucleoside phosphonates: past, present and future: bridging chemistry to hiv, hbv, hcv, hpv, adeno-, herpes-, and poxvirus infections: the phosphonate bridge. 2007;73(7):911-22. 69. de clercq ejcmr. clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of dna virus and retrovirus infections. 2003;16(4):569-96. 70. anderson j, schiffer c, lee s-k, swanstrom r. viral protease inhibitors. antiviral strategies: springer; 2009. p. 85-110. 71. piliero pjjeooid. atazanavir: a novel hiv-1 protease inhibitor. 2002;11(9):1295-301. 72. chrusciel ra, strohbach jwjctimc. non-peptidic hiv protease inhibitors. 2004;4(10):1097-114. 73. vergani b, rusconi sjdir. tipranavir in the protease inhibitors arena. 2011;11(4):291-3. 74. zalman l, brothers m, dragovich p, zhou r, prins t, worland s, et al. inhibition of human rhinovirus-induced cytokine production by ag7088, a human rhinovirus 3c protease inhibitor. 2000;44(5):1236-41. 75. basler cf, krogan nj, leung dw, amarasinghe gkjar. virus and host interactions critical for filoviral rna synthesis as therapeutic targets. 2019;162:90-100. 76. gubareva lv, kaiser l, hayden fgjtl. influenza virus neuraminidase inhibitors. 2000;355(9206):827-35. 77. mcnicholl ir, mcnicholl jjjaop. neuraminidase inhibitors: zanamivir and oseltamivir. 2001;35(1):57-70. 78. burnham aj, baranovich t, govorkova eajar. neuraminidase inhibitors for influenza b virus infection: efficacy and resistance. 2013;100(2):520-34. 79. babu ys, chand p, bantia s, kotian p, dehghani a, el-kattan y, et al. bcx1812 (rwj-270201): discovery of a novel, highly potent, orally active, and selective influenza neuraminidase inhibitor through structure-based drug design. 2000;43(19):3482-6. 80. markland w, mcquaid t, jain j, kwong ajaa, chemotherapy. broadspectrum antiviral activity of the imp dehydrogenase inhibitor vx-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon. 2000;44(4):859-66. 81. parker wbjvr. metabolism and antiviral activity of ribavirin. 2005;107(2): 165-71. 82. gish rgjjoac. treating hcv with ribavirin analogues and ribavirin-like molecules. 2006;57(1):8-13. 83. huggins j, zhang z-x, bray mjtjoid. antiviral drug therapy of filovirus infections: s-adenosylhomocysteine hydrolase inhibitors inhibit ebola virus in vitro and in a lethal mouse model. 1999;179(supplement_1):s240-s7. 84. picazo e, giordanetto fjddt. small molecule inhibitors of ebola virus infection. 2015;20(2):277-86. 85. markland w, mcquaid t, jain j, kwong a. broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon. antimicrob agents chemother. 2000;44(4):859-66. 86. parker wb. metabolism and antiviral activity of ribavirin. virus res. 2005;107(2):165-71. 87. dimitrov ds. virus entry: molecular mechanisms and biomedical applications. nat re microbiol. 2004;2(2):109-22. 88. gescher k, kühn j, hafezi w, louis a, derksen a, deters a, et al. inhibition of viral adsorption and penetration by an aqueous extract from rhododendron ferrugineum l. as antiviral principle against herpes simplex virus type-1. fitoterapia. 2011;82(3):408-13. 89. tao yj, ye q. rna virus replication complexes. plos pathogens. 2010;6(7). 90. chin lw, cheng y-w, lin s-s, lai y-y, lin l-y, chou m-y, et al. anti-herpes simplex virus effects of berberine from coptidis rhizoma, a major component of a chinese herbal medicine, ching-wei-san. arch virol. 2010;155(12):1933-41. 91. flexner c. hiv-protease inhibitors. new engl j med. 1998;338(18):1281-93. 92. huff jr. hiv protease: a novel chemotherapeutic target for aids. j med chem. 1991;34(8):2305-14. 93. lü j-m, yan s, jamaluddin s, weakley sm, liang z, siwak eb, et al. ginkgolic acid inhibits hiv protease activity and hiv infection in vitro. med sci monitor int med j exp clin res. 2012;18(8):br293. 94. pommier y, johnson aa, marchand c. integrase inhibitors to treat hiv/aids. nat rev drug discov. 2005;4(3):236-48. 95. suedee a, tewtrakul s, panichayupakaranant p. anti-hiv-1 integrase compound from pometia pinnata leaves. pharm biol. 2013;51(10):1256-61. 96. zhao q, chen x-y, martin c. scutellaria baicalensis, the golden herb from the garden of chinese medicinal plants. sci bull. 2016;61(18):1391-8. 21 review a narrative review of herbal preparations against rna virusessaeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 97. gubareva lv, kaiser l, hayden fg. influenza virus neuraminidase inhibitors. the lancet. 2000;355(9206):827-35. 98. mcauley j, gilbertson b, trifkovic s, brown le, mckimm-breschkin j. influenza virus neuraminidase structure and functions. front microbiol. 2019;10:39. 99. xue h, zhang d-k, yu-ming g, wu-wen f, qin d, zhang c-e, et al. screening and evaluation of commonly-used anti-influenza chinese herbal medicines based on anti-neuraminidase activity. chin j nat med. 2016;14(10):794-800. 100. hudak ka, wang p, tumer ne. a novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped rna template. rna. 2000;6(3):369-80. 101. kaur i, gupta r, puri m. ribosome inactivating proteins from plants inhibiting viruses. virol sin. 2011;26(6):357-65. 102. thabti i, albert q, philippot s, dupire f, westerhuis b, fontanay s, et al. advances on antiviral activity of morus spp. plant extracts: human coronavirus and virus-related respiratory tract infections in the spotlight. molecules. 2020;25(8):1876. 103. signer j, jonsdottir h, albrich w, strasser m, züst r, ryter s. in vitro antiviral activity of echinaforce®, an echinacea purpurea preparation, against common cold coronavirus 229e and highly pathogenic mers-cov and sars-cov. virology. 2020. 104. ngoutane mfopa a, corona a, eloh k, tramontano e, frau a, boyom ff, et al. uvaria angolensis as a promising source of inhibitors of hiv-1 rt-associated rna-dependent dna polymerase and rnase h functions. nat prod res. 2018;32(6):640-7. 105. ulasli m, gurses sa, bayraktar r, yumrutas o, oztuzcu s, igci m, et al. the effects of nigella sativa (ns), anthemis hyalina (ah) and citrus sinensis (cs) extracts on the replication of coronavirus and the expression of trp genes family. mol biol rep. 2014;41(3):1703-11. 106. notka f, meier g, wagner rjar. concerted inhibitory activities of phyllanthus amarus on hiv replication in vitro and ex vivo. 2004;64(2):93-102. 107. yamai m, tsumura k, kimura m, fukuda s, murakami t, kimura yjb, biotechnology,, et al. antiviral activity of a hot water extract of black soybean against a human respiratory illness virus. 2003;67(5):1071-9. 108. zakay-rones z, thom e, wollan t, wadstein j. randomized study of the efficacy and safety of oral elderberry extract in the treatment of influenza a and b virus infections. j int med res. 2004;32(2):132-40. 109. pantev a, ivancheva s, staneva l, serkedjieva jjzfnc. biologically active constituents of a polyphenol extract from geranium sanguineum l. with anti-influenza activity. 2006;61(7-8):508-16. 110. huang kl, lai yk, lin cc, chang jm. inhibition of hepatitis b virus production by boehmeria nivea root extract in hepg2 2.2.15 cells. world j gastroenterol. 2006;12(35):5721-5. 111. chang js, liu hw, wang kc, chen mc, chiang lc, hua yc, et al. ethanol extract of polygonum cuspidatum inhibits hepatitis b virus in a stable hbvproducing cell line. antiviral res. 2005;66(1):29-34. 112. felipe am, rincao vp, benati fj, linhares re, galina kj, de toledo ce, et al. antiviral effect of guazuma ulmifolia and stryphnodendron adstringens on poliovirus and bovine herpesvirus. biol pharm bull. 2006;29(6):1092-5. 113. parida m, upadhyay c, pandya g, jana ajjoe. inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type-2 replication. 2002;79(2):273-8. 114. micol v, caturla n, pérez-fons l, más v, pérez l, estepa ajar. the olive leaf extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus (vhsv). 2005;66(2-3):129-36. 115. lee-huang s, zhang l, huang pl, chang yt, huang pl. anti-hiv activity of olive leaf extract (ole) and modulation of host cell gene expression by hiv-1 infection and ole treatment. biochem biophys res commun. 2003;307(4):1029-37. 116. mpiana pt, ngbolua k-t-n, tshibangu d, kilembe j, mwanangombo d, inkoto c, et al. aloe vera (l.) burm. f. as a potential anti-covid-19 plant: a mini-review of its antiviral activity. 2020. 117. sun z, yu c, wang w, yu g, zhang t, zhang l, et al. aloe polysaccharides inhibit influenza a virus infection-a promising natural anti-flu drug. front microbiol. 2018;9:2338. 118. reichling j, schnitzler p, suschke u, saller r. essential oils of aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties–an overview. complement med res. 2009;16(2):79-90. 119. dhifi w, bellili s, jazi s, bahloul n, mnif w. essential oils’ chemical characterization and investigation of some biological activities: a critical review. medicines. 2016;3(4):25. 120. loizzo mr, saab am, tundis r, statti ga, menichini f, lampronti i, et al. phytochemical analysis and in vitro antiviral activities of the essential oils of seven lebanon species. 2008;5(3):461-70. 121. wu q-f, wang w, dai x-y, wang z-y, shen z-h, ying h-z, et al. chemical compositions and anti-influenza activities of essential oils from mosla dianthera. 2012;139(2):668-71. 122. tseliou m, pirintsos sa, lionis c, castanas e, sourvinos g. antiviral effect of an essential oil combination derived from three aromatic plants (coridothymus capitatus (l.) rchb. f., origanum dictamnus l. and salvia fruticosa mill.) against viruses causing infections of the upper respiratory tract. j herbal med. 2019;17-18:100288. 123. garozzo a, timpanaro r, bisignano b, furneri p, bisignano g, castro ajliam. in vitro antiviral activity of melaleuca alternifolia essential oil. 2009;49(6): 806-8. 124. abu-jafar a, huleihel mjijocv. antiviral activity of eucalyptus camaldulensis leaves ethanolic extract on herpes viruses infection. 2017;1:1-9. 125. adeniyi cba, lawal to, mahady gbjpb. in vitro susceptibility of helicobacter pylori to extracts of eucalyptus camaldulensis and eucalyptus torelliana. 2009;47(1):99-102. 126. el-baz fk, mahmoud k, el-senousy wm, darwesh o, el gohary ajijpsrr. antiviral–antimicrobial and schistosomicidal activities of eucalyptus camaldulensis essential oils. 2015;31(1):262-8. 127. chung msjfs, biotechnology. antiviral activities of artemisia princeps var. orientalis essential oil and its α-thujone against norovirus surrogates. 2017;26(5):1457-61. 128. sinico c, de logu a, lai f, valenti d, manconi m, loy g, et al. liposomal incorporation of artemisia arborescens l. essential oil and in vitro antiviral activity. 2005;59(1):161-8. 129. duschatzky cb, possetto ml, talarico lb, garcía cc, michis f, almeida nv, et al. evaluation of chemical and antiviral properties of essential oils from south american plants. 2005;16(4):247-51. 130. garcia c, talarico l, almeida n, colombres s, duschatzky c, damonte ejpraijdtp, et al. virucidal activity of essential oils from aromatic plants of san luis, argentina. 2003;17(9):1073-5. 131. brochot a, guilbot a, haddioui l, roques cjm. antibacterial, antifungal, and antiviral effects of three essential oil blends. 2017;6(4):e00459. 132. astani a, reichling j, schnitzler pjpraijdtp, derivatives teonp. comparative study on the antiviral activity of selected monoterpenes derived from essential oils. 2010;24(5):673-9. 133. astani a, reichling j, schnitzler pje-bc, medicine a. screening for antiviral activities of isolated compounds from essential oils. 2011;2011. 134. ibrahim na, el-hawary ss, mohammed m, farid m, abdel-wahed nam, ali m, et al. chemical composition, antiviral against avian influenza (h5n1) virus and antimicrobial activities of the essential oils of the leaves and fruits of fortunella margarita, lour. swingle, growing in egypt. j appl pharm sci. 2015;5:006-12. 135. pourghanbari g, nili h, moattari a, mohammadi a, iraji a. antiviral activity of the oseltamivir and melissa officinalis l. essential oil against avian influenza a virus (h9n2). virusdisease. 2016;27(2):170-8. 136. swamy mk, sinniah ur. a comprehensive review on the phytochemical constituents and pharmacological activities of pogostemon cablin benth.: an aromatic medicinal plant of industrial importance. molecules. 2015;20(5):8521-47. 137. li yc, peng sz, chen hm, zhang fx, xu pp, xie jh, et al. oral administration of patchouli alcohol isolated from pogostemonis herba augments protection against influenza viral infection in mice. int immunopharmacol. 2012;12(1):294-301. 138. wu h, li b, wang x, jin m, wang g. inhibitory effect and possible mechanism of action of patchouli alcohol against influenza a (h2n2) virus. molecules. 2011;16(8):6489-501. 139. roy s, chaurvedi p, chowdhary a. evaluation of antiviral activity of essential oil of trachyspermum ammi against japanese encephalitis virus. pharmacognosy res. 2015;7(3):263-7. 140. lee-huang s, zhang l, huang pl, chang y-t, huang pljb, communications br. anti-hiv activity of olive leaf extract (ole) and modulation of host cell gene expression by hiv-1 infection and ole treatment. 2003;307(4):1029-37. 141. cella m, riva d, coulombié f, mersich sjradm. virucidal activity presence in trichilia glabra leaves. 2004;36(3):136-8. 142. hayashi k, imanishi n, kashiwayama y, kawano a, terasawa k, shimada y, et al. inhibitory effect of cinnamaldehyde, derived from cinnamomi cortex, on the growth of influenza a/pr/8 virus in vitro and in vivo. antiviral res. 2007;74(1):1-8. 143. salem mm, werbovetz ka. isoflavonoids and other compounds from psorothamnus arborescens with antiprotozoal activities. j nat prod. 2006;69(1):43-9. 144. zhou j, xie g, yan x. encyclopedia of traditional chinese medicines. isolat compound ab. 2011;1:455. 22 review a narrative review of herbal preparations against rna viruses saeideh momtaz et al. j contemp med sci | vol. 7, no. 1, january-february 2021: 6 – 22 145. yi l, li z, yuan k, qu x, chen j, wang g, et al. small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells. j virol. 2004;78(20):11334-9. 146. ul qamar mt, alqahtani sm, alamri ma, chen l-l. structural basis of sarscov-2 3clpro and anti-covid-19 drug discovery from medicinal plants. 2020. 147. yu m-s, lee j, lee jm, kim y, chin y-w, jee j-g, et al. identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13. bioorg med chem lett. 2012;22(12):4049-54. 148. li s-y, chen c, zhang h-q, guo h-y, wang h, wang l, et al. identification of natural compounds with antiviral activities against sars-associated coronavirus. 2005;67(1):18-23. 149. cinatl j, morgenstern b, bauer g, chandra p, rabenau h, doerr hjtl. glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus. 2003;361(9374):2045-6. 150. jo s, kim h, kim s, shin dh, kim ms. characteristics of flavonoids as potent mers‐cov 3c‐like protease inhibitors. chem biol drug design. 2019;94(6):2023-30. 151. liu h, ye f, sun q, liang h, li c, lu r, et al. scutellaria baicalensis extract and baicalein inhibit replication of sars-cov-2 and its 3c-like protease in vitro. biorxiv. 2020. 152. esposito f, carli i, del vecchio c, xu l, corona a, grandi n, et al. sennoside a, derived from the traditional chinese medicine plant rheum l., is a new dual hiv-1 inhibitor effective on hiv-1 replication. phytomedicine. 2016;23(12):1383-91. 153. yang c-m, cheng h-y, lin t-c, chiang l-c, lin c-cjjoe. the in vitro activity of geraniin and 1, 3, 4, 6-tetra-o-galloyl-β-d-glucose isolated from phyllanthus urinaria against herpes simplex virus type 1 and type 2 infection. 2007;110(3):555-8. 154. sanna c, rigano d, cortis p, corona a, ballero m, parolin c, et al. onopordum illyricum l., a mediterranean plant, as a source of anti hiv-1 compounds. plant biosyst int j dealing aspects plant bio. 2018;152(6):1274-81. 155. tsai yc, lee cl, yen hr, chang ys, lin yp, huang sh, et al. antiviral action of tryptanthrin isolated from strobilanthes cusia leaf against human coronavirus nl63. biomolecules. 2020;10(3). 156. schwarz s, sauter d, wang k, zhang r, sun b, karioti a, et al. kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus. planta med. 2014;80(2-3):177-82. 157. haq fu, roman m, ahmad k, rahman su, shah sma, suleman n, et al. artemisia annua: trials are needed for covid-19. phytother res. 2020. 158. cheng pw, ng lt, chiang lc, lin cc. antiviral effects of saikosaponins on human coronavirus 229e in vitro. clin exp pharmacol physiol. 2006;33(7):612-6. 159. kim hj, lee js, woo er, kim mk, yang bs, yu yg, et al. isolation of virus-cell fusion inhibitory components from eugenia caryophyllata. planta med. 2001;67(3):277-9. 160. ovenden sp, yu j, wan ss, sberna g, tait rm, rhodes d, et al. globoidnan a: a lignan from eucalyptus globoidea inhibits hiv integrase. phytochemistry. 2004;65(24):3255-9. 161. chen h, jie c, tang lp, meng h, li xb, li yb, et al. new insights into the effects and mechanism of a classic traditional chinese medicinal formula on influenza prevention. phytomed int j phytother phytopharmacol. 2017;27:52-62. 162. wang c, cao b, liu q-q, zou z-q, liang z-a, gu l, et al. oseltamivir compared with the chinese traditional therapy maxingshigan–yinqiaosan in the treatment of h1n1 influenza: a randomized trial. ann intern med. 2011;155(4):217-25. 163. zhang t, xiao m, wong c-k, mok k-pc, zhao x, ti h, et al. sheng jiang san, a traditional multi-herb formulation, exerts anti-influenza effects in vitro and in vivo via neuraminidase inhibition and immune regulation. bmc complement alternat med. 2018;18(1):150. 164. runfeng l, yunlong h, jicheng h, weiqi p, qinhai m, yongxia s, et al. lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (sars-cov-2). pharmacol res. 2020;156:104761. 165. hu k, guan w-j, bi y, zhang w, li l, zhang b, et al. efficacy and safety of lianhuaqingwen capsules, a repurposed chinese herb, in patients with coronavirus disease 2019: a multicenter, prospective, randomized controlled trial. phytomed int j phytother phytopharmacol. 2020:153242. 166. ma q, yu q, xing x, liu s, shi c, luo j. san wu huangqin decoction, a chinese herbal formula, inhibits influenza a/pr/8/34 (h1n1) virus infection in vitro and in vivo. viruses. 2018;10(3). 167. kiran g, karthik l, shree devi ms, sathiyarajeswaran p, kanakavalli k, kumar km, et al. in silico computational screening of kabasura kudineer official siddha formulation and jacom against sars-cov-2 spike protein. j ayurv integr med. 2020. 168. mishra nn, agarwal a, moitra t, polachira sk, nair r, gupta sk. anti-hiv-1 activity and safety profile of a polyherbal gel formulation as a candidate microbicide. j herb med. 2019;17-18:100284. 169. zhong l, yang w, lam wc, bian z-x, wong v, cho s, et al. potential targets for treatment of coronavirus disease 2019 (covid-19): a review of qing-feipai-du-tang and its major herbs. am j chin med. 2020;48. 170. yang l, li y-t, miao j, wang l, fu h, li q, et al. network pharmacology studies on the effect of chai-ling decoction in coronavirus disease 2019. trad med res. 2020;5(3):145-59. 171. tai zy, tay l, goh rm, zhou rs, wong lh, wong po. mixed chinese herbs and western medicine for novel coronavirus disease 2019 (covid-19): a mixed method review. medrxiv. 2020. 172. jassim saa, naji ma. novel antiviral agents: a medicinal plant perspective. j appl microbiol. 2003;95(3):412-27. 173. ang l, song e, lee hw, lee ms. herbal medicine for the treatment of coronavirus disease 2019 (covid-19): a systematic review and meta-analysis of randomized controlled trials. j clin med. 2020;9(5):1583. this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.896 256 j contemp med sci | vol. 6, no. 6, november–december 2020: 256–261 review issn 2413-0516 introduction migration as a global phenomenon that could be highly motivated by improving individual’s perceptions about better opportunities and quality of life in other affluent countries.1 in 2018, about 257 million people in the world migrated from their home lands, half of them migrating from lowand middle-income countries (lmics).2 migration of healthcare workers is one of the most significant reasons contributing to drain of human resources from healthcare systems particularly in lmics.3 human resource as a significant and key part of health systems can influence the quantity and quality of healthcare services.4 world health organization (who) raised concern about a global shortage of 18 million healthcare workers by 2030, which might have additional negative effect on resource poor countries.5 active recruitment of healthcare workers, mainly from lmics have been the compensation strategy of some high-income countries.6 about 25 percent of physicians working in the usa, canada, australia, and the uk are international medical graduates, with half of them having migrated from low-income countries.7 australia is classified as a country with highest number of foreign dentists between oecd countries.8 one out of four dentists in australia is an international dental graduate mainly from developing countries.9 decision for migration can be influenced by various factors mostly described with a simple model of push and pull, defined as factors driving people away from their home country and factors attracting them by a foreign country.10 financial incentives, professional development, and better quality of life were indicated as the main driving factors for healthcare workers migrating from lmics.11 the most significant push factors, as reported by south-african doctors, were insecurity, high level of crimes, and racial conflicts.12,13 ambition of working and studying in a country with a developed healthcare system were among the most significant pull factors influenced healthcare workers’ decisions for migration to the uk.14 more than half of the iranian health workers who intended to migrate were concerned about structural and professional factors such as unfavorable educational environment and inter-professional inequity.15 healthcare worker migration could influence both source and destination countries. however, the source countries are most affected by its negative impacts due to loss of skilled human resources.16 this could influence the quality and quantity of healthcare provision, increasing workload for the remaining workforce along with healthcare inequalities especially in deprived area.17 in 2010, who introduced a code of practice for moral guidance of recruiting the workforces for health sector.18 in this code, member countries were encouraged to collaborate in improving research programs about migration of the healthcare forces.18 given the importance of migration of physicians and dentists on global burden of disease as the key members of factors influencing the migration intention of health professionals in lowandmiddle income countries: critical review with a theoretical model sara hajian1, shahram yazdani2, mohammadpooyan jadidfard3, mohammadhossein khoshnevisan4 1preventive dentistry research center, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran. 2school of medical education, shaheed beheshti university of medical sciences, tehran, iran. 3school of dentistry, shaheed beheshti university of medical sciences, tehran, iran. 4head of preventive dentistry research center, research institute of dental sciences, shahid beheshti university of medical sciences, tehran, iran. corresponding author: mohammad hossein khoshnevisan ( e-mail: khoshmh@gmail.com) key words: dental graduates, medical graduates, brain drain, migration, lowand middle-income country (submitted: 14 september 2020 – revised version received: 28 september 2020 – accepted: 16 october 2020 – published online: 26 december 2020) abstract objective migration of healthcare workers could result in shortage of human resources and rising inequalities in service provision in resource poor countries. the aim of this review was to determine the factors influencing the migration decisions of medical and dental graduates migrating from lowand middle-income countries as well as introducing a practical model for health professional’s migration. methods google scholar and pubmed were searched together with relevant journals for english studies from january 2005 to january 2020. the original studies which evaluated the motivational factors of dental and medical graduates migrating from lowor middle-income countries were included. the migration model was developed by investigating the factors and frameworks of selected studies. results twenty-five articles met the inclusion criteria. push and pull theory was the most popular way to describe the driving factors of migration. these factors were classified into three: (i) macro-, (ii) meso-, and (iii) micro-level with eight key domains. poor income, unfavorable socioeconomic situation, political instability, lack of professional and educational opportunities together with family and personal concerns are found as strong common reasons perpetuating migration. conclusion despite the fact that health workers migrate for different reasons, they follow a same route for decision to stay or leave their home countries. unfulfillment of expectations in mother land in addition to media reconstructed reality of life in foreign land can develop a positive attitude for better quality of life improvement after migration. once individuals could overpass their national identity and barriers of migration, the final decision toward migration would be more feasible. 257 original factors influencing the migration intention of health professionals in lowandmiddle income countriessara hajian j contemp med sci | vol. 6, no. 6, november–december 2020: 256–261 fig. 1 the paper selection process based on prisma 2009 flowchart. healthcare provider teams, studies focused on rate and reasons of health professional migration through modeling were limited. thus, through this study we aim to review available literature to model the factors affecting migration of physicians and dentists from lmics who act as a supply source for overseas doctors. materials and methods a critical review was conducted by defining the scope of the review, identifying the sources of relevant data, reviewing the literature, and applying the literature to the present study.19 the population, issue, context, outcome (pico) elements were defined and variations and combinations of keywords were used by appropriate boolean operators (table 1). electronic databases such as pubmed, google scholar, and relevant journals were systematically searched for english language publications from january 2005 to january 2020. the inclusion criteria were: (1) studies stated factors affecting migration decisions of physicians and/or dentist. (2) the study participants were from a lowor middle-income country. (3) original research both qualitative and/or quantitative that assessed dental and/or medical graduates among their participants. articles with mixed source countries including highand lowor middle-income countries as well as studies assessing mobilization of physicians and dentists within european and other high-income countries were excluded. titles and abstracts were screened by two authors (sa and mj) for possible inclusion in the study following the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines.20 appropriate studies were assessed with regards to the inclusion and exclusion criteria. full texts of selected studies were carefully shortlisted regarding the eligibility criteria and methodological aspects. original articles investigated the migration of dental or medical graduates from lowand middle-income countries or proposed a model of migration were included. after reviewing the full texts, all authors were involved in analysis and reaching consensus on the factors emerging from the data. the influencing factors of migration categorized into macro(national factors), meso(professional factors) and micro(personal factors) levels.21 two reviews and one study of dentists’ migration were also added to our results because of their useful data and models. after explaining frameworks and models of included studies and writing our critics, we introduced a theoretical model of migration to explain how physicians and dentists decided to migrate. results from 1117 studies identified through electronic searching, 272 related abstracts were considered for further evaluation after applying the eligibility criteria. by reading 74 full texts, a total of 25 studies met the criteria to be included in the review (fig. 1). an overview of numbers of studies with regards to the source country, type of study. and professional groups participated in the study were provided in table 2. the selected studies reported different push and pull factors but they were mostly under common key domains. all domains were categorized into three groups of macro-, meso-, and micro-levels based on the framework of young’s model and illustrated in table 3.21 macro-level factors related to national factors include health system, political, economic, and social factors. meso-level factors included professional specific factors focusing on training and career opportunities and job condition. micro-level factors pointed to personal factors and individual attitudes toward migration including personal fulfilment and family factors. professional factors including training and job progression opportunities as well as higher income were the most reported factors driving migration. most of the studies mentioned motivational factors in a descriptive way reporting push and pull factors as positive and negative factors of origin and destination countries. overall, three original studies were found presenting a conceptual or theoretical framework about migration of dental or medical graduates, which are discussed in the following. table 1. the population, issue, context, outcomes (pico) and the related keywords of the study the population, issue, context, outcomes (pico) main keywords population medical and dental graduates health profession or medical or medicine or doctor or dentists or dental or oral health issues migration motivations migration or immigration or brain drain context lowand middle-income countries low income or middle income or developing outcomes migration model model or modelling or framework 258 original factors influencing the migration intention of health professionals in lowandmiddle income countries sara hajian j contemp med sci | vol. 6, no. 6, november–december 2020: 256–261 oberoi et al. addressed the migration reasons of southafrican doctors through an adopted conceptual framework which divided push and pull factors into two “endogenous” and “exogenous” categories with regards to the factors in or out of the health system and health profession.22 the results show that low wages, lack of job satisfaction, lack of professional development, poor working conditions, and risk of contracting aids were among the most significant endogenous factors of migration.22 while, lack of quality of life, high level of crimes, civil conflicts, political instability, and social pressures were counted as the most significant exogenous factors.22 in this study “stick” factors – the reasons why a person decided to stay in a home country before considering push and pull factors of migration – and “stay” factors – the reasons why a person decided to stay in a foreign country – have also been investigated.22 the stay factors referred to further education and pursuit of specialized qualification along with children’s familiarity with school setting.22 thus, considering stick factors as important factors before looking at push and pull factors of migration would be beneficial. akl et al. drew a model through investigating the reasons behind lebanese medical students’ intention to migrate.41 factors playing independent and significant roles in migration were highlighted in the model including residency training, future professional career, and political conditions.41 societal expectations, marketing of studying abroad advertising by doctors with experience of training abroad and prevalent culture of migration among family and friends living abroad or residents retraining abroad were also among the most important driving factors of migration.41 the factors inhibiting table 2. numbers of included articles by their source country, world bank income groups, type of study and type of study participants. source country (who regions) world bank income groups type of study professional groups middle east and north africa: 7 south asia: 7 sub-saharan africa & east asia and pacific: 9 latin america and the caribbean: 2 low income: 3 lower middle income: 11 upper middle income: 11 qualitative study: 8 quantitative study: 13 mixed methods: 4 medical graduates: 16 dental graduates: 1 health workers: 8 table 3. migration motives of dental and medical graduates in lowand middle-income countries. domain factors and references macro-level: national factors health system poor income22-35, selective system of students and academic members34, oversaturation of job market25 economic poor economic condition and devaluation of national currency15,30,34, high costs of living31,36 political insecurity and high level of crime12,22,25,30,32,34,36-40, political instability22,25,28,29,39,41,42, terrorism27, freedom of expression34,40, high level of corruption12,25,36,9favoritism27,28,39, lack of meritocracy34, poor law and order enforcement35 social gender inequalities34, ethnic and racial discriminations12, low quality of social services, culture of migration and social pressure22,24-27,40,41, poor social rights25, religious conflicts27,30,34 meso-level: professional factors educational environment and professional opportunities unavailability of enough post graduate training opportunities22,24,25,28,29,31,35,39-44, limitation of positions in a particular specialty and academic career25, unfair competition for training position25, inexplicit curriculum25,32, poor research opportunities and networking15,25,28,34, limitation on career opportunities22,27,32,33,35,37,39,41,45, poor access to enhanced technology23,34, role modeling of instructors24, poor quality of training26,27,30,34,38, lack of using optimal knowledge and experiences of graduates34 work conditions poor work environment and infrastructures15,22,26-29,31-33,36,37, staff shortage12,25, workload25,27,32,33,37, job dissatisfaction22,26,27,32, risk of contracting a serious illness12,22,37, poor resource availability and equipment quality12, management issues and appreciation27,28, poor work ethos12, inter-professional inequity15,34, professional prestige and respect for foreign-trained professionals15,25,32,36,45 micro-level: personal factors personal fulfilment adventure37, better quality of life15,22,27,43, financial gain (welfare)37,43,45, learning a new language34, desire to settle abroad15,26,27,34, desire to change immediate circumstances37 family concerns better future and education for children15,23,28,34,36,37,39, partner or parent decision for migration27, having a family living abroad27,32,38,39,41,42 259 original factors influencing the migration intention of health professionals in lowandmiddle income countriessara hajian j contemp med sci | vol. 6, no. 6, november–december 2020: 256–261 migration from countries of origin to countries of destination were termed “repel & retain” factors which were classified into four: (i) individual, (ii) social, (iii) occupational, and (iv) political categories.41 the inhibitive role of certain barriers such as financial problems, equivalency exams, and visa process as well as the role of social pressure and marketing of abroad training as mediating factors affecting the migration decision of medical graduates have been highlighted.41 this model draw attention to the barriers and facilitators which play a pivotal role in the final decision of migration. although, the findings could not show a clear causality relationship between push– pull factors and the decision of migration. in a study of factors motivating dental practitioners migration from pakistan, local and foreign push and pull factors were reported for source and destination countries, respectively.35 significant local push factors were dissatisfaction with law and order, salary, job opportunities, while family responsibilities reported the most important local pull for migration.35 meanwhile, enhancing knowledge and skills, financial gains, and better working environment considered as major foreign pull factors.35 participants indicated their concern about foreign push factors such as racism and intense competition.35 the time relationship between individuals ‘attention to motivational factors and inhibitors is not well defined. it is not clear when a person thinks about push and pulls or barriers and facilitators of migration. to reach a common language, we introduce a model considering mentioned factors and frameworks in addition to further steps a person should take in order to reach a final decision for migration (fig. 2). first of all, individuals have usually started weighing up the push and pull factors of home and destination countries after experiencing a major dissatisfaction in their own social, political, economic, and professional life. although some personal reasons such as migration of other family members or a sense of adventure could be exceptions. when needs and desires which build a profile of expectations are not fulfilled in home countries and on the other hand, marketing abroad and media reconstructed reality of life in destination land set up a high perceived quality of life after migration, a person would hold a positive attitude toward migration. if individuals’ value framework including national identity and social commitment are not strong enough for retention, the intention for migration leads to an important decision of migration. not to mention, there are some migration barriers on accreditation and equivalency process, job opportunity, financing, and ability to obtain visas which may affect this decision. as if the barriers pull over, migration could be facilitated or expedited. it is noteworthy to mention that globalization and understanding better opportunities in other affluent countries may cast a shadow over the whole process of decision making in migration of health professionals. fig. 2 a theoretical model for dental and medical graduates migrating from lowand middleincome countries. 260 original factors influencing the migration intention of health professionals in lowandmiddle income countries sara hajian j contemp med sci | vol. 6, no. 6, november–december 2020: 256–261 discussion physicians and dentists’ migration from lmics could not only affect health systems regarding healthcare provisions and quality of care, but also lead to some inevitable losses of human and financial resources. in this review, some significant motivational factors of migration such as national, professional, and personal factors were categorized into three levels of macro, meso and micro, respectively. most of the articles explained the migration reasons in a descriptive way using the push and pull model of migration. we tried to organize driving factors by adding some time sequences between different steps of the migration process as illustrated by a schematic model. dohlman et al. used maslow’s hierarchy of human needs to reach a common language on physician migration.46 in this model, the motivating factors were categorized into five levels: (i) physiological needs, (ii) safety needs, (iii) social needs, (iv) esteem needs, and (v) self-actualization needs.46 from 19 included studies, physical and financial security needs (level 2), a desire to improve educational or professional opportunities (level 4), and “being all you can be” (level 5) were mentioned most.46 the lower level of needs is considered more important as it must first be met in order for a person to think about the next levels while a person could reach esteem needs without having basic needs. moreover, the discrimination between the esteem needs and self-actualization in this study is not clearly defined. while the desire for educational and professional development opportunities were considered equivalent to self-esteem, the research opportunities and career opportunities were mentioned for self-actualization needs. it should be a better detailed explanation for categorizing each factor. the positive point would be the emphasis on the role of higher level needs in migration decisions of elite health workers as they could easily afford the basic needs. blacklock et al. addressed the health workers’ reasons for migration from africa by systematic review of qualitative studies.47 push factors were divided into five categories: (i) economic, (ii) workplace, (iii) health system, (iv) others’ understanding, and (v) expectations and social setting. the pull factors were divided into two groups of “attractiveness of working and living abroad through exchange of information” and “vivid improvements in workplace, economic, social setting, and family factors”.47 the study introduced six lines of arguments through meta-ethnographic synthesis that include: (1) struggle to realize unmet material expectations of self, family and society, 2) strain and emotion, interpersonal discord, and insecurity in workplace, 3) fear from threats to personal or family safety, in and out of workplace, 4) absence of adequate professional support and development, 5) desire for professional prestige and respect, 6) conviction that hopes and goals for the future will be fulfilled overseas.47 this study contributes to the current knowledge of reasons behind migration of health workforce by providing new theoretical explanation of migration through meta-ethnographic synthesis. the study is not only limited to listing the typical motivating factors but also the six lines of arguments draw our attention to sociopsychological aspects of migration. dentists may consider different factors for their migration regarding the private nature of their practice and duration of dental training compared to medical doctors. balasubramanian et al. conducted a study on international dentists migrating to australia.9 by taking the conceptual model known as “explanatory framework” for this study, interest in migration was illustrated through four subordinate themes: (i) “being good at something”, (ii) “feelings of being let down”, (iii) “a novel experience,” and (iv) “influenced by someone”. a superordinate theme called, “global interconnectedness” was found common among the participants.9 this model emphasizes on complicated nature of migration stimuli and signifies the role of social networks and communities in the globalized world. higher levels of needs, desire for respect and feelings of being let down, reminded us of the different desires of highly skilled individuals which should prioritize in planning strategies for migration. in our model, we also try to mention the role of social pressure and media on marketing abroad and establishing the culture of migration in developing countries. however, detailed push and pull or repel and retain factors are not illustrated in the model mainly due to the country specific nature of these factors. conclusion in this study, driving factors of migration were classified into three levels of macro, meso and micro and eight domains. factors such as the desire for higher income, better job, educational opportunities, security, and better quality of life for children were mentioned most. low perceived quality of life in source country which is highly affected by social pressure besides high perception of quality of life in destination country mostly mediated by media reconstructed reality, can push a person into thinking about migration. a weak national identity and high capacity for overcoming migration barriers such as financial support can lead health professionals to leave their home countries. acknowledgments this study was supported by the preventive research center, research institute of dental sciences, dental school, shaheed beheshti university of medical sciences, tehran, iran. the authors would like to thank dr. ali kazemian and dr. abbas zabihzadeh for their kind help in developing the migration model. conflict of interest disclosure the author(s) declared no conflicts of interest in this study. references 1. aluttis c, bishaw t, frank mw. the workforce for health in a globalized context-global shortages and international migration. global health action. 2014;7:23611. 2. global migration trends factsheet. berlin. global migration data analysis centre, iom; 2017. accessed accessed 13 sep 2017. 3. lofters a, slater m, thulien n. the “brain drain”: factors influencing physician migration to canada. health. 2013;5(01):125. 4. the world health report 2006: working together for health. world health organization; 2006. 5. limb m. world will lack 18 million health workers by 2030 without adequate investment, warns un. bmj (clin res ed.). sep 22 2016;354:i5169. 6. robinson m, clark p. forging solutions to health worker migration. the lancet. 2008;371(9613):691-693. 7. chen pg, mn-s, berg d, gozu a, rulisa s, curry la. international medical graduates in the usa: a qualitative study on perceptions of physician migration. bmj open. 2011:1-8. 261 original factors influencing the migration intention of health professionals in lowandmiddle income countriessara hajian j contemp med sci | vol. 6, no. 6, november–december 2020: 256–261 8. immigrant health workers in oecd countries in the broader context of highly skilled migration. in: oecd international migration outlook 2007. paris: oecd publishing, 161–228. 9. balasubramanian m, brennan ds, spencer aj, short sd. the ‘global interconnectedness’ of dentist migration: a qualitative study of the lifestories of international dental graduates in australia. health policy plan. 2014;30(4):442-450. 10. lee es. a theory of migration. demography. 1966;3(1):47-57. 11. willis-shattuck m, bidwell p, thomas s, wyness l, blaauw d, ditlopo p. motivation and retention of health workers in developing countries: a systematic review. bmc health services res. dec 4 2008;8:247. 12. bidwell p, laxmikanth p, blacklock c, et al. security and skills: the two key issues in health worker migration. global health action. 2014;7:24194. 13. witt j. addressing the migration of health professionals: the role of working conditions and educational placements. bmc public health. nov 18 2009;9 suppl 1:s7. 14. davda ls, gallagher je, radford dr. migration motives and integration of international human resources of health in the united kingdom: systematic review and meta-synthesis of qualitative studies using framework analysis. human resour health. jun 27 2018;16(1):27. 15. asadi h, ahmadi b, nedjat s, sari aa, gorji ha, zalani gs. factors affecting intent to immigration among iranian health workers in 2016. electron phys. jun 2017;9(6):4669-4677. 16. stilwell b, diallo k, zurn p, vujicic m, adams o, dal poz m. migration of health-care workers from developing countries: strategic approaches to its management. bull world health organ. 2004;82:595-600. 17. naicker s, plange-rhule j, tutt rc, eastwood jb. shortage of healthcare workers in developing countries--africa. ethnicity dis. spring 2009;19(1 suppl 1):s1-60-64. 18. dayrit m, taylor a, yan j, et al. who code of practice on the international recruitment of health personnel. bull world health organ. oct 2008;86(10):739. 19. carnwell r, daly w. strategies for the construction of a critical review of the literature. nurse educ pract. 2001;1(2):57-63. 20. moher d, shamseer l, clarke m, et al. preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015 statement. systemat rev. 2015;4(1):1. 21. young r. motivations and experience of health professionals who migrate to the united kingdom. evaluation of international recruitment of health professionals in england. j health services res policy. 2010;15(4):195-203. 22. oberoi ss, lin v. brain drain of doctors from southern africa: brain gain for australia. austr health rev publ austr hosp assoc. feb 2006;30(1):25-33. 23. astor a, akhtar t, matallana ma, et al. physician migration: views from professionals in colombia, nigeria, india, pakistan and the philippines. social sci med (1982). dec 2005;61(12):2492-2500. 24. hagopian a, ofosu a, fatusi a, et al. the flight of physicians from west africa: views of african physicians and implications for policy. social sci med (1982). oct 2005;61(8):1750-1760. 25. akl ea, maroun n, major s, et al. why are you draining your brain? factors underlying decisions of graduating lebanese medical students to migrate. social sci med (1982). mar 2007;64(6):1278-1284. 26. syed na, khimani f, andrades m, ali sk, paul r. reasons for migration among medical students from karachi. med educ. jan 2008;42(1):61-68. 27. sheikh a, naqvi sh, sheikh k, naqvi sh, bandukda my. physician migration at its roots: a study on the factors contributing towards a career choice abroad among students at a medical school in pakistan. global health. dec 15 2012;8:43. 28. fouad ya, fahmy ym, abdel hady sm, elsabagh ae. egyptian future physicians are packing to leave but may be willing to return. int health. may 2015;7(3):190-194. 29. kizito s, mukunya d, nakitende j, et al. career intentions of final year medical students in uganda after graduating: the burden of brain drain. bmc med educ. 2015;15(1):122. 30. hossain n, shah n, shah t, lateef sb. physicians’ migration: perceptions of pakistani medical students. j coll phys surg--pak jcpsp. aug 2016;26(8):696-701. 31. tomblin murphy g, mackenzie a, waysome b, et al. a mixed-methods study of health worker migration from jamaica. human resour health. jun 30 2016;14(suppl 1):36. 32. castro-palaganas e, spitzer dl, kabamalan mm, et al. an examination of the causes, consequences, and policy responses to the migration of highly trained health personnel from the philippines: the high cost of living/ leaving-a mixed method study. human resour health. mar 31 2017;15(1):25. 33. walton-roberts m, runnels v, rajan si, et al. causes, consequences, and policy responses to the migration of health workers: key findings from india. human resour health. apr 5 2017;15(1):28. 34. asadi h, ahmadi b, nejat s, et al. factors influencing the migration of iranian healthcare professionals: a qualitative study. plos one. 2018;13(6):e0199613. 35. firdous sn, naqvi smzh, akhter m. factors affecting migration abroad of dental practitioners from karachi: a cross-sectional survey. jpma j pak med assoc. 2019;69(10). 36. labonte r, sanders d, mathole t, et al. health worker migration from south africa: causes, consequences and policy responses. human resour health. dec 3 2015;13:92. 37. mm b. reasons for doctor migration from south africa. sa fam pract. 2009;51(3):211-215. 38. al-khalisi n. the iraqi medical brain drain: a cross-sectional study. int j health serv. 2013;43(2):363-378. 39. bleeker h. factors influencing guyanese health worker migration to canada. univ toronto med j. 2016;93(3):27. 40. schumann m, maaz a, peters h. doctors on the move: a qualitative study on the driving factors in a group of egyptian physicians migrating to germany. global health. jan 7 2019;15(1):2. 41. akl ea, maroun n, li ck, grant bj, schünemann hj. factors influencing lebanese medical students’ decisions to train abroad: evaluation of a conceptual framework. open public health j. 2012;5:19-27. 42. poppe a, jirovsky e, blacklock c, et al. why sub-saharan african health workers migrate to european countries that do not actively recruit: a qualitative study post-migration. global health action. 2014;7:24071. 43. de silva nl, samarasekara k, rodrigo c, samarakoon l, fernando sd, rajapakse s. why do doctors emigrate from sri lanka? a survey of medical undergraduates and new graduates. bmc res notes. dec 16 2014;7:918. 44. balasubramanian m, spencer aj, short sd, watkins k, chrisopoulos s, brennan ds. characteristics and practice profiles of migrant dentist groups in australia: implications for dental workforce policy and planning. int dent j. jun 2015;65(3):146-155. 45. imran n, azeem z, haider, ii, amjad n, bhatti mr. brain drain: post graduation migration intentions and the influencing factors among medical graduates from lahore, pakistan. bmc res notes. oct 17 2011;4:417. 46. dohlman l, dimeglio m, hajj j, laudanski k. global brain drain: how can the maslow theory of motivation improve our understanding of physician migration? int j environ res public health. 2019;16(7):1182. 47. blacklock c, ward am, heneghan c, thompson m. exploring the migration decisions of health workers and trainees from africa: a meta-ethnographic synthesis. social sci med (1982). jan 2014;100:99-106. https://doi.org/10.22317/jcms.v6i6.897 this work is licensed under a creative commons attribution-noncommercial 3.0 unported license which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.